Targeting the polyamine pathway with transition-state analogue inhibitors of 5′-methylthioadenosine phosphorylase

Article abstract of DOI:10.1021/jm0306475

The polyamine biosynthetic pathway is a therapeutic target for proliferative diseases because cellular proliferation requires elevated levels of polyamines. A byproduct of the latter stages of polyamine biosynthesis (the synthesis of spermidine and spermi

Full text of DOI:10.1021/jm0306475

J . Med. Chem. 2004, 47, 3275-3281
3275
Ta r getin g th e P olya m in e P a th w a y w ith Tr a n sition -Sta te An a logu e In h ibitor s of
5′-Meth ylth ioa d en osin e P h osp h or yla se
Gary B. Evans,† Richard H. Furneaux,† Vern L. Schramm,‡ Vipender Singh,‡ and Peter C. Tyler*,†
Carbohydrate Chemistry, Industrial Research Limited, P.O. Box 31310, Lower Hutt, New Zealand, and Department of
Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461
Received December 30, 2003
The polyamine biosynthetic pathway is a therapeutic target for proliferative diseases because
cellular proliferation requires elevated levels of polyamines. A byproduct of the latter stages
of polyamine biosynthesis (the synthesis of spermidine and spermine) is 5′-methylthioadenosine
(MTA). In humans, MTA is processed by 5′-methylthioadenosine phosphorylase (MTAP) so
that significant amounts of MTA do not accumulate. Potent inhibitors of MTAP might allow
the buildup of sufficient levels of MTA to generate feedback inhibition of polyamine biosynthesis.
We have designed and synthesized a family of potential transition-state analogue inhibitors of
MTAP on the basis of our knowledge of the transition-state structure of purine nucleoside
phosphorylase and the assumption that it is likely the two enzymes share a common catalytic
mechanism. Several of the inhibitors display slow-onset tight-binding properties, consistent
with them being transition-state analogues, with the most potent having a dissociation constant
of 166 pM.
In tr od u ction
in human hepatoma cells in vitro.14 However, in vivo
in humans, MTA is rapidly degraded by 5′-methylthio-
adenosine phosphorylase (MTAP) so that no significant
concentration of MTA is known to accumulate. MTAP
is the only enzyme in humans that processes MTA, and
human genomic studies indicate only a single gene locus
for the enzyme. Its phosphorolysis reaction produces
5-methylthio-D-ribose-1-phosphate and adenine (Figure
2).15,16 Potent inhibitors of MTAP might be expected to
raise the intracellular concentration of MTA sufficiently
to achieve feedback inhibition of polyamine biosynthesis
and have antiproliferative activity.
A report of the crystal structure of human MTAP at
1.7 Å has identified a high degree of structural homology
with human purine nucleoside phosphorylase (PNP),
and it is likely that these enzymes share common
catalytic mechanisms.17 Knowledge of the transition-
state structure of PNP18-20 has allowed us to design
transition-state analogues that are extremely potent
inhibitors of PNP.21-31 The first-generation inhibitor,
immucillin-H (1, Chart 1), has a K/i ) 56 pM against
human PNP. Assuming that the transition state for
phosphorolysis of MTA by MTAP is similar to that of
PNP, then 5′-methylthio-immucillin-A (2, Chart 1)
becomes a desirable target as a potential transition-
state analogue inhibitor of human MTAP. The immu-
cillins are proving to be effective pharmacophores on
the basis of their affinity for the target enzyme, chemical
and metabolic stability, and biological availability.32-34
Here we report the synthesis of 2 and a family of its
structural analogues as potential transition-state ana-
logue inhibitors for MTAP. Their inhibitory properties
against human MTAP extend to a dissociation constant
of 166 pM and establish these compounds as powerful
inhibitors for human MTAP while at the same time
delineating some structure-activity relationships. We
have described in an initial report the inhibitory proper-
ties of some of these compounds.35,41
The polyamines putrescine, spermidine, and spermine
play important roles in all mammalian cells, protozoa,
bacteria, and fungi. They are involved in replication,
transcription, and translation, with their roles in growth-
related processes being best defined.1-3 Cellular prolif-
eration requires an increased level of polyamine bio-
synthesis, and compared to quiescent cells, rapidly
dividing cells have elevated polyamine pools. For this
reason, the polyamine biosynthetic pathway has been
identified as a therapeutic target for the treatment of
proliferative diseases such as cancer and parasitic
infections.4-8 So far, the main focus has been on inhibi-
tors of ornithine decarboxylase, an enzyme operating
early in the polyamine biosynthetic pathway. However,
rapid turnover of this target enzyme and dose-limiting
ototoxicity with ornithine decarboxylase inhibitors such
as difluromethylornithine have limited the applications
of this otherwise promising drug.
The pathway for the latter stages of polyamine
biosynthesis involves the action of spermidine and
spermine synthases on decarboxylated S-adenosylme-
thionone to effect chain elongation through donation of
propylamine residues in the synthesis of spermidine and
spermine (Figure 1). A product of each of these steps is
5′-methylthioadenosine (MTA). In principle, therefore,
accumulation of MTA could lead to feedback inhibition
of spermidine and spermine synthases and thus down-
regulation of polyamine biosynthesis. Elevated MTA in
vitro is known to have a cytostatic effect and to have
antiproliferative activity as well as being a product
inhibitor of both spermidine and spermine synthases.9-13
Additionally, MTA has been shown to be proapoptoptic
* To whom correspondence should be addressed. Phone: 64-4-
9313062. Fax: 64-4-9313055. E-mail: p.tyler@irl.cri.nz.
† Industrial Research Ltd.
‡ Albert Einstein College of Medicine.
10.1021/jm0306475 CCC: $27.50 © 2004 American Chemical Society
Published on Web 05/04/2004

3276 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 12
Evans et al.
F igu r e 1. Polyamine biosynthesis from ornithine to spermine.
F igu r e 2. Phosphorolysis of methylthioadenosine catalyzed by MTAP.
Ch a r t 1
Sch em e 1
iminoheptononitrile (7), whereby the deazaadenine
moiety is built up in a standard manner.22 This hep-
tononitrile 7 can be synthesized in good yield from the
nitrone 8,36 which is generated from the readily avail-
able iminoribitol derivative 9 (Scheme 2).22,37
Our previous attempts to effect nucleophilic displace-
ments at the 5′-position of imino-C-nucleosides had
resulted in participation of the deazapurine moiety
without formation of the desired product. Consequently,
we decided to prepare the 5′-methylthio-immucillin-A
(2) and other 5′-substituted thio-immucillins by synthe-
sizing the corresponding 7-substituted thioheptononi-
Resu lts a n d Discu ssion
Syn th esis of In h ibitor s. Immucillin-H (1) is most
conveniently prepared by a convergent route in which
the lithiated deazapurine derivative 3 is added to the
imine 4, affording adduct 5 in high yield (Scheme 1).23
The corresponding deazaadenine compound, immucil-
lin-A (6, Chart 1), is unavailable by this method, as
9-lithio-9-deazaadenine derivatives are not stable enough
under the conditions required for addition to this imine,
even when the reaction is promoted by Lewis acids.36
Immucillin-A can be prepared by elaboration of the 2,5-

Targeting the Polyamine Biosynthetic Pathway
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 12 3277
Sch em e 2a
Ch a r t 3
a
Reagents: (i) SeO2, H2O2; (ii) LiCH2CN, THF, -70 °C; (iii)
Zn, HOAc; (iv) (Boc)2O.
Ch a r t 2
Sch em e 3a
a
Reagents: (i) NaH, EtOCHO, DMF; (ii) H2NCH2CN; (iii)
triles and then elaborating them into the desired imino-
C-nucleosides. Thus, desilylation of 7 afforded alcohol
10 that on mesylation gave 11 and, following displace-
ment reactions using the sodium salts of the appropriate
thiols, gave the 7-thioheptononitrile derivatives 12-23
(Chart 2). Treatment of the hydroxyethyl compound 15
with DAST afforded the 2-fluoroethyl derivative 24,
while methylation of the alcohol 10 generated the
O-methyl ether 25 (Chart 2). The 7-deoxyheptononitrile
26 (Chart 2) was prepared by displacement of mesylate
11 with sodium iodide, followed by treatment with
tributyltin hydride. Oxidation of alcohol 10 with the
Dess-Martin periodinane generated the corresponding
aldehyde, which was treated with ethylene triphenyl-
phosphorane followed by catalytic hydrogenation to
afford the nonononitrile 27 (Chart 2).
The heptononitriles 12-26 and nonononitrile 27 were
individually converted into the imino-C-nucleosides 2
and 28-42 (Chart 3) by way of a standard sequence of
reactions used for the construction of the 9-deazaad-
enine moiety.22,38 For example, treatment of the meth-
ylthio derivative 12 with sodium hydride and ethyl
formate effected functionalization of the active methyl-
ene to give the enol 43 (Scheme 3). Exposure of this
material to aminoacetonitrile led to the enamine 44, and
subsequent treatment with methylchloroformate and
DBU caused pyrrole formation with N1 protected as the
carbamate. Addition of methanol to the reaction solution
provided the unprotected pyrrole 45. Finally, reaction
with formamidine generated the deazaadenine moiety,
MeOCOCl, DBU, CH2Cl2, then add MeOH; (iv) formamidine,
EtOH, reflux; (v) aqueous HCl, MeOH.
and acid deprotection gave rise to the 5′-methylthio-
immucillin-A (2).
Biologica l Resu lts. (i) En zym a tic Assa ys. Reac-
tion mixtures (1.0 mL) containing 100 mM potassium
phosphate, pH 7.4, 50 mM KCl, 0.1-2 mM dithiothrei-
tol, methylthioadenosine at 0.2 or 0.25 mM, and variable
concentrations of inhibitor were warmed to 25 °C in a
temperature-controlled spectrophotometer, and reac-
tions were initiated with 1-10 nM purified human
MTAP. Activity was monitored by the decrease in
absorbance at 274 nm equivalent to 1.6 mM-1 cm-1. The
equilibrium constant for this reaction is unfavorable
(Keq ≈ 0.001) for phosphorolysis, so care must be taken
to accurately estimate intital rates, even with the high
phosphate concentration.
(ii) In h ibition Stu d ies. Inhibition by transition-
state analogue inhibitors often involves the two-step
process of slow-onset tight-binding inhibition. The first
step involves reversible competitive binding at the
catalytic site: E + I T EI, characterized by Ki as the
dissociation constant. In the second step, the EI complex
undergoes a conformational change to tighten the bind-
ing: EI T E*I. This second step is usually slow relative
to EI formation and equilibration. The overall equilib-
rium dissociation constant, K/i , is defined by the overall
equilibrium dissociation: E + I T E*I. Experimentally,
inhibition constants were estimated by two different

3278 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 12
Evans et al.
Ta ble 1. Inhibition of Human MTAP by Immucillin-A Series
methods. In the first, the initial reaction rates (t f 0)
were fitted to the equation for competitive inhibition to
give Ki, and the final reaction rates (after slow onset
was complete) were fitted to the equation for competitive
inhibition to give K/i . These methods are valid for
conditions where initial and final rates are clearly
defined and delineated and the inhibitor concentration
is >10 times the enzyme concentration.24 Under condi-
tions of high inhibitor concentration, initial rates are
difficult to measure because the tight-binding phase
occurs rapidly. Inhibition curves were also fitted to the
general integrated rate equation:
Analoguesa
inhibition constants
compd
R
Ki (nM)
K/i (nM)
2
methylthio
ethylthio
n-propylthio
(2-hydroxyethyl)thio
phenylthio
(4-fluorophenyl)thio
(4-chlorophenyl)thio
(4-methylphenyl)thio
(3-chlorophenyl)thio
(3-methylphenyl)thio
benzylthio
1-naphthylthio
(2-fluoroethyl)thio
methoxy
26 ( 0.8
9.2 ( 1.0
4.0 ( 0.6
56 ( 4.0
3.6 ( 0.4
6.4 ( 0.6
1.0 ( 0.5
0.266 ( 0.03
0.214 ( 0.01
14 ( 1.2
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
P ) νst + (νo - νs)(1 - e-kt)/k
1.0 ( 0.1
2.0 ( 0.2
where νo, νs, and k represent respectively initial rate,
final steady-state rate, and first-order rate constant for
attainment of the final rate. The value of k increases
hyperbolically as a function of inhibitor concentration,
such that
0.576 ( 0.076 0.166 ( 0.024
4.4 ( 1.2
6.4 ( 0.4
1.39 ( 0.14
26 ( 2.0
90 ( 16.0
20 ( 2.0
134 ( 10
720 ( 360
44 ( 4.0
0.640 ( 0.018
nd
0.628 ( 0.034
nd
nd
3.2 ( 0.3
nd
nd
nd
1 + (I/ (K/i (1 + A/Ka))
k ) k6
H
ethyl
1 + (I/(Ki(1 + A/Ka))
Ki is the initial dissociation constant, and K/ is the equilibri-
um dissociation constant obtained after slow-onsiet inhibition. ND
indicates that no slow-onset phase was observed.
a
where k6 is the rate of conversion of E*I to EI, and I
and A are inhibitor and substrate concentrations.39
The 5′-methylthio-immucillin-A (2) is a powerful
inhibitor of MTAP and, after slow onset, has K/i ) 1.03
methylthio group that distinguishes the reaction of
MTAP from other purine nucleoside phosphorylases and
is key for recognition of its biological substrate. For 41,
without the 5′-methylthio, the Ki value of 360 nM is only
7-fold less than the Km value and does not cause a slow-
onset inhibition phase.
nM. The Km/K/ value of 4800, together with the chem-
i
ical similarity to the proposed transition-state structure,
supports the hypothesis that features of the transition
state are being captured with this molecule. When the
methyl group is extended to ethyl (as in 28), the binding
affinity increases to give K/i ) 266 pM and a Km/K/
i
The K/i values for the 5′-arylthio- and 5′-alkylthio-9-
deazaadenyliminoribitols identify a new class of power-
ful inhibitors for this central enzyme of polyamine
metabolism. Kinetic isotope effect measurements to
characterize the transition-state structure of MTAP are
in progress (V. Singh and V. L. Schramm) and confirm
that MTAP is similar to all other known purine nucleo-
side N-ribosyl transferases in being characterized by
ribooxacarbenium character at the transition state.40
The compounds listed in Table 1 resemble the transition
state by having the positive charge on the ribosyl
analogue and the elevated pKa at N7. These features
have been demonstrated to be important transition-
state mimics of the closely related purine nucleoside
phosphorylases.25 The structures of the most powerful
inhibitors (28, 29, and 33) are of interest in terms of
the X-ray crystal structure of human MTAP.35 The 5′-
methylthio region is surrounded by the side chains from
five hydrophobic amino acids (Thr18, Phe177, Val233,
Val236, and Leu279B, from the adjacent subunit) and
His137B from a loop on the adjacent subunit. We
hypothesize that the ethyl and propyl groups of 28 and
29 show improved binding by more favorable interac-
tions with these hydrophobic groups. However, the
presence of the single His in this cluster also provides
the environment for a favorable H-bond interaction. The
enhanced binding of the 5′-(4-chlorophenyl)thio (33)
relative to 5′-(3-chlorophenyl)thio (35) derivative sug-
gests a specific orientation for this interaction, which
remains for future structural exploration.
value of 18 800, while the n-propyl analogue 29 has a
binding affinity similar to that of 28. 2-Hydroxyethyl
(30) and 2-fluoroethyl (39) compounds are weaker
inhibitors at 14 and 3.2 nM, respectively, establishing
the hydrophobic character of the methyl/ethyl binding
region on the enzyme. Replacement of the sulfur of 2
with oxygen (40) or a methylene group (42) resulted in
a considerable decrease in inhibitory potency to 134 and
44 nM, respectively, and no slow-onset phase of inhibi-
tion that characterizes the more powerful of the MTAP
inhibitors was observed. In view of these results, it was
surprising that the 5′-phenylthio (31) and 5′-(4-chlo-
rophenyl)thio (33) derivatives displayed affinity equal
or superior to that of the parent compound 2, with Ki/
values of 1.0 nM and 166 pM, respectively. The 5′-(4-
fluorophenyl)thio (32) and 5′-(4-methylphenyl)thio (34)
derivatives are both within a factor of 2 in affinity
relative to the 5′-phenylthio (31) derivative, with Ki/
values of 2 nM and 640 pM, respectively. The 5′-(3-
methylphenyl)thio compound 36 shows affinity similar
to that of this group, with K/i ) 628 pM. In contrast,
the 5′-(3-chlorophenyl)thio (35), the 5′-benzylthio (37),
and the 5′-naphthylthio (38) derivatives were weaker
inhibitors that did not cause slow-onset inhibition and
gave Ki values of 6.4, 26, and 90 nM, respectively. Even
though we classify these as “weak” inhibitors, it should
be noted that these compounds bind 56-780-fold more
tightly than substrate, and the MTA substrate for this
enzyme binds relatively tightly, with Km ) 5.0 µM. An
interesting compound (41) in this series lacks the 5′-

Targeting the Polyamine Biosynthetic Pathway
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 12 3279
concentrated to dryness, and the crude material was purified
by chromatography. A solution of the product in methanol (4x
mL) and 4 M HCl (4x mL) was allowed to stand for 4 h and
then concentrated to dryness. Trituration with 2-propanol gave
solid product as a bishydrochloride in ∼50% overall yield.
The following compounds were prepared in the same man-
ner.
(1S)-1-(9-Dea za a d en in -9-yl)-1,4-d id eoxy-1,4-im in o-5-
m eth ylth io-D-r ibitol Bish yd r och lor id e (2). The solid de-
composed at 223-225 °C without melting. 1H NMR: δ 8.33
(s, 1H), 7.97 (s, 1H), 4.90 (d, J ) 8.4 Hz, 1H), 4.75 (m, 1H),
4.38 (t, J ) 4.2 Hz, 1H), 3.87 (m, 1H), 3.05 (dd, J ) 14.4, 5.7
Hz, 1H), 2.91 (dd, J ) 14.4, 9.3 Hz, 1H), 2.11 (s, 3H). 13C
NMR: δ 149.4 (C), 143.6 (CH), 139.1 (C), 133.0 (CH), 113.0,
105.6 (C), 73.2, 72.5 63.6, 56.3 (CH), 33.5 (CH2), 14.6 (CH3).
Anal. (C12H19Cl2N5O2S): C, H, Cl, N, S.
Con clu sion s
Using the knowledge gained from our work on the
transition state of purine nucleoside phosphorylase, we
have designed and synthesized putative transition-state
analogues of human 5′-methylthioadenosine phospho-
rylase. Some of these compounds are extremely potent
inhibitors and display slow-onset tight-binding charac-
teristics that are consistent with them being transition-
state analogues.
Exp er im en ta l Section
Aluminum-backed silica gel sheets (Merck or Riedel de
Haen) were used for TLC. Column chromatography was
performed on silica gel (230-400 mesh, Merck). Chromatog-
raphy solvents were distilled prior to use. Anhydrous solvents
were obtained from Aldrich or Acros. NMR spectra were
recorded at 300 MHz (1H) and 75 MHz (13C) in D2O unless
otherwise indicated.
(1S)-1-(9-Dea za a d en in -9-yl)-1,4-d id eoxy-5-et h ylt h io-
1,4-im in o-D-r ibitol Bish yd r och lor id e (28). Mp: 204-212
1
°C dec. H NMR: δ 8.37 (s, 1H), 8.00 (s, 1H), 4.94 (d, J ) 8.6
Hz, 1H), 4.78 (dd, J ) 5.2, 8.6 Hz, 1H), 4.42 (t, J ) 4.8 Hz,
1H), 3.91-3.84 (m, 1H), 3.15 (dd, J ) 6.0, 14.5 Hz, 1H), 2.96
(dd, J ) 9.3, 14.5 Hz, 1H), 2.65 (d, J ) 7.4 Hz, 1H), 2.60 (d,
J ) 7.4 Hz, 1H), 1.22 (t, J ) 7.4 Hz, 3H). 13C NMR: δ 149.4
(C), 143.6 (CH), 139.4 (C), 133.0 (CH), 113.1, 105.8 (C), 73.2,
72.6, 64.1, 56.4 (CH), 30.9, 25.7 (CH2), 14.2 (CH3). Anal.
(C13H21Cl2N5O2S): C, H, Cl, N, S.
In h ibition Assa ys. The enzymatic activity and inhibition
assays for MTAP have been described recently.35
N-ter t-Bu toxycar bon yl-3,6-im in o-4,5-O-isopr opyliden e-
7-O-m et h a n esu lfon yl-2,3,6-t r id eoxy-D-a llo-h ep t on on i-
tr ile (11). Tetrabutylammonium fluoride (1 M in THF, 75 mL)
was added to a solution of 722 (19.1 g, 44.8 mmol) in THF (50
mL), and the resulting solution was allowed to stand for 0.5
h. Chloroform (350 mL) was added, and the solution was
washed with water (×2), dried, and concentrated to dryness.
Chromatography afforded N-tert-butoxycarbonyl-3,6-imino-4,5-
O-isopropylidene-2,3,6-trideoxy-D-allo-heptononitrile (10) (13.6
g, 43.6 mmol, 97%) as a syrup. A solution of some of this
material (1.0 g, 3.2 mmol) in dichloromethane (15 mL)
containing diisopropylethylamine (1.12 mL, 6.3 mmol) was
treated with methanesulfonyl chloride (0.37 mL, 4.8 mmol) at
0 °C, and then the solution was stirred at room temperature
for 1 h. Normal processing and chromatography afforded
(1S)-1-(9-Deazaaden in -9-yl)-1,4-dideoxy-5-n -pr opylth io-
1,4-im in o-D-r ibitol Bish yd r och lor id e (29). Mp: 213-215
1
°C dec. H NMR: δ 8.37 (s, 1H), 8.00 (s, 1H), 4.94 (d, J ) 8.6
Hz, 1H), 4.78 (dd, J ) 5.2, 8.6 Hz, 1H), 4.42 (t, J ) 4.7 Hz,
1H), 3.91-3.84 (m, 1H), 3.12 (dd, J ) 6.0, 14.5 Hz, 1H), 2.96
(dd, J ) 9.2, 14.5 Hz, 1H), 2.60 (t, J ) 7.2 Hz, 2H), 1.64-1.52
(m, 2H), 0.92 (t, J ) 7.3 Hz, 3H). 13C NMR: δ 149.4 (C), 143.6
(CH), 139.4 (C), 133.0 (CH), 113.1, 105.8 (C), 73.2, 72.6, 64.2,
56.4 (CH), 33.7, 31.3, 22.6 (CH2), 13.0 (CH3). Anal. (C14H23
Cl2N5O2S): C, H, Cl, N, S.
-
(1S)-1-(9-Dea za a d en in -9-yl)-1,4-d id eoxy-5-(2-h yd r oxy-
eth yl)th io-1,4-im in o-D-r ibitol Bish yd r och lor id e (30). The
solid charred at ∼220 °C without melting. 1H NMR: δ 8.38 (s,
1H), 8.00 (s, 1H), 4.95 (d, J ) 8.5 Hz, 1H), 4.79 (dd, J ) 5.2,
8.5 Hz, 1H), 4.43 (t, J ) 4.6 Hz, 1H), 3.94-3.87 (m, 1H), 3.74
(t, J ) 6.0 Hz, 2H), 3.18 (dd, J ) 5.8, 14.5 Hz, 1H), 3.01 (dd,
J ) 9.2, 14.5 Hz, 1H), 2.81-2.76 (m, 2H). 13C NMR: δ 149.4
(C), 143.6 (CH), 139.4 (C), 133.0 (CH), 113.1, 105.8 (C), 73.2,
72.6, 64.3 (CH), 60.8 (CH2), 56.5 (CH), 34.2, 31.7 (CH2). Anal.
(C13H21Cl2N5O3S): C, H, Cl, N, S.
1
syrupy title compound 11 (1.12 g, 2.87 mmol, 90%). H NMR
(CDCl3): δ 4.75 (dd, J ) 5.7, 1.4 Hz, 1H), 4.61 (brs, 1H), 4.38
(brd, J ) 5.7 Hz, 2H), 4.19 (m, 2H), 3.07 (s, 3H), 2.81 (m, 2H),
1.50 (s, 12H), 1.35 (s, 3H). 13C NMR: δ 154.0, 117.5, 113.3,
83.3, 82.4, 81.3, 68.6, 64.2, 61.5, 60.7, 37.7, 28.6, 27.6, 25.6,
21.8. HRMS (MH+): calcd for C16H27N2O7S, 391.1539; found,
391.1539.
Gen er a l P r oced u r e for th e Syn th esis of 5′-Su bstitu ted
Th io-im m u cillin -A Com p ou n d s 2 a n d 28-39. A solution
of N-tert-butoxycarbonyl-3,6-imino-4,5-O-isopropylidene-7-O-
methanesulfonyl-2,3,6-trideoxy-D-allo-heptononitrile (x mmol)
in DMF (2x mL) was added to a solution of the appropriate
sodium thiolate [prepared by adding sodium hydride (1.8x
mmol) to a solution of the thiol (2x mmol) in DMF (4x mL)],
and the resulting solution was stirred for 3 h. Toluene was
added, and the mixture was washed twice with water, dried,
and concentrated to dryness. Chromatography afforded the
corresponding 7-thioheptononitriles 12-24. This material in
THF (8x mL) and ethyl formate (x mL) was treated with
sodium hydride (4x mmol), and the mixture was stirred for 2
h. Acetic acid (0.5x mL) and chloroform (20x mL) were added,
and the mixture was washed with water, dried, and concen-
trated to dryness. Sodium acetate (8x mmol) and aminoaceto-
nitrile bisulfate (4x mmol) were added to a solution of the crude
product in methanol (8x mL), and the mixture was stirred at
room temperature for 2 d (or heated under reflux for 2-3 h)
and then was concentrated to dryness. Chloroform (20x mL)
was added, and the mixture was washed with water, dried,
and concentrated to dryness. The crude residue in dichlo-
romethane (12x mL) was treated with DBU (x mL) and methyl
chloroformate (0.25x mL), and the solution was stirred at room
temperature for 18 h. Methanol (4x mL) was then added, and
after 1 h the solution was washed with 2 M HCl and aqueous
sodium bicarbonate, dried, and concentrated to dryness. Chro-
matography then afforded the corresponding pyrroles. This
material in ethanol (8x mL) containing formamidine acetate
(2x mmol) was heated under reflux for 4 h. The solution was
(1S)-1-(9-Dea za a d en in -9-yl)-1,4-d id eoxy-1,4-im in o-5-
p h en ylth io-D-r ibitol Bish yd r och lor id e (31). Mp: 180-182
1
°C. H NMR: δ 8.30 (s, 1H), 7.96 (s, 1H), 7.44-7.25 (m, 5H),
4.88 (d, J ) 8.3 Hz, 1H), 4.76 (dd, J ) 5.0, 8.3 Hz, 1H), 4.41 (t,
J ) 4.6 Hz, 1H), 3.75 (m, 1H), 3.54 (dd, J ) 5.7, 14.8 Hz, 1H),
3.35 (dd, J ) 9.2, 14.8 Hz, 1H). 13C NMR (D2O): δ 149.1 (C),
143.4 (CH), 139.5 (C), 133.0 (CH), 132.9 (C), 130.9, 130.0, 128.1
(CH), 113.0, 105.9 (C), 73.2, 72.6, 63.8, 56.7 (CH), 33.8 (CH2).
Anal. (C17H21Cl2N5O2S): C, H, Cl, N, S.
(1S)-1-(9-Deazaaden in -9-yl)-1,4-dideoxy-5-(4-flu or oph en -
yl)th io-1,4-im in o-D-r ibitol Bish yd r och lor id e (32). The
1
solid charred at 240 °C without melting. H NMR: δ 8.32 (s,
1H), 7.97 (s, 1H), 7.50-7.43 (m, 2H), 7.12-7.04 (m, 2H), 4.89
(d, J ) 8.5 Hz, 1H), 4.76 (dd, J ) 5.0, 8.5 Hz, 1H), 4.40 (t, J )
4.5 Hz, 1H), 3.74-3.67 (m, 1H), 3.47 (dd, J ) 5.9, 14.8 Hz,
1H), 3.30 (dd, J ) 9.2, 14.8 Hz, 1H). 13C NMR: δ 162.8 (d,
J C,F ) 245 Hz, C), 149.1 (C), 143.4 (CH), 139.6 (C), 134.1 (d,
J C,F ) 8.4 Hz, CH), 133.0 (CH), 127.9 (C), 116.9 (d, J C,F ) 22
Hz, CH), 113.0, 105.8 (C), 73.2, 72.5, 63.7, 56.6 (CH, 35.0 (CH2).
Anal. (C17H20Cl2FN5O2S): C, H, Cl, N, S.
(1S)-1-(9-Dea za a d en in -9-yl)-5-(4-ch lor op h en yl)th io-1,4-
d id eoxy-1,4-im in o-D-r ib it ol Bish yd r och lor id e (33). The
solid charred at 260-270 °C without melting. 1H NMR: δ 8.32
(s, 1H), 7.97 (s, 1H), 7.40 (d, J ) 8.7 Hz, 2H), 7.34 (d, J ) 8.7
Hz, 2H), 4.89 (d, J ) 8.3 Hz, 1H), 4.77 (dd, J ) 5.0, 8.3 Hz,
1H), 4.42 (t, J ) 4.7 Hz, 1H), 3.75 (m, 1H), 3.54 (dd, J ) 5.7,
14.8 Hz, 1H), 3.35 (dd, J ) 9.2, 14.8 Hz, 1H). 13C NMR: δ
149.0 (C), 143.3 (CH), 133.6 (C), 133.0, 132.4 (CH), 131.6 (C),

3280 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 12
Evans et al.
129.8 (CH), 113.0, 105.9 (C), 73.3, 72.6, 63.6, 56.7 (CH), 34.0
(CH2). Anal. (C17H20Cl3N5O2S): C, H, Cl, N, S.
hydride (0.10 g, 60%, 2.5 mmol). The mixture was stirred for
2 h and then quenched with ethanol. Chloroform was added,
and normal processing afforded a syrup. This material was
treated as described above in the preparation of 2 to give title
compound 40. Mp: 175-180 °C. 1H NMR: δ 8.44 (s, 1H), 8.05
(s, 1H), 5.01 (d, J ) 8.9 Hz, 1H), 4.81 (dd, J ) 4.8, 8.9 Hz,
1H), 4.48 (dd, J ) 3.4, 4.8 Hz, 1H), 4.00 (m, 1H), 3.85 (dd, J )
5.4, 11.2 Hz, 1H), 3.79 (dd, J ) 3.9, 11.2 Hz, 1H), 3.43 (s, 3H).
13C NMR: δ 149.8 (C), 143.9 (CH), 138.6 (C), 132.9 (CH), 113.0,
105.5 (C), 73.8, 71.2 (CH), 68.9 (CH2), 64.3 (CH), 59.1, (CH3),
55.9 (CH). Anal. (C12H19Cl2N5O3): C, H, Cl, N.
(1S)-1-(9-Dea za a d en in -9-yl)-1,4-d id eoxy-1,4-im in o-5-(4-
m eth ylp h en yl)th io-D-r ibitol Bish yd r och lor id e (34). The
1
solid charred at 250 °C without melting. H NMR: δ 8.31 (s,
1H), 7.96 (s, 1H), 7.33 (d, J ) 8.2 Hz, 2H), 7.17 (d, J ) 8.2 Hz,
2H), 4.88 (d, J ) 8.4 Hz, 1H), 4.77 (dd, J ) 5.0, 8.4 Hz, 1H),
4.40 (t, J ) 4.5 Hz, 1H), 3.73 (m, 1H), 3.48 (dd, J ) 5.8, 14.8
Hz, 1H), 3.30 (dd, J ) 9.1, 14.8 Hz, 1H). 13C NMR: δ 149.0
(C), 143.3 (CH), 139.6, 139.0 (C), 133.0, 131.6, 130.6 (CH),
129.0, 113.0, 105.9 (C), 73.3, 72.6, 63.9, 56.8 (CH), 34.4 (CH2),
20.5 (CH3). Anal. (C18H23Cl2N5O2S): C, H, Cl, N, S.
(1S)-1-(9-Dea za a d en in -9-yl)-1,4-im in o-1,4,5-tr id eoxy-D-
r ibitol Bish yd r och lor id e (41). Sodium iodide (1.3 g, 8.6
mmol) was added to a solution of 11 (0.50 g, 1.28 mmol) in
acetone (10 mL), and the mixture was heated under reflux for
24 h and then concentrated to dryness. The residue was
partitioned between chloroform and water, and the organic
phase was dried and concentrated to dryness. A solution of
the residue in benzene (10 mL) was treated with tributyltin
hydride (1.0 mL, 3.7 mmol), and the solution was heated under
reflux for 2 h and then concentrated to dryness. The residue
in ether was stirred for 1 h with 10% aqueous KF, and the
organic layer was dried and concentrated to dryness. Chro-
matography afforded a syrup (0.25 g) of, presumably, N-tert-
butoxycarbonyl-3,6-imino-4,5-O-isopropylidene-2,3,6,7-tetra-
deoxy-D-allo-heptononitrile. This material was treated as
described above in the preparation of 2 to give 41. Mp: 198-
(1S)-1-(9-Dea za a d en in -9-yl)-5-(3-ch lor op h en yl)th io-1,4-
d id eoxy-1,4-im in o-D-r ib it ol Bish yd r och lor id e (35). The
1
solid charred at 230 °C without melting. H NMR: δ 8.30 (s,
1H), 7.96 (s, 1H), 7.41 (d, J ) 0.5 Hz, 1H), 7.34-7.22 (m, 3H),
4.89 (d, J ) 8.2 Hz, 1H), 4.77 (dd, J ) 5.0, 8.1 Hz, 1H), 4.42 (t,
J ) 4.8 Hz, 1H), 3.80-3.73 (m, 1H), 3.57 (dd, J ) 5.7, 14.8
Hz, 1H), 3.36 (dd, J ) 9.2, 14.8 Hz, 1H). 13C NMR: δ 148.9
(C), 143.3 (CH), 139.6, 135.1, 134.8 (C), 133.1, 131.1, 129.9,
128.8, 128.0 (CH), 113.0, 105.9 (C), 73.3, 72.6, 63.6, 56.8 (CH),
33.6 (CH2). Anal. (C17H20Cl3N5O2S): C, H, Cl, N, S.
(1S)-1-(9-Dea za a d en in -9-yl)-1,4-d id eoxy-1,4-im in o-5-(3-
m eth ylp h en yl)th io-D-r ibitol Bish yd r och lor id e (36). The
solid charred at 250-260 °C without melting. 1H NMR: δ 8.30
(s, 1H), 7.97 (s, 1H), 7.24 (m, 3H), 7.12 (m, 1H), 4.89 (d, J )
8.3 Hz, 1H), 4.77 (dd, J ) 5.0, 8.3 Hz, 1H), 4.41 (t, J ) 4.5 Hz,
1H), 3.76 (m, 1H), 3.53 (dd, J ) 5.7, 14.8 Hz, 1H), 3.33 (dd,
J ) 9.1, 14.8 Hz, 1H), 2.26 (s, 3H). 13C NMR: δ 149.0 (C), 143.3
(CH), 140.4, 139.6 (C), 133.0 (CH), 132.7 (C), 131.2, 129.8,
128.8, 127.8 (CH), 113.0, 105.9 (C), 73.3, 72.6, 63.8, 56.7 (CH),
33.8 (CH2), 20.8 (CH3). Anal. (C18H23Cl2N5O2S): C, H, Cl, N,
S.
1
200 °C. H NMR: δ 8.41 (s, 1H), 8.04 (s, 1H), 4.96 (d, J ) 8.5
Hz, 1H), 4.88 (dd, J ) 4.8, 8.5 Hz, 1H), 4.31 (t, J ) 4.5 Hz,
1H), 3.87 (dq, J ) 4.2, 7.1 Hz, 1H), 1.54 (d, J ) 7.1 Hz, 3H).
13C NMR: δ 149.5 (C), 143.7 (CH), 139.2 (C), 132.8 (CH), 113.1,
106.2 (C), 74.5, 73.2, 60.8, 56.2 (CH), 16.0 (CH3). Anal. (C11H17
Cl2N5O2): C, H, Cl, N.
-
(1S)-1-(9-Dea za a d en in -9-yl)-1,4-im in o-1,4,5,6,7-p en t a -
d eoxy-D-r ibo-h ep titol Bish yd r och lor id e (42). Dess-Mar-
tin periodinane (1.42 g, 3.35 mmol) was added to a stirred
solution of 10 (0.7 g, 2.24 mmol) in dichloromethane (20 mL).
After 0.5 h, the mixture was concentrated to dryness. Ether
(20 mL) was added, and the mixture was washed twice with
1:1 saturated aqueous NaHCO3/10% aqueous Na2S2O3, dried,
and concentrated to dryness. A solution of the crude product
in dry THF (8 mL) was added to the red solution obtained after
adding n-butyllithium (3.4 mL, 1.6M, 5.44 mmol) to a suspen-
sion of ethyl triphenylphosphonium iodide (2.44 g, 5.84 mmol)
in THF (25 mL) and stirring for 0.5 h. The resulting dark
solution was stirred for 0.5 h and then diluted with petroleum
ether (100 mL) and washed with water. Normal processing
afforded, after chromatography, syrupy material which was
stirred in ethanol with 10% Pd/C under hydrogen for 2.5 h.
Removal of the solids and solvent and chromatography af-
forded a syrup (0.28 g), which was treated as above in the
preparation of 2 to give 42. Mp: 206-215 °C dec. 1H NMR: δ
8.33 (s, 1H), 7.95 (s, 1H), 4.84 (d, J ) 8.9 Hz, 1H), 4.28 (t, J )
4.7 Hz, 1H), 3.67-3.56 (m, 1H), 1.87-1.66 (m, 2H), 1.46-1.33
(m, 2H), 0.88 (t, J ) 7.3 Hz, 3H). 13C NMR: δ 149.7 (C), 143.8
(CH), 138.8 (C), 132.9 (CH), 113.1, 105.6 (C), 73.0, 72.9, 65.1,
(1S)-1-(9-Dea za a d en in -9-yl)-5-ben zylth io-1,4-d id eoxy-
1,4-im in o-D-r ibitol Bish ydr och lor ide (37). The solid charred
at 235 °C without melting. 1H NMR: δ 8.34 (s, 1H), 7.94 (s,
1H), 7.37-7.24 (m, 5H), 4.89 (d, J ) 8.4 Hz, 1H), 4.31 (t, J )
4.7 Hz, 1H), 3.83 (s, 2H), 3.78-3.71 (m, 1H), 2.98 (dd, J ) 6.3,
14.6 Hz, 1H), 2.90 (dd, J ) 8.8, 14.6 Hz, 1H). 13C NMR: δ
149.2 (C), 143.5 (CH), 138.1 (C), 133.0, 129.4, 128.0 (CH),
113.0, 105.9 (C), 73.2, 72.6, 63.9, 56.6 (CH), 35.6, 30.8 (CH2).
Anal. (C18H23Cl2N5O2S): C, H, Cl, N, S.
(1S)-1-(9-Dea za a d en in -9-yl)-1,4-d id eoxy-1,4-im in o-5-(1-
n a p h th yl)th io-D-r ibitol Bish yd r och lor id e (38). The solid
charred at 270 °C without melting. 1H NMR (MeOH-d4/DMSO-
d6): δ 7.83 (s, 1H), 7.68 (d, J ) 7.9 Hz, 1H), 7.45 (s, 1H), 7.27-
7.14 (m, 3H), 6.96-6.80 (m, 3H), 4.19 (d, J ) 8.6 Hz, 1H), 3.96
(dd, J ) 5.1, 8.6 Hz, 1H), 2.96-2.74 (m, 3H). 13C NMR (MeOH-
d4/DMSO-d6): δ 143.4, 133.5 (C), 131.9 (CH), 131.6, 129.9 (C),
128.9, 128.1, 127.6, 126.3, 125.9, 125.1, 123.8 (CH), 111.8 (C),
72.1, 71.7, 62.7, 55.3 (CH), 32.8 (CH2). Anal. (C21H23Cl2N5-
O2S): C, H, Cl, N, S.
(1S)-1-(9-Dea za a d en in -9-yl)-1,4-d id eoxy-5-(2-flu or oeth -
yl)th io-1,4-im in o-D-r ibitol Bish yd r och lor id e (39). A solu-
tion of 15 (1.0 g) in dry chloroform (10 mL) was treated with
DAST (0.71 mL). The solution was allowed to stand for 16 h
and then was washed with water and aqueous NaHCO3, dried,
and concentrated to dryness. Chromatography afforded syrupy
24 (0.558 g). This material was converted into the title
compound by the same sequence of reactions descirbed above
in the preparation of 2 to give a solid 39 (0.307 g). The solid
55.9 (CH), 32.8, 19.4 (CH2), 13.2 (CH3). Anal. (C13H21
Cl2N5O2): C, H, Cl, N.
-
Su p p or tin g In for m a tion Ava ila ble: Elemental analyses
for target compounds 2 and 28-42. This material is available
free of charge via the Internet at http://pubs.acs.org.
1
charred at 210-220 °C without melting. H NMR: δ 8.38 (s,
1H), 8.00 (s, 1H), 4.95 (d, J ) 8.5 Hz, 1H), 4.79 (dd, J ) 5.2,
8.5 Hz, 1H), 4.66 (dt, J ) 5.6, 46.9 Hz, 2H), 4.43 (t, J ) 4.6
Hz, 1H), 3.94-3.88 (m, 1H), 3.22 (dd, J ) 5.9, 14.5 Hz, 1H),
3.09-2.86 (m, 3H). 13C NMR: δ 149.4 (C), 143.6 (CH), 139.5
(C), 133.1 (CH), 113.1, 105.8 (C), 84.2 (d, J C,F ) 164 Hz, CH2),
73.2, 72.6, 64.2, 56.5 (CH), 31.9 (CH2), 31.9 (d, J C,F ) 20 Hz,
CH2). Anal. (C13H20Cl2FN5O2S): C, H, Cl, N, S.
(1S)-1-(9-Dea za a d en in -9-yl)-1,4-d id eoxy-1,4-im in o-5-O-
m eth yl-D-r ibitol Bish yd r och lor id e (40). The silyl ether 7
(0.5 g, 1.17 mmol) was desilylated as described above in the
preparation of 11. A solution of the crude product in THF was
treated with methyl iodide (0.125 mL, 2 mmol) and sodium
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