Synthesis of fluorinated analogues of SJG-136 and their DNA-binding potential

Article abstract of DOI:10.1016/j.bmcl.2004.08.050

A series of C2-exo-fluorounsaturated pyrrolobenzodiazepines (PBDs) have been synthesized. These C2-exo-fluorounsaturated PBD dimers have exhibited remarkable DNA-binding affinity.

Full text of DOI:10.1016/j.bmcl.2004.08.050

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5699–5702
Synthesis of fluorinated analogues of SJG-136 and their
DNA-binding potential
Ahmed Kamal,a,* P. S. M. M. Reddy,a D. Rajasekhar Reddy,a E. Laxmana and
Y. L. N. Murthyb
aDivision of Organic Chemistry, Indian Institute of Chemical Technology, Hyderabad 500007, India
bDepartment of Organic Chemistry, Andhra University, Visakhapatnam 530003, India
Received 29 June 2004; accepted 9 August 2004
Available online 17 September 2004
Abstract—A series of C2-exo-fluorounsaturated pyrrolobenzodiazepines (PBDs) have been synthesized. These C2-exo-fluorounsatu-
rated PBD dimers have exhibited remarkable DNA-binding affinity.
Ó 2004 Elsevier Ltd. All rights reserved.
There is a growing interest in agents, such as pyr-
rolo[2,1-c][1,4]benzodiazepines (PBDs), that can recog-
nize and bond to specific sequences of DNA. They are
potential gene regulators with possible therapeutic
applications in the treatment of genetic disorders,
including some cancers, as selective antiinfective agents,
and as probes and tools for use in molecular biology.1
PBDs are a group of naturally occurring antitumor
antibiotics, members of which include anthramycin,
tomaymycin, neothramycins A and B, sibiromycin,
mazethramycin, chicamycin, prothracarcin, DC-81,
and dextochrysin.2 The PBDs interact within the minor
groove of DNA by forming covalent bond between their
elecrophilic C11-position and the exocyclic C2–NH2
moiety of a guanine residue.3 Bonding occurs in se-
quence specific fashion with a preference for PuGPu mo-
tifs. Typical examples of PBD natural products such as
DC-81 (1) and tomaymycin (2) have the same substitu-
tions in the aromatic A-ring, although 2 has an addi-
tional C2-exo unsaturation. In order to understand the
structure–activity relationship studies many synthetic
analogues have been prepared mainly with modifica-
tions and substitutions in the aromatic A-ring and
not much attention has been given to the C-ring
modifications.
A novel sequence selective PBD dimer (SJG-136, 5) has
been developed that comprises of C2-exo-methylene-
substituted DC-81 subunits tethered through their C8
positions via an inert propanedioxy linker that has
exhibited efficient minor groove interstrand cross-link-
ing property.4,5 This compound shows significant in vivo
anticancer activity and has been selected for clinical tri-
als.6 It is known in the literature that in case of PBD di-
mers, they span approximately six base pairs of DNA
and in which the sequence selectivity also increases
(e.g., purine–GATC–pyrimidine)7 in comparison to the
PBD monomers that have sequence selectivity for three
base pairs. Therefore, design and synthesis of symme-
trical dimers of PBD has been considered interesting
from the point of view of DNA interaction.
In the literature, a number of antiviral and antitu-
mor agents have been developed in which fluorine sub-
stitution has played a key role in their biological
activity. For example, the discovery of 20-difluoro mod-
ified gemcitabine exhibited promising biological activity
such as antitumor and antiviral activity, which is being
used clinically for the treatment of various solid tum-
ors.8,9 Therefore, the importance of fluorine substitu-
tion in pharmaceutical development is evident in a
large number of fluorinated compounds that have been
approved by the Food and Drug Administration
(FDA) as drugs.10,11 In view of this, it has been consid-
ered of interest to design molecules with fluorine substi-
tutions in the pyrrolo[2,1-c][1,4]benzodiazepine ring
system.
Keywords: C2-exo-fluorounsaturated pyrrolobenzodiazepine; DNA-
binding ability.
*
Corresponding author. Tel.: +91 40 27193157; fax: +91 40 27193189;
e-mail addresses: ahmedkamal@ins.iictnet.com; ahmedkamal@iict.
res.in
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.08.050

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A. Kamal et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5699–5702
10
11
9
N
N
N
HO
HO
HO
H
H
H
8
11a
1
5
N
N
CH3
F
N
H3CO
H3CO
H3CO
7
2
6
4
3
O
O
O
F
3
DC-81 (1)
Tomaymycin (2)
N
N
O
O
O
H
H
N
n
N
OCH3 H3CO
R
R
O
R = H, n = 1 (DSB-120, 4a)
R = F, n = 1-3 (4b)
N
N
O
O
H
H
H
N
N
OCH3 H3CO
O
O
SJG-136 (5)
N
O
O
N
H
(CH2)n
6a n = 3
6b n = 4
6c n = 5
N
F
OCH3
H3CO
N
F
O
O
F
F
Figure 1.
HO
HO
HO
ii
i
COOH
CO2CH3
CO2CH3
N
N
N
H
H HCl
BOC
7
8
9
iii
F
F
O
F
F
iv
v
CO2CH3
CO2CH3
CO2CH3
N
N
N
H
12
BOC
11
BOC
10
BnO
NO2
OH
vi
H3CO
NO2
O
BnO
NO2
BnO
CO2CH3
vii
ix
CHO
viii
N
H3CO
F
F
N
H3CO
F
O
O
14
F
13
NO2
BnO
NO2
HO
CH(SEt)2
CH(SEt)2
x
N
H3CO
F
F
N
H3CO
F
F
O
15
O
16
NH2
HO
CH(SEt)2
xi
3
N
H3CO
F
F
O
17
Scheme 1. Reagents and conditions: (i) SOCl2, MeOH; (ii) Boc2O, 2N NaOH, THF–H2O (2:1), rt; (iii) trichloroisocyanuric acid, Tempo, CH2Cl2;
(iv) CF2Br2, HMPT, Zn, THF; (v) TFA, CH2Cl2; (vi) (COCI)2, DMF, THF, Et3N, THF; (vii) DIBAL-H, CH2Cl2, ꢀ78°C, 45min, 80–85%; (viii)
EtSH–TMSCl, CHCl3, 24h, rt, 90%; (ix) EtSH–BF3OEt2, CH2Cl2, 12h, rt, 80%; (x) SnCl2Æ2H2O, CH3OH, reflux, 2h, 80–85%; (xi) HgCl2–CaCO3,
CH3CN–H2O, 12h, rt, 68–86%.

A. Kamal et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5699–5702
5701
Therefore, on the basis of this presumption and our
NO2
N
O
16
HO
CH(SEt)2
earlier efforts on the design and development of struc-
turally modified PBDs,12 we herein report the synthesis
of C2-exo-difluoromethylene analogue of compound 1
and fluorinated analogues of SJG-136 (6) to explore
their DNA-binding affinity in comparison with their
non-fluorinated counterparts. Moreover, we have
recently reported the synthesis of C2-fluorinated PBD
analogues (4b) and their DNA-binding potential13
(Fig. 1).
H3CO
F
F
i
NO2
O2N
N
O
O
CH(SEt)2
(EtS)2HC
(H2C)n
N
F
F
F
OCH3
H3CO
18a-c
The synthesis of C2-exo-difluoromethylene DC-81 (3)
has been carried out by employing commercially availa-
ble L-trans-4-hydroxyproline (7), which is N-protected
by Boc via its methyl ester to give compound 9 in quan-
titative yield. The oxidation of 2-hydroxy group of com-
pound 9 with trichloroisocyanuric acid and Tempo gives
the key precursor with C2-ketone (10) in excellent
yield.14 This has been converted to its C2-exo-fluoro-
methylene compound 11 employing dibromodifluoro-
methane in the presence of HMPT, Zn.15 This upon
deprotection of Boc provides the required precursor
methyl (2S)-4-difluoromethylidenepyrrolidine-2-carboxy-
late (12). The other precursor 4-benzyloxy-5-methoxy-
2-nitrobenzoic acid has been prepared by earlier
reported methods16 and is coupled to compound 12
via its acid chloride to give methyl (2S)-N-(4-benzyl-
oxy-5-methoxy-2-nitrobenzoyl)-4-difluoromethylidene-
pyrrolidine-2-carboxylate (13). Reduction by DIBAL-H
gives the corresponding aldehyde (14), which upon
EtSH protection followed by deprotection of benzyl
group by using EtSH–BF3OEt2 and subsequent reduc-
tion with tin chloride provides the key intermediate ami-
no thioacetal (17). This upon deprotective cyclization
affords the target C2-exo-difluoromethylene DC-81 (3)
as shown in Scheme 1.17
O
O
F
ii
NH2
H2N
N
O
O
CH(SEt)2
(EtS)2HC
(H2C)n
OCH3
N
O
F
F
F
F
H3CO
19a-c
O
iii
6a-c
n = 3,4 and 5
Scheme 2. Reagents and conditions: (i) Dibromoalkanes, K2CO3,
acetone, 36–48h, reflux, 89–91%; (ii) SnCl2Æ2H2O, CH3OH, reflux, 2h,
70–72%; (iii) HgCl2–CaCO3, CH3CN–H2O, 12h (or) Bi(OTf)3ÆxH2O,
CH2Cl2–H2O, 4h, rt.
DNA molar ratio 1:5. In case of C2-exo-difluoromethyl-
ene DC-81 (3) helix melting temperature marginally
increased after 18h of incubation in comparison to nat-
urally occurring DC-81. It is observed from the data one
of the fluoro substituted PBD dimer 6a stabilizes the
double-stranded CT-DNA in an efficient manner. This
compound with a three carbon alkane spacer elevates
The synthesis of C2/C20-exo-difluoromethylene PBD di-
mers has been carried out by the etherification of (2S)-
N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)-4-difluorome-
thylidenepyrrolidine-2-carboxaldehyde diethyl thio-
acetal (16) with dibromoalkanes to provide 18a–c.
These upon reduction with SnCl2Æ2H2O gave the corre-
sponding aminodiethyl thioacetals (19a–c). Deprotec-
tion of these amino thioacetals by usual literature
methods employing HgCl2–CaCO3 provided the re-
quired products 6a–c in 60–65% yields. However,
amino thioacetal functionality of 19a–c has also been
deprotected by employing bismuth triflate to afford
the desired C2/C20-exo-difluoromethylene PBD dimers
6a–c in 65–70% yields (Scheme 2).17 The use of cata-
lytic amount of bismuth triflate in biphasic system
for such deprotections has been earlier investigated
in our laboratory.18 It is interesting to note that this
procedure not only provides better yields with rela-
tively easier work up procedure but more importantly
the products are free from trace impurities of mercuric
salts.
Table 1. Thermal denaturation data for C2/C20-exo-fluoromethylene
PBDs with calf thymus (CT) DNA
Compounds [PBD]:[DNA] molar ratiob
DTm (°C)a after
incubation at 37°C
0h
18h
36h
3
1:5
1:5
1:5
1:5
1:5
1:5
1:5
1.0
1.7
2.2
6a
6b
6c
4a
5c
1
20.8 24.6 24.9
2.1 5.2 6.5
16.2 19.2 21.3
10.2 15.1 15.7
25.7 33.6
0.3
0.7
0.7
a For CT-DNA alone at pH7.00 0.01, Tm = 69.6°C 0.01 (mean
value from 10 separate determinations), all DTm values are 0.1–
0.2°C.
b For a 1:5 molar ratio of [PBD]/[DNA], where CT-DNA concentra-
tion = 100lM and ligand concentration = 20lM in aqueous sodium
phosphate buffer [10mM sodium phosphate + 1mM EDTA,
pH7.00 0.01].
The DNA-binding ability for these C2-exo-difluoro-
methylene PBDs and their dimers has been examined
by thermal denaturation studies using calf thymus
(CT) DNA. These studies were carried out at PBD/
c Literature values.5

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the helix melting temperature of the CT-DNA by a
remarkable 24.6°C after incubation at 37°C for 18h.
In the same experiment the DC-81 dimer (DSB-120)
gives a DTm of 15.1°C. On the other hand, DTm of com-
pound 6c having five carbon alkane spacer is 19.2°C
after incubation for 18h at 37°C. It is interesting to
observe that, both the PBD dimers 6a and 6c the DTm
values are higher when the length of alkyl chain spacers
is three and five but they are not as high as SJG-136 as
illustrated in Table 1. However, compound 6b with a
four carbon alkane spacer shows comparatively lower
DTm value (5.2°C) after 18h of incubation. Probably
because of unfavorable fit of this molecule within the
minor groove of host DNA duplex. Therefore, it
appears that the linker length in these dimers is probably
modulating the DNA reactivity potential.
In summary, new C2-exo-difluoromethylene DC-81 and
its dimers have been synthesized that exhibit remarkable
DNA-binding ability. The detailed mechanistic and
molecular modeling studies for these fluorinated PBDs
are in progress.
Acknowledgements
One of the authors P.S.M.M.R. is grateful to CSIR,
New Delhifor the award of research fellowshpi .
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