EA041296B1 - DRUG DELIVERY CONJUGATES AND METHODS FOR TREATMENT OF DISEASES CAUSED BY PSMA-EXPRESSING CELLS - Google Patents

Description

Cross-references to related applications

Pursuant to 35 U.S.C. § 119(e), this application claims priority from U.S. Provisional Application Serial No. 61/726991, filed Nov. 15, 2012, U.S. Provisional Application Serial No. 61/788382, filed March 15, 2013, and U.S. Provisional Application Serial No. 61/875971 filed September 10, 2013, all of which are hereby incorporated by reference in their entirety.

Technical field

The present invention described herein relates to the diagnosis, imaging and/or treatment of pathogenic cell populations. In particular, the present invention relates to the diagnosis, imaging and/or treatment of diseases caused by PSMA expressing cells, such as prostate cancer cells, using compounds capable of targeting PSMA expressing cells.

Background and brief description of the invention

The prostate gland is the male reproductive organ, and its function is to produce and store seminal fluid, which provides nutrients and a liquid medium for the survival of spermatozoa introduced into the vagina during reproduction. Like other tissues, the prostate gland can give rise to either malignant (cancerous) or benign (non-cancerous) tumors. In fact, prostate cancer is one of the most common types of cancer in men in Western society and is the second major form of malignancy among American men. Current treatments for prostate cancer include hormone therapy, radiation therapy, surgery, chemotherapy, photodynamic therapy, and combination therapy. However, many of these types of treatments have a negative impact on the patient's quality of life, especially for men over 50 with a diagnosis of prostate cancer. For example, the use of hormonal drugs is often accompanied by side effects such as osteoporosis and liver damage. Such side effects could be mitigated by the use of types of treatment that are more selective or specific for the tissue responsible for the disease state and avoid non-target tissues such as bone or liver.

Prostate specific membrane antigen (PSMA) is a biomarker that is overexpressed in prostate cancer. PSMA is overexpressed in malignant tissues of the prostate compared to other organs of the human body such as the kidneys, proximal small intestine, and salivary glands. PSMA is also expressed on newly formed vessels in many non-prostate solid tumors, including cancers of the lung, colon, breast, kidney, liver, and pancreas, but not on normal vessels. PSMA is also minimally expressed in the brain. PSMA is a membrane-bound type II cell surface glycoprotein with a molecular weight of ~110 kDa, including an intracellular segment (amino acids 1-18), a transmembrane domain (amino acids 19-43) and an extensive extracellular domain (amino acids 44-750). While the functions of the intracellular segment and the transmembrane domain are currently considered unimportant, the extracellular domain is involved in several different activities. For example, PSMA plays a role in the central nervous system where it metabolizes N-acetyl-aspartylglutamate (NAAG) into glutamic and N-acetylaspartic acid. PSMA also plays a role in the proximal small intestine where it removes γ-linked glutamate from poly-γ-glutamic folate and α-linked glutamate from peptides and small molecules. However, the specific function of PSMA in prostate cancer cells remains unclear.

Unlike many other membrane-bound proteins, PSMA undergoes rapid internalization into the cell in the same way as cell surface-bound receptors such as vitamin receptors. PSMA is internalized through clathrin-lined pits and can then either return to the cell surface or move to lysosomes. Accordingly, diagnostic, imaging, and therapeutic agents can target PSMA for delivery to cells expressing PSMA, such as prostate cancer cells.

This document describes compounds that can bind to PSMA. Also described herein are compounds capable of targeting PSMA to deliver diagnostic, imaging, and therapeutic agents. Also described herein are compounds and compositions, as well as methods and their use for the diagnosis, imaging and treatment of diseases caused by pathogenic cell populations that express or overexpress PSMA.

Surprisingly, the conjugates described herein were found to exhibit high affinity for PSMA. The compounds described herein have also been found to be effective in the treatment of diseases caused by pathogenic cells that express PSMA, such as prostate cancer cells.

In one exemplary embodiment of the present invention, PSMA binding conjugates are described herein for drug delivery of the formula

- 1 041296

BL-(D) _n or their pharmaceutically acceptable salts, where B contains the structure

where L is a polyvalent linker containing 1) C ₁ -C ₄ -alkylene or C ₁ -C ₄ alkylenecarbonyl, substituted with an aryl C1-C ₄ -alkyl group, where aryl is phenyl or 2naphthyl, and 2) C3-C8- cycloalkylene, C ₁ -C ₄ -alkylene-C3-C8-cycloalkyl or C3-C8-cycloalkylenecarbonyl; where L forms urea, thiourea, amide or thioamide with lysine B; where D contains a radioactive metal isotope coordinated into a chelate group; and where n is 1.

In another illustrative embodiment, described herein are PSMA binding conjugates for drug delivery of the formula

B-L-D or a pharmaceutically acceptable salt thereof;

where B contains the structure

where L is a polyvalent linker, where L forms an amide with lysine, and where L contains: 1) C 1 -C ₄ -alkylene or C 1 -C ₄ -alkylenecarbonyl, substituted with an aryl-C 1 -C ₄ -alkyl group, where aryl represents phenyl or 2-naphthyl, and 2) C3-C8-cycloalkylene, _C1 - _C4- alkylene-C3-C8-cycloalkyl or C3-C8-cycloalkylenecarbonyl; and where D contains a radioactive metal isotope coordinated to a chelate group.

It should be understood that each combination of different embodiments of each of the B, L, D and n described herein forms illustrative embodiments of the conjugates of the present invention, whether such different embodiments of each of the B, L, D species, subgenus or kind.

Brief description of graphic materials

In FIG. 1 shows relative affinity () PMPA, 1.0 (normalized); (·) DUPA, 0.05 (19 times lower); (o) EC 1067, 30X; (□) EC1069 22X and (▼) EC1080 6X in 10% serum/FDRPMI against PSMA.

In FIG. 2 shows relative affinity () PMPA, 1.0 (normalized); (·) EC1100, 20X; (▼)EC1168, 17X; (A) EC1169, 7X and (p) EC1170, 7X in 10% serum/FDRPMI against PSMA.

In FIG. 3 shows the dose effect and IC50 for EC1169 on LNCaP cells (2-72 h) as determined by incorporating ³ H-thymidine into cells in vitro.

In FIG. 4 shows the dose and IC50 effect for (▼)EC1718, (♦)EC1677, (A)EC1719, (>)EC1720 and ()EC1721 on LNCaP cells (2-72h) as determined by incorporation ^{of 3} H-thymidine into cells in vitro.

In FIG. 5 shows the in vivo efficacy of EC1169 (c), EC1550 ( ) and EC1551 (), each at 2 µmol/kg, TIW (thrice weekly), 2 weeks, compared to medium treated controls (♦), when treated xenografts of the LNCaP tumor.

In FIG. 6 shows that EC1169 (c), EC1550 (·), and EC1551 (), each at 2 µmol/kg, TIW, 2 weeks, compared to vehicle-treated controls (♦), showed no general toxicity to animals.

In FIG. 7 shows the in vivo efficacy of EC1584 (▼) and EC1588 (A), each at 2 µmol/kg, TIW, 2 weeks, compared to medium-treated controls (·) in treatment of LNCaP tumor xenografts.

In FIG. 8 shows that EC1584 (▼) and EC1588 (A), each at 2 µmol/kg, TIW, 2 weeks, compared to media-treated controls (·), showed no general toxicity to animals.

In FIG. 9 shows in vivo efficacy of EC1169 (·) at 2 µmol/kg, TIW, 2 weeks, versus docetaxel, at 10 mg/kg, BIW, 2 weeks, MTD (▼), and each versus controls, treated with medium (), when processing LNCaP tumor xenografts.

In FIG. 10 shows that EC1169 (·) administered at 2 µmol/kg, TIW, 2 weeks, exhibited much less overall animal toxicity compared to docetaxel administered at 10 mg/kg, BIW, 2 weeks, MTD ( ▼).

In FIG. 11 shows in vivo efficacy of () EC1718; (A) EC1720; (▼) EC1721; (♦) EC1719 and (o)

- 2 041296

EC1677, each administered at 2 µmol/kg, TIW, 2 weeks; compared to media-treated controls (·) when treated with LNCaP tumor xenografts.

In FIG. 12 shows that () EC1718; (G) EC 1720; (▼) EC1721; (♦) EC1719 and (o) EC1677; compared to medium-treated controls (·), showed no general toxicity to animals.

Detailed description

Some illustrative embodiments of the present invention are described in the following numbered paragraphs.

1. Formula conjugate

B-L-(D)n or a pharmaceutically acceptable salt thereof;

where B contains the structure

where L is a polyvalent linker containing 1) C ₁ -C ₄ alkylene or C 1 -C 4 alkylene carbonyl substituted with an aryl C 1 -C ₄ alkyl group, where aryl is phenyl or 2 naphthyl, and

2) C ₃ -C ₈ -cycloalkylene, C1 -C ₄ -alkylene-C ₃ -C ₈ -cycloalkyl or C ₃ -C ₈ -cycloalkylenecarbonyl;

where L forms urea, thiourea, amide or thioamide with lysine B;

where D contains a radioactive metal isotope coordinated into a chelate group; and where n is 1.

2. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein B is selected from the group consisting of:

from:

3. The conjugate according to claim 1 or

a pharmaceutically acceptable salt, wherein B is selected from the group consisting of

from:

4. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein B is selected from the group consisting of

- 3 041296

5. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein L forms an amide with lysine.

6. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein L contains a chain of at least 7 atoms.

7. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein L contains a chain of at least 8 atoms.

8. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein L contains a chain of at least 9 atoms.

9. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein L contains a chain of at least 10 atoms.

10. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the linker contains C1-C4 alkylene carbonyl.

11. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the aryl is 2naphthyl.

12. A conjugate according to claim 1, or a pharmaceutically acceptable salt thereof, wherein B-L contains a biradical of the formula

13. Formula conjugate

or a pharmaceutically acceptable salt thereof; where B contains the structure where L is a polyvalent linker, where L forms an amide with lysine, and where L contains:

1) C ₁ -C ₄ -alkylene or C ₁ -C ₄ -alkylenecarbonyl, substituted with aryl-C ₁ -C ₄ -alkyl group, where aryl is phenyl or 2-naphthyl, and

2) C ₃ -C ₈ -cycloalkylene, C ₁ -C ₄ -alkylene-C ₃ -C ₈ -cycloalkyl or C ₃ -C ₈ -cycloalkylenecarbonyl; and where D contains a radioactive metal isotope coordinated to a chelate group.

14. The conjugate of claim 13, or a pharmaceutically acceptable salt thereof, wherein the aryl is 2naphthyl.

15. The conjugate of claim 13, or a pharmaceutically acceptable salt thereof, wherein B is selected from the group consisting of:

- 4 041296

16. The conjugate of claim 13, or a pharmaceutically acceptable salt thereof, wherein B is selected from the group consisting of:

17. The conjugate of claim 13, or a pharmaceutically acceptable salt thereof, wherein B is selected from the group consisting of:

In listing the above and following sets of embodiments and claims, it is to be understood that all possible combinations of features and all possible subgenera and subcombinations are described. For example, it should be understood that if B is limited to a linking ligand containing the urea of L-lysine and L-glutamate, L may be limited to a linker containing one or more aspartic acid biradicals, or alternatively containing a cysteine biradical, or alternatively containing L- Asp-L-Asp-L-Cys(S-S), etc. Similarly, if B is limited to a linking ligand containing urea or thiourea of lysine and glutamate or one or more carboxylic acid derivatives thereof, L may be limited to a linker containing a diradical of the formula O-C(O)-N. Other combinations, sub-genera and sub-combinations are also described in the paragraphs.

A drug is a radioactive isotope of a metal coordinated into a chelate group. Exemplary radioactive metal isotopes include technetium, rhenium, gallium, gadolinium, indium, copper, and the like, including the isotopes ¹¹¹ In, ^99m Tc, ⁶⁴ Cu, ⁶⁷ Cu, ⁶⁷ Ga, ⁶⁸ Ga, and the like. Additional illustrative examples of radionuclide imaging tools are described in US patent No. 7128893, the disclosure of which is incorporated herein by reference. Additional illustrative chelate groups are tripeptide or tetrapeptides, including, without limitation, tripeptides with the formula

- 5 041296

where R is at each occurrence independently selected from H, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, alkenyl, alkynyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, and the like, each of which is optionally substituted. It should be understood that one R includes a heteroatom such as nitrogen, oxygen, or sulfur, and is the point of attachment of the linker L. By way of illustration, the following chelate groups are described:

where X is oxygen, nitrogen or sulfur, and where X is attached to the linker L, and n is an integer from 1 to about 5.

As used herein, the term linker includes a chain of atoms that connects two or more functional moieties to form a conjugate. By way of illustration, the atomic chain is selected from C, N, O, S, Si, and P, or C, N, O, S, and P, or C, N, O, and S. The atomic chain covalently links the various functionalities of the conjugate, such as binding ligands, drugs, diagnostics, imaging agents, and the like. The linker can be of various lengths, for example, in the range from about 2 to about 100 atoms in a continuous backbone. The atoms used in the formation of the linker can be combined in all chemically relevant ways, such as chains of carbon atoms forming alkylene, alkenylene and alkynylene groups, and the like; chains of carbon and oxygen atoms forming ethers, polyoxyalkylene groups, or when combined with carbonyl groups forming esters and carbonates, etc.; chains of carbon and nitrogen atoms forming amines, imines, polyamines, hydrazines, hydrazones, or when combined with carbonyl groups, forming amides, ureas, semicarbazides, carbazides, and the like; chains of carbon, nitrogen, and oxygen atoms forming alkoxyamines, alkoxylamines or, when combined with carbonyl groups, forming urethanes, amino acids, acyloxylamines, hydroxamic acids, and the like; as well as many others. Furthermore, it should be understood that the chain-forming atoms in each of the above exemplary embodiments may be either saturated or unsaturated, thus forming single, double or triple bonds, such that, for example, the radicals that are included in the linker, may be alkanes, alkenes, alkynes, imines, and the like. In addition, it should be understood that the atoms forming the linker may also cyclize with each other or may be part of a cyclic structure to form bivalent ring structures that form the linker, including cycloalkanes, cyclic ethers, cyclic amines and other heterocycles, arylenes , heteroarylenes, etc. in the linker. In the case of the latter arrangement, it should be understood that the length of the linker can be determined along any path through one or more ring structures. By way of illustration, the linker length is determined by the shortest path through each of the ring structures. It should be understood that linkers may optionally be substituted at any one or more of the open valences along the atomic chain, for example, with optional substituents on any of the carbon, nitrogen, silicon or phosphorus atoms. It should also be understood that a linker can connect two functional moieties to form a conjugate at any open valency, and it is not necessary that either of the two functional moieties forming a conjugate be attached at any apparent end of the linker.

In another embodiment, the linker (L) contains a radical of the formula

where m1, m2, m3, n, p, q, and r are integers each independently selected from the range of 0 to about 8, provided that at least one of m1, m2, m3, n, p , q and r is not equal to 0; AA is an amino acid; and medicines optional

- 6 041296 dynes to one or more of the (*) atoms. It should be understood that drugs can be attached directly or attached through additional parts of the linker (L). In another embodiment, AA is a naturally occurring amino acid in either natural or non-natural configuration. In another embodiment, one or more AAs are hydrophilic amino acids. In another embodiment, one or more AAs are Asp and/or Arg. In another embodiment, the integer n is 1 or greater. In another embodiment, the integer n is 2 or greater. In another embodiment, the integer n is 3 or greater. In another embodiment, the integer n is 4 or greater. In another embodiment, the integer n is 5 or greater. In another aspect, the integer q is 1 or greater. In another embodiment, the integer m1 is 1 or greater. In another embodiment, the integer m1 is 1. In another embodiment, the integer m2 is 1 or greater. In another embodiment, the integer m2 is 1. In another embodiment, the integer m3 is 1 or greater. In another embodiment, the integer m3 is 1. In another embodiment, the integer p is 1 or greater. In another embodiment, integer p is 1. In another embodiment, integer p is 2. In another embodiment, integer q is 1 or greater. In another embodiment, the integer q is 1. In another embodiment, the integer q is 2. In another embodiment, the integer r is 1 or greater. In another embodiment, the integer r is 1. In another embodiment, the integer r is 2.

It should be understood that all combinations of the above embodiments are described herein. For example, in another embodiment, n is 1 or greater and m1 is one or greater; or n is 1 or greater, m1 is 1, and q is 1, and so on. For example, in another embodiment, n is 1 or greater and m2 is one or greater; or n is 2 or greater, m2 is 1, and q is 1; or n is 2 or greater, m3 is 1, q is 1, and p is 1, and so on. For example, in another embodiment, n is 1 or greater and m1 is one or greater; or n is 2 or greater, m3 is 1, and q is 1; or n is 2 or greater, m2 is 1, q is 1, and p is 1; or n is 2 or greater, m1 is 1, q is 1, and r is 1; or n is 2 or greater, m3 is 1, q is 1, p is 1, and r is 1, and so on.

In another embodiment, the polyvalent linker includes one or more divalent hydrophilic radicals as described herein, which may also be referred to as spacer linkers. It should be noted that the arrangement and/or orientation of the various hydrophilic linkers may be linear or branched, or both. For example, hydrophilic linkers can form the backbone of a linker that forms a conjugate between a ligand and one or more drugs. Alternatively, the hydrophilic portion of the linker may be lateral to, or attached to, a backbone of a chain of atoms connecting binding ligand B to one or more drugs D. In the case of the latter location, the hydrophilic portion may be proximal or distal to the skeletal chains of atoms.

In another embodiment, the linker is typically linear and the hydrophilic groups are typically aligned to form a chain-like linker in the conjugate. In other words, the hydrophilic groups form some or all of the backbone of the linker in such an embodiment of the linear linker.

In another embodiment, the linker is branched with hydrophilic groups. In such a branched linker embodiment, the hydrophilic groups may be proximal to the skeleton or distal to the skeleton. In each of these arrangements, the linker is typically more spherical or cylindrical in shape. In another embodiment, the linker is shaped like a bottle brush. In another embodiment, the backbone of the linker is formed by linear rows of amides, and the hydrophilic portion of the linker is formed by a parallel arrangement of branching side chains, such as when linking monosaccharides, sulfonates, and the like, as well as their derivatives and analogs.

It should be understood that the linker (L) may be neutral or ionizable under certain conditions, such as the physiological conditions encountered in vivo. For ionizable linkers, under the chosen conditions, the linker can deprotonate to form a negative ion, or alternatively become protonated to form a positive ion. It should be noted that more than one deprotonation or protonation event may occur. In addition, it should be understood that the same linker can be deprotonated and protonated to form internal salts or zwitterionic compounds.

In another embodiment, the hydrophilic spacer linkers are neutral, and in particular neutral under physiological conditions, wherein the linkers are not significantly protonated or deprotonated. In another embodiment, the hydrophilic spray

- 7 041296 sulfur linkers can be protonated with one or more positive charges. It should be understood that the ability to protonate depends on the conditions. In one aspect, the conditions are physiological conditions and the linker is protonated in vivo. In another embodiment, the spacers include both regions that are neutral and regions that can be protonated with one or more positive charges. In another embodiment, the spacers include both regions that can be deprotonated with one or more negative charges and regions that can be protonated with one or more positive charges. It should be understood that in this latter embodiment, zwitterions or internal salts may be formed.

In another embodiment, the regions of the linkers that can be deprotonated with a negative charge include carboxylic acids such as aspartic acid, glutamic acid and longer chain carboxylic acid groups, as well as sulfuric acid esters such as sulfuric acid alkyl esters. . In another embodiment, the linker regions that can be positively protonated include amino groups, such as polyaminoalkylenes, including ethylenediamines, propylenediamines, butylenediamines, and the like, and/or heterocycles, including pyrrolidines, piperidines, piperazines. , and other amino groups, each of which is optionally substituted. In another embodiment, the regions of the linkers that are neutral include polyhydroxyl groups such as sugars, carbohydrates, saccharides, inositols, and the like, and/or polyester groups, such as polyoxyalkylene groups, including polyoxyethylene, polyoxypropylene, and the like. P.

In another embodiment, the hydrophilic spacer linkers described herein include those formed primarily from carbon, hydrogen, and oxygen and having a carbon to oxygen ratio of about 3:1 or less, or about 2:1 or less. In another embodiment, the hydrophilic linkers described herein include a plurality of ether functional groups. In another embodiment, the hydrophilic linkers described herein include multiple hydroxyl functional groups. Illustrative moieties and radicals that can be used to form such linkers include polyhydroxyl compounds such as carbohydrates, polyester compounds such as polyethylene glycol units, and acidic groups such as carboxylic and alkylsulfuric acids. In one variation, oligoamide spacers and the like can also be included in the linker.

Exemplary bivalent hydrophilic linkers include carbohydrates, such as sugar peptides as described herein, that are characterized by both peptide and sugar features; glucuronides, which can be inserted via [2+3] Huisgen cyclization, also known as click chemistry; β-alkyl glycosides such as 2-deoxyhexapyranoses (2-deoxyglucose, 2-deoxyglucuronide, etc.) and β-alkylmannopyranosides. Exemplary PEG groups include defined lengths ranging from about 4 to about 20 PEG groups. Exemplary sulfuric acid alkyl esters can also be introduced by click chemistry directly into the skeleton. Exemplary oligoamide spacers include EDTA and DTPA spacers, β-amino acids, and the like.

In another embodiment, the polyvalent linker L contains one or more polyesters, such as linkers of the following formulas:

where m in each case is independently selected from an integer from 1 to about 8; p is an integer selected from 1 to about 10; and n in each occurrence is independently selected from an integer from 1 to about 3. In one aspect, m in each occurrence is independently 1 to about 3. In another aspect, n in each occurrence is 1. In another aspect, p is independently in each occurrence from 4 to about 6. By way of illustration, polypropylene polyesters corresponding to the above are covered by this document and can be included in conjugates as hydrophilic spacer linkers. In addition, it should be noted that mixed polyethylene and polypropylene polyesters can be included in the conjugates as hydrophilic spacer linkers. In addition, cyclic variations of the aforementioned polyester compounds, such as those comprising tetrahydrofuranyl, 1,3-dioxanes, 1,4-dioxanes, and the like, span

- 8 041296 are subject to this document.

In another embodiment, the polyvalent linker L contains a plurality of hydroxyl functional groups, such as linkers, which include monosaccharides, oligosaccharides, polysaccharides, and the like. It should be understood that spacer linkers containing polyhydroxyl contain a plurality of -(CROH)- groups, where R is hydrogen or alkyl.

In another embodiment, the multivalent linker L contains one or more of the following moieties:

where R is H, alkyl, cycloalkyl or arylalkyl; m is an integer from 1 to about 3; n is an integer from 1 to about 5 or 2 to about 5, p is an integer from 1 to about 5, and r is selected from an integer from 1 to about 3. In one aspect, integer n is 3 or 4 In another aspect, the integer p is 3 or 4. In another aspect, the integer r is 1.

In another embodiment, the multivalent linker L contains one or more of the following moieties:

where R is H, alkyl, cycloalkyl or arylalkyl; m is an integer from 1 to about 3; n is an integer from 1 to about 5 or 2 to about 5, p is an integer from 1 to about 5, and r is selected from an integer from 1 to about 3. In one aspect, integer n is 3 or 4 In another aspect, the integer p is 3 or 4. In another aspect, the integer r is 1.

In another embodiment, the multivalent linker L contains one or more of the following cyclic polyhydroxyl groups:

- 9 041296

where n is an integer from 2 to about 5, p is an integer from 1 to about 5, and r is an integer from 1 to about 4. In one aspect, the integer n is 3 or 4. In another aspect, the integer the p number is 3 or 4. In another aspect, the integer r is 2 or 3. It is to be understood that all stereochemical forms of such linker sections are encompassed herein. For example, in the above formulas, the section may be derived from ribose, xylose, glucose, mannose, galactose, or another sugar and retains the stereochemical arrangements of the pendant hydroxyl and alkyl groups present in these molecules. In addition, it should be understood that the above formulas also cover various deoxy compounds. By way of illustration, compounds of the following formulas are covered:

where n is equal to or less than r, for example, if r is 2 or 3, n is 1 or 2, or 1, 2, or 3, respectively.

In another embodiment, the polyvalent linker L contains one or more polyhydroxyl radicals of the following formula:

where each n and r is selected from an integer from 1 to about 3. In one aspect, the linker includes one or more polyhydroxy compounds of the following formulas:

It should be understood that all stereochemical forms of such linker sections are covered by this document. For example, in the above formulas, a section can be derived from ribose, xylose, glucose, mannose, galactose, or another sugar and retains the stereochemical arrangements

- 10 041296 side hydroxyl and alkyl groups present in these molecules.

In another embodiment, the polyvalent linker L contains one or more polyhydroxyl groups that are located at a certain distance from the backbone of the linker. In one embodiment, such carbohydrate groups or polyhydroxyl groups are coupled to the backbone via a triazole group to form triazole-linked hydrophilic spacer linkers. By way of illustration, the linker includes fragments of the following formulas:

where n, m, and r are each independently selected from an integer from 1 to about 5. In one illustrative aspect, m is each independently 2 or 3. In another aspect, r is each 1. In another aspect, n is each case is 1. In one variation, the group connecting the polyhydroxyl group to the linker backbone is a different heteroaryl group, including, but not limited to, pyrrole, pyrazole, 1,2,4-triazole, furan, oxazole, isoxazole, thienyl, thiazole , isothiazole, oxadiazole, etc. Similarly, bivalent 6-membered ring heteroaryl groups are covered. Other variations on the above exemplary hydrophilic spacer linkers include oxyalkylene groups, for example, with the following formulas:

where n and r are each independently selected from an integer from 1 to about 5; and p is selected from an integer from 1 to about 4.

In another embodiment, the multivalent linker L contains one or more carbohydrate groups or polyhydroxyl groups linked to the backbone via an amide group to form amide-linked hydrophilic spacer linkers. By way of illustration, such linkers include fragments of the following formulas:

where n is selected from an integer from 1 to about 3, and m is selected from an integer from 1 to about 22. In one illustrative aspect, n is 1 or 2. In another illustrative aspect, m is selected from from about 6 to about 10, in as illustration 8. In one variation, the group connecting the polyhydroxyl group to the linker backbone is a different functional group, including, without limitation, esters, ureas, carbamates, acylhydrazones, and the like. Similarly, cyclic variations are covered. Other variations on the above exemplary hydrophilic spacer linkers include oxyalkylene groups, for example, with the following formulas:

- 11 041296

where n and r are each independently selected from an integer from 1 to about 5; and p is selected from an integer from 1 to about 4.

In another embodiment, the multivalent linker L contains one or more of the following moieties:

where R is H, alkyl, cycloalkyl or arylalkyl; m is independently selected from an integer from 1 to about 3; n is an integer from 1 to about 6, p is

- 12 041296 is an integer from 1 to about 5, and r is selected from an integer from 1 to about 3. In one variation, the integer n is 3 or 4. In another variation, the integer p is 3 or 4. In another variation the integer r is 1.

In another embodiment, the multivalent linker L contains one or more of the following moieties:

where R is H, alkyl, cycloalkyl or arylalkyl; m is independently selected from an integer from 1 to about 3; n is an integer from 2 to about 6, p is an integer from 1 to about 5, and r is selected from an integer from 1 to about 3. In one variation, the integer n is 3 or 4. In another variation, the integer r is 3 or 4. In another variation, the integer r is 1.

In another embodiment, the multivalent linker L contains one or more of the following moieties:

where m is independently selected from an integer from 1 to about 3; n is an integer from 1 to about 6, p is an integer from 1 to about 5, and r is selected from an integer from 1 to about 3. In one variation, the integer n is 3 or 4. In another variation, the integer r is 3 or 4. In another variation, the integer r is 1.

In another embodiment, the multivalent linker L contains one or more of the following moieties:

where m is independently selected from an integer from 1 to about 3; n is an integer

- 13 041296 a number from 2 to about 6, p is an integer from 1 to about 5, and r is selected from an integer from 1 to about 3. In one variation, the integer n is 3 or 4. In another variation, the integer p is 3 or 4. In another variation, the integer r is 1.

In another embodiment, the multivalent linker L contains one or more of the following moieties:

he he he where m is independently selected from an integer from 1 to about 3; n is an integer from 1 to about 6, p is an integer from 1 to about 5, and r is selected from an integer from 1 to about 3. In one variation, the integer n is 3 or 4. In another variation, the integer r is 3 or 4. In another variation, the integer r is 1.

In another embodiment, the multivalent linker L contains a combination of a backbone and branching lateral motifs, such as those illustrated by the following formulae:

where n is in each case independently selected from an integer from 0 to about 3. The above formulas are intended to represent cyclic sugars with 4, 5, 6 or even more members. In addition, it should be understood that the above formulas can be modified to typical deoxysugars, where one or more of the hydroxy groups represented in the formulas are replaced by hydrogen, alkyl or amino. In addition, it should be understood that the corresponding carbonyl compounds are covered by the above formulas, where one or more of the hydroxyl groups are oxidized to the corresponding carbonyl. In addition, in this exemplary embodiment, the pyranose includes both a carboxyl and amino functionality, and (a) can be inserted into the skeleton, and (b) can provide a synthetic backbone for the branching side chains in variations of this embodiment. Any of the side hydroxyl groups can be used to attach other chemical moieties, including additional sugars, to form the corresponding oligosaccharides. Other variations of this embodiment are also covered, including the insertion of pyranose or other sugar into the skeleton at a single carbon, i.e. spiro-arrangement, on the geminal pair of carbons and similar arrangements. For example, one or two ends of the linker, or drug D, or binding ligand B can be connected to the sugar to be inserted into the backbone in 1.1; 1.2; 1.3; 1.4; 2.3 or other location.

In another embodiment, the hydrophilic spacer linkers described herein include those formed primarily from carbon, hydrogen, and nitrogen and having a carbon to nitrogen ratio of about 3:1 or less, or about 2:1 or less. In one aspect, the hydrophilic linkers described herein include a plurality of amino functional groups.

In another embodiment, the polyvalent linker L contains one or more amino groups of the following formulas:

where n is in each case independently selected from an integer from 1 to about 3. In one aspect, the integer n in each case is independently 1 or 2. In another aspect, the integer n in each case is 1.

In another embodiment, the polyvalent linker L contains one or more sulfuric acid esters, such as a sulfuric acid alkyl ester. By way of illustration, the linker is characterized by the following formula(s):

where n in each case is independently selected from an integer from 1 to about 3. By way of illustration, n in each case is independently 1 or 2.

It should be understood that in such polyhydroxyl, polyamine carboxylic acid, sulfuric acid, and the like linkers that include free hydrogen atoms bonded to heteroatoms, one or more of such free hydrogen atoms may be protected by an appropriate hydroxyl, amino, or acid protecting group, respectively, or , alternatively, can be blocked as appropriate prodrugs, the latter of which are selected for a particular application, such as prodrugs that release the parent drug under normal or specific physiological conditions.

In another embodiment, the polyvalent linker contains one or more of the following bivalent radicals:

- 15 041296

where n is an integer from 2 to about 5, p is an integer from 1 to about 5, and r is an integer from 1 to about 4, as described above.

In addition, it should be understood that in the above embodiments, open positions such as (*) atoms are locations for attachment of the binding ligand (B) or any drug (D) to be delivered. In addition, it should be understood that such attachment of one or both B and any D may be direct or may be through an intermediate linker containing one or more of the radicals described herein. In addition, the (*) atoms can form releasable linkers with any drug D or other parts of the linker L.

In another embodiment, the hydrophilic spacer linker contains one or more carbohydrate-containing or polyhydroxy group-containing linkers. In another embodiment, the hydrophilic spacer linker contains at least three carbohydrate-containing or polyhydroxyl group-containing linkers. In another embodiment, the hydrophilic spacer linker contains one or more carbohydrate-containing or polyhydroxy group-containing linkers and one or more aspartic acids. In another embodiment, the hydrophilic spacer linker contains one or more carbohydrate-containing or polyhydroxy group-containing linkers and one or more glutamic acids. In another embodiment, the hydrophilic spacer linker contains one or more carbohydrate-containing or polyhydroxy group-containing linkers, one or more glutamic acids, one or more aspartic acids, and one or more beta-aminoalanines. In a number of variations, in each of the above embodiments, the hydrophilic spacer linker also includes one or more cysteines. In other variations, in each of the above embodiments, the hydrophilic spacer linker also includes at least one arginine.

In another embodiment, the polyvalent linker L includes a hydrophilic spacer linker containing one or more bivalent 1,4-piperazines that are included in a chain of atoms connecting at least one binding ligand (L) to at least one drug (D). In one variation, the hydrophilic spacer linker includes one or more carbohydrate-containing or polyhydroxy group-containing linkers. In another variation, the hydrophilic spacer linker includes one or more carbohydrate-containing or polyhydroxy group-containing linkers and one or more aspartic acids. In another variation, the hydrophilic spacer linker includes one or more carbohydrate-containing or polyhydroxy group-containing linkers and one or more glutamic acids. In a number of variations, in each of the above embodiments, the hydrophilic spacer linker also includes one or more cysteines. In other variations, in each of the above embodiments, the hydrophilic spacer linker also includes at least one arginine.

In another embodiment, the hydrophilic spacer linker contains one or more oligoamide hydrophilic spacers such as, but not limited to, aminoethylpiperazinylacetamide.

In another embodiment, the polyvalent linker L comprises a hydrophilic spacer linker containing one or more triazole-linked carbohydrate-containing or polyhydroxy group-containing linkers. In another embodiment, the hydrophilic spacer linker contains one or more amide-linked carbohydrate-containing or polyhydroxy group-containing linkers. In another embodiment, the hydrophilic spacer linker contains one or more PEG groups and one or more cysteines. In another embodiment, the hydrophilic spacer linker contains one or more EDTE derivatives.

In another embodiment, the polyvalent linker L comprises a bivalent radical of the formula

- 16 041296

where * denotes the point of attachment to folate, and ** denotes the point of attachment to the drug; and each F and G is independently 1, 2, 3, or 4, as described.

In another embodiment, the polyvalent linker L comprises a trivalent radical of the formula

where *, **, *** each denotes the point of attachment to the folate receptor binding moiety B and one or more drugs D. It will be understood that if there are fewer drugs, *, **, *** are replaced by a hydrogen or a heteroatom . Each F and G is independently 1, 2, 3, or 4; a W ¹ is NH or O as described. In another aspect, m1 is 0 or 1.

In any of the embodiments described herein, heteroatomic linkers can also be included in the polyvalent linker L, such as -NR1R2-, oxygen, sulfur, and the formulas -(NHR1NHR2)-, -SO-, -(SO2)- and -N (R3)O-, wherein R1, R2 and R3 are each independently selected from hydrogen, alkyl, aryl, arylalkyl, substituted aryl, substituted arylalkyl, heteroaryl, substituted heteroaryl, and alkoxyalkyl. It should be understood that heteroatomic linkers can be used to covalently attach any of the radicals described herein, including drug D radicals, to a polyvalent linker, ligand B radicals to a polyvalent linker, or various bi- and polyvalent radicals that form a polyvalent linker. L.

Illustrative additional bivalent radicals that can be used to form linker moieties are as follows.

- 17 041296

BUT ₂ C _about 1 l CO ₂ H * --- o η ₂ ν^,νη ΗΝ Γ ο CO ₂ H HO ₂ C. Ί* o CO ₂ H A ly. , II o O /^N^Y^OR V-^, OR * o R= R=H, alkyl, acyl .SH * g about HO ₂ C Ν y * but ₂ A < ^ΝΗ * il * o 0 ζ°γΎ *yOR OR Er = n, alkyl, acyl O II ⁿ A. / CO ₂ H S * .l° Q-. ABOUT nh ₂ Ύ o CO ₂ H * ^S * CO ₂ H \u003d ^{: <} Au * • ^s Y η”. O NO ₂ C Ν γ* J /ΝΗ NO ₂ SG < * * O NO ₂ C Ν ho ₂ c ^J < ^ΝΗ * ^N g 0 O II ⁿ A. / CO ₂ H * h ° 1 ^Ν * CO ₂ H * O O / O,, O.A _k zγΌΡ *N 1 OR R - H, alkyl, acyl o VA* AnY^or /А θ' * N R = H, alkyl, acyl O _n * ^N \ / ¹ % / CO ₂ H VT S * Xo BUT ₂ C o * \ ^N * HO ₂ C _o 1 l *N CO ₂ H O CO ₂ H o BUT ₂ C-o HO ₂ C. * N^* o DHg V-i OR * ABOUT R=R = H, alkyl, acyl CO ₂ H Ι'^Ιχ^Ν* O Η ₂ Ν^ΝΗ ΗΝ *\^Ν* O H ₂ N^NH ΗΝ. * Ν ||* 0 CO ₂ H γ o νη ₂ γ o ,νη ₂ * ^Ν fl Ο CO ₂ H * Ν^^ίι* Oh ,SH * ABOUT .SH Ν Α* * II ο υ°Α ^ν * Y^OR OR R= R = H, alkyl, acyl CO ₂ H A.,s* *N O η = 0-3 ο ο η = 0-3 * ° About About η = 1-3 *°o V> I Ν-Ηίη ABOUT n = 1-3

- 18 041296

In another embodiment, the polyvalent linker L is a releasable linker.

As used herein, the term releasable linker refers to a linker that includes at least one bond that can be broken under physiological conditions if the compounds described herein are delivered to or within a target cell. The linker itself may include one or more cleavable, cleavable or erodible bonds or form one or more cleavable, cleavable or erodible bonds with the PSMA binding ligand (B) and/or with one or more drugs (D). However, it should be appreciated that the releasable linkers described herein are advantageously not cleaved, cut or destroyed until the conjugate containing the releasable linker is at or near the intended target site. Accordingly, the releasable linkers described herein generally do not include those linkers that have bonds that are substantially cleavable, cleavable, or degradable under non-target conditions or in non-target tissues. Likewise, the releasable linkers described herein do not include those linkers that include bonds that are substantially cleavable, cleavable, or degradable only under non-physiological conditions.

The term releasable linker does not generally refer simply to a bond that is labile in vivo, such as in serum, plasma, gastrointestinal tract or liver, unless these systems are the target of a cell surface receptor binding ligand. However, after delivery and/or selective targeting, the released linkers can be cleaved by any process that involves the destruction of at least one bond in the linker or in the covalent attachment of the linker to B or any D under physiological conditions, for example, in the presence of one or more pH labile, acid labile, base labile, oxidation labile, metabolically labile, biochemically labile and/or enzymatically labile bonds. It should be noted that such physiological conditions leading to bond disruption do not necessarily involve a biological or metabolic process, but may instead

- 19 041296 include a standard chemical reaction, such as a hydrolysis reaction, for example, at physiological pH, or be the result of compartmentalization in a cell organelle, such as an endosome, having a lower pH than that in the cytosol.

It should be understood that a cleavable bond can connect two adjacent atoms within a releasable linker and/or connect other linkers with B and/or any D, as described herein, at either end of the releasable linker. In the event that a cleavable bond joins two adjacent atoms within a released linker, upon breaking the bond, the released linker breaks down into two or more fragments. Alternatively, in the event that a cleavable bond is located between a releasable linker and another moiety, such as an additional heteroatom, a spacer linker, another releasable portion of the linker, either D or B, upon cleavage of the bond, the releasable linker is separated from the other moiety. It should be understood that a linker is a releasable linker if it forms a cleavable, cleavable, or destructible bond with one or more of the drugs (D) and is capable of delivering the drug (D) in a traceless manner, and the drug (D) does not include any -or the remainder of the conjugate.

Exemplary radicals themselves include a cleavable bond or form a cleavable bond with B and/or any D, hemiacetals and their sulfur variations, acetals and their sulfur variations, hemiaminals, aminals, and the like. or may be formed from methylene moieties substituted with at least one heteroatom, such as 1-alkoxyalkylene, 1alkoxycycloalkylene, 1-alkoxyalkylenecarbonyl, 1-alkoxycycloalkylenecarbonyl, and the like. Exemplary releasable linkers described herein include polyvalent linkers which include carbonylarylcarbonyl, carbonyl(carboxyaryl)carbonyl, carbonyl(biscarboxyaryl)carbonyl, haloalkylenecarbonyl, and the like. Exemplary releasable linkers described herein include polyvalent linkers which include alkylene(dialkylsilyl), alkylene(alkylarylsilyl), alkylene(diarylsilyl), (dialkylsilyl)aryl, (alkylarylsilyl)aryl, (diarylsilyl)aryl, and the like. Exemplary releasable linkers described herein include hydroxycarbonyloxy, oxycarbonyloxyalkyl, sulfonyloxy, oxysulfonylalkyl, and the like. Exemplary releasable linkers described herein include polyvalent linkers which include iminoalkylidenyl, carbonylalkylideniminyl, iminocycloalkylidenyl, carbonylcycloalkylideniminyl, and the like. Exemplary releasable linkers described herein include polyvalent linkers which include alkylenethio, alkylenearylthio, and carbonylalkylthio, and the like. Each of the above moieties is optionally substituted with an X2 substituent as defined herein.

Substituents X2 may be alkyl, alkoxy, alkoxyalkyl, hydroxy, hydroxyalkyl, amino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, halogen, haloalkyl, sulfhydrylalkyl, alkylthioalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, carboxy, carboxyalkyl, alkylcarboxylate , alkylalkanoate, guanidinealkyl, R4-carbonyl, R5carbonylalkyl, R6-acylamino, and R7-acylaminoalkyl, wherein R4 and R5 are each independently selected from amino acids, amino acid derivatives, and peptides, and wherein R6 and R7 are each independently selected from amino acids, amino acid derivatives, and peptides. In this embodiment, the heteroatom linker can be nitrogen, and the X2 substituent and the heteroatom linker, taken together with the releasable linker to which they are attached, can form a heterocycle.

The heterocycles may be pyrrolidines, piperidines, oxazolidines, isoxazolidines, thiazolidines, isothiazolidines, pyrrolidinones, piperidinones, oxazolidinones, isoxazolidinones, thiazolidinones, isothiazolidinones and succinimides.

Exemplary releasable linkers include ketals, acetals, hemiaminals, and aminals formed from methylene, 1-alkoxyalkylene, 1-alkoxycycloalkylene, 1-alkoxyalkylenecarbonyl, and 1-alkoxycycloalkylenecarbonyl radicals, esters and amides formed from carbonylarylcarbonyl, carbonyl(carboxyaryl)carbonyl, carbonyl(carboxyaryl)carbonyl( )carbonyl and haloalkylenecarbonyl, oxysilanes and aminosilanes formed from alkylene(dialkylsilyl), alkylene(alkylarylsilyl), alkylene(diarylsilyl), (dialkylsilyl)aryl, (alkylarylsilyl)aryl and (diarylsilyl)aryl radicals, oxycarbonyloxy, oxycarbonyloxyalkyl, sulfonyloxy, oxysulfonylalkyl radicals , iminoalkylidenyl, carbonylalkylideniminyl, iminocycloalkylidenyl, carbonylcycloalkylideniminyl, alkylenethio, alkylenearylthio, and carbonylalkylthio, each of which is optionally substituted.

Additional exemplary releasable linkers include hydrazones, acylhydrazone orthoformates, and carbamoyl derivatives.

Additional exemplary releasable linkers include disulfides and activated thioethers.

In any of the embodiments described herein, the release linker may include an oxygen atom bonded to methylene, 1-alkoxyalkylene, 1-alkoxycycloalkylene, 1-alkoxyalkylenecarbonyl, and 1-alkoxycycloalkylenecarbonyl with form

- 20 041296 acetal or ketal, where each of the fragments is optionally substituted by substituent X2, as defined herein. Alternatively, the methylene or alkylene is substituted with an optionally substituted aryl.

In any of the embodiments described herein, the release linker may include a nitrogen atom bonded to methylene, 1-alkoxyalkylene, 1-alkoxycycloalkylene, 1-alkoxyalkylenecarbonyl, and 1-alkoxycycloalkylenecarbonyl to form a hemiaminal ester or aminal, where each of the moieties is optionally substituted with an X2 substituent as defined in this document. Alternatively, the methylene or alkylene is substituted with an optionally substituted aryl.

In any of the embodiments described herein, the released linker may include an oxygen atom bonded to a sulfonylalkyl to form an alkylsulfonate.

In any of the embodiments described herein, the release linker may include a nitrogen atom bonded to iminoalkylidenyl, carbonylalkylideniminyl, iminocycloalkylidenyl, and carbonylcycloalkylideniminyl to form a hydrazone, each of which is optionally substituted with an X2 substituent as defined herein. In an alternative configuration, the hydrazone can be acylated with a carboxylic acid derivative, an orthoformate derivative, or a carbamoyl derivative to form releasable linkers containing various acylhydrazones.

In any of the embodiments described herein, the release linker may include an oxygen atom bonded to alkylene(dialkylsilyl), alkylene(alkylarylsilyl), alkylene(diarylsilyl), (dialkylsilyl)aryl, (alkylarylsilyl)aryl, and (diarylsilyl)aryl with forming a silanol, each of which is optionally substituted with an X2 substituent, as defined herein.

In any of the embodiments described herein, the release linker may include a nitrogen atom bonded to a carbonylarylcarbonyl, carbonyl(carboxyaryl)carbonyl, carbonyl(biscarboxyaryl)carbonyl to form an amide or, alternatively, an amide to the nitrogen of the drug.

In any of the embodiments described herein, the release linker may include an oxygen atom bonded to a carbonylarylcarbonyl, carbonyl(carboxyaryl)carbonyl, carbonyl(biscarboxyaryl)carbonyl to form an ester or, alternatively, an ester to the oxygen atom of the drug.

It should be understood that the bivalent spacer linkers may be combined in any chemically relevant manner, either directly or via a heteroatom intermediate to construct the releasable linkers described herein. It should also be understood that the nature of the arrangement of the spacer and heteroatomic linkers determines at what point the released linker will be cleaved off in vivo. For example, two spacer linkers that have sulfur atoms at their ends, when combined, form a disulfide, which is a cleavable bond in the released linker thus formed.

For example, in another embodiment, the polyvalent linker contains a moiety that is 3-thiosuccinimid-1-ylalkyloxymethyloxy, where methyl is optionally substituted with alkyl or substituted aryl.

In another embodiment, the polyvalent linker contains 3-thiosuccinimide-1ylalkylcarbonyl, where the carbonyl forms an acylazaridine with the drug.

In another embodiment, the polyvalent linker contains a moiety that is 1-alkoxycycloalkyleneoxy.

In another embodiment, the polyvalent linker contains an alkyleneaminocarbonyl (dicarboxylarylene) carboxylate.

In another embodiment, the polyvalent linker contains a dithioalkylcarbonyl hydrazide, where the hydrazide forms a hydrazonone with the drug.

In another embodiment, the polyvalent linker contains 3-thiosuccinimide-1ylalkylcarbonylhydrazide, wherein the hydrazide forms hydrazonone with the drug.

In another embodiment, the polyvalent linker contains 3thioalkylsulfonylalkyl(disubstituted silyl)oxy, wherein the disubstituted silyl is substituted with alkyl or optionally substituted aryl.

In another embodiment, the polyvalent linker contains a plurality of spacer linkers selected from the group consisting of naturally occurring amino acids and their stereoisomers.

In another embodiment, the polyvalent linker contains 2-dithioalkyloxycarbonyl, where the carbonyl forms a carbonate with the drug.

In another embodiment, the polyvalent linker contains 2dithioarylalkyloxycarbonyl, where the carbonyl forms a carbonate with the drug and the aryl is optionally substituted.

- 21 041296

In another embodiment, the polyvalent linker contains 4dithioarylalkyloxycarbonyl, where the carbonyl forms a carbonate with the drug and the aryl is optionally substituted.

In another embodiment, the polyvalent linker contains a 3-thiosuccinimide-1ylalkyloxyalkyloxyalkylidene wherein the alkylidene forms a hydrazone with the drug, each alkyl being independently selected and the oxyalkyloxy optionally substituted with alkyl or optionally substituted aryl.

In another embodiment, the polyvalent linker contains 2dithioalkyloxycarbonylhydrazide.

In another embodiment, the polyvalent linker contains a 2- or 3-dithioalkylamino, where the amino forms a vinylogic amide with the drug.

In another embodiment, the polyvalent linker contains 2-dithioalkylamino, where amino forms a vinylogic amide with the drug and alkyl is ethyl.

In another embodiment, the polyvalent linker contains 2- or 3-dithioalkylaminocarbonyl, wherein the carbonyl forms a drug carbamate.

In another embodiment, the polyvalent linker contains 2-dithioalkylaminocarbonyl, where the carbonyl forms a drug carbamate. In another aspect, alkyl is ethyl.

In another embodiment, the polyvalent linker contains 2-dithioalkyloxycarbonyl, where the carbonyl forms a drug carbamate. In another aspect, alkyl is ethyl.

In another embodiment, the polyvalent linker contains 2dithioarylalkyloxycarbonyl, where the carbonyl forms a carbamate or carbamoylaziridine with the drug.

In another embodiment, the polyvalent linker contains 4-dithioarylalkyloxycarbonyl, where the carbonyl forms a carbamate or carbamoylaziridine with the drug.

In another embodiment, the multivalent linkers described herein contain bivalent radicals of the formulas where n is selected from an integer from 1 to about 4; each of Ra and Rb is independently selected from the group consisting of hydrogen and alkyl, including lower alkyl, such as C ₁ -C ₄ -alkyl, which is optionally branched; or Ra and Rb taken together with the attached carbon atom form a carbocyclic ring; R is an optionally substituted alkyl group, an optionally substituted acyl group, or a suitably selected nitrogen protecting group; and (*) denotes attachment points for drug, vitamin, imaging agent, diagnostic agent, other bivalent linkers, or other portions of the conjugate.

In another embodiment, the multivalent linkers described herein contain bivalent radicals of the formulas where m is selected from an integer from 1 to about 4; R is an optionally substituted alkyl group, an optionally substituted acyl group, or a suitably selected nitrogen protecting group; and (*) denotes points of attachment for the drug, vitamin, imaging agent, diagnostic agent, other bivalent linkers, or other portions of the conjugate.

In another embodiment, the polyvalent linkers described herein contain bivalent radicals of formulas

- 22 041296

where m is selected from an integer from 1 to about 4; R represents an optionally substituted alkyl group, an optionally substituted acyl group or a suitably selected nitrogen protecting group; and (*) denotes points of attachment for the drug, vitamin, imaging agent, diagnostic agent, other bivalent linkers, or other portions of the conjugate.

In another embodiment, the compounds described herein contain one or more linker radicals selected from the formulas

where X is NH, O or S.

In another embodiment, the multivalent linkers described herein contain a radical with the formula

In another embodiment, the multivalent linkers described herein contain a radical with the formula

where X is a heteroatom such as a nitrogen, oxygen or sulfur atom, 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 inductively or by using resonance on an aryl ring such as alkoxy and the like, and the sign (*) denotes points of attachment. It should be noted that other substituents may be present on the aryl ring, on the benzyl carbon, on the alkanoic acid, or on the methylene bridge, including, but not limited to, hydroxy, alkyl, alkoxy, alkylthio, halogen, and the like.

In another embodiment, the polyvalent linkers described herein contain radicals selected from carbonyl, thionocarbonyl, alkylene, cycloalkylene, alkylenecycloalkyl, alkylenecarbonyl, cycloalkylenecarbonyl, carbonylalkylcarbonyl, 1-alkylenesuccinimide-3yl, 1-(carbonylalkyl)succinimid-3-yl, alkylenesulfoxyl, sulfonylalkyl, alkylenesulfoxylalkyl, alkylenesulfonylalkyl, carbonyltetrahydro-2H-pyranyl, carbonyltetrahydrofuranyl, 1(carbonyltetrahydro-2H-pyranyl)succinimide-3-yl, and 1-(carbonyltetrahydrofuranyl)succinimide-3yl, wherein each of said spacer linkers is optionally substituted with one or more substituents X ¹ ;

where each substituent X ¹ is independently selected from the group consisting of alkyl, alkoxy, alkoxyalkyl, hydroxy, hydroxyalkyl, amino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, halogen, haloalkyl, sulfhydrylalkyl, alkylthioalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, carboxy, carboxyalkyl, alkylcarboxylate, alkylalkanoate, guanidinealkyl, R4-carbonyl, R5-carbonylalkyl, R6-acylamino and R7acylaminoalkyl, where each of R4 and R5 is independently selected from the group consisting of an amino acid, an amino acid derivative and a peptide, and where each of R6 and R7 is independently selected from the group consisting of an amino acid, an amino acid derivative and a peptide.

It should be understood that the compounds described herein may contain one or more chiral centers or may otherwise exist as multiple stereoisomers. It should be understood that in one embodiment, the present invention described herein is not limited to any particular stereochemical condition.

- 23 041296 viem, and that the compounds and compositions, methods, uses and drugs that include them may be optically pure or may be any kind of stereoisomeric mixtures, including racemic and other mixtures of enantiomers, other mixtures of diastereomers, etc. P. It should also be understood that such mixtures of stereoisomers may include a single stereochemical configuration at one or more chiral centers, while they may include mixtures of stereochemical configurations at one or more other chiral centers.

Similarly, the compounds described herein may include geometric centers such as cis-, trans-, E- and Z-double bonds. It should be understood that in another embodiment, the present invention described herein is not limited to any particular condition regarding geometric isomer, and that the compounds and compositions, methods, uses, and medicinal preparations in which they are incorporated may be pure or may be any of a number of mixtures of geometric isomers. It should also be understood that such mixtures of geometric isomers may include one configuration at one or more double bonds, while they may include mixtures of geometric structures at one or more other double bonds.

In each of the above and each of the following embodiments, it is also to be understood that the formulas include and are not only all pharmaceutically acceptable salts of the compounds, but also include any and all hydrates and/or solvates of the compounds of the formulas. It should be noted that certain functional groups, such as hydroxy, amino groups, and the like, form complexes and/or coordination compounds with water and/or various solvents in various physical forms of the compounds. Accordingly, the above formulas are to be understood as describing such hydrates and/or solvates, including pharmaceutically acceptable solvates.

In each of the above and each of the following embodiments, it is also to be understood that the formulas include and represent each possible isomer, such as stereoisomers and geometric isomers, either alone or in any and all possible mixtures. In each of the above and each of the following embodiments, it is also to be understood that the formulas include and are any and all crystalline forms, partially crystalline forms, and non-crystalline and/or amorphous forms and co-crystals of the compounds.

In another embodiment, the compounds described herein can be internalized into target pathogenic cells by binding to PSMA. In particular, PSMA binds selectively and/or specifically to the conjugate and internalization can occur, for example by PSMA-mediated endocytosis. After internalization, conjugates containing a releasable linker can complete drug delivery to the target cell. Without wishing to be bound by a particular theory, it is believed herein that in cases where a drug is toxic to normal cells or tissues, such a delivery system can reduce toxicity against such non-target cells and tissues because the released linker remains largely or completely intact, as long as the compounds described herein are delivered to the target cells. Accordingly, the compounds described herein act intracellularly by delivering the drug to an intracellular biochemical process, which in turn reduces the effect of the unconjugated drug on healthy cells and tissues of the host animal.

The conjugates described herein can be used in both human and veterinary clinical medicine. Thus, the host animal carrying the pathogenic cell population to be treated with the compounds described herein may be a human or, in the case of veterinary use, may be a laboratory, farm, domestic, or wild animal. The present invention can be applied to animal hosts, including but not limited to humans, laboratory animals such as rodents (e.g., mice, rats, hamsters, etc.), rabbits, monkeys, chimpanzees, pets such as dogs, cats and rabbits, farm animals 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 drug delivery conjugate compounds described herein may be administered in combination therapy with any other known drug, whether or not the additional drug is targeted. Illustrative additional drugs include, without limitation, peptides, oligopeptides, retro-inverted oligopeptides, proteins, protein analogs in which at least one non-peptide bond replaces the peptide bond, apoproteins, glycoproteins, enzymes, coenzymes, enzyme inhibitors, amino acids and their derivatives, receptors and other membrane proteins; antigens and antibodies against them; haptens and antibodies against them; hormones, lipids, phospholipids, liposomes; toxins; antibiotics; analgesics; bronchodilators; beta blockers; antimicrobial agents; antihypertensive drugs; cardiovascular drugs, including antiarrhythmic drugs,

- 24 041296 cardiac glycosides, antianginal agents and vasodilators; drugs for the central nervous system, including stimulants, psychotropic drugs, anti-manic drugs and depressants; antiviral agents; antihistamines; anti-cancer agents, including chemotherapeutic agents; tranquilizers; antidepressants; H-2 antagonists; anticonvulsants; anti-nausea drugs; prostaglandins and prostaglandin analogs; muscle relaxants; anti-inflammatory substances; stimulants; decongestants; antiemetics; diuretics; antispastic agents; anti-asthma drugs; drugs against Parkinson's disease; expectorants; antitussives; mucolytics; and mineral and nutritional supplements.

As used herein, the term alkyl includes a chain of carbon atoms that is optionally branched. As used herein, the terms alkenyl and alkynyl include a carbon chain that is optionally branched and includes at least one double bond or triple bond, respectively. It should be understood that alkynyl may also include one or more double bonds. In addition, it should be understood that in certain embodiments, the implementation of alkyl mainly has a limited length, including C1-C ₂₄ , C1-C ₁₂ , C1-C ₈ , C1C ₆ and C1-C ₄ and C ₂ -C ₂₄ , C ₂ -C ₁₂ , C ₂ -C ₈ , C ₂ -C ₆ , and C ₂ -C ₄ and the like, by way of illustration, such alkyl groups with specifically limited length, including C1- C ₈ , C1-C ₆ , and C1-C4 and C ₂ -C ₈ , C ₂ -C ₆ , and C ₂ -C ₄ and the like, may be referred to as lower alkyl. In addition, it should be understood that in certain embodiments, each of the alkenyl and/or alkynyl can advantageously have a limited length, including C ₂ -C ₂₄ , C ₂ -C ₁₂ , C ₂ -C ₈ , C ₂ -C ₆ , and C ₂ -C ₄ , and C ₃ -C ₂₄ , C ₃ -C ₁₂ , C ₃ -C ₈ , C ₃ -C ₆ , and C ₃ -C ₄ , etc. By way of illustration, such alkenyl and/or alkynyl groups of particularly limited length, including C ₂ -C ₈ , C ₂ -C ₆ , and C ₂ -C ₄ , and C ₃ -C ₈ , C ₃ -C ₆ , and C ₃ -C ₄ and the like, may be referred to as lower alkenyl and/or alkynyl. It should be noted herein that shorter alkyl, alkenyl and/or alkynyl groups may render the compound less lipophilic and thus will have different pharmacokinetic behavior. In the embodiments of the present invention described herein, it should be understood in each case that the reference to alkyl refers to alkyl as defined herein, and optionally to lower alkyl. In the embodiments of the present invention described herein, it should be understood in each case that the reference to alkenyl refers to alkenyl as defined herein, and optionally to lower alkenyl. In the embodiments of the present invention described herein, it should be understood in each case that reference to alkynyl refers to alkynyl as defined herein, and optionally to lower alkynyl. Illustrative alkyl, alkenyl and alkynyl groups are, without limitation, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, 2-pentyl, 3-pentyl, neopentyl, hexyl, heptyl , octyl, etc., and the corresponding groups containing one or more double and/or triple bonds, or a combination thereof.

As used herein, the term alkylene includes a bivalent chain of carbon atoms, which is optionally branched. As used herein, the terms alkenylene and alkynylene include a bivalent chain of carbon atoms, which is optionally branched and includes at least one double bond or triple bond, respectively. It should be understood that alkynylene may also include one or more double bonds. In addition, it should be understood that in certain embodiments, the implementation, alkylene, mainly has a limited length, including C1-C ₂₄ , C1-C ₁₂ , C1-C ₈ , C1-Q, and C1-C ₄ , and C ₂ -C ₂₄ , C ₂ -C ₁₂ , C ₂ -C ₈ , C ₂ -C ₆ , and C ₂ -C ₄ , etc. By way of illustration, such alkylene groups with specifically limited length, including C1-C8, C ₁ -C6, and C1-C ₄ , and C ₂ -C ₈ , C ₂ -C ₆ , and C ₂ -C ₄ and the like, may be referred to as lower alkylene. In addition, it should be understood that in certain embodiments, each of the alkenylene and/or alkynylene advantageously may have a limited length, including C2-C24, C2-C12, C2-C8, C2-C6, and C2-C4, and C3 -C24, C ₃ -C ₁₂ , C ₃ -C ₈ , C ₃ -C ₆ , and C ₃ -C ₄ , etc. By way of illustration, such alkenylene and/or alkynylene groups of particularly limited length, including C ₂ -C ₈ , C ₂ -C ₆ , and C ₂ -C ₄ , and C ₃ -C ₈ , C ₃ -C ₆ , and C ₃ -C ₄ and the like, may be referred to as lower alkenylene and/or alkynylene. It should be noted herein that shorter alkylene, alkenylene and/or alkynylene groups may render compounds less lipophilic and thus exhibit different pharmacokinetic behavior. In the embodiments of the present invention described herein, it is to be understood in each instance that references to alkylene, alkenylene, and alkynylene refer to alkylene, alkenylene, and alkynylene as defined herein, and optionally to lower alkylene, alkenylene, and alkynylene. Illustrative alkyl groups are, without limitation, methylene, ethylene, n-propylene, isopropylene, n-butylene, isobutylene, sec-butylene, pentylene, 1,2-pentylene, 1,3-pentylene, hexylene, heptylene, octylene, etc. P.

As used herein, the term cycloalkyl includes a chain of carbon atoms, which is optionally branched, where at least part of the chain is cyclic. It should be understood that cycloalkylalkyl is a type of cycloalkyl. It should be understood that cycloalkyl can be

- 25 041296 polycyclic. Exemplary cycloalkyl includes, without limitation, cyclopropyl, cyclopentyl, cyclohexyl, 2-methylcyclopropyl, cyclopentylethyl-2-yl, adamantyl, and the like. As used herein, the term cycloalkenyl includes a chain of carbon atoms, which is optionally branched, and includes at least one double bond, where at least part of the chain is cyclic. It should be understood that one or more double bonds may be in the cyclic portion of the cycloalkenyl and/or the non-cyclic portion of the cycloalkenyl. It should be understood that each of cycloalkenylalkyl and cycloalkylalkenyl is a type of cycloalkenyl. It should be understood that cycloalkyl may be polycyclic. Illustrative cycloalkenyl includes, without limitation, cyclopentenyl, cyclohexylethen-2-yl, cycloheptenylpropenyl, and the like. It should also be understood that the chain forming cycloalkyl and/or cycloalkenyl is preferably of limited length, including C ₃ -C ₂₄ , C3-C12, C3C ₈ , C ₃ -C ₆ and C ₅ -C ₆ . It should be noted herein that shorter alkyl and/or alkenyl chains forming cycloalkyl and/or cycloalkenyl, respectively, can render compounds less lipophilic and thus will have different pharmacokinetic behavior.

As used herein, the term heteroalkyl includes a chain of atoms that includes both carbon atoms and at least one heteroatom and is optionally branched. Illustrative heteroatoms include nitrogen, oxygen, and sulfur. In certain variations, exemplary heteroatoms also include phosphorus and selenium. As used herein, the term cycloheteroalkyl, including heterocyclyl and heterocycle, includes a chain of atoms that includes both carbon atoms and at least one heteroatom, such as heteroalkyl, and is optionally branched, wherein at least a portion of the chain is cyclic. . Illustrative heteroatoms include nitrogen, oxygen, and sulfur. In certain variations, exemplary heteroatoms also include phosphorus and selenium. Exemplary cycloheteroalkyl includes, without limitation, tetrahydrofuryl, pyrrolidinyl, tetrahydropyranyl, piperidinyl, morpholinyl, piperazinyl, homopiperazinyl, quinuclidinyl, and the like.

As used herein, the term aryl includes monocyclic and polycyclic aromatic carbocyclic groups, each of which may be optionally substituted. Illustrative aromatic carbocyclic groups described herein include, without limitation, phenyl, naphthyl, and the like. As used herein, the term heteroaryl includes aromatic heterocyclic groups, each of which may be optionally substituted. Illustrative aromatic heterocyclic groups include, without limitation, pyridinyl, pyrimidinyl, pyrazinyl, triazinyl, tetrazinyl, quinolinyl, quinazolinyl, quinoxalinyl, thienyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, triazolyl, benzimidazolyl, benzoxazolyl, benzothiazolyl , benzisothiazolyl and the like.

As used herein, the term amino includes the NH2 group, alkylamino, and dialkylamino, where the two alkyl groups in the dialkylamino may be the same or different, i. alkylalkylamino. By way of illustration, amino includes methylamino, ethylamino, dimethylamino, methylethylamino, and the like. In addition, it should be understood that when amino is modified by another term or by another term, such as aminoalkyl or acylamino, the above variations of the term amino are thus included. By way of illustration, aminoalkyl includes H ₂ N-alkyl, methylaminoalkyl, ethylaminoalkyl, dimethylaminoalkyl, methylethylaminoalkyl, and the like. By way of illustration, acylamino includes acylmethylamino, acylethylamino, and the like.

As used herein, the term amino and its derivatives includes amino as defined herein and alkylamino, alkenylamino, alkynylamino, heteroalkylamino, heteroalkenylamino, heteroalkynylamino, cycloalkylamino, cycloalkenylamino, cycloheteroalkylamino, cycloheteroalkenylamino, arylamino, arylalkylamino, arylalkenylamino, arylakynylamino , heteroarylamino, heteroarylalkylamino, heteroarylalkenylamino, heteroarylkynylamino, acylamino, and the like, each of which is optionally substituted. The term amino derivative also includes urea, carbamate, and the like.

As used herein, the term amino acid generally refers to beta, gamma, and longer amino acids, such as the amino acids of formula

-N(R)-(CR'R)q-C(O)- where R is hydrogen, alkyl, acyl, or a suitable nitrogen protecting group, R' and R are hydrogen or a substituent, each independently selected at each occurrence , and q is an integer such as 1, 2, 3, 4, or 5. By way of illustration, R' and/or R are independently, without limitation, hydrogen or side chains present in naturally occurring amino acids, such as methyl, benzyl, hydroxymethyl, thiomethyl, carboxyl, carboxylmethyl, guanidinopropyl, and the like. and their derivatives and protected derivatives. The above formula includes all stereoisomeric variations. For example, the amino acid may be selected from asparagine, aspartic acid, cysteine, glutamic acid, lysine, glutamine, arginine, serine, ornithine, threonine, and the like.

- 26 041296

As used herein, the term amino acid derivative generally refers to an amino acid, as defined herein, in which the amino group and/or the side chain, or both, have been substituted. Exemplary amino acid derivatives include prodrugs and amino and/or side chain protecting groups such as amine, amide, hydroxy, carboxylic acid and sulfur-containing prodrugs and protecting groups. Additional exemplary amino acid derivatives include substituted amino acid variations as described herein, such as, without limitation, ethers and esters of hydroxyl groups, amides, carbamates, and ureas of amino groups, esters, amides, and cyano derivatives of carboxyl groups, and the like.

As used herein, the term hydroxy and its derivatives include OH and alkyloxy, alkenyloxy, alkynyloxy, heteroalkyloxy, heteroalkenyloxy, heteroalkynyloxy, cycloalkyloxy, cycloalkenyloxy, cycloheteroalkyloxy, cycloheteroalkenyloxy, aryloxy, arylalkyloxy, arylalkenyloxy, arylalkynyloxy, heteroaryloxy, heteroarylalkyloxy, heteroarylalkenyloxy, heteroaryloxy, acyloxy and the like, each of which is optionally substituted. The term hydroxy derivative also includes carbamate and the like.

Как применяется в настоящем документе, термин тио и его производные включает SH и алкилтио, алкенилтио, алкинилтио, гетероалкилтио, гетероалкенилтио, гетероалкинилтио, циклоалкилтио, циклоалкенилтио, циклогетероалкилтио, циклогетероалкенилтио, арилтио, арилалкилтио, арилалкенилтио, арилалкинилтио, гетероарилтио, гетероарилалкилтио, гетероарилалкенилтио, гетероарилалкинилтио, acylthio and the like, each of which is optionally substituted. The term thio derivative also includes thiocarbamate and the like.

Как применяется в настоящем документе, термин ацил включает формил и алкилкарбонил, алкенилкарбонил, алкинилкарбонил, гетероалкилкарбонил, гетероалкенилкарбонил, гетероалкинилкарбонил, циклоалкилкарбонил, циклоалкенилкарбонил, циклогетероалкилкарбонил, циклогетероалкенилкарбонил, арилкарбонил, арилалкилкарбонил, арилалкенилкарбонил, арилалкинилкарбонил, гетероарилкарбонил, гетероарилалкилкарбонил, гетероарилалкенилкарбонил, гетероарилалкинилкарбонил, ацилкарбонил и т .p., each of which is optionally substituted.

As used herein, the term carbonyl and its derivatives includes the C(O), C(S), C(NH) group and its substituted amino derivatives.

As used herein, the term carboxylic acid and its derivatives includes the CO2H group and its salts and its esters and amides and CN.

As used herein, the term sulfinic acid or derivative thereof includes SO2H, and its salts, and its esters, and amides.

As used herein, the term sulfonic acid or derivative thereof includes SO3H, and its salts, and its esters, and amides.

Как применяется в настоящем документе, термин сульфонил включает алкилсульфонил, алкенилсульфонил, алкинилсульфонил, гетероалкилсульфонил, гетероалкенилсульфонил, гетероалкинилсульфонил, циклоалкилсульфонил, циклоалкенилсульфонил, циклогетероалкилсульфонил, циклогетероалкенилсульфонил, арилсульфонил, арилалкилсульфонил, арилалкенилсульфонил, арилалкинилсульфонил, гетероарилсульфонил, гетероарилалкилсульфонил, гетероарилалкенилсульфонил, гетероарилалкинилсульфонил, ацилсульфонил и т.п ., each of which is optionally substituted.

As used herein, the term phosphinic acid or derivative thereof includes P(R)O2H, and its salts, and its esters, and amides, where R is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heteroalkyl, heteroalkenyl, cycloheteroalkyl , cycloheteroalkenyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, each of which is optionally substituted.

As used herein, the term phosphonic acid or derivative thereof includes PO3H2, and its salts, and its esters, and amides.

As used herein, the term hydroxylamino and its derivatives includes NHOH and alkyloxy-NH, alkenyloxy-NH, alkynyloxy-NH, heteroalkyloxy-NH, heteroalkenyloxy-NH, heteroalkynyloxy-NH, cycloalkyloxy-NH, cycloalkenyloxy-NH, cycloheteroalkyloxy-NH, cycloheteroalkenyloxy-NH, aryloxy-NH, arylalkyloxy-NH, arylalkynyloxy-NH, arylalkynyloxy-NH, heteroaryloxy-NH, heteroarylalkyloxy-NH, heteroarylalkenyloxy-NH, heteroarylalkynyloxy-NH, acyloxy, and the like, each of which is optionally substituted.

As used herein, the term hydrazino and its derivatives include alkylNHNH, alkenyl-NHNH, alkynyl-NHNH, heteroalkyl-NHNH, heteroalkenyl-NHNH, heteroalkynylNHNH, cycloalkyl-NHNH, cycloalkenyl-NHNH, cycloheteroalkyl-NHNH, cycloheteroalkenylNHNH, aryl-NHNH, arylalkyl-NHNH, arylalkenyl-NHNH, arylalkynyl-NHNH, heteroaryl-NHNH, heteroarylalkyl-NHNH, heteroarylalkenyl-NHNH, heteroarylalkynyl-NHNH, acyl-NHNH, and the like, each of which is optionally substituted.

The expression optionally substituted, as used herein, includes the substitution of hydrogen atoms with other functional groups on a radical that is optionally substituted. Such other functional groups, by way of illustration, include, without limitation, amino, hydroxyl, halogen, thiol, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl,

- 27 041296 heteroaryl, heteroarylalkyl, heteroarylheteroalkyl, nitro, sulfonic acids and their derivatives, carboxylic acids and their derivatives, and the like. By way of illustration, any of amino, hydroxyl, thiol, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl, heteroarylheteroalkyl, and/or sulfonic acid are also optionally substituted.

As used herein, the terms optionally substituted aryl and optionally substituted heteroaryl include substitution of hydrogen atoms with other functional groups in aryl or heteroaryl that are optionally substituted. Such other functional groups, by way of illustration, include, without limitation, amino, hydroxy, halogen, thio, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl, heteroarylheteroalkyl, nitro, sulfonic acids and their derivatives, carboxylic acids and their derivatives, etc. By way of illustration, any of amino, hydroxy, thio, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl, heteroarylheteroalkyl, and/or sulfonic acid are optionally substituted.

Illustrative substituents include, without limitation, the radical -(CH ₂ ) _x Z ^x where x is an integer 0-6 and Z ^x is selected from halo, hydroxy, alkanoyloxy, including €1-€6alkanoyloxy, optionally substituted aroyloxy , alkyl, including C1-C6-alkyl, alkoxy, including C1-C6-alkoxy, cycloalkyl, including C3-C8-cycloalkyl, cycloalkoxy, including C3-C8-cycloalkoxy, alkenyl, including C2- C6-alkenyl, alkynyl, including C2-C6-alkynyl, haloalkyl, including C1-C6-haloalkyl, haloalkoxy, including C1-C6-haloalkoxy, halocycloalkyl, including C3-C8-halocycloalkyl, halocycloalkoxy, including C3-C8 halocycloalkoxy, amino, C1-C6-alkylamino, (C1-C6-alkyl)(C1-C6-alkyl)amino, alkylcarbonylamino, N-(C1-C6-alkyl)alkylcarbonylamino, aminoalkyl, C1-C6- alkylaminoalkyl, (C1-C6alkyl)(C1-C6alkyl)aminoalkyl, alkylcarbonylaminoalkyl, N-(C1-C6alkyl)alkylcarbonylaminoalkyl, cyano and nitro; or Z ^x is selected from -CO2R ⁴ and -CONR5R ⁶ where R ⁴ , R ⁵ and R ⁶ are each independently selected from hydrogen, C1-C ₆ -alkyl, aryl-C ₁ -C ₆ -alkyl and heteroaryl -C ₁ -C ₆ -alkyl.

As used herein, the term leaving group refers to a reactive functional group that forms an electrophilic site at the atom to which it is attached, whereby nucleophiles can be added to the electrophilic site at the atom. Exemplary leaving groups include, without limitation, halogens, optionally substituted phenols, acyloxy groups, sulfonic groups, and the like. It should be understood that such leaving groups may be in alkyl, acyl, and the like. Such leaving groups may also be referred to herein as activating groups, such as when the leaving group is present on the acyl. In addition, traditional peptide, amide, and ester coupling agents such as, but not limited to, PyBop, BOP-Cl, BOP, pentafluorophenol, isobutyl chloroformate, and the like, form various intermediates that include a leaving group as defined herein. , at the carbonyl group.

As used herein, the term radical in relation to, for example, a PSMA binding or targeting ligand and/or an independently selected drug refers to a PSMA binding or targeting ligand and/or an independently selected drug, as described herein, wherein one or more atoms or groups such as a hydrogen atom, or an alkyl group on a heteroatom, and the like, are removed to provide a radical for conjugation to the polyvalent linker L.

The term prodrug, as used herein, generally refers to any compound that, when introduced into a biological system, produces a biologically active compound by one or more spontaneous chemical reactions, enzyme-catalyzed chemical reactions and/or metabolic chemical reactions, or a combination thereof. In vivo, the prodrug is typically acted upon by an enzyme (such as esterases, amidases, phosphatases, and the like), simple biochemical processes, or another in vivo process to release or recover more of the pharmacologically active drug. This activation can occur through the action of an endogenous host enzyme or a non-endogenous enzyme that is administered to the host before, after, or during administration of the prodrug. Additional details on the use of prodrugs are described in US patent No. 5627165; and in Pathalk et al., Enzymic protecting group techniques in organic synthesis, Stereosel. Biocatal. 775-797 (2000). It should be noted that the prodrug is predominantly converted to the parent drug once the goal such as targeted delivery, safety, stability, etc. is achieved, followed by the subsequent rapid removal of the released prodrug-forming group residues.

Prodrugs can be prepared from the compounds described herein by attaching groups that are ultimately cleaved in vivo to one or more functional groups present on the compound, such as -OH-, -SH, -CO ₂ H, -NR2 . Illustrative prodrugs include, without limitation, carboxylate esters where the group is alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, acyloxyalkyl, alkoxycarbonyloxyalkyl, and hydroxyl, thiol, and amine esters where the attached group is acyl.

- 28 041296 group, alkoxycarbonyl, aminocarbonyl, phosphate or sulfate. Exemplary esters, also referred to as active esters, include, without limitation, 1-indanyl, N-hydroxysuccinimide; acyloxyalkyl groups such as acetoxymethyl, pivaloyloxymethyl, β-acetoxyethyl, βpivaloyloxyethyl, 1-(cyclohexylcarbonyloxy)prop-1-yl, (1-aminoethyl)carbonyloxymethyl and the like; alkoxycarbonyloxyalkyl groups such as ethoxycarbonyloxymethyl, αethoxycarbonyloxyethyl, β-ethoxycarbonyloxyethyl and the like; dialkylaminoalkyl groups including di-lower alkylaminoalkyl groups such as dimethylaminomethyl, dimethylaminoethyl, diethylaminomethyl, diethylaminoethyl and the like; 2-(alkoxycarbonyl)-2-alkenyl groups such as 2(isobutoxycarbonyl)pent-2-enyl, 2-(ethoxycarbonyl)but-2-enyl and the like; and lactone groups such as phthalidyl, dimethoxyphthalidyl and the like.

Additional exemplary prodrugs contain a chemical moiety, such as an amide or phosphorus group, that functions to increase the solubility and/or stability of the compounds described herein. Additional exemplary amino prodrugs include, without limitation, (C ₃ -C ₂₀ )alkanoyl; halo(C ₃ -C ₂₀ )alkanoyl; (C ₃ -C ₂ o) alkenoyl; (C ₄ C7) cycloalkanoyl; (C ₃ -C6)cycloalkyl( _C2 -C1b)alkanoyl; optionally substituted aroyl such as unsubstituted aroyl or aroyl substituted with 1-3 substituents selected from the group consisting of halogen, cyano, trifluoromethanesulfonyloxy, (C1-C ₃ )alkyl and (C ₁ -C ₃ )alkoxy, each of which is optional optionally substituted with one or more 1-3 halogen atoms; optionally substituted aryl(C ₂ -C ₁₆ )alkanoyl; and optionally substituted heteroaryl(C ₂ -C ₁₆ )alkanoyl such as an aryl or heteroaryl radical which is unsubstituted or substituted with 1-3 substituents selected from the group consisting of halogen, (C ₁ -C ₃ )alkyl and (C ₁ -C ₃ )alkoxy, each optionally further substituted with 1-3 halogen atoms; and optionally substituted heteroarylalkanoyl with one to three heteroatoms selected from O, S and N in the heteroaryl moiety and 2 to 10 carbon atoms in the alkanoyl moiety, such as a heteroaryl radical that is unsubstituted or substituted with 1-3 substituents selected from the group consisting of halogen, cyano, trifluoromethanesulfonyloxy, (C1-C ₃ )alkyl and (C1C ₃ )alkoxy, each optionally further substituted with 1-3 halogen atoms. The groups shown are illustrative, not exhaustive, and may be prepared using conventional methods.

It should be understood that prodrugs themselves may not have significant biological activity, but instead undergo one or more spontaneous chemical reactions, enzyme-catalyzed chemical reactions and/or metabolic chemical reactions, or a combination thereof, after administration in vivo to form the compound described herein. a document that is biologically active or is a precursor of a biologically active compound. However, it should be noted that in some cases the prodrug is biologically active. It should also be noted that prodrugs can often serve to improve drug efficacy or safety by improving oral bioavailability, pharmacodynamic half-life, and the like. Prodrugs also refer to derivatives of the compounds described herein that include groups that simply mask undesirable drug properties or improve drug delivery. For example, one or more of the compounds described herein may exhibit an undesirable property that is advantageously blocked or minimized, which may become pharmacological, pharmaceutical, or pharmacokinetic barriers to drug use in medical practice, such as poor oral absorption of the drug. cavities, lack of site specificity, chemical instability, toxicity and unacceptability for the patient (bad taste, smell, pain at the injection site, etc.) and others. As used herein, it should be understood that a prodrug or other strategy using reversible derivatives may be useful in optimizing drug use in medical practice.

It should be understood that in each case disclosed herein, reference to a range of integers for any variable includes the range referred to, each individual member in the range, and each possible subrange for that variable. For example, mentioning that n is an integer from 0 to 8 describes this range, the individual and selectable values 0, 1, 2, 3, 4, 5, 6, 7, and 8, such as n being 0, or n is 1, or n is 2, and so on.

In addition, mentioning that n is an integer from 0 to 8 also describes absolutely all sub-ranges, each of which can serve as the basis for an additional embodiment, such as n is an integer from 1 to 8, from 1 to 7, 1 to 6, 2 to 8, 2 to 7, 1 to 3, 2 to 4, etc.

As used herein, the term composition generally refers to any product containing certain ingredients in certain amounts, as well as any product that is obtained, directly or indirectly, as a result of combinations of certain

- 29 041296 ingredients in certain quantities. It should be understood that the compositions described herein may be prepared from the isolated compounds described herein, or from salts, solutions, hydrates, solvates, and other forms of the compounds described herein. It should also be understood that compositions can be prepared from various amorphous, non-amorphous, partially crystalline, crystalline and/or other morphological forms of the compounds described herein. It should also be understood that compositions may be prepared from various hydrates and/or solvates of the compounds described herein. Therefore, such pharmaceutical compositions, in which the compounds described herein are mentioned, should be understood as including each or any combination of various morphological forms and/or solvate or hydrate forms of the compounds described herein. In addition, it should be understood that the compositions can be obtained from various co-crystals of the compounds described herein.

By way of illustration, the compositions may include one or more carriers, diluents and/or excipients. The compounds described herein, or compositions containing them, may be formulated in a therapeutically effective amount in any conventional dosage form suitable for the methods described herein. The compounds described herein, or compositions containing them, including such formulations, can be administered via a wide variety of conventional routes for the methods described herein and a wide variety of dosage formats using known procedures (see generally , Remington: The Science and Practice of Pharmacy, ( ^21st ed., 2005)).

The term therapeutically effective amount, as used herein, refers to that amount of an active compound or pharmaceutical agent that produces a biological or medical response in a tissue system in an animal or human, as determined by the researcher, veterinarian, physician, or other clinician, which includes relieving symptoms of the disease or disorder being treated. In one aspect, a therapeutically effective amount is one that can treat or alleviate a disease or symptoms of a disease with an acceptable benefit/risk ratio applicable to any therapeutic treatment. However, it should be understood that the total daily use of the compounds and compositions described herein may be determined by the attending physician as part of a thorough medical evaluation. The specific therapeutically effective dose level for any particular patient will depend on a number of factors, including the disorder being treated and the severity of the disorder; the activity of the particular compound used; the particular composition used; age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the particular compound used; duration of treatment; drugs used in combination or incidentally with the particular compound used; and the like, well known to the researcher, veterinarian, physician, or other clinician of skill in the art.

It should also be noted that a therapeutically effective amount, whether referred to as monotherapy or combination therapy, is preferably chosen in consideration of any toxicity or other undesirable side effect that may occur during administration of one or more of the compounds described herein. In addition, it should be appreciated that the co-therapies described herein may involve the administration of lower doses of compounds exhibiting such toxicity or other undesirable side effect, such lower doses resulting in toxicity below threshold values or lower within the therapeutic window than would otherwise be administered in the absence of concomitant therapy.

In addition to the illustrative dosages and dosing protocols described herein, it should be understood that the effective amount of any or mixture of the compounds described herein can be readily determined by the attending physician or physician using known techniques and/or by observing the results. obtained under similar conditions. In determining the effective amount or dose, the treating diagnostician or physician will take into account a variety of factors, including, without limitation, the type of mammal, including humans, size, age and general health, the specific disease or disorder involved, the degree of involvement, or the severity of the disease. or disturbances, response of the individual patient, particular compound administered, route of administration, bioavailability characteristics of the administered drug, dosing regimen selected, use of concomitant medicinal products, and other relevant conditions.

The dosage of each compound of the claimed combinations depends on several factors, including: the route of administration, the condition being treated, the severity of the condition, whether the condition is being treated or prevented, and the age, weight, and health of the person being treated. In addition, pharmacogenomic information (the effect of the genotype on the pharmacokinetic, pharmacodynamic or efficacy profile of a therapeutic agent) about a particular patient can influence the applied dosage.

- 30 041296

It should be understood that in the methods described herein, the individual components of the co-administration or combination can be entered using any suitable route, simultaneously, together, sequentially, separately or in a single pharmaceutical composition. If the co-administered compounds or compositions are administered in separate dosage forms, the number of administered dosages per day for each compound may be the same or different. The compounds or compositions can be administered by the same or different routes of administration. The compounds or compositions can be administered in concurrent or interleaved schedules, at the same or different time points during the course of therapy, in parallel in divided or single forms.

The term administration as used herein includes all means of administering the compounds and compositions described herein to a patient, including but not limited to oral (po), intravenous (iv), intramuscular (im), subcutaneous (sc), transdermal , inhalation, buccal, ocular, sublingual, vaginal, rectal, etc. The compounds and compositions described herein may be administered in unit dosage forms and/or formulations containing conventional non-toxic, pharmaceutically acceptable carriers, adjuvants and/or media.

Exemplary formats for oral administration include tablets, capsules, tinctures, syrups, and the like.

Illustrative routes for parenteral administration include intravenous, intraarterial, intraperitoneal, epidural, intraurethral, intrasternal, intramuscular, and subcutaneous, as well as any other parenteral routes known in the art.

By way of illustration, administration includes topical application, as when administered topically to a disease, lesion, or defect, or to a specific organ or tissue system. Exemplary topical administration may be performed during open surgery or other procedures when the site of the disease, lesion, or defect is accessible. Alternatively, topical administration can be carried out using parenteral delivery, where the compound or compositions described herein are deposited locally in a lesion without a general distribution to multiple other non-target sites in the patient being treated. In addition, it should be noted that topical administration can be carried out directly into the lesion or locally into the surrounding tissue. Similar variations regarding local delivery to specific tissue types such as organs and the like are also described herein. By way of illustration, compounds can be administered directly to the nervous system, including, but not limited to, intracerebral, intraventricular, intracerebroventricular, intrathecal, intracisternal, intraspinal, and/or perispinal routes of administration by delivery via intracranial or intravertebral needles and/or catheters with pumping devices. or without them.

Depending on the disease, as described herein, on the route of administration and/or whether the compounds and/or compositions are administered topically or systemically, a wide range of acceptable dosages is contemplated herein, including doses ranging from approximately 1 mcg/kg to approximately 1 g/kg. Doses may be single or divided and may be administered according to a variety of protocols, including q.d., b.i.d., t.i.d. or even every other day, once a week, once a month, once a quarter, etc. In each of these cases, it should be understood that the therapeutically effective amounts described herein correspond to the example of administration or, alternatively, the total daily, weekly, monthly, or quarterly dose, as determined by the dosing protocol.

When preparing pharmaceutical compositions from the compounds described herein, a therapeutically effective amount of one or more compounds in any of the various forms described herein can be mixed with one or more excipients, diluted with one or more excipients, or enclosed in such a carrier that may be in the form of a capsule, sachet, paper or other container. Excipients may serve as a diluent and may be solid, semi-solid or liquid materials which act as a base, carrier or vehicle for the active ingredient. Thus, formulation compositions may be in the form of tablets, dragees, powders, lozenges, sachets, cachets, tinctures, suspensions, emulsions, solutions, syrups, aerosols (both solid and liquid), ointments, soft and hard gelatin capsules, suppositories, sterile injectable solutions and sterile packaged powders. Compositions may contain from about 0.1% to about 99.9% active ingredients, depending on the dose and dosage form chosen.

The effective use of the compounds, compositions and methods described herein for the treatment or amelioration of diseases caused by pathogenic cells expressing PSMA can be based on animal models, such as murine, canine, porcine animal models of the disease, as well as animal models based on primates. that are not related to humans. For example, it is understood that prostate cancer in humans may be characterized by loss of function and/or development of symptoms, all of which can be induced in animals such as mice and other replacement test animals. In particular, the mouse models described herein, which are subcutaneously

- 31 041296 implanted cancer cells, such as LNCaP, can be used to evaluate the compounds, methods of treatment and pharmaceutical compositions described herein, with the determination of therapeutically effective amounts described herein.

The compounds, linkers, intermediates and conjugates described herein can be prepared using conventional methods, including those described in International Patent Application Publication Nos. WO 2009/002993, WO 2004/069159, WO 2007/022494 and WO 2006/012527 and US Patent Application No. 13/837539 (filed March 15, 2013). The disclosure of each of the above documents is incorporated herein by reference in its entirety.

Each of the publications cited in this document is incorporated herein by reference.

The following reference examples are provided by way of illustration.

Reference examples

Reference example. Compound 104 In a 250 ml round bottom flask H-Glu(OtBu)-OtBu-HCl (1) (4.83 g, 16.3 mmol) and 4-nitrophenyl horformate (102) (3.47 g, 17.2 mmol) was dissolved in dichloromethane (50 ml) and stirred in an ice bath under argon. Diisopropylethylamine (6.28 ml, 36.1 mmol) was slowly added dropwise and the reaction mixture was stirred in an ice bath for 5 minutes, then warmed to room temperature and stirred for 30 minutes. HLys(Z)-OtBu-HCl (103) (7.01 g, 18.8 mmol) was added in portions followed by diisopropylethylamine (6.54 ml, 37.5 mmol) added dropwise and stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure, then purified by silica gel chromatography in 10-100% ethyl acetate/petroleum ether to give 104 (8.76 g, 86%, ESI m/z=622.54 [M+H] +).

Reference example. Compound 105.104 (8.76 g, 14.1 mmol) was dissolved in anhydrous methanol (100 ml) and added slowly along the walls to a 250 ml round bottom flask containing palladium on charcoal, 10 wt% (100 mg). A balloon containing hydrogen gas was attached to the flask using a three-way stopcock and the atmosphere of the flask was removed under reduced pressure, then replaced with hydrogen gas (3x), then stirred at room temperature under a hydrogen gas atmosphere for 1 h. untreated celite (~20 g) and stirred for 5 minutes. The reaction mixture was filtered and concentrated under reduced pressure to give 105 (6.86 g, quantitative yield, ESI m/z = 488.46 [M+H] ⁺ ).

Reference example. Compound 107 Boc-4-aminomethylphenylacetic acid (106) (2.00 g, 7.5 mmol) was dissolved in a solution of trifluoroacetic acid (9.75 ml) and triisopropylsilane (0.25 ml) and stirred at room temperature for 30 min, then concentrated under reduced pressure and co-evaporated with dichloromethane (3x), then placed under vacuum to give 4-aminomethylphenylacetic acid (107) (quantitative).

Reference example. Compound 108 A solution of 105

- 32 041296 (2.45 g, 5.0 mmol) and diisopropylethylamine (0.88 ml, 5.0 mmol) in dry dimethylformamide (10 ml) and the reaction mixture was stirred at room temperature for 30 min under argon. The reaction mixture was cooled in an ice bath and a suspension of 7 (~1.25 g, ~7.5 mmol) and diisopropylethylamine (1.76 ml, 10.1 mmol) in dry dimethylformamide (10 ml) was slowly added dropwise to the reaction vessel, then the reaction mixture was warmed to room temperature and stirred for 30 min under argon. The reaction mixture was purified by preparative HPLC in 10-100% acetonitrile/0.1% formic acid to give 8 (0.56 g, 16%, 1H NMR consistent with structure 108; ESI m/z = 679.50 [M+H ]+).

Reference example. Obtaining a protected ligand 7, including a combination group.

Reference example. Peptide 109.

Table 1

Reagents for the synthesis of peptide 109

Reagent mmol Equivalents Molecular weight (g/mol) quantity H-Cys(4methoxytrityl)-2chlorotrityl resin 0.87 1.0 F shoc-Asp(OtBu)-OH 2x 1.74 2x2.0 411.5 716 mg RuVOR 2 x 1.73 2x2.0 520.39 900 mg diisopropylethylamine 2x3.48 2x4.0 129.25 (d = 0.742 g/ml) 606 µl

The peptide synthesis vessel was loaded with N-cis(4-methoxytrityl)-2-chlorotrityl resin (0.87 mmol) and washed with isopropyl alcohol (3x10 ml) followed by dimethylformamide (3x10 ml). Fmoc-Asp(OtBu)-OH (2.0 eq) in dimethylformamide, diisopropylethylamine (4.0 eq) and PyBOP were then added to the vessel.

- 33 041296 (2.0 eq.). Argon was bubbled in for 1 h, the coupling solution was decanted and the resin was washed with dimethylformamide (3x10 ml) and isopropyl alcohol (3x10 ml). Kaiser tests were performed to evaluate the completion of the reaction. Deprotection of the Fmoc group was performed using 20% piperidine in dimethylformamide (3x10 ml) before each combination of amino acids. The above sequence was repeated to complete the 2 coupling steps. The resin was dried in an argon atmosphere for 30 min.

O

but ₂ s nn co ₂ h

110

Reference example. Peptide 110.

table 2

Reagents for the synthesis of peptide 110

Reagent mmol Equivalents Molecular weight (g/mol) quantity Fmoc-Asp(OtBu)Asp(OtBu)-Cys(Mmt)2-C1TC-resin 0.18 1.0 108 0.22 1.2 678.81 150 mg RuVOR 0.37 2.0 520.39 191 mg diisopropylethylamine 0.74 4.0 129.25 (d = 0.742 g/ml) 128 µl

A peptide synthesis vessel was charged with 109 (0.18 mmol) and washed with isopropyl alcohol (3x10 ml) followed by dimethylformamide (3x10 ml). Deprotection of the Fmoc group was performed using 20% piperidine in dimethylformamide (3x10 ml). Kaiser tests were performed to evaluate the completion of the reaction. 108 (1.2 eq) in dimethylformamide, diisopropylethylamine (4.0 eq) and PyBOP (2.0 eq) were then added to the vessel. Argon was bubbled in for 1 h, the coupling solution was decanted and the resin was washed with dimethylformamide (3x10 ml) and isopropyl alcohol (3x10 ml). Kaiser tests were performed to evaluate the completion of the reaction. The peptide was cleaved from the resin using a cleavage mixture consisting of dithiothreitol (114 mg, 0.74 mmol) dissolved in trifluoroacetic acid solution (19 ml), H2O (0.5 ml), triisopropylsilane (0.5 ml). One third of the cleavage mixture was introduced and argon was sparged for 30 minutes. The cleavage mixture was poured into a clean flask. The resin was bubbled 2 more times with additional cleavage mixture, each time for 30 minutes, and poured into a clean flask. The confluent cleavage mixture was then concentrated and purified by preparative HPLC in 030% acetonitrile/0.1% formic acid to give 110 (66.9 mg, 43%, 1H NMR consistent with structure 110; ESI m/z = 844.57 [ M+H]+).

Reference example. Similarly, the following compounds are prepared as described herein:

- 34 041296

- 35 041296

Reference example. EC1169 (connection 112). In a 25 ml round bottom flask, 16 (47 mg, 0.04 mmol) was dissolved in dimethyl sulfoxide (2 ml). A solution of 110 (36 mg, 0.04 mmol) in 20 mM sodium phosphate buffer (2 ml), pH 7 was added dropwise, stirred at room temperature with argon bubbling for 30 min. The reaction mixture was purified by preparative HPLC (10-100% acetonitrile/50 mM NH4CO3, pH 7) to give 112 (56.6 mg, 74%, ¹ H NMR consistent with EC1169 structure; ESI m/z = 895.58 [M +2Н] ²⁺ ).

- 36 041296

Reference example. Synthesis of 3-nitro-2-disulfenylethanol 2

A three-necked 500 ml flask was dried and purged with argon, then fitted with an addition funnel. 3-nitro-2-sulfenylchloridepyridine 1 (5.44 g, 27.11 mmol, 1.4 eq) was added to the flask and dissolved in 200 ml CH ₂ Cl ₂ . The solution was cooled to 0°C. Mercaptoethanol (1.33 ml, 18.98 mmol) was diluted with 50 ml CH ₂ Cl ₂ and placed in an addition funnel. Then a solution of 2-merkatpoethanol was added slowly dropwise over 15 min. The progress of the reaction was monitored by TLC (Rf 0.4 in 5% CH ₃ OH/CH ₂ Cl ₂ ). The solvent was removed under reduced pressure and dried. The crude product was purified through silica gel (5% CH ₃ OH/CH ₂ C1 ₂ ). The fractions were collected and the solvent was removed by rotary evaporation and dried. 3.4 g of 3-nitro-2-disulfenylethanol 2 were obtained (yield 77%).

Reference example. Synthesis of 4-nitrophenyl-(3'-nitropyridin-2'-yl)disulfenethyl carbonate 3

A 250 ml round bottom flask was dried and purged with argon. 3-nitro-2-disulfenylethanol 2 (3.413 g, 14.69 mmol) was added and dissolved in 45 ml CH ₂ Cl ₂ . 4-nitrophenyl chloroformate (3.663 g, 17.63 mmol, 1.2 eq.) was added along with triethylamine (2.9 ml, 20.57 mmol, 1.4 eq.) and the mixture was stirred under argon overnight. The mixture was concentrated under reduced pressure and dried. The residue was purified with silica (30% EtOAc/petroleum ether) and the fractions were collected, the solvent was removed under reduced pressure and dried. 2.7 g of 4-nitrophenyl-(3'-nitropyridin-2'-yl)disulfenethyl carbonate 3 (47% yield) were obtained.

Reference example. Synthesis of (3'-nitropyridyl-2'-yl)disulfanylethyl ester of 2-(Vostubutyrosine(Tut))hydrazinecarboxylic acid 6

5

THF 3

10.67 g (33 mmol) Boc-Tut acid 4 was dissolved in 100 ml anhydrous THF, 17.24 g (33 mmol) PyBop and 17.50 ml (99 mmol, 3.0 eq.) DIPEA were added. The reaction mixture was stirred for several minutes, 1.0 ml (31.68 mmol, 0.96 eq.) of hydrazine was added and stirred for 15 minutes. LC-MS analysis (X-Bridge shield RP18 column, 3.5 µm; gradient 10%-100% acetonitrile over 6 min, buffer pH 7.4) confirmed the formation of hydrazide 5. Added 14.47 g (36.3 mmol , 1.1 eq.) 4-nitrophenyl(3'-nitropyridin-2'-yl)disulfenethyl carbonate 2. The resulting clear solution was stirred at room temperature for 24 h. LC-MS analysis (X-Bridge shield column RP18, 3 .5 µm; 30%-100% acetonitrile gradient over 9 min, buffer pH 7.4) showed >98% conversion. The reaction mixture was diluted with EtOAc (~1.0 L), washed with sat. NH4C1 (400 ml), sat. NaHCO ₃ solution (3x300 ml) and brine (300 ml). The organic layers were dried over Na ₂ SO ₄ (100 g) and concentrated under reduced pressure. The crude product was loaded onto a Teledyne Redisep Gold silica gel column and eluted with MeOH/CH ₂ Cl ₂ (330 g column; 0-10% gradient) using a CombiFlash chromatography system. The fractions were collected and the solvent was removed under reduced pressure and dried. 16.10 g of 2-(BocTut)hydrazinecarboxylic acid (3'-nitropyridyl-2'-yl)disulfanylethyl ester 6 (82% yield) were obtained.

- 37 041296

Reference example. Synthesis of azidomethylbutyrate dipeptide 9

Dipeptide 7 (10.83 g, 27.25 mmol) was dissolved in 100 ml dichloromethane and imidazole (2.05 g, 1.1 eq.) was added. The reaction mixture was stirred at room temperature to dissolve all solids and cooled in an ice bath for 10 minutes. TESCl (4.8 mL, 1.05 eq.) was added dropwise at 0° C., stirred under argon and warmed to room temperature over 1.5 h. TLC (3:1 hexanes/EtOAc) showed complete conversion. The reaction mixture was filtered to remove the HCl imidazole salt. 125 ml of dichloromethane was added to the filtrate, and the resulting solution was extracted with 250 ml of brine. The brine layer was extracted with 125 ml dichloromethane. The combined organic phase was washed with 250 ml of brine, separated, dried over 45.2 g of Na ₂ SO ₄ and filtered. The resulting solution was concentrated under reduced pressure, co-evaporated with toluene (2x5 ml) and dried under high vacuum overnight to give 14.96 g of crude product 8.

The crude product 8 was used without further purification. The TES-protected dipeptide was dissolved in 100 ml THF (anhydrous, no inhibitors), cooled to -45°C and stirred at -45°C for 15 min before KHMDS (0.5 M in toluene, 61 ml, 1 .05 equiv.). After the addition of KHMDS was completed, the reaction mixture was stirred at -45° C. for 20 min and chloromethyl butyrate (4.4 ml, 1.1 eq.) was added. The reaction mixture was stirred at -45° C. for an additional 20 minutes. The reaction was stopped with 25 ml of MeOH and warmed to room temperature. 250 ml EtOAc and 250 ml brine were added to the reaction mixture and the organic phase was separated. The solvent was evaporated to reduce the volume of the solution. The solution was passed through 76.5 g of silica into a 350 ml fritted glass funnel. The silica plug was washed with 500 ml EtOAc/petroleum ether (1:4). The filtrate and washings were concentrated to an oily residue and dried under high vacuum to give 16.5 g of product 9 as a light yellow wax.

Reference example. Synthesis of tripeptide methyl ester 10

10

Starting from 16.5 g of alkylated dipeptide 9 (26.97 mmol), N-methylpipecolinate (MEP) (5.51 g, 1.4 eq.) and pentafluorophenol (7.63 g, 1.5 eq.). NMP (115 ml) was then added followed by EDC (7.78 g, 1.5 eq.). The mixture was stirred at room temperature overnight. 16.5 g of alkylated dipeptide 9 was dissolved in 16.5 ml of NMP, the solution was transferred to a hydrogenation flask, the residual 9 was washed with 8 ml of NMP and transferred to a hydrogenation flask. Dry 10% Pd/C (1.45, 0.05 eq.) was added. The reaction mixture was evacuated/displaced with hydrogen 3 times and the flask was shaken in an atmosphere of hydrogen (~35 psi) for 3.5 hours. The reaction mixture was analyzed by HPLC. The reaction mixture was filtered through 40 g of celite in a 350 ml fritted glass funnel and washed with 250 ml of EtOAc. The filtrate and washings were transferred to a separating funnel and washed with a solution of 1% NaHCO ₃ /10% NaCl (200 mlx3). The organic layer was isolated and dried over 45.2 g Na ₂ SO ₄ . The solution was filtered and evaporated with rotation under reduced pressure. A sticky amber residue was obtained and dried under high vacuum overnight to give 19.3 g of crude product. The crude product was dissolved in 10 ml dichloromethane, divided into two portions and purified using a 330 g Teledyne Redisep Silica Gold column. The combined fractions from the two purifications were evaporated and dried under high vacuum to give 7.64 g of 10 as a pale yellow solid (overall yield: 39% over 3 steps from compound 7).

- 38 041296

Reference example. Synthesis of tripeptide acid 11

Methyl ester 10 (6.9 g, 9.7 mmol) was dissolved in 1,2-dichloroethane (193 mL) and added to a round bottom flask equipped with a magnetic arm and condenser. To this solution was added trimethyltin hydroxide (24.6 g, 14 eq.). The mixture was heated at 70° C. for 5 hours. LC-MS analysis indicated that the desired product had been formed and <15% of the starting methyl ester 10 remained. The reaction mixture was cooled in an ice bath for 30 min. The resulting precipitate was then removed by filtration. The filtrate was stored overnight at -20°C. Then the filtrate was divided into two portions and each was subjected to the chromatography procedure carried out as follows.

Each batch was concentrated under reduced pressure and then placed under high vacuum for 30 minutes. The concentrate was then immediately dissolved in acetonitrile (95 ml). To this solution was then added an ammonium bicarbonate solution (95 ml; 50 mm, pH 7). This solution was loaded onto a Biotage SNAP C18 reverse phase cartridge (400 g, KP-C18-HS) and eluted with 50 mM ammonium bicarbonate and acetonitrile (1:1 to 100% ACN) using the Biotage chromatography system. Fractions were analyzed by LC-MS. Pure fractions were combined and ACN was removed under reduced pressure. The resulting aqueous suspension was extracted with EtOAc (3X). The combined organic layers were washed with brine, dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. Purification of two portions resulted in pure 11 (4.6 g, 65%).

Reference example. Synthesis of acetyltripeptide acid 13

In a round bottom flask, tripeptide acid 11 (3.9 g, 5.6 mmol) was dissolved in anhydrous THF (23 ml). To this solution was added complex 3 HF-TEA (1.8 ml, 2 eq.). The reaction mixture was stirred at room temperature for 1 hour. LC-MS analysis showed complete conversion to the desired product des-TES 12. The solvent was removed under reduced pressure and the residue was placed under high vacuum for 40 minutes. The resulting residue was then dissolved in pyridine (26 ml) and acetic anhydride (7.9 ml, 15 eq.) and DMAP (25 mg) were added. The reaction mixture was stirred at room temperature for 1 hour. LC-MS analysis showed complete conversion to the desired acetyltripeptide acid 13. Then a 1:1 solution of 1,4-dioxane/water (150 ml) was added to the reaction mixture. The reaction mixture was stirred for 1 h, after which the solvents were removed by evaporation with rotation in high vacuum. Toluene was added to the residue and the solvent was removed in vacuo (80 ml, 3X). The resulting crude 13 was dried under high vacuum overnight. Then the crude material was dissolved in ACN (72 ml). Then added sodium phosphate buffer (50 mm, pH 7.8, 288 ml) and the pH of the resulting suspension was brought to neutral using saturated sodium bicarbonate solution. This solution was loaded onto a Biotage SNAP C18 reverse phase cartridge (400 g, KP-C18-HS) and eluted with water and acetonitrile (20% ACN-65% ACN) using a Biotage chromatography system. Fractions were analyzed by LC-MS. Pure fractions were pooled, ACN was removed and the aqueous solution was placed in a lyophilizer resulting in purified acetyl tripeptide 13 (2.5 g, 71%).

- 39 041296

Reference example. Synthesis of (3'-nitropyridyl-2'-yl)disulfanylethyl ester of 2-(tubulisin B)hydrazinecarboxylic acid 16.

Activated Boc-Tut fragment 6 (2.63 g, 4.42 mmol, 1.1 eq.) was treated with TFA/CH ₂ Cl ₂ (42 ml; 1:1) and stirred for 30 minutes. LC-MS analysis (X-Bridge shield RP18 column, 3.5 µm; gradient 10-100% acetonitrile over 6 min, buffer pH 7.4) confirmed product formation. TFA was removed under reduced pressure, co-evaporated with CH ₂ Cl ₂ (3x30 ml) and the activated Tut derivative 14 was dried in high vacuum for 18 hours. In another flask, tripeptide acid 13 (2.51 g, 4.02 mmol) was dissolved in 70 ml CH ₂ Cl ₂ (anhydrous) and 1.48 g (8.04 mmol, 2.0 eq.) pentafluorophenol in 5 ml CH ₂ Cl ₂ was added, followed by 8.74 g (20.1 mmol, 5 0 eq.) DCC resins. The resulting reaction mixture was stirred at room temperature for 20 h. LC-MS analysis (X-Bridge shield RP18 column, 3.5 µm; gradient 10-100% acetonitrile over 6 min, buffer pH 7.4) showed a conversion of >99 %. The DCC resin was filtered off, CH ₂ Cl ₂ was removed under reduced pressure, and pentafluorophenol-activated product 15 was dried under high vacuum for 10 minutes. The residue was dissolved in 16.7 ml DMF and DIPEA (12.6 ml, 72.36 mmol, 18.0 eq.) was added. The Tut salt of trifluoroacetic acid 14 in DMF (8.5 ml) was added slowly over 5 minutes. The resulting clear solution was stirred at room temperature for 1 hour. LC-MS analysis (X-Bridge shield RR18 column, 3.5 µm; gradient 10-100% acetonitrile over 6 minutes, buffer pH 7.4) confirmed product formation. The reaction mixture was diluted with EtOAc (700 ml), washed with brine (300 ml, 2x100 ml), dried over Na ₂ SO ₄ (75 g), concentrated and dried for 15 h. The crude product was dissolved in CH ₂ Cl ₂ ( 25 ml) and loaded onto a Teledyne Redisep Gold silica gel column and eluted with MeOH/CH ₂ Cl ₂ (330 g column; 0-5% gradient) using a Combiflash chromatography system. The fractions were collected and the solvent was removed by rotary evaporation and dried. 3.91 g of 2-(tubulisin B)hydrazinecarboxylic acid (3'nitropyridyl-2'-yl)disulfanylethyl ester 16 were obtained (89% yield).

Reference example. Obtaining (pyrid-2-yl)disulfanylethyl ester of 2-(tubulizin B)hydrazinecarboxylic acid 3

Reference example. Similarly, the following compounds are prepared as described herein:

- 40 041296

EC1555

EC1568

Reference example. Additional tubulisins described herein can be isolated from natural sources, including, without limitation, from the cultivation of bacteria and other organisms. Alternatively, the tubulisins described herein can be prepared according to conventional methods, including, without limitation, those described in PCT International Publication Nos. US Patent Application Serial No. 13/841078, the disclosures of each of which are incorporated herein by reference in their entirety.

Reference example. Alternative preparation of EC1169 (compounds 112)

1) Fmoc-L-Asp( ^OfBu )-OH, ? RuVOP, DIPEA, DMF

2) Piperidine, DMF

Pts^O 3) Fmoc-L-AspiO'BiO-OH,

A RuVOP, DIPEA, DMF

4) Piperidine, DMF

5) Fmoc-4-aminomethyl-acetic acid,

RuVOP, DIPEA, DMF

6) Piperidine, DMF

7) 7, DIPEA, DMF

8) TIPS, H ₂ O, DTT, TFA

buffer, pH 7; 3/MeOH

----------------► EC1169 (connection 112)

Reference example. The following representative exemplary compounds are described to better illustrate the present invention as described herein and may be prepared according to the synthetic methods described for the examples above and/or using conventional methods.

- 41 041296

EC 1069

_CO2H

nh ₂ oh cooh

EC1183

EC1192 (C78H112N14O28S3, exact wt.: 1788.69, mol. wt.: 1790.00

EC1197 (С77Н110N14O28S3; exact mass: 1774.68; mol. mass: 1775.97)

EC1241 (С79Н114N14O28S3, exact weight: 1802.71, mol. weight: 1804.03)

EC1268 (C78H112N14O28S3, exact weight: 1788.69, mol. weight: 1790.00)

- 42 041296

- 43 041296

- 44 041296

- 45 041296

EC1719 (C79H116N14O27S3, exact wt.: 1788.73, mol. wt.: 1790.04)

EC1720 (C80H118N14O27S3, exact weight: 1802.75, mol. weight: 1804.07)

EC1721 (C81H120N14O27S3, exact weight: 1816.76, molecular weight: 1818.09)

Method example. Relative affinity analysis for PSMA. LNCaP cells are seeded in 12-well Corning Cell-BIND plates and allowed to form adherent monolayers overnight in RPMI/HIFCS. The used incubation medium is replaced with RPMI supplemented with 10% HIFCS and containing a standard PSMA binding ligand such as 100 nM 3HPMPA or a competitor compound such as EC0652, Re-EC652 or 99mTc-EC0652 in the absence or presence of increasing concentrations of test compound, such as unlabeled PMPA, or a compound described herein such as EC1169 or EC1568, a negative control intermediate lacking PSMA binding ligand, which is used as a negative control. Cells are incubated for 1 hour at 37° C. and then washed three times with 0.5 ml PBS. Five hundred microliters of 1% sodium dodecyl sulfate in PBS is added to each well; after 5 min. cell lysates are collected, transferred into separate tubes or vials containing 5 ml of scintillation cocktail, and then their radioactivity is counted. Cells exposed only to a standard PSMA binding ligand such as 3H-PMPA or a competitor compound such as 99mTc-EC0652 in FFRPMI (no competitor) are considered negative controls, while cells exposed to a standard PSMA binding ligand such both 3H-PMPA as well as 1 mM unlabeled PMPA or a competitive compound such as 99mTc-EC0652 as well as Re-EC0652 serve as positive controls. The number of disintegrations per minute (DPM) measured in the last samples (representing non-specific label binding) is subtracted from the DPM values from all samples. Relative affinities are defined as the reciprocal molar ratio of a compound required to remove 50% of a standard PSMA binding ligand such as 3H-PMPA or a competitive compound such as 99mTc-EC0652 bound to PSMA on LNCaP cells and the relative affinity of the standard PSMA binding a ligand such as PMPA or a competitor compound such as ReEC0652 is considered to be 1 for PSMA.

Method example. Dose effect analysis on PSMA+ LNCaP cells. LNCaP cells are seeded in 24-well Corning Cell-BIND plates and allowed to form nearly confluent monolayers overnight in RPMI/HIFCS. 30 minutes before the addition of a test compound, such as the compound described herein, used media is aspirated from all wells and replaced with fresh RPMI. After one wash with 1 ml of fresh RPMI/HIFCS, 1 ml of medium containing increasing concentrations of test compound is placed in each well (four wells per sample). Cells treated with test compound are pulsed for 2 h at 37°C, washed four times with 0.5 ml of medium and then purified in 1 ml of fresh medium for up to 70 h. The used medium is aspirated from all wells and replaced with fresh medium containing 5 µCi/ml 3H-thymidine. After an additional 4 hours incubation at 37° C., the cells are washed three times with 0.5 ml PBS and then treated with 0.5 ml very cold 5%

- 46 041296 trichloroacetic acid per well. After 15 min. trichloroacetic acid is sucked off and the cells are solubilized by adding 0.5 ml of 0.25 N. sodium hydroxide for 15 min. Four hundred and fifty microliters of each solubilized sample is transferred to scintillation vials containing 3 ml Ecolume scintillation cocktails and then counted in a liquid scintillation counter. The end results, presented in tabular form, are expressed as the percentage of 3 H-thymidine incorporation compared to untreated controls.

Method example. In vivo activity against PSMA+ expressing tumor implanted in mice. Four to seven week old male nu/nu mice (Harlan Sprague Dawley, Inc., Indianapolis, IN) are housed on a standard 12 hour light-dark cycle and fed ad libitum Rodent Food No. 2918 (Harlan Teklad, Madison, Wisconsin) for throughout the experiment. LNCaP cells are grown in RPMI in 10% HIFCS at 37°C in a humidified atmosphere of 5% CO ₂ /95% air, harvested and resuspended on ice in Matrigel solution (50% RPMI + 50% Matrigel at high concentration, BD#354248) to a final concentration of 1x10 ⁶ cells/50 µl. The cell solution and injection needles (28 gauge) are kept on ice prior to injection and 50 µl of the cell solution is injected into the subcutaneous layer of the dorsal central region. Mice are divided into groups of five, seven or nine, and freshly prepared solutions of test compounds are injected into the lateral tail vein under sterile conditions in phosphate-buffered saline (PBS) with a volume of 200 μl. Intravenous (iv) treatments are typically started when LNCaP tumors reach a volume of approximately 100-150 mm ³ . Mice in the control groups do not receive any treatment. The growth of each p.k. tumors are monitored by measuring the tumor three times a week during treatment and twice a week thereafter until a volume of 1500 mm ³ is reached. Tumors are measured in two perpendicular directions using a vernier caliper and their volumes are calculated as 0.5xLxW2 where L = dimension of the longest axis in mm and W = dimension of the axis perpendicular to L in mm. As a general quantitative measure of general toxicity, changes in body weight are determined on the same schedule as measurements of tumor volume. For each mouse, the maximum % weight loss due to treatment on any given day is determined. Animal survival is monitored daily. Animals in agony (or unable to reach food or water) are euthanized by CO ₂ asphyxiation.

Reference example. The relative affinity of the compounds described herein compared to the PSMA, DUPA and PMPA inhibitors. PMPA is reportedly one of the highest affinity ligands or the highest affinity ligand for PSMA. The data in FIG. 1 and FIG. 2 show that the compounds described herein exhibit higher affinity for PSMA than even PMPA.

He

PMPA DUPA

EU0652

Surprisingly, the ligands described herein have been found to have a higher affinity for PSMA than the highest known ligand, PMPA. In addition, it was unexpectedly found in this document that the conjugates of the ligands described herein have even higher affinity for PSMA.

Binding data for additional exemplary compounds described herein are shown in the following table.

- 47 041296

Reference example Relative binding affinity of PSMA (fold increase compared to PMPA=1.0) EC1080 6 EU 1067 thirty EU1100 20 EC1167 AND EC1168 17 EC1170 7 EU 1069 22 EC1183 9 EU1241 1.1 EC1303 7 EC1307 28 EC1308 20 EC1310 10 EU1584 6 EC1568 (negative control) 0

Reference example. Dose effect of the compounds described herein on PSMA+ LNCaP cells. Using a standard ³ H-thymidine incorporation assay as a quantitative measure of cytotoxicity, the data in FIG. 3 show that EC1169 exhibits dose-dependent cytotoxicity against cells in vitro with an IC ₅₀ of 13 nM. Corresponding dose-dependent cytotoxicity and IC50 values for (▼) EC1718, IC50 17.9 nM; (♦) EC 1677, IC50 20.9 nM; (A) EC1719, IC50 37.5 nM; (·) EC1720, IC50 54.2 nM; () EC1721, IC50 65.6 nM are shown in FIG. 4.

Reference example. The effect of the additional compounds described herein on LNCaP cells (2-72 h), as determined by in vitro incorporation of ³ H-thymidine into cells, is shown in the following table.

Reference example % incorporation of ³ H-thymidine EU 1069 13 nM EU1268 59.1 EU1385 184 EU1386 57 EU1387 24 EU1388 12 EU1437 thirty EU1550 22 EU1551 20 EU1452 22 EU1584 33 EU1588 42

Reference example. Activity of the compounds described herein against PSMA+ tumors in vivo. As shown in FIG. 5 treatment of nude mice bearing PSMA positive human LNCaP xenografts with EC1169 (c), EC1550 (·) and EC1551 (), each at 2 µmol/kg, TIW, 2 weeks, resulted in complete responses in all tested animals. Each compound was compared to media treated controls (♦). A complete response is observed if the tumor does not show any net growth during the treatment period of 14 days (the vertical dotted line indicates the last day of treatment). As described herein, it should be understood that the implants contain cancer cells in a matrix (total volume 100-150 mm ³ ). Since the matrix remains during the entire observation period, the reduction in tumor size cannot always be determined using an external measurement. It was also surprisingly found that treatment with the compounds described herein resulted in a cure. For example, EC1169 resulted in a cure in 2/7 of the animals tested. Cure is observed if the tumor does not grow during the entire observation period of 85 days. The data shown in FIG. 5 are the means of measurements for each cohort. Therefore, it should be understood that the increase in tumor volume starting at approximately day 40-45 represents re-growth in the remaining test animals.

Reference example. General toxicity of the compounds described in this document. As shown in FIG. 6, the observed efficacy of EC1169(c), EC1550("), and EC1551(), was in the absence of weight loss or degeneration of major organ tissues.

Reference example. Activity of the compounds described herein against PSMA+ tumors in vivo. Similarly, as shown in FIG. 7, treatment of nude mice bearing PSMA-positive human LNCaP xenografts with EC1584 (▼) and EC1588 (A), each at 2 µmol/kg, TIW, 2 weeks, resulted in complete responses in all tested animals. Each compound was compared to vehicle-treated controls (·). It was also unexpectedly found that treatment with EC1588 led to a cure in 3/7 of the tested animals.

-

Claims (2)

Reference example. General toxicity of the compounds described in this document. As shown in FIG. 8, the observed efficacy of EC1584 (▼) and EC1588 (A) was in the absence of weight loss or tissue degeneration of major organs. Reference example. The activity of the compounds described herein in relation to PSMA+ tumors in comparison with traditional chemotherapeutic agents. As shown in FIG. 9, it was found that treatment of LNCaP tumor-bearing mice with docetaxel (the most potent chemotherapeutic agent approved for prostate cancer) at 10 mg/kg, BIW, 2 weeks, MTD (▼), provided only limited anti-cancer activity and showed only 1/4 cure, even when administered at its MTD. In addition, as shown in FIG. 10, the observed limited efficacy of docetaxel was accompanied by high overall toxicity, as evidenced by severe weight loss (18%). EC1169 administered at 2 µmol/kg, TIW, 2 weeks (·), was more active and less toxic than docetaxel against PSMA+ LNCaP tumors. In FIG. 9 shows that treatment with EC1169 resulted in a complete response in all tested animals and resulted in 2/5 cures. In FIG. 10 also shows that the higher potency exhibited by EC1169 was not accompanied by significantly lower toxicity than docetaxel, providing a significantly wider therapeutic window. The potency of each compound was compared to a medium-treated control (). Reference example. In vivo efficacy () EC1718; (L) EC1720; (▼) EC1721; (♦) EC1719 and (o) EC1677 compared to (·) untreated controls is shown in FIG. 11. All compounds were administered at 2 μmol/kg, TIW for 2 weeks starting on day 21 post tumor implantation (PTI). The dotted dotted line shows the last day of treatment. The data indicate that the compounds described herein were effective in reducing tumor growth in vivo compared to untreated animals. In addition, () EC1718 resulted in a 1/7 cure; (▼) EC1721 resulted in a 1/7 cure; (♦) EC1719 resulted in 2/7 cures and (o) EC1677 resulted in 4/7 cures where no tumor regrowth was observed in these animals during the observation period. In addition, the compounds described herein did not show general toxicity in test animals, as shown in FIG. 12. Without wishing to be bound by a particular theory, it is believed herein that the weight change observed in FIG. 12, in the case of EC1718 around day 81 is associated with effects of tumor size. Reference example. The specificity of the compounds described in this document. PSMA-negative KB tumors did not respond significantly to EC1169 therapy, supporting the claim that the compounds described herein exhibit targeted specificity for PSMA-expressing cells. Reference example. hematological toxicity. The conjugates described herein demonstrate significantly improved hematological toxicity. EC1169, EC1584 and EC1588 were administered to rats i.v. at the rate of 0.33 and 0.51 µmol/kg, twice a week (BIW) for 2 weeks. Hematological toxicity in erythrocytes and leukocytes was significantly lower than in untreated controls. CLAIM 1. Formula conjugate BL-(D) _n or a pharmaceutically acceptable salt thereof; where B contains the structure where L is a polyvalent linker containing: 1) C ₁ -C ₄ alkylene or C 1 -C ₄ alkylenecarbonyl substituted with an aryl C 1 -C ₄ alkyl group, where aryl is phenyl or 2-naphthyl, and 2) C ₃ -C ₈ -cycloalkylene, C ₁ -C ₄ -alkylene-C ₃ -C ₈ -cycloalkyl or C ₃ -C ₈ -cycloalkylenecarbonyl; where L forms urea, thiourea, amide or thioamide with lysine B; where D represents the radical of a drug for the treatment of diseases caused by cells expressing PSMA, which contains a radioactive metal isotope coordinated to a chelate group; and where n is 1. 2. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein B is selected from the group consisting of: - 49 041296 3. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein B is selected from the group consisting of: COOH nn n 4. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein B is selected from the group consisting of: 5. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein L forms an amide with lysine. 6. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein L contains a chain of at least 7 atoms. 7. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein L contains a chain of at least 8 atoms. 8. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein L contains a chain of at least 9 atoms. 9. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein L contains a chain of at least 10 atoms. 10. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the linker contains a C ₁ -C ₄ alkylene carbonyl. 11. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the aryl is 2naphthyl. 12. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein B-L contains a biradical of the formula - 50 041296 13. The conjugate of the formula or its pharmaceutically acceptable salt; where B contains the structure where L is a polyvalent linker, where L forms an amide with lysine, and where L contains: 1) C1- _C4 alkylene or C1- _C4 alkylenecarbonyl substituted with an aryl-C1- _C4 alkyl group, where aryl is phenyl or 2-naphthyl, and 2) C ₃ -C 8 -cycloalkylene, C 1 -C ₄ -alkylene-C ₃ -C 8 -cycloalkyl or C ₃ -C 8 -cycloalkylenecarbonyl; and where D is a radical of a drug for the treatment of diseases caused by cells expressing PSMA, which contains a radioactive metal isotope coordinated to a chelate group. 14. Naphthyl conjugate. 15. Conjugate of cabbage soup from: 13 or a pharmaceutically acceptable salt thereof, wherein aryl is 2 of claim 13, or a pharmaceutically acceptable salt thereof, wherein B is selected from the group consisting of 16. Shchi conjugate from: 13 or a pharmaceutically acceptable salt thereof, where B is selected from the group consisting of -