Design and synthesis of potent "sulfur-free" transition state analogue inhibitors of 5′-methylthioadenosine nucleosidase and 5′-methylthioadenosine phosphorylase

Article abstract of DOI:10.1021/jm100898v

5′-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) is a dual substrate bacterial enzyme involved in S-adenosylmethionine (SAM) related quorum sensing pathways that regulates virulence in many bacterial species. MTANs from many bacteria are directly involved in the quorum sensing mechanism by regulating the synthesis of autoinducer molecules that are used by bacterial communities to communicate. In humans, 5′-methylthioadenosine phosphorylase (MTAP) is involved in polyamine biosynthesis as well as in purine and SAM salvage pathways and thus has been identified as an anticancer target. Previously we have described the synthesis and biological activity of several aza-C-nucleoside mimics with a sulfur atom at the 5′ position that are potent E. coli MTAN and human MTAP inhibitors. Because of the possibility that the sulfur may affect bioavailability, we were interested in synthesizing "sulfur-free" analogues. Herein we describe the preparation of a series of "sulfur-free" transition state analogue inhibitors of E. coli MTAN and human MTAP that have low nano-to picomolar dissociation constants and are potentially novel bacterial anti-infective and anticancer drug candidates.

Full text of DOI:10.1021/jm100898v

6730 J. Med. Chem. 2010, 53, 6730–6746
DOI: 10.1021/jm100898v
Design and Synthesis of Potent “Sulfur-Free” Transition State Analogue Inhibitors of
50-Methylthioadenosine Nucleosidase and 50-Methylthioadenosine Phosphorylase
Alistair I. Longshaw,† Florian Adanitsch,† Jemy A. Gutierrez,‡ Gary B. Evans,*,† Peter C. Tyler,† and Vern L. Schramm‡
†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 July 18, 2010
50-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) is a dual substrate bacterial
enzyme involved in S-adenosylmethionine (SAM) related quorum sensing pathways that regulates
virulence in many bacterial species. MTANs from many bacteria are directly involved in the quorum
sensing mechanism by regulating the synthesis of autoinducer molecules that are used by bacterial
communities to communicate. In humans, 50-methylthioadenosine phosphorylase (MTAP) is involved
in polyamine biosynthesis as well as in purine and SAM salvage pathways and thus has been identified as
an anticancer target. Previously we have described the synthesis and biological activity of several aza-C-
nucleoside mimics with a sulfur atom at the 50 position that are potent E. coli MTAN and human MTAP
inhibitors. Because of the possibility that the sulfur may affect bioavailability, we were interested in
synthesizing “sulfur-free” analogues. Herein we describe the preparation of a series of “sulfur-free”
transition state analogue inhibitors of E. coli MTAN and human MTAP that have low nano- to picomolar
dissociation constants and are potentially novel bacterial anti-infective and anticancer drug candidates.
Introduction
MTAN is a major route for the metabolism of methylthio-
adenosine (MTA) and S-adenosylhomocysteine (SAH) in
bacteria. MTA is a byproduct of the formation of the AI-1
family of autoinducers (acylhomoserine lactones, for exam-
ple, hydroxybutanoyl-L-homoserine lactone) that are believed
to be responsible for intraspecies communication.12 MTAN
subsequently catalyzes the hydrolysis of MTA to adenine and
5-methylthio-D-ribose, which in turn is recycled into SAM.
The MTAN-catalyzed hydrolysis of SAH leads to S-ribosyl-
homocysteine (SRH) which is a biochemical precursor to
AI-2, a family of AIs important in interspecies bacterial com-
munications.13 Thus, inhibition of MTAN is expected to
cause the accumulation of MTA, resulting in product inhibi-
tion of AI-1 production, and directly block the formation of
SRH, the precursor of AI-2.
Human MTAP effects phosphorolysis of MTA and is
involved in polyamine biosynthesis and purine salvage path-
ways. MTA is a byproduct formed during the biosynthesis
of the polyamines spermidine and spermine, which have an
important role in growth-related processes.14 MTAP is the
only known enzyme involved in the recycling of MTA in
humans, and it has been proposed that inhibition of MTAP
could increase MTA concentration to cause feedback inhibi-
tion of polyamine biosynthesis resulting in potential antipro-
liferative activity, which has made MTAP a target for the
design of novel anticancer drugs.15,16 We have previously
reported a variety of transition state inhibitors of MTAP,
one of which is currently in preclinical trials for the treatment
of head and neck tumors.8,17
The determination of enzymatic transition states is a powerful
technique used to provide information on both the geometric
and electronic features of the transition state and thus provides
a blueprint for putative enzyme inhibitors incorporating transi-
tion state features.1 This process has been applied to a number of
N-ribosyl transferases, including purine nucleoside phosphory-
lase (PNPa),2 50-methylthioadenosine phosphorylase (MTAP),3
and 50-methylthioadenosine/S-adenosylhomocysteine nucleo-
sidase (MTAN),4-6 and has led to the development of extremely
potent inhibitors that incorporate transition state features.1,7-9
MTAN is a dual substrate enzyme that is found in bacteria but
not mammals and is involved in polyamine and S-adenosyl-
methionine (SAM) quorum sensing pathways. Many bacteria
utilize quorum sensing signaling molecules known as autoindu-
cers (AIs) to communicate with each other. The production and
detection of AIs coordinate some gene expression and regulate
processes beneficial to microbial communities. Quorum sensing
is a potentially useful target for bacterial anti-infective agents, as
it has been shown that several mutant bacterial strains defective
in quorum sensing are less virulent,10,11 and as inhibition of
quorum sensing is nonlethal to bacteria, the potential for the
development of resistance is reduced when compared to more
traditional anti-infective agents.
*To whom correspondence should be addressed. Phone: þ64-4-
9313000. Fax: þ64-4-9313055. E-mail: g.evans@irl.cri.nz.
a Abbreviations: AI, autoinducer; 9-DAA, 9-deazaadenine; DADMe,
50-deaza-10-aza-20-deoxy-10-(9-methylene); DIAD, diisopropyl azodicar-
boxylate; ImmA, immucillin-A; MT, methylthio; MTA, methylthioadeno-
sine; MTAN, 50-methylthioadenosine/S-adenosylhomocysteine nucleosidase;
MTAP, 50-methylthioadenosine phosphorylase; PNP, purine nucleoside
phosphorylase; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethio-
nine; SRH, S-ribosylhomocysteine.
The transition states for the hydrolysis of MTA by Escherichia
coli MTAN and phosphorolysis of MTA by human MTAP have
been solved using a combination of kinetic isotope effects and
computational modeling. In the case of E. coli it has been shown
r
pubs.acs.org/jmc
Published on Web 08/18/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6731
Scheme 1a
Figure 1. Reaction catalyzed by MTAP and MTANs with MTA as
the substrate. In the case of MTAP the attacking nucleophile is
phosphate, whereas in MTANs it is water.
a Reagents: (a) Boc2O, MeOH, 0 °C; (b) benzyl chloroformate, Et3N,
CH2Cl2, 0 °C f room temperature; (c) (PCy3)2Cl2RudCHPh, CH2Cl2,
room temperature; (d) (i) NBS, DMSO, H2O, room temperature, (ii)
NaOH, MeOH, 0 °C f room temperature.
rapidly generate a series of hydroxypyrrolidines, via the addi-
tion of Grignard reagents to epoxides meso-10 and meso-11.
Diallylamine was protected as either its tert-butylcarbamate 6
or benzyloxycarbamate 7 and subjected to a ring closing
metathesis reaction using Grubb’s first generation catalyst.25,26
Pyrrolines 8 and 9 were converted into the corresponding
epoxides meso-10 and meso-11 via a two step bromohydrin/
epoxidation sequence, in good yields (Scheme 1).
With the key epoxypyrrolidines in hand, ring-opening
of the epoxide ring with various alkyl, cycloalkyl, and
aryl Grignard reagents was investigated. Pyrrolidine sub-
units (()-20 - (()-22, (()-24, (()-26 were prepared by the
copper(I) catalyzed addition of the corresponding Grignard
reagent into epoxide meso-11, followed by hydrogenolysis of
the N-protecting group. The deprotected amines were trea-
ted with formaldehyde and 9-DAA to afford the correspond-
ing Mannich products (()-28 - (()-30, (()-32, and (()-34.
Compounds (()-15, (()-17, and (()-19 could not be isolated
cleanly from the ring-opening reactions and were therefore used
as crude compounds and purified after subsequent steps to gene-
rate immucillins (()-31, (()-33, and (()-35 (Scheme 2).
The vinyl and allyl-substituted DADMe-immucillins
(()-40 and (()-41 were prepared in an analogous manner
by addition of vinylmagnesium bromide or allylmagne-
sium chloride in the presence of copper(I) bromide di-
methyl sulfide complex to N-Boc protected epoxide meso-
10 to give pyrrolidines (()-36 and (()-37. Subsequent
deprotection with 36% aqueous HCl in methanol affor-
ded the corresponding pyrrolidine subunits (()-38 and
(()-39, which were coupled to 9-DAA to generate (()-40
and (()-41, respectively (Scheme 3).
Figure 2. Known enantiopure transition state analogue inhibitors
of MTANs (3R,4S)-1, (3R,4S)-2, and (3R,4S)-3 and target racemic
“sulfur-free” inhibitors (()-4.
to have a late dissociative SN1 transition state with extensive
cleavage of the bond between adenine and the ribosyl moiety,4
and for human MTAP it has been shown to be late dissociative
SN1 with a zero N-glycosidic bond order.3 In MTAP the
nucleophile responsible for the cleavage of the N-glycosidic bond
is phosphate, while for E. coli MTAN the nucleophile is water
(Figure 1).
Previously we have reported the synthesis of potent transi-
tion state analogue inhibitors, based on a chiral 4-substituted-
3-hydroxypyrrolidine motif, of which several exhibit binding
affinities in the femtomolar range against E. coli MTAN and
picomolar against MTAP.18,19 Recently it has been shown that
three of these compounds, MT-, EtT-, and BuT-DADMe-
immucillin (3R,4S)-1, (3R,4S)-2, and (3R,4S)-3, respectively
(Figure 2), disrupt AI production in Vibrio cholerae and E. coli
and cause reduction in biofilm formation.20
In vivo screening of some of these chiral femtomolar
inhibitors indicated that the sulfur atom may adversely affect
bioavailability.21 Herein we report the synthesis of a new
family of transition state analogue inhibitors of MTAN and
MTAP in which the sulfur atom has been removed. These
racemic “sulfur-free” DADMe-immucillins (()-4 (Figure 2)
have also been found to be potent inhibitors of the E. coli
MTAN and human MTAP with dissociation constants in the
low nano- to picomolar range and show potential as new anti-
infective and anticancer drug candidates. These simplified
inhibitors are easier and cheaper to prepare than the pre-
viously reported chiral compounds, and the removal of the
sulfur atom may offer improved pharmacological properties.
Ethynyl DADMe-immucillin (()-45 was synthesized in
four steps from epoxide meso-10. Addition of trimethylsilyl-
acetylene to meso-10 in the presence of n-butyllithium and
boron trifluoride etherate27 gave (()-42. Treatment with
methanolic HCl gave (()-43, which underwent a Mannich
reaction with 9-DAA to give crude (()-44 which was directly
ꢀ
deprotected under Zemplen conditions to give the desired
Results and Discussion
DADMe-immucillin (()-45 after purification (Scheme 4).
Having prepared a series of alkyl, cycloalkyl, and aromatic
substituted immucillins, we investigated what other biologically
relevant substituents would be tolerated by the target enzymes,
for example, 1,4-disubstituted 1-H-1,2,3-triazoles.28
Tsuzuki and co-workers reported the ring-opening of epoxide
meso-10 with sodium azide.29 We repeated this and subjected the
resulting azide (()-46 and trimethylsilylacetylene to the Sharp-
less copper(I) catalyzed azide-alkyne cycloaddition (CuAAC)
conditions (CuSO4, sodium ascorbate, t-BuOH, H2O, room
Synthesis. Compounds (()-4 should be readily available
by a Mannich reaction between a suitably substituted hydroxy-
pyrrolidine, 9-DAA, and formaldehyde as described for the
corresponding chiral immucillins.22 Hydroxypyrrolidines of
the type required for the racemic “sulfur-free” DADMe-
immucillins (()-4 have been previously reported to be
formed via the nucleophile opening of epoxypyrroli-
dines.23,24 We decided to use a modification of the procedure
described by Hansen and Bols,23 as this would allow us to

6732 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Longshaw et al.
Scheme 2a
a Reagents: (a) RMgX, CuBr DMS, THF, -30 °C; (b) Pd (10 wt % on carbon), H2, MeOH, room temperature; (c) formaldehyde, 9-DAA, 1,4-dioxane,
H2O, room temperature.
3
Scheme 3a
a Reagents: (a) RMgX, CuBr DMS, THF, -30 °C; (b) 36% aq HCl, MeOH, room temperature; (c) formaldehyde, 9-DAA, 1,4-dioxane, H2O, room
temperature.
3
temperature),30,31 but this was unsuccessful. However, the de-
sired transformation could be achieved by simply refluxing the
reagents in toluene. The crude mixture was treated with tetra-
butylammonium fluoride to give TMS-triazole (()-47 and the
desired desilylated triazole (()-48 in good yield. Removal of the
nitrogen protecting group was achieved under our standard
conditions (HCl/MeOH), and then pyrrolidine (()-49 was sub-
jected to the Mannich reaction to afford DADMe-immucillin
(()-50 (Scheme 5).
Scheme 4a
With ethynylpyrrolidine (()-42 in hand we attempted the
formation of the NH-1,2,3-triazol-4-ylpyrrolidine subunit, an
isomer of triazole subunit (()-48. Selective removal of the TMS
group in (()-42 with tetrabutylammonium fluoride gave
(()-51. Attempts to form the NH-1,2,3-triazol-4-yl compound
from the resulting terminal alkyne present in (()-51 using
recently reported methodology with either sodium azide32 or
“protected” azides33 were unsuccessful. However, the N-sub-
stituted triazol-4-ylpyrrolidine (()-52 was prepared by reaction
of (()-51 with benzylazide under Sharpless CuAAC condi-
tions.31 Removal of the Boc protecting group gave (()-53
which was coupled to 9-DAA using the standard Mannich
conditions to afford (()-54 (Scheme 6).
a Reagents: (a) trimethylsilylacetylene, n-BuLi, BF3 OEt2, THF,
3
-78 °C f room temperature; (b) 36% aq HCl, MeOH, room tempera-
ture; (c) formaldehyde, 9-DAA, 1,4-dioxane, H2O, room temperature;
(d) NaOMe, MeOH, room temperature.
As we had easy access to allylpyrrolidine (()-37, we investi-
gated the thiol-ene “click reaction”.34 Although counter to our

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6733
major objective of eliminating sulfur from our putative inhibi-
tors, this reaction would allow us to probe the effect of
increasing the length of the linker between the sulfur moiety
and the pyrrolidine ring of the DADMe-immucillins and
also demonstrate another synthetic strategy capable of gene-
rating large libraries of compounds via “click chemistry”. Thus
allylpyrrolidine (()-37 was treated with benzylmercaptan and
1,10-azobis(cyanocyclohexane) in 1,4-dioxane at 90 °C35 to give
(()-55. The crude product was treated with 36% aqueous HCl
in methanol to afford (()-56, which underwent a subsequent
Mannich reaction to give the desired DADMe-immucillin
(()-57 (Scheme 7).
The use of copper salts to assist in the ring-opening of
epoxides is known in some cases to give a mixture of the cis
and the expected trans isomers;23,36 thus, it was necessary to
confirm the relative stereochemistry of our pyrrolidines.
X-ray crystallography was not applicable, as none of the
relevant intermediates or final DADMe-immucillin com-
pounds were crystalline; therefore, known chiral amine
(3R,4R)-58,37 was converted into Et-DADMe-immucillin
(3R,4S)-69 via a modification of a previously reported
route.19 This provided material with known relative stereo-
chemistry between the 3- and 4-position for direct compar-
ison with the corresponding racemic compound (()-28.
Selective protection of the secondary hydroxyl group of
amine (3R,4R)-58 was achieved in three steps. Oxidation of
the primary hydroxyl with Dess-Martin periodinane re-
agent gave aldehyde (3R,4S)-62 which was methylenated
under standard Wittig conditions. Hydrogenation of the
olefinic group, followed by concomitant removal of both
protecting groups, gave enantiopure amine (3R,4S)-67. A
Mannich reaction with 9-DAA gave the enantiopure
DADMe-immucillin (3R,4S)-69. As Bu-DADMe-immucillin
(()-29 was the most potent racemic inhibitor of both E. coli
MTAN and human MTAP, the same sequence was used to
prepare enantiopure Bu-DADMe-immucillin (3R,4S)-70 by
substituting n-propyltriphenylphosphonium bromide as the
Wittig reagent (Scheme 8).
Scheme 5a
a Reagents: (a) NaN3, 1,4-dioxane, H2O, 100 °C; (b) (i) trimethylsilyl-
acetylene, toluene, 110 °C; (ii) TBAF (1 M in THF), THF, room tempe-
rature; (c) 36% aq HCl, MeOH; (d) formaldehyde, 9-DAA, 1,4-dioxane,
H2O, room temperature.
The 1H and 13C NMR data for enantiopure amine
(3R,4S)-67 and DADMe-immucillin (3R,4S)-69 were iden-
tical to those of their racemic equivalents, compounds (()-20
and (()-28 respectively. To further confirm the relative
stereochemical assignment of the C-3 and C-4 substituents
in (()-20 and (()-28, the hydroxyl group in pyrrolidine
(()-12 was subjected to a Mitsunobu inversion protocol38
by treatment with benzoic acid in the presence of DIAD and
triphenylphosphine to give inverted ester (()-71 in good
yield. Saponification of the benzoate followed by hydroge-
nation of the Cbz group gave pyrrolidine (()-73 which was
converted into racemic cis-Et-DADMe-immucillin (()-74
(Scheme 9). Comparison of the NMR data of pyrrolidine
cis-(()-73 with trans-(()-20 and (3R,4S)-67 as well as
DADMe-immucillin cis-(()-74 with trans-(()-28 and (3R,4S)-
69 showed clear differences. In particular the 13C NMR reso-
nance for C-3 in trans-Et-DADMe-immucillin (()-28
(77.7 ppm) was further downfield from that for the cis isomer
(()-74 (72.5 ppm). All the DADMe-immucillins generated via
the copper(I)-catalyzed Grignard ring-opening of epoxides
meso-10 or meso-11 have the C-3 resonance between 76.7 and
Scheme 6a
a Reagents: (a) TBAF (1 M in THF), THF, room temperature; (b)
benzyl azide, sodium ascorbate, CuSO4, t-BuOH, H2O, room tempera-
ture; (c) 36% aq HCl, MeOH, room temperature; (d) formaldehyde,
9-DAA, 1,4-dioxane, H2O, room temperature.
Scheme 7a
a Reagents: (a) 1,10-azobis(cyanocyclohexane), benzylmercaptan, 1,4-dioxane, 90 °C; (b) 36% aq HCl, MeOH, room temperature; (c) formaldehyde,
9-DAA, 1,4-dioxane, H2O, room temperature.

6734 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Longshaw et al.
Scheme 8a
a Reagents: (a) (Bu)2SnO, toluene, 110 °C, then BzCl, 5 °C f room temperature; (b) TBDMSCl, imidazole, DMF, room temperature; (c) NaOMe,
MeOH, room temperature; (d) Dess-Martin periodinane, CH2Cl2, room temperature; (e) Ph3PCH3Br or Ph3PCH2CH2CH3Br, n-BuLi, THF, -78 °C
f room temperature; (f) Pd (10 wt % on carbon) or Pd(OH)2 (20 wt % on carbon), H2 (1 atm), EtOH, room temperature; (g) TFA or 36% aq HCl in
MeOH, room temperature; (h) formaldehyde, 9-DAA, 1,4-dioxane, H2O, room temperature (for (3R,4S)-69) or 85 °C (for (3R,4S)-70).
Scheme 9a
a Reagents: (a) benzoic acid, DIAD, PPh3, THF, 0 °C f room temperature; (b) K2CO3, EtOH, H2O, 100 °C; (c) Pd (10 wt % on carbon), H2, MeOH,
room temperature; (d) formaldehyde, 9-DAA, 1,4-dioxane, H2O, room temperature.
78.1 ppm, indicating a trans arrangement of substituents at C-3
and C-4.
substituents (e.g., alkyl (()-28, olefinic (()-40 and (()-41,
and alkynyl (()-45) as well as the highly branched alkyl
group (()-31 bound less well. The N-linked triazole group
(()-50 (Ki = 2 nM) did not bind as well as the C-linked benzyl
group (()-54 (Ki = 64 pM).
For human MTAP, Bu-DADMe-immucillin (()-29 was
the best inhibitor in this family of compounds (Ki = 0.8 nM)
with a comparable affinity to its sulfur-containing counter-
part (Ki* = 1.4 nM), also suggesting that sulfur was not a
necessary component of human MTAP inhibitors. Although
MTAN prefers bulky substituents, human MTAP preferred
the short alkyl and olefinic groups (()-28, (()-40 and (()-41.
Comparisons of inhibitors for MTAN and MTAP are best
made from the Km/Ki ratio to provide comparative substrate
and inhibitor interaction energies. For racemic Et-DADMe-
immucillin (()-28, the Ki values were 0.84 and 1.7 nM for
E. coli MTAN and human MTAP, respectively. The respective
Inhibition Studies. The inhibition of Escherichia coli
MTAN and human MTAP was evaluated with racemic
immucillins (()-28 - (()-35, (()-40, (()-41, (()-45, (()-50,
(()-54, (()-57, and (()-74 and enantiopure immucillins
(3R,4S)-69 and (3R,4S)-70 (Table 1).
E. coli MTAN showed broad tolerance for a variety of
substituents in the C-4 position of the pyrrolidine ring and
bound most of the compounds tightly with dissociation
constants in the picomolar range. The diversity of functional
groups accommodated at this position reaffirms the struc-
tural flexibility of the E. coli MTAN 50 binding pocket.9 The
Bu-DADMe compound (()-29 gave the tightest binding
(Ki* value of 9 pM) and was equivalent to its sulfur isostere
(compound 2, Ki* = 8 pM).18 Thus, the presence of sulfur is
not necessary for activity against E. coli MTAN. Shorter

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6735
Table 1. Inhibition Constants for the Interaction of Immucillins with E. coli MTAN and Human MTAPa and the Ratio of the Two Values in Order To
Provide a Qualitative Indication of the Therapeutic Window for the Treatment of an E. coli Infection
a Ki is the dissociation constant for the first step in E þ I T EI T EI*, the two-step binding characteristic of slow-onset tight-binding inhibition.
/ indicates slow onset binding and is the overall dissociation constant for E þ I T EI*. In cases where no / is shown, then slow-onset inhibition was not
observed. b (-) Indicates that no inhibition was observed at 2.5 μM.

6736 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Longshaw et al.
Km/Ki ratios are 512 and 3105 for E. coli MTAN and human
MTAP. Thus, the inhibitor is a better competitor for the
catalytic site of human MTAP than for E. coli MTAN by a
factor of 6. The same is true for inhibitors with short olefinic
groups (()-40 and (()-41. For (()-40, the Km/Ki ratios are 662
and 1649, while they are 1228 and 1759 for (()-41, favored
more by human MTAP than by E. coli MTAN. For the rest of
the inhibitors, the Km/Ki ratio suggests that E. coli MTAN has a
higher affinity for these compounds relative to substrate than
does human MTAP.
Single enantiomers are usually more potent than racemic
mixtures,39 as is the case for the inhibition constants for
compounds (3R,4S)-69 and (()-28 and (3R,4S)-70 and
(()-29 with E. coli MTAN and human MTAP. Likewise,
the trans-Et-DADMe-immucillin (()-28 is a better inhibitor
of both E. coli MTAN and human MTAP than cis-Et-DADMe-
immucillin (()-74 which confirms that the trans stereochemistry
is preferred as a transition state analogue.
TFA in H2O/MeOH gradient through a Phenomenex Synergi
4 μm polar-RP 80A, 250 mm ꢀ 30 mm column and Agilent 1100
photodiode array detector. Thin layer chromatography was per-
formed on glass-backed silica gel plates (Merck). Column chro-
matography was performed on silica gel (Davisil LC60A 40-
63 μm). Anhydrous solvents were obtained from Aldrich. Methyl-
triphenylphosphonium bromide was azeotropically dried with
toluene and stored in vacuo over P2O5 prior to use. All other
reagents were used as received. All final compounds had a purity
of >95% as assessed by analytical HPLC.
Chemistry. tert-Butyl Diallylcarbamate (6).25 Di-tert-butyl
dicarbonate (42.2 g, 193 mmol) was added, portionwise, to
a solution of diallylamine (20 mL, 162 mmol) in methanol
(500 mL) at 0 °C. After complete addition the reaction mixture
was allowed to warm to room temperature, stirred for 1 h, then
concentrated under reduced pressure. Dry flash chromato-
graphy of the residue (gradient 0-50% EtOAc in Petrol)
afforded 6 as a colorless oil (31.9 g, 99%). 1H NMR (500 MHz,
CDCl3): δ = 5.80-5.72 (m, 2H), 5.13-5.09 (m, 4H), 3.80 (br s,
4H), and 1.45 ppm (s, 9H).
tert-Butyl 3-Pyrroline-1-carboxylate (8).23 Grubb’s first gene-
ration catalyst (920 mg, 1.1 mmol) was added to a solution of
tert-butyl diallylcarbamate (6) (31.9 g, 162 mmol) in CH2Cl2
(370 mL). The reaction mixture was stirred for 17 h and then
concentrated under reduced pressure. Dry flash chromatogra-
phy of the residue (gradient: 0-70% EtOAc in Petrol) afforded
8 as a pale yellow oil (19.9 g, 73%). 1H NMR (500 MHz,
CDCl3): δ = 5.80-5.74 (m, 2 H), 4.14-4.08 (m, 4H), and 1.48
ppm (s, 9H). 13C NMR (125 MHz, CDCl3): δ = 154.3, 125.9,
125.8, 79.3, 53.1, 52.8 (rotamers), and 28.5 ppm. ESI-HRMS for
C9H15NONa [MNa]þ calcd, 192.1000; found, 192.0997.
DADMe-immucillin (()-57 is a relatively potent inhibitor
of bacterial MTAN; however, increasing the linker between
the benzyl thiol moiety and the pyrrolidine ring from one
carbon (Ki* = 0.5 pM)18 to three carbons as in (()-57 (Ki* =
54 pM) is detrimental to activity against E. coli MTAN.
Although a similar reduction in activity against human
MTAP was also observed, the magnitude of the Km/Ki ratio
of (()-57 (7963 for MTAN vs 74 for MTAP) reveals that this
is one of the more selective inhibitors for the bacterial
enzyme.
In terms of a therapeutic window a highly selective bacter-
ial MTAN inhibitor is the benzyltriazole compound (()-54
which inhibited E. coli MTAN with a Ki* of 64 pM without
inhibiting human MTAP even at 2.5 μM. Interestingly, the
N-linked triazole compound (()-50 was a weaker inhibitor
than (()-54 for the bacterial enzyme but a better inhibitor for
human MTAP. Again, this suggests that the E. coli enzyme
50-binding pocket is able to accommodate bulkier aromatic
groups much better than its human orthologue or better
than other MTAN isozymes.9 Exploiting these enzymatic
subtleties may prove useful in designing clinically selective
antibacterials.
tert-Butyl 3,4-Epoxypyrrolidine-1-carboxylate (meso-10).23
N-Bromosuccinimide (8.05 g, 45 mmol) was added portionwise
over 10 min to a solution of olefin (8) (6.4 g, 38 mmol) in DMSO
(40 mL) and water (2 mL) at 0 °C. The reaction mixture was
warmed to room temperature, stirred for 2.5 h, and then further
N-bromosuccinimide (2 g, 11 mmol) was added. After the
mixture was stirred for a further 2 h, the reaction was quenched
by the addition of water (50 mL) and then extracted into EtOAc
(4 ꢀ 50 mL). The combined organic phase was washed with
brine (2 ꢀ 50 mL), dried (MgSO4), filtered, and concentrated
under reduced pressure. The residue was dissolved in MeOH
(100 mL), cooled to 0 °C, and then an aqueous solution of
NaOH (23 mL, 46 mmol, 2 M) was added in one portion. The
mixture was warmed to room temperature, stirred for 17.5 h,
and then the MeOH was removed under reduced pressure. The
residue was partitioned between water (100 mL) and EtOAc
(100 mL) and extracted with EtOAc (3 ꢀ 100 mL). The
combined organic phase was washed with brine (2 ꢀ 100 mL),
dried (MgSO4), filtered, and concentrated under reduced pres-
sure. Flash chromatography of the residue (1:9 then 2:8, EtOAc/
Petrol) afforded meso-10 as a pale yellow oil (4.54 g, 64% over
the two steps). 1H NMR (500 MHz, CDCl3): δ = 3.81 (d, J =
12.9 Hz, 1H), 3.74 (d, J = 12.8 Hz, 1H), 3.66 (d, J = 5.3 Hz, 2H),
3.31 (dd, J = 12.6, 6.2 Hz, 2H), and 1.44 ppm (s, 9H). 13C NMR
(125 MHz, CDCl3): δ = 154.8, 79.8, 55.6, 55.1 (rotamers), 47.3,
46.9 (rotamers), and 28.4 ppm. ESI-HRMS for C9H15NO3Na
[MNa]þ calcd, 208.0950; found, 208.0955.
Conclusions
A series of “sulfur-free” transition state analogues of E. coli
MTAN and human MTAP have been designed and synthe-
sized on the basis of their dissociative transition state struc-
tures. These racemic compounds can be rapidly prepared in a
simple manner. We have demonstrated that the removal of
the sulfur moiety from the transition state analogues is not
detrimental to their activity against these enzymes. Further
investigation into the bioavailability and other pharmacolo-
gical properties of these potent “sulfur-free” inhibitors will be
conducted and reported in due course.
Benzyl Diallylcarbamate (7).26 A solution of diallylamine
(15 mL, 120 mmol) in CH2Cl2 (150 mL) was cooled to 0 °C,
and Et3N (23 mL, 160 mmol) and then benzyl chloroformate
(20 mL, 140 mmol) were added dropwise. The reaction mixture
was slowly allowed to warm to room temperature and stirred for
16 h and then quenched with water (200 mL). The phases were
separated, and the aqueous phase was extracted with CH2Cl2
(3 ꢀ 200 mL). The combined organic phase was washed with
brine (200 mL), dried (MgSO4), filtered, and concentrated under
reduced pressure. Flash chromatography (gradient: 2-10%
EtOAc in Petrol) of the residue afforded 7 as a pale yellow oil
Experimental Section
General. NMR spectra were recorded on a Bruker Avance III
spectrometer at 500 MHz (1H) or 125 MHz (13C). Spectra were
measured in either CDCl3 or CD3OD using Me4Si as internal
reference. High resolution accurate mass determinations were
performed on a Waters Q-Tof Premier mass spectrometer under
electrospray ionization conditions. Analytical HPLC was per-
formed on an Agilent 1100 instrument, eluting with 0.1% w/w
TFA in H2O/MeOH gradient through a Phenomenex Synergi
4 μm polar-RP 80A, 250 mm ꢀ 4.6 mm column. Preparative
HPLC was performed using a Gilson 321 pump, eluting 0.1% w/w
1
(25.9 g, 92%). H NMR (500 MHz, CDCl3): δ = 7.35-7.29

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6737
(m, 5H), 5.77 (br s, 2H), 5.17-5.13 (br m, 6H), and 3.88 ppm
(br s, 4H).
CD3OD): δ = 3.94-3.91 (m, 1H), 3.23-3.19 (m, 1H),
2.98-2.95 (m, 1H), 2.78-2.76 (m, 1H), 2.51-2.47 (m, 1H),
1.87-1.81 (m, 1H), 1.54-1.46 (m, 1H), 1.32-1.23 (m, 1H), and
0.96 ppm (t, J = 7.4, 3H). 13C NMR (125 MHz, CD3OD): δ =
78.1, 54.7, 51.6, 51.0, 26.3, and 12.9 ppm. ESI-HRMS for
C6H14NO [MH]þ calcd, 116.1075; found, 116.1070.
Benzyl 3-Pyrroline-1-carboxylate (9).26 Grubb’s first genera-
tion catalyst (173 mg, 0.21 mmol) was added to a solution of
benzyl diallylcarbamate (7) (6.78 g, 29 mmol) in CH2Cl2 (300
mL). The reaction mixture was stirred for 16 h, and then further
catalyst was added (340 mg, 0.4 mmol). The reaction mixture
was stirred for a further 16 h and then concentrated under
reduced pressure. Flash chromatography (1:9 then 2:8, EtOAc/
Petrol) afforded 9 as a yellow oil (5.74 g, 94%). 1H NMR (500
MHz, CDCl3): δ = 7.39-7.29 (m, 5 H), 5.82-5.80 (m, 1H),
5.77-5.75 (m, 1H), 5.17 (s, 2H), and 4.22-4.18 ppm (m, 4H).
Benzyl 3,4-Epoxypyrrolidine-1-carboxylate (meso-11).26 N-
Bromosuccinimide (5.47 g, 31 mmol) was added to a solution
of olefin (9) (5.02 g, 25 mmol) in DMSO (65 mL) and water (3.4
mL) at 0 °C. The reaction mixture was warmed to room
temperature, stirred for 1.5 h, and then further N-bromosucci-
nimide (1.1 g, 6.2 mmol) was added. After the mixture was
stirred for a further 3.5 h, the reaction was quenched by the
addition of water (150 mL) and then extracted into EtOAc (3 ꢀ
150 mL). The combined organic phase was washed with brine
(3 ꢀ 100 mL), dried (MgSO4), filtered, and concentrated under
reduced pressure. The residue was dissolved in MeOH (80 mL),
cooled to 0 °C, and then an aqueous solution of NaOH (37 mL,
37 mmol, 1 M) was added in one portion. The mixture was
warmed to room temperature, stirred for 5 h, then the MeOH
was removed under reduced pressure. The residue was diluted
with water (100 mL) and extracted with EtOAc (3 ꢀ 200 mL).
The combined organic phases were washed with brine (200 mL),
dried (MgSO4), filtered, and concentrated under reduced pres-
sure. Flash chromatography of the residue (3:7 then 4:6, EtOAc/
Petrol) afforded meso-11 as a pale yellow oil (3.69 g, 68% over
the two steps). 1H NMR (500 MHz, CDCl3): δ = 7.35-7.30 (m,
5H), 5.11 (dd, J = 17.9, 12.4 Hz, 2H), 3.87 (dd, J = 25.0, 12.9
Hz, 2H), 3.68 (d, J = 5.1 Hz, 2H), and 3.39 ppm (ddd, J = 12.8,
7.6, 0.8 Hz, 2H). 13C NMR (125 MHz, CDCl3): δ = 155.3,
136.6, 128.5, 128.0, 127.9, 67.0, 55.5, 54.9 (rotamers), 47.5 and
47.2 (rotamers) ppm. ESI-HRMS for C12H13NO3Na [MNa]þ
calcd, 242.0793; found, 242.0791.
(()-trans-1-[(9-Deazaadenin-9-yl)methyl]-4-ethyl-3-hydroxy-
pyrrolidine [(()-28]. Formaldehyde (120 μL, 1.5 mmol, 37 wt %
solution in water) followed by 9-deazaadenine (102 mg, 0.9
mmol) was added to a solution of (()-20 (116 mg, 0.87 mmol) in
1,4-dioxane (1.6 mL) and water (3.2 mL). The reaction mixture
was stirred at room temperature for 15 h, absorbed onto silica,
and eluted down a silica column using a gradient 5-30% (7 N
NH3 in MeOH) in CH2Cl2. The crude product was collected,
concentrated, and subjected to flash chromatography (5:4.9:0.1
then 5:4.8:0.2, CH2Cl2/MeOH/28% aqueous NH4OH) to
1
afford (()-28 as a pale yellow solid (175 mg, 77%). H NMR
(500 MHz, CD3OD): δ = 8.16 (s, 1H), 7.49 (s, 1H), 3.87-3.78
(m, 3H), 3.07 (dd, J = 9.6, 8.1 Hz, 1H), 2.76 (dd, J = 10.4, 6.3
Hz, 1H), 2.70 (dd, J = 10.4, 4.0 Hz, 1H), 2.19 (dd, J = 9.7, 7.9
Hz, 1H), 1.91-1.84 (m, 1H), 1.59-1.50 (m, 1H), 1.38-1.28 (m,
1H), and 0.92 ppm (t, J = 7.4 Hz, 3H). 13C NMR (125 MHz,
CD3OD): δ = 152.1, 151.0, 147.0, 130.1, 115.1, 112.5, 77.4, 62.4,
59.4, 50.4, 49.1, 27.1, and 12.9 ppm; ESI-HRMS for C13H20N5O
[MH]þ calcd, 262.1668; found, 262.1664.
(()-Benzyl trans-4-Butyl-3-hydroxypyrrolidine-1-carboxylate
[(()-13]. A solution of epoxide (meso-11) (80 mg, 0.4 mmol) and
CuBr DMS (7 mg, 0.03 mmol) in THF (3 mL) was cooled to
3
-30 °C. n-Butylmagnesium chloride (1 mL, 2 mmol, 2 M
solution in THF) was added dropwise over 10 min, keeping
the internal temperature below -25 °C. After complete addition
the mixture was allowed to warm to -15 °C over 45 min and
then quenched with 10% aqueous NH4Cl solution (10 mL) and
EtOAc (20 mL). The mixture was stirred at room temperature
for 75 min, and then the layers were separated. The aqueous
phase was extracted with EtOAc (2 ꢀ 20 mL), and the combined
organic extracts were dried (MgSO4), filtered, and concentrated
under reduced pressure. Flash chromatography of the residue
(4:6 then 1:1, EtOAc/Petrol) afforded (()-13 as a pale yellow oil
(72 mg, 71%). 1H NMR (500 MHz, CDCl3): δ = 7.38-7.32 (m,
5H), 5.13 (s, 2H) 4.06-4.03 (m, 1H), 3.70-3.65 (m, 2H),
3.34-3.27 (m, 1H), 3.19-3.12 (m, 1H), 2.08-1.98 (m, 1H),
1.64 (br s, 1H), 1.51-1.44 (m, 1H), 1.36-1.15 (m, 5H), and
0.91-0.88 ppm (m, 3H). 13C NMR (125 MHz, CDCl3): δ =
155.1, 136.8, 128.5, 127.9, 127.8, 75.4, 74.6 (rotamers), 66.8,
53.0, 52.6 (rotamers), 49.6, 49.4 (rotamers), 46.2, 45.7
(rotamers), 31.2, 29.9, 29.7(rotamers), 22.7, and 13.9 ppm.
ESI-HRMS for C16H23NO3Na [MNa]þ calcd, 300.1576; found,
300.1584.
(()-Benzyl trans-4-Ethyl-3-hydroxypyrrolidine-1-carboxylate
[(()-12]. A solution of epoxide (meso-11) (467 mg, 2.13 mmol)
and CuBr DMS (53 mg, 0.26 mmol) in THF (20 mL) was cooled
3
to -30 °C. Ethylmagnesium bromide (10 mL, 10 mmol, 1 M
solution in THF) was added dropwise over 20 min, keeping the
internal temperature below -30 °C. After complete addition the
mixture was allowed to warm to -15 °C over 50 min and then
quenched with 10% aqueous NH4Cl solution (20 mL) and
EtOAc (40 mL). The mixture was stirred at room temperature
for 45 min, and then the layers were separated. The aqueous
phase was extracted with EtOAc (3 ꢀ 50 mL). The combined
organic phases were dried (MgSO4), filtered, and concentrated
under reduced pressure. Flash chromatography of the residue
(3:7 then 4:6, EtOAc/Petrol) afforded (()-12 as a pale yellow oil
(()-trans-4-Butyl-3-hydroxypyrrolidine [(()-21]. Palladium
(10 mg, 0.01 mmol, 10 wt % on carbon) was added to a solution
of (()-13 (70 mg, 0.3 mmol) in MeOH (4 mL) under argon. The
reaction mixture was placed under a hydrogen atmosphere and
stirred for 1.5 h and then filtered through Celite and concen-
trated under reduced pressure. Flash chromatography of the
residue (5:4.8:0.2 then 5:4.5:0.5, CH2Cl2/MeOH/28% aqueous
NH4OH) afforded (()-21 as a yellow oil (20 mg, 54%). 1H
NMR (500 MHz, CD3OD): δ = 4.19-4.17 (m, 1H), 3.55 (dd,
J = 11.7, 7.3 Hz, 1H), 3.41 (dd, J = 12.3, 5.1 Hz, 1H), 3.16 (dd,
J = 12.3, 3.1 Hz, 1H), 3.02 (dd, J = 11.7, 5.8 Hz, 1H), 2.23-2.17
(m, 1H), 1.55-1.49 (m, 1H), 1.40-1.28 (m, 5H) and 0.95-0.92
ppm (m, 3H). 13C NMR (125 MHz, CD3OD): δ = 75.3, 52.4,
50.0, 47.3, 31.9, 31.0, 23.6, and 14.3 ppm. ESI-HRMS for
C8H18NO [MH]þ calcd, 144.1388; found, 144.1390.
1
(370 mg, 70%). H NMR (500 MHz, CDCl3): δ = 7.35-7.28
(m, 5H), 5.11 (s, 2H), 3.99 (br s, 1H,), 3.69-3.60 (m, 2H), 3.29
(td, J = 11.3, 4.1 Hz, 1H), 3.16-3.08 (m, 2H), 1.99-1.92 (m,
1H), 1.53-1.47 (m, 1H), 1.24-1.17 (m, 1H), and 0.93 ppm (dd,
J = 12.4, 7.3 Hz, 3H). 13C NMR (125 MHz, CDCl3): δ = 155.3,
136.9, 128.5, 128.0, 127.9, 67.0, 66.9 (rotamers), 53.1, 52.7
(rotamers), 49.4, 49.2 (rotamers), 48.0, 47.4 (rotamers), 24.4,
and 12.3 ppm. ESI-HRMS for C14H19NO3Na [MNa]þ calcd,
272.1263; found, 272.1269.
(()-trans-4-Ethyl-3-hydroxypyrrolidine [(()-20]. Palladium
(20 mg, 0.02 mmol, 10 wt % on carbon) was added to a solution
of (()-12 (270 mg, 1.1 mmol) in MeOH (10 mL) under argon.
The reaction mixture was placed under a hydrogen atmosphere
and stirred for 1 h and then filtered through Celite and con-
centrated under reduced pressure. Flash chromatography of the
residue (5:4:1, CH2Cl2/MeOH/28% aqueous NH4OH) afforded
(()-20 as a yellow oil (70 mg, 56%). 1H NMR (500 MHz,
(()-trans-4-Butyl-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-
pyrrolidine [(()-29]. Formaldehyde (95 μL, 1.2 mmol, 37 wt %
solution in water) followed by 9-deazaadenine (100 mg, 0.7
mmol) was added to a solution of (()-21 (88 mg, 0.66 mmol) in
1,4-dioxane (1.2 mL) and water (2.5 mL). The reaction mixture
was stirred at room temperature for 66 h, absorbed onto silica,

6738 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Longshaw et al.
and eluted down a silica column using a gradient 10-20% (7 N
NH3 in MeOH) in CH2Cl2. The crude product was collected,
concentrated, and subjected to flash chromatography (5:4.8:0.2,
CH2Cl2/MeOH/28% aqueous NH4OH) to afford (()-29 as a
pale yellow solid (89 mg, 47%). 1H NMR (500 MHz, CD3OD):
δ = 8.16 (s, 1H), 7.5 (s, 1H), 3.86-3.79 (m, 3H), 3.07 (dd, J =
9.5, 8.0 Hz, 1H), 2.76 (dd, J = 10.4, 6.3 Hz, 1H), 2.71 (dd, J =
10.4, 4.0 Hz, 1H), 2.19 (dd, J = 9.7, 8.0 Hz, 1H), 1.98-1.91 (m,
1H), 1.55-1.48 (m, 1H), 1.35-1.25 (m, 5H), and 0.89 ppm (t, J
= 6.9 Hz, 3H). 13C NMR (125 MHz, CD3OD): δ = 152.1,
151.0, 147.0, 130.1, 115.2, 112.4, 77.7, 62.3, 59.7, 49.0, 48.5, 34.0,
31.5, 23.8, and 14.3 ppm. ESI-HRMS for C15H24N5O [MH]þ
calcd, 290.1981; found, 290.1989.
(()-Benzyl trans-3-Hydroxy-4-(penta-3-yl)pyrrolidine-1-
carboxylate [(()-15]. A solution of epoxide (meso-11) (230 mg,
1.05 mmol) and CuBr DMS (28 mg, 0.22 mmol) in THF (10 mL)
3
was cooled to -30 °C (internal temperature). 3-Pentylmagnesium
bromide (2.3 mL, 4.6 mmol, 2 M solution in ether) was added
dropwise over 10 min. After complete addition the mixture was
allowed to warm to -20 °C over 30 min and then quenched with
10% aqueous NH4Cl solution (40 mL) and EtOAc (40 mL). The
mixture was stirred at room temperature for 1 h, and then the layers
were separated. The aqueous phase was extracted with EtOAc (3 ꢀ
40 mL), and the combined organic layers were dried (MgSO4),
filtered, and concentrated under reduced pressure. Flash chroma-
tography of the residue (2:8, EtOAc/Petrol) afforded (()-15 and an
unknown corunning minor impurity as a pale yellow oil (195 mg).
1H NMR (500 MHz, CDCl3): δ= 7.37-7.29 (m, 5H), 5.11 (s, 2H),
4.20-4.14 (m, 1H), 3.73-3.69, (m, 1H), 3.67-3.59 (m, 1H),
3.29-3.23 (m, 1H), 3.19-3.14 (m, 1H), 2.14-2.07 (m, 1H),
1.42-1.38 (m, 3H), 1.30-1.20 (m, 2H), and 0.90-0.85 ppm
(m, 6H). 13C NMR (125 MHz, CDCl3): δ = 154.9, 136.9, 128.5,
127.9, 127.8, 73.3, 72.5 (rotamers), 67.4, 66.8 (rotamers), 53.5, 53.1
(rotamers), 48.4, 47.7, 47.4 (rotamers), 41.2, 23.1, 22.4 (rotamers),
and 10.9 ppm. ESI-HRMS for C17H25NO3Na [MNa]þ calcd,
314.1732; found, 314.1731 (for clarity only the data for (()-15 are
quoted).
(()-Benzyl trans-3-Hydroxy-4-isobutylpyrrolidine-1-carboxy-
late [(()-14]. A solution of epoxide (meso-11) (203 mg, 0.93
mmol) and CuBr DMS (30 mg, 0.15 mmol) in THF (8 mL) was
3
cooled to -30 °C. Isobutylmagnesium bromide (2.3 mL, 4.6
mmol, 2 M solution in THF) was added dropwise over 10 min,
keeping the internal temperature below -27 °C. After complete
addition the mixture was allowed to warm to -15 °C over 80 min
and then quenched with 10% aqueous NH4Cl solution (20 mL)
and EtOAc (50 mL). The mixture was stirred at room tempera-
ture for 45 min, and then the layers were separated. The aqueous
phase was extracted with EtOAc (2 ꢀ 50 mL), and the combined
organic layers were dried (MgSO4), filtered, and concentrated
under reduced pressure to afford crude (()-14 (193 mg, 75%).
This material was used in the next step without further purifica-
tion. 1H NMR (300 MHz, CDCl3): δ = 7.38-7.29 (m, 5H), 5.13
(s, 2H), 4.04-3.98 (m, 1H), 3.74-3.64 (m, 2H), 3.34-3.25 (m,
1H), 3.17-3.09 (m, 1H), 2.17-2.06 (m, 1H), 1.70-1.58 (m, 2H),
1.36-1.25 (m, 1H), 1.20-1.08 (m, 1H), and 0.91 ppm (t, J = 6.6
Hz, 6H). 13C NMR (125 MHz, CDCl3): δ = 155.1, 136.9, 128.5,
127.9, 127.9, 75.8, 75.0 (rotamers), 52.9, 52.5 (rotamers), 49.7, 49.5
(rotamers), 44.2, 43.6 (rotamers), 40.7, 26.3, 26.2 (rotamers), 23.2,
and 22.1 ppm. ESI-HRMS for C16H23NO3Na [MNa]þ calcd,
300.1576; found, 300.1575.
(()-trans-3-Hydroxy-4-(penta-3-yl)pyrrolidine [(()-23]. Palla-
dium (35 mg, 0.03 mmol, 10 wt % on carbon) was added to a
solution of crude (()-15 (195 mg, 0.9 mmol) in MeOH (10 mL)
under argon. The reaction mixture was placed under a hydrogen
atmosphere and stirred for 1 h and then filtered through Celite
andconcentratedunder reduced pressure. Flashchromatography
of the residue (5:4.9:0.1 then 5:4.8:0.2, CH2Cl2/MeOH/28%
aqueous NH4OH) afforded (()-23 as a pale yellow gum (27
1
mg, 16% over two steps). H NMR (500 MHz, CD3OD): δ =
4.07 (m, 1H), 3.15 (dd, J = 11.5, 8.2 Hz, 1H), 2.89 (dd, J = 12.1,
5.8 Hz, 1H), 2.80 (dd, J = 12.1, 3.3 Hz, 1H), 2.50 (dd, J = 11.5,
8.5 Hz, 1H), 1.94 (q, J = 8.2, 4.4 Hz, 1H), 1.54-1.39 (m, 3H),
(()-trans-3-Hydroxy-4-isobutylpyrrolidine [(()-22]. Palladium
(20mg, 0.02 mmol, 10 wt% on carbon) was addedtoa solutionof
crude (()-14 (190 mg, 0.7 mmol) in MeOH (10 mL) under argon.
The reaction mixture was placed under a hydrogen atmosphere
and stirred for 2 h and then filtered through Celite and concen-
trated under reduced pressure. Flash chromatography of the
residue (gradient 5:4.8:0.2 to 5:4:1, CH2Cl2/MeOH/28% aqueous
NH4OH) afforded (()-22 as a yellow oil (50 mg, 49%). 1H NMR
(500 MHz, CD3OD): δ = 4.16-4.13 (m, 1H), 3.57 (dd, J = 11.7,
7.3 Hz, 1H), 3.41 (dd, J = 12.3, 5.1 Hz, 1H), 3.16 (dd, J = 12.3,
3.1 Hz, 1H), 3.00 (dd, J = 11.7, 5.8 Hz, 1H), 2.32-2.26 (m, 1H),
1.69-1.61 (m, 1H), 1.39-1.34 (m, 1H), 1.23-1.18 (m, 1H), and
0.95 ppm (dd, J = 10.5, 6.6 Hz, 6H). 13C NMR (125 MHz,
CD3OD): δ = 75.6, 52.5, 50.2, 45.3, 41.4, 27.4, 23.2, and 22.6
ppm. ESI-HRMS for C8H18NO [MH]þ calcd, 144.1388; found,
144.1382.
(()-trans-1-[(9-Deazaadenin-9-yl)methyl]-3-hydroxy-4-isobutyl-
pyrrolidine [(()-30]. Formaldehyde (75 μL, 0.9 mmol, 37 wt %
solution in water) followed by 9-deazaadenine (52 mg, 0.4 mmol)
was added to a solution of (()-22 (49 mg, 0.34 mmol) in 1,4-
dioxane (0.6 mL) and water (1.2 mL). The reaction mixture was
stirred at room temperature for 17 h, absorbed onto silica, and
eluted down a silica column with 10% (7 N NH3 in MeOH) in
CH2Cl2. The crude product was collected, concentrated, and
subjected to flash chromatography (5:4.9:0.1, CH2Cl2/MeOH/
28% aqueous NH4OH) to afford (()-30 as an off-white solid
(42 mg, 42%). 1H NMR (500 MHz, CD3OD): δ = 8.16 (s, 1H),
7.49 (s, 1H), 3.86-3.78 (m, 3H), 3.09-3.05 (m, 1H), 2.75 (dd, J =
10.4, 6.3 Hz, 1H), 2.70 (dd, J = 10.3, 3.9 Hz, 1H), 2.18-2.14 (m,
1H), 2.09-2.02 (m, 1H), 1.61-1.52 (m, 1H), 1.42-1.36 (m, 1H),
1.23-1.17 (m, 1H), and 0.88 ppm (t, J = 6.7 Hz, 6H). 13C NMR
(125 MHz, CD3OD): δ = 152.1, 150.1, 147.0, 130.1, 115.1, 112.6,
78.1, 62.3, 59.8, 49.1, 46.2, 43.9, 27.7, 23.6, and 22.7 ppm. ESI-
HRMS for C15H24N5O [MH]þ calcd, 290.1981; found, 290.1979.
1.32-1.21 (m, 2H), and 0.91 ppm (dt, J = 12.9, 7.4 Hz, 6H). 13
C
NMR (125 MHz, CD3OD): δ = 76.7, 56.0, 52.1, 50.5, 43.4, 24.4,
23.6, 11.4, and11.0 ppm. ESI-HRMSfor C9H20NO[MH]þ calcd,
158.1545; found, 158.1540.
(()-trans-1-[(9-Deazaadenin-9-yl)methyl]-3-hydroxy-4-(penta-
3-yl)pyrrolidine [(()-31]. Formaldehyde (25 μL, 0.3 mmol,
37 wt % solution in water) followed by 9-deazaadenine (30 mg,
0.22 mmol) was added to a solution of (()-23 (27 mg, 0.17 mmol)
in 1,4-dioxane (0.6 mL) and water (0.6mL). The reaction mixture
was stirred at room temperature for 17 h, absorbed onto silica,
and eluted down a silica column with a gradient of 5-20% (7 N
NH3 in MeOH) in CH2Cl2. The crude product was collected,
concentrated, and subjected to flash chromatography (5:4.9:0.1,
CH2Cl2/MeOH/28% aqueous NH4OH) to afford (()-31 as a
pale yellow solid (24 mg, 33%). 1H NMR (500 MHz, CD3OD):
δ = 8.16 (s, 1H), 7.50 (s, 1H), 3.99-3.96 (m, 1H), 3.84 (d, J =
13.4 Hz, 1H), 3.77 (d, J = 13.4 Hz, 1H), 3.04 (t, J = 8.8 Hz, 1H),
2.79 (dd, J = 10.4, 2.3 Hz, 1H), 2.62 (dd, J = 10.4, 6.4 Hz, 1H),
2.17 (t, J = 9.5 Hz, 1H), 2.04-1.98 (m, 1H), 1.51-1.37 (m, 3H),
1.27-1.19 (m, 2H), 0.89 (t, J = 7.4, 3H), and 0.84 ppm (t, J =
7.1, 3H). 13C NMR (125MHz, CD3OD): δ = 152.1, 151.0, 147.0,
130.1, 115.2, 112.5, 75.8, 63.3, 58.2, 51.3, 49.1, 44.3, 24.0, 23.6,
11.3, and 10.7 ppm. ESI-HRMS for C16H26N5O [MH]þ calcd,
304.2137; found, 304.2137.
(()-Benzyl trans-4-Cyclopropyl-3-hydroxypyrrolidine-1-car-
boxylate [(()-16]. A solution of epoxide (meso-11) (283 mg,
1.3 mmol) and CuBr DMS (43 mg, 0.21 mmol) in THF (10 mL)
3
was cooled to -30 °C (internal temperature). Cyclopropyl-
magnesium bromide (10 mL, 5.0 mmol, 0.5 M solution in THF)
was added dropwise over 25 min. After complete addition the
mixture was allowed to slowly warm to -15 °C over 45 min and
then quenched with 10% aqueous NH4Cl solution (20 mL) and
EtOAc (50 mL). The mixture was stirred at room temperature
for 40 min, and then the layers were separated. The aqueous

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6739
phase was extracted with EtOAc (3 ꢀ 50 mL), and the combined
organic phases were dried (MgSO4), filtered, and concentrated
under reduced pressure. Flash chromatography of the residue
(3:7 then 4:6, EtOAc/Petrol) afforded (()-16 as a pale yellow oil
stirred for 1 h, and then further catalyst (50 mg) was added. The
reaction mixture was placed under a hydrogen atmosphere for a
further 1 h and then filtered through Celite and concentrated
under reduced pressure. Flash chromatography of the residue
(5:4.6:0.4, CH2Cl2/MeOH/28% aqueous NH4OH) afforded
crude (()-25 (43 mg) which was used without further purifica-
tion and characterization in the next step.
1
(311 mg, 92%). H NMR (500 MHz, CDCl3): δ = 7.38-7.29
(m, 5H), 5.13 (s, 2H), 4.25-4.22 (m, 1H), 3.75-3.70 (m, 1H),
3.69-3.63 (m, 1H), 3.40-3.28 (m, 1H), 1.94-1.85 (m, 1H),
1.48-1.43 (m, 1H), 0.60-0.44 (m, 3H), 0.30-0.25 (m, 1H), and
0.16-0.10 ppm (m, 1H). 13C NMR (125 MHz, CDCl3): δ =
155.1, 136.9, 128.5, 128.0, 127.9, 75.8, 75.0 (rotamers), 66.9,
53.3, 52.8 (rotamers), 51.3, 50.5 (rotamers), 49.2, 49.1 (rota-
mers), 12.4, 12.2 (rotamers), 3.4, 3.4 (rotamers), 3.1 and 3.0
(rotamers) ppm. ESI-HRMS for C15H19NO3Na [MNa]þ calcd,
284.1263; found, 284.1260.
(()-trans-4-Cyclopentyl-1-[(9-deazaadenin-9-yl)methyl]-3-
hydroxypyrrolidine [(()-33]. Formaldehyde (35 μL, 0.4 mmol,
37 wt % solution in water) followed by 9-deazaadenine (42 mg,
0.3 mmol) was added to a solution of crude (()-25 (43 mg) in 1,4-
dioxane (1 mL) and water (1 mL). The reaction mixture was
stirred at room temperature for 94 h, absorbed onto silica, and
eluted down a silica column with a gradient of 5-30% (7 N NH3
in MeOH) in CH2Cl2. The crude product was collected, con-
centrated, and subjected to flash chromatography (5:4.95:0.05,
CH2Cl2/MeOH/28% aqueous NH4OH) to afford (()-33 as an
(()-trans-4-Cyclopropyl-3-hydroxypyrrolidine [(()-24]. Palla-
dium (25 mg, 0.02 mmol, 10 wt % on carbon) was added to a
solution of (()-16 (310 mg, 1.2 mmol) in MeOH (20 mL) under
argon. The reaction mixture was placed under a hydrogen atmo-
sphere and stirred for 1 h and then filtered through Celite and
concentrated under reduced pressure. Flash chromatography of
the residue (5:4.6:0.4, CH2Cl2/MeOH/28% aqueous NH4OH)
afforded (()-24 as an off-white solid (126 mg, 84%). 1H NMR
(500 MHz, CD3OD): δ = 4.30 (dt, J = 5.0, 2.7 Hz, 1H), 3.51 (dd,
J = 11.7, 7.3 Hz, 1H), 3.43 (dd, J = 12.4, 4.7 Hz, 1H), 3.17 (dd,
J = 12.3, 2.2 Hz, 1H), 3.11 (dd, J = 11.7, 4.6 Hz, 1H), 1.61-1.55
(m, 1H), 0.71-0.63 (m, 1H), 0.57-0.48 (m, 1H), 0.33-0.29 (m,
1H), and 0.21-0.17 ppm (m, 1H). 13C NMR (125 MHz, CD3-
OD): δ = 76.4, 52.9, 52.6, 50.0, 13.2, 4.2, and 3.6 ppm. ESI-
HRMS for C7H14NO [MH]þ calcd, 128.1075; found, 128.1082.
(()-trans-4-Cyclopropyl-1-[(9-deazaadenin-9-yl)methyl]-3-
hydroxypyrrolidine [(()-32]. Formaldehyde (50 μL, 0.6 mmol,
37 wt % solution in water) followed by 9-deazaadenine (60 mg,
0.45 mmol) was added to a solution of (()-24 (51 mg, 0.40 mmol)
in1,4-dioxane (1 mL) andwater (1mL). The reactionmixturewas
stirred at room temperature for 41 h, absorbed onto silica, and
eluted down a silica column with a gradient of 5-30% (7 N NH3
in MeOH) in CH2Cl2. The crude product was collected, concen-
trated, and subjected to flash chromatography (5:4.5:0.5, CH2Cl2/
MeOH/28% aqueous NH4OH) to afford (()-32 as an off-white
solid (59 mg, 54%). 1H NMR (500 MHz, CD3OD): δ = 8.16 (s,
1H), 7.45 (s, 1H), 4.05 (dt, J = 6.4, 4.1 Hz, 1H), 3.85 (d, J = 13.4
Hz, 1H), 3.80 (d, J = 13.4 Hz, 1H), 3.02 (dd, J = 9.6, 8.1 Hz, 1H),
2.82 (dd, J = 10.5, 6.4 Hz, 1H), 2.68 (dd, J = 10.4, 3.9 Hz, 1H),
2.36 (dd, J = 9.8, 7.5 Hz, 1H), 1.38 (ddd, J = 16.9, 7.8, 4.3 Hz,
1H), 0.75-0.68 (m, 1H), 0.46-0.37 (m, 2H), 0.28-0.24 (m, 1H),
and 0.08-0.03 ppm (m, 1H). 13C NMR(125 MHz, CD3OD):δ =
152.1, 151.0, 147.0, 130.1, 115.2, 112.5, 77.4, 62.6, 59.1, 53.6, 49.1,
14.5, 3.8, and 3.5 ppm. ESI-HRMS for C14H20N5O [MH]þ calcd,
274.1668; found, 274.1666.
1
off-white solid (28 mg, 10%, over three steps). H NMR (500
MHz, CD3OD): δ = 8.16 (s, 1H), 7.50 (s, 1H), 3.97-3.94 (m,
1H), 3.85 (d, J = 13.4 Hz, 1H), 3.80 (d, J = 13.4 Hz, 1H), 3.06
(dd, J = 9.6, 8.1 Hz, 1H), 2.76-2.68 (m, 2H), 2.39 (dd, J = 9.7,
8.1 Hz, 1H), 1.89-1.50 (m, 8H), 1.34-1.30 (m, 1H) and
1.21-1.08 ppm (m, 1H). 13C NMR (125 MHz, CD3OD): δ =
152.1, 151.0, 147.0, 130.2, 115.2, 112.4, 76.7, 63.0, 59.1, 54.2,
49.1, 44.7, 32.2, 31.8, and 26.1 (2C) ppm. ESI-HRMS for
C16H23N5ONa [MNa]þ calcd, 324.1800; found, 324.1802.
(()-Benzyl trans-4-(Cyclohexylmethyl)-3-hydroxypyrrolidine-1-
carboxylate [(()-18]. A solution of epoxide (meso-11) (243 mg, 1.1
mmol) and CuBr DMS (50 mg, 0.22 mmol) in THF (10 mL) was
3
cooled to -30 °C (internal temperature). Cyclohexylmethylmag-
nesium bromide (11 mL, 5.5 mmol, 0.5 M solution in THF) was
added dropwise over 20 min. After complete addition the mixture
was allowed to warm to -25 °C over 30 min and then quenched
with 10% aqueous NH4Cl solution (20 mL) and EtOAc (20 mL).
The mixture was stirred at room temperature for 30 min, and then
the layers were separated. The aqueous phase was extracted with
EtOAc (3 ꢀ 30 mL), and the combined organic phases were dried
(MgSO4), filtered, and concentrated under reduced pressure. Flash
chromatography of the residue (gradient 2:8 to 4:6, EtOAc/Petrol)
1
afforded (()-18 as an off-white solid (286 mg, 81%). H NMR
(500 MHz, CDCl3): δ = 7.40-7.30 (m, 5H), 5.13 (s, 2H), 4.01 (t,
J = 4.8 Hz, 1H), 3.72-3.64 (m, 2H), 3.33-3.26 (m, 1H), 3.16-
3.10 (m, 1H), 2.19-2.11 (m, 1H), 1.74-1.64 (m, 6H), 1.36-1.07
(m, 6H), and 0.95-0.81 ppm (m, 2H). 13C NMR (125 MHz,
CDCl3): δ = 155.1, 137.0, 128.5, 128.0, 127.9, 75.9, 75.1 (rota-
mers), 66.8, 52.9, 52.5 (rotamers), 49.8, 49.5 (rotamers), 43.5, 43.0
(rotamers), 39.4, 39.2 (rotamers), 35.8, 35.7 (rotamers), 34.0, 32.9,
26.5, 26.2, and 26.2 ppm. ESI-HRMS for C19H27NO3Na [MNa]þ
calcd, 340.1895; found, 340.1895.
(()-Benzyl trans-4-Cyclopentyl-3-hydroxypyrrolidine-1-car-
boxylate [(()-17]. Cyclopentyl bromide (0.5 mL, 4.7 mmol)
was added dropwise to a suspension of magnesium (221 mg,
9 mmol) in THF (10 mL), activated with 1,2-dibromoethane.
After complete addition the reaction mixture was stirred for
1 h at room temperature and then added dropwise, over 10 min,
to a solution of epoxide (meso-11) (230 mg, 1 mmol) and
(()-trans-4-(Cyclohexylmethyl)-3-hydroxypyrrolidine [(()-26].
Palladium (20 mg, 0.02 mmol, 10 wt % on carbon) was added to
a solution of (()-18 (286 mg, 0.9 mmol) in MeOH (10 mL) under
argon. The reaction mixture was placed under a hydrogen atmo-
sphere and stirred for 1 h, and then further catalyst (20 mg, 0.02
mmol, 10 wt % on carbon) was added. The reaction mixture was
stirred under a hydrogen atmosphere for a further 1 h and then
filtered through Celite and concentrated under reduced pressure.
Flash chromatography of the residue (5:4.8:0.2 and then 5:4.5:0.5,
CH2Cl2/MeOH/28% aqueous NH4OH) afforded (()-26 as an off-
white solid (115 mg, 70%). 1H NMR (500 MHz, CD3OD): δ =
3.88 (q, J = 4.2 Hz, 1H), 3.20 (dd, J = 11.2, 7.6 Hz, 1H), 2.97 (dd,
J = 12.0, 5.5 Hz, 1H), 2.76 (dd, J = 12.0, 3.4 Hz, 1H), 2.45 (dd,
J = 11.2, 6.4 Hz, 1H), 2.07-2.01 (m, 1H), 1.80-1.65 (m, 5H),
1.39-1.10 (m, 6H), and 0.97-0.86 ppm (m, 2H). 13C NMR (125
MHz, CD3OD): δ = 78.8, 54.5, 52.1, 46.2, 41.6, 37.4, 35.1, 34.3,
27.7, and 27.4 (2 ꢀ C) ppm. ESI-HRMS for C11H22NO [MH]þ
calcd, 184.1701; found, 184.1696.
CuBr DMS (55 mg, 0.3 mmol) in THF (10 mL) at -30 °C
3
(internal temperature). The reaction mixture was stirred for
75 min and then quenched with 10% aqueous NH4Cl solution
(20 mL) and EtOAc (20 mL). The biphasic mixture was stirred
for 1 h and then the layers were separated, and the aqueous
phase was extracted with EtOAc (2 ꢀ 50 mL). The combined
organic phases were dried (MgSO4), filtered, and concentrated
under reduced pressure to afford the crude (()-17 as a pale
yellow oil, which was used immediately in the next step without
characterization.
(()-trans-4-Cyclopentyl-3-hydroxypyrrolidine [(()-25]. Palla-
dium (50 mg, 0.05 mmol, 10 wt % on carbon) was added to a
solution of crude (()-17 in MeOH (20 mL) under argon. The
reaction mixture was placed under a hydrogen atmosphere and
(()-trans-4-(Cyclohexylmethyl)-1-[(9-deaza-adenin-9-yl)methyl]-
3-hydroxypyrrolidine [(()-34]. Formaldehyde (35 μL, 0.4 mmol,
37 wt % solution in water) followed by 9-deazaadenine (42 mg,

6740 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Longshaw et al.
0.31 mmol) was added to a solution of (()-26 (51 mg, 0.28 mmol) in
1,4-dioxane (1.5 mL) and water (1.5 mL). The reaction mixture was
stirred at room temperature for 67 h, absorbed onto silica, and
eluted down a silica column with a gradient of 5-30% (7 N NH3 in
MeOH) in CH2Cl2. The crude product was collected, concentrated,
and subjected to flash chromatography (5:4.9:0.1, CH2Cl2/MeOH/
28% aqueous NH4OH) to afford (()-34 as a pale yellow solid (42
mg, 46%). 1H NMR (500 MHz, CD3OD): δ=8.16 (s, 1H), 7.49(s,
1H), 3.86-3.78 (m, 3H), 3.08-3.05 (m, 1H), 2.75 (dd, J = 10.4, 6.4
Hz, 1H), 2.70 (dd, J = 10.4, 4.0 Hz, 1H), 2.16 (dd, J = 9.5, 8.1 Hz,
1H), 2.11-2.01 (m, 1H), 1.75-1.62 (m, 5H), 1.43-1.38 (m, 1H),
1.28-1.11 (m, 5H), and 0.95-0.81 ppm (m, 2H). 13C NMR (125
MHz, CD3OD): δ = 152.1, 151.1, 147.0, 130.1, 115.1, 112.5, 78.1,
62.2, 59.8, 49.1, 45.8, 42.4, 37.4, 35.1, 34.2, 27.7, and 27.4 (2 ꢀ C)
ppm. ESI-HRMS for C18H28N5O [MH]þ calcd, 330.2294; found,
330.2297.
(()-tert-Butyl trans-3-Hydroxy-4-vinylpyrrolidine-1-carboxylate
[(()-36].23 A solution of epoxide (meso-10) (331 mg, 1.8 mmol)
and CuBr DMS (73 mg, 0.35 mmol) in THF (15 mL) was cooled
3
to -30 °C (internal temperature). Vinylmagnesium bromide (8 mL,
8.0 mmol, 1 M solution in THF) was added dropwise over
15 min. After complete addition the reaction was allowed to
slowly warm to -10 °C over 1 h and then quenched with 10%
aqueous NH4Cl solution (20 mL) and EtOAc (50 mL). The
mixture was stirred at room temperature for 1 h, and then the
layers were separated. The aqueous phase was extracted with
EtOAc (3 ꢀ 50 mL), and the combined organic phases were
dried (MgSO4), filtered, and concentrated under reduced pres-
sure to afford (()-36 as a pale yellow oil (286 mg, 75%). No
further purification was necessary. 1H NMR (500 MHz,
CDCl3): δ = 5.74-5.67 (m, 1H), 5.20 (d, J = 17.2 Hz, 1H),
5.15 (d, J = 10.4 Hz, 1H), 4.14-4.08 (m, 1H), 3.73-3.61 (m,
2H), 3.29-3.17 (m, 2H), 2.71-2.65 (m, 1H), 2.71 (br s, 1H),
and 1.46 ppm (s, 9H). 13C NMR (125 MHz, CDCl3): δ = 154.5,
136.3, 117.3, 79.5, 74.5, 74.2 (rotamers), 52.2, 51.9 (rotamers), 50.5,
49.9 (rotamers), 48.8, 48.3 (rotamers), and 28.5 ppm. ESI-HRMS
for C11H19NO3Na [MNa]þ calcd, 236.1263; found, 236.1263.
(()-trans-3-Hydroxy-4-vinylpyrrolidine [(()-38].23 Aqueous
HCl (36%, 1 mL) was added to a solution of (()-36 (286 mg,
1.34 mmol) in MeOH (20 mL). The reaction mixture was
concentrated under reduced pressure and then azeotroped with
MeOH (20 mL) followed by toluene (20 mL). Flash chroma-
tography of the residue (5:4.6:0.4, CH2Cl2/MeOH/28% aqu-
eous NH4OH) afforded (()-38 as a brown oil (95 mg, 63%). 1H
NMR (500 MHz, CD3OD): δ = 5.78 (ddd, J = 17.3, 10.4, 7.9
Hz, 1H), 5.12 (dt, J = 17.3, 1.5 Hz, 1H), 5.06 (ddd, J = 10.4, 1.5,
1.0 Hz, 1H), 4.03 (dt, J = 5.6, 4.3 Hz, 1H), 3.21 (dd, J = 11.4, 7.6
Hz, 1H), 3.03 (dd, J = 11.9, 5.7 Hz, 1H), 2.77 (dd, J = 11.8, 4.1
Hz, 1H), 2.68 (dd, J = 11.3, 6.7 Hz, 1H), and 2.61-2.56 ppm (m,
1H). 13C NMR (125 MHz, CD3OD): δ = 139.4, 116.1, 78.3,
54.6, 53.5, and 51.3 ppm. ESI-HRMS for C6H12NO [MH]þ
calcd, 144.0919; found, 114.0911.
(()-Benzyl trans-3-Hydroxy-4-phenylpyrrolidine-1-carboxylate
[(()-19]. A solution of epoxide (meso-11) (149 mg, 0.68 mmol)
and CuBr DMS (30 mg, 0.15 mmol) in THF (5.6 mL) was cooled
3
to -30 °C. Phenylmagnesium bromide (1.5 mL, 1.5 mmol, 1 M
solution in THF) was added dropwise over 10 min, keeping the
internal temperature below -25 °C. After complete addition the
mixture was allowed to warm to -15 °C over 90 min and then
cooled back to -30 °C, and further phenylmagnesium bro-
mide (1.5 mL, 1.5 mmol, 1 M solution in THF) was added. The
reaction mixture was allowed to warm to -20 °C over 1 h and
then quenched with 10% aqueous NH4Cl solution (20 mL)
and EtOAc (20 mL). The mixture was stirred at room tem-
perature for 30 min, and then the layers were separated. The
aqueous phase was extracted with EtOAc (3 ꢀ 20 mL), and the
combined organic phases were dried (MgSO4), filtered, and
concentrated under reduced pressure. Flash chromatography
of the residue (gradient 3:7 to 1:1, EtOAc/Petrol) afforded a
mixture of (()-19 and unreacted epoxide (meso-11) as a pale
yellow oil (162 mg, 2:1, (()-19/meso-11) which was used
without characterization.
(()-trans-1-[(9-Deazaadenin-9-yl)methyl]-3-hydroxy-4-vinyl-
pyrrolidine [(()-40]. Formaldehyde (45 μL, 0.56 mmol, 37 wt %
solution in water) followed by 9-deazaadenine (61 mg, 0.46
mmol) was added to a solution of (()-38 (42 mg, 0.37 mmol) in
1,4-dioxane (1 mL) and water (1 mL). The reaction mixture was
stirred at room temperature for 17 h, absorbed onto silica, and
eluted down a silica column with a gradient of 5-20% (7 N NH3 in
MeOH) in CH2Cl2. The crude product was collected, concen-
trated, and subjected to flash chromatography (5:4.5:0.5, CH2Cl2/
MeOH/28% aqueous NH4OH) to afford (()-40 as an off-white
solid (55 mg, 57%). 1H NMR (500 MHz, CD3OD): δ = 8.16 (s,
1H), 7.49 (s, 1H), 5.79 (ddd, J = 17.2, 10.3, 8.0 Hz, 1H), 5.06 (dt,
J = 17.2, 1.4 Hz, 1H), 4.99 (dq, J = 10.3, 1.0 Hz, 1H), 4.01-3.98
(m, 1H), 3.87 (d, J = 13.5 Hz, 1H), 3.82 (d, J = 13.5 Hz, 1H), 3.05
(dd, J = 9.8, 8.1 Hz, 1H), 2.86 (dd, J = 10.4, 6.7 Hz, 1H), 2.73 (dd,
J = 10.4, 4.5 Hz, 1H), 2.65-2.59 (m, 1H), and 2.42 ppm (dd, J =
9.9, 8.3 Hz, 1H). 13C NMR (125 MHz, CD3OD): δ = 152.1, 151.0,
147.0, 139.7, 130.3, 116.0, 115.2, 112.1, 77.2, 61.9, 58.7, 52.9, and
49.1 ppm. ESI-HRMS for C13H18N5O [MH]þ calcd, 260.1511;
found, 260.1511.
(()-trans-3-Hydroxy-4-phenylpyrrolidine [(()-27]. Palladium
(25 mg, 0.02 mmol, 10 wt % on carbon) was added to a solution
of (()-19/meso-11 (2: 1) (160 mg) in MeOH (12 mL) under
argon. The reaction mixture was placed under a hydrogen
atmosphere and stirred for 75 min and then filtered through
Celite and concentrated under reduced pressure. Flash chroma-
tography of the residue (5:4.8:0.2 then 5:4.6:0.4, CH2Cl2/
MeOH/28% aqueous NH4OH) afforded (()-27 as a yellow oil
1
(39 mg, 35%, over two steps). H NMR (500 MHz, CD3OD):
δ = 7.32-7.26 (m, 4H), 7.22-7.19 (m, 1H), 4.30-4.27 (m, 1H),
3.46 (dd, J = 11.5, 8.2 Hz, 1H), 3.18 (dd, J = 11.9, 6.0 Hz, 1H),
3.12 (td, J = 8.2, 5.4 Hz, 1H), and 2.92-2.88 ppm (m, 2H). 13
C
NMR (125 MHz, CD3OD): δ = 142.7, 129.7, 128.5, 127.8, 80.0,
55.3, 54.9, and 53.6 ppm. ESI-HRMS for C10H14NO [MH]þ
calcd 164.1075; found, 164.1068.
(()-trans-1-[(9-Deazaadenin-9-yl)methyl]-3-hydroxy-4-phenyl-
pyrrolidine [(()-35]. Formaldehyde (35 μL, 0.4 mmol, 37 wt %
solution in water) followed by 9-deazaadenine (39 mg, 0.24
mmol) was added to a solution of (()-27 (30 mg, 0.22 mmol) in
1,4-dioxane (0.4 mL) and water (0.8 mL). The reaction mixture
was stirred at room temperature for 17 h, absorbed onto silica,
and eluted down a silica column with a gradient of 10-20% (7 N
NH3 in MeOH) in CH2Cl2. The crude product was collected,
concentrated, and subjected to flash chromatography (5:4.9:0.1
then 5:4.8:0.2, CH2Cl2/MeOH/28% aqueous NH4OH) to afford
(()-35 as a white solid (38 mg, 55%). 1H NMR (500 MHz,
CD3OD): δ = 8.17 (s, 1H), 7.52 (s, 1H), 7.26 (br s, 2H), 7.25 (br s,
2H), 7.18-7.15 (m, 1H), 4.28-4.25(m, 1H), 3.89 (q, J = 13.4 Hz,
2H), 3.28-3.25 (m, 1H), 3.16 (td, J = 8.7, 5.7 Hz, 1H), 3.00 (dd,
J = 10.3, 6.8 Hz, 1H), 2.85 (dd, J = 10.3, 4.3 Hz, 1H), and 2.64
ppm (t, J = 9.3 Hz, 1H). 13C NMR (125 MHz, CD3OD): δ =
152.1, 151.0, 147.0, 143.6, 130.1, 129.5, 128.6, 127.5, 115.2, 112.6,
79.3, 62.8, 61.2, 54.9, and 49.1 ppm. ESI-HRMS for C17H19N5O-
Na [MNa]þ calcd, 332.1487; found, 332.1490.
(()-tert-Butyl trans-4-Allyl-3-hydroxypyrrolidine-1-carboxy-
late [(()-37].23 A solution of epoxide (meso-10) (227 mg, 1.2
mmol) in diethyl ether (2.6 mL) was added dropwise to a
solution of allylmagnesium chloride (1.4 mL, 2.8 mmol, 2 M
solution in THF) in diethyl ether (4.4 mL) at 0 °C (internal
temperature). The reaction mixture was stirred at 0 °C for 15
min and then warmed to room temperature. After 90 min the
reaction mixture was quenched by the addition of saturated
aqueous NH4Cl solution (20 mL) and then extracted with
EtOAc (3 ꢀ 50 mL). The combined organic phases were washed
with brine (2 ꢀ 50 mL), dried (MgSO4), filtered, and concen-
trated under reduced pressure. Flash chromatography of the
residue (1:9, EtOAc/Petrol) afforded (()-37 as a pale yellow oil
1
(138 mg, 50%). H NMR (500 MHz, CDCl3): δ = 5.82-5.75

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6741
(m, 1H), 5.09-5.04 (m, 2H), 4.04 (dd, J = 10.1, 4.8 Hz, 1H),
3.64-3.54 (m, 2H), 3.26-3.21 (m, 1H), 3.11-3.05 (m, 1H),
2.24-2.19 (m, 1H), 2.17-2.11 (m, 1H), 2.07-1.98 (m, 1H), and
1.45 ppm (s, 9H). 13C NMR (125 MHz, CDCl3): δ = 154.8,
135.8, 116.7, 79.5, 74.7, 74.0 (rotamers), 52.7, 52.4 (rotamers),
49.0, 48.7 (rotamers), 45.5, 45.0 (rotamers), 35.6, and 28.7 ppm.
ESI-HRMS for C12H21NO3Na [MNa]þ calcd, 250.1419; found,
250.1416.
was subsequently azeotroped with methanol (20 mL) followed by
toluene (20 mL) and then absorbed on to silica and eluted down a
flash column (5:4.8:0.2, CH2Cl2/MeOH/28% aqueous NH4OH)
to afford (()-43 as a yellow solid (276 mg, 89%). 1H NMR (500
MHz, CD3OD): δ = 4.28 (dt, J = 5.0, 2.8 Hz, 1H), 3.35 (dd, J =
11.2, 7.4 Hz, 1H), 3.09 (dd, J = 12.2, 5.0 Hz, 1H), 2.87-2.78 (m,
3H), and 0.13 ppm (s, 9H). 13C NMR (125 MHz, CD3OD): δ =
107.6, 87.6, 79.0, 54.8, 52.8, 41.8, and 0.0 ppm. ESI-HRMS for
C9H18NOSi [MH]þ calcd, 184.1158; found, 184.1161.
(()-trans-4-Allyl-3-hydroxypyrrolidine [(()-39].23 Aqueous
HCl (36%, 1 mL, 33 mmol) was added to a stirred solution of
(()-37 (59 mg, 0.26 mmol) in methanol (5 mL). The reaction
mixture was concentrated under reduced pressure and subse-
quently azeotroped with methanol (10 mL) followed by toluene
(10 mL). The residue was absorbed onto silica and eluted down a
flash column (5:4.5:0.5, CH2Cl2/MeOH/28% aqueous NH4OH)
(()-trans-1-[(9-Deazaadenin-9-yl)methyl]-3-hydroxy-4-((tri-
methylsilyl)ethynyl)pyrrolidine [(()-44]. Formaldehyde (55 μL,
0.69 mmol, 37 wt % solution in water) followed by 9-deaza-
adenine (73 mg, 0.54 mmol) was added to a solution of 45
(81 mg, 0.44 mmol) in 1,4-dioxane (2.5 mL) and water (2.5 mL).
The reaction mixture was stirred at room temperature for 66 h
and then concentrated under reduced pressure. Crude (()-44
was used directly in the next step without characterization.
(()-trans-1-[(9-Deazaadenin-9-yl)methyl]-4-ethynyl-3-hydroxy-
pyrrolidine [(()-45]. Sodium methoxide (10 μL, 0.05 mmol, 30 wt %
solution in methanol) was added to a solution of crude (()-44
(145 mg, 0.44 mmol) in methanol (5 mL). The reaction mixture was
stirred at room temperature for 2.5 h, and then further sodium
methoxide (10 μL, 0.05 mmol, 30 wt % solution in methanol) was
added. After being stirred for a further 17 h, the reaction mixture
was absorbed onto silica and eluted down a silica column (5:4.95:
0.05, CH2Cl2/MeOH/28% aqueous NH4OH) to afford (()-45
(29 mg, 25%). A sample was purified by preparative HPLC to
1
to afford (()-39 as a yellow oil (40 mg, 95%). H NMR (500
MHz, CD3OD): δ = 5.87-5.79 (m, 1H), 5.08-5.00 (m, 2H),
3.95-3.93 (m, 1H), 3.15 (dd, J = 11.5, 7.0 Hz, 1H), 2.95 (dd, J =
12.0, 5.5 Hz, 1H), 2.74 (dd, J = 12.0, 3.4 Hz, 1H), 2.48 (dd, J =
11.6, 5.5 Hz, 1H), 2.24-2.19 (m, 1H), and 2.04-1.96 ppm (m,
2H). 13C NMR (125 MHz, CD3OD): δ = 136.7, 116.7, 77.4,
54.3, 51.0, 48.3, and 37.3 ppm. ESI-HRMS for C7H14NO
[MH]þ calcd, 128.1075; found, 128.1072.
(()-trans-4-Allyl-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-
pyrrolidine [(()-41]. Formaldehyde (44 μL, 0.54 mmol, 37 wt %
solution in water) followed by 9-deazaadenine (44 mg, 0.33
mmol) was added to a solution of (()-39 (40 mg, 0.32 mmol) in
1,4-dioxane (0.6 mL) and water (1.2 mL). The reaction mixture
was stirred at room temperature for 17 h, absorbed onto silica,
and eluted down a silica column with a gradient of 5-40% (7 N
NH3 in MeOH) in CH2Cl2. The crude product was collected,
concentrated, and subjected to flash chromatography (5:4.9:0.1,
CH2Cl2/MeOH/28% aqueous NH4OH) to afford (()-41 as an off-
white solid (20 mg, 24%). 1H NMR (500 MHz, CD3OD): δ= 8.16
(s, 1H), 7.49 (s, 1H), 5.83-5.74 (m, 1H), 5.05-4.95 (m, 2H), 3.89
(dt, J = 6.3, 4.1 Hz, 1H), 3.85 (d, J = 13.4 Hz, 1H), 3.80 (d, J =
13.6 Hz, 1H), 3.02 (dd, J = 9.9, 7.6 Hz, 1H), 2.80 (dd, J = 10.4, 6.4
Hz, 1H), 2.68 (dd, J = 10.4, 4.1 Hz, 1H), 2.30-2.24 (m, 2H), and
2.10-2.02 ppm (m, 2H). 13C NMR (125 MHz, CD3OD): δ =
152.1, 151.0, 147.0, 138.0, 130.1,116.3, 115.1, 112.5, 76.8, 62.2, 59.0,
49.1, 48.0, and 38.2 ppm. ESI-HRMS for C14H20N5O [MH]þ
calcd, 274.1668; found, 274.1661.
1
analytical purity as a TFA salt. H NMR (500 MHz, CD3OD):
δ = 8.47 (s, 1H), 8.07 (s, 1H), 4.68 (s, 2H), 4.53 (quin, J = 2.2 Hz.
1H), 3.91 (dd, J = 11.8, 7.4 Hz, 1H), 3.74 (dd, J = 12.3, 4.1 Hz,
1H), 3.59 (dd, J = 11.7, 4.1 Hz, 1H), 3.49 (d, J = 12.4 Hz, 1H),
3.22-3.19 (m, 1H), and 2.78 ppm (d, J = 2.6 Hz, 1H). 13C NMR
(125 MHz, CD3OD): δ = 159.2, 152.2, 145.3, 136.0, 114.1, 104.3,
81.2, 75.9, 75.1, 60.5, 58.1, 50.2, and 39.2 ppm (resonance signals
due to CF3COOH have not been quoted). ESI-HRMS for
C13H16N5O [MH]þ calcd, 258.1355; found, 258.1355.
(()-tert-Butyl trans-4-Azido-3-hydroxypyrrolidine-1-carboxylate
[(()-46].29 Sodium azide (1.02 g, 15.7 mmol) was added to a solu-
tion of epoxide (meso-10) (1.0 g, 5.4 mmol) in 1,4-dioxane
(9 mL) and water (1.8 mL). The resulting suspension was heated
to 100 °C for 65 h and then cooled to 0 °C, and water (20 mL) was
added. The mixture was extracted with EtOAc (3 ꢀ 50 mL), and the
combined organic phases were washed with brine (50 mL), dried
(MgSO4), filtered, and concentrated under reduced pressure. Flash
chromatography of the residue (3:7, then 4:6, EtOAc/Petrol)
afforded (()-46 as a pale yellow oil (1.21 g, 98%). 1H NMR (500
MHz, CDCl3): δ = 4.23 (br s, 1H), 3.93 (br s, 1H), 3.70-3.66 (m,
1H), 3.60-3.56 (m, 1H), 3.46-3.16 (m, 3H), and 1.46 ppm (s, 9H).
13C NMR (125 MHz, CDCl3): δ = 154.6, 80.3, 74.1, 73.3
(rotamers), 65.4, 64.9 (rotamers), 51.9, 51.6 (rotamers), 48.7,
48.2 (rotamers), and 28.4 ppm. ESI-HRMS for C9H16N4O3Na
[MNa]þ calcd, 251.1120; found, 251.1121.
(()-tert-Butyl trans-3-Hydroxy-4-(5-(trimethylsilyl)-1H-1,2,3-
triazol-1-yl)pyrrolidine-1-carboxylate [(()-47]. Trimethylsilylace-
tylene (2.2 mL, 15.6 mmol) was added to a solution of (()-46
(730 mg, 3.2 mmol) in toluene (35 mL). The resulting mixture was
heated to reflux for 88 h and then allowed to cool and concen-
trated under reduced pressure to afford a mixture of (()-47 and
(()-48. This mixture was used directly in the next step without
characterization.
(()-tert-Butyl trans-3-Hydroxy-4-(1H-1,2,3-triazol-1-yl)-
pyrrolidine-1-carboxylate [(()-48]. The mixture of (()-47 and
(()-48 was taken up in THF (20 mL), and TBAF (4.8 mL, 4.8
mmol, 1 M solution in THF) was added. The reaction mixture
was stirred for 4 h, and then further TBAF (1.6 mL, 1.6 mmol,
1 M solution in THF) was added. The mixture was stirred for a
further 16 h and then partitioned between EtOAc (30 mL) and
water (30 mL). The layers were separated, and the aqueous
phase was extracted with EtOAc (2 ꢀ 50 mL). The combined
organic phases were washed with brine (2 ꢀ 50 mL), dried
(()-tert-Butyl trans-3-Hydroxy-4-((trimethylsilyl)ethynyl)-
pyrrolidine-1-carboxylate [(()-42]. n-Butyllithium (4.6 mL,
6.0 mmol, 1.3 M solution in hexanes) was added, over 5 min, to
a solution of trimethylsilylacetylene (1.1 mL, 7.8 mmol) in THF
(8.5 mL) at -78 °C. After 30 min BF3 OEt2 (1.4 mL, 11.4 mmol)
3
was added over 5 min. After the mixture was stirred for a further
30 min at -78 °C, a solution of epoxide (meso-10) (550 mg,
3.0 mmol) in THF (10 mL) was added. The reaction mixture was
stirred at -78 °C for 1.5 h and then allowed to warm to room
temperature. The mixture was stirred for 16 h and then quenched
by the addition of saturated aqueous NH4Cl solution (20 mL) and
then partitioned between water (50 mL) and EtOAc (50 mL). The
layers were separated, and the aqueous phase was extracted with
EtOAc (3 ꢀ 50 mL). The combined organic phases were washed
with brine (2 ꢀ 50 mL), dried (MgSO4), filtered, and concentrated
under reduced pressure. Flash chromatography of the residue (2:8,
EtOAc/Petrol) afforded (()-42 as a yellow gum (478 mg, 57%). 1H
NMR (500 MHz, CDCl3): δ = 4.33 (q, J = 4.7, 1H), 3.77-3.65
(br m, 2H), 3.48-3.36 (br m, 1H), 3.32-3.22 (br m, 1H), 2.90 (br s,
1H), 1.58 (br s, 1H), 1.46 (s, 9H), and 0.15 ppm (s, 9H). 13C NMR
(125 MHz, CDCl3): δ = 154.7, 104.7, 87.8, 79.8, 75.5, 74.7 (rota-
mers), 52.4, 52.1 (rotamers), 49.9, 49.4 (rotamers), 39.2, 38.7
(rotamers), 28.5, and 0.0 ppm. ESI-HRMS for C14H25NO3NaSi
[MNa]þ calcd, 306.1501; found, 306.1505.
(()-trans-3-Hydroxy-4-((trimethylsilyl)ethynyl)pyrrolidine
[(()-43]. Aqueous HCl (36%, 1 mL, 33 mmol) was added to a
solution of (()-42 (478 mg, 1.7 mmol) in methanol (20 mL)
and then concentrated under reduced pressure. The residue

6742 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Longshaw et al.
(MgSO4), filtered, and concentrated under reduced pressure.
Flash chromatography of the residue (100% EtOAc) afforded
(()-48 as a pale yellow gum (510 mg, 63%, over 2 steps) and
recovered (()-47 as a pale yellow oil (140 mg, 14%). (()-48. 1H
NMR (500 MHz, CDCl3): δ = 7.63 (d, J = 6.8 Hz, 1H), 7.58 (s,
1H), 5.44 (br s, 1H), 4.93-4.89 (m, 1H), 4.60 (d, J = 15.9 Hz, 1H),
4.00 (dd, J = 9.5, 7.2 Hz, 1H), 3.77-3.67 (m, 2H), 3.36 (dd, J =
11.8, 4.6 Hz, 1H), and 1.38 ppm (s, 9H). 13C NMR (125 MHz,
CDCl3): δ = 154.4, 154.2 (rotamers), 133.6, 123.1, 80.4, 74.2, 73.4
(rotamers), 65.4, 64.9 (rotamers), 51.7, 51.1 (rotamers), 48.8, 48.4
(rotamers), and 28.4 ppm. ESI-HRMS for C11H18N4O3Na[MNa]þ
37.8, 37.2 (rotamers), and 28.4 ppm. ESI-HRMS for C11H17NO3-
Na [MNa]þ calcd, 234.1106; found, 234.1108.
(()-tert-Butyl trans-4-(1-Benzyl-1H-1,2,3-triazol-4-yl)-3-
hydroxypyrrolidine-1-carboxylate [(()-52]. Sodium ascorbate
(14 mg, 0.07 mmol) and then copper(II) sulfate (20 μL, 0.02
mmol, 1.0 M aqueous solution) were added to a solution of
(()-51 (122 mg, 0.6 mmol) andbenzylazide(111 mg, 0.8mmol)in
t-BuOH (1 mL) and water (1 mL). After being stirred at room
temperature for 18.5 h, the mixture was partitioned between
water (10 mL) and EtOAc (10 mL). The layers were separated,
and the aqueous phase was extracted with EtOAc (2 ꢀ 20 mL).
The combined organic phases were washed with 5% aqueous
NH4OH solution (2 ꢀ 20 mL) and brine (20 mL), dried (MgSO4),
filtered, and concentrated under reduced pressure. Flash chro-
matography of the residue (gradient 50-100% EtOAc in Petrol)
1
calcd, 277.1277; found, 277.1275. (()-47. H NMR (500 MHz,
CDCl3): δ = 7.51 (s, 1H), 4.92-4.78 (m, 2H), 4.49 (br s, 1H),
4.10-4.03 (m, 1H), 3.89-3.79 (m, 2H), 3.44 (br s, 1H), 1.47 (s, 9H)
and 0.29 ppm (s, 9H). 13C NMR (125 MHz, CDCl3): δ = 155.6,
155.4 (rotamers), 147.7, 129.5, 81.5, 75.5, 74.6 (rotamers), 66.5, 66.0
(rotamers), 52.6, 52.1 (rotamers), 50.2, 49.7 (rotamers), 29.6, and 0.0
ppm. ESI-HRMS for C14H26N4O3NaSi [MNa]þ calcd, 349.1672;
found, 349.1669.
1
afforded (()-52 as a yellow oil (114 mg, 57%). H NMR (500
MHz, CDCl3): δ = 7.34-7.30 (m, 4H), 7.22-7.21 (m, 2H), 5.44
(s, 2H), 4.43 (br d, J = 38 Hz, 1H), 4.18 (d, J = 11.0 Hz, 1H), 3.83
(dd, J = 11.0, 7.6 Hz, 1H), 3.67-3.58 (m, 1H), 3.51-3.44 (m,
1H), 3.41-3.33 (m, 1H), 3.29-3.25 (m, 1H), and 1.40 ppm (s,
9H). 13C NMR (125 MHz, CDCl3): δ = 154.6, 154.5 (rotamers),
147.2, 147.0 (rotamers), 134.5, 129.1, 128.8, 128.1, 120.9, 79.6,
74.9, 74.1 (rotamers), 54.2, 52.2, 51.9 (rotamers), 49.1, 48.6 (rota-
mers), 43.5, 43.0 (rotamers), and 28.5 ppm. ESI-HRMS for
C18H24N4O3Na [MNa]þ calcd, 367.1746; found, 367.1747.
(()-trans-4-(1-Benzyl-1H-1,2,3-triazol-4-yl)-3-hydroxypyrro-
lidine [(()-53]. Aqueous HCl (36%, 500 μL, 16 mmol) was added
to a solution of (()-52 (114 mg, 0.3 mmol) in methanol (10 mL).
The reaction mixture was concentrated under reduced pressure
and then azeotroped with methanol (2 ꢀ 20 mL) followed by
toluene (10 mL). Flash chromatography of the residue (20%
(7N NH3 in MeOH) in CH2Cl2) afforded (()-53 as an off-white
solid (60 mg, 74%). 1H NMR (500 MHz, CD3OD): δ = 7.81 (s,
1H), 7.38-7.31 (m, 5H), 5.55 (s, 2H), 4.36 (dt, J = 5.7, 3.9 Hz,
1H), 3.43 (dd, J = 11.4, 7.8 Hz, 1H), 3.29-3.25 (m, 1H), 3.14
(dd, J = 12.1, 5.7 Hz, 1H), 2.97 (dd, J = 11.4, 6.5 Hz, 1H), and
2.85 ppm (dd, J = 12.0, 3.7 Hz, 1H). 13C NMR (125 MHz,
CD3OD): δ = 149.8, 136.8, 130.0, 129.6, 126.2, 123.1, 78.9, 55.1,
54.9, 52.4, and 46.8 ppm. ESI-HRMS for C13H17N4O [MH]þ
calcd, 245.1402; found, 245.1401.
(()-trans-3-Hydroxy-4-(1H-1,2,3-triazol-1-yl)pyrrolidine
[(()-49]. Aqueous HCl (36%, 1 mL, 33 mmol) was added to a
solution of (()-48 (500 mg, 2.0 mmol) in methanol (25 mL). The
reaction mixture was concentrated under reduced pressure and
subsequently azeotroped with methanol (2 ꢀ 20 mL) followed by
toluene (10 mL). Flash chromatography of the residue (5:4.6:0.4,
CH2Cl2/MeOH/28% aqueous NH4OH) afforded (()-49 as an
off-white foam (185 mg, 61%). 1H NMR (500 MHz, CD3OD):
δ = 8.06 (d, J = 0.9 Hz, 1H), 7.75 (d, J = 0.9 Hz, 1H), 4.94-4.91
(m, 1H), 4.54-4.51 (m, 1H), 3.58 (dd, J = 12.5, 7.3 Hz, 1H),
3.37-3.34 (m, 1H), 3.27 (dd, J = 12.6, 4.7 Hz, 1H), and 2.92 ppm
(dd, J = 12.2, 3.9 Hz, 1H). 13C NMR (125 MHz, CD3OD): δ =
134.5, 125.4, 78.7, 69.8, 54.7, and 52.3 ppm. ESI-HRMS for
C6H11N4O [MH]þ calcd, 155.0933; found, 155.0931.
(()-trans-1-[(9-Deazaadenin-9-yl)methyl]-3-hydroxy-4-(1H-
1,2,3-triazol-4-yl)pyrrolidine [(()-50]. Formaldehyde (75 μL,
0.9 mmol, 37 wt % solution in water) followed by 9-deaza-
adenine (100 mg, 0.75 mmol) was added to a solution of (()-49
(96 mg, 0.62 mmol) in 1,4-dioxane (1.5 mL) and water (1.5
mL). After being stirred for 66 h the reaction mixture was
absorbed onto silica and eluted down a silica column with a
gradient of 10-20% (7 N NH3 in MeOH) in CH2Cl2. The
crude product was collected, concentrated, and subjected to
flash chromatography (5:4.98:0.02, CH2Cl2/MeOH/28% aqu-
eous NH4OH) to afford (()-50 (58 mg, 31%). A sample was
purified by preparative HPLC to analytical purity as a TFA
salt. 1H NMR (500 MHz, CD3OD): δ = 8.45 (s, 1H), 8.14 (d,
J = 0.8 Hz, 1H), 8.08 (s, 1H), 7.80 (d, J = 0.7 Hz, 1H), 5.33 (dt,
J = 7.3, 2.8 Hz, 1H), 4.82 (s, 2H), 4.66 (quin, J = 2.2 Hz, 1H),
4.33 (dd, J = 13.2, 7.5 Hz, 1H), 4.15 (dd, J = 13.2, 3.6 Hz, 1H),
3.92 (dd, J = 12.4, 4.5 Hz, 1H), and 3.66 ppm (dd, J = 12.3, 1.7
Hz, 1H). 13C NMR (125 MHz, CD3OD): δ = 152.0, 145.1,
139.9, 136.1, 135.1, 126.6, 114.1, 104.6, 75.8, 66.6, 60.3, 57.2, and
49.9 ppm (resonance signals due to CF3COOH have not been
quoted). ESI-HRMS for C13H17N8O [MH]þ calcd, 301.1525;
found, 301.1530.
(()-tert-Butyl trans-4-Ethynyl-3-hydroxypyrrolidine-1-carboxy-
late [(()-51]. Tetrabutylammonium fluoride (3 mL, 3 mmol, 1.0 M
solution in THF) was added dropwise to a stirred solution of
(()-42 (569 mg, 2 mmol) in THF (15 mL). After being stirred for
1 h at room temperature, the reaction mixture was quenched by
the addition of water (100 mL) and then extracted with EtOAc
(3 ꢀ 75 mL). The combined organic phase was washed with
brine (80 mL) and then dried (MgSO4), filtered, and concen-
trated under reduced pressure to afford crude (()-51 as a
yellow oil (420 mg, 99%). No further purification was neces-
sary. 1H NMR (500 MHz, CDCl3): δ = 4.26 (dd, J = 8.4, 3.8
Hz, 1H), 3.63-3.56 (m, 2H), 3.39-3.32 (m, 1H), 3.22 (t, J =
11.5 Hz, 1H), 2.83 (br s, 1H), 2.11 (br s, 1H), and 1.37 ppm (s,
9H). 13C NMR (125 MHz, CDCl3): δ = 154.7, 82.7, 79.9, 75.0,
74.2 (rotamers), 71.3, 52.3, 52.1 (rotamers), 49.7, 49.2 (rotamers),
(()-trans-4-(1-Benzyl-1H-1,2,3-triazol-4-yl)-1-[(9-deazaadenin-
9-yl)methyl]-3-hydroxypyrrolidine[(()-54]. Formaldehyde (35μL,
0.44 mmol, 37 wt % solution inwater) followed by 9-deazaadenine
(40 mg, 0.30 mmol) was added to a solution of (()-53 (60 mg,
0.25 mmol) in 1,4-dioxane (0.6 mL) and water (1.2 mL). The
reaction mixture was stirred at room temperature for 17 h, and
then further formaldehyde (20μL, 0.25 mmol, 37 wt % solution in
water) wasadded. After being stirredfor 60 h, the reactionmixture
was absorbed onto silica and eluted down a silica column with a
gradient of 10-30% (7 N NH3 in MeOH) in CH2Cl2. The crude
product was collected, concentrated, and subjected to flash chro-
matography (5:4.9:0.1, CH2Cl2/MeOH/28% aqueous NH4OH)
toafford(()-54 as an off-white solid(49 mg, 51%). 1H NMR (500
MHz, CD3OD): δ = 8.14 (s, 1H), 7.79 (s, 1H), 7.48 (s, 1H), 7.37-
7.29 (m, 5H), 5.53 (s, 2H), 4.33-4.30 (m, 1H), 3.89 (d, J = 13.4
Hz, 1H), 3.84 (d, J = 13.4 Hz, 1H), 3.25-3.21 (m, 1H), 2.96 (dd,
J = 10.3, 6.8 Hz, 1H), 2.77 (dd, J = 10.3, 4.0 Hz, 1H), and 2.68-
2.65 ppm (m, 1H). 13C NMR (125 MHz, CD3OD): δ = 152.1,
151.0, 150.3, 147.0, 136.8, 130.0 (2 C), 129.6, 129.1, 123.1, 115.2,
112.7, 77.8, 62.2, 59.4, 54.9, 48.9, and 46.2 ppm. ESI-HRMS for
C20H23N8O [MH]þ calcd, 391.1995; found, 391.1994.
(()-tert-Butyl trans-4-[3-(Benzylthio)propyl]-3-hydroxypyrro-
lidine-1-carboxylate [(()-55]. 1,10-Azobis(cyanocyclohexane) (20
mg, 0.08 mmol) was added to a solution of (()-37 (245 mg, 1.1
mmol) and benzyl mercaptan (1.9 mL, 16 mmol) in 1,4-dioxane
(1.9 mL). The reaction mixture was heated to 90 °C for 22 h, with
further 1,10-azobis(cyanocyclohexane) (32 mg, 0.1 mmol) being
added at intervals of 3, 5, and 6 h. The mixture was allowed to cool
and concentrated under reduced pressure. Flash chromatography
of the residue (1:9 then 1:1, EtOAc/Petrol) afforded a 5:1 mixture

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6743
of (()-55/(()-37 as a colorless oil (272 mg). 1H NMR (500 MHz,
CDCl3): δ = 7.31-7.22 (m, 5H), 3.96 (br s, 1H), 3.70 (s, 1H),
3.62-6.52 (m, 2H), 3.25-3.16 (m, 1H), 3.03-2.98 (m, 1H),
2.43-2.41 (m, 2H), 2.21-1.95 (m, 2H), 1.61-1.51 (m, 2H), 1.45
(s, 9H), and 1.30-1.24 ppm (m, 1H). 13C NMR (125 MHz,
CDCl3): δ = 154.6, 138.5, 128.8, 128.5, 127.0, 79.4, 75.5, 74.7
(rotamers), 52.8, 52.5 (rotamers), 49.4, 48.9 (rotamers), 45.9, 45.4
(rotamers), 36.4, 35.7 (rotamers), 31.3, 30.6, 28.5, and 27.3 ppm.
ESI-HRMS for C19H29NO3NaS [MNa]þ calcd, 374.1766; found,
374.1761.
(()-trans-4-[3-(Benzylthio)propyl]-3-hydroxypyrrolidine [(()-56].
Aqueous HCl (36%, 500 μL, 16 mmol) was added to a solution of
(()-55/(()-37 (272 mg, 5:1) in methanol (10 mL). The reaction
mixture was concentrated under reduced pressure and then azeo-
troped with methanol (2 ꢀ 20 mL) followed by toluene (10 mL).
Flash chromatography of the residue (5:4.6:0.4, CH2Cl2/MeOH/
28% aqueous NH4OH) afforded (()-56 as a yellow oil (115 mg,
60% over two steps). 1H NMR (500 MHz, CD3OD): δ = 7.33-
7.28 (m, 4H), 7.23-7.20 (m, 1H), 4.14 (s, 1H), 3.72 (s, 2H), 3.53-
3.49 (m, 1H), 3.38-3.35 (m, 1H), 3.14 (d, J = 12.3 Hz, 1H), 2.96
(dd, J = 11.6, 5.5 Hz, 1H), 2.47-2.44 (m, 2H), 2.16 (br s, 1H),
1.64-1.50 (m, 3H), and 1.39-1.32 ppm (m, 1H). 13C NMR (125
MHz, CD3OD): δ= 140.3, 130.0, 129.5, 128.0, 75.1, 52.5, 49.9, 47.0,
37.1, 32.1, 31.2, and 28.5 ppm. ESI-HRMS for C14H22NOS [MH]þ
calcd, 252.1422; found, 252.1417.
the aqueous phase was extracted with toluene (2 ꢀ 50 mL). The
combined organic phases were washed with water (2 ꢀ 50 mL) and
brine (2 ꢀ 50 mL), dried (MgSO4), filtered, and concentrated under
reduced pressure to afford (3R,4R)-60 as a yellow oil (3.31 g, 99%).
No further purification was required. 1H NMR (500 MHz, CDCl3):
δ = 8.03-8.01 (m, 2H), 7.60-7.56 (m, 1H), 7.47-7.43 (m, 2H),
4.37-4.33 (m, 1H), 4.28-4.20 (m, 2H), 3.73-3.57 (m, 2H), 3.33-
3.17 (m, 2H), 2.51 (quin, J = 6.3 Hz, 1H), 1.46 (s, 9H), 0.85 (s, 9H),
and 0.05 ppm (s, 6H).
(3R,4R)-tert-Butyl 3-(tert-Butyldimethylsilyloxy)-4-(hydroxy-
methyl)pyrrolidine-1-carboxylate [(3R,4R)-61].40 Sodium meth-
oxide (1.8 mL, 7.9 mmol, 25 wt % in methanol) was added to a
solution of (3R,4R)-60 (3.31 g, 7.6 mmol) in methanol (10 mL).
The reaction mixture was stirred at room temperature for 3 h and
then diluted with chloroform (40 mL). The mixture was washed
with water (2 ꢀ 20 mL), dried (MgSO4), filtered, and concen-
trated under reduced pressure. Flash chromatography of the
residue (60% EtOAc in Petrol) afforded (3R,4R)-61 as a yellow
oil (1.2 g, 47%). 1H NMR (500 MHz, CDCl3): δ = 4.20-4.14
(m, 1H), 3.71-3.51 (m, 4H), 3.18-3.10 (m, 2H), 2.31-2.23 (m,
1H), 1.46 (s, 9H), 0.88 (s, 9H), 0.08 (s, 3H), and 0.07 ppm (s, 3H).
13C NMR (125 MHz, CDCl3): δ = 154.6, 79.4, 72.8, 72.1 (rota-
mers), 62.5, 62.4 (rotamers), 53.2, 52.5 (rotamers), 48.9, 48.3
(rotamers), 46.4, 45.9 (rotamers), 28.5, 25.8 (rotamers), 17.9,
-4.7, and -4.9 ppm.
(()-trans-4-[3-(Benzylthio)propyl]-1-[(9-deazaadenin-9-yl)-
methyl]-3-hydroxypyrrolidine [(()-57]. Formaldehyde (40 μL,
0.5 mmol, 37 wt % solution in water) followed by 9-deaza-
adenine (46 mg, 0.3 mmol) was added to a solution of (()-56
(75 mg, 0.3 mmol) in 1,4-dioxane (0.8 mL) and water (0.8 mL).
After being stirred for 16 h, the reaction mixture was absor-
bed onto silica and eluted down a silica column with a
gradient of 5-50% (7 N NH3 in MeOH) in CH2Cl2. The
crude product was collected, concentrated, and subjected to
flash chromatography (5:4.95:0.05 then 5:4.8:0.2, CH2Cl2/
MeOH/28% aqueous NH4OH) to afford (()-57 as an off-
white solid (23 mg, 20%). 1H NMR (500 MHz, CD3OD): δ =
8.16 (s, 1H), 7.48 (s, 1H), 7.30-7.23 (m, 4H), 7.18-7.15 (m,
1H), 3.83-3.79 (m, 3H), 3.67 (s, 2H), 3.00 (t, J = 8.3 Hz, 1H),
2.74 (dd, J = 10.4, 6.4 Hz, 1H), 2.67 (dd, J = 10.3, 4.0 Hz,
1H), 2.38 (t, J = 7.0 Hz, 1H), 2.13 (dd, J = 9.6, 8.0 Hz, 1H),
1.92-1.86 (m, 1H), 1.97-1.48 (m, 3H), and 1.37-1.30 ppm
(m, 1H). 13C NMR (125 MHz, CD3OD): δ = 152.1, 151.0,
147.0, 140.3, 130.1, 130.0, 129.4, 127.8, 115.2, 112.6, 77.6,
62.4, 59.5, 49.1, 48.2, 36.9, 33.4, 32.2, and 28.9 ppm. ESI-
HRMS for C21H28N5OS [MH]þ calcd, 398.2015; found,
398.2013.
(3R,4R)-tert-Butyl 4-(Benzoyloxymethyl)-3-hydroxypyrroli-
dine-1-carboxylate [(3R,4R)-59].40 A solution of (3R,4R)-5837
(4.10 g, 19 mmol) and dibutyltin oxide (5.17 g, 21 mmol) in
toluene (60 mL) was refluxed in a Dean-Stark apparatus for 1 h
and then cooled to 5 °C. Benzoyl chloride (2.2 mL, 19 mmol) was
added dropwise while the internal temperature was kept below
10 °C. After being stirred at room temperature for 17 h, the
reaction mixture was concentrated under reduced pressure.
Flash chromatography of the residue (40% EtOAc in Petrol)
afforded (3R,4R)-59 as a yellow oil (2.48 g, 41%). 1H NMR (500
MHz, CDCl3): δ = 8.03 (d, J = 7.6 Hz, 2H), 7.58 (t, J = 7.4 Hz,
1H), 7.47-7.44 (m, 2H), 4.41-4.37 (m, 1H), 4.35-4.26 (m, 2H),
3.76-3.67 (m, 2H), 3.36-3.25 (m, 2H), 2.60-2.52 (m, 1H), 2.10
(br s, 1H), and 1.46 ppm (s, 9H). 13C NMR (125 MHz, CDCl3):
δ = 166.6, 154.6, 133.2, 129.7, 129.6, 128.5, 79.7, 72.1, 71.4
(rotamers), 64.1, 64.0 (rotamers), 52.7, 52.5 (rotamers), 46.9,
46.4 (rotamers), 45.7, 45.2 (rotamers), and 28.5 ppm.
(3R,4S)-tert-Butyl 3-(tert-Butyldimethylsilyloxy)-4-formylpyrro-
lidine-1-carboxylate [(3R,4S)-62].40 Alcohol (3R,4R)-61 (1.1 g,
3.3 mmol) was added to a suspension of Dess-Martin periodinane
(1.54 g, 3.6 mmol) in CH2Cl2 (30 mL), and the reaction mixture was
stirred at room temperature for 2.5 h. The reaction mixture was
diluted with ether (150 mL) and washed with a 1:1 solution of
saturated aqueous sodium hydrogen carbonate solution:10% aqu-
eous sodium thiosulfate solution (2 ꢀ 100 mL) and then dried
(MgSO4), filtered, and concentrated under reduced pressure. Flash
chromatography of the residue (1:9 then 2:8, EtOAc/Petrol) affor-
ded (3R,4S)-62 as a pale yellow oil (980 mg, 90%). 1H NMR (500
MHz, CDCl3): δ = 9.67 (s, 1H), 4.54-4.52 (m, 1H), 3.68-3.53 (m,
3H), 3.19 (br s, 1H), 2.96 (br s, 1H), 1.43 (s, 9H), 0.86 (s, 9H), and
0.06 ppm (s, 6H). 13C NMR (125 MHz, CDCl3): δ = 199.7, 154.2,
79.8, 71.5, 70.9 (rotamers), 59.1, 58.4 (rotamers), 53.6, 53.0 (rota-
mers), 43.5, 28.4, 25.6, 17.9, -4.8, and -4.9 ppm.
(3R,4S)-tert-Butyl 3-(tert-Butyldimethylsilyloxy)-4-vinylpyrro-
lidine-1-carboxylate [(3R,4S)-63]. n-Butyllithium (4.3 mL, 6.8 mmol,
1.6 M solution in hexanes) was added dropwise to a stirred
suspension of methyltriphenylphosphonium bromide (2.44 g, 6.8
mmol) in THF (10 mL) at 0 °C. After 30 min the suspension was
added to a solution of (3R,4S)-62 (977 mg, 3.0 mmol) in THF
(10mL) at-78 °C. After being stirred at -78 °C for 1 h, the reaction
mixture was warmed to room temperature, stirred for 2.5 h, and
then quenched with water (30 mL) and extracted with CH2Cl2
(100 mL). The combined organic phases were washed with saturated
aqueous sodium hydrogen carbonate solution (50 mL) and brine
(50 mL) and then dried (MgSO4), filtered, and concentrated under
reduced pressure. Flash chromatography of the residue (1:9, EtOAc/
1
Petrol) afforded (3R,4S)-63 as a colorless oil (867 mg, 89%). H
NMR (500 MHz, CDCl3): δ = 5.67 (ddd, J = 17.2, 10.4, 7.8 Hz,
1H), 5.13-5.07 (m, 2H), 3.99 (q, J = 5.9 Hz, 1H), 3.64-3.49 (m,
2H), 3.22-3.07 (m, 2H), 2.67-2.60 (m, 1H), 1.45 (s, 9H), 0.86 (s,
9H), and 0.04 ppm (s, 6H). 13C NMR (125 MHz, CDCl3): δ =
154.5, 136.6, 116.8, 79.3, 75.8, 74.8 (rotamers), 52.8, 52.3 (rotamers),
50.8, 50.0 (rotamers), 48.5, 48.0 (rotamers), 28.5, 25.7, 18.0,
and -4.8 ppm. ESI-HRMS for C17H33NO3NaSi [MNa]þ
calcd, 350.2127; found, 350.2128.
(3R,4S)-tert-Butyl 3-(tert-Butyldimethylsilyloxy)-4-ethylpyrro-
lidine-1-carboxylate [(3R,4S)-65]. Palladium (100 mg, 0.9 mmol,
10 wt % on carbon) was added to a solution of (3R,4S)-63
(870 mg, 2.7 mmol) in ethanol (25 mL) under an argon atmos-
phere. The reaction mixture was placed under a hydrogen atmos-
phere and stirred for 15 h and then filtered through Celite and
concentrated under reduced pressure to afford (3R,4S)-65 (770
(3R,4R)-tert-Butyl 4-(benzoyloxymethyl)-3-(tert-butyldimethyl-
silyloxy)pyrrolidine-1-carboxylate [(3R,4R)-60].40 tert-Butylchloro-
dimethylsilane (2.33 g, 15 mmol) was added to a stirred solution of
(3R,4R)-59 (2.48 g, 7.7 mmol) and imidazole (2.1 g, 31 mmol) in
DMF (4 mL). After 17 h the reaction mixture was diluted with
toluene (50 mL) and water (50 mL). The phases were separated, and

6744 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Longshaw et al.
mg, 88%). No further purification was necessary. 1H NMR (500
MHz, CDCl3): δ = 3.90 (d, J = 5.4 Hz, 1H), 3.59 (br s, 1H),
3.54-3.48 (m, 1H), 3.08 (br s, 1H), 3.00-2.94 (m, 1H), 1.90 (br s,
1H), 1.45 (s, 9H), 1.23-1.13 (m, 2H), 0.93 (t, J = 7.5 Hz, 3H),
0.88 (s, 9H), 0.06 (s, 3H), and 0.05 ppm (s, 3H). 13C NMR (125
MHz, CDCl3): δ = 154.7, 79.1, 75.5, 74.8 (rotamers), 53.4, 52.6
(rotamers), 49.0, 48.5(rotamers), 48.3, 47.6 (rotamers), 28.5, 25.7,
24.3, 18.0, 12.2, -4.7, and -4.8 ppm. ESI-HRMS for C17H35-
NO3NaSi [MNa]þ calcd, 352.2284; found, 352.2288.
(3R,4S)-4-Ethyl-3-hydroxypyrrolidine [(3R,4S)-67]. A solu-
tion of (3R,4S)-65 (770 mg, 2.3 mmol) in trifluoroacetic acid
(20 mL, 260 mmol) was stirred at room temperature for 17 h and
then concentrated under reduced pressure. The residue was
dissolved in water (50 mL) and washed with chloroform (2 ꢀ
50 mL). The aqueous phase was absorbed onto silica and eluted
down a silica column (5:4.5:0.5, CH2Cl2/MeOH/28% aqueous
NH4OH) to afford (3R,4S)-67 as a yellow oil (205 mg, 76%). 1H
NMR (500 MHz, CD3OD): δ = 3.96 (dt, J = 5.4, 3.5 Hz, 1H),
3.26 (dd, J = 11.4, 7.6 Hz, 1H), 3.01 (dd, J = 12.1, 5.4 Hz, 1H),
2.82 (dd, J = 12.0, 3.4 Hz, 1H), 2.55 (dd, J = 11.4, 6.2 Hz, 1H),
1.91-1.84 (m, 1H), 1.55-1.46 (m, 1H), 1.33-1.24 (m, 1H), and
0.97 ppm (t, J = 7.4 Hz, 3H). 13C NMR (125 MHz, CD3OD):
δ = 77.7, 54.4, 51.3, 50.7, 26.1, and 12.8 ppm. ESI-HRMS for
C6H14NO [MH]þ calcd, 116.1075; found, 116.1080. [R]2D1 þ5.04
(c 1.15, MeOH).
(3R,4S)-1-[(9-Deazaadenin-9-yl)methyl]-4-ethyl-3-hydroxy-
pyrrolidine [(3R,4S)-69]. Formaldehyde (53 μL, 0.7 mmol,
37 wt % solution in water) followed by 9-deazaadenine (54 mg,
0.4 mmol) was added to a solution of (3R,4S)-67 (44 mg, 0.38
mmol) in 1,4-dioxane (0.7 mL) and water (1.4 mL). The reaction
mixture was stirred at room temperature for 66 h, absorbed onto
silica, and eluted down a silica column using a gradient 5-30% (7 N
NH3 in MeOH) in CH2Cl2. The crude product was collected,
concentrated, and subjected to flash chromatography (5:4.5:0.5,
CH2Cl2/MeOH/28% aqueous NH4OH) to afford (3R,4S)-69 as an
off-white solid (67 mg, 67%). 1H NMR (500 MHz, CD3OD): δ =
8.15 (s, 1H), 7.49 (s, 1H), 3.86-3.77 (m, 3H), 3.06 (dd, J = 9.5, 8.2
Hz, 1H), 2.75 (dd, J = 10.4, 6.3 Hz, 1H), 2.69 (dd, J = 10.4, 4.0 Hz,
1H), 2.18 (dd, J = 9.7, 7.9 Hz, 1H), 1.91-1.84 (m, 1H), 1.59-1.50
(m, 1H), 1.38-1.28 (m, 1H), and 0.92 ppm (t, J = 7.4 Hz, 3H). 13C
NMR (125 MHz, CD3OD): δ = 152.1, 151.0, 147.0, 130.1, 115.1,
112.6, 77.4, 62.4, 59.4, 50.4, 49.2, 27.1, and 12.9 ppm. ESI-HRMS
for C13H20N5O [MH]þ calcd, 262.1668; found, 262.1665.
[R]2D1 þ3.48 (c 1.03, MeOH).
The reaction mixture was then filtered through Celite and concen-
trated under reduced pressure to afford, presumably, (3R,4S)-
tert-butyl-4-(butyl)-3-(tert-butyldimethylsilyloxy)pyrrolidine-
1-carboxylate [(3R,4S)-66] as a colorless oil. 1H NMR confirmed
the absence of any olefinic protons, and compound (3R,4S)-66
was committed to the next step without further characterization
or purification. Aqueous HCl (36%, 1 mL, 12 mmol) was added
to a solution of (3R,4S)-66 (270 mg, 0.76 mmol) in methanol
(2 mL) and the resulting solution concentrated under reduced
pressure. The resulting residue was dissolved in 36% aqueous
HCl (1 mL, 12 mmol) and concentrated under reduced pressure
and the resulting residue partitioned between water (10 mL) and
CHCl3 (5 mL). The water layer was washed again with CHCl3
(5 mL) and concentrated under reduced pressure to afford the
hydrochloride salt of (3R,4S)-68 as a white foam (136 mg, 100%).
1H NMR(500 MHz, D2O):δ = 4.18-4.14 (m, 1H), 3.47 (dd, J =
11.9, 7.4 Hz, 1H), 3.34 (dd, J = 12.7, 5.3 Hz, 1H), 3.11 (dd, J =
12.7, 2.9 Hz, 1H), 2.93 (dd, J = 11.9, 6.1 Hz, 1H), 2.16-2.08 (m,
1H), 1.42-1.31 (m, 1H), 1.25-1.14 (m, 5H) and 0.75 ppm (t, J =
7.1 Hz, 3H). 13C NMR (125 MHz, D2O): δ = 73.9, 51.1, 48.9,
45.3, 30.1, 29.1, 21.9, and 13.3 ppm. ESI-HRMS for C8H18NO
[MH]þ calcd, 144.1388; found, 144.1383.
(3R,4S)-4-Butyl-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-
pyrrolidine [(3R,4S)-70]. Formaldehyde (86 μL, 1.1 mmol,
37 wt % solution in water) followed by 9-deazaadenine (112 mg,
0.84 mmol) was added to a solution of (3R,4S)-68 (100 mg, 0.56
mmol) in 1,4-dioxane (1 mL) and water (2 mL). The reaction
mixture was warmed to 85 °C, and after 1 h the crude reaction
mixture was absorbed onto silica and eluted down a silica column
using a gradient 5-30% (7 N NH3 in MeOH) in CH2Cl2. The crude
product was collected, concentrated, and subjected to flash
chromatography (5:4.5:0.5, CH2Cl2/MeOH/28% aqueous NH4OH)
to afford (3R,4S)-70 as an off-white solid (90 mg, 56%). 1H NMR
(500 MHz, CD3OD): δ = 8.17 (s, 1H), 7.49 (s, 1H), 3.86-3.83
(m, 1H), 3.81 (q, J = 13.2 Hz, 2H), 3.05 (dd, J = 9.6, 8.0 Hz, 1H),
2.74 (dd, J = 10.4, 6.3 Hz, 1H), 2.69 (dd, J = 10.4, 4.0 Hz, 1H),
2.17 (dd, J = 9.7, 8.0 Hz, 1H), 1.98-1.90 (m, 1H), 1.57-1.47 (m,
1H), 1.34-1.24 (m, 5H), and 0.89 ppm (t, J = 6.9 Hz, 3H). 13
C
NMR (125 MHz, CD3OD): δ = 152.1, 151.0, 147.0, 130.1, 115.1,
112.6, 77.8, 62.4, 59.7, 49.1, 48.6, 34.0, 31.5, 23.8, and 14.4 ppm.
ESI-HRMS for C15H24N5O [MH]þ calcd, 290.1981; found,
290.1988.
(()-Benzyl cis-3-(Benzoyloxy)-4-ethylpyrrolidine-1-carboxylate
[(()-71]. Benzoic acid (430 mg, 3.5 mmol) and triphenylphosphine
(909 mg, 3.5 mmol) were added to a stirred solution of (()-12
(719 mg, 2.8 mmol) in THF (24 mL). The reaction mixture was
cooled to -10 °C, and DIAD (680 μL, 3.5 mmol) was added
dropwise over 10 min. After being stirred at -10 °C for 45 min, the
reaction mixture was warmed to room temperature and stirred
for 22 h and then concentrated under reduced pressure. Flash
chromatography of the residue (1:9 then 2:8, EtOAc/Petrol)
(3R,4S)-tert-Butyl-4-(but-1-enyl)-3-(tert-butyldimethylsilyloxy)-
pyrrolidine-1-carboxylate [(3R,4S)-64]. n-Butyllithium (2.7 mL, 3.8
mmol, 1.4 M solution in hexanes) was added dropwise to a stirred
suspension of n-propyltriphenylphosphonium bromide (1.743 g,
4.52 mmol) in THF (20 mL) at 0 °C. After 20 min the suspension
was cooled to -40 °C and a solution of (3R,4S)-62 (497 mg, 1.5
mmol) in THF (10 mL) was added and the resulting mixture was
allowedtowarmto-10 °C and kept at that temperature for 30 min.
The reaction mixture was then quenched with water (50 mL) and
extracted with ethyl acetate (50 mL). The organic layer was
separated and washed with water (50 mL) and brine (50 mL) and
then dried (MgSO4), filtered, and concentrated under reduced
pressure. Flash chromatography of the resulting residue (1:19,
1
afforded (()-71 as a pale colorless oil (995 mg, 98%). H NMR
(500 MHz, CDCl3): δ = 8.12-8.00 (m, 2H), 7.60-7.56 (m, 1H),
7.49-7.28 (m, 7H), 5.57-5.53 (m, 1H), 5.20-5.09 (m, 2H),
3.89-3.67 (m, 3H), 3.29 (dt, J = 19.1, 10.7 Hz, 1H), 2.35-2.24
(m, 1H), 1.68-1.46 (m, 2H), and 0.98-0.94 ppm (m, 3H). 13C
NMR (125 MHz, CDCl3): δ = 166.0, 165.9, 154.8, 136.9, 136.7,
133.5, 133.3, 133.2, 130.2, 130.0, 129.9, 129.7, 128.5, 128.4, 127.9,
74.7, 73.8 (rotamers), 67.0, 66.9 (rotamers), 53.3, 53.0 (rotamers),
49.8, 49.5 (rotamers), 45.1, 44.3 (rotamers), 20.1, and 12.4 ppm.
ESR-HRMS for C21H23NO4Na [MNa]þ calcd, 376.1525; found,
376.1521.
1
EtOAc/Petrol) afforded (3R,4S)-64 as an oil (300 mg, 56%). H
NMR (500 MHz, CDCl3): δ= 5.51 (dt, J = 10.8, 7.3 Hz, 1H), 5.10
(br t, J = 10.1 Hz, 1H), 3.99-3.91 (m, 1H), 3.67-3.58 (m, 1H),
3.56-3.49 (m, 1H), 3.17-2.89 (m, 3H), 2.17 - 2.00 (m, 2H), 1.45 (s,
9H), 0.97 (t, J= 7.6 Hz, 3H), 0.87 (s, 9H), and 0.04 ppm (s, 6H). 13
C
NMR (125 MHz, CDCl3): δ = 154.6, 135.0, 127.6, 79.3, 76.3, 75.6
(rotamers), 53.0, 52.5 (rotamers), 49.6, 49.2 (rotamers), 45.2, 44.5
(rotamers), 28.6, 25.8, 21.0, 18.1, 14.4, and -4.8 ppm. ESI-HRMS
for C19H37NNaO3Si [MNa]þ calcd, 378.2440; found, 378.2438.
(3R,4S)-4-Butyl-3-hydroxypyrrolidine [(3R,4S)-68]. A sus-
pension of (3R,4S)-64 (260 mg, 0.73 mmol) and Pearlman’s
catalyst (50 mg, cat., 20% b/w) in ethanol (5 mL) was stirred
under an atmosphere of hydrogen at room temperature for 18 h.
(()-Benzyl cis-4-Ethyl-3-hydroxypyrrolidine-1-carboxylate
[(()-72]. A solution of K2CO3 (583 mg, 4.2 mmol) in water (20
mL) was added to a solution of (()-71 (995 mg, 2.8 mmol) in
ethanol (40 mL). The resulting mixture was heated to reflux for
90 min and then allowed to cool and was concentrated under
reduced pressure. The residue was partitioned between DCM
(50 mL) and water (50 mL). The layers were separated, and
the aqueous phase was extracted with EtOAc (3 ꢀ 50 mL).

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6745
The combined organic phases were washed with brine (2 ꢀ
50 mL), dried (MgSO4), filtered, and concentrated under re-
duced pressure. Flash chromatography of the residue (3:7,
EtOAc/Petrol) afforded (()-72 as a yellow oil (476 mg, 68%).
1H NMR (500 MHz, CDCl3): δ = 7.35-7.29 (m, 5H),
5.15-5.08 (m, 2H), 4.23 (br s, 1H), 3.67-3.47 (m, 3H), 3.14
(dd, J = 10.8, 3.6 Hz, 1H), 2.24 (br d, J = 45 Hz, 1H), 2.01-1.94
(m, 1H), 1.61-1.53 (m, 1H), 1.51-1.43 (m, 1H), and 0.98-0.94
ppm (m, 3H). 13C NMR (125 MHz, CDCl3): δ = 155.2, 155.0,
136.9, 128.4, 127.9, 127.8, 71.7, 70.7 (rotamers), 66.8, 66.7
(rotamers), 55.5, 55.0 (rotamers), 49.0, 48.8 (rotamers), 46.0,
45.3 (rotamers), 19.6, and 12.4 ppm. ESI-HRMS for C14H19-
NO3Na [MNa]þ calcd, 272.1263; found, 272.1268.
(()-cis-4-Ethyl-3-hydroxypyrrolidine [(()-73]. Palladium (10
mg, 0.01 mmol, 10 wt % on carbon) was added to a solution of
(()-72 (146 mg, 0.5 mmol) in MeOH (10 mL) under argon. The
reaction mixture was placed under a hydrogen atmosphere and
stirred for 1 h and then filtered through Celite and concentrated
under reduced pressure. Flash chromatography of the residue
(gradient 10-40% (7 N NH3 in MeOH) in DCM) afforded
(()-73 as a yellow oil (42 mg, 62%). 1H NMR (500 MHz,
CD3OD): δ = 4.17 (td, J = 4.5, 1.4 Hz, 1H), 3.04 (dd, J = 12.2,
4.4 Hz, 1H), 2.99 (dd, J = 10.5, 7.9 Hz, 1H), 2.84 (dd, J = 12.2,
1.3 Hz, 1H), 2.61 (t, J = 10.6 Hz, 1H), 1.86-1.79 (m, 1H),
1.65-1.56 (m, 1H), 1.46-1.37 (m, 1H), and 0.98 ppm (t, J = 7.4
Hz, 3H). 13C NMR (125 MHz, CD3OD): δ = 73.2, 56.0, 50.6,
48.6, 21.0, and 13.3 ppm. ESI-HRMS for C6H14NO [MH]þ
calcd, 116.1075; found, 116.1077.
(()-cis-1-[(9-Deazaadenin-9-yl)methyl]-4-ethyl-3-hydroxy-
pyrrolidine [(()-74]. Formaldehyde (35 μL, 0.4 mmol, 37 wt %
solution in water) followed by 9-deazaadenine (52 mg, 0.4 mmol)
was added to a solution of (()-73 (32 mg, 0.3 mmol) in 1,4-dioxane
(1 mL) and water (1 mL). The reaction mixture was stirred at room
temperature for 68 h, absorbed onto silica, and eluted down a silica
column using a gradient 10-50% (7 N NH3 in MeOH) in CH2Cl2.
The crude product was collected, concentrated, and subjected to
flash chromatography (5:4.9:0.1 then 5:4.8:0.2, CH2Cl2/MeOH/
28% aqueous NH4OH) to afford (()-74 as an off-white solid
(45 mg, 62%). 1H NMR (500 MHz, CD3OD): δ = 8.16 (s, 1H),
7.49 (s, 1H), 4.20 (td, J = 5.8, 3.3 Hz, 1H), 3.89 (s, 2H), 3.17 (dd,
J = 10.9, 5.5 Hz, 1H), 2.95 (dd, J = 9.4, 7.5 Hz, 1H), 2.57 (dd, J =
10.9, 3.3 Hz, 1H), 2.41 (t, J = 9.9 Hz, 1H), 2.00-1.92 (m, 1H),
1.61-1.53 (m, 1H), 1.38-1.28 (m, 1H), and 0.92 ppm (t, J =
7.5 Hz, 3H). 13C NMR (125 MHz, CD3OD): δ = 152.1, 151.0,
147.0, 130.1, 115.1, 112.7, 72.5, 63.0, 58.3, 49.4, 46.6, 21.4, and 13.2
ppm. ESI-HRMS for C13H20N5O [MH]þ calcd, 262.1668; found,
262.1663.
Protein Expression and Inhibition Assays. E. coli MTAN and
human MTAP used in the biological evaluation of the com-
pounds were expressed as IPTG-inducible His-tagged recombi-
nant enzymes purified using Ni-NTA chromatography. Enzyme
inhibition assays were carried out using a xanthine oxidase-
coupling enzyme that converts the adenine product of the
MTAN and MTAP reactions to 2,8-dihydroxyadenine moni-
tored at 293 nm (ε293 = 15.2 mM-1 cm-1). The 1 mL reaction
mixture consisted of 100 mM HEPES, pH 7.4, 50 mM KCl,
250 μM MTA, 100 mM potassium phosphate (only for the MTAP
reaction), and varying concentrations of inhibitor, in the presence
of xanthine oxidase (Sigma). The reaction was initiated by addition
of 4 nM E. coli MTAN or 10 nM human MTAP and monitored for
60-90 min at 25 °C. Controls without inhibitor and without
enzyme were included in the experiment. Ki values were determined
using the following equation for competitive inhibition:
inhibition constant and inhibitor concentration, respectively. If [I] is
less than 10 times the enzyme concentration, the following correc-
tion is applied:
ꢀ
ꢁ
v00
I0 ¼ I - 1 -
Et
v0
where I0 is the effective inhibitor concentration to be used in the
equation for competitive inhibition above, and Et is the final
enzyme concentration. In some instances, a second slower rate
phase is observed in progress curves of tight binding inhibitors.
These steady state rates are taken instead (v0s/vs) and used in the
inhibition equation above to determine the overall inhibition
constant Ki*, which reflects the slow onset inhibition of the
enzyme-inhibitor complex.
Acknowledgment. This work was supported by the New
Zealand Foundation for Research, Science and Technology
and the U.S. National Institutes of Health Grant GM41916.
The authors would like to thank Dr. Keith Clinch for helpful
discussions regarding this manuscript.
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