Design and synthesis of novel pyrrolo[2,1-c][1,4]benzodiazepine- lexitropsin conjugates

Full text of DOI:10.1021/jo9812072

290
J . Org. Chem. 1999, 64, 290-292
stereochemistry at the C11a position provides a right-
Design a n d Syn th esis of Novel
P yr r olo[2,1-c][1,4]ben zod ia zep in e-
Lexitr op sin Con ju ga tes
handed molecular twist, when viewed from the C-ring
toward the A-ring, which enables the PBD to assume a
snug fit in the minor groove of DNA.3 Molecular model-
ing, solution NMR, fluorimetry, and DNA footprinting
experiments reveal that the PBDs recognize a three base-
pair motif with a preference for 5′PuGuPu sequences.1c
Since the synthesis of the PBD ring system itself is
problematic, due to the labile N10-C11 imine moiety
(carbinolamine or methyl ether equivalent), much of the
literature to date has focused on the development of new
synthetic strategies.4,5 In contrast, very little has been
reported about modifications on the PBD ring system or
conjugation with carriers.6 Recently, a C8-linked PBD
dimer was prepared7 which forms a symmetric inter-
strand cross-link with duplex DNA involving a four-base
pair bonding site but spanning six DNA base pairs
overall.8 Although some efforts to date have been directed
at different modifications on the PBD ring system, no
attempt has been made to link the PBD ring system with
other well-established DNA groove binders such as
distamycin (1) and netropsin (2). The latter agents bind
to four or more consecutive A-T base pairs.9 Agents that
combine the features of minor groove binding and DNA
sequence selectivity, together with the ability to form
covalent attachment to the DNA, have proven to have
clinical potential in the treatment of human malignan-
cies.10 We herein report the design, synthesis and char-
acterization of a series of novel PBD-lexitropsin conju-
gates in order to probe the combined effect of both
moieties on DNA sequence selective binding ability and
cytotoxicity.
Yalamati Damayanthi, B. S. Praveen Reddy, and
J . William Lown*
Department of Chemistry, University of Alberta,
Edmonton, AB, Canada T6G 2G2
Received J une 22, 1998
Pyrrolo[2,1-c][1,4]benzodiazepines (PBDs), a group of
potent naturally occurring antitumor antibiotics from
various Streptomyces species, are of considerable interest
because of their potential as antitumor agents, gene
regulators, and DNA probes.1 Well-known members of
this group include anthramycin, tomaymycin, the neo-
thramycins A and B, sibiromycin, chicamycin, and DC-
81.2,4 The cytotoxicity and antitumor activity of these
agents are attributed to their property of sequence-
selective covalent binding to the N2 of guanine in the
minor groove of duplex DNA via an acid-labile aminal
bond to the electrophilic imine at the N10-C11 position.
The N10-C11 carbinolamine form, A, may exist in the
equivalent imine, C, or carbinolamine methyl ether form,
B, depending on the precise structure of the compound
and the method of isolation (Figure 1).4b,16 The (S)-
(1) (a) Dervan, P. B. Science 1989, 232, 464. (b) Hurley, L. H. J .
Med. Chem. 1989, 32, 2027. (c) Thurston, D. E.; Thompson, A. S. Chem.
Br. 1990, 26, 767.
(2) Hurley, L. H. J . Antibiot. 1977, 30, 349. (b) Schimizu, K.;
Kawamoto, I.; Tomita, F.; Morimoto, M.; Fujimoto, K. J . Antibiot. 1982,
35, 992.
Molecular modeling studies suggested that C8-linked
PBD dimers have greater isohelicity with the minor11
groove of DNA compared with the C7-linked dimers.
Thurston et al. reported that the C8-linked dimer, DSB-
120 (3) (Figure 2) forms an irreversible interstrand cross-
link between two guanine bases within the minor groove
via their exocyclic N2 atoms, and it spans six base pairs,
(3) (a) Thurston, D. E. Advances in the study of pyrrolo[2,1-c]1, 4]-
benzodiazepine (PBD) Antitumour Antibiotics in Molecular Aspects of
Anticancer Drug-DNA Interactions; Neidle, S., Waring, M. J ., Eds.;
The Macmillan Press: Riverside, NJ , 1993; Vol. 1, pp 54-88. (b)
Remers, W. A. In The chemistry of Antitumour Antibiotics; Wiley: New
York, 1988; Vol. 2, pp 28-92. (c) Hurley, L. H.; Pertrusek, R. L. Nature
1979, 282, 529.
(4) (a) Lown, J . W.; J oshua, A. V. Biochem Pharmacol. 1979, 28,
2017. (b) Thurston, D. E.; Bose, D. S. Chem. Rev. 1994, 94, 433. (c)
Molina, P.; Diaz, I.; Tarraga, A. Tetrahedron 1995, 51, 5617. (d) Kamal,
A.; Rao, N. V. Chem. Commun. 1996, 385. (e) Kamal, A.; Reddy, B. S.
P.; Reddy, B. S. N. Tetrahedron Lett. 1996, 37, 6803.
(5) (a) Kamal, A.; Damayanthi, Y.; Reddy, B. S. N.; Lakminarayana,
B.; Reddy, B. S. P. Chem. Commun. 1997, 1015. (b) Kamal, A.; Reddy,
B. S. N.; Reddy, B. S. P. Bioorg. Med. Chem. Lett. 1997, 7, 1825.
(6) (a) Thurston, D. E.; J ones, G. B.; Davis, M. E. J . Chem. Soc.,
Chem. Commun. 1990, 874. (b) Liegeois, J . F.; Rogister, F. A.;
Bruhwyler, J .; Damas, J .; Nguyen, T. P.; Inarejos, M.; Chleide, E. M.
G.; Mercier, M. G. A.; Delarge, J . E. J . Med. Chem. 1994, 37, 519.
(7) Bose, D. S.; Thompson, A. S.; Smellie, M.; Berardini, M. D.;
Hartley, J . A.; J enkins, T. C.; Neidle, S.; Thurston, D. E. J . Chem.
Soc., Chem. Commun. 1992, 1518.
(8) J enkins, T. C.; Hurley, L. H.; Neidle, S.; Thurston, D. E. J . Med.
Chem. 1994, 37, 4529.
(9) (a) Lown, J . W. J . Mol. Recogn. 1994, 7, 79. (b) Guo, D.; Gupta,
R.; Lown, J . W. Anti-Cancer Drug Design 1993, 8, 369. (c) Fontana,
M.; Lestingi, M.; Mondello, C.; Braghetti, A.; Montecucco, A.; Ciarrochi,
G. Anti-Cancer Drug Design 1992, 7, 131.
(10) (a) Rao, K. E.; Lown, J . W. Chem. Res. Toxicol. 1990, 3, 262.
(b) Rao, K. E.; Lown, J . W. Biochemistry 1992, 31, 12076. (c) Rao, K.
E.; Shea, R. G.; Yadagiri, B.; Lown, J . W. Anti-Cancer Drug Design
1990, 5, 3. (d) Xie, G.; Gupta, R.; Lown, J . W. Anti-Cancer Drug Design
1995, 10, 389.
actively recognizing a central 5′-GATC sequence.12
A
variety of lexitropsin conjugates of other cytotoxic agents
which are minor groove binders with improved selectivi-
ties have been reported and are summarized in a recent
review.13 In view of the commonly observed enhanced
activity and selectivity of the parent drugs when conju-
gated with lexitropsins and the intrinsic activity of DSB-
120, we attempted to conjugate certain lexitropsins with
the PBD nucleus through the C8-position with a suitable
linker. The natural product DC-81 (4) was chosen as the
PBD unit, and an efficient synthetic pathway was
developed (Scheme 1) to provide the various PBD-
lexitropsin conjugates 5a -c. The new compounds are
designed to effect sequence-selective binding with duplex
DNA.
The overall synthetic strategy is shown in Scheme 1.
The lexitropsin unit was prepared according to the
(11) Farmer, J . D.; Rudnicki, S. M. J r.; Suggs, J . W. Tetrahedron
Lett. 1988, 29, 5105.
(12) Bose, D. S.; Thompson, A. S.; Ching, J .; Hartley, J . A.; Berardini,
M. D.; J enkins, T. C.; Neidle, S.; Hurley, L. H.; Thurston, D. E. J . Am.
Chem. Soc. 1992, 114, 4939.
(13) Sondhi, S. M.; Reddy, B. S. P.; Lown, J . W. Curr. Med. Chem.
1997, 4, 313.
(14) (a) Gupta, R. Xie, G.; Lown, J . W. Gene 1994, 149, 81. (b) Gupta,
R.; Wang, H.; Huang, L.; Lown, J . W. Anti-Cancer Drug Design 1994,
10, 25. (c) Dervan, P. B.; Baird, E. E. J . Am. Chem. Soc. 1996, 118,
6141.
(15) Langley, D. R.; Thurston, D. E. J . Org. Chem. 1987, 52, 91.
(16) Kopka, M. L.; Goodsell, D. S.; Baikalov, I.; Grzeskowaik, K.;
Cascio, D.; Dickerson, R. E. Biochemistry 1994, 33, 13593.
10.1021/jo9812072 CCC: $18.00 © 1999 American Chemical Society
Published on Web 12/09/1998

Notes
J . Org. Chem., Vol. 64, No. 1, 1999 291
F igu r e 1. Equilibrium of N10-C11 carbinolamine form (A), imine (C), and carbinolamine methyl ether (B) and possible mechanism
of the formation of PBD-DNA adduct.
F igu r e 2.
previously described method.14 A versatile approach has
been developed to join the lexitropsin unit with the PBD
unit, i.e., through an amidic linkage. The five-atom length
of the spacer between lexitropsin and PBD unit is judged
as optimal.7 The deprotective cyclization method is
adopted to obtain the conjugated PBD imines. The
compounds 5a -c were prepared to examine the effect of
the number of pyrrole units (n ) 1, 2, 3) on binding
affinity and cytotoxicity of the PBD system.
The synthesis was started with coupling of an ap-
propriate spacer with the commercially available vanillin
(6) which serves as “A” ring of the PBD unit. The reaction
of ethyl bromobutyrate with vanillin in the presence of
K2CO3 in dry acetone under reflux conditions produced
ester 7 which upon nitration gave the corresponding nitro
compound 8 in about 85% yield. The aldehydic group of
compound 8 was oxidized to the corresponding carboxylic
acid 9 by sulfamic acid and sodium chlorite, and then
coupled with (2S)-pyrrolidine-2-carboxaldehyde diethyl
thioacetal (10)15 via the acid chloride. The resulting nitro
ester 11 upon hydrolysis produced nitro acid 12 which
was then coupled with the amine moiety of lexitropsin,
(14a -c) with EDCI in dry DMF;14 the latter amino
compounds are prepared by hydrogenation of the corre-
sponding nitro lexitropsins 13a -c.14 The DCC coupling
resulted in poor yield, whereas the EDCI coupling
resulted in 65-70% yield of 15a -c. Compounds 15a -c
on reduction with the H2/Pd-C followed by deprotection4
with HgCl2/HgO in aqueous acetonitrile at room temper-
ature produced the corresponding imines 5a -c in 40-
45% yield. Since the conjugation of the lexitropsin
moieties with PBD resulted in a high polarity of the
imines, this necessitated the use of methanol in combina-
tion with chloroform as eluent during the purification of
imines by column chromatography, and therefore the
product was obtained as a mixture of imine and methyl
ether. The presence of both the forms is confirmed by both
NMR and mass spectra. Attempts were made to convert

292 J . Org. Chem., Vol. 64, No. 1, 1999
Notes
Sch em e 1a
a
(i) K2CO3, THF, Br(CH2)3COOEt; (ii) HNO3/SnCl4, DCM; (iii) sodium chlorite/sulfamic acid, H2O; (iv) SOCl2/C6H6, rt, 2 h; (v) pyrrolidine-
2-carboxaldehyde diethyl thioacetal (10), Et3N, DCM, 0 °C, 1 h; (vi) 2 N NaOH solution, EtOH, 50 °C, 18 h; (vii) H2/Pd-C, MeOH, 2 h;
(viii) lexitropsin amine14 (14a -c) (n ) 1-3), HOBT, EDCI, dry DMF, rt, 14 h, (ix) H2/Pd-C, MeOH; (x) HgCl2/HgO, aq CH3CN, rt, 14 h.
this mixture into either exclusively the methyl ether form
or imine form, but the compounds are soluble only in a
mixture of chloroform and methanol. The insolubility of
the compounds in either of the solvents alone did not
allow conversion into only one form. Attempts were made
to isolate the imines by using chloroform and Hunig’s
base, but without use of methanol, the compounds do not
elute by flash column chromatography. The methyl ether
form 6a -c may consist two diastereoisomers of 11S,11aS
and 11R,11aS, as observed in the case of tomaymycin.17
But, this diastereoisomer formation could not be deter-
mined by NMR because of the complexity of NMR
spectra. The final compounds were isolated as pale yellow
crystalline compounds in 28-30% overall yields.
(overall five-atom spacer). The conjugation is achieved
by amidic linkage by coupling amine of the lexitropsin
unit with the acid moiety of the linker attached to the
PBD system. We have synthesized the first members of
PBD-lexitropsin conjugates in order to examine their
sequence-selective binding affinity with duplex DNA.
Efforts are underway to prepare complexes of these
conjugates with DNA for X-ray diffraction analysis, and
their results will be disclosed in due course.
Ack n ow led gm en t. The research was supported by
a grant (to J .W.L.) from the Natural Sciences and
Engineering Research Council of Canada.
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures and compound characterization data for the total
synthesis of 5a -c; copies of NMR and mass spectra (29 pages).
This material is contained in libraries on microfiche, im-
mediately follows this article in the microfilm version of the
journal, and can be ordered from the ACS; see any current
masthead page for ordering information.
In summary, we have described a versatile and con-
venient strategy for the design and synthesis of the first
examples of PBD-lexitropsin conjugates bonded through
the C8 position with a suitable linker of three carbons
(17) Cheatham, S.; Kook, A.; Hurley, L. H.; Barkley, M. D.; Remers,
W. J . Med. Chem. 1988, 31, 583.
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