Synthesis of Second-Generation Transition State Analogues of Human Purine Nucleoside Phosphorylase

Article abstract of DOI:10.1021/jm030305z

Purine nucleoside phosphorylases (PNPs) catalyze nucleophilic displacement reactions by migration of the cationic ribooxacarbenium carbon between the fixed purine and phosphate nucleophiles. As the phosphorolysis reaction progresses along the reaction coo

Full text of DOI:10.1021/jm030305z

J . Med. Chem. 2003, 46, 5271-5276
5271
Syn th esis of Secon d -Gen er a tion Tr a n sition Sta te An a logu es of Hu m a n P u r in e
Nu cleosid e P h osp h or yla se
Gary B. Evans,*,† Richard H. Furneaux,† Andrzej Lewandowicz,‡ Vern L. Schramm,‡ and Peter C. Tyler†
Carbohydrate Chemistry Team, Industrial Research Limited, P.O. Box 31310, Lower Hutt, New Zealand, and Department of
Biochemistry, Albert Einstein College of Medicine of Yeshiva University, 1300 Morris Park Avenue, Bronx, New York 10461
Received J une 24, 2003
Purine nucleoside phosphorylases (PNPs) catalyze nucleophilic displacement reactions by
migration of the cationic ribooxacarbenium carbon between the fixed purine and phosphate
nucleophiles. As the phosphorolysis reaction progresses along the reaction coordinate, the
distance between the purine and carbocation increases and the distance between carbocation
and phosphate anion decreases. Immucillin-H and Immucillin-G have been shown previously
to be potent inhibitors of PNP. We now report the synthesis of a second generation of stable
transition state analogues, DADMe-Immucillins 2, 3, and 4, with increased distance between
ribooxacarbenium and purine mimics by incorporation of a methylene bridge between these
groups. These compounds are potent inhibitors with equilibrium dissociation constants as low
as 7 pM against human PNP. Stable chemical analogues of enzymatic transition states are
necessarily imperfect since they lack the partial bond character of the transition state. The
immucillins and DADMe-Immucillins represent approaches from the product and reaction side
of the transition state.
In tr od u ction
kinetic isotope effects (Figure 1). The characteristics of
the transition state, i.e., the ribooxacarbenium ion
character in the ribosyl group and the elevated pKa for
the purine-leaving group with little participation of the
phosphate ion, have been partially captured by the
Immucillins. To better mimic the cationic charge which
develops at the anomeric carbon and the dissociative
transition state, we designed DADMe-Immucillin-H in
which the nitrogen atom is now at the anomeric carbon,
the ring oxygen is replaced with a methylene group, and
the ribosyl mimic and the deazapurine have increased
separation through insertion of a methylene bridge.
In a recent publication we described the potent
inhibition of Mycobacterium tuberculosis PNP21 by the
second-generation versions of the Immucillins, the
DADME-Immucillins. The DADMe-Immucillins mimic
structures further along the phosphorolysis reaction
coordinate than the Immucillins. We report here the
first synthesis of the DADMe-Immucillins 2, 3, and 4
and their activity against human PNP.
Recently we reported the design,1-3 synthesis,4-6 and
biological activity of the Immucillins, a series of high
affinity transition state analogue inhibitors of bovine,7-10
malarial,11,12 Mycobacterium tuberculosis,13,14 and hu-
man PNPs (purine nucleoside phosphorylases).7,15 In
humans, a genetic deficiency in PNP leads to a build-
up of deoxyguanosine triphosphate (dGTP) in human
T-cells and results in cell death for proliferating T-cells.
The human T-cell is unique in this regard because of a
combination of high deoxycytidine kinase activity (which
phosphorylates deoxyguanosine) and a relatively low
nucleotidase activity which allows dGTP to accumu-
late.16,17 The implication has been18,19 that inhibition of
PNP provides a mechanism to suppress T-cell prolifera-
tion in human beings and this prediction is being bourn
out in human clinical studies using Immucillin-H (as
BCX-1777).20 Perturbation of T-cell population through
the inhibition of PNP provides new avenues for the
treatment of T-cell-mediated disorders.19
Resu lts a n d Discu ssion
Syn th esis. The 1′-aza sugar (3R,4R)-3-hydroxy-4-
(hydroxymethyl)pyrrolidine (1) was first reported by
J aeger and Biel as a mixture of stereoisomers.22 Bols
et al. reported the enzymatic chiral resolution of racemic
3,4-trans-3-hydroxy-4-(hydroxymethyl)pyrrolidine23 and
the first asymmetric synthesis of 1 was reported by
Ichikawa et al.24 We prepared 1 by a modification of the
method of Filichev and Pedersen in 12 steps from
D-xylose,25 in which 5 was protected by tert-butoxycar-
bonylation to afford 6, rather than Fmoc protection
(Scheme 1). Periodate cleavage of compound 6 followed
by reduction of the resulting aldehyde with sodium
borohydride yielded (3R,4R)-N-tert-butoxycarbonyl-3-
hydroxy-4-hydroxymethylpyrrolidine (7). Protection of
the hydroxyl groups of 7 was effected by treatment with
The transition state structures for the bovine PNP-
catalyzed phosphorolysis of inosine have been solved by
* Corresponding author. Fax +64-4-9313055; e-mail g.evans@
irl.cri.nz.
† Industrial Research Limited.
‡ Albert Einstein College of Medicine of Yeshiva University.
10.1021/jm030305z CCC: $25.00 © 2003 American Chemical Society
Published on Web 10/23/2003

5272 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24
Evans et al.
aqueous ammonia to remove the remaining N-7 hy-
droxymethyl residue.
In h ibition of P u r in e Nu cleosid e P h osp h or yla se
by DADMe-Im m u cillin s. The human genetic defi-
ciency of PNP causes a specific T-cell immunodefi-
ciency.31 Pharmacologic intervention in T-cell prolifera-
tion is desirable in T-cell cancers, autoimmune diseases,
and tissue transplant rejection.18 The first generation
PNP inhibitor Immucillin-H is in clinical trials against
T-cell cancers.20 Experiments with xenograft models
with mice have established that Immucillin-H is as
effective as cyclosporin in mouse models of tissue
transplant rejection and that combinations of Immucil-
lin-H and cyclosporin are more effective than each
individual agent.32 Pharmokinetic studies indicate that
immunological, anticancer, and pharmacokinetic prop-
erties of anti-PNP agents will be key to their develop-
ment as anticancer and autoimmune agents.33
F igu r e 1. Phosphorolysis of inosine catalyzed by PNP with
features of the transition state and selected features for the
Michaelis complexes.
Biological effectiveness of anti-PNP agents in vivo
depends on the complete inhibition of the target enzyme.
Therefore inhibitors with even higher specificity and
affinity than the Immucillins remain attractive targets.
The first generation inhibitors Immucillin-H, Immucil-
lin-G, and 8-Aza-Immucillin-H are 56, 42, and 180 pM
inhibitors, respectively, for the human enzyme7,8 whereas
DADMe-Immucillin-H (2), DADMe-Immucillin-G (3),
and 8-Aza-DADMe-Immucillin-H (4) are 16, 7, and 2000
pM inhibitors of human PNP, respectively (Table 1).
Compared to Immucillin-H, the current clinical agent,
DADMe-Immucillin-G (3), exhibits an approximately
8-fold increase in affinity. 8-Aza-DADMe-Immucillin-H
(4) is a less potent inhibitor than 8-Aza-Immucillin-H.
Administration of tight-binding transition state ana-
logues as pharmacophores has the potential to target
the enzyme of interest with high specificity. The biologi-
cal half-life is closely related to the residence time on
the catalytic site of the target enzyme. Residence time
is dominated by the off-rate, although on-rates can also
contribute.34 The increased affinity of DADMe-Immu-
cillin-G (3) is highly significant in terms of pharmacol-
ogy. It implies an 8-fold increased time for biological
effectiveness and therefore an 8-fold change in dose
scheduling. Finally, the DADMe-Immucillins have only
two asymmetric centers compared to the four centers
in Immucillin-H. This is expected to improve the
synthetic efficiency for these potential anti-T-cell agents.
sodium hydride and benzyl bromide to afford the diben-
zyl ether 8. Deprotection of compounds 7 and 8 could
be achieved by treatment with concentrated hydrochlo-
ric acid in methanol to afford hydrochloride salts 1 and
9, respectively.
Initially, attempts were made to construct the de-
azapurines via the de novo approach first published by
Kline et al.26-28 and adapted by ourselves to synthesize
the Immucillins,5 which starts by constructing the
propionitrile adduct 10 (Scheme 2). However on at-
tempted reaction with either Bredereck’s reagent or
ethyl formate and sodium hydride 10 reverted to 9 as
the free base, presumably via â elimination. Therefore
alternative routes to the desired compounds were
investigated.
The reductive aminations of the hydrochloride salts
of 1 or 9 with an appropriately formylated deazapurine
were investigated, as the convergent nature of this
reaction was attractive. Formylation of the previously
reported bromodeazapurines compounds 11,15 12,29 and
1330 was achieved via lithium-halogen exchange to
afford the formyl derivatives 14, 15, and 16, respec-
tively, in good yields (Scheme 3).
With the formyl group in place, we investigated the
coupling of these compounds to the dibenzyl ether 9 and
(3R,4R)-3-hydroxy-4-(hydroxymethyl)pyrrolidine (1) it-
self by reductive amination. The protected amine hy-
drochloride 9 was allowed to react with aldehyde 14 in
the presence of sodium cyanoborohydride in methanol
to afford 17 as the sole product in good yield (Scheme
4). Removal of the tert-butyl protecting group of com-
pound 17 using TFA in DCM yielded 18. Similarly, the
amine hydrochloride 1 reacted with aldehydes 14 and
16 under the same conditions to afford the correspond-
ing protected DADMe-Immucillins 19 and 20, respec-
tively.
Con clu sion s
This report establishes that extended geometry for
transition state analogue of PNP can more efficiently
capture the features of the transition state for human
PNP. Immucillin-H is a substrate-like transition state
mimic while DADMe-ImmH is more closely related to
products. The significance of this knowledge is that
relatives of both early and late transition state ana-
logues can now be explored in attempts to ascend higher
on the slopes toward the transition state. Both inhibitors
are efficient analogues in the specific case of PNP. The
Km for the human enzyme described here is 38 µM,
giving a Km/Ki* ratio for Immucillin-H and DADMe-
Immucillin-G of 679 000 and 5 590 000, respectively.
Deprotection could be achieved via treatment with
concentrated hydrochloric acid in methanol at reflux,
or in the case of compound 20 at room temperature, to
afford compounds 2, 3, and 4, (Scheme 5).
Improved yields of DADMe-Immucillin-H (2) were
achieved via the mild acid hydrolysis of the tert-butyl
group followed by catalytic hydrogenolysis of the re-
maining benzyl groups and subsequent treatment with
Exp er im en ta l Section
Gen er a l. NMR spectra were recorded on a Bruker AC-300
instrument at 300 MHz (1H) or 75 MHz (13C). Normally,
spectra were measured in CDCl3 with Me4Si as internal

Human Purine Nucleoside Phosphorylase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24 5273
Sch em e 1a
a
Reagents: (a) Boc2O, MeOH, room temp., 73%; (b) NaIO4, EtOH, 0 °C; (c) NaBH4, EtOH, 0 °C, 92% for two steps; (d) BnBr, NaH,
DMF, 0 °C, 96%; (e) MeOH, cHCl, room temp, quantitative.
Sch em e 2a
and 71.4, 65.4, 52.1 and 51.7, 48.0 and 47.4, 46.7 and 46.3,
28.5. HRMS (M-H-) calcd for C11H20NO5: 2246.1341. Found
246.1348.
(3R,4R)-N-ter t-Bu toxyca r bon yl-3-h yd r oxy-4-h yd r oxy-
m eth ylp yr r olid in e (7). (3R,4S)-N-tert-Butoxycarbonyl-3-hy-
droxy-4-[(1S)-1,2-dihydroxyethyl]pyrrolidine (6) (3.4 g, 13.7
mmol) in ethanol (50 mL) was added dropwise to a stirred
a
Reagents: (a) 3-Bromopropiononitrile, Hunigs base, acetoni-
trile, room temp.
solution of sodium periodate (3.4 g, 16 mmol) in water (25 mL)
while maintaining the reaction temperature at 0 °C. The
reaction was left an additional 20 min after which time sodium
borohydride (2.0 g, excess) was added portionwise while again
ensuring the reaction temperature was maintained at 0 °C.
On complete addition the solid was filtered and washed with
ethanol (50 mL), and the filtrate was concentrated in vacuo
to afford a syrup. Chromatography afforded 7 (2.74 g, 92%) as
Sch em e 3a
1
a syrup. H NMR δ 4.24 (m, 1H), 3.61 (m, 4H), 3.25 (m, 1H),
3.12 (m, 1H), 2.24 (m, 1H), 1.45 (s, 9H). 13C NMR δ 155.2, 80.2,
(73.2, 72.4), 63.1, (53.1, 52.7), (48.6, 48.1), (47.0, 46.5), 28.9.
HRMS (MH+) calcd for C10H20NO4: 218.1392. Found 218.1392.
(3R,4R)-N-ter t-Bu toxyca r bon yl-3-ben zyloxy-4-ben zyl-
oxym eth ylp yr r olid in e (8). Sodium hydride (140 mg, 60% oil
dispersion, 3.7 mmol) was added portionwise to a stirred
solution of benzyl bromide (300 µL, 2.8 mmol) and 7 (200 mg,
0.92 mmol) in DMF (10 mL) at 0 °C. On complete addition,
the resulting suspension was allowed to warm to room temp,
diluted with toluene (100 mL), washed with water (50 mL)
and brine (50 mL), dried (MgSO4), and concentrated in vacuo
to afford a syrup. Chromatography afforded 8 (350 mg, 96%)
as an oil, which was used in the next step without purification.
(3R,4R)-3-Ben zyloxy-4-ben zyloxym eth ylpyr r olidin e Hy-
d r och lor id e (9). Hydrochloric acid (2 mL, 1 M) was added to
a solution of 8 (500 mg, 1.3 mmol) in methanol (2 mL) and
the resulting mixture stirred for 1 h at 40 °C. On completion
the reaction was concentrated in vacuo to afford 9 as the
a
Reagents: (a) nBuLi, THF, -78 °C; (b) DMF, -78 °C, room
temp.
reference; when D2O was the solvent, acetone (δ 1H, 2.20; 13C,
33.2) was used as an internal reference. High-resolution
accurate mass determinations were performed by Hort Re-
search Ltd., Palmerston North, N. Z., 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 FAB conditions in a
glycerol or nitrobenzyl alcohol matrix. Melting points were
1
hydrochloride salt (370 mg, 100%). H NMR δ 7.35-7.21 (m,
determined on
a Reichert hot stage microscope and are
10H), 4.48 (m, 4H), 4.08 (d, J ) 2.9 Hz,1H), 3.53 (m, 1H), 3.44
(m, 3H), 3.24 (m, 1H), 2.65 (m, 1H). 13C NMR δ 138.0, 137.6,
128.9, 128.8, 128.3, 128.2, 79.3, 73.7, 71.9, 68.7, 49.6, 46.4, 44.8.
(3R,4R)-3-Hyd r oxy-4-h yd r oxym eth ylp yr r olid in e Hy-
d r och lor id e (1‚HCl). Hydrochloric acid (5 mL, 12 M) was
added dropwise to a stirred solution of 7 (2.3 g, 10.6 mmol) in
methanol (5 mL) at room temperature. After 1 h the reaction
was concentrated in vacuo to afford 1‚HCl (1.63 g, 100%) as
an oil. 1H NMR δ 4.43 (quintet, J ) 3.0 Hz, 1H), 3.46 (dd, J )
12.7, 5.1 Hz,1H), 3.29 (dd, J ) 12.7, 2.2 Hz,1H), (m, 1H), 3.18
(dd, J ) 12.3, 5.8 Hz,1H), 2.49 (m, 1H). 13C NMR δ 71.9, 60.9,
52.1, 47.9, 46.6. 1H and 13C NMR of the free base were identical
to those reported in the literature.25
uncorrected. Aluminum-backed silica gel sheets (Merck or
Reidel de Haen) were used for thin-layer chromatography.
Column chromatography was performed on silica gel (230-
400 mesh, Merck). Chromatography solvents were distilled
prior to use.
Ch em istr y. (3R,4S)-N-ter t-Bu toxyca r bon yl-4-[(1S)-1,2-
d ih yd r oxyet h yl]-3-h yd r oxyp yr r olid in e (6). Di-tert-butyl
dicarbonate (4.54 g, 20.8 mmol) was added portionwise to a
stirred solution of (3R,4S)-4-[(1S)-1,2-dihydroxyethyl]-3-hy-
droxypyrrolidine25 (2.80 g, 18.9 mmol) in methanol at room
temp. On complete addition the reaction was then left for 1 h
and concentrated in vacuo. Chromatography afforded (3R,4S)-
N-tert-butoxycarbonyl-4-[(1S)-1,2-dihydroxyethyl]-3-hydroxy-
pyrrolidine (6) (3.4 g, 73%). 1H NMR δ 4.36 (q, J ) 7.5 Hz,
1H), 3.73-3.54 (m, 6H), 3.18-3.04 (m, 2H), 2.15 (brs, 1H), 1.45
(s, 9H). 13C NMR (many of the peaks are doubled due to slow
interconversion of rotamers) δ 154.8, 80.0, 73.2 and 72.9, 72.4
7-Ben zyloxym eth yl-6-O-ter t-bu tyl-9-d ea za -9-for m ylh y-
p oxa n th in e (14). 7-Benzyloxymethyl-9-bromo-6-O-tert-butyl-
9-deaza-hypoxanthine15 (11) (400 mg, 1.02 mmol) was dis-
solved in diethyl ether (10 mL) and anisole (5 mL) and cooled
to -78 °C. n-Butyllithium (600 µL, 2.5 M) was then added
dropwise at such a rate as to maintain the reaction temper-

5274 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24
Evans et al.
Sch em e 4a
a
Reagents: (a) NaCNBH3, MeOH, room temp.. (b) TFA, DCM, room temp.
Sch em e 5a
a
Reagents: (a) cHCl, MeOH; (b) i, TFA, DCM; ii, H2, Pd(OH)2, EtOH.
Ta ble 1. Inhibition Constants for the Interaction of
Immucillins with Human PNP
layers were combined, washed with brine, dried (MgSO4),
filtered, and concentrated in vacuo to afford a solid residue.
The solid was triturated with ethanol to afford 15 (280 mg,
72%) as a white solid. Mp 172-174 °C. 1H NMR δ 10.25 (s,
1H), 7.79 (s, 1H), 7.30-7.21 (m, 13H), 6.85-6.82 (m, 5H), 5.62
human PNPa
inhibitor
Immucillin-H
Ki (pM)
Ki* (pM)
(s, 2H), 5.44 (s, 2H), 4.84 (s, 4H), 4.45 (s, 2H), 3.79 (s, 6H). 13
C
3300 ( 200
540 ( 100
1400 ( 200
1100 ( 120
163 ( 25
56 ( 15
42 ( 6
180 ( 20
16 (1.4
6.8 ( 1.2
no slow onset
NMR δ 185.5, 159.4, 159.1, 156.5, 153.9, 136.9, 136.7, 134.9,
Immucillin-G
131.5, 129.5, 128.9, 128.5, 128.3, 128.0, 117.3, 114.2, 111.0,
8-Aza-Immucillin-H
DADMe-ImmH 2
DADMe-ImmG 3
8-aza-DADMe-ImmH 4
78.4, 71.0, 67.9, 55.7, 49.5. HRMS (MH+) calcd for C38H37
N4O5: 629.2764. Found 629.2749.
-
2000 ( 50
8-Aza -9-d ea za -9-for m yl-6-O-m eth yl-8-tetr a h yd r op yr a -
n ylh yp oxa n th in e (16). n-Butyllithium (0.7 mL, 2.4 M) was
added dropwise to a stirred solution of 8-aza-9-bromo-9-deaza-
6-O-methyl-8-N-tetrahydropyranylhypoxanthine30 (13) (530
mg, 1.7 mmol) in THF (20 mL) at -78 °C under an inert
atmosphere. The reaction was stirred for an additional 30 min
at -78 °C, and then DMF (1.0 mL) was added and the reaction
was allowed to warm to room temp. The reaction was quenched
with water (50 mL) and extracted with toluene (2 × 100 mL).
The organic layers were combined, washed with brine, dried
(MgSO4), filtered, and concentrated in vacuo to afford a solid
residue. Chromatography afforded 16 as a solid. 1H NMR δ
10.43 (s, 1H), 8.71 (s, 1H), 6.55 (dd, J ) 10.0, 2.7 Hz, 1H),
4.25 (s, 3H), 4.13 (m, 1H), 3.83 (dt, J ) 10.8, 2.8 Hz), 2.53-
1.65 (m, 7H). 13C NMR δ 177.0, 161.5, 154.5, 143.9, 130.2,
128.9, 87.0, 67.4, 53.5, 28.7, 23.7, 21.2. HRMS (M+) calcd for
a
Ki* is the dissociation constant for E + I h EI*.
ature below -70 °C and the resulting solution left for 30 min
at -78 °C. Dimethylformamide (100 µL) was then added, and
the reaction was left stirring for an additional 30 min and then
quenched with water and allowed to warm to room temp. The
reaction was diluted with ethyl acetate (100 mL), washed with
water (30 mL) and brine (30 mL), dried (MgSO4), and concen-
trated in vacuo to afford a syrup. Purification by chromatog-
raphy afforded 14 (270 mg, 78%) as a solid. Mp 100-101 °C.
1H NMR δ 10.29 (s, 1H), 8.62 (s, 1H), 7.98 (s, 1H), 7.34-7.22
(m, 5H), 5.79 (s, 2H), 4.53 (s, 2H), 1.71 (s, 9H). 13C NMR δ
184.8, 156.63, 152.6, 150.0, 136.7, 136.6, 128.9, 128.5, 127.8,
118.4, 84.4, 78.3, 71.0, 29.0. HRMS (MH+) calcd for C19H21
N3O3: 340.1661. Found 340.1652.
-
C
12H14N4O3: 262.1066. Found 262.1068.
7-Ben zyloxym eth yl-6-O-ben zyl-9-d ea za -9-for m yl-2-N-
bis(4-m eth oxyben zyl)gu a n in e (15). n-Butyllithium (0.5 mL,
1.5 M) was added dropwise to a stirred solution of 7-benzyl-
oxymethyl-6-O-benzyl-9-bromo-9-deaza-2-N-bis(4-methoxy-
benzyl)guanine29 (12) in diethyl ether (6 mL) and anisole (3
mL) at -80 °C under an inert atmosphere. The reaction was
stirred for an additional 30 min at -80 °C, and then DMF
(1.0 mL) was added and the reaction was allowed to warm to
room temp. The reaction was quenched with water (50 mL)
and extracted with chloroform (2 × 100 mL). The organic
(3R,4R)-1-[(7-Ben zyloxym eth yl-9-d ea za h yp oxa n th in -9-
yl)m et h yl]-3-b en zyloxy-4-b en zyloxym et h ylp yr r olid in e
(18). Sodium cyanoborohydride (100 mg, 1.59 mmol) was added
to a stirred solution of 14 (220 mg, 0.64 mmol) and 9‚HCl (190
mg, 0.57 mmol) in methanol (5 mL) which was stirred
overnight at room temp. The reaction was then concentrated
in vacuo to afford 17 as a syrup, which was not further
characterized. The syrup was redissolved in DCM (2 mL) and
TFA (0.5 mL) was added. The resulting solution was stirred

Human Purine Nucleoside Phosphorylase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24 5275
for 1 h at room temp and then concentrated in vacuo to afford
a solid residue, which was redissolved in water. The aqueous
phase was washed with DCM (×2) and concentrated in vacuo.
Chromatography of the resulting residue afforded 18 (202 mg,
63%) as a solid. 1H NMR δ 7.87 (1H, s), 7.32 (1H, s), 7.31-
7.23 (m, 5H), 5.89 (s, 2H), 4.56 (s, 2H), 4.50 (s, 2H), 4.48 (s,
2H), 4.47 (s, 2H), 3.87 (m, 2H), 3.81 (q, J ) 13.4 Hz, 2H), 3.43
(d, J ) 7.1 Hz, 2H), 3.01 (t, J ) 8.1 Hz, 1H), 2.79 (d, J ) 4.7
Hz, 1H), 2.55 (m, 1H), 2.36 (m, 1H). 13C NMR δ 156.2, 145.8,
141.8, 138.9, 138.8, 137.6, 131.4, 128.8, 128.7, 128.7, 128.3,
128.2, 128.1, 128.0, 128.8, 117.9, 115.7, 81.3, 77.1, 73.5, 72.1,
71.4, 70.8, 60.0, 56.4, 48.6, 45.9. HRMS (MH+) calcd for
J ) 9.1 Hz, 1H), 4.17-3.94 (m, 4H), 4.12 (s, 3H), 3.67-3.52
(m, 2H), 2.94-2.79 (m, 2H), 2.66-2.52 (m, 2H), 2.35-2.09 (m,
2H), 1.70-1.56 (m, 2H). 13C NMR δ 162.6, 152.2, (140.1, 140.0),
133.5, 131.6, (87.0, 86.9), 74.3, (68.3, 68.2), (64.3, 64.2), 62.6,
(56.2, 56.1), 54.5, (50.6, 50.7), (47.7, 47.6), 29.7, 25.2, 21.8.
HRMS (MH+) calcd for C17H25N5O4: 364.1985. Found 364.1982.
(3R,4R)-1-[(8-Aza -9-d ea za h yp oxa n th in -9-yl)m eth yl]-3-
h yd r oxy-4-h yd r oxym et h ylp yr r olid in e (4). Concentrated
hydrochloric acid (1 mL, 12 M) was added to a solution of 20
(50 mg, 0.14 mmol) in methanol. The solution was stirred
overnight and then concentrated in vacuo to afford a solid
residue which was triturated with methanol and filtered to
1
C
34H37N4O4: 565.2815. Found 565.2799.
afford 4 (38 mg, 92%) as a solid. Mp 264-266 °C. H NMR δ
8.13 (s, 1H), 4.35 (d, J ) 2.7 Hz, 1H), 3.86 (m, 1H), 3.66-3.43
(m, 2H), 3.55 (d, J ) 5.7 Hz, 2H), 3.10 (m, 1H), 2.44 (brs, 1H).
13C NMR δ 154.7, 145.4, 137.1, 134.7, 128.6, 71.4, 60.6, 60.6,
55.0, 48.0, 47.9. HRMS (MH+) calcd for C11H16N5O3: 266.1253.
Found 266.1248. Anal. (C11H15N5O3.HCl) C, H, N, Cl.
Biology. P r otein P r ep a r a tion . Human PNP was recloned
into T7/NT-TOPO vector and expressed in BL21(DE3) E. coli.
(to be published elsewhere).
(3R,4R)-1-[(9-deazah ypoxan th in -9-yl)m eth yl]-3-h ydr oxy-
4-h yd r oxym eth ylp yr r olid in e (2). Compound 18 (120 mg,
0.21 mmol) and Pearlman’s catalyst (120 mg, 20% Pd(OH)2
on C) were suspended in ethanol (3 mL) and acetic acid (1 mL)
and vigorously stirred under an atmosphere of hydrogen for
24 h at r.t.. The reaction was then filtered through Celite and
concentrated in vacuo to afford a solid, which was redissolved
in 7N methanolic ammonia, allowed to stand at room temp.
for 1 h and then concentrated in vacuo. Chromatography and
ion exchange of the resulting residue afforded 2 (38 mg, 68%)
Deter m in a tion of Kin etic a n d In h ibition Con sta n ts.
Continuous assays for PNP coupled the production of hypox-
anthine to uric acid by xanthine oxidase.7 PNP (1.4 nM) was
added to inosine (1 mM) and xanthine oxidase (60 mU/mL) in
50 mM KPO4 buffer pH 7.4 (25 °C). The increase in absorbance
was monitored at 293 nm (ꢀ293 ) 12.9 mM-1). For Ki and Ki*
analysis, the amounts of enzyme and inhibitor were adjusted
to give an absorbance change < 1.0 for the full analysis. Initial
inhibition (Ki) was determined by fitting to the equation for
competitive inhibition: ui ) (kcatS)/(Km(1+I/ Ki) + S), where
ui, kcat, Km, and Ki are initial rates, catalytic turnover,
Michaelis constant, and inhibitor dissociation constant, re-
spectively. The dissociation constant for tight bound complex
(Ki*) was determined by the same equation after the slow-
onset equilibrium had been achieved. Valid analysis requires
inhibitor to be present at >10× enzyme concentration.34
Inhibitor release studies used an inhibitor/enzyme subunit
molar ratio of 1.10 and 1.09 for DADMe-ImmH and Immucil-
lin-H, respectively. A solution of PNP (12.6 µL of 96 µM) was
incubated 5.5 h (25 °C) with 105 µM Immucillin-H or 108 µM
DADMe-Immucillin-H. The mixture was diluted 500 000 times,
by adding 1 µL to 500 µL of 20 mM Tris HCl, pH ) 7.5 buffer,
and then 1 µL of this dilution was added directly to 1 mL of
reaction mixture containing 1 8 mM inosine and 60 mU/mL
xanthine oxidase in 50 mM KPO4, pH ) 7.5 buffer. The
inhibitor release studies were performed in the absence and
presence of phosphate at 50 mM.
1
as a white solid with mp 248-250 °C. H NMR δ 7.81 (s,1H),
7.34 (s,1H), 3.97 (brs, 1H), 3.65 (s, 2H), 3.53 (m, 1H), 3.44 (m,
1H), 2.93 (t, J ) 9.0 Hz, 1H), 2.77 (m, 1H), 2.60 (1H, m), 2.33
(t, J ) 7.1 Hz, 1H), 2.12 (brs, 1H).13C NMR δ 155.8, 144.1,
142.8, 130.0, 117.3, 111.1, 72.9, 62.7, 60.2, 54.8, 48.9, 47.3.
HRMS (MH+) calc. for C12H16N4O3: 265.1301. Found 265.1302.
Anal. Calc. (C12H16N4O3.1/2H2O) C, H, N.
(3R,4R)-1-{[6-O-Ben zyl-7-ben zyloxym eth yl-9-d ea za -2-
bis(4-m eth oxyben zyl)gu a n in -9-yl]m eth yl}-3-h yd r oxy-4-
h yd r oxym eth ylp yr r olid in e (19). Sodium cyanoborohydride
(200 mg, 3.0 mmol) was added to a stirred solution of 15 (530
mg, 0.84 mmol) and 1‚HCl (163 mg, 1.06 mmol) in methanol
(10 mL) and the mixture was then stirred overnight at room
temp.. The reaction mixture was absorbed onto silica and
concentrated in vacuo. Chromatography of the resulting
residue afforded 19 (430 mg, 70%) as a white solid. Mp 98-
1
100 °C. H NMR 7.49 (s, 1H), 7.35-7.12 (s, 14H), 6.81 (d, J )
8.5 Hz, 4H), 5.59 (s, 2H), 5.47 (s, 2H), 4.85-4.73 (m, 4H), 4.44
(s, 2H), 4.23-4.12 (m, 3H), 3.75 (s, 6H), 3.50-3.35 (m, 3H),
3.20 (dd, J ) 12.0, 5.0 Hz, 1H), 3.08 (d, J ) 12.0 Hz, 1H), 2.95
(dd, J ) 11.4, 5.4 Hz, 1H), 2.24 (brs, 1H). 13C NMR δ 159.0,
158.4, 156.7, 153.1, 137.6, 137.0, 135.0, 131.5, 129.4, 128.9,
128.7, 128.4, 128.3, 128.1, 128.0, 125.7, 114.3, 110. 6, 105.1,
78.1, 73.1, 70.8, 68.0, 62.2, 60.7, 55.7, 54.8, 49.2, 48.9. HRMS
(MH+) calcd for C43H48N5O6: 730.3605. Found 730.3629.
(3R,4R)-1-{[9-d ea za gu a n in -9-yl]m et h yl}-3-h yd r oxy-4-
h yd r oxym eth ylp yr r olid in e (3). cHCl (2 mL) was added
dropwise to a solution of 19 (370 mg, 0.5 mmol) in methanol
(4 mL) and the resulting solution heated at reflux for 4 h. The
reaction was cooled to room temp and then concentrated in
vacuo. The resulting residue was partitioned between water
and chloroform and separated and the water layer concen-
trated in vacuo. Silica gel and ion exchange chromatography
of the resulting residue afforded 3 (39 mg, 28%) as a white
Ack n ow led gm en t. This work was supported by
research grant GM41916 from the NIH and by the New
Zealand Foundation for Research, Science & Technology
Contract.
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solid. Mp 223-225 °C. H NMR δ 7.18 (s, 1H), 4.03-3.98 (m,
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1
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