N-alkoxysulfamide, N-hydroxysulfamide, and sulfamate analogues of methionyl and isoleucyl adenylates as inhibitors of methionyl-tRNA and isoleucyl-tRNA synthetases

Article abstract of DOI:10.1016/S0960-894X(03)00020-9

A series of sulfamate surrogates of methionyl and isoleucyl adenylate have been investigated as MetRS and IleRS inhibitors by modifications of the sulfamate linker and adenine moieties. The discovery of 2-iodo Ile-NHSO₂-AMP (58) as a potent Escherichia coli IleRS inhibitor revealed that a significant hydrophobic interaction between the 2-substituent of Ile-NHSO₂-AMP and the adenine binding site of IleRS provided its high potency to the enzyme.

Full text of DOI:10.1016/S0960-894X(03)00020-9

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1087–1092
N-Alkoxysulfamide, N-Hydroxysulfamide, and Sulfamate
Analogues of Methionyl and Isoleucyl Adenylates as Inhibitors
of Methionyl-tRNA and Isoleucyl-tRNA Synthetases
Jeewoo Lee,a,* Sung Eun Kim,a Ji Young Lee,a Su Yeon Kim,a Sang Uk Kang,a
Seung Hwan Seo,a Moon Woo Chun,a Taehee Kang,b
Soo Young Choib and Hea Ok Kimc
aLaboratory of Medicinal Chemistry, RIPS, College of Pharmacy, Seoul National University, Seoul 151-742, South Korea
bImagene Co., Ltd., #406 Biotechnology Incubating Center, Seoul National University, Seoul, South Korea
cDivision of Chemistry and Molecular Engineering, Seoul National University, Seoul, South Korea
Received 6November 2002; revised 6January 2003; accepted 7 January 2003
Abstract—A series of sulfamate surrogates of methionyl and isoleucyl adenylate have been investigated as MetRS and IleRS inhib-
itors by modifications of the sulfamate linker and adenine moieties. The discovery of 2-iodo Ile-NHSO2-AMP (58) as a potent
Escherichia coli IleRS inhibitor revealed that a significant hydrophobic interaction between the 2-substituent of Ile-NHSO2-AMP
and the adenine binding site of IleRS provided its high potency to the enzyme.
# 2003 Elsevier Science Ltd. All rights reserved.
The aminoacyl-tRNA synthetases (aaRSs) are a family
of enzymes that precisely attach amino acids to tRNA
molecules containing the corresponding anticodon. The
resulting aminoacyl-tRNAs are substrates for trans-
lation in protein synthesis and are pivotal in determining
how the genetic code is interpreted as amino acids.1 The
enzymes have been regarded as novel bacterial targets,
particularly for the treatment of antibiotic-resistant
bacterial strains, such as MRSA and VRE.2ꢀ4 Since the
activity of aminoacyl-tRNA synthetases is essential in
all living organisms, the clinical application of the enzyme
inhibitors primarily depends on the selectivity to the
pathogen synthetases over the human cell counterparts.
antibiotic candidates, such as chemical stability, tight
binding, and pathogen selectivity. Since the mixed
anhydride acylphosphate bond of the intermediate is
readily susceptible to hydrolysis, its substitution with
non-hydrolyzable bioisosteres has been most exten-
sively investigated to find potent aminoacyl adenylate
inhibitors. To date, alkylphosphate (II),5ꢀ7 ester (III,
aa-CO2–AMP),8ꢀ10 amide (IV, aa-CONH–AMP),8
hydroxamate (V-VI, aa-CON(OH)–AMP),8ꢀ10 sulfa-
mate (VII, aa-NHSO3–AMP),5,6,11ꢀ14 and sulfamide
(VIII, aa-NHSO2NH–AMP)6 surrogates have been used
to replace the labile acylphosphate of the aminoacyl
adenylate (Fig. 1). Among them, the sulfamate moiety
has exhibited both tight binding affinity and stability to
a variety of aaRSs, such as AlaRS,11 ArgRS,5 HisRS,5
IleRS,12 ProRS,13 SerRS,14 ThrRS,5 and TyrRS.6 Par-
ticularly, an isoleucyl sulfamate analogue bearing the
dihydroxytetrahydropyran moiety of pseudomonic acid
had exceptionally ultrapotent binding affinity, with a
value in the femtomolar range for binding to the
S. aureus IleRS.12 As a similar strategy to modify an
acylphosphate linkage of an aminoacyl adenylate, we
recently reported that ester (III), amide (IV), and hydro-
xamate analogues (V, VI) of methionyl and isoleucyl
adenylate, as stable surrogates of aminoacyl adenylate,
Aminoacyl adenylate (aa-AMP, I), an intermediate in
the enzymatic reaction, has been used as a starting
prototype to discover novel aaRS inhibitors because of
its tighter binding affinity than the substrates, amino
acid and ATP, which is generally greater by two or three
orders of magnitude. Most modifications of aminoacyl
adenylates have focused on several important issues as
*Corresponding author. Tel.:+82-2-880-7846; fax:+82-2-888-0649;
e-mail: jeewoo@snu.ac.kr
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00020-9

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J. Lee et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1087–1092
The synthesis of N-alkoxy sulfamide (9, 10) analogues,
as sulfamate surrogates, is outlined in Scheme 1.
O-Adenosylhydroxylamine (3), as an intermediate for
the synthesis of N-adenosyloxy sulfamide, was prepared
from the commercially available 20,30-isopropylidene-
adenosine (1) by the Mitsunobu reaction with N-hydroxy-
phthalimide, followed by the hydrazine reaction, as
reported previously.8 Treatment of 3 with N-(carbo-
benzyloxy)sulfamoyl chloride15 and subsequent cataly-
tic hydrogenation provided the sulfamate intermediate
5. The condensation of 5 with N-Boc methionine and
N-Boc isoleucine by DCC afforded the N-alkoxy-
sulfamate analogues (7, 8), whose protecting groups
were hydrolyzed under acidic conditions to produce the
final N-methionyl(or isoleucyl)-N0-adenosyloxy sulfa-
mide compounds, 9 and 10, respectively. For the synth-
esis of the N-hydroxy sulfamide (16, 17) analogues
shown in Scheme 2, the Mitsunobu reaction of
20,30-isopropylideneadenosine (1) with O-benzylhydroxy-
lamine produced the O-benzyl-N-adenosylhydroxy-
lamine (11), which was converted to the final
N-methionyl (or isoleucyl)-N0-adenosyl-N0-hydroxy sul-
famides (16, 17) by following a sequence similar to that
described in Scheme 1. In order to investigate the struc-
ture–activity relationship toward the adenine moiety in
the sulfamate analogues of methionyl and isoleucyl
adenylate, we synthesized several 2-substituted ana-
logues, as shown in Schemes 3 and 4. 2-Chloro adeno-
sine is commercially available, and 2-iodo-6-methylamino
adenosine was prepared according to the literature.16
2-Iodo, 2-ethynyl, and 2-hexynyl substituted analogues
of adenosine were synthesized by the modified method
of Matsuda et al.17 The key iodo-intermediate 21 was
prepared from 2-amino-6-chloropurine in 3 steps. The
Figure 1.
have moderate to potent inhibitory activities,8ꢀ10 which
were much lower than those of the corresponding sul-
famate analogues (VII).
As part of our continuing effort to find potent and
pathogen-selective inhibitors of MetRS and IleRS as
potential antibacterial candidates, we herein describe
the N-alkoxysulfamide (IX, aa-NHSO2NHO–AMP)
and N-hydroxysulfamide (X, aa-NHSO2N(OH)–AMP)
analogues of methionyl and isoleucyl adenylates, as
sulfamate surrogates, and a SAR investigation of the
adenine moiety of sulfamate (XI, aa-NHSO3–AMP)
analogues, which provides crucial information regard-
ing the interaction of the isoleucyl adenylate with
IleRS.
Scheme 1. Reagents and conditions: (a) N-hydroxyphthalimide, DEAD, PPh3, THF, 83%; (b) N2H4, EtOH, reflux, 96%; (c) BnO2CNHSOCl,
NEt3, THF–CH2Cl2, 81%; (d) H2, Pd–C, MeOH, 97%; (e) BocNCH(R)CO2H, DCC, DMAP, CH2Cl2, 99% for Met, 96% for Ile; (f) CF3CO2H,
anisole, CH2Cl2, 82% for Met, 98% for Ile.

J. Lee et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1087–1092
1089
Scheme 2. Reagents and conditions: (a) BnONH–HCl, DEAD, PPh3, THF, 98%; (b) BnO2CNHSOCl, NEt3, THF–CH2Cl2, 77%; (c) H2, Pd–C,
MeOH, 93%; (d) BocNCH(R)CO2H, DCC, DMAP, CH2Cl2, 68% for Met, 96% for Ile; (e) CF3CO2H, anisole, CH2Cl2, 74% for Met, 75% for Ile.
palladium-catalyzed cross-coupling reaction of 21 with
alkynes provided the 2-alkyne substituted adenosines,
22 and 23. The adenosine analogues, 21–26, were acet-
onided to the 20,30-O-isopropylidenes 27–31, and the
5-hydroxyls were reacted with sodium hydride and sulfa-
moyl chloride as previously reported13,18,19 to furnish the
5-O-sulfamoylated adenosines 32–37. Finally, the ami-
noacylation of 32–37 with methionine or isoleucine fol-
lowed by acidic hydrolysis afforded the methionyl (50–55)
and isoleucyl (56–61) sulfamate analogues, respectively.
and IleRS, respectively (Table 1). The inhibitory activ-
ities were determined by measuring the decrease of the
aminoacylation product, the [35S]methionyl E. coli-
tRNAMet or [3H]isoleucyl E. coli-tRNAIle, in the pre-
sence of different chemical concentrations as previously
reported.20 Sulfamate analogues of methionyl adenylate
(Met-NHSO3–AMP) (50) and isoleucyl adenylate (Ile-
NHSO3–AMP) (56) were evaluated as references in our
assay protocol, and showed values of IC50=0.007 and
0.098 mM, respectively. The replacement of the sulfa-
mate oxygen with –NH-O- provided the N-alkoxy-
sulfamide analogues (9, 10), which exhibited much
lower inhibitory activities than the corresponding sulfa-
mates (50, 56), but similar activities as the ester and
hydroxamate surrogates reported previously.9 The
N-hydroxysulfamides (16, 17) resulting from the repla-
cement of the sulfamate oxygen with –N(OH)– atte-
nuated the inhibition of MetRS and IleRS as compared
to the N-alkoxysulfamides. Since the sulfamate ana-
logues of methionyl and isoleucyl adenylate had shown
the most potent inhibitory activities, as compared to the
other isosteres studied thus far, we wanted to investigate
the SAR of their adenine region by creating aminoacyl
adenylates with different adenine derivatives. Four
functional groups, including chloro, iodo, ethynyl, and
hexynyl groups were introduced at the 2-position of the
sulfamate analogues of the methionyl and isoleucyl
adenylates. In the methionyl adenylate analogues, the
2-substituted analogues (51, 52, 54, 55) showed moder-
ate inhibitory activities, with a range of IC50=1.1–7.4
mM, but were less potent than the unsubstituted parent
compound (50). Among the isoleucyl adenylate ana-
logues, the 2-chloro (57) analogue exhibited moderate
inhibition, while the 2-iodo (58) and 2-ethynyl (60)
analogues showed significantly improved inhibitory
activity to IleRS, with values of IC50=0.014 and 0.039
mM, which were 7- and 2.5-fold more potent than the
parent compound (56), respectively. However, the sub-
stitution with a more extended hexynyl to afford 61 led
The synthesized methionyl and isoleucyl adenylate ana-
logues were evaluated as inhibitors of E. coli MetRS
Scheme 3. Reagents and conditions: (a) (i) HMDS, (NH4)2SO4, reflux;
(ii) O-tribenzoylribosyl-1-acetate, TMSOTf, ClCH2CH2Cl, 83%; (b)
I2, CH2I2, isoamyl nitrite, CuI, reflux, 85%; (c) NH3, MeOH, 83%; (d)
23: (i) HCꢁCTMS, (PPh3)2PdCl2, CuI, Et3N, DMF, 80 ꢂC, 84%; (ii)
NH3, MeOH, 87%; (e) 24: (i) HCꢁC(CH2)3CH3, (PPh3)2PdCl2, CuI,
Et3N, DMF, 80 ꢂC, 89%.

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J. Lee et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1087–1092
Scheme 4. Reagents and conditions: (a) camphorsulfonic acid, acetone, 65–75%; (b) NH2SO2Cl, NaH, DME, 60–80%; (c) BocNCH(R)CO2H,
DCC, DMAP, CH2Cl2, 85–95%; (d) CF3CO2H, CH2Cl2-H2O, 45–65%.
to a dramatic loss in the inhibitory activity. 6-Methyla-
tion of the 2-iodo analogues (52, 58) to furnish 53 and
59 was ineffective for the enzyme inhibition, as they
showed 160 and 1600-fold reductions, respectively. This
finding suggested that the 6-amino group is crucial to
interact with the IleRS active site, where the two amino
hydrogens form hydrogen bonds with two carbonyl
oxygens in the enzyme, as previously examined in the
complex between Ile-AMS and the X-ray structure of
the Thermus thermophilus IleRS.21
The high potency of the 2-iodo (58) and 2-ethynyl (60)
analogues of Ile-NHSO3–AMP suggested that IleRS
may contain a cavity to afford such bulky groups. This
finding prompted us to search for the presence of such a
pocket in IleRS. Recently, the structural basis for the
recognition of an isoleucyl-adenylate by IleRS was
identified by the crystal structure of the IleRS from
Thermus thermophilus in complex with the sulfamide,
Ile-AMS (Ile-NHSO2NH–AMP). Careful examination
of the adenine binding site of Ile-AMS with the enzyme
revealed a small hole (circle with arrows) around the
2-position of adenine, as shown in Figure 2a. The
docking study with the 2-iodo sulfamate analogue (58)
was performed on the X-ray model above, and the
modeled results are shown in Figure 2b (ball and stick)
and c (spacefill).22 The modeling study revealed that the
2-iodo atom (violet color) of 58 was accomodated by
the cavity shown in Figure 2a very well, in which the
Gly56, Gln59, Gly551, His581, Gly582, Leu583, Ile584,
and Arg628 amino acids were present within a 5 A
range from the iodo moiety. This docking result con-
firmed that the enhanced potency of 58 resulted from its
better binding to the active site of IleRS by accomodating
the 2-iodo moiety into the hole. Also, the dimensions of
the hole were not sufficient to hold a bulkier group, like
the hexynyl group of 61, which explains its lower inhibi-
tory activity. Nevertheless, the reduced inhibitory activ-
ity of the 2-chloro analogue (57) cannot be explained by
this model, and its investigation is still under way.
Table 1. Inhibitory activities of the synthesized compounds toward
E. coli MetRS and IleRS
Number
X
R1
H
R2
H
IC50
19.7
Met-AMP
9
16N(OH)
NHO
H
H
106
50
51
52
53
54
55
O
O
O
O
O
O
H
H
H
CH3
H
H
Cl
I
0.007
7.4
1.2
191
1.1
I
CꢁCH
H
CꢁC(CH2)3CH3
1.9
Ile-AMP
10
17
56O
57
58
59
6
NHO
N(OH)
H
H
H
H
H
CH3
H
H
0.098
Cl
I
I
12.8
132
H
O
O
O
0
5.5
0.014
22.2
C0.039
1.1
In summary, the potent MetRS and IleRS inhibitions
by the sulfamate analogues of Met-AMP and Ile-AMP
were optimized by modifications of the sulfamate linker
O
O
ꢁHCH
6
1
ꢁC(CH2)3CH3
C

J. Lee et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1087–1092
1091
Figure 2. Docking model of (a) Ile-NHSO2NH–AMP21 and (b, c) 2-iodo analogue of Ile-NHSO3–AMP (58) with T. thermophilus IleRS.
and the adenine moiety. Replacement of the sulfamate
oxygen with N–O groups, to furnish N-hydroxy and
N-alkoxy sulfamide, caused marked reductions in the
inhibitory potency to E. coli MetRS and IleRS,
respectively. However, the modification of the adenine
group in the sulfamate surrogates led to a highly
potent IleRS inhibitor, the 2-iodo analogue (58). A
docking study of 58 into the X-ray structure of the T.
thermophilus IleRS indicated that the potent inhibition
by 58 resulted from its snug fit within a hydrophobic
cavity around the 2-position of adenine in Ile-NHSO3–
AMP.
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Acknowledgements
This work was supported by a grant (HMP-00-CH-
15-0014) from the Ministry of Health & Welfare,
R.O.K.

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22. The 2-iodo compound (58) was modeled in the T.
thermophilus IleRS active site, using the X-ray structure of the
Ile-AMS (Ile-NHSO2NH–AMP) complex in ref 21, with the
Ile-AMS molecule as a guide for the docking. The complex
was energy-minimized with SYBYL 6.5, using the Tripos force
field. All computational work was done on a Silicon Graphics
O2 R10000 workstation.