A novel, broad-spectrum anticancer compound containing the imidazo[4,5-e][1,3]diazepine ring system

Article abstract of DOI:10.1016/j.bmcl.2010.06.061

Synthesis and broad-spectrum anticancer activity of a novel heterocyclic compound 1 containing the title imidazo[4,5-e][1,3]diazepine ring system have been reported. The compound shows potent in vitro antitumor activity with low micromolar IC₅₀'s against prostate, lung, breast, and ovarian cancer cell lines tested. The long alkyl chain attached to the six-position of the heterocyclic ring of 1 appears to be necessary for the observed biological activity.

Full text of DOI:10.1016/j.bmcl.2010.06.061

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4386–4389
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A novel, broad-spectrum anticancer compound containing
the imidazo[4,5-e][1,3]diazepine ring system
Min Xie a, Ravi K. Ujjinamatada a, Mariola Sadowska b, Rena G. Lapidus b, Martin J. Edelman b,
Ramachandra S. Hosmane a, ,
*
a Laboratory for Drug Design and Synthesis, Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, 1000 Hilltop Circle,
Baltimore, Maryland 21250, USA
b University of Maryland Marlene and Stewart Greenbaum Cancer Center, 22 South Greene Street, Baltimore, Maryland 21201, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis and broad-spectrum anticancer activity of a novel heterocyclic compound 1 containing the title
imidazo[4,5-e][1,3]diazepine ring system have been reported. The compound shows potent in vitro anti-
tumor activity with low micromolar IC50’s against prostate, lung, breast, and ovarian cancer cell lines
tested. The long alkyl chain attached to the six-position of the heterocyclic ring of 1 appears to be nec-
essary for the observed biological activity.
Received 10 May 2010
Revised 9 June 2010
Accepted 10 June 2010
Available online 15 June 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Imidazo[4,5-e][1,3]diazepine analogue
Synthesis
In vitro screening
Broad-spectrum anticancer activity
Prostate, lung, breast and ovarian cancers
NC
NC
Cancer is the second leading cause of death worldwide after
heart disease, claiming more than half a million deaths in US alone
in 2009.1 The major causes of cancer deaths are those related to
lung (30%), prostate (9%) and colorectum (9%) in men, and lung
(26%), breast (15%), colorectum (9%), and ovarian (5%) cancers in
women.1,2 Deaths from cancer worldwide are projected to con-
tinue rising with an estimated 12 million deaths in the year
2030.2 Surgery, radiation, and chemotherapy are the principal
modes of cancer treatment.3 Newer agents, specifically targeted
against detectable molecular abnormalities in certain tumors,
and which minimize damage to normal cells, are emerging as valu-
able therapeutics.4–9 Despite this progress, the majority of patients
diagnosed with these major malignancies will die of their disease
and therefore, there is a need for new agents with novel mecha-
nisms of action. Though much effort has been focused on the devel-
opment of novel tyrosine kinase inhibitors and antibodies directed
at signal transduction,10–15 exploration of new compounds direc-
ted against ‘traditional’ targets of DNA and tubulin continues to
be important.16,17
N
N
NC
NC
N
Cl
DMF/K2CO3
88%
+
O
N
H
O
3
2
NH
NH2
TFA / Rt
4h
NH
NH2HNO3
)
H
(C
H
C
N
N
N
N
4
CH3(CH2)17NH
99%
NaOMe/MeOH
NH
57%
O
5
NH2
N
NH2
N
N
N
N
N
CH3(CH2)17NH
CH3(CH2)17NH
N
NH2
1
N
H
NH
1a
NH2
NH*HNO3
In this Letter, we describe the synthesis and broad-spectrum
anticancer activity of a novel heterocycle (1) containing the title
MeOH, Reflux
85%
N
N
+
+
CH3(CH2)17 NH2
R
N
H
N
N
H
6
H2N
NH*HNO3
4
8
7
* Corresponding author. Tel.: +1 410 381 0005; fax: +1 410 455 1148.
E-mail address: hosmane@umbc.edu (R.S. Hosmane).
Recently retired.
Scheme 1.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.061

M. Xie et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4386–4389
4387
Figure 1. Antitumor activity of compound 1 in vitro against (A) A549 (lung), (B) H460 (lung), (C) MCF-7 (breast), (D) MDA-MB-231 (breast), (E) OVCAR-3 (ovarian), and (F)
PC-3 (prostate) cell lines. Each compound was tested at nine different concentrations, and each drug dilution was repeated four times. Cells treated with DMSO (equiv
volume) were used as a vehicle control.
dimethylformamide, catalyzed by potassium carbonate, to afford 3
in 88% yield. Compound 3 was reacted with octadecyl-guanidinium
nitrate (4), freshly prepared by the reaction of octadecylamine (6)
with 3,5-dimethyl-1-pyrozolylamidinium nitrate (7), to produce
the ring-closed product 5 in 57% yield. The deprotection of 5 was car-
ried out by treatment with trifluoroacetic acid at room temperature
for four hours, which gave the target product 1 in quantitative yield.
Compound 1 was completely characterized18 by 1H and 13C NMR, IR,
and mass spectral data, as well as elemental microanalyses.
Compound 1 can exist in several different tautomeric forms,
including but not limited to 1a, 1b, and 1c as shown (Scheme 2).
The 1H NMR of the product in DMSO-d6 showed five
D2O-exchangeable protons, three of which appeared as a broad
singlet each at d 7.79, 7.69 and 7.38 and the remaining two
appeared as a triplet overlapped with a singlet centered at d
7.45. As structure 1a contains a free imidazole NH, which normally
imidazo[4,5-e][1,3]diazepine ring system. Compound 1 is easy to
synthesize from commercially available starting materials, can be
conveniently scaled up to multigram quantities, is a stable, crystal-
line solid, soluble in aqueous acid, and was found to be highly ac-
tive in vitro against all of the cancer cell lines tested, which include
lung, breast, prostate, and ovarian cancers.
NH2
N
N
N
N
H3C-(H2C)17
N
H
NH2
1
Compound
1
was synthesized18 in three steps starting from
4,5-dicyanoimidazole (2) (Scheme 1). The NH group of the latter
was protected by reaction with p-methoxybenzyl chloride in

4388
M. Xie et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4386–4389
NH2
NH2
H
NH2
NH2
NH
N
N
NC
NC
N
N
N
N
NaOMe/MeOH
82%
N
N
N
N
N
N
N
N
N
R-H2C N
CH3CH2-NH
R-H2C-HN
R-H2C-HN
+
NH2
N
H
N
N
H
2
3
NH2
1b
NH
NH2
9
NH2
2
10
1a
1c
R= (CH2)16-CH3
Scheme 3.
80 oC 25 oC
shorter alkyl chain, namely an ethyl group. Thus, compound 9
was synthesized in 82% yield by direct condensation of 4,5-dicy-
anoimidazole with ethylguanidine (Scheme 3). Compound 9 was
fully characterized as before using spectral and microanalytical
data.
H(d)
H(c)
N
N
N
N
R-H2C N
Compound 9 was screened in vitro as before against six cancer
H(e)
N
cell lines. The compound was found to be inactive (IC50 >800
against PC-3 (prostate) and MCF-7 (breast) cancer cell lines, and
only weakly active against A549 (IC50 = 38 M) (lung) and H460
(IC50 = 22 M) (lung), and OVCAR-3 (ovarian) cancer cell lines.
lM)
N
H(a)
H(b)
l
1c
l
These results suggest that the long alkyl chain attached at the
six-position of the heterocyclic ring plays a significant role in the
observed biological activity.
Scheme 2.
In conclusion, we have discovered a novel, broad-spectrum
antitumor compound that shows potent in vitro activity with
low micromolar IC50’s against all six cancer cell lines tested. The
cytotoxicity (CC50) of 1 to normal cells is at least at a four fold high-
er concentration than the therapeutic concentration levels. The
long alkyl chain attached to the six-position of the heterocyclic
ring of 1 appears to be necessary for the observed biological activ-
ity as compound 9 with an ethyl group failed to show good activity
under the same experimental conditions. Further studies of struc-
ture–activity relationships (SAR) to enhance potency and decrease
toxicity as well as mechanistic explorations of antitumor activity of
1 are currently in progress.
Acknowledgment
This research was supported by a grant (#1R01 GM087738-
01A1) from the National Institute of General Medical Sciences of
the National Institutes of Health.
Figure 2. Effect of compound 1 on immortalized breast cancer cell line MCF10A.
WST-1 cell proliferation assay, MCF-10A, 1250 cells treated with 1 for 72 h.
References and notes
is expected to appear at >10 d, and since structure 1c lacks the H(e)
proton to exhibit a triplet by coupling with an adjacent CH2 group,
either of the tautomeric forms 1a or 1b does not match the ob-
served NMR data. On the other hand, structure 1c is consistent
with the observed data in that the two NH protons in each of the
two NH2 groups would be magnetically different because of the
anticipated H-bonding with the imidazole N atoms, resulting in
four different signals, and the remaining NH would be a triplet
through coupling with the adjacent CH2 group. Indeed, variable
temperature NMR experiments conducted at 10° intervals between
25 and 80 °C confirmed this assignment by revealing gradual
coalescence of signals at d 7.79 and 7.69 (tentatively Ha and Hb
protons) and at 7.45 and 7.38 (tentatively He, Hc, and Hd).
Compound 1 was screened in vitro against six cancer cell lines,
including A549 and H460 (lung cancer), MCF-7 and MDA-MB-231
(breast cancer), OVCAR-3 (ovarian cancer), and PC-3 (prostate
cancer). The results are graphically represented in Figure 1.
In order to study the effect of 1 on normal cell growth and
proliferation, we also determined the CC50 value (cytotoxicity) of
1, employing the immortalized normal breast cell line MCF10A.
The results are shown in Figure 2.
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www.who.int/mediacentre/factsheets/fs297/en/index.html.
3. Lee, A. Options for Cancer Treatment: Surgery, Chemotherapy, and Radiation,
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Cespedes, R.; Atkins, J.; Lippman, S.; Carlin, S.; Ryan, A.; Szczepanek, C.;
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7. Jordan, V. C. Br. J. Pharmacol. 2006, 147, S269.
8. Love, S. Study Finds New Use for Raloxifene: Reducing Breast Cancer in High-
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9. Targeted Chemotherapy—Fighting Cancer without the Side Effects, 2009.
http://www.gizmag.com/isoflow-isolate-tumour-chemotherapy/12820.
10. Shawver, L. K.; Slamon, D.; Ullrich, A. Cancer Cell 2002, 1, 117.
11. Nahta, R.; Hortobagyi, G. N.; Esteva, F. J. Expert Opin. Invest. Drugs 2003, 12, 909.
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17. Jiang, N.; Wang, X.; Yang, Y.; Dai, W. Mini-Rev. Med. Chem. 2006, 6, 885.
18. Experimental: Organic synthesis procedure: Preparation and physico-chemical
properties of the compounds are as follows:
As part of a preliminary structure–activity relationship study,
we became interested in exploring the role of the long hydrophobic
chain attached to the seven-membered heterocyclic ring of 1. To
this end, we synthesized an analogue of 1 containing a much
1-(4-Methoxybenzyl)-4,5-dicyanoimidazole (3):
dicyanoimidazole (1.49 g, 12 mmol) and potassium carbonate (1.99 g,
A
suspension of 4,5-

M. Xie et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4386–4389
4389
14.4 mmol, 1.2 equiv) in anhydrous DMF (15 mL) was stirred for 5 min. To the
reaction mixture was added p-methoxybenzyl chloride (1.99 mL, 14.4 mmol).
The reaction mixture was stirred at room temperature overnight. The reaction
mixture was evaporated to leave 5 mL dimethyl-formamide. Ice was added to
the residue to remove excess potassium carbonate, the solution was stirred for
1 h, leaving the precipitate which was filtered and washed successively with
water and methanol. The reaction afforded a white solid (88%). 1H NMR
(DMSO-d6): d 8.457 (s, 1H, imidazole-CH), 7.26 (d, J = 8.7 Hz, 2H, Ph-H), 6.94 (d,
J = 8.7 Hz, 2H, Ph-H), 5.36 (s, 2H, benzyl-CH2), 3.72 (s, 3H, methoxy-CH3). Anal.
Calcd for C13H10N4O: C, 65.54; H, 4.23; N, 23.52. Found: C, 65.45; H, 4.26; N,
23.46.
NH), 3.29 (m, 2H, CH2-NH), 1.49 (m, 2H, CH2CH2NH), 1.23(s, 30H, 15xCH2),
0.86(t, J = 6.6 Hz, 3H, CH3); 13C NMR (DMSO, 400 MHz): d 161.53, 159.72,
159.16, 136.50, 135.69, 41.87, 31.83, 29.57, 29.39, 29.24, 27.00, 22.64, and
14.50; UV (lmax) 249.5 nm (e = 4.35 Â 104, 280.5 nm (sh) (e = 2.07 Â 104); MS
(ESI): 430 (MH+); HRMS (FAB) Calcd for C24H43N7: m/z 430.3658; found
430.3651; Anal. Calcd for C24H43N7Á2H2O: C, 61.90; H, 10.17; N, 21.05. Found:
C, 61.87; H, 10.25; N, 20.83.
8-Imino-4,6-diamino-N6-ethyl-1,8-dihydroimidazo[4,5-e][1,3]diazepine (9): Fine
sodium metal was placed in
a flame dried apparatus and anhydrous
methanol (15 mL) was added and stirred at room temperature for 10 min
in argon atmosphere. Then ethyl guanidine hemisulfate 1.092 g (0.008 mol)
was added to the above sodium methoxide solution. This reaction mixture
was stirred at room temperature for 1 h. Separated sodium sulfate was
removed under centrifugation at 4 °C and then ethyl guanidine solution was
added to the solution of dicyanoimmidazole 0.708 g (0.006 mol) in
anhydrous methanol (15 mL). This reaction mixture was refluxed for 72 h.
Reaction mixture was brought to room temperature and separated solid was
filtered and washed with cold methanol. 1H NMR (DMSO-d6, 400 MHz) d
7.58 (br s, 4H, NH2, D2O exchangeable), 7.55 (s, 1H, imidazole CH), 3.28 (q,
J = 7.32 Hz, 2H, CH2), 1.02 (t, J = 7.32 Hz, 3H, CH3); 13C NMR (DMSO-d6,
400 MHz) d 161.3, 159.8, 159.2, 149.7, 136.4, 135.6, 36.5, 15.3; MS (ESI) m/z
206 (MH+); Anal. Calcd for C8H11N7: C, 46.82; H, 5.40; N, 47.78. Found: C,
46.70; H, 5.69; N, 48.01.
1-(p-Methoxybenzyl)-8-imino-4,6-diamino-N6-octadecyl-1,8-dihydroimidazo[4,5-
e][1,3]diazepine (5): In
a flame-dried, two-necked round bottom flask,
anhydrous methanol (30 mL), 3,5-dimethyl-1-pyrozolylamidinium nitrate
(1.05 g, 5 mmol), and octadecylamine (3.0 g, 5 mmol) was added. The
reaction mixture was refluxed for 5 h. Then the solvent was evaporated
under reduced pressure to dryness. The octadecylguanidine nitrate was
recrystallized from methanol in a quantitative yield. Anhydrous methanol
(10 mL) was added to a flame-dried, two-necked round bottom flask equipped
with a nitrogen gas inlet and a condenser, and fresh sodium metal (0.15 g,
5 mmol) was added to methanol and stirred for half an hour. The
octadecylguanidine nitrate prepared previously was added to the freshly
prepared sodium methoxide reaction mixture. After 0.5 h, the reaction mixture
was transferred to a centrifuge tube to remove the sodium nitrate. The residue
was transferred back to another flame-dried flask, 4,5-dicyano-1-p-
methoxybenzyl-imidazole (0.83 g, 4 mmol) was added to the flask, and the
reaction mixture was refluxed overnight. The reaction mixture was evaporated
to dryness and the residue was recrystallized from anhydrous methanol to
afford 1.26 g of a white solid. Yield 57%. 1H NMR (DMSO-d6): d 8.0 (s, 1H,
imidazole-CH), 7.41 (br, 1H, NH), 7.20 (d, J = 8.7 Hz, 2H, Ph-H), 7.10 (br, 2H,
NH2), 6.83 (d, J = 8.7 Hz, 2H, Ph-H), 6.30 (br, 1H, NH), 5.68 (s, 2H, benzyl-CH2),
3.67 (s, 3H, –OCH3), 1.37 (m, 2H, CH2-NH), 1.19 (s, 32H, CH2s), 0.81 (t,
J = 6.4 Hz, 3H, CH3). MS (ESI): 550 (MH+).
Biological screening procedure: New anticancer compounds were tested using
following cell lines: lung cancer: A549 and H460; breast cancer: MCF-7 and
MDA-MB-231; ovarian cancer: OVCAR-3 and prostate cancer: PC-3. Cells
(0.5–1.7 Â 103 cells/50
ll/well) were seeded in RPMI + 10% FBS in 96-well
plate the day before adding the drug dilutions. DMSO was used to dissolve
all compounds (stock 3–200 mM). Each compound was tested at nine
different concentrations: 100, 50, 10, 5, 1, 0.5, 0.1, 0.05 and 0.01 lM, final.
Each drug dilution for each drug was tested in four-replicates within each
experiment, and each experiment was repeated 1Â or 2Â. Cells treated with
DMSO (equiv volume) were used as a ‘vehicle control’. After addition of the
drug, cells were cultured for 72 h at 37 °C, 5% CO2. The experiment was
terminated by adding WST-1 cell proliferation reagent (Roche, Mannheim,
Germany) to each well and additional incubation for 4 h at 37 °C, 5% CO2.
The colorimetric readouts of cellular metabolic activity was performed by
measuring absorbance at 450–690 nm using a Synergy HT Multi-Detection
Microplate Reader and GEN5 software (Bio-Tek, Winoski, VT). Data analysis
and IC50 calculation was done using GRAPHPAD PRISM software, v.5 (La Jolla, CA).
8-Imino-4,6-diamino-N6-octadecyl-1,8-dihydroimidazo[4,5-e][1,3]diazepine (1):
Compound 5 (96.6 mg, 0.176 mmol) was added to a 25 mL round bottom
flask with TFA (5 mL), and the reaction mixture was stirred for 3 h at room
temperature. It was evaporated to dryness, the residue was treated with
saturated sodium bicarbonate, filtered and washed with cold methanol to
afford a white solid (75.5 mg, 99%). 1H NMR (DMSO, 400 MHz): d 7.80(s, 1H,
NH), 7.70(s, 1H, NH), 7.52(s, 1H, imidazole-CH), 7.45(m, 2H, 2NH), 7.40(s,1H,