An efficient solid-phase synthesis of biologically important DNA-interactive pyrrolo[2,1-c][1,4]benzodiazepine dimers (DSB-120) and their C2-fluorinated analogues

Article abstract of DOI:10.1016/j.tetlet.2006.07.016

A facile method for the solid-phase synthesis of pyrrolo[2,1-c][1,4]benzodiazepine dimers has been developed. p-Nitrophenyl carbonate Wang resin attached to 2-amino-5-methoxy-methyl benzoate has been utilized as the resin-bound starting material and these reactions are monitored by FT-IR spectroscopy of resin beads.

Full text of DOI:10.1016/j.tetlet.2006.07.016

Tetrahedron Letters 47 (2006) 6553–6556
An efficient solid-phase synthesis of biologically important
DNA-interactive pyrrolo[2,1-c][1,4]benzodiazepine dimers
(DSB-120) and their C2-fluorinated analogues
Ahmed Kamal,* N. Shankaraiah, V. Devaiah and K. Laxma Reddy
Biotransformation Laboratory, Division of Organic Chemistry, Indian Institute of Chemical Technology, Hyderabad 500 007, India
Received 19 May 2006; revised 26 June 2006; accepted 7 July 2006
Available online 31 July 2006
Abstract—A facile method for the solid-phase synthesis of pyrrolo[2,1-c][1,4]benzodiazepine dimers has been developed. p-Nitro-
phenyl carbonate Wang resin attached to 2-amino-5-methoxy-methyl benzoate has been utilized as the resin-bound starting material
and these reactions are monitored by FT-IR spectroscopy of resin beads.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Solid-phase combinatorial chemistry has recently
emerged as a useful tool for the generation of libraries
of new molecules with different biological profiles.1 In
recent years, solid-phase heterocyclic chemistry has
expanded rapidly, and numerous methods have been
reported.2 As part of our continuing effort to develop
new heterocyclic solid-phase strategies for synthesizing
nitrogen-rich heterocyclic compounds based on pyr-
rolobenzodiazepines, we required libraries of the dimers
of pyrrolobenzodiazepines for lead generation against
certain disease targets, particularly cancer.3
of the covalently bonded DNA strand. Many members
of the PBD family such as DC-81 1, tomaymycin 2,
SJG-136 3, DSB-120 4a (n = 1) and C2-fluoro-substi-
tuted PBD dimers 4d–f exert their biological activity
through covalent binding via their guanine residue with-
in the minor groove of DNA (Fig. 1). Some of these
compounds have reached various stages of clinical trials
but have not progressed due to problems including
cardiotoxicity or lack of efficacy.9
A number of antiviral and antitumour agents have been
developed in which a fluorine substituent has played a
The pyrrolo[2,1-c][1,4]benzodiazepines (PBD)s are a
family of sequence-selective DNA-interactive anti-
tumour antibiotics derived from Streptomyces species.4
These compounds bind within the minor groove of
DNA, forming a covalent aminal bond between the
C11-position of the central B-ring and the N2-amino
group of guanine base.5,6 The cytotoxic and antitumour
activity of PBDs are attributed to their ability to form
covalent DNA adducts. Molecular modelling, solution
NMR, fluorometry and DNA footprinting experiments
have shown that these molecules have preferred selectiv-
ity for Pu–G–Pu sequences,7,8 and can be oriented with
their A-rings pointed either towards the 30- or the 50-end
10
9
A
6
11
N
N
HO
HO
H
H
8
B11a
1
C
N
7
N
H3CO
H3CO
5
2
4
3
O
O
2
1
N
N
O
O
H
H
H
N
N
OCH3 H3CO
O
O
3
N
N
O
O
H
n
N
Keywords: Solid-phase synthesis; Pyrrolo[2,1-c][1,4]benzodiazepine
dimers; DSB-120; Swern oxidation.
N
OCH3 H3CO
R
R
O
O
4a-c, R= H
4d-f, R= F
*
Corresponding author. Tel.: +91 40 27193157; fax: +91 40
27193189; e-mail addresses: ahmedkamal@iict.res.in; ahmedkamal@
iictnet.org
n= 1-3
Figure 1.
0040-4039/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.07.016

6554
A. Kamal et al. / Tetrahedron Letters 47 (2006) 6553–6556
key role in their biological activity.10 In earlier studies,
attempts were made to enhance the sequence selectivity
and antitumour potency by synthesizing C7- or C8-
linked PBD dimers,11,12 for example, DC-81 dimer
(DSB-120), which is one of the most potent irreversible
interstrand cross-linking agents. Recently, SJG-136 3,
an analogue of DSB-120 was reported to be significantly
more cytotoxic than DSB-120 across a number of can-
cer cell lines and this PBD dimer is presently under
phase I clinical trials.13 In continuation of our earlier
efforts on the structural modifications of PBDs14 and
their dimers,15 we were interested in the development
of solid-phase methodologies, particularly for the PBD
dimers.
Table 1. Yields and molecular ions observed for PBD dimers 4a–f
Entry
n
R
Yield (%)a
FABMSb
4a
4b
4c
4d
4e
4f
1
2
3
1
2
3
H
H
H
F
F
F
62
68
63
56
58
55
533
547
561
569
583
597
a Based on initial loading of the p-nitrophenyl carbonate Wang resin.
b Parent ion observed as (M+H)+.
H2N
H3CO
O
O
NH2
OCH3
n
OCH3 H3CO
n= 1-3
5
O
O
In this letter, we report for the first time, the solid-phase
synthesis of PBD dimers such as DSB-120 and its
C2-fluoro-substituted analogues. In our previous inves-
tigations we developed solid-phase procedures for the
synthesis of DC-81 and other related PBD monomers
in addition to several solution-phase methodologies.16
It has been observed that in most solution-phase proce-
dures for the preparation of PBD dimers there are prob-
lems relating to solubility. Therefore, the development
of solid-phase protocols for PBD dimers is of immense
importance as it could address the solubility difficulties
encountered during work-up.
O
O
O
i
6
NO2
O
O
O
O
9
O
HN
H3CO
O
O
NH
OCH3
n
OCH3 H3CO
ii
O
O
7
The starting material 5 was prepared by employing the
procedure described in our earlier study,17 which in-
volved bis-coupling 2-nitro-4-hydroxy 5-methoxy-
methyl benzoate with a dihaloalkane and subsequent
reduction of the nitro group. It is interesting to note
from the literature that there are site–site interactions
on using resins, which depend on the percentage amount
of cross-linking polystyrene.18 Usually, as the percent-
age of cross-linking decreases and as the functionaliza-
tion increases, the site–site interactions also increase.
In the present study, we have employed (1% DVB) poly-
styrene for the synthesis of the target compound based
on pyrrolo[2,1-c][1,4]benzodiazepine dimers. A stirred
solution of 5 was treated with p-nitrophenyl carbonate
Wang resin 6 using 1-hydroxybenzotriazole (HOBt)
and diisopropylethylamine (DIPEA) in CH2Cl2–DMF
(2:1) to give 7. After loading, the unreacted free hydr-
oxyl groups of the Wang resin were capped with acetic
anhydride/triethylamine in dichloromethane. Hydroly-
sis of the methyl esters afforded the corresponding
acid,19 8, which was coupled with pyrrolidinemethanol
9 in the presence of EDCI and HOBt to provide 11.
Swern oxidation20 of 11 then gave 12 via an oxidative
cyclization process. However, Thurston et al. had re-
ported a similar oxidation method using Dess–Martin
periodinane for the preparation of PBD monomers.21
Finally, the resin was cleaved22 using TFA (50%) to
afford the target product 423 via loss of the hydroxyl
groups and the products were formed in moderate yields
as shown in Table 1. Similarly, C2-fluoro-substituted
pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimers were
also synthesized as shown in Scheme 1, employing
fluoro-substituted pyrrolidinemethanol 10f, which was
itself prepared from hydroxy L-proline 10a as shown
in Scheme 2.
O
O
NH
OH
O
HN
HO
O
O
n
OCH3 H3CO
HO
8
O
O
iii
R=H
NH
10f R=F
R
O
O
O
O
OH
R
HO
HN
NH
O
O
n
N
N
O
OCH3 H3CO
iv
R
11
O
O
O
O
N
O
HO
H
OH
H
N
N
O
O
n
N
OCH3 H3CO
v
R
R
12
O
O
N
N
O
O
H
H
n
N
N
OCH3 H3CO
R
R
4a-c, R= H
4d-f, R= F
O
O
Scheme 1. Reactions and conditions: (i) HOBt, DIPEA, CH2Cl2/
DMF (2:1) 6 h, rt; (ii) 1 N NaOH, 1,4-dioxane, 80 ꢁC, 12 h; (iii) EDCI,
HOBt, 15–24 h, rt; (iv) DMSO, (COCl)2, Et3N, CH2Cl2, ꢀ78 ꢁC, 2 h;
(v) TFA/CH2Cl2 (1:1), 2 h, rt.
In a typical synthesis, a suspension of p-nitrophenyl car-
bonate Wang resin 6 (2.00 g, 1.2 mmol/g, 100–200 mesh

A. Kamal et al. / Tetrahedron Letters 47 (2006) 6553–6556
6555
COOH
COOCH3
COOCH3
(ii)
(i)
HN
10a
HN
Boc-N
OH
OH
OH
10b
10c
(iii)
CH2OH
CH2OH
COOCH3
F
(v)
(iv)
HN
10f
Boc-N
Boc-N
F
F
10e
10d
Scheme 2. Reagents and conditions: (i) SOCl2, MeOH, 0 ꢁC, rt, 6 h; (ii) Boc-anhydride, Et3N, DMAP, CH2Cl2, 0 ꢁC, rt, overnight; (iii) DAST,
CH2Cl2, ꢀ78 ꢁC, 12 h; (iv) LiBH4, THF, 0 ꢁC, 12 h; (v) TFA, CH2Cl2, rt, 6 h.
and 1% DVB) in CH2Cl2–DMF (2:1, 20 mL) was stirred
for 30 min. A solution of 5 (0.360 g, 0.80 mmol), HOBt
(0.649 g, 4.80 mmol) and DIPEA (1.39 mL, 4.80 mmol)
in CH2Cl2–DMF (2:1, 10 mL) was added to the swollen
resin and stirring was continued at room temperature
for 6 h. The derivatized resin 7 was then filtered, rinsed
with DMF (2 · 15 mL), CH2Cl2 (2 · 15 mL), MeOH
(2 · 15 mL), ether (2 · 15 mL) and dried in vacuo. To
a suspension of 7 in dioxane (30 mL) was added 1 N
NaOH solution (10 mL) and the reaction heated at
100 ꢁC for 12 h. On cooling, the resin 8 was filtered
and rinsed with water (2 · 15 mL), water/dioxane (1:9,
2 · 15 mL), MeOH (2 · 15 mL), CH2Cl2 (2 · 15 mL),
Et2O (2 · 15 mL) and dried in vacuo. To a suspension
of resin 8 in DMF (20 mL), EDCI (0.918 g, 4.80 mmol),
HOBt (0.649 g, 4.80 mmol) and pyrrolidinemethanol 9
(0.32 mL, 129.25 mmol) were added and the reaction
mixture was stirred for 15–24 h at room temperature.
The resin was then filtered and washed with DMF
(2 · 15 mL), DMF/water (8:2, 2 · 15 mL), MeOH (2 ·
15 mL), MeOH/water (9:1, 2 · 15 mL), MeOH (2 · 15
mL), CH2Cl2 (2 · 15 mL), Et2O (2 · 15 mL) and dried
in vacuo. To a suspension of resin 11 in CH2Cl2
(15 mL) was added DMSO (0.91 mL, 12.80 mmol),
(COCl)2 (0.57 mL, 6.40 mmol), Et3N (2.23 mL, 16.00
mmol) and the reaction stirred at ꢀ78 ꢁC for 2 h. The
oxidized resin was filtered, rinsed with CH2Cl2 (2 ·
15 mL), DMSO (2 · 15 mL), MeOH/water (8:2, 2 ·
15 mL), MeOH (2 · 15 mL), CH2Cl2 (2 · 15 mL) and
Et2O (2 · 15 mL), then dried in vacuo. A suspension
of resin 12 in (15 mL) TFA/CH2Cl2 (50%) was allowed
to stir for 2 h then filtered. This procedure was repeated
once again to ensure complete cleavage of the product
from the resin. The combined supernatant was diluted
with water (20 mL) and neutralized by the addition of
solid NaHCO3. The organic phase was separated and
washed with water, brine and dried over Na2SO4 and
evaporated under reduced pressure. The crude products
4a–f were purified by column chromatography through
silica gel (60–120 mesh) with CHCl3/MeOH (95:5) to give
55–68% yields of dimers 4a–f. The product from each
step was confirmed by NMR and FT-IR and also by
comparison with the products obtained in solution-phase.
their C2-flourinated analogues has been demonstrated.
This methodology is also highly amenable for the gener-
ation of combinatorial libraries of PBD dimers with
diversity in the C-rings.
Acknowledgements
The authors N.S., V.D. and K.L.R. are grateful to
CSIR, New Delhi, for the award of Research
fellowships.
References and notes
1. (a)Combinatorial Chemistry; Jung, G., Ed.; Wiley-VCH:
Weinhein, 1998; (b) Handbook of Combinatorial Chemis-
try; Nicolaou, K. C., Hanko, R., Hartwig, W., Eds.;
Wiley-VCH: Weinheim, 2002; (c) Booth, S.; Hermkens, P.
H.; Ottenheijm, H. C. J.; Rees, D. C. Tetrahedron 1998,
54, 15385–15443; (d) Sammelson, R. E.; Kurth, M. J.
Chem. Rev. 2001, 101, 137; (e) Franzen, R. G. J. Comb.
Chem. 2000, 2, 195–214.
2. (a) Kamal, A.; Reddy, K. L.; Devaiah, V.; Shankaraiah,
N.; Reddy, D. R. Mini-Rev. Med. Chem. 2006, 6, 53–69;
(b) Kamal, A.; Reddy, K. L.; Devaiah, V.; Shankaraiah,
N.; Rao, M. V. Mini-Rev. Med. Chem. 2006, 6, 71–89.
3. (a) Thurston, D. E.; Bose, D. S.; Thompson, A. S.;
Howard, P. W.; Leoni, A.; Croker, S. J.; Jenkins, T. C.;
Neidle, S.; Hartley, J. A.; Hurley, L. H. J. Org. Chem.
1996, 61, 8141–8147; (b) Kamal, A.; Ramesh, G.; Laxman,
N.; Ramulu, P.; Srinivas, O.; Neelima, K.; Kondapi, A.
K.; Sreenu, V. B.; Nagarajaram, H. A. J. Med. Chem.
2002, 45, 4679–4688.
4. Neidle, S.; Thurston, D. E. In New Targets for Cancer
Chemotherapy; Kerr, D. J., Workmann, P., Eds.; CRC
Press: London, UK, 1994; p 159.
5. Thurston, D. E. Advances in the Study of Pyrrolo[2,1-c]-
[1,4]-benzodiazepine (PBD) Antitumour Antibiotics.
Molecular Aspects of Anticancer Drug–DNA Interactions;
Macmillan Press Ltd.: London, UK, 1993; pp 54–88.
6. Petrusek, R. L.; Uhlenhopp, E. L.; Duteau, N.; Hurley,
L. H. J. Biol. Chem. 1982, 257, 6207–6216.
7. Hurley, L. H.; Reck, T.; Thurston, D. E.; Langley, D. R.;
Holden, K. G.; Hertzberg, R. P.; Hoover, J. R. E.;
Gallagher, G., Jr.; Faucette, L. F.; Mong, S. M.; Johnson,
R. K. Chem. Res. Toxicol. 1988, 1, 258–268.
In conclusion, an efficient and practical solid-phase syn-
thesis of pyrrolo[2,1-c][1,4]benzodiazepine dimers and
8. Boyd, F. L.; Stewart, D.; Remers, W. A.; Barkley, M. D.;
Hurley, L. H. Biochemistry 1990, 29, 2387–2403.

6556
A. Kamal et al. / Tetrahedron Letters 47 (2006) 6553–6556
9. Remers, W. A. In The Chemistry of Antitumor Antibiotics;
1843; (c) Kamal, A.; Devaiah, V.; Reddy, K. L.;
Shankaraiah, N. Adv. Synth. Catal. 2006, 348, 249–254;
(d) Kamal, A.; Shankaraiah, N.; Reddy, K. L.; Devaiah,
V. Tetrahedron Lett. 2006, 47, 4253–4257; (e) Kamal, A.;
Reddy, P. S. M. M.; Reddy, D. R. Tetrahedron Lett.
2002, 43, 6629–6631; (f) Kamal, A.; Ramana, K. V.;
Ankati, H. B.; Ramana, A. V. Tetrahedron Lett. 2002,
43, 6861–6863.
Wiley: New York, 1988; Vol. 2, pp 28–92.
10. Kamal, A.; Reddy, P. S. M. M.; Reddy, D. R.; Laxman,
E.; Murthy, Y. L. N. . Bioorg. Med. Chem. Lett. 2004, 14,
5699–5702.
11. (a) Farmer, J. D.; Rudnicki, S. M.; Suggs, J. W.
Tetrahedron Lett. 1988, 29, 5105–5108; (b) Farmer,
J. D.; Gustafson, G. R.; Conti, A.; Zimmt, M. B.; Suggs,
J. W. Nucleic Acids Res. 1991, 19, 899–903.
12. (a) Bose, D. S.; Thompson, A. S.; Ching, J.; Hartley, J. A.;
Berardini, M. D.; Jenkins, T. C.; Neidle, S.; Hurley, L. H.;
Thurston, D. E. J. Am. Chem. Soc. 1992, 114, 4939–4941;
(b) Bose, D. S.; Thompson, A. S.; Smellie, M.; Berardini,
M. D.; Hartley, J. A.; Jenkins, T. C.; Neidle, S.; Thurston,
D. E. J. Chem. Soc., Chem. Commun. 1992, 14, 1518–
1520.
13. Thurston, D. E.; Bose, D. S.; Howard, P. W.; Jenkins, T.
C.; Leoni, A.; Baraldi, P. G.; Guiotto, A.; Cacciari, B.;
Kelland, L. R.; Foloppe, M.-P.; Rault, S. J. Med. Chem.
1999, 42, 1951–1964.
17. Kamal, A.; Rao, N. V. Tetrahedron Lett. 1995, 36, 4299–
4302.
18. (a) Farrall, J. M.; Frechet, J. M. J. J. Am. Chem. Soc.
1978, 100, 7998–7999; (b) Scott, T. L.; Rebek, J.;
Ovsyanko, L.; Sims, L. C. J. Am. Chem. Soc. 1977, 99,
625–626; (c) Pulko, I.; Krajnc, P. Acta Chim. Slov. 2005,
52, 215–223.
19. Kamal, A.; Reddy, G. S. K.; Raghavan, S. Bioorg. Med.
Chem. Lett. 2001, 11, 387–389.
20. Cheng, W.-C.; Kurthu, M. J. J. Org. Chem. 2002, 67,
4387–4391.
21. Berry, J. M.; Howard, P. W.; Thurston, D. E. Tetrahedron
Lett. 1996, 41, 6171–6174.
14. (a) Kamal, A.; Babu, A. H.; Ramana, A. V.; Ramana, K.
V.; Bharathi, E. V.; Kumar, M. S. Bioorg. Med. Chem.
Lett. 2005, 15, 2621–2623; (b) Kamal, A.; Srinivas, O.;
Ramulu, P.; Ramesh, G.; Kumar, P. P. Bioorg. Med.
Chem. Lett. 2003, 13, 3577–3581; (c) Kamal, A.; Ramulu,
P.; Srinivas, O.; Ramesh, G.; Kumar, P. P. Bioorg. Med.
Chem. Lett. 2004, 14, 4791–4794.
22. Advanced ChemTech Handbook of Combinatorial and
Solid-Phase Organic Chemistry—DA Guide to Principles,
Products and Protocols; Bennett, W. D., Christensen, J.
W., Hamaker, L. K., Peterson, M. L., Rhodes, M. R.,
Saneii, H. H., Eds.; Advanced Chemtech: Louisville, KY,
1998; p 356.
15. (a) Kamal, A.; Reddy, P. S. M. M.; Reddy, D. R.;
Laxman, E. Bioorg. Med. Chem. 2006, 14, 385–394; (b)
Kamal, A.; Reddy, P. S. M. M.; Reddy, D. R. Bioorg.
Med. Chem. Lett. 2004, 14, 2669–2672.
16. (a) Kamal, A.; Reddy, K. L.; Devaiah, V.; Shankaraiah,
N. Synlett 2004, 2533–2536; (b) Kamal, A.; Reddy, K.
L.; Devaiah, V.; Shankaraiah, N. Synlett 2004, 1841–
23. Analysis of PBD dimer 4b: 1H NMR (200 MHz, CDCl3) d
2.09–1.96 (m, 10H), 2.40–2.25 (m, 4H), 3.88–3.48 (m, 4H),
3.93 (s, 6H), 4.28–4.10 (m, 4H), 6.82 (s, 2H), 7.50 (s, 2H),
7.66 (d, J = 4.68 Hz, 2H); 13C NMR (50 MHz, CDCl3) d
24.2, 25.7, 29.6, 46.7, 53.7, 56.1, 65.4, 110.7, 111.6, 120.3,
140.6, 147.8, 150.6, 162.4, 164.6; FABMS, m/z for
C30H34N4O6 [M+H]+: 547.