Pharmaceutical compositions comprising macrolide diastereomers, methods of their synthesis and therapeutic uses

Abstract

The disclosure relates to compositions comprising diastereomer of a macrolide exhibiting improved therapeutic profile in the context of inhibiting cell proliferation compared to the corresponding compositions comprising mixture of diastereomers. The disclosure further provides drug-ligand conjugates formed using diastereomer of the macrolide. The disclosure also provides novel method of preparation of diastereomer of the macrolide and their therapeutic uses.

Claims

1. A diastereomer of Formula I ##STR00032## wherein the diastereomer is characterized by a .sup.1H NMR characterized by delta shifts of (300 MHz, CDCl.sub.3) δ 6.85 (d, 1H, J=4 Hz), 6.72 (m, 1H), 6.65 (d, 1H, J=4 Hz), 6.44 (dd, 1H, J=15 Hz, 11 Hz), 6.25 (s, 1H), 5.67 (dd, 1H, J=16 Hz, 9 Hz), 5.41 (m, 1H), 4.79 (d, 1H, J=11 Hz), 4.30 (t, 1H, J=11 Hz), 3.72 (m, 2H), 3.51 (d, 1H, J=9 Hz), 3.37 (m, 4H), 3.27 (m, 1H), 3.23 (s, 3H), 3.16-2.99 (m, 4H), 2.85 (m, 7H), 2.62 (m, 3H), 2.39 (ddd, 1H, J=19 Hz, 12 Hz, 4 Hz), 2.18 (br m, 2H), 1.77 (br m, 3H), 1.66 (s, 3H), 1.60-1.47 (m, 4H), 1.31 (m, 6H), 1.05 (m, 2H), and 0.82 (s, 3H); and wherein the diastereomer characterized by the delta shifts is present in a diastereomeric excess of more than 90%, and wherein the diastereomeric excess is based on a composition comprising a mixture of at least two diastereomers having different stereochemical configurations at the chiral carbon that is bound to the sulfur atom of Formula I.

2. A compound of Formula I ##STR00033## wherein the compound is characterized by a .sup.1H NMR characterized by delta shifts of (300 MHz, CDCl.sub.3) δ 6.85 (d, 1H, J=4 Hz), 6.72 (m, 1H), 6.65 (d, 1H, J=4 Hz), 6.44 (dd, 1H, J=15 Hz, 11 Hz), 6.25 (s, 1H), 5.67 (dd, 1H, J=16 Hz, 9 Hz), 5.41 (m, 1H), 4.79 (d, 1H, J=11 Hz), 4.30 (t, 1H, J=11 Hz), 3.72 (m, 2H), 3.51 (d, 1H, J=9 Hz), 3.37 (m, 4H), 3.27 (m, 1H), 3.23 (s, 3H), 3.16-2.99 (m, 4H), 2.85 (m, 7H), 2.62 (m, 3H), 2.39 (ddd, 1H, J=19 Hz, 12 Hz, 4 Hz), 2.18 (br m, 2H), 1.77 (br m, 3H), 1.66 (s, 3H), 1.60-1.47 (m, 4H), 1.31 (m, 6H), 1.05 (m, 2H), and 0.82 (s, 3H); and wherein the compound characterized by the delta shifts is present in a diastereomeric excess of more than 90%, and wherein the diastereomeric excess is based on a composition comprising a mixture of at least two diastereomers having different stereochemical configurations at the chiral carbon that is bound to the sulfur atom of Formula I; prepared by a process comprising the steps of (a) contacting (i) a compound of Formula III ##STR00034## wherein R.sub.1 and R.sub.2 are hydrogen; and n is one; (ii) a compound of Formula IV ##STR00035##  wherein Y.sup.1 is ##STR00036## (iii) silica gel; (iv) a diluent comprising an organic solvent and water; and (b) isolating the compound of Formula I.

3. An antibody-drug conjugate prepared by contacting a compound of Formula I ##STR00037## wherein the compound is characterized by a .sup.1H NMR characterized by delta shifts of (300 MHz, CDCl.sub.3) δ 6.85 (d, 1H, J=4 Hz), 6.72 (m, 1H), 6.65 (d, 1H, J=4 Hz), 6.44 (dd, 1H, J=15 Hz, 11 Hz), 6.25 (s, 1H), 5.67 (dd, 1H, J=16 Hz, 9 Hz), 5.41 (m, 1H), 4.79 (d, 1H, J=11 Hz), 4.30 (t, 1H, J=11 Hz), 3.72 (m, 2H), 3.51 (d, 1H, J=9 Hz), 3.37 (m, 4H), 3.27 (m, 1H), 3.23 (s, 3H), 3.16-2.99 (m, 4H), 2.85 (m, 7H), 2.62 (m, 3H), 2.39 (ddd, 1H, J=19 Hz, 12 Hz, 4 Hz), 2.18 (br m, 2H), 1.77 (br m, 3H), 1.66 (s, 3H), 1.60-1.47 (m, 4H), 1.31 (m, 6H), 1.05 (m, 2H), and 0.82 (s, 3H); and wherein the compound characterized by the delta shifts is present in a diastereomeric excess of more than 90%, and wherein the diastereomeric excess is based on a composition comprising a mixture of at least two diastereomers having different stereochemical configurations at the chiral carbon that is bound to the sulfur atom of Formula I; with an antibody or antigen-binding fragment thereof, wherein the compound of Formula I is prepared by a process comprising the steps of (a) contacting (i) a compound of Formula III ##STR00038## wherein R.sub.1 and R.sub.2 are hydrogen; and n is one; (ii) a compound of Formula IV ##STR00039##  wherein Y.sup.1 is ##STR00040## (iii) silica gel; (iv) a diluent comprising an organic solvent and water; and (b) isolating the antibody-drug conjugate of Formula I.

4. The antibody-drug conjugate of claim 3, wherein the antibody or antigen-binding fragment thereof specifically binds a tumor-associated antigen and further wherein the tumor-associated antigen is selected from the group consisting of AFP, ALK, BAGE proteins, β-catenin, brc-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, caspase-8, CD20, CD40, CDK4, CEA, CLEC12A, cMET, CTLA4, cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2/Her2, ErbB3, ErbB4, ETV6-AML, EphA2, Fra-1, FOLR1, GAGE proteins, GD2, GD3, GloboH, glypican-3, GM3, gp100, Her2, HLA/B-raf, HLA/k-ras, HLA/MAGE-A3, hTERT, IGF1R, LGR5, LMP2, MAGE proteins (e.g., MAGE-1, -2, -3, -4, -6, and -12), MART-1, mesothelin, ML-IAP, Muc1, Muc16 or CA-125, MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PDGFR-α, PDGFR-β, PDGF-A, PDGF-B, PDGF-C, PDGF-D, PLAC1, PRLR, PRAME, PSMA, FOLH1, RAGE proteins, Ras, RGS5, Rho, SART-1, SART-3, Steap-1, Steap-2, survivin, TAG-72, TGF-β, TMPRSS2, Tn, TNFRSF17, TRP-1, TRP-2, tyrosinase, and uroplakin-3.

5. A process for preparing a compound of Formula I ##STR00041## wherein the compound of Formula I is characterized by a .sup.1H NMR characterized by delta shifts of (300 MHz, CDCl.sub.3) δ 6.85 (d, 1H, J=4 Hz), 6.72 (m, 1H), 6.65 (d, 1H, J=4 Hz), 6.44 (dd, 1H, J=15 Hz, 11 Hz), 6.25 (s, 1H), 5.67 (dd, 1H, J=16 Hz, 9 Hz), 5.41 (m, 1H), 4.79 (d, 1H, J=11 Hz), 4.30 (t, 1H, J=11 Hz), 3.72 (m, 2H), 3.51 (d, 1H, J=9 Hz), 3.37 (m, 4H), 3.27 (m, 1H), 3.23 (s, 3H), 3.16-2.99 (m, 4H), 2.85 (m, 7H), 2.62 (m, 3H), 2.39 (ddd, 1H, J=19 Hz, 12 Hz, 4 Hz), 2.18 (br m, 2H), 1.77 (br m, 3H), 1.66 (s, 3H), 1.60-1.47 (m, 4H), 1.31 (m, 6H), 1.05 (m, 2H), and 0.82 (s, 3H); and wherein the compound characterized by the delta shifts is present in a diastereomeric excess of more than 90%, and wherein the diastereomeric excess is based on a composition comprising a mixture of at least two diastereomers having different stereochemical configurations at the chiral carbon that is bound to the sulfur atom of Formula I; the process comprising the steps of (a) contacting a compound of Formula III ##STR00042## wherein R.sub.1 and R.sub.2 are hydrogen; and n is one; (ii) a compound of Formula IV ##STR00043##  wherein Y.sup.1 is ##STR00044## (iii) silica gel; (iv) a diluent comprising an organic solvent and water; and (b) isolating the compound of Formula (I).

6. The process of claim 5, wherein the organic solvent comprises a polar aprotic solvent.

7. The process of claim 6, wherein the polar aprotic solvent is acetonitrile.

8. The process of claim 5, wherein the molar ratio of the starting material having Formula III and the compound of Formula IV is from about 1:1 to about 1:3.

9. The process of claim 5, wherein the organic solvent and water are present in a ratio from about 1:1 to about 4:1, or from about 1:1 to about 10:1.

10. The process of claim 9, wherein the organic solvent is acetonitrile and the ratio of acetonitrile to water is about 4:1.

11. The process of claim 5, further comprising contacting the compound of Formula I of step (b) with an antibody or antigen-binding fragment thereof, to obtain an antibody-drug conjugate.

12. The antibody-drug conjugate of claim 11, wherein the antibody or antigen-binding fragment thereof specifically binds a tumor-associated antigen and further wherein the tumor-associated antigen is selected from the group consisting of AFP, ALK, BAGE proteins, β-catenin, brc-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, caspase-8, CD20, CD40, CDK4, CEA, CLEC12A, cMET, CTLA4, cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2/Her2, ErbB3, ErbB4, ETV6-AML, EphA2, Fra-1, FOLR1, GAGE proteins, GD2, GD3, GloboH, glypican-3, GM3, gp100, Her2, HLA/B-raf, HLA/k-ras, HLA/MAGE-A3, hTERT, IGF1R, LGR5, LMP2, MAGE proteins MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-6, MAGE-12, MART-1, mesothelin, ML-IAP, Muc1, Muc16 or CA-125, MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PDGFR-α, PDGFR-β, PDGF-A, PDGF-B, PDGF-C, PDGF-D, PLAC1, PRLR, PRAME, PSMA, FOLH1, RAGE proteins, Ras, RGS5, Rho, SART-1, SART-3, Steap-1, Steap-2, survivin, TAG-72, TGF-β, TMPRSS2, Tn, TNFRSF17, TRP-1, TRP-2, tyrosinase, and uroplakin-3.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 illustrates an .sup.1H-NMR spectrum of Maytansin-3-N-methyl-L-alanine-propanamidyl-3-thio-3-succinimidyl-N-methylcyclohexyl-4-trans-carboxysuccinate (5). The .sup.1H-NMR spectrum is consistent with a single diastereomer present in at least 95% diastereomeric excess since the spectrum is not complicated by resonances attributable to the other diastereomer. (For comparative purposes Example 2 sets forth the .sup.1H-NMR spectrum of the mixture of diastereomers).

(2) FIG. 2 illustrates an .sup.1H-NMR spectrum of mixture of diastereomers of Maytansin-3-N-methyl-L-alanine-propanamidyl-3-thio-3-succinimidyl-N-methylcyclohexyl-4-trans-carboxysuccinate (6).

(3) FIG. 3 illustrates that in SKBR3 cells the single diastereomer compound conjugate HER2-5 (in vitro and in vivo lots) possessed an IC50 value of 0.3 nM versus 0.9 nM for the mixture of diastereomer conjugate HER2-6.

(4) FIG. 4 illustrates that in BT474 cells the single diastereomer compound conjugate HER2-5 (in vitro) in possessed an IC.sub.50 value of 4.6 nM while the HER2-5 (in vivo) lot had an IC.sub.50 value of 4.0 nM versus 11.6 nM for the mixture of diastereomer conjugate HER2-6.

(5) FIG. 5 illustrates that in NCI-N87 cells the single diastereomer compound conjugate HER2-5 (in vitro) possessed an IC.sub.50 value of 0.6 nM while the HER2-5 (in vivo) lot had an IC.sub.50 value of 0.4 nM versus 1.0 nM for the mixture of diastereomer conjugate HER2-6.

(6) FIG. 6 illustrates that in HEK293/hEGFRvIII cells the single diastereomer compound conjugate EGFRvIII-5 possessed an IC.sub.50 value of 0.4 nM while the mixture of diastereomer conjugate EGFRvIII-6 had an IC.sub.50 value of 0.5 nM.

(7) FIG. 7 illustrates that in MMT/hEGFRvIII cells the single diastereomer compound conjugate EGFRvIII-5 possessed an IC.sub.50 value of 0.5 nM while the mixture of diastereomer conjugate EGFRvIII-6 had an IC.sub.50 value almost 20 fold higher at 9.8 nM.

(8) FIG. 8 illustrates that in U251/hEGFRvIII cells the single diastereomer compound conjugate EGFRvIII-5 possessed an IC.sub.50 value of 2.4 nM while the mixture of diastereomer conjugate EGFRvIII-6 had an IC.sub.50 value of 3.3 nM.

(9) FIG. 9 illustrates that in Ovcar-3 cells the single diastereomer compound conjugate MUC16-5 possessed an IC.sub.50 value of 6.3 nM while the mixture of diastereomer conjugate MUC16-6 had an IC.sub.50 value of 16.0 nM.

(10) FIG. 10 illustrates that in PC3/MUC16 long cells the single diastereomer compound conjugate MUC16-5 in possessed an IC.sub.50 value of 0.34 nM while the mixture of diastereomer conjugate MUC16-6 had an IC.sub.50 value at 0.80 nM.

(11) FIG. 11 illustrates tumor growth curves in mice following dosing with HER2-5 and control reagents. Mice received PBS vehicle (.square-solid.), 300 ug/kg DM1-SMe (.circle-solid.) and Isotype Control mAb at 15 mg/kg (.Math.). Mice also received HER2 mAb (.box-tangle-solidup.), HER2-5 (Δ) and Isotype Control-5 (∇) at doses of 1 mg/kg (A), 5 mg/kg (B) and 15 mg/kg (C). Mice received 3 once weekly doses of conjugates and control agents as indicated by the black arrows (↑. Groups are N=8, Mean±SE.).

(12) FIG. 12 illustrates change in tumor volume following dosing with HER2-5 and control reagents at termination of the PBS vehicle control group on Day 79 post tumor implantation. Individual tumor sizes are shown for each dosing group. Mice received PBS vehicle (.square-solid.), 300 ug/kg DM1-SMe (.circle-solid.) and Isotype Control mAb at 15 mg/kg (.Math.). Mice also received HER2 mAb (.box-tangle-solidup.), HER2-5 (Δ) and Isotype Control-5 (∇) at doses of 1 mg/kg (A), 5 mg/kg (B) and 15 mg/kg (C). Groups are N=8, Mean±SD.

(13) FIG. 13 illustrates percentage change in animal weights following dosing with HER2-5 and control reagents. Mice received PBS vehicle (.square-solid.), 300 ug/kg DM1-SMe (.circle-solid.) and Isotype Control mAb at 15 mg/kg (.Math.). Mice also received HER2 mAb (.box-tangle-solidup.), HER2-5 (Δ) and Isotype Control-5 (∇) at doses of 1 mg/kg (∇), 5 mg/kg (B) and 15 mg/kg (C). Mice received 3 once weekly doses of conjugates and control agents as indicated by the black arrows (T. Groups are N=8, Mean±SE.).

EXAMPLES

(14) Experimental Details

(15) Proton NMR spectra (for compounds that could not be detected by UV) were acquired on a Varian Inova 300 MHz instrument, while mass spectra were collected on an Agilent 1100 series LC/MSD with electrospray ionization source and quadrupole ion trap analyzer. All conjugates were analyzed using a Bruker ultraFleXtreme MALDI-TOF-TOF mass spectrometer. All starting materials and solvents were purchased commercially and used without purification, unless otherwise noted.

Example 1

Synthesis of Maleimidylmethyl-4-trans-cyclohexylcarboxy-succinate (3): Maleimidylmethyl-4-trans-cyclohexanecarboxylic acid (2)

(16) The title compound was prepared in two steps (Step A and Step B) using modified versions of the methods described by Marnett et al. (J. Med. Chem., 1996, 39, 1692-1670).

(17) Step A:

(18) A solution of trans-4-aminomethylcyclohexane carboxylic acid (7.00 g, 44.5 mmol) in 1,4-dioxane (70 mL) was treated with maleic anhydride (4.89 g, 49.9 mmol) and stirred at ambient temperature for 48 h. The reaction was evaporated in vacuo to a white solid that can be stored or carried on to the next step without further purification. .sup.1H NMR (300 MHz, DMSO-d.sub.6) δ 9.11 (m, 1H), 6.44 (d, 1H, J=13 Hz), 6.24 (d, 1H, J=13 Hz), 3.05 (t, 2H, J=6 Hz), 2.13 (tt, 1H, J=12 Hz, 4 Hz), 1.90 (m, 2H), 1.75 (m, 2H), 1.44 (m, 1H), 1.28 (m, 2H), 0.96 (m, 2H).

(19) Step B:

(20) The maleamic acid from Step A (36.8 g, 144 mmol) and sodium acetate (13.6 g, 165 mmol) were dissolved in acetic anhydride (368 mL), sealed in a glass reaction vessel, and heated to 120° C. for 3 hours. The cooled reaction mixture (a black syrup) was poured onto water (3 L), stirred, and extracted with dichloromethane. The organic layer was dried over Na.sub.2SO.sub.4, filtered over a sintered glass funnel, and the clear filtrate evaporated and dried under high vacuum giving the title compound as a yellow solid (7.00 g, 20%). .sup.1H NMR (300 MHz, CDCl.sub.3) δ 6.73 (s, 2H), 3.40 (d, 2H, J=7 Hz), 2.28 (m, 1H), 2.06 (m, 2H), 1.75 (m, 3H), 1.42 (m, 2H), 1.03 (m, 2H).

Maleimidylmethyl-4-trans-cyclohexanecarboxysuccinate (3)

(21) The product of the preceding step B (10.0 g, 42.1 mmol) was dissolved in dichloromethane (50 mL) under Ar, treated with N-hydroxysuccinimide (7.27 g, 63.2 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDAC, 12.4 g, 64.5 mmol), and the reaction was stirred at ambient temperature overnight. The resulting brown solution was diluted with dichloromethane, washed with water and brine, dried over Na.sub.2SO.sub.4, filtered over a sintered glass funnel, and the filtrate concentrated and dried in vacuo. This product was then dissolved in hot ethyl acetate, treated with activated charcoal (1.5 g), filtered, and the filtrate cooled. Filtration of the crystalline product, washing with cold ethyl acetate, and suction drying then gave the title compound as a tan solid (5.52 g, 39%). .sup.1H NMR (300 MHz, CDCl.sub.3) δ 6.72 (s, 2H), 3.42 (m, 2H), 2.85 (br s, 4H), 2.56 (tt, 1H, J=12 Hz, 4 Hz), 2.18 (m, 2H), 1.80 (m, 2H), 1.70 (m, 1H), 1.56 (m, 2H), 1.09 (m, 2H).

(22) ##STR00026##

Example 2

Maytansin-3-N-methyl-L-alanine-propanamide-3-thiol (4)

(23) The title compound, known in the art as DM1, was prepared using a modified version of the method described by Whitesides et al. (J Org. Chem., 1991, 56, 2648-2650). Maytansin-3-N-methyl-L-Ala-(3-methyldisulfanyl) propanamide (DM1-SMe, 2.42 g, 3.09 mmol, prepared in a manner similar to Ho and Carrozzella, U.S. Pat. Appl. 2007/0037972 A1) was dissolved in acetonitrile (30 mL), treated with a solution of tris(2-carboxyethyl)phosphine hydrochloride (8.23 g, 28.7 mmol) in water (30 mL), the pH was raised to ca. 3 with the addition of saturated aqueous NaHCO.sub.3 (5 mL), the flask was purged with Ar, and the reaction was stirred at ambient temperature under a rubber septum (vented due to effervescence). After 2 h, the reaction was treated with brine (ca. 100 mL), bubbled with Ar for 5 minutes (to remove the free methylmercaptan), and the phases separated. The aqueous phase was extracted twice with ethyl acetate (EtOAc), saturated with NaCl, and extracted twice more with EtOAc. The combined organic layers were then dried over Na.sub.2SO.sub.4, filtered, and the filtrate concentrated and dried in vacuo to give the title compound as a white solid (2.24 g, 98%). .sup.1H NMR (300 MHz, CDCl.sub.3/CD.sub.3OD) δ 6.76 (d, 1H, J=1.5 Hz), 6.63 (d, 1H, J=11 Hz), 6.59 (d, 1H, J=1.5 Hz), 6.35 (m, 2H), 5.59 (dd, 1H, J=15 Hz, 9 Hz), 5.36 (q, 1H, J=6.5 Hz), 4.68 (dd, 1H, J=12 Hz, 3 Hz), 4.21 (t, 1H, J=10 Hz), 3.92 (s, 3H), 3.60 (d, 1H, J=13 Hz), 3.42 (d, 1H, J=9 Hz), 3.29 (s, 3H), 3.14 (s, 3H), 3.05 (d, 1H, J=13 Hz), 2.95 (d, 1H, J=10 Hz), 2.77 (s, 3H), 2.75-2.47 (m, 6H), 2.11 (dd, 1H, J=14 Hz, 3 Hz), 1.58 (s, 3H), 1.47 (d, 1H, J=14 Hz), 1.40 (m, 1H), 1.22 (m, 6H), 0.73 (s, 3H). MS (ESI, pos.): calc'd for C.sub.35H.sub.48ClN.sub.3O.sub.10S, 737.3; found 738.3 (M+H), 720.3 (M−H.sub.2O+H).

Maytansin-3-N-methyl-L-alanine-propanamidyl-3-thio-3-succinimidyl-N-methylcyclohexyl-4-trans-carboxysuccinate (5)

(24) The following procedure describes a new method not known in the art. The product of the preceding step (4, 2.23 g, 3.02 mmol) and Maleimidylmethyl-4-cyclohexanecarboxysuccinate (3, 1.50 g, 4.49 mmol) were dissolved in 4:1 acetonitrile/water (75 mL), treated with fine silica gel scraped from a preparative TLC plate (11.2 g), the flask purged with Ar, and the mixture stirred at ambient temperature under rubber septum. After 18 hours, more 3 (0.77 g, 2.3 mmol) and acetonitrile (MeCN, 10 mL) were added, and the reaction stirred an additional 6 hours. The mixture was filtered over Celite, solids washed with MeCN and ethyl acetate (EtOAc), and the filtrate concentrated in vacuo to a gold solid, which was purified by flash column chromatography on an 120 g silica gel cartridge (50-100% 1:1 EtOAc/MeCN in dichloromethane over 33 min, 75 mL/min). Evaporation and drying of the pure column fractions in vacuo gave the title compound as a cream-colored solid (2.09 g). Concentration of the impure fractions and repurification on an 80 g silica gel cartridge as above gave an additional batch of cream-colored solid (0.22 g), and brought the total yield of title compound to 2.31 g (71%). .sup.1H NMR (300 MHz, CDCl.sub.3) δ 6.85 (d, 1H, J=4 Hz), 6.72 (m, 1H), 6.65 (d, 1H, J=4 Hz), 6.44 (dd, 1H, J=15 Hz, 11 Hz), 6.25 (s, 1H), 5.67 (dd, 1H, J=16 Hz, 9 Hz), 5.41 (m, 1H), 4.79 (d, 1H, J=11 Hz), 4.30 (t, 1H, J=11 Hz), 3.72 (m, 2H), 3.51 (d, 1H, J=9 Hz), 3.37 (m, 4H), 3.27 (m, 1H), 3.23 (s, 3H), 3.16-2.99 (m, 4H), 2.85 (m, 7H), 2.62 (m, 3H), 2.39 (ddd, 1H, J=19 Hz, 12 Hz, 4 Hz), 2.18 (br m, 2H), 1.77 (br m, 3H), 1.66 (s, 3H), 1.60-1.47 (m, 4H), 1.31 (m, 6H), 1.05 (m, 2H), 0.82 (s, 3H). MS (ESI, pos.): calc'd for C.sub.51H.sub.66ClN.sub.5O.sub.16S, 1071.4; found 1072.4 (M+H), 1054.9 (M−H.sub.2O+H); [α].sup.20.sub.589 nm=−52.4 (c=0.00301, MeOH). See FIG. 1 for .sup.1H-NMR of single diastereomer.

(25) ##STR00027##

Example 3

Mixture of diastereomers of Maytansin-3-N-methyl-L-alanine-propanamidyl-3-thio-3-succinimidyl-N-methylcyclohexyl-4-trans-carboxysuccinate (6)

(26) A sample of the mixed stereoisomers of 5 was synthesized according to US Patent Application 20100129314, Example XI. .sup.1H NMR (300 MHz, CDCl.sub.3) δ 6.85-6.6 (m), 6.4 (m), 6.1 (m), 5.8-5.4 (m), 5.2 (m), 4.92-4.79 (m), 4.4-4.1 (m), 4.03 (s), 3.82 (m), 3.8-2.2 (m), 2.1 (m), 2.07 (s), 2.0-0.8 (m). MS (ESI, pos.): calc'd for C.sub.51H.sub.66ClN.sub.5O.sub.16S, 1071.4; found 1072.4 (M+H). See FIG. 2 for .sup.1H-NMR of mixture of diastereomers.

Example 4

Racemic Maytansin-3-N-methyl-L-alanine-propanamidyl-3-thio-3-succinimidyl-N-methylcyclohexyl-4-trans-carboxylic acid (8)

(27) A solution of trans-1,4-(maleimidomethyl) cyclohexane-1-carboxylic acid 7 (167 mg, 0.701 mmol) in 1, 2-dimethoxyethane (8 mL) was added to a solution of 4 (340 mg, 0.461 mmol) in 1, 2-dimethoxyethane (15 mL). The mixture was then treated with pH 7.5 buffer (20 mL) and a few drops of saturated aqueous NaHCO.sub.3 to maintain the pH. The reaction mixture was stirred overnight at room temperature under argon and then concentrated under reduced pressure. The crude residue was purified by reverse phase chromatography using a C18 column, 20-40 micron column (100 g), eluting with a gradient (10 95% over 25 mins) of acetonitrile (0.1% AcOH) in water (0.1% AcOH), and lyophilized to give 8 (330 mg, 0.338 mmol, 73% yield) as a white solid. MS m/z: 977.2 [MH+], 957.2 [M−18], 999.2 [M+Na]; Purity: >98% (by LC/MS).

(28) ##STR00028##

Example 5

Chiral Separation of 8 to Single Diastereomers (R*)-Maytansin-3-N-methyl-L-alanine-propanamidyl-3-thio-3-succinimidyl-N-methylcyclohexyl-4-trans-carboxylic acid (9) and (S*)-Maytansin-3-N-methyl-L-alanine-propanamidyl-3-thio-3-succinimidyl-N-methylcyclohexyl-4-trans-carboxylic acid (10) (S* and R* Represent a Single Stereoisomer of Unknown Chirality)

(29) The diastereomeric mixture of compounds 8 (20 mg) was dissolved in 0.5 ml of acetonitrile and separated using semi-prep Chiral column (Chirlacel OJ, Solvent system, 6:1:1 Hexanes:IPA:Ethanol) to afford 3.5 mg of 10 as the faster-running compound, MS m/z: 977.2 [MH+], 957.2 [M−18], 999.2 [M+Na]; Purity: >95% (by LC/MS), RT=32 min and 4.6 mg of 9 as the slower-running compound, MS m/z: 977.2 [MH+], 957.2 [M−18], 999.2 [M+Na]; Purity: >95% (by LC/MS), Rf=48 min.

(30) ##STR00029##

Example 6

Synthesis of (S*)-Maytansin-3-N-methyl-L-alanine-propanamidyl-3-thio-3-succinimidyl-N-methylcyclohexyl-4-trans-carboxylic acid (11)

(31) A solution of 10 (2.5 mg, 0.0026 mmol) was dissolved in dichloromethane (1 mL), then treated with N-hydroxysuccinimide (NHS, 6.0 mg, 0.052 mmol) and 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide hydrochloride (EDCI, 13 mg, 0.065 mmol). The reaction mixture was stirred overnight at room temperature under argon, washed with water followed by brine, dried over anhydrous sodium sulfate, and filtered. The solvent was evaporated under reduced pressure to give the crude residue, which was purified by reverse phase chromatography using a C18, 20-40 micron column (30 g), eluting with a gradient (10-95% over 18 min) of acetonitrile (0.1% AcOH) in water (0.1% AcOH), and lyophilized to afford 11. MS m/z: 1073.2 [MH+], 1054.4[M−18]; Purity: 95% (by LC/MS).

(32) ##STR00030##

Example 7

(R*)-Maytansin-3-N-methyl-L-alanine-propanamidyl-3-thio-3-succinimidyl-N-methylcyclohexyl-4-trans-carboxylicacid (12)

(33) A solution of 9 (3, mg, 0.003 mmol) was dissolved in dichloromethane (1 mL), then treated with N-hydroxysuccinimide (NHS, 3.0 mg, 0.026 mmol) and 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide hydrochloride (EDCI, 7 mg, 0.036 mmol). The reaction mixture was stirred overnight at room temperature under argon, washed with water followed by brine, dried over anhydrous sodium sulfate, and filtered. The solvent was evaporated under reduced pressure to give the crude residue, which was purified by reverse phase chromatography using C18 column, 20-40 micron (15 g), eluting with a gradient (10-95% over 18 min) of acetonitrile (0.1% AcOH) in water (0.1% AcOH), lyophilized to afford 12. MS m/z: 1073.2 [MH+], 1054.4[M−18]; Purity: 95% (by LC/MS).

(34) ##STR00031##

Example 8

(35) Conjugate Preparation and Characterization.

(36) Two different maytansine-linker compositions prepared according to the previous Examples (Compound 5 and Compound 6) were conjugated to various anti-tumor antigen monoclonal antibodies. Compound 5 comprises predominantly a single diastereomer of the linker-DM1 cytotoxic compound, whereas Compound 6 comprises a mixture of various linker-DM1 diastereomers. The antibodies used in this Example were an anti-HER2 antibody having variable regions derived from humAb4D5-8 from Carter et al, PNAS 1992 89 4285, an anti-EGFRvIII antibody having variable regions derived from clone 131 from WO2013075048 A1, and an anti-MUC16 antibody having variable regions derived from clone 3A5 from WO2007001851.

(37) The antibodies were expressed in CHO cells and purified by Protein A. A non-binding isotype control antibody derived from an infectious disease antigen having no relation to oncology was also used in this Example.

(38) The antibodies (10 mg/ml) in 50 mM HEPES, 150 mM NaCl, pH 8.0, and 10% (v/v) DMA were conjugated with a 6 fold excess of compound 5 or 6 for 1 hour at ambient temperature. The conjugates were purified by size exclusion chromatography and sterile filtered. Protein and linker payload concentrations were determined by UV spectral analysis and MALDI-TOF mass spectrometry. Size-exclusion HPLC established that all conjugates used were >95% monomeric, and RP-HPLC established that there was <0.5% unconjugated linker payload. Yields are reported in Table 1 based on protein. All conjugated antibodies were analyzed by UV for linker payload loading values according to Hamblett et al, Cancer Res 2004 10 7063 and by mass difference, native versus conjugated. The results are summarized in Table 1.

(39) TABLE-US-00001 TABLE 1 ε252 nm (cm.sup.−1 M.sup.−1) ε280 nm (cm.sup.−1 M.sup.−1) Compound 5 26790 5700 6* 26790 5700 Antibody HER2 81847 215388 EGFRvIII 79579 209420 MUC16 88671 248380 Isotype Control 81718 233480 Antibody Payload:Antibody Payload:Antibody Conjugate (UV) (MS) Yield % HER2-5 (in vivo) 2.7 2.7 66 HER2-5 (in vitro) 3.1 2.4 75 HER2-6 (in vitro) 2.9 2.4 70 EGFRvIII-5 2.8 2.3 56 EGFRvIII-6 2.9 2.2 56 MUC16-5 2.4 2.0 76 MUC16-6 2.3 2.1 96 Isotype Control-5 3.3 3.3 67 *Extinction coefficients were used from compound 5

Example 9

(40) In Vitro Cytotoxicity Assays.

(41) Cells were seeded in PDL-coated 96 well plates at 10,000 (SK-BR-3 and NCI-N.sub.87), 15,000 (BT-474), 3000 (Ovcar-3 and PC3/Muc16), 2000 (HEK293/hEGFRvIII), 1500 (U251/hEGFRvIII), or 400 (MMT/hEGFRvIII) cells per well in complete growth media and grown overnight. For cell viability curves, serially diluted antibody-drug conjugates or free payload were added to the cells at final concentrations ranging from 1 μM to 1 pM and incubated for 3 days. Each concentration was run in duplicate and reported with the respective standard deviation. Cells were incubated with CCK8 (Dojindo) for the final 1-3 h and the absorbance at 450 nm (OD.sub.450) was determined on a Flexstation3 (Molecular Devices). Background OD.sub.450 levels from digitonin (40 nM) treated cells were subtracted from all wells and viability is expressed as a percentage of the untreated controls. IC.sub.50 values were determined from a four-parameter logistic equation over a 10-point response curve (GraphPad Prism). All curves and IC.sub.50 values are corrected for payload equivalents based on the loading from the MALDI-TOF experiment.

(42) In SKBR3 cells (breast cancer line), natively expressing HER2 at 607 fold above isotype control binding, the single diastereomer compound conjugate HER2-5 (in vitro and in vivo lots) possessed an IC.sub.50 value of 0.3 nM versus 0.9 nM for the mixture of diastereomer compound conjugate HER2-6 (Table 2, FIG. 3). A small in vitro lot was conjugated first and only used for cell proliferation assays, while a larger in vivo lot was then conjugated and used for both in vitro and in vivo experiments. The naked HER2 antibody had little anti-proliferation activity.

(43) In BT474 cells (breast cancer line), natively expressing HER2 at 426 fold above isotype control binding, the single diastereomer compound conjugate HER2-5 (in vitro) possessed an IC.sub.50 value of 4.6 nM while the HER2-5 (in vivo) lot had an IC.sub.50 value of 4.0 nM versus 11.6 nM for the mixture of diastereomer compound conjugate HER2-6 (Table 2, FIG. 4). The naked HER2 antibody had little anti-proliferation activity.

(44) In NCI-N87 cells (breast cancer line), natively expressing HER2 at 869 fold above isotype control binding, the single diastereomer compound conjugate HER2-5 (in vitro) possessed an IC.sub.50 value of 0.6 nM while the HER2-5 (in vivo) lot had an IC.sub.50 value of 0.4 nM versus 1.0 nM for the mixture of diasteromer compound conjugate HER2-6 (Table 2, FIG. 5). The naked HER2 antibody had little anti-proliferation activity.

(45) In HEK293/hEGFRvIII cells, expressing hEGFRvIII at 360 fold above isotype control binding, both conjugates (single and mixture of diastereomer) exhibited IC.sub.50 values of 0.4 nM (Table 3, FIG. 6). The naked EGFRvIII antibody had little anti-proliferation activity.

(46) In MMT/hEGFRvIII cells, expressing hEGFRvIII at 280 fold above isotype control binding, the single diastereomer compound conjugate EGFRvIII-5 possessed an IC.sub.50 value of 0.5 nM while the mixture of diastereomer compound conjugate EGFRvIII-6 had an IC.sub.50 value of 9.8 nM (Table 2, FIG. 7). The naked EGFRvIII antibody had little anti-proliferation activity.

(47) In U251/hEGFRvIII cells, expressing hEGFRvIII at 165 fold above isotype control binding, the single diastereomer compound conjugate EGFRvIII-5 possessed an IC.sub.50 value of 2.4 nM while the mixture of diastereomer compound conjugate EGFRvIII-6 had an IC.sub.50 value of 3.3 nM (Table 3, FIG. 7). The naked EGFRvIII antibody had little anti-proliferation activity.

(48) In Ovcar-3 cells (ovarian cancer line), natively expressing MUC16 at 373 fold above isotype control binding, the single diastereomer compound conjugate MUC16-5 possessed an IC.sub.50 value of 6.3 nM while the mixture of diasteromer compound conjugate MUC16-6 had an IC.sub.50 value of 16.0 nM (Table 4, FIG. 8). The naked Muc16 antibody had little anti-proliferation activity.

(49) In PC3/MUC16 cells, expressing MUC16 at 105 fold above isotype control binding, the single diastereomer compound conjugate MUC16-5 possessed an IC.sub.50 value of 0.34 nM while the mixture of diastereomer compound conjugate MUC16-6 had an IC.sub.50 value of 0.8 nM (Table 4, FIG. 9). The naked Muc16 antibody had little anti-proliferation activity.

(50) In FIGS. 3, 4, and 5 maytansin-3-N-methyl-L-Ala-(3-methyldisulfanyl) propanamide (DM1-SMe, prepared according to Ho and Carrozzella, U.S. Pat. Appl. 2007/0037972 A1) was chosen to represent the payload in these assays. Compound 4 would be too reactive to use in vitro or in vivo, thereby giving unreliable results.

(51) The in vitro results are summarized in Tables 2-4 on a target basis below. This Example demonstrates that anti-tumor antibody-drug conjugates comprising the single diastereomer drug of the present invention, in most cases, exhibited greater in vitro killing potency than the corresponding antibody-drug conjugates comprising mixture of diastereomers. For the targets analyzed, the single diastereomer antibody-drug conjugates were typically on the order of 2- to 3-fold more potent than the corresponding mixture conjugates, depending on the particular cell lines tested.

(52) TABLE-US-00002 TABLE 2 Antibody SKBR3 BT474 N87 Conjugate IC50 (nM) IC50 (nM) IC50 (nM) HER2-5 (in vivo) 0.3 4.0 0.4 HER2-5 (in vitro) 0.3 4.6 0.6 HER2-6 (in vitro) 0.9 11.6 1.0

(53) TABLE-US-00003 TABLE 3 Antibody HEK293/hEGFRvIII MMT/hEGFRvIII U251/hEGFRvIII Conjugate IC50 (nM) IC50 (nM) IC50 (nM) EGFRvIII-5 0.4 0.5 2.4 EGFRvIII-6 0.5 9.8 3.3

(54) TABLE-US-00004 TABLE 4 Antibody Ovcar-3 PC3/MUC16 Conjugate IC50 (nM) IC50 (nM) MUC16-5 6.3 0.34 MUC16-6 16.0 0.80

Example 10

(55) In Vivo Studies.

(56) To determine the in vivo efficacy of the anti-HER2 single-diastereomer conjugate (“HER2-5”), studies were performed in mice bearing HER2+ gastric cancer xenografts, as efficacy had been previously reported in this model by Barok et al (Barok M et al, Can Letters 2011). Specifically, 5×10.sup.6 NCI-N87 cells (ATCC CRL-5822) were implanted subcutaneously into the lower right flank of CB-17 SCID mice (Taconic). Once tumors had reached an average volume of 150 mm.sup.3, mice were randomized in to groups of eight and dosed with HER2-5 or control reagents. Control reagents included PBS vehicle, free DM1-SMe, isotype control, isotype control-5, or HER2. Mice received once weekly doses for three weeks and tumor volumes and body weights were monitored twice weekly throughout the study. Conjugates were dosed at 1, 5 and 15 mg/kg, as these doses had been shown to be effective in previous in vivo studies by Lewis-Phillips et al (Lewis-Phillips G et al., Can Res 2008).

(57) In the current N87 tumor model, HER2-5 demonstrated clear anti-tumor efficacy, with doses of 5 and 15 mg/kg leading to significant decreases in initial tumor volume and eradication of some tumors at the higher dose (FIGS. 11 and 12). A significant delay in tumor growth relative to control agents was also observed in the 1 mg/kg dose level. No adverse events were observed following dosing, with mice receiving HER2-5 demonstrating robust weight gain throughout the study (FIG. 13).