Hrmiasterlin derivatives and uses thereof

Description of the invention

The invention relates to hemiasterline derivatives, a pharmaceutical composition containing such a derivative as an active ingredient and the use of hemiasterline derivatives for the preparation of a medicament.

Hemiasterlin (1) was first isolated from a Hemiasterella minor sponge (Demospongiae class; Hadromedidia order; Hemiasterllidae family) harvested at Sodwana Bay, South Africa (see Kashman et al., US 5,661,175). Hemiasterline has been reported to have antitumor activity against a variety of cell lines including human lung cancer, human colon cancer, and human melanoma.

<img file="PL219737B1_D0001.tif" />

After the initial isolation and characterization of this compound, additional hemiasterlins were isolated and several hemiasterlin derivatives were synthesized and their biological activity was also tested. Recently, hemiasterline and some of its analogs have been reported to have antimitotic activity and are therefore useful in the treatment of certain cancers (see US No. 6,153,590 and PCT application WO 99/32509). However, only a limited number of hemiasterline analogs were obtained, half of which were natural products alone, isolated from Cymbastela sp. Or compounds obtained by modification of natural products. Accordingly, the number and types of derivatives that could be obtained and assessed for biological activity was limited.

Clearly there remains a need to develop synthetic methods making it possible to obtain and test the therapeutic effect of various new hemiasterline derivatives, especially those that cannot be obtained by modifying the natural product. It would also be of particular interest to develop new compounds with an advantageous therapeutic profile in vivo (i.e., safe and effective, while maintaining stability in biological media).

As stated above, there remains a need to develop new hemiasterline analogues to evaluate their action as therapeutic agents for the treatment of cancer.

Thus, the invention relates to hemiasterline derivatives having the formula:

<img file="PL219737B1_D0002.tif" />

and pharmaceutically acceptable salts, esters or ester salts thereof; where g is 0, 1, 2 or 3;

Χ2 is C (= O);

R2 is hydrogen, an unsubstituted aliphatic, or an unsubstituted or substituted heteroaliphatic group;

each of R5 and R9a is hydrogen and each of R6, R7, R8a and R10a is independently aliphatic, the aliphatic group may be substituted or unsubstituted, linear or branched, cyclic or acyclic;

each of RL1 and RL2 is independently hydrogen, and

PL 219 737 B1

Q is OR^Q'or NO^q'R^qwhere each of the R.^Q'and r^Q is independently hydrogen, or an aliphatic or heteroaliphatic group, or R^Q'and r^Q together with the nitrogen atom to which they are attached may form a heteroalicyclic group;

wherein the aliphatic groups contain 1-10 carbon atoms, i.e. they are C groups_1-10 aliphatic, heteroaliphatic groups contain 1-10 carbon atoms, i.e. they are C groups_1-10 heteroaliphatic and heteroalicyclic groups contain 3 - 7 carbon atoms, ie they are C3-7 heteroalicyclic groups, and wherein the heteroaliphatic and heteroalicyclic groups contain 1 - 3 heteroatoms selected from oxygen, sulfur and nitrogen atoms.

Compounds having the following stereochemistry are preferred:

<img file="PL219737B1_D0003.tif" />

and pharmaceutically acceptable salts, esters or ester salts thereof.

Furthermore, compounds of the above formula are preferred, wherein each of R6, R7, R8a and R10a is independently C1-6 alkyl, wherein the C1-6 alkyl may be substituted or unsubstituted, linear or branched, cyclic or acyclic.

Furthermore, compounds of the above formula are preferred, wherein R2 is hydrogen or unsubstituted, linear or branched, cyclic or acyclic C1-6 alkyl.

In addition, compounds of the above formula are preferred, in which R2 is hydrogen, methyl, ethyl, propyl, butyl, pentyl, tert-butyl, isopropyl, -CH (CH3) CH2CH3, -CH (CH3) CH2CH2CH3, -CH2CH (CH3 ) 2, -CH (CH3) CH (CH3) 2, -C (CH3) 2CH2CH3, -CH (CH3) cyclobutyl, -CH (Et) 2, -CH (CHhC = CH, cyclohexyl, cyclopentyl, cyclobutyl or cyclopropyl.

Among the above-mentioned compounds, those in which Q is selected from the group consisting of

<img file="PL219737B1_D0004.tif" />

with each occurrence r is 0, 1 or 2; whereby in each case of the occurrence of R.^Q1 and r^Q2 are independently hydrogen, or substituted or unsubstituted

A linear or branched cyclic or acyclic alkyl or heteroalkyl group; and pharmaceutically acceptable salts, esters or ester salts thereof.

Of the above-mentioned compounds, those in which Q is preferred are preferable

<img file="PL219737B1_D0005.tif" />

and pharmaceutically acceptable salts, esters or ester salts thereof.

Compounds represented by formulas selected from among are particularly preferred

<img file="PL219737B1_D0006.tif" />

PL 219 737 B1

<img file="PL219737B1_D0007.tif" />

and pharmaceutically acceptable salts, esters or ester salts thereof. The compound represented by the formula below is especially preferred

<img file="PL219737B1_D0008.tif" />

In addition, compounds wherein Q is an optionally substituted cyclic nitrogen-containing cyclic group are preferred; these compounds having the following formula:

<img file="PL219737B1_D0009.tif" />

wherein each occurrence of A, B, D or E is independently CHR^and, each occurrence being hydrogen or -C (= O) Rⁱⁱⁱ, where in each case of occurrence of R.ⁱⁱⁱ is an aliphatic or heteroaliphatic group;

N and A, A and B, B and D, D and E, and E and N are independently linked by a single or double bond, as valence permits; aa, b, d and e are independently 0, 1, 2, 3, 4, 5, 6 or 7, with the sum of a, b, d and e being 4-7.

Among the above compounds, compounds having the following stereochemistry are preferable:

<img file="PL219737B1_D0010.tif" />

and pharmaceutically acceptable salts, esters or ester salts thereof.

Among the above compounds, those in which R2 is hydrogen or C1-6 alkyl are preferable.

Also preferred among the above compounds are those in which each of R6, R7, R8a and R10a is independently C1-6 alkyl, wherein the C1-6 alkyl may be substituted or unsubstituted, linear or branched, cyclic or acyclic, and saturated or unsaturated .

In addition, compounds of the above formulas in which R6 is t-butyl, R7 and R10a are each methyl, and R8a are isopropyl are each preferred.

PL 219 737 B1

Among the above compounds, also preferred are those in which each of a, b, d and e is 1; each of B and D is CH2; and each of A and E is independently CH2, CH (C = O) R^and, CH (C = O) OR^and or CH (C = O) NO^andRⁱⁱ; whereby in each case of the occurrence of R.^and and rⁱⁱ are independently hydrogen, C1-6 alkyl, or C1-6 heteroalkyl; wherein the C1-6 alkyl and C1-6 heteroalkyl may be substituted or unsubstituted, linear or branched, cyclic or acyclic, and saturated or unsaturated.

Moreover, compounds of the above-mentioned formula in which R2 is methyl, R6 is t-butyl, R are preferred₇ is methyl, R._8a is isopropyl and Q is ORQ 'or NRQ'RQ ", wherein R^q'and r^Q independently represent hydrogen, C._1-6 alkyl or heteroalkyl, or R^Q'and r^Q, together with the nitrogen atom to which they are attached form a heterocyclic group.

In addition, compounds represented by formulas selected from are preferred

<img file="PL219737B1_D0011.tif" />

where Q is OH or OEt;

<img file="PL219737B1_D0012.tif" />

Among the above compounds, those represented by the formula are preferable

PL 219 737 B1

<img file="PL219737B1_D0013.tif" />

where Q is OH or OEt; and pharmaceutically acceptable salts, esters or ester salts thereof.

Among the above compounds, those represented by the formula are preferable

<img file="PL219737B1_D0014.tif" />

wherein Q is OH or OEt.

Among the above compounds, the compound represented by the formula is more preferable

<img file="PL219737B1_D0015.tif" />

and a pharmaceutically acceptable salt, ester or ester salt thereof.

Among the above compounds, the compound represented by the formula is most preferred

<img file="PL219737B1_D0016.tif" />

Further, the invention relates to a pharmaceutical composition comprising an active ingredient, a pharmaceutically acceptable carrier or diluent, the feature of which is that it comprises the above-defined hemiasterline derivative as active ingredient, optionally in the form of a pharmaceutically acceptable salt, ester or ester salt thereof, and optionally further comprises an additional therapeutic agent. .

Preferably, the pharmaceutical composition comprises a compound of formula as active ingredient

<img file="PL219737B1_D0017.tif" />

or a pharmaceutically acceptable salt, ester or ester salt thereof.

PL 219 737 B1

Preferably, the pharmaceutical composition comprises an amount of the compound effective to inhibit the growth of tumor cells in vitro.

Preferably, the pharmaceutical composition comprises the compound in an amount effective for regression of the tumor in vivo.

In addition, the invention relates to the above-defined hemiasterline derivatives, optionally in the form of their pharmaceutically acceptable salts, esters or ester salts, and a pharmaceutically acceptable carrier or diluent and optionally an additional therapeutic agent for use in the treatment of cancer.

Preferably, the compounds are for use in the treatment of cancer of the prostate, breast, colon, bladder, cervix, skin, testes, kidney, ovary, stomach, brain, liver, pancreas or esophagus, or lymphoma, leukemia, or multiple myeloma.

Preferably the compounds are for use in the treatment of cancer which is a solid tumor.

Preferably the compounds are for use in the treatment of a tumor which is a tumor other than a solid tumor.

Furthermore, the invention relates to the use of the above-defined hemiasterline derivatives, optionally in the form of their pharmaceutically acceptable salts, esters or ester salts, for the manufacture of a medicament for the treatment of cancer.

The use of hemiasterline derivatives for the manufacture of a medicament for the treatment of cancer of the prostate, breast, colon, bladder, cervix, skin, testes, kidney, ovary, stomach, brain, liver, pancreas or esophagus, or lymphoma, leukemia or multiple myeloma is preferred.

Furthermore, the use of hemiasterline derivatives for the preparation of a medicament for the treatment of a solid tumor neoplasm is preferred.

Furthermore, the use of hemiasterline derivatives for the preparation of a medicament for the treatment of a tumor which is a non-solid tumor is preferred.

The formulas below illustrate several exemplary types of compounds of the invention. Additional compounds are described in the examples section.

<img file="PL219737B1_D0018.tif" />

Other compounds of the invention will become apparent to the person who reads the description. A number of important subclasses of relationships require separate mention; these subclasses include the following subclasses of compounds in which:

i. R2 is hydrogen or unsubstituted, linear or branched, cyclic or acyclic, or saturated or unsaturated lower alkyl, or substituted or unsubstituted linear or branched, cyclic or acyclic, saturated or unsaturated heteroalkyl;

ii. R2 is unsubstituted, linear or branched, cyclic or acyclic, saturated or unsaturated lower alkyl, or unsubstituted or substituted, linear or branched, cyclic or acyclic, saturated or unsaturated heteroalkyl;

iii. R2 is unsubstituted, linear or branched, cyclic or acyclic, saturated or unsaturated lower alkyl;

iv. R2 is methyl, ethyl, propyl, butyl, pentyl, t-butyl, i-propyl, -CH (CH3) CH2CH3, -CH (CH3) CH2CH2CH3, -CH2CH (CH3) 2, -CH (CH3) CH (CH3) 2, -CH (CH3) 2CH2CH3, -CH (CH3) cyclobutyl,

-CH (Et)₂, -CH (CH₃)₂CCH, cyclohexyl, cyclopentyl, cyclobutyl or cyclopropyl;

PL 219 737 B1

v. R2 is hydrogen;

vi. R2 is hydrogen or methyl;

vii. R2 is methyl;

viii. R6 is optionally substituted, linear or branched, cyclic or acyclic, or saturated or unsaturated lower alkyl;

ix. R6 is methyl, ethyl, propyl, butyl, pentyl, t-butyl, i-propyl, -CH (CH3) CH2CH3, CH2CH (CH3) 2, cyclohexyl, cyclopentyl, cyclobutyl or cyclopropyl;

x. R6 is t-butyl;

xi. The carbon atom with R6 is in the S configuration;

xii. R7 is substituted or unsubstituted, linear or branched, cyclic or acyclic, or saturated or unsaturated lower alkyl;

xiii. R7 is methyl;

a) R8a is substituted or unsubstituted, linear or branched, cyclic or acyclic, or saturated or unsaturated lower alkyl;

b) R8a is isopropyl;

c) the carbon atom bearing R8a is in the S configuration;

d) R10a is hydrogen, or substituted or unsubstituted, linear or branched, cyclic or acyclic, or saturated or unsaturated lower alkyl; or

e) R10a is methyl;

xv. Q is OR^Q', NO^Q'R^Q"Or a moiety selected from the group consisting of:

<img file="PL219737B1_D0019.tif" />

xvi.

Q2 ^Q2 are independently hydrogen or a substituted or unsubstituted linear or branched cyclic or acyclic alkyl or heteroalkyl group; and R.^Q'and r^Q are independently hydrogen or a substituted or unsubstituted linear or branched cyclic or acyclic alkyl or heteroalkyl; or R.^Q'and r^Q, together with the nitrogen atom to which they are attached form a substituted or unsubstituted heterocyclic group; and / or Q is OR^Q'and NO^q'R^q or a moiety selected from the group consisting of:

PL 219 737 B1

<img file="PL219737B1_D0020.tif" />

wherein at each occurrence r is 0, 1 or 2; and R.^Q'and r^Q are independently hydrogen or a substituted or unsubstituted linear or branched cyclic or acyclic alkyl or heteroalkyl; or R.^Q'and r^Q, together with the nitrogen atom to which they are attached form a substituted or unsubstituted heterocyclic group.

A reader of the text will understand that relationships of particular interest include those that share the common characteristics of one or more of the above subclasses. Some of these subclasses are illustrative of the following types of relationships:

- compounds of the following formula (and their pharmaceutically acceptable derivatives):

<img file="PL219737B1_D0021.tif" />

wherein g, R9a, R10a, RL1 and RL2 are as defined above and in subclasses; R2 is unsubstituted linear or branched lower alkyl; R6 is optionally substituted linear or branched lower alkyl; and Q is OR^Q'or NO^q'R^q where r^Q'is hydrogen or lower alkyl, or R^Q'and r^Qtogether with the nitrogen atom to which they are attached form a substituted or unsubstituted heterocyclic group, each of the above alkyls being substituted or unsubstituted, linear or branched, cyclic or acyclic.

Further, it should be understood that within each of the subgroups of compounds described above, various other subclasses are of particular interest, including, but not limited to, classes i. To xvi. described above, and the classes, subclasses, and species of compounds described above and in the examples. In certain embodiments of compounds of the subgroups of compounds described above, R2 is methyl, isopropyl, s-butyl, or -CH (CH3) CH (CH3) 2. In certain embodiments of compounds of the subgroups of compounds described above, R6 is t-butyl or isopropyl. In certain embodiments of the compounds of the subgroups of compounds described above, R2 is methyl, isopropyl, s-butyl or -CH (CH3) -CH (CH3) 2 and R6 is t-butyl or isopropyl. In certain exemplary embodiments of the subgroups described above, R2 is methyl and R6 is t-butyl. In certain exemplary embodiments of compounds of the subgroups of compounds described above, R2 is isopropyl and R6 is t-butyl. In certain exemplary embodiments of compounds of the subgroups of compounds described above, R2 is s-butyl and R6 is t-butyl or isopropyl. In certain exemplary embodiments for compounds of the subgroups of compounds described above, R2 is -CH (CH3) CH (CH3) 2 and R6 is t-butyl.

Certain of the above compounds may contain one or more asymmetric centers and thus may exist in different isomeric forms, e.g., as stereoisomers and / or diastereoisomers. It should be understood that the invention includes every possible isomer, such as a geometric isomer, optical isomer, stereoisomer, and tautomer, based on an asymmetric carbon atom that may exist in the structures of the compounds of the invention, and mixtures of such isomers, and is not limited by any particular stereochemistry. shown for compounds disclosed herein. Furthermore, it should be appreciated that the absolute stereochemistry of some of the compounds mentioned in the Examples section has not been established and that when these compounds have been assigned stereochemistry, it should be taken into account that the stereochemistry is uncertain and indicates that the diastereomeric set is present for these compounds and / or that the diastereoisomer is isolated in pure form. Therefore, the compounds and their pharmaceutical agents may be in the form of a single enantiomer, diastereoisomer, or geometric isomer, or they may be in the form of a mixture of stereoisomers. In certain embodiments, the compounds of the invention are enantiomerically pure compounds. In certain other embodiments, there are mixtures of stereoisomers or diastereoisomers.

In addition, certain compounds described may contain one or more double bonds, which may exist as the Z or E isomer, unless otherwise stated. Thus, the compounds of the invention may exist as individual isomers, substantially free of other isomers, and alternatively as mixtures of different isomers, e.g. racemic mixtures of stereoisomers. The specific compounds of the invention described above may also exist as tautomers. In addition to the above-mentioned compounds per se, the invention also includes pharmaceutically acceptable derivatives of such compounds, and compositions containing one or more compounds of the invention and one or more pharmaceutically acceptable excipients or additives.

The compounds of the invention may be obtained by crystallization under various conditions, and may exist as a single polymorph or combination of polymorphs of the compound. For example, different polymorphs may be identified and / or prepared using different solvents or different mixtures of recrystallization solvents; by carrying out crystallization at different temperatures; or by using various methods of cooling, ranging from very fast to very slow cooling during crystallization. Polymorphs can also be obtained by heating or melting a compound followed by gradual or rapid cooling. The presence of polymorphs can be detected by solid sample NMR spectroscopy, IR spectroscopy, differential scanning calorimetry, X-ray powder and / or other techniques. Thus, the invention includes the compounds of the invention, their derivatives, their tautomeric forms, their stereoisomers, their polymorphs, their pharmaceutically acceptable salts, their pharmaceutically acceptable solvates, and pharmaceutically acceptable agents containing them.

As outlined above, the invention provides novel compounds with a range of biological properties. The compounds of the invention exhibit biological activity suitable for the treatment of diseases or other disorders such as proliferative diseases, including, but not limited to, cancer. In addition, the compounds of the invention may also find use in the prevention of blood vessel restenosis following trauma, such as angioplasty, and in the insertion of stents.

The compounds of the invention include those compounds specifically set forth above and described and illustrated in part by the various classes, subgroups, and species disclosed elsewhere in this specification.

Additionally, the invention provides pharmaceutically acceptable derivatives of the compounds of the invention in the form of pharmaceutically acceptable salts, esters or salts of such esters, and the use of such derivatives of the compounds to treat a subject, their pharmaceutical compositions, or any of these compounds in combination with one or more additional therapeutic agents. . As used herein, the term "pharmaceutically acceptable derivatives" refers to any pharmaceutically acceptable salt, ester or salt of such ester, such compound as well as any other adduct or derivative which, when administered to a patient, is capable of providing (directly or indirectly) a compound or metabolite thereof as described herein, or the rest. Therefore, pharmaceutically

Acceptable derivatives include, but are not limited to, prodrugs. A prodrug is a derivative of a compound, typically with significantly reduced pharmacological activity, that contains an additional moiety that is prone to removal in vivo to provide the parent molecule as a pharmacologically active ingredient. An example of a prodrug is an ester that is cleaved in vivo to provide a compound of interest. Prodrugs of the various compounds, and materials and methods for derivatizing the parent compounds to produce prodrugs are known and can be used in the present invention. Certain exemplary pharmaceutical compositions and pharmaceutically acceptable derivatives will be discussed in more detail below.

Numerous suitable prodrug groups, and information regarding their selection, synthesis, and use, are well known. Examples of prodrug groups of interest include, but are not limited to, prodrug groups that can be attached to functional groups containing a primary or secondary amine group. Examples of such prodrug groups are as follows:

<img file="PL219737B1_D0022.tif" />

₁

R¹ = any natural or unnatural amino acid

Synthesis of prodrug groups - see Borchardt RT et al., J. Org. Chem. 1997, 43, 3641-3652

OO

<img file="PL219737B1_D0023.tif" />

R¹ = C1-C4 alkyl, cycloalkyl, oxyalkyl, aminoalkyl, etc. R2 = any natural or unnatural amino acid

For the synthesis of prodrug groups see Zhou XX et al., PCT WO99 / ​​51613

<img file="PL219737B1_D0024.tif" />

R¹, R² = any natural or unnatural amino acid

For the synthesis of prodrug groups see Ezra A. et al., J. Med. Chem. 2000, 43, 3641-3652

Other examples of prodrug groups of interest include groups that can be attached to hydroxyl-containing functional groups. Such prodrug groups are well known and readily identifiable by one of ordinary skill in the art. The invention encompasses any prodrug form of the described compounds.

Certain compounds of the invention and definitions of specific functional groups are also described in more detail below. For the purposes of the invention, the chemical elements are identified according to the Periodic Table of the Elements, CAS Revision, Handbook of Chemistry and Physics, 75th Edition, inside cover, and specific functional groups are generally defined and described herein. The general principles of organic chemistry, as well as specific functional groups and their reactivity, are further described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999. Furthermore, one skilled in the art should understand that the synthetic methods described herein use various protecting groups. As used herein, the term "protecting group" means that a particular functional group, e.g. O, S or N is temporarily blocked such that in the case of a polyfunctional compound the reaction can be carried out selectively at a different reaction center. In preferred embodiments, the protecting group reacts selectively in good yield to provide a protected substrate that is stable to the anticipated reactions; the protecting group must be selectively removable in good yield using readily available, preferably non-toxic reagents which do not attack other functional groups; the protecting group forms readily derivatizable derivatives (more preferably without the formation of new stereogenic centers); moreover, a group

The protective device exhibits minimal additional functionality to avoid introducing additional reaction sites. In particular, oxygen, sulfur, nitrogen and carbon protecting groups can be used. For example, in certain embodiments detailed herein, certain exemplary oxygen protecting groups are used. Such oxygen protecting groups include, but are not limited to, methyl ethers, substituted methyl ethers (e.g. MOM (methoxymethyl ether), MTM (methylthiomethyl ether), BOM (benzyloxymethyl ether), PMBM (p-methoxybenzyloxymethyl ether) to name a few), substituted ethyl ethers, substituted benzyl ethers, silyl ethers (e.g. trimethylsyl) TMS (ether) , TES (triethylsilyl ether), TIPS (triisopropylsilyl ether), TBDMS (t-butyldimethylsilyl ether), tribenzylsilyl ether, TBDPS (t-butyldiphenylsilyl ether) to name a few), esters (e.g. formate, acetate, benzoate (Bz), trifluoroacetate, dichloroacetate to name a few), carbonates, cyclic acetals and ketals. In certain other exemplary embodiments, nitrogen protecting groups are used. Such nitrogen protecting groups include, but are not limited to, carbamates (including methyl, ethyl and substituted ethyl carbamates (e.g., Troc) to name a few) amides, cyclic imide derivatives, N-alkyl and N-arylamines, imine derivatives and enamine derivatives. Certain other exemplary protecting groups are set out in the specification in detail, but it should be understood that the invention is not limited to these protecting groups; rather, a variety of additional equivalent protecting groups can be identified based on the above criteria, and used in the invention. In addition, a wide variety of protecting groups are described in "Protective Groups in Organic Synthesis" 3rd edition, Greene, TW and Wuts, PG, Ed., John Wiley &amp; Sons, New York: 1999.

It should be appreciated that the compounds described may be substituted with multiple substituents or functional moieties. In general, the term "substituted", whether or not preceded by the term "optionally", and substituents included in the formulas of the invention refer to the replacement of hydrogen atoms in a given structure with the radical of the appropriate substituent. When more than one position may be substituted in a given structure by more than one substituent selected from a particular group, the substituents at each position may be the same or different. As used herein, the term "substituted" includes all acceptable substituents on organic compounds. Broadly speaking, acceptable substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic organic substituents. For the purposes of the invention, heteroatoms such as nitrogen atoms may contain hydrogen substituents and / or any acceptable organic substituents mentioned herein which fill the valences of the heteroatoms. Furthermore, the invention is by no means limited by the permissible substituents of organic compounds. Combinations of substituents and variables envisaged by the invention are preferably such that they result in the formation of stable compounds useful e.g. in the treatment of cancer. As used herein, the term "stable" preferably refers to compounds which exhibit sufficient stability to make them, and which maintain the integrity of the compound for a period of time sufficient to be detected, and preferably for a period of time sufficient to be useful for the purposes specifically described.

As used herein, the term "aliphatic" includes both saturated and unsaturated, straight chain (or unbranched) or branched aliphatic hydrocarbons, optionally substituted with one or more functional groups. As one skilled in the art will appreciate, "aliphatic" is intended to include, but is not limited to, alkyl, alkenyl, and alkynyl. Accordingly, as used herein, the term "alkyl" includes straight and branched alkyl groups. An analogous concept applies to other generic terms such as "alkenyl", "alkynyl" and the like. In addition, as used herein, the terms "alkyl", "alkenyl", "alkynyl" and the like include both substituted and unsubstituted groups. In some embodiments, the term "lower alkyl" is used herein to denote those alkyl groups (cyclic, acyclic, substituted, unsubstituted, branched, or unbranched) having 1-6 carbon atoms.

In some embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-20 aliphatic carbon atoms. In certain other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-10 aliphatic carbon atoms. In still other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-8 aliphatic carbon atoms. In further other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-6 aliphatic carbon atoms. In still other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-4 carbon atoms. Therefore

Exemplary aliphatic groups include, but are not limited to, e.g., methyl, ethyl, n-propyl, isopropyl, allyl, n-butyl, s-butyl, isobutyl, t-butyl, n-pentyl, s-pentyl, isopentyl , t-pentyl, n-hexyl, s-hexyl and the like, which may bear one or more substituents. Alkenyl groups include, but are not limited to, e.g., ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, etc. Representative alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (propargyl), 1-propynyl, etc.

As used herein, the term "alicyclic" refers to compounds that combine the properties of aliphatic and cyclic compounds, and includes, but is not limited to, cyclic or polycyclic aliphatic hydrocarbons and bridged cycloalkyl compounds, optionally substituted with one or more functional groups. As one skilled in the art will appreciate, "alicyclic" is intended to include, but is not limited to, cycloalkyl, cycloalkenyl, and cycloalkynyl groups, optionally substituted with one or more functional groups. Accordingly, exemplary alicyclic groups include, but are not limited to, e.g. cyclopropyl, -CH2-cyclopropyl, cyclobutyl, -CH2-cyclobutyl, cyclopentyl, -CH2-cyclopentyl-n, cyclohexyl, -CH2-cyclohexyl, cyclohexenylethyl, cyclohexanylethyl, norbornyl and the like which may also contain one or more substituents.

As used herein, the term "alkoxy" (or "alkyloxy") or "thioalkyl" refers to an alkyl group, as defined previously, attached to the parent molecular moiety through an oxygen atom or through a sulfur atom. In some embodiments, the alkyl contains 1-20 aliphatic carbon atoms. In certain other embodiments, the alkyl contains 1-10 aliphatic carbon atoms. In still other embodiments, the alkyl, alkenyl, and alkynyl as used in the invention contain 1-8 aliphatic carbon atoms. In still other embodiments, the alkyl contains 1-6 aliphatic carbon atoms. In still other embodiments, the alkyl contains 1-4 aliphatic carbon atoms. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, t-butoxy, neopentoxy, and n-hexoxy. Examples of thioalkyls include, but are not limited to, methylthio, ethylthio, propylthio, isopropylthio, n-butylthio and the like.

The term "alkylamino" refers to a group of the formula -NHR '. Wherein R 'is alkyl, as defined herein. The term "aminoalkyl" refers to a group of the formula NH2R'-, wherein R 'is alkyl, as defined herein. In some embodiments, the alkyl contains 1-20 aliphatic carbon atoms. In certain other embodiments, the alkyl contains 1-10 aliphatic carbon atoms. In still other embodiments, the alkyl, alkenyl, and alkynyl as used in the invention contain 1-8 aliphatic carbon atoms. In still other embodiments, the alkyl contains 1-6 aliphatic carbon atoms. In still other embodiments, the alkyl contains 1-4 aliphatic carbon atoms. Examples of alkylamino groups include, but are not limited to, methylamino, ethylamino, isopropylamino, and the like.

Some examples of substituents on the above-described aliphatic (and other) moieties in the compounds of the invention include, but are not limited to, an aliphatic group; alicyclic; heteroaliphatic; heteroalicyclic; aryl; heteroaryl; alkylaryl; alkylheteroaryl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; an alkylthio group; an arylthio group; a heteroalkylthio group; a heteroarylthio group; F; Cl Br; AND; -OH; -NO2; -CN; -CF3; -CH2CF3; -CHCl2; -CH2OH; -CH2CH2OH; -CH2NH2; -CH2SO2CH3; -C (O) Rx; -CO2 (Rx); -CON (R_X) 2 -OC (O) R_X; -OCO2R_X; -OCON (Rxh; -N (Rx) 2; -S (OhR_x; -NR_X(CO) R_Xwherein for each occurrence of R x independently includes, but is not limited to, an aliphatic, alicyclic, heteroaliphatic, heteroalicyclic, aryl, heteroaryl, alkylaryl, or alkylheteroaryl group, any of aliphatic, alicyclic, heteroaliphatic, heteroalicyclic, aryl, heteroaliphatic, hetero-cyclic or aryl, as described above and herein may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic and any of the aryl and heteroaryl substituents described above and herein may be substituted or unsubstituted. Additional examples of generally useful substituents are exemplified by the specific forms provided in the examples described.

As used herein, the terms "aryl" and "heteroaryl" generally refer to stable mono- or polycyclic, heterocyclic, polycyclic and polyheterocyclic unsaturated groups having preferably 3-14 carbon atoms, each of which may be substituted or unsubstituted. Further, it should be understood that the aryl and heteroaryl groups as defined herein may be attached via an aliphatic, alicyclic, heteroaliphatic, heteroalicyclic, alkyl or heteroalkyl group and thus also include - (aliphatic) aryl, - (heteroaliphatic) aryl, - (aliphatic) heteroaryl, - (heteroaliphatic) heteroaryl, - (alkyl) aryl, - (heteroalkyl) aryl, - (heteroalkyl) aryl, and - (heteroalkyl) heteroaryl. Accordingly, in the specification of the phrases "aryl or heteroaryl" and "aryl, heteroaryl, - (aliphatic) aryl, - (heteroaliphatic) aryl, - (aliphatic) heteroaryl, - (heteroaliphatic) heteroaryl, - (alkyl) aryl, - ( heteroalkyl) aryl, - (heteroalkyl) aryl, and - (heteroalkyl) heteroaryl "are interchangeable. The substituents include, but

Not exclusively, any of the aforementioned substituents, eg, substituents listed with respect to aliphatic groups or other groups disclosed herein, provide for the formation of a stable compound. In certain embodiments of the invention, "aryl" refers to a mono- or bicyclic carbon ring system containing one or two aromatic rings, including, but not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, and the like. In certain embodiments of the invention, the term "heteroaryl" as used herein refers to a cyclic aromatic group having 5-10 ring atoms of which one ring atom is selected from S, O and N; 0, 1 or 2 ring atoms are additional heteroatoms independently selected from S, O and N; and the remaining ring atoms are carbon atoms, the group being linked to the rest of the molecule via any ring-forming atoms, such as e.g. pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isoxazolyl, thiadiazolyl, oxadiazolyl, thi- phenyl, furanyl, quinolinyl, isoquinolinyl, etc.

It should be understood that aryl and heteroaryl groups (including bicyclic aryl groups) may be substituted or unsubstituted, substitution including the replacement of 1, 2 or 3 of their hydrogen atoms with any one or more of the following groups, including but not limited to: aliphatic groups; alicyclic; heteroaliphatic; heteroalicyclic; aryl; heteroaryl; alkylaryl; alkylheteroaryl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; Cl; Br; AND; -OH; -NO2; -CN; -CF3; -CH2CF3; -CHCl2; -CH2OH; -CH2CH2OH; -CH2NH2; -CH2SO2CH3; -C (O) Rx; -CO2 (Rx); -CON (Rx) 2; -OC (O) Rx; -ABOUT WHAT₂R_x; -OCON (R_x)₂; -N (R_x)₂; -S (O)₂R_X; -NR_X(CO) R_X, with R._x independently includes, but is not limited to, an aliphatic, alicyclic, heteroaliphatic, heteroalicyclic, aryl, heteroaryl, alkylaryl, or alkylheteroaryl group, and any of the aliphatic, alicyclic, heteroaliphatic, heteroalicyclic, aryl, heteroaryl, or alkylionic, and heteroaryl, or alkylionic, and heteroaryl substituents described above may be or unsubstituted, branched or unbranched, cyclic or acyclic, and any of the aryl or heteroaryl substituents described above and herein may be substituted or unsubstituted. Additional examples of generally useful substituents are exemplified by the specific forms provided in the examples described.

In this specification, the term "cycloalkyl" refers in particular to groups having 3-7, preferably 3-10 carbon atoms. Useful cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like which, like other aliphatic, heteroaliphatic, or heterocyclic moieties, are optionally substituted with substituents including, but not limited to, aliphatic substituents; alicyclic; heteroaliphatic; heteroalicyclic; aryl; heteroaryl; alkylaryl; alkylheteroaryl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; Cl; Br; AND; -OH; -NO2; -CN; -CF3; -CH2CF3; -CHCl2; -CH2OH; -CH2CH2OH; -CH2NH2; -CH2SO2CH3; -C (O) Rx; -CO2 (Rx); -CON (Rx) 2;

-OC (O) R_x; -ABOUT WHAT₂R_x; -OCON (R_x)₂; -N (R_x)₂; -S (O)₂R_x; -NR_x(CO) R_x, with R._xindependently includes, but is not limited to, an aliphatic, alicyclic, heteroaliphatic, heteroalicyclic, aryl, heteroaryl, alkylaryl, or alkylheteroaryl group, and any of the aliphatic, alicyclic, heteroaliphatic, heteroalicyclic, aryl, heteroaryl, and alkylhearyl, and alkylhearyl, and the above-described substituents may be or unsubstituted, branched or unbranched, cyclic or acyclic, and any of the aryl or heteroaryl substituents described above and herein may be substituted or unsubstituted. Additional examples of generally useful substituents are exemplified by the specific forms provided in the examples described.

As used herein, the term "heteroaliphatic group" refers to aliphatic groups in which one or more carbon atoms in the main chain is replaced with a heteroatom. Accordingly, the term "heteroaliphatic group" refers to an aliphatic chain containing one or more, for example, oxygen, sulfur, nitrogen, phosphorus or silicon atoms, instead of carbon atoms. Heteroaliphatic groups can be branched or linear, unbranched. In some embodiments, heteroaliphatic groups are substituted by independently replacing one or more of their hydrogen atoms with one or more groups including, but not limited to, an aliphatic group; an alicyclic group; a heteroaliphatic moiety; a heteroalicyclic group; aryl; heteroaryl; alkylaryl; alkylheteroaryl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; an alkylthio group; an arylthio group; a heteroalkylthio group; a heteroarylthio group; F; Cl; Br; AND; -OH; -NO2; -CN -CF3; -CH2CF3; -CHCl2; -CH2OH; -CH2CH2OH; -CH2NH2 -CH2SO2CH3; -C (O) R_X; -CO2 (Rx); -CON (Rx)₂; -OC (O) Rx; -OCO2Rx; -OCON (Rxh; -NCR2;

PL 219 737 B1

-S (O)₂R_x; -NR_x(CO) R_xwhere in each case R._x independently includes, but is not limited to, aliphatic, alicyclic, heteroaliphatic, heteroalicyclic, aryl, heteroaryl, alkylaryl, or alkylheteroaryl, where any of the aliphatic, alicyclic, heteroaliphatic, heteroalicyclic, alkylaryl or alkylhetero substituents depicted above may be depicted above or unsubstituted, branched or unbranched, cyclic or acyclic, and any of the aryl or heteroaryl substituents described above and herein may be substituted or unsubstituted. Additional examples of generally useful substituents are exemplified by the specific forms provided in the examples described.

As used herein, the term "heteroalicyclic" refers to compounds that combine the properties of heteroaliphatic and cyclic compounds, including, but not limited to, saturated and unsaturated mono- or polycyclic heterocycles such as morpholino, pyrrolidinyl, furanyl, thiofuranyl, pyrrolyl and the like, optionally substituted with one or more functional groups.

In this specification, the terms "halogen" and "halogen" refer to an atom selected from fluorine, chlorine, bromine and iodine.

The term "haloalkyl" denotes an alkyl as defined above having 1, 2 or 3 halogen atoms attached, e.g. a group such as chloromethyl, bromoethyl, trifluoromethyl and the like.

As used herein, the terms "heterocycloalkyl" or "heterocyclyl" refer to non-aromatic 5-, 6- or 7-membered rings or polycyclic groups, including, but not limited to, bi- or tricyclic groups containing fused 6-membered rings, with 1-3 heteroatoms independently selected from oxygen, sulfur and nitrogen, wherein (i) each 5-membered ring contains 0-1 double bond and each 6-membered ring has 0-2 double bonds, (ii) the nitrogen and sulfur heteroatoms are optionally oxidized, (iii) the nitrogen heteroatom is optionally quaternized, and (iv) any of the above heterocyclic rings may be fused to an aryl or heteroaryl ring. Representative heterocycles include, but are not limited to, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazoIidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazoIidinyl, and tetrahydrofuryl. In some embodiments, "substituted heterocycloalkyl or heterocyclyl" is used, ie, heterocycloalkyl or heterocyclyl, as defined above, substituted by independently replacing 1, 2, or 3 hydrogen atoms with groups including, but not limited to, aliphatic substituents; alicyclic; heteroaliphatic; heteroalicyclic; aryl; heteroaryl; alkylaryl; alkylheteroaryl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; Cl; Br; AND; -OH; -NO2; -CN; -CF3; -CH2CF3; -CHCl2; -CH2OH; -CH2CH2OH; -CH2NH2; -CH2SO2CH3; -C (O) Rx; -CO2 (Rx); -CON (Rx) 2; -OC (O) Rx; -OCO2Rx; -OCON (Rx) 2; -N (Rx) 2; -S (O) 2Rx; -NRx (CO) Rx, wherein for each occurrence of Rx independently includes, but is not limited to, an aliphatic, alicyclic, heteroaliphatic, heteroalicyclic, aryl, heteroaryl, alkylaryl, or alkylheteroaryl substituent, and any of the aliphatic, alicyclic, heteroaliphatic, heteroaliphatic, heteroaliphatic, , heteroaryl, alkylaryl, or alkylheteroaryl described above and may be substituted or unsubstituted, branched or unbranched in the specification, cyclic or acyclic, and any of the aryl or heteroaryl substituents described above and herein are optionally substituted. Additional examples of generally useful substituents are exemplified by the specific forms provided in the examples described.

As used herein, the terms "aliphatic", "heteroaliphatic", "alkyl", "alkenyl", "alkynyl", "heteroalkyl", "heteroalkenyl", "heteroalkynyl" and the like include substituted and unsubstituted, saturated and unsaturated, and linear and branched groups. Similarly, the terms "alicyclic group", "heteroalicyclic group", "heterocycloalkyl", "heterocyclyl" and the like include substituted and unsubstituted and saturated and unsaturated groups. In addition, the terms "aliphatic (aryl)", "heteroaliphatic (aryl)", "aliphatic (heteroaryl)", "heteroaliphatic (heteroaryl)", "alicyclic (aryl)", "heteroalicyclic (aryl)", "alicyclic (heteroaryl)" , "Heteroalicyclic (heteroaryl)", "-alkyl (aryl)", "heteroalkyl (aryl)", "-alkyl (heteroaryl)", "heteroalkyl (heteroaryl)" etc. refer to substituted and unsubstituted, saturated and unsaturated (i.e. non-aromatic part of the grouping). In addition, the terms "cycloalkyl", "cycloalkenyl", "cycloalkynyl", "heterocycloalkyl", "heterocycloalkenyl", "heterocycloalkynyl", "aryl", "heteroaryl" and the like include substituted and unsubstituted groups unless otherwise stated.

Methods of synthesis

According to the invention, any of the available techniques can be used to prepare or prepare the compounds of the invention or compositions containing them. For example, various solution phase synthesis methods may be used, such as the methods detailed below. Alternatively

Or in addition, the compounds of the invention may be prepared using any of the known variations of combinatorial techniques, parallel synthesis, and / or solid phase synthesis methods.

Examples of synthetic methods for the preparation of exemplary types of compounds of the invention and reference compounds are provided below in detail in Schemes 1-12 and in the Examples section. It should be understood that the methods described can be applied to any of the disclosed compounds and their equivalents. Moreover, the reagents and starting materials are well known to those skilled in the art. While the following schemes represent some exemplary compounds, it should be appreciated that other analogs of the invention will be obtained from the use of alternative starting materials. For example, compounds are described below where X2 is C = O, R5 is hydrogen, R6 is t-butyl, and R7 is methyl; however, it should be appreciated that alternative starting materials and / or intermediates may be used to obtain compounds in which e.g. X2 is C = O and R5-R7 may be groups other than those shown, such as alkyl, heteroalkyl, aryl, heteroaryl and the like. It should also be appreciated that available known techniques may be used to prepare compounds or compositions containing them. One skilled in the art will recognize that suitable synthetic methods are not limited to those shown in Schemes 1-12 below, and that any suitable known synthetic methods can be used to prepare the compounds of the invention.

In certain embodiments, compounds of the invention have the general formula (I ') shown in scheme 1, where R, R' and Q are aliphatic, heteroaliphatic, aryl, or heteroaryl groups. In preferred embodiments, R, R 'and Q are groups listed in classes and subclasses. Examples of preferred R, R 'and Q structures are shown in Scheme 1.

Scheme 1

<img file="PL219737B1_D0025.tif" />

Examples of such compounds include, but are not limited to, compounds in which:

RG = H, Me, Et or forms a 5-6 membered ring with RH1

RH1 = H, Me, Et or forms a 5-6 membered ring with RG

RH2 = H, CO2H, CO2Me, CONH2, CONHMe, CONHMe2, CONHBn, CH2OMe

RG1 = H, Me or forms a 5-6 membered ring with RG2

RG2 = H or forms a 5-6 membered ring with RG1

RG3 = Η, CO2H, CO2Me, CONH2, CONHMe, CONHMe2, CONHBn, CH2OMe

PL 219 737 B1

Scheme 2 shows the synthesis of reference compounds of general formula 11. As shown in Scheme 2, the dipeptide core can be constructed from N-Boc-N-methylvalinal (2) and N-Boc-t-Ieucine (4). The N-terminal group in compounds (R 'in scheme 1) can be introduced via (S) -N-Boc-neophenylalanine (6). As shown in Scheme 2, different synthesis methods allow the preparation of different analogs, e.g. carboxylic esters of general formula 7, carboxylic acid 8 or amides of general formula 11. It will be apparent to the reader of the text that other known synthetic methods can be used to prepare other derivatives.

<img file="PL219737B1_D0026.tif" />

<img file="PL219737B1_D0027.tif" />

Examples of compounds of this type include, but are not limited to, compounds in which: RG1 = H, Me or forms a 5-6 membered ring with RG2 RG2 = H or forms a 5-6 membered ring with RG1

PL 219 737 B1

RG3 = H, CO2H, CO2Me, CONH2, CONHMe, CONHMe2, CONHBn, CH2OMe.

An exemplary synthesis to intermediate 6 is shown in Scheme 3. This method produces (S) -N-Boc-neo-phenylalanine (6) in an overall yield of 20%.

<img file="PL219737B1_D0028.tif" />

Schemes 4-6 illustrate the synthesis of exemplary types of reference compounds (e.g., aminoesters, amino acids, aminoamides, and N-acetylaminoamides of general formulas 18, 20, and 23, respectively, as shown in Scheme 4; see also aminoesters, amino acids, aminoamides, and N-acetylamides, respectively). -acetylaminoamides of general formulas 25, 26 and 27, respectively, in scheme 5). In some embodiments, R can be a nitrogen-containing heteroalkyl (see schemes 4 and 5) or an unsaturated oxygen-containing heteroalkyl (see scheme 6). While Schemes 4-6 show compounds having an N-terminal group derived from (S) -N-Boc-neo-phenylalanine (6), one skilled in the art will understand that many different organic groups other than those shown in Schemes 4-6 can be used in the construction of such compounds. Similarly, Schemes 4-6 show compounds in which the C-terminal group may be a carboxylic ester, carboxylic acid, or amide.

PL 219 737 B1

<img file="PL219737B1_D0029.tif" />

PL 219 737 B1

Examples of such compounds include, but are not limited to, compounds in which:

R10b = H, Me or forms a 5-6 membered ring with R11b

R11b = H or forms a 5-6 membered ring with R10b

RG = H, Me or forms a 5-6 membered ring with RH1

RH1 = H forms a 5-6 membered ring with RG

RH2 = H, CO2H, CO2Me, CONH2, CONHMe, CONHMe2, CONHBn, CH2OMe

<img file="PL219737B1_D0030.tif" />

Examples of compounds of this type include, but are not limited to, compounds in which: RG = forms a 5- or 6-membered ring with RH1 RH1 = forms a 5- or 6-membered ring with RG R_H2 = CO₂Me, CONH₂

<img file="PL219737B1_D0031.tif" />

PL 219 737 B1

<img file="PL219737B1_D0032.tif" />

Schemes 7, 9, and 10 show the synthesis of exemplary types of reference compounds (e.g., aminoesters, amino acids, and aminoamides of general formulas 42, 43, and 45, respectively, as shown in Scheme 7). Scheme 8 shows the synthesis of exemplary types of compounds of the present invention. In some embodiments, the compounds contain an N-terminal heterocyclic group with a nitrogen atom. For example, a heterocyclic group may be a piperidine ring (schemes 7, 8, and 9) or a thiazolidine ring (scheme 10). Examples of other suitable groups are given in the Examples section below or will be apparent to those skilled in the art. As shown above, R can be a nitrogen-containing heteroalkyl (scheme 7) or an unsaturated alkyl (scheme 8, 9 and 10).

PL 219 737 B1

Scheme 7

Boc g co₂h

LiOH _r

THF, MeOH aminoesters

HCl R_g (⁴⁴)

NMM, DEPC, DMF room temperature amino amides amino acids (43)

CO, Me HCl, MeOH

NMM, HOAt, Boc

C1H "N

CO, Me

CMC, DMF

WHAT

Me

Cl

CO.H

NMM, HOAt,

CMC, DMF

WHAT

Me

NaBH

MeOH / THF

Dress

Martin

THF

WHAT

Me

HN dichloroethane mol

4A sieve triacetoxyborohydride

2) sodium

PL 219 737 B1

Examples of such compounds include, but are not limited to, compounds in which:

R10b = H, Me or forms a 5-6 membered ring with R11b R11b = H or forms a 5-6 membered ring with R10b RG = H, Me, OMe or forms a 5-6 membered ring with RH1 RH1 = H, and -Pr or forms a 5-6 membered ring with RG

RH2 = OH, OMe, OBn, Oi-Pr, O-cyclo-Bu, O-cyclo-Pent, O-cyclo-HeX, NH2, NHBn, NH (2-Naphthyl). Scheme 8

<img file="PL219737B1_D0033.tif" />

PL 219 737 B1

Conditions: a) K2CO3, CH3I, DMF; (b) TMS-diazomethane, MeOH, CH2Cl2; (C) DIBAL, PhCH3, -78 ° C; (d) Ph3P = C (CH3) CO2Et, CH2Cl2; (e) HCl in 1,4-dioxane; (f) BOC-Tie-OH, CMC, HOAt, NMM, DMF; (g) N-methylpipecolinic acid, CMC, HOAt, NMM, DMF; (h) LiOH, aqueous MeOH solution; (i) HCl * L-Pro-OMe, DEPC, NMM, DMF

Scheme 9

<img file="PL219737B1_D0034.tif" />

Me

<img file="PL219737B1_D0035.tif" />

<img file="PL219737B1_D0036.tif" />

only one enantiomer is shown

Boc and

<img file="PL219737B1_D0037.tif" />

(2,4-anti) only one '78.5% enantiomer is shown

<img file="PL219737B1_D0038.tif" />

PL 219 737 B1

Scheme 9 cont.

<img file="PL219737B1_D0039.tif" />

<img file="PL219737B1_D0040.tif" />

It will be understood by those skilled in the art that the exemplary heterocyclic starting materials shown in schemes 7-10 (particularly compounds 38, 60 or 65) may be replaced with acyclic α-amino acid groups to provide reference compounds as illustrated below in scheme 11:

PL 219 737 B1

Scheme 11

<img file="PL219737B1_D0041.tif" />

Examples of such compounds include, but are not limited to, compounds in which:

R1 = H or Me

R3 = Me, Et or forms a 5-6 membered ring with R4 R4 = Me, Et or forms a 5-6 membered ring with R3 R10b = H, M or forms a 5-6 membered ring with R11b R11b = H or forms 5-6 membered ring with R10b RG = H, Me or forms 5-6 membered ring with RH1 RH1 = H, Me forms 5-6 membered ring with RG

RH2 = H, CO2H, CO2Me, CONH2, CONHMe, CONHMe2, CONHBn, CH2OMe.

Reaction of diethylglycine (72) with amine hydrochloride 49 yields the N-terminal gem diethyl ethyl ester 73 or the corresponding carboxylic acid 74 after hydrolysis under suitable conditions (Scheme 12).

PL 219 737 B1

<img file="PL219737B1_D0042.tif" />

It should be understood that each of the reactions depicted in Schemes 2-12 above may be performed using the reagents and under the conditions described for the synthesis of the various types of exemplary compounds described above, or may be modified using other available reagents or starting materials. For example, various conditions for amide formation, esterification, hydrolysis, and functional group introduction into the aromatic ring are known and may be used in the process of the invention. See generally, March, Advanced Organic Chemistry, 5th Edition, John Wiley &amp; Sons, 2001; and "Comprehensive Organic Transformations, a guide to functional group preparations, Richard C. Larock, VCH publishers, 1999.

As noted above, it should be understood that the invention is not limited in scope to the compounds mentioned in the description. Synthetic strategies or starting materials other than those described herein may be used to prepare the compounds of the invention. Furthermore, it should be appreciated that any of the ingredients / starting materials used in the synthesis of the compounds of the invention can be modified either prior to the synthesis or after the preparation of the peptide construct. As used herein, the term "modifying" or "modifying" means reacting a compound of the invention, as defined herein, at one or more reactive sites in order to modify a functional group or introduce a functional group. For example, when an aromatic ring is included in a compound, the aromatic ring may be modified (before or after the reaction) to introduce a functional group (e.g. in the case of a hydrogen atom present, a halogen atom or other functional group may be introduced) or the functional group may be modified (e.g., in the case where an aromatic ring contains hydroxyl, such an aromatic ring may be modified by reaction with a hydroxyl protecting reagent, or it may be converted into an aliphatic or heteroaliphatic moiety). Various schemes are generally provided below to enable the reader of the text to synthesize various analogs by modifying intermediates or modifying a peptide construct.

PL 219 737 B1

In some embodiments, the preparation of chemically diverse derivatives can be achieved by modifying the C-terminal group of the compounds. For example, when the C-terminal group is a carboxylic acid moiety, examples of chemical transformations suitable to achieve such modifications include, but are not limited to, reduction to the appropriate aldehyde or alcohol, amidation, Wittig reaction, decarboxylation, esterification, nucleophilization, ketone conversion, imines, hydrazones, azides, etc. Examples of suitable transformations for the reference compounds are shown in schemes 13 and 14. One skilled in the art will understand that possible chemical transformations useful for modifying the compounds of the invention are not limited to the methods shown in Schemes 1-14, but that rather, any suitable known synthetic method may be used to achieve such chemical transformations.

Scheme 13

<img file="PL219737B1_D0043.tif" />

<img file="PL219737B1_D0044.tif" />

PL 219 737 B1

MA

<img file="PL219737B1_D0045.tif" />

<img file="PL219737B1_D0046.tif" />

PL 219 737 B1

Research applications, formulation and administration

Compounds of the invention can be evaluated by any available known assay to identify compounds with established biological activity. These can be, for example, cellular or non-cellular assays, in vivo or in vitro, in high or low throughput format, etc. In certain exemplary embodiments, the compounds of the invention are tested in assays to identify compounds having cytotoxic or growth inhibitory activity in vitro, or causing tumor regression and / or tumor growth inhibition in vivo.

Compounds of particular interest according to the invention are those which:

they exhibit cytotoxic and / or growth inhibitory activity on tumor cell lines maintained in vitro or in animal studies using an accepted scientific tumor cell xenograft model;

preferably they cause tumor regression in vivo; show low sensitivity to MDR;

they show low cytotoxicity to normal, non-dividing cells; and / or exhibit a favorable therapeutic profile (e.g. with regard to safety, efficacy and durability).

As detailed in the examples herein, in in vitro testing of the ability of compounds to inhibit the growth of tumor cell lines, certain compounds of the invention exhibit IC values₅₀ &lt;10 μΜ. In other embodiments, compounds of the invention exhibit IC values₅₀ &lt;5 μΜ. In other embodiments, compounds of the invention exhibit IC values₅₀ &lt;1 μΜ. In other embodiments, compounds of the invention exhibit IC50 values ​​&lt;750 nM. In other embodiments, compounds of the invention exhibit IC50 values ​​&lt;500 nM. In other embodiments, compounds of the invention exhibit IC50 values ​​&lt;250 nM. In other embodiments, compounds of the invention exhibit IC50 values ​​&lt;100 nM. In other embodiments, compounds of the invention exhibit IC50 values ​​&lt;50 nM. In other embodiments, compounds of the invention exhibit IC50 values ​​&lt;25 nM. In other embodiments, compounds of the invention exhibit IC50 values ​​&lt;10 nM. In other embodiments, compounds of the invention exhibit IC50 values ​​&lt;7.5 nM. In other embodiments, compounds of the invention exhibit IC50 values ​​&lt;5 nM. In other embodiments, compounds of the invention exhibit IC50 values ​​&lt;2.5 nM. In other embodiments, compounds of the invention exhibit IC50 values ​​&lt;1 nM. In other embodiments, compounds of the invention exhibit IC50 values ​​&lt;0.75 nM. In other embodiments, compounds of the invention exhibit IC50 values ​​&lt;0.5 nM. In other embodiments, compounds of the invention exhibit IC50 values ​​&lt;0.25 nM. In other embodiments, compounds of the invention exhibit IC50 values ​​&lt;0.1 nM. In certain embodiments, compounds of the invention exhibit IC50 values ​​for inhibiting growth of cultured human tumor cells from 0.1 nM - 10 nM.

In certain other embodiments, compounds of the invention exhibit low sensitivity to MDR. In certain exemplary embodiments, inventive compounds exhibit a ratio [MDR-positive cell growth inhibition] / [MDR-negative cell growth inhibition] (i.e., resistance index) &lt;10. In certain exemplary embodiments, inventive compounds exhibit a resistance index &lt;9. In exemplary embodiments, the compounds of the invention exhibit a resistance index &lt;8. In certain exemplary embodiments, inventive compounds exhibit a resistance index of &lt;7.

In certain exemplary embodiments, the compounds of the invention exhibit a resistance index &lt;6.

In certain exemplary embodiments, the compounds of the invention exhibit a resistance index of &lt;5.

In certain exemplary embodiments, the compounds of the invention exhibit a resistance index of &lt;4.

In certain other embodiments, compounds of the invention exhibit low cytotoxicity to non-dividing, normal cells. In certain exemplary embodiments, the compounds of the invention exhibit little or no cytotoxicity to non-dividing, normal cells at concentrations&gt; 1000 times the concentration at which they inhibit the growth of cancer cells. In certain exemplary embodiments, the compounds of the invention exhibit little or no cytotoxicity to non-dividing, normal cells at concentrations ranging from 1-10 µΜ.

In certain embodiments, the compounds of the invention are stable in mouse serum.

In certain embodiments, the compounds of the invention exhibit a low rate of reversibility of mitotic blocking. In certain embodiments, compounds of the invention exhibit mitotic blocking reversibility rates ranging from 1 to about 30. In some embodiments, compounds of the invention exhibit mitotic blocking reversibility rates ranging from 1 to about 25. In certain embodiments, compounds of the invention exhibit mitotic blocking reversibility rates

In some embodiments, compounds of the invention exhibit mitotic blocking reversibility rates ranging from 1 to about 15. In some embodiments, compounds of the invention exhibit mitotic blocking reversibility rates ranging from 1 to about 10 In certain embodiments, compounds of the invention exhibit mitotic blocking reversibility rates ranging from 1 to about 5. In certain embodiments, compounds of the invention exhibit mitotic blocking reversibility rates ranging from 1 to about 3.

In certain embodiments, the compounds of the invention cause tumor regression in vivo. In certain exemplary embodiments, the compounds of the invention cause tumor regression in vivo in corresponding mouse xenograft tumor models. In certain exemplary embodiments, the compounds of the invention cause a tumor size reduction to less than 70% when initiating the compound administration in an appropriate tumor cell xenograft model. In certain exemplary embodiments, compounds of the invention bring about a tumor size reduction to less than 65% when initiating administration of the compound in an appropriate tumor cell xenograft model. In certain exemplary embodiments, compounds of the invention bring about a tumor size reduction to less than 60% when initiating the compound administration in an appropriate tumor cell xenograft model. In certain exemplary embodiments, compounds of the invention bring about a tumor size reduction to less than 55% when initiating administration of the compound in an appropriate tumor cell xenograft model. In certain exemplary embodiments, compounds of the invention bring about a tumor size reduction to less than 50% when initiating administration of the compound in an appropriate tumor cell xenograft model. In certain exemplary embodiments, compounds of the invention cause tumor regression in certain multi-drug resistant xenograft models.

In certain exemplary embodiments, the compounds of the invention cause tumor growth inhibition in vivo. In certain exemplary embodiments, the compounds of the invention cause significant inhibition of tumor growth in suitable tumor cell xenograft models. In certain exemplary embodiments, the compounds of the invention cause significant inhibition of tumor growth in suitable multi-drug resistant tumor cell xenograft models. In certain exemplary embodiments, compounds of the invention inhibit tumor growth in treated animals by&gt; 50% as compared to control animals (i.e., size of "treated" tumor &lt;50%; size of "control" tumor; or T / C value &lt;50%) in appropriate tumor cell xenograft models. In certain embodiments, compounds of the invention exhibit T / C values ​​&lt;70%. In certain embodiments, inventive compounds exhibit T / C values ​​&lt;65%. In certain embodiments, compounds of the invention exhibit T / C values ​​&lt;60%. In certain embodiments, inventive compounds exhibit T / C values ​​&lt;55%.

In certain embodiments, compounds of the invention inhibit the growth of human tumor cells in vitro, show low sensitivity to MDR (i.e., low index of resistance), show low cytotoxicity to non-dividing normal cells, show stability in mouse serum, show a low rate of mitotic blockade reversibility. cause tumor regression in vivo, and / or inhibit tumor growth in vivo.

In certain embodiments, compounds of the invention inhibit the growth of human tumor cells in vitro, show low sensitivity to MDR (i.e., low index of resistance), show low cytotoxicity to non-dividing normal cells, show stability in mouse serum, show a low rate of mitotic blockade reversibility. they cause tumor regression in vivo, and inhibit tumor growth in vivo.

In certain embodiments, the compounds of the invention exhibit one or more of the following properties:

exhibit IC50 values ​​for growth inhibition of cultured human tumor cells in the range of 0.1 nM - 10 nM;

have a resistance index of preferably &lt;10, preferably 5-9, preferably &lt;8, preferably &lt;7, preferably &lt;6, preferably &lt;5, more preferably &lt;4;

they show little or no cytotoxicity to non-dividing, normal cells at concentrations ranging from 1 to 10 µΜ;

are stable in mouse serum;

They show mitotic blocking reversibility rates ranging from 1 to about 30, preferably from 1 to about 25, preferably from 1 to about 20, preferably from 1 to about 15, preferably from 1 to about 10, preferably from 1 to about 25. 5, most preferably about 1-3;

cause a reduction in tumor size to less than 70%, preferably less than 65%, preferably less than 60%, preferably less than 55%, most preferably less than 50% of the size at the initiation of administration of the compound in the respective tumor cell xenograft models; and / or cause a significant inhibition of tumor growth in an appropriate tumor cell xenograft model (e.g. have a T / C value of preferably &lt;70%, preferably &lt;65%, preferably &lt;60%, preferably &lt;55%, most preferably &lt;50%).

In certain embodiments, compounds of the invention exhibit the following properties: exhibit IC50 values ​​for inhibition of growth of cultured human tumor cells in the range of 0.1 nM - 10 nM;

have a resistance index of preferably &lt;10, preferably &lt;9, preferably &lt;8, preferably &lt;7, preferably &lt;6, preferably &lt;5, more preferably &lt;4;

they show little or no cytotoxicity to non-dividing, normal cells at concentrations ranging from 1 to 10 µΜ;

are stable in mouse serum;

show blocking reversibility indexes ranging from 1 to about 30, preferably from 1 to about 25, preferably from 1 to about 20, preferably from 1 to about 15, preferably from 1 to about 10, preferably from 1 to about 5, most preferably about 1 - 3;

cause a reduction in tumor size to a value of 70%, preferably less than 65%, preferably less than 60%, preferably less than 55%, most preferably less than 50% of the size at the initiation of administration of the compound in the respective tumor cell xenograft models; and cause significant inhibition of tumor growth in an appropriate tumor cell xenograft model (e.g. have a T / C value of preferably &lt;70%, preferably &lt;65%, preferably &lt;60%, preferably &lt;55%, most preferably &lt;50%).

Examples of compounds exhibiting the desired properties include ER-807102, ER-807974, ER-808368, ER-808662, ER-808824, and salts thereof (see table below).

As explained above, the compounds of the invention exhibit tumor cell growth inhibitory activity. Therefore, the compounds of the invention are useful in the treatment of a variety of disorders including, but not limited to, glioblastoma, retinoblastoma, breast cancer, cervical cancer, colorectal cancer, leukemia, lung cancer (including, but not limited to, small cell carcinoma). lung disease), melanoma, multiple myeloma, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, and stomach cancer to name a few. In certain embodiments, the compounds of the invention are useful in the treatment of solid and non-solid tumors. In still other interesting embodiments, the compounds of the invention are particularly useful in the treatment of breast cancer, prostate cancer, colorectal cancer, lung cancer, leukemia, and lymphoma.

In an embodiment, the method comprises administering a therapeutically effective amount of a compound or a pharmaceutically acceptable derivative thereof to a subject in need thereof (including, but not limited to, a human or animal). In some embodiments, the compounds of the invention are useful in the treatment of cancer (including, but not limited to, glioblastoma, retinoblastoma, breast cancer, cervical cancer, colorectal cancer, leukemia, lymphoma, lung cancer (including but not limited to small cell lung cancer), melanoma and / or skin cancer, multiple myeloma, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, prostate cancer and gastric cancer, bladder cancer, uterine cancer, kidney cancer, testicular cancer, brain cancer, liver cancer or cancer of the esophagus).

Pharmaceuticals

As outlined above, the invention provides novel compounds with biological properties useful in the treatment of cancer. In certain embodiments, certain compounds described herein act as tumor growth inhibitors, and thus are useful in treating cancer and in inhibiting tumor growth, and in killing tumor cells. In certain embodiments, compounds of the invention are useful in the treatment of solid tumors or non-solid tumors. In still other interesting embodiments, the compounds of the invention are useful in the treatment of glioblastoma, retinoblastoma, breast cancer, cervical cancer, colorectal cancer, leukemia, lymphoma, lung cancer (including but not limited to small cell lung cancer), melanoma , multiple myeloma, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, prostate cancer and stomach cancer to name a few. Relationships

The present invention also finds utility in preventing blood vessel restenosis following trauma, such as angioplasty and stent insertion.

Accordingly, in another embodiment, the invention provides pharmaceutical compositions comprising any of the compounds described herein (or a prodrug, pharmaceutically acceptable salt, or other pharmaceutically acceptable derivative thereof) and optionally containing a pharmaceutically acceptable carrier. In some embodiments, the compounds are capable of inhibiting the growth or killing of cancer cells. In some embodiments, such agents optionally further comprise one or more additional therapeutic agents. Alternatively, a compound of the invention may be administered to a patient in need thereof in conjunction with the administration of one or more other therapeutic agents. For example, a cytotoxic or anti-neoplastic agent suitable for the treatment of cancer, as further described herein, may be used as an additional therapeutic agent to be administered with or incorporated into a pharmaceutical agent containing a compound of the invention, or it may be one of the many approved agents. by the Food and Drug Administration, which will eventually be approved for the treatment of immune disorders or cancer. Furthermore, it should be appreciated that the compounds according to the invention can be used in therapy in free form or, where appropriate, in the form of a pharmaceutically acceptable derivative thereof. Pharmaceutically acceptable derivatives in accordance with the invention include, but are not limited to, pharmaceutically acceptable salts, esters, salts of such esters, or a prodrug or other adduct or derivative of a compound of the invention which, when administered to a patient in need thereof, will be capable of delivering, directly or indirectly, the described herein. the compound or its metabolite or the rest.

As used herein, the term "pharmaceutically acceptable salt" refers to those salts which are, within the scope of significant medical judgment, suitable for use in contact with human and lower animal tissues, with little or no undue toxicity, irritation, allergic response, etc., commensurate with reasonable the benefit-risk ratio. Pharmaceutically acceptable salts of amines, carboxylic acids, and other types of compounds are well known. For example, SM Berge et al. Describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977). Salts may be formed in situ during the final isolation and purification of the compounds of the invention, or separately by reacting the free base or free acid moiety with a suitable reagent as generally described below. For example, the free base moiety can be reacted with an appropriate acid. In addition, when the compounds of the invention contain an acidic group, suitable pharmaceutically acceptable salts thereof may include metal salts such as alkali metal salts, e.g. sodium or potassium salts; and alkaline earth metal salts, e.g. calcium or magnesium salts. Examples of pharmaceutically acceptable non-toxic acid addition salts include salts of the amine group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid. , tartaric acid, citric acid, succinic acid, or malonic acid, or by other known methods such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphate, camphorsulfonate, citrate, cyclopentanpropionate, digluconate, dodecyl sulfate, ethanesulfptonate, glucosulfonate, glucosulfonate, glucosulfate, glucosulfate heptanoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectate, persulfate, 3-phenylepropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocoluate, thiocoluate, thiocoluate, thiocoluate undecanoate, valerate, etc. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. Further pharmaceutically acceptable salts include, when appropriate, non-toxic ammonium and ammonium salts, quaternary ammonium salts, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and arylsulfonate.

In addition, as used herein, the term "pharmaceutically acceptable ester" refers to esters that hydrolyze in vivo and includes those compounds that break down readily in the human body leaving the parent compound or a salt thereof. Suitable ester groups include, for example, those derived from pharmaceutically acceptable aliphatic carboxylic acids, especially acids

Are alkane, alkene, cycloalkane and alkanedioic, wherein each alkyl or alkenyl suitably contains no more than 6 carbon atoms. Examples of suitable esters include formates, acetates, propionates, butyrates, acrylates, and ethyl succinates.

Furthermore, as used herein, the term "pharmaceutically acceptable prodrugs" refers to those prodrugs of the compounds of the invention which, to the extent of significant medical judgment, are suitable for use in contact with human and lower animal tissues without undue toxicity, irritation, allergic response, etc. commensurate with reasonable medical judgment. the benefit-risk ratio, and effective for their intended use, and the bipolar forms, where possible, of the compounds of the invention. The term "prodrug" refers to compounds which are readily converted in vivo to form the parent compound of the above formula, for example by blood hydrolysis. For a thorough discussion, see T. Higuchi and V. Stella, Prodrugs as Novel Delivery Systems, Vol. 14 ACS Symposium Series, and Edward B. Roche, Ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987.

As described above, the pharmaceutical compositions of the invention additionally contain a pharmaceutically acceptable carrier which, as used herein, includes any and all solvents, diluents or other liquid carrier, dispersing or suspending agents, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like to fit the particular dosage form desired. Remington's Pharmaceutical Sciences, 16th edition, EW Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in formulating pharmaceutical compositions and known techniques for their preparation. Except where the carrier medium is incompatible with the compounds of the invention, e.g. by causing an adverse biological effect or otherwise adversely affecting any of the other ingredients of the pharmaceutical composition, its use is contemplated as being within the scope of the invention. Some exemplary ingredients that can serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatine; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil, sesame oil; olive oil; corn oil and soybean oil; glycols; such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, separating agents, coating agents, sweeteners, flavorings and flavors, preservatives and antioxidants can be also present in the composition, according to the judgment of the person preparing the recipe.

Uses and formulations of the compounds of the invention

As more fully described herein, the invention generally provides compounds useful in the treatment of cancer and proliferative disorders.

As outlined above, certain compounds described above act as tumor growth inhibitors and are therefore useful in treating cancer and in inhibiting tumor growth and killing tumor cells. The compounds of the invention are useful in a method of inhibiting tumor growth and / or tumor metastasis. The method comprises administering a therapeutically effective amount of a compound or a pharmaceutically acceptable derivative thereof to a subject in need thereof (including, but not limited to, a human or animal). In certain embodiments, compounds of the invention are useful in the treatment of solid tumors or non-solid tumors. In still other interesting embodiments, the compounds of the invention are useful in the treatment of glioblastoma, retinoblastoma, breast cancer, cervical cancer, colorectal cancer, leukemia, lymphoma, lung cancer (including but not limited to small cell lung cancer), melanoma , multiple myeloma, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, prostate cancer and stomach cancer to name a few.

As outlined in more detail herein, the invention generally provides compounds useful in the treatment of cancer, especially solid and non-solid tumors. In particular, certain compounds of the invention have been shown to inhibit the growth of certain tumor cell lines in vitro, as further described herein, and are useful in the treatment of cancer, including solid and non-solid tumors.

PL 219 737 B1

As discussed above, the compounds of the invention also find use in preventing blood vessel restenosis following trauma, such as angioplasty and stenting. For example, it is contemplated that the compounds of the invention will be useful as a coating for implantable medical devices such as tubes, artificial fistulas, catheters, artificial implants, pins, electrical implants such as pacemakers, and in particular arterial or venous stents, including expandable stents. balloons. In some embodiments, compounds of the invention may be associated with an implantable medical device or, alternatively, may be passively adsorbed onto the surface of an implantable device. In certain other embodiments, the compounds of the invention may be formulated to be contained or releasable by a medical or surgical device or implant, such as, e.g., stents, surgical threads, indwelling catheters, prostheses, and the like.

In certain exemplary embodiments, the compounds of the invention may be used as a coating on stents. The stent is typically an open tubular structure containing an array (or arrangements) of holes extending from the outer surface of the stent into its lumen. Stents are commonly made of biocompatible metal materials, with systems incised at the surface by a laser device. The stent may be electropolished to minimize surface irregularities as such irregularities can trigger an adverse biological response. However, stents can still stimulate a foreign body response, leading to thrombosis or restenosis. To avoid these complications, various stent coatings and compositions have been proposed in the literature, both to reduce the incidence of these and other complications and to restore the effects of the tissue itself or by delivering the therapeutic compound to the lumen. For example, the effects of drugs with antiproliferative and anti-inflammatory activity as coatings on stents have been evaluated and promising results have been obtained with respect to the prevention of restenosis (see e.g. Presbitero P. et al., "Drug eluting stents do they make the difference?", Minerva Cardioangiol, 2002, 50 (5): 431-442; Ruygrok PM et al., "Rapamycin in cardiovascular medicine", Intern. Med. J., 2003, 33 (3): 103-109; and Marx SO

et al., "Bench to bedside: the development of rapamycin and its application to stent restenosis." Circulation, 2001, 104 (8): 852 - 855). Accordingly, without wishing to be bound by any particular theory, it is believed that the compounds of the present invention having antiproliferative activity may be used as coatings on stents and / or in drug delivery devices to stents, inter alia for the prevention of restenosis. Various compositions and methods are known for coating stents and / or local drug delivery to stents to prevent restenosis (see, e.g., U.S. Patent Nos. 6,517,889, 6,273,913, 6,258,121, 6,251,136, 6,248,127, 6231600, 6203551, 6153252, 6071305, 5891507 , 5,837,313 and U.S. Published Patent Application No. US2001 / 0027340). For example, stents can be coated with polymer-drug conjugates by dipping the stent in a polymer-drug solution or by spraying the stent with such a solution. In an embodiment, suitable materials for implantable devices include biocompatible and non-toxic materials that can be selected from metals such as nickel-titanium alloys, steel or biocompatible polymers, hydrogels, polyurethanes, polyethylenes, ethylene-vinyl acetate copolymers, and the like in some embodiments. according to the invention, a stent is coated for insertion into an artery or vein after balloon angioplasty.

The compounds of the invention can be used in a method of inhibiting arterial restenosis or obstruction of an artery following a vascular injury, comprising administering to a subject in need thereof a composition comprising a compound of the invention coupled to an appropriate polymer or polymeric material. In implementing the method, the subject may be e.g. a patient with coronary bypass surgery, after vascular surgery, after organ transplant or after vascular or any other arterial angioplasty, and the composition may be administered directly, intravenously, or even as a coating on a stent implanted at the site of a vascular injury.

Surgical or medical implants and devices, including stents and implants, are coated or otherwise constructed to contain and / or release any of the disclosed compounds of the invention. In some embodiments, the compounds exhibit anti-proliferative activity. In certain other embodiments, the compounds inhibit the proliferation of smooth muscle cells. Representative examples of implants and surgical or medical devices include cardiovascular devices (e.g. implantable venous catheters, intravenous ports, canaled venous catheters, long-term infusion lines or ports, including hepatic artery infusion catheters, pacemaker lines, implantable defibrillators); neurological / neurosurgical devices (e.g. artificial ventricular peritoneal fistulas, artificial ventricular atrial fistulas, nerve stimulation devices, plasters and implants for the dura mater for the prevention of epidural fibrosis after IaminekPL 219 737 B1 tom, continuous subarachnoid infusion devices); gastrointestinal devices (e.g. permanently inserted catheters, feeding tubes, portal-systemic artificial fistulas, artificial ascites fistulas, peritoneal implants for drug administration, peritoneal dialysis catheters, implantable mesh for hernia, suspensions or permanent implants to prevent postoperative adhesions, including mesh) ; genitourinary devices (e.g. uterine implants, including intrauterine devices (IUDs) and devices to prevent endometrial hyperplasia, fallopian tube implants, including reversible sterilization devices, tubal stents, artificial sphincters and periurethral incontinence implants, ureteral stents, indwelling catheters, components enlarging bladder or wraps or splints for suturing a ruptured vas deferens); ophthalmological implants (e.g. multino implants and other implants for neovascular glaucoma, drug-eluting contact lenses for pterygia, splints in case of sac-nasal anastomosis failure, drug-eluting contact lenses for corneal neovascularization, implants for diabetic retinopathy, drug-eluting contact lenses for transplants high-risk corneas); otolaryngological devices (e.g. bone implants, splints of the eustachian tube or stents in the case of chronic exudative otitis media or chronic otitis media as an alternative to transmembrane drains); plastic surgery implants (e.g. prevention of fibrous contracture in response to gel or saline containing breast implants for sub-pectoral or sub-glandular approach, or after mastectomy or chin implants) and orthopedic implants (e.g. cement orthopedic prostheses).

Implants and other surgical or medical devices may be coated (or otherwise adapted to release) the compositions of the invention by a variety of methods, including, for example: (a) direct attachment to an implant or device of a compound or composition of the invention (e.g. by spraying a polymer / drug film onto the implant or device, or by dipping the implant or device in a polymer / drug solution, or by other covalent or non-covalent means); (b) by coating the implant or device with a substance, such as a hydrogel, which in turn will absorb a compound or composition of the invention; (c) weaving a suture coated with a compound or composition of the invention (or a polymer itself formed into a suture) into the implant or device; (d) by inserting an implant or device into a sleeve or mesh, containing or coated therewith a compound or composition of the invention; (e) making the implant or device alone with a compound or composition of the invention; or (f) otherwise adapting the implant or device to deliver a compound of the invention. In certain embodiments, the composition should adhere tightly to the implant or device during storage and insertion. The compound or composition of the invention preferably also should not degrade during storage, prior to insertion, or after heating to body temperature after being placed in the body (when required). In addition, it should preferably coat the implant or device smoothly and uniformly, with even distribution of the compound of the invention but without altering the contours of the stent. In preferred embodiments of the invention, the implant or device should provide for an even, predictable, sustained release of the compound or composition of the invention into the tissue surrounding the implant or device upon insertion. In the case of vascular sten, in addition to the above properties, the composition should not render the stent thrombogenic (the ability to clot blood) or cause significant blood flow turbulence (more than that caused by the stent itself when uncoated).

For stents, a wide variety of stents may be designed to contain and / or release the compounds or compositions of the invention, including oesophageal stents, gastrointestinal stents, vascular stents, biliary stents, colon stents, pancreatic stents, ureteral and urethral stents, and urethral stents. lacrimal, eustachian tube stents, fallopian tube stents, and tracheal / bronchial stents (see, e.g., U.S. Patent No. 6,515,016). Stents can be readily obtained from commercial sources or made by known techniques. Representative examples include the stents described in US Patent No. 4,768,523, entitled "Hydrogel Adhesive"; in US Patent No. 4,776,337, entitled "Expandable Intraluminal Graft and Method and Apparatus for Implanting and Expandable Intraluminal Graft"; in US Patent No. 5,041,126, entitled "Endovascular Stent and Delivery System"; in US Patent No. 5,052,998, entitled "Indwelling Stent and Method of Use"; in US Patent No. 5,064,435, entitled "Self Expanding Prosthesis Having Stable Axial Length"; in US Patent No. 5,089,606, entitled "Water-insoluble Polysaccharide Hydrogel Foam for Medical Applications"; in description

U.S. Patent No. 5,147,370, entitled "Nitinol Stent for Hollow Body Conduits"; in US Patent No. 5,176,626 entitled "Indwelling Stent"; in US Patent No. 5,213,580, entitled "Biodegradable Polymeric Endoluminal Sealing Process"; and in US Patent No. 5,328,471, entitled "Method and Apparatus for Treatment of Focal Disease in Hollow Tubular Organs and Other Tissue Lumens".

As discussed above, a stent coated (or otherwise adapted to be released) with the compositions of the invention can be used to eliminate vascular obstruction and prevent restenosis and / or reduce the rate of restenosis. In accordance with other aspects of the invention, stents coated (or otherwise adapted to be released) with the compositions of the invention are intended to increase the lumen of the channels in the body. In particular, a stent having a generally tubular structure and a surface coated (or otherwise adapted to be released) with a compound or composition of the invention may be inserted into the channel such that the channel expands. In certain embodiments, a stent coated (or otherwise adapted to release) the compositions of the invention can be used to eliminate obstruction of the biliary, gastrointestinal, esophageal, tracheobronchial, oval, or vascular systems.

In another embodiment, the compounds of the invention find use in the treatment of cancer, comprising administering a therapeutically effective amount of a compound of the invention described herein to a subject in need thereof. In certain embodiments, the compounds of the invention are useful in the treatment of solid and non-solid tumors. It will be appreciated that the compounds and agents of the invention may be administered in any amount and by any route of administration that is effective in treating cancer. Accordingly, as used herein, the phrase "effective amount" refers to an amount of an agent sufficient to kill or inhibit the growth of tumor cells, or to an amount sufficient to reduce the growth of tumor cells. The exact amount required varies from one subject to another, depending on the species, age, and general condition of the subject, the severity of the disease, the particular anticancer agent, its mode of administration, etc. The compounds of the invention are preferably formulated in dosage unit form for ease of administration and uniformity of dosage. As used herein, the expression "dosage unit form" refers to a physically discrete unit of therapeutic agent appropriate for the patient to be treated. However, it should be appreciated that the total daily amount of the compounds and compositions of the present invention will be determined by the attending physician within the scope of significant medical judgment. The specific therapeutically effective dose level for any particular patient or organism will depend on a number of factors, including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition used; the age, body weight, general health, sex and nutrition of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; duration of treatment; drugs used in combination or concomitantly with the specific compound employed; and similar factors well known in medicine (see, e.g., Goodman and Gilman's, "The Pharmacological Basis of Therapeutics", 10th edition, A. Gilman, J. Hardman, and L. Limbird, eds., McGraw-Hill Press, 155-173 , 2001).

Implants or other surgical or medical devices may be coated (or otherwise adapted to release) with the compounds and compositions of the invention. The method of preventing restenosis comprises inserting a generally tubular stent into an occluded blood vessel whose surface is coated (or otherwise adapted to be released) with a compound or composition of the invention such that the obstruction is eliminated and the compound or composition of the invention is provided in amounts. sufficient to prevent restenosis and / or reduce the rate of restenosis. The method of preventing restenosis comprises inserting a generally tubular stent into an occluded blood vessel whose surface is coated (or otherwise adapted to be released) with a compound or composition of the invention such that the obstruction is eliminated and the compound or composition of the invention is provided in amounts. sufficient to inhibit the proliferation of smooth muscle cells.

The method of increasing the lumen of a lumen in an organism comprises inserting into the lumen a stent having a generally tubular structure whose surface is coated (or otherwise adapted to be released) with a compound or composition of the invention such that the lumen is enlarged. In some embodiments, the lumen of the body's lumen is enlarged to eliminate obstruction in the biliary, gastrointestinal, esophagus, tracheo / bronchial system, fallopian tube, or vascular system.

The method of eliminating obstruction of the biliary system includes inserting a generally tubular stent into the bile duct, the surface of which is coated (or otherwise

A compound or composition according to the invention such that the obstruction of the biliary system is eliminated. In short, tumor overgrowth in the common bile duct causes progressive, life-threatening cholestatic jaundice. In general, the biliary system that drains bile from the liver into the duodenum is most often blocked by (1) a tumor composed of cells in the bile duct (bile duct cancer), (2) a tumor that affects the bile duct (e.g., pancreatic cancer), or (3) a tumor causing outside pressure and compression of the bile duct (e.g. enlarged lymph nodes). Both primary biliary tumors and other neoplasms that can compress the bile ducts can be treated with stents, implants, and other surgical or medical devices that may be coated (or otherwise adapted to be released) with the compositions of the invention. One example of primary biliary tumors is adenocarcinomas (also referred to as Klatskin tumors when they are located at a bifurcation of the common hepatic duct). Such neoplasms are also referred to as bile carcinomas, common bile duct carcinomas or biliary adenocarcinomas. Benign tumors that affect the bile duct (e.g. biliary adenoma) and, in rare cases, squamous cell carcinomas of the bile duct and gallbladder adenocarcinomas, can also cause constriction of the bile ducts and therefore cause obstruction of the biliary system. Compression of the biliary system is most often caused by cancers of the liver and pancreas, which compress and thereby block the ducts. Most pancreatic cancers come from the cells of the pancreatic ducts. It is a very lethal form of cancer (5% of all cancer deaths; 26,000 new cases annually in the US) with an average survival of 6 months and a 1-year survival rate of only 10%. When such tumors are located in the head of the pancreas, they often obstruct the biliary system, significantly worsening the patient's quality of life. While all types of pancreatic cancers are generally referred to as "pancreatic cancer," there are histological subtypes including adenocarcinoma, adenocarcinoma, adenocystic carcinoma, and acinar adenocarcinoma. Liver cancers, as outlined above, can also cause constriction of the biliary system and thus obstruction of the bile ducts.

The bile stent may first be inserted into the bile duct in one of several ways: from the upper end by inserting the needle through the abdominal wall and through the liver (percutaneous transhepatic cholangiogram or "PTC"); from the lower end by catheterization of the bile duct through an endoscope inserted through the mouth, stomach and duodenum (endoscopic retrograde cholangiogram or "ERCP"); or by direct incision during a surgical procedure. In some embodiments, pre-insertion, PTC, ERCP, or direct visualization during surgery is performed to determine the correct position of the inserted stent. The guide wire is then guided through the defect and a delivery catheter slides over it, allowing the stent to be inserted in its folded form. When the diagnostic test was PTC, the guide wire and delivery catheter are inserted through the abdominal wall, while when the baseline test was ERCP, the stent can be inserted through the oral cavity. The stent is positioned under radiological, endoscopic or direct visual control, paying particular attention to placing it exactly in the stricture in the bile duct. The delivery catheter is then removed, leaving the stent standing as a scaffold to hold the bile duct open. Another cholangiogram may be taken to ensure that the stent is properly positioned.

The method for eliminating an obstruction of the esophagus comprises inserting an esophageal stent into the esophagus, having a generally tubular structure whose surface is coated (or otherwise adapted to release) with a compound or composition of the invention such that the esophageal obstruction is eliminated. In short, the esophagus is an empty tube through which food and liquids are transported from the mouth to the stomach. Esophageal cancer or infiltration by cancer that occurs in nearby organs (e.g. stomach or lung cancer) makes it impossible to swallow food or saliva. In some embodiments, a preliminary examination, typically involving barium swallowing or an endoscopy, is performed to determine the appropriate position of an inserted stent. The catheter or endoscope can then be inserted through the mouth and the guide wire is pushed through the blockage site. The stent delivery catheter is advanced around the guide wire under radiological or endoscopic guidance and the stent is positioned exactly at the site of the esophageal stricture. Post-insertion testing, usually barium swallowing, may be performed to confirm proper stent placement.

The method for eliminating a colon obstruction includes inserting a colon stent into the colon, which has a generally tubular structure whose surface is coated (or otherwise adapted).

To be released) with a compound or composition of the invention such that the obstruction of the esophagus is eliminated. Briefly, the colon is an empty tube through which digested food and waste materials are transported from the small intestine to the anus. Cancer of the rectum and / or colon or infiltration by neoplasms appearing in adjacent organs (e.g., uterine, ovarian, bladder cancer) makes it impossible to remove feces from the intestine. In some embodiments, a preliminary examination, typically a barium infusion or a colonoscopy, is performed to determine the appropriate position of an inserted stent. The catheter or endoscope can then be inserted through the anus and the guide wire is pushed through the blockage site. The stent delivery catheter is advanced around the guide wire under radiological or endoscopic guidance and the stent is positioned exactly at the site of the stricture in the colon or rectum. Post-insertion testing, usually a bar-infusion X-ray, may be performed to confirm proper stent placement.

The method for eliminating a laryngeal / bronchial obstruction involves inserting a laryngeal / bronchial stent into the larynx or bronchi of a generally tubular structure whose surface is coated (or otherwise adapted to be released) with a compound or composition according to the invention such that the laryngeal / bronchial obstruction is eliminated. . In short, the larynx and bronchi are the tubes through which air flows from the mouth and nose to the lungs. A blockage of the larynx by cancer, infiltration by a tumor that occurs in nearby organs (e.g. lung cancer), or collapse of the larynx or bronchi due to softening of the cartilage (weakening of the cartilage rings) makes it impossible to breathe. In some embodiments, a pre-stent insertion examination, typically an endoscopy, is performed to determine the appropriate position of the stent being inserted. The catheter or endoscope can then be inserted through the mouth and the guide wire is pushed through the blockage site. The stent delivery catheter slides around the guide wire to allow the stent to be inserted in its folded form. The stent is placed under radiological or endoscopic guidance to position it accurately in the constriction. The delivery catheter can then be removed, leaving the stent standing as a self-supporting scaffold. Post-insertion examination, usually bronchoscopy, may be performed to confirm correct placement.

The method for eliminating urethral obstruction comprises inserting a urethral stent into the urethra having a generally tubular structure whose surface is coated (or otherwise adapted to be released) with a compound or composition of the invention such that the urethral obstruction is eliminated. In short, the urethra is the tube for emptying the bladder through the penis. Externally induced narrowing of the urethra as it passes through the prostate due to an enlarged prostate occurs in almost all men over the age of 60 and causes progressive difficulty in urinating. In some embodiments, a preliminary examination, typically an endoscopy or urethrogram, is first performed to establish the appropriate position of the insertion stent that is above the external urinary sphincter at the lower end and adjacent to the jet with the bladder neck at the upper end. The endoscope or catheter is then inserted through the opening in the penis and the guide wire pushed into the bladder. The stent delivery catheter is advanced around the guide wire to allow stent insertion. The delivery catheter is then removed so that the stent expands into place. Post-insertion testing, usually endoscopy or retrograde urethrogram, may be performed to confirm proper stent placement.

The method for eliminating vascular obstruction includes inserting a vascular stent into a blood vessel, the generally tubular structure whose surface is coated (or otherwise adapted to be released) with a compound or composition of the invention such that the vessel obstruction is eliminated. Briefly, stents in a wide variety of blood vessels, both in arteries and veins, to prevent recurrence of failed angioplasty site stenosis, to treat stenoses that could fail with angioplasty, and to treat postoperative stenosis (e.g., transplant stenosis). for dialysis). Suitable sites include, but are not limited to, the iliac, renal, and coronary arteries, the superior vena cava, and dialysis grafts. In some embodiments, angiography is first performed to locate the stent insertion site. This is usually accomplished by injecting radiopaque through a catheter inserted into an artery or vein during x-ray examinations. The catheter can then be inserted percutaneously or surgically into the femoral artery, brachial artery, femoral vein, or brachial vein and directed to the appropriate blood vessel by routing it through the bloodstream with fluoroscopic tracking. The stent can then be brought into place

Vascular stenosis. The post-insertion angiogram can also be used to confirm proper stent placement.

Furthermore, after formulating with a suitable pharmaceutically acceptable carrier in the desired dose, the pharmaceutical compositions of the invention can be administered to humans and animals orally, rectally, parenterally, intraventricularly, vaginally, intraperitoneally, topically (e.g. as powders, ointments or drops), buccal, as a spray. mouth or nose etc. depending on the severity of the infection being treated. In some embodiments, the compounds of the invention can be administered at doses of about 0.001-50 mg / kg, about 0.01-25 mg / kg, or about 0.1-10 mg / kg of the subject's body weight per day, one or more times per day, to achieve the desired therapeutic effect. Furthermore, it should be appreciated that a patient may be administered doses of less than 0.001 mg / kg or greater than 50 mg / kg (e.g. 50-100 mg / kg). In some embodiments, the compounds are administered orally or parenterally.

Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredients, liquid dosage forms may contain commonly used inert diluents, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cotton, peanut oil, corn, sprout, olive oil, castor and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycols and sorbitan fatty acid esters, and mixtures thereof. Besides inert diluents, the oral compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, and perfuming agents.

Injectables, for example sterile injectable aqueous or oleaginous suspensions, may be formulated in a known manner with suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may further be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Acceptable diluents and solvents that can be used include water, USP Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are often used as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

Injectables may be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which may be dissolved or dispersed in sterile water or other sterile injection medium before use.

In order to prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This can be achieved by the use of a liquid suspension or a crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends on its rate of dissolution, which in turn may depend on crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is achieved by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. The rate of drug release can be controlled depending on the drug to polymer ratio and the nature of the particular polymer used. Examples of other biodegradable polymers include (poly (orthoesters) and poly (anhydrides). Depot injectable forms are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of the invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or suppository wax which are solid at ambient temperature but liquid at body temperature. so that they melt in the rectum or vaginal cavity and release the active ingredient.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In solid dosage forms, the active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and / or a) fillers or fillers such as starches, lactose, sucrose, glucose, mannitol and silicic acid. , b) binders such as e.g. carboxymethyl cellulose, algae42

Nanates, gelatin, polyvinylpyrrolidinone, sucrose and acacia, c) humectants such as glycerin d) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate e) dissolution retarding agents such as paraffin; f) absorption accelerators such as quaternary ammonium compounds g) wetting agents such as e.g. cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethyl glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also contain buffering agents.

Solid compositions of a similar type can also be used as fillers in soft and hard filled gelatin capsules using excipients such as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like. Solid dosage forms, tablets, dragees, capsules, pills, and granules can be used. be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulation art. They may optionally contain opacifying agents and may also be of such a composition that they release the active ingredient (s) only or preferentially in a certain part of the intestinal tract, possibly in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type can also be used as fillers in soft and hard filled gelatin capsules using excipients such as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like.

The active ingredients can also be in micro-encapsulated form with one or more of the excipients set out above. The solid dosage forms, tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings, and other coatings well known in the pharmaceutical formulation art. In such solid dosage forms, the active ingredient may be admixed with at least one inert diluent, such as sucrose, lactose, and starch. Such dosage forms may also contain, as is commonly practiced, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such as magnesium stearate and microcrystalline cellulose. For capsules, tablets, and pills, the dosage forms can also contain buffering agents. They may optionally contain opacifying agents and may also be of such a composition that they release the active ingredient (s) only or preferentially in a certain part of the intestinal tract, possibly in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

Dosage forms for the topical or transdermal administration of a compound of the invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active ingredient is mixed under sterile conditions with a pharmaceutically acceptable carrier and any required preservatives or buffers. Ophthalmic formulations, ear drops, and eye drops are also contemplated as being within the scope of the invention. The invention further contemplates the use of transdermal patches that have the added benefit of providing controlled delivery of a compound to the body. Such dosage forms are made by dissolving or dispensing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by the use of a rate-regulating membrane or by dispersing the compound in a polymer matrix or gel.

It will be appreciated that the compounds and pharmaceutical compositions of the invention may be formulated and used in combination therapies, meaning that the compounds and pharmaceutical compositions may be formulated or administered simultaneously, before or after one or more other therapeutic or medical procedures desired. The particular combination of therapies (therapeutic agents or procedures) to be used in a combination therapy regimen will take into account the compatibility of the desired therapeutic agents and / or procedures and the desired therapeutic effect to be achieved. It should also be realized that the applied therapies may provide the desired effect on the same disorder (e.g. a compound of the invention may be co-administered with another anti-cancer agent) or may provide other effects (e.g., combating any adverse effects). For example, other anti-cancer therapies or agents that may be useful in combination with the anti-cancer agents of the invention include surgery, radiation therapy (including by way of example only, γ radiation, neutron beam radiation, electron beam radiation therapy, proton therapy, brachytherapy, and systemic isotope administration. radioactive substances to replace only a few), hormone therapy, use of biological response modifiers (interferons, interleukins and tumor necrosis factor (TNF) to name a few), hyperthermia and cryotherapy, measures to alleviate any undesirable side effects (e.g. antiemetics) and other approved chemotherapeutic drugs, including, but not limited to, alkylating drugs (mechlorethamine, chlorambucil, cyclophosphamide, melphalan, ifosfamide), antimetabolites (methotrexate), purine antagonists and pyrimidine antagonists (6-mercaptopurine, 5-ferabilouracil, gemcitabine), mitotic spindle poisons (vinblastine, vincristine, vinorelbine, paclitaxel), podophyllotoxins (etoposide, irinotecan, topotecan), antibiotics (doxorubicin, bleomycin, mitomycin), nitrosoureas (carmustine, lomustine), inorganic ions (cisplatin, carboplatin), enzymes (asparaginase) and hormones (tamoxifen, Ieuprolide, fIutamide and megestrol) to name a few. For a more detailed discussion of updated cancer therapies, see The Merck Manual, 17th ed. 1999. See also the National Cancer Institute (NCI) website (www.nci.nih.gov) and the Food and Drug Administration (FDA) website for a list of FDA-approved cancer drugs (www.fda.gov/cder/cancer/druglistframe - see Annex A).

In certain embodiments, the pharmaceutical compositions of the invention further comprise one or more therapeutically active (e.g., chemotherapeutic and / or palliative) ingredients. As used herein, the term "palliative" refers to a treatment that is focused on alleviating the symptoms of a disease and / or the side effects of a therapeutic regimen, but which does not lead to a cure. Palliative treatment includes e.g. Also, chemotherapy, radiation therapy, and surgery can be used palliatively (e.g., to reduce symptoms without healing; e.g., to shrink tumors and reduce pressure, bleeding, pain, and other symptoms of cancer).

Treatment kits

In other aspects, the invention relates to a kit for conveniently and efficiently carrying out the methods of the invention. Generally, the pharmaceutical pack or kit comprises one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Such kits are particularly suitable for the administration of solid oral forms such as tablets or capsules. Such a kit preferably comprises a number of unit doses, and may also contain a packet of the doses arranged in the order of their intended use. Booster elements, e.g. in the form of numbers, letters or other characters, or with a calendar insert may be used as needed to indicate which days in the treatment regimen during which the dose can be taken. Alternatively, placebo doses or calcium dietary supplements, in a form similar to or different from the dose of the pharmaceutical compositions, may be included to provide a dose kit to be taken daily. The container (s) may also be accompanied by information in a form prescribed by a governmental agency regulating the production, use, or sale of pharmaceutical products, indicating that the agency has authorized the manufacture, use, or sale of the drug for human administration.

The following representative examples are intended to help illustrate the invention and are not intended to limit the scope of the invention nor are they so interpreted.

The following examples contain important additional information, exemplary embodiments, and guidance on how variations can be made to the practice of the invention in its various forms and equivalents.

Experimental part

One of ordinary skill in the art has at his disposal the recognized literature on peptide chemistry to outline, in conjunction with the information contained herein, guidance on synthetic strategies, protecting groups, and other ingredients and methods useful in the synthesis of the compounds of the invention.

The various sources cited herein provide useful background information regarding the preparation of compounds similar to the described compounds of the invention or appropriate intermediates, as well as information regarding the formulation, use and administration of these compounds that may be of interest.

In addition, one skilled in the art can follow the specific guidance and examples provided in this document for various exemplary compounds and intermediates for their synthesis.

The compounds of the invention and their preparation will be better understood from the examples which illustrate certain methods of making or using these compounds. However, it should be understood that these examples do not limit the invention. Variants of the invention, now known or further developed, are considered to fall within the scope of the invention as described herein and as claimed below.

PL 219 737 B1

According to the invention, any of the available techniques may be used to prepare or manufacture the compounds of the invention or compositions containing them. For example, various solution phase synthesis methods may be used, such as detailed below. Alternatively or additionally, the compounds of the invention may be prepared using any of the known variations of combinatorial techniques, parallel synthesis, and / or solid phase synthesis methods.

It will be appreciated as described below that various compounds of the invention can be synthesized by the methods described. The starting materials and reagents used to prepare such compounds are available from commercial suppliers such as Aldrich Chemical Company (Milwaukee, WI), Bachem (Torrance, CA), Sigma (St. Louis, MO) or are prepared by methods well known to those skilled in the art according to the procedures described in such reference works as Fieser and Fieser 1991, "Reagents for Organic Synthesis", Volumes 1-17, John Wiley and Sons, New York, NY, 1991; Rodd 1989 "Chemistry of Carbon Compounds", Vol. 1-5 and Supplements, Elsevier Science Publishers, 1989; "Organic Reactions", volumes 1-40, John Wiley and Sons, New York, NY, 1991; March 2001, "Advanced Organic Chemistry", 5. edition, John Wiley and Sons, New York, NY; and Larock 1990, "Comprehensive Organic Transformations: A Guide to Functional Group Preparations", 2nd Edition, VCH Publishers. These schemes simply illustrate certain methods by which the compounds of the present invention can be synthesized, and various modifications may be made to the schemes that will occur to those skilled in the art based upon the disclosure given herein.

The starting materials, intermediates, and compounds of the invention can be isolated and purified by known techniques, including filtration, distillation, crystallization, chromatography, and the like. They can be characterized by a variety of known methods, including physical constants and spectral data.

Certain examples of the invention are listed below with the referenced compound numbers.

<td>Relationship</td><td>Building</td>

<td>ER-803840 (Hemiasterlina) - reference compound</td><td>oh toto ^{Me 0}N toC OH^{H 0}</td>

<td>ER-806718</td><td>H-Cl about the track ^Me ° AA</td>

<td>ER-806735</td><td>H — Cl -_at about AA ^{Me 0}AHA</td>

<td>ER-806748</td><td>H-Cl "0 AA ^{Me 0}óAa ^</td>

<td>ER-806749</td><td>H-CI. o track w® o 0 'ϊΗτχτ ™</td>

<td>ER-807078 single diastereoisomer</td><td>Me OA Me</td>

PL 219 737 B1

<img file="PL219737B1_D0047.tif" />

PL 219 737 B1

<td>Relationship</td><td colspan="7">Building</td>

<td>ER-807370</td><td colspan="2">Me 0</td><td> 0</td><td> 7</td><td>ζ about</td><td>Me</td><td>WHAT₂Et</td>

<td></td><td></td><td>Me</td><td>c</td><td></td><td>k</td><td>Me</td><td></td>

<td>ER-807371</td><td></td><td>AND,</td><td></td><td>n</td><td></td><td></td><td>_XWHAT₂H.</td>

<td></td><td></td><td> □</td><td></td><td></td><td>about</td><td>Λ. ¹</td><td></td>

<td></td><td>Me</td><td> 0 '</td><td></td><td>r</td><td>Me</td><td></td><td>° CO₂Me</td>

<td>ER-807800</td><td> 0</td><td>Χ'Ν</td><td>J.</td><td>Y about</td><td>Y</td><td>YY /</td><td></td>

<td>ER-807860</td><td colspan="2">Mo ₀cY</td><td></td><td> 0</td><td>Me</td><td> 0</td><td>WHAT₂t-Bu N ^ \</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>ABOUT</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>ys</td>

<td>ER-807861</td><td>Me AND</td><td>p </td><td></td><td>Y</td><td>Me</td><td> 0</td><td>° · ν-ΝΗ</td>

<td></td><td> 0</td><td>Yn H.</td><td></td><td>Y 0</td><td>Y</td><td></td><td>Ό</td>

<td></td><td></td><td>Me AND</td><td> 0</td><td>s</td><td>Y</td><td>Me</td><td> 0</td>

<td>ER-807874</td><td>c</td><td>y</td><td>X</td><td>H.</td><td>Y 0</td><td>s</td><td>&lt;Υοβ</td>

<td></td><td></td><td>Me</td><td> 0</td><td>s</td><td>Y</td><td>Me</td><td> 0</td>

<td>ER-807875</td><td>c</td><td>y</td><td>Jk</td><td>H.</td><td>Y about</td><td>λγ ^</td><td>Υγοβ</td>

<td></td><td></td><td>Me</td><td> 0</td><td>s</td><td>Y</td><td>Me</td><td> 0</td>

<td>ER-807880</td><td>c</td><td>y</td><td>Jk</td><td>H.</td><td>Y 0</td><td>X</td><td>ΥόΕ (</td>

<td></td><td></td><td>Me</td><td> 0</td><td>s</td><td>k</td><td>Me</td><td> 0</td>

<td>ER-807884</td><td></td><td>y</td><td>Λ</td><td>'h</td><td>7 about</td><td>s</td><td>OH</td>

PL 219 737 B1

<td>Relationship</td><td colspan="2">Building</td>

<td>ER-807885</td><td colspan="2">Me 0 Y Me o ΥΛϊ 5 ΥΜ</td>

<td>ER-807886</td><td colspan="2">M® 0 '' J Me 0 5 yM</td>

<td>ER-807911</td><td>Me o cy</td><td>'Me 0 po₂H. \ Y ϊ Yj O</td>

<td>ER-807961</td><td>Y θ Y</td><td>Y Me O ΑαλΑ »⁰</td>

<td>ER-807974</td><td colspan="2">'' 'Ύ' about Me 0 γΑ $ γγΛ⁰</td>

<td>ER-807981</td><td> «^β 0 Υ-</td><td>'' - '' Me 0 V; Yo</td>

<td>ER-807982</td><td>· Ο Μ Y?</td><td>^X Me 0.-OMe τΥΥ</td>

<td>ER-807983</td><td>«Ο 0&gt; Exh</td><td>Me 0 CO, Me ΥΧ ...... Y OH</td>

<td>ER-808031</td><td colspan="2">Y 0 'YY' Me 0 γ^Νγ^ζ^ Ν '^ γγ^Ν&gt; ^ Υγ^Λ^ ΟΕί</td>

<td>ER-808032</td><td colspan="2">Me θ _{Me 0}Y ^ ayY¹'⁰”</td>

PL 219 737 B1

<td>Relationship</td><td colspan="7">Building</td>

<td></td><td>Me |</td><td>about ^x</td><td></td><td>Me</td><td></td><td> 0</td><td></td>

<td>ER-808034</td><td></td><td>yN</td><td></td><td>, Ν_χ</td><td>γγ</td><td></td><td>OH</td>

<td> -</td><td></td><td>H.</td><td> 0</td><td></td><td></td><td></td><td></td>

<td></td><td>Me</td><td colspan="2">o A /</td><td>Me</td><td></td><td> 0</td><td></td>

<td>ER-808035</td><td>c</td><td>AND</td><td colspan="2">AND'⁰ Y</td><td>behind</td><td></td><td>^XOH</td>

<td></td><td>Me |</td><td> 0 ^X</td><td colspan="2">OMe γ Me</td><td></td><td> 0</td><td></td>

<td>ER-808139</td><td></td><td></td><td></td><td>N</td><td>/ y</td><td></td><td>OEt</td>

<td></td><td></td><td>H.</td><td> 0</td><td></td><td></td><td></td><td></td>

<td></td><td>Me</td><td>about </td><td>AND</td><td>Me</td><td></td><td> 0</td><td></td>

<td>ER-808140</td><td>AND</td><td>AND"'</td><td></td><td>AND</td><td>/ s.</td><td></td><td>OEt</td>

<td></td><td>AND</td><td>H.</td><td>T. 0</td><td></td><td>Y</td><td></td><td></td>

<td></td><td>Me AND</td><td>about ^v</td><td>AND</td><td>Me</td><td></td><td> 0</td><td></td>

<td>ER-808141</td><td></td><td>An '</td><td>T.</td><td>s</td><td>AA</td><td>AND</td><td>OEt</td>

<td></td><td>AND</td><td>H.</td><td> 0</td><td>J.</td><td>at</td><td></td><td></td>

<td></td><td>Me</td><td>about ^s</td><td colspan="2">OMe Y Me</td><td></td><td> 0</td><td></td>

<td>ER-808145</td><td>.N ^</td><td>ANDⁿ</td><td></td><td>, N</td><td>yy</td><td></td><td>OH</td>

<td></td><td></td><td>H.</td><td> 0</td><td></td><td></td><td></td><td></td>

<td></td><td>Me</td><td>about </td><td>AND</td><td>Me</td><td></td><td> 0</td><td></td>

<td>ER-808146</td><td>AND</td><td>ANDⁿ</td><td>AND</td><td>Ά</td><td>zz</td><td></td><td>^ OH</td>

<td></td><td>AND</td><td>and H.</td><td> 0</td><td></td><td>Y</td><td></td><td></td>

<td></td><td>Me</td><td> 0</td><td>AND</td><td>Me</td><td></td><td> 0</td><td></td>

<td>ER-808147</td><td></td><td>An</td><td></td><td> .&lt;</td><td>AA</td><td></td><td>OH</td>

<td></td><td>AND</td><td>JH</td><td>AND 0</td><td>Y</td><td>ABOUT</td><td></td><td></td>

<td></td><td>Me AND</td><td>H 7</td><td>Me</td><td></td><td> 0</td><td></td><td>^ -OH</td>

<td>ER-808161</td><td>Λ</td><td>M.</td><td></td><td colspan="2">γγΧ '</td><td></td><td></td>

<td></td><td>at</td><td>H.</td><td>⁰ Y</td><td></td><td></td><td></td><td></td>

<td></td><td>Me AND</td><td> 0</td><td colspan="2">Y 'Me</td><td></td><td> 0</td><td></td>

<td>ER-808166</td><td></td><td>H.</td><td colspan="2">° ^ A</td><td>Ύ-</td><td></td><td>Ό</td>

<td></td><td>Me and</td><td> 0</td><td colspan="2">Me</td><td></td><td> 0</td><td></td>

<td>ER-808167</td><td>Υ^Νγ_χ</td><td>rA</td><td>\ Y^N</td><td></td><td>γ</td><td colspan="2"></td>

<td></td><td></td><td>H.</td><td> 0 _</td><td></td><td></td><td colspan="2">H.</td>

PL 219 737 B1

<td>Relationship</td><td colspan="4">Building</td>

<td></td><td>Me AND</td><td>⁰</td><td>it's Me |</td><td> 0</td>

<td>ER-808168</td><td></td><td>H.</td><td>here-there</td><td>J k</td>

<td>ER-808169</td><td>Me 1 / N</td><td> 0 *^ZS'N^XH.</td><td>this \ zs \ / </td><td>IX / χγ -^H.</td>

<td></td><td>Me AND</td><td>V</td><td>Me</td><td> 0</td>

<td>ER-808170</td><td>this</td><td>Π</td><td>this</td><td>it ^ -_N^ toto \ to \ H.</td>

<td></td><td>Me</td><td>0 this</td><td>^X Me</td><td>0 ^ .- ΟΜθ</td>

<td>ER-808171</td><td>.N</td><td>* ^ lto H.</td><td>⁰ this"</td><td>νΧ</td>

<td></td><td></td><td></td><td></td><td>OMe</td>

<td></td><td>Me</td><td>° is ^</td><td>'Me</td><td>&lt;? pity</td>

<td>ER-808172</td><td></td><td>H.</td><td>toto * 0</td><td>tontoU 1 ^H. ÓH</td>

<td></td><td>Me</td><td>⁰</td><td>^x Me</td><td>ϋ (⁸ )</td>

<td>ER-808173</td><td></td><td>/this H.</td><td>1 , / N \ /⁰ this-</td><td>toto ^_Nto — s H.</td>

<td></td><td>Me 1</td><td></td><td>this</td><td>0 Me II i</td>

<td>ER-808174</td><td> 0</td><td>AJ</td><td>AND⁰ χ '</td><td>this -_{N Me}^H.</td>

<td></td><td>Me 1</td><td> 0</td><td>this^e</td><td> 0</td>

<td>ER-808175</td><td>c</td><td>AND</td><td>this about /</td><td>here</td>

<td></td><td>Me AND</td><td>about it</td><td>Me</td><td>About ν ^</td>

<td>ER-808176</td><td></td><td>this H.</td><td>⁰ it ^</td><td>this</td>

<td></td><td>Me 1</td><td>ABOUT</td><td>it's Me AND</td><td> 0 —°^H.</td>

<td>ER-808177</td><td></td><td>H.</td><td>L.N about it</td><td>toó</td>

<td></td><td>ί Me j 1</td><td> °</td><td>Me 1</td><td> 0</td>

<td>ER-808178</td><td></td><td>H.</td><td>⁰ it ^</td><td>J it</td>

<td></td><td></td><td></td><td></td><td> \=/</td>

PL 219 737 B1

<td>Relationship</td><td colspan="5">Building</td>

<td>ER-808179</td><td>about Y Y¹</td><td colspan="2">Me⁰ YX</td><td> 0</td><td>"WHAT₂Me WITH</td>

<td></td><td></td><td>Y 'i'⁶</td><td></td><td> 0</td><td>, .- OMe</td>

<td>ER-808180</td><td>ΛΑ</td><td>AND</td><td>AA</td><td></td><td></td>

<td></td><td>M. ^H.</td><td>about Y</td><td></td><td></td><td></td>

<td></td><td>Me o '</td><td colspan="2">'Y Me</td><td></td><td>about</td>

<td>ER-808181</td><td>JL Γ YN II H.</td><td> 0</td><td></td><td>Yy</td><td>JL, Me &lt;N 1 Me</td>

<td></td><td> “· 0 ^x</td><td>X Me</td><td></td><td>ABOUT</td><td>° Y-NHj</td>

<td>ER-808182</td><td>.N. JL. 1 ^N1 JH</td><td>ABOUT _χ</td><td>-Ay,</td><td></td><td>ń</td>

<td></td><td>M. ₀ \</td><td>/ 'Me</td><td></td><td> 0</td><td>Y-OMe</td>

<td>ER-808183</td><td>.N. JL. 1 ^NII H.</td><td> 0</td><td>AA</td><td></td><td>AND</td>

<td>ER-808206</td><td>?. &lt;y</td><td>Λ</td><td>Me -^Nyy</td><td>Ά</td><td>0 A ^ OEt</td>

<td></td><td>y ΐ</td><td>Y</td><td>Me</td><td></td><td>about</td>

<td>ER-808209</td><td></td><td>AND</td><td>X</td><td>Y,</td><td>Υ'ΌΕΙ</td>

<td>ER-808210</td><td>? · γ</td><td>Λ</td><td>Me</td><td></td><td>ABOUT X ^ OEt</td>

<td>ER-808216</td><td>Me 0 --N. [1 ^NIJ H</td><td colspan="2">Y ^ Me 1 AND _with\ / OY \</td><td>'Y</td><td>ΛΑ</td>

<td>ER-808217</td><td>yr</td><td></td><td></td><td> 0</td><td>AND</td>

<td></td><td>æA</td><td>Ύ⁰ /</td><td>γ ^ 'Ύ</td><td></td><td>Ν'Χ,</td>

PL 219 737 B1

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<td>ER-808218</td><td>Υ? Ί</td><td></td><td>Me 1</td><td>0 V »</td><td></td>

<td></td><td>ITN II H.</td><td> 0</td><td> -^N'-YY</td><td>Y</td><td></td>

<td></td><td>Y</td><td>Y</td><td>Me</td><td> 0</td><td></td>

<td>ER-808221</td><td>and JL</td><td></td><td>and</td><td></td><td></td>

<td></td><td>Γ τ</td><td>with</td><td>γγ</td><td> Y ^ 'oh</td><td></td>

<td></td><td></td><td></td><td>⁰ χΑ</td><td></td><td></td>

<td></td><td>Y</td><td>Y</td><td>Me</td><td>about</td><td></td>

<td>ER-808222</td><td></td><td>you</td><td>Vv</td><td> '^&lt;ΪΥ'ΟΗ</td><td></td>

<td></td><td></td><td></td><td>⁰ ^ A</td><td></td><td></td>

<td></td><td>Y i</td><td> &gt;</td><td>Me</td><td> 0</td><td></td>

<td>ER-808223</td><td></td><td></td><td>Yy</td><td></td><td></td>

<td></td><td></td><td></td><td>⁰ AND-</td><td></td><td></td>

<td></td><td>Ύ j</td><td></td><td>With 'Me 1</td><td> 0</td><td></td>

<td>ER-808224</td><td></td><td>N</td><td></td><td>ΥΥ ^ 'ΟΕΙ</td><td></td>

<td></td><td></td><td></td><td>about</td><td></td><td></td>

<td></td><td>Y.</td><td></td><td>A Me 1</td><td>ABOUT</td><td></td>

<td>ER-808225</td><td></td><td></td><td></td><td>ΎΥΥ&gt; Η</td><td></td>

<td></td><td></td><td></td><td>0 Ay</td><td></td><td></td>

<td></td><td>Yf</td><td></td><td>^X Me 1</td><td rowspan="2">IX YSr '·'</td><td></td>

<td>ER-808226</td><td colspan="2">-,ON &lt;YN</td><td>Y^M.Y ^</td><td></td>

<td></td><td colspan="2">H.</td><td>⁰ AND</td><td></td><td></td>

<td></td><td>Ϊ '°</td><td colspan="2">YP Me</td><td> 0</td><td></td>

<td>ER-808248</td><td>about^AND</td><td>WITH</td><td>γ</td><td>Ύ | Χόε *</td><td></td>

<td></td><td></td><td colspan="2">Me</td><td> 0</td><td></td>

<td>ER-808249</td><td> 0</td><td></td><td>YY</td><td></td><td></td>

<td></td><td>Me 0</td><td></td><td>Me</td><td>0 Y-^0H</td><td></td>

<td>ER-808251</td><td>.Ł and JL. IN II H.</td><td>c</td><td>/AND</td><td>different</td><td></td>

<td></td><td>Y p</td><td>r</td><td>Me</td><td>0 ° V-OH</td><td></td>

<td>ER-808253</td><td>y = -</td><td> 0</td><td></td><td>different</td><td></td>

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<td>ER-808301</td><td>Y</td><td>° O ^ isr</td><td>Me 1 .___ χΝ</td><td></td><td>'^ K ^ -Ph 0 V.</td>

<td></td><td></td><td></td><td>⁰ AND'</td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td>

<td>ER-808302</td><td>Y</td><td>⁰ ABOUT</td><td>Me |</td><td></td><td>0 ° A.</td>

<td></td><td></td><td>H.</td><td>o A</td><td>Άγ</td><td>^ANDabout</td>

<td></td><td></td><td></td><td></td><td></td><td>Rh and</td>

<td></td><td></td><td></td><td></td><td></td><td> 0</td>

<td>ER-808303</td><td></td><td></td><td></td><td></td><td>to-Ph</td>

<td></td><td>Y</td><td>° O toto H.</td><td>Me 1 Y ^ N⁰ AND</td><td>Ά</td><td>o ° A^ANDabout</td>

<td>ER-808304</td><td>Y</td><td>° A AND H.</td><td>A Me 1 \ A \ xo χΑ</td><td>-AND</td><td>'~ N --- \ \ _- Pli o ° Y °^Λό</td>

<td>ER-808305</td><td>Y</td><td>0 A. Ά H.</td><td>A Me 1 to / N,. ^⁰ AND</td><td></td><td>at Ά^Λό</td>

<td>ER-808306</td><td>Y</td><td>0 A.</td><td>f Me |</td><td></td><td>.this</td>

<td></td><td>AA</td><td>AND H.</td><td>oh</td><td>Ay</td><td>^Λό</td>

<td>ER-808307</td><td>Y</td><td>° A</td><td>A Me AND</td><td></td><td>.this</td>

<td></td><td>.N</td><td>H.</td><td>Α'-ά⁰ AND</td><td></td><td>^ANDabout</td>

<td>ER-808308</td><td>Y</td><td> 0 ^X** ^ A H.</td><td>A Me and⁰ AND</td><td></td><td>.this Y</td>

PL 219 737 B1

<td>Relationship</td><td>Building</td>

<td>ER-808309</td><td>Y Υ '”' 0 '' 'Y Me 0 V- · ® Y y * y^Λγ^χΝ&gt;^{/ ΐ&gt; ϊ}γ^&lt;'^Ν\</td>

<td>ER-808328</td><td>ΥΥ * "^and 0 'y Me 0 ÓYYYÓ</td>

<td>ER-808329</td><td>0 ^ y-Ph “· 0 Ύ Me 0 (Χ-ipró</td>

<td>ER-808330</td><td>ęO γ "O y Me 0 ° 'r-⁰C ( ⁵ ΪΛΊ ó</td>

<td>ER-808331</td><td>ęO Me 0 'y' Me 0 ° V- ° about</td>

<td>ER-808332</td><td>...... oh OY Me O 'i-'⁰YFFyyFó</td>

<td>ER-808333</td><td>oh 0 y Me 0 γΛγχ-ηό</td>

<td>ER-808334</td><td>0 Y Me 0 Mo 0 y- ° ΥπΎυ ó</td>

<td>ER-808335</td><td>- ov Me o Ύ Me 0 V- ° -&gt; · .JL 4. -k JL. -Λ YJj ^N iD Ly</td>

PL 219 737 B1

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<td>Relationship</td><td>Building</td>

<td>ER-808344</td><td></td>

<td>ER-808345</td><td>0 Y Me 0 ° V ° cyykyó</td>

<td>ER-808357 single diastereoisomer</td><td>^ y '° ^m® ° γΎγ</td>

<td>ER-808358 single diastereoisomer</td><td>^ 'yy °' 'X ^m® ° ¢ 6 jj / k "'</td>

<td>ER-808359 single diastereoisomer</td><td>0 Μ O Ο^Λ5'ϊΧτ^Λ“'</td>

<td>ER-808366 single diastereoisomer</td><td>o Υ “· · u ό'ΧΛ "</td>

<td>ER-808367</td><td>^ ~ y ° ^Μβ ° 0^^s 5 ΧΧ⁰</td>

<td>ER-808368</td><td>'' 'yy ° X ^μ® ^θ</td>

<td>ER-808389</td><td>'' 'Ύ' 0 Υ '' Me 0 yysy-a</td>

<td>ER-808390</td><td>'^ γ^Χ 0 '' Υ Me 0</td>

PL 219 737 B1

<img file="PL219737B1_D0050.tif" />

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<td>ER-808402</td><td>Y</td><td>X Me α⁰ Α</td><td>Ac</td><td></td>

<td>ER-808403</td><td>AX .N Jt J 1 ^NII H.</td><td>f Me 1⁰ Α</td><td colspan="2">θ ^ ΟΜβ Αό</td>

<td>ER-808404</td><td>Yy .N. JL 1 ^N| 'H</td><td>X Me 1⁰ Υ</td><td colspan="2">ο, -^0ΗΑό</td>

<td>ER-808475 single diastereoisomer</td><td>σ</td><td>ο Α Α</td><td> Me rA⁰ aa</td><td> 0 ^^&gt;61</td>

<td>ER-808476 single diastereoisomer</td><td>,,,, σ</td><td>ο aL AND</td><td>Me rA⁰ ΥΥ</td><td>Ο 'Α »</td>

<td>ER-808477 single diastereoisomer</td><td></td><td></td><td>Me υΑ⁰ αα</td><td>Ο A ^ OEt</td>

<td>ER-808478 single diastereoisomer</td><td></td><td>Α</td><td>^Χ Me υΑ⁰ Α \</td><td>0 A ^ oEt</td>

<td>ER-808479</td><td>Λ ° γ</td><td>Λ</td><td>Me Ά</td><td>0 X) Et</td>

<td>ER-808480</td><td>Ie ' .κ AL Γ ^ΝII Η</td><td>Ά Me 1 0</td><td> 0</td><td>WHAT</td>

<td>ER-808481</td><td>γ · Ί .ν Α. Α Γ^ΝII Η</td><td>Me 1 \ ζ^Ν\ ζ ⁰ ΧΑ</td><td> 0</td><td>,WHAT₂Et</td>

<td>ER-808482f</td><td>ύί Al JL Α Γ Ν II Η</td><td>^Χ Me 1 \ ζ^Ν\/ ⁰ Υυ</td><td> 0</td><td>^ COjEt</td>

PL 219 737 B1

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<td></td><td>Tr</td><td>A Me 1</td><td>c</td><td></td><td></td>

<td>ER-808483</td><td>rn II H.</td><td>YX oh</td><td>^&gt; A</td><td>A''A Y ^ OH</td><td></td>

<td></td><td>Yn</td><td>Me</td><td> 0</td><td>COjt-Bu</td><td></td>

<td>ER-808484</td><td></td><td>Y^NY⁰ AND</td><td>άΑ</td><td>X AND</td><td></td>

<td></td><td>Yf</td><td>Ά Me 1</td><td></td><td>0 COjMb</td><td></td>

<td>ER-808485</td><td> 1 ^N</td><td>A Αχ</td><td>A ^ A</td><td>^ nA.</td><td></td>

<td></td><td></td><td>⁰ r</td><td></td><td></td><td></td>

<td> -</td><td>Yr</td><td>A Μ® 1</td><td></td><td>' WHAT_witht-Bu</td><td></td>

<td>ER-808486</td><td></td><td>y /ⁿy</td><td>Αχ</td><td>YA</td><td></td>

<td></td><td></td><td>⁰ /</td><td></td><td></td><td></td>

<td></td><td>yp</td><td>AND ^m® 1</td><td></td><td>about what₂m «</td><td></td>

<td>ER-808487</td><td> 1 ^N1 JH</td><td>Ax^Nx or</td><td> :</td><td>about</td><td></td>

<td>ER-808488</td><td>ΥΐΊ Γ N II H.</td><td>Me 1 o A '</td><td>ABOUT yA</td><td>/WHAT₂H. nYy</td><td></td>

<td></td><td>Yp</td><td>Me 1</td><td> 0</td><td></td><td></td>

<td>ER-808489</td><td> 1 ^N</td><td></td><td>Yf</td><td>'on</td><td></td>

<td></td><td></td><td>⁰ AND'</td><td></td><td>Y ^ Yy_C.q₂h</td><td></td>

<td></td><td>Yf</td><td>A Me 1</td><td></td><td>0 COjMe</td><td></td>

<td>ER-808490</td><td>Γ i ^N</td><td>YA</td><td>aa</td><td>tA</td><td></td>

<td></td><td></td><td>⁰ P.</td><td></td><td></td><td></td>

<td>ER-808491</td><td>P.</td><td>and Y</td><td>^X Me</td><td> 0</td><td></td>

<td>single diastereoisomer</td><td>Γ τ</td><td>^Λ'π'</td><td>AND</td><td>'' '/' OH</td><td></td>

<td></td><td>YJ</td><td></td><td>⁰ AND</td><td></td><td></td>

<td>ER-808492</td><td>p</td><td>and Y</td><td>^X Me</td><td>ABOUT</td><td></td>

<td>single diastereoisomer</td><td>AND</td><td></td><td>AND</td><td>Y / ΌΗ</td><td></td>

<td></td><td>AND</td><td></td><td>⁰ AND</td><td></td><td></td>

<td>ER-808493</td><td></td><td></td><td>Me</td><td> 0</td><td></td>

<td>single diastereoisomer</td><td>AND</td><td rowspan="2"></td><td>Y^N</td><td>Υ'Υ'όη</td><td></td>

<td></td><td>Yr</td><td>⁰ s</td><td></td><td></td>

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PL 219 737 B1

<td>Relationship</td><td>Building</td>

<td>ER-808609</td><td>Y 5 Y ff A, L _c.„</td>

<td>ER-808610</td><td>° Ά^- in ° 0^As iVA "</td>

<td>ER-808656 single diastereoisomer</td><td>A 0 Y «. 0 0^ΛΑ AA</td>

<td>ER-808662 single diastereoisomer</td><td>5 AA "</td>

<td>ER-808674</td><td>Z ° Y r ° τ ^ίΎΑ “</td>

<td>ER-808676</td><td>'' Ά ⁰ AND' ^Me 9 ^N Y,</td>

<td>ER-808677</td><td>γ o 'Me 0</td>

<td>ER-808678</td><td>Yj? “I Cj^! ϊ Yi “</td>

<td>ER-808679</td><td>Y 0 Y 0 Y Me 0 (AA AA "</td>

<td>ER-808680</td><td>Y 0 Q Me 0 q^andss yA "</td>

PL 219 737 B1

<td>Relationship</td><td>Building</td>

<td>ER-808681</td><td>AO T 0 Y Me 0 DĄSj</td>

<td>ER-808682</td><td>top 0 / Me 0 cH Ύ d "¹</td>

<td>ER-808683</td><td>| Here is '' Me 0</td>

<td>ER-808684</td><td>'' 'β o γ * o ddftid¹'”</td>

<td>ER-808685</td><td>Y 0 | o Y Me o Cj η and νΫ¹</td>

<td>ER-808686</td><td>Y 0 T 0 Y Me O</td>

<td>ER-808687</td><td>γ o 9 «, o £ jss Xj “</td>

<td>ER-808688</td><td>Y o 0V Me 0 Cj dxd °</td>

<td>ER-808689</td><td>0 / Me 0 toto⁸ ϊ υΉ</td>

<td>ER-808690</td><td>° A ^Me ° Cd ΐ YY¹'”</td>

PL 219 737 B1

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<td>ER-808693</td><td>''ABOUT ⁰ 'A Me 0 YAAA</td>

<td>ER-808694</td><td>A ° ^ A «β ° YAAA</td>

<td>ER-808695</td><td>A ^ ° ά i ** ° ΥΑχΆ</td>

<td>ER-808696</td><td>Y o Ą Me 0 AAA? A</td>

<td>ER-808697</td><td>A ° C ° A-ΑχΑ ™</td>

<td>ER-808698</td><td>AND ⁰ A Me 0 Ca Χχτ¹'⁰</td>

<td>ER-808699</td><td>Y 0 Ą Me 0 YAAA</td>

<td>ER-808700</td><td>AND ⁰ Ά Me o YYcA</td>

<td>ER-808706 single diastereoisomer</td><td>A ° i ^M.® ° YA^NrA °</td>

<td>ER-808707 single diastereoisomer</td><td>'j' 0 k ^ Young O YYxA "</td>

<td>ER-808708 single diastereoisomer</td><td>-V 0 YA Υ »</td>

PL 219 737 B1

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<td>ER-80870 single diastereoisomer</td><td>C.</td><td>Y</td><td>0 Λ</td><td>ί</td><td>γ 1 " Υύ⁰ Υ ^</td><td></td><td> 0</td><td>OEt</td>

<td>ER-808710 single diastereoisomer</td><td colspan="2">Y G.</td><td>0 Λ</td><td>Η</td><td>(f Υύ⁰ υ '</td><td>Ύ, -</td><td> 0</td><td>OEt</td>

<td>ER-808731</td><td>with</td><td>Ί</td><td> 0</td><td>Ν '</td><td>'X Μβ 1 Υ \ γΝ</td><td></td><td>ο</td><td>OH</td>

<td></td><td></td><td></td><td></td><td></td><td>ο</td><td></td><td></td><td></td>

<td>ER-808732</td><td>y</td><td></td><td>0 Λ</td><td></td><td>κ *</td><td></td><td>ο</td><td>OH</td>

<td></td><td>k</td><td>k</td><td></td><td></td><td>⁰ Υ ^</td><td></td><td></td><td></td>

<td>ER-808774 single diastereoisomer</td><td colspan="2">Y 0</td><td>about Λ</td><td>Η</td><td>γ Μβ 1 Υ * Υχ-^Ν«. Ο γΧ</td><td></td><td> 0</td><td>OH</td>

<td>ER-808775 single diastereoisomer</td><td colspan="2">Y 0</td><td>0 Λ</td><td colspan="2">μ. ys</td><td></td><td>ο</td><td>OH</td>

<td>ER-808777 single diastereoisomer</td><td colspan="2"> 0</td><td>0 Λ</td><td>c Η</td><td>Υ Υύ ο X '</td><td></td><td>ο</td><td>OH</td>

<td>ER-808779 single diastereoisomer</td><td colspan="2">Y 0</td><td>0 Λ</td><td></td><td>And " Υύ⁰ Υ '</td><td></td><td> 0</td><td>OH</td>

<td>ER-808780 single diastereoisomer</td><td colspan="2">Y 0</td><td>0 Λ</td><td>Η</td><td>(Μβ Υύ Ο Υ</td><td>-Ύγ.</td><td> 0</td><td>OH</td>

<td>ER-808815 single diastereoisomer</td><td colspan="2">γ σ</td><td>0 Λ</td><td></td><td>Μβ ν-τ⁰ Υ ^</td><td></td><td> 0</td><td>OEt</td>

PL 219 737 B1

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<td>ER-808816 single diastereoisomer</td><td>'Ύ σ</td><td> 9</td><td>AND⁰</td><td>0 ^ pY ^ OEt</td>

<td>ER-808817 single diastereoisomer</td><td>YS</td><td></td><td>Y r V '⁰</td><td>0 Ρ ^, Υ '' ΌΕ1</td>

<td>ER-808824</td><td>ογ</td><td>H.</td><td>Me 1⁰ Y \</td><td> 0 ''^;·&gt; · 'ΌΗ</td>

<td>ER-808825</td><td></td><td> 0 ^X'N' H.</td><td>Y Me¹ABOUT</td><td>0 ^^ Υ'όη</td>

<td>ER-808826 single diastereoisomer</td><td></td><td>about ^xH.</td><td>'' Ύ Me&gt; A ^ / N⁰ S.</td><td>0 Y ^ Y ^ OH</td>

<td>ER-808861</td><td>Υ; V .n. JL 1 Γ ^{N x}IJ H</td><td>Me 1 9 Y</td><td>0 Y% A</td><td>AFTER N H.</td>

<td>ER-808862</td><td>Yn and N II H.</td><td colspan="2">Y Me 1⁰</td><td>SMe &gt; Y &lt;-Y / OH H.</td>

<td>ER-808863</td><td>Y?</td><td colspan="2">P Me YyY-⁰</td><td>⁰A /^F.Ν ΥΎ H.</td>

<td>ER-808864</td><td></td><td>AND 0</td><td>Me</td><td>P. Y 'N H.</td>

<td>ER-808865</td><td>YjT ^ N. A 1^NII H.</td><td>Me 1⁰</td><td>0 ppA</td><td>Ν '^^^' ΟΗ H.</td>

<td>ER-808867</td><td>Yr , -N. JL 1 ^NII H.</td><td> 0</td><td>Me 1</td><td>AA H.</td>

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<td>ER-808868</td><td>υγ Yk 1 ^NII H.</td><td>Me I and Α ^ Χ 0 _x</td><td colspan="2">about γγ</td><td colspan="2">.Τ Η</td>

<td>ER-808869</td><td>with</td><td>^X Me yS 0 Y&gt;</td><td>Y</td><td>0 ΥΖ 1</td><td></td><td>^, 0 τ&gt;</td>

<td>ER-808870</td><td>Yn 1 ^N</td><td>Y Me 1 kA</td><td>γγ</td><td>0 υ ^ ν</td><td></td><td>sY^0H</td>

<td></td><td></td><td>° Y</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td>f</td><td>'' R</td><td></td>

<td>ER-808871</td><td>Yr</td><td>Y Me 1 J.</td><td></td><td> 0</td><td>c</td><td rowspan="2"> ...</td>

<td></td><td>A and ^^nZ</td><td></td><td>γγ</td><td>Υ</td><td></td>

<td></td><td></td><td>o γ</td><td></td><td></td><td></td><td></td>

<td>ER-808872</td><td>ya --N JL J and N 11 H.</td><td>r Me 1 kx * &lt;⁰ AND</td><td>ζγ</td><td>0 τΥ Η</td><td></td><td>Ο</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td>Υ</td>

<td>ER-808873</td><td>Ts)</td><td>Y Me 1</td><td></td><td>Λ J</td><td></td><td>Α</td>

<td></td><td></td><td></td><td>γ.</td><td>^Λν^;</td><td></td><td>χ^θΗ</td>

<td></td><td></td><td>o γ</td><td></td><td></td><td></td><td></td>

<td>ER-808874</td><td>oy</td><td>Y Me γ \</td><td>γ</td><td>0 γ</td><td> )</td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td>No J</td>

<td>ER-808876</td><td>vn Yk A AND ^NIJ H</td><td>Me 1⁰ Y ^</td><td>γγ</td><td colspan="2">0 Ζ^Ν</td><td>χ ^ Χ^ΡΚ</td>

<td>ER-808877</td><td>T.</td><td>γ \ ..... Λ ·</td><td>γγ</td><td>Λ χ</td><td> 5</td><td>νο₂ 5</td>

<td>ER-808878</td><td>YjA</td><td>Me 1</td><td>AND γγ</td><td> 1</td><td></td><td>Π γγ</td>

<td></td><td></td><td>⁰ AND\</td><td></td><td></td><td></td><td></td>

<td>ER-808880</td><td>YY --N. JL J and N 1 JH</td><td>Y Me 1 k / N⁰ Y</td><td>γγ</td><td>ο k</td><td>Ph</td><td>:about₂Et</td>

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<td>ER-808881</td><td>'d ° ° Cj i tod "</td>

<td>ER-808882</td><td>0 Me o^{NNN Ph}CN</td>

<td>ER-808883</td><td>γ ° γ “θ ° /ⁿAy -Jy χΆ / to Αύ y - ^. Χ-Ά /track^fABOUT Cl</td>

<td>ER-808884</td><td>γ ° γ ^{It has 0} ίγ γ^Ν n&gt; /ⁿdJ ^H. jj, = 1 ^H.</td>

<td>ER-808885</td><td>or^Phγ ° toto ^m® ° θγ-θ todY</td>

<td>ER-808888</td><td>0 ^ A Me 0 ΥγΧ Α ^ \ / γΑ νΆγΑ LJ ^H. - 1 ^H. &lt;JL AA AA AA ^_Br</td>

<td>ER-808890</td><td>~ 'd ⁰ Here ^{Me 0}Y ^N Y ** '' n 'Ar' ^N Yto 'n' '' toYto dH ^H. ol ^M. dto-</td>

<td>ER-808891</td><td>Ύ ^ 0 XA Me 0 σ «τ / to Od</td>

<td>ER-808893</td><td>dd ° γ * ι *^{β 0} IN^N 'c^N h /</td>

<td>ER-808895</td><td>Y o toto ^{Me 0} F.^hγΛτΉΎ toto o ^0H</td>

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<td>ER-808896</td><td>Yn</td><td>this</td><td>Me</td><td></td><td>⁰ 1</td>

<td></td><td>U "</td><td>AND about</td><td></td><td></td><td>YY '' / H.</td>

<td>ER-808897</td><td>Yn .hi Jl X 1 T. ^N</td><td></td><td>Me 1 , N</td><td></td><td>0 X N</td>

<td></td><td>H.</td><td> 0</td><td></td><td></td><td>kh</td>

<td>ER-808898</td><td>YlA 'toto tok A 1 ^NIJ H</td><td> 0</td><td>Me 1 "N</td><td></td><td>ΥΥ ΌΗ</td>

<td>ER-808899</td><td> 1 ^NII H.</td><td> 0</td><td>Me 1 .N</td><td></td><td>.OH Vy H.</td>

<td>ER-808900</td><td>Tsó . VAJ and^Nand JH</td><td> 0</td><td>Me 1 / N.</td><td>γ</td><td>O HO ^^ l tok .o N H.</td>

<td>* ER-808901</td><td>ya .h and JL X IN II H.</td><td> 0</td><td>Me 1 "N</td><td></td><td>0 so who³» N H.</td>

<td>ER-808902</td><td>k? r .hi tok Jk 1 ^N| ; H.</td><td> 0</td><td>Me 1</td><td></td><td>xY H.</td>

<td>ER-808903</td><td>Y 1 .hi tok 1 N. 1 ' ^H.</td><td></td><td>MC 1 ..N. 0 _χ</td><td></td><td>ABOUT . X .Me toto ^ N H.</td>

<td>ER-808904</td><td>Ye .N. Jk 1 ^NII H.</td><td></td><td>Me 1 n_x &gt;</td><td colspan="2">about YY ^ nY kY O</td>

<td>ER-808905</td><td rowspan="2">Y ;, .N Jk is 1 ^N1 i ^H.</td><td></td><td>Me 1</td><td></td><td>⁰ 1</td>

<td></td><td> 0</td><td></td><td>this</td><td>H.</td>

<td></td><td></td><td></td><td></td><td></td><td>SMe</td>

<td>ER-808906</td><td>AJ</td><td></td><td>Me</td><td>Ύ</td><td>Vy</td>

<td></td><td>kJ ^H.</td><td> 0</td><td></td><td> 1</td><td>^H. 0</td>

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<td></td><td>Ys</td><td></td><td></td><td>Me 1</td><td> 0</td><td></td>

<td>ER-808907</td><td> ^-^NxAL</td><td>'N^WITH</td><td></td><td>_WITHN.</td><td>ΎΥΊΓ</td><td>'^' COjEt</td>

<td> -</td><td></td><td>H.</td><td> 0</td><td></td><td>1 M</td><td></td>

<td></td><td>A ° i, u</td><td></td><td></td><td>Me 1</td><td> 0</td><td></td>

<td>ER-808908</td><td>λΑ</td><td>X</td><td></td><td>AND</td><td>Ay</td><td>XOat-eu</td>

<td></td><td></td><td rowspan="2">H.</td><td></td><td></td><td>H.</td><td></td>

<td></td><td>about</td><td> 0</td><td></td><td></td><td></td>

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<td></td><td>And and?</td><td></td><td></td><td>Me</td><td>about</td><td></td>

<td>ER-808909</td><td>.nAL</td><td>AND</td><td></td><td>1 &lt;N</td><td>yAn '</td><td>X</td>

<td></td><td></td><td rowspan="2">H.</td><td></td><td></td><td>H.</td><td></td>

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<td></td><td>Ie</td><td></td><td></td><td>Me 1</td><td> 0</td><td></td>

<td>ER-808910</td><td></td><td>PT</td><td></td><td>^N\ Y</td><td>ΎΥ ^ Ύ</td><td></td>

<td></td><td></td><td>H.</td><td> 0</td><td>JL</td><td>^H.</td><td>LU</td>

<td></td><td>Yf</td><td></td><td></td><td>Me 1</td><td> 0</td><td rowspan="2">Άυ '^εν</td>

<td>ER-808911</td><td></td><td>AND</td><td></td><td>_WITHN</td><td>Y &lt;Y</td>

<td></td><td></td><td>H.</td><td> 0</td><td></td><td colspan="2">s, ¹ AND^CN</td>

<td></td><td>-about</td><td></td><td></td><td>Me |</td><td> 0</td><td></td>

<td>ER-808913</td><td></td><td>^XN '</td><td></td><td></td><td>YYY</td><td>Α, οη</td>

<td></td><td></td><td>H.</td><td> 0</td><td></td><td>H.</td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td>^0H</td>

<td>ER-808914</td><td>Ie</td><td></td><td></td><td>Me 1</td><td>ABOUT</td><td>AT</td>

<td></td><td>AND</td><td>Ν ''</td><td></td><td></td><td>YY'N</td><td></td>

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<td></td><td>Y</td><td> 0</td><td></td><td>^X Me 1</td><td> 0</td><td></td>

<td>ER-808915</td><td></td><td></td><td></td><td></td><td>ΑτΧ</td><td></td>

<td></td><td></td><td></td><td>H.</td><td> 0</td><td></td><td>AND</td>

<td></td><td>Y «</td><td></td><td></td><td>Me 1</td><td> 0</td><td></td>

<td>ER-808916</td><td>_withn JL</td><td>N '</td><td></td><td>, N</td><td>Υγν '</td><td>ΎΛ,</td>

<td></td><td></td><td>H.</td><td> 0</td><td></td><td>H.</td><td>Ly</td>

<td></td><td>A θ</td><td></td><td></td><td>Me 1</td><td> 0</td><td>.oh</td>

<td>ER-808917</td><td>X τ</td><td>'Ν' '</td><td></td><td></td><td>YfY</td><td>X, .oh</td>

<td></td><td></td><td rowspan="2">K.</td><td></td><td></td><td>H.</td><td rowspan="2">Rh</td>

<td></td><td>AND</td><td> 0</td><td></td><td></td>

<td></td><td>A s</td><td></td><td></td><td>Me</td><td> 0</td><td>y&gt;</td>

<td>ER-808918</td><td>A. AL</td><td></td><td></td><td rowspan="2">ⁿand</td><td></td><td> =</td>

<td></td><td></td><td>N ' H.</td><td>about</td><td colspan="2">Υ Ό</td>

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<td>ER-808919</td><td>Y 0 YY Me 0 AND ^N Ύ Ν ^N '', / 'Y' N^H. and A. ¹</td>

<td>ER-808920</td><td>0 Y Me 0 P.^Nx ^ x JL.Ń ΡΆ, Ρ ^ Χ A Z ^ ΐ'Κ ^ Ζ YY ^ AN COjMe^H. 5 and [ ^H.</td>

<td>ER-808921</td><td>0 Y Me 0 Ph Λα X Ap &lt;Y Ν YY Y4 ^H. o 'i ^H.</td>

<td>ER-808922</td><td>'' 'Υ 0 Y Me O Y ^N γΎ ^N 'uA ^N Ύγ 'n ΆΥΥ Η II = H. Y ° p ¹ YP_NOl</td>

<td>ER-808923</td><td>Υ,^ph z and Jk X Υχ Λ A Y Υν γ / Υ _N What₂m «^H. at 1 ^M.</td>

<td>ER-808987</td><td>with ° có ιρΛ "</td>

<td>ER-808988</td><td>Pp</td>

<td>ER-808990</td><td>XX o 'Υ γ o</td>

<td>ER-809040 single diastereoisomer</td><td>r p AA aa YYN 'j °^E.*</td>

<td>ER-809041</td><td>O · γ · 'Me 0 ¢ 4 ϊ Ύγ ^ ™ ·</td>

<td>ER-809043</td><td>Υΐ YX j pA A p λ, P Y ^N | Χ '/ X OH</td>

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<td>ER-809044</td><td></td>

<td>ER-809045 single diastereoisomer</td><td>γ | 0 'Υ Me 0</td>

<td>ER-809046</td><td>° C t * ° Cj^s j yS "</td>

<td>ER-809054</td><td>γ o γ _M.. about Yk. YVYó ⁱⁿ</td>

<td>ER-809055</td><td>MeS?^H.Y 0 Y Me 0 V^H.γΡγΥΛΟ</td>

<td>ER-809056</td><td>F. Y 0 Υ Μβ O VH YYYó</td>

<td>ER-809057</td><td>. V Y 0 Y Me 0 ° V- '^NHYySYó</td>

<td>ER-809058</td><td>Υ O Υ Μ, 0 ^Ον «Η) Y = u χτΥ "</td>

<td>ER-809059</td><td>k YOY M. OY^NHYYjYó</td>

<td>ER-809060</td><td>γ Υ ° YM ° ° Y^NHYYYó</td>

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<td>ER-809061</td><td>V 0 Ύ Me 0 y «^H.γγγό</td>

<td>ER-809062</td><td>for Y;? γ? ·? V \. dido</td>

<td>ER-809063</td><td>γ H O 0 ° ° ° Ύ '^νηγΥΥό</td>

<td>ER-809064</td><td>γ ΗΗ 0 tok Me Ο ° 'V'^NHγΑγό</td>

<td>ER-809065</td><td>for γ / V 9 Y «· ⁰ V^NHYCyCYó</td>

<td>ER-809066</td><td>&lt;d^H.d^-'o γ ^m® ° ° ύ^νηΥΥχΙό</td>

<td>ER-809067</td><td>° Y V 0 Y Me 0 V '^NMV^! !! Yó</td>

<td>ER-809068</td><td>Ph Cl 0 Ί Me 0 ° Ą-^NHνΥγν</td>

<td>ER-809069</td><td>γ »ψ -, vO'- YWó</td>

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<td></td><td></td><td></td><td></td><td> /</td>

<td></td><td></td><td></td><td></td><td></td>

<td>ER-809070</td><td>yy</td><td>p</td><td></td><td>V₀ ° y-NH</td>

<td></td><td>Γ</td><td>T.</td><td>γγ</td><td>ΎΡ</td>

<td></td><td>M. ^H.</td><td>about</td><td></td><td></td>

<td>ER-809071</td><td>P.</td><td>^X Me P.</td><td>PP</td><td>yCOjEt □ νΥ ^ ΊΊ · Ύ \</td>

<td></td><td>of the Penal Code ^H.</td><td>° p ^</td><td></td><td></td>

<td>ER-809072</td><td></td><td>AND</td><td>γγ</td><td>^Phχ ^ουρ. ΎΡ</td>

<td></td><td>of the Penal Code ^H.</td><td>about P</td><td></td><td></td>

<td>ER-809073</td><td>Ace '</td><td>AND</td><td>γγ</td><td>Κ ° V \ Ύ \</td>

<td></td><td>of the Penal Code ^H.</td><td>⁰ P.</td><td></td><td></td>

<td></td><td></td><td></td><td></td><td>α J.</td>

<td>ER-809074</td><td>PI</td><td> Me</td><td> 0</td><td>yÓ % -νη</td>

<td></td><td>Γ Γ P</td><td>Ύ</td><td>γΥ</td><td>^si | Y \</td>

<td></td><td>of the Penal Code ^H.</td><td>⁰ PP</td><td></td><td></td>

<td>ER-809075</td><td>ρ-</td><td>vol</td><td></td><td>• γρη 0 ° ν^ΝΗ</td>

<td></td><td>Γ rs</td><td>T. ^x</td><td>γγ</td><td></td>

<td></td><td>of the Penal Code ^H.</td><td>⁰ P.</td><td></td><td></td>

<td>ER-809076</td><td>Ύ</td><td>Y</td><td>γγ</td><td>Ph o Y ° Ύ | Υ \</td>

<td></td><td></td><td></td><td></td><td>F.</td>

<td></td><td></td><td></td><td></td><td>about</td>

<td>ER-809077</td><td>OC</td><td>Dr.</td><td></td><td>0 Γ 0%. - NH</td>

<td></td><td>US '</td><td>T. </td><td>γγ</td><td>Υ'νΡ</td>

<td></td><td>AT ^H.</td><td>⁰ P.</td><td></td><td></td>

<td></td><td></td><td></td><td></td><td>Y</td>

<td>ER-809078</td><td>p</td><td>t Γ</td><td></td><td>0 Γ ABOUT ^AT? ..- NH</td>

<td></td><td>e.g.</td><td>T. </td><td>ΡΡζ</td><td>Υ ^ 'Ν'Ά</td>

<td></td><td>of the Penal Code ^H.</td><td>about P</td><td></td><td></td>

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<td>ER-809079</td><td>'' 'Y 0 γγ Ma O' 'ί; -^ΜΗΥΛΆυ</td>

<td>ER-809080</td><td>Λ YOU 0 A Me 0 V-^NHΧΥχΑό</td>

<td>ER-809081</td><td>, -Ο</td>

<td>ER-809082</td><td>Cn TT 0 'Υ Me Ο ° V-^NHΑΥχΥό</td>

<td>ER-809083</td><td>Υ Ο Υ Me Ο ΧΥγΥό</td>

<td>ER-809084</td><td>ci._ γ. υ ». νΟ'θ Υ ΥγΥό</td>

<td>ER-809085</td><td>^ΡΥ 1 ο ' ^0Μ· Υ 0 A Me 0 V-^NHXτ 'υ</td>

<td>ER-809086</td><td>Ph ΗοΆ, "Rh Υ Ο Υυ Me 0 ° 'Υ^ΝΗίΥΥίγΥο</td>

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<td>ER-809087</td><td>ΥοΨ OZZZó</td>

<td>ER-809088</td><td>.Ph TT o 'A Me 0% · -^ΝΖΆΖό &lt;</td>

<td>ER-809089</td><td>..OH Ύ o _Μβ at ANH °^H.ΖΑζ ^ 'Ο</td>

<td>ER-809090</td><td>OK OH With ° Ύ ^Has ° ° v^nhcjsiZTó</td>

<td>ER-809091</td><td>HO γ 0 Y M. O ° yT AND⁸ ϊΖΤό</td>

<td>ER-809092</td><td>THT 1 *! ° V^NY WITH^s saAó</td>

<td>ER-809093</td><td>OH; Z ^ OA ^{Me 0} ° γ.ΝΗ CTZjAó</td>

<td>ER-809094</td><td>Me Z 0 Z Me 0 Z CZITZó</td>

<td>ER-809095</td><td>ABOUT WITH 5ZZ</td>

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<td>ER-809096</td><td>γ</td><td>Ψ Me AND⁰ this</td><td>O ° V-Ńh tototoY_N-this</td>

<td></td><td></td><td></td><td>MeS WHAT₂Me</td>

<td>ER-809097</td><td>YH</td><td>^X Me</td><td>0 ° V '^NH</td>

<td></td><td>Γ τ r</td><td>γ^Νν</td><td>χΧ — nA</td>

<td></td><td>this ^H.</td><td>here it is ^</td><td></td>

<td>ER-809098</td><td>IN</td><td> Me_YY</td><td>—.COjEt and v^N'«· Ύ-Ύγ</td>

<td></td><td>this ^H.</td><td>about this'</td><td></td>

<td>ER-809099</td><td>Y</td><td>Me AND</td><td>^ .CO₂t-Bu o ° V^NHtoto n it</td>

<td>ER-809100</td><td>From</td><td>Me</td><td>MeS. ^^ Sy ^ COjEt o ° Ύ</td>

<td></td><td></td><td>γΥ</td><td>γ \ - \</td>

<td></td><td>this ^H.</td><td>⁰ this*</td><td></td>

<td>ER-809101</td><td>Jk</td><td>^X Me YY</td><td>CN 0 k^{0 &lt;}this^Nthis ^ \ II f CN toto n it</td>

<td></td><td></td><td>⁰ toY</td><td></td>

<td>ER-809102</td><td>Ay</td><td>k</td><td>/ OH Y 0 this^NHύΥνΛ</td>

<td></td><td>this ^H.</td><td>⁰ this"</td><td></td>

<td>ER-809103</td><td>Jk</td><td>^X Me γΥ</td><td>Y 0 ° ν-^ΝΗtoto n A.</td>

<td></td><td>this ^H.</td><td>⁰ toto</td><td></td>

<td></td><td></td><td></td><td></td>

<td>ER-809104</td><td>AND"'</td><td>Y Me course</td><td>o ° V-^NH</td>

<td></td><td>AT ²</td><td>Y⁰ this</td><td></td>

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<td>ER-809105</td><td>d-</td><td>A Me γ⁰ this</td><td>this o ° v-Ńh</td>

<td>ER-809106</td><td></td><td>this⁰ this</td><td>0 z ^ to-Nto</td>

<td>ER-809107</td><td>about</td><td>Me ? Y⁰ toto</td><td>γ) o V *^H.</td>

<td>ER-809108</td><td>this</td><td>^X Me this⁰ AND</td><td>Ph HO- ^ Y.to. O / 0 o V-^NH'/ jto-_NzX</td>

<td>ER-809109</td><td>d-</td><td>A Me γ⁰ this</td><td>and Va Yto / toto ^</td>

<td>ER-809110</td><td>this</td><td>^X Me this oh toto</td><td>this \ ^ CO₂Me 0 ° Y-NH^αΑνΥ</td>

<td>ER-809111</td><td>d</td><td>this⁰ AND</td><td>0 / about ^θ% -Ά Z ^ to ^ Nto</td>

<td>ER-809112</td><td>this</td><td>A Me this⁰ AND</td><td>OH this₀ this_NH</td>

<td>ER-809113</td><td>di</td><td>Me with ^</td><td>toto 0 ° V-MH</td>

PL 219 737 B1

<img file="PL219737B1_D0052.tif" />

PL 219 737 B1

<td>Relationship</td><td colspan="3">Building</td>

<td></td><td></td><td></td><td>XJ</td>

<td>ER-809123</td><td>Y</td><td>vol_r- About γ.</td><td>Yr 0 V.^NHaYnA,</td>

<td>ER-809124</td><td>Y</td><td>1 ». r ⁰ /</td><td>About Υ and V '·· yyyYy</td>

<td></td><td></td><td></td><td>about-</td>

<td>ER-809125</td><td></td><td>^X Me γ⁰ Y ^</td><td>or 0V-^NH. υΥλ</td>

<td>ER-809126</td><td>Y</td><td>k Y '⁰ Y</td><td>'9 0 ° V^NHy ^ YY γ</td>

<td>ER-809127</td><td>Y</td><td>⁰ Y</td><td>about V 0V-^NHaYY</td>

<td>ER-809128</td><td>γ</td><td>⁰ Y</td><td>at Ύ₀^υν-ΝΗ ΆγΖ &lt;_Ν-\</td>

<td>ER-809129</td><td>Y^l</td><td>^x Me Y⁰ / Ϋ</td><td>Υ and V '" νΥΛ</td>

<td></td><td></td><td></td><td>G)</td>

<td>ER-809130</td><td>Y</td><td>Me V⁰ Y</td><td>_about Y-NH ΥΥζΎΥ</td>

PL 219 737 B1

<td>Relationship</td><td colspan="4">Building</td>

<td>ER-809131</td><td>AND?</td><td>^X Me Y &lt;⁰ AND</td><td>0 aA</td><td>ο A ° ν-^ΝΗon.</td>

<td>ER-809132</td><td>r</td><td>^X Me Yv</td><td>0 γργ</td><td>. with α^νηΑ</td>

<td></td><td>AT ^H.</td><td>⁰ AND</td><td></td><td></td>

<td></td><td></td><td></td><td></td><td>Α</td>

<td>ER-809133</td><td>Yr</td><td>kr</td><td> 0</td><td>0 Γ ρΝΗ</td>

<td></td><td>AND</td><td>γ ^x⁰ P '</td><td>PAP</td><td>4Α.</td>

<td></td><td></td><td></td><td></td><td>Me</td>

<td></td><td></td><td></td><td></td><td>“Υ</td>

<td>ER-809134</td><td rowspan="2">r</td><td></td><td> 0</td><td>□ Α “Γ, ΝΗ</td>

<td></td><td>γ^Νν</td><td>ΆγΑ</td><td>'νΑ</td>

<td></td><td>Ά ^H.</td><td>0 A-</td><td></td><td></td>

<td>ER-809135</td><td>from</td><td>^X Me yX⁰ P ^</td><td>0 ΑγΑ</td><td>V \ L νΑ</td>

<td>ER-809136</td><td></td><td>Me yS⁰ AND</td><td>about ΆγΑ</td><td>'νΑ.</td>

<td>ER-809137</td><td>ABOUT</td><td> ¢⁰ p</td><td>0 υΆρ</td><td>° ν-^ΝΗ</td>

<td>ER-809138</td><td>V ° s</td><td>Me 1</td><td> 0</td><td>. ζ V^NH</td>

<td></td><td>AND</td><td>Y^NY 0 ^ P,</td><td>ΆρΆ</td><td>νΑ</td>

PL 219 737 B1

<td>Relationship</td><td colspan="5">Building</td>

<td>ER-809139</td><td>Ts</td><td>J.</td><td>Y r</td><td> 0</td><td>° V-nL ^</td>

<td></td><td></td><td>^%N </td><td>Y&gt;</td><td>xX</td><td>'νΑ</td>

<td></td><td>AND</td><td></td><td>⁰ X</td><td></td><td></td>

<td>ER-809140</td><td>and Jt</td><td></td><td>C "</td><td> 0</td><td>ο Ο</td>

<td></td><td></td><td></td><td>ϊ τ</td><td>AA</td><td>'ir \</td>

<td></td><td>AND</td><td></td><td>⁰ x</td><td></td><td></td>

<td>ER-809141</td><td>V ° N Jl</td><td>jl</td><td>Me</td><td> 0</td><td></td>

<td></td><td>r V</td><td></td><td>γΥ</td><td>χΑ</td><td>νΑ</td>

<td></td><td>AND</td><td></td><td>⁰ X</td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td> 9</td>

<td>ER-809142</td><td>V ° and 1</td><td>y</td><td>A Γ</td><td> 0</td><td>0 Γ Ύ-ΝΗ</td>

<td></td><td>Γ τ</td><td></td><td>γΥ</td><td>άΧ'-</td><td>νΧ)</td>

<td></td><td>AND</td><td></td><td>⁰ X</td><td></td><td></td>

<td>ER-809143</td><td>V ° N JL</td><td>yl</td><td>Me</td><td> 0</td><td></td>

<td></td><td>r τ</td><td></td><td>γΥ</td><td>αΧ ^ -</td><td>νΑ</td>

<td></td><td>AND</td><td></td><td>⁰ X '</td><td></td><td></td>

<td>ER-809144</td><td rowspan="2">A ° rY</td><td>X</td><td>C r</td><td> 0</td><td>AND</td>

<td></td><td>Χ-</td><td>V pp</td><td>γχΧ</td><td>-Λ '</td>

<td></td><td>AND</td><td></td><td>⁰ X</td><td></td><td></td>

<td>ER-809145</td><td>Ie</td><td></td><td>? r</td><td> &lt;</td><td>. 9 Ύ-ΝΗ</td>

<td></td><td>Γ t</td><td>X</td><td>T.</td><td>Αγ</td><td>ΥΑ</td>

<td></td><td>AND</td><td></td><td>⁰ AND</td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td>Υ</td>

<td>ER-809146</td><td>Y ⁰</td><td></td><td>^X Me</td><td> 0</td><td>Ąnh</td>

<td></td><td>AND</td><td>AT</td><td>Λ</td><td>χη</td><td>-ζ</td>

<td></td><td>AND</td><td></td><td>⁰ AND</td><td></td><td></td>

PL 219 737 B1

<td>Relationship</td><td>Building</td>

<td>ER-809147</td><td>WITH Z 0 Y M. 0 ° VH ZzzZ</td>

<td>ER-809148</td><td></td>

<td>ER-809149</td><td>Z ° Y «° V^NHWith at</td>

<td>ER-809150</td><td>, Y 'Z 0 γ Ma 0 Y *^NHZZZó</td>

<td>ER-809151</td><td>, WITH ZAZKó</td>

<td>ER-809152</td><td>AA</td>

<td>ER-809153</td><td>Q Z θ Y * ° ° Y^H.ΖΑγγ</td>

<td>ER-809154</td><td>WITH ⁰ Y Mo ASS</td>

PL 219 737 B1

<td>Relationship</td><td>Building</td>

<td>ER-809155</td><td>Y Y 0 Y Me 0 V ™ γγγό</td>

<td>ER-809156</td><td>Y 0 Ύ Me 0 ° YsPyAÓ</td>

<td>ER-809157</td><td>Y 0 Y Me o V * γΡγνΑό</td>

<td>ER-809158</td><td>γ Y 0 Y Me 0 ° Y^H.YYYÓ</td>

<td>ER-809159</td><td>Y 0 Ύ Me YrySYó</td>

<td>ER-809160</td><td>MeOjC, A o ό “· ° γγχΑό</td>

<td>ER-809161</td><td> O Ύ Me o</td>

PL 219 737 B1

<td>Relationship</td><td colspan="3">Building</td>

<td>ER-809162</td><td>this</td><td>Me Ά</td><td>0 Άη</td>

<td>ER-809163</td><td>HO</td><td>^X Me Y^Nlife</td><td rowspan="2">this</td>

<td></td><td>u -</td><td>Ϊ i.e.</td>

<td></td><td rowspan="2"> 99='</td><td>f Γ</td><td> 0</td>

<td>ER-809164</td><td>τ vto</td><td>AoH</td>

<td></td><td></td><td>⁰ this</td><td></td>

<td></td><td rowspan="2"> 99»'</td><td>C r</td><td>about</td>

<td>ER-809165</td><td>ΎΥΑ 0,_Ί</td><td>to'OH</td>

<td>ER-809166</td><td>V</td><td>it's Me toto⁰ \</td><td>0 Aaqh</td>

<td>ER-809167</td><td>Of ' And 1 N. II H.</td><td>A Me 1 Y^NVto ⁰ this</td><td>0 A ^ oh</td>

<td>ER-809240</td><td>di</td><td>Me 0 '' Α ·</td><td> \-^OHΝ'Α</td>

<td></td><td></td><td>Me 1</td><td>about</td>

<td>ER-809241</td><td rowspan="2">IN II H.</td><td></td><td>^ OEt</td>

<td></td><td>0 A, I</td><td></td>

<td>ER-809242</td><td>this</td><td>^X Me rtor ⁰ \ ¹</td><td>0 OEt</td>

<td>ER-809243</td><td>d?</td><td>^X Me tTt</td><td>about about.</td>

PL 219 737 B1

<img file="PL219737B1_D0053.tif" />

PL 219 737 B1

<img file="PL219737B1_D0054.tif" />

PL 219 737 B1

General reaction procedures:

Unless otherwise stated, the reaction mixtures were mixed with a stir bar with a magnetic drive. The inert atmosphere is either dry argon or dry nitrogen. Reactions were monitored by thin layer chromatography (TLC), proton nuclear magnetic resonance, or high performance liquid chromatography (HPLC), an appropriately prepared sample of the reaction mixture.

Abbreviations of some known organic reagents are listed below:

BOC or BOC2O: Di-tert-butyl dicarbonate

CMC: 1-Cyclohexyl-3- (2-morpholinoethyl) carbodiimide meto-p-toluenesulfonate

DCM: Dichloromethane

DEPC: Diethylphosphoryl cyanide (diethyl cyanophosphate)

DIBAL: Diisobutylaluminum hydride

DIEA: Diisopropylethylamine

DMF: N, N-Dimethylformamide

DMSO: Dimethylsulfoxide

Ether: Diethyl ether

HBTU: O- (1-H-benzotriazol-1-yl) -N, N, N, N-tetramethyluronium hexafluorophosphate

HOAt: 1-Hydroxy-7-azabenzotriazole

LAH: Lithium aluminum hydride

MSA: Methanesulfonic acid

NMM: N-Methylmorpholine

TBME: Tert-Butyl Methyl Ether

TFA: Trifluoroacetic acid

THF: Tetrahydrofuran

TMEDA: Tetramethylethylenediamine

General processing procedures

Unless specifically stated, reaction mixtures were cooled to room temperature or below, and then quenched as necessary with water or saturated aqueous ammonium chloride solution. The desired products were extracted by partitioning between water and an appropriate water-immiscible solvent (e.g., ethyl acetate, dichloromethane, diethyl ether). The extracts containing the desired product were washed appropriately with water and then with saturated brine. In the event that the product-containing extract was judged to contain residual oxidants, the extract was washed with 10% sodium thiosulfate in a saturated aqueous sodium bicarbonate solution prior to the above-described washing procedure. In cases where the product-containing extract was judged to contain residual acids, the extract was washed with a saturated aqueous sodium bicarbonate solution prior to the above-described washing procedure (except where the desired product was acidic in nature). In cases where the product-containing extract was judged to contain residual bases, the extract was washed with a 10% aqueous citric acid solution prior to the above-described washing procedure (except where the desired product was alkaline). After washing, the extracts containing the desired product were dried over anhydrous magnesium sulfate and then filtered. The crude product was isolated by removing the solvent (s) on a rotary evaporator under reduced pressure at an appropriate temperature (typically below 45 ° C).

In the case where triphenylphosphine oxide was the major by-product of the reaction, the reaction mixture was added directly to a large volume of hexane with vigorous stirring. The precipitate of triphenylphosphine oxide was filtered off, and the filtrate was treated in the usual way.

General cleaning procedures:

Chromatographic purification refers to flash column chromatography on silica using a single solvent or mixture of solvents as eluent, or HPLC on a C18 column. Eluates containing the appropriately purified desired product were combined and concentrated under reduced pressure at an appropriate temperature (typically below 45 ° C) to a constant weight. The final compounds were prepared for biological studies by a) dissolving in 50% acetonitrile in water, filtering and placing in vials, followed by freeze drying under high vacuum; or b) dissolving in methanol, filtering and placing in vials, followed by concentration to dryness in a vacuum centrifuge evaporator.

PL 219 737 B1

Example 1 (reference example): Preparation of 18 aminoesters, 20 amino acids and 23 amino acids

<img file="PL219737B1_D0055.tif" />

<img file="PL219737B1_D0056.tif" />

To a solution of compound 12 (205 mg) in DMF (3.8 ml) at room temperature, was added (S) -N-Boc-neo-phenylalanine (6) (140 mg), NMM (0.30 ml), HOAt (0.124 g) and CMC (1.16 g). The reaction mixture was shaken at room temperature for 24 h. Work-up with water followed by chromatographic purification afforded compound 13 (153 mg, 61%).

Preparation of compound 14

<img file="PL219737B1_D0057.tif" />

To a solution of compound 13 (153 mg) in methanol (20 ml) at 0 ° C, sodium borohydride (3.18 g) was added portionwise with shaking for 3 days. The temperature of the reaction mixture was kept in the range of 0-5 ° C. After the reaction mixture solidified, THF was added to aid stirring. The reaction mixture was allowed to warm to room temperature, then cooled back to 0 ° C and worked up as usual to afford compound 14 (140 mg, 96%).

PL 219 737 B1

Preparation of compound 15

<img file="PL219737B1_D0058.tif" />

Dess-Martin periodate (204 mg) was added to a solution of compound 14 (50 mg) THF (3 mL) at room temperature in one portion. The resulting suspension was stirred vigorously for 4.5 h. Work-up with water afforded crude compound 15 (50 mg) which was used immediately in the next step without purification.

General procedure for the preparation of aminoesters 18

<img file="PL219737B1_D0059.tif" />

To a solution of 15 (1 eq.) In an appropriate volume of 1,2-dichloroethane at room temperature, 4A molecular sieves (crushed and dried) (with a weight equal to the weight of the amine hydrochloride) were added. An appropriately selected amine hydrochloride (16) (10 eq.) Was added with vigorous stirring, followed by sodium triacetoxyborohydride (1.5 eq.). The reaction mixture was stirred at an appropriate temperature (20 ° -50 ° C) until compound 15 had reacted satisfactorily. Work-up with water followed by chromatographic purification afforded the corresponding N-Boc aminoester 17. Deprotection of the N-Boc group under suitable conditions would yield the corresponding compound with the N-terminal free amino group 18.

General Procedure for Amino Acid Making 20

<img file="PL219737B1_D0060.tif" />

To a solution of the N-Boc-aminoester 17 in the appropriate mixture of THF and methanol, 1M lithium hydroxide solution (10-50 equivalents) was added. When the N-Boc-aminoester 17 was satisfactorily hydrolyzed, the reaction mixture was subjected to aqueous workup. N-Boc-amino acid 19 was purified by chromatography. Deprotection of the N-Boc group under suitable conditions would yield the corresponding compound with the N-terminal free amino group 20.

PL 219 737 B1

General procedure for the preparation of amino amides 23

<img file="PL219737B1_D0061.tif" />

NMM (20 eq.) Was added to a solution of N-Boc-amino acid 19 in DMF at room temperature. An appropriately selected amine hydrochloride (21) (20 eq.) Was added followed by DEPC (20 eq.). When the N-Boc-amino acid 19 reacted satisfactorily, the N-Boc-aminoamide 22 was isolated by direct chromatographic purification of the reaction mixture or by work-up with water followed by chromatographic purification. Deprotection of the N-Boc group under suitable conditions would yield the corresponding compound with the N-terminal free amino group 23.

Example 2 (reference example): Preparation of N-acetylaminoamides 27

<img file="PL219737B1_D0062.tif" />

Preparation of compound 24

<img file="PL219737B1_D0063.tif" />

To a solution of aldehyde 13 (50 mg) in 1,2-dichloroethane (2 ml) at room temperature, 4A molecular sieves (crushed and dried) (50 mg) were added. With vigorous stirring, glycine methyl ester hydrochloride (120 mg) was added followed by sodium triacetoxyborohydride (205 mg). The reaction mixture was stirred at 40 ° C for 2 hours. Work-up with water followed by chromatographic purification afforded compound 24 (31 mg, 46%).

Preparation of compound 25

<img file="PL219737B1_D0064.tif" />

PL 219 737 B1

To a solution of compound 24 (5.5 mg) in DMF (0.4 ml) at room temperature, pyridine (0.006 ml) was added followed by acetic anhydride (0.004 ml). The reaction mixture was shaken for 3 hours at room temperature and then concentrated in vacuo to dryness. The residue was dissolved in saturated HCl in methanol (1 ml) and allowed to stand at room temperature for 15 minutes.

The reaction mixture was concentrated in vacuo to afford compound 25 (4 mg, 90%).

Preparation of compound 26

<img file="PL219737B1_D0065.tif" />

To a solution of compound 25 (3.35 mg) in methanol (0.2 ml), 1 M lithium hydroxide solution (0.118 ml) was added. The reaction mixture was stirred at room temperature for 5 h. Chromatographic purification followed by work-up with methanolic HCl afforded the hydrochloride of 26 (1.95 mg, 61%).

General Procedure for the Preparation of N-Acetylaminoamides 27

<img file="PL219737B1_D0066.tif" />

NMM (20 eq.) Was added to a solution of compound 26 (1 eq.) In DMF at room temperature. An appropriately selected amine hydrochloride (21) (20 eq.) Was added followed by DEPC (20 eq.). When compound 26 reacted satisfactorily, N-acetylaminoamide (27) was isolated by direct chromatographic purification of the reaction mixture.

Example 3 (reference example): Preparation of compound 33

<img file="PL219737B1_D0067.tif" />

Preparation of compound 28

<img file="PL219737B1_D0068.tif" />

PL 219 737 B1

To a solution of 3b (1.94 g) in dry DCM (20 ml) at 0 ° C under an inert atmosphere, a 1 M solution of DIBAL (32 ml) was added dropwise. The reaction mixture was stirred at 0 ° C for 2.5 h, then methanol (4.4 ml) was added dropwise, followed by the addition of saturated ammonium chloride solution (8.8 ml). DCM (200 ml) was added and the reaction mixture was vigorously stirred at room temperature for 30 min. After filtration followed by concentration in vacuo, crude compound 28 (1.08 g, 65%) was obtained.

<img file="PL219737B1_D0069.tif" />

Sodium hydride (60% dispersion in mineral oil; 160 mg) was added portionwise to a solution of compound 28 (207 mg) in THF (5 ml) at 0 ° C under an inert atmosphere. The reaction mixture was stirred at 0 ° C for 45 min then ethyl bromoacetate (0.47 ml) was added. The reaction mixture was allowed to warm to room temperature. Work-up with water followed by chromatographic purification afforded the intermediate Boc-compound (185 mg, 67%). Intermediate Boc-compound (139 mg) was dissolved in ethanol (2 ml) and saturated HCl in ethanol (2 ml) was added. The reaction mixture was allowed to stand at room temperature for 10 min, then concentrated in vacuo to dryness to give compound 29 (114 mg).

Preparation of compound 30

<img file="PL219737B1_D0070.tif" />

To a solution of compound 29 (114 mg) in DMF (1.8 ml) at room temperature was added (S) -N-Boc-tert-leucine (4) (283 mg), NMM (0.135 ml), HOAt (56 mg) and CMC (518 mg). The reaction mixture was shaken at room temperature for 16 h. Work-up with water followed by chromatographic purification afforded the intermediate Boc-compound (42 mg, 22%). Intermediate Boc-compound (42 mg) was dissolved in saturated HCl in ethanol (5 ml) and allowed to stand at room temperature for 10 min. Concentration in vacuo afforded compound 30 (37 mg).

Preparation of compound 31

<img file="PL219737B1_D0071.tif" />

To a solution of compound 30 (24 mg) in DMF (0.26 ml) at room temperature, was added (S) -N-Boc-neo-phenylalanine (6) (38 mg), NMM (0.014 ml), HOAt (8 , 3 mg) and CMC (52 mg). The reaction mixture was shaken at room temperature for 16 h. Work-up with water followed by chromatographic purification afforded the intermediate Boc-compound (38 mg, 64%). Intermediate Boc-compound (38 mg) was dissolved in saturated HCl in ethanol (5 ml) and allowed to stand at room temperature for 10 min. Concentration in vacuo afforded 31 as its hydrochloride salt.

PL 219 737 B1

Preparation of compound 32

<img file="PL219737B1_D0072.tif" />

1 M lithium hydroxide (0.5 ml) was added to a solution of compound 31 (4 mg) in ethanol (2 ml). The reaction mixture was stirred at room temperature for 1.5 h. Work-up with water followed by chromatographic purification afforded compound 32 (2.9 mg, 76%).

Preparation of compound 33

<img file="PL219737B1_D0073.tif" />

NMM (3.8 µL), pyrrolidine (2.8 µL) and DEPC (5.2 µL) were added to a solution of compound 32 (1.9 mg) in DMF (70 µL) at room temperature. The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was purified by chromatography to afford compound 33 (1.2 mg, 58%).

Example 4 (reference example): Preparation of 42 aminoesters, 43 amino acids and 45 amino amides

<img file="PL219737B1_D0074.tif" />

(45)

PL 219 737 B1

Preparation of compound 39

<img file="PL219737B1_D0075.tif" />

To a solution of compound 12 (1.25 g) in DMF (21 ml) at room temperature, (R) -N-methylpipekin hydrochloride (38) (0.38 g), NMM (1.4 ml), HOAt ( 0.575 g) and CMC (5.37 g). The reaction mixture was shaken at room temperature for 24 h. Work-up with water gave compound 39 (0.511 g, 63%).

<img file="PL219737B1_D0076.tif" />

To a solution of compound 39 (0.8 g) in methanol (8 ml) at 0 ° C, sodium borohydride (7.9 g) was added portionwise over 3 days. The temperature of the reaction mixture was kept in the range of 0 ° - 5 ° C. After the reaction mixture solidified, THF was added to aid stirring. The reaction mixture was allowed to warm to room temperature before it was cooled back to 0 ° C and quenched with saturated sodium bicarbonate solution. Treatment with water gave compound 40.

Preparation of compound 41

<img file="PL219737B1_D0077.tif" />

Dess-Martin periodate (225 mg) was added to a solution of compound 40 (50 mg) THF (3 mL) at room temperature in one portion. The resulting suspension was stirred vigorously for 4 h. Work-up with water gave crude compound 41 (55 mg) which was used immediately in the next step without purification.

General procedure for the preparation of aminoesters with an N-terminal N-heterocyclic group 42

<img file="PL219737B1_D0078.tif" />

To a solution of compound 41 (300 mg) in 1,2-dichloroethane (10 ml) at room temperature, 4A molecular sieves (crushed and dried) (1.5 g) were added. Amino acid ester hydrochloride (16) (10 eq.) Was added and the reaction mixture was stirred vigorously for ~ 10 minutes.

PL 219 737 B1

Sodium triacetoxyborohydride (290 mg) was added in one portion and the reaction mixture was stirred vigorously at room temperature. When compound 41 reacted satisfactorily, the reaction mixture was worked up with water. N-terminal N-heterocyclic aminoesters 42 were purified by chromatography except where not necessary.

General procedure for producing amino acids with an N-terminal N-heterocyclic group 43

<img file="PL219737B1_D0079.tif" />

To a solution of the N-terminal N-heterocyclic aminoesters (42) in the appropriate mixture of THF and methanol, 1 M lithium hydroxide solution (10-50 equivalents) was added. When the aminoesters with the N-terminal N-heterocyclic group 42 were satisfactorily hydrolyzed, the reaction mixture was worked up with water. The amino acid with the N-terminal N-heterocyclic group 43 was purified by chromatography except when not necessary.

General Procedure for the Preparation of Aminoamides with an N-Terminal N-Heterocyclic Group 45

<img file="PL219737B1_D0080.tif" />

NMM (20 eq.) Was added to an N-terminal N-heterocyclic 43 W DMF solution of the amino acid at room temperature. An appropriately selected amine hydrochloride (44) (20 eq.) Was added followed by DEPC (20 eq.). When the amino acid with the N-terminal N-heterocycle 43 reacted satisfactorily, the amino amide with the N-terminal N-heterocycle 45 was isolated by direct chromatographic purification of the reaction mixture or by work-up followed by chromatographic purification.

Example 5: Preparation of compounds 51 and 52

<img file="PL219737B1_D0081.tif" />

<img file="PL219737B1_D0082.tif" />

PL 219 737 B1

Preparation of compound 47:

BOC

Me ^ N ^ .CO₂Me

<img file="PL219737B1_D0083.tif" />

Procedure a.

Compound 46 (1.0405 g, 4.4984 mmol) was dissolved in DMF (8.0 mL). K2CO3 (0.6258 g, 4.5279 mmol) was added. Methyl iodide (0.6 mL, 9.6379 mmol) was added. The milky suspension was stirred at room temperature under nitrogen for 3 days. Conventional water treatment afforded ester 47 as a colorless oil (1.0590 g, 96%).

Preparation of compound 2:

Me and

<img file="PL219737B1_D0084.tif" />

(2)

Compound 47 (0.9447 g, 3.8509 mmol) was dissolved in toluene (15 mL) and the solution was cooled to -78 ° C under nitrogen. DIBAL (6.0 mL, 6.00 mmol, 1.0 M in hexanes) was added via a syringe over 5 minutes. The solution was stirred for 1 h and the reaction was quenched with MeOH (1.0 mL) at -78 ° C. The bath was removed and 5.0 ml of saturated potassium sodium tartrate solution was added. The mixture was stirred for about 1 h and filtered through ceIit. The filtrate was washed with H 2 O and brine then dried over Na 2 SO 4, filtered and evaporated to afford compound 2 (0.8413 g, 101%) pure enough for use in the next step.

Preparation of compound 3b:

<img file="PL219737B1_D0085.tif" />

Compound 2 (0.8413 g, 3.8509 mmol) was dissolved in CH2Cl2 (5.0 mL) and triphenyl (carboethoxyethylidene) phosphate (1.8212 g, 5.0254 mmol) was added. The solution was stirred overnight at room temperature under nitrogen atmosphere. The solution was evaporated and the residue was diluted with EtOAc (70 ml) and washed with H 2 O (2 x 25 ml) and brine (25 ml) then dried over Na 2 SO 4, filtered and evaporated to an oil. Purification by flash chromatography on SiO2 (FC) gave pure 3b (0.7863 g, 68%).

Preparation of compound 48:

<img file="PL219737B1_D0086.tif" />

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Compound 3b (0.7863 g, 2.6262 mmol) was dissolved in CH2Cl2 (1.0 mL) and triethylsilane (0.460 mL, 2.880 mmol) was added. Trifluoroacetic acid (TFA) (2.5 ml) was added at room temperature. After 30 minutes (when the reaction was complete by HPLC), the solution was evaporated to give a solid (1.1307 g). This solid was dissolved in CH3CN (about 10 mL) and 5.5 N HCl (2.4 mL, 13.2 mmol) was added. Evaporation gave the hydrochloride compound 48 (0.618 g, 100%).

Preparation of compound 5b:

<img file="PL219737B1_D0087.tif" />

Compound 48 (0.390 g, 1.6543 mmol), LN-BOC-t-butylglycine (1.0106 g, 4.3694 mmol), CMC (1.9704 g, 4.6518 mmol), HOAt (0.5905 g , 4.3384 mmol) and NMM (0.490 mL, 4.4567 mmol) were combined and DMF (4.0 mL) was added. The solution was stirred at room temperature under nitrogen atmosphere for 25 h. The solution was diluted with EtOAc (70 mL) and washed with H 2 O (2 x 25 mL), aqueous phosphate buffer pH 7.2 (25 mL), H 2 O (25 mL) and brine (25 mL) then dried over MgSO 4, filtered. and evaporated to give a solid which was purified by FC to afford compound 5b (0.4239 g, 62%).

Preparation of compound 49:

<img file="PL219737B1_D0088.tif" />

Compound 5b (0.1159 g, 0.2809 mmol) was dissolved in CH2Cl2 (3.0 mL) and triethylsilane (0.050 mL, 0.3130 mmol) was added. Trifluoroacetic acid (TFA) (2.5 ml) was added at room temperature. After 30 minutes (when the reaction was complete by HPLC), the solution was evaporated to give a solid. This solid was dissolved in CH3CN (about 5 mL) and 5.5 N HCl (0.3 mL, 1.65 mmol) was added. Evaporation gave the hydrochloride compound 49 (0.0662 g, 100%).

Step 2: Preparation of Compound 51:

Preparation of compound 50:

<img file="PL219737B1_D0089.tif" />

Compound 49 (0.0774 g, 0.2219 mmol), (R) -N-methylpipecolinic acid (0.0705 g, 0.3925 mmol), CMC (0.1752 g, 0.4136 mmol), HOAt (0 0.0344 g, 0.2527 mmol) and NMM (0.063 mL, 0.5730 mmol) were combined and DMF (2.0 mL) was added. The solution was stirred at room temperature under nitrogen for 20 h. The solution was directly purified by RP HPLC (reverse phase) to afford compound 50 (0.0989 g, 81%).

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Preparation of compound 51:

<img file="PL219737B1_D0090.tif" />

Compound 50 (0.0989 g, 0.2086 mmol) was dissolved in 1: 1 H 2 O / MeOH (14 mL) at room temperature. LiOH (0.0537 g, 2.2422 mmol) was added. The suspension was stirred at room temperature for 19 h. The solution was acidified with 5.5 N HCl (0.50 mL) and purified by RP HPLC to give the TFA salt of compound 11 (0.0978 g, 90%). This salt was dissolved in CH3CN (ca. 5 ml), 5.5 N HCl (ca. 1 ml, 5.5 mmol) was added and evaporated to give the hydrochloride salt of compound 51 (0.0667 g, 72%).

Step 2: Preparation of Compound 52:

<img file="PL219737B1_D0091.tif" />

Compound 51 (0.0062 g, 0.0139 mmol) and L-proline methyl ester hydrochloride (0.0263 g, 0.1588 mmol) were dissolved in DMF (1.0 mL) at room temperature under a nitrogen atmosphere. DEPC (0.017 mL, 0.1120 mmol) was added via a syringe. NMM (0.025 mL, 0.2274 mmol) was added via syringe. The solution was stirred overnight, quenched with H 2 O (1.0 mL), and the mixture was purified by RP HPLC to give the TFA salt of compound 52. This was dissolved in CH3CN (approx. 3 mL), 5.5 N HCl (0.10 mL, 0.55 mmol) was added and evaporated to give compound 52 hydrochloride (0.0078 g, 100%).

Example 6 (reference example): Preparation of compound 62a

<img file="PL219737B1_D0092.tif" />

Preparation of compound 54

<img file="PL219737B1_D0093.tif" />

(54)

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To a solution of 4-methylpiperidine (53) (600 μl, 5.0 mmol) in MeOH (20 ml) was added Et₃N (770 µ ^ 5.5 mmol) followed by Boc₂O (1.2 g, 5.5 mmol) at 0 ° C. After 15 minutes, the reaction mixture was warmed to room temperature and stirred overnight. The reaction solution was then diluted with H 2 O and extracted several times with ether. The ether extracts were combined, dried over Mg2SO4, filtered, and concentrated to afford compound 54 (926.5 mg) quantitatively as a colorless oil.

Preparation of compound 55

<img file="PL219737B1_D0094.tif" />

A solution of compound 54 (926.5 mg, 5.0 mmol) in Et2O (10.5 mL) was cooled to -78 ° C and TMEDA (755 μ ^ 5.0 mmol) was added followed by slow addition of 1.3 M of the s-butyllithium in cyclohexane (4.6 mL, 6.0 mmol) over 30 minutes. The reaction solution was then warmed to -20 ° C and held at that temperature for 30 minutes, after which the solution was cooled back to -78 ° C and purged with carbon dioxide gas for 15 minutes. The reaction solution was then slowly warmed to 0 ° C and poured into a biphasic mixture of 1 N HCl (100 mL) and EtOAc (50 mL). The reaction solution was then extracted with EtOAc several times. The EtOAc extracts were combined, dried over Mg2SO4, filtered and concentrated to afford compound 55 (1.07 g) in 89% yield as a colorless oil (mixture of 2 cis enantiomers).

Preparation of compound 59a

<img file="PL219737B1_D0095.tif" />

(59a)

To a solution of compound 55 (292 mg, 1.2 mmol) in CH₂CI₂ (2.4 ml) TFA (2.4 ml) was added at 0 ° C. After 15 minutes, the reaction solution was warmed to room temperature and stirred for 3 hours. The reaction mixture was then concentrated in vacuo to afford compound 59a (309 mg) quantitatively as a light yellow oil.

Preparation of compound 59b

<img file="PL219737B1_D0096.tif" />

(59b)

100

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Step 1: Preparation of Compound 56

<img file="PL219737B1_D0097.tif" />

K2CO3 (663 mg, 4.8 mmol) was added to a solution of compound 55 (780 mg, 3.2 mmol) in DMF (6.4 mL) followed by MeI (300 µL, 4.8 mmol). The reaction solution was stirred overnight. The reaction mixture was then diluted with H 2 O and extracted several times with ether. The ether extracts were combined, dried over Mg2SO4, filtered and concentrated in vacuo. Purification of the residue by silica gel chromatography (4% EtOAc in hexanes) provided 535 mg (65%) of compound 56 as a colorless oil.

<img file="PL219737B1_D0098.tif" />

To a solution of compound 56 (463 mg, 1.8 mmol) in MeOH (2.6 mL) was added 25 wt. a solution of NaOMe in MeOH (100 µl). The solution was stirred overnight. The reaction mixture was then diluted with H 2 O and extracted several times with ether. The ether extracts were combined, dried over Mg2SO4, filtered and concentrated in vacuo. Purification of the residue by silica gel chromatography (4% EtOAc in hexanes) provided 363.6 mg (79%) of racemic 57 as a colorless oil.

Step 3: Preparation of Compound 58

<img file="PL219737B1_D0099.tif" />

KOH pellets (786 mg, 14 mmol) were added to a solution of compound 57 (360 mg, 1.4 mmol) in a 2: 1 mixture of H 2 O (2.75 mL) and EtOH (5.50 mL) and the reaction solution was stirred at temperature. room until reaction is complete by TLC. The reaction mixture was then diluted with H 2 O and extracted several times with ether. The ether extracts were combined, dried over Mg2SO4, filtered, and concentrated to afford compound 58 (341 mg) quantitatively as a white solid.

Step 4: Preparation of compound 59b

TFA (2.4 ml) was added to a solution of compound 58 (292 mg, 1.2 mmol) in CH2Cl2 (2.4 ml) at 0 ° C. After 15 minutes, the reaction solution was warmed to room temperature and stirred for 3 hours. The reaction mixture was then concentrated in vacuo to afford compound 59b (309 mg) quantitatively as a light yellow oil.

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101

Preparation of compounds 60a and 60b

<img file="PL219737B1_D0100.tif" />

Pd (OH) 2 (75 mg) was added to a solution of compound 59a (or 59b) (283 mg, 1.1 mmol) in MeOH (5 mL) followed by 37 wt. formaldehyde solution in H.₂O (300 μΙ). Gas H was introduced₂ (balloon pressure) and the reaction mixture was stirred under an atmosphere of H 2 overnight. The reaction solution was then filtered through a bed of celite and concentrated to afford compound 60a (or 60b) (173 mg) quantitatively as a white solid.

Preparation of compounds 61a and 61b

<img file="PL219737B1_D0101.tif" />

To a solution of 60a and 60b (11.0 mg, 0.07 mmol) in CH₂CI₂ (350 µ) HBTU (40 mg, 0.11 mmol) and DIEA (37 µL, 0.21 mmol) were added. After 5 minutes, the amine 49 (22.0 mg, 0.07 mmol) was added. The reaction mixture was stirred for 30 minutes, filtered and concentrated. Purification of the residue by chromatography on silica gel (2% EtOH in CH2Cl2) gave 15.1 mg (96%) of each of diastereoisomers 61a and 61b as colorless oils.

Preparation of compound 62a

<img file="PL219737B1_D0102.tif" />

To a solution of diastereoisomer 61a (9.0 mg, 0.02 mmol) in a mixture of 2: 1H₂O (80 µL) and EtOH (160 µL) were added LiOH · H₂O (840 mg, 0.20 mmol). The reaction solution was stirred overnight. The reaction mixture was then acidified with 1 N HCl to pH = 6.00. The solution was then extracted several times with CH2Cl2. The CH2Cl2 extracts were combined, dried over Mg2SO4, filtered and concentrated to give 62a (8.4 mg) quantitatively as a white solid.

Example 7 (reference example): Preparation of compound 67b

<img file="PL219737B1_D0103.tif" />

102

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Preparation of compound 64

<img file="PL219737B1_D0104.tif" />

Benzaldehyde (233 mg, 2.2 mmol) was added to a suspension of L-penicillamine (63) (300 mg, 2.0 mmol) in methanol (10 mL) followed by sodium bicarbonate (336 mg, 4.0 mmol). The mixture was refluxed with stirring for 16 h. After cooling to room temperature, it was acidified to pH 5 with 1N HCl and extracted 3 times with ethyl acetate. The organic phase was concentrated to afford the crude product 64 (469 mg, 99%) as a yellow solid.

Preparation of compound 65

<img file="PL219737B1_D0105.tif" />

To a solution of crude compound 64 (47 mg, 0.2 mmol) of THF (1 mL) was added 37% formaldehyde in water (49 µL, 0.6 mmol) followed by NaBH4 (38 mg, 0.6 mmol). The mixture was stirred at room temperature for 24 h. After acidification to pH 5 and extraction with ethyl acetate, the organic phase was dried and concentrated to give the crude product 65 (67 mg,&gt; 100%).

Preparation of compounds 66a and 66b

<img file="PL219737B1_D0106.tif" />

To a mixture of 65 (29 mg, 0.115 mmol), amine hydrochloride 49 (15 mg, 0.043 mmol), CMC (55 mg, 0.129 mmol), and HOAt (3 mg, 0.022 mmol) was added DMF (0.5 ml) followed by NMM (6 mL, 0.055 mmol). The mixture was stirred at room temperature for 24 h. The reaction was quenched by adding water (0.5 mL) and methanol (0.5 mL). Products 66a (32%) and 66b (75%) were obtained after RP HPLC separation (0-100% B in 30 minutes. A: 5% MeCN + 0.15% TFA in H2O; B: 0.15% TFA in MeCN) and lyophilization.

Preparation of compounds 67b

<img file="PL219737B1_D0107.tif" />

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103

Aqueous LiOH (1 M, 0.5 mL) was added to a solution of compound 66b (4 mg, 0.0073 mmol) in methanol (0.5 mL). The mixture was stirred for 16 h and acidified with 1N HCl. Product 67b (2.79 mg, 74%) was obtained after RP HPLC purification and lyophilization.

Example 8 (reference example): Preparation of compound 74

<img file="PL219737B1_D0108.tif" />

Preparation of compound 69

<img file="PL219737B1_D0109.tif" />

A solution of di-tert-butyl dicarbonate (436 mg, 2.0 mmol) in dioxane (1.0 mL) was added to a solution of diethylglycine (68) (131 mg, 1.0 mmol) in 1N NaOH (1.5 mL). The mixture was stirred for 16 h. It was acidified to pH 3 with 1N HCl and extracted 3 times with ethyl acetate. The organic phases were combined, dried and concentrated to give the crude product 69 (135 mg, 58%).

Preparation of compound 70

<img file="PL219737B1_D0110.tif" />

To a solution of the crude product 69 (135 mg, 0.58 mmol) in MeOH (0.5 mL) and THF (0.5 mL) was added trimethylsilyl diazomethane (2M in hexanes, 2.0 mmol). The solution was stirred at room temperature for 1 h. Evaporation gave crude product 70 (0.58 mmol).

Preparation of compound 71

<img file="PL219737B1_D0111.tif" />

To a mixture of sodium hydride (160 mg 60%, 4 mmol) in DMF (1 mL) was added a solution of compound 70 (0.58 mmol) in DMF (1 mL) followed by methyl iodide (188 µL, 3 mmol). The mixture was stirred at room temperature for 24 h. Water was added to quench the reaction. The product 71 (118 mg, 78% 2 steps) was extracted with ethyl acetate and purified by flash column chromatography (silica, ethyl acetate / hexanes).

104

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Preparation of compound 72

<img file="PL219737B1_D0112.tif" />

A solution of compound 71 (118 mg, 0.46 mmol) in concentrated HCl (1 ml) was stirred at room temperature for 24 h. The product 72 was obtained by evaporating the volatiles.

Preparation of compound 73

<img file="PL219737B1_D0113.tif" />

To a mixture of compound 72 (30 mg, 0.166 mmol), amine hydrochloride 49 (39 mg, 0.166 mmol), CMC (141 mg, 0.332 mmol), and HOAt (14 mg, 0.103 mmol) was added DMF (1.5 ml) and then NMM (6 mL, 0.128 mmol). The mixture was stirred at room temperature for 24 h. The reaction was quenched by adding water (0.5 mL) and methanol (0.5 mL). Product 73 (27 mg, 34%) was obtained after RP HPLC separation (0-100% B in 30 minutes. A: 5% MeCN + 0.15% TFA in H2O; B: 0.15% TFA in MeCN) and lyophilization.

Preparation of compound 74

<img file="PL219737B1_D0114.tif" />

LiOH in water (1 M, 0.5 mL) was added to a solution of compound 73 (18 mg) in methanol (0.5 mL). The mixture was stirred for 16 h then acidified with 1 N HCl. Product 74 (12.3 mg, 73%) was obtained after RP HPLC purification and lyophilization.

Example 9 (reference example): Preparation of compound 78

<img file="PL219737B1_D0115.tif" />

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105

Preparation of compound 76

<img file="PL219737B1_D0116.tif" />

To a solution of compound 75 (123 mg) in dry DCM (1 ml) at 0 ° C under an inert atmosphere, 1 M DIBAL (1.6 ml) was added dropwise. The reaction mixture was stirred at 0 ° C for 2 h before it was allowed to warm to 10 ° C, then re-cooled to 0 ° C. Methanol (0.22 ml) was added dropwise followed by a saturated ammonium chloride solution (0.44 ml). DCM (20 ml) was added and the reaction mixture was vigorously stirred at room temperature for 30 minutes. Filtration followed by concentration in vacuo afforded compound 76 (73 mg, 65%).

Preparation of compound 77

<img file="PL219737B1_D0117.tif" />

Dess-Martin periodate (3.1 mg) was added to a solution of compound 76 (3 mg) in acetonitrile (0.6 ml). The reaction mixture was stirred at room temperature for 1 h before it was diluted with diethyl ether (2 mL). The resulting slurry was filtered through a 0.25 µm PTFE syringe filter and concentrated in vacuo to give crude compound 77 (4 mg).

Preparation of compound 78

<img file="PL219737B1_D0118.tif" />

Ethyl carboethoxymethylidene triphenylphosphate (21 mg) was added to a solution of compound 77 (3 mg) in DCM (0.5 ml) at room temperature. The reaction mixture was stirred at room temperature for 16 h and then concentrated in vacuo to dryness. Chromatographic purification provided compound 78 (1.48 mg, 44%).

Example 10 (reference example): Preparation of compound 81

<img file="PL219737B1_D0119.tif" />

106

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Preparation of compound 79

<img file="PL219737B1_D0120.tif" />

To a solution of 7b (10 mg) in dry DCM (0.5 ml) at 0 ° C under an inert atmosphere, 1 M DIBAL (0.085 ml) was added dropwise. The reaction mixture was stirred at 0 ° C for 1.5 h before methanol (0.012 mL) was added dropwise followed by a saturated ammonium chloride solution (0.024 mL). DCM (5 ml) was added and the reaction mixture was vigorously stirred at room temperature for 20 minutes. Filtration followed by concentration in vacuo afforded crude compound 79 (9 mg, 95%).

Preparation of compound 80

<img file="PL219737B1_D0121.tif" />

Sodium bicarbonate (3.6 mg) and Dess-Martin periodate (7.2 mg) were added to the solution of compound 79 (5 mg) THF (0.5 ml).

The reaction mixture was stirred at room temperature for 3 h and then concentrated in vacuo to afford crude compound 80.

Preparation of compound 81

<img file="PL219737B1_D0122.tif" />

To a solution of compound 80 (4.8 mg) in ethanol (0.5 ml) at room temperature, hydroxylamine hydrochloride (4 mg) and sodium acetate (6 mg) were added. The reaction mixture was stirred at 40 ° C for 1.5 h and then dry. The residue was dissolved in DCM (0.2 ml), TFA (0.2 ml) was added and allowed to stand at room temperature for 10 minutes. Concentration in vacuo to dryness followed by chromatographic purification afforded compound 881 (2.04 mg).

Example 11 (reference example): Preparation of compound 87

<img file="PL219737B1_D0123.tif" />

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107

Preparation of compound 84

<img file="PL219737B1_D0124.tif" />

Sodium hydride (65% dispersion in mineral oil; 144 mg) was added portionwise to a solution of compound 28 (335 mg) in THF (10 ml) at 0 ° C under an inert atmosphere. The reaction mixture was stirred at 0 ° C for 30 minutes then methyl iodide (0.405 ml) was added. The reaction mixture was allowed to warm to room temperature and stirred at room temperature for 3 h. Work-up with water followed by chromatographic purification afforded compound 84 (254 mg, 72%). Preparation of compound 85

<img file="PL219737B1_D0125.tif" />

Saturated HCl in methanol (5 mL) was added to compound 84 (189 mg). The reaction mixture was allowed to stand at room temperature for 2 h and then concentrated in vacuo to dryness to afford compound 85 (145 mg).

Preparation of compound 86

<img file="PL219737B1_D0126.tif" />

(S) -N-Boc-t-leucine (483 mg), NMM (0.230 ml), HOAt (95 mg) and CMC were added to a solution of compound 85 (14 5 mg) in DMF (3 ml) at room temperature (884 mg). The reaction mixture was shaken at room temperature for 16 h. Work-up with water followed by chromatographic purification afforded the intermediate Boc-compound (249 mg, 93%). Intermediate Boc-compound (60 mg) was dissolved in methanol (1 ml), saturated HCl in methanol (3 ml) was added and allowed to stand at room temperature for 30 minutes. Concentration in vacuo afforded compound 86 (49 mg).

Preparation of compound 87

<img file="PL219737B1_D0127.tif" />

To a solution of compound 86 (49 mg) in DMF (0.44 ml) at room temperature, was added (S) -N-Boc-neo-phenylalanine (94 mg), NMM (34 µL), HOAt (21 mg) and CMC (130 mg). Reaction mixture

108

The mixture was shaken at room temperature for 16 h. Work-up with water followed by chromatographic purification afforded the intermediate Boc-compound (41 mg, 47%). Intermediate Boc-compound (5.5 mg) was dissolved in DCM (1 ml) and TFA (1 ml) was added. The reaction mixture was allowed to stand at room temperature for 30 minutes and then concentrated to dryness in vacuo. The residue was dissolved in saturated HCl in methanol (1 mL) and allowed to stand at room temperature then concentrated in vacuo to afford compound 87 (4.39 mg, 89%).

Example 12 (reference example): Preparation of compound 91

<img file="PL219737B1_D0128.tif" />

Preparation of compound 88

<img file="PL219737B1_D0129.tif" />

Methanesulfonyl chloride (0.207 ml) was added dropwise to a solution of compound 28 (344 mg) in 0.5 M Hunnig's base in DCM (8 ml) at 0 ° C under an inert atmosphere. After stirring the reaction at 0 ° C for 1.5 h, it was worked up with water followed by chromatographic purification to give the mesylate intermediate (444 mg). The intermediate mesylate was dissolved in DMSO (2 ml) and sodium azide (258 mg) was added. The reaction mixture was heated at 40 ° C for 6 h. Water treatment afforded compound 88 (306 mg, 82%).

Preparation of compound 89

<img file="PL219737B1_D0130.tif" />

Compound 88 (140 mg) was dissolved in DCM (1 ml) and TFA (1 ml) was added. The reaction mixture was allowed to stand at room temperature for 30 minutes and then concentrated to dryness in vacuo. The residue was dissolved in saturated HCl in methanol (1 mL) and allowed to stand at room temperature then concentrated in vacuo to give compound 89 (109 mg).

<img file="PL219737B1_D0131.tif" />

To a solution of compound 89 (109 mg) in DMF (2 ml) at room temperature was added (S) -N-Boc-t-leucine (347 mg), NMM (0.165 ml), HOAt (68 mg) and CMC ( 635 mg). The reaction mixture was stirred

PL 219 737 B1

109 at room temperature for 16 h. Work-up with water followed by chromatographic purification afforded the intermediate Boc-compound (173mg, 87%). Intermediate Boc-compound (51 mg) was dissolved in methanol (1 ml), saturated HCl in methanol (3 ml) was added and allowed to stand at room temperature for 30 minutes. Concentration in vacuo afforded compound 90 (43 mg).

Preparation of compound 91

<img file="PL219737B1_D0132.tif" />

(S) -N-Boc-neo-phenylalanine (79 mg), NMM (28 μθ, HOAt (17 mg) and CMC ( 108 mg) The reaction mixture was shaken at room temperature for 16 h. Work-up with water followed by chromatographic purification gave the intermediate Boc-compound (88 mg). Intermediate Boc-compound (88 mg) was dissolved in saturated HCl in methanol (5 ml) and allowed to stand at room temperature for 30 minutes then concentrated in vacuo to afford compound 91 (70 mg, 89%).

Example 13: General procedure for the preparation of compounds with a C-terminal acid group:

<img file="PL219737B1_D0133.tif" />

R2 = Me or Et

R1 = see examples below

<img file="PL219737B1_D0134.tif" />

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To a solution of the appropriate methyl or ethyl ester (e.g., compound 7b) in the appropriate mixture of methanol and tetrahydrofuran at room temperature, 1 M aqueous lithium hydroxide solution (10-50 equivalents) was added. The reaction mixture was stirred or shaken or allowed to stand at room temperature until the starting ester was satisfactorily hydrolyzed. Normal work-up followed by chromatographic purification afforded the desired compound with a C-terminal acid group (e.g. compound 82).

Example 14: Preparation of ER-807974

<img file="PL219737B1_D0135.tif" />

Preparation of ER-807641

<img file="PL219737B1_D0136.tif" />

While stirring into a solution of N-Boc-N-Me-L-valine (200 g, 0.86 mol), N, O-dimethylhydroxylamine (92.8 g, 0.95 mol, 1.1 eq.) And DIEA ( 316.3 mL, 1.82 mol, 2.1 eq.) In CH 3 CN (2 L) at 0 ° C was added HBTU (360.7 g, 0.95 mol, 1.1 eq.) In portions. The solution was stirred an additional 0 ° C for 15 min and then 1 h at 25 ° C. The reaction was monitored by TLC (Heptane / EtOAc 1: 1) and deemed complete when no compound 46 was observed. The solution was concentrated on the rotary evaporator then diluted with TBME (1 L). The organic solution was washed with HCl (IN, 500 ml), water (250 ml), NaHCO3 (saturated, 250 ml), and brine (250 ml). The organic solution was dried over MgSO4 (~ 120 g). The solution was filtered through a bed of silica gel (~ 200 g) and concentrated. ER-807641 crude amide was used without any further purification.

Preparation of ER-808993

<img file="PL219737B1_D0137.tif" />

To a solution of the amide ER-807641 (207 g, 755 mmol, 1 eq.) In dry THF (2070 ml) at -78 ° C, a solution of LiAlH was added while stirring₄ (1.0 M / THF, 754 mL, 755 mmol, 1.0 eq.). The solution was stirred at -78 ° C for 1 h. The reaction was quenched at -78 ° C by adding the reaction solution to the Na slurry.₂SO₄»10H₂O (243 g) in TBME (1.5 liters). The slurry was allowed to warm to ~ 15 ° C and then filtered through a pad of celite. The filtrate was concentrated and the crude aldehyde ER-808993 was obtained as a clear oil which was used without further purification: 157.9 g (97%).

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Preparation of ER-808995-01

<img file="PL219737B1_D0138.tif" />

Part A:

While stirring, to a solution of the aldehyde ER-808993 (138 g, 641 mmol, 1 eq.) In dry THF (1.4 L) at 25 ° C was added Ph3P = CMeCO2Et (256 g, 705.1 mmol, 1.1 eq.) . The solution was stirred at room temperature for 18 h. At this time, the reaction was incomplete. The solution was heated to reflux for 5 h when TLC showed the aldehyde had reacted. The solution was cooled to room temperature and heptane (1.5 L) was added. Precipitation of the Ph3P = O by-product was observed. The mixture was filtered through a pad of silica gel (200 g). The filtrate was concentrated to a minimum volume (-50 mL) and the residue was dissolved in EtOAc (800 mL).

Hello B:

MSA (80 ml) was added to a stirred solution of the crude ER-808994 in EtOAc (800 mL). The mixture was stirred at room temperature for 45 minutes (until complete by TLC). The MSA aminoester salt was extracted from the organic solution with water (2 x 300 mL). The aqueous layer was neutralized to pH 7-8 with saturated NaHCO solution₃ (300 ml). The resulting solution was extracted with EtOAc (2 x 400 mL), washed with brine (300 mL), dried over MgSO4, and filtered. HCl (gas) was bubbled through the EtOAc solution of the free aminoester and the precipitated ER-808995 was filtered off under a N2 atmosphere.

Preparation of ER-803921-01

<img file="PL219737B1_D0139.tif" />

While stirring into the solution of ER-808995 (61.2 g, 259.6 mmol, 1 eq.), N-Boc-tBu-Gly-OH (90.1 g, 389.4 mmol, 1.5 eq.) And DIEA HBTU (147.7 g, 389.4 mmol, 1.5 eq.) Was added (158 mL, 906.6 mmol, 3.5 eq.) In dry DCM (612 mL) at 25 ° C. The solution was stirred at room temperature for 4 h. After concentration, the solid residue was suspended in TBME (250 ml). The mixture was filtered through a bed of silica gel (~ 120 g) and the filtrate was washed with aq. HCl (IN, 200 ml), water (200 ml) and NaHCO3 (saturated, 200 ml). The organic layer was dried over MgSO4, filtered and concentrated. The N-Boc-Aminoester ER-808996 is isolated as an oil. The obtained intermediate was dissolved in EtOAc (120 ml) and MSA (75 ml) was added. The solution was stirred at room temperature for 1 h when the reaction was judged to be complete by TLC. The MSA aminoester salt was extracted with water (2 x 250 mL),

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The mixture was then neutralized with a NaOH solution (about 50%, 300 mL) to a pH of ~ 8-9. The free amine was extracted with TBME (2 x 30 ml). The combined organic solutions were washed with water (200 ml) and brine (200 ml). After drying over MgSO4 and filtration, bubbles of HCl (gas) were bubbled through the solution to give ER-803921 hydrochloride as a white solid, collected by filtration at about 5 ° C.

Preparation of ER-808998

<img file="PL219737B1_D0140.tif" />

While stirring, a suspension of D-pipecolinic acid (100.0 g, 0.77 mol, 1 eq.) And Pd (OH) 2 (20 wt.% Pd, 10 g) in MeOH / acetone (2: 1 v / v, 1 eq.) 5 liters)₂ dorniano (H2 60 lb / in²) for 24 h. The reaction was monitored by TLC (ethanol) and deemed complete when no D-pipecolinic acid was observed. The mixture was filtered through a bed of celite (~ 50 g). The clear filtrate was concentrated to approximately 100 ml and TBME (50 ml) was added. ER-808998 was filtered off as a white crystalline solid in 88% yield.

Preparation of ER-807961

<img file="PL219737B1_D0141.tif" />

N-iPr-pipecolinic acid ER-808998 (3.7 g, 21.8 mmol, 1.3 eq.) and HBTU (8.3 g, 21.8 mmol, 1.3 eq.) in 50 mL of DCM was added dropwise DIEA ( 7.3 ml, 41.9 mmol, 2.5 eq) at 25 ° C. The mixture was stirred for 18 h (overnight) when the reaction was judged to be complete by TLC (heptane / EtOAc 1: 1). The mixture was concentrated in vacuo and TBME (50 ml) was added. The "thick" oil residue was separated from the ethereal solution by filtration through a pad of celite. The filtrate was washed with an aqueous HCl solution (1M, 3 x 25 ml). The combined aqueous phases were neutralized with NH₄OH to pH 8 - 9 in the presence of EtOAc (25 mL). The aqueous layer was separated and extracted with TBME (25 ml). The combined organic phase was washed with brine then dried over MgSO4, filtered and concentrated to afford the tripeptide-aminoester ER-807961 in 93% yield.

Preparation of ER-807974

<img file="PL219737B1_D0142.tif" />

THF / H 2 O (50 mL) was added LiOH (3.50 g, 83.8 mmol) and the mixture was stirred at room temperature for 20 h. The reaction was monitored by TLC (ethanol) and deemed complete when ER-807961 was not observed. The suspension was acidified with H 2 SO 4 (~ 0.50 mL) to pH 7. The mixture was extracted with EtOAc (3 x 25 mL). The combined organic solution was washed with brine (20 mL), dried over MgSO4,

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113 filtered and concentrated. The residue was triturated with TBME to give 1.8 g (83%) of ER-807974 free base as a thick oil.

Example 15: Preparation of ER-808367

<img file="PL219737B1_D0143.tif" />

Preparation of 2Z

<img file="PL219737B1_D0144.tif" />

Pd (OH) 2 (175 mg) was added to a suspension of D-pipecolinic acid 1Z (750 mg, 5.81 mmol) in MeOH (23.2 ml) and 2-butanone (11.6 ml). H 2 gas (balloon pressure) was introduced and the reaction was stirred under H 2 atmosphere overnight. The reaction solution was then filtered through a bed of celite and concentrated to yield a crude white solid. The crude product was flash chromatographed (SiO2) eluting with 100% EtOH. Compound 2Z (721 mg, white solid) was obtained as a mixture of diastereoisomers in 67% yield.

Preparation of 3Z and 4Z compounds

<img file="PL219737B1_D0145.tif" />

K2CO3 (728 mg, 5.27 mmol) and p-nitrobenzyl bromide (1.1 g, 5.27 mmol) were added to a solution of 2Z (650 mg, 3.51 mmol) in DMF (8.8 mL). The reaction mixture was stirred overnight. The reaction solution was diluted with water and extracted several times with diethyl ether. The ether extracts were combined, washed with water and brine. The solution was dried over MgSO4, filtered, and concentrated in vacuo. The crude mixture of diastereoisomers was then separated by flash chromatography, eluting with 8% EtOAc in hexanes to give each diastereoisomer as a pale yellow oil. Compound 3Z (360 mg) was made in 32% yield, Rf = 0.590 (SiO2) using 30% EtOAc in hexanes. Compound 4Z (652 mg) was made in 58% yield, Rf = 0.482 (SiO2) with 30% EtOAc in hexanes.

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<img file="PL219737B1_D0146.tif" />

Pd (OH) 2 (50 mg) was added to a solution of 3Z (320 mg, 1.0 mmol) in MeOH (10 mL). H 2 gas (balloon pressure) was introduced and the reaction mixture was stirred under H 2 atmosphere for 3 hours. The reaction solution was then filtered through a bed of celite and concentrated to give ER-809439 (185 mg) as a white solid quantitatively. Compound ER-809439, Rf = (SiO2, 0.292, 100% EtOH).

Preparation of ER-809447

A similar procedure was used as for the preparation of ER-809439. Compound ER-809447, Rf = (SiO2, 0.292, 100% EtOH).

Preparation of ER-808357

<img file="PL219737B1_D0147.tif" />

Compound 49 (9.6 mg, 0.031 mmol), Ns-Butylpipecolinic acid ER-809439 (5.2 mg, 0.028 mmol) and HBTU (12.9 mg, 0.034 mmol) were combined. DMF (0.28 mL) was added followed by DIEA (14.9 mL, 0.084 mmol). The solution was stirred at room temperature under nitrogen atmosphere for 20 h. The solution was purified directly by RP HPLC to give the TFA salt of ER-808357 (13.6 mg, 82%).

Preparation of ER-808367

<img file="PL219737B1_D0148.tif" />

(0.072 ml / 0.144 ml) at room temperature. LiOH (7.5 g, 0.18 mmol) was added. The suspension was stirred at room temperature for 19 hours. The solution was directly purified by RP HPLC to give the TFA salt of ER-808367 (10.1 mg, quantitative).

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Example 16: Preparation of ER-808368

<img file="PL219737B1_D0149.tif" />

<img file="PL219737B1_D0150.tif" />

A similar procedure was used as for the preparation of ER-808357.

Preparation of ER-808368

<img file="PL219737B1_D0151.tif" />

A similar procedure was used as for the preparation of ER-808367.

Example 17: Preparation of ER-808662

<img file="PL219737B1_D0152.tif" />

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Preparation of 5Z

<img file="PL219737B1_D0153.tif" />

To a suspension of 1Z D-pipecolinic acid (1.00 g, 7.74 mmol) in MeOH (31 mL) and 3-methyl-2-butanone (15.5 mL) was added Et3N (1.1 mL) and Pd (OH ) 2 (250 mg). H 2 gas (balloon pressure) was introduced and the reaction was stirred under H 2 atmosphere overnight. The reaction solution was then filtered through a bed of celite and concentrated to yield a crude white solid. The crude product was flash chromatographed (SiO2) eluting with 100% EtOH. Compound 5Z (377.9 mg, white solid) was obtained as a single diastereoisomer in 24.5% yield. Rf = (SiO2, 0.280, 100% EtOH).

Preparation of ER-808656

<img file="PL219737B1_D0154.tif" />

A similar procedure was used as for the preparation of ER-808357. Preparation of ER-808662

<img file="PL219737B1_D0155.tif" />

A similar procedure was used as for the preparation of ER-808367. Example 18: Preparation of ER-808824

<img file="PL219737B1_D0156.tif" />

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<img file="PL219737B1_D0157.tif" />

Compound 48 (325.5 mg, 1.38 mmol), LN-BOC-valine (300.0 mg, 1.38 mmol) and HBTU (628.3 mg, 1.66 mmol) were combined. CH2Cl2 (7 mL) was added followed by DIEA (0.72 mL, 4.14 mmol). The solution was stirred at room temperature under nitrogen for 1 hour. The solution was concentrated in vacuo and the crude product was purified by flash chromatography (SiO2) eluting with 4% EtOAc in hexanes. Compound 6Z (476.8 mg) was obtained as a colorless oil with a yield of 86.7%.

Preparation of 7Z

<img file="PL219737B1_D0158.tif" />

Compound 6Z (450 mg, 1.13 mmol) was dissolved directly in 4N HCl / dioxane (2.8 mL). The reaction mixture was stirred overnight then concentrated in vacuo to give compound 7Z (374.8 mg) as a white solid quantitatively.

Preparation of ER-808815

<img file="PL219737B1_D0159.tif" />

A similar procedure was used as for the preparation of ER-808357. Preparation of ER-808824

<img file="PL219737B1_D0160.tif" />

A similar procedure was used as for the preparation of ER-808367.

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Example 19: Biological tests:

In certain embodiments, the in vitro and in vivo activity of the compounds of the invention has been tested. Screening methods included standard in vitro cell growth inhibition assays using a panel of human tumor cell lines, the mitotic blockade reversibility assay U937 (ATCC accession number CRL 1593), murine serum stability assay, MDR assay, and cytotoxicity assay. In certain other embodiments, compounds of the invention have been evaluated in an in vivo tumor xenograft growth inhibition assay.

The in vitro efficacy was assessed in the MDA-MB-435 cell growth inhibition assay, and active compounds (IC50 &lt;20 nM) were assessed in the reversibility, MDR and stability assays in mouse serum. In addition, the active compounds were tested in the IMR-90 cytotoxicity assay and in additional cell growth inhibition assays on a group of human tumor cell lines, both solid and non-solid tumors.

Cell Growth Inhibition Assay: Cultured human tumor cells (including breast, prostate, colon, lung, leukemia, lymphoma, and others) were seeded in 96-well plates and cultured in the constant presence of test compounds for 72 or 96 hours. Human cell lines used in this cell growth inhibition assay included, but were not limited to, the following solid tumor cell lines and non-solid tumor cell lines: colorectal cancer cells DLD-1 (ATCC accession number CCL-221), prostate cancer cells DU 145 (ATCC accession number HTB-81), non-small cell lung cancer H460, colorectal cancer cells HCT-15 (ATCC accession number CCL-225) , HEL erythroleukemia cells, HL-60 promyelocytic leukemia cells (ATCC accession number CCL-240), leukemias K562 (ATCC accession number CCL-243), LOX melanoma, MDA-MB-435 breast cancer cells, U937 lymphoma cells (ATCC accession number CRL 1593), pancreatic cancer PANC-1 (ATCC accession number CRL-1469), colorectal cancer HCC-2998 (NCI-Frederick Cancer DCTD Tumor / Cell Line Repository), colorectal cancer HCT 116 (ATCC accession number CCL-247), colorectal cancer HT-29 (ATCC accession number HTB-38), colorectal cancer LoVo (ATCC accession number CCL-229), colorectal cancer SW-480 (ATCC accession number CCL-228), colorectal cancer SW-620 (ATCC accession number CCL-227) and colorectal cancer COLO-205 (ATCC accession number CCL-222). For monolayer cultures, growth was assessed using modifications (Amin et al., Cancer Res., 47: 6040-6045, 1987) of the methylene blue microculture assay (Finlay et al., Anal. Biochem., 139: 272-277). , 1984). Absorbances at 620 and 405 nm were measured in a Titertek Multiscan MCC / 340 plate reader and the absorbances at 405 nm were subtracted from the absorbances at 620 nm. For suspension cultures, growth was assessed using a 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide test (Mosmann et al., J. Immunol. Methods, 65: 55-63, 1983). ), modified as follows. After incubation for 4 days with the test compounds, sterile filtered 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (final concentration 0.5 mg / ml) was added to each well and the plates were incubated. at 37 ° C for 4 h. Acidic isopropanol (0.1 N HCl in isopropanol, 150 mL) was then added to each well and the resulting formazan crystals were dissolved by gentle stirring. Absorbances at 540 nm were measured in a Titertek Multiscan MCC / 340 plate reader.

A mitotic blocking reversibility test was performed as described (see US 6,214,865 B1, B. Littelfield et al., 04/10/2001).

Briefly, U937 (ATCC accession number CRP 1593) was exposed to the compounds at various concentrations for 12 hours. Compounds were washed away and cells were allowed to recover for an additional 10 hours to recover. Cells were centrifuged and fixed overnight in 70% ethanol. Cells were washed in PBS, incubated with RNase A and stained with propidium iodide. Single channel flow cytometry was performed on a Becton Dickinson FACScan; data collection and analysis was performed using Becton Dickinson CELLQuest software. Doublet cases were eliminated from the analysis by appropriate gating of the primary FL2-W / FL2-A plots before analyzing the histograms for DNA content (measured as FL2-A).

Determination of in vitro activity using the MDR assay. This is a modification of the known cell growth inhibition assay described above. Two cultured human tumor cell lines were used: MDR-negative human uterine sarcoma cells MES-SA (ATCC accession number CRL-1976) and MDR positive human uterine sarcoma cells MES-SA / Dx5 (ATCC accession number CRL-1977). Cells were seeded in 96-well microtiter plates at 7,500 cells / well. Cells were incubated with or without the test compounds for 96 hours. Cell growth was assessed using a modified (Amin

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119 et al., Cancer Res., 47: 6040-6045, 1987) methylene blue microculture trials (Finlay et al., Anal. Biochem., 139: 272-277, 1984). Absorbances at 620 and 405 nm were measured on a Titertek Multiscan MCC / 340 plate reader and the absorbances at 405 nm were subtracted from the absorbances at 620 nm. The concentrations of the compound that inhibited cell growth of 50% were calculated and used to evaluate the sensitivity of the MDR compounds (multi-drug resistance or P-glycoprotein mediated drug efflux). In some cases, a different pair of cell lines was used: MDR-negative P388 / S murine leukemia cells and MDR-positive P388 / VMDRC.04 murine leukemia cells. Cells were seeded in 96-well microtiter plates at 4000 cells / well. Cells were incubated with or without the test compounds for 72 hours. Cell growth was assessed using a 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide assay (Mosmann et al., J. Immunol. Methods, 65: 55-63, 1983), modified as follows. After incubation for 3 days with the test compounds, sterile filtered 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (final concentration 0.5 mg / ml) was added to each well and the plates were incubated at 37 ° C for 4 h. Acidic isopropanol (0.1N HCl in isopropanol, 150 ml) was then added to each well and the resulting formazan crystals were dissolved by gentle stirring. Absorbances at 540 nm were measured in a Titertek Multiscan MCC / 340 plate reader.

The resistance to esterase degradation was determined in a mouse serum stability test. The enzymatic activity of mouse serum can inactivate the compounds in vivo, despite their promising in vitro activity. The modified cell growth inhibition known assay described above was used to determine the resistance of test compounds to degradation by esterase. The human breast cancer cell line MDA-MB-435 or the human prostate cancer cell line DU 145 was used. Cells were seeded in 96-well microtiter plates at 7,500 cells / well. Test compounds were incubated in 100% mouse serum or normal growth medium for 6 hours at 37 ° C before adding test compounds to the cells in the cell growth inhibition assay. Test compounds were then added to 96-well microtiter plates containing the cells. Cells were incubated with or without the test compounds for 96 hours. Cell growth was assessed using a modified (Amin et al., Cancer Res., 47: 6040-6045, 1987) methylene blue microculture assay (Finlay et al., Anal. Biochem., 139: 272-277, 1984). Absorbances at 620 and 405 nm were measured in a Titertek Multiscan MCC / 340 plate reader. The ability of the test compounds to inhibit cell growth upon exposure of the compounds to mouse serum esterases was assessed.

Cytotoxicity test. To test the toxicity of compounds against normal, non-dividing cells, inactive, normal human IMR-90 fibroblasts (ATCC accession number CCL-186) were used. IMR-90 cells were plated in 96-well microtiter plates and grown to confusion (for 72 hours). After culturing for 72 hours, the cells were washed and the medium was changed from normal medium with 10% fetal bovine serum to medium with a lower serum concentration (0.1%). Cells were allowed to rest by incubating in 0.1% serum growth medium for an additional 72 hours. Cells were incubated with test compounds for 24 hours. Cellular ATP levels were measured using the ViaLight HS kit (LumiTech Ltd). The cytotoxic compound, carbonyl cyanide, was used as a positive cytotoxicity control in all trials.

Determination of in vivo anti-tumor activity in mice. In vivo tumor xenograft studies were conducted in immunocompromised (nude) mice. Mice (female Ncr without thymus) were subcutaneously implanted with human tumor xenografts (including MDA-MB-435 breast carcinoma, colorectal colorectal carcinoma COLO-205, HCT-15, HCT-116, HCC2998, HT-29, SW-620, DLD-1, LoVo, LOX melanoma, H522 lung, PANC-1 pancreas). After the xenografts have reached an average volume of 75-200 mm³ or 400-600 mm³, animals were weighed and randomized into groups of 8-10 on the first day of compound administration. Test compounds were administered intravenously or intraperitoneally. Tumor and body weights were measured twice a week.