Block copolymers for stable micelles.

(54) Title: BLOCK COPOLYMERS FOR STABLE MICELS. (54) Title: BLOCK COPOLYMERS FOR STABLE MICELLES.

(57) Summary

The present invention relates to the field of polymer chemistry and more particularly to multi-block copolymers and micelles comprising the same. The compositions herein are useful for drug delivery applications.

(57) Abstract

The present invention relates to the field of polymer chemistry and more particularly to multiblock copolymers and micelles comprising the same. Compositions herein are useful for drug-delivery applications.

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PATENT TITLE NO. 340992 _SE_ would know w kowmM

Mexican Institute of Industrial Property

Owner (s): INTEZYNE TECHNOLOGIES, INC.

Address: 3720 Spectrum Boulevard, Suite 104, Tampa, Florida, 33612, USA

Denomination: BLOCK COPOLYMERS FOR STABLE MICELLAS.

Classification: lnt.CI.8: A61K31 / 337; A61K47 / 34; C08G63 / 48

Inventor (s): KEVIN SILL; ADAM CARIE; JOSEPH EDWARD SEMPLE; TOMAS VOJKOVSKY

Number:

MX / a / 2014/011980

REQUEST

International filing date of March 2013

PRIORITY

Country:

US

US

Validity: Twenty years

Date:

April 2012 June 14, 2012

Number:

61/622,755

61/659,841

Expiration Date: March 15, 2033

The reference patent is granted based on articles 1, 2, fraction V, 6 fraction III, and 68 of the Industrial Property Law.

In accordance with article 23 of the Industrial Property Law, this patent has a validity of twenty non-extendable years, counted from the date of filing of the international application and will be subject to the payment of the fee to maintain the rights in force. .

Whoever endorses this title does so based on the provisions of articles β * liaceones lll and 7 ° bis 2 of the Industrial Property Law (Mario Oficed de la Federación (D OF) 06/27/1801, amended ef 02 / 08/1994 10/25/1996, 12/26/1987, 05/17/1999, 01/26/2004, 06/16/2006 ¾ 01/25/2906, 05/06/2009, 01/06/2010 , 1NMB2MQ, * * βΜ10. 27Ί31®βΤ2 and 04/09/2012); Articles 1® 3rd fraction V subsection a), 4th and 12th fractions I and lll of the Regulations of the Mexican Institute of Industrial Property (DOF 14/12 / 19®, amended on 07/01/2002, 15/07 / 2004; 07/28/2004 and 09/07/2007); Articles 1, 3, 4, 5, section V, Section a), 16 sections I and III and 30 of the Organic Statute of the Mexican Institute of Industrial Property! (DDF. 12/27/1999, amended on 10/10/2002, S9f0? F2004, 04 ^ (2004 and 09/13/2007); 1, 3 and 5 Clause a) of the Agreement that delegates powers to the Deputy Directors General, Coordinator, Divisional Directors, Holders of the Regional Offices, Divisional Deputy Directors, Departmental Coordinators and other subordinates of the Mexican Institute of Industrial Property. (DOF, 12/15/1999, amended on 02/04/2000, 07/29/2004, 08/04/2004 and 09/13/2007).

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Issue Date: August 3, 2016

THE DIVISIONAL DIRECTOR OF PATENTS

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NAHANNY CANAL REYES

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Arenal No. 55C. 1st floor.

Coi Pueble Santa María Tepepan.

Xochirniico. C P. 16020,

Mexico City

Teí (55) 53 34 07 00 www.impi qob ni

MX / 201 S / 61542

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Field of the Invention

The present invention relates to the field of polymer chemistry and more particularly to multi-block copolymers and uses thereof.

Background of the Invention

The development of new therapeutic agents has dramatically improved the quality of life and survival rate of patients suffering from a variety of disorders. However, innovations for drug delivery are necessary to improve the success rate of these treatments. Specifically, delivery systems are still required to effectively minimize the premature excretion and / or metabolism of therapeutic agents and to deliver these agents specifically to diseased cells thereby reducing their toxicity to healthy cells.

Rationally designated nanoscopic drug carriers, or nanovectors, offer a promising procedure for achieving these goals because of their inherent ability to overcome many biological barriers. However, its multi-functionality allows the incorporation of cell target groups, diagnostic agents, and a multitude of drugs into a single delivery system. The

REF .: 251186 polymer micelles, formed by the assembly

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Functional amphiphilic block copolymers represent a remarkable type of multifunctional nanovector.

Polymer micelles are particularly attractive because of their ability to deliver large payloads of a variety of drugs (eg, small molecule, protein, and DNA / RNA therapeutics), their improved in vivo stability compared to other colloidal carriers (for example, liposomes), and its nanoscopic size which allows passive accumulation in diseased tissues, such as solid tumors, due to its improved retention and permeation effect (EPR, for its acronym in English). Using appropriate surface functionality, the polymer micelles are further decorated with target groups to the cell and permeation enhancers that can actively target diseased cells and aid in cell entry, resulting in improved cell-specific delivery.

While self-assembly represents a convenient method for bottom-up design of nanovectors, the forces that drive and sustain the assembly of polymer micelles are concentration-dependent and inherently reversible. In clinical applications, where polymer micelles are rapidly diluted after administration, this reversibility,

IMPI together with high concentrations of componerl'fce®TG «sjangu ^J OF THE ROHEDAD

INDUSTRIAL micelle destabilizers (eg protein,

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lipids, and phospholipids), often leading to premature dissociation of drug-loaded micelles before the active or passive target is effectively achieved. In order for polymer micelles to fully reach their cell-directed potential and exploit their contemplated multi-functionality, in vivo circulation time must be improved. Drug delivery vehicles are required, which are infinitely stable for post-administration dilution, can bypass biological barriers (eg, reticuloendothelial system (RES) uptake, and delivery drugs in response to physiological environment found in diseased tissues, such as solid tumors.

Brief Description of the Figures Figures la and Ib. Schematic illustrations representing the triblock copolymer and polymer micelle of the present invention.

Figure 2. Schematic illustrations showing the preparation of drug-loaded micelles.

Figure 3. Schematic illustrations showing the crosslinking of a drug loaded micelle with metal ions.

Figure 4. Schematic illustrations depicting the crosslinked drug-loaded micelle of the

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MFXICAN INSTITUTE OF INDUSTRIAL PROPERTY.

encapsulation present invention.

Figure 5. Validation of daunorubicin by dialysis of the non-withdrawn formulation Τ 'mg / ml (black bar) and 0.2 mg / mL (white bar) for 6 hours against phosphate buffer at pH 8.

Figure 6. Verification of iron-dependent crosslinking by dialysis at 0.2 mg / mL in phosphate buffer at pH for 6 hours.

Figure 7. Verification of time dependence on iron-mediated crosslinking by dialysis at 0.2 mg / mL in phosphate buffer at pH 8 for 6 hours.

Figure 8. The uncrosslinked sample was reconstituted at 2 0 mg / ml and pH adjusted to 3, 4, 5, 6, 7, 7.4, and 8 to determine the iron-mediated crosslinking pH dependence. Samples were diluted to 0.2 mg / mL and dialyzed against 10 mM phosphate buffer at pH 8 for 6 hours.

Figure 9. pH dependent release of the crosslinked daunorubicin formulation dialyzed against 10 mM phosphate buffer at pH adjusted to 3, 4, 5, 6, 7,

7.4 and 8 for 6 hours.

Figure 10. Salt-dependent release of the crosslinked daunorubicin formulation at 0.2 mg / mL dialyzed against 10 mM phosphate buffer with NaCl concentrations of 0, 10, 50, 100, 200, 300, 400 or 500 mM.

Figure 11. DLS histogram demonstrating the

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particle size distribution for la-lñhrAuTacJi

MEXICAN INSTITUTE OF PROPERTY. ,. ,, -. INDUSTRIAL crosslinked aminopterm.

Figure 12. Verification of ecapsulation by dialysis of the previous formulation (20 mg / mL, black bar) and below (0.2 mg / mL, white bar) CMC.

Figure 13. Verification of crosslinking and pH-dependent release of aminopterin formulation at 0.2mg / mL by dialysis in 10mM phosphate buffer for 6 hours.

Figure 14. Cell viability for A549 lung cancer cells treated with free aminopterin, uncrosslinked aminopterin formulation, crosslinked aminopterin formulation, uncrosslinked empty micelle vehicle, and crosslinked empty micelle vehicle.

Figure 15. Cell viability for OVCAR3 ovarian cancer cells treated with free aminopterin, uncrosslinked aminopterin formulation, crosslinked aminopterin formulation, uncrosslinked empty micelle vehicle, and crosslinked empty micelle vehicle.

Figure 16. Cell viability for pancreatic PANC-1 cells (folate receptor +) treated with free aminopterin, uncrosslinked aminopterin formulation, crosslinked aminopterin formulation, uncrosslinked empty micelle vehicle, and crosslinked empty micelle vehicle.

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Figure 17. Cell viabilityj Jé,

MEXICAN INSTITUTE pancreatic cancer BxPC3 (free folatfif aminopterin receptor, formulation dg arainopterin na.

crosslinked, crosslinked aminopterin formulation, uncrosslinked empty micelle vehicle and crosslinked empty micelle vehicle.

Figure 18. Concentration of SN-38 in the rat plasma compartment from IT-141 formulation (NHOH; 127C) compared to IT-141 formulation (Asp; 127E) at 10 mg / kg.

Figure 19. Rat pharmacokinetics of SN-38 formulations.

Figure 20. pH dependent release of crosslinked cabizataxel formulation dialyzed against 10mM phosphate buffer at pH adjusted at 3, 4, 5, 6, 7, 7.4 and 8 for 6 hours.

Figure 21. Free daunorubucine pharmacokinetics and daunorubicin formulations in rats.

Figure 22. Plasma levels in cabizataxel rat after administration of the crosslinked and cabizataxel-free cabizataxel formulation.

Figure 23. Anti-tumor efficacy of cross-linked SN-39 formulations in a HCT-116 xenograft model.

Figure 24. Biodistribution of aminopterin from crosslinked aminopterin formulations in a model of

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xeno graft OVCAR-3.

Figure 25. Anti-tumor efficacy of formulations

Crosslinked aminopterin IMPIs in a MFE296 xenograft model.

Detailed description of the invention

one. General description:

In accordance with one embodiment, the present invention provides a micelle comprising a multiple block copolymer which comprises a polymeric hydrophilic block, optionally a crosslinkable or crosslinked poly (amino acid block), and a D, L poly (amino acid) block hydrophobic blend, characterized in that such a micelle has an inner core, optionally a crosslinkable or crosslinked outer core, and a hydrophilic coating. It will be appreciated that the polymeric hydrophilic block corresponds to the hydrophilic shell, the optionally crosslinkable or crosslinked poly (amino acid block) corresponds to the optionally crosslinked outer core, and the hydrophobic poly (amino acid) D, L-mixed block corresponds to the inner core.

The hydrophobic L-mixed poly (amino acid) block D, as described herein, consists of a mixture of D and L enantiomers to facilitate encapsulation of hydrophobic portions. It is well established that amino acid homopolymers and copolymers,

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consisting of a single stereoisomer, pué ^ áh ™

INDUSTRIAL secondary structures such as the α-helix or β-lamina. See a-Aminoacid-N-Caroboxi-Anhydrides and Related Heterocycles, HR Kricheldorf, Springer-Verlag, 1987. For example, poly (L-benzyl glutamate) typically has an α-helical conformation; however this secondary structure can be altered by a change in solvent or temperature (see Advances in Protein Chemistry XVI, P. Urnes and P. Doty, Academic Press, New York 1961). The secondary structure can also be altered by the incorporation of structurally different amino acids such as amino acids that form β-sheets (for example, proline) or through the incorporation of amino acids with different stereochemistry (for example, mixture of D and L stereoisomers), which result in poly (amino acids) with a random spiral conformation. See Sakai, R .; Ikeda; S .; Isemura, T. Bull Chem. Soc. Japan 1969, 42, 1332-1336, Paolillo, L .; Temussi, PA; Bradbury,

MS; Crane-Robinson, C. Biopolimers 1972, 11, 2043-2052, and

Cho, I .; Kim, JB; Jung, HJ Polymer 2003, 44, 5497-5500.

While methods for influencing the secondary structure of poly (amino acids) have been known for some time, it has been surprisingly discovered that block copolymers possessing a random spiral conformation are particularly useful for encapsulating

IMPI hydrophobic molecules and nanoparticles when industrial similar block polymers that possess a helical segment. See US Patent Application 20080274173. Without wishing to be bound by any particular theory, it is believed that providing block copolymers having a spiral-spiral shape allows efficient packaging and loading of hydrophilic portions within the nucleus of the micelle, while the spherical demands of a rod shape- Spiraling for a helix containing block copolymer results in less effective encapsulation.

The hydrophobic forces driving the aqueous assembly of colloidal drug carriers, such as polymer micelles and liposomes, are relatively weak, and these assembled structures dissociate below a finite concentration known as the critical micelle concentration (CMC, by their acronym in English). The CMC value of polymer micelles is of greater importance in clinical applications because drug-loaded colloidal carriers are diluted in the blood stream after administration and rapidly reach concentrations below CMC (μΜ or less). This dilution effect will lead to dissociation of the micelle and drug release out of the target area and any benefits associated with micelle size (EPR effect) or active target will be lost. While

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IMPI that a lot of research through ^ D ^ * ^ *? ^

INDUSTRIAL focused on identifying polymer micelles with ultra-low CMC values ​​(nM or less), Maysinger (Savic et. Al, Langmuir, 2006, p3570-3578) and Schiochet (Lu et. Al, Macromolecules, 2011, p6002- 6008) have redefined the concept of a biologically relevant CMC showing that the CMC values ​​for polymer micelles change by two orders of magnitude when the CMC values ​​in saline are compared with and without serum.

In addition to their core shell morphology, polymer micelles can be modified to allow passive and active cell targeting to maximize the benefits of current and future therapeutic agents. Because drug-loaded micelles typically have diameters greater than 20nm, they have dramatically increased circulation time when compared to independent drugs due to minimized renal separation. This unique feature of nanovectors and polymeric drugs leads to selective accumulation in diseased tissue, especially cancerous tissue due to enhanced permeation and retention effect (EPR). The effect of EPR is a consequence of the disorganized nature of the tumor vasculature, resulting in increased permeability of polymer therapeutics and drug retention at the tumor site. In addition to the cell target

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IMPI passive due to the effect of EPR, the micelles are aliisenasiia &amp; o

INDUSTRIAL _ actively target tumor cells through the chemical binding of target groups to the periphery of the micelle. Incorporation of such groups is most often accomplished through hydrophilic block end group functionalization using chemical conjugation techniques. As viral particles, target group functionalized micelles use ligand-receptor interactions to control the spatial distribution of micelles after administration, further improving cell-specific delivery of therapeutics. In cancer therapy, target groups are designed to interact with receptors that are overexpressed in cancerous tissue relative to normal tissue such as folic acid, oligopeptides, sugars, and monoclonal antibodies. See Pan, D .; Turner, JL; Wooley, KL Chem. Commun. 2003, 2400-2401; Gabizon, A .;

Shmeeda, H .; Horowitz, AT; Zalipsky, S. Adv. Drug Deliv. Rev. 2004, 56, 1177-1202; Reynolds, Ρ. N.; Dmitriev, I .;

Curiel, DT Vector. Gene Ther. 1999, 6, 1336-1339; Derycke,

ASL; Kamuhabwa, A .; Gijsens, A .; Roskams, T .; De Vos, D .; Kasran, A .; Huwyler, J .; Missiaen, L .; from Witte, PAM

T J. Nat. Cancer Inst. 2004, 96, 1620-30; Nasongkla, N.,

Shuai, X., Ai, H .; Weinberg, BDP, J.; Boothman, DA; Gao, J. Angew. Chem. Int. Ed. 2004, 43, 6323-6327; Jule, E .; Nagasaki, Y .; Kataoka, K. Bioconj. Chem. 2003, 14, 177-186;

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IMPI

MEXICAN INSTITUTE

Stubenrauch, K.; Gleiter, S .; Brinkmann, U .;

Lilie, H. Biochem. J. 2001, 356, 867-873; JCurschus, F. C;

Kleinschmidt, M.; Fellows, E .; Dornmair, K.; Rudolph, R.; Lilie, H.; Jenne, FROM FEBS Lett. 2004, 562, 87-92; and Jones, SD; Marasco, WA Adv. Drug Del. Rev. 1998, 31, 153-170.

Despite the large volume of work in micellar drug carriers, little effort has been focused on improving their in vivo stability for dilution. One potential reason is that the true effects on dilution of the micelle in vivo are not fully realized until larger minimal studies are used. Because the metabolism of mice is much higher than that of larger animals, they can receive considerably higher doses of toxic drugs when compared to larger animals such as rats or dogs. Therefore, when the drug-loaded micelles are administered and fully diluted through the entire blood volume, the corresponding polymer concentration will always be higher in the mouse model. Therefore, it may be highly desirable to prepare a micelle that is stabilized (crosslinked) for dilution within the biological medium.

In the present invention, the optionally crosslinkable or crosslinked poly (amino acid block) is comprised of chemical functionality that strongly binds or

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coordinates with metal ions. A special example &amp; 'í ^ Sfiióf ^' ch

INDUSTRIAL hydroxamic acids and iron (III). Another example are ortho-substituted hydroxybenzene groups (catechols) with iron. Portions of both hydroxamic acid and catechol are common in siderophores, high-affinity iron chelating agents produced by microorganisms. Additionally, it has been reported that hydroxamic acid modified poly (acrylates) can form a crosslinked gel after treatment with iron (III) (Rosthauser and Winston,

Macromolecules, 1981, p538-543). Without wishing to be bound by any particular theory, it is believed that the incorporation of high affinity metal chelating groups such as hydroxamic acids and catechols into the outer nucleus of the micelle, after treatment with a metal ion will result in a micelle that is stable at dilution in the biological medium.

Previous work has used carboxylic acids to interact with metal ions to provide micelle stability. See Request for

US Patent 2006-0240092. It has been surprisingly discovered that the use of hydroxamic acid modified polymers is effective in reversibly stabilizing the polymer micelle for dilution within the biological medium. This hydroxamic acid chemistry has been shown to be particularly effective when encapsulating a drug that has one or more functionalities.

Chemical IMPIs known to bind to iron (poUNse ™

INDUSTRIAL

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carboxylic). Without wishing to be bound by any particular theory, it is believed that the metal ions used to stabilize the micelle will potentially bind to high affinity metal chelating groups such as hydroxamic acids and catechols, resulting in a stabilized micelle. Furthermore, the chelation reaction between iron (III) and hydroxamic acid portions precedes within seconds, allowing a rapid crosslinking step.

2. Definitions:

The compounds of this invention include those generally described above, and are further illustrated by the modalities, sub-modalities, and species described herein. As used herein, the following definitions should apply unless otherwise indicated. For purposes of this invention, the chemical elements are identified in accordance with Table

Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75^ava· Ed. Additionally, general principles of organic chemistry are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito: 1999, and March's Advanced Organic Chemistry,

5^th Ed., Ed .: Smith, MB and March, J., John Wiley &amp; Sons, New

York: 2001, the full contents of which are thus incorporated by reference.

As used herein / ^ DseÍFKOPiiifíerl

INDUSTRIAL

IMPI

MEXICAN INSTITUTE · _F D ^ AFROPIElUé INDUSTRIAL sequential polymerization, and variations thereof, are

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refers to the method where, after a first monomer (eg, NCA, lactam, or imide) is incorporated into the polymer, thereby forming an amino acid block, a second monomer (eg, NCA, lactam, or imide ) is added to the reaction to form a second amino acid block, in which the process can be continued in a similar way to introduce additional amino acid blocks into the resulting multi-block copolymers.

As used herein, the term "multi-block copolymer" refers to a polymer comprising one portion of synthetic polymer and two or more portions of poly (amino acid). Such multi-block copolymers include those having the WXX format.¹, where W is a portion of synthetic polymer and X and X¹ they are poly (amino acid) chains or amino acid blocks. In certain embodiments, the multi-block copolymers of the present invention are triblock copolymers. As described herein, one or more of the amino acid blocks may be mixed blocks, meaning that these blocks may contain a mixture of amino acid monomers thereby creating multiple block copolymers of the present invention. In some modalities,

IMPI multiple block copolymers of pnes © in &lt;toexicbHEy

OF THE PROPERTY

INDUSTRIAL

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they comprise a mixed amino acid block and are tetrablock copolymers.

One skilled in the art will recognize that a monomer repeating unit is defined by parentheses around the repeating monomer unit. The number (or letter representing a numerical range) to the lower right of the parenthesis represents the number of monomer units that are present in the polymer chain. In the case where only one monomer represents the block (for example, a homopolymer), the block will be denoted only by the parentheses. In the case of a mixed block, the multiple monomers comprise a single continuous block. It will be understood that the square brackets will define a portion or block. For example, a block may consist of four individual monomers, each defined by its own individual series of parentheses and number of repeating units present. All four sets of parentheses will be enclosed by a series of square brackets, denoting that all four of these monomers will be combined in random, or near-random, in order to understand the mixed block.

For clarity, the randomly mixed block of [BCADDCBADABCDABC] could be represented in abbreviated form by [(A) ₄ (B) ₄ (C) ₄ (D) ₄] .

As used herein, the repeating unit

IMPI of monomer described above is an industrial value represents the average number of monomer units that comprise the polymer chain. For example, a polymer represented by (A) io corresponds to a polymer consisting of ten monomer units A bound together. One of ordinary skill in the art will recognize that the number 10 in this case will represent a distribution of numbers with an average of 10. The amplitude of this distribution is represented by the polydispersity index (PDI). A PDI of 1.0 represents a polymer where each chain length is exactly the same (eg, a protein). A PDI of 2.0 represents a polymer where the chain lengths have a Gaussian distribution. The polymers of the present invention typically possess a PDI of less than

1.20.

As used herein, the term "triblock copolymer" refers to a polymer comprising one portion of synthetic polymer and two portions of poly (amino acids).

As used herein, the term "tetrablock copolymer" refers to a polymer comprising one portion of synthetic polymer and either two portions of poly (amino acid), wherein 1 portion of poly (amino acid) is a mixed block or a polymer comprising one portion of synthetic polymer and three portions of poly (amino acids).

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As used herein.

IMPI

MEXICAN INSTITUTE »HELA PROPIEDAD, I el termintPL'si'fcftuc

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internal as applied to a micel of the pitLent iii ^ i clean lining, refers to the center of the micelle formed by the hydrophobic mixed poly (amino acid) block D, L-mixed. In accordance with the present invention, the inner core is not crosslinked. By way of illustration, in a triblock polymer of the W-X'-X format, as described above, the inner core corresponds to block X.

As used herein, the term "outer core" as applied to a micelle of the present invention refers to the layer formed by the first poly (amino acid) block. The outer core falls between the inner core and the hydrophilic shell. In accordance with the present invention, the outer core is either crosslinkable or crosslinked. By way of illustration, in a triblock polymer of the W-X'-X format, as described above, the outer core corresponds to block X '. It is contemplated that block X 'may be a mixed block.

As used herein, the terms drug-loaded and encapsulated, and derivatives thereof, are used interchangeably. In accordance with the present invention, a "drug-loaded micelle" refers to a micelle having a drug, or therapeutic agent, located within the nucleus having a drug, or therapeutic agent, located within the nucleus of the micelle. In certain cases, the drug or therapeutic agent is located

IMPJt

MEXICAN INSTITUTE OF PROPERTY

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_,,. . _ 'INDUSTRIAL „***.

between the nucleus and the hydrophilic crown This is also referred to as a drug, or therapeutic agent, itself encapsulated within the micelle.

As used herein, the term "polymeric hydrophilic block" refers to a polymer that is not a poly (amino acid) and is hydrophilic in nature. Such hydrophilic polymers are well known in the art and include polyethylene oxide (also referred to as polyethylene glycol or PEG), and derivatives thereof, poly (Nvinyl-2-pyrolidone), and derivatives thereof, poly (Nisopropylacrylamide), and derivatives of the same, poly (hydroxyethyl acrylate), and derivatives thereof, poly (hydroxylethyl methacrylate), and derivatives thereof, and polymers of N- (2-hydroxypropoyl) methacrylamide (HMPA) and derivatives thereof.

As used herein, the term poly (amino acid) or amino acid block refers to a covalently linked amino acid chain wherein such a monomer is an amino acid unit. Such amino acid units include natural and unnatural amino acids. In certain embodiments, each amino acid unit of the optionally crosslinkable or crosslinked poly (amino acid block) is in the L configuration. Such poly (amino acids) include those that have adequately protected functional groups. By

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For example, amino acid monomers can t

OF THE PROPERTY

INDUSTRIAL _ hydroxyl or amino, which are optionally protected by a hydroxyl protecting group or an amine protecting group, as appropriate. Such suitable hydroxyl protecting groups and amine protecting groups are described in more detail herein below. As used herein, an amino acid block comprises one or more monomers or a series of two or more monomers. In certain embodiments, an amino acid block comprises one or more monomers such that the total block is hydrophilic. In still other embodiments, the amino acid blocks of the present invention include random amino acid blocks, i.e. blocks comprising a mixture of amino acid residues.

As used herein, the term L-mixed poly (amino acid) block D refers to a poly (amino acid) block where the poly (amino acid) consists of a mixture of amino acids in both D-configurations - and L. In certain embodiments, the poly (amino acid) block D, Lmixed is hydrophilic. In other embodiments, the L, mixed poly (amino acid) block consists of a mixture of D-hydrophobic amino acid side chain groups and L-configured hydrophilic amino acids such that the total poly (amino acid) block it comprises is hydrophilic .

ΙΜΡΪ

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INSTITUI

Exemplary poly (amino acids) include

INDUSTRIAL glutamate), poly (benzyl aspartate), poly (L-leucine-cotyrosine), poly (D-leucine-co-tyrosine), poly (L-phenylalanine-tyrosine), poly (D-phenylalanine-co-tyrosine), poly (Lleucine-coaspartic acid), poly (D-leucine-coaspartic acid), poly (L-phenylalanine-co-aspartic acid), poly (Dphenylalanine-co-aspartic acid).

As used herein, the phrase "natural amino acid side chain group" refers to the side chain group of any of the 20 naturally occurring amino acids in proteins. For clarity, the -CH3 side chain group could represent the amino acid alanine. Such natural amino acids include the non-polar or hydrophobic amino acids, glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline. Cysteine ​​is sometimes classified as nonpolar or hydrophobic and other times as polar. Natural amino acids also include hydrophilic or polar amino acids, such as tyrosine, serine, threonine, aspartic acid (also known as aspartate, when loaded), glutamic acid (also known as glutamate, when loaded), asparagine, and glutamine. Certain polar or hydrophilic amino acids have charged side chains. Such charged amino acids include lysine, arginine, and histidine. One of ordinary skill in the art might recognize that

IMPI protection of an amino acid side chain © i ° iED®lÉoi3r

INDUSTRIAL hydrophilic can provide such a non-polar amino acid. By

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For example, a properly protected hydroxyl tyrosine group can provide such a hydrophobic and nonpolar tyrosine by virtue of protecting the hydroxyl group.

As used herein, the phrase "unnatural amino acid side chain group" refers to amino acids not included in the list of 20 naturally occurring amino acids in proteins, as described above. Such amino acids include the D-isomer of any of the 20 naturally occurring amino acids. Unnatural amino acids also include homoserin, ornithine, and thyroxine. Other unnatural amino acid side chains are well known to one of ordinary skill in the art and include unnatural aliphatic side chains. Other unnatural amino acids include modified amino acids, including those that are N-alkylated, cyclized, phosphorylated, acetylated, amidated, azidylated, labeled, and the like.

As used herein, the term tacticity refers to the stereochemistry of the poly (amino acid) hydrophobic block. A poly (amino acid) block consisting of a single stereoisomer (eg, all L-isomer) is referred to as isotactic. A poly (amino acid) consisting of a random incorporation of monomers of

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IMPI amino acid D and L is referred to as an industrial poly (amino acid) polymer, with alternating stereochemistry (eg, ... DLDLDL ...) is referred to as a syndiotactic polymer. The tacticity of the polymer is described in more detail in the Principles of Polymerization, 3rd Ed., G. Odian, John Wiley &amp; Sons, New York: 1991, the entire contents of which are thus incorporated by reference.

As used herein, the phrase "living polymer chain end" refers to the term resulting from a polymerization reaction which maintains the ability to further react with an additional monomer or with a polymerization terminator.

As used herein, the term "termination" refers to binding to a terminal group with one end of the polymer chain by the reaction of a living polymer with an appropriate compound. Alternatively, the term "termination" can refer to binding to a terminal group with or derived from the hydroxyl or amine end of the polymer chain.

As used herein, the term polymerization terminator is used interchangeably with the term polymerization termination agent and refers to a compound that reacts with one end of the living polymer chain to provide a polymer with a terminal group. Alternatively, the term "polymerization terminator" may refer to a compound aseas® i \

OF INDUSTRIAL PROPERTY

IMPI:

iwguero aseas® i OF INDUSTRIAL PROPERTY with a hydroxyl or amine end, or derived from it, of the

<img file="MX340992B_D0032.tif" />

polymer chain, to provide a polymer with a terminal group.

As used herein, the term "polymerization initiator" refers to a compound, which reacts with, or whose free anion or base reactive with, the desired monomer in a manner which results in polymerization of such monomer. In certain embodiments, the polymerization initiator is the compound that reacts with an alkylene oxide to provide a polyalkylene oxide block. In other embodiments, the polymerization initiator is an amine salt as described herein. In certain embodiments, the polymerization initiator is a trifluoroacetic acid amine salt.

The term aliphatic or aliphatic group, as used herein, denotes a hydrocarbon moiety that can be straight chain (i.e. unbranched), branched, or cyclic (including fused, bridged, and spiro-fused polycyclic) and it can be completely saturated or it can contain one or more unsaturation units, but which are not aromatic. Unless otherwise specified, aliphatic groups contain 1-20 carbon atoms. In some embodiments, the aliphatic groups contain 1-10 carbon atoms. In other modalities,

<img file="MX340992B_D0033.tif" />

aliphatic groups contain 1-8 carb atoms &lt;D ^ u_Toíi¡nic4í¡9

FROM INDUSTRIAL PROPERTY other modalities, aliphatic groups contain 1-6 carbon atoms, and in still other modalities aliphatic groups contain 1-4 carbon atoms. Aliphatic groups include, but are not limited to, linear or branched alkyl, alkenyl, and alkynyl groups, and hybrids thereof such as (cycloalkyl) alkyl, (cycloalkenyl) alkyl, or (cycloalkyl) alkenyl.

The term heteroatom means one or more of oxygen, sulfur, nitrogen, phosphorous, or silicon. This includes any oxidized form of nitrogen, sulfur, phosphorous, or silicon; the quaternized form of any basic nitrogen, or; a substitutable nitrogen from a heterocyclic ring including = N- as in 3,4-dihydro-2H-pyrrolyl, -NH- as in pyrrolidinyl, or = N (R^F) - as in N-substituted pyrrolidinyl.

The term unsaturated, as used herein, means that a portion has one or more unsaturation units.

As used herein, the term "bivalent straight or branched, saturated or unsaturated, C1-C12 hydrocarbon chain" refers to bivalent alkylene, alkenylene and alkynylene chains that are straight or branched as defined herein.

The term aryl used alone or as part of a larger portion as in aralkyl, aralkoxy,

Anlalkoxyalkyl uiftas, refers to systems ^{, ν}·&gt; Φθτο

DELA IKOF.'EDAD industrial

<img file="MX340992B_D0034.tif" />

moncyclic, bicyclic, and tricyclic having a total of five to fourteen elements in the ring, where at least one ring in the system is aromatic and where each ring in the system contains three to seven elements in the ring. The term aryl can be used interchangeably with the term aryl ring.

As described herein, the compounds of the invention may contain optionally substituted portions. In general, the term substituted, whether optionally preceded by the term or not, means that one or more hydrogens of the designated portion are replaced with a suitable substituent. Unless otherwise indicated, an optionally substituted group may have a suitable substituent at any substitutable position in the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group , the substituent can be either the same or different in any position. Substituent combinations contemplated by this invention are preferably those that result in the formation of stable or chemically feasible compounds. The term stable, as used herein, refers to compounds that are not substantially altered when subjected to conditions

IMPI to allow its production, detection, and,

INDUSTRIAL modalities, their recovery, purification, and use for one or more of the purposes described herein.

Monovalent substituents on a substitutable carbon atom from an optionally substituted group are independently halogen; - (CH₂) o-4R °; - (CH₂) 0-4OR⁰ ; -0 (CH₂) or-₄C (0) 0R °; - (CH₂) o-4CH (OR °)₂; - (CH₂) or-₄SR °; - (CH₂)or-₄Ph, which can be substituted with R °; - (CH₂) 0-4O (CH₂) o-iPh which can be replaced with R °; -CH = CHPh, which can be substituted with R °; -NOT₂; -CN; -N3; - (CH₂) 0-4N (R °) ₂;

- (CH₂) 0-4N (R °) C (0) R °; -N (R °) C (S) R °; - (CH₂) 0-4N (R °) C (O) NR °₂ ; N (R^OR) C (S) NR °₂; - (CH₂) 0-4N (R °) C (O) OR °; -N (R °) N (R °) C (O) R °;

N (R °) N (R °) C (O) NR °₂; -N (R °) N (R °) C (0) 0R °; - (CH₂) ₀-₄C (0) R °;

C (S) R °; - (CH₂) o-4C (0) OR °; - (CH₂) 0-4C (0) SR °; - (CH₂) or-₄C (0) OSiR °₃ ;

- (CH₂) 0-4OC (0) R °; -0C (0) (CH₂)or-₄SR-, SC (S) SR °; - (CH₂)or-₄SC (0) R °;

- (CH₂) o-4C (0) NR °₂; -C (S) NR °₂; -C (S) SR °; -SC (S) SR °, (CH₂) 0-4OC (0) NR^or2 ; -C (O) N (OR °) R °; -C (O) C (O) R °; -C (O) CH₂C (O) R °;

-C (N0R °) R °; - (CH₂) 0-4SSR⁰ ; - (CH₂) 0-4S (O) ₂R °; - (CH₂) 0-4S (0) ₂0R °; (CH₂) 0-4OS (0) ₂R °; -S (0)₂NR °₂; - (CH₂) 0-4S (O) R °; -N (R °) S (O)₂NR °₂; N (R °) S (0)₂R °; -N (0R °) R °; -C (NH) NR °₂; -P (O)₂R °; -P (O) R °₂; -0

P (O) R °₂; -0P (0) (0R °)₂; SiR ° 3; - (C1-4 alkylene straight or branched) 0-N (R °)₂; or - (straight or branched C1-4 alkylene) C (0) 0-N (R °) 2, where each R ° can be substituted as defined below and is independently hydrogen, C1-6 aliphatic, -CH₂Ph, -0 (CH₂) o-iPh, or a ring

<img file="MX340992B_D0035.tif" />

IMPI aryl, saturated, partially unsaturated, of

IÑOC'STRIAL having 0-4 heteroatoms independently selected to

<img file="MX340992B_D0036.tif" />

from nitrogen, oxygen, or sulfur, or, despite the definition above, two independent occurrences of R °, taken together with their intervention atom (s), form a saturated mono- or bicyclic aryl ring, partially unsaturated from 3 to 12 elements having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which can be substituted as defined below.

Monovalent substituents on R ° (or the ring formed by taking two independent occurrences of R ° together with their intervention atoms), are independently halogen, - (CH2) o-2R ·, - (haloR ·), - (CH2) or- 20H, - (CH₂) 0-2OR ·, (CH₂) or-₂CH (OR *)₂; -0 (haloR ·), -CN, -N₃, - (CH₂) 0-2C (O) R ·, (CH₂)or-₂C (0) OH, - (CH₂)or-₂C (0) OR ·, - (CH₂)or-₂SR ·, - (CH₂)or-₂SH, (CH₂)or-₂NH2, - (CH₂) 0-2NHR ·, - (CH₂) or-₂NR *₂, -NO2, -YES * 3, -ear *₃, C (O) SR · - (straight or branched C1-4 alkylene) C (O) OR ·, or -SSR where each R * is unsubstituted or where halo is preceded by only one or more halogens, and is independently selected from C1-4 aliphatic, CH₂Ph, -O (CH₂) o-iPh, or a 5-6 membered saturated, partially unsaturated aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Such divalent substituents on a R ° saturated carbon atom include = 0 and = S.

• - «a» ngr ^ -y.2 «r. ·

<img file="MX340992B_D0037.tif" />

Divalent substituents on an saturated α from an optionally substituted group include the following: = 0, = S, = NNR *₂, = NNHC (O) R \ ~~ = NNHC (0) 0R *, = NNHS (O)₂R ', = NR *, = N0R, -O (C (R *₂) )₂-₃0-, or -S (C (R *₂) )₂-₃S-, where each independent occurrence of R * is selected from hydrogen, aliphatic Ci-₆ which can be substituted as defined below, or a 5-6 membered unsaturated, saturated, partially unsaturated aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Divalent substituents that are attached to suitable neighborhood carbons of an optionally substituted group include: -O (CR *₂) 2-3O-, where each independent occurrence of R * is selected from hydrogen, C1-6 aliphatic which can be substituted as defined below, or a 5-6 saturated, partially unsaturated, unsubstituted aryl ring elements having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. A tetravalent substituent that binds to neighborhood substitutable methylene carbons from an optionally substituted group is the hexacarbonyl dicobalt group represented by

<img file="MX340992B_D0038.tif" />

when it is represented with the methylenes which it carries.

Suitable substituents in the group »irir £ ¡abac &lt;so

OF THE mCFÍE.-AD INDUSTRIAL

IM.Pi

OF THE INDUSTRIAL COMPANY include halogen, -R ·, - (haloR ·), -OH, -OR ·, -0 (haloR ·),

<img file="MX340992B_D0039.tif" />

CN, -C (O) OH, -C (O) OR ·, -NH₂, -NHR ·, -NR »₂, or not₂, where each R · is unsubstituted or where halo is preceded by only one or more halogens, and is independently aliphatic Ci-₄, -CH₂Ph, -O (CH₂) o-iPh, or a 5-6 membered saturated, partially unsaturated aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

Suitable substituents on a substitutable nitrogen from an optionally substituted group include -R^F, -NR^F2, C (O) Rt, -C (O) OR^F, -C (O) C (O) R^T, -C (0) CH2C (0) R^F, -S (O) 2R^F,

YES ^ NRh, -C (S) NR^F2, -C (NH) NR^F2, or -N (R^F) S (0) ₂R^F; where each R¹ is independently hydrogen, aliphatic Ci-6 which can be substituted as defined below, -OPh unsubstituted, or a 5-6 membered saturated, partially unsaturated, unsubstituted aryl ring having 0-4 heteroatoms independently selected from nitrogen , oxygen, or sulfur, or, despite the definition above, two independent occurrences of R^F, taken together with their intervention atom (s) form a 3-12 unsubstituted, saturated, partially unsaturated, mono- or bicyclic aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

Suitable substituents in the group &lt;

<img file="MX340992B_D0040.tif" />

R^T they are independently halogen, -R ·, - (haloR ·), -OH,

OR ·, -0 (haloR ·), -CN, -C (O) OH, -C (O) OR ·, -NH₂, -NHR ·, -NR *₂, or not₂, where each R · is unsubstituted or where halo is preceded by only one or more halogens, and is independently aliphatic Ci-₄, -CH₂Ph, -O (CH₂) ₀-iPh, or a 5-6 membered, partially unsaturated, saturated aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

Protected hydroxyl groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, TW Greene and PG

M. Wuts, 3^ra edition, John Wiley &amp; Sons, 1999, the entirety of which is incorporated herein by reference. Examples of suitably protected hydroxyl groups further include, but are not limited to, esters, carbonates, sulfonate allylesters, ethers, silyl ethers, alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers. Examples of suitable esters include formates, acetates, proprionates, pentanoates, chronates, and benzoates. Specific examples of suitable esters include formate, benzoyl formate, chloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenylpropionate, (ethylenedithio) pentanoate, p-chlorophenoxyacetate,

4-oxopentanoate,

34,4pivaloate (trimethylacetate), crotonate, 4-methoxy-crotonate, benzoate,

INDUSTRIAL

IMPI

INlVS i RlAL

2,4,6-trimethylbenzoate. Examples of carbonates include 9-

<img file="MX340992B_D0041.tif" />

fluorenylmethyl, ethyl, 2,2,2-trichlorethyl, 2 (trimethylsilyl) ethyl, 2- (phenylsulfonyl) ethyl, vinyl, allyl, and p-nitrobenzyl carbonate. Examples of silyl ethers include trimethylsilyl, triethylsilyl, t-butyldimethylsilyl.

t-butyldiphenylsilyl, triisopropylsilyl ether, other butyloxycarbonylamino methyloxycarbonylamino, trialkylsilyl ethers. Examples of alkyl ethers include methyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, and allyl ether, or derivatives thereof. Alkoxyalkyl ethers include acetals such as methoxymethyl, methylthiomethyl, (2-methoxyethoxy) methyl, benzyloxymethyl, beta (trimethylsilyl) ethoxymethyl, and tetrahydropyran-2-yl ether. Examples of arylalkyl ethers include benzyl, pmethoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, pnitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, pcyanobenzyl, 2- and 4-picolyl ethers.

Protected amines are well known in the art and include those described in detail in Greene (1999). Monoprotected amines further include, but are not limited to, aralkylamines, carbamates, allylamines, amides, and the like. Examples of mono-protected amino moieties include t (-NHBOC), ethyloxycarbonylamino, trichloroethyloxycarbonylamino, allyloxycarbonylamino (-NHAloc), benzyloxocarbonylamino

IMPI,,. ,,. , INSTITUTO MEXICANO '(-NHCBZ), alilamino, bencilammo de la ι κοπΕ &amp; βΝ.

fluorenylmethylcarbonyl (-NHFmoc), formamido, acetamido, chloroacetamido, dichloroacetamido, trichloroacetamido, trifluoroacetamido, benzamido, t and the like. Di-protected amines

<img file="MX340992B_D0042.tif" />

Phenylacetamido, butyldiphenylsilyl, include amines that are substituted with two substituents independently selected from those described above as mono-protected amines, and further include cyclic amines, such as phthalimide, maleimide, succinimide, and the like. Di-protected amines also include pyrroles and the like, 2,2,5,5-tetramethyl [1,2,5] azadisylolidine and the like, and azide.

Protected aldehydes are well known in the art and include those described in detail in Greene (1999). Protected aldehydes further include, but are not limited to, acyclic acetals, cyclic acetals, hydrazones, imines, and the like. Examples of such groups include dimethylacetal, diethylacetal, diisopropylacetal, dibenzyl acetal, bis (2-nitrobenzyl) acetal, 1,3-dioxanes, 1,3-dioxolanes, semicarbazones, and derivatives thereof.

Protected carboxylic acids are well known in the art and include those described in detail in Greene (1999). Protected carboxylic acids include, but are not limited to, optionally substituted Ci-6 aliphatic esters, optionally aryl esters

ΙΜΡΙ

MEXICAN INSTITUTE activatedegriíoMJ a

INDUSTRIAL

<img file="MX340992B_D0043.tif" />

substituted, silyl esters, hydrazide esters, and the like. Examples of such ester groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, benzyl, and phenyl ester, where each group is optionally substituted. Additional protected carboxylic acids include oxazolines and orthoesters.

Protected thiols are well known in the art and include those described in detail in Greene (1999). Protected thiols further include, but are not limited to, disulfides, thioethers, silylthioethers, thioesters, thiocarbonates, and thiocarbamates, and the like. Examples of such groups include, but are not limited to, alkylthioethers, benzyl and substituted benzylthioethers, triphenylmethylthioethers, and trichloroethoxycarbonylthioesters, to name but a few.

Unless stated otherwise, structures represented herein also mean including all isomeric (eg, enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, Z and E double bond isomers, and conformational Z and E isomers. Therefore, unique as well as diastereomeric, and enantiomeric stereochemical mixtures isomers, also (or geometric conformational) of the present compounds are within

ΙΜΡΙ @

MEXICAN INSTITUTE of the scope of the invention. Unless it was said ^ us'áeS oSk2! Thus, all tautomeric forms of the gompuaGtoe ..... of ·, the invention are within the scope of the invention.

Additionally, unless stated otherwise, the structures represented herein also mean including compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a carbon enriched with¹³C- or ¹⁴C- are within the scope of this invention. Such compounds are useful, for example, as in neutron scattering experiments, as probes or analytical tools in biological assays.

As used herein, the term detectable portion is used interchangeably with the term tag and refers to any portion capable of being detected (eg, primary tags and secondary tags). A detectable portion or label is the radical of a detectable compound.

Primary labels include portions containing radioisotopes (eg portions containing ³²P, ³³P, ³⁵Yes, or ¹⁴C), mass labels, and fluorescent labels, and are reporter groups that generate signal which can be detected without additional modifications.

IMPI »*!

MEXICAN INSTITUTE

Other primary labels include for positxon emission tomography_ which include molecules that contain radioisotopes (eg ¹⁸F) or ligands with bound radioactive metals (eg, ⁶²Cu).

In other embodiments, the primary labels are magnetic resonance imaging contrast agents such as gadolinium particles, gadolinium chelates, or iron oxide (eg, Fe3C&gt; 4 and Fe2O₃). Similarly, semiconductor nanoparticles (eg, cadmium selenide, cadmium sulfide, cadmium telluride) are useful as fluorescent labels. Other metal nanoparticles (eg, colloidal gold) also serve as primary labels.

Secondary labels include portions such as biotin, or protein antigens, that require the presence of a second compound to produce a detectable signal. For example, in the case of a biotin tag, the second compound may include conjugates of the enzyme streptavidin. In the case of an antigen tag, the second compound may include an antibody enzyme conjugate. Additionally, certain fluorescent groups can act as secondary labels by transferring energy to another compound or group in a non-radiative fluorescent resonance energy transfer (FRET) process, causing the second compound or group

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX340992B_D0044.tif" />

then generate the signal that is detected.

Unless otherwise indicated, radioisotope-containing poveians are optionally substituted hydrocarbon groups containing at least one radioisotope. Unless otherwise indicated, radioisotope-containing portions contain from 1-40 carbon atoms and one radioisotope. In certain embodiments, radioisotope-containing portions contain from 1-20 carbon atoms and one radioisotope.

The terms fluorescent label, fluorescent group, fluorescent compound, fluorescent dye, and fluorescent gold, as used herein, refer to compounds or portions that absorb light energy at a defined excitation wavelength and emit light energy at a different wavelength. Examples of fluorescent compounds include, but are not limited to:

tints Alexa Fluor (Alexa Fluor 350, Alexa Fluor 488, Alexa

Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660 and Alexa Fluor 680), tints

AMCA, AMCA-S, BODIPY (BODIPY FL, BODIPY R6G, BODIPY TMR,

BODIPY TR, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570,

BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665), Carboxydamine 6G, Carboxy-X-rhodamine (ROX), Cascade Blue, Cascade Yellow, Coumarin 343, Cyanide dyes (Cy3, Cy5,

Cy5.5),

Dapoxil,

Dansilo, Mexican institute

Dialkylaminocoumarin, 4 ', 5' -Dichloro-2 ^{one D},^ANDYXraffiÉáfe fluoresceina, DM-NERF, Eosina, Eritrosina, Fluoresqeína, FAM. Hydroxycoumarin, DyesIR (IRD40, IRD 700, IRD 800), JOE,

Lissamine Rhodamine B, Marina Blue, Methoxy Coumarin, Naphtho Fluorescein, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, PyMPO, Pyrene, Rhodamine B, Rhodamine

6G, Rhodamine Green, Rhodamine Red, Rodol Green, 2 ', 4', 5 ', 7'Tetra-bromosulfone-fluorescein, Tetramethyl-rhodamine (TMR),

Carboxytetramethylrodamine (TAMRA), Texas Red, Texas Red-X.

The term substrate, as used herein, refers to any macromolecular material or complex to which a functionalized final group of a block copolymer can be attached. Examples of commonly used substrates include, but are not limited to, glass surfaces, silica surfaces, plastic surfaces, metal surfaces, surfaces that contain a chemical or metallic coating, membranes (eg, nylon, polysulfone, silica), micro-beads (for example, latex, polystyrene, or other polymer), porous polymer matrices (for example, polyacrylamide gel, polysaccharide, polymethacrylate), macromolecular complexes (for example, protein, polysaccharide).

The term hydroxamic acid, as used herein, refers to a portion containing a hydroxamic acid functional group (-CO-NH-OH). The structure is

IMPI

<img file="MX340992B_D0045.tif" />

represented by and can also be represented by

OR

H

One skilled in the art will recognize that the dotted bond represents the point of attachment to the rest of the molecule.

The term hydroxamate, as used herein, refers to a portion that contains either hydroxamic acid or an N-substituted hydroxamic acid. Due to the

N-substitution, there are two separate locations for chemical bonding, as shown by the R and R 'groups here the hydroxamates of

Λν-^οη

I

They can also be represented by fA-^oh or

Oh

N

I

R '

TO_n.OH in the present.

The term catechol, as used herein, refers to a substituted ortho-dihydroxybenzene derivative.

Two different isomeric conformations are represented by

<img file="MX340992B_D0046.tif" />

mw., ------.

<img file="MX340992B_D0047.tif" />

raiglUf.

Catechol is also known as pyrocatechol and benzene-1,2-diol.

3. Description of Exemplary Modalities:

A. Multi-block copolymers

In certain embodiments, the multi-block copolymer comprises a hydrophilic poly (ethylene glycol) block, a hydroxamic acid containing poly (amino acid) block, and a hydrophobic poly (amino acid) block characterized in that the resulting micelle has an inner core, an outer core containing hydroxamic acid, and a hydrophilic shell. It will be appreciated that the hydrophilic poly (ethylene glycol) block corresponds to the hydrophilic coating, the stabilizing hydroxamic acid containing poly (amino acid) block corresponds to the hydroxamic acid containing outer core, and the hydrophobic poly (amino acid) block corresponds to the core internal.

In other embodiments, the multi-block copolymer comprises a hydrophilic poly (ethylene glycol) block, a catechol-containing poly (amino acid) block, and a hydrophobic poly (amino acid) block characterized in that the resulting micelle has an inner core, a outer core containing catechol, and a hydrophilic coating. It will be appreciated that

IMPI

INDUSTRIAL

<img file="MX340992B_D0048.tif" />

Hydrophilic poly (ethylene glycol) corresponds to the hydrophilic coating, the stabilizing catechol-containing poly (amino acid) block corresponds to the catechol-containing outer core, and the hydrophobic poly (amino-acid) block corresponds to the inner core.

In certain embodiments, the multi-block copolymer comprises a hydrophilic poly (ethylene glycol) block, a hydroxamate-containing poly (amino acid) block, and a hydrophobic poly (amino acid) block characterized in that the resulting micelle has an inner core, a outer nucleus containing hydroxamate, and a hydrophilic shell. It will be appreciated that the hydrophilic poly (ethylene glycol) block corresponds to the hydrophilic shell, the stabilizing hydroxamate-containing poly (amino acid) block corresponds to the hydroxamate-containing outer core, and the hydrophobic poly (amino acid) block corresponds to the inner core.

In certain embodiments, the multi-block copolymer comprises a hydrophilic poly (ethylene glycol) block, a hydroxamic acid-containing poly (amino acid) block, and a hydrophobic poly (D) L-mixed block characterized in that the resulting micelle It has an inner core, an outer core containing hydroxamic acid, and a hydrophilic coating. It will be appreciated that the

IMPI hydrophilic poly (ethylene glycol) block runs ^ p ^ i * | ^ «* N ^

INDUSTRIAL hydrophilic coating, poly (amino acid) block containing

<img file="MX340992B_D0049.tif" />

Stabilizing hydroxamic acid corresponds to the outer core containing hydroxamic acid, and the hydrophobic poly (amino acid) block D, L-mixed corresponds to the inner core.

In other embodiments, the multi-block copolymer comprises a hydrophilic poly (ethylene glycol) block, a catechol-containing poly (amino acid) block, and a hydrophobic poly, D-L-mixed block characterized in that the resulting micelle has an inner core, an outer core containing catechol, and a hydrophilic coating. It will be appreciated that the hydrophilic poly (ethylene glycol) block corresponds to the hydrophilic coating, the stabilizing catechol-containing poly (amino acid) block corresponds to the catechol-containing outer core, and the hydrophobic poly (amino acid) block D corresponds to the inner core.

In certain embodiments, the multi-block copolymer comprises a hydrophilic poly (ethylene glycol) block, a hydroxamate-containing poly (amino acid) block, and a hydrophobic D, L-mixed poly (amino acid) block characterized in that the resulting micelle has an inner core, an outer core containing hydroxamate, and a hydrophilic coating. It will be appreciated that the

IMPIÍS

<img file="MX340992B_D0050.tif" />

hydrophilic poly (ethylene glycol) block cor: ¿^^ g $$ 9 ^ o ^; h

INDUSTRIAL hydrophilic coating, the stabilizing hydroxamate-containing poly (amino acid) block corresponds to the hydroxamate-containing outer core, and the hydrophobic D-L-mixed poly (amino acid) block corresponds to the inner core.

In certain embodiments, the present invention provides a triblock copolymer of formula I:

<img file="MX340992B_D0051.tif" />

where:

n is 20-500;

x is 3 to 50;

y is 5 to 100;

R^x it is a portion containing hydroxamate or catechol;

R^and is selected from one or more natural or unnatural amino acid side chain groups such that the total block is hydrophobic;

R¹ is -Z (CH2CH2Y) p (CH₂) _tR³, where:

Z is -0-, -NH-, -S-, -C = C-, or -CH₂-;

each Y is independently -0- or -Sp is 0-10;

<img file="MX340992B_D0052.tif" />

t is 0-10; and

R³ is hydrogen, -N_3/ -CN, -NH₂, -CH₃,

TO O. , 0&gt; ^ a portion of chain cyclooctin, a mono-protected amine, a protected di-amine, an optionally protected aldehyde, an optionally protected hydroxyl, an optionally protected carboxylic acid, an optionally protected thiol, or an optionally substituted selected group starting from a 5-8 element, aliphatic, partially unsaturated, saturated aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, an 8-10 element, partially unsaturated, saturated bicyclic aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a detectable portion;

Q is a valence bond or a straight or branched, saturated or unsaturated, bivalent C1-12 hydrocarbon chain, where 0-6 methylene units of Q are independently replaced by -Cy-, -O-, -NH-, - S-,

-0C (0) -, -C (0) 0-, -C (0) -, -SO-, -S0₂-, -NHSO2-, -SO2NH-, NHC (O) -, -C (O) NH-, -OC (O) NH-, or -NHC (O) O-, where:

-Cy- is an optionally 5-8 element bivalent, partially unsaturated, saturated aryl ring

IMPI

<img file="MX340992B_D0053.tif" />

substituted they have 0-4 heteroatoms

INDUSTRIAL independently from nitrogen, oxygen, or sulfur, or an optionally substituted 8-10 membered saturated, bivalent, partially unsaturated bicyclic aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur;

R² is a mono-protected amine, a protected di-amine, N (R⁴) 2, -NR⁴C (O) R⁴, -NR⁴C (0) N (R⁴) 2, -NR⁴C (O) OR⁴, or NR⁴SW₂R⁴; and each R⁴ is independently hydrogen or an optionally substituted group selected from a 5-8 membered, unsaturated, aliphatic, saturated aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a bicyclic aryl ring saturated, partially unsaturated, 8-10 elements having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a detectable portion, or:

two R⁴ at the same nitrogen atom they are taken in conjunction with such a nitrogen atom to form an optionally substituted 4-7 element, partially unsaturated, aryl or saturated ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In accordance with another modality, the present

INSTITUTO MEXICANO invention provides compounds of formula T^{THE A},! $ o &lt;$$ t @ described above, where such compounds have a polydispersity index (PDI) of 1.0

1.2. In accordance with another embodiment, the present invention provides compounds of formula I, as described above, wherein such compound has a polydispersity index (PDI) of 1.01 to 1.10. In accordance with yet another embodiment, the present invention provides compounds of formula I, as described above, wherein such compound has a polydispersity index (PDI) of 1.10 to 1.20. In accordance with other embodiments, the present invention provides compounds of formula I having a PDI of less than 1.10.

As defined generally above, n is 20 to 500. In certain embodiments, the present invention provides compounds where n is 225. In other embodiments, n is 40 to 60. In other embodiments, n is 60 to

90. In still other modes, n is 90 to 150. In other ways, n is 150 to 200. In some ways, n is 200 to 300, 300 to 400, or 400 to 500. In still other ways, n is 250 to 280 In other modalities, n is 300 to 375. In other modalities, n is 400 to 500. In certain modalities, n is selected from 50 + 10. In other modalities, n is selected from 80 ± 10, 115 ± 10, 180 + 10, 225 + 10, or 275 ± 10.

IMPI. , INSTITL

In certain modes, x is 3 to 50.de

MEXICAN INSTITUTE ^--Bnxcr © 3ker INDUSTRIAL

<img file="MX340992B_D0054.tif" />

modalities, x is 10. In other modalities, _x is 20. According to still another modality, x is 15. In other modalities, x is 5. In other modalities, x is selected from 5 ± 3, 10 + 3 , 10 ± 5, 15 + 5, or 20 + 5.

In certain modes, y is 5 to 100. In certain modes, y is 10. In other modes, y is 20. Pursuant to yet another mode, y is 15. In other portions R³ it is a substituted alkynyl group. When such portion substituted, substituents in modalities, y is 30. In other modalities, y is selected from 10 ± 3, 15 ± 3, 17 ± 3, 20 + 5, or 30 ± 5.

In certain modalities, the R portion³ of group R¹ of formula I is -N3.

In other modalities, the R portion³ of group R¹ of formula I is -CH3.

In some embodiments, the R portion³ of group R¹of formula I is hydrogen.

In certain modalities, the R portion³ of group R¹of formula I is an optionally substituted aliphatic group. Examples include methyl, t-butyl, 5-norbomen-2-yl, octan-5-yl, acetylenyl, trimethylsilylacetylenyl, triisopropylsilylacetylenyl, and t-butyldimethylsilylacetylenyl. In some embodiments, such portion R³it is an optionally substituted alkyl group. In other embodiments, such or optionally alkenyl R³ is an aliphatic group R³ include CN, N3,

ΙΜΡΙ

Slfeül

<img file="MX340992B_D0055.tif" />

trimethylsilyl, triisopropylsilyl, t-butyldimeipheailsiiJ £ aii3ASiQ

11 (THE I INDUSTRIAL PROPERTY methylpropiolamido, N-methyl-4-acetylenylanilino, N-methyl-4-acetylenylbenzoamido, bis- (4-ethinyl-benzyl) -amino, dipropargi lamino, di -hex- 5 - inil - amino, di-pent -4-ynyl-amino, di-but-3-ini 1-amino, prcpargiloxy, hex-5-ynyloxy, pent-4-ynyloxy, di-but-3-ynyloxy, N-methylpropargylamino, N-methyl-hex- 5-ynyl-amino, N-methyl-pent-4-ynyl-amino, N-methyl-but-3-ynyl-amino, 2-hex-5-ynyldisulfanyl, 2-pent-4-ynyldisulfanyl, 2-but-3-ynyldisulfanyl, and 2-propargildisulfanyl. In certain modalities, group R¹ it is 2- (N-methyl-N (ethynylcarbonyl) amino) ethoxy, 4-ethynylbenzyloxy, or 2- (4ethynylphenoxy) ethoxy.

In certain modalities, the R portion³ of group R¹ of formula I is an optionally substituted aryl group. Examples include optionally substituted phenyl and optionally substituted pyridyl. When such portion R³ is a substituted aryl group, substituents on R³ include CN, N₃, NO2, CH₃, -CH2N3, -CH = CH₂, -C ^ ch, Br, I, F, bis- (4-ethynyl-benzyl) amino, dipropargylamino, di-hex-5-ynyl-amino, di-pent-4-ynylamino, di-but-3-ynyl -amino, propargyloxy, hex-5-ynyloxy, pent-4-ynyloxy, di-but-3-ynyloxy, 2-hex-5-ynyloxyethyldisulfanyl, 2-pent-4-ynyloxy-ethyldisulfanyl, 2-but-3-ynyloxy-ethyldisulfanyl , 2-propargyloxy-ethyldisulfanyl, bisbenzyloxy-methyl, [1,3] dioxolan-2-yl, and [1,3] dioxan-2-yl.

In other modalities, the R portion³ of group R¹ of formula I is a protected aldehyde group. In certain

IMPI modalities the aldehyde portion protected^lNS1SS2 ^ íi '^

INDUSTRIAL

<img file="MX340992B_D0056.tif" />

acyclic acetal, a cyclic acetal, a hydrazone, or an imine. Groups R³ Exemplary include dimethylacetal, diethylacetal, diisopropylacetal, dibenzyl acetal, bis (2-nitrobenzyl) acetal, 1,3-dioxane, 1,3-dioxolane, and semicarbazone. In certain modalities, R³ it is an acyclic acetal or a cyclic acetal. In other modalities, R³ it is a dibenzyl acetal.

In still other modalities, the R portion³ of group R¹of formula I is a protected carboxylic acid group. In certain embodiments, the protected carboxylic acid portion of R³ it is an optionally substituted ester selected from Ci-6 aliphatic or aryl, or a silyl ester, an activated ester, an amide, or a hydrazide. Examples of such ester groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, benzyl, and phenyl ester. In other embodiments, the protected carboxylic acid portion of R³ it is an oxazoline or an orthoester. Examples of such portions of protected carboxylic acid include oxazolin-2-yl and 2-methoxy- [1,3] dioxin-2-yl. In certain modalities, group R¹ it is oxazolin-2-ylmethoxy or 2oxazolin-2-yl-l-propoxy.

In still other modalities, the R portion³ of group R¹ Formula I is a detectable portion. Of

<img file="MX340992B_D0057.tif" />

in accordance with an aspect of the invention, lá '^ SSí ^ ÍSSiSSR * iwni KTBUi group R¹ of formula I is a fluorescent portion. Such fluorescent portions are well known in the art and include coumarins, quinolones, benzoisoquinolones, hostasol, and rhodamine dyes, to name but a few. Exemplary fluorescent portions of group R³ from R¹ They include anthracene-9-yl, pyrene-4-yl, 9-H-carbazol-9-yl, rhodamine carboxylate B, coumarin carboxylate 343. In certain embodiments, the R portion³ of group R¹ Formula I is a detectable portion selected from:

<img file="MX340992B_D0058.tif" />

HO

<img file="MX340992B_D0059.tif" />

NH

0 · ^ NH

0 'NH

In certain modalities, the R portion³ of group R¹ Formula I is a suitable group for Clic chemistry. The

<img file="MX340992B_D0060.tif" />

Click reactions tend to involve reagents

INDUSTRIAL energy (spring loaded) with well-defined reaction coordinates, giving rise to events that form wide-ranging selective bonds. Examples include nucleophilic entrapment of chain ring electrophiles (epoxide, aziridines, aziridinium ions, episulfonium ions), certain forms of carbonyl reactivity (aldehydes and hydrazines or hydroxylamines, for example), and various types of cycloaddition reactions. The 1,3-dipolar cycloaddition of azide-alkyl is one such reaction. Clic chemistry is known in the art and one of ordinary skill in the art will recognize that certain portions R³ of the present invention are suitable for Clic chemistry.

Compounds of formula I having R³ Portions suitable for Clic chemistry are useful for conjugating such compounds to biological systems or macromolecules such as proteins, viruses, and cells, to name a few. The Clic reaction is known to proceed rapidly and selectively under physiological conditions. In contrast, most conjugation reactions are carried out using the primary amine functionality in proteins (eg, lysine or protein end group). Because most proteins contain a multitude of lysines and arginines, such conjugation occurs uncontrollably at multiple sites on the protein. This

IMPI ____ Τ .5.ΛΊ.ΤΎ:

4tuto-m3xS: 1íI¿ £

OF INDUSTRIAL PROPERTY

<img file="MX340992B_D0061.tif" />

it is particularly troublesome when. l »&amp;_TuToXfcft; waa arginines are located around the active site of an enzyme or other biomolecule. Thus, another embodiment of the present invention provides a method for conjugating the R groups.¹ from a compound of formula I to a macromolecule using Clic chemistry. Still another embodiment of the present invention provides a macromolecule conjugated to a compound of formula I via group R¹.

In accordance with one modality, the R portion³ of group R¹ of formula I is an azide-containing group. In accordance with another modality, the R portion³ of group R¹ of formula I is an alkyne-containing group. In certain modalities, the R portion³ of group R¹ of formula I has a terminal alkyne moiety. In other modalities, the R portion³ of group R¹ Formula I is an alkyl moiety having an electron-attracting group. Therefore, in such modalities, the portion R³ of group R¹ of formula I is

E where E is an electron-attracting group and y is

0-6. Such electron-attracting groups are known by one of ordinary skill in the art. In certain embodiments, E is an ester. In other modalities, the R portion³ of the group

R¹ of formula I is

<img file="MX340992B_D0062.tif" />

where E is an electron-attracting group, such as a group -C (0) 0- ey is 0-6.

Certain metal-free Click portions are known in the literature. Examples include 4-dibenzocyclooctinol (DIBO)

<img file="MX340992B_D0063.tif" />

(from Ning et. al; Angew Chem Int Ed, 2008, 47,

2253); difluorinated cyclooctins (DIFO or DFO)

<img file="MX340992B_D0064.tif" />

(de Codelli, et. al .; J. Am. Chem. Soc. 2008, 130,

11486-11493.); biarylazacyclooctinone (BARAC)

<img file="MX340992B_D0065.tif" />

(de Jewett et. al; J. Am. Chem. Soc. 2010, 132,

3688); or bicyclononine (BCN)

<img file="MX340992B_D0066.tif" />

IMPI®

MEXICAN INSTITUTE OF PROPERTY

INDUSTRIAL

<img file="MX340992B_D0067.tif" />

(de Dommerholt, et. al .; Angew Chem Int Ed, 2010, 49, 9422-9425). The preparation of metal-free click PEG derivatives is described in US Application Number

13 / 601,606, the full contents of which are thus incorporated by reference.

In accordance with one modality, the R portion³ of group R¹ Formula I is a Metal Free Click portion. In another embodiment, the R portion³ of group R¹ of formula I is a portion of chain substituted cyclooctin. In certain modalities, the R portion³ of group R¹ Formula I is a Metal Free Click portion selected from:

<img file="MX340992B_D0068.tif" />

As generally defined above, Q is a valence bond or a Ci-i hydrocarbon chain₂ straight or branched, saturated or unsaturated, bivalent, where 0-6 methylene units of Q are independently replaced by -Cy-, -0-, -NH-, -S-, -0C (0) -, -C ( 0) 0-, -C (0) -, -SO-, S0₂-, -NHSO2-, -SO2NH-, -NHC (O) -, -C (0) NH-, -OC (O) NH-, or

IMPI

MEXICAN INSTITUTE OF PROPERTY

NHC (O) O-, where -Cy- is an aryl ring usertnira

<img file="MX340992B_D0069.tif" />

partially unsaturated, optionally substituted bivalent, of -5-8 »1 catheters having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a bivalent, partially unsaturated, bivalent, 8-10 membered bicyclic ring optionally substituted, having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In certain embodiments, Q is a valence bond. In other embodiments, Q is a bivalent, saturated Cl12 alkylene chain, where 0-6 methylene units of

Q are independently replaced by -Cy-, -O-, -NH-, S-, -0C (0) -, -C (0) 0-, or -C (O) -, where -Cy- is a Optionally substituted 5-8 element, bivalent, partially unsaturated, saturated aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a bivalent, partially unsaturated, bivalent, bicyclic ring of 8-10 optionally substituted elements, having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In certain embodiments, Q is -Cy- (i.e., a Ci alkylene chain where the methylene unit is replaced by -Cy-), where -Cy- is a bivalent, partially unsaturated, saturated aryl ring of 5- 8 elements optionally substituted

<img file="MX340992B_D0070.tif" />

independently selected heteroatoms — at — split do. nitrogen, oxygen, or sulfur. In accordance with one aspect of the present invention, -Cy- is an optionally substituted bivalent aryl group. In accordance with another aspect of the present invention, -Cy- is an optionally substituted bivalent phenyl group. In other embodiments, -Cy- is an optionally substituted bivalent, 5-8 membered carbocyclic ring. In still other embodiments, -Cyes is an optionally substituted bivalent, 5-8 membered saturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Exemplary -Cy- groups include bivalent rings selected from phenyl, pyridyl, pyrimidinyl, cyclohexyl, cyclopentyl, or cyclopropyl.

As defined above, R^x it is a portion containing hydroxamate or catechol. In certain modalities, R^x it is a portion containing hydroxamic acid. In other modalities, R^x It is a portion that contains catechol. In certain modalities, R^x is selected from

HO.

<img file="MX340992B_D0071.tif" />

OR

In certain modalities, R^x selected from

<img file="MX340992B_D0072.tif" />

<img file="MX340992B_D0073.tif" />

OH OH OH

<td>How</td><td>it defines</td><td colspan="2">previously Ry</td><td colspan="2">select a</td>

<td>starting from one</td><td colspan="2">or more chain groups</td><td>side</td><td>of</td><td>amino acid</td>

<td>natural or not</td><td>natural of</td><td>Way that</td><td colspan="2">the block</td><td>total is</td>

<td>hydrophobic.</td><td>Such groups</td><td>chain</td><td>side</td><td>of</td><td>amino acid</td>

<td>hydrophobic</td><td colspan="2">include a chain</td><td>side</td><td>of</td><td>tyrosine</td>

<td>optionally</td><td>protected,</td><td>a chain</td><td colspan="2">side</td><td>from serina</td>

<td>optionally</td><td>protected,</td><td>a chain</td><td>side</td><td colspan="2">threonine</td>

<td>optionally</td><td>protected,</td><td colspan="3">phenylalanine, alanine</td><td>valine</td>

leucine, tryptophan, proline, benzyl and alkyl glutamates, or benzyl and alkyl aspartates or mixtures thereof. One of ordinary skill in the art will recognize that protection of a hydrophobic or polar amino acid side chain can provide such a non-polar amino acid. For example, a suitably protected hydroxyl tyrosine group can provide such a nonpolar tyrosine and

IMPI hydrophobic by virtue of protecting the group K3Seobo ¿eÍ? G&gt;. protecting groups for the hydroxyl, amino, and thiol, and carboxylate functional groups of are as described herein. Furthermore, one of ordinary skill in the art will recognize that hydrophilic and hydrophobic amino acid chains can be combined so that the total block is hydrophilic. For example, a majority of leucine side chain groups can be combined with a majority of aspartic acid side chain groups where the resulting block is pure hydrophobic. Such mixtures of amino acid side chain groups include tyrosine and leucine, tyrosine and phenylalanine, serine and phenylalanine, aspartic acid and phenylalanine, glutamic acid and phenylalanine, tyrosine and benzyl glutamate, serine and benzyl glutamate, aspartic acid and benzyl glutamate, glutamic acid. and benzyl glutamate, aspartic acid and leucine, and glutamic acid and leucine.

In some embodiments, Ry consists of a mixture of three natural or unnatural amino acid side chain groups such that the total block is hydrophobic. Such ternary mixtures of amino acid side chain groups include, but are not limited to: leucine, tyrosine, and aspartic acid; leucine, tyrosine, and glutamic acid;

phenylalanine, tyrosine, and aspartic acid; or phenylalanine, tyrosine, and glutamic acid.

In other modalities, Ry consists of

INDUSTRIAL

ΙΜΡΙί

INDUSTRIAL D-hydrophobic and L-hydrophilic side chain groups of

<img file="MX340992B_D0074.tif" />

amino acid such that the total poly (amino acid) block comprising Ry is hydrophobic and is a mixture of D- and L-configured amino acids. Such mixtures of amino acid side chain groups include L-tyrosine and D-leucine, L-tyrosine and D-phenylalanine, L-serine and D-phenylalanine, Aspartic acid and D-phenylalanine, L-glutamic acid and Dphenylalanine, L- tyrosine and D-benzyl glutamate, L-serine and Dbenzyl glutamate, L-aspartic acid and D-benzyl glutamate, Glutamic acid and D-benzyl glutamate, L-aspartic acid and Dleucine, and L-glutamic acid and D-leucine. The ratio (Hydrophobic to L-hydrophilic) of such mixtures include any of 6: 1, 5: 1, 4: 1, 3: 1, 2: 1, 1: 1, 1: 2, 1: 3, 1: 4 ;

1: 5, and 1: 6.

As generally defined above, group R² of formula I is a mono-protected amine, a protected di-amine, -NHR⁴, -N (R⁴) 2, -NHC (O) R⁴, -NR⁴C (O) R⁴, NHC (0) NHR⁴, NHC (O) N (R⁴) 2, -NR⁴C (O) NHR⁴, NR⁴C (0) N (R⁴)₂, -NHC (O) OR⁴,

NR⁴C (0) OR⁴, -NHSO2R⁴, or -NR⁴S02R⁴, where each R⁴ is independently an optionally substituted group selected from a 5-8 membered, unsaturated, aliphatic, saturated aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, an aryl ring

IMPI®

<img file="MX340992B_D0075.tif" />

INSTITUTO MEXICANO bicyclic saturated, partially unsaturated, ^{OF THE}^ uswl8elements having 0-5 heteroatoms ...... ar - ^ - i onaHn.g independently from nitrogen, oxygen, or sulfur, or a detectable portion, or two R⁴ at the same nitrogen atom they are taken in conjunction with such a nitrogen atom to form an optionally substituted 4-7 element, partially unsaturated, aryl or saturated ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In certain modalities, group R² of formula I is -NHR⁴ or -N (R⁴) 2 where each R⁴ it is an optionally substituted aliphatic group. A group R⁴ Exemplary is 5norbornen-2-yl-methyl. In accordance with yet another aspect of the present invention, group R^2nd of formula I is -NHR⁴ where R⁴ it is a C3 aliphatic group substituted with N3. Examples include -CH2N3. In some modalities, R⁴ it is an optionally substituted C1-6 alkyl group. Examples include methyl, ethyl, propyl, butyl, pentyl, hexyl, 2- (tetrahydropyran-2-ylcsxi) ethyl, pyridin-2-yldisulfanylmethyl, methyldisulfanylmethyl, (4-acetylenyl-phenyl) methyl, 3- (methoxycarbonyl) -prqp-2-ynyl, methoxycarbonyl 2- (N-Methyl-N- (4-acetylenylphenyl) carbonylamino) ethyl, 2-phthalimidoethyl, 4-bromobenzyl, 4-chlorobenzyl, 4fluorobenzyl, 4-yodcbenzyl, 4-propargyloxybenzyl, 2-nitrobenzyl, 4 (bis-4- acetylenylbenzyl) aminomethyl-benzyl, 4-propargyloxybenzyl, 4-dipropargilamino-benzyl, 4- (2-propargiloxi61

IΜ ΡI

INSTITUTO MEXICANO etildisulfañil) benzil,

2-propargiloxy-etTW 'FROPTEDaD

INDUSTRIAL propargildisulfanyl-ethyl, 4-propargyloxy-butyl, 2- (N-methylN-propargylamino) ethyl, and 2- (2-dipropargylaminoethoxy) -ethyl. In other modalities, R⁴ it is an optionally substituted C2-6 alkenyl group. Examples include vinyl, allyl, crotyl, 2-propenyl, and but-3-enyl. When group R⁴ is a substituted aliphatic group, substituents on R⁴ They include N3, CN, and halogen. In certain modalities, R⁴ is -CH2CN, CH2CH2CN, -CH2CH (OCH3) 2, 4- (bisbenzyloxymethyl) phenylmethyl, and the like.

In accordance with another aspect of the present invention, group R² of formula I is -NHR⁴ where R⁴ is an alkynyl C group₂-6 optionally substituted. Examples include -CC = CH, -CH₂C = CH, -CH₂CsCCH₃, and -CH₂CH₂C = CH.

In certain modalities, group R² of formula I is

-NHR⁴ where R⁴ it is an optionally substituted 5-8 element aryl ring. In certain modalities, R⁴ it is optionally substituted phenyl or optionally substituted pyridyl. Examples include phenyl, 4-tbutoxycarbonylaminophenyl, 4-azidomethylphenyl, 4propargyloxyphenyl, 2-pyridyl, 3-pyridyl, and 4-pyridyl. In certain modalities, R^2nd is 4-t-butoxycarbonylaminophenylamino,

4-azidomethylphenamino, or 4-propargyloxyphenylamino.

In certain modalities, group R^2nd of formula I is

-NHR⁴ where R⁴ it is an optionally substituted phenyl ring.

Substituents on the phenyl ring

<img file="MX340992B_D0076.tif" />

halogen; - (CH₂)₀-₄R °; - (CH₂) ₀-₄OR °; - (CH₂) ₀-₄CH (0R °) ₂;

(CH₂)or-₄SR °; - (CH₂) o-4Ph, which can be substituted with R °; - (CH₂) 0-4O (CH₂) o-iPh which can be replaced with R °; -CH = CHPh, which can be substituted with R °; -N0₂; -CN; -N₃; - (CH₂) ₀-₄N (R °) ₂ ; - (CH₂) ₀-₄N (R °) C (0) R °; -N (R °) C (S) R °; (CH₂) or-₄N (R °) C (0) NR °₂ ; -N (R °) C (S) NR °₂; - (CH₂) 0-4N (R °) C (0) 0R °; N (R °) N (R °) C (0) R °; -N (R °) N (R °) C (0) NR °₂ ; -N (R °) N (R °) C (0) 0R °; (CH₂) or-₄C (0) R °; -C (S) R °; - (CH₂) ₀-₄C (O) 0R °; - (CH₂) ₀-₄C (0) SR °; (CH₂) 0-4C (0) OSíR °₃ ; - (CH₂) or-₄OC (0) R °; - (CH₂) ₀-₄SC (0) R °;

(CH₂) or-₄C (0) NR °₂; -C (S) NR °₂; - (CH₂) ₀-₄OC (0) NR °₂; -C (0) N (0R °) R °; C (O) C (O) R °; -C (0) CH₂C (0) R °; -C (NOR °) R °; - (CH₂)₀-₄SSR °;

(CH₂) or-₄S (0) ₂R °; - (CH₂) ₀-₄S (0) ₂0R °; - (CH₂) ₀-₄OS (0) ₂R °; -SW)₂NR °₂;

- (CH₂) ₀-₄S (0) R °; -N (R °) S (0)₂NR °₂; -N (R °) S (0)₂R °; -N (OR °) R °; C (NH) NR °₂; -P (O)₂R °; -P (O) R °₂; -0 P (O) R °₂; YES °₃; where each independent occurrence of R ° is as defined in the present supra. In other modalities, group R^2nd of formula

I is -NHR⁴ where R⁴ it is phenyl substituted with one or more optionally substituted C 1-6 aliphatic groups. In still other modalities, R⁴ it is vinyl substituted phenyl, allyl, acetylenyl, -CH₂N₃, -CH₂CH₂N₃, -CH₂ChCCH₃, or -CH₂C = CH.

In certain modalities, group R² of formula I is -NHR⁴ where R⁴ is phenyl substituted with N₃, N (R °)₂, CO₂R °, or C (0) R ° where each R ° is independently as defined herein above.

IMPI

<img file="MX340992B_D0077.tif" />

In certain modalities, group R² dd^NS^^^^ gI

INDUSTRIAL

-N (R⁴)₂ where each R⁴ is independently an optionally substituted group selected from aliphatic, phenyl, naphthyl, a 5-6-membered aryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8- bicyclic aryl ring 10 elements having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a detectable portion.

In other modalities, group R² of formula I is N (R⁴)₂ where the two R groups⁴ they are taken in conjunction with such a nitrogen atom to form an optionally substituted 4-7 membered, partially unsaturated, aryl or saturated ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In accordance with another modality, the two groups R⁴ they are taken together to form a 5-6 element saturated or partially unsaturated ring having a nitrogen where such ring is substituted with one or two oxo groups. Such groups R^2nd include, but are not limited to, phthalimide, maleimide, and succinimide.

In certain modalities, group R² of formula I is a mono-protected or di-protected amino group. In certain modalities R^2nd it is a mono-protected amine. In certain modalities R^2nd is a mono-protected amine selected to

IM ΡI from aralkylamines, carbamates, allylamine SVC ^ S'ÍR ^. ^ &amp; AKÍ ^^ '^

INDUSTRIAL ΧΞ '-' βί ^ ϊ '¹

Exemplary mono-protected portions include t-butyloxycarbonylamino, methyloxycarbonylamino, allyloxycarbonylamino, ethyloxycarbonylamino, trichloroethyloxy-carbonylamino, benzyl-yloxycarbonylamino, benzylamino, fluoroyl-methylcarbonylamino, trichloroyl, acetamido, trichloroyl, acetamido, trichloroidoyl, In other modalities R^2nd it is a protected diamine. Exemplary di-protected amino portions include di-benzylamino, di-allylamino, phthalimide, maleimido, succinimido, pyrrolo, 2,2,5, 5-tetramethyl [1,2,5] azadisylolidino, and azido. In certain modalities, the R portion^2nd it is phthalimid. In other modalities, the R portion^2nd it is mono- or di-benzylamino or mono- or di-allylamino.

In certain embodiments, the present invention provides a triblock copolymer of formula II:

<img file="MX340992B_D0078.tif" />

II where:

n is 20-500;

<td>m</td><td>is</td><td> 0,</td><td> 1, 0</td>

<td>X</td><td>is</td><td> 3</td><td>to 50;</td>

<td>and</td><td>is</td><td> 5</td><td>to 100;</td>

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX340992B_D0079.tif" />

Ry is selected from one or more natural or unnatural amino acid side chain groups such that the total block is hydrophobic;

R¹ is -Z (CH2CH2Y) p (CH₂) _tR³, where:

Z is -O-, -NH-, -S-, -C = C-, or -CH₂-;

each Y is independently -0- or -S-;

p is 0-10;

t is 0-10; and

R³ is hydrogen, -N₃, -CN, -NH2, -CH₃,

<img file="MX340992B_D0080.tif" />

a chain cyclooctin moiety, a mono-protected amine, a protected di-amine, an optionally protected aldehyde, an optionally protected hydroxyl, an optionally protected carboxylic acid, an optionally protected thiol, or an optionally substituted group selected from a aliphatic, 5-8 element, partially unsaturated, saturated aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, an aril ring

<img file="MX340992B_D0081.tif" />

IMPI, _χ . INSTITUTO MEXICANO bicyclic saturated, partially more saturated? ^^^ ·¹* 'elements having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a detectable portion.

In certain embodiments, a triblock copolymer of formula II is selected from the following exemplary compounds shown in Table 1,

<img file="MX340992B_D0082.tif" />

where n is 20 to 500, x is 3 to 50, y 'is 3 to 50, and is 3 to 50.

Table 1.

<td>Compound #</td><td>R¹</td><td>R^already</td><td>_Rand b</td>

<td> 1</td><td>ch₃or.....</td><td>Ύ</td><td>-ΊΧ</td>

<td> 2</td><td>ch₃or.....</td><td>Ύ</td><td>^zYh</td>

<td> 3</td><td>ch₃or.....</td><td>-Ύ</td><td>-Ύ^Η 0</td>

<td> 4</td><td>CH₃OR.....</td><td>Ύ</td><td>-Y-OH</td>

<td> 5</td><td>CH₃OR.....</td><td> ^0</td><td>'Ό- „</td>

<img file="MX340992B_D0083.tif" />

<img file="MX340992B_D0084.tif" />

<img file="MX340992B_D0085.tif" />

<img file="MX340992B_D0086.tif" />

<img file="MX340992B_D0087.tif" />

<img file="MX340992B_D0088.tif" />

IMPI?

<td rowspan="2"> 79</td><td rowspan="2"> /</td><td rowspan="2"></td><td>OH&gt; £ THE IkCFIEDA 'INDUSTRY or</td>

<td></td>

<td> 80</td><td></td><td> '•^0-0</td><td>.- ^ OH</td>

In certain embodiments, a triblock copolymer of formula II is

<img file="MX340992B_D0089.tif" />

In certain embodiments, a triblock copolymer of formula II is

<img file="MX340992B_D0090.tif" />

In certain embodiments, a Formula II triblock copolymer is

Oh

<img file="MX340992B_D0091.tif" />

In certain embodiments, the present invention provides a triblock copolymer of formula III:

<img file="MX340992B_D0092.tif" />

<img file="MX340992B_D0093.tif" />

oy

where:

n is 20-500;

month 0, 1, or 2;

x is 3 to 50;

y is 5 to 100;

Ry is selected from one or more natural or unnatural amino acid side chain groups such that the total block is hydrophilic;

R¹ is -Z (CH2CH2Y) p (CH₂) tR³, where:

Z is -O-, -NH-, -S-, -ChC-, or -CH₂-;

each Y is independently -0- or -S-;

p is 0-10;

t is 0-10; and

R³ is hydrogen, -N3, -CN, -NH2, -CH3,

Ο&gt; Λ O 'a portion of chain cyclooctin, a mono-protected amine, a protected di-amine, an optionally protected aldehyde, an optionally protected hydroxyl, an optionally protected carboxylic acid, an optionally protected thiol, or an optionally substituted group selected from

<img file="MX340992B_D0094.tif" />

INSTITUTO MEXICANO DE LA PROPERTY a 5-8 Alloy, partially unsaturated, saturated aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a partially unsaturated, saturated bicyclic aryl ring of 8 -10 elements having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a detectable portion.

In certain embodiments, a triblock copolymer of formula III is selected from the following exemplary compounds as shown in Table 2,

<img file="MX340992B_D0095.tif" />

Where n is 20 to 500, xes3a50, y 'is 3 to 50, and y' 'is 3 to 50.

Table 2

<td> 81</td><td>ch₃or.....</td><td>Ύ</td><td> -70.,</td>

<td> 82</td><td>ch₃or.....</td><td>Ύ</td><td></td>

<td> 83</td><td>ch₃or.....</td><td>Ύ</td><td>-V^H 0</td>

<td> 84</td><td>ch₃or.....</td><td>Ύ</td><td> 0</td>

<td> 85</td><td>ch₃or.....</td><td>• ~ o</td><td>Q „</td>

<img file="MX340992B_D0096.tif" />

IMPI

<td> 86</td><td>CH₃OR.....</td><td>'X</td><td>_INDUSTRIAL Χ) Η</td>

<td> 87</td><td>ch₃or.....</td><td>X</td><td>• -Ύ^0ΗΟ</td>

<td> 88</td><td>ch₃or.....</td><td>• χ</td><td>ΧθΗ</td>

<td> 89</td><td>ch₃or.....</td><td>..'- γΟ ^ χΟ 0</td><td>'α.</td>

<td> 90</td><td>CH₃OR</td><td>..-- γθν ^ Ο 0</td><td>Χ&gt; Η</td>

<td> 91</td><td>ch₃or.....</td><td> 0</td><td>• -V^H 0</td>

<td> 92</td><td>ch₃or.....</td><td> 0</td><td> 0</td>

<td> 93</td><td>CH₃OR</td><td></td><td>Ό ,.</td>

<td> 94</td><td>ch₃or.....</td><td>Χοχ</td><td>Χ) Η</td>

<td> 95</td><td>ch₃or.....</td><td>X0</td><td>• -ν^Η 0</td>

<td> 96</td><td>CH₃OR.....</td><td></td><td>ΧθΗ</td>

<td> 97</td><td>N₃—°-·</td><td>'Ύ</td><td>Ό-οπ</td>

<td> 98</td><td>Ν ^ Χ</td><td>'Ύ</td><td>'' '^ ΌΗ</td>

<td> 99</td><td>N ^⁰·</td><td>'Ύ</td><td>-Ύ^0Η 0</td>

<img file="MX340992B_D0097.tif" />

<img file="MX340992B_D0098.tif" />

• ι · ιι ·&gt; ι », ι» «« ί ·! ».? * Ι.ϋτχ_ ^ η

<img file="MX340992B_D0099.tif" />

IMPI

<td rowspan="2"> 140</td><td rowspan="2">OR,.....</td><td rowspan="2">,. ·· -γ ° ν / 0 0</td><td>O INDUSTRIAL --A&gt; h</td>

<td></td>

<td> 141</td><td>'ΓΧ</td><td></td><td>former</td>

<td> 142</td><td>Oo .....</td><td></td><td>^{/ X}Oh</td>

<td> 143</td><td>Oo .....</td><td></td><td> ,.·_Ύ° η 0</td>

<td> 144</td><td>former</td><td> -^0-0</td><td>-Ah</td>

<td> 145</td><td> /</td><td>AND</td><td>x „</td>

<td> 146</td><td></td><td>AND</td><td>^{/ X}Oh</td>

<td> 147</td><td></td><td>Ύ</td><td>-x^0H 0</td>

<td> 148</td><td></td><td>Ύ</td><td>-TO_H</td>

<td> 149</td><td>and</td><td>YQ</td><td>'X „</td>

<img file="MX340992B_D0100.tif" />

<img file="MX340992B_D0101.tif" />

<img file="MX340992B_D0102.tif" />

IMPI

<img file="MX340992B_D0103.tif" />

<img file="MX340992B_D0104.tif" />

<img file="MX340992B_D0105.tif" />

<img file="MX340992B_D0106.tif" />

provides a triblock copolymer of formula IV:

<img file="MX340992B_D0107.tif" />

where:

n is 20-500;

m is 0, 1, or 2;

x is 3 to 50;

y is 5 to 100;

Ry is selected from one or more natural or unnatural amino acid side chain groups such that the total block is hydrophobic;

R¹ is -Z (CH₂CH₂Y) p (CH₂) tR³, where:

Z is -0-, -NH-, -S-, -C = C-, or -CH₂-;

each Y is independently -0- or -S-;

p is 0-10;

IMPI

MEXICAN INSTITUTE E'E LA PROWIDAD INDUSTRIAL

<img file="MX340992B_D0108.tif" />

t is 0-10; and _

R³ is hydrogen, -N3, -CN, -NH2, -CH3,

<img file="MX340992B_D0109.tif" />

a chain cyclooctin moiety, a mono-protected amine, a protected di-amine, an optionally protected aldehyde, an optionally protected hydroxyl, an optionally protected carboxylic acid, an optionally protected thiol, or an optionally substituted group selected from a aliphatic, 5-8 element, partially unsaturated, saturated aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, an 8-10 element, partially unsaturated, saturated bicyclic aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a detectable portion.

B. Target Group Union

Compounds of any of the formulas I, II, III, and IV that have R portions³ Suitable for Clic chemistry are useful for conjugation of such compounds to biological systems or macromolecules such as peptides, proteins, viruses, and cells, to name but a few. The Click reaction

IMPIAS!

MEXICAN INSTITUTE OF PROPERTY is known for proceeding quickly and select ivamé ¥ ft ^^klAfca · physiological conditions. In contrast, most of the conjugation reactions are carried out using the primary amine functionality in the proteins (eg, lysine or protein end group). Because most proteins contain a multitude of lysines and arginines, such conjugation occurs uncontrollably at multiple sites on the protein. This is particularly problematic when lysines or arginines are located around the active site of an enzyme or other biomolecule.

Thus, another embodiment of the present invention provides a method for conjugating the R groups.¹ from a compound of any of the formulas I, II, III, and IV to a macromolecule by Clic chemistry. Still another embodiment of the present invention provides a macromolecule conjugated to a compound of any of formulas I, II, III, and IV by the group R¹.

After incorporation of the poly (amino acid) block moieties into the multiple block copolymer of the present invention resulting in a multiple block copolymer of the WX-X 'form, the other end group functionality, corresponding to portion R¹ of any of formulas I, II, III, and IV can be used to bind to target groups for cell-specific delivery including, but not limited to,

IMPI 3 ^^,

INSTITUTO MEX ICANO, ri&gt; 'union of target groups for specific supply ¥ i¿12 ^ T ^ ié cell that includes, but is not limited

-ΡΧΩίΧίΐΙ ^ oligopeptides, antibodies, monosaccharides, oligosaccharides, vitamins, or other small biomolecules. Such target groups include, but are not limited to monoclonal and polyclonal antibodies (eg, IgG, IgA, IgM, IgD, IgE antibodies), sugars (eg, mannose, mannose-6-phosphate, galactose), proteins (eg, Transimerin), oligopeptides (eg, cyclic and acyclic RGD containing oligopeptides), and vitamins (eg, folate). Alternatively, portion R¹ of any of formulas I, II, III, and IV is attached to a biomolecule, drug, cell, or other substrate.

In other modalities, the R portion¹ of any of the formulas I, II, III, and IV is bound to biomolecules which promote cell entry and / or endosomal escape. Such biomolecules include, but are not limited to, oligopeptides containing protein transduction domains such as the HIV Tat peptide sequence (GRKKRRQRRR) or oligoarginine (RRRRRRRRR). Oligopeptides which undergo conformational changes in varying pH environments such as oligohistidine (HHHHH) also promote cell entry and endosomal escape.

In other modalities, the R portion¹ of any of the formulas I, II, III, and IV is attached to portions

<img file="MX340992B_D0110.tif" />

IMPI

Detectable MEXICAN INSTITUTE, such as dyes or fluorescent labels &amp; asnupa positron emission tomography that inrlnypn. mniÁpnlag gii »contain radioisotopes (eg¹⁸F) or ligands with radioactive metal bonds (eg, ⁶²Cu). In other modalities, the R portion¹ of any of the formulas I, II, III, and IV is bound to magnetic resonance imaging contrast agents such as gadolinium particles, gadolinium chelates, or iron oxide (eg Fe3C&gt; 4 and Fe2C&gt; 3) . In other modalities, the R portion¹ of any of the formulas I, II, III, and IV is attached to a semiconductor nanoparticle such as cadmium selenide, cadmium sulfide, or cadmium telluride or attached to other metal nanoparticles such as colloidal gold. In other modalities, the R portion¹ of any of formulas I, II, III, and IV is attached to natural or synthetic surfaces, cells, viruses, dyes, drugs, chelating agents, or used for incorporation into hydrogels or other tissue scaffolds.

In one embodiment, the R portion¹ of any of formulas I, II, III, and IV is an alkyne or a terminal alkyl derivative which is capable of undergoing [3 + 2] cycloaddition reactions with molecules and biomolecules bearing complementary azide. In another embodiment, the R portion¹ of any of the formulas I, II, III, and IV is an azide or an azide derivative which is capable of undergoing [3 + 2] cycloaddition reactions with molecules and biomolecules that carry

IMPIfé,. _,. . . ,. x MEXICAN INSTITUTE alkyne complementary (that is, Clic chemistry).^MLA..property

INDUSTRIAL

<img file="MX340992B_D0111.tif" />

Clic chemistry has become a popular bioconjugation method due to its high reactivity and selectivity, even in a biological medium. See Kolb, HC; Finn,

MG; Sharpless, KB Angew. Chem. Int. Ed. 2001, 40, 20042021; and Wang, Q.; Chan, TR; Hilgraf, R.; Fokin, VV; Sharpless, KB; Finn, MGJ Am. Chem. Soc. 2003, 125, 3192-3193. In addition, currently available recombinant techniques allow the introduction of non-canonical azides and amino acids that carry alkynes into proteins, cells, viruses, bacteria, and other biological entities that consist of or contain proteins. See Link, AJ; Vink, Μ. KS;

Tirrell, DAJ Am. Chem. Soc. 2004, 126, 10598-10602;

Deiters, A .; Cropp, TA; Mukherji, M .; Chin, JW; yerson, C; Schultz, PGJ Am. Chem. Soc. 2003, 125, 11782-11783.

In another embodiment, the [3 + 2] cycloaddition reaction of the nanovectors bearing azide or acetylene and biomolecules bearing complementary azide or acetylene, are catalyzed by transition metals. Copper-containing molecules that catalyze the Clic reaction include, but are not limited to, copper bromide (CuBr), copper chloride (CuCl), copper sulfate (CuSO₄), copper iodide (Cul), [Cu (MeCN)₄] (OTf), and [Cu (MeCN)₄] (PFs). Ligands that bind to organic and inorganic meta can be used in conjunction with metal catalysts and include,

<img file="MX340992B_D0112.tif" />

IMPI

INSTITUTO MEXICANO but not limited to, sodium ascorbate, tris (triazolyl) amine, tris (carboxy ethyl) phosphine (TCEP), and sulfonated batophenanthroline ligands.

In another embodiment, the R portion¹ of any of the formulas I, II, III, and IV is a hydrazine or hydrazide derivative which is capable of reacting with aldehyde or ketone containing biomolecules to form hydrazone linkages. In another embodiment, the R portion¹ of any of formulas I, II, III, and IV is an aldehyde or ketone derivative which is capable of reacting with biomolecules containing a hydrazine or hydrazide derivative to form hydrazone bonds.

In another embodiment, the R portion¹ of any of the formulas I, II, III, and IV is a hydroxylamine derivative which is capable of reacting with aldehyde or ketone containing biomolecules. In another embodiment, the R portion¹ of any of the formulas I, II, III, and IV is an aldehyde or ketone which is capable of reacting with biomolecules containing a hydroxylamine, or a hydroxylamine derivative.

In yet another embodiment, portion R¹ of any of the formulas I, II, III, and IV is an aldehyde or ketone derivative which is capable of reacting with biomolecules containing primary or secondary amines to form imine bonds. In another embodiment, the R portion¹

IMPI

MEXICAN INSTITUTE OF FRCTIEDAD

<img file="MX340992B_D0113.tif" />

of any of the formulas I, II, III, and IV is a primary or secondary amino acid which is capable of reacting with biomolecules containing an aldehyde or ketone functionality to form imine linkages. It will be appreciated that the imine linkages can further be converted to stable amine linkages by treatment with a reducing agent (eg, lithium aluminum hydride, sodium borohydride, sodium cyanoborohydride, etc.).

In yet another embodiment, portion R¹ of any of the formulas I, II, III, and IV is an amine (primary or secondary) or alcohol which is capable of reacting with biomolecules containing activated esters (eg, 4-nitrophenol ester, N-hydroxysuccinimide, pentafluorophenyl ester , ortho-pyridylthioester), to form amide or ester linkages. In still other modalities, the R portion¹ of any of formulas I, II, III, and IV is an activated ester which is capable of reacting with biomolecules possessing amine (primary or secondary) or alcohols to form amide or ester linkages.

In still other modalities, the R portion¹ of any of formulas I, II, III, and IV is an amine or alcohol which is attached to carboxylic acid functional biomolecules using a coupling agent. In still other modalities, the R portion¹ of any of the formulas I, II, III, and IV is an acid functionality

OHR

IMPI 63¾¾

INSTITUTO MEXICANO carboxylic which is linked to biomolecules and amine or alcohol functionality using —v Hp coupling. Such coupling agents include, but are not limited to, carbodiimides (eg, l-ethyl-3- (3dimethylaminopropyl) -carbodiimide (EDC), diisopropylcarbodiimide (DIC), dicyclohexylcarbodiimide (DCC)), aminium or phosphonium derivatives (by example, PyBOP, PyAOP, TBTU, HATU, HBTU), or a combination of 1-hydroxybenzotriazole (HOBt) and an amino or phosphonium derivative.

In another embodiment, the R portion¹ of any of the formulas I, II, III, and IV is an electrophile such as a maleimide, a maleimide derivative, or a bromoacetamide derivative, which is capable of reaction with biomolecules containing thiols or amines. In another embodiment, the R portion¹ of any of formulas I, II, III, and IV is a nucleophile such as an amine or thiol which is capable of or reacts with biomolecules containing electrophilic functionality such as maleimide, a maleimide derivative, or a bromoacetamide derivative.

In still other modalities, the R portion¹ of any of formulas I, II, III, and IV is a portion of ortho-pyridyl disulfide which undergoes disulfide exchange with biomolecules containing thiol functionality. In still other modalities, the R portion¹ from any of

IMPI

<img file="MX340992B_D0114.tif" />

MEXICAN INSTITUTE formulas I, II, III, and IV is a thiol or deri - \? Ado / '¿£ 3) £&gt; l which undergoes disulfide exchange with biomolecules containing ortho-pyridyl disulfide functionality. It will be appreciated that such exchange reactions will result in a disulfide bond, which is reversible in the presence of a reducing agent (eg glutathione, dithiothreitol (DTT), etc.).

In certain embodiments, micelles of the present invention are mixed micelles comprising one or more compounds of formulas I, II, III, and IV. It will be appreciated that mixed micelles having different R groups¹, as described herein, can be conjugated to multiple other compounds and / or macromolecules. For example, a mixed micelle of the present invention may have a group R¹ Suitable for Clic chemistry and other R group¹suitable for covalent bonding through a variety of coupling reactions. Such mixed micelle can be conjugated to different compounds and / or macromolecules by these different R groups.¹. Such conjugation reactions are well known to one of ordinary skill in the art and include those described herein.

In certain embodiments, the present invention provides a triblock copolymer of formula V:

<img file="MX340992B_D0115.tif" />

where each of Q, x, y, n, R^x, RY and R² it is as defined above and as described in the classes and subclasses herein, both individually and in combination;

J is independently a valence bond or a straight or branched, saturated or unsaturated, bivalent C1-12 hydrocarbon chain, where 0-6 methylene units of are independently replaced by -Cy-, -O-, -NH-, - S-, -OC (O) -, -C (0) 0-, -C (O) -, -SO-, - SO2-, -NHSO2-, -SO2NH-,

NHC (O) -, -C (O) NH-, -OC (O) NH-, or -NHC (O) O-, where: -Cy- is a bivalent, partially unsaturated, saturated aryl ring of 5 -8 optionally substituted elements having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an optionally substituted 8-10 element saturated, partially unsaturated bicyclic aryl ring having 0-5 selected heteroatoms independently from nitrogen, oxygen, or sulfur; each T is

IMPI

Mexican INSTITUTE independently a target group. ^OF^ CSstpjal

<img file="MX340992B_D0116.tif" />

As generally described above, T is a target group. Such target groups are described in detail in US Patent Application Publication Number 2009/01 10662, published April 30, 2009, all of which is hereby incorporated by reference. Additional target groups are described in detail in US Patent Application No. 13 / 415,910, filed March 9, 2012, all of which is thus incorporated by reference.

In certain embodiments, group J is a valence bond as described above. In certain embodiments, group J is a methelene group. In other embodiments, group J is a carbonyl group. In certain embodiments, group J of formula V is a valence bond. In other modalities, group J is represented by a portion in Table 3.

IMPI

MEXICAN INSTITUTE OF FEOEIEDAD «INDUSTRIAL

<img file="MX340992B_D0117.tif" />

Table 3

<img file="MX340992B_D0118.tif" />

C. Formation of Mi ce la

Amphiphilic multi-block copolymers, as described herein, can self-assemble in aqueous solution to form nano-sized structures and

ΪΜΡΙ

<img file="MX340992B_D0119.tif" />

MEXICAN INSTITUTE micro. In water, these copolymers of blpqueá^{&gt; ELA}1fi | ^^ p amphiphilics are assembled by multimolecular micelization when presented in solution above the critical concentration of the micelle (CMC). Without wishing to be bound by any particular theory, it is believed that the hydrophobic poly (amino acid) portion or block of the copolymer, collapses to form the micellar nucleus, while the hydrophilic PEG block forms a peripheral crown and imparts solubility in water. In certain embodiments, the multi-block copolymers according to the present invention possess different hydrophobic and hydrophilic segments that form micelles. Furthermore, these multiple block polymers optionally comprise a poly (amino acid) block which contains functionality for crosslinking. It will be appreciated that this functionality is found in the corresponding amino acid side chain.

D. Drug Load

In accordance with one embodiment, the present invention provides a micelle comprising a triblock copolymer which comprises a polymeric hydrophilic block, optionally a crosslinkable or crosslinked poly (amino acid block), and a D, L- poly (amino acid) block. hydrophobic mixing, characterized in that such a micelle has an inner core, optionally a crosslinkable or crosslinked outer core, and a hydrophilic coating. How I know

II

<img file="MX340992B_D0120.tif" />

disclosed herein, micelles of the pres§ñ $ EeA are especially useful for encapsulating therapeutic agents. In certain modalities the therapeutic agent is hydrophilic.

Without wishing to be bound by any particular theory, it is believed that the accommodation of structurally diverse therapeutic agents within a micelle of the present invention is accomplished by adjusting the hydrophobic poly (amino acid) D, L-mixed block, i.e., the block that Ry understands. As discussed above, the hydrophobic mixture of D and L stereoisomers provide a poly (amino acid) block with a random spiral conformation thereby enhancing encapsulation of hydrophobic drugs.

Hydrophobic small molecule drugs suitable for loading on micelles of the present invention are well known in the art. In certain embodiments, the present invention provides a drug-loaded micelle as described herein. In other embodiments, the present invention provides a drug-loaded micelle as described herein, wherein the drug is a hydrophobic drug selected from those described herein, infra.

As used herein, the terms hydrophobic small molecule drugs, molecule drugs

IMPIOS small, therapeutic agent, and agents

INDUSTRIAL hydrophobic are all interchangeable.

In accordance with another embodiment, the present invention provides a drug-loaded micelle comprising a triblock copolymer of formula I and a therapeutic agent.

In accordance with another embodiment, the present invention provides a drug-loaded micelle comprising a triblock copolymer of formula I and a hydrophobic therapeutic agent.

In other embodiments, the present invention provides a system comprising a triblock copolymer of formula I and a hydrophobic therapeutic agent. In another embodiment, the present invention provides a system comprising a triblock copolymer of any of formulas I, II, III, or IV, either individually or in combination, and a

In yet another hydrophobic therapeutic agent. In the embodiment, the present invention provides a system comprising a triblock copolymer of formula II and a hydrophobic therapeutic agent.

In some embodiments, the present invention provides a micelle, having a suitable hydrophobic therapeutic agent encapsulated therein, comprising a multi-block copolymer of

IMPI

MEXICAN INSTITUTE OF PROPERTY formula I and a multiple block copolymer^F^ T ^^{TO THE} ¿T formula V, where each of the · ΤόπιιΐιϊΉ Γ 'and

<img file="MX340992B_D0121.tif" />

formula V are as defined above and described herein, wherein the ratio of formula I to formula V is between 1000: 1 and 1: 1. In other modes, the ratio is 1000: 1, 100: 1,

50: 1, 33: 1, 25: 1, 20: 1, 10: 1, 5: 1, or 4: 1. In still other modes, the ratio is between 100: 1 and 25: 1.

In some embodiments, the present invention provides a micelle, having a hydrophobic therapeutic agent encapsulated therein, comprising a multiple block copolymer of formula II and a multiple block copolymer of formula V, wherein each of formula II and formula V are as defined above and described herein, wherein the ratio of formula II to formula V is between 1000: 1 and 1: 1. In other modes, the ratio is 1000: 1, 100: 1,

50: 1, 33: 1, 25: 1, 20: 1, 10: 1, 5: 1, or 4: 1. In still other modes, the ratio is between 100: 1 and 25: 1.

Modalities with respect to each of the R groups¹, R^2nd, Q, R^x, Ry, n, m, and m 'of formula I are as described in various classes and subclasses, both individually and in combination, herein.

In certain embodiments, the present invention provides a drug-loaded micelle, as described

IMPI »

INSTITUTO MEXICANO DE LA HiOFlEDAO here, where the drug is a taxane. industrial

Taxanes are well-known in literature and are natural products produced by plants of the Taxus genus. The mechanism of action is stabilization by microtubule, thus inhibiting mitosis. Many taxanes are barely completely insoluble in water.

<img file="MX340992B_D0122.tif" />

HO

<img file="MX340992B_D0123.tif" />

<img file="MX340992B_D0124.tif" />

Epothilones are a group of molecules that have been shown to be microtubule stabilizers, a mechanism similar to paclitaxel (Bollag DM et al. Cancer Res. 1995, 55, 2325-2333).

Biochemical studies demonstrate that epothilones compete for the same binding site (Kowalski RJ, Giannakakou P, Hamel E. J Biol Chem. 1997, 272, 25342541). An advantage of epothilones is that they exert a much greater cytotoxic effect in cells that overexpress PGP compared to paclitaxel. Exemplary epothilones are shown below.

100

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL IROTIED.AO

<img file="MX340992B_D0125.tif" />

<img file="MX340992B_D0126.tif" />

O OH O

Epothilone A

<img file="MX340992B_D0127.tif" />

<img file="MX340992B_D0128.tif" />

Epothilone C

<img file="MX340992B_D0129.tif" />

<img file="MX340992B_D0130.tif" />

In certain embodiments, the present invention provides a drug-loaded micelle, as described herein, wherein the drug is paclitaxel.

In certain embodiments, the present invention provides a drug-loaded micelle, as described herein, wherein the drug is docetaxel.

In certain embodiments, the present invention provides a drug-loaded micelle, as described herein, wherein the drug is cabazitaxel.

In certain embodiments, the present invention provides a drug-loaded micelle, as described herein, wherein the drug is an epothilone.

In certain embodiments, the present invention provides a drug-loaded micelle, as described herein, wherein the drug is Epothilone B or Epothilone D.

101

<img file="MX340992B_D0131.tif" />

IMPI

INSTm? TC

In certain embodiments, the present invention &lt;9H ^ gjjf | j ^ c a drug-loaded micelle, as described herein, wherein the drug is Epothylan A or Epothilone C.

Vinca alkaloids are well known in the literature and are a number of anti-fungal agents. Vinca alkaloids include vinblastine, vincristine, vindesine, and vinorelbine, and work to prevent microtubule formation. Exemplary vinca alkaloids are shown below.

Oh

<img file="MX340992B_D0132.tif" />

Vincristine

<img file="MX340992B_D0133.tif" />

<img file="MX340992B_D0134.tif" />

Vindesine

<img file="MX340992B_D0135.tif" />

OR

Vinorelbina

In certain embodiments, the present invention provides a drug-loaded micelle, as described herein, wherein the drug is a vinca alkaloid.

102

<img file="MX340992B_D0136.tif" />

IMPI

MEXICAN INSTITUTE OF PROPERTY

In certain embodiments, the present provides a drug-loaded micelle, ComO ~ SS deyurlbe herein, wherein the drug is vinorelbine.

Berberine is well known in the literature and shows pharmaceutical effects in a range of applications including antibacterial and oncology applications. The anti-tumor activity of berberine and associated derivatives is described in Hoshi, et. to the. Gann, 1976, 67, 321-325.

Specifically, berberubin and berberubin ester derivatives are shown to have increased anti-tumor activity relative to berberine. The structures of berberine and berberubin are shown below.

<img file="MX340992B_D0137.tif" />

<img file="MX340992B_D0138.tif" />

Berberubin

In certain embodiments, the present invention provides a drug-loaded micelle, as described herein, wherein the drug is berberine.

In certain embodiments, the present invention provides a drug-loaded micelle, as described herein, wherein the drug is berberubin.

Camptothecin from the plant anti-tumor alkaloid (CPT) is a broad-spectrum anticancer agent that

103

<img file="MX340992B_D0139.tif" />

IMPI

The Mexican INSTITUTE OF LA directs DNA topoisomerase I. Although CPT has shown promising antitumor activity in vitro and "in¹ Vi V¿5, rió has been used clinically due to its low therapeutic efficacy and severe toxicity. Among the CPT analogues, irinotecan hydrochloride (CPT-11) has recently been shown to be active against colorectal, lung, and ovarian cancer. CPT-11 itself is a prodrug and is converted to 7-ethyl-10-hydroxy-CPT (known as SN-38), a biologically active metabolite of CPT-11, by carboxylesterases in vivo. A number of camptothecin derivatives are in development, the structures of which are shown below.

<img file="MX340992B_D0140.tif" />

In certain embodiments, the present invention

104

ΪΜΡΙ provides a drug-loaded micelle,

INDUSTRIAL herein, where the drug is camptothecin.

<img file="MX340992B_D0141.tif" />

In certain embodiments, the present invention provides a drug-loaded micelle, as described herein, wherein the drug is SN-38.

In certain embodiments, the present invention provides a drug-loaded micelle, as described herein, wherein the drug is S39625.

In certain embodiments, the present invention provides a drug-loaded micelle, as described herein, wherein the drug is an anthracycline.

Various anthracycline derivatives have been produced and found in clinical use for the treatment of leukemias, Hodgkin lymphoma, as well as cancers of the bladder, breast, stomach, lung, ovaries, thyroid, and soft tissue sarcoma. Such anthracycline derivatives include daunorubicin (also known as Daunomycin or daunomycin cerubidine), doxorubicin (also known as DOX, hydroxidaunorubicin, or adriamycin), epirubicin (also known as Elence or Farmorubicinine), idarubicin (also known as 4-desmetoxidans) or

Idamicin), and valrubicin (also known as Ntrifluoroacetyladriamycin-14-valerate or Valstar). Anthracyclines are typically prepared as an ammonium salt (eg, hydrochloride salt) to improve the

105

<img file="MX340992B_D0142.tif" />

In certain embodiments, the present invention provides a drug-loaded micelle, as described herein, wherein the drug is daunorubicin.

In certain embodiments, the present invention provides a drug-loaded micelle, as described herein, wherein the drug is doxorubicin.

Aminopterin is well known in the literature and is a folic acid analog that is an antineoplastic agent. Aminopterin functions as an enzyme inhibitor competing for the folate binding target of the enzyme dihydrofolate reductase. The structure of aminopterin is shown below.

<img file="MX340992B_D0143.tif" />

Aminopterin

IMPI

MEXICAN INSTITUTE OF THE INDUSTRIAL FROP1EOAD

<img file="MX340992B_D0144.tif" />

HO O

In certain embodiments, the present invention provides a drug-loaded micelle, as described herein, wherein the drug is aminopterin.

Platinum-based therapeutics are well known in the literature. Platinum therapeutics are widely used in oncology and act to cross-link DNA which results in cell death (apoptosis). Carboplatin, picoplatin, cisplatin, and oxaliplatin are exemplary platinum therapeutics and the structures are shown below.

CK_r...^NH3 cr ^ nh₃

Cisplatin

<img file="MX340992B_D0145.tif" />

<img file="MX340992B_D0146.tif" />

Oxaliplatin

<img file="MX340992B_D0147.tif" />

In certain embodiments, the present invention provides a drug-loaded micelle, as described herein, wherein the drug is picoplatin.

In certain embodiments, the present invention provides a drug-loaded micelle, as described herein, wherein the drug is a platinum therapeutic.

107

Suitable small molecule drugs &amp; t ^ JTc ^^^ o

INDUSTRIAL

IMPI

INDUSTRIAL micelles of the present invention are well known in the

<img file="MX340992B_D0148.tif" />

technique. In certain embodiments, the present invention provides a drug-loaded micelle as described herein, wherein the drug is a hydrophobic drug selected from pain relievers, anti-inflammatory agents, HDAC inhibitors, mitotic inhibitors, microtubule stabilizers, intercalators of DNA, topoisomerase inhibitors, anthelmintics, anti-arrhythmic agents, antibacterial agents, anti-viral agents, anticoagulants, anti-depressants, anti-diabetics, anti-epileptics, antifungal agents, anti-gout agents, anti-hypertensive agents, anti-materials, anti-migraine agents, anti-muscarinic agents, anti-neoplastic agents, agents that improve erectile dysfunction, immunosuppressants, agents anti-protozoa, anti-thyroid agents, anxiolytic agents, sedatives, hypnotics, neuroleptics, β blockers, cardiac inotropic agents, corticosteroids, diuretics, antiparquinsonian agents, gastro-intestinal agents, histamine receptor antagonists, keratolyptics, lipid regulating agents, anti-anginal agents, Cox inhibitors, leukotriene inhibitors, macrolides, muscle relaxants, nutritional agents, opioid analgesics, protease inhibitors, sex hormones, stimulants, relaxants muscle, anti-osteoporosis agents, anti-obesity agents, cognition enhancers, anti-continence agents

108 . IMPI urinary, prostatic anti-hypertrophy agents benignssnTvá ^ iSasho

INDUSTRIAL PROPERTY essential, non-essential fatty acids, and mixtures thereof.

<img file="MX340992B_D0149.tif" />

In other embodiments, the hydrophobic drug is selected from one or more analgesics, anti-bacterial agents, anti-viral agents, anti-inflammatory agents, anti-depressants, anti-diabetics, anti-epileptics, anti-hypertensive agents, anti-agents migraine, immunosuppressants, anxiolytic agents, sedatives, hypnotics, neuroleptics, β-blockers, gastro-intestinal agents, lipid regulating agents, anti-anginal agents, Cox-2 inhibitors, leukotriene inhibitors, macrolides, muscle relaxants, opioid pain relievers, protease inhibitors, sex hormones, cognition enhancers, urinary incontinence agents, and mixtures thereof.

In accordance with one aspect, the present invention provides a micelle, as described herein, loaded with a hydrophobic drug selected from any one or more of an Exemestance (aromasin), Camptosar (irinotecan), Elence (epirubicin), Femara (Letrozole), Gleevac (imatinib mesylate), Lentaron (formestane), Citadren / Orimeten (aminoglutethimide), Temodar, Prosear (finasteride), Viadur (leuprolide), Nexavar (Sorafenib), Kitril (Granisetron), Taxotero (Docetaxel), Taxol (paclitaxel), Kitril (Granisetron), Vesanoid) tretinoin) (retina A), XELODA (Capecitabine), Arimidax

109

IMPI

<img file="MX340992B_D0150.tif" />

(Anastrozole), Casodex / Cosudex (Bicalutamide'PV'ra ™ £?

INDUSTRIAL (Fulvestrant), Iressa (Gefitinib), Nolvadex, Istubal, Valodex (tamoxifen citrate), Tomudex (Raltitrexed), Zoladex (goserelin acetate), Leustatin (Cladribine), Velcade (bortezomib), Milotarg (ozogamycin, gemtuz Alimta (pemetrexed), Gemzar (gemcitabine hydrochloride), Rituxan (rituximab), Revlimid (lenalidomide), Thalomid (thalidomide), Alkeran (melphalane), and derivatives thereof.

E. Crosslinking Chemistry

In certain embodiments, the present invention provides crosslinked micelles with ionic or hydrophobic therapeutic agents effectively encapsulated at pH 7.4 (blood) but dissociates and releases the drug at target acidic pH values ​​ranging from 5.0 (endosomal pH) to 6.8 (pH of extracellular tumor). In still other modes, the pH value can be adjusted between 4.0 and 7.4. These target pH nanovectors will dramatically improve cancer-specific delivery of chemotherapeutic agents and minimize the harmful side effects commonly found with powerful chemotherapy drugs. In addition, the use of chemicals which can be adjusted to dissociate across a range of pH values ​​make these drug-loaded micelles applicable in the treatment of solid tumors and malignancies that have become resistant to the drug.

110

IMPI

In certain modalities, the presefTESIL? ρμβ &amp; μβικ

INDUSTRIAL provides a drug-loaded micelle comprising a triblock copolymer, wherein such a micelle has a drug-loaded inner core, a crosslinked outer core, and a hydrophilic coating, wherein the triblock copolymer is of formula VI:

<img file="MX340992B_D0151.tif" />

R² it is as defined above and as described in the classes and subclasses herein, both individually and in combination;

M is a metal ion;

Each R^T is independently selected from either -JT or -Z (CH₂CH₂AND) _P (CH₂) _tR³, where:

Z is -O-, -S-, -CaC-, or -CH₂-;

each Y is independently -0- or -S-;

p is 0-10;

t is 0-10; and

R³ is -N3, -CN, a mono-protected amine, a di111

IMPIO protected amine, a protected aldehyde, «kiituto ^^ iox

INDUSTRIAL protected, a protected carboxylic acid, a protected thiol,

<img file="MX340992B_D0152.tif" />

a 9-30 element crown ether, or an optionally substituted group selected from a 5-8 element, aliphatic, partially unsaturated, saturated aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, an 8-10 membered partially unsaturated, saturated bicyclic aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a detectable moiety;

Q is a valence bond or a straight or branched, saturated or unsaturated, bivalent C1-12 hydrocarbon chain, where 0-6 methylene units of Q are independently replaced by -Cy-, -0-, -NH-, - S-, OC (O) -, -C (O) O-, -C (O) -, -SO-, -SO2-, -NHSO2-, -SO2NH-, NHC (O) -, -C (O ) NH-, -OC (O) NH-, or -NHC (O) O-, where:

-Cy- is an optionally substituted 5-8 membered, partially unsaturated, saturated aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or a bivalent, partially unsaturated, bicyclic saturated aryl ring , optionally substituted 8-10 element, having 0-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur;

112

PROPERTY ~ industrial

<img file="MX340992B_D0153.tif" />

In certain modalities, M is

OF THE OWNER

INDUST »_ modalities, M is zinc. In another embodiment, M is nickel, cobalt, copper, or platinum. In other embodiments, M is calcium or aluminum. In still other embodiments, M is strontium, manganese, platinum, palladium, silver, gold, cadmium, chromium, indium, or lead.

In certain embodiments, the present invention provides a drug-loaded micelle comprising a triblock copolymer, wherein such a micelle has a drug-loaded inner core, a crosslinked outer core, and a hydrophilic coating, wherein the triblock copolymer is formula VII:

<img file="MX340992B_D0154.tif" />

VII where each of Q, J, T, M, m, y, n, R^and and R^T is as defined above and as described in classes

113

IMPI

<img file="MX340992B_D0155.tif" />

and subclasses of the present, both individualiffélS ^ reíLiMuisj

INDUSTRIAL combination;

x¹ is 1-20; and x² is 0-20.

In certain embodiments, the present invention provides a drug-loaded micelle comprising a triblock copolymer, wherein such a micelle has a drug-loaded inner core, a crosslinked outer core, and a hydrophilic coating, wherein the triblock copolymer is formula VIII:

HO OH

<img file="MX340992B_D0156.tif" />

HO OH

HIV where each of Q, J, T, M, m, y, x¹, x², n, RY

114 and R^T is as defined above and as seNSToteseEKxidse * * OF PROPERTY

INDUSTRIAL

ΙΜΡΙ Ol aNSTdterseHflOite® f &amp; í

PROPERTY OR * »INDUSTRIAL classes and subclasses herein, both individually and in combination.

In certain embodiments, the present invention provides a drug-loaded micelle comprising a triblock copolymer, wherein such a micelle has a drug-loaded inner core, a crosslinked outer core, and a hydrophilic coating, where the triblock copolymer is formula IX:

<img file="MX340992B_D0157.tif" />

where each of

M, x¹ x- = yn is as defined above and as described in classes and

115

<img file="MX340992B_D0158.tif" />

combination subclasses;

and¹ is and² is

To be drug loaded, the crosslinked micelle of the present invention is comprised of tens to hundreds of polymer chains. Because the fact that only two polymer chains linked by a metal ion are represented in any of formulas VI, VII, VIII, or IX, it will be understood that the polymer micelle is comprised of many more polymer chains that are not represented by ease of presentation.

In other embodiments, the present invention provides a system comprising a triblock copolymer of formula I, a hydrophobic therapeutic agent, and a metal ion. In another embodiment, the present invention provides a system comprising a triblock copolymer of any of formulas I, II, III, and IV, either individually or in combination, a hydrophobic therapeutic agent, and a metal ion. In yet another embodiment, the present invention provides a system comprising a triblock copolymer of formula II, a hydrophobic therapeutic agent, and a metal ion.

In other embodiments, the present invention

116 provides a system comprising a

INDUSTRIAL

IMPI

INDUSTRIAL triblock of formula VI and a hydrophobic therapeutic agent.

<img file="MX340992B_D0159.tif" />

In another embodiment, the present invention provides a system comprising a triblock copolymer of any of formulas VI, VII, VIII, and IX, either individually or in combination, and a hydrophobic therapeutic agent. In yet another embodiment, the present invention provides a system comprising a triblock copolymer of formula VII and a hydrophobic therapeutic agent.

In some embodiments, the present invention provides a system comprising a triblock copolymer of formula XI and a hydrophobic therapeutic agent.

The ultimate goal of metal mediated crosslinking is to ensure the stability of the micelle when diluted in the blood (pH 7.4) followed by rapid dissolution and release of the drug in response to a finite pH change such as that found in an environment tumor.

In one aspect of the invention, a drug-loaded micelle is crosslinked by a portion of hydroxamic acid. Hydroxamic acids as described above chelate certain metals as described in

Rosthauser et. to the. Macromolecules 1981, 14, 538-543 and in Miller Chemical Reviews 1989, 89, 1563-1579 (later Miller). This chelation chemistry is shown in Reaction Scheme 1.

117

<img file="MX340992B_D0160.tif" />

Reaction scheme 1

ΙΜΡΠ

Divalent Metal

X -OH + MX₂RN ²

H

Hⁿ fan-⁰

H

Oh

H

ΗΟ '^Νγ^Π

O + 2X

Trivalent Metal

OR ³ fan'^0H + mx₃

H

An-^oh

H

R

AHIST ^ 0, I

Oh '' M '' OH

Item

HO-NH or ^^R + 3X

TO

Accordingly, the addition of a metal ion to a drug-loaded micelle of the present invention could result in chelation of the metal ions by hydroxamic acid, providing a cross-linked, drug-loaded micelle. Metal ions are selected from, but not limited to: iron, nickel, cobalt, zinc, calcium, copper, strontium, platinum, palladium, vanadium, manganese, and titanium.

One skilled in the art will recognize that the M group of formula VI, VII, or VIII can be either a divalent or trivalent metal ion. It is also recognized that the structures of formula VI, VII, or VIII, for clarity, are represented using a divalent metal ion. In the case of a trivalent metal ion as described in Reaction Scheme 1, it is understood that there may be three hydroxamic acid or catechol groups attached to a single metal ion.

118

IMPI _ _x MEXICAN INSTITUTE Γ ^ · * “Οίϊί * \ Λ

In one aspect of the invention, a drug mica ^ .iapRtsBfflnsraa '^ - ^ U' 'is crosslinked by a portion of catechol. The catechols, as described above, are metal complex ions as represented in Reaction Scheme 2. Chelation of catechols with metal ions is also described in Miller. Accordingly, the addition of a metal ion to a drug-loaded micelle of the present invention could result in chelation of the metal ions by hydroxamic acid, providing a cross-linked, drug-loaded micelle. Metal ions are selected from, but not limited to: iron, nickel, cobalt, zinc, calcium, copper, strontium, vanadium, manganese, and titanium.

Reaction scheme 2.

Divalent Metal

<img file="MX340992B_D0161.tif" />

R

In certain embodiments, the present invention provides a crosslinked drug-loaded micelle, as described herein, wherein the polymer is

119 '' f / V '⁰

<img file="MX340992B_D0162.tif" />

MEXICAN INSTITUTE ^ χ, · ^ ύϋι · &lt;.-^;' 2

OF INDUSTRIAL PROPERTY

IMPI

In certain embodiments, the present invention provides a crosslinked drug loaded micelle, as described herein, wherein the polymer is .oh

<img file="MX340992B_D0163.tif" />

In certain embodiments, the present invention provides a crosslinked drug-loaded micelle, as described herein, wherein the polymer is

120

<img file="MX340992B_D0164.tif" />

IMPI 0¾¾

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

In certain embodiments, the present invention provides a crosslinked drug-loaded micelle, as described herein, wherein the drug is a taxane.

In certain embodiments, the present invention provides a crosslinked drug-loaded micelle, as described herein, wherein the drug is paclitaxel.

In certain embodiments, the present invention provides a crosslinked drug-loaded micelle, as described herein, wherein the drug is docetaxel.

In certain embodiments, the present invention provides a crosslinked drug loaded micelle, as described herein, wherein the drug is cabazitaxel.

In certain embodiments, the present invention

121

IMPI. . . J J-, INSTITUTO MEXICANO provides a crosslinked drug-loaded micelle, such as E is® «oe®scr^c r =&gt; INDUSTRIAL herein, where the drug is an epothiline. _

<img file="MX340992B_D0165.tif" />

In certain embodiments, the present invention provides a crosslinked drug loaded micelle, as described herein, wherein the drug is Epothilone B or Epothilone D.

In certain embodiments, the present invention provides a cross-linked drug-loaded micelle, as described herein, wherein the drug is Epothilone A or Epothilone C.

In certain embodiments, the present invention provides a crosslinked drug-loaded micelle, as described herein, wherein the drug is a vinca alkaloid.

In certain embodiments, the present invention provides a crosslinked drug-loaded micelle, as described herein, wherein the drug is vinorelbine.

In certain embodiments, the present invention provides a crosslinked drug loaded micelle, as described herein, wherein the drug is berberine.

In certain embodiments, the present invention provides a crosslinked drug-loaded micelle, as described herein, wherein the drug is berberubin.

In certain embodiments, the present invention provides a crosslinked drug-loaded micelle, as described herein, wherein the drug is a camptothecin.

In certain embodiments, the present invention

122

IΜ ΡI provides a drug-loaded micelle, retmoTOÍai ^ a ^ ANncd ^^ a ^^^

OF THE PRO PIE

INDUSTRIAL is described herein, where the drug is SN-38.

In certain embodiments, the present invention provides a crosslinked drug-loaded micelle, as described herein, wherein the drug is S39625.

In certain embodiments, the present invention provides a crosslinked drug-loaded micelle, as described herein, wherein the drug is an anthracycline.

In certain embodiments, the present invention provides a crosslinked drug-loaded micelle, as described herein, wherein the drug is daunorubicin.

In certain embodiments, the present invention provides a crosslinked drug-loaded micelle, as described herein, wherein the drug is doxorubicin.

In certain embodiments, the present invention provides a crosslinked drug-loaded micelle, as described herein, wherein the drug is aminopterin.

In certain embodiments, the present invention provides a crosslinked drug-loaded micelle, as described herein, wherein the drug is picoplatin.

In certain embodiments, the present invention provides a crosslinked drug-loaded micelle, as described herein, wherein the drug is a platinum therapeutics.

123

IMPI

Four. General Methods for Providing nTo- ^ c ^^^ p

INDUSTRIAL

<img file="MX340992B_D0166.tif" />

of the Present Invention

The multi-block copolymers of the present invention are prepared by methods known to one of ordinary skill in the art. In general, such multi-block copolymers are prepared by sequentially polymerizing one or more cyclic amino acid monomers in a hydrophilic polymer having a terminal amine wherein such polymerization is initiated by such amine. In certain embodiments, such polymerization occurs by ring-opening polymerization of the cyclic amino acid monomers. In other embodiments, the cyclic amino acid monomer is an NCA, lactam, or imide amino acid.

Reaction scheme 3

<img file="MX340992B_D0167.tif" />

<img file="MX340992B_D0168.tif" />

<img file="MX340992B_D0169.tif" />

Reaction Scheme 3 above represents a

124

IMPI ^ -5 »

MEXICAN INSTITUTE general method to prepare block polymers ^DíiW ^ X ^^ Jes¡3! ®f^!'*‘^-tothe present invention. A macroinitiator of formula A is treated with a first amino acid NCA to form a compound of formula B having a first amino acid block. The second amino acid NCA is added to the living polymer of formula B to give a triblock copolymer of formula C having two different amino acid blocks. Each of R¹, R², n, Q, R^x, RX, x, e, and the groups represented in Reaction Scheme 3 are as defined and described in the classes and subclasses, individually and in combination, herein.

One step in the preparation of a compound of formula I comprises terminating the living polymer chain end of the compound of formula C with a polymerization terminator to provide a compound of formula I. One of ordinary skill in the art will recognize that the terminator polymerization provides group R² of formula I. Therefore, modalities directed to group R² of formula I as set forth above and herein, are also directed at the polymerization terminator itself, and similarly, embodiments are directed at the polymerization terminator, as set forth above and herein, are also directed at group R² of formula I.

As described above, compounds of formula I are prepared from compounds of formula C by treatment with a terminating agent. One of

125

<img file="MX340992B_D0170.tif" />

IMPI

MEXICAN INSTITUTE,,. . , 'MLA PROPERTY ordinary skill in the art may reconu'we »*' · compounds of formula I are also easily) propagated or directly from compounds of formula C. One of ordinary skill in the art might also recognize that the above method for preparing a compound of formula I can be performed as a synthesis of a melting pot of compounds of formula I that use the living polymer chain end to incorporate the group R² of formula I. Alternatively, the compounds of formula I can also be prepared in a multistage form. For example, the living polymer chain end of a compound of formula C can be quenched to provide an amino group which can then be further derivatized, in accordance with known methods, to provide a compound of formula I.

One of ordinary skill in the art will recognize that a variety of polymerization terminating agents are for the present invention. Such polymerization terminating agents include any group containing R²capable of reacting with the living polymer chain end of a compound of formula C, or the free base amino group of formula C, to provide a compound of formula I. Thus, polymerization terminating agents include anhydrides, and other acylating agents, and groups containing a leaving LG group that is subjected to nucleophilic displacement.

126

IMPI

MEXICAN INSTITUTE Al

DE LAJRPPIETAD τ ^ ·&gt; - * · *&gt; ** w? ^

Alternatively, the EBsarámaiAia compounds can be coupled to groups containing áid ^ —eaadaQxáJicxx. to form an imide of it. Thus, it is contemplated that the amine group of formula C can be coupled with a carboxylic acid moiety to provide compounds of formula I wherein R² is -NHC (O) R⁴. Such coupling reactions are well known in the art. In certain embodiments, coupling is accomplished with a

<td rowspan="2">coupling. Such technique and include,</td><td colspan="2">reagents</td><td rowspan="2">are DCC</td><td rowspan="2">well known in and EDC, among others.</td><td rowspan="2">the In</td>

<td>by</td><td>example,</td>

<td>other modalities,</td><td>the</td><td>portion</td><td>of</td><td>carboxylic acid</td><td>is</td>

<td>activated for use</td><td>in</td><td colspan="2">the reaction</td><td>coupling.</td><td>Such</td>

Activation includes formation of an acyl halide, use of a Mukaiyama reagent, and the like. These methods, and others, are known to one of ordinary skill in the art, for example, see Advanced Organic Chemistry, Jerry March, 5^th Ed., Pp. 351-357, John Wiley and Sons, NY

A suitable leaving group that is subjected to nucleophilic displacement is a chemical group that is easily displaced by a desired incoming chemical moiety. Suitable leaving groups are well known in the art, eg see March. Such leaving groups include, but are not limited to, halogen, alkoxy, sulfonyloxy, optionally substituted alkylsulfonyloxy, optionally substituted alkenylsulfonyloxy, arylsulfonyloxy

127

<img file="MX340992B_D0171.tif" />

Optionally substituted IMPI and portions of diazoni ^ TnE ^ empLas

OE INDUSTRIAL PROPERTY suitable leaving groups include chlorine, iodine, bromine, fluoro, methanesulfonyloxy (mesyloxy), tosyloxy, triflyloxy, nitrophenylsulfonyloxy (nosiloxy), and bromo-phenylsulfonyloxy (brosiloxy).

In accordance with an alternative modality, the leaving group can be generated in situ within the reaction medium. For example, a leaving group may be generated in situ from a precursor of such a compound wherein said precursor contains a group readily placed by such a leaving group in situ.

Alternatively, when group R² of formula I is a protected mono- or di-amine, the protecting group (s) are removed and such functional group can be derivatized or protected with a different protecting group. It will be appreciated that removal of any protecting group from group R² Formula I is performed by methods for such a protection group. Such methods are described in detail in

Green.

In other modalities, group R² of formula I is incorporated by derivatization of the amino group of formula C by anhydride coupling, optionally in the presence of a base as appropriate. One of ordinary skill in the art will recognize that anhydride polymerization terminating agents containing an azide, an aldehyde, a hydroxyl, an alkyl, and other groups,

128

IMPI r-. . jj_η . MEXICAN INSTITUTE or protected forms thereof, may be uaadi® »¡Er&gt; ría^c industrial incorporate such azide, such aldehyde, such protected hydroxyl,

<img file="MX340992B_D0172.tif" />

such alkyne, and other groups in group R² of compounds of formula I. It will also be appreciated that such anhydride polymerization terminating agents are also suitable for terminating the end of the living polymer chain of a compound of formula C, or free base thereof. Such anhydride polymerization terminating agents include, but are not limited to, those set forth in Table 3 below.

Table 3. Representative Anhydride Polymerization Terminating Agents oo

A-1 OO

OO hAAh or OI or OI or O

Λ₀Λ ^ AqA ^ · ^ Λ_ο,

<img file="MX340992B_D0173.tif" />

A-7

A-8

A-9

A-6 At, or

V1-4 '' 1-4

<img file="MX340992B_D0174.tif" />

OO o

A-15

<img file="MX340992B_D0175.tif" />

In certain embodiments, the hydrophilic polymer block is poly (ethylene glycol) (PEG) having an amine

129 terminal (PEG macroinitiator)

East

<img file="MX340992B_D0176.tif" />

p mac ro * e * fi¡ * agfe &amp; o

OF INDUSTRIAL PROPERTY

<img file="MX340992B_D0177.tif" />

initiates the polymerization of NCAs to provide the multi-block copolymers of the present invention. Such synthetic polymers having a terminal amine group are known in the art and include PEG amines. PEG amines can be obtained by deprotection of a properly protected PEG amine. The preparation of such suitably protected PEG-amines and methods for deprotecting them are described in detail in the Patent Application.

American series number 11 / 256,735, filed on October 24, 2005, and published as US 20060142506 on 29

June 2006, the entirety of which is thus incorporated herein by reference.

As described in US20060142506, suitably protected PEG-amines can be formed by terminating the living polymer chain end of a PEG with a terminating agent containing a suitably protected amine. Accordingly, in other embodiments, the terminating agent has a suitably protected amino group where the protecting group is acid labile.

Alternatively, synthetic polymers having a terminal amine can be prepared from synthetic polymers containing terminal functional groups that can be converted to amines by known synthetic routes. In certain modalities, the conversion of functional groups

130

IMPI

OF INDUSTRIAL PROPERTY

<img file="MX340992B_D0178.tif" />

terminals to the amine is conducted in an etaraanuTBiaattéifei ^c nt ii pnnpiFnin only. In other embodiments, the conversion of the terminal functional groups to the amine is accomplished by means of a multi-step sequence. In yet another embodiment, a protected amine initiator can be used to polymerize the ethylene oxide then terminated with an appropriate functional group to form the R group.¹ of formula I. The protected amine initiator can then be deprotected to provide the free amine for subsequent polymerization.

Functional group transformations that provide amines or protected amines are well known in the art and include those described in Larock, RC, Comprehensive Organic Transformations, John Wiley &amp; Sons, New York, 1999.

Reaction scheme 4

AND

ΌΗ (a)

<img file="MX340992B_D0179.tif" />

F

M

A n + 1

Reaction Scheme 4 above shows an exemplary method for preparing the bifunctional PEGs used for

131

IMPI prepare multi-block copolymers

INDUSTRIAL invention. In step (a), polymerization initiator E is treated with a base to form F. A variety of bases are suitable for the reaction in step (a). Such bases include, but are not limited to, potassium naphthalenide, diphenylmethylpotassium, triphenylmethyl potassium, and potassium hydride. In step (b), the resulting anion is treated with ethylene oxide to form polymer G. Polymer G is then quenched with a terminating agent in step (c) to form group R¹ of polymer H. Exemplary terminating agents for Polymer G can be found in Table 4. Polymer H can be transformed in step (d) to a compound of formula A by deprotection of the dibenzyl amine group by hydrogenation.

Table 4. Exemplary PEG Termination Agents h₃c._l

D-1

D-5

Na

D-2

D-6

Cr^s'

D-9

D-10

Na

D-3

OR

D-7 n

D-11

D-4

D-8 'SL

D-12

In accordance with another embodiment, the present invention provides a method for preparing a micelle that

132

<img file="MX340992B_D0180.tif" />

indiÍÍtrial comprises a multiple block copolymer the box

<img file="MX340992B_D0181.tif" />

a polymeric hydrophilic block, optionally a crosslinkable or crosslinked poly (amino acid block), and a hydrophobic poly (amino acid) block, characterized in that such a micelle has an inner core, an optionally crosslinkable or crosslinked outer core, and a hydrophilic coating, such method comprises the stages of:

(a) provide a multiple block copolymer of formula I:

R '^ °

<img file="MX340992B_D0182.tif" />

and where each of R¹, R², Q, R^x, Ry, n, x, and y groups of formula I, are as described in various classes and subclasses, both individually and in combination, herein, (b) combining such a compound of formula I with a therapeutic agent; and (c) treating the resulting micelle with a crosslinking reagent to crosslink R^x.

In one embodiment, the drugs are loaded into the inner core of the micelle by adding an aliquot of a copolymer solution in water to the drug to be incorporated. For example, a base solution of the drug is made in a polar organic solvent and allowed to evaporate, and then

133 add the copolymer / water solution

In otr &amp; is-

<img file="MX340992B_D0183.tif" />

OF INDUSTRIAL PROPERTY

<img file="MX340992B_D0184.tif" />

The drug is incorporated using an oil-in-water emulsion technique. In this case, the drug is dissolved in an organic solvent and added dropwise to the micelle solution in water, and the drug is incorporated into the micelle during evaporation of the solvent. In another embodiment, the drug is dissolved with the copolymer in a common polar organic solvent and dialyzed against water or other aqueous medium. See Alien, C; Maysinger, D .; Eisenberg A. Colloid Surface B 1999,

16, 3-27.

5. Uses, Methods and Compositions

As described herein, micelles of the present invention can encapsulate a wide variety of therapeutic agents useful for treating a wide variety of diseases. In certain embodiments, the present invention provides a drug-loaded micelle, as described herein, wherein such a micelle is useful in treating the disorder for which the drug is known to treat. In accordance with one embodiment, the present invention provides a method of treating one or more disorders selected from pain, inflammation, arrhythmia, arthritis (rheumatoid or osteoarthritis), atherosclerosis, restenosis, bacterial infection, viral infection, depression, diabetes, epilepsy , fungal infection, gout, hypertension, malaria, migraine, cancer or other disorder

134 prolif erative, erectile dysfunction, a traelg ^ ro ^ gHCAN ^

INDUSTRIAL

IMPIgg&gt; »

INDUSTRIAL thyroid, neurological disorders and hormone-related diseases, Parkinson's disease,

Huntingon, Alzheimer's disease, a gastrointestinal disorder, allergy, an autoimmune disorder, such as asthma or psoriasis, osteoporosis, obesity and comorbidities, a cognitive disorder, stroke, AIDS-associated dementia, amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease) , multiple sclerosis (MS), schizophrenia, anxiety, bipolar disorder, tauopoty, a lesion of the peripheral nerve or spinal cord, myocardial infarction, cardiomyocyte hypertrophy, glaucoma, an attention deficit disorder (ADD or ADHD), a sleep disorder, reperfusion / ischemia, an angiogenic disorder, or urinary incontinence, which comprises administering to a patient a micelle comprising a multiple block copolymer which comprises a hydrophilic block polymeric, optionally a crosslinkable or crosslinked poly (amino acid block), and a hydrophobic, D, L-mixed, poly (amino acid block) characterized in that such a micelle has a drug-loaded inner core, optionally a crosslinkable or crosslinked outer core, and a hydrophilic shell, wherein such micelle encapsulates a therapeutic agent suitable for treating such a disorder.

In other embodiments, the present invention

135

<img file="MX340992B_D0185.tif" />

provides a method of treating one or

IMPI selected from autoimmune disease, inflammatory disease, metabolic disorder, psychiatric disorder, diabetes, angiogenic disorder, tauopoty, neurological or neurodegenerative disorder, spinal cord injury, glaucoma, baldness, or cardiovascular disease, comprising administering to a patient a multiple block copolymer which comprises a polymeric hydrophilic block, optionally a crosslinkable or crosslinked poly (amino acid block), and a hydrophobic, D-L-mixed poly (amino acid block) characterized in that such a micelle has a drug-loaded inner core, optionally a crosslinkable or crosslinked outer core, and a hydrophilic coating, wherein such a micelle encapsulates a therapeutic agent suitable for treating such a disorder.

In certain embodiments, drug-loaded micelles of the present invention are useful in treating cancer. Accordingly, another aspect of the present invention provides a method of treating cancer in a patient comprising administering to a patient a multiple block copolymer which comprises a polymeric hydrophilic block, optionally a crosslinkable or crosslinked poly (amino acid block), and a D, L-mixed, hydrophobic poly (amino acid block) characterized in that such a micelle has a nucleus

136

<img file="MX340992B_D0186.tif" />

drug-loaded internal, optionally a

INDUSTRIAL PROPERTY crosslinkable or crosslinked, and a hydrophilic coating, in genitourinary, neuroblastoma, where such micelle encapsulates a chemotherapeutic agent. In accordance with another embodiment, the present invention relates to a method of treating a cancer selected from the breast, ovary, cervix, prostate, testes, esophagus, larynx, glioblastoma, stomach, skin, keratocanthoma, lung, squamous cell carcinoma, large cell carcinoma, small cell carcinoma, adenocarcinoma of the lung, bone, colon, adenoma, pancreas, adenocarcinoma, thyroid, follicular carcinoma, undifferentiated carcinoma, papillary carcinoma, seminoma, melanoma, sarcoma, carcinoma of the bladder, carcinoma of the liver, and bile ducts, carcinoma of the kidney, myeloid disorders, lymphoid disorders, Hodgkin, hair cells, oral cavity and pharynx (oral), lips, tongue, mouth, pharynx, small intestine, colon-rectum, large intestine, rectum, central nervous system and brain, and leukemia, comprising administering a micelle in accordance with the present invention wherein such micelle encapsulates a chemotherapeutic agent suitable for treating such cancer.

P-glycoprotein (Pgp, also called multiple drug resistance protein) is found in the plasma membrane of higher eukaryotes where it is responsible for the export triggered by ATP hydrolysis

137 of hydrophobic molecules. In animals, Pgp

<img file="MX340992B_D0187.tif" />

<img file="MX340992B_D0188.tif" />

OTO MEX1CAH OF INDUSTRIAL PROPERTY important in the excretion of, and protection from, environmental toxins, when expressed in the plasma membrane of cancer cells, can lead to chemotherapy failure preventing hydrophobic chemotherapeutic drugs from reaching their targets within cells . However, Pgp is known to transport chemotherapeutic drugs outside of tumor cells. In accordance with one aspect, the present invention provides a method of delivering a hydrophobic chemotherapeutic drug to a cancer cell while preventing, or decreasing, the excretion of Pgp from such a chemotherapeutic drug, comprising administering a drug-loaded micelle comprising a polymer Block Drug of the Present Invention Loaded with a Hydrophobic Chemotherapeutic Drug. Such hydrophobic chemotherapeutic drugs are well known in the art and include those described herein.

Compositions

In accordance with another embodiment, the invention provides a composition comprising a pharmaceutically acceptable micelle of this invention or a derivative thereof and a pharmaceutically acceptable carrier, adjuvant, or vehicle. In certain embodiments, the composition of this invention is formulated for administration to a patient in need of such a composition.

138

<img file="MX340992B_D0189.tif" />

In other embodiments, the composition of this is formulated for oral administration to a patient.

<img file="MX340992B_D0190.tif" />

'.' li

FROM THE FROMEPAD

INDUSTRIAL "*

The term patient, as used herein, means an animal, preferably a mammal, and most preferably a human.

The term "pharmaceutically acceptable carrier, adjuvant, or vehicle" refers to a non-toxic carrier, adjuvant, or vehicle that does not destroy the pharmacological activity of the compound with which it is formulated.

Pharmaceutically acceptable carriers, adjuvants, or carriers that can be used in the compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffering substances such like phosphates, glycine, sorbic acid, potassium sorbate, mixtures of partial glycerides of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium phosphate hydrogen, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, polyvinylpyrrolidone trisilicate, polyethylene glycol, carboxymethyl cellulose polyacrylates, waxes, polyethylenepolyoxypropylene block polymers, polyethylene glycol and grease.

magnesium, cellulose base, sodium,

Acceptable and fair sales of the

139

Compuestos Μ Ρ T compounds of this invention include that the ^ (^ e ^ jv ^ .dAs \&gt; &lt;g ^

OF THE PROPERTY

INDUSTRIAL ~ pharmaceutically acceptable organic and inorganic acids and bases. Examples of acid salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydrochloride, 2-hydroxy ethanesulfonate, lactate, maleate, malonate, methanesulfonate, 2naphthalenesulfonate, nicotinate, nitrate, oxalate, palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, tosylate and undecanoate. Other acids, such as oxalic, while not being the same pharmaceutically acceptable, can be used in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable acid addition salts.

Salts derived from suitable bases include alkali metal salts (eg, sodium and potassium), alkaline earth metal (eg, magnesium), ammonia, and N + (Ci-4-alkyl) 4. This invention also contemplates the quatemization of any of the basic nitrogen-containing groups of the compounds described herein. Dispersible or soluble products in oil or water can be obtained by such quatemization.

140

<img file="MX340992B_D0191.tif" />

OF THE INDUSTRIAL PROPERTY

The compositions herein are to be administered orally, parenterally, by intracranial spray.

administered by inhalation, topically, rectally, nasally, orally, vaginally, or through an implanted reservoir. The term parenteral as used herein includes subcutaneous, intravenous, intramuscular, intraarticular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional injection or infusion techniques. Preferably, the compositions are orally, intraperitoneally, or intravenously. The sterile injectable forms of the compositions of this invention can be aqueous or oleaginous suspensions. These suspensions can be formulated in accordance with techniques known in the art using dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally used as a solvent or suspending medium.

For this purpose, any soft fixed oil can be used including mono- or di-glycerides. The

141 fatty acids, such as oleic acid and

INDUSTRIAL

IMPI bhbkjsms »

INDUSTRIAL glycerides are useful in the preparation of injectables, such as

<img file="MX340992B_D0192.tif" />

they are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oily solutions or suspensions can also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms that include emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans, and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of solid, liquid, or other pharmaceutically acceptable dosage forms, may also be used for the purposes of the formulation.

The pharmaceutically acceptable compositions of this invention can be administered orally in any orally acceptable dosage form, including, but not limited to, capsules, tablets, suspensions, or aqueous solutions. In the case of tablets for oral use, commonly used carriers include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form,

142 useful diluents include lactose and alw

<img file="MX340992B_D0193.tif" />

) MW®ANCm OF PROPERTY

INDUSTRIAL

<img file="MX340992B_D0194.tif" />

dry. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring, or coloring agents may also be added. In certain embodiments, pharmaceutically acceptable compositions of the present invention are enterically coated.

Alternatively, the pharmaceutically acceptable compositions of this invention can be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and will therefore fuse in the rectum to release the drug. Such materials include cocoa butter, beeswax, and polyethylene glycols.

The pharmaceutically acceptable compositions of this invention can also be administered topically, especially when the goal of treatment includes areas or organs readily accessible for topical application, including diseases of the eye, skin, or lower intestinal tract. Suitable topical formulations are easily prepared for each of these areas or organs.

143

IMPI

Topical application for tracto^NSTI¿|^,Lower industrial j ^ e ^ $ h can be performed in a rectal suppository formulation (see above) or in an enema formulation. Topically transdermal patches can also be used.

For topical applications, the pharmaceutically acceptable compositions can be formulated in an ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax, and water. Alternatively, the pharmaceutically acceptable compositions can be formulated in a lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester waxes, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water.

For ophthalmic use, the pharmaceutically acceptable compositions can be formulated as micronized suspensions in sterile, pH-adjusted saline, isotonic, or, preferably, as solutions in sterile, pH-adjusted, isotonic saline, either with or without a

<img file="MX340992B_D0195.tif" />

144

IMPIOS, INSTITUTO MEXICANO il benct ^^ o) preservative such as chloride

Alternatively, for ophthalmic uses, the pharmaceutically acceptable compositions can be formulated in an ointment such as petrolatum.

The pharmaceutically acceptable compositions of this invention can also be administered by nasal spray or inhalation. Such compositions are prepared in accordance with techniques well known in the art of pharmaceutical formulation and can be prepared as solutions in saline, using benzyl alcohol or other preservatives, absorption promoters to improve bioavailability, fluorocarbons, and / or other solubilizing or dispersing agents. conventional.

In certain embodiments, the pharmaceutically acceptable compositions of this invention are formulated for oral administration.

The amount of the compounds of the present invention that can be combined with the carrier materials to produce a composition in a single dosage form will vary depending on the host treated, the particular mode of administration. Preferably, the compositions should be formulated such that a dosage of between 0.01 - 100 mg / kg body weight / day of the drug can be administered to a patient receiving these compositions.

145

<img file="MX340992B_D0196.tif" />

OF INDUSTRIAL PROPERTY

<img file="MX340992B_D0197.tif" />

It will be appreciated that two if icac ioneg_NSTITThe drugs used for the encapsulated drug are contemplated by the present invention. In certain embodiments, a patient is administered with a drug-loaded micelle of the present invention wherein the dosage of the drug is equivalent to that typically administered for such a drug. In other embodiments, a patient is administered with a drug-loaded micelle of the present invention wherein the dosage of the drug is less than what is typically administered for such a drug.

It should be understood that a specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the activity of the specific compound employed, age, body weight, general health, sex, diet, time of administration, speed of excretion, combination drug, and the judgment of the attending physician and the severity of the particular disease being treated. The amount of a compound of the present invention in the composition will also depend on the particular compound in the composition.

In order that the described invention may be more fully understood, the following examples are set forth. It will be understood that these examples are for illustrative purposes only and are not to be construed as limiting this invention in any way.

146

IMPI

EXEMPLIFICATION

As generally described above,

INSTITUI O MEXICANO DE LA PROFIEDAI · »INDUSTRIAL

<img file="MX340992B_D0198.tif" />

Multi-block copolymers of the present invention are prepared using the heterobifunctional PEGs described herein and in US Patent Application Serial No. 11 / 256,735, filed October 24, 2005, published as W02006 / 047419 May 4, 2006 , and published as US 20060142506 on June 29, 2006, the entirety of which is thus incorporated herein by reference. The preparation of multiple block polymers in accordance with the present invention is performed by methods known in the art, including those described in detail in US Patent Application Serial No. 11 / 325,020, filed January 4, 2006, published as W02006 / 74202 on July 13, 2006 and published as US 20060172914 on August 3, 2006, the entirety of which is thus incorporated herein by reference.

In each of the following Examples, where a corresponding amino acid, or NCA is designated D, then such amino acid,

NCA is of the corresponding, D-configuration. Where no such designation is mentioned, then such amino acid, or corresponding NCA, is of the L-configuration.

147

General Methods:

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL PXOFIEDAD

<img file="MX340992B_D0199.tif" />

Particle Size Analysis. Dynamic light scattering was used with a Wyatt Dynapro plate reader to determine the particle sizes of the crosslinked and uncrosslinked formulations. The solutions of the formulations were made at 1 mg / mL in 150 mM NaCl. The samples were centrifuged at 2000 RPM for 5 minutes, and then 3 00 pL each added to one well of a 96-well plate in triplicate for analysis. 10 acquisitions per cavity with acquisition times of 30 seconds and laser auto-attenuation were used to collect the data.

Encapsulation Verification Dialysis. The uncrosslinked formulation was dissolved in 3.5 mL of 10 mM phosphate buffer at pH 8 at 20 mg / mL, and at 0.2 mg / mL. 3 mL of the samples were added to dialysis balls of 3500 molecular weight reference value, and the remaining 0.5 mL were added to HPLC vials for the pre-dialysis samples. The dialysis bags were placed in 300 mL of 10 mM PB at pH 8 and shaken for 6 hours. Aliquots were then taken from inside the dialysis bags and HPLC analysis was used to determine the peak areas of the drug from the pre-dialysis and post-dialysis samples. The areas were then used to calculate the% of post-dialysis drug remaining.

148

Analysis and Dependent Crosslinking of the θΙ'χβκνζτοΛί

MEXICAN INSTITUTE

<img file="MX340992B_D0200.tif" />

Uncrosslinked formulation was reconstituted in water, at- 20 mg / mL with either 0.1, 0.25, 0.5, 0.75, 1, 2.5, 5, 7.5, or 10 mM of iron (III) chloride and allowed to stir overnight at room temperature. The samples were then diluted to 0.2 mg mL in 10 mM phosphate buffer at pH 8, with a final volume of 5 mL. Aliquots of 1.5 mL were taken as pre-dialysis samples for HPLC analysis, then 3 mL of each sample was added to a dialysis bag of reference value 3500 MWC and dialyzed against 10 mM phosphate buffer at pH 8 by 6 hours. After 6 hours the samples were removed from inside the dialysis bags and analyzed by HPLC. The post-dialysis peak area for each sample was divided by the pre-dialysis peak areas and multiplied by 100 to convert the remaining percentage.

Time Dependent Analysis and Crosslinking. The uncrosslinked formulation was reconstituted in water at 20 mg / mL, and 50 pL was diluted in 4.95 mL for the uncrosslinked sample. A 500 mM base solution of iron (III) chloride was then added to the uncrosslinked solution for a final concentration of 10 mM of iron (III) chloride. This was used as the crosslinked base solution, where 50 aliquots were taken at 5 minutes, 30 minutes, 1 hour, 2 hours, 4 hours and 16 hours and diluted to 0.2 mg mL in 10 mM of

149

IMPI phosphate buffer pH 8, with a volume

INDUSTRIAL

1.5 mL aliquots were taken as pre-dialysis samples

<img file="MX340992B_D0201.tif" />

for HPLC analysis, then 3 mL of each sample was added to a 3500 MWC reference value dialysis bag and dialyzed against 10 mM phosphate buffer pH 8 for 6 hours. After 6 hours, the samples were removed from inside the dialysis bags and analyzed by HPLC. The post-dialysis peak area for each sample was divided by the pre-dialysis peak areas and multiplied by 100 to convert the remaining percentage.

PH Dependent Analysis and Crosslinking. The uncrosslinked formulation was reconstituted in water at 20 mg / mL with 10 mM of iron (III) chloride at pH 3, 4, 5, 6, 7, 7.4 and 8. The samples were allowed to incubate for 10 minutes after reconstitution and pH adjustment, and then diluted to 0.2 mg mL in 10 mM phosphate buffer at pH 8, with a final volume of 5 mL. Aliquots of 1.5 mL were taken as pre-dialysis samples for HPLC analysis, then 3 mL of each sample was added to a dialysis bag of reference value 3500 MWC and dialysed against 10 mM phosphate buffer at pH 8 per 6 hours. After 6 hours the samples were removed from inside the dialysis bags and analyzed by HPLC. The post-dialysis peak area for each sample was divided by the peak areas

150

IΜ ΡI aiNS'CjROTwextoajr Js para'NS'Cjisnweictoajr ') DELA INDUSTRIAL PROPERTY of Formulations was reconstituted in pre-dialysis and multiplied by 100 remaining percentage.

PH Dependent Release

Reticulated. The uncrosslinked formulation water at 20 mg / mL with 10 mM iron (III) chloride, pH adjusted to 8.0 with NaOH and allowed to stir overnight at room temperature. The next day the sample was diluted to 0.2 mg / mL 3n 10 mM phosphate buffer at pH 3, 4, 5, 6, 7, 7.4 and 8, with a final volume of 5 mL per sample. Aliquots of 1.5 mL were taken as pre-dialysis samples for HPLC analysis, then 3 mL of each sample was added to a dialysis bag of reference value 3500 MWC and dialyzed against 10 mM phosphate buffer at pH 8 by 6 hours. After 6 hours the samples were removed from inside the dialysis bags and analyzed by HPLC. The post-dialysis peak area for each sample was divided by the pre-dialysis peak areas and multiplied by 100 to convert the remaining percentage.

PH Dependent Release of Non-Crosslinked Formulations. The uncrosslinked formulation was reconstituted in water at 2 0 mg / mL, the pH was adjusted to 8.0 with NaOH, and allowed to stir overnight at room temperature. The next day the sample was diluted to 0.2 mg / mL in 10 mM phosphate buffer pH 3, 4, 5, 6, 7, 7.4 and 8, with a final volume

151

IMPI of 5 mL per sample. 1.5 mL aliquots s4^NCT6áS3féÉ # ÉfÉ £ Í

INDUSTRIAL pre-dialysis samples for HPLC analysis, then 3 mL of each sample were added to a 3500 MWC reference value dialysis bag and dialyzed against 10 mM phosphate buffer pH 8 for 6 hours. After 6 hours the samples were removed from inside the dialysis bags and analyzed by HPLC. The post-dialysis peak area for each sample was divided by the pre-dialysis peak areas and multiplied by 100 to convert the remaining percentage.

Salt-Dependent Release of Crosslinked Formulations. The uncrosslinked formulation was reconstituted in water at 20 mg / mL with 10 mM of iron (III) chloride, the pH was adjusted to 8.0 with NaOH, and allowed to stir for 10 minutes. The sample was then diluted to 0.2 mg / mL in 10 mM phosphate buffer at pH 8 with increased NaCl concentration from 0 to 10, 50, 100, 200, 300, 400 and 500 mM with a final volume of 5 mL per shows. Aliquots of 1.5 mL were taken as pre-dialysis samples for HPLC analysis, then 3 mL of each sample was added to a dialysis bag of reference value 3500 MWC and dialyzed against 10 mM phosphate buffer at pH 8 with the corresponding salt concentration for 6 hours. After 6 hours the samples were removed from inside the dialysis bags and analyzed by HPLC. The peak area

-iias.

<img file="MX340992B_D0202.tif" />

152

ΙΜΡΙ post-dialysis for each sample was divided by pre-dialysis and multiplied by 100 to convert the remaining percentage.

In Vitro Cytotoxicity of Formulations of

Aminopterin. Cells originally purchased from ATCC (A549, Panc-1, OVCAR3, and BXPC-3) were seeded into 96-well tissue culture plates to be 50% confluent for 24 hours. Cells were incubated at 37 ° C with 5.0% CO2. Cells were treated with escalated doses of free aminopterin, crosslinked aminopterin formulation, non-crosslinked aminopterin formulation, and non-micelle formulations.

<img file="MX340992B_D0203.tif" />

<td>drug loaded 24</td><td>hours</td><td>after the</td><td colspan="2">strewn with</td>

<td>license plate. Aminopterin</td><td>free</td><td>dissolved</td><td>in</td><td>DMSO and it</td>

<td>administered to cells</td><td>with a</td><td>total volume</td><td>of</td><td>DMSO equal to</td>

<td>0 less than 0.0025%.</td><td>The</td><td>formulations</td><td>of</td><td>micelles</td>

resuspended in biological grade water. Dilutions were made in deep cavity plates with cell medium and water or DMS (for free aminopterin only) equalizing the displaced volume. The incubation medium was aspirated from the 96 well plates and 100 pL of each dilution were added to the wells in triplicate and incubated for hours at 37 ° C with 5.0% CO2. The crosslinked and uncrosslinked drug-free micelle formulations were administered at the highest four doses and were calculated at comparative concentrations of mg / ml at concentrations of

153

<img file="MX340992B_D0204.tif" />

drug-loaded micelles of vehicle ds5TiTÍ © u ^^ j (^

INDUSTRIAL

After 72 hours of incubation, the plates were allowed to cool to room temperature and 25 pL of the cellglo titrator were added to each well. The plates were briefly shaken for mixing and the luminescence readings were read on a plate reader. Luminescence readings for triplicate doses are averaged and divided by the average luminescence readings from untreated cells on the same plate to calculate the% viable cells per dose.

Formulation Method A. The polymer was dissolved in water at a concentration of 5 mg / mL by stirring and heating at 40 ° C for approximately 30 minutes. Sucrose was then added to the 5 mg / mL polymer solution and stirred at room temperature until homogeneous. The solution was then allowed to cool to room temperature while stirring. The active pharmaceutical ingredient (API) was dissolved in organic solvent only below the solubility limit. The API / organic solution was then added to the polymer / sucrose solution while mixing at 10,000 RPM for approximately 30 seconds, or until a homogeneous emulsion results. The solution was then processed in a single pass through a microfluidizer with an operating pressure of approximately 1620 kg / cm²(23,000 PSI) with the outlet current cooled by a bath

154

IMPI cold water. The solution was then passed to

INDUSTRIAL 0.22 micron dead end filter, then processed by ultrafiltration using tangential flow filtration until a total of 4 times the original volume of the sucrose buffer was exchanged and the final concentration of the polymer in solution was approximately 20 mg / mL . Iron (III) chloride was then added to the formulation for a final concentration of 10mM. The pH of the solution was then adjusted to 6.0 with NaOH and stirred at room temperature for 4 hours. A volume of buffer containing a cryopreservative agent at 20 mg / mL was then added to the solution, and then concentrated down again to approximately 20 mg / mL of polymer concentration. The solution was then frozen at -40 ° C and lyophilized.

Formulation Method B. The polymer was dissolved in water at a concentration of 2 mg / mL by stirring and heating at 40 ° C for approximately 30 minutes. The solution was then allowed to cool to room temperature while stirring. The active pharmaceutical ingredient (API) was dissolved in organic solvent only below the solubility limit. The API / organic solution was then added to the polymer / sucrose solution while mixing at 10,000 RPM for approximately 30 seconds, or until a homogeneous emulsion results. The solution was then stirred overnight in a fume hood to allow the

<img file="MX340992B_D0205.tif" />

155

<img file="MX340992B_D0206.tif" />

organic solution evaporate. To the next dSfe-Liá- -rsolu

The MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY was then passed through a 0.22 micron dead end filter, and then processed by ultrafiltration using tangential flow filtration to concentrate the sample from 2 mg / mL to approximately 20 mg / mL. Iron (III) chloride was then added to the formulation for a final concentration of 10mM. The pH of the solution was then adjusted to 6.0 with NaOH and stirred at room temperature for 4 hours. The solution was then frozen at -40 ° C and lyophilized.

Analysis of Weight Load of Formulation SN-38.

Weight loading was determined by comparing a standard SN3 8 curve at a known formulation concentration by HPLC analysis. SN38 was dissolved in methanol ranging from 30 pg / mL to 150 pg / mL, and the formulation was dissolved at 5 mg / mL in methanol. The amount of SN-38 in the formulation is then converted to% based on the known amount of the formulation used (ie, 5 mg / mL).

Daunorubicin Formulation Weight Load Analysis. Weight loading was determined by comparing a daunorubicin standard curve at a known formulation concentration by HPLC analysis. Daunorubicin was dissolved in methanol ranging from 40 pg / mL to 200 pg / mL, and the formulation was dissolved at 2 mg / mL in methanol.

156

IMPI

<img file="MX340992B_D0207.tif" />

MEXICAN INSTITUTE 'í ~^TO

The amount of daunorubicin in the formulation converted to% based on the known amount of formulation used (i.e. 2 mg / mL).

Load Analysis of Formulation Weight

Aminopterin. Weight loading was determined by comparing a standard aminopterin curve at a known formulation concentration by HPLC analysis. Aminopterin was dissolved in mobile phase HPLC (60% acetonitrile, 40% 10 mM phosphate buffer pH 8) in a range from 40 pg / mL to 200 pg / mL, and the formulation was dissolved at 5 mg / mL on mobile phase HPLC. The amount of aminopterin in the formulation is then converted to% based on the known amount of formulation used (ie, 5 mg / mL).

Berberine Formulation Weight Load Analysis. Weight loading was determined by comparing a standard berberine curve to a known concentration of formulation by HPLC analysis. Berberine was dissolved in methanol ranging from 40 pg / mL to 200 pg / mL, and the formulation was dissolved at 5 mg / mL in methanol. The amount of berberine in the formulation was then converted to% based on the known amount of the formulation used (ie, 5 mg / mL).

Weight Loading Analysis of the Cabazitaxel Formulation. Weight load was determined by comparing a

157

IMPI

INSTITUTO MEXICANO f cabazitaxel standard curve at a formulation concentration by HPLC analysis. The ca.ká.Zj,, SS dissolved in methanol ranging from 40 pg / mL to 200 pg / mL, and the formulation dissolved at 10 mg / mL in methanol. The amount of cabazitaxel in the formulation was then converted to% based on the known amount of the formulation used (ie, 10 mg / mL).

Weight Loading Analysis of the Epothilone D Formulation. Weight loading was determined by comparing a standard curve of epothilone D at a known concentration of formulation by HPLC analysis. Epothilone D was dissolved in methanol ranging from 40 pg / mL to 200 pg / mL, and the formulation was dissolved at 10 mg / mL in methanol. The amount of Epothilone then converted into the formulation was based on the known amount of the formulation used (ie, 10 mg / mL).

General Pharmacokinetic Experiments in Rat. Surgically modified Sprague-Dawley rats with jugular vein catheters were purchased from Harian Laboratories, Dublin, VA. The formulations were dissolved in water with 150 mM NaCl for a final concentration of

158

IMPI typically 10 mg API per kg body weight

<img file="MX340992B_D0208.tif" />

for approximately 1 minute, followed by a jet wash of approximately 250 heparinized saline. The time points for blood collection after administration of the test article were as follows: 1, 5, 15 minutes, 1, 4, 8 and 24 hours. Approximately 250 blood per time point were collected in K3-EDTA blood collection tubes followed by a jet wash of approximately 250 heparinized saline. The blood was then centrifuged at 2000 RPM for 5 minutes to isolate the plasma. Plasma was then collected and instantly frozen until processed for HPLC analysis. The samples were prepared for analysis by first thawing the plasma samples at room temperature. 50 pL of plasma were added to a 2 mL eppendorf tube of

150 pL of extraction solution (0.1% phosphoric acid in methanol, 5 pg / mL of internal standard). The samples were then vortexed for 10 minutes and centrifuged for 10 minutes at 13,000 RPM. The supernatant was then transferred into HPLC vials, then analyzed by HPLC. API quantification was determined using a standard curve of the API formulation in rat plasma compared to

159 samples collected from rats in isadaoMi nc IA l'B

IMPI isadao Monjibc

OF THE INDUSTRIAL I'RflHEDAD

<img file="MX340992B_D0209.tif" />

weather.

Example 1

<img file="MX340992B_D0210.tif" />

Cl

KgCOg

Ethanol

<img file="MX340992B_D0211.tif" />

^N—Oh

Dibenzylamino Ethanol. Benzyl chloride (278.5g,

2.2 mol), amine ethanol (60 mL, 1 mol), potassium carbonate (283.1 g, 2.05 mol) and ethanol (2 L) were mixed together in a 3 1 3-neck flask, equipped with a head shaker, a condenser and a glass stopper. The apparatus was heated to reflux for 36 hrs, after which the insoluble solid was filtered through a medium frit. The filtrate was recovered and ethanol was removed by rotary evaporation. The viscous liquid was dissolved again in ether, the solid suspension was removed by filtration and extracted twice against water. The ether solution was maintained and the aqueous layer was extracted twice with dichloromethane (2x400 mL). Fractions were recombined, dried over MgSCU, shaken over carbon black for 15 min, and filtered through a pad of celite. It was removed

Dichloromethane and the solid were dissolved again in a minimum amount of ether (combined volume of 30 0 mL with the

160

<img file="MX340992B_D0212.tif" />

IMPI

MEXICAN INSTITUTE first fraction of ether, 300 mL). ML) were added and the solution was heated slightly to complete dissolution of the product. The solution was then slightly cooled, placed in the refrigerator (+ 4 ° C) overnight, and white crystals were obtained. Recrystallization was carried out a second time. 166.63g, 69% yield. NMR^XH (de-DMSO) δ 7.39-7.24 (10H), 4.42 (1H), 3.60 (4H), 3.52 (2H), 2.52 (2H).

Example 2

<img file="MX340992B_D0213.tif" />

<img file="MX340992B_D0214.tif" />

Dibenzylamino-PEG-methoxy. An apparatus consisting of a 4L jacketed 3 neck polymerization flask equipped with a glass magnetic stir bar and an insulated thermal jacketed addition funnel was evacuated to 1.33 Pa (10mTorr) then filled with argon. The reaction flask was charged with N, N-dibenzylaminoethanol (4.28g, 17.7mmol) and 50% solid KH in paraffin wax (1.70g, 21.2mmol) under a gentle stream of argon gas. Anhydrous THF, about 2L, was introduced into the reaction flask and the mixture was stirred under Argon at room temperature for 16 h. The resulting suspension was cooled to 10

161

<img file="MX340992B_D0215.tif" />

Mr

IMPI ° C, and the addition funnel under vacuum was added to^{Of the}-i¿i8&gt; st &amp; x2. Ss condensed ethylene oxide gas in the chilled funnel, up to 225 mL (4.8 mol) of liquid EO was collected. The liquid ethylene oxide in the condensation funnel was added in one portion in the reaction mixture. The reaction mixture was shaken in a closed flask at 10 ° C for 6 hours, then at 20 ° C for 16 hours. The polymerization was completed by raising the temperature to 30 ° C for 16 hours, then to 40 ° C for 2 days. The reaction mixture was cooled to 25 ° C, then methyl iodide (1.6 mL) was added together and the mixture was stirred at 25 ° C for 10 hours. Excess unreacted potassium hydride was then destroyed by addition of ethanol (99%, 100 mL). After 30 min, the quenched reaction mixture was transferred into a large beaker, and the polymer product was precipitated by adding ethyl ether (8 L). The precipitated product was collected by filtration on a large Buchener funnel and then dried in vacuo. The yield was 215.1 g of a white solid. Aqueous CPG showed M_n of 12. OkDa and a POI of 1.01. ¹H-NMR (d6-DMSO,

00MHz): 7,344 (m, 8H), 7,225 (m, 4H), 3,681 (m, 8H), 3,507 (m, approx. 1000H), 3,320 (m, 6H + water signal), 3,237 (s,

3H), 2.551 (t, 6.0Hz, 2H).

162

Example 3

IMPI

MEXICAN INSTITUTE OF THE INDUSTRIAL EKOHEQAD

<img file="MX340992B_D0216.tif" />

<img file="MX340992B_D0217.tif" />

mPEG-amine. The

Pd (OH)₂

H₂OR

Hg

<img file="MX340992B_D0218.tif" />

mPEG-dibenzylamine product from

Example 3 (214.0 g) was dissolved in deionized water (IL). Pearlman's Catalyst 13.2g (20% Pd Carbon Hydroxide, Aldrich) suspension in deionized water (150 mL) was activated by stirring under a hydrogen balloon at room temperature. The hydrogen in the flask was replaced with nitrogen, the mPEG dibenzylamino starting material solution was added to the catalyst suspension and the flask was evacuated, then filled with hydrogen (repeated 3 times). The hydrogenation was then continued at room temperature under a hydrogen balloon for 2% days, at which point ¹H-NMR indicated complete disappearance of benzyl signals. Solid sodium chloride (350 g) was added to the reaction mixture and the mixture was stirred for half a day under nitrogen, the consumed catalyst was removed by filtration and rinsed thoroughly with brine. The combined filtrates were made alkaline (at ca. pH 11) by addition of a small volume of 1M NaOH and extracted with dichloromethane (4 x 0.7 L). The combined extracts were dried with anhydrous sodium carbonate, filtered, and

163 concentrated on rotary evaporator to approx.

<img file="MX340992B_D0219.tif" />

<img file="MX340992B_D0220.tif" />

excess ether (8L). The precipitated product was collected by filtration and dried in vacuo to provide 202.5 g of a bulky white snow solid.^XH-NMR (dg-DMSO, 4 00MHz): 3,681 (m, 8H), 3,507 (m, approx. 1000H), 3,341 (m, 4H + water signal), 3,238 (s, 3H), 2,634 (t, 5.7Hz, 2H).

Example 4

HO i

Cl

OR

D-Leucine NCA

HD-Leu-OH (100g, 0.76mol) was suspended in 1L of anhydrous THF and heated to 50 ° C while stirring vigorously. Phosgene (20% in toluene) (500 mL, 1 mol) was added to the amino acid suspension. After lh 20min, the amino acid dissolved, forming a clear solution. The solution was concentrated in the rotoevaporator, transferred into a beaker, and hexane was added to precipitate the product. The white solid was isolated by filtration and dissolved in toluene (~ 700 mL) with a small amount of THF (~ 60 mL). The solution was filtered over a Celite pad to remove any insoluble material. An excess of hexane (~ 4 L) was added to the filtrate to precipitate the product. The NCA was isolated by

164

I JMt ΡI filtration and dried in vacuo. (91g, 79% of

INDUSTRIAL isolated D-Leu NCA as a white, crystalline solid. NMR¹H (d6DMSO) δ 9.13 (1H), 4.44 (1H), 1.74 (1H), 1.55 (2H), 0.90 (6H) ppm.

Example 5

<img file="MX340992B_D0221.tif" />

or

Λ

CI ^ XI

<img file="MX340992B_D0222.tif" />

OR

or.

THF

NCA tert-Butyl aspartate. Η-Asp (OBu) -OH (120g, 0.63mol) was suspended in 1.2L of anhydrous THF and heated to 50 ° C while stirring vigorously. Phosgene (20% in toluene) (500 mL, 1 mol) was added to the amino acid suspension. After lh 30min, the amino acid dissolved, forming a clear solution. The solution was concentrated in the rotoevaporator, transferred into a beaker, and hexane was added to precipitate the product. The white solid was isolated by filtration and dissolved in anhydrous THF. The solution was filtered over a Celite pad to remove any insoluble material. An excess of hexane was added to precipitate the product. The NCA was isolated by filtration and dried in vacuo. 93g (68%) of NCA Asp (OBu) was isolated as a white, crystalline solid. ^ -H NMR (d₆-DMSO) δ 8.99 (1H), 4.61 (1H), 2.93 (1H), 2.69 (1H), 1.38 (9H) ppm.

165

<img file="MX340992B_D0223.tif" />

NCA of Benzyl Tyrosine. Η-Tyr (OBzl) -OH (140g, 0.52mol) was suspended in 1.5L of anhydrous THF and heated to 50 ° C while stirring vigorously. Phosgene (20% in toluene) (5 0 0 mL, 1 mol) was added to the amino acid suspension by means of cannulation. The amino acid dissolved during the course of approx. lh 30, forming a pale yellow solution. The solution was first filtered through a Buchner equipped with Whatman # 1 paper to remove any of the particles still in suspension. The solution was then concentrated by rotary evaporation, transferred into a beaker, and hexane was added to precipitate the product. The off-white solid was isolated by filtration and dissolved in anhydrous THF (~ 600 mL). The solution was filtered over a Celite pad to remove any insoluble material. An excess of hexane (~ 6 L) was added to the filtrate to precipitate the product. The NCA was isolated by filtration and dried in vacuo. 114.05 g, 74.3% of NCA Tyr (OBzl) was isolated

166

<img file="MX340992B_D0224.tif" />

like a whitish powder. NMR³H (d6-DMSO) δ

7.29 (5H), 7.12-7.07 (2H), 6.98-6.94 (2H), (1H), 3.05-2.88 (2H) ppm.

IMPI

5.06 (2H), 4.74

Example 7

<img file="MX340992B_D0225.tif" />

NCA Phenylalanine. HL-Phe-OH (20.0 g, 132 mmol) was suspended in 300 mL of anhydrous THF and heated to 50 ° C. Phosgene (20% in toluene) (90 mL, 182 mmol) was added to the amino acid suspension, and the amino acid dissolved over the course of approx. 1 hr, forming a cloudy solution. The solution was filtered through a paper filter (Whatman # 1), concentrated by rotary evaporation, transferred into a beaker, and hexane was added to precipitate the product. The white solid was isolated by filtration and dissolved in anhydrous THF. The solution was filtered over a Celite pad to remove any insoluble material. An excess of hexanes was added to the filtrate while stirring with a spatula. The NCA was isolated by filtration and dried in vacuo. 20.0 g (86% yield) of D-PheNCA was isolated as a white, crystalline solid. NMR^Ί-Η (d6-DMSO) δ

9.09 (1H), 7.40-7.08 (5H), 4,788 (1H), 3,036 (2H) ppm.

167

<img file="MX340992B_D0226.tif" />

<img file="MX340992B_D0227.tif" />

IMPI

Example 8

CO_?Bn

<img file="MX340992B_D0228.tif" />

H₂n co¿h hr

NCA L-benzylglutamate. H-Glu (OBn) -OH Vacuum-dried (71.2 g, 300.0 mmol) was suspended in 900 mL of anhydrous THF. Phosgene (20% in toluene) (210 mL, 42 0 mmol) was added to the amino acid suspension at room temperature and after ten minutes, the mixture was heated to 50 ° C. The amino acid dissolved during the course of approx. 1 hr, forming a clear solution. The solution was slightly cooled and concentrated in the rotoevaporator. Fresh anhydrous THF (400 mL) was added to the residue and the solution was again evaporated in the rotoevaporator to give a colorless solid, which was dissolved in 300 mL of anhydrous THF, a 4L beaker was transferred and precipitated by addition slow 1.5 L heptane anhydrous. The anhydrous NCA was isolated by suction filtration and dried in vacuo. 75.31 g (95.4% yield) of NCA Glu (OBn) was isolated as a colorless, crystalline solid. NMR^XH (CDC1₃) δ 7.36 (5H), 6.40 (1H),

5.14 (2H), 4.40 (1H), 2.60 (2H), 2.22 (2H).

168

<img file="MX340992B_D0229.tif" />

Example 9

<img file="MX340992B_D0230.tif" />

H₂N X0₂H

COCI_Z, 50 ° C, THF, .toluene

1.25hr

00₂Bn k

φθ

NCA D-benzylglutamate. Using the same method and reaction scale as in Example 8 and substituting Hd-Glu (OBn) OH as the starting material, reaction with phosgene for 1.25 hours at 50 ° C provided 75.53 g (Yield = 95.6%) of NCA d-Glu (OBn) as a colorless, crystalline solid. NMR¹H (CDCI3): identical to Example 8.

Example 10/270

one. Toluene, 60 C azeotrope in vacuo

2. Glu (OBn) NCA d-Glu (OBn) NCA,

NMP. TA, 18hr

3. d-PheNCA, Tyr (OBn) NCA TA&gt; 35 ° C, 48 hr

Four. ACaO, NMM DMAP, RT, 1 day

<img file="MX340992B_D0231.tif" />

mPEG12K-b-Poly- (d-Glu (OBn) 5-co-Glu (OBn) 5) -b-Poly

169 (Tyr (OBn) 30-co-d-Pheio) -Ac. m-PEG12k-NH₂, (119 weighed in a 2L round bottom flask,

IMPIféS ^ _ INÍTiTUTQMSXICANO J .7 g,

-³ INDUSTRIAL oven-dried, dissolved in toluene (1 L), and dried by azeotropic distillation. After distillation to dryness, the polymer was left under vacuum for three hours. The flask was subsequently filled with N₂, was emptied again under reduced pressure, and dry N-methylpyrrolidone (NMP) (1100 mL) was introduced by cannula. The mixture was briefly heated to 40 ° C to facilitate dissolution, and then cooled again to 25 ° C. NCA Glu (OBn) (13.16 g, 50.0 mmol) and NCA d-Glu (OBn) (13.16 g, 50.0 mmol) were added to the flask, and the reaction mixture was allowed to stir for 16 hours at room temperature under nitrogen gas. . Then, NCA d-Phe (19.12 g, 100 mmol) and NCA Tyr (OBn) (89.19 g, 300 mmol) were added and the solution was allowed to stir at 35 ° C for 48 hours at which point the reaction was complete ( GPC, DMF / 0.1% LiBr). The solution was cooled to room temperature and acetic anhydride (10.21 g, 100 mmol, 9.45 mL), N-methylmorpholine (NMM) (11.13 g, 110 mmol, 12.1 mL) and dimethylaminopyridine (DMAP) (1.22 g, 10.0 mmol) were added. ). Stirring was continued for 1 day at room temperature. The polymer was precipitated from diethyl ether (14 L) and isolated by filtration, washed with 500 mL portions of fresh diethyl ether, and dried in vacuo to give the block copolymer as a fine, almost colorless powder (214.7 g , Yield = 92.3%). IH NMR (d₆-DMSO) δ 8.42-7.70 (theo. 50H, obs. 47H),

7.30 (theo. 250H, obs. 253H), 6.95 (theo. 120H, obs. 122H),

170

IMPI

5.10-4.85 (theo. 80H, obs. 80H), 4.65-4.20 (industrial

56H), 3.72-3.25 (theo. 1087H, obs. 1593H), 3.05-2.45 (theo.

80H, obs. 83H), 2.44-1.60 (theo. 40H, obs. 42H).

<img file="MX340992B_D0232.tif" />

<img file="MX340992B_D0233.tif" />

mPEG12K-b-Poli- [d-Glu (OBn) s-co-Glu (OBn) ₅] -bPoli (Tyr (OH) 30-co-d-Pheio) -Ac. mPEG12K-b-Poli- (d-Glu (OBn)₅-coGlu (OBn) 5) -b-Poly (Tyr (OBn) 30-co-d-Pheio) -Ac from Example 10 (151.3 g, 6.5 mmol) and pentamethylbenzene (86.1 g, 0.58 mol) dissolved in 1400 mL trifluoroacetic acid (TFA). The reaction was quickly stirred for six hours at room temperature. The TFA was removed on a rotary evaporator with the temperature of the water bath not exceeding 35 ° C. The resulting hard paste was dissolved in 800 mL of dry THF and the crude product was precipitated in 12 L of diethyl ether

171 while cooling to -30 ° C. The solid BeffirtcbteaELfcje

DELA INDUSTRIAL PROPERTY

IMPI peoBuwteaELfcje DELA PROPERTY INDUSTRIAL collected by filtration, dissolved again in 500 mL of

<img file="MX340992B_D0234.tif" />

Dry THF and reprecipitated in 3 L of diethyl ether. An almost colorless, odorless, spongy polymer was obtained after drying the product overnight in vacuo (126.0 g, Yield = 94.2%). NMR Ή (dg-DMSO) δ 9.09 (theo. 30H, obs. 29.4H), 8.50-7.75 (theo. 50H, obs.

52.7H), 7.40-6.45 (teo. 220H, obs. 220H), 5.04 (teo. 20H, obs.

17.5H), 4.70-4.20 (theo. 50H, obs. 54.5H), 3.91-3.05 (theo.

1087H, obs. 1391H), 3.03-2.10 (teo. 80H, obs. 91H), 2.09-1.50 (teo. 40H, obs. 46H).

270

<img file="MX340992B_D0235.tif" />

270

CU ^ NHOH

H

I

<img file="MX340992B_D0236.tif" />

O ^ NHOH mPEGl2K-b-Poli- [d-Glu (NHOH) s-co-Glu (NHOH) ₅] -bPoli (Tyr (OH) ₃o-co-d-Pheio) -Ac. mPEG12K-b-Poli- [d-Glu (OBn)₅-coGlu (OBn) 5] -b-Poly (Tyr (OH) 30-co-d-Pheio) -Ac (113.3 g, 5.5 mmol)

172

<img file="MX340992B_D0237.tif" />

it was dissolved in 113 0 mL of dry THF and treated c &lt;w¡TSoluc¿órí ^J OF THE PROPERTY

INDUSTRIAL hydroxylamine (50% aqueous, 2.20 mol, 146 mL) and 1,5,7triazabicyclo [4.4.0] dec-5-ene (TBD, 2.30 g, 16.5 mmol). The resulting slightly cloudy solution was stirred at 50 ° C for 19 hours under N₂, cooled to room temperature and diluted with 1130 mL of MeOH. The crude product was precipitated from 8 L of diethyl ether while cooling to -30 ° C. The resulting solid was collected by filtration, redissolved in a mixture of 250 mL of dry THF and 125 mL of acetone, treated with acetic acid (4.72 g, 79 mmol, 4.5 mL), heated under reflux for five minutes, and then allowed to stir at room temperature for 1.5 hours. The product was precipitated by the addition of 2 L of diethyl ether, collected by suction filtration, washed with portions of fresh diethyl ether, and dried overnight in vacuo to provide 106.3 g (Yield = 97.5%) of foamy polymer , almost colorless. NMR¹H (d6-DMSO δ 9.12 (teo. 30H, obs. 30H), 8.80-7.75 (teo. 50H, obs. 38.4H), 7.15 (teo. 50H, obs. 50H), 6.80 (teo. 120H, obs. 120H), 4.65-4.05 (teo. 50H, obs. 50.4H), 3.80-3.15 (teo. 1087H, obs. 1360H), 3.00-2.20 (teo. 80H, obs. 79H), 2.15-1.60 (teo. 40H , obs. 40H).

173

<img file="MX340992B_D0238.tif" />

\ /270

Example 13

one. Toluene, 60 ° C, azeotrope in vacuo

2. Glu (OBn) NCA, tl-GIlljOBlONCA,

ΝΜΡ, ΤΑ, 16 hr

3. d-LeuNCA, Asp (OtBu) NCA, Tyr (OBn) NCA, TA, 48 lu; after 35'C, 23 hr

Four. Ac₂Or, NMM,

V DMAP, TA, 11hr

<img file="MX340992B_D0239.tif" />

mPEG12K-b-Poli- (d-Glu (OBn) ₅-co-Glu (OBn) ₅) -b-Poli (dLeus-co-Asp (OtBu) ιο-co-Tyr (OBn) 25) -Ac. Using the general protocol detailed in Example 10 and substituting the appropriate NCA starting materials, a crude polymer was provided which was precipitated with 12 volumes of diethyl ether, then dichloromethane / diethyl ether was precipitated again: 1.12. After filtration and drying in vacuo, the title compound (Yield =

93.9%) was obtained as a fine, colorless, odorless solid.

174

<img file="MX340992B_D0240.tif" />

Example 14

270

CO₂Bn

<img file="MX340992B_D0241.tif" />

270

<img file="MX340992B_D0242.tif" />

mPEG12K-b-Poli- (d-Glu (OBn) ₅ -co-Glu (OBn) ₅) -b-Poli (dLeus-co-Asp (OH) ιο-co-Tyr (OH) 25) -Ac. Using the method of Example 11 and substituting mPEG12K-b-Poly- (d-Glu (OBn)₅-coGlu (OBn) 5) -b-Poly (d-Leus-co-Asp (OtBu) 10-co-Tyr (OBn) 25) -Ac as starting material, reaction for three hours, 15 minutes at room temperature provided the title product (Yield = 97.0%) as a colorless, odorless, fluffy polymer.

175

Example 15

IMPI

DELAPI'.GTECaD MEXICAN INSTITUTE

INDUSTRIAL

<img file="MX340992B_D0243.tif" />

<img file="MX340992B_D0244.tif" />

r ⁵¹

V °

COgBn

HO

NHgOH, HA THF, TBD, ° C, 12 hr

<img file="MX340992B_D0245.tif" />

HO

OH mPEG12K-b-Poli- (d-Glu (NHOH) ₅-co-Glu (NHOH) ₅) -bPoli (d-Leus-co-Asp (OH) ιο-σο-Tyr (OH) 25) -Ac. mPEG12K-b-Poli- (dGlu (OBn) 5-co-Glu (OBn)₅) -b-Poly (d-Leus-co-Asp (OH) 10-coTyr (OH) 25) -Ac (20.81 g, 1.0 mmol) was dissolved in 210 mL of THF and treated with hydroxylamine solution (50% aqueous, 0.80 mol, 53.0 mL) and 1,5,7-triazabicyclo [4.4.0] dec-5-ene (TBD, 0.84 g, 6.0 mmol). The resulting slightly cloudy solution was stirred at 50 ° C for 17 hours under N₂, cooled to room temperature and diluted with 210 mL of MeOH. The crude product was precipitated with 1 L of diethyl ether, filtered, washed with portions of fresh diethyl ether, and dried overnight in vacuo (Yield = 93.3%, hydroxylamine salt)

176

<img file="MX340992B_D0246.tif" />

like a colorless, fine polymer.

Example 16/270

one. Toluene, 60 "C azeotrope in vacuo

2. Glu (OBn) NCA, d-Glu (OBn) NCA,

NMP.TA. 22 hl

3. d-LeuNCA, Asp (OtBu) NCA, 35 "C, 24hr

Four. ACj.0, NMM,

DMAP, TA

<img file="MX340992B_D0247.tif" />

<img file="MX340992B_D0248.tif" />

mPEG12K-b-Poli- (d-Glu (OBn) 5-co-Glu (OBn) 5) -b-Poly (dLeu3o-co-Asp (OtBu) 10) -Ac. Using the general protocol detailed in Example 10 and substituting the appropriate NCA starting materials, a crude polymer was provided which was precipitated with 30 volumes of diethyl ether / heptane: 6.1, then precipitated again from dichloromethane / diethyl ether: 1 ,twenty. After filtration and drying in vacuo, the title compound (Yield = 90.7%) was obtained as a colorless, odorless creamy solid.

177

<img file="MX340992B_D0249.tif" />

270

<img file="MX340992B_D0250.tif" />

TFA, CHjC ^: 1.1; TA, 2hr

I

4 &lt;M₀^ ü

270

CO₂Bn

<img file="MX340992B_D0251.tif" />

mPEG12K-b-Poli- (d-Glu (OBn) s-co-Glu (OBn) ₅) -b-Poly (dLeu3o-co-Asp (OH) io) -Ac. Using the method of Example 11, substituting mPEG12K-b-Poly- (d-Glu (OBn) s-co-Glu (OBn)₅) -bPoli (d-Leu3o-co-Asp (OtBu) 10) -Ac as starting material and omitting PMB, reaction for two hours at room temperature provided the title product (Yield = 97.4%) as a spongy polymer, colorless.

178

<img file="MX340992B_D0252.tif" />

<img file="MX340992B_D0253.tif" />

Example 18

<img file="MX340992B_D0254.tif" />

NHgOH, HaO, THF.TBD, ° C, 17 hr

<img file="MX340992B_D0255.tif" />

270

CONHOH

<img file="MX340992B_D0256.tif" />

CONHOH

<img file="MX340992B_D0257.tif" />

mPEGl2K-b-Poli- (d-Glu (NHOH) ₅-co-Glu (NHOH) ₅) -bPoli (d-Lemo-co-Asp (OH) io) -Ac. Using the method of Example 12 and substituting mPEG12K-b-Poly- (d-Glu (0Bn) 5-co-Glu (0Bn) s) -bPoli (d-Leu3o-co-Asp (OH) io) -Ac as material Starting, reaction for 17 hours at 50 ° C provided the title product (Yield = 96.4%, hydroxylamine salt) as a fine, colorless polymer.

179

<img file="MX340992B_D0258.tif" />

<img file="MX340992B_D0259.tif" />

Example 19

270

NHa

one. Toluene, 50®C azeotrope in vacuo

2. GMOMNCA, NMP, TA.-20hf

3. (HWfCA. Tyr (Oen) NCA. 35⁰C, -48hr

Four. AfeO (10 "to NMM (11" X DMAP (leq), RT, ~ 18hf

5. -axBtgO was added,

<img file="MX340992B_D0260.tif" />

<img file="MX340992B_D0261.tif" />

then CHjCI * £ ^ 0: -1.14 mPEG12K-b-Poli- (Glu (OBn) xo) -b-Poli (d-Phe2o-coTyr (OBz) 20) -Ac. Using the general protocol detailed in Example 10 and substituting the appropriate NCA starting materials, a crude polymer was provided which was precipitated with 9 volumes of diethyl ether, then precipitated again from dichloromethane / diethyl ether: 1.14. After filtration and drying in vacuo, the title compound (Yield = 89%) was obtained as a colorless, odorless creamy solid.

Example 20

<img file="MX340992B_D0262.tif" />

<img file="MX340992B_D0263.tif" />

trifluoroacetic acid - * ¥

<img file="MX340992B_D0264.tif" />

<img file="MX340992B_D0265.tif" />

mPEG12K-b-Poli- (Glu (OBn) io) -b-Poli (d-Phe2o-co-Tyr2o) Ac. Using the method of Example 11, substituting mPEG12K-b180

IaMPI

Poly- (Glu (OBn) 10) -b-Poly (d-Phe₂₀-co-Tyr (OBz) ₂₀) Mexican INSTITUTE M IA PROPERTY starting material and making it react by CtféWo

<img file="MX340992B_D0266.tif" />

At room temperature the title product (Yield = 87%) was provided as a colorless, fluffy polymer.

Example 21

Y / 8 »« jf 1

<img file="MX340992B_D0267.tif" />

<img file="MX340992B_D0268.tif" />

Ο Ό

H / F-OH

CO

MFI λ / ZTO

<img file="MX340992B_D0269.tif" />

mPEG12K-b-Poli- (Glu (NHOH) ₁₀) -b-Poly (d-Phe₂₀-coTyr₂o) -Ac. Using the . method of Example 12 and substituting mPEG12K-b-Poly- (Glu (OBn) io) -b-Poly (d-Phe₂o-co-Tyr₂₀) -Ac as a starting material, reaction for 17 hours at 50 ° C provided the title product (Yield = 94%, hydroxylamine salt) as a fine, colorless polymer.

181

Example 22

IMPI

MEXICAN INSTITUTE [&gt; E THE INDUSTRIAL RROnEDAD

<img file="MX340992B_D0270.tif" />

\ /270

one. Toluene, 60 * C azeotropoin vacuo

2. GlulOBiONC A. (IGliilOBioNCA.

NMP. TA, 18 hr

3. (l-PheNCA, Tyr (OBn) NCA, TA -&gt; 35'C, 48hr

Four. ACaO, NMM,

DMAP, TA, 1 day

<img file="MX340992B_D0271.tif" />

mPEG12K-b-Pol¡- (d-Glu (OBn) 5-co-Glii (OBn) 5) - £&gt; -Pol¡ (Tyr (OBn) 3o-co-d-Pheio) -Ac

Synthesis of mPEG12K-b-Poli- (d-Glu (OBn) s-coGlu (OBn) 5) -b-Poly (Tyr (OBn) 30-co-d-Pheio) -Ac. m-PEGlOkNH₂ (119.7 g, 10.0 mmol, Example 3) was weighed into an oven dried 2L round bottom flask, dissolved in toluene (1 L), and dried by azeotropic distillation. After distillation to dryness, the polymer was left under vacuum for three hours. The flask was subsequently filled with N₂, was emptied again under reduced pressure, and dry N-methylpyrrolidone (NMP) (1100 mL) was introduced per cannula. The mixture was briefly heated to 40 ° C to facilitate dissolution

182 _χ I] Μ [Ρ I @ 8 ^ 5 'and then cooled again to 25 ° C. S®_s-TiTagregA'EO) ^ tíS ^ í \ ^

OF INDUSTRIAL PROPERTY

Glu (OBn) (13.16 g, 50.0 mmol) and NCA d-Glu (OBn) (13.16 g, 50.0 mmol) in the flask, and the reaction mixture was allowed to stir for 16 hours at room temperature under nitrogen gas. Then, NCA d-Phe (19.12 g, 100 mmol) and NCA Tyr (OBn) (89.19 g, 300 mmol) were added and the solution was allowed to stir at 35 ° C for 48 hours at which point the reaction was complete ( GPC, DMF / 0.1% LiBr). The solution was cooled to room temperature and acetic anhydride (10.21 g, 100 mmol, 9.45 mL), N-methylmorpholine (NMM) (11.13 g, 110 mmol, 12.1 mL) and dimethylaminopyridine (DMAP) (1.22 g, 10.0 mmol) were added. ). Stirring was continued for 1 day at room temperature. The polymer was precipitated from diethyl ether (14 L) and isolated by filtration, washed with 500 mL portions of fresh diethyl ether, and dried in vacuo to give the block copolymer as a fine, almost colorless powder (214.7 g , Yield = 92.3%). NMR^τΗ (d₆-DMSO) δ 8.42-7.70 (theo. 50H, obs. 47H),

7.30 (theo. 250H, obs. 253H), 6.95 (theo. 120H, obs. 122H),

5.10-4.85 (teo. 80H, obs. 80H), 4.65-4.20 ((teo. 50H, obs.

56H), 3.72-3.25 (theo. 1087H, obs. 1593H), 3.05-2.45 (theo.

80H, obs. 83H), 2.44-1.60 (theo. 40H, obs. 42H).

183

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX340992B_D0272.tif" />

Example 23

<img file="MX340992B_D0273.tif" />

TFA, pentamethylbenzene (PMB), TA

<img file="MX340992B_D0274.tif" />

mPEG12K-b-Pol¡- [d-Glu (OBn) 5-co-Glu (OBn)₅] -6-Poli- (Tyr (OH) 3o-co-d-Pheio) -Ac

Synthesis of mPEG12K-b-Poli- [d-Glu (OBn) s-coGlu (OBn) 5] -b-Poli- (Tyr (OH) 30-co-d-Pheio) -Ac. mPEG12K-jb-Poli- [dGlu (OBn) 5-co-Glu (OBn) 5] -b-Poli- (Tyr (OBn) 30-co-d-Pheio) -Ac (151.3 g, 6.5 mmol) and pentamethylbenzene (86.1 g, 0.58 mol) were dissolved in 1400 mL of trifluoroacetic acid (TFA). The reaction was quickly stirred for six hours at room temperature. The TFA was removed on a rotary evaporator with the

<td>temperature of</td><td>Water bath</td><td>than</td><td>not</td><td>exceeds</td><td>35 ° C. Pasta</td>

<td>resulting hard</td><td>dissolved</td><td>in</td><td> 800</td><td>mL of</td><td>Dry THF and the</td>

<td>raw product</td><td>rushed</td><td>in</td><td> 12</td><td>L of</td><td>diethyl ether</td>

184 while cooling&gt; C. The

<img file="MX340992B_D0275.tif" />

solid

OF INDUSTRIAL PROPERTY

<img file="MX340992B_D0276.tif" />

Collected by filtration, re-dissolved in 500 mL of dry THF and re-precipitated in 3 L of diethyl ether. An almost colorless, odorless, spongy polymer was obtained after drying the product overnight in vacuo (126.0 g, Yield = 94.2%). NMR * Η (d₆-DMSO) δ 9.09 (theo.

30H, obs. 29.4H), 8.50-7.75 (teo. 50H, obs. 52.7H), 7.40-6.45 (teo. 220H, obs. 220H), 5.04 (teo. 20H, obs. 17.5H), 4.704.20 (teo. 50H , obs. 54.5H), 3.91-3.05 (teo. 1087H, obs.

1391H), 3.03-2.10 (teo. 80H, obs. 91H), 2.09-1.50 (teo. 40H, obs. 46H).

Example 24

<img file="MX340992B_D0277.tif" />

mPEG12K-í&gt; Poly- [d-Glu (NHOH) 5-co-Glu (NHOH)₅] -d-Poli- (Tyr (OH) 3o-co-d-Pheio) -Ac

185

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX340992B_D0278.tif" />

Synthesis of mPEG12K-b-PolyGlu (NH0H)_s] -b-Poli- (Tyr (OH) 30-co-d-Pheio) -Ac. mPEG12K-b-Poly [d-Glu (OBn)₅-co-Glu (OBn) ₅] -b-Poli- (Tyr (OH) ₃o-co-d-Pheio) -Ac (113.3 g, 5.5 mmol) was dissolved in 113.0 mL of dry THF and treated with hydroxylamine solution (50% aqueous, 2.2 mol, 146 mL) and 1.5, 7-triazabicyclo [4.4.0] dec-5-ene (TBD, 2.30 g,

16.5 mmol). The resulting slightly cloudy solution was stirred at 50 ° C for 19 hours under N₂, cooled to room temperature and diluted with 113 0 mL MeOH. The crude product was precipitated from 8 L of diethyl ether while cooling to -30 ° C. The resulting solid was collected by filtration, redissolved in a mixture of 250 mL of dry THF and 125 mL of acetone, treated with acetic acid (4.72 g, 79 mmol,

4.5 mL), heated at reflux for five minutes, and then allowed to stir at room temperature for 1.5 hours. The product was precipitated by the addition of 2 L of diethyl ether, collected by suction filtration, washed with portions of fresh diethyl ether, and dried overnight in vacuo to provide 106.3 g (Yield = 97.5%) of foamy polymer , almost colorless. NMR¹H (d6-DMSO δ 9.12 (teo. 30H, obs. 30H), 8.80-7.75 (teo. 50H, obs. 38.4H), 7.15 (teo. 50H, obs. 50H), 6.80 (teo. 120H, obs. 120H), 4.65-4.05 (teo. 50H, obs. 50.4H), 3.80-3.15 (teo. 1087H, obs. 1360H), 3.00-2.20 (teo. 80H, obs. 79H), 2.15-1.60 (teo. 40H , obs. 40H).

186 _|-_ΤΓί|_ΓΤ~ «-« - Μπ · ηττν? i¿aa;

<img file="MX340992B_D0279.tif" />

^Μθ/ θ '~ 4'^Ο· - ^ ΝΗ_; \ /270

Example 25

IMPI

one. Toluene, 60'C azeotrope in vacuo

2. Asp (OtBu) NCA, NMP, TA, 5 hr

3. d-Glu (Obn) NCA, Tyr (OBn) NCA, 35'C, 60 lw

Four. ACjgO, NMM, DMAP, TA, 4 hr

<img file="MX340992B_D0280.tif" />

mPEG12K-0-Pol¡- (Asp (OtBu) io - &amp; - Pol¡- (Tyr (OBn) 2o-co-d-Glu (OBn) 2o-Ac

Synthesis of mPEG12K-b-Poli- (Asp (OtBu) io-b-Poly (Tyr (OBn) 20-co-d-Glu (OBn) 2o-Ac. Using the protocol detailed in Example 22, replacing the NMP solvent with dichloromethane: DMF: 10, 1, and substituting the appropriate NCA starting materials, the title compound (Yield = 93.9%) was prepared as a colorless, fine, odorless solid.

! H NMR (de-DMSO) δ 8.42-7.85 (teo. 50H, obs. 51H), 7.30 (teo.

200H, obs.l98H), 6.98 (theo. 80H, obs. 72H), 5.15-4.85 (theo.

80H, obs. 80H), 4.68-4.20 (theo. 50H, obs. 46H), 3.72-3.25 (theo. 1087H, obs. 1415H), 3.05-1.50 (theo. 120H, obs. 114H),

1.35 (theo. 90H, obs. 76H).

187

IMPI

Mexican Institute of Industrial Property

<img file="MX340992B_D0281.tif" />

Example 26

<img file="MX340992B_D0282.tif" />

TFA, peirtamethylhencene TA 'f

<img file="MX340992B_D0283.tif" />

mPEG12K-hPol¡- (Asp (OH) io-f&gt; Pol¡- (Tyr (OH)₂o-co-d-Glu (OBn) 2o-Ac Synthesis of mPEGl2K-b-Poli- (Asp (OH) io-b-Poly (Tyr (OH) 2o-co-d-Glu (OBn) 2o-Ac. Using the method of Example 23 and substituting mPEG12K-b-Poli- (Asp (OtBu) io-jb-Poli- (Tyr (OBnbo-co-dGlu (OBn) 2o-Ac as starting material, the reaction for three hours, 15 minutes at room temperature and precipitation of a mixture of dichloromethane, diethyl ether: 1.8.5 provided the title product (Yield = 98.9%) as a fine, colorless, odorless polymer. NMR Ή (de-DMSO) δ 12.38 (theo . 10H, obs. 9H), 9.13 (theo. 20H, obs. 17H),

8.40-7.80 (theo. 50H, obs. 43H), 7.32 (theo. 100H, obs. 82H),

6.80 (theo. 80H, obs. 83H), 5.04 (theo. 40H, obs. 34.2H), 4.604.20 (theo. 50H, obs. 55H), 3.80-3.20 (theo. 1087H, obs.

1100H), 2.95-1.45 (theo. 140H, obs. 154.6H)

188 itjttzs .;

<img file="MX340992B_D0284.tif" />

<img file="MX340992B_D0285.tif" />

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL FROriEDAD

<img file="MX340992B_D0286.tif" />

<img file="MX340992B_D0287.tif" />

mPEG12K-bPol¡- (Asp (OH) io-bPol¡- (Tyr (OH) 2o-ccMd-Glu (NHOH)₂o-ac

Synthesis of mPEG12K-b-Poli- (Asp (OH) io-b-PolimPEG12K-b-Poli- (Asp (OH) io-b (20.81 g, 1.0 mmol) se (Tyr (OH) ₂or-_co-d-Glu (NHOH) ₂₀-Ac.

Poly- (Tyr (OH) 20-co-d- Glu (OBn) 20- Ac dissolved in 210 mL of THF and treated with hydroxylamine solution (50% aqueous, 0.80 mol, 53.0 mL) and 1,5,7triazabicyclo [4.4.0] dec-5-ene (TBD, 0.84 g, 6.0 mmol) The resulting slightly cloudy solution was stirred at 50 ° C for 17 hours under N₂, cooled to room temperature and diluted with 210 mL of MeOH. The crude product was precipitated with 1 L of diethyl ether, filtered, washed with portions of fresh diethyl ether, and dried overnight in vacuo to provide 19.68 g (Yield = 98.5%) of colorless, fluffy polymer as salt hydroxylamine. A portion of the

189

<img file="MX340992B_D0288.tif" />

Hydroxylamine salt (10.0 g) was dissolved in 1 L of tert-butyl / water, treated with ammonium carbonate (3.33 g), lyophilized to provide the salt form of the native carboxylic acid (quantitative yield) as a colorless, odorless, fluffy solid. NMR^XH (¿U-DMSO, hydroxylamine salt) δ 9.08 (teo. 2OH, obs. 10H), 6.80 (teo. 80H, obs. 80H), 4.60-4.02 (teo. 50H, obs. 54.7H), 3.80- 3.15 (theo. 1087H, obs. 1211H), 2.90 (theo.

H, obs. 45H), 2.80-1.50 (theo. 100H, obs. 120H). The spectrum showed solvent residues that affect the integration of the upfield region.

Example 28/270

one. Toluene, 60 ° C azeotropobr vacuo

2. Gto (OBn) NCA, d-Glu (OBn) NCA,

NMP.TA. 16 hr

3. d-leuNCA, Asp (0t8u) NCA, Tyr (OBn) NCA, TA, 48hr after 35 * C. 23 hr

Four. ACjO, NMM,

DMAP, TA, 11 hr

<img file="MX340992B_D0289.tif" />

mPEG12K-¿»-Poli- (d-Glu (OBn) 5-co-Glu (OBn) 5) -¿&gt; -Pol¡ (d-Leu5-co-Asp (OtBu) io-co-Tyr (OBn) 25 ) -Ac

Synthesis of mPEG12K-b-Poli- (d-Glu (OBn) 5-coGlu (OBn) 5) -b-Poly (d-Leus-co-Asp (OtBu) 10-co-Tyr (OBn) 25) -Ac .

Using the general protocol detailed in Example 22 and

190 substitute

IMPI taking the starting materials of

OF industrial PROPERTY

<img file="MX340992B_D0290.tif" />

provided a crude polymer that was precipitated with 12 volumes of diethyl ether, then precipitated again from dichloromethane / diethyl ether: 1.12. After filtration and drying in vacuo, the title compound (Yield = 89.2%) was obtained as a fine, colorless, odorless solid. NMR³Η (cU-DMSO) δ 8.52-7.75 (theo. 50H, obs.

<td>49H),</td><td> 7.35</td><td>(theo. 175H, obs.</td><td>198H), 7.11</td><td>(theo. 50 H,</td><td>obs. 49H),</td><td> 6.80</td>

<td>(teo.</td><td>50H,</td><td>obs. 50H), 5.10-4</td><td>_:.75 (theo. 70</td><td>H, obs. 75H),</td><td> 4.70-4.15</td><td>(teo.</td>

<td>50H,</td><td>obs.</td><td>56H), 3.72-3.25</td><td>(theo. 1087H,</td><td>obs. 1580H),</td><td> 3.05-1.65</td><td>(teo.</td>

<td>110H,</td><td>obs.</td><td>144H), 1.58-0.55</td><td>(theo. 135H</td><td>, obs. 155H)</td><td></td><td></td>

<img file="MX340992B_D0291.tif" />

<img file="MX340992B_D0292.tif" />

mPEG12K-Z&gt; Pol¡- (d-Glu (OBn) 5-co-Glu (OBn) 5) -b-Pol¡ (d-Leu5-co-Asp (OH) io-co-Tyr (OH) 25) -Ac

Synthesis of mPEG12K-b-Poly- (d-Glu (OBn) 5-coGlu (OBn) 5) -b-Poly (d-Leus-co-Asp (OH) 10-co-Tyr (OH) 25) -Ac . Using the method of Example 23 and substituting mPEG12K-b-Poly- (dGlu (OBn) 5-co-Glu (OBn) 5) -b-Poly (d-Leus-co-Asp (OtBu) 10-co191

Tyr (OBn) 25) -Ac as starting material the

<img file="MX340992B_D0293.tif" />

<img file="MX340992B_D0294.tif" />

OF THE PRCFiE-AD

INDUSTRIAL

<img file="MX340992B_D0295.tif" />

hours, 15 minutes at room temperature and precipitation of a mixture of dichloromethane, diethyl ether: 1.24 followed by dichloromethane, diethyl ether: 1.12 provided the title product (Yield = 97.0%) as a colorless, odorless, spongy polymer . NMR Ή (ck-DMSO)) δ 9.4-8.5 (theo. 35H, obs. 34H), 8.40-7.75 (theo. 50H, obs. 61H), 7.357.15 (theo. 50H, obs. 43H), 6.98 ( Theo. 50 H, Obs. 49H), 6.60 (Theo. 50H, Obs. 50H), 5.04 (Theo. 20H, Obs. 18H), 4.65-4.10 (Theo. 50H, Obs.

58H), 3.80-3.20 (teo. 1087H, obs. 1367H, contains masked H2O peak), 3.00-2.15 (teo. 90H, obs. 95H), 2.05-1.70 (teo.

20H, obs. 26H), 1.63-0.57 (theo. 45 H, obs. 45H).

Example 30

<img file="MX340992B_D0296.tif" />

<img file="MX340992B_D0297.tif" />

mPEG12K-d-Poli- (d-Glu (NHOH) 5-co-Glu (NHOH) 5) -h-Pol¡ (d-Leu5-co-Asp (OH) io-co-Tyr (OH)₂5) -Ac

Synthesis of mPEG12K-b-Poly- (d-Glu (OBn) 5-coGlu (OBn) s) -b-Poly (d-Leus-co-Asp (OH) 10-co-Tyr (OH) 25) -Ac . Using

192 the method of Example 27 and substituting mPEG12K-b-Poíi^s-^IíSx &lt;^ ÉÍC ^ n

INDUSTRIAL

IMPI βΚΒΕΒΒ »»

INDUSTRIAL

Glu (OBn) 5) -b-Poly (d-Leu5-co-Asp (OH) io-co-Tyr (OH) 25) -Ac as a

<img file="MX340992B_D0298.tif" />

After starting, reaction for 12 hours at 50 ° C provided the title product (Yield = 93.3%, hydroxylamine salt) as a fine, colorless polymer. N NMR (cU-DMSO) δ 9.4-8.5 (theo. 35H, obs. 34H),

8.60-7.75 (theo. 50H, obs. 43H), 7.2-6.85 (theo. 50H, obs.

55H), 6.60 (theo. 50 H, obs. 50H), 4.60-4.00 (theo. 50H, obs.

41H), 3.80-3.00 (teo. 1087H, obs. 1174H, contains H peak₂0 masked), 3.00-1.65 (theo. 110H, obs. 124H), 1.63-0.57 (theo. 45 H, obs. 40H).

Example 31 \ / 270

one. Toluene, 60 ° C, azeotrope in vacuo

2. Glu (OBn) NCA, d-Glu (OBn) NCA,

NMP, TA, 22hr

3. d-LeuNCA, Asp (GtBu) NCA, ° C, 24 hl

Four. Ac20, NMM,

DMAP, TA

270

CO₂Bn

<img file="MX340992B_D0299.tif" />

mPEG12K- £ »-Poli- (d-Glu (OBn) 5-co-Glu (OBn) 5) -í» -Poli (d-Leu30-co-Asp (OtBu) io) -Ac

Synthesis of mPEG12K-b-Poli- (d-Glu (0Bn)₅-coGlu (OBn) 5) -b-Poly (d-Leu3o-co-Asp (OtBu) 10) -Ac. Using the

193

<img file="MX340992B_D0300.tif" />

<img file="MX340992B_D0301.tif" />

general protocol detailed in Example .2l_JNsnTui,^_{to go}

OF THE PROPERTY

INDUSTRIAL _ The appropriate NCA starting materials were provided with a crude polymer which was precipitated with 30 volumes of diethyl ether / heptane: 6.1, then precipitated again from dichloromethane / diethyl ether: 1.20. After filtration and drying in vacuo, the title compound (Yield =

90.7%) was obtained as a colorless, odorless creamy solid. NMR³H (d₄_MeOH) δ 7.31 (teo. 50H, obs. 66H), 5.04 (teo. 20H, obs. 20H), 4.45-3.97 (teo. 50H, obs. 37H), 3.95-3.25 (teo.

1087H, obs. 1876H), 3.05-0.80 (theo. 420H, obs. 313H).

Example 32

<img file="MX340992B_D0302.tif" />

<img file="MX340992B_D0303.tif" />

NHj¡OH, HaO, THF, TBD, ° C, 12 hr

<img file="MX340992B_D0304.tif" />

mPEG12K-b-Pol¡- (cl-Glu (NHOH) 5-co-Glu (NHOH) 5) -ú-Pol (d-Leu5-co-Asp (OH) io-co-Tyr (OH) 25) - Ac Synthesis of mPEG12K-b-Poli- (d-Glu (NHOH)₅-coGlu (NHOH) s) -b-Poly (d-Leus-co-Asp (OH) ιο-co-Tyr (OH) 25) -Ac. Using

194

<img file="MX340992B_D0305.tif" />

IMPI the method of Example 27 and substituting mPEiSí ^ SSxMc ^ éSígi

INDUSTRIAL

Glu (OBn) 5-co-Glu (OBn) 5) -b-Poly (d-Leus-co-Asp (OH) 10-coTyr (OH) 25) -Ac as starting material, reaction for 12 hours at 50 ° C provided the title product (Yield = 93.3%, hydroxylamine salt) as a colorless polymer,

<td>fine.</td><td>NMR ^XH (ds-DMSO)</td><td>δ 9.4-8.5 (theo.</td><td>35H,</td><td>obs.</td><td>34H),</td><td> 8.60-</td>

<td> 7.75</td><td>(theo. 5 OH, obs.</td><td>43H), 7.2-6.85</td><td>(teo.</td><td>50H,</td><td>obs.</td><td>55H),</td>

<td> 6.60</td><td>(theo. 50 H, obs.</td><td>50H), 4.60-4.00</td><td>(teo.</td><td>50H,</td><td>obs.</td><td>41H),</td>

3.80-3.00 (teo. 1087H, obs. 1174H, contains masked H20 peak), 3.00-1.65 (teo. 110H, obs. 124H), 1.63-0.57

Itheus. 45 H, obs. 40H).

I

Example 33

CO_?Bn

270

<img file="MX340992B_D0306.tif" />

TFA, CH₂CÍ2: 1.1: RT, 2hr

M ©

270

<img file="MX340992B_D0307.tif" />

r

CO₂8n mPEG12K-b-Pol¡- (d-Glu (OBn) 5-co-Glu (OBn) s) -b-Pol¡ (d-Leu30-co-Asp (OH) io) -Ac

Synthesis of mPEG12K-b-Poli- (d-Glu (0Bn) s-coGlu (OBn) s) -b-Poly (d-Leu3o-co-Asp (OH) io) -Ac. Using the method

195 from Example 23, substituting mPEG12K-b-Pol Glu (OBn) 5) -b-Poly (d-Leu3O-co-Asp (OtBu) 10) -Ac

<img file="MX340992B_D0308.tif" />

As a starting material and omitting PMB, reaction for two hours at room temperature and precipitation of dichloromethane, diethyl ether: 1, 13 provided the title product (Yield = 97.4%) as a colorless, fluffy polymer. NMR^τΗ (d₄_MeOH) δ 7.31 (teo. 50H, obs. 61H), 5.04 (teo. 20H, obs. 20H), 4.45-3.97 (teo. 50H, obs. 29H), 3.95-3.25 (teo.

1087H, obs. 1542H), 3.05-0.80 (theo. 330H, obs. 258H).

Example 34

<img file="MX340992B_D0309.tif" />

NHgOH, HjO, THF, TBO, _v 50 ° C, 17 hr

<img file="MX340992B_D0310.tif" />

<img file="MX340992B_D0311.tif" />

mPEG12K - /&gt; Pol¡- (d-Glu (NHOH) 5-co-Glu (NHOH) 5) -6-Pol¡ (d-Leu30-co-Asp (OH) io) -Ac

Synthesis of mPEG12K-b-Poly- (d-Glu (NHOH) 5-coGlu (NHOH) 5) -b-Poly (d-Leu3o-co-Asp (OH) 10) -Ac. Using the method of Example 27 and substituting mPEG12K-b-Poly- (d-Glu (OBn) 5-co196

Glu OBn ₅ -b-Poli d-Leu₃o- co - Asp (OH) i₀ ) -βΚΒτυτο Mexican

OF INDUSTRIAL PROPERTY

IMPI ftSÜJTUTO MEXICANO OE IA INDUSTRIAL PROPERTY starting material, reaction for 17 hours at 50 ° C

<img file="MX340992B_D0312.tif" />

provided the title product (Yield 96.4%, hydroxylamine salt) as a fine, colorless polymer. NMR^XH (d6-DMSO) δ 8.8-7.2 (theo. 70H, obs. 67H), 4.55-3.85 (theo. 50H, obs. 50H), 3.80-3.30 (theo. 1087H, obs. 1520H), 3.29-2.60 ( theo. 60H, obs.

H), 2.42-0.70 (theo. 270H, obs. 278H).

Example 35

<img file="MX340992B_D0313.tif" />

mPEG12K-d-Poli- [d-Glu (NHOH) 5-co-Glu (NHOH) 5] -fa-Poli- (Tyr (OH) 3o-co-d-Pheio) -Ac

Synthesis of mPEG12K-b-Poly- [d-Glu (NHOH)₅-coGlu (NHOH) 5] -b-Poly- (Tyr (OH) 30-co-d-Pheio) -Ac. mPEG12K-b-Poli197 [d-Glu (OBn)₅-co-Glu (OBn) 5] -jb-Poli- (Tyr (OH) 30

INDUSTRIAL

IMPI

INDUSTRIAL (30.86 g, 1.50 mmol) was dissolved in 310 mL of THF

<img file="MX340992B_D0314.tif" />

It was dried and treated with hydroxylamine solution (50% aqueous, 0.60 mol, 39.7 mL) and 1,5,7-triazabicyclo [4.4.0] dec-5-ene (TBD, 626.4 mg, 4.5 mmol). The resulting slightly cloudy solution was stirred at room temperature for 69 hours under N2 and diluted with 310 mL of MeOH. The crude product was precipitated from

L of diethyl ether while cooling to -30 ° C resulting solid was collected by filtration, redissolved in a mixture of 150 mL of dry THF and 100 mL of acetone, treated with acetic acid (1.26 g, 21.0 mmol, 1.2 mL ) and then allowed to stir at room temperature for 2 hours. The product was precipitated by adding 1.5 L of diethyl ether, collected by suction filtration, washed with portions of fresh diethyl ether, and dried overnight in vacuo to provide 29.41 g (Yield = 98.9%) of foamy polymer , almost colorless. NMR¹H (d6-DMSO): identical to Example 24.

198

ΚΒβΕΜ552ώ £ 3Ε5χς ^ 3Γ?

Example 36

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MEXICAN INSTITUTE OF THE INDUSTRIAL PRCHEDAB

<img file="MX340992B_D0315.tif" />

\ /270

one. Toluene, 60 ° C, azeotrope in vacuo

2. Glu | OBii) NCA, (l-Gki (OBn) NCA,

NMP. RT

3. (1-PheNCA. Tyi (OBn) NCA, TA -&gt; 35 ° C

Four. Ac20. NMM,

DMAP.TA

<img file="MX340992B_D0316.tif" />

Synthesis of mPEG12K-b-Poli- (d-Glu (OBn) 5-coGlu (OBn) 5) -b-Poli- (Tyr (OBn) 25-co-d-Pheis) -Ac. Using the protocol detailed in Example 24 and substituting the appropriate NCA starting materials, a crude polymer was provided which was precipitated with 10 volumes of diethyl ether. After filtration and drying in vacuo, the title compound (Yield = 83.6%) was obtained as a fine, colorless, odorless solid. NMR¹H (d6-DMSO) δ 8.42-7.80 (theo. 50H, obs. 43H), 7.42-6.68 (theo. 350H, obs. 350H), 5.104.80 (theo. 70H, obs. 73H), 4.65-4.20 ( theo. 50H, obs. 50H), 3.75-3.25 (theo. 1087H, obs. 1755H), 3.01-2.30 (theo. 80H, obs.

85H), 2.02-1.60 (theo. 40H, obs. 38H).

199

IMPI

MEXICAN INSTITUTE OF PROPERTY

INDUSTRIAL

<img file="MX340992B_D0317.tif" />

Example 37

<img file="MX340992B_D0318.tif" />

mPEG12K-b-Pol¡- [d-Glu (OBn) 5-co-Glu (OBn) 5] -b-Pol¡- (Tyr (OH) 25-co-d-Phei5) -Ac

Synthesis of mPEG12K-b-Poli- [d-Glu (OBn) 5-coGlu (OBn) 5] -b-Poli- (Tyr (OH) 25-co-d-Pheis) -Ac. Using the method of Example 23 and substituting mPEG12K-b-Poli- [d-Glu (0Bn) s-coGlu (OBn) 5] -b-Poli- (Tyr (OBn) 25-co-d-Phei5) -Ac as Starting material, reaction for 5.25 hours at room temperature provided the title product (Yield = 99.3%) as a fine, colorless, odorless polymer. NMR¹H (d6-DMSO) δ 9.09 (teo. 25H, obs. 22H), 8.40-7.75 (teo. 50H, obs. 49H), 7,406.50 (teo. 225H, obs. 225H), 5.04 (teo. 20H, obs. 21H), 4,654.20 (teo. 50H, obs. 54H), 3.81-3.20 (teo. 1087H, obs.

1613H), 3.05-2.10 (theo. 80H, obs. 90H), 2.05-1.63 (theo. 40H,

200

<img file="MX340992B_D0319.tif" />

one. NHaOH, H₂O, TBD. THF, TA

2. THF, acetone (3,2), i HOAc (cat), TA

<img file="MX340992B_D0320.tif" />

mPEG12K-í? -Pol¡- [d-Glu (NHOH) 5-co-Glu (NHOH) 5] -6-Pol¡- (Tyr (OH) 25-co-d-Phei5) -Ac

Synthesis of mPEG12K-b-Poli- [d-Glu (NHOH) 5-coG1u (NHOH)₅] -b-Poli- (Tyr (OH) 25-co-d-Pheis) -Ac. mPEG12K-b-Poly [d-Glu (OBn) s-co-Glu (OBn) 5] -b-Poli- (Tyr (OH) 25-co-d-Phei₅) -Ac (51.23 g, 2.50 mmol) was dissolved in 515 mL of dry THF and treated with hydroxylamine solution (50% aqueous, 1.00 mol, 66.3 mL, 40 equiv / portion of ester Bn) and 1.5, 7-triazabicyclo [4.4.0] dec-5-ene (TBD, 1,044 g, 7.5 mmol, 0.3 equiv.). The resulting slightly cloudy solution was stirred at temperature

201

IMPI ambient for 108 hours under N₂ and diluted ccftF ^^ J ^^ gd industrial

The crude product was precipitated from 6 L of diethyl ether. The resulting solid was collected by filtration, redissolved in a mixture of 300 mL of dry THF and 200 mL of acetone, treated with acetic acid (2.25 g, 37.5 mmol, 2.15 mL), and then allowed to stir at room temperature for 2.5 hours. The product was precipitated by adding 3 L of diethyl ether, collected by suction filtration, washed with portions of fresh diethyl ether, and dried overnight in vacuo to provide 45.16 g (Yield = 91.5%) of the compound of the Title as a foamy, almost colorless polymer with a slight odor of acetic acid. NMR²Η (d6-DMSO) δ 9.35-8.85 (teo. 45H, obs. 28H), 8.42-7.75 (teo. 50H, obs.

37H), 7.37-6.46 (teo. 175H, obs. 164H), 4.65-4.00 (teo. 50H, obs. 50H), 3.82-3.07 (teo. 1087H, obs. 1708H, contains H peak₂0 masked), 3.05-2.20 (teo. 80H, obs. 84H), 2,181.63 (teo. 40H, obs. 68H, contains HOAc residues).

<img file="MX340992B_D0321.tif" />

202

<img file="MX340992B_D0322.tif" />

I

270

<img file="MX340992B_D0323.tif" />

<img file="MX340992B_D0324.tif" />

1.NH_?OH, HjO.TBD, THF, TA • 2. THF, acetone (3,2), HOAc (cat), TA

I

270

<img file="MX340992B_D0325.tif" />

mPEG12K-h-Pol¡- [d-Glu (NHOH) 5-co-Glu (NHOH)₅] - / 7-Poly- (Tyr (OH) 25-co-d-Phei5) -Ac

Synthesis of mPEG12K-b-Poli- [d-Glu (NH0H) 5-coGlu (NHOH) 5] -b-Poli- (Tyr (OH) 25-co-d-Phei5) -Ac. Using the method of Example 38 and increasing the concentration of hydroxylamine (80 equiv / Ester Bn), the reaction for 65 hours at room temperature and development as above provided the title product (Yield = 87.8%) as a colorless polymer, Fine with a slight smell of acetic acid. ^ -H NMR (dg-DMSO): identical to Example 38.

203

Example 40

IMPI

MEXICAN INSTITUTE OF THE industrial KOPIEDAD

<img file="MX340992B_D0326.tif" />

<img file="MX340992B_D0327.tif" />

one. NHjOH, HfeO.Í-OH-pir, THF, RT

2. THF, acetone (3,2),

HOAc (cat), TA

<img file="MX340992B_D0328.tif" />

mPEG12K-í&gt; PolÍ- [d-Glu (NHOH) 5-co-Glu (NHOH) 5] -6-Pol¡- (Tyr (OH) 25-co-d-Phei5) -Ac

Synthesis of mPEG12K-b-Poli- [d-Glu (NHOH) 5-coGlu (NHOH) 5] -h-Poli- (Tyr (OH) 25-co-d-Pheis) -Ac. Using the method of Example 39 and substitution of TBD with 2-hydroxypyridine (0.3 equiv.), The reaction for 137 hours at room temperature and development as above, provided the title product (Yield = 91.2%) as a fine, colorless polymer with a slight odor of acetic acid. NMR²H (d₆-DMSO): identical to Example 38.

204

<img file="MX340992B_D0329.tif" />

I

Example 41

<img file="MX340992B_D0330.tif" />

one. NHaOH, HgO, 2-OH-pir, THF. TA

2. THF, acetone (3.2), HOAc (cat), TA

I

270

CK -NHOH

<img file="MX340992B_D0331.tif" />

TO

CT ^ HOH mPEG12K - /&gt; - Pol¡- [d-Glu (NHOH) 5-co-Glu (NHOH) 5] -6-Pol¡- (Tyr (OH) 25-co-d-Phe₁5) -Ac

Synthesis of mPEG12K-b-Poli- [d-Glu (NHOH) s-coGlu (NHOH) 5] -b-Poli- (Tyr (OH) 25-co-d-Pheis) -Ac. Using the method of Example 38 and substitution of TBD with 2-hydroxypyridine (0.3 equiv.), Reaction at 50 ° C for 24.5 hours and development as above provided the title product (Yield = 91.2%) as a colorless polymer, Fine with a slight odor of acetic acid. ^ -H NMR (d6-DMSO): identical to Example 38.

205

Example 42

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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX340992B_D0332.tif" />

^M®'AO^x^ and ° '^^xNH2 \ / 270

one. Toluene, 60 ° C, azeotrope in vacuo

2. GlujOBiONCA. d-GUHOBlONCA.

NMP. RT

3. d-PheNCA, Tyr (OBn) NCA, TA -&gt; 35 ° C

Four. ACjO, NMM,

DMAP.TA

<img file="MX340992B_D0333.tif" />

mPEG12K-bPol¡- (d-Glu (OBn) 5-co-Glu (OBn) 5) -ó-Poli- (Tyr (OBn) 25-co-d-Phei5) -Ac

Synthesis of mPEG12K-b-Poli- (d-Glu (OBn)₅-co15 Glu (OBn) 5) -b-Poli- (Tyr (OBn) 25-co-d-Phei5) -Ac. Using the protocol detailed in Example 36 and substituting the appropriate NCA starting materials, a crude polymer was provided which was precipitated with 5 volumes of isopropanol. After filtration and drying in vacuo, the title compound (Yield = 84.2%) was obtained as a fine, colorless, odorless solid. NMR¹H (d6-DMSO): identical to

Example 36.

206

<img file="MX340992B_D0334.tif" />

<img file="MX340992B_D0335.tif" />

Example 43

<img file="MX340992B_D0336.tif" />

<img file="MX340992B_D0337.tif" />

mPFG12K-í&gt; -Pol¡- [d-Glu (OBn) 5-co-Glu (OBn) 5] -h-Poli- (Tyr (OH) 25-co-d-Phei5) -Ac

Synthesis of mPEG12K-b-Poli- [d-Glu (OBn) 5-co15 Glu (OBn) 5] -b-Poli- (Tyr (OH) 25-co-d-Pheis) -Ac. Using the method of Example 37, the reaction of mPEG12K-b-Poly- [d-Glu (OBn)₅co-Glu (OBn) 5] -b-Poli- (Tyr (OBn) 25-co-d-Phei₅) -Ac with PMB in TFA for four hours at room temperature and precipitation of a mixture of chlorobutane, TBME: 1.3 provided the title product (Yield = 93.1%) as a fine, colorless, odorless polymer. NMR^XH (ds-DMSO): identical to Example 37.

207

<img file="MX340992B_D0338.tif" />

one. NHjOH, HgO, UOH, THF, RT

2. THF, acetone (3.2) ,, HOAc (cat), TA

270

H, ΝΗΟΗ

<img file="MX340992B_D0339.tif" />

O ^ NHOH mPEG12K-fa-Pol¡- [d-Glu (NHOH) 5-co-Glu (NHOH) 5] -¿&gt; -Pol¡- (Tyr (OH)₂₅-co-d-Phei₅) -Ac

Synthesis of mPEG12K-b-Poly- [d-Glu (NHOH) 5-coGlu (NHOH)_S] -b-Poli- (Tyr (OH) 25-co-d-Phei5) -Ac. mPEG12K-b-Poly [d-Glu (OBn) 5-co-Glu (OBn) 5] -b-Poli- (Tyr (OH) 25-co-d-Pheis) -Ac (4.10 g, 0.20 mmol) se Dissolved in 41 mL of dry THF and treated with hydroxylamine solution (50% aq, 40.0 mmol, 2.65 mL, 20 equiv./Bn ester portion) and lithium hydroxide monohydrate (84.0 mg, 2.0 mmol, 1.0 equiv./ Portion of ester Bn). The resulting pale yellow solution was stirred at room temperature for 22 hours under N2 and diluted with 41 mL of IPA. The crude product was precipitated from 160 mL of TBME with rapid stirring. The resulting solid was collected

208

<img file="MX340992B_D0340.tif" />

<img file="MX340992B_D0341.tif" />

INSTITUTO MEXICANO was filtered by drying in vacuo, and a mixture of 24 mL of dry THF and 16 mL of acetone was dissolved. The solution was treated with acetic acid (0.18 g, 3.00 mmol, 0.17 mL), briefly heated under reflux, and allowed to stir at room temperature for 15 hours. The product was precipitated by adding volumes of TEME, collected by suction filtration, washed with fresh portions of TEME, and dried overnight in vacuo to provide 3.62 g (Yield = 91.7%) of the title compound as a foamy, almost colorless polymer. NMR¹H (d6DMSO): identical to Example 38.

<img file="MX340992B_D0342.tif" />

<img file="MX340992B_D0343.tif" />

mPEG12K-h-Pol¡- [d-Glu (NHOH) 5-co-Glu (NHOH) 5] -í&gt; Pol¡- (Tyr (OH) 25-cod-Phei5) -Ac

Synthesis of mPEG12K-b-Poli- [d-Glu (NHOH) s-co209

IMPL

Glu (NHOH)₅] -b-Poli- (Tyr (OH) 25-co-d-Pheis) -Ac. Usáífí ^ L · ^^

INDUSTR described above in Example 44, mPEG12K-b-Poly- [d-

<img file="MX340992B_D0344.tif" />

Glu (OBn) 5-co-Glu (OBn) 5] -b-Poly- (Tyr (OH) 25-co-d-Pheis) -Ac was converted to the title compound using lithium hydroxide monohydrate (2.0 equiv./ Portion of ester Bn). The reaction time was 18 hours. The crude product was precipitated from 16 volumes of IPA and the resulting solid was treated with THF, acetone, and acetic acid as detailed in Example 44. After precipitation of two volumes of TBME, filtration, and drying in vacuo, the title compound (Yield = 96.2%) was obtained as a fine, colorless solid. NMR¹H (d6-DMSO): identical to Example 38.

<img file="MX340992B_D0345.tif" />

Example 46

<img file="MX340992B_D0346.tif" />

one. NH₂OH, HaO, LiOH, MeOH, TA

2. THF. acetone (3,2i ,, HOAc (cat), TA

<img file="MX340992B_D0347.tif" />

mPEG12K-á-Pol¡- [d-Glu (NHOH) 5-co-Glu (NHOH)₅] -b-Poli- (Tyr (OH) 25-co-d-Phei₆) -Ac

210

Synthesis of mPEG12K-b-Pol i- [dDE INDUSTRIAL PROPERTY

<img file="MX340992B_D0348.tif" />

Glu (NH0H)_s] -h-Poli- (Tyr (OH) 25-co-d-Pheis) -Ac. mPEG12K-b-Poly [d-Glu (OBn) 5-co-Glu (OBn)₅] -b-Poly- (Tyr (OH) 25 -co-d-Pheis) -Ac (2.05 g, 0.10 mmol) was dissolved in 21 mL of methanol and treated with hydroxylamine solution (50% aqueous, 20 .0 mmol, 1.32 mL, 20 equiv / Portion of ester Bn) and 1M lithium hydroxide solution (1.0 mL, 1.0 mmol, 1.0 equiv / Portion of ester Bn). The resulting pale yellow solution was stirred at room temperature for 22 hours under N2 and then an additional portion of 1M lithium hydroxide solution (1.0 mL, 1.0 mmol, 1.0 equiv./Bn ester portion) was added. After an additional 24 hours, the crude product precipitated from 160 mL of TBME. The resulting solid was collected by filtration, dried in vacuo, and dissolved again in a mixture of 12 mL of dry THF and 8 mL of acetone. The solution was treated with acetic acid (0.21 g, 3.50 mmol, 0.20 mL), briefly heated under reflux, and allowed to stir at room temperature for 16 hours. The product was precipitated by adding 4 mL of TBME, collected by suction filtration, washed with fresh portions of TBME, and dried overnight in vacuo to provide 1.87 g (Yield = 94.9%) of the title compound like a foamy, almost colorless polymer. NMR^XH (ds-DMSO): identical to Example 38.

211

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL rsOFIEDAD

<img file="MX340992B_D0349.tif" />

Example 47

<img file="MX340992B_D0350.tif" />

one. NHaOH, HjO, UOH, THF, TA

2. THF, acetone (3.2)., HOAc (cat), TA

<img file="MX340992B_D0351.tif" />

mPEG12K-d-Poli- [d-Glu (NHOH) 5-co-Glu (NHOH) 5] -o-Poli- (Tyr (OH)₂5-co-d-Phei₅) -Ac

Synthesis of mPEG12K-b-Poli- [d-Glu (NHOH) s-coGlu (NHOH) 5] -b-Poli- (Tyr (OH) 25-co-d-Ph.eis) -Ac. Using the method described above in Example 44, mPEG12K-b-Poli- [dGlu (OBn) 5-co-Glu (OBn) 5] -b-Poli- (Tyr (OH) 25-co-d-Pheis) - Ac was converted to the title compound using lithium hydroxide solution (0.5 equiv / Portion of ester Bn). The reaction time was 72 hours. The solution was diluted with one volume of IPA, and the crude product was precipitated from two volumes of TBME. The resulting solid was treated with THF, acetone, and acetic acid as detailed in Example 44.

212

After filtration, and (Colorless Yield. NMR ^ΧΗ two volume precipitation mLMBI

MEXICAN INSTITUTE OF PROPERTY

<img file="MX340992B_D0352.tif" />

drying in vacuo, the compound aeT ^ titu 91.1%) was obtained as a STÓlldó 'filio, (d6-DMSO): identical to Example 38.

Example 48

<img file="MX340992B_D0353.tif" />

one. NHjOH, H_ZO, KOH, THF, TA

2. THF, acetone (3,2), · HOAc (cat), TA

<img file="MX340992B_D0354.tif" />

mPEG12K-bPol¡- [d-Glu (NHOH) 5-co-Glu (NHOH} 5] - ^ Pol¡- (Tyr (OH)₂5-co-d-Phei5) -Ac

Synthesis of mPEG12K-b-Poly- [d-Glu (NHOH)₅-coGlu (NHOH) 5] -b-Poli- (Tyr (OH) 25-co-d-Phei5) -Ac. Using the method described above in Example 47, mPEG12K-b-Poli- [dGlu (OBn) 5-co-Glu (OBn) 5] -b-Poli- (Tyr (OH) 25-co-d-Phei₅) -Ac was converted to the title compound using IM potassium hydroxide solution (2.0 equiv / portion of ester Bn). The reaction time was 6 hours. The development provided the

213

<img file="MX340992B_D0355.tif" />

title compound (Yield = 92.4%) fine, colorless. NMR¹H (d6-DMSO): identical to

Example 49 cLMhPÍ

MEXICAN INSTITUTE OF THE FRCP1EPA0

Exemplify^NnW.^{TO THE}^I nh₂ \ /270

one. Toluene, 60'C azeotrope in vacuo

2. Glu (OBn) NCA, (l-Glu (OBn) NCA.

0¾¾. NMP: 1,1, TA

3. (1-PheNCA, Tyr (OBn) NCA, TA -&gt; 35'C

Four. AcgO, NMM,

DMAP, TA

<img file="MX340992B_D0356.tif" />

mPEG12K-í&gt; Poli- (d-Glu (OBn) 3.5-co-Glu (OBn) 3.5) -b-Pol¡ (Tyr (OBn) 25-co-d-Pheis) -Ac

Synthesis of mPEG12K-b-Poli- (d-Glu (OBn) 3.s-coGlu (OBn) 3.5) -b-Poly (Tyr (OBn) 25-co-d-Pheis) -Ac. m-PEG10k-NH2, (59.86 g, 5.0 mmol) was weighed into a round bottom flask of

Kiln-dried IL was dissolved in toluene (450 mL), and dried by azeotropic distillation. After distillation to dryness, the polymer was left under vacuum for 16 hours. The flask was subsequently filled with N2, emptied again under reduced pressure, and dry N-methylpyrrolidone (NMP, 250 mL) and then dichloromethane (250 mL) were introduced per cannula. The mixture was briefly heated to

214

<img file="MX340992B_D0357.tif" />

<img file="MX340992B_D0358.tif" />

<img file="MX340992B_D0359.tif" />

40 ° C to facilitate dissolution and despijj ^^ g ^ Hgni

OF INDUSTRIAL PROPERTY again at 25 ° C. NCA Glu (OBn) (4.61 g, 17.5 mmol) and NCA d-Glu (OBn) (4.61 g, 17.5 mmol) were added to the flask, and the reaction mixture was allowed to stir for 24 hours at room temperature under nitrogen gas . Then, NCA dPhe (14.34 g, 75.0 mmol) and NCA Tyr (OBn) (37.16 g, 125.0 mmol) were added and the solution was allowed to stir at room temperature for three days and then heated at 35 ° C for 7 hours dot in which the reaction was completed (GPC, DMF / 0.1% LiBr). The solution was cooled to room temperature and acetic anhydride (5.11 g, 50.0 mmol, 4.80 mL), N-methylmorpholine (NMM) (5.56 g, 55.0 mmol, 6.1 mL) and dimethylaminopyridine (DMAP) (0.61 g, 5.0 mmol) were added. Stirring was continued for 18 hours at room temperature and the dichloromethane was removed on the rotary evaporator. The polymer was precipitated from isopropanol (2.6 L) and isolated by filtration, washed with 500 mL portions of fresh isopropanol, and dried in vacuo to give the block copolymer as a fine, almost colorless powder (102.40 g, Yield). =

92.6%). N NMR (de-DMSO) δ 8.42-7.80 (theo. 47H, obs. 44H),

7.35 (theo. 75H, obs. 75H), 7.28-6.65 (theo. 125H, obs. 125H),

5.10-4.84 (teo. 64H, obs. 59H), 4.64-4.20 (teo. 47H, obs.

39H), 3.72-3.25 (theo. 1087H, obs. 16713H), 3.00-2.20 (theo.

80H, obs. 88H), 2.03-1.60 (theo. 28H, obs. 27H).

215

Example 50

IMPI

MEXICAN INSTITUTE OF PROPERTY

INDUSTRIAL

<img file="MX340992B_D0360.tif" />

\ /270

one. Toluene, 60'C azeotrope in vacuo

2. GIlKOBlONCA. d-Gílii {OBn) NCA,

CHgCfj. NMP: 1.3, TA

3. d-PheNCA, Tyr (OBn) NCA, TA -&gt; 35'C

Four. Ac¿0, NMM,

DMAP, TA

<img file="MX340992B_D0361.tif" />

mPEG12K-b-Pol¡- (d-Glu (OBn) 3.5-co-Glu (OBn) 3.5) -6-Pol¡ (Tyr (OBn) 25-co-d-Pheis) -Ac

Synthesis of mPEG12K-b-Poli- (d-Glu (OBn) 3.5-σοGlu (OBn) 3.5) -b-Poly (Tyr (OBn) 25-co-d-Phei5) -Ac. Using the protocol detailed in Example 49 with dry N-methylpyrrolidone (NMP, 125 mL) and dichloromethane (375 mL) as solvents provided a crude polymer that was precipitated with 5 volumes of isopropanol. After filtration and drying in vacuo, the title compound (Yield = 96.5%) was obtained as a fine, colorless, odorless solid. NMR^τΗ (d6DMSO): identical to Example 49.

216

IMPI

<img file="MX340992B_D0362.tif" />

<img file="MX340992B_D0363.tif" />

<img file="MX340992B_D0364.tif" />

r

COgBn

one. NH₂OH H₂O, KOH, THF, TA

2. THF, acetone (3,2), HOAc (cat), TA

<img file="MX340992B_D0365.tif" />

mPEG12K-h-Pol¡- [d-Glu (NHOH) 5-co-Glu (NHOH) 5] -b-Pol¡- (Tyr (OH) 25-co-d-Phei5) -Ac

Synthesis of mPEG12K-b-Poli- [d-Glu (NHOH) 5-coGlu (NHOH) 5] -b-Poli- (Tyr (OH) 25-co-d-Pheis) -Ac. mPEG12K-b-Poly [d-Glu (OBn)₅-co-Glu (OBn) 5] -b-Poli- (Tyr (OH) 25-co-d-Pheis) -Ac (4.10 g, 0.20 mmol) was dissolved in 41 mL of THF and treated with hydroxylamine solution (2.65 mL, 40.0 mmol) and IM Potassium Hydroxide (2.0 mL, 2.0 mmol, 1.0 equiv / Portion of Bn ester). The resulting slightly cloudy pink solution was stirred at room temperature for 42 hours under N₂ and then diluted with acetone (58.1 g, 1.0 mol, 74 mL). Acetic acid (2.40 g, 40.0 mmol, 2.3 mL) was added, the solution

217

<img file="MX340992B_D0366.tif" />

four hours. The product is mL) using vigorous stirring.

For another 30 minutes, the mixture was briefly warmed to room temperature by precipitating with TBME (300. After stirring, filtration and drying in vacuo afforded the title compound (Yield = 92.9%) as a colorless, fine solid.

N NMR (d6-DMSO): identical to Example 38.

Example 52

I

270

<img file="MX340992B_D0367.tif" />

<img file="MX340992B_D0368.tif" />

mPEG12K-b-Pol¡- [d-Glu (NHOH) 5-co-Glu (NHOH) 5] -b-Poli- (Tyr (OH) 25-co-d-Phei5) -Ac

Synthesis of mPEG12K-b-Poly- [d-Glu (NHOH)₅-coGlu (NHOH) 5] -b-Poli- (Tyr (OH) 25-co-d-Pheis) -Ac. Using the method described above in Example 51, mPEG12K-b-Poli- [dGlu (OBn) 5-co-Glu (OBn) 5] -b-Poli- (Tyr (OH) 25-co-d-Pheis) - Ac se

218

IMPI

OF INDUSTRIAL PROPERTY

<img file="MX340992B_D0369.tif" />

converted to the title compound usandQ_NsTn6faltt © ¿W3n 1M lithium hydroxide (2.0 equiv / portion of ester Bn). The reaction time was 6 hours. Development as above and dilution with IPA (1 volume) followed by precipitation with TBME (3 volumes) provided the title compound (Yield = 90.6%) as a fine, colorless solid. NMR^ΧΗ (d6-DMSO): identical to Example 38.

<img file="MX340992B_D0370.tif" />

270

O. NHOH

<img file="MX340992B_D0371.tif" />

cr NHOH mPEG12K-6-Pol¡- [d-Glu (NHOH) 5-co-Glu (NHOH) 5] -b-Pol¡- (Tyr (OH)₂5-co-d-Phei5) -Ac

Synthesis of mPEG12K-b-Poli- [d-Glu (NHOH) s-coGlu (NHOH) 5] -b-Poli- (Tyr (OH) 25-co-d-Pheis) -Ac Using the method described above in Example 52, mPEG12K-b-Poli- [dGlu (OBn) 5-co-Glu (OBn) 5] -Jb-Poli- (Tyr (OH) 25-co-d-Pheis) -Ac was

219

IMPI

INSTITUTO MEXICANO converted the title compound using solid lithium hydroxide (2.0 equiv. / Portion of .ester Bn). The reaction time was 6 hours. Development provided the title compound (Yield = 99.2%) as a fine, colorless solid. NMR²Η (d6-DMSO): identical to Example 38.

<img file="MX340992B_D0372.tif" />

270

Example 54

<img file="MX340992B_D0373.tif" />

TFA, pentainethylhencene (PMB), TA

I

270

<img file="MX340992B_D0374.tif" />

mPEG12K-b-Pol¡- [d-Glu (OBn) 3.5-co-Glu (OBn) 3.5] -h-Pol¡- (Tyr (OH) 25-co-d-Phei5) -Ac

Synthesis of mPEG12K-b-Poli- [d-Glu (OBn) 3.5-coGlu (OBn) 3.5] -b-Poli- (Tyr (OH) 25-co-d-Pheis) -Ac. Using the method of Example 37, the reaction of mPEG12K-b-Poli- [d-Glu (OBn) 3.5co-Glu (OBn) 3.5] -b-Poli- (Tyr (OBn) 25-co-d-Pheis) -Ac with PMB in TFA for 3.5 hours at room temperature and precipitation of a mixture of dichloromethane, TBME: 1.7 provided the product

220 Title (Yield = 96.1%) as colorless, odorless. NMR¹H (d6-DMSO) δ 9.

22H), 8.46-7.79 (theo. 47H, obs. 48H), 7.

obs. 229H), 5.04 (theo. 14H, obs. 13H), 4 obs. 47H), 3.81-3.15 (theo. 1087H, obs.

(theo. 80H, obs. 78H), 2.06-1.62 (theo. 40H,

Example 55 a

IMPI poSajmé ^énx df industrial

<img file="MX340992B_D0375.tif" />

(theo. 25H, obs.

40-6.45 (teo. 210H, .65-4.20 (teo. 47H,

1308H), 3.03-2.10 obs. 27H).

<img file="MX340992B_D0376.tif" />

270

<img file="MX340992B_D0377.tif" />

mPEG12K-h-Pol¡- [d-Glu (NHOH) 5-co-Glu (NHOH) 5] -b-Pol¡- (Tyr (OH) 25-co-d-Phei5) -Ac

Synthesis of mPEG12K-b-Poli- [d-Glu (NHOH) s-coGlu (NHOH) 5] -b-Poli- (Tyr (OH) 25-co-d-Phei5) -Ac. Using the method described in Example 51 above, mPEG12K-b-Poli- [dGlu (OBn) 5-co-Glu (OBn) 5] -b-Poli- (Tyr (OH) 25-co-d-Phei₅) -Ac was converted to the title compound using solution of

221 4M sodium hydroxide (2 equiv / Serving

<img file="MX340992B_D0378.tif" />

OF THE (INDUSTRIAL ROPIEDAD

<img file="MX340992B_D0379.tif" />

reaction time was 4 hours. The solution was diluted with acetone (0.30 volumes based on the total reaction mixture volume) and acetic acid (1.0 equiv./hydroxylamine) was added. After 14 hours, the crude product was precipitated from three volumes of TBME, stirred for three days, and filtered. The filtered cake was washed with TBME (50 mL), TBME, IPA: 20, 1 (50 mL) and dried in vacuo to provide the title compound (Yield = 93.5%) as a fine, colorless solid with a slight odor acetic acid. NMR^XH (d6-DMSO): identical to Example 38.

<img file="MX340992B_D0380.tif" />

<img file="MX340992B_D0381.tif" />

mPEG12K-d-Pol¡- [d-Glu (NHOH) 3.5-co-Glu (NHOH) 3.5] -b-Pol¡- (Tyr (OH) 25-co-d-Phei5) -Ac

Synthesis of mPEG12K-b-Poly- [d-Glu (NHOH)₃.5-coGlu (NHOH) 3.5] -b-Poly- (Tyr (OH) 25-co-d-Pheis) -Ac. Using the method described above in Example 52, mPEG12K-b222

Poly- [d-Glu (OBn) ₃.5-co-Glu (OBn) 3.5] -b-Poli- (Tyr (OHhs-

<img file="MX340992B_D0382.tif" />

converted to the title compound using solid lithium hydroxide monohydrate (2.0 equiv / Portion of ester Bn). The reaction time was 6 hours. The development provided the title compound (Yield = 94.9%) as a fine, colorless solid with a slight acetic acid odor. NMR Ή (de-EMSO) δ 10.2-9.2 (teo. 25H, obs. 19H), 8.52-7.90 (teo. 47H, obs 38H), 7.40-6.49 (teo. 175H, obs. 175H), 4.63-4.00 (theo. 47H, obs. 42H), 3.84-3.11 (theo. 1087H, obs. 1496H, contains masked H2O peak), 3.00-2.20 (teo. 80H, obs. 78H), 2.16-1.60 (teo. 28H, obs.

~ 26H, contains overlapping peak HOAc at δ 1.69).

Example 57

<img file="MX340992B_D0383.tif" />

<img file="MX340992B_D0384.tif" />

mPEG12K-b-Pol¡- [d-Glu (NHOH) 3.5-co-Glu (NHOH) 3.5] -b-Poli- (Tyr (OH) 25-co-d-Phei₅) -Ac

Glu (NHOH) 3.5] -b-Poly- (Tyr (OH) 25-co-d-Pheis) -Ac.

Using the

Synthesis of mPEG12K-b-Poli- [d-Glu (NHOH) 3.5-co223 method described above in

IMPI

Example Π'δΦντ'ο toBSSA ^J Dt THE PROPERTY

INDUSTRIAL

<img file="MX340992B_D0385.tif" />

Poly- [d-Glu (OBn) 3.5-co-Glu (OBn) 3.5] -b-Poly- (Tyr (OH) 25-co-dPhei₅) -Ac was converted to the title compound using 10M sodium hydroxide solution (2.0 equiv / Portion of ester Bn).

The reaction time was 3 hours. The development provided the title compound (Yield = 85.8%) as a fine, colorless solid with a slight acetic acid odor. ^ -H NMR (d6-DMSO): identical to Example 56.

Example 58

I

NH₂ \ /270

one. Toluene, 60 ° C, azeotrope in vacuo

2. GliiíOBnlNCA.

(l-Giu (OBn) NCA,

CHjCIfc DM AC: 2.1, TA, 22 hr

3. d-PheNCA, Asp (OtBu) NCA, Tyr (OBn) NCA,

TA-&gt; 35 ° C, 22 hr

Four. Ac20, NMM,

DMAP, RT

<img file="MX340992B_D0386.tif" />

mPEG12K-d-Pol¡- (d-Glu (OBn) 5-co-Glu (OBn) 5) -b-Poli (d-Phei5-co-Asp (OtBu) 5-co-Tyr (OBn) 2o) - Ac

Synthesis of mPEG12K-b-Poli- (d-Glu (OBn) 5-coGlu (OBn) 5) -b-Poli (d-Pheis-co-Asp (OtBu) 5-co-Tyr (OBn) 20) -Ac .

Using the method detailed in Example 49 with dichloromethane

224

IMPI anhydrous (2 parts) and industrial N, N-dimethylacetamide as solvents and replacing NCA building blocks

<img file="MX340992B_D0387.tif" />

Appropriate provided a crude polymer which was precipitated with 5 volumes of isopropanol. After filtration and drying in vacuo, the title compound (Yield = 95.4%) was obtained as a fine, colorless, odorless solid. NMR Ή (de-DMSO) δ 8.57-7.75 (teo. 50H, obs. 47H), 7.41-6.67 (teo. 305 H, obs. 305H), 5.10-4.85 (teo. 60 H, obs.

59H), 4.70-4.18 (theo. 50H, obs. 49H), 3.72-3.25 (theo. 1087H, obs. 1131H), 3.05-2.20 (theo. 80H, obs. 100H), 2.05-1.58 (theo.

40H, obs. 25H), 1.38-1.20 (theo. 45H, obs. 40H).

Example 59 \ / 270

one. Toluene, 60'C, azeotrope in vacuo

2. GlutOBmNCA, d-Giu (OBn) NCA,

CHjCIj, DM AC: 2.1, TA, 23 hr

3. d-LeuNCA, Asp (OtBu | NCA, TyrfOBniNCA.

TA -&gt; 35 ° C. 21 hr

Four. ACjO, NMM,

DMAP, TA

<img file="MX340992B_D0388.tif" />

<img file="MX340992B_D0389.tif" />

mPEG12K-6-Poli- (d-Glu (OBn) 5-co-Glu (OBn) 5) -í) -Poli (d-Leui5-co-Asp (OtBu) 5-co-Tyr (OBn) 2o) - Ac mPEG12K-b-Poly- (d-Glu (OBn) s-co-Glu (OBn) 5) -b-Poly - (d225

<img file="MX340992B_D0390.tif" />

Leuis-co-Asp (OtBu) s-co-Tyr (OBn) 20) -Ac. Using tNSTmeXatlncAÍ »® detailed in Example 58 and replacing the appropriate NCA building blocks yielded a crude polymer which was precipitated with 5 volumes of isopropanol. After filtration and drying in vacuo, the title compound (Yield = 95.5%) was obtained as a fine, colorless, odorless solid. NMR^XH (d6-DMSO) δ 8.45-7.78 (teo. 50H, obs. 47H), 7.45-6.67 (teo. 230H, obs. 230H), 5.10-4.80 (teo. 60 H, obs. 59H), 4.65-4.00 (theo. 50H, obs. 52H), 3.70-3.25 (theo.

1087H, obs. 1196H), 3.05-2.55 (theo. 40H, obs. 41H), 2.48-2.30 (theo. 40H, obs. 33H), 2.05-1.71 (theo. 40H, obs. 25H), 1.691.02 (theo. 60H, obs. 65H), 0.95-0.55 (theo. 90H, obs. 83H).

Example 60

<img file="MX340992B_D0391.tif" />

I.NHzOH, HaO.NaOH,

THF, TA

2. Acetone, HOAc (catl.

Oh

I'

OH mPEG12K-b-Pol¡- [d-Glu (NHOH) 3.5-co-Glu (NHOH) 3.5] -b-Poli- (Tyr (OH) 25-co-d-Phei5) -Ac

Synthesis of mPEG12K-b-Poli- [d-Glu (NHOH) 3.5-co226

Glu (NHOH) 3.5] -b-Poly- (Tyr (OH) 25-co-d-Pheis) -Ac.

method described above in Example

<img file="MX340992B_D0392.tif" />

Poly- [d-Glu (OBn) 3.5-co-Glu (OBn) 3.5] -b-Poly- (Tyr (OH) 25-co-dPheis) -Ac was converted to the title compound using 4M sodium hydroxide solution (2.0 equiv / portion of ester Bn). The reaction time was 16 hours. Development with three times the normal volume of IPA followed by precipitation with TBME provided the title compound (Yield = 92.7%) as a fine, pale creamy solid with a slight odor of acetic acid.

Example 61

I

one. Toluene, «TC azeotropo in vacuo

2. Glu (OBn) NCA, d-Glu (QBn) NCA, CH_ZCI_{Z &lt;} OMAC; 2.1, TA

3. d-Tyr (OBn) NCA, Tyr (OBn) NCA, TA-&gt; 35 ° C

Four. Ac_zO, NMM, DMAP, TA

<img file="MX340992B_D0393.tif" />

mPEG12K-b-Pol¡- (d-Glu (OBn) 5-co-Glu (OBn) 5) -b-Pol¡- (d-Tyr (OBn) 2o-co-Tyr (OBn) 2o) -Ac

Synthesis of mPEG12K-b-Poli- (d-Glu (OBn) 5-σοGlu (OBn) 5) -b-Poly - (d-Tyr (OBn) 20-co-Tyr (OBn) 20) -Ac.

Using the

227

<img file="MX340992B_D0394.tif" />

mixed reaction solvent method detiwhl ^ cdoncAen »PROPERTY

INDUSTRIAL

IMPI

PaiTlactexicAer

OF INDUSTRIAL PROPERTY

Example 58 and substituting the appropriate NCA building blocks provided a crude polymer that precipitated with 9 volumes of isopropanol. After filtration and drying in vacuo, the title compound (Yield = 96.6%) was obtained as a fine, colorless, odorless solid. NMR¹H (άβDMSO) δ 8.44-7.80 (teo. 50H, obs. 47H), 7.40-6.75 (teo. 410H, obs. 410H), 5.11-4.84 (teo. 100H, obs. 94H), 4.60-4.20 (teo.

50H, obs. 52H), 3.70-3.25 (theo. 1087H, obs. 1605H), 3.00-2.28 (theo. 80H, obs. 95H), 2.03-1.60 (theo. 40H, obs. 31H).

Example 62

CO ^ Bn

H

270

ΧΓ

<img file="MX340992B_D0395.tif" />

TFA, pentamethylbenzene TA, 3.5 hr

<img file="MX340992B_D0396.tif" />

mPEG12K-b-Pol¡- (d-Glu (OBn) 5-co-Glu (OBn) 5) -b-Pol¡ (d-Leui5-co-Asp (OH) 5-co-Tyr (OH) 2o) -Ac

Synthesis of mPEG12K-b-Poli- (d-Glu (OBn) s-co ·

Glu (OBn) 5) -b-Poli- (dL ul5-co-Asp (OH) 5-co-Tyr (OH) 20) -Ac.

228

Using the method of Example 54, the reaction

<img file="MX340992B_D0397.tif" />

Poly- (d-Glu (OBn) s-co-Glu (OBn) ₅) -b-Poli- (d-Leuis-co-Asp (OtBu) 5co-Tyr (OBn) 20) -Ac with PMB in TFA for 3.5 hours at room temperature and precipitation of a mixture of dichloromethane, TBME: 1.6 provided the title product (Yield = 95.5%) as a fine, colorless, odorless polymer. N NMR (deDMSO) δ 9.15 (theo. 20H, obs. 18H), 8.43-7.60 (theo. 50H, obs.

47H), 7.40-6.45 (theo. 130H, obs. 130H), 5.04 (theo. 20H, obs.

13H), 4.65-4.00 (theo. 50H, obs. 48H), 3.85-3.15 (theo. 1087H, obs. 1334H), 3.01-2.10 (theo. 80H, obs. 80H), 2.05-1.65 (theo.

40H, obs. 42H), 1.63-0.55 (theo. 90H, obs. 75H).

I

I

Example 63 '4^{Οχ /}) Ο

27(

<img file="MX340992B_D0398.tif" />

one. NHsOH, HjO, KOH, THF, TA

270

THF, acetone (3,2), HOAc (cat), TA

<img file="MX340992B_D0399.tif" />

Oh

Synthesis of mPEG12K-b-Poly- [d-Glu (NHOH) 5-coOH mPEG12K-bPol¡- [d-Glu (NHOH) 5-co-Glu (NHOH)₅] -bPol¡- (Tyr (OH) 25-co-d-Phei₅) -Ac

229

<img file="MX340992B_D0400.tif" />

IMPI

Glu (NHOH) 5] -b-Poly- (Tyr (OH) 25-co-d-Pheis) -Ac. They use SoLAeiFgret described above in Example 47, mPEG12K-b-Poli- [dGlu (OBn) 5-co-Glu (OBn) 5] -b-Poli- (Tyr (OH) 25-co-d-Pheis) -Ac is converted to the title compound using solid potassium hydroxide (2.0 equiv / Portion of ester Bn) previously dissolved in the hydroxylamine solution. The reaction time was 5.5 hours. The development provided the title compound (Yield = 74.0%) as a fine, colorless solid with a slight acetic acid odor. NMR^XH (d6-DMSO): identical to Example 38.

Example 64

I

OBn

270

<img file="MX340992B_D0401.tif" />

OBn

COjBn

TFA, pentamethylbenzene (PHB), TA

<img file="MX340992B_D0402.tif" />

mPEG12K-í? -Pol¡- (d-Glu (OBn) 5-co-Glu (OBn) 5) -b-Pol¡- (d-Tyr (OH) 2o-co-Tyr (OH) 2o) -Ac

Synthesis of mPEG12K-b-Poli- (d-Glu (OBn) 5-coGlu (OBn) 5) -b-Poli- (d-Tyr (OH) 20-co-Tyr (OH) 20) -Ac. Using the method of Example 54, the reaction of mPEG12K-b-Poly- (dGlu (OBn) 5-co-Glu (OBn) 5) -b-Poli- (d-Tyr (OBn) 20-co-Tyr (OBn ) 20) -Ac with PMB in TFA for 4.5 hours at room temperature and precipitation

230

IMPI ^ of a mixture of dichloromethane, TBME: 1.5 provided eT ^ praáuató ^ Ztg ^^ title (Yield = 97.7%) as an odorless solid © r - á »e © lGKa_w£ iüQ_J__

NMR Ή (de-DMSO) δ 9.1 (theo. 40H, obs. 33H), 8.36-7.77 (theo. 50H, obs.

52H), 7.40-6.45 (teo. 210H, obs. 234H), 5.04 (teo. 20H, obs. 17H),

4.60-4.20 (teo. 50H, obs. 50H), 4.02-3.15 (teo. 1087H, obs. 1384H, contains darkened water peak), 3.00-2.10 (teo. 80H, obs. 78H), 2.06-1.62 (teo 40H, obs. 39H).

Example 65

<img file="MX340992B_D0403.tif" />

mPEG12K-b-Pol¡- (d-Glu (NHOH) 5-co-Glu (NHOH) 5) -bPol¡ (d-Leui5-co-Asp (OH) 5-co-Tyr (OH)₂o) -Ac

Synthesis of mPEG12K-b-Poli- (d-Glu (NHOH) 5-coGlu (NHOH) 5) -b-Poli- (d-Leuis -co-Asp (OH) 5 -co-Tyr (OH) 20) - Ac.

Using the method described above in Example 52, mPEG12K-b-Poli- (d-Glu (OBn) ₅-co-Glu (OBn) 5) -b-Poly- (d-Leuis-coAsp (OH) 5-co-Tyr (OH) 20) -Ac was converted to the title compound using lithium hydroxide monohydrate (2.0 equiv. / Portion of ester

231

Bn). The reaction time was 15 hours. The development M ^ o ^ ig ^ gone

INDUSTRIAL

ΙΜΡΙ

INDUSTRIAL precipitation with IPA, TBME provided the title compound

<img file="MX340992B_D0404.tif" />

(Yield = quantitative) as a fine, colorless solid with a slight odor of acetic acid. NMR^XH (d6-DM¡SO) δ 10.2-9.0 (teo. 40H, obs. 31H), 8.65-7.75 (teo. 50H, obs. 37H), 7.27-6.50 (teo. 80H, obs.

80H), 4.61-4.00 (theo. 50H, obs. 58H), 3.90-3.15 (theo. 1087H, obs.

1356H), 3.02-2.20 (theo. 80H, obs. 100H), 2.40-1.70 (theo. 40H, obs. ~ 47H, contains peak overlap HOAc at δ 1.69), 1.630.55 (theo. 105H, obs. 96H ).

Example 66

<img file="MX340992B_D0405.tif" />

mPEG12K-b-Poli- (d-Glu (NHOH) 5-co-Glu (NHOH) 5) -b-Poli- (d-Tyr (OH) 2o-co-Tyr (OH)₂o) -Ac

Synthesis of mPEG12K-b-Poli- (d-Glu (NHOH)₅-coGlu (NHOH) ₅) -b-Poli- (d-Tyr (OH) ₂o-co-Tyr (OH) ₂o) -Ac. Using the method described above in Example 52, mPEG12K-bPoli- (d-Glu (OBn) 5-co-Glu (OBn) 5) -b-Poli- (d-Tyr (OH) 20-co232

Tyr (OH) 20) -Ac was converted to the compound of

INDUSTRIAL

IMPI ¥ 8S? Affi £ SS

INDUSTRIAL Lithium Hydroxide Monohydrate (2.0 Equiv. / Portion of Ester

<img file="MX340992B_D0406.tif" />

Bn). The reaction time was 5.5 hours. Development followed by precipitation with IPA, TBME provided the title compound (Yield = 93.2%) as a fine, colorless solid with a slight acetic acid odor. NMR Ή (cU-EMSO) δ 9.55 (teo. 40H, obs. 26H), 8.45-7.90 (teo. 50H, obs. 34H), 7.37-6.51 (teo. 160H, obs.

166H), 4.55-4.10 (teo. 50H, obs. 50H), 3.80-3.20 (teo. 1087H, obs. 1269H, contains darkened water peak), 3.00-2.20 (teo. 80H, obs. 108H), 2.18- 1.60 (theo. 40H, obs. 39H, contains HOAc peak overlap at δ 1.69).

I

<img file="MX340992B_D0407.tif" />

<img file="MX340992B_D0408.tif" />

mPEG12K-b-Pol¡- [d-Glu (OBn) 5-co-Glu (OBn) 5] -fa-Pol¡- (Tyr (OH)₂5-co-d-Phei5) -Ac

Synthesis of mPEG12K-b-Poli- [d-Glu (OBn)₅-coWMM · UV * ·· - ·· ....

233

<img file="MX340992B_D0409.tif" />

Glu (OBn) 5] -b-Poli- (Tyr (OH) 25-co-d-Pheis) -Ac. UsaffS ^ T ^^^,? * ^

INDUSTRIAL

IMPI «asme®

INDUSTRIAL from Example 43, the reaction of mPEG12K-b-Poly- [d-Glu (OBn) ₅co-Glu (OBn) 5] -b-Poly- (Tyr (OBn) 25-co-d-Phei5) -Ac with PMB in TFA for 3.5 hours at room temperature gave a crude product, which was dissolved in dichloromethane ( 2 volumes) and then precipitated from TBME (5 volumes). Filtration and drying in vacuo provided the title product (Yield

93.1%) as a fine, colorless, odorless solid. NMR¹H (d6DMSO): identical to Example 37.

Example 68

<img file="MX340992B_D0410.tif" />

one. NHjOH, HgO, KOH, THF, TA

2. THF, acetone (3,2), HOAc (cat), TA

I

270

O ^ NHOH

H

<img file="MX340992B_D0411.tif" />

O ^ NHOH mPEG12K-6-Poly- [d-Glu (NHOH) 5-co-Glu (NHOH)₅] -6-Poli- (Tyr (OH) 25-co-d-Phei5) -Ac

MPEG12K-b-Poli- [d-Glu (NHOH) 5-co234 Synthesis

IMPI

Glu (NHOH) ₅] -b-Poli- (Tyr (OH) ₂₅-co-d-Phei₅) -Ac. ιηΕΐ! ®β&gt;

INDUSTRIAL [d-Glu (OBn) ₅-co-Glu (OBn) 5] -b-Poli- (Tyr (OH) 25-co-d-Phei₅) -Ac (38.94 g, 1.90 mmol) was dissolved in 390 mL of THF and treated with hydroxylamine solution (25.2 mL, 380.0 mmol) and 4M potassium hydroxide solution (9.5 mL, 38.0 mmol, 2.0 equiv / Serving ester Bn). The resulting slightly cloudy pale yellow solution was stirred at room temperature for 5.5 hours under N₂ and then diluted with acetone (220.7 g, 3.8 mol, 280 mL). Acetic acid (22.82 g, 380.0 mmol, 21.7 mL) was added, the solution was briefly heated under reflux, and then stirred at room temperature for 18 hours. The solution was diluted with 280 mL of acetone and the product was precipitated by adding TBME (5 L) and diethyl ether (IL) using vigorous mechanical stirring. After cooling to -25 ° C and additional stirring for 30 minutes, filtration and drying in vacuo afforded the title compound (35.98 g, Yield = 87.3%) as a fine, colorless solid with a slight odor of acetic acid. NMR¹H (d₆-DMS0): identical to Example 38.

<img file="MX340992B_D0412.tif" />

235

Example 69

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX340992B_D0413.tif" />

MePEGIIk-OH

one. Toluene. 55-60 ° C, azeotrope in vacuo

2. Glu (OBn) NCA, d-Glu (OBn) NCA,

CH2CI2, DHAC: 2.1, TA

3. d-PheNCA, Tyr &lt;OBn) NCA, TA-&gt; 35'C

Four. AC₂Or, NMM,

DHAP, TA

CO_zBn

I'm ^ '^^/jo^{z / Sv}' ^N

249

<img file="MX340992B_D0414.tif" />

OBn mPEGHK-bPol¡- (d-Glu (OBn)₅-co-Glu (OBn) 5) -bPol¡- (d-Phei5-co-Tyr (OBn) 25) -Ac

Synthesis of mPEGHK-b-Poli- (d-Glu (OBn) s-coGlu (OBn) 5) -b-Poli- (d-Pheis-co-Tyr (OBn) 25) -Ac. Using the dichloromethane, DMAC co-solvent method detailed in Example 58 with m-PEGllk-NH2 (1.10 kg, 100.0 mmol) and the appropriate NCA building blocks provided a crude polymer solution in DMAC that precipitated with 8 volumes of isopropanol. After filtration, the crude product was suspended in 5 volumes of isopropanol for two hours. The resulting solid was filtered, washed with IPA / Et2O, fresh Et2Ü and then dried in an oven at

236

<img file="MX340992B_D0415.tif" />

IMPIíC tHSTITUTC MEXICANO M THE PROPERTY vacuum overnight to provide 213 0 g yield) of product as a solid odorless. ^XH-NMR (d₆-DMSO) δ 8.45-7.85 (theo. 50H, obs. 50H),

7.45-6.60 (teo. 350H, obs. 350H), 5.10-4.84 (teo. 70H, obs.

68H), 4.65-4.20 (theo. 50H, obs. 48H), 3.72-3.25 (theo. 1000H, obs. 1120H), 3.05-2.55 (theo. 50H, obs. 49H), 2.44-1.60 (theo.

70H, obs. 68H).

I

270

Example 70

<img file="MX340992B_D0416.tif" />

<img file="MX340992B_D0417.tif" />

mPEG12K-b-Pol¡- [d-Glu (OBn) 5-co-Glu (OBn) 5] -d-Pol¡- (Tyr (OH) 25-co-d-Phei₅) -Ac

Synthesis of mPEG12K-b-Poli- [d-Glu (0Bn) s-coGlu (OBn) 5] -b-Poli- (Tyr (OH) 25-co-d-Pheis) -Ac. Using the method of Example 37, the reaction of mPEG12K-b-Poly- [d-Glu (OBn)₅co-Glu (OBn) 5] -b-Poli- (Tyr (OBn) 25-co-d-Phei5) -Ac with PMB in TFA

237

<img file="MX340992B_D0418.tif" />

IMPI

INSTITUTO MEXICANO for three hours at room temperature and the dichloromethane mixture, TBME: 1.5, gave the title product (Yield = 92.7%) as a fine, colorless, odorless polymer. NMR^ΤΗ (d6-DMSO) identical to Example 37.

Example 71

<img file="MX340992B_D0419.tif" />

1, NHaOH, HjO, KOH, THF, TA

2. THF, acetone (3,2), HOAc (cat), TA

<img file="MX340992B_D0420.tif" />

mPEG12K-í&gt; Pol¡- [d-Glu (NHOH)₅-co-Glu (NHOH) 5] -b-Pol¡- (Tyr (OH) 25-co-d-Phei5) -Ac

Synthesis of mPEG12K-b-Poli- [d-Glu (NHOH) s-coGlu (NHOH) 5] -b-Poli- (Tyr (OH) as-co-d-Pheis) -Ac. Using the method described in Example 52, mPEG12K-b-Poli- [d-Glu (OBn) 5-coGlu (OBn) 5] -b-Poli- (Tyr (OH) 25-co-d-Pheis) -Ac was converted to the title compound using hydroxylamine solution (5 equiv / portion of ester) and potassium hydroxide solution

238

<img file="MX340992B_D0421.tif" />

ϊ Μ ΡI s * ”

MEXICAN INSTITUTE

4Μ (2.0 equiv / portion of ester Bn). The rem ^ Ei time was 5.25 hours. The development of acetone / áe ido-aeétie © ^ -.

IPA, TBME: 1, 2 precipitation and further crushing of the cake filtered with IPA, TBME: 1, 2 and vacuum drying provided the title compound (Yield = 89.9%) as a cream colored solid with a slight acidic odor acetic. NMR^XH (d6-DMSO): identical to Example 38.

Example 72

<img file="MX340992B_D0422.tif" />

one. NHtOH, HA KOH, THF, TA

2. THF, acetone (3,2), HOAc (cay, TA

I

270

<img file="MX340992B_D0423.tif" />

NHOH mPEG12K-¿&gt; -Pol¡- [d-Glu (NHOH) 5-co-Glu (NHOH) 5] -f) -Pol¡- (Tyr (OH) 25-co-d-Phei5) -Ac

Synthesis of mPEG12K-b-Poli- [d-Glu (NH0H) 5-coGlu (NHOH) 5] -b-Poli- (Tyr (OH) 25-co-d-Pheis) -Ac. Using the method described in Example 71, mPEG12K-b-Poly- [d-Glu (OBn)₅-co239

<img file="MX340992B_D0424.tif" />

IMPI

MEXICAN INSTITUTE

Glu (OBn) ₅] -Jb-Poli- (Tyr (OH) 25-co-d-Pheis) -Ac was composed of the title using bi d-nov-i 1 ajpjna solution (10 equiv / portion of ester) and hydroxide solution of 4M potassium (2.0 equiv./Port of ester Bn). The reaction time was 5.5 hours. Acetone / acetic acid development, IPA precipitation, TBME: 1, 4 and vacuum drying provided the title compound (Yield = 82.9%) as a fine, colorless solid with a slight acetic acid odor. NMR¹H (d6-DMSO): identical to Example 38.

Example 73

one. Toluene, «TC Azeotmpo, 3 hr.

2. NMP, via cannula

3. Glu (oBn) NCA, d-Glu (oBn) NCA 18 hr, TA

Four. d-Phe NCA, Tyr &lt;oBn) NCA, Asp (oTbu) NCA 48 hr, TA - * 35 ° C

5. NMM, DMAP, AcANH ONTA

COjBo

<img file="MX340992B_D0425.tif" />

and ¡t

CO₂Bn

OBn

IO

<img file="MX340992B_D0426.tif" />

mPEG12K-fc-Poli- (d-Glu (OBn) 5-co-Glu (OBn) 5) -á-Pol¡ (Tyr (OBn) io-co-d-Phe2o-co-Asp (oTbu) io-Ac

Synthesis

Glu (OBn) 5) -b-Poly of mPEG12K-b-Poli- (d-Glu (OBn)₅-co (Tyr (OBn) io-co-d-Phe2o-co-Asp (oTbu) 10-Ac.

240 mPEG12K-NH2 prepared in the same way as ¡f

<img file="MX340992B_D0427.tif" />

OF INDUSTRIAL PROPERTY

3, Weighed (30g, 2.5mmol) into a round bottom flask of

500 Clean mL and completely dissolved in toluene and dried by azeotropic distillation. Toluene was collected in a second nitrogen-cooled 500 mL round-bottom flask using a single glass bridge. The resulting solid was allowed to dry completely for three hours. To the recently distilled dry solid, Nmethylpyrrolidine was added via cannula and transferred under vacuum. This mixture was allowed to dissolve completely before the addition of NCA. The NCA as prepared from Example 8 and Example 9 was therefore weighed into a clean two-necked round bottom flask NCA Glu (oBn) (2.87g) NCA d-Glu (oBn) (2.87g) and emptied for one hour before this solid was completely dissolved in NMP, and then cannulated in the flask containing the PEG. This polymerization was stirred at room temperature and monitored by GPC (DMF, .1% LiBr) to ensure completion (approximately 16hrs). Upon completion of the polymerization of this first block of NCA, the second addition of NCA was performed in the same manner as the first, and consists of dPhe (9.5g) from Example 7, Tyr (oBn) (7.4g) from Example 6 , and Asp (otBu) (5.38g) from Example 5. This was allowed to polymerize at room temperature for two hours and then heated to 35 ° C until complete (approximately 24hrs). Once

241 confirmed by GPC, mL, 25 mmol), DMAP

IMPI added N-Methyl-MorfolinS ^^^^ g industrial

(.3 g, 2.5 mmol), and acetic anhydride

<img file="MX340992B_D0428.tif" />

(2.5g, 2.36mL, 25mmol), the reaction solution was stirred overnight. This reaction mixture was poured into a two liter beaker with a magnetic stir bar, and diethyl ether was added slowly until a white precipitate was observed. This solid was filtered and washed in a medium porosity sintered glass frit. This solid was dried in vacuo, characterized with iH NMR and GPC. (yield = 74.8%, 40 grams). NMR¹H (d6-DMSO) δ 8.427.70, 7.30, 6.95, 5.10-4.9, 4.65-4.20, 3.77-3.25, 3.05-2.45,

2.44-1.60, 1.38-1.22.

Met

Synthesis

Example 74

COaBn f '\

<img file="MX340992B_D0429.tif" />

QBn

TFA, PMB TA 3Hr

P'v 'iQ-COgBn Z

<img file="MX340992B_D0430.tif" />

OH oX% \ of

OH mPEG12K-b-Poli- (d-Glu (OBn) s-co242

Glu (OBn) 5) -b-Poly (Tyrio-co-d-Phe2o-co-Aspio-Ac.

industrial

Protected triblock industrial "rasas" IMPI (Example 73) weighed (30 g, 1.4 mmol)

<img file="MX340992B_D0431.tif" />

in a clean 500 mL beaker and dissolved in trifluoroacetic acid. Pentamethylbenzene (4g, 26.98mmol) was added and stirred with a magnetic stir bar. The reaction mixture was stirred for two hours and monitored by NMR for complete removal of the benzylic protecting groups on tyrosine and t-butyl group on aspartate. After the end of this protection, the solution was precipitated in cold diethyl ether. This solid was then filtered on a medium fritted glass frit and dissolved again in methylene chloride and again precipitated from cold ether and filtered. This solid (24.7g, Yield = 88.4%) was dried in vacuo γ was characterized. NMR¹H (d6-DMSO) δ 9.09, 8.50-7.75,

7.40-6.45, 5.03, 4.70-4.20, 3.91-3.05, 3.03-2.10, 2.09-1.50.

243

<img file="MX340992B_D0432.tif" />

Example 75

one. Tolerates, 0O ° C Azeotrope 3 Hr.

2. NHP, via cannula

3. GluíoBn) NGA, d-Glu (oBn) NGA 18 hr, TA.

Four. d-Leu NGA, TyrfoBn) NGA Asp (oTbu) NCA 48 hr, RT 35 ° C

5. NMM, DMAP, AcANH Ο.Ν., Τ.Α.

I'

<img file="MX340992B_D0433.tif" />

CO ^ Bn

Synthesis of mPEG12K-b-Poli- (d-Glu (oBn) 5-coGlu (oBns) -b-Poly (Tyr (OBn) 10-co-d-Leu2o-co-Asp (oTbu) 10-Ac. Using the general protocol of Example 73 and substituting appropriate NCA starting materials, resulted in the crude polymer, this was precipitated with about 10 volumes diethyl ether After filtration and drying the title compound (Yield = 80.2%) was collected as a solid colorless.

NMR ³Η (de-DMSO) δ 8.50-7.75, 7.40-6.6, 5.03, 4.70-4.20, 3.693.09, 3.03-2.10, 2.09-1.50, 1.43-1.25, 0.85-0.62.

244

Example 76

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX340992B_D0434.tif" />

<img file="MX340992B_D0435.tif" />

OBn

TFA, PMB 3 Hr, TA

'1

<img file="MX340992B_D0436.tif" />

COjBn

<img file="MX340992B_D0437.tif" />

Oh

<img file="MX340992B_D0438.tif" />

<img file="MX340992B_D0439.tif" />

Synthesis of mPEG12K-b-Poly- (d-Glu (oBn) 5-coGlu (oBn) 5-b-Poly (Tyr (OBn) io-co-d-Leu2o-co-Asp (oTbu) io) -Ac.

Thirty-four grams of the protected triblock polymer (Example 75) was weighed into a clean 500 mL beaker and dissolved in trifluoroacetic acid (500 mL). To this solution (4 g, 27 mmol) was added pentamethylbenzene and stirred with a magnetic stir bar. Thirty minutes after the addition of pentamethylbenzene, a solution precipitate was observed. The reaction mixture was stirred for 2.5 hours and monitored by NMR for complete removal of the benzylic protecting groups on tyrosine and t-butyl group on aspartate. After the end of this

245 protection, the solution was submitted

<img file="MX340992B_D0440.tif" />

To obtain a thick paste, it was dissolved again in methylene chloride and then precipitated in cold diethyl ether. This solid was then filtered on a medium fritted glass frit and dissolved again in methylene chloride and again precipitated in cold ether and filtered. This solid was dried under vacuum and characterized. NMR¹H (d6DMSO) δ 9.09, 8.50-7.75, 7.45-6.55, 5.03, 4.65-4.00, 3.693.09, 3.03-2.10, 2.09-1.50, 0.85-0.55.

Example 77

<img file="MX340992B_D0441.tif" />

Oh

eleven. THF, hydroxy mine TBD, TA, 48 hr 2. Acetone acetic acid TAON

HOHN

<img file="MX340992B_D0442.tif" />

Oh

Synthesis of mPEG12K-b- (d-Glu (NHOH) s-co-Glu (NHOH) -bPoli (Tyr (OH) io-co-d-Leu2o-co-Aspio) -Ac. Triblock ester

<img file="MX340992B_D0443.tif" />

<img file="MX340992B_D0444.tif" />

246 '..... (Example 76) was weighed (20 g, 0.96 mmol) in an erf^NSt ^ '¿^ i¡ ^ cg! »z industrial 500 mL round bottom clean and 2 00 mL of tetrahydrofuran was added and completely dissolved. To this solution of thirty equivalents of hydroxylamine (1.9 mL, 28 mmol) and

0.5 g of TBD Catalyst was stirred under nitrogen at 50 ° C overnight. The term was verified by NMR¹H. This solution was mixed with 100 mL methanol and precipitated with diethyl ether (approximately 7 volumes). This white solid was collected by filtration and washed with fresh diethyl ether. The solid collected afterwards was dissolved in acetone and a catalytic amount of acetic acid was allowed to stir overnight. The solution was poured into a clean two-liter beaker, and diethyl ether was added slowly to the solution with stirring. NMR^XH (d6-DMSO) δ

9.4-8.6, 8.51-7.77, 7.44-7.57, 6.96, 6.56, 4.52-4.00, 3.753.29, 3.03-2.45, 2.08-1.21, 0.95-0.57.

247

<img file="MX340992B_D0445.tif" />

Example 78

Xh₂

one. Tolero, C0 ° C Azeotrope 3Hr,

2. NMP, via cannula

3. Glu (oBn) NCA, d-Clu (oBn) NCA -IShre., TA

Four. d ~ Phe NCA Tyi (oBn) NCA, 48hre., TA-&gt; 35 ° C

5. NMM, DMAP, AcANH Ο.Ν., Τ.Α

<img file="MX340992B_D0446.tif" />

OBn

Synthesis of mPEG12K-b- (d-Glu (OBn) 5-co-Glu (OBn) 5) -bPoli (Tyr (OBn) 30-co-d-Pheio) -Ac. The first block of the copolymer was prepared using the same scale and procedure as in Example 73. At the end of this first NCA block, a second addition of NCA d-Phe NCA (4.78 g, 25 mmol) prepared in the same manner as in Example 7, and from NCA Tyr (oBn) (22.29 g, 75 mmol) from the procedure in Example 6. This solution was allowed to polymerize at room temperature for two hours and then heated at 35 ° C until complete (approximately 48hrs). Once confirmed by GPC, N-Methyl-Morpholine (2.5 g, 2.7 mL, 25 mmol), DMAP (.3) was added.

248 g, 2.5 mmol), and Acetic Anhydride (2.5 g, 2.3 industrial

IMPI

INDUSTRIAL the reaction solution was stirred overnight. East

<img file="MX340992B_D0447.tif" />

Capped polymer was developed in the same manner as in Example 73. (yield = 79.6%) approximately 40 grams. NMR^XH (de-DMSO) δ 8.46-7.72, 7.44-6.57, 5.10-4.80, 4.62-4.13,

3.74-3.23, 3.03-2.77, 2.62-2.21, 2.02-1.56 (solvent impurities).

Example 79

I

CO₂Bn

<img file="MX340992B_D0448.tif" />

OBn

COsBn Z ~ \ w

<img file="MX340992B_D0449.tif" />

CO₂Bn

WH \ j X ^ N4j χ

<img file="MX340992B_D0450.tif" />

Synthesis of mPEG12K-b-Poly- (d-Glu (oBn) ¡-coGlu (oBn) 5-b-Poly (Tyr (OH) 30-co-d-Pheio) -Ac. The Protected Triblock Copolymer ( from Example 78) was weighed (34 g, 1.46

249

<img file="MX340992B_D0451.tif" />

mmol) in a 500 mL beaker^INS1^ J) g ^^ &lt;g ^ jy &lt;

INDUSTRIAL 'dissolved in trifluoroacetic acid (500 mL). To this solution (4 g, 27 mmol) was added pentamethylbenzene and stirred with a magnetic stir bar. Thirty minutes after the addition of pentamethylbenzene, a solution precipitate was observed. The reaction mixture was stirred for 2.5 hours and monitored by NMR for complete removal of the benzyl protecting groups on tyrosine. After the end of this protection (3 hrs) the solution was subjected to a rotary evaporator until obtaining a thick paste, it was dissolved again in methylene chloride and then precipitated in cold diethyl ether. This solid was then filtered on a medium fritted glass frit and dissolved again in methylene chloride and again precipitated in cold ether and filtered. This solid was dried in vacuo and characterized. NMR (de-DMSO) δ 9.10, 8.38-7.77, 7.39-6.73, 6.59, 5.03, 4.643.79, 3.71-3.30, 2.98-2.56, 2.02-1.62.

-« _

250

<img file="MX340992B_D0452.tif" />

Example 80

1Μ P .1

MEXICAN INSTITUTE

<img file="MX340992B_D0453.tif" />

Synthesis of mPEG12K-.b-Poly- (d-Glu (NHOH) 5-coGlu (NHOH) 5) -b-Poly (Tyr (OH) 30-co-d-Pheio) -Ac. 20g of triblock ester (From Example 79) was weighed into a clean 500 mL round bottom flask and 2 00 mL of tetrahydrofuran was added and completely dissolved. To this solution of ten equivalents of hydroxylamine, and 1,5,7-triazabicyclo [4.4.0] dec-5-ene (TBD, .5g, 3.5 mmol) was stirred under nitrogen at room temperature. The term was verified by H NMR * (48Hrs). This solution was mixed with 100 mL methanol and this

251 solution was poured into a beaker Id ^r UXSTinToSLí.OsO

ΓΙΐΛΓ, ν.-υΓΑΟ INDCSTU / J, liters. Methyl tert-butyl ether (about 5 volume

<img file="MX340992B_D0454.tif" />

It was slowly added to the solution with stirring. The resulting white éólldb ”was then collected in a medium frit and dried in vacuo. (17.34 g, Yield = 90%). NMR¹H (άβDMSO) δ 9.10-8.65, 8.39-7.78, 7.28-6.75, 6.80, 6.59, 4.594.31, 3.75-3.13, 3.00-2.57, 2.16-1.57.

Example 81

one. Tolero, «TC Azeotiopo 3 Hr

2. NMP, via cannula

GlutoBní NCA, d-Glu (oBn) NCA Ws .. TA

4.d4&gt; he NCA, Tyr (oBn) NCA, 48hrs., AT 35 ° C

9, PWH DMAP.AcANH 'F ΟΧ, TA

<img file="MX340992B_D0455.tif" />

OBn

Synthesis of mPEG12K-b-Poli- (d-Glu (OBn) 1.5-coGlu (OBn) is) -b-Poly (Tyr (OBn) 25-co-d-Pheis) -Ac.

mPEG12KNH₂ prepared in the same manner as in Example 3 (25 g, 2.08 mm) was weighed into a round bottom flask, two

252 1000 mL necks, oven dried, clean and

<img file="MX340992B_D0456.tif" />

toluene (300mL) with heating and dried by azeotropic distillation. After distillation to dryness, the polymer was left under vacuum for three hours. The flask was subsequently filled with N2, emptied again under reduced pressure, and dry N-methylpyrrolidone (NMP) (250 mL) was introduced via cannula. The mixture was briefly heated to 40 ° C to facilitate dissolution, and then cooled to 25 ° C. NCA Glu (OBn) (.82 g, 3.1 mmol) made in the same manner as in Example 8, and NCA d-Glu (OBn) (.82 g, 3.1 mmol) from Example 9 were added to the flask directly, and the reaction mixture was allowed to stir for 18 hours at room temperature under nitrogen gas. Then, NCA d-Phe (5.97 g, 31.25 mmol) from Example 7, and NCA Tyr (OBn) (15.49 g, 52.08 mmol) prepared from Example 6, and then added to the solution and stirred for 2 hours, then It was heated at 35 ° C for 48 hours at which point the reaction was complete (GPC, DMF / 0.1% LiBr). The solution was cooled to room temperature and acetic anhydride (2.04 g, 20 mmol, 1.88 mL), N-methylmorpholine (NMM) (2.23 g, 22 mmol, 2.47 mL) and dimethylaminopyridine (DMAP) (.24 g, 2.0) were added. mmol). Stirring was continued for 1 day at room temperature. The polymer was precipitated from 10: 1 diethyl ether: heptane (2.5 L) and isolated by filtration, washed with 100 mL portions of fresh diethyl ether, and dried in vacuo to give the copolymer

253 en bloc as a whitish powder, fine = 90.3%). NMR^XH (de-DMSO) δ 9.26-9.04, (39.sT

IMPI gn i'íteaada.

INDUSTRIAL

<img file="MX340992B_D0457.tif" />

8.36-7.75, 7.41-7.25,

6.97, 6.60, 5.04, 4.59-4.13, 3.81-3.13,

2.96-2.76, 2.75-2.57,

2.43-2.12, 2.00-1.45.

Example 82

<img file="MX340992B_D0458.tif" />

Oh

Synthesis of mPEG12K-b-Poli- (d-Glu (OBn) 15-coGlu (OBn) 15) -b-Poly (Tyr (OH) 25-σο-d-Pheis) -Ac. The polymer of Example 81 was deprotected using the general method of Example 74 only by stoichiometric adjustment. Once complete (3 hrs) the solution was subjected to a rotary evaporator until a thick paste was obtained and then it was dissolved again in dichloromethane and precipitated in cold diethyl ether, collected by filtration and dried in vacuo. This

254

<img file="MX340992B_D0459.tif" />

IMPI

INSTITUTO MEXICANO reaction provided 22 g of dry material

76.92%). NMR^XH (dg-DMSO) δ 9.09, 8.50-7.75, 7.35-6.45. 5.04, _

4.70-4.20, 3.91-3.05, 3.03-2.10, 2.09-1.50.

Example 83

I

<img file="MX340992B_D0460.tif" />

one. THF, Hydroxylamine, TBD, TA24hr

2. Acetone, HOAc TA 5hr

OR'

<img file="MX340992B_D0461.tif" />

ηΟην '^Λ;'Ό

OH mPEGl2K-b-Poli- (d-Glu (NHOH) 1.5-CO-GluCNHOH) is) -b-Poli (Tyr (OH) 25-co-d-Pheis) -Ac

Synthesis of mPEG12K-b-Poli- (d-Glu (NHOH) i.₅-co-Glu (NHOH) 1.5) b-Poly (Tyr (OH) 25-co-d-Pheis) -Ac. The polymer of Example 82 (13.2g,

0.705 mmol) was completely dissolved in 160 mL of THF with heating, this solution was allowed to cool to room temperature before adding 1,5,7-triazabicyclo [4.4.0] dec-5-ene (TBD, 0.3g, 2.2τπηο1 ) followed by hydroxylamine (50% water solution, 25mL, 378mmol) this solution was stirred at room temperature for 24 hours. Methanol (80 mL) was added and then precipitated with ether of

255 methyl tert-butyl, collected by filtration¹,^STD ^^,p'Sá4 «2Xi

INDUSTRIAL

IMPI

INDUSTRIAL in acetone. Acetic acid was added to this solution

<img file="MX340992B_D0462.tif" />

acetone and stirred for 5 hours. The solution was evaporated until almost dry, redissolved in methylene chloride and precipitated in MTBE, collected by filtration and dried in vacuo (12.1 g, Yield = 92.8%). NMR^XH (dg-DMSO) δ 9.11,

8.34-7.75, 7.37-7.05, 6.92, 6.58 4.60-4.32, 3.81-3.12, 2.992.57, 2.49-2.32, 2.10-1.73.

Example 84 ^IfO^XX4th ^ 'NH₂

one. Toluene, «TC Azeotropo 3 Hr

2. NMP, via cannula

3. Qkí (GBn&gt; NCA, d-Oki (QBn) NCA

1Shr $., TA

d-Phe NCA, TyhoQn) NCA, 48htS „TA ~&gt; 35^FC

5, NMM, OMAP, AcANH 'r ON, TA

<img file="MX340992B_D0463.tif" />

OBn mPEG12K-b-Poli- (d-Glu (OBn) 2.5- co-Glu (OBn) 2.5) -b-Poli (Tyr (OBn) ₂5-co-d-Phei₅) -Ac

Synthesis of mPEG12K-b-Poli- (d-Glu (OBn) 2.s-co256

Glu (OBn) 2.s) -b-Poly

<img file="MX340992B_D0464.tif" />

(Tyr (OBn) ₂5i * e © Hd »Bbea5

OF INDUSTRIAL PROPERTY

<img file="MX340992B_D0465.tif" />

mPEG12KNH₂ prepared in the same method as in Example 3, lll —-. . ..._z - I (25g, 2.08mm) was weighed into a clean, oven dried, two necked round neck flask and dissolved in toluene (300mL) with heating and dried by azeotropic distillation. After distillation to dryness, the polymer was left under vacuum for three hours. The flask was subsequently filled with N₂, was emptied again under reduced pressure, and dry N-methylpyrrolidone (NMP) (250 mL) was introduced by cannula. The mixture was briefly heated to 40 ° C to facilitate dissolution, and then cooled to 25 ° C. NCA Glu (OBn) (1.37 g, 5.2 mmol) and NCA d-Glu (OBn) (1.37 g, 5.2 mmol) were added to the flask, and the reaction mixture was allowed to stir for 18 hours at room temperature under nitrogen gas. . After completion of the first block of NCA, NCA d-Phe (5.97 g, 31.25 mmol) prepared in the same manner as in Example 79, and NCA Tyr (OBn) (15.49 g, 52.08 mmol) from Example 6, were added and the solution was allowed to stir at room temperature for two hours at 35 ° C for 48 hours, at which point the reaction was complete (GPC, DMF / 0.1% LiBr). The solution was cooled to room temperature and acetic anhydride (2.04 g, 20 mmol, 1.88 mL), Nmethylmorpholine (NMM) (2.23 g, 22 mmol, 2.47 mL) and dimethylaminopyridine (DMAP) (.24 g, 2.0 mmol) were added. . Stirring was continued for 1 day at room temperature. The polymer is

257

<img file="MX340992B_D0466.tif" />

precipitated from diethyl ether, heptane 10: 1 (2.5

INSTITUTO MEXICANt DE LA PROPIEDAI was filtered, washed with 100 mL portions of diethTH98 '* ether. NMR Ή (cU-DMSO) δ 9.10

8.37-7.83, 7.39-7.21, 6.95, 6.56, 5.02, 4.61-4.34, 4.32-4.20, 3.713.25, 2.94-2.59, 2.40-2.10, 1.96-1.45.

Example 85

<img file="MX340992B_D0467.tif" />

Oh

Synthesis of mPEG12K-b-Poli- (d-Glu (OBn) 2.5-coGlu (OBn) 2.5) -Jb-Poli (Tyr (OH) 25-co-d-Pheis) -Ac. Using the general method of Example 74 and adjusting the stoichiometry, the

258

<img file="MX340992B_D0468.tif" />

INSTITUTO MEXICANO V&gt; - ~ D Ε IA I RO FIE DA O polymer from Example 84 was deprotected (32 g, complete time (3 Hrs.) The solution was subjected to rotoevapÓffáSót obtaining a thick paste and then dissolved again in DCM · 7 precipitated from cold diethyl ether, collected by filtration, and dried in vacuo This reaction provided 27g of dry material (94.2%). ^XH (de-DMSO) δ 9.09, 8.50-7.75, 7.35-6.45, 5.04, 4.70-4.20, 3.91-3.05,

3.03-2.10, 2.09-1.50.

Example 86

<img file="MX340992B_D0469.tif" />

V

Oh

<img file="MX340992B_D0470.tif" />

one. THF, H id rene lamina, TBD, TA 24 hr

2. Acetone, HOAc TA 5 hr

<img file="MX340992B_D0471.tif" />

<img file="MX340992B_D0472.tif" />

Synthesis of mPEG12K-b-Poly- (d-Glu (NHOH) 2.5-coGlu (NHOH) 2.5) -b-Poly (Tyr (OH) 2.5-co-d-Pheis) -Ac. The polymer of

259

<img file="MX340992B_D0473.tif" />

Example 85 (20 g, 1 mmol) was completely dissolved ^^ ipljo

THF MEXICAN INSTITUTE OF PROPERTY with heating, this solution was left at room temperature before adding 1.5, 'ν ^ ΙχιΔϊϋΐυΐοίυΙ'υ ·· [4.4.0] dec-5-ene (TBD, .5 g , 3.6 mmol), followed by hydroxylamine (50% water solution, 30mL, 545 mmol), this solution was stirred at room temperature for 24 hours. Methanol (80 mL) was added and then precipitated with methyl tert-butyl ether, collected by filtration, and dissolved in acetone. Acetic acid was added to this acetone solution and stirred for 5 hours, then this solution was subjected to a rotary evaporator until almost dry, dissolved again in methylene chloride and precipitated in MTBE, collected by filtration and dried in vacuo (18 g,

Yield = 91.7%). NMR^XH (d₆-DMSO) δ 9.11, 8.34-7.75, 7.15,

6.80, 4.60-4.32, 3.81-3.12, 2.99-2.32, 1.93-1.83).

260

Example 87

<img file="MX340992B_D0474.tif" />

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX340992B_D0475.tif" />

d.Totuene, 60 ° C Azeotrope 3Hrs,

2. NMP, via cannula

3. GlufoBn) NGA, d-Glu (oBn) NCA 18 hr., .TA

Four. d-Phe NGA, Tyr ^ oBn) NGA, 48hr., TA—&gt; 35 ° C

5. NMM, DMAP, AcANH Ο.Ν., Τ.Α.

<img file="MX340992B_D0476.tif" />

<img file="MX340992B_D0477.tif" />

OBn

Synthesis of mPEG12K-b-Poli- (d-Glu (OBn) 3.s-coGlu. (OBn) 3 .5) -b-Poly (Tyr (OBn) 25-co-d-Pheis) -Ac.

mPEG12KNH2 prepared in the same way as in the

Example 3 (25g, 2.08mm) was weighed into a clean, oven dried, two necked round neck flask and dissolved in toluene (300mL). East

<td>polymer</td><td>I know</td><td>prepared from the</td><td>same way as in</td><td>the</td>

<td>Example</td><td> 1 .</td><td>NCA Glu (OBn)</td><td>(1.92 g, 7.3 mmol)</td><td>of 1</td>

<td>Example</td><td>8 and</td><td>NCA d-Glu (OBn)</td><td>(1.92 g, 7.3 mmol)</td><td>of the</td>

<td>Example</td><td> 9,</td><td>were added to</td><td>flask, and mixing</td><td>of</td>

reaction allowed to stir for 18 hours at temperature

261

IMPI

MEXICAN INSTITUTE ^;and Axle 7 and NCA

<img file="MX340992B_D0478.tif" />

,. - MEXICAN INSTITUTE environment under nitrogen gas. Later^to&amp; &lt;gg ^ cjMEpr

NCA d-Phe (5.97 g, 31.25 mmol)

Tyr (OBn) (15.49 g, 52.08 mmol) prepared in the same manner as in Example 6, and the solution was allowed to stir at room temperature for 2 hours and then heated at 35 ° C for 48 hours at which point the reaction was complete (GPC, DMF / 0.1% LiBr). The solution was cooled to room temperature and acetic anhydride (2.04 g, mL), N-methylmorpholine (NMM) (2.23 g,

0 mmol, 1.88

2 mmol, 2.47 mL) and dimethylaminopyridine (DMAP) (.24 g, 2.0 mmol).

Stirring was continued for ambient. The polymer was dietetic 1 ico: heptane 10: 1 (2.5 days a precipitated

L) The ether ether was isolated by filtration, washed with 100 mL portions of fresh diethyl ether, and dried in vacuo to give the block copolymer as a fine, almost colorless powder (37.0 g, Yield = 80.6% ). NMR^XH (d6-DMSO) δ 9.08 8.42-7.70, 7.29, 6.96, 6.58, 5.10-4.85, 4.65-4.20,

3.71-3.25, 2.94-2.59, 2.40-2.10, 1.97-1.50.

262

<img file="MX340992B_D0479.tif" />

Synthesis of mPEG12K-b-Poly- (d-Glu (OBn) 3.s-coGlu (OBn) 3.5) -b-Poly (Tyr (OH) 2.5-co-d-Pheis) -Ac. The polymer of Example 87 was deprotected using the general method of Example 74 only by stoichiometric adjustment (32 g, 1.61 mmol). Once complete (3 Hrs.) The solution was subjected to a rotary evaporator until a thick paste was obtained and then it was dissolved again in DCM and precipitated in cold diethyl ether, collected by filtration and dried in vacuo. This reaction provided 23 g of dry material (80%). NMR³H (d6DMSO) δ 9.09, 8.50-7.75, 7.35-6.45, 5.04, 4.70-4.20, 3.913.05, 3.03-2.10, 2.09-1.50.

263

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX340992B_D0480.tif" />

Example 89

<img file="MX340992B_D0481.tif" />

\

Oh

one. THF, Hydraxitamin, TBD, TA24hr

2. Acetone, HOAc, _r TA, 5 hr

<img file="MX340992B_D0482.tif" />

^ -NHOH

<img file="MX340992B_D0483.tif" />

<img file="MX340992B_D0484.tif" />

<img file="MX340992B_D0485.tif" />

Oh

Synthesis of mPEG12K-b-Poly- (d-Glu (NHOH) 3.5-coGlu (NHOH) 3.5) -Jb-Poly (Tyr (OH) 25-co-d-Pheis) -Ac. The polymer (20g, 1mmol) from Example 88 was completely dissolved in 160mL THF with heating, this solution was allowed to cool to room temperature before adding 1,5,7-triazabicyclo [4.4.0] dec-5-ene (TBD, .5g, 3.6mmol), followed by hydroxylamine (50% water solution, 30mL, 545mmol) this solution was stirred at room temperature for 24 hours. Methanol (80 mL) was added and then precipitated with methyl tert-butyl ether, collected by filtration, and dissolved

254

<img file="MX340992B_D0486.tif" />

acetone and stirred overnight. The solution was subjected to a rotary evaporator until almost dry, was dissolved again in methylene chloride and precipitated in MTBE, collected by filtration and dried in vacuo (18.1 g, Yield = 92.9%). NMR¹H (de-DMSO) δ 9.11, 8.34-7.75, 7.15, 6.80, 4.60-4.32,

3.81-3.12, 2.99-2.32, 1.93-1.83.

Example 90

one. Tote no, 80 ° C Azeotrope 3Hrs.

2. HUP, via cannula

3. Glu (oBn) NCA, d-Glu (oBn) NCA 18hre „TA

Four. d ~ Phe NCA. Tyi (oBn) NCA, 48hr., .TA -&gt; 35 ° C

5. NMM, DMAP, AcANH ON, TA.

’&gt;

<img file="MX340992B_D0487.tif" />

OBn

Synthesis of mPEG12K-b-Poly- (d-Glu (OBn) s-coGlu (OBn) 5) -b-Poly (Tyr (OBn) 25-co-d-Pheis) -Ac.

mPEG12KNH2 prepared by the same method as in Example 3, was weighed (25g, 2.08mm) into a round bottom flask of

265 two 1000 mL necks dried in a clean oven

<img file="MX340992B_D0488.tif" />

OF INDUSTRIAL PROPERTY

<img file="MX340992B_D0489.tif" />

in toluene (300mL). This polymer was prepared in the same manner as in Example 73. NCA Glu (OBn) (2.74 g, 10.4 mmol) prepared in the same manner as in Example 8, and NCA d-Glu (OBn) (2.74) were then added. g, 10.4 mmol) prepared in the same manner as in Example 9, into the flask, and the reaction mixture was allowed to stir for 18 hours at room temperature under nitrogen gas. Then, NCA dPhe (5.97 g, 31.25 mmol) from Example 7 and NCA Tyr (OBn) (15.49 g, 52.08 mmol) prepared from the method in Example 6 were added and the solution was allowed to stir at room temperature for 2 hours and it was then heated at 35 ° C for 48 hours at which point the reaction was complete (GPC, DMF / 0.1% LiBr). The solution was cooled to room temperature and acetic anhydride (2.04 g, 20 mmol, 1.88 mL), Nmethylmorpholine (NMM) (2.23 g, 22 mmol, 2.47 mL) and dimethylaminopyridine (DMAP) (.24 g, 2.0 mmol) were added. . Stirring was continued for 1 day at room temperature. The polymer was precipitated from 10: 1 diethyl ether: heptane (2.5 L) and isolated by filtration, washed with 100 mL portions of fresh diethyl ether, and dried in vacuo to give the block copolymer as an almost colorless powder , fine (38.48 g, Yield = 81.4%). NMR¹H (de-DMSO) δ 9.08 8.42-7.70,

7.29, 6.97, 5.11-4.84, 4.65-4.20, 3.72-3.25, 3.05-2.45, 2.441.59.

266

<img file="MX340992B_D0490.tif" />

27&lt;5

<img file="MX340992B_D0491.tif" />

Example 91

<img file="MX340992B_D0492.tif" />

CO₂Bn

<img file="MX340992B_D0493.tif" />

ÓBn

1.PMB, TFA, TA, 3 Hre *

<img file="MX340992B_D0494.tif" />

Oh

IMPI

INSTITUTO MEXÍC4NO M LA EROME DAD INDUSTRIAL

<img file="MX340992B_D0495.tif" />

Synthesis of mPEG12K-b-Poly- (d-Glu (OBn) s-coGlu (OBn) 5) -b-Poly (Tyr (OH) 25-CO-d-Pheis) -Ac Using the general method of Example 74 only by stoichiometry adjustment this polymer was deprotected (32 g, 1.56 mmol). Once complete (3 Hrs.) The solution was subjected to a rotary evaporator until a thick paste was obtained and then it was dissolved again in DCM and precipitated in cold diethyl ether, collected by filtration and dried in vacuo. This reaction provided 27g of dry material (93.6%). NMR ³Η (d6-DMSO) δ

9.09, 8.50-7.75, 7.35-6.45, 5.04, 4.70-4.20, 3.91-3.05, 3.03267

<img file="MX340992B_D0496.tif" />

Example 92

2.10, 2.09-1.50.

<img file="MX340992B_D0497.tif" />

\

Oh

Synthesis of mPEG12K-b-Poli- (d-Glu (NHOH) ₃.s-coGlu (NHOH) 3.5) -b-Poly (Tyr (OH) 25-co-d-Pheis) -Ac The polymer (18 g, .88 mmol) from Example 91 was completely dissolved in 160 mL of THF with Heating, this solution was allowed to cool to room temperature before 1,5,7triazabicyclo [4.4.0] dec-5-ene (TBD, 0.5 g, 3.6 mmol) was added, followed by hydroxylamine (50% water solution, 30mL, 545 mmol) this solution was stirred at room temperature for 24 hours. Methanol (80 mL) was added and then precipitated with ether of

268

IMPI

MEXICAN INSTITUTE methyl tert-butyl was collected by filtration and in acetone. Acetic acid was added to this acetone solution and stirred for 5 hours and then developed, the solution was subjected to a rotary evaporator until almost dry, dissolved again in methylene chloride and MTBE precipitated, collected by filtration and dried in vacuo (16.7

Yield = 96.3%).

<img file="MX340992B_D0498.tif" />

of

I know in

g.

Example 93 nh₂

one. Toluene, 60 ° C Azeotrope 3Hrs.

2. NMP, via cannula

3. Glu (oBn) NCA, d-Glu (oBn) NCA 18hrs., TA

Four. d-Phe ANCA, Tyr (oBn) NCA, 48hrs., TA -&gt; 35 ° C

5. NMM, DMAP, AcANH V Ο.Ν ,, ΤΑ.

<img file="MX340992B_D0499.tif" />

Synthesis of mPEG12K-b-Poli- (d-Glu (OBn)? .S-coGlu (OBn) 7.5) -b-Poly (Tyr (OBn) 25-co-d-Pheis) -Ac mPEG12KNH₂ (25 g,

269 iMPlgM

<img file="MX340992B_D0500.tif" />

2.08 mm) prepared in the same manner as in e was weighed into a clean, oven dried, two necked, 1000 mL round bottom flask and dissolved in toluene (300mL). This polymer was prepared in the same way as in the

Example 1. Glu (OBn) NCA (2.74 g, 10.4 mmol) prepared in the same way as in Example 8 and d-Glu (OBn) NCA (2.74 g, 10.4 mmol) from Example 9, were added to the flask, and The reaction mixture was allowed to stir for 18 hours at room temperature under nitrogen gas. Then d-Phe NCA (5.97 g, 31.25 mmol) from Example 7 and Tyr (OBn) NCA (15.49 g, 52.08 mmol) from Example 6 were added and the solution was allowed to stir at room temperature for 2 hours and then it was Heated at 35 ° C for 48 hours at which point the reaction was complete (GPC, DMF / 0.1% LiBr). The solution was cooled to room temperature and acetic anhydride (2.04 g, 20 mmol, 1.88 mL), N-methylmorpholine (NMM) (2.23 g, 22 mmol, 2.47 mL) and dimethylaminopyridine (DMAP) (.24 g, 2.0) were added. mmol). Stirring was continued overnight at room temperature. The polymer was precipitated from 10: 1 diethyl ether: heptane (2.5 L) and isolated by filtration, washed with 100 mL portions of fresh diethyl ether, and dried in vacuo to give the block copolymer as an almost colorless powder (37.0 g,

Yield = 74.66%). NMR Ή (d₆-DMSO) δ 9.10 8.42-7.71,

7.27, 6.97, 5.11-4.85, 4.65-4.20, 3.72-3.25, 3.05-2.45, 2.451.60.

270

<img file="MX340992B_D0501.tif" />

<img file="MX340992B_D0502.tif" />

Oh

Synthesis of mPEG12K-b-Poly- (d-Glu (OBn) 7.5-coGlu (OBn) 7.5) -b-Poly (Tyr (OH) 25-co-d-Pheis) -Ac Using the general method of Example 74 only by stoichiometry adjustment this polymer was deprotected (32 g, 1.48 mmol). Once complete (3 Hrs.) The solution was subjected to a rotary evaporator until a thick paste was obtained and then it was dissolved again in DCM and precipitated in cold diethyl ether, collected by filtration and dried in vacuo. This reaction provided 24g of dry material (82.8%). NMR^XH (d6-DMSO) δ 9.09, 8.50-7.75, 7.35-6.45, 5.04, 4.70-4.20, 3.91-3.05, 3,032.10, 2.09-1.50.

271

<img file="MX340992B_D0503.tif" />

Example 95

Mei

Ίο '

Μι

COZBn

<img file="MX340992B_D0504.tif" />

COjBn

one. THF, Hidrnxilainina, TBD.TA 24 hr

2. Acetone, HOAc, TA, 5 hr / 9

15' \ 2»

<img file="MX340992B_D0505.tif" />

Oh

I

ΙΟ.

<img file="MX340992B_D0506.tif" />

X

Η0ΗΝΌ

Synthesis of mPEG12K-b-Poly- (d-Glu (NHOH) 3.s-coGlu (NHOH) 3.5) -b-Poly (Tyr (OH) 25-CO-d-Pheis) -Ac. The polymer (19 g, .88 mmol) prepared in Example 94 was completely dissolved in 160 ml of THF with heating, this solution was allowed to cool to room temperature before 1,5,7-triazabicyclo [4.4.0] dec- was added. 5-ene (TBD, .5 g, 3.6 mmol), followed by hydroxylamine (50% water solution, 30mL, 545 mmol) this solution was stirred at room temperature for 24 hours. Methanol (80 mL) was added and then precipitated with methyl tert-butyl ether, collected by filtration, and

272 dissolved in acetone. Acid added

INDUSTRIAL

IMPI

ÍNsrjwyo mexicanos - I- -Wg-M PROWEDAI?

INDUSTRIAL acetone solution and stirred for 5 hours. The solution is

<img file="MX340992B_D0507.tif" />

Rotovaporated until almost dry, dissolved again in methylene chloride and precipitated in MTBE, collected by filtration and dried in vacuo (16.4 g, Yield = 91.1%). NMR³H (d₆-DMSO) δ 9.11, 8.34-7.75, 7.15,

6.80, 4.60-4.32, 3.81-3.12, 2.99-2.32, 1.93-1.83.

Example 96

one. Toluene, 60 ° C Azeotrope 3 Hrs.

2. NMP, via cannula

3. Glu (oBn) NCA, d-Glu (oBn) NCA 18 hrs. TA

Four. d-Phe NCA. Tyr (oBn) NCA, hrs., TA -&gt; 35 ° C

5. NMM, DMAP, AcANH V ON, TA

<img file="MX340992B_D0508.tif" />

OBn

Synthesis of mPEG12K-b-Poli- (d-Glu (OBn) io-coGlu (OBn) _i0) -b-Poly (Tyr (OBn) 25-co-d-Pheis) -Ac mPEG12KNH₂ (25g, 2.08mm) prepared in the same manner as in Example 3,

273 weighed in a round bottom flask with two cuíéí'J ^^ l ^ 'í

INDUSTRIAL

IMPI «rasfigaas

INDUSTRIAL oven-dried, clean and dissolved in toluene (300mL)

<img file="MX340992B_D0509.tif" />

This polymer was prepared in the same manner as in Example 1. Glu (OBn) NCA (5.48g, 20.8mmol) prepared in the same manner as in Example 8, and d-Glu (OBn) NCA (5.48g, 2 0.8 mmol) prepared by the method in Example 9, were added to the flask directly, and the reaction mixture was allowed to stir for 16 hours at room temperature under nitrogen gas. Then, d-Phe NCA (5.97 g, 31.25 mmol) from Example 7 and Tyr (OBn) NCA (15.49 g, 52.08 mmol) from Example 6 were added and the solution was allowed to stir at room temperature for 2 hours and then it was heated at 35 ° C for 48 hours at which point the reaction was complete (GPC, DMF / 0.1% LiBr). The solution was cooled to room temperature and acetic anhydride (2.04 g, 20 mmol, 1.88 mL), N-methylmorpholine (NMM) (2.23 g, 22 mmol, 2.47 mL) and dimethylaminopyridine (DMAP) (.24 g, 2.0 mmol) were added. . Stirring was continued for 1 day at room temperature. The polymer was precipitated from 10: 1 diethyl ether: heptane (2.5 L) and isolated by filtration, washed with 100 mL portions of fresh diethyl ether, and dried in vacuo to give the block copolymer as a powder almost colorless, fine (38.9 g, Yield = 75.23%). NMR^XH (d₆-DMSO) δ 9.08, 8.40-7.65,

7.35-7.25, 6.99, 6.76, 5.10-4.85, 4.65-4.20, 3.72-3.25, 3.062.45, 2.34-1.59.

274

<img file="MX340992B_D0510.tif" />

Synthesis of mPEG12K-b-Poli- (d-Glu (OBn) io-coGlu (OBn) io) -b-Poly (Tyr (OH) 25-co-d-Pheis) -Ac Using the general method of Example 74 only by stoichiometry adjustment this polymer was deprotected (32 g, 1.41 mmol). Once complete (3 Hrs.) The solution was subjected to a rotary evaporator until a thick paste was obtained and then it was dissolved again in DCM and precipitated in cold diethyl ether, collected by filtration and dried in vacuo. This reaction provided 27g of dry material (92.8%). NMR ³H (d6-DMSO) δ 9.09, 8.50-7.75, 7.35-6.45, 5.04, 4.70-4.20, 3.91-3.05, 3,032.10, 2.09-1.50.

275

Example 98

IMPI

MEXICAN INSTITUTE OF PROPERTY

INDUSTRIAL

I\

CO₂Bn / ....... \ ve

MeV ^ '

<img file="MX340992B_D0511.tif" />

270

one. THF, Hydroxylamine, TDB, TA, 24 Hrs.

2. Acetone, HOAc, TA 5 Hrs

V-nhoh

270

Xf (

<img file="MX340992B_D0512.tif" />

Ίκ A.,

You &amp; no Ά _w &lt;- io / · wj

HCHNT)

Synthesis of mPEG12K-b-Poly- (d-Glu (NHOH) io-coGlu (NHOH) io) -b-Poly (Tyr (OH) 25-co-d-Pheis) -Ac The polymer (20 g, 0.88 mmol ) of Example 98 was completely dissolved in 160mL of THF with heating, this solution was allowed to cool to room temperature before 1,5,7triazabicyclo [4.4.0] dec-5-ene (TBD, .5 g, 3.6) was added mmol), followed by hydroxylamine (50% water solution, 30mL, 545 mmol), this solution was stirred at room temperature for 24 hours. Methanol (80 mL) was added and then precipitated with methyl tert-butyl ether, collected by filtration, and dissolved

276

<img file="MX340992B_D0513.tif" />

in acetone. Acetic acid was added to it.

TlTuTiTWtdcXNó

OF THE PROPERTY

INDUSTRIAL _ acetone and stirred for 5 hours and then developed. The solution was rotary evaporated until almost dry, redissolved in methylene chloride and precipitated in MTBE, collected by filtration and dried in vacuo (17.2 g, Yield = 92.1%). NMR^XH (d_s-DMSO) δ 9.1 1, 8.33-7.69,

7.15, 6.98, 6.79, 5.06-4.85, 4.60-4.32, 3.81-3.19, 2.99-2.32,

2.03-1.59.

Example 99 x'7G

one. Toluene Azeotrope,

60'C, 3 Hrs

2. Glu (oBn) NCA, d-Glu (oBn) NCA TA, 15 Hrs, NMP

3. d-Phe NCA, Tyr (oBn) NCA TA -&gt; 35 ° C 48 Hrs.

Four. NMM, AcAnh, DMAP TA, 16 Hrs

V

<img file="MX340992B_D0514.tif" />

OBn

Synthesis of mPEG12K-b-Poli- (d-Glu (OBn) 3.s-coGlu (OBn) 3.5) -b-Poli (Tyr (OBn) zs-co-d-Pheis) -Ac mPEG12KNH₂ (45.34 g, 3.78 mmol) prepared by the same method detailed in

277 —Ua-a ^ r ...

Example 3 Weighed in a Bottom Flask

<img file="MX340992B_D0515.tif" />

OF INDUSTRIAL PROPERTY

<img file="MX340992B_D0516.tif" />

1000 mL necks, oven dried, clean and dissolved in toluene (300mL). This polymer was prepared in the same manner as in Example 73. Glu (OBn) NCA (3.5 g, 13.29 mmol) from the method detailed in Example 8, and d-Glu (OBn) NCA (3.5 g, 13.29 mmol) ) From the method detailed in Example 9, they were added to the flask, and the reaction mixture was allowed to stir for 16 hours at room temperature under nitrogen gas. Then, d-Phe NCA (10.89 g, 56.9 mmol) from the method detailed in Example 7, and Tyr (OBn) NCA (28.24 g, 94.98 mmol) from the method detailed in Example 6, were added and the The solution was allowed to stir at 35 ° C for 4-8 hours at which point the reaction was complete (GPC, DMF / 0.1% LiBr). The solution was cooled to room temperature and acetic anhydride (3.88 g, 37.8 mmol, 3.58 mL), Nmethylmorpholine (NMM) (3.76 g, 37.8 mmol, 4.16 mL) and dimethylaminopyridine (DMAP) (0.47 g, 3.8 mmol) were added. Stirring was continued for 1 day at room temperature. The polymer was precipitated from 10: 1 diethyl ether: heptane (2.5 L) and isolated by filtration, washed with 100 mL portions of fresh diethyl ether, and dried in vacuo to give the block copolymer as an almost colorless powder , fine (68.22 g, Yield = 82.2%). NMR^ΧΗ (de-DMSO) δ 8.43-7.84, 7.30,

6.98, 6.97-6.65, 5.04, 4.98- 4.80, 4.66-4.16, 3.72-3.21,

3.01-2.76, 2.74-2.56, 2.41-2.26, 2.23-2.10, 2.01-1.58.

278

IMPI

Mexican Institute of Industrial Property

<img file="MX340992B_D0517.tif" />

Example 100

<img file="MX340992B_D0518.tif" />

Oh

Synthesis of mPEG12K-b-Poly- (d-Glu (OBn) 3.5-coGlu (OBn) 3.5) -b-Poly (Tyr (GHhs-co-d-Pheis) -Ac Using the general method of Example 74 only by adjustment stoichiometrically this polymer was deprotected (60 g, 2.73 mmol). Once complete (5 Hrs.) The solution was subjected to a rotary evaporator until obtaining a thick paste and then it was dissolved again in DCM and precipitated in cold diethyl ether, collected by filtration, washed several times with fresh 200 mL portions of ether diethyl cold and dried in vacuo. This reaction provided 43.8g of dry material (81.4%). NMR²Η (d6-DMSO) δ 9.04, 8.38-7.73, 7.38-6.73, 5.04, 4.62-4.19, 3.82-3.27,

3.02-2.76, 2.75-2.56, 2.42-2.226, 2.20-1.61, 1.08 (solvent, ether).

279

<img file="MX340992B_D0519.tif" />

Synthesis of mPEG12K-b-Poli- (d-Glu (NHOH) ₃.₅-coGlu (NHOH) 3.5) -b-Poly (Tyr (OH) 25-co-d-Pheis) -Ac The polymer (40 g, 1 mmol) from Example 100, was completely dissolved in 700 mL of THF with heating, This solution was allowed to cool to room temperature before triazabicyclo [4.4.0] dec-5-ene followed by hydroxylamine (50% mmol) this solution was stirred for hours. Isopropanol was added with methyl tert-butyl ether, to which was added 1,5,7 (TBD, 1.5 g, 10.8 mmol), water solution, 45 mL, 817.5 at room temperature per 24 (200 mL) and then precipitated, collected by filtration, and

280 it was dissolved in acetone (500 mL). Added

INDUSTRIAL

IΜ Ρ1

INDUSTRIAL ^ β_Σ! Ξ—, ν drowned out overnight.

mL) to this acetone solution and 9

The solution was rotary evaporated until almost dry, redissolved in methylene chloride and precipitated in MTBE, collected by filtration and dried in vacuo (33.5 g, Yield = 86%). NMR^XH (d₆-DMSO) δ 9.03, 8.37-7.70, 7.366.72, 6.68-6.42, 4.64-4.14, 3.73-3.10, 3.00-2.76, 2.71-2.56,

2.42-2.27, 2.21-1.61.

Example 101A

<img file="MX340992B_D0520.tif" />

one. Toluene Azeotrope,

60 ° C 3 Hrs

2. Glu (oBn) NCA, d-Glu (oBn) NCA TA, 16 Hrs, NMP

3. d-Phe NCA, Tyr (oBn) NCA TA, -&gt; 35 ° C48Hrs

Four. NMM, AcAnh, DMAP TA, 16 Hrs

COjBn /

<img file="MX340992B_D0521.tif" />

OBn

Synthesis

Glu (0Bn) 3.s) -b-Pol from mPEGll.5K-b-Poli- (d-Glu (OBn) 3.5-co (Tyr (OBn) 25-co-d-Pheis) -Ac mPEGll.5KNH₂ (15

281

IMPI

<img file="MX340992B_D0522.tif" />

v &lt;'- v_v·

INSTITUTO MEXICANu g, 1.3 mmol) prepared by the same method as éñ * ú ^ ro | W&gt; e with the exception of molecular weight, weighed in a 1000 mL two-necked round bottom flask, oven dried, clean and dissolved in toluene (300mL). This polymer was prepared in the same manner as in Example 73. Glu (OBn) NCA (1.2g, 4.56mmol) prepared by the same method as in Example 8, and d-Glu (OBn) NCA (1.2g, 4.56mmol) prepared by the same method as in Example 9, were They added to the flask, and the reaction mixture was allowed to stir for 16 hours at room temperature under nitrogen gas. Then, d-Phe NCA (2.88 g, 19.56 mmol) prepared with the same method as in Example 7, and Tyr (OBn) NCA (8.26 g, 32.60 mmol) prepared with the same method as in Example 6, were added and the solution directly, allowed to stir at room temperature for 2 hours and then heated at 35 ° C for 48 hours at which point the reaction was complete (GPC, DMF / 0.1% LiBr). The solution was cooled to room temperature and acetic anhydride (1.34 g, 13 mmol, 1.23 mL), Nmethylmorpholine (NMM) (1.3 g, 13 mmol, 1.43 mL) and dimethylaminopyridine (DMAP) (0.16 g, 1.3 mmol) were added. Stirring was continued for 16 hours at room temperature. The polymer was precipitated from 10: 1 diethyl ether: heptane (2.5 L) and isolated by filtration, washed with 100 mL portions of fresh diethyl ether, and dried in vacuo to give the block copolymer as an almost colorless powder , fine (26 g,

282

Yield = 82.2%). NMR^XH (d6-DMSO) δ ιν &amp; ι: γΑΌ «7ιο &amp; 3,

FROM THE P «r'rt £ nA £&gt; INIKJSTMM

IMPI ινΘίτ &amp; Θ wEÜicxftS DE LA P «r'rt £ nA £&gt;

INIKJSTMM

7.16-6.98, 6.83-6.64, 5.06-4.79, 4.62-4.18, 3.71-3.21, 2.98-

<img file="MX340992B_D0523.tif" />

2.78, 2.75-2.58, 2.42-2.25, 2.22-2.13, 1.99-1.70.

Example 102

J &amp; 2 'NH?

one. Toluene Azeotrope,

60 ° C 3 Hrs

2. Glu (oBn) NCA, d-Glu (oBn) NCA TA, 16 Hrs, 1: 1 NMP: DCM

3. d-Phe NCA, Tyr (oBn) NCA TA. -&gt; 35 ° C 48 Hrs

Four. NMM, AcAnh, DMAP TA, 16 Hrs.

CO? Bn

<img file="MX340992B_D0524.tif" />

OBn