Synthesis, anticancer activity, and SAR analyses of compounds containing the 5:7-fused 4,6,8-triaminoimidazo[4,5-e][1,3]diazepine ring system This paper is dedicated to Dr. Stewart W. Schneller, Professor of Chemistry, Auburn University, Alabama, on the occasion of his 75th birthday in early 2017

Article abstract of DOI:10.1016/j.bmc.2016.03.015

Described herein are our limited structure-activity relationship (SAR) studies on a 5:7-fused heterocycle (1), containing the 4,6,8-triaminoimidazo[4,5-e][1,3]diazepine ring system, whose synthesis and potent broad-spectrum anticancer activity we reported a few years ago. Our SAR efforts in this study are mainly focused on judicial attachment of substituents at N-1 and N⁶-positions of the heterocyclic ring. Our results suggest that there is some subtle correlation between the substituents attached at the N-1 position and those attached at the N⁶-position of the heterocycle. It is likely that there is a common hydrophobic binding pocket on the target protein that is occupied by the substituents attached at the N-1 and N⁶-positions of the heterocyclic ligand. This pocket appears to be large enough to hold either a C-18 alkyl chain of N⁶ and no attachment at N-1, or a combined C-10 at N⁶ and a CH₂Ph at N-1. Any alkyl chain shorter or longer than C-10 at N⁶ with a CH₂Ph attached at N-1, would result in decrease of biological activity.

Full text of DOI:10.1016/j.bmc.2016.03.015

Accepted Manuscript
Synthesis, anticancer activity, and SAR analyses of compounds containing the
5:7-fused 4,6,8-triaminoimidazo[4,5-e][1,3]diazepine ring system
Min Xie, Rena G. Lapidus, Mariola Sadowska, Martin J. Edelman,
Ramachandra S. Hosmane
PII:
S0968-0896(16)30164-X
http://dx.doi.org/10.1016/j.bmc.2016.03.015
BMC 12864
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
18 January 2016
26 February 2016
6 March 2016
Please cite this article as: Xie, M., Lapidus, R.G., Sadowska, M., Edelman, M.J., Hosmane, R.S., Synthesis,
anticancer activity, and SAR analyses of compounds containing the 5:7-fused 4,6,8-triaminoimidazo[4,5-e]
[1,3]diazepine ring system, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.
2016.03.015
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Bioorganic & Medicinal Chemistry
journal h omepage: www.els evier. com
Synthesis, anticancer activity, and SAR analyses of compounds containing the 5:7-
fused 4,6,8-triaminoimidazo[4,5-e][1,3]diazepine ring system
Min Xiea,c, Rena G. Lapidusb, Mariola Sadowskab, Martin J. Edelmanb, and Ramachandra S. Hosmanea,║,ψ,
*
a
Laboratory for Drug Design & Synthesis, Department of Chemistry & Biochemistry, University of Maryland,
Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland, 21250, USA.
b
Translational Core Laboratory, University of Maryland Marlene & Stewart Greenbaum Cancer Center, 22 South
Greene Street, Baltimore, Maryland 21201, USA
Department of Radiology and Radiological Science, Johns Hopkins University School of Medicine, Traylor 338,
c
Baltimore, MD 21205, USA
ARTICLE INFO
ABSTRACT
Described herein are our limited structure-activity relationship (SAR) studies on a 5:7-
Article history:
Received
Received in revised form
Accepted
fused heterocycle (1), containing the 4,6,8-triaminoimidazo[4,5-e][1,3]diazepine ring system,
whose synthesis and potent broad-spectrum anticancer activity we reported a few years ago.
Our SAR efforts in this study are mainly focused on judicial attachment of substituents at N-1
and N6-positions of the heterocyclic ring. Our results suggest that there is some subtle
correlation between the substituents attached at the N-1 position and those attached at the N6-
position of the heterocycle. It is likely that there is a common hydrophobic binding pocket on
the target protein that is occupied by the substituents attached at the N-1 and N6-positions of the
heterocyclic ligand. This pocket appears to be large enough to hold either a C-18 alkyl chain of
N6 and no attachment at N-1, or a combined C-10 at N6 and a CH2Ph at N-1. Any alkyl chain
shorter or longer than C-10 at N6 with a CH2Ph attached at N-1,would result in decrease of
biological activity.
Available online
Keywords:
Organic synthesis and medicinal chemistry
Triaminomidazo[4,5-e][1,3]diazepines
Anti-cancer activity
Lung, breast, prostate and ovarian cancers
In vitro screening
Structure-activity relationship (SAR) studies
DDX3 as a potential target
2015 Elsevier Ltd. All rights reserved.
1. Introduction
We have been involved for decades in the design, synthesis,
and biochemical as well as biological investigations of ring-
Tremendous efforts and resources worldwide have been invested
for over a century in the prevention, diagnosis, and treatment of
cancer, the second leading cause of mortality in North America
and Europe after heart disease, claiming a total of 1,658,370 new
cancer cases and 589,430 cancer deaths in the United States alone
in 2015.1 While the paradigms of anticancer drug discovery
have lately shifted toward molecularly targeted therapeutics due
to significant advances made in molecular biology, genomics and
proteomics, the conventional approach to drug discovery and
development based on identification of compounds with
considerable cytotoxic or cytostatic activity on multiple human
tumor cell lines with minimum toxicity to the host still remains
the single most successful route.2 The latter approach can either
be simply serendipitous or comprise of judicious design of
inhibitors of metabolic pathways that are crucial to cell division.2
expanded heterocycles (REHs) and nucleosides (RENs) with
broad spectrum antiviral/anticancer activities.3-18
These
compounds possess the characteristic 5:7-fused imidazodiazepine
ring system, and thus mimic the natural 5:6-fused purines or their
nucleoside counterparts. A host of RENs were confirmed to be
inhibitors of a wide variety of viral NTPases/helicases, including
but not limited to those of the West Nile virus (WNV), hepatitis
C virus (HCV), and Japanese encephalitis virus.13, 14
Two leading RENs were recently demonstrated to be potent
inhibitors of a human RNA helicase hDDX3 in vitro.3, 12 Since
hDDX3 over-expression inhibits apoptosis and promotes EMT
(epithelial mesenchymal transition), one of the pre-requisites for
cell transformation, cancer invasion and metastasis,2 inhibition of
hDDX3 has potentially beneficial implications in cancer
chemotherapy.19 The hypothesis concerning anticancer drugs
through inhibition of hDDX3 by RENs/REHs gained further
support when one of the 5:7:5-fused REHs, designed by
molecular modeling using the reported crystal coordinates of
hDDX3,20, 21 proved to be an excellent inhibitor of hDDX3 both
in vitro and in vivo,19, 21, 22 and is currently a viable drug candidate
for lung cancer chemotherapy.19, 23, 24
║ This paper is dedicated to Dr. Stewart W. Schneller, Professor of
Chemistry, Auburn University, Alabama, on the occasion of his
75th birthday.
ψ Retired
* Corresponding author. Tel: 561-791-6675
E-mail address: hosmane@umbc.edu (R. S. Hosmane).

The crude products were purified by recystallization from
toluene.
A few years ago, we reported the synthesis and broad-
spectrum anticancer activity of another novel REH (1) containing
the 5:7-fused imidazo[4,5-e][1,3]diazepine ring skeleton with a
long C-18 alkyl chain attached to the N6-position of the
heterocycle.25 Compound 1, a stable, crystalline solid, soluble in
aqueous acid and easy to synthesize and scale up
NC
NC
N
X
N
N
K2CO3/DMF
R1
+
RT, overnight
N
H
NC
NC
NH2
OH
4
5
N
4
5
N
R1
3
N
3
N
2. R1=H
3. R1=OMe
4. R1=F
6
6
2
2
H3C-(H2C)17
N
H3C-(H2C)17 N
H
N
N
N
7
N
7
8
8
OH
1
1
OH
O
NH2
1A
1
Scheme 2: Preparation of 4,5-dicyanoimidazole analogs
HO
The general method for the synthesis of target 5:7-fused
OH
from commercially available starting materials, was found to be
highly active in vitro against all of the cancer cell lines tested,
including lung, breast, prostate, and ovarian cancers, with IC50
values ranging 1-2 µM.25 The attachment of a long C-18 alkyl
chain to N6 of 1 was simply a serendipitous exercise based solely
on the earlier observed potent hDDX3 inhibition by two RENs
(see 1A), containing analogous ring structures with a long C-18
alkyl chain at N6 of each, but with hydroxyls replacing the amino
groups at positions 4 and 8 and with a ribose moiety attached to
one of the imidazole nitrogens.12 We describe herein our
preliminary structure-activity relationship studies on 1 by mainly
focusing on judicial attachment of substituents at N-1 and N6-
positions of the heterocyclic ring. These compounds fall under
general structural formulas represented by I and II as shown:
heterocycles with different substitutions at N6 involved
condensation of 1-substituted-4,5-dicyanoimidazole (2, 3, or 4)
with the appropriately substituted guanidine derivatives. The
latter were obtained by condensation of the corresponding alkyl-
or aralkylamine with 3,5-dimethyl-1-pyrazolylformamidinium
nitrate, following the literature procedure (Scheme 3).14 In
addition, some of these substituted guanidines (e.g., methyl-,
ethyl-, and octylguanidine) are commercially available as their
HCl or H2SO4 salts, and can be purchased directly for use in the
subsequent cyclization step.
NH2
+
N
MeOH, Reflux
R2 NH2
N
+
R2
N
NH HNO3
N
N
H
H
R2 =C2-C18-Alkyl, C4-Aralkyl
H2N
NH HNO3
NH2
NH2
N
N
N
N
N
N
N
N
R2
N
R
NH
Scheme 3: Synthesis of the appropriately substituted guanidines
from amines
NH2
NH2
The reaction was done in methanol under refluxing conditions.
Since the byproduct 3,5-dimethylpyrazole is inert in the
subsequent ring-closure reaction, the reaction mixture could be
directly carried on to the next step after removal of the solvent on
a rotary evaporator.
I
II
R1
R1=H, OMe, F
R2=H, C2-C18 alkyl; C2-C4 aralkyl
R= C2H5; C8H17; C18H37
Since structure II can apparently exist in multiple tautomeric
forms as shown in Scheme 1, the formula shown
The
final
condensation25
of
1-substituted-4,5-
dicyanoimidazole 2, 3, or 4 with the appropriate guanidinium salt
in a solution of sodium methoxide in anhydrous methanol at
reflux for 5-6 hours gave the desired imidazo[4,5-
NH
NH2
NH2
N
N
N
N
N
N
N
N
N
N
N
N
R2 HN
R2
N
R2 NH
e][1,3]diazepines as outlined in Scheme 4.
NH2
NH2
NH2
NH
NH2
NC
N
N
N
N
N
N
NaOMe/MeOH
Reflux
R1
R1
R1
R2
R2
N
NH HNO3
N
+
IIb
IIc
IIa
H
NC
NH2
Scheme 1: Multiple tautomeric forms of structural formula II
above is only tentative. No further investigations were
conducted, such as N-15/C-13 labeling studies, to assign any
specific tautomeric form for the compounds represented by
formula II. While several tautomeric forms can also theoretically
exist for structure I, the 1H NMR data of all compounds
represented by formula I strongly supported the 4,6,8-triamino
structure as drawn above.
R1
R1
2. R1=H
3. R1=OMe
4. R1=F
5: R2=(CH2)4Ph, R1=H
6: R2=(CH2)2Ph, R1=H
7: R2=(CH2)17CH3, R1=H
8: R2=(CH2)13CH3, R1=H
9: R2=(CH2)11CH3, R1=H
10: R2=(CH2)9CH3, R1=H
11: R2=(CH2)7CH3, R1=H
12: R2=H, R1=H
13: R2=CH2CH3, R1=H
14: R2=(CH2)3CH3, R1=H
15: R2=(CH2)5CH3, R1=H
16: R2=(CH2)17CH3, R1=OMe
17: R2=(CH2)7CH3, R1=OMe
18: R2=H, R1=OMe
19: R2=CH2CH3, R1=OMe
20: R2=(CH2)3CH3, R1=OMe
21: R2=(CH2)5CH3, R1=OMe
22: R2=(CH2)7CH3, R1=F
2. Chemistry
The synthesis of target heterocycles started with the
commercially available 4,5-dicyano imidazole which was easily
converted to 1-substituted benzyl-4,5-dicyanoimidazole (2, 3 and
4) by benzylation at N-126 (Scheme 2) in high yield (80-90%).
Scheme 4: Synthesis of ring-expanded heterocyles with benzyl
substituent at N-1 and different alkyl or arakyl substituents at N6

NH2
NH2
We then turned our attention to synthesize analogues of 1,
containing no substituents at the N-1 position, with lower than
C18-alkyl homologues attached to the N6-position. In this regard,
direct condensation of 4,5-dicyanoimidazole with the
appropriately substituted guanidine was attempted. While we
were successful in synthesizing the parent (23a)26 and the ethyl
analogue (23 b) by this route (Scheme 5), the method failed
N
N
N
N
Pd(OH)2/C
N
N
N
N
H
Ph(CH2)4
N
Ph(CH2)4
N
Glacial CH3CO2H
NH2
NH2
Ph
24
5
Scheme 6: Synthesis of ring-expanded heterocycle (24) by
debenzylation of 5
NH2
NH2
N NH HNO3
NC
NC
While hydrogenation on Pd/C was successful and gave a high
yield of the product, the reaction took too long to complete. An
alternative, more efficient route was to employ the p-
methoxybenzyl group for the N-1 protection as it could be easily
removed in less than 2 hours upon treatment with trifluoroacetic
acid.25, 27 Compounds 1 and 25 were synthesized by this method
from 16 and 17, respectively (Scheme 7).
N
N
N
N
N
NaOMe/MeOH
Reflux
+
R
R
HN
H
N
H
NH2
a; R=H
b; R=Et
23
a; R=H
b; R=Et
Scheme 5: Synthesis of ring-expanded heterocycles (23) by
direct condensation of 4,5-dicyanoimidazole with guanidine and
ethylguanidine
NH2
NH2
N
N
N
N
N
N
N
N
TFA, RT
2 h
R2
N
R2
NH
to provide the desired products with substituted guanidines
containing longer than C2-alkylamine chains, and largely resulted
in intractable materials. Therefore, the heterocyclic bases with a
longer than ethyl alky chain attached to the N6-position needed to
be synthesized by sequential protection-deprotection steps
involving the N-1 atom of the imidazole ring. The procedure
involved initial attachment of a benzyl or a p-methoxybenzyl
protecting group at N-1 of 4,5-dicyanoimidazole, subsequent
condensation with the appropriate guanidine derivative, followed
by removal of the benzyl or p-methoxybenzyl protecting group.
Thus, compound 24 was synthesized by debenzylation of 5 using
Palladium hydroxide/C in glacial acetic acid (Scheme 6).
NH2
NH2
OMe
1: R2=(CH2)17CH3;
25: R2=(CH2)7CH3;
16: R2=(CH2)17CH3;
17: R2=(CH2)7CH3;
Scheme 7: Synthesis of ring-expanded heterocycles 1 and 25
from 16 and 17, respectively, by removal of the N-1 p-
methoxybenzyl protecting group with trifluoroacetic acid
Table 1: Anti-cancer activity of target ring-expanded heterocycles with different alkyl and arakyl chains at the N-1 and N6- positions
NH2
N
N
N
R2 N
N
R1
NH2
Compd.
No.
IC50 (µM)
R1
R2
A549
H460
MCF7
OVCAR3
PC3
MDA-MB231
H
(CH2)17CH3
(CH2)4Ph
1.85 ± 0.21
15.30±3.96
20.40±1.84
5.3 ± 0
1.75 ± 0.25
18.80±0.10
36.65±1.05
5.4 ± 0
3.53 ± 1.94
8.80±0.57
10.40±6.93
8.30±0.14
4.10±2.55
1.40±0.14
4.55±1.77
3.80±0.82
3.00±0.57
>100
3.10 ± 2.7
37.33±3.32
31.70±18.10
4.95±0.07
5.50±3.39
3.95±1.63
4.98±1.85
3.75±1.48
4.3 ± 0
2.61 ± 0.84
>75 (3)
1.43 ± 0.40
16.65±0.07
17.95±0.64
5.40±0.14
5.85±0.64
2.05±0.21
4.75±0.78
2.55±0.07
3.60±0.71
NE
1
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
5
(CH2)2Ph
13.05±6.43
5.45±0.21
4.20±3.54
5.15±0.21
5.93±2.39
5.90±028
3.80±0.57
>100
6
(CH2)17CH3
(CH2)13CH3
(CH2)9CH3
(CH2)7CH3
(CH2)7CH3
7
4.25±1.20
1.8 ± 0
6.75±2.45
3.20±1.10
7.45±0.15
4.20±1.70
4.70 ± 0
22
8
10
11
17
22
23b
25
3.83±1.76
5.70±1.56
5.90±1.70
38
p-OMe-
CH2Ph
p-F-CH2Ph (CH2)7CH3
H
H
CH2CH3
NE
(CH2)7CH3
NE
NE
NE
NE
NE
NE
NE: not effective

position as in 1A. This is important since 1A was the general
structure for both compounds that we biochemically explored for
inhibition of hDDX3 as described earlier.12 The mere fact that
the activity was found with a hydrophobic benzyl group attached
at N-1, as in a series of compounds reported in this paper, does
not in itself negate the possibility of existence of a hydrophilic
pocket also lying in the vicinity of N-1 on the protein surface,
which might be occupied by the N-1 sugar moiety. This is
because the phenyl group of the benzyl substituent in compounds
5-22 can effectively swing around toward C-8 and partly occupy
the same hydrophobic pocket where the long N6-alkyl chain falls.
If this proves to be the case, then it might be worthwhile
exploring further SAR studies with a sugar moiety attached to N-
1 and various hydrophobic groups attached at N8 of 1 or O8 of
1A.
3. Biological Activity
In order to investigate anti-cancer activity of the target ring-
expanded heterocycles, six cancer cell lines from four major
cancers were chosen, including A549 and H460 (lung cancer),
MCF-7 and MDA-MB-231 (breast cancer), OVCAR-3 (ovarian
cancer), and PC-3 (prostate cancer). Heterocyclic bases with no
substitution at the N-1 position (1, 23, and 25) or with benzyl
substitution at N-1 (5, 6, 7, 8, 10, and 11) but with different alkyl
group at the N6 position were screened in vitro against the
mentioned six cancer cell lines. The tests were also conducted on
heterocycles containing the same substituents at the N6 position
but with different substituents at the N-1 position, including
benzyl-, p-methoxybenzyl, and p-fluorobenzyl groups (11, 17,
22). The screening data are collected in Table 1.
Comparison of biological activities of 1 and 7, containing
the same length (C-18) alkyl chain at the N6-position, suggest
that the introduction of a benzyl group at the 1-position lowers
the activity by ~3-4 fold. Reducing the length of the N6-alkyl
chain to C-14, as in 8, does not significantly alter the biological
activity profile compared to that of 7. However, a drastic change
in activity is apparent when the N6-alkyl chain was further
shortened to C-10, as in 10. The biological activity of the latter
was almost equal, better, or comparable to that of 1. When the
N6-alkyl chain was further reduced to C-8, as in 11, the activity
goes down by ~2-3 fold in four of the six cell lines screened, and
is now nearly comparable to that of 8. Introduction of a p-
methoxy- or p-fluoro substituent to the benzyl group at the 1-
position, as in 17 or 22, did not significantly alter the biological
profiles as compared to those of 8 and 11. On the other hand,
shortening of the N6-alkyl chain to either C-2 or C-8, along with
removal of the benzyl group at the 1-position, as in 23 or 25,
resulted in total loss of biological activity. Considerable loss of
biological activity also occurred when the N6-position was
substituted with a C-4-Ph or C-2-Ph chain, as in 5 or 6.
5. Experimental
5.1. Synthetic Proceedures
5.1.1 General: The H and 13C NMR spectra were recorded on
1
a JEOL ECX-400 MHz spectrometer operating at 400 MHz for
1H and 100 MHz for 13C NMR, respectively. The Chemical shift
data are reported with reference to Me4Si (internal standard) for
1H and 13C NMR spectra. The data are reported in the following
format: Chemical shift, multiplicity (s = singlet, d = doublet, dd =
doublet of doublet, t = triplet, q= quartet, m = multiplet, coupling
constants, integration and assignment. Elemental microanalyses
were performed by Atlantic Microlab, Inc., Norcross, Georgia.
Mass spectra were recorded using electrospray ionization (ESI) at
the Mass Spectral Facility, Department of chemistry and
Biochemistry, UMBC. Thin layer chromatography was
performed on Merck Kieselgel 60 GF254 (0.2 mm thickness).
Dry solvents were either purchased or prepared as follows:
methanol, methylene chloride, and acetonitrile were distilled over
calcium hydride and stored over molecular sieves (type 3Å).
Starting materials were purchased from Aldrich Chemical Co.,
Acros, and Fischer. Bulk solvents were purchased from Pharmco.
Deuterated solvents were obtained from Cambridge Isotope
Labeling Co. All solvents were reagent grade and were purchased
from VWR Scientific or Fisher Scientific.
4. Conclusions
The above biological data appear to suggest that there is
some subtle correlation between the substituents attached at the
N-1 position and those attached at the N6-position. The activity
observed when there is a C-10 alkyl chain at N6 and a CH2Ph
substituent at N-1, as in 10, was almost equal to that when there
was a C-18 alkyl chain at N6 and no substituent at N-1, as in 1.
Lengthening of the C-10 alkyl chain at N6 to C-14, as in 8, or
shortening to C-8, as in 11, results in 2-4 fold decrease in
activity. Furthermore, the activity of 11 does not seem to be
greatly affected, with an exception of breast cancer cell lines,
when either a methoxy or a fluoro group was introduced at the p-
position of the N-1 benzyl group. These results suggest that there
is a common hydrophobic pocket in the target protein that is
occupied by the hydrophobic substituents attached at the N-1 and
N6-positions of the heterocyclic ligand. This pocket is large
enough to hold either a C-18 alkyl chain of N6 and no attachment
at N-1, as in 1, or a combined C-10 at N6 and a CH2Ph at N-1, as
in 10. A p-methoxy or a p-fluoro group attached to the CH2Ph at
N-1, as in 17 or 22, must be falling outside this pocket to render
any significant effect on the biological activity. Also, with a
CH2Ph attached at N-1, any alkyl chain shorter or longer than C-
10 at N6 would result in improper fit of this hydrophobic pocket,
lowering the biological activity.
5.1.2.
1-(4-Methoxybenzyl)-4,5-dicyanoimidazole (3). A
suspension of 4,5-dicyanoimidazole (1.49 g, 12 mmol) and
potassium carbonate (1.99 g, 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
dimethylformamide. Ice was added to the residue to remove
excess potassium carbonate, and the solution was stirred for 1h,
leaving a precipitate which was filtered and washed successively
with water and methanol. The reaction afforded a white solid
1
(0.92 g, 89%). H NMR (DMSO-d6): δ 8.49 (s, 1H, imidazole-
CH), 7.30 (d, J=8.7 Hz, 2H, Ph-H), 6.97 (d, J=8.7 Hz, 2H, Ph-H),
5.39 (s, 2H, benzyl CH2), 3.75 (s, 3H, OCH3); 13C NMR (DMSO-
d6): δ 159.5, 143.4, 129.7, 126.4, 121.9, 114.4, 112.4, 111.7,
108.5, 55.2, 50.3. Anal. Calcd for C13H10N4O: C, 65.54; H, 4.23;
N, 23.52. Found: C, 65.45; H, 4.26; N, 23.46.
5.1.3.
1-(4-Fluorobenzyl)-4,5-dicyanoimidazole (4).
A
mixture of anhydrous potassium carbonate (1.82 g, 10 mmol),
4,5-Dicyanoimidazole (1.42 g, 10 mmol) and DMF (50 mL) was
stirred in a 100 mL round-bottom flask equipped with a drying
tube for 10 minutes. 4-Fluorobenzyl bromide (1.60 mL, 10
The above SAR exercise, however, does not address the
issue of the potential effect of a ribose moiety attached to the N-1

1
mmol) was added and the reaction mixture was stirred overnight
at room temperature for completion. The reaction mixture was
evaporated under vacuum to leave a bit of DMF to which was
added ice water and the mixture was stirred for 2 hours to make
the product precipitate out. The reaction mixture was filtered to
afford a white solid (1.98 g, 58%). H NMR (DMSO-d6): δ 8.50
(s, 1H, imidazole -CH), 7.41 (m, 2H, Ph-H), 7.25 (m, 2H, Ph-H),
5.48 (s, 2H, benzyl CH2).
decylguanidine nitrate (12.5% yield). H NMR (DMSO-d6): δ
8.06 (s, 1H, imidazole-CH), 7.42 (s, 1H, C=NH), 7.33-7.16 (m,
7H, 5Ph-H + NH2), 6.35 (t, J=5.3 Hz, 1H, -NHCH2), 5.81 (s, 2H,
benzyl CH2), 3.04 (m, 2H, -CH2 NH), 1.40 (s, 2H, -CH2CH2NH),
1.22 (m, 22H, CH2s in the middle), 0.85 (t, J=6.9 Hz, 3H, CH3);
13C NMR (DMSO-d6): δ 156.8, 156.4, 155.2, 140.4, 138.1, 132.2,
132.1, 128.5, 127.5, 127.3, 48.0, 41.1, 31.3, 29.1, 28.8, 28.7,
26.5, 26.4, 22.1, 13.9; MS (ESI): m/z 464 (MH+). HRMS (FAB)
Calcd for C27H42N7: m/z 464.3502. Found 464.3497.
1
5.1.4.
1-Benzyl-4,8-diamino-6-(4-phenylbutylimino)-1,6-
dihydroimidazo[4,5-e][1,3]diazepine (5). Anhydrous methanol
(15 mL) was added to a flame-dried two-necked round bottom
flask which was equipped with a drying tube and a nitrogen gas
inlet. While cooling the flask in an ice-water bath, sodium metal
(4.35 mmol, 0.10 g) was added and stirred for half an hour. 1-(4-
Phenylbutyl)guanidine nitrate (6.0 mmol, 1.25 g) was added to
the freshly prepared sodium methoxide solution and the reaction
mixture was stirred for another half an hour. The reaction mixture
was transferred to a centrifuge tube to allow precipitation of
sodium nitrate. After the sodium nitrate had been removed, the
reaction mixture was transferred to the flame-dried round bottom
flask, and 1-benzyl-4,5-dicyanoimidazole (2)26 (3.75 mmol, 0.78
g) and anhydrous methanol (25 mL) were added. The reaction
mixture was heated to reflux for 5 hours and and the solvent was
evaporated to dryness.The residue was recrystallized from
5.1.8.
1-Benzyl-4,8-diamino-6-(dodecylimino)-1,6-
dihydroimidazo[4,5-e][1,3] diazepine (9). It was prepared using
the procedure described above for 5, but using 226 and
1
tetradecylguanidine nitrate (31 % yield). H NMR (DMSO-d6): δ
8.06 (s, 1H, imidazole-CH), 7.42 (s, 1H, C=NH), 7.32-7.22 (m,
5H, 5Ph-H), 7.15 (s, 2H, NH2) 6.35 (t, J=5.5 Hz, 1H, -NHCH2),
5.81 (s, 2H, benzyl CH2), 3.04 (m, 2H, -CH2NH), 1.40 (m, 2H, -
CH2CH2NH), 1.22 (m, 18H, CH2s in the center), 0.85 (t, J=6.9
Hz, 3H, CH3); 13C NMR (DMSO-d6): δ 156.8, 156.4, 155.2,
140.5, 138.1, 132.2, 132.1, 128.5, 127.5, 127.3, 48.1, 41.1, 31.3,
29.1, 29.0, 28.8, 28.7, 26.5, 22.1, 14.0. MS (ESI): m/z 436
(MH+). Anal. Calcd for C25H37N70.5H2O: C, 67.53; H, 8.61; N,
22.05. Found: C, 67.94; H, 8.46; N, 22.09.
5.1.9.
1-Benzyl-4,8-diamino-6-(decylimino)-1,6-
1
anhydrous methanol to afford a white solid (0.40 g, 25%). H
dihydroimidazo[4,5-e][1,3] diazepine (10). It was prepared
using the procedure described above for 5, but using 226 and
NMR (DMSO-d6): δ 8.06 (s, 1H, imidazole-CH), 7.44 (s, 1H,
C=NH), 7.31-7.12 (m, 12H, 10Ph-H + NH2), 6.42 (br, 1H, -
NHCH2), 5.80 (s, 2H, benzyl CH2), 3.10 (m, 2H, -CH2NH), 2.55
(s, 2H, -CH2Ph), 1.54-1.42 (m, 4H, CH2s in the center); 13C NMR
(DMSO-d6): δ 157.40, 157.3, 156.6, 142.9, 141.5, 138.0, 133.1,
132.6, 129.4, 128.9, 128.5, 127.9, 126.4, 49.2, 41.5, 35.4, 28.9;
MS (ESI): m/z 400 (MH+). Anal. Calcd for C23H25N7: C, 69.15;
H, 6.31; N, 24.54. Found: C, 68.87; H, 6.24; N, 24.49.
1
decylguanidine nitrate (22.5% yield). H NMR (DMSO-d6): δ
8.06 (s, 1H, imidazole -CH), 7.42 (s, 1H, C=NH), 7.33-7.15 (m,
7H, 5Ph-H + NH2), 6.35 (t, J=4.8 Hz, 1H, -NHCH2), 5.81 (s, 2H,
benzyl CH2), 3.04 (m, 2H, -CH2NH), 1.39 (m, 2H, -CH2CH2NH),
1.22 (m, 14H, CH2s in the center), 0.84 (t, J=6.8 Hz, 3H, CH3);
13C NMR (DMSO-d6): δ 156.8, 156.4, 155.2, 140.5, 138.1, 132.2,
132.1, 128.5, 127.5, 127.3, 48.1, 41.2, 31.3, 29.0, 28.9, 28.7,
26.5, 22.1, 13.9; MS (ESI): MS (ESI): m/z 408 (MH+). Anal.
Calcd for C23H33N7: C, 67.78; H, 8.16; N, 24.06. Found: C,
67.91, H, 8.22, N, 24.16.
5.1.5.
1-Benzyl-4,8-diamino-6-(3-phenylpropylimino)-
1,6-dihydroimidazo[4,5-e][1,3]diazepine (6). It was prepared
employing the procedure described above for 5, but using 226 and
1-(3-phenylpropyl)guanidine nitrate (20% yield). 1H NMR
(DMSO-d6): δ 8.07 (s, 1H, imidazole-CH), 7.44 (s, 1H, C=NH),
7.30-7.12 (m, 12H, 10Ph-H + NH2), 6.46 (s, J=5.0 Hz, 1H, -
NHCH2), 5.81 (s, 2H, benzyl CH2), 3.09 (m, 2H, -CH2NH), 2.55
(t, J=7.8 Hz, 2H, -CH2Ph), 1.75 (m, 2H, CH2 in the center); 13C
NMR (DMSO-d6): δ 156.9, 156.4, 155.2, 142.0, 140.5, 138.0,
132.2, 132.1, 128.5, 128.3, 128.2, 127.5, 127.3, 125.6, 48.1, 40.9,
32.7, 30.6; MS (ESI): m/z 386 (MH+). Anal. Calcd for
C22H23N70.5H2O: C, 66.98; H, 6.13; N, 24.86. Found: C, 67.36;
H, 5.88; N, 24.49.
5.1.10.
1-Benzyl-4,8-diamino-6-(octylimino)-1,6-
dihydroimidazo[4,5-e][1,3]diazepine (11). It was prepared
using the procedure described above for 5, but using 226 and
1
octylguanidine nitrate (35% yield). H NMR (DMSO-d6): δ 8.06
(s, 1H, imidazole -CH), 7.42 (s, 1H, C=NH), 7.33- 7.15(m, 7H,
5Ph-H + NH2), 6.34 (t, J=5.5 Hz, 1H, -NHCH2), 5.81 (s, 2H,
benzyl CH2), 3.05 (m, 2H, -CH2NH), 1.39 (m, 2H, -CH2CH2NH),
1.25 (m, 10H, CH2s in the center), 0.84 (t, J=6.9 Hz, 3H, CH3);
13C NMR (DMSO-d6): δ 156.8, 156.4, 155.2, 140.5, 138.1, 132.2,
132.1, 128.5, 127.5, 127.3, 48.1, 41.2, 31.3, 28.8, 28.7, 28.6,
26.5, 22.1, 14.0; MS (ESI): m/z 380 (MH+). Anal. Calcd for
C21H29N7: C, 66.46; H, 7.70; N, 25.84. Found: C, 66.20; H, 7.68;
N, 25.49.
5.1.6.
1-Benzyl-4,8-diamino-6-(octadecylimino)-1,6-
dihydroimidazo[4,5-e][1,3]diazepine (7). It was prepared
employing the procedure described above for 5, but using 226 and
1
octadecylguanidine nitrate (48% yield). H NMR (DMSO-d6): δ
5.1.11
1-Benzyl-4,8-diamino-6-imino-1,6-
8.06 (s, 1H, imidazole-CH), 7.41 (s, 1H, C=NH), 7.31-7.15 (m,
7H, 5Ph-H + NH2), 6.34 (t, J=5.3 Hz, 1H, -NHCH2), 5.81 (s, 2H,
benzyl CH2), 3.04 (m, 2H, -CH2NH), 1.40 (m, 2H, -CH2CH2NH),
1.22 (m, 30H, CH2s in the center), 0.85 (t, J=6.9 Hz, 3H, CH3);
13C NMR (DMSO-d6): δ 156.8, 156.4, 155.2, 140.4, 138.1, 132.2,
132.1, 128.5, 127.5, 127.3, 48.0, 41.1, 31.3, 29.0, 28.8, 28.7,
26.5, 22.1, 13.9; MS (ESI): m/z 520 (MH+). HRMS (FAB) Calcd
for C31H50N7:m/z 520.4123; found 520.4115.
dihydroimidazo[4,5-e][1,3]diazepine (12). It was prepared
using the procedure described above for 5, but using 226 (0.95 g,
4.6 mmol) and guanidine hydrochloride (0.48 g, 5 mmol) with a
sodium methoxide solution (25 % wt in methanol, 2.3 mL, 10
1
mmol) in 25 mL methanol (69% yield). H NMR (DMSO-d6): δ
8.10 (s, 1H, imidazole -CH), 7.42- 7.20 (m, 8H, 5Ph-H +
NH2+NH), 6.20-5.85 (b, 2H, -NH2), 5.82 (s, 2H, benzyl CH2).
13C NMR (DMSO-d6): δ 157.6, 157.3, 156.5, 140.6, 138.0, 132.3,
132.2, 128.6, 127.5, 127.4, 48.1; MS (ESI): m/z 268 (MH+).
HRMS (FAB) Calcd for C13H14N7: m/z 268.1311. Found
268.1302.
5.1.7.
1-Benzyl-4,8-diamino-6-(tetradecylimino)-1,6-
dihydroimidazo[4,5-e][1,3]diazepine (8). It was prepared using
the procedure described above for 5, but using 226 and tetra

evaporated to dryness and the residue was recrystallized from
anhydrous methanol to afford a white solid (1.26 g, 57% yield).
1H NMR (DMSO-d6): δ 8.03 (s, 1H, imidazole-CH), 7.44 (br, 1H,
NH), 7.24 (d, J=8.7 Hz, 2H, Ph-H), 7.14 (br, 2H, NH2), 6.85 (d,
J=8.7Hz, 2H, Ph-H), 6.33 (br, 1H, NH), 5.71 (s, 2H, benzyl-
CH2), 3.70 (s, 3H, -OCH3), 3.05 (m, 2H, -CH2NH2) 1.40 (m, 2H,
-CH2CH2NH), 1.22 (s, 30H, CH2s in the center), 0.84 (t, J=
6.4Hz, 3H, CH3); 13C NMR (DMSO-d6): δ 158.7, 156.8, 156.5,
155.1, 140.2, 132.2, 131.9, 130.0, 129.1, 113.9, 55.0, 47.5, 41.2,
31.3, 29.1, 28.9, 28.7, 26.5, 22.1, 13.9; MS (ESI): m/z 550
(MH+). HRMS (FAB) Calcd for C32H52N7O: m/z 550.4233.
Found 550.4231.
5.1.12.
1-Benzyl-4,8-diamino-6-(ethylimino)-1,6-
dihydroimidazo[4,5-e][1,3] diazepine (13). It was prepared
using the procedure described above for 5, but using 226 (0.60 g,
2.88 mmol) and ethylguanidine hemisulfate (0.54 g, 4 mmol)
with sodium methoxide solution (25 % wt in methanol, 1.8 mL, 8
1
mmol) in 25 mL methanol (96% yield). H NMR (DMSO-d6): δ
8.07 (s, 1H, imidazole -CH), 7.47 (s, 1H, C=NH) 7.37- 7.10 (m,
7H, 5Ph-H + NH2), 6.35 (t, J= 5.0 Hz, 1H, -NHCH2), 5.82 (s, 2H,
benzyl CH2), 3.09 (m, 2H, -NHCH2), 1.0 (t, J=7.1Hz, 3H, CH3);
13C NMR (DMSO-d6): δ 156.9, 156.4, 155.0, 140.5, 138.1, 132.2,
132.1, 128.6, 127.5, 127.3, 48.1, 35.8, 14.5; MS (ESI): m/z 296
(MH+). HRMS (FAB) Calcd for C15H18N7: m/z 296.1624. Found
296.1627.
5.1.16.
1-(4-Methoxybenzyl)-4,8-diamino-6-(octylimino)-
1,6-dihydroimidazo[4,5-e][1,3]diazepine (17). It was prepared
using the procedure described above for 16, but using 3 and
octylguanidine hemisulfate (81% yield).1H NMR (DMSO-d6): δ
8.04 (s, 1H, imidazole-CH), 7.47 (s, 1H, NH), 7.25 (d, J=8.5 Hz,
2H, 2Ph-H), 7.19-7.15 (s, 2H, NH2), 6.86 (d, J=8.5 Hz, 2H, 2Ph-
H), 6.34 (t, J=5.0 Hz, 1H, -NHCH2), 5.72 (s, 2H, benzyl-CH2),
3.70 (s, 3H, -OCH3), 3.06 (m, 2H, -CH2NH), 1.41 (2H, m, -
CH2CH2NH), 1.22 (s, 10H, CH2s in the center), 0.83 (t, J=6.6 Hz,
3H, CH3); 13C NMR (DMSO-d6): δ158.7, 156.8, 156.5, 155.2,
140.2,132.2,132.0, 130.0, 129.1,113.9, 55.0,47.5, 41.2, 39.7,
31.3, 28.8, 28.7, 28.7, 26.6, 22.1, 13.9; MS (ESI): m/z 410
(MH+). Anal. Calcd for C22H31N7O0.5H2O: C, 63.13; H, 7.71;
N, 23.43. Found: C, 63.22; H, 7.49; N, 23.57.
5.1.13.
1-Benzyl-4,8-diamino-6-(butylimino)-1,6-
dihydroimidazo[4,5-e][1,3]diazepine (14). It was prepared
using the procedure described above for 5, but using 226 (1.04 g,
5 mmol) and butylguanidine nitrate (10 mmol) which was
prepared by 10 mmol butyl amine and 10 mmol 3,5-dimethyl-1-
pyrazolylformamidinium nitrate and directly applied to this
reaction without futher purification. The reaction mixture was
rotary evapored to leave 5-10 mL of methanol and cooled down
to let the product precipitate out of the solution. The solid was
filtered out to get the pure product (68% yield). 1H NMR
(DMSO-d6): δ 8.06 (s, 1H, imidazole -CH), 7.45 (s, 1H, C=NH)
7.39- 7.12 (m, 7H, 5Ph-H + NH2), 6.35 (b, 1H, -NHCH2), 5.82 (s,
2H, benzyl CH2), 3.08 (m, 2H, -NHCH2), 1.40 (m, 2H, -
NHCH2CH2), 1.12 (quartet of triplet, J1=J2= 7.4 Hz, 2H, -
NHCH2CH2CH2), 0.84 (t, J=7.4Hz, 3H, -CH3); 13C NMR
(DMSO-d6): δ 156.8, 156.4, 155.2, 140.5, 138.1, 132.2, 132.1,
128.5, 127.5, 127.3, 48.1, 40.8, 30.9, 19.7, 13.8; MS (ESI) m/z
324 (MH+). HRMS (FAB) Calcd for C17H22N7: m/z 324.1937.
Found 324.1945.
5.1.17.
1-(4-Methoxybenzyl)-4,8-diamino-6-imino-1,6-
dihydroimidazo[4,5-e][1,3] diazepine (18). It was prepared
using the procedure described above for 16, but using 3 and
guanidine hydrochloride (94% yield).1H NMR (DMSO-d6): δ
8.11 (s, 1H, imidazole-CH), 7.45 (s, 2H, NH2), 7.34 (s, 1H, NH),
7.23 (d, J=8.7 Hz, 2H, 2Ph-H), 6.87 (d, J=8.7 Hz, 2H, 2Ph-H),
6.34 (t, J=5.0 Hz, 1H, -NHCH2), 5.9-6.3 (b, 2H, -NH2) 5.71 (s,
1H, benzyl-CH2), 3.71 (s, 3H, -OCH3); 13C NMR (DMSO-d6): δ
158.8, 157.6, 157.4, 156.6, 140.4, 132.3,132.1, 130.0, 129.2,
113.9, 55.1, 47.6; MS (ESI): m/z 298 (MH+). HRMS (FAB)
Calcd for C14H16N7O:m/z 298.1416. Found 298.1416.
5.1.14.
1-Benzyl-4,8-diamino-6-(hexylimino)-1,6-
dihydroimidazo[4,5-e][1,3]diazepine (15). It was prepared
using the procedure described above for 5, but using 226 and
1
hexylguanidine nitrate (65% yield). H NMR (DMSO-d6): δ 8.06
(s, 1H, imidazole -CH), 7.43 (s, 1H, C=NH) 7.36- 7.12 (m, 7H,
5Ph-H + NH2), 6.35 (b, 1H, -NHCH2), 5.81 (s, 2H, benzyl CH2),
3.05 (m, 2H, -NHCH2), 1.40 (m, 2H, -NHCH2CH2), 1.22 (s, 6H,
CH2s in the center), 0.83 (t, J=6.4Hz, 3H, -CH3); 13C NMR
(DMSO-d6): δ 157.2, 157.1, 156.1, 141.2, 138.2, 132.8, 132.6,
129.2, 128.3, 127.8, 48.8, 41.5, 31.5, 29.1, 26.6, 22.5, 14.4; MS
(ESI) m/z 352 (MH+). HRMS (FAB) Calcd for C19H26N7: m/z
352.2250. Found 352.2252.
5.1.18.
1-(4-Methoxybenzyl)-4,6-diamino-6-(ethylimino)-
1,6-dihydroimidazo [4,5-e][1,3]diazepine (19). It was prepared
using the procedure described above for 16, but using 3 and
ethylguanidine hemisulfate (90% yield).1H NMR (DMSO-d6): δ
8.04 (s, 1H, imidazole-CH), 7.49 (s, 1H, NH), 7.24 (d, J=8.7 Hz,
2H, 2Ph-H), 7.18 (b, 2H, NH2), 6.87 (d, J=8.7 Hz, 2H, 2Ph-H),
6.33 (b, 1H, -NHCH2), 5.71 (s, 2H, benzyl-CH2), 3.71 (s, 3H, -
OCH3), 3.09 (m, 2H, -CH2NH), 1.01 (t, J=7.3 Hz, 3H, CH3); 13
C
5.1.15
1-(4-Methoxy)benzyl-4,6-diamino-6-
(octadecylimino)-1,6-dihydroimidazo[4,5-e][1,3]diazepine
(16). 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) were
added. The reaction mixture was refluxed for 5h. Then the
solvent was evaporated under reduced pressure to dryness. The
product octadecylguanidine nitrate was recystallized from
methanol in 90% yield and directly used for the ring-closure step.
Anhydrous methanol (10 mL) was added to a flame-dried, two-
necked round bottom flask equipped with a nitrogen gas inlet and
a condenser, to which sodium metal (0.15 g, 5 mmol) was added
and the mixture was stirred for half an hour. The
octadecylguanidine nitrate prepared previously was added to the
above sodium methoxide/methanol solution. After 0.5h, the
reaction mixture was transferred to a centrifuge tube to remove
the sodium nitrate. The residue was transferred to another flame-
dried flask containing 3 (0.83 g, 4 mmol), and the reaction
mixture was refluxed overnight. The reaction mixture was
NMR (DMSO-d6): δ 158.8, 156.8, 156.7, 155.1, 140.4, 132.3,
132.1, 130.0, 129.2, 114.1, 55.2, 47.7, 41.2, 35.8, 14.6; MS
(ESI): m/z 326 (MH+). HRMS (FAB) Calcd for C16H20N7O: m/z
326.1729. Found 326.1732.
5.1.19.
1-(4-Methoxybenzyl)-4,8-diamino-6-(butylimino)-
1,6-dihydroimidazo[4,5-e][1,3]diazepine (20). It was prepared
using the procedure described above for 16, but using 3 and
1
butylguanidine nitrate (80% yield). H NMR (DMSO-d6): δ 8.04
(s, 1H, imidazole-CH), 7.48 (s, 1H, NH), 7.23 (d, J=8.7 Hz, 2H,
2Ph-H), 7.15 (s, 2H, NH2), 6.87 (d, J=8.7 Hz, 2H, 2Ph-H), 6.36
(b, 1H, -NHCH2), 5.71 (s, 2H, benzyl-CH2), 3.71 (s, 3H, -OCH3),
3.07 (m, 2H, -CH2NH), 1.4 (m, 2H, -CH2CH2NH), 1.24 (m, 2H,
CH2CH3), 0.84 (t, J=7.3 Hz, 3H, CH3); 13C NMR (DMSO-d6): δ
159.3, 157.3, 157.1, 156.3, 141.0,132.9,132.4, 130.2, 129.6,
114.5, 55.6, 48.3, 41.3, 31.4, 20.2, 14.3; MS (ESI): m/z 354

(MH+). HRMS (FAB) Calcd for C18H23N7O: m/z 354.2042.
Found 354.2042.
(m, 2H, -CH2CH2NH), 1.45 (m, 2H, -CH2CH2Ph); 13C NMR
(DMSO-d6): δ 174.3, 161.0, 159.2, 158.7, 149.2, 142.3, 136.0,
135.2, 128.3, 128.2, 125.6, 41.0, 34.9, 28.8, 28.4; MS (ESI): m/z
310 (MH+). HRMS (FAB) Calcd for C16H20N7: m/z 310.1775.
Found 310.1776.
5.1.20.
1-(4-Methoxybenzyl)-4,6-diamino-6-(hexylimino)-
1,6-dihydroimidazo [4,5-e][1,3]diazepine (21). It was prepared
using the procedure described above for 16, but using 3 and
1
hexylguanidine nitrate (47% yield). H NMR (DMSO-d6): δ 8.04
5.1.24.
4,6-Diamino-6-(octylimino)-1,6-
(s, 1H, imidazole-CH), 7.45 (s, 1H, NH), 7.24 (d, J=8.7 Hz, 2H,
2Ph-H), 7.14 (s, 2H, NH2), 6.86 (d, J=8.7 Hz, 2H, 2Ph-H), 6.34
(b, 1H, -NHCH2), 5.71 (s, 2H, benzyl-CH2), 3.71 (s, 3H, -OCH3),
3.05 (m, 2H, -CH2NH), 1.4 (m, 2H, -CH2CH2NH), 1.22 (m, 6H,
CH2s in the center), 0.83 (t, J=6.4 Hz, 3H, CH3); 13C NMR
(DMSO-d6): δ 159.2, 157.0, 156.7, 156.4, 141.1, 133.2, 131.7,
129.9, 129.4, 114.3, 55.4, 48.2, 41.5, 31.4, 29.1, 26.5, 22.4, 14.3;
MS (ESI): m/z 382 (MH+). HRMS (FAB) Calcd for C20H28N7O:
m/z 382.2355. Found 382.2360.
dihydroimidazo[4,5-e][1,3]diazepine (25). Compound 17 (896
mg, 2.2 mmol) was placed in a round-bottom flask, and 5 ml of
TFA was added The reaction mixture was stirred for 4 hours. The
reaction mixture was evaporated to dryness, and cooled in an ice
bath while saturated sodium bicarbonate was used to neutralize
the residue. White solid precipitated out, which was filtered and
1
washed with water and mathanol to yield 600 mg (95%). H
NMR (DMSO-d6): δ 8.25 (s, 1H, NH), 8.09 (s, 1H, NH), 7.91(s,
1H, imidazole-CH), 7.83(s, 2H, 2NH), 3.29 (s, 2H, -CH2NH),
1.62 (2H, s, -CH2CH2NH), 1.25 (s, 10H, CH2s in the center), 0.84
(s, 3H, CH3); 13C NMR (DMSO-d6): δ 161.4, 157.6, 156.9, 145.3,
133.7, 133.3, 41.6, 31.5, 29.1, 28.9, 26.6, 22.3, 14.2; MS (ESI):
m/z 290 (MH+). HRMS (FAB) Calcd for C14H24N7: m/z 290.2093.
Found 290.2094.
5.1.21.
1-(4-Fluorobenzyl)-4,8-diamino-6-(octylimino)-1,6-
dihydroimidazo[4,5-e][1,3]diazepine (22). It was prepared
using the procedure described above for 16, but using 4 and
1
octylguanidine nitrate (29% yield). H NMR (DMSO-d6): δ 8.08
(s, 1H, imidazole-CH), 7.43 (s, 1H, NH), 7.33(m, 2H, 2Ph-H),
7.20-7.12 (m, 4H, 2Ph-H+2NH), 6.36 (t, J=5.5 Hz, 1H, -
NHCH2), 5.78 (s, 2H, benzyl-CH2), 3.05 (m, 2H, -CH2 NH), 1.40
(m, 2H, -CH2CH2NH), 1.25 (s, 10H, 5CH2s in the center), 0.84 (t,
J=6.6 Hz, 3H, CH3); 13C NMR (DMSO-d6): δ 162.7, 160.3,
156.8, 156.4, 155.2, 140.3, 134.3, 132.3, 131.9, 129.7, 129.6,
115.4, 115.2, 47.3, 39.5, 31.3, 28.8, 28.7, 28.7, 26.5, 22.1, 13.9;
MS (ESI): m/z 398 (MH+). Anal. Calcd for C21H28N7F0.5H2O:
C, 62.05; H, 7.19; N, 24.12. Found: C, 62.26; H, 7.23; N, 24.20.
5.1.25.
4,6-Diamino-6-(octadecylimino)-1,6-
dihydroimidazo[4,5-e][1,3]diazepine (1).25 This compound,
whose synthesis we reported earlier,25 was prepared by an
improved procedure as follows. Compound 16 (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 3h at room
temperature. It was evaporated to dryness, the residue was treated
with saturated sodium bicarbonate, and the precipitate formed
was filtered and washed with cold methanol to afford a white
1
solid (75.5 mg, 99% yield). HNMR (DMSO-d6): δ 7.80(s, 1H,
5.1.22.
4,6-Diamino-6-(ethylimino)-1,6-
NH), 7.70(s, 1H, NH), 7.52(s, 1H, imidazole-CH), 7.45(m, 2H,
2NH), 7.40(s,1H, NH), 3.29 (m, 2H, -CH2NH), 1.49 (m, 2H, -
CH2CH2NH), 1.23(s, 30H, CH2s in the center), 0.86(t, J=6.6 Hz,
3H, CH3); 13C NMR (DMSO): δ 161.0, 159.2, 158.6, 149.1,
136.0, 135.2, 41.3, 31.3, 29.0, 28.9, 28.7, 26.5, 22.1, 14.0. HRMS
(FAB) Calcd for C24H44N7:m/z 430.3658; found 430.3651.
dihydroimidazo[4,5-e][1,3]diazepine (23b). To a flame dried
apparatus containing anhydrous methanol (15 mL) was added
freshly cut sodium metal (0.18 g, 8 mmol) and stirred at room
temperature for 10 min in argon atmosphere. Then ethyl
guanidine hemisulfate 1.1 g (8 mmol) was added to the above
sodium methoxide solution. This reaction mixture was stirred at
room temperature for 1 h. Separated sodium sulfate was removed
o
5.2. Biological Proceedures
under centrifugation at 4 C and then ethyl guanidine solution
was added to the solution of dicyanoimmidazole 0.708 g (6
mmol) 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
New compounds were biologically screened for anti-cancer
activity using the following 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 assay was performed at the Greenbaum Cancer Center of the
University of Maryland School of Medicine, Baltimore. Cells
(0.5–1.7x103 cells/50 l/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 M, final. Each drug dilution for
each drug was tested in four-replicates within each experiment,
and each experiment was repeated 1 or 2 times. Cells treated with
DMSO (equiv volume) were used as a “vehicle control”. After
1
cold methanol (82% yield). H NMR (DMSO-d6) δ 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) δ 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.
5.1.23.
4,6-Diamino-6-(4-phenylbutylimino)-1,6-
dihydroimidazo[4,5-e][1,3]diazepine (24). Compound 5 (0.28
mmol, 0.1124g), palladium hydroxide on carbon (0.35 mmol,
525 mg), and 10 mL glacial acetic acid were added to the
hydrogenation bottle. The reaction mixture was hydrogenated for
18 hours at 40 psi pressure. The reaction mixture was filtrated
through a celite pad to remove the palladium-carbon complexes.
The filtrate was evaperated to leave 1 to 2 ml of acetic acid and
neutralized with sodium carbonate. The precipitate formed was
filtered and washed with methanol to yield a white solid (90%
o
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 oC, 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
calculations were done using GraphPad Prism software, v.5 (La
Jolla, CA)).
1
yield). H NMR (DMSO-d6): δ 7.84 (s, 1H, NH), 7.72 (s, 1H,
NH), 7.52(s, 1H, imidazole-CH), 7.51-7.43 (br, 2H,
1NHCH2+1NH), 7.39 (s, 1H, NH), 7.28-7.12 (m, 5H, Ph-H), 3.07
(t, J=6.8 Hz, 2H, -CH2NH), 2.6 (t, J=6.8 Hz, 2H, -CH2Ph) 1.54

23. Hosmane, R. S.; Raman, V.; Kumar, R. Application: WO
Patent 2009-US5273
2010039187, 2010, 175 pp.
Acknowledgments
The research was supported by a grant to RSH (#1R01
GM087738-01A1) from the National Institute of General
24. Hosmane, R. S.; Raman, V.; Kumar, R. US Patent#
Medical Sciences of the National Institutes of Health.
8518901 B2, 2013.
25. Xie, M.; Ujjinamatada, R. K.; Sadowska, M.; Lapidus, R.
G.; Edelman, M. J.; Hosmane, R. S. Bioorg Med Chem Lett 2010,
20, 4386.
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Graphical Abstract
Synthesis, anticancer activity, and SAR analyses of
compounds containing the 5:7-fused 4,6,8-triaminoimidazo-
[4,5-e][1,3]diazepine ring system
Min Xie, Rena G. Lapidus, Mariola Sadowska, Martin Edelman, and Ramachandra S. Hosmane*
Structure-activity relationship (SAR) studies on title heterocycles containing the general structural formulas I and II have
been reported.
NH2
NH2
NH2
NC
NC
N
N
R1=OMe
N
N
HNO3
NH
N
N
N
N
R2
N
N
N
H
R2
N
R
NH
NaOMe/MeOH
Reflux
TFA, RT
2h
NH2
NH2
R1
I
II
R1
R1=H, OMe, F
R2=H, C2-C18 alkyl; C2-C4 aralkyl
R= C2H5; C8H17; C18H37