A novel transition state analog inhibitor of guanase based on azepinomycin ring structure: Synthesis and biochemical assessment of enzyme inhibition

Article abstract of DOI:10.1016/j.bmcl.2010.11.109

Synthesis and biochemical inhibition studies of a novel transition state analog inhibitor of guanase bearing the ring structure of azepinomycin have been reported. The compound was synthesized in five-steps from a known compound and biochemically screened

Full text of DOI:10.1016/j.bmcl.2010.11.109

Bioorganic & Medicinal Chemistry Letters 21 (2011) 756–759
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A novel transition state analog inhibitor of guanase based on azepinomycin
ring structure: Synthesis and biochemical assessment of enzyme inhibition
,
⇑
Saibal Chakraborty, Niti H. Shah, James C. Fishbein, Ramachandra S. Hosmane
Laboratory for Drug Design & Synthesis, Department of Chemistry & Biochemistry, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis and biochemical inhibition studies of a novel transition state analog inhibitor of guanase bear-
ing the ring structure of azepinomycin have been reported. The compound was synthesized in five-steps
from a known compound and biochemically screened against the rabbit liver guanase. The compound
Received 1 November 2010
Revised 21 November 2010
Accepted 23 November 2010
Available online 27 November 2010
exhibited competitive inhibition profile with a Ki of 16.7 0.5
l
M.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Synthesis
Biochemical screening
Inhibition of guanine deaminase (guanase)
Imidazo[4,5-e][1,4]diazepine-5,8-dione ring
system
Azepinomycin analog
Guanine deaminase (EC 3.5.4.3, Guanase, or GDA), is an enzyme
that hydrolyzes guanine to form xanthine that is unsuitable for
DNA/RNA buildup.1 This enzyme has been found in normal or
transformed human organs and sera.2 One of the approaches for
antiviral/anticancer therapy is to design structural mimics of natu-
ral guanine as nucleic acid building blocks, with an anticipation
that such analogs would be incorporated into DNA/RNA of virus
or cancer cells, interrupting their normal replicative processes.3 In-
deed, there are some known guanine mimics, such as 6-thiogua-
nine4,5 and 8-azaguanine,6 both of which are potent anticancer
compounds. However, they both are believed to be substrates for
the enzyme guanase, which converts them into their respective
guanase activity.8,10,11 Even though no direct cause and effect rela-
tionship has been established between increased guanase activity
and post-transfusional hepatitis (PTH), it is reasonable to assume
that the guanase level is responsible, in part, for the development
of PTH. There are only a few and scattered reports on the specific
physiological or metabolic role of guanase. Nonetheless, there are
important observations made on this enzyme as reported in the lit-
erature. For example, it is known that high serum guanase activity
is a clear biochemical indicator of organ rejection in liver trans-
plant patients.12 It has also been shown that patients with multiple
sclerosis (MS) have significantly elevated levels of guanase activity
in their cerebral spinal fluid (CSF), and that there is a clear correla-
tion between the extent of disability and the level of guanase
activity.13
Another important aspect of guanase activity is its involvement
in cancerous tissues. It has long been known that carcinogenic pro-
cesses and the activities of some enzymes in cancer tissues and
cells are strongly interrelated. In this regard, it is important to con-
sider reports of abnormal levels of guanase activity in various can-
cer tissues. An increase in guanase activity has been reported in
lung,14 gastric,15 kidney,16–18 and breast cancer tissues.19 It is sug-
gested that this difference in activity is a physiological attempt of
the cancer cell to regulate the guanine and/or xanthine level, which
is needed by cancer cells to accelerate their salvage metabolic path-
way activity. Since the de novo pathway is mostly employed by
normal cells for replication, a guanase inhibitor can preferentially
check the growth of cancer cells without affecting the normal cells.
Furthermore, guanase plays an important role in the detoxification
inactive forms 6-thioxanthine and 8-azaxanthine, respectively.7
A
potent guanase inhibitor would restore the original potency of
these anticancer compounds.
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 as a causative fac-
tor. There are several documented reports on detection of abnor-
mally high levels of serum guanase activity in patients with liver
diseases like hepatitis.8–10 Therefore, the elevated enzyme activity
has been suggested as a marker of hepatitis and hepatoma.10 There
are also various reports of patients developing hepatitis C when
they are transfused with blood containing high levels of serum
⇑
Corresponding author. Tel.: +1 410 381 0005; fax: +1 410 455 1148.
E-mail address: hosmane@umbc.edu (R.S. Hosmane).
Recently retired.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.11.109

S. Chakraborty et al. / Bioorg. Med. Chem. Lett. 21 (2011) 756–759
757
process of high amounts of toxic guanine produced from acceler-
ated metabolism in the cancerous tissues. In any event, a guanase
inhibitor can trigger cell death either due to cytotoxicity of the
accumulating metabolites as proposed in the case of adenosine
deaminase (ADA)20–23 or through depletion of the nucleotide pool
necessary for cell growth.
While many studies of guanase inhibition have been reported
both from this lab24–30 and others,31–35 guanase is still one of the
least studied enzymes of the nucleic acid metabolism, although it
has been isolated from rabbit liver,1 human liver,36 and rat brain37
years ago.
to effectively mimic the tetrahedral intermediate (2) of the gua-
nase-mediated conversion of guanine (1) to xanthine (3).
With the above rationale, we report here the design, synthesis,
and biochemical evaluation of our current target molecule, namely,
3-benzyl-4,5,7,8-tetrahydro-6-hydroxymethyl-6-[(benzyloxycar-
bonyl)amino]imidazo[4,5-e][1,4]diazepine-5,8-dione (10). Com-
pound 10 meets the criteria outlined above. It has the required
geminal carbon at position-6 as described except that the OH
group had to be removed one atom further from the geminal junc-
tion in order to avoid any potential elimination of water with
anchimeric assistance from one of the two NH groups attached
to the junction. For the same reason, the exonuclear NH2 had to
be protected by a benzyloxycarbonyl (CBZ) group. A benzyl group
has been introduced at position-3 in order to create a hydrophobic
environment in its vicinity, which has been suggested to enhance
the enzyme–ligand interactions, based on our earlier studies on
this enzyme.26,28 Attachment of a benzyl group has further scope
for introduction of a variety of substituents on the phenyl ring with
defined dimensions, and thus can act as both hydrophobic and
dimensional probes. Finally, the introduction of a carbonyl group
at position-5 of the heterocycle, coupled with movement of the
6-hydroxy group away from the ring by an additional carbon atom,
would allow excellent coordination of the inhibitor with Zn+2 to
form a stable, six-membered ring structure. The latter would use
two of the four metal coordination sites, while the other two would
be occupied by two of the three original amino acid residues at the
enzyme active site. This arrangement would further strengthen the
interaction of azepinomycin with the active site Zn+2 ion.
Azepinomycin38,39 is a naturally occurring inhibitor of guanase,
which has so far been studied only in tissue culture systems. So,
while its IC50 value (the concentration of the drug required to inhi-
bit 50% of the guanase activity) is known to be ꢀ10À5 M,38 the Ki of
azepinomycin against guanase has never been determined bio-
chemically against the isolated pure enzyme. In our continued ef-
forts to discover a potent guanase inhibitor, we report here the
synthesis and biochemical studies of an analog azepinomycin that
more closely resembles the putative transition state of the en-
zyme-catalyzed reaction.
O
1
HN
6
N
8
7
2
HO
3
5
4
N
H
N
H
Azepinomycin
The recently solved crystal structure of guanase40 from Bacillus
subtilis suggests that the enzyme-catalyzed reaction (Fig. 1) is as-
sisted by an active site zinc metal ion (Zn+2), which forms a tetra-
hedral complex with His-53, Cys-83, and Cys-86 of the protein,
along with an isolated water molecule. Glutamate-55 serves as a
proton shuttle, abstracting a proton from the zinc-activated water
to form the necessary hydroxide nucleophile, while also enabling
protonation at the N-3 site of guanine, thus resulting in the forma-
tion of the tetrahedral intermediate 2, as shown. Glu-55 also assists
in protonation of the NH2 group of 2, facilitating the elimination of
a molecule of ammonia to form the final product xanthine.
Azepinomycin may interrupt the above hydrolytic process via
coordination of its own OH group at position-6 with the active site
zinc, displacing the crucial active site water molecule involved in
hydrolysis. The observed weak inhibitory characteristics of azepino-
mycinmay involve multiplefactors, including, but not limitedto, the
fact that it does not represent the true transition state analog lacking
the geminal hydroxy-amino groups attached to its 6-position so as
O
H
N
HO
O
N
NH
O
N
O
N
H
10
The target 10 was synthesized (Scheme 1) in five-steps com-
mencing from compound 5, which has been reported from this lab-
oratory several years ago.41 The sequential in situ bromination of
the malonate carbon of 5, elimination of HBr, followed by conju-
gate addition of ammonia, gave 6 in 74% yield. The free NH2 of 6
was protected as a benzyloxycarbonyl (CBZ) derivative by treat-
ment with CBZ-Cl in the presence of Hünig’s base to produce 7 in
84% yield. The nitro group of 7 was reduced with zinc and acetic
acid to obtain 8 in 87% yield. Compound 8 was ring-closed using
potassium t-butoxide in dimethylformamide to yield 9 in 46%
yield. Finally, the ester group of 9 was selectively reduced to a pri-
mary alcohol using LiH-9BBN42 to obtain the target 10 in 39% yield.
All new compounds were completely characterized by 1H and
13CNMR and mass spectral data, coupled with either elemental
microanalyses or high resolution mass spectral (HRMS) data.43
Guanase inhibition studies with target 10 were conducted using
guanase from rabbit liver (MP Biochemicals) at 25 °C and pH 7.4 by
spectroscopically measuring the rate of hydrolysis of the substrate
guanine at kmax 245 nm. The change in optical density at kmax
245 nm per unit time is a measure of the guanase activity. A total
of seven different concentrations of the substrate, ranging from 5
O
Cys-86 Cys-83
7
Zn+2
6
1
5
N
HN
His-53
+
8
2
9
O
O
Glu55
N
H
H
H
H2N
N
3
4
O
Guanine (1)
Guanase
O
O
to 40
was either 20 or 40
l
M, was employed for each inhibitor concentration that
M, while the amount of enzyme in each assay
- NH3
N
N
HN
HN
l
Zn+2
O
was 7.7 Â 10À3 unit. The Lineweaver–Burk plots (1/V vs 1/S) (see
Fig. 2) were used to calculate KM, Vmax, and Ki. Our biochemical re-
sults showed that Km of enzyme with guanine as substrate was
9.5 Â 10À6 M. The target compound 10 showed competitive inhibi-
tion with a Ki of 16.70 0.48 Â 10À6 M.
N
H
N
H
N
H
N
H
O
H2N
(2)
Xanthine (3)
Figure 1. Zn+2-assisted hydrolysis of guanine to xanthine, catalyzed by guanase.

758
S. Chakraborty et al. / Bioorg. Med. Chem. Lett. 21 (2011) 756–759
O
O
O
O
Cl
+
O
O
(1) NaH/THF/Br2
(2) liq. NH3
H2N
O
O
O
O
O
Hunig's Base
HN
HN
74%
84%
O
N
N
O2N
O2N
N
N
5
6
O
O
O
O
NH
KOt-Bu/DMF
Zn/AcOH/EtOH
O
O
O
O
NH
O
O
O
O
87%
N
46%
N
HN
HN
O
O
H2N
O2N
N
N
8
7
O
H
O
O
O
N
H
N
HO
N
O
NH
O
N
O
LiH-9BBN
39%
NH
O
N
N
O
N
O
H
N
H
10
9
Scheme 1. Synthesis of the target compound 10.
In conclusion, we have designed, synthesized, and biochemi-
Acknowledgments
cally evaluated a novel, putative transition state analog inhibitor
of guanase. While the observed Ki in micromolar range is respect-
able, additional structure–activity relationship (SAR) studies would
be necessary to further enhance the inhibitory potential. Such
studies could involve introduction of various alkyl, aryl, or aralkyl
substituents at N-4, N-6, and/or N-7 of 10 or attachment of atoms
or groups with + or À inductive effects at various positions of the
two exonuclear phenyl rings.
This research was supported by a grant (#1R01 GM087738-
01A1) from the National Institute of General Medical Sciences of
the National Institutes of Health.
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Figure 2. Lineweaver–Burk plot of target compound 10.

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135.9, 136.3, 136.9, 140.5, 155.1, 160.0, 164.6; HRMS (FAB), calcd. for
C
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appeared. It was then filtered through Celite followed by washing with
ethanol. Evaporation of ethanol gave 8, which was further purified by column
chromatography (care should be taken to remove the acetic acid completely
otherwise it can make the ring closure step problematic). Yield 87%, (1.8 g,
3.4 mmol); Rf 0.17 (chloroform/methanol, 5:0.1); 1H NMR (DMSO-d6): d 1.11 (t,
6H, CH3), 4.18 (m, 4H, CH2), 5.03 (s, 2H, OBn), 5.09 (s, 2H, Bn), 5.9 (s, 2H, NH2
21. Tung, R.; Silber, R.; Quagliata, F.; Conklyn, M.; Gottesman, J.; Hirscholrn, R. J.
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+ Imd CH), 8.07 (s, 1H,
carbamate NH, exchangeable with D2O), d 8.1 (s, 1H, amide NH, exchangeable
with D2O); 13C NMR (DMSO-d6): d 14.2, 46.2, 63.3, 66.5, 69.9, 111.1, 127.8,
128.1, 128.2, 128.4, 128.2, 129.2, 131.4, 136.8, 137.2, 144.4, 154.8, 163.2,
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26H29N5O7: 523.20691 (M+); obsd m/z
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43. Experimental: Organic synthesis procedure: Preparation and physico-chemical
properties of the compounds are as follows: Diethyl 2-amino-2-[N-(1-benzyl-5-
524.21452 (MH+). 3-Benzyl-4,5,7,8-tetrahydro-6-(benzyloxycarbonyl)amino-6-
ethoxycarbonylimidazo[4,5-e][1,4]diazepine-5,8-dione (9): potassium tert-
butoxide (24 mg, 0.21 mmol) was taken in a dry flask with nitrogen line and
5 mL of dry DMF was added and stirred vigorously. To the mixture was added 8
(70 mg, 0.13 mmol). The heterogeneous mixture immediately turns orange.
The mixture was vigorously stirred for 2 h and was monitored by TLC. Care
should be taken as one of the by-products is very close to the starting material
in TLC using ethyl acetate as the solvent system. The resulting yellow liquid
was concentrated under vacuum and neutralized and extracted with ethyl
acetate. It is then purified by column chromatography using chloroform/
methanol (10:1) as the solvent system. Triturating with ether gave 9 as a
yellow product (30 mg, 0.06 mmol), yield 46%; Rf 0.46 (ethylacetate/methanol,
2:1); 1H NMR (DMSO-d6): d 1.09 (t, 3H, CH3), 4.09 (q, 2H, CH2), 5.05 (m, 2H,
OBn), 5.2 (s, 2H, Bn), 6.8 (d, 1H, NH), 7.24–7.34 (m, 11H, Ar), 7.7 (d, 1H, NH,
exchangeable with D2O), 7.8 (1H); 13C NMR (DMSO-d6): d 14.2, 47.5, 62.7, 66.7,
99.9, 114.7, 127.7, 128.1, 128.5, 128.7, 128.9, 129.4, 135.9, 136.7, 138.6, 140.7,
150.9, 156.2, 156.9, 167.8; anal. calcd for C24H24N5O6.75: C, 60.37; H, 4.86; N,
14.67. Found: C, 58.38; H, 4.74; N, 13.96; HRMS (FAB), calcd for C24H23N5O6:
477.16469 (M+); obsd m/z 478.17203 (MH+). 3-Benzyl-4,5,7,8-tetrahydro-6-
hydroxymethyl-6-[(benzyloxycarbonyl)-amino]imidazo[4,5-e][1,4]diazepine-5,8-
dione (10): compound 9 (280 mg, 0.58 mmol) was taken in a dry flask under
nitrogen atmosphere. It was dissolved in 10 mL of freshly distilled THF. It was
kept in ice-cold condition. To the cooled, well stirred solution was added drop
wise through syringe 1 M solution of LiH-9BBN in THF (0.75 mL). The mixture
was allowed to stir for an hour before quenching with 10 mL ethyl acetate and
evaporated to dryness with small portion of silica gel. Column chromatography
was done with 2% methanol in chloroform. The proper fractions were collected
and evaporated to obtain 10 a was white solid (0.1 g, 0.23 mmol), yield 39%; Rf
0.26 (ethylacetate/methanol, 2:1); 1H NMR (DMSO-d6): d 3.76 (m, 1H + 1H,
CH2), 5.02 (m, 2H + 1H, OBn + OH, OH exchangeable with D2O), 5.2 (s, 2H, Bn),
6.5 (m, NH, 1H), 7.28–7.36 (m, 10H, Ar+), 8.28 (1H, Imidazole), 12.3 (s, NH, 1H,
exchangable with D2O); 13C NMR (DMSO-d6): d 47.2, 60.7, 66.2, 79.7, 115.4,
127.8, 128.3, 128.4, 128.8, 129.3, 136.9, 137.2, 137.5, 140, 151.4, 155.6; HRMS
(FAB), calcd for C22H20O5N5: 434.14413 (M+); obsd m/z 435.15205 (MH+).
Biochemical inhibition procedure: guanine deaminase from rabbit liver was
purchased from MP biochemicals as a suspension in 3.2 M (NH4)2SO4, pH 6,
nitroimidazolyl-4-carbonyl)amino]malonate (6): in
a flame-dried two-neck
round bottomed flask, NaH (0.68 g, 17.2 mmol) was added under nitrogen
environment. Dry THF (20 mL) was then added and the flask was then cooled
and maintained at À78 °C (with dry ice acetone mixture). The starting material
5 (3.5 g, 8.6 mmol) was added quickly in one portion and was allowed to stir
for 10 min before bromine (liquid) (0.95 mL, 17.2 mmol) was added. After
10 min liq ammonia (2 mL) (excess) was added. The reaction mixture was
allowed to stir for 1 h and then allowed to come to room temperature. After
rotary evaporating the solvent it is neutralized in cold, filtered and extracted in
dichloromethane.
Further
purification
was
done
using
column
chromatography using 1–3% methanol in chloroform. Hexane wash gives
yellow product. Yield 74% (2.7 g, 6.6 mmol). 1H NMR (DMSO-d6): d 1.13 (t, 6H,
CH3), 4.17 (m, 4H, CH2), 5.06 (s, 2H, Bn), 6.0 (s, 2H, exchangeable with D2O,
NH2), 7.2–7.32 (m, 6H, Ar + Imd CH), 7.9 (s, 1H, exchangeable with D2O,
CONH); 13C NMR (DMSO-d6): d 14.3, 46.2, 62.7, 83.9, 111.0, 127.8, 128.1, 129.1,
131.4, 137.1, 144.7, 163.2, 165.9; anal. calcd C, 51.55; H, 5.05; N, 16.70. Found:
C, 51.44; H, 5.09; N, 16.57. Diethyl 2-(benzyloxycarbonyl)amino-2-[N-(1-benzyl-
5-nitroimidazolyl-4-carbonyl)amino]-malonate (7): compound
6
(2.3 g,
activity = 10 mg/mL, 0.06 lmol/mL. Freshly prepared solutions of Guanine and
5.48 mmol) was taken in dry flask and dissolved in dry THF under
a
Tris–HCl buffer (0.05 M) were used for the of biochemical studies. Enzyme
solution could be kept at room temperature for a day’s run. Twenty-five
milligram of Guanine was dissolved in 1 mL NaOH (1 N) and volume made to
100 mL in volumetric flask with Tris–HCl buffer (0.05 M). The solution was
kept at room temperature overnight. Next day, the solution was filtered and
concentration was measured by UV spectrophotometer using the molar
extinction coefficient 10,700 at k 243 nm. Sixty microliter of enzyme solution
was used in each 1 mL cuvette and was quickly shaken before taking the
nitrogen. To the stirred solution, benzylchloroformate (0.8 mL, 5.6 mmol) and
Hunig’s base (2.8 mL, 16.4 mmol) were added in iced condition. The reaction
mixture was allowed to stir in iced condition to room temperature overnight.
After the reaction was complete, the solvent was evaporated and the residue
was neutralized in cold and extracted in dichloromethane. It was further
purified using column chromatography with 1% methanol in chloroform. Yield
83% (2.5 g, 4.5 mmol). Rf 0.34 (chloroform/methanol, 5:0.1); 1H NMR (DMSO-
d6): d 1.12 (t, 6H, CH3), 4.17 (m, 4H, CH2), 5.05 (s, 2H, Bn), 5.5 (s, 2H, OBn), 7.21–
7.39 (m, 10H, Ar), 8.2 (s, 1H, Imd CH), 8.4 (s, 1H, carbamate NH, D2O
exchangeable), 9.4 (s, 1H, amide NH, D2O exchangeable); 13C NMR (DMSO-d6):
reading against air. Tris–HCl buffer was used as
measurements.
a reference for all