A β-fluoroamine inhibitor of purine nucleoside phosphorylase

Article abstract of DOI:10.1021/jm800792b

The potent immucillin purine nucleoside phosphorylase (PNP) inhibitors F-DADMe-ImmH [(3S,4S)-3], and [(3R,4R)-3] are synthesized in seven steps. Cycloaddition to a fluoroalkene and an enzymic resolution are the key features of the construction of the fluoropyrrolidines 11, from which the immucillins are assembled by use of a three-component Mannich reaction. Slow-onset binding constants (K_i*) for [(3S,4S)-3] and [(3R,4R)-3] with human PNP are 0.032 and 1.82 nM, respectively. F-DADMe-ImmH [(3S,4S)-3] exhibits oral availability in mice at doses as low as 0.2 mg/kg.

Full text of DOI:10.1021/jm800792b

5880
J. Med. Chem. 2008, 51, 5880–5884
A ꢀ-Fluoroamine Inhibitor of Purine Nucleoside Phosphorylase
Jennifer M. Mason,†,* Andrew S. Murkin,‡ Lei Li,‡ Vern L. Schramm,‡ Graeme J. Gainsford,† and Brian W. Skelton§
Carbohydrate Chemistry Team, Industrial Research Limited, P O Box 31310, Lower Hutt, New Zealand, Department of Biochemistry, Albert
Einstein College of Medicine of YeshiVa UniVersity, 1300 Morris Park AVenue, Bronx, New York 10461, Chemistry, M313, School of
Biomedical, Biomolecular and Chemical Sciences, UniVersity of Western Australia, Crawley 6006, Australia
ReceiVed June 30, 2008
The potent immucillin purine nucleoside phosphorylase (PNP) inhibitors F-DADMe-ImmH [(3S,4S)-3], and
[(3R,4R)-3] are synthesized in seven steps. Cycloaddition to a fluoroalkene and an enzymic resolution are
the key features of the construction of the fluoropyrrolidines 11, from which the immucillins are assembled
by use of a three-component Mannich reaction. Slow-onset binding constants (Ki/) for [(3S,4S)-3] and
[(3R,4R)-3] with human PNP are 0.032 and 1.82 nM, respectively. F-DADMe-ImmH [(3S,4S)-3] exhibits
oral availability in mice at doses as low as 0.2 mg/kg.
Introduction
Purine nucleoside phosphorylase (PNPa) is a purine-metab-
olizing enzyme that catalyzes the phosphorolysis of purine
nucleosides to ribose or deoxyribose 1-phosphate and a nucleo-
base.1 Individuals with a congenital deficiency of PNP exhibit
an unusual and characteristic impairment of their immune system
in that they have little or no T-cell immunity but retain normal
B-cell function.2 The biochemical link between PNP and T-cell
deficiencies is the failure to degrade deoxyguanosine and its
conversion to deoxyguanosine triphosphate (dGTP) in activated
T-cells.3,4 Such T-cells require DNA synthesis for cell division
and excess dGTP acts to inhibit ribonucleotide-diphosphate
reductase, causing unbalanced deoxynucleotide pools and the
induction of apoptosis.5,6 Therefore, PNP inhibitors are potential
Figure 1. Structures of some immucillins and a precursor amine.
therapeutics for the control of T-cell proliferation without
affecting humoral immunity. We have reported the design and
propranolols for cytochrome oxidase enzymes,22,23 increased
synthesis of a series of transition state analogue inhibitors of
oral absorption of fluorinated piperidine and piperazine indoles,24
and changes in metabolism and tissue distribution of amphet-
amines.25
As part of our ongoing program of synthesis of transition-
state analogue inhibitors of N-ribosylphosphorylases and hy-
drolases and the use of these compounds as therapeutic
agents,9,10 we now report the synthesis of fluoro-DADMe-
Immucillin-H [(3S,4S)-3], and its enantiomer.
PNP.7-10 Two of these, Immucillin-H (1, ImmH, Forodesine)11,12
and DADMe-Immucillin-H (2, DADMe-ImmH, BCX-3408,
R-3421)13 (Figure 1), are currently in clinical trials against T-cell
cancers and autoimmune diseases.14,15
Since the 1980s, fluorine substituents have become wide-
spread and important drug components.16 Organofluorine affects
most adsorption, distribution, metabolic, and excretion properties
as well as potency at the site of interest.17 In the case of amines,
the effects of ꢀ- or γ-fluorine substitution are largely the result
of a change in the degree of protonation at physiological pH
because such substitution lowers basicity.18 For example, the
pKas of protonated ethylamines are: 10.7 for ethylamine, 9.0
for 2-fluoroethylamine, and 5.7 for trifluoroethylamine.16 Also,
fluorine substitution at the 3-position of a piperidine ring lowers
the pKa by over 2 units.19 There are many studies connecting
the decrease in pKa associated with ꢀ-fluorination of aliphatic
amines with changes in biological properties. Some examples
include: reduced affinity of fluoroisoquinolines for the R2-
adrenoceptor,20 increased activity of lung N-methyltransferase
for fluoroalkylarylamines,21 changes in affinity and activity of
Results and Discussion
DADMe-Immucillin-H (2) is most easily assembled by means
of a three-component Mannich reaction between 9-deazahy-
poxanthine, formaldehyde, and pyrrolidine 4.26 This method,
which is tolerant of variations in both the amine and 9-dea-
zapurine, has provided us with many variants of 2 and so was
an obvious choice for our synthesis of 3.9,27 We chose to initially
pursue a synthesis of the racemate, with the required fluoro-
pyrrolidine (()-11 (Scheme 1) to be constructed through a
cycloaddition of azomethine ylid 6 to the known fluoroacry-
late 7.28,29
The 1,3-dipolar cycloaddition between azomethine ylids and
alkenes is a potent method for the construction of substituted
pyrrolidines with defined regio- and stereochemistry. Such a
cycloaddition is the key step in one of the routes to diol 4,30,31
and a plethora of other examples can be found in the
literature.32-34 The cycloaddition reaction is accelerated by
electron withdrawing substituents on the alkene,35 and so
* To whom correspondence should be addressed. Phone: +64-4-931-
3000. Fax: +64-4-931-3055. E-mail: j.mason@irl.cri.nz.
†
Industrial Research Limited.
‡
Albert Einstein College of Medicine of Yeshiva University.
University of Western Australia.
§
a Abbreviations: PNP, purine nucleoside phosphorylase; CAL-B, C.
antarctica lipase B.
10.1021/jm800792b CCC: $40.75
2008 American Chemical Society
Published on Web 08/23/2008

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5881
Scheme 1. Synthesis of the Racemate and Enantiomers of F-DADMe-ImmH [(()-3, (3S,4S)-3, and (3R,4R)-3]a
a Reagents (a) TFA, CH2Cl2, 0°C, 64% from 7; (b) LiBH4, MeOH, Et2O, 0 °C then HCl, 50 °C, 80%; (c) H2, Pd-C, (Boc)2O, MeOH, 100%; (d) HCl,
then 9-deazahypoxanthine, CH2O, NaOAc, water-dioxane, 100 °C, 40%; (e) NaOEt, EtOH, rt, 55%; (f) CAL-B, vinyl acetate, tert-BuOMe, 50 °C, 31%
[(3S,4R)-8] and 30% [(3R,4S)-9].
fluoroalkenes are suitable substrates for synthesis of 3- and
4-fluoropyrrolidines. However, this approach has only recently
received attention.36
Z-Ethyl 3-acetoxy-2-fluoroacrylate (7) was synthesized, fol-
lowing the literature method, from ethyl fluoroacetate.28 Z-
configuration was assigned on the basis of the H-F coupling
constant (3JH,F ) 18 Hz).29,37 Cycloaddition between ester 7
and the azomethine ylid 6, generated in situ from N-methoxym-
ethyl-N-(trimethylsilylmethyl)benzylamine (5), gave pyrrolidine
ester (()-8 in good yield.30 A cis arrangement of the fluoro
and acetoxy substituents in pyrrolidine (()-8, as would be
expected from a concerted cycloaddition to enol acetate 7, was
assumed at this point and was later confirmed by crystal structure
(vide infra).
Reduction of ester (()-8 (LiBH4, 80%) gave crystalline diol
(()-10, which was converted to N-Boc-pyrrolidinol (()-12 in
quantitative yield by hydrogenolysis in the presence of di-tert-
butyl dicarbonate. Pyrrolidine (()-11, produced either by
hydrogenolysis of (()-10 or hydrolysis of (()-12, participated
in a three-component Mannich reaction with 9-deazahypoxan-
thine and formaldehyde to give the target compound (()-3 in
moderate yield.
We then turned our attention to the synthesis of the enan-
tiomerically pure forms of 3. An enzymic transacetylation is a
Figure 2. ORTEP40 drawing of (3R,4R)-10.
key step in one of the published syntheses of nonracemic
pyrrolidine diol 4, and so we applied this method to the
4-fluoropyrrolidines (3S,4S)- and (3R,4R)-11.38 Pyrrolidine (()-8
was first deacetylated, in moderate yield, with sodium ethoxide
to give secondary alcohol (()-9. Acetylation catalyzed by an
immobilized form of lipase B of Candida antarctica (CAL-B)
gave moderate yields (31%) of alcohol (+)-9 [(3R,4S)-9, vide
infra] and acetate (+)-8 [(3S,4R)-8]. The enantiomeric excess
(ee) of alcohol (+)-9 was determined to be 90% by HPLC.
Reduction of (+)-8 with lithium borohydride gave crystalline
N-benzylpyrrolidinol (3R,4R)-10. A crystal structure (Figure 2)
determined both the absolute stereochemistry of this compound
and the cis relationship of the fluoro and hydroxy substituents
that had been established during the initial cycloaddition.39
Alcohol (3R,4S)-9 was also reduced with lithium borohydride
to N-benzylpyrrolidinol (3S,4S)-10. Both benzylpyrrolidinols
were purified by crystallization from ethyl acetate-hexanes. The
N-Boc-pyrrolidinols (3S,4S)- and (3R,4R)-12, synthesized from
these crystals by hydrogenolysis in the presence of di-tert-butyl
dicarbonate, both had ee greater than 95%; some enhancement

5882 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Brief Articles
Table 1. Inhibition of Human and Plasmodial PNP by the Racemate and Enantiomers of F-DADMe-ImmH [(()-3, (3S,4S)-3, and (3R,4R)-3]
human PNP P. falciparum PNP
Ki (nM)
Ki/ (nM) a
Ki (nM)
Ki/ (nM)
(()-3
0.50 ( 0.07
0.33 ( 0.11
18 ( 2
0.066 ( 0.009
0.032 ( 0.010
1.82 ( 0.08
23.3 ( 1.4
19 ( 4
3.6 ( 0.5
2.63 ( 0.18
260 ( 20
(3S,4S)-3
(3R,4R)-3
DADMe-ImmH (2)
L-DADMe-ImmH
2070 ( 160
1.10 ( 0.12b
0.38 ( 0.03d
0.011 ( 0.001b
0.50 ( 0.04 c
1700 ( 300 e
80 ( 7 e
a Ki is the dissociation constant for the first step in E + I T EI T EI/, the two-step binding characteristic of slow-onset, tight-binding inhibition. Ki/ is
the overall dissociation constant for E + I T EI/. In cases where only Ki is reported, no slow-onset inhibition was observed. b Ref 42. c Ref 41. d Ref 43.
e Ref 38.
component Mannich reaction. F-DADMe-ImmH [(3S,4S)-3] is
a potent inhibitor of human PNP. It is orally available in mice
at 0.2 mg/kg but has decreased oral availability and duration of
action relative to DADMe-ImmH (2).
Experimental Section
cis-1-((9-Deazahypoxanthin-9-yl)methyl)-4-fluoro-4-hydroxy-
methylpyrrolidin-3-ol [(()-3]. HCl (37%, 0.2 mL) was added to
a solution of pyrrolidinol (()-10 (31 mg, 0.13 mmol) in methanol
(5 mL), and the whole was concentrated to dryness under reduced
pressure. The residue was suspended in methanol and again
Figure 3. Bioavailability of F-DADMe-ImmH in mice. Enzyme
concentrated to dryness. Water (1.5 mL), dioxane (0.3 mL), NaOAc
(16.2 mg, 1.5 equiv), 9-deazahypoxanthine44 (36 mg, 2 equiv), and
formaldehyde solution (37%, 22 µL, 2 equiv) were added, and the
mixture was heated at 100 °C overnight. Chromatography of the
residue obtained when this solution was evaporated to dryness, using
methanolic ammonia (2 M, 35%) in CH2Cl2 as eluant, gave the
crude title compound (()-3 as a cream-colored foam (15 mg, 40%).
1H NMR (of hydrochloride) (D2O) δ 8.31 (s, 1H), 7.79 (s, 1H),
4.60 (s, 2H), 4.45 (m, 1H), 3.9-3.7 (m, 5H), 3.49 (m, 1H). 13C
NMR (of hydrochloride) (D2O) δ 155.2, 144.3, 141.8, 132.7, 118.7,
104.5, 100.5 (d, J ) 186 Hz), 69.6 (d, J ) 17 Hz), 61.3 (d, J ) 27
Hz), 57.8 (d, J ) 25 Hz), 56.2, 49.7. 19F NMR (D2O) δ -176.
HRMS for C12H16N4O3F [M + H]+ calcd, 283.1206; found,
283.1208. This material was further purified by HPLC.
activity was assayed following administration of 14 nmol by oral
treatment (9) or intraperitoneal injection ([).
of ee having occurred upon crystallization. Deprotection fol-
lowed by Mannich reaction gave the target compounds (3S,4S)-
and (3R,4R)-3.
Inhibition Studies. F-DADMe-ImmH [(3S,4S)-3] is a potent
inhibitor of human purine nucleoside phosphorylase, having a
slow-onset dissociation constant (Ki/) of 32 pM, binding 3-fold
weaker than DADMe-ImmH (2) (Table 1). As expected, the
enantiomer [(3R,4R)-3] does not bind as tightly to the enzyme,
but it is still a nanomolar inhibitor and, by comparison of its
Ki/ to the Ki of L-DADMe-ImmH, is about five times less potent
than its nonfluorinated counterpart. Plasmodium falciparum
(malaria) PNP binds these compounds less well, but this is not
surprising because this enzyme has repeatedly shown relatively
poor affinity for immucillins.41
(3S,4S)-1-((9-Deazahypoxanthin-9-yl)methyl)-4-fluoro-4-hy-
droxymethyl pyrrolidin-3-ol [(3S,4S)-3]. (3S,4S)-12 (10 mg, 0.043
mmol) was deprotected and coupled with 9-deazahypoxanthine44
as described for the racemate to give crude title compound (3S,4S)-3
1
Bioavailability. F-DADMe-ImmH [(3S,4S)-3] was admin-
istered to mice orally or by intraperitoneal (ip) injection with a
single dosage of 14 nmol. Complete inhibition of PNP catalytic
activity was observed in the ip injection group within 30 min,
yielding a t1/2 of less than 15 min for the onset of inhibition
(Figure 3). For the orally treated group, the t1/2 of onset was
20 h, suggesting that F-DADMe-ImmH was slowly absorbed
from the mouse gastrointestinal tract. Despite slow uptake, 80%
inhibition of blood PNP with a single oral dose of 0.2 mg/kg is
adequate for oral delivery. Both ip and oral groups regained
50% PNP catalytic activity at about 70-75 h, demonstrating
long-term action for both routes of administration. In control
experiments, mice were treated by oral and ip administration
with 14 nmol of DADMe-ImmH (2). The t1/2 of PNP inhibition
for oral administration of 2 was 90 min and no regaining of
catalytic activity was observed after 80 h for either administra-
tion route (Supporting Information). Thus, the bioavailability
and duration of action for F-DADMe-ImmH [(3S,4S)-3] is less
than that of DADMe-ImmH (2).
(8 mg, 67%), which was further purified by HPLC. H NMR (of
hydrochloride) identical to the racemate. HRMS for C12H16N4O3F
[M + H]+ calcd, 283.1206; found, 283.1201.
(3R,4R)-1-((9-Deazahypoxanthin-9-yl)methyl)-4-fluoro-4-hy-
droxymethyl pyrrolidin-3-ol [(3R,4R)-3]. (3R,4R)-12 (10 mg,
0.043 mmol) was deprotected and coupled with 9-deazahypoxan-
thine44 as described for the racemate to give crude title compound
1
(3R,4R)-3 (8 mg, 67%), which was further purified by HPLC. H
NMR (of hydrochloride) identical to the racemate. HRMS for
C12H16N4O3F [M + H]+ calcd, 283.1206; found, 283.1204.
Bioavailability of F-DADMe-ImmH [(3S,4S)-3]. Two male
BALB/c mice (∼25 g, purchased from the National Cancer Institute)
were fasted overnight. One mouse was treated orally with a single
dosage of 14 nmol of F-DADMe-ImmH [(3S,4S)-3], and the same
dosage was administered by intraperitoneal (ip) injection to the other
mouse. Blood samples (5 µL) were taken at intervals, mixed with
the same volume of 1% heparin and 0.3% Triton X-100 in PBS,
and stored at 4 °C before assays. Erythrocyte PNP catalytic activity
was monitored spectrophotometrically by adding 1 µL of the blood
sample to the reaction mixture containing 50 mM potassium
phosphate (pH 7.4), 1 mM inosine, and 60 mU of xanthine oxidase.
PNP catalytic activity of each sample was normalized for protein
concentration. The normalized rate at each time point was divided
by the normalized rate prior to inhibitor administration and plotted
against time. The time (t1/2) required for 50% of the PNP catalytic
activity to be lost and the time (t1/2) required for 50% regain of
PNP catalytic activity were interpolated from the plot.
Conclusions
We have synthesized F-DADMe-ImmH [(3 S,4 S)-3] in seven
steps. Cycloaddition to a fluoroalkene and an enzymic resolution
are the key features of the construction of the fluoropyrrolidine
11, from which the target is assembled by use of a three-

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5883
Zimmerli, D.; Schneider, J.; Diederich, F.; Kansy, M.; Muller, K.
Predicting and tuning physiochemical properties in lead optimisation:
amine basicities. ChemMedChem 2007, 2, 1100–1115.
Acknowledgment. This work was supported by research
grant GM41916 from the NIH and by a contract from the New
Zealand Foundation for Research, Science & Technology. We
thank Dr H. Wong for NMR spectra and Drs. K. Clinch, G. B.
Evans, and P. C. Tyler for helpful discussions.
(19) Jensen, H. H.; Lyngbye, L.; Jensen, A.; Bols, M. Stereoelectronic
substituent effects in polyhydroxylated piperidines and hexahydropy-
radazines. Chem.sEur. J. 2002, 8, 1218–1226.
(20) Grunewald, G. L.; Seim, M. R.; Lu, J.; Makboul, M.; Criscione, K. R.
Application of the Goldilocks effect to the design of potent and
selective inhibitors of phenylethanolamine N-methyltransferase: bal-
ancing pKa and steric effects in the optimization of 3-methyl-1,2,3,4-
tetrahydroisoquinoline inhibitors by ꢀ -fluorination. J. Med. Chem.
2006, 49, 2939–2952.
Supporting Information Available: Experimental details for
compounds (()-8, (()-9, (()-10, (()-12, (3S,4R)-8, (3R,4S)-9,
(3S,4S)-10, (3S,4S)-12, (3R,4R)-10, and (3R,4R)-12; 1H NMR
spectra of (()-8, (()-9, (()-12, (3S,4R)-8, (3R,4S)-9, (3S,4S)-10,
(3S,4S)-12, (3R,4R)-10, (3R,4R)-12, (()-3, (3S,4S)-3, and (3R,4R)-
3; HPLC data for (()-3, (3S,4S)-3, and (3R,4R)-3; details of X-ray
crystallographic structure determination of (3R,4R)-10; details of
inhibition assays; bioavailability of DADMe-ImmH (2). This
material is available free of charge via the Internet at http://
pubs.acs.org.
(21) Fuller, R. W.; Roush, B. W. Influence of pKa on the methylation of
arylalkylamines by rabbit lung N-methyltransferase. Res. Commun.
Chem. Pathol. Pharmacol. 1975, 735–738.
(22) Upthagrove, A. L.; Nelson, W. L. Importance of Amine pKa and
distribution coefficient in the metabolism of fluorinated propranolol
derivatives. Preparation, identification of metabolite regioisomers, and
metabolism by CYP2D6. Drug Metab. Dispos. 2001, 29, 1377–1388.
(23) Upthagrove, A. L.; Nelson, W. L. Importance of Amine pKa and
distribution coefficient in the metabolism of fluorinated propranolol
analogs: metabolism by CYP1A2. Drug Metab. Dispos. 2001, 29, 1389–
1395.
(24) van Niel, M. B.; Collins, I.; Beer, M. S.; Broughton, H. B.; Cheng,
S. K. F.; Goodacre, S. C.; Heald, A.; Locker, K. L.; MacLeod, A. M.;
Morrison, D.; Moyes, C. R.; O’Connor, D.; Pike, A.; Rowley, M.;
Russell, M. G. N.; Sohal, B.; Stanton, J. A.; Thomas, S.; Verrier, H.;
Watt, A. P.; Castro, J. L. Fluorination of 3-(3-(piperidin-1-yl)propy-
l)indoles and 3-(3-(piperazin-1-yl)propyl)indoles gives selective human
5-HT1D receptor ligands with improved pharmacokinetic profiles.
J. Med. Chem. 1999, 42, 2087–2104.
(25) Fuller, R. W.; Molloy, B. B.; Parli, C. J. Effect of ꢀ-ꢀ-difluoro
substitution on the metabolism and pharmacology of amphetamines.
In Psychopharmacology, Sexual Disorders, and Drug Abuse; Proceed-
ings of the Symposium at the Congress of the Collegium Internationale
Neuro-Psychopharmacologicum, 8th ed.; Ban,T. A. , Ed. North-
Holland: Amsterdam, 1973; pp 615-624.
(26) Evans, G. B.; Furneaux, R. H.; Tyler, P. C.; Schramm, V. L. Synthesis
of a transition state analogue inhibitor of purine nucleoside phospho-
rylase via the Mannich reaction. Org. Lett. 2003, 5, 3639–3640.
(27) Singh, V.; Evans, G. B.; Lenz, D. H.; Mason, J. M.; Clinch, K.; Mee,
S.; Painter, G. F.; Tyler, P. C.; Furneaux, R. H.; Lee, J. E.; Howell,
P. L.; Schramm, V. L. Femtomolar transition state analogue inhibitors
of 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase from
Escherichia coli. J. Biol. Chem. 2005, 280, 18265–18273.
(28) Bergmann, E. D.; Shahak, I.; Gruenwald, I. Organic fluorine com-
pounds. Part XXXIX. Reactions of R-fluoro-ꢀ-keto-esters. J. Chem.
Soc. C 1967, 220, 6–2207.
References
(1) Krenitsky, T. A. Purine nucleoside phosphorylase: kinetics, mechanism,
and specificity. Mol. Pharmacol. 1967, 3, 526–536.
(2) Markert, M. L. Purine nucleoside phosphorylase deficiency. Immu-
nodefic. ReV. 1991, 3, 45–81.
(3) Chan, T. Deoxyguanosine toxicity on lymphoid cells as a cause for
immunosuppression in purine nucleoside phosphorylase deficiency.
Cell 1978, 14, 523–530.
(4) Cohen, A.; Gud as, L. J.; Ammann, A. J.; Staal, G. E.; Martin, D. W.
Deoxyguanosine triphosphate as a possible toxic metabolite in the
immunodeficiency associated with purine nucleoside phosphorylase
deficiency. J. Clin. InVest 1978, 61, 1405–1409.
(5) Ullman, B.; Clift, S. M.; Gudas, L. J.; Levinson, B. B.; Wormsted,
M. A.; Martin, D. W. Alterations in deoxyribonucleotide metabolism
in cultured cells with ribonucleotide reductase activities refractory to
feedback inhibition by 2′-deoxyadenosine triphosphate. J. Biol. Chem.
1980, 255, 8308–8314.
(6) Bantia, S.; Ananth, S. L.; Parker, C. D.; Horn, L. L.; Upshaw, R.
Mechanism of inhibition of T-acute lymphoblastic leukemia cells by
PNP inhibitor BCX-1777. Int. Immunopharmacol. 2003, 3, 879–887.
(7) Evans, G. B.; Furneaux, R. H.; Lewandowicz, A.; Schramm, V. L.;
Tyler, P. C. Exploring structure-activity relationships of transition
state analogues of human purine nucleoside phosphorylase. J. Med.
Chem. 2003, 46, 3412–3423.
(8) Lewandowicz, A.; Tyler, P. C.; Evans, G. B.; Furneaux, R. H.;
Schramm, V. L. Achieving the ultimate physiological goal in transition
state analogue inhibitors for purine nucleoside phosphorylase. J. Biol.
Chem. 2003, 278, 31465–31468.
(9) Evans, G. B.; Furneaux, R. H.; Greatrex, B.; Murkin, A. S.; Schramm,
V. L.; Tyler, P. C. Azetidine based transition state analogue inhibitors
of N-ribosyl hydrolases and phosphorylases. J. Med. Chem. 2008, 51,
948–956.
(29) Imbeaux-Oudette, M.; Elkik, E. Formyl fluoro ce´tones et esters. 1.
Pre´paration et de´rive´s ace´tyle´s. Bull. Soc. Chem. Fr. 1975, 1165–1169.
(30) Karlsson, S.; Ho¨gberg, H. E. Synthesis of enantiomerically pure
4-substituted pyrrolidin-3-ols via asymmetric 1,3-dipolar cycloaddition.
Tetrahedron: Asymmetry 2001, 12, 1977–1982.
(31) Kotian, P. L.; Lin, T. H.; El Kattan, Y.; Chand, P. A practical large-scale
synthesis of (3R,4R)-4-(hydroxymethyl)pyrrolidin-3-ol via asymmetric 1,3-
dipolar cycloaddition. Org. Process Res. DeV. 2005, 9, 193–197.
(32) Coldham, I.; Hufton, R. Intramolecular dipolar cycloaddition reactions
of azomethine ylides. Chem. ReV. 2005, 105, 2765–2809.
(33) Pandey, G.; Banerjee, P.; Gadre, S. R. Construction of enantiopure
pyrrolidine ring system via asymmetric [3 + 2]-cycloaddition of
azomethine ylides. Chem. ReV. 2006, 106, 4484–4517.
(34) Pellissier, H. Asymmetric 1,3-dipolar cycloadditions. Tetrahedron
2007, 63, 3235–3285.
(10) Taylor, E. A.; Clinch, K.; Kelly, P. M.; Li, L.; Evans, G. B.; Tyler,
P. C.; Schramm, V. L. Acyclic ribooxacarbenium ion mimics as
transition state analogues of human and malarial purine nucleoside
phosphorylases. J. Am. Chem. Soc. 2007, 129, 6984–6985.
(11) Kicska, G. A.; Long, L.; Horig, H.; Fairchild, C.; Tyler, P. C.;
Furneaux, R. H.; Schramm, V. L.; Kaufman, H. L. Immucillin H, a
powerful transition-state analog inhibitor of purine nucleoside phos-
phorylase, selectively inhibits human T lymphocytes. Proc. Natl. Acad.
Sci. U.S.A. 2001, 98, 4593–4598.
(12) Ravandi, F.; Gandhi, V. Novel purine nucleoside analogues for T-cell-
lineage acute lymphoblastic leukaemia and lymphoma. Expert Opin.
InVest. Drugs 2006, 15, 1601–1613.
(35) Padwa, A. Intermolecular 1,3-Dipolar Cycloadditions. In Comprehen-
siVe Organic Synthesis, Vol. 4, 1st ed.; Trost, B. M., Fleming, I. ,
Eds. Pergamon: Elmsford, NY, 1991; pp 1069-1109.
(13) Evans, G. B.; Furneaux, R. H.; Tyler, P. C.; Schramm, V. L. Synthesis
of a transition state analogue inhibitor of purine nucleoside phospho-
rylase via the Mannich reaction. Org. Lett. 2003, 5, 3639–3640.
(14) Gandhi, V.; Kilpatrick, J. M.; Plunkett, W.; Ayres, M.; Harman, L.; Du,
M.; Bantia, S.; Davisson, J.; Wierda, W. G.; Faderl, S.; Kantarjian, H.;
Thomas, D. A proof-of-principle pharmacokinetic, pharmacodynamic, and
clinical study with purine nucleoside phosphorylase inhibitor Immucillin-H
(BCX-1777, forodesine). Blood 2005, 106, 4253–4260.
(15) BioCryst Pharmaceuticals Inc. http://www.biocryst.com. Accessed July
2008.
(16) Bohm, H.-J.; Banner, D.; Kuhn, B.; Muller, K.; Obst-Sander, U.; Stahl,
M.; Bendels, S.; Kansy, M. Fluorine in medicinal chemistry. Chem-
BioChem 2004, 5, 637–643.
(36) Bonini, B. F.; Boschi, F.; Franchini, M. C.; Fochi, M.; Fini, F.;
Mazzanti, A.; Ricci, A. First 1,3-dipolar cycloaddition of azomethine
ylides with (E)-ethyl 3-fluoroacrylate: regio- and stereoselective
synthesis of enantiopure fluorinated prolines. Synlett 2006, 543–546.
(37) Sato, M.; Kaneko, C.; Iwaoka, T.; Kobayashi, Y.; Iida, T. Synthesis
of 5-fluoro-1,3-dioxin-4-ones: versatile building blocks of fluorinated
compounds. J. Chem. Soc., Chem. Commun. 1991, 699–700.
(38) Clinch, K.; Evans, G. B.; Fleet, G. W. J.; Furneaux, R. H.; Johnson, S. W.;
Lenz, D. H.; Mee, S. P. H.; Rands, P. R.; Schramm, V. L.; Ringia,
E. A. T.; Tyler, P. C. Syntheses and bioactivities of the L-enantiomers of
two potent transition state analogue inhibitors of purine nucleoside
phosphorylases. Org. Biomol. Chem. 2006, 4, 1131–1139.
(17) Muller, K.; Faeh, C.; Diederich, F. Fluorine in pharmaceuticals: looking
(39) CCDC 684082 contains the supplementary crystallographic data for
this paper. These data can be obtained free of charge from The
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif.
beyond intuition. Science 2007, 317, 1881–1886.
(18) Morgenthaler, M.; Schweizer, E.; Hoffman-Roder, A.; Benini, F.;
Martin, R. E.; Jaeschke, G.; Wagner, B.; Fischer, H.; Bendels, S.;

5884 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Brief Articles
(40) Farrugia, L. J. ORTEP-3 for Windows, a version of ORTEP-III
with a graphical user interface (GUI). J. Appl. Crystallogr. 1997,
30, 565.
(41) Lewandowicz, A.; Ringia, E. A. T.; Ting, L. M.; Kim, K.; Tyler,
P. C.; Evans, G. B.; Zubkova, O. V.; Mee, S.; Painter, G. F.; Lenz,
D. H.; Furneaux, R. H.; Schramm, V. L. Energetic mapping of
transition state analogue interactions with human and Plasmodium
falciparum purine nucleoside phosphorylases. J. Biol. Chem. 2005,
280, 30320–30328.
in the transition state of human purine nucleoside phosphorylase.
Biochemistry 2007, 46, 5038–5049.
(43) Rinaldo-Matthis, A.; Murkin, A. S.; Ramagopal, U. A.; Clinch, K.;
Mee, S. P. H.; Evans, G. B.; Tyler, P. C.; Furneaux, R. H.; Almo,
S. C.; Schramm, V. L. L-Enantiomers of transition state analogue
inhibitors bound to human purine nucleoside phosphorylase. J. Am.
Chem. Soc. 2008, 130, 842–844.
(44) Furneaux, R. H.; Tyler, P. C. Improved syntheses of 3H,5H-
pyrrolo[3,2-d]pyrimidines. J. Org. Chem. 1999, 64, 8411–8412.
(42) Murkin, A. S.; Birck, M. R.; Rinaldo-Matthis, A.; Shi, W.; Taylor,
E. A.; Almo, S. C.; Schramm, V. L. Neighboring group participation
JM800792B