Technical Field

The present disclosure provides antibody drug conjugates (Formula I) comprising a Dolastatin derivative moiety of Formula II as the drug component.

Background

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 ells.

Summary The present disclosure provides a compound comprising a Dolastatin derivative

(IV)

wherein Y is OH, or NH2,

R ₄ is OH, NH ₂ , F, CI, Br, I, OR ₅ , wherein R ₅ is C1-C4 alkyl.

The present disclosure further provides an antibody drug-conjugate having the structure of Formula I:

(I)

or a pharmaceutically acceptable salt thereof,

wherein:

Ab is a monoclonal antibody

L ¹ is a connector

L ² is a linker

wherein Y is O, or NH, the wavy line indicates the point of attachment,

O O

X is -CH ₂ N ₃ or ^¾ H " o O , wherein R is C1-C8 alkyl, C3-C6 cyclic alkyl, aryl or heteroaryl.

n is an integer from 1-8.

Preferably, L ² is selected from the group consisting of an amino acid, peptide, - (CH ₂ )n-, -(CH ₂ CH ₂ 0) _n -, p-aminobenzyl (PAB), Val-Cit (Citrulline)-PAB, Val-Ala-PAB, Ala-Ala-Asn-PAB, or combinations thereof. Preferably, -L -L ² is selected from the group

Preferably, Ab-L -L ² 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:

(I)

or a pharmaceutically acceptable salt thereof,

wherein:

Ab is a monoclonal antibody

L ¹ is a connector

L ² is a linker

wherein Y = O, or NH, the wavy line indicates the point of attachment wherein R is C1-C8 alkyl, C3-C6 cyclic alkyl, aryl or heteroaryl.

n is an integer from 1-8, comprising

reactin a compound of formula III with a Lys on an Ab

wherein G is selected from the group consisting of -F, -CI,- Br, -I, -N ₃ , -OR, SR, -ONRR, RC(=0)0-, and RSO2-O-; and

R is optionally substituted alkyl, or optionally substituted aryl.

m = 0, or 1.

Brief Description of the Figures

Figure 1 shows a single dose of conjugate 16 administered to BALB/c nude mice (n=8) by intravenous administration.

Figure 2 shows a single dose of conjugate 16 administered to BALB/c nude mice (n=8) by intravenous administration.

Figure 3 shows pictures of the mice 35 days after treatment.

Figure 4A shows in vitro activity of ADC-23 (anti-Her2 antibody) in a group of tumor cell lines.

Figure 4B shows in vitro activity of ADC- 16 (anti-Her2 antibody) in a group of tumor cell lines.

Figure 5 shows in vivo efficacy of ADC-65, ADC-23 and ADC- 19 in various xenograft tumor models.

Figure 6A and 6B shows a single dose of conjugate 16 and 19 administered to BALB/c nude mice (n=8) by intravenous administration.

Detailed Description

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. Table 1. Examples of structures of drug-linker moieties for Lys conjugation onto an antibody.

Table 2. Examples of structures of drug-linker compounds (for Cys conjugation) to be conjugated onto a hinge region of an IgG class antibody.

Compound ID Structures

Definitions

Abbreviations are defined as follows:

Ac Acetyl

aq. Aqueous

BOC or Boc tert-Butoxycarbonyl

BrOP bromo tris(dimethylamino) phosphonium hexafluorophosphate

Bu n-Butyl

°C Temperature in degrees Centigrade

Cit Citrulline

DCM methylene chloride

DEPC Diethylcyanophosphonate

DIC diisopropylcarbodiimide

DIEA Diisopropylethylamine

DMA N,N-Dimethylacetamide

DMF N,N-Dimethylformamide

EDC l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

Et Ethyl

EtOAc Ethyl acetate

Eq Equivalents

Fmoc 9-Fluorenylmethoxycarbonyl

g Gram(s)

h Hour (hours)

HATU 2-(lH-7-azabenzotriazol-l-yl)-l, l,3,3-tetramethyl uronium hexafluorophosphate

ΗΟΒΤ N-Hydroxybenzotriazole

HOSu N-Hydroxysuccinimide

HPLC High-performance liquid chromatography LC/MS Liquid chromatography-mass spectrometry

Me Methyl

MeOH Methanol

MeCN Acetonitrile

mL Milliliter(s)

MS mass spectrometry

PAB p-aminobenzyl

RP-HPLC reverse phase HPLC

rt room temperature

t-Bu tert-Butyl

TEA Triethylamine

Tert, t tertiary

TFA Trifluoracetic acid

THF Tetrahydrofuran

TLC Thin-layer chromatography

Microliter(s)

General synthesis procedure - Formation of an activated ester (e.g. NHS) from an acid An acid was dissolved in DCM (methylene chloride) and DMF (Ν,Ν' dimethyl formamide) was added to aid dissolution if necessary. N-hydroxysuccinimide (1.5 eq) was added, followed by EDC.HC1 (l-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.

Example 1

Preparation of Compound 1

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. NaHCCb (1 mL). The mixture was stirred at room temperature for 30 min, then acidified with IN aq. HC1 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).

Example 2

Preparation of compound 2

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. NaHCCb (1 mL) was added and the mixture was stirred at room temperature for 10 min. The reaction was acidified with HO Ac and concentrated. The crude product was purified by RP-HPLC to give compound 2 (138 mg, 68%). MS m/z 1020.6 (M+H).

Example 3

Preparation of compound 4

Preparation of compound 38:

To compound 37 (261 mg, 0.52 mmol) in 6 mL of DMF was added HATU (217 mg, 0.57 mmol), DIEA (362 μΐ,, 2.08 mmol), and amine 36 (213 mg, 0.52 mmol). The mixture was stirred for 30 min, then 400 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).

Preparation of compound 40:

To compound 39 (37 mg, 0.15 mmol) in 4 mL of DMF was added HATU (59 mg,

0.15 mmol), DIEA (108 μί, 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).

Preparation of compound 4:

To compound 41 (85 mg, 0.12 mmol) in 2 mL of DMF was added HATU (48 mg, 0.12 mmol), DIEA (83 μί, 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).

Example 4

Preparation of compound 6

Preparation of compound 46:

To compound 41 (1000 mg, 1.67 mmol) in 20 mL of DMF was added HATU (640 mg, 1.68 mmol), DIEA (870 μί, 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).

Preparation of compound 47:

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 μί, 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).

Preparation of compound 49:

To compound 47 (125 mg, 0.12 mmol) in 4 mL of DMF was added HOBt (7 mg, 0.05 mmol), DIEA (21 μί, 0.12 mmol), and amine 48 (40 mg, 0.12 mmol). The mixture was stirred for 16 h, then 200 μΐ, 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).

Preparation of compound 6:

To compound 49 (30 mg, 0.027 mmol) in 2 mL of DCM was added DIEA (15 uL,

0.086 mmol), DIEA (50 μί,, 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).

Example 5

Pre aration of com ound 7.

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).

Example 6

Preparation of compound 8.

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 μί). 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] ⁺ .

Example 7

Preparation of compound 9.

Preparation of compound 52:

To compound 46 (120 mg, 0.12 mmol) in 3 mL of DMF was added K ₂ C0 ₃ (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).

Preparation of compound 53:

To compound 52 (92 mg, 0.1 mmol) in 2 mL of DMF was added HATU (38 mg, 0.1 mmol), DIEA (70 μί, 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).

Preparation of compound 9:

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 μΐ,, 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).

Example 8

To compound 37 (130 mg, 0.26 mmol) in 3 mL of DMF was added HATU (110 mg, 0.29 mmol), DIEA (175 μί, 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 μί, 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 |jL). 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). Example 9.

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.

Example 10

This example shows in vivo efficacy of ADC 16 (an anti-Her2 antibody conjugate) in a Subcutaneous N87 Xenograft Model. Figure 1 shows a single dose of conjugate 16 administered to BALB/c nude mice by intravenous administration. There were 8 mice in each group and total 6 groups of mice were studied: 3 groups were injected with T-DM1

(Trastuzumab - DM1 conjugate) at different doses; 2 groups were injected with ADC 16 at different doses; and one vehicle control. All the drugs were administered in the same manner (single dose). A single dose of ADC- 16 iv. at 1 mg/kg or 3 mg/kg outperformed T-DM1 at 3 mg/kg or 10 mg/kg respectively. 3 mg/kg ADC- 16 completely inhibited tumor growth up to 100 days.

Example 11

This example shows in vivo safety of ADC 16 (an anti-Her2 antibody conjugate) in a Subcutaneous N87 Xenograft Model. Figure 2 shows a single dose of conjugate 16 administered to BALB/c nude mice by intravenous administration. There were 8 mice in each group and total 7 groups of mice were studied: 3 groups were injected with T-DM1

(Trastuzumab - DM1 conjugate) at different doses; 3 groups were injected with ADC 16 at different doses; and one vehicle control. All the drugs were administered in the same manner (single dose). A single dose of ADC- 16 iv. at 1 mg/kg, 3 mg/kg or 10 mg/kg did not retard body weight gain. The difference of the body weights between T-DM1 and ADC- 16 groups were caused by the difference of tumor weight. Figure 3 shows pictures of the mice 35 days after treatment. Example 12

This example (Figure 4A) shows ADC-23 induces equivalent or stronger antiproliferative activity in breast cancer cell lines, compared to MMAE conjugates. In these studies, the cells were all treated with either ADC-23 or MMAE conjugates for 3 d. IC50 is determined as the concentration that showed 50% inhibition of cell growth.

Example 13

This example (Figure 4B) shows ADC- 16 induces equivalent or stronger antiproliferative activity in breast cancer cell lines, compared to MMAE conjugates. In the above studies, the cells were all treated with either ADC-16 or MMAE conjugates for 3 d. IC50 is determined as the concentration that showed 50% inhibition of cell growth.

Example 14

This example (Figure 5) shows the in vivo efficacy of ADC-65, ADC-23 and ADC- 19 in LoVo (Colon), MDA-MB-468 (Breast), BxPC-3 (Pancreatic), PA-1 (Ovarian) and H1975 NSCLC xenograft nude mice. All ADCs were given as single dose via iv. at indicated concentrations. The ADCs tested outperformed MMAF in most cases at the same level, and completely inhibited tumor growth by single dose.

Example 15

This example shows in vivo safety and efficacy of ADC 19 (an anti-Her2 antibody conjugate) in a Subcutaneous N87 Xenograft Model. Figure 6A and 6B shows a single dose of conjugate 19 administered to BALB/c nude mice by intravenous administration. There were 8 mice in each group and total 3 groups of mice were studied: 1 group of mice was injected with ADC 16; 1 group of mice was injected with ADC 19; and one vehicle control. All the drugs were administered in the same manner (single dose). A single dose of ADC- 19 iv. at 2 mg/kg was comparable to that of ADC-16 at the same dose and completely inhibited tumor growth up to 49 days and did not retard body weight gain that was comparable to ADC-16.

Example 16

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.

Example 17

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.