Influence of a Basic Side Chain on the Properties of Hypoxia-Selective Nitro Analogues of the Duocarmycins: Demonstration of Substantial Anticancer Activity in Combination with Irradiation or Chemotherapy

Article

pubs.acs.org/jmc

Inﬂuence of a Basic Side Chain on the Properties of Hypoxia-

Selective Nitro Analogues of the Duocarmycins: Demonstration of

Substantial Anticancer Activity in Combination with Irradiation or

Chemotherapy

Moana Tercel,* Ho H. Lee, Sunali Y. Mehta,^‡Jean-Jacques Youte Tendoung,^§Sally Y. Bai,^∥

H. D. Sarath Liyanage, and Frederik B. Pruijn

Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019,

Auckland 1142, New Zealand

S

* Supporting Information

ABSTRACT: A new series of nitro analogues of the duocarmycins

was prepared and evaluated for hypoxia-selective anticancer activity.

The compounds incorporate 13 diﬀerent amine-containing side chains

designed to bind in the minor groove of DNA while spanning a wide

range of base strength from pK_a9.64 to 5.24. The most favorable in

vitro properties were associated with strongly basic side chains, but the

greatest in vivo antitumor activity was found for compounds

containing a weakly basic morpholine. This applies to single-agent

activity and for activity in combination with irradiation or chemo-

therapy (gemcitabine or docetaxel). In combination with a single dose

of γ irradiation 50 at 42 μmol/kg eliminated detectable clonogens in some SiHa cervical carcinoma xenografts, and in

combination with gemcitabine using a well-tolerated multidose schedule, the same compound caused regression of all treated

A2780 ovarian tumor xenografts. In the latter experiment, three of seven animals receiving the combination treatment were

completely tumor free at day 100.

(ADCs)¹³or small molecule-drug conjugates,¹⁴and removal

INTRODUCTION

■

of the phenol functional group to give precursors designed for

The duocarmycins, exempliﬁed by duocarmycin SA (1, Figure

activation by cytochrome P450 oxidation.¹⁵

1), comprise a small group of extremely cytotoxic natural

In an alternative structural modiﬁcation the phenol of the

seco precursor can be replaced with an amino functional group.

products that alkylate adenine in the minor groove of DNA.^1,2

Several synthetic analogues showed very promising preclinical

This has been reported for a number of diﬀerent alkylating

antitumor activity,^3,4and four examples advanced to clinical

subunits¹⁶⁻¹⁸but has been studied in most depth for aminoCBI

trial.⁵⁻⁸However, all proved to be myelotoxic and lacking in

7.¹⁹We have provided evidence that aminoCBIs share many of

eﬃcacy at tolerated doses.

Subsequent eﬀorts have sought to improve the therapeutic

index by introducing a level of tumor-selective delivery or

release, aided by an understanding of the duocarmycin

mechanism of action. This is illustrated in Figure 1 for

1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one (CBI, 3), a

widely used synthetic variant of the alkylating subunit which,

when linked to appropriate minor groove-binding side chains R,

the properties of their phenol analogues; particular examples

alkylate DNA sequence selectively in AT-rich regions²⁰and

form exclusively the N3 adenine adduct,²¹with the S-

enantiomer showing superior DNA alkylation eﬃciency.²⁰We

have also shown that aminoCBIs react with nucleophiles in

dilute aqueous solution exclusively via the cyclopropyl

intermediate 8, an imine analogue of 3.²²Intermediate 8

forms readily from 7 under physiological conditions but is

extremely reactive because it is fully protonated. Nevertheless, a

direct comparison of 7 and 2 (where R is the 5,6,7-

trimethoxyindole (TMI) side chain found in duocarmycin

retains similar cytotoxic potency to that of the natural

products.⁹In the presence of DNA, 3 undergoes selective

attack by N3 of adenine to generate a thermally unstable

adduct; this depurinates on heating to release 4. The active

alkylating agent 3 can be formed by ring closure of a seco

precursor 2. Modiﬁcations of the phenol in 2 which prevent

SA) showed very similar cytotoxicity across a cell line panel,

especially for the more potent S enantiomers.²²

AminoCBIs have also been employed as the active species in

several tumor-selective prodrug strategies. Various carbamates

form the basis of potentially tumor-selective prodrug

ring closure also prevent DNA alkylation and can therefore

strategies.¹⁰Examples of these strategies include phenol

glycosides,¹¹N-acyl O-amino phenol derivatives,¹²phenol

Received: April 11, 2017

carbamates as payloads for antibody-drug conjugates

A

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to advantage with a variety of existing therapies, an outlook

supported in preclinical and in some clinical trials,^32,33but to

date a HAP has yet to achieve registration as an anticancer

agent.

In preclinical experiments, we showed that 6 (R = TMI) is

selectively toxic to some tumor cell lines in the absence of

oxygen and that the corresponding adduct 5 can be isolated

from cells incubated with 6 under hypoxic but not oxic

conditions.²¹The introduction of electron-withdrawing sub-

stituents, particularly sulfonamides or carboxamides in the 7-

position of the nitroCBI, provided analogues that were more

eﬃciently reduced by endogenous reductases and exhibited

substantial hypoxic cytotoxicity ratios (HCRs) in all tumor cell

lines tested.³⁴This substituent in turn provided a convenient

site for the introduction of a phosphate “preprodrug” that

conferred excellent water solubility while being rapidly cleaved

after iv administration.³⁵The particular example 9 is highly

active against hypoxic cells in human tumor xenografts at well-

tolerated doses in mice and provides a substantial increase in

tumor growth delay when combined with γ irradiation.

Although 9 is racemic and the enantiomers diﬀer at least 20-

fold in potency, there is no enantioselectivity in the hypoxia-

selective activation step in vitro; both enantiomers show clean

metabolism from nitro to amino in the presence of hypoxic

tumor cells and selectively kill cells under hypoxic conditions.³⁶

Hypoxia-selective metabolism of 9 was subsequently demon-

strated in a panel of 23 human tumor cell lines in a study which

identiﬁed three ﬂavoreductases capable of performing the one-

electron reductive activation step.³⁷

In the development of 9, a set of ﬁve diﬀerent minor groove-

binding side chains was examined.³⁵One of these, 5-(2-

dimethylaminoethoxy)indole (DEI), the only example with a

basic group in the side chain, outperformed the others in terms

of both HCR in vitro and activity against radioresistant tumor

cells in animal models. Superior hypoxic selectivity of DEI

versus TMI side chains had previously been noted with a series

of substituted nitroCBIs.³⁴However, a basic side chain is not

always preferred; when the leaving group of a nitroCBI is

changed from chloride to sulfonate, strong hypoxia-selective

activity is sometimes associated with neutral (TMI) rather than

basic (DEI) side chains.^38,39Because of these observations, and

to build on the promising antitumor activity of 9, we decided to

explore the inﬂuence of a wide range of basic side chains on the

in vitro and in vivo antitumor properties of nitroCBIs related to

9.

Figure 1. Structure of the natural product duocarmycin SA, DNA

alkylation pathway illustrated with 3 (a synthetic variant of the

alkylating subunit), and the hypoxia-selective mechanism of action of

nitroCBI prodrugs. R is a side chain that can bind in the minor groove

of DNA; examples include TMI (5,6,7-trimethoxyindole) and DEI [5-

(2-(dimethylamino)ethoxy)indole]. NitroCBIs do not undergo

spirocyclization but are reduced selectively in the absence of oxygen

to the corresponding aminoCBIs which alkylate DNA by a mechanism

analogous to that of the phenol congeners.

have been designed as ADC payloads^23,24or for activation

following reduction by exogenous enzymes²⁵or in the presence

of ferrous iron.²⁶We have proposed an alternative strategy with

nitroCBIs of general structure 6. In this scheme, the strongly

electron-withdrawing nitro substituent suppresses spirocycliza-

tion but is reduced by endogenous reductases via an initial 1-

electron oxygen-sensitive step to ultimately yield the corre-

sponding aminoCBI 7. NitroCBIs are thus examples of

hypoxia-activated prodrugs (HAPs), compounds designed to

take advantage of the low oxygen concentrations commonly

found in tumors but not in healthy tissues.^27,28Hypoxic cells in

tumors have been linked to multiple types of treatment

resistance^29,30(to radiotherapy, to some forms of chemo-

therapy, and more recently to immunotherapy³¹) in part

because the selective pressures of a hypoxic microenvironment

drive the development of a more aggressive and metastatic

phenotype. The promise of HAPs is that they may be combined

RESULTS AND DISCUSSION

■

Chemistry. The structures of the new compounds prepared,

together with relevant reference compounds, are collected in

Table 1. The compounds fall into three groups: nitroCBI

prodrugs, aminoCBI cytotoxins, and phosphate preprodrugs of

the nitroCBIs. The ﬁrst two are used to assess in vitro

properties because their alcohol side chain (Y = H) is

compatible with cell permeability, while the highly polar and

water-soluble phosphates are used for in vivo evaluation. In

addition to TMI and DEI 13 other side chains (A−M in Table

1) were explored, all of which carry a basic substituent. Eight of

the new side chains are indoles (A−H), each functionalized at

the 5-position. Incorporating a substituent at this site which

projects toward the base of the DNA minor groove can have a

substantial inﬂuence on cytotoxicity, and a variety of such

substituents are known that generate potent CBI⁴⁰and

aminoCBI analogues.⁴¹In addition, three cinnamic acid-based

B

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where the aromatic system was replaced with a methylene

linker to a morpholine base.

Table 1. Structures of New and Reference Compounds

Carboxylic acids corresponding to the selected side chains

were commercially available in one case (M) or previously

reported for three others (D,⁴⁰E,⁴²G⁴²). For the remaining

side chains, the required acids were prepared using standard

methods as shown in Scheme 1. The basic substituent was

generally introduced via Mitsunobu reaction or alkylation of a

phenol (B, F, I−L) or by acylation of an aniline (H). In

addition, side chain A was accessed using a Fischer indole

synthesis and side chain C by displacing the bromide of a

suitably protected (bromomethyl)indole intermediate using

dimethylamine.

Incorporation of the new side chains into nitroCBIs and

aminoCBIs bearing a 7-sulfonamide substituent is shown in

Scheme 2, and the corresponding routes to 7-carboxamides are

presented in Scheme 3. In general, these schemes make use of

previously described intermediates and methods. For example,

nitroCBIs 12−14 and 16−19 were prepared by 1-ethyl-3-(3-

(dimethylamino)propyl)carbodiimide hydrochloride (EDCI)-

mediated coupling of the alkylating subunit 87³⁵to the

appropriate side chain acid under acidic conditions (in the

presence of toluenesulfonic acid). This approach is generally

high-yielding, with the only diﬃculty being the tendency of the

product to eliminate HCl, giving an exomethylene byproduct³⁶

hard to separate from the desired material, particularly if basic

conditions are used to remove the excess acid side chain (see

Supporting Information). A slight modiﬁcation of this method

employs a TBDMS protecting group on the alkylating subunit,

previously reported in the synthesis of the enantiomers of 11 to

give more soluble and more easily puriﬁed intermediates.³⁶For

the current application, TBDMS-protected sulfonamide and

carboxamide subunits 89 and 92 were prepared as shown and

used to make 6 nitroCBI analogues (four sulfonamide and two

carboxamide examples), with a subsequent HCl-mediated

deprotection of the TBDMS group. An alternative approach

for the amide coupling involved preparation of the acid chloride

of the side chain using thionyl chloride, which gave good yields

of the desired products in the two cases it was employed

(sulfonamides 90i and 90l). AminoCBIs were prepared from

the nitroCBIs by hydrogenation over PtO₂, followed by HCl

treatment to remove the TBDMS protecting group if present.

This method was not suitable for the cinnamate side chains

because of competing saturation of the double bond. Instead,

for two examples, conversion to the aminoCBI (39 and 40) was

achieved in moderate yield using selective reduction with Zn

and NH₄Cl.

nitroCBI

aminoCBI

phosphate

X = NO₂,

Y = H

X = NH₂,

Y = H

X = NO₂,

Y = P(O) (OH)₂

a

R

pK_a

sulfonamides

b

c

TMI

10

na

29

45

b

c

DEI

11

8.70

9.64

9.35

8.84

5.24

6.47

7.16

7.45

8.86

6.39

7.15

7.58

6.30

6.59

30

9

A

12

13

14

15

16

17

18

19

20

21

22

23

24

31

32

33

34

35

36

37

38

39

40

46

47

48

49

50

51

52

53

54

55

56

57

58

B

C

D

E

F

G

H

I

J

Synthesis of water-soluble phosphates suitable for in vivo

experiments also followed previous methods using the

protected t-Bu phosphate esters 94³⁵and 96³⁵as starting

materials (Scheme 4). The amide bond to the side chain was

again formed using EDCI, with the single exception of side

chain L, for which the acid chloride was employed, giving the

desired amide in high yield. Final deprotection to produce the

free phosphates was achieved using TFA (46−58), except for

two examples where HCl was employed. This proceeded

smoothly for 61, but in the case of 60, acid exposure also gave

an impurity which was tentatively identiﬁed as the correspond-

ing N-(2-chloroethyl) carboxamide.

K

L

41

42

M

carboxamides

b

c

TMI

25

na

b

c

DEI

26

43

59

E

I

27

28

6.47

6.39

44

60

61

a

b

Calculated using ACD/Laboratories software ACD/pK_av12.01. The

structures of the TMI and DEI side chains are illustrated in Figure 1.

Reference compounds previously reported.³⁵

c

Base Strength. The relative basicity of the side chains was

assessed by calculating pK_afor the nitroCBIs 11−24 and 27

and 28 using ACD software.⁴³Not surprisingly, the nitroCBI 7-

substituent (sulfonamide versus carboxamide) had no inﬂuence

side chains were investigated (I−K, again substituted in a

position known to be compatible with cytotoxic potency⁴¹),

along with a benzoic acid derivative L and a single example M,

C

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a

presence of σ-acceptor ether or electron-withdrawing carbox-

amide substituents in the γ- or especially β- or α-positions.⁴⁴

Overall the set provides a large range of pK_a, with examples

more basic than DEI (those with side chains A−C, H) and less

basic (D−G, I−M) to the point where limited protonation

would be expected even in the acidic microenvironment often

found in solid tumors.⁴⁵

Scheme 1. Synthesis of Side Chain Acids

In Vitro Cytotoxicity. Cytotoxicity was determined using

an antiproliferative assay in two human tumor cell lines, the

cervical carcinoma SiHa and the colon carcinoma HT29, for

which reference data were available for the TMI and DEI

analogues (Figure 2 and Supporting Information, Tables S1

and S2). In general, most of the new aminoCBIs retained

strong cytotoxic activity with IC₅₀s in SiHa in the low nM

range, suggesting that even the bulkier basic substituents

(containing diethylamine or cyclic amine groups) were

compatible with binding in the DNA minor groove. While a

protonated side chain might be expected to inﬂuence

cytotoxicity by a number of mechanisms (e.g., aﬃnity to

negatively charged DNA or sequestration in acidic organelles),

there was no obvious trend in aminoCBI toxicity with

calculated pK_a. Instead, side chains L and M, which share the

morpholine common to several other side chains, stand out as

generating less cytotoxic aminoCBIs (about 100 and 250 times

less toxic than DEI, respectively), perhaps because of a poorer

ﬁt with the minor groove (L) or decreased binding aﬃnity in

the absence of an aromatic group (M). In addition, side chains

G and H also tend to generate slightly weaker aminoCBI

cytotoxicity. A similar observation was noted for an aminoCBI

with a side chain indole 5-NHCOMe substituent⁴¹even though

an amide in this position mimics a structural motif found in

some of the natural products. AminoCBI toxicity in the two cell

lines was very strongly correlated (Supporting Information,

Figure S2), indicating a common mechanism of action,

although IC₅₀s were on average 4.2-fold higher in HT29, in

keeping with this being the most resistant of 14 cell lines

surveyed against reference aminoCBI 30.³⁵As expected, there

was no dependence in either cell line on oxic versus hypoxic

incubation conditions for aminoCBI cytotoxicity (i.e., HCRs

close to 1, see Supporting Information, Table S2).

All nitroCBIs exhibited oxic IC₅₀s in the low μM range in

both cell lines, with little dependence on the structure of the

side chain, thus showing good deactivation of cytotoxicity in the

prodrug form. In contrast, the cytotoxicity of nitroCBIs

incubated under hypoxic conditions was much more variable.

For some side chains, the shift from oxic to hypoxic conditions

gave hardly any enhancement in cytotoxicity (e.g., I, J), while

for others sizable HCRs were observed (e.g., A, B, C; HCRs of

27 to 68 for 12−14 in the two cell lines) but none superior to

those for the two DEI reference compounds 11 and 26 in

HT29 (Supporting Information, Table S1). In HT29, the

greatest hypoxic selectivity was clearly associated with strongly

basic side chains (all HCRs >10 were found for the ﬁve

nitroCBIs with pK_a≥ 8.7), and in each of these cases the

nitroCBI became as potent, or nearly so, as the corresponding

aminoCBI under hypoxic conditions (Figure 2). In SiHa, the

nitroCBIs rarely achieved equipotency with aminoCBIs under

hypoxia but HCRs >10 were observed for a larger selection of

side chains, including some of much weaker base strength.

Despite the discrepancy between absolute HCRs between the

two cell lines, nitroCBI cytotoxicity under hypoxia was again

highly correlated between them (Supporting Information,

Figure S3), with the more basic side chains clustered at the

a

Conditions: (a) NaNO₂, HCl; (b) ethyl 2-methylacetoacetate,

NaOAc, then HCl; (c) KOH, then HCl; (d) 3-(dimethylamino)-1-

propanol, DEAD, PPh₃; (e) AcCl, NaH; (f) NBS, AIBN; (g)

dimethylamine; (h) H₂, Pd/C; (i) 3-morpholino-1-propanol, DEAD,

PPh₃; (j) 3-(dimethylamino)propanoic acid hydrochloride, EDCI; (k)

4-(2-chloroethyl)morpholine, NaH; (l) 81 (prepared by reaction of

morpholine with 1,3-dibromopropane), NaH; (m) 1,2-dibromoethane,

NaH; (n) 1-methylpiperazine then HCl; (o) 4-(2-chloroethyl)-

morpholine.

on the calculated base strength. The observed trends in pK_a

match expectations based on an increase of basicity with Et

versus Me amine substitution and a decrease of basicity in the

D

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a

Scheme 2. Synthesis of Sulfonamide-Substituted Nitro- and AminoCBIs

a

Side chain R is deﬁned in Table 1. Conditions: (a) EDCI, RCO₂H·HCl, toluenesulfonic acid, DMA; (b) (tert-butyldimethylsilyloxy)ethylamine, i-

PrNEt₂, then Cs₂CO₃; (c) RCO₂H, SOCl₂, DMF, then 89, i-PrNEt₂; (d) HCl; (e) H₂, PtO₂; (f) Zn, NH₄Cl. For aminoCBI 40 (R = J), the amide

formation and Zn-mediated nitro group reduction were done sequentially in a one-pot reaction in 39% overall yield. For intermediates 90, the lower

case letter refers to the corresponding side chain R, e.g., 90d has R = D.

μmol/kg). These doses were not achievable for one of the new

nitroCBI phosphates: iv administration of 53 caused death

within a few seconds at 13 μmol/kg, and this compound was

therefore dropped from further study. Notably, 52, which

diﬀers only by the removal of one methylene from the side

chain (and a consequent reduction in pK_afrom 8.86 to 7.45)

was well-tolerated, with no toxicity noted at the highest dose

tested (75 μmol/kg).

Scheme 3. Synthesis of Carboxamide-Substituted Nitro- and

AminoCBIs

a

Formulation for iv administration was achieved using PBS

containing 2−4 equiv of NaHCO₃, which gave solutions with

pH in the range 6.8−8.0. Previously, this approach produced a

more than 1000-fold increase in aqueous solubility for

phosphate 9 compared to alcohol 11. However, some of the

new phosphates proved to be less water-soluble, and the target

concentration of 7.5 mM (required to dose mice at 75 μmol/

kg) was not achieved in four cases (for details, see Supporting

Information, Tables S3 and S4).

Antitumor activity in combination with irradiation was

assessed using a previously reported excision assay³⁵as outlined

in Figure 3A. A large single dose of γ irradiation was used to

eliminate the well-oxygenated tumor cell population, with 15

Gy causing on average a 2.0 and 1.2 log reduction in the

number of colony-forming cells that could be isolated from

SiHa and H460 xenografts, respectively. The nitroCBI

phosphates were administered 5 min after irradiation to avoid

any potential for radiosensitization. Thus, any further reduction

in the residual radioresistant population after the combination

treatment indicates activity of the nitroCBI against hypoxic

tumor cells in vivo. This is illustrated in Figure 3B for the new

analogues in gray compared to 9 and 59 in black (and white for

45, which was not tested in H460). Although there was some

variation on repeat testing (see Supporting Information, Tables

S3 and S4), there are clearly several analogues with superior

HAP activity compared to the reference compounds. The most

marked activity is seen for the sulfonamides 50, 51, and 54

(side chains E, F, and I, respectively), which outperform 9 on

an equimolar basis in both tumor models. Notably, these

compounds share a morpholine in the side chain and are not

the compounds that might have been predicted to be highly

active on the basis of their in vitro properties; the

corresponding nitroCBI alcohols 16, 17, and 20 were not

a

Side chain R is deﬁned in Table 1. Conditions: (a) (tert-

butyldimethylsilyloxy)ethylamine, i-PrNEt₂, pyBOP, then Cs₂CO₃;

(b) EDCI, RCO₂H·HCl, toluenesulfonic acid, DMA; (c) HCl; (d) H₂,

PtO₂. For intermediates 93, the lower case letter refers to the

corresponding side chain R, e.g., 93e has R = E.

more potent end of the spectrum, possibly indicating a link

between basicity and the extent of reductive metabolism in

vitro.

Antitumor Activity in Combination with Irradiation. In

our previous evaluation of 9 in nude mice, a sporadic acute

toxicity was noted in some animals treated at doses >100

μmol/kg.³⁵Similar observations were made for some of the

new nitroCBI phosphates, making it diﬃcult to determine

MTDs. The new compounds were therefore compared on an

equimolar basis. Two xenografts were chosen, SiHa and the

lung carcinoma H460, both characterized as containing a

moderate hypoxic fraction when grown as sc xenografts.⁴⁶

These xenografts diﬀered in their response to the hypoxia-

selective activity of 9,³⁵and accordingly the new analogues

were compared at a lower dose in the more sensitive SiHa (42

μmol/kg) and a higher dose in the more resistant H460 (75

E

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a

Scheme 4. Synthesis of NitroCBI Phosphates

a

Side chain R is deﬁned in Table 1. Conditions: (a) EDCI, RCO₂H·HCl, toluenesulfonic acid, DMA; (b) 86, SOCl₂, DMF, then 94, i-PrNEt₂, (c)

TFA; (d) HCl. For intermediates 95 and 97, the lower case letter refers to the corresponding side chain R, e.g., 95a has R = A.

remarkable in terms of either HCR or potency under hypoxic

conditions (Figure 2). Ranking the best compounds is diﬃcult

in SiHa because their activity at the dose used is approaching

the limit of the dynamic range of the assay (about 1 in 100000

surviving cells). This is indicated in Figure 3B, with the arrows

identifying complete sterilization of the tumor sample (no

colonies detected even at the highest plating concentration) for

three of ﬁve animals receiving 50 or 54 in combination with

irradiation. In H460, 54 appears particularly eﬀective, especially

considering that solubility limited the dose of this compound to

56 μmol/kg. In contrast, there are several compounds that are

not signiﬁcantly active against hypoxic cells in H460 and only

weakly active in SiHa, a group that includes 57 and 58 (side

chains L and M associated with weakly cytotoxic aminoCBIs)

but also 46 and 48 with strongly basic side chains (A and C)

that generated the most favorable in vitro properties.

Remarkably, 55 also falls within this group even though it

diﬀers only by the insertion of one methylene (side chain J

versus I) from the highly active 54. A comparison of

sulfonamides and carboxamides also provides unexpected

results. Previously, with the DEI side chain, we observed

somewhat stronger HAP activity in SiHa for carboxamide 59

compared to sulfonamide 9,³⁵but with the new examples,

sulfonamides 50 and 54 clearly outperform the carboxamide

analogues 60 and 61 in both SiHa and H460.

As well as in combination with irradiation, single-agent

activity of the nitroCBI phosphates was assessed in comparison

to control tumors. Highly signiﬁcant activity in both tumor

models was associated with side chains E, F, and I (Supporting

Information, Tables S3 and S4), with 50, 51, 54, and 60 all

causing more than 1 log of cell kill in SiHa (Figure 3C). This

represents a considerably greater proportion than the hypoxic

fraction of this xenograft,⁴⁶implying nitroCBI toxicity to well-

oxygenated tumor cells. Possible mechanisms include a low

level of reductive activation in these cells or a bystander eﬀect

in which aminoCBI produced in hypoxic zones diﬀuses to and

kills cells in surrounding better-oxygenated regions of the

Figure 2. Cytotoxicity of nitroCBIs and aminoCBIs in two human

tumor. Because we have observed evidence for each mechanism

in in vitro experiments,^21,36it is possible that a combination of

carcinoma cell lines. Cell monolayers were exposed to the compounds

for 4 h under oxic or hypoxic conditions, and proliferation was

both is in action in these tumors. Nevertheless, if single-agent

measured 5 days later in comparison to untreated control cells. Side

activity against the entire tumor cell population is compared to

chain structures are shown in Table 1.

hypoxia-selective activity against the radioresistant fraction

(Figure 3C), there is clearly much greater activity against the

F

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latter. The appearance of data points exclusively in the upper

left portion of this graph is strong evidence supporting a

hypoxia-selective mechanism of action for this class of

compounds in vivo.

For a subset of compounds activity in combination with

irradiation was also examined in other human tumor xenografts

(Figure 4 and Supporting Information, Tables S5 −S7). The

Figure 4. Activity of nitroCBI phosphates against hypoxic cells in lung,

colon, and prostate cancer xenografts. Eﬃcacy for a subset of nitroCBI

phosphates was assessed using the excision assay illustrated in Figure 3.

Doses used were 75, 56, and 30 μmol/kg for H1299, HCT116, and

22Rv1, respectively. Arrows indicate the number of tumors/group for

which no surviving clonogens were detected after the combination

treatment of irradiation (15 Gy) plus nitroCBI.

lung carcinoma cell line H1299 is relatively insensitive to

aminoCBI 11³⁵but as a xenograft has an extremely high

hypoxic fraction,⁴⁶whereas colon carcinoma HCT116 and

prostate carcinoma 22Rv1 are quite sensitive to 11³⁵in vitro

and as xenografts display a moderate hypoxic fraction slightly

larger than that of SiHa and H460.⁴⁶Highly signiﬁcant activity

was observed in combination with irradiation for all compounds

tested, but once again the morpholine-containing side chains of

50 and 51 conferred superior activity to that of the stronger

bases of the DEI and B side chains (9, 47, 59). At the doses

used, 50 and 51 with irradiation eliminated detectable

clonogens in several H1299 and HCT116 tumors, and even

at the low dose of 30 μmol/kg, 50 was signiﬁcantly active

against hypoxic cells in 22Rv1 tumors.

Antitumor Activity in Combination with Chemo-

therapy. We have previously shown that combination of 9

with irradiation can lead to substantial tumor growth delay³⁵

but have not to date reported combination of nitroCBIs with

chemotherapy despite the recognition that hypoxic tumor cells

are resistant to many types of cytotoxic treatment.²⁹For

example, limited drug penetration to slowly proliferating

hypoxic cells is considered to contribute to resistance to both

gemcitabine⁴⁷and docetaxel,⁴⁸and for this reason these agents

have been investigated in combination with other HAPs in

preclinical⁴⁹⁻⁵¹and clinical studies.^33,52Here we investigate the

ability of either 9 or 50 to enhance the growth delay activity

provided by gemcitabine or docetaxel in three diﬀerent

Figure 3. Antitumor activity in combination with irradiation. (A)

Experimental design. Immunocompromised mice bearing SiHa or

H460 xenografts were treated with or without (a) a large single dose of

γ irradiation (15 Gy) and 5 min later with (b) an iv dose of nitroCBI

phosphate 9, 45−51, or 53−61. After 18 h (c) the tumor was excised,

dissociated, and plated (d) to determine the number of surviving

clonogens. Group sizes were n = 3 for control and nitroCBI alone and

n = 5 for radiation alone and radiation plus nitroCBI. Hypoxic

(radioresistant) cell kill is the additional cell kill for the combination

treatment compared to radiation alone. (B) Eﬃcacy of nitroCBIs

against hypoxic cells in the two xenograft models. Circles represent

sulfonamides and squares carboxamides with the side chain structures

given in Table 1. The TMI compound 45 (white circle) was not tested

in H460. Standard doses of 42 and 75 μmol/kg were used for SiHa

and H460, respectively, with exceptions noted in the Supporting

Information. Where repeat experiments were performed single

representative examples are shown. Arrows indicate the number of

tumors/group for which no surviving clonogens were detected after

combination treatment. (C) Comparison of single agent activity (x-

axis) versus activity against radioresistant cells (y-axis) for all

experiments (including replicates) in SiHa. Symbols as for (B) apart

from highlighting of 50 in red.

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Figure 5. Antitumor activity in combination with chemotherapy. Growth delay curves are displayed as median relative tumor volume (RTV,

calculated until the ﬁrst animal in each group passes end point) for black (control), green (chemotherapy alone), blue (nitroCBI alone), or red

(combination) treatment groups. Doses for: (A) gemcitabine 237 μmol/kg, 9 42 and 56 μmol/kg (dashed and solid lines respectively), n = 4−5; (B)

gemcitabine 133 μmol/kg, 9 56 μmol/kg, n = 6−7; (C) gemcitabine 100 μmol/kg, 50 15 μmol/kg, n = 7; (D) docetaxel 32 μmol/kg, 50 14 μmol/

kg, n = 6−7. Treatment schedule is indicated with arrows and complete responses/group (no tumor at end of observation period at 100−120 days)

noted in colored text. Acute deaths occurred in A (four mice) and B (one mouse) (for details, see Experimental Section); these animals were

excluded from the analysis.

xenograft models (Figure 5, Supporting Information, Figure S4

and Tables S8, S9).

on repeat dosing); these animals were excluded from analysis.

Otherwise, the treatment was well-tolerated; although there was

a trend to greater body weight loss after combination

treatment, none of the groups had an average body weight

nadir signiﬁcantly diﬀerent from that of the controls

(Supporting Information, Table S9).

The schedule for administration of gemcitabine (four ip

doses every 3 days, q3d × 4) optimizes its activity against sc

xenografts.⁵³Mice bearing established SiHa tumors were

treated with gemcitabine (237 μmol/kg) in combination with

two diﬀerent doses of 9 and tumor growth followed as relative

tumor volume (RTV) over time (Figure 5A). In this

experiment, gemcitabine alone had little activity while 9 alone

gave a period of tumor stasis followed by regrowth some days

after the treatment period was completed. The survival

advantage was calculated on the basis of time to quadrupling

of the initial tumor volume (RTV4, Kaplan−Meier analyses are

presented in the Supporting Information), showing that 9 had

signiﬁcant single-agent activity at both doses used. The

combination treatment was very eﬀective, causing regressions

in all treated tumors (more durable at the higher dose of 9), a

signiﬁcant growth delay compared to gemcitabine alone (an

extra 39 days at the higher dose of 9, p = 0.004), and several

complete responses in which the tumors became undetectable

and remained so until the experiment was terminated at >100

days. Although the dose of 9 was limited to 42 and 56 μmol/kg,

sporadic acute toxicity was still seen on four occasions (mostly

Gemcitabine was also combined with 9 in A2780 ovarian

tumors (Figure 5B). This is a very rapidly growing xenograft

with a high hypoxic fraction⁴⁶and marked sensitivity to

gemcitabine.⁵³For this reason, the gemcitabine dose was

reduced to 133 μmol/kg, which produced substantial

regressions and one complete response with little associated

toxicity (maximum body weight loss of 5%, although one

mouse was culled with >15% weight loss). In contrast, 9 alone

was inactive and in some cases the tumors passed the end point

before the treatment schedule could be completed. However,

the combination treatment was dramatically active. A single

mouse in this group was excluded following acute toxicity, but

the remaining six generated complete responses. In these cases,

all tumors became undetectable 8−23 days after treatment

commenced, and the animals remained in good health and

completely tumor-free for the remainder of the 100 days.

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Two further growth delay experiments were conducted to

investigate 50, a representative of the new nitroCBIs with a

morpholine-containing side chain. Given the excellent activity

of 50 in combination with irradiation, and in an eﬀort to avoid

any acute toxicity, considerably lower doses of this compound

were employed. The dose of gemcitabine was also further

reduced to 100 μmol/kg for the treatment of A2780 (Figure

5C). At this level, gemcitabine provided a similar growth delay

to that observed in Figure 5B but without any complete

responses. Single-agent 50 at 15 μmol/kg (3.7 times lower dose

than 9 in Figure 5B) had no eﬀect on tumor growth, but once

again the combination gave striking results, universal tumor

regression, an additional 14 day median growth delay (p =

0.001), and in three of seven cases lasting complete responses.

These results were obtained without any acute toxicity or any

signiﬁcant body weight losses. 50 was also combined with

docetaxel in the aggressive prostate cancer model 22Rv1

(Figure 5D). Antitumor activity of docetaxel is relatively

schedule-independent,⁵⁴and the convenient and widely used⁵⁰

qw × 2 schedule was employed. At 32 μmol/kg, docetaxel

produced short-term tumor stasis and a 13-day growth delay

compared to controls but at a cost of signiﬁcant body weight

loss. There was little activity for 50 alone, but combination with

docetaxel induced several regressions and a signiﬁcantly

increased growth delay compared to single-agent docetaxel

(17 days, p = 0.007). Concomitant with the low dose of 50,

there was no acute toxicity and no measurable eﬀect of

nitroCBI on body weight.

under hypoxia and the highest HCRs were associated with the

most basic side chains, particularly A−C and the previously

reported DEI. NitroCBIs with these side chains became as

cytotoxic, or nearly so, as the corresponding aminoCBIs when

incubated under hypoxic conditions. This suggests eﬃcient

metabolic activation for these compounds, even in HT29,

which was one of the least proﬁcient cell lines at converting

reference nitroCBI 11 to aminoCBI 30.³⁷

The ability of the new nitroCBI phosphates to eliminate

hypoxic tumor cells in vivo was investigated in combination

with irradiation (Figure 3). Despite the clear preference for

strongly basic side chains in vitro, the greatest activity in vivo

was associated with weakly basic morpholine-containing side

chains E, F, and I, particularly in combination with the

sulfonamide-substituted alkylating subunit. Moderate single

doses of 50 and 51 were suﬃcient to eliminate detectable

clonogens in the radioresistant subpopulation of some SiHa

tumors (and other xenografts, Figure 4), and to cause

considerably more hypoxic cell killing than the reference

compound 9 in the more resistant H460 tumor model.

Discrepancies between in vitro and in vivo data have been

noted previously in HAP development. For example, for

nitroCBIs containing a benzylsulfonate leaving group, the

standout activity in vivo was found for a compound with only a

moderate HCR in vitro.³⁹In a more comprehensive study of

281 analogues of the HAP tirapazamine, no correlation was

found between HCR or hypoxic potency in vitro and the ability

to kill hypoxic tumor cells in vivo.⁵⁵Instead, the in vitro data

was used as a screen to eliminate less interesting candidates. A

model was then developed that speciﬁcally included extrava-

scular transport and PK properties and was able to predict the

extent of HAP activity in vivo. Interestingly, the two best

tirapazamine analogues to emerge from this work both

incorporated a morpholine-containing side chain.

Evaluation of the new nitroCBIs in vivo also provided

evidence of single-agent activity that was once again particularly

marked for side chains E, F, and I. Single-agent activity could be

a consequence of a bystander eﬀect or a low level of reductive

activation in better-oxygenated cells. Whatever the mechanism,

the greater sensitivity of the radioresistant rather than bulk

population of tumor cells to all nitroCBIs tested (Figure 3C) is

compelling evidence of hypoxia-selective activity for this class of

compounds in vivo.

One attractive feature of a HAP approach is its potential to

be combined to therapeutic advantage with many standard

treatments that spare hypoxic cells. Radiotherapy clearly falls

within this category, as do several chemotherapy agents such as

gemcitabine and docetaxel. We present here the ﬁrst evidence

that nitroCBIs can potentiate the tumor growth delay activity of

both these agents (Figure 5). In three diﬀerent xenograft

models, coadministration of 9 or 50 signiﬁcantly increased the

growth delay provided by chemotherapy alone, in several cases

leading to complete elimination of the tumor. Signiﬁcant

additional growth delay is observed even for xenografts in

which the nitroCBI has no activity as a single agent; this is

consistent with a relatively small hypoxic tumor fraction that

nevertheless harbors cells resistant to chemotherapy and

capable of repopulating the tumor after challenge with

chemotherapy alone.

CONCLUSIONS

■

This study was prompted by the observation that the strongest

hypoxia-selective activity, both in vitro and in vivo, for a series

of nitroCBIs was associated with the only DNA minor groove-

binding side chain that contained a basic rather than neutral

substituent.³⁵In the present study, we describe 13 new side

chains (A to M in Table 1) which contain a variety of basic

groups spanning a pK_arange from a strongly basic 9.64 to an

eﬀectively neutral 5.24, as compared to the previously reported

DEI side chain with pK_a8.70. The necessary side chain acids

were synthesized by straightforward routes (Scheme 1) and

used to prepare a set of nitroCBI prodrugs and aminoCBI

cytotoxins for in vitro analysis and water-soluble phosphate

preprodrugs for administration to tumor-bearing mice. This

work was done with racemic alkylating subunits rather than the

more potent S enantiomers because our previous studies had

shown no enantioselectivity in the crucial reductive activation

step.³⁶

Evaluation in vitro (Figure 2) showed that most of the new

side chains were able to deliver strongly cytotoxic aminoCBIs

and that cytotoxicity was more dependent on structural features

other than that of the particular basic substituent. All

aminoCBIs with side chains based on a 5-substituted indole

or 4-substituted styrene generated submicromolar IC₅₀s after

brief exposure to two diﬀerent human carcinoma cell lines even

though these structures covered the widest range of pK_a. In

contrast, two side chains sharing a morpholine but attached to a

phenyl ring (L) or directly to the alkylating subunit (M)

generated considerably less cytotoxic aminoCBIs, presumably

because of weaker binding to the minor groove. Some

dependence on the nature of the basic substituent was however

noted in the in vitro properties of the nitroCBIs. While all of

these prodrugs demonstrated good deactivation (relatively

weak cytotoxicity under oxic conditions), the greatest potency

A possible limitation for nitroCBIs is the sporadic acute

toxicity that was previously observed following large single

doses of 9 and seen in the present study for some animals

treated with multiple lower doses of the same compound. For

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requires C, 51.58; H, 5.34; N, 10.02. Found: C, 51.51; H, 5.30; N,

10.07.

this reason, growth delay experiments with 50, which at an

equimolar level was more eﬀective than 9 in combination with

irradiation, were conducted at further reduced doses. This

approach completely eliminated acute toxicity while preserving

excellent antitumor activity. Until further information is

obtained (PK characterization, and a basis for toxicity

comparisons other than MTDs), it is diﬃcult to identify the

best candidate from the nitroCBIs so far prepared. Never-

theless, we note that 50 has very favorable HAP properties, and

in addition to the information presented here, there is

preliminary evidence suggesting utility against triple-negative

breast cancer⁵⁶and for the development of clickable analogues

suitable as biomarkers for preclinical investigation of the

mechanism of action.⁵⁷Further studies of 50 and related

compounds are in progress with a view to selection of a lead

compound for clinical development.

1-(Chloromethyl)-3-(5-(3-(dimethylamino)propoxy)-1H-indole-2-

carbonyl)-N-(2-hydroxyethyl)-5-nitro-2,3-dihydro-1H-benzo[e]-

indole-7-sulfonamide (13). Prepared by general method A from 87

(91 mg, 0.24 mmol) and 67·HCl (99 mg, 0.33 mmol). Filtration of the

product was slow, resulting in elimination of HCl to give an

1

exomethylene byproduct that was apparent in the H NMR of the

crude product (in DMSO-d₆diagnostic signals centered at δ 6.3, 5.8,

and 5.5 in a 1:1:2 integral ratio), comprising about 10% of the material.

The crude product (115 mg, 77%) was dissolved in DCM (4 mL) and

MeOH (2 mL), and HCl-saturated MeOH (2 mL) was added. The

precipitated solid was ﬁltered oﬀ and dried, then dissolved in DMF (3

mL) and precipitated by the addition of EtOAc. The solid was ﬁltered

oﬀ and dried to give the hydrochloride salt of 13 as a yellow powder

(77 mg, 49%): mp 224−228 °C. HPLC purity 93.8%. ¹H NMR

(DMSO-d₆) δ 11.78 (d, J = 1.7 Hz, 1H), 9.82 (br s, 1H), 9.29 (s, 1H),

8.85 (d, J = 1.7 Hz, 1H), 8.44 (d, J = 8.9 Hz, 1H), 8.03 (dd, J = 8.9, 1.7

Hz, 1H), 7.93 (t, J = 5.9 Hz, 1H), 7.44 (d, J = 8.9 Hz, 1H), 7.21 (d, J =

1.7 Hz, 1H), 7.19 (d, J = 2.3 Hz, 1H), 6.97 (dd, J = 8.9, 2.4 Hz, 1H),

5.00−4.93 (m, 1H), 4.76−4.61 (m, 3H), 4.19−4.12 (m, 2H), 4.08 (t, J

= 6.0 Hz, 2H), 3.42−3.36 (m, 2H), 3.28−3.20 (m, 2H), 2.90−2.85 (m,

2H), 2.81 (s, 6H), 2.20−2.11 (m, 2H). HRMS (FAB) calcd for

C₂₉H₃₃³⁵ClN₅O₇S (MH⁺) m/z 630.17892, found 630.17846. Calcd for

C₂₉H₃₃³⁷ClN₅O₇S (MH⁺) m/z 632.17597, found 632.17615.

1-(Chloromethyl)-3-(5-((dimethylamino)methyl)-1H-indole-2-

carbonyl)-N-(2-hydroxyethyl)-5-nitro-2,3-dihydro-1H-benzo[e]-

indole-7-sulfonamide (14). Prepared by general method A from 87

(150 mg, 0.39 mmol) and 71·HCl (120 mg, 0.47 mmol) to give 14 as

a yellow−orange solid (225 mg, 98%): mp 242−245 °C (dec). HPLC

purity 92.8%. ¹H NMR (DMSO-d₆) δ 11.83 (s, 1H), 9.30 (s, 1H), 8.85

(d, J = 1.6 Hz, 1H), 8.43 (d, J = 8.9 Hz, 1H), 8.02 (dd, J = 8.9, 1.7 Hz,

1H), 7.91 (t, J = 5.9 Hz, 1H), 7.59 (s, 1H), 7.46 (d, J = 8.5 Hz, 1H),

7.31−7.22 (m, 2H), 4.98 (dd, J = 10.8, 9.7 Hz, 1H), 4.73 (dd, J = 11.0,

2.4 Hz, 1H), 4.70−4.62 (m, 2H), 4.20−4.10 (m, 2H), 3.48 (s, 2H),

3.37 (q, J = 6.0 Hz, 2H), 2.87 (q, J = 6.0 Hz, 2H), 2.18 (s, 6H). Anal.

C₂₇H₂₈ClN₅O₆S·¹/₂H₂O requires C, 54.50; H, 4.91; N, 11.77. Found:

C, 54.61; H, 4.93; N, 11.79.

EXPERIMENTAL SECTION

■

General Chemistry Methods. All ﬁnal products were analyzed by

reverse-phase HPLC (Alltima C18 5 μm column, 150 mm × 3.2 mm;

Alltech Associated, Inc., Deerﬁeld, IL) using an Agilent HP1100

equipped with a diode array detector. Mobile phases were gradients of

80% acetonitrile/20% H₂O (v/v) in 45 mM ammonium formate at pH

3.5 and 0.5 mL/min. Purity of ﬁnal products was determined by

monitoring at 330 200 nM. The following ﬁnal products had purity

≥95%: 12, 15−17, 19−21, 24, 27, 28, 35, 36, 38, 40−42, 44, 47, 49−

57, 60. For those cases where purity was <95% (13, 14, 18, 22, 23,

31−34, 37, 39, 46, 48, 58, 61), the chromatograms (at 330 200 nm

and at λ_max16 nm), along with identiﬁed impurities, are presented in

the Supporting Information.

Solvents were distilled prior to use by common laboratory methods.

Petroleum ether refers to the fraction with a bp = 40−60 °C. Organic

solutions were dried over anhydrous Na₂SO₄or MgSO₄. Column

chromatography was carried out on silica gel (Merck 230−400 mesh).

Melting points were determined on an Electrothermal 2300 melting

point apparatus. NMR spectra were obtained on a Bruker Avance 400

spectrometer at 400 MHz for ¹H and 100 MHz for ¹³C spectra. HRMS

was performed with a Bruker micrOTOF-QII or an Agilent 6530B

Accurate Mass Q-TOF mass spectrometer. Combustion analyses were

carried out in the Campbell Microanalytical Laboratory, University of

Otago, Dunedin, New Zealand.

General Method B, TBDMS Ether Cleavage. A solution of the

TBDMS ether in DCM or MeOH or dioxane was treated with HCl

(1.25 M in MeOH, 5−10 equiv), and the mixture was stirred at rt for

30 min to 2 h. The mixture was diluted with EtOAc to precipitate the

product, which was ﬁltered oﬀ either directly or after standing at 5 °C

overnight. The product was washed with EtOAc and dried.

General Method A, EDCI-Mediated Amide Formation. A

mixture of the appropriate indoline (87,³⁵89, 92, 94,³⁵or 96³⁵), side

chain acid hydrochloride salt (1.2 to 1.5 equiv), EDCI (4 equiv), and

anhyd TsOH (0.15 to 0.4 equiv) in DMA was stirred at rt for 1−5 h.

Reactions with the cinnamic acids 80, 82, and 84 were incomplete at

this stage and required the addition of a second batch of acid, EDCI,

and TsOH and stirring overnight. The mixtures were cooled in an ice

bath and cold alkali (generally 5% NaHCO₃or 2% Na₂CO₃) added.

The precipitated solids were ﬁltered oﬀ, washed with water, and dried.

1-(Chloromethyl)-3-(5-(2-(diethylamino)ethoxy)-1H-indole-2-car-

bonyl)-N-(2-hydroxyethyl)-5-nitro-2,3-dihydro-1H-benzo[e]indole-

7-sulfonamide (12). Prepared by general method A from 1-

(chloromethyl)-N-(2-hydroxyethyl)-5-nitro-2,3-dihydro-1H-benzo[e]-

indole-7-sulfonamide (87) (100 mg, 0.26 mmol) and 64·HCl (97 mg,

0.31 mmol). The crude product was obtained as an oil which was

dissolved in MeOH (5 mL). This solution was stirred with HCl in

MeOH (1.25M, 1.0 mL), and EtOAc was added to precipitate the

product, which was collected, washed with EtOAc, and dried to give

the hydrochloride salt of 12 as a yellow−orange solid (151 mg, 85%):

1-(Chloromethyl)-3-(5-(dimethylamino)-1H-indole-2-carbonyl)-

N-(2-hydroxyethyl)-5-nitro-1,2-dihydro-3H-benzo[e]indole-7-sulfo-

namide (15). Prepared by general method B from 90d (185 mg, 0.27

mmol) to give the hydrochloride salt of 15 as a yellow−orange solid

(142 mg, 87%): mp >300 °C. HPLC purity 97.3%. ¹H NMR (DMSO-

d₆) δ 12.61 (br s, 1H), 12.18 (br s, 1H), 9.29 (s, 1 H), 8.86 (br d, J =

1.6 Hz, 1H), 8.45 (d, J = 8.9 Hz, 1H), 8.10 (br s, 1H), 8.04 (dd, J =

8.9, 1.7 Hz, 1H), 7.75−7.55 (m, 2H), 7.42 (s, 1H), 4.99 (br t, J = 10.6

Hz, 1H), 4.77−4.62 (m, 2H), 4.21−4.09 (m, 2H), 3.39 (t, J = 6.2 Hz,

2H), 2.99 (s, 6H), 2.88 (br q, J = 6.1 Hz, 2H), 1H obscured by water

peak. Anal. C₂₆H₂₆ClN₅O₆S·HCl·H₂O requires C, 49.85; H, 4.67; N,

11.18. Found: C, 50.07; H, 4.69; N, 10.88.

1-(Chloromethyl)-N-(2-hydroxyethyl)-3-(5-(2-morpholinoe-

thoxy)-1H-indole-2-carbonyl)-5-nitro-1,2-dihydro-3H-benzo[e]-

indole-7-sulfonamide (16). Prepared by general method A from 87

(88 mg, 0.23 mmol) and 5-(2-morpholinoethoxy)-1H-indole-2-

carboxylic acid hydrochloride⁴²(104 mg, 0.32 mmol) to give 16 as

a yellow solid (150 mg, 100%): mp 131−135 °C. HPLC purity 96.0%.

¹H NMR (DMSO-d₆) δ 11.73 (d, J = 1.7 Hz, 1H), 9.29 (s, 1H), 8.85

1

mp 204−206 °C (dec). HPLC purity 97.0%. H NMR (DMSO-d₆) δ

(d, J = 1.7 Hz, 1H), 8.43 (d, J = 8.9 Hz, 1H), 8.03 (dd, J = 8.9, 1.7 Hz,

1H), 7.91 (t, J = 5.6 Hz, 1H), 7.41 (d, J = 8.9 Hz, 1H), 7.21 (d, J = 1.8

Hz, 1H), 7.19 (d, J = 2.3 Hz, 1H), 6.96 (dd, J = 8.9, 2.4 Hz, 1H),

5.01−4.94 (m, 1H), 4.73 (dd, J = 10.9, 2.4 Hz, 1H), 4.68−4.62 (m,

1H), 4.67 (t, J = 5.6 Hz, 1H), 4.20−4.10 (m, 4H), 3.64−3.58 (m, 4H),

3.39 (q, J = 6.0 Hz, 2H), 2.87 (q, J = 6.2 Hz, 2H), 2.73 (t, J = 5.7 Hz,

2H), ca. 2.52−2.46 (m, 4H, partially obscured by DMSO peak). Anal.

11.83 (s, 1H), 9.80 (s, 1H), 9.29 (s, 1H), 8.86 (d, J = 1.7 Hz, 1H), 8.44

(d, J = 8.9 Hz, 1H), 8.03 (dd, J = 8.9, 1.7 Hz, 1H), 7.93 (t, J = 5.9 Hz,

1H), 7.46 (d, J = 8.9 Hz, 1H), 7.30−7.20 (m, 2H), 7.02 (dd, J = 8.9,

2.3 Hz, 1H), 4.97 (t, J = 10.1, 1H), 4.77−4.60 (m, 3H), 4.43−4.29 (m,

2H), 4.20−4.07 (m, 2H), 3.60−3.48 (m, 2H), 3.38 (q, J = 5.9 Hz,

2H), 3.31−3.17 (m, partially obscured by water peak, 4H), 2.87 (q, J =

6.1 Hz, 2H), 1.27 (t, J = 7.1 Hz, 6H). Anal. C₃₀H₃₄ClN₅O₇S·HCl·H₂O

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C₃₀H₃₂ClN₅O₈S·³/₄H₂O requires C, 53.65; H, 5.03; N, 10.43. Found:

C, 53.79; H, 4.85; N, 10.37.

1-(Chloromethyl)-N-(2-hydroxyethyl)-3-(5-(3-morpholinopro-

poxy)-1H-indole-2-carbonyl)-5-nitro-1,2-dihydro-3H-benzo[e]-

indole-7-sulfonamide (17). Prepared by general method A from 87

(87 mg, 0.23 mmol) and 74·HCl (123 mg, 0.36 mmol) to give 17 as a

yellow solid (153 mg, 100%): mp 130−134 °C. HPLC purity 95.0%.

¹H NMR (DMSO-d₆) δ 11.72 (d, J = 1.7 Hz, 1H), 9.29 (s, 1H), 8.85

(d, J = 1.6 Hz, 1H), 8.44 (d, J = 8.9 Hz, 1H), 8.04 (dd, J = 8.9, 1.7 Hz,

1H), 7.91 (t, J = 5.9 Hz, 1H), 7.42 (d, J = 8.9 Hz, 1H), 7.22 (d, J = 1.8

Hz, 1H), 7.16 (d, J = 2.3 Hz, 1H), 6.95 (dd, J = 8.9, 2.4 Hz, 1H),

5.01−4.94 (m, 1H), 4.72 (dd, J = 10.9, 2.4 Hz, 1H), 4.69−4.63 (m,

1H), 4.67 (t, J = 5.6 Hz, 1H), 4.21−4.10 (m, 2H), 4.04 (t, J = 6.4 Hz,

2H), 3.63−3.57 (m, 4H), 3.39 (q, J = 6.0 Hz, 2H), 2.88 (q, J = 6.1 Hz,

2H), 2.47 (t, J = 7.2 Hz, 2H), 2.43−2.36 (m, 4H), 1.96−1.88 (m, 2H).

Anal. C₃₁H₃₄ClN₅O₈S·H₂O requires C, 53.95; H, 5.26; N, 10.15.

Found: C, 53.97; H, 5.21; N, 10.29.

from 90j (112 mg, 0.145 mmol) to give the hydrochloride salt of 21 as

a yellow−orange solid (98 mg, 97%): mp 262−263 °C (dec). HPLC

1

purity 97.9%. H NMR (DMSO-d₆) δ 10.73 (br s, 1H), 9.35 (s, 1H),

8.83 (br d, J = 1.6 Hz, 1H), 8.38 (d, J = 8.9 Hz, 1H), 8.01 (dd, J = 8.9,

1.7 Hz, 1H), 7.92 (t, J = 5.8 Hz, 1H), 7.82 (d, J = 8.8 Hz, 2H), 7.74 (d,

J = 15.3 Hz, 1H), 7.11 (d, J = 15.3 Hz, 1H), 7.05 (d, J = 8.8 Hz, 2H),

4.75−4.57 (m, 3H), 4.16 (t, J = 6.04 Hz, 2H), 4.09 (br d, J = 4.0 Hz,

2H), 4.04−3.71 (m, 4H), 3.54−3.00 (m, 8H), 2.86 (q, J = 6.1 Hz,

2H), 2.28−2.13 (m, 2H). Anal. C₃₁H₃₅ClN₄O₈S·HCl·¹/₂H₂O requires

C, 52.84; H, 5.29; N, 7.95. Found: C, 52.98; H, 5.33; N, 7.97.

1-(Chloromethyl)-N-(2-hydroxyethyl)-3-(4-(2-(4-methylpiperazin-

1-yl)ethoxy)benzoyl)-5-nitro-1,2-dihydro-3H-benzo[e]indole-7-sul-

fonamide (22). Prepared by general method B from 90k (98 mg, 0.13

mmol) to give the dihydrochloride salt of 22 as a yellow−orange solid

1

(93 mg, 100%): mp 225 °C (dec). HPLC purity 94.8%. H NMR

(DMSO-d₆) δ 11.10 (v br s, 1H), 9.35 (br s, 1H), 8.83 (br d, J = 1.6

Hz, 1H), 8.39 (d, J = 8.9 Hz, 1H), 8.02 (dd, J = 8.9, 1.6 Hz, 1H), 7.92

(t, J = 5.9 Hz, 1H), 7.83 (d, J = 8.7 Hz, 2H), 7.74 (d, J = 15.3 Hz, 1H),

7.13 (d, J = 15.3 Hz, 1H), 7.08 (d, J = 8.7 Hz, 2H), 4.72−4.59 (m,

3H), 4.39 (br s, 2H), 4.08 (br d, J = 4.0 Hz, 2H), 3.38 (t, J = 6.2 Hz,

2H), 3.35−2.91 (m, 10H), 2.93−2.75 (m, 5H). Anal. C₃₁H₃₆ClN₅O₇S·

2HCl·H₂O requires C, 49.70; H, 5.38; N, 9.35. Found: C, 50.14; H,

5.32; N, 9.03.

N-(2-(1-(Chloromethyl)-7-(N-(2-hydroxyethyl)sulfamoyl)-5-nitro-

2,3-dihydro-1H-benzo[e]indole-3-carbonyl)-1H-indol-5-yl)-2-

(dimethylamino)acetamide (18). Prepared by general method A from

87 (200 mg, 0.52 mmol) and 5-(2-(dimethylamino)acetamido)-1H-

indole-2-carboxylic acid hydrochloride⁴²(185 mg, 0.62 mmol) to give

18 as a yellow−orange solid (305 mg, 88%): mp 260 °C (dec). HPLC

1

purity 93.3%. H NMR (DMSO-d₆) δ 11.81 (br d, J = 1.4 Hz, 1H),

1-(Chloromethyl)-N-(2-hydroxyethyl)-3-(4-(2-morpholinoethoxy)-

benzoyl)-5-nitro-1,2-dihydro-3H-benzo[e]indole-7-sulfonamide

(23). Prepared by general method B from 90l (65.5 mg, 0.089 mmol)

to give the hydrochloride salt of 23 as an orange solid (45 mg, 82%):

9.74 (s, 1H), 9.30 (s, 1H), 8.86 (d, J = 1.6 Hz, 1H), 8.44 (d, J = 8.9

Hz, 1H), 8.13 (s, 1H), 8.01 (dd, J = 8.9, 1.6 Hz, 1H), 7.92 (t, J = 6.0

Hz, 1H), 7.50−7.41 (m, 2H), 7.30 (br d, J = 2.0 Hz, 1H), 4.98 (br t, J

= 10.8 Hz, 1H), 4.74 (br dd, J = 11.5, 2.4 Hz, 1H), 4.71−4.63 (m,

2H), 4.15 (br d, J = 4.3 Hz, 2H), 3.39 (q, J = 6.0 Hz, 2H), 3.23 (br s,

2H), 2.88 (q, J = 6.0 Hz, 2H), 2.39 (s, 6H). Anal. C₂₈H₂₉ClN₆O₇S·

H₂O requires C, 51.97; H, 4.83; N, 12.99. Found: C, 52.30; H, 4.63;

N, 12.71.

1

mp 203−206 °C (dec). HPLC purity 94.0%. H NMR (DMSO-d₆) δ

10.75 (br s, 1H), 8.91 (br s, 1H), 8.84 (br d, J = 1.6 Hz, 1H), 8.40 (d, J

= 8.9 Hz, 1H), 8.02 (dd, J = 8.9, 1.7 Hz, 1H), 7.93 (t, J = 6.0 Hz, 1H),

7.72 (d, J = 8.8 Hz, 2H), 7.17 (d, J = 8.8 Hz, 2H), 4.63 (br dd, J = 10.9,

9.4 Hz, 1H), 4.56−4.44 (m, 3H), 4.17 (br dd, J = 11.1, 1.8 Hz, 1H),

4.12−3.95 (m, 3H), 3.88−3.75 (m, 2H), 3.68−3.48 (m, 4H), 3.44−

3.15 (m, 6H, partially obscured by water peak), 2.87 (q, J = 6.0, Hz,

2H). Anal. C₂₈H₃₂Cl₂N₄O₈S·¹/₂H₂O requires C, 50.61; H, 5.01; N,

8.43. Found: C, 50.60; H, 5.03; N, 8.15.

N-(2-(1-(Chloromethyl)-7-(N-(2-hydroxyethyl)sulfamoyl)-5-nitro-

2,3-dihydro-1H-benzo[e]indole-3-carbonyl)-1H-indol-5-yl)-3-

(dimethylamino)propanamide (19). Prepared by general method A

from 87 (150 mg, 0.39 mmol) and 77·HCl (146 mg, 0.47 mmol).

After the addition of cold aq Na₂CO₃(2%), the mixture was extracted

with DCM (2 × 250 mL) and the extracts were dried and ﬁltered. HCl

(1.25 M in MeOH, 1.0 mL) was added, and the mixture was

evaporated at 25 °C. The resulting oil was dissolved in MeOH (2 mL),

and the solution was diluted with EtOAc. The precipitated solid was

ﬁltered oﬀ, washed with EtOAc, and dried to give the hydrochloride

salt of 19 as a yellow−orange solid (185 mg, 70%): mp 225−227 °C

1-(Chloromethyl)-N-(2-hydroxyethyl)-3-(3-morpholinopropano-

yl)-5-nitro-1,2-dihydro-3H-benzo[e]indole-7-sulfonamide (24). Pre-

pared by general method B from 90m (110 mg, 0.17 mmol) to give

the hydrochloride salt of 24 as an orange solid (45 mg, 46%): mp 193

1

°C (dec). HPLC purity 96.1%. H NMR (C₅D₅N) δ ca. 11.1 (v br s,

1H), 9.84 (t, J = 5.6 Hz, 1H), 9.66 (br s, 1H), 9.47 (d, J = 1.6 Hz, 1H),

8.36 (dd, J = 8.9, 1.6 Hz, 1H), 8.18 (d, J = 8.9 Hz, 1H), 4.61−4.39 (m,

3H), 4.13 (dd, J = 11.3, 3.3 Hz, 1H), 4.04 (t, J = 5.6 Hz, 2H), 3.98 (dd,

J = 11.3, 8.0 Hz, 1H), 3.79 (t, J = 4.6 Hz, 4H), 3.61 (q, J = 5.6 Hz,

2H), 3.05−2.86 (m, 4H), 2.59 (t, J = 4.6 Hz, 4H), 1H obscured by

water peak. HRMS (FAB) calcd for C₂₂H₂₈³⁵ClN₄O₇S (MH⁺) m/z

527.1367, found 527.1361. Calcd for C₂₂H₂₈³⁷ClN₄O₇S (MH⁺) m/z

529.1338, found 529.1357.

1

(dec). HPLC purity 95.8%. H NMR (DMSO-d₆) δ 11.84 (s, 1H),

10.18 (s, 1H), 9.65 (s, 1H), 9.29 (s, 1H), 8.85 (d, J = 1.6 Hz, 1H), 8.44

(d, J = 8.9 Hz, 1H), 8.07 (br s, 1H), 8.03 (dd, J = 8.9, 1.7 Hz, 1H),

7.93 (t, J = 5.9 Hz, 1H), 7.46 (d, J = 8.8 Hz, 1H), 7.41 (dd, J = 8.9, 1.9

Hz, 1H), 7.30 (d, J = 1.7 Hz, 1H), 4.97 (t, J = 10.2, 1H), 4.78−4.61

(m, 3H), 4.21−4.09 (m, 2H), 3.46−3.34 (m, 4H), 2.91−2.83 (m, 4H),

2.81 (s, 6H). Anal. C₂₉H₃₁ClN₆O₇S·HCl·H₂O requires C, 49.93; H,

4.91; N, 12.05. Found: C, 50.31; H, 5.09; N, 11.95.

1-(Chloromethyl)-N-(2-hydroxyethyl)-3-(5-(2-morpholinoe-

thoxy)-1H-indole-2-carbonyl)-5-nitro-1,2-dihydro-3H-benzo[e]-

indole-7-carboxamide (27). Prepared by general method B from 93e

(83 mg, 0.113 mmol) to give the hydrochloride salt of 27 as a yellow−

orange solid (69 mg, 93%): mp 214−217 °C. HPLC purity 99.1%. ¹H

NMR (DMSO-d₆) δ 11.80 (s, 1H), 10.70 (s, 1H), 9.16 (s, 1H), 8.82

(br d, J = 1.3 Hz, 1H), 8.78 (t, J = 5.6 Hz, 1H), 8.30 (d, J = 8.8 Hz,

1H), 8.16 (dd, J = 8.8, 1.5 Hz, 1H), 7.46 (d, J = 8.9 Hz, 1H), 7.28 (br

s, 1), 7.22 (br d, J = 1.6 Hz, 1H), 7.04 (br dd, J = 8.9, 2.0 Hz, 1H), 4.96

(br t, J = 10.1 Hz, 1H), 4.70 (br dd, J = 10.9, 2.3 Hz, 1H), 4.68−4.59

(m, 1H), 4.43 (s, 2H), 4.19−4.07 (m, 2H), 4.05−3.71 (m, 4H), 3.69−

3.48 (m, 5H), 3.40 (q, J = 5.9 Hz, 2H), 3.36 (m, 4H). Anal.

C₃₁H₃₂ClN₅O₇·HCl·¹/₂H₂O requires C, 55.78; H, 5.13; N, 10.49.

Found: C, 55.65; H, 5.12; N, 10.43.

(E)-1-(Chloromethyl)-N-(2-hydroxyethyl)-3-(3-(4-(2-

morpholinoethoxy)phenyl)acryloyl)-5-nitro-1,2-dihydro-3H-benzo-

[e]indole-7-sulfonamide (20). Prepared by general method B from

90i (142 mg, 0.187 mmol) to give the hydrochloride salt of 20 as a

yellow−orange solid (127 mg, 99%): mp 280−282 °C (dec). HPLC

1

purity 97.7%. H NMR (DMSO-d₆) δ 10.75 (br s, 1H), 9.35 (s, 1H),

8.83 (d, J = 1.6 Hz, 1H), 8.38 (d, J = 8.9 Hz, 1H), 8.02 (dd, J = 8.9, 1.6

Hz, 1H), 7.92 (t, J = 6.0 Hz, 1H), 7.84 (d, J = 8.7 Hz, 2H), 7.75 (d, J =

15.3 Hz, 1H), 7.14 (d, J = 15.3 Hz, 1H), 7.09 (d, J = 8.7 Hz, 2H),

4.72−4.60 (m, 3H), 4.49 (br s, 2H), 4.10 (br d, J = 4.1 Hz, 2H), 3.99

(br d, J = 13.8, 2H), 3.80 (br t, J = 11.7 Hz, 2H), 3.63−3.49 (m, 4H),

3.39 (t, J = 6.2 Hz, 2H, partially obscured by water peak), 3.30−3.16

(m, 2H, partially obscured by water peak), 2.87 (q, J = 6.0 Hz, 2H).

HRMS (FAB) calcd for C₃₀H₃₄³⁵ClN₄O₈S (MH⁺) m/z 645.1785,

found 645.1787. Calcd for C₃₀H₃₄³⁷ClN4O₈S (MH⁺) m/z 647.1756,

found 647.1770. Anal. C₃₀H₃₃ClN₄O₈S·HCl·H₂O·¹/₂EtOAc requires

C, 51.68; H, 5.42; N, 7.53. Found: C, 51.41; H, 5.31; N, 7.50.

(E)-1-(Chloromethyl)-N-(2-hydroxyethyl)-3-(3-(4-(3-

morpholinopropoxy)phenyl)acryloyl)-5-nitro-1,2-dihydro-3H-

benzo[e]indole-7-sulfonamide (21). Prepared by general method B

(E)-1-(Chloromethyl)-N-(2-hydroxyethyl)-3-(3-(4-(2-

morpholinoethoxy)phenyl)acryloyl)-5-nitro-1,2-dihydro-3H-benzo-

[e]indole-7-carboxamide (28). Prepared by general method B from

93i (97 mg, 0.134 mmol) to give the hydrochloride salt of 28 as a

yellow−orange solid (87 mg, 100%): mp 239−241 °C (dec). HPLC

1

purity 98.1%. H NMR (DMSO-d₆) δ 10.81 (br s, 1H), 9.22 (s, 1H),

8.82−8.74 (m, 2H), 8.24 (d, J = 8.8 Hz, 1H), 8.13 (dd, J = 8.8, 1.5 Hz,

1H), 7.84 (d, J = 8.7 Hz, 2H), 7.74 (d, J = 15.3 Hz, 1H), 7.18−7.05

K

DOI: 10.1021/acs.jmedchem.7b00563

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(m, 3H), 4.72−4.58 (m, 3H), 4.48 (br s, 2H), 4.09 (br d, J = 4.2 Hz,

2H), 3.98 (br d, J = 12.0, 2H), 3.81 (br t, J = 11.7 Hz, 2H), 3.66−3.47

(m, 5H), 3.40 (q, J = 5.9 Hz, 2H), 3.26 (br s, 4H). Anal.

C₃₁H₃₃ClN₄O₇·HCl·H₂O requires C, 56.11; H, 5.47; N, 8.44. found:

C, 56.08; H, 5.42; N, 8.44.

General Method C, Hydrogenation of NitroCBIs. A solution of

the nitroCBI in THF with PtO₂was hydrogenated at 40−50 psi for 30

min to 4 h. The catalyst was ﬁltered oﬀ through Celite, the ﬁltrate was

evaporated, and the residue was triturated with EtOAc. The solid was

ﬁltered oﬀ and dried.

calcd for C₂₆H₂₉³⁵ClN₅O₄S (MH⁺) m/z 542.1628, found 542.1618.

Calcd for C₂₆H₂₉³⁷ClN₅O₄S (MH⁺) m/z 544.1599, found 544.1607.

5-Amino-1-(chloromethyl)-N-(2-hydroxyethyl)-3-(5-(2-morpholi-

noethoxy)-1H-indole-2-carbonyl)-1,2-dihydro-3H-benzo[e]indole-7-

sulfonamide (35). Prepared by general method C from 16 (49.6 mg,

0.075 mmol) to give 35 as a pale-green solid (35 mg, 74%): mp 220−

1

225 °C (dec). HPLC purity 98.6%. H NMR (DMSO-d₆) δ 11.57 (s,

1H), 8.54 (d, J = 1.6 Hz, 1H), 7.94 (d, J = 8.9 Hz, 1H), 7.82 (s, 1H),

7.74 (dd, J = 8.8, 1.7 Hz, 1H), 7.45 (br s, 1H), 7.40 (d, J = 8.9 Hz,

1H), 7.18 (d, J = 2.3 Hz, 1H), 7.09 (d, J = 1.2 Hz, 1H), 6.93 (dd, J =

8.9, 2.4 Hz, 1H), 6.29 (s, 2H), 4.76 (dd, J = 10.8, 9.0 Hz, 1H), 4.65 (t,

J = 5.4 Hz, 1H), 4.53 (dd, J = 10.9, 1.8 Hz, 1H), 4.21−4.14 (m, 1H),

4.12 (t, J = 5.8 Hz, 2H), 4.01 (dd, J = 11.0, 3.1 Hz, 1H), 3.80 (dd, J =

11.0, 7.7 Hz, 1H), 3.63−3.58 (m, 4H), 3.40 (q, J = 6.0 Hz, 2H), 2.84

(br t, J = 6.2 Hz, 2H), 2.72 (t, J = 5.8 Hz, 2H), ca. 2.52−2.46 (m, 4H,

partially obscured by DMSO peak). Anal. C₃₀H₃₄ClN₅O₆S requires C,

57.37; H, 5.46; N, 11.15. Found: C, 57.12; H, 5.36; N, 11.00.

5-Amino-1-(chloromethyl)-N-(2-hydroxyethyl)-3-(5-(3-morpholi-

nopropoxy)-1H-indole-2-carbonyl)-1,2-dihydro-3H-benzo[e]indole-

7-sulfonamide (36). Prepared by general method C from 17 (52.6 mg,

0.078 mmol) to give 36 as a pale-green solid (38 mg, 76%): mp 220−

225 °C (dec). HPLC purity 95.0%. ¹H NMR (DMSO-d₆) δ 11.56 (d, J

= 1.5 Hz, 1H), 8.54 (d, J = 1.6 Hz, 1H), 7.94 (d, J = 8.9 Hz, 1H), 7.81

(s, 1H), 7.74 (dd, J = 8.9, 1.7 Hz, 1H), 7.49−7.44 (m, 1H), 7.40 (d, J =

8.9 Hz, 1H), 7.16 (d, J = 2.3 Hz, 1H), 7.09 (d, J = 1.5 Hz, 1H), 6.92

(dd, J = 8.9, 2.4 Hz, 1H), 6.30 (s, 2H), 4.76 (dd, J = 10.7, 9.0 Hz, 1H),

4.65 (t, J = 5.4 Hz, 1H), 4.53 (dd, J = 10.9, 1.7 Hz, 1H), 4.21−4.15 (m,

1H), 4.06−3.98 (m, 3H), 3.82 (dd, J = 11.0, 7.7 Hz, 1H), 3.63−3.56

(m, 4H), 3.39 (q, J = 6.0 Hz, 2H), 2.88−2.81 (m, 2H), 2.46 (t, J = 7.2

Hz, 2H), 2.42−2.36 (m, 4H), 1.95−1.87 (m, 2H). Anal.

C₃₁H₃₆ClN₅O₆S requires C, 57.98; H, 5.65; N, 10.91. Found: C,

57.70; H, 5.70; N, 11.10.

5-Amino-1-(chloromethyl)-N-(2-hydroxyethyl)-3-(5-(2-

(dimethylamino)acetamido)-1H-indole-2-carbonyl)-1,2-dihydro-

3H-benzo[e]indole-7-sulfonamide (37). Prepared by general method

C from 18 (60 mg, 0.095 mmol) to give 37 as a yellow solid (38 mg,

68%): mp 287 °C (dec). HPLC purity 92.8%. ¹H NMR (DMSO-d₆) δ

11.65 (br d, J = 1.3 Hz, 1H), 9.63 (s, 1H), 8.54 (br d, J = 1.5 Hz, 1H),

8.08 (s, 1H), 7.94 (d, J = 8.8 Hz, 1 H), 7.81 (s, 1 H), 7.74 (dd, J = 8.8,

1.6 Hz, 1 H), 7.51−7.38 (m, 3 H), 7.16 (br d, J = 2.0 Hz, 1H), 6.29 (s,

1H), 4.76 (br dd, J = 10.8, 9.2 Hz, 1H), 4.65 (t, J = 5.6 Hz, 1H), 4.53

(br dd, J = 10.9, 1.7 Hz, 1H), 4.24−4.13 (m, 1H), 3.98 (dd, J = 10.9,

3.0 Hz, 1H), 3.83 (dd, J = 10.9, 7.5 Hz, 1H), 3.39 (q, J = 6.1 Hz, 2H),

3.13 (br s, 2H), 2.84 (q, J = 6.2 Hz, 2H), 2.34 (s, 6H). Anal.

C₂₈H₃₁ClN₆O₅S·¹/₂H₂O requires C, 55.30; H, 5.30; N, 13.82. Found:

C, 55.25; H, 5.16; N, 13.53.

N-(2-(5-Amino-1-(chloromethyl)-7-(N-(2-hydroxyethyl)-

sulfamoyl)-2,3-dihydro-1H-benzo[e]indole-3-carbonyl)-1H-indol-5-

yl)-3-(dimethylamino)propanamide (38). Prepared by general

method C from 19 (42 mg, 0.065 mmol) to give 38 as a yellow

solid (30 mg, 76%): mp 320 °C (dec). HPLC purity 95.5%. ¹H NMR

(DMSO-d₆) δ 11.62 (s, 1H), 9.93 (s, 1H), 8.54 (d, J = 1.5 Hz, 1H),

8.05 (d, J = 1.5 Hz, 1H), 7.93 (d, J = 8.9 Hz, 1H), 7.81 (s, 1H), 7.73

(dd, J = 8.9, 1.6 Hz, 1H), 7.48−7.38 (m, 2H), 7.34 (dd, J = 8.9, 1.8 Hz,

1H), 7.15 (d, J = 1.5 Hz, 1H), 6.29 (s, 2H), 4.77 (dd, J = 10.7, 9.1 Hz,

1H), 4.63 (t, J = 5.6 Hz, 1H), 4.52 (dd, J = 10.8, 1.7 Hz, 1H), 4.22−

4.12 (m, 1H), 3.98 (dd, J = 10.5, 3.0 Hz, 1H), 3.82 (dd, J = 11.0, 7.5

Hz, 1H), 3.38 (q, J = 6.1, Hz, 2H), 2.83 (q, J = 6.2 Hz, 2H), 2.60 (t, J

= 7.0 Hz, 2H), 2.45 (t, J = 7.0 Hz, 2H), 2.21 (s, 6H). Anal.

C₂₉H₃₃ClN₆O₅S·³/₄H₂O requires C, 55.58; H, 5.55; N, 13.41. Found:

C, 55.55; H, 5.55; N, 13.15.

(E)-5-Amino-1-(chloromethyl)-N-(2-hydroxyethyl)-3-(3-(4-(2-

morpholinoethoxy)phenyl)acryloyl)-1,2-dihydro-3H-benzo[e]-

indole-7-sulfonamide (39). Saturated aq NH₄Cl (4 mL) and then Zn

powder (450 mg) were added to a stirred solution of 90i (107 mg,

0.141 mmol) in acetone (8 mL) and water (4 mL). The mixture was

stirred at rt for 1 h then ﬁltered through Celite, and the Celite pad was

rinsed with acetone. The combined ﬁltrates were concentrated to

remove acetone, and the aqueous residue was extracted with DCM

(×2). The extracts were washed with water and then brine and then

dried and evaporated. The residue was dissolved in dioxane (1.5 mL)

5-Amino-1-(chloromethyl)-3-(5-(2-(diethylamino)ethoxy)-1H-in-

dole-2-carbonyl)-N-(2-hydroxyethyl)-2,3-dihydro-1H-benzo[e]-

indole-7-sulfonamide (31). Prepared by general method C from 12

(34 mg, 0.053 mmol) to give 31 as a yellow solid (16.4 mg, 50%): mp

283−286 °C (dec). HPLC purity 93.1%. ¹H NMR (DMSO-d₆) δ

11.56 (s, 1H), 8.54 (d, J = 1.7 Hz, 1H), 7.92 (d, J = 8.9 Hz, 1H), 7.80

(s, 1H), 7.72 (dd, J = 8.8, 1.7 Hz, 1H), 7.42 (t, J = 6.0 Hz, 1H), 7.38

(d, J = 8.9 Hz, 1H), 7.15 (d, J = 2.3 Hz, 1H), 7.08 (br d, J = 1.5 Hz,

1H), 6.91 (dd, J = 8.8, 2.3 Hz, 1H), 6.29 (s, 2H), 4.79−4.70 (m, 1H),

4.63 (t, J = 5.6 Hz, 1H), 4.52 (dd, J = 10.9, 1.8 Hz, 1H), 4.21−4.10 (m,

1H), 4.07−3.95 (m, 3H), 3.79 (dd, J = 11.0, 7.6 Hz, 1H), 3.38 (q, J =

6.1 Hz, 2H), 2.90−2.76 (m, 4H), 2.68−2.46 (m, partially obscured by

DMSO peak, 4H) 1.00 (t, J = 7.0 Hz, 6H). HRMS (FAB) calcd for

C₃₀H₃₇ClN₅O₅S (MH⁺) m/z 614.2203, found 614.2204.

5-Amino-1-(chloromethyl)-3-(5-(3-(dimethylamino)propoxy)-1H-

indole-2-carbonyl)-N-(2-hydroxyethyl)-2,3-dihydro-1H-benzo[e]-

indole-7-sulfonamide (32). A solution of NaHCO₃(32 mg, 0.38

mmol) in water (3 mL) was added to a suspension of 13·HCl (38.5

mg, 0.058 mmol) in THF (4 mL) at 0 °C, and the mixture was stirred

at this temperature until all the solid dissolved. The mixture was

diluted with water and extracted with EtOAc (×2), and the extracts

were washed with brine and then dried and evaporated. The resulting

free base was hydrogenated using general method C to give 32 as a

pale-green solid (26 mg, 75%): mp 235−240 °C (dec). HPLC purity

92.1%. ¹H NMR (DMSO-d₆) δ 11.56 (d, J = 1.7 Hz, 1H), 8.54 (d, J =

1.7 Hz, 1H), 7.93 (d, J = 8.9 Hz, 1H), 7.81 (s, 1H), 7.74 (dd, J = 8.9,

1.7 Hz, 1H), 7.44 (t, J = 5.9 Hz, 1H), 7.40 (d, J = 8.9 Hz, 1H), 7.15 (d,

J = 2.3 Hz, 1H), 7.09 (d, J = 1.7 Hz, 1H), 6.91 (dd, J = 8.9, 2.4 Hz,

1H), 6.29 (s, 2H), 4.75 (dd, J = 10.8, 9.0 Hz, 1H), 4.65 (t, J = 5.6 Hz,

1H), 4.53 (dd, J = 10.9, 1.8 Hz, 1H), 4.20−4.14 (m, 1H), 4.05−3.96

(m, 3H), 3.81 (dd, J = 11.0, 7.7 Hz, 1H), 3.39 (q, J = 6.1 Hz, 2H), 2.84

(q, J = 6.2 Hz, 2H), 2.39 (t, J = 7.1 Hz, 2H), 2.16 (s, 6H), 1.90−1.83

(m, 2H). HRMS (FAB) calcd for C₂₉H₃₅³⁵ClN₅O₅S (MH⁺) m/z

600.20474, found 600.20449. Calcd for C₂₉H₃₅³⁷ClN₅O₅S (MH⁺) m/z

602.20179, found 602.20117.

5-Amino-1-(chloromethyl)-3-(5-((dimethylamino)methyl)-1H-in-

dole-2-carbonyl)-N-(2-hydroxyethyl)-2,3-dihydro-1H-benzo[e]-

indole-7-sulfonamide (33). Prepared by general method C from 14

(50 mg, 0.085 mmol) to give 33 as a yellow solid (16.4 mg, 50%): mp

1

290 °C (dec). HPLC purity 87.7%. H NMR (DMSO-d₆) δ 11.67 (s,

1H), 8.52 (br d, J = 1.3 Hz, 1H), 7.92 (d, J = 8.8 Hz, 1H), 7.81 (s,

1H), 7.74 (dd, J = 8.8, 1.6 Hz, 1H), 7.56 (s, 1H), 7.48−7.40 (m, 2H),

7.21 (dd, J = 8.5, 1.4 Hz, 1H), 7.16 (br d, J = 1.2 Hz, 1H), 6.29 (s,

2H), 4.75 (dd, J = 10.8, 9.0 Hz, 1H), 4.63 (t, J = 5.6 Hz, 1H), 4.53 (br

dd, J = 11.0, 1.7 Hz, 1H), 4.21−4.11 (m, 1H), 4.03−3.96 (m, 1H),

3.84−3.76 (m, 1H), 3.46 (s, 2H), 3.38 (q, J = 6.1 Hz, 2H), 2.83 (q, J =

6.2 Hz, 2H), 2.16 (s, 6H). HRMS (FAB): calcd for C₂₇H₃₁ClN₅O₄S

(MH⁺) m/z 556.1785, found 556.1786.

5-Amino-1-(chloromethyl)-3-(5-(dimethylamino)-1H-indole-2-

carbonyl)-N-(2-hydroxyethyl)-1,2-dihydro-3H-benzo[e]indole-7-sul-

fonamide (34). Prepared by general method C followed by general

method B from 90d (60 mg, 0.088 mmol) to give the hydrochloride

salt of 34 as a yellow solid (42 mg, 82%): mp >300 °C. HPLC purity

1

93.1%. H NMR (DMSO-d₆) δ 12.70 (br s, 1H), 12.10 (s, 1H), 8.56

(br d, J = 1.6 Hz, 1H), 8.14 (br s, 1H), 7.97 (d, J = 8.8 Hz, 1H), 7.86

(br s, 1H), 7.76 (dd, J = 8.8, 1.6 Hz, 1H), 7.65 (br s, 2H), 7.50 (br t, J

= 5.5 Hz, 1H), 7.32 (br d, J = 1.9 Hz, 1H), 4.79 (br dd, J = 10.7, 9.1

Hz, 1H), 4.52 (br dd, J = 10.9, 1.8 Hz, 1H), 4.26−4.18 (m, 1H), 4.01

(dd, J = 11.0, 3.1 Hz, 1H), 3.83 (dd, J = 10.1, 7.5 Hz, 1H), 3.39 (t, J =

6.3 Hz, 2H), 3.21 (s, 6H), 2.84 (br q, J = 5.9 Hz, 2H). HRMS (FAB)

L

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and MeOH (0.75 mL), and HCl (4 M in MeOH, 1 mL) was added.

The resulting mixture was stirred at rt for 30 min, concentrated, and

diluted with DCM (2 mL). The mixture was cooled at 0 °C, and ice-

cold aq NaHCO₃(10%, 10 mL) was added, followed by water (10

mL). The mixture was stirred for 30 min and then extracted with

DCM (×2). The extracts were washed with water and then brine and

then dried and evaporated. The residue was dissolved in the minimum

quantity of MeOH, cooled to 0 °C, and diluted with Et₂O. After

stirring at 0 °C for 30 min, the resulting precipitate was ﬁltered oﬀ and

washed with water and then Et₂O to provide 39 as a yellow powder

(38 mg, 44%): mp 225−230 °C (dec). HPLC purity 93.4%. ¹H NMR

(DMSO-d₆) δ 8.51 (d, J = 1.5 Hz, 1H), 7.89−7.84 (m, 2H), 7.76−7.68

(m, 3H), 7.63 (d, J = 15.3 Hz, 1H), 7.41 (br s, 1H), 7.06 (d, J = 15.6

Hz, 1H), 7.01 (d, J = 8.8 Hz, 2H), 6.25 (s, 2H), 4.63 (t, J = 5.6 Hz,

1H), 4.51−4.38 (m, 2H), 4.16−4.02 (m, 3H), 3.95 (dd, J = 10.9, 2.9

Hz, 1H), 3.79 (dd, J = 11, 8 Hz, 1H), 3.61−3.56 (m, 4H), 3.38 (q, J =

12, 6.3 Hz, 2H), 2.85−2.81 (m, 2H), 2.71 (t, J = 5.8 Hz, 2H), 2.53−

2.46 (4H obscured by DMSO peak). Anal. C₃₀H₃₅ClN₄O₆S requires

C, 58.58; H, 5.73; N, 9.11. Found: C, 58.31; H, 5.63; N, 8.74.

(E)-5-Amino-1-(chloromethyl)-N-(2-hydroxyethyl)-3-(3-(4-(3-

morpholinopropoxy)phenyl)acryloyl)-1,2-dihydro-3H-benzo[e]-

indole-7-sulfonamide (40). A mixture of 87 (60 mg, 0.155 mmol),

82·HCl (101 mg, 0.31 mmol), EDCI (180 mg, 3.63 mmol), and

toluenesulfonic acid (2.6 mg, 0.0155 mmol) in DMA (6 mL) was

stirred at rt overnight. Acetone (15 mL) and water (5 mL) were

added, followed by saturated aq NH₄Cl (2 mL) and Zn powder (145

mg). The suspension was stirred at rt for 2 h then ﬁltered through

Celite, and the Celite pad was rinsed with acetone. The combined

ﬁltrates were concentrated to remove most of acetone. The aqueous

residue was cooled in an ice-bath, diluted with cool aq NaHCO₃(10

mL) and water (10 mL), and then extracted with DCM. The organic

layer was washed successively with water (×2) and brine and then

dried and evaporated. The residue was dissolved in the minimum

quantity of MeOH, and the solution was cooled to 0 °C and diluted

with Et₂O. After stirring at 0 °C for 30 min, the precipitate was ﬁltered

oﬀ and washed with water and Et₂O to provide 40 as a yellow powder

(38 mg, 39%): mp 225−230 °C (dec). HPLC purity 97.1%. ¹H NMR

(DMSO-d₆) δ 8.51 (d, J = 1.5 Hz, 1H), 7.89−7.86 (m, 2H), 7.76−7.69

(m, 3H), 7.61 (d, J = 15.3 Hz, 1H), 7.40 (t, J = 5.4 Hz, 1H), 7.08−6.95

(m, 3H), 6.25 (s, 2H), 4.63 (t, J = 5.6 Hz, 1H), 4.49−4.38 (m, 2H),

4.19−4.04 (m, 2H), 3.95 (dd, J = 11.3 Hz, 1H), 3.82−3.44 (m, 1H),

3.01−3.55 (m, 4H), 2.87−2.79 (m, 2H), 2.38−2.31 (m, 7H), 1.91−

1.82 (m, 2H), 2H not observed. Anal. C₃₁H₃₇ClN₄O₆S·¹/₂Et₂O

requires C, 59.49; H, 6.35; N, 8.41. Found: C, 59.65; H, 6.09; N, 8.21.

5-Amino-1-(chloromethyl)-N-(2-hydroxyethyl)-3-(4-(2-

morpholinoethoxy)benzoyl)-1,2-dihydro-3H-benzo[e]indole-7-sul-

fonamide (41). Prepared by general method C followed by general

method B from 90l (42 mg, 0.057 mmol) to give 41 as a gray−brown

solid (31 mg, 78%). HPLC analysis showed that this material was 86%

pure. Puriﬁcation by column chromatography (CHCl₃then

CHCl₃:MeOH 9:1 then CHCl₃:MeOH:NH₃10:50:0.25) gave a

yellow oil which was dissolved in dioxane (1 mL) and treated with

HCl (4 M in dioxane, 0.2 mL). The mixture was diluted with EtOAc

(20 mL), stirred at 0 °C for 5 min, and then left to stand at 5 °C

overnight. The precipitated solid was ﬁltered oﬀ, washed with EtOAc,

and dried to give 41 as a beige solid (12.9 mg, 34%): mp 265−268 °C.

HPLC purity 97.1%. ¹H NMR (C₅D₅N) δ 11.10 (br s, 1H), 9.39 (d, J

= 1.6 Hz, 1H), 9.23 (t, J = 5.8 Hz, 1H), 8.22 (dd, J = 8.8, 1.6 Hz, 1H),

7.95−7.76 (m, 4H), 7.08 (d, J = 8.8 Hz, 2H), 4.53−4.40 (m, 2H), 4.16

(t, J = 5.7 Hz, 2H), 4.03−3.95 (m, 4H), 3.82−3.71 (m, 5H), 3.50 (q, J

= 5.8 Hz, 2H), 2.78 (t, J = 5.7 Hz, 2H), 2.61−2.53 (m, 4H). HRMS

(FAB) calcd for C₂₈H₃₄³⁵ClN₄O₆S (MH⁺) m/z 589.1887, found

589.1886. Calcd for C₂₈H₃₄³⁷ClN₄O₆S (MH⁺) m/z 591.1858, found

591.1874.

1H), 7.91 (d, J = 8.9 Hz, 1H), 7.86 (s, 1H), 7.73 (dd, J = 8.9, 1.6 Hz,

1H), 7.49 (br s, 1H), 4.33 (t, J = 10.2 Hz, 1H), 4.22−4.14 (m, 2H),

4.03−3.95 (m, 2H), 3.84−3.78 (m, 2H), 3.72−3.62 (m, 2H), 3.51−

3.34 (m, 6H), 3.20−2.91 (m, 4H), 2.82 (t, J = 6.2 Hz, 2H), 3H

obscured by water peak. HRMS (FAB) calcd for C₂₂H₃₀³⁵ClN₄O₅S

(MH⁺) m/z 497.1625, found 497.1615. Calcd for C₂₂H₃₀³⁷ClN₄O₅S

(MH⁺) m/z 499.1596, found 499.1588.

5-Amino-1-(chloromethyl)-N-(2-hydroxyethyl)-3-(5-(2-morpholi-

noethoxy)-1H-indole-2-carbonyl)-1,2-dihydro-3H-benzo[e]indole-7-

carboxamide (44). Prepared by general method C followed by general

method B from 93e (39.4 mg, 0.054 mmol) to give the hydrochloride

salt of 44 as a pale-green solid (30 mg, 89%): mp >300 °C. HPLC

purity 95.3%. ¹H NMR (C₅D₅N) δ 12.98 (s, 1H), ca. 11.0 (v br s, 1H),

9.59 (br d, J = 1.3 Hz, 1H), 9.35 (t, J = 5.4 Hz, 1H), 8.55 (br s, 1H),

8.43 (br dd, J = 8.7, 1.5 Hz, 1H), 7.86 (d, J = 8.7 Hz, 1H), 7.68 (d, J =

8.8 Hz, 1H), 7.41 (d, J = 2.3 Hz, 1H), 7.34−7.24 (m, 2H), 4.93 (br dd,

J = 10.8, 1.7 Hz, 1H), 4.73 (br t, J = 8.8 Hz, 1H), 4.36 (t, J = 5.7 Hz,

2H), 4.23−4.12 (m, 3H), 4.10−3.96 (m, 3H), 3.87−3.75 (m, 4H),

3.65 (dd, J = 10.9, 9.5 Hz, 1H), 2.93 (t, J = 5.7 Hz, 2H), 2.73−2.63 (m,

4H), 3H obscured by water peak. HRMS (FAB) calcd for

C₃₁H₃₅³⁵ClN₅O₅(MH⁺) m/z 592.2327, found 592.2318. Calcd for

C₃₁H₃₅³⁷ClN₅O₅(MH⁺) m/z 594.2297, found 594.2310.

General Method D, Phosphate tert-Butyl Ester Cleavage.

TFA (10 equiv) was added to a suspension of the phosphate ester in

DCM, and the mixture was stirred at rt for 16−25 h. The mixture was

evaporated, the residue was resuspended in DCM and evaporated once

more, and the residue was triturated with EtOAc. The solid was

ﬁltered oﬀ and dried.

2-(1-(Chloromethyl)-3-(5-(2-(diethylamino)ethoxy)-1H-indole-2-

carbonyl)-5-nitro-2,3-dihydro-1H-benzo[e]indole-7-sulfonamido)-

ethyl Dihydrogen Phosphate (46). Prepared by general method D

from 95a (482 mg, 0.576 mmol) to give the triﬂuoroacetate salt of 46

as a yellow solid (452 mg, 94%): mp 215 °C (dec). HPLC purity

93.6%. ¹H NMR (DMSO-d₆) δ 11.82 (s, 1H), 9.28 (s, 1H), 8.86 (d, J

= 1.6 Hz, 1H), 8.42 (d, J = 8.9 Hz, 1H), 8.24 (br s, 1H), 8.03 (dd, J =

8.9, 1.6 Hz, 1H), 7.45 (d, J = 8.9 Hz, 1H), 7.26 (d, J = 2.2 Hz, 1H),

7.23 (d, J = 1.3 Hz, 1H), 7.01 (dd, J = 8.9, 2.3 Hz, 1H), 4.94 (t, J =

10.1 Hz, 1H), 4.77−4.59 (m, 2H), 4.32 (t, J = 4.9 Hz, 2H), 4.20−4.07

(m, 2H), 3.79 (q, J = 6.6 Hz, 2H), 3.57−3.49 (m, 2H), 3.25 (br q, J =

6.9 Hz, 4H), 3.02 (br t, J = 5.5 Hz, 2H), 1.25 (t, J = 7.2 Hz, 6H), 3H

not observed. Anal. C₃₀H₃₅ClN₅O₁₀PS·³/₄CF₃CO₂H requires C,

46.73; H, 4.45; N, 8.65. Found: C, 46.77; H, 4.48; N, 8.35.

2-(1-(Chloromethyl)-3-(5-(3-(dimethylamino)propoxy)-1H-in-

dole-2-carbonyl)-5-nitro-2,3-dihydro-1H-benzo[e]indole-7-

sulfonamido)ethyl Dihydrogen Phosphate (47). Prepared by general

method D from 95b (144 mg, 0.18 mmol) to give the triﬂuoroacetate

salt of 47 as a pale-yellow solid (141 mg, 98%): mp 148−151 °C.

1

HPLC purity 96.7%. H NMR (DMSO-d₆) δ 11.79 (s, 1H), 9.7 (v br

s, 1H), 9.27 (s, 1H), 8.86 (d, J = 1.6 Hz, 1H), 8.43 (d, J = 8.9 Hz, 1H),

8.30 (br s, 1H), 8.02 (dd, J = 8.9, 1.7 Hz, 1H), 7.37 (d, J = 8.8 Hz,

1H), 7.21 (d, J = 1.6 Hz, 1H), 7.15 (s, 1H), 6.89 (br d, J = 9.1 Hz,

1H), 5.00−4.91 (m, 1H), 4.74−4.60 (m, 2H), 4.18−4.02 (m, 4H),

3.81 (q, J = 6.6 Hz, 2H), 3.29−3.23 (m, 2H), 3.07−3.00 (m, 2H), 2.83

(s, 6H), 2.17−2.09 (m, 2H). Anal. C₂₉H₃₃ClN₅O₁₀PS·CF₃CO₂H

requires C, 45.18; H, 4.16; N, 8.50. Found C, 44.96; H, 4.23; N, 8.79.

2-(1-(Chloromethyl)-3-(5-((dimethylamino)methyl)-1H-indole-2-

carbonyl)-5-nitro-2,3-dihydro-1H-benzo[e]indole-7-sulfonamido)-

ethyl Dihydrogen Phosphate (48). Prepared by general method D

from 95c (476 mg, 0.612 mmol) to give the triﬂuoroacetate salt of 48

as a yellow−orange solid (425 mg, 89%): mp 212−215 °C (dec).

1

HPLC purity 92.8%. H NMR (DMSO-d₆) δ 11.98 (s, 1H), 9.20 (s,

1H), 8.83 (s, 1H), 8.55 (br s, 1H), 8.33 (d, J = 8.8 Hz, 1H), 8.01 (dd, J

= 8.8, 1.6 Hz, 1H), 7.75 (s, 1H), 7.41−7.11 (m, 3H), 4.94 (t, J = 10.0

Hz, 1H), 4.71−4.50 (m, 2H), 4.30−4.02 (m, 4H), 3.92−3.78 (m, 2H),

3.19−3.01 (m, 2H), 2.67 (s, 6H), 3H not observed. Anal.

(C₂₇H₂₉ClN₅O₉PS·³/₄CF₃CO₂H) requires C, 45.55; H, 3.99; N,

9.32. Found: C, 45.66; H, 4.21; N, 9.35.

2-(1-(Chloromethyl)-3-(5-(dimethylamino)-1H-indole-2-carbon-

yl)-5-nitro-1,2-dihydro-3H-benzo[e]indole-7-sulfonamido)ethyl Di-

hydrogen Phosphate (49). Prepared by general method D from

95d (350 mg, 0.458 mmol) to give the triﬂuoroacetate salt of 49 as a

5-Amino-1-(chloromethyl)-N-(2-hydroxyethyl)-3-(3-morpholino-

propanoyl)-1,2-dihydro-3H-benzo[e]indole-7-sulfonamide (42).

Prepared by general method C followed by general method B from

90m (50 mg, 0.078 mmol) to give the hydrochloride salt of 42 as a

gray−white solid (30 mg, 71%): mp 225 °C (dec). HPLC purity

1

95.0%. H NMR (DMSO-d₆) δ 10.64 (br s, 1H), 8.53 (d, J = 1.6 Hz,

M

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yellow−orange solid (305 mg, 87%): mp >300 °C. HPLC purity

96.9%. ¹H NMR (C₅D₅N) δ 12.93 (s, 1H), 9.67 (s, 1H), 9.46 (br d, J

= 1.4 Hz, 1H), 8.40 (br dd, J = 8.8, 1.5 Hz, 1H), 8.18 (d, J = 8.8 Hz,

1H), 7.76 (v br s, 4H), 7.66 (d, J = 8.9 Hz, 1H), 7.37 (br d, J = 1.5 Hz,

1H), 7.27−7.17 (m, 2H), 5.08 (dd, J = 10.8, 2.5 Hz, 1H), 5.02 (t, J =

9.8 Hz, 1H), 4.63−4.46 (m, 3H), 4.19 (dd, J = 11.3, 3.4 Hz, 1H), 4.04

(dd, J = 11.3, 8.1 Hz, 1H), 3.72 (t, J = 5.2 Hz, 2H), 2.93 (s, 6H). Anal.

(C₂₆H₂₇ClN₅O₉PS·³/₄CF₃CO₂H) requires C, 44.78; H, 3.79; N, 9.50.

Found: C, 44.94; H, 4.19; N, 9.25.

2-(1-(Chloromethyl)-3-(5-(2-morpholinoethoxy)-1H-indole-2-car-

bonyl)-5-nitro-1,2-dihydro-3H-benzo[e]indole-7-sulfonamido)ethyl

Dihydrogen Phosphate (50). Prepared by general method D from

95e (164 mg, 0.19 mmol) to give the triﬂuoroacetate salt of 50 as a

yellow solid (158 mg, 98%): mp 166−169 °C. HPLC purity 97.8%. ¹H

NMR (DMSO-d₆) δ 11.84 (s, 1H), ca. 10.7 (v br s, 1H), 9.29 (s, 1H),

8.87 (d, J = 1.7 Hz, 1H), 8.45 (d, J = 8.9 Hz, 1H), 8.27−8.20 (m, 1H),

8.03 (dd, J = 8.9, 1.7 Hz, 1H), 7.44 (d, J = 8.9 Hz, 1H), 7.26 (d, J = 2.2

Hz, 1H), 7.24 (d, J = 1.8 Hz, 1H), 7.02 (dd, J = 8.9, 2.3 Hz, 1H),

5.01−4.94 (m, 1H), 4.71 (dd, J = 10.8, 2.1 Hz, 1H), 4.68−4.62 (m,

1H), 4.34−4.29 (m, 2H), 4.19−4.09 (m, 2H), 3.87−3.76 (m, 6H),

3.26−3.13 (m, 4H), 3.06−3.00 (m, 2H), 2H not observed. Anal.

(C₃₀H₃₃ClN₅O₁₁PS·CF₃CO₂H) requires C, 45.11; H, 4.02; N, 8.22.

Found: C, 44.72; H, 4.36; N, 8.57.

2-(1-(Chloromethyl)-3-(5-(3-morpholinopropoxy)-1H-indole-2-

carbonyl)-5-nitro-1,2-dihydro-3H-benzo[e]indole-7-sulfonamido)-

ethyl Dihydrogen Phosphate (51). Prepared by general method D

from 95f (162 mg, 0.19 mmol) to give the triﬂuoroacetate salt of 51 as

a pale-yellow powder (165 mg, 100%): mp 165−168 °C. HPLC purity

98.4%. ¹H NMR (DMSO-d₆) δ 11.82 (d, J = 1.6 Hz, 1H), ca. 9.7 (v br

s, 1H), 9.30 (s, 1H), 8.87 (d, J = 1.6 Hz, 1H), 8.44 (d, J = 8.9 Hz, 1H),

8.20 (t, J = 5.9 Hz, 1H), 8.02 (dd, J = 8.9, 1.7 Hz, 1H), 7.44 (d, J = 8.9

Hz, 1H), 7.23 (d, J = 1.7 Hz, 1H), 7.20 (d, J = 2.2 Hz, 1H), 6.97 (dd, J

= 8.9, 2.4 Hz, 1H), 5.02−4.93 (m, 1H), 4.71 (dd, J = 10.9, 2.2 Hz,

1H), 4.68−4.62 (m, 1H), 4.20−3.97 (m, 6H), 3.81 (q, J = 6.5 Hz,

2H), 3.34−3.28 (m, 2H), 3.20−3.08 (m, 2H), 3.02 (q, J = 5.9 Hz,

2H), 2.21−2.10 (m, 2H), 4H not observed. Anal. (C₃₁H₃₅ClN₅O₁₁PS·

CF₃CO₂H·H₂O) requires C, 44.83; H, 4.33; N, 7.92. Found: C, 44.96;

H, 4.41; N, 8.20.

Hz, 1H), 8.14 (d, J = 8.9 Hz, 1H), 7.74 (d, J = 8.8 Hz, 2H), 7.16 (d, J

= 15.2 Hz, 2H), 7.10 (d, J = 8.8 Hz, 2H), 4.80 (br dd, J = 10.8, 2.6 Hz,

1H), 4.65 (br t, J = 10.0 Hz, 1H), 4.59−4.51 (m, 2H), 4.50−4.42 (m,

1H), 4.21−4.13 (m, 3H), 4.01 (br dd, J = 11.3, 8.3 Hz, 1H), 3.73 (br t,

J = 4.6 Hz, 6H), 2.76 (t, J = 5.7 Hz, 2H), 2.53 (t, J = 4.6 Hz, 4H). Anal.

(C₃₀H₃₄ClN₄O₁₁PS·¹/₂CF₃CO₂H) requires C, 47.61; H, 4.45; N, 7.16.

Found: C, 47.69; H, 4.65; N, 6.97.

(E)-2-(1-(Chloromethyl)-3-(3-(4-(3-morpholinopropoxy)phenyl)-

acryloyl)-5-nitro-1,2-dihydro-3H-benzo[e]indole-7-sulfonamido)-

ethyl Dihydrogen Phosphate Triﬂuoroacetate (55). Prepared by

general method D from 95j (218 mg, 0.256 mmol) to give the

triﬂuoroacetate salt of 55 as an orange solid (177 mg, 81%): mp 215

1

°C (dec). HPLC purity 97.0%. H NMR (DMSO-d₆) δ ca. 10.7 (v br

s, 1H), 9.33 (br s, 1H), 8.84 (d, J = 1.6 Hz, 1H), 8.36 (d, J = 8.9 Hz,

1H), 8.23 (br s, 1H), 8.01 (dd, J = 8.9, 1.6 Hz, 1H), 7.76 (d, J = 8.7

Hz, 2H), 7.70 (d, J = 15.2 Hz, 1H), 7.08 (d, J = 15.2 Hz, 1H), 6.97 (d,

J = 8.7 Hz, 2H), 4.70−4.55 (m, 3H), 4.17−4.02 (m, 4H), 4.85−3.66

(m, 6H), 3.10−2.82 (m, 8H), 2.12−2.00 (m, 2H). Anal.

(C₃₁H₃₆ClN₄O₁₁PS·¹/₂CF₃CO₂H) requires C, 48.28; H, 4.62; N,

7.04. Found: C, 47.94; H, 4.85; N, 6.80.

2-(1-(Chloromethyl)-3-(4-(2-(4-methylpiperazin-1-yl)ethoxy)-

benzoyl)-5-nitro-1,2-dihydro-3H-benzo[e]indole-7-sulfonamido)-

ethyl Dihydrogen Phosphate (56). Prepared by general method D

from 95k (1.57 g, 1.85 mmol) to give the triﬂuoroacetate salt of 56 as

a yellow−orange solid (1.37 g, 87%): mp 230 °C (dec). HPLC purity

1

95.0%. H NMR (DMSO-d₆) δ 9.35 (br s, 1H), 9.35 (br s, 1H), 8.85

(br d, J = 1.6 Hz, 1H), 8.38 (d, J = 8.9 Hz, 1H), 8.13 (t, J = 5.9 Hz,

1H), 8.02 (dd, J = 8.9, 1.6 Hz, 1H), 7.82 (d, J = 8.8 Hz, 2H), 7.74 (d, J

= 15.2 Hz, 1H), 7.11 (d, J = 15.2 Hz, 1H), 7.04 (d, J = 8.8 Hz, 2H),

4.12−4.58 (m, 3H), 4.21 (t, J = 5.2 Hz, 2H), 4.09 (br d, J = 4.9 Hz,

2H), 3.82 (q, J = 6.2 Hz, 2H), 3.51−2.85 (m, 12H), 2.77 (s, 3H), 2H

not observed. Anal. (C₃₁H₃₇ClN₅O₁₀P·1¹/₂CF₃CO₂H·EtOAc) requires

C, 45.77; H, 4.70; N, 7.02. Found: C, 45.72; H, 4.67; N, 7.09.

2-(1-(Chloromethyl)-3-(4-(2-morpholinoethoxy)benzoyl)-5-nitro-

1,2-dihydro-3H-benzo[e]indole-7-sulfonamido)ethyl Dihydrogen

Phosphate (57). Prepared by general method D from 95l (458 mg,

0.565 mmol) to give the triﬂuoroacetate salt of 57 as a yellow solid

1

(424 mg, 92%): mp 194 °C (dec). HPLC purity 96.7%. H NMR

(DMSO-d₆) δ ca. 10.6 (v br s, 1H), 8.90 (br s, 1H), 8.85 (br d, J = 1.6

Hz, 1H), 8.39 (d, J = 8.9 Hz, 1H), 8.16 (br s, 1H), 8.02 (d, J = 8.9, 1.7

Hz, 1H), 7.71 (d, J = 8.8 Hz, 2H), 7.16 (d, J = 8.8 Hz, 2H), 4.64 (dd, J

= 10.9, 9.4 Hz, 1H), 4.52−4.43 (m, 1H), 4.37 (t, J = 5.0 Hz, 2H), 4.17

(dd, J = 11.1, 1.7 Hz, 1H), 4.11−3.99 (m, 2H), 3.86−3.72 (m, 6H),

3 . 3 2 ( b r s , 2 H ) , 3 . 1 5 − 2 . 9 7 ( m , 6 H ) . A n a l .

(C₂₈H₃₂ClN₄O₁₁PS·³/₄CF₃CO₂H) requires C, 45.16; H, 4.21; N,

7.14. Found: C, 45.43; H, 4.49; N, 6.94.

2-(1-(Chloromethyl)-3-(3-morpholinopropanoyl)-5-nitro-1,2-di-

hydro-3H-benzo[e]indole-7-sulfonamido)ethyl Dihydrogen Phos-

phate (58). Prepared by general method D from 95m (407 mg,

0.566 mmol) to give the triﬂuoroacetate salt of 58 as a yellow−orange

solid (388 mg, 95%): mp 192 °C (dec). HPLC purity 94.6%. ¹H NMR

(C₅D₅N) δ ca. 10.7 (v br s, 1H), 9.65 (br s, 1H), 9.44 (br d, J = 1.5

Hz, 1H), 8.38 (dd, J = 8.9, 1.6 Hz, 1H), 8.13 (d, J = 8.9 Hz, 1H),

4.61−4.38 (m, 5H), 4.15 (dd, J = 11.3, 3.2 Hz, 1H), 3.97 (dd, J = 11.3,

8.1 Hz, 1H), 3.76 (t, J = 4.5 Hz, 4H), 3.71 (t, J = 5.2 Hz, 2H), 3.04−

2.81 (m, 4H), 2.56 (t, J = 4.5 Hz, 4H), 3H not observed. Anal.

(C₂₂H₂₈ClN₄O₁₀PS·CF₃CO₂H) requires C, 39.98; H, 4.05; N, 7.77.

Found: C, 39.91; H, 4.08; N, 7.54.

2-(1-(Chloromethyl)-3-(5-(2-morpholinoethoxy)-1H-indole-2-car-

bonyl)-5-nitro-1,2-dihydro-3H-benzo[e]indole-7-carboxamido)ethyl

Dihydrogen Phosphate (60). HCl (saturated solution in DCM, 1.0

mL) was added to a solution of 97e (197 mg, 0.242 mmol) in DCM

(10 mL), and the mixture was stirred at rt for 22 h and then

evaporated. EtOAc (50 mL) was added, and the mixture was stirred at

0 °C for 2 h. The solid was ﬁltered oﬀ, washed with EtOAc, and dried

to give crude 60 as the hydrochloride salt (172 mg, 96%). LC-MS

analysis showed the crude product contained 85% of the desired 60

and 13% N-(2-chloroethyl)-1-(chloromethyl)-3-(5-(2-morpholinoe-

thoxy)-1H-indole-2-carbonyl)-5-nitro-1,2-dihydro-3H-benzo[e]indole-

7-carboxamide. The crude product was dissolved in the minimum

amount of DMSO (3 mL), and DCM (20 mL) was added slowly. The

2-(1-(Chloromethyl)-3-(5-(2-(dimethylamino)acetamido)-1H-in-

dole-2-carbonyl)-5-nitro-1,2-dihydro-3H-benzo[e]indole-7-

sulfonamido)ethyl Dihydrogen Phosphate (52). Prepared by general

method D from 95g (348 mg, 0.424 mmol) to give the triﬂuoroacetate

salt of 52 as a yellow−orange solid (321 mg, 92%): mp 215−218 °C

1

(dec). HPLC purity 98.2%. H NMR (DMSO-d₆) δ 11.85 (s, 1H),

10.39 (s, 1H), 9.27 (s, 1H), 8.86 (d, J = 1.6 Hz, 1H), 8.37 (br s, 1H),

8.10 (br d, J = 1.5 Hz, 1H), 8.03 (dd, J = 8.9, 1.6 Hz, 1H), 7.40 (d, J =

8.8 Hz, 1H), 7.38−7.25 (m, 2H), 4.94 (t, J = 10.2 Hz, 1H), 4.69 (br

dd, J = 10.8, 1.9 Hz, 1H), 4.67−4.57 (m, 1H), 4.20−4.07 (m, 2H),

3.87−3.77 (m, 2H), 3.11−3.02 (m, 2H), 2.78 (s, 6H), 3H not

observed. Anal. (C₂₈H₃₀ClN₆O₁₀PS·³/₄CF₃CO₂H) requires C, 44.59;

H, 3.90; N, 10.58. Found: C, 44.19; H, 4.11; N, 10.41.

2-(1-(Chloromethyl)-3-(5-(3-(dimethylamino)propanamido)-1H-

indole-2-carbonyl)-5-nitro-2,3-dihydro-1H-benzo[e]indole-7-

sulfonamido)ethyl Dihydrogen Phosphate (53). Prepared by general

method D from 95h (476 mg, 0.57 mmol) to give the triﬂuoroacetate

salt of 53 as a yellow solid (438 mg, 92%): mp 215−218 °C (dec).

1

HPLC purity 95.4%. H NMR (DMSO-d₆) δ 11.70 (s, 1H), 10.25 (s,

1H), 9.14 (s, 1H), 8.83 (s, 1H), 8.52 (br s, 1H), 8.24 (br s, 1H), 8.06

(br s, 1H), 8.00 (dd, J = 8.8, 1.5 Hz, 1H), 7.27 (br s, 1H), 7.12 (br s,

1H), 6.89 (br s, 1H), 4.88−4.79 (m, 1H), 4.65−4.43 (m, 2H), 4.12−

3.96 (m, 2H), 3.86−3.76 (m, 2H), 3.37 (t, J = 7.2 Hz, 2H), 3.12−3.04

(m, 2H), 2.85−2.73 (m, 8H), 3 H not observed. Anal.

(C₂₉H₃₂ClN₆O₁₀PS·³/₄CF₃CO₂H) requires C, 45.30; H, 4.08; N,

10.39. Found: C, 45.65; H, 4.34; N, 10.43.

(E)-2-(1-(Chloromethyl)-3-(3-(4-(2-morpholinoethoxy)phenyl)-

acryloyl)-5-nitro-1,2-dihydro-3H-benzo[e]indole-7-sulfonamido)-

ethyl Dihydrogen Phosphate (54). Prepared by general method D

from 95i (366 mg, 0.437 mmol) to give the triﬂuoroacetate salt of 54

as a yellow solid (292 mg, 80%): mp 212 °C (dec). HPLC purity

98.3%. ¹H NMR (C₅D₅N) δ ca. 10.7 (v br s, 1H), 9.81 (br s, 1H), 9.46

(d, J = 1.6 Hz, 1H), 8.39 (dd, J = 8.9, 1.6 Hz, 1H), 8.22 (d, J = 15.2

N

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Journal of Medicinal Chemistry

Article

resulting mixture was stirred at rt for 1 h, and the precipitated solid

was ﬁltered oﬀ, washed with DCM, and dried to give the

hydrochloride salt of 60 as a yellow−orange solid (153 mg, 85%),

mp 225 °C (dec). HPLC purity 98.1%. ¹H NMR (DMSO-d₆) δ 11.80

(br d, J = 1.6 Hz, 1H), ca. 11.0 (v br s, 3H), 9.17 (s, 1H), 8.97 (t, J =

5.5 Hz, 1H), 8.84 (br d, J = 1.3 Hz, 1H), 8.32 (d, J = 8.8 Hz, 1H), 8.16

(dd, J = 8.8, 1.5 Hz, 1H), 7.46 (d, J = 8.9 Hz, 1H), 7.27 (br d, J = 2.3

Hz, 1H), 7.22 (br d, J = 1.6 Hz, 1H), 4.96 (br t, J = 10.2 Hz, 1H),

4.75−4.58 (m, 2H), 4.41 (t, J = 4.9 Hz, 2H), 4.20−3.77 (m, 8H),

3.64−3.12 (m, 8H). Anal. (C₃₁H₃₃ClN₅O₁₀·HCl·H₂O) requires C,

49.22; H, 4.80; N, 9.26. Found: C, 49.14; H, 4.83; N, 9.17.

EtOAc:MeOH 9:1, 8:2, 7:3, 6:4) followed by recrystallization from aq

EtOH gave 66 as cream crystals (0.57 g, 34%): mp 118−120 °C. H

1

NMR (DMSO-d₆) δ 11.67 (s, 1H), 7.33 (d, J = 8.9 Hz, 1H), 7.09 (d, J

= 2.3 Hz, 1H), 7.03 (s, 1H), 6.90 (dd, J = 8.9, 2.5 Hz, 1H), 4.32 (q, J =

7.1 Hz, 2H), 3.97 (t, J = 6.5 Hz, 2H), 2.36 (t, J = 7.3 Hz, 2H), 2.14 (s,

6H), 1.85 (quin, J = 6.8 Hz, 2H), 1.33 (t, J = 7.1 Hz, 3H). Anal.

C₁₆H₂₂N₂O₃requires C, 66.19; H, 7.64; N, 9.65. Found: C, 65.95; H,

7.85; N, 9.79.

5-(3-(Dimethylamino)propoxy)-1H-indole-2-carboxylic Acid (67).

A solution of KOH (430 mg, 7.7 mmol) in water (10 mL) was added

to a solution of 66 (543 mg, 1.9 mmol) in EtOH (20 mL), and the

mixture was stirred at reﬂux for 5 min and then cooled to rt. The

EtOH was evaporated, and the aq portion was neutralized by the

addition of aq HCl (2N, 3.8 mL). The mixture was allowed to stand at

4 °C overnight, and the white precipitate was ﬁltered oﬀ and dried

(475 mg, 97% as free base). This solid was stirred with HCl-saturated

dioxane (30 mL) for 2 h. The solid was ﬁltered oﬀ and dried to give

the hydrochloride salt of 67 as a white solid (407 mg, 73%): mp 229−

231 °C. ¹H NMR (DMSO-d₆) δ 12.8 (v br s, 1H), 11.61 (s, 1H), 10.2

(v br s, 1H), 7.34 (d, J = 8.9 Hz, 1H), 7.12 (d, J = 2.4 Hz, 1H), 7.00−

6.97 (m, 1H), 6.91 (dd, J = 8.9, 2.4 Hz, 1H), 4.05 (t, J = 6.0 Hz, 2H),

3.25−3.19 (m, 2H), 2.79 (s, 6H), 2.18−2.10 (m, 2H). Anal.

C₁₄H₁₈N₂O₃·HCl·¹/₄H₂O requires C, 55.45; H, 6.48; N, 9.24.

Found: C, 55.57; H, 6.34; N, 9.25.

(E)-2-(1-(Chloromethyl)-3-(3-(4-(2-morpholinoethoxy)phenyl)-

acryloyl)-5-nitro-1,2-dihydro-3H-benzo[e]indole-7-carboxamido)-

ethyl Dihydrogen phosphate (61). HCl (saturated solution in DCM,

10 mL) was added to a solution of phosphate ester 97i (190 mg, 0.237

mmol) in DCM (10 mL), and the mixture was stirred at rt for 18 h

and then evaporated. EtOAc was added, and the mixture was stirred at

0 °C for 2 h. The solid was ﬁltered oﬀ, washed with EtOAc, and dried

to give the hydrochloride salt of 61 as a yellow−orange solid (147 mg,

86%): mp 229 °C (dec). HPLC purity 93.8%. ¹H NMR (DMSO-d₆) δ

ca. 10.6 (v br s, 1H), 9.23 (br s, 1H), 8.97 (br t, J = 4.7 Hz, 1H), 8.82

(br d, J = 0.9 Hz, 1H), 8.26 (d, J = 8.8 Hz, 1H), 8.14 (br d, J = 8.7 Hz,

1H), 7.83 (d, J = 8.5 Hz, 2H), 7.73 (d, J = 15.3 Hz, 1H), 7.12 (d, J =

15.3 Hz, 1H), 7.08 (d, J = 8.5 Hz, 2H), 4.72−4.56 (m, 3H), 4.39 (br s,

2H), 4.12−3.95 (m, 4H), 3.80 (br s, 4H), 3.55 (q, J = 5.6 Hz, 2H),

3.37 (br s, 2H), 3.15 (br s, 4H). Anal. (C₃₁H₃₄ClN₄O₁₀P·HCl·¹/₄H₂O)

requires C, 51.07; H, 4.89; N, 7.68. Found: C, 51.00; H, 4.90; N, 7.68.

Ethyl 5-(2-(Diethylamino)ethoxy)-1H-indole-2-carboxylate (63).

4-(2-(Diethylamino)ethoxy)aniline (62)⁵⁸was subjected to a Fischer

indole synthesis, as described for the morpholine analogue⁴²to give 63

as an orange oil (77%). ¹H NMR (DMSO-d₆) δ 11.68 (s, 1H), 7.33 (d,

J = 8.9 Hz, 1H), 7.11 (d, J = 2.4 Hz, 1H), 7.04−7.01 (m, 1H), 6.90

(dd, J = 8.9, 2.4 Hz, 1H), 4.32 (q, J = 7.1 Hz, 2H), 4.00 (t, J = 6.2 Hz,

2H), 2.77 (t, J = 6.2 Hz, 2H), 2.55 (q, J = 7.1 Hz, 4H), 1.33 (t, J = 7.1

Hz, 3H), 0.80 (t, J = 7.1 Hz, 6H). HRMS (EI) calcd for C₁₇H₂₄N₂O₃

(M⁺) m/z 304.1787, found 304.1790. 63 was treated with 4 M HCl in

dioxane to give the corresponding hydrochloride salt as a beige solid:

mp 95−97 °C. Anal. C₁₇H₂₄N₂O₃·HCl requires C, 59.90; H, 7.39; N,

8.22. Found: C, 59.97; H, 7.55; N, 7.83.

Ethyl 1-Acetyl-5-methyl-1H-indole-2-carboxylate (69). NaH (60%

in oil, 1.42 g, 59.0 mmol) was added in portions to a stirred solution of

ethyl 5-methyl-1H-indole-2-carboxylate (68) (10.0 g, 49.2 mmol) in

DMF (40 mL) at rt, and the mixture was stirred for 15 min. The

reaction ﬂask was cooled in an ice bath, and AcCl (4.89 mL, 68.9

mmol) was added dropwise over 5 min. The mixture was stirred at rt

for 3 h and then poured into ice−water (400 mL) and extracted with

EtOAc (500 mL). The extract was dried and evaporated to give an oil

that was puriﬁed by column chromatography (EtOAc:petroleum ether

1

1:20) to give 69 (10.8 g, 89%) as a yellow oil. H NMR (CDCl₃) δ

8.01 (d, J = 8.6 Hz, 1H), 7.41−7.37 (m, 1H), 7.29−7.22 (m, 2H), 4.40

(q, J = 7.1 Hz, 2H), 2.6 (s, 3H), 2.44 (s, 3H), 1.41 (s, 3H). HRMS

(FAB) calcd for C₁₄H₁₆NO₃(MH⁺) m/z 246.1130, found 246.1130.

Ethyl 5-((Dimethylamino)methyl)-1H-indole-2-carboxylate (70).

NBS (12.0 g, 67.6 mmol) and AIBN (1.51 g, 9.2 mmol) were added to

a solution of 69 (10.8 g, 44.1 mmol) in CCl₄(150 mL), and the

mixture was stirred at reﬂux for 3 h. The mixture was cooled, diluted

with DCM (300 mL), and washed with water (500 mL). The organic

layer was separated, dried, and evaporated to give a yellow oil which

was dissolved in DMF (70 mL) and cooled to °C. Gaseous

dimethylamine was bubbled into this solution for 10 min, and then

the mixture was stirred at rt for 18 h. The mixture was partitioned

between EtOAc (400 mL) cold aq Na₂CO₃(2%). The EtOAc layer

was separated, dried, and evaporated to give an amber oil which was

puriﬁed by column chromatography (DCM:MeOH 10:1) to give 70 as

5-(2-(Diethylamino)ethoxy)-1H-indole-2-carboxylic Acid (64).

KOH (9.8 g, 175 mmol) was added to a suspension of 63 (17.7 g,

58.2 mmol) in MeOH (120 mL) and water (60 mL), and the mixture

was stirred at rt for 3 h. The MeOH was evaporated, and the aqueous

residue was diluted with water (150 mL). The mixture was acidiﬁed

with concd HCl at 0 °C to pH 6−7. The solid was collected, washed

with cold water (3 × 25 mL), and dried to give 64 (16.1 g, 100%): mp

1

238−240 °C. H NMR (DMSO-d₆) δ 11.52 (s, 1H), 7.31 (d, J = 8.9

Hz, 1H), 7.10 (d, J = 2.4 Hz, 1H), 6.97−6.92 (m, 1H), 6.88 (dd, J =

8.9, 2.4 Hz, 1H), 4.04 (t, J = 6.0 Hz, 2H), 2.89 (t, J = 6.0 Hz, 2H), 2.65

(q, J = 7.1 Hz, 4H), 1.02 (t, J = 7.1 Hz, 6H), 1H not observed. Anal.

C₁₅H₂₀N₂O₃·¹/₂H₂O requires C, 63.14; H, 7.42; N, 9.82. Found: C,

62.93; H, 7.32; N, 9.90. 64 (15.8 g, 57.4 mmol) was treated with 4 M

HCl in dioxane to give the corresponding hydrochloride salt (13.6 g,

76%): mp 198−200 °C (dec). ¹H NMR (DMSO-d₆) δ 12.86 (s, 1H),

11.66 (s, 1H), 10.11 (s, 1H), 7.37 (d, J = 8.9 Hz, 1H), 7.19 (d, J = 2.4

Hz, 1H), 7.04−6.99 (m, 1H), 6.97 (dd, J = 8.9, 2.4 Hz, 1H), 4.34 (t, J

= 4.9 Hz, 2H), 3.55−3.47 (m, 2H), 3.28−3.14 (m, 4H), 1.25 (t, J = 7.2

Hz, 6H). Anal. C₁₅H₂₁ClN₂O₃·H₂O requires C, 54.46; H, 7.01; N,

8.47. Found: C, 54.55; H, 7.04; N, 8.44.

1

a pale-amber gum (2.61 g, 24%). H NMR (CDCl₃) δ 8.86 (s, 1H),

7.58 (s, 1H), 7.37 (d, J = 8.5 Hz, 1H), 7.31 (dd, J = 8.5, 1.5 Hz, 1H),

7.21−7.15 (m, 1H), 4.40 (q, J = 7.1 Hz, 2H), 3.52 (s, 2H), 2.27 (s,

6H), 1.41 (t, J = 7.1 Hz, 3H). 70 was treated with 4 M HCl in dioxane

and EtOAc to give the corresponding hydrochloride salt as a pale

brown solid: mp 133−135 °C. Anal. C₁₄H₁₈N₂O₂·HCl·H₂O requires

C, 55.90; H, 7.04; N, 9.31. Found: C, 55.53; H, 6.91; N, 9.11.

5-((Dimethylamino)methyl)-1H-indole-2-carboxylic Acid (71).

KOH (1.57 g, 28.1 mmol) was added to a mixture of 70 (2.3 g,

9.35 mmol), MeOH (40 mL), and water (20 mL), and the mixture

was stirred at rt for 5 h. The MeOH was evaporated, and the aqueous

residue was stirred with petroleum ether for 15 min. The petroleum

ether layer was separated and discarded. The aqueous layer was

acidiﬁed with concd HCl to pH ca. 6−7 at 0 °C. The solid was ﬁltered

oﬀ, washed with cold water (3 × 5 mL), and dried to give 71 (1.43 g,

70%): mp 266−268 °C (dec). ¹H NMR (DMSO-d₆) δ 11.58 (s, 1H),

7.52 (s, 1H), 7.37 (d, J = 8.5 Hz, 1H), 7.19 (dd, J = 8.5, 1.5 Hz, 1H),

7.01−6.96 (m, 1H), 3.54 (s, 2H), 2.22 (s, 6H), 1H not observed. 71

was treated with 4 M HCl in dioxane to give the corresponding

hydrochloride salt: mp 273−275 °C. Anal. C₁₂H₁₄N₂O₂·HCl requires

C, 56.58; H, 5.94; N, 11.00. Found: C, 56.51; H, 5.91; N, 10.78.

Ethyl 5-(3-(Dimethylamino)propoxy)-1H-indole-2-carboxylate

(66). DEAD (1.52 g, 8.7 mmol) was added dropwise to a solution

of 3-(dimethylamino)-1-propanol (1.03 mL, 8.7 mmol), ethyl 5-

hydroxy-1H-indole-2-carboxylate (65) (1.45 g, 5.8 mmol), and

triphenylphosphine (2.29 g, 8.7 mmol) in dry THF (40 mL) while

cooling in a water bath. The solution was stirred at rt for 4 h. The

mixture was diluted with EtOAc, washed with water (×2), and then

extracted with aq HCl (2N, ×2). The acidic extracts were cooled in an

ice bath and basiﬁed with concd aq NH₃. The mixture was extracted

with EtOAc (×2), and the extracts were dried and evaporated to give a

light-brown oil. Puriﬁcation by column chromatography (EtOAc then

O

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Article

Ethyl 5-(3-Morpholinopropoxy)-1H-indole-2-carboxylate (73).

Pd/C (5%) was added to a solution of ethyl 5-(benzyloxy)-1H-

indole-2-carboxylate (72) (5.10 g, 17.3 mmol) in EtOAc (40 mL),

THF (40 mL), and EtOH (30 mL). The mixture was hydrogenated at

50 psi for 17 h, then ﬁltered through Celite. The ﬁltrate was

evaporated to give crude ethyl 5-hydroxy-1H-indole-2-carboxylate as a

gray solid. A portion of this material (5.51 mmol) was dissolved in

THF (40 mL) with triphenylphosphine (2.17 g, 8.3 mmol) and 4-(3-

hydroxypropyl)morpholine (1.20 g, 8.3 mmol). DEAD (1.44 g, 8.3

mmol) was added dropwise, and the mixture was stirred at rt for 2.5 h.

The THF was evaporated, and the residue was partitioned between

EtOAc and water. The EtOAc layer was washed with brine (×3) and

extracted with aq HCl (2N, ×2). The combined acid extracts were

cooled to 0 °C, basiﬁed with concd aq NH₃, and extracted with EtOAc

(×3). The combined organic extracts were dried and evaporated to

give a tan solid. Recrystallization from EtOH gave 73 as pale-yellow

crystals (0.86 g, 47%): mp 152−155 °C. ¹H NMR (DMSO-d₆) δ 11.68

(s, 1H), 7.33 (d, J = 8.9 Hz, 1H), 7.10 (d, J = 2.3 Hz, 1H), 7.03 (s,

1H), 6.91 (dd, J = 8.9, 2.4 Hz, 1H), 4.33 (q, J = 7.1 Hz, 2H), 3.99 (t, J

= 6.4 Hz, 2H), 3.60−3.56 (m, 4H), 2.43 (t, J = 7.2 Hz, 2H), 2.39−2.35

(m, 4H), 1.93−1.84 (m, 2H), 1.33 (t, J = 7.1 Hz, 3H). Anal.

C₁₈H₂₄N₂O₄requires C, 65.04; H, 7.28; N, 8.43. Found: C, 64.70; H,

7.58; N, 8.56.

5-(3-Morpholinopropoxy)-1H-indole-2-carboxylic Acid (74). A

solution of KOH (651 mg, 11.6 mmol) in water (10 mL) was

added to a suspension of 73 (811 mg, 2.44 mmol) in EtOH (20 mL).

The mixture was stirred at reﬂux for 10 min then cooled to rt. The

EtOH was evaporated, and the aqueous residue was neutralized by the

addition of aq HCl (2N, 5.80 mL, 11.6 mmol). An oil separated that

solidiﬁed on standing. The solid was ﬁltered oﬀ and dried, then

suspended in dioxane (30 mL) and treated with HCl-saturated dioxane

(10 mL). After stirring at room temperature for 3 h, the solid was

ﬁltered oﬀ and dried to give the hydrochloride salt of 74 as a white

solid (824 mg, 99%): mp 246−249 °C. ¹H NMR (DMSO-d₆) δ 12.81

(br s, 1H), 11.61 (s, 1H), 10.69 (br s, 1H), 7.34 (d, J = 8.9 Hz, 1H),

7.13 (d, J = 2.3 Hz, 1H), 6.99 (dd, J = 2.0, 0.6 Hz, 1H), 6.91 (dd, J =

8.9, 2.4 Hz, 1H), 4.06 (t, J = 6.0 Hz, 2H), 4.01−3.92 (m, 2H), 3.85−

3.74 (m, 2H), 3.53−3.43 (m, 2H), 3.16−3.03 (m, 2H), 2.24−2.14 (m,

2H), 2H obscured by water peak observed after D₂O exchange. Anal.

C₁₆H₂₀N₂O₄·HCl·¹/₂H₂O requires C, 54.94; H, 6.34; N, 8.01. Found:

C, 55.00; H, 6.62; N, 7.64.

(t, J = 7.1 Hz, 2H), 2.47 (t, J = 7.1 Hz, 2H), 2.24 (s, 6H), 1H not

observed. Anal. C₁₄H₁₇N₃O₃·³/₄H₂O requires C, 58.22; H, 6.46; N,

14.55; found: C, 57.88; H, 6.57; N, 14.47. 77 (2.38 g, 8.65 mmol) was

treated with 4 M HCl in dioxane to give the corresponding

1

hydrochloride salt (2.71 g, 100%): mp 221−223 °C (dec). H NMR

(DMSO-d₆) δ 12.85 (br s, 1H), 11.68 (s, 1H), 10.18 (s, 1H), 9.95 (br

s, 1H), 7.99 (br s, 1H), 7.37 (d, J = 8.8 Hz, 1H), 7.34 (dd, J = 8.8, 1.8

Hz, 1H), 7.04 (d, J = 1.6 Hz, 1H), 3.37 (t, J = 7.1 Hz, 2H), 2.85 (t, J =

7.1 Hz, 2H), 2.79 (s, 6H).

(E)-Methyl 3-(4-(2-Morpholinoethoxy)phenyl)acrylate (79). A

mixture of 4-(2-chloroethyl)morpholine hydrochloride (6.42 g, 34.5

mmol), NaOH (1.52 g, 38.1 mmol), water (24 mL), and toluene (26

mL) was stirred at 0 °C and saturated with solid NaCl. The toluene

layer was separated, and the alkaline solution was extracted with

toluene (4 × 15 mL). The combined toluene extracts were dried over

KOH and used for the following reaction. NaH (60% in oil, 1.28 g,

32.0 mmol) was added in portions to a solution of (E)-methyl 3-(4-

hydroxyphenyl)acrylate (78) (5.43 g, 30.5 mmol) in dry ether (100

mL) and stirred under nitrogen at rt. After the addition was complete,

the mixture was stirred for a further 2 h. Most of the ether was

evaporated, petroleum ether (100 mL) was added, and the mixture was

stirred for 5 min. The petroleum ether was decanted, and the process

was repeated once more. To the residue was added toluene (26 mL),

followed by the toluene solution of 4-(2-chloroethyl)morpholine

prepared above. The mixture was stirred at reﬂux for 92 h and then

cooled and ﬁltered through a short column of neutral Al₂O₃eluting

with hot toluene. The combined eluates were washed successively with

cold aq NaHCO₃and then with water, then dried and evaporated to

1

give 79 as a colorless solid (6.66 g, 75%): mp 61−62 °C. H NMR

(CDCl₃) δ 7.64 (d, J = 16.0 Hz, 1H), 7.51−7.45 (m, 2H), 6.93−6.88

(m, 2H), 6.31 (d, J = 16.0 Hz, 1H), 4.14 (t, J = 5.7 Hz, 2H), 3.79 (s,

3H), 3.77−3.70 (m, 4H), 2.81 (t, J = 5.7 Hz, 2H), 2.61−2.55 (m, 4H).

Anal. C₁₆H₂₁NO₄requires C, 65.96; H, 7.27; N, 4.81. Found: C, 65.98;

H, 7.33; N, 4.82.

(E)-3-(4-(2-Morpholinoethoxy)phenyl)acrylic Acid (80). A mixture

of 79 (6.58 g, 22.6 mmol), MeOH (40 mL), water (20 mL), and KOH

(3.16 g, 56.5 mmol) was stirred at 20 °C for 21 h. The mixture was

evaporated at 35 °C to remove the MeOH. The resulting alkaline

mixture was washed with CH₂Cl₂, and the aqueous layer was ﬁltered.

The alkaline ﬁltrate was acidiﬁed with concd HCl at 0 °C. The

resulting precipitate was ﬁltered oﬀ, washed with 6 N HCl, and dried

to give the hydrochloride salt of 80 as a colorless solid (5.90 g, 83%):

mp 252−254 °C. ¹H NMR (DMSO-d₆) δ 12.22 (br s, 1H), 10.90 (br

s, 1H), 7.69 (d, J = 8.8 Hz, 2H), 7.56 (d, J = 16.0 Hz, 1H), 7.05 (d, J =

8.8 Hz, 2H), 6.41 (d, J = 16.0 Hz, 1H), 4.46 (br s, 2H), 4.06−3.73 (m,

4H), 3.67−3.42 (m, 4H), 3.28−3.14 (m, 2H). Anal. C₁₅H₁₉NO₄·

HCl·¹/₃H₂O requires C, 56.34; H, 6.51; N, 4.38. Found: C, 56.49; H,

6.46; N, 4.42.

4-(3-Bromopropyl)morpholine (81). A solution of morpholine

(8.71 g, 100 mmol) in EtOAc (500 mL) was added dropwise over 3 h

to a solution of 1,3-dibromopropane (20.2 g, 100 mmol) in EtOAc

(500 mL) at 0 °C. The mixture was stirred at 0 °C for 5 h then at rt for

a further 110 h. The solid was ﬁltered oﬀ and washed with EtOAc (3 ×

100 mL). The combined ﬁltrates were washed successively with cold

aq Na₂CO₃(2%) and water (×2) and then dried. The resulting

solution was acidiﬁed with HCl (4 M in dioxane, ca. 10 mL) and then

evaporated to dryness. The residue was dried to give the hydrochloride

salt of 81 as a colorless solid (5.1 g, 21%): mp 134−136 °C. ¹H NMR

(CDCl₃) δ 13.37 (br s, 1H), 4.30 (br s, 2H), 4.00 (br s, 2H), 3.52 (t, J

= 6.0 Hz, 2H), 3.45 (br s, 2H), 3.15 (t, J = 8.0 Hz, 2H), 2.91 (s, 2H),

2.63−2.51 (m, 2H). Anal. C₇H₁₄BrNO·HCl requires C, 34.38; H,

6.18; N, 5.73. Found: C, 34.54; H, 6.20; N, 5.72.

Ethyl 5-(3-(Dimethylamino)propanamido)-1H-indole-2-carboxy-

late (76). A mixture of ethyl 5-amino-1H-indole-2-carboxylate (75)⁴²

(2.74 g, 13.4 mmol), 3-(dimethylamino)propanoic acid hydrochloride

(3.09 g, 20.1 mmol), and EDCI (7.71 g, 40.2 mmol) in DMA (50 mL)

was stirred at rt for 18 h. The mixture was cooled in an ice bath, and

EtOAc (500 mL) and water (500 mL) were added. The aq layer was

basiﬁed with aq Na₂CO₃(2%), and the EtOAc layer was separated.

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

combined EtOAc layers were dried and evaporated to give a brown

solid, which was puriﬁed by column chromatography (DCM:EtOAc

1:1 then DCM:MeOH 10:1) to give 76 as a light-brown foam (3.17 g,

1

78%). H NMR (CDCl₃) δ 10.78 (s, 1H), 8.82 (s, 1H), 7.98 (s, 1H),

7.37−7.30 (m, 2H), 7.17 (d, J = 1.5 Hz, 1H), 4.40 (q, J = 7.1 Hz, 2H),

2.68 (t, J = 7.3 Hz, 2H), 2.53 (t, J = 7.3 Hz, 2H), 2.40 (s, 6H), 1.41 (t,

J = 7.1, 3H). HRMS (FAB) calcd for C₁₆H₂₂N₃O₃(MH⁺) m/z

304.1661, found 304.1659.

5-(3-(Dimethylamino)propanamido)-1H-indole-2-carboxylic

Acid (77). KOH (1.73 g, 30.9 mmol) was added to a suspension of 76

(3.12 g, 10.3 mmol) in MeOH (40 mL) and water (20 mL), and the

mixture was stirred at rt for 3 h. The MeOH was evaporated, and the

aqueous residue was diluted with water (15 mL) and acidiﬁed with

concd HCl at 0 °C to pH 6−7. The solvent was evaporated to give a

brown residue, which was extracted with DCM:MeOH (5:1) several

times. The extracts were evaporated to give a gray solid (3.29 g), which

was stirred with water (20 mL) at 0 °C for 2 h. The precipitate was

collected, washed with cold water (5 × 3 mL), and dried to give 77 as

the free base (2.39 g, 85%): mp 215−217 °C. ¹H NMR (DMSO-d₆) δ

11.54 (s, 1H), 9.90 (s, 1H), 7.96 (d, J = 1.5 Hz, 1H), 7.33 (d, J = 8.8

Hz, 1H), 7.27 (dd, J = 8.8, 1.9 Hz, 1H), 6.97 (d, J = 1.4 Hz, 1H), 2.65

(E)-3-(4-(3-Morpholinopropoxy)phenyl)acrylic Acid (82). A mix-

ture of the hydrochloride salt of 81 (8.44 g, 34.5 mmol), NaOH (1.52

g, 38.1 mmol), water (24 mL), and toluene (26 mL) was stirred at 0

°C and saturated with solid NaCl. The toluene layer was separated,

and the alkaline solution was extracted with toluene (4 × 15 mL). The

combined toluene extracts were dried over KOH and used for the

following reaction. A toluene solution of the sodium salt of 78 (5.39 g,

30.3 mmol) was prepared as described in the preparation of 79. To

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Article

this was added the toluene solution of 4-(3-bromopropyl)morpholine,

and the mixture was stirred at reﬂux for 46 h, then cooled and ﬁltered

through a short column of neutral Al₂O₃, eluting with hot toluene. The

combined eluates were washed successively with cold aq NaHCO₃and

water and then dried and evaporated to give a beige solid. The solid

was treated with KOH (4.25 g, 75.8 mmol) in MeOH (60 mL) and

water (30 mL) at rt for 20 h. The mixture was evaporated at 35 °C to

remove the MeOH then washed with DCM and ﬁltered. The alkaline

ﬁltrate was acidiﬁed with concd HCl at 0 °C to pH < 1. The

precipitated solid was ﬁltered oﬀ, washed with 6 N HCl, and dried to

give the hydrochloride salt of 82 as a colorless solid (7.2 g, 73%): mp

225−227 °C. ¹H NMR (DMSO-d₆) δ 12.19 (br s, 1H), 11.22 (s, 1H),

7.64 (d, J = 8.7 Hz, 2H), 7.54 (d, J = 16.0 Hz, 1H), 6.98 (d, J = 8.7 Hz,

2H), 6.40 (d, J = 16.0 Hz, 1H), 4.12 (t, J = 6.1 Hz, 2H), 4.03−3.76 (m,

4H), 3.53−3.00 (m, 6H), 2.33−2.14 (m, 2H). Anal. C₁₆H₂₁NO₄·HCl

requires C, 58.62; H, 6.77; N, 4.27. Found: C, 58.49; H, 6.94; N, 4.25.

(E)-Methyl 3-(4-(2-(4-Methylpiperazin-1-yl)ethoxy)phenyl)-

acrylate (83). NaH (60% in oil, 0.77 g, 32.0 mmol) was added to a

solution of 78 (5.4 g, 30.5 mmol) in dry ether (100 mL), and the

mixture was stirred under nitrogen at rt for 6 h. The ether was

evaporated, and DMA (20 mL) was added, followed by 1,2-

dibromoethane (13.2 mL, 153 mmol). The mixture was stirred at

130−135 °C for 21 h and then cooled to rt. More NaH (60% in oil,

0.77 g, 32.0 mmol) and 1,2-dibromoethane (13.2 mL, 153 mmol) were

added, and the mixture was stirred at 150−160 °C for 5 h, then cooled

and diluted with toluene (100 mL). The mixture was ﬁltered, and the

ﬁlter pad was washed with hot toluene several times. The combined

ﬁltrates were washed successively with cold water, cold aq NaHCO₃,

and again with water. The organic layer was dried and evaporated to

give an oil which was dissolved in DMF (30 mL) and stirred with 1-

methylpiperazine (14.7 mL, 133 mmol) at rt for 3 h. The mixture was

diluted with EtOAc (200 mL) and then stirred with aq Na₂CO₃(2%)

at 0 °C for 5−10 min. The organic layer was separated, washed with

water (×2), and then dried and evaporated. The resulting oil was

dissolved in EtOAc, and the solution was treated with HCl (4 M in

dioxane, 17 mL, 68 mmol). The mixture was stirred and then left to

stand at 5 °C overnight. The solid was ﬁltered oﬀ, washed with EtOAc

several times, and dried to give the hydrochloride salt of 83 as a beige

successively with cold aq NaHCO₃and water and then dried and

evaporated to give crude methyl 4-(2-morpholinoethoxy)benzoate

(18.5 g) as an amber oil. This product was contaminated with excess 4-

(2-chloroethyl)morpholine. The crude product was treated with KOH

(9.8 g, 175 mmol) in a mixture of MeOH (100 mL) and water (50

mL) at rt for19 h. The mixture was concentrated at 35 °C to remove

MeOH. The alkaline remainder was washed with DCM and ﬁltered.

The ﬁltrate was acidiﬁed with concd HCl at 0 °C. The resulting

precipitate was ﬁltered oﬀ and dried to give the hydrochloride salt of

1

86 as a colorless solid (10.9 g, 39%): mp 266−268 °C. H NMR

(DMSO-d₆) δ 12.66 (br s, 1H), 11.06 (br s, 1H), 7.96−7.89 (m, 2H),

7.12−7.06 (m, 2H), 4.57−4.43 (m, 2H), 3.96 (br d, J = 12.4 Hz, 2H),

3.81 (br t, J = 12.0 Hz, 2H), 3.65−3.43 (m, 4H), 3.27−3.14 (m, 2H).

Anal. C₁₄H₁₈ClNO₄requires C, 54.26; H, 6.31; N, 4.87. Found: C,

54.00; H, 6.38; N, 4.90.

N-(2-(tert-Butyldimethylsilyloxy)ethyl)-1-(chloromethyl)-5-nitro-

1,2-dihydro-3H-benzo[e]indole-7-sulfonamide (89). 88³⁴(1.84 g,

4.03 mmol) and i-Pr₂NEt (0.84 mL, 4.85 mmol) were added to a

solution of 2-((tert-butyldimethylsilyl)oxy)ethylamine (850 mg, 4.85

mmol) in THF (100 mL), and the mixture was stirred at 0 °C for 40

min. Cs₂CO₃(1.31 g, 4.03 mmol) and MeOH (50 mL) were added,

and the mixture was stirred at rt for 45 min then allowed to stand at 5

°C for 14 h. The mixture was concentrated under reduced pressure at

25 °C to remove most of the THF. Ice−water was added, and the

mixture was stirred at 0 °C for 1 h 30 min. The solid was ﬁltered oﬀ,

washed with cold water, and dried to give 89 as a red−orange solid

(2.0 g, 100%): mp 127−129 °C. ¹H NMR (CDCl₃) δ 8.92 (d, J = 1.6

Hz, 1H), 7.90 (dd, J = 8.9, 1.6 Hz, 1H), 7.79 (d, J = 8.9 Hz, 1H), 7.73

(s, 1H), 4.88 (t, J = 5.8 Hz, 1H), 4.40 (br s, 1H), 4.17−4.08 (m, 1H),

4.05−3.95 (m, 2H), 3.83−3.74 (m, 1H), 3.68 (t, J = 5.2 Hz, 2H), 3.60

(dd, J = 11.1, 10.0 Hz, 1H), 3.18−3.11 (m, 2H), 0.84 (s, 9H), 0.00 (s,

6H). Anal. C₂₁H₃₀ClN₃O₅SSi requires C, 50.44; H, 6.05; N, 8.40.

Found: C, 50.71; H, 6.35; N, 8.32.

N-(2-(tert-Butyldimethylsilyloxy)ethyl)-1-(chloromethyl)-3-(5-(di-

methylamino)-1H-indole-2-carbonyl)-5-nitro-1,2-dihydro-3H-

benzo[e]indole-7-sulfonamide (90d). Prepared by general method A

from 89 (195 mg, 0.39 mmol) and 5-(dimethylamino)-1H-indole-2-

carboxylic acid hydrochloride (113 mg, 0.47 mmol) to give 90d as a

1

brown−orange solid (268 mg, 100%): mp 125 °C (dec). H NMR

solid (5.47g, 48%): mp 235−237 °C (dec). H NMR (DMSO-d₆) δ

(DMSO-d₆) δ 11.53 (br d, J = 1.6 Hz, 1H), 9.30 (s, 1H), 8.85 (d, J =

1.6 Hz, 1H), 8.43 (d, J = 8.9 Hz, 1H), 8.06−7.95 (m, 2H), 7.38 (d, J =

9.0 Hz, 1H), 7.15 (br d, J = 1.8 Hz, 1H), 7.04 (dd, J = 9.0, 2.3 Hz,

1H), 6.92 (br d, J = 2.2 Hz, 1H), 4.96 (br t, J = 10.2 Hz, 1H), 4.72 (br

dd, J = 10.9, 2.4 Hz, 1H), 4.69−4.61 (m, 1H), 4.20−4.08 (m, 2H),

3.55 (t, J = 6.1 Hz, 2H), 2.91 (q, J = 6.1 Hz, 2H), 2.79 (s, 6H), 0.788

(s, 9H), −0.046 (s, 6H). HRMS (ESI) calcd for C₃₂H₄₁ClN₅O₆SSi

(MH⁺) m/z 686.2230, found 686.2232.

11.78 (br s, 2H), 7.71 (d, J = 8.8 Hz, 2H), 7.64 (d, J = 16.0 Hz, 1H),

7.05 (d, J = 8.8 Hz, 2H), 6.52 (d, J = 16.0 Hz, 1H), 4.44 (poorly

resolved t resolved after D₂O exchange, J = 4.9 Hz, 2H), 3.71 (s, 3H),

3.52−3.31 (m, partially obscured by water peak, revealed after D₂O

exchange, 10H), 2.81 (s, 3H). Anal. C₁₇H₂₆Cl₂N₂O₃·¹/₂H₂O requires

C, 52.86; H, 7.05; N, 7.25. Found: C, 52.79; H, 6.98; N, 6.94.

(E)-3-(4-(2-(4-Methylpiperazin-1-yl)ethoxy)phenyl)acrylic Acid

(84). 83 (5.18 g, 13.7 mmol) was treated with KOH (3.5 g, 62

mmol) in a mixture of MeOH (30 mL) and water (15 mL) at rt for 29

h. The mixture was evaporated at 35 °C to remove the MeOH. The

alkaline remainder was washed with DCM and ﬁltered. The ﬁltrate was

acidiﬁed with concd HCl at 0 °C and left to stand at 5 °C overnight.

The resulting precipitate was ﬁltered oﬀ, washed with cold 6 N HCl (3

× 1 mL), and dried to give hydrochloride salt of 84 as a beige solid

(E)-N-(2-(tert-Butyldimethylsilyloxy)ethyl)-1-(chloromethyl)-3-(3-

(4-(2-morpholinoethoxy)phenyl)acryloyl)-5-nitro-1,2-dihydro-3H-

benzo[e]indole-7-sulfonamide (90i). A mixture of 80·HCl (68.1 mg,

0.24 mmol), DMF (one drop), and SOCl₂(2 mL) was stirred at reﬂux

for 10 min. The mixture was cooled and evaporated to dryness. The

reaction ﬂask was immersed in an ice bath and 89 (108 mg, 0.215

mmol) was added, followed by a mixture of DMA (2 mL) and i-

Pr₂NEt (0.041 mL, 0.237 mmol). The resulting mixture was stirred at

0 °C for 2 h. Dilute aq NaHCO₃(5%, 7 mL) was added, followed by

water (7 mL), and the mixture was stirred at 0 °C for 10 min. The

precipitated solid was ﬁltered oﬀ, washed with cold water, then dried

and triturated with EtOAc:petroleum ether (1:10) to give 90i as a

yellow−orange solid (128 mg, 79%): mp 203−205 °C. ¹H NMR

(CDCl₃) δ 9.42 (br s, 1H), 8.99 (d, J = 1.6 Hz, 1H), 8.03 (dd, J = 8.9,

1.6 Hz, 1H), 7.95 (d, J = 8.9 Hz, 1H), 7.90 (d, J = 15.2 Hz, 1H), 7.58

(d, J = 8.8 Hz, 2H), 6.96 (d, J = 8.8 Hz, 2H), 6.74 (d, J = 15.2 Hz,

1H), 4.96 (t, J = 5.9, 1H), 4.64 (dd, J = 10.7, 2.4 Hz, 1H), 4.56 (br t, J

= 9.7 Hz, 1H), 4.35−4.26 (m, 1H), 4.18 (t, J = 5.7, 2H), 3.95 (br dd, J

= 11.4, 3.3 Hz, 1H), 3.80−3.72 (m, 4H), 3.68 (t, J = 5.1 Hz, 2H), 3.63

(br dd, J = 11.4, 9.5 Hz, 1H), 3.22−3.13 (m, 2H), 2.84 (t, J = 5.7 Hz,

2H), 2.65−2.55 (m, 4H), 0.833 (s, 9H), −0.008 (s, 6H, obscured by

TMS). Anal. C₃₆H₄₇ClN₄O₈SSi requires C, 56.94; H, 6.24; N, 7.38.

Found: C, 57.09; H, 6.14; N, 7.27.

1

(2.97 g, 60%): mp 264−267 °C (dec). H NMR (DMSO-d₆) δ 11.89

(br s, 3H), 7.67 (d, J = 8.8 Hz, 2H), 7.56 (d, J = 16.0 Hz, 1H), 7.05 (d,

J = 8.8 Hz, 2H), 6.41 (d, J = 16.0 Hz, 1H), 4.46 (unresolved t resolved

after D₂O exchange, J = 4.8 Hz, 2H), 3.71 (s, 3H), 3.57−3.30 (m,

partially obscured by water peak, revealed after D₂O exchange, 10H),

2.82 (s, 3H). Anal. C₁₆H₂₄Cl₂N₂O₃·¹/₂H₂O requires C, 51.62; H, 6.77;

N, 7.53. Found: C, 51.77; H, 6.63; N, 7.17.

4-(2-Morpholinoethoxy)benzoic Acid (86). A mixture of 4-(2-

chloroethyl)morpholine hydrochloride (20.4 g, 110 mmol), NaOH

(4.85 g, 121 mmol), water (75 mL), and toluene (83 mL) was stirred

at 0 °C and saturated with solid NaCl. The toluene layer was separated

and the aqueous layer extracted with toluene (4 × 33 mL). The

combined toluene extracts were dried over solid KOH and then added

to a mixture of sodium 4-(methoxycarbonyl)phenolate (85) (16.9 g,

97 mmol) and toluene (83 mL). The mixture was stirred at reﬂux for 4

h. The mixture was cooled and ﬁltered, and the ﬁlter pad was washed

with hot toluene several times. The combined ﬁltrates were washed

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(E)-N-(2-(tert-Butyldimethylsilyloxy)ethyl)-1-(chloromethyl)-3-(3-

(4-(3-morpholinopropoxy)phenyl)acryloyl)-5-nitro-1,2-dihydro-3H-

benzo[e]indole-7-sulfonamide (90j). Prepared by general method A

from 89 (90 mg, 0.18 mmol) and 82·HCl (71 mg, 0.22 mmol) to give

concentrated at 25 °C to remove most of the THF. Ice−water was

added, and the mixture was stirred at 0 °C for 5 h 30 min. The

resulting solid was ﬁltered oﬀ, washed with cold water, and dried.

Puriﬁcation by column chromatography (DCM:EtOAc 5:1) gave 92 as

a red solid (212 mg, 88%): mp 156−158 °C. ¹H NMR (CDCl₃) δ 8.75

(br d, J = 1.4 Hz, 1H), 8.02 (dd, J = 8.8, 1.7 Hz, 1H), 7.77 (d, J = 8.8

Hz, 1H), 7.71 (s, 1H), 6.67 (br s, 1H), 4.26 (s, 1H), 4.14−4.07 (m,

1H), 4.01−3.91 (m, 2H), 3.87−3.77 (m, 3H), 3.67−3.55 (m, 3H),

0.92 (s, 9H), 0.11 (s, 6H). Anal. C₂₂H₃₀ClN₃O₄Si requires C, 56.94;

H, 6.52; N, 9.06. Found: C, 57.07; H, 6.67; N, 9.20.

N-(2-(tert-Butyldimethylsilyloxy)ethyl)-1-(chloromethyl)-3-(5-(2-

morpholinoethoxy)-1H-indole-2-carbonyl)-5-nitro-1,2-dihydro-3H-

benzo[e]indole-7-carboxamide (93e). Prepared by general method A

from 92 (126 mg, 0.27 mmol) and 5-(2-morpholinoethoxy)-1H-

indole-2-carboxylic acid hydrochloride⁴²(107 mg, 0.33 mmol) to give

93e as a yellow solid (193 mg, 97%): mp 124 °C. ¹H NMR (CDCl₃) δ

9.38 (br s, 1H), 9.28 (s, 1H), 8.79 (br d, J = 1.3 Hz, 1H), 8.15 (dd, J =

8.8, 1.6 Hz, 1H), 7.94 (d, J = 8.8 Hz, 1H), 7.38 (d, J = 8.9 Hz, 1H),

7.14 (br d, J = 2.3 Hz, 1H), 7.09−7.04 (m, 2H), 6.79 (t, J = 5.2 Hz,

1H), 4.92 (dd, J = 11.3, 2.1 Hz, 1H), 4.81 (br t, J = 9.8 Hz, 1H), 4.39−

4.29 (m, 1H), 4.18 (t, J = 5.7 Hz, 1H), 3.99 (dd, J = 11.4, 3.2 Hz, 1H),

3.86 (t, J = 5.2 Hz, 2H), 3.82−3.74 (m, 4H), 3.71−3.59 (m, 3H), 2.86

(t, J = 5.7 Hz, 2H), 2.67−2.58 (m, 4H), 0.94 (s, 9H), 0.12 (s, 6H).

Anal. C₃₇H₄₆ClN₅O₇Si·¹/₃H₂O requires C, 59.86; H, 6.34; N, 9.43.

Found: C, 59.76; H, 6.41; N, 9.58.

1

90j as a yellow−orange solid (136 mg, 98%): mp 130 °C. H NMR

(CDCl₃) δ 9.41 (br s, 1H), 8.98 (br d, J = 1.5 Hz, 1H), 8.03 (dd, J =

8.9, 1.7 Hz, 1H), 7.94 (d, J = 8.9 Hz, 1H), 7.90 (d, J = 15.2 Hz, 1H),

7.57 (d, J = 8.7 Hz, 2H), 6.94 (d, J = 8.7 Hz, 2H), 6.73 (d, J = 15.2 Hz,

1H), 4.97 (t, J = 5.8, 1H), 4.63 (dd, J = 10.7, 2.4 Hz, 1H), 4.56 (br t, J

= 9.7 Hz, 1H), 4.44−4.24 (m, 1H), 4.09 (t, J = 6.3, 2H), 3.94 (br dd, J

= 11.4, 3.3 Hz, 1H), 3.79−3.56 (m, 7H), 3.16 (q, J = 5.4 Hz, 2H),

2.58−2.43 (m, 6H), 2.05−1.95 (m, 2H), 0.832 (s, 9H), −0.013 (s, 6H,

obscured by TMS). Anal. C₃₇H₄₉ClN₄O₈SSi requires C, 57.46; H,

6.39; N, 7.24. Found: C, 57.80; H, 6.45; N, 7.30.

N-(2-(tert-Butyldimethylsilyloxy)ethyl)-1-(chloromethyl)-3-(4-(2-

(4-methylpiperazin-1-yl)ethoxy)benzoyl)-5-nitro-1,2-dihydro-3H-

benzo[e]indole-7-sulfonamide (90k). Prepared by general method A

from 89 (90 mg, 0.18 mmol) and 84·HCl (78 mg, 0.22 mmol) to give

1

90k as a yellow−orange solid (118 mg, 85%): mp 202 °C (dec). H

NMR (CDCl₃) δ 9.42 (br s, 1H), 8.98 (br d, J = 1.6 Hz, 1H), 8.03 (dd,

J = 8.8, 1.6 Hz, 1H), 7.95 (d, J = 8.8 Hz, 1H), 7.91 (d, J = 15.2 Hz,

1H), 7.57 (d, J = 8.7 Hz, 2H), 6.95 (d, J = 8.7 Hz, 2H), 6.73 (d, J =

15.2 Hz, 1H), 4.99 (t, J = 5.8 Hz, 1H), 4.63 (dd, J = 10.8, 2.5 Hz, 1H),

4.45 (br t, J = 9.7 Hz, 1H), 4.34−4.26 (m, 1H), 4.17 (t, J = 5.8 Hz,

2H), 3.94 (dd, J = 11.4, 3.3 Hz, 1H), 3.69 (t, J = 5.0 Hz, 2H), 3.62 (dd,

J = 11.4, 9.5 Hz, 1H), 3.22−3.13 (m, 2H), 2.85 (t, J = 5.8 Hz, 2H),

2.73−2.42 (m, 8H), 2.30 (s, 3H), 0.84 (s, 9H), −0.008 (s, 6H,

obscured by TMS). Anal. C₃₇H₅₀ClN₅O₇SSi requires C, 57.53; H,

6.52; N, 9.07. Found: C, 57.51; H, 6.52; N, 8.86.

(E)-N-(2-(tert-Butyldimethylsilyloxy)ethyl)-1-(chloromethyl)-3-(3-

(4-(2-morpholinoethoxy)phenyl)acryloyl)-5-nitro-1,2-dihydro-3H-

benzo[e]indole-7-carboxamide (93i). Prepared by general method A

from 92 (80 mg, 0.17 mmol) and 80·HCl (65 mg, 0.21 mmol) to give

N-(2-(tert-Butyldimethylsilyloxy)ethyl)-1-(chloromethyl)-3-(4-(2-

morpholinoethoxy)benzoyl)-5-nitro-1,2-dihydro-3H-benzo[e]-

indole-7-sulfonamide (90l). A mixture of 86·HCl (68.1 mg, 0.237

mmol), DMF (one drop), and SOCl₂(2 mL) was stirred at reﬂux for

10 min. The mixture was cooled and evaporated to dryness. The ﬂask

was immersed in an ice bath and 89 (108 mg, 0.215 mmol) was added,

followed by a mixture of DMA (2 mL) and i-Pr₂NEt (0.041 mL, 0.237

mmol). The mixture was stirred at 0 °C for 1 h. Aqueous NaHCO₃

(5%, 4 mL) was added, followed by water (4 mL), and the mixture was

stirred at 0 °C for 10 min. The solid was ﬁltered oﬀ, washed with cold

water, and dried to give 90l as an orange solid (147 mg, 93%): mp

86−89 °C. ¹H NMR (CDCl₃) δ 9.00 (br d, J = 1.5 Hz, 1H), 8.76 (br s,

1H), 8.83 (dd, J = 8.9, 1.7 Hz, 1H), 7.95 (d, J = 8.9 Hz, 1H), 7.64 (d, J

= 8.8 Hz, 2H), 7.02 (d, J = 8.8 Hz, 2H), 4.95 (t, J = 5.9, 1H), 4.55−

4.41 (m, 2H), 4.25−4.12 (m, 3H), 3.88 (dd, J = 11.4, 3.4 Hz, 1H),

3.80−3.62 (m, 7H), 3.20−3.13 (m, 2H), 2.86 (t, J = 5.7 Hz, 2H),

2.65−2.56 (m, 4H), 0.84 (s, 9H), 0.002 (s, 6H, obscured by TMS).

Anal. C₃₄H₄₅ClN₄O₈SSi requires C, 55.68; H, 6.18; N, 7.64. Found: C,

55.63; H, 6.30; N, 7.64.

N-(2-(tert-Butyldimethylsilyloxy)ethyl)-1-(chloromethyl)-3-(3-

morpholinopropanoyl)-5-nitro-1,2-dihydro-3H-benzo[e]indole-7-

sulfonamide (90m). Prepared by general method A from 89 (195 mg,

0.39 mmol) and 3-morpholinopropanoic acid hydrochloride (92 mg,

0.47 mmol) to give 90m as a yellow-orange solid (229 mg, 92%): mp

135−137 °C. ¹H NMR (DMSO-d₆) δ 9.23 (br s, 1H), 8.82 (br d, J =

1.5 Hz, 1H), 8.37 (d, J = 9.0 Hz, 1H), 8.02−7.95 (m, 2H), 4.63−4.47

(m, 2H), 4.37 (br d, J = 8.5 Hz, 1H), 4.12−4.00 (m, 2H), 3.58 (t, J =

4.6 Hz, 4H), 3.54 (t, J = 6.1 Hz, 2H), 2.91 (t, J = 6.1 Hz, 2H), 2.87−

2.65 (m, 4H), 2.44 (br t, J = 4.4 Hz, 4H), 0.78 (s, 9H), −0.06 (s, 6H).

HRMS (ESI) calcd for C₂₈H₄₂ClN₄O₇SSi (MH⁺) m/z 641.2226,

found 641.2225.

1

93i as a yellow−orange solid (121 mg, 97%): mp 164−165 °C. H

NMR (CDCl₃) δ 9.35 (br s, 1H), 8.75 (br d, J = 1.3 Hz, 1H), 8.12 (dd,

J = 8.8, 1.6 Hz, 1H), 7.93−7.86 (m, 2H), 7.58 (d, J = 8.8 Hz, 2H), 6.95

(d, J = 8.8 Hz, 2H), 6.78−6.69 (m, 2H), 4.63 (dd, J = 10.8, 2.4 Hz,

1H), 4.53 (t, J = 9.7 Hz, 1H), 4.33−4.25 (m, 1H), 4.17 (t, J = 5.7 Hz,

2H), 3.97 (dd, J = 10.4, 3.2 Hz, 1H), 3.85 (t, J = 5.2 Hz, 2H), 3.75 (br

t, J = 4.7 Hz, 4H), 3.69−3.56 (m, 3H), 2.83 (t, J = 5.7 Hz, 2H), 2.59

(br t, J = 4.7 Hz, 4H), 0.93 (s, 9H), 0.11 (s, 6H). Anal.

C₃₇H₄₇ClN₄O₇Si requires C, 61.44; H, 6.55; N, 7.75. fFund: C,

61.31; H, 6.66; N, 7.99.

Di-tert-Butyl (2-(1-(Chloromethyl)-3-(5-(2-(diethylamino)ethoxy)-

1H-indole-2-carbonyl)-5-nitro-2,3-dihydro-1H-benzo[e]indole-7-

sulfonamido)ethyl)phosphate (95a). Prepared by general method A

from di-tert-butyl 2-(1-(chloromethyl)-5-nitro-2,3-dihydro-1H-benzo-

[e]indole-7-sulfonamido)ethyl phosphate (94)³⁵(387 mg, 0.67 mmol)

and 64·HCl (272 mg, 0.87 mmol) to give 95a as a yellow−orange

1

solid (521 mg, 93%): mp 225−227 °C (dec). H NMR (CDCl₃) δ

9.36 (s, 2H), 9.00 (d, J = 1.5 Hz, 1H), 8.07 (dd, J = 8.9, 1.6 Hz, 1H),

7.98 (d, J = 8.9 Hz, 1H), 7.37 (d, J = 8.9 Hz, 1H), 7.14 (d, J = 2.2 Hz,

1H), 7.09−7.01 (m, 2H), 6.00 (br s, 1H), 4.90 (dd, J = 10.7, 2.2 Hz,

1H), 4.82 (t, J = 10.7 Hz, 1H), 4.40−4.31 (m, 1H), 4.27−4.15 (m,

2H), 4.12−4.02 (m, 2H), 3.95 (dd, J = 11.5, 3.4 Hz, 1H), 3.65 (dd, J =

11.5, 9.0 Hz, 1H), 3.32 (t, J = 4.6 Hz, 2H), 3.06 (br s, 2H), 2.92−2.74

(m, 4H), 1.47 (s, 9H), 1.46 (s, 9H), 1.17 (t, J = 7.1 Hz, 6H). Anal.

C₃₈H₅₁ClN₅O₁₀PS·¹/₂H₂O requires C, 53.99; H, 6.20; N, 8.29. Found:

C, 53.77; H, 6.07; N, 8.00.

Di-tert-butyl (2-(1-(Chloromethyl)-3-(5-(3-(dimethylamino)-

propoxy)-1H-indole-2-carbonyl)-5-nitro-2,3-dihydro-1H-benzo[e]-

indole-7-sulfonamido)ethyl)phosphate (95b). Prepared by general

method A from 94 (179 mg, 0.31 mmol) and 67·HCl (120 mg, 0.40

mmol). The crude product was triturated with acetone to give 95b as a

pale-yellow solid (157 mg, 62%): mp 195−198 °C. ¹H NMR (DMSO-

d₆) δ 11.76 (d, J = 1.6 Hz, 1H), 9.30 (s, 1H), 8.87 (d, J = 1.7 Hz, 1H),

8.46 (d, J = 8.9 Hz, 1H), 8.20 (t, J = 5.7 Hz, 1H), 8.01 (dd, J = 8.9, 1.7

Hz, 1H), 7.41 (d, J = 8.9 Hz, 1H), 7.22 (d, J = 1.8 Hz, 1H), 7.16 (d, J

= 2.3 Hz, 1H), 6.95 (dd, J = 8.9, 2.4 Hz, 1H), 5.01−4.93 (m, 1H), 4.73

(dd, J = 10.9, 2.3 Hz, 1H), 4.69−4.62 (m, 1H), 4.20−4.10 (m, 2H),

4.02 (t, J = 6.4 Hz, 2H), 3.83 (q, J = 6.3 Hz, 2H), 3.09−3.04 (m, 2H),

2.39 (t, J = 7.1 Hz, 2H), 2.16 (s, 6H), 1.92−1.85 (m, 2H), 1.36 (s,

18H). Anal. C₃₇H₄₉ClN₅O₁₀PS requires C, 54.04; H, 6.01; N, 8.52.

Found: C, 53.94; H, 5.84; N, 8.44.

N-(2-(tert-Butyldimethylsilyloxy)ethyl)-1-(chloromethyl)-5-nitro-

1,2-dihydro-3H-benzo[e]indole-7-carboxamide (92). (Benzotriazol-

1-yloxy)tripyrrolidinophosphonium hexaﬂuorophosphate (pyBOP,

352 mg, 0.675 mmol) was added to a solution of 1-(chloromethyl)-

5-nitro-3-(2,2,2-triﬂuoroacetyl)-1,2-dihydro-3H-benzo[e]indole-7-car-

boxylic acid (91)³⁵(209 mg, 0.519 mmol), 2-((tert-

butyldimethylsilyl)oxy)ethylamine (109 mg, 0.62 mmol), and i-

Pr₂NEt (0.27 mL, 1.56 mmol) in THF (20 mL) at 0 °C and the

mixture was stirred at this temperature for 1 h. Cs₂CO₃(339 mg, 1.04

mmol) and MeOH (10 mL) were added, and the mixture was stirred

at rt for 2 h then allowed to stand at 5 °C overnight. The mixture was

R

DOI: 10.1021/acs.jmedchem.7b00563

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Di-tert-butyl (2-(1-(Chloromethyl)-3-(5-((dimethylamino)methyl)-

1H-indole-2-carbonyl)-5-nitro-2,3-dihydro-1H-benzo[e]indole-7-

sulfonamido)ethyl)phosphate (95c). Prepared by general method A

from 94 (387 mg, 0.67 mmol) and 71·HCl (222 mg, 0.87 mmol) to

give 95c as a yellow−orange solid (494 mg, 95%): mp 242−245 °C

(dec). ¹H NMR (DMSO-d₆) δ 11.70 (s, 1H), 9.30 (s, 1H), 8.87 (br d,

J = 1.6 Hz, 1H), 8.45 (d, J = 8.9 Hz, 1H), 8.15 (t, J = 5.8 Hz, 1H), 8.02

(dd, J = 8.9, 1.7 Hz, 1H), 7.60 (s, 1H), 7.46 (d, J = 8.5 Hz, 1H), 7.32−

7.21 (m, 2H), 5.03−4.91 (m, 1H), 4.75−4.60 (m, 2H), 4.18−4.05 (m,

2H), 3.83 (q, J = 7.0 Hz, 2H), 3.53 (br s, 2H), 3.05 (q, J = 5.8 Hz,

2H), 2.21 (s, 6H), 1.36 (s, 9H), 1.35 (s, 9H). Anal. C₃₅H₄₅ClN₅O₉PS

requires C, 54.02; H, 5.83; N, 9.00. Found: C, 53.61; H, 5.90; N, 9.10.

Di-tert-butyl 2-(1-(Chloromethyl)-3-(5-(dimethylamino)-1H-in-

dole-2-carbonyl)-5-nitro-1,2-dihydro-3H-benzo[e]indole-7-

sulfonamido)ethyl)phosphate (95d). Prepared by general method A

from 94 (289 mg, 0.50 mmol) and 5-(dimethylamino)-1H-indole-2-

carboxylic acid hydrochloride (151 mg, 0.63 mmol) to give 95d as a

yellow−brown solid (371 mg, 97%): mp >300 °C. ¹H NMR (DMSO-

d₆) δ 11.53 (br d, J = 1.5 Hz, 1H), 9.30 (s, 1H), 8.86 (br d, J = 1.6 Hz,

1H), 8.44 (d, J = 8.9 Hz, 1H), 8.14 (t, J = 5.8 Hz, 1H), 8.02 (dd, J =

8.9, 1.7 Hz, 1H), 7.38 (d, J = 9.0 Hz, 1H), 7.15 (br d, J = 1.7 Hz, 1H),

7.03 (dd, J = 9.0, 2.3 Hz, 1H), 6.92 (br d, J = 2.2 Hz, 1H), 4.96 (t, J =

10.2 Hz, 1H), 4.72 (dd, J = 10.9, 2.4 Hz, 1H), 4.70−4.58 (m, 1H),

4.21−4.07 (m, 2H), 3.83 (m, 2H), 3.06 (q, J = 5.8 Hz, 2H), 2.88 (s,

6H), 1.358 (s, 9H), 1.355 (s, 9H). Anal. C₃₄H₄₃ClN₅O₉PS requires C,

53.44; H, 5.67; N, 9.16. Found: C, 53.32; H, 5.84; N, 9.11.

Di-tert-butyl 2-(1-(Chloromethyl)-3-(5-(2-morpholinoethoxy)-1H-

indole-2-carbonyl)-5-nitro-1,2-dihydro-3H-benzo[e]indole-7-

sulfonamido)ethyl)phosphate (95e). Prepared by general method A

from 94 (182 mg, 0.31 mmol) and 5-(2-morpholinoethoxy)-1H-

indole-2-carboxylic acid hydrochloride⁴²(134 mg, 0.40 mmol). The

crude product was triturated with acetone to give 95e as a yellow solid

(176 mg, 66%): mp 200−204 °C (dec). ¹H NMR (DMSO-d₆) δ 11.77

(d, J = 1.6 Hz, 1 H), 9.30 (s, 1H), 8.86 (d, J = 1.7 Hz, 1H), 8.45 (d, J =

8.9 Hz, 1H), 8.20 (t, J = 5.9 Hz, 1H), 8.00 (dd, J = 8.9, 1.7 Hz, 1H),

7.39 (d, J = 8.9 Hz, 1H), 7.21 (d, J = 1.7 Hz, 1H), 7.08 (d, J = 2.3 Hz,

1H), 6.95 (dd, J = 8.9, 2.4 Hz, 1H), 5.01−4.94 (m, 1H), 4.71 (dd, J =

10.9, 2.2 Hz, 1H), 4.69−4.63 (m, 1H), 4.20−4.09 (m, 4H), 3.83 (q, J

= 6.3 Hz, 2H), 3.63−3.58 (m, 4H), 3.06 (q, J = 5.8 Hz, 2H), 2.73 (t, J

= 5.7 Hz, 2H), ca. 2.52−2.46 (m, 4H, partially obscured by DMSO

peak), 1.35 (s, 18H). Anal. C₃₈H₄₉ClN₅O₁₁PS requires C, 53.68; H,

5.81; N, 8.24. Found: C, 53.59; H, 5.61; N, 8.19.

Di-tert-butyl (2-(1-(Chloromethyl)-3-(5-(3-(dimethylamino)-

propanamido)-1H-indole-2-carbonyl)-5-nitro-2,3-dihydro-1H-

benzo[e]indole-7-sulfonamido)ethyl)phosphate (95h). Prepared by

general method A from 94 (387 mg, 0.67 mmol) and 77·HCl (271

mg, 0.87 mmol) to give 95h as a yellow−orange solid (507 mg, 91%):

mp 237−240 °C (dec). ¹H NMR (DMSO-d₆) δ 11.79 (s, 1H), 9.96 (s,

1H), 9.30 (s, 1H), 8.87 (d, J = 1.6 Hz, 1H), 8.45 (d, J = 8.9 Hz, 1H),

8.15 (t, J = 5.6 Hz, 1H), 8.09 (br s, 1H), 8.02 (dd, J = 8.9, 1.7 Hz, 1H),

7.42 (d, J = 8.8 Hz, 1H), 7.35 (dd, J = 8.8, 1.8 Hz, 1H), 7.29 (d, J = 1.5

Hz, 1H), 5.02−4.92 (m, 1H), 4.76−4.61 (m, 2H), 4.19−4.10 (m, 2H),

3.83 (q, J = 7.0 Hz, 2H), 3.06 (q, J = 5.6 Hz, 2H), 2,59 (t, J = 7.0 Hz,

2H), 2,45 (t, J = 7.0 Hz, 2H), 2.21 (s, 6H), 1.36 (s, 9H), 1.35 (s, 9H).

Anal. C₃₇H₄₈ClN₆O₁₀PS requires C, 53.20; H, 5.79; N, 10.06. Found:

C, 53.07; H, 5.82; N, 10.01.

(E)-Di-tert-butyl 2-(1-(Chloromethyl)-3-(3-(4-(2-

morpholinoethoxy)phenyl)acryloyl)-5-nitro-1,2-dihydro-3H-benzo-

[e]indole-7-sulfonamido)ethyl)phosphate (95i). Prepared by general

method A from 94 (290 mg, 0.50 mmol) and 80·HCl (2 batches of

198 mg, 0.60 mmol) to give 95i as a yellow−orange solid (395 mg,

1

94%): mp 245−248 °C (dec). H NMR (CDCl₃) δ 9.41 (br s, 1H),

8.97 (d, J = 1.6 Hz, 1H), 8.50 (dd, J = 8.9, 1.6 Hz, 1H), 7.96 (d, J = 8.9

Hz, 1H), 7.91 (d, J = 15.2 Hz, 1H), 7.58 (d, J = 8.7 Hz, 2H), 6.96 (d, J

= 8.7 Hz, 2H), 6.74 (d, J = 15.2 Hz, 1H), 5.95 (t, J = 5.7 Hz, 1H), 4.63

(br dd, J = 10.8, 2.5 Hz, 1H), 4.56 (br t, J = 9.8 Hz, 1H), 4.34−4.26

(m, 1H), 4.18 (t, J = 5.6, 2H), 4.12−4.04 (m, 2H), 3.96 (br dd, J =

11.5, 3.2 Hz, 1H), 3.75 (br t, J = 4.6 Hz, 4H), 3.62 (br dd, J = 11.4, 9.9

Hz, 1H), 3.33 (br q, J = 5.4 Hz, 2H), 2.85 (br t, J = 5.6 Hz, 2H), 2.65−

2.57 (m, 4H), 1.46 (s, 9H), 1.45 (s, 18H). Anal.

C₃₈H₅₀ClN₄O₁₁PS·¹/₂H₂O requires C, 53.93; H, 6.07; N, 6.62.

Found: C, 53.83; H, 5.99; N, 6.53.

(E)-Di-tert-butyl 2-(1-(Chloromethyl)-3-(3-(4-(2-

morpholinopropoxy)phenyl)acryloyl)-5-nitro-1,2-dihydro-3H-

benzo[e]indole-7-sulfonamido)ethyl)phosphate (95j). Prepared by

general method A from 94 (183 mg, 0.32 mmol) and 82·HCl (2

batches of 125 mg, 0.38 mmol) to give 95j as a yellow−orange solid

1

(236 mg, 87%): mp 230 °C (dec). H NMR (CDCl₃) δ 9.41 (br s,

1H), 8.97 (br d, J = 1.6 Hz, 1H), 8.04 (dd, J = 8.9, 1.7 Hz, 1H), 7.94

(d, J = 8.9 Hz, 1H), 7.90 (d, J = 15.2 Hz, 1H), 7.58 (d, J = 8.8 Hz,

2H), 6.95 (d, J = 8.8 Hz, 2H), 6.73 (d, J = 15.2 Hz, 1H), 5.92 (t, J =

5.7 Hz, 1H), 4.62 (dd, J = 10.8, 2.5 Hz, 1H), 4.55 (br t, J = 9.7 Hz,

1H), 4.34−4.23 (m, 1H), 4.14−4.02 (m, 4H), 3.94 (br dd, J = 11.4, 3.3

Hz, 1H), 3.73 (br t, J = 4.6 Hz, 4H), 3.62 (dd, J = 11.4, 9.4 Hz, 1H),

3.32 (br q, J = 5.4 Hz, 2H), 2.59−2.42 (m, 6H), 2.06−1.93 (m, 2H),

1.46 (s, 9H), 1.45 (s, 9H). Anal. C₃₉H₅₂ClN₄O₁₁PS requires C, 55.02;

H, 6.16; N, 6.58. Found: C, 54.77; H, 6.32; N, 6.63.

Di-tert-butyl 2-(1-(Chloromethyl)-3-(5-(3-morpholinopropoxy)-

1H-indole-2-carbonyl)-5-nitro-1,2-dihydro-3H-benzo[e]indole-7-

sulfonamido)ethyl)phosphate (95f). Prepared by general method A

from 94 (176 mg, 0.30 mmol) and 74·HCl (156 mg, 0.45 mmol). The

crude product was triturated with acetone to give 95f as a pale-yellow

solid (175 mg, 66%): mp 200−203 °C (dec). ¹H NMR (DMSO-d₆) δ

11.76 (d, J = 2.0 Hz, 1H), 9.30 (s, 1H), 8.87 (d, J = 1.7 Hz, 1H), 8.46

(d, J = 8.9 Hz, 1H), 8.20 (t, J = 5.9 Hz, 1H), 8.02 (dd, J = 8.9, 1.7 Hz,

1H), 7.42 (d, J = 8.9 Hz, 1H), 7.23 (d, J = 1.7 Hz, 1H), 7.16 (d, J = 2.3

Hz, 1H), 6.95 (dd, J = 8.9, 2.4 Hz, 1H), 5.02−4.95 (m, 1H), 4.74 (dd,

J = 10.9, 2.3 Hz, 1H), 4.70−4.63 (m, 1H), 4.20−4.11 (m, 2H), 4.03 (t,

J = 6.3 Hz, 2H), 3.84 (q, J = 6.3 Hz, 2H), 3.62−3.56 (m, 4H), 3.05 (q,

J = 5.8 Hz, 2H), 2.46 (t, J = 7.2 Hz, 2H), 2.41−2.36 (m, 4H), 1.35 (s,

18H). Anal. C₃₉H₅₁ClN₅O₁₁PS requires C, 54.20; H, 5.95; N, 8.10.

Found: C, 54.13; H, 5.91; N, 7.82.

Di-tert-butyl 2-(1-(Chloromethyl)-3-(4-(2-(4-methylpiperazin-1-

yl)ethoxy)benzoyl)-5-nitro-1,2-dihydro-3H-benzo[e]indole-7-

sulfonamido]ethyl)phosphate (95k). Prepared by general method A

from 94 (1.16 g, 2.00 mmol) and 84·HCl (2 batches of 0.87 g, 2.40

mmol) to give 95k as a yellow−orange solid (1.58 g, 93%): mp 118−

120 °C (dec). ¹H NMR (CDCl₃) δ 9.41 (br s, 1H), 8.98 (br d, J = 1.3

Hz, 1H), 8.04 (dd, J = 8.8, 1.4 Hz, 1H), 7.95 (d, J = 8.8 Hz, 1H), 7.90

(d, J = 15.2 Hz, 1H), 7.57 (d, J = 8.6 Hz, 2H), 6.94 (d, J = 8.6 Hz,

2H), 6.73 (d, J = 15.2 Hz, 1H), 5.96 (br s, 1H), 4.63 (dd, J = 10.7, 2.3

Hz, 1H), 4.55 (br t, J = 9.7 Hz, 1H), 4.32−4.23 (m, 1H), 4.17 (t, J =

5.8 Hz, 2H), 4.12−4.02 (m, 2H), 3.95 (dd, J = 11.4, 3.2 Hz, 1H), 3.62

(dd, J = 11.3, 9.5 Hz, 1H), 3.32 (br s, 2H), 2.85 (t, J = 5.8 Hz, 2H),

2.66 (br s, 4H), 2.52 (br s, 4H), 2.32 (s, 3H), 1.46 (s, 9H), 1.45 (s,

9H). Anal. C₃₉H₅₃ClN₅O₁₀S·¹/₂H₂O requires C, 54.51; H, 6.33; N,

8.15. Found: C, 54.52; H, 6.34; N, 7.91.

Di-tert-butyl 2-(1-(Chloromethyl)-3-(4-(2-morpholinoethoxy)-

benzoyl)-5-nitro-1,2-dihydro-3H-benzo[e]indole-7-sulfonamido)-

ethyl)phosphate (95l). A mixture of 86·HCl (136 mg, 0.474 mmol),

DMF (one drop), and SOCl₂(3 mL) was stirred at reﬂux for 10 min.

The mixture was cooled and evaporated to dryness. The residue was

cooled in an ice bath, and 94 (248 mg, 0.430 mmol) was added,

followed by a mixture of DMA (3 mL) and i-Pr₂NEt (0.082 mL, 0.474

mmol). The reaction mixture was stirred at 0 °C for 1 h. Aqueous

NaHCO₃(5%, 6 mL) was added, followed by water (6 mL), and the

mixture was stirred at 0 °C for 10 min. The precipitated solid was

ﬁltered oﬀ, washed with cold water, and dried to give 95l as a yellow−

Di-tert-butyl 2-(1-(Chloromethyl)-3-(5-(2-(dimethylamino)-

acetamido)-1H-indole-2-carbonyl)-5-nitro-1,2-dihydro-3H-benzo-

[e]indole-7-sulfonamido)ethyl)phosphate (95g). Prepared by general

method A from 94 (258 mg, 0.45 mmol) and 5-(2-(dimethylamino)-

acetamido)-1H-indole-2-carboxylic acid hydrochloride⁴²(167 mg, 0.56

mmol) to give 95g as a yellow−orange solid (355 mg, 97%): mp 235−

1

238 °C (dec). H NMR (DMSO-d₆) δ 11.80 (br d, J = 1.5 Hz, 1H),

9.63 (s, 1H), 8.87 (br d, J = 1.6 Hz, 1H), 8.46 (d, J = 8.9 Hz, 1H),

8.21−8.12 (m, 2H), 8.02 (dd, J = 8.9, 1.7 Hz, 1H), 7.44 (br d, J = 1.1

Hz, 2H), 7.30 (br d, J = 2.1 Hz, 1H), 4.98 (t, J = 10.8 Hz, 1H), 4.74

(dd, J = 11.0, 2.4 Hz, 1H), 4.73−4.62 (m, 1H), 4.22−4.11 (m, 2H),

3.83 (q, J = 6.3 Hz, 2H), 3.12−3.03 (m, 4H), 2.32 (s, 6H), 1.358 (s, 9

H), 1.355 (s, 9H). Anal. C₃₆H₄₆ClN₆O₁₀PS·¹/₂H₂O requires C, 52.08;

H, 5.71; N, 10.12. Found: C, 51.87; H, 5.58; N, 10.08.

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1

orange solid (326 mg, 94%): mp 113−115 °C. H NMR (CDCl₃) δ

8.99 (br d, J = 1.5 Hz, 1H), 8.75 (br s, 1H), 8.05 (dd, J = 8.9, 1.7 Hz,

1H), 7.95 (d, J = 8.9 Hz, 1H), 7.64 (d, J = 8.8 Hz, 2H), 7.02 (d, J = 8.8

Hz, 2H), 5.95 (t, J = 5.7 Hz, 1H), 4.54−4.41 (m, 2H), 4.23 (t, J = 5.4

Hz, 2H), 4.21−4.12 (m, 1H), 4.11−4.04 (m, 2H), 3.88 (dd, J = 11.5,

3.4 Hz, 1H), 3.83−3.72 (m, 4H), 3.66 (dd, J = 11.5, 8.3 Hz, 1H), 3.33

(br q, J = 5.6 Hz, 2H), 2.93−2.82 (m, 2H), 2.63 (br s, 4H), 1.47 (s,

9H), 1.46 (s, 9H). Anal. C₃₆H₄₈ClN₄O₁₁PS·¹/₃H₂O requires C, 52.91;

H, 6.00; N, 6.86. Found: C, 52.89; H, 6.06; N, 6.83.

mice were randomized to treatment groups when tumors reached a

mean diameter of 8−10 mm. In each experiment, mice received vehicle

alone (PBS, n = 3), nitroCBI alone (dissolved in PBS containing 2−4

equiv of NaHCO₃and administered iv through the lateral tail vein, n =

3), radiation alone (15 Gy, whole body cobalt-60 gamma irradiation, n

= 5), or radiation followed 5 min later by nitroCBI (n = 5).

Antitumor Activity by Growth Delay Assay. Growth delay

assays were performed as previously described.⁴⁹Tumors were grown

in the ﬂank of female mice by sc inoculation of cells from tissue culture

(as above), and mice were randomized to treatment groups when

tumors reached treatment size (mean volume of 250, 160, and 200

mm³for SiHa, A2780, and 22Rv1 tumors, respectively). Gemcitabine

was dissolved in saline (0.9% NaCl) solution and docetaxel in

polysorbate 80 (40 mg/mL) then diluted 14-fold with 13% (w/w)

ethanol in water, and each was administered ip. NitroCBIs 9 and 50

were dissolved in PBS containing 2−4 equiv of NaHCO₃and

administered iv through the lateral tail vein. Doses are reported in

μmol/kg, conversion to mg/kg is as follows: gemcitabine 100, 133, 237

μmol/kg (26, 35, 62 mg/kg); docetaxel 32 μmol/kg (26 mg/kg); 9 42,

56 μmol/kg (34, 45 mg/kg); 50 14, 15 μmol/kg (12, 13 mg/kg).

Tumor volume was calculated as π(L × w²)/6, where L is the major

axis and w is the perpendicular minor axis. Animals were culled 100−

120 days after start of treatment or when the mean tumor diameter

exceeded 15 mm. Acute deaths were noted in experiments A and B

(see Figure 5) as follows: (A) 9 at 42 μmol/kg (one mouse after third

iv dose), 9 at 56 μmol/kg (one mouse after fourth iv dose),

gemcitabine plus 9 at 56 μmol/kg (one mouse after ﬁrst iv dose and 1

mouse after fourth iv dose); (B) gemcitabine plus 9 (one mouse after

fourth iv dose). These animals were replaced to maintain group sizes

and were excluded from the growth delay analyses. The biological end

point used in the Kaplan−Meier analyses (Supporting Information,

Figure S4) was the time required for the tumors to quadruple their

volume relative to initial volume at the start of the treatment (i.e.,

RTV4). Statistically signiﬁcant diﬀerences for pairwise comparisons

were determined using Log-Rank test with SigmaPlot v13 (Systat

Software, Inc.) (Supporting Information, Table S8).

Di-tert-butyl 2-(1-(Chloromethyl)-3-(3-morpholinopropanoyl)-5-

nitro-1,2-dihydro-3H-benzo[e]indole-7-sulfonamido)ethyl)-

phosphate (95m). Prepared by general method A from 94 (194 mg,

0.34 mmol) and 3-morpholinopropanoic acid hydrochloride (85 mg,

0.44 mmol) to give 95m as a yellow−orange solid (219 mg, 91%): mp

107−110 °C. ¹H NMR (CDCl₃) δ 9.31 (br s, 1H), 8.97 (br d, J = 1.5

Hz, 1H), 8.03 (dd, J = 8.9, 1.7 Hz, 1H), 7.93 (d, J = 8.9 Hz, 1H), 5.95

(br t, J = 5.3 Hz, 1H), 4.50−4.36 (m, 2H), 4.30−4.20 (m, 1H), 4.10−

4.02 (m, 2H), 3.92 (dd, J = 11.5, 3.3 Hz, 1H), 3.74 (t, J = 4.6 Hz, 4H),

3.62 (dd, J = 11.4, 8.9 Hz, 1H), 3.32 (br q, J = 5.1 Hz, 2H), 2.94−2.66

(m, 4H), 2.57 (br t, J = 4.2 Hz, 4H), 1.46 (s, 9H), 1.45 (s, 9H). Anal.

C₃₀H₄₄ClN₄O₁₀PS·¹/₂H₂O requires C, 49.48; H, 6.23; N, 7.69. Found:

C, 49.22; H, 6.28; N, 7.50.

Di-tert-butyl 2-(1-(Chloromethyl)-3-(5-(2-morpholinoethoxy)-1H-

indole-2-carbonyl)-5-nitro-1,2-dihydro-3H-benzo[e]indole-7-

carboxamido)ethyl)phosphate (97e). Prepared by general method A

from 96 (143 mg, 0.26 mmol) and 5-(2-morpholinoethoxy)-1H-

indole-2-carboxylic acid hydrochloride⁴²(104 mg, 0.32 mmol) to give

1

97e as a yellow−orange solid (213 mg, 99%): mp 130−132 °C. H

NMR (CDCl₃) δ 9.34 (br s, 1H), 9.24 (s, 1H), 8.93 (br d, J = 1.3 Hz,

1H), 8.20 (dd, J = 8.8, 1.6 Hz, 1H), 8.06 (t, J = 4.9 Hz, 1H), 7.92 (d, J

= 8.8 Hz, 1H), 7.38 (d, J = 8.9 Hz, 1H), 7.14 (br d, J = 2.3 Hz, 1H),

7.10−6.99 (m, 2H), 4.91 (dd, J = 10.8, 2.1 Hz, 1H), 4.80 (br t, J = 9.7

Hz, 1H), 4.38−4.13 (m, 5H), 3.98 (dd, J = 11.5, 3.3 Hz, 1H), 3.86−

3.72 (m, 6H), 3.61 (dd, J = 11.4, 9.5 Hz, 1H), 2.87 (t, J = 5.6 Hz, 2H),

2.70−2.58 (m, 4H), 1.503 (s, 9H), 1.502 (s, 9H). Anal.

C₃₉H₄₉ClN₅O₁₀P·¹/₃H₂O requires C, 57.11; H, 6.10; N, 8.54.

Found: C, 57.02; H, 6.06; N, 8.44.

(E)-Di-tert-butyl 2-(1-(Chloromethyl)-3-(3-(4-(2-

morpholinoethoxy)phenyl)acryloyl)-5-nitro-1,2-dihydro-3H-benzo-

[e]indole-7-carboxamido)ethyl)phosphate (97i). Prepared by general

method A from 96 (176 mg, 0.33 mmol) and 80·HCl (2 batches of

123 mg, 0.39 mmol) to give 97i as a yellow−orange solid (211 mg,

81%): mp 128−131 °C. ¹H NMR (CDCl₃) δ 9.30 (br s, 1H), 8.90 (br

d, J = 1.4 Hz, 1H), 8.17 (dd, J = 8.7, 1.4 Hz, 1H), 8.01 (t, J = 4.6 Hz,

1H), 7.94−7.86 (m, 2H), 7.58 (d, J = 8.7 Hz, 2H), 6.96 (d, J = 8.7 Hz,

2H), 6.76 (d, J = 15.2 Hz, 1H), 4.62 (dd, J = 10.8, 2.1 Hz, 1H), 4.52

(br t, J = 9.7 Hz, 1H), 4.32−4.20 (m, 3H), 4.17 (t, J = 5.7 Hz, 2H),

3.96 (dd, J = 11.4, 3.1 Hz, 1H), 3.81 (q, J = 4.6 Hz, 2H), 3.74 (br t, J =

4.6 Hz, 4H), 3.59 (br t, J = 10.6 Hz, 1H), 2.84 (t, J = 5.7 Hz, 2H), 2.60

(br t, J = 4.5 Hz, 4H), 1.50 (s, 18H). Anal. C₃₉H₅₀ClN₄O₁₀P·¹/₂H₂O

requires C, 57.81; H, 6.34; N, 6.91. Found: C, 57.51; H, 6.10; N, 6.95.

Calculation of pK_a. Approximated apparent pK_as were calculated

for nitroCBIs using ACD/Laboratories software ACD/pK_av12.01.⁴³

In Vitro Cytotoxicity. The sources of the cell lines used in this

study are as previously described.³⁷All lines were conﬁrmed to be

Mycoplasma-free. Inhibition of proliferation of log-phase monolayers

was assessed in 96-well plates as previously described.⁵⁹The drug

exposure time was 4 h under aerobic or hypoxic conditions (5% CO₂

in air or nitrogen, respectively), followed by sulforhodamine B staining

5 days later. The IC₅₀was determined by interpolation as the drug

concentration required to inhibit cell density to 50% of that of the

untreated controls on the same plate.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.jmed-

chem.7b00563.

■

S

Information on purity of ﬁnal products, tabulated IC₅₀

data for 10−28 and 29−44 in HT29 and SiHa, tabulated

data for all excision assays shown in Figures 3 and 4, and

Kaplan−Meier analyses and body weight data for all

tumor growth delay experiments shown in Figure 5

(PDF)

Molecular formula strings (CSV)

AUTHOR INFORMATION

Corresponding Author

*Phone: +64 9 923 6843. E-mail: m.tercel@auckland.ac.nz.

ORCID

■

Moana Tercel: 0000-0001-8443-2801

Present Addresses

^‡Sunali Y. Mehta, Department of Pathology, University of

Otago, 56 Hanover Street, Dunedin 9054, New Zealand.

^§Jean-Jacques Youte Tendoung, StrainChem, Inserm U 1240,

58 rue Montalembert, BP 184 63005 Clermont-Ferrand,

France.

Animal Experiments. All animal studies were approved by the

University of Auckland Animal Ethics Committee (approval numbers

R256 and CR590) and were conducted using CD1-Foxn1^nu/nu(nude)

mice.

Antitumor Activity by Excision Assay. Excision assays were

performed as previously described.^34,35Tumors were grown in the

ﬂank of male mice by sc inoculation of cells from tissue culture (10⁷

for SiHa and H460, 5 × 10⁶for H1299, HCT116, and 22Rv1), and

^∥Sally Y. Bai, APAC Oncology Medical Aﬀairs, Merck & Co.,

Inc., 8 Biomedical Grove, Neuros Building, Singapore 138665.

Notes

The authors declare no competing ﬁnancial interest.

T

DOI: 10.1021/acs.jmedchem.7b00563

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(13) Elgersma, R. C.; Coumans, R. G. E.; Huijbregts, T.; Menge, W.

M. P. B.; Joosten, J. A. F.; Spijker, H. J.; de Groot, F. M. H.; van der

Lee, M. M. C.; Ubink, R.; van den Dobbelsteen, D. J.; Egging, D. F.;

Dokter, W. H. A.; Verheijden, G. F. M.; Lemmens, J. M.; Timmers, C.

M.; Beusker, P. H. Design, synthesis, and evaluation of linker-

duocarmycin payloads: toward selection of HER2-targeting antibody-

drug conjugate SYD985. Mol. Pharmaceutics 2015, 12, 1813−1835.

(14) Krall, N.; Pretto, F.; Decurtins, W.; Bernardes, G. J. L.; Supuran,

C. T.; Neri, D. A small-molecule drug conjugate for the treatment of

carbonic anhydrase IX expressing tumors. Angew. Chem., Int. Ed. 2014,

53, 4231−4235.

ACKNOWLEDGMENTS

■

We are grateful for the assistance of Sisira Kumara for HPLC

and solubility/formulation studies, Susan Pullen, Rita Patel, and

Wouter van Leeuwen for in vitro experiments, and Sarah

McManaway, Caroline McCulloch, and Chenbo Wu for in vivo

studies. This work was funded by the Foundation for Research,

Science and Technology (UOAX0211, UOAX0703), the

Health Research Council (12/529, 14/538), and the Auckland

Division of the Cancer Society of New Zealand.

(15) Sheldrake, H. M.; Travica, S.; Johansson, I.; Loadman, P. M.;

Sutherland, M.; Elsalem, L.; Illingworth, N.; Cresswell, A. J.; Reuillon,

T.; Shnyder, S. D.; Mkrtchian, S.; Searcey, M.; Ingelman-Sundberg,

M.; Patterson, L. H.; Pors, K. Re-engineering of the duocarmycin

structural architecture enables bioprecursor development targeting

CYP1A1 and CYP2W1 for biological activity. J. Med. Chem. 2013, 56,

6273−6277.

(16) Tercel, M.; Gieseg, M. A.; Milbank, J. B.; Boyd, M.; Fan, J. Y.;

Tan, L. K.; Wilson, W. R.; Denny, W. A. Cytotoxicity and DNA

interaction of the enantiomers of 6-amino-3-(chloromethyl)-1-[(5,6,7-

trimethoxyindol-2-yl)carbonyl]indoline (amino-seco-CI-TMI). Chem.

Res. Toxicol. 1999, 12, 700−706.

ABBREVIATIONS USED

■

ADC, antibody-drug conjugate; CBI, 1,2,9,9a-

tetrahydrocyclopropa[c]benz[e]indol-4-one; DEI, 5-(2-

dimethylaminoethoxy)indole; EDCI, 1-ethyl-3-(3-

(dimethylamino)propyl)carbodiimide hydrochloride; HAP,

hypoxia-activated prodrug; HCR, hypoxic cytotoxicity ratio;

RTV, relative tumor volume; TMI, 5,6,7-trimethoxyindole

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DOI: 10.1021/acs.jmedchem.7b00563

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

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DOI: 10.1021/acs.jmedchem.7b00563

J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

Influence of a Basic Side Chain on the Properties of Hypoxia-Selective Nitro Analogues of the Duocarmycins: Demonstration of Substantial Anticancer Activity in Combination with Irradiation or Chemotherapy

DOI: 10.1021/acs.jmedchem.7b00563

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