Analogs of iso-azepinomycin as potential transition-state analog inhibitors of guanase: Synthesis, biochemical screening, and structure-activity correlations of various selectively substituted imidazo[4,5-e][1,4]diazepines

Article abstract of DOI:10.1016/j.bmc.2013.06.069

Guanase is an important enzyme of the purine salvage pathway of nucleic acid metabolism and its inhibition has beneficial implications in viral, bacterial, and cancer therapy. The work described herein is based on a hypothesis that azepinomycin, a heterocyclic natural product and a purported transition state analog inhibitor of guanase, does not represent the true transition state of the enzyme-catalyzed reaction as closely as does iso-azepinomycin, wherein the 6-hydroxy group of azepinomycin has been translocated to the 5-position. Based on this hypothesis, and assuming that iso-azepinomycin would bind to guanase at the same active site as azepinomycin, several analogs of iso-azepinomycin were designed and successfully synthesized in order to gain a preliminary understanding of the hydrophobic and hydrophilic sites surrounding the guanase binding site of the ligand. Specifically, the analogs were designed to explore the hydrophobic pockets, if any, in the vicinity of N1, N3, and N4 nitrogen atoms as well as O⁵ oxygen atom of iso-azepinomycin. Biochemical inhibition studies of these analogs were performed using a mammalian guanase. Our results indicate that (1) increasing the hydrophobicity near O⁵ results in a negative effect, (2) translocating the hydrophobicity from N3 to N1 also results in decreased inhibition, (3) increasing the hydrophobicity near N3 or N4 produces significant enhancement of inhibition, (4) increasing the hydrophobicity at either N3 or N4 with a simultaneous increase in hydrophobicity at O⁵ considerably diminishes any gain in inhibition made by solely enhancing hydrophobicity at N3 or N4, and (5) finally, increasing the hydrophilic character near N3 has also a deleterious effect on inhibition. The most potent compound in the series has a K_i value of 8.0 ± 1.5 μM against rabbit liver guanase.

Full text of DOI:10.1016/j.bmc.2013.06.069

Accepted Manuscript
Analogs of Iso-Azepinomycin as Potential Transition-State Analog Inhibitors
of Guanase: Synthesis, Biochemical Screening, and Structure-Activity Corre‐
lations of Various Selectively Substituted Imidazo[4,5-e][1,4]diazepines
Saritha Tantravedi, Saibal Chakraborty, Niti H. Shah, James C. Fishbein,
Ramachandra S. Hosmane
PII:
S0968-0896(13)00609-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.06.069
BMC 10962
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
9 May 2013
17 June 2013
28 June 2013
Please cite this article as: Tantravedi, S., Chakraborty, S., Shah, N.H., Fishbein, J.C., Hosmane, R.S., Analogs of
Iso-Azepinomycin as Potential Transition-State Analog Inhibitors of Guanase: Synthesis, Biochemical Screening,
and Structure-Activity Correlations of Various Selectively Substituted Imidazo[4,5-e][1,4]diazepines, Bioorganic
& Medicinal Chemistry (2013), doi: http://dx.doi.org/10.1016/j.bmc.2013.06.069
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com
Analogs of Iso-Azepinomycin as Potential Transition-State Analog Inhibitors of
Guanase: Synthesis, Biochemical Screening, and Structure-Activity Correlations of
Various Selectively Substituted Imidazo[4,5-e][1,4]diazepines
Saritha Tantravedi, Saibal Chakraborty, Niti H. Shah, James C. Fishbein, and Ramachandra S. Hosmane*,†
Laboratory for Drug Design & Synthesis, Department of Chemistry & Biochemistry, University of Maryland,
Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland, 21250, USA.
ARTICLE INFO
ABSTRACT
Guanase is an important enzyme of the purine salvage pathway of nucleic acid metabolism and its
inhibition has beneficial implications in viral, bacterial, and cancer therapy. The work described
herein is based on a hypothesis that azepinomycin, a heterocyclic natural product and a purported
transition state analog inhibitor of guanase, does not represent the true transition state of the enzyme-
catalyzed reaction as closely as does iso-azepinomycin, wherein the 6-hydroxy group of azepinomycin
has been translocated to the 5-position. Based on this hypothesis, and assuming that iso-azepinomycin
would bind to guanase at the same active site as azepinomycin, several analogs of iso-azepinomycin
were designed and successfully synthesized in order to gain a preliminary understanding of the
hydrophobic and hydrophilic sites surrounding the guanase binding site of the ligand. Specifically, the
analogs were designed to explore the hydrophobic pockets, if any, in the vicinity of N1, N3, and N4
nitrogen atoms as well as O5 oxygen atom of iso-azepinomycin. Biochemical inhibition studies of
these analogs were performed using a mammalian guanase. Our results indicate that (1) increasing the
hydrophobicity near O5 results in a negative effect, (2) translocating the hydrophobicity from N3 to N1
also results in decreased inhibition, (3) increasing the hydrophobicity near N3 or N4 produces
significant enhancement of inhibition, (4) increasing the hydrophobicity at either N3 or N4 with a
simultaneous increase in hydrophobicity at O5 considerably diminishes any gain in inhibition made by
solely enhancing hydrophobicity at N3 or N4, and (5) finally, increasing the hydrophilic character near
N3 has also a deleterious effect on inhibition. The most potent compound in the series has a Ki value of
8.0 ±1.5 M against rabbit liver guanase.
Article history:
Received
Received in revised form
Accepted
Available online
Keywords:
Organic synthesis and medicinal chemistry
Inhibitors of guanase
Imidazo[4,5-e][1,4]diazepines
Iso-Azepinomycin analogs
Biochemical inhibition studies
Structure-activity relationship (SAR) studies
2013 Elsevier Ltd. All rights reserved.
was further demonstrated that inhibition of guanase could be
caused selectively in pathogens without seriously affecting the
1. Introduction
Guanase or guanine deaminase (EC 3.5.4.3) is an important
metalloenzyme in the salvage pathway of purine metabolism,
catalyzing the hydrolytic deamination of guanine to xanthine.1, 2
Inhibition of guanase has beneficial implications in viral,
bacterial, and cancer therapy.3-8 While many studies of guanase
inhibition have been reported both from our lab9-16 and others,17-27
it is still one of the least explored enzymes of nucleic acid
metabolism, although it has been isolated from rabbit liver,28
human liver,29 and rat brain30 many years ago.
nucleic acid biosynthesis of the host mammals that largely
employ the de novo pathway.31 A potent guanase inhibitor will
also be an excellent tool for studying a number of disease-related
metabolic processes in which the enzyme is involved either as an
indicative or a causative factor. There are several documented
reports on detection of abnormally high levels of serum guanase
activity in patients with liver diseases like hepatitis. Increased
levels of hepatitis C have been observed when patients with HCV
are transfused with blood containing high levels of serum
guanase activity.8, 32 It is further known that high serum guanase
activity is a clear biochemical indicator of organ rejection in liver
transplant patients.33 In addition, patients with multiple sclerosis
have significantly elevated levels of guanase activity in their
cerebrospinal fluid, and a clear correlation was established
between the extent of disability and the level of guanase activity.5
A variety of pathogens are dependent upon the host’s purine
salvage pathway for the nucleotides necessary for their survival,
as they do not possess a de novo purine biosynthetic route.31 It
________
* Corresponding author. Tel: 561-791-6675
E-mail address: hosmane@umbc.edu (R. S. Hosmane).
† Retired.
Another important aspect of guanase activity is its
involvement in cancerous tissues. It has long been known that
carcinogenic processes and the activities of some enzymes in

cancer tissues and cells are strongly interrelated. In this regard, it
is important to consider reports of abnormal levels of guanase
activity in various cancer tissues in lung,34 kidney,4 and breast
cancer tissues.3 It is suggested that this difference in activity is a
physiological attempt of the cancer cell to regulate the guanine
and /or xanthine level, which are needed by cancer cells to
is considerably less likely in azepinomycin as it involves the
lactam nitrogen, which cannot provide as efficient anchimeric
assistance as an amino nitrogen of the previous case in
elimination of a water molecule to form an imine. Nonetheless,
we were optimistic that the corresponding alkoxy derivatives of
iso-azepinomycin, especially with long alkyl chains, would have
reasonable stability to explore some important preliminary
information on the hydrophobic or hydrophilic residues
surrounding the ligand in the enzyme active site.
accelerate their salvage metabolic pathway activity.
The
alternate de novo metabolic pathway is mostly employed by
normal cells for replication. So, a guanase inhibitor could
discriminately check the growth of cancer cells without affecting
the normal cells. In view of these reasons, it is timely and
important to search for a suitable guanase inhibitor that may
assist in exploring guanase as a novel chemotherapeutic target as
well as in understanding the specific physiological and
biochemical role played by guanase in a number of metabolic
disorders in which guanase is suspected to be involved.
Therefore, assuming that iso-azepinomycin would bind to
guanase at the same active site as azepinomycin, the present work
is aimed at gaining
a preliminary understanding of the
hydrophobic and hydrophilic sites surrounding the guanase
binding site of iso-azepinomycin. This is a starting point in any
new drug design targeting an enzyme. In particular we intend to
explore the hydrophobic or hydrophilic pocket, if any, in the
vicinity of N1, N3, and N4 nitrogen atoms as well as O5 oxygen
atom of iso-azepinomycin. To this end, we set out to design and
synthesize eleven analogs of iso-azepinomycin (see Figure 2)
2. Hypothesis, Specific Aims, and Rationale
Azepinomycin is a naturally occurring moderate inhibitor of
guanase, isolated from the culture filtrates of Streptomyces sp.
MF718-03.35 It is also a purported transition state analog
inhibitor of the enzyme-catalyzed reaction with an IC50 of ~ 5 M
O
7
1
N
8 NH
in tissue cultures,35 and a Ki of ~2.5 M against an islolated
mammalian enzyme from rabbit liver.12 However, when one
closely looks at the reaction catalyzed by guanase, which is the
hydrolysis of guanine to xanthine via the aminol intermediate
(see Figure 1), it is apparent that the carbon and nitrogen atoms
involved in the hydrolysis reaction are C2 and N3, respectively,
and not C2 and N1.
2
6
5
N
H
3
N
OH
H4
Iso-azepinomycin
O
O
NH
N
N
NH
N
N
The hydrolysis apparently involves the imine nitrogen N3
instead of the lactam nitrogen N1. Therefore, if one extrapolates
this to azepinomycin as the purported transition state analog and
iso-azepinomycin as a hypothetical transition state analog
(Figure 1), it is the latter that more closely resembles the
transition state as it can be regarded as an aminol formed from
the hydrolysis of an imine precursor.
N
H
N
H
OR
OR
R'
1; R=H
5; R=CH3; R'=OCH3
6; R=CH2CH3; R'=OCH3
7; R=CH3; R'=F
2; R=CH3
3; R=CH2CH3
4; R=CH2CH2CH2CH3
O
O
O
1
1
+ H2O
1
NH
H3CO
- NH3
N
N
N
NH
NH
2 NH2
2
2
Guanase
N
H
N
H
H3CO
N
H
N3 NH2
Guanine
N
H
3 OH
N3
O
O
N
H
O
NH
OCH3
N
Xanthine
NH
N
N
Intermediate
O
N
H
NH
N
N
N
OR
O
O
N
H
7
7
NH
8 NH
1
N
1
N
8
OCH3
2
2
OH
6
6
10; R=CH3
11; R=CH2CH3
9
8
N
H
3
N
H
3
5
N
N
5
OH
H4
H4
Figure 2:The target analogs of iso-azepinomycin
Azepinomycin
Iso-azepinomycin
Figure 1: Comparison of the intermediate of the guanase-
catalyzed hydrolysis reaction with the purported and proposed
transition state analog inhibitors azepinomycin and iso-
azepinomycin
The biochemical inhibition data of the proposed analogs 1-4
against guanase are anticipated to throw some light on the
hydrophobic environment, if any, in the vicinity of N3 and O5 of
the ligand in the enzyme binding site, while analogs 5-7 would
serve as probes to map spatial and electronic environments
beyond where the phenyl group is seated on the protein.
Likewise, inhibition data of analogs 8 and 9 would provide
important information on the hydrophobic pockets, if any, in the
vicinity of N1 and N4, while compound 10 would assess if the
enhanced hydrophobicity at both N1 and N4 would have an
additive or deleterious effect on the enzyme inhibion. If the
hydrophobic effect indeed turns out to be additive, then analog
Therefore, the work described herein is based on the above
hypothesis that iso-azepinomycin could be a better analog than
azepinomycin in mimicking the true transition state of the
guanase-catalyzed deaminase reaction of guanine to xanthine.
However, we realized early on that there was one potentially
serious stability problem in realizing such a compound. This is
because of the potentially facile equilibrium existing between an
imine and an aminol in iso-azepinomycin. Such an equilibrium

11 would determine if further enhancement of hydrophobicity at
was preferred among all the eluting solvent systems attempted
afforded methyl analog 2 (65% yield), ethyl analog 3 (40%) and
butyl analog 4 (22%).
O5 would fair well for the overall enzyme inhibition.
3. Chemistry
Synthesis of the target analogs 1-4 is outlined in Scheme 1.
The synthesis commenced with 1-benzyl-5-nitroimidazole-4-
carboxylic acid (12),36 which was converted into the desired
amide (13) in 86% yield through activation with 1,1’-
carbonyldiimidazole (CDI), followed by treatment with
aminoacetaldehyde dimethyl acetal. The nitro group of 13 was
reduced under neutral reaction conditions using Pd-C/H2 in
methanol to obtain 14 in 54% yield. The reaction of 14 with 1N
A logical starting material for the synthesis of target
compounds 5, 6, and 7 would be an analog of 12 containing a
methoxy or fluoro substituent at the para position of the phenyl
ring. Surprisingly, a thorough examination of the literature
revealed that despite their simple structures, neither 16a nor 16b
Scheme 2) was known. Therefore, compounds 16a and 16b
were synthesized starting from 4(5)-methyl-5(4)-nitro-1H-
imidazole (17), employing procedures well-established in our
lab.37
MeO
MeO
MeO
MeO
O
O
Ph
HOOC
O2N
N
H
N
H
CH3
NO2
p-OCH3PhCH2Cl or p-FPhCH2Cl
N
N
N
N
CDI/ THF
PhCHO
N
N
N
H
H
Piperidine
NO2
K2CO3/DMF, 85o
91-95%
C
H2N
O2N
N
N
N
100o-110o
77%
C
Pd-C/ H2
5 h
MeO
18
17
NH2
86%
MeO
54%
14
13
12
Ph
NO2
N
N
N
N
1N HCl, rt
overnight
~100%
Column Chromatography
(Silica gel)
+
ROH, 1N HCl,
50OC, 24h
NO2
Ph
R
O
O
N
N
R
20
19
HN
HN
N
a; R=OCH3, 50%
b; R=F, 63%
a; R=OCH3, 41%
b; R=F, 32%
O
HN
ROH, 1N HCl
rt, 6h
RO
N
HO
N
N
N
H
H
Ph
COOH
NO2
N
N
N
N
N
H
(1)
KMnO4
N
2N NaOH
NO2
2; R= Methyl
3; R= Ethyl
4; R= Butyl
(65%)
(2) Aq. HCl
58-61%
1
R
15
(40%)
(22%)
R
19
16
a; R=OCH3, 61%
b; R=F, 58%
a; R=OCH3
b; R=F
Scheme 1. Synthesis of the target analogs 1-4
Scheme 2. Synthesis of the Key Intermediates 16a and 16b
aqueous HCl at room temperature overnight resulted in the
complete conversion to the ring closed product 1 as revealed by
1H NMR analysis. While compound 1 was reasonably stable in
an aqueous acidic environment, it was quite unstable and
decomposed under neutral reaction conditions, Thus, all our
attempts to isolate 1 in a pure solid form were futile, although we
The styrylation of 17 using benzaldehyde and piperidine gave
the 4(5)-styryl-5(4)-nitroimidazole (18) as a yellow solid in 77%
yield. The latter was then benzylated using p-methoxybenzyl
chloride or p-fluorobenzyl chloride and potassium carbonate in
DMF at 85° C to yield a mixture of regioisomers, namely, 1-p-
methoxybenzyl-5-nitro-4-styrylimidazole, 19a (50% yield) and
1-p-methoxybenzyl-4-nitro-5-styrylimidazole, 20a (41%), or 1-p-
fluorobenzyl-5-nitro-4-styrylimidazole, 19b (63%) and 1-p-
fluoro-benzyl-4-nitro-5-styrylimidazole, 20b (32%), respectively.
The two isomers in each case were separated by silica gel column
1
were able to obtain the necessary H and 13C NMR data, as well
as its high resolution mass spectral data, from the crude reaction
mixture in D2O, all of which were consistent with structure 1.
Apparently, the hydroxy group of 1 at position 5 is very labile
and presumably undergoes facile elimination reaction via
anchimeric assistance from the adjacent NH at the 4-position to
form a highly reactive iminium ion 15. The latter can readily
undergo nucleophilic addition reactions with reactive species
present in the medium, including but not limited to self
condensation reaction with another molecule of 1, to form an
intractable material. Support for this notion was obtained by
treatment of the crude acidic reaction mixture of 1, obtained from
14, with methanol, ethanol or butanol at room temperature for 6
hours, which gave the corresponding target compounds 2, 3, or 4,
respectively. It was also possible to obtain the target compounds
through direct reaction of 14 with the corresponding alcohols in
1N HCl at 50° C for 24 hours. The purification of the products
was, however, a little tricky as they readily underwent exchange
reactions with methanol when it was used either alone or as part
of a mixture of an eluting solvent in silica gel column
chromatography. Unfortunately, the target compounds could not
be eluted off the column without using at least a minimum
amount of a protic solvent such as methanol. The use of a
mixture of chloroform: methanol: ammonium hydroxide (5:2:0.2)
1
chromatography and were distinguished from each other by H
NMR chemical shifts of the N-alkyl and H-2 protons, both of
which are slightly more deshielded in 19 as compared with those
of 20. This is due to the combined inductive and resonance
effects of the nitro group attached at position 4 or 5 of the
imidazole ring. As shown in Figure 3, the electron-withdrawing
nitro group in 19 is conjugated to position-2 of the imidazole
ring, thus causing H-2 to be deshielded. Such a conjugation is not
possible in 20. Likewise, the electron-withdrawing inductive
effect of the nitro group upon the benzyl CH2 is expected to be
more pronounced in 19 as compared with 20, due to close
proximity of the two functional groups in the former. This
1
regioisomeric structural assignment based on the H NMR data
has further been corroborated by single crystal X-ray diffraction
analysis of 1-methyl-4-nitro-5-styrylimidazole reported from this

H
C
column chromatography was somewhat tricky as they readily
underwent exchange reactions when different alcohols other than
what is dictated by the alkoxy substituent in the product was used
for elution. The product would not move down the silica gel
column unless an appropriate combination of alcohol and
ammonium hydroxide, along with chloroform or any other non-
protic solvent, was used for elution. The use of chloroform:
methanol: ammonium hydroxide (10:1:0.05) or chloroform:
ethanol: ammonium hydroxide (10:1:0.05) solvent system,
respectively, afforded 5 or 6 in 57% or 36% yield, respectively,
while the use of chloroform: methanol: ammonium hydroxide
(12:1:0.05) gave 7 in 42% yield.
Synthesis of target 8 (Scheme 4) commenced with 20a, whose
synthesis has been described earlier. Compound 20a was
oxidized to the corresponding carboxylic acid 25 using the same
procedure as described earlier for the conversion of 19 to 16 (cf.
Scheme 2)
NO2
CHPh
CHPh
N
N
N
N
C
O
N
O
H
R
R
19
R=OCH3 or F
20
Figure 3: Resonance effect in the two regioisomeric products 19
and 20
laboratory.38 Finally, oxidation of the 19a and 19b with aqueous
potassium permanganate gave the desired 16a and 16b, in 61%
and 58% yields, respectively.
1-p-Methoxy-
carboxylic acid (16a or 16b) was further condensed with
aminoacetaldehyde dimethylacetal, employing CDI as
or
1-p-fluorobenzyl-5-nitroimidazole-4-
a
condensing agent (Scheme 3) to obtain the amide 21a or 21b in
83% or 85% yield, respectively. The nitro group in each was then
reduced under neutral conditions using Pd-C/H2 in methanol to
obtain the corresponding amine 22a or 22b in 76% or 58% yield,
respectively. Reaction of 22a or 22b with 1N aqueous HCl at
NO2
NO2
N
N
N
N
O
O
NH2
KMnO4
2N NaOH
COOH
Ph
54%
CDI/THF
87%
H3CO
H3CO
20a
25
O
O
NH2
O
NH2
NO2
COOH
NO2
N
N
N
N
N
O
O
O
O
H
H
N
N
Pd-C/H2
1N HCl; rt
overnight
N
N
N
O
N
H
O
NO2
O
O
MeOH
65%
CDI/THF
83-85%
H3CO
H3CO
16
21
R
R
26
27
a; R=OCH3 (83%)
b; R=F (85%)
a; R=OCH3
b; R=F
Pd-C/H2
MeOH
5 h
58-76%
H
N
OH
NH
N
N
H
N
OCH3
O
MeOH, 1N HCl
rt, 5h
N
N
O
NH
N
N
1N HCl, rt
overnight
O
NH
NH
N
N
O
+
O
N
N
O
N
H
52%
N
H3CO
OH
H
N
O
8
OCH3
H3CO
28
H
NH2
R
23
22
5
R
R
5; R=OCH3
7; R=F
a; R=OCH3 (76%)
b; R=F (58%)
a; R=OCH3
b; R=F
Scheme 4: Synthesis of target analog 8
Compound 25 was carried through the same sequence of steps as
described earlier for the conversion of 16 to targets 5-7 (cf.
Scheme 3). The H NMR of the crude solid isolated from the
ROH/1N HCl
50o C, 24h
1
ROH/1N HCl
rt; 6 h
reaction of 27 with aqueous HCl overnight indicated complete
conversion to 28, but no attempts were made to purify the
product because of the known instability and prior experience
with difficulties associated with purification of such compounds
in our lab, including but not limited to 23. The target 8 was
obtained in a pure form after column chromatography in 52%
yield.
O
NH
OR'
N
N
O
NH
N
N
N
H
N
H
R
5, R =OCH3, R'=CH3 (57%)
6, R = OCH3, R'=CH2CH3 (36%)
7, R=F, R'=CH3 (42%)
R
24
Synthesis of target 9 proceeded from 18, which was converted to
the desired starting material N-(2,2-dimethoxyethyl)-5-nitro-1H-
imidazole-4-carboxamide (30)39 via 5-nitro-1H-imidazole-4-
carboxylic acid (29),37 employing the procedures reported from
Scheme 3: Synthesis of the target analogs 5, 6 and 7
39
our lab several years ago.37,
Compound 30 was reduced by
room temperature overnight resulted in the complete conversion
to a mixture of the ring closed compounds 23a or 23b and 5 or 7,
respectively. When the latter mixture was treated with excess
methanol and stirred at room temperature for several hours, the
target 5 or 7 was formed exclusively. Similarly, when the mixture
23 and 5 was treated with excess ethanol, the corresponding ethyl
analog 6 was formed. Apparently, the reaction proceeds through
an iminium ion intermediate 24, which undergoes nucleophilic
addition reactions with methanol and ethanol to form the
observed target products 5 and 6, respectively. Compound 5, 6
or 7 could also be directly synthesized from 22 by reaction with
corresponding methanol or ethanol using 1N aqueous HCl at 50
0C for 2 hours. Purification of the resulting analogs using
catalytic hydrogenation over Pd-C at 60 psi to the amine 31 in
50% yield. The 5-amino group of 31 was benzylated by
sequential reactions with benzaldehyde, catalyzed by TFA,
followed by reduction with sodium cyanoborohydride to obtain
32 in 43% yield. Compound 32 was ring-closed to form 33 upon
reaction with 1N HCl at room temperature for 8 hours. Without
further purification, the crude solid 33 obtained after evaporation
of the solvent was stirred with excess methanol using catalytic
amounts of 1N HCl for 8 hours at room temperature, which gave
the desired 9 in 38% yield.

O
Ph
substrate guanine at λmax 245 nm. The change in optical density
per unit time is a measure of the guanase activity. A total of 5-6
different concentrations of the substrate, ranging from 5 to 40
μM, was employed for each inhibitor concentration that was
either 10, 20, or 30 μM. The Lineweaver-Burk plots (1/V versus
1/S) were generated and used to calculate KM, Vmax, and Ki.24
Our biochemical results showed that KM of enzyme with guanine
as substrate is 5.5 x 10-6 M. A representative Lineweaver-Burk
plot is shown for compound 2 (Figure 4). The Ki data for all the
target compounds are collected in Table 1, and the detailed
kinetic data, graphs, and calculations associated with biochemical
inhibition studies all target compounds (2-11) have been
provided in the Supplementary Data available from the online
version of the Journal.
COOH
NO2
N
O
KMnO4
1. SOCl2
N
N
N
H
O
N
H
N
H
N
H
1N NaOH
80%
NO2
NO2
O
2.
NH2
O
18
30
29
76%
Pd-C/H2
MeOH
50%
60 psi
O
O
1. PhCHO/TFA
MeOH
O
O
N
O
1N HCl, rt
8 h
N
N
H
N
H
O
N
H
NH2
N
H
NH
2. NaCNBH3
43%
32
31
O
NH
O
N
MeOH/1N HCl (cat.)
NH
N
N
H
N
OH
N
H
N
rt, 6h
38%
OCH3
33
9
Scheme 5: Synthesis of target 9
Synthesis of targets 10 and 11 was accomplished (Scheme 6)
starting from 27 (cf. Scheme 4), which was converted into the
corresponding N-benzyl derivative 34 in 93% yield using an
analogous procedure as described above for the conversion of 31
to 32. Treatment of 34 with 1N HCl at room temperature
overnight, evaporation of volatiles, followed by reaction of the
residue with methanol or ethanol, catalyzed by 1N HCl at room
temperature for 8 hours, provided the target analog 10 or 11 in
38% or 31% yield, respectively.
Figure 4: Lineweaver-Burk plot for guanase inhibition study of
target compound 2.
NH2
H
O
N
N
(1) TFA/MeOH
rt, overnight
O
NH
N
N
Table 1. Biochemical inhibition (Ki) data of target compounds 2-
11 against rabbit liver guanase. For comparison, the inhibition
data of azepinomycin, reported by us recently,12 has been
included.
N
O
O
H
N
O
O
+
(2) NaBH3CN
rt, 8 h
O
H3CO
27
34
93%
H3CO
1N HCl
General Chemical
Target Compounds
Ki (µM)
rt, overnight
Structure
O
NH
N
N
1; R=H
2; R=CH3
3; R=CH2CH3
4; R=CH2CH2CH2CH3
--
40±1
47±2
54±3
N
OR
NH
OH
NH
OCH3
NH
OR
N
ROH, 1N HCl
rt, 8 h
H
N
O
N
N
N
N
N
+
N
N
O
O
O
H3CO
H3CO
H3CO
NH
N
N
10; R = CH3 (38%)
11; R = CH2CH3 (31%)
10
35
5; R=CH3; R’=OCH3
6; R=CH2CH3; R’=OCH3
7; R=CH3; R’=F
21±2
33.4±2.5
39.7±2
N
H
OR
Scheme 6: Synthesis of targets 10 and 11
R'
All isolable intermediates and products were fully
H3CO
1
characterized by H NMR, 13C NMR, and MS data, coupled with
elemental microanalyses and/or HRMS data
O
4. Biochemistry
47.5±1
8.8±0.8
8
9
NH
N
N
Guanase inhibition studies of target compounds 2-11 were
N
H
conducted using guanase from rabbit liver (MP Biochemicals) at
OCH3
o
25 C and pH 7.4 by spectroscopically measuring the rate of
O
hydrolysis of the substrate guanine at λmax 245 nm. Stock
solutions of the substrate, enzyme and inhibitors were prepared
using 0.05 M Tris buffer (pH 7.4). The enzyme kinetics were
followed by measuring decrease in absorbance per minute of the
NH
N
N
H
N
OCH3

H3CO
LC-Quadrupole ion trap spectrometer. High resolution mass
spectra were recorded a Bruker Daltonics (Billerica, MA) Apex
IV FTICR. Melting points were recorded on Hoover capillary
melting point apparatus and are uncorrected. All the reagents
were purchased from Sigma-Aldrich or Fisher Pharmaceuticals.
Anhydrous solvents were purchased in the highest purity
available and used without further purification or drying. Bulk
solvents were purchased from Pharmco. Deuterated solvents
were purchased from Cambridge Isotope Labeling Co. All
reactions sensitive to air or moisture were performed in an inert
atmosphere of nitrogen.
O
N
10; R=CH3
11; R=CH2CH3
26.5±1.5
43.8±0.6
NH
OR
N
N
O
7
1
8 NH
6 OH
N
2
Azepinomycin
2.5±0.6
N
H
3
N
5
H4
6.2. Organic Synthesis
5. Conclusions
6.2.1. 1-Benzyl-N-(2,2-dimethoxyethyl)-5-nitro-1H-imidazole
-4-carboxamide (13)
As stated under Rationale (see section 2 above), the scope of
the present work is limited to exploring the hydrophobic
environment, if any, near N1, N3, N4, and O5 sites of iso-
azepinomycin. Based on the observed enzyme inhibition data,
we conclude that (a) increasing the hydrophobicity near O5 has a
negative effect on inhibition as revealed by the Ki data of targets
2-4, (b) further enhancement of hydrophobicity beyond the
phenyl group at N3, as in target 5, favors inhibition, but any
simultaneous enhancement of hydrophobic character at O5, as in
target 6, would diminish the gain made by increased
hydrophobicity at N3, (c) increasing the hydrophilic character
with concurrent decrease in hydrophobicity, for example with a
fluoro substituent attached to the phenyl group at N3, as in target
7, brings down the potency of inhibition, (d) enhanced
hydrophobicity at N1, as in target 8, also has deleterious effect on
inhibition, (e) on the other hand, increasing the hydrophobicity at
N4, as in target 9, has a large positive effect on inhibition, (f)
simultaneous increase of hydrophobicity at both N4 and N1, as in
target 10, diminishes any gain made by increasing the
hydrophobicity only at N4, and finally (g) any simultaneous
increase of hydrophobicity near O5, results in large negative
effect on inhibition
A
mixture of 12 (2.0 g, 8.1 mmol) and 1, 1’-
carbonylimidazole (2.0 g, 12.3 mmol) in dry THF (60 mL) was
refluxed in a flame-dried three-neck flask kept under a nitrogen
atmosphere. When the mixture turned clear it was allowed to cool
to room temperature. Amino acetaldehyde dimethylacetal (1 mL,
10 mmol) was added to the cooled solution and stirred for 2 h.
The reaction was monitored for completion. The solvent was
rotary evaporated to dryness and ice-water was added and stirred
overnight. Then the product was extracted with chloroform and
washed with water. The organic layer was rotary evaporated and
purified by silica-gel column chromatography with EtOAc : Hex
(1:1) as the eluting solvent system. The appropriate fraction was
1
collected to give 13. Yield 2.31g, 86%; HNMR (DMSO-d6) δ
8.5 (t, 1H, NH, exchangeable with D2O), 8.24 (s, 1H, Imid-H2),
7.3 – 7.16 (m, 5H, Ar-H), 5.5 (s, 2H, Ar-H), 4.4 (t,1H, acetal-H),
3.29 (d, J = 5.52 Hz, 2H, CH2), 3.24 (s, 6H, 2xCH3); 13C-NMR
(CDCl3) δ 40.9, 51.5, 54.5, 102.6, 127.8, 129.2, 129.4, 133.2,
136.8, 159.4; MS (ESI) m/z 335 (MH+); Anal. Calcd for
C15H18N4O5: C, 53.89, H, 5.43, N 16.76; found C, 53.86, H, 5.42,
N, 16.80.
In summary, our results suggest that there is a large
hydrophobic pocket in the vicinity of N3 and N4 as revealed by
the guanase inhibition data of compounds 5 and 9. It is not yet
clear, however, if simultaneous increase of hydrophobicity at
both N3 and N4 would have additive or diminished effect on
inhibition. The best compound in the series so far is compound 9
containing a phenyl substituent at N4, which shows a Ki of ~8
M. Although the inhibitory potency of 9 is somewhat lower than
the natural guanase inhibitor azepinomycin with a Ki of ~2.5 M,
it is not too unreasonable to hope that further structure-activity
relationship studies on 5 and 9, for example, by further increasing
hydrophobicity at their respective N3 and/or N4 position, would
lead to more potent inhibitors of guanase, and not to mention that
much yet needs to be explored about the possible hydrophilic
pocket surrounding N1 and the hydrophilic/ hydrophobic pockets
surrounding N7 and O8 sites of iso-azepinomycin.
6.2.2. 5-Amino-1-benzyl-N-(2,2-dimethoxyethyl)-1H-imidazo
le-4-carboxamide (14)
A mixture of 13 (2.3 g, 6.7 mmol) and Pd-C (10%) (200 mg)
in absolute methanol (50 mL) was hydrogenated in a Parr
hydrogenator at 40 psi for 5 h. The reaction mixture was filtered
through Celite, and the filtrate evaporated to dryness on a rotary
evaporator. The yellow semi-solid residue was triturated with
diethyl ether to obtain 14 as a yellowish white solid. Yield 1.1 g,
1
54%; HNMR (DMSO-d6) δ 7.33–7.16, 7H), 5.7 (s, 2H, NH2 ,
exchangeable with D2O), 5.04 (s, 2H, CH2), 5.0 (s, 2H, NH2,
exchangeable with D2O), 4.1-4.0 (q, 4H, 2xCH2), 3.65 (s, 3H,
OMe), 4.43 (t, 1H, acetal-H), 3.24 (d, J = 5.52 Hz, 2H, CH2),
3.20 (s, 6H, 2xCH3); 13C-NMR (CDCl3) δ 40.1, 47.4, 54.3,
103.1, 115.2, 126.8, 128.6, 129.4, 130.4, 134.6, 141.9, 165; MS
(ESI) m/z 305 (MH+); Anal. Calcd for C15H20.5N4O3.25: C, 59.20,
H, 6.62, N, 18.41; found C, 58.68, H, 6.64, N, 18.31.
6.2.3.
3-Benzyl-5-hydroxy-4,5,6,7-tetrahydroimidazo[4,5-e]
6. Experimental
6.1 General
[1,4]diazepin-8(3H)-one (1)
Compound 14 (0.8g, 2.6 mmol) was taken in a clean flask and
20 mL of 1N HCl was added and stirred at room temperature for
10 h. The color of the solution turned from red to light yellow.
The acidic solution was concentrated under vacuum and then was
neutralized to pH 7. The neutral solution was kept in the cold (4
°C) for complete precipitation. The precipitate was then gravity
1
The H and 13C NMR spectra were recorded on a JEOL-400
1
NMR spectrometer, operating at 400MHz for H and 100 MHz
for 13C NMR, respectively. Thin layer chromatography was
performed on Merck Kieselgel 60 F254 (0.2 mm thickness). Flash
chromatography was performed using 32-63 mesh silica gel.
Mass spectra were recorded on a Brucker Daltonics Esquire-3000
1
filtered and washed with water. Mass spectral and H NMR data

of the aqueous filtrate shows exclusive formation of 1. The latter,
however, decomposes in neutral aqueous medium over time. The
following are the spectral and analytical data of the crude
reaction mixture containing 1: 1HNMR (D2O) δ 3.25 (d, J= 14.64
Hz, 1H), 3.66 (m, 1H), 5.32 (d, J= 5.52 Hz, 2H), 5.37 (d, J= 5.52
Hz, 1H), 7.49–7.34 (m, 5H), 8.51 (s, 1H); 13C-NMR (D2O) δ
44.4, 47.1, 74.4, 112.0, 126.9, 128.3, 129.2, 135.0, 135.6, 140.8,
168.7; HRMS (FAB) Calcd. for C13H14O2: m/z 259.1190 (MH+);
obsd. m/z 259.1188.
31.6, 44.1, 46.7, 65.8, 80.1, 114.2, 127.6, 128.0, 129.0, 133.7,
137.2, 139.3, 166.7; MS (ESI) m/z 315 (MH+); HRMS (FAB)
Calcd. for C17H23N4O2: 315.1815 (MH+); obsd. m/z 315.1811.
6.2.7. 5(4)-Nitro-4(5)-styrylimidazole (18)
To a stirred suspension of 4(5)-methyl-5(4)-nitroimidazole (5
g, 0.04 mol) in benzaldehyde was added piperidine (3.95 g, 0.04
o
mol). The mixture was heated to 110 C for 2.5 h. When the
reaction was complete and solid started separating, 100 mL
ethanol was added to the reaction mixture. The mixture was
allowed to cool to room temperature and filtered to obtain a
yellow solid which was washed with ethanol and dried to get a
6.2.4. 3-Benzyl-5-methoxy-4,5,6,7-tetrahydroimidazo[4,5-e]
[1,4]diazepin-8(3H)-one (2)
o
1
Compound 14 (0.2g, 0.6 mmol) was taken in a clean flask to
which was added 1mL of 1N HCl and 20 mL of methanol and the
mixture was stirred overnight at 50 °C for 24 h. The resulting
solution was evaporated and purified by column chromatography,
using chloroform: methanol: ammonium hydroxide (5:2:0.2) as
the eluting solvent system. Evaporating the appropriate fractions
gave 2 as a light yellow colored solid. Yield 0.46g, 65%, mp
pure compound. Yield 6.5 g, 77%; mp: 258–260 C; H NMR
(DMSO-d6) δ 7.90 (s, 1H, Imid-H), 7.68-7.36 (m, 7H, CH=CH,
Ar-H); 13C-NMR (DMSO-d6) δ 143.73, 136.33, 136.10, 135.30,
130.93, 129.76, 129.65, 127.54, 114.64; HRMS (FAB), Calcd.
for C11H9N3O2: 216.0767 (MH+); obsd. m/z 216.0769.
6.2.8. 1-(4-Methoxybenzyl)-5(4)-nitro-4(5)-styryl-1H-imidazo
le (19a & 20a)
~185°C,
Rf=0.46
(chloroform:
methanol:
ammonium
1
hydroxide:5:2:0.2); HNMR (DMSO-d6) δ (2.98, dd, J= 3.64 Hz,
1H), 3.1 (s, 3H), 3.37 (m, 1H), 4.6 (t, J= 5.2 Hz, 1H), 5.08 (d, J=
16.08 Hz, 1H), 5.11 (d, J= 16.08 Hz, 1H), 7.01 (m, NH,
exchangeable with D2O), 7.15-7.35 (m, Aromatic + imidazole
H’s, 5H+1H), 7.41 (m, NH, exchangeable with D2O); 13C-NMR
(DMSO-d6) δ 44.2, 47.3, 54.2, 80.7, 113.3, 128.1, 128.4, 129.1,
134.8, 136.9, 139.5, 167.4; MS (ESI) m/z 273 (MH+); Anal.
Calcd for C14H16.25N4O2.125: C 61.75, H 5.92, N 20.52; found C
61.12, H 5.90, N 20.10; HRMS (FAB) Calcd. for C14H17N4O2:
273.1346 (MH+); obsd. m/z 273.1345.
To a solution of 18 (4 g, 18 mmol) in hot DMF was slowly
added anhydrous potassium carbonate (3.73 g, 27 mmol). To this
solution was added p-methoxybenzyl chloride (3.38 g, 21 mmol).
The mixture was heated at 85 oC for 5 h, cooled to room
temperature and filtered. The filtrate was concentrated under
reduced pressure to remove DMF. The residual thick solution
was poured into ice and extracted with chloroform. The organic
layer was dried over anhydrous sodium sulfate and was further
purified
by
column
chromatography
using
400:1
chloroform/methanol as eluting solvent system to obtain pure
compounds:
6.2.5.
3-Benzyl-5-ethoxy-4,5,6,7-tetrahydroimidazo[4,5-e]
o
Compound 19a: Yield 3.0 g, 50%; mp: 105-108 C; Rf=0.5
[1,4]diazepin-8(3H)-one (3)
1
(chloroform/methanol 400:1); H NMR (CDCl3) δ 7.79-7.70 (m,
2H, Ar-H), 7.60-7.57 (m, 2H, Ar-H), 7.50 (s, 1H, Imid-H), 7.17-
7.14 (m, 2H, Ar-H), 6.89-6.85 (m, 2H, CH=CH), 5.44 (s, 2H,
benzylic-CH2), 3.78 (s, 3H, -OCH3); 13C-NMR (CDCl3): δ
159.99, 144.08, 140.45, 137.28, 136.32, 133.98, 129.38, 129.17,
128.89, 127.65, 126.36, 117.88, 114.63, 55.43, 51.68; HRMS
(FAB) Calcd. for C19H17N3O3: 335.1263 (MH+); obsd m/z
336.1328; Anal. Calcd. for C19H17N3O3: C, 68.05, H, 5.11, N,
12.53; found, C, 68.35, H, 5.09, N, 12.37.
Compound 14 (0.2g, 0.6 mmol) was taken in a clean flask to
which was added 1mL of 1N HCl and 20 mL of ethanol and
stirred overnight at 50 °C for 24 h. The resulting solution was
evaporated and the residue was purified by column
chromatography,
using
chloroform:methanol:ammonium
hydroxide (5:2:0.2) as the eluting solvent system. Evaporating
the appropriate fractions gave 3 as a light yellow solid. Yield
0.07 g, 40%, Rf 0.61 (chloroform:methanol: ammonium
1
Compound 20a: Yield 2.5 g, 41%; mp: 115-119 oC; Rf=0.23;
1H NMR (CDCl3) δ 7.43-7.33 (m, 7H, Ar-H), 7.11-7.07 (m, 3H,
Ar-H, imid-H), 6.93-6.91 (m, 2H, CH=CH), 5.23 (s, 2H,
benzylic-CH2), 3.84 (s, 3H, -OCH3); 13C-NMR (CDCl3) δ 160.04,
138.45, 136.37, 135.64, 130.21, 129.59, 129.43, 129.03, 128.72,
128.49, 126.10, 114.94, 113.03, 55.48, 50.33; HRMS (FAB)
Calcd. for C19H17N3O3: 336.1342 (MH+); obsd. m/z 336.1345.
hydroxide: 5:2:0.2); HNMR (DMSO-d6) δ 1.00 (t, 3H), 2.97 (d,
1H), 3.34–3.41 (m, 2H+1H), 4.76 (t, 1H), 5.04–5.16 (m, 2H),
7.19–7.41 (m, 5H+1H+NH), 7.71 (s, NH, exchangeable with
D2O); 13C-NMR (DMSO-d6) δ 15.5, 44.0, 46.6, 61.4, 80.2, 114.3,
127.6, 128.0, 129.0, 133.7, 137.2, 139.3, 166.5; MS (ESI) m/z
287 (MH+); HRMS (FAB) Calcd. for C15H19N4O2: 287.1502
(MH+); obsd. m/z 287.1500.
6.2.9
(E)-1-(4-Fluorobenzyl)-5-nitro-4-styryl-1H-imidazole
6.2.6.
3-Benzyl-5-butoxy-4,5,6,7-tetrahydroimidazo[4,5-e]
(19b) and (E)-1-(4-fluorobenzyl)-4-nitro-5-styryl-1H-imida
zole (20b)
[1,4]diazepin-8(3H)-one (4)
Compound 14 (0.2 g, 0.6 mmol) was taken in a clean flask to
which was added 1mL of 1N HCl and 20 mL of butanol and the
mixture was stirred overnight at 50 °C for 24 h. The resulting
solution was evaporated and the residue was purified by column
chromatography, using chloroform: methanol:ammonium
hydroxide (5:1:0.1) as the eluting solvent system. Evaporating
the appropriate fractions gave 4 as a light yellow solid. Yield
0.04 g, 22%; Rf=0.75 (chloroform: methanol: ammonium
To a solution of 18 (4 g, 18.5 mmol) in hot DMF was slowly
added anhydrous potassium carbonate (3.828 g, 27.7 mmol). To
this solution was added p-fluorobenzyl chloride (3.2 g, 22.2
o
mmol). The mixture was heated at 85 C for 5 h, cooled to room
temperature and filtered. The filtrate was concentrated under
reduced pressure to remove DMF. The thick solution was poured
onto ice and extracted with chloroform. The organic layer was
dried over anhydrous sodium sulfate and the two regioisomers
were separated by column chromatography using 400:1
chloroform/methanol as eluting solvent system to obtain pure
compounds.
1
hydroxide: 5:2:0.2); HNMR (DMSO-d6) δ 0.79 (t, 3H), 1.37–
1.17 (m, 4H), 2.98 (d, 1H), 3.42–3.27 (m, 2H+1H), 4.72 (t, 1H),
5.08–5.14 (m, 2H), 7.16–7.39 (m, 5H+1H+NH), 7.54 (s, NH,
exchangeable with D2O); 13C-NMR (DMSO-d6) δ 14.2, 19.3,

o
Compound 19b: Yield 3.8g, 63%; mp: 134-137 C; Rf =0.71
7.27 (m, 2H, Ar-H), 7.23-7.19 (m, 2H, Ar-H), 5.50 (s, 2H,
benzylic- CH2); 13C-NMR (DMSO-d6) δ 162.90, 161.90, 140.68,
136.50, 134.49, 131.95, 130.22, 116.37, 116.15, 50.00; Anal.
Calcd. for C12H11N3O5 : C, 49.82, H, 3.04, N, 15.84; found, C,
50.05, H, 3.01, N, 15.59.
(chloroform/methanol, 100:1); 1H NMR (CDCl3) δ 7.80-7.70 (m,
2H, Ar-H), 7.60-7.58 (m, 2H, Ar-H), 7.55 (s, 1H, Imid-H), 7.38-
7.31 (m, 3H, Ar-H), 7.20-7.16 (m, 2H, Ar-H), 7.06-7.01 (m, 2H,
CH=CH), 5.47 (s, 2H, benzylic-CH2); 13C-NMR (CDCl3) δ
164.20, 161.73, 144.31, 140.63, 137.72, 136.33, 130.54, 129.65,
129.39, 129.03, 127.78, 117.82, 116.52, 116.30, 51.41; HRMS
(FAB) Calcd. for C19H17N3O3: 324.1142 (MH+); obsd. m/z
324.1144 (MH+); Anal. Calcd. for C19H17N3O3 : C, 66.87, H, 4.36,
N, 13.00; found, C, 67.07, H, 4.35, N, 13.04.
6.2.12. 1-(4-Methoxybenzyl)-N-(2,2-dimethoxyethyl)-5-nitro-
1H-imidazole-4-carboxamide (21a)
A
mixture of 16a (2 g, 7.2 mmol) and 1,1’-
Compound 20b: Yield: 1.2 g, 32%; Rf =0.56 (chloroform/
methanol, 100:1); (1H NMR (CDCl3) δ 7.39 (s, 1H, Imid-H),
7.33-7.28 (m, 4H, Ar-H), 7.07-7.02 (m, 4H, Ar-H), 5.24 (s, 2H,
benzylic-CH2), 2.88-2.80 (d, 1H, CH=CH, J = 18.76 Hz ), 2.53-
2.47 (d, 1H, CH=CH, J = 18.76 Hz ). 13C-NMR (CDCl3) δ
164.11, 161.63, 138.59, 136.40, 129.71, 129.06, 128.81, 128.72,
127.21, 116.79, 116.69, 116.57, 112.81, 49.97.
carbonyldiimidazole (1.16 g, 7.2 mmol) in dry THF (10 mL) was
refluxed in a flame-dried three-neck flask kept under a nitrogen
atmosphere. When the mixture turned clear it was allowed to cool
to room temperature. Aminoacetaldehyde dimethylacetal (1.17
mL, 10.8 mmol) was added to the cooled solution and allowed to
stir for 2 h. The reaction was monitored by TLC for completion.
The solvent was rotary evaporated to dryness; ice-cold water was
added and the mixture was stirred overnight. Then the product
was extracted with chloroform and washed with water and dried
over anhydrous magnesium sulfate. The organic layer was
evaporated to obtain a thick oily syrup. The latter was further
subjected to silica gel column chromatography using 1:1
hexanes/ethyl acetate as the eluting solvent system to obtain 21a
6.2.10. 1-(4-Methoxybenzyl)-5-nitro-1H-imidazole-4-carboxy
lic Acid (16a)
Finely powdered 19a (3.0 g, 9 mmol) was placed in a 250 mL
round-bottomed flask equipped with a magnetic stirring bar. Ice-
cold water (36 mL) was added, followed by 2N sodium
hydroxide (7.5 mL) and crushed ice (75 g). The temperature
inside the flask was maintained between 0-5 oC. Finely powdered
potassium permanganate (6.0 g) was added slowly over a period
of 1 h and the reaction mixture was stirred vigorously for 36 h.
The dark brown reaction mixture was filtered in vacuo and the
solid sludge (manganese dioxide) was thoroughly washed with
hot water. The light yellow filtrate was acidified with
concentrated hydrochloric acid to pH 1, when a light yellow solid
precipitated out. The solid was filtered, air dried for 24 h, and
suspended in ether with stirring for a few minutes to dissolve the
by-product, benzoic acid. An off-white solid which remained was
filtered in vacuo, washed with ether and dried over phosphorous
pentoxide for 24 h to obtain 16a as a pure compound. Yield 1.5
1
as a pure compound. Yield: 2.2 g, 83%; Rf =0.73; H NMR
(CDCl3) δ 7.37 (s, 1H, Imid-H), 7.18 (t, 1H, -CONH) 7.14-7.09
(m, 2H, Ar-H), 6.89-6.85 (m, 2H, Ar-H), 5.29 (s, 2H, benzylic-
CH2), 4.46-4.43 (t, 1H, -CH, J1,2 = 5.04 Hz, J2,3 = 5.6 Hz), 3.78
(s, 3H, -OCH3), 3.56-3.53 (t, 2H, CH2, J1,2 = 5.96 Hz, J2,3 = 5.48
Hz), 3.39 (s, 6H, 2xOCH3); 13C-NMR (CDCl3) δ 160.31, 159.48,
136.60, 136.54, 133.94, 129.68, 124.99, 114.84, 102.62, 55.45,
54.58, 51.24, 40.96; HRMS (FAB) Calcd. for C16H20N4O6:
365.1455 (MH+); obsd. m/z 365.1447; Anal. Calcd. for
C16H20N4O6 : C, 52.74, H, 5.53, N, 15.38; found, C, 52.46, H,
5.47, N, 15.57.
6.2.13.
1-(4-Fluorobenzyl)-N-(2,2-dimethoxyethyl)-5-nitro-
1H-imidazole-4-carboxamide (21b)
o
1
g, 61%; mp: 162-166 C; H NMR (DMSO-d6) δ 8.22 (s, 1H,
Imid-H), 7.19-7.14 (m, 2H, Ar-H), 6.94-6.91 (m, 2H, Ar-H), 5.43
(s, 2H,benzylic- CH2), 3.73 (s, 3H, -OCH3); 13C-NMR (DMSO-
d6) δ 162.87, 159.71, 127.42, 129.57, 114.76, 140.41, 135.00,
138.12, 55.66, 50.31; HRMS (FAB) Calcd. for C12H11N3O5:
277.0699 (MH+); obsd m/z 278.0778; Anal. Calcd. for
C12H11N3O5 : C, 51.99, H, 4.00, N, 15.16; found, C, 51.63, H,
4.03, N, 14.82.
A
mixture of 16b (3 g, 11.3 mmol) and 1,1’-
carbonyldiimidazole (3.08 g, 11.3 mmol) in dry THF (10 mL)
was refluxed in a flame-dried three-neck flask kept under
nitrogen atmosphere. When the mixture turned clear it was
allowed to cool to room temperature. Aminoacetaldehyde
dimethylacetal (1.83 mL, 17 mmol) was added to the cooled
solution and the mixture was allowed to stir for 2 h. The reaction
was monitored for completion. The solvent was removed by
rotary evaporation, ice-cold water was added and the resulting
mixture was stirred overnight. Then the product was extracted
with chloroform and washed with water and dried over
anhydrous magnesium sulfate. The organic layer was evaporated
to obtain a thick oily syrup. This was further subjected to silica
gel column chromatography using 1:1 hexanes/ethyl acetate as
the eluting solvent system to obtain pure 21b. Yield: 3.4 g, 85%;
mp: 86-89 C; Rf =0.63 (hexanes/ethyl acetate, 1:1); H NMR
(CDCl3) δ 7.43 (s, 1H, Imid-H), 7.19-7.14 (m, 3H, Ar-H, -
CONH), 7.07-7.03 (m, 2H, Ar-H), 5.35 (s, 2H, benzylic-CH2),
4.47-4.44 (t, 1H, -CH, J1,2 = 5.04 Hz, J2,3 = 5.48 Hz), 3.57-3.55 (t,
2H, -CH2, J1,2 = 1.84 Hz, J2,3 = 5.96 Hz), 3.40 (s, 6H, 2xOCH3);
13C-NMR (CDCl3) δ 159.32, 136.63, 134.17, 129.91, 129.83,
129.10, 116.69, 116.48, 102.59, 54.59, 50.86, 40.97; HRMS
(FAB) Calcd. for C16H20N4O6: 353.1255 (MH+); obsd. m/z
353.1257.
6.2.11. 1-(4-Fluorobenzyl)-5-nitro-1H-imidazole-4-carboxylic
Acid (16b)
Finely powdered 19b (5 g, 15 mmol) was placed in a 250 mL
round bottomed flask equipped with a magnetic stirring bar. Ice-
cold water (60 mL) was added, followed by 2N sodium
hydroxide (12.5 mL) and crushed ice (75 g). The temperature
inside the flask was maintained between 0-5 oC. Finely powdered
potassium permanganate (10 g) was added slowly over a period
of 1 h and the reaction mixture was stirred vigorously for 36 h.
The dark brown reaction mixture was filtered in vacuo and the
solid sludge (manganese dioxide) was thoroughly washed with
hot water. The light yellow filtrate was acidified with
concentrated hydrochloric acid to pH 1, when a light yellow solid
precipitated out. The solid was filtered, air dried for 24 h, and
suspended in ether with stirring for few minutes to dissolve the
by-product, benzoic acid. An off-white solid which remained was
filtered in vacuo, washed with ether and dried over phosphorous
pentoxide for 24 h to obtain pure 16b. Yield: 2.4 g, 58%; mp:
o
1
6.2.14.
5-Amino-1-(4-methoxybenzyl)-N-(2,2’-dimethoxy
o
1
168-170 C; H NMR (DMSO-d6) δ 8.24 (s, 1H, Imid-H), 7.31-
ethyl)-1H-imidazole-4-carboxamide (22a)

1N HCl (2 mL) was added to 22a (0.1 g, 0.3 mmol) and the
mixture was stirred over-night. Solvent was removed on a rotary
evaporator. To this was added ethanol (2 mL) and about 0.1 mL
of 1N HCl and stirred at room temperature for about 6 h. The
reaction was complete based on the TLC analysis using 10:1:0.01
chloroform/ethanol/ammonium hydroxide. Volatiles were
removed to obtain a thick, oily liquid. This was further purified
by column chromatography using 10:1:0.05 chloroform/ethanol/
ammonium hydroxide solvent system to obtain 6 as a pure white
A mixture of 21a (2 g, 5.48 mmol) and Pd-C (0.2 g) in
absolute methanol (30 mL) was hydrogenated in a Parr
hydrogenator at 55 psi for 5 h. The reaction mixture was filtered
through Celite, and the filtrate was evaporated to dryness on a
rotary evaporator to obtain a thick oily syrup. This was further
triturated with hexane to obtain 22a as a pure white compound.
Yield: 1.4 g, 76%; mp: 62-65 oC; Rf = 0.14 (hexanes/ethylacetate,
1
1:1); H NMR (CDCl3) δ 7.08-7.05(m, 2H, Ar-H), 7.01 (s, 1H,
1
Imid-H), 6.86-6.85 (m, 3H, Ar-H), 6.81 (t, 1H, -CONH) 4.86 (s,
2H, benzylic-CH2), 4.61 (s, 2H, NH2), 4.44-4.41 (t, 1H, -CH, J1,2
= 5.48 Hz, J2,3 = 5.52 Hz), 3.78 (s, 3H, -OCH3), 3.52-3.48 (t, 2H,
-CH2, J1,2 & J2,3 = 5.96 Hz), 3.38 (s, 6H, 2xOCH3); 13C-NMR
(CDCl3) δ 165.02, 159.81, 141.99, 130.34, 128.39, 126.39,
115.28, 114.77, 103.14, 55.45, 54.54, 47.15, 40.17; HRMS
(FAB) Calcd. for C16H22N4O4: 335.1713 (MH+); obsd. m/z
335.1705; Anal. Calcd. for C16H22N4O4.0.25 H2O : C, 56.71, H,
6.69, N, 16.53; found, C, 56.71, H, 6.81, N, 16.41.
solid. Yield: 0.034 g, 35.66%; H NMR (DMSO-d6) δ 7.34 (d,
1H, NH, J = 5.04 Hz), 7.29 (s, 1H, imidazole-H), 7.16 – 7.14 (m,
2H, Ar-H), 6.99-6.98 (m, 1H, -CONH), 6.91-6.87 (m, 2H, Ar-H),
5.03-4.93 (dd, 2H, benzylic-CH2), 4.74-4.71 (t, 1H, -CH, J1,2
=
5.04 Hz, J2,3 = 5.04 Hz), 3.71 (m, 4H, -OCH3 & CH), 3.45-3.36
(m, 2H, -OCH2), 2.99-2.95 (dd, 1H, -CH), 1.01 (t, 3H, -CH3, J1,2
= 7.36 Hz, J2,3 = 6.84 Hz); 13C-NMR (DMSO-d6): δ 166.22,
159.26, 138.89, 133.26, 129.20, 128.92, 114.49, 108.70, 80.30,
61.39, 55.64, 46.08, 43.75, 15.47; HRMS (FAB) Calcd. for
C16H20N4O3: 317.1608 (MH+); obsd. m/z 317.1608.
6.2.15. 5-amino-1-(4-Fluorobenzyl)-N-(2,2-dimethoxyethyl)-
1H-imidazole-4-carboxamide (22b)
6.2.18.
3-(4-Fluorobenzyl)-5-methoxy-4,5,6,7-tetrahydro
imidazo[4,5-e][1,4]diazepin-8(3H)-one (7)
A mixture of 21b (3 g, 8.5 mmol) and Pd-C (0.3 g) in absolute
methanol (50 mL) was hydrogenated in a Parr hydrogenator at 40
psi for 5 h. The reaction mixture was filtered through Celite, and
the filtrate was concentrated on a rotary evaporator to obtain a
thick oily syrup. This was further triturated with hexane to obtain
22b as a pure white compound. Yield: 1.6 g, 58%; mp: 58-60 oC;
To 22b (0.1 g, 0.3 mmol) was added 1N HCl (2 mL), and the
mixture was stirred overnight. Solvent was removed by rotary
evaporation. To the residue was added methanol (2 mL) and
about 0.1 mL of 1N HCl, and the mixture was stirred at room
temperature for about 6 h. The reaction was complete based on
the TLC analysis using 8:1:0.01 chloroform/methanol/
ammonium hydroxide. Volatiles were removed and the resulting
reaction mixture was further purified by silica gel column
1
Rf =0.41 (hexanes/ethyl acetate, 1:1); H NMR (CDCl3) δ 7.13-
7.02 (m, 5H, Ar-H, Imid-H), 6.82 (t, 1H, -CONH), 4.92 (s, 2H,
benzylic-CH2), 4.62 (s, 2H, NH2), 4.44-4.41 (t, 1H, -CH, J1,2
&
J2,3 = 5.48 Hz), 3.52-3.49 (t, 2H, -CH2, J1,2 & J2,3 = 5.48 Hz), 3.39
(s, 6H, 2xOCH3); 13C-NMR (CDCl3) δ 164.94, 141.74, 130.40,
130.24, 128.73, 116.56, 116.34, 115.54, 103.14, 54.33, 46.81,
40.18; HRMS (FAB) Calcd. for C16H22N4O4: 323.1513 (MH+);
obsd. m/z 323.1515.
chromatography
using
12:1:0.05
chloroform/methanol/
ammonium hydroxide solvent system to obtain 7 as a pure white
solid. Yield: 0.038 g, 42%; mp: 192-195 oC; Rf=0.24
(chloroform/methanol/ammonium hydroxide, 10:1:0.05); 1H
NMR (MeOH-d4) δ 7.47 (s, 1H, Imid-H), 7.27 – 7.23 (m, 2H, Ar-
H), 7.16-7.10 (m, 2H, Ar-H), 5.24-5.5.20 (d, J=16 Hz, 1H,
benzylic-CH2), 5.15-5.11 (d, J=9 Hz, 1H, benzylic-CH2), 4.78 (d,
J=5.52 Hz, 1H, -CONH), 3.68-3.62 (dd, 1H, -CH), 3.38 (s, 3H, -
OCH3), 3.22 (s, 2H, CH2), 3.19-3.15 (d, J=4 Hz, 1H, -NH); 13C-
NMR (MeOH-d4) δ = 167.54, 163.64, 161.33, 139.54, 133.68,
128.79, 128.70, 115.46, 81.90, 48.08, 46.09, 43.51; HRMS
(FAB) Calcd. for C15H18N4O3: 291.1251 (MH+); obsd. m/z
291.1238.
6.2.16.
3-(4-Methoxybenzyl)-5-methoxy-4,5,6,7-tetrahydro
imidazo[4,5-e][1,4]diazepin-8(3H)-one (5)
1N HCl (2 mL) was added to compound 22a (0.1 g, 0.3
mmol), and the mixture was stirred overnight. Solvent was
removed on a rotary evaporator. To the residue was added
methanol (2 mL) and about 0.1 mL of 1N HCl, and the mixture
was stirred at room temperature for about 6 h. The reaction was
complete based on the TLC analysis using 8:1:0.01
chloroform/methanol/ammonium hydroxide. Volatiles were
removed when a thick oily liquid was obtained. This was further
purified by column chromatography using 12:1:0.05
chloroform/methanol/ammonium hydroxide solvent system to
obtain 5 as a pure white solid. Yield: 0.05 g, 57%; mp: 155-158
oC; Rf=0.19 (chloroform/methanol/ammonium hydroxide,
6.2.19.
1-(4-Methoxybenzyl)-4-nitro-1H-imidazole-5-carbo
xylic Acid (25)
Finely powdered 20a (4 g, 11 mmol) was placed in a 250 mL
round bottomed flask equipped with a magnetic stirring bar. Ice-
cold water (36 mL) was added, followed by 2N sodium
hydroxide (10 mL) and crushed ice (75 g). The temperature
inside the flask was maintained between 0-5 oC. Finely powdered
potassium permanganate (8 g) was added slowly over a period of
1 h and the reaction mixture was stirred vigorously for 36 h. The
dark brown reaction mixture was filtered in vacuo and the solid
sludge (manganese dioxide) was thoroughly washed with hot
water. The light yellow filtrate was acidified with concentrated
hydrochloric acid to pH 1, when a light yellow solid precipitated
out. The solid was filtered, air dried for 24 h, and suspended in
ether with stirring for few minutes to dissolve the by-product,
benzoic acid. An off-white solid which remained was filtered in
vacuo, washed with ether and dried over phosphorous pentoxide
for 24 h to obtain pure 25. Yield: 1.8 g, 54%; mp: 153-156 oC; 1H
NMR (DMSO-d6) δ 8.12 (s, 1H, Imid-H), 7.22-7.19 (m, 2H, Ar-
H), 6.94-6.90 (m, 2H, Ar-H), 5.35 (s, 2H,benzylic- CH2), 3.73 (s,
1
10:1:0.05); H NMR (DMSO-d6) δ 7.41-7.40 (d, J=4 Hz, 1H, -
NH), 7.30 (s, 1H, Imid-H), 7.18 – 7.15 (m, 2H, Ar-H), 7.01-6.99
(m, 1H, -CONH), 6.92-6.89 (m, 2H, Ar-H), 5.04-5.00 (d, J=16
Hz, 1H, benzylic-CH2), 4.98-4.95 (d, J=12 Hz, 1H, benzylic-
CH2), 4.63-4.61 (t, 1H, -CH, J1,2 = 5.48 Hz, J2,3 = 5.04 Hz), 3.72
(s, 3H, -OCH3) 3.70 (s, 1H, CH), 3.13 (s, 3H, -OCH3), 2.99-2.94
(dd, 1H, -CH); 13C-NMR (DMSO-d6) δ 163.40, 159.25, 138.0,
131.0, 129.90, 129.16, 114.90, 114.51, 82.29, 55.65, 53.69,
40.39, 40.59; HRMS (FAB) Calcd. for C15H18N4O3: 303.1451
(MH+); obsd. m/z 303.1446.
6.2.17. 3-(4-Methoxybenzyl)-5-ethoxy-4,5,6,7-tetrahydroimid
azo[4,5-e][1,4]diazepin-8(3H)-one (6)

3H, -OCH3); 13C-NMR (DMSO-d6): δ 159.95, 159.70, 138.11,
129.78, 128.10, 114.72, 55.68, 50.01; HRMS (FAB) Calcd. for
C12H11N3O5: 278.0771 (MH+); obsd. m/z 278.0775.
H), 7.51-7.50 (d, J=5.04 Hz, 1H, -NH), 7.19 – 7.13 (m, 3H, -
CONH & Ar-H), 6.86-6.83 (m, 2H, Ar-H), 5.61-5.57 (d, J=14.64
Hz, 1H, benzylic-CH2), 5.14-5.10 (d, J=14.64 Hz, 1H, benzylic-
CH2), 4.55-4.52 (t, 1H, -CH, J1,2 & J2,3 = 5.04 Hz), 3.70 (s, 3H, -
OCH3), 3.18 (s, 3H, -OCH3), 2.86-2.81 (dd, 1H, -CH); 13C-NMR
(DMSO-d6): δ 164.18, 159.05, 149.36, 140.49, 131.08, 129.33,
114.27, 103.67, 82.35, 55.56, 53.48, 48.65, 43.20; HRMS (FAB)
Calcd. for C15H18N4O3: 303.1451 (MH+); obsd. m/z 303.1448.
6.2.20. 1-(4-Methoxybenzyl)-N-(2,2-dimethoxyethyl)-4-nitro-
1H-imidazole-5-carboxamide (26)
A mixture of 25 (2 g, 7.2 mmol) and 1,1’-carbonyldiimidazole
(1.16 g, 7.2 mmol) in dry THF (10 mL) was refluxed in a flame-
dried three-neck flask kept under nitrogen atmosphere. When the
mixture turned clear it was allowed to cool to room temperature.
Aminoacetaldehyde dimethylacetal (1.17 mL, 10.8 mmol) was
added to the cooled solution which was allowed to stir for 2 h.
The reaction was monitored for completion. The solvent was
removed by rotary evaporation and to the residue was added ice-
cold water (10 mL) and the mixture was stirred overnight. Then
the product was extracted with chloroform and washed with
water and dried over anhydrous magnesium sulfate. The organic
layer was evaporated to obtain a thick oily syrup. This was
further subjected to column chromatography using 1:1
hexanes/ethyl acetate as the eluting solvent system to obtain pure
6.2.23.
5-Amino-N-(2,2-dimethoxyethyl)-1H-imidazole-4-
carboxamide (31)
A mixture of 30 (0.5 g, 2 mmol) and Pd-C (0.05 g) in absolute
methanol (10 mL) was hydrogenated in a Parr hydrogenator at 60
psi for 5 h. The reaction mixture was filtered through Celite, and
the filtrate was concentrated on a rotary evaporator to obtain 31
as
a
thick oily syrup. Yield: 0.2 g, 50%, Rf 0.51
1
(chloroform/methanol, 10:1); H NMR (CDCl3) δ 7.02 (s, 1H,
Imid-H), 6.98 (t, 1H, -CONH), 5.14 (s, 2H, NH2), 4.39-4.36 (t,
1H, -CH, J1,2 = 5.48 & J2,3 = 5.04 Hz), 3.48-3.45 (t, 2H, -CH2, J1,2
= 5.96 & J2,3 = 5.48 Hz), 3.32 (s, 6H, 2xOCH3); 13C-NMR
(CDCl3): δ 165.14, 144.35, 129.17, 111.46, 103.2, 54.45, 40.48;
HRMS (FAB) Calcd. for C8H15N4O3: 215.1138 (MH+); obsd. m/z
215.1135.
o
26. Yield: 2.3 g, 87%; mp: 125-127 C; Rf 0.68 (hexanes/ethyl
1
acetate, 1:1); H NMR (CDCl3) δ 7.75 (t, 1H, -CONH), 7.40 (s,
1H, Imid-H), 7.17-7.15 (m, 2H, Ar-H), 6.88-6.85 (m, 2H, Ar-H),
5.40 (s, 2H, benzylic-CH2), 4.43-4.40 (t, 1H, -CH, J1,2 = 5.48 Hz,
J2,3 = 5.04 Hz), 3.78 (s, 3H, -OCH3), 3.56-3.53 (t, 2H, -CH2, J1,2
=
6.2.24. 5-(Benzylamino)-N-(2,2-dimethoxyethyl)-1H-imidazo-
le-5-carboxamide (32)
5.48 Hz, J2,3 = 5.52 Hz), 3.39 (s, 6H, 2xOCH3); 13C-NMR
(CDCl3): δ 160.10, 157.63, 140.50, 136.69, 129.89, 126.28,
123.76, 114.63, 102.18, 55.42, 54.49, 51.51, 41.46; HRMS
(FAB) Calcd. for C16H20N4O6: 365.1455 (MH+); obsd. m/z
365.1444.
To 31 (0.1 g, 0.5 mmol) in methanol was added benzaldehyde
(0.06 g, 0.6 mmol) and trifluoroacetic acid (0.07 g, 0.6 mmol)
under a nitrogen atmosphere and the mixture was stirred at room
temperature overnight. When the starting material was consumed
as determined by TLC analysis using a 10:1 chloroform/methanol
solvent system, sodium cyanoborohydride (0.04 g, 0.6 mmol)
was added to the reaction mixture. The reaction was stirred for
about 8 h and the reaction was completed based on the TLC
analysis using 10:1 chloroform/methanol solvent system.
Solvents were removed in vacuo and the product was isolated by
silica gel column chromatography using 15:1 chloroform/
methanol solvent system to obtain 32 as a white solid. Yield:
6.2.21.
4-Amino-1-(4-Methoxybenzyl)-N-(2,2’-dimethoxy
ethyl)-1H-imidazole-5-carboxamide (27)
A mixture of 26 (2 g, 5.48 mmol) and Pd-C (0.2 g) in absolute
methanol (30 mL) was hydrogenated in a Parr hydrogenator at 40
psi for 5 h. The reaction mixture was filtered through Celite, and
the solvent was removed on a rotary evaporator to yield a thick
oily syrup. This was further triturated with hexane to obtain 27 as
a white solid. Yield: 1.2 g, 65%; mp: 95-98 oC; Rf 0.13
(hexanes/ethylacetate, 1:2); 1H NMR (CDCl3) δ 7.16 (s, 1H,
Imid-H), 7.13-7.09 (m, 2H, Ar-H), 6.85-6.81 (m, 2H, Ar-H), 6.77
(t, 1H, -CONH), 5.33 (s, 2H, benzylic-CH2), 4.37-4.35 (t, 1H, -
CH, J1,2 & J2,3 = 5.04 Hz), 3.95 (s, 2H, NH2), 3.76 (s, 3H, -
OCH3), 3.46-3.44 (t, 2H, -CH2, J1,2 = 5.92 Hz, J2,3 = 5.04 Hz),
3.33 (s, 6H, 2xOCH3); 13C-NMR (CDCl3): δ 161.49, 159.49,
149.20, 137.01, 128.94, 128.45, 114.33, 109.21, 102.75, 55.36,
54.35, 50.08, 40.36; HRMS (FAB) Calcd. for C16H22N4O4:
335.1713 (MH+); obsd. m/z 335.1704.
1
0.08 g, 43.3%; Rf 0.13 (chloroform/methanol, 10:1); H NMR
(CDCl3): δ 7.37-7.33 (m, 5H, Ar-H), 6.95 (s, 1H, Imid-H), 6.79
(t, 1H, CONH), 4.45-4.42 (t, 1H, CH, J1,2 = 5.04 & J2,3 = 5.48
Hz), 4.39 (s, 2H, benzylic CH2), 3.53-3.5 (t, 2H, CH2, J1,2 = 5.48
Hz, J2,3 = 5.96 Hz), 3.38 (s, 6H, -OCH3), 3.33 (s, 1H, NH); 13C-
NMR (CDCl3): δ 163.7, 138.26, 130.1, 129.15, 128.94, 128.47,
127.96, 127.15, 103.19, 54.36, 48.7, 40.25; HRMS (FAB) Calcd.
for C15H20N4O3: 305.1608 (MH+); obsd. m/z 305.1599.
6.2.25.
4-Benzyl-5-methoxy-4,5,6,7-tetrahydroimidazo[4,5-
e][1,4]diazepin-8(3H)-one (9)
6.2.22. 5-Methoxy-1-(4-methoxybenzyl)-4,5,6,7-tetrahydro-
To 32 (0.05 g, 0.16 mmol) was added 1N HCl (2 mL) and the
mixture was stirred overnight. When the starting material was
consumed as determined by TLC analysis using 10:1:0.01
chloroform/methanol/ammonium hydroxide as eluent, the solvent
was removed in vacuo. Methanol (2 mL) and 0.1 mL of 1N HCl
were added and the mixture was stirred for 6 h. Volatiles were
removed and the resulting reaction mixture was further purified
by column chromatography using 30:1:0.01 chloroform/
methanol/ammonium hydroxide solvent system to obtain 9 as a
pure white solid. Yield: 0.016 g, 37.5%; Rf 0.36
(chloroform/methanol/ammonium hydroxide, 10:1:0.01); 1H
NMR (DMSO-d6) δ 12.12 (s, 1H, Imid-NH), 7.44 (s, 1H, Imid-
H), 7.30-7.26 (m, 5H, Ar-H), 7.2 (m, 1H, -CONH), 5.14-5.10 (d,
1H, benzylic CH, J1,2 = 15.6 Hz), 4.71-4.69 (d, 1H, benzylic CH,
imidazo[4,5-e][1,4]diazepin-8(1H)-one (8)
To 27 (0.1 g, 0.3 mmol) was added 1N HCl (2 mL) and the
mixture was stirred overnight. Solvent was removed by rotary
evaporation. To the residue was added methanol (2 mL) and
about 0.1 mL of 1N HCl, and the resulting mixture was stirred at
room temperature for 5h. The reaction was complete based on the
TLC analysis using 8:1:0.01 chloroform/methanol/ammonium
hydroxide. Volatiles were removed and the resulting reaction
mixture was further purified by column chromatography using
12:1:0.05 chloroform/methanol/ammonium hydroxide solvent
system to obtain 8 as a pure white solid. Yield: 0.047 g, 52%;
mp: 138-141o C; Rf1 0.38 (chloroform/methanol/ammonium
hydroxide, 10:1:0.05); H NMR (DMSO-d6) δ 7.63 (s, 1H, Imid-

J1,2 = 5.96 Hz), 4.56-4.52 (d, 1H, -CH, J1,2 = 16.04 Hz), 3.53-3.50
(m, 1H, -CH), 3.17 (s, 3H, -OCH3), 2.98-2.95 (dd, 1H, -CH); 13C-
NMR (DMSO-d6) δ 163.72, 147.16, 140.17, 136.03, 128.72,
127.99, 127.19, 105.89, 87.30, 54.13, 53.65, 42.57; HRMS (ESI)
Calcd. for C14H16N4O2: 273.1346 (MH+); obsd. m/z 273.1340.
product was isolated by silica gel column chromatography using
chloroform/ethanol/ammonium hydroxide (25:1:0.1) solvent
system to obtain 11 as an off-white solid. Yield: 0.03 g, 31%; 1H
NMR (DMSO-d6) δ 7.70 (s, 1H, imid-H), 7.35 (m, 1H, -CONH),
7.28-7.17 (m, 7H, Ar-H), 6.87-6.85 (m, 2H, Ar-H), 5.16-5.15
(dd, 2H, benzylic CH2), 5.02-4.67 (dd, 2H, benzylic CH2), 4.58-
4.54 (d, 1H, -CH, J = 15.56 Hz), 3.72 (s, 4H, -OCH3, -CH), 3.48-
3.36 (m, 2H, -OCH2), 2.94-2.89 (dd, 1H, -CH), 1.05 (t, 3H, -
CH3); 13C-NMR (DMSO-d6) δ 164.24, 159.11, 149.43, 140.02,
139.63, 130.89, 129.43, 128.70, 127.98, 127.16, 114.33, 104.27,
86.72, 61.73, 55.56, 52.79, 48.92, 41.97, 15.78; HRMS (ESI)
Calcd. for C23H26N4O3: 407.2077 (MH+); obsd. m/z 407.2084.
6.2.26. 1-(4-Methoxybenzyl)-4-(benzylamino)-N-(2,2-dimetho
xyethyl)-1H-imidazole-5-carboxamide (34)
To 27 (0.1 g, 0.3 mmol) in methanol was added benzaldehyde
(0.038 g, 0.36 mmol) and trifluoroacetic acid (0.04 g, 0.36 mmol)
under a nitrogen atmosphere and the mixture was stirred at room
temperature overnight. When the starting material was consumed
as determined by TLC analysis using 1:1 hexanes/ethyl acetate
solvent system, sodium cyanoborohydride (0.028 g, 0.45 mmol)
was added to the reaction mixture. The reaction mixture was
stirred for about 8 h when the reaction was completed based on
the TLC analysis using 1:1 hexanes/ethyl acetate solvent system.
Solvents were removed in vacuo and the product was isolated by
column chromatography using 2:1 hexanes/ethyl acetate solvent
system to obtain 34 as a white solid. Yield: 0.12 g, 93%; Rf 0.12
6.3. Biochemical Studies
Target analogs of iso-azepinomycin were screened in vitro as
inhibitors of guanase from rabbit liver in Tris buffer (pH 7.4) at
250 C by spectrophotometric measurements of the rate of
hydrolysis of the substrate guanine at λmax 245 nm. Stock
solutions of the substrate, enzyme and inhibitors were prepared
using 0.05 M Tris buffer (pH 7.4). The enzyme kinetics were
followed by measuring the change in optical density (decrease in
absorbance) per minute of the substrate guanine. By keeping the
concentration of the inhibitor constant, and varying the
concentration of the substrate, a set of kinetic data was obtained.
Additional sets of data were generated using different
concentrations of the inhibitor.
1
(hexanes/ethyl acetate, 2:1); H NMR (CDCl3) δ 7.39 (s, 1H,
imid-H), 7.36-7.25 (m, 5H, Ar-H), 7.13-7.11 (m, 2H, Ar-H),
6.87-6.84 (m, 2H, Ar-H), 6.69 (t, 1H, CONH), 5.34 (s, 2H,
benzylic CH2), 4.44 (s, 2H, benzylic CH2), 4.31-4.28 (t, 1H, CH,
J1,2 & J2,3 = 5.04 Hz), 3.77 (s, 3H, -OCH3), 3.46 (s, 1H, NH),
3.43-3.40 (t, 2H, CH2, J1,2 = 5.48 Hz, J2,3 = 5.52 Hz), 3.24 (s, 6H,
-OCH3); 13C-NMR (DMSO-d6) δ 160.98, 159.82, 150.76, 139.04,
136.22, 128.92, 128.65, 128.02, 127.45, 127.25, 114.60, 108.52,
102.67, 55.41, 54.39, 50.67, 49.62, 40.42; HRMS (FAB) Calcd.
for C23H28N4O4: 425.2183 (MH+); obsd. m/z 425.2187.
The following are the principles and procedures employed for
the biochemical assay: (1) The change in optical density at λmax
245 nm per unit time is the measure of the guanine deaminase
activity. The Michaelis constant (KM) of the guanine was
determined for each of the target compounds. (2) Solutions: (a)
Tris-HCl buffer, pH= 7.4, 0.05 M (7.88 g of Tris-HCl was
dissolved in 950 mL of deionized distilled water, the pH was
adjusted to 7.4 using 1N NaOH, and then diluted to 1000 mL
using deionized distilled water). (b) Guanine solution: 30 – 40
mg of guanine was dissolved in 1N NaOH solution and diluted to
100 mL using 0.05 M Tris-buffer solution. The solution was
filtered to get clear solution and the concentration of the stock
solution was calculated from its UV absorbance. (c) Substrate
concentration in each assay was in the range of 5-40 µM. (d)
Inhibitor solution were made either in DMSO or methanol. (e)
The inhibitor concentration in each assay was 10 – 20 µM. (f) In
our laboratory, guanase of activity 0.1278 U/mL was used for
biochemical studies. A 60 µL volume of the enzyme solution
used in each assay corresponds to 0.0077 units of the enzyme.
Guanine deaminase from rabbit liver was purchased from M P
Biochemicals as a suspension in 3.2 M (NH4)2SO4, pH 6.0,
activity = 10mg/mL, 0.06 U/mg and 0.6 U/mL. 0.1278 U/mL
equals to 2.13 mg/mL in protein concentration. To calculate the
volume of enzyme to be used from the stock enzyme solution, the
following formula was employed: 10 mg/mL x (volume of stock
enzyme solution) mL = 2.13 mg/mL x (volume of the enzyme
solution to be made) mL. (3) Spectrophotometric measurements:
Wavelength, 245 nm, final volume 1.0 mL, light path 1 cm,
temperature 25°C, read against air. The enzymatic hydrolysis was
~2 mins long. The data collected from the linear part of the plot,
ranging from 0.1 – 0.6 sec, corresponds to the initial velocity.
The mean of the initial velocity (V) was calculated from two data
sets and was used to generate values of 1/V in the Y-axis of
Lineweaver-Burk plot. (4) Procedure: A mixture of calculated
amount of guanine solution and calculated amount of inhibitor
solution were mixed in a 1 mL cuvette and diluted with Tris
buffer to make up to a volume of 940 µL. The Tris buffer was
used as reference. In each case the concentration of the enzyme
used was kept constant. After adding 60 µL of the enzyme
6.2.27.
1-(4-Methoxybenzyl)-4-benzyl-5-methoxy-4,5,6,7-
tetrahydroimidazo[4,5-e][1,4]diazepin-8(1H)-one (10)
To 34 (0.12 g, 0.28 mmol) was added 1N HCl (2 mL) and the
mixture was stirred overnight. When the starting material was
consumed as determined by TLC analysis using 10:1:0.01
chloroform/methanol/ammonium hydroxide as the solvent
system, the solvent was removed in vacuo. Methanol (2 mL) and
0.1 mL of 1N HCl were added and the mixture was stirred for 6
h. Volatiles were removed and the resulting reaction mixture was
further purified by silica gel column chromatography using
50:1:0.05 chloroform/methanol/ ammonium hydroxide solvent
system to obtain 10 as a pure white solid. Yield: 0.042 g, 38%; Rf
0.38 (chloroform/methanol/ammonium hydroxide, 10:1:0.01); 1H
NMR (CDCl3) δ 7.28-7.24 (m, 8H, imid-H and Ar-H), 6.84-6.82
(m, 2H, Ar-H), 5.58-5.55 (m, 2H, -CONH and –CH), 5.38-5.25
(dd, 2H, benzylic CH2), 4.60-4.51 (dd, 2H, benzylic CH2), 3.76
(s,3H, -OCH3), 3.46-3.39 (m, 1H, -CH), 3.27 (s, 3H, -OCH3),
3.15-3.09 (dd, 1H, -CH); 13C-NMR (CDCl3) δ 164.62, 159.34,
150.07, 139.12, 138.63, 129.53, 129.01, 128.62, 127.76, 127.24,
114.15, 104.68, 87.31, 55.35, 54.82, 55.33, 50.32, 42.85; HRMS
(ESI) Calcd. for C22H24N4O3: 393.1921 (MH+); obsd. m/z
393.1920.
6.2.28. 1-(4-Methoxybenzyl)-4-benzyl-5-ethoxy-4,5,6,7-tetra-
hydroimidazo[4,5-e][1,4]diazepin-8-(1H) (11)
To 34 (0.1 g, 0.23 mmol) was added 1N HCl (2 mL) and the
mixture was stirred over-night. When the starting material was
consumed as determined by TLC analysis using 10:1:0.01
chloroform/methanol/ammonium hydroxide solvent system, the
solvent was removed in vacuo and to this residue was added
ethanol (2 mL) and 0.1 mL of 1N HCl and the mixture was
stirred for 6 h. The solvent was evaporated to dryness and the

solution to the cuvette, the solution was mixed thoroughly. The
biochemical reaction was completed in about 2 mins. For lower
concentrations of the substrate, the reaction was even faster. The
reaction mixture was quickly and carefully mixed to make sure
there were no bubbles. The reaction was followed at 25°C by
measuring the decrease in absorbance at λ= 245 nm using Cary-
50 UV spectrophotometer. The data were plotted as 1/V vs 1/[S]
(standard Lineweaver-Burk plots) using Microsoft Word Excel
2007. Computed Ki values have been collected in Table 1 in the
text. The details of enzyme kinetics on all target compounds
have been provided in the Supplementary Data.
21. Lucacchini, A.; Bazzichi, L.; Biagi, G.; Livi, O.; Segnini,
D. Ital. J. Biochem. 1982, 31, 153.
22. Lucacchini, A.; Montali, U.; Segnini, D. Ital. J. Biochem.
1977, 26, 27.
23. Lucacchini, A.; Segnini, D.; Da Settimo, A.; Livi, O. Ital. J.
Biochem. 1979, 28, 194.
24. Meyer, R. B., Jr.; Skibo, E. B. J. Med. Chem. 1979, 22,
944.
25. Saxena, A. K.; Ahmad, S.; Shanker, K.; Bhargava, K. P.;
Kishor, K. Pharmazie 1980, 35, 16.
26. Saxena, A. K.; Ahmad, S.; Shanker, K.; Kishor, K.
Pharmacol. Res. Commun. 1984, 16, 243.
27. Silipo, C.; Hansch, C. J. Med. Chem. 1976, 19, 62.
28. Lewis, A. S.; Glantz, M. D. J Biol Chem 1974, 249, 3862.
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Acknowledgments
This research was supported by a grant (#1R01 GM087738-
01A1) from the National Institute of General Medical Sciences of
the National Institutes of Health.
31. Somoza, J. R.; Skillman, A. G., Jr.; Munagala, N. R.;
Oshiro, C. M.; Knegtel, R. M. A.; Mpoke, S.; Fletterick, R. J.;
Kuntz, I. D.; Wang, C. C. Biochemistry 1998, 37, 5344.
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Supplementary Data
Supplementary data on details of enzyme kinetics including
associated tables and graphs (12 pages) are available from the
online version of the journal.
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Analogs of Iso-Azepinomycin as Potential Transition-
State Analog Inhibitors of Guanase: Synthesis,
Biochemical Screening, and Structure-Activity
Correlations of Various Selectively Substituted
Imidazo[4,5-e][1,4]diazepines
Saritha Tantravedi, Saibal Chakraborty, Niti H. Shah, James C. Fishbein, and Ramachandra S. Hosmane*
O
O
O
O
R'
N
O
7
R''
1
N
8 NH
NH
N
NH
N
NH
NH
N
2
6
N
R'
N
3
N
H
N
N
H
N
5
N
N
R'
OH
N
H
H
H4
N
R'
OR
OR
OR
OR
Iso-azepinomycin