Exploration of Pyrrolobenzodiazepine (PBD)-Dimers Containing

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Exploration of Pyrrolobenzodiazepine(PBD)-dimers Containing Disulfide-based Prodrugs as Payloads for Antibody-Drug Conjugates Zhonghua Pei, Chunjiao Chen, Jinhua Chen, Josefa dela Cruz-Chuh, Reginald Delarosa, Yuzhong Deng, Aimee Fourie-O’Donohue, Isabel Figueroa, Jun Guo, Weiwei Jin, Cyrus Khojasteh, Katherine R Kozak, Brandon Latifi, James Lee, Guangmin Li, Eva Lin, Liling Liu, Jiawei Lu, Scott Martin, Carl Ng, Trung Nguyen, Rachana Ohri, Gail Lewis Phillips, Thomas H Pillow, Rebecca K Rowntree, Nicola J Stagg, David Stokoe, Sheila Ulufatu, Vishal A. Verma, John Wai, Jing Wang, Keyang Xu, Zijin Xu, Hui Yao, Shang-Fan Yu, Donglu Zhang, and Peter S. Dragovich Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00431 • Publication Date (Web): 24 Jul 2018 Downloaded from http://pubs.acs.org on July 25, 2018

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Molecular Pharmaceutics

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Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Exploration of Pyrrolobenzodiazepine(PBD)-dimers Containing Disulfide-based Prodrugs as Payloads for Antibody-Drug Conjugates Zhonghua Pei,†,‡ Chunjiao Chen,║ Jinhua Chen,§ Josefa dela Cruz-Chuh,† Reginald Delarosa,† Yuzhong Deng,† Aimee Fourie-O’Donohue,† Isabel Figueroa,† Jun Guo,† Weiwei Jin,║ S. Cyrus Khojasteh,† Katherine R. Kozak,† Brandon Latifi,† James Lee,† Guangmin Li,† Eva Lin,† Liling Liu,† Jiawei Lu,║ Scott Martin,† Carl Ng,† Trung Nguyen,† Rachana Ohri,† Gail Lewis Phillips,† Thomas H. Pillow,† Rebecca K. Rowntree,† Nicola J. Stagg,† David Stokoe,†,┴ Sheila Ulufatu,† Vishal A. Verma,† John Wai,§ Jing Wang,§ Keyang Xu,† Zijin Xu,§ Hui Yao,§ Shang-Fan Yu,† Donglu Zhang,† Peter S. Dragovich*,† †

Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA Present address: Ideaya Biosciences, 7000 Shoreline Court, South San Francisco, CA 94080, USA § Wuxi Apptec, 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai 200131, China ║ WuXi Biologics, 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai 200131, China ┴Present address: Calico Inc., 1170 Veterans Blvd, South San Francisco, CA, 94080, USA ‡

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Abstract A number of cytotoxic pyrrolobenzodiazepine(PBD) monomers containing various disulfide-based prodrugs were evaluated for their ability to undergo activation (disulfide cleavage) in vitro in the presence of either glutathione (GSH) or cysteine (Cys). A good correlation was observed between in vitro GSH stability and in vitro cytotoxicity toward tumor cell lines. The prodrug-containing compounds were typically more potent against cells with relatively high intracellular GSH levels (e.g., KPL-4 cells). Several antibody-drug conjugates (ADCs) were subsequently constructed from pyrrolobenzodiazepine(PBD) dimers that incorporated selected disulfide-based prodrugs. Such HER2 conjugates exhibited potent antiproliferation activity against KPL-4 cells in vitro in an antigen-dependent manner. However, the disulfide prodrugs contained in the majority of such entities were surprisingly unstable toward whole blood from various species. One HER2-targeting conjugate that contained a thiophenol-derived disulfide prodrug was an exception to this stability trend. It exhibited potent activity in a KPL-4 in vivo efficacy model that was approximately 3-fold weaker than that displayed by the corresponding parent ADC. The same prodrug-containing conjugate demonstrated a 3-fold improvement in mouse tolerability properties in vivo relative to the parent ADC which did not contain the prodrug.

Keywords: prodrug, disulfide, antibody-drug conjugate, pyrrolobenzodiazepine



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INTRODUCTION Tumors frequently experience high levels of oxidative stress and have evolved various biological mechanisms to cope with and potentially exploit such conditions.1-3 For example, the levels of reduced glutathione (GSH), a promiscuous, naturally-occurring antioxidant,4 are often elevated in tumors relative to non-cancerous tissues.5 Numerous recent drug-delivery approaches have sought to utilize these differing GSH levels as a means to selectively release various cytotoxic cargos in tumors without affecting non-malignant cells.613

As part of our on-going efforts to develop next-generation antibody-drug conjugates

(ADCs),14-16 we were interested in similarly attempting to exploit high tumor GSH levels as a means to prepare conjugates with improved therapeutic potential relative to entities that we previously studied. Specifically, we sought to identify prodrugs of cytotoxic ADC payloads that could be selectively activated by the high reducing potential present in many intratumor environments following targeted, antibody-mediated delivery. In this report, we describe our efforts to design and synthesize such novel entities and also provide some initial details regarding their in vitro and in vivo biological profiling. We envisioned constructing ADCs derived from the highly potent cytotoxic pyrrolobenzodiazepine(PBD)-dimer 117 in which one of the compound’s reactive imine moieties18 was masked as a disulfide-containing carbamate group and the other was utilized for antibody attachment purposes via another carbamate and a cleavable linker (compound 2, Scheme 1). Following antigen binding and internalization into a targeted tumor cell, the linker portion of 2 was anticipated to undergo lysosome-mediated cleavage and subsequent self-immolation to unmask one of the imine moieties present in compound 1 (i.e., conversion of 2 to 3, Scheme 1). Multiple ADCs containing similarly linked PBD-dimer payloads have previously demonstrated strong in vivo efficacy,19,20 and these results provided high


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Scheme 1.

confidence that the antibody attachment and release strategy described above would afford robust intracellular levels of the released payload 3. However, in contrast to typical PBDdimer payloads produced intracellularly following ADC-mediated delivery, intermediate 3 is a PBD-dimer prodrug that is expected to exhibit attenuated cytotoxicity properties relative to dimeric molecules such as 1.21 Consistent with this hypothesis, we previously demonstrated that a compound related to structure 4 (R1-R2 = cyclopropyl) exhibited significantly reduced binding to DNA relative to the PBD-dimer 1.22 We also envisioned that the disulfide moiety present in 3 would undergo reductive cleavage in the intracellular environment and ensuing self-immolation to produce the corresponding PBD-dimer (i.e., the conversion of 3 to 4 to 1; Scheme 1). Our prior studies of highly efficacious disulfide-linked ADCs provided good precedent that the described transformation could efficiently occur,23,24 although some uncertainty existed regarding the exact cellular compartment in which disulfide reduction took place.25-30 Ideally, high concentrations of the potent PBD-dimer 1 would only be generated from 3 in strongly reducing intracellular environments (i.e., in tumor cells). This 4 ACS Paragon Plus Environment


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selective production of 1 was anticipated to help minimize damage caused to non-cancerous tissues by conjugates such as 2 in addition to any antigen-related selectivity benefits provided by the targeted ADC-mediated delivery.

Importantly, we expected that the steric and

electronic environments associated with the disulfide bonds present in compounds such as 2 and 3 would need to be carefully modulated so as to ensure (1) efficient cleavage in tumor cells, (2) significantly attenuated cleavage in non-tumor tissue, and (3) good blood/plasma stability during ADC circulation in vivo. Accordingly, we began our exploration of these prodrugs by seeking to identify disulfides that met these three criteria.

METHODS The majority of the disulfide-containing PBD-monomer compounds described in this work were prepared by the three related methods depicted in Scheme 2. These routes were somewhat interchangeable and all worked relatively well. The particular synthetic route used to make a given target compound was sometimes influenced by the sterics associated with the various carbamate precursors and whether these entities contained a UV-active chromophore. In the first method, aniline 5031 was converted to a variety of carbamates containing various disulfides derived from 5-nitropyridine-2-thiol (52a-52g). The disulfide-containing alcohols required for these conversions (51a-51g) were prepared as depicted in Scheme 3. Many of these entities were known in the literature (51a-51d),22,31,32 and the remainder were synthesized from the corresponding aldehydes using the two-step procedure shown in Scheme 3 (method A, compounds 51e-51g).33 Removal of the silyl protecting groups present in selected intermediates 52 under acidic conditions provided the corresponding primary alcohols (53), and these entities were subsequently oxidized using the Dess-Martin periodinane34,35 to hemi-aminals 54 (Method 1). It was important to closely monitor the progress of these oxidation reactions as prolonged exposure to the Dess-Martin reagent led to


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over-oxidation of the carbamate-protected hemi-aminal moieties present in the target compounds to give the corresponding carbamate-protected amides.

Treatment of

intermediates 54 with a variety of thiols (R3SH) then afforded the target compounds. The final compounds that were prepared by this process are listed in Scheme 2, and the R1, R2, and R3 groups associated with intermediates 52, 53, and 54 correspond to those listed in Figure 1. Scheme 2. Preparation of target compounds 6-28.a N O2N

S

S

R1 R3S TBDMSO H2N H N

S

R2 O O TBDMSO H HN

OH

R1

R2

O

51a-51g

O

a

Cmpds 6, 7, 8, 9, 10, 11, 12, 13, 14, 17, 19, 20, 22, 23, 24, 25

N

O

O

50

52a-52g

O

Method 1

O

b

N O2N

S

S

R1 R3S

R2

O O HO H HN

Cmpds 15, 16, 18

S

OH

R1

Method 2

R2

51j-51m

N

d

a

Method 3

O

R3SH

R3S

Cmpds 21, 26, 27, 28

O2N

S

S

R1

53

S

R1 R2 O O RO HN H N

R2 O

c

6-28

R3SH

d

O

O

HO H

O N

N

O

b

O O

N

O

c

O O

55 R = TBDMS

54

56 R = H

a

Reagents and conditions: TBDMS = tBu(CH3)2Si. (a) triphosgene, Et3N or pyridine, CH2Cl2, 0 °C; (b) HOAc/THF/H2O (3/2/1, v/v/v); (c): Dess-Martin periodinane, CH2Cl2; (d): R3SH, CH2Cl2.

Alternatively, several R3SH thiols were condensed with other selected intermediates 52 to provide the mixed disulfides 55 (Method 2). Removal of the silyl protecting groups present in 55 followed by oxidation of the resulting primary alcohols afforded the target 6 ACS Paragon Plus Environment


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compounds. Final compounds 15, 16, and 18 were synthesized by this method and the R1, R2, and R3 groups associated with intermediates 55 and 56 correspond to those depicted in Figure 1. Scheme 3. Preparations of disulfides 51a-51m.a

R3 S S

OH

R1

R2

R1

R2

a

H

H

b

(R)-CH3

H

Preparation

R3 N

Ref 31

O2N

Refs 22, 32

c

Cyclopropyl

d

Cyclobutyl

e

Cyclopentyl

f

Cyclohexyl

ga

CH2OCH2CH2

" " " " " "

h

Cyclobutyl

Et

Method B

i

Cyclopentyl

Et

Method B

j

Cyclohexyl

Et

Method C

k

CH3

CH3

Et

Method C

l

CH3

CH3

iPr

Method C

m

CH3

CH3

tBu

Method C

51

Ref 22 Ref 22 Method A Method A Method A

a

Racemic

N

N

O2N O

A

R1

a, b

HS

H

NO2

51e-51g

OH

R1

R2

S S

R2

c

57 N

B

O2N

S

d

S

OH

R1

51h-51i

R2

52d-52f

C

e

HS R1

OH

51j-51m

R2

a

Reagents and conditions: (a) S2Cl2, MTBE; (b) LiAlH4, THF; (c) CH2Cl2/CH3OH, 25 °C; (d) EtSH, CH2Cl2; (e) I2, R3SH, CH3OH. 7 ACS Paragon Plus Environment

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In the final preparation method (Method 3), intermediate 5031 was transformed into additional disulfide intermediates 55 using the disulfide-containing alcohols 51j-51m. These entities were prepared by oxidative condensation of the corresponding mercapto-alcohols with various R3 thiols (method C, Scheme 3). As described above for Method 2, the silyl protecting groups were subsequently removed from the intermediates 55 to give the corresponding alcohols, and these entities were then oxidized to afford the final products. As shown in Scheme 2, final compounds 21 and 26-28 were prepared using Method 3, and the R1, R2, and R3 groups associated with intermediates 55 and 56 correspond to those depicted in Figure 1. Two additional disulfides (51h and 51i) employed in the synthesis of linkerdrugs 33 and 34 below were prepared by condensing the corresponding 2-pyridyl-4-nitrodisulfides 51d and 51e with ethane thiol (method B, Scheme 3). The two PBD-monomer compounds containing thiophenol-derived disulfides were synthesized by the method shown in Scheme 4. Coupling of aniline 5031 with alcohol 58a in a manner related to that described above in Scheme 2 for the preparation of intermediates 52 and 55 provided the corresponding carbamate 59a.

Subsequent removal of the silyl

protecting group present in 59a, oxidation, and disulfide exchange with either iPrSH or tBuSH respectfully afforded the target compounds 29 and 30 via intermediates 60a and 61 (Scheme 4). Use of benzyl alcohol in lieu of alcohol 58a in the derivatization of aniline 50 gave carbamate 59b. This intermediate was transformed into target compound 31 via the same deprotection/oxidation sequence used to convert 59a to 61. Disulfide 58a was prepared in one step by condensation of (4-mercaptophenyl)methanol with 1,2-bis(5-nitropyridin-2yl)disulfane (Scheme 4, method D). A related disulfide (58b) required for the synthesis of linker-drug 35 below was also generated from (4-mercaptophenyl)methanol and tBuSH.



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Scheme 4. Preparation of target compounds 29-31 (Method 4).a R4

R4

O O TBDMSO H HN

58a or BnOH

O

O O HO H HN

b

O

50 N

a

N

O O

O O

60a R4 = SS(2-Pyridyl-4-NO2)

59a R4 = SS(2-Pyridyl-4-NO2) 59b R4 = H

60b R4 = H c

c R4

31 O HO H

O N

N

O

iPrSH or tBuSH

29-30

d

O O

61 R4 = SS(2-Pyridyl-4-NO2)

N O2N

HS

N S S

NO2

d

R4

58a R4 = SS(2-Pyridyl-4-NO2)

D

58b R4 = SStBu

or OH

tBuSH, e

OH

a

Reagents and conditions: TBDMS = tBu(CH3)2Si. (a) triphosgene, Et3N, CH2Cl2; (b) HOAc/THF/H2O (3/2/1, v/v/v); (c) Dess-Martin periodinane, CH2Cl2; (d) CH2Cl2; (e) I2, EtOH.


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Scheme 5. Preparations of linker-drugs 32-36.a TBDMSO H2N H N

O

O

O

O

O

OTBDMS H

NH2 N

O

O

62

One of: 51a, 51h, 51i, 58b, or BnOH

N

N

O

OH

64

O

TBDMSO H HN

H N

N H

O

O

R O

O

H N

O

a

NH2 NH

O

O

O

O

NH2

OTBDMS H N

O

O O2N

N

63a R =

S

b

O

S

NH

N

63b R =

63c R =

S S

NH2

O

S O

O

H N

S

N H

O

H N O

R

63d R =

S

63e R =

S

S

c

O O R1O HN H

O

N

63f R =

S

O

O

O

O

O

O

e

32-36

OR1 NH H N O

65b-65f R1 = TBDMS

d

66b-66f R1 = H (R = same as 63b-f)

a

Reagents and conditions: TBDMS = tBu(CH3)2Si. (a) triphosgene, Et3N, CH2Cl2; (b) iPrSH, CH2Cl2; (c) triphosgene, Et3N, CH2Cl2, then compound 64, Et3N, DMF; (d) HOAc/THF/H2O(3/2/1, v/v/v); (e) IBX, DMSO, 40-50 °C.

The preparation of linker-drugs 32-36 is depicted in Scheme 5. In this method, one aniline group present in the PBD-dimer 6236,37 was converted to the disulfide-containing carbamate intermediates 63a and 63c-63e via coupling with alcohols 51a, 51h, 51i, or 58b. Disulfide exchange was subsequently performed with intermediate 63a and iPrSH to give 63b.

The free aniline group present in intermediates 63b-63e was then coupled with



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compound 6438-40 to give the bis-carbamate-containing compounds 65b-65e. Removal of both silyl protecting groups present in these entities followed by oxidation of the resulting primary alcohols (66b-66e) afforded the desired linker-drugs 32-35. Use of benzyl alcohol in lieu of alcohols 51a, 51h, 51i, or 58b in the derivatization of bis-aniline 62 afforded the benzyl-carbamate-containing intermediate 63f. This entity was converted to linker-drug 36 via a sequence analogous to that used to prepare linker-drugs 32-35 from compounds 65b65e.

The preparation of linker-drug 37 is described in reference 31. General Experimental Criteria.

All solutions were aqueous unless otherwise

indicated. The purity of all compounds submitted for biological assessment was determined to be ≥90% by HPLC. 1H NMR spectra were recorded on a Varian or Brucker spectrometer. Chemical shifts are reported in ppm relative to tetramethylsilane as an internal standard. Silica gel for column chromatography was typically (40−63 µm, 60 Å). All solvents used were reagent-grade or better purity. The R3SH thiols employed in the Scheme 2 and Scheme 3 syntheses were commercially available. LCMS condition #1: mobile phase: 1.5 mL / 4 L TFA in water (solvent A) and 0.75 mL / 4 L TFA in acetonitrile (solvent B), using the elution gradient 5%-95% (solvent B) over 0.8 minutes and holding at 95% for 0.4 minutes at a flow rate of 1.5 mL/min; column: MK RP18e 25-2 mm; wavelength: UV 220 nm and 254 nm; column temperature: 50 °C; MS ionization: ESI.

LCMS condition #2: experiments

performed on an Agilent 1290 UHPLC coupled with Agilent MSD (6140) mass spectrometer using ESI as ionization source. The LC separation used a Phenomenex XB-C18, 1.7 µm, 50 × 2.1 mm column with a 0.4 mL/minute flow rate. Mobile phase A was water and mobile phase B was acetonitrile (both with 0.1% formic acid). The gradient consisted of 2 - 98% solvent B over 7 min then holding at 98% solvent B for 1.5 min followed by equilibration for 1.5 min. LC column temperature was 40 °C. UV absorbance was collected at 220 nm and 254 nm and mass spec full scan was applied to all experiments. 11 ACS Paragon Plus Environment

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Compound 6. Method 1. Representative example. A mixture of compound 5031 (70 mg, 0.17 mmol) and Et3N (88 mg, 0.86 mmol) in anhydrous CH2Cl2 (2.0 mL) was added slowly to a solution of triphosgene (52 mg, 0.17 mmol) and 4 Å powdered molecular sieves (150 mg) in CH2Cl2 (5.0 mL) at 0 °C. The reaction mixture was stirred at 20 °C for 1 h. After the mixture was concentrated, it was dissolved in CH2Cl2 (5.0 mL) and a solution of compound 51a31 (44 mg, 0.19 mmol) and Et3N (52 mg, 0.51 mmol) in CH2Cl2 (3.0 mL) was added dropwise at 20 °C. The resulting mixture was stirred for 1 h at 20 °C, then the mixture was concentrated under reduced pressure. The residue was purified by prep-TLC (EtOAc:petroleum ether, 1:2, Rf = 0.5) to afford compound 52a (90 mg, 79%) as a yellow oil. LCMS #1: RT = 1.08 min, m/z = 665.1 [M+1]+. 1H NMR (400 MHz, CDCl3) δ 9.28 (d, J = 4.0 Hz, 1H), 9.15 (br s, 1H) 8.49 (dd, J = 8.4 Hz, 1H), 7.97 (d, J = 8.8 Hz, 1H), 7.82 (s, 1H), 6.85 (s, 1H), 5.00-4.92 (m, 2H), 4.70 (br s, 1H), 4.43-4.39 (m, 2H) 4.35-4.33 (m, 1H), 4.104.00 (m, 1H), 3.94 (s, 3H), 3.85 (s, 3H), 3.83-3.75 (m, 1H), 3.72-3.56 (m, 1H), 3.14 (t, J = 6.0 Hz, 2H), 2.80-2.65 (m, 2H), 0.89 (s, 9H), 0.05 (s, 6H). A solution of compound 52a (90 mg, 0.14 mmol) in THF (2.0 mL) was added to a mixture of HOAc (2.0 mL) and water (2.0 mL) at 20 °C. The mixture was then stirred for 16 h at 40 °C. The reaction mixture was diluted with EtOAc (10 mL) and was washed with water (5 mL x 2) and aq. NaHCO3 (5 mL x 2). The organic layer was dried over Na2SO4, filtered, and was concentrated. The residue was purified by flash chromatography on silica gel (0-10% CH3OH in CH2Cl2) to afford 53a (74 mg, 99%) as a yellow oil. LCMS #1: RT = 0.87 min, m/z = 573.0 [M+Na]+.

1

H NMR (400 MHz, CDCl3) δ 9.27 (d, J = 2.8 Hz, 1H),

8.75 (br, 1H) 8.47 (dd, J = 8.8, 2.8 Hz, 1H), 7.95 (d, J = 8.8 Hz, 1H), 7.76 (s, 1H), 6.85 (s, 1H) 5.05-4.97 (m, 2H), 4.70 (br, 1H), 4.42-4.36 (m, 2H), 4.22 (d, J = 14.4 Hz, 1H), 4.15-4.04 (m, 1H), 3.94 (s, 3H) 3.88 (s, 3H), 3.82-3.70 (m, 2H), 3.42-3.27 (m, 1H), 3.15 (t, J = 6.4 Hz, 2H), 3.02 (t, J = 5.2 Hz, 1H), 2.85-2.78 (m, 1H), 2.52-2.44 (m, 1H). 12 ACS Paragon Plus Environment


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Dess-Martin periodinane (69 mg, 0.16 mmol) and powdered 4Å molecular sieves (150 mg) were added sequentially in portions to compound 53a (60 mg, 0.11 mmol) in CH2Cl2 (5 mL) at 20 °C.

The resulting mixture was stired for 1 h at 20 °C then was

quenched with saturated NaHCO3 and Na2SO3 solution (1:1, 6 mL). The mixture was then extracted with EtOAc (5 mL x 3), and the combined organic layers were dried over Na2SO4, filtered, and concentrated. The residue was purified by prep-TLC (8% CH3OH in CH2Cl2, Rf = 0.6) to afford compound 54a (50 mg, 83%) as a yellow solid. LCMS #1: RT = 0.78 min, m/z = 549.2 [M+1]+. 1H NMR (400 MHz, CDCl3) δ 9.27 (s, 1H), 8.32 (dd, J = 8.8, 2.8 Hz, 1H), 7.65 (d, J = 8.8 Hz, 1H), 7.23 (s, 1H), 6.87 (s, 1H), 5.58 (d, J = 10.0 Hz, 1H), 5.20-5.14 (m, 2H), 4.44-4.39 (m, 1H), 4.30 (d, J = 16 Hz, 1H), 4.14-4.06 (m, 2H), 3.93 (s, 3H), 3.92 (s, 3H), 3.76-3.63 (m, 2H), 3.03-2.94 (m, 3H), 2.75-2.71 (m, 1H). Ethanethiol (57 mg, 0.91 mmol) was added dropwise to a solution of 54a (50 mg, 0.09 mmol) in CH2Cl2 (10.0 mL) at 30 °C. The reaction mixture was stirred at 30 °C for 16 h, then MnO2 (100 mg) was added and the mixture stirred for another 20 min. MnO2 was removed by filtration, and the filtrate was concentrated. The residue was purified by prepTLC (8% CH3OH in CH2Cl2, Rf = 0.5) to afford compound 6 (35 mg, 84%) as a white solid. LCMS #1: RT = 0.68 min, m/z = 477.0 [M+Na]+.

1

H NMR (400 MHz, CDCl3) δ 7.22 (s,

1H), 6.74 (s, 1H), 5.59-5.57 (m, 1H), 5.18-5.11 (m, 2H), 4.57-4.45 (m, 1H), 4.30 (d, J = 16.0 Hz, 1H), 4.15 (m, J = 16.0 Hz, 2H), 3.94 (s, 6H), 3.64 (t, J = 10.1 Hz, 1H), 3.55 (d, J = 4.2 Hz, 1H), 2.97-2.83 (m, 2H), 2.81-2.61 (m, 4H), 1.28 (t, J = 7.6 Hz, 3H). Compound 7. Method 1. LCMS #2: RT = 1.72 min, m/z = 471 [M+1]+. 1H NMR (400 MHz, DMSO-d6) δ 7.06 (s, 1H), 6.79 (d, J = 4.9 Hz, 1H), 6.62 (s, 1H), 5.37 (dd, J = 9.7, 5.8 Hz, 1H), 5.13 (d, J = 7.0 Hz, 2H), 4.88-4.80 (m, 1H), 4.45-4.34 (m, 1H), 4.14-4.05 (m, 1H), 4.02-3.93 (m, 1H), 3.80 (s, 6H), 3.79-3.78 (m, 1H), 3.60 (dq, J = 14.2, 6.5 Hz, 2H), 3.49-3.40 (m, 1H), 2.94-2.69 (m, 4H), 2.54 (s, 1H). 13 ACS Paragon Plus Environment

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Compound 8. Method 1. LCMS #2: RT = 3.05 min, m/z = 485 [M+1]+. 1H NMR (400 MHz, DMSO-d6) δ 7.06 (s, 1H), 6.79 (s, 1H), 6.68-6.56 (m, 1H), 5.37 (dd, J = 9.7, 5.9 Hz, 1H), 5.13 (d, J = 7.2 Hz, 2H), 4.88 (t, J = 5.7 Hz, 1H), 4.37 (dd, J = 11.7, 6.0 Hz, 1H), 4.17-3.93 (m, 3H), 3.80 (s, 6H), 3.51-3.41 (m, 2H), 2.95-2.80 (m, 4H), 1.19 (d, J = 6.9 Hz, 3H). Compound 9. Method 1. LCMS #2: RT = 2.42 min, m/z = 469 [M+1]+. 1H NMR (400 MHz, DMSO-d6) δ 7.06 (s, 1H), 6.80 (s, 1H), 6.62 (s, 1H), 5.37 (dd, J = 9.6, 5.4 Hz, 1H), 5.13 (d, J = 7.0 Hz, 2H), 4.37 (dt, J = 12.2, 6.3 Hz, 1H), 4.14-3.93 (m, 4H), 3.80 (s, 6H), 3.45 (t, J = 9.3 Hz, 1H), 3.01-2.83 (m, 3H), 1.23-1.16 (m, 6H). Compound 10. Method 1. LCMS #2: RT = 2.56 min, m/z = 483 [M+1]+. 1H NMR (400 MHz, DMSO-d6) δ 7.06 (s, 1H), 6.79 (s, 1H), 6.62 (s, 1H), 5.37 (dd, J = 9.7, 5.9 Hz, 1H), 5.16-5.09 (m, 2H), 4.35 (dt, J = 12.2, 6.3 Hz, 1H), 4.10 (d, J = 16.0 Hz, 1H), 3.98 (d, J = 16.0 Hz, 2H), 3.80 (s, 6H), 3.44 (t, J = 9.3 Hz, 1H), 2.88 (t, J = 12.6 Hz, 3H), 2.54 (s, 1H), 1.25 (s, 9H), 0.08 (s, 1H). Compound 11. Method 1. LCMS #2: RT = 2.40 min, m/z = 469 [M+1]+. 1H NMR (400 MHz, DMSO-d6) δ 7.07 (s, 1H), 6.82 (s, 1H), 6.63 (s, 1H), 5.38 (dd, J = 9.7, 5.9 Hz, 1H), 5.13 (d, J = 6.7 Hz, 2H), 4.21 (dd, J = 11.1, 6.2 Hz, 1H), 4.10 (d, J = 16.0 Hz, 1H), 3.97 (d, J = 16.1 Hz, 2H), 3.81 (d, J = 2.3 Hz, 6H), 3.45 (t, J = 9.3 Hz, 1H), 3.05 (s, 1H), 2.94-2.83 (m, 1H), 2.70-2.53 (m, 3H), 2.45 (p, J = 1.9 Hz, 1H), 1.16 (t, J = 7.2 Hz, 3H), 1.08-1.02 (m, 2H). Compound 12. Method 1. LCMS #2: RT = 2.16 min, m/z = 499 [M+1]+. 1H NMR (400 MHz, DMSO-d6) δ 7.07 (s, 1H), 6.82 (s, 1H), 6.63 (br s, 1H), 5.38 (d, J = 9.7 Hz, 1H), 5.13 (d, J = 6.8 Hz, 2H), 4.22 (dd, J = 11.1, 6.2 Hz, 1H), 4.10 (d, J = 15.9 Hz, 1H), 3.97 (d, J



Page 16 of 56

= 15.4 Hz, 2H), 3.81 (s, 6H), 3.50-3.44 (m, 2H), 3.22 (s, 3H), 2.94-2.75 (m, 3H), 1.05 (d, J = 6.9 Hz, 3H). Compound 13. Method 1. LCMS #2: RT = 2.08 min, m/z = 497 [M+1]+. 1H NMR (400 MHz, DMSO-d6) δ 7.08 (s, 1H), 6.82 (s, 1H), 6.65 (s, 1H), 5.38 (d, J = 9.7 Hz, 1H), 5.13 (d, J = 6.6 Hz, 2H), 4.77 (s, 3H), 4.45-4.34 (m, 1H), 4.26-4.06 (m, 3H), 4.02-3.93 (m, 1H), 3.81 (s, 6H), 3.45 (t, J = 9.3 Hz, 1H), 3.09 (s, 1H), 2.94-2.83 (m, 1H), 2.62-2.53 (m, 1H), 1.04 (d, J = 6.9 Hz, 2H), -0.03 to -0.13 (m, 1H). Compound 14. Method 1. LCMS #2: RT = 2.24 min, m/z = 525 [M+1]+. 1H NMR (400 MHz, DMSO-d6) δ 7.07 (s, 1H), 6.82 (s, 1H), 6.61 (s, 1H), 5.37 (d, J = 9.6 Hz, 1H), 5.13 (d, J = 7.0 Hz, 2H), 4.23 (dd, J = 11.1, 6.0 Hz, 1H), 4.10 (d, J = 15.7 Hz, 1H), 4.02-3.83 (m, 4H), 3.81 (s, 6H), 3.45 (t, J = 9.3 Hz, 1H), 3.02 (dt, J = 11.1, 4.1 Hz, 1H), 2.94-2.83 (m, 2H), 2.59-2.52 (m, 2H), 1.95-1.76 (m, 2H), 1.55-1.37 (m, 2H), 1.27-1.21 (m, 1H), 1.05 (d, J = 6.9 Hz, 3H). Compound 15. Method 2. Representative example. A mixture of triphosgene (53.6 mg, 0.18 mmol) and powdered 4Å molecular sieves (150 mg) in CH2Cl2 (5 mL) was added to a solution of compound 5031 (74 mg, 0.18 mmol) and Et3N (92 mg, 0.90 mmol) in CH2Cl2 (5.0 mL). After the reaction mixture was stirred for 1 h at 25 °C, it was concentrated and the residue was dissolved in CH2Cl2 (5.0 mL). A solution of compound 51b32 (53 mg, 0.21 mmol) and Et3N (54 mg, 0.54 mmol) in CH2Cl2 (5 mL) was added dropwise at 25 °C, and the reaction mixture was stirred for 1 h at 25 °C. The mixture was then concentrated, and the residue was purified by prep-HPLC (Xtimate C18 150 x 25 mm x 5 µm, acetonitrile 80100/0.23% formic acid in water) to afford 52b (79 mg, 65%) as a white solid. LCMS #1 RT = 1.17 min, m/z = 679.1 [M+1]+. 1H NMR (400 MHz, CDCl3) δ 9.24 (d, J = 2.8 Hz, 1H), 8.53 (d, J = 8.8, 2.0 Hz, 1H), 8.03 (d, J = 9.2 Hz, 1H), 7.82 (s, 1H), 6.85 (s, 1H), 4.99-4.92


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(m, 2H), 4.70 (brs, 1H), 4.28-4.24 (m, 2H), 4.13-4.08 (m, 2H), 3.93 (s, 4H), 3.85 (s, 3H), 3.74-3.67 (m, 1H), 3.36-3.28 (m, 1H), 2.73 (br, 2H), 1.41 (d, J = 6.8 Hz, 3H), 0.89 (s, 3H), 0.05 (s, 6H). To a mixture of 52b (78.0 mg, 0.1100 mmol) in CH2Cl2 (5.0 mL) was added 2propanethiol (88 mg, 1.15 mmol). The mixture was stirred under N2 at 25 °C for 16 h, then MnO2 (100 mg) was added stirring was continued for another 20 min. MnO2 was removed by filtration, and the filtrate was concentrated. The residue was purified by prep-TLC (5% CH3OH in CH2Cl2, Rf = 0.6) to afford 55b (51 mg, 74%) as a white solid. LCMS #1: RT = 1.21 min, m/z = 621.1 [M+Na]+. 1H NMR (400 MHz, CDCl3) δ 8.92 (br, 1H), 7.85 (s, 1H), 6.82 (s, 1H), 5.00-4.92 (m, 2H), 4.66 (br, 1H), 4.35-4.31 (m, 1H), 4.22 (d, J = 16 Hz, 1H), 4.14-4.09 (m, 2H), 3.94 (s, 3H), 3.85 (s, 4H), 3.66-3.63 (m, 1H), 3.13-2.95 (m, 2H), 2.71 (brs, 2H), 1.35-1.30 (m, 9H), 0.89 (s, 9H), 0.04 (s, 6H). To a mixture of 55b (50 mg, 0.08 mmol) in water (1.0 mL) and THF (1.0 mL) was added HOAc (1.5 mL) at 25 °C. After the reaction mixture was stirred at 40 °C for 15 h, it was diluted with EtOAc (10 mL) and washed sequentially with water (5 mL × 2) and aq. NaHCO3 (5 mL × 2). concentrated.

The organic layer was dried over Na2SO4, filtered, and was

The residue was purified by flash chromatography on silica gel (0-10%

CH3OH in CH2Cl2) to afford 56b (32 mg, 77%) as a white solid. LCMS #1: RT = 0.91 min, m/z = 485.0 [M+1]+. 1H NMR (400 MHz, CDCl3) δ 8.54 (br, 1H), 7.75 (s, 1H), 6.83 (s, 1H), 5.03 (brs, 1H), 4.95 (br, 1H), 4.67 (br, 1H), 4.33-4.29 (m, 1H), 4.17-4.13 (m 3H), 3.93 (s, 3H), 3.87 (s, 3H), 3.75-3.63 (m, 1H), 3.44-3.35 (m, 1H), 3.12-2.97 (m, 2H), 2.78-2.76 (m, 1H), 2.49-2.44 (m, 1H), 1.35-1.30 (m, 9H). To a solution of 56b (15 mg, 0.03 mmol) in CH2Cl2 (4.0 mL) was added Dess-Martin periodinane (18 mg, 0.04 mmol) and powdered 4Å molecular sieves (100 mg). The mixture



Page 18 of 56

was stirred at 25 °C for 1 h then was quenched with saturated NaHCO3 and Na2SO3 solution (1:1, 3.0 mL). The mixture was subsequently extracted with EtOAc (5 mL x 3), and the combined organic layers were dried over Na2SO4, filtered, and concentrated. The residue was purified by prep-TLC (50% EtOAc in petroleum ether, Rf = 0.3) to afford compound 15 (8.0 mg, 54%) as a white solid. LCMS #1: RT = 0.88 min, m/z = 483.2 [M+1]+. 1H NMR (400 MHz, CDCl3) δ 7.22 (s, 1H), 6.77 (s, 1H), 5.59 (d, J = 8.8 Hz, 1H), 5.15 (br, 2H), 4.354.27 (m, 2H), 4.16 (d, J = 16.4 Hz, 1H), 4.08-4.04 (m, 1H), 3.94 (s, 6H), 3.64 (t, J = 8.0 Hz, 1H), 3.51 (brs, 1H), 2.99-2.90 (m, 3H), 2.74-2.70 (m, 1H), 1.27-1.20 (m, 9H). Compound 16. Method 2. LCMS #2: RT = 2.70 min, m/z = 497 [M+1]+. 1H NMR (400 MHz, DMSO-d6) δ 7.07 (s, 1H), 6.81 (s, 1H), 6.65 (d, J = 5.9 Hz, 1H), 5.38 (dd, J = 9.7, 5.9 Hz, 1H), 5.19-5.04 (m, 2H), 4.20 (dd, J = 11.2, 5.9 Hz, 1H), 4.10 (d, J = 15.8 Hz, 1H), 4.04-3.91 (m, 2H), 3.80 (s, 6H), 3.45 (t, J = 9.3 Hz, 1H), 3.04-2.82 (m, 2H), 2.58-2.53 (m, 1H), 1.23 (s, 9H), 1.03 (d, J = 6.9 Hz, 3H). Compound 17. Method 1. LCMS #2: RT = 1.57 min, m/z = 528 [M+1]+. 1H NMR (400 MHz, DMSO-d6) δ 7.06 (s, 1H), 6.98 (s, 1H), 5.44 (d, J = 9.6 Hz, 1H), 5.13 (d, J = 4.6 Hz, 2H), 4.46 (s, 1H), 4.10 (d, J = 15.9 Hz, 2H), 3.97 (d, J = 15.7 Hz, 1H), 3.83 (s, 3H), 3.80 (s, 6H), 2.89 (s, 4H), 0.99 (d, J = 7.0 Hz, 3H). Compound 18. Method 2. LCMS #1: RT = 0.83 min, m/z = 481 [M+1]+. 1H NMR (400 MHz, CDCl3) δ 7.21 (s, 1H), 6.80 (s, 1H), 5.57 (d, J = 9.6 Hz, 1H), 5.14 (s, 2H), 4.68 (d, J = 11.2 Hz, 1H), 4.32-4.28 (m, 1H), 4.17-4.13 (m, 1H), 3.93 (s, 6H), 3.73-3.62 (m, 2H), 2.95-2.89 (m, 1H), 2.73-2.67 (m, 2H), 1.25-1.21 (m, 4H), 1.08-0.81 (m, 5H). Compound 19. Method 1. LCMS #2: RT = 2.61 min, m/z = 495 [M+1]+. 1H NMR (400 MHz, DMSO-d6) δ 7.08 (s, 1H), 6.82 (s, 1H), 6.64 (d, J = 6.0 Hz, 1H), 5.45-5.36 (m, 1H), 5.13 (d, J = 7.2 Hz, 2H), 4.30 (d, J = 11.5 Hz, 1H), 4.10 (d, J = 15.8 Hz, 1H), 3.97 (d, J 17 ACS Paragon Plus Environment

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= 13.7 Hz, 2H), 3.80 (s, 6H), 3.46 (td, J = 9.5, 1.8 Hz, 1H), 2.89 (dd, J = 15.8, 9.2 Hz, 1H), 2.62-2.52 (m, 2H), 1.91 (s, 6H), 1.63 (s, 1H), 1.13 (t, J = 7.5 Hz, 3H). Compound 20. Method 1. LCMS #2: RT = 2.48 min, m/z = 509 [M+1]+. 1H NMR (400 MHz, DMSO-d6) δ 7.08 (s, 1H), 6.82 (s, 1H), 6.73-6.60 (m, 1H), 5.40 (d, J = 7.7 Hz, 1H), 5.13 (d, J = 7.2 Hz, 2H), 4.22 (d, J = 11.0 Hz, 1H), 4.10 (d, J = 15.9 Hz, 1H), 4.01-3.84 (m, 2H), 3.81 (s, 6H), 3.46 (t, J = 9.2 Hz, 1H), 2.88 (dd, J = 15.9, 9.3 Hz, 1H), 2.55 (dd, J = 4.1, 2.3 Hz, 2H), 1.81-1.39 (m, 10H), 1.12 (t, J = 7.3 Hz, 3H). Compound 21. Method 3. LCMS #1: RT = 0.89 min, m/z = 523 [M+1]+. 1H NMR (400 MHz, CDCl3) δ 7.22 (s, 1H), 6.81 (s, 1H), 5.59 (d, J = 10.0 Hz, 1H), 5.15 (brs, 2H), 4.44 (d, J = 10.8 Hz, 1H), 4.33-4.28 (m, 1H), 4.17-4.14 (m, 1H), 3.94 (s, 6H), 3.75 (d, J = 10.4 Hz, 1H), 3.64 (t, J = 8.8 Hz, 1H), 3.50 (s, 1H), 2.97-2.88 (m, 1H), 7.74-2.72 (m, 1H), 2.72-2.59 (m, 1H), 1.61-0.85 (m, 13H). Compound 22. Method 1. LCMS #1: RT = 0.66 min, m/z = 525 [M+1]+. 1H NMR (400 MHz, CDCl3) δ 7.23 (s, 1H), 6.85 (s, 1H), 5.65 (d, J = 9.6 Hz, 1H), 5.15 (s, 2H), 4.55 (d, J = 11.2 Hz, 1H), 4.31-4.27 (m, 1H), 4.18-4.15 (m, 1H), 3.95 (s, 7H), 3.77-3.64 (m, 5H), 2.92-2.86 (m, 2H), 2.74-2.70 (m, 1H), 2.58-2.51 (m, 1H), 1.81-1.74 (m, 4H), 1.60-1.48 (m, 4H). Compound 23. Method 1. LCMS #2: RT = 1.95 min, m/z = 511 [M+1]+. 1H NMR (400 MHz, DMSO-d6) δ 7.08 (s, 1H), 6.83 (s, 1H), 6.64 (s, 1H), 5.39 (d, J = 9.7 Hz, 1H), 5.13 (d, J = 7.1 Hz, 2H), 4.34 (d, J = 11.5 Hz, 1H), 4.10 (d, J = 16.0 Hz, 1H), 3.98 (t, J = 15.3 Hz, 2H), 3.81 (d, J = 2.4 Hz, 6H), 3.78-3.72 (m, 1H), 3.63 (d, J = 6.1 Hz, 1H), 3.54 (s, 1H), 3.46 (t, J = 9.2 Hz, 1H), 2.89 (dd, J = 15.7, 9.4 Hz, 1H), 1.96-1.72 (m, 3H), 1.20-1.06 (m, 3H).



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Compound 24. Method 1. LCMS #2: RT = 1.99 min, m/z = 511 [M+1]+. 1H NMR (400 MHz, DMSO-d6) δ 7.08 (s, 1H), 6.83 (s, 1H), 6.64 (s, 1H), 5.39 (d, J = 9.7 Hz, 1H), 5.13 (d, J = 7.2 Hz, 2H), 4.34 (d, J = 11.3 Hz, 1H), 4.10 (d, J = 15.9 Hz, 1H), 3.98 (t, J = 15.5 Hz, 2H), 3.81 (d, J = 1.9 Hz, 7H), 3.63 (d, J = 6.1 Hz, 1H), 3.54 (s, 1H), 3.46 (t, J = 9.1 Hz, 1H), 2.89 (dd, J = 15.9, 9.4 Hz, 1H), 1.96-1.72 (m, 3H), 1.20 -1.06 (m, 3H). Compound 25. Method 1. LCMS #1: RT = 0.58 min, m/z = 527 [M+1]+. 1H NMR (400 MHz, CDCl3) δ 7.23 (s, 1H), 6.79 (s, 1H), 5.64 (d, J = 10.0 Hz, 1H), 5.16 (s, 2H), 4.624.60 (m, 1H), 4.31-4.28 (m, 1H), 4.18-4.10 (m, 1H), 3.95-3.85 (m, 11H), 3.71-3.62 (m, 5H), 2.92-2.86 (m, 2H), 2.74-2.67 (m, 1H), 2.61-2.52 (m, 1H), 2.03-1.84 (m, 2H). Compound 26: Method 3. Representative example. A mixture of compound 5031 (50 mg, 0.12 mmol) and Et3N (37 mg, 0.37 mmol) in CH2Cl2 (3.0 mL) was added to a solution of triphosgene (40 mg, 0.13 mmol) in CH2Cl2 (5.0 mL) at 25 °C. The reaction mixture was stirred for 1 h at 25 °C then was concentrated. The residue was dissolved in CH2Cl2 (5.0 mL) and a solution of compound 51k (41 mg, 0.25 mmol) in pyridine (29 mg, 0.25 mmol) and CH2Cl2 (5.0 mL) was added dropwise. The resulting mixture was stirred for 1 h at 25 °C then was concentrated under reduced pressure. The residue was purified by flash column chromatography (0-50% EtOAc in petroleum ether, Rf = 0.5) to afford compound 55k (45 mg, 57%) as a colorless oil. LCMS #1: RT = 1.21 min, m/z = 621.2 [M+Na]+. 1H NMR (400 MHz, CDCl3) δ 8.87 (br, 1H), 7.84 (s, 1H), 6.81 (s, 1H), 4.99 (s, 1H), 4.92 (br, 1H), 4.64-4.57 (m, 1H), 4.22-4.07 (m, 4H), 3.93 (s, 3H), 3.84 (s, 3H), 3.75-3.55 (m, 1H), 2.73-2.67 (m, 4H), 1.34 (s, 6H), 1.28 (t, J = 7.2 Hz, 3H), 0.87 (s, 9H), 0.03 (s, 6H). A solution of 55k (45 mg, 0.08 mmol), water (4.0 mL), and acetic acid (6.0 mL)was stirred at 10 °Cfor 16 h. The mixture was concentrated to a residue, and the pH was adjusted to about 8 with saturated NaHCO3 solution (10 mL). The resulting mixture was extracted


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with EtOAc (20 mL x 3), and the combined organic layers were washed sequentially with water (20 mL) and saturated brine (20 mL x 2), then dried over Na2SO4, filtered and concentrated. The residue was purified by column chromatography (0-10 % CH3OH in CH2Cl2, Rf = 0.6) to afford 56k (20 mg, 55%) as a colorless oil. LCMS #1: RT = 0.93 min, m/z = 507.1 [M+Na]+. 1H NMR (400 MHz, CD3OD) δ 7.20 (s, 1H), 7.00 (s, 1H), 5.01-4.93 (m, 2H), 4.56 (s, 1H), 4.50 (br, 1H), 4.12-4.01 (m, 4H), 3.85 (s, 6H), 3.69-3.61 (m, 2H), 2.772.67 (m, 4H), 1.32 (s, 6H), 1.28-1.25 (t, J = 7.6 Hz, 3H). To a solution of 56k (20 mg, 0.04 mmol)in anhydrous CH2Cl2 (2.0 mL) was added Dess-Martin periodinane (26 mg, 0.06 mmol). The reaction mixture was stirred at 25 °C for 1 h whereupon TLC (8% CH3OH in CH2Cl2, Rf = 0.6) showed that the desired product was generated. The mixture was diluted with H2O (5.0 mL) followed by aq. Na2SO3 solution (5.0 mL) and aq. NaHCO3 solution (5.0 mL). The resulting mixture was extracted with EtOAc (20 mL × 3), and the combined organic layers were dried over Na2SO4, filtered, and were concentrated. The residue was then purified by prep-TLC (8% CH3OH in CH2Cl2) to give compound 26 (19 mg, 50%) as a white solid. LCMS #1: RT = 0.87 min, m/z = 505.1 [M+Na]+. 1H NMR (400 MHz, CDCl3) δ 7.22 (s, 1H), 6.77 (s, 1H), 5.58 (d, J = 9.6 Hz, 1H), 5.14 (s, 2H), 4.38-4.28 (m, 2H), 4.14 (d, J = 16.4 Hz, 1H), 3.93 (s, 6H), 3.73 (d, J = 10.4 Hz, 1H), 3.66-3.61 (m, 1H), 3.47 (brs, 1H), 2.95-2.89 (m, 1H), 2.73-2.62 (m, 3H), 1.25 (t, J = 7.6 Hz, 3H), 1.15 (s, 3H), 1.08 (s, 3H). Compound 27. Method 3. LCMS #2: RT = 2.69 min, m/z = 497 [M+1]+. 1H NMR (400 MHz, DMSO-d6) δ 7.08 (s, 1H), 6.82 (s, 1H), 6.64 (d, J = 6.0 Hz, 1H), 5.49-5.33 (m, 1H), 5.13 (d, J = 7.3 Hz, 2H), 4.21-4.05 (m, 2H), 4.04-3.91 (m, 1H), 3.81 (s, 4H), 3.79-3.72 (m, 1H), 3.46 (t, J = 9.3 Hz, 1H), 3.31 (s, 3H), 2.99-2.76 (m, 2H), 2.63-2.51 (m, 1H), 1.380.94 (m, 12H).



Page 22 of 56

Compound 28. Method 3. LCMS #2: RT = 2.78 min, m/z = 511 [M+1]+. 1H NMR (400 MHz, DMSO-d6) δ 7.08 (s, 1H), 6.81 (s, 1H), 6.63 (d, J = 5.9 Hz, 1H), 5.39 (dd, J = 9.7, 5.9 Hz, 1H), 5.13 (d, J = 5.7 Hz, 2H), 4.07 (dd, J = 19.9, 13.3 Hz, 2H), 3.96 (d, J = 15.9 Hz, 1H), 3.81 (d, J = 2.2 Hz, 5H), 3.77-3.70 (m, 1H), 3.50-3.43 (m, 1H), 2.89 (dd, J = 15.4, 9.5 Hz, 1H), 1.17 (s, 10H), 1.11-0.98 (m, 5H). Compound 29. Method 4. Representative example. A mixture of compound 5031 (300 mg, 0.74 mmol) and Et3N (75 mg, 0.74 mmol) in CH2Cl2 (10 mL) was added to a solution of triphosgene (88 mg, 0.30 mmol) in CH2Cl2 (5 mL) at 0 °C over 15 min. The reaction mixture was stirred for 1 h at 0 °C, and a solution of compound 58a (217 mg, 0.74 mmol) and Et3N (224 mg, 2.21 mmol) in CH2Cl2 (10 mL) was then added dropwise at 0 °C. The resulting mixture was stirred for 1 h at 0 °C, then was concentrated under reduced pressure. The residue was purified by prep-TLC (EtOAc:petroleum ether, 1:2, Rf = 0.5) to afford compound 59a (286 mg, 50%) as a yellow oil. LCMS #1: RT = 1.01 min, m/z = 727.2 [M+1]+. A solution of compound 59a (286 mg, 0.39 mmol) in THF (3 mL) was added to a mixture of HOAc (5.0 mL) and water (2.0 mL) at 20 °C. The mixture was stirred for 16 h at that temperature, then was heated to 40 °C for an additional 16 h.

The mixture was

concentrated to a residue, and the pH was adjusted to about 8 with saturated NaHCO3 solution (10 mL). The mixture was then extracted with EtOAc (10 mL x 3) and the combined extracts were washed with saturated brine (5 mL x 2), dried over Na2SO4, filtered and concentrated. The residue was purified by prep-TLC (EtOAc:petroleum ether, 2:1, Rf = 0.4) to afford 60a (206 mg, 80%) as yellow oil. LCMS #1: RT = 0.80 min, m/z = 613.1 [M+1]+. Dess-Martin periodinane (55 mg, 0.13 mmol) was added in portions to compound 60a (40 mg, 0.070 mmol) in CH2Cl2 (5 mL) at 0 °C. After warning to 20 °C and stiring for 2 h,


Page 23 of 56 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the mixture was quenched with saturated NaHCO3 and Na2SO3 solution (1:1, 6 mL). The mixture was then extracted with EtOAc (5 mL x 3), and the combined organic layers were dried over Na2SO4, filtered, and concentrated. The residue was purified by prep-TLC (5% CH3OH in CH2Cl2, Rf = 0.6) to afford compound 61 (19 mg, 45%) as a yellow solid. LCMS #1: RT =0.89 min, m/z = 611.4 [M+1]+.

1

H NMR (400 MHz,CDCl3) δ 9.30 (s, 1H), 8.40

(dd, J = 2.4 Hz, 8.8 Hz, 1H), 7.83 (d, J = 8.8 Hz, 1H), 7.45 (d, J = 7.6 Hz, 2H), 7.17 (s, 1H), 7.15 (d, J = 7.6 Hz, 2H), 6.54 (s, 1H), 5.56 (d, J = 10 Hz, 1H), 5.23 (d, J = 12.8 Hz, 1H), 5.15 (s, 2H), 4.95-4.92 (m, 1H), 4.31-4.28 (m, 1H), 4.16-4.12 (m, 1H), 3.93 (s, 3H), 3.73 (s, 3H), 3.63 (t, J = 9.2 Hz, 1H), 2.96-2.89 (m, 1H), 2.73-2.69 (m, 1H). iPrSH (700 mg, 9.2 mmol) was added dropwise to a solution of compound 61 (40 mg, 0.070 mmol) in CH2Cl2 (5.0 mL) at 23 °C. The reaction mixture was stirred at that temperature for 16 h, then MnO2(100 mg) was added and stirring was continued for another 20 min. MnO2 was removed by filtration, and the filtrate was concentrated. The residue was purified by prep-TLC twice (5% CH3OH in CH2Cl2, Rf = 0.6) and lyophilized to afford 29 (22 mg, 63%) as a white solid. LCMS #1: RT = 0.92 min, m/z = 531.1 [M+1]+. 1H NMR (400 MHz, CDCl3) δ 7.47 (d, J = 8.0 Hz, 2H), 7.23 (s, 1H) , 7.17 (d, J = 7.6 Hz, 2H), 6.51 (s, 1H), 5.57 (d, J = 9.6 Hz, 1H), 5.32 (d, J = 12.8 Hz, 1H), 5.15-5.13 (m, 2H), 4.84 (d, J = 12.0 Hz, 1H), 4.33-4.29 (m, 1H), 4.18-4.14 (m, 1H), 3.94 (s, 3H), 3.70 (s, 3H), 3.67-3.62 (m, 1H), 3.08-3.04 (m, 1H), 2.98-2.88 (m, 1H), 2.72-2.68 (m, 1H), 1.29 (d, J = 6.4 Hz, 6H). Compound 30. Method 4. LCMS #1: RT = 0.94 min, m/z = 545.1 [M+1]+. 1H NMR (400 MHz, CDCl3) δ 7.50 (d, J = 7.6 Hz, 2H), 7.22 (s, 1H) , 7.15 (d, J = 7.2 Hz, 2H), 6.51 (s, 1H), 5.59-5.57 (m, 1H), 5.38 (d, J = 12.0 Hz, 1H), 5.151 (brs, 2H), 4.83 (d, J = 12.4 Hz, 1H), 4.33-4.29 (m, 1H), 4.18-4.14 (m, 1H), 3.94 (s, 3H), 3.69 (s, 3H), 3.67-3.62 (m, 1H), 3.51 (brs, 1H), 2.96-2.90 (m, 1H), 2.74-2.70 (m, 1H), 1.31 (s, 9H).



Compound 31. Method 4. LCMS #2: RT = 4.51 min, m/z = 425.2 [M+1]+.

Page 24 of 56

1

H

NMR (400 MHz, DMSO-d6) δ 7.46-7.15 (m, 5H), 7.06 (s, 1H), 6.76 (s, 1H), 6.64 (d, J = 6.0 Hz, 1H), 5.40 (dd, J = 9.7, 6.0 Hz, 1H), 5.22 (d, J = 12.6 Hz, 1H), 5.13 (d, J = 8.3 Hz, 2H), 4.89 (d, J = 12.6 Hz, 1H), 4.16-4.06 (m, 1H), 3.98 (d, J = 15.6 Hz, 1H), 3.79 (s, 4H), 3.70 (s, 3H), 3.51-3.42 (m, 1H), 2.89 (dd, J = 15.7, 9.4 Hz, 1H). Compound 51e.

Method A.

Representative example. To a solution of

cyclopentanecarbaldehyde (9.0 g, 92 mmol) in MTBE (30 mL) at 25 °C was added S2Cl2 (7.4 g, 55 mmol). The reaction mixture was then heated to 55 °C and was stirred at that temperature for 16 h under nitrogen. After cooling to ambient temperature, the solvent was removed under vacuum and the residue was purified via silica gel column chromatography (EtOAc:petroleum ether, 1:80) to give 1,1'-disulfanediyldicyclopentanecarbaldehyde (5.5 g, 46%) as brown oil. 1H NMR (300 MHz, CDCl3) δ 9.23 (s, 2H), 2.41-1.51 (m, 16H). To a solution of 1,1'-disulfanediyldicyclopentanecarbaldehyde (8.5 g, 32.9 mmol) in THF (60 mL) at 25 °C was added LiAlH4 (2.5 g, 65.8 mmol) in portions. After completion of the addition, the reaction mixture was stirred at ambient temperature for 2 h. The mixture was subsequently acidified to pH = 6 by careful addition of 3 M HCl and was then extracted with EtOAc (2 x 150 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated.

The residue was purified by silica gel column chromatography

(EtOAc:petroleum ether, 1:20) to give (1-mercaptocyclopentyl)methanol (5.5 g, 63%) as yellow oil. 1H NMR (300 MHz, CDCl3) δ 3.51 (s, 2H), 2.04 (s, 1H), 1.85-1.67 (m, 7H), 1.64 (s, 1H). A mixture of (1-mercaptocyclopentyl)methanol (3.5 g, 26.5 mmol) and 1,2-bis(5nitropyridin-2-yl)disulfane (12.3 g, 39.8 mmol) in CH3OH (50 mL) was stirred at ambient temperature for 16 h under nitrogen. After the reaction was completed, the mixture was


Page 25 of 56 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


concentrated under vacuum, and the residue was purified by silica gel column chromatography (EtOAc:petroleum ether, 1:10) to give compound 51e (1.9 g, 25%) as a yellow solid. 1H NMR (400 MHz, DMSO-d6): δ 9.24-9.20 (m, 1H), 8.58 (dd, J = 8.9, 2.7 Hz, 1H), 8.17 (dd, J = 8.9, 0.5 Hz, 1H), 5.18 (t, J = 5.5 Hz, 1H), 3.40 (d, J = 5.5 Hz, 2H), 1.631.82 (m, 8H). Compound 51f. Method C. 1H NMR (400 MHz, CDCl3): δ 9.31 (s, 1H), 8.33 (dd, J = 8.8, 2.4 Hz, 1H), 7.58 (d, J = 8.8 Hz, 1H), 3.34 (d, J = 5.6 Hz, 2H), 1.86-1.84 (m, 2H), 1.67-1.46 (m, 7H), 1.36-1.26 (m, 1H). Compound 51g. To a solution of (tetrahydrofuran-3-yl)methanol (10 g, 98 mmol ) in CH2Cl2 (300 mL) was added Dess-Martin periodinane (46 g, 108 mmol) in portions. The reaction mixture was stirred at 25 °C for 2 h then was filtered. The filtrate was concentrated under vacuum, and the residue was purified by silica gel column chromatography (EtOAc:petroleum ether, 1:7) to give tetrahydrofuran-2-carbaldehyde (4.8 g, 49%) as a colorless oil.

1

H NMR (300 MHz, CDCl3) δ 9.65 (d, J = 2.4 Hz, 1H), 3.96-3.71 (m, 4H),

2.20 (dd, J = 12.3, 7.4 Hz, 2H). This material was converted to compound 51g using Method A. 1H NMR (400 MHz, DMSO-d6) δ 9.32 (d, J = 2.5 Hz, 1H), 8.37 (dd, J = 8.8, 2.6 Hz, 1H), 7.60 (d, J = 8.8 Hz, 1H), 4.12-3.84 (m, 4H), 3.56 (d, J = 1.5 Hz, 2H), 2.18-2.00 (m, 2H). Compound 51h. Method B. Representative example. Ethanethiol (2.74 g, 44.1 mmol) was added to a mixture of 51d (400 mg, 1.47 mmol) in CH2Cl2 (10 mL) at 25 °C. The reaction mixture was heated to 40 °C and was stirred at that temperature for 30 h. After cooling to 25 °C, the reaction mixture was slurried with MnO2 (0.20 g) for 5 min and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by prepTLC (100% CH2Cl2, Rf = 0.5) to give compound 51h (110 mg, 42%) as a colorless oil. 1H



Page 26 of 56

NMR (400 MHz, CDCl3) δ 3.74 (s, 2H), 2.75-2.70 (m, 2H), 2.13-1.87 (m, 6H), 1.84 (s, 1H), 1.30 (t, J = 7.6 Hz, 3H). Compound 51i. Method C. Representative example. I2 (2.33 g, 9.2 mmol) was added slowly to a solution of (1-mercaptocyclopentyl)methanol (500 mg, 3.78 mmol) and ethanethiol (2.73 mL, 37.8 mmol) in CH3OH (30 mL) at 0 °C. The mixture was stirred at 8 °C for 1 h. then was concentrated under reduced pressure. The residue was dissolved in CH2Cl2 (80 mL), and was washed with sat. Na2S2O3 solution (80 mL x 3). The organic layer was dried over Na2SO4, filtered, and concentrated and the residue was purified by prep-TLC (EtOAc:petroleum ether, 1:5, Rf = 0.4) to give the desired product (300 mg, 41%) as a colorless oil.

1

H NMR (400 MHz, CDCl3) δ 3.57 (d, J = 4.0 Hz, 2H), 2.77-2.72 (m, 2H),

1.79-1.68 (m, 8H), 1.31 (t, J = 7.2 Hz, 3H). Compound 51j. Method C. 1H NMR (400 MHz, CDCl3) δ 3.53 (d, J = 4.4 Hz, 2H), 2.73-2.67 (m, 2H), 1.76-1.63 (m, 2H), 1.62-1.55 (m, 8H), 1.31 (t, J = 6.8 Hz, 3H). Compound 51k. Method C. 1H NMR (400 MHz, CDCl3) δ 3.50 (s, 2H), 2.75-2.70 (m, 2H), 1.39-1.33 (m, 3H), 1.31 (s, 6H). Compound 51l. A solution of propane-2-thiol (2.5 g, 32.8 mmol) in CH2Cl2 (40 mL was added dropwise with stirring to a solution of 1-chloro-1H-1,2,3-benzotriazole (7.26 g, 47.3 mmol) and 1H-1,2,3-benzotriazole (3.74 g, 31.4 mmol) in CH2Cl2 (200 mL) at -78 oC. The resulting solution was stirred at -78 oC for 1 h then was warmed to -20 oC. A solution of 2-methyl-2-sulfanylpropan-1-ol (5.0 g, 47.1 mmol) in CH2Cl2 (20 mL) was subsequently added dropwise with stirring at -20 oC, and the resulting solution was warmed to 25 oC and stirred overnight. The reaction was quenched by the addition of saturated aqueous Na2S2O3 (200 mL) and the resulting mixture was sequentially with washed with 1 x 200 mL of saturated aqueous NaHCO3 (1 x 200 mL) and brine (1 x 200 mL). The organic phase was 25 ACS Paragon Plus Environment

Page 27 of 56 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


dried over Na2SO4, filtered, and was concentrated under vacuum. The residue was purified by flash-Prep-HPLC to afford compound 51l (3.1 g) as light yellow oil. 1H NMR (300 MHz, CDCl3) δ 3.49 (s, 2H), 3.01-2.92 (m, 1H), 1.33-1.29 (m, 12H). Compound 51m. Prepared in a manner analogous to that described for compound 51l but using 2-methylpropane-2-thiol in place of propane-2-thiol.

1

H NMR (300 MHz,

CDCl3) δ 3.43 (s, 2H), 1.34 (s, 9H), 1.29 (s, 6H). Compound 58a. 1,2-Bis(5-nitropyridin-2-yl)disulfane (2.2 g, 7.1 mmol) in CH2Cl2 (15 mL) was added to a solution of (4-mercaptophenyl)methanol (940 mg, 6.7 mmol) in CH2Cl2 (10 mL) dropwise under N2 over 15 min at 25 °C. The mixture was stirred for another 1 h at 25 °C, whereupon TLC analysis showed a new product (33% EtOAc in petroleum ether, Rf = 0.5). MnO2 (1.0 g, 11.5 mmol) was added, and the suspensions was stirred for another 10 min at 25 °C. The yellow reaction mixture became colorless during this time. Manganese dioxide was filtered off and filtrate was concentrated, and treated with EtOH (5.0 mL). The resulting precipitate was filtered to remove the disulfide. The filtrate was concentrated and the residue was purified by chromatography on silica (solvent gradient: EtOAc:petroleum ether, gradient elution, 1:2 to 1:1) to afford compound 58a (1.1 g, 54.1%) as a yellow solid. LCMS #1: RT = 0.73 min, m/z = 294.8 [M+1]+. Compound 58b. 2-Methylpropane-2-thiol (2.01 mL, 17.85 mmol) was added to a solution of (4-mercaptophenyl)methanol (250 mg, 1.78 mmol) in 95% EtOH (10 mL) at 25 °C. The mixture was cooled to 0 °C, and a solution of iodine (200 mg, 0.79 mmol) in 95% EtOH (10 mL) was added drop-wise until the color of the mixture changed from colorless to brown. After the reaction mixture was stirred for 2 h at 0 °C, saturated NaHCO3 (2.0 mL) was added until the pH was greater than 7. The mixture was then concentrated in vacuo. The residue was dissolved in EtOAc (20 mL) and washed sequentially with 10% NaHCO3 (3 x 15



mL) and brine.

Page 28 of 56

The organic layer was dried over Na2SO4, filtered, and was concentrated.

The residue was purified by flash column chromatography (EtOAc:petroleum ether, gradient elution, 0:100 to 1:2) to afford compound 58b (280 mg, 69%) as yellow oil. 1H NMR (400 MHz, CDCl3) δ 7.51 (d, J = 8.4 Hz, 2H), 7.23 (d, J = 8.4 Hz, 2H), 4.57 (s, 2H), 2.40 (br, 1H), 1.29 (s, 9H). Linker-Drug 32. Representative example of linker-drug synthesis. To a solution of triphosgene (192 mg, 0.65 mmol) in CH2Cl2 (5.0 mL) at 0 °C was added a solution of 51a (300 mg, 1.29 mmol) and pyridine (306 mg, 3.87 mmol) in CH2Cl2 (5 mL). The mixture was stirred at 8 °C for 10 min then was added dropwise to a solution of 62 (1.43 mg, 1.68 mmol) and pyridine (306 mg, 3.87 mmol) in CH2Cl2 (5.0 mL) at 0 °C. The reaction mixture was stirred at 8 °C for 30 min then was concentrated under reduced pressure. The residue was purified by flash column chromatography (EtOAc:petroleum ether, 1:1) to give 63a (0.50 g, 33%) as a yellow oil. LCMS #1: RT =1.08 min, m/z = 1111.7 [M+1]+. To a solution of 63a (300 mg, 0.27 mmol) in CH2Cl2 (10 mL) was added iPrSH (206 mg, 2.7 mmol) at 0 °C. The mixture was stirred at 8 °C for 10 min, then MnO2 (100 mg) was added and the resulting suspension was stirred for 5 min and filtered. The filtrate was concentrated to give the crude product, which was purified by flash column chromatography (EtOAc:petroleum ether, 1:1) to give 63b (210 mg, 75%) as a yellow solid. LCMS #1: RT =1.27 min, m/z = 1031.5 [M+1]+. To a solution of triphosgene (30 mg, 0.10 mmol) in CH2Cl2 (5.0 mL) was added a solution of 63b (210 mg, 0.20 mmol) and Et3N (62 mg, 0.61 mmol) in CH2Cl2 (5 mL) at 0 °C. The mixture was stirred at 8 °C for 30 min, then a solution of 64 (140 mg, 0.24 mmol) and Et3N (62 mg, 0.61 mmol) in DMF (5 mL) was added dropwise. The mixture was stirred at 8 °C for 12 h, then was concentrated under reduced pressure. Purification of the residue by


Page 29 of 56 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


flash column chromatography (8% CH3OH in CH2Cl2) gave 65b (140 mg, 42%) as a yellow oil. LCMS #1: RT =1.25 min, m/z = 816.1 [M/2+1]+. A solution of 65b (140 mg, 0.09 mmol) in acetic acid (3.0 mL), THF (2.0 mL) and water (2.0 mL) was stirred at 8 °C for 8 h. The mixture was added to EtOAc (60 mL), washed with water (50 mL x 3), sat. NaHCO3 (50 mL), brine (50 mL). The organic layer was dried over Na2SO4, filtered, and was concentrated to give crude 66b (120 mg, 100%) as a white solid. LCMS #1: RT =0.79 min, m/z =701.0 [M/2+1]+. To a solution of 66b (130 mg, 0.09 mmol) in DMSO (3.0 mL) was added IBX (130 mg, 0.46 mmol) at 10 °C. The reaction mixture was warmed to 50 °C and was maintained at that temperature for 48 h. The mixture was purified by prep-HPLC (acetonitrile 35–65% / 0.225% FA in water) to give 32 (10 mg, 8%) as a white solid. LCMS #1: RT = 0.73 min, m/z =1397.9 [M+1]+. Linker-Drug 33. LCMS #1: RT = 0.76 min, m/z = 1423.4 [M+1]+. Linker-Drug 34. LCMS #1: RT = 0.76 min, m/z = 1437.7 [M+1]+. Linker-Drug 35. LCMS #1: RT = 0.81 min, m/z = 737.1 [M/2+1]+. Linker-Drug 36. LCMS #1: RT = 0.73 min, m/z = 677.5 [M/2+1]+.



Page 30 of 56

RESULTS As shown in Figure 1 and Table 1, we systematically examined the propensity of various disulfide-containing prodrugs of the cytotoxic PBD-monomer 541 to undergo reductive cleavage in the presence of either glutathione (GSH) or cysteine (Cys). The use of PBD-monomers in these studies in lieu of the more potent PBD-dimers (e.g., 1) greatly simplified the preparation of the tested molecules but still enabled the assessment of cytotoxic outcomes in cell culture experiments. Glutathione is a key intracellular reducing agent,42 and the selected concentration (4.0 mM) was chosen to mimic its average intracellular levels reported in many diverse tumors.5 Our own assessments of GSH levels in various cancer cell lines were consistent with these reported data (see Supporting Information, Table S1). Cysteine is similarly believed to be the primary reducing agent present in human plasma.43-46 However, we found that utilization of Cys concentrations (30 µM) that were close to those found in circulation in vivo (8-45 µM)43-46 did not adequately stratify the various disulfides in the in vitro cleavage experiments. Accordingly, we also employed a somewhat higher Cys concentration (200 µM) to better differentiate the stability of the various disulfides under study.

To make these in vitro assessments relatively

conservative, a short incubation time (1 h) was employed for GSH cleavage work while a much longer time period was used for the Cys evaluations (24 h). Thus, an ideal disulfide prodrug would display efficient cleavage in the short GSH assessment (suggesting rapid intracellular release) and excellent longer-term stability in the presence of Cys (indicating good stability in plasma). It was also recognized, however, that these in vitro studies were surrogates for actual in vivo outcomes and that correlation between all of them and measured in vivo performance would eventually need to be established. As depicted in Table 1, prodrugs containing little steric bulk on either side of the disulfide bond were highly labile in both the GSH and Cys (200 µM) stability assessments


Page 31 of 56 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(compounds 6 and 7). As expected, increasing the steric congestion at the disulfide terminus improved both stability parameters (compounds 9 and 10), but these outcomes were somewhat attenuated by inclusion of a heteroatom in this region of the prodrug structure (compound 8). The latter result is likely due to the influence of the heteroatom on the pKa of the corresponding thiol that comprises the disulfide bond (i.e., more acidic thiol = less stable disulfide). Addition of a single methyl group to the non-terminal side of the disulfide bond improved Cys stability but not GSH cleavage outcomes (compare 11 with 6). As was observed with compounds 6-10 above, increasing the terminal steric bulk of 11 dramatically improved prodrug stability (compounds 15 and 16).

Similarly, analogs of 11 bearing

heteroatoms at the disulfide termini were all less stable than the former molecule regardless of the associated sterics (compare 11 with 12-14). We also prepared a Cys-derived disulfide related to 11 to benchmark the stability of intermediates expected to be generated intracellular by disulfide-linked ADCs (compound 17).23-24

As shown in Table 1, this

compound was highly labile in both the GSH and Cys (200 µM) assessments. In addition to the molecules described above, we examined many prodrugs that contained two non-terminal substituents adjacent to the disulfide bond. Compounds bearing two geminal methyl groups were highly stable toward both GSH and Cys if a branched R3 substituent was also present in the molecule (27-28, compare to 26). Spirocyclic analogs displayed attenuated (18), equivalent (19, 21), or improved stability properties (20) relative to the gem-dimethyl analog 26 depending on spirocyclic ring size (cyclopropyl = least stable).



Page 32 of 56

Table 1.

Cmpd

% Disulfide Remaininga

Cell IC50 (nM)b italics = intracell. [GSH] (mM) A2058 WSU KPL-4 (ND) (1.4) (12-44) 57 107 285

5

GSH 4.0 mM NA

Cys 30 µM NA

Cys 200 µM NA

6

0

94

18

83

221

7

0

66

0

ND

8

0

ND

48

9

56

100

10

88

11

Prodrug Shiftc WSU

KPL-4

NA

NA

296

2.1

1.0

ND

ND

ND

ND

ND

ND

ND

ND

ND

92

635

401

772

3.7

2.7

100

99

5593

3836

1461

36

5.1

0

98

57

31

112

201

1.0

0.70

12

0

1

0

ND

ND

ND

ND

ND

13

1

93

30

ND

ND

ND

ND

ND

14

0

0

0

ND

ND

ND

ND

ND

15

98

100

99

304

1077

1012

10

3.6

16

100

100

100

>20000

>10000

>10000

>90

>35

17

0

77

0

ND

ND

ND

ND

ND

18

9

100

19

3026

1862

28

6.5

19

21

99

86

675 75

434

320

4.1

1.1

20

68

100

91

395

1350

1058

13

3.7

21

44

98

55

272

1147

1321

11

4.6

22

59

100

87

ND

ND

ND

ND

ND

23

0

97

14

ND

ND

ND

ND

ND

24

0

98

48

ND

ND

ND

ND

ND

25

0

94

0

ND

ND

ND

ND

ND

26

39

100

57

ND

450

346

4.2

1.2

27

100

100

100

1827

3512

1913

33

6.7

28

100

100

100

8109

>10000

>10000

>90

>35

29

0

42

4

ND

ND

ND

ND

ND

30

82

99

85

567

2807

806

26

2.8

NA NA NA >20000 >10000 >10000 NA NA 31 Disulfide cleavage in presence of indicated concentration of GSH or Cys; time points: GSH = 1 h, Cys = 24 h. See Supporting Information for additional details. b Antiproliferation activity of given compound in indicated cell line. WSU = WSU-DLCL2. See Supporting Information for additional details. c Ratio of prodrug IC50 to compound 5 IC50 (in same cell line). NA = not applicable. ND = not determined. Color-codings based on the prodrug design strategies described in the text are provided to help visualize outcomes and trends: green = desired/favorable, red = not desired/unfavorable, yellow = intermediate/unknown. a


Page 33 of 56 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1.

Once again, inclusion of heteroatoms at various locations in the prodrug structures had a deleterious effect on the resulting GSH and Cys stability outcomes (compounds 23-25). We also examined two compounds containing thiophenol-derived disulfides (29 and 30) that were expected to generate PBD-monomer 5 following disulfide cleavage via an immolation mechanism that was distinct from that depicted in Scheme 1 (see Scheme S1 in the



Page 34 of 56

Supporting Information).47 The former molecule was highly labile in both the GSH and Cys assessments, but the latter compound was significantly more stable. The results depicted in Table 1 paralleled observations by others regarding the ability of dithiothreitol to reduce similarly substituted disulfide-containing ADC linkers in vitro.48 They also enabled the prioritization of the various prodrugs for subsequent cell-based assessments, although we did not know the precise correlation of the in vitro stability data to in vivo outcomes. In order to increase the likelihood of obtaining prodrugs that would be stable during in vivo circulation in the context of an ADC, we conservatively focused the next phase of biological characterization primarily on molecules that displayed relatively high stability in the 200 µM Cys cleavage assessment (>50% remain after 24 h). As shown in Table 1, the cytotoxic properties of several prodrugs selected based on the above criteria were evaluated in vitro against multiple cancer cell lines.

To help

benchmark the outcomes of these experiments, the parent PBD-monomer (5) was included in the assessments along with compounds 6 and 18 (labile disulfide controls) and a negative control containing a non-cleavable carbamate moiety (31). Importantly, the media employed in these cell culture experiments was purposefully depleted of Cys so as to minimize the effects of extracellular disulfide cleavage on the resulting outcomes (see Supporting Information for more details). In the A2058 cell line, a reasonable correlation was observed between cell potency and GSH disulfide stability values with the more stable molecules showing the weakest antiproliferation properties and the greatest potency shifts relative to parent compound 5 (Figure S1A; for unknown reasons, compound 15 was an exception to this trend).

These results illustrated the importance of efficient prodrug cleavage (and

presumably subsequent liberation of 5) in achieving potent cytotoxic properties. As expected based on this requirement, the non-cleavable negative control compound (31) was inactive toward the A2058 cells. Interestingly, the cyclopropyl-containing prodrug (18) exhibited


Page 35 of 56 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


relatively weak antiproliferation activity in spite of efficient disulfide cleavage in the GSH assessment.

This outcome is presumably a result of poor linker immolation following

disulfide cleavage and is analogous to observations made with a related PBD-dimer molecule in our earlier studies.22

Collectively, the initial cell culture data indicated that the disulfide

prodrug structure could influence the cytotoxic properties of the associated molecules. We therefore conducted additional cell-based assessments to better characterize the relationships between these two parameters. We next profiled the selected disulfides in two cell lines that were chosen due to their widely differing intracellular concentrations of GSH [WSU-DLCL2 = 1.4 mM (low GSH), KPL-4 = 12-44 mM (high GSH); see Supporting Information for method employed to measure such levels]. As was the case with the A2058 studies above, the media employed in these cell culture experiments was carefully selected to minimize the propensity for extracellular disulfide cleavage to occur (see Supporting Information for more details). As depicted in Table 1 and Figure S1B, all of the prodrugs examined displayed smaller potency shifts relative to parent compound 5 when tested against the KPL-4 cell line (high GSH) as compared to the outcomes observed with the WSU-DLCL2 cells (low GSH). These data were consistent with the stronger reducing environment present inside the KPL-4 cells producing larger quantities of cytotoxic 5 during the course of the experiments relative to the levels of 5 generated in the WSU-DLCL2 cells. Assuming the GSH levels present in the latter cells were reasonable surrogates for those found in the majority of non-cancerous tissues (see Table S2, Supporting Information),49,50 the results also suggested that prodrugs which displayed large activation differences between high and low GSH environments might exhibit enhanced (i.e., selective) cytotoxic activity toward highly reducing tumors as compared to their activity against normal cells.51 Related, albeit small, impacts of GSH levels on disulfide prodrug activation were also observed in engineered stable cell lines via



Page 36 of 56

shRNA modulation of the Nrf2 protein (see Supporting Information, Table S3 and Figures 6A-6D). With the above data in hand, we selected several disulfide prodrugs for incorporation into PBD-dimer-containing antibody drug conjugates analogous to compound 2 above (Scheme 1). We focused these selections on disulfides that displayed the following in vitro parameters when incorporated into PBD-monomers related to compound 5: (1) good stability in the Cys (200 µM) assay (suggestive of good blood/plasma stability), (2) a low potency shift in the KPL-4 cell assessments (indicative of good activation/cleavage in strong reducing environments), (3) a potency shift in the WSU-DLCL2 cell line that was significantly larger than that observed in the KPL-4 cells (suggesting poor activation/cleavage in weak reducing environments), and/or (4) acceptable absolute KPL-4 potency (antiproliferation IC50 1000

84

91

94

30

85

HER2-hc-37

HC-A140

1.9

0.5

NA

NA

NA

NA

NA

CD22-hc-37

HC-A140

2.0

>1000

NA

NA

NA

NA

NA

HER2-hc-36

HC-A140

2.0

>1000

100

100

100

31

ND

CD22-hc-36

HC-A140

2.0

>1000

100

100

100

31

ND

CNJ Name (antigen-sitelinker-drug)

Site

HER2-lc-32

LC-K149

HER2-lc-33

a

DAR

b

a

Location of antibody residue mutated to Cys for conjugation to linker-drugs. LC = lightchain, HC = heavy chain. See reference 23 for details regarding residue numbering. b Drug-antibody ratio. c Antiproliferation activity. See Supporting Information for additional details. d Stability of prodrug-containing conjugates in whole-blood derived from various species. Data are averages of 2 assessments. See Supporting Information for additional details. e Unconjugated PBD-monomer prodrug that corresponds to PBD-dimer linker-drug (see Table 1 for additional information). f Stability of unconjugated PBD-monomer prodrug in presence of 200 µM Cys over 24 h (see Table 1 for additional information). NA = not applicable. ND = not determined. As shown in Table 2, the HER2-targeting conjugates prepared from compounds 32-35 exhibited varying degrees of cytotoxic activity when evaluated against the HER2-expressing KPL-4 breast cancer cell line. Conjugates containing the two spirocyclic prodrugs exhibited in vitro potencies that were similar to the activity observed for the control ADC (compare HER2-lc-33 and HER2-lc-34 with HER2-lc-37, Table 2 and Figure S7). In contrast, potency 38 ACS Paragon Plus Environment


Page 40 of 56

attenuations relative to control ADC HER2-lc-37 were noted for the other prodrug-containing LC-K149 conjugates that were tested (compare HER2-lc-32 and HER2-lc-35 with HER2-lc37, Table 2 and Figure S7). The observed potency shifts for these conjugates did not perfectly parallel those measured above for the corresponding PBD-monomer prodrugs in the KPL-4 cell line (compounds 19, 20, and 30; Table 1). These differences may result from the use of PBD-dimers in the conjugate-based experiments, the associated antibody-mediated delivery, or other unknown factors. In vitro potency outcomes observed for the HC-A140 conjugates closely matched those noted for the LC-K149 counterparts (compare HER2-hc-34 with HER2-lc-34, HER2-hc-35 with HER2-lc-35, and HER2-hc-37 with HER2-lc-37, Table 2). These data were consistent with our prior observations that LC-K149 and HC-A140 conjugates tended to display similar activities in cell-based assessments.55,56 As expected, HC-A140 control conjugates CD22-hc-35 and CD22-hc-37 which did not recognize the HER2 antigen displayed significantly weaker antiproliferation activities relative to the corresponding HER2-targeting ADCs in the KPL-4 experiments (HER2-hc-35 and HER2-hc37, respectively). Importantly, the HER2-targeting HC-A140 conjugate containing a noncleavable prodrug was also inactive in the in vitro potency assay (HER2-hc-36, Table 2). This result demonstrated the importance of achieving efficient disulfide prodrug cleavage in order to elicit meaningful in vitro antiproliferation outcomes. Collectively, the in vitro studies performed with the conjugatesdepicted in Table 2 identified several entities that, based on their antigen-specific KPL-4 antiproliferation properties, appeared to be good candidates for progression into in vivo efficacy studies. As a prelude to conducting in vivo work with the aforementioned ADCs, we evaluated the stability of many of them toward whole blood derived from several different species. As expected based on our prior experiences with such assessments, the maleimidederived connections between the corresponding linker-drugs (compounds 32-35) and the Cys-


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engineered antibodies were highly stable in these experiments (

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