USOO9662402B2

(12) United States Patent (10) Patent No.: US 9,662.402 B2 Vlahov et al. (45) Date of Patent: May 30, 2017

(54) DRUG DELIVERY CONJUGATES 4,337,339 A 6, 1982 Farina et al.

CONTAINING UNNATURAL AMNO ACDS 3. A f 3. SE"Cal C. a.l AND METHODS FOR USING 4.691,024 A 9, 1987 Shirahata 4,713,249 A 12/1987 Schroder (71) Applicant: ENDOCYTE, INC., West Lafayette, IN 4,801,688 A 1/1989 Laguzza et al. (US) 4.866, 180 A 9/1989 Vyas et al. 4,870,162 A 9, 1989 Trouet et al. (72) Inventors: Iontcho Radoslavov Vlahov, West S-36 A 22, SHA Lafayette, IN (US); Christopher Paul 5,100,883.W-1 A 3, 1992 Schiehsera C a. Leamon, West Lafayette, IN (US) 5,108,921. A 4, 1992 Low et al. 5,118,677 A 6/1992 Caufield (73) Assignee: Endocyte, Inc., West Lafayette, IN 5,118,678 A 6/1992 Kao et al. (US) 5,120,842 A 6/1992 Failli et al. 5,130,307 A 7, 1992 Falli et al. 5,138,051 A 8, 1992 H tal. (*) Notice: Subject to any disclaimer, the term of this 5,140,104 A 8, 1992 SE et al. patent is extended or adjusted under 35 5,151,413 A 9, 1992 Caufield et al. U.S.C. 154(b) by 0 days. 5,169,851 A 12/1992 Hughes et al. 5,194,447 A 3, 1993 Kao (21) Appl. No.: 14/.435.919 5,221,670 A 6/1993 Caufield 9 5,233,036 A 8/1993 Hughes (22) PCT Filed: Oct. 15, 2013 5,258,389 A 11/1993 Goulet et al. e - a 9 (Continued) 86) PCT NO.: PCT/US2013/065079 (86) O FOREIGN PATENT DOCUMENTS S 371 (c)(1), (2) Date: Apr. 15, 2015 CA 2372841 11 2000 CA 2376175 12/2000 (87) PCT Pub. No.: WO2014/062697 (Continued) PCT Pub. Date: Apr. 24, 2014 OTHER PUBLICATIONS (65) Prior Publication Data Beil, Prevention, updated on Mar. 29, 2010, available via internet at: US 2015/O2582O3 A1 Sep. 17, 2015 http://www.nbcnews.com/id/35874922/ns/health-diet and nutri s tion/t/your-breakfast-giving-you-canceri.V40lrflV21.* O O Adessi et al., Current Medicinal Chemistry, 2002, 9,963-978.* Related U.S. Application Data Agoston E.S. et al., “Vitamin D Analogs as Anti-Carcinogenic (60) Provisional application No. 61/714,565, filed on Oct Agents.” Anti-Cancer Agents in Medicinal Chemistry, 2006; 6(1): 16,s 2012, provisional application No. 6877,317.01/s/ ?. s Anderson' et al., “Potocytosis: Sequestration and transport of Small filed on Sep. 13, 2013, provisional application No. molecules by caveolae.” Science, 1992; 255; 410-411. 61/865,382, filed on Aug. 13, 2013, provisional Antony A.C., “Folate receptors.” Annu Rev Nutr, 1996; 16:501-21. application No. 61/790,234, filed on Mar. 15, 2013. Antony A.C., “The biological chemistry of folate receptors,” Blood, 1992; 79(11):2807-2820. (51) Int. Cl. Antony A.C. et al., “Studies of the Role of a Particulate Folate A6 IK 38/08 (2006.01) binding Protein in the Uptake of 5-Methyltetrahydrofolate by Cul A6 IK 47/48 (2006.01) tured Human KB Cells,” J. Biological Chem., 1985; 260(28): C07K 7/06 (2006.01) 14911-7. (52) U.S. Cl. (Continued) CPC. A61K 47/48092(2013,01); (2013.01); CozK A61K 706 (2013.01)47/48107 Primary Examiner Hasan Ahmed (58) Field of Classification Search Assistant Examiner - Kaipeen Yang CPC ...... A61K 47/48107; A61K 47/48092; CO7K (74) Attorney, Agent, or Firm — Barnes & Thornburg 7/06 LLP See application file for complete search history. (57) ABSTRACT (56) References Cited Described herein are drug delivery conjugates for targeted therapy. In particular, described herein are drug delivery U.S. PATENT DOCUMENTS conjugates that include polyvalent linkers comprising one or 2.515.483. A 7, 1950 Wolf et al. more unnatural amino acids that are useful for treating 2.816,110 A 12/1957 Sletzinger et al. cancers and inflammatory diseases. The invention described 3,387,001 A 6/1968 Hargrove et al. herein pertains to drug delivery conjugates for targeted 3,392,173 A 7/1968 Hargrove et al. therapy. In particular, the invention described herein pertains SE6 A 33 E. (al. tO drug delivery conjugates that include polyvalent linkers 4,166,810 A 9, 1979 Cullinan et al. compr1S1ng one or more unnatural amino acids. 4,203,898 A 5, 1980 Cullinan et al. 4,316,885. A 2f1982 Rakhit 2 Claims, 22 Drawing Sheets US 9,662.402 B2 Page 2

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s & & se c e s is 3uble M Apogul e6ueup's, US 9,662,402 B2 1. 2 DRUG DELIVERY CONUGATES thereof. In another embodiment, methods for using the CONTAINING UNNATURAL AMNO ACDS compounds and pharmaceutical compositions for treating AND METHODS FOR USING patients or host animals with cancer, inflammation, and the like are also described herein. In one aspect, the methods CROSS REFERENCE TO RELATED include the step of administering one or more of the com APPLICATIONS pounds and/or compositions described herein to a patient with cancer, inflammation, and the like. In another aspect, This application is a U.S. national stage application under the methods include administering a therapeutically effec 35 U.S.C. S371 (b) of International Application No. PCT/ tive amount of the one or more compounds and/or compo US2013/065079 filed Oct. 15, 2013, and claims priority 10 sitions described herein for treating patients with cancer, under 35 U.S.C. S 119(e) to U.S. Provisional Application Ser. inflammation, and the like. In another embodiment, uses of No. 61/714,565 filed Oct. 16, 2012, U.S. Provisional Appli the compounds and compositions in the manufacture of a cation Ser. No. 61/790,234 filed Mar. 15, 2013, U.S. Pro medicament for treating patients with cancer, inflammation, visional Application Ser. No. 61/865.382 filed Aug. 13, and the like are also described herein. In one aspect, the 2013, and U.S. Provisional Application Ser. No. 61/877,317 15 medicaments include a therapeutically effective amount of filed Sep. 13, 2013. The disclosures of all the above refer the one or more compounds and/or compositions for treating enced applications are incorporated herein by reference in a patient with cancer, inflammation, and the like. their entirety. BRIEF DESCRIPTION OF THE DRAWINGS TECHNICAL FIELD FIG. 1A shows the relative affinity of EC1669 in KB cells, The invention described herein pertains to drug delivery 1 h at 37° C. FIG. 1B shows the relative affinity of EC1669 conjugates for targeted therapy. In particular, the invention in CHO-3 cells, 1 h at 37° C. described herein pertains to drug delivery conjugates that FIG. 2 shows the cytostatic effect of EC1669 on include polyvalent linkers comprising one or more unnatural 25 RAW264.7 cells, as determined by XTT cell viability at 2h amino acids. and 72 h. FIG. 3A shows in vivo activity of EC1456 against KB BACKGROUND AND SUMMARY OF THE tumors in nu/nu mice dosed at 1 umol/kg three times per INVENTION week (M/W/F) (TIW) for two consecutive weeks (O), 30 compared to EC1456 co-dosed with EC0923 at 100 umol/kg The mammalian immune system provides a means for the (AA), and untreated (PBS) controls (). The dotted vertical recognition and elimination of pathogenic cells. Such as line represents the day of the final dose. FIG. 3B shows that tumor cells, and other invading foreign pathogens. While the EC1456 did not result in any observable whole animal immune system normally provides a strong line of defense, toxicity as determined by animal body weight. there are many instances where pathogenic cells, such as 35 FIG. 4A shows the activity of EC1663 in nu/nu mice cancer cells, and other infectious agents evade a host bearing s.c. KB tumors, where EC1663 was administered i.v. immune response and proliferate or persist with concomitant starting on Day 7 with 0.5 umol/kg (A), three times per host pathogenicity. Chemotherapeutic agents and radiation week (M/W/F) for a 2 week period, and compared to therapies have been developed to eliminate, for example, untreated controls (), N=5 animals per cohort. Dotted replicating neoplasms. However, many of the currently 40 vertical line-day of final dosing day. FIG. 4A shows 4/4 PRs available chemotherapeutic agents and radiation therapy in test animals. FIG. 4B shows that EC1663 did not exhibit regimens have adverse side effects because they lack suffi significant host animal toxicity. cient selectivity to preferentially destroy pathogenic cells, FIG. 5A shows the activity of EC1456 against established and therefore, may also harm normal host cells, such as cells subcutaneous MDA-MB-231 tumors. Animals bearing s.c. of the hematopoietic system, and other non-pathogenic cells. 45 MDA-MB-231 tumors (94-145 mm) were treated i.v. start The adverse side effects of these anticancer drugs highlight ing on Day 17 with 2 umol/kg (panel A) of EC1456 (O), the need for the development of new therapies selective for three times per week (M/W/F) for a 2 week period, and pathogenic cell populations and with reduced host toxicity. compared to untreated animals (), as shown in FIG. 5A. It has been discovered herein that drug delivery conju N=5 animals per cohort. Dotted vertical line-day of final gates that include polyvalent linkers formed from one or 50 dose. FIG. 5B shows that EC1456 did not cause gross whole more unnatural amino acids are efficacious in treating patho animal toxicity as determined by % weight change. genic cell populations, and exhibit low host animal toxicity. FIG. 6A shows the activity of EC1456 in animals bearing In one illustrative and non-limiting embodiment of the s.c. KB-CR2000 (cisplatin resistant) tumors (98-148 mm3). invention, described herein are compounds of the formula where EC1456 was administered i.v. starting on Day 6 with 55 2 umol/kg (O), three times per week (M/W/F) for a 2 week period, or with 3 mg/kg of cisplatin (A), twice per week wherein each of B, L, D, and X are as defined in the various (T/Th) for a 2 week period, and compared to untreated embodiments and aspects described herein. controls (), N=5 animals per cohort. Dotted vertical In another embodiment, pharmaceutical compositions line-day of final dosing day. FIG. 6B shows that EC1456 did containing one or more of the compounds are also described 60 not exhibit significant host animal toxicity. In contrast, herein. In one aspect, the compositions include a therapeu cisplatin treatment resulted in Substantial host animal tox tically effective amount of the one or more compounds for icity during the dosing period. treating a patient with cancer, inflammation, and the like. It FIG. 7 shows the in vivo efficacy of EC1496 against is to be understood that the compositions may include other adjuvant-induced arthritis. The arrows indicate the treatment components and/or ingredients, including, but not limited to, 65 days. (a) healthy control, (b) untreated control, (c) EC1496, other therapeutically active compounds, and/or one or more (d) EC1496+excess EC0923 (comparator/competition com carriers, diluents, excipients, and the like, and combinations pound). US 9,662,402 B2 3 4 FIG. 8A shows the in vivo efficacy of EC1669 against FIG. 15 shows the in vivo efficacy of EC1496 against arthritis, (a) healthy control, (a) untreated control, (b) EAE. Individual EAE scores from untreated animals and EC1669 (375 nmol/kg), (c) EC1669+500x EC0923. FIG.8B animals treated with EC1496 and EC1496 plus EC0923 shows that EC1669 does not exhibit whole animal toxicity, every other day starting on day 8 after EAE induction are shown, and compared to untreated control. (a) untreated control, (b) EC1669 (375 nmol/kg), (c) FIG. 16A shows the pharmacokinetics of EC1496 (500 EC1669+500x EC0923, (d) healthy control. nmol/kg, s.c.), and in vivo production of aminopterin and FIG. 9A shows the in vivo efficacy of EC1669 against aminopterin hydrazide. FIG. 16B shows the pharmacokinet arthritis, as determined by paw swelling. FIG.9B shows the ics of ECO746 (comparator compound, 500 nmol/kg, s.c.), in vivo efficacy of EC1669 against arthritis, as determined and in vivo production of aminopterin and aminopterin by bone radiography. 10 hydrazide. FIG. 10A shows the in vivo efficacy of EC1669 alone, and FIG. 17 shows the pharmacokinetic biodistribution of EC 1669 plus CellCept combination co-therapy in AIA rats, H-EC1669, panels (A), (C), and (E); and H-methotrexate where day 0 is 9 days post induction, and the arrows indicate in mice, panels (B), (D), and (F). Test compounds were treatment days, (a) healthy control, (b) untreated control, (c) administered to Balb/c mice at 500 nmol/kg, s.c. EC1669 (1000 nmol/kg, siw, sc), (d) CellCept TM (30 mg/kg, 15 FIG. 18 shows the comparison of RBC uptake of po, qdx5), (e) EC1669+CellCeptTM, FIG. 10B shows the H-EC1669 () and H-MTX (V) in mice, as a measure of whole animal toxicity compared to control for each of the radioactivity over time. FIG. 19 shows the relative whole animal toxicity between administration protocols. (b) EC1496 (3 umol/kg) and (c) EC0746 (comparator com FIG. 11 shows the in vivo efficacy of EC1669 alone, and pound, 3 umol/kg)), and compared to vehicle control (a) EC 1669 plus CellCept combination co-therapy in AIA rats, when dosed BIW for 2 weeks in folate deficient rats. as determined by paw Swelling. FIG. 20 shows the maximum tolerated dose (MTD) of FIG. 12A shows the in vivo efficacy of EC1669 against EC1456 compared to vehicle controls. Vehicle control (O), EAU (total uveitis scores for both eyes). Animals are treated EC1456 at 0.33 umol/kg (), EC1456 at 0.41 umol/kg (A), with EC1669 (), EC1669 plus EC0923 (D), and MTX (0) 25 EC1456 at 0.51 umol/kg (V), and EC1456 at 0.67 umol/kg every other day starting on day 8 after EAU induction or (0). from untreated animals (O). Day 0 is 8 days post induction, and the arrows indicate treatment days. FIG. 12B shows that DETAILED DESCRIPTION EC 1669 does not cause whole animal toxicity. FIG. 13A shows the in vivo efficacy of EC1496 against Several illustrative embodiments of the invention are EAU (total uveitis scores for both eyes), (a) uveitis untreated 30 described by the following clauses: control, (b) EC1496 (375 nmol/kg), (c) EC1496+excess A compound of the formula B-L(D), or a pharmaceuti EC0923. FIG. 13B shows the in vivo efficacy of EC1496 cally acceptable salt thereof, wherein B is a radical of a cell against EAU, as determined by histology. Surface receptor binding and/or targeting ligand, D is in each FIG. 14A shows the in vivo efficacy of EC1669 against instance a radical of an independently selected drug, X is an EAE, (a) untreated EAE control, (b) EC1669 (250 nmol/kg), 35 integer selected from 1, 2, 3, 4 and 5; and L is a polyvalent (c) EC1669+excess EC0923. Animals are treated every releasable linker comprising one or more unnatural amino other day (as indicated by arrows) starting on day 8 after acids; and where B is covalently attached to L, and L is EAE induction, and compared to untreated control. FIG. covalently attached to each D; and 14B shows the percent changes in body weight (B), aver where the compound is not any one of or any Subgroup or aged for each group. subset of the following formulae

US 9,662,402 B2 10

Á000?I

O Oº--CS

ponunuoo CHN NIH ~^pH

NSS ºr H| NN NO NIH ~~ US 9,662,402 B2 11 12 88000€I

O

ponunuoo

US 9,662,402 B2 15 16

09ZOOFI

ponunuoo

O

N H O H N ! O

O US 9,662,402 B2 17 18 and/or where the compound is not of the following formula

ECO153 HN YN OH

Nin

sulluSa O O CO2H H N-- N ~us N H H 1N-ns-n- O COH 5. H2

and/or where the compound is not any one of or any subgroup or subset of the following formulae

US 9,662,402 B2 21 22

0£000EI

ponunuoo

O

O

US 9,662,402 B2 27 28 and/or where the compound is not any one of or any The compound of any one of the preceding clauses subgroup or subset of the following formulae wherein the ligand is a folate comprising D-glutamyl, also

ECOO82 CO2H O HOC O H H N N CO2H O N N H H N O O HN s N S H a H.N. N. N. NH in- NH2 ECOO83

CO2H O HOC O 1 H H N N. CO2H O N N H H N O O HN n N S H a HN ls N N NH

ECOO84

O HOC O 1

ins,

and/or where the compound is not of the following referred to herein as D-folate, or pteroyl-D-glutamic acid. It formula is to be understood herein that when B is a radical of

ECOO60 HO

S Ows" - HN N N S Y S O HN 2 N -CO2H O N O O N

E NH E H 1. N-1sE O O COH NH O COH

and/or any combination of the foregoing; D-folate, the included D-glutamyl portion of B is not part of or any pharmaceutically acceptable salt thereof. the linker L. The compound of any one of the preceding clauses The compound of the preceding clause wherein B-L(D) 60 wherein B is an unnatural folate radical of the formula is capable of binding to the cell surface receptor. The compound of any one of the preceding clauses O CO2H wherein the ligand is a vitamin receptor binding ligand. O N The compound of any one of the preceding clauses H O wherein the ligand is a folate receptor binding ligand. 65 HN N N The compound of any one of the preceding clauses ls a H wherein the ligand is a folate. HN N N US 9,662,402 B2 29 30 The compound of any one of the preceding clauses The compound of any one of the preceding clauses wherein the ligand is folic acid. wherein L further comprises one or more divalent polyoxy The compound of any one of the preceding clauses radicals. wherein B is a radical of the formula The compound of any one of the preceding clauses wherein L further comprises two or more divalent polyoxy radicals. O CO2H The compound of any one of the preceding clauses wherein L further comprises three or more divalent polyoxy O H N-- radicals. N O 10 HN n N The compound of any one of the preceding clauses H ls a wherein L further comprises four or more divalent polyoxy H.N. N. N. radicals. The compound of any one of the preceding clauses wherein L further comprises one or more divalent polyhy The compound of any one of the preceding clauses 15 droxy radicals. wherein at least one unnatural amino acid has the D-con The compound of any one of the preceding clauses figuration. wherein L further comprises two or more divalent polyhy The compound of any one of the preceding clauses droxy radicals. wherein at least one unnatural amino acid is selected from The compound of any one of the preceding clauses D-alanine, D-aspartic acid, D-asparagine, D-cysteine, wherein L further comprises three or more divalent polyhy D-glutamic acid, D-phenylalanine, D-histidine, D-isoleu droxy radicals. cine, D-lysine, D-leucine, D-methionine, D-proline, D-glu The compound of any one of the preceding clauses tamine, D-arginine, D-serine, D-threonine, D-valine, wherein L further comprises four or more divalent polyhy D-tryptophan, D-tyrosine, and D-ornithine, and any amino 25 droxy radicals. acid derivatives thereof. The compound of any one of the preceding clauses The compound of any one of the preceding clauses wherein at least one unnatural amino acid comprises a wherein at least one unnatural amino acid is selected from polyhydroxy radical. D-aspartic acid, D-asparagine, D-cysteine, D-glutamic acid, The compound of any one of the preceding clauses D-histidine, D-lysine, D-methionine, D-glutamine, D-argi 30 wherein at least two unnatural amino acids comprise a nine, D-serine, D-threonine, D-tryptophan, D-tyrosine, and polyhydroxy radical. D-ornithine, and any amino acid derivatives thereof. The compound of any one of the preceding clauses The compound of any one of the preceding clauses wherein at least three unnatural amino acids comprise a wherein at least one unnatural amino acid is selected from polyhydroxy radical. D-aspartic acid, D-asparagine, D-cysteine, D-glutamic acid, 35 The compound of any one of the preceding clauses D-histidine, D-lysine, D-glutamine, D-arginine, D-serine, wherein at least four unnatural amino acids comprise a D-threonine, D-tryptophan, and D-ornithine, and any amino polyhydroxy radical. acid derivatives thereof. The compound of any one of the preceding clauses wherein at least one of the polyhydroxy radicals is of the The compound of any one of the preceding clauses formula wherein at least one unnatural amino acid is selected from 40 D-aspartic acid, D-cysteine, D-glutamic acid, D-lysine, D-arginine, D-serine, and D-ornithine, and any amino acid derivatives thereof. where n is selected from 1, 2, 3, 4, 5, and 6. The compound of any one of the preceding clauses The compound of any one of the preceding clauses wherein L comprises two or more unnatural amino acids. 45 wherein n is selected from 1, 2, 3, and 4. The compound of any one of the preceding clauses The compound of any one of the preceding clauses wherein L comprises three or more unnatural amino acids. wherein n is selected from 3 and 4. The compound of any one of the preceding clauses The compound of any one of the preceding clauses wherein L comprises four or more unnatural amino acids. wherein n is 3. The compound of any one of the preceding clauses 50 The compound of any one of the preceding clauses wherein L further comprises one or more disulfides. wherein L comprises a divalent polyglutamic acid radical, The compound of any one of the preceding clauses where at least one glutamic acid forms an amide with an wherein at least one disulfide comprises L-cysteinyl. aminopolyhydroxy radical. The compound of any one of the preceding clauses The compound of any one of the preceding clauses wherein at least one disulfide comprises D-cysteinyl. 55 wherein L comprises a divalent polyglutamic acid radical, The compound of any one of the preceding clauses where at least two glutamic acids form an amide with an wherein L further comprises one or more divalent hydro aminopolyhydroxy radical. philic radicals. The compound of any one of the preceding clauses The compound of any one of the preceding clauses wherein L comprises a divalent polyglutamic acid radical, wherein L further comprises two or more divalent hydro 60 where at least three glutamic acids form an amide with an philic radicals. aminopolyhydroxy radical. The compound of any one of the preceding clauses The compound of any one of the preceding clauses wherein L further comprises three or more divalent hydro wherein L comprises a divalent polyglutamic acid radical, philic radicals. where at least four glutamic acids form an amide with an The compound of any one of the preceding clauses 65 aminopolyhydroxy radical. wherein L further comprises four or more divalent hydro The compound of any one of the preceding clauses philic radicals. wherein at least one of the glutamic acids is D-glutamic acid. US 9,662,402 B2 31 32 The compound of any one of the preceding clauses The compound of any one of the preceding clauses wherein at least two of the glutamic acids is D-glutamic wherein L comprises a divalent radical of the formula acid. The compound of any one of the preceding clauses wherein at least three of the glutamic acids is D-glutamic The compound of any one of the preceding clauses acid. wherein L-D comprises a radical of the formula The compound of any one of the preceding clauses wherein at least four of the glutamic acids is D-glutamic acid. The compound of any one of the preceding clauses 10 The compound of any one of the preceding clauses wherein at least one of the glutamic acids is unsubstituted wherein L-D comprises a radical of the formula D-glutamic acid. The compound of any one of the preceding clauses wherein at least two of the glutamic acids is unsubstituted The compound of any one of the preceding clauses D-glutamic acid. 15 wherein X is 3. The compound of any one of the preceding clauses The compound of any one of the preceding clauses wherein at least three of the glutamic acids is unsubstituted wherein X is 2. D-glutamic acid. The compound of any one of the preceding clauses The compound of any one of the preceding clauses wherein X is 1. wherein at least four of the glutamic acids is unsubstituted The compound of any one of the preceding clauses D-glutamic acid. wherein at least one drug is a cytotoxic agent. The compound of any one of the preceding clauses The compound of any one of the preceding clauses wherein L comprises a divalent poly(D-glutamic acid) radi wherein at least one drug is a cancer treating agent. cal, where at least one glutamic acid forms an amide with an The compound of any one of the preceding clauses aminopolyhydroxy radical. 25 wherein at least one drug is a Vinca alkaloid. The compound of any one of the preceding clauses The compound of any one of the preceding clauses wherein L comprises a divalent poly(D-glutamic acid) radi wherein at least one drug is desacetylvinblastine monohy cal, where at least two glutamic acids form an amide with an drazide. aminopolyhydroxy radical. The compound of any one of the preceding clauses The compound of any one of the preceding clauses 30 wherein at least one drug is a tubulysin. wherein L comprises a divalent poly(D-glutamic acid) radi The compound of any one of the preceding clauses cal, where at least three glutamic acids form an amide with wherein at least one drug is tubulysin A. an aminopolyhydroxy radical. The compound of any one of the preceding clauses The compound of any one of the preceding clauses wherein at least one drug is tubulysin B. wherein L comprises a divalent poly(D-glutamic acid) radi 35 The compound of any one of the preceding clauses cal, where at least four glutamic acids form an amide with wherein at least one drug is tubulysin A hydrazide. an aminopolyhydroxy radical. The compound of any one of the preceding clauses The compound of any one of the preceding clauses wherein at least one drug is tubulysin B hydrazide. wherein L comprises a divalent radical of the formula The compound of any one of the preceding clauses (K-L), where K is a divalent D-glutamic acid radical, L is 40 wherein at least one drug is a tubulysin where the Tuv a divalent L-glutamic acid radical that forms an amide with residue includes an ether aminal. an aminopolyhydroxy radical, and d is 1, 2, 3, or 4. The compound of any one of the preceding clauses The compound of the preceding clause wherein d is 2, 3, wherein at least one drug is a tubulysin hydrazide where the or 4. TuV residue includes an ether aminal. The compound of the preceding clause wherein d is 3 or 45 The compound of any one of the preceding clauses 4. wherein at least one drug is a inflammation-treating agent. The compound of the preceding clause wherein d is 3. The compound of any one of the preceding clauses The compound of any one of the preceding clauses wherein at least one drug is an anti-inflammatory agent. wherein at least one of the aminopolyhydroxy radicals is of The compound of any one of the preceding clauses the formula 50 wherein at least one drug is a dihydrofolate reductase inhibitor. NH-CH2-(CHOH)), CH-OH The compound of any one of the preceding clauses where m is selected from 1, 2, 3, 4, 5, and 6. wherein at least one drug is aminopterin or methotrexate. The compound of any one of the preceding clauses The compound of any one of the preceding clauses wherein at least one of the aminopolyhydroxy radicals is of 55 wherein at least one drug is an aminopterin. the formula The compound of any one of the preceding clauses wherein at least one drug is an inhibitor of mammalian target of rapamycin (mTOR). where m is selected from 1, 2, 3, 4, 5, and 6; and R is H, alkyl, cycloalkyl, or arylalkyl. The compound of any one of the preceding clauses 60 wherein at least one drug is sirolimus (rapamycin), temsi The compound of any one of the preceding clauses rolimus, everolimus, or ridaforolimus. wherein m is selected from 1, 2, 3, and 4. The compound of any one of the preceding clauses The compound of any one of the preceding clauses wherein at least one drug is not T-2 mycotoxin. wherein m is selected from 3 and 4. The compound of any one of the preceding clauses The compound of any one of the preceding clauses 65 wherein at least one drug is not a duocarmycin. wherein L comprises a divalent radical of the formula The compound of any one of the preceding clauses wherein at least one drug is not a mitomycin. US 9,662,402 B2 33 34 The compound of any one of the preceding clauses The compound of any one of the preceding clauses wherein at least one drug is not desacetylvinblastine mono hydrazide. wherein at least one D is a radical of the formula

AcO N-1 O

NH X-K O) ~s O AcO N-1 O -cri ---,

25 The compound of any one of the preceding clauses wherein at least one D is a radical of the formula

50 The compound of any one of the preceding clauses wherein at least one D is a radical of the formula

HO

O AC g O N N N \ -->J US 9,662,402 B2 35 36 -continued HO

E O w N \ Ny-n-r s s N O u

O AcO N-1 O N s w N y-n-r N O ii \ S u O O

where n=1, 2, 3, 4, 5, or 6, or alternatively, n=1, 2, or 3, or The compound of any one of the preceding clauses alternatively, n=2 or 3. wherein at least one D is a radical of the formula

HO

O Aco N-1 o N Y HN N \ S.-->J N US 9,662,402 B2 37 38 -continued HO

O Acg N1 O N N- s HN w N \ yr- N O S O O

in 1's--

where n=1, 2, 3, 4, 5, or 6, or alternatively, n=1, 2, or 3, or to or reacting with a nucleic acid or a DNA transcription alternatively, n=2 or 3. factor, or a prodrug thereof. The compound of any one of the preceding clauses * The compound of any one of the preceding clauses wherein at least one drug is a compound capable of binding wherein B-L is a radical of the formula

US 9,662,402 B2 39 40 The compound of any one of the preceding clauses wherein B-L is a radical of the formula

2O The compound of any one of the preceding clauses wherein B-L is a radical of the formula

The compound of any one of the preceding clauses wherein B-L is a radical of the formula

US 9,662,402 B2 41 42 The compound of any one of the preceding clauses wherein the compound is of the formula EC1456

N. a C CO2H H Q H Q 1 H Q CO2H NH ar s1N O Na-N-N-N-N-N-Na-S-S-N-6 N NH \- ) 6 N H r H r H r H O 1a1s HNSNHNS N N Orl, NH O1l. NH O l. NH O ROH OH OH HO'N\OH HO'N\OH HO'N\OH HO HO HO OH HO HO or a pharmaceutically acceptable salt thereof. The compound of any one of the preceding clauses wherein the compound is not of the formula EC1456

C CO2H H Q H 9 H Q CO2H S-NH N.y^ : O Na-N-N-N-N-N-NS-S-SN-6 N NH \, JJ HN Ns1 N r H H r H O -n-sO 1. H H.N. N. N. OY NH OY NH O1 NH OH OH OH HO'N\OH HO'N\OH HO'N\OH HO HO HO OH HO HO

35 or a pharmaceutically acceptable salt thereof. The compound of any one of the preceding clauses wherein the compound is of the formula EC1496

COH CO2H H 2N NNH; C CO2H H Q - H 9 - H C CO2H H. O H Nu I HN ON-Ns1N O''''''',O O O 's--OO H a 6 NH J., ii HONO H.N. N. N. OY NH OY NH OY NH OH OH OH HO'N\OH HO'N\OH HONAOH HO HO HO OH HO HO or a pharmaceutically acceptable salt thereof. The compound of any one of the preceding clauses wherein the compound is not of the formula EC1496 CO2H CO2H H 2N NNH; Q CO2H H Q - H Q - H Q CO2H O H Nu I HNNNs1NO O'''''''',O O O 's--OO H 1s. (5 NH J. H HONO HN N N Or NH O NH O NH OH OH OH IONAOH HO'N\OH HON4OH HO HO HO US 9,662,402 B2 43 44 or a pharmaceutically acceptable salt thereof. The compound of any one of the preceding clauses wherein the compound is of the formula EC 1669 ol ot H al NNH; C CO2H H Q H Q H Q CO2H O H Nui HN O Ns1N cy''''',O O O 's--OO H HO-so () NH H.N.J.l. N. N. ii O1 NH O1 NH O1 NH OH OH OH O'N\OH HO'N\OH HO'N\OH HO HO HO OH HO HO or a pharmaceutically acceptable salt thereof. The compound of any one of the preceding clauses wherein the compound is not of the formula EC 1669

C OH O COH - O u HNNNs1N N ~y N r N ls fi O's l. Hoso O HN N N luO NH Or NH O NH OH OH OH HOY OH HON' OH HOY OH HO HO HO OH HO HO or a pharmaceutically acceptable salt thereof. 35 The method or composition or unit dose or use of any one A pharmaceutical composition comprising a compound of of the preceding clauses wherein the cancer is drug resistant any one of the preceding clauses in combination with one or CaCC. more carriers, diluents, or excipients, or a combination The method or composition or unit dose or use of any one thereof. 40 of the preceding clauses wherein the cancer is a platinum A unit dose or unit dosage form composition comprising resistant cancer. a therapeutically effective amount of one or more com The method or composition or unit dose or use of any one pounds of any one of the preceding clauses, optionally in of the preceding clauses wherein the cancer is a cisplatin combination with one or more carriers, diluents, or excipi resistant cancer. ents, or a combination thereof. 45 The method or composition or unit dose or use of any one A composition for treating cancer or inflammation in a of the preceding clauses wherein the cancer is an ovarian host animal, the composition comprising a therapeutically CaCC. effective amount of one or more compounds of any one of The method or composition or unit dose or use of any one the preceding clauses; or a pharmaceutical composition of the preceding clauses wherein the cancer is a drug comprising a therapeutically effective amount of one or 50 resistant ovarian cancer. more compounds of any one of the preceding clauses, The method or composition or unit dose or use of any one optionally further comprising one or more carriers, diluents, of the preceding clauses wherein the cancer is a cisplatin or excipients, or a combination thereof. resistant ovarian cancer. A method for treating cancer or inflammation in a host The method or composition or unit dose or use of any one animal, the method comprising the step of administering to 55 of the preceding clauses wherein the cancer is a platinum the host animal a composition comprising a therapeutically resistant ovarian cancer, such as NCI/ADR-RES or NCI/ effective amount of one or more compounds of any one of ADR-RES related ovarian cancer. the preceding clauses; or a pharmaceutical composition The method or composition or unit dose or use of any one comprising one or more compounds of any one of the of the preceding clauses wherein the cancer is a platinum preceding clauses, optionally further comprising one or 60 resistant ovarian cancer, such as IGROVCDDP or more carriers, diluents, or excipients, or a combination IGROVCDDP related ovarian cancer. thereof. The method or composition or unit dose or use of any one Use of one or more compounds of any one of the of the preceding clauses wherein the cancer is a breast preceding clauses, optionally in combination with one or CaCC. more carriers, diluents, or excipients, or a combination 65 The method or composition or unit dose or use of any one thereof, in the manufacture of a medicament for treating a of the preceding clauses wherein the cancer is a drug cancer or inflammation in a host animal. resistant breast cancer. US 9,662,402 B2 45 46 The method or composition or unit dose or use of any one The method or composition or unit dose or use of any one of the preceding clauses wherein the cancer is a triple of the preceding clauses wherein the cancer is a hepatocel negative breast cancer, such as MDA-MB-231 or MDA lular carcinoma or cancer. MB-231 related breast cancer. The method or composition or unit dose or use of any one 5 of the preceding clauses wherein the cancer is a non-Small An intermediate for preparing a compound of any one of cell lung cancer. the preceding clauses of the formula CO2H CO2H O CO2H O s- O s- O CO2H "Y- L

HN N N O O O

ls r HN N N O

or a pharmaceutically acceptable salt thereof, wherein L is a leaving group. An intermediate for preparing a compound of any one of the preceding clauses of the formula COH COH - O - O O CO2H 5 CO2H

H N ------N- HN n N 1s a r O D. O D. O D. H.N. N. N. O NH O NH O NH

or a pharmaceutically acceptable salt thereof, wherein M is hydrogen or a cation. An intermediate for preparing a compound of claim 1 of the formula

CO2H CO2H

O CO2H o - o - CO2H H H H N O N Nulls N r N ulN H H H -- N O O O HN n N H HN ls N - N 2 US 9,662,402 B2 47 48 or a pharmaceutically acceptable salt thereof, wherein L is a animals. In accordance with the invention pathogenic cells' leaving group. means cancer cells, infectious agents such as bacteria and In another embodiment, the compounds described herein viruses, bacteria- or virus-infected cells, activated macro can be internalized into the targeted pathogenic cells by phages capable of causing a disease state, other pathogenic binding to the corresponding cell Surface receptor. In par cells causing inflammation, any other type of pathogenic ticular, vitamin receptors, such as folate receptors, selec cells that uniquely express, preferentially express, or over tively and/or specifically bind the vitamin, and internaliza express vitamin receptors or receptors that bind vitamins tion can occur, for example, through receptor-mediated and/or vitamin receptor binding ligands, and any other type endocytosis. Once internalized, the releasable linker of pathogenic cells that uniquely express, preferentially included in the compounds described herein allows for the 10 delivery of the drug cargo to the interior of the target cell, express, or overexpress high affinity folate receptors or thus decreasing toxicity against non-target tissues because receptors that bind folates and/or folate receptor binding the releasable linker remains substantially or completely ligands. Pathogenic cells can also include any cells causing intact until the compounds described herein are delivered to a disease state for which treatment with the compounds the target cells. Accordingly, the compounds described 15 described herein results in reduction of the symptoms of the herein act intracellularly by delivering the drug to an intra disease. For example, the pathogenic cells can be host cells cellular biochemical process, a decrease the amount of that are pathogenic under some circumstances Such as cells unconjugated drug exposure to the host animals healthy of the immune system that are responsible for graft versus cells and tissues. host disease, but not pathogenic under other circumstances. In another embodiment, compounds described herein that Thus, the population of pathogenic cells can be a cancer include a folate receptor binding ligand exhibit greater cell population that is tumorigenic, including benign tumors specificity for the folate receptor compared to the corre and malignant tumors, or it can be non-tumorigenic. The sponding compounds that do not include at least one unnatu cancer cell population can arise spontaneously or by Such ral amino acid. In another embodiment, compounds processes as mutations present in the germline of the host described herein that include afolate receptor binding ligand 25 animal or Somatic mutations, or it can be chemically show high activity for folate receptor expressing cells. In virally-, or radiation-induced. The invention can be utilized another embodiment, compounds described herein exhibit to treat Such cancers as carcinomas, sarcomas, lymphomas, potent in vitro and in vivo activity against pathogenic cells, Hodgekin’s disease, melanomas, mesotheliomas, Burkitt's Such as KB cells, including cisplatin resistant KB cells, lymphoma, nasopharyngeal carcinomas, leukemias, and NC1/ADR-RES-C1, cells, IGROV1 cells, and MDA-MB 30 myelomas. The cancer cell population can include, but is not 231 cells. In another embodiment, compounds described herein that include a folate receptor binding ligand do not limited to, oral, thyroid, endocrine, skin, gastric, esophageal, show significant binding to folate receptor negative cells. In laryngeal, pancreatic, colon, bladder, bone, ovarian, cervi another embodiment, compounds described herein that cal, uterine, breast, testicular, prostate, rectal, kidney, liver, include a folate receptor binding ligand enter cells prefer 35 and lung cancers. entially or exclusively via the high affinity folate receptors, In another embodiment, the method or pharmaceutical Such as folate receptor alpha (C.) and/or folate receptor beta composition of any one of the preceding embodiments (B). In another embodiment, compounds described herein wherein the disease is selected from the group consisting of generally do not substantially enter cells via passive trans arthritis, including rheumatoid arthritis and osteoarthritis, port, such as via the reduced folate carrier (RFC). In another 40 glomerulonephritis, proliferative retinopathy, restenosis, embodiment, compounds described herein exhibit lower ulcerative colitis, Crohn's disease, fibromyalgia, psoriasis host animal toxicity compared to compounds that do not and other inflammations of the skin, osteomyelitis, include at least one unnatural amino acid. In another Sjögren's syndrome, multiple Sclerosis, diabetes, atheroscle embodiment, compounds described herein exhibit greater rosis, pulmonary fibrosis, lupus erythematosus, Sarcoidosis, serum stability compared to compounds that do not include 45 systemic sclerosis, organ transplant rejection (GVHD) and at least one unnatural amino acid. In another embodiment, chronic inflammations is described. compounds described herein are cleared rapidly compared to The drug can be any molecule capable of modulating or compounds that do not include at least one unnatural amino otherwise modifying cell function, including pharmaceuti acid. In another embodiment, compounds described herein cally active compounds. Illustrative drugs include, but are are cleared primarily via renal clearance compared to 50 not limited to, peptides, oligopeptides, retro-inverso oligo hepatic clearance. peptides, proteins, protein analogs in which at least one The compounds described herein can be used for both non-peptide linkage replaces a peptide linkage, apoproteins, human clinical medicine and Veterinary applications. Thus, glycoproteins, enzymes, coenzymes, enzyme inhibitors, the host animal harboring the population of pathogenic cells amino acids and their derivatives, receptors and other mem and treated with the compounds described herein can be 55 brane proteins; antigens and antibodies thereto; haptens and human or, in the case of veterinary applications, can be a antibodies thereto; hormones, lipids, phospholipids, lipo laboratory, agricultural, domestic, or wild animal. The pres Somes; toxins; antibiotics; analgesics; bronchodilators; beta ent invention can be applied to host animals including, but blockers; antimicrobial agents; antihypertensive agents; car not limited to, humans, laboratory animals such rodents diovascular agents including antiarrhythmics, cardiac (e.g., mice, rats, hamsters, etc.), rabbits, monkeys, chimpan 60 glycosides, antianginals and vasodilators; central nervous Zees, domestic animals such as dogs, cats, and rabbits, system agents including stimulants, psychotropics, antiman agricultural animals such as cows, horses, pigs, sheep,goats, ics, and depressants; antiviral agents; antihistamines; cancer and wild animals in captivity Such as bears, pandas, lions, drugs including chemotherapeutic agents; tranquilizers; tigers, leopards, elephants, Zebras, giraffes, gorillas, dol anti-depressants; H-2 antagonists; anticonvulsants; antinau phins, and whales. 65 seants; prostaglandins and prostaglandin analogs; muscle The invention is applicable to populations of pathogenic relaxants; anti-inflammatory Substances; immunosuppres cells that cause a variety of pathologies in these host sants, stimulants; decongestants; antiemetics: diuretics; anti US 9,662,402 B2 49 50 spasmodics; antiasthmatics; anti-Parkinson agents; expecto and their derivatives, receptors and other membrane pro rants; cough Suppressants; mucolytics; and mineral and teins, antigens and antibodies thereto, haptens and antibod nutritional additives. ies thereto, hormones, lipids, phospholipids, liposomes, tox Further, the drug can be one that is cytotoxic, enhances ins, antibiotics, analgesics, bronchodilators, beta-blockers, tumor permeability, inhibits tumor cell proliferation, pro antimicrobial agents, antihypertensive agents, cardiovascu motes apoptosis, decreases anti-apoptotic activity in target lar agents including antiarrhythmics, cardiac glycosides, cells, is used to treat diseases caused by infectious agents, antianginals, vasodilators, central nervous system agents enhances an endogenous immune response directed to the including stimulants, psychotropics, antimanics, and depres pathogenic cells, or is useful for treating a disease state sants, antiviral agents, antihistamines, cancer drugs includ caused by any type of pathogenic cell. Additional illustrative 10 ing chemotherapeutic agents, tranquilizers, anti-depressants, drugs include adrenocorticoids and corticosteroids, alkylat H-2 antagonists, anticonvulsants, antinauseants, prostaglan ing agents, antiandrogens, antiestrogens, androgens, dins and prostaglandin analogs, muscle relaxants, anti-in aclamycin and aclamycin derivatives, estrogens, antime flammatory Substances, stimulants, decongestants, antiemet tabolites such as cytosine arabinoside, purine analogs, ics, diuretics, antispasmodics, antiasthmatics, anti pyrimidine analogs, and methotrexate, buSulfan, carbopla 15 Parkinson agents, expectorants, cough Suppressants, tin, chlorambucil, cisplatin and other platinum compounds, mucolytics, and mineral and nutritional additives. tamoxiphen, taxol, paclitaxel, paclitaxel derivatives, Taxo In another embodiment, at least one additional composi tereR), cyclophosphamide, daunomycin, rhizoxin, T2 toxin, tion comprising a therapeutic factor can be administered to plant alkaloids, prednisone, hydroxyurea, teniposide, mito the host in combination or as an adjuvant to the above mycins, discodermolides, microtubule inhibitors, epoth detailed methodology, to enhance the drug delivery conju ilones, tubulysins, cyclopropylbenzelindolone, seco-cyclo gate-mediated elimination of the population of pathogenic propyl benzelindolone, O—Ac-seco-cyclopropyl benze cells, or more than one additional therapeutic factor can be indolone, bleomycin and any other antibiotic, nitrogen administered. The therapeutic factor can be selected from a mustards, nitroSureas, Vinca alkaloids, such as Vincristine, compound capable of stimulating an endogenous immune vinblastine, Vindesine, Vinorelbine and analogs and deriva 25 response, a chemotherapeutic agent, or another therapeutic tive thereof such as deacetylvinblastine monohydrazide factor capable of complementing the efficacy of the admin (DAVLBH), colchicine, colchicine derivatives, allocolchi istered drug delivery conjugate. The method of the invention cine, thiocolchicine, trityl cysteine, halicondrin B. dolasta can be performed by administering to the host, in addition to tins such as dolastatin 10, amanitins such as C.-amanitin, the above-described conjugates, compounds or composi camptothecin, irinotecan, and other camptothecin deriva 30 tions capable of stimulating an endogenous immune tives thereof, geldanamycin and geldanamycin derivatives, response (e.g. a cytokine) including, but not limited to, estramustine, nocodazole, MAP4, colcemid, inflammatory cytokines or immune cell growth factors such as interleukins and proinflammatory agents, peptide and peptidomimetic 1-18, stem cell factor, basic FGF, EGF, G-CSF, GM-CSF, signal transduction inhibitors, and any other drug or toxin. FLK-2 ligand, HILDA, MIP-1C, TGF-C., TGF-B, M-CSF, Other drugs that can be included in the conjugates described 35 IFN-C, IFN-?3. IFN-y, soluble CD23, LIF, and combinations herein include rapamycins, such as Sirolimus or everolimus, thereof. penicillins, cephalosporins, Vancomycin, erythromycin, Therapeutically effective combinations of these factors clindamycin, rifampin, chloramphenicol, aminoglycoside can be used. In one embodiment, for example, therapeuti antibiotics, gentamicin, amphotericin B, acyclovir, trifluri cally effective amounts of IL-2, for example, in amounts dine, ganciclovir, Zidovudine, amantadine, ribavirin, and 40 ranging from about 0.1 MIU/m/dose/day to about 15 MIU/ any other antimicrobial compound. m/dose/day in a multiple dose daily regimen, and IFN-O. In another embodiment, the drug is selected from cryp for example, in amounts ranging from about 0.1 MIU/m/ tophycins, bortezomib, thiobortezomib, tubulysins, aminop dose/day to about 7.5 MIU/m/dose/day in a multiple dose terin, rapamycins, paclitaxel, docetaxel, doxorubicin, daily regimen, can be used along with the drug delivery daunorubicin, everolimus, C.-amanatin, Verucarin, didemnin 45 conjugates to eliminate, reduce, or neutralize pathogenic B, geldanomycin, purvalanol A, ispinesib, budesonide, dasa cells in a host animal harboring the pathogenic cells tinib, epothilones, maytansines, and tyrosine kinase inhibi (MIU-million international units; m—approximate body tors, including analogs and derivatives of the foregoing. Surface area of an average human). In another embodiment In another embodiment, the compounds described herein IL-12 and IFN-O. are used in the above-described therapeu include at least two drugs (D), which are illustratively 50 tically effective amounts for interleukins and interferons, selected from Vinca alkaloids, cryptophycins, bortezomib, and in yet another embodiment IL-15 and IFN-C. are used in thiobortezomib, tubulysins, aminopterin, rapamycins, such the above described therapeutically effective amounts for as everolimus and sirolimus, paclitaxel, docetaxel, doxoru interleukins and interferons. In an alternate embodiment bicin, daunorubicin, C.-amanatin, Verucarin, didemnin B, IL-2, IFN-O. or IFN-Y, and GM-CSF are used in combination geldanomycin, purvalanol A, ispinesib, budesonide, dasat 55 in the above described therapeutically effective amounts. inib, epothilones, maytansines, and tyrosine kinase inhibi The invention also contemplates the use of any other effec tors, including analogs and derivatives of the foregoing. In tive combination of cytokines including combinations of one variation, the drugs (D) are the same. In another other interleukins and interferons and colony stimulating variation, the drugs (D) are different. factors. The drug delivery conjugate compounds described herein 60 Chemotherapeutic agents, which are, for example, cyto can be administered in a combination therapy with any other toxic themselves or can work to enhance tumor permeability, known drug whether or not the additional drug is targeted. are also suitable for use in the method of the invention in Illustrative additional drugs include, but are not limited to, combination with the drug delivery conjugate compounds. peptides, oligopeptides, retro-inverso oligopeptides, pro Such chemotherapeutic agents include adrenocorticoids and teins, protein analogs in which at least one non-peptide 65 corticosteroids, alkylating agents, antiandrogens, antiestro linkage replaces a peptide linkage, apoproteins, glycopro gens, androgens, aclamycin and aclamycin derivatives, teins, enzymes, coenzymes, enzyme inhibitors, amino acids estrogens, antimetabolites such as cytosine arabinoside, US 9,662,402 B2 51 52 purine analogs, pyrimidine analogs, and methotrexate, chain in each of the foregoing illustrative embodiments may buSulfan, carboplatin, chlorambucil, cisplatin and other be either Saturated or unsaturated, thus forming single, platinum compounds, tamoxiphen, taxol, paclitaxel, pacli double, or triple bonds, such that for example, alkanes, taxel derivatives, TaxotereR), cyclophosphamide, daunomy alkenes, alkynes, imines, and the like may be radicals that cin, rhizoxin, T2 toxin, plant alkaloids, prednisone, are included in the linker. In addition, it is to be understood hydroxyurea, teniposide, mitomycins, discodermolides, that the atoms forming the linker may also be cyclized upon microtubule inhibitors, epothilones, tubulysin, cyclopropyl each other or be part of cyclic structure to form divalent benzelindolone, seco-cyclopropyl benzelindolone, cyclic structures that form the linker, including cyclo O—Ac-seco-cyclopropyl benzelindolone, bleomycin and 10 alkanes, cyclic ethers, cyclic amines, and other heterocycles, any other antibiotic, nitrogen mustards, nitroSureas, Vincris arylenes, heteroarylenes, and the like in the linker. In this tine, vinblastine, and analogs and derivative thereof Such as latter arrangement, it is to be understood that the linker deacetylvinblastine monohydrazide, colchicine, colchicine length may be defined by any pathway through the one or derivatives, allocolchicine, thiocolchicine, trityl cysteine, more cyclic structures. Illustratively, the linker length is Halicondrin B. dolastatins such as dolastatin 10, amanitins 15 Such as C.-amanitin, camptothecin, irinotecan, and other defined by the shortest pathway through the each one of the camptothecin derivatives thereof, geldanamycin and geldan cyclic structures. It is to be understood that the linkers may amycin derivatives, estramustine, nocodazole, MAP4, col be optionally Substituted at any one or more of the open cemid, inflammatory and proinflammatory agents, peptide Valences along the chain of atoms, such as optional Sub and peptidomimetic signal transduction inhibitors, and any stituents on any of the carbon, nitrogen, silicon, or phos other art-recognized drug or toxin. Other drugs that can be phorus atoms. It is also to be understood that the linker may used in accordance with the invention include penicillins, connect the two or more functional parts of a molecule to cephalosporins, Vancomycin, erythromycin, clindamycin, form a conjugate at any open Valence, and it is not necessary rifampin, chloramphenicol, aminoglycoside antibiotics, gen that any of the two or more functional parts of a molecule tamicin, amphotericin B, acyclovir, trifluridine, ganciclovir, 25 forming the conjugate are attached at any apparent end of the Zidovudine, amantadine, ribavirin, maytansines and analogs linker. and derivatives thereof, gemcitabine, and any other art In another embodiment, a folate-linker radical is recognized antimicrobial compound. described having the following formula

NH-6 O COH (1, AA O N pi N N O iii. HN ls 2 R HN N N

As used herein, the term “linker' includes is a chain of wherein m, n, and q are integers that are independently atoms that connects two or more functional parts of a selected from the range of 0 to about 8: AA is an amino acid, molecule to form a conjugate. Illustratively, the chain of R" is hydrogen, alkyl, or a nitrogen protecting group, and 45 drugs are optionally attached at the () atoms. In one aspect, atoms is selected from C, N, O, S, Si, and P. or C, N, O, S, AA is a naturally occurring amino acid of either the natural and P. C, N, O, and S. The chain of atoms covalently or unnatural configuration. In another aspect, one or more of connects different functional capabilities of the conjugate, AA is a hydrophilic amino acid. In another aspect, one or Such as binding ligands, drugs, diagnostic agents, imaging more of AA is Asp and/or Arg. In another aspect, the integer agents, and the like. The linker may have a wide variety of 50 n is 1 or greater. In another aspect, the integer n is 2 or lengths, such as in the range from about 2 to about 100 atoms greater. In another aspect, the integer n is 3 or greater. In another aspect, the integer n is 4 or greater. In another aspect, in the contiguous backbone. The atoms used in forming the the integer n is 5 or greater. In another aspect, the integer q linker may be combined in all chemically relevant ways, is 1 or greater. In another aspect, the integer q is 1. In another Such as chains of carbon atoms forming alkylene, alk aspect, the integer m is 1 or greater. In another aspect, the enylene, and alkynylene groups, and the like; chains of 55 integer m is 1. In another aspect, R' is hydrogen. The drugs carbon and oxygen atoms forming ethers, polyoxyalkylene and optionally additional linkers and additional receptor groups, or when combined with carbonyl groups forming binding ligands may be connected to the above formula at esters and carbonates, and the like; chains of carbon and the free NH side chains of the 2,0)-diaminoalkanoic acid nitrogen atoms forming amines, imines, polyamines, hydra fragments, or at the terminal carboxylate as indicated by the 60 free valences therein. It is to be understood that every Zines, hydrazones, or when combined with carbonyl groups combination of the foregoing aspects is described herein as forming amides, ureas, semicarbazides, carbazides, and the further illustrative embodiments of the invention. For like; chains of carbon, nitrogen, and oxygen atoms forming example, in another embodiment, n is 1 or greater, and m is alkoxyamines, alkoxylamines, or when combined with car one or greater, or n is 1 or greater, m is 1, and q is 1; and so bonyl groups forming urethanes, amino acids, acyloxylam 65 forth. ines, hydroxamic acids, and the like; and many others. In In another embodiment, a folate-linker radical is addition, it is to be understood that the atoms forming the described having the following formula US 9,662,402 B2 53 54

N: O CO2H S*

O N pi H AA N Sl p N O H iii. CO:s HN n N O 2 ls 2 R1 HN N N wherein m, n, q, and p are integers that are independently 1 or greater. In another aspect, the integer m is 1. In another selected from the range of 0 to about 8: AA is an amino acid, aspect, R' is hydrogen. The drugs and optionally additional R" is hydrogen, alkyl, or a nitrogen protecting group, and 15 linkers and additional receptor-binding ligands may be con drugs are optionally attached at the () atoms. In one aspect, nected to the above formula at the free NH side chains of the AA is as a naturally occurring amino acid of either the 2.()-diaminoalkanoic acid fragments, at the cysteinyl thiol natural or unnatural configuration. In another aspect, one or groups, or at the terminal carboxylate, as indicated by the more of AA is a hydrophilic amino acid. In another aspect, free valences therein. It is to be understood that every one or more of AA is Asp and/or Arg. In another aspect, the 20 combination of the foregoing aspects is described herein as integer n is 1 or greater. In another aspect, the integer n is 2 further illustrative embodiments of the invention. For or greater. In another aspect, the integer n is 3 or greater. In example, in another embodiment, n is 1 or greater, and m is another aspect, the integer n is 4 or greater. In another aspect, one or greater, or n is 2 or greater, m is 1, and q is 1; or n the integer n is 5 or greater. In another aspect, the integers is 2 or greater, m is 1. q is 1, and p is 1; and so forth. q and/or p are 1 or greater. In another aspect, the integer 25 In another embodiment, a folate-linker radical is integers q and/or pare 1. In another aspect, the integer m is described having the following formula

N: S* O CO2H (1, H O (1. AA N O N in NN N CO* H N O iii. )r HN n O *O ls 2 R1 HN N N wherein m, n, q, p, and rare integers that are independently 40 In another embodiment, the polyvalent linker includes R"selected is hydrogen, from the alkyl,range ofor 0a tonitrogen about 8: protecting AA is an aminogroup, acid, and herein,one or alsomore called divalent linkers hydrophilic or spacer linkers.radicals, It isas appreciated described drugs are optionally attached at the () atoms. In one aspect, that the arrangement and/or orientation of the various hydro AA is as a naturally occurring amino acid of either the philic linkers may be in a linear or branched fashion, or both. natural or unnatural configuration. In another aspect, one or as For example, the hydrophilic linkers may form the backbone more of AA is a hydrophilic amino acid. In another aspect, one or more of AA is Asp and/or Arg. In another aspect, the of the linker forming the conjugate between the ligand and integer n is 1 or greater. In another aspect, the integer n is 2 the one or more drugs. Alternatively, the hydrophilic portion or greater. In another aspect, the integer n is 3 or greater. In of the linker may be pendant to or attached to the backbone another aspect, the integer n is 4 or greater. In another aspect, of the chain of atoms connecting the binding ligand B to the the integer n is 5 or greater. In another aspect, the integers one or more drugs D. In this latter arrangement, the hydro q and/or p and/or r are 1 or greater. In another aspect, the philic portion may be proximal or distal to the backbone integers q and/or p and/or r are 1. In another aspect, the chain of atoms. integer m is 1 or greater. In another aspect, the integer m is In another embodiment, the linker is more or less linear, 1. In another aspect, R' is hydrogen. The drugs and option and the hydrophilic groups are arranged largely in a series to ally additional linkers and additional receptor-binding 55 ------ligands may be connected to the above formula at the free form a chain-like linker in the conjugate. Said another way, NH side chains of the 2.()-diaminoalkanoic acid fragments, the hydrophilic groups form some or all of the backbone of at the cyteinylthiol groups, at the serinyl hydroxy groups, or the linker in this linear embodiment. at the terminal carboxylate, as indicated by the free valences In another embodiment, the linker is branched with therein. It is to be understood that every combination of the 60 hydrophilic groups. In this branched embodiment, the foregoing aspects is described herein as further illustrative hydrophilic groups may be proximal to the backbone or embodiments of the invention. For example, in another distal to the backbone. In each of these arrangements, the embodiment, n is 1 or greater, and m is one or greater, or n linker is more spherical or cylindrical in shape. In one is 2 or greater, m is 1, and q is 1; or n is 2 or greater, m is variation, the linker is shaped like a bottle-brush. In one 1, q is 1, and p is 1; or n is 2 or greater, m is 1. q is 1, and 65 aspect, the backbone of the linker is formed by a linear series r is 1; or n is 2 or greater, m is 1. q is 1. p is 1, and r is 1: of amides, and the hydrophilic portion of the linker is and so forth. formed by a parallel arrangement of branching side chains, US 9,662,402 B2 55 56 Such as by connecting monosaccharides, Sulfonates, and the lidines, piperidines, piperazines, and other amino groups, like, and derivatives and analogs thereof. each of which is optionally substituted. In another embodi It is understood that the linker may be neutral or ionizable ment, the regions of the linkers that are neutral include poly under certain conditions, such as physiological conditions hydroxyl groups. Such as Sugars, carbohydrates, saccharides, inositols, and the like, and/or polyether groups, such as encountered in vivo. For ionizable linkers, under the polyoxyalkylene groups including polyoxyethylene, poly selected conditions, the linker may deprotonate to form a oxypropylene, and the like. negative ion, or alternatively become protonated to form a In one embodiment, the hydrophilic spacer linkers positive ion. It is appreciated that more than one deproto described herein include are formed primarily from carbon, nation or protonation event may occur. In addition, it is hydrogen, and oxygen, and have a carbon/oxygen ratio of understood that the same linker may deprotonate and pro 10 about 3:1 or less, or of about 2:1 or less. In one aspect, the tonate to form inner salts or Zwitterionic compounds. hydrophilic linkers described herein include a plurality of In another embodiment, the hydrophilic spacer linkers are ether functional groups. In another aspect, the hydrophilic neutral, an in particular neutral under physiological condi linkers described herein include a plurality of hydroxyl tions, the linkers do not significantly protonate nor depro functional groups. Illustrative fragments and radicals that tonate. In another embodiment, the hydrophilic spacer link 15 may be used to form such linkers include polyhydroxyl ers may be protonated to carry one or more positive charges. compounds such as carbohydrates, polyether compounds It is understood that the protonation capability is condition Such as polyethylene glycol units, and acid groups such as dependent. In one aspect, the conditions are physiological carboxyl and alkyl Sulfuric acids. In one variation, oligo conditions, and the linker is protonated in vivo. In another amide spacers, and the like may also be included in the embodiment, the spacers include both regions that are neu linker. tral and regions that may be protonated to carry one or more Illustrative divalent hydrophilic linkers include carbohy positive charges. In another embodiment, the spacers drates Such as saccharopeptides as described herein that include both regions that may be deprotonated to carry one include both a peptide feature and Sugar feature; glu or more negative charges and regions that may be protonated curonides, which may be incorporated via 2+3 Huisgen to carry one or more positive charges. It is understood that 25 cyclization, also known as click chemistry; B-alkyl glyco in this latter embodiment that Zwitterions or inner salts may sides, such as of 2-deoxyhexapyranoses (2-deoxyglucose, be formed. 2-deoxyglucuronide, and the like), and B-alkyl mannopyra In one aspect, the regions of the linkers that may be nosides. Illustrative PEG groups include those of a specific deprotonated to carry a negative charge include carboxylic length range from about 4 to about 20 PEG groups. Illus acids, such as aspartic acid, glutamic acid, and longer chain 30 trative alkyl sulfuric acid esters may also be introduced with carboxylic acid groups, and sulfuric acid esters, such as click chemistry directly into the backbone. Illustrative oli alkyl esters of Sulfuric acid. In another aspect, the regions of goamide spacers include EDTA and DTPA spacers, B-amino the linkers that may be protonated to carry a positive charge acids, and the like. include amino groups, such as polyaminoalkylenes includ In another embodiment, the polyvalent linker L comprises ing ethylene diamines, propylene diamines, butylene 35 one or more polyethers, such as the linkers of the following diamines and the like, and/or heterocycles including pyrol formulae:

O

NH O Y), Q Y), Q H s N s N N HO 1sH H O )n S r O HOC )n HOC

H H -N-N-N-N-N-N-N-N-N-N-N-N-p r N Y. s1” US 9,662,402 B2 57 58 where m is an integer independently selected in each -continued instance from 1 to about 8; p is an integer selected 1 to about 10; and n is an integer independently selected in each O H gOH instance from 1 to about 3. In one aspect, m is independently : N in each instance 1 to about 3. In another aspect, n is 1 in each 5 S instance. In another aspect, p is independently in each instance about 4 to about 6. Illustratively, the corresponding HO 1-4N. polypropylene polyethers corresponding to the foregoing are OH contemplated herein and may be included in the conjugates OH as hydrophilic spacer linkers. In addition, it is appreciated HO that mixed polyethylene and polypropylene polyethers may 10 p be included in the conjugates as hydrophilic spacer linkers. Further, cyclic variations of the foregoing polyether com HOC )m O CO2H pounds, such as those that include tetrahydrofuranyl, 1.3- H dioxanes, 1,4-dioxanes, and the like are contemplated N S 15 : N herein. H N * N In another embodiment, the polyvalent linker L comprises a plurality of hydroxyl functional groups, such as linkers that O tion, incorporate monosaccharides, oligosaccharides, polysaccha R rides, and the like. It is to be understood that the polyhy p droxyl containing spacer linkers comprises a plurality of HOC —(CROH)—groups, where R is hydrogen or alkyl. )m O In another embodiment, the polyvalent linker L comprises H N COH one or more of the following fragments: : N E H S 25 O on NN1y-N, HO H O H HO N : OH : N1 p H 30 HOC tion.R p O H : N 35

N 1." HO H OH

OH 40 HO wherein 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 from 2 to about 5. p is an integer from 1 to about 5, and 45 r is an integer selected from 1 to about 3. In one aspect, the integer n is 3 or 4. In another aspect, the integer p is 3 or 4. HO In another aspect, the integer r is 1. In another embodiment, the polyvalent linker L comprises HO one or more of the following fragments: 50

H N O : H N : 55 H toR it. p toR it. O p H CO2H : N s 60 O CO2H S H 1N-N. N S HO H OH : 1-4N. (HOCH) OH 65 HO US 9,662,402 B2

-continued -continued HOC O H COH )m O CO2H : N H N S S : N n N 1N1 n : N : H H * HO H O (HOCH) O R 10 HO OH wherein 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 from 2 to about 5. p is an integer from 1 to about 5, and r is an integer selected from 1 to about 3. In one aspect, the 15 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 polyvalent linker L comprises one or more of the following cyclic polyhydroxyl groups:

25

30 HO OH

OH

HO 35

40

45 HOC O H CO2H N E : N S

50 O H HO O HO N H 55 HO OH

HO OH HOC

: N 60 H

65 US 9,662,402 B2 61 62 -continued wherein in and rare each an integer selected from 1 to about HOC 3. In one aspect, the linker includes one or more polyhy O droxyl compounds of the following formulae: H N CO2H : N E H S O N 1N1 n : s: O OH s: O OH HO H Z Z O O O - N 10 HO HN NH OH HO OH p

OH wherein n is an integer from 2 to about 5. p is an integer from 15 1 to about 5, and r is an integer from 1 to about 4. In one O OH O OH aspect, the integer n is 3 or 4. In another aspect, the integer 4 21 p is 3 or 4. In another aspect, the integer r is 2 or 3. It is understood that all stereochemical forms of such sections of HO N1 HO OH the linkers are contemplated herein. For example, in the H OH HN above formula, the section may be derived from ribose, n Xylose, glucose, mannose, galactose, or other Sugar and retain the stereochemical arrangements of pendant hydroxyl and alkyl groups present on those molecules. In addition, it is to be understood that in the foregoing formulae, various 25 deoxy compounds are also contemplated. Illustratively, compounds of the following formulae are contemplated: 21 O OH

30 NN OH H OH

It is understood that all stereochemical forms of such 35 sections of the linkers are contemplated herein. For example, in the above formula, the section may be derived from ribose, Xylose, glucose, mannose, galactose, or other Sugar and retain the stereochemical arrangements of pendant 40 hydroxyl and alkyl groups present on those molecules. In another embodiment, the polyvalent linker L comprises one or more polyhydroxyl groups that are spaced away from the backbone of the linker. In one embodiment, such car bohydrate groups or polyhydroxyl groups are connected to 45 the back bone by a triazole group, forming triazole-linked hydrophilic spacer linkers. Illustratively, the linker includes fragments of the following formulae:

50 HO HO

O HO

55 O OH wherein n is equal to or less than r, Such as when r is 2 or O 3. n is 1 or 2, or 1, 2, or 3, respectively. In another embodiment, the polyvalent linker L comprises ). one or more polyhydroxyl radicals of the following formula: N 60 : MN . Ns (1. N / |-d). (OH) 65 US 9,662,402 B2 63 64 -continued -continued OH OH OH OH OH OH OH OH O O HO O HO O HO O OH HO O OH O O OH O O OH O

). O 10 N N 9. : (r l, MN Ns O (1. N S 9. 15 H N 1. N S al N N N N O O s 2 N 2 i )n O : 4 N M. HOC Ns O N lu : pi rs Sri HOC O )n O 25 HOC wherein n, m, and rare integers and are each independently selected in each instance from 1 to about 5. In one illustra tive aspect, m is independently 2 or 3 in each instance. In wherein in and r are integers and are each independently another aspect, r is 1 in each instance. In another aspect, in selected in each instance from 1 to about 5; and p is an is 1 in each instance. In one variation, the group connecting 30 integer selected from 1 to about 4. the polyhydroxyl group to the backbone of the linker is a In another embodiment, the polyvalent linker L comprises different heteroaryl group, including but not limited to, one or more carbohydrate groups or polyhydroxyl groups pyrrole, pyrazole, 1,2,4-triazole, furan, oxazole, isoxazole, connected to the back bone by an amide group, forming thienyl, thiazole, isothiazole, oxadiazole, and the like. Simi amide-linked hydrophilic spacer linkers. Illustratively, such larly, divalent 6-membered ring heteroaryl groups are con 35 linkers include fragments of the following formulae: templated. Other variations of the foregoing illustrative hydrophilic spacer linkers include oxyalkylene groups, such as the following formulae: HO HO 40 HO

45

50 COH O

HO CO2H 55

O m( O

60 : NH

65 US 9,662,402 B2 65 66 -continued -continued OH OH HO HO HO HO r HO O O "YSz O 5 m( O

10

Ns NH 15 a 1 : 1. wherein n is an integer selected from 1 to about 3, and m is n( s N an integer selected from 1 to about 22. In one illustrative r H aspect, n is 1 or 2. In another illustrative aspect, m is selected CO2H O from about 6 to about 10, illustratively 8. In one variation, the group connecting the polyhydroxyl group to the back bone of the linker is a different functional group, including but not limited to, esters, ureas, carbamates, acylhydrazones, and the like. Similarly, cyclic variations are contemplated. Other variations of the foregoing illustrative hydrophilic 25 wherein in and r are integers and are each independently spacer linkers include oxyalkylene groups. Such as the selected in each instance from 1 to about 5; and p is an following formulae: integer selected from 1 to about 4. In another embodiment, the polyvalent linker L comprises 30 one or more of the following fragments: HO HO HO

35

40

45 pi 1" N

O H : N : N 1. H O O )m p p HN O

60 HO : NH Ns ng OH OH pi ( N. HO rH 65 CO2H O OH

US 9,662,402 B2 70 -continued -continued

HOC )m CO2H

N : S S : -- N N N-n-rin. H O O R N- p 10 HN

HOC 15

HOC )m O 25 COH H N S : N 1YN. O wherein R is H. alkyl, cycloalkyl, or arylalkyl: m is an independently selected integer from 1 to about 3; n is an O )m integer from 2 to about 6, p is an integer from 1 to about 5.

and r is an integer selected 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. 35 In another embodiment, the polyvalent linker L comprises one or more of the following fragments:

40 wherein R is H. alkyl, cycloalkyl, or arylalkyl: m is an independently selected integer from 1 to about 3; n is an 45 integer from 1 to about 6, p is an integer from 1 to about 5. and r is an integer selected 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 polyvalent linker L comprises 50 one or more of the following fragments:

55

60 HO OH

OH HO 65

US 9,662,402 B2 74 -continued -continued

HOC COH

: S : HN os- N H (H2C) H

10

15

HOC ) O 25 iii. CO2H H N S : HN wherein m is an independently selected integer from 1 to O about 3; n is an integer from 2 to about 6, p is an integer from 30 1 to about 5, and r is an integer selected from 1 to about 3. O )m 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 polyvalent linker L comprises 35 one or more of the following fragments:

O H : N 40 N 1" O m H wherein m is an independently selected integer from 1 to about 3; n is an integer from 1 to about 6, p is an integer from HN p 1 to about 5, and r is an integer selected from 1 to about 3. In one variation, the integer n is 3 or 4. In another variation, HO the integer p is 3 or 4. In another variation, the integer r is OH 1. O In another embodiment, the polyvalent linker L comprises 50 HO one or more of the following fragments:

55

60

65 OH US 9,662,402 B2 75 76 -continued -continued

HOC

10

15

2O HOC N

25 : NH

HO OH 30 HO

OH

35

40 HOC

: N 45 H

50

55

wherein m is an independently selected integer from 1 to 60 about 3; n is an integer from 1 to about 6, p is an integer from 1 to about 5, and r is an integer selected 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. 65 In another embodiment, the polyvalent linker L comprises a combination of backbone and branching side motifs Such as is illustrated by the following formulae US 9,662,402 B2 78

In addition, in this illustrative embodiment, the pyranose includes both carboxyl and amino functional groups and (a) can be inserted into the backbone and (b) can provide synthetic handles for branching side chains in variations of this embodiment. Any of the pendant hydroxyl groups may be used to attach other chemical fragments, including addi tional Sugars to prepare the corresponding oligosaccharides. Other variations of this embodiment are also contemplated, including inserting the pyranose or other Sugar into the 10 backbone at a single carbon, i.e. a spiro arrangement, at a geminal pair of carbons, and like arrangements. For example, one or two ends of the linker, or the drug D, or the binding ligand B may be connected to the Sugar to be wherein n is an integer independently selected in each 15 inserted into the backbone in a 1,1; 1.2: 13; 14; 2.3, or other instance from 0 to about 3. The above formula are intended arrangement. to represent 4, 5, 6, and even larger membered cyclic Sugars. In another embodiment, the hydrophilic spacer linkers In addition, it is to be understood that the above formula may described herein include are formed primarily from carbon, be modified to represent deoxy Sugars, where one or more of hydrogen, and nitrogen, and have a carbon/nitrogen ratio of the hydroxy groups present on the formulae are replaced by 20 about 3:1 or less, or of about 2:1 or less. In one aspect, the hydrogen, alkyl, or amino. In addition, it is to be understood hydrophilic linkers described herein include a plurality of that the corresponding carbonyl compounds are contem amino functional groups. plated by the above formulae, where one or more of the In another embodiment, the polyvalent linker L comprises hydroxyl groups is oxidized to the corresponding carbonyl. one or more amino groups of the following formulae:

H N --~~ O E 1 n-rrN-s-s-s-s O CO2H O H N 1 n-1N O CO2H O CO2H O

COH O CO2H O COH

CO2H CO2H O ( , O ( ),

CO2H CO2H :

S CO2H Y), O ( pi H HOC 1N N pi S-r N-n- ls.CO2H --CCO2H O r

CO2H O COH O N Crrrr US 9,662,402 B2 79 80 -continued H H

pi pi

: CO2H O NO O NN 1SN1 COH O COH O

: CO2H O r Sr.'ss :s-N- N1N N- O COH O COH O CO2H H H where n is an integer independently selected in each instance It is understood, that in Such polyhydroxyl, polyamino, from 1 to about 3. In one aspect, the integer n is indepen carboxylic acid, Sulfuric acid, and like linkers that include dently 1 or 2 in each instance. In another aspect, the integer free hydrogens bound to heteroatoms, one or more of those n is 1 in each instance. free hydrogen atoms may be protected with the appropriate In another embodiment, the polyvalent linker L comprises hydroxyl, amino, or acid protecting group, respectively, or one or more Sulfuric acid esters, such as an alkyl ester of sulfuric acid. Illustratively, the linker includes the following alternatively may be blocked as the corresponding pro drugs, the latter of which are selected for the particular use, formula(e): 25 Such as pro-drugs that release the parent drug under general or specific physiological conditions. HO In another embodiment, the polyvalent linker comprises ye one or more of the following divalent radicals: o?\ O 30

()). N /N Ns 1 N

pi N1 s s H 40 HOC O

45

HQeÓ 2 O HOye O o?\ 50 O

S )n ()). N N 55 M.N | N2N N (1 N M S O 1 N

60 pi 1sH HN1 COH 6 )n O HOC where n is an integer independently selected in each instance 65 from 1 to about 3. Illustratively, n is independently 1 or 2 in each instance. US 9,662,402 B2 81 82 wherein n is an integer from 2 to about 5. p is an integer from connecting at least one of the binding ligands (L) with at 1 to about 5, and r is an integer from 1 to about 4, as least one of the drugs (D). In one variation, the hydrophilic described above. spacer linker includes one or more carbohydrate containing It is to be further understood that in the foregoing embodi or polyhydroxyl group containing linkers. In another varia ments, open positions, such as () atoms are locations for 5 tion, the hydrophilic spacer linker includes one or more attachment of the binding ligand (B) or any drug (D) to be carbohydrate containing or polyhydroxyl group containing delivered. In addition, it is to be understood that such linkers and one or more aspartic acids. In another variation, attachment of either or both of B and any D may be direct the hydrophilic spacer linker includes one or more carbo or through an intervening linker comprising one or more of 10 hydrate containing or polyhydroxyl group containing linkers the radicals described herein. In addition, () atoms may and one or more glutamic acids. In a series of variations, in form releasable linkers with any drug D, or other portion of the linker L. each of the foregoing embodiments, the hydrophilic spacer In another embodiment, the hydrophilic spacer linker linker also includes one or more cysteines. In another series comprises one or more carbohydrate containing or polyhy 15 of variations, in each of the foregoing embodiments, the droxyl group containing linkers. In another embodiment, the hydrophilic spacer linker also includes at least one arginine. hydrophilic spacer linker comprises at least three carbohy In another embodiment, the hydrophilic spacer linker drate containing or polyhydroxyl group containing linkers. comprises one or more oligoamide hydrophilic spacers, such In another embodiment, the hydrophilic spacer linker com as but not limited to aminoethylpiperazinylacetamide. prises one or more carbohydrate containing or polyhydroxyl In another embodiment, the polyvalent linker L includes group containing linkers, and one or more aspartic acids. In a hydrophilic spacer linker comprising one or more triazole another embodiment, the hydrophilic spacer linker com linked carbohydrate containing or polyhydroxyl group con prises one or more carbohydrate containing or polyhydroxyl taining linkers. In another embodiment, the hydrophilic group containing linkers, and one or more glutamic acids. In 25 spacer linker comprises one or more amide linked carbohy another embodiment, the hydrophilic spacer linker com drate containing or polyhydroxyl group containing linkers. prises one or more carbohydrate containing or polyhydroxyl In another embodiment, the hydrophilic spacer linker com group containing linkers, one or more glutamic acids, one or prises one or more PEG groups and one or more cysteines. more aspartic acids, and one or more beta amino alanines. In 30 In another embodiment, the hydrophilic spacer linker com a series of variations, in each of the foregoing embodiments, prises one or more EDTE derivatives. the hydrophilic spacer linker also includes one or more In another embodiment, the polyvalent linker L includes cysteines. In another series of variations, in each of the a divalent radical of the formula

N ins 1N1

foregoing embodiments, the hydrophilic spacer linker also wherein * indicates the point of attachment to a folate and includes at least one arginine. ** indicates the point of attachment to a drug; and F and G In another embodiment, the polyvalent linker L includes as are each independently 1, 2, 3 or 4 are described. a hydrophilic spacer linker comprising one or more divalent In another embodiment, the polyvalent linker L includes 1,4-piperazines that are included in the chain of atoms a trivalent radical of the formula US 9,662,402 B2 83 84

wherein *, *, *** each indicate points of attachment to the -continued folate receptor binding moiety B, and the one or more drugs O D. It is to be understood that when there are fewer drugs, *, O O : SH **, *** are substituted with hydrogen or a heteroatom. F and 30 Gare each independently 1, 2, 3 or 4; and W' is NH or O O is described. In another aspect, m' is 0 or 1. OR : : In any of the embodiments described herein heteroatom linkers can also be included in the polyvalent linker L, such OR O as NR'R' , oxygen, sulfur, and the formulae 35 : O —(NHR'NHR)-, -SO —(SO)—, and - N(R)O , R = H, alkyl, acyl wherein R', R, and Rare each independently selected from O hydrogen, alkyl, aryl, arylalkyl, Substituted aryl, Substituted O O O arylalkyl, heteroaryl, Substituted heteroaryl, and alkoxy- HOC r 1. alkyl. It is to be understood that the heteroatom linkers may " be used to covalently attach any of the radicals described u NH herein, including drug radicals D to the polyvalent linker, HOC OR ligand radicals B to the polyvalent linker, or various di and : OR polyvalent radicals that from the polyvalent linker L 45 : R = H, alkyl, acyl Illustrative additional bivalent radicals that can be used to O form part of the linker are as follows. O O COH HN NH : gr N1 21 N1\. HOC t CO2H Y 50 O : lu. : : HN S: O NH2 O CO2H CO2H 55 : : S* : : : :

O O 1N O CO2H CO2H 60 O HOC N ~. : N- S.N HO2C : S ! NH N: N. : HOC

: : : N : : : 65 O O O O

US 9,662,402 B2 87 88 -continued -continued

: S 1n-S-N" HOC : r : S N CO2H y 21 O r N: 10 : S N O

Y 15 A-4 "S O :: O N

O HOC O y-sixts. O: CO2H 'co.H S S O: 25 ex Ys. ex ns1N1

The polyvalent linker L is a releasable linker. As used herein, the term “releasable linker” refers to a 30 linker that includes at least one bond that can be broken under physiological conditions when the compounds described herein are delivered to or inside of the target cell. Accordingly, the term releasable linker does not generally refer simply to a bond that is labile in Vivo, Such as in serum, 35 plasma, the gastrointestinal tract, or liver, unless those systems are the target for the cell Surface receptor binding ligand. However, after delivery and/or selective targeting, releasable linkers may be cleaved by any process that includes at least one bond being broken in the linker or at the 40 covalent attachment of the linker to B or any D under * S physiological conditions, such as by having one or more pH-labile, acid-labile, base-labile, oxidatively labile, meta bolically labile, biochemically labile, and/or enzyme-labile bonds. It is appreciated that such physiological conditions 45 resulting in bond breaking do not necessarily include a biological or metabolic process, and instead may include a standard chemical reaction, such as a hydrolysis reaction, for example, at physiological pH, or as a result of compartmen talization into a cellular organelle Such as an endosome 50 having a lower pH than cytosolic pH. It is understood that a cleavable bond can connect two adjacent atoms within the releasable linker, and/or connect other linkers with B, and/or any D, as described herein, at any ends of the releasable linker. In the case where a 55 cleavable bond connects two adjacent atoms within the releasable linker, following breakage of the bond, the releas able linker is broken into two or more fragments. Alterna tively, in the case where a cleavable bond is between the releasable linker and another moiety, such as an additional 60 heteroatom, a spacer linker, another releasable portion of the linker, any D, or B, following breakage of the bond, the releasable linker is separated from the other moiety. Illustrative radicals that themselves include a cleavable bond, or form a cleavable bond with B and/or any D 65 hemiacetals and Sulfur variations thereof, acetals and Sulfur variations thereof, hemiaminals, aminals, and the like, or which can be formed from methylene fragments substituted US 9,662,402 B2 89 90 with at least one heteroatom, Such as 1-alkoxyalkylene, In any of the embodiments described herein, the releas 1-alkoxycycloalkylene, 1-alkoxyalkylenecarbonyl, able linker may include nitrogen bonded to carbonylaryl 1-alkoxycycloalkylenecarbonyl, and the like. Illustrative carbonyl, carbonyl(carboxyaryl)carbonyl, carbonyl(biscar releasable linkers described herein include polyvalent link boxyaryl)carbonyl to form an amide, or alternatively an ers that include carbonylarylcarbonyl, carbonyl(carbox amide with a drug nitrogen. yaryl)carbonyl, carbonyl(biscarboxyaryl)carbonyl, In any of the embodiments described herein, the releas haloalkylenecarbonyl, and the like. Illustrative releasable able linker may include oxygen bonded to carbonylarylcar linkers described herein include polyvalent linkers that bonyl, carbonyl(carboxyaryl)carbonyl, carbonyl (biscarbox include alkylene(dialkylsilyl), alkylene(alkylarylsilyl), alky yaryl)carbonyl to forman ester, or alternatively an ester with lene(diarylsilyl), (dialkylsilyl)aryl, (alkylarylsilyl)aryl, (dia 10 drug oxygen. rylsilyl)aryl, and the like. Illustrative releasable linkers It is to be understood that the bivalent spacer linkers may described herein include oxycarbonyloxy, oxycarbonyloxy be combined in any chemically relevant way, either directly alkyl, sulfonyloxy, oxysulfonylalkyl, and the like. Illustra or via an intervening heteroatom to construct the releasable tive releasable linkers described herein include polyvalent 15 linkers described herein. It is further understood that the linkers that include iminoalkylidenyl, carbonylalkylidenimi nature of the arrangement of spacer and heteroatom linkers nyl, iminocycloalkylidenyl, carbonylcycloalkylideniminyl, defines where the releasable linker will cleave in vivo. For and the like. Illustrative releasable linkers described herein example, two spacer linkers that terminate in a Sulfur atom include polyvalent linkers that include alkylenethio, alkyle when combined form a disulfide, which is the cleavable nearylthio, and carbonylalkylthio, and the like. Each of the bond in the releasable linker formed thereby. foregoing fragments is optionally Substituted with a Sub For example, in another embodiment, the polyvalent stituent X’, as defined herein. linker comprises a 3-thiosuccinimid-1-ylalkyloxymethyloxy The substituents X can be alkyl, alkoxy, alkoxyalkyl, moiety, where the methyl is optionally substituted with alkyl hydroxy, hydroxyalkyl, amino, aminoalkyl, alkylamino or substituted aryl. alkyl, dialkylaminoalkyl, halo, haloalkyl, Sulfhydrylalkyl, 25 In another embodiment, the polyvalent linker comprises a alkylthioalkyl, aryl, substituted aryl, arylalkyl, substituted 3-thiosuccinimid-1-ylalkylcarbonyl, where the carbonyl arylalkyl, heteroaryl, Substituted heteroaryl, carboxy, car forms an acylaziridine with the drug. boxyalkyl, alkyl carboxylate, alkyl alkanoate, guanidino In another embodiment, the polyvalent linker comprises a alkyl, R'-carbonyl, R-carbonylalkyl, R-acylamino, and 1-alkoxycycloalkylenoxy moiety. R7-acylaminoalkyl, wherein R* and R are each indepen 30 In another embodiment, the polyvalent linker comprises dently selected from amino acids, amino acid derivatives, an alkyleneaminocarbonyl(dicarboxylarylene)carboxylate. and peptides, and wherein RandR are each independently In another embodiment, the polyvalent linker comprises a selected from amino acids, amino acid derivatives, and dithioalkylcarbonylhydrazide, where the hydrazide forms an peptides. In this embodiment the heteroatom linker can be hydrazone with the drug. nitrogen, and the substituent X and the heteroatom linker 35 can be taken together with the releasable linker to which In another embodiment, the polyvalent linker comprises a they are bound to form an heterocycle. 3-thiosuccinimid-1-ylalkylcarbonylhydrazide, where the The heterocycles can be pyrrolidines, piperidines, oxazo hydrazide forms a hydrazone with the drug. lidines, isoxazolidines, thiazolidines, isothiazolidines, pyr In another embodiment, the polyvalent linker comprises a rolidinones, piperidinones, oxazolidinones, isoxazolidino 40 3-thioalkylsulfonylalkyl (disubstituted silyl)oxy, where the nes, thiazolidinones, isothiazolidinones, and Succinimides. disubstituted silyl is substituted with alkyl or optionally In any of the embodiments described herein, the releas substituted aryl. able linker may include oxygen bonded to methylene, In another embodiment, the polyvalent linker comprises a 1-alkoxyalkylene, 1-alkoxycycloalkylene, 1-alkoxyalkylen plurality of spacer linkers selected from the group consisting ecarbonyl, and 1-alkoxycycloalkylenecarbonyl to form an 45 of the naturally occurring amino acids and stereoisomers acetal or ketal, wherein each of the fragments is optionally thereof. substituted with a substituent X’, as defined herein. Alter In another embodiment, the polyvalent linker comprises a natively, the methylene or alkylene is substituted with an 2-dithioalkyloxycarbonyl, where the carbonyl forms a car optionally-substituted aryl. bonate with the drug. In any of the embodiments described herein, the releas 50 In another embodiment, the polyvalent linker comprises a able linker may include oxygen bonded to sulfonylalkyl to 2-dithioarylalkyloxycarbonyl, where the carbonyl forms a form an alkylsulfonate. carbonate with the drug and the aryl is optionally Substi In any of the embodiments described herein, the releas tuted. able linker may include nitrogen bonded to iminoalkylide In another embodiment, the polyvalent linker comprises a nyl, carbonylalkylideniminyl, iminocycloalkylidenyl, and 55 4-dithioarylalkyloxycarbonyl, where the carbonyl forms a carbonylcycloalkylideniminyl to forman hydraZone, each of carbonate with the drug, and the aryl is optionally Substi which is optionally substituted with a substituent X’, as tuted. defined herein. In an alternate configuration, the hydrazone In another embodiment, the polyvalent linker comprises a may be acylated with a carboxylic acid derivative, an 3-thiosuccinimid-1-ylalkyloxyalkyloxyalkylidene, where orthoformate derivative, or a carbamoyl derivative to form 60 the alkylidene forms an hydraZone with the drug, each alkyl releasable linkers containing various acylhydrazones. is independently selected, and the oxyalkyloxy is optionally In any of the embodiments described herein, the releas substituted with alkyl or optionally substituted aryl. able linker may include oxygen bonded to alkylene(dialkyl In another embodiment, the polyvalent linker comprises a silyl), alkylene(alkylarylsilyl), alkylene(diarylsilyl), (di 2-dithioalkyloxycarbonylhydrazide. alkylsilyl)aryl, (alkylarylsilyl)aryl, and (diarylsilyl)aryl to 65 In another embodiment, the polyvalent linker comprises a form a silanol, each of which is optionally substituted with 2- or 3-dithioalkylamino, where the amino forms a vinylo a substituent X’, as defined herein. gous amide with the drug. US 9,662,402 B2 91 92 In another embodiment, the polyvalent linker comprises a and () indicates points of attachment for the drug, vitamin, 2-dithioalkylamino, where the amino forms a vinylogous imaging agent, diagnostic agent, other bivalent linkers, or amide with the drug, and the alkyl is ethyl. other parts of the conjugate. In another embodiment, the polyvalent linker comprises a In another embodiment, the polyvalent linkers described 2- or 3-dithioalkylaminocarbonyl, where the carbonyl forms 5 herein comprise divalent radicals of formulae (IV) a carbamate with the drug. In another embodiment, the polyvalent linker comprises a 2-dithioalkylaminocarbonyl, where the carbonyl forms a (IV) carbamate with the drug. In another aspect, the alkyl is ethyl. In another embodiment, the polyvalent linker comprises a 10 2-dithioalkyloxycarbonyl, where the carbonyl forms a car bamate with the drug. In another aspect, the alkyl is ethyl. In another embodiment, the polyvalent linker comprises a Cl-, : 2-dithioarylalkyloxycarbonyl, where the carbonyl forms a carbamate or a carbamoylaziridine with the drug. 15 In another embodiment, the polyvalent linker comprises a 4-dithioarylalkyloxycarbonyl, where the carbonyl forms a carbamate or a carbamoylaziridine with the drug. Cl In another embodiment, the polyvalent linkers described herein comprise divalent radicals of formulae (II) S

O N: (II) Ra Ra iii. r 25 O ^- O s: "S Ns pi O O O O Ra Rb Ra Rb Clu 30 "S O N* "S O S" Ns pi Ns pi r O O where m is an integer selected from 1 to about 4: R is an optionally substituted alkyl group, an optionally Substituted where n is an integer selected from 1 to about 4: R* and R' 35 acyl group, or a Suitably selected nitrogen protecting group; are each independently selected from the group consisting of and () indicates points of attachment for the drug, vitamin, hydrogen and alkyl, including lower alkyl such as C-C, imaging agent, diagnostic agent, other divalent linkers, or alkyl that are optionally branched; or R and R are taken other parts of the conjugate. together with the attached carbonatom to form a carbocyclic In another embodiment, the compounds described herein ring; R is an optionally Substituted alkyl group, an optionally 40 comprise one or more radicals selected from the formulae: Substituted acyl group, or a Suitably selected nitrogen pro tecting group; and () indicates points of attachment for the drug, Vitamin, imaging agent, diagnostic agent, other biva O O lent linkers, or other parts of the conjugate. In another embodiment, the polyvalent linkers described 45 -N-- -N-- herein comprise divalent radicals of formulae (III) N-- O N--O (III) 50 O

55 ins Or's Cl-,*S 5 C1 O S !l O N: O S* 60 O S O S O 1. where m is an integer selected from 1 to about 4: R is an 65 optionally substituted alkyl group, an optionally Substituted O acyl group, or a suitably selected nitrogen protecting group; US 9,662,402 B2 93 94 bonyltetrahydrofuranyl, 1-(carbonyltetrahydro-2H-pyranyl) -contigued Succinimid-3-yl, and 1-(carbonyltetrahydrofuranyl)succin n S O imid-3-yl, wherein each of said spacer linkers is optionally substituted with one or more substituents X'; wherein each substituent X" is independently selected ! 1s. from the group consisting of alkyl, alkoxy, alkoxyalkyl, hydroxy, hydroxyalkyl, amino, aminoalkyl, alkylamino S alkyl, dialkylaminoalkyl, halo, haloalkyl, Sulfhydrylalkyl, alkylthioalkyl, aryl, substituted aryl, arylalkyl, substituted 10 arylalkyl, heteroaryl, Substituted heteroaryl, carboxy, car boxyalkyl, alkyl carboxylate, alkyl alkanoate, guanidino alkyl, R-carbonyl, R-carbonylalkyl, R-acylamino, and R’-acylaminoalkyl, wherein R* and R are each indepen Crd Crs. dently selected from the group consisting of an amino acid, 15 an amino acid derivative, and a peptide, and wherein R and R" are each independently selected from the group consist ing of an amino acid, an amino acid derivative, and a peptide. r The compounds described herein may contain one or more chiral centers, or may otherwise be capable of existing wherein X is NH, O, or S. as multiple stereoisomers. It is to be understood that in one embodiment, the invention described herein is not limited to In another embodiment, the polyvalent linkers herein any particular Stereochemical requirement, except where described comprise a radical having the formula: specifically indicated, and that the compounds, and compo 25 sitions, methods, uses, and medicaments that include them O may be optically pure, or may be any of a variety of Stereoisomeric mixtures, including racemic and other mix tures of enantiomers, other mixtures of diastereomers, and the like. It is also to be understood that such mixtures of Clu r NH or 30 Stereoisomers may include a single stereochemical configu Y O ration at one or more chiral centers, while including mix O tures of stereochemical configuration at one or more other chiral centers. Similarly, the compounds described herein may include 35 geometric centers, such as cis, trans, E, and Z double bonds. Clu r NH It is to be understood that in another embodiment, the R-O O invention described herein is not limited to any particular geometric isomer requirement, and that the compounds, and compositions, methods, uses, and medicaments that include Another embodiment, the polyvalent linkers described 40 them may be pure, or may be any of a variety of geometric herein comprise a radical of having the formula: isomer mixtures. It is also to be understood that Such mixtures of geometric isomers may include a single con figuration at one or more double bonds, while including R O mixtures of geometry at one or more other double bonds. 45 As used herein, the term “cell surface receptor binding or targeting ligand generally refers to compounds that bind to and/or target receptors that are found on cell Surfaces, and in O cr's particular those that are found on, over-expressed by, and/or ----> preferentially expressed on the Surface of pathogenic cells. 50 Illustrative ligands include, but are not limited to, Vitamins where X is an heteroatom, Such as nitrogen, oxygen, or and vitamin receptor binding compounds. Sulfur, n is an integer selected from 0, 1, 2, and 3, R is Illustrative vitamin moieties include carnitine, inositol, hydrogen, or a Substituent, including a substituent capable of lipoic acid, pyridoxal, ascorbic acid, niacin, pantothenic stabilizing a positive charge inductively or by resonance on acid, folic acid, riboflavin, thiamine, biotin, vitamin B, and the aryl ring, Such as alkoxy, and the like, and the symbol () 55 the lipid soluble vitamins A, D, E and K. These vitamins, indicates points of attachment. It is appreciated that other and their receptor-binding analogs and derivatives, consti Substituents may be present on the aryl ring, the benzyl tute the targeting entity from which a radical can be formed carbon, the alkanoic acid, or the methylene bridge, including for covalent attachment to the polyvalent linker L. Illustra but not limited to hydroxy, alkyl, alkoxy, alkylthio, halo, and tive biotin analogs that bind to biotin receptors include, but the like. 60 are not limited to, biocytin, biotin sulfoxide, oxybiotin, and In another embodiment, the polyvalent linkers described the like. herein comprise radicals selected from carbonyl, thionocar Illustrative folic acid analogs that bind to folate receptors bonyl, alkylene, cycloalkylene, alkylenecycloalkyl, alkyle include, but are not limited to folinic acid, pteropolygluta necarbonyl, cycloalkylenecarbonyl, carbonylalkylcarbonyl, mic acid, and folate receptor-binding pteridines such as 1 alkylenesuccinimid-3-yl, 1-(carbonylalkyl)Succinimid-3- 65 tetrahydropterins, dihydrofolates, tetrahydrofolates, and y1, alkylenesulfoxyl, sulfonylalkyl, alkylenesulfoxylalkyl, their deaza and dideaza analogs. The terms “deaza' and alkylenesulfonylalkyl, carbonyltetrahydro-2H-pyranyl, car "dideaza' analogs refer to the art-recognized analogs having US 9,662,402 B2 95 96 a carbon atom Substituted for one or two nitrogen atoms in As used herein, the term “amino acid refers generally to the naturally occurring folic acid structure, or analog or beta, gamma, and longer amino acids, such as amino acids derivative thereof. For example, the deaza analogs include of the formula: the 1-deaza, 3-deaza, 5-deaza, 8-deaza, and 10-deaza ana logs of folate, folinic acid, pteropolyglutamic acid, and where R is hydrogen, alkyl, acyl, or a suitable nitrogen folate receptor-binding pteridines such as tetrahydropterins, protecting group, R and R" are hydrogen or a Substituent, dihydrofolates, and tetrahydrofolates. The dideaza analogs each of which is independently selected in each occurrence, include, for example, 1,5-dideaza, 5,10-dideaza, 8.10 and q is an integer Such as 1, 2, 3, 4, or 5. Illustratively, R dideaza, and 5,8-dideaza analogs of folate, folinic acid, and/or R" independently correspond to, but are not limited pteropolyglutamic acid, and folate receptor-binding pteri 10 to, hydrogen or the side chains present on naturally occur dines Such as tetrahydropterins, dihydrofolates, and tetrahy ring amino acids, such as methyl, benzyl, hydroxymethyl, drofolates. Other folates useful as complex forming ligands thiomethyl, carboxyl, carboxylmethyl, guanidinopropyl, and for this invention are the folate receptor-binding analogs the like, and derivatives and protected derivatives thereof. aminopterin, amethopterin (also known as methotrexate), The above described formula includes all stereoisomeric N'-methylfolate, 2-deamino-hydroxyfolate, deaza analogs 15 variations. For example, the amino acid may be selected Such as 1-deazamethopterin or 3-deazamethopterin, and from asparagine, aspartic acid, cysteine, glutamic acid, 3',5'-dichloro-4-amino-4-deoxy-N'-methylpteroylglutamic lysine, glutamine, arginine, serine, ornitine, threonine, and acid (dichloromethotrexate). The foregoing folic acid ana the like. logs and/or derivatives are conventionally termed “folates.” As used herein, the term “amino acid derivative' gener reflecting their ability to bind with folate-receptors, and such ally refers to an amino acid as defined herein where either, or both, the amino group and/or the side chain is Substituted. ligands when conjugated with exogenous molecules are Illustrative amino acid derivatives include prodrugs and effective to enhance transmembrane transport, such as via protecting groups of the amino group and/or the side chain, folate-mediated endocytosis as described herein. Such as amine, amide, hydroxy, carboxylic acid, and thio Additional analogs of folic acid that bind to folic acid prodrugs and protecting groups. Additional Illustrative receptors are described in US Patent Application Publication 25 amino acid derivatives include substituted variations of the Serial Nos. 2005/0227985 and 2004/0242582, the disclo amino acid as described herein, such as, but not limited to, sures of which are incorporated herein by reference. Illus ethers and esters of hydroxy groups, amides, carbamates, tratively, such folate analogs have the general formula: and ureas of amino groups, esters, amides, and cyano derivatives of carboxylic acid groups, and the like. 30 As used herein, the terms “tubulysin' and “tubulysins' X R6 R7 R6 R7 refer generally to tetrapeptide compounds of the formula N1 N1W No1 24. N. e YT-R (A') N / O Y l N4 1- S-(L) N 35 R Y Z. O S HN V M N N N sN N wherein X and Y are each independently selected from the H O --R group consisting of halo, R, OR, SR, and NRR: )n O V W 2 U, V, and W represent divalent moieties each indepen 40 dently selected from the group consisting of —(R')C=, —N= -(R)C(R') , and - N(R') : Q is selected and pharmaceutical salts thereof, where n is 1-3: from the group consisting of C and CH; T is selected from V is H, OR, or halo, and W is H, OR, or alkyl, where the group consisting of S, O, N, and —C=C-: R’ is independently selected in each instance from H. alkyl, A' and A are each independently selected from the group 45 and C(O)R, where R is alkyl, cycloalkyl, alkenyl, aryl, or consisting of oxygen, Sulfur, —C(Z)— —C(Z)O— —OC arylalkyl, each of which is optionally Substituted; providing that R is not H when both V and W are OR: or V and W are taken together with the attached carbon to form a carbonyl: 50 X—H, C alkyl, alkenyl, each of which is optionally C-C alkylene, and C-C alkyeneoxy, where Z is oxygen substituted, or CHQR'; where Q is —O , or - S : or sulfur, R—H, C, alkyl, alkenyl, aryl, or C(O)R'; and R'—C. R" is selected from the group consisting of hydrogen, alkyl alkenyl, aryl, or heteroaryl, each of which is optionally halo, C-C alkyl, and C-C alkoxy; R. R. R. R. R. substituted; R. R. R', and R' are each independently selected from 55 Z is alkyl and Y is O; or Z is alkyl or C(O)R, and Y is the group consisting of hydrogen, halo, C-C alkyl, C-C, absent, where R is alkyl, CF, or aryl; alkoxy, C-C alkanoyl, C-C alkenyl, C-C alkynyl, R" is H, or R' represents 1 to 3 substituents selected from (C-C alkoxy)carbonyl, and (C-C alkylamino)carbonyl: halo, nitro, carboxylate or a derivative thereof, cyano, Rand R7 are each independently selected from the group hydroxyl, alkyl, haloalkyl, alkoxy, haloalkoxy, and OR, consisting of hydrogen, halo, C-C alkyl, and C-C2 60 where R is hydrogen or optionally substituted aryl, a phenol alkoxy; or, Rand R7 are taken together to form a carbonyl protecting group, a prodrug moiety, alkyl, arylalkyl, C(O) group: R* and R'' are each independently selected from the R", P(O)(OR), or SOR, where R7 and R are indepen group consisting of hydrogen, halo, C-C alkyl, and dently selected in each instance from H. alkyl, alkenyl, C-C alkoxy; or R'' and R'' are taken together to form a cycloalkyl, heterocyclyl, aryl, and arylalkyl, each of which carbonyl group; 65 is optionally substituted, or R is a metal cation; and L is a divalent linker as described herein; and R is OH or a leaving group, or R forms a carboxylic acid n, p, r, S and t are each independently either 0 or 1. derivative, such as an acylhydrazide. US 9,662,402 B2 97 98 Conjugates of each of the foregoing tubulysins are the foregoing description is a description of the variation described herein. In one variation, Z is methyl. In another where Z is methyl, and R' is H; where R' is OR at C(4), and variation, R' is H. In another variation, R' is OR at C(4), R is H; where Z is methyl, R' is OR at C(4), R is H, and where R is H, alkyl, or COR". In another variation, V is H. X=CHOR: and the like. and W is OC(O)R. In another variation, X=CHQR. In Natural tubulysins are generally linear tetrapeptides con another variation, X=CHOR. In another variation, R is sisting of N-methylpipecolic acid (Mep), isoleucine (Ile), an alkyl or alkenyl. In another variation, R is C(O)R''. In unnatural aminoacid called tubuvaline (TuV), and either an another variation, R'-optionally substituted C, alkyl. In unnatural aminoacid called tubutyrosine (Tut, an analog of another variation, R'-C alkyl. In another variation, R tyrosine) or an unnatural aminoacid called tubuphenylala forms an acylhydrazide. It is to be understood that the 10 nine (Tup, an analog of phenylalanine). In another embodi foregoing description is an explicit description of each ment, naturally occurring tubulysins, and analogs and chemically possible combination of variations of the general derivatives thereof, of the following general formula are tubulysin structure. For example, it is to be understood that described

30 and pharmaceutical salts thereof, where R, R', and R'' are as described in the various embodiments herein. Conjugates of each of the foregoing tubulysins are described herein. In another embodiment, conjugates of naturally occurring tubulysins of the following general formula are described r

N Clu) H O ÖAc RI

Factor R10 R (CH3)2CHCH2 OH CH3(CH2)2 OH CHCH- OH (CH3)2CHCH2 H CH3(CH2)2 H CHCH H (CH)2C=CH OH CH H CH OH

and pharmaceutical salts thereof. 60 In another embodiment, compounds are described herein where the conjugate is formed at the terminal carboxylic acid group or the terminal acylhydrazine group of each of the tybulysins described herein. As used herein, the term “a rapamycin' is understood to 65 include Sirolimus (rapamycin), temsirolimus, everolimus, and ridaforolimus, and related compounds, and compounds of the formula US 9,662,402 B2 99 100

As used herein, the term “alkylene' includes a divalent chain of carbon atoms, which is optionally branched. As used herein, the term “alkenylene' and “alkynylene’ includes a divalent chain of carbon atoms, which is option ally branched, and includes at least one double bond or triple bond, respectively. It is to be understood that alkynylene may also include one or more double bonds. It is to be further understood that in certain embodiments, alkylene is advantageously of limited length, including C-C, C-C, 10 C-Cs, C-C, and C-C Illustratively, such particularly limited length alkylene groups, including C-Cs, C-C, and C-C may be referred to as lower alkylene. It is to be further understood that in certain embodiments alkenylene and/or alkynylene may each be advantageously of limited length, 15 including C-C2, C-C2, C2-Cs. C-C and C2-C. Illus tratively, such particularly limited length alkenylene and/or alkynylene groups, including C-Cs. C-C, and C-C may be referred to as lower alkenylene and/or alkynylene. It is appreciated herein that shorter alkylene, alkenylene, and/or alkynylene groups may add less lipophilicity to the com and pharmaceutically acceptable salts thereof, wherein pound and accordingly will have different pharmacokinetic Y is OR or OCHCHOR; behavior. In embodiments of the invention described herein, one of R. R. or R is a bond connected to L; and it is to be understood, in each case, that the recitation of the other two of R', R, and R are independently alkylene, alkenylene, and alkynylene refers to alkylene, selected in each case from the group consisting of hydrogen, 25 alkenylene, and alkynylene as defined herein, and optionally optionally Substituted heteroalkyl, prodrug forming group, lower alkylene, alkenylene, and alkynylene. Illustrative and C(O)R’, where R' is in each instance independently alkyl groups are, but not limited to, methylene, ethylene, selected from the group consisting of hydrogen, and alkyl, n-propylene, isopropylene, n-butylene, isobutylene, Sec-bu alkenyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, ary tylene, pentylene, 1.2-pentylene, 1,3-pentylene, hexylene, lalkyl, heteroaryl, and heteroarylalkyl, each of which is 30 heptylene, octylene, and the like. optionally substituted is described. As used herein, the term “cycloalkyl includes a chain of As used herein, the term “alkyl includes a chain of carbon atoms, which is optionally branched, where at least carbon atoms, which is optionally branched. As used herein, a portion of the chain is cyclic. It is to be understood that the term “alkenyl and “alkynyl' includes a chain of carbon cycloalkylalkyl is a subset of cycloalkyl. It is to be under 35 stood that cycloalkyl may be polycyclic. Illustrative atoms, which is optionally branched, and includes at least cycloalkyl include, but are not limited to, cyclopropyl. one double bond or triple bond, respectively. It is to be cyclopentyl, cyclohexyl, 2-methylcyclopropyl, cyclopenty understood that alkynyl may also include one or more leth-2-yl, adamantyl, and the like. As used herein, the term double bonds. It is to be further understood that in certain “cycloalkenyl includes a chain of carbon atoms, which is embodiments, alkyl is advantageously of limited length, 40 optionally branched, and includes at least one double bond, including C-C, C-C, C-Cs. C-C and C-C Illus where at least a portion of the chain in cyclic. It is to be tratively, such particularly limited length alkyl groups, understood that the one or more double bonds may be in the including C-Cs, C-C, and C-C may be referred to as cyclic portion of cycloalkenyl and/or the non-cyclic portion lower alkyl. It is to be further understood that in certain of cycloalkenyl. It is to be understood that cycloalkenylalkyl embodiments alkenyl and/or alkynyl may each be advanta 45 and cycloalkylalkenyl are each Subsets of cycloalkenyl. It is geously of limited length, including C-C2, C-C2, C2-Cs. to be understood that cycloalkyl may be polycyclic. Illus C-C, and C-C. Illustratively, such particularly limited trative cycloalkenyl include, but are not limited to, cyclo length alkenyl and/or alkynyl groups, including C-Cs. pentenyl, cyclohexylethen-2-yl, cycloheptenylpropenyl, and C-C, and C-C may be referred to as lower alkenyl and/or the like. It is to be further understood that chain forming alkynyl. It is appreciated herein that shorter alkyl, alkenyl, 50 cycloalkyl and/or cycloalkenyl is advantageously of limited and/or alkynyl groups may add less lipophilicity to the length, including C-C2, C-C2, Ca-Cs. Cs-Co. and Cs-Co. compound and accordingly will have different pharmacoki It is appreciated herein that shorter alkyl and/or alkenyl netic behavior. In embodiments of the invention described chains forming cycloalkyl and/or cycloalkenyl, respectively, herein, it is to be understood, in each case, that the recitation may add less lipophilicity to the compound and accordingly of alkyl refers to alkyl as defined herein, and optionally 55 will have different pharmacokinetic behavior. lower alkyl. In embodiments of the invention described As used herein, the term "heteroalkyl includes a chain of herein, it is to be understood, in each case, that the recitation atoms that includes both carbon and at least one heteroatom, of alkenyl refers to alkenyl as defined herein, and optionally and is optionally branched. Illustrative heteroatoms include lower alkenyl. In embodiments of the invention described nitrogen, oxygen, and Sulfur. In certain variations, illustra herein, it is to be understood, in each case, that the recitation 60 tive heteroatoms also include phosphorus, and selenium. As of alkynyl refers to alkynyl as defined herein, and optionally used herein, the term "cycloheteroalkyl including hetero lower alkynyl. Illustrative alkyl, alkenyl, and alkynyl groups cyclyl and heterocycle, includes a chain of atoms that are, but not limited to, methyl, ethyl, n-propyl, isopropyl. includes both carbon and at least one heteroatom, Such as n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, 2-pentyl, heteroalkyl, and is optionally branched, where at least a 3-pentyl, neopentyl, hexyl, heptyl, octyl, and the like, and 65 portion of the chain is cyclic. Illustrative heteroatoms the corresponding groups containing one or more double include nitrogen, oxygen, and Sulfur. In certain variations, and/or triple bonds, or a combination thereof. illustrative heteroatoms also include phosphorus, and sele US 9,662,402 B2 101 102 nium. Illustrative cycloheteroalkyl include, but are not lim nyl, heteroarylalkenylcarbonyl, heteroarylalkynylcarbonyl, ited to, tetrahydrofuryl, pyrrolidinyl, tetrahydropyranyl, pip acylcarbonyl, and the like, each of which is optionally eridinyl, morpholinyl, piperazinyl, homopiperazinyl, substituted. quinuclidinyl, and the like. As used herein, the term “carbonyl and derivatives As used herein, the term “aryl' includes monocyclic and thereof includes the group C(O), C(S), C(NH) and substi polycyclic aromatic carbocyclic groups, each of which may tuted amino derivatives thereof. be optionally substituted. Illustrative aromatic carbocyclic As used herein, the term "carboxylic acid and derivatives groups described herein include, but are not limited to, thereof includes the group COH and salts thereof, and phenyl, naphthyl, and the like. As used herein, the term esters and amides thereof, and CN. 10 The term “optionally substituted as used herein includes "heteroaryl' includes aromatic heterocyclic groups, each of the replacement of hydrogen atoms with other functional which may be optionally substituted. Illustrative aromatic groups on the radical that is optionally substituted. Such heterocyclic groups include, but are not limited to, pyridinyl, other functional groups illustratively include, but are not pyrimidinyl, pyrazinyl, triazinyl, tetrazinyl, quinolinyl, qui limited to, amino, hydroxyl, halo, thiol, alkyl, haloalkyl, nazolinyl, quinoxalinyl, thienyl, pyrazolyl, imidazolyl, 15 heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl, het oxazolyl, thiazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, eroarylalkyl, heteroarylheteroalkyl, nitro, Sulfonic acids and thiadiazolyl, triazolyl, benzimidazolyl, benzoxazolyl, benz derivatives thereof, carboxylic acids and derivatives thereof, thiazolyl, benzisoxazolyl, benzisothiazolyl, and the like. and the like. Illustratively, any of amino, hydroxyl, thiol, As used herein, the term “amino” includes the group NH, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, alkylamino, and dialkylamino, where the two alkyl groups in heteroaryl, heteroarylalkyl, heteroarylheteroalkyl, and/or dialkylamino may be the same or different, i.e. alkylalky Sulfonic acid is optionally Substituted. lamino. Illustratively, amino includes methylamino, ethyl As used herein, the terms “optionally substituted aryl amino, dimethylamino, methylethylamino, and the like. In and “optionally substituted heteroaryl' include the replace addition, it is to be understood that when amino modifies or ment of hydrogen atoms with other functional groups on the is modified by another term, such as aminoalkyl, or acy 25 aryl or heteroaryl that is optionally substituted. Such other lamino, the above variations of the term amino are included functional groups illustratively include, but are not limited therein. Illustratively, aminoalkyl includes HN-alkyl, meth to, amino, hydroxy, halo, thio, alkyl, haloalkyl, heteroalkyl, ylaminoalkyl, ethylaminoalkyl, dimethylaminoalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl, methylethylaminoalkyl, and the like. Illustratively, acy heteroarylheteroalkyl, nitro, sulfonic acids and derivatives lamino includes acylmethylamino, acylethylamino, and the 30 thereof, carboxylic acids and derivatives thereof, and the like. like. Illustratively, any of amino, hydroxy, thio, alkyl, As used herein, the term “amino and derivatives thereof haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, het includes amino as described herein, and alkylamino, alk eroaryl, heteroarylalkyl, heteroarylheteroalkyl, and/or sulfo enylamino, alkynylamino, heteroalkylamino, heteroalk nic acid is optionally Substituted. enylamino, heteroalkynylamino, cycloalkylamino, cycloalk 35 Illustrative substituents include, but are not limited to, a enylamino, cycloheteroalkylamino, radical—(CH), Z, where x is an integer from 0-6 and Z cycloheteroalkenylamino, arylamino, arylalkylamino, aryl is selected from halogen, hydroxy, alkanoyloxy, including alkenylamino, arylalkynylamino, heteroarylamino, het C-C alkanoyloxy, optionally substituted aroyloxy, alkyl, eroarylalkylamino, heteroarylalkenylamino, heteroarylalky including C-C alkyl, alkoxy, including C-C alkoxy, nylamino, acylamino, and the like, each of which is 40 cycloalkyl, including C-C cycloalkyl, cycloalkoxy, includ optionally substituted. The term “amino derivative” also ing C-C cycloalkoxy, alkenyl, including C-C alkenyl, includes urea, carbamate, and the like. alkynyl, including C-C alkynyl, haloalkyl, including As used herein, the term “hydroxy and derivatives C-C haloalkyl, haloalkoxy, including C-C haloalkoxy, thereof includes OH, and alkyloxy, alkenyloxy, alkynyloxy, halocycloalkyl, including C-Cs halocycloalkyl, halocy heteroalkyloxy, heteroalkenyloxy, heteroalkynyloxy, 45 cloalkoxy, including C-Cs halocycloalkoxy, amino, C-C, cycloalkyloxy, cycloalkenyloxy, cycloheteroalkyloxy, alkylamino, (C-C alkyl)(C-C alkyl)amino, alkylcarbo cycloheteroalkenyloxy, aryloxy, arylalkyloxy, arylalkeny nylamino, N-(C-C alkyl)alkylcarbonylamino, amino loxy, arylalkynyloxy, heteroaryloxy, heteroarylalkyloxy, alkyl, C-C alkylaminoalkyl, (C-C alkyl)(C-C alkyl) heteroarylalkenyloxy, heteroarylalkynyloxy, acyloxy, and aminoalkyl, alkylcarbonylaminoalkyl, N-(C-C alkyl) the like, each of which is optionally substituted. The term 50 alkylcarbonylaminoalkyl, cyano, and nitro; or Z is selected “hydroxy derivative' also includes carbamate, and the like. from -COR and – CONRR, where R. R. and Rare As used herein, the term “thio and derivatives thereof each independently selected in each occurrence from hydro includes SH, and alkylthio, alkenylthio, alkynylthio, het gen, C-C alkyl, aryl-C-C alkyl, and heteroaryl-C-C, eroalkylthio. heteroalkenylthio. heteroalkynylthio. alkyl. cycloalkylthio, cycloalkenylthio, cycloheteroalkylthio. 55 As used herein the term “radical with reference to, for cycloheteroalkenylthio, arylthio, arylalkylthio, arylalkenyl example, the cell Surface receptor binding and/or targeting thio, arylalkynylthio, heteroarylthio, heteroarylalkylthio. ligand, and/or the independently selected drug, refers to a heteroarylalkenylthio, heteroarylalkynylthio, acylthio, and cell Surface receptor binding and/or targeting ligand, and/or the like, each of which is optionally substituted. The term an independently selected drug, as described herein, where “thio derivative” also includes thiocarbamate, and the like. 60 one or more atoms or groups, such as a hydrogen atom, or As used herein, the term “acyl includes formyl, and an alkyl group on a heteroatom, and the like, is removed to alkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, het provide a radical for conjugation to the polyvalent linker L. eroalkylcarbonyl, heteroalkenylcarbonyl, heteroalkynylcar Such ligand radicals and drug radicals may also be referred bonyl, cycloalkylcarbonyl, cycloalkenylcarbonyl, cyclohet herein as ligand analogs and drug analogs, respectively. eroalkylcarbonyl, cycloheteroalkenylcarbonyl, 65 As used herein, the term “leaving group' refers to a arylcarbonyl, arylalkylcarbonyl, arylalkenylcarbonyl, aryl reactive functional group that generates an electrophilic site alkynylcarbonyl, heteroarylcarbonyl, heteroarylalkylcarbo on the atom to which it is attached such that nucleophiles US 9,662,402 B2 103 104 may be added to the electrophilic site on the atom. Illustra disease or disorder being treated. In one aspect, the thera tive leaving groups include, but are not limited to, halogens, peutically effective amount is that which may treat or optionally Substituted phenols, acyloxy groups, Sulfonoxy alleviate the disease or symptoms of the disease at a rea groups, and the like. It is to be understood that Such leaving sonable benefit/risk ratio applicable to any medical treat groups may be on alkyl, acyl, and the like. Such leaving ment. However, it is to be understood that the total daily groups may also be referred to herein as activating groups, usage of the compounds and compositions described herein Such as when the leaving group is present on acyl. In may be decided by the attending physician within the scope addition, conventional peptide, amide, and ester coupling of Sound medical judgment. The specific therapeutically agents, such as but not limited to PyBop, BOP-C1, BOP effective dose level for any particular patient will depend pentafluorophenol, isobutylchloroformate, and the like, 10 upon a variety of factors, including the disorder being form various intermediates that include a leaving group, as treated and the severity of the disorder; activity of the defined herein, on a carbonyl group. specific compound employed; the specific composition It is to be understood that in every instance disclosed employed; the age, body weight, general health, gender and herein, the recitation of a range of integers for any variable diet of the patient: the time of administration, route of describes the recited range, every individual member in the 15 administration, and rate of excretion of the specific com range, and every possible Subrange for that variable. For pound employed; the duration of the treatment; drugs used example, the recitation that n is an integer from 0 to 8, in combination or coincidentally with the specific compound describes that range, the individual and selectable values of employed; and like factors well known to the researcher, 0, 1, 2, 3, 4, 5, 6, 7, and 8. Such as n is 0, or n is 1, or n is veterinarian, medical doctor or other clinician of ordinary 2, etc. In addition, the recitation that n is an integer from 0 skill. to 8 also describes each and every Subrange, each of which The term “administering as used herein includes all may for the basis of a further embodiment, such as n is an means of introducing the compounds and compositions integer from 1 to 8, from 1 to 7, from 1 to 6, from 2 to 8, from described herein to the patient, including, but are not limited 2 to 7, from 1 to 3, from 2 to 4, etc. to, oral (po), intravenous (iv), intramuscular (im), Subcuta As used herein, the term “composition' generally refers to 25 neous (sc), transdermal, inhalation, buccal, ocular, Sublin any product comprising the specified ingredients in the gual, vaginal, rectal, and the like. The compounds and specified amounts, as well as any product which results, compositions described herein may be administered in unit directly or indirectly, from combinations of the specified dosage forms and/or formulations containing conventional ingredients in the specified amounts. It is to be understood nontoxic pharmaceutically-acceptable carriers, adjuvants, that the compositions described herein may be prepared 30 and vehicles. from isolated compounds described herein or from salts, Illustrative formats for oral administration include tablets, solutions, hydrates, solvates, and other forms of the com capsules, elixirs, syrups, and the like. pounds described herein. It is appreciated that certain func Illustrative routes for parenteral administration include tional groups, such as the hydroxy, amino, and like groups intravenous, intraarterial, intraperitoneal, epidurial, intrau form complexes and/or coordination compounds with water 35 rethral, intrasternal, intramuscular and Subcutaneous, as well and/or various solvents, in the various physical forms of the as any other art recognized route of parenteral administra compounds. It is also to be understood that the compositions tion. may be prepared from various amorphous, non-amorphous, Depending upon the disease as described herein, the route partially crystalline, crystalline, and/or other morphological of administration and/or whether the compounds and/or forms of the compounds described herein. It is also to be 40 compositions are administered locally or systemically, a understood that the compositions may be prepared from wide range of permissible dosages are contemplated herein, various hydrates and/or solvates of the compounds described including doses falling in the range from about 1 lug/kg to herein. Accordingly, such pharmaceutical compositions that about 1 g/kg. The dosages may be single or divided, and may recite compounds described herein are to be understood to administered according to a wide variety of protocols, include each of, or any combination of the various mor 45 including q.d., b.i.d. t.i.d., or even every other day, once a phological forms and/or solvate or hydrate forms of the week, once a month, once a quarter, and the like. In each of compounds described herein. In addition, it is to be under these cases it is understood that the therapeutically effective stood that the compositions may be prepared from various amounts described herein correspond to the instance of co-crystals of the compounds described herein. administration, or alternatively to the total daily, weekly, Illustratively, compositions may include one or more 50 month, or quarterly dose, as determined by the dosing carriers, diluents, and/or excipients. The compounds protocol. described herein, or compositions containing them, may be The term “prodrug as used herein generally refers to any formulated in a therapeutically effective amount in any compound that when administered to a biological system conventional dosage forms appropriate for the methods generates a biologically active compound as a result of one described herein. The compounds described herein, or com 55 or more spontaneous chemical reaction(s), enzyme-cata positions containing them, including such formulations, may lyzed chemical reaction(s), and/or metabolic chemical reac be administered by a wide variety of conventional routes for tion(s), or a combination thereof. In Vivo, the prodrug is the methods described herein, and in a wide variety of typically acted upon by an enzyme (such as esterases, dosage formats, utilizing known procedures (see generally, amidases, phosphatases, and the like), simple biological Remington: The Science and Practice of Pharmacy, (21 ed., 60 chemistry, or other process in vivo to liberate or regenerate 2005)). the more pharmacologically active drug. This activation The term “therapeutically effective amount’ as used may occur through the action of an endogenous host enzyme herein, refers to that amount of active compound or phar or a non-endogenous enzyme that is administered to the host maceutical agent that elicits the biological or medicinal preceding, following, or during administration of the prod response in a tissue system, animal or human that is being 65 rug. Additional details of prodrug use are described in U.S. sought by a researcher, veterinarian, medical doctor or other Pat. No. 5,627, 165; and Pathalk et al., Enzymic protecting clinician, which includes alleviation of the symptoms of the group techniques in organic synthesis, Stereosel. Biocatal. US 9,662,402 B2 105 106 775-797 (2000). It is appreciated that the prodrug is advan- It is understood that the prodrugs themselves may not tageously converted to the original drug as soon as the goal, possess significant biological activity, but instead undergo Such as targeted delivery, safety, stability, and the like is one or more spontaneous chemical reaction(s), enzyme achieved, followed by the subsequent rapid elimination of catalyzed chemical reaction(s), and/or metabolic chemical the released remains of the group forming the prodrug. reaction(s),vivo to produce or a combination the compound thereof described after administration herein that inis Prodrugs may be prepared from the compounds described biologically active or is a precursor of the biologically active herein by attaching groups that ultimately cleave in vivo to compound. However, it is appreciated that in Some cases, the one or more functional groups present on the compound, prodrug is biologically active. It is also appreciated that such as —OH , —SH, —COH, -NR. Illustrative pro- prodrugs may often serves to improve drug efficacy or safety drugs include but are not limited to carboxylate esters where 10 through improved oral bioavailability, pharmacodynamic the group is alkyl, aryl, arylalkyl, heteroaryl, heteroarylal- half-life, and the like. Prodrugs also refer to derivatives of kyl, acyloxyalkyl, alkoxycarbonyloxyalkyl as well as esters the compounds described herein that include groups that of hydroxyl, thiol and amines where the group attached is an simply mask undesirable drug properties or improve drug acyl group, an alkoxycarbonyl, aminocarbonyl, phosphate or delivery. For example, one or more compounds described sulfate. Illustrative esters, also referred to as active esters, 1s herein may exhibit an undesirable property that is advanta include but are not limited to 1-indanyl, N-oxysuccinimide: geously blocked or minimized may become pharmacologi acyloxyalkyl groups Such as acetoxymethyl, pivaloyloxym- cal, pharmaceutical, O pharmacokinetic barriers in clinical ethyl, 3-acetoxyethyl, B-pivaloyloxyethyl, 1-(cyclohexylcar- drug application, such as low oral drug absorption, lack of bonyloxy)prop-1-yl, (1-aminoethyl)carbonyloxymethyl, site specificity, chemical instability, toxicity, and poor and the like: alkoxycarbonyloxyalkyl groups, such as patient acceptance (bad taste, odor, pain at injection site, and ethoxycarbonyloxymethyl, C.-ethoxycarbonyloxyethylf- the like), and others. It is appreciated herein that a prodrug, ethoxycarbonyloxyethyl, and the like; dialkylaminoalkyl or other strategy using reversible derivatives, can be useful groups, including di-lower alkylamino alkyl groups, such as in the optimization of the clinical application of a drug. dimethylaminomethyl, dimethylaminoethyl, diethylamin- The compounds, linkers, intermediates, and conjugates omethyl, diethylaminoethyl, and the like: 2-(alkoxycarbo- described herein may be prepared using conventional pro nyl)-2-alkenyl groups Such as 2-(isobutoxycarbonyl) pent- 25 CeSSes, including those described in International Patent 2-enyl, 2-(ethoxycarbonyl)but-2-enyl, and the like: and Publication Nos. WO 2009/002993, WO 2004/069159, WO lactone groups such as phthalidyl, dimethoxyphthalidyl, and 2007/022494, and WO 2006/012527, and U.S. patent appli the like. cation Ser. No. 13/837,539 (filed Mar. 15, 2013). The Further illustrative prodrugs contain a chemical moiety, disclosures of each of the foregoing are herein incorporated such as an amide or phosphorus group functioning to 30 by reference in their entirety. increase solubility and/or stability of the compounds Each publications cited herein is incorporated herein by described herein. Further illustrative prodrugs for amino reference. groups include, but are not limited to, (C-C)alkanoyl; The following examples further illustrate specific halo-(C-Co.)alkanoyl; (C-C)alkenoyl; (C-C)cycloal- embodiments of the invention; however, the following illus kanoyl; (C-C)-cycloalkyl (C-C)alkanoyl: optionally 35 trative examples should not be interpreted in any way to Substituted aroyl. Such as unsubstituted aroyl or aroyl Sub- limit the invention. stituted by 1 to 3 substituents selected from the group consisting of halogen, cyano, trifluoromethaneSulphony- EXAMPLES loxy, (C-C)alkyl and (C-C)alkoxy, each of which is optionally further substituted with one or more of 1 to 3 40 Compound Examples halogen atoms; optionally Substituted aryl (C-C)alkanoyl and optionally substituted heteroaryl(C-C)alkanoyl. Such The compounds described herein may be prepared using as the aryl or heteroaryl radical being unsubstituted or the process and syntheses described herein, as well as using substituted by 1 to 3 substituents selected from the group general organic synthetic methods. In particular, methods for consisting of halogen, (C-C)alkyl and (C-C)alkoxy, each preparing the compounds are described in U.S. patent appli of which is optionally further substituted with 1 to 3 halogen 5 cation publication 2005/0002942, the disclosure of which is atoms; and optionally Substituted heteroarylalkanoyl having incorporated herein by reference. one to three heteroatoms selected from O, S and N in the heteroaryl moiety and 2 to 10 carbon atoms in the alkanoyl Example moiety, such as the heteroaryl radical being unsubstituted or Substituted by 1 to 3 substituents selected from the group 50 General formation of folate-peptides. The folate-contain consisting of halogen, cyano, trifluoromethaneSulphony- ing peptidyl fragment Pte-Glu-(AA)-NH(CHR)COH (3) loxy, (C-C)alkyl, and (C-C)alkoxy, each of which is is prepared by a polymer-Supported sequential approach optionally further substituted with 1 to 3 halogen atoms. The using standard methods, such as the Fmoc-strategy on an groups illustrated are exemplary, not exhaustive, and may be acid-sensitive Fmoc-AA-Wang resin (1), as shown in the prepared by conventional processes. following Scheme:

Scheme O O H R-NH a, b N a, c, a, d e, f O-Wang --> R-NH-(AA) O-Wang --> --> (n times) R2 O R2 1 2 US 9,662,402 B2 107 108 -continued O O H -o-r'sN HN N N O R NY n NH HN 2 N

O 3 (a) 20% piperidine/DMF; (b) Fmoc-AA-OH, PyBop, DIPEA, DMF; (c) Fmoc-GlucC-t-Bu)-OH, PyBop, DIPEA, DMF; (d) 1. N'(TFA)- Pte-OH; PyBop, DIPEA, DMSO, (e) TFAA, (CH2SH), i-PrSiH: (f) NH4OH, pH 10.3.

It is to be understood that unnatural amino acids may be cysteine, aspartate, and the like may also be used in the included in the foregoing process using the appropriate folate-peptide synthesis described herein. Further, gamma, starting materials. delta, or longer homologous amino acids may also be In this illustrative embodiment of the processes described included as starting materials in the folate-peptide synthesis herein, R is Fmoc, R is the desired appropriately-protected described herein. Further, amino acid analogs having amino acid side chain, and DIPEA is diisopropylethylamine. homologous side chains, or alternate branching structures, Standard coupling procedures, such as PyBOP and others Such as norleucine, isovaline, B-methyl threonine, B-methyl described herein or known in the art are used, where the cysteine, Bf3-dimethyl cysteine, and the like, may also be coupling agent is illustratively applied as the activating 25 included as starting materials in the folate-peptide synthesis reagent to ensure efficient coupling. Fmoc protecting groups described herein. are removed after each coupling step under standard condi The coupling sequence (steps (a) & (b)) involving Fmoc tions, such as upon treatment with piperidine, tetrabutylam AA-OH is performed “n” times to prepare solid-support monium fluoride (TBAF), and the like. Appropriately pro peptide (2), where n is an integer and may equal 0 to about tected amino acid building blocks, such as Fmoc-Glu-OtBu, 30 100. Following the last coupling step, the remaining Fmoc Fmoc-D-Glu-OtBu, N'-TFA-Pte-OH, and the like, are group is removed (step (a)), and the peptide is sequentially used, as described in the Scheme, and represented in step (b) coupled to a glutamate derivative (step (c)), deprotected, and by Fmoc-AA-OH. Thus, AA refers to any amino acid coupled to TFA-protected pteroic acid (step (d)). Subse starting material, that is appropriately protected. It is to be quently, the peptide is cleaved from the polymeric Support understood that the term amino acid as used herein is 35 upon treatment with trifluoroacetic acid, ethanedithiol, and intended to refer to any reagent having both an amine and a triisopropylsilane (step (e)). These reaction conditions result carboxylic acid functional group separated by one or more in the simultaneous removal of the t-Bu, t-Boc, and Trt carbons, and includes the naturally occurring alpha and beta protecting groups that may form part of the appropriately amino acids, as well as amino acid derivatives and analogs protected amino acid side chain. The TFA protecting group of these amino acids. In particular, amino acids having side 40 is removed upon treatment with base (step (f)) to provide the chains that are protected, such as protected serine, threonine, folate-containing peptidyl fragment (3).

EC119 HN N N Ys H HN 2 N CO2H CO2H SH N O O O H H H O N N N N N N CO2H O HOC O P l O P n HOC NH 1. NH2

LCMS ESI IM+H: 1046; Partial "H NMR (D.O, 300 MHz): 8 8.68 (s, 1H, FAH-7), 7.57 (d. 2H, J=8.4 Hz, FA H-12 & 16), 6.67 (d. 2H, J=9 HZ, FAH-13 & 15), 4.40-4.75 65 (series of m, 5H), 4.35 (m. 2H), 4.16 (m. 1H), 3.02 (m. 2H), 2.55-2.95 (series of m, 8H), 2.42 (m, 2H), 2.00-2.30 (m, 2H), 1.55-1.90 (m, 2H), 1.48 (m, 2H) ppm. US 9,662,402 B2 109 110 Example The corresponding compounds containing one or more D-amino acids may also be prepared, such as the following: EC1213

Ys H HN 2 N CO2H C OH SH N O O O H H H O N N N N N N CO2H

O HOC O P O P N HOC

NH 1. NH2

LCMS ESI, M+HI''') 1046. Partial 1H-NMR (DMSO) 6 (ppm): 8.6 (s), 7.5 (d), 6.6 (d), 3.8-4.6 (m), 2.8-3.2 (m), 2.2-2.8 (m), 1-2.2 (m) ECO835

MS (ESI, M+H")=1046.5. Partial 1H-NMR (DMSO) & (ppm): 8.6 (s), 7.5 (d), 6.6 (d), 3.8-4.6 (m), 2.8-3.2 (m), 2.2-2.8 (m), 1-2.2 (m) EC819 Ys HN 2 COH2 COH2 SH N O 1 O 1 O H H H O N-- N N N N N CO2H H H H

HOC

HN NH2

MS (ESI, M+H")=1046.4. Partial 1H-NMR (DMSO) & 6s (ppm): 8.6 (s), 7.6 (d), 6.6 (d), 4-4.6 (m), 3.4-3.8 (m), 3-3.15 (m), 1-2.8 (m)

US 9,662,402 B2 113 114

Nes H HN 2 N CO2H CO2H SH N O 1 O O H s H H N N N COH E H H H O HOC O O isYHO.c

NH ins, and YN's HN 2 COH2 COH2 SH N O 1 O 1 O H H H O N N N N N N CO2H. H H H 5 iOC O O S. HOC

NH

HN NH2

Example 30 Preparation of tubulysin hydrazides. Illustrated by pre paring EC0347 (TubB-H).

ECO347 HO AcO N-1 O

O N : -CNS !N O N ~sO

N,N-Diisopropylethylamine (DIPEA, 6.1 uL) and isobutyl chloroformate (3.0 uL) were added with via syringe in so Example tandem into a solution of tubulysin B (0.15 mg) in anhy drous EtOAc (2.0 mL) at -15° C. After stirring for 45 Synthesis of Coupling Reagent EC0311 minutes at -15° C. under argon, the reaction mixture was cooled down to -20°C. and to which was added anhydrous hydrazine (5.0 uL). The reaction mixture was stirred under 55 argon at -20°C. for 3 hours, quenched with 1.0 mM sodium N HCI phosphate buffer (pH 7.0, 1.0 mL), and injected into a NN Ne preparative HPLC for purification. Column: Waters XTerra p1Nus Her Prep MSCs 10 um, 19x250 mm; Mobile phase A: 1.0 mM N -Q Ns N f sodium phosphate buffer, pH 7.0; Mobile phase B: acetoni- 60 O O trile; Method: 10% B to 80% B over 20 minutes, flow Na rate=25 mL/min. Fractions from 15.14-15.54 minutes were p1Nus collected and lyophilized to produce EC0347 as a white H.N. -R N-O solid (2.7 mg). The foregoing method is equally applicable 6s N O for preparing other tubulysin hydrazides by the appropriate selection of the tubulysin starting compound. US 9,662,402 B2 115 116 DIPEA (0.60 mL) was added to a suspension of HOBt -continued OCO-(CH) SS-2-pyridine HCl (685 mg, 91%) in anhydrous DCM (5.0 mL) at 0°C., stirred under argon for 2 minutes, and to which was added anhydrous hydrazine (0.10 mL). The reaction mixture was stirred under argon at 0° C. for 10 minutes and room temperature for an additional C 30 minutes, filtered, and the filtrate was purified by flash NN "N- chromatography (silica gel, 2% MeOH in DCM) to afford EC0311 as a clear thick oil (371 mg), solidified upon PN-s, He Standing. 10 Y-k s-all O Example Preparation of Tubulysin Disulfides (Stepwise Process)

O Na NHN-k pNu-s, s-N h O

OH

30

Illustrated for EC0312. DIPEA (36 uL) and isobutyl chlo 35 -continued roformate (13 uI) were added by Syringe in tandem into a solution of tubulysin B (82 mg) in anhydrous EtOAc (2.0 mL) at -15°C. After stirring for 45 minutes at -15°C. under argon, to the reaction mixture was added a solution of EC0311 in anhydrous EtOAc (1.0 mL). The resulting solu 40 tion was stirred under argon at -15° C. for 15 minutes and room temperature for an additional 45 minutes, concen trated, and the residue was purified by flash chromatography (silica gel, 2 to 8% MeOH in DCM) to give EC0312 as a N white Solid (98 mg). The foregoing method is equally 45 n applicable for preparing other tubulysin derivatives by the 21 appropriate selection of the tubulysin starting compound.

50

Example To a solution of doxorubicin (100 mg, 0.184 mmol) and 2-benzotriazole-1-yl-(oxycarbonyloxy)-ethyldisulfanyl 55 pyridine (77.8 mg, 0.184 mmol) in DCM (4 ml) was added DIPEA (0.064 ml, 0.368 mmol.). The reaction was allowed to stir for 2 hours. TLC (10% MeOH in DCM) indicated that the reaction was complete. DCM was removed under 60 reduced pressure and purified on SiO, column (10% MeOH in DCM) to yield pure product (90 mg, 65%). LCMS (ESI): (M+H)" Calculated for CHNOS, 757.17. found 757.30, H NMR (300 MHz, CDC1/CDOD): 8 8.44 (brs, 1H), 8.00 (d. 1H), 7.65-7.82 (m, 3H), 7.38 (d. 1H), 7.18 (br 65 s, 1H), 5.45 (s, 1H), 5.25 (s.3H), 4.70 (m, 2H), 4.3 (m. 1H), 4.22-3.90 (m, 2H), 3.75 (s, 1H), 3.62 (s, 1H), 3.35-2.90 (m, 2H), 2.45-2.10 (m, 2H), 1.85 (m, 5H), 1.32 (d. 3H). US 9,662,402 B2 117 118 Example Tubulysin B pyridyldisulfide.

O 1 a 6Ac OH

Similarly, Tubulysin B pyridyldisulfide is prepared as described herein. 2O Example

EC1577 COH COH

O CO2H O O H H H N N N O N N N O D. O O NH

EC1577 MS (ESI, M+H")=1681. Partial 'H NMR (DO): 8.96 (s), 7.65 (d), 6.81 (d), 4.66 (s), 4.40-4.15 (m), 3.90-3.54 (m), " 3.50-3.18 (m), 2.97-2.90 (m), 2.51-1.80 (m). Example General Synthesis of Disulfide Containing Tubulysin Conjugates.

B-L-SH +

N N 1a 1S N N O US 9,662,402 B2 119 120 -continued

OAc N-1 O

L S -cr) 1'N's 1N1)- Y es

Illustrated with pyridinyl disulfide derivatives of certain naturally occurring tubulysins, where R' is H or OH, and R', is alkyl or alkenyl. A binding ligand-linker intermediate 15 containing a thiol group is taken in deionized water (ca. 20 mg/mL, bubbled with argon for 10 minutes prior to use) and the pH of the suspension was adjusted by saturated NaHCO (bubbled with argon for 10 minutes prior to use) to about 6.9 (the Suspension may become a solution when the pH increased). Additional deionized water is added (ca. 20-25%) to the solution as needed, and to the aqueous solution is added immediately a solution of EC0312 in THF (ca. 20 mg/mL). The reaction mixture becomes homogenous 25 quickly. After stirring under argon, e.g. for 45 minutes, the reaction mixture is diluted with 2.0 mM sodium phosphate buffer (pH 7.0, ca 150 volume percent) and the THF is removed by evacuation. The resulting Suspension is filtered and the filtrate may be purified by preparative HPLC (as 30 described herein). Fraction are lyophilized to isolate the conjugates. The foregoing method is equally applicable for preparing other tubulysin conjugates by the appropriate selection of the tubulysin starting compound. Example 35 EC 1663 and EC1664. The following additional com pounds are preparable using the methods and processed described herein: 40

45

50

55

60

65 US 9,662,402 B2 121 122

OH

US 9,662,402 B2 123 124 Example EC1426 is prepared according to the following process.

TESC1 -- Imidazole DCM Step 1

O.5M KHMDS O OTES Chloromethylbutyrate --->THF N.7. N 2N COMe Step 2 COMe ''' / w -->O ECO997 EC1004

F Step 3 EDC, PFP-OH Pol/C, H2 w OH NMP w O F NMP N N Silica Chromatography O F F

F

O OTES MeSnOH O OTES DCE H N w .. N 2N. Step 4 N a N CO2H ep

w O X w 1.--> EC1006

1) EtN-3HF Step 52) Ac2O, DMAP, Py 3) C18 Chromatography EC1422 ECO607 HNNH, PyBOP DIPEA, THF

O O OAc H BocN w NH2 w N, s' N1 9 '' S X-coil -- 1) THFStep 6 w O 2) Silica Chromatography --N HO EC1008 US 9,662,402 B2 125 126 -continued O BOCHNOC w w N1 ON-1Ns 1Ne.S N O ON N

HO EC1426

Example EC1456 is prepared according to the following process. US 9,662,402 B2 127 128

N 2 S

N

ro-OS US 9,662,402 B2 129 130

ponu?uoo US 9,662,402 B2 131 132 Example N'-TFA Protected EC1454 is prepared according to the following process.

1) EC0475, 2) Fmoc-D-Glut OtBu)-OH, 3) EC0475, 4) Fmoc-D-Glut OtBu)-OH, 5) EC0475, 6) Fmoc-D-Glu-OtBu, and 7) N10-TFA-Pte-OH (CSO S-Trityl-D-cysteine-2-chlorotrityl resin

O COH

O N H N O HN s N

HN lsN N 2 O1. CF

N10-TFA EC1454

45 Example EC1454 is prepared according to the following process.

O N N O O O HN n N Na2CO3 He C18 Chrom.

HN 1s N N a O1. CF O

N1 O-TFA EC1454 US 9,662,402 B2 133 134 -continued

EC1454

EC1454: MS (ESI, M+2H)=840.90, M+H"=1681.3. Partial 1H-NMR (DMSO) 8 (ppm): 8.6 (s), 7.6 (d), 6.6 (d), 4.45 (s), 4.35 (t), 4.15-4.3 (m), 3.3-3.6 (m), 3.25 (m), 3.0 (m), 2.7-2.9 (m), 2-2.3 (m), 1.6-2 (m). EC1415

O COH H N O H N O D

HN N N

45 EC1415: M+H=1709.69, M+2H*=855.22. Partial -continued H NMR (DO, 300 MHz) 8(ppm): 8.6 (s, 1H), 7.45 (d. 2H), 0.5M 6.5 (d. 2H), 4.5 (s. 2H), 4.3-4.1 (m, 6H), 3.95 (t, 1H), 3.8-3.4 O OTES cNER, (m. 19H), 3.4-2.95 (m, 7H), 2.4-1.7 (m, 26H), 1.6 (m, 1H), 50 N3 N SR y 1.25 (s. 2H), 1.05 (s, 3H). N e coMe Thir S / Step 2 Example w EC1004 is prepared according to the following process. ECO997

OTES

TESC1 60 2N He Imidazole COMe DCM S / Step 1

EC1458 65 O --> EC1004 US 9,662,402 B2 135 136 Into a round bottomed flask equipped with magnetic stir bar Into an appropriately sized hydrogenation flask place and temperature probe dipeptide EC1458, imidazole, and R N-methyl pipecolinate (MEP), pentafluorophenol, methylene chloride is added. Once all the solids have N-methyl pyrrolidinone (NMP), and ethyl dimethylamino dissolved, the solution is cooled using an ice bath. Chloro propyl carbodiimide (EDC). The mixture is stirred for at triethylsilane (TESC1) is added drop wise and the ice bath is least 16 h. EC1004 dissolved in N-methyl pyrrolidinone removed. The reaction is monitored for completion. A (NMP) and 10 wt % Pd/C are added. The reaction mixture second portion of chlorotriethylsilane and/or imidazole is is stirred/shaken under hydrogen pressure until the reaction is complete by LC analysis. The Pd/C is removed by added if necessary. The imidazole HCl salt is removed by filtration through celite. The celite is washed with ethyl filtration and methylene chloride is added. The organics are acetate and the combined organics are washed three times washed with a saturated solution of sodium chloride (brine), 10 with a 1% sodium bicarbonate? 10% sodium chloride solu the aqueous layer is back extracted once with methylene tion. The organic layer is dried over Sodium sulfate and chloride, and the combined organic layers are washed with concentrated on a rotary evaporator. The residue is dissolved brine. The organic layer is dried over sodium sulfate and in DCM and purified by Silica gel chromatography using concentrated on a rotary evaporator. The residue is dissolved ethyl acetate and petroleum ether as eluents. Fractions are in tetrahydrofuran (THF) and cooled to approximately -45° 15 collected, checked for purity, combined and dried on a rotary C. A solution of potassium bis(trimethylsilyl)amide evaporator. The EC1005 oil is assayed by LC and stored in (KHMDS) in toluene is added drop wise. With stirring, a freezer until use. chloromethyl butyrate is added and the reaction is moni tored. The reaction is quenched with methanol and then Example ethyl acetate and brine are added. The aqueous layer is discarded and the organics are washed once with brine. The EC1008 is prepared according to the following process. organic layer is concentrated on a rotary evaporator and the oily residue is passed through a short plug of silica gel. The 1) MeSnOH plug is washed with a 20% solution of ethyl acetate in H O OTES DCE petroleum ether. The combined organics are concentrated on 25 Ns' N. N a N 2) NaHF a rotary evaporator until distillation ceases. The crude COMe y Hs EC1004 oil is analyzed by LC and NMR and stored in a O ' N SA 3) Ac2O, freezer until use. w O DMAP

O1-n Steps 4-5 Example 30 EC1005 EC1005 is prepared according to the following process.

Pol/C, H2 2N NMP

COMe Silica Chrom. Step 3

EC1004

s OH EDC, w O w PFP-OH -e- O NMP O F MEP

H V. N,

O

EC1005