Design and efficient synthesis of novel DNA interstrand cross-linking agents: C2-linked pyrrolo[2,1-c][1,4]benzodiazepine dimers

Article abstract of DOI:10.1055/s-1999-2769

The design and facile synthesis of C2-linked pyrrolo[2,1-c][1,4]benzodiazepines (1a-c) are described. The compounds are prepared with varying degrees of linker length in order to probe the structural requirements for optimal DNA interstrand cross-linking.

Full text of DOI:10.1055/s-1999-2769

1112
LETTER
Design and Efficient Synthesis of Novel DNA Interstrand Cross-Linking
Agents: C2-Linked Pyrrolo[2,1-c][1,4]benzodiazepine Dimers
B. S. Praveen Reddy, Yalamati Damayanthi, J. William Lown*
Department of Chemistry, University of Alberta, Edmonton, AB, Canada T6G 2G2
Fax +1 (780)-492-8231; E-mail: annabelle.wiseman@ualberta.ca
Received 13 April 1999
terstrand cross-link. They showed that the tomaymycin
Abstract: The design and facile synthesis of C2-linked pyrrolo[2,1-
tail is close to the floor of the minor groove, while the five
c][1,4]benzodiazepines (1a-c) are described. The compounds are
membered ring of DSB-120 is more shallowly immersed,
perhaps due to strain from cross-linking with a very short
prepared with varying degrees of linker length in order to probe the
structural requirements for optimal DNA interstrand cross-linking.
The products formed are exclusively of the E-configuration and this linker unit. These results prompted us to design the syn-
is the first report on the synthesis of these kind of dimers.
thesis of tail to tail dimers of the PBD molecule, in order
to probe and optimize the selectivity and DNA binding ef-
ficiency. Considerable literature is available on various
PBD conjugates and head to head dimers but this is the
first report on synthesis of tail to tail PBD dimers. As part
of our continuing efforts on the studies of synthetic DNA
minor groove binding agents bearing more than one type
of reactive center, we recently synthesized a novel type of
lexitropsin-pyrrolobenzodiazepine hybrids,7 to study their
sequence selectivity and binding efficiency. Now we
herein report the efficient synthesis of bifunctional DNA
alkylating C2-linked PBD dimers in order to probe the
DNA cross-linking efficiency and structural requirements
for the optimum interstrand cross-linking as well as cyto-
toxicity.
Key words: minor groove binders, pyrrolo[2,1-c][1,4]benzodiaz-
epine, lexitropsins, cross-linking agent, bisalkylators
There has been increasing interest in the synthesis of
DNA-sequence selective agents for the last decade. It has
been observed that an increasing range of DNA sequences
may be recognized by small molecules which have DNA
binding properties including pyrrolo[2,1-c][1,4]benzodi-
azepines (PBDs), and lexitropsins. PBDs, are a group of
naturally occuring antibiotics, examples of which include
anthramycin, tomaymycin, sibiromycin and the neothra-
mycins A and B (Fig. 1).1 A key feature of these mole-
cules with respect to their mechanism of action is the N10-
C11 carbinolamine (or imine equivalent). Nucleophilic at-
tack by the 2-NH2 of a guanine base at the C11 position of
PBD forms a covalent adduct in the minor groove of
DNA.1 Furthermore, the PBDs bind to DNA sequence-
selectively2 and have potential not only as antitumor
agents but as gene regulators and probes of DNA struc-
ture.3
Because of the unreactive nature of the hydroxy group at
the C2 position in the PBD moiety, it is very difficult to
functionalize at that position in contrast to the case of
DSB-120.8 That is possibly why no attempt has been
found in the literature to prepare C2 linked dimers by an
ether linkage. We designed instead the synthesis of C2-
linked dimers to proceed via peptide bond formation. This
versatile approach makes the synthesis straightforward
and convenient. Compounds, (1a-c) were made according
to the routes described in schemes 1 and 2. The precursor
PBD-acid was synthesized according to scheme 1. The
coupling of 2-nitrobenzoic acid (2) via its acid chloride
with the methyl-(2S)-trans-4-hydroxycarboxylate ester
hydrochloride (3) gave methyl-(2S)-N-(2-nitrobenzoyl)-
4-hydroxypyrrolidine-2-carboxylate (4) in 75-80% yield.
Reduction of nitroester (4) with DIBAL-H to produce the
corresponding aldehyde, followed by protection of the al-
dehydic group with ethanethiol to give rise to the corre-
sponding diethylthioacetal compound (5). Several
unsuccessful attempts were made to oxidize the C-2 hy-
droxy group with various oxidizing agents including pyri-
dinium chlorochromate and Jones reagent. In all these
attempts, the yield of the isolated product was less than
10%. The Swern oxidation with DMSO, oxalylchloride,
and Et3N afforded no better results. Finally, satisfactory
results were obtained with the reagent DMSO/Ac2O. With
this reagent, the oxidation of C2-hydroxy group was
achieved under mild conditions and afforded an approxi-
OH
H
OCH3
H
N
11
10
HO
9
H
N
H3C
8
7
11a
1
Head
Tail
H3CO
N
2
N
5
6
4
CONH2
O
O
3
tomaymycin
anthramycin
H
N
H
N
O
O
(CH2)n
N
N
N
H
N
H
O
O
1 a-c (n = 3, 4, 5)
Figure 1
In an earlier study, Wang et al.5 showed that two tomay-
mycin molecules can be covalently bound to a 12 mer du-
plex DNA, where the drug molecules are on opposite
strands six base pairs apart. Later, Mountzouris et al.6
demonstrated that DSB-120 forms a guanine-guanine in-
Synlett 1999, No. 07, 1112–1114 ISSN 0936-5214 © Thieme Stuttgart · New York

LETTER
Design and Efficient Synthesis of Novel DNA Interstrand Cross-Linking Agents
1113
mately 60% yield of the corresponding oxo compound
(6). The principal side product isolated in this oxidation
reaction was the acetylated compound which, upon hy-
drolysis, led to the recovery of the starting hydroxy com-
pound (5) quantitatively. The Wittig reaction of the keto
compound (6) with methyl(triphenylphosphoranyl-
idine)acetate afforded the methyl ester in 80% yield. In
this reaction, the (E) ester9 (7) was obtained exclusively
(vide infra) which upon hydrolysis with ethanolic NaOH
produced the corresponding acid compound (8) in 90%
yield.
CH(SEt)2
NO2
O
+
H2N
NH2
(CH2)n
N
OH
9 a-c ( n = 3, 4, 5)
O
8
i
(EtS)2HC
O2N
CH(SEt)2
NO2
O
O
(CH2)n
N
N
N
N
H
H
O
O
10 a-c
COOMe
NO2
NO2
i, ii
N
ii, iii
COOH
OH
O
2
4
1 a-c
iii, iv
Scheme 2: (i) EDCI, HOBT, DMF, r.t.; (ii) Pd/C, H2, 50 psi, MeOH, 2h
(iii) HgCl2, HgO, 25% aq CH3CN
CH(SEt)2
CH(SEt)2
NO2
NO2
v
N
N
O
OH
Assigning the configuration of the Wittig product: The
Wittig reaction of the compound (6) resulted in the forma-
tion of exclusively one product (7).9 A proton decoupling
experiment was performed on the corresponding acid (8)
NMR spectrum. Double irradiation on the d 5.9 ethylenic
proton signal affected only one proton signal correspond-
ing to that at the C4 position and there is no effect on any
other proton, which confirms the E-configuration of the
Wittig product (Fig. 2).10
O
O
6
5
vi
CH(SEt)2
NO2
CH(SEt)2
NO2
O
O
vii
O
N
OMe
N
OH
O
7
8
Scheme 1: (i) SOCl2,C6H6,r.t,2-3h; (ii) trans-4-Hydroxy-L-prolin methyl
ester hydrochloride (2),Et3N,CH2Cl2,0 oC; (iii) DIBAL-H, CH2Cl2,r.t,
20-24h. ; (iv) EtSH,(CH3)3SiCl,CH2Cl2,r.t.,20-24h; (v) DMSO,Ac2,r.t.,
ethylenic
Ha
H
H
H
N
NO2
O
18h; (vi)(Ph)3P=CHCOOMe,C6H6, reflux, 12h;(vii) Ethanolic NaOH,∆,12h
H
H
Hb
2
N
O
H
Ha
C2H5S
H
SC2H5
Hb
E
- form
The formation of the dimeric PBDs is shown in scheme 2.
Two moles of the acid (8) were coupled with one mole of
one of the diamines (9a-c) (n = 3, 4, 5 respecively), with
EDCI and HOBT to afford the corresponding nitrodithi-
olane dimers (10a-c)$, with different lengths of linker
chain. The yields in the coupling steps were good and ap-
proximately 60-65% compared with the coupling using
DCC and HOBT (yield is 20-25%). Hydrogenation of
10a-c with H2/Pd-C afforded 100% conversion of nitro
compounds to the corresponding amino compounds
which were then subjected to deprotective cyclization
with HgCl2/HgO. The latter conditions produced dimeric
PBD imines (1a-c)# in 30-35% isolated yields. These
dimeric compounds, linked via peptide bonds, were com-
paratively more polar than the corresponding monomers
such as DC-81. Purification by column chromatography
with 10% MeOH in CH2Cl2 resulted in an equilibrium
mixture of imine and carbinolamine methyl ether.
Ha
O
H
NH
2
NO2
O
N
H
H
Hb
H
ethylenic
H
Ha
C2H5S
H
SC2H5
Hb
Z
- form
Figure 2
In conclusion, in this communication, we describe the de-
sign and synthesis of novel C2-linked PBD dimers. The
products are formed exclusively with the E-configuration.
The compounds were prepared with varying degrees of
linker length in order to probe the DNA sequence selectiv-
Synlett 1999, No. 07, 1112–1114 ISSN 0936-5214 © Thieme Stuttgart · New York

1114
B. S. P. Reddy et al.
LETTER
ity and structural requirements for optimal DNA cross- 1a-c are between 0.2 to 0.3 in dichloromethane: methanol:
linking. The biological studies of these compounds are in aq ammonia, 9.0:1.0:0.2), so the reaction mixture was fil-
progress and the results will be published in due course.
tered and directly charged on to a small silica column. The
HgCl2 was eluted first with ethyl acetate and then the sol-
vent system was changed to 5% methanol in dichlo-
romethane. The product was obtained after evoparation
of the solvent as pale yellow compound. spectral data for
Acknowledgement
The research was supported by a grant (to JWL) from the Natural
Sciences and Engineering Research Council of Canada.
o
1
1a : mp. 73 C (dec.); H NMR (CD3OD, 360 MHz): d
1.62-1.70 (m, 2H), 3.05-3.12 (m, 4H), 3.40-3.48 (m, 4H),
4.20-4.72 (m, 6H + OCH3 of methyl ether form), 5.70-
5.80 (m, 2H), 6.70-6.78 (m, 2H), 7.20-7.32 (m, 2H), 7.45-
7.68 (m, 4H), 7.80 (d, 2H, J = 4.4 Hz, imine proton), 7.90-
References and Notes
$ General procedure 10a: To a solution of 8 (200 mg,
0.4872 mmol, 1 eq) in dry DMF, EDCI (93 mg, 0.4872
mmol), HOBT (66 mg, 0.4872 mmol) and 1,3-diamino-
propane (18 mg, 0.2436 mmol, 0.5 eq) (9a) were added
under nitrogen atmosphere and stirred for 6 h. When TLC
indicated the absence of starting material, DMF was re-
moved under reduced pressure. The dark residue was dis-
soloved in ethyl acetate and washed with satd. NaHCO3
solution (2 x 10 ml) and then with water (1 x 10 ml). The
organic layer was dried under anhydrous Na2SO4 and con-
centrated under reduced pressure. The resulting solid was
purified by silica gel flash column chromatography using
ethyl acetate : Hexane (1:2) as eluent. spectral data for
10a : mp. 65-67 oC; 1H NMR (CDCl3, 360 MHz): d 1.24-
1.38 (m, 6H), 1.50-1.60 (m, 2H), 2.65-2,82 (m, 4H),
3.05-3.10 (m, 3H), 3.82-3.88 (d, J = 6.0 Hz, 1H), 4.02-
4.08 (dd, J1 = 4.0 Hz, J2 = 0.5 Hz, 1H), 4.50 (d, J = 1.2 Hz,
1H), 5.42 (bs, 1H), 5.95 (s, 1H), 6.42 (t, 1H), 7.42-7.45
(m, 1H), 7.50-7.60 (m, 1H), 7.65-7.80 (m, 1H), 8.15-8.20
(m, 1H); MS m/z 859.4 (M+1), 846, 611, 510, 412, 331,
237, 135.
+
1
8.10 (m, 2H); MS m/z 551.5 (M , imine), 308.8, 237.0,
134.9, 119.1; ES+ Calcd. 551.2406, found 551.2399.
References
(1) Reddy, B. S. P. ; Sondhi, S. M. ; Lown, J. W. Pharmacology
and Therapeutics (In press).
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York, 1988, PP 28-92; Thurston, D. E. "Advances in the Study
of Pyrrolo[2,1-c][1,4]benzodiazepine (PBD Antitumor
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Inter,actions, Eds, Neidle, S. and Waring, M. J. Macmillan
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H. J. Med. Chem. , 1994, 37 , 3132.
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# General Procedure 1a: To a solution of 10a (170 mg,
1976 mol) in methanol was added 10 % Pd/C (100 mg)
and the compound was hydrogenated in a Parr shaker at
50 psi pressure for 2 hrs. TLC indicated the complete con-
sumption of starting material, and the reaction mixture
was filtered on a small celite bed to remove Pd/C and then
the filtrate was concentrated under reduced pressure to re-
move methanol completely. The amine was a pale yellow
compound to which 10 ml of 25% acetonitrile in water to-
gether with HgCl2 (118 mg) and HgO (117 mg). The reac-
tion mixture was stirred for 12 h. TLC indicated the
(10) B. S. P. Reddy, and J. W. Lown, (unpublished results).
Article Identifier:
completion of the reaction, (the Rf values for the products, 1437-2096,E;1999,0,07,1112,1114,ftx,en;S00799ST.pdf
Synlett 1999, No. 07, 1112–1114 ISSN 0936-5214 © Thieme Stuttgart · New York