8-Aza-immucillins as transition-state analogue inhibitors of purine nucleoside phosphorylase and nucleoside hydrolases

Article abstract of DOI:10.1021/jm0203332

The 8-aza-immucillins (8-aza-9-deazapurines linked from C9 to C1 of 1,4-dideoxy-1,4-iminoribitol) have been designed as transition-state analogues of the reactions catalyzed by purine nucleoside phosphorylase and nucleoside hydrolases. Syntheses of the 8-aza-immucillin analogues of inosine and adenosine are described. They are powerful inhibitors of the target enzymes with equilibrium dissociation constants as low as 42 pM.

Full text of DOI:10.1021/jm0203332

J . Med. Chem. 2003, 46, 155-160
155
8
-Aza -im m u cillin s a s Tr a n sition -Sta te An a logu e In h ibitor s of P u r in e Nu cleosid e
P h osp h or yla se a n d Nu cleosid e Hyd r ola ses
,
†
†
†
‡
§
Gary B. Evans,* Richard H. Furneaux, Graeme J . Gainsford, J ohn C. Hanson, Gregory A. Kicska,
§
§
†
Antony A. Sauve, Vern L. Schramm, and Peter C. Tyler
Carbohydrate Chemistry, Industrial Research Limited, P.O. Box 31-310, Lower Hutt, New Zealand, Department of
Biochemistry, Albert Einstein College of Medicine of Yeshiva University, 1300 Morris Park Avenue, Bronx, New York 10461,
and Brookhaven National Laboratory, Upton, New York 11973
Received J uly 30, 2002
The 8-aza-immucillins (8-aza-9-deazapurines linked from C9 to C1 of 1,4-dideoxy-1,4-iminor-
ibitol) have been designed as transition-state analogues of the reactions catalyzed by purine
nucleoside phosphorylase and nucleoside hydrolases. Syntheses of the 8-aza-immucillin
analogues of inosine and adenosine are described. They are powerful inhibitors of the target
enzymes with equilibrium dissociation constants as low as 42 pM.
In tr od u ction
We have an ongoing interest in the design and
synthesis of purine nucleoside phosphorylase (PNP)
inhibitors because such compounds are expected to have
therapeutic potential for the control of undesirable T-cell
proliferation. The importance of PNP in T-cell prolifera-
tion has been demonstrated in patients with inherited
PNP deficiency, where T-cell levels and their response
to stimuli are dramatically reduced. However, these
1
,2
patients have relatively normal B-cell function. PNP
inhibition leads to an accumulation of deoxyguanosine
(dG), which is metabolized to deoxyguanosine triphos-
phate (dGTP) in T-cells where it causes inhibition of
ribonucleotide reductase and, consequently, DNA syn-
thesis.2 The human T-cell is unique in this selectivity
because of high deoxycytidine kinase levels, which
phosphorylate dG, and the relatively low nucleotidase
activity, which allows dGTP to accumulate.2
Undesirable activation and proliferation of T-cells are
associated with several human disease states including
psoriasis, rheumatoid arthritis, T-cell leukemias and
lymphomas, transplant tissue rejection, and other type
,3
known of several purine phosphoribosyltransferases.9
In addition to powerful inhibition of their target en-
zymes, the immucillins have been useful in crystal-
lography by providing complexes with closed and or-
dered catalytic sites. Properties of inhibition and catalytic
site contacts support the proposal that these complexes
are closely related to the structures at the transition
IV autoimmune disorders. Consequently PNP inhibition
has become a target for drug design.4,5
The transition-state structures of the enzyme-cata-
lyzed phosphorolysis of inosine and the hydrolysis of
inosine and adenosine have been solved by analysis of
1
3,17,21
states.
Early studies on transition-state analogues of AMP
6
N-ribosylhydrolase revealed that formycin 5′-phosphate
kinetic isotope effects. The ribooxacarbenium character
2
4,25
6
is a powerful inhibitor.
As part of ongoing studies
of the transition state can be reproduced in stable
analogues by the combination of a 9-deazapurine and
an iminoribitol. Using this information, we have de-
signed and synthesized transition-state analogue inhibi-
tors of PNP, protozoan nucleoside hydrolases, and
into the structure-activity relationship of the immuci-
llins, we were interested in how 8-aza-immucillins 7 and
8
might perform as inhibitors of PNP and nucleoside
hydrolases. The extra nitrogen would be expected to
phosphoribosyltransferases.7
-23
modify the pK of the 7-NH residue, an important site
Immucillins 1 and 2 are
a
for substrate-enzyme interaction in both PNP and
nucleoside hydrolases.
We report here the first synthesis of 8-aza-immucillins
exceedingly potent inhibitors of PNP, and 1 is in clinical
trials for the control of T-cell leukemia. Additionally 1-3
are powerful inhibitors of nucleoside hydrolases, and the
7
and 8 and their in vitro activity against bovine and
5
′-phosphates 4 and 5 are the most potent inhibitors
human PNP and two nucleoside hydrolases.
*
To whom correspondence should be addressed. Phone: 64-4-569-
0
000. Fax +64-4-569-0055. E-mail g.evans@irl.cri.nz.
Resu lts a n d Discu ssion
†
Industrial Research Limited.
Brookhaven National Laboratory.
Albert Einstein College of Medicine of Yeshiva University.
‡
Syn th esis. The immucillins have all been synthe-
sized by way of imine 9 (Scheme 1). Thus, immucillin-H
§
1
0.1021/jm0203332 CCC: $25.00 © 2003 American Chemical Society
Published on Web 11/28/2002

1
56 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1
Evans et al.
Sch em e 1
(
1) may be readily prepared by a direct condensation of
Deprotection of the diethyl acetal was achieved with
a mixture of glacial acetic acid and 10% aqueous
hydrochloric acid, and the resulting acetylenic aldehyde
was treated in situ with hydrazine hydrate under reflux
to afford a pyrazole. The crude product was acetylated
to give the peracetylated pyrazole derivative 18 in good
overall yield.
2
6
the lithiated deazapurine derivative 10 with imine 9.
The 9-deazaguanosine and -adenosine analogues 2 and
3
as lithiated 9-deazaadenine and 9-deazaguanine deriva-
tives undergo decomposition either in concert with or
before addition to the imine 9. Similarly, attempts to
prepare 7 by addition of the lithiated pyrazolopyrimi-
2
7
are better prepared via the acetonitrile adduct 11
Nitration of 18 was achieved using ammonium nitrate
2
6
dine derivative 12 to the imine 9 were unsuccessful.
and trifluoroacetic anhydride (TFAA) in trifluoroacetic
By analogy with reported work on the synthesis of
3
1
acid (TFA) to afford a 2,4-dinitropyrazole, which was
treated without purification with potassium cyanide in
a mixture of ethanol and ethyl acetate to afford the
formycin (13) and pyrazofurin (14) from the acetylene
C-glycoside (15),
an appropriate lithiated alkyne to imine 9 would afford
material that could be transformed into the desired
products.
2
8-31
we envisaged that the addition of
2
-cyano-3-nitropyrazole 19 in excellent yield. Reduction
of the nitro group of 19 to give the desired aminopyra-
zole 20 proved to be problematic because standard
hydrogenation conditions either were ineffective or
produced side products. This may in part be due to the
reported instability of the Troc group under hydro-
3
2
genolysis conditions. However, zinc in acetic acid
efficiently reduced the nitro group, although these
conditions also removed the Troc group to give 20. The
acetate protecting groups were removed at this stage
because some of the reagents required for later steps
resulted in their partial loss. Deacetylation under
Zemplen conditions followed by reprotection of the
iminoribitol nitrogen as the tert-butyl carbamate gave
the key intermediate 21.
Hydrolysis of nitrile 21 was effected by treatment
with hydrogen peroxide and potassium carbonate in
DMSO, affording amide 22 in good yield. Deprotection
of 22 with dilute HCl afforded the seco-immucillin
analogue 23 as its hydrochloride salt. Treatment of 21
with formamidine acetate in refluxing ethanol followed
by dilute HCl afforded the 8-aza-immucillin-A homo-
logue 8 as its hydrochloride salt. Similar treatment of
Addition of lithiated 3,3-diethoxy-1-propyne to the
imine 9 (Scheme 2) at 10-15 °C in diethyl ether afforded
the aza-C-glycoside 16. Reaction temperatures above 15
°C led to the rapid decomposition of the lithiated alkyne,
but below 10 °C little or no addition to the imine was
seen. With careful control of the temperature, the
reaction could be carried out on a multigram scale and
in acceptable yield. Treatment of 16 with 2,2,2-trichlo-
roethyl chloroformate afforded 17. The Troc protecting
group was selected because it is stable at both extremes
of pH necessary for subsequent manipulations and
under nitration conditions.
2
2 afforded the 8-aza-immucillin-H homologue 7 as its
hydrochloride salt.
X-r a y Sin gle-Cr ysta l An a lysis of 8-Aza -im m u ci-
llin -H (7). An X-ray diffraction analysis of 8-aza-
immucillin-H (7) (Figure 1) confirmed its structure,
providing proof that the product derived by addition of

8
-Aza-immucillins
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1 157
Sch em e 2a
a
Reagents: (a) 3,3-diethoxy-1-lithiopropyne, diethyl ether, 10-15° C, 46%; (b) 2,2,2-trichloroethyl chloroformate, Hunigs base, CH2Cl2,
°C f room temp, 88%; (c) glacial acetic acid, 10% HCl, room temp, then N2H4, reflux, then Ac2O, Py, room temp, 65%; (d) TFAA, TFA,
0
NH4NO3, 0 °C f room temp, then KCN, EtOAc, EtOH, room temp, 93%; (e) Zn, HOAc, room temp, 68%; (f) NaOMe, MeOH, room temp,
+
8
4%, then Boc2O, MeOH, room temp, 71%; (g) H2O2, K2CO3, DMSO, room temp, 61%; (h) H3O , room temp, 100%; (i) formamidine acetate,
+
ethanol, reflux, then H3O , room temp.
Ta ble 1. Inhibition Constants for the Interaction of
Immucillins with Human and Bovine PNPs
human PNP
* (pM)
(s-1)
bovine
inhibitor
K
i
k
6
K
i
* (pM)
6
k )
(s-1
72 ( 26 a (1.5 ( 0.6) × 10
29 ( 8 a (1.5 ( 0.6) × 10
104 ( 31 (1.3 ( 0.1) × 10
-3
-3
a
a
23 ( 5 a (4 ( 2) × 10
-5 a
1
2
7
30 ( 6 a (1.9 ( 0.2) × 10
42 ( 6 (2.1 ( 0.1) × 10
60 ( 50 (1.9 ( 0.1) × 10
-4
-2
-2
a
-2
96 ( 16 (1.3 ( 0.1) × 10-
3
2
3
a
Values taken from ref 12.. The rate constant k6 describes the
conformational change from the tightly bound EI* complex to the
less tightly bound EI complex for the equilibrium E + I h EI yk6
\z
EI*. Ki* is the dissociation constant for E + I h EI*.
human and bovine enzymes by approximately a factor
of 2. The similar inhibition of these two enzymes arises
from common transition-state interactions, since the
human and bovine enzymes share 87% identity in the
amino acid sequence, and in the X-ray crystal struc-
tures, contacts to substrate and product analogues in
human and bovine enzymes are identical within experi-
mental error. The seco-analogue 23 has surprisingly
robust activity considering that the rigid structure of
the purine ring has been lost. The major inhibitory
forces between PNP and the immucillins therefore arise
from bonds readily achieved even with rotation of the
carboxamide group. The N-3 in 1, 2, and 7 can interact
only as an H-bond acceptor, while in 23 it is restricted
to H-bond donation. The similar affinity indicates that
enzymatic interaction with N-3 plays a minor role in
Ki*.
F igu r e 1. X-ray single-crystal analysis of 8-aza-immucillin-H
(7).
the lithiated alkyne to imine 9 possessed the required
â-stereochemistry.
In h ibition of P u r in e Nu cleosid e P h osp h or yla ses
by 8-Aza -im m u cillin s. Inhibition of bovine and human
PNP by 8-aza-immucillin-H 7 and 23 were tested and
compared to immucillin-H 1 and immucillin-G 2 (Table
3
3
1
). For the human enzyme, 1, 2, 7, and 23 all inhibit
within a factor of 4, with Ki* values of 29-104 pM. The
same series for the bovine enzyme reveals Ki* values of
2
3-60 pM. The pyrazolo group, as opposed to a pyrolo
group, weakens the equilibrium binding to both the

1
58 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1
Evans et al.
Ta ble 2. Inhibition Constants for the Interaction of
Immucillins with IU and IAG Nucleoside Hydrolases
NMR spectra were recorded on a Bruker AC-300 instrument
1
13
at 300 MHz ( H) or 75 MHz ( C). Normally, spectra were
measured in CDCl with Me Si as the internal reference; when
O was the solvent, acetone ( H, δ 2.20; C, δ 33.2) was used
3
4
IU nucleoside hydrolase
IAG nucleoside hydrolase
1
13
D
2
Ki* or Ki (µM)
Ki* or Ki (µM)
as the internal reference. High-resolution accurate mass
determinations were performed by Hort Research Ltd., Palm-
erston North, New Zealand, on a VG70-250S double-focusing
magnetic sector mass spectrometer under chemical ionization
conditions using isobutane or ammonia as the ionizing gas or
under high-resolution fast atom bombardment conditions in
a glycerol or nitrobenzyl alcohol matrix. Melting points were
determined on a Reichert hot stage microscope and are
uncorrected. Column chromatography was performed on silica
gel (230-400 mesh, Merck). Chromatography solvents were
distilled prior to use.
1
3
2
7
8
2
0.042 a
0.024 a
a
a
0.0072
0.0009
a
a
4
0.003
0.023
b
1.6 ( 0.8
0.15 ( 0.1
4.8 ( 0.7
13.9 ( 0.8
14.0 ( 1.0
3.7 ( 0.5
b
3b
a
Values taken from ref 15. Compounds 1, 3, and 24 cause tight
b
binding inhibition characterized by Ki*. Compounds 7, 8, and 23
are competitive inhibitors where Ki represents dissociation con-
stants for E + I h EI.
Immucillin-G 2 remains the most powerful inhibitor
known for human PNP. Changes in pKa values at the
pyrazolo nitrogens and the hydrogen bonding capacity
of the exocyclic oxygen are likely to be responsible for
the small decrease in affinity of 7 relative to immucil-
lin-G 2 and immucillin-H 1.
2
. Ch em istr y. (1S)-5-O-ter t-Bu tyldim eth ylsilyl-1,4-dide-
oxy-1-(3,3-diethoxyprop-1-ynyl)-1,4-imino-2,3-O-isopropylidene-
D-r ibitol (16). A solution of (1S)-5-O-tert-butyldimethylsilyl-
2
6
1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol (6.0 g,
1.0 mmol) in petroleum ether (100 mL) was stirred with
2
N-chlorosuccinimide (3.6 g, 27.0 mmol) for 1 h. The solids were
removed by filtration, and the solvent was removed in vacuo.
The resulting residue was dissolved in dry THF (100 mL), and
the solution was cooled to -78 °C. Lithium tetramethylpip-
eridide (75 mL, 0.4 M in THF) was added dropwise, and on
completion the reaction was diluted with petroleum ether (200
mL) and washed with water (100 mL) and brine (100 mL).
The organic layer was dried, concentrated, and chromato-
graphed to afford imine 9 (4.70 g) as a colorless oil. The imine
8
-Aza-immucillin-A 8 was, as expected, inactive against
both PNP enzymes. Adenosine is not a substrate for
these PNP enzymes but is a weak substrate for bacterial
PNPs.
In h ibition of Nu cleosid e Hyd r ola ses by 8-Aza -
im m u cillin s. Compounds 7, 8, and 23 were tested as
inhibitors of the nonspecific inosine-uridine nucleoside
hydrolase (IU-NH) from Crithidia fasciculata and of
the purine-specific inosine-adenosine-guanosine nu-
cleoside hydrolase (IAG-NH) from Trypanosoma brucei
brucei in comparison to immucillin-H 1, immucillin-A
9
was dissolved in diethyl ether (75 mL), and the resulting
solution was then added via cannula to one of the lithiated
,3-diethoxypropynes [prepared by the dropwise addition of
3
n-butyllithium (24.0 mL, 57.6 mmol) to a solution of 3,3-
diethoxypropyne (2.08 mL, 40 mmol) in dry diethyl ether (150
mL) at -78 °C, followed by stirring for 15 min] at -78 °C.
The reaction mixture was stirred for 10 min at -78 °C and
then allowed to warm to 15 °C and held at this temperature
for 1 h. After this time, the reaction was quenched by the
addition of water (5 mL) and the mixture was partitioned into
water and ethyl acetate. The organic phase was dried, con-
centrated, and chromatographed to afford alkyne (16) (4.00 g,
3
, and 24 (Table 2). For the enzyme from C. fasciculata,
9
1
.7 mmol, 46%) as an oil. IR νmax: 3448 (NH), 2243 (CtC), 1462,
-
1 1
3 2
372, 1254 cm . H NMR: δ 5.19 [1H, s, (CH O) CH], 4.60
(
1H, dd, J 2,3 ) 6.3, J 1,2 ) 3.2 Hz, H-2), 4.50 (1H, dd, J 2,3 ) 6.3,
3,4 ) 2.8 Hz, H-3), 3.87 (1H, d, J 1,2 ) 3.2 Hz, H-1), 3.59 (6H,
m, OCH , H-5), 3.18 (1H, dd, J 4,5 ) 5.9, J 3,4 ) 2.8 Hz, H-4),
.42, 1.25 [2 × 3H, s, C(CH ], 1.14 (2 × 3H, t, J ) 4.8 Hz,
OCH CH ), 0.83 [9H, s, C(CH
NMR: δ 113.1 [OC(CH ], 91.3 [CH(OEt)
CdC), 82.5 (C-3), 79.8 (CdC), 66.7 (C-4), 63.2 (C-5), 60.8
OCH CH ), 55.5 (C-1), 27.0, 25.0 [C(CH ], 25.9 [C(CH ],
8.3 [C(CH ], 15.1 (OCH CH ), -5.5, -5.4 (SiCH ). HRMS
M ) calcd for C21 Si: 414.2676. Found: 414.2684.
(1S)-5-O-ter t-Bu tyld im eth ylsilyl-1,4-d id eoxy-1-(3,3-d i-
et h oxyp r op -1-yn yl)-1,4-im in o-2,3-O-isop r op ylid en e-N-
2,2,2-tr ich lor oeth oxycar bon yl)-D-r ibitol (17). 2,2,2-Trichlo-
7
, 8, and 23 are poorer inhibitors by 1-3 orders of
J
magnitude, with inhibition constants from 0.15 to 4.8
µM. The same series for the T. brucei brucei enzyme
reveals Ki values of 14.0, 13.9, and 3.7 µM, all signifi-
cantly weaker than for immucillin-H 1, immucillin-A
2
1
3 2
)
1
3
2
3
3
)
3
], 0.08 (6H, s, SiCH
3
).
C
3
)
2
2
], 86.7 (C-2), 85.0
(
(
1
3
, and 24.
2
3
3
)
2
3 3
)
3
)
3
2
3
3
Con clu sion s
+
(
H39NO
5
8
-Aza-immucillins are powerful inhibitors for mam-
malian PNPs with Ki* values near 100 pM. The PNP
isozyme specificity is similar for 8-aza-immucillins and
the parent immucillins. 8-Aza-immucillin-A 8, as ex-
pected, was inactive against human and bovine PNP
and had weaker equilibrium binding to the nucleoside
hydrolase enzymes than 3 by 3-4 orders of magnitude.
Perturbation of N-7 interactions with target enzymes
by introduction of the pyrazolo group has little effect
on PNPs but has profound effects on the nucleoside
hydrolases. These features will be useful in the design
of transition-state analogues for specific N-ribosyltrans-
ferases.
(
roethyl chloroformate (3.0 mL, 22 mmol) was added dropwise
to a solution of amine 16 (6.0 g, 14.5 mmol) and Hunig’s base
(7.5 mL, 43 mmol) in dichloromethane (50 mL) at 0 °C. The
resulting solution was allowed to warm to room temperature,
stirred for an additional 30 min, and then partitioned between
chloroform and 10% HCl. The organic layer was further
washed with water and brine, dried, concentrated, and chro-
matographed to afford 17 (7.5 g, 12.7 mmol, 88%) as a syrup.
1
H NMR (C
6
D
6
): δ 5.22 [1H, brs, (CH
CCl ), 4.46 (1H, brs, H-1), 3.80 (6H,
, H-5), 3.46 (1H, m, H-4), 1.35, 1.12 [2 × 3H, brs,
], 1.14 (2 × 3H, brs, OCH CH ), 0.97 [9H, s, C(CH ],
). C NMR (C ): δ 152.6 (NCO),
], 96.1 (CCl ), 91.8 [CH(OEt) ], 86.0, 84.9
CCl ), 67.3,
), 56.6, 56.4 (C-1),
], 18.5 [C(CH ], 15.3
3 2
O) CH], 4.96 (2H, m,
H-2, H-3), 4.55 (2H, s, CH
m, OCH
C(CH
0
1
(C-2), 83.0, 82.1 (C-3), 82.9, 81.6 (CdC), 75.1 (OCH
66.9 (C-4), 62.6, 62.2 (C-5), 61.0 (OCH CH
27.1, 25.0 [C(CH ], 26.1 [C(CH
2
3
2
3
)
2
2
3
3 3
)
1
3
.13, 0.11 (2 × 3H, s, SiCH
3
6 6
D
Exp er im en ta l Section
12.4 [OC(CH
3
)
2
3
2
1
. Gen er a l. Each new compound migrated as a single
2
3
discrete component on thin-layer chromatography using alu-
minum-backed silica gel sheets (Merck or Reidel de Haen).
2
3
3
)
2
3
)
3
3 3
)

8
-Aza-immucillins
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1 159
+
+
(
Cl
OCH
2
CH
3
), -5.5, -5.4 (SiCH
3
). HRMS (M ) calcd for C24
H
40
-
× COCH
3
). HRMS (MH ) calcd for C15
Found: 366.1424.
H
20
N
5
O
6
: 366.1414.
3
NO
7
Si: 587.1640. Found: 587.1541.
(
1S)-2,3,5-Tr i-O-a cetyl-1-(1-a cetyl-1H-p yr a zol-3-yl)-1,4-
(1S)-1-(4-Am in o-5-cya n o-1H-p yr a zol-3-yl)-1,4-d id eoxy-
1,4-im in o-D-r ibitol. A catalytic amount of sodium methoxide
was added to a stirred solution of 20 (110 mg, 0.3 mmol) in
methanol (3 mL) at room temperature. After 30 min, the
reaction was complete and the product was preabsorbed onto
silica and purified by chromatography to afford (1S)-1-(4-
d id eoxy-1,4-im in o-N-(2,2,2-tr ich lor oeth oxyca r bon yl)-D-
r ibitol (18). Acetal (17) (7.5 g, 12.8 mmol) was dissolved in
glacial acetic acid (125 mL) and 10% HCl (30 mL), and the
mixture was stirred at room temperature for 1 h. To this, a
solution of hydrazine hydrate (7.5 mL, 155 mmol) in glacial
acetic acid (60 mL) was added dropwise over 10 min. The
resulting solution was heated at reflux for 16 h and concen-
trated in vacuo to afford a dark-brown oil. The crude product
was redissolved in pyridine (60 mL), acetic anhydride (30 mL)
was added, and the resulting solution was stirred for 16 h at
room temperature. Solvent was removed in vacuo, and the
crude residue was redissolved in ethyl acetate (500 mL),
washed with 10% HCl (100 mL), water (100 mL), and brine
amino-5-cyano-1H-pyrazol-3-yl)-1,4-dideoxy-1,4-imino-D-ribi-
1
tol (60 mg, 0.25 mmol, 84%). H NMR (MeOH-d
4
): δ 4.30 (1H,
d, J ) 7.2 Hz, H-1), 4.05-3.94 (2H, m, H-2, H-3), 3.65 (2H, dt,
1
3
J ) 11.1, J ) 5.1 Hz, H-5), 3.27 (1H, q, J ) 4.5 Hz, H-4).
NMR (MeOH-d ): δ 134.7, 134.7, 134.1 (C-3′, C-4′, C-5′), 114.9
(CN), 77.9 (C-2), 74.0 (C-3), 66.6 (C-4), 64.9 (C-5), 59.5 (C-1).
C
4
+
9 13 5 3
HRMS (MH ) calcd for C H N O : 239.1018. Found: 239.1028.
(1S)-1-(4-Am in o-5-cya n o-1H -p yr a zol-3-yl)-N-(ter t-b u -
toxyca r bon yl)-1,4-d id eoxy-1,4-im in o-D-r ibitol (21). Di-tert-
butyl dicarbonate (100 mg, 0.46 mmol) was added to a stirred
solution of (1S)-1-(4-amino-5-cyano-1H-pyrazol-3-yl)-1,4-dideoxy-
1,4-imino-D-ribitol (60 mg, 0.25 mmol) in methanol (5 mL) at
room temperature. After 1 h, the reaction was complete and
the reaction mixture was concentrated in vacuo and chromato-
graphed to afford 21 (60 mg, 0.18 mmol, 71%), which was used
in the subsequent step without characterization.
(
1
(
(
100 mL), dried, concentrated, and chromatographed to afford
8 (4.5 g, 8.3 mmol, 65%) as a syrup. H NMR (C D ): δ 7.95
6 6
1
1H, brd, J 10.5 Hz, H-5′), 6.36 (1H, brd, J 10.5 Hz, H-4′), 5.95
2H, m, H-2, H-3), 5.33 (1H, d, J ) 26.3 Hz, H-1), 4.60 (2H, m,
H-5), 4.40 (2H, m, CH
2
CCl
). C NMR (C
), 154.4, 154.1 (C-3′), 153.6 (NCO), 129.2 (C-5′), 109.2,
08.6 (C-4′), 95.7 (CCl ), 75.2 (OCH CCl ), 75.2, 74.1 (C-2),
3.0, 72.3 (C-3), 62.3, 62.0 (C-5), 61.0, 60.5 (C-1), 60.3, 60.1
3
), 1.68 (3H, s, NCOCH
3
), 1.67 (9H,
1
3
s, OCOCH
COCH
3
6 6
D
): δ 169.6, 169.3, 169.1, 168.8
(
1
7
(
Cl
3
3
2
3
(
1S)-1-(3-Am in o-2-ca r b oxa m id o-1H -p yr a zol-3-yl)-1,4-
+
C-4),21.1, 20.1 (4 × COCH
3
). HRMS (MH ) calcd for C19
: 542.0500. Found: 542.0513.
1S)-2,3,5-Tr i-O-a cet yl-1-(5-cya n o-4-n it r o-1H-p yr a zol-
23
H -
d id eoxy-1,4-im in o-D-r ibitol (23). Hydrogen peroxide (0.3
mL) was added dropwise to a solution of 21 (60 mg, 0.18 mmol)
and potassium carbonate (60 mg) in DMSO (1.0 mL), and the
mixture was stirred for 1 h. The mixture was diluted with
water (50 mL) and lyophilized, redissolved in water, preab-
sorbed onto silica, concentrated, and chromatographed to
afford a product (42 mg) that was dissolved in 2 M HCl (5 mL).
The solution was allowed to stand at room temperature for 5
min, and then the solvent was removed to afford the pyrazole
3
3 9
N O
(
3
-yl)-1,4-d id eoxy-1,4-im in o-N-(2,2,2-tr ich lor oeth oxyca r -
bon yl)-D-r ibitol (19). Trifluoroacetic anhydride (12.0 mL, 85
mmol) was added dropwise to a stirred solution of 18 (4.5 g,
8
.3 mmol) and ammonium nitrate (6.8 g, 85 mmol) in trifluo-
roacetic acid (100 mL) at 0 °C. The resulting solution was
allowed to warm to room temperature and stirred for an
additional 3 h. The reaction was then diluted with chloroform,
washed with water until the pH of the aqueous layer was
neutral, dried, and concentrated in vacuo. The crude product
thus isolated was committed to the next step without further
purification. A solution of the 2,4-dinitro compound (5.0 g) in
ethanol (45 mL) and ethyl acetate (45 mL) was added dropwise
over 5 min to a stirred solution of potassium cyanide (6.6 g,
C-nucleoside 23 as its hydrochloride salt (28 mg, 0.11 mmol,
1
61%). H NMR (D
2
O): δ 4.93 (1H, d, J ) 7.7 Hz, H-1), 4.66
(1H, m, H-2), 4.43 (1H, t, J ) 4.0 Hz, H-3), 3.92 (3H, m, H-4,
H-5). 13C NMR (D
O): δ 163.3 (CONH ), 134.4, 133.2, 118.5
(pyrazole), 73.9 (C-2), 71.0 (C-3), 66.1 (C-4), 58.9 (C-5), 55.6
2
2
+
(C-1). HRMS (MH ) calcd for C
258.1203.
9 16 5 4
H N O : 258.1202. Found:
1
01 mmol) in ethanol (120 mL) and water (30 mL). Following
(1S)-1-(7-Am in o-1H-p yr a zolo[4,3-d ]p yr im id in -3-yl)-1,4-
d id eoxy-1,4-im in o-D-r ibitol (8‚HCl). A solution of 21 (0.16
g, 0.47 mmol) in ethanol (5 mL) was stirred with formamidine
acetate (0.06 g, 0.58 mmol) under reflux for 30 min. The
solution was preabsorbed onto silica, and the solvent was
removed in vacuo and chromatographed to afford a product
(90 mg) that was dissolved in 2 M HCl (5 mL). The mixture
was allowed to stand at room temperature for 5 min. The
solvent was removed in vacuo to afford (1S)-1-(7-amino-1H-
pyrazolo[4,3-d]pyrimidin-3-yl)-1,4-dideoxy-1,4-imino-D-ribitol‚
HCl (8‚HCl) (76 mg, 0.25 mmol, 53%) as a hygroscopic white
solid (recrystallized from MeOH/acetonitrile) that melted at
216 °C. [R]D -9.2° (c 0.5, MeOH). IR νmax: 3448 (NH), 1676,
an additional 5 min at room temperature, the reaction mixture
was neutralized with acetic acid and diluted with ethyl acetate
(
500 mL), washed with water (100 mL) and brine (100 mL),
dried, concentrated in vacuo, and chromatographed to afford
9 (4.4 g, 7.7 mmol, 93%) as a syrup. IR ν-max: 3345 (NH), 2229.8
1
1
1
(
6
CN), 1741 (CO), 1376, 1235, 1048 cm . H NMR (C
.04 (1H, d, J ) 6.0 Hz, H-1), 5.69 (1H, brs, H-2), 5.42 (1H,
brs, H-3), 4.54-3.75 (5H, m, H-4, H-5, CH CCl ), 1.83, 1.79,
): δ 172.8, 169.8, 169.6
), 153.4 (NCO), 143.1, 134.5, 123.7 (C-3′, C-4′, C-5′),
11.0 (CN), 94.9 (CCl ), 75.4 (OCH CCl ), 75.4 (C-2), 71.8 (C-
), 63.6 (C-5), 62.9 (C-4), 57.1 (C-1), 20.6, 20.1, 19.9 (COCH ).
570.0198. Found:
6 6
D ): δ
2
3
1
3
1
(
1
3
.71 (3H, s, COCH
3 6 6
). C NMR (C D
COCH
3
3
2
3
3
+
-1
1
HRMS (MH ) calcd for C18
H
18Cl
3
N
5
O
10
:
1612, 1405, 1129 cm . H NMR (D O): δ 8.42 (1H, s, H-5′),
2
5
70.0181.
1S)-2,3,5-Tr i-O-a cetyl-1-(4-a m in o-5-cya n o-1H-p yr a zol-
-yl)-1,4-d id eoxy-1,4-im in o-D-r ibitol (20). Zinc dust (1.5 g)
5.20 (1H, d, J ) 6.5 Hz, H-1), 4.80 (1H, t, J ) 4.6 Hz, H-2),
4.50 (1H, t, J ) 4.6 Hz, H-3), 4.01-3.89 (3H, m, H-4, H-5a,-
5b). 13C NMR: δ 151.0 (C), 145.3 (C-5′), 134.9 (C), 132.9 (C),
(
3
was suspended in glacial acetic acid (10 mL) with stirring, and
a solution of pyrazole 19 (570 mg, 1 mmol) in glacial acetic
acid (5 mL) was then added dropwise over a 5 min period. After
a further 10 min, the zinc salts were removed by filtration and
washed with ethyl acetate. The combined filtrate was concen-
trated in vacuo, redissolved in chloroform, washed with
saturated sodium bicarbonate, water and brine, dried, con-
centrated in vacuo, and chromatographed to afford 20 (250 mg,
123.4 (C), 73.9 (C-2), 70.9 (C-3), 65.6 (C-4), 58.8 (C-5), 57.3
+
(C-1). HRMS (MH ) calcd for C10
H
15
N
6
O
3
: 267.1206. Found:
267.1208. Anal. (C10
14 6 3
H N O ‚2HCl) C, H, N, Cl.
(1S)-1-(7-Hyd r oxy-1H-p yr a zolo[4,3-d ]p yr im id in -3-yl)-
1,4-d id eoxy-1,4-im in o-D-r ibitol (7‚HCl). A solution of 22
(0.05 g, 0.19 mmol) in ethanol (5 mL) was stirred with
formamidine acetate (0.05 g, 0.48 mmol) under reflux for 1 h.
The solution was preabsorbed onto silica, and the solvent was
removed in vacuo and chromatographed to afford a product
(40 mg) that was dissolved in 2 M HCl (5 mL). The mixture
was allowed to stand at room temperature for 5 min. The
solvent was removed in vacuo to afford (1S)-1-(7-hydroxy-1H-
pyrazolo[4,3-d]pyrimidin-3-yl)-1,4-dideoxy-1,4-imino-D-ribitol‚
HCl (7‚HCl) (31 mg, 0.10 mmol, 54%) as a white solid
0
1
J
.68 mmol, 68%) as a syrup. IR νmax: 3345 (NH), 2229.8 (CN),
741 (CO), 1376, 1235, 1048 cm-1. H NMR: δ 5.16 (1H, dd,
23 ) 4.9, J 12 ) 3.5 Hz, H-2), 4.99 (1H, dd, J 3,4 ) 7.2, J 3,4 ) 4.9
1
Hz, H-3), 4.51 (1H, d, J 1,2 ) 3.5 Hz, H-1), 4.23 (2H, d, J 4,5
)
4
2
1
7
.6 Hz, H-5), 3.77 (1H, dt, J 3,4 ) 7.2, J 4,5 ) 4.6 Hz, H-4), 2.16,
13
.10, 2.10 (COCH
3
3
). C NMR: δ 171.5, 171.2, 170.3 (COCH ),
33.0, 133.0, 129.3 (C-3′, C-4′, C-5′), 113.5 (CN), 75.9 (C-2),
(recrystallized from MeOH) that decomposed at 265 °C. [R]
-25.0° (c 0.1, MeOH). IR νmax: 3380 (NH), 1718, 1672, 1593,
D
2.1 (C-3), 65.1 (C-5), 59.0 (C-4), 57.5 (C-1), 21.2, 21.2, 21.0 (3

1
60 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1
131, 1031 cm-1. 1H NMR (D
Evans et al.
1
2
O): δ 8.09 (1H, s, H-5′), 5.11
(14) Miles, R. W.; Tyler, P. C.; Furneaux, R. H.; Bagdassarian, C.
K.; Schramm, V. L. Biomedical and Health Research; Frey, P.
A., Northrop, D. B., Eds.; IOS Press: Amsterdam, 1999; pp 32-
(
(
1H, d, J ) 6.2 Hz, H-1), 4.78 (1H, t, J ) 5.0 Hz, H-2), 4.46
1H, t, J ) 5.0 Hz, H-3), 3.96-3.88 (3H, m, H-4, H-5a,5b). 13
C
4
7.
NMR: δ 154.8 (C), 144.9 (C-5′), 137.3 (C), 137.1 (C), 128.6 (C),
7
(
15) Miles, R. W.; Tyler, P. C.; Evans, G. B.; Furneaux, R. H.; Parkin,
D. W.; Schramm, V. L. Biochemistry 1999, 38, 13147-13154.
16) Furneaux, R. H.; Schramm, V. L.; Tyler, P. C. Bioorg. Med.
Chem. 1999, 7, 2599-2606.
(17) Shi, W.; Li, C. M.; Tyler, P. C.; Furneaux, R. H.; Cahill, S. M.;
Girvin, M. E.; Grubmeyer, C.; Schramm, V. L.; Almo, S. C.
Biochemistry 1999, 38, 9872.
3.7 (C-2), 71.0 (C-3), 65.2 (C-4), 58.8 (C-5), 57.9 (C-1). HRMS
+
(
MH ) calcd for C10
‚HCl) C, H, N, Cl.
. Biology. Enzymatic assays used the absorbance change
as inosine is converted to hypoxanthine or the coupled assay
14 5 4
H N O : 268.1046. Found: 268.1055. Anal.
(
10 14 5 4
(C H N O
3
1
2
of xanthine oxidase to convert hypoxanthine to uric acid. Slow
3
4
(18) Kicska, G. A.; Long, L.; H o¨ rig, H.; Fairchild, C.; Tyler, P. C.;
Furneaux, R. H.; Schramm, V. L.; Kaufman, H. L. Proc. Natl.
Acad. Sci. U.S.A. 2001, 98, 4593-4598.
onset inhibition was analyzed by curve fitting. Human and
bovine PNP were purchased from Sigma, and nucleoside
hydrolases were prepared from expression of the appropriate
(
(
(
19) Basso, L. A.; Santos, D. S.; Shi, W.; Furneaux, R. H.; Tyler, P.
1
5
DNA in E. coli overexpression vectors.
C.; Schramm, V. L.; Blanchard, J . S. Biochemistry 2001, 40,
8
196-8203.
Ack n ow led gm en t. This work was supported by
Research Grant GM41916 from the NIH. The work at
Brookhaven National Laboratory was supported by the
U.S. Department of Energy under Contract AC02-
20) Shi, W.; Basso, L. A.; Santos, D. S.; Tyler, P. C.; Furneaux, R.
H.; Blanchard, J . S.; Almo, S. C.; Schramm, V. L. Biochemistry
2
001, 40, 8204-8215.
21) Federov, A.; Shi, W.; Kicska, G.; Federov, E.; Tyler, P. C.;
Furneaux, R. H.; Hanson, J . C.; Gainsford, G. J .; Larese, J . Z.;
Schramm, V. L.; Almo, S. C. Biochemistry 2001, 40, 853-860.
22) Kicska, G. A.; Tyler, P. C.; Evans, G. B.; Furneaux, R. H.; Kim,
K.; Schramm, V. L. J . Biol. Chem. 2002, 277, 3219-3225.
23) Kicska, G. A.; Tyler, P. C.; Evans, G. B.; Furneaux, R. H.;
Schramm, V. L.; Kim, K. J . Biol. Chem. 2002, 277, 3226-3231.
24) DeWolf, W. E., J r.; Fullin, F. A.; Schramm, V. L. J . Biol. Chem.
9
8CH10886.
(
(
(
(
Su p p or tin g In for m a tion Ava ila ble: X-ray analysis of 7
and a table of hydrogen bond contact parameters. This
material is available free of charge via the Internet at http://
pubs.acs.org.
1
979, 254, 10868-10875.
25) Leung, H. B.; Schramm, V. L. J . Biol. Chem. 1980, 255, 10867-
0874.
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