Inhibition of adenosine deaminase by analogues of adenosine and inosine, incorporating a common heterocyclic base, 4(7)-amino-6(5)H-imidazo[4,5-d] pyridazin-7(4)one

Article abstract of DOI:10.1021/jm700931t

Four nucleoside analogues (1-4) containing a common heterocyclic base, 4(7)-amino-6(5)H-imidazo[4,5-d]pyridazin-7(4)one, were screened against calf-intestine adenosine deaminase. Compounds 1 and 3 with K_i values of 10-12 μM are more than four t

Full text of DOI:10.1021/jm700931t

694
J. Med. Chem. 2008, 51, 694–698
Inhibition of Adenosine Deaminase by Analogues of Adenosine and Inosine, Incorporating a
Common Heterocyclic Base, 4(7)-Amino-6(5)H-imidazo[4,5-d]pyridazin-7(4)one
Ravi K. Ujjinamatada,† Pornima Phatak,‡ Angelika M. Burger,‡ and Ramachandra S. Hosmane*,†,‡
Laboratory for Drug Design and Synthesis, Department of Chemistry and Biochemistry, UniVersity of Maryland, Baltimore County, 1000
Hilltop Circle, Baltimore, Maryland 21250, and Marlene and Stewart Greenbaum Cancer Center, Experimental Therapeutics Program,
UniVersity of Maryland School of Medicine, 655 West Baltimore Street, Baltimore, Maryland 21201
ReceiVed July 30, 2007
Four nucleoside analogues (1-4) containing a common heterocyclic base, 4(7)-amino-6(5)H-imidazo[4,5-
d]pyridazin-7(4)one, were screened against calf-intestine adenosine deaminase. Compounds 1 and 3 with Ki
values of 10–12 µM are more than four times as potent inhibitors of ADA compared with 2 and 4, with Ki
values of 51–52 µM. Also, 3 is not a substrate of ADA. Nucleosides 3 and 4 also exhibit moderate in vitro
activity against breast cancer cell lines, while all four are only minimally or nontoxic to the normal cells.
Scheme 1. Hydrolysis of Adenosine to Inosine Catalyzed by
Adenosine Deaminase (ADA)
Introduction
Adenosine deaminase (ADA) is an important metabolic
enzyme that plays a crucial role in many human disorders
involving the immune system and function.1–4 Inhibition of
adenosine deaminase for immunosuppression has been the
objective of many investigations for the selective treatment of
lympho- proliferative malignancies.5–7 However, because ADA
is also an important essential enzyme, its inhibition cannot be
for an unlimited duration or extent. Therefore, the most desirable
inhibitors of ADA would not only be potent, but also be
considerably short-acting and easily reversible.8–12 In this
context, while there exist a host of known ADA inhibitors,2,13–20
many, if not all, suffer from one or the other drawbacks,
including too high potency with total irreversibility or too long
duration of action.10,11,21 For example, coformycin22–24 and
pentostatin (2′-deoxycoformycin),20,25,26 the two naturally oc-
curring, strongest known inhibitors of ADA with Ki values in
the 10-11-12 M range,27–30 have not been clinically as successful
as anticipated because of their associated toxicity,11,12,31 with
an exception of pentostatin in the treatment of hairy cell
leukemia.32–36 The coformycins are believed to be extremely
tight-binding, transition state analogue inhibitors of ADA,27–30
which lead to the total demise of the enzyme upon binding.10
So, the search must continue for ADA inhibitors with optimal
potency combined with a short duration of action and ready
reversibility. Our laboratory has been involved in such a quest
for some time,2,8,9,37–39 and this work represents our continued
effort in that direction.
with nearly equal efficiency. As we have already reported the
synthesis of 1 and 2 recently,40 only the synthesis of ribose
analogues 3 and 4 have been described here.
We have recently reported the synthesis and self-comple-
mentary base-pairing properties of two novel nucleoside ana-
logues 1 and 2 containing the title heterocyclic ring system.40
Because of their structural resemblance to adenosine and inosine,
respectively, compounds 1 and 2 were considered as potential
substrate and product analogues of the reaction catalyzed by
ADA (see Scheme 1). Therefore, it seemed logical to biochemi-
cally screen 1 and 2, along with their respective ribose
counterparts 3 and 4, as likely substrates or inhibitors (antime-
tabolites) of ADA. ADA catalyzes the hydrolysis of both
adenosine and 2′-deoxyadenosine into their respective inosines
Chemistry. The synthesis of the target nucleosides 3 and 4
(Scheme 2) commenced with ethyl 5(4)-cyano-1H-imidazole-
4(5)carboxylate (5).41 The Vorbrüggen ribosylation42,43 of the
latter with 1-O-acetyl-2,3,5-tri-O-benzoyl-ꢀ-D-ribofuranose (6)
at 5 °C yielded the two regioisomeric nucleosides 7 and 8 in
67 and 18%, respectively. The isomers were separated by flash
chromatography using a mixture of 5:1 hexanes/ethyl acetate,
but their structures were not assigned until after ring closure
with hydrazine in the next step. It was easier to distinguish them
from each other via a structural match of one of the isomers to
the known compound 3, which was independently synthesized
by an altogether different route by Berry et al.44 several years
ago, starting from an imidazole-fused oxadiazole. Compounds
* To whom correspondence should be addressed. Tel.: +1 410 455 2520.
Fax: +1 410 455 1148. E-mail: hosmane@umbc.edu.
†
University of Maryland.
University of Maryland School of Medicine.
‡
10.1021/jm700931t CCC: $40.75
2008 American Chemical Society
Published on Web 01/04/2008

Brief Article
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3 695
Scheme 2. Synthesis of Nucleosides 3 and 4
Figure 1. Lineweaver–Burk plot of compound 1.
Figure 2. Lineweaver–Burk plot of compound 2.
Figure 3. Lineweaver–Burk plot of compound 3.
7 and 8 were independently reacted with excess t-butylamine
in anhydrous methanol at room temperature to obtain 9 and 10
in 87 and 92% yield, respectively. The two compounds were
separately reacted with hydrazine at 5 °C for 30 min, followed
by heating with sodium ethoxide in ethanol at reflux for 18–28
h to yield the respective target nucleosides 3 and 4. While the
use of excess hydrazine with 9 did not affect the outcome of
the reaction, it produced an unexpected bis-adduct 11 in the
case of 10.
that we reported earlier.8,9,37,39 All studies were carried out at
25 °C and pH 7.4 by spectroscopic measurements of the rate
of hydrolysis of the substrate adenosine into product inosine at
λ
max 265 nm. The change in optical density at λmax 265 nm per
unit time was used as a measure of the enzyme activity. Seven
different concentrations of the substrate adenosine, ranging from
30 to 60 µM, were employed for each inhibitor concentration
that was either 5 or 15 µM, while the amount of enzyme in
each assay was 2 × 10-3 unit. The Lineweaver–Burk plots (1/V
vs 1/S), shown in Figures 1–4, were used to calculate the Ki
values, which are listed in Table 1.
Therefore, only 1 equiv of hydrazine had to be employed in
the reaction with 10 to obtain the desired product 4. Both 3
and 4 were fully characterized by H NMR, 13C NMR, IR,
HRMS, and elemental microanalytical data. The physicochem-
ical data for 3 are completely consistent with those reported by
Berry et al.44
1
The biochemical screening results clearly suggest that, out
of the four nucleosides 1–4, two of them, namely, 1 and 3, which
structurally resemble the substrate adenosine, have Ki values
ranging 10–12 µM, while the other two, namely, 2 and 4, which
mimic inosine, the product of ADA-mediated hydrolysis, possess
Ki values in the 51–52 µM range, suggesting that 1 and 3 are
more than 4 times as potent as 2 and 4. Also, the ribose
Results and Discussion
Compounds 1-4 and 11 were assessed for inhibitory activity
against ADA from calf intestine (Sigma), employing the protocol

696 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3
Brief Article
UV profile of the ADA reaction product was noticeably different
than that of the expected product. Therefore, we conclude that 3
is not a substrate of ADA or an antimetabolite of adenosine.
Finally, nucleosides 1–4 were screened in vitro for anticancer
activity as well as toxicity in human breast cell lines. The MTT
assay was used to determine the drug effects on cell proliferation
using the breast cancer cell lines MCF7, while toxicity was assessed
using the normal breast cell lines MCF10A, following the assay
procedure as described by Alley et al.46 The results are collected
in Table 2 and are graphically represented in Figure 5(A-D). The
data reveal that while 3 and 4, both of which are ribose analogues,
show moderate anticancer activity, all four nucleosides are
minimally or nontoxic to the normal breast cell lines.
Figure 4. Lineweaver–Burk plot of compound 4.
Experimental Section
Table 1. Inhibition Constants (Ki) for Compounds 1-4 and 11 against
Calf Intestine ADA
Ethyl 4-Cyano-1-(2,3,5-tri-O-benzoyl-ꢀ-D-ribofuranosyl)imi-
dazole-5-carboxylate (7) and Ethyl 5-Cyano-1-(2,3,5-tri-O-
benzoyl-ꢀ-D-ribofuranosyl)imidazole-4-carboxylate (8). A solu-
tion of 5-cyano-1H-imidazole-4-carboxylic acid ethyl ester (5;
3.30 g, 20 mmol) and 1-O-acetyl-2,3,5-tri-O-benzoyl-ꢀ-D-ribofura-
nose (10.08 g, 20 mmol) in anhyd acetonitrile (150 mL) was placed
into a flame-dried, three-necked, round-bottomed flask under an
argon atmosphere. The solution was stirred at 5 °C for 15 min.
Then hexamethyldisilazine (10.69 g, 14 mL, 33 mmol), chlorotri-
methylsilane (7.85 g, 9.2 mL, 36 mmol), and trifluoromethane
sulfonic acid (10.8 g, 6.39 mL, 36 mmol) were consecutively added
to the above solution. The resulting solution was stirred at 5 °C
for 1.5 h. The reaction mixture was brought to room temperature
and acetonitrile was evaporated under reduced pressure. The residue
was dissolved in chloroform (500 mL), and the solution was washed
with 10% sodium bicarbonate solution (3 × 150 mL) and water (3
× 100 mL). The chloroform layer was dried in anhydrous sodium
sulfate, and finally, regioisomers 7 and 8 were separated through
column chromatography using 5:1 hexanes/ethyl aceate solvent
system. Characterization data of 7 and 8 was as follows. Compound
7: Yield 8.17 g, 67%; mp 153 °C; Rf 0.23 (3:1 hexane/ethyl acetate);
cmpd
Ki
(µM)
1
2
3
4
11
12.14 ( 0.31 52.01 ( 1.6 10.62 ( 0.89 51.44 ( 3.26 26.54 ( 0.39
Table 2. Cytotoxic Effects of Nucleosides 1–4 in Cancer (MCF7) and
Normal (MCF10A) Breast Cell Lines
MCF7 (human breast
cancer cell line)
IC50 (µM)
MCF10A (normal human
breast cell line)
cmpd No.
IC50 (µM)
1
2
3
4
>100 ( 0
>100 ( 0
>100 ( 0
82.5 ( 3.54
82.5 ( 3.54
97.5 ( 3.54
27.5 ( 3.54
23.5 ( 2.1
analogues 3 and 4 were only marginally superior to their
respective 2′-deoxy counterparts 1 and 2, as anticipated.
Finally, because of its close structural similarity to adenosine,
it was of interest to see if 3 also acted as a substrate of ADA to
form the corresponding dicarbonyl compound HMC-HO5,
whose synthesis, along with its 2′-OMe analogue HMC-HO4,
was reported by us several years ago.45 In that case, 3 would
be considered an antimetabolite of the enzyme’s natural substrate
adenosine. It was convenient to monitor the product formation of
the ADA reaction with 3 through a simple comparison with the
UV λmax of the authentic product. Our results show that while the
IR 2244, 1726, 1601, 1262, 1247 cm-1 1H NMR (CDCl3, 400
;
MHz) δ 8.15 (s, 1H, imidazole CH), 8.06–7.25 (m, 15H, ArH),
6.89–6.88 (d, J ) 2.4 Hz, 1H, 1′-H), 5.85–5.84 (d, J ) 3.6 Hz,
2H, 4′,3′-H), 4.88–4.84 (m, 2H, 5′, 5′H), 4.74–4.68 (dd, J ) 4.2
Hz, 1H, 2′H), 4.39–4.32 (q, J ) 7.5 Hz, 2H, CH2), 1.65–1.34 (t, J
) 6.9 Hz, 3H, CH3); 13C NMR (CDCl3, 100 MHz) δ 165.5, 164.5,
164.1, 157.3, 138.5, 133.2, 133.2, 133.1, 129.3, 129.2, 129.1, 128.4,
128.01, 127.9, 127.9, 127.3, 121.1, 112.6, 88.4, 80.1, 76.8, 76.4,
76.0, 75.2, 69.6, 62.3, 62.0; Mass spectrum (FAB) m/z 610 (MH+);
Anal. (C33H27N3O9) C, H, N. Compound 8: Yield 2.2 g, 18%; mp
81 °C; Rf 0.12 (3:1 hexane/ethyl acetate); IR 2236, 1723, 1601,
λ
max (264.9 nm) of the starting material had changed to a slightly
higher wavelength in the product, it fell significantly short of the
expected λmax (275 nm) of the product. Furthermore, the general
1262, 1179 cm-1 1H NMR (CDCl3, 400 MHz) δ 8.07 (s, 1H,
;
Figure 5. Cytotoxic effects of nucleosides 1-4 in cancer (MCF7) and normal (MCF10A) breast cell lines.

Brief Article
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3 697
imidazole CH), 8.05–7.34 (m, 15H, ArH), 6.30 (d, J ) 6.3 Hz,
1H, 1′H), 5.95–5.92 (dd, J ) 3.3 Hz, 1H, 4′-H), 5.82 (t, J ) 6.3
Hz, 1H, 3′-H), 4.92–4.83 (m, 2H, 5′, 5′H), 4.77–4.72 (dd, J ) 3.3
Hz, 1H, 2′H), 4.43–4.35 (q, J ) 6.9 Hz, 2H, CH2), 1.39–1.35 (t, J
) 7.2 Hz, 3H, CH3); 13C NMR (CDCl3, 100 MHz) δ 165.4, 164.6,
164.4, 159.3, 141.5, 137.6, 133.5, 133.3, 133.1, 129.4, 129.2, 129.1,
128.4, 128.2, 128.1, 127.8, 127.2, 108.7, 87.8, 81.5, 76.8, 76.9,
76.5, 76.1, 74.9, 70.8, 62.9, 61.3; Mass spectrum (FAB) m/z 610
(MH+); Anal. (C33H27N3O9) C, H, N.
The reaction mixture was stirred at ice-cold temperature for 30 min.
Then methanol and unreacted hydrazine hydrate were removed
under high vacuum. The residue was refluxed in ethanol with a
catalytic amount of sodium methoxide (8 mg in 2 mL ethanol).
After 75 h of refluxing with vigorous stirring, the reaction mixture
was brought to room temperature and the separated gummy solid
was filtered and washed with ethanol. Yield 0.17 g, 54%; mp
194–196 °C; IR 3487, 3352, 3324, 3271, 1643, 1621, 1571, 1476,
1403, 1282, 1055 cm-1; Rf 0.25 (1:1:0.3 chloroform/methanol/
ammonium hydroxide); 1H NMR (DMSO-d6, 400 MHz) δ 9.39 (s,
1H, NH), 8.27 (s, 1H, imidazole CH), 6,63 (s, 2H, NH2), 6.15 (d,
J ) 2.9 Hz, 1H, 1′H), 5.50 (s, 1H, OH), 5.20 (bs, 2H, NH2), 5.14
(s, 1H, OH), 4.89 (s, 1H, OH), 4.45 (d, J ) 3.6 Hz,1H, CH), 4.03
(s, 2H, NH2), 3.89 (m,1H, CH), 3.75 (m, 1H, CH), 3.60 (m, 2H,
CH + CH); 13C NMR (DMSO-d6, 100 MHz) δ 163.0, 140.1, 137.0,
132.3, 128.6, 90.4, 84.3, 69.7, 60.4, 41.2; Anal. (C10H17N7O5 ·0.75
CH3OH) C, H, N; HRMS (FAB) calcd for C10H18N7O5, 316.1369
(MH+); observed, m/z 316.1363 (MH+).
Methyl 4-Cyano-1-(ꢀ-D-ribofuranosyl)imidazole-5-carboxy-
late (9). Ethyl 4-cyano-1-(2,3,5-tri-O-benzoyl-ꢀ-D-ribofuranosyl)im-
idazole-5-carboxylate (7; 6.1 g, 0.01 mol) was taken in anhyd
methanol (50 mL), and t-butylamine (7.3 g, 10.5 mL, 0.10 mol)
was added. The reaction mixture was stirred at room temperature
for 48 h. Methanol was removed under vacuum and the residue
was purified by silica gel flash chromatography. First chloroform
was used to remove the deprotected benzoyl groups and then 10:1
chloroform/methanol was used to collect the methyl ester 9. Yield
2.47 g, 87%; mp 159 °C; Rf 0.21 (10:1 chloroform/methanol); IR
3322, 3142, 2247, 1713, 1537, 1479, 1226 cm-1; 1H NMR (DMSO-
d6, 400 MHz) δ 8.69 (s, 1H, imidazole CH), 6.19 (d, J ) 2.3 Hz,
1H, 1′H), 5.56 (d, J ) 5.0 Hz, 1H, OH), 5.26 (t, J ) 4.6 Hz, 1H,
OH), 5.04 (d, J ) 6.4 Hz, 1H, OH), 4.13–4.05 (m, 2H, CH2),
3.95–3.93 (m, 1H, CH), 3.89 (s, 3H, CH3), 3.79–3.76 (s, 1H, CH),
3.64–3.60 (m, 1H, CH); 13C NMR (DMSO-d6, 100 MHz) δ 158.2,
141.3, 128.6, 119.4, 114.6, 91.7, 84.7, 76.3, 68.5, 59.8, 53.25; Mass
spectrum (FAB) m/z 284 (MH+); Anal. (C11H13N3O6) C, H, N.
Methyl 5-Cyano-1-(ꢀ-D-ribofuranosyl)imidazole-4-carboxy-
late (10). Ethyl 5-cyano-1-(2,3,5-tri-O-benzoyl-ꢀ-D-ribofurano-
syl)imidazole-4-carboxylate (8; 3.5 g, 0.005 mol) was taken in
anhyd methanol, t-butylamine (3.65 g, 5.25 mL, 0.05 mol) was
added, and the reaction mixture was stirred at room temperature
for 48 h. Methanol was evaporated under reduced pressure and the
pale yellow residue was purified by silica gel flash column
chromatography. Initially, chloroform was used to remove the
deprotected benzoyl groups and then 10:1 chloroform/methanol was
used to collect the title product 10. Yield 1.3 g, 92%; mp 56 °C; Rf
0.10 (10:1 chloroform/methanol); IR 3322, 3141, 2247, 1712, 1537,
1479, 1226 cm-1; 1H NMR (DMSO-d6, 400 MHz) δ 8.48 (s, 1H,
imidazole CH), 5.72 (d, J ) 3.6 Hz, 1H, 1′H), 5.70 (d, J ) 2.7 Hz,
1H, OH), 5.34 (d, J ) 5.0 Hz, 1H, OH), 5.09 (t, J ) 5.4 Hz, 1H,
OH), 4.35–4.32 (m, 1H, CH), 4.08–4.05 (m, 2H, CH2), 3.8 (s, 3H,
CH3), 3.64–3.60 (m, 2H, CH + CH); 13C NMR (DMSO-d6, 100
MHz) δ 160.9, 141.0, 140.6, 110.4, 107.8, 90.8, 86.8, 75.6, 70.4,
61.1, 52.7; Mass spectrum (FAB) m/z 284 (MH+); Anal.
(C11H13N3O6. 0.125 CH3OH) C, H, N.
7-Amino-1-(ꢀ-D-ribofuranosyl)-1H-imidazo[4,5-d]pyridazin-
4-(5H)-one (4). To a solution of hydrazine (0.016 g, 0.5 mmol) in
anhyd ethanol (10 mL), methyl 5-cyano-(1-ꢀ-D-ribofuranosyl)-
imidazole-4-carboxylate (10; 0.142 g, 0.5 mmol) was added and
the reaction solution was refluxed for 28 h. The separated solid
was filtered and washed with ethanol and recrystallized from
methanol. Yield 53 mg, 38%; Rf 0.51 (1:1:0.3 chloroform/methanol/
ammonium hydroxide); mp 234–236 °C; IR 3503, 3421, 3356,
1
3238, 1661, 1627, 1564, 1481, 1416, 1290, 1052 cm-1; H NMR
(DMSO-d6, 400 MHz) δ 11.63 (s, 1H, NH), 8.65 (s, 1H, imidazole
CH), 6.38 (d, J ) 5.04 Hz, 1H, 1′H), 5.84 (s, 2H, NH2), 5.50 (d,
J ) 5.04 Hz, 1H, OH), 5.16 (m, 2H, OH + OH), 4.38 (dd, J )
5.04 Hz, 1H, CH), 4.12 (dd, J ) 4.6 Hz,1H, CH), 3.98 (dd, J )
3.68 Hz, 1H, CH), 3.70 (m, 1H, CH), 3.59 (m, 1H, CH); 13C NMR
(DMSO-d6, 100 MHz) δ 154.6, 146.9, 145.6,136.1, 125.5, 88.6,
85.9, 70.5, 61.4, 41.1; Anal. (C10H13N5O5 ·2H2O) C, H, N; HRMS
(FAB) calcd for C10H14N5O5, 284.0995 (MH+); observed, m/z
284.0997 (MH+).
In Vitro Cell Cytotoxicity Assay. The MTT (3-(4, 5-dimeth-
ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma, St. Louis,
MO) assay was used to measure drug effects on normal and cancer
cell proliferation following the procedure as described by Alley et
al.46 In brief, 2000 cells/well were seeded into 96-well plates (Nunc,
U.S.A.) in complete RPMI1640 media and cells allowed to attach
overnight at 37 °C in 5% CO2. Drugs were dissolved in DMSO
(as 20 mM stock solutions) and added in final concentrations
ranging between 0.0001 to 100 µM in replicates of eight. Assays
were terminated after 5 days of continuous exposure to drug by
adding MTT. The resulting purple formazan was measured at 550
nm using a Synergy HT Multi-Detection Microplate Reader and
KC4 software (Bio-Tek, Winooski, VT). Growth inhibition was
4-Amino-1-(ꢀ-D-ribofuranosyl)-3H-imidazo[4,5-d]pyridazin-
7-(6H)-one (3). To a solution of methyl 4-cyano-1-(ꢀ-D-ribofura-
nosyl)imidazole-5-carboxylate (9; 0.568 g, 0.002 mol) in anhyd
methanol (15 mL), hydrazine hydrate (2 mL) was added. The
reaction mixture was stirred at ice cold temperature for 30 min.
Then methanol and unreacted hydrazine hydrate were removed
under high vacuum. Residue was refluxed in ethanol with a catalytic
amount of sodium methoxide (8 mg in 2 mL ethanol). After 16 h
of refluxing with vigorous stirring, the reaction mixture was brought
to room temperature, and the separated solid was filtered and
washed with ethanol. Yield 0.41 g, 72%; mp 242–244 °C; Rf 0.56
(1:1:0.3 chloroform/methanol/ammonium hydroxide); IR 3427,
assessed as inhibitory concentration 50% (IC50) and 100% (IC100
)
compared to vehicle treated controls and relative to cell growth at
the time of drug addition (day 0). Three independent experiments
were performed.
Acknowledgment. This paper is dedicated to Professor
Stewart W. Schneller, Dean of Science and Mathematics,
Auburn University, on the occasion of his 65th birthday. The
research was supported in part by grants (#9 R01 AI55452 and
#1 R21 AI071802) from the National Institute of Allergy and
Infectious Diseases (NIAID) of the National Institutes of Health,
Bethesda, Maryland, a pilot grant from the University of
Maryland Greenbaum Cancer Center (UMGCC), and an unre-
stricted grant from Nabi Biopharmaceuticals, Rockville, Maryland.
3337, 3266, 3192, 1669, 1630, 1569, 1475, 1414, 1287, 1050 cm-1
;
1H NMR (DMSO-d6, 400 MHz) δ 11.62 (s, 1H, NH), 8.64 (s, 1H,
imidazole CH), 6.38 (d, J ) 4.1 Hz, 1H, 1′H), 5.83 (s, 2H, NH2),
5.55 (s, 1H, OH), 5.20 (bs, 2H, OH + OH), 4.37 (t, J ) 5.0 Hz,1H,
CH), 4.13 (t, J ) 4.1 Hz,1H, CH), 3.94–3.93 (m, 1H, CH),
3.69–3.54 (m, 2H, CH + CH); 13C NMR (DMSO-d6, 100 MHz) δ
154.6, 145.5, 143.08, 136.18, 125.61, 89.36, 86.01, 75.9, 70.2, 61.4;
Anal. (C10H13N5O5 ·H2O) C, H, N; HRMS (FAB) calcd for
C10H14N5O5, 284.0995 (MH+); found, m/z 284.0995 (MH+).
5-Carbamimidoyl-1-(ꢀ-D-ribofuranosyl)-1H-imidazole-4-car-
bohydrazide (11). To a solution of methyl 5-cyano-1-(ꢀ-D-
ribofuranosyl)imidazole-4-carboxylate (10; 0.284 g, 0.001 mol) in
anhyd methanol (15 mL), hydrazine hydrate (2 mL) was added.
Supporting Information Available: General Experimental
Procedure and elemental microanalyical data on all new compounds.
This material is available free of charge via the Internet at http://
pubs.acs.org.

698 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3
Brief Article
unusual nucleoside of microbial origin. J. Am. Chem. Soc. 1974, 96,
4327–4328.
(23) Nakamura, H.; Koyama, G.; Umezawa, H.; Iitaka, Y. The crystal and
molecular structure of coformycin. Acta Crystallogr., Sect. B: Struct.
Sci. 1976, 32, 1206–1212.
(24) Ohno, M.; Yagisawa, N.; Shibahara, S.; Kondo, S.; Maeda, K.;
Umezawa, H. Synthesis of coformycin. J. Am. Chem. Soc. 1974, 96,
4326–4327.
(25) Baker, D. C.; Putt, S. R. A total synthesis of pentostatin, the potent
inhibitor of adenosine deaminase. J. Am. Chem. Soc. 1979, 101, 6127–
6128.
References
(1) Kurz, L. C.; Frieden, C. Atomic structure of adenosine deaminase
complexed with a transition-state analog: Understanding catalysis and
immunodeficiency mutations. Chemtracts: Biochem. Mol. Biol. 1991,
2, 276–278.
(2) Hosmane, R. S. Coformycin and analogues as inhibitors of adenosine
deaminase. Modified Nucleosides: Synthesis and Applications; Re-
search Signpost: Trivendrum, India, 2002; pp 133–151.
(3) Cristalli, G.; Costanzi, S.; Lambertucci, C.; Lupidi, G.; Vittori, S.;
Volpini, R.; Camaioni, E. Adenosine deaminase: Functional implica-
tions and different classes of inhibitors. Med. Res. ReV. 2001, 21, 105–
128.
(4) Juliusson, G. Immunological and genetic abnormalities in chronic
lymphocytic leukaemia. Impact of the purine analogues. Drugs 1994,
47 Suppl 6, 19–29.
(5) Smyth, J. F.; Poplack, D. G.; Holiman, B. J.; Leventhal, B. G.; Yarbro,
G. Correlation of adenosine deaminase activity with cell surface
markers in acute lymphoblastic leukemia. J. Clin. InVest. 1978, 62,
710–712.
(6) Smyth, J. F.; Prentice, H. G.; Proctor, S.; Hoffbrand, A. V. Deoxy-
coformycin in the treatment of leukemias and lymphomas. Ann. N.Y.
Acad. Sci. 1985, 451, 123–128.
(7) Aldrich, M. B.; Blackburn, M. R.; Kellems, R. E. The importance of
adenosine deaminase for lymphocyte development and function.
Biochem. Biophys. Res. Commun. 2000, 272, 311–315.
(8) Hong, M.; Hosmane, R. S. Irreversible, tight-binding inhibition of
adenosine deaminase by coformycins: Inhibitor structural features that
contribute to the mode of enzyme inhibition. Nucleosides Nucleotides
1997, 16, 1053–1057.
(9) Reayi, A.; Hosmane, R. S. Inhibition of adenosine deaminase by novel
5:7 fused heterocycles containing the imidazo[4,5-e][1,2,4]triazepine
ring system: A structure-activity relationship study. J. Med. Chem.
2004, 47, 1044–1050.
(10) Agarwal, R. P.; Cha, S.; Crabtree, G. W.; Parks, R. E., Jr. Coformycin
and deoxycoformycin: Tight-binding inhibitors of adenosine deami-
nase. Chem. Biol. Nucleosides Nucleotides, [Pap. Symp.] 1978, 159–
197.
(11) Agarwal, R. P. Deoxycoformycin toxicity in mice after long-term
treatment. Cancer Chemother. Pharmacol. 1980, 5, 83–87.
(12) Major, P. P.; Agarwal, R. P.; Kufe, D. W. Deoxycoformycin:
Neurological toxicity. Cancer Chemother. Pharmacol. 1981, 5, 193–
196.
(13) Agarwal, R. P.; Spector, T.; Parks, R. E., Jr. Tight-binding inhibitors.
IV. Inhibition of adenosine deaminases by various inhibitors. Biochem.
Pharmacol. 1977, 26, 359–367.
(14) Cristalli, G.; Franchetti, P.; Grifantini, M.; Vittori, S.; Lupidi, G.; Riva,
F.; Bordoni, T.; Geroni, C.; Verini, M. A. Adenosine deaminase
inhibitors. Synthesis and biological activity of deaza analogues of
erythro-9-(2-hydroxy-3-nonyl)adenine. J. Med. Chem. 1988, 31, 390–
393.
(15) Antonini, I.; Cristalli, G.; Franchetti, P.; Grifantini, M.; Martelli, S.;
Lupidi, G.; Riva, F. Adenosine deaminase inhibitors. Synthesis of
deaza analogues of erythro-9-(2-hydroxy-3-nonyl)adenine. J. Med.
Chem. 1984, 27, 274–278.
(16) Da Settimo, F.; Primofiore, G.; La Motta, C.; Taliani, S.; Simorini,
F.; Marini, A. M.; Mugnaini, L.; Lavecchia, A.; Novellino, E.;
Tuscano, D.; Martini, C. Novel, highly potent adenosine deaminase
inhibitors containing the pyrazolo[3,4-d]pyrimidine ring system.
Synthesis, structure-activity relationships, and molecular modeling
studies. J. Med. Chem. 2005, 48, 5162–5174.
(17) Harriman, G. C.; Poirot, A. F.; Abushanab, E.; Midgett, R. M.;
Stoeckler, J. D. Adenosine deaminase inhibitors. Synthesis and
biological evaluation of C1′ and nor-C1′ derivatives of (+)-erythro-
9-(2(S)-hydroxy-3(R)-nonyl)adenine. J. Med. Chem. 1992, 35, 4180–
4184.
(26) Chan, E.; Putt, S. R.; Showalter, H. D. H.; Baker, D. C. Total synthesis
of (8R)-3-(2-deoxy-b-D-erythro-pentofuranosyl)-3,6,7,8-tetrahydroimi-
dazo[4,5-d][1,3]diazepin-8-ol (pentostatin), the potent inhibitor of
adenosine deaminase. J. Org. Chem. 1982, 47, 3457–3464.
(27) Kati, W. M.; Wolfenden, R. Contribution of a single hydroxyl group
to transition-state discrimination by adenosine deaminase: Evidence
for an “entropy trap” mechanism. Biochemistry 1989, 28, 7919–7927.
(28) Kati, W. M.; Wolfenden, R. Major enhancement of the affinity of an
enzyme for a transition-state analog by a single hydroxyl group.
Science 1989, 243, 1591–1593.
(29) Frick, L.; Wolfenden, R.; Smal, E.; Baker, D. C. Transition-state
stabilization by adenosine deaminase: structural studies of its inhibitory
complex with deoxycoformycin. Biochemistry 1986, 25, 1616–1621.
(30) Wolfenden, R.; Wentworth, D. F.; Mitchell, G. N. Influence of
substituent ribose on transition state affinity in reactions catalyzed by
adenosine deaminase. Biochemistry 1977, 16, 5071–5077.
(31) Margolis, J.; Grever, M. R. Pentostatin (Nipent): A review of potential
toxicity and its management. Semin. Oncol. 2000, 27, 9–14.
(32) Dillman, R. O. Pentostatin (Nipent) in the treatment of chronic
lymphocyte leukemia and hairy cell leukemia. Expert ReV. Anticancer
Ther. 2004, 4, 27–36.
(33) Tallman, M. S. Current treatment strategies for patients with hairy
cell leukemia. ReV. Clin. Exp. Hematol. 2002, 6, 389–400.
(34) Tallman, M. S.; Hoffman, M. A.; Peterson, L. C. Current concepts in
the management of hairy cell leukemia. Basic Clin. Oncol. 2001, 26,
567–591.
(35) Ogawa, K.; Shichishima, T.; Nakamura, N.; Maruyama, Y. Induction
of apoptosis in vivo and in vitro in hairy cell leukemia treated by
deoxycoformycin. Tohoku J. Exp. Med. 2000, 192, 87–98.
(36) Spiers, A. S. D. Deoxycoformycin (pentostatin): Clinical pharmacol-
ogy, role in the chemotherapy of cancer, and use in other diseases.
Haematologia 1996, 27, 55–84.
(37) Hosmane, R. S.; Hong, M. How important is the N-3 sugar moiety in
the tight-binding interaction of coformycin with adenosine deaminase.
Biochem. Biophys. Res. Commun. 1997, 236, 88–93.
(38) Hosmane, R. S. Ring-expanded (“fat”) nucleosides as broad-spectrum
anticancer and antiviral agents. Curr. Top. Med. Chem. 2002, 2, 1093–
1109.
(39) Reayi, A.; Hosmane, R. S. Inhibitors of adenosine deaminase:
Continued studies of structure-activity relationships in analogues of
coformycin. Nucleosides, Nucleotides Nucleic Acids 2004, 23, 263–
271.
(40) Ujjinamatada, R. K.; Paulman, R. L.; Ptak, R. G.; Hosmane, R. S.
Nucleosides with self-complementary hydrogen-bonding motifs: Syn-
thesis and base-pairing studies of two nucleosides containing the
imidazo[4,5-d]pyridazine ring system. Bioorg. Med. Chem. 2006, 14,
6359–6367.
(41) Subrayan, R. P.; Thurber, E. L.; Rasmussen, P. G. Synthesis and
assignments of regioisomeric cyanoimidazole esters. Tetrahedron 1994,
50, 2641–2656.
(42) Vorbruggen, H.; Krolikiewicz, K.; Bennua, B. Nucleoside syntheses.
XXII. Nucleoside synthesis with trimethylsilyl triflate and perchlorate
as catalysts. Chem. Ber. 1981, 114, 1234–1255.
(43) Vorbruggen, H.; Bennua, B. Nucleoside syntheses. XXV. A
new simplified nucleoside synthesis. Chem. Ber. 1981, 114, 1279–
1286.
(44) Berry, D. A.; Wotring, L. L.; Drach, J. C.; Townsend, L. B.
Synthesis and biological activity of 4-amino-1-(ꢀ-D-ribofurano-
syl)imidazo[4,5-d]pyridazin-7-one. Nucleosides Nucleotides 1994,
13, 2001–2011.
(45) Borowski, P.; Lang, M.; Haag, A.; Schmitz, H.; Choe, J.; Chen,
H.-M.; Hosmane, R. S. Characterization of imidazo[4,5-d]pyridazine
nucleosides as modulators of unwinding reaction mediated by west
nile virus NTPase/helicase: Evidence for activity on the level of
substrate and/or enzyme. Antimicrob. Agents Chemother. 2002, 46,
1231–1239.
(46) Alley, M. C.; Scudiero, D. A.; Monks, A.; et al. Feasibility of drug
screening with panels of human tumor cell lines using a microculture
tetrazolium assay. Cancer Res. 1988, 48, 589–601.
(18) Vittori, S.; Camaioni, E.; Costanzi, S.; Volpini, R.; Lupidi, G.; Cristalli,
G. Structure-activity relationships of adenosine deaminase inhibitors.
Nucleosides Nucleotides 1999, 18, 741–742.
(19) Wang, L.; Hosmane, R. S. A unique ring-expanded acyclic nucleoside
analogue that inhibits both adenosine deaminase (ADA) and guanine
deaminase (GDA; Guanase): Synthesis and enzyme inhibition studies
of 4,6-diamino-8H-1-hydroxyethoxymethyl-8-iminoimidazo[4,5-e]-
[1,3]diazepine. Bioorg. Med. Chem. Lett. 2001, 11, 2893–2896.
(20) Woo, P. W. K.; Dion, H. W.; Lange, S. M.; Dahl, L. F.; Durham,
L. J. Novel adenosine and ara-A deaminase inhibitor, (R)-3-(2-deoxy-
b-D-erythro-pentofuranosyl) -3,6,7,8-tetrahydroimidazo[4,5-d][1,3]di-
azepin-8-ol. J. Heterocycl. Chem. 1974, 11, 641–643.
(21) Goday, A.; Simmonds, H. A.; Webster, D. R.; Morris, G. S. EHNA
is a poor inhibitor of deoxyadenosine catabolism in cultured human
lymphocytes. ReV. Esp. Fisiol. 1985, 41, 49–54.
(22) Nakamura, H.; Koyama, G.; Iitaka, Y.; Ohno, M.; Yagisawa, N.;
Kondo, S.; Maeda, K.; Umezawa, H. Structure of coformycin, an
JM700931T