Synthesis and biological evaluation of antibody conjugates of phosphate prodrugs of cytotoxic DNA alkylators for the targeted treatment of cancer

Article

pubs.acs.org/jmc

Synthesis and Biological Evaluation of Antibody Conjugates of

Phosphate Prodrugs of Cytotoxic DNA Alkylators for the Targeted

Treatment of Cancer

Robert Yongxin Zhao,* Hans K. Erickson, Barbara A. Leece, Emily E. Reid, Victor S. Goldmacher,

John M. Lambert, and Ravi V. J. Chari

ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451, United States

S

* Supporting Information

ABSTRACT: The synthesis and biological evaluation of

phosphate prodrugs of analogues of 1 (CC-1065) and their

conjugates with antibodies are described. The phosphate group

on the 1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one

(CBI) portion of the compounds confers enhanced solubility

and stability in aqueous solutions. In the presence of

phosphatases, these compounds convert into active DNA-

alkylating agents. The synthesis of the prodrugs was achieved

sequentially through coupling of CBI with a bis-indolyl moiety, followed by attachment of a thiol-containing linker, and

conversion of the hydroxyl group of CBI into a phosphate prodrug. The linkers incorporated into the prodrugs enable

conjugation to an antibody via either a stable disulfide or thioether bond, in aqueous buffer solutions containing as little as 5%

organic cosolvent, resulting in exclusively monomeric and stable antibody-cytotoxic prodrug conjugates. Two disulfide-containing

linkers differing in the degree of steric hindrance were used in antibody conjugates to test the effect of different rates of

intracellular disulfide cleavage and effector release on biological activity. The prodrugs can be converted to the active cytotoxic

compounds through the action of endogenous phosphatases. Antibody−prodrug conjugates displayed potent antigen-selective

cytotoxic activity in vitro and antitumor activity in vivo.

The design of synthetic analogues of 1, such as adozelesin,

carzelesin, and bizelesin, overcame the delayed toxicity

issue.^15,16Thus, these analogues proceeded to clinical

evaluation but were found to have limited therapeutic activity

and their development was discontinued.¹⁷A simpler cyclo-

propabenzindole (CBI) subunit developed by Boger and co-

workers¹⁸to replace the alkylating CPI subunit of 1 gave

compounds that were chemically more stable (∼4-fold),

biologically more potent (∼4-fold), and considerably more

synthetically accessible.¹⁹A simplified analogue of 1 containing

the CBI subunit, called DC1, has been explored in our

laboratory as an effector moiety for conjugation with an

antibody (Figure 1).⁹DC1 is about 1000-fold more cytotoxic

than commonly used anticancer drugs, such as doxorubicin,

methotrexate, and vincristine.²⁰It binds to the minor groove of

DNA, followed by alkylation of adenine residues by its CBI

component.¹³A conjugate of DC1 with the humanized anti-

CD19 antibody B4, huB4−DC1, was found to be highly

cytotoxic to CD19-expressing cell lines, with IC₅₀values in the

picomolar range. The conjugate was selective in its cytotoxicity

being at least 1000-fold less cytotoxic toward antigen negative

cells. In human tumor xenograft models in immunodeficient

mice, huB4−DC1 was found to be superior in its antitumor

INTRODUCTION

■

For cancer treatment, it is highly desirable to selectively target

malignant cells and not healthy tissues. One approach that has

been advanced through a series of preclinical and clinical

studies is to use antibodies that recognize tumor-associated

antigens expressed on the surface of tumor cells to selectively

target cytotoxic agents to these cells.¹⁻³In this approach, a

cytotoxic agent is covalently linked to a tumor-targeted

monoclonal antibody (mAb) forming an antibody−drug

conjugate (ADC). Upon binding to cell surface antigens, a

typical ADC is internalized via antigen-mediated endocytosis

and then transported to lysosomes, where enzymes or reducing

agents facilitate release of the cytotoxic effector molecule which

then kills the tumor cells. A number of ADCs containing a

highly potent cytotoxic compound are currently in various

stages of preclinical and clinical development. The cytotoxic

agents used include the antimicrotubule agents, the maytansi-

noids (DM1 and DM4)¹⁻⁴and the auristatins,^5,6the DNA

damaging agents, calicheamicin,^7,8and analogues of 1 and

duocarmycin.⁹⁻¹¹

The compound 1 bearing a cyclopropapyrroloindole

pharmacophore (CPI) is a very potent antitumor antibiotic

isolated from Streptomyces sp. in the late 1970s.¹²The synthetic

and mechanistic aspects of 1 and its derivatives have been

extensively studied by Boger and co-workers.^13,14Compound 1

was not developed because it exhibited delayed toxicity in mice.

Received: September 26, 2011

Published: December 8, 2011

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improved therapeutic efficacy in vivo compared to the parent

compounds.^28,29A few of these prodrugs were conjugated with

antibodies, and the resulting conjugates displayed antigen-

specific cytotoxicity in vitro.²⁶However, most of these

prodrugs, in particular the phenyl, piperazino, and piperidino

carbamates, display low solubility in aqueous buffer solution in

the pH range (6−8) typically used in antibody-conjugation

reactions. Moreover, the activation of other piperazino or

piperidino carbamate prodrugs, such as irinotecan, by human

carboxylesterases is reported to be inefficient,³⁰and interpatient

variability in the level of enzyme activity has been reported.³¹

Thus, the level of carbamate activation observed in preclinical

models (e.g., mice or rats) may not translate to the human

situation.

There are a few examples of anticancer drugs, unrelated to 1,

that have been converted into water-soluble prodrugs. The

anticancer drug, etoposide phosphate, is an example of a water-

soluble prodrug that has a phenolic phosphate protecting group

and is rapidly converted into the active moiety during

circulation in humans, presumably through hydrolysis by

endogenous alkaline phosphatase.³²Thus, a phosphate group

appeared to be a good choice for protecting the phenolic

hydroxyl of the CBI unit. Here we report prodrugs of DC1 in

which seco-CBI is protected with a phosphate group. It has

been previously reported that protected seco-CBI compounds

generally do not alkylate DNA.³³We found that these prodrugs

were stable in aqueous solutions at physiological pH but were

easily converted into active drugs in the presence of

phosphatases. These prodrug conjugates were highly potent

in killing cells in vitro, suggesting that the prodrugs were

activated inside target cells, presumably by endogenous

phosphatases.

Figure 1. Structures of CC-1065 (1),¹²DC1 (2), DC41 (3), and

huB4−DC1 conjugate.⁹DC1 (2): R = H, R₁= H, n = 1; DC41 (3): R

= H, R₁= CH₃, n = 2; B4−DC1 conjugate: R = SCH(CH₃)CON-

(CH₃)(CH₂)₃CON-mAb, R₁= H, n = 1.

activity to the clinically used anticancer drugs doxorubicin,

vincristine, and cyclophosphamide.⁹Antibody−DC1 conjugates

were not developed further for two main reasons, instability

and poor solubility of the DC1 component in aqueous

solutions. In physiological buffers, the seco-form of DC1

(seco-CBI) is spontaneously converted via Winstein cycliza-

tion²¹to the active cyclopropyl form, which then reacts with

water, resulting in opening of the cyclopropyl ring yielding the

inactive hydroxy compound (Scheme 1). The poor solubility of

a

Scheme 1. The Property of CBI

RESULTS

■

DC1 comprises three different subunits: a DNA-alkylating unit

CBI (A), binding unit bis-indole (B), and a linker unit for

conjugation (C) to antibodies. CBI ((1,2,9,9a-

tetrahydrocyclopropa[c]benz[e]indol-4-one) is the key precur-

sor required for the synthesis of DC1 and its derivatives.³⁴The

synthesis of CBI (Scheme 2) began with the commercially

available 1,3-dihydroxynaphthlene 4, which was treated with

ammonia at high temperature to generate the unstable 3-

aminonaphthelenol, which was converted without purification

to the bis-BOC protected 1-hydroxy-3-naphthylamine (5) using

excess BOC anhydride in the presence of a base in 30% yield.

Regioselective electrophilic iodination of 5 with N-iodosucci-

nimide and a catalytic amount of TsOH at reduced temperature

gave the iodide 6 as the sole identifiable product in 86% yield.

Deprotonation of the carbamate 6, using NaH in DMF,

followed by N-alkylation of the resulting anion with

commercially available (E:Z)-1,3-dichloropropene. gave a 93%

yield of the (1:2) E:Z isomers of vinyl chloride 7, the desired

precursor for the key aryl radical cyclization. A deoxygenated

solution of iodide 7 in dry benzene, refluxed for 3 h in the

presence of one equivalent of tri-N-butyltin hydride and AIBN

as the catalyst, gave the desired, fully protected alkylating

subunit 8 in 94% yield as a mixture of optical isomers. The

cyclized product 8 is formed in a highly chemoselective

manner; the reaction is believed to proceed via an initial

preferential homolysis of the weaker aryl C−I bond in 7 to

generate an aryl radical which undergoes a preferred 5-exotrig

intramolecular cyclization onto the tethered vinyl chloride

acceptor to give 8.³⁵Resolution of the isomers of 8 was readily

a

Conditions: (a) phophatases; (b) chemical phosphorylation; (c)

aqueous buffer; (d) hydrolysis by H₂O; (e) DNA alkylation.

DC1 complicated the conjugation efforts, necessitating the use

of 20% organic cosolvents which led to significant aggregation

of the conjugates. Therefore, a prodrug of DC1 has been

sought that would (i) remain stable in aqueous formulation and

during circulation in vivo, (ii) convert into the active

cyclopropyl form only upon reaching the target tumor cell

(this might limit the extent of its inactivation in the absence of

target DNA, and also might reduce toxic side effects), and (iii)

have improved solubility in aqueous solutions.

Several prodrugs of various analogues of 1 have been

reported in which the seco phenol was protected with a labile

group, such as carbamoyl,^10,22,23glycosyl,²⁴O-(acylamino),²⁵

peptidyl,²⁶and carbonyl.^23,27Some of these prodrugs exhibited

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a

Scheme 2. Synthesis of CBI

a

Conditions: (a) (1) NH₃(l), 135 °C, 14 h, (2) BOC₂O, DIPEA, 30%; (b) NIS/TsOH, −40 °C to RT, 4 h, 86%; (c) NaH/DMF, ClCHCH−

CH₂Cl, 0 °C, 4 h, 93%; (d) (l) Bu₃SnH/AIBN, phH/80 °C, 3 h, 94%, (2) chiral OD column, 20% IPA/hexane; (e) HCl (conc), EtOAc, 95%.

accomplished by chiral chromatography on a preparative

Chiralcel OD column providing multigram quantities of both

enantiomers (+)-8a (t_R= 18.5 min) and (−)-(S)-8b (t_R= 35.8

min, >99% ee) in a single run. The slower eluting

(−)-enantiomer of 8b was assigned to be the desired natural

(S)-configuration as it exhibited the more potent biological

activity, and its DNA-alkylation selectivity was identical with

that of the natural products. Acid-mediated (20% HCl in ethyl

acetate) deprotection of (−)-(1S)-8b removed both BOC

groups quantitatively to give the CBI salt 9, which was directly

used for the synthesis of DC1. The incorporation of these

synthetic improvements provided CBI salt (9) in six steps and

overall 21% conversion.

gave the bis-indole carboxylic acid 20 in 90% yield with no

detected cleavage of any of the amide bonds. Coupling of the

free acid of 20 with seco-CBI 9 using EDC in DMA provided

the bis indolyl-seco-CBI compound 21 in 75% yield. Reduction

of the nitro group in 21 with hydrogen over Pd/C under mildly

acidic conditions provided the amino-bis-indolyl-seco-CBI

compound 22 which was then coupled with the different

linkers (12−15) to form DC1−SR2 (23−26, respectively).

Initial efforts to reduce 21 with TiCl₃in an acetone/water

mixture led to poor yields of DC1−SR2 products due to

incomplete reduction of 21 and the instability of both 22 and

DC1−SR2 in aqueous solutions. Also the separation of DC1−

SR2 from the mixtures of 21 and 22 was tedious and it had to

be conducted in a mixed solvent system containing DMA or

DMF through normal phase HPLC purification. The linkable

compound 2 (DC1) can be generated from the reduction of 23

(DC1SMe) or 24 (DC1SPy) by either DTT or TCEP or from

the hydrolysis of 25 (DC1Ac) by NH₂OH in DMA containing

a small amount of a weak acid buffer. At high pH (>7.5), DC1−

SR2, exemplified by compound 23, readily underwent

spirocyclic hydrolysis with displacement of the chloride of the

CBI subunit to form the cyclopropylcycloheadienone com-

pound 27 (DC10SMe), which would undergo N-3 alkylation

with an adenine residue and thus form a DNA adduct.³³

Because of challenges encountered in the purification of DC1−

SR2 from compound 22, an alternative strategy involving a

change of the sequence of amide bond coupling to prepare

DC1−SR2 was explored. Thus, the linker was first coupled to

the bis-indolyl moiety followed by coupling of the CBI unit.

5-Nitroindole-2-carboxylic acid 17 was converted to the tert-

butyl ester 28 in 85% yield utilizing oxalyl chloride and

potassium tert-butoxide in tetrahydrofuran (Scheme 5). Initial

efforts on direct transesterification of the commercial available

ethyl ester 16 to the t-butyl ester 28, or from ethyl ester 19 to t-

butyl ester 30 utilizing published t-BuOH and sulfated SiO₂

method³⁶were unsuccessful. Pd/C catalyzed reduction of nitro

group in 28 with hydrogen provided the amino ester 29 in

quantitative yield. Amide bond coupling of 29 with 5-

nitroindole-2-carboxylic acid 17 with TBTU in DMA provided

the nitro-bis-indolyl ester 30 in 89% yields. Reduction of the

nitro group in 30 by catalytic hydrogenation, followed by

coupling of the resulting amino compound 31 with either linker

12 or 15, provided linkable bis-indoles 32 and 33 in 80% and

82% yields, respectively. The t-butyl esters of 32 and 33 were

hydrolyzed by 20% TFA in dichloromethane in the presence of

a catalytic amount of Et₃SiH to give the carboxylic acids 34 and

35 in 85−92% yields after crystallization. Coupling of 34 or 35

The linkers chosen to connect analogues of 1 to a

monoclonal antibody were prepared using either commercially

available 3-mercaptopropionic acid (10) or 4-mercapto-4,4-

dimethyl-butanoic acid (11) prepared from isobutylene

sulfide.⁴As depicted in Scheme 3, the reactive free thiols of

a

Scheme 3. Synthesis of DCx Linkers

a

Condition: MEOH/H₂O, pH 6−7.5, CH₃SSO₂CH₃, or PySSPy, or

Ac₂O.

these two linkers were protected as methylthio, acetyl, or

pyridine-2-ylthio derivatives by reaction with an excess of

CH₃SSO₂CH₃, Ac₂O, or aldrithiol-2 (PySSPy), respectively, in

a neutral aqueous buffer.

The synthesis of DC1 was accomplished through an initial

amide bond coupling of the CBI subunit with the di-indole

moiety followed by coupling with the linker (Scheme 4).

Commercially available ethyl 5-nitroindole-2-carboxylate 16

was either hydrolyzed to the carboxylic acid 17 or reduced to

ethyl 5-aminoindole-2-carboxylate 18 in excellent yields.

Condensation of 17 and 18 in the presence of O-

(benzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoro-

borate (TBTU) in N,N-dimethyl acetamide (DMA) provided

80% yield of the bis-indolyl ester 19 after simple filtration and

washing with water and methanol. Alkaline hydrolysis of the

ester in 19 with a mixture of 1.0 M NaOH and DMA at 60 °C

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a

Scheme 4. Synthesis of DC1−SR2

a

Conditions: (a) NaOH/THF, 94%; (b) H₂/Pd/C, THF, 97%; (c) 17, TBTU/DMA, 80%; (d) NaOH/DMA, 90%; (e) 9, EDC/DMA, 75%; (f)

H₂/Pd/C, DMA, 90%; (g) 12, 13, 14, 15, EDC/DMA; (h) DTT/DMA/TCEP, pH 6 for 23, 24, 26, NH₂OH/DMA pH 6, for 25; (i) from 23, 5%

NaHCO₃/THF, 80%.

a

Scheme 5. Alternative Strategy for Synthesis of DC1SMe and DC41SMe

a

Conditions: (a) (1) (COCl)₂, (2) t-BuOK, 85%; (b) H₂/Pd/C, THF, 97%; (c) 16, TBTU/DMA, 89%; (d) H₂/Pd/C/DMA, 92%; (e) 12 or 15,

EDC/DMA, ∼80%; (f) 20% TFA/DCM/Et₃SiH (cat), ∼90%; (g) 9, EDC/DMA, ∼75%.

with seco-CBI 9 in the presence of EDC in DMA provided 23

(DC1SMe) and 26 (DC41SMe) in 70−80% yields. Addition-

ally compounds 23 and 26 were easily separated from

compounds 34 and 35 using SiO₂chromatography. Thus the

method in Scheme 5 was more favorable than the one in

Scheme 4 for the large scale production of DC1−SR2.

Compounds 23 and 26 were converted to the phosphate

prodrugs 40 (DC4) and 41 (DC44), respectively, as shown in

Scheme 6. Treatment of the phenols 23 or 26 with

dibenzylphosphate, carbon tetrachloride, and base (DIPEA)

in a mixture of THF and CH₃CN provided the DC1−

dibenzylphosphates (36, 37). Removal of the benzyl protecting

groups and cleavage of the disulfide bonds of DC1−

dibenzylphosphates (36, 37) by methanesulfonic acid with

the aid of DTT, provided 40 (DC4) and 41 (DC44).

Alternatively reaction of 23 or 26 with phosphorus oxychloride

in the presence of base (DIPEA) provided DC1−dichlor-

ophosphates which hydrolyzed spontaneously at pH 4−6 to

form 38 (DC4SMe) and 39 (DC44SMe), respectively.

Reduction of 38 and 39 by TCEP or DTT in DMA containing

pH 5−7 buffers provided 40 (DC4) and 41 (DC44) in high

yields (>90%).

Solubility and Stability. To achieve conjugation of a

cytotoxic agent to an antibody and provide a reasonable yield,

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a

Scheme 6. Synthesis of DC4 and DC44

a

Conditions: (a) HP(O)(OBn)₂, CCl₄, THF/CH₃CN, DIPEA; (b) CH₃SO₃H/DTT; (c) (1) POCl₃/THF/CH₃CN/DIPEA, (2) NaH₂PO₄/H₂O;

(d) TCEP or DTT, pH 5−7.

the conjugation reaction should be performed in a solvent in

which they both are fully dissolved. Because antibodies can only

be dissolved in either aqueous solutions or in a solution

containing a small amount of a water-miscible organic solvent,

the choice of an acceptable solvent is limited. An antibody of

the IgG type has a molecular weight of approximately 150000,

and in our experience its optimal concentration in the

conjugation reaction should be around 3 mg/mL (∼2 × 10⁻⁵

M). To achieve a cytotoxic agent per antibody ratio (CAR) of

3, at least 3 equiv of the cytotoxic agent should be added per

mole of the antibody. Thus the minimum required solubility for

the cytotoxic agent is at least 6 × 10⁻⁵M. We found that the

effectors 23, 24, 25, 26, 38, 39, 40, and 41 were not soluble in

water, toluene, ethyl acetate, hexane, chloroform, dichloro-

methane, ethanol, or methanol but were soluble in polar

aprotonic solvents, such as DMSO, DMA, and DMF. DC1SMe

(23) and DC41SMe (26) were also soluble in THF, dioxane,

and acetone. The phosphate prodrugs (38, 39, 40, and 41) are

readily soluble in a mixture of water containing as little as 5% of

a water miscible organic solvent, such as THF, acetone,

dioxane, DMSO, DMA, or DMF. Because the thiol-bearing

compounds 2 (DC1), 3 (DC41), 40 (DC4), and 41 (DC44)

are prone to oxidation in aqueous solution, particularly in the

presence of a trace of heavy metal salt, the methyl disulfide

moieties 23 (DC1SMe), 26 (DC41SMe), 38 (DC4SMe), and

39 (DC44SMe) were used instead for the solubility study.

While compounds 23 and 26 were not soluble in a buffer

mixture containing up to 20% organic solvents (Table 1), the

correspondent phosphate prodrugs 38 and 39 were soluble in

buffers containing 5−20% DMA and were over 3000-fold more

soluble than their respective precursors 23 and 26.

Table 1. The Solubility of DCx Compounds

solubility (μM) at pH 7.0

100%

buffer

5% DMA,

95% buffer

10% DMA,

90% buffer

20% DMA,

80% buffer

compd

23

(DC1SMe)

<0.1

<1

3000

<1

1.0

4700

1.0

3.0

>6000

3.0

38

(DC4SMe)

19

26

(DC41SMe)

<0.1

19

39

(DC44SMe)

2900

4600

>6000

after storage for one year at room temperature in a phosphate

buffer containing 5% DMA at pH ∼ 7. Next, we tested if 38, 39,

40, and 41 would be dephosphorylated by commercially

available mammalian (human, bovine, rabbit, calf, and porcine)

acid or alkaline phosphatases. All of the tested enzymes readily

dephosphorylated 38, 39, 40, and 41 at pH 7.4. In particular,

38 and 39 were completely converted to 23 and 26,

respectively, by alkaline phosphatase from bovine liver, bovine

kidney, rabbit intestine, or porcine mucosa. Following

dephosphorylation, the newly formed compound 23 readily

converted into the active cyclopropyl form 27 (as detected by

HPLC Figure 2).

Antibody Conjugates. DC4 (40) and DC44 (41) were

conjugated to the humanized monoclonal antibody huB4³⁸and

to the humanized antibody huC242 (its parental murine C242

antibody was described previously).³⁹These antibodies

recognize the tumor-associated antigen CD19 and CanAg,

respectively, which are expressed on many cancer cell lines. The

preparation of ADCs through disulfide linkage using the

antibody modifying agents N-succinimidyl-4-(2-pyridyldithio)

butyrate (SPDB) and N-succinimidyl 4-(2-pyridyldithio)-

pentanoate (SPP), or through a thioether linkage using N-

succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate

(SMCC) was performed as previously described for maytansi-

Like the CBI-based agents of 1 detailed previously,³⁷23 and

26 are readily converted to the ring-closed cyclopropyl forms in

aqueous solutions. In contrast, the phosphate prodrugs (38, 39,

40, and 41) proved to be very stable such that no more than

3% of cyclopropyl ring formation was detected by HPLC even

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Table 2. DC Drugs Cytotoxicity: IC₅₀(pM) against Ramos,

Namalwa, HL60/s, and COLO 205 Cancer Cells

Ramos

cells

Namalwa

cells

HL60/s

cells

compd

COLO 205

21 (DC0-NO₂)

22 (DC0-NH₂)

23 (DC1SMe)

24 (DC1Spy)

25 (DC1SAc)

26 (DC41SMe)

27 (DC10SMe)

38 (DC4SMe)

39 (DC44SMe)

nd

5

3

7

3

nd

30 10

32

22

5

10

2

8

250 85

nd

50 20

47 33

90 20

20 14

21 10

50 25

45 25

nd

18

5

25

12

5

220 70

nd

Figure 2. HPLC analysis of the hydrolysis of DC4SMe (0.2 μmol) by

an alkaline phosphatase from bovine liver (2 units) in a phosphate-

buffered saline, pH 7.5, containing 5% DMA at 37 °C. (a) Without a

phosphatase, (b) after 2 h incubation with the phosphatase, (c) after 4

h incubation with the phosphatase.

15 10

1900

2000

80

12

5

2900

1800

>3000

nd

2800

30

1900

nd

38 (DC4SMe +

a

phosphatase )

39 (DC44Me +

90

25

nd

noid conjugates.⁴In general, the antibodies were modified with

∼6 equiv of either SPP, SPDB, or SMCC. Uncoupled linkers

were removed by gel filtration over a small Sephadex-based

desalting column. To remove any noncovalently bound free

cytotoxins from the conjugates, in most cases, a second-step

purification was applied using a Porapak column⁹that was

found to selectively remove the noncovalently bound cytotoxic

compound. However, the use of Porapak columns significantly

reduced the yields of the antibody−DCx conjugates because

the Porapak resin also bound the conjugates, although to a

lesser degree than the free DCx compounds (data not shown).

The molar ratios of linker to antibody incorporated using

SPDB or SPP was between 3.5−5.0. Because compounds 40

(DC4) and 41 (DC44) are not soluble in 100% aqueous

buffers, their conjugation to a linker-modified antibody, as well

as subsequent purification by size exclusion chromatography,

were performed in a phosphate-buffered saline, containing 5−

20% DMA. DC4 and DC44 were used at 1.7 equiv of the

conjugated linker. The amount of any residual free effector

moiety remaining in the purified conjugate that was not

covalently bound to the antibody was determined by first

capping any free thiol with N-ethyl maleimide (NEM) then

precipitating protein with addition of acetone and analyzing

supernates by HPLC. The antibody−DC4 and antibody−

DC44 conjugates were monomeric, soluble in the buffered

aqueous solution, and stable for over one year upon storage at 4

°C.

In Vitro Cytotoxicity of DC1 Derivatives. We found that

the compounds that contained free thiol were not very

cytotoxic (data not shown), possibly due to the formation of

mixed disulfides with a cystine in the cell culture medium

resulting in charged compounds that would diffuse poorly

across cellular membranes.⁴Therefore, we used the compounds

whose sulfurhydryl groups were protected by either a

methyldisulfide or a thioacetate to determine their in vitro

cytotoxicity. In these cytotoxicity experiments, human cancer

cell lines Ramos, Namalwa, HL60/s, and COLO 205 were

exposed to these compounds for 72 h, and then the surviving

fractions of cells was determined by their ability to form

colonies (clonogenic assay). The disulfide-containing phos-

phate prodrugs 38 and 39 were evaluated both in the absence

and in the presence of an acid phosphatase and compared with

their parent compounds 23 and 26. As shown in Table 2, DC1

derivatives 21, 22, 23, 24, 25, and 26 were found to be highly

potent with IC₅₀values in the 1 pM to 10 pM range on Ramos,

Namalwa, and HL60/s cells, and in the 100 pM range when

tested on COLO 205 cells. The IC₅₀value of 23 is consistent

a

phosphatase )

a

Alkaline phosphatase from bovine liver.

with that previously reported.⁹Compound 24 (DC1SPy) was

slightly less potent than 23 and 25, possibly due to reaction of

its pyridyl disulfide moiety with a thiol compound present

either in the cells or in cell culture medium. The prodrugs 38

and 39 were 20- to 160-fold less cytotoxic (IC₅₀of 2 nM) than

their parental compounds 23 and 26, but following incubation

with an acid phosphatase for 1.5 h, their cytotoxicity was fully

restored. HPLC analysis confirmed the complete conversion of

these prodrugs to the corresponding active cytotoxic moieties.

In Vitro Cytotoxicity of Antibody−Drug Conjugates.

To test if an antibody conjugate of a phosphate prodrug would

have cytotoxic activity, we conjugated DC4 (40) to the

humanized C242 antibody via either a disulfide-containing

linker (SPP or SPDB) or via a noncleavable SMCC linker.

Neither of these modifications damaged the binding affinity of

the antibody to CanAg-positive cells, as evaluated by an indirect

flow cytometric assay (Supporting Information, Figure S3). As

shown in Figure 3, all three conjugates displayed high

cytotoxicity in vitro toward the antigen-expressing COLO

205 cells with IC₅₀values between 0.7 and 1.6 pM (antibody

concentration) in the presence of a phosphatase and between

2.1 and 6.0 pM in the absence of a phosphatase, the latter

values indicating that the prodrug moiety was dephosphory-

lated inside the cell. The conjugates were at least 30000-fold

less cytotoxic toward the antigen-negative A375 cells (Figure

3). The cleavability of the linker in these three conjugates did

not appear to affect the in vitro cytotoxicity of the conjugates.

The conjugates were more potent (IC₅₀values in the picomolar

range) than the free DC1SMe (23), which has an IC₅₀value of

250 pM for antigen-positive COLO 205 cells, suggesting that

the antibody enhanced the delivery of the effectors. The modes

of internalization of the free prodrugs and their conjugates

differ. The free cytotoxic agent is expected to penetrate the cell

via diffusion across the plasma membrane, and this diffusion is

likely to slow down when the cytotoxic agent possesses a highly

charged and hydrated phosphate group. Hydrolysis of the

phosphate should enhance diffusion of the resulting non-

charged hydrophobic cytotoxic molecules inside the cells, which

is consistent with the 20- to 160-fold enhancement of their

cytotoxicity upon acid phosphatase treatment. In contrast,

antigen-mediated internalization of conjugates via endocytosis

seems to be not dependent on the charge of the effector

moiety, as reflected by only a modest (2- to 5-fold)

enhancement of their cytotoxicity upon acid phosphatase

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Figure 3. Cytotoxicity of conjugates of huC242−SPDB−DC4, huC242−SPP−DC4, and huC242−SMCC−DC4 against Colo 205 (Ag+) and A 375

(Ag−) cells in the presence and absence of an acid phosphatase from bovine prostate.

treatment. This small different in potency may simply reflect a

difference in rate of cell killing between an ADC wherein the

“payload” is already activated before it binds to cells versus an

ADC that requires the payload to be activated by intracellular

enzymes following binding and internalization.

To test whether antibody−DC4 conjugates targeting

antigens other than CanAg would also be potent, we

conjugated DC4 to the humanized B4 antibody (huB4). This

antibody recognizes CD19, an antigen found on lymphomas

and leukemias of B cell lineage.⁴⁰Two huB4−DC4 conjugates

were prepared, one conjugate with the disulfide-containing SPP

linker, and the other conjugate with the noncleavable SMCC

linker. In addition, we also prepared a conjugate bearing a

hindered disulfide bond (huB4−SPP−DC44), 41, and a

disulfide-linked conjugate with the parent drug DC1 (2)

(huB4−SPP−DC1). The latter served as a positive control,

since in our previous studies we had established that DC1

conjugate was highly active and selective in killing CD19-

positive cells.⁹HuB4−SPP−DC4 displayed high potency in

vitro toward CD19-expressing Ramos cells both in the absence

and in the presence of acid phosphatase, with IC₅₀values (in

protein concentration) of 11 pM and 7.5 pM, respectively. This

activity of the conjugate was antigen-selective because it was at

least 200-fold less potent toward antigen-negative HL60/s cells,

both with and without phosphatase treatment. This prodrug

conjugate was only slightly less active than the corresponding

nonprodrug conjugate, huB4−SPP−DC1 (IC₅₀= 1.4 pM

toward Ramos cells) (Table 3), HuB4−SPP−DC44, which

bears a more hindered disulfide bond than huB4−SPP−DC4,

and the noncleavable B4−SMCC−DC4 displayed much poorer

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Table 3. Cytotoxicity of B4−SPP−DCx Conjugates against

Ramos (Ag+) and HL60/s (Ag−) Cells

IC₅₀(pM)

acid phosphatase

treatment

Ramos cell (Ag

+)

HL60/s cells

(Ag−)

compd

B4-SPP-DC4

B4-SPP-DC44

no

11.0 2.0

7.5 2.0

400

>3000

>1500 500

>1800

yes

B4-SMCC-

DC4

1200

2600

B4-SPP-DC1

no

1.4

1300

potency, with IC₅₀values of 0.4 nM and 1.2 nM respectively,

against CD19-expressing Ramos cells, and IC₅₀of 1.8 and 2.6

nM, respectively, against CD19-negative HL60/s cells. One

explanation for the poorer potency of these two conjugates

might be incomplete activation by intracellular phosphatases

upon their internalization by the targeted tumor cells. To test

this hypothesis, we examined the yield of dephosphorylated

prodrugs in these conjugates by treatment with an acid

phosphatase. HuB4−SPP−DC4 and huB4−SPP−DC44 (∼5

μmol) were incubated with a large excess (∼10 units) of a

bovine acid phosphatase in phosphate-buffered saline, pH 6.5 at

37 °C for 24 h, denatured by acetone precipitation, and the

linked DC4 and DC44 moieties were released by TCEP

reduction. HPLC analysis showed that in both cases

approximately 59% of the free DCx moieties were released

from the conjugates by TCEP. The small molecule weight

compounds released from huB4−SPP−DC4 consisted of 48%

of DC1 and 52% of nonconverted DC4, while those released

from huB4−SPP−DC44 consisted of 33% of DC1, the rest

being nonconverted DC44. Thus a considerable amount of

antibody-bound prodrug was not activated in these two

conjugates even after extensive exposure to a phosphatase.

The poor conversion rate coupled with a slower rate of cleavage

of the more hindered linker in huB4−SPP−DC44 may account

for the poorer potency of huB4−SPP−DC44 relative to the

huB4−SPP−DC4 conjugate.

In Vivo Antitumor Activity. The antitumor activities of

huB4−SPP−DC4 and huB4−SPP−DC44 conjugates were

evaluated in SCID mice bearing CD19-positive xenograft

tumors. Animals with established subcutaneous xenograft

Ramos tumors were treated with either huB4−SPP−DC4

(DC4 dose of 75 μg/kg, qd ×5), or huB4−SPP−DC44 (DC44

dose of 75 μg/kg, qd ×5), unconjugated DC4 (75 μg/kg, qd

×5), unconjugated DC44 (75 μg/kg, qd ×5), or with

phosphate-buffered saline vehicle (control), administered

intravenously, and the tumor growth was monitored. As

shown in Figure 4, the tumors in the control group of mice

grew aggressively. The two prodrug conjugates, huB4−SPP−

DC4 and huB4−SPP−DC44 were active and delayed tumor

growth by 38 and 17 days, respectively. The corresponding

unconjugated prodrugs DC4 and DC44 were inactive in this

model, with the tumor growth rates tracking with that of the

control mice.

Figure 4. Comparisons of antitumor activities of free DC4, DC44, and

conjugates of huB4−SPP−DC4 and huB4−SPP−DC44 on Ramos

xenografts.

aqueous buffer, the conjugation was highly inefficient. To

improve this conjugation, we were interested in developing a

prodrug strategy that would increase both the aqueous

solubility and stability of these analogues of 1 through the

incorporation of a phosphate moiety. The ideal position for

incorporation of this substituent is the phenol group of the CBI

subunit as it would serve two purposes: (a) improve the

hydrophilicity of the molecule and thus its water solubility and

(b) improve the stability in physiological buffers by preventing

the undesired premature cyclization reaction to form a

cyclopropyl ring, which would then react with water causing

loss of activity.

A key step in the synthesis of these phosphate prodrugs is the

resolution of the enantiomers of seco(-)CBI during the CBI

synthesis. The previously published procedure¹⁸used two

different protecting groups for the seco-CBI, a t-BOC for the

amino moiety, and a benzyl for the phenolic group. Resolution

of the enantiomers on a chiral HPLC column was inefficient

and not amenable to scale up. Through a slight modification of

this procedure we found that using a di-t-BOC protected seco-

CBI (8) resulted in a vastly improved separation of the

enantiomers on a chiral HPLC column (Supporting Informa-

tion, Figure S3) and allowed for the easier accumulation of

larger amounts of material. In addition, using the same t-BOC

protecting group for the amine and phenolic groups reduced

the number of protection and deprotection steps, thus

increasing the efficiency and overall yield of the process.

For the synthesis of linkable bis-indolyl CBI drugs, two

different approaches were explored. In the first approach

(Scheme 4), the bis-indole unit was first attached to seco-CBI,

and the linker was connected in the last step. However, the

insolubility of bis-indolyl compounds resulted in a difficult

separation of the desired compound 23, 24, 25, or 26 from the

amino bis-indolyl seco-CBI precursor 22. Thus, column

chromatography required the use of polar aprotic cosolvents

along with a large column size, resulting in product streaking

and poor resolution of separation. Thus synthetic Scheme 5,

wherein the linker was attached to the bis-indolyl unit in the

first step, followed by incorporation of the seco-CBI moiety in

the final step, was preferred. In this case, the difference in

DISCUSSION

■

Previously we have known that DC1 conjugated via a disulfide

bond to the huB4 antibody (huB4−DC1) was a highly

efficacious immunoconjugate resulting in complete regression

in the majority of animals in a Namalwa xenograft model.

However, due to the lack of solubility and stability of DC1 in

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Scheme 7. The Possible Metabolism of mAb−SPP−DC4, mAb−SPP−DC44, and mAb−SMCC−DC4

polarity between the desired bis-indolyl-seco-CBI products and

the corresponding bis-indole precursors allowed for a facile

separation.

organic cosolvents were completely removed by dialysis and

stable for an extended period of time when stored at 4 °C.

Our in vitro studies with the unconjugated prodrugs indicate

that these unnatural phenolic phosphate esters are not active

against all tested tumor cell lines but can be fully activated by

mammalian phosphatases (Table 2). In vitro studies with two

different ADCs of these prodrugs demonstrated potent and

good antigen-selective killing of target cells without the

addition of exogenous phosphatases, suggesting that tumor

cells possess phosphatases that are capable of activating the

ADC once delivered into the cell. On the basis of our previous

studies on the mechanism of intracellular activation of ADCs

comprising of maytansinoid drugs,⁴¹we propose that

phosphatases do not act on the intact ADC of the prodrug

but on a cellular metabolite. Thus, upon internalization into the

cell, the disulfide-linked antibody−SPP−DC4 conjugate would

undergo lysosomal degradation of the antibody component to

give a lysine−SPP−DC4 metabolite (Scheme 7), which is then

enzymatically dephosphorylated to produce the lysine−SPP−

DC1 metabolite. This metabolite can directly alkylate DNA or

can first undergo disulfide reduction to give DC1 followed by

alkylation of DNA. In the case of the noncleavable thioether-

linked conjugate, antibody−SMCC−DC4, the sole metabolite

would be lysine−SMCC−DC4, which would be dephosphory-

lated to lysine−SMCC−DC1. The huC242−SMCC−DC4

conjugate is quite potent in vitro, suggesting that the proposed

metabolite lysine−SMCC−DC1 is capable of binding to, and

In the development of ADCs, the aqueous solubility and

stability of the cytotoxic agent used are two key factors in

determining the efficiency of conjugation and the usefulness of

the conjugate, respectively. The extreme insolubility of the bis-

indolyl-seco-CBI compounds, such as DC1 (2) in water

hampered the efficient production of ADCs. In our earlier

attempts, conjugation of DC1 with an antibody could only be

performed in a buffer containing 20% organic cosolvents, which

led to a significant amount of antibody aggregation. In addition,

because of the propensity of the DC1 component to undergo

hydrolytic inactivation in water, the conjugate had to be stored

in a frozen or lyophilized state. Thus, we sought a prodrug

approach that would address the issue of aqueous solubility and

stability while also ensuring conversion to the active drug under

physiological conditions. Our data shows that the choice of a

phosphate prodrug fulfilled all these requirements. Thus,

incorporation of a charged phosphate group into the cytotoxic

agent dramatically improved the solubility of the compound

under conjugation conditions. For example, in the presence of

as little as 5% of a water-miscible organic cosolvent, such as

DMA or DMF, the solubility of the prodrugs was improved

over 3000 times. The resulted ADCs with these phosphate

prodrugs were soluble in physiological buffers even after the

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to 1:4 EtOAc/hexane) and crystallization with EtOAc/ethanol/hexane

provided 33.66 g (30%) of the title compound (5). ¹H NMR (CDCl₃,

400 MHz) 8.14 (d, 1H, J = 8.1 Hz), 7.66 (d, 1H, J = 8.1 Hz), 7.43 (dd,

1H, J = 6.8, 8.2 Hz), 7.35 (dd, 1H, J = 6.8, 8.2 Hz), 7.22 (d, 1H, J = 1.8

Hz), 7.15 (br, 1H, NH), 6.69 (s, 1H), 1.59 (s, 9H), 1.37 (s, 9H). ¹³C

NMR (CDCl₃) 153.71, 152.9, 136.11, 135.20, 128.12, 128.01, 126.81,

126.03, 123.61, 107.94, 102.95, 82.98, 82.10, 28.93, 27.69. ESI MS m/z

382.52 (M + Na)⁺. HRMS (C₂₀H₂₅NO₅+ Na), m/z+ 382.1630, calcd

382.1641 (M + Na)⁺.

alkylating a DNA sequence. We did find that another ADC,

huB4−SMCC−DC4, was not very potent in vitro. One

explanation for the differential potency of the huC242−

SMCC−DC4 and huB4−SMCC−DC4 conjugate toward

their respective target cells could lie in the relative level of

antigen expression: the CanAg antigen that binds to huC242 is

expressed at a high level (>10⁶antigens/cell) on the surface of

the target COLO 205 cells, while the antigen for huB4 on

Ramos cells is expressed at a much lower level (<10⁵antigens/

cell). Thus, a threshold level of active metabolite in the cytosol

sufficient to cause cell killing is probably not achieved with the

huB4−SMCC−DC4 conjugate. In vivo, both the huB4−SPP−

DC4 and the huB4−SPP−DC44 conjugates displayed anti-

tumor activity by slowing tumor growth. The respective free

prodrugs were inactive in this model demonstrating the benefits

of antibody-mediated delivery of the charged molecules. For

the two conjugates, the relative disulfide bond strength appears

to play a role in the level of antitumor activity. The huB4−

SPP−DC44, which has a more sterically hindered disulfide

bond than the corresponding DC4 conjugate, was less active,

suggesting that the more labile disulfide bond of the DC4

conjugate makes it more susceptible to release of DC4 at the

tumor. The phosphate-containing DC4 prodrug can undergo

activation to DC1 which then, in addition to killing the targeted

cell, can also diffuse into neighboring cell causing bystander

killing resulting in an enhanced antitumor effect.⁴²Our study

suggests that a phosphate prodrug may be a suitable choice for

ADCs of effector molecules that do not possess sufficient water

solubility or stability.

2-N-(tert-Butyloxycarbonyl)amino-4-O-(tert-butyloxycarbonyl)-

oxy-1-iodo-naphthalene (6). 3-N-(tert-Butyloxycarbonyl)amino-1-O-

(tert-butyloxycarbonyl)-1-naphthol (5) (24.50 g, 68.24 mmol) and N-

iodosuccinimide (17.70 g, 74.73 mmol) were dissolved in THF/

CH₃OH (250 mL, 1:1). After the solution was stirred at −40 °C under

Ar in the dark for 5 min, TsOH (0.86 g 4.52 mmol) was added. The

reaction mixture was stirred under Ar in the dark at −40 °C for 2 h

and then at room temperature for 2 h. The mixture was diluted with

Et₂O (800 mL), washed successively with saturated aqueous NaHCO₃

and saturated aqueous NaCl, dried over MgSO₄, filtered, and

concentrated in vacuo. Flash chromatography on SiO₂(EtOAc/

hexane 1:10) and crystallization with ethanol/ethyl acetate/hexane

afforded 28.46 g (86%) of the title compound (6). R_f= 0.48 (10%

1

EtOAc/hexane). H NMR (CDCl₃, 400 MHz) 8.27 (d, 1H, J = 8.0

Hz), 7.98 (dd, 1H, J = 1.5, 8.1 Hz), 7.83 (s, 1H), 7.55 (m, 2H), 7.18

(br, 0.8H, NH), 1.62 (m, 18H). MS m/z 508.36 (M + Na)⁺. HRMS

m/z+ 508.0575, calcd 508.0597

2-[N-(tert-Butyloxycarbonyl)-N-(E,Z-3-chloro-2′-propen-1′-yl)-

amino]-4-O-(tert-butyloxycarbonyl)oxy-1-iodo-naphthalene (7). To

2-N-(tert-butyloxycarbonyl)amino-4-O-(tert-butyloxycarbonyl)oxy-1-

iodo-naphthalene (6) (940 mg,1.86 mmol) in 20 mL of dry DMF was

added NaH (150 mg of 60% in mineral oil, 3.75 mmol) under Ar.

After the mixture was stirred at 0 °C for 30 min, E,Z-1,3-

dichloropropene (1.50 mL, 14.57 mmol) was added. The reaction

mixture was stirred at 0 °C under Ar for 2 h, then neutralized with 1.0

M NaH₂PO₄, extracted with EtOAc, dried with dried over MgSO₄,

filtered, and concentrated in vacuo. Flash silica gel chromatography

(ethyl acetate/hexane 1:9) afforded 1.01 g (93%) of the vinyl chloride

compound (7). R_fZ= 0.37, R_fE= 0.32 (1:8 EtOAc/hexane) (E:Z vinyl

chloride rotamers). ¹H NMR (CDCl₃, 400 MHz) 8.26 (d, 2H, J = 7.7

Hz), 7.96 (m, 2H), 7.59 (br, 4H), 7.20 (s, 1H), 7.16 (s, 1H), 6.17−

6.07 (m, 4H), 4.64 (dd, 1H, J = 6.2, 15.2 Hz), 4.53 (dd, 1H, J = 6.2,

14.7 Hz), 4.31 (dd, 1H, J = 6.0, 15.0 Hz), 3.84 (dd, 1H, J = 7.5, 15.0

Hz), 1.58 (S, 9H), 1.33 (s, 9H). ¹³C NMR (CDCl₃) 153.78, 151.08,

150.98, 133.31, 133.29, 128.66, 128.61, 127.50, 127.41, 126.41, 121.68,

119.03, 84.22, 84.11, 80.99, 77.20, 28.20, 27.66. MS m/z+ 582.8 (M +

Na)⁺, 598.03 (M + K)⁺. HRMS m/z+ 582.0491 (M + Na)⁺, calcd

582.0520

5-(O-tert-Butyloxycarbonyl)oxy-3-[N-(tert-butyloxycarbonyl)-

amino-1-(chloromethyl)-1,2-dihydro-3H-benz(e)indole (8). To a

solution of the aryl iodide (7) (1.36 g, 2.43 mmol) in dry benzene

(100 mL) were added tri-N-butyltin hydride (0.70 mL, 2.52 mmol)

and catalytic ammount of 2,2′-azobis(isobutyronitrile) (AIBN) (30

mg, 0.18 mmol). The mixture was stirred under Ar at room

temperature for 30 min and then refluxed at 80 °C for 2 h. The

reaction mixture was cooled, and the solvent was removed in vacuo.

Trituration of the crude oil with hexane provided a solid which was

filtered and washed with hexane to give the title compound 8 (∼93%

pure) as an off-white solid. Direct purification, after removal of the

solvent, by silica gel flash chromatography (ethyl acetate/hexane 1:9)

afforded 1.01 g (94%) of the indoline compound (8). R_f= 0.34 (1:9

EtOAc/hexane). ¹H NMR (CDCl₃) 8.12 (br, 1H), 7.91 (d, 1H, J = 8.4

Hz), 7.69 (d, 1H, J = 8.4 Hz), 7.50 (dt, 1H, J = 1.0, 6.9, 7.0 Hz), 7.37

(dt, 1H, J = 0.9, 6.9, 6.9 Hz), 4.27 (br, 1H), 4.12 (t, 1H, J = 9.0 + 10.0

Hz), 3.99 (m, 1H), 3.90 (dd, 1H, J = 2.4, 11.0 Hz), 3.45 (t, 1H, J =

10.8 +10.8 Hz), 1.58 (S, 18H). ¹³C NMR (CDCl₃) 152.27, 151.84,

147.99, 130.17, 127.62, 124.33, 122.46, 122.22, 108.95, 83.78, 52.80,

46.13, 28.36, 27.79. MS m/z 456.9 (M + Na)⁺. HRMS m/z 456.1550,

calcd 456.1554 (M + Na)⁺.

EXPERIMENTAL SECTION

■

Melting points were measured using an Electrothermal apparatus and

are uncorrected. NMR spectra were recorded on a Bruker

AVANCE400 (400 MHz) spectrometer. Chemical shifts are reported

in ppm relative to TMS as an internal standard. Low-resolution mass

spectra were obtained using a Bruker Esquire 3000 system. Ultraviolet

spectra were recorded on a Hitachi U1200 spectrophotometer.

Analytical HPLC was performed using a Beckman Coulter system

GOLD 168 variable wavelength detector. The analytical HPLC

columns were Alltech’s Altima C18 column, Vydac analytical C-18

column (both are 4.6 mm × 150 mm 10 μm) and a Chiralcel’s OD 4.6

mm × 250 mm chiral column. Preparative HPLC was performed on R

& S Technology Zonator system equipped with a Hitachi UV detector,

using a self-packed Chiralcel OD 7.5 mm × 50 cm column. Thin layer

chromatography was performed on analytical GF silica gel TLC plates.

Silica gel for flash column chromatography was from Baker. All

solvents used were reagent grade or HPLC grade. All the tested

compounds (21, 22, 23, 24, 25, 26, 27, 38, 39, 40, and 41) were over

95% pure as determined by HPLC.

Synthesis of CBI. 3-N-(tert-Butyloxycarbonyl)amino-1-O-(tert-

butyloxycarbonyl)-1-naphthol (5). Naphthoresorcinol (4) (50.0 g,

0.312 mol) was dissolved in liquid ammonia (200 mL) at −78 °C.

This solution was sealed in a 1 L steel bomb containing a glass linear.

The reaction mixture was kept at 135 10 °C and 1300 psi for 14 h

with vigorous stirring. The vessel was allowed to cool to 60 °C, and

the NH₃was released slowly. The remaining traces of NH₃were

removed by coevaporation of THF (2 × 150 mL) under a stream of

argon at 60 °C. Di-tert-butyl dicarbonate (175 g, 0.801 mol) in dry

THF (300 mL) and N,N-diisopropylethylamine (140 mL, 0.803 mol)

were successively added to the bomb. The bomb was resealed, and the

contents were warmed at 100 °C with stirring for 4 h. The bomb was

cooled to room temperature and opened and the residue partitioned

between saturated aqueous NaCl (800 mL) and EtOAc (500 mL).

The aqueous phase was extracted with EtOAc (200 mL × 2). The

combined organic layers were dried (MgSO₄), filtered, and

concentrated under reduced pressure. Chromatography on SiO₂(1:8

Resolution of (8). The mixture of enantiomers of (8) (1.5 g in 20

mL of EtOAc) were resolved on a HPLC preparative column (7.5 cm

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× 50 cm, packed with Diacel Chiralcel OD gel in our laboratory)

eluted with 15% i-PrOH-hexane at 180 mL/min with an R & S

Technology Zonator system. The enantiomers eluted with retention

times of 18.5 min [8a (+) enantiomer] and 35.8 min [8b (−) natural

(1S) enantiomer]. ent 8b (−)-(1S): [α]²⁵= −49.6° (c = 5.25 CHCl₃).

The resolution is R_s= 2(t_RB− t_RA)/(w_B+ w_A), where t_RB= retention

time of solute B, t_RA= retention time of solute A, w_B= Gaussian curve

width of solute B, w_A= Gaussian curve width of solute A. (see the

Supporting Information data).

5-Hydroxy-3-amino-1-[S]-(chloromethyl)-1,2-dihydro-3H-benz-

(e)indole (9). To a solution of 5-(O-tert-butyloxycarbonyl)oxy-3-[N-

(tert-butyloxycarbonyl)amino-1-[S]-(chloromethyl)-1,2-dihydro-3H-

benz(e)indole (8b) (400 mg, 0.923 mmol) in 10 mL of 1:5 HCl

(conc)/ethyl acetate was added 0.05 mL of triethylsilane. After stirring

for 3 h under Ar, the mixture was diluted with 10 mL of 1:1 CH₂Cl₂/

toluene and evaporated to dryness 252 mg (93%). The dry solid was

again coevaporated three times with CH₂Cl₂/toluene and used directly

for coupling to di-indole compounds without further purification

(∼92% pure). ¹H NMR (DMSO-d₆, 400 MHz) 10.93 (s, 0.8 H), 8.18

(d, 1H, J = 8.6 Hz), 7.92 (d, 1H, J = 8.3 Hz), 7.61 (t, 1H, J = 7.9 + 7.2

Hz), 7.48 (t, 1H, J = 7.6 + 7.4 Hz), 6.86 (s, 1H), 4.27 (br, 1H), 4.04

(dd, 1H, J = 3.1, 11.0 Hz), 3.93−3.79 (m, 3H). MS m/z 234.78 (M +

H)⁺. C₁₃H₁₃ClNO HRMS (C₁₃H₁₃ClNO + H), m/z+ 234.0686, calcd

234.0657 (M + H)⁺.

The Synthesis of DC1 Linkers. General Procedure for

Synthesis of Linkers 12, 13, 14, and 15. To the solution of the

mercapto acids (10) or (11) (20 mmol) in a mixture of 100 mL of 100

mM NaH₂PO₄, pH 7.5, and 30 mL of THF at 0 °C was added the

agents [either methylmethanethiosulfonate (50 mmol) or Aldrithiol-2

(80 mmol) or acetic anhydride (40.0 mmol)] in THF (60 mL). After

addition, the reaction mixture was stirred at 0 °C for 1 h and room

temperature for 1−2 h and concentrated to ∼100 mL in vacuo. The

mixture then was washed twice with CH₂Cl₂(2 × 50 mL), and the

aqueous solution was acidified to pH = 3.0 with 2 M HCl and then

extracted with EtOAc (4 × 100 mL). The EtOAc layers were

combined, dried over MgSO₄, filtered, concentrated, and purified on

silica gel chromatography (ethyl acetate/hexane/acetic acid 3:1:0.05%)

to afford the linkers 12, 13, 14, and 15.

organic layers from the extractions. Drying over MgSO₄, filtration,

concentration in vacuo, and crystallization with THF/ethyl acetate/

hexane afforded 20.5 g (94%) of 5-nitroindole-2-carboxylic acid. H

NMR (DMSO), 11.50 (s, 1H), 7.20 (d, 1H, J = 8.4 Hz), 6.85 (s, 1H),

6.70 (m, 2H); MS m/z−205.2 (M − H). HRMS (C₉H₆N₂O₄− H),

m/z− 205.0262, calcd 205.0249 (M − H)⁻.

1

Ethyl 5-Aminoindole-2-carboxylate (18). A 500 mL Parr hydro-

genation bottle was charged with ethyl 5-nitroindole-2-carboxylate

(16) (5.0 g, 21.36 mmol), Pd/C (0.3 g, 10% of Pd, 50% wet),

CH₃OH/THF (150 mL, 1:4 v/v), and purged with H₂. The reaction

mixture was shaken with 40 psi H₂overnight. The catalyst was

removed by filtration, and the solvent was evaporated to give 4.2 g

(97%) of the title compound as brown solid. ¹H NMR (CDCl₃), 8.77

(s, 1H), 7.26 (s, 1H), 7.23 (t, 1H, J = 0.8 Hz), 7.21 (d, 1H, J = 0.7

Hz), 7.03 (dd, 1H, J = 0.7, 1.5 Hz), 6.93 (dd, 1H, J = 0.7, 1.6 Hz), 6.80

(dd, 1H, J = 2.2, 8.6 Hz), 4.38 (dd, 2H, J = 7.2, 14.3 Hz), 1.40 (t, 3H, J

= 7.2 Hz). ¹³C NMR (CDCl₃) 162.02, 140.30, 138.14, 131.87, 128.45,

127.77, 117.12, 112.50, 107.36, 105.86, 60.87, 14.41. MS m/z− 205.20

(M + H). HRMS m/z+ 227.0791 (M + Na)⁺, calcd 227.0797. This

product is unstable and it was directly used for next step.

Ethyl 5-(5′-Nitroindol-2′-yl-carbonyl amino)indole-2-carboxylate

(19). To a mixture of 5-nitroindole-2-carboxylic acid (17) (1.02 g, 5.00

mmol) and ethyl 5-aminoindole-2-carboxylate (18) (1.02 g, 4.95

mmol) in DMA (30 mL) were added TBTU (4.00 g, 12.40 mmol) and

DIPEA (0.3 mL, 1.72 mmol) under Ar. The reaction mixture was

stirred overnight, concentrated, and the mixture was diluted with 30

mL of ethyl acetate and 150 mL of NaHCO₃(satd), and the solid was

suspended between the two layers. The solid compound was filtered,

washed with water, and then resuspended with 1 M NaH₂PO₄, pH 3.0,

filtered, washed again with water and 10% methanol in water, dried

under oil pump vacuum to afford 1.55 g (80%) of the title compound

(19). R_f= 0.31 (1:2 THF/hexane). ¹H NMR (DMSO), 12.45 (s, 1H),

11.90 (s, 1H), 10.43 (s, 1H), 8.77 (d, 1H, J = 1.9 Hz), 8.15 (s, 1H),

8.13 (dd, 1H, J = 2.2, 9.1 Hz), 7.70 (s, 1H), 7.61 (m, 2H), 7.46 (d, 1H,

J = 8.9 Hz), 7.18 (s, 1H), 4.35 (dd, 2H, J = 7.1, 14.1 Hz), 1.35 (t, 3H, J

= 7.1 Hz). ¹³C NMR (DMSO), 161.22, 158.68, 141.32, 139.50,

135.37, 134.60, 131.47, 128.01, 126.56, 126.38, 119.92, 119.27, 118.59,

113.27, 112.87, 112.60, 107.77, 105.69, 60.43, 14.31. MS m/z 443.85

(M + Na)⁺. HRMS m/z + 415.1012 (M + Na)⁺, calcd 415.1019.

5-(5′-Nitroindol-2′-yl-carbonyl amino)indole-2-carboxylic Acid

(20). To ethyl 5-(5′-nitroindol-2′-yl-carbonyl amino)indole-2-carbox-

ylate (19) (1.26 g, 3.20 mmol) in 35 mL of DMA was added 1.0 g of

NaOH in 10 mL of H₂O. After stirring for 1 h, the mixture was

concentrated and coevaporated three times with 10 mL of H₂O at 40

°C. The solution was diluted with cold CH₂OH and H₂O, adjusted pH

to 3 with HCl (conc), and a solid was precipitated. The solid was

filtered, washed with 10% methanol in water, and dried under oil-

pump vacuum to afford 1.06 g (90%) of the title compound (20). ¹H

NMR (DMSO), 12.48 (s, 1H), 11.75 (s, 1H), 10.44 (s, 1H), 8.77 (s,

1H), 8.15 (s, 1H), 8.10 (d, 1H, J = 9.3 Hz), 7.69 (s, 1H), 7.60 (m,

2H), 7.44 (d, 1H, J = 8.9 Hz), 7.10 (s, 1H). ¹³C NMR (DMSO),

161.91, 158.66, 141.32, 139.52, 135.45, 134.44, 131.26, 128.01, 126.72,

126.39, 119.47, 119.25, 118.02, 113.24, 112.88, 112.48, 107.23, 105.71.

ESI MS m/z 386.66 387.85 (M + Na)⁺. HRMS m/z+ 387.0697 (M +

Na)⁺, calcd 387.0706.

3-(Methyldithio)propionic Acid (12). Yield 2.61 g (86%); R_f= 0.31

(1:100:300 HOAc/EtOAc/hexane). ¹H NMR (CDCl₃), 11.23 (b,

1H), 2.94 (m, 2H), 2.82 (m, 2H), 2.41 (s, 3H). ¹³C NMR, 178.34,

33.99, 31.95, 23.17. MS m/z 153.38 (M + H)⁺. HRMS (C₄H₈O₂S₂+

Na), m/z+ 174.9845, calcd 174.9863 (M + Na)⁺.

1

3-(Pyridin-2-yldisulfanyl)propanoic Acid (13). 3.42 g (80%); H

NMR (CD₃COCD₃), 8.45 (ddd, 1H, J = 1.3, 2.7, 4.8 Hz), 7.80 (m,

2H), 7.21 (m, 1H), 3.09 (t, 2H, J = 6.9 Hz), 2.55 (t, 2H, J = 6.9 Hz),

¹³C NMR, 172.66, 160.12, 150.46, 138.23, 121.81, 120.10, 34.57,

33.87; MS m/z 238.4 (M + Na)⁺; HRMS (C₈H₉O₂S₂+ Na), m/z+

237.9972, calcd 237.9950 (M + Na)⁺.

1

3-(Acetylthio)propanoic Acid (14). Yield 2.60 g (88%). H NMR

(CDCl₃), 11.90 (b, 1H), 3.04 (t, 2H, J = 7.0 Hz), 2.68 (t, 2H, J = 6.9

Hz), 2.31 (s, 3H). ¹³C NMR, 190.10, 173.34, 32.19, 31.25, 30.09. MS

m/z 171.8 (M + Na)⁺. HRMS (C₅H₈O₃S + Na), m/z+ 171.0092, calcd

171.0068 (M + Na)⁺.

4-Methyl-4-(methyldisulfanyl)pentanoic Acid (15). Yield 3.22 g

(83%); R_f= 0.3 (1:200:400 HOAc/EtOAc/hexane). ¹H NMR

(CDCl₃), 2.48 (m, 2H), 2.41 (s, 3H), 1.95 (m, 2H), 1.31 (s, 6H).

¹³C NMR, 179.15, 50.52, 35.73, 29.93, 25.21, 18.47. MS m/z− 193.1

1-[S]-(Chloromethyl)-5-hydroxy-3-{{5-[5′-nitroindol-2′-yl-carbon-

yl amino]indole-2-carbonyl}-1,2-dihydro-3H-benz[e]indole (21). To

a solution of 5-hydroxy-3-amino-1-[S]-(chloromethyl)-1,2-dihydro-

3H-benz(e)indole, hydrochloride salt (9) (200 mg, 0.74 mmol),

[fresh prepared from 5-(O-tert-butyloxycarbonyl)oxy-3-[N-(tert-

butyloxycarbonyl)amino-1-[S]-(chloromethyl)-1,2-dihydro-3H-benz-

(e)indole, (7)] and 5-[5′-nitroindol-2′-yl-carbonyl amino]indole-2-

carboxylic acid (20) (250 mg, 0.69 mmol) in 30.0 mL of DMA was

added EDC (400 mg, 0.20 mmol) under Ar. After stirring overnight,

two drops of 50% acetic acid were added to the mixture and the

mixture was evaporated to dryness, purified on silica gel chromato-

graphic column (40% THF in toluene) to afford 301 mg (75%) of

(M − H). HRMS (C₇H₁₄O₂S₂+ Na), m/z+ 217.0333, calcd

217.03358 (M + Na)⁺.

The Synthesis of Bis-indole Component of DC1. 5-Nitro-

indole-2-carboxylic Acid (17). To a stirred solution of ethyl-5-

nitroindole-2-carboxylate (16) (25.0 g, 106.8 mmol) in 500 mL of

THF-methanol (1:1, v/v) at room temperature was added a solution

of NaOH (40 g, 1.0 mmol) in 300 mL of water. The resulting deep-

red−brown solution was stirred for 3 h and then quenched by

acidification to pH 1 with dilute HCl. The precipitated product was

collected by vacuum filtration, and the remaining dissolved product

was extracted with THF/ethyl acetate (1:2, v/v, 2 × 400 mL). The

precipitate was dissolved in THF, and its solution was combined with

1

DC0NO₂(21). H NMR (DMF-d₇) 12.54 (s, 1H), 11.73 (s, 1H),

10.60 (s, 1H), 10.58 (s, 1H), 8.80 (d, 1H, J = 2.3 Hz), 8.42 (d, 1H, J =

1.9 Hz), 8.25 (d, 1H, J = 8.5 Hz), 8.19 (dd, 1H, J = 2.1, 9.1 Hz), 8.09

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(br, 1H), 7.95 (d, 1H, J = 8.3 Hz), 7.82 (d, 1H, J = 1.5 Hz), 7.79 (d,

1H, J = 9.1 Hz), 7.74 (dd, 1H, J = 2.0, 8.9 Hz), 7.62 (d, 1H, J = 8.8

Hz), 7.58 (dt, 1H, J = 1.7, 7.0 + 7.0 Hz), 7.42 (dt, 1H, J = 1.2, 7.0 + 7.0

Hz), 7.33 (d, 1H, J = 1.7 Hz), 4.91 (t, 1H, J = 11.0 Hz), 4.77 (dd, 1H, J

= 2.1, 11.1 Hz), 4.33 (m, 1H), 4.13 (dd, 1H, J = 3.1, 11.1 Hz), 3.97

(dd, 1H, J = 7.9, 11.1 Hz). ¹³C NMR 163.35, 161.48, 160.05, 155.79,

142.98, 137.18, 135.03, 133.22, 133.16, 131.50, 128.85, 128.45, 128.11,

124.62, 124.02, 123.76, 120.33, 119.36, 118.70, 116.45, 114.00, 113.08,

106.97, 105.02, 101.53. MS m/z 602.96 (M + Na)⁺, 604.78, 603.81,

618.64 (M + K)⁺, 620.48. HRMS m/z+ 602.1215 (M + Na)⁺, calcd

602.1207.

196.07, 170.01, 162.85, 161.51, 160.86, 155.77, 143.91, 135.09, 134.86,

133.93, 133.55, 133.02, 131.48, 128.84, 128.77, 128.42, 124.41, 123.89,

123.75, 120.26, 116.45, 113.92, 113.32, 113.28, 112.54, 106.92, 104.72,

101.63, 56.38, 48.53, 43.09, 38.65, 29.60. MS m/z 702.66 (M + Na)⁺.

HRMS C₃₆H₃₀ClN₅O₅S 702.1542 (M + Na), calcd 702.1554.

1-[S]-(Chloromethyl)-5-hydroxy-3-{{5-[5′-(4″-methyldithio-3″,3″-

dimethyl butyryl)indol-2′-yl-carbonyl amino]indole-2-yl}carbonyl}-

1,2-dihydro-3H-benz[e]indole (26) (DC41SMe). Yield 52.5 mg (65%);

R_f= 0.40 (2:3 THF/toluene). ¹H NMR (DMF-d₇) 11.68 (dd, 2H, J =

1.6, 6.7 Hz), 10.58 (s, 1H), 10.25 (s, 1H), 9.92 (s, 1H), 8.41 (d, 1H, J

= 1.9 Hz), 8.25 (d, 1H, J = 8.0 Hz), 8.22 (d, 1H, J = 1.8 Hz), 8.10 (s,

1H), 7.94 (d, 1H, J = 8.3 Hz), 7.72 (dd, 1H, J = 2.0, 8.8 Hz), 7.60−

7.52 (m, 3H), 7.48−7.39 (m, 3H), 7.29 (d, 1H, J = 1.7 Hz), 4.89 (dd,

1H, J = 8.7, 11.0 Hz), 4.75 (dd, 1H, J = 2.0, 10.9 Hz), 4.31 (m, 1H),

4.13 (dd, 1H, J = 3.3, 11.0 Hz), 3.96 (dd, 1H, J = 7.8, 11.1 Hz), 2.53

(m, 2H), 2.48 (s, 3H), 2.03 (m, 2H), 1.34 (s, 6H). ¹³C NMR 171.72,

163.30, 161.45, 160.83, 155.72, 143.85, 135.04, 134.81, 134.04, 133.50,

132.96, 131.42, 128.80, 128.75, 128.38, 124.36, 124.13, 123.83, 123.70,

120.21, 118.77, 116.38, 113.88, 113.30, 113.25, 112.47, 108.52, 106.87,

104.20, 101.71, 67.74, 56.33, 48.48, 42.99, 37.67, 33.51, 24.78 21.93.

MS m/z+ 764.0 (M + K)⁺, 748.0 (M + Na)⁺, 728.1 (M + K − Cl)⁺,

712.1(M + Na − Cl)⁺; m/z− 724.1 (M − H), 688.2 (M − Cl − H).

HRMS C₃₈H₃₆ClN₅O₄S₂748.1784 (M + Na), calcd 748.1795.

General Procedure for DC1−SR2 (23, 24, 25, 26,). A flask was

charged with 1-[S]-(chloromethyl)-5-hydroxy-3-{{5-[5′-nitroindol-2′-

yl-carbonyl amino]indole-2-yl}carbonyl}-1,2-dihydro-3H-benz[e]-

indole (21) (∼58 mg, 0.1 mmol), Pd/C (30 mg, 10% Pd, 50%

wet), HCl (conc) (15 μL), and DMA (25 mL). After the air was

removed by vacuum suction, H₂was conducted through hydrogen

balloon overnight. The mixture was filtered through Celite and the

solution was evaporated to give ∼50 mg (∼90%) of a brown solid

(22), which was used directly without further purification. To this

solid amine compound in 10 mL of DMA was added 0.12 mmol of the

linker (either 12 or 13 or 14 or 15) and 0.35 mmol of EDC under Ar.

After stirred overnight, two drops of 50% acetic acid was added, the

mixture was evaporated to dryness, and purified by preparative silica

gel TLC (10% DMA, 30% THF, 60% toluene) to afford DC₁−SR₂.

1 - [ S ] - ( C h l o r o m e t h y l ) - 5 - h y d r o x y - 3 - { { 5 - [ 5 ′ - ( 3 ″ -

methyldithiopropionyl)indol-2′-yl-carbonyl amino]indole-2-yl}-

carbonyl}-1,2-dihydro-3H-benz[e]indole (23) (DC1SMe). Yield 48.1

5-(3-(Methyldisulfanyl)propanamido)-N-(2-((9aS)-4-oxo-2,4,9,9a-

tetrahydro-1H-benzo[e]cyclopropa[c]indole-2-carbonyl)-1H-indol-

5-yl)-1H-indole-2-carboxamide (27) (DC10SMe). 1-[S]-(Chloro-

methyl)-5-hydroxy-3-{{5-[5′-(3″-methyldithiopropionyl)indol-2′-yl-

carbonyl amino]indole-2-yl}carbonyl}-1,2-dihydro-3H-benz[e]indole

(23) (15 mg, 21.9 mmol) in THF (8 mL) was 5% NaHCO₃(2

mL). After stirred at room temperature for 1 h, the mixture was diluted

with EtOAc, and the aqueous phase separated and extracted twice with

EtOAc/THF (2:1). The organic layers were combined, dried

(Na₂SO₄), filtered, concentrated, and purified on short SiO₂column

eluted with THF/CH₂Cl₂(1:2 to 1:1) to afford 11.5 mg (83% yield)

1

mg (63%); R_f= 0.40 (3:7 acetone/toluene). H NMR (CD₃COCD₃)

10.91 (s, 1H), 10.88 (s, 1H), 9.64 (s, 1H), 9.56 (s, 1H), 9.27 (s, 1H),

8.35 (d, 1H, J = 1.9 Hz), 8.25 (d, 1H, J = 8.0 Hz), 8.17 (d, 1H, J = 1.9

Hz), 8.07 (s, 1H), 7.88 (d, 1H, J = 8.3 Hz), 7.64 (dd, 1H, J = 2.0, 8.1

Hz), 7.58−7.50 (m, 3H), 7.38−7.35 (m, 2H), 7.31 (d, 1H, J = 1.7 Hz),

7.26 (d, 1H, J = 1.7 Hz), 4.86 (dd, 1H, J = 8.7, 11.0 Hz), 4.80 (dd, 1H,

J = 2.3, 10.9 Hz), 4.30 (m, 1H), 4.07 (dd, 1H, J = 3.1, 11.0 Hz), 3.83

(dd, 1H, J = 8.4, 11.2 Hz), 3.09 (t, 2H, J = 7.1 Hz), 2.83 (t, 2H, J = 7.1

Hz), 2.45 (s, 3H). ¹³C NMR 169.56, 161.10, 160.43, 155.13, 143.50,

134.78, 134.46, 133.55, 133.34, 133.03, 132.57, 131.21, 128.80, 128.69,

128.21, 124.22, 124.02, 123.53, 123.44, 120.16, 118.79, 116.45, 113.91,

113.02, 112.95, 112.73, 106.78, 103.72, 101.63, 56.01, 47.73, 43.10,

37.25, 34.01, 23.00. MS m/z 706.66 (M + Na)⁺. HRMS

C₃₅H₃₀ClN₅O₄S₂706.1306 (M + Na), calcd 706.1325

1

of the title compound. H NMR (DMF-d₇) 11.69 (s, 1H), 11.67 (s,

1H), 10.58 (s, 1H), 10.24 (s, 1H), 9.92 (s, 1H), 8.40 (d, 1H, J = 1.9

Hz), 8.25 (d, 1H, J = 8.3 Hz), 8.22 (d, 1H, J = 1.8 Hz), 8.10 (s, 1H),

7.95 (d, 1H, J = 8.3 Hz), 7.73 (dd, 1H, J = 2.0, 8.1 Hz), 7.61−7.52 (m,

3H), 7.47 (d, 1H, J = 1.8 Hz), 7.45−7.39 (m, 2H), 7.20 (s, 1H), 4.75

(dd, 1H, J = 2.1, 10.7 Hz), 3.98−3.95 (m, 1H), 3.83−3.78 (m, 1H),

2.94 (t, 2H, J = 7.1 Hz), 2.75 (m, 1H), 2.58 (t, 2H, J = 7.1 Hz), 2.46

(s, 3H), 1.55 (m, 1H), 1.27 (m, 1H). ¹³C NMR 201.37, 171.03,

162.80, 161.82, 160.90, 156.77, 144.96, 135.11, 134.88, 133.96, 133.77,

133.62, 131.49, 128.89, 128.80, 128.52, 124.42, 123.91, 123.77, 120.27,

117.05, 113.93, 113.37, 113.29, 112.55, 106.93, 104.73, 101.65, 58.30,

43.09, 38.69, 37.55, 29.60, 27.90, 21.02. MS m/z 670.40 (M + Na)⁺.

HRMS C₃₅H₂₉N₅NaO₄S₂670.1672 (M + Na), calcd 670.1559.

t-Butyl 5-Nitroindole-2-carboxylate(28). To a stirred solution of 5-

nitroindole-2-carboxylic acid (17) (12.8 g, 61.2 mmol) in 200 mL of

dry THF under Ar was added oxalyl chloride (12.0 mL, 137.5 mmol),

followed by 0.1 mL of DMF, which caused a vigorous evolution of gas.

After stirring for 40 min, the reaction mixture was evaporated to

dryness. The resulting solid was redissolved in 150 mL of dry THF

and cooled at ∼30 °C under Ar. A solution of potassium t-butoxide

(1.0 M in THF, 140 mL, 140 mmol) was then added dropwise over 45

min, and stirring was continued for an additional 45 min. The reaction

was quenched with 600 mL of water, neutralized with few drops of

H₃PO₄, and extracted with ethyl acetate (3 × 400 mL). The organic

extracts were washed with saturated aqueous NaHCO₃and water, and

then dried over MgSO₄, filtered, concentrated, and crystallized with

ethanol/hexane to afford 9.62 g (85%) of the title compound (28). R_f

1-[S]-(Chloromethyl)-5-hydroxy-3-{{5-[5′-(3″-pyridi-2′-yl-

dithiopropionyl)indol-2′-yl-carbonyl amino]indole-2-yl}carbonyl}-

1,2-dihydro-3H-benz[e]indole (24) (DC1SPy). Yield 50.6 mg (61%),

1

R_f= 0.35 (1:2 THF/toluene). H NMR (DMF-d₇) 11.87 (br, 1H),

11.68 (s, 2H), 10.58 (s, 1H), 10.25 (s, 1H), 10.04 (s, 1H), 8.51 (m,

1H), 8.40 (d, 1H, J = 1.8 Hz), 8.25 (d, 1H, J = 7.9 Hz), 8.20 (d, 1H, J

= 1.9 Hz), 8.10 (s, 1H), 7.94 (d, 1H, J = 8.3 Hz), 7.86 (m, 2H), 7.71

(dd, 1H, J = 2.0, 8.1 Hz), 7.60 − 7.52 (m, 3H), 7.48 (d, 1H, J = 1.5

Hz), 7.42 (m, 2H), 7.30−7.26 (m, 2H), 4.90 (dd, 1H, J = 8.7, 11.0

Hz), 4.75 (dd, 1H, J = 2.3, 10.9 Hz), 4.32 (m, 1H), 4.13 (dd, 1H, J =

3.2, 11.0 Hz), 3.96 (dd, 1H, J = 8.4, 11.2 Hz), 3.24 (t, 2H, J = 7.1 Hz),

2.80 (t, 2H, J = 7.1 Hz). ¹³C NMR 169.70, 161.50, 160.95, 160.85,

155.76, 150.78, 143.91, 138.78, 135.14, 134.86, 134.14, 133.72, 133.02,

129.30, 128.84, 128.78, 128.43, 124.41, 124.18, 123.88, 123.74, 122.20,

120.32, 116.43, 113.92, 113.38, 113.29, 112.63, 106.92, 104.26, 101.75,

56.37, 48.53, 43.04, 36.97, 35.57. MS m/z 748.6 (M + H)⁺. HRMS

C₃₉H₃₁ClN₆O₄S₂747.1611 (M + H)⁺, calcd 747.1615.

1 - [ S ] - ( C h l o r o m e t h y l ) - 5 - h y d r o x y - 3 - { { 5 - [ 5 ′ - ( 3 ″ -

acetylthiopropionyl)indol-2′-yl-carbonyl amino]indole-2-yl}-

carbonyl}-1,2-dihydro-3H-benz[e]indole (25) (DC1SAc). Yield 47.2

1

= 0.35 (1:5 ethyl acetate/hexane). H NMR (CDCl₃), 11.63 (s, 1H),

1

mg (62%); R_f= 0.38 (1:2 THF/toluene). H NMR (DMF-d₇) 11.68

8.66 (dd, 1H, J = 0.5, 1.3 Hz), 8.20 (dd, 1H, J = 0.5, 9.0 Hz), 7.48 (dd,

1H, J = 0.5, 9.1 Hz), 7.28 (dd, 1H, J = 0.9, 11.1 Hz), 1.63 (s, 9H). ¹³C

NMR 160.39, 142.12, 138.11, 132.10, 126.78, 120.22, 119.83, 111.98,

109.82, 82.91, 28.26. ESI MS m/z 285.43 (M + Na)⁺. HRMS

C₁₃H₁₄N₂O₄285.0859 (M + Na), calcd 285.0851.

t-Butyl 5-Aminoindole-2-carboxylate (29). A 500 mL of Parr

hydrogenation bottle was charged with t-butyl 5-nitroindole-2-

carboxylate (28) (5.80 g, 22.14 mmol), Pd/C (0.6 g, 10% Pd, 50%

(s, 1H), 11.67 (s, 1H), 10.58 (s, 1H), 10.25 (s, 1H), 9.88 (s, 1H), 8.37

(d, 1H, J = 1.9 Hz), 8.25 (m, 1H), 8.22 (d, 1H, J = 1.9 Hz), 8.10 (s,

1H), 7.94 (d, 1H, J = 8.3 Hz), 7.73 (dd, 1H, J = 2.0, 8.1 Hz), 7.60−

7.50 (m, 3H), 7.45−7.39 (m, 3H), 7.30 (d, 1H, J = 1.7 Hz), 4.90 (dd,

1H, J = 8.7, 11.0 Hz), 4.77 (dd, 1H, J = 2.1, 10.7 Hz), 4.32 (m, 1H),

4.14 (dd, 1H, J = 3.1, 11.0 Hz), 3.96 (dd, 1H, J = 8.4, 11.2 Hz), 3.07 (t,

2H, J = 7.1 Hz), 2.68 (t, 2H, J = 7.1 Hz), 2.35 (s, 3H). ¹³C NMR

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1

wet), THF (150 mL), and purged with H₂. The reaction mixture was

shaken with 50 psi H₂overnight. The catalyst was removed by

filtration, and the solvent was evaporated to give 4.98 g (97%) of the

toluene). H NMR (DMF-d₇), 11.71 (s, 1H), 11.64 (s, 1H), 10.21 (s,

1H), 9.91 (s, 1H), 8.29 (d, 1H, J = 2.0 Hz), 8.20 (d, 1H, J = 1.8 Hz),

7.72 (dd, 1H, J = 2.0, 8.9 Hz), 7.51 (dd, 2H, J = 0.76, 9.0 Hz), 7.51

(dd, 1H, J = 0.76, 9.0 Hz), 7.44 (m, 2H), 7.12 (dd, 1H, J = 0.8, 2.0

Hz), 2.53 (m, 2H), 2.48 (s, 3H), 2.02 (m, 2H), 1.60 (s, 9H), 1.33 (s,

6H). ¹³C NMR 171.32, 162.91, 161.54,, 160.43, 135.32, 134.65,

133.62, 133.57, 133.22, 130.63, 128.36, 127.87, 120.16, 118.38, 113.34,

113.05, 112.91, 112.08, 108.03, 103.82, 81.56, 51.51, 37.30, 33.12,

28.31, 27.74, 25.10. MS m/z+ 589.1 (M + Na)⁺, 605.1 (M + K)⁺. m/

z− 565.3 (M − H)⁻. HRMS for C₂₉H₃₄N₄O₄S₂, 589.1932 (M + Na),

calcd 589.1919.

1

title compound (29) as brown solid. H NMR (DMSO), 11.42 (s,

1H), 7.18 (d, 1H, J = 8.3 Hz), 6.83 (s, 1H), 6.71 (s, 1H), 6.67 (d, 1H, J

= 8.4 Hz), 1.62 (s, 9H). ESI MS m/z 255.40 (M + Na)⁺. This product

is unstable and it was directly used for next step.

t-Butyl 5-(5′-Nitroindol-2′-yl-carbonyl amino)indole-2-carboxy-

late (30). To the solution of 5-nitroindole-2-carboxylic acid (17) (4.50

g, 21.81 mmol) and t-butyl 5-aminosindole-2-carboxylate (29) (4.98 g,

21.45 mmol) in 200 mL of DMA were added TBTU (10.5 g, 32.70)

and DIPEA (2.0 mL, 45.83 mmol) of under Ar. The reaction mixture

was stirred overnight, concentrated, diluted with ethyl acetate and

NaHCO₃(satd) and a suspended solid was formed. The solid

compound was filtered, washed with water, and then resuspended with

1 M NaH₂PO₄, pH 3.0, filtered, washed with water and 10% methanol

in water again, and dried over oil vacuum pump to afford 8.40 g (89%)

5-[5′-(3″-Methyldithiopropionyl)indol-2′-yl-carbonyl amino]-

indole-2-carboxylic Acid (34). A mixture of t-butyl 5-[5′-(3″-

methyldithiopropionyl)indol-2′-yl-carbonyl amino]indole-2-carboxy-

late (32) (300 mg, 0.57 mol) and Et₃SiH (20 μL, 0.12 mmol) in 30

mL of CH₂Cl₂was added 7.0 mL of TFA and the mixture to become

clear solution. After stirring for 0.5 h, the reaction mixture was diluted

with 25 mL of toluene. The mixture was evaporated to dryness and

crystallized with THF/toluene/hexane to yield of 245 mg (92%) of the

1

of the title compound (30). R_f= 0.31 (1:2 THF/hexane). H NMR

(DMSO), 12.43 (s, 1H), 11.69 (s, 1H), 10.41 (s, 1H), 8.77 (d, 1H, J =

2.2 Hz), 8.13 (dd, 2H, J = 2.3, 9.0 Hz), 7.64 (t, 2H, J = 9.2 Hz), 7.47

(d, 1H, J = 8.9 Hz), 7.08 (s, 1H), 1.59 (s, 9H). ¹³C NMR (DMSO),

161.48, 159.53, 142.19, 140.38, 136.30, 135.27, 132.28, 130.30, 127.43,

127.25, 120.57, 120.12, 114.08, 113.74, 108.22, 106.64, 81.74, 28.84.

ESI MS m/z 443.85 (M + Na)⁺. HRMS C₂₂H₂₀N₄O₅443.1314 (M +

Na), calcd 443.1331.

t-Butyl 5-(5′-Aminoindole-2′-yl-carbonyl amino)indole-2-carbox-

ylate (31). A 250 mL of Parr hydrogenation bottle was charged with t-

butyl 5-(5′-nitroindol-2′-yl-carbonyl amino)indole-2-carboxylate (30)

(2.40 g, 5.71 mmol), Pd/C (0.3 g, 10% Pd, 50% wet), DMA (50 mL),

and purged with hydrogen. The reaction mixture was shaken with 40

psi H₂overnight, filtrated through Celite, and evaporated over an oil

pump to give 2.05 g (92%) of the title compound (31) as brown solid.

¹H NMR (DMSO), 11.75, (s, 1H), 11.67 (s, 1H), 10.17 (s, 1H), 8.10

1

title compound (34). H NMR (DMSO), 11.71 (s, 1H), 11.61 (s,

1H), 10.10 (s, 1H), 9.92 (s, 1H), 8.11 (d, 1H, J = 1.9 Hz), 8.02 (d, J =

1.7 Hz), 7.55 (dd, 1H, 2.0, 11.0 Hz), 7.42 (d, 1H, J = 8.8 Hz), 7.39 (d,

1H, J = 8.8 Hz), 7.34 (d, 1H, J = 2.0 Hz), 7.31 (dd, 1H, J = 2.0, 8.8

Hz), 7.08 (d, 1H, J = 1.3 Hz), 3.06 (t, 2H, J = 7.0 Hz), 2.75 (t, 2H, J =

7.0 Hz), 2.45 (s, 3H). ¹³C NMR (DMSO), 168.70, 162.79, 159.47,

134.37, 133.56, 132.44, 131.98, 131.64, 126.96, 126.75, 119.62, 117.74,

113.04, 112.46, 112.35, 111.44, 107.36, 103.37, 36.03, 33.01. MS

490.81 (M + Na)⁺. HRMS for C₂₂H₂₀N₄O₄S₂491.0810, cal: 491.0824.

5-[5′-(4″-Methyldithio-3″,3″-dimethyl butyryl)indol-2′-yl-carbon-

yl amino]indole-2-carboxylic Acid (35). A mixture of t-butyl 5-[5′-

(4″-Methyldithio-3″,3″-dimethyl butyryl)indol-2′-yl-carbonyl amino]-

indole-2-carboxylate (33) (150 mg, 0.26 mmol) and Et₃SiH (5 μL

0.031 mmol) in CH₂Cl₂(21 mL) was added 5.0 mL of TFA and the

mixture became clear. After stirring for 1 h, the reaction mixture was

diluted with 25 mL of toluene. The mixture was evaporated to dryness

and crystallized with THF/toluene/hexane to yield of 125 mg (90%)

(d, 1H, J = 1.2 Hz), 7.59 (t, 2H, J = 8.8 Hz), 7.45 (m, 1H), 7.35 (m,

1H), 7.17 (dd, 1H, J = 0.8, 8.0 Hz), 7.06 (d, 1H, J = 2.0 Hz), 1.57 (s,

9H). ESI MS m/z 413.40 (M + Na)⁺. HRMS for C₂₂H₂₂N₄O₃,

413.1598 (M + Na), calcd 413.1590. This product is unstable and it

was directly used for next step.

1

of the title compound (35). H NMR (DMF-d₇), 13.17 (br, 0.75H,

COOH), 11.70 (s, 1H), 11.64 (s, 1H), 10.21 (s, 1H), 9.91 (s, 1H),

8.31 (d, 1H, J = 1.6 Hz), 8.20 (d, J = 1.0 Hz), 7.71 (dd, 1H, J = 2.0, 8.9

Hz), 7.52 (dd, 1H, J = 8.0, 9.7 Hz), 7.46 (d, 1H, J = 1.5 Hz), 7.42 (dd,

1H, J = 1.9, 8.8 Hz), 7.13 (dd, 1H, J = 1.3 Hz), 3.06 (t, 2H, J = 7.0

Hz), 2.54 (m, 2H), 2.50 (s, 3H), 2.03 (m, 2H), 1.33 (s, 6H); ¹³C

NMR (DMSO), 171.32, 163.50, 162.91, 160.42, 135.46, 134.65,

133.65, 133.57, 133.14, 130.23, 128.36, 128.03, 120.05, 118.37, 113.43,

113.07, 112.91, 112.08, 108.10, 103.81, 67.93, 51.51, 37.30, 33.12,

27.74, 25.11; MS m/z- 509.1 (M-H)⁻, m/z+ 550.6 (M + K + H)⁺;

HRMS for C₂₅H₂₆N₄O₄S₂511.1466 (M + H)⁺, cal: 511.1474.

1 - [ S ] - ( C h l o r o m e t h y l ) - 5 - h y d r o x y - 3 - { { 5 - [ 5 ′ - ( 3 ″ -

methyldithiopropionyl)indol-2′-yl-carbonyl amino]indole-2-yl}-

carbonyl}-1,2-dihydro-3H-benz[e]indole (23) (DC1SMe). To a

solution of 5-hydroxy-3-amino-1-[S]-(chloromethyl)-1,2-dihydro-3H-

benz(e)indole, hydrochloride salt (9) (55 mg, 0.20 mmol) and 5-[5′-

(3″-methyldithiopropionyl)indol-2′-yl-carbonyl amino]indole-2-car-

boxylic acid (34) (100 mg, 0.21 mmol) in 7.0 mL of DMA was

added EDC (120 mg, 0.62 mmol) under Ar. After stirring overnight,

the mixture was evaporated to dryness, purified by silica chromatog-

raphy (25% to 70% THF in toluene) and crystallized with THF/

toluene/hexane to afford 116 mg (85%) of DC1SMe (23). R_f= 0.40

(3:7 acetone/toluene); (the NMR data and HRMS are the same

described above).

1-[S]-(Chloromethyl)-5-hydroxy-3-{{5-[5′-(4″-methyldithio-3″,3″-

dimethyl butyryl)indol-2′-yl-carbonyl amino]indole-2-yl}carbonyl}-

1,2-dihydro-3H-benz[e]indole (26) (DC41SMe). To the dry salt of 5-

hydroxy-3-amino-1-[S]-(chloromethyl)-1,2-dihydro-3H-benz(e)indole,

hydrochloride salt (9) (67.2 mg, 0.25 mmol) was added 5-[5′-(4″-

methyldithio-3″,3″-dimethyl butyryl)indol-2′-yl-carbonyl amino]indole-

2-carboxylic acid (35) (122 mg, 0.239 mmol), 10 mL of DMA and

EDC (220 mg, 1.14 mmol) under Ar. After stirred overnight, the

mixture was evaporated to dryness, purified by silica gel chromatog-

raphy (25% to 70% THF in toluene) and crystallized with THF/

t-Butyl 5-[5′-(3″-Methyldithiopropionyl)indol-2′-yl-carbonyl

amino]indole-2-carboxylate (32). To a solution of t-butyl 5-(5′-

aminoindol-2′-yl-carbonyl amino)indole-2-carboxylate (31) (2.00 g,

5.12 mmol) in 30 mL of DMA was added of 3-(methyldithio)-

propionic acid (12) (0.90 g, 5.92 mmol) and EDC (3.0 g, 15.33

mmol). After stirring under Ar overnight, the mixture was diluted with

70 mL of 1.0 M NaH₂PO₄, pH 6.0, and extracted with THF/ethyl

acetate (1:1, 6 × 100 mL). The organic layers were combined, dried

over MgSO₄, filtered, evaporated, purified with silica gel chromatog-

raphy (1:3 acetone/toluene) and crystallized with THF/hexane to

yield 2.15 g (80%) of the title compound (32). mp = 279−283 (dec),

R_f= 0.31 (1:3 THF/toluene). ¹H NMR (CD₃COCD₃), 10.75 (d, 2H,

J = 3.07 Hz), 9.50 (s, 1H), 9.14 (s, 1H), 8.20 (d, 1H, J = 2.0 Hz), 8.14

(d, 1H, J = 1.8 Hz), 7.62 (dd, 1H, J = 2.0, 8.9 Hz), 7.46 (dd, 2H, J =

0.7, 8.1 Hz), 7.34 (dd, 1H, J = 2.0, 10.8 Hz), 7.26 (d, 1H, J = 1.5 Hz),

7.07 (dd, 1H, J = 0.9, 2.1 Hz), 3.05 (t, 2H, J = 7.1 Hz), 2.76 (t, 2H, J =

7.0 Hz), 2.42 (s, 3H), 1.57 (s, 9H). ¹³C NMR 169.42, 161.58, 160.32,

135.31, 134.76, 133.56, 133.40, 133.12, 130.86, 128.72, 128.27, 120.27,

118.75, 113.69, 113.09, 113.02, 112.69, 108.27, 103.58, 81.66, 37.28,

34.00, 28.41. MS m/z 547.88 (M + Na)⁺. HRMS for C₂₆H₂₈N₄O₄S₂,

547.1457 (M + Na), calcd 547.1450.

t-Butyl 5-[5′-(4″-Methyldithio-3″,3″-dimethyl butyryl)indol-2′-yl-

carbonyl amino]indole-2-carboxylate (33). To a solution of t-butyl

5-(5′-aminoindol-2′-yl-carbonyl amino)indole-2-carboxylate (31) (301

mg, 0.78 mmol) in 30 mL of DMA was added 4-methyldithio-3,3-

dimethyl butyric acid (15) (155 mg, 0.79 mmol) and EDC (206 mg,

1.07 mmol). After stirring under Ar overnight, the mixture was diluted

with 70 mL of 1.0 M NaH₂PO₄, pH 6.0, and extracted with THF/

EtOAc (1:1, 4 × 70 mL). The organic layers were combined, dried

over MgSO₄, filtered, evaporated, purified with silica gel chromatog-

raphy (1:3 THF/toluene), and crystallized with THF/hexane to yield

344 mg (78%) of the title compound (33). R_f= 0.30 (1:3 THF/

778

dx.doi.org/10.1021/jm201284m | J. Med. Chem. 2012, 55, 766−782

Journal of Medicinal Chemistry

Article

toluene/hexane to afford 117 mg (83%) of DC41SMe (26). R_f= 0.40

(2:3 THF/Toluene); (The NMR data and HRMS are the same

described above).

purified on a C-18 chromatography column, eluted with water/THF,

and crystallized with THF/H₂O/CH₃OH to afford 81.5 mg (84%) of

the title compound (DC44SMe, 39). H NMR (DMF-d₇) 11.75 (s,

1

1-[S]-(Chloromethyl)-5-dibenzylphosphonoxy-3-{{5-[5-(3-

methyldithiopropionyl)indol-2-yl-carbonyl amino]indole-2-yl}-

carbonyl}-1,2-dihydro-3H-benz[e]indole (DC4−dibenzylphosphate,

36). To a solution of 1-[S]-(chloromethyl)-5-hydroxy-3-{{5-[5′-(3″-

methyldithiopropionyl)indol-2′-yl-carbonyl amino]indole-2-yl}-

carbonyl}-1,2-dihydro-3H-benz[e]indole (23) (DC1SMe) (50 mg,

0,073 mmol) in 10 mL of THF/CH₃CN (1:1) was added CCl₄(100

μL, 1.036 mmol), DIPEA (55.0 μL, 0.316 mmol), dibenzylphosphite

(0.100 mL, 0.452 mmol), and DMAP (0.2 mg, 0.0016 mmol) under

Ar. After stirring under Ar overnight, the mixture was diluted with 5

mL of 1.0 M NaH₂PO_4,pH 4.0, and 10 mL of ethyl acetate. The

organic layer was separated, and the aqueous solution was extracted

with THF/EtOAc (1:1, 4 × 25 mL). The organic layers were

combined, dried over MgSO₄, filtered, evaporated, and purified with

silica gel chromatography (3:7 acetone/toluene) to afford 62 mg

(89%) of the title compound (36). (R_f= 0.37 acetone/toluene 1:2).

¹H NMR (DMF-d₇) 11.84 (s, 1H), 11.74 (s, 1H), 10.31 (s, 1H), 10.04

(s, 1H), 8.77 (s, 1H), 8.44 (s, 1H), 8.22 (s, 1H), 8.10 (t, 2H, J = 7.3

Hz), 7.74 (dd, 1H, J = 1.7, 8.8 Hz), 7.66−7.61 (m, 2H), 7.55−7.29 (m,

15H), 5.37 (t, 4H, J = 7.5 Hz), 5.01 (m, 2H), 4.84 (dd, 1H, J = 1.9,

10.9 Hz), 4.51 (m, 1H), 4.20 (dd, 1H, J = 3.2, 10.9 Hz), 4.11 (dd, 1H,

J = 6.9, 11.1 Hz), 3.13 (t, 2H, J = 7.2 Hz), 2.87 (t, 2H, J = 7.1 Hz),

2.50 (s, 3H). ¹³C NMR 169.52, 161.18, 160.41, 147.88, 147.20, 136.74,

136.67, 134.68, 134.56, 133.70, 133.36, 133.20, 132.11, 130.83, 129.25,

129.23, 129.21, 129.19, 128.84, 128.78, 128.62, 128.42, 128.32, 127.90,

124.28, 124.22, 123.96, 123.33, 122.87, 120.03, 118.32, 113.51, 112.94,

112.12, 108.42, 106.87, 70.75, 70.69, 67.91, 55.90, 47.96, 42.59, 37.02,

34.04, 23.08. ³¹P NMR −4.49; MS m/z 966.17 (M + Na)⁺, 968.14 (M

+ 2 + Na), 967.17. HMMS m/z for C₄₉H₄₃ClN₅O₇PS₂966.1957, (M +

Na)⁺, calcd 966.1928.

1H), 11.68 (s, 1H), 10.25 (s, 1H), 9.92 (s, 1H), 8.73 (s, 1H), 8.41 (s,

1H), 8.31 (d, 1H, J = 7.6 Hz), 8.20 (s, 1H), 7.71 (d, 1H, J = 8.3 Hz),

7.59 (m, 2H), 7.53−7.43 (m, 4H), 7.31 (s, 1H), 4.94 (m, 1H), 4.80 (d,

1H, J = 10.2 Hz), 4.41 (m, 1H), 4.15 (m, 1H), 4.00 (dd, 1H, J = 7.8,

11.0 Hz), 2.53 (m, 2H), 2.48 (s, 3H), 2.03 (m, 2H), 1.34 (s, 6H). ¹³C

NMR 171.33, 170.31, 161.00, 160.42, 142.93, 134.65, 134.49, 133.68,

133.56, 133.15, 132.38, 130.71, 128.43, 128.35, 128.09, 124.86, 124.43,

123.55, 120.70, 118.36, 112.92, 112.88, 112.07, 107.56, 106.96, 106.64,

103.89, 69.02, 61.30, 50.30, 42.97, 37.29, 33.13, 25.10, 23.35. MS m/z

804.30 (M − H)⁻, 806.30. HMMS m/z for C₃₈H₃₈ClN₅O₇PS₂

804.1461 (M − H)⁻, calcd 804.1482

1-[S]-(Chloromethyl)-5-phosphonoxy-3-{{5-[5-(3-

mercaptopropionyl)indol-2-yl-carbonyl amino]indole-2-yl}-

carbonyl}-1,2-dihydro-3H-benz[e]indole (40, DC4). A solution of

TCEP (30 mg, 0.104 mmol) in 2 mL of H₂O was adjusted to pH 6.5−

7.0 with addition of NaHCO₃powder. To the solution was added 1-

[ S ] - ( c h l o r o m e t h y l ) - 5 - p h o s p h o n o x y - 3 - { { 5 - [ 5 - ( 3 -

methyldithiopropionyl)indol-2-yl-carbonyl amino]indole-2-yl}-

carbonyl}-1,2-dihydro-3H-benz[e]indole (38, DC4SMe) (26 mg,

0.034 mmol) in 3 mL of DMA/H₂O (1:1). After stirring for 2 h

under Ar, a few drops of 10% H₃PO₄were added to adjust pH to ∼4.0.

The mixture was evaporated and purified by preparative HPLC (c18,

20 mm × 250 mm, v = 8.0 mL/min., mobile phase: A, 0.01% acetic

acid in H₂O; B, 50% DMA in CH₃CN (metal free); time table, 0−10′,

5% of B; to 20′, 20% of B; to 50′, 50% of B. The DC4 (40) was eluted

out at 30−35 min. The fractions were pooled, concentrated, and dried

over the oil vacuum pump to yield 22 mg (89%) of the title

1

compound. H NMR (DMF-d₇) 11.76 (s, 1H), 11.69 (s, 1H), 10.26

(s, 1H), 10.02 (s, 1H), 8.78 (s, 1H), 8.41 (s, 1H), 8.29 (d, 1H, J = 8.3

Hz), 8.21 (s, 1H), 7.79 (d, 1H, J = 5.1 Hz), 7.60−7.43 (m, 6H), 7.27

(s, 1H), 4.96 (t, 1H, J = 9.2 Hz), 4.80 (d, 1H, J = 10.6 Hz), 4.42 (m,

1H), 4.23 (dd, 1H, J = 2.2, 9.4 Hz), 4.06 (dd, 1H, J = 7.4, 10.6 Hz),

3.12 (t, 2H, J = 7.1 Hz), 2.88 (t, 2H, J = 7.1 Hz). HMMS m/z for

C₃₄H₂₈ClN₅O₇PS 716.1130 (M − H)⁻, calcd 716.1136.

Alternative Procedure. A solution of 1-[S]-(chloromethyl)-5-

dibenzylphosphonoxy-3-{{5-[5-(3-methyldithiopropionyl)indol-2-yl-

carbonyl amino]indole-2-yl}carbonyl}-1,2-dihydro-3H-benz[e]indole

(36) (20 mg, 0.021 mmol), 5 mL of methylsulfonic acid, thiolphenol

(100 μL, 0.94 mmol) and DTT (15 mg, 0.097 mmol) was stirred 4 h

under Ar. After the reaction was indicated completion by HPLC, the

mixture was diluted with toluene, evaporated, and purified by the

preparative HPLC as above-described to yield 7 mg (45%) of the DC4

(40).

1-[S]-(Chloromethyl)-5-phosphonoxy-3-{{5-[5-(3-

methyldithiopropionyl)indol-2-yl-carbonyl amino]indole-2-yl}-

carbonyl}-1,2-dihydro-3H-benz[e]indole (38, DC4SMe). 1-[S]-

(Chloromethyl)-5-hydroxy-3-{{5-[5′-(3″-methyldithiopropionyl)indol-

2′-yl-carbonyl amino]indole-2-yl}carbonyl}-1,2-dihydro-3H-benz[e]-

indole (23) (DC1SMe) (50 mg, 0.073 mmol) was dissolved in a

mixture of THF (5 mL), CH₃CN (4 mL), and DMA (0.5 mL) under

Ar. To the mixture was added POCl₃(80 μL, 0.858 mmol), DIPEA

(150 μL, 0.862 mmol), and DMAP (5 mg, 0.040 mmol). After stirring

for 2 h, both TLC and HPLC indicated that DC1SMe was completed

consumed. Then 5 mL of 1.0 M NaH₂PO₄, pH 4, was added, and the

mixture was stirred overnight. The mixture was concentrated, purified

with C-18 chromatography column eluted with water/THF, and

crystallized with THF/H₂O/CH₃OH to afford 47 mg (84%) of the

1-[S]-(Chloromethyl)-5-phosphonoxy-3-{{5-[5-(4″-mercapto-

3″,3″-dimethyl butyryl)indol-2-yl-carbonyl amino]indole-2-yl}-

carbonyl}-1,2-dihydro-3H-benz[e]indole (41, DC44). A solution of

TCEP (106 mg, 0.367 mmol) in 5 mL of H₂O was adjusted to pH

6.5−7.0 with addition of NaHCO₃powder. To the solution was added

1-[S]-(chloromethyl)-5-phosphonoxy-3-{{5-[5-(4″-methyldithio-3″,3″-

dimethyl butyryl)indol-2-yl-carbonyl amino]indole-2-yl}carbonyl}-1,2-

dihydro-3H-benz[e]indole (39, DC44SMe) (65 mg, 0.076 mmol) in

10 mL of DMA/H₂O (1:1) followed added 50 mg of DTT. After

stirring for 12 h under Ar, a few drops of 10% H₃PO₄were added to

pH 4.0. The mixture was evaporated, coevaporated 3 times with

DMA/toluene (2:1, 30 mL), and purified with preparative HPLC (c18,

20 mm × 250 mm, v = 8.0 mL/min, mobile phase: A, 0.01% HOAc in

H₂O; B, 50% DMA in CH₃CN; time table, 0−10′, 5% B; to 20′, 20%

B; to 50′, 50% B. The DC44 was eluted out at 30′−35′. The DC44

fractions were pooled, concentrated, and dried over the oil vacuum

1

title compound (DC4SMe, 38). H NMR (DMF-d₇) 11.77 (s, 1H),

11.70 (s, 1H), 10.26 (s, 1H), 10.02 (s, 1H), 8.74 (s, 1H), 8.42 (s, 1H),

8.30 (d, 1H, J = 7.6 Hz), 8.22 (s, 1H), 7.72 (d, 1H, J = 8.3 Hz), 7.59

(m, 2H), 7.55−7.43 (m, 4H), 7.33 (s, 1H), 4.96 (t, 1H, J = 9.8 Hz),

4.81 (d, 1H, J = 10.2 Hz), 4.42 (m, 1H), 4.18 (m, 1H), 4.05 (dd, 1H, J

= 7.8, 11.0 Hz), 3.11 (t, 2H, J = 7.0 Hz), 2.87 (t, 2H, J = 7.1 Hz), 2.49

(s, 3H). ¹³C NMR 169.96, 161.45, 160.85, 143.10, 135.12, 134.93,

134.15, 133.81, 133.58, 132.80, 131.60, 128.86, 128.78, 128.02, 124.50,

124.05, 120.22, 118.61, 113.97, 113.40, 113.32, 112.57, 108.02, 104.27,

56.18, 48.60, 43.21, 37.47, 34.49, 23.53. ³¹P NMR −3.37; ESI MS m/

z⁻762.20 (M − H). HMMS m/z for C₃₅H₂₇ClN₅O₇PS₂762.1021 (M

− H)⁻, calcd 762.1013.

1-[S]-(Chloromethyl)-5-phosphonoxy-3-{{5-[5-(4″-methyldithio-

3″,3″-dimethyl butyryl)indol-2-yl-carbonyl amino]indole-2-yl}-

carbonyl}-1,2-dihydro-3H-benz[e]indole (39, DC44SMe). 1-[S]-

(Chloromethyl)-5-hydroxy-3-{{5-[5′-(4″-methyldithio-3″,3″-dimethyl

butyryl)indol-2′-yl-carbonyl amino]indole-2-yl}carbonyl}-1,2-dihydro-

3H-benz[e]indole (26) (85 mg, 0.117 mmol) in a mixture of 3.5 mL of

THF, 4 mL of CH₃CN at 0 °C was added DIPEA (100 μL, 0.575

mmol) and POCl₃(90 μL, 0.96 mmol) under argon. After stirring for

2.5 h, both TLC and HPLC showed that DC₄₁SMe was completely

consumed. Five mL of 1.0 M NaH₂PO_4,pH 7.0 was added, and the

mixture was stirred at 0 °C overnight. The mixture was concentrated,

1

pump to yield 56 mg (91%) of the title compound. H NMR (DMF-

d₇) 11.75 (s, 1H), 11.68 (s, 1H), 10.25 (s, 1H), 9.92 (s, 1H), 8.73 (s,

1H), 8.41 (s, 1H), 8.31 (d, 1H, J = 7.6 Hz), 8.20 (s, 1H), 7.71 (d, 1H, J

= 8.3 Hz), 7.59 (m, 2H), 7.53−7.43 (m, 4H), 7.31 (s, 1H), 4.94 (m,

1H), 4.80 (d, 1H, J = 10.2 Hz), 4.41 (m, 1H), 4.15 (m, 1H), 4.00 (dd,

1H, J = 7.8, 11.0 Hz), 2.53 (m, 2H), 2.03 (m, 2H), 1.34 (s, 6H). ¹³C

NMR 171.33, 170.31, 161.00, 160.42, 142.93, 134.65, 134.49, 133.68,

133.56, 133.15, 132.38, 130.71, 128.43, 128.35, 128.09, 124.86, 124.43,

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123.55, 120.70, 118.36, 112.92, 112.88, 112.07, 107.56, 106.96, 106.68,

103.89, 69.32, 61.30, 50.30, 42.97, 37.29, 33.13, 25.10. MS m/z 804.1

(M − H)⁻, 805.2, 806.2. HMMS m/z for C₃₇H₃₅ClN₅O₇PS₂758.1642

(M − H)⁻, calcd 758.1605.

Solubility Test. The DC compound (∼1.5 mg) in the appropriate

solvent was sonicated for 45 min, then centrifuged at 1400g for 45

min. The concentration of the compound in the supernatants was

measured by UV/vis spectrometry at 340 nm. At pH 7.0, DC₁−SR₂

(23, 24, 25, and 26) ε = 41500 M⁻¹cm⁻¹; the phosphate prodrugs (38,

39, 40, and 41) ε = 34500 cm⁻¹M⁻¹. For in the buffers containing 5%

or less DMA, stock solutions (50 mM) of the subject compounds (23,

26, 38, 39) in DMA was diluted 20−100 times with the buffers, then

the same sonication, centrifuged, and UV measurement procedure

were followed.

HPLC Analysis. HPLC analysis of the hydrolysis of DC4SMe (0.2

μmol) by an alkaline phosphatase from bovine liver (2 units) in a pH

7.5 phosphate buffer at 37 °C was performed using a Vydac analytical

C-18 column (length, 150 mm; i.d., 4.6 mm; particle size, 10 μm)

operating at 25 °C and at a flow rate of 1 mL/min, eluting with

gradients of water (containing 0.5% acetic acid) (A) and acetonitrile/

DMA (1:1) (B): 0 min, 20% B; 5 min, 20% B; 5−30 min, 20−65% B;

30−35 min, 65−95%. Under these conditions, compounds 38

(DC4SMe), 27, and 23 eluted with retention time of 12, 23, and 27

min, respectively.

HPLC monitoring the hydrolysis of DC4 by a phosphatase was

performed using a Vydac analytical C-18 column (length, 150 mm; i.d.,

4.6 mm; particle size, 10 μm) at a flow rate of 1 mL/min, eluting with

a gradient of 1% acetic acid in water (A) and acetonitrile/DMA (1:1)

(B): 0 min, 5% B; 5 min, 5% B; 5−30 min, 5−75% B.

the conjugates was the same as previous reported.⁹The final

concentration of the conjugates was determined spectrophotometri-

cally using the known extinction coefficients for the antibody (ε_280nm

=

217560 M⁻¹cm⁻¹) and for the DCx compounds (ε_325nm= 33500 M⁻¹

cm⁻¹and ε_280nm= 22500 M⁻¹cm⁻¹) at pH 7.0.

To analyze any residual free DCx compounds in the conjugates, two

aliquots of the conjugates, were either added a solution of N-ethyl

maleimide (NEM) (20 equiv) or a buffer, respectively, and diluted 2−

3-fold with cold acetone. The resulting solutions were vortexed briefly

and placed on dry ice for at least 1 h then centrifuged at 14000g for 45

min. The supernatants were concentrated and analyzed by HPLC. A

internal standard, which either used NEM-adducted DC44

(DC44NEM) when analyzing DC4, or used DC4NEM when

analyzing DC44, or used DC4-acetamide, was added to the samples

for HPLC quantification.

Activation of Prodrugs and the Conjugates by Phospha-

tases. The phosphatases used to test the activation of the prodrugs

were acid phosphatase from prostatic acid phosphatase from bovine

prostate (P6409), prostatic acid phosphatase from bovine semen

(P3147), prostatic acid phosphatase from human semen (P1649),

wheat germ (Sigma P3627), alkaline phosphatase from bovine

(P8361), alkaline phosphatase from bovine intestinal mucosa

(P7640), alkaline phosphatase from bovine kidney (P4653), alkaline

phosphatase from bovine liver (P7034), alkaline phosphatase from calf

intestine (P7923), alkaline phosphatase from canine intestine (P8639),

alkaline phosphatase from porcine intestine mucosa (P4002), alkaline

phosphatase from porcine kidney (P4439), alkaline phosphatase from

rabbit intestine (P2256), alkaline phosphatase from human placenta

(P3895), alkaline phosphatase from calf intestine mucosa (P79390). In

general, 0.2−5.0 unit of above individual phosphatase was added to 0.5

mg of free DC4 or DC44 drug in phosphate-buffered saline, pH 6.5 or

7.5, with 10% DMA. The mixture was incubated at room temperature

or 37 °C for 0−24 h and analyzed by HPLC. Each antibody−DCx

drug conjugate (500 μL) was activated with 1−1.7 units of acid

phosphatase in PBS, pH 6.5 containing 10% DMA. The mixtures were

incubated overnight at 37 °C. A portion (100 μL) of each sample was

removed, mixed with acetone (200 μL), and the DCx moiety was

released by addition of TCEP (4 equiv) and incubation for 1−2 h, and

then analyzed by HPLC.

In Vitro Cytotoxicity Assays. The cell lines used in cytotoxicity

assays were Namalwa (human Burkitt’s lymphoma, ATCC CRL-

1432), Ramos (human Burkitt’s lymphoma), A subclone of the human

acute promyelocytic leukemia HL60 cell line, HL-60/s,⁴³COLO 205

(human colon adenocarcinoma, ATCC CCL-222), and A375 (human

malignant melanoma, ATCC CRL-1619). Cell cultures were

maintained in RPMI 1640 supplemented with 10% heat-inactivated

fetal bovine serum (FBS) in a humidified incubator at 37 °C, 6% CO₂.

The cytotoxicity clonogenic assay was described previously.⁴⁴Briefly,

the test cell lines were plated into 6-well culture dishes at a constant

number of 1000−5000 cells per well and incubated with varying

concentrations (0−3 nM) of a test-agent for 72 h. The medium was

then aspirated from the plates and replaced with fresh medium, and

the cultures were allowed to grow and form colonies for a total of 7−

10 days after plating. Cells were then fixed and stained with 0.2%

crystal violet in 10% formalin/PBS, and the colonies were counted.

Plating efficiency of nontreated cells (medium alone) was determined

by dividing the number of colonies counted by the number of cells

plated. The surviving fraction of cells exposed to a toxic agent was

determined by dividing the number of colonies in wells that were

exposed to the agent by the number of colonies in the control wells.

Antitumor Activity In Vivo. The in vivo efficacy of conjugates of

DC4 (40) and DC44 (41) and their conjugates with the huB4

antibody was evaluated in a human Burkitt’s lymphoma tumor

xenograft model established with Ramos cells. Five-week-old female

CB 17 SCID mice (36 animals, supplied by Taconic Farms, Inc.) were

inoculated subcutaneously in the area under the right shoulder with

Ramos human lymphoma carcinoma cells (5 × 10⁶cells/mouse) in 0.1

mL of serum-free medium. The tumors were grown for 7 days to an

average size of 100 mm³. The animals were then randomly divided into

five groups (six animals per group). The first group of mice served as

HPLC analysis of the target compounds (21, 22, 23, 24, 25, 26, 27,

38, 39, 40, 41) was performed using an Alltech’s Altima C18 column

(length, 150 mm; i.d., 4.6 mm; particle size, 10 μm) at a flow rate of 1

mL/min, eluting with a gradient of 1% acetic acid in water (A) and

acetonitrile/DMA (1:1) (B): 0 min, 15% B; 5 min, 15% B; 5−30 min,

15−80% B; 30−35 min, 80−95% B.

Preparation of Antibody Conjugates with DC4 (40) and

DC44 (41). DC4 and DC44 conjugations were conjugated to

humanized C242 (huC242) and humanized B4 (huB4) antibodies in

two steps. First, the antibody was modified with either N-succinimidyl-

4-(2-pyridyldithio) butyrate (SPDB) or 4-(2-pyridyldithio)pentanoic

acid-N-hydroxysuccinimide ester (SPP) to incorporate a linker bearing

a pyridyl disulfide moiety, or antibody was modified with N-

succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate

(SMCC) to introduce a linker bearing a maleimido group, following

procedures described in the preparation of maytansinoid conjugates.^3,4

In general, the antibodies were modified with ∼6 equiv of either SPP

or SPDB or SMCC in phosphate buffer A (0.1 M potassium

phosphate buffer, pH 7.5) with either 5% DMA or 5% DMSO for 90

min at room temperature. Noncoupled linkers were removed by gel

filtration over a Nap 25 column equilibrated in 0.1 M potassium

phosphate buffer, pH 7. The ratios of the linker versus antibody

resulting from reaction of the antibody with SPDB and SPP were

determined by measure UV absorbance at both 325 and 280 nm (the

linker chromophore absorbance versus mAb absorbance). On average,

3.5−5.0 linkers were incorporated per antibody molecule. The SMCC

linker has no unique chromophore by which its level of incorporation

concentration can be determined by UV once coupled to an antibody.

Therefore, an assumption was made that the reaction yielded four

equiv of SMCC incorporated per antibody, similar to the level of

incorporation for the other linkers. In the second step, 1.7 equiv of

DC4 or DC44 over incorporated linker was added to the purified

linker-modified antibody solutions in sodium phosphate buffer, pH

6.5, containing 5−20% DMA (v/v), and the mixture was incubated at

room temperature for 3 h. Then ∼5 equiv of 1 mM of N-

ethylmaleimide was added to quench any unreacted free thiol group

on DC4 or DC44. The mixtures were purified by size exclusion on

Sephadex G-25 columns, equilibrated with 50−100 mM sodium

phosphate buffer, pH 6.5, containing 5−20% DMA. In most cases, the

second step purification of using a Porapak column (Waters Corp, part

no. 35683) to remove noncovalent bound free DCx compounds from

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the control group and was treated with the phosphate-buffered saline

vehicle. The remaining four groups were treated with either huB4−

SPP−DC4 (DC4 dose of 75 μg/kg, qd ×5), or huB4−SPP−DC44

(DC44 dose of 75 μg/kg, qd ×5), or DC4 (75 μg/kg, qd ×5) or DC44

(75 μg/kg, qd ×5), administered intravenously. Three dimensions of

the tumor were measured twice weekly using the LabCat system

(Innovative Programming Associates, Inc. Princeton, NJ), and the

tumor volumes were calculated using the formula tumor volume =

¹/₂(length × width × height). The weight of the animals was also

measured twice per week. A mouse was sacrificed when any one of the

following criteria was met: (1) loss of body weight of more than 20%

from pretreatment weight, (2) tumor volume larger than 1500 mm³,

(3) too sick to reach food and water, or (4) skin necrosis. A mouse

was considered to be tumor-free if no tumor was palpable.

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1494−1524.

ASSOCIATED CONTENT

■

S

* Supporting Information

HPLC monitoring activation of phosphate prodrugs with a

phosphatase, chiral HPLC separation of CBI and binding assays

of the antibody-DC4 drug conjugates, and analytical data

(HPLC purity data) for target compounds. This material is

available free of charge via the Internet at http://pubs.acs.org.

(12) Hanka, L. J.; Dietz, A.; Gerpheide, S. A.; Kuentzel, S. L.; Martin,

D. G. CC-1065 (NSC-298223), a new antitumor antibiotic.

Production, in vitro biological activity, microbiological assays and

taxonomy of the producing microorganism. J. Antibiot. (Tokyo) 1978,

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(14) MacMillan, K. S.; Boger, D. L. Fundamental relationships

between structure, reactivity, and biological activity for the

duocarmycins and CC-1065. J. Med. Chem. 2009, 52, 5771−5780.

(15) Carter, C. A.; Waud, W. R.; Li, L. H.; DeKoning, T. F.;

McGovren, J. P.; Plowman, J. Preclinical antitumor activity of bizelesin

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AUTHOR INFORMATION

■

Corresponding Author

*Phone: (781)895-0600. Fax: (781)895-0610. E-mail: robert.

zhao@immunogen.com.

ACKNOWLEDGMENTS

■

We thank our former colleague Hongsheng Xie (now at Shire

Pharmaceutical, Lexington, MA) for his contribution, and

Wayne C. Widdison and Michael L. Miller, both at

ImmunoGen, for reading this manuscript.

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ABBREVIATIONS USED

■

CBI, 5-hydroxy-3-amino-1-[S]-(chloromethyl)-1,2-dihydro-3H-

benz(e)indole; CAR, cytotoxic agent per antibody ratio; DTT,

dithiothreitol; DMA, dimethylacetamide; DCx, DC4 and/or

DC44; mAb, monoclonal antibody; NEM, N-ethyl maleimide;

PBS, phosphate-buffered saline; SMCC, succinimidyl-4-(N-

maleimidomethyl)cyclohexane-1-carboxylate; SPP, N-succini-

midyl 4-(2-pyridyldithio)pentanoate; SPDB, N-succinimidyl 4-

(2-pyridyldithio)butanoate; TBTU, O-(benzotriazol-1-yl)-

N,N,N′,N′-tetramethyluronium tetrafluoroborate; TCEP, Tris-

(2-carboxyethyl)phosphine

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Synthesis and biological evaluation of antibody conjugates of phosphate prodrugs of cytotoxic DNA alkylators for the targeted treatment of cancer

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Article abstract of DOI:10.1021/jm201284m

Full text of DOI:10.1021/jm201284m

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