Vitamin receptor binding drug delivery conjugates

ES 2 395 082 T3

DESCRIPTION

Folate-vinblastine conjugate as a medicine.

Cross reference to related requests

The present application claims priority under title 35 of the United States Code, § 119 (e) for the United States Patent Application Serial No. 60 / 442,845, entitled Vitamin-Receptor Binding Drug Delivery Conjugates , filed January 27, 2003, United States Patent Application Serial No. 60 / 492,119, entitled Vitamin-Receptor Binding Drug Delivery Conjugates, filed August 1, 2003, and US Patent Application Serial No. 60 / 516,188, entitled Vitamin-Receptor Binding Drug Delivery Conjugates, filed October 31, 2003.

Field of the invention

The present invention relates to compositions and their use in the targeted delivery of drugs. More specifically, the invention is directed to conjugates for delivering drugs to the vitamin-binding receptor for use in treating disease states caused by populations of pathogenic cells, and to pharmaceutical compositions manufactured from the foregoing.

Background of the invention

The mammalian immune system provides a means of recognizing and killing tumor cells, other pathogenic cells, and invading foreign pathogens. Although the immune system is normally a strong line of defense, there are many cases in which cancer cells, other pathogenic cells, and invading foreign pathogens can elude the host's immune response and proliferate or persist with consequent host pathogenesis. Chemotherapeutic agents and radiation therapies have been developed to eliminate, for example, replicating neoplasms. However, many of the currently available chemotherapeutic agents and radiation therapy regimens have adverse side effects because they not only act to kill pathogenic cells, but also affect normal host cells, such as cells of the hematopoietic system. The adverse side effects of these anticancer drugs highlight the need to develop new treatments that are selective for the pathogenic cell population and with less toxicity for the host.

Researchers have developed therapeutic protocols to kill pathogenic cells by targeting cytotoxic compounds at these cells. Many of these protocols use antibody-conjugated toxins that bind to antigens that are uniquely or excessively expressed by pathogenic cells in an attempt to minimize delivery of the toxin to normal cells. With this approach, several immunotoxins have been developed consisting of antibodies directed against specific antigens on pathogenic cells, the antibodies being bound to toxins such as ricin, Pseudomonas exotoxin, diphtheria toxin, and tumor necrosis factor. These immunotoxins target pathogenic cells, such as tumor cells, which contain the specific antigens recognized by the antibody (Olsnes, S., Immunol Today, 10, pp. 291-295, 1989; Melby, EL, Cancer Res., 53 (8), pp. 1755-1760, 1993; Better, MD, PCT publication number WO 91/07418, published May 30, 1991).

Another approach to targeting drugs against pathogenic cell populations, such as cancer cells or foreign pathogens, in a host is to enhance the host's immune response against pathogenic cells to avoid the need to administer compounds that may also show independent toxicity to the host. One reported immunotherapy strategy is to bind antibodies, eg, genetically engineered multimeric antibodies, to the surface of tumor cells to present the constant region of the antibodies on the cell surface and induce tumor cell death by different processes mediated by the immune system (De Vita, VT, Biologic Therapy of Cancer, 2^to ed. Philadelphia, Lippincott, 1995; Soulillou, JP, US Patent 5,672,486). However, these approaches have been complicated by difficulties in defining tumor specific antigens.

Summary of the invention

In an attempt to develop effective treatments specific to pathogenic cells and with minimized toxicity to normal cells, conjugates have been developed to deliver drugs to the vitamin-binding receptor. The present invention can be applied to pathogenic cell populations that uniquely express, preferably express, or overexpress folate receptors or receptors that bind to folate derivatives or analogs.

The invention relates to a conjugate for administering drugs having the formula

ES 2 395 082 T3

<img file="ES2395082T3_D0001.tif" />

The invention further relates to a pharmaceutical composition comprising the above drug delivery conjugate and a pharmaceutically acceptable carrier, diluent or excipient thereof.

The invention further relates to the use of the above drug delivery conjugate or the above composition in the manufacture of a drug for treating cancer in a patient. In a related aspect, the invention relates to the above drug delivery conjugate or the above composition in a method for treating cancer in a patient.

In some embodiments of the invention, the cancer is selected from the group consisting of oral, thyroid, endocrine, skin, gastric, esophageal, laryngeal, pancreatic, colon, bladder, bone, ovarian, cervical, uterine cancer. , breast, testicular, prostate, rectal, kidney, liver and lung. One specific cancer is ovarian cancer. Another specific cancer is breast cancer. Another specific additional cancer is lung cancer.

The invention relates to the above subject matter. However, the following is included for the purpose of further illustration and providing supporting information regarding the claimed subject matter.

A drug delivery conjugate comprises a vitamin receptor binding moiety, a bivalent linker, and a drug. As used herein, V refers to a vitamin receptor binding moiety and includes vitamins, and vitamin receptor binding analogs or derivatives thereof, and the term "vitamin or analog or derivative of itself ”refers to vitamins and analogs and derivatives thereof that are capable of binding to vitamin receptors. As used herein, D refers to drugs, and includes analogs and derivatives thereof. The vitamin, or the analog or derivative thereof, is covalently bound to the bivalent linker (L), and the drug, or the analog or derivative thereof, is also covalently bound to the bivalent linker (L). The bivalent linker (L) can comprise several linkers. For example, the bivalent linker (L) may comprise one or more components selected from spacer linkers (ls), releasable linkers (lr), and heteroatom linkers (Ih), and combinations thereof, in any order.

Conjugates for drug delivery that illustrate this embodiment include:

VLD

V- (lr) cD

V- (ls) aD

V- (ls) a- (lr) cD

V- (lr) c- (ls) aD

V- (lH) b- (lr) cD

V- (lr) c- (lH) bD

V- (lH) d- (lr) c- (lH) eD

V- (ls) a- (lH) b- (lr) cD

V- (lr) c- (lH) - (ls) aD

V- (lH) d- (ls) a- (lr) c- (lH) eD

V- (lH) d- (lr) c- (ls) a- (lH) eD

V- (lH) d- (ls) a- (lH) b- (lr) c- (lH) eD

V- (lH) d- (lr) c- (lH) b- (ls) a- (lH) eD

V- (ls) a- (lr) c- (lH) bD

ES 2 395 082 T3

V - [(ls) a- (lH) b] d- (lr) c- (lH) eD where a, b, c, d, and e are each independently 0, 1, 2, 3, or 4 , (ls), (Ih), and (lr) are as defined herein, V is a vitamin, or analog or derivative thereof, and D is a drug, or analog or derivative thereof, and where the bivalent L encompasses one of a variety of (ls), (Ih), and (lr), in any order and any combination. It is understood that the above examples of the bivalent linker L are intended to illustrate the wide variety of assemblies between (Ih), (ls), and (lr) that the bivalent linker encompasses.

Each of the release, heteroatom, and spacer linkers is understood to be bivalent. It should be further understood that the connectivity of each of the different spacer linkers, heteroatom and releasable, and between the different spacer linkers, heteroatom and releasable and D and / or V, as defined herein, may occur in any of the spacer linkers, heteroatom and releasable atoms, and do not necessarily occur at any apparent end of any of the different spacer linkers, heteroatom and releasable. For example, in the exemplary embodiment where the bivalent linker is:

<img file="ES2395082T3_D0002.tif" />

that is, where the bivalent linker L is - (lH) - (ls) s- (lr-lH) 2-D, where (Ih) is nitrogen, (l_s) s is Ala-Glu-Lys-AspAsp, and (Ιγ-Ιη> 2 is - (CH2) 2-SS- (CH2) 2-OC (O) -O-, respectively, the linker (lr-lH> 2 is connected to the middle part of the linker (ls) s.

The drug delivery conjugate may comprise a vitamin receptor binding moiety, a bivalent linker (L), and a drug, and the bivalent linker (L) comprises one or more heteroatom linkers (1H). The vitamin receptor binding moiety may be covalently attached to the bivalent linker (L) via a first heteroatom linker (IH) d and the drug may be covalently attached to the bivalent linker (L) via a second heteroatom linker (IH) and . The bivalent linker (L) may also comprise one or more spacer linkers and releasable linkers, wherein the spacer linkers and releasable linkers may be covalently linked to each other in a different manner to a third heteroatom linker (IH) b. Said drug delivery conjugate is illustrated as follows:

V- (lH) d- (ls) a- (lH) b- (lr) c- (lH) eD where a, b, c, d, and e are each independently 0, 1, 2, 3, or 4, (ls), (Ih), and (lr), and V and D are as defined herein, and wherein the bivalent linker L encompasses (ls), (Ih), and (lr ) as illustrated.

The drug delivery conjugate may comprise a vitamin receptor binding moiety, a bivalent linker (L), and a drug, and the bivalent linker (L) may comprise a heteroatom linker (Ih). The vitamin receptor binding moiety can be a vitamin, or an analog or derivative thereof, and the drug can include analogs or derivatives thereof. The vitamin, or the analog or derivative thereof, may be covalently bound to the bivalent linker (L) and the drug, or the analog or derivative thereof, may be covalently bound to the bivalent linker (L). The bivalent linker (L) may also comprise spacer linkers and releasable linkers, and spacer linkers and releasable linkers may be covalently bonded to each other differently than by the heteroatom linker. A drug delivery conjugate is illustrated as follows:

V- (ls) a- (lH) b- (lr) cD where a, b, and c are each independently 0, 1, 2, 3, or 4, (ls), (Ih), and (lr ), and V and D are as defined herein, and wherein the bivalent linker L encompasses (ls), (Ih), and (lr) as illustrated.

A conjugate for delivering drugs to the vitamin binding receptor may also have the general formula VL-D. In this embodiment, L is constructed with one or more linkers (lr) c, (ls) a, and (lH) b, and combinations thereof, in any order, where (lr) is a releasable linker, ( ls) is a spacer linker, (Ih) is a heteroatom linker, and a, b, and c are each independently 0, 1, 2, 3, or 4, V is a vitamin, or an analog or derivative thereof , and D is a drug, or an analog or a derivative thereof. It will be appreciated that the drug delivery conjugates described herein may include a bivalent linker having more than one spacer linker, releasable linker, or heteroatom linker. For example, bivalent linkers are contemplated that include two or more releasable linkers (lr). Furthermore, configurations of such releasable linkers include bivalent linkers in which the releasable linkers are attached.

ES 2 395 082 T3 covalently with each other, and wherein the releasable linkers are separated from each other by one or more heteroatom linkers and / or spacer linkers.

A vitamin binding receptor drug delivery conjugate may also have the general formula VL-D where L is a bivalent linker comprising (ls) a and (lH) b, and combinations thereof in any order, in the that (ls) a and (lH) b, and V and D are as defined herein. In this embodiment, the drug in the drug delivery conjugate can be a hapten such as fluorescein or nitrophenyl.

A conjugate for drug delivery to the vitamin-binding receptor may also have the general formula VL-D, where L is a bivalent linker comprising (l_s) a, (lH) b, and (lr) c, and combinations thereof in any order, in which (l_s) a and (lH) b, and V and D are as defined herein, and wherein at least one of (lr) is not a disulfide. It will be appreciated that the bivalent linkers of this embodiment with more than one (lr), ie where c is greater than 1, may include a disulfide releasable linker in addition to one or more other releasable linkers.

In one aspect of different conjugates for delivering drugs to the vitamin-binding receptor, the bivalent linker comprises a heteroatom linker, a spacer linker, and a releasable linker taken together to form 3-thiosuccinimid-1-ylalkyloxymethyloxy, wherein the methyl is optionally substituted by a substituted alkyl or aryl.

In another aspect, the bivalent linker comprises a heteroatom linker, a spacer linker, and a releasable linker taken together to form 3-thiosuccinimid-1-ylalkylcarbonyl, wherein the carbonyl forms an acylaridine with the drug, or analog or derivative of the drug. same.

In another aspect, the bivalent linker comprises a heteroatom linker, a spacer linker, and a releasable linker taken together to form 1-alkoxycycloalkyleneoxy.

In another aspect, the bivalent linker comprises a spacer linker, a heteroatom linker, and a releasable linker taken together to form alkylene aminocarbonyl (dicarboxylarylene) carboxylate.

In another aspect, the bivalent linker comprises a releasable linker, a spacer linker, and a releasable linker taken together to form dithioalkylcarbonylhydrazide, wherein the hydrazide forms a hydrazone with the drug, or analog or derivative thereof.

In another aspect, the bivalent linker comprises a heteroatom linker, a spacer linker, and a releasable linker taken together to form 3-thiosuccinimid-1-ylalkylcarbonylhydrazide, wherein the hydrazide forms a hydrazone with the drug, or analog or derivative of the same.

In another aspect, the bivalent linker comprises a heteroatom linker, a spacer linker, a heteroatom linker, a spacer linker, and a releasable linker taken together to form 3-thioalkylsulfonylalkyl (disubstituted silyl) oxyl, wherein the disubstituted silyl is substituted by optionally substituted alkyl or aryl.

In another aspect, the bivalent linker comprises a plurality of spacer linkers selected from the group consisting of natural amino acids and stereoisomers thereof.

In another aspect, the bivalent linker comprises a releasable linker, a spacer linker, and a releasable linker taken together to form 3-dithioalkyloxycarbonyl, wherein the carbonyl forms a carbonate with the drug, or analog or derivative thereof.

In another aspect, the bivalent linker comprises a releasable linker, a spacer linker, and a releasable linker taken together to form 3-dithioarylalkyloxycarbonyl, wherein the carbonyl forms a carbonate with the drug, or analog or derivative thereof, and the aryl is optionally substituted.

In another aspect, the bivalent linker comprises a heteroatom linker, a spacer linker, a releasable linker, a spacer linker, and a releasable linker taken together to form 3-thiosuccinimid-1ylalkyloxyalkyloxyalkylidene, wherein the alkylidene forms a hydrazone with the drug. , or analog or derivative thereof, each alkyl is independently selected, and the oxyalkyloxy is optionally substituted by alkyl or optionally substituted aryl.

In another aspect, the bivalent linker comprises a releasable linker, a spacer linker, and a releasable linker taken together to form 3-dithioalkyloxycarbonylhydrazide.

In another aspect, the bivalent linker comprises a releasable linker, a spacer linker, and a releasable linker taken together to form 3-dithioalkylamino, wherein the amino forms a vinyl amide with the drug, or analog or derivative thereof.

In another aspect, the bivalent linker comprises a releasable linker, a spacer linker, and a releasable linker taken together to form 3-dithioalkylamino, wherein the amino forms a vinyl amide with the drug,

ES 2 395 082 T3 or analog or derivative thereof, and the alkyl is ethyl.

In another aspect, the bivalent linker comprises a releasable linker, a spacer linker, and a releasable linker taken together to form 3-dithioalkylaminocarbonyl, wherein the carbonyl forms a carbamate with the drug, or analog or derivative thereof.

In another aspect, the bivalent linker comprises a releasable linker, a spacer linker, and a releasable linker taken together to form 3-dithioalkylaminocarbonyl, wherein the carbonyl forms a carbamate with the drug, or analog or derivative thereof, and the alkyl is ethyl.

In another aspect, the bivalent linker comprises a releasable linker, a spacer linker, and a releasable linker taken together to form 3-dithioarylalkyloxycarbonyl, wherein the carbonyl forms a carbamate or a carbamoylaziridine with the drug, or analog or derivative thereof.

In one aspect, linkers, releasable, spacers, and heteroatoms can be arranged such that upon cleavage of a bond in the bivalent linker, the released functional groups chemically aid in the breaking or cleavage of other bonds, also called breaking or cleavage. with anchimeric aid. An exemplary embodiment of such a bivalent linker or part thereof includes compounds having the formula:

<img file="ES2395082T3_D0003.tif" />

where X is a heteroatom, such as nitrogen, oxygen, or sulfur, n is an integer selected from 0, 1, 2, and 3, R is hydrogen, or a substituent, including a substituent capable of stabilizing a positive charge by induction or resonance on the aryl ring, such as alkoxy, and the symbol (*) indicates attachment points for additional spacer, heteroatom, or releasable linkers, conforming the linker bivalent, or alternatively for drug attachment, or analog or derivative thereof, or the vitamin, or analog or derivative thereof. It will be appreciated that other substituents may be present on the aryl ring, benzyl carbon, alkanoic acid, or methylene bridge, including hydroxyl, alkyl, alkoxy, alkylthio, and halo. Assisted cleavage can include mechanisms involving benzyl intermediates, benzene intermediates, lactone cyclization, oxonium intermediates, and beta-elimination. It will further be appreciated that, in addition to post-cleavage cleavage of the releasable linker, initial cleavage of the releasable linker may be facilitated by an anchimeric assist mechanism.

An intermediate of the conjugate for delivering drugs to the vitamin binding receptor may comprise a vitamin receptor binding moiety, a bivalent linker, having a first end and a second end, and a coupling group. The vitamin receptor binding moiety can be a vitamin, or an analog or derivative thereof, and the coupling group is a nucleophile, an electrophile, or a precursor thereof. The vitamin receptor binding moiety may be covalently attached to the bivalent linker at the first end of the bivalent linker, and the coupling group may be covalently attached to the bivalent linker at the second end of the bivalent linker, and the bivalent linker may comprise one or more spacer linkers, releasable linkers, and heteroatom linkers, and combinations thereof, in any order.

An intermediate of the conjugate for delivering drugs to the vitamin-binding receptor may also comprise a bivalent linker, having a first end and a second end, a drug, or an analog or derivative thereof, and a coupling group. The bivalent linker may comprise one or more components selected from spacer linkers, releasable linkers, and heteroatom linkers, as described herein. The coupling group may be covalently attached to the bivalent linker at the first end of the bivalent linker, and the drug or analog or derivative thereof may be covalently attached to the bivalent linker at the second end of the bivalent linker. Furthermore, the coupling group can be a nucleophile, an electrophile, or a precursor thereof, capable of forming a covalent bond with a vitamin receptor binding moiety, where the vitamin receptor binding moiety is a vitamin, or an analog or a derivative thereof.

In another exemplary embodiment of the vitamin binding receptor conjugate intermediate described herein, the coupling group may be a Michael acceptor, and the bivalent linker includes a releasable linker having the formula - C (O) NHN =, -NHC (O) NHN =, or CH2C (O) NHN =. In an illustrative aspect of the conjugate intermediate for drug delivery to the vitamin-binding receptor described herein, the coupling group and the bivalent linker are taken together to form a compound having the formula:

ES 2 395 082 T3

<img file="ES2395082T3_D0004.tif" />

or a protected derivative thereof, wherein D is the drug, or an analog or derivative thereof, capable of forming a hydrazone as illustrated herein; and n is an integer such as 1, 2, 3, or 4. In another illustrative aspect of the conjugate intermediate for delivering drugs to the vitamin-binding receptor described herein, the vitamin, or an analog or derivative of itself includes an alkylthiol nucleophile.

In another exemplary embodiment of the conjugate intermediate for delivering drugs to the vitamin binding receptor described herein, the coupling group is a heteroatom, such as nitrogen, oxygen, or sulfur, and the bivalent linker includes one or more heteroatom linkers and one or more spacer linkers that covalently connect the vitamin or analog or derivative thereof to the coupling group. In an illustrative aspect, the vitamin-binding receptor drug delivery conjugate intermediate described herein includes a compound having the formula:

<img file="ES2395082T3_D0005.tif" />

or a protected derivative thereof, wherein X is oxygen, nitrogen, or sulfur, and m is an integer such as 1, 2, or 3, and wherein V, ls, and Ih are as defined herein document.

In another illustrative aspect, the vitamin-binding receptor drug delivery conjugate intermediate described herein includes a compound having the formula:

HOjC '__

V-lj — NH XH, or a protected derivative thereof, where X is nitrogen or sulfur, where V and ls are as defined herein.

In another illustrative aspect, the vitamin-binding receptor drug delivery conjugate intermediate described herein includes a compound having the formula:

<img file="ES2395082T3_D0006.tif" />

or a protected derivative thereof, wherein Y is hydrogen or a substituent, for example an electrophilic substituent including nitro, cyano, halo, alkylsulfonyl, and a carboxylic acid derivative, and wherein V and ls are as shown. have been defined in this document.

In another exemplary embodiment of the conjugate intermediate for drug delivery to the vitamin-binding receptor described herein, the coupling group is a Michael acceptor, and the bivalent linker includes one or more heteroatom linkers and one or more plus spacer linkers that covalently connect the vitamin or analog or derivative thereof to the coupling group. In an illustrative aspect of the conjugate intermediate for drug delivery to the vitamin-binding receptor described herein, the coupling group and the bivalent linker are taken together to form a compound having the formula:

<img file="ES2395082T3_D0007.tif" />

or a protected derivative thereof, where X is oxygen, nitrogen, or sulfur, and m and n are independently selected integers, such as 1, 2, or 3, and where V, ls, and Ih are as defined in This document. In another illustrative aspect of the conjugate intermediate for delivering drugs to the vitamin-binding receptor described herein, the drug, or an analog or derivative thereof, includes an alkylthiol nucleophile.

In another illustrative aspect of the drug delivery conjugate to the vitamin-binding receptor intermediate described herein, the intermediate includes compounds of the formulas:

ES 2 395 082 T3

<img file="ES2395082T3_D0008.tif" />

or protected derivatives thereof, wherein V is the vitamin, or an analog or derivative thereof, AA is an amino acid, selected by way of example from natural amino acids or stereoisomers thereof, X is nitrogen oxygen, or sulfur, Y is hydrogen or a substituent, for example an electrophilic substituent, including, nitro, cyano, halo, alkylsulfonyl and a carboxylic acid derivative, m and n are independently selected integers, such as 1, 2, or 3, and p is an integer such as 1, 2, 3, 4, or 5. AA can also be any other amino acid, such as an amino acid having the general formula:

-N (R) - (CR'R) qC (O) where R is hydrogen, alkyl, acyl, or a suitable nitrogen protecting group, R 'and R are hydrogen or a substituent, each of which is has independently selected at each occurrence, and q is an integer such as 1, 2, 3, 4, or 5. By way of example, R 'and / or R independently correspond to hydrogen or the side chains present in natural amino acids, such as methyl, benzyl, hydroxymethyl, thiomethyl, carboxyl, carboxylmethyl, guanidinopropyl, and derivatives and protected derivatives thereof. . The formulas described above include all stereoisomeric variants. For example, the amino acid can be selected from asparagine, aspartic acid, cysteine, glutamic acid, lysine, glutamine, arginine, serine, ornithine, and threonine. In another illustrative aspect of the conjugate for delivering drugs to the vitamin-binding receptor intermediate described herein, the drug, or an analog or derivative thereof, includes an alkylthiol nucleophile.

In another exemplary embodiment, a process for preparing a compound having the formula is described:

<img file="ES2395082T3_D0009.tif" />

or a protected derivative thereof, wherein L is a linker comprising (lr) c, (ls) a, and (ta) b, and combinations thereof; and D is a drug, or an analog or derivative thereof, capable of forming a hydrazone, where (lr) c, (ls) a, and (lH) b, and V are as defined herein , a process that includes the stages of:

(a) reacting a compound having the formula:

<img file="ES2395082T3_D0010.tif" />

or a protected derivative thereof, with a compound having the formula:

<img file="ES2395082T3_D0011.tif" />

or a protected derivative thereof to form a thiosuccinimide derivative; and (b) forming a hydrazone derivative of the drug, or an analog or derivative thereof, with the thiosuccinimide derivative.

In another exemplary embodiment, a process for preparing a compound having the formula is described:

<img file="ES2395082T3_D0012.tif" />

wherein L is a linker comprising (lr) c, (ls) a, and (ta) b, and combinations thereof; and where D is the drug, or an analog or derivative thereof, capable of forming a hydrazone, and (lr) c, (ls) a, and (lH) b, and V are as

ES 2 395 082 T3 have been defined in this document, the process comprises the stage of:

reacting a compound that has the formula:

<img file="ES2395082T3_D0013.tif" />

or a protected derivative thereof, with a compound having the formula:

<img file="ES2395082T3_D0014.tif" />

or a protected derivative thereof.

In another exemplary embodiment, a pharmaceutical composition is disclosed. The pharmaceutical composition comprises a drug delivery conjugate, and a pharmaceutically acceptable carrier therefor.

In another exemplary embodiment, a drug delivery conjugate is disclosed for use in a method of killing a pathogenic cell population in a host animal harboring the pathogenic cell population wherein members of the pathogenic cell population have a site accessible binding site for a vitamin, or an analog or derivative thereof, and wherein the binding site is uniquely expressed, overexpressed, or preferentially expressed by pathogenic cells.

Brief description of the drawings

Fig. 1 shows inhibition of M109 tumor growth by EC112 (Example 9c).

Fig. 2 shows the effect of EC 112 (Example 9c) on the body weight of the animal.

Fig. 3 shows inhibition of M109 tumor growth by EC105 (Example 10a).

Fig. 4 shows the effect of EC105 (Example 10a) on the body weights of the animals.

Fig. 5 shows the lack of inhibition of 4T1 tumor growth by EC105 (Example 10a).

Fig. 6 shows inhibition of M109 tumor growth by EC145 (Example 16b).

Fig. 7 shows inhibition of M109 tumor growth by EC140 (Example 17a).

Fig. 8 shows inhibition of L1210 tumor growth by EC136 (Example 10b).

Figs. 9-16 shows the inhibition of cellular DNA synthesis by EC135, EC136, EC137, EC138, EC140, EC145, EC158, and EC159 (Examples 17b, 10b, 16a, 10c, 17a, 16b, 14e, and 15, respectively) .

Detailed description of the invention

The invention relates to a conjugate for administering a drug having the formula

<img file="ES2395082T3_D0015.tif" />

The invention further relates to a pharmaceutical composition comprising the above conjugate for drug delivery and a pharmaceutically acceptable carrier, diluent or excipient therefor.

The invention further relates to the use of the above conjugate for administering drug or the above composition in the manufacture of a medicament for treating cancer in a patient. In a related aspect, the invention relates to the above conjugate for drug delivery or the above composition for use in a method of treating cancer in a patient.

In some embodiments of the invention the cancer has been selected from the group consisting of oral, thyroid, endocrine, skin, gastric, esophageal, laryngeal, pancreatic, colon, bladder, bone, ovarian, cervical, uterine cancer. , breast, testicular, prostate, rectal, kidney, liver and lung. One specific cancer is ovarian cancer. Another specific cancer is breast cancer.

The invention relates to the above subject matter. However, the following is included for the purpose of further illustration and providing supporting information regarding the claimed subject matter.

ES 2 395 082 T3

An exemplary embodiment relates to a vitamin-binding receptor drug delivery conjugate comprising a vitamin receptor-binding moiety, a bivalent linker (L), and a drug wherein the vitamin-binding receptor moiety The vitamin and the drug are each linked to the bivalent linker (L), optionally via a heteroatom linker. The bivalent linker (L) comprises one or more spacer linkers, heteroatom linkers, and releasable (ie, cleavable) linkers, and combinations thereof, in any order.

The term "releasable linker" as used herein refers to a linker that includes at least one linkage that can be cleaved under physiological conditions (e.g., a pH-labile, acid-labile, acid-labile bond oxidation, or enzyme labile). It will be appreciated that such physiological conditions that result in bond breaking include the usual chemical hydrolysis reactions that occur, for example, at physiological pH, or as a result of compartmentalisation in a cellular organelle such as an endosome having a lower pH. at cytosolic pH.

It is understood that a cleavable bond can connect two adjacent atoms within the releasable linker and / or connect other linkers or V and / or D, as described herein, at either or both ends of the releasable linker. In the case where a cleavable bond connects two adjacent atoms within the releasable linker, upon breaking of the bond, the releasable linker breaks into two or more fragments. Alternatively, where the cleavable linker is between the releasable linker and another moiety, such as a heteroatom linker, a spacer linker, another releasable linker, the drug, or analog or derivative thereof, or the vitamin, or Analogue or derivative thereof, upon breaking of the bond, the releasable linker is separated from the other moiety.

The lability of the cleavable bond can be adjusted by, for example, substitutional changes at or near the cleavable bond, such as including alpha branching adjacent to a cleavable disulfide bond, increasing the hydrophobicity of silicon substituents on a moiety having a silicon bond. -oxygen that can be hydrolyzed, analogously to the alkoxyl groups that are part of a ketal or acetal that can be hydrolyzed.

Conjugates to deliver drug to the vitamin-binding receptor can be used to treat disease stages characterized by the presence of a population of pathogenic cells in the host where members of the population of pathogenic cells have an accessible binding site for a vitamin, or an analog or derivative thereof, wherein the binding site is uniquely expressed, overexpressed, or preferentially expressed by pathogenic cells. Selective killing of pathogenic cells is mediated by binding of the vitamin moiety of the conjugate to deliver drugs to the vitamin-binding receptor or carrier or other surface-presented protein that specifically binds to the vitamin, or analog or derivative of the itself, and that it is uniquely expressed, overexpressed, or preferentially expressed by pathogenic cells. A surface-presented protein that is uniquely expressed, overexpressed, or preferentially expressed by pathogenic cells is a receptor not present or present at low concentrations on non-pathogenic cells providing a means for the selective killing of pathogenic cells. .

For example, surface-expressed vitamin receptors, such as the high-affinity receptor for folate, are over-expressed on cancer cells. All epithelial cancers of the ovary, mammary gland, colon, lung, nose, throat, and brain have been reported to express elevated levels of the folate receptor. In fact, more than 90% of all human ovarian tumors are known to express large amounts of this receptor. Accordingly, drug delivery conjugates can be used to treat a variety of tumor cell types, as well as other types of pathogenic cells, such as infectious agents, that preferentially express vitamin receptors and therefore have sites of surface accessible binding for vitamins, or vitamin analogs or derivatives.

In addition to the vitamins described herein, it will be appreciated that other ligands can be coupled to the drugs and linkers described and contemplated herein to form ligand-linker-drug conjugates capable of facilitating drug delivery to a desired target. These other ligands, in addition to the vitamins and their described analogs and derivatives, can be used to form drug delivery conjugates capable of binding to target cells. In general, any ligand of a cell surface receptor can be advantageously used as a targeting ligand for which a linker-drug conjugate can be prepared. Other exemplary ligands contemplated herein include peptide ligands identified in library screening, tumor growth specific peptides, tumor cell specific aptamers, tumor cell specific carbohydrates, tumor cell specific polyclonal or monoclonal antibodies, Fab fragments or scFv (i.e. single chain variable region) of antibodies such as, a Fab fragment of an antibody directed against EphA2 or other proteins specifically expressed or accessible only in metastatic cancer cells, small organic molecules derived from combinatorial libraries, growth factors such as EGF, FGF, insulin, and growth factors insulin analogs, and homologous polypeptides, somatostatin and its analogs, transferrin, lipoprotein complexes, bile salts, selectins, steroid hormones, Arg-Gly-Asp-containing peptides, retinoids, various galectins, opioid receptor 8 ligands, cholecystokinin A receptor ligands, specific ligands for angiotensin AT1 or AT2 receptors, proliferator-activated λ receptor ligands

ES 2 395 082 T3 peroxisomes (3-lactam type antibiotics such as penicillin, small organic molecules including antimicrobial drugs and other molecules that specifically bind to a receptor preferentially expressed on the surface of tumor cells or of an infectious organism, antimicrobials and other drugs designed to fit the binding site of a particular receptor based on the crystal structure of the receptor or other cell surface protein, Ligands of tumor antigens or other molecules preferentially expressed on the surface of tumor cells, or fragments of any of these molecules. An example of a specific tumor antigen that could act as a binding site for ligand-immunogen conjugates includes extracellular epitopes from a member of the ephrin family of proteins, such as EphA2. EphA2 expression is restricted to cell-cell junctions in normal cells, but EphA2 is distributed over the entire cell surface in metastatic tumor cells. Thus, EphA2 in metastatic cells would be accessible to bind to, for example, a Fab fragment of an antibody conjugated to an immunogen, whereas the protein would not be accessible to bind to the Fab fragment in normal cells, resulting in a specific ligandoimmunogenic conjugate for metastatic cancer cells.

The use of combinations of ligand-linker-drug conjugates to maximize targeting of pathogenic cells for elimination is further contemplated.

Exemplary drug delivery conjugates are as follows:

VLD

V- (l_r)CD

V- (ls) aD

V- (ls) a- (lr) cD

V- (lr) c- (ls) aD

V- (lH) b- (lr) dD

V- (lr) c- (lH) bD

V- (lH) d- (lr) c- (lH) eD

V- (ls) a- (lH) b- (lr) cD

V- (lr) c- (lH) b- (ls) -D

V- (lH) d- (ls) a- (lr) c- (lH) cD

V- (lH) d- (lr) c- (ls) a- (lH) eD

V- (lH) d- (ls) a- (lH) b- (lr) c- (lH) eD

V- (lH) d- (lr) c- (lH) b- (ls) a- (lH) eD

V- (ls) a- (lr) c- (lH) bD

V - [(ls) a- (lH) b] d- (lr) c- (lH) eD where a, b, c, d, and e are each independently 0, 1, 2, 3, or 4 , (l_s) is a spacer linker, (Ih) is a heteroatom linker, and (lr) is a releasable linker, V is a vitamin, or analog or derivative thereof, and D is a drug, or analog or derivative thereof, and in which the bivalent L encompasses one of a variety of (ls), (Ih), and (lr), in any order and in any combination. It is understood that the above examples of the bivalent linker L are intended to illustrate the wide variety of constructs of Ih, ls, and lr that the bivalent linker encompasses.

In an exemplary embodiment of the VLD drug delivery conjugate, wherein V is the vitamin folic acid, L is not ethylenediamine, according to the formula:

<img file="ES2395082T3_D0016.tif" />

I_________ V --------- II— L —ILdJ

In another exemplary embodiment of the VLD drug delivery conjugate, wherein V is the vitamin folic acid, and D is the drug mitomycin C; L is not L-Cys- (S-thioethyl), according to the formula:

ES 2 395 082 T3

<img file="ES2395082T3_D0017.tif" />

L is not L-Asp-L-Arg-L-Asp- L-Cys- (S-thioethyl), according to the formula:

<img file="ES2395082T3_D0018.tif" />

L is not L-Arg-L-Cys- (S-thioethyl-L-Ala-L-Gly-OH, according to the formula:

<img file="ES2395082T3_D0019.tif" />

Conjugates for drug delivery are also contemplated in which the vitamin, or analog or derivative thereof, is attached to a releasable linker that is attached to the drug via a spacer linker. Furthermore, both the drug and the vitamin, or analog or derivative thereof, may each be linked by spacer linkers, wherein the spacer linkers are attached to each other by a releasable linker. Furthermore, both the drug and the vitamin, or analog or derivative thereof, can each be attached to releasable linkers, wherein the releasable linkers are attached to each other by a spacer linker. The heteroatom linker can be located between any two linkers, or between any linker and the vitamin or analog or derivative, or between any linker and the drug or analog or derivative. All possible permutations and combinations are also contemplated.

An exemplary embodiment provides a conjugate for delivering drugs to the vitamin-binding receptor. The drug delivery conjugate consists of a vitamin receptor binding moiety, a bivalent linker (L), and a drug. The vitamin receptor binding moiety is a vitamin, or an analog or derivative thereof, capable of binding to vitamin receptors, and the drug includes analogs or derivatives thereof with pharmacological activity. The vitamin, or the analog or derivative thereof, is covalently attached to the bivalent linker (L), and the drug, or the analog or derivative thereof, is also covalently attached to the bivalent linker (L). The bivalent linker (L) comprises one or more spacer linkers, releasable linkers, and heteroatom linkers, and combinations thereof, in any order. For example, the heteroatom linker can be nitrogen and the releasable linker and the heteroatom linker can be taken together to form a divalent radical comprising alkylene-aziridin-1-yl, alkylenecarbonylaziridin-1-yl, carbonylalkylaziridin-1-yl, alkylene sulfoxylaziridin- 1-yl, sulfoxylalkylaziridin-1-yl, sulfonylalkylaziri-din-1-yl, or alkylenesulfonylaziridin-1-yl, wherein each of the releasable linkers is optionally substituted by a substituent X², as defined below.

Alternatively, the heteroatom linkers can be nitrogen, oxygen, sulfur, and the formulas (NHR¹NHR²) -, -SO-, - (SO2) -, and -N (R³) O-, in which R¹, R², and R³ each is independently selected from hydrogen, alkyl, aryl, arylalkyl, substituted aryl, substituted arylalkyl, heteroaryl, substituted heteroaryl, and alkoxyalkyl. In another embodiment, the heteroatom linker can be oxygen, the spacer linker can be 1-alkylenesuccinimid-3-yl, optionally substituted by an X substituent.¹, as defined below, and the releasable linkers may be methylene, 1-alkoxyalkylene, 1-alkoxycycloalkylene, 1-alkoxyalkylenecarbonyl, 1alkoxycycloalkylenecarbonyl, wherein each of the releasable linkers is optionally substituted by

ES 2 395 082 T3 a substituent X², as defined below, and wherein the spacer linker and the releasable linker are each attached to the heteroatom linker to form a succinimid-1-yl-alkyl acetal or ketal.

Spacer linkers can be carbonyl, thienecarbonyl, alkylene, cycloalkylene, alkylenecycloalkyl, alkylenecarbonyl, cycloalkylenecarbonyl, carbonylalkylcarbonyl, 1-alkylenesuccinimid-3-yl, 1 (carbonylalkyl) succinimid-3-yl, alkylenesullyl-2-alkylene-alkylaxylafoxyl, sulfonylaxylafoxyhydroxy -pyranyl, carbonyltetrahydrofuranyl, 1- (carbonyltetrahydro-2H-pyranyl) succinimid-3-yl, and 1 (carbonyltetrahydrofuranyl) succinimid-3-yl, wherein each of the spacer linkers is optionally substituted by a substituent X¹, as defined below. In this embodiment, the heteroatom linker may be nitrogen and the spacer linkers may be alkylenecarbonyl, cycloalkylenecarbonyl, carbonylalkylcarbonyl, 1- (carbonylalkyl) succinimid-3-yl, wherein each of the spacer linkers is optionally substituted by a substituent X¹, as defined below, and the spacer linker is attached to the nitrogen to form an amide. Alternatively, the heteroatom linker can be sulfur, and the spacer linkers can be alkylene and cycloalkylene, where each of the spacer linkers is optionally substituted by carboxyl, and the spacer linker is attached to sulfur to form a thiol. In another embodiment, the heteroatom linker can be sulfur, and the spacer linkers can be 1-alkylenesuccinimid-3-yl and 1- (carbonylalkyl) succinimid-3-yl, and the spacer linker is attached to sulfur to form a succinimid- 3-ylthiol.

As an alternative to the above-described embodiments, the heteroatom linker can be nitrogen and the releasable linker and heteroatom linker can be taken together to form a divalent radical comprising alkylene-aziridin-1-yl, carbonylalkylaziridin-1-yl, sulfoxylalkylaziridin-1 -yl, or sulfonylalkylaziridin-1-yl, wherein each of the releasable linkers is optionally substituted by an X substituent², as defined below. In this alternate embodiment, the spacer linkers may be carbonyl, thienocarbonyl, alkylenecarbonyl, cycloalkylenecarbonyl, carbonylalkylcarbonyl, 1- (carbonylalkyl) succinimid-3-yl, wherein each of the spacer linkers is optionally substituted by an X substituent¹, as defined below, and wherein the spacer linker is attached to the releasable linker to form an aziridinamide.

The X substituents¹ They can be alkyl, alkoxy, alkoxyalkyl, hydroxyl, hydroxyalkyl, amino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, halo, haloalkyl, sulfhydrylalkyl, alkylthioalkyl, aryl, substituted aryl, arylalkyl, carboxycaryl substituted arylalkyl, heteroaryl, heteroxycaryl substituted arylalkyl, heteroaryl, heteroboxyl , alkyl, guanidinalkyl alkanoate, R⁴-carbonyl, R⁵-carbonylalkyl, R⁶-acylamino, and R⁷-acylaminoalkyl, in which R⁴ and R⁵ each have been independently selected from amino acids, amino acid derivatives, and peptides, and in which R⁶ and R⁷ each have been independently selected from amino acids, amino acid derivatives, and peptides. In this embodiment the heteroatom linker can be nitrogen and the substituent X¹ and the heteroatom linker can be taken together with the spacer linker to which they are attached to form a heterocycle.

Releasable linkers can be methylene, 1-alkoxyalkylene, 1-alkoxycycloalkylene, 1-alkoxyalkylenecarbonyl, 1-alkoxycycloalkylenecarbonyl, carbonylarylcarbonyl, carbonyl (carboxyaryl) carbonyl, carbonyl (biscarboxyaryl) carbonyl, haloalkylene dialkylenecarbonyl, alkalylenyl (alkylaryl) dialkylenecarbonyl, alkylenyl (alkylaryl) (diarylsilyl), (dialkylsilyl) aryl, (alkylarylsilyl) aryl, (diarylsilyl) aryl, oxycarbonyloxy, oxycarbonyloxyalkyl, sulfonyloxy, oxysulfonylalkyl, iminoalkylidenyl, carbonylalkylideneiminyl, iminocycloalkylidenyl, carbonylcycloalkylideneiminyl, alkylenthio, alkylenearylthio, and carbonylalkylthio, wherein each of the releasable linkers is optionally substituted by an X substituent², as defined below.

In the above embodiment, the heteroatom linker may be oxygen, and the releasable linkers may be methylene, 1-alkoxyalkylene, 1-alkoxycycloalkylene, 1-alkoxyalkylenecarbonyl, and 1-alkoxycycloalkylenecarbonyl, wherein each of the releasable linkers is substituted in a manner optional by an X substituent², as defined below, and the releasable linker is attached to the oxygen to form an acetal or ketal. Alternatively, the heteroatom linker may be oxygen, and the releasable linker may be methylene, where the methylene is substituted by an optionally substituted aryl, and the releasable linker is attached to oxygen to form an acetal or ketal. Furthermore, the heteroatom linker can be oxygen, and the releasable linker can be sulfonylalkyl, and the releasable linker is attached to oxygen to form an alkylsulfonate.

In another embodiment of the foregoing embodiment of the releasable linker, the heteroatom linker may be nitrogen and the releasable linkers may be iminoalkylidenyl, carbonylalkylideneiminyl, iminocycloalkylidenyl, and carbonylcycloalkylideneiminyl, wherein each of the releasable linkers is optionally substituted by an X substituent.², as defined below, and the releasable linker is attached to the nitrogen to form a hydrazone. In an alternative configuration, the hydrazone can be acylated with a carboxylic acid derivative, an orthoformate derivative, or a carbamoyl derivative to form various releasable acylhydrazone linkers.

Alternatively, the heteroatom linker may be oxygen, and the releasable linkers may be alkylene (dialkylsilyl), alkylene (alkylarylsilyl), alkylene (diarylsilyl), (dialkylsilyl) aryl, (alkylarylsilyl) aryl, and (diarylsilyl) aryl, wherein each one of the releasable linkers is optionally substituted by an X substituent², as defined below, and the releasable linker is attached to oxygen to form a silanol.

ES 2 395 082 T3

In the embodiment of the above releasable linker, the drug may include a nitrogen atom, the heteroatom linker may be nitrogen, and the releasable linkers may be carbonylarylcarbonyl, carbonyl (carboxyaryl) carbonyl, carbonyl (biscarboxyaryl) carbonyl, and the releasable linker may be attached to the nitrogen of the heteroatom to form an amide, and also attached to the nitrogen of the drug to form an amide.

In the embodiment of the above releasable linker, the drug may include an oxygen atom, the heteroatom linker may be nitrogen, and the releasable linkers may be carbonylarylcarbonyl, carbonyl (carboxyaryl) carbonyl, carbonyl (biscarboxyaryl) carbonyl, and the releasable linker may be attached to the nitrogen linker of the heteroatom to form to form an amide, and also attached to the oxygen of the drug to form an ester.

The X substituents² They can be alkyl, alkoxy, alkoxyalkyl, hydroxyl, hydroxyalkyl, amino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, halo, haloalkyl, sulfhydrylalkyl, alkylthioalkyl, aryl, substituted aryl, arylalkyl, carboxycaryl substituted arylalkyl, heteroaryl, heteroxycaryl substituted arylalkyl, heteroaryl, heteroboxyl , alkyl, guanidinalkyl alkanoate, R⁴-carbonyl, R⁵-carbonylalkyl, R⁶-acylamino, and R⁷-acylamino alkyl, where R⁴ and R⁵ each have been independently selected from amino acids, amino acid derivatives and peptides, and in which R⁶ and R⁷ each has been independently selected from amino acids, amino acid derivatives and peptides. In this embodiment, the heteroatom linker can be nitrogen and the substituent X² and the heteroatom linker can be taken together with the releasable linker to which they are attached to form a heterocycle.

Heterocycles can be pyrrolidines, piperidines, oxazolidines, isoxazolidines, thiazolidines, isothiazolidines, pyrrolidinones, piperidinones, oxazolidinones, isoxazolidinones, thiazolidinones, isothiazolidinones, and succinimides.

The drug may be mitomycin, a mitomycin derivative, or a mitomycin analog, and in this embodiment, the releasable linkers may be carbonylalkylthio, carbonyltetrahydro-2H-pyranyl, carbonyltetrahydrofuranyl, 1 (carbonyltetrahydro-2H-pyranyl) succinimid-3-yl, and 1- (carbonyltetrahydrofuranyl) succinimid-3-yl, wherein each of the releasable linkers is optionally substituted by a substituent X², and wherein the aziridine of mitomycin is attached to the releasable linker to form an acylaziridine.

The drug may include a nitrogen atom and the releasable linker may be haloalkylenecarbonyl, optionally substituted by an X substituent.², and the releasable linker is attached to the nitrogen of the drug to form an amide.

The drug may include an oxygen atom, and the releasable linker may be haloalkylenecarbonyl, optionally substituted by an X substituent.², and the releasable linker is attached to the oxygen of the drug to form an ester.

The drug may include a nitrogen atom with a double bond and, in this embodiment, the releasable linkers may be alkylenecarbonylamino and 1- (alkylenecarbonylamino) succinimid-3-yl, and the releasable linker may be attached to the nitrogen of the drug to form a hydrazone.

The drug may include a sulfur atom and, in this embodiment, the releasable linkers may be alkylene and carbonylalkylthio, and the releasable linker may be attached to the sulfur of the drug to form a disulfide.

The vitamin can be folate, which includes a nitrogen, and in this embodiment the spacer linkers can be alkylenecarbonyl, cycloalkylenecarbonyl, carbonylalkylcarbonyl, 1-alkylenesuccinimid-3-yl, 1 (carbonylalkyl) succinimid-3-yl, wherein each of spacer linkers is optionally substituted by an X substituent¹, and the spacer linker is attached to the folate nitrogen to form an imide or an alkylamide.

In this embodiment, the substituents X¹ They can be alkyl, hydroxyalkyl, amino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, sulfhydrylalkyl, alkylthioalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, carboxyl, carboxyalkyl, guanidinalkyl, R⁴-carbonyl, R⁵-carbonylalkyl, R⁶-acylamino, and R⁷-acylaminoalkyl, in which R⁴ and R⁵ each have been independently selected from amino acids, amino acid derivatives and peptides, and in which R⁶ and R⁷ each has been independently selected from amino acids, amino acid derivatives and peptides.

The term "alkyl" as used herein refers to a monovalent straight chain of carbon atoms that may optionally be branched, such as methyl, ethyl, propyl, and 3-methylpentyl.

The term "cycloalkyl" as used herein refers to a monovalent chain of carbon atoms, a part of which forms a ring, such as cyclopropyl, cyclohexyl, and 3-ethylcyclopentyl.

The term "alkylene" as used herein refers to a linear bivalent chain of carbon atoms that may optionally be branched, such as methylene, ethylene, propylene, and 3-methylpentylene.

The term "cycloalkylene" as used herein refers to a bivalent chain of atoms.

ES 2 395 082 T3 of carbon, a part of which forms a ring, such as cycloprop-1,1-diyl, cycloprop-1,2-diyl, cyclohex-1,4-diyl, 3-ethylcyclopent-1,2-diyl , and 1-methylenecyclohex-4-yl.

The term "heterocycle" as used herein refers to a monovalent chain of carbon atoms and hetero atoms, wherein the hetero atoms have been selected from nitrogen, oxygen, and sulfur, a part of which, including at least one hetero atom , forms a ring such as aziridine, pyrrolidine, oxazolidine, 3-methoxypyrrolidine, and 3-methylpiperazine.

The term "alkoxy" as used herein refers to alkyl as defined herein combined with a final oxygen, such as methoxy, ethoxy, propoxy, and 3-methylpentoxy.

The term "halo" or "halogen" refers to fluoro, chloro, bromo, and iodo.

The term "aryl" as used herein refers to a mono or polycyclic aromatic ring of carbon atoms such as phenyl and naphthyl.

The term "heteroaryl" as used herein refers to a mono or polycyclic aromatic ring of carbon atoms and at least one heteroatom selected from nitrogen, oxygen, and sulfur, such as pyridinyl, pyrimidinyl, indolyl, and benzoxazolyl.

The term "substituted aryl" or substituted heteroaryl "as used herein refers to an aryl or heteroaryl substituted by one or more substituents selected as halo, hydroxy, amino, alkyl or dialkylamino, alkoxy, alkylsulfonyl, cyano, and nitro. .

The term "iminoalkylidenyl" as used herein refers to a divalent alkylene-containing radical as defined herein and a nitrogen atom, wherein the carbon at the end of the alkylene is linked by a double bond. to the nitrogen atom, as in the formulas - (CH) = N-, (CH2) 2 (CH) = N-₁ and -CH2C (Me) = N-.

The term amino acid as used herein refers generally to aminoalkylcarboxylates, where the alkyl radical is optionally substituted by alkyl, hydroxyalkyl, sulfhydrylalkyl, aminoalkyl, carboxyalkyl, and the like, including groups corresponding to natural amino acids such such as serine, cysteine, methionine, aspartic acid, and glutamic acid.

The term "arylalkyl" refers to aryl as defined herein substituted by an alkylene group, as defined herein, such as benzyl, phenethyl, and α-methylbenzyl.

It should be understood that the terms described above can be combined to generate chemically relevant groups, such as alkoxyalkyl "in reference to methyloxymethyl, and ethyloxymethyl, and haloalkoxyalkyl" in reference to trifluoromethyloxyethyl, and 1,2-difluoro-2-chloroeth-1-yloxypropyl .

The term "amino acid derivative" as used herein refers generally to aminoalkylcarboxylate, wherein the amino radical or carboxylate radical are each optionally substituted by alkyl, carboxylalkyl, or alkylamino, or optionally protected; and the intermediate divalent alkyl fragment is optionally substituted by alkyl, hydroxyalkyl, sulfhydrylalkyl, aminoalkyl, or carboxyalkyl, including groups corresponding to the side chains of natural amino acids, such as those found in serine, cysteine, methionine, aspartic acid and glutamic acid.

The term "peptide" as used herein refers generally to series of amino acids and amino acid analogs and derivatives covalently linked together by amide bonds.

The releasable linker includes at least one bond that can be cleaved or cleaved under physiological conditions (eg, a pH labile, acid labile, oxidation labile, or enzyme labile bond). The cleavable link (s) may be present within a cleavable linker and / or at one or both ends of a cleavable linker. It will be appreciated that the lability of the cleavable bond can be adjusted by including functional groups or fragments within the bivalent linker L that can aid or facilitate such bond breaking, also referred to as anchimeric aid. In addition, it will be appreciated that additional functional groups or fragments may be included within the bivalent linker L that may aid or facilitate breakage of the linker from the releasable linker.

Illustrative mechanisms of releasable linker bond cleavage include oxonium-assisted cleavage as follows:

ES 2 395 082 T3

<img file="ES2395082T3_D0020.tif" />

wherein Z is the vitamin, or analog or derivative thereof, or the drug, or analog or derivative thereof, or each is a vitamin moiety, or drug together with other parts of the bivalent linker, such as a vitamin moiety or drug that includes one or more spacer linkers, heteroatom linkers, and / or other releasable linkers. In this embodiment, the acid-catalyzed removal of the carbamate leads to the release of CO2 and the nitrogen-containing moiety attached to Z, and the formation of a benzyl cation, which can be entrapped by water or other Lewis base.

Another illustrative mechanism for cleaving connected or contained bonds between releasable linkers, which may be part of the bivalent linker L, includes the following beta-removal and beta-removal vinyloga mechanisms:

<img file="ES2395082T3_D0021.tif" />

A + COa + HjN-Z¹

<img file="ES2395082T3_D0022.tif" />

wherein X is a nucleophile, GSH, glutathione, or a bioreductive agent, and any of Z or Z 'is the vitamin, or analog or derivative thereof, or the drug, or analog or derivative thereof, a vitamin moiety , or drug together with other parts of the bivalent linker. It will be appreciated that the cleavage of the bond can also occur by acid catalyzed removal of the carbamate moiety which can be anchymerically assisted thanks to the stabilization provided by either the aryl group of the sulfur in beta, or by the disulfide shown in the previous examples. In such variations of this embodiment, the releasable linker is the carbamate moiety.

Another illustrative mechanism involves a rearrangement of the releasable, spacer, and heteroatom linkers so that subsequent to the cleavage of a bond in the bivalent linker, the release of the functional groups chemically aids in the breaking or cleavage of additional bonds, also called cleavage. or breakage with anchimeric aid. An exemplary embodiment of such a bivalent linker or part thereof includes compounds having the formula:

<img file="ES2395082T3_D0023.tif" />

wherein X is a heteroatom, such as nitrogen, oxygen, or sulfur, n is an integer selected from 0, 1, 2, and 3, R is hydrogen, or a substituent, a substituent capable of stability including a positive charge inductively or by resonance of the aryl ring, such as alkoxy, and any of Z or Z 'is the vitamin, or analog or derivative thereof, or the drug, or analog or derivative thereof, or a vitamin residue, or drug along with other parts of the bivalent linker. It will be appreciated that other substituents may be present on the aryl ring, benzyl carbon, carbamate nitrogen, alkanoic acid, or methylene bridge, including hydroxyl, alkyl, alkoxy, alkylthio, and holo. Aided cleavage can include mechanisms involving benzyl intermediates, benzene intermediates, lactone cyclization, oxonium intermediates, and beta-elimination. It will further be appreciated that, in addition to post-cleavage cleavage of the releasable linker, initial cleavage of the releasable linker may be facilitated by an anchimeric assist mechanism.

In this embodiment, hydroxyalkanoic acid, which can be cyclized, facilitates cleavage of the methylene bridge, by for example an oxonium ion, and facilitates bond cleavage or subsequent cleavage upon cleavage of the releasable linker. Alternatively, oxonium ion-assisted acid-catalyzed cleavage of the methylene bridge can result in a cascade of fragmentation of this exemplary bivalent linker, or fragment thereof. Alternatively, acid-catalyzed hydrolysis of the carbamate can facilitate beta-removal of the acid.

ES 2 395 082 T3 hydroxyalkanoic, which can cyclize, and facilitate cleavage of the methylene bridge, for example, by an oxonium ion. It will be appreciated that other chemical bond breaking or cleavage mechanisms under metabolic, physiological or cellular conditions described herein can initiate such a fragmentation cascade. It will be appreciated that other chemical bond breaking or cleavage mechanisms under metabolic, physiological or cellular conditions described herein can initiate such a fragmentation cascade.

The drug delivery conjugates described herein can be prepared by synthetic procedures known in the art. The synthetic procedures have been chosen depending on the selection of the heteroatom linkers, and the functional groups present in the spacer linkers and the releasable linkers. In general, reactions relevant to bond formation have been described in Richard C. Larock, Comprehensive Organic Transformations, a guide to functional group preparations, VCH Publishers, Inc. New York (1989), and in Theodora E. Greene &amp; Peter GM Wuts, Protective Groups in Organic Synthesis, 2nd edition, John Wiley &amp; Sons, Inc New York (1991).

General formation of esters and amides.

For example, if the heteroatom linker is a nitrogen atom and the end functional group present on the spacer linker or the releasable linker is a carbonyl group, the required amide group can be obtained by coupling reactions or acylation reactions of the corresponding carboxylic acid or derivative, wherein L is a suitably chosen leaving group such as halo, triflate, pentafluorophenoxy, trimethylsilyloxy, succinimide-N-oxyl, and an amine, as illustrated in Scheme 1.

Scheme 1

<img file="ES2395082T3_D0024.tif" />

'nh ♦ ho₂c - cr

RO ^ C-

<img file="ES2395082T3_D0025.tif" />

Coupling reagents include DCC, EDC, RRDQ, CGI, HBTU, TBTU, HOBT / DCC, HOBT / EDC, BOP-Cl, PyBOP, and PyBroP. Alternatively, the parent acid can be converted to an activated carbonyl derivative, such as an acid chloride, an N-hydroxysuccinimidyl ester, and a pentafluorophenyl ester. The amide-forming reaction can also be carried out in the presence of a base, such as triethylamine, diisopropylethylamine, and N, N-dimethyl-4-aminopyridine. Suitable solvents to form amides described herein include CH2Cl2, CHCl₃, THF. Suitable solvents to form amides described herein include CH2Cl2, CHCl₃, THF, DMF, DMSO, acetonitrile, and EtOAc. By way of example, amides can be prepared at temperatures in the range of between about -15 ° C to about 80 ° C, or between about 0 ° C to about 45 ° C. Amides can be formed from, for example, nitrogen-containing aziridine rings, carbohydrates, and α-halogenated carboxylic acids. Exemplary carboxylic acid derivatives useful for forming amides include compounds having the formula:

<img file="ES2395082T3_D0026.tif" />

where n is an integer such as 1, 2, 3, or 4.

Similarly, if the heteroatom linker is an oxygen atom and the end functional group present on the spacer linker or the releasable linker is a carbonyl group, the required ester group can be obtained by coupling reactions of the corresponding carboxylic acid or derivative, and an alcohol.

Coupling reagents include DCC, EDC, CDI, BOP, PyBOP, isopropenyl chloroformate, EEDQ, DBAD, and PPh.₃. Solvents include CH2Cl2, CHCl₃, THF, DMF, DmSO, acetonitrile, and EtOAc. The bases include triethylamine, diisopropylethylamine, and N, N-dimethyl-4-aminopyridine. Alternatively, the parent acid can be converted to an activated carbonyl derivative, such as an acid chloride, an N-hydroxysuccinimidyl ester, and a pentafluorophenyl ester.

General formation of ketals and acetals.

Furthermore, if the heteroatom linker is an oxygen atom, and the functional group present on the spacer linker or the releasable linker is 1-alkoxyalkyl, the necessary ketal or acetal group can be formed by ketal and acetal formation reactions of the corresponding alcohols and an enol ether, as illustrated in

ES 2 395 082 T3

<img file="ES2395082T3_D0027.tif" />

Scheme 2.

Scheme 2

<img file="ES2395082T3_D0028.tif" />

Solvents include alcohols, CH2CL, CHCl₃, THF, diethyl ether, DMF, DMSO, acetonitrile, and EtOAc. The formation of said ketals and acetals can be carried out with acid catalysis. If the heteroatom linker comprises two oxygen atoms, and the releasable linker is methylene, optionally substituted by a group X² As described herein, the necessary symmetric acetal or ketal group can be formed by way of example by ketal and acetal formation reactions from the corresponding alcohols and an aldehyde or ketone, as illustrated in Scheme 3 .

Scheme 3

<img file="ES2395082T3_D0029.tif" />

Alternatively, if the methylene has been substituted by an optionally substituted aryl group, the necessary acetal or ketal can be prepared in steps, where L is a suitably selected leaving group such as halo, trifluoroacetoxy, and triflate, as illustrated in Scheme 4. The procedure illustrated in Scheme 4 is a conventional preparation, and generally follows the procedure reviewed by RR Schmidt et al., Chem. Rev., 2000, 100, 4423-42.

Scheme 4

<img file="ES2395082T3_D0030.tif" />

<img file="ES2395082T3_D0031.tif" />

The resulting arylalkyl is treated with an oxidizing agent, such as DDQ, to generate an oxonium intermediate which is subsequently treated with another alcohol to generate the acetal or ketal.

General formation of succinimides

Furthermore, if the heteroatom linker is, for example, a nitrogen, oxygen, or sulfur atom, and the functional group present on the spacer linker or the releasable linker is a succinimide derivative, the resulting carbon-heteroatom bond can be formed. by a Michael addition of the corresponding amine, alcohol, or thiol, and a maleimide derivative, where X is the heteroatom linker, as illustrated in Scheme 5.

<img file="ES2395082T3_D0032.tif" />

Solvents to carry out the Michael addition include TBF, EtOAc, CH2Cl2, DMF, DMSO, and H2O. The formation of these Michael adducts can be carried out by adding equimolar amounts of bases, such as triethylamine, Hünig's base, or by adjusting the pH of the aqueous solutions to 6.0-7.4. It will be appreciated that if the heteroatom linker is an oxygen or nitrogen atom, the reaction conditions can be adjusted to facilitate the Michael addition, such as, for example, using higher reaction temperatures, adding catalysts, using solvents. more polar such as DMF, or DMSO, and activating the maleimide with silylating agents.

ES 2 395 082 T3

General silyloxy formation.

Also, if the heteroatom linker is an oxygen atom, and the functional group present on the spacer linker or the releasable linker is a silyl derivative, the necessary silyloxyl group can be formed by reacting the corresponding silyl derivative with an alcohol, wherein L is a suitably selected leaving group such as halo, trifluoroacetoxy, or triflate, as illustrated in Scheme 6.

Scheme 6 »| • I

-°^H * (T * · --- Silyl derivatives include suitably functionalized silyl derivatives such as vinyl sulfonoalkyl diaryl, or diaryl, or alkyl aryl silyl chloride. Instead of a vinylsulfonoalkyl group, a β-chloroethylsulfonoalkyl precursor can be used. Any aprotic solvent Anhydrous and any nitrogen-containing bases can serve as the reaction medium The temperature range used in this transformation can vary between -78 ° C and 80 ° C.

General formation of hydrazones.

Furthermore, if the heteroatom linker is a nitrogen atom, and the functional group present on the spacer linker or the releasable linker is an iminyl derivative, the necessary hydrazone group can be formed by reacting the corresponding aldehyde or ketone, and a hydrazine or acylhydrazine derivative, as illustrated in Scheme 7, equations (1) and (2).

Scheme 7

<td>X / F-NHa</td><td>&gt; v</td><td> _</td><td>N — N / \\ _.</td>

<td></td><td> 4</td><td></td><td>⁷ (i)</td>

<td> -</td><td>NH_to</td><td>. v</td><td></td>

<td></td><td></td><td>H</td><td>/ “V.</td>

<td>H% 'N — NHj * y—</td><td> —-</td><td>N — N / x \ ._ /</td><td> ' (2)</td>

Solvents that can be used include THF, EtOAc, CH2Cl2, CHCl3, CCI4, DMF, DMSO, and MeOH. The temperature range used in this transformation varies between 0 ° C and 80 ° C. Any acid catalyst such as mineral acid, H3CCOOH, F3CCOOH, p-TsOHN2O, and pyridinium p-toluenesulfonate can be used. In the case of the acylhydrazone of equation (2), the acylhydrazone can be prepared by initially acylating hydrazine with a suitable carboxylic acid, or derivative, as generally described above in Scheme 1, and subsequently reacting the acylhydrazide with the corresponding aldehyde or ketone to form the acylhydrazone. Alternatively, the hydrazone functionality can be formed initially by reacting hydrazine with the corresponding aldehyde or ketone. The resulting hydrazone can then be acylated with a suitable carboxylic acid, or derivative, as generally further described in Scheme 1.

General disulfide formation.

Furthermore, if the heteroatom linker is a sulfur atom, and the functional group present on the releasable linker is an alkylenthiol derivative, the necessary disulfide group can be formed by reacting the corresponding alkylsulfonylthioalkyl or arylsulfonylthioalkyl derivative, or the corresponding derivative of heteroaryldithioalkyl such as a pyridin-2-yldithioalkyl derivative with an alkylenthiol derivative, as illustrated in Scheme 8.

ES 2 395 082 T3

Scheme 8

<img file="ES2395082T3_D0033.tif" />

<img file="ES2395082T3_D0034.tif" />

Solvents that can be used are THF, EtOAc, CH2Cl2, CHCl₃, CCl4, DMF, and DMSO. The temperature range used in this transformation can vary between 0 ° C and 80 ° C. The necessary alkylsulfonylthioalkyl or arylsulfonylthioalkyl derivative can be prepared using protocols known in the art, and also according to the procedure of Ranasinghe and Fuchs, Synth. Commun. 18 (3), 227-32 (1988).

Other processes for preparing asymmetric dialkyl disulfides are based on a transthiolation of asymmetric heteroarylalkyl disulfides such as 2-thiopyridinyl, 3-nitro-2-thiopyridinyl, and similar disulfides, with alkyl thiol, as described in WO 88 / 01622, European patent application with no. 0116208A1, and US Patent No. 4,691,024.

General formation of carbonates.

Furthermore, if the heteroatom linker is an oxygen atom, and the functional group present on the spacer linker or the releasable linker is an alkoxycarbonyl derivative, the necessary carbonate group can be formed by reacting the corresponding hydroxy-substituted compound with a derivative. activated alkoxycarbonyl in which L is a suitable leaving group, as illustrated in Scheme 9.

Scheme 9

<img file="ES2395082T3_D0035.tif" />

Solvents that can be used are THF, EtOAc, CH2Cl2, CHCl₃, CCl4, DMF, and DMSO. The temperature range used in this transformation can vary between 0 ° C and 80 ° C. To facilitate the reaction, any basic catalyst can be used, such as an inorganic base, an amino base, and a polymer-binding base.

General formation of semicarbazones.

Furthermore, if the heteroatom linker is a nitrogen atom, and the functional group present on one spacer linker or the releasable linker is an iminyl derivative, and the functional group present on the other spacer linker or the other releasable linker is a derivative For alkylamino or arylaminocarbonyl, the necessary semicarbazone group can be formed by reacting the corresponding aldehyde or ketone, and a derivative of semicarbazide, as illustrated in Scheme 10.

Scheme 10

<img file="ES2395082T3_D0036.tif" />

Solvents that can be used are THF, EtOAc, CH2Cl2, CHCl₃, CCl4, DMF, DMSO, and MeOH. The temperature range used in this transformation can vary between 0 ° C and 80 ° C. Any acid catalyst can be used such as mineral acid, H₃CCOOH, F₃CCOOH, p-TsOH-H2O, and pyridinium p-toluenesulfonate. Furthermore, to form the semicarbazone, the hydrazone functionality can be formed initially by reacting hydrazine with the corresponding aldehyde or ketone. The resulting hydrazone can then be acylated with an isocyanate or a carbamoyl derivative such as a carbamoyl halide, to form the semicarbazone. Alternatively, the corresponding semicarbazide can be formed by reacting hydrazine with an isocyanate or carbamoyl derivative, such as a carbamoyl halide to form a semicarbazide. Subsequently, the semicarbazide can be reacted with the corresponding aldehyde or ketone to form the semicarbazone.

ES 2 395 082 T3

General formation of sulfonates.

Furthermore, if the heteroatom linker is an oxygen atom, and the functional group present on the spacer linker or the releasable linker is a sulfonyl derivative, the necessary sulfonate group can be formed by reacting the corresponding hydroxysubstituted compound with a sulfonyl derivative. activated where L is a suitable leaving group such as halo, as illustrated in Scheme 11.

Scheme 11

<img file="ES2395082T3_D0037.tif" />

Solvents that can be used are THF, EtOAc, CH2CL, CHCl3, and CCL.

The temperature range used in this transformation can vary between 0 ° C and 80 ° C. Any basic catalyst, such as an inorganic base, an amino base, and a polymer-binding base can be used to facilitate the reaction.

General formation of folate peptides.

The peptidyl fragment containing folate Pte-Glu- (AA) n-NH (CHR2) CO2H () is prepared by a polymer-supported sequential approach using standard procedures, such as the Fmoc strategy on Fmoc-AA-Wang resins () , acid sensitive, as shown in Scheme 12.

Scheme 12

<img file="ES2395082T3_D0038.tif" />

<img file="ES2395082T3_D0039.tif" />

or (a) piperidine / 20% DMF; (b) Fmoc-AA-OH, PyBop, DIPEA, DMF;

(c) Fmoc-Glu (O- t-Bu) -OH, PyBop, DIPEA, DMF; (d) 1. N^,0(TFA) -Pte-OH; PyBop, DIPEA, DMSO; (e) TFAA, (CH2SH) 2, / -PraSiH; (f) NH4OH, pH 10.3.

In this exemplary embodiment of the procedures described herein, R1 is Fmoc, R2 is the suitably protected side chain of the desired amino acid, and DIPEA is diisopropylethylamine. Standard coupling procedures, such as PyBOP and others described herein or known in the art, are used where the coupling agent is applied by way of example as an activating reagent to ensure sufficient coupling. The Fmoc protecting the groups is removed after each coupling step under standard conditions, such as by treatment with piperidine, and tetrabutylammonium fluoride (TBAF). Properly protected amino acid building blocks are used, such as Fmoc-Glu- OtBu, and N¹⁰TFA-Pte-OH, as described in Scheme 12, and represented in step (b) by Fmoc-AAOH. Thus, AA refers to any amino acid starting material that is adequately protected. It will be understood that the term amino acid as used herein is intended to refer to any reagent that has an amine and carboxylic acid functional group separated by one or more carbons, and includes the natural alpha and beta amino acids, as well as derivatives and analogs of said amino acids, In particular, amino acids that have side chains that are protected, such as serine, threonine, cysteine ​​and aspartate, they can also be used in the synthesis of the folate peptides described herein. In addition, homologous gamma, delta, or longer amino acids can also be included as starting materials in the synthesis of folate peptides described herein. In addition, amino acid analogs that have homologous side chains, or alternative branched structures, such as norleucine, isovaline, β-methyl threonine, β-methyl cysteine, and β, β-dimethyl cysteine ​​can also be included as

ES 2 395 082 T3 starting materials in the synthesis of folate peptides described herein.

The coupling sequence (steps (a) and (b)) involving Fmoc-AA-OH is performed n times to prepare peptide 2 on solid support, where n is an integer and can be equal to 0 to about 100 After the last coupling step, the remaining Fmoc group is removed (step (a)), and the peptide is coupled sequentially with a glutamate derivative (step (c)), deprotected, and coupled to TFA-protected pteroic acid (stage (d). Subsequently, the peptide is cleaved from the polymeric support after treatment with trifluoroacetic acid, ethanediol and triisopropylsilane (step (e)). These reaction conditions result in the simultaneous removal of the protecting groups t-Bu, t-Boc, and Trt that can form part of the suitably protected amino acid side chain. The TFA protecting group is removed by base treatment (step (f)) to give the folate-containing peptidyl fragment 3.

Spacer and releasable linkers and heteroatom linkers can be combined in different ways. By way of example, the linkers are linked to each other differently than a heteroatom linker, such as alkylene-amino-alkylenecarbonyl, and alkylene-thio-carbonylalkylsuccinimid-3-yl, as further illustrated by the following formulas, in that the integers x and y are 1, 2, 3, 4, or 5:

<img file="ES2395082T3_D0040.tif" />

<img file="ES2395082T3_D0041.tif" />

Another exemplary embodiment of the linkers described herein includes releasable linkers that are cleaved under the conditions described herein by a chemical mechanism involving beta removal. In one aspect, such releasable linkers include beta-thio, beta-hydroxyl, and beta-amino substituted carboxylic acids and derivatives thereof, such as esters, amides, carbonates, carbamates, and ureas. In another aspect, such releasable linkers include 2-thio-aryl and 4-thio-aryl esters, carbamates, and carbonates.

In addition, the binding of the vitamin or drug to the heteroatom linker drug can be accomplished by a reactive functional group present on the drug or vitamin that has been converted to a heteroatom linker, such as by converting the aclamycinketone to the corresponding hydrazone, and the conversion of folic acid to the corresponding amide, as shown in the following formula:

<img file="ES2395082T3_D0042.tif" />

The bivalent linker (L) comprises one or more components selected from spacer linkers, releasable linkers, heteroatom linkers, and combinations thereof in any order. For example, spacer linkers, releasable linkers, and heteroatom linkers, and combinations thereof, shown in Tables 1 and 2 are contemplated.

Asterisks present in the following structures, as well as those shown in Tables 1 and 2, identify exemplary binding sites for additional spacer, releasable, or heteroatom linkers, or for the drug or vitamin component of the conjugate to deliver drugs to the vitamin-binding receptor. It will be understood that the bivalent linker L comprises one or more spacer linkers, releasable linkers, and heteroatom linkers, including those shown in Tables 1 and 2, and such spacer linkers, releasable linkers, and heteroatom linkers may be combined in any order. to form the bivalent linker L.

ES 2 395 082 T3

Table 1. Linkers contemplated by way of example, and combinations of some spacer and heteroatom linkers.

<td>ho₂c _or.The</td><td>co₂h</td><td>h₂n ^ nh HN s 0</td><td>COj-H</td>

<td> *1* 0</td><td>co₂h \ 0</td><td>* Ό ] R = H, alkyl, acyl</td><td>^, SH * Ύ or</td>

<td>Η0₂σ^Λ-Ν '^Λγ °</td><td>0 OR R = H, alkyl, acyl</td><td>• rV'A ''⁰⁰’</td><td>•TO-</td>

<td>r * X * 1st</td><td>co₂h</td><td>co₂h</td><td>TO-</td>

<td>HO_ZC --- HO₂TO OR</td><td>ho₂AA^h *^hMl * o</td><td>F ^HΑ ^ / γΝ ^ οο₂Η i #</td><td>co₂h Y 0</td>

<td>OR A / U * •ACE R = H, alkyl, acyl i</td><td>i ^ oj. R = H, alkyl, acyl</td><td>? H S » ⁰</td><td>X or</td>

<td>Χζ</td><td>H ° ^ o .TO-'·</td><td>co₂h * Ιχ&gt; γΝ * 0</td><td>co₂h TO * N || 0</td>

ES 2 395 082 T3 (continued)

<td>HOjC,</td><td>HO_ZC.</td>

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

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

<td>HlL</td><td>HsN ^ NH A.</td>

<td>* M_N *</td><td>* NY</td>

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

<td>zNH₂</td><td>.nh₂</td>

<td>V</td><td> *^N ll *</td>

<td>or</td><td>TO</td>

<td>, -SH</td><td>_-SH</td>

<td></td><td>YOU*</td>

<td> 0</td><td>* TO</td>

<td>. P γ</td><td>V '</td>

<td>or</td><td> '0</td>

<td> 11 = 0-3</td><td>n = 0-3</td>

<td>W GO OR * V R = H, alkyl, acyl</td><td>co₂h s i · '' s. 4· T</td>

<td>COaH * m N *</td><td>V * 0</td>

<td>CO2H</td><td>Y * N 'Y</td>

<td>Υ ° γΑ 'y ^ OR OR R = H, alkyl, bye</td><td>C0₂H</td>

<td>„PP ME n = 1-3</td><td> '^Νχ ^ o n = 1-3</td>

ES 2 395 082 T3

Table 2. Linkers contemplated by way of example, and combinations of some releasable and heteroatom linkers.

<td>Λ</td><td></td><td> *^Νγ ^ ν *^&lt;'CO₂H</td><td></td>

<td>OCC ° HOíC - ^^^ COíH</td><td>* rA ^^{: i}^ CO₂H</td><td></td><td></td>

<td></td><td></td><td> *^NVY * ^ * HO_toC °</td><td> 5</td>

<td>O ^ -N * * θ u</td><td>TO</td><td>* N '^^XOR*</td><td></td>

<img file="ES2395082T3_D0043.tif" />

Conjugates for drug delivery can also be prepared from intermediates. In an exemplary embodiment, a compound of the formula can be prepared:

VLZ¹ in which Z¹ it is an electrophile, nucleophile or a precursor, suitable to facilitate the binding of the drug, or analog or derivative thereof.

In one respect, Z¹ it can be a leaving group that allows the drug to be attached by a nucleophilic moiety present in the drug, or an analog or derivative thereof, such as a heteroatom, eg nitrogen.

In another aspect, Z¹ it can be a nucleophile such as a heteroatom, for example nitrogen capable of displacing a leaving group present in the drug, or an analog or derivative thereof, such as a carboxylic acid derivative,

ES 2 395 082 T3 for example, an acid chloride.

In another aspect, Z¹ It can be a precursor, such as a nitro group capable of being transformed into a nucleophilic nitrogen by a reduction reaction, or an ester capable of being transformed into an electrophilic acid chloride by hydrolysis and sequential chlorination. It will be appreciated that Z¹ it can be a heteroatom linker.

In another exemplary embodiment, drug delivery conjugates can be prepared from intermediates such as the following:

Z²-LD where Z¹ it is an electrophile, nucleophile or a precursor, suitable to facilitate the binding of the vitamin, or analog or derivative thereof.

In one respect, Z¹ it can be a leaving group that allows the vitamin to be attached via a nucleophilic moiety present in the vitamin, or an analog or derivative thereof, such as a heteroatom, eg nitrogen.

In another aspect, Z¹ it can be a nucleophile such as a heteroatom, for example nitrogen capable of displacing a leaving group present on the vitamin, or analog or derivative thereof, such as a carboxylic acid derivative, for example an acid chloride.

In another aspect, Z¹ It can be a precursor, such as a nitro group capable of being transformed into a nucleophilic nitrogen by a reduction reaction, or an ester capable of being transformed into an electrophilic acid chloride by hydrolysis and sequential chlorination. It will be appreciated that Z¹ it can be a heteroatom linker.

In another exemplary embodiment, the bivalent linker (L) can be independently synthesized, and subsequently linked to the vitamin and drug in later steps, such as intermediate preparing a compound of the formula:

Z¹-LZ² in which Z¹ and Z² each have been independently selected and are as defined above.

Conjugates for drug delivery can be used in both human clinical medicine and veterinary applications. Thus, the host animal that harbors the pathogenic cell population and that is treated with the conjugates to deliver drug to the vitamin-binding receptor may be a human or, in the case of veterinary applications, it may be a laboratory, agricultural animal. , domestic or wild. Conjugates can be applied to host animals including humans, laboratory animals such as rodents (eg, mice, rats, hamsters, etc.), rabbits, monkeys, chimpanzees, domestic animals such as dogs, cats, and rabbits, animals. agricultural such as cows, horses, pigs, sheep, goats, and captive wild animals such as bears, pandas, lions, tigers, leopards, elephants, zebras, giraffes, gorillas, dolphins, and whales.

The foregoing includes the application to populations of pathogenic cells that can cause different pathologies in said host animals.

Pathogenic cells means cancer cells, infectious agents such as bacteria and viruses, cells infected with bacteria and viruses, active macrophages capable of causing a disease state, and any other type of pathogenic cells that uniquely express, preferably express or overexpress receptors of vitamin or receptors that bind to vitamin analogs or derivatives. Pathogenic cells can also include any cell that causes a disease state for which treatment with the conjugates to deliver drug to the vitamin-binding receptor results in a reduction in symptoms of the condition. For example, pathogenic cells can be host cells that are pathogenic in certain circumstances such as cells of the immune system that are responsible for graft-versus-host disease, but are not pathogenic in other circumstances.

Thus, the pathogenic cell population can be a cancer cell population that is tumorigenic, including benign tumors and malignant tumors, or that is non-tumorigenic. The cancer cell population can arise spontaneously or due to processes such as mutations present in the germ line of the host animal or somatic mutations, or it can be induced, chemically, virally or by radiation. Conjugates can be used to treat cancers such as carcinomas, sarcomas, lymphomas, Hodgkin's disease, melanomas, mesotheliomas, Burkitt's lymphoma, nasopharyngeal carcinomas, leukemias, and myelomas. Cancer cell population may include oral, thyroid, endocrine, skin, gastric, esophageal, laryngeal, pancreatic, colon, bladder, bone, ovarian, cervical, uterine, breast, testicular, prostate, rectal, kidney, liver and lung.

In embodiments where the pathogenic cell population is a cancer cell population, the effect of conjugate administration is a therapeutic response as measured by reduction or elimination of tumor mass, or inhibition of tumor cell proliferation. In the case of a tumor, removal may be a

ES 2 395 082 T3 elimination of cells from the primary tumor or cells that have metastasized or are in the process of separating from the primary tumor. Prophylactic use of the conjugate is also contemplated to deliver drugs to the vitamin-binding receptor to prevent return of the tumor after removal by any therapeutic approach including surgical removal of the tumor, radiation therapy, chemotherapy, or treatment with biological agents. The prophylactic use can be the initial use of the conjugate to administer drug, such as a use in a daily multidose therapeutic regimen, and / or it can be an additional use or series of uses after an interval of days or months after the initial treatment or treatments. . Accordingly, the elimination of any of the pathogenic cell populations in response to use according to the present invention includes reduction in the number of pathogenic cells, inhibition of proliferation of pathogenic cells, a prophylactic treatment that prevents the return of pathogenic cells, or a response to use on pathogenic cells that results in a reduction in symptoms of the disease.

In cases where cancer cells are to be killed, the conjugates can be used in conjunction with surgical removal of the tumor, radiation therapy, chemotherapy, or treatment with biological agents such as other immunotherapies including monoclonal antibody treatment, treatment with immunomodulatory agents, adoptive transfer. of immune effector cells, treatment with hematopoietic growth factors, cytokines and vaccination.

Conjugates can also be applied to populations of pathogenic cells that cause a variety of infectious conditions. For example, the conjugates can be applied to such pathogenic cell populations such as bacteria, fungi, including yeast, viruses, virus-infected cells, mycoplasma, and parasites. Infectious organisms that can be treated with the drug delivery conjugates are any infectious organisms known in the art to cause pathogenesis in an animal, including organisms such as bacteria that are gram-negative or gram-positive cocci or rods. For example, Proteus species, Klebsiella species, Providencia species, Yersinia species, Erwinia species, Enterobacter species, Salmonella species, Serratia species, Aerobacter species, Escherichia species, Pseudomonas species, Shigella species , Vibrio species, Aeromonas species, Campylobacter species, Streptococcus species, Staphylococcus species, Lactobacillus species, Micrococcus species, Moraxella species, Bacillus species, Clostridium species, Corynebacterium species, Eberthella species, Micrococcus species, Mycobacterium species, Neisseria species, Haemophilus species, Bacteroides species, Listeria species, Erysipelothrix species, Acinetobacter species, Brucella, Pasteurella species, Vibrio species, Flavobacterium species, Fusobacterium species, Streptobacillus species, Calymmatobacterium species, Legionella species, Treponema species, Borrelia species, Leptospira species, Actinomyces species, Nocardia species, Rickettsia species, and any other bacterial species that produces a disease in a host that can be treated with the conjugates for administration of drug.

Of particular interest are bacteria that are resistant to antibiotics such as Streptococcus species and Staphylococcus species resistant to antibiotics, or bacteria that are susceptible to antibiotics but cause recurrent infections treated with antibiotics so that organisms can eventually develop resistance. . Bacteria that are susceptible to antibiotics but cause recurrent infections treated with antibiotics so that organisms may eventually develop resistance can be treated with drug delivery conjugates in the absence of antibiotics, or in combination with lower doses of antibiotics than are would normally be administered to a patient, to prevent the development of these antibiotic resistant strains of bacteria.

Viruses, such as DNA and RNA viruses, can also be treated. These viruses include DNA viruses such as papillomavirus, parvovirus, adenovirus, herpesvirus, and vaccinia virus, and RNA virus such as sand virus, coronavirus, rhinovirus, respiratory syncytial virus, influenza virus, picornavirus, paramyxovirus, reoviruses, retrovirus, lentivirus, and rhabdovirus.

The conjugates can also be applied to any fungus, including yeast, mycoplasma species, parasites, or other infectious organisms that can cause a disease in animals. Examples of fungi that can be treated with the methodology and compositions include fungi that grow like molds or are yeast-like, including, for example, fungi that cause ailments such as dermatophytosis, histoplasmosis, blastomycosis, aspergillosis, cryptococcosis, sporotrichosis, cocidioidomycosis, paracocidioidomycosis , mucormycosis, chromoblastomycosis, dermatophytosis, protothecosis, fusariosis, pityriasis, mycetoma, paracocidioidomycosis, pheohifomycosis, pseudalescheriasis, sporotrichosis, trichosporosis, pneumocyst infection, and candidiasis.

Conjugates can also be used to treat parasitic infections including infections caused by cestodes, such as Taenia, Hymenolepsis, Diphillobothrium, and Echinococcus species, trematodes, such as Fasciolopsis, Heterophyes, Metagonimus, Clonorchis, Fasciola, Paragonimus, and Schitosoma species, nematodes, such as Enterobius, Trichuris, Ascaris, Hookworm, Necator, Strongiloides, Trichinella, Wuchereria, Brugia, Loa Onchocerca, and Dracunculus species, amoebas such as Naegleria and Acanthamoeba species, and protozoa, such as Plasmodium, Trypanosoma, Leishmania, Toxoplasma, Entamoeba, Giardia, Isospora, Cryptosporidium, and Enterocytozoon species.

ES 2 395 082 T3

The pathogenic cells targeted by the drug delivery conjugates can also be cells harboring endogenous pathogens, such as cells infected with viruses, mycoplasma, parasites, or bacteria, if these cells preferentially express vitamin receptors.

In one embodiment, conjugates for drug delivery to the vitamin-binding receptor can be internalized into target pathogenic cells upon binding of the vitamin moiety to a vitamin receptor, vitamin transporter, or other surface-presented proteins that specifically bind. to the vitamin and that they are preferentially expressed in pathogenic cells. Such internalization can occur, for example, by receptor-mediated endocytosis. If the drug delivery conjugate contains a releasable linker, the vitamin moiety and the drug can dissociate intracellularly and the drug can act on its intracellular target.

In an alternative embodiment, the vitamin moiety of the drug delivery conjugate can bind to the pathogen cell by bringing the drug into close association with the surface of the pathogen cell. The drug can then be released by cleavage of the releasable linker. For example, the drug can be released via a disulfide isomerase protein if the releasable linker is a disulfide group. The drug can then be taken up by the pathogenic cell to which the conjugate is bound to deliver drugs to the vitamin-binding receptor, or the drug can be taken up by another pathogen cell in close proximity thereto. Alternatively, the drug can be released by a disulfide isomerase protein within the cell if the releasable linker is a disulfide group. The drug can also be released by a hydrolytic mechanism, such as acid-catalyzed hydrolysis, as described above for some beta-elimination mechanisms, or by anchimerically aided cleavage by an oxonium ion or lactonium ion producing mechanism. The selection of the releasable linker (s) will determine the mechanism by which the drug will be released from the conjugate. It will be appreciated that such selection can be predefined by the conditions under which the drug conjugate is to be used.

In another embodiment, if the linker does not comprise a releasable linker, the vitamin moiety of the drug delivery conjugate can bind to the pathogenic cell by placing the drug on the surface of the pathogenic cell to target the pathogenic cell for attack by others. molecules capable of binding the drug.

Alternatively, in this embodiment, the drug delivery conjugates can be internalized into the target cells upon binding and the vitamin and drug moiety can remain intracellularly associated with the drug displaying its effects without dissociating from the vitamin moiety.

In yet another exemplary embodiment, or in combination with the above-described embodiments, the conjugate for delivering drugs to the vitamin-binding receptor may act via a mechanism independent of cellular vitamin receptors. For example, conjugates for drug delivery can bind to soluble vitamin receptors present in serum or target serum proteins such as albumin, resulting in prolonged circulation of the conjugates relative to the unconjugated drug, and an increase in the activity of the conjugates with respect to the pathogenic cell population relative to the unconjugated drug.

In another illustrative embodiment, a vitamin-binding receptor drug delivery conjugate is provided with the general formula VLD. L is selected from (ls) a and (lH) b, and combinations thereof, wherein (ls) a, (lH) b, and V are as defined herein, and D is a drug. such as an immunogen. The immunogen can be a hapten, for example fluorescein or dinitrophenyl. In this embodiment, the vitamin-binding receptor drug delivery conjugate binds to the surface of pathogenic cells and "tags" the cells with the immunogen, thereby eliciting an immune response directed toward the targeted population of pathogenic cells. Antibodies administered to the host in an immunization or passive antibodies existing in the host system from an already existing, innate or acquired immunity, bind to the immunogen and elicit endogenous immune responses. Antibodies that bind to the cell-bound vitamin-immunogen conjugate result in complement-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity, opsonization and phagocytosis of antibodies, death or quiescence of signal cells by antibody-induced receptor clustering, or any other humoral or cellular immune response stimulated by an antibody that binds to the ligand-immunogen conjugates bound to cells. In cases where an immunogen can be recognized directly by immune cells without prior antibody opsonization, direct death of the pathogenic cells can be achieved. This embodiment has been described in more detail in US Patent Application Serial No. 09 / 822,379. It will be appreciated that in certain variations of this embodiment where the drug is an immunogen, the bivalent linker may also include releasable linkers, as described above, such as a conjugate for delivering drugs to the vitamin-binding receptor of the general formula VLD where L is selected from (ls) a, (lH) b, (lr) c, and combinations thereof where (ls) is a spacer linker, (Ih) is a heteroatom linker, (Ir) is a releasable linker, V is a vitamin, or an analog or derivative thereof, and a, b, and c are integers.

Conjugates for drug delivery to the vitamin binding receptor described herein comprise a vitamin receptor binding moiety, a bivalent linker (L), a drug, and optionally,

ES 2 395 082 T3 heteroatom linkers for attaching the binding moiety to the vitamin receptor and the drug to the bivalent linker (L). The bivalent linker (L) may comprise a spacer linker, a releasable (ie, cleavable) linker, and a heteroatom linker, or combinations thereof.

The vitamin binding receptor drug delivery conjugates described herein can be formed with a wide variety of vitamins or vitamin analogs / derivatives that bind to a vitamin receptor, linkers, and drugs. Conjugates for drug delivery are capable of selectively targeting a population of pathogenic cells in a host animal due to the preferential expression of a vitamin receptor accessible for binding to a vitamin on pathogenic cells. Exemplary vitamin moieties include carnitine, inositol, lipoic acid, pyridoxal, ascorbic acid, niacin, pantothenic acid, folate, riboflavin, thiamine, biotin, vitamin B12, and the fat-soluble vitamins A, D, E, and K. These vitamins, and their receptor-binding analogs and derivatives, constitute the targeting entity that can be coupled to the drug via the bivalent linker (L) to form the conjugates for drug delivery to the vitamin-binding receptor described herein. document. Thus, the term "vitamin" includes vitamin analogs and / or derivatives (eg, pteroic acid which is a derivative of folate, biotin analogs such as biocytin, biotin sulfoxide, oxybiotin, and other compounds that bind to the biotin receptor). It will be appreciated that vitamin analogs or derivatives can mean a vitamin incorporating a heteroatom whereby the vitamin analog or derivative is covalently linked to the bivalent linker (L).

Exemplary vitamin moieties include folic acid, biotin, riboflavin, thiamine, vitamin B12, and analogs and derivatives that bind receptor- of these vitamin molecules, and other vitamin-related receptor-binding molecules. Exemplary embodiments of vitamin analogs and / or derivatives include folate analogs and derivatives such as folinic acid, pteropoliglutamic acid, and pteridines that bind to the folate receptor, such as tetrahydropterins, dihydrofolates, tetrahydrofolates, and their deaza and dideaza analogs. . The terms "deaza" and "dideaza" analogs refer to analogs known in the art that have one carbon atom substituted by one or two carbon atoms in the structure of natural folic acid, or analog or derivative thereof. For example, deaza analogs include the folate analogs 1-deaza, 3-deaza, 5-deaza, 8-deaza, and 10-deaza. Dideaza analogs include, for example, the folate 1,5-dideaza, 5,10-dideaza, 8,10-dideaza, and 5,8-dideaza analogs. Other folates useful as complexing ligands are the folate receptor-binding analogs aminopterin, amethopterin (methotrexate), N¹⁰-methylfolate, 2-deaminohydroxyfolate, analogs such as 1deazamethopterine or 3-deazamethopterine, and 3 ', 5'-dichloro-4-amino-4-deoxy-N-methylpteroylglutamic acid (dichloromethhotrexate). The above analogs and derivatives of folic acid are conventionally called "folates", reflecting their ability to bind to folate receptors, and such ligands, when conjugated to exogenous molecules, are effective in enhancing transmembrane transport, such as through mediated endocytosis. by folate, as described herein. Other suitable ligands capable of binding to folate receptors to initiate endocytotic transport mediated by the folate receptor include anti-idiotypic folate receptor antibodies. An exogenous molecule is used in a complex with an anti-idiotypic folate receptor antibody to trigger transmembrane transport of the complex.

Exemplary embodiments of vitamin analogs and derivatives also include biotin analogs and derivatives such as biocytin, biotin sulfoxide, oxybiotin, and other compounds that bind to the biotin receptor. It will be appreciated that other analogs and derivatives of other vitamins described herein are also contemplated herein. In one embodiment, vitamins that can be used in the drug delivery conjugates described herein include those that bind to specifically expressed vitamin receptors on activated macrophages, such as the folate receptor, which binds folate, or an analog or derivative thereof, as described herein.

The vitamin binding site can include receptors for any vitamin molecule or a derivative or analog thereof, capable of specifically binding to a receptor in which the receptor or other protein is uniquely expressed, overexpressed, or preferentially expressed by a population of pathogenic cells. A surface-expressed protein uniquely expressed, overexpressed, or preferentially expressed by pathogenic cells is typically a receptor that is either absent, or present in lower amounts, on non-pathogenic cells providing a means of selectively eliminating pathogens. pathogenic cells. Conjugates for drug delivery to the vitamin-binding receptor may be capable of binding with high affinity to receptors on cancer cells or other pathogenic cell types. This high binding affinity may be inherent in the vitamin moiety, or the binding affinity may be enhanced by the use of a chemically modified vitamin, (ie, an analog or derivative).

The drug can be any molecule capable of modulating or otherwise modifying cell function, including pharmaceutically active compounds. Suitable molecules can include: peptides, oligopeptides, retroinverted oligopeptides, proteins, protein analogs in which at least one non-peptide bond replaces a peptide bond, apoproteins, glycoproteins, enzymes, coenzymes, enzyme inhibitors, amino acids and their derivatives, receptors and other membrane proteins; antigens and antibodies against the above; haptens and antibodies against the above; hormones, lipids, phospholipids, liposomes; toxins; antibiotics; analgesics; bronchodilators; beta-blockers; antimicrobial agents, antihypertensive agents, cardiovascular agents including antiarrhythmics, cardiac glycosides, antianginals, and vasodilators; central nervous system agents including stimulants, psychotropics, antimaniacs, and depressants; antiviral agents; antihistamines; drugs

ES 2 395 082 T3 against cancer including chemotherapeutic agents; tranquilizers, antidepressants, H-2 antagonists; anticonvulsants; antiemetics; prostaglandins and prostaglandin analogs; muscle relaxants; anti-inflammatory substances; stimulants, decongestants; antiemetics; diuretics; antispasmodics; antiasthmatics; anti-Parkinson's agents; expectorants; cough suppressants; mucolytics; and nutritive mineral additives.

Furthermore, the drug can be any drug known in the art that is cytotoxic, enhances tumor permeability, inhibits tumor cell proliferation, stimulates apoptosis, decreases antiapoptotic activity in target cells, is used to treat ailments caused by infectious agents. , potentiates an endogenous immune response directed at pathogenic cells, or that is useful to treat a pathological state produced by any type of pathogenic cell. Suitable drugs for use include adrenocorticoids and corticosteroids, alkylating agents, antiandrogens, antiestrogens, androgens, aclamycin and aclamycin derivatives, estrogens, antimetabolites such as cytosine arabinoside, purine analogs, pyrimidine analogs, and methotrexate, busulfan, carboplatin, chloral cisplatin and other platinum compounds, tamoxifen, taxol, paclitaxel, derivatives of paclitaxel, Taxotere®, cyclophosphamide, daunomycin, rhizoxin, T2 toxin, plant alkaloids, prednisone, hydroxyurea, teniposide, mitomycins, discodermolides, microtubule inhibitors, epothilones, tubulysin, cyclopropyl benz [e] indolone, seco-cyclo-propylbenc [e] indolone, O-Ac-seco-cyclopropyl benz [e] indolone , bleomycin and other antibiotics, nitrogen mustards, nitrosureas, vincristine, vinblastine, and analogues and derivatives thereof such as deacetylvinblastine monohydrazide, colchicine, colchicine derivatives, allocolchicine, thiocolchicine, trityl cysteine, halichondrine B, dolastatins such as dolastatin 10, amanitins such as αamanitin, camptothecin, irinotecan, and other camptothecin derivatives of the foregoing, geldanamycin and geldanamycin derivatives, estramustine, nocodazole, MAP4, colcemide, inflammatory agents, and proinflammatory agents and signal transduction inhibitor peptidomimetics and any other drug or toxin known in the art. Other drugs that may be used include penicillins, cephalosporins, vancomycin, erythromycin, clindamycin, rifampin, chloramphenicol, aminoglycoside antibiotics, gentamicin, amphotericin B, acyclovir, trifluridine, ganciclovir, zidovudine, amantadine, any other known antibavirin compound, and any other known antibavirin compound, technique.

In one embodiment, drugs for use remain stable in serum for at least 4 hours. In another embodiment, the drugs have an IC50 in the nanomolar range, and, in another embodiment, the drugs are soluble in water. If the drug is not soluble in water, the bivalent linker (L) can be derivatized to enhance water solubility. The term "drug" also means any drug analog or derivative described herein above, including dolastatins such as dolastatin 10, the amanitins such as αamanitin, camptothecins, and irinotecan, and other camptothecin and irinotecan derivatives of the foregoing. It will be appreciated that the drug analog or derivative can mean a drug incorporating a heteroatom through which the drug analog or derivative is covalently linked to the bivalent linker (L).

Conjugates for drug delivery to the vitamin-binding receptor may comprise a vitamin receptor-binding moiety, a bivalent linker (L), a drug, and optionally, heteroatom linkers to bind the vitamin receptor-binding moiety. vitamin and drug to the bivalent linker (L). It will be appreciated that a vitamin analog or derivative can mean a vitamin that incorporates a heteroatom whereby the vitamin analog or derivative is covalently linked to the bivalent linker (L). Thus, the vitamin can be covalently attached to the bivalent linker (L) via a heteroatom linker, or a vitamin analog or derivative (ie, incorporating a heteroatom) may be directly attached to the bivalent linker (L). Similarly, a drug derivative or analog can mean a drug incorporating a heteroatom whereby the drug analog or derivative is covalently attached to the bivalent linker (L). Thus, the drug can be covalently linked to the bivalent linker (L) via a heteroatom linker, or a drug analog or derivative (ie, incorporating a heteroatom) can be directly linked to the bivalent linker (L). The bivalent linker (L) may comprise a spacer linker, a releasable (ie, cleavable) linker, and a heteroatom linker for attaching the spacer linker to the releasable linker in conjugates containing both types of linkers.

Thus, the bivalent linker (L) may comprise an element for associating the vitamin with the drug, such as by connecting with heteroatom linkers (i.e. spacer arms or bridging molecules), or by direct covalent bonding between the bivalent linker ( L) and the vitamin or drug analog or derivative. Any association element should not prevent the binding of the vitamin, or vitamin receptor binding derivative or analog, to the vitamin receptor on the cell membrane for the method of the present invention to work.

In general, any way to form a conjugate between the bivalent linker (L) and the vitamin, or analog or derivative thereof, between the bivalent linker (L) and the drug, or analog or derivative thereof, can be used, including any heteroatom linkers involved. Similarly, any method known in the art can be used to form a conjugate between the spacer linker, the releasable linker, and the heteroatom linker to form the bivalent linker (L). The conjugate can be formed by direct conjugation of any of these molecules, for example, by hydrogen, ionic, or covalent bonding. Covalent bonding can occur, for example, by the formation of amide, ester, disulfide, or imino bonds between acidic, aldehyde, hydroxyl, amino, sulfhydryl, or hydrazo groups.

The detachable and / or releasable linker (i.e., cleavable linker) can be any linker

ES 2 395 082 T3 biocompatible. The cleavable linker may be, for example, a linker susceptible to cleavage under reducing or oxidizing conditions present in cells, a pH-sensitive linker which may be an acid-labile or base-labile linker, or a linker that can be cleaved by biochemical or metabolic processes, such as an enzyme-labile linker.

Typically, the spacer and / or releasable linker comprises from about 1 to about 30 carbon atoms, more typically from about 2 to about 20 carbon atoms. Low molecular weight linkers (ie, those having a molecular weight of about 30 to about 300) are typically employed. The precursors of such linkers are typically selected to have nucleophilic or electrophilic functional groups, or both, optionally in a form protected with a protecting group that is easily cleaved to facilitate their use in the synthesis of the intermediate species.

Pharmaceutical compositions comprising an amount of conjugate for delivering drugs to the vitamin-binding receptor effective to kill a population of pathogenic cells in a host animal are also contemplated when administered in one or more doses. The drug delivery conjugate is preferably administered to the host animal parenterally, eg, intradermally, subcutaneously, intramuscularly, intraperitoneally, intravenously, or intrathecally. Alternatively, the drug delivery conjugate can be administered to the host animal by other medically useful processes, such as orally, and any suitable effective dose and dosage form can be used, including sustained release dosage forms.

Examples of parenteral dosage forms include aqueous solutions of the active ingredient, in isotonic saline, 5% glucose, or other well-known pharmaceutically acceptable liquid carriers, such as liquid alcohols, glycols, esters, and amides. The parenteral dosage form may be in the form of a lyophilisate to reconstitute comprising the dose of the conjugate for administering drug. In one aspect of the present embodiment, any number of sustained release dosage forms known in the art may be administered such as, for example, the biodegradable carbohydrate matrices described in US Patent Nos. 4,713,249; 5,266,333; and 5,417,982 or, alternatively, a slow pump (eg, an osmotic pump) can be used.

At least one additional composition comprising a therapeutic factor or an adjunct to the procedures detailed above can be administered to the host to enhance the conjugate-mediated elimination of the pathogenic cell population to deliver drug, or more than one therapeutic factor can be administered. additional. The therapeutic factor can be selected from a compound capable of stimulating an endogenous immune response, a chemotherapeutic agent, or another therapeutic factor capable of complementing the efficacy of the conjugate to deliver administered drug. The use may involve administering to the patient, in addition to the conjugates described above, compounds or compositions capable of stimulating an endogenous immune response (eg, a cytokine), including cytokines or immune cell growth factors such as interleukins 1-18, factors stem cell, basic FGF, EGF, G-CSF, GM-CSF, FLK-2 ligand, HILDA, MIP-1a, TGF-α, TGF-β, M-CSF, IFN-α, IFN-β, IFN- γ, soluble CD23, LIF, and combinations thereof.

Therapeutically effective combinations of these factors can be used. In one embodiment, for example, therapeutically effective amounts of IL-2 can be used, for example, in amounts between about 0.1 MIU / m²/ dose / day and approximately 15 MIU / m²/ dose / day in a multi-dose daily regimen, and IFN-α, for example, in amounts between about 0.1 MIU / m²/ dose / day at approximately 7.5 MIU / m²/ dose / day in a daily multiple dose schedule in conjunction with the administration of drug conjugates to kill, reduce or neutralize pathogenic cells in a host animal harboring the pathogenic cells (MIU = million international units; m² = approximate surface body area of ​​an average human). In another embodiment IL-12 and IFN-α are used in the therapeutically effective amounts described above for interleukins and interferons, and in yet another embodiment, IL-15 and IFN-α are used in the therapeutically effective amounts described above for interleukins and interferons. . In an alternative embodiment, IL-2, IFN-α or IFN-γ, and GM-CSF are used in combination with the therapeutically effective amounts described above. The use of any other effective combination of cytokines including combinations with other interleukins and interferons and colony stimulating factors is also contemplated.

Chemotherapeutic agents that are, for example, cytotoxic by themselves, or that can act to enhance tumor permeability, are also suitable for combining with the use of drug delivery conjugates. Said chemotherapeutic agents adrenocorticoids and corticosteroids, alkylating agents, antiandrogens, antiestrogens, androgens, aclamycin and derivatives of aclamycin, estrogens, antimetabolites such as cytosine arabinoside, purine analogs, pyrimidine analogs, and methotrexcylato, busulfan, carbohydrate and others platinum compounds, tamoxifen, taxol, paclitaxel, paclitaxel derivatives, Taxotere®, cyclophosphamide, daunomycin, rhizoxin, T2 toxin, plant alkaloids, prednisone, hydroxyurea, teniposide, mitomycins, discodermolides, microtubule inhibitors, epothilones,, tubulysin, cyclopropyl benz [e] indolone, seco-cyclo-propylbenc [e] indolone, O-Ac-seco-cyclopropyl benz [e] indolone, bleomycin and other antibiotics, nitrogen mustards, nitrosureas, vincristine, vinblastine, and analogues and derivatives thereof such as deacetylvinblastine monohydrazide, colchicine, colchicine derivatives, allocolchicine, thiocolchicine, trityl

ES 2 395 082 T3 Cysteine, Halichondrine B, dolastatins such as dolastatin 10, amanitins such as α-amanitin, camptothecin, irinotecan, and other camptothecin derivatives of the foregoing, geldanamycin and geldanamycin derivatives, estramustine, nocodazolide, MAP4, colcemustine, nocodazolide, MAP4, inflammatory and pro-inflammatory agents, signal transduction inhibitor peptides and peptidomimetics, and any other drug or toxin known in the art. Other drugs that may be used include penicillins, cephalosporins, vancomycin, erythromycin, clindamycin, rifampin, chloramphenicol, aminoglycoside antibiotics, gentamicin, amphotericin B, acyclovir, trifluridine, ganciclovir, zidovudine, amantadine, any other known antibavirin compound, and any other known antibavirin compound. technique.

The therapeutic factor can be administered to the host animal before, after or at the same time as the conjugates to administer drug to the vitamin-binding receptor and the therapeutic factor can be administered as part of the same composition containing the conjugate to administer drug or as part of a composition other than the conjugate for drug delivery. Any such therapeutic composition containing the therapeutic factor at a therapeutically effective dose can be used.

Additionally, more than one type of conjugate can be used to deliver drug. For example, the host animal can be treated with conjugates of different vitamins, but the same, drug (eg, folate-mitomycin conjugates and vitamin B12-mitomycin conjugates) with a simultaneous dosing protocol. In other embodiments, the host animal can be treated with conjugates comprising the same vitamins bound to different drugs, or to different vitamins bound to different drugs. For example, the host animal can be treated with a folate-mitomycin conjugate and a folate-cisplatin conjugate, or with a folate-mitomycin conjugate and a vitamin B12-cisplatin conjugate. Furthermore, drug delivery conjugates with the same or different vitamins, and the same or different drugs comprising several vitamins and several drugs could be used as part of the same drug delivery conjugate.

The daily unit dose of the conjugate to administer drug can vary significantly depending on the state of the host, the disease state being treated, the molecular weight of the conjugate, its route of administration and tissue distribution, and the possible simultaneous use of other treatments. therapeutics such as radiation therapy. The effective amount to be administered to a patient is based on the body surface area, weight of the patient, and medical evaluation of the patient's condition. Effective doses may range, for example, from about 1 ng / kg to about 1 mg / kg, from about 1 pg / kg to about 500 pg / kg, and from about 1 pg / kg to about 100 pg / kg.

Any effective regimen for using the drug delivery conjugates can be used. For example, drug delivery conjugates can be used in unit doses, or they can be divided and administered in a regimen of several daily doses. In addition, a staged regimen, eg, one to three days a week, can be used as an alternative to daily treatment, and such an intermittent or staged daily regimen is considered equivalent to daily treatment. In one embodiment, the host is treated with multiple injections of the drug delivery conjugate to kill the pathogenic cell population. In one embodiment, the host receives several injections (preferably about 2 to about 50 times) with the conjugate to deliver drug, eg, at 12-72 hour or 48-72 hour intervals. Additional injections of the conjugate can be administered to deliver drug to the patient within days or months of the initial injection (s), and the initial injections prevent recurrence of the disease state caused by the cells.

In one embodiment, vitamins, or analogs or derivatives thereof that can be used in conjugates for drug delivery include those that bind to specifically expressed receptors on activated macrophages, such as the folate-binding receptor for folate. , or an analog or derivative thereof. Folate-bound conjugates, for example, can be used to kill or suppress the activity of activated macrophages that cause disease states in a host. Such macrophage targeting conjugates, when administered to a patient suffering from an activated macrophage mediated disease state, act to concentrate and associate the conjugated drug in the activated macrophage population to kill activated macrophages or suppress macrophage function. Elimination, reduction, or inactivation of the activated macrophage population acts to arrest or reduce the activated macrophage-mediated pathogenesis characteristic of the disease states being treated. Exemplary conditions known to be mediated by activated macrophages include rheumatoid arthritis, ulcerative colitis, Crohn's disease, psoriasis, osteomyelitis, multiple sclerosis, atherosclerosis, pulmonary fibrosis, sarcoidosis, systemic sclerosis, transplanted organ rejection (GVHD), and inflammations. Chronicles. Administration of the drug delivery conjugate is continued until the symptoms of the disease state are reduced or eliminated.

Conjugates for drug delivery administered to kill activated macrophages or suppress the function of activated macrophages can be administered parenterally to the animal or patient suffering from the disease state, eg, intradermally, subcutaneously, intramuscularly, intraperitoneally, or intravenously in combination with a vehicle. pharmaceutically acceptable. Alternatively, the drug delivery conjugates can be administered to the animal or patient by other medically useful processes, and effective doses can be administered in normal or sustained release dosage forms. The therapeutic procedure can be used alone or in combination with other therapeutic procedures for the treatment of disease states mediated by activated macrophages.

ES 2 395 082 T3

The following Drug Delivery Conjugates are illustrative of Drug Delivery Conjugates.

These drug delivery conjugates can be prepared by the procedures described herein in addition to protocols known in the art.

<img file="ES2395082T3_D0044.tif" />

<img file="ES2395082T3_D0045.tif" />

<img file="ES2395082T3_D0046.tif" />

ES 2 395 082 T3

<img file="ES2395082T3_D0047.tif" />

<img file="ES2395082T3_D0048.tif" />

In addition, the following drug delivery conjugates are also illustrative of drug delivery conjugates. The accompanying synthetic procedures are illustrative of those that can be used to prepare the drug delivery conjugates described herein.

<img file="ES2395082T3_D0049.tif" />

To a solution of diacetoxyscirpenol (DAS) in acetonitrile was added 1.0 eq. of 1,2,4,5-benzene tetracarboxylic dianhydride followed by 1 eq. Hünig's base. The reaction mixture was stirred for 1.5 hours under argon and at room temperature. If part of the DAS remains unreacted, add 0.2 eq. more of the dianhydride and stirring was continued for 1 hour. A solution of 1.2 eq. pteroyl hydrazide (prepared according to J. Am. Chem. Soc., 1997, 119, 10004) in anhydrous DMSO, followed by 1.0 eq. Hünig's base. The reaction mixture was stirred for 1 hour and precipitated with diethyl ether. The resulting precipitate was further purified by preparative HPLC.

<img file="ES2395082T3_D0050.tif" />

As generally described in Example 10a, Boc-hydrazide is reacted with succinic anhydride, and the resulting product is reacted with 5-amino-bis-indolyl-seco-CBI in the presence of EDC as a condensing agent. Removal of Boc and formation of the hydrazone with free levulinic acid provide, upon activation with NHS-ester, a reactive partner for the Pte-y-Glu-Asp-Arg-Asp-Dap-OH peptide fragment. This peptide fragment has been prepared by a polymer-supported sequential approach using the Fmoc strategy starting from Fmoc-Dap (Boc) -Wang resin as generally described in Scheme 12.

ES 2 395 082 T3

<img file="ES2395082T3_D0051.tif" />

Fmoc-hydrazide is reacted with 3- (2-pyridyldithio) propionic acid to give Fmoc-hydrazido- [3- (2-pyridyldithio) propionate]. Reaction with a mitomycin C derivative (mitomycin C, N- (CH2) 2SH) results in a mitomycin C derivative containing a disulfide. Removal of Fmoc by standard protocols and formation of the hydrazone with free levulinic acid provide, upon activation with NHS-ester, a reactive partner for the Pte-y-Glu-Dap-OH peptide fragment, which can be prepared as follows. described generally in Scheme 12.

In each compound presented herein, the stereochemistry of the amino acids used to form the linker can optionally be selected from the natural L configuration, or the non-natural D configuration. Each example has been characterized by NMR, NMR, MS, and / or UV spectroscopy. and / or HPLC as indicated. the selected characteristic signals have been reported as appropriate.

Comparative Example 1

<img file="ES2395082T3_D0052.tif" />

Diethylenetriamine folic acid y-amide, (DETA-folate) was synthesized according to a procedure described by P. Fuchs et al., J. Am. Chem. Soc., 1997, 119, 10004. This compound (100mg) was dissolved in 2 ml of 0.1N HCl. The resulting solution was added to a solution of K2PtCl4 (158 mg) in 1 ml of 0.1N HCl with stirring. 3 ml of DMSO was added and stirring was continued for 3 days, the solution was filtered, and the filtrate was precipitated in acetonitrile to give 170 mg of a yellow powder; MS (MALDI) 1249.92, 1286.27; NMR¹H (D2O) δ 1.05 (t, 1H), 2.3 (t, 2H), 3.1 (t, 2H), 4.45 (m, 1H), 6.65 (d, 2H), 7.5 (d, 2H), 8.65 (s, 1H).

Comparative example 2a

<img file="ES2395082T3_D0053.tif" />

The peptidyl fragment N¹⁰-TFA-Pte-Glu-Glu-Lys-OH containing N-protected folate¹⁰-trifluoroacetyl was prepared by a polymer-supported sequential approach using the Fmoc strategy, as generally described in Scheme 12. It was synthesized on the acid-sensitive Fmoc-Lys (Boc) -Wang resin. PyBop was applied as an activating reagent to ensure sufficient coupling with low amino acid equivalents. Fmoc protecting groups were removed after each coupling step under standard conditions (20% piperidine in DMF). Fmoc-Glu-OtBu and N¹⁰-TFA-Pte-OH were used as protected amino acid building blocks. After the last assembly step, the peptide was cleaved from the polymeric support by treatment with trifluoroacetic acid, ethanediol and triisopropylsilane. This reaction also achieved the simultaneous removal of the protecting groups t-Bu and t-Boc. The crude peptide was purified by preparative HPLC to give N¹⁰-TFA-Pte-YGluyGlu-Lys-OH as the TFA salt. A solution of 81 mg (0.1 mmol) of the peptide in 2 ml of DMSO was treated with 15 µl (0.11 mmol) Et₃N and 35 mg (0.1 mmol) of mitomycin A. Mitomycin A can be prepared from mitomycin C according to the procedures of M. Matsui, Y. Yamada, K. Uzu, and T. Hirata, J. Antibiot. 21, 189-198 (1968) and D. Vias, D. Benigni, R Partyka, and T. Doile, J. Org. Chem. 51, 4307-4309 (1986)

The reaction mixture was stirred for 48 h at room temperature and the solvent was removed by lyophilization. Unless otherwise indicated, all solvent evaporations were carried out under reduced pressure. Finally, the trifluoroacetyl protecting group was removed in aqueous ammonium hydroxide (pH = 10.0) and the product was precipitated in acetonitrile to give 102 mg of the conjugate as a yellow solid; NMR¹H (D2O) δ 2.45 (q, 1H), 2.95 (m, 2H), 3.35 (dd, 1H), 3.5 (d, 1H), 6.5 (d, 2H), 7.55 (d, 2H), 8.55 (s, 1H).

ES 2 395 082 T3

Comparative Example 2b

<img file="ES2395082T3_D0054.tif" />

The peptidyl fragment N¹⁰-TFA-Pte-Glu-Cys-OH containing N-protected folate¹⁰-trifluoroacetyl was prepared by a polymer-supported sequential approach using the Fmoc strategy, as generally described in Scheme 12, and described in Example 2a. Cystamine was reacted with mitomycin A (see Matsui et al., J. Antibiot. 21, 189-198 (1968); Vias et al., J. Org. Chem. 51, 4307-4309 (1986)) giving the mitomycin derivative C containing a disulfide having a free amino group at the end, which was coupled with levulinic acid, followed by subsequent reaction of the carbonyl group with a derivatized acyl hydrazide with maleimide. The resulting Michael acceptor reaction with N¹⁰-TFA-Pte-Glu-Cys-OH provided the final conjugate after removal of the trifluoroacetic protecting group with aqueous ammonium hydroxide (pH = 10.0), and precipitation in acetonitrile; MS (MALDI) 1059.04, 1148.44, 1225.32, 1300.8; NMR¹H (D2O) δ 1.8 (d, 2H), 1.9 (s, 1H), 2.3 (q, 1h), 2.45 (q, 1H), 2.9 (1, 1H), 3.35 (dd, 1H), 4.45 (s, 1H), 4.5 (dd, 1H), 6.65 (d, 2H), 7.55 (d, 2H), 8.6 (s , 1 HOUR).

Comparative example 3

<img file="ES2395082T3_D0055.tif" />

The maleimido intermediate p-methoxybenzylidene acetal of toxin T-2 was synthesized starting from commercial N- (2-hydroxyethyl) maleimide. Its hydroxyl group was reacted with p-methoxybenzyl chloride (1.2 eq.) In the presence of silver (I) oxide (2 eq.) As a weak base in methylene chloride. The crude product was purified on a silica column. Oxidative treatment of the resulting p-methoxybenzyl ether with 1.5 eq. of 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) in the presence of the OH T-2 containing toxin (1 eq.) gave the desired p-methoxybenzylidene acetal by the stabilized p-methoxybenzylcarbenium acetal intermediate.

The other reaction partner, Pte-y-Glu-Arg-Asp-Cys-OH, was prepared by a polymer supported sequential approach using the Fmoc strategy. H-Cys (4-methoxytrityl) -2-chlorotrityl-acid sensitive resin was synthesized on the resin. PyBop was applied as an activating reagent to ensure sufficient coupling with low amino acid equivalents. Fmoc-Asp (OtBu) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Glu-OtBu), and N¹⁰-TFA-Pte-OH were used as protected amino acid building blocks. Fmoc protecting groups were removed after each coupling step under standard conditions (20% piperidine in DMF). After the last assembly step, the peptide was cleaved from the polymeric support by treatment with trifluoroacetic acid, ethanediol and triisopropylsilane. This reaction also achieved the simultaneous removal of the protecting groups t-Bu and t-Boc. The crude peptide was purified by preparative HPLC to give N¹⁰-TFA-Pte-y-Glu-Arg-Asp-Cys-OH. The trifluoroacetyl protecting group was removed in aqueous ammonium hydroxide (pH = 10.0).

Finally, the target folate-linked acetal-p-methoxybenzylidene drug conjugate was prepared by mixing under argon a buffered aqueous solution (pH = 7.0) of the peptide with an equimolar acetonitrile solution of the maleimide-containing acetal of toxin T- 2. After stirring at room temperature for 1 hour, the final conjugate was subjected to preparative HPLC, and gave a yellow powder after lyophilization of the collected fraction; MS (m + H)⁺ 1541.3; NMR¹H (DMSO-O6) δ 0.1 (s, 1H), 0.55 (d, 2H), 0.9 (dd, 3H), 1.65 (s, 1H), 2.0 (d, 1H ), 3.75 (d, 2H), 5.25 (d, 1H), 6.65 (d, 2H), 6.9 (d, 2H), 7.3 (t, 2H), 7.65 (d, 2H), 8.65 (s, 1H).

ES 2 395 082 T3

Comparative Example 4a

<img file="ES2395082T3_D0056.tif" />

Comparative Example 4b

<img file="ES2395082T3_D0057.tif" />

Comparative example 4c

<img file="ES2395082T3_D0058.tif" />

The compounds of Examples 4a, 4b, and 4c have been prepared according to the procedure generally described in Example 3, except that acylaziridine was prepared by acylation (see Scheme 1) of mitomycin A with M- ( commercially available suitable alkanoic acid) maleimide.

Comparative Example 5

<img file="ES2395082T3_D0059.tif" />

The reaction of trans-4-aminocyclohexanol hydrochloride with an equimolar amount of Fmoc-OSu in the presence of 2.2 eq. by NaHCO₃ as base, and acetonitrile / water (1/1) as solvent gave the M-Fmoc protected amino alcohol which was oxidized to the corresponding N-Fmoc protected amino ketone under Swern conditions (Synthesis, 1981, 165). Ketalization with 4 eq. Of methyl orthoformate and a catalytic amount of trifluoroacetic acid gave an N-Fmoc protected aminoketal in quantitative yield. Treatment of this ketal with equimolar amounts of trimethylsilyl trifluoromethanesulfonate and 2,4,6-tri-i-butyl-pyridine resulted in the 4-Fmocaminocyclohexyl enol ether product. In the next step, the drug, toxin T-2, was treated with a four-fold excess of the enol ether in the presence of molecular sieves (3A) and catalytic amounts of trifluoroacetic acid. The resulting asymmetric mixed ketal was purified on silica gel. The Fmoc protecting group was removed by treatment with resin-bound piperidine in DMF. The liberated amino group was reacted with 1.1 eq. of maleimidoacetic acid-NHS-ester in the presence of 1.1 eq. Hünig's base. The maleimide-containing ketal of the T-2 toxin was purified on silica gel.

The folate-containing peptide fragment, Pte-and-Glu-p-Dap-Asp-Cys-OH, was prepared by a polymer-supported sequential approach using the Fmoc strategy, as generally described in Scheme 12. It was synthesized on the acid-sensitive Wang resin loaded with Fmoc-L-Cys (Trt) -OH. PyBop was applied as an activating reagent to ensure sufficient coupling with low amino acid equivalents. Fmoc protecting groups were removed after each coupling step under standard conditions (20% piperidine in DMF). Fmoc-Asp (OtBu) -OH, Boc-Dap (Fmoc) -OH, Fmoc-Glu-OtBu, and V ° -TFA-Pte-OH were used as protected amino acid building blocks. After the last assembly step, the peptide was cleaved from the polymeric support by treatment with trifluoroacetic acid, ethanediol and triisopropylsilane. This reaction also achieved the simultaneous removal of the LBu, t-Boc, and trityl protecting groups. Finally, the trifluoroacetyl moiety was removed in aqueous ammonium hydroxide to give the desired thiol-containing peptide. The crude peptide was purified by preparative HPLC.

ES 2 395 082 T3

Finally, the target ketal-linked folate drug conjugate was prepared by mixing under argon a buffered aqueous solution (pH = 7.0) of the peptide with an equimolar acetonitrile solution of the maleimide-containing acetal of the T-2 toxin. After stirring at room temperature for 1 hour, the final conjugate was subjected to preparative HPLC, and gave a yellow powder after lyophilization of the collected fraction; ES MS (mH) - 1474.5, (m + H)⁺ 1476.2, (m + Na)⁺ 1498,3.

Comparative Example 6

<img file="ES2395082T3_D0060.tif" />

Ethylenediamine folic acid, γ-amide (EDA-folate) was synthesized according to a procedure described by P. Fuchs et al. (J. Am. Chem. Soc., 1997, 119, 10004; see for example the synthesis of compound 52 described therein.

EDA-folate (600 mg) was suspended in 5 ml of anhydrous DMSO. After stirring for 4 hours at 60 ° C the resulting solution was cooled to 20 ° C and 4 eq. 1,2,4,5-benzenetetracarboxylic dianhydride (BTCA anhydride). After 5 min the reaction mixture was poured into well stirred anhydrous acetonitrile. The resulting precipitate was isolated by centrifugation to give 657 mg of BTCA (monoanhydride) -EDA-folate.

Solid BTCA (monoanhydride) -EDA-folate (1.5 eq.) Was added to a well stirred solution of daunomycin in dry DMSO. After stirring for a further 14 hours, 50% of the daunomycin remained unreacted (HPLC), then 1.5 eq. BTCA (monoanhydride) -EDA-folate. After stirring for a further four hours, all of the daunomycin had been consumed. Two new peaks with close retention times were observed on the HPLC profile representing the two regioisomers of the final conjugate. The crude product was isolated after precipitation from acetonitrile and further purified by reverse phase HPLC. The structure of the product was in accordance with the ES MS (mH)^- 1227,1.

Comparative Example 7

<img file="ES2395082T3_D0061.tif" />

Under argon and at 0 ° C, to a well stirred solution of 250 mg (0.25 mmol) of paclitaxel and 130 μΙ (0.73 mmol) of Hünig's base in 4 ml of anhydrous dichloromethane, 85 μΙ (0 , 8 mmol) of Alloc-Cl. Stirring was continued for a further 12 hours, and the product was isolated by conventional extraction techniques. This white powder, 2'-alloc-paclitaxel, was used in the next step without further purification.

In this step, 108 mg (0.117 mmol) of 2'-alloc-paclitaxel was dissolved in 1.0 ml of anhydrous acetonitrile. Under argon with stirring, 25 mg (0.117 mmol) of 1,2,4,5-benzenetetracarboxylic dianhydride (BTCA anhydride) and 21 µΙ (0.120 mmol) of Hünig's base were added to this solution. Stirring was continued for a further 2.5 hours. In a separate reaction flask, 52 mg of EDA-folate was stirred at 60 ° C until all material dissolved (ca. 60 min). After cooling to room temperature, the above reaction mixture was added to this solution and stirring was continued for a further 2 hours. The reaction mixture was added dropwise to a well stirred acetonitrile / diethyl ether mixture (20:80). The yellow precipitate was separated by centrifugation and further purified by preparative HPLC. The structure of the product was in agreement with the NMR spectra¹H 1D and 2D (COZY); ES MS (m + H)⁺ 1555,5.

ES 2 395 082 T3

Comparative Example 8

<img file="ES2395082T3_D0062.tif" />

A mixture of 1.0 eq. of aclamycin, 2.0 eq. of hydrazido- [3- (2-pyridyldithio) propionate] (SPDP-hydrazone) and a few crystals of pyridinium p-toluenesulfonate were dissolved under argon with stirring in anhydrous methanol. The reaction mixture was stirred at room temperature for 8 hours. The solvent was evaporated to dryness. The residue was purified on a silica gel column pretreated with 1.5% triethylamine in chloroform / methanol (90:10). The aclamycin acyl hydrazone obtained was dissolved in a minimal amount of acetonitrile. An equimolar amount of Pte-and-Glu-Cys-OH (dissolved in water and adjusted to pH = 6.8) was added slowly and under argon to the resulting solution. The preparation of Pte-and-Glu-Cys-OH is analogous to the procedure described in Example 2a, and generally described in Scheme 12. The disulfide exchange reaction took place in 10 min. The reaction mixture was slowly added to excess acetonitrile and the resulting precipitate was isolated after centrifugation. The precipitate was resuspended once more in acetonitrile and after stirring for 15 min it was separated by centrifugation. After drying under high vacuum overnight, the final conjugate was sufficiently pure (HPLC); ES MS (m + H⁺) 1474,1.

Comparative Example 9

<img file="ES2395082T3_D0063.tif" />

A mixture of 1.0 eq. of aclamycin and 1.2 eq. of β-maleimidopropionic acid TFA was dissolved under argon with stirring in anhydrous methanol. The reaction mixture was stirred at room temperature for 1 hour. The solvent was evaporated to dryness. The residue was passed through a short column of silica gel pretreated with 15% triethylamine in chloroform / methanol (90:10). In a separate flask, the Pte-y-Glu-y-Glu-Cys-OH peptide fragment was dissolved in water under argon adjusting the pH to 6.8. The preparation of Pte-y-Glu-y-Glu-Cys-OH is analogous to the procedure described in Example 2a, and described generally in Scheme 12. To the resulting yellowish solution, the maleimidohydrazone of aclamycin dissolved in a minimal amount of methanol. The reaction mixture was stirred for 1 hour under argon. Methanol was removed and the residue was purified by HPLC on a preparative column, followed by lyophilization; ES MS (m + H)⁺ 1722,3.

ES 2 395 082 T3

Comparative Example 9b

<img file="ES2395082T3_D0064.tif" />

<img file="ES2395082T3_D0065.tif" />

Comparative Example 9c

<img file="ES2395082T3_D0066.tif" />

The compounds of Examples 9b and 9c were prepared from doxorubicin derivatives (14-hydroxidaunomycin) according to the procedure generally described in Example 9a.

Comparative Example 10a

<img file="ES2395082T3_D0067.tif" />

The 5- (M-Boc) amino analog of the potent cytotoxic drug bis-indolyl-seco-1,2,9,9a-tetrahydrocyclopropa [c] benz [e] indol-4-one (bis-indolyl-seco-CBI) was prepared according to a slight modification of the procedure first described by D. Boger et al., J. Org. Chem., 1992, 57, 2873

The peptide fragment, Pte-y-Glu-Asp-Arg-Asp-Cys-OH, was prepared by a polymer supported sequential approach using the Fmoc strategy on the H-Cys (4-methoxytrityl) -2-chlorotrityl resin. -acid sensitive resin, as generally described in Scheme 12. PyBop was applied as an activating reagent to ensure sufficient coupling with low amino acid equivalents. Fmoc-Asp (OtBu) -OH, Fmoc-Arg (Pbf) OH, Fmoc-Glu-OtBu), and V ° -TFA-Pte-OH were used as protected amino acid building blocks. Fmoc protecting groups were removed after each coupling step under standard conditions (20% piperidine in DMF). After the last assembly step, the peptide was cleaved from the polymeric support by treatment with trifluoroacetic acid, ethanediol and triisopropylsilane. This reaction also achieved the simultaneous removal of the protecting groups t-Bu and t-Boc. The crude peptide was purified by preparative HPLC to give A /¹⁰-TFA-Pte-Y-GluAsp-Arg-Asp-Cys-OH. The trifluoroacetyl protecting group was removed in aqueous ammonium hydroxide (pH = 10.0).

ES 2 395 082 T3

Lev-Val-OH was synthesized by a standard protocol that included condensation of valine methyl ester hydrochloride with levolinic acid in the presence of EDC and Hünig's base followed by hydrolysis of the methyl ester with lithium hydroxide and water.

The final assembly of the complex conjugate began with the elimination of the N-Boc group of 5- (N-Boc) amino-bisindolyl-seco-CBI and coupling of the liberated amino group with the carboxyl functionality of Lev-Val-OH in the presence of EDC. The formation of the acyl hydrazone was carried out by reacting the ketone functionality of the levolinic moiety with 1.2 eq. of β-maleimidopropionic acid TFA in tetrahydrofuran. After purification by chromatography (silica gel, THF / hexane = 1/1) the product of the above reaction was dissolved in DMSO. To this solution, under argon, 0.9 eq. of Pte-y-Glu-Asp-Arg-Asp-Cys-OH and the reaction mixture was stirred for 18 hours. The solvent was removed by lyophilization and the residue was purified by HPLC.

Comparative Example 10b

<img file="ES2395082T3_D0068.tif" />

Comparative Example 10c

<img file="ES2395082T3_D0069.tif" />

<img file="ES2395082T3_D0070.tif" />

The compounds of Examples 10c and 10c were prepared from 5- (N-Boc) amino-bis-indolyl-secoCBI derivatives according to the procedure generally described in Example 10a.

Comparative Example 11

<img file="ES2395082T3_D0071.tif" />

In the presence of potassium carbonate, the S-alkylation of 2-mercaptoethanol was carried out with allyl bromide. The hydroxyl group of the resulting allyl β-hydroxyethyl sulfide was exchanged for chloride by thionyl chloride. Oxidation of this product with hydrogen peroxide in the presence of acetic anhydride J. Am. Chem. Soc., 1950, 72, 59) resulted in an allyl β-chloroethyl sulfone. Reaction of this product with chlorodimethylsilane in the presence of a catalytic amount of hydrogenhexachloroplatinate (IV) and elevated temperature gave 3 (β-chloroethyl sulfonyl) propyldimethylsilyl chloride after distillation. This chlorosilane silylated the hydroxyl group of strongly cytotoxic compound rhizoxin using 1 eq. of pyridine as a base. Treatment of the β-chloroethyl sulfone moiety of this molecule with an excess of triethylamine resulted in gentle β-removal of hydrogen chloride with formation of the respective vinyl sulfone.

The other reaction partner, the Pte-y-Glu-Arg-Asp-Cys-OH peptide fragment, was prepared by a polymer-supported sequential approach using an Fmoc protocol, as generally described in Example 2a and in Scheme 12.

Final assembly of the complex conjugate was achieved by a Michael addition of the peptide fragment group to the vinyl sulfone moiety of the rhizoxin-linked silicon linker. The reaction medium for this

ES 2 395 082 T3 transformation was 50:50 acetonitrile / water (pH = 7.2). After stirring at room temperature for 24 hours, the final conjugate was isolated after HPLC on a preparative column; ES MS (m + H)⁺ 1631.6; (mH)^- 1629,6.

Comparative Example 12

<img file="ES2395082T3_D0072.tif" />

This silicon-bound rhizoxin conjugate was synthesized by the protocol described in Example 11, except that commercial chloromethylphenylsilane was used in place of chlorodimethylsilane.

Comparative Example 13

General preparation of compounds containing a disulfide bond from cys.

Thiosulfonates 4 (1 eq.), Prepared according to the procedure of Ranasinghe and Fuchs, Synth. Commun. 18 (3), 227-32 (1988) were reacted with drugs, drug analogs or drug derivatives 5 (1 eq.) To prepare the thiosulfonates of drug 6 as solution in methanol, as shown in Scheme 13 . R is alkyl or aryl, L is a suitable leaving group, such as halogen and pentafluorophenyl, n is an integer between 1 and 4, and X is -O-, -NH-, -C (O) are O-, or -C (O) NH-. The conversion was suitably followed by observing the disappearance of each starting material by TLC (silica gel; CHCl₃/ MeOH = 9/1). Scheme 13

<img file="ES2395082T3_D0073.tif" />

The peptidyl fragment containing folate Pte-Glu- (AA) n-Cys-OH (9) was prepared by a polymer-supported sequential approach using the strategy with Fmoc on the resin (7) Fmoc-Cys (Trt) -Wang acid sensitive, as shown in Scheme 14. R1 is Fmoc, R2 is trityl, and DIPEA is diisopropylethylamine. PyBop was applied as an activating agent to ensure sufficient coupling. Fmoc protecting groups were removed after each coupling step under standard conditions. Properly protected amino acid building blocks were used, such as Fmoc-Glu-OtBu, and M¹⁰-TFA-Pte-OH, as described in Scheme 14, and represented in step (b) by Fmoc-AA-OH. Thus, AA refers to any amino acid starting material that is adequately protected. The coupling sequence (steps (a) and (b)) involving FmocAA-OH are performed n times to prepare peptide 8 on solid support, where n is an integer and can be equal to 0 to about 100. After the last coupling step, the remaining Fmoc group is removed, and the peptide is sequentially coupled to a glutamate derivative (step (c)), deprotected, and coupled to TFA-protected pteroic acid (step (d)). Subsequently, the peptide is cleaved from its polymeric support after treatment with trifluoroacetic acid, ethanediol, and triisopropylsilane (step (e)). These reaction conditions result in the simultaneous removal of the protecting groups t-Bu, t-Boc, and Trt. The TFA protecting group is removed after base treatment (step (f)) to give the Cys-containing, folate-containing peptidyl fragment 9

ES 2 395 082 T3

Scheme 14

<img file="ES2395082T3_D0074.tif" />

(a) piperidine 20% DMF /; (b) Fmoc-AA-OH, PyBop, DIPEA, DMF; (c) Fmoc-Glu (Ot-Bu) -OH, PyBop, DIPEA, DMF;

(d) 1. N¹⁰(TFA) -Pte-OH; PyBop, DIPEA, DMSO; (e) TFAA, (CH2SH) 2, / -PraSiH; (f) NH4OH, pH 10.3.

Drug conjugates were prepared by reacting the folate derivative 9 (0.9-0.95 eq.) With the thiosulfonate of drug 6 in deionized water (0.04 M, pH adjusted to 7 with NaHCO₃ 0.1 N) under argon for about 30 minutes, forming a disulfide bond. After evaporation of the methanol in vacuo, the conjugate can be purified by preparative HPLC (Prep Novapak Hr C18 19 X 300 mM column; mobile phase (A) -1.0 mM phosphate buffer, pH = 6; organic phase (B) -acetonitrile ; gradient conditions from 99% A and 1% B to 50% A and 50% B in 30 minutes, flow rate = 15 ml / minute).

Comparative Example 14a

<img file="ES2395082T3_D0075.tif" />

NMR ¹H (DMSO-de) δ 4.7 (d, 1H), 4.95 (t, 1H), 6.7 (d, 4H), 6.9 (t, 1H), 7.95 (d, 2H ), 8.1 (d, 2H), 8.2 (m, 1H), 8.3 (s, 1H), 8.4 (s, 1H), 8.7 (s, 1H), 10.2 (s, 1H), 11.8 (d, 2H).

Comparative Example 14b

<img file="ES2395082T3_D0076.tif" />

ES MS (mH) '1436.4, (m + H)⁺ 1438,3.

Comparative Example 14c

<img file="ES2395082T3_D0077.tif" />

NMR ¹H (DMSO-d₆/ D2O) δ 1.0 (s, 1H), 1.1 (s, 1H), 1.6 (s, 1H), 1.8 (s, 1H), 2.1 (s, 1H), 2 , 25 (s, 3H), 2.65 (dd, 2H), 3.7 (d, 1H), 4.4 (t, 1H), 4.55 (q, 2H), 4.6 (d, 2H), 4.95 (d, 1H), 5.9 (t, 1H), 6.15 (s, 1H), 6.6 (d, 2H), 7.85 (d, 2H), 7, 95 (d,

ES 2 395 082 T3

2H), 8.6 (s, 1H), 8.95 (d, 1H).

Comparative Example 14d

<img file="ES2395082T3_D0078.tif" />

NMR ¹H (DMSOWD2O) δ 1.0 (s, 1H), 1.1 (s, 1H), 1.65 (s, 1H), 2.1 (s, 1H), 2.25 (s, 3H), 3.6 (t, 2H), 3.7 (d, 1H), 4.4 (t, 1H), 4.6 (d, 1H), 4.95 (d, 1H), 5.9 (t , 1H), 6.2 (s, 1H), 6.6 (d, 7.95 (d, 2H), 8.6 (s, 1H), 9.1 (d, 2H).

2.6 (dd, 2H), 3.25 (dd, 1H),

2H), 7.7 (t, 1H), 7.9 (d, 2H),

Comparative Example 14e

<img file="ES2395082T3_D0079.tif" />

NMR ¹H (DMSOWD2O) δ 10.85 (d, 2H), 1.05 (d, 2H), 1.2 (d, 2H), 1.7 (d, 1H), 5.7 (dd, 1H), 6.65 (d, 2H), 7.6 (d, 2H), 7.95 (s, 1H), 8.65 (s, 1H).

2H), 3.95 (d, 1H), 4.05 (dd, 1H), 5.4 (dd,

Comparative Example 14f

<img file="ES2395082T3_D0080.tif" />

ES MS (m + H)⁺ 1487.23; NMR¹H (DMSOWD2O) δ 0.9 (t, 2H), 1.3 (t, 2H), 2.15 (t, 2H), 3.2 (dd, 1H, 4.0 (t, 1H), 4 , 15 (q, 1H), 5.3 (s, 2H), 5.5 (s, 2H), 6.6 (d, 2H), 7.0 (s, 1H), 7.4 (m, 2H), 7.55 (d, 2H), 8.0 (d, 2H), 8.6 (s, 1H).

Examples 14a, 14b, 14c, 14d, 14e, and 14f were prepared by the following general procedure. To a well stirred solution of the corresponding drug containing an -OH group (1 eq. In dry CH2CL or dry THF) was added under argon 6- (trifluoromethyl) benzotriazolyl 2- (2'-pyridyldithio) ethyl carbonate (1.3 eq.) and NN-dimethylaminopyridine (1.5 eq.). The reaction mixture was stirred for 3 h, and the pyridyldithium derivatized drug was isolated by silica gel chromatography (> 65% for each example). The corresponding peptidyl fragment (0.5 eq.), Prepared according to the general approach detailed in Scheme 12, was dissolved in DMSO. The pyridyldithium derivatized drug was added to the clear yellow solution. After 30 min, the reaction was completed and the conjugate purified by HPLC. In the case of Example 14e, the peptidyl fragment Pte-GluAsp-Arg-Asp-Asp-Cys-OH was first dissolved in water, and the pH of the solution was adjusted to 2.5 with 0.1 N HCl , causing precipitation of the peptidyl fragment. The peptidyl fragment was collected by centrifugation, dried and dissolved in DMSO for further reaction with the pyridyldithium derivatized drug.

ES 2 395 082 T3

Comparative Example 15

<img file="ES2395082T3_D0081.tif" />

SN 38 (10-hydroxy-7-ethylcamptothecin) intermediate 4- (2-pyridinyldithio) benzylcarbonate was prepared according to the procedure described by P. Senter et al., J. Org. Chem. 1990, 55, 2875 The peptidyl fragment Pte-Glu-AspArg-Asp-Cys-OH was dissolved in DMSO, and pyridyldithium derivatized drug was added to the clear yellow solution. After 30 min, the reaction was complete and the conjugate was purified by HPLC; ES MS (m + H)⁺ 1425.38; NMR¹H (DMSO-d₆/ D2O) δ 0.9 (t), 1.15 (t), 3.9 (t), 4.0 (t), 4.25 (t), 5.1 (m), 5.2 ( s), 5.4 (s), 6.55 (d),

7.25 (d), 7.35 (d), 7.5 (d), 7.9 (d), 8.55 (s).

Comparative Example 16a

<img file="ES2395082T3_D0082.tif" />

Working example 16b

<img file="ES2395082T3_D0083.tif" />

The compounds of Examples 16a and 16b were prepared from the peptidyl fragment Pte-Glu-Asp-Arg-AspAsp-Cys-OH, prepared according to the general procedure described in Scheme 12. The Michael addition of this fragment Peptidyl to the maleimido derivative of seco-CBI-bis-indole resulted in the folate conjugates of Example 16b. The maleimido derivative of seco-CBI-bis-indole, and pyridyldithium activated vinblastine thiosulfonate and intermediates were prepared using the procedures described herein for other examples.

Comparative Example 17a

<img file="ES2395082T3_D0084.tif" />

Deacetylvinblastine monohydrazide (1 eq.) (See Barnett et al., J. Med. Chem., 1978, 21, 88) was treated with freshly distilled THF with 1 eq. of trifluoroacetic acid. After stirring for 10 min the solution was treated with 1.05 eq. of N- (4-acetylphenyl) maleimide. The acyl hydrazone formation was complete in 45 min and the solvent was evaporated. The peptidyl fragment Pte-Glu-Asp-Arg-Asp-Asp-Cys-OH (0.85 eq.), Prepared according to the general approach

ES 2 395 082 T3 detailed in Scheme 12, was dissolved in water, and the pH was adjusted to 2.5 with 0.1N HCl, causing the peptide to precipitate. The peptidyl fragment was collected by centrifugation, dried, and dissolved in DMSO. Hünig's base (15 eq.) And Michel's adduct of acyl hydrazone were added to the resulting clear yellow solution. After 1 hr, the final conjugate was purified by HPLC.

Comparative Example 17b

<img file="ES2395082T3_D0085.tif" />

Comparative Example 17c

<img file="ES2395082T3_D0086.tif" />

Examples 17b and 17c were prepared according to the procedure described in Example 17a with the corresponding peptidyl fragments and monohydrazide derivatives of CBI.

The compounds of Examples 18-41 have been prepared according to the procedure generally described in Example 13. Examples 18-41 were characterized by electrospray mass spectroscopy (ES MS), and other spectroscopic techniques including 1D NMR and 2D, and UV.

Comparative Example 18

<img file="ES2395082T3_D0087.tif" />

ES MS (m + H)⁺ 1071.9, (m + Na)⁺ 1093.9; NMR¹H (D2O) δ 2.6 (t, 4H), 2.7 (t, 4H), 4.15 (s, 2H), 5.45 (s, 2H), 7.75 (d, 2H), 8.15 (d, 2H), 8.9 (s, 1H).

Comparative Example 19

<img file="ES2395082T3_D0088.tif" />

UV (nm) 233 (max), 255, 280; NMR¹H (D2O, NaOD, CD3CN) δ 1.15 (d, 3H), 2.3 (s, 3H), 3.6 (s, 1H), 3.85 (s, 3H), 4.9 (s , 1H), 5.3 (s, 1H), 6.5 (d, 2H), 7.3 (m, 1H), 7.5 (d, 2H), 7.65 (d, 2H), 8 , 4 (s, 1H).

ES 2 395 082 T3

Comparative Example 20

<img file="ES2395082T3_D0089.tif" />

ES MS (mH) '935.6, (m + H)⁺ 937.4, (m + Na)⁺ 959,5.

Comparative Example 21

<img file="ES2395082T3_D0090.tif" />

NMR ¹H (D2O, NaOD, CD3CN) δ 0.1 (s, 1H), 1.1 (s, 3H), 1.2 (s, 3H), 1.75 (s, 3H), 1.9 (s , 3H), 2.05 (s, 3H), 2.35 (s, 3H), 3.3 (dd, 2H), 3.8 (d, 1H), 4.3 (q, 2H), 4 , 9 (d, 1H), 5.1 (d, 1H), 5.4 (q, 1H), 5.55 (d, 1H), 5.65 (d, 1H), 6.1 (t, 1H), 6.35 (s, 1H), 6.9 (d, 2H), 7.9 (d, 2H), 8.15 (d, 2H), 8.7 (s, 1H).

Comparative Example 22

<img file="ES2395082T3_D0091.tif" />

Comparative Example 23

<img file="ES2395082T3_D0092.tif" />

ES MS (mH) '1136.5.

Comparative Example 24

<img file="ES2395082T3_D0093.tif" />

ES MS (mH) '1136.3, (m + H)⁺ 1138,0.

Comparative Example 25

<img file="ES2395082T3_D0094.tif" />

ES MS (m + H)⁺ 1382.3, (m + Na)⁺ 1405,4.

ES 2 395 082 T3

Comparative Example 26

<img file="ES2395082T3_D0095.tif" />

ES MS (mH) '1379.2, (m + H)⁺ 1381,2.

Comparative Example 27

<img file="ES2395082T3_D0096.tif" />

ES MS (mH)^- 949.2; NMR¹H (D2O) δ 1.55 (s, 3H), 1.95 (m, 2H), 2.05 (s, 3H), 2.45 (s, 3H), 2.75 (dd, 2H), 2.95 (dd, 2H), 3.05 (s, 3H), 3.3 (dd, 2H), 3.35 (d, 2H), 3.45 (t, 2H), 4.85 (q , 2H), 6.5 (d, 2H), 7.45 (d, 2H), 8.5 (s, 1H).

Comparative Example 28

<img file="ES2395082T3_D0097.tif" />

NMR ¹H (DMSO-de) δ 1.5 (s), 2.25 (t), 2.75 (m), 3.9 (q), 4.6 (d), 4.85 (t), 6 , 6 (d), 7.6 (d), 7.9 (d), 8.15 (d), 8.25 (t), 8.65 (s), 8.7 (m), 9, 3 (m), 10.2 (t).

Comparative Example 29

<img file="ES2395082T3_D0098.tif" />

ES 2 395 082 T3

ES MS (mH) '1413.5, (m + H)⁺ 1415,3.

Comparative Example 30

<img file="ES2395082T3_D0099.tif" />

ES MS (m + H)⁺ 1530.2; NMR¹H (DMSOWD2O) δ 1.2 (s, 1H), 2.9 (t, 1H), 3.65 (t, 1H), 4.15 (t, 1H), 4.25 (t, 1H), 4.35 (t, 1H), 6.7 (d, 2H), 7.0 (s, 1H), 8.1 (d, 2H), 8.25 (s, 1H), 8.7 (s , 1 HOUR).

Comparative Example 31

<img file="ES2395082T3_D0100.tif" />

NMR ¹H (DMSO-U) δ 1.75 (s, 1H), 1.85 (s, 1H), 2.1 (t, 2H), 4.3 (t, 1H), 4.6 (d, 1H ), 4.9 (t, 1H), 6.6 (d, 2H), 8.15 (s, 2H), 8.6 (s, 1H).

Comparative Example 32

<img file="ES2395082T3_D0101.tif" />

ES MS (m + H)⁺ 1408,4.

Comparative Example 33

<img file="ES2395082T3_D0102.tif" />

ES MS (mH) '1491.1, (m + H)⁺ 1493.1; NMR¹H (DMSOWD2O) δ 4.15 (q, 1H), 4.6 (d, 1H), 4.9 (t, 1H), 6.6 (d, 2H),

7.25 (s, 1H), 7.4 (d, 1H), 7.9 (d, 1H), 7.95 (d, 2H), 8.15 (d, 2H), 8.6 (s , 1 HOUR).

Comparative Example 34

<img file="ES2395082T3_D0103.tif" />

NMR ¹H (DMSOWD2O) δ 2.1 (t, 2H), 2.75 (q, 2H), 4.3 (t, 1H), 4.65 (d, 1H), 4.9 (t, 1H), 6.6 (d, 2H), 7.9 (d, 1H, 8.0 (d, 2H), 8.2 (t, 2H), 8.6 (s, 1H).

Comparative Example 35

ES 2 395 082 T3

<img file="ES2395082T3_D0104.tif" />

Comparative Example 36

<img file="ES2395082T3_D0105.tif" />

ES MS (m + H)⁺ 1680.4; NMR¹H (DMSOWD2O) δ 0.3 (s, 3H), 0.35 (s, 3H), 1.05 (s, 9H), 2.15 (t, 2H), 4.15 (t, 1H), 4. 5 85) t, 1H), 6.6 (d, 2H), 7.55 (t, 4H), 7.9 (d, 1H), 8.0 (s, 1H), 8.05 ( d, 1H), 8.15 (s, 1H), 8.6 (s, 1H).

Comparative Example 37

<img file="ES2395082T3_D0106.tif" />

<img file="ES2395082T3_D0107.tif" />

<img file="ES2395082T3_D0108.tif" />

HOjC

NMR ¹H (DMSOWD2O) δ (s, 1H), 8.6 (s, 1H).

(s, 3H)

1.8 (s, 1H),

4.55 (d, 1H), 4.8 (t, 1H), 6.6 (d, 2H), 7.8 (d, 1H), 8.1 (d, 1H), 8.15

Comparative Example 38

<img file="ES2395082T3_D0109.tif" />

Comparative Example 39

<img file="ES2395082T3_D0110.tif" />

ES 2 395 082 T3

Comparative Example 40

<img file="ES2395082T3_D0111.tif" />

Comparative Example 41

<img file="ES2395082T3_D0112.tif" />

Comparative Example 42

Inhibition of tumor growth in ec112-treated mice

The antitumor activity of the compounds of Examples 9b (EC111) and 9c (EC 112), in which the drug is daunorubicin, administered to animals bearing tumors intravenously (iv) was evaluated in Balb / c mice with M109 tumors subcutaneous. Four days after tumor inoculation into the subcutaneous tissue of the right armpit with 1 x 10⁶ M109 cells, mice (5 / group) were injected iv twice a week for 4 weeks with 2-10 pmol / kg of the compound of either Example 9b or Example 9c or with unconjugated daunorubicin or PBS. Tumor growth was measured with calipers at 3-day or 4-day intervals in each treatment group. Tumor volumes were calculated using the equation V = axb²/ 2, where a is the length of the tumor and b is the width expressed in millimeters. The body weight of the animal was also determined in 3-day or 4-day intervals.

As shown in Figs. 1 and 2, treatment with the compound of Example 9c was effective in retarding the growth of M109 tumors without apparent toxicity (based on the body weight of the animals). Unconjugated doxorubicin also provided an antitumor response, but with concomitant toxicity based on body weights.

Comparative Example 43

Inhibition of tumor growth in ec105-treated mice

The protocol was as described in Example 42 except that the compound of Example 10a (EC105) in which the drug is bis-indolyl-seco-CBI was used. The compound of Example 10a was injected at a dose of 0.3 pmol / kg. Similarly, two subcutaneous tumor models were tested including model M109 (positive for folate receptor) and model 4T1 (negative for folate receptor), and in some animals a 67-fold excess of folate was injected simultaneously (20 pmmol / kg; FA) together with the conjugate (ie, the compound of Example 10a).

A surprising antitumor response was observed with the compound of Example 10a with no apparent toxicity based on the body weight of the animals (see Figs. 3 and 4). The antitumor response with the compound of Example 10a was blocked with an excess of free folate, demonstrating the specificity of the response (see Fig. 3). As shown in Fig. 5, no antitumor activity was observed in the 4T1 (negative for folate receptor) model, again demonstrating the specificity of the response.

Work Example 44

Inhibition of tumor growth in mice treated with ec145

The antitumor activity of the compound of Example 16b (EC145), whose drug is deacetylvinblastine monohydrazide, administered to animals bearing tumors intravenously (iv) was evaluated in Balb / c mice with subcutaneous M109 tumors. Approximately 11 days after tumor inoculation into the subcutaneous tissue of the right armpit with 1 x 10⁶ M109 cells (mean tumor volume at to = 60 mm³), the mice (5 / group) were injected iv twice a week (BIW), for 3 weeks with 1500 nmol / kg of EC145 or with an equal volume dose of PBS (control). Tumor growth was measured with calipers at 2-day or 3-day intervals in each treatment group. Tumor volumes were calculated using the equation V = axb²/ 2, where a is the length of the tumor and b is the width expressed in millimeters.

As shown in Fig. 6, EC 145 treatment was effective in retarding the growth of M109 tumors compared to the growth of M109 tumors in saline-treated animals.

ES 2 395 082 T3

Comparative Example 45

Inhibition of tumor growth in ec140-treated mice

The antitumor activity of the compound of Example 17a (EC140), the drug of which is deacetylvinblastine monohydrazide, administered to animals bearing tumors intravenously (iv) was evaluated in Balb / c mice with subcutaneous M109 tumors. Approximately 11 days after tumor inoculation into the subcutaneous tissue of the right armpit with 1 x 10⁶ M109 M109 cells (mean tumor volume at to = 60 mm³), the mice (5 / group) were injected iv three times a week (TIW), for 3 weeks with 1500 nmol / kg of EC140 or with an equal volume dose of PBS (control). Tumor growth was measured with calipers at 2-day or 3-day intervals in each treatment group. Tumor volumes were calculated using the equation V = axb²/ 2, where a is the length of the tumor and b is the width expressed in millimeters.

As shown in Fig. 7, EC 140 treatment was effective in retarding the growth of M109 tumors compared to the growth of M109 tumors in saline-treated animals.

Comparative Example 46

Inhibition of tumor growth in ec136-treated mice

The antitumor activity of the compound of Example 10b (EC136), whose drug is CBI, administered to animals bearing tumors intravenously (iv) was evaluated in DBA mice with subcutaneous L1210A tumors. Approximately 5 days after inoculation of 0.25 x 10⁵ L1210A cells in the subcutaneous tissue of the right armpit (mean tumor volume at ~ 50 mm³; 5 mice / group) the animals were injected iv three times a week (TIW), for 3 weeks with 400 nmol / kg of EC136 or with an equal volume dose of PBS (control). Tumor growth was measured with calipers at 2-day or 3-day intervals in each treatment group. Tumor volumes were calculated using the equation V = axb²/ 2, where a is the length of the tumor and b is the width expressed in millimeters.

As shown in Fig. 8, EC136 treatment was effective in retarding the growth of L1210A tumors compared to the growth of L1210A tumors in saline-treated animals.

Comparative and working example 47

Inhibition of cellular DNA synthesis by various drug-conjugates

The compounds of Examples 17b, 10b, 16a, 10c, 17a, 16b, 14e, and 15 (EC135, EC136, EC137, EC138, EC140, EC145, EC158, and EC159, respectively) were evaluated using an in vitro cytotoxicity assay. which predicts the drug's ability to inhibit the growth of folate receptor-positive KB cells. The compounds were comprised of folate bound to the respective chemotherapeutic drug, prepared according to the protocols described in the present specification. KB cells were exposed for up to 7 h at 37 ° C to the indicated concentrations of folate-drug conjugate (see the x-axes of the graphs shown in Figs. 9-16) in the absence or presence of an excess of at least 100 times of folic acid. The cells were then rinsed once with fresh culture medium, and incubated in fresh culture medium for 72 hours at 37 ° C. Cell viability was evaluated by an incorporation assay of³H-thymidine.

As shown in Figs. 9-16, it was possible to measure dose-dependent cytotoxicity and, in most cases, CIs0 values ​​(concentration of drug conjugate necessary to reduce the incorporation of³Hthymidine in the recently synthesized DNA by 50%) were in the low nanomolar range. Furthermore, the cytotoxicities of these conjugates were reduced in the presence of excess free folic acid, indicating that the observed cell death was mediated by binding to the folate receptor.

Similar results were obtained in this type of assay using EC158 and cell lines including IGROV (known cell line), A549-Clone-4 (A549 cells transfected with human folate receptor cDNA), New Line-01 (Line-01 cell mutant selected in vivo by receptor receptor expression), M109, 4T1 Clone-2 (4T1 cells transfected with murine folate receptor cDNA), and HeLa cells.