The effect of C2-fluoro group on the biological activity of DC-81 and its dimers

Article abstract of DOI:10.1016/j.bmcl.2004.02.063

C2-Fluoro substituted pyrrolobenzodiazepines (PBDs) have been synthesized that exhibit potential anticancer activity in a number of human tumour cell lines. These C2-fluoro substituted PBDs also exhibit significant DNA-binding ability.

Full text of DOI:10.1016/j.bmcl.2004.02.063

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2669–2672
The effect of C2-fluoro group on the biological activity of DC-81
and its dimers
Ahmed Kamal,* P. S. M. M. Reddy and D. Rajasekhar Reddy
Biotransformation Laboratory, Division of Organic Chemistry, Indian Institute of Chemical Technology, Hyderabad 500007, India
Received 27 October 2003; accepted 10 February 2004
Abstract—C2-Fluoro substituted pyrrolobenzodiazepines (PBDs) have been synthesized that exhibit potential anticancer activity in
a number of human tumour cell lines. These C2-fluoro substituted PBDs also exhibit significant DNA-binding ability.
Ó 2004 Elsevier Ltd. All rights reserved.
10
N
11a
5
A number of pyrrolo[2,1-c][1,4]benzodiazepines (PBDs)
possessing antibiotic and antitumour activities have
been isolated from Streptomyces species.1 Later exten-
sive studies have been devoted towards the structural
modifications of the ring system particularly by linking
this ring system to other DNA-interactive moieties2
apart from the synthesis of different type of dimers of
this class of compounds.3 Several naturally occurring
PBDs that have been isolated exhibit remarkable cyto-
toxicity particularly with substitutions in the C-ring.4 In
order to understand the structure–activity relationship
studies many synthetic analogues have been prepared
mainly with modifications and substitutions in the aro-
matic A-ring and not much attention has been given to
the C-ring modifications. Recently, C2-aryl substituted
PBDs have been prepared that have exhibited selective
cytotoxicity at the sub-nanomolar level towards mela-
noma and ovarian cancer cell lines.5 Furthermore, the
discovery of the antimetabolic properties of fluoroacetic
acid6 and fluorouracil7 initiated interest incorporating
fluorine into other structures for exploring the effect on
the anticancer properties such as in case of combre-
tastatin A-4.8 There are also some recent reports in the
literature wherein fluorinated analogues have improved
the biological activity profile of some pharmacologically
important compounds.9;10
9
6
11
8
7
N
HO
RO
H
H
1
N
N
H3CO
H3CO
2
4
F
3
O
1
O
2 a. R = H; 2-F (α)
b. R = benzyl;2-F (α)
c. R= H; 2-F (β)
N
O
N
H
H
O
N
OCH3
N
O
H3CO
O
3
N
O
O
N
H
O
H
(CH2)n
N
OCH3
N
H3CO
4 a-c
F
F
O
where n = 3, 4, 5
Our interest in the design and synthesis of PBDs has led
to the development of some new PBD hybrids with
potential antitumour activity. In continuation of these
efforts, it has been considered of interest to prepare C2-
fluoro substituted DC-81 and its dimers. During the
course of this work a report has appeared on the syn-
thesis of C2-fluorinated A-ring unsubstituted PBDs with
some enhancement in cytotoxicity and marginal increase
of DTm values in comparison to DC-81.11
The preparation of these C2-fluoro substituted PBDs
has been carried out by employing (2S)-N-[4-benzyloxy-
5-methoxy-2-nitrobenzoyl]-4-fluoropyrrolidine-2-carb-
oxylate 6 as one of the starting material and this has
Keywords: C2-Fluoropyrrolobenzodiazepines; Cytotoxicity and DNA-
binding affinity.
* Corresponding author. Tel.: +91-40-27193157; fax: +91-40-271931-
89; e-mail: ahmedkamal@iict.ap.nic.in
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.02.063

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A. Kamal et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2669–2672
been prepared by literature method,12 which upon de-
NO2
NO2
BnO
BnO
CO2CH3
OH
CO2CH3
F
benzylation gives 9. Further, these upon reduction and
followed by deprotection of aminothioacetal precursors
(10 and 11) afford the target C2-fluoro substituted PBDs
2a–b as shown in Scheme 1.13
i
N
N
H3CO
H3CO
O
O
6
5
ii
The synthesis of the dimers has been carried out by the
etherification of (2S)-N-(4-hydroxy-5-methoxy-2-nitro-
benzoyl)-4-fluoropyrrolidine-2-carboxaldehyde diethyl
thioacetal 9 with dibromoalkanes to provide 12a–c.
Further, these upon reduction and followed by depro-
tection of aminothioacetal precursors (13a–c) afford the
desired C2-fluorinated PBD dimers 4a–c in good yields
(Scheme 2).13
NO2
NO2
BnO
BnO
CHO
CH(SEt)2
F
iii
N
H3CO
N
H3CO
F
O
7
O
8
iv
v
Representative compounds 2a, 2b, 4a and 4c (Table 1)
have been evaluated for the primary anticancer activity
in the standard three-cell line panel consisting of the
NCI-H460 (lung), MCF7 (breast), SF-268 (CNS). These
fluorinated PBDs were also evaluated for in vitro anti-
cancer activity in human 60-cell line panel. Amongst
these 2b and 4c have exhibited promising anticancer
activity. The GI50 value of compounds 2b and 4c (Table
2) against leukemia cancer (CCRF-CEM and SR) and
renal (UO-31) cell lines is <0.01 lM whereas in CNS
cancer panel in which SF-268, SF-295 cell lines affected,
GI50 values are 0.01, <0.01, 0.03 and <0.01 lM,
respectively. Compounds 2a, 2b and 4c exhibited cyto-
toxic potency against breast cancer cell line HS-578T
with GI50 values of 0.01 and <0.01 lM. The in vitro
cytotoxicity (IC50) for the naturally occurring DC-8114 is
0.38, 0.33 and 0.1 lM whereas for DC-81 dimer (DSB-
120)14 is 0.01, 0.0005 and 0.003 lM in L1210, PC6 and
CH1 cell lines, respectively. The in vitro cytotoxicity
(IC50) reported11 for A-ring unsubstituted 2a-fluoro-
PBD is 1.1, 0.22 and 1.9 lM in A2780, CH1 and L1210
cell lines, respectively, whereas for 2b-fluoro-PBD it is
0.43, 0.076 and 0.64 lM in A2780, CH1 and L1210 cell
lines.
NH2
BnO
CH(SEt)2
F
NO2
H3CO
N
HO
CH(SEt)2
O
H3CO
N
O
11
F
9
vi
2 b
v
NH2
HO
CH(SEt)2
F
H3CO
N
O
10
vi
2 a
Scheme 1. Reagents and conditions: (i) DAST, CH2Cl2, )78 °C, 12 h,
rt, 85%; (ii) DIBAL-H, CH2Cl2, )78 °C, 45 min, 80–85%; (iii) EtSH–
TMSCl, CHCl3, 24 h, rt, 90%; (iv) EtSH–BF3OEt2, CH2Cl2, 12 h, rt,
75%; (v) SnCl2Æ2H2O, CH3OH, reflux, 2 h, 80–85%; (vi) HgCl2–
CaCO3, CH3CN–H2O, 12 h, rt, 68–86%.
been obtained by the treatment of the (2S)-N-(4-benzyl-
oxy-5-methoxy-2-nitrobenzoyl)-4-hydroxypyrrolidine-2-
carboxylate 5 by DAST in CH2Cl2 (Scheme 1). Whereas,
(2S)-N-(4-benzyloxy-5-methoxy-2-nitrobenzoyl)-4-fluoro-
pyrrolidine-2-carboxaldehyde diethyl thioacetal 8 has
The DNA-binding ability of these new C2-fluorinated
PBDs and their dimers have been investigated by ther-
mal denaturation studies using calf thymus (CT) DNA
NO2
HO
O2N
N
O
NO2
O
CH(SEt)2
F
CH(SEt)2
F
(EtS)2HC
F
(H2C)n
i
H3CO
N
OCH3
N
H3CO
O
9
O
O
12
ii
H2N
O
NH2
O
CH(SEt)2
(EtS)2HC
F
(H2C)n
N
OCH3
N
H3CO
F
O
13
O
iii
4 a-c
Scheme 2. Reagents and conditions: (i) dibromoalkanes, K2CO3, acetone, 36–48 h, reflux, 89–91%; (ii) SnCl2Æ2H2O, CH3OH, reflux, 2 h, 70–72%;
(iii) HgCl2–CaCO3, CH3CN–H2O, 12 h, rt, 65–70%.

A. Kamal et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2669–2672
2671
Table 1. In vitro one dose primary anticancer assaya of C2-fluorinated
PBDs
Table 3. Thermal denaturation data for fluorinated PBDs with
CT-DNA
PBD
Growth percentages
Compounds [PBD]:[DNA]
molar ratiob
DTm (°C)a after incubation at
37 °C for
(Lung)
NCI-H460
(Breast)
MCF7
(CNS)
SF-268
0 h
18 h
36 h
2a
2b
4a
4c
0
1
0
0
0
0
0
0
0
19
0
2a
2b
2c
4a
4b
4c
3
1:5
1:5
1:5
1:5
1:5
1:5
1:5
1:5
0.3
1.0
2.0
2.2
2.5
1.1
0.6
1.5
4.9
2.25
6.16
13.8
17.4
0
3.1
4.6
12.3
16.0
15.4
0.7
a One dose of 2a, 2b, 4a and 4c at 10ꢀ4 M concentration.
14.2
10.2
0.3
1
a For CT-DNA alone at pH 7.00 0.01, Tm ¼ 69:8 °C 0.01 (mean
value from 10 separate determinations), all DTm values are 0.1–
0.2 °C.
Table 2. In vitro cytotoxicity of compounds 2a, 2b, 4a and 4c in
selected human cancer cell lines
Cancer panel/cell
line
GI50 (lM)
b For a 1:5 molar ratio of [PBD]/[DNA], where CT-DNA concentra-
tion ¼ 100 lM and ligand concentration ¼ 20 lM in aqueous sodium
phosphate buffer (10 mM sodium phosphate+1 mM EDTA,
pH 7.00 0.01).
2a
2b
4a
4c
Leukemia
CCRF-CEM
SR
0.30
0.20
<0.01
<0.01
––
1.54
<0.01
<0.01
molecular modeling studies for these fluorinated PBDs
are in progress and further the effect of fluorination for
the C2/C20-exo unsaturated PBDs is also under investi-
gation.
Colon
SW-620
1.19
0.41
<0.01
<0.01
4.2
0.02
Non-small cell
lung
NCI-H522
1.88
<0.01
CNS
Acknowledgements
SF-268
SF-295
0.35
0.32
0.01
0.03
2.2
1.96
<0.01
<0.01
We thank the National Cancer Institute, Maryland,
USA for the in vitro anticancer assay in human cancer
cell lines. One of the authors P.S.M.M.R. is grateful to
CSIR, New Delhi for the award of research fellowship.
Renal
UO-31
0.17
0.01
<0.01
<0.01
2.40
––
<0.01
<0.01
Breast
HS-578T
References and notes
at pH 7.0, incubated at 37 °C. In case of C2a-fluorinated
DC-81 (2a) the helix melting temperature has marginally
increased after 18 h of incubation in comparison to
naturally occurring DC-81. The C8-benzylated C2-
fluorinated DC-81 (2b) exhibited further enhancement in
the melting temperatures. Similarly, C2b-fluorinated
DC-81 (2c) exhibited slightly higher DNA melting
temperatures compared to the a-isomer. On the other
hand in case of C2-fluorinated dimers it is interesting to
observe that C2-fluorinated dimer (4a) shows lower
DNA melting temperatures compared to DC-81 dimer
(3), while as the linker length increases from 3 to 5 the
helix melting temperature of CT-DNA increases to
16 °C after incubation of 18 h for compound 4c as
shown in Table 3. It is well known in case of pyr-
rolobenzodiazepine compounds that substitution in A-
ring have often exhibited higher DNA melting and in
vitro antitumour activity, and this is true in case of
present study when compared to the report on C2-
fluorinated A-ring unsubstituted PBDs.11
1. Kamal, A.; Rao, M. V.; Laxman, N.; Ramesh, G.; Reddy,
G. S. K. Curr. Med. Chem.––Anti-Cancer Agents 2002, 2,
215.
2. (a) Kamal, A.; Ramesh, G.; Ramulu, P.; Srinivas, O.;
Rehana, T.; Sheelu, G. Bioorg. Med. Chem. Lett. 2003, 13,
3451; (b) Kamal, A.; Ramulu, P.; Srinivas, O.; Ramesh, G.
Bioorg. Med. Chem. Lett. 2003, 13, 3517; (c) Kamal, A.;
Srinivas, O.; Ramulu, P.; Ramesh, G.; Kumar, P. P.
Bioorg. Med. Chem. Lett. 2003, 13, 3577; (d) Zhou, Q.;
Duan, W.; Simmons, D.; Shayo, Y.; Raymond, M. A.;
Dorr, R. T.; Hurley, L. H. J. Am. Chem. Soc. 2001, 123,
4865; (e) Tercel, M.; Stribbling, S. M.; Sheppard, H.; Siim,
B. G.; Wu, K.; Pullen, S. M.; Botting, K. J.; Wilson, W.
R.; Denny, W. A. J. Med. Chem. 2003, 46, 2132; (f)
Reddy, B. S. P.; Damayanthi, Y.; Reddy, B. S. N.; Lown,
W. J. Anti-Cancer Drug Des. 2000, 15, 225; (g) Kamal, A.;
Laxman, N.; Ramesh, G.; Srinivas, O.; Ramulu, P. Bioorg.
Med. Chem. Lett. 2002, 12, 1917; (h) Kamal, A.; Reddy, B.
S. N.; Reddy, G. S. K.; Ramesh, G. Bioorg. Med. Chem.
Lett. 2002, 12, 1933.
3. (a) 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; (b)
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; (c) Kamal, A.;
Ramulu, P.; Srinivas, O.; Ramesh, G. Bioorg. Med. Chem.
Lett. 2003, 13, 3955.
In summary new C2-fluorinated DC-81 and its dimers
have been synthesized that exhibits significant DNA-
binding ability. Moreover, these new fluorinated com-
pounds possess in vitro anticancer activity in a number
of human cancer cell lines. The detailed mechanistic and

2672
A. Kamal et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2669–2672
4. (a) Tozuka, Z.; Takasugi, H.; Takaya, T. J. Antibiot. 1983,
36, 276; (b) Tozuka, Z.; Yazawa, H.; Murata, M.; Takaya,
T. J. Antibiot. 1983, 36, 1699.
5. (a) Cooper, N.; Hagan, D. R.; Tiberghein, A.; Ademefun,
T.; Matthews, C. S.; Howard, P. W.; Thurston, D. E.
Chem. Commun. 2002, 1764; (b) Kang, G.-D.; Howard, P.
W.; Thurston, D. E. Chem. Commun. 2003, 1688.
6. Peters, R. Endeavour 1954, 13, 147.
7. Duschinsky, R.; Pleven, E.; Heidelberger, C. J. Am. Chem.
Soc. 1957, 79, 4559.
8. Lawrence, N. J.; Hepworth, L. A.; Rennison, D.;
McGown, A. T.; Hadfield, J. A. J. Fluorine Chem. 2003,
123, 101.
Tertyshnikova, S.; Weaver, D.; Yeola, S.; Zoeckler, M.;
Sinz, M. W. J. Med. Chem. 2003, 46, 3778.
11. OꢀNeil, I. A.; Thompson, S.; Kalindjian, S. B.; Jenkins,
T. C. Tetrahedron Lett. 2003, 44, 7809.
12. Thurston, D. E.; Murty, V. S.; Langley, D. R.; Jones,
G. B. Synthesis 1990, 81.
13. Spectral data for compound 2a: 1H NMR (CDCl3) d 2.58–
2.76 (m, 2H), 3.56 (m, 3H), 3.98 (s, 3H), 5.35 (dt, 1H,
J ¼ 53:0, 5.2 Hz), 6.95 (s, 1H), 7.56 (s, 1H), 7.82 (d, 1H,
1
J ¼ 4:2 Hz); MS 264 (M+H)þÅ. Compound 4c: H NMR
(CDCl3) d 1.58–1.81 (m, 4H), 1.90–2.01 (m, 2H), 2.38–2.50
(m, 4H), 3.08–3.24 (m, 6H), 3.95 (s, 6H), 4.01–4.20
(m, 4H), 5.39 (dt, 2H, J ¼ 52:5, 5.3 Hz), 6.81 (s, 2H),
7.49 (s, 2H), 7.83 (d, 2H, J ¼ 4:4 Hz); FABMS 596
(M+H)þ.
9. Ming Pu, Y.; Torok, D. S.; Ziffer, H. J. Med. Chem. 1995,
38, 4120.
10. Wu, Y.-J.; Davis, C. D.; Dworetzky, S.; Fitzpatrick, W.
C.; Harden, D.; He, H.; Knox, R. J.; Newton, A. E.;
Philip, T.; Polson, C.; Sivarao, D. V.; Sun, L.-Q.;
14. 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.