A critical electrostatic interaction mediates inhibitor recognition by human asparagine synthetase

Article abstract of DOI:10.1016/j.bmc.2009.07.071

The first sulfoximine-based inhibitor of human asparagine synthetase (ASNS) with nanomolar potency has been shown to suppress proliferation of asparaginase-resistant MOLT-4 cells in the presence of l-asparaginase. This validates literature hypotheses concerning the viability of human ASNS as a target for new drugs against acute lymphoblastic leukemia and ovarian cancer. Developing structure-function relationships for this class of human ASNS inhibitors has proven difficult, however, primarily because of the absence of rapid synthetic procedures for constructing highly functionalized sulfoximines. We now report conditions for the efficient preparation of these compounds by coupling sulfoxides and sulfamides in the presence of a rhodium catalyst. Access to this methodology has permitted the construction of two new adenylated sulfoximines, which were expected to exhibit similar binding affinity and better bioavailability than the original human ASNS inhibitor. Steady-state kinetic characterization of these compounds, however, has revealed the importance of a localized negative charge on the inhibitor that mimics that of the phosphate group in a key acyl-adenylate reaction intermediate. These experiments place an important constraint on the design of sulfoximine libraries for screening experiments to obtain ASNS inhibitors with increased potency and bioavailability.

Full text of DOI:10.1016/j.bmc.2009.07.071

Bioorganic & Medicinal Chemistry 17 (2009) 6641–6650
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A critical electrostatic interaction mediates inhibitor recognition by human
asparagine synthetase
b,
Hideyuki Ikeuchi a, Megan E. Meyer b, Yun Ding b, , Jun Hiratake a, , Nigel G. J. Richards
*
*
a Institute for Chemical Research, Kyoto University, Kyoto 611-011, Japan
b Department of Chemistry, University of Florida, Gainesville, FL 32611-7200, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The first sulfoximine-based inhibitor of human asparagine synthetase (ASNS) with nanomolar potency
has been shown to suppress proliferation of asparaginase-resistant MOLT-4 cells in the presence of
Received 12 June 2009
Revised 26 July 2009
Accepted 28 July 2009
Available online 3 August 2009
L-asparaginase. This validates literature hypotheses concerning the viability of human ASNS as a target
for new drugs against acute lymphoblastic leukemia and ovarian cancer. Developing structure–function
relationships for this class of human ASNS inhibitors has proven difficult, however, primarily because of
the absence of rapid synthetic procedures for constructing highly functionalized sulfoximines. We now
report conditions for the efficient preparation of these compounds by coupling sulfoxides and sulfamides
in the presence of a rhodium catalyst. Access to this methodology has permitted the construction of two
new adenylated sulfoximines, which were expected to exhibit similar binding affinity and better bio-
availability than the original human ASNS inhibitor. Steady-state kinetic characterization of these com-
pounds, however, has revealed the importance of a localized negative charge on the inhibitor that
mimics that of the phosphate group in a key acyl-adenylate reaction intermediate. These experiments
place an important constraint on the design of sulfoximine libraries for screening experiments to obtain
ASNS inhibitors with increased potency and bioavailability.
Keywords:
Leukemia
Sulfoximine synthesis
Enzyme inhibitors
Transition state analogs
Asparagine synthetase
Structure–activity relationship
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
surroundings.8,9 Thus, in the presence of ASNase, which hydrolyzes
L
-asparagine, ASNase-sensitive leukemic blasts undergo cell death,
Acute lymphoblastic leukemia (ALL) is the most common form
of childhood cancer, with an annual incidence in the USA of four
cases per 100,000 children. A variety of clinical protocols have been
developed and substantial success in treating the disease has been
achieved, resulting in a cure rate of approximately 80%.1 The en-
perhaps from impaired protein synthesis.8
Further support for this hypothesis is provided by the observa-
tion that ASNase-resistance is correlated with the up-regulation of
glutamine-dependent asparagine synthetase (ASNS), the enzyme
responsible for de novo asparagine biosynthesis (Scheme 1),10 in
response to asparagine depletion.11 One prediction of this model
is that increased levels of cellular ASNS are the cause of ASNase-
resistance in lymphoblasts because sufficient asparagine is made
available as a result of increased intracellular asparagine biosyn-
thesis.8,12,13 Support for this hypothesis has come from studies
employing transformed cell lines. For example, in vitro studies
with U937 cells demonstrated that ASNase-sensitive cells can be
made resistant by persistent exposure to increasing levels of sub-
lethal doses of the drug, and constitutive expression of ASNS in a
human ALL cell line (MOLT-4) conferred ASNase-resistance onto
sensitive parental cells.14 Additional evidence has been provided
from recent work by our group, which demonstrated that incuba-
tion of ASNase-resistant MOLT-4 cells with ASNase and the
adenylated sulfoximine derivative 1 (Fig. 1) suppresses cell prolif-
eration.15 Compound 1 is a potent inhibitor of human ASNS
(hASNS),15 and both classes of bacterial asparagine synthetase,16,17
because it is stable analog of the transition state for the reaction of
ammonia (released in the glutaminase site of ASNS) with an acyl-
zyme
hydrolysis of
L
-asparagine amidohydrolase (ASNase), which catalyzes the
L-asparagine to yield
L
-aspartate and ammonia,2 is a
key component in the majority of the clinical treatments for
ALL,3 albeit that this drug has a relatively narrow therapeutic index
and hypersensitivity reactions occur in up to two-thirds of patients
receiving intensive ASNase therapy.4,5 It has been shown that incu-
bation of ASNase-sensitive cancer cell lines with ASNase leads to
cell cycle arrest and subsequent apoptosis even though the enzyme
is not taken up by the leukemic blasts.6,7 Although the basis for the
effectiveness of ASNase in treating childhood ALL remains the sub-
ject of some debate, one widely accepted explanation postulates
that malignant lymphocytes must import L-asparagine from their
* Corresponding authors. Tel./fax: +81 774 38 3231 (J.H.); tel.: +1 352 392 3601;
fax: +1 352 846 2095 (N.G.J.R.).
E-mail addresses: hiratake@scl.kyoto-u.ac.jp (J. Hiratake), richards@qtp.ufl.edu,
richards@chem.ufl.edu (N.G.J. Richards).
Present address: GlaxoSmithKline, 830 Winter Street, Waltham, MA 02451, USA.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.07.071

6642
H. Ikeuchi et al. / Bioorg. Med. Chem. 17 (2009) 6641–6650
Scheme 1. Overview of the reaction catalyzed by human glutamine-dependent asparagine synthetase, showing the structure of the acyl-adenylate intermediate and the
transition state for asparagine formation.
lated adenosylsulfamate derivative 4 because cyclization to the
corresponding cycloadenosine can often occur due to the leaving
group ability of the sulfamate moiety.21,22
In this paper we now report the synthesis of the adenylated sul-
foximines 3 and 4, and their ability to inhibit hASNS relative to sul-
foximine 1. Somewhat unexpectedly in light of our previous work
with acylsulfamate 2, the ability of 3 to inhibit the enzyme is re-
duced by several orders of magnitude while 4 is not a hASNS inhib-
itor. These results suggest that the negative charge on the
phosphate is an essential recognition element in the adenylated
sulfoximine 1, which is consistent with computational models.
2. Chemistry
Although the original synthetic route to 1 was relatively effi-
cient, proceeding in good overall yield from S-methylcysteine,16
the construction of the sulfoximine functional group was accom-
plished using O-mesitylsulfonylhydroxylamine (MSH) or sodium
Figure 1. Chemical structures of the adenylated sulfoximine derivative 1 and the
azide as nitrogen donors, which are both potentially dangerous
acylsulfamate 2, which are nanomolar human ASNS inhibitors, together with the
iminating agents when employed on a large scale.23–25 We were
target sulfoximine derivatives 3 and 4.
therefore interested in exploring alternate strategies for the prep-
aration of these highly functionalized molecules. In addition, we
adenylate intermediate in the synthetase site of the enzyme
(Scheme 1). Despite having a high affinity for human ASNS, the
adenylated sulfoximine 1 must be incubated with ASNase-sensi-
required an approach that might be amenable to making large li-
braries of functionalized sulfoximines for use in high-throughout
screening assays, that is, one that was capable of yielding the de-
sired sulfoximine moiety from simple, readily available precursors.
We therefore decided to exploit a recent method for coupling
sulfoxides with sulfamides in the presence of iodosylbenzene and
either a rhodium26 or iron catalyst.27 Our synthesis therefore began
with (i) commercially available 20,30-O-isopropylideneadenosine,28
and (ii) the protected sulfoxide 7, which was prepared as a mixture
tive cells at 100–1000 lM concentration before it can exert its bio-
logical effects.15 Although a number of factors may be responsible
for the need to use high concentrations of sulfoximine to see its
biological effects, our working hypothesis is that the ionized
groups in 1 preclude its facile entry into cells. The preparation of
modified ‘pro-drug’ variants of 1 containing phosphate protecting
groups that are labile within cells represents one solution to this
problem.18 The modification of the relatively difficult synthetic se-
quence used to obtain 1 to prepare suitably functionalized analogs
seemed a less attractive prospect to the development of novel
compounds containing more drug-like functional groups that
might substitute for the phosphate group.19 Moreover, we antici-
pated that the preparation of such structures would permit the
use of simpler synthetic transformations than those used in the
original synthesis of 1.16 In previous work,20 we had shown that
replacing the phosphate by a sulfamate group gave an acyl-adenyl-
ate analog 2 (Fig. 1) that inhibited hASNS with micromolar po-
tency, and limited structure–function studies showed the
importance of the amino and carboxylate groups in recognition
and binding to the synthetase active site. We therefore decided
to prepare the functionalized sulfoximine 3 (Fig. 1) to determine
the effects on hASNS inhibition. This target, in which an amine con-
nects the sulfur to the ribose, was selected in preference to the re-
of four diastereoisomers from S-methyl-L
-cysteine (Scheme 2).16
Although we expected to obtain a mixture of epimers at the sulfur
center when the thioether was oxidized to the sulfoxide, complete
racemization at C occurred under the conditions used to intro-
a
duce the t-butyl ester. The desired sulfoxide 7 was therefore ob-
tained as a mixture of four diastereoisomers, which was used
without further purification in subsequent coupling steps. 20,30-
O-Isopropylideneadenosine was converted in three steps to pro-
tected adenosylamine 10, which was then reacted with Z-sulfa-
moyl chloride. Catalytic hydrogenation to remove the benzyl
protecting group from the resulting adduct gave the desired adeno-
sylsulfamide derivative 12 in 59% overall yield from 20,30-O-isopro-
pylideneadenosine (Scheme 2). Attempts to couple intermediates 7
and 12 using published conditions, that is, using Rh2(OAc)4 as a
catalyst with 1,4-dioxane as solvent, gave the protected sulfoxim-
ine 17 in only 4% yield (Table 1). Moreover, these reagents failed to
yield any of the desired sulfoximine when Fe(acac)3 was used as

H. Ikeuchi et al. / Bioorg. Med. Chem. 17 (2009) 6641–6650
6643
Scheme 2. Reagents and conditions: (a) (Boc)2O, NaOH, Et2O–H2O, rt, 100%; (b) DCC, DMAP, tBuOH, CH2Cl2, 0 °C to rt, 90%; (c) NaIO4, THF–H2O, rt, 93%; (d)
diphenylphosphoryl azide, DBU, 1,4-dioxane, rt, then NaN3, 15-crown-5, 100 °C, 91%; (e) (Boc)2O, DMAP, Et3N, DMF, 0 °C to rt, 82%; (f) H2, Pd/C, MeOH–H2O, rt, 100%; (g) Z-
sulfamoyl chloride, Et3N, 0 °C to rt, 82%; (h) H2, Pd/C, MeOH–H2O, rt, 88%; (i) TBDMS-Cl, imidazole, 0 °C to rt, 100%; (j) (Boc)2O, DMAP, Et3N, DMF, 0 °C to rt, 91%; (k) TBAF, THF,
rt, 100%; (l) H2NSO2Cl, Et3N, DMF, 0 °C to rt, 87%; (m) iodosylbenzene, Rh2(esp)2, 4 Å molecular sieves, CH3CN, 36 °C, 75% (X = NH) or 85% (X = O); (n) 5:4:1 TFA–CH2Cl2–H2O
rt, 74% (X = NH) or 71% (X = O).
the catalyst. By considering the details of the Rh-catalyzed imina-
tion mechanism (Scheme 3), we were able to identify several mod-
ifications to our initial coupling conditions that raised the yield of
the target compound to 75% (Table 1). First, Rh2(esp)2 was used in
place of Rh2(OAc)4 because the steric bulk of the
a,a, ,
a0 a0-tetra-
methyl-1,3-benzenedipropionate ligands would prevent coordina-
tion of the rhodium center (with concomitant inactivation of the
catalyst) by nitrogen atoms in the adenine ring. Another important
problem was rhodium-catalyzed intramolecular C–H bond amina-
tion of both sulfamide 12 and sulfamate 16 (Scheme 4),29 which
competed with intermolecular imination. The solution was to
employ an excess of the sulfoxide component 7 in order to increase
the rate of sulfoximine formation. The overall rate of coupling was
also accelerated by the addition of 4 Å molecular sieves to remove
water generated from iodosylbenzene, and increasing the reaction
temperature gave improved yields of the sulfoximine targets.
Scheme 3. Schematic overview of the rhodium-catalyzed imination reaction.
zyme was obtained by expression in Sf9 insect cells, and purified
by metal-affinity chromatography, as reported previously.32 The
effect of sulfoximine 3 (as a mixture of four diastereoisomers) on
glutamine-dependent asparagine synthesis was assayed by mea-
suring the rate of inorganic pyrophosphate (PPi) production.33
Somewhat unexpectedly, these experiments showed that 3 was
much less effective at inhibiting human ASNS than the adenylated
sulfoximine 1, even though it still exhibited slow-onset kinetics
(Fig. 2).34 Similar behavior was observed when ammonia was used
3. Enzyme assays
Having obtained the adenylated sulfoximine 3 (Fig. 1), we
investigated its ability to inhibit human ASNS using well-estab-
lished steady-state kinetic protocols.17,30,31 The recombinant en-
in place of
L-glutamine as the nitrogen source (data not shown).
After establishing that 3 did not inhibit the coupling enzymes used
Table 1
Yield of adenylated sulfoximine 17 upon coupling reaction conditions
7 (equiv)
12 (equiv)
Catalyst (20 mol %)a
Solvent
Temp (°C)
4 Å Sievesb
Yield (%)
1
1
1
1
5
5
3
1.5
1.5
1.5
1.5
1
Fe(acac)3
Rh2(OAc)4
Rh2(OAc)4
Rh2(esp)2
Rh2(esp)2
Rh2(esp)2
Rh2(esp)2
CH3CN
1,4-Dioxane
CH3CN
25
25
25
25
25
25
36
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+
NDc
4
14
20
40
50
75
CH3CN
Dry CH3CN
Dry CH3CN
Dry CH3CN
1
1
+
a
b
c
Esp—a,a,a’,a’-tetramethyl-1,3-benzenedipropionic acid.
Indicates whether sieves were present (+) or absent (ꢀ) in the reaction mixture.
Not determined.

6644
H. Ikeuchi et al. / Bioorg. Med. Chem. 17 (2009) 6641–6650
Scheme 4. By-products formed by the rhodium-catalyzed, intramolecular C–H insertion reaction.
to detect PPi (data not shown), the observed kinetic behavior was
analyzed using a standard kinetic model in which it is assumed
that the inhibitor binds to the free enzyme, as demonstrated for
the structurally related sulfoximine 1.16 When glutamine was em-
ployed as the nitrogen source, curve fitting gave values of
assayed the sulfamate analog 4 from the protected sulfamate 16
(Scheme 2). To our surprise, kinetic experiments showed that the
sulfamate 4 had essentially no effect on either the glutamine- or
ammonia-dependent synthetase activity of human ASNS at con-
centrations up to 1 mM, at least on the basis of PPi production.
Control experiments were also undertaken, which established that
neither 3 nor 4 perturbed the 1:1 Asn:PPi ratio by stimulating ATP
hydrolysis (data not shown).
20 10
lM and 10
6
l
M for KI and Kꢁ, respectively. As discussed
I
elsewhere,16,35,36 there is considerable evidence for a strong ste-
reochemical dependence of tight-binding inhibition in the case of
functionalized sulfoximines. For example, only a single enantiomer
of a given sulfoximine derivative exhibits potent inhibitor activity
in experiments where diastereoisomeric and/or enantiomeric mix-
tures of sulfoximines have been separated.37,38 Additional support
for this assumption is provided by experimental39,40 and computa-
tional studies41 showing that hydrogen bonding interactions stabi-
lize a single epimer of the tetrahedral intermediate formed when
nucleophiles react with activated carbonyl groups. Assuming that
only one of the four diastereoisomers of 3 is active, then the KIꢁ
value demonstrates that the sulfamide derivative is three orders
of magnitude less effective an inhibitor of human ASNS than the
adenylated sulfoximine 1. No chromatographic conditions were
identified under which the diastereoisomers of the adenylated sul-
foximines 3, or 17 could be separated, and although it was possible
to separate the diastereoisomers of sulfoxide derivative 7, we
decided to examine the ability of other sulfoximine analogs to
inhibit human ASNS in light of the substantial decrease in the KIꢁ
value for 3 relative to that of 1. Thus, given that there are numer-
ous examples of potent inhibitors in which a phosphate moiety has
been replaced by either sulfamide or sulfamate functional
groups,42–44 including acylsulfonamide 2,20 we prepared and
Assuming that diastereoisomers of compounds 1 and 3 having
the same relative stereochemistry bind in the same fashion to
the synthetase site of ASNS, these observations appear consistent
with a model in which the negative charge on the phosphate group
of the acyl-adenylate intermediate is an essential element in ligand
recognition by the synthetase site of human ASNS. There are sev-
eral reasons, in addition to those discussed above, that support
the idea that the active forms of 1 and 3 bind in similar, if not iden-
tical, orientations. For example, it seems certain that the adenosyl
moieties of 1 and 3 bind within the ATP pocket, which is very
tightly defined in the AS-B crystal structure because of the need
to form hydrogen bonds between the purine and the backbone
amides of a conserved valine, thereby ensuring that the enzyme
uses ATP rather than GTP as a substrate.45 Prior work has also
shown that ASNS binds the amino and carboxylate groups of aspar-
tate in order to recognize this substrate with high specificity.46 If
the negative charge on the phosphate group of the acyl-adenylate
intermediate is essential for ligand recognition, then the adenylat-
ed sulfoximine 1 can bind to the enzyme with high affinity because
it contains an ionized phosphoramidate moiety. The acylsulfamate
and sulfamide derivatives 2 and 3, can only form this electrostatic
interaction within the synthetase site, however, if they bind as
their negatively charged conjugate bases (Fig. 3). As a result, if
the pKa of the acylated sulfamate NH in 2 is lower than that of
the sulfamide NH in 3, the former compound would be expected
to be a more potent human ASNS inhibitor. That this is the case
seems evident from qualitative considerations of resonance stabil-
ization of the conjugate bases of 2 and 3, but the likely differences
in pKa can be placed on a more quantitative footing through recent
studies of sulfamate inhibitors for the adenylating enzyme MtbA,47
which is present in Mycobacterium tuberculosis.48 Thus, the pKa of
the N-benzoyl derivative 19 (Fig. 5) is reported to be 2.8,47 suggest-
ing that the pKa of the N-acylsulfamate 2 is probably in the range of
4–5 as the carbonyl group of the latter compound is less electron-
Figure 2. Glutamine-dependent production of PPi in the presence of increasing
amounts of the functionalized sulfoximine 3: open circles, 0
10 M; open diamonds, 50 M; crosses, 100 M; closed diamonds, 500
triangles, 1 mM; closed circles, 10 M N-adenylated sulfoximine 1. Solid lines
represent the best fit lines using Eq. 1 (see Section 5).
l
M; open squares,
l
l
l
lM; closed
l
Figure 3. Conjugate bases of the functionalized acylsulfamate 2 (top) and sulfam-
ide 3 (bottom).

H. Ikeuchi et al. / Bioorg. Med. Chem. 17 (2009) 6641–6650
6645
deficient. Similarly, the NH pKa of the electron-deficient, function-
alized sulfamate 3 is probably in the range of 9–10 given that those
of sulfanilamide and methanesulfonamide are 10.43 and 10.87. Fi-
nally, our model also explains the inability of functionalized sul-
foximine
4 to exhibit significant inhibitory activity against
human ASNS. Hence, the absence of suitably positioned ionizable
groups in 4 meaning that this compound can only interact with
the enzyme in its neutral form at the solution pH values employed
in our assays.
Figure 5. Structure of N-benzoylsulfamate 19 for which the pKa of the sulfamate
NH proton has been established.47
Our hypothesis is reinforced by considering a computational
model in which the acyl-adenylate intermediate (Scheme 1) is
bound within the synthetase site of Escherichia coli AS-B (Fig. 4).
Although this model was built from the crystal structure of AS-B
(Ding, unpublished results), which is an ortholog of human ASNS,
all residues within the active site are strictly conserved in the bac-
terial and human enzymes. In this model, the phosphate moiety
interacts with the side chain of Lys-449 (AS-B numbering), which
corresponds to Lys-466 of the human enzyme. This residue is
invariant in known glutamine-dependent asparagine synthetases,
suggesting that this electrostatic interaction is critical for ATP
binding and/or catalysis. The importance of this active site lysine
is underscored by the fact that efforts to obtain site-directed AS-
B mutants at this position have only given enzymes lacking any
synthetase activity (Meyer, unpublished data). Thus, we conclude
that potent ASNS inhibitors must possess functional groups that
can mimic the negative charge of the phosphate group when
bound within the synthetase site so as to maintain this interaction
with the lysine side chain. This is an important finding for (i) the
design of sulfoximine-based libraries of small molecules with
which to explore the extent to which the adenylate moiety can
be replaced by other heterocycles, and (ii) the development of
ASNS inhibitors that can easily be taken up into leukemia cells.
enzyme,36 including glutathione synthetase,49
synthetase,50 -glutamyl transpeptidase,51 and asparagine synthe-
c-glutamylcysteine
c
tase.15,16 This protocol also enables the creation of sulfoximine
libraries that can be screened for ASNS inhibition using high-
throughput methods and optimized by rational discovery strate-
gies.52 Perhaps more importantly, we conclude that potent ASNS
inhibitors must possess functionality that can mimic the negative
charge of the phosphate group so as to form a favorable electro-
static interaction with the conserved lysine when bound within
the synthetase site. On this point, we note that previous work
using modified acylsulfamates has already shown that similar elec-
trostatic interactions involving the charged amino and carboxylate
moieties of aspartic acid contribute a considerable amount of bind-
ing energy.20 The importance of electrostatics in inhibitor recogni-
tion is therefore established, which is significant finding for the
development of highly cell-permeable ASNS inhibitors that can
be used clinically in the treatment of leukemia and ovarian
cancer.53,54
5. Experimental
5.1. General
All chemicals were obtained commercially and used without
further purification unless otherwise stated. N,N-dimethylformam-
ide (DMF), 1,4-dioxane, acetonitrile and CH2Cl2, triethylamine
(Et3N) were purchased from Wako Pure Chemical Industries (Osa-
ka, Japan), dried by distillation from CaH2 and stored over 4 Å
molecular sieves. Melting points (mp) were recorded using a Met-
tler FP62 melting point apparatus and are corrected. Optical rota-
tions were measured using a Horiba automatic polarimeter SEPA-
200. 1H NMR spectra were recorded on a JEOL JNM-AL 300 at
300 MHz. In CDCl3, DMSO-d6 and acetone-d6, 1H chemical shifts
are reported in ppm (d) downfield of tetramethylsilane as an inter-
nal reference (d 0.0), except for measurements in D2O for which so-
dium 3-(trimethylsilyl)propanesulfonate was used as an internal
standard (d 0.0). Splitting patterns are abbreviated as follows: s,
singlet, d, doublet, t, triplet, q, quartet, and m, multiplet. Infrared
spectra were determined using a Horiba FT-720 spectrophotome-
ter, and mass spectra were recorded on a JEOL JMS 700 (high res-
olution) spectrometer. Elemental analyses were performed on a
Yanaco MT-5 apparatus. Reactions were monitored with analytical
thin layer chromatography (TLC) on Silica Gel 60-F254 plates
(Merck). Compounds were purified by flash column chromatogra-
4. Conclusions
Conditions have been developed that will permit the facile
construction of highly functionalized sulfoximines from readily
available sulfoxide and sulfamide precursors. Such compounds
are proven inhibitors of several biologically important classes of
phy on silica gel 60 N (Kanto Kagaku, 40–50 lm), or by medium-
pressure, reverse-phase column chromatography on Diaion
HP20SS resin (Mitsubishi Chemical Corporation) using a Yamazen
YFLC System (Yamazen Co., Osaka, Japan).
5.1.1. 2-(N-tert-Butoxycarbonyl)amino-S-methylcysteine (5)
S-Methyl-L-cysteine (5.1 g, 37.7 mmol) was protected by a
known procedure (Boc2O, NaOH, tert-BuOH-H2O) to give partially
racemized 5 as a colorless oil (8.8 g, quant.): ½a D17
ꢀ14.6 (c 1.05,
ꢂ
Figure 4. The interaction between the Lys-449 side chain and the phosphate group
of the acyl-adenylate intermediate (both shown in ‘stick’ representations) in a
computational model of the AS-B/Gln/acyl-adenylate complex (Ding, unpublished).
The protein backbone is shown by the blue ribbon. Color scheme: C, green; H,
white; N, blue; O, red; P, orange. This image was generated in PYMOL.65
AcOH) [lit.55 ꢀ24.6 (c 0.705, AcOH)]; IR (NaCl, neat) mmax 3600–
3318 (br), 2978, 2925, 1720, 1693, 1666, 1511, 1394, 1369, 1336,
1299, 1249, 1164, 1056, 1025, 960, 914, 860, 777, 744, 669, 572,

6646
H. Ikeuchi et al. / Bioorg. Med. Chem. 17 (2009) 6641–6650
478, 443 cmꢀ1; 1H NMR (300 MHz, CDCl3) dH 1.46 [s, 9H, (CH3)3CO-
CON], 2.16 (s, 3H, SCH3), 2.94–3.10 (m, 2H, CH2S), 4.57 (m, 1H,
proton), 5.42 (br d, J = 6.9 Hz, 1H, NH), 8.53 (br s, 1H, COOH); Anal.
Calcd for C9H17NO4S: C, 45.94; H, 7.28; N, 5.95. Found: C, 45.82; H,
7.43; N, 5.81. HRMS (FAB, glycerol) calcd for C9H18NO4S (MH+)
236.0957, found 236.0954.
and evaporation of the solvent gave a residue that was re-dissolved
in CHCl3 (150 mL). The CHCl3 solution was washed successively
with water (50 mL) and sat. NaCl (50 mL), and then dried over
Na2SO4. After filtration, and CHCl3 was evaporated to give a brown
oil, which was purified by flash column chromatography on silica
gel (EtOH/CHCl3, 1:9) to afford the azide 8 as an amorphous, yellow
solid that could be used in subsequent steps without further puri-
fication (5.4 g, 100%): IR (KBr) mmax 3309, 3154, 3133, 2989, 2935,
2105 (N3), 1675, 1646, 1600, 1473, 1417, 1375, 1328, 1301,
1249, 1213, 1155, 1118, 1078, 1010, 931, 869, 798, 715, 690,
a
-
5.1.2. tert-Butyl 2-(N-tert-butoxycarbonyl)amino-3-methylsulfa-
nylpropanoate (6)
A mixture of 2-(N-tert-butoxycarbonyl)amino-S-methylcysteine
(5) (8.88 g, 37.7 mmol), 4-(N,N-dimethylamino)pyridine (1.38 g,
11.3 mmol), and 2-methyl-2-propanol (3.36 g, 45.3 mmol) in dry
CH2Cl2 (50 mL) was cooled at 0 °C under an Ar atmosphere. N,N0-
Dicyclohexylcarbodiimide (8.72 g, 42.3 mmol) was added to the
solution, and the resulting reaction mixture was stirred initially
at 0 °C for 2 h and then overnight at room temperature. After filtra-
tion to remove dicyclohexylurea, the filtrate was evaporated and
the residue dissolved in EtOAc (100 mL). This solution was filtrated
again, and the filtrate washed successively with 5% aq KHSO4
(30 mL), water (30 mL) and satd NaCl (30 mL). After drying over
Na2SO4, filtration and evaporation of the solvent under reduced
pressure gave an oil that was purified by flash column chromatog-
raphy on silica gel (EtOAc/hexane, 1:5) to afford the racemic sulfide
6 as a colorless oil (9.9 g, 90%): IR (NaCl, neat) mmax 3372, 2977,
2931, 1716, 1498, 1455, 1367, 1309, 1249, 1222, 1155, 1054,
649, 595, 538, 511, 412 cmꢀ1 1H NMR (300 MHz, CDCl3) dH 1.40
;
(s, 3H, isopropylidene), 1.62 (s, 3H, isopropylidene), 3.56 (dd,
J = 5.4 and 12.9 Hz, 1H) and 3.61 (dd, J = 6.3 and 12.9 Hz, 1H) (50-
CH2), 4.39 (dt, J = 3.6 and 5.9 Hz, 1H, 40-H), 5.07 (dd, J = 3.6 and
6.3 Hz, 1H, 30-H), 5.47 (dd, J = 2.4 and 6.3 Hz, 1H, 20-H), 5.65 (br s,
2H, adenyl NH2), 6.11 (d, J = 2.4 Hz, 1H, 10-H), 7.92 (s, 1H, adenyl
2-H), 8.37 (s, 1H, adenyl 8-H); HRMS (FAB, p-nitrobenzyl alcohol)
calcd for C13H17N8O3 (MH+) 333.1426, found 333.1424.
5.1.5. 50-Azido-N6,N6-bis(tert-butoxycarbonyl)-50-deoxy-20,30-O-
isopropylideneadenosine (9)
4-(N,N-Dimethylamino)pyridine (349 mg, 2.85 mmol) and dry
Et3N (4.33 g, 5.96 mL, 42.8 mmol) were added to a solution of azide
8 (4.74 g, 14.3 mmol) in dry DMF (100 mL) at 0 °C under an Ar
atmosphere. Boc2O (12.45 g, 57.1 mmol) was then added to this
solution, and the mixture stirred at 0 °C for 1 h and at room tem-
perature for 4 h. The reaction mixture was filtered and evaporated
to give a residual oil that was purified by flash column chromatog-
raphy on silica gel (EtOAc/hexane, 1:1). The desired azide 9 was ob-
tained as an amorphous solid (6.24 g, 82%): IR (KBr) mmax 3324,
3170, 2983, 2937, 2103 (N3), 1789, 1758, 1731, 1600, 1577,
1498, 1454, 1419, 1371, 1336, 1278, 1253, 1211, 1106, 1039,
1010, 943, 846, 777, 669, 632, 557, 503, 464, 435 cmꢀ1 1H NMR
;
(300 MHz, CDCl3) dH 1.45 [s, 9H, (CH3)3COCON], 1.48 [s, 9H,
(CH3)3CO], 2.15 (s, 3H, SCH3), 2.89 and 2.92 (2 ꢃ dd, 2H, J = 5.4
and 13.1 Hz, CH2S), 4.41 (m, 1H,
a-proton), 5.34 (br d, J = 7.2 Hz,
1H, NH); Anal. Calcd for C13H25NO4S: C, 53.58; H, 8.65; N, 4.81.
Found: C, 53.48; H, 8.58; N, 4.94. HRMS (FAB, glycerol) calcd for
C13H26NO4S (MH+) 292.1588, found 292.1583.
914, 850, 811, 775, 734, 646, 590, 559. 509, 462, 433 cmꢀ1 1H
;
5.1.3. (R,S)-tert-Butyl 2-(N-tert-butoxycarbonyl)amino-3-(R,S)-
methylsulfinylpropanoate (7)
NMR (300 MHz, CDCl3) dH 1.41 (s, 3H, isopropylidene), 1.46 [s,
18H, 2 ꢃ (CH3)3CO], 1.64 (s, 3H, isopropylidene), 3.57–3.63 (m,
2H, 50-CH2), 4.41 (q, J = 3.6 Hz, 1H, 40-H), 5.06 (dd, J = 3.6 and
6.3 Hz, 1H, 30-H), 5.44 (dd, J = 2.4 and 6.3 Hz, 1H, 20-H), 6.19 (d,
J = 2.4 Hz, 1H, 10-H), 8.21 (s, 1H, adenyl 2-H), 8.89 (s, 1H, adenyl
8-H); Anal. Calcd for C23H32N8O7: C, 51.87; H, 6.06; N, 21.04.
Found: C, 51.75; H, 6.05; N, 20.98. HRMS (FAB, glycerol) calcd for
C23H33N8O7 (MH+) 533.2475, found 533.2471.
The sulfide 6 (10 g, 34.3 mmol) was dissolved in THF–H2O (5:2,
70 mL) and was cooled at 0 °C before the addition of an aqueous
solution (30 mL) of NaIO4 (8.8 g, 41.1 mmol). The mixture was stir-
red at ambient temperature overnight and then filtered. After re-
moval of THF under reduced pressure, EtOAc (100 mL) was added
to the resulting aqueous solution, which was then washed succes-
sively with water (30 mL) and sat. NaCl (30 mL). After drying over
Na2SO4 and filtration, the filtrate was evaporated to give the sulf-
oxide 7 as a 5:4 mixture of two diastereomers (9.79 g, 93%, color-
less oil): IR (NaCl, neat) mmax 3400–3200 (br), 2979, 2933, 2815,
1716, 1523, 1455, 1392, 1367, 1278, 1251, 1155, 1049, 1024,
5.1.6. 50-Amino-N6,N6-bis(tert-butoxycarbonyl)-50-deoxy-20,30-
O-isopropylideneadenosine (10)
50-Azido-N6,N6-bis(tert-butoxycarbonyl)-50-deoxy-20,30-O-iso-
propylideneadenosine (9) (9.4 g, 17.7 mmol) and 10% Pd on carbon
(wet, Degussa type E101 NE/W, Aldrich) were suspended in 9:1
MeOH–H2O (250 mL). Hydrogen gas was passed through the mix-
ture at ambient temperature for 5 h, and completion of reaction, as
monitored by TLC (eluant: CHCl3/MeOH, 10:1), the mixture was fil-
tered through Hyflo Super-Cel, and the filtrate was evaporated to
give crude amine 10 as an amorphous solid (8.94 g), which was
contaminated with approximately 10% of 50-N-Boc-N6-Boc-adeno-
sine. This material 10 was used in the next step without further
purification (8.94 g, 100%): 1H NMR (300 MHz, CDCl3) dH 1.40 (s,
3H, isopropylidene), 1.47 [s, 18H, 2 ꢃ (CH3)3CO], 1.64 (s, 3H, iso-
propylidene), 2.92–3.08 (m, 2H, 50-CH2), 4.26–4.30 (m, 1H, 40-H),
5.02 (dd, J = 3.6 and 6.6 Hz, 1H, 30-H), 5.44 (dd, J = 3.0 and 6.6 Hz,
1H, 20-H), 6.13 (d, J = 3.0 Hz, 1H, 10-H), 8.23 (s, 1H, adenyl 2-H),
8.87 (s, 1H, adenyl 8-H); HRMS (FAB, p-nitrobenzylalcohol) calcd
for C23H35N6O7 (MH+) 507.2570, found 507.2567.
971, 943, 846, 813, 750, 686, 576, 565, 497, 458, 431 cmꢀ1 1H
;
NMR (300 MHz, DMSO-d6) dH 1.39 and 1.40 [2 ꢃ s, 9H, (CH3)3CO-
CON], 1.405 and 1.413 [2 ꢃ s, 9H, (CH3)3CO], 2.577 and 2.584
(2 ꢃ s, 3H, SCH3), 2.93 (dd, J = 7.8 and 13.2 Hz), 2.98 (m) and 3.19
(dd, J = 5.9 and 13.1 Hz) (2H, CH2S@O), 4.15 (dt, J = 5.7 and
8.7 Hz) and 4.23 (dt, J = 6.0 and 7.8 Hz) (1H,
a-proton), 7.37 and
7.46 (2 ꢃ br d, J = 8.1 Hz, 1H, NH); Anal. Calcd for C13H25NO5S: C,
50.79; H, 8.20; N, 4.56. Found: C, 50.57; H, 8.28; N, 4.48. HRMS
(FAB, glycerol) calcd for C13H26NO5S (MH+) 308.1533, found
308.1532.
5.1.4. 50-Azido-50-deoxy-20,30-O-isopropylideneadenosine (8)
20,30-O-Isopropylideneadenosine (5.0 g, 16.3 mmol) was sus-
pended in dry 1,4-dioxane (50 mL) at room temperature under
an Ar atmosphere. Diphenylphosphoryl azide (9.45 g, 7.4 mL,
32.5 mmol) and DBU (7.64 g, 7.5 mL, 48.8 mmol) were added drop-
wise to the suspension, and the reaction mixture stirred at ambient
temperature for 4 h. Sodium azide (5.29 g, 81.4 mmol) and 15-
crown-5 (0.359 g, 0.323 mL, 1.63 mmol) were added, and the mix-
ture heated at 110 °C for 4 h under an Ar atmosphere. Filtration
5.1.7. 50-[(N-Benzyloxycarbonyl)sulfamoylamino]-N6,N6-bis(tert-
butoxycarbonyl)-50-deoxy-20,30-O-isopropylideneadenosine (11)
Benzyl alcohol (1.39 g, 1.33 mL, 12.9 mmol) was added drop-
wise to a stirred solution of chlorosulfonyl isocyanate (1.82 g,

H. Ikeuchi et al. / Bioorg. Med. Chem. 17 (2009) 6641–6650
6647
1.12 mL, 12.9 mmol) in dry CH2Cl2 (20 mL) at 0 °C, and the mixture
was stirred at 0 °C for 1 h under an Ar atmosphere. After comple-
tion of the reaction, as judged by 1HNMR (dH 4.615 and 5.319 for
PhCH2O proton for benzyl alcohol and N-benzyloxycarbonylsulfa-
moyl chloride, respectively), the resulting solution was added via
cannula to an ice-cold solution of the crude amine 10 (5.93 g,
11.7 mmol) and dry Et3N (2.48 g, 3.42 mL, 23.4 mmol) in dry
CH2Cl2 (60 mL). The mixture was stirred at ambient temperature
overnight, before being evaporated under reduced pressure to give
a residue that was dissolved in EtOAc (100 mL). This solution was
washed successively with 5% KHSO4 (40 mL), water (30 mL) and
sat. NaCl (30 mL). After drying over Na2SO4 and filtration, evapora-
tion of the solvent gave an amorphous solid, which was purified by
flash column chromatography on silica gel (EtOAc/hexane, 3:2) to
afford 11 as an amorphous solid (6.94 g, 82%): 1H NMR
(300 MHz, CDCl3) dH 1.37 (s, 3H, isopropylidene), 1.48 [s, 18H,
2 ꢃ (CH3)3CO], 1.64 (s, 3H, isopropylidene), 3.41–3.57 (m, 2H, 50-
CH2), 4.58 (m, 1H, 40-H), 5.03 (d, J = 12 Hz, 1H, PhCH2O), 5.05 (dd,
J = 1.5 and 6.3 Hz, 1H, 30-H), 5.14 (d, J = 12 Hz, 1H, PhCH2O), 5.23
(dd, J = 4.8 and 6.3 Hz, 1H, 20-H), 5.86 (d, J = 4.8 Hz, 1H, 10-H),
7,28–7.36 (m, 6H, Ph and SO2NHCH2), 8.06 (s, 1H, adenyl 2-H),
9.06 (s, 1H, adenyl 8-H), 9.28 (d, J = 9.6 Hz, 1H, CONHSO2); Anal.
Calcd for C31H41N7O11S: C, 51.73; H, 5.74; N, 13.62. Found: C,
51.70; H, 5.84; N, 13.56. HRMS (FAB, glycerol) calcd for
C31H42N7O11S (MH+) 720.2666, found 720.2654.
(300 MHz, CDCl3) dH 0.014 (s, 3H, CH3Si), 0.02 (s, 3H, CH3Si),
0.845 (s, 9H, (CH3)3CSi), 1.41 (s, 3H, isopropylidene), 1.64 (s, 3H,
isopropylidene), 3.76 (dd, J = 4.2 and 11.1 Hz, 1H, 50-CH2), 3.88
(dd, J = 3.9 and 11.1 Hz, 1H, 50-CH2), 4.26 (q, J = 2.4 Hz, 1H, 40-H),
4.96 (dd, J = 2.4 and 6.0 Hz, 1H, 30-H), 5.28 (dd, J = 2.4 and 6.0 Hz,
1H, 20-H), 5.50 (br s, 2H, adenyl NH2), 6.17 (d, J = 2.4 Hz, 1H, 10-
H), 8.05 (s, 1H, adenyl 2-H), 8.39 (s, 1H, adenyl 8-H); Anal. Calcd
for C19H31N5O4Si: C, 54.13; H, 7.41; N, 16.01. Found: C, 54.04; H,
7.51; N, 16.21. HRMS (FAB, glycerol) calcd for C19H32N5O4Si
(MH+) 422.2225, found 422.2216.
5.1.10. N6,N6-Bis(tert-butoxycarbonyl)-50-O-tert-butyldimeth-
ylsilyl-20,30-O-isopropylidene-adenosine (14)
50-O-tert-Butyldimethylsilyl-20,30-O-isopropylideneadenosine (13)
(7 g, 16.6 mmol) was dissolved in dry DMF (150 mL) together with
4-(N,N-dimethylamino)pyridine (0.41 g, 3.49 mmol) and dry Et3N
(3.53 g, 4.86 mL, 34.9 mmol) under an Ar atmosphere. Boc2O
(7.61 g, 34.9 mmol) was added to this solution at 0 °C, and the mix-
ture was stirred initially at 0 °C for 1 h and then at room tempera-
ture for 3 h. The reaction mixture was filtered and evaporated
under a high vacuum (1 mmHg) to give an oily residue, which
could be purified by flash column chromatography on silica gel
(EtOAc/hexane, 1:2) to give 14 as a colorless solid (9.4 g, 91%):
1H NMR (300 MHz, CDCl3) dH 0.031 [s, 6H, (CH3)2Si], 0.86 [s, 9H,
(CH3)3CSi], 1.42 (s, 3H, isopropylidene), 1.44 (s, 18H, 2 ꢃ (CH3)3-
CO), 1.66 (s, 3H, isopropylidene), 3.79 (dd, J = 3.9 and 11.1 Hz,
1H, 50-CH2), 3.90 (dd, J = 3.6 and 11.4 Hz, 1H, 50-CH2), 4.46 (q,
J = 3.2 Hz, 1H, 40-H), 4.96 (dd, J = 2.7 and 6.0 Hz, 1H, 30-H), 5.23
(dd, J = 2.7 and 6.0 Hz, 1H, 20-H), 6.26 (d, J = 2.4 Hz, 1H, 10-H),
8.35 (s, 1H, adenyl 2-H), 8.89 (s, 1H, adenyl 8-H); Anal. Calcd for
C29H47N5O8Si: C, 56.02; H, 7.62; N, 11.26. Found: C, 56.22; H,
7.61; N, 11.21. HRMS (FAB, glycerol) calcd for C29H48N5O8Si
(MH+) 622.3266, found 622.3280.
5.1.8. N6,N6-Bis(tert-butoxycarbonyl)-50-deoxy-20,30-O-isopropyl-
idene-50-sulfamoylamino adenosine (12)
The protected sulfamide 11 (6.43 g, 8.94 mmol) and 10% Pd on
carbon (wet, Degussa type E101 NE/W, Aldrich) were suspended
in 9:1 MeOH–H2O (250 mL). Hydrogen gas was passed through
the mixture at ambient temperature for 2 h, and on completion
of the reaction, as monitored by TLC (eluant: EtOAc/hexane, 3:2),
the mixture was filtered through Hyflo Super-Cel, and the filtrate
evaporated to give crude 12 as an amorphous solid. The crude
product was purified by flash column chromatography on silica
gel (EtOAc/hexane, 2:1) to afford sulfamide 12 as an amorphous
solid (4.59 g, 88%): IR (KBr) mmax 1798, 1758, 1735, 1602, 1581,
1500, 1455, 1421, 1371, 1336, 1276, 1253, 1214, 1159, 1106,
5.1.11. N6,N6-Bis(tert-butoxycarbonyl)-20,30-O-isopropylidenead-
enosine (15)
N6,N6-Bis(tert-butoxycarbonyl)-50-O-tert-butyldimethylsilyl-20,30-
O-isopropylideneadenosine (14) (9.00 g, 14.5 mmol) was dissolved
in dry THF (100 mL), and tetrabutylammonium fluoride (TBAF,
1.0 M solution in THF, 21 mL, 21.7 mmol) added to the solution
at room temperature under an Ar atmosphere. The mixture was
stirred for 3 h at room temperature before being evaporated to give
a colorless oil, which was purified by flash column chromatogra-
phy on silica gel (acetone/hexane, 1:2) to give 15 as an amorphous
solid (7.33 g, 100%): IR (KBr) mmax 3509–3369 (br), 3116, 3089,
2983, 2937, 2881, 1789, 1758, 1735, 1602, 1579, 1498, 1455,
1419, 1371, 1336, 1276, 1253, 1213, 1141, 1110, 1083, 950, 850,
983, 941, 852, 775, 696, 644, 590, 549, 464 cmꢀ1
;
1H NMR
(300 MHz, CDCl3) dH 1.38 (s, 3H, isopropylidene), 1.48 [s, 18H,
2 ꢃ (CH3)3CO], 1.64 (s, 3H, isopropylidene), 3.50 (dd, J = 3.0 and
5.9 Hz, 2H, 50-CH2), 4.55 (br s, 1H, SO2NH2), 4.58 (m, 1H, 40-H),
5.13 (dd, J = 2.4 and 6.5 Hz, 1H, 30-H), 5.34 (dd, J = 4.8 and 6.3 Hz,
1H, 20-H), 5.90 (d, J = 4.5 Hz, 1H, 10-H), 7.74 (br t, J = 5.7 and
5.6 Hz, 1H, SO2NHCH2), 8.11 (s, 1H, adenyl 2-H), 8.96 (s, 1H, adenyl
8-H); Anal. Calcd for C23H35N7O9Sꢄ0.3 EtOAc: C, 47.49; H, 6.15; N,
16.01. Found: C, 47.29; H, 5.97; N, 16.01. HRMS (FAB, p-nitrobenzyl
alcohol) calcd for C23H36N7O9S (MH+) 586.2297, found 586.2295.
775, 646, 588, 559, 511, 462 cmꢀ1 1H NMR (300 MHz, CDCl3) dH
;
1.39 (s, 3H, isopropylidene), 1.47 [s, 18H, 2 ꢃ (CH3)3CO], 1.66 (s,
3H, isopropylidene), 3.80 (dd, J = 1.8 and 11.1 Hz), 3.84 (dd,
J = 2.1 and 11.4 Hz) and 4.00 (dt, J = 1.5 and 12.9 Hz) (2H, 50-CH2),
4.56 (s, 1H, OH), 5.13 (dd, J = 1.2 and 6.0 Hz, 1H, 40-H), 5.22 (dd,
J = 5.1 and 5.4 Hz, 1H, 20-H), 5.40 (dd, J = 2.4 and 11.1 Hz, 1H, 30-
H), 5.94 (d, J = 4.8 Hz, 1H, 10-H), 8.12 (s, 1H, adenyl 2-H), 8.84 (s,
1H, adenyl 8-H); Anal. Calcd for C23H33N5O8: C, 54.43; H, 6.55; N,
13.80. Found: C, 54.42; H, 6.64; N, 13.51. HRMS (FAB, glycerol)
calcd for C23H34N5O8 (MH+) 508.2409, found 508.2399.
5.1.9. 50-O-tert-Butyldimethylsilyl-20,30-O-isopropylideneadeno-
sine (13)
Imidazole (2.83 g, 40.7 mmol) and tert-butyldimethylsilyl chlo-
ride (2.95 g, 19.5 mmol) were added to a solution of 20,30-O-isopro-
pylideneadenosine (5.00 g, 16.3 mmol) in dry DMF (50 mL) at 0 °C
under an Ar atmosphere. The mixture was stirred at 0 °C for 2 h
and then at room temperature overnight before being filtered
and evaporated under a high vacuum (1 mmHg). 2:1 EtOAc/H2O
(300 mL) was added to the resulting residue. The organic layer
was separated, washed successively with water (100 mL) and satd
NaCl (50 mL), and dried over Na2SO4. Filtration and evaporation of
the solvent under reduced pressure yielded 13 as a colorless solid
(6.81 g, 100%): IR (KBr) mmax 3363, 3273, 3120, 3050, 2985, 2935,
2857, 2749, 2682, 1918, 1679, 1646, 1604, 1571, 1508, 1473,
1415, 1373, 1330, 1303, 1255, 1207, 1132, 1081, 1008, 964, 906,
5.1.12. N6,N6-Bis(tert-butoxycarbonyl)-20,30-O-isopropylidene-
50-O-sulfamoyladenosine (16)
N6,N6-Bis(tert-butoxycarbonyl)-20,30-O-isopropylideneadenosine
(15) (2.4 g, 4.73 mmol) and dry Et3N (0.493 g, 0.679 ml, 4.87 mmol)
were dissolved in dry DMF (10 mL) and the solution cooled to 0 °C
under an Ar atmosphere. Sulfamoyl chloride (20)56 (1.31 g,
11.3 mmol) was added to the solution, and the reaction mixture
was stirred at 0 °C for 2 h and then at room temperature overnight.
860, 838, 773, 717, 659, 638, 592, 541, 511 cmꢀ1
;
1H NMR

6648
H. Ikeuchi et al. / Bioorg. Med. Chem. 17 (2009) 6641–6650
The reaction mixture was evaporated, and the residue was diluted
with EtOAc (50 mL). This solution was then washed successively
with water (30 mL) and satd NaCl (30 mL) before being dried over
Na2SO4. The EtOAc solution was filtered, and the filtrate was evap-
orated to give sulfamate (16) as an amorphous solid that was used
3.29, 3.311 and 3.338 (3 ꢃ s, 3H, SCH3), 3.83–4.48 (m, 5H CH2S,
50-CH2, -proton), 4.62 (m, 1H, 40-H), 5.13–5.16 (m, 1H, 30-H),
a
5.31–5.40 (m, 1H, 20-H), 5.64 (br, 1H, CONH), 6.27 (d, J = 2.7 Hz,
1H, 10-H), 8.323 and 8.331 (2 ꢃ s, 1H, adenyl 2-H), 8.880 and
8.888 (2 ꢃ s, 1H, adenyl 8-H); Anal. Calcd for C36H57N7O15S2: C,
48.47; H, 6.44; N, 10.99. Found: C, 48.69; H, 6.54; N, 10.77. HRMS
(FAB, p-nitrobenzyl alcohol) calcd for C36H58N7O15S2 (MH+)
892.3435, found 892.3430.
without further purification (2.4 g, 87%): IR (KBr)
1789, 1731, 1604, 1581, 1496, 1455, 1373, 1338, 1278, 1253, 1213,
1186, 1139, 1110, 998, 850, 809, 775, 646, 593, 553, 514, 466 cmꢀ1
mmax 2981, 2938,
;
1H NMR (300 MHz, CDCl3) dH 1.43 (s, 3H, isopropylidene), 1.50 [s,
18H, 2 ꢃ (CH3)3CO], 1.64 (s, 3H, isopropylidene), 4.22 (dd, J = 4.5
and 10.8 Hz, 1H, 50-CH2), 4.30 (dd, J = 3.3 and 11.1 Hz, 1H, 50-
CH2), 4.6–4.7 (m, 1H, 40-H), 4.83 (br s, 2H, SO2NH2), 5.08 (dd,
J = 2.1 and 6.0 Hz, 1H, 30-H), 5.72 (dd, J = 1.5 and 6.0 Hz, 1H, 20-
H), 6.26 (d, J = 1.5 Hz, 1H, 10-H), 8.26 (s, 1H, adenyl 2-H), 8.90 (s,
5.1.15. (R,S)-2-Amino-3-{N-[(50-deoxyadenosin-50-yl)amin-
osulfonyl]-S-methyl-(R,S)-sulfonimidoyl}propanoic acid (3)
A suspension of the fully protected sulfoximine (17) (910 mg,
1.02 mmol) in 5:1:4 TFA–H2O–CH2Cl2 (10 mL) was stirred at ambi-
ent temperature for 5 h, and then evaporated to dryness to give a
residual syrup that was purified by reversed phase, medium-pres-
sure column chromatography on Diaion HP20SS eluting with a lin-
ear gradient of THF in H2O (0–99%). The eluant was monitored with
UV (254 nm), and the desired functionalized sulfoximine eluted at
79:21 H2O/THF. UV positive-fractions were then combined, and
1H, adenyl 8-H); HRMS (FAB, glycerol) calcd for C23H35N6O10
S
(MH+) 587.2134, found 587.2124.
5.1.13. (R,S)-tert-Butyl 2-(N-tert-butoxycarbonyl)amino-3-(N-
{[N6,N6-bis(tert-butoxycarbonyl)-50-deoxy-20,30-O-isopropylide-
neadenosin-50-yl]aminosulfonyl}-S-methyl-(R,S)-sulfonimi-
doyl) propanoate (17)
lyophilization afforded (3) as
a mixture of diastereomers
(374 mg, 74%, colorless solid): IR (KBr) mmax 3600–3000 (br),
1648, 1604, 1508, 1477, 1419, 1378, 303, 1216 (O@S@N), 1139,
1079 (S@O), 997, 962 (S@N), 898, 848, 798, 728, 680, 647, 599,
The protected adenosine (12) (1.5 g, 2.56 mmol) and (R,S)-tert-
butyl 2-(N-tert-butoxycarbonyl)amino-3-(R,S)-methylsulfinylpro-
panoate (7) (2.11 g, 6.86 mmol) were dissolved in dry CH3CN
(30 mL) before the addition of 4 Å molecular sieves (1 g), Rh2(esp)2
(389 mg, 0.512 mmol) and iodosylbenzene (1.18 g, 5.38 mmol).
The reaction mixture was stirred overnight at 36 °C under an Ar
atmosphere before being filtered and evaporated to yield a brown
oil. Purification of this material using flash column chromatogra-
phy on silica gel (CH3CN/toluene, 1:4) gave the desired sulfoximine
(17) as a mixture of diastereomers (1.71 g, 75%, amorphous solid):
586, 539, 462 cmꢀ1 1H NMR (300 MHz, D2O) dH 3.30–3.40 (m,
;
2H, 50-CH2), 3.487, 3.496, 3.513 and 3.522 (4 ꢃ s, 3H, SCH3),
3.80–4.40 (m, 4H CH2S,
a
-proton, 40-H), 4.54–4.81 (m, 2H, 30-H
and 20-H), 5.92 (dd, J = 3.0 and 6.2 Hz, 1H, 10-H), 8.14 (s, 1H, adenyl
2-H), 8.170 and 8.176 (2 ꢃ s, 1H, adenyl 8-H); Anal. Calcd for
C14H22N8O8S2ꢄ1.4H2O: C, 32.36; H, 4.81; N, 21.56. Found: C,
32.36; H, 4.77; N, 21.88. HRMS (FAB, glycerol) calcd for
C14H23N8O8S2 (MH+) 495.1083, found 495.1088.
IR (KBr)
mmax 3600–3386 (br), 2981, 2937, 1798, 1727, 1602, 1500,
1455, 1371, 1328, 1276, 1253, 1214 (O@S@N), 1151, 1103, 1079
5.1.16. (R,S)-2-Amino-3-[N-(adenosin-50-yl)-50-O-sulfonyl-S-me-
thyl-(R,S)-sulfonimidoyl]propanoic acid (4)
(S@O), 977, 941 (S@N), 850, 775, 646, 588, 509, 468, 437 cmꢀ1
;
1H NMR (300 MHz, CDCl3) dH 1.38 (s, 3H, isopropylidene), 1.43–
1.47 [m, 36H, 3 ꢃ (CH3)3COCON and (CH3)3CO], 1.64 (s, 3H, isopro-
pylidene), 3.293 and 3.317 and 3.349 (3 ꢃ s, 3H, SCH3), 3.50–3.56
A suspension of the fully protected sulfoximine (18) (220 mg,
0.247 mmol) in TFA–H2O–CH2Cl2 (5:1:4, 10 mL) was stirred at
ambient temperature for 6 h. The resulting mixture was evapo-
rated to dryness. The residual syrup was purified by reversed
phase medium-pressure column chromatography on Diaion
HP20SS eluting with a linear gradient of THF in H2O (0–99%).
The eluant was monitored with UV (254 nm). The final com-
pound was eluted at H2O/THF, 79:21. The UV positive-fractions
were combined and lyophilized to afford the final product (4)
as a mixture of diastereomers (394 mg, 71%): IR (KBr) mmax
3465–3349 (br), 2929, 1698, 1644, 1606, 1506, 1481, 1417,
1384, 1338, 1220 (O@S@N), 1164, 1095 (S@O), 1024, 978, 948
(m, 2H, 50-CH2), 3.80–4.30 (m, 2H CH2S), 4.38–4.53 (m, 1H,
a-pro-
ton), 4.60 (m, 1H, 40-H), 5.18–5.20 (m, 1H, 30-H), 5.29–5.40 (m, 1H,
20-H), 5.60 (m, 1H, CONH), 5.89 (2 ꢃ d, J = 4.2 and 4.1 Hz, 1H, 10-H),
7.60–7.82 (m, 1H, SO2NHCH2), 8.08 (s, 1H, adenyl 2-H), 8.980 and
8.988 (2 ꢃ s, 1H, adenyl 8-H); Anal. Calcd for C36H58N8O14S2: C,
48.53; H, 6.56; N, 12.58. Found: C, 48.64; H, 6.51; N, 12.51. HRMS
(FAB, p-nitrobenzyl alcohol) calcd for C36H59N8O14S2 (MH+)
891.3596, found 891.3597.
5.1.14. tert-Butyl (R,S)-2-(N-tert-butoxycarbonyl)amino-3-{N-
[(N6,N6-bis(tert-butoxycarbonyl)-20,30-O-isopropylideneadenosin-
50-yl)-50-O-sulfonyl]-S-methyl-(R,S)-sulfonimidoyl}propanoate
(18)
(S@N), 889, 823, 723, 676, 647, 592, 538, 472, 410 cmꢀ1
;
1H
NMR (300 MHz, D2O) dH 3.441 and 3.470 (2 ꢃ s, 3H, SCH3),
3.80–4.40 (m, 8H, 50-CH2, CH2S,
a
-proton, 40-H, 30-H, 20-H), 5.97
(d, J = 3.9 Hz, 1H, 10-H), 8.10 (s, 1H, adenyl 2-H), 8.18 (s, 1H, ade-
nyl 8-H); HRMS (FAB, glycerol) calcd for C14H22N7O9S2 (MH+)
496.0923, found 496.0912.
The functionalized sulfamate (16) (1.23 g, 2.10 mmol) was
dissolved in dry CH3CN (30 mL) together with the diastereomeric
mixture of sulfoxide (7) (2.00 g, 6.50 mmol). 4 Å Molecular sieves
(1 g), Rh2(esp)2 (319 mg, 0.42 mmol) and iodosylbenzene
(693 mg, 3.2 mmol) were then added, and the resulting mixture
stirred overnight at 36 °C under an Ar atmosphere. Filtration
and evaporation of the solvent under reduced pressure gave a
brown oil, which was purified by flash column chromatography
on silica gel (CH3CN/toluene, 1:3) to give the sulfoximine (18)
as a mixture of diastereomers (1.59 g, 85%, amorphous solid): IR
(KBr) mmax 3415–3384 (br), 2981, 2937, 1789, 1724, 1600, 1575,
1500, 1455, 1394, 1371, 1338, 1213 (O@S@N), 1164, 1106, 989,
5.1.17. Sulfamoyl chloride (20)56
Chlorosulfonyl isocyanate (14.7 g, 104 mmol) was placed in a
dry three-necked flask equipped with a CaCl2 drying tube, and for-
mic acid (3.96 mL, 104 mmol) added dropwise via syringe with
cooling in an ice bath (CAUTION: This reaction is vigorous and
effervescence is observed). After the addition was complete, the
mixture was stirred at room temperature until no more gas was
evolved. After 1 h, colorless crystals accumulated in the flask, and
dry benzene (30 mL) was added before the removal of insoluble
material by filtration under an Ar atmosphere. Evaporation of the
solution under reduced pressure gave 20 as colorless crystals
(11.2 g, 93%): mp 36.2–37.1 °C (corrected), lit.56 mp 40 °C.
948 (S@N), 850, 825, 775, 680, 646, 592, 512, 468 cmꢀ1 1H
;
NMR (300 MHz, CDCl3) dH 1.40–1.50 [m, 39H, isopropylidene,
3 ꢃ (CH3)3COCON and (CH3)3CO], 1.65 (s, 3H, isopropylidene),

H. Ikeuchi et al. / Bioorg. Med. Chem. 17 (2009) 6641–6650
6649
Kik6
k5 þ k6
5.2. Steady-state kinetic assays
Kiꢁ
¼
ð4Þ
Unless otherwise stated, all chemicals and reagents were pur-
chased from Sigma Aldrich (St. Louis, MO) and were of the highest
purity available. Protein concentrations were determined using the
Bradford Assay1 (Pierce, Rockford, IL),57 and are corrected as previ-
Experiments employing an HPLC-based assay31 were performed to
measure the amount of -asparagine produced under the conditions
L
of the inhibitor assay, which confirmed that neither 3 nor 4 was
affecting the 1:1 stoichiometry of human ASNS-catalyzed Asn:PPi
production. Briefly, recombinant human ASNS (2 lg) was incubated
at 37 °C for 20 min in 100 mM EPPS, pH 8.0, buffer containing
0.5 mM ATP, 100 mM NH4Cl, 10 mM MgCl2, 10 mM aspartic acid,
and 1 mM of either sulfoximine 3 or 4 (1 mL total volume). The
reaction was then quenched with TCA to a final concentration of
4%. Following enzyme precipitation, the solution was neutralized
ously described.31
L-Glutamine was recrystallized prior to use in all
kinetic assays.31 The ability of 3 and 4 to inhibit human ASNS was
measured by determining their effect on PPi production, under
steady-state conditions, using a continuous assay in which product
formation is coupled to NADH consumption (340 nm) (Sigma tech-
nical bulletin BI-100). In experiments employing the sulfamide
derivative 3, the compound at a number of different concentrations
using 10 M NaOH, and a portion of the reaction mixture (40
was taken for derivatization in a mixture of 800 M Na2CO3 buffer,
pH 9 (80 L), DMSO (20 L), and a solution of DNFB dissolved in
absolute EtOH (60 L). This reaction was incubated at 50 °C for
lL)
(0, 10, 50, 100, 500, and 1000
25 mM -Gln and the pyrophosphate reagent (350
EPPS, pH 8, containing 10 mM MgCl2 (1 mL final volume). The reac-
tion was initiated by the addition of ASNS (2 g), and the produc-
lM) was incubated with 0.5 mM ATP,
l
L
l
L) in 100 mM
l
l
l
l
50 min before the addition of glacial AcOH and separation by re-
tion of pyrophosphate monitored spectrophotometrically at 37 °C
for 20 min. Identical conditions were employed for the sulfamate
derivative 4 except that progress curves were generated at 25 °C
because this compound underwent cyclization to form the cyclo-
adenosine at higher temperatures. Control experiments using
known amounts of PPi demonstrated that the assay reagent was
not affected by the presence of either functionalized sulfoximine.
The sets of progress curves were analyzed by fitting the data,
using the Kaleidagraph v3.5 software package (Synergy, Reading,
PA), to Eq. 1:34
verse-phase HPLC on a Varian C18 Microscorb column using a step
gradient of 40 mM formic acid, pH 3.6, and CH3CN. DNP-L-aspara-
gine was detected at 365 nm and quantified by comparison to a
commercially available standard at known concentration.
5.3. Molecular modeling
Full details of the model for ASNS complexed to various reaction
intermediate and inhibitors will be published elsewhere, although
coordinates for the AS-B/glutamine/acyl-adenylate complex are
available from the authors on request. Briefly, homology modeling
tools in SYBYL 6.2 (Tripos) were used to add two missing loop seg-
ments (Ala-251 to Gln-266, and Gly-423 to Gly-425) to one of
the monomers in the high-resolution crystal structure of E. coli
AS-B (1CT9).45 This gave an initial model of the bacterial enzyme
(residues 1–516) bound to glutamine and AMP. Of the three uranyl
ions in the original crystal structure, the one coordinated with the
side chains of Asp-238 and Asp-351 was modified to Mg2+ because
site-directed mutagenesis studies had shown that Asp-238 medi-
ates metal binding (Boehlein, unpublished results). The remaining
uranyl ions were deleted before the addition of hydrogen atoms
and energy minimization using the CHARM software package58
(version 22 parameter set).59 All calculations employed a General-
ized-Born continuum solvation potential.60
ð
vo
ꢀ
vss
Þ
½PPiꢂ ¼
v
sst þ
ð1 ꢀ eꢀkt
Þ
ð1Þ
k
where [PPi] is the concentration of inorganic pyrophosphate formed
at time t, vo and vss are the initial and steady-state rates, respec-
tively, and k is the apparent first-order rate constant for isomeriza-
tion of EI to EI*. By analogy to the known behavior of the adenylated
sulfoximine 1, we assumed that the compounds bound to free en-
zyme and were competitive with respect to ATP, as shown in the
following kinetic model:
k1 [ATP]
k7
E
E.ATP
E
k2
The bound AMP was graphically modified to ATP in a conforma-
tion that mimicked that observed in the active site of Streptomyces
clavuligerus b-lactam synthetase (BLS),61 an enzyme that is clearly
k4
k3 [I]
evolutionarily related to ASNS.62 In this model, the b- and
c-phos-
k5
k6
phate groups of ATP interacted with the ‘pyrophosphate loop’ mo-
tif,63 as seen in structures of both BLS54 and GMP synthetase.64
After energy refinement of this AS-B/Gln/MgATP complex using
CHARMM, aspartate was built into the structure at the cognate po-
sition to that at which substrate binds in BLS,61 in the conformation
E.I
EI*
delineated in previous studies employing constrained analogs of
aspartic acid.46 In this structure, the side chain carboxylate was
also positioned for in-line attack on the -phosphate of ATP. Subse-
quent energy minimization then yielded a model for the AS-B/Gln/
Asp/MgATP complex, and graphical manipulation then yielded a
model of the acyl-adenylate bound in the synthetase active site to-
gether with MgPPi.
L-
Having obtained values for k, vo and vss at each concentration of
the functionalized sulfoximine 3, this information was then used to
compute an estimate of k6, using Eq. 2:
c
vss
K6
ð2Þ
¼ k v0
With an estimate of k6, values of Ki and k5 could be determined by
fitting the variation of k with the concentration of 3 using Eq. 3:
ꢀ
ꢁ
Acknowledgements
I=Ki
k ¼ k6 þ k5
ð3Þ
ð1 þ ½ATPꢂ=Ka þ I=KiÞ
This work was supported, in part, by a grant from the Chiles
Endowment Biomedical Research Program of the Florida Depart-
ment of Health, and a Grant-in-Aid for Scientific Research from Ja-
pan for the Promotion of Science. Funding for a pre-doctoral
where I is the concentration of the inhibitor, Ka is taken to be
0.1 mM,30 and [ATP] = 0.5 mM. The overall inhibition constant, KIꢁ
was then computed using Eq. 4:

6650
H. Ikeuchi et al. / Bioorg. Med. Chem. 17 (2009) 6641–6650
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