Synthesis of the first examples of A-C8/C-C2 amide-Linked pyrrolo[2,1-c][1,4]benzodiazepine dimers

Article abstract of DOI:10.1016/S0960-894X(03)00436-0

We report the synthesis of novel A-C8/C-C2 amide-linked pyrrolo[2,1-c][1,4]benzodiazepine dimers (4a and 4b) via convergent routes. These compounds lack the potent DNA interstrand cross-linking ability and resultant pronounced cytotoxicity of the known A-

Full text of DOI:10.1016/S0960-894X(03)00436-0

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2277–2280
Synthesis of the First Examples of A-C8/C-C2 Amide-Linked
Pyrrolo[2,1-c][1,4]benzodiazepine Dimers
Stephen J. Gregson, Philip W. Howard and David E. Thurston*
Cancer Research UK Gene Targeted Drug Design Research Group, The School of Pharmacy,
University of London, 29/39 Brunswick Square, London WC1N 1AX, UK
Received 10 February 2003; accepted 28 April 2003
Abstract—We report the synthesis of novel A-C8/C-C2 amide-linked pyrrolo[2,1-c][1,4]benzodiazepine dimers (4a and 4b) via
convergent routes. These compounds lack the potent DNA interstrand cross-linking ability and resultant pronounced cytotoxicity
of the known A-C8/A-C80 linked dimers (e.g., 2a–b).
# 2003 Elsevier Science Ltd. All rights reserved.
The pyrrolobenzodiazepines are a family of tricyclic
antitumour antibiotics that interact in the minor groove
of DNA forming monocovalent adducts.1ꢀ4 The first
PBD dimer (1) comprising two unsubstituted PBD units
joined through their A-C7/A-C70 positions was reported
by Suggs and co-workers in 19885,6 (Fig. 1). Dimers with
this linkage had only weak DNA cross-linking activity
and no cytotoxicity data were reported. The first dimer
with an A-C8/A-C80 linkage (2a) was reported7ꢀ9 in 1992.
Dimers of this type have significant DNA interstrand
cross-linking activity, and pronounced in vitro cytotoxi-
city and in vivo antitumour activity. One example (2b,
SJG-136)10,11 has been selected for clinical evaluation.
More recently, Lown and co-workers reported12 three
C-C2/C-C20 linked dimers (3a–c). These are less cyto-
toxic than the A-C8/A-C80 dimers but their cross-linking
efficiency has not been reported. The examples cited
above spurred us to explore the C-C2/A-C80 linkage by
targeting the novel dimers 4a and 4b to obtain further
SAR data and to potentially allow the synthesis of
longer PBD oligomers by coupling a series of PBD units
together in this orientation.
In order to construct prototype C–A amide-linked PBD
dimers of type 4, it was first necessary to synthesise the
C8-amino and C2-methylenecarboxy PBD building
Figure 1.
*Corresponding author: Tel.: +44-207-753-5932; fax: +44-207-753-5935; e-mail: david.thurston@ulsop.ac.uk
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00436-0

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S. J. Gregson et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2277–2280
blocks. Synthesis of the right-hand portion of the C7-des-
methoxy dimer 4a began by N-protecting commercially
available 4-nitroanthranilic acid 5 as its allyl carba-
mate 6 (Scheme 1). This was coupled without further
purification to (S)-(+)-2-pyrrolidinemethanol to pro-
vide 7 which was reduced using SnCl2 in refluxing
methanol. The resulting aniline 8 was then N-Fmoc
protected in good yield using 9-fluorenylmethyl chloro-
formate in the presence of aqueous sodium carbonate
and THF. The orthogonal dicarbamate 9 was sub-
jected to Swern conditions to provoke B-ring cyclisa-
tion in excellent yield. Treatment of the cyclised
compound 10 with N,N-dimethylamine in MeOH
removed the Fmoc protecting group to provide the
key C7-des-methoxyaniline 11.
The aniline coupling partner (18) for the C7-methoxy
dimer 4b was synthesised via a similar route. Known
Scheme 1. (a) AllocCl, pyridine, CH2Cl2, 0 ꢁC, 75%; (b) TBTU, (S)-(+)-2-pyrrolidinemethanol, DIPEA, DMF, 40%; (c) SnCl2.H2O, MeOH, Á,
74%; (d) FmocCl, aq Na2CO3, THF, 0 ꢁC, 76% (e) (COCl)2, DMSO, TEA, CH2Cl2, ꢀ45 ꢁC, 76%; (f) HNMe2, MeOH, 80%.
Scheme 2. (a) f-HNO3, 0 ꢁC, 90%; (b) DCC, HOBt, CH2Cl2, 0 ꢁC then (S)-(+)-2-pyrrolidinemethanol, CH2Cl2, ꢀ20ꢁC, 78%; (c) H2, 45 psi, 10 % Pd/C,
EtOH, quant; (d) AllocCl, pyridine, CH2Cl2, 0 ꢁC, 81%; (e) pyridinium dichromate, 4 A sieves, CH2Cl2, 51%; (f) aq K2CO3, MeOH, CH2Cl2, 70 %.

S. J. Gregson et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2277–2280
2279
Scheme 3. (a) LiOH.H2O, MeOH, H2O, 0 ꢁC, 89%; (b) DIC, HOBT, CH2Cl2, 0 ꢁC then 11 or 18, CH2Cl2, 40 % 2( 1a), 67% (21b); (c) Pd(PPh3)4,
PPh3, pyrrolidine, CH2Cl2, 70 % (4a), 77% (4b).
trifluoroacetylamino benzoic acid 1213 was nitrated in
high yield using fresh f-HNO3 at 0 ꢁC (maxm 3 g scale; 5
min reaction time) to provide nitro acid 13 (Scheme 2).
This was coupled to (S)-(+)-2-pyrrolidinemethanol
using DCC/HOBt to provide the amide 14 in good
yield. Reduction of the nitro group furnished aniline 15
which was subsequently N-protected (16) upon treat-
ment with allyl chloroformate in the presence of pyri-
dine. The best yield for oxidation of 16 was achieved
using pyridinium dichromate to give the N10-Alloc
protected PBD 17 in 51% yield. Key C7-methoxy ani-
line 18 was obtained in high yield after cleavage of the
trifluoroacetlyl protecting group.
molecules of type 4a and 4b compared to 1, 2 and 3,
they may be forced into a non-isohelical conformation
not conductive to binding in the DNA minor groove.
To investigate this possibility we are currently synthe-
sising C-C2/A-C8 linked dimers where the nitrogen
moiety of the amide group is separated from the A-ring
by an alkoxy chain, thus allowing more flexibility
between the PBD units.
Acknowledgements
This work was supported by Cancer Research UK
(Grant numbers: C180/A1060 [previously SP1938/0402]
and SP1938/0401 to D.E.T.). Professor John Hartley
and Ms. Anzu Hamaguchi (UCL) and Dr. Lloyd Kel-
land (previously at the Institute for Cancer Research,
Sutton, UK) are thanked for evaluating cross-linking
activity and cytotoxicity, respectively.
The PBD C2-tethered acid 20 is common to both dimers
and was synthesised by hydrolysing known PBD-ester
1914 in good yield (Scheme 3). The Alloc protected C–A
linked PBD dimers were synthesised in moderate (40%
for C7-des-methoxy 21a) and good (67% for C7-meth-
oxy 21b) yields. Although several coupling methods
were attempted for 21a, the yield could not be improved
upon. Treatment of 21a–b with palladium(0) in the pre-
sence of pyrrolidine cleaved the Alloc protecting groups
to give the novel N10–C11 imine PBD dimers 4a15 and
4b.16
References and Notes
1. Thurston, D. E. Advances in the Study of Pyrrolo[2,1-c]-
[1,4]benzodiazepine (PBD) Antitumour Antibiotics. In Mole-
cular Aspects of Anticancer Drug-DNA Interactions; Neidle, S.,
Waring, M. J, Eds.; Macmillan: London, UK, 1993; Vol. 1, p
54.
2. Thurston, D. E.; Bose, D. S. Chem. Rev. 1994, 94, 433.
3. Thurston, D. E. Br. J. Cancer 1999, 80, 65.
4. Kamal, A.; Rao, M. V.; Laxman, N.; Ramesh, G.; Reddy,
G. S. K. Curr. Med. Chem. Anti-Cancer Agents 2002, 2, 215.
5. Farmer, J. D.; Rudnicki, S. M.; Suggs, J. W. Tetrahedron
Lett. 1988, 29, 5105.
4a and 4b were evaluated for in vitro cytotoxicity and
DNA cross-linking ability. Neither molecule demon-
strated significant cytotoxicity in a number of human
tumour cell lines (e.g., 4a: IC50>25 mM in A2780,
A2780cis, CH1, CH1cis and SKOV-3). Similarly, both
molecules are poor cross-linking agents (e.g., 4a
approximately 200-fold less efficient than 2b). These
observations suggest that the A-C8/A-C80 linkage is
optimal for DNA cross-linking and cytotoxicity com-
pared to A-C7/A-C70, C-C2/C-C20 or C-C2/A-C8 lin-
kages. Although the C-C2/A-C8 linkage of 4a and 4b
may be inherently sub-optimal for DNA interaction,
preliminary modelling studies have suggested an alter-
native explanation. Due to the relatively short linker in
6. Farmer, J. D.; Gustafson, G. R.; Conti, A.; Zimmt, M. B.;
Suggs, J. W. Nucleic Acids Res. 1991, 19, 899.
7. Bose, D. S.; Thompson, A. S.; Ching, J. S.; Hartley, J. A.;
Berardini, M. D.; Jenkins, T. C.; Neidle, S.; Hurley, L. H.;
Thurston, D. E. J. Amer. Chem. Soc. 1992, 114, 4939.
8. Bose, D. S.; Thompson, A. S.; Smellie, M.; Berardini,
M. D.; Hartley, J. A.; Jenkins, T. C.; Neidle, S.; Thurston,
D. E. Chem. Commun. 1992, 1518.

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S. J. Gregson et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2277–2280
9. 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.
10. Gregson, S. J.; Howard, P. W.; Jenkins, T. C.; Kelland,
L. R.; Thurston, D. E. Chem. Commun. 1999, 797.
11. Gregson, S. J.; Howard, P. W.; Hartley, J. A.; Brooks,
N. A.; Adams, L. J.; Jenkins, T. C.; Kelland, L. R.; Thurston,
D. E. J. Med. Chem. 2001, 44, 737.
126.3, 119.6, 118.8, 117.9, 111.4, 109.9, 56.2, 54.0, 53.8, 46.7,
37.5, 37.2, 29.6, 24.2; MS (FAB), m/z (relative intensity) 514
(M+.+H, 97), 279 (43), 271 (19), 216 (23), 192 (53), 180 (100),
149 (21), 135 (17), 112 (28), 91 (59), 80(36), 73 (91), 57 (80);
IR (NUJOL) 3315 (br), 2924, 2854, 1682, 1624, 1598, 1511,
1455, 1378, 1246, 1214, 1128, 1071, 1010, 965, 872, 827, 722,
665 cmꢀ1; HRMS [M+H]+. calcd for C28H28N5O5 m/z
514.2090, Found (FAB) m/z 514.2096; [a]2D0=+624.0( c 0.03,
CHCl3).
12. Reddy, B. S. P.; Damayanthi, Y.; Reddy, B. S. N.; Lown,
J. W. Anti-Cancer Drug Des. 2000, 15, 225.
1
16. Data for 4b: H NMR (250MHz, CDCl3): d 8.28 (s, 1H),
13. Jones, P.; Villeneuve, G. B.; Fei, C. P.; DeMarte, J.; Hag-
garty, A. J.; Nwe, K. T.; Martin, D. A.; Lebuis, A. M.; Fin-
kelstein, J. M.; Gour-Salin, B. J.; Chan, T. H.; Leyland-Jones,
B. R. J. Med. Chem. 1998, 41, 3062.
7.86 (d, 1H, J=3.94 Hz), 7.69 (d, 1H, J=4.46 Hz), 7.53 (s, 1H),
7.50(s, 1H), 7.05 (s, 1H), 6.82 (s, 1H), 4.61–4.45 (m, 1H), 4.38–
4.21 (m, 1H), 3.95–3.78 (m, 9H), 3.60–3.48 (m, 2H), 3.34–3.00
(m, 4H), 2.37–2.18 (m, 2H), 2.10–1.78 (m, 2H); 13C NMR
(62.9MHz, CDCl3): d 167.2, 164.4, 162.5, 161.4, 151.9, 147.8,
146.1, 140.6, 140.4, 129.9, 127.1, 122.9, 118.9, 117.9, 111.5, 110.3,
109.8, 56.2, 56.1, 53.9, 53.6, 46.7, 37.6, 37.4, 29.6, 24.1; MS
(FAB), m/z (relative intensity) 544 (M+.+H., 100), 413 (83), 391
(89), 329 (54), 307 (91), 289 (81), 272 (77), 246 (56); IR (CHCl3)
3329 (br), 3011, 2930, 2867, 1725, 1691, 1602, 1511, 1454, 1434,
14. Gregson, S. J.; Howard, P. W.; Barcella, S.; Nakamya, A.;
Jenkins, T. C.; Kelland, L. R.; Thurston, D. E. Bioorg. Med.
Chem. Lett. 2000, 10, 1849.
1
15. Data for 4a: H NMR (400 MHz, CDCl3): d 7.89 (d, 1H,
J=8.24 Hz), 7.78–7.44 (m, 3H), 7.40–7.25 (m, 2H), 6.91 (s,
1H), 6.77 (s, 1H), 4.28–4.10(m, 1H), 3.89 and 3.85 (s ꢂ2, 6H),
3.83–3.60(m, 2H), 3.55–3.46 (m, 1H), 3.34–3.10(m, 4H),
2.36–1.60(m, 4H); 13C NMR (100.5 MHz, CDCl3): d 168.4,
164.8, 162.9, 161.5, 152.0, 147.8, 146.7, 141.2, 140.6, 131.2,
1387, 1343, 1264, 1215, 1128, 1073, 1019, 969, 874, 664 cmꢀ1
;
HRMS [M+H]+. calcd for C29H30N5O6 m/z 544.2196, Found
(FAB) m/z 544.2209; [a]2D3=+405.9 (c 0.10, CHCl3).