Second generation transition state analogue inhibitors of human 5′-methylthioadenosine phosphorylase

Article abstract of DOI:10.1021/jm050269z

The polyamine biosynthetic pathway is a therapeutic target for proliferative diseases because cellular proliferation requires elevated levels of polyamines. A byproduct of the synthesis of spermidine and spermine is 5′-methylthioadenosine (MTA). In humans

Full text of DOI:10.1021/jm050269z

J. Med. Chem. 2005, 48, 4679-4689
4679
Second Generation Transition State Analogue Inhibitors of Human
5′-Methylthioadenosine Phosphorylase
Gary B. Evans,*,† Richard H. Furneaux,† Dirk H. Lenz,† Gavin F. Painter,† Vern L. Schramm,‡
Vipender Singh,‡ and Peter C. Tyler†
Carbohydrate Chemistry, Industrial Research Limited, P. O. Box 31310, Lower Hutt, New Zealand, and Department of
Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461
Received March 23, 2005
The polyamine biosynthetic pathway is a therapeutic target for proliferative diseases because
cellular proliferation requires elevated levels of polyamines. A byproduct of the synthesis of
spermidine and spermine is 5′-methylthioadenosine (MTA). In humans MTA is processed by
5′-methylthioadenosine phosphorylase (MTAP) so that significant amounts of MTA do not
accumulate. Products of the MTAP reaction (adenine and 5-methylthio-R-D-ribose-1-phosphate)
are recycled to S-adenosylmethionine, the precursor for polyamine synthesis. Potent inhibitors
of MTAP might allow the build-up of sufficient levels of MTA to generate feedback inhibition
of polyamine biosynthesis and/or reduce S-adenosylmethionine levels. We recently reported
the design and synthesis of a family of potent transition state analogue inhibitors of MTAP.
We now report the synthesis of a second generation of stable transition state analogues with
increased distance between the ribooxocarbenium ion and purine mimics. These compounds
are potent inhibitors with equilibrium dissociation constants as low as 10 pM. The first and
second generation inhibitors represent synthetic approaches to mimic early and late features
of a dissociative transition state.
Introduction
antiproliferativeactivity.8-12 Thishasmadethepolyamine
biosynthetic pathway a therapeutic target for the treat-
ment of proliferative diseases such as cancer and
parasitic infections.13-18 A less-recognized feature of the
polyamine pathway is the recycling of 5-methylthio-R-
D-ribose-1-phosphate to methionine and adenine to ATP,
the precursors of S-adenosylmethionine. These steps
complete the cycle of the polyamine pathway since
S-adenosylmethionine is the precursor for polyamine
synthesis.
Recently we reported the design,1 synthesis,2 and
biological activity1,2 of a first generation series of inhibi-
tors of human methylthioadenosine phosphorylase
(MTAP), the 5′-alkylthio and 5′-arylthio immucillins.
MTAP is a key enzyme in the catabolism of methyl-
thioadenosine (MTA), a byproduct of polyamine synthe-
sis (Figure 1).
In humans it is proposed that inhibition of MTAP and
accumulation of methylthioadenosine (MTA) could lead
to feedback inhibition of spermidine and spermine
synthases and thus down-regulate polyamine biosyn-
thesis. The polyamines putrescine, spermidine, and
spermine play important roles in all mammalian cells,
protozoa, bacteria, and fungi. They are involved in
replication, transcription and translation, but their roles
in growth-related processes are best defined.3-5 Cellular
proliferation requires increased levels of polyamines,
and compared to quiescent cells, rapidly dividing cells
have elevated polyamine pools.
In humans MTA is rapidly degraded by MTAP so that
no significant concentration of MTA is known to ac-
cumulate. MTAP is the only enzyme in humans that
processes MTA, and human genomic studies indicate
only a single gene locus for the enzyme. The phospho-
rolysis reaction that it catalyses produces 5-methylthio-
R-D-ribose-1-phosphate and adenine (Figure 2).6,7 Potent
inhibitors of MTAP might be expected to raise the
intracellular concentration of MTA sufficiently to achieve
feed-back inhibition of polyamine biosynthesis and have
The transition state for the reaction catalyzed by
human MTAP was inferred through our knowledge of
the transition state for human purine nucleoside phos-
phorylase (PNP)19-21 and confirmed by transition state
analysis of human MTAP (Figure 2).22 The character-
istics of an early transition state for an SN1 mechanism
i.e., the ribooxocarbenium ion character in the 5′-
methylthioribosyl group and the elevated pKa for the
purine leaving group with little participation of the
phosphate ion, have been partially captured by the first
generation inhibitors. To better mimic the cationic
charge which develops at the anomeric carbon of a fully
dissociative transition state, we designed a second
generation of inhibitors in which the nitrogen atom was
moved to the position of the anomeric carbon, the ring
oxygen was replaced by a methylene group, and the
ribosyl mimic and the deazapurine were increased in
separation through insertion of a methylene bridge.23
Herein we report the synthesis of a family of these
second generation transition state analogue inhibitors
of human MTAP. Their inhibitory properties against
human MTAP extend to dissociation constants of 10 pM
and establish these compounds as powerful inhibitors
for human MTAP while at the same time delineating
some structure-activity relationships. We have de-
* To whom correspondence should be addressed. Phone: 64-4-
9313040. Fax: 64-4-9313055. E-mail: g.evans@irl.cri.nz.
† Industrial Research Limited.
‡ Albert Einstein College of Medicine.
10.1021/jm050269z CCC: $30.25 © 2005 American Chemical Society
Published on Web 06/21/2005

4680 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 14
Evans et al.
Figure 1. Polyamine biosynthesis from ornithine to spermine.
Figure 2. Phosphorolysis of methylthioadenosine catalyzed by MTAP, including the putative transition state, and the general
structures of the first and second generation inhibitors of MTAP.
scribed the inhibitory properties of some of these
compounds in an initial report that includes the X-ray
crystal structure of human MTAP with one of these
inhibitors.1 Other members of this family have been
shown to be powerful inhibitors of E. coli 5’-methyl-
thioadenosine nucleosidase.24
in good overall yield. Reductive amination of 2 with
7-benzyloxymethyl-6-O-tert-butyl-9-deaza-9-formylhy-
poxanthine (3) using sodium cyanoborohydride could be
carried out in an analogous manner to that previously
described, to afford compound 4.23 Conversion of the
protected deazahypoxanthine to a deazaadenine moiety
was achieved via a series of synthetic steps which
initially involved acetylation of the secondary hydroxyl,
followed by removal of the tert-butyl group through acid
deprotection to afford 5. Compound 5 was then treated
with refluxing phosphoryl chloride and the C-6 chlorine
atom resultant in the product was displaced with
concomitant removal of the acetate by heating with
methanolic ammonia in a sealed tube to yield 6 contain-
ing the deazaadenine moiety. Global deprotection of 6
through treatment with cHCl, followed by conversion
Results and Discussion
Synthesis. The tert-butoxycarbonyl-protected amine
(1), prepared as previously described, was used for the
preparation of the 9-deazaadenine derivatives 7 and 12
(Scheme 1).23 Sulfonation of 1 with methanesulfonyl
chloride and Hunigs base in CH2Cl2 at 0 °C afforded
predominantly the primary mono-methanesulfonate,
and this was readily displaced with sodium thiomethox-
ide, followed by removal of the tert-butoxycarbonyl
protecting group, to provide the amine hydrochloride 2

Transition State Analogue Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 14 4681
Scheme 1a
a Reagents: (a) MsCl, iPR2NEt, CH2Cl2, 0 °C f room temp; (b) NaSMe or NaSBn, DMF, room temp; (c) cHCl, MeOH, room temp; (d)
NaCNBH3, MeOH, room temp; (e) Ac2O, DMAP, Et3N, CH2Cl2, room temp; (f) TFA, CH2Cl2, room temp; (g) POCl3, reflux; (h) 7 N NH3
in MeOH, 110 °C; (i) cHCl, reflux; (j) NH4OH, room temp.
Scheme 2a
Scheme 3a
a Reagents: (a) 37% aq formaldehyde, NaOAc, dioxane, H2O,
95 °C.
To study the SAR of this class of inhibitors, the MeS
group was replaced with alternative substituents. The
requisite thio-substituted amine hydrochlorides 17-26
were prepared from compound 1 in an analogous fashion
to that used for compounds 2 and 8 (Scheme 4).
O-Alkyl-substituted amine hydrochlorides 27 and 28
were prepared via selective protection of the primary
hydroxyl of 1 as a tert-butyldiphenylsilyl ether to afford
45 (Scheme 5). MOM protection of the secondary hy-
droxyl group yielded 46 and then removal of the silyl
protecting group afforded 47. Methylation or benzylation
of the primary hydroxyl of 47 afforded compounds 48
and 49, and removal of the tert-butoxycarbonyl protect-
ing group afforded amine hydrochlorides 27 and 28,
respectively.
The chain-extended deoxygenated amines 29 and 30
were synthesized via the aldehyde 50, obtained as a
precursor during the synthesis of amine 1 (Scheme 6).
The Wittig reaction between compound 50 and the
appropriate ylid afforded the individual compounds 51
and 52 as E/Z mixtures of stereoisomers. Hydrogenation
of compounds 51 and 52 followed by removal of the tert-
butoxycarbonyl protecting group afforded the amines 29
and 30, respectively.
a Reagents: (a) NaCNBH3, MeOH, room temp; (b) 7 N NH3 in
MeOH, 110 °C; (c) cHCl, MeOH, room temp.
of the subsequent hydrochloride salt to the free base,
afforded the target compound 7.
Similarly displacement of the monomesylate of com-
pound 1 with sodium thiobenzyloxide afforded, after
deprotection, the S-benzyl derivative 8. This was con-
verted into compound 12 via compounds 9, 10, and 11.
The final deprotection of 11 to give 12 proceeded in poor
yield (12%), but the reaction was not further investi-
gated.
For the preparation of the 8-aza-9-deazaadenine
derivative 16 the benzylthio amine hydrochloride 8 was
also reductively aminated with 8-aza-9-deaza-9-formyl-
6-methoxy-8-tetrahydropyranylpurine (13)23 under stan-
dard conditions to yield 14 (Scheme 2). Displacement
of the C-6 methoxy group was readily achieved in this
case with 7 N ammonia in methanol in a sealed tube at
130 °C to afford 15. Global deprotection yielded the
target compound 16, the 8-aza homologue of 12.
Subsequently it was found that compounds 7 and 12
could be synthesized in a more direct manner via the
Mannich reaction between the amine hydrochlorides 2
and 8, 9-deazaadenine and formaldehyde (Scheme 3,)
but we were unable to prepare compound 16 using this
strategy.25
The Mannich reaction of the amine hydrochlorides
17-29 with 9-deazaadenine and formaldehyde (Scheme
4) allowed their rapid conversion into compounds 31-
43 all of which were potent inhibitors of human MTAP
(Table 1). The final analogue 44 was prepared via
coupling with 6-chloro-9-deazapurine and displacement
of the chlorine atom with ammonia followed by global

4682 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 14
Evans et al.
Scheme 4a
a Reagents: (a) 9-Deazaadenine, 37% formaldehyde, NaOAc, dioxane, H2O, 95 °C; (b) 6-chloro-9-deazapurine, 37% formaldehyde, NaOAc,
dioxane, H2O, 95 °C; (c) 7 N NH3 in MeOH, 130 °C.
Scheme 5a
a Reagents: (a) TBDPSCl, Im, DMF, room temp; (b) MOMBr, iPr2NEt, CH2Cl2; (c) TBAF, THF; (d) NaH, RI, THF; (e) 4 M HCl, dioxane.
Scheme 6a
a Reagents: (a) MeCHdPPh3 or C6H5CHdPPh3, THF, 0 °C; (b) H2, Pd/C, EtOH; (c) HCl, MeOH, room temp.
deprotection. Use of the Mannich reaction has enabled
the synthesis of gram quantities of candidate MTAP
inhibitors for in vivo trials.
equilibrium dissociation constant: E + I T E*I. Ex-
perimentally, inhibition constants were estimated from
two time periods of the reaction rate curve. In the first,
the initial reaction rates (t f 0) were fitted to the
equation for competitive inhibition to give Ki, and the
final reaction rates (after slow-onset was complete) were
fitted to the equation for competitive inhibition to give
Ki*. These methods are valid for conditions where initial
and final rates are clearly defined and delineated and
the inhibitor concentration is >10× enzyme concentra-
tion.26 Under high inhibitor (I) conditions, initial rates
are difficult to measure because the tight-binding phase
occurs rapidly. Inhibition curves were also fitted to the
general integrated rate equation: P ) νst + (νo -νs)(1
Biological Results. Inhibition Studies. Inhibition
of MTAP by the transition state analogue inhibitors
described here normally involves the two-step process
of slow-onset tight-binding inhibition. The first step
involves reversible competitive binding at the catalytic
site: E + I T EI characterized by Ki as the dissociation
constant. In the second step the EI complex undergoes
a conformational change to tighten the binding: EI T
E*I. This second step is usually slow relative to EI
formation and equilibration. The overall equilibrium
dissociation constant Ki* is defined by the overall

Transition State Analogue Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 14 4683
Table 1. Inhibition of Human MTAP by Second Generation
raphy solvents were distilled prior to use. Anhydrous solvents
were obtained from Aldrich or Acros. NMR spectra were
recorded at 300 MHz (1H) and 75 MHz (13C) in D2O unless
otherwise indicated.
Immucillinsa
compd
R
Ki (nM)
Ki* (nM)
MTA
Km ) 5000 nM
26.0 ( 0.8
1.7 ( 0.2
1.4 ( 0.2
55 ( 4
0.65 ( 0.08
1.30 ( 0.04
0.9 ( 0.1
0.28 ( 0.05
0.76 ( 0.07
1.5 ( 0.1
0.36 ( 0.07
2.0 ( 0.5
0.81 ( 0.11
2.0 ( 0.1
14 ( 2
(3R,4S)-3-Hydroxy-4-methylthiomethylpyrrolidine Hy-
drochloride (2). Methanesulfonyl chloride (800 µL, 10 mmol)
was added dropwise to a solution of Hunig’s base (3.5 mL, 20
mmol) and (3R,4R)-1-tert-butoxycarbonyl-3-hydroxy-4-(hy-
droxymethyl)pyrrolidine (1) (2.2 g, 10 mmol) in CH2Cl2 (30 mL)
at 0 °C, and the resulting solution allowed to warm to room
temp. The reaction mixture was diluted with CH2Cl2, washed
with water and brine, dried (MgSO4), and concentrated in
vacuo. The resulting residue was purified by chromatography
(5% MeOH in CH2Cl2) to afford, presumably, (3R,4R)-1-tert-
butoxycarbonyl-3-hydroxy-4-(mesyloxymethyl)pyrrolidine (2.1
g) as an oil. Without further characterization, the product (900
mg) was dissolved in DMF (10 mL) and stirred with sodium
thiomethoxide (400 mg, 5.7 mmol) at room temp overnight.
The reaction was diluted with toluene, washed with water and
brine, dried (MgSO4), and concentrated in vacuo. The resulting
residue was purified by chromatography (ethyl acetate) to
afford, presumably, (3R,4S)-1-tert-butoxycarbonyl-3-hydroxy-
4-(methylthiomethyl)pyrrolidine (600 mg, 2.4 mmol) as a
syrup, which was not further characterized but dissolved in
MeOH (5.0 mL) and cHCl (1.0 mL) and concentrated in vacuo
to afford (3R,4S)-3-hydroxy-4-(methylthiomethyl)pyrrolidine
hydrochloride (2) (442 mg, 49% overall yield for three steps)
MT-ImmA
1.0 ( 0.5
0.09 ( 0.01
0.7 ( 0.2
7
SMe
12
16
31
32
33
34
35
36
37
38
39
40
41
42
43
44
SBn
ND
SEt
SnPr
SiPr
SnBu
SCy
SPh
SPh-4-Cl
SPh-3-Cl
SPh-4-F
S-4-Py
OMe
0.034 ( 0.003
0.12 ( 0.01
0.26 ( 0.03
0.11 ( 0.02
0.37 ( 0.03
0.17 ( 0.02
0.010 ( 0.005
0.27 ( 0.15
0.16 ( 0.01
0.160 ( 0.015
8.0 ( 0.7
OBn
Et
Bn
85 ( 7
22 ( 2
>2000
42 ( 4
3.0 ( 0.4
ND
a Ki is the initial dissociation constant, and Ki* is the equilib-
rium dissociation constant obtained after slow-onset inhibition.
ND indicates that no slow-onset phase was observed.
- e-kt)/k where P, νo, νs, and k represent, respectively,
product formed, initial rate, final steady-state rate, and
first-order rate constant for attainment of the final rate.
The value of k increases hyperbolically as a function of
inhibitor concentration such that k ) k6[1 + (I/(Ki*(1 +
A/Ka))]/[1 + (I/(Ki(1 + A/Ka))] where k6 is the rate of
conversion of E*I to EI and I and A are inhibitor and
substrate concentrations.27
The 5′-MT-DADMe-ImmA (7) is a powerful inhibitor
of MTAP and, after slow onset, has Ki* ) 90 pM. The
Km/Ki value of 55 555 for compound 7, together with the
chemical similarity to the proposed transition-state
structure, supports the hypothesis that features of the
transition state are being captured with this molecule.
Extension of the methyl group shows a similar trend to
that previously reported2 with the Ki* for the ethyl
analogue 31 falling to 34 pM with a Km/Ki* value of
147 058. Weaker inhibitors are found as the chain
length is either extended or branched as in compounds
31-35. The inhibitory potency of the 8-aza analogue of
12, compound 16, is around 80 times less. In the
5′-arylthio series the 4-chlorophenyl analogue 37 is the
most powerful of the inhibitors found with a Ki* ) 10
pM and a Km/Ki* value of 500 000. Substitution of the
sulfur atom of compound 7 with either an oxygen, as in
compound 41, or a carbon atom, as in compound 43,
affords inhibitors significantly less active with Ki*
values of 8 and 3 nM, respectively.
1
as a syrup. H NMR (D2O) 4.40 (q, J ) 3.1 Hz, 1H), 3.67 (dd,
J ) 12.0, 6.6 Hz, 1H), 3.50 (dd, J ) 12.8, 5.1 Hz, 1H), 3.28
(dd, J ) 12.8, 3.0 Hz, 1H), 3.22 (dd, J ) 8.7, 3.4 Hz, 1H), 2.73-
2.66 (m, 1H), 2.58-2.50 (m, 2H). 13C NMR (D2O) δ 73.5, 51.5,
48.6, 45.2, 34.3, 14.9. HRMS (MH+) calcd for C6H14NOS:
148.0796. Found 148.0803.
(3R,4S)-1-[(7-Benzyloxymethyl-6-tert-butoxy-9-deazapu-
rin-9-yl)methyl]-3-hydroxy-4-methylthiomethylpyrroli-
dine (4). Sodium cyanoborohydride (200 mg, 3.2 mmol) was
added to a stirred solution of 323 (800 mg, 2.32 mmol) and 2
(550 mg, 3.00 mmol) in methanol (10 mL), and the mixture
was stirred overnight at room temp. The crude reaction
mixture was absorbed onto silica and purified by chromatog-
raphy (10% MeOH in CH2Cl2) to afford 4 (1.10 g, 78%) as a
1
gum. H NMR (CDCl3) δ 8.48 (s, 1H), 7.54 (s, 1H), 7.33-7.23
(m, 5H), 5.75 (s, 2H), 4.50 (s, 2H), 4.12 (m, 1H), 4.02 (s, 2H),
3.30 (dd, J ) 9.9, 7.5 Hz, 1H), 2.95 (m, 2H), 2.64 (dd, J ) 12.7,
7.1 Hz, 1H), 2.52-2.38 (m, 3H), 2.07 (s, 3H), 1.70 (s, 9H). 13C
NMR (CDCl3) δ 156.4, 150.3, 150.3, 137.5, 133.3, 128.8, 128.2,
127.8, 117.1, 111.7, 83.5, 77.5, 76.1, 70.4, 61.4, 58.2, 48.8, 47.4,
37.3, 29.0, 16.0. HRMS (MH+) calcd for C34H38N5O3S: 564.2975.
Found 564.2976.
(3R,4S)-3-Acetoxy-1-[(7-benzyloxymethyl-9-deazahypo-
xanthin-9-yl)methyl]-4-methylthiomethylpyrrol-idine (5).
Acetic anhydride (1 mL, excess) was added dropwise to a
solution of compound 4 (1.1 g, 2.3 mmol), DMAP (30 mg, cat.),
and Et3N (2 mL, excess) in CH2Cl2 (20 mL) at room temp..
After 15 min, the reaction mixture was diluted with CH2Cl2,
washed with satd NaHCO3, water, and brine, dried (MgSO4),
and concentrated in vacuo. The resulting residue was purified
by chromatography (5% MeOH in CH2Cl2) to afford a syrupy
product (1.35 g), which was not further characterized. TFA (5
mL) was added dropwise to a solution of the syrup in CH2Cl2
(20 mL) at room temp and the mixture was then concentrated
in vacuo. The resulting residue was dissolved in CH2Cl2 and
washed with satd NaHCO3 and water, dried (MgSO4), and
concentrated in vacuo to afford 5 (800 mg, 76% for two steps)
as a foam. 1H NMR (CDCl3) δ 7.80 (s, 1H), 7.65 (s, 1H), 7.25-
7.20 (m, 5H), 5.82 (s, 2H), 5.11 (brs, 1H), 4.56 (s, 2H), 4.47 (s,
2H), 3.80-3.59 (m, 3H), 3.32 (brs, 1H), 2.80-2.69 (m, 2H), 2.57
(dd, J ) 13.0, 8.3 Hz, 1H), 2.07 (s, 3H), 2.05 (s, 3H).13C NMR
(CDCl3) ?δ 170.7, 155.4, 145.8, 143.3, 137.2, 134.2, 128.8, 128.3,
128.1, 118.1, 106.9, 77.4, 75.7, 71.2, 57.0, 55.8, 48.1, 43.5, 35.0,
21.0, 16.1. HRMS (MH+) calcd for C23H29N4O4S: 457.1910.
Found 457.1915.
Conclusions
A series of second generation transition state ana-
logues of human 5′-methylthioadenosine phosphorylase
have been designed and synthesized based on its dis-
sociative transition state structure. Many of these
compounds are potent inhibitors and display slow-onset
tight binding characteristics that are consistent with
action as transition state analogues.
Experimental Section
Aluminum-backed silica gel sheets (Merck or Riedel de
Haen) were used for TLC. Column chromatography was
performed on silica gel (230-400 mesh, Merck). Chromatog-
(3R,4S)-1-[(7-Benzyloxymethyl-9-deazaadenin-9-yl)-
methyl]-3-hydroxy-4-methylthiomethylpyrrolidine (6).

4684 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 14
Evans et al.
Compound 5 (800 mg, 1.75 mmol) was dissolved in POCl3 and
heated under reflux for 1 h. The resulting solution was
concentrated in vacuo and evaporated with toluene (×2) to
afford a solid residue. Without further purification, the residue
was dissolved in 7 N NH3 in MeOH (15 mL) and heated in a
sealed tube at 110 °C overnight. The reaction was concentrated
in vacuo, and the resulting residue purified by chromatography
(15% MeOH in CH2Cl2) to afford 6 (500 mg, 69%) as a syrup.
1H NMR (d4-MeOH) δ 8.24 (s, 1H), 7.85 (s, 1H), 7.29-7.26 (m,
5H), 5.74 (s, 2H), 4.63 (s, 2H), 4.43 (s, 2H), 4.26-4.22 (m, 1H),
3.70 (dd, J ) 11.4, 6.9 Hz, 1H), 3.49 (dd, J ) 12.1, 5.6 Hz,
1H), 3.29 (dd, J ) 12.3, 3.3 Hz, 1H), 3.16 (dd, J ) 11.4, 6.1
Hz, 1H), 2.76-2.67 (m, 1H), 2.50-2.44 (m, 2H), 2.07 (s, 3H).
13C NMR (CDCl3) δ 153.4, 153.0, 149.9, 138.1, 137.0, 130.0,
129.7, 129.3, 116.5, 106.5, 79.5, 75.0, 72.3, 60.9, 57.8, 50.1, 47.6,
36.6, 16.0. HRMS (MH+) calcd for C21H28N5O2S: 414.1964.
Found 414.1970.
(10). Acetic anhydride (1 mL, excess) was added dropwise to
a solution of 9 (1.16 g, 2.12 mmol), DMAP (30 mg, cat.), and
Et3N (2 mL, excess) in CH2Cl2 (20 mL) at room temp. After 15
min, the reaction mixture was diluted with CH2Cl2, washed
with satd aq NaHCO3, water, brine, dried (MgSO4), and
concentrated in vacuo. The resulting residue was purified by
chromatography (ethyl acetate) to afford a syrup (1.35 g). TFA
(5 mL) was added dropwise to a solution of the syrup in CH2-
Cl2 (20 mL) at room temp and concentrated in vacuo. The
resulting residue was redissolved in CH2Cl2 and washed with
satd aq NaHCO3 and water, dried (MgSO4), and concentrated
in vacuo to afford 9 (900 mg, 80% for two steps) as a foam. 1H
NMR (CDCl3) δ 7.90 (s, 1H), 7.33 (s, 1H), 7.28-7.17 (m, 10H),
5.91 (s, 2H), 4.85 (brs, 1H), 4.58 (s, 2H), 3.86-3.74 (m, 2H),
3.68 (s, 2H), 3.16-3.11 (m, 1H), 2.84-2.80 (m, 2H), 2.71 (dd,
J ) 11.4, 4.6 Hz, 1H), 2.50-2.36 (m, 2H), 2.27-2.21 (m, 1H),
2.00 (s, 3H). 13C NMR (CDCl3) δ 171.3, 156.2, 145.8, 141.9,
138.6, 137.5, 131.4, 129.2, 128.8, 128.3, 128.2, 127.4, 117.9,
115.2, 78.9, 77.0, 70.9, 59.8, 58.7, 48.3, 45.1, 36.9, 34.4, 21.5.
HRMS (MH+) calcd for C29H33N4O4S: 533.2223. Found
533.2236.
(3R,4S)-1-[(7-Benzyloxymethyl-9-deazaadenin-9-yl)-
methyl]-4-benzylthiomethyl-3-hydroxypyrrolidine (11).
Compound 10 (900 mg, 1.7 mmol) was dissolved in POCl3 (15
mL) and heated at reflux for 1 h. The resulting solution was
concentrated in vacuo and evaporated with toluene (×2) to
afford a solid residue. Without further purification the residue
was dissolved in 7 N NH3 in MeOH (15 mL) and heated in a
sealed tube at 130 °C overnight. The reaction mixture was
cooled to room temp and concentrated in vacuo and the
resulting residue purified by chromatography (10% MeOH in
CH2Cl2) to afford 11 (720 mg, 87% yield for two steps) as a
syrup. 1H NMR (CDCl3) δ 8.32 (s, 1H), 7.67 (s, 1H), 7.35-7.25
(m, 10H), 5.52 (s, 2H), 4.56 (s, 2H), 4.23 (s, 2H), 3.68 (s, 2H),
3.54-3.48 (m, 1H), 3.22 (d, J ) 3.4 Hz, 2H), 2.83 (brs, 1H),
2.63-2.45 (m, 4H). 13C NMR (CDCl3) δ 152.2, 151.6, 149.5,
138.2, 135.7, 134.5, 129.2, 129.1, 128.9, 128.8, 128.2, 127.5,
115.2, 107.4, 77.6, 74.9, 70.7, 60.2, 57.3, 48.5, 46.9, 46.3, 36.7,
33.5, 23.1.
(3R,4S)-4-Benzylthiomethyl-1-[(9-deazaadenin-9-yl)-
methyl]-3-hydroxypyrrolidine (12). Compound 11 (330 mg,
0.7 mmol) was dissolved in a solution of MeOH (4 mL) and
cHCl (4 mL) and heated under reflux for 90 min.. The reaction
mixture was cooled to room temp, diluted with water (50 mL),
and washed with CHCl3 (×2) and the aqueous layer concen-
trated in vacuo, followed by codistillation with water (×2). The
resulting residue was dissolved in aq cNH4OH and concen-
trated in vacuo and the residue purified by chromatography
to afford 12 (30 mg, 12%) as a solid. Mp 126-128 °C. 1H NMR
(d4-MeOH) δ 8.17 (s, 1H), 7.46 (s, 1H), 7.26-7.16 (m, 5H),
3.93-3.90 (m, 1H), 3.83-3.74 (m, 2H), 3.68 (s, 2H), 3.03-2.97
(m, 1H), 2.80 (dd, J ) 10.2, 6.4 Hz, 1H), 2.66-2.58 (m, 2H),
2.38 (dd, J ) 12.5, 8.9 Hz, 1H), 2.30 (dd, J ) 9.5, 7.2 Hz, 1H),
2.20-2.14 (m, 1H). 13C NMR (d4-MeOH) δ 152.5, 151.4, 147.4,
140.4, 130.4, 130.4, 129.8, 128.3, 115.5, 112.9, 77.3, 62.7, 59.2,
49.3, 48.6, 37.5, 35.6. HRMS (MH+) calcd for C19H24N5OS:
370.1702. Found 370.1694. Anal. (C19H23N5OS.2HCl‚H2O) C,
H, N, Cl, S.
(3R,4S)-1-[(8-Aza-9-deazaadenin-9-yl)methyl]-4-ben-
zylthiomethyl-3-hydroxypyrrolidine Hydrochloride (16).
Sodium cyanoborohydride (20 mg, 0.32 mmol) was added to a
stirred solution of 13 (180 mg, 0.52 mmol) and (3R,4S)-4-
benzylthiomethyl-3-hydroxypyrrolidine hydrochloride (8) (95
mg, 0.37 mmol) in methanol (5 mL), and stirring was continued
overnight at room temp. The crude reaction mixture was
absorbed onto silica and purified by chromatography (12:4:
0.5 CH2Cl2:MeOH:NH4OH v/v/v) to afford, presumably, (3R,4S)-
1-[(8-aza-9-deazaadenin-9-yl)methyl]-4-benzylthiomethyl-3-
hydroxypyrrolidine hydrochloride (14) as a foam (80 mg, 46%),
which was committed to the next step without further char-
acterization. Compound 14 was dissolved in 7 N NH3 in MeOH
(15 mL) and heated in a sealed tube at 110 °C overnight. The
resulting solution was cooled to room temp and concentrated
in vacuo and the resulting residue purified by chromatography
(3R,4S)-1-[(9-Deazaadenin-9-yl)methyl]-3-hydroxy-4-
methylthiomethylpyrrolidine (7). Compound 6 (150 mg,
0.37 mmol) was dissolved cHCl (5 mL) and the resulting
solution heated under reflux for 90 min.. The reaction was
cooled to room temp, diluted with water (50 mL), and washed
with CHCl3 (×2), and the aqueous layer was concentrated in
vacuo, followed by codistillation with water (×2). The resulting
residue was redissolved in aq cNH4OH and concentrated in
vacuo and the residue purified by chromatography (12:4:0.5
CH2Cl2:MeOH:NH4OH v/v/v) to afford 7 (69 mg, 65%) as a
1
solid. Mp 108-110 °C. H NMR (D2O) δ 7.96 (s, 1H), 7.31 (s,
1H), 4.00-3.95 (m, 1H), 3.74 (s, 2H), 3.05 (dd, J ) 10.5, 7.9
Hz, 1H), 2.88 (dd, J ) 11.1, 6.2 Hz, 1H), 2.71 (dd, J ) 11.1,
4.0 Hz, 1H), 2.49 (dd, J ) 13.0, 6.7 Hz, 1H), 2.40-2.24 (m,
2H), 2.16-2.13 (m, 1H), 1.93 (s, 3H). 13C NMR (D2O) δ 150.5,
150.1, 145.4, 130.4, 113.6, 108.33, 75.0, 59.8, 56.6, 47.4, 45.9,
35.9, 14.8. HRMS (MH+) calcd for C13H20N5OS: 294.1389.
Found 294.1394. Anal. (C13H19N5OS‚4/3H2O) C, H, N, S.
(3R,4S)-4-Benzylthiomethyl-3-hydroxypyrrolidine Hy-
drochloride (8). (3R,4R)-1-tert-Butoxycarbonyl-3-hydroxy-4-
mesyloxymethylpyrrolidine (1.10 g, 3.7 mmol) was dissolved
in DMF (2 mL) and added dropwise to a solution of benzyl
mercaptan (870 µL, 7.4 mmol) and NaH (270 mg, 60% oil
dispersion, 6.8 mmol) in DMF (10 mL) and stirred at room
temp for 1 h. The reaction mixture was diluted with toluene,
washed with water then brine, dried (MgSO4), and concen-
trated in vacuo. The resulting residue was purified by chro-
matography (30% ethyl acetate in petroleum ether) to afford
(3R,4S)-4-benzylthiomethyl-1-tert-butoxycarbonyl-3-hydroxy-
pyrrolidine as a syrup. Without further characterization, the
syrup was dissolved in MeOH (5.0 mL) and cHCl (1.0 mL) and
concentrated in vacuo to afford 8 (730 mg, 76% overall yield
for two steps). 1H NMR (D2O) δ 7.40-7.27 (m, 5H), 4.26-4.22
(m, 1H), 3.74 (s, 2H), 3.56 (dd, J ) 12.4, 7.2 Hz, 1H), 3.37 (dd,
J ) 12.8, 5.2 Hz, 1H), 3.21(dd, J ) 12.8, 3.0 Hz, 1H), 3.07 (dd,
J ) 12.4, 5.5 Hz, 1H), 2.61-2.52 (m, 1H), 2.47-2.34 (m, 2H).
13C NMR (D2O) δ 138.7, 129.5, 129.3, 127.9, 73.5, 51.5, 48.5,
45.4, 35.9, 31.8. (MH+) calcd for C12H18NOS: 224.1109. Found
224.1102.
(3R,4S)-1-[(7-Benzyloxymethyl-6-tert-butoxy-9-deazapu-
rin-9-yl)methyl]-4-benzylthiomethyl-3-hydroxypyrroli-
dine (9). Sodium cyanoborohydride (200 mg, 3.2 mmol) was
added to a stirred solution of 323 (800 mg, 2.32 mmol) and 8
(570 mg, 2.2 mmol) in methanol (10 mL), and the mixture was
stirred overnight at room temp. The crude reaction mixture
was absorbed onto silica and purified by chromatography (5%
MeOH in CH2Cl2) to afford 9 (1.16 g, 96%) as a syrup. 1H NMR
(CDCl3) δ 8.45 (s, 1H), 7.62 (s, 1H), 7.27-7.22 (m, 10H), 5.75
(s, 2H), 4.51 (s, 2H), 4.15 (s, 2H), 3.67 (s, 2H), 3.38 (dd, J )
10.7, 7.0 Hz, 1H), 3.12-3.02 (m, 2H), 2.69-2.63 (m, 1H), 2.54-
2.49 (m, 1H), 2.44-2.39 (m, 2H), 1.70 (s, 9H). 13C NMR (CDCl3)
δ 156.6, 150.6, 150.0, 138.3, 137.5, 134.4, 129.3, 129.0, 128.8,
128.2, 127.8, 117.1, 109.0, 83.9, 77.9, 75.2, 70.7, 60.5, 57.7, 49.1,
47.0, 36.8, 33.7, 29.0. (MH+) calcd for C31H49N4O3S: 547.2743.
Found 547.2723.
(3R,4S)-3-Acetoxy-1-[(7-benzyloxymethyl-9-deazahy-
poxanthin-9-yl)methyl]-4-benzylthiomethylpyrrolidine

Transition State Analogue Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 14 4685
(5% MeOH in CH2Cl2) to afford, presumably, (3R,4S)-1-[8-aza-
9-deaza-8-(tetrahydropyran-2-yl)-adenin-9-yl]methyl-3-hydroxy-
4-benzylthiomethylpyrrolidine (15) (67 mg, 87%) as a foam.
The product was not characterized further but dissolved in
methanol (2.0 mL) and cHCl (2 mL) and concentrated in vacuo
and the resulting residue triturated with 2-propanol to afford
1.61-1.51 (m, 2H), 1.40-1.33 (m, 2H), 0.86 (t, J ) 7.3 Hz, 3H).
13C NMR (D2O) δ 73.5, 51.5, 48.6, 45.7, 32.2, 31.7, 31.3, 21.7,
13.3. HRMS (MH+) calcd for C9H20NOS: 190.1260. Found
190.1257.
(3R,4S)-4-Cyclohexylthiomethyl-3-hydroxypyrrol-
idine Hydrochloride (21). Following the general procedure
(vide supra), mesylate (565 mg, 1.91 mmol) was processed to
afford the intermediate (3R,4S)-1-tert-butoxycarbonyl-4-cyclo-
hexylthiomethyl-3-hydroxypyrrolidine (428 mg, 71%) as a
syrup.. NMR (CDCl3): δ: 4.17-4.09 (m, 1H), 3.72-3.60 (m,
2H), 3.28-3.18 (m, 1H), 3.15-3.09 (m, 1H), 2.74-2.64 (m, 2H),
2.60-2.53 (m, 1H), 2.32-2.23 (m, 1H), 1.96 (m, 2H), 1.81-
1.77 (m, 2H), 1.66-1.61 (m, 1H), 1.45 (s, 9H), 1.35-1.24 (m,
5H). 13C NMR (CDCl3): δ: (note that some peaks are doubled
due to slow conversion of rotamers) 154.91, 79.94, (75.57,
74.72), (52.80, 52.55), (49.70, 49.43), (46.24, 45.41), 44.26,
33.99, 33.93, 31.90, 28.87, 26.43, 26.12. The intermediate
compound was converted to the title compound 21 as described
in the general method (vide supra).
1
16 (41 mg, 75%) as a white solid. H NMR (d4-MeOH) δ 8.35
(s, 1H), 7.29 (m, 5H), 4.75 (s, 2H), 4.22 (brs, 1H), 3.82 (m, 1H),
3.73 (s, 2H), 3.58 (m, 1H), 3.46 (m, 1H), 3.30 (m, 2H), 2.62
(dd, J ) 12.2, 5.6 Hz, 1H), 2.48-2.31 (m, 2H). 13C NMR (d4-
MeOH) δ 153.7, 152.0, 139.9, 138.8, 135.1, 130.4, 130.0, 128.5,
124.6, 74.6, 61.4, 58.4, 50.4, 47.7, 37.4, 33.4. (MH+) calcd for
C18H23N6OS: 371.1654. Found 371.1670.
Procedures for the Synthesis of (3R,4S)-4-(Alkyl-,
Aralkyl-, and Aryl-thiomethyl)-3-hydroxypyrrolidines.
General Preparative Method. 4-Substituted-4-thiopyrro-
lidines were prepared from compound 1 essentially following
the method detailed for the preparation of 2, except where
modifications are noted. In cases when the sodium thiolate
was not directly available it was preformed by treating a
stirred mixture of NaH (2.85 mmol) in DMF (5 mL) at 0 °C
with the appropriate thiol (2.85 mmol). After stirring the
mixture for 10 min, a solution of the intermediate mesylate
(450 mg, 1.53 mmol) was added as a solution in DMF (5 mL)
and the mixture was stirred at RT until the complete con-
sumption of the mesylate was observed (0.5-4 h) by TLC. The
tert-butoxycarbonyl-protected pyrrolidine intermediate was
purified by chromatography (0-5% MeOH in CH2Cl2), and
then a methanolic solution of the purified tert-butoxycarbonyl-
protected pyrrolidine intermediate, following purification by
chromatography, was treated with cHCl and concentrated in
vacuo to afford the title compounds 17-26 (vide infra).
(3R,4S)-4-Ethylthiomethyl-3-hydroxypyrrolidine Hy-
drochloride (17). Following the general procedure (vide
supra), mesylate (260 mg, 0.88 mmol) was processed to afford
the title compound 17 (100 mg, 58%) as a syrup. 1H NMR (D2O)
δ 4.30-4.24 (m, 1H), 3.53 (dd, J ) 12.3, 7.2 Hz, 1H), 3.37 (dd,
J ) 12.8, 5.2 Hz, 1H), 3.14 (dd, J ) 12.8, 3.1 Hz, 1H), 3.07
(dd, J ) 12.2, 5.7 Hz, 1H), 2.65-2.55 (m, 1H), 2.46 (q, J ) 7.4
Hz, 2H), 2.40-2.30 (m, 2H) 1.09 (t, J ) 7.4 Hz, 3H). 13C NMR
(D2O) δ 73.6, 51.5, 48.6, 45.6, 31.7, 26.0, 14.4. HRMS (MH+)
calcd for C7H16NOS: 162.0947. Found 162.0952.
(3R,4S)-3-Hydroxy-4-phenylthiomethylpyrrolidine Hy-
drochloride (22). Following the general procedure (vide
supra), mesylate (300 mg, 1.00 mmol) was processed to afford
1
22 (692 mg, 0.69 mmol) as a syrup. H NMR (D2O) δ 7.51-
7.24 (m, 5H), 4.38-4.34 (m, 1H), 3.56 (dd, J ) 12.2, 7.7 Hz,
1H), 3.45 (dd, J ) 12.6, 5.2 Hz, 1H), 3.26-3.00 (m, 3H), 2.88
(dd, J ) 13.7, 8.3 Hz, 1H), 2.48-2.37 (m, 1H). 13C NMR (D2O)
δ 134.5, 130.5, 129.9, 127.6, 73.4, 51.5, 48.4, 45.5, 34.3. HRMS
(MH+) calcd for C11H16NOS: 210.0953. Found 210.0948.
(3R,4S)-4-(4-Chlorophenylthiomethyl)-3-hydroxypyr-
rolidine Hydrochloride (23). Following the general proce-
dure (vide supra), mesylate (245 mg, 0.83 mmol) was processed
to afford 23 (212 mg, 0.76 mmol, 91%) as a syrup. 1H NMR
(d4-MeOH) 7.51-7.39 (m, 2H), 7.35-7.31 (m, 2H), 4.38-4.33
(m, 1H), 3.59 (dd, J ) 12.0, 7.6 Hz, 1H), 3.47 (dd, J ) 12.4,
4.9 Hz, 1H), 3.28-3.18 (m, 3H), 2.93 (dd, J ) 13.6, 9.0 Hz,
1H), 2.49-2.38 (m, 1H). 13C NMR (D2O) 135.7, 134.1, 132.9,
130.8, 74.8, 52.9, 49.6, 47.5, 35.8. HRMS (MH+) calcd for
C11H15NOSCl: 244.0557. Found 244.0546.
(3R,4S)-4-(3-Chlorophenylthiomethyl)-3-hydroxypyr-
rolidine Hydrochloride (24). Following the general proce-
dure (vide supra), mesylate (300 mg, 1.02 mmol) was processed
1
to afford 24 (192 mg, 0.685 mmol, 67%) as a syrup. H NMR
(D2O) 7.33-7.15 (m, 4H), 4.39-4.35 (m, 1H), 3.59 (dd, J ) 12.2,
7.7 Hz, 1H), 3.47 (dd, J ) 12.7, 5.1 Hz, 1H), 3.28-3.04 (m,
3H), 2.89 (dd, J ) 13.7, 8.3 Hz, 1H), 2.49-2.41 (m, 1H). 13C
NMR (D2O) 136.9, 134.7, 131.0, 129.3, 128.1, 127.2, 73.4, 51.5,
48.4, 45.3, 34.0. HRMS (MH+) calcd for C11H15NOSCl: 244.0557.
Found 244.0556.
(3R,4S)-4-(4-Fluorophenylthiomethyl)-3-hydroxypyr-
rolidine Hydrochloride (25). Following the general proce-
dure (vide supra), mesylate (281 mg, 0.951 mmol) was pro-
cessed to afford 25 (160 mg, 0.607 mmol, 64%) as a syrup. 1H
NMR (D2O) 7.49-7.42 (m, 2H), 7.19-7.06 (m, 2H), 4.41-4.36
(m, 1H), 3.60 (dd, J ) 12.3, 7.7 Hz, 1H), 3.48 (dd, J ) 12.8,
5.2 Hz, 1H), 3.27-3.16 (m, 2H), 3.07 (dd, J ) 13.8, 6.9 Hz,
1H), 2.88 (dd, J ) 13.8, 8.3 Hz, 1H), 2.45-2.38 (m, 1H). 13C
NMR (D2O) 164.2, 160.9, 133.7, 133.6, 129.4, 116.9, 116.6, 73.3,
51.5, 48.4, 45.5, 35.5. HRMS (MH+) calcd for C11H15NOSF:
228.0853. Found 228.0856.
(3R,4S)-3-Hydroxy-4-(4-pyridylthiomethyl)pyrrol-
idine Hydrochloride (26). Following the general procedure
(vide supra), mesylate (348 mg, 1.18 mmol) was processed to
afford 26 (105 mg, 0.426 mmol, 36%) as a syrup. 1H NMR (D2O)
8.42 (d, J ) 7.2 Hz, 2H), 7.82 (d, J ) 7.2 Hz, 2H), 4.51-4.46
(m, 1H), 3.74 (dd, J ) 12.4, 7.8 Hz, 1H), 3.57 (dd, J ) 12.8,
5.5 Hz, 1H), 3.44 (dd, J ) 13.6, 7.3 Hz, 1H) 3.34-3.22 (m, 3H),
2.78-2.60 (m, 1H). 13C NMR (D2O) 164.1, 139.4, 122.9, 73.4,
51.4, 48.4, 44.3, 31.3. HRMS (MH+) calcd for C10H15N2OS:
211.0900. Found 211.0908.
(3R,4R)-1-tert-Butoxycarbonyl-4-[(tert-butyldiphenyl-
silyloxy)methyl]-3-hydroxypyrrolidine (45). tert-Butyl-
diphenylsilyl chloride (0.740 mL, 2.85 mmol) was added
dropwise to a stirred solution of the diol 1 (560 mg, 2.58 mmol)
and imidazole (270 mg, 3.97 mmol) in DMF (12 mL) cooled to
(3R,4S)-3-Hydroxy-4-(1-propylthiomethyl)pyrrol-
idine Hydrochloride (18). Following the general procedure
(vide supra), mesylate (264 mg, 0.89 mmol) was processed to
afford the title compound 18 (139 mg, 0.66 mmol, 73%) as a
1
syrup. H NMR (D2O) 4.41-4.37 (m, 1H), 3.67 (dd, J ) 12.3,
7.2 Hz, 1H), 3.50 (dd, J ) 12.8, 5.2 Hz, 1H), 3.27 (dd, J ) 12.8,
3.1 Hz, 1H), 3.21 (dd, J ) 12.2, 5.6 Hz, 1H), 2.76-2.71 (m,
1H), 2.61-2.50 (m, 4H), 1.59 (sextet, J ) 7.3 Hz), 0.95 (t, J )
7.3 Hz, 3H). 13C NMR (D2O) 73.6, 51.5, 48.6, 45.7, 34.1, 32.1,
22.7, 13.1. HRMS (MH+) calcd for C8H18NOS: 176.1104. Found
176.1106.
(3R,4S)-3-Hydroxy-4-(2-propylthiomethyl)pyrrol-
idine Hydrochloride (19). Following the general procedure
(vide supra), mesylate (565 mg, 1.91 mmol) was processed to
afford the intermediate compound (3R,4S)-1-tert-butoxycar-
bonyl-3-hydroxy-4-(2-propylthiomethyl)pyrrolidine (490 mg,
97%) as a syrup. 1H NMR (CDCl3): δ: 4.18-4.11 (m, 1H),
3.72-3.60 (m, 2H), 3.28-3.18 (m, 1H), 3.13 (dd, J ) 11.1, 6.7
Hz, 1H), 2.95 (sept., J ) 6.7 Hz, 1H), 2.69-2.50 (m, 2H), 2.33-
2.22 (m, 1H), 1.46 (s, 9H), 1.29, 1.27 (s, 3H each). 13C NMR
(CDCl3): δ: (note that some peaks are doubled due to slow
conversion of rotamers) 154.93, 79.97, (75.48, 74.64), (52.81,
52.55), (49.69, 49.42), (46.13, 45.35), 35.71, 32.32, 28.87, 23.73,
23.69. The intermediate compound was converted to the title
compound 19 as described in the general method (vide supra).
(3R,4S)-4-Butylthiomethyl-3-hydroxypyrrolidine Hy-
drochloride (20). Following the general procedure (vide
supra), mesylate (438 mg, 1.48 mmol) was processed to afford
20 (284 mg, 1.25 mmol, 84%) as a syrup. 1H NMR (D2O) δ
4.40-4.36 (m, 1H), 3.66 (dd, J ) 12.3, 7.2 Hz, 1H), 3.48 (dd, J
) 12.8, 5.2 Hz, 1H), 3.26 (dd, J ) 12.8, 3.1 Hz, 1H), 3.21 (dd,
J ) 12.2, 5.6 Hz, 1H), 2.76-2.70 (m, 1H), 2.62-2.50 (m, 4H),

4686 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 14
Evans et al.
0 °C. After stirring for 2 h, the reaction mixture was diluted
with toluene (100 mL) and washed with water (25 mL) and
brine (25 mL), and the organic layer was dried (MgSO4) and
concentrated in vacuo to yield the crude product. Purification
by chromatography (20-50% ethyl acetate in petroleum ether)
afforded 45 (837 mg, 1.84 mmol, 71%) as an oil. 1H NMR
(CDCl3) 7.70-7.62 (m, 4H), 7.48-7.38 (m, 6H), 4.30-4.20 (m,
1H), 3.75 - 3.45 (m, 4H), 3.30-3.05 (m, 2H), 2.32-2.25 (m,
2H), 1.44 (s, 9H), 1.06 (s, 9H). 13C NMR (CDCl3) 154.9, 135.9,
133.4, 130.3, 128.2, 79.8, 73.9, 73.0, 64.7, 53.1, 52.8, 48.7, 48.0,
46.8, 46.4, 28.9, 27.3, 19.6. HRMS (MH+) calcd for C26H38NO4-
Si: 456.2565. Found 456.2531.
to afford 49 (108 mg, 0.31 mmol, 88%) as an oil. 1H NMR
(CDCl3) 7.40-7.20 (m, 5H) 4.64 (s, 2H), 4.51 (s, 2H), 4.15-
4.05 (m, 1H) 3.61-3.15 (m, 6H), 3.34 (s, 3H), 2.58-2.46 (m,
1H), 1.45 (s, 9H). 13C NMR (CDCl3) 155.0, 138.4, 128.8, 128.1,
128.1, 128.0, 96.1, 79.7, 77.9, 76.9, 73.6, 69.9, 55.9, 51.5, 50.9,
47.4, 46.9, 45.2, 44.3, 28.9. HRMS (MH+) calcd for C19H30-
NO5: 352.2119. Found 352.2132.
(3R,4R)-4-Benzyloxymethyl-3-hydroxypyrrolidine Hy-
drochloride (28). HCl (4 M in dioxane, 2.5 mL) was added
to a stirred solution of 49 (107 mg, 0.304 mmol) in MeOH. After
14 h at room temp, the solvent was removed in vacuo to yield
the crude product which was purified by chromatography (20-
35% 7 N NH3/MeOH in CH2Cl2) to afford 28 (51 mg, 0.25 mmol,
(3R,4R)-1-tert-Butoxycarbonyl-4-[(tert-butyldiphenyl-
silyloxy)methyl]-3-methoxymethoxypyrrolidine (46). Bro-
momethyl methyl ether (0.420 mL, 5.2 mmol) was added
dropwise to a stirred solution of 45 (787 mg, 1.73 mmol) and
diisopropylethylamine (1.50 mL, 8.47 mmol) in toluene (50
mL). The reaction mixture was heated to 70 °C for 3 h. After
cooling to room temp, the reaction mixture was subjected to
standard aqueous workup to yield the crude product which
was purified by chromatography (20-30% ethyl acetate in
petroleum ether) to afford 46 (841 mg, 1.68 mmol, 97%) as an
1
82%) as a hygroscopic glass. H NMR (D2O, free base) 7.32-
7.15 (m, 5H), 4.36 (s, 2H), 3.92-3.85 (m, 1H), 3.35 (dd, J )
9.8, 7.0 Hz, 1H), 3.24 (dd, J ) 9.8, 7.8 Hz, 1H), 2.97 (dd, J )
11.8, 7.9 Hz, 1H), 2.75 (dd, J ) 12.4, 5.5 Hz, 1H), 2.57 (dd, J
) 12.4, 3.4 Hz, 1H), 2.36 (dd, J ) 11.8, 5.7 Hz, 1H), 2.15-2.05
(m, 1H). 13C NMR (D2O) 137.6, 129.0, 128.8, 128.6, 74.7, 73.1,
71.0, 53.2, 48.0, 47.6. HRMS (MH+) calcd for C12H18NO2:
208.1332. Found 208.1329.
(3R,4S)-3-Hydroxy-4-(1-propyl)pyrrolidine Hydrochlo-
ride (29). To a suspension of ethyltriphenylphosphonium
bromide (2.9 g, 6.93 mmol) in dry THF (15 mL) under argon
at 0 °C was added n-BuLi in THF (1.6 M, 4.0 mL, 6.40 mmol)
and the deep red solution left stirring without cooling for 10
min. After re-cooling to 0 °C, the aldehyde 50 (580 mg, 2.7
mmol) in THF (10 mL) was added and the mixture stirred at
room temp for 12 h. The reaction was then quenched with
water (1 mL) and extracted with dichloromethane (100 mL).
The organic phase was washed with saturated aqueous
NaHCO3 (15 mL) then water (15 mL), dried (MgSO4), and
concentrated in vacuo. The residue was subjected to chroma-
tography (30% ethyl acetate in petroleum ether) to afford,
presumably, (3R,4S)-N-tert-butoxycarbonyl-3-hydroxy-4-pro-
penyl-pyrrolidine (51) (165 mg, 27%) as a syrup. Without
further characterization, the alkene (165 mg, 0.73 mmol) was
dissolved in ethanol (10 mL), 10% Pd/C (60 mg) was added,
and the suspension was stirred under an atmosphere of
hydrogen for 3 h. After filtration, the solvent was removed in
vacuo to afford, presumably, (3R,4S)-N-tert-butoxycarbonyl-
3-hydroxy-4-(1-propyl)pyrrolidine as a syrup (172 mg, 100%),
which was committed to the next step without further puri-
fication. To a solution of the intermediate tert-butoxycarbonyl-
protected amine (172 mg, 0.75 mmol) in methanol (10 mL) was
added cHCl (4 mL) and the solution stirred at 40 °C for 30
min. After removal of the solvent in vacuo and azeotroping
with toluene, the title compound 29 was obtained as a syrup
1
oil. H NMR (CDCl3) 7.70-7.62 (m, 4H), 7.48-7.38 (m, 6H),
4.61 (s, 2H), 4.20-4.15 (m, 1H) 3.60-3.20 (m, 6H), 3.30 (s,
3H), 2.43-2.38 (m, 1H), 1.45 (s, 9H), 1.06 (s, 9H). 13C NMR
(CDCl3) 154.9, 135.9, 133.7, 130.2, 128.5, 128.1, 96.0, 95.9, 79.6,
76.7, 63.5, 55.8, 51.5, 50.9, 47.3, 46.8, 46.6, 46.4, 28.9, 27.2,
19.6, 14.6. HRMS (MH+) calcd for C28H42NO5Si: 500.2827.
Found 500.2821.
(3R,4R)-1-tert-Butoxycarbonyl-4-hydroxymethyl-3-
methoxymethoxypyrrolidine (47). Tetrabutylammonium
fluoride (1 M in THF, 1.90 mL, 1.90 mmol) was added dropwise
to a stirred solution of 46 (779 mg, 1.56 mmol) in THF (20
mL). After 2 h at room temp, the solvent was removed in vacuo
to yield a residue which was purified by chromatography (70%
ethyl acetate in petroleum ether) to afford 47 (264 mg, 1.01
mmol, 65%) as a thin film. 1H NMR (CDCl3) 4.66 (s, 2H), 4.15-
4.05 (m, 1H) 3.60-3.48 (m, 4H), 3.37 (s, 3H), 3.37-3.10 (m,
2H), 2.78-2.60 (m, 1H), 2.45-2.35 (m, 1H), 1.45 (s, 9H). 13C
NMR (CDCl3) 155.0, 96.2, 79.9, 78.1, 77.2, 62.6, 55.9, 51.5, 50.9,
47.1, 46.9, 46.5, 46.3, 28.8. HRMS (MH+) calcd for C12H24-
NO5: 262.1654. Found 262.1796.
(3R,4R)-1-tert-Butoxycarbonyl-3-methoxymethoxy-4-
methoxymethylpyrrolidine (48). Sodium hydride (60%
dispersion in mineral oil, 42 mg, 1.05 mmol) was added to a
stirred solution of 47 (173 mg, 0.66 mmol) and iodomethane
(0.100 mL, 1.59 mmol) in DMF (10 mL) cooled to 0 °C. After
1 h at room temp, the reaction mixture was subjected to a
standard aqueous workup and purified by chromatography
(20-35% ethyl acetate in petroleum ether) to afford 48 (147
mg, 0.534 mmol, 81%) as an oil. 1H NMR (CDCl3) 4.65 (s, 2H),
4.12-4.02 (m, 1H), 3.63-3.52 (m, 2H), 3.37 (s, 3H), 3.34 (s,
3H), 3.37-3.10 (m, 4H), 2.52-2.41 (m, 1H), 1.45 (s, 9H). 13C
NMR (CDCl3) 154.9, 96.1, 79.7, 77.9, 72.7, 59.3, 55.9, 51.4, 50.9,
47.4, 46.9, 45.1, 44.2, 28.9. HRMS (MH+) calcd for C13H26-
NO5: 276.1806. Found 276.1813.
(3R,4R)-3-Hydroxy-4-methoxymethylpyrrolidine Hy-
drochloride (27). cHCl (1 mL) was added to a stirred solution
of 48 (152 mg, 0.552 mmol) in MeOH (3 mL). After 4 h at room
temp, the solvent was removed in vacuo to yield the crude
product which was azetroped with D2O (×2) to afford 27 (87
mg, 0.552 mmol, 100%) as a hygroscopic glass. 1H NMR (D2O)
4.30-4.26 (m, 1H), 3.52-3.28 (m, 4H), 3.22 (s, 3H), 3.15-3.00
(m, 2H), 2.48-2.37 (m, 1H). 13C NMR (D2O) 72.1, 71.6, 58.8,
52.0, 46.7, 45.7. HRMS (MH+) calcd for C6H14NO2: 132.1019.
Found 132.1012.
(3R,4R)-4-Benzyloxymethyl-1-tert-butoxycarbony-3-
methoxymethoxypyrrolidine (49). Sodium hydride (60%
dispersion in mineral oil, 28 mg, 0.70 mmol) was added to a
stirred solution of 47 (91 mg, 0.35 mmol) and benzyl bromide
(0.10 mL, 0.84 mmol) in DMF (5 mL) cooled to 0 °C. After 1 h
at room temp, the reaction mixture was subjected to a standard
aqueous workup to yield the crude product which was purified
by chromatography (20-40% ethyl acetate in petroleum ether)
1
(138 mg, 100%). H NMR (d4-MeOH): δ: 4.20-4.16 (m, 1H),
3.59-3.52 (m, 1H), 3.44-3.39 (m, 1H), 3.19-3.14 (m, 1H),
3.05-2.99 (m, 1H), 2.23-2.17 (m, 1H), 1.55-1.28 (m, 4H),
0.98-0.94 (m, 3H). 13C NMR (d4-MeOH): δ: 75.65, 52.82,
50.30, 47.45, 34.69, 22.30, 14.78.
(3R,4S)-3-Hydroxy-4-phenylethylpyrrolidine Hydro-
chloride (30). To a suspension of benzyltriphenylphospho-
nium bromide (1.75 g, 4.97 mmol) in dry THF (10 mL) under
argon at 0 °C was added n-BuLi in THF (1.6 M, 2.33 mL, 3.73
mmol), and the deep red solution was stirred without cooling
for 10 min. After recooling to 0 °C, the aldehyde 50 (335 mg,
1.56 mmol) in THF (5 mL) was added and the mixture stirred
at room temp for 12 h. The reaction was then quenched with
water (1 mL) and extracted with dichloromethane (100 mL).
The organic phase was washed with saturated aqueous
NaHCO3 (15 mL) and water (15 mL), dried (MgSO4), and
concentrated in vacuo. The residue was subjected to chroma-
tography (30% ethyl acetate in petroleum ether) to afford,
presumably, compound 52 as a 1:3 E/Z mixture of stereoiso-
mers (290 mg, 64%), which was committed to the next step
without further characterization. To 52 (290 mg, 1.00 mmol)
in ethanol (20 mL) was added 10% Pd/C (250 mg), and the
suspension was stirred under an atmosphere of hydrogen for
12 h. After filtration, the solvent was removed in vacuo to
afford, presumably, (3R,4S)-N-tert-butoxycarbonyl-3-hydroxy-
4-(2-phenylethyl)pyrrolidine (254 mg 87%), which was com-

Transition State Analogue Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 14 4687
mitted to the next step without further characterization. To a
solution of the tert-butoxycarbonyl-protected amine (254 mg,
0.87 mmol) in methanol (10 mL) was added cHCl (4 mL) and
the solution stirred at 40 °C for 30 min. After removal of the
solvent in vacuo and azeotroping with toluene, the title
compound 30 was obtained as a solid (202 mg, quantitative).
1H NMR (d4-MeOH): δ: 7.14 (m, 5H), 4.22 (m, 1H), 3.52 (dd,
J ) 11.8, 7.4 Hz, 1H), 3.39 (dd, J ) 12.3, 4.9 Hz, 1H), 3.14
(dd, J ) 12.3, 2.8 Hz, 1H), 3.02 (dd, J ) 11.8 Hz, 1H), 2.71 (m,
2H), 2.20 (m, 1H), 1.84 (m, 1H), 1.62 (m, 1H). 13C NMR (d4-
MeOH): δ: 142.94, 129.93, 129.89, 127.56, 75.56, 52.90, 48.55,
47.28, 35.18, 34.44.
General Procedure for the Preparation of Compounds
(31-44) Using the Mannich Reaction. Pyrrolidine hydro-
chloride (17-30) (1.0 mol equiv) and sodium acetate (1.0 mol
equiv) were dissolved in water and 1,4-dioxane (4:1 v/v, 2 mL
per mmol), and to the solution was added aqueous formalde-
hyde (1.0-1.5 mol equiv) and deazaadenine (0.8-1.5 mol
equiv). The reaction was stirred at 95 °C for 1 h and then
cooled to room temp. Silica gel (1.0 g per mmol of pyrrolidine)
was added, and the mixture was evaporated to dryness.
Purification by chromatography on silica gel, using gradient
elution with CH2Cl2:MeOH:NH4OH (95:5:1f80:20:1 v/v/v) as
the eluent, afforded the compounds 31-44 as the free base or
partial acetic acid salt, which was converted to the HCl salt
by addition and evaporation of excess concentrated HCl, unless
stated otherwise.25
(3R,4S)-1-[(9-Deaza-adenin-9-yl)methyl]-4-ethylthio-
methyl-3-hydroxypyrrolidine (31). Following the general
procedure for the Mannich reaction (vide supra), pyrrolidine
hydrochloride 17 (99 mg, 0.50 mmol) was processed to afford
31 (92 mg, 60%) as the syrupy free base. 1H NMR (d4-MeOH)
8.16 (s, 1H), 7.52 (s, 1H), 4.00-3.82 (m, 3H), 3.12 (dd, J ) 9.9,
7.9 Hz, 1H), 2.92 (dd, J ) 10.5, 6.3 Hz, 1H), 2.76-2.68 (m,
2H), 2.55-2.41 (m, 4H), 2.25-2.15 (m, 1H), 1.21 (t, J ) 7.4
Hz, 3H). 13C NMR (d4-MeOH) δ 152.5, 151.5, 147.3, 130.7,
115.6, 112.1, 77.0, 62.4, 59.1, 49.4, 48.8, 35.5, 27.2, 15.5. HRMS
(MH+) calcd for C14H21N5OS: 308.1540. Found 308.1535. Anal.
(C14H21N5OS‚1/2H2O) C, H, N, S.
4H), 0.90 (t, J ) 7.3 Hz, 3H). 13C NMR (d4-MeOH) δ 152.5,
151.4, 147.4, 130.5, 115.6, 112.6, 77.1, 62.6, 59.2, 49.4, 49.0,
36.1, 33.3, 33.2, 23.3, 14.4. HRMS (MH+) calcd for C16H25N5-
OS: 336.1853. Found 336.1850. Anal. (C16H25N5OS) C, H, N,
S.
(3R,4S)-4-Cyclohexylthiomethyl-1-[(9-deaza-adenin-9-
yl)methyl]-3-hydroxypyrrolidine (35). Following the gen-
eral procedure for the Mannich reaction (vide supra), pyrro-
lidine hydrochloride 21 (192 mg, 0.685 mmol) was processed
to afford 35 (144 mg, 34%) as the hydrochloride salt. 1H NMR
(MeOH-d4): δ: 8.15 (s, 1H), 7.50 (s, 1H), 3.97-3.92 (m, 1H),
3.82 (dd, J ) 19.1, 13.4 Hz, 2H), 3.06-3.00 (m, 1H), 2.84 (dd,
J ) 10.3, 6.4 Hz, 1H), 2.75 (dd, J ) 12.5, 5.9 Hz, 1H), 2.67-
2.58 (m, 2H), 2.48 (dd, J ) 12.5, 9.3 Hz, 1H), 2.37 (dd, J )
9.8, 7.2 Hz, 1H), 2.20-2.08 (m, 1H), 1.94-1.92 (m, 2H), 1.74-
1.72 (m, 2H), 1.60-1.58 (m, 1H), 1.36-1.19 (m, 5H). 13C NMR
(MeOH-d4): δ: 152.48, 151.35, 147.32, 130.50, 115.48, 112.74,
77.21, 62.62, 59.18, 49.38, 49.26, 45.10, 35.27, 35.20, 34.18,
27.48, 27.39. HRMS (MH+) calcd for C18H27N5OS: 362.2009.
Found 362.2016. Anal. (C18H27N5OS‚2HCl‚H2O) C, H, N, S, Cl.
(3R,4S)-1-[(9-Deaza-adenin-9-yl)methyl]-3-hydroxy-4-
phenylthiomethylpyrrolidine (36). Following the general
procedure for the Mannich reaction (vide supra), pyrrolidine
hydrochloride 22 (210 mg, 0.86 mmol) was processed to afford
36 (175 mg, 57%) as the hydrochloride salt. 1H NMR (d4-
MeOH) δ 8.22 (s, 1H), 7.74 (s, 1H), 7.33-7.15 (m, 2H), 4.43 (s,
2H), 4.26 (m, 1H), 3.62 (dd, J ) 11.7, 7.9 Hz, 2H), 3.48 (dd, J
) 12.0, 5.6 Hz, 1H), 3.25 (t, dd, J ) 12.0, 3.3 Hz, 1H), 3.15 (m,
2H), 2.85 (dd, J ) 13.5, 9.1 Hz, 1H), 2.43 (m, 1H). 13C NMR
(d4-MeOH) δ 152.9, 152.1, 146.8, 136.8, 132.7, 131.4, 130.6,
128.1, 115.8, 106.3, 74.9, 60.6, 57.4, 49.7, 47.7, 36.3. HRMS
(MH+) calcd for C18H21N5OS: 356.1545. Found 356.1542. Anal.
(C18H21N5OS‚3HCl‚2H2O) C, H, N, S, Cl.
(3R,4S)-4-(4-Chlorophenylthiomethyl)-1-[(9-deaza-ad-
enin-9-yl)methyl]-3-hydroxypyrrolidine (37). Following
the general procedure for the Mannich reaction (vide supra),
pyrrolidine hydrochloride 23 (180 mg, 0.64 mmol) was pro-
cessed to afford 37 (180 mg, 72%) as the hydrochloride salt.
1H NMR (d4-MeOH) δ 8.25 (s, 1H), 7.84 (s, 1H), 7.35-7.23 (m,
5H), 4.54 (s, 2H), 4.30 (m, 1H), 3.74 (dd, J ) 11.9, 7.9 Hz, 1H),
3.59 (dd, J ) 12.2, 5.6 Hz, 1H), 3.40-3.15 (m, 4H), 2.89 (dd, J
) 13.5, 9.1 Hz, 1H), 2.47 (brs, 1H), 1.98 (s, 3H). 13C NMR (d4-
MeOH) δ 153.0, 151.8, 146.1, 135.7, 134.0, 133.2, 132.2, 130.7,
115.7, 105.5, 74.6, 60.4, 57.3, 49.2, 47.7, 36.1, 23.0. HRMS
(MH+) calcd for C18H20ClN5OS: 390.1155. Found 390.1264.
Anal. (C18H20N5ClOS‚2H2O‚2HCl) C, H, N, Cl, S.
(3R,4S)-4-(3-Chlorophenylthiomethyl)-1-[(9-deaza-ad-
enin-9-yl)methyl]-3-hydroxypyrrolidine (38). Following
the general procedure for the Mannich reaction (vide supra),
pyrrolidine hydrochloride 24 (192 mg, 0.69 mmol) was pro-
cessed to afford 38 (139 mg, 0.357 mmol, 52%) as the
hydrochloride salt. 1H NMR (d4-MeOH) δ 8.16 (s, 1H), 7.46 (s,
1H), 7.25-7.05 (m, 4H), 4.01-3.97 (m, 1H), 3.87-3.76 (m, 2H),
3.18 (dd, J ) 12.9, 5.9 Hz, 1H), 2.99 (dd, J ) 9.8, 7.9 Hz, 1H),
2.94-2.86 (m, 2H), 2.64 (dd, J ) 10.2, 4.3 1H), 2.41 (dd, J )
9.9, 7.0 Hz, 1H), 2.26 - 2.15 (m, 1H). 13C NMR (d4-MeOH) δ
152.5, 151.4, 147.4, 140.7, 136.1, 131.6, 130.4, 129.8, 128.6,
127.4, 115.5, 112.8, 77.1, 62.6, 58.9, 49.3, 48.7, 37.4. HRMS
(MH+) calcd for C18H20N5OClS: 390.1150. Found 390.1142.
Anal. (C18H20ClN5OS‚2HCl‚1/2H2O) C, H, N, S, Cl.
(3R,4S)-1-[(9-Deaza-adenin-9-yl)methyl]-3-hydroxy-4-
(1-propylthiomethyl)pyrrolidine (32). Following the gen-
eral procedure for the Mannich reaction (vide supra) pyrroli-
dine hydrochloride 18 (70 mg, 0.33 mmol) was processed to
1
afford 32 (62 mg, 58%) as the syrupy free base. H NMR (d4-
MeOH) δ 8.17 (s, 1H), 7.50 (s, 1H), 4.00-3.79 (m, 3H), 3.08
(dd, J ) 9.8, 7.9 Hz, 1H), 2.86 (dd, J ) 10.3, 6.4 Hz, 1H), 2.72-
2.62 (m, 2H), 2.50-2.38 (m, 4H), 2.22-2.12 (m, 1H), 1.55
(sextet, J ) 7.3 Hz, 2H), 0.95 (t, J ) 7.3 Hz, 3H). 13C NMR
(d4-MeOH) δ 152.5, 151.4, 147.4, 130.5, 115.6, 112.7, 77.2, 62.6,
59.2, 49.4, 49.0, 36.1, 35.6, 24.3, 14.1. HRMS (MH+) calcd for
C15H23N5OS: 322.1696. Found 322.1709. Anal. (C15H23N5OS‚
1/2H2O) C, H, N, S.
(3R,4S)-1-[(9-Deaza-adenin-9-yl)methyl]-3-hydroxy-4-
(2-propylthiomethyl)pyrrolidine (33). Following the gen-
eral procedure for the Mannich reaction (vide supra), pyrro-
lidine hydrochloride 19 was processed to afford 33 (183 mg,
1
38%) as the hydrochloride salt. H NMR (MeOH-d4): δ: 8.16
(s, 1H), 7.49 (s, 1H), 3.99-3.94 (m, 1H), 3.82 (dd, J ) 18.7,
13.4 Hz, 1H), 3.04 (dd, J ) 9.7, 7.9 Hz, 1H), 2.95-2.82 (m,
2H), 2.75 (dd, J ) 12.5, 6.0 Hz, 1H), 2.66 (dd, J ) 10.3, 4.2
Hz, 1H), 2.50 (dd, J ) 12.5, 9.1 Hz, 1H), 2.38 (dd, J ) 9.7, 7.1
Hz, 1H), 2.21-2.10 (m, 1H), 1.23, 1.22 (2s, 3H each). 13C NMR
(MeOH-d4): δ: 152.48, 151.38, 147.40, 130.45, 115.54, 112.90,
77.29, 62.66, 59.26, 49.32, 49.09, 36.49, 34.66, 24.19. HRMS
(MH+) calcd for C15H23N5OS: 322.1696:. Found 322.1701. Anal.
(C15H23N5OS‚2HCl‚2H2O) C, H, N, Cl, S.
(3R,4S)-1-[(9-Deaza-adenin-9-yl)methyl]-4-(4-fluorophen-
ylthiomethyl)-3-hydroxypyrrolidine (39). Following the
general procedure for the Mannich reaction (vide supra),
pyrrolidine hydrochloride 25 (149 mg, 0.57 mmol) was pro-
cessed to afford 39 (55 mg, 0.15 mmol, 27%) as the hydrochlo-
ride salt. 1H NMR (d4-MeOH) δ 8.16 (s, 1H), 7.46 (s, 1H), 7.40-
7.30 (m, 2H), 7.00-6.90 (m, 2H), 4.02-3.97 (m, 1H), 3.86-
3.75 (m, 2H), 3.11 (dd, J ) 12.9, 5.9 Hz, 1H), 3.00 (t, J ) 8.7
Hz, 1H), 2.90-2.75 (m, 2H), 2.65-2.59 (m, 1H), 2.41-2.32 (m,
1H), 2.20-2.10 (m, 1H). 13C NMR (d4-MeOH) δ 165.5, 162.0,
152.5, 151.4, 147.4, 134.1, 134.0, 133.1, 130.4, 117.4, 117.1,
115.5, 112.9, 77.2, 62.7, 59.0, 49.3, 48.8, 39.1. HRMS (MH+)
calcd for C18H20N5OFS: 374.1445. Found 374.1438. Anal.
(C18H20FN5OS‚2HCl‚2H2O) C, H, N, S.
(3R,4S)-4-(1-Butylthiomethyl)-1-[(9-deaza-adenin-9-yl)-
methyl]-3-hydroxypyrrolidine (34). Following the general
procedure for the Mannich reaction (vide supra), pyrrolidine
hydrochloride 20 (125 mg, 0.55 mmol) was processed to afford
34 (97 mg, 52%) as the syrupy free base. 1H NMR (d4-MeOH)
δ 8.16 (s, 1H), 7.50 (s, 1H), 3.99-3.79 (m, 3H), 3.08 (dd, J )
9.7, 7.9 Hz, 1H), 2.87 (dd, J ) 10.3, 6.4 Hz, 1H), 2.75-2.69
(m, 2H), 2.51-2.38 (m, 4H), 2.22-2.12 (m, 1H), 1.55-1.32 (m,

4688 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 14
Evans et al.
(3R,4S)-1-[(9-Deaza-adenin-9-yl)methyl]-3-hydroxy-4-
(4-pyridylthiomethyl)pyrrolidine (40). Following the gen-
eral procedure for the Mannich reaction (vide supra), pyrro-
lidine hydrochloride 26 (81 mg, 0.33 mmol) was processed to
afford 40 (53 mg, 0.15 mmol, 45%) as the hydrochloride salt.
1H NMR (D2O), free base, 8.43 (d, J ) 7.2 Hz, 1H), 8.42 (s,
1H), 8.00 (s, 1H), 7.77 (d, J ) 7.2 Hz, 1H), 4.64 (s, 2H), 4.52-
4.47 (m, 1H), 3.94 (dd, J ) 12.1, 8.0 Hz, 1H), 3.67 (dd, J )
12.6, 5.7 Hz, 1H), 3.50-3.15 (m, 4H), 2.78-2.64 (m, 1H). 13C
NMR (D2O) 163.9, 150.2, 144.6, 139.5, 135.4, 122.8, 113.2,
102.7, 73.0, 59.0, 55.9, 48.1, 44.4, 31.5. HRMS (MH+) calcd
for C17H20N6OS: 357.1492. Found 357.1509. Anal. (C17H20N6-
OS‚3HCl‚2H2O) C, H, N, S, Cl.
(3R,4R)-1-[(9-Deaza-adenin-9-yl)methyl]-3-hydroxy-4-
methoxymethylpyrrolidine (41). Following the general
procedure for the Mannich reaction (vide supra), pyrrolidine
hydrochloride 27 (87 mg, 0.52 mmol) was processed to afford
41 (85 mg, 60%) as the hydrochloride salt. 1H NMR (d4-MeOH)
δ 8.19 (s, 1H), 7.63 (s, 1H), 4.18-4.05 (m, 3H), 3.40-2.28 (m,
3H), 3.30 (s, 3H), 3.10 (dd, J ) 11.0, 5.7 Hz, 1H), 2.95 (dd, J
) 11.0, 3.3 Hz, 1H), 2.77 (dd, J ) 10.8, 6.7 Hz, 1H), 2.41-2.29
(m, 1H). 13C NMR (d4-MeOH) δ 152.7, 151.8, 147.2, 131.6,
115.7, 109.5, 74.3, 74.1, 62.2, 59.6, 56.5, 49.4, 49.0. HRMS
(MH+) calcd for C13H19N5O2: 278.1612. Found 278.1626. Anal.
(C13H19N5O2‚2HCl‚0.5H2O) C, H, N, Cl.
(3R,4R)-4-(Benzyloxymethyl)-1-[(9-deaza-adenin-9-yl)-
methyl]-3-hydroxypyrrolidine (42). Following the general
procedure for the Mannich reaction (vide supra), pyrrolidine
hydrochloride 28 (55 mg, 0.23 mmol), was processed to afford
42 (18 mg, 22%) as the hydrochloride salt. 1H NMR (d4-MeOH)
δ 8.17 (s, 1H), 7.55 (s, 1H), 7.30-7.20 (m, 5H), 4.46 (bs, 2H),
4.10-4.00 (m, 3H), 3.55-3.38 (m, 2H), 3.23-3.18 (m, 1H), 2.98
(dd, J ) 10.7, 5.8 Hz, 1H), 2.85 (dd, J ) 10.7, 3.4 Hz, 1H),
2.68 (dd, J ) 10.4, 6.9 Hz, 1H), 2.38-2.30 (m, 1H). 13C NMR
(d4-MeOH) δ 152.6, 151.7, 147.2, 139.9, 131.3,129.8, 129.3,
129.1, 115.6, 110.4, 74.5, 74.3, 71.9, 62.3, 56.6, 49.4, 49.0. Anal.
(C19H23N5O2‚2HCl‚0.75H2O) C, H, N, Cl.
Enzymatic Assays. Reaction mixtures of 1.0 mL contained
100 mM potassium phosphate (Sigma) pH 7.4, 50 mM KCl
(Sigma), 0.1 to 2 mM dithiothreitol (Sigma), 5’-methylthioad-
enosine (Sigma) at 0.25 or 0.30 mM, and 0.5 units of xanthine
oxidase (Sigma) and variable concentrations of inhibitor. After
warming to 25 °C in a temperature-controlled spectrophoto-
meter (Cary 300 Bio spectrophotometer from Varian), reactions
were initiated with 1 to 10 nM of purified human MTAP.
Activity was monitored by the increase in absorbance at 294
nm equivalent to 15.2 mM-1 cm-1. The coupled reaction is
irreversible and measures the formation of 2,8-dihydroxyad-
enine from adenine.
Acknowledgment. This work was supported by
research grant GM41916 from the NIH and by the New
Zealand Foundation for Research, Science, & Technol-
ogy.
Supporting Information Available: Analytical data.
This material is available free of charge via the Internet at
http://pubs.acs.org.
References
(1) Singh, V.; Shi, W., Evans, G. B., Tyler, P. C.; Furneaux, R. H.;
Almo, S. C.; Schramm, V. L. Picomolar Transition State Ana-
logue Inhibitors of Human 5′-Methylthioadenosine Phosphoryl-
ase and X-ray Structure with MT-Immucillin-A. Biochemistry
2004, 43, 9-18.
(2) Evans, G. B.; Furneaux, R. H.; Schramm, V. L.; Singh, V.; Tyler,
P. C. Targeting the Polyamine Pathway with Transition-State
Analogue Inhibitors of 5′-Methylthioadenosine Phosphorylase.
J. Med. Chem. 2004, 47, 3275-3281.
(3) Cohen, S. S. A Guide to the Polyamines; Oxford University
Press: New York: 1998.
(4) Tabor, C. W.; Tabor, H. Polyamines. Annu. Rev. Biochem. 1984,
53, 749-790.
(5) Thomas, T.; Thomas, T. J. Polyamines in cell growth and cell
death: molecular mechanisms and therapeutic applications.
Cell. Mol. Life Sci. 2001, 58, 244-258.
(6) Ragione, F. D.; Carteni-Farina, M.; Gragnaniello, V.; Schettino,
M. I.; Zappia, V. Purification and characterization of 5′-deoxy-
5′-methylthioadenosine phosphorylase from human placenta. J.
Biol. Chem. 1986, 261, 12324-12329.
(3R,4S)-1-[(9-Deazaadenin-9-yl)methyl]-3-hydroxy-4-
(1-propyl)pyrrolidine (43). Following the general procedure
for the Mannich reaction (vide supra), pyrrolidine hydrochlo-
ride 29 was processed to afford 43 (58 mg, 31%) as the
(7) Toorchen, D.; Miller, R. L. Purification and characterization of
5′-deoxy-5′-methylthioadenosine (MTA) phosphorylase from hu-
man liver. Biochem. Pharmacol. 1991, 41, 2023-2030.
(8) Wolford, R. W.; MacDonald, M. R.; Zehfus, B.; Rogers, T. J.;
Ferro, A. J. Effect of 5′-methylthioadenosine and its analogues
on murine lymphoid cell proliferation. Cancer Res. 1981, 41,
3035-3039.
(9) Williams-Ashman, H. G.; Seidenfeld, J.; Galletti, P. Trends in
the biochemical pharmacology of 5′-deoxy-5′-methylthioadeno-
sine. Biochem. Pharmacol. 1982, 31, 277-288.
(10) Pegg, A. E.; Borchardt, R. T.; Coward, J. K. Effects of inhibitors
of spermidine and spermine synthesis on polyamine concentra-
tions and growth of transformed mouse fibroblasts. Biochem. J.
1981, 194, 79-89.
(11) Pajula, R.-L.; Raina, A. Methylthioadenosine, a potent inhibitor
of spermine synthase from bovine brain. FEBS Lett. 1979, 99,
343-345.
(12) Carteni-Farina, M.; Cacciapuoti, G.; Porcelli, M.; Ragione, F. D.;
Lancieri, M.; Geraci, G.; Zappia, V. Studies on the metabolic
effects of methylthioformycin. Biochim. Biophys. Acta 1984, 805,
158-164 and references therein.
(13) Marton, L. J.; Pegg, A. E. Polyamines as targets for therapeutic
intervention. Annu. Rev. Pharmacol. Toxicol. 1995, 35, 55-91.
(14) Seiler, N.; Atanassov, C. L.; Raul, F. Polyamine metabolism as
target for cancer chemoprevention. Int. J. Oncol. 1998, 13, 993-
1006.
(15) Mu¨ller, S.; Coombs, G. H.; Walter, R. D. Targeting polyamines
of parasitic protozoa in chemotherapy. Trends Parasitol. 2001,
17, 242-249.
(16) Belting, M.; Borsig, L.; Fuster, M. M.; Brown, J. R.; Persson, L.;
Fransson, L.-A. Tumor attenuation by combined heparan sulfate
and polyamine depletion. Proc. Natl. Acad. Sci. 2002, 99, 371-
376.
1
hydrochloride salt. H NMR (MeOH-d4): δ: 8.18 (s,1H), 7.51
(s,1H), 3.91-3.85 (m, 3H), 3.10 (dd, J ) 9.6, 8.0 Hz, 1H), 2.82-
2.72 (m, 2H), 2.22 (dd, J ) 9.6, 8.0 Hz, 1H), 2.04-1.95 (m,
1H), 1.56-1.44 (m, 1H), 1.39-1.21 (m, 3H), 0.92-0.87 (m, 3H).
13C NMR (MeOH-d4): δ: 152.52, 151.45, 147.37, 130.59,
115.55, 112.50, 77.94, 62.63, 59.94, 49.55, 48.59, 36.89, 22.67,
14.91. HRMS (MH+) calcd for C14H21N5O: 276.1819. Found
276.1822. Anal. (C14H21N5O‚2HCl‚2H2O) C, H, N, Cl.
(3R,4S)-1-[(9-Deazaadenin-9-yl)methyl]-3-hydroxy-4-
(2-phenylethyl)pyrrolidine (44). Following the general
procedure for the Mannich reaction (vide supra), pyrrolidine
hydrochloride 30 was processed to afford, presumably, (3R,4S)-
1-[(6-chloro-9-Deazapurin-9-yl)methyl]-3-hydroxy-4-(2-phenyl-
ethyl)pyrrolidine (70 mg, 38%), Without further characteriza-
tion this was dissolved in 7 N ammonia in methanol (4 mL)
and heated in a sealed tube at 130 °C for 3 h at which point
the reaction was cooled to room temp and concentrated in
vacuo. Chromatography (CH2Cl2:MeOH:NH4OH 80:20:1 v/v/
v) of the resulting residue afforded 44 as a syrup (29 mg, 44%).
1H NMR (D2O): δ ppm: 8.40 (s, 1H), 7.83 (s, 1H), 7.26 (m,
5H), 4.33 (m, 4H), 4.07 (m, 1H), 3.80 (m, 2H), 2.75 (m, 2H),
2.37 (m, 1H), 1.90 (m, 1H), 1.66 (m, 1H). 13C NMR (D2O): δ
149.54, 144.47, 142.40, 133.05, 129.05, 128.85, 126.51, 113.64,
103.48, (74.87, 72.96), (55.34, 54.87), (52.59, 52.09), (45.96,
43.65), 33.30, 32.94, 32.33. C19H23N5O: (MH+): calc.: 338.19754,
found:338.19794.
Biology. Protein Preparation. Human MTAP was ex-
pressed in E. coli BL21(DE3) using the pQE32 expression
vector (provided by Drs. Carson and Rosenbech of Scripps
Institute La Jolla, CA). Protein with a His-6-N-terminal label
was purified by Ni-NTA (Qiagen) chromatography, concen-
trated, and stored at -80 °C. Details are provided in ref 1.
(17) Gupta, S.; Ahmad, N.; Marengo, S. R.; MacLennan, G. T.;
Greenberg, N. M.; Mukhtar, H. Chemoprevention of prostate
carcinogenesis by R-difluoromethylornithine in TRAMP mice.
Cancer Res. 2000, 60, 5125-5133.
(18) Subhi, A. L.; Tang, B.; Balsara, B. R.; Altomare, D. A.; Testa, J.
R.; Cooper, H. S.; Hoffman, J. P.; Meropol, N. J.; Kruger, W. D.
Clin. Cancer Res. 2004, 10, 7290-7296.

Transition State Analogue Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 14 4689
(19) Kline, P. C.; Schramm, V. L. Purine nucleoside phosphorylase.
Inosine hydrolysis, tight binding of the hypoxanthine intermedi-
ate, and third-the-sites reactivity. Biochemistry 1992, 31, 5964-
5973.
(20) Kline, P. C.; Schramm, V. L. Purine nucleoside phosphorylase.
Catalytic mechanism and transition state analysis of the arse-
nolysis reaction. Biochemistry 1993, 32, 13212-13219.
(21) Kline, P. C.; Schramm, V. L. Pre-steady-state transition state
analysis of the hydrolytic reaction catalyzed by purine nucleoside
phosphorylase. Biochemistry 1995, 34, 1153-1162.
(22) Singh, V.; Schramm, V. L. 2005, unpublished results.
(23) Evans, G. B.; Furneaux, R. H.; Lewandowicz, A.; Schramm, V.
L.; Tyler, P. C. Synthesis of Second Generation Transition State
Analogues of Human Purine Nucleoside Phosphorylase J. Med.
Chem. 2003, 46, 5271-5276.
(24) 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-adenosylho-
mocysteine nucleosidase from Escherichia coli. J. Biol. Chem.
2005, 280, 18265-18273.
(25) Evans, G. B.; Furneaux, R. H.; Schramm, V. L.; Tyler, P. C
Synthesis of a Transition State Analogue Inhibitor of Purine
Nucleoside Phosphorylase via the Mannich Reaction. Org. Lett.
2003, 5, 2639-2640.
(26) Miles, R. W.; Tyler, P. C.; Furneaux, R. H.; Bagdassarian, C.
K.; Schramm, V. L. One-third-the-sites transition state inhibitors
for purine nucleoside phosphorylase. Biochemistry 1998, 37,
8615-8621.
(27) Morrison, J. F.; Walsh, C. T. The behavior and significance of
slow-binding enzyme inhibitors. Adv. Enzymol. 1988, 61, 201-301.
JM050269Z