Synthesis of aryl-substituted naphthalene-linked pyrrolobenzodiazepine conjugates as potential anticancer agents with apoptosis-inducing ability

DOI: 10.1002/cmdc.201100207

Synthesis of Aryl-Substituted Naphthalene-Linked

Pyrrolobenzodiazepine Conjugates as Potential Anticancer

Agents with Apoptosis-Inducing Ability

Ahmed Kamal,*^[a]M. Kashi Reddy,^[a]M. Janaki Ramaiah,^[a]Y. V. V. Srikanth,^[a]Rajender,^[a]

V. Santosh Reddy,^[a]G. Bharath Kumar,^[a]S. N. C. V. L. Pushpavalli,^[a]Indira Bag,^[a]

Aarti Juvekar,^[b]Subrata Sen,^[b]Surekha M. Zingde,^[b]and Manika Pal-Bhadra*^[a]

A

library of new aryl-substituted naphthalene C8-linked

tivity relating to cell-cycle distribution, these PBD conjugates

induce G₀/G₁-phase arrest and also cause an increase in the

levels of p53 and caspase-9 proteins, followed by apoptotic

cell death. One conjugate in particular is the most promising

candidate of the series, with the potential to be selected for

further studies, either alone or in combination with existing an-

ticancer therapies.

pyrrolo[2,1-c][1,4]benzodiazepine (PBD) conjugates with vari-

ous linker architectures were designed, synthesized, and evalu-

ated for their anticancer activity against a panel of 11 human

cancer cell lines. All 32 conjugates show anticancer potential,

with some of them exhibiting particularly high activity (0.01–

0.19 mm). Thermal denaturation studies showed effective DNA

binding capacity relative to DC-81. In assays for biological ac-

Introduction

Cancer is a major global health problem, represent-

ing the second-leading cause of death worldwide.^[1]

Improvements in treatment and prevention have led

to a decrease in cancer deaths, but the number of

new diagnoses continues to rise. According to infor-

mation from the World Health Organization (WHO), it

is estimated that there will be 12 million deaths from

cancer in 2030. Over the last few years, a number of

chemotherapeutic drugs have been developed to

treat cancer, and these include DNA binding and al-

kylating agents. The pyrrolobenzodiazepines (PBDs)

are a family of naturally occurring antibiotics such as

anthramycin, chicamycin, mazethramycin, porothra-

mycin A, sibiromycin, tomaymycin, oxotomaymycin,

prothracarcin, and DC-81 (1), all of which have antitu-

mor activity. These compounds are isolated from vari-

Figure 1. Structures of naturally occurring DC-81 (1), 6-(3,4-dimethoxyphenyl)-2-naphthol

(2), and aryl-substituted naphthalene PBD conjugates 3a–x and 4a–h.

ous Streptomyces species,^[2]and they bind in the

minor groove of DNA, spanning three base pairs and

forming a covalent bond between their electrophilic

N10–C11 imine moiety and the N2 position of a gua-

nine base.^[3,4]PBDs are tricyclic molecules, which in most cases

contain a stereogenic center at the C11a position (Figure 1).

Over the past few years, various types of conjugates, in which

a known antitumor compound or some simple active moiety

tethered to a PBD, have been designed, synthesized, and eval-

uated for biological activity.^[5–9]Wang and co-workers recently

synthesized indole and enediyne PBD conjugates as potential

antitumor agents and explained the correlation between anti-

tumor activity and apoptosis.^[10,11]

[a] Dr. A. Kamal, M. K. Reddy, Dr. M. J. Ramaiah, Y. V. V. Srikanth, Rajender,

V. S. Reddy, G. B. Kumar, Dr. S. N. C. V. L. Pushpavalli, I. Bag,

Dr. M. Pal-Bhadra

Chemical Biology Laboratory

Indian Institute of Chemical Technology, Hyderabad 500607 (India)

Fax: (+91)40-2719-3189

E-mail: ahmedkamal@iict.res.in

manika@iict.res.in

[b] Dr. A. Juvekar, Dr. S. Sen, Dr. S. M. Zingde

Advanced Centre for Treatment, Research & Education

in Cancer (ACTREC) Kharghar, Navi Mumbai 410208 (India)

For the last few years our research group has been involved

in the development of new synthetic strategies for the prepa-

Supporting information for this article is available on the WWW under

http://dx.doi.org/10.1002/cmdc.201100207.

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A. Kamal, M. Pal-Bhadra, et al.

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ration of the PBD ring system,^[12–14]as well as in the design and

synthesis of structurally modified PBDs and their conju-

gates.^[15–20]More recently, we have described some of the PBD

conjugates that demonstrate potent apoptotic activity through

a mitochondria-mediated pathway.^[21–23]Moreover, several pi-

perazine-containing molecules useful as chemotherapeutic

agents have been reported over the past few years.^[24,25]It is

role in cytotoxicity, cell-cycle effects, and apoptosis by using

the MCF-7 human breast cancer cell line. Representative struc-

tures of DC-81 (1), 6-(3,4-dimethoxyphenyl)-2-naphthol (2, con-

jugate partner), and their conjugates 3a–x and 4a–h are

shown in Figure 1.

well known that this heterocyclic backbone could act on vari- Results and Discussion

ous pharmacological targets and display anticancer,^[26,27]calci-

Chemistry

um-channel-blocking,^[28]and histamine antagonist proper-

ties.^[29]Furthermore, piperazine-containing PBDs have also

shown significant anticancer activity with improved bioavaila-

bility.^[21,30,31]

The synthesis of aryl-substituted naphthalene precursors 8a–x

and 16a–h is outlined in Scheme 1. Commercially available 6-

bromo-2-naphthol (5) was treated with phenylboronic acids

6a–h in the presence of aqueous sodium bicarbonate to

afford compounds 7a–h via Suzuki palladium-catalyzed cross-

coupling reaction; these compounds, upon etherification with

various dibromoalkanes using potassium carbonate, produced

the corresponding naphthalene precursors 8a–x. For the prep-

aration of the other precursors, isovanillic acid (9) was treated

with thionyl chloride using catalytic amounts of N,N-dimethyl-

formamide to produce the acid chloride, which was coupled

with N-Boc-piperazine in the presence of triethylamine to give

the corresponding Boc-protected compound 10; this, upon de-

protection with trifluoroacetic acid, provided compound 11.

Ethyl-6-bromo-2-napthoate (12) was allowed to react with bor-

onic acids 6a–h using sodium bicarbonate to afford the aryl-

substituted naphthalenes 13a–h. Upon hydrolysis with sodium

hydroxide and treatment with thionyl chloride, these provided

the corresponding acid chlorides 14a–h, which were coupled

with compound 11 using triethylamine to furnish the naphtha-

The naphthalene skeleton is an important building block for

a large number of clinical drugs^[32,33]that possess a variety of

biological activities such as anti-inflammatory,^[34,35]cardiovascu-

lar,^[36]antibacterial,^[37]and anticancer.^[38–40]Naphthalene-based

resveratrol analogues are the most effective compounds in

causing apoptosis in a human breast cancer cell line and show

considerable antiproliferative effects.^[41,42]Thus, due to the di-

verse range of pharmacological activities of naphthalene and

its derivatives, we decided to explore various aryl-substituted

naphthalene derivatives as pharmacophores in the design of

PBD conjugates.

In continuation of these efforts, aryl-substituted naphthalene

derivatives were linked to the C8-position of the PBD scaffold

(DC-81, 1) through stable alkane linkers and also by incorporat-

ing a piperazine moiety. This led to a new library of PBD conju-

gates that were evaluated for their anticancer potential. In

view of their promising activity, we decided to investigate their

Scheme 1. Reagents and conditions: a) Pd(PPh₃)₄, aq. NaHCO₃, toluene/EtOH (3:1), 80–908C, 2–4 h, (71–90%); b) dibromoalkanes, anhyd. K₂CO₃, dry acetone,

reflux, 12–24 h, (78–88%); c) SOCl₂, DMF, dry CH₂Cl₂, 08C!RT, 2 h, (85%); d) N-Boc-piperazine, Et₃N, dry CH₂Cl₂, 08C!RT, 4 h, (72%); e) TFA, dry CH₂Cl₂, 08C!

RT, 2 h, (93%); f) 1n NaOH, EtOH, 70–808C, 1–2 h, (91–95%); g) SOCl₂, dry CH₂Cl₂, 08C!RT, 2–4 h, (90%); h) Et₃N, dry CH₂Cl₂, RT, 2–4 h, (65–70%).

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ChemMedChem 2011, 6, 1665 – 1679

PBD Conjugates as Anticancer Agents

lene intermediates 15a–h. Finally, etherification of compounds

15 with 1,3-dibromopropane in the presence of potassium car-

bonate afforded the aryl-substituted naphthalene precursors

16a–h.

The synthesis of PBD conjugates 3a–x and 4a–h is illustrat-

ed in Scheme 2. Compound 17, (2S)-N-[4-hydroxy-5-methoxy-

2-nitrobenzoyl]pyrrolidine-2-carboxaldehyde diethylthioacetal

was prepared from vanillin by employing our previously re-

ported procedure.^[12–20]Coupling of compound 17 with aryl-

tive to those with an even number of carbons (n=2) in the

linker.

Restriction endonuclease digestion assay

The DNA melting temperature studies indicated that the aryl-

substituted PBD conjugates exhibit significant DNA binding af-

finity. To further confirm their interaction with DNA, a protec-

tion assay using restriction endonuclease digestion was per-

formed. The PBD compounds

are effective as DNA binding

agents particularly at G-rich se-

quences.^[43]Therefore, to deter-

mine the binding activity of

these PBD conjugates relative to

naturally occurring PBD com-

pounds such as DC-81 (1), re-

striction enzyme digestion (RED)

assays were carried out for com-

pounds with particular promise,

such as 3g and 4g, alongside

compound 1. In this assay, a

pBR322 vector with a BamHI re-

striction site (5’-GjGATCC-3’, in

which ’j’ denotes the site of

cleavage) flanked by G-rich re-

Scheme 2. Reagents and conditions: a) anhyd. K₂CO₃, dry acetone, reflux, 12–24 h, (72–83%); b) SnCl₂·2H₂O, MeOH,

reflux, 4–6 h; c) HgCl₂, CaCO₃, CH₃CN/H₂O (4:1), RT, 8–12 h, (50–73% yield over two steps).

gions was used. The experiment

is based on the principle that

any compound binding at the

substituted naphthalene precursors 8a–x and 16a–h in the

presence of potassium carbonate provided the corresponding

nitrothioacetols 18a–x and 19a–h, which upon reduction with

tin(II) chloride dihydrate followed by deprotection and cycliza-

tion using mercury(II) chloride/calcium carbonate, afforded the

target compounds 3a–x and 4a–h; see Table 1 for structures.

BamHI site should impede endonuclease activity toward the

DNA substrate by BamHI. Details of the experimental protocol

are described in a study reported earlier by our research

group.^[15]For the assays reported herein, we used various test

compound concentrations ranging from 1 to 8 mm. Com-

pounds 3g and 4g were observed to effectively bind G-rich re-

gions of DNA along the minor groove at 4 and 8 mm, whereas

DC-81 (1) did not show binding, as can been seen from the re-

sults shown in Figure 2. The inhibitory activity of these PBD

conjugates is ranked in the following order: 4g > 3g > 1,

and is in agreement with the DNA binding ability as deter-

mined by thermal denaturation studies. These assays clearly in-

dicate that the linking of the PBD scaffold with other conju-

gate partners significantly enhances the binding potential. It

has been well established that the PBD scaffold forms a cova-

lent bond with N2 of a guanine base in the DNA, while the

other (naphthalene) component of this new conjugate is likely

to associate with DNA through noncovalent interactions.

Indeed, similar observations were made in previous studies

with related PBD conjugates.^[44,45]

Biological activity

DNA interaction: thermal denaturation studies

The DNA binding ability of the new PBD conjugates 3a–x and

4a–h was investigated by thermal denaturation studies using

calf thymus (CT) duplex DNA at pH 7.0, using protocols report-

ed in our previous work.^[22,23]These studies were carried out at

a DNA/ligand molar ratio of 1:5. The melting temperature

(DT_m) for each compound was examined after 0 and 18 h incu-

bation at 378C. All conjugates 3a–x and 4a–h caused an in-

crease in CT-DNA DT_mvalues in the respective ranges of 1.5–

4.0 and 2.8–4.28C relative to the naturally occurring DC-81 (1)

and conjugate partners (2 and 15g). Interestingly, conjugates

3g, 3h, 4g, and 4h elevate the melting temperature by 4.0,

3.8, 4.2, and 4.18C, respectively, after 18 h incubation at 378C

as shown in Table 1. Conjugates 4 show slightly higher DT_m

values than conjugates 3, probably due to the presence of a

piperazine moiety in the linker. Most of the conjugates in

series 3 containing a linker with an odd number of methylene

units (n=1 or 3) show slightly higher DNA binding affinity rela-

Anticancer activity

These aryl-substituted naphthalene PBD conjugates 3a–x and

4a–h were evaluated for their anticancer activity in selected

human cancer cell lines of lung, breast, oral, colon, prostate,

ovarian, and cervical tissue by using the sulforhodamine B

(SRB) method. Conjugates that exhibit GI₅₀ꢀ10^ꢁ5m are consid-

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A. Kamal, M. Pal-Bhadra, et al.

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on the phenyl moiety of aryl-

substituted naphthalene deriva-

tives, all the 3,4-dimethoxy- (3g,

3o, 3w, and 4g) and 3,4,5-trime-

thoxy- (3h, 3p, 3x, and 4h) con-

taining aryl-substituted naphtha-

lene PBD conjugates showed

significant anticancer activity

against all 11 human cancer cell

lines relative to other conju-

gates. The PBD conjugates with

an odd number (n=1 or 3) of

methylene units in the linker ex-

hibited greater activity than their

counterparts with an even

number of units (n=2) in the

linker. PBD conjugates with pro-

pyloxy linkers showed slightly

better activity than those with

pentyloxy linkers. Furthermore,

we observed that the incorpora-

tion of piperazine along with an

isovanillin moiety in the linker

between the aryl naphthalene

and PBD subunits (series 4) pro-

duced compounds with activity

similar to that of the other PBD

conjugates. Among all the com-

pounds synthesized, conjugates

3a–h, 3q, 3s, 3w, 3x, and 4a–h

exhibited significant anticancer

activity, with GI₅₀values ranging

from 0.01 to 0.19 mm against the

panel of 11 human cancer cell

lines.

Table 1. A library of aryl-substituted naphthalene PBD conjugates (3a–x and 4a–h) and their thermal denatu-

ration data with calf thymus (CT)-DNA.

DT_m[8C]^[a]

Compd

R¹

R²

R³

n

0 h

18 h

3a

3b

3c

3d

3e

3 f

3g

3h

3i

3j

3k

3l

3m

3n

3o

3p

3q

3r

H

OCH₃

H

OCH₃

H

OCH₃

H

OCH₃

–

H

F

CF₃

H

Cl

OCH₃

H

1

2

3

–

2.4

2.0

2.6

1.7

2.2

2.8

3.2

3.0

2.1

1.9

1.8

1.6

1.8

2.1

3.5

3.3

2.1

1.5

2.1

1.7

2.0

2.6

3.2

2.9

2.8

3.0

2.8

3.1

3.0

3.7

3.5

0.3

0.2

0.3

3.0

2.2

2.6

1.9

2.8

3.1

4.0

3.8

2.5

2.1

1.8

1.9

1.8

2.5

3.8

3.5

2.5

1.8

2.4

1.7

2.5

2.8

3.7

3.5

3.1

2.8

3.3

3.5

3.7

3.6

4.2

4.1

0.7

0.2

0.5

OCF₃

OCH₃

N(CH₃)₂

H

OCH₃

F

CF₃

OCF₃

OCH₃

N(CH₃)₂

H

OCH₃

F

Cl

OCH₃

H

Cl

OCH₃

H

CF₃

3s

3t

OCF₃

OCH₃

N(CH₃)₂

H

OCH₃

F

3u

3v

3w

3x

4a

4b

4c

4d

4e

4 f

CF₃

H

OCF₃

OCH₃

N(CH₃)₂

H

OCH₃

–

MTT assays were carried out

for eight potent PBD conjugates

(3 f–3i, 3s, 3w, 4d, and 4g) to

identify any cytotoxic effects in

MCF-7 cells at 4 mm for 24 h.

These PBD conjugates show

higher cytotoxicity than DC-81

(1) and the conjugate partner 2;

among them, compounds 3g,

3h, and 4g exhibited particularly

high activity, as shown in

Figure 3.

Cl

4g

4h

DC-81 (1)

2

OCH₃

–

OCH₃

15g

H

[a] Determined at a PBD/DNA molar ratio of 1:5, for which [CT-DNA]=100 mm and [ligand]=20 mm in aqueous

sodium phosphate buffer (10 mm sodium phosphate, 1 mm EDTA, pH 7.00ꢂ0.01), after incubation at 378C for

either 0 or 18 h; for CT-DNA alone at pH 7.00ꢂ0.01, T_m=69.68Cꢂ0.01 (mean value from 10 separate determi-

nations); all T_mvalues are ꢂ0.05–0.158C.

ered to be active on the respective cell lines. All 32 conjugates

exhibited significant anticancer activity, with GI₅₀values rang-

Effect of PBD conjugates on cell cycle

ing from 0.01 to 3.41 mm; the positive control compounds,

adriamycin and DC-81 (1), showed GI₅₀values toward these

cell lines in the ranges of 0.1–7.25 and 0.1–2.37 mm, respective-

ly, and the results are listed in Table 2. Interestingly, all the new

conjugates showed significant activity in the breast (Zr-75-1,

MCF-7) and ovarian (A2780) cancer cell lines. Some interesting

trends have been observed for these conjugates in structure–

activity relationship (SAR) studies. With respect to substitution

To investigate the mechanism underlying the cytotoxic effect

of these PBD conjugates on cell-cycle progression in the MCF-

7 human breast cancer cell line, the DNA content in cell nuclei

was measured by flow cytometry. Treatment of MCF-7 cells

with PBD conjugates 3g, 3h, and 4g at 4 mm for 24 h caused

87.02, 84.17, and 82.42% accumulation of cells in G₀/G₁phase,

respectively. For comparison, DC-81 (1) and conjugate partner

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ChemMedChem 2011, 6, 1665 – 1679

PBD Conjugates as Anticancer Agents

Figure 3. Effect of PBD conjugates on cell viability (in vitro cytotoxicity).

MCF-7 cells were treated with conjugates 3 f–3i, 3s, 3w, 4d, and 4g at

4 mm for 24 h in 96-well plates seeded with 10000 cells per well. OD read-

ings were taken at l 570 nm to determine percent cell viability after treat-

ment with the compounds. Here DC-81 (1) and the conjugate partner 2

were used as positive controls; C=negative control (untreated cells). Each

experiment was repeated three times, and error bars represent standard de-

viations, derived from three independent experiments.

Figure 2. RED assay with DC-81 (1) at top; the middle and bottom panels

depict RED assay results for compounds 3g and 4g. Compound concentra-

tions are indicated; ’ctrl’ refers to uncut vector DNA.

2 caused respective G₀/G₁-phase accumulations of 68.14 and

59.80%. Therefore, the increase in the percentage of cells in

G₀/G₁phase clearly shows that these conjugates cause G₁-

phase arrest in MCF-7 cells. Conjugate 3g appears to be the

most effective conjugate in effecting cell-cycle arrest, as evi-

dent from the data in Table 3. The percentage of apoptotic (G₀

phase) cells in the presence of

test compound at 4 mm, as well

as at each compound’s respec-

tive GI₅₀and IC₅₀concentrations,

are listed in Table 4.

Table 2. GI₅₀values for PBD conjugates 3a–x and 4a–h in selected human cancer cell lines.

GI₅₀[mm]^[a]

Compd

ZR-75-1^[b]A549^[c]A2780^[d]Hop62^[c]KB^[e]SiHa^[f]Gurav^[c]MCF-7^[b]Colo205^[g]DWD^[e]PC3^[h]

3a

3b

3c

3d

3e

3 f

3g

3h

3i

3j

3k

3l

3m

3n

3o

3p

3q

3r

0.02

0.03

0.13

0.05

0.13

0.09

0.01

0.02

0.14

0.17

0.18

0.19

0.18

0.17

0.02

0.13

0.09

0.12

0.08

0.09

0.02

0.06

0.11

0.13

0.14

0.08

0.13

0.11

0.01

0.03

1.79

0.13

0.19

0.14

0.11

0.13

1.18

1.47

1.24

1.28

1.49

1.29

1.18

1.21

0.13

0.14

0.12

0.13

0.14

0.13

0.14

0.12

0.17

0.02

0.16

0.17

0.01

7.25

0.16

0.08

0.05

0.09

0.13

0.07

0.01

0.17

0.19

0.18

0.19

0.17

0.19

0.08

0.16

0.15

0.14

0.05

0.01

0.04

0.14

0.17

0.11

0.07

0.17

0.05

0.01

0.05

0.16

0.14

0.13

0.14

0.17

0.16

0.11

0.14

0.19

2.63

2.26

0.19

2.32

0.16

0.19

0.17

0.18

0.16

0.17

0.13

0.14

0.16

0.17

0.16

0.13

0.16

0.17

0.11

0.14

0.15

0.15 0.16

0.17 0.17

0.16 0.18

0.16 0.17

0.16 0.18

0.15 0.09

0.01 0.06

0.11 0.08

1.26 1.01

3.27 2.82

3.05 2.52

2.48 3.19

2.22 2.81

3.12 0.19

1.21 2.19

1.27 2.21

0.18 0.16

0.18 1.90

0.16 0.15

0.15 0.16

0.15 0.18

0.17 0.17

0.15 0.14

0.17 0.17

0.18 0.19

0.17 0.19

0.16 0.14

0.15 0.17

0.17 0.18

0.14 0.11

0.17 0.18

0.17 0.17

0.17

0.14

0.17

0.16

0.01

0.17

2.06

0.18

2.92

2.00

0.17

1.42

1.10

0.16

1.80

0.15

0.14

0.17

0.14

0.18

0.17

0.12

0.17

0.15

0.17

0.16

0.07

0.04

0.11

0.08

0.04

0.03

0.01

0.02

0.11

0.17

0.19

0.18

0.17

0.12

0.14

0.11

0.17

0.09

0.15

0.13

0.14

0.06

0.11

0.13

0.05

0.13

0.11

0.02

0.11

0.17

0.13

0.14

0.18

0.17

0.18

0.17

0.01

0.02

2.27

3.11

3.13

2.71

3.33

3.41

2.70

2.72

0.18

2.40

0.18

0.15

2.00

2.18

0.16

0.19

0.17

0.15

0.17

0.14

0.17

0.03

0.16

0.14

0.11

0.17

0.14

0.15

0.02

0.01

0.18

2.30

0.19

2.30

0.19

0.17

0.15

0.18

0.15

0.14

0.15

0.09

0.16

0.18

0.16

0.18

0.13

0.17

0.10

1.49

0.09

0.17

0.16

0.17

0.08

0.07

0.08

1.16

2.48

2.10

2.20

2.48

1.20

1.16

1.32

0.17

2.00

0.17

2.00

0.18

0.11

0.14

0.17

0.19

0.17

0.18

0.17

0.05

0.17

1.81

0.20

Effect of PBD conjugates on the

expression of p53 and caspase-9

The activation of p53 plays an

important role in the induction

of

apoptosis

by

various

agents,^[46]and it is well known

that p53 is a tumor-suppressor

gene, the product of which

exerts its antiproliferative effects

through its function as a se-

quence-specific DNA binding

transcription factor. To determine

the effect of PBD conjugates on

the expression of the p53-de-

pendent apoptotic pathway,

MCF-7 cells were treated with

conjugates 3g, 3h, and 4g at

4 mm for 24 h, and Western blot

analysis was carried out with a

p53-specific antibody. The levels

of p53 were up-regulated in the

presence of all conjugates tested

relative to DC-81 (1) and conju-

gate partner 2 (Figure 4a). Simi-

lar results were observed in the

results of ELISA, as shown in Fig-

ure 4b. Interestingly, the in-

crease in the level of p53 protein

was more prominent for conju-

3s

3t

3u

3v

3w

3x

4a

4b

4c

4d

4e

4 f

4g

4h

ADR^[i]

DC-81 (1) 2.37

[a] Values represent the mean of three determinations. [b] Breast cancer. [c] Lung cancer. [d] Ovarian cancer.

[e] Oral cancer. [f] Cervical cancer. [g] Colon cancer. [h] Prostate cancer. [i] Adriamycin.

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Table 3. Cell-cycle distribution of MCF-7 cells with DC-81 (1) [4 mm], con-

jugate partner 2, and PBD conjugates 3g, 3h, and 4g.^[a]

Compd

G₀/G₁

S

G₂/M

Control

DC-81 (1)

2

3g

3h

52.09ꢂ1.38

68.14ꢂ0.13

59.80ꢂ0.67

87.02ꢂ0.27

84.17ꢂ2.00

82.42ꢂ0.64

10.39ꢂ0.40

8.48ꢂ0.22

10.34ꢂ0.40

4.33ꢂ0.32

5.32ꢂ0.35

5.05ꢂ0.13

37.51ꢂ0.93

23.37ꢂ0.33

29.85ꢂ0.28

8.70ꢂ0.10

10.50ꢂ1.80

12.53ꢂ0.57

4g

[a] Values represent the mean ꢂSD of at least three experiments, each

performed in triplicate.

Table 4. Percentage of apoptosis (G₀cells) with DC-81 (1), conjugate

partner 2, and PBD conjugates 3g, 3h, and 4g.^[a]

G₀cells [%]

Compd

at 4 mm

at GI₅₀

at IC₅₀

Control

DC-81 (1)

2

3g

3h

3.72ꢂ0.25

4.24ꢂ0.01

3.33ꢂ0.14

15.26ꢂ0.21

7.25ꢂ0.34

4.55ꢂ0.45

1.13ꢂ0.41

1.67ꢂ0.28

1.57ꢂ0.40

4.30ꢂ0.64

2.66ꢂ0.51

1.83ꢂ0.26

2.76ꢂ0.25

3.57ꢂ0.36

1.56ꢂ0.46

11.10ꢂ1.10

5.34ꢂ0.57

2.66ꢂ0.12

4g

[a] Values represent the mean ꢂSD of at least three experiments, each

Figure 4. a) Effect of PBD conjugates on the expression of p53 and procas-

pase-9. MCF-7 cells were treated with DC-81 (1), 2, 3g, 3h, and 4g at 4 mm

for 24 h. Cell lysates were collected, and protein expression levels were de-

termined by Western blot analysis using specific antibodies; b-actin was

used as loading control. Control (ctrl) lanes: untreated. b) Effect of PBD con-

jugates on the expression p53. MCF-7 cells were treated with the indicated

compounds at 4 mm for 24 h. Cell lysates were collected and observed for

p53 protein expression levels using a p53 ELISA kit. c) Effect of PBD conju-

gates on caspase-9 protein activity. MCF-7 cells were treated with the indi-

cated compounds at 4 mm for 24 h and then subjected to fluorimetry as de-

tailed in the Experimental Section. Here DC-81 (1) was used as the positive

control with compound 2 as the conjugate partner.

performed in triplicate.

gate 3g, as evident in Figure 4a and 4b, thus indicating that

such PBD conjugates suppress tumor cell proliferation.

Given the observed increase in p53 levels in the presence of

these PBD conjugates, we wanted to shed light on the molecu-

lar mechanism behind the induction of apoptosis. The MCF-7

cell line lacks endogenous caspase-3,^[47]whereas caspase-9

plays an important role in mediating drug-induced apopto-

sis.^[48]Thus the role of caspases was examined in MCF-7 cells

treated with conjugates 3g, 3h, and 4g at 4 mm for 24 h. Cell

lysates were analyzed for active caspase-9 expression levels by

a fluorescence-based caspase-9 assay. Up-regulation of cas-

pase-9 was effected by these conjugates relative to both con-

trols, DC-81 (1) and conjugate partner 2, as shown in Fig-

ure 4c. A similar observation was made by Western blot analy-

sis, indicating a degradation of procaspase-9 and production

of active caspase-9; such activity is ranked in the order 3g >

3h > 4g as shown in Figure 4a. Apoptosis is more prominent

for all three conjugates relative to both controls, and is most

prominent in the presence of 3g, as evident in Figure 4.

treated with conjugate 3g, which is more prominent than for

cells treated with positive control, DC-81 (1), as apparent in

Figure 5b. These results further confirm the apoptotic-inducing

ability of such PBD conjugates.

Conclusions

In summary, a library of aryl-substituted naphthalene–PBD con-

jugates were synthesized and evaluated for their anticancer

potential. Thermal denaturation studies showed that these

conjugates exhibit higher DNA binding affinity than the natu-

rally occurring PBD, DC-81 (1). All these PBD conjugates 3a–x

and 4a–h showed significant anticancer activity, with GI₅₀

values ranging from 0.01 to 3.41 mm, in comparison with some

previously reported PBD conjugates such as phosphonate-

linked PBD conjugates (GI₅₀values in the 0.17–30.50 mm

range),^[19]1,2,3-triazole-linked PBD conjugates (GI₅₀values in

the 0.13–30.50 mm range),^[20]and triazolobenzothiadiazine-

linked PBD conjugates (GI₅₀values in the 0.22–30.30 mm

range).^[49]Moreover, from the MTT proliferation assay, the three

most promising conjugates 3g, 3h, and 4g showed higher cy-

totoxicity in MCF-7 cells. Flow cytometry (FACS) analysis

Effect of PBD conjugates on DNA

A terminal transferase dUTP nick end labeling (TUNEL) assay

was carried out with a view to understand the effects of these

PBD conjugates on DNA by treating MCF-7 cells with DC-81 (1)

and conjugate 3g at 4 mm for 24 h. Confocal microscopy re-

vealed DNA fragmentation (green FITC staining) in the pres-

ence of conjugate 3g as well as DC-81 (1), but not in untreat-

ed cells (Figure 5a). Moreover, Hoechst DNA staining also indi-

cated the formation of apoptotic bodies (blebbing) in cells

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ChemMedChem 2011, 6, 1665 – 1679

PBD Conjugates as Anticancer Agents

helix–coil transition temperatures (T_m) were determined from the

maxima in d(A₂₆₀)/dT derivative plots. Results for each conjugate

are shown as the mean ꢂSD from at least three determinations

and are corrected for the effects of MeOH co-solvent using a linear

correction term.^[51]Ligand-induced alterations in DNA melting be-

havior are given by DT_m=T_m(DNA+PBD)ꢁT_m(DNA alone), where

the T_mvalue for PBD-free CT-DNA is 68.5ꢂ0.001; the fixed PBD/

DNA ratio used did not result in binding saturation of the host

DNA duplex for any compound examined.

Cell line maintenance

The MCF-7 (human breast cancer) cell line was obtained from the

American Type Culture Collection (ATCC), USA. MCF-7 cells were

maintained in Dulbecco’s modified Eagle’s medium (DMEM; Invitro-

gen), supplemented with 10% fetal calf serum and 100 UmL^ꢁ1

penicillin and 100 mgmL^ꢁ1streptomycin sulfate (Sigma). The cells

were maintained at 378C in a humidified atmosphere containing

5% CO₂.

Figure 5. a) Effect of PBD conjugates on DNA fragmentation. MCF-7 cells

were treated with DC-81 (1) and conjugate 3g at 4 mm for 24 h. DNA frag-

mentation was monitored by primary antibody binding to fragmented DNA,

followed by detection with FITC-labeled secondary antibody. Apoptotic cells

are thus stained green. Here DC-81 was used as positive control; 3g is the

most effective conjugate of this series. Control cells yield only background

color. The apoptotic nature of the compounds is apparent by fragmented

nuclei in the compound-treated cells. b) Effect of PBD conjugates on apop-

tosis. MCF-7 cells were treated with DC-81 and conjugate 3g at 4 mm for

24 h. Hoechst 33258 was used to stain the nuclei of both control and conju-

gate 3g-treated cells. Here DC-81 was used as positive control; 3g is the

most effective conjugate in this series. As above, the apoptotic nature of the

compounds is apparent by fragmented nuclei in compound-treated cells.

Cell viability (MTT assay)

Cell viability was assessed by MTT assay using the Vybrant MTT cell

proliferation assay kit (Invitrogen). The assay is based on the ability

of viable cells to reduce MTT to insoluble formazan crystals by mi-

tochondrial dehydrogenase. Briefly, MCF-7 cells were seeded in a

96-well plate (TPP) at a cell density of 10000 cells per well. After in-

cubation overnight, the cells were treated with compounds DC-81

(1), 2, 3 f–3i, 3s, 3w, 4d, and 4g at 4 mm for 24 h. The medium

was then discarded and replaced with fresh medium (100 mL) fol-

lowed by addition of 10 mL MTT dye. Plates were incubated at

378C for 2 h. The resulting formazan crystals were solubilized in

100 mL extraction buffer (SDS). The optical density (OD) was read

at l 570 nm using Multimode Varioscan FLASH (Thermo Fischer Sci-

entific).

showed a greater cell population in the G₀/G₁phase, indicating

that the PBD conjugates possess the ability to induce apopto-

sis. To understand the mechanism of cell death, further biologi-

cal assays such as Western blot analyses of p53, procaspase-9,

and caspase-9 were carried out. The results indicated an up-

regulation of p53 and caspase-9 upon treatment with conju-

gates 3g, 3h, and 4g. Furthermore, TUNEL assays and Hoechst

staining further confirmed apoptosis effected by conjugate 3g.

This investigation reveals that linking of aryl-substituted naph-

thalene derivatives to the PBD ring system enhances anticanc-

er activity. Based on the results of the studies reported herein,

it is evident that conjugate 3g is a suitable candidate for fur-

ther detailed studies either alone or in combination with exist-

ing therapies.

Cell cycle analysis

MCF-7 cells (5ꢁ10⁵) were seeded in a 60 mm Petri dish and were

allowed to grow for 24 h. Compounds DC-81 (1), 2, 3g, 3h, and

4g (each at 4 mm) were added to the culture medium, and the

cells were incubated for an additional 24 h. Cells were harvested

with trypsin/EDTA, fixed with ice-cold 70% EtOH at 48C for 30 min,

washed with PBS and incubated with 1 mgmL^ꢁ1RNase A solution

(Sigma) at 378C for 30 min. Cells were collected by centrifugation

at 2000 rpm (Heraeus Sorvall swinging bucket rotor (model #

75002000), max speed: 4700 rpm, Heraeus Multifuge 1S-R, Thermo

Scientific) for 5 min and further stained with 250 mL DNA staining

solution [10 mg propidium iodide (PI), 0.1 mg trisodium citrate,

and 0.03 mL Triton X-100 dissolved in 100 mL sterile Milli-Q water

at room temperature for 30 min in the dark]. The DNA content of

20000 events was measured by flow cytometry (DAKOCYTOMA-

TION, Beckman Coulter, Brea, CA, USA). Histograms were analyzed

using Summit software.

Experimental Section

Biological assays

Thermal denaturation studies

Compounds 3a–x and 4a–h were subjected to DNA thermal melt-

ing (denaturation) studies with duplex calf thymus DNA (CT-DNA)

using a modified published procedure.^[50]Working solutions were

produced by appropriate dilution in aqueous buffer (10 mm

Na₂HPO₄/NaH₂PO₄, 1 mm Na₂EDTA, pH 7.00ꢂ0.01) containing CT-

DNA (100 mm in phosphate) and the PBD (20 mm); these were pre-

pared by the addition of concentrated PBD solutions in MeOH to

obtain a fixed PBD/DNA molar ratio of 1:5. The DNA/PBD solutions

were incubated at 378C for 0 h prior to analysis; samples were

monitored at l 260 nm using a Beckman DU-7400 spectrophotom-

eter fitted with a high-performance temperature controller. Heat-

ing was applied at a rate of 18Cmin^ꢁ1in the 40–908C range. DNA

Protein extraction and Western blot analysis

Total cell lysates from cultured MCF-7 cells after compound treat-

ment [DC-81 (1), 2, 3g, 3h, and 4g] as described above were ob-

tained by lysing the cells in ice-cold RIPA buffer (1ꢁPBS, 1% NP-

40, 0.5% sodium deoxycholate, and 0.1% SDS) containing

100 mgmL^ꢁ1PMSF, 5 mgmL^ꢁ1aprotinin, 5 mgmL^ꢁ1leupeptin,

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A. Kamal, M. Pal-Bhadra, et al.

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5 mgmL^ꢁ1pepstatin, and 100 mgmL^ꢁ1NaF. After centrifugation at

12000 rpm (fixed angle rotor (model # 75003348), max speed:

22000 rpm, Heraeus Multifuge 1S-R, Thermo Scientific) for 10 min,

the protein content in the supernatant was quantified by the Brad-

ford method (Bio-Rad) using a Multimode Varioscan instrument

(Thermo Fischer Scientific). Protein (50 mg per lane) was applied to

a 12% SDS polyacrylamide gel. After electrophoresis, the protein

was transferred to a polyvinylidine difluoride (PVDF) membrane

(Amersham Biosciences). The membrane was blocked at room tem-

perature for 2 h in TBS containing 0.1% Tween-20 (TBST) contain-

ing 5% blocking powder (Santa Cruz Biotechnology). The mem-

brane was washed with TBST for 5 min, and primary antibody was

added and incubated at 48C overnight. Mouse monoclonal b-actin

antibody and rabbit polyclonal procaspase-9 antibody were pur-

chased from Imgenex. Mouse monoclonal p53 antibody was pur-

chased from Santa Cruz Biotechnology. After three TBST washes,

the membrane was incubated with corresponding horseradish per-

oxidase (HRP)-labeled secondary antibody (1:2000; Santa Cruz) at

room temperature for 1 h. Membranes were washed with TBST

three times for 15 min, and the protein blots were visualized with

a Super Signal West Pico chemiluminescence reagent (Thermo

Fischer Scientific). Blot images were collected by X-ray autoradiog-

raphy.

Chemistry

All chemicals and reagents were purchased from Sigma–Aldrich

(St. Louis, MO, USA), Lancaster (Alfa Aesar, Johnson Matthey Com-

pany, Ward Hill, MA, USA) or Spectrochem Pvt. Ltd. (Mumbai,

India), and were used without further purification. Reactions were

monitored by TLC, performed on glass plates containing silica gel

60 GF₂₅₄, and visualized by UV light or iodine indicator. Column

chromatography was performed with Merck 60–120 mesh silica

1

gel. H NMR and ¹³C NMR spectra were recorded on Gemini Varian

VXR Unity (200 MHz) or Bruker UXNMR/XWIN-NMR (300 MHz) in-

struments. Chemical shifts (d) are reported in ppm relative to the

peak for (CH₃)₄Si as an internal standard, and coupling constants

are reported in Hz. High-resolution mass spectra (HRMS) were re-

corded on a QSTAR XL Hybrid MS–MS mass spectrometer. ESIMS

data were recorded on a Micromass Quattro LC instrument using

ESI⁺software with a capillary voltage of 3.98 kV and an ESI positive

ion trap detector. Melting points were determined with an Electro-

thermal melting point apparatus and are uncorrected.

General procedure for synthesis of 7a–h (Suzuki coupling)

A catalytic amount (0.02–0.04 mmol) of Pd(PPh₃)₄and aqueous 2m

NaHCO₃(1 mL) were added to a solution of 6-bromo-2-naphthol

(5, 223 mg, 1 mmol) in toluene (10 mL). A solution of substituted

phenylboronic acids (6a–h, 1 mmol) in 3 mL EtOH was added and

stirred at RT under N₂. After 10 min, the solution was heated at 80–

908C for 2–4 h. TLC showed the disappearance of starting materi-

als, and the reaction mixture was cooled to RT and extracted with

EtOAc (3ꢁ30 mL). The organic phases were combined, washed

with H₂O and brine, dried over anhydrous Na₂SO₄, and the solvent

was removed under reduced pressure to obtain the crude product.

This residue was purified by column chromatography using EtOAc

and hexane to afford compounds 7a–h.

p53 ELISA

Enzyme-linked immunorsorbent assays (ELISA) for p53 were con-

ducted with the p53 ELISA kit from Alexis Biochemical. MCF-7 cells

were treated with compounds DC-81 (1), 2, 3g, 3h, and 4g at

4 mm for 24 h. Cell lysates were isolated and added to microplate

wells containing p53 antibody. Biotin-conjugated anti-human p53

monoclonal antibody (100 mL) was then added. After the incuba-

tion period and washing steps, bound p53 was detected by using

streptavidin–HRP secondary antibody (150 mL). The colored prod-

uct obtained was detected by measuring OD at l 450 nm; the OD

is directly proportional to the amount of p53 protein present in

the sample.

General procedure for synthesis of 8a–x

Anhydrous K₂CO₃(4 mmol) and various dibromoalkanes (3 mmol)

were added to a solution of compounds 7a–h (1 mmol) in dry ace-

tone (10 mL), and the reaction mixture was held at reflux for 12–

24 h. After completion of the reaction, K₂CO₃was removed by suc-

tion filtration, and the solvent was evaporated under reduced pres-

sure to obtain the crude product. This was further purified by

column chromatography using EtOAc and hexane to afford com-

pounds 8a–x.

Caspase-9 assay

The Apoalert caspase-9/6 fluorescent assay kit (Clonetech, CA,

USA) was used according to the manufacturer’s recommendations.

MCF-7 cells were treated with compounds DC-81 (1), 2, 3g, 3h,

and 4g at 4 mm as obtained from FACS analysis. Here the substrate

used is LEHD–AMC, which was added to the cell lysates, and incu-

bation was carried out at 378C for 1 h. Readings were taken at

l_ex400 nm and l_em505 nm.

tert-Butyl-4-(3-hydroxy-4-methoxybenzoyl)-1-piperazinecarboxy-

late (10): A catalytic amount of DMF (2–4 drops) was added to a

stirred solution of 3-hydroxy-4-methoxybenzoic acid (isovanillic

acid, 168 mg, 1 mmol) and SOCl₂(0.18 mL, 2.5 mmol) in dry CH₂Cl₂

(5 mL), and the mixture was stirred for 2 h under N₂. The solvent

was evaporated under vacuum, the resulting acid chloride was

washed with dry CH₂Cl₂(3ꢁ10 mL), and the solvent was evaporat-

ed under reduced pressure to obtain the product 3-hydroxy-4-me-

thoxybenzoyl chloride as a light-yellow oil. Owing to potential sta-

bility problems, this product was taken directly to the next step.

Et₃N (0.14 mL, 2 mmol) was added dropwise to a stirred solution of

N-Boc-piperazine (186 mg, 1 mmol) in dry CH₂Cl₂(10 mL) at 08C

over 10 min; 3-hydroxy-4-methoxybenzoyl chloride (220 mg,

1.2 mmol) dissolved in dry CH₂Cl₂(5 mL) was then added at the

same temperature. The reaction was brought to RT and stirred for

another 2 h. After completion of the reaction, the solvent was

evaporated and extracted with CH₂Cl₂(3ꢁ20 mL), washed with

aqueous NaHCO₃(2m) followed by brine, separated, and dried

TUNEL assay

Terminal transferase dUTP nick end labeling (TUNEL) assays were

conducted with the Apoalert DNA fragmentation assay kit (Clone-

tech). Apoptosis-induced nuclear DNA fragmentation was deter-

mined using this assay, which was conducted according to the

manufacturer’s recommendations and is based on the principle of

terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick

end labeling. TdT catalyzes the incorporation of fluorescein–dUTP

at the free 3’-hydroxy ends of fragmented DNA. Flourescein-la-

beled DNA can be detected by confocal microscopy.

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PBD Conjugates as Anticancer Agents

over anhydrous Na₂SO₄. The solvent was evaporated under re-

duced pressure and further purified by column chromatography

using with EtOAc and hexane (1:1) as eluent to afford compound

10 as a white solid.

(3 mmol). The crude product was purified by column chromatogra-

phy using EtOAc and hexane to afford compounds 16a–h.

(3-Hydroxy-4-methoxybenzoyl)-1-piperazine (11): Trifluoroacetic

acid (TFA; 0.9 mL, 13 mmol) was added slowly to compound 10

(336 mg, 1 mmol) in dry CH₂Cl₂(25 mL) at 08C, and the reaction

mixture was stirred at RT for 2 h. After completion, the reaction

mixture was concentrated, and the resulting compound was sus-

pended in aqueous NaHCO₃(2m, 20 mL), the solution was extract-

ed into CH₂Cl₂(3–30 mL), and the combined organic phases were

washed with H₂O and brine (2ꢁ30 mL), dried over anhydrous

Na₂SO₄, and the solvent was evaporated under reduced pressure

to obtain the product. This compound was used without further

purification for next step to afford compound 11 as a light-yellow

oil.

General procedure for synthesis of 18a–x and 19a–h

Anhydrous K₂CO₃(552 mg, 4 mmol) and naphthalene precursors

(8a–x and 16a–h 1 mmol) were added to a solution of (2S)-N-[4-

hydroxy-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxaldehyde di-

ethylthioacetal (17, 400 mg, 1 mmol) in dry acetone (20 mL). The

reaction mixture was held at reflux for 12–24 h. After completion

of the reaction as indicated by TLC, K₂CO₃was removed by suction

filtration, and the solvent was removed under vacuum. The crude

product was further purified by column chromatography using

EtOAc and hexane to afforded pure nitro compounds 18a–x and

19a–h.

General procedure for synthesis of 13a–h

General procedure for synthesis of 3a–x and 4a–h

Compounds 13a–h were prepared by following the method de-

scribed for the preparation of compound 7 (Suzuki cross-coupling

reaction), employing compound 12 (279 mg, 1 mmol) and 6a–h

(1 mmol). The crude product was purified by column chromatogra-

phy using EtOAc and hexane to afford compounds 13a–h.

SnCl₂·2H₂O (1 mmol) was added to a solution of nitro compounds

(18a–x and 19a–h, 0.5 mmol) in MeOH (20 mL) and held at reflux

for 2–4 h. The MeOH was evaporated under vacuum, and the

aqueous layer was carefully adjusted to pH 8 with a solution of

10% NaHCO₃and then extracted with EtOAc (3ꢁ30 mL). The com-

bined organic phase was dried over anhydrous Na₂SO₄and evapo-

rated under vacuum to afford the aminodiethyl thioacetal, which

due to potential stability problems, was taken directly to the next

step. A solution of aminodiethyl thioacetal (0.5 mmol), HgCl₂

(1.13 mmol), and CaCO₃(1.23 mmol) in CH₃CN/H₂O (4:1) was stirred

slowly at RT until TLC indicated complete loss of starting material

(12 h). The reaction mixture was diluted with EtOAc (30 mL) and fil-

tered through a Celite bed. The clear-yellow organic supernatant

was washed with 5% NaHCO₃(20 mL) and brine (20 mL), and the

combined organic phase was dried over anhydrous Na₂SO₄. The or-

ganic layer was evaporated under vacuum and purified by column

chromatography using MeOH/CHCl₃to give the final products 3a–

x and 4a–h.

General procedure for synthesis of 14a–h

Aqueous NaOH (1n, 1 mL) was added dropwise to a stirred solu-

tion of ethyl-6-(substituted phenyl)-2-naphthoate (13a-h, 1 mmol)

in EtOH (10 mL) at RT, and the reaction mixture was held at reflux

(70–808C) for 1–2 h. After completion of the reaction, the mixture

was acidified to pH 7 with 12n HCl and extracted with CH₂Cl₂(3ꢁ

30 mL), dried over Na₂SO₄, and the EtOH solvent was removed

under reduced pressure to obtain the acid product as a white solid

(91–95% yield). DMF (2–3 drops) was added to a stirred suspension

of acid compound (1 mmol) and SOCl₂(0.18 mL, 2.5 mmol) in dry

CH₂Cl₂(5 mL), and stirring was continued at RT for 2–4 h under N₂.

The solvent was evaporated under reduced pressure to obtain the

product (14a–h) as a pale-yellow oil, which, due to potential stabil-

ity problems, was used directly in the next step.

7-Methoxy-8-{6-(4-fluorophenyl)-2-naphthyl]oxy)propoxy}-

(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzo-

diazepine-5-one (3a): White solid (170 mg, 65% yield): R_f=0.3

(2% MeOH/CHCl₃); mp: 69–718C; [a]²_D⁷=+198.0 (c=0.1, CHCl₃); IR

(KBr): n_max=3317 (br), 2929, 2873, 1605, 1507, 1431, 1253, 1202,

;

1056, 839 cm^{ꢁ1 1}H NMR (300 MHz, CDCl₃): d=7.91 (s, 1H, ArH),

General procedure for synthesis of 15a–h

Et₃N (0.14 mL, 2 mmol) was added dropwise to a stirred solution of

compound 11 (186 mg, 1 mmol) in dry CH₂Cl₂(10 mL) at 08C over

10 min; acid chloride (14a–h, 1.2 mmol) dissolved in dry CH₂Cl₂

(5 mL) was then added at the same temperature. The reaction was

brought to RT and stirred for another 2–4 h. After completion of

the reaction, the solvent was evaporated and extracted with

CH₂Cl₂(3ꢁ20 mL). The reaction mixture was washed with aqueous

NaHCO₃(2m) followed by brine, separated, and dried over anhy-

drous Na₂SO₄. The solvent was removed under reduced pressure to

obtain crude product, which was further purified by column chro-

matography with EtOAc and hexane to afford the pure compounds

15a–h.

7.73 (d, J=3.8 Hz, 1H, imine-H), 7.59–7.71 (m, 4H, ArH), 7.52 (s, 1H,

ArH), 7.28 (s, 1H, ArH), 7.11–7.21 (m, 4H, ArH), 6.89 (s, 1H, ArH),

4.25–4.37 (m, 4H, 2ꢁOCH₂), 3.86 (s, 3H, OCH₃), 3.43–3.83 (m, 3H,

NCH, NCH₂), 2.36–2.49 (m, 2H, CH₂), 1.94–2.15 ppm (m, 4H, 2ꢁ

CH₂); (ESI) MS: m/z 525 [M+1]⁺; HRMS (ESI m/z) for C₃₂H₃₀N₂O₄F,

calcd 525.2451, found 525.2440 [M+1]⁺.

7-Methoxy-8-{6-(4-trifluoromethyl)phenyl)-2-naphthyl]oxy)pro-

poxy}-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzo-

diazepine-5-one (3b): White solid (201 mg, 70% yield): R_f=0.3

(2.5% MeOH/CHCl₃); mp: 84–868C; [a]²_D⁷=+155.0 (c=0.1, CHCl₃);

IR (KBr): n_max=3325 (br), 2959, 2876, 1609, 1508, 1433, 1326, 1255,

1

1200, 1119, 1066, 843 cm^ꢁ1; H NMR (200 MHz, CDCl₃): d=7.98 (s,

1H, ArH), 7.76–7.85 (m, 4H, ArH), 7.74 (s, 2H, ArH), 7.72 (d, J=

3.3 Hz, 1H, imine-H), 7.28 (s, 1H, ArH), 7.21 (s, 2H, ArH), 7. 15–7.19

(m, 1H, ArH), 6.44 (s, 1H, ArH), 4.24–4.37 (m, 4H, 2ꢁOCH₂), 3.87 (s,

3H, OCH₃), 3.42–3.82 (m, 3H, NCH, NCH₂), 2.35–2.48 (m, 2H, CH₂)

1.92–2.11 ppm (m, 4H, 2ꢁCH₂); (ESI) MS: m/z 575 [M+1]⁺; HRMS

(ESI m/z) for C₃₃H₃₀N₂O₄F₃calcd 575,1328, found 575.1321 [M+1]⁺.

General procedure for synthesis of 16a–h

Compounds 16a–h were prepared according to the method de-

scribed for the general procedure for the synthesis of 8a–x, em-

ploying compounds 15a–h (1 mmol) and 1,3-dibromopropane

ChemMedChem 2011, 6, 1665 – 1679

ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemmedchem.org

1673

A. Kamal, M. Pal-Bhadra, et al.

MED

7-Methoxy-8-{6-(4-trifluoromethoxy)phenyl)-2-naphthyl]oxy)pro-

poxy}-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzo-

diazepine-5-one (3c): White solid (200 mg, 68% yield): R_f=0.3

(3% MeOH/CHCl₃); mp: 75–778C; [a]²_D⁷=+103.0 (c=0.1, CHCl₃); IR

(KBr): n_max=3329 (br), 2934, 2866, 1607, 1507, 1464, 1259, 1206,

3.49–3.69 (m, 2H, NCH₂), 2.39–2.46 (m, Hz, 2H, CH₂), 2.24–2.31 (m,

2H, CH₂), 2.00–2.09 ppm (m, 2H, CH₂); ¹³C NMR (75 MHz, CDCl₃):

d=164.4, 162.3, 157.9, 150.5, 147.6, 142.8, 140.4, 134.7, 134.5,

133.8, 129.9, 129.6, 128.9, 127.3, 127.0, 126.8, 125.5, 125.3, 125.1,

120.1, 119.3, 111.4, 110.4, 106.3, 65.3, 64.1, 55.9, 53.5, 46.5, 29.4,

28.9, 24.0 ppm; (ESI) MS: m/z 542 [M+1]⁺; HRMS (ESI m/z) for

C₃₂H₃₀N₂O₄Cl calcd 542.2156, found 542.2160 [M+1]⁺.

1054, 847 cm^ꢁ^{1 1}H NMR (200 MHz, CDCl₃): d=7.93 (s, 1H, ArH),

;

7.79 (d, 2H, J=8.5 Hz, ArH), 7.72 (s, 1H, ArH), 7.68 (s, 2H, ArH), 7.64

(d, J=4.3, Hz, 1H, imine-H), 7.52 (s 1H, ArH), 7.31 (d, J=8.5 Hz, 2H,

ArH), 7.14–7.22 (m, 2H), 6.88 (s, 1H, ArH), 4.23–4.38 (m, 4H, 2ꢁ

OCH₂), 3.95 (s, 3H, OCH₃), 3.49–3.88 (m, 3H, NCH, NCH₂), 2.32–2.46

(m, 2H, CH₂), 1.89–2.14 ppm (m, 4H, 2ꢁCH₂); ¹³C NMR (75 MHz,

CDCl₃): d=164.5, 162.3, 156.9, 151.0, 148.3, 144.8, 139.8, 137.0,

134.8, 133.7, 129.5, 128.9, 128.3, 127.3, 125.5, 121.1, 120.0, 119.3,

112.3, 111.6, 110.4, 106.3, 96.5, 65.4, 64.2, 56.0, 53.5, 46.4, 29.4, 24.6,

23.0 ppm; (ESI) MS: m/z 591 [M]⁺; (ESI) MS: m/z 591 [M+1]⁺;

HRMS (ESI m/z) for C₃₃H₃₀N₂O₅F₃calcd 591.1429, found 591.1423

[M+1]⁺.

7-Methoxy-8-{6-(3,4-dimethoxyphenyl)-2-naphthyl]oxy)propoxy}-

(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzo-

diazepine-5-one (3g): White solid (186 mg, 66% yield): R_f=0.3

(3.5% MeOH/CHCl₃); mp: 88–908C; [a]²_D⁷=+174.0 (c=0.1, CHCl₃);

IR (KBr): n_max=3377 (br), 2932, 2869, 1602, 1510, 1460, 1257, 1219,

1168, 1023, 853 cm^ꢁ^{1 1}H NMR (300 MHz, CDCl₃): d=7.91 (s, 1H,

;

ArH), 7.73–7.84 (m, 2H, ArH), 7.70 (s, 1H, ArH), 7.64 (d, J=4.4 Hz,

1H, imine-H), 7.52(s, 1H, ArH), 7.13–7.24 (m, 3H, ArH), 6.98 (dd, J=

2.9 Hz, 5.8 Hz, 1H, ArH), 6.88 (s, 1H, ArH), 6.76 (s, 1H, ArH), 4.26–

4.39 (m, 4H, 2ꢁOCH₂), 3.98 (s, 3H, OCH₃), 3.95 (s, 6H, 2ꢁOCH₃),

3.75–3.85 (m, 1H, NCH), 3.49–3.64 (m, 2H, NCH₂) 2.20–2.48 (m, 4H,

2ꢁCH₂), 1.97–1.20 ppm (m, 2H, CH₂); ¹³C NMR (75 MHz, CDCl₃):

d=164.4, 162.3, 156.6, 150.7, 149.1, 148.3, 147.6, 140.5, 136.0,

134.0, 133.3, 129.4, 129.0, 128.1, 127.0, 125.8, 125.4, 124.8, 120.1,

119.2, 114.6, 111.4, 110.3, 106.4, 65.4, 64.1, 56.0, 55.8, 53.7, 46.5,

29.4, 28.9, 24.0 ppm; (ESI) MS: m/z 567 [M+1]⁺; HRMS (ESI m/z)

for C₃₄H₃₅N₂O₆calcd 567.2314, found 567.2335 [M+1]⁺.

7-Methoxy-8-{6-(4-methoxyphenyl)-2-naphthyl]oxy)propoxy}-

(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzo-

diazepine-5-one (3d): White solid (166 mg, 62% yield): R_f=0.3

(3% MeOH/CHCl₃); mp: 86–888C; [a]²_D⁷=+137.0 (c=0.1, CHCl₃); IR

(KBr): n_max=3375 (br), 2930, 2861, 1604, 1506, 1461, 1247, 1178,

1023, 836 cm^ꢁ^{1 1}H NMR (200 MHz, CDCl₃): d=7.91 (s, 1H, ArH),

;

7.76–7.80 (m, 1H, ArH), 7.73 (d, J=8.1, 1H, ArH), 7.68 (d, J=8.1,

1H, ArH), 7.64 (d, J=3.6 Hz, 1H, imine-H), 7.62 (s, 2H, ArH), 7.52 (s,

1H, ArH), 7.12–7.19 (m, 2H, ArH), 6.98–7.04 (m, 2H, ArH), 6.88 (s,

1H, ArH), 4.27–4.38 (m, 4H, 2ꢁOCH₂), 3.95 (s, 3H, OCH₃), 3.86 (s,

3H, OCH₃), 3.74–3.81 (m, 1H, NCH), 3.53–3.68 (m, 2H, NCH₂), 2.35–

2.48 (m, 2H, CH₂), 2.20–2.31 (m, 2H, CH₂), 1.98–2.09 ppm (m, 2H,

CH₂); ¹³C NMR (75 MHz, CDCl₃): d=164.5, 162.3, 158.9, 156.5,

150.5, 147.6, 140.5, 135.8, 133.5, 133.2, 129.4, 129.1, 128.0, 127.0,

125.7, 124.7, 120.1, 119.1, 114.1, 111.5, 110.5, 106.4, 65.4, 64.1, 56.0,

55.2, 53.5, 46.5, 29.4, 29.0, 24.0 ppm; (ESI) MS: m/z 537 [M+1]⁺;

HRMS (ESI m/z) for C₃₃H₃₃N₂O₅calcd 537.3776, found 537.3768 [M+

1]⁺.

7-Methoxy-8-{6-(3,4,5-trimethoxyphenyl)-2-naphthyl]oxy)pro-

poxy}-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzo-

diazepine-5-one (3h): White solid (202 mg, 68% yield): R_f=0.3

(4% MeOH/CHCl₃); mp: 92–948C; [a]²_D⁷=+178.0 (c=0.1, CHCl₃); IR

(KBr): n_max=3359 (br), 2929, 2876, 1602, 1509, 1432, 1266, 1209,

1157, 853 cm^ꢁ^{1 1}H NMR (300 MHz, CDCl₃): d=7.91 (s, 1H, ArH),

;

7.80 (d, J=6.0 Hz, 1H, ArH), 7.77 (d, J=4.5 Hz, 1H, ArH), 7.67 (s,

1H, ArH), 7.64 (d, J=3.7 Hz, 1H, imine-H), 7.52 (s, 1H, ArH) 7.15–

7.21 (m, 2H, ArH), 6.86–6.90 (m, 3H, ArH), 4.27–4.38 (m, 4H, 2ꢁ

OCH₂), 3.97 (s, 6H, 2ꢁOCH₃), 3.95 (s, 3H, OCH₃), 3.91 (s, 3H, OCH₃),

3.54–3.82 (m, 3H, NCH, NCH₂), 2.40–2.47 (m, 2H, CH₂), 2.23–2.37

(m, 2H, CH₂), 1.99–2.11 ppm (m, 2H, CH₂); ¹³C NMR (75 MHz,

CDCl₃): d=164.5, 162.3, 156.8, 153.4, 151.2, 150.5, 147.7, 140.4,

137.1, 136.4, 133.6, 129.4, 129.0, 127.1, 125.8, 125.3, 120.2, 119.3,

111.5, 110.5, 106.4, 104.3, 65.4, 64.2, 60.9, 56.1, 53.6, 46.6, 29.6, 24.1,

22.6 ppm; (ESI) MS: m/z 597 [M+1]⁺; HRMS (ESI m/z) for

C₃₅H₃₇N₂O₇calcd 597.2862, found 597.2848 [M+1]⁺.

7-Methoxy-8-{6-(4-N,N-dimethylamino)phenyl)-2-naphthyl]oxy)-

propoxy}-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]-

benzodiazepine-5-one (3e): White solid (164 mg, 60% yield): R_f=

0.3 (3.0% MeOH/CHCl₃); mp: 89–918C; [a]²_D⁷=+181.0 (c=0.1,

CHCl₃); IR (KBr): n_max=3312 (br), 2927, 1605, 1503, 1433, 1256,

1200, 1057, 806 cm^{ꢁ1 1}H NMR (300 MHz, CDCl₃): d=7.90 (s, 1H,

;

ArH), 7.66–7.77 (m, 3H, ArH), 7.62 (d, J=3.7 Hz, 1H, imine-H), 7.60

(s, 2H, ArH), 7.52 (s, 1H, ArH), 7.12–7.16 (m, 2H, ArH), 6.89 (s, 2H,

ArH), 6.85 (s, 1H, ArH), 4.27–4.36 (m, 4H, 2ꢁOCH₂), 3.94 (s, 3H,

OCH₃), 3.75–3.90 (m, 1H, NCH), 3.52–3.66 (m, 2H, NCH₂) 3.01 (s,

6H, 2ꢁN(CH₃)₂), 2.40–2.48 (m, 2H, CH₂), 2.21–2.29 (m, 2H, CH₂),

1.99–2.07 ppm (m, 2H, CH₂); ¹³C NMR (75 MHz, CDCl₃): d=164.5,

162.3, 156.3, 150.5, 149.7, 147.6, 140.5, 136.3, 132.9, 129.3, 129.0,

127.8, 127.6, 126.9, 125.6, 123.9, 120.1, 118.9, 112.8, 111.4, 110.4,

106.4, 65.4, 64.1, 56.0, 53.5, 46.5, 40.5, 29.4, 29.0, 24.0 ppm; (ESI)

MS: m/z 550 [M+1]⁺; HRMS (ESI m/z) for C₃₄H₃₆N₃O₄calcd

550.2967, found 550.2961 [M+1]⁺.

7-Methoxy-8-{6-(4-fluorophenyl)-2-naphthyl]oxy)butoxy}-(11aS)-

1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one

(3i): White solid (188 mg, 70% yield): R_f=0.3 (2.0% MeOH/CHCl₃);

mp: 78.5–80.58C; [a]_D²⁷=+163.0 (c=0.1, CHCl₃); IR (KBr): n_max

=

3335 (br), 2928, 2873, 1603, 1507, 1433, 1253, 1202, 1166,

1

838 cm^ꢁ1; H NMR (300 MHz, CDCl₃): d=7.91 (d, 1H, ArH), 7.78 (d,

J=8.3 Hz, 2H ArH), 7.67 (d, J=3.0 Hz, 1H, imine-H), 7.62–7.65 (m,

3H, ArH), 7.52 (s, 1H, ArH), 7.13–7.19(m, 4H, ArH), 6.84 (s, 1H, ArH),

4.13–4.24 (m, 4H, 2ꢁOCH₂), 3.93 (s, 3H, OCH₃), 3.77–3.81 (m, 1H,

NCH) 3.56–3.75 (m, 2H, NCH₂), 2.28–2.36 (m, 2H, CH₂), 2.03–2.24

(m, 4H, 2ꢁCH₂), 1.60–1.72 ppm (m, 2H, CH₂); ¹³C NMR (75 MHz,

CDCl₃): d=164.5, 163.8, 162.3, 160.5, 156.9, 150.6, 147.6, 140.4,

137.2, 135.1, 133.5, 129.4, 128.6, 127.1, 125.6, 125.3, 120.0, 119.3,

115.6, 111.4, 110.3, 106.2, 68.5, 67.3, 56.0, 53.6, 46.5, 29.4, 25.8, 25.6,

24.0 ppm; (ESI) MS: m/z 539 [M+1]⁺; HRMS (ESI m/z) for

C₃₃H₃₂N₂O₄F calcd 539.2862, found 539.2848 [M+1]⁺.

7-Methoxy-8-{6-(3-chlorophenyl)-2-naphthyl]oxy)propoxy}-

(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzo-

diazepine-5-one (3 f): White solid (191 mg, 70% yield): R_f=0.3 (2%

MeOH/CHCl₃); mp: 80–828C; [a]²_D⁷=+210.0 (c=0.1, CHCl₃); IR

(KBr): n_max=3356 (br), 2928, 2873, 1597, 1507, 1431, 1254, 1200,

1169, 851 cm^ꢁ^{1 1}H NMR (300 MHz, CDCl₃): d=7.94 (s, 1H, ArH),

;

7.76–7.80 (m, 2H, ArH), 7.66–7.69 (m, 2H, ArH), 7.63 (d, J=4.5 Hz,

1H, imine-H), 7.57 (d, J=7.5 Hz, 1H, ArH), 7.52 (s 1H, ArH), 7.30–

7.41 (m, 2H, ArH), 7.16–7.21 (m, 2H, ArH), 6.88 (s 1H, ArH), 4.28–

4.38 (m, 4H, 2ꢁOCH₂), 3.95 (s, 3H, OCH₃), 3.77–3.87 (m, 1H, NCH),

7-Methoxy-8-{6-(4-trifluoromethyl)phenyl)-2-naphthyl]oxy)bu-

toxy}-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzo-

diazepine-5-one (3j): White solid (199 mg, 68% yield): R_f=0.3

(2.5% MeOH/CHCl₃); mp: 91–938C; [a]²_D⁷=+140.5 (c=0.1, CHCl₃);

1674

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ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

ChemMedChem 2011, 6, 1665 – 1679

PBD Conjugates as Anticancer Agents

IR (KBr): n_max=3326 (br), 2960, 2866, 1606, 1509, 1432, 1326, 1254,

2H, ArH), 7.52(s, 1H, ArH), 7.13–7.19 (m, 5H, ArH), 6.85 (s, 1H, ArH),

4.12–4.25 (m, 4H, 2ꢁOCH₂), 3.93 (s, 3H, OCH₃), 3.78–3.82 (m, 1H,

NCH) 3.58–3.74 (m, 2H, NCH₂), 2.27–2.35 (m, 2H, CH₂), 2.02–2.25

(m, 4H, 2ꢁCH₂), 1.22–1.33 ppm (m, 2H, CH₂); ¹³C NMR (75 MHz,

CDCl₃): d=164.5, 162.3, 157.1, 150.6, 147.6, 142.8, 140.4, 134.6,

134.4, 133.9, 129.9, 129.6, 128.8, 127.2, 127.0, 126.8, 125.6, 125.4,

125.1, 120.0, 119.4, 111.4, 110.3, 106.2, 68.4, 67.3, 56.0, 53.5, 46.5,

29.4, 25.8, 25.6, 24.0 ppm; (ESI) MS: m/z 556 [M+1]⁺; HRMS (ESI

m/z) for C₃₃H₃₂N₂O₄Cl calcd 556.2312, found 556.2321 [M+1]⁺.

1

1202, 1120, 1056, 841 cm^ꢁ1; H NMR (300 MHz, CDCl₃): d=7.93 (s,

1H, ArH), 7.78 (d, J=8.3 Hz, 1H, ArH), 7.71 (s, 1H, ArH), 7.68 (d, J=

3.7 Hz, 1H, imine-H), 7.52 (s, 1H, ArH), 7.27–7.38 (m, 3H, ArH), 7.15

(d, J=7.5 Hz, 1H, ArH), 6.98 (s, 1H, ArH), 6.82–6.85 (m, 3H, ArH),

4.12–4.23 (m, 4H, 2ꢁOCH₂), 3.95 (s, 3H, OCH₃), 3.76–3.87 (m, 1H,

NCH), 3.57–3.74 (m, 2H, NCH₂), 2.27–2.34 (m, 2H, CH₂), 2.05–2.25

(m, 4H, 2ꢁCH₂), 1.61–1.73 ppm (m, 2H, CH₂); (ESI) MS: m/z 589

[M+1]⁺; HRMS (ESI m/z) for C₃₄H₃₂N₂O₄F₃calcd 589.4628, found

589.4619 [M+1]⁺.

7-Methoxy-8-{6-(3,4-dimethoxyphenyl)-2-naphthyl]oxy)butoxy}-

(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzo-

diazepine-5-one (3o): White solid (182 mg, 63% yield): R_f=0.3

(3.5% MeOH/CHCl₃); mp: 70–728C; [a]²_D⁷=+168.0 (c=0.1, CHCl₃);

IR (KBr): n_max=3372 (br), 2931, 2867, 1606, 1509, 1459, 1257, 1220,

1170, 1023, 851 cm^ꢁ^{1 1}H NMR (300 MHz, CDCl₃): d=7.94 (s, 1H,

;

ArH), 7.91(s, 1H, ArH), 7.79 (d, J=7.3 Hz, 1H, ArH), 7.76 (s, 1H,

ArH), 7.70 (d, J=7.5 Hz, 1H, ArH), 7.68 (d, J=4.3 Hz, 1H, imine-H),

7.52(s, 1H, ArH), 7.19–7.24(m, 2H, ArH), 7.15 (s, 1H, ArH), 6.99 (t,

J=7.5 Hz, 1H, ArH), 6.82 (s, 1H, ArH), 4.11–4.26 (m, 4H, 2ꢁOCH₂),

3.99 (s, 3H, OCH₃), 3.96 (s, 3H, OCH₃), 3.95 (s, 3H, OCH₃), 3.78–3.88

(m, 1H, NCH), 3.56–3.75 (m, 2H, NCH₂), 2.27–2.35 (m, 2H, CH₂),

1.88–2.21 (m, 4H, 2ꢁCH₂), 1.20–1.34 ppm (m, 2H, CH₂); (ESI) MS:

m/z 581 (M+23)⁺; HRMS (ESI m/z) for C₃₅H₃₇N₂O₆calcd 581.2573,

found 581.2566 [M+1]⁺.

7-Methoxy-8-{6-(4-trifluoromethoxy)phenyl)-2-naphthyl]oxy)bu-

toxy}-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzo-

diazepine-5-one (3k): White solid (211 mg, 70% yield): R_f=0.3

(3.0% MeOH/CHCl₃); mp: 86–888C; [a]²_D⁷=+168.0 (c=0.1, CHCl₃);

IR (KBr): n_max=3331 (br), 2956, 2856, 1604, 1508, 1421, 1320, 1265,

1

1197, 1120, 1045, 840 cm^ꢁ1; H NMR (300 MHz, CDCl₃): d=7.94 (s,

1H, ArH), 7.79 (d, J=8.3 Hz, 1H, ArH), 7.72 (s, 1H, ArH), 7.68 (d, J=

3.7, 1H, imine-H), 7.52 (s, 1H, ArH), 7.29–7.37 (m, 3H, ArH), 7.16 (d,

J=7.5 Hz, 1H, ArH), 6.99 (s, 1H, ArH), 6.81–6.86 (m, 3H, ArH), 4.11–

4.22 (m, 4H, 2ꢁOCH₂), 3.94 (s, 3H, OCH₃), 3.77–3.88 (m, 1H, NCH),

3.48–3.55 (m, 2H, NCH₂), 2.24–2.33 (m, 2H, CH₂), 2.07–2.28 (m, 4H,

2ꢁCH₂), 1.64–1.75 ppm (m, 2H, CH₂); (ESI) MS: m/z 605 [M+1]⁺;

HRMS (ESI m/z) for C₃₄H₃₂N₂O₅F₃calcd 605.6155, found 605.6147

[M+1]⁺.

7-Methoxy-8-{6-(4-methoxyphenyl)-2-naphthyl]oxy)butoxy}-

(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzo-

7-Methoxy-8-{6-(3,4,5-trimethoxyphenyl)-2-naphthyl]oxy)bu-

toxy}-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzo-

diazepine-5-one (3p): White solid (201 mg, 66% yield): R_f=0.3

(4.0% MeOH/CHCl₃); mp: 90–928C; [a]²_D⁷=+150.0, (c=0.1, CHCl₃);

IR (KBr): n_max=3354 (br), 2929, 2869, 1607, 1507, 1460, 1429, 1270,

diazepine-5-one (3l) White solid (170 mg, 62% yield): R_f=0.3

(3.0% MeOH/CHCl₃); mp: 82–848C; [a]²_D⁷=+135.0 (c=0.1, CHCl₃);

IR (KBr): n_max=3336 (br), 2932, 2863, 1597, 1521, 1421, 1326, 1255,

1

1202, 1132, 1025, 820 cm^ꢁ1; H NMR (300 MHz, CDCl₃): d=7.91 (s,

1124, 1010, 851 cm^ꢁ^{1 1}H NMR (300 MHz, CDCl₃): d=7.92 (s, 1H,

;

1H, ArH), 7.74–7.78 (m, 2H, ArH), 7.69 (d, J=3.6, 1H, imine-H),

7.61–7.67 (m, 2H, ArH) 7.52 (s, 1H, ArH), 7.14–7.16 (m, 2H, ArH),

7.02 (s, 1H, ArH), 7.00 (s, 1H, ArH), 6.84 (s, 1H, ArH) 4.12–4.25 (m,

4H, 2ꢁOCH₂), 3.93 (s, 3H, OCH₃), 3.87 (s, 3H, OCH₃), 3.74–3.81 (m,

1H, NCH), 3.50–3.62 (m, 2H, NCH₂), 2.31–2.41 (m, 2H, CH₂), 2.04–

2.27 (m, 4H, 2ꢁCH₂), 1.61–1.74 ppm (m, 2H, CH₂); ¹³C NMR

(75 MHz, CDCl₃): d=162.3, 158.9, 156.8, 155.1, 152.9, 149.6, 136.5,

133.7, 130.9, 129.4, 129.0, 128.1, 127.1, 125.9, 125.8, 124.8, 119.2,

114.2, 111.5, 110.4, 107.5, 106.3, 68.5, 67.3, 56.1, 55.3, 53.6, 46.6,

29.5, 26.0, 25.6, 24.1 ppm; (ESI) MS: m/z 551 [M+1]⁺; HRMS (ESI

m/z) for C₃₄H₃₅N₂O₅calcd 551.2808, found 551.2801 [M+1]⁺.

ArH), 7.77–7.83 (m, 2H, ArH), 7.77 (d, J=8.3 Hz, 1H, ArH), 7.67 (d,

J=8.3 Hz, 1H, imine-H), 7.52 (s, 1H, ArH), 7.16 (s, 2H, ArH), 6.87 (s,

2H, ArH), 6.84 (s, 1H, ArH), 4.16–4.30 (m, 4H, 2ꢁOCH₂), 3.96 (s, 6H,

2ꢁOCH₃), 3.93 (s, 3H, OCH₃), 3.91 (s, 3H, OCH₃), 3.74–3.86 (m, 1H,

NCH), 3.53–3.73 (m, 2H, NCH₂) 2.27–2.36 (m, 2H, CH₂), 2.01–2.18

(m, 4H, 2ꢁCH₂), 1.64–1.70 ppm (m, 2H, CH₂); ¹³C NMR (75 MHz,

CDCl₃): d=164.5, 162.3, 157.0, 153.4, 150.7, 147.7, 140.5, 137.2,

136.3, 133.7, 129.5, 128.9, 127.1, 125.9, 125.6, 125.3, 120.1, 119.4,

111.4, 110.3, 106.3, 104.4, 68.5, 67.4, 60.9, 56.1, 53.6, 46.6, 29.5, 25.9,

25,6, 24.1 ppm; (ESI) MS: m/z 611 [M+1]⁺; HRMS (ESI m/z) for

C₃₆H₃₉N₂O₇calcd 611.6961, found 611.6953 [M+1]⁺.

7-Methoxy-8-{6-(4-N,N-dimethylamino)phenyl)-2-naphthyl]oxy)-

butoxy}-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzo-

diazepine-5-one (3m): White solid (168 mg, 60% yield): R_f=0.3

(3% MeOH/CHCl₃); mp: 86–888C; [a]²_D⁷=+178.0 (c=0.1, CHCl₃); IR

(KBr): n_max=3332 (br), 2960, 2874, 1607, 1508, 1420, 1337, 1251,

7-Methoxy-8-{6-(4-fluorophenyl)-2-naphthyl]oxy)pentoxy}-

(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzo-

diazepine-5-one (3q): White solid (187 mg, 68% yield): R_f=0.3

(2.0% MeOH/CHCl₃); mp: 70–728C; [a]²_D⁷=+141.0 (c=0.1, CHCl₃);

IR (KBr): n_max=3329 (br), 2932, 2869, 1605, 1505, 1466, 1251, 1202,

1165, 839 cm^ꢁ^{1 1}H NMR (300 MHz, CDCl₃): d=7.87 (s, 1H, ArH),

;

1

1202, 1120, 1056, 830 cm^ꢁ1; H NMR (300 MHz, CDCl₃): d=7.91 (s,

1H, ArH), 7.69–7.76 (m, 4H, ArH), 7.66 (d, J=3.7 Hz, 1H, imine-H),

7.60 (s, 1H, ArH), 7.52 (s, 1H, ArH), 7.12–7.16 (m, 2H, ArH), 6.85–

6.89 (m, 3H, ArH), 4.27–4.36 (m, 4H, 2ꢁOCH₂), 3.93 (s, 3H, OCH₃),

3.75–3.88 (m, 1H, NCH), 3.52–3.66 (m, 2H, NCH₂), 3.02 (s, 6H,

N(CH₃)₂), 2.40–2.44 (m, 2H, CH₂), 2.21–2.30 (m, 4H, 2ꢁCH₂), 1.98–

2.08 ppm (m, 2H, CH₂); (ESI) MS: m/z 564 [M+1]⁺; HRMS (ESI m/z)

for C₃₅H₃₈N₃O₄calcd 564.6860, found 564.6851 [M+1]⁺.

7.74 (d, J=8.3 Hz, 2H, ArH), 7.63–7.65 (m, 2H, ArH), 7.62 (d, J=

4.2 Hz, 1H, imine-H), 7.60 (s, 1H, ArH), 7.49(s, 1H, ArH), 7.09–

7.17(m, 4H, ArH), 6.78 (s, 1H, ArH), 4.05–4.17 (m, 4H, 2ꢁOCH₂),

3.94 (s, 3H, OCH₃), 3.77–3.86 (m, 1H, NCH), 3.55–3.72 (m, 2H,

NCH₂), 2.26–2.32 (m, 2H, CH₂), 1.90–2.10 (m, 6H, 3ꢁCH₂), 1.68–

1.79 ppm (m, 2H, CH₂); ¹³C NMR (75 MHz, CDCl₃): d=164.5, 163.8,

162.2, 160.5, 157.0, 150.7, 147.6, 140.4, 137.1, 135.1, 133.5, 129.4,

128.8, 127.1, 125.6, 125.2, 120.0, 119.9, 115.4, 111.4, 110.3, 106.2,

68.6, 67.5, 56.0, 53.5, 46.5, 29.4, 28.7, 28.5, 24.0, 22.5 ppm; (ESI) MS:

m/z 553 [M+1]⁺; HRMS (ESI m/z) for C₃₄H₃₄N₂O₄F calcd 553.2764,

found 553.2775 [M+1]⁺.

7-Methoxy-8-{6-(3-chlorophenyl)-2-naphthyl]oxy)butoxy}-(11aS)-

1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one

(3n): White solid (194 mg, 70% yield): R_f=0.3 (2.0% MeOH/CHCl₃);

mp: 74–768C; [a]²_D⁷=+124.0 (c=0.1, CHCl₃); IR (KBr): n_max=3323

(br), 3058, 2931, 2871, 1597, 1507, 1498, 1253, 1202, 1125,

7-Methoxy-8-{6-(4-trifluoromethyl)phenyl)-2-naphthyl]oxy)pen-

toxy}-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzo-

diazepine-5-one (3r): White solid (183 mg, 61% yield): R_f=0.3

1

850 cm^ꢁ1; H NMR (300 MHz, CDCl₃): d=7.92 (s, 1H, ArH), 7.79 (d,

J=8.3 Hz, 2H, ArH), 7.68 (d, J=4.1 Hz, 1H, imine-H), 7.63–7.66 (m,

ChemMedChem 2011, 6, 1665 – 1679

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www.chemmedchem.org

1675

A. Kamal, M. Pal-Bhadra, et al.

MED

(2.5% MeOH/CHCl₃); mp: 88–908C; [a]²_D⁷=+131.0 (c=0.1, CHCl₃);

IR (KBr): n_max=3383 (br), 2936, 2872, 1606, 1506, 1466, 1328, 1260,

7-Methoxy-8-{6-(3-chlorophenyl)-2-naphthyl]oxy)pentoxy}-

(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzo-

diazepine-5-one (3v): White solid (190 mg, 67% yield): R_f=0.3

(2.0% MeOH/CHCl₃); mp: 88–908C; [a]²_D⁷=+167.0 (c=0.1, CHCl₃);

IR (KBr): n_max=3333 (br), 3058, 2933, 2868, 1598, 1498, 1429, 1252,

1202, 1170, 850 cm^ꢁ^{1 1}H NMR (300 MHz, CDCl₃): d=7.89 (s, 1H,

;

ArH), 7.74 (d, J=8.3 Hz, 2H, ArH), 7.68 (d, J=4.2 Hz, 1H, imine-H),

7.62–7.66 (m, 2H, ArH), 7.60 (s, 1H, ArH), 7.51 (s, 1H, ArH), 7.10–

7.16 (m, 4H, ArH), 6.78 (s, 1H, ArH), 4.06–4.16 (m, 4H, 2ꢁOCH₂),

3.94 (s, 3H, OCH₃), 3.77–3.87 (m, 1H, NCH), 3.53–3.72 (m, 2H,

NCH₂), 2.27–2.33 (m, 2H, CH₂), 1.91–2.09 (m, 6H, 3ꢁCH₂), 1.71–

1.78 ppm (m, 2H, CH₂); ¹³C NMR (75 MHz, CDCl₃): d=164.5, 162.3,

157.2, 150.7, 147.6, 142.9, 140.4, 134.6, 134.5, 133.9, 129.9, 129.5,

128.8, 127.3, 127.0, 126.8, 125.6, 125.4, 125.1, 120.2, 119.4, 111.4,

110.3, 106.2, 68.6, 67.6, 56.1, 53.6, 46.5, 29.5, 28.8, 28.5, 24.0,

22.5 ppm; (ESI) MS: m/z 570 [M+1]⁺; HRMS (ESI m/z) for

C₃₄H₃₄N₂O₄Cl calcd 570.3898, found 570.3891 [M+1]⁺.

1120, 843 cm^ꢁ1

;

¹H NMR (300 MHz, CDCl₃): d=7.98 (s, 1H, ArH),

7.77–7.84 (m, 4H, ArH), 7.69–7.75 (m, 3H, ArH), 7.67 (d, J=3.8 Hz,

1H, imine-H), 7.52 (s, 1H, ArH), 7.15–7.21 (m, 2H, ArH), 6.82 (s, 1H,

ArH), 4.05–4.21 (m, 4H, 2ꢁOCH₂), 3.95 (s, 3H, OCH₃), 3.78–3.87 (m,

1H, NCH), 3.55–3.74 (m, 2H, NCH₂), 2.28–2.36 (m, 2H, CH₂), 1.91–

2.10 (m, 6H, 3ꢁCH₂), 1.61–1.78 ppm (m, 2H, CH₂); ¹³C NMR

(75 MHz, CDCl₃): d=164.5, 162.3, 157.4, 150.7, 148.1, 147.6, 144.5,

140.4, 134.5, 134.1, 129.6, 128.8, 127.4, 127.2, 125.9, 125.5, 125.3,

120.0, 119.6, 111.4, 110.3, 106.2, 68.6, 67.6, 56.0, 53.6, 46.5, 29.4,

28.8, 28.5, 24.0, 22.5 ppm; (ESI) MS: m/z 603 [M+1]⁺; HRMS (ESI

m/z) for C₃₅H₃₄N₂O₄F₃calcd 603.2732, found 603.2743 [M+1]⁺.

7-Methoxy-8-{6-(4-trifluoromethoxy)phenyl)-2-naphthyl]oxy)pen-

toxy}-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzo-

diazepine-5-one (3s): White solid (225 mg, 73% yield): R_f=0.3

(3% MeOH/CHCl₃); mp: 72–748C; [a]²_D⁷=+182.0 (c=0.1, CHCl₃); IR

(KBr): n_max=3324 (br), 2936, 2871, 1603, 1507, 1432, 1260, 1205,

1164, 845 cm^ꢁ1; H NMR (300 MHz, CDCl₃): d=7.93 (s, 1H, ArH),

7.79 (dd, J=2.2 Hz, 6.7 2H, Hz, ArH), 7.72 (s, 1H), 7.69 (s, 1H), 7.67

(d, J=4.3 Hz, 1H, imine-H), 7.64 (s, 1H, ArH), 7.53 (s, 1H, ArH), 7.31

(d, J=8.3 Hz, 2H, ArH), 7.18 (dd, J=2.2 Hz, 9.0 Hz, 2H, ArH), 6.82 (s,

1H, ArH), 4.04–4.20 (m, 4H, 2ꢁOCH₂), 3.95 (s, 3H, OCH₃), 3.78–3.88

(m, 1H, NCH), 3.56–3.74 (m, 2H, NCH₂), 2.27–2.36 (m, 2H, CH₂),

1.91–2.08 (m, 6H, 3ꢁCH₂), 1.69–1.77 ppm (m, 2H, CH₂); ¹³C NMR

(75 MHz, CDCl₃): d=164.5, 162.3, 157.1, 150.7, 148.2, 147.6, 140.4,

139.8, 134.6, 133.7, 129.5, 128.8, 128.2, 127.2, 125.5, 121.1, 119.9,

119.4, 112.2, 111.3, 110.2, 106.1, 96.5, 68.5, 67.5, 56.0, 53.5, 46.5,

29.4, 28.7, 28.5, 24.0, 22.5 ppm; (ESI) MS: m/z 619 [M+1]⁺; HRMS

(ESI m/z) for C₃₅H₃₄N₂O₅F₃calcd 619.2681, found 619.2661 [M+1]⁺.

7-Methoxy-8-{6-(3,4-dimethoxyphenyl)-2-naphthyl]oxy)

pen-

toxy}-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodi-

azepine-5-one (3w): White solid (207 mg, 70% yield): R_f=0.3

(3.5% MeOH/CHCl₃); mp: 73–758C; [a]²_D⁷=+162.0 (c=0.1, CHCl₃);

IR (KBr): n_max=3326 (br), 2929, 2870, 1602, 1511, 1458, 1256, 1220,

1167, 1022, 850 cm^ꢁ^{1 1}H NMR (300 MHz, CDCl₃): d=7.91 (s, 1H,

;

ArH), 7.75–7.84 (m, 3H, ArH), 7.68 (d, J=4.1 Hz, 1H, imine-H), 7.52

(s, 1H, ArH), 7.18–7.24 (m, 3H, ArH), 7.15 (s, 1H, ArH), 6.98 (t, J=

8.3 Hz, 1H, ArH), 6.82 (s, 1H, ArH), 4.10–4.15 (m 4H, 2ꢁOCH₂), 3.99

(s, 3H, OCH₃), 3.97 (s, 3H, OCH₃), 3.95 (s, 3H, OCH₃), 3.79–3.87 (m,

1H, NCH), 3.56–3.74 (m, 2H, NCH₂), 2.28–2.33 (m, 2H, CH₂), 1.85–

2.10 (m, 6H, 3ꢁCH₂), 1.64–1.75 ppm (m, 2H, CH₂); ¹³C NMR

(75 MHz, CDCl₃): d=164.5, 162.8, 156.8, 150.7, 149.1, 148.3, 147.7,

140.5, 135.9, 134.1, 133.4, 129.3, 129.0, 128.1, 127.0, 125.8, 125.3,

124.8, 124.2, 119.2, 114.8, 111.5, 110.4, 106.3, 68.7, 67.6, 56.0, 55.8,

53.9, 46.5, 30.6, 29.4,28.8, 24.0, 22.5 ppm; (ESI) MS: m/z 595 [M+

1]⁺; HRMS (ESI m/z) for C₃₆H₃₉N₂O₆calcd 595.3070, found 595.3079

[M+1]⁺.

7-Methoxy-8-{6-(4-methoxyphenyl)-2-naphthyl]oxy)pentoxy}-

(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzo-

diazepine-5-one (3t): White solid (191 mg, 68% yield): R_f=0.3 (3%

MeOH/CHCl₃); mp: 78–808C; [a]²_D⁷=+178.0 (c=0.1, CHCl₃); IR

(KBr): n_max=3328 (br), 2926, 2865, 1604, 1506, 1460, 1247, 1179,

1124, 1022, 834 cm^ꢁ^{1 1}H NMR (300 MHz, CDCl₃): d=7.91 (s, 1H,

;

ArH), 7.77 (d, J=8.0 Hz, 2H, ArH), 7.68 (d, J=4.1 Hz, 1H, imine-H),

7.60–7.66 (m, 3H, ArH), 7.52 (s, 1H, ArH), 7.11–7.19 (m, 2H, ArH),

7.02 (d, J=8.0 Hz, 2H, ArH), 6.84 (s, 1H), 4.04–4.20 (m, 4H, 2ꢁ

OCH₂), 3.95 (s, 3H, OCH₃), 3.87 (s, 3H, OCH₃), 3.69–3.78 (m, 1H,

NCH), 3.59–3.66 (m, 2H, NCH₂), 2.25–2.35 (m, 2H, CH₂), 1.86–2.10

(m, 6H, 3ꢁCH₂), 1.64–1.81 ppm (m, 2H, CH₂); ¹³C NMR (75 MHz,

CDCl₃): d=164.5, 162.2, 158.8, 156.7, 150.7, 147.6, 140.4, 135.7,

133.5, 133.2, 129.3, 129.0, 128.0, 127.0, 125.6, 124.6, 119.9, 119.1,

114.1, 111.3, 110.2, 106.2, 68.6, 67.5, 56.0, 55.2, 53.5, 46.5, 29.5, 28.8,

28.5, 24.0, 22.5 ppm; (ESI) MS: m/z 565 [M+1]⁺; HRMS (ESI m/z)

for C₃₅H₃₇N₂O₅calcd 565.2964, found 565.2950 [M+1]⁺.

7-Methoxy-8-{6-(3,4,5-trimethoxyphenyl)-2-naphthyl]oxy)pen-

toxy}-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodi-

azepine-5-one (3x): White solid (193 mg, 62% yield): R_f=0.3 (4.0%

MeOH/CHCl₃); mp: 78–808C; [a]²_D⁷=+144.0 (c=0.1, CHCl₃); IR

(KBr): n_max=3352 (br), 2930, 2870, 1597, 1504, 1459, 1430, 1262,

1124, 1009, 838 cm^{ꢁ1 1}H NMR (300 MHz, CDCl₃): d=7.91 (s, 1H,

;

ArH), 7.74–7.82 (m, 3H, ArH), 7.69 (d, J=4.1 Hz, 1H, imine-H), 7.52

(s, 1H, ArH), 7.19 (d J=8.2 Hz, 1H, ArH), 7.15 (s, 1H, ArH), 6.88 (s,

2H, ArH), 6.82 (s, 1H, ArH), 4.07–4.17 (m, 4H, 2ꢁOCH₂), 3.96 (s, 6H,

2ꢁOCH₃), 3.94 (s, 3H, OCH₃), 3.91 (s, 3H, OCH₃), 3.77–3.86 (m, 1H,

NCH), 3.54–3.73 (m, 2H, NCH₂), 2.26–2.34 (m, 2H, CH₂), 1.90–2.09

(m, 6H, 3ꢁCH₂), 1.68–1.77 ppm (m, 2H, CH₂); ¹³C NMR (75 MHz,

CDCl₃): d=164.5, 162.3, 157.0, 153.9, 150.7, 147.6, 140.4, 137.1,

136.2, 133.6, 129.4, 128.9, 127.1, 125.9, 125.8, 125.2, 120.0, 119.3,

111.4, 110.3, 106.2, 104.3, 68.7, 67.6, 60.8, 56.1, 53.5, 46.5, 29.4, 28.8,

28.5, 24.0, 22.5 ppm; (ESI) MS: m/z 625 [M+1]⁺; HRMS (ESI m/z)

for C₃₇H₄₁N₂O₇calcd 625.3175, found 625.3162 [M+1]⁺.

7-Methoxy-8-{6-(4-N,N-dimethylamino)phenyl)-2-naphthyl]oxy)-

pentoxy}-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c]-

[1,4]benzodiazepine-5-one (3u): White solid (184 mg, 64% yield):

R_f=0.3 (3% MeOH/CHCl₃); mp: 82–848C; [a]²_D⁷=+192.0 (c=0.1,

CHCl₃); IR (KBr): n_max=3366 (br), 2929, 2871, 1607, 1509, 1432,

7-Methoxy-8-{6-(4-fluorophenyl)-2-naphthyl]{4-[3-(3-oxypro-

poxy)-4-methoxybenzoyl]piperazino}methanone}-(11aS)-

1

1250, 1205, 1154, 850 cm^ꢁ1; H NMR (300 MHz, CDCl₃): d=7.90 (s,

1H, ArH), 7.75 (d, J=9.0 Hz, 2H, ArH), 7.68 (d, J=3.7 Hz, 1H, imine-

H), 7.62 (d, J=8.8 Hz, 2H, ArH), 7.52 (s, 1H, ArH), 7.10–7.16 (m, 3H),

6.90 (d, J=8.8 Hz, 2H, ArH), 6.81 (s 1H, ArH) 4.06–4.18 (m, 4H, 2ꢁ

OCH₂), 3.95 (s, 3H, OCH₃), 3.76–3.88 (m, 1H, NCH), 3.53–3.73 (m,

2H, NCH₂), 3.02 (s, 6H, N(CH₃)₂) 2.28 (m, 2H, CH₂), 1.87–2.10 (m,

6H, 3ꢁCH₂), 1.62–1.80 ppm (m, 2H, CH₂); (ESI) MS: m/z 578 [M+

1]⁺; HRMS (ESI m/z) for C₃₆H₄₀N₃O₄calcd 578.3280, found 578.3287

[M+1]⁺.

1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one

(4a): White solid (211 mg, 55% yield): R_f=0.3 (4.0% MeOH/CHCl₃);

mp: 130–1328C; [a]²_D⁷=+153.0 (c=0.1, CHCl₃); IR (KBr): n_max=3422

(br), 2927, 1627, 1512, 1430, 1261, 1220, 1013, 817 cm^ꢁ^{1 1}H NMR

;

(300 MHz, CDCl₃): d=8.01 (s, 1H, ArH), 7.93–7.98 (m, 3H, ArH),

7.76 (d, J=9.0 Hz, 1H, ArH), 7.70 (d, J=4.3 Hz, 1H, imine-H), 7.64–

7.68 (m, 2H, ArH), 7.54 (s, 1H, ArH), 7.52 (s, 1H, ArH), 7.19 (t, J=

8.3 Hz, 2H ArH), 7.05 (s 1H, ArH), 7.00 (d, J=8.3 Hz, 1H, ArH), 6.85–

1676

www.chemmedchem.org

ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

ChemMedChem 2011, 6, 1665 – 1679

PBD Conjugates as Anticancer Agents

6.89 (m, 2H, ArH), 4.21–4.33 (m, 4H, 2ꢁOCH₂), 3.93 (s, 3H, OCH₃),

3.87 (s, 3H, OCH₃), 3.80–3.85 (m, 1H, NCH), 3.50–3.78 (m, 10H,

N(CH₂-CH₂)₂N, NCH₂), 2.27–2.43 (m, 4H, 2ꢁCH₂) 2.02–2.10 ppm (m,

2H, CH₂); ¹³C NMR (75 MHz, CDCl₃): d=170.5, 170.3, 164.2, 162.3,

157.8, 151.0, 148.2, 147.7, 143.4, 140.4, 138.9, 133.9, 132.3, 131.6,

128.9, 128.8, 128.6, 127.0, 126.8, 126.4, 125.3, 124.6, 120.5, 120.2,

115.9, 115.6, 112.7, 111.4, 110.9, 110.5, 65.5, 65.2, 56.0, 53.6, 46.6,

42.4, 29.5, 28.9, 24.0 ppm; (ESI) MS: m/z 771 [M+1]⁺; HRMS (ESI

m/z) for C₄₅H₄₄N₄O₇F calcd 771.8439, found 771.8431 [M+1]⁺.

(5% MeOH/CHCl₃); mp: 115–1178C; [a]²_D⁷=+134.0 (c=0.1, CHCl₃);

IR (KBr): n_max=3416 (br), 2924, 1608, 1512, 1431, 1261, 1220, 1135,

1014, 814 cm^ꢁ^{1 1}H NMR (500 MHz, CDCl₃): d=7.99 (s, 1H, ArH),

;

7.87–7.92 (m, 3H, ArH), 7.80 (d, J=8.3 Hz, 1H, ArH), 7.64–7.67 (m,

2H, Hz, ArH), 7.63 (d, J=3.8 Hz, 1H, imine-H), 7.51 (s, 1H, ArH),

7.47 (d, J=8.3 Hz, 1H, ArH), 7.04 (s, 1H, ArH), 7.00 (d, J=8.3 Hz,

1H, ArH), 6.84–6.87 (m, 4H, ArH), 4.22–4.34 (m, 4H, 2ꢁOCH₂), 3.92

(s, 3H, OCH₃), 3.87 (s, 3H, OCH₃), 3.77–3.84 (m, 1H, NCH), 3.55–3.75

(m, 10H, N(CH₂-CH₂)₂N, NCH₂), 3.03 (s, 6H, N(CH₃)₂), 2.26–2.41 (m,

4H, 2ꢁCH₂), 2.02–2.08 ppm (m, 2H, CH₂); ¹³C NMR (75 MHz,

CDCl₃): d=170.8, 164.5, 162.4, 151.0, 148.5, 147.7, 140.4, 140.0,

134.3, 131.0, 131.4, 128.6, 128.4, 127.9, 126.9, 126.2, 124.3, 123.7,

120.5, 112.7, 111.4, 110.9, 110.4, 65.5, 56.0, 53.6, 46.5, 40.4, 29.6,

28.9, 24.0 ppm; (ESI) MS: m/z 796 [M+1]⁺; HRMS (ESI m/z) for

C₄₇H₅₀N₅O₇calcd 796.3972, found 796.3966 [M+1]⁺.

7-Methoxy-8-{6-(4-trifluoromethylphenyl)-2-naphthyl]{4-[3-(3-

oxypropoxy)-4-methoxybenzoyl]piperazino}methanone}-(11aS)-

1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one

(4b): White solid (205 mg, 50% yield): R_f=0.3 (4.5% MeOH/CHCl₃);

mp: 125–1278C; [a]²_D⁷=+178.0 (c=0.1, CHCl₃); IR (KBr): n_max=3420

(br), 2929, 1629, 1514, 1436, 1251, 1221, 1113, 820 cm^ꢁ1; H NMR

(300 MHz, CDCl₃): d=8.06 (s, 1H, ArH), 7.95–7.00 (m, 2H, ArH),

7.89 (d, J=7.3 Hz, 1H, ArH), 7.78 (s, 1H, ArH), 7.75 (s, 1H, ArH), 7.72

(s, 1H, ArH), 7.65 (d, J=3.7 Hz, 1H, imine-H), 7.52–7.59 (m, 3H,

ArH), 7.36 (s 1H, ArH), 7.34 (s, 1H, ArH), 6.95–7.06 (m, 2H, ArH),

6.86 (s, 1H, ArH), 4.20–4.34 (m, 4H, 2ꢁOCH₂), 3.93 (s, 3H, OCH₃),

3.89 (s, 3H, OCH₃), 3.77–3.86 (m, 1H, NCH), 3.42–3.74 (m, 10H,

N(CH₂-CH₂)₂N, NCH₂), 2.24–2.39 (m, 4H, 2ꢁCH₂) 2.00–2.11 ppm (m,

2H, CH₂); (ESI) MS: m/z 821 [M+1]⁺; HRMS (ESI m/z) for

C₄₆H₄₄N₄O₇F₃calcd 821.8514, found 821.8505 [M+1]⁺.

7-Methoxy-8-{6-(3-chloromethoxyphenyl)-2-naphthyl]{4-[3-(3-

oxypropoxy)-4-methoxybenzoyl]piperazino}methanone}-(11aS)-

1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one

(4 f): White solid (216 mg, 55% yield): R_f=0.3 (4.0% MeOH/CHCl₃);

mp: 118–1208C; [a]²_D⁷=+180.0 (c=0.1, CHCl₃); IR (KBr): n_max=3421

(br), 2929, 1637, 1516, 1430, 1260, 1156, 1014, 817 cm^{ꢁ1 1}H NMR

;

(300 MHz, CDCl₃): d=8.02 (s, 1H, ArH), 7.94–7.97 (m, 3H, ArH),

7.77 (s, 1H, ArH), 7.70 (d, J=3.4 Hz, 1H, imine-H), 7.64–7.69 (m, 4H,

ArH), 7.55 (s, 1H, ArH), 7.51 (s, 1H, ArH), 7.16–7.21 (t, J=8.3 Hz, 1H,

ArH), 7.06 (s 1H, ArH), 7.01 (d, J=7.5 Hz, 1H, ArH), 6.88 (d, J=

8.3 Hz, 1H, ArH), 4.22–4.35 (m, 4H, 2ꢁOCH₂), 3.92 (s, 3H, OCH₃),

3.87 (s, 3H, OCH₃), 3.79–3.82 (m, 1H, NCH), 3.48–3.74 (m, 10H,

N(CH₂-CH₂)₂N, NCH₂), 2.01–2.13 (m, 4H, 2ꢁCH₂), 1.95–2.07 ppm (m,

2H, CH₂); (ESI) MS: m/z 788 [M+1]⁺; HRMS (ESI m/z) for

C₄₆H₄₄N₄O₇Cl calcd 788.2985, found 788.2976 [M+1]⁺.

7-Methoxy-8-{6-(4-trifluoromethoxyphenyl)-2-naphthyl]{4-[3-(3-

oxypropoxy)-4-methoxybenzoyl]piperazino}methanone}-(11aS)-

1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one

(4c): White solid (217 mg, 52% yield): R_f=0.3 (5% MeOH/CHCl₃);

mp: 120–1228C; [a]²_D⁷=+141.0 (c=0.1, CHCl₃); IR (KBr): n_max=3444

(br), 2932, 1626, 1513, 1430, 1260, 1220, 1172, 1014, 818 cm^ꢁ1

;

¹H NMR (300 MHz, CDCl₃): d=8.04 (s, 1H, ArH), 7.91–7.99 (m, 2H,

ArH), 7.88 (t, J=7.4 Hz, 1H, ArH), 7.79 (s, 1H, ArH), 7.76 (s, 1H,

ArH), 7.72 (s, 1H, ArH), 7.66 (d, J=3.7 Hz, 1H, imine-H), 7.51–7.58

(m, 3H, ArH), 7.37 (s 1H, ArH), 7.33 (s, 1H, ArH), 6.96–7.05 (m, 2H,

ArH), 6.87 (s, 1H, ArH), 4.19–4.33 (m, 4H, 2ꢁOCH₂), 3.92 (s, 3H,

OCH₃), 3.88 (s, 3H, OCH₃), 3.78–3.84 (m, 1H, NCH), 3.46–3.76 (m,

10H, N(CH₂-CH₂)₂N, NCH₂), 2.26–2.47 (m, 4H, 2ꢁCH₂), 1.98–

2.12 ppm (m, 2H, CH₂); (ESI) MS: m/z 837 [M+1]⁺; HRMS (ESI m/z)

for C₄₆H₄₄N₄O₈F₃calcd 837.4508, found 837.4501 [M+1]⁺.

7-Methoxy-8-{6-(3,4-dimethoxyphenyl)-2-naphthyl]{4-[3-(3-oxy-

propoxy)-4-methoxybenzoyl]piperazino}methanone}-(11aS)-

1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one

(4g): White solid (203 mg, 50% yield): R_f=0.3 (5% MeOH/CHCl₃);

mp: 138–1408C; [a]²_D⁷=+156.0 (c=0.1, CHCl₃); IR (KBr): n_max=3420

(br), 2926, 1627, 1514, 1429, 1257, 1176, 1016, 814 cm^ꢁ^{1 1}H NMR

;

(300 MHz, CDCl₃): d=8.01 (s, 1H, ArH), 7.92–7.95 (m, 3H, ArH),

7.81 (s, 1H, ArH), 7.76 (s, 1H, ArH), 7.67 (d, J=3.6 Hz, 1H, imine-H),

7.52 (s, 1H, ArH), 7.12–7.24 (m, 4H, ArH), 6.98 (dd, J=2.9 Hz,

5.8 Hz, 1H, ArH), 6.88 (s, 1H, ArH), 6.76 (s, 1H, ArH), 4.20–4.33 (m,

4H, 2ꢁOCH₂), 4.00 (s, 3H, OCH₃), 3.95 (s, 3H, OCH₃), 3.92 (s, 3H,

OCH₃), 3.87 (s, 3H, OCH₃), 3.76–3.84 (m, 1H, NCH), 3.50–3.74 (m,

10H, N(CH₂-CH₂)₂N, NCH₂), 2.06–2.33 (m, 4H, 2ꢁCH₂), 2.00–

2.12 ppm (m, 2H, CH₂); ¹³C NMR (75 MHz, CDCl₃): d=170.5, 170.3,

162.3, 159.3, 150.9, 150.5, 149.1, 148.8, 148.1, 139.7, 134.0, 133.2,

131.9, 131.4, 131.2, 128.7, 128.2, 127.0, 126.8, 126.4, 126.2, 124.8,

124.6, 124.3, 120.4, 120.1, 119.6, 114.2, 112.6, 111.4, 110.8, 110.4,

65.4, 65.2, 55.9, 55.2, 53.8, 46.4, 41.6, 29.5, 28.2, 23.9 ppm; (ESI) MS:

m/z 813 [M+1]⁺; HRMS (ESI m/z) for C₄₇H₄₉N₄O₉calcd 813.3157,

found 813.3148 [M+1]⁺.

7-Methoxy-8-{6-(4-methoxyphenyl)-2-naphthyl]{4-[3-(3-oxypro-

poxy)-4-methoxybenzoyl]piperazino}methanone}-(11aS)-

1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one

(4d): White solid (215 mg, 55% yield): R_f=0.3 (5% MeOH/CHCl₃);

mp: 141–1438C; [a]²_D⁷=+173.0 (c=0.1, CHCl₃); IR (KBr): n_max=3426

(br), 2927, 1608, 1512, 1432, 1258, 1220, 1178, 1014, 815 cm^ꢁ1

;

¹H NMR (300 MHz, CDCl₃): d=8.01 (s, 1H, ArH), 7.90–7.96 (m, 3H,

ArH), 7.66 (d, J=4.1 Hz, 1H, imine-H), 7.64–7.70 (m, 3H, ArH), 7.52 (s,

1H, ArH), 7.48 (s, 1H, ArH), 6.98–7.06 (m, 4H, ArH), 6.88. (s 1H, ArH),

6.85 (s, 1H, ArH), 4.19–4.35 (m, 4H, 2ꢁOCH₂), 3.92 (s, 3H, OCH₃),

3.88 (s, 6H, 2ꢁOCH₃), 3.80–3.86 (m, 1H, NCH), 3.50–3.79 (m, 10H,

N(CH₂-CH₂)₂N, NCH₂), 2.26–2.46 (m, 4H, 2ꢁCH₂), 2.00–2.13 ppm (m,

2H, CH₂); ¹³C NMR (75 MHz, CDCl₃): d=170.6, 170.4, 164.5, 162.3,

159.4, 151.0, 150.5, 148.2, 147.7, 140.4, 139.6, 134.1, 132.8, 131.8,

131.3, 128.8, 128.5, 128.3, 127.1, 126.9, 126.3, 124.7, 124.4, 120.5,

120.2, 114.3, 112.7, 111.4, 110.9, 110.2, 65.5, 65.3, 56.0, 55.9, 55.3,

53.6, 46.6, 29.5, 28.9, 24.1 ppm; (ESI) MS: m/z 783 [M+1]⁺; HRMS

(ESI m/z) for C₄₇H₄₇N₄O₈calcd 783.3656, found 783.3643 [M+1]⁺.

7-Methoxy-8-{6-(3,4,5-trimethoxyphenyl)-2-naphthyl]{4-[3-(3-

oxypropoxy)-4-methoxybenzoyl]piperazino}methanone}-(11aS)-

1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one

(4h): White solid (227 mg, 54% yield): R_f=0.3 (6% MeOH/CHCl₃);

mp: 104–1068C; [a]²_D⁷=+170.0 (c=0.1, CHCl₃); IR (KBr): n_max=3432

(br), 2929, 1628, 1513, 1429, 1261, 1127, 1013, 819 cm^ꢁ^{1 1}H NMR

;

(300 MHz, CDCl₃): d=8.01 (s, 1H, ArH), 7.91–7.98 (m, 3H, ArH),

7.77 (dd, J=1.7 Hz, 6.6 Hz, 1H, ArH), 7.66 (d, J=4.3 Hz, 1H, imine-

H), 7.51 (s, 1H, ArH), 6.97–7.05 (m, 3H, ArH), 6.91 (s, 2H, ArH), 6.88

(s, 1H, ArH), 6.86 (s, 1H, ArH), 4.20–4.34 (m, 4H, 2ꢁOCH₂), 3.97 (s,

6H, 2ꢁOCH₃), 3.92 (s, 6H, 2ꢁOCH₃), 3.87 (s, 3H, OCH₃), 3.77–3.83

(m, 1H, NCH), 3.50–3.75 (m, 10H, N(CH₂-CH₂)₂N, NCH₂), 2.28–2.43

7-Methoxy-8-{6-(4-N,N-dimethylaminophenyl)-2-naphthyl]{4-[3-

(3-oxypropoxy)-4-methoxybenzoyl]piperazino}methanone}-

(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzo-

diazepine-5-one (4e): White solid (214 mg, 54% yield): R_f=0.3

ChemMedChem 2011, 6, 1665 – 1679

ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemmedchem.org

1677

A. Kamal, M. Pal-Bhadra, et al.

MED

(m, 4H, 2ꢁCH₂) 1.99–2.09 ppm (m, 2H, CH₂); ¹³C NMR (75 MHz,

CDCl₃): d=170.6, 170.5, 164.5, 162.4, 160.9, 153.5, 151.1, 148.3,

140.5, 140.1, 136.4, 134.0, 132.2, 131.7, 129.9, 128.9, 128.6, 127.1,

126.9, 126.6, 125.4, 124.6, 124.2, 120.5, 120.2, 112.7, 111.5, 110.9,

110.4, 104.6, 65.5, 65.3, 56.1, 55.9, 53.6, 50.6, 46.6, 29.6, 28.9,

24.1 ppm; (ESI) MS: m/z 843 [M+1]⁺; HRMS (ESI m/z) for

C₄₈H₅₁N₄O₁₀calcd 843.3527, found 843.2521 [M+1]⁺.

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Synthesis of aryl-substituted naphthalene-linked pyrrolobenzodiazepine conjugates as potential anticancer agents with apoptosis-inducing ability

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