Patent Application
20170190736
The present disclosure provides antibody drug conjugates (Formula I) comprising a Dolastatin derivative moiety of Formula II as the drug component.
Dolastatins, such as natural product Dolastatin 10, and its synthetic derivatives Monomethyl Auristatin E (MMAE) and Monomethyl Auristatin F (MMAF) are products that show potent antineoplastic and tubulin inhibitory property. Because of their high toxicity, the direct use of Dolastatins as therapeutic agents has not been effective. Instead, they were conjugated to an antibody for targeted delivery to kill cancer cells.
The present disclosure provides a compound comprising a Dolastatin derivative moiety of Formula IV:
wherein Y is OH, or NH2,
R4 is OH, NH2, F, Cl, Br, I, OR5, wherein R5 is C1-C4 alkyl.
The present disclosure further provides an antibody drug-conjugate having the structure of Formula I:
AbL1-L2-D)n (I)
or a pharmaceutically acceptable salt thereof,
wherein:
Ab is a monoclonal antibody
L1 is a connector
L2 is a linker
D is an active agent having the structure of Formula II
wherein Y is O, or NH, the wavy line indicates the point of attachment,
X is —CH2N3 or
wherein R is C1-C8 alkyl, C3-C6 cyclic alkyl, aryl or heteroaryl.
n is an integer from 1-8.
Preferably, L2 is selected from the group consisting of an amino acid, peptide, —(CH2)n—, —(CH2CH2O)n—, p-aminobenzyl (PAB), Val-Cit (Citrulline)-PAB, Val-Ala-PAB, Ala-Ala-Asn-PAB, or combinations thereof. Preferably, -L1-L2 is selected from the group consisting of
Preferably, Ab-L1-L2 is selected from the group consisting of
The present disclosure further provides a method for synthesizing an antibody drug-conjugate having the structure of Formula I:
AbL1-L2-D)n (I)
or a pharmaceutically acceptable salt thereof,
wherein:
Ab is a monoclonal antibody
L1 is a connector
L2 is a linker
D is an active agent having the structure of Formula II
wherein Y═O, or NH, the wavy line indicates the point of attachment
X is —CH2N3 or
wherein R is C1-C8 alkyl, C3-C6 cyclic alkyl, aryl or heteroaryl.
n is an integer from 1-8, comprising
reacting a compound of formula III with a Lys on an Ab
wherein G is selected from the group consisting of —F, —Cl, —Br, —I, —N3, —OR, SR, —ONRR, RC(═O)O—, and RSO2—O—; and
R is optionally substituted alkyl, or optionally substituted aryl.
m=0, or 1.
The present disclosure provides compounds and conjugates, such as ADC (antibody drug conjugates), wherein a linker moiety that is peptide based has an attaching point at its C terminal which reacts with either Cys or Lys on an antibody in a controlled fashion. For Lys conjugation, for example, the DAR (drug antibody ratio) is 2. The DAR (drug antibody ratio) of the majority of conjugate is 4, when conjugation occurred on Cys.
Abbreviations are defined as follows:
Formation of an activated ester (e.g. NHS) from an acid An acid was dissolved in DCM (methylene chloride) and DMF (N,N′ dimethyl formamide) was added to aid dissolution if necessary. N-hydroxysuccinimide (1.5 eq) was added, followed by EDC.HCl (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) (1.5 eq). The reaction mixture was stirred at room temperature for 1 h until most of the acid was consumed. The progress of the reaction was monitored by RP-HPLC. The mixture was then diluted with DCM and washed successively with citric acid (aq. 10%) and brine. The organic layer was dried and concentrated to dryness. The crude product was optionally purified by RP-HPLC or silica gel column chromatography.
To a crude solution of compound 47 (0.1 mmol) in THF (3 mL) was added a solution of piperidine 4-carboxylic acid (60 mg) in sat. aq. NaHCO3 (1 mL). The mixture was stirred at room temperature for 30 min, then acidified with 1N aq. HCl to pH=4-5. The reaction mixture was concentrated and the residue was purified by reverse phase HPLC to give compound 1 as a white powder after lyophilization (68 mg). MS m/z 1020.7 (M+H).
Compound 52 (185 mg, 0.2 mmol) was dissolved in DCM/DMF (5/1, v/v, 5 mL).
EDC.HCl (0.5 mmol) and HOSu (0.3 mmol) were added. The mixture was stirred at room temperature for 30 min. HPLC analysis confirmed that all of compound 52 was consumed. The reaction was diluted with DCM (50 mL) and washed with brine. The organic layer was concentrated to 1 mL and diluted with acetonitrile/water (6/4, v/v, 3 mL). A solution of pyrrolidine 3-carboxylic acid (60 mg) in sat. aq. NaHCO3 (1 mL) was added and the mixture was stirred at room temperature for 10 min. The reaction was acidified with HOAc and concentrated. The crude product was purified by RP-HPLC to give compound 2 (138 mg, 68%). MS m/z 1020.6 (M+H).
To compound 37 (261 mg, 0.52 mmol) in 6 mL of DMF was added HATU (217 mg, 0.57 mmol), DIEA (362 μL, 2.08 mmol), and amine 36 (213 mg, 0.52 mmol). The mixture was stirred for 30 min, then 400 μL of piperidine was added and stirred for 10 min. The mixture was evaporated and purified by HPLC to give compound 38 (171 mg, 60%). MS m/z 548.3 (M+H).
To compound 39 (37 mg, 0.15 mmol) in 4 mL of DMF was added HATU (59 mg, 0.15 mmol), DIEA (108 μL, 0.6 mmol), and amine 38 (102 mg, 0.15 mmol). The mixture was stirred for 30 min, then evaporated to dryness. The residue was dissolved in 2 mL of DCM, then 1 mL of TFA was added and stirred for 10 min. The mixture was evaporated and purified by HPLC to give compound 40 (94 mg, 78%). MS m/z 673.4 (M+H).
To compound 41 (85 mg, 0.12 mmol) in 2 mL of DMF was added HATU (48 mg, 0.12 mmol), DIEA (83 μL, 0.48 mmol), and amine 40 (94 mg, 0.12 mmol). The mixture was stirred for 30 min, then a solution of 90 mg of NaOH in 1 mL of water was added and stirred for 30 min. The mixture purified by HPLC to give compound 4 (86 mg, 58%). MS m/z 1239.7 (M+H).
To compound 41 (1000 mg, 1.67 mmol) in 20 mL of DMF was added HATU (640 mg, 1.68 mmol), DIEA (870 μL, 5.00 mmol), and amine 45 (535 mg, 1.67 mmol). The mixture was stirred for 30 min, then evaporated and purified by HPLC to give compound 46 (1140 mg, 70%). MS m/z 865.5 (M+H).
To compound 46 (500 mg, 0.57 mmol) in 10 mL of DMA was added bis(p-nitropenyl)carbonate (210 mg, 0.69 mmol), and DIEA (35 μL, 0.2 mmol). The mixture was stirred for 18 h, then 100 mL of ether was added and the precipitate was collected by filtration to give compound 47 (500 mg, 85%). MS m/z 1030.6 (M+H).
To compound 47 (125 mg, 0.12 mmol) in 4 mL of DMF was added HOBt (7 mg, 0.05 mmol), DIEA (21 μL, 0.12 mmol), and amine 48 (40 mg, 0.12 mmol). The mixture was stirred for 16 h, then 200 μL of piperidine was added and stirred for 10 min. The mixture was evaporated and purified by HPLC to give compound 49 (72 mg, 60%). MS m/z 1005.6 (M+H).
To compound 49 (30 mg, 0.027 mmol) in 2 mL of DCM was added DIEA (15 μL, 0.086 mmol), DIEA (50 μL, 0.288 mmol), and anhydride 50 (19 mg, 0.027 mmol). The mixture was stirred for 30 min, then evaporated and purified by HPLC to give compound 6 (32 mg, 88%). MS m/z 1347.5 (M+H).
Compound 7 was synthesized from compound 49 (0.1 mmol) and anhydride 63 (0.1 mmol) as described for the synthesis of compound 6. Yield: 79%. MS m/z 1296.8 (M+H).
To a solution of compound 47 (0.1 mmol) in THF (3 mL) was added a solution of compound 64 (0.15 mmol, 67 mg) in acetonitrile/water (1/1, v/v, 1 mL), followed by DIEA (50 μL). After 30 min, the reaction was acidified and concentrated. The residue was purified by reverse phase HPLC to give compound 8 as a white solid (87 mg). MS m/z 1243.6 [M+H]+.
To compound 46 (120 mg, 0.12 mmol) in 3 mL of DMF was added K2CO3 (118 mg, 0.85 mmol), and bromoacetate 51 (35 mg, 0.18 mmol). The mixture was stirred for 16 h, then evaporated. The residue was dissolved in 2 mL of DCM, filtered, and 2 mL of TFA was added. After 20 min the mixture was evaporated and purified by HPLC to give compound 52 (92 mg, 83%). MS m/z 923.5 (M+H).
To compound 52 (92 mg, 0.1 mmol) in 2 mL of DMF was added HATU (38 mg, 0.1 mmol), DIEA (70 μL, 0.4 mmol), and boc-hydrazine (15 mg, 0.12 mmol). The mixture was stirred for 30 min, then evaporated to dryness. The residue was dissolved in 2 mL of DCM, then 1 mL of TFA was added and stirred for 10 min. The mixture was evaporated and purified by HPLC to give compound 53 (82 mg, 78%). MS m/z 937.5 (M+H).
To compound 54 (53 mg, 0.156 mmol) in 2 mL of DCM was added DIC (10 mg, 0.078 mmol) and stirred for 10 min. Then DIEA (54 μL, 0.312 mmol) and amine 53 (82 mg, 0.078 mmol) was added and the mixture was stirred for 15 min. The mixture was evaporated and purified by HPLC to give compound 9 (62 mg, 63%). MS m/z 1260.5 (M+H).
Preparation of Compound 13
To compound 37 (130 mg, 0.26 mmol) in 3 mL of DMF was added HATU (110 mg, 0.29 mmol), DIEA (175 μL, 1 mmol), and amine 36 (110 mg, 0.27 mmol). The mixture was stirred for 30 min, then concentrated to dryness. The residue was then treated with TFA/DCM (1/4, v/v, 5 mL) for 30 min. The mixture was evaporated and purified by HPLC to give compound 66 (108 mg, 65%). MS m/z 670.5 (M+H).
To compound 41 (85 mg, 0.12 mmol) in 2 mL of DMF was added HATU (48 mg, 0.12 mmol), DIEA (83 μL, 0.48 mmol), and amine 66 (94 mg, 0.12 mmol). The mixture was stirred for 30 min, then piperidine (0.2 mL) was added and stirred for 30 min. The mixture was concentrated and purified by HPLC to give compound 67 (87 mg, 63%). MS m/z 1028.7 (M+H).
To a solution of compound 67 (57 mg, 0.05 mmol) and acid 68 (22 mg) in DCM/DMF (3/1, v/v, 4 mL) was added PyBrOP (0.055 mmol) and DIEA (35 μL). The mixture was stirred at room temperature for 30 min and then concentrated to about 2 mL. The residue was purified by reverse phase HPLC to give compound 13 (41 mg). MS m/z 1425.7 (M+H).
This example provides the results of EC50 assays of the designated drug conjugated antibodies measured in vitro in specified cells. The antibody used was an anti-HER2 IgG class of antibody.
This example shows in vivo efficacy of ADC 16 (an anti-Her2 antibody conjugate) in a Subcutaneous N87 Xenograft Model.
This example shows in vivo safety of ADC 16 (an anti-Her2 antibody conjugate) in a Subcutaneous N87 Xenograft Model.
This example (
This example (
This example (
This example shows in vivo safety and efficacy of ADC 19 (an anti-Her2 antibody conjugate) in a Subcutaneous N87 Xenograft Model.
This example shows the general conjugation procedure for synthesizing antibody drug conjugates 16, 17, 19, and 64. To a solution of 0.5-50 mgs/mL of antibody in buffer at pH 6.0-9.0 with 0-30% organic solvent, was added 0.1-10 eq of activated drug linker conjugate (1, or 2,
Abbreviations are defined as follows:
Formation of an activated ester (e.g. NHS) from an acid An acid was dissolved in DCM (methylene chloride) and DMF (N,N′ dimethyl formamide) was added to aid dissolution if necessary. N-hydroxysuccinimide (1.5 eq) was added, followed by EDC.HCl (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) (1.5 eq). The reaction mixture was stirred at room temperature for 1 h until most of the acid was consumed. The progress of the reaction was monitored by RP-HPLC. The mixture was then diluted with DCM and washed successively with citric acid (aq. 10%) and brine. The organic layer was dried and concentrated to dryness. The crude product was optionally purified by RP-HPLC or silica gel column chromatography.
To a crude solution of compound 47 (0.1 mmol) in THF (3 mL) was added a solution of piperidine 4-carboxylic acid (60 mg) in sat. aq. NaHCO3 (1 mL). The mixture was stirred at room temperature for 30 min, then acidified with 1N aq. HCl to pH=4-5. The reaction mixture was concentrated and the residue was purified by reverse phase HPLC to give compound 1 as a white powder after lyophilization (68 mg). MS m/z 1020.7 (M+H).
Compound 52 (185 mg, 0.2 mmol) was dissolved in DCM/DMF (5/1, v/v, 5 mL). EDC.HCl (0.5 mmol) and HOSu (0.3 mmol) were added. The mixture was stirred at room temperature for 30 min. HPLC analysis confirmed that all of compound 52 was consumed. The reaction was diluted with DCM (50 mL) and washed with brine. The organic layer was concentrated to 1 mL and diluted with acetonitrile/water (6/4, v/v, 3 mL). A solution of pyrrolidine 3-carboxylic acid (60 mg) in sat. aq. NaHCO3 (1 mL) was added and the mixture was stirred at room temperature for 10 min. The reaction was acidified with HOAc and concentrated. The crude product was purified by RP-HPLC to give compound 2 (138 mg, 68%). MS m/z 1020.6 (M+H).
To compound 37 (261 mg, 0.52 mmol) in 6 mL of DMF was added HATU (217 mg, 0.57 mmol), DIEA (362 μL, 2.08 mmol), and amine 36 (213 mg, 0.52 mmol). The mixture was stirred for 30 min, then 400 μL of piperidine was added and stirred for 10 min. The mixture was evaporated and purified by HPLC to give compound 38 (171 mg, 60%). MS m/z 548.3 (M+H).
To compound 39 (37 mg, 0.15 mmol) in 4 mL of DMF was added HATU (59 mg, 0.15 mmol), DIEA (108 μL, 0.6 mmol), and amine 38 (102 mg, 0.15 mmol). The mixture was stirred for 30 min, then evaporated to dryness. The residue was dissolved in 2 mL of DCM, then 1 mL of TFA was added and stirred for 10 min. The mixture was evaporated and purified by HPLC to give compound 40 (94 mg, 78%). MS m/z 673.4 (M+H).
To compound 41 (85 mg, 0.12 mmol) in 2 mL of DMF was added HATU (48 mg, 0.12 mmol), DIEA (83 μL, 0.48 mmol), and amine 40 (94 mg, 0.12 mmol). The mixture was stirred for 30 min, then a solution of 90 mg of NaOH in 1 mL of water was added and stirred for 30 min. The mixture purified by HPLC to give compound 4 (86 mg, 58%). MS m/z 1239.7 (M+H).
To compound 41 (1000 mg, 1.67 mmol) in 20 mL of DMF was added HATU (640 mg, 1.68 mmol), DIEA (870 μL, 5.00 mmol), and amine 45 (535 mg, 1.67 mmol). The mixture was stirred for 30 min, then evaporated and purified by HPLC to give compound 46 (1140 mg, 70%). MS m/z 865.5 (M+H).
To compound 46 (500 mg, 0.57 mmol) in 10 mL of DMA was added bis(p-nitropenyl)carbonate (210 mg, 0.69 mmol), and DIEA (35 μL, 0.2 mmol). The mixture was stirred for 18 h, then 100 mL of ether was added and the precipitate was collected by filtration to give compound 47 (500 mg, 85%). MS m/z 1030.6 (M+H).
To compound 47 (125 mg, 0.12 mmol) in 4 mL of DMF was added HOBt (7 mg, 0.05 mmol), DIEA (21 μL, 0.12 mmol), and amine 48 (40 mg, 0.12 mmol). The mixture was stirred for 16 h, then 200 μL of piperidine was added and stirred for 10 min. The mixture was evaporated and purified by HPLC to give compound 49 (72 mg, 60%). MS m/z 1005.6 (M+H).
To compound 49 (30 mg, 0.027 mmol) in 2 mL of DCM was added DIEA (15 μL, 0.086 mmol), DIEA (50 μL, 0.288 mmol), and anhydride 50 (19 mg, 0.027 mmol). The mixture was stirred for 30 min, then evaporated and purified by HPLC to give compound 6 (32 mg, 88%). MS m/z 1347.5 (M+H).
Compound 7 was synthesized from compound 49 (0.1 mmol) and anhydride 63 (0.1 mmol) as described for the synthesis of compound 6. Yield: 79%. MS m/z 1296.8 (M+H).
To a solution of compound 47 (0.1 mmol) in THF (3 mL) was added a solution of compound 64 (0.15 mmol, 67 mg) in acetonitrile/water (1/1, v/v, 1 mL), followed by DIEA (50 μL). After 30 min, the reaction was acidified and concentrated. The residue was purified by reverse phase HPLC to give compound 8 as a white solid (87 mg). MS m/z 1243.6 [M+H]+.
To compound 46 (120 mg, 0.12 mmol) in 3 mL of DMF was added K2CO3 (118 mg, 0.85 mmol), and bromoacetate 51 (35 mg, 0.18 mmol). The mixture was stirred for 16 h, then evaporated. The residue was dissolved in 2 mL of DCM, filtered, and 2 mL of TFA was added. After 20 min the mixture was evaporated and purified by HPLC to give compound 52 (92 mg, 83%). MS m/z 923.5 (M+H).
To compound 52 (92 mg, 0.1 mmol) in 2 mL of DMF was added HATU (38 mg, 0.1 mmol), DIEA (70 μL, 0.4 mmol), and boc-hydrazine (15 mg, 0.12 mmol). The mixture was stirred for 30 min, then evaporated to dryness. The residue was dissolved in 2 mL of DCM, then 1 mL of TFA was added and stirred for 10 min. The mixture was evaporated and purified by HPLC to give compound 53 (82 mg, 78%). MS m/z 937.5 (M+H).
To compound 54 (53 mg, 0.156 mmol) in 2 mL of DCM was added DIC (10 mg, 0.078 mmol) and stirred for 10 min. Then DIEA (54 μL, 0.312 mmol) and amine 53 (82 mg, 0.078 mmol) was added and the mixture was stirred for 15 min. The mixture was evaporated and purified by HPLC to give compound 9 (62 mg, 63%). MS m/z 1260.5 (M+H).
Preparation of Compound 13
To compound 37 (130 mg, 0.26 mmol) in 3 mL of DMF was added HATU (110 mg, 0.29 mmol), DIEA (175 μL, 1 mmol), and amine 36 (110 mg, 0.27 mmol). The mixture was stirred for 30 min, then concentrated to dryness. The residue was then treated with TFA/DCM (1/4, v/v, 5 mL) for 30 min. The mixture was evaporated and purified by HPLC to give compound 66 (108 mg, 65%). MS m/z 670.5 (M+H).
To compound 41 (85 mg, 0.12 mmol) in 2 mL of DMF was added HATU (48 mg, 0.12 mmol), DIEA (83 μL, 0.48 mmol), and amine 66 (94 mg, 0.12 mmol). The mixture was stirred for 30 min, then piperidine (0.2 mL) was added and stirred for 30 min. The mixture was concentrated and purified by HPLC to give compound 67 (87 mg, 63%). MS m/z 1028.7 (M+H).
To a solution of compound 67 (57 mg, 0.05 mmol) and acid 68 (22 mg) in DCM/DMF (3/1, v/v, 4 mL) was added PyBrOP (0.055 mmol) and DIEA (35 μL). The mixture was stirred at room temperature for 30 min and then concentrated to about 2 mL. The residue was purified by reverse phase HPLC to give compound 13 (41 mg). MS m/z 1425.7 (M+H).
This example provides the results of EC50 assays of the designated drug conjugated antibodies measured in vitro in specified cells. The antibody used was an anti-HER2 IgG class of antibody.
This example shows in vivo efficacy of ADC 16 (an anti-Her2 antibody conjugate) in a Subcutaneous N87 Xenograft Model.
This example shows in vivo safety of ADC 16 (an anti-Her2 antibody conjugate) in a Subcutaneous N87 Xenograft Model.
This example (
This example (
This example (
This example shows in vivo safety and efficacy of ADC 19 (an anti-Her2 antibody conjugate) in a Subcutaneous N87 Xenograft Model.
This example shows the general conjugation procedure for synthesizing antibody drug conjugates 16, 17, 19, and 64. To a solution of 0.5-50 mgs/mL of antibody in buffer at pH 6.0-9.0 with 0-30% organic solvent, was added 0.1-10 eq of activated drug linker conjugate (1, or 2, or 3, or 4, or 5, or 62) in a manner of portion wise or continuous flow. The reaction was performed at 0-40° C. for 0.5-50 hours with gentle stirring or shaking, monitored by HIC-HPLC. The resultant crude ADC product underwent necessary down-stream steps of desalt, buffet changes/formulation, and optionally, purification, using the state-of-art procedures. The ADC product was characterized by HIC-HPLC, SEC, RP-HPLC, and optionally LC-MS.
This example shows a general conjugation procedure for synthesizing antibody drug conjugates 21, 22, 23, 24, 28, and 65. To a solution of antibody, 0.5-50 mgs/mL, in a certain buffet at pH 5.0-9.0, such as PBS, was added 0.5-100 eq of reducing agent such as TCEP and DTT. The reduction was performed at 0-40° C. for 0.5-40 hours with gentle stirring or shaking, and then the reducing agent was removed by column or ultrafiltration. To the reduced antibody, 0.5-50 mgs/mL, in a certain buffet at pH 5.0-9.0, such as PBS, with 0-30% of organic co-solvent such as DMA, was added 0.5-10 eq of the drug-linker reactant (selected from compound 6-15, or 63). The reaction was conducted at 0-40° C. for 0.5-40 hours with gentle stirring or shaking, monitored by HIC-HPLC. The resultant crude ADC product underwent necessary down-stream steps of desalt, buffet changes/formulation, and optionally, purification, using the state-of-art procedures. The final ADC product was characterized by HIC-HPLC, SEC, RP-HPLC, and optionally LC-MS.
This patent application claims priority to pending U.S. provisional patent application 62/108,894 filed 28 Jan. 2015.
| Number | Date | Country | |
|---|---|---|---|
| 62108894 | Jan 2015 | US |
