Synthesis and anticancer activity of new hydroxamic acid containing 1,4-benzodiazepines

Article abstract of DOI:10.1021/ol900210h

By employing an intramolecular Pd(0)-mediated ring opening of an acylnitroso-derived cycloadduct, new hydroxamic acid containing benzodiazepines have been synthesized and have demonstrated biological activity in MCF-7 and PC-3 tumor cell lines. Subsequent

Full text of DOI:10.1021/ol900210h

ORGANIC
LETTERS
2009
Vol. 11, No. 7
1575-1578
Synthesis and Anticancer Activity of
New Hydroxamic Acid Containing
1,4-Benzodiazepines
Lawrence P. Tardibono, Jr. and Marvin J. Miller*
Department of Chemistry and Biochemistry, UniVersity of Notre Dame,
Notre Dame, Indiana 46556
mmiller1@nd.edu
Received February 1, 2009
ABSTRACT
By employing an intramolecular Pd(0)-mediated ring opening of an acylnitroso-derived cycloadduct, new hydroxamic acid containing
benzodiazepines have been synthesized and have demonstrated biological activity in MCF-7 and PC-3 tumor cell lines. Subsequent N-O bond
reduction of the hydroxamate has provided access to amide analogues for SAR studies. During the course of our syntheses, an intermediate
oxazoline N-oxide was isolated and gave insight into the mechanism of the key Pd(0)-mediated reaction.
1,4-Benzodiazepines are important biomolecules with a wide
array of biological activities and therapeutic functions.
Benzodiazepines are primarily known for their actions in the
central nervous system. In addition to their established
anxiolytic activites, 1,4-benzodiazepines also demonstrate
activities as antibiotic,1 antimalarial,2 and anti-HIV3 agents.
Additionally, there have been several reports of benzodiaz-
which has antiproliferative effects through the regulation of
the c-myc protein (Figure 1).
epines as anticancer agents,4 including BMS-214662,4a
a
known farnesyltransferase (FTase) inhibitor, and Bz-423,4b
(1) Thuston, D. E.; Bose, D. S. Chem. ReV. 1994, 94, 433.
(2) Nallan, L.; Bauer, K. D.; Bendale, P.; Rivas, K.; Yokoyama, K.;
Horne´y, C. P.; Pendyala, P. R.; Floyd, D.; Lombardo, L. J.; Williams, D. K.;
Hamilton, A.; Sebti, S.; Windsor, W. T.; Weber, P. C.; Buckner, F. S.;
Chakrabarti, D.; Gelb, M. H.; Van Voorhis, W. C. J. Med. Chem. 2005,
48, 3704.
Figure 1. Benzodiazepines with anticancer activity.
(3) Kukla, M. J.; Breslin, H. J.; Diamond, C. J.; Grous, P. P.; Ho, C. Y.;
Miranda, M.; Rodgers, J. D.; Sherrill, R. G.; De Clercq, E.; Pauwels, R.;
Andries, K.; Moens, L. J.; Janssen, M. A. C.; Janssen, P. A. J. J. Med.
Chem. 1991, 34, 3187.
Herein we report the synthesis of 1,4-benzodiazepines via
a Pd(0)-mediated ring opening of cycloadduct 3a and
subsequent elaboration and evaluation of their anticancer
activity in MCF-7 and PC-3 tumor cell lines. In contrast to
the benzodiazepines disclosed in the literature, the benzo-
diazepines synthesized from cycloadduct 3a contain a
hydroxamate embedded within the core structure. This
(4) (a) Hunt, J. T.; Ding, C. Z.; Batorsky, R.; Bednarz, M.; Bhide, R.;
Cho, Y.; Chong, S.; Chao, S.; Gullo-Brown, J.; Guo, P.; Kim, S. H.; Lee,
F. Y. F.; Leftheris, K.; Miller, A.; Mitt, T.; Patel, M.; Penhallow, B. A.;
Ricca, C.; Rose, W. C.; Schmidt, R.; Slusarchyk, W. A.; Vite, G.; Manne,
V. J. Med. Chem. 2000, 43, 3587. (b) Sundberg, T. B.; Ney, G. M.;
Subramanian, C.; Opipari, A. W.; Glick, G. D. Cancer Res. 2006, 66, 1775.
(c) Dourlat, J.; Liu, W.; Greash, N.; Garbay, C. Bioorg. Med. Chem. Lett.
2007, 17, 2527.
10.1021/ol900210h CCC: $40.75
Published on Web 03/11/2009
2009 American Chemical Society

presents the possibility of direct metal binding with the
benzodiazepine ring system and makes these compounds
potential inhibitors of metalloenzymes such as the afore-
mentioned farnesyltransferase protein.5 Whereas BMS-
214662 contains an imidazole group that coordinates to
zinc(II) in the FTase active site,6 our benzodiazepine
derivatives may potentially bind to the active site of FTase
through a monoanionic bidentate chelation of the hydrox-
amate to the zinc(II) ion.
Acylnitroso-derived hetero-Diels-Alder adducts, gener-
ated from the reaction of transient acylnitroso species with
cyclopentadiene, are synthetically important precursors to a
variety of bioactive molecules.7 Pd(0)-mediated ring openings
of acylnitroso-derived cycloadducts have been employed to
provide syn-1,4-disubstituted cyclopentene derivatives.8 Con-
struction of the appropriately functionalized cycloadducts 3a
and 3b began with commercially available 2-nitrobenzoic
acid 1 (Scheme 1), which was coupled with O-benzyl
Figure 2. X-ray structure of 4 with thermal ellipsoids drawn at
50% probability.
cycloadditions.9 After isolation and purification, resubjection
of nitrone 4 to Pd(OAc)2 and PPh3 in refluxing THF gave
benzodiazepine 5 in 30% yield.10
Scheme 1
Treatment of cycloadduct 3a with 3.5 mol % of polymer-
bound triphenylphosphine-Pd(0) in refluxing THF for 2 h
facilitated direct conversion to benzodiazepine 5 in an
optimized 75% yield.11 Several palladium reagents were
explored and the polymer-bound triphenylphosphine-Pd(0)
emerged as the most practical due to the ease of product
isolation12 and increased yield. Direct transformation of
adduct 3a to benzodiazepine 5 was ideal for further elabora-
tion to biologically relevant molecules.
hydroxylamine and subsequently deprotected and reduced
under hydrogenolysis conditions to yield hydroxamic acid
2. In situ oxidation of hydroxamic acid 2 in the presence of
cyclopentadiene afforded the anthranilic acid-based cycload-
duct, which was reacted with the appropriate sulfonyl
chloride to give cycloadducts 3a and 3b in 47% and 73%
yield, respectively.
A plausible mechanism for the formation of benzodiaz-
epine 5 is proposed in Scheme 3. The hydroxamate carbonyl
Scheme 3
Treatment of cycloadduct 3a with Pd(OAc)2 and PPh3 at
40 °C for 10 min generated nitrone 4 (Scheme 2). The
Scheme 2
oxygen may act as a nucleophile and allow for reversible
attack on π-allyl complex 6 to form nitrone 4. Cyclization
structure was confirmed by X-ray crystallographic analysis
(Figure 2). To our knowledge, this is the first reported
example of the formation of an oxazoline N-oxide from an
acylnitroso-derived hetero-Diels-Alder adduct. Oxazoline
N-oxides are versatile synthons and have been used in [3+2]
(7) (a) Jiang, M. X.-W.; Warshakoon, N. C.; Miller, M. J. J. Org. Chem.
2005, 70, 2824. (b) Li, F.; Brogan, J. B.; Gage, J. L.; Zhang, D.; Miller,
M. J. J. Org. Chem. 2004, 69, 4538.
(8) Mulvihill, M. J.; Surman, M. D.; Miller, M. J. J. Org. Chem. 1998,
63, 4874.
(9) Dirat, O.; Kouklovsky, C.; Langlois, Y. Org. Lett. 1999, 1, 753.
(10) Low yield due to difficulty with purification. The yield would be
expected to be higher with polymer-bound Pd(0).
(5) Bell, I. M. J. Med. Chem. 2004, 47, 1869.
(6) Reid, T. S.; Beese, L. S. Biochemistry 2004, 43, 6877.
1576
Org. Lett., Vol. 11, No. 7, 2009

to the [5,5] system (path A) is faster than formation of the
[7,5] system. Prolonged reaction times and/or heat would
allow Pd(0) complexation with 4, which would reintroduce
the intermediate π-allyl complex 6. Intramolecular proton
transfer to complex 7 followed by irreversible nucleophilic
attack (path B) of the sulfonamide nitrogen provides the
thermodynamically preferred benzodiazepine 5. Further
evidence that this pathway could be operative was found
when the dinitrobenzenesulfonamide group was replaced with
a 2-thiophenesulfonamide.
Scheme 5
Treatment of cycloadduct 3b with Pd(OAc)2 and PPh3 at
40 °C for 10 min resulted in formation of nitrone 8 in 83%
yield (Scheme 4). Prolonged heating of 3b in the pre-
Scheme 4
sence of various palladium reagents (Pd(OAc)2, Pd(dba)2,
Pd2(dba)3·CHCl3, PS-PPh3Pd), phosphine ligands, solvents,
and bases resulted in either no reaction or decomposition of
the reaction mixture. Isolation and purification of nitrone 8
followed by reexposure to Pd(0) was also unsuccessful. Since
the pKa of the sulfonamide NH is significantly higher in
cycloadduct 3b (compared to 3a), the essential proton transfer
was expected to be less facile and the benzodiazepine would
be unable to form. Still, formation of 9 was of particular
interest considering its structural similarity to the known
anticancer compound BMS-214662.4a
treated with several different sulfonyl chlorides. Reaction
with 2-thiophenesulfonyl chloride was first explored in an
effort to prepare compound 9. Unfortunately, compound 11
was completely unreactive with several sulfonyl chlorides,
including 2-thiophenesulfonyl chloride, dinitrobenzene sul-
fonyl chloride, and tosyl chloride.15 To derivatize 11, we
focused on reductive aminations, as aldehydes are unhindered
and strongly electrophilic.
Unable to convert cycloadduct 3b directly to the benzo-
diazepine core, we turned our attention to an alternative
multistep route toward functionalized benzodiazepines. Thus,
benzodiazepine 5 was treated with TBSCl in pyridine to give
protected hydroxamate 10 in good yield (Scheme 5). Upon
protection of the hydroxamate, attention was turned to
functionalization of the 4-amino position. Treatment of
benzodiazepine 10 with excess n-propylamine at rt allowed
for the mild deprotection of the dinitrobenzenesulfonamide
to afford core 11.13 The resulting deep yellow dinitro-N-
propylaniline byproduct was easily separated from 11 by
column chromatography. Alternatively, mercaptoacetic acid
was evaluated for the deprotection of the dinitrobenzene-
sulfonamide, but gave inconsistent results.14
Benzodiazepine 11 was treated with various aldehydes in
the presence of acetic acid and molecular sieves to form the
corresponding iminium species, which were subsequently
reduced with sodium triacetoxyborohydride to give protected
benzodiazepines 12a-e. Removal of the TBS group with
CsF in MeOH and purification with iron-free silica gel16
provided hydroxamate-containing benzodiazepines 13a-e in
32-48% yield over two steps (Scheme 5).
An important functionality present in benzodiazepines
13a-e is the hydroxamic acid. This group was anticipated
to play a role in the biological activity of these compounds.
Therefore, it was important to synthesize several analogues
that do not contain the hydroxamate. Benzodiazepine 5 was
deprotected with n-propylamine in 70% yield (Scheme 6).
Benzodiazepine 14 was then subjected to stoichiometric
titanocene chloride reduction conditions,17 which produced
To test the reactivity of the relatively hindered and electron
deficient 4-amino position, benzodiazepine 11 was first
(11) Unoptimized yield with Pd(OAc)2/PPh3/THF/∆ was 56%. See:
Surman, M. D.; Mulvihill, M. J.; Miller, M. J. Org. Lett. 2002, 4, 139.
(12) Simply filtering the resin and concentrating the filtrate afforded a
yellow solid, which upon trituration with CH2Cl2 gave product 5 as a pure
white powder.
(13) Fukuyama, T.; Cheung, M.; Jow, C.; Hidai, U.; Kant, T. Tetrahe-
dron Lett. 1997, 38, 5831.
(14) Decomposition of mercaptoacetic acid due to oxidation and
occasional deprotection of TBS group proved problematic.
(15) Several bases, solvents, and temperatures were exhaustively
explored.
(16) The procedure for the preparation of iron free silica gel may be
found in the Supporting Information.
Org. Lett., Vol. 11, No. 7, 2009
1577

Scheme 6
Table 1. Results of Anticancer Screeningsa
% inhibition at 20 µM
IC50 (µM)
PC-3 MCF-7
compd
PC-3
MCF-7
5
100
32
31
<10
16
<10
11
96
92
90
15
22
39
29
13
4
3
8
1.8
13a
13b
13c
13d
13e
16a
16b
<10
a Trichostatin A was used as the positive control (MCF-7 IC50 ) 16
nM, PC-3 IC50 ) 160 nM).
MCF-7 cell lines. Benzodiazepine 13b has shown low
micromolar activity and is a suitable lead for further SAR
studies. The exact mechanism of action for this new class
of 1,4-benzodiazepines is currently unknown. Enzyme assays
and other means to determine the mechanism of action are
under investigation.
amide 15 in 60% yield. Benzodiazepine 15 proved more
amenable to the same reductive amination conditions (NaB-
H(OAc)3/AcOH) and analogues 16a,b were synthesized in
improved yield.
Acknowledgment. We gratefully acknowledge Mrs. Patty
Miller (University of Notre Dame) for performing MCF-7
and PC-3 cellular assays and Dr. Bruce Noll (Univeristy of
Notre Dame) and Dr. Allen Oliver (Univeristy of Notre
Dame) for X-ray crystal determination and data manipulation.
We also thank Nonka Sevova (Univeristy of Notre Dame)
for mass spectroscopic analyses, Dr. Jaroslav Zajicek (Univer-
isty of Notre Dame) for NMR assistance, and Dr. Jed Fisher
(Univeristy of Notre Dame) for helpful discussions. We
acknowledge the University of Notre Dame and NIH
(GM068012 and GM075855) for support of this work.
Several benzodiazepines demonstrated growth-inhibitory
activity in MCF-7 (breast cancer) and PC-3 (prostate cancer)
tumor cell assays (Table 1). Compounds 5, 13a, and 13b
reached the low micromolar range of inhibition against
MCF-7 cells. In general, all compounds showed significantly
improved inhibitory activity against the MCF-7 cell line. The
substituent at the aniline nitrogen of the benzodiazepine
seems to play a rather important role in determining activity.
On the basis of the results of compounds 16a,b, the
hydroxamate has also been demonstrated to be a necessary
functionality for activity in this class of benzodiazepines.
In summary, we have reported a new class of benzodiaz-
epines that have shown encouraging biological activity in
Supporting Information Available: General methods,
1
experimental details, and H and 13C NMR spectra for 3b,
4, 8, 10, 11, 13a-e, 14, 15, and 16a,b. This material is
available free of charge via the Internet at http://pubs.acs.org.
(17) Cesario, C.; Tardibono, L.; Miller, M. J. J. Org. Chem. 2009, 74,
448.
OL900210H
1578
Org. Lett., Vol. 11, No. 7, 2009