A sulfoximine-based inhibitor of human asparagine synthetase kills l-asparaginase-resistant leukemia cells

Article abstract of DOI:10.1016/j.bmc.2012.07.047

An adenylated sulfoximine transition-state analogue 1, which inhibits human asparagine synthetase (hASNS) with nanomolar potency, has been reported to suppress the proliferation of an l-asparagine amidohydrolase (ASNase)-resistant MOLT-4 leukemia cell line (MOLT-4R) when l-asparagine is depleted in the medium. We now report the synthesis and biological activity of two new sulfoximine analogues of 1 that have been studied as part of systematic efforts to identify compounds with improved cell permeability and/or metabolic stability. One of these new analogues, an amino sulfoximine 5 having no net charge at cellular pH, is a better hASNS inhibitor (K_I= 8 nM) than 1 and suppresses proliferation of MOLT-4R cells at 10-fold lower concentration (IC₅₀ = 0.1 mM). More importantly, and in contrast to the lead compound 1, the presence of sulfoximine 5 at concentrations above 0.25 mM causes the death of MOLT-4R cells even when ASNase is absent in the culture medium. The amino sulfoximine 5 exhibits different dose-response behavior when incubated with an ASNase-sensitive MOLT-4 cell line (MOLT-4S), supporting the hypothesis that sulfoximine 5 exerts its effect by inhibiting hASNS in the cell. Our work provides further evidence for the idea that hASNS represents a chemotherapeutic target for the treatment of leukemia, and perhaps other cancers, including those of the prostate.

Full text of DOI:10.1016/j.bmc.2012.07.047

Bioorganic & Medicinal Chemistry 20 (2012) 5915–5927
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A sulfoximine-based inhibitor of human asparagine synthetase kills
L-asparaginase-resistant leukemia cells
a
b
a
a
b
Hideyuki Ikeuchi , Yong-Mo Ahn , Takuya Otokawa , Bunta Watanabe , Lamees Hegazy ,
a,
⇑
b,
⇑
Jun Hiratake , Nigel G. J. Richards
Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan
a
b
Department of Chemistry & Chemical Biology, IUPUI, 402 N. Blackford St. Indianapolis, IN 46202, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
An adenylated sulfoximine transition-state analogue 1, which inhibits human asparagine synthetase
Received 23 May 2012
Revised 19 July 2012
Accepted 24 July 2012
Available online 7 August 2012
(
hASNS) with nanomolar potency, has been reported to suppress the proliferation of an
dohydrolase (ASNase)-resistant MOLT-4 leukemia cell line (MOLT-4R) when -asparagine is depleted in
the medium. We now report the synthesis and biological activity of two new sulfoximine analogues of
that have been studied as part of systematic efforts to identify compounds with improved cell perme-
L-asparagine ami-
L
1
ability and/or metabolic stability. One of these new analogues, an amino sulfoximine 5 having no net
Keywords:
⁄
charge at cellular pH, is a better hASNS inhibitor (K
I
= 8 nM) than 1 and suppresses proliferation of
Acute lymphoblastic leukemia
Asparagine synthetase
Transition-state analogue inhibitor
Sulfoximine
Structure–activity relationship
MOLT-4 leukemia cell line
MOLT-4R cells at 10-fold lower concentration (IC50 = 0.1 mM). More importantly, and in contrast to the
lead compound 1, the presence of sulfoximine 5 at concentrations above 0.25 mM causes the death of
MOLT-4R cells even when ASNase is absent in the culture medium. The amino sulfoximine 5 exhibits differ-
ent dose-response behavior when incubated with an ASNase-sensitive MOLT-4 cell line (MOLT-4S), sup-
porting the hypothesis that sulfoximine 5 exerts its effect by inhibiting hASNS in the cell. Our work
provides further evidence for the idea that hASNS represents a chemotherapeutic target for the treatment
of leukemia, and perhaps other cancers, including those of the prostate.
L
-Asparagine amidohydrolase
Cell death
Ó 2012 Elsevier Ltd. All rights reserved.
1
. Introduction
sensitivity is associated with a requirement for malignant lympho-
cytes to import L
-asparagine from their surroundings.9 Although
,10
Substantial success has been achieved in the clinical treatment
this idea has generated some controversy, there have been reports
of a correlation between the appearance of ASNase resistance and
of acute lymphoblastic leukemia (ALL), which is the most common
1
form of childhood cancer. A majority of current protocols include
the up-regulation of glutamine-dependent asparagine synthetase
-asparagine amidohydrolase (ASNase),2 which cata-
,3
(ASNS),
11–13
which mediates de novo asparagine biosynthesis
In vitro adaptive studies employing U937 cells in
the enzyme
L
9
,14
lyzes the hydrolysis of
nia.
L
-asparagine to yield
On the other hand, ASNase has
therapeutic index with up to two thirds of patients exhibiting a
L
-aspartate and ammo-
(Scheme 1).
4
,5
a
relatively narrow
which ASNase-sensitive cells became resistant by persistent expo-
sure to sub-lethal doses of ASNase have also been reported.15 Per-
haps the strongest evidence for the importance of ASNS expression
has been obtained, however, from experiments in which an ASN-
ase-sensitive MOLT-4 (MOLT-4S) leukemia cell line was rendered
resistant to the presence of ASNase in the growth medium merely
by transformation with a plasmid from which the hASNS gene was
6,7
hypersensitive immune response. Another significant problem
with the use of ASNase in chemotherapy is that 10–12% of patients
who initially go into remission undergo subsequent relapse due to
the emergence of ASNase-resistant leukemic blasts.8
The molecular basis for the sensitivity of leukemic blasts to
ASNase remains undetermined, in part because of the absence of
small molecules that can be used to probe the cellular mechanisms
that give rise to ASNase sensitivity and the suppression of cell pro-
liferation. One widely-accepted hypothesis postulates that ASNase
1
5
constitutively expressed. The likely importance of human ASNS
(hASNS) in other forms of cancer is suggested by recent work
showing that this enzyme is a biomarker for ovarian cancer.1
More direct methods for examining the role of ASNS expression
in ASNase-resistance, such as siRNA knockdown experiments on
ASNase-resistant MOLT-4 (MOLT-4R) leukemia cell lines have, to
the best of our knowledge, not yet been reported. However, very
recent work has shown that siRNA knockdown of hASNS expres-
sion inhibited the growth of both androgen-responsive lymph
6,17
⇑
Corresponding authors. Tel.: +81 774 38 3231; fax: +81 774 38 3229 (J.H.); tel.:
1 317 274 6624; fax: +1 317 274 4701 (N.G.J.R.).
E-mail addresses: hiratake@scl.kyoto-u.ac.jp (J. Hiratake), richards@chem.ufl.e-
du, richards@qtp.ufl.edu, ngrichar@iupui.edu (N.G.J. Richards).
+
0
968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.07.047

5
916
H. Ikeuchi et al. / Bioorg. Med. Chem. 20 (2012) 5915–5927
Scheme 1. Overview of the reaction catalyzed by human glutamine-dependent asparagine synthetase, showing the structure of the b-aspartyl-AMP intermediate and the
transition state for asparagine formation.
node and prostate cancer cell lines, and cells isolated from a castra-
tion-resistant (MDA PCa 180-30) prostate cancer xenograft when
We now report syntheses of two new adenylated sulfoximines 5
and 6 (Fig 1), in which the -carboxy and -amino groups are re-
a
a
grown in media depleted of
L
-asparagine.18
moved from the hASNS inhibitor 1, respectively. These changes
have allowed us to not only elucidate the relative contributions
of these ionizable groups to the affinity of the parent sulfoximine
1 for the hASNS synthetase active site but also examine how over-
all molecular charge might be correlated with sulfoximine bio-
availability and cell-based activity. The amino sulfoximine 5,
In an important step towards understanding whether ASNase-
resistance arises from an increased ability of leukemic blasts to
up-regulate L-asparagine biosynthesis, we recently showed that
the adenylated sulfoximine 1 (Fig. 1), which is a stable analogue
of the transition-state for ammonia addition to b-aspartyl-AMP
19
intermediate (Scheme 1), inhibited hASNS with nanomolar po-
which has no net charge at cellular pH, is a better hASNS inhibitor
2
0
⁄
tency. More importantly, sulfoximine 1 suppresses the prolifera-
(K
I
= 8 nM) and suppresses proliferation of MOLT-4R cells at 10-
tion of ASNase-resistant MOLT-4 (MOLT-4R) cells in a dose-
fold lower concentration (IC50 = 0.1 mM) than sulfoximine 1. More-
over, in an exciting observation for the investigation of molecular
mechanisms underlying the sensitivity of leukemic blasts to ASN-
ase, sulfoximine 5 induces cell death in MOLT-4R cells at sub-milli-
molar concentrations irrespective of the presence of ASNase in the
external medium. Although the exact mechanism by which sulfox-
imine 5 induces cell death remains to be established, MOLT-4R and
MOLT-4S cells exhibit different dose–response behaviors when
incubated with sulfoximine 5, supporting the hypothesis that sul-
foximine 5 exerts its effect by inhibiting hASNS in the cell.
dependent manner, but only at 100–1000
lM concentrations and
when -asparagine is depleted in the external medium by ASN-
L
2
0
ase. The need to use high concentrations of sulfoximine 1 to sup-
press cell proliferation is likely due to ionizable groups on the
molecule that result in an overall negative charge at cellular pH
and limit cell permeability (Fig. 1). Experiments using compounds
2
and 3 (Fig. 1), in which the phosphate moiety is replaced by a
sulfamide or sulfamate functional group, however, revealed that
the negatively charged phosphate is essential for hASNS inhibi-
2
1
tion. Examination of the crystal structure of Escherichia coli
22
asparagine synthetase B (AS-B) reveals a critical electrostatic
interaction between the phosphate moiety of inhibitor 1 and a con-
served, catalytically important lysine (Lys-449 and Lys-466 for AS-
B and hASNS, respectively).21 In support of this hypothesis, the N-
acylsulfamide 4 (Fig. 1), in which dissociation of an acidic NH pro-
ton produces a resonance-stabilized negative charge on the sulfa-
mate oxygen, inhibits hASNS with micromolar potency.23
2
. Chemistry
Synthetic routes to adenylated sulfoximines 5 and 6 were based
1
9
on that used to prepare the parent compound 1. Given that the
0
negatively charged 5 -phosphate of adenylic acid is essential for
tight binding to hASNS, this moiety was attached by N-phosphor-
ylation of pre-formed sulfoximines 10 and 16, themselves pre-
Figure 1. Chemical structures of adenylated sulfoximine 1, sulfamide analogue 2, sulfamate analogue 3, acylsulfamate 4, and the target set of sulfoximine derivatives 5 and 6.

H. Ikeuchi et al. / Bioorg. Med. Chem. 20 (2012) 5915–5927
5917
pared from the corresponding sulfoxides 9 (Scheme 2) and 15
Scheme 3), respectively. Despite attempts to perform the efficient
amidate intermediate under these conditions requires the
2
8
(
exclusion of moisture, and this was accomplished by running
the coupling reaction in anhydrous CH CN over 4 Å molecular
imination of these sulfoxides in a safe manner, for example by
using a rhodium-catalyzed reaction with trifluoroacetamide and
3
sieves. The adenylated sulfoximines 12 and 17 could then be ob-
tained, in good yields, albeit as a 1:1 mixture of diastereoisomers
at the sulfur stereogenic center, after in situ oxidation of the
P(III) intermediates formed in the initial coupling reaction. Inter-
estingly, the P-N bond, once formed with sulfoximine nitrogen
(S@N), was hydrolytically stable even under highly acidic condi-
tions (Schemes 2 and 3), probably because of the weak basicity
of the sulfoximine nitrogen. Chromatographic conditions to sepa-
rate the diastereoisomers of sulfoximines 5 and 6 (or their precur-
sors 13 and 18) could not be identified.
2
4
PhI(OAc)
2
,
the best reagent for preparing the unsubstituted sul-
foximines 10 and 16 proved to be O-mesitylsulfonylhydroxylamine
2
5,26
(
MSH).
[Caution: MSH is potentially explosive and extreme
care must be taken in handling and storing this hazardous reagent
particularly in large amounts (>ca. 1 g)]. The need to perform the
imination using MSH constrained the choice of protecting groups
for sulfoxides 9 and 15 because strongly acidic mesitylsulfonic acid
is formed as a by-product of the reaction. We therefore selected the
2
,2,2-trichloroethyl carbamate group (Troc) for masking the a-
amino group, and allyl esters to protect the
a-carboxy and the
phosphate groups.
3
. Enzyme assays
N-adenylation of sulfoximines 10 and 16 was carried out by
reaction with protected adenosyl phosphite 11 under standard
phosphoramidite coupling conditions using tetrazole as a cata-
With sulfoximines 5 and 6 in hand, we investigated their ability
to inhibit hASNS following well-established steady-state kinetic
2
7
2
0,29
lyst. Formation of the acid sensitive P–N bond in the phosphor-
procedures.
Hence, the effect of sulfoximines 5 and 6 (each
Scheme 2. Synthetic route to amino sulfoximine 5.

5
918
H. Ikeuchi et al. / Bioorg. Med. Chem. 20 (2012) 5915–5927
Scheme 3. Synthetic route to carboxy sulfoximine 6.
as a mixture of two diastereoisomers) on hASNS-catalyzed aspara-
gine synthesis was assayed by measuring the rate of inorganic
suggesting that inhibitor 5 binds strongly to an enzyme conforma-
tion in which the N-terminal glutaminase site is occupied by either
pyrophosphate (PP
i
) production.30 The recombinant human en-
substrate glutamine or a thioester intermediate.
11,37,38
Experi-
zyme was obtained by expression in Sf9 insect cells, and purified
by metal-affinity chromatography, as reported previously.3 All ki-
netic assays employed diastereoisomeric mixtures of 5 and 6 in
which each of the sulfoximines was a 1:1 mixture of epimers at
the sulfur stereogenic center. As discussed in detail else-
ments examining the effect of various nucleotides on the glutamin-
ase activity of Escherichia coli asparagine synthetase B (AS-B) are
consistent with conformational cross-talk between the glutamin-
1
3
9
ase and synthetase active sites of the enzyme. Future efforts to
establish which diastereoisomer of 5 binds to the hASNS synthe-
tase site with highest affinity will require access to the intermedi-
ate sulfoxide 9 in optically pure form, perhaps by the asymmetric
2
0,32,33
where,
considerable evidence exists for a strong stereochem-
ical dependence of any tight-binding inhibition that is observed for
functionalized sulfoximines.3
4,35
oxidation of sulfide 8. In contrast to the improved activity of ami-
40
When recombinant hASNS was incubated with ATP,
MgCl and either glutamine (Fig. 2A) or ammonia (Fig. 2B) as a
nitrogen source, the amino sulfoximine 5 acted as a slow-onset,
L
-aspartate,
no sulfoximine 5, the carboxy sulfoximine 6 was a relatively poor
⁄
2
hASNS inhibitor (Fig. 3), exhibiting ‘uncorrected’ K
I
values of only
800 and 300 nM when the nitrogen source was glutamine or
ammonia, respectively. Control experiments established that nei-
ther compound inhibited the coupling enzymes in the pyrophos-
phate reagent used for the continuous assay (data not shown).
Given the close resemblance of all three sulfoximine derivatives
1, 5, and 6, and their conformational flexibility, we anticipated that
the inhibitory diastereoisomers of all three compounds would bind
in a similar fashion within the synthetase active site of hASNS. This
idea was qualitatively investigated by positioning the diastereoiso-
3
6
20,21
tight-binding inhibitor. As in previous studies,
the kinetic
data were analyzed using a simple model in which the functional-
ized sulfoximine competes with ATP for free hASNS and showed
that 5 was a more potent hASNS inhibitor than 1, with an overall
⁄
inhibition constant (K
I
) of 8 nM when
L
-glutamine was used as
⁄
the nitrogen source (Table 1). The K
uncorrected on the basis of an assumed concentration of the ac-
tive isomer) was increased four-fold under the same conditions
when ammonia was employed in place of -glutamine (Table 1),
I
value for sulfoximine 5
(
2
1
L
mers of 5 and 6 within a model of Escherichia coli AS-B, which we

H. Ikeuchi et al. / Bioorg. Med. Chem. 20 (2012) 5915–5927
5919
Figure 2. (A) Glutamine-dependent production of PP
inhibitor 5 (0–5 M): open squares, 0 M; open circles, 0.2
.5 M; open triangles, M; filled squares, M; filled circles,
Ammonia-dependent production of PPi in the presence of the inhibitor 5 (0–
M): open squares, 0 M; open circles, 0.4 M; open diamonds, 0.7 M; open
triangles, 1 M; filled squares, 2.5 M; filled circles, 5 M; filled diamonds 10 M.
In both figures, the solid lines represent those of best fit to Eq. (1) (see Section 5).
i
in the presence of the
M; open diamonds,
M. (B)
Figure 3. (A) Glutamine-dependent production of PP
inhibitor 6 (0–80 M): open squares, 0
25 M; open triangles, 40 M; filled squares, 60
Ammonia-dependent production of PP in the presence of the inhibitor 6 (0–
20 M): open squares, 0 M; open circles, 2 M; open diamonds, 5 M; open
triangles, 8 M; filled squares, 15 M; filled circles, 20 M. In both figures, the solid
i
in the presence of the
l
l
l
l
l
M; open circles, 5
lM; open diamonds,
0
l
1
l
2
l
5
l
l
l
l
M; filled circles, 80 lM. (B)
i
1
0
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
lines represent those of best fit to Eqn. (1) (see Section 5).
2
1
solvated system using the CHARMM27 force field,42 augmented
by parameters for the sulfoximine moiety obtained using the
CGenFF software package. In these models Mg(II) was bound at
its usual site, i.e. coordinated to the side chain of Asp-238 (AS-B
numbering), and we assumed that neither 5 nor 6 would be bound
have developed from X-ray structural information (Fig. 4A). Mod-
els (AS-B/glutamine/inhibitor/Mg2 ) were constructed for each of
the hASNS inhibitors (two diastereoisomers of each sulfoximine 5
and 6), and placed within an octahedral box of TIP3 water mole-
+
4
3
4
1
cules. Subsequent energy minimization was performed on the
Table 1
Steady-state kinetic parameters for the slow-onset inhibition of hASNS by the adenylated sulfoximine derivatives 1, 5 and 6.a
(nM)b
k
5
(s
ꢀ1
)
k
6
(s
ꢀ1
)
⁄
(nM)b
K
I
Sulfoximine
Nitrogen source
-Gln
K
I
1c
1c
1000 ± 200
2.8 ꢁ 10
3.0 ꢁ 10
ꢀ3
2.6 ꢁ 10
ꢀ5
24 ± 3
L
ꢀ3
ꢀ5
ꢀ5
NH
4
Cl
280 ± 40
490 ± 25
7.1 ꢁ 10
2.5 ± 0.3
8 ± 2
1.7 ꢁ 10ꢀ
3
2.6 ꢁ 10
5
L-Gln
ꢀ
3
ꢀ4
ꢀ4
5
6
4
NH Cl
450 ± 30
4500 ± 300
7.3 ꢁ 10
5.7 ꢁ 10
8.9 ꢁ 10
4.5 ꢁ 10
33 ± 5
840 ± 140
2.0 ꢁ 10ꢀ
3
L-Gln
3.9 ꢁ 10ꢀ
3
ꢀ4
310 ± 70
6
4
NH Cl
3040 ± 80
a
Reaction mixtures consisted of human ASNS (4
l
g), 0.5 mM ATP, 10 mM
L
-aspartate, either 25 mM
L
-glutamine or 100 mM NH
4
Cl as nitrogen source, and the pyro-
⁄
phosphate reagent (350 L) dissolved in 100 mM EPPS buffer, pH 8.0, containing 10 mM MgCl
l
2
(1 mL total volume). K
I
and K
I
are the initial and overall inhibition constant,
36
respectively.
b
All inhibition constants reported in this table are not corrected on the basis of an assumed concentration for the active diastereoisomer.
Values for the adenylated sulfoximine 1, determined at 37 °C, have been published previously, and are included here for ease of comparison. All inhibition constants for
c
20
functionalized sulfoximines 5 and 6 were determined at 25 °C.

5
920
H. Ikeuchi et al. / Bioorg. Med. Chem. 20 (2012) 5915–5927
at the same time as inorganic pyrophosphate (PP
i
). This computa-
enzyme.22,45 It is believed that the methyl group attached to the
sulfur atom mimics the ammonia molecule as it undergoes reac-
tional strategy provided a qualitative understanding of differences
in the interaction of conserved residues in the synthetase active
site with functional groups on the diastereoisomers of sulfoximine
2
0
tion with b-aspartyl-AMP (Scheme 1). In contrast, the absence
of the -amino group in sulfoximine 1 (giving the carboxy sulfox-
a
5
(Fig. 5A and B) and 6 (Fig. 5C and D). For example, the amino
imine 6) results in a reorientation of the synthetase site side chains
so that the carboxylate of the diastereoisomer of 6 that is likely to
represent the true inhibitor, based on the orientation of the methyl
group connected to the stereogenic (R)-sulfur center, interacts with
a conserved lysine residue (Lys-376 in AS-B) (Fig. 5C). The Lys-449/
phosphate interaction is also maintained in this model of the com-
plex and the side chain of Glu-348 re-orients to form a hydrogen
group in both diastereoisomers of 5 forms a complementary inter-
action with Glu-348 (a residue that has been shown to play a role
4
4
in mediating the formation of the b-aspartyl-AMP intermediate ),
and the critical electrostatic interaction between the phosphate
group and Lys-449 is maintained in both complexes.21 Altering
the stereochemistry at the stereogenic sulfur center does, however,
change the interaction of the amino group with other carboxylate
side chains (Fig. 5A and B). On this point, we note that the diaste-
reoisomer in which the sulfoximine has an (R)-configuration
0
bond to the 2 -hydroxy group on ribose. The hypothesis that the
other diastereoisomer of 6 is inactive is supported by (i) the possi-
ble interaction of Lys-449 with the carboxylate group rather than
0
(Fig. 5A) represents the structure in which the methyl substituent
the 5 -phosphate of sulfoximine 6, and (ii) a reorientation of Glu-
on sulfur points towards the ammonia tunnel present in the
348 to form a hydrogen bond with Ser-346 rather than the ribose
Figure 4. (A) Cartoon representation of the homology model of the AS-B/
glutaminase and the C-terminal synthetase domain are colored blue and yellow, respectively. Bonds in the
synthetase domain) ligands are indicated by lines. (B) The electrostatic potential in the synthetase active site of the AS-B model rendered on the van der Waals surface of
residues located within 0.4 nm of the b-aspartyl-AMP intermediate (Leu-232, Leu-233, Ser-234, Asp-238, Ser-239, Ser-270, Phe-271, Ala-272, Pro-279, Met-332, Ser-346, Gly-
L
-glutamine/b-aspartyl–AMP/MgPP
i
complex, rendered in Chimera.65 Residues in the N-terminal
-glutamine (glutaminase domain), b-aspartyl-AMP and MgPP
L
i
(
4
5
3
47, Glu-348, Asp-351, Glu-352, Tyr-357, Lys-376, Asp-384, Arg-387, Gly-456 and Ly-449). The potential was computed using the Poisson-Boltzmann equation using all
66
atoms in the uncomplexed protein, and is visualized using the VMD software package. Regions of positive and negative electrostatic potential are colored blue and red,
respectively. Quantitative values at 300 K can be computed by multiplying those shown in the legend by 0.026 J/mol/C.

H. Ikeuchi et al. / Bioorg. Med. Chem. 20 (2012) 5915–5927
5921
Figure 5. Putative interactions between conserved residues in the ASNS synthetase active site with the four diastereoisomers of sulfoximines 5 (A and B) and 6 (C and D).
Color scheme: C, grey; H, white; N, blue; O, red; P, orange; S, yellow. Image was rendered using Chimera.65
ring, presumably due to electrostatic repulsion with the carboxyl-
ate of sulfoximine 6 (Fig. 5D).
source, we investigated whether this compound could also stop
5
1–53
the growth of the MOLT-4R leukemia cell line.
We note that
These qualitative changes do not seem to provide an adequate
explanation as to why the putative active diastereoisomers of 5
and 6 (Fig. 5A and C) should exhibit such large differences in affin-
ity (Table 1). We therefore examined alternate hypotheses given
that sulfoximine 5 has no net charge while 6 is present as a dianion
under the conditions of our assay (pH 8). First, we calculated the
electrostatic potential of the free enzyme in aqueous solution by
this line was obtained by growing MOLT-4S cells in media contain-
ing increasing amounts of the enzyme ASNase, and not by plasmid
transformation and constitutive expression of the ASNS gene. As in
our experiments on the biological activity of sulfoximine 1,20 these
MOLT-4R cells were transferred to growth medium containing
10–1000 lM concentrations of the sulfoximine 5 and their survival
after 48 h was assayed using a dye-based method to estimate
46
54
solving the Poisson–Boltzmann equation, using the algorithms
changes in the number of viable cells. Once again, micro- to
implemented in the APBS software package.4
7,48
The protein struc-
millimolar concentrations of the hASNS inhibitor were used
because of concerns about the cell permeability of compound. As
ture used in these calculations was that obtained by removing b-
2
0
aspartyl-AMP and inorganic pyrophosphate from our original mod-
expected from our previous work, sulfoximine 5 showed a weak
2
1
el of the AS-B/
L
-glutamine/b-aspartyl-AMP/MgPP
i
complex.
dose-dependent ability to suppress MOLT-4R proliferation at con-
These calculations show that a large portion of the synthetase ac-
tive site surface carries a negative electrostatic potential with only
two specific regions of positive potential to stabilize the carboxyl-
ate and phosphate moieties of the b-aspartyl-AMP intermediate
centrations up to 130 lM in the absence of ASNase (Fig. 6A). This
represents an approximate 7-fold gain in suppression activity
relative to sulfoximine 1 in the presence of ASNase,21 suggesting
an increased bioavailability of sulfoximine 5 and an intracellular
interaction with hASNS. In an important contrast to our findings
for sulfoximine 1, however, the number of viable MOLT-4R cells
(Fig. 4B). Thus the differential affinities of 5 and 6 might be associ-
ated with their overall charge. In addition, differences in the initial
dissociation constants (K
I
) for 5 and 6 (Table 1) could also arise
was markedly decreased when 5 was present at 250
tration, and this compound caused the death of almost all the
MOLT-4R cells at higher concentrations (>500 M). These biologi-
cal effects could be amplified to a small extent by depleting
asparagine in the culture medium by the addition of 1 U/mg ASN-
ase; cell proliferation was suppressed when 5 was added at
lM concen-
from desolvation effects,4
9,50
that is, if the dianion of sulfoximine
6
is better solvated in water than 5 then its relative affinity for
l
the synthetase site will be decreased. Definitive insights await
the characterization of additional sulfoximine derivatives using
both experimental and quantitative computational methods.
L-
1
30
l
M concentration (Fig. 6B). Furthermore the ability of 5 to
M of compound 5
4
. Cell based assays
cause cell death was also enhanced with 250 l
being sufficient to kill almost all the MOLT-4R cells (Fig. 6B). How-
ever, the magnitude of the shift in dose response for the activity of
5 with the MOLT-4R cell line in the presence and absence of
Given that the amino sulfoximine 5 inhibited hASNS with low
nanomolar affinity in assays where L-glutamine was the nitrogen

5
922
H. Ikeuchi et al. / Bioorg. Med. Chem. 20 (2012) 5915–5927
Figure 6. The effect of incubating the amino sulfoximine 5 on the proliferation of MOLT-4 leukemia cells in the presence and absence of 1U
ASNase). (A) MOLT-4R cells without ASNase; (B) MOLT-4R cells with ASNase; (C) MOLT-4S cells without ASNase. Cell proliferation is defined as the number of viable cells
after 48 h incubation expressed as the ratio to the initial number of cells (t = 0). Error bars represent the standard deviation of triplicate experiments.
L-asparagine amidohydrolase
(
ASNase was much smaller than that observed for sulfoximine 1.20
It is possible that this observation is associated with the impact of
ASNase on the ability of the cells to proliferate. Thus, careful exam-
ination of the data suggests that compound 5 does have a slightly
greater ability to reduce proliferation when present at 0.13 mM but
this is masked by the significant reduction in cell growth caused by
deletion of asparagine in the medium. At concentrations of
(Fig. 1), this compound may possess better cell permeability be-
cause of its zero net charge at neutral pH. The improved activity
of 5 might also arise from its increased stability to metabolic trans-
formations within the cell, such as depurination or hydrolysis, but
the structural similarity of 1 and 5 seems to argue against this
hypothesis. Clearly, more work will be required to examine the
ability of these functionalized sulfoximines to pass through the cell
membrane, and the chemical pathways by which they might be
broken down within the cytosol. Compound 5 also causes cell
death in ASNase-resistant MOLT-4 cells at concentrations higher
0
.06 mM, and below, 5 seems to have essentially no effect whether
ASNase is present or not. While this observation might argue for
the hypothesis that 5 must be present at 0.1 mM before being able
to enter the cells, it could also reflect the fact that cells with a much
reduced rate of proliferation require much lower levels of endoge-
nous/exogenous asparagine to survive. In order to evaluate
whether these biological effects were correlated with the presence
of hASNS in the MOLT-4R cell lines, we incubated sulfoximine 5
with MOLT-4S cells derived from the same parent cell line as the
MOLT-4R cells. Prior work has established that MOLT-4S cells ex-
press hASNS in considerably lower amounts than those found in
than 250
exogenous
l
L
M, with this effect being augmented by depletion of
-asparagine. Although we cannot rule out the possibil-
ity that MOLT-4R cell death might arise from the interaction of 5
with other cellular targets (e.g. aminoacyl tRNA synthetases) when
present in the medium at millimolar concentrations, the results re-
ported in this paper provide new evidence to support the idea that
ASNS represents an intriguing, and druggable, target for the clinical
treatment of acute lymphoblastic leukemia. This conclusion would
be greatly strengthened by siRNA experiments to knockdown hAS-
NS concentrations, such as those reported for prostate cancer-de-
5
,55
MOLT-4R cells,
which means that higher concentrations of 5
should be required to suppress the proliferation of the MOLT-4S
cells. In fact, compound 5 appeared to have little effect on the
1
8
rived cell lines. It is also possible that hASNS inhibitors might
permit the use of lower levels of ASNase in current therapeutic
protocols with a concomitant reduction in the side-effects associ-
MOLT-4S cells at concentrations below 130
er activity in suppression of cell proliferation at concentrations of
50 M and 500 M (Fig. 6C) when compared with the results of
lM, and exhibited low-
3
,58
2
l
l
ated with ASNase administration.
Finally, recent findings show-
experiments using the MOLT-4R cells (Fig. 6A). These results pro-
vide a support for a possible link between hASNS activity and cell
death: the MOLT-4R cell line is highly dependent on its own hASNS
ing the importance of hASNS for the growth of both androgen-
responsive lymph node and prostate cancer cell lines and cells iso-
lated from a castration-resistant (MDA PCa 180–30) prostate can-
activity for access to
L-asparagine supply and is accordingly more
cer xenograft suggest that analogs of sulfoximine
5 might
susceptible to inhibition of this enzyme. However, millimolar con-
centrations of 5 once again caused significant levels of MOLT-4S
cell death, raising the possibility that this compound binds to other
cellular targets at high concentration. Given that the parent sulfox-
imine 1 can also inhibit the ammonia-dependent Escherichia coli
represent lead compounds for identification of novel therapies
against solid tumors, including those of the prostate.
1
8
6
6
. Experimental
.1. General
1
9
AS-A, which is evolutionarily related to aspartyl tRNA synthe-
5
6,57
tase,
it is possible that 5 might be capable of binding to, and
inhibiting, human aspartyl-tRNA synthetase. Further work, per-
haps using sulfoximines functionalized with adenosine mimics
that bind specifically to hASNS, will be required to rule out the lat-
ter possibility.
All chemicals were obtained commercially and used without
further purification unless otherwise stated. N,N-Dimethylform-
amide (DMF), 1,4-dioxane, acetonitrile (CH CN), CH Cl , tetrahy-
drofuran (THF) and triethylamine (Et N) were purchased from
Wako Pure Chemical Industries (Osaka, Japan). These solvents
were dried by distillation from CaH , and stored over 4 Å molecular
3
2
2
3
5
. Conclusions
2
sieves prior to use. Melting points (mp) were recorded using a Met-
1
We have identified the first functionalized sulfoximine (5) that
tler FP62 melting point apparatus and are corrected. H NMR and
3
1
inhibits the glutamine-dependent synthetase activity of human
ASNS with low nanomolar potency. As judged by its ability to sup-
press the proliferation of an ASNase-resistant MOLT-4 cell line at
reduced concentrations compared to the parental sulfoximine 1
P NMR spectra were recorded on a JEOL JNM-AL 300 (300 MHz
for H). C NMR spectra were recorded on a JEOL LAMBDA
1
13
1
3
1
(100 MHz for C) under a complete H decoupling condition. In
3 6 6 H C
CDCl , DMSO-d and acetone-d , ) and C (d ) chemical
1H (d
13

H. Ikeuchi et al. / Bioorg. Med. Chem. 20 (2012) 5915–5927
5923
shifts are reported downfield of tetramethylsilane as an internal
reference (d 0.0). For measurements in D O, sodium 3-(trimethyl-
silyl)propanesulfonate was used as an internal standard (d 0.0).
over Na
oil was purified by column chromatography on silica gel (CHCl
MeOH, 19:1 to 10:1) to afford sulfoximine 10 as a colorless oil
(1.97 g, 65%): IR (NaCl, neat) max 3400–3200 (br), 2979, 2933,
2815, 1716, 1523, 1455, 1392, 1367, 1278, 1251, 1155, 1049,
2 4
SO and filtration, the filtrate was evaporated. The residual
2
3
/
Splitting patterns are abbreviated as follows: s, singlet; d, doublet;
m
t, triplet, q; quartet; m, multiplet. 31P chemical shifts (d
) are re-
as an external standard (d 0.0).
P
ꢀ1
ported downfield of 85% H
3
PO
4
P
1024, 971, 943, 846, 813, 750, 686, 576, 565, 497, 458, 431 cm ;
1
Infrared spectra were measured using a Horiba FT-720 Fourier
Transform infrared spectrometer. Mass spectra were recorded on
a high resolution JEOL JMS 700 spectrometer. Elemental analyses
were performed using a Yanaco MT-5 apparatus. Reactions were
monitored by analytical thin layer chromatography (TLC) on silica
gel 60 F254 plates (Merck). Compounds were purified by column
H NMR (300 MHz, CDCl
2H, CH S), 3.79 (dt, 2H, J = 6.0 and 6.0 Hz, NHCH
2H, Cl CCH ), 6.15 (br t, 1H, carbamate NH), 8.5 (br s, 1H, S@NH);
3
) d
H
3.07 (s, 3H, SCH
3
), 3.29–3.41 (m,
2
2
CH S), 4.73 (s,
2
3
2
+
6 3 2 3
HRMS (FAB, p-nitrobenzyl alcohol) calcd for C H12Cl N O S (MH )
296.9634, found 296.9634.
chromatography on 40–50
l
m silica gel 60 N (Kanto Kagaku), or
6.1.4. (R,S)-Allyloxy(N,N-diisopropylamino)(2’,3’-O-isopropyl-
ideneadenosin-5’-yloxy) phosphine (11)
The phosphine 11 was synthesized in a yield of 63% after puri-
fication by silica gel column chromatography (acetone/hexane, 2:1
by reverse-phase, medium pressure, column chromatography (Ni-
hon Büchi) on either Diaion HP20SS resin (Mitsubishi Chemical
Corporation) or a COSMOSIL 5C18-PAQ column (Nacalai Tesque).
3
containing 5% Et N) according to the method as described previ-
1
9
6
.1.1. 2,2,2-Trichloroethyl N-2-(S-methylsulfanyl)ethylcarbamate
ously (Koizumi, M. et al. J. Am. Chem. Soc. 1999, 121, 5799–
5800; Supplementary data). The method for the preparation of
allyloxy[bis(diisopropylamino)]phosphine is modified as follows:
(
8)
A
solution of 2-sulfanylethylamine hydrochloride (5.21 g,
4
5.9 mmol) and NaOH (5.5 g, 138 mmol) in EtOH (100 mL) was
PCl
cooled to 0 °C before diisopropylamine (40.4 g, 400 mmol) in anhy-
drous CH CN (10 mL) was added over a 15 min period with vigor-
3 3
(7.87 g, 57.3 mmol) in anhydrous CH CN (250 mL) was
stirred for 10 min at 0 °C. Methyl iodide (6.84 g, 48.2 mmol) was
then added dropwise to the mixture over a period of 30 min at
3
0
°C. After stirring for 3 h at ambient temperature, 2,2,2-trichloroe-
ous stirring. The mixture was stirred at 0 °C for 30 min and then at
ambient temperature for 20 h. The resulting reaction mixture was
re-cooled to ꢀ20 °C, and allyl alcohol (3.33 g, 57.3 mmol) was
added dropwise over 1 hr with vigorous stirring. The mixture
was stirred at ambient temperature for 19 h, and the reaction mix-
ture was filtered. The filtrate was evaporated, and the residual oil
purified by distillation under reduced pressure (bp 90–95 °C at
thy chloroformate (11.4 g, 53.8 mmol) was added dropwise at 0 °C.
The mixture was stirred at ambient temperature overnight and fil-
tered. The solvent was evaporated under reduced pressure, and the
residual oil was purified by column chromatography on silica gel
(
EtOAc/hexane, 1:5 to 1:3) to afford the sulfide 8 as a colorless
oil (11.3 g, 93%): IR (NaCl, neat) max 3338, 2950, 2929, 1731,
720, 1525, 1434, 1403, 1361, 1322, 1247, 1197, 1143, 1083,
m
1
1
0.8 mmHg) to give allyloxy[bis(diisopropylamino)]phosphine as a
ꢀ1
1
1
049, 1033, 958, 902, 815, 767, 721, 651, 568, 476, 449 cm
) d 2.13 (s, 3H, SCH ), 2.68 (t, 2H,
S), 3.46 (dt, 2H J = 6.3 and 6.3 Hz, NHCH CH S),
CCH ), 5.35 (br s, 1H, NH); HRMS (FAB, p-nitrobenzyl
alcohol) calcd for C
;
H
colorless oil (10.8 g, 65%): H NMR (300 MHz, CDCl
3
) d
ꢁ 2}, 3.54 {double septet,
ꢁ 2}, 4.08–4.13 (m, 2H,
), 5.07–5.33 (m, 2H, CH@CH ), 5.89–6.03 (m, 1H,
122.4.
H
1.17 {dd,
NMR (300 MHz, CDCl
J = 6.5 Hz, CH
.74 (s, 2H, Cl
3
H
3
J = 6.6 and 4.5 Hz, 24H, N[CH(CH
J = 10.8 and 6.6 Hz, 4H, N[CH(CH
OCH CH@CH
CH@CH
3 2 2
) ]
) ]
3 2 2
2
2
2
4
3
2
2
2
2
+
31
6
H11Cl
3
NO
2
S (MH ) 265.9576, found 265.9576.
2
). P NMR (121 MHz, CDCl
) d
3 P
6
.1.2. 2,2,2-Trichloroethyl N-2-[S-methyl-(R,S)-sulfinyl]ethyl-
6.1.5. 2,2,2-Trichloroethyl 2-{N-[(allyloxy)(2’,3’-O-isopropyl-
ideneadenosin-5’-yloxy)-(R,S)-phosphoryl]- S-methyl-(R,S)-
sulfonimidoyl}ethylcarbamate (12)
carbamate (9)
The sulfide 8 (11.3 g, 42.4 mmol) was dissolved in aqueous tet-
rahydrofuran (5:2 THF: H
the addition of an aqueous solution (30 mL) of NaIO
5.2 mmol). The mixture was stirred overnight at ambient temper-
ature and filtered. After the removal of THF under reduced pres-
sure, EtOAc (100 mL) was added to the residue to extract the
product. The EtOAc extracts were washed successively with water
2
O, 70 mL) and was cooled to 0 °C before
The phosphine 11 (2.40 g, 4.86 mmol) and the sulfoximine 10
(1.97 g, 6.62 mmol) were mixed in dry CH CN (40 mL) over 4Å
3
4
(11.8 g,
5
molecular sieves (0.5 g) under an Ar atmosphere. Tetrazole
(0.461 g, 6.62 mmol) was added, and the mixture stirred at ambi-
ent temperature under Ar for 2 h before the addition of tert-butyl
hydroperoxide (1.41 g, 15.7 mmol). After stirring overnight, the
reaction mixture was evaporated and the residue purified by col-
(
2 4
30 mL) and sat. NaCl (30 mL). After drying over Na SO and filtra-
tion, evaporation gave the sulfoxide 9 as a pale yellow oil (10.9 g,
3
umn chromatography on silica gel (CHCl /MeOH, 19:1 to 4:1) to af-
9
0% yield): IR (NaCl, neat)
m
max 3400–3200 (br), 2979, 2933,
ford the N-adenylated sulfoximine 12 as a mixture of four
2
1
815, 1716, 1523, 1455, 1392, 1367, 1278, 1251, 1155, 1049,
diastereoisomers (2.68 g, 78%, amorphous solid): IR (NaCl, neat)
ꢀ1
024, 971, 943, 846, 813, 750, 686, 576, 565, 497, 458, 431 cm
;
mmax 3400–3200 (br), 2979, 2933, 2815, 1716, 1523, 1455, 1392,
1
H NMR (300 MHz, CDCl
.12 and 3.29–3.32 (3 ꢁ m, 2H, CH
NHCH CH S), 4.73 (s, 2H, Cl CCH
3
) d
H
2.67 (s, 3H, SCH
S), 3.74–3.78 (m, 2H,
), 6.00–6.11 (2 ꢁ br s, 1H, NH);
3
), 2.82–2.90, 3.03–
1367, 1278, 1251, 1155, 1049, 1024, 971, 943, 846, 813, 750,
ꢀ
1
1
3
2
686, 576, 565, 497, 458, 431 cm
1.40 and 1.64 (2 ꢁ s, 2 ꢁ 3H, isopropylidene), 3.23, 3.24, 3.28
and 3.30 (4 ꢁ s, 3H, SCH ), 3.33–5.36 (m, 17H, 5’-CH , 4’-H, 3’-H,
2’-H, NHCH CH S, CH CH@CH , Cl CCH2, adenyl 6-NH ), 5.83–
.00 (m, 1H, CH@CH ), 6.06–6.44 (m, 2H, 1’-H, NHCO), 8.29–8.49
(8 ꢁ s , 2H, adenyl 2-H and 8-H); C NMR (100 MHz, CDCl
3 H
; H NMR (300 MHz, CDCl ) d
2
2
3
2
+
HRMS (FAB, glycerol) calcd for C
found 281.9528.
6
H
11Cl
3
NO
3
S (MH ) 281.9525,
3
2
2
2
2
2
3
2
6
2
1
3
6
.1.3. 2,2,2-Trichloroethyl N-2-[S-methyl-(R,S)-sulfonimidoyl]-
ethylcarbamate (10)
To a solution of the sulfoxide 9 (2.88 g, 10.2 mmol) in dry
3 C
) d
25.33 and 25.35; 27.14 and 27.17; 35.22; 35.59, 35.63 and 35.69;
43.76, 43.81, 43.95 and 44.00; 55.72 and 55.76; 66.17 and 66.44;
67.47, 67.53, 67.57 and 67.62; 74.55; 81.16 and 81.25; 84.91;
85.08, 85.14, 85.38 and 85.52; 90.55, 90.72, 90.90 and 91.16;
95.4; 114.37, 114.40, 114.42 and 114.46 (adenine, C-5); 118.01,
118.06, 118.13 and 118.16 (C-8); 119.30, 119.40, 119.43 and
119.48 (C-4); 132.61 and 132.69 (C-2); 139.40, 139.43, 139.54
and 139.65 (C-6), 149.40 and 149.53; 153.23 and 153.27; 154.99,
2
5,26
CH
3.29 g, 15.3 mmol) under an Ar atmosphere, and the mixture
was stirred at ambient temperature for 4 days. The reaction mix-
ture was evaporated, and the residual oil was dissolved in CHCl
MeOH (10:1, 77 mL). The resulting solution was washed succes-
sively with sat. NaHCO (30 mL) and sat. NaCl (30 mL). After drying
3
CN was added O-mesitylsulfonylhydroxylamine (MSH)
(
3
/
3

5
924
H. Ikeuchi et al. / Bioorg. Med. Chem. 20 (2012) 5915–5927
1
55.04, 155.42 and 155.48 (C=O) [two or four sets of peaks corre-
washed successively with water (30 mL) and sat. NaCl (30 mL).
The water layer was extracted with CHCl
(50 mL ꢁ 2), and the
combined organic layers were dried over Na SO and filtered.
The filtrate was evaporated to give the allyl ester 14 as a pale yel-
sponding to four diastereoisomers were observed for each of a total
of 22 carbons]; P NMR (121 MHz, CDCl
3
3
3
1
) d
3 p
2.58, 2.85, 3.36 and
PS
2
4
.36. HRMS (FAB, p-nitrobenzyl alcohol) calcd for C22H32Cl N O
3 7 9
+
1
(
MH ) 706.0790, found 706.0776.
low oil (4.25 g, 55%): H NMR (300 MHz, CDCl
SCH ), 2.66 (t, 2H, J = 6.8 Hz, CH S), 2.76 (t, 2H, J = 6.8 Hz, CH
4.56–4.66 (m, 2H, OCH CH@CH ), 5.24 (dq, 1H, J = 10.5 and 1.2 Hz,
CH CH@CHH), 5.33 (dq, 1H, J = 17.4 and 1.5 Hz, CH CH=CHH), 5.92
(ddt, 1H, J = 17.3, 10.4 and 6.0 Hz, CH CH@CH ); HRMS (FAB, glyc-
S (MH ) 161.0637, found 161.0637.
) d
3 H
2.13 (s, 3H,
3
2
2
CO),
6
.1.6. Morpholine salt of 2,2,2-trichloroethyl 2-{N-[hydroxy-
2
2
(
2’,3’-O-isopropylideneadenosin-5’-yloxy)phosphoryl]- S-
2
2
methyl-(R,S)-sulfonimidoyl}ethylcarbamate (13)
2
2
+
The phosphoryl allyl ester 12 (1.05 g, 1.49 mmol) and morpho-
7 13 2
erol) calcd for C H O
line (0.65 g, 7.43 mmol) were dissolved in dry THF (10 mL) under
an Ar atmosphere. To this solution was added Pd(PPh
3
)
4
(0.343 g,
6.1.9. Allyl 3-[S-methyl-(R,S)-sulfinyl]propanoate (15)
2
The sulfide 14 (4.25 g, 26.5 mmol) was dissolved in THF–H O
0
.297 mmol) under an Ar atmosphere. The mixture was stirred at
ambient temperature for 0.5 h and was evaporated to dryness.
The residual oil was purified by reversed phase column chromatog-
raphy on Diaion HP20SS eluting with a linear gradient of THF in
(5:1, 60 mL) and was cooled at 0 °C before the addition of an aque-
ous solution (10 mL) of NaIO (7.38 g, 34.5 mmol). The mixture was
stirred at ambient temperature overnight and was filtered. After
removal of THF under reduced pressure, CHCl (80 mL) was added
to the residual aqueous mixture, and the organic layer was washed
successively with water (30 mL) and sat. NaCl (30 mL). The water
4
H
2
O (0 to 99%). The eluant was monitored with UV (254 nm),
and the desired compound was eluted at 53:47 H O/THF. UV posi-
tive-fractions were combined and lyophilized to afford compound
3 as a mixture of two diastereomeric salts of morpholine (1.06 g,
3
2
1
layer was extracted with CHCl
ganic layers were dried over Na
evaporated to give the racemic sulfoxide 15 as an yellow oil
(4.13 g, 88%): IR (NaCl, neat) max, 2983, 2925, 2892, 1785, 1648,
1419, 1378, 1348, 1240, 1186, 1145, 1031 (S@O), 991, 948, 777,
3
(50 mL ꢁ 2), and the combined or-
1
quant., pale yellow amorphous solid): H NMR (300 MHz, DMSO-
) d
1.32 and 1.54 (2 ꢁ s, 2 ꢁ 3H, isopropylidene), 2.98 (m, 4H,
morpholine NCH ), 3.1–3.6 and 3.7–3.8
ꢁ 2), 3.08 (s, 3H, SCH
m, 6H, NHCH CH S, 5’-CH ), 3.73 (m, 4H, morpholine OCH
ꢁ 2),
.37 (m, 1H, 4’-H), 4.79 (s, 2H, Cl CH ), 5.06 (br d, 1H, J = 6.3 Hz,
’-H), 5.31 (dd, 1H, J = 3.3 and 6.0 Hz, 2’-H), 6.14 (d, J = 3.3 Hz,
H, 1’-H), 7.29 (br s, 2H, adenyl 6-NH ), 8.16 (s, 1H, adenyl 2-H),
.3–8.4 (br m, 1H, CONH), 8.51 (s, 1H, adenyl 8-H); P NMR
) d 0.291 and 0.358. HRMS (FAB, glycerol)
2
SO and filtered. The filtrate was
4
d
6
H
2
3
m
(
2
2
2
2
ꢀ1
1
4
3
1
8
3
2
696, 559, 507 cm
SCH ), 2.80–3.13 (m, 4H, CH
OCH CH@CH ), 5.22–5.40 (m, 2H, CH@CH
CH@CH ); HRMS (FAB, glycerol) calcd for
177.0586, found 177.0589.
;
H NMR (300 MHz, CDCl
CH S), 4.59–4.72 (m, 2H,
), 5.86–5.98 (m, 1H,
3 H
) d 2.61 (s, 3H,
3
2
2
2
2
2
2
31
+
2
7 13 3
C H O S (MH )
(
121 MHz, DMSO-d
6
p
+
3 7 9
calcd for C19H28Cl N O PS (MH ) 666.0472, found 666.0487.
6
.1.10. Allyl 3-[S-methyl-(R,S)-sulfonimidoyl]propanoate (16)
To a solution of the sulfoxide 15 (1.40 g, 7.94 mmol) in dry
6
.1.7. 2-Aminoethyl-[S-methyl-(R,S)-sulfonimidoyl]-N-
adenylate (5)
The N-adenylated sulfoximine 13 (0.99 g, 1.50 mmol) was sus-
pended in TFA-H O-CH Cl (5:1:4, 20 mL) and was stirred at ambi-
ent temperature overnight to give a clear solution. The reaction
mixture was evaporated to dryness to give a syrupy residue, which
was then re-dissolved in AcOH (20 mL) before the addition of zinc
powder (1 g, 15.3 mmol). The mixture was stirred at ambient tem-
perature overnight and was evaporated to dryness to give a resid-
ual syrup that was purified by reverse phase MPLC
chromatography on Diaion HP20SS eluting with a linear gradient
3
CH CN (20 mL) was added MSH (2.56 g, 11.9 mmol) under an Ar
atmosphere. The mixture was stirred at ambient temperature for
4 days until completion of the reaction. The reaction mixture was
evaporated, and the residual oil was dissolved in EtOAc (50 mL).
2
2
2
The solution was washed successively with sat. NaHCO
and sat. NaCl (30 mL). After drying over Na SO and filtration, the
filtrate was evaporated. The residual oil was purified by column
chromatography on silica gel (CHCl /MeOH, 19:1 to 10:1) to afford
sulfoximine 16 as a pale brown oil (1.28 g, 84%): IR (NaCl, neat)
max 3606–3093 (br), 1731, 1646, 1606, 1565, 1490, 1452, 1417,
1378, 1338, 1241, 1201 (O@S@N), 1087, 1024 (S=O), 989, 933,
3
(30 mL)
2
4
3
m
of THF in H
2
O (0 to 99%). The eluant was monitored with UV
O/
ꢀ1
1
(
254 nm), and the desired compound was eluted at 99:1 H
2
842, 788, 748, 680, 582, 518, 468, 431, 410 cm
(300 MHz, CDCl ) d 2.60 (br s, 1H, NH), 2.93 (t, 2H, J = 7.4 Hz,
CH CH S), 3.02 (s, 3H, SCH ), 3.46 (t, 2H, J = 7.2 Hz, CH CH S),
4.64 (dt, 2H, J = 6.0 and 1.2 Hz, OCH CH=CH ), 5.27 (dq, 1H,
J = 10.5 and 1.2 Hz, CH@CHH), 5.34 (dq, 1H, J = 15.0 and 1.5 Hz,
CH@CHH), 5.92 (ddt, 1H, J = 15.0, 10.5 and 6.0 Hz, CH CH@CH );
S (MH ) 192.0691, found
;
H NMR
THF. The UV positive-fractions were combined and lyophilized to
3
H
afford the final product 5 as a mixture of two diastereoisomers
2
2
3
2
2
(
3
0.67 g, 90%, colorless solid): 1H NMR (300 MHz, DMSO-d
.31 (br s, 3H, SCH ), 3.61–4.11 (m, 7H, NH CH CH S, 5’-CH
3 2 2 2 2
6
) d
, 4’-
H), 4.19 (dd, 1H, J = 3.6 and 4.5 Hz, 3’-H), 4.59 (dd, 1H, J = 5.1 and
H
2
2
2
2
+
5
6
.1 Hz, 2’-H), 5.94 (d, J = 5.7 Hz, 1H, 1’-H), 7.60 (br s, 2H, adenyl
-NH
), 8.22 and 8.31 (2 ꢁ s, 1H, adenyl 2-H), 8.35 and 8.40
HRMS (FAB, glycerol) calcd for C
192.0691.
7 3
H14NO
2
13
(
(
(
2 ꢁ s, 1H, adenyl 8-H); C NMR (100 MHz, DMSO-d
6
) d
S(@O)(@N)], 63.39
ribose, C-5’), 70.55 (C-3’), 73.79 (C-2’), 83.38 (C-4’), 87.38 (C-1’),
C
33.20
+
CH
2
NH
3
), 42.86 [S(@O)(@N)–CH
3
], 50.59 [CH
2
6.1.11. Allyl 3-{N-[(allyloxy)(2’,3’-O-isopropylideneadenosin-5’-
yloxy)-(R, S)-phosphoryl]- S-methyl-(R, S)-
1
1
18.31 (adenine, C-5), 140.46 (C-8), 149.23 (C-4), 152.79 (C-2),
sulfonimidoyl}propanoate (17)
The reactive phosphine 11 (1.68 g, 3.40 mmol) and the sulfox-
imine 16 (0.94 g, 4.92 mmol) and 4 Å molecular sieves (1.0 g) were
3
1
54.17 (C-6); P NMR (121 MHz, DMSO-d
6
) d
p
1.45. HRMS (FAB,
+
glycerol) calcd for C13
H
23
7
N O
7
PS (MH ) 452.1117, found 452.1115.
3
mixed in dry CH CN (30 mL) under an Ar atmosphere. Tetrazole
6
.1.8. Allyl 3-(S-methylsulfanyl)propanoate (14)
Allyl bromide (7.02 g, 58.0 mmol) was added dropwise under an
Ar atmosphere to an ice-cooled, vigorously stirred suspension of 3-
methylsulfanylpropanoic acid (5.80 g, 48.3 mmol), K CO (7.34 g,
3.1 mmol) in dry acetone (40 mL). After completion of the addi-
tion of allyl bromide, the mixture was heated under reflux for
4 h. The reaction mixture was filtered and evaporated to give a
residual yellow oil. CHCl (80 mL) was added to the residue and
(0.344 g, 4.92 mmol) was added, and the resulting mixture was
stirred at ambient temperature under an Ar atmosphere for 2 h be-
fore the addition of tert-butyl hydroperoxide (0.972 g, 7.55 mmol).
After overnight stirring, the reaction mixture was evaporated, and
the residue purified by column chromatography on silica gel
2
3
5
(CHCl
of four diastereoisomers (1.54 g, 76%, amorphous solid): IR (NaCl,
neat) max 3586–3264 (br), 2989, 2938, 1735, 1644, 1600, 1506,
3
/MeOH, 19:1 to 4:1) to afford sulfoximine 17 as a mixture
1
3
m

H. Ikeuchi et al. / Bioorg. Med. Chem. 20 (2012) 5915–5927
5925
1
1
4
475, 1423, 1376, 1330, 129, 1245, 1214 (O@S@N), 1157, 1076,
assay (Pierce),59 and
L
8
-glutamine was recrystallized prior to use
3
016 (S=O), 993, 927, 863, 786, 728, 680, 649, 580, 549, 511, 460,
in all kinetic assays. The inhibition of human ASNS by 5 and 6
was measured by determining their effect on PPi production under
steady-state conditions, using a continuous assay in which PPi for-
mation is enzyme-coupled to NADH consumption (340 nm) (pyro-
phosphate reagent, Sigma technical bulletin BI-100). Assay
mixtures in which 25 mM
source contained sulfoximine 5 at various fixed concentrations
(0, 0.2, 0.5, 1, 2 and 5 M) and 0.5 mM ATP, 10 mM -Asp, and
the pyrophosphate reagent (350 L) in 100 mM EPPS buffer, pH
8.0, containing 10 mM MgCl (1 mL total volume). Reactions were
initiated by the addition of hASNS (4 g), and pyrophosphate pro-
duction was monitored spectrophotometrically at 25 °C over a per-
iod of 20 min. When 100 mM NH Cl was the nitrogen source,
ꢀ1
1
49, 403 cm
;
H NMR (300 MHz, CDCl
) d
3 H
1.39 and 1.63
CH S), 3.19,
), 4.09–4.30
(
3
(
2 ꢁ s, 2 ꢁ 3H, isopropylidene), 2.93–3.01 (m, 2H, CH
.22, 3.27 (3 ꢁ s, 3H, SCH ), 3.49–3.80 (m, 2H, 5’-CH
m, 2H, CH CH S), 4.38–4.63 (m, 6H, COOCH
, 4’-H, 3’-H), 5.08–5.40 (m, 5H, COOCH
2
2
3
2
2
2
2
CH@CH
2
2
2
,
,
,
POCH
2
CH@CH
2
2
2
2
CH=CH
2
2’-H), 5.80–5.97 (m, 2H, COOCH CH@CH
L-glutamine was used as the nitrogen
POCH
POCH
2
CH@CH
CH@CH
,
2
), 6.14 (br s, 2H, NH
2
), 6.22 (br s, 1H, 1’-H), 8.19
l
L
31
(
s, 1H, adenyl 2-H), 8.36 (s, 1H, adenyl 8-H); P NMR (121 MHz,
CDCl ) d 2.44, 2.60, 2.66, 3.52. HRMS (FAB, p-nitrobenzyl alcohol)
calcd for C23
l
3
p
2
+
H
34
N
6
O
9
PS (MH ) 601.1848, found 601.1844.
l
6
.1.12. Bis-morpholine salt of 3-{N-[(2’,3’-O-Isopropyl-
4
ideneadenosin-5’-yloxy)phosphoryl]-S-methyl-(R,S)-sulfo-
nimidoyl}propanoic acid (18)
reaction mixtures were identical to those outlined above except
for the fixed concentrations of sulfoximine 5 (0, 0.4, 0.7, 1, 2.5, 5
The allyl ester 17 (1.53 g, 2.55 mmol) and morpholine (2.21 g,
and 10 lM). Each data point represents the average of duplicate
2
5.5 mmol) were dissolved in dry THF (20 mL) under an Ar atmo-
experiments. Identical procedures were employed to characterize
sphere. To this solution was added Pd(PPh (0.59 g, 0.51 mmol)
3
)
4
the interaction of sulfoximine 6 with hASNS except for the fixed
under an Ar atmosphere. The mixture was stirred at ambient tem-
perature for overnight. The reaction mixture was evaporated to
dryness. The residue oil was purified by reversed phase, med-
ium-pressure column chromatography on ODS eluting with a lin-
concentrations of 6, which were 0, 5, 25, 40, 60 and 80
0, 2, 5, 8, 15 and 20 M for the glutamine- and ammonia-depen-
dent synthetase reactions, respectively. Control experiments using
known amounts of PP demonstrated that the assay pyrophosphate
lM, and
l
i
ear gradient of MeOH in H
monitored with UV (254 nm), and the desired product 18 eluted
at 30:70 H O/MeOH. UV positive-fractions were then combined
and lyophilized to afford 18 as a mixture of two diastereomers
1.33 g, quant, yellow oil): 1H NMR (300 MHz, DMSO-d
) d 1.33
and 1.54 (2 ꢁ s, 2 ꢁ 3H, isopropylidene), 2.92 (d, 2H, J = 6.6 Hz,
CH CH S), 3.03 (br s, 3H, SCH ), 3.41–3.82 (m, 4H, CH CH S, 5’-
CH ), 4.37 (m, 1H, 4’-H), 5.00–5.17 (m, 1H, J = 6.3, 3’-H), 5.32 (m,
H, 2’-H), 6.14 (br d, J = 3.0 Hz, 1H, 1’-H), 7.29 (br s, 2H, NH ),
.16 (s, 1H, adenyl 2-H), 8.49 (s, 1H, adenyl 8-H); P NMR
) d 0.02, 5.59. HRMS (FAB, glycerol) calcd for
PS (MH ) 521.1222, found 521.1227.
2
O (0 to 100%). The eluant was
reagent was not affected by the presence of either sulfoximine 5 or
6.
2
The four sets of progress curves (Figs. 2 and 3) were analyzed by
fitting the data to the following equation, using the Kaleidagraph
36
(
6
H
v3.5 software package (Synergy):
ð
m
0
ꢀ
m
ss
Þ
kt
½PP
i
ꢂ ¼
msst þ
ð1 ꢀ eꢀ
Þ
ð1Þ
2
2
3
2
2
k
2
where [PP
at time t,
i
] is the concentration of inorganic pyrophosphate formed
and ss are the initial and steady-state rates, respec-
1
8
(
2
3
1
v
o
v
tively, and k is the apparent first-order rate constant for isomeriza-
tion of EI to EI . By analogy to the known kinetic behavior of the
121 MHz, DMSO-d
H N O
17 26 6 9
6
+
p
⁄
C
adenylated sulfoximine 1, it was assumed that the inhibitors 5
and 6 bound to the free enzyme being competitive with respect
to ATP, as shown in the following kinetic model:
6
.1.13. 3-{N-[(Adenosin-5’-yloxy)(hydroxy)phosphoryl]-(R,S)-S-
methylsulfonimidoyl} propanoic acid (6)
A suspension of compound 18 (1.34 g, 2.57 mmol) in 5:1:4 TFA–
H O–CH Cl (30 mL) was stirred at ambient temperature overnight
2 2 2
to give a clear solution. The reaction mixture was evaporated to
dryness to give a residual syrup that was purified by reversed
phase, medium-pressure column chromatography on ODS eluting
with a linear gradient of MeOH in H
was monitored with UV (254 nm), and the desired sulfoximine 6
eluted at 60:40 H O/MeOH. UV positive-fractions were combined
2
O (0 to 100%). The eluant
2
and triturated in EtOAc (10 mL). The suspension was filtrated,
and the precipitate was washed with EtOAc (20 mL) to afford the
final product as a mixture of two diastereomers 6 (1.23 g, quant,
1
colorless solid): H NMR (300 MHz, DMSO-d
6
) d
, CH
H, 4’-H), 4.55–4.66 (m, 1H, 3’-H) , 5.52 (m, 2H, 2’-H), 5.92 (d,
), 8.15 (s, 1H, adenyl 2-H),
.34 (s, 1H, adenyl 8-H); 13C NMR (100 MHz, DMSO-d
) d 27.62
CH COOH), 41.64 and 42.85 [S(@O)(@N)–CH ], 51.15 and 51.19
CH S(@O)(@N)], 63.34 (ribose, C-5’), 70.58 (C-3’), 73.53 (C-2’),
3.27 (C-4’), 87.04 (C-1’), 118.93 (adenine, C-5), 139.50 (C-8),
H
2.73–2.80 (m,
2
H, CH
2
CH
2
S), 3.00–4.07 (m, 7H, 5’-CH
2
, SCH
3
2
CH S), 4.18 (m,
2
1
J = 5.7 Hz, 1H, 1’-H), 7.27 (br s, 2H, NH
2
Having values for k,
the inhibitors, this information was then used to calculate an esti-
mate of k , using equation (2):
o
v and vss in hand at each concentration of
8
6
C
(
[
2
3
6
2
m
m
ss
k
6
¼ k
ð2Þ
8
1
o
3
1
49.56 (C-4), 152.27 (C-2), 155.71 (C-6), 171.68 (C@O); P NMR
) d 1.903. HRMS (FAB, glycerol) calcd for
PS (MH ) 481.0907, found 481.0907.
With an estimate of k
6
, values of K
I
and k
5
were determined by
(
C
121 MHz, DMSO-d
6
+
p
fitting the variation of k with [I] using equation (3):
14 22 6 9
H N O
ꢀ
ꢁ
I=K
ð1 þ ½ATPꢂ=K
I
K ¼ k
6
þ k
5
:
ð3Þ
6
.2. Steady-state kinetic assays
a
þ I=K Þ
I
Unless otherwise stated, all chemicals and reagents were pur-
chased from Sigma Aldrich and were of the highest purity avail-
able. Protein concentrations were determined using the Bradford
where I is the concentration of the inhibitor, K
I
is the initial inhibi-
3
9
tion constant, K
a
is taken to be 0.1 mM, and [ATP] = 0.5 mM. Plots
showing the variation in k with sulfoximine concentration for the

5
926
H. Ikeuchi et al. / Bioorg. Med. Chem. 20 (2012) 5915–5927
four sets of progress curves (Figs. 2 and 3) are provided in support-
Education, Culture, Sports, Science and Technology of Japan (J.H).
These studies were initiated through the provision of a grant from
the Chiles Endowment Biomedical research program of the Florida
Department of Health (N.G.J.R.) and we thank the University of
Florida for additional studentship support (Y.A. and L.H.). Molecu-
lar graphics and analyses were performed with the UCSF Chimera
package. Chimera is developed by the Resource for Biocomputing,
Visualization, and Informatics at the University of California, San
Francisco, with support from the National Institutes of Health (Na-
tional Center for Research Resources grant 2P41RR001081, Na-
tional Institute of General Medical Sciences grant 9P41GM103311).
⁄
ing information. The overall inhibition constant K
I
was computed
using equation (4):
I 6
K k
ꢃ
K ¼
:
ð4Þ
I
k
5
þ k
6
6
.3. Cell-based assays
MOLT4R and MOLT4S cell cultures were maintained with RPMI
640 medium (Hyclone), supplemented with heat-inactivated 10%
1
FBS (Hyclone) and 100 U/mL penicillin-streptomycin (Mediatech
Inc). The suspension cultures were incubated in 95% air with 5%
CO at 37 °C. Twenty-four hours before all experiments, cells were
2
Supplementary data
collected by centrifugation for 3 min at 250 g, rinsed twice with
phosphate buffered saline (0.15 M NaCl, 10 mM sodium phosphate,
pH 7.4) and re-suspended in fresh medium before being seeded
into 96-well plates at an initial density of 50,000 cells/well. The
cells were incubated with sulfoximine 5 at concentrations varying
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.07.047.
References and notes
from 10–1000
lM in the presence or absence of 1 U/mL of ASNase
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3
.
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VERSAmax Microplate Reader (Molecular Diagnostics). The mean
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1
2. Leslie, M.; Case, M. C.; Hall, A. G.; Coulthard, S. A. Br. J. Haematol. 2006, 132, 740.
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tyl-AMP/MgPP
, which we have described previously.21 Each sul-
foximine was superimposed onto bound b-aspartyl-AMP. The PP
L
-glutamine/b-aspar-
145.
i
1
1
5. Aslanian, A. M.; Fletcher, B. S.; Kilberg, M. S. Biochem. J. 2001, 357, 321.
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i
moiety bound to the Mg(II) ion and the b-aspartyl-AMP intermedi-
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1
1
7. Lorenzi, P. L.; Reinhold, W. C.; Rudelius, M.; Gunsior, M.; Shankavaram, U.;
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60
structure using the HBUILD algorithm, which was then solvated
in an octahedral box of TIP3P41 water molecules after the addition
of ions to give a system of zero net charge. The final model was
then obtained by energy minimization using the steepest descents
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1
2
2
2
9. Koizumi, M.; Hiratake, J.; Nakatu, T.; Kato, H.; Oda, J. J. Am. Chem. Soc. 1999, 121,
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1
package. These calculations employed the CHARMM27 force
4
2
field with missing parameters for the sulfoximine ligand being
43
obtained from the CGenFF software package. Electrostatic inter-
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2
2033.
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12 Å cutoff) and a force-switching function was employed for
2
2
4. Okamura, H.; Bolm, C. Org. Lett. 2004, 6, 1305.
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3
3
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6
3,64
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(
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