Benzoic acid and pyridine derivatives as inhibitors of Trypanosoma cruzi trans-sialidase

Article abstract of DOI:10.1016/j.bmc.2006.12.024

Benzoic acid and pyridine derivatives inhibit recombinant trans-sialidase from Trypanosoma cruzi with I₅₀ values between 0.4 and 1 mM. The best compounds, 4-acetylamino-3-hydroxymethylbenzoic acid and 5-acetylamino-6-aminopyridine-2-carboxylic

Full text of DOI:10.1016/j.bmc.2006.12.024

Bioorganic & Medicinal Chemistry 15 (2007) 2106–2119
Benzoic acid and pyridine derivatives as inhibitors of
Trypanosoma cruzi trans-sialidase
Joao Neres,a Pascal Bonnet,a,ꢀ Philip N. Edwards,a Pravin L. Kotian,c
˜
Alejandro Buschiazzo,b Pedro M. Alzari,b Richard A. Brycea and Kenneth T. Douglasa,*
aSchool of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester, M13 9PL, UK
bUnite´ de Biochimie Structurale, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris, France
cBioCryst Pharmaceuticals, Inc., 2190 Parkway Drive, Birmingham, AL 35244, USA
Received 3 October 2006; revised 7 December 2006; accepted 13 December 2006
Available online 15 December 2006
Abstract—Benzoic acid and pyridine derivatives inhibit recombinant trans-sialidase from Trypanosoma cruzi with I50 values between
0.4 and 1 mM. The best compounds, 4-acetylamino-3-hydroxymethylbenzoic acid and 5-acetylamino-6-aminopyridine-2-carboxylic
acid, provide new leads to inhibitors not containing the synthetically complex sialic acid structure. The weak inhibition by such
compounds contrasts with their much stronger inhibition of neuraminidase from Influenza virus.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
(TcTS), a parasite membrane-associated protein which
catalyses the transfer of sialic acid molecules from host
cell-surface glycoconjugates to its own surface mucin-
like glycoproteins, seems to be a key enzyme to T. cruzi
infective/invasive ability.4–6 Thus, the developmental
stage-specific expression of trans-sialidase is an impor-
tant virulence factor of T. cruzi. As the trans-sialidases
are unique to the parasite, TcTS has been discussed as
a potential target for anti-Chagas drug design.7,8 It is
not possible to test the potential validity of this enzyme
as a drug target by gene-knockout experiments as the
TcTS protein family is coded for by 140 genes in the
genome.9 However, there are good biochemical reasons
to believe that processes involving TcTS are vital to the
parasite.
Chagas’ disease, widely distributed in Central and South
America with 13 million persons infected and 100 mil-
lion people at risk, is estimated to cause 14,000 deaths
annually. This disease is caused by Trypanosoma cruzi,
a protozoan parasite transmitted to humans by haemat-
ophagous bugs or directly by transfusion of infected
blood.1 The only established drugs for the acute phase
of infection are nifurtimox and benznidazole, with few
other candidates yet on the clinical horizon.2 The prob-
lem of resistance, both of the parasite to drug at molec-
ular level and of the vector to insecticide, further adds to
the gloomy prospects.3 With this in mind there has been
considerable interest in trying to develop novel
approaches and targets for drug design.
TcTS specifically (and preferentially) transfers sialic acid
linked to saccharides or glycoconjugates by a-2,3 bonds
to terminal b-galactosyl residues in acceptor molecules.
However, in the absence of an appropriate acceptor,
TcTS can also act as a sialidase, hydrolysing the donor
substrate and releasing free sialic acid. Both activities
(sialidase and trans-sialidase) are associated with the
same active site in TcTS, therefore assays based on sialic
acid transfer or hydrolysis can be used to monitor TcTS
activity.10–13
A key problem in the pathogenicity of T. cruzi is its abil-
ity to evade host immune responses. Surface sialylation
plays a central role in this and in the host cell adhesion/
invasion mechanism, and provides a possible entry to
novel therapeutics against this disease. This parasite is
unable to synthesise sialic acids de novo. Trans-sialidase
Keywords: Trans-sialidase; Inhibitors; Chagas disease; Neuraminidase.
*
Corresponding author. Tel.: +44 161 275 2371; fax: +44 161 275
2481; e-mail: Ken.Douglas@manchester.ac.uk
ꢀ
There are no known specific chemical inhibitors of
TcTS, nor drugs known to act against it. Thus, chemical
Present address: Johnson & Johnson Pharmaceutical Research &
Development, Turnhoutseweg 30, 2340 Beerse, Belgium.
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.12.024

J. Neres et al. / Bioorg. Med. Chem. 15 (2007) 2106–2119
2107
evidence of the enzyme as a target is lacking, and the
present contribution reports initial studies to discover
novel inhibitory molecular architectures based on the
availability of the crystal structure for this enzyme.10
The crystal structure of TcTS shows two domains; the
N-terminal catalytic domain is connected through a long
a-helix to a C-terminal, lectin-like domain. The active
site shows several of the conserved features of microbial
sialidases, the main features being the arginine triad that
binds the carboxylate group and a hydrophobic pocket
that binds the N-acetyl group in sialic acid.10
Away from complex, sugar frameworks, the only report
of TcTS inhibitors of which we are aware is pyridoxal
phosphate (7, non-competitive inhibition, Ki 7.3 mM).21
It has been pointed out that although strong inhibitors
have been discovered against Influenza virus enzyme
based on DANA, inhibitor design against the sialidases
or trans-sialidases of bacteria or trypanosomes has been
much less straightforward.22 In view of this we now re-
port the first generation of simple, non-sugar based
inhibitors of TcTS that may lead to biological tools or
potential leads for drug design. We address at this stage
the sialic acid-binding site in TcTS.
The few inhibitors reported for TcTS are weak and have
complex chemical structures. 2-Deoxy-2,3-didehydro-D-
N-acetylneuraminic acid (DANA, 1, Fig. 1), a potent
inhibitor of Influenza neuraminidase, inhibits TcTS with
a reported Ki of 12.3 mM.14 The GM3 ganglioside 2 is a
good donor substrate for TcTS, but when the sialic acid
residue is modified, namely at C4 (deoxy or methoxy),
C7 (deoxy) and C8 (deoxy or methoxy), it becomes a
partial inhibitor of the trans-sialidase reaction, at rela-
tively high inhibitor/substrate ratios.13
One initial approach was to use a framework that had
proved successful in the inhibition of Influenza virus
neuraminidase.23–27 This involved the replacement of
the tetrahydropyran ring of sialic acid by simpler cyclic
structures such as benzene or pyridine. With this ap-
proach, the relative chemical sensitivity and complex
stereochemistry of the sialic acid or DANA-derived
class of inhibitors is avoided, and one can begin to ad-
dress issues such as bioavailability and other biologically
important molecular properties. The compounds detect-
ed in this way also serve as initial frameworks to subse-
quently decorate with acceptor site ligand structures. We
report TcTS inhibition data for a series of benzoic acid
and pyridine-carboxylic acid derivatives (I and II, full
structures are given in Tables 1 and 2, respectively),
along with other carboxylic acid frameworks.
2,3-Difluorosialic acid (3) inactivates TcTS time-depen-
dently, through a covalent bond with the hydroxyl
group of Tyr342. However, complete inactivation re-
quires very high concentrations (20 mM) and the en-
zyme spontaneously recovers activity after removal of
excess inactivator.15 3-Fluoro-sialosyl fluoride also
covalently labels tyrosine for the sialidase from
T. rangeli (ꢀ70% sequence identity for 640 aminoac-
ids).16 Although the enzymes from T. cruzi and
T. rangeli show some similar features they behave dis-
tinctly from each other in their structural acceptance
of various inhibitor frameworks.17 Lactitol (4) and other
lactose (acceptor substrate) analogues were reported to
inhibit TcTS activity towards conventional substrates
both in vitro and in vivo, again at relatively high concen-
trations of lactitol (mM range). However, these com-
pounds do not inhibit the catalytic activity of TcTS
but act as lpreferential acceptors when compared with
the conventional b-galactosides.18,19 Recently, two
2. Results
2.1. Molecular modelling
cyclohexenephosphonate monoalkyl esters
4.7 mM) and 6 (I50 = 5.7 mM) were also reported as
5 (I50 =
2.1.1. Structure-based de novo design. The GRID
program28 was used to identify favourable energy
weak inhibitors.20
Figure 1. Structure of reported TcTS inhibitors.

2108
J. Neres et al. / Bioorg. Med. Chem. 15 (2007) 2106–2119
Table 1. Inhibition of TcTS by benzoic acid derivatives I
R1
O
R2
HO
R3
I
Code
R1
R2
R3
Activity
% inhibition at 1 mM ( SD)
I50 (mM)
28
29
30
31a
32a
33
34
35
36
37
38
39
19
18
8
H
CH2OH
NHCOCH3
NHCOCH3
NHCOCH3
NHCOCH3
NHCOCH3
NHCOCH3
NHCOCH3
NHCOCH3
NHCOCH3
NHCOCH3
CONHCH3
NHSOCH3
NHCOCH2Ph
NHCOCH2Ph
NHCH2Ph
H
H
H
H
H
H
H
H
H
H
0
—
—
0.54
—
—
—
—
—
—
—
—
0.76
—
1.2
—
—
—
—
—
—
—
—
—
—
CH2CH2OH
CH2CH(OH)CH2OH
CH2CH(OH)CH2OH
CH2CH2NH2
CH2CONH2
CH@NOH
N@C(NH2)2
N@C(NH2)2
N@C(NH2)2
N@C(NH2)2
NH2
16
17
0
5
2
25
0
5
24
41
0
6
7
CH2CH2OH
H
—
29
—
54 6b
H
9
H
N@C(NH2)2
N@C(NH2)2
N@C(NH2)2
N@C(NH2)2
H
H
H
9
c
4
40
41
42
43
44
45
46
47
CH2CH2NH2
CH2OH
CH(OH)CH2NH2
C@NOH
N@C(NH2)2
C@NOH
CH2CH2OH
H
NHSO2CH3
NHSO2CH3
NHSO2CH3
H
11
17
4
2
7
N@C(NH2)2
N@C(NH2)2
N@C(NH2)2
N@C(NH2)2
H
46
d
H
NH2
35
0
4
0
NHCOCH2
N
N
O
48
49
NHCOCH3
NH2
H
H
—
—
0.58
0.74
O
N
NHCH2
24
25
27
N@C(NH2)2
NH2
NHCOCH2NH2
SCH2Ph
SCH2Ph
SCH2Ph
H
H
H
43 6e
—
—
—
0.70
1.0
a Compounds 31 and 32 are enantiomers.
b Concentration tested was 0.5 mM.
c Apparent activation (19% increase in activity).
d Idem (31%).
e Concentration tested was 0.4 mM.
Table 2. Inhibition of TcTS by pyridine-2-carboxylic acid derivatives II
R1
R2
R3
O
N
HO
II
Code
R1
R2
R3
Activity
% inhibition at 1 mM ( SD)
I50 (mM)
50
51
52
53
54
H
NHCOCH3
NHCOCH3
NHCOCH3
NHSO2CH3
NHCSCH3
H
22
14
—
6
6
—
—
H
NH2
H
NH2
H
0.44
—
H
25 12
—
H
H
0.54

J. Neres et al. / Bioorg. Med. Chem. 15 (2007) 2106–2119
2109
interaction regions in the active site between different
functional group probes. As expected, the very strong
interaction (ꢁ19 kcal/mol) of the carboxylate probe
was detected in the region close to the arginine triad,
as well as a favourable interaction with the cationic side
chain of Arg93, as shown in Figure 2A where the struc-
ture of 2-deoxy-2,3-didehydro-D-N-acetylneuraminic
acid (DANA) is superimposed in the crystallographic
conformation in complex with TcTS.10 The hydropho-
bic pocket formed by Trp120, Tyr113 and Val95 was
clearly shown by various hydrophobic probes, including
the methyl probe (Fig. 2B), and the DRY (hydrophobic
probe) or the aromatic CH probe (not shown). This is
the region which accommodates the N-acetyl group of
DANA or sialic acid. Free space is available in the
hydrophobic pocket, apparently enough to enclose
groups as large as a phenyl ring.
These results indicated target regions for positively
charged groups, such as guanidino or protonated ami-
no-acylamino groups (e.g., glycylamino), and also for
hydrophobic groups to fit into the hydrophobic pocket.
Benzoic acid derivatives were designed, replacing the su-
gar ring of sialic acid by a more synthetically accessible
benzene ring, which would allow easier variation of the
different substituent groups. This approach had proven
successful for Influenza neuraminidase, with several
benzoic acid-based sialic acid mimetics inhibiting the en-
zyme with I50 values in the low micromolar range, some
even stronger.23,29,30 Therefore, benzoic acid-derived
structures were designed to contain these two functions
meta and para to the carboxylate group. The structures
of the designed compounds and all other compounds
tested during the present work were docked into the ac-
tive site using DOCK 4.0,31 with ligand flexibility. Gen-
erally, the functional groups in the top-ranked docked
poses obtained corresponded to the respective regions
predicted by the GRID program.
The positively charged amidine probe, like other proton-
ated nitrogen probes, gave essentially two regions with a
strong interaction energy of ꢁ13 kcal/mol, between res-
idues Glu230 and Asp96, and near Asp51 (Fig. 2C).
Favourable binding positions at approximately
ꢁ10 kcal/mol (Fig. 2D) were predicted for neutral NH
or NH2 probes in several regions of the active site,
namely close to residues Glu230, Asp96 and Gln195
(binding region for the glycerol chain of DANA).
Figure 3 shows the docked structures of 25 and 29 in the
active site. The benzyl group of 25 was placed inside the
hydrophobic pocket, adopting the favourable position
predicted by GRID, and the amino group was hydro-
gen-bonded to both Asp96 and Glu230 (Fig. 3A). Dock-
ing of 29 gave a pose where the hydroxyl group was
Figure 2. Favourable interaction energy surfaces (shown in green) obtained from the GRID program in the active site of TcTS. (A) Carboxylate
probe (ꢁ19.0 kcal/mol) also showing DANA (crystallographic pose;10) (B) methyl probe (ꢁ4.0 kcal/mol); (C) neutral sp3 NH probe (ꢁ10.5 kcal/mol);
(D) cationic amidine probe (ꢁ13.5 kcal/mol).

2110
J. Neres et al. / Bioorg. Med. Chem. 15 (2007) 2106–2119
Scheme 1. The methyl ester 10 was prepared to avoid
side-reactions of the carboxylate group. Compound 11
was successfully synthesised in good yield (77%) by
refluxing 10 in DMF for 70 h in the presence of ex-
cess benzyl bromide, even though the amine in the
starting material was strongly deactivated by the o-ni-
tro and p-carboxylic ester. Synthesis of 15 by reacting
10 with phenylacetyl chloride was more straightfor-
ward. Reduction of the nitro groups of 11 and 15
proceeded without problem using transfer hydrogena-
tion, following the procedure described by Singh
et al.32 Whereas 17 was synthesised by reaction of
16 with cyanamide and HCl with 52% yield, the syn-
thesis of 14 was performed with the strerically more
demanding reagent, 1,3-bis(tert-butoxycarbonyl)-2-
methyl-2-thiopseudourea, due to the presence of the
additional benzyl-substituted amine group in 12. The
t-Boc groups of 13 were efficiently removed using tri-
ethylsilane and TFA in reasonable yield (68%),
according to a method previously developed in our
laboratory.33 Hydrolysis of the methyl esters in
NaOH yielded the target compounds in the neutral
form (zwitterionic for 8 and 18).
Synthesis of 24, 25 and 27 (Scheme 2) started from the
methyl ester of 4-chloro-3-nitrobenzoic acid (20) with
introduction of the benzylmercaptyl group. Compound
24 was obtained after introduction of the guanidino
group using the conditions used for 18, followed by
methyl ester hydrolysis. Fmoc-protected glycine was
used to synthesise 26 from 22 in the presence of DCC.
Fmoc group removal with diethylamine followed by es-
ter hydrolysis gave 27.
Figure 3. Docked conformation of compounds 25 (A), superimposed
with the GRID favourable contour energy surface for the aromatic CH
probe at ꢁ3.7 kcal/mol) and 29 (B) in the active site of TcTS.
hydrogen-bonded to both Gln195 and Glu230 and the
amide nitrogen with Asp96 (Fig. 3B). Commonly to
the observed for most benzoic acid derivatives studied
in this work, in both structures the carboxylate group
was in close interaction with the three arginine residues
in the triad.
2.3. TcTS inhibition
TcTS inhibition screening results including I50 values for
a series of substituted benzoic acid and pyridinecarboxy-
lic acid derivatives are given in Tables 1 and 2, respec-
tively. Inhibition results and structures for other
compounds tested, including furosemide (55), are shown
in Figure 5. The percentage inhibition at 1 mM concen-
tration is the average of at least three independent
experiments.
2.1.2. Virtual screening. Virtual screening of the Asinex
database using DOCK produced numerous structurally
diverse hits potentially containing negatively charged
groups, such as carboxylate, sulfonamide and sulfone
functions, predominantly positioned close to the argi-
nine triad. Many also contained a positively charged
group, usually interacting with one of the anionic resi-
dues in the active site, and/or a hydrophobic moiety,
which was located in the hydrophobic pocket. Thus,
the results of the screen are in line with the types of
interactions seen in the preceding modelling work. Thir-
ty-two highly ranked compounds with varied structural
features were purchased from Asinex, with special atten-
tion given to benzoic acid and benzimidazole deriva-
tives. However, only five of these compounds inhibited
TcTS to some extent at 1 mM, namely 48, 49 (Table 1)
and 58–60 (Fig. 5). Results from virtual screening hits
other than those shown in Table 1 (47–49) and Figure
5 (57–60) are not shown.
The inhibition by 29 was studied in greater detail to
determine the inhibition pattern of diagnostic plots.
The data are shown in Figure 4A–C. The lines, drawn
by linear least squares regression analysis, are not fit-
ted to either mixed or non-competitive kinetic inhibi-
tion models for this display. Fitting the data by
non-linear least squares analysis to the respective
equations for non-competitive and mixed inhibition
models gave the following results, which were not
significantly different, according to the statistical F
test, performed in GRAFIT.34 Non-competitive
inhibition: Ki 0.33 0.03 mM, Km 0.73 0.11, Vmax
10,806 870 fluorescence units (reduced v2 71,671);
mixed inhibition: Ki 0.37 0.17 mM, K0i 0.31
0.13 mM, Km 0.75 0.17, Vmax 11,011 1352
fluorescence units (reduced v2 75,068). Either model
confirms the weak inhibition with Ki values around
300 lM.
2.2. Synthesis
Benzoic acids 8, 18 and 19 were synthesised from
4-amino-3-nitrobenzoic
acid
(9),
according
to

J. Neres et al. / Bioorg. Med. Chem. 15 (2007) 2106–2119
2111
Scheme 1. Reagents and conditions: (a) MeOH, concd H2SO4, D; (b) PhCH2Br, DMF, D; (c) PhCH2COCl, CHCl3, D; (d) H2NNH2, 10% Pd/C,
EtOH; (e) H2NCN, concd HCl, EtOAc, D; (f) 1—1 M NaOH; 2—HCl; (g) H3CSC[@NCO2C(CH3)3]NHCO2C(CH3)3, Et3N, HgCl2, DMF; (h) TFA,
Et3SiH, DCM.
3. Discussion
neuraminidase, with no apparent correlation between
the two. For example, the stronger Influenza enzyme
inhibitors 36, 38, 43 and 51 had I50 values of 2.5, 5, 80
and 70 lM, respectively, against this enzyme.23,24 How-
ever with TcTS, inhibition was much weaker, with 36
and 43 inhibiting 40–50% of the activity at 1 mM and
51 only 14% inhibition at the same concentration, and
38 with an I50 of 0.76 mM. Conversely, stronger TcTS
inhibitors 29 and 52, with I50 of approximately
0.5 mM, were weaker inhibitors of the Influenza en-
zyme.23,24 Compound 54 had a similar I50 for both en-
zymes (ꢀ0.4–0.5 mM).24 In general, the strong
inhibition that some aromatic carboxylates could
achieve for the Influenza enzyme (with I50 values as
low as 2.5 lM for 36) was not observed for TcTS. The
strongest TcTS inhibitors here described only showed
I50 values in 0.4–1 mM region. The remaining aromatic
Sialic acid derivatives/mimetics were highly successful in
the design of inhibitors for the related Influenza neur-
aminidase, with the discovery of potent inhibitors, two
of which are now in the market for the treatment of
Influenza virus infections.35,36 However, we have found
in the present studies that the behaviour of the enzymes
from Influenza and trypanosome is very different in re-
spect of recognition of inhibitor structures.
In the present work, we used a sialic acid hydrolysis as-
say, previously validated in our laboratory,11 to assess
the inhibition of TcTS by the described compounds.
We found a substantial difference in the behaviours of
the substituted aromatic carboxylic acids from Tables
1 and 2 towards TcTS compared with the Influenza

2112
J. Neres et al. / Bioorg. Med. Chem. 15 (2007) 2106–2119
Scheme 2. Reagents and conditions: (a) PhCH2SH, Na2CO3, D; (b) Fe, AcOH, EtOH, D; (c) 1—1 M NaOH; 2—HCl; (d) Fmoc-Gly-OH, DCC,
DMF; (e) Et2NH, EtOAc, D; (f) H2NCN, concd HCl, EtOAc, D.
0.4
0.3
0.2
0.1
0.05
0
0.1
0.3
0.6
1
0.0023
0.0018
0.0013
0.0008
0.0003
0.1
0.3
0.6
1
0.002
0.0015
0.001
0.0005
0
0.0004
0.0003
0.0002
0.0001
0.0000
A
B
C
-0.4 -0.3 -0.2 -0.1
0
0.1 0.2 0.3 0.4 0.5
0
0.1
0.2
0.3
0.4
-2
-0.0002
0
2
4
6
8
10
12
-0.0005
[I] mM
1/[MuNANA]
[I] mM
Figure 4. Graphical determination of the type of inhibition for compound 29. (A) Dixon plot; (B) Lineweaver-burk plot; (C) Cornish-Bowden plot.
Points are experimental; lines are by linear least squares regression analysis.
compounds are also weak inhibitors of Influenza neur-
aminidase, with I50 above 0.1 mM, in most cases even
higher than 1 mM.23,24,37
(33) or a guanidino group (36). Replacing R2 = NHC-
OCH3 of 36 by NHCOCH2Ph (18) improved the per-
centage inhibition at 1 mM slightly, and going to
R2 = NHCH2Ph (8) substantially weakened inhibition,
although this could be recovered by the SCH2Ph
replacement (24). For the R2 = SCH2Ph family, replac-
ing the R1 guanidino group of 24 by NH2 (25) or
Regarding TcTS inhibition of compounds in Table 1
with R2 = NHCOCH3, it is clear that we have been un-
able to recruit the potential cationic binding site of Fig-
þ
þ
ure 2C (Glu230 and Asp96) either using CH2CH2NH3
NHCOCH2NH3 (27) probably weakened the inhibi-

J. Neres et al. / Bioorg. Med. Chem. 15 (2007) 2106–2119
2113
NH2
HN
O
HN
H
N
N
OH
HOOC
Cl
N
N
HOOC
HOOC
SO2NH2
55
56
57
I50 0.67 mM
17 5% inhibition at 1 mM
No inhibition at 1 mM
O
HN
HO
O
O
O
OH
O
OH
HO
H
N
NH
HOOC
HOOC
O
O
S
HN
O
NH2
61
No inhibition at 1 mM
O
NH2
58
31 2% inhibition at 1 mM
59
14 8% inhibition at 1 mM
60
20 2% inhibition at 1 mM
Figure 5. Structures and TcTS inhibition of other compounds tested.
tion slightly (with I50 values of 0.70 mM for 25 and
1.0 mM for 27).
acid hydrolysis for neuraminidase and sialic acid trans-
fer for TcTS.10 Another remarkable difference is the
dynamic behaviour of TcTS during substrate binding
and catalysis, where occupancy of the sialic acid-binding
site was demonstrated to promote a conformational
change that modulates the affinity for the acceptor sugar
substrate.10 Similar conformational changes are expect-
ed to take place upon inhibitor binding to the sialic acid-
binding site. Although the nature and extent of these
inhibitor-induced changes cannot be assessed in the ab-
sence of experimental structural information on TcTS-
inhibitor complexes, it is plausible to suggest that they
could be responsible for the lower sensitivity of trans-
sialidase for the general sialidase inhibitor DANA14
and might explain, at least in part, why it is difficult to
obtain good TcTS inhibitors.
The inhibition by 29 is consistent with either non-com-
petitive inhibition (Ki 0.33 0.03 mM) or mixed inhibi-
tion (Ki 0.37 0.17 mM, K0i 0.31 0.13 mM) with really
very little to discriminate the models. In either case the
inhibition is weak with Ki values around 300 lM only.
The introduction of a pyridine (Table 2) instead of a
benzene nucleus (Table 1) made little difference to the
strength of inhibition of TcTS (compare 28 and 50).
Compound 51 inhibits the Influenza enzyme with an
I50 of 0.07 mM24 but is much weaker with TcTS. Com-
pound 61 is very strong against the Influenza enzyme
(with I50 in the low nanomolar range), indeed reaching
phase III clinical trials,38 but was only a poor inhibitor
of TcTS.
In particular, both the low accessibility and the flexible
nature of the active site may account for the low activity
of the aromatic carboxylate derivatives (Tables 1 and 2),
since their docked structures fitted well in the active site
of TcTS, with the guanidino and other positively
charged groups interacting with the side chains of
Glu230 and Asp96. Furthermore, these structures do
not explore the acceptor substrate binding site in TcTS,
which has been discussed as a potential target for TcTS
inhibition and is targeted by lactitol and its derivatives,
which prevent the sialylation of lactose or lactosamine
by TcTS.10,18,22
From the above data, it is clear that the Influenza and
TcTS enzymes respond very differently to inhibitor
structure. This can be explained not only by the struc-
tural differences between the two enzymes, but also by
a different dynamic behaviour of sialidases and trans-
sialidases.14,17,39 Comparison of the crystal structures
of Influenza neuraminidase (1F8B40) and TcTS shows
many common features in the sialic acid-binding region,
namely the arginine triad and Asp/Glu residues in
approximately the same regions of the active site. How-
ever, the active site of the Influenza enzyme contains a
greater number of negatively charged residues
(Glu119, Asp151, Glu227, Glu276 and Glu277) when
compared with TcTS (Asp59, Asp96, Glu230), plus
there is one extra arginine residue (Arg53) in the latter.
Furthermore, the active site of the Influenza enzyme
(and indeed those of all structurally characterized sialid-
ases, including the closely related T. rangeli enzyme) is
easily accessible, with a greater solvent exposure than
that in TcTS, where the active site is more hydrophobic.
This is probably one key feature that distinguishes the
different nature of the enzymatic activities, namely sialic
The clinically used diuretic compound 55 (furosemide,
Lasixꢁ) also inhibits TcTS, but, with an I50 of
0.67 mM, any TcTS-based dose would be unacceptable
in man. However, the furan ring region of 55 is docked
against the acceptor substrate site in our docking analy-
sis, thus providing an extra interaction with the active
site compared with the other aromatic derivatives; fur-
ther exploration of the SAR in this region is desirable.
In conclusion, the range of aromatic derivatives evaluat-
ed in the present work as TcTS inhibitors has shown the

2114
J. Neres et al. / Bioorg. Med. Chem. 15 (2007) 2106–2119
possibility of inhibiting TcTS with this framework.
Some of these compounds inhibit TcTS with I50 values
in the high lM range (Ki value of ꢀ300 lM for com-
pound 29), and while this is not of itself strong inhibi-
tion, they are the strongest inhibitors reported to date,
with the exception of the extremely complex modified
GM3 ganglioside derivatives. Inhibition of TcTS by
furosemide and the respective docked structure show
that there is a possibility of including a substituent ortho
to the carboxylate group in the aromatic ring, which
binds the acceptor substrate binding site, thus offering
room for improvement.
Miracle accessory. Electron ionisation (EI) and chemical
ionisation (CI) mass spectra were recorded on a
Micromass Trio 2000. Electrospray ionisation (ES) mass
spectra were obtained using a Micromass Platform.
High-resolution mass spectra were recorded using a
Thermo Finnigan Mat 95 XP spectrometer. Elemental
analyses were recorded on an EA 1108 Elemental
Analyser (Carlo Erba Instruments) and were within
0.4% of the calculated values unless otherwise noted.
Compounds 29–46, 50–54, 56 and 61 were from previ-
ous studies by BioCryst Pharmaceuticals Inc. working
on Influenza neuraminidase inhibitors.23,24,37,38 Com-
pounds 28 and 55 were purchased from Sigma–Aldrich
(Gillingham, Dorset, UK) and 47–49 and 57–60 from
Asinex Ltd, Moscow, Russia. Compound 10 was syn-
thesised following a literature procedure43 and the
remaining compounds were synthesised as described
below.
4. Experimental
4.1. Molecular modelling
The crystal structure of the TcTS–DANA complex10
(PDB Accession code 1MS8) was used. Hydrogens were
added using WHATIF,41 with optimization of hydro-
gen bonds and determination of correct protonation
state of histidine residues. The GREATER module of
the GRID program28 was used with default settings
4.2.1. Methyl 4-benzylamino-3-nitrobenzoate (11). A
mixture of 10 (8.0 g, 41 mmol), benzyl bromide
(13.5 mL, 113 mmol) and DMF (15 mL) was heated at
70 ꢂC for 3 days. The reaction mixture was diluted with
CH2Cl2 (30 mL) and 1,4-diazabicyclo[2,2,2]octane
(15.7 g, 94 mmol) added slowly with stirring at room
temperature. Stirring was continued for 2 h and solvent
was then evaporated in vacuo. The resulting residue was
partitioned between EtOAc (100 mL) and 1 M citric
acid (94 mL). The aqueous layer was washed with
EtOAc (70 mL), the organic layers combined, dried over
anhydrous Na2SO4 and evaporated to dryness in vacuo
to give 11 (9.3 g, 79%) as a yellow solid: mp 98–100 ꢂC
(recryst from EtOH), lit. 98 ꢂC.44 1H NMR (DMSO-
d6): dH 3.81 (s, 3 H, OCH3), 4.70 (d, J = 6.2 Hz, 2H,
C6H5CH2), 7.00 (d, J = 9.1 Hz, 1H, H5), 7.24–7.40 (m,
5H, C6H5CH2), 7.9 (dd, J = 2.0 and 9.1 Hz, 1H, H6),
8.64 (d, J = 2.1 Hz, 1H, H2), 9.10* (t, J = 6.1 Hz, 1H,
NH). 13C NMR (DMSO-d6): dC 46.1 (C6H5CH2), 52.4
(OCH3), 115.6 (C5), 116.4 (C1), 127.3 (C20 and C60),
127.5 and 128.7 (C2 and C40), 129.0 (C30 and C50),
131.2 (C3), 135.9 (C6), 138.2 (C10), 147.6 (C4), 165.1
(COOCH3). MS (CI) m/z 287 ([M+H]+, 100). HR-EI-
MS (m/z) [M]+ calcd for C15H14N2O4: 286.0954. Found:
286.0960. Anal. Calcd for C15H14N2O4: C, 62.9; H, 4.9;
N, 9.8. Found: C, 63.0; H, 4.6; N, 9.6.
˚
(grid resolution of 4 planes per A) to screen the active
site for energetically favourable binding regions for dif-
ferent functional group probes, thus giving further indi-
cations about the regions to target with newly designed
inhibitors. Virtual screening on the Asinex Gold
(ꢀ200,000 compounds) and Platinum (ꢀ100,000 com-
pounds) databases (Asinex Ltd, Moscow, Russia) was
performed with DOCK 4.0,31 in two steps: the first step
was rigid-body docking of all structures in the Asinex
databases, with ligand minimization, testing a maxi-
mum of 1000 orientations for each molecule followed
by minimization of the ligand docked pose. The DOCK
4.0 intermolecular scoring function was used to rank
the ligands.31 The top 320 scoring hits were then re-
docked, now allowing flexibility of the ligand structure,
using the torsion drive option for the conformational
search and testing a maximum of 1000 orientations.
Selection of compounds for in vitro inhibition assays
was performed by visual inspection of the docked con-
formations resulting from the flexible-docking, using
Sybyl 6.8.42 Both virtual screening and de novo design
targeted the sialic acid-binding site in TcTS. De novo
design focused on benzoic acid derivatives with differ-
ent substituents, as sialic acid mimetics. All compounds
tested were docked in the active site using the flexible-
docking algorithm as described for the second step of
the virtual screening.
4.2.2. Methyl 3-amino-4-benzylaminobenzoate (12). A
suspension of 11 (9 g, 31.4 mmol) in EtOH (25 mL)
was added to a mixture of 10% Pd–C (3 g) and 5%
HCl (7 mL). Hydrazine hydrate (85%, 4.4 mL,
120 mmol) dissolved in EtOH (10 mL) was then added
drop-wise and the resulting mixture stirred for 2 h at
room temperature. The reaction mixture was diluted
with EtOH and EtOAc (20 mL of each), Pd–C was re-
moved by filtration through Celiteꢁ and the filtrate con-
centrated under vacuum. The resulting residue was
purified by column chromatography starting elution
with CH2Cl2 with an increasing gradient of CH2Cl2:
EtOAc. Fractions containing the product were
combined and the solvent removed in vacuo. The
residue obtained was recrystallised from MeOH yielding
12 as off-white crystals (4.9 g, 61%): mp 129–130 ꢂC, lit.
4.2. Chemistry: general procedures
Melting points, recorded in open capillary tubes in a
Griffin melting point apparatus, and are uncorrected.
1H NMR and 13C NMR spectra were obtained in
CDCl3 or DMSO-d6 with Me4Si as internal standard
on a Bruker Avance-300 Spectrometer operating at
300 MHz for 1H NMR and 75 MHz for 13C NMR.
Exchangeable protons indicated by an asterisk were
identified by D2O shake. IR spectra were recorded on
neat samples on a JASCO FT/IR 4100 with a PIKE/

J. Neres et al. / Bioorg. Med. Chem. 15 (2007) 2106–2119
2115
125 ꢂC.44 1H NMR (DMSO-d6): dH 3.70 (s, 3H, OCH3),
4.38 (d, J = 5.8 Hz, 2H, C6H5CH2NH), 4.85* (s, 2H,
NH2), 5.96* (br t, J = 5.7 Hz, 1H, C6H5CH2NH), 6.35
(d, J = 8.3 Hz, 1H, H5), 7.10 (dd, J = 1.9 and 8.3 Hz,
1H, H6), 7.19 (d, J = 2.0 Hz, 1H, H2), 7.21–7.36 (m,
5H, C6H5CH2NH). 13C NMR (DMSO-d6): dC 46.6
(C6H5CH2NH), 51.5 (OCH3), 108.9 and 114.8 (C2 and
C5), 117.3 (C1), 120.9 (C6), 127.1 (C40), 127.5 (C20,
C60), 128.7 (C30 and C50), 134.4 (C3), 139.9 and 140.6
(C4 and C10), 167.1 (COOCH3). MS (ES+) m/z 311
([M+Na+MeOH]+, 100), 279 ([M+Na]+, 10), 257
([M+H]+, 23). Anal. Calcd for C15H16N2O2: C, 70.3;
H, 6.3; N, 10.9. Found: C, 69.9; H, 6.2; N, 10.9.
126.7 (C20, C40 and C60), 128.2 (C30 and C50), 130.3
and 130.9 (C2 and C6), 138.9 (C10), 149.1 (C4), 156.8
(NH@C(NH2)2), 165.7 (COOCH3). MS (ES+) m/z 299
([M+H]+, 100). HR-ES-MS (m/z) [M+H]+ calcd for
C16H19N4O2: 299.1503. Found: 299.1500.
4.2.5. 4-Benzylamino-3-guanidino-benzoic acid (8). A
mixture of 14 (80 mg, 0.19 mmol), 1 M NaOH (10 mL)
and THF (1 mL) was stirred at 40 ꢂC for 3 h. The resulting
solution was concentrated in vacuo, diluted with water
and the pH was adjusted to ꢀ7 with 1 N HCl. The precip-
itate that formed was collected by filtration and dried,
yielding 8 as a white solid (0.040 g, 73%). 1H NMR
(DMSO-d6): dH 4.37 (br d, 2H, C6H5CH2NH), 6.14* (br
s, 1H, C6H5CH2NH), 6.44 (d, J = 8.5 Hz, 1H, H5), 7.19
(t, J = 7.3 Hz, 1H, H40), 7.28 (t, J = 7.3 Hz, 2H, H30 and
H50), 7.40 (d, J = 7.3 Hz, 2H, H20 and H60), 7.53–7.56
(m, 2H, H2 and H6), 7.92* (br s, 4H, NH@C(NH2)2),
11.32* (br s, 1 H, NH@C(NH2)2). 13C NMR (DMSO-
d6): dC 45.9 (C6H5CH2), 109.7 (C5), 119.9 and 126.4 (C1
and C3), 126.5 (C40), 126.9 (C20 and C60), 128.1 (C30
and C50), 128.6 and 129.2 (C2 and C6), 139.6 (C10),
145.4 (C4), 157.3 (NH@C(NH2)2), 170.9 (COOH). MS
(ES+) m/z 285 ([M+H]+, 100), 308 ([M+Na]+, 48). HR-
ES-MS (m/z) [M+H]+ calcd for C15H17N4O2: 285.1346.
Found: 285.1347.
4.2.3. Methyl 4-benzylamino-3-[N0,N00-di(tert-butoxycar-
bonyl)guanidino]benzoate (13). To an ice-cold suspension
of 12 (1 g, 3.9 mmol) in dry DMF (7 mL) were added
with stirring triethylamine (1.1 mL, 7.8 mmol), 1,3-bis-
(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea (1.7 g,
5.9 mmol) and HgCl2 (1.6 g, 5.9 mmol). The ice bath
was removed after 20 min and stirring continued at
room temperature for 7 h. The reaction mixture was
diluted with EtOAc (20 mL) and filtered through Cel-
iteꢁ. Solvent was removed in vacuo and the residue ob-
tained purified by column chromatography, starting
with CH2Cl2 with an increasing gradient of CH2Cl2:
EtOAc. Fractions containing the product were com-
bined and solvent was removed in vacuo to give 13 as
4.2.6. Methyl 3-nitro-4-phenylacetylaminobenzoate (15).
A mixture of 10 (1 g, 5.1 mmol) and phenylacetyl chlo-
ride (1.35 mL, 10.2 mmol) in CHCl3 (2 mL) was stirred
at 100 ꢂC for 2 h. Acetonitrile (6 mL) was added to the
reaction mixture, which was heated until the suspended
solid dissolved. Water (1.5 mL) was added and the solu-
tion partitioned between CH2Cl2 (15 mL) and saturated
NaHCO3 solution (15 mL). The organic layer was sepa-
rated, washed with water (2· 15 mL), dried over anhy-
drous Na2SO4 and solvent removed in vacuo. The
residue was recrystallised from MeOH, yielding 15
(0.96 g, 60%) as a yellow solid: mp 129–130 ꢂC. 1H
NMR (CDCl3): dH 3.86 (s, 2H, CH2C6H5), 3.94 (s,
3H, OCH3), 7.35–7.47 (m, 5H, CH2C6H5), 8.25 (dd,
J = 2.0 and 8.9 Hz, 1H, H6), 8.83 (d, J = 2.0 Hz, 1H,
H2), 8.94 (d, J = 8.9 Hz, 1H, H5), 10.48* (s, 1H,
CONH). 13C NMR (CDCl3): dC 46.30 (CH2C6H5),
53.02 (OCH3), 121.92 (C5), 125.42 (C1), 127.86 and
128.60 (C2 and C40), 129.88 (C30 and C50), 130.07
(C20 and C60), 133.21 and 135.93 (C3 and C4), 136.81
(C5), 138.52 (C10), 165.04 (COOCH3), 170.87 (CONH).
MS (CI) m/z 315 ([M+H]+, 100). HR-EI-MS (m/z) [M]+
calcd for C16H14N2O5: 314.0903. Found: 314.0908.
Anal. Calcd for C16H14N2O5: C, 61.1; H, 4.5; N, 8.9.
Found: C, 61.2; H, 4.3; N, 8.9.
1
a white solid (1.2 g, 62%): mp 135–138 ꢂC. H NMR
(CDCl3): dH 1.44 (s, 9H, C(CH3)3), 1.54 (s, 9H,
C(CH3)3), 3.84 (s, 3H, OCH3), 4.43 (d, J = 5.8 Hz, 2H,
C6H5CH2NH), 5.64* (t, J = 5.7 Hz, 1H, C6H5CH2NH),
6.64 (d, J = 8.4 Hz, 1H, H5), 7.25–7.37 (m, 5H,
C6H5CH2NH), 7.76–7.81 (m, 2H, H2 and H6), 9.97*
(s,
1H,
NHC(@NR)NHR),
11.61*
(s,
1H,
NHC(@NR)NHR). 13C NMR (CDCl3): dC 28.5 and
28.6 [2· (CH3)3], 48.1 (C6H5CH2), 52.1 (OCH3), 80.2
and 84.4 [2· C(CH3)3], 112.2 (C5), 119.0 (C1), 122.2
(C3), 127.6 (C40), 127.8 (C20 and C60), 128.7 (C6),
129.1 (C30 and C50), 130.5 (C3), 138.8 (C10), 147.7
(C4), 153.7 and 155.0 (2· C@O t-Boc), 163.5
(NHC(@NR)NHR), 167.2 (COOCH3). MS (ES+) m/z
499 ([M+H]+, 100). Anal. Calcd for C26H34N4O6Æ0.25-
H2O: C, 62.1; H, 6.9; N, 11.1. Found: C, 62.2; H, 6.5;
N, 11.1.
4.2.4. Methyl 4-benzylamino-3-guanidino-benzoate (14).
To a solution of 13 (250 mg, 0.5 mmol) in dichlorometh-
ane (2 mL) were added trifluoroacetic acid (1 mL,
13 mmol) and triethylsilane (0.4 mL, 2.5 mmol). The
reaction mixture was stirred at room temperature for
6 h with exclusion of moisture. Solvent was removed
in vacuo, the residue triturated with chloroform and col-
lected by filtration, and dried giving 14 as a white solid
(0.14 g, 68%): mp 119–121 ꢂC (recryst from MeOH/
4.2.7. Methyl 3-amino-4-phenylacetylaminobenzoate (16).
A suspension of 15 (0.7 g, 2.23 mmol) in EtOH (20 mL)
was added to a mixture of 10% Pd–C (0.7 g) and 5%
HCl (0.51 mL). Hydrazine hydrate (64%, 0.44 mL,
9.16 mmol) dissolved in ethanol (3 mL) was added
drop-wise, and the mixture stirred for 1 h at room tem-
perature, diluted with ethanol and ethyl acetate (20 mL
of each) and the Pd–C removed by filtration through
Celiteꢁ. The filtrate was evaporated in vacuo to give
16 as a white solid (0.52 g, 82%): mp 179–181 ꢂC (recryst
1
Et2O). H NMR (DMSO-d6): dH 3.75 (s, 3H, OCH3),
4.45 (d, J = 6.0 Hz, 2H, C6H5CH2NH), 6.61 (d,
J = 8.4 Hz, 1H, H5), 7.00* (t, J = 6.0 Hz, 1H,
C6H5CH2NH), 7.21–7.36 (m, 9H, C6H5 and NH@
C(NH2)2), 7.56 (d, J = 1.8 Hz, 1H, H2), 7.68 (dd,
J = 1.8 and 9.0 Hz, 1H, H6), 9.05* (s, 1H, NH@
C(NH2)2). 13C NMR (CDCl3): dC 45.4 (C6H5CH2),
51.4 (OCH3), 110.6 (C5), 116.2 and 118.5 (C1 and C3),

2116
J. Neres et al. / Bioorg. Med. Chem. 15 (2007) 2106–2119
1
from MeOH). H NMR (DMSO-d6): dH 3.70 (s, 2H,
CH2C6H5), 3.79 (s, 3H, OCH3), 5.22* (s, 2H, NH2),
7.15 (dd, J = 1.7 and 8.3 Hz, 1H, H6), 7.38 (d,
J = 1.7 Hz, 1H, H2), 7.48 (d, J = 8.3 Hz, 1H, H5),
7.24–7.35 (m, 5H, CH2C6H5), 9.46* (s, 1H, CONH).
13C NMR (DMSO-d6): dC 42.7 (CH2C6H5), 51.7
(OCH3), 116.3 and 117.2 (C2 and C6), 123.8 (C5),
126.2 (C1), 126.4 (C1 and C40), 127.7 (C4), 128.2 (C30
and C50), 129.1 (C20 and C60), 135.9 (C10), 140.8 (C3),
166.2 (COOCH3), 169.3 (CONH). MS (CI) m/z 285
([M+H]+, 49). HR-EI-MS (m/z) [M]+ calcd for
C16H16N2O3: 284.1161. Found: 284.1160. Anal. Calcd
for C16H16N2O3: C, 67.6; H, 5.7; N, 9.9. Found: C,
67.8; H, 5.5; N, 9.7.
dH 3.70 (s, 2H, C6H5CH2CONH), 5.18* (s, 2H, NH2),
7.12 (d, J = 8.2 Hz, 1H, H5), 7.23–7.34 (m, 6H,
H2 and C6H5CH2CONH), 7.43 (d, J = 8.2 Hz, 1H,
H6), 9.50* (s, 1H, C6H5CH2CONH), 12.56* (br s, 1H,
COOH). 13C NMR (DMSO-d6): dC 42.7 (C6H5COCH2),
116.6, 117.4 and 123.7 (C2, C5 and C6), 126.4 (C40),
127.2 and 127.8 (C1 and C4), 128.2 (C20 and C60),
129.0 (C30 and C50), 136.0 (C10), 140.7 (C3), 167.4
(C6H5CH2CONH), 169.2 (COOH). MS (ESꢁ) m/z 269
([MꢁH]ꢁ, 100). HR-EI-MS (m/z) [M+Na]+ calcd
for C15H14N2O3Na: 293.0897. Found: 293.0893.
4.2.11. Methyl 3-nitro-4-benzylmercaptobenzoate (21).
To a mixture of benzyl mercaptan (3.3 mL, 27.8 mmol),
Na2CO3 (3.25 g, 30.6 mmol) and water (10 mL) was
added methyl 4-chloro-3-nitrobenzoate (5 g, 23.2 mmol)
in ethanol (40 mL). The mixture was refluxed for 2 h and
diluted with water (40 mL). The solid thus formed was
filtered off, washed with n-hexane and dried, yielding
21 as a yellow solid (6.56 g, 93%): mp 136–138 ꢂC (re-
cryst from MeOH), lit. 138 ꢂC.45 1H NMR (DMSO-
d6): dH 3.90 (s, 3H, OCH3), 4.45 (s, 2H, C6H5CH2S),
7.28–7.40 (m, 3H, H20, H40 and H60), 7.47–7.50 (m,
2H, H30 and H50), 7.90 (d, J = 8.6 Hz, 1H, H5), 8.17
(dd, J = 1.9 and 8.6 Hz, 1H, H6), 8.64 (d, 1H,
J = 1.9 Hz, H2). 13C NMR (DMSO-d6): dC 36.0
(C6H5CH2S), 52.6 (OCH3), 126.1 (C1), 126.4, 127.6
and 127.8 (C2, C5 and C40), 128.6 (C20 and C60),
129.3 (C30 and C50), 133.4 (C6), 134.9 (C4), 142.8
(C10), 144.5 (C3) and 164.3 (COOCH3). MS (CI) m/z
321 ([M+NH4]+, 100), (EI) m/z 303 ([M]+, 21). Anal.
Calcd for C15H13NO4S: C, 59.4; H, 4.3; N, 4.6; S,
10.6. Found: C, 59.1; H, 4.3; N, 4.6; S, 10.3.
4.2.8. Methyl 3-guanidino-4-phenylacetylaminobenzoate
(17). A mixture of 16 (0.47 g, 1.65 mmol), cyanamide
(1.4 g, 33 mmol) and concd HCl (0.15 mL) in EtOAc
(15 mL) was refluxed for 6 h. The reaction mixture
was diluted with EtOAc (30 mL) and partitioned with
K2CO3 solution (15 mL, half of saturation concentra-
tion). The organic layer was washed with water, dried
over Na2SO4, filtered and the solvent evaporated in vac-
uo. The residue obtained was recrystallised from EtOH
yielding 17 (0.28 g, 52%) as a white solid: mp 182–
184 ꢂC. 1H NMR (DMSO-d6): dH 3.73 (s, 2H,
CH2C6H5), 3.77 (s, 3H, OCH3), 5.48* (s, 4H,
N@C(NH2)2), 7.24–7.40 (m, 6H, H6 and CH2C6H5),
7.46 (d, J = 1.9 Hz, 1H, H2), 8.23 (d, J = 8.4 Hz, 1H,
H5), 9.10* (s, 1H, CONH). 13C NMR (DMSO-d6): dC
43.98 (CH2C6H5), 51.56 (OCH3), 116.75, 121.18 and
121.21 (C2, C5 and C6), 123.65 (C1), 126.72 (C40),
128.50 (C30 and C50), 129.28 (C20 and C60), 135.25
and 136.11 (C4 and C10), 139.06 (C3), 154.39
(N@C(NH2)2), 166.35 (COOCH3), 168.62 (CONH).
MS (CI) m/z 327 ([M+H]+, 100), 267 ([MꢁNH@
C(NH2)2]+, 45). HR-EI-MS (m/z) [M]+ calcd for
C17H18N4O3: 326.1379. Found: 326.1386. Anal. Calcd
for C17H18N4O3Æ0.6H2O: C, 60.4; H, 6.0; N, 16.6.
Found: C, 60.2; H, 5.5; N, 16.9.
4.2.12. Methyl 3-amino-4-benzylmercaptobenzoate (22).
A solution of 21 (5 g, 16.5 mmol) in EtOH (10 mL) was
treated with iron dust (7.9 g, 137 mmol) and 20% aque-
ous acetic acid (2.8 mL) refluxed for 30 min with vigor-
ous stirring. The reaction mixture was diluted with
EtOAc (40 mL) and filtered over Celiteꢁ. The filtered
solution was washed with 5% aqueous NaHCO3
(40 mL), followed by water (2· 40 mL), and dried over
anhydrous Na2SO4. The solvent was evaporated in vac-
uo and the residue obtained was recrystallised from
MeOH to give 22 as an off-white solid (4.0 g, 88%): mp
76–77 ꢂC lit. 76–78 ꢂC.45 1H NMR (DMSO-d6): dH 3.79
(s, 3H, OCH3), 4.10 (s, 2H, C6H5CH2S), 5.46* (s, 2H,
NH2), 7.05 (dd, J = 1.9 and 9.0 Hz, 1H, H6), 7.19–7.28
(m, 6H, H5 and C6H5CH2S), 7.32 (d, J = 1.9 Hz, 1H,
H2). 13C NMR (DMSO-d6): dC 36.3 (C6H5CH2S), 51.8
(OCH3), 114.3 and 116.6 (C2 and C6), 122.7 (C4),
127.0 (C40), 128.2 (C20 and C60), 128.8 (C30 and C50),
128.9 (C1), 131.7 (C5), 137.4 (C10), 147.7 (C3), 166.3
(COOCH3). MS (CI) m/z 274 ([M+H]+, 100). Anal.
Calcd for C15H15NO2S: C, 65.9; H, 5.5; N, 5.1; S, 11.7.
Found: C, 66.0; H, 5.6; N, 5.1; S, 11.4.
4.2.9. 3-Guanidino-4-phenylacetylaminobenzoic acid (18).
A mixture of 17 (80 mg, 0.18 mmol), NaOH 1 M
(0.6 mL) and THF (0.4 mL) was stirred at room temper-
ature for 2 h. The pH of the resulting clear solution was
adjusted to ꢀ7 with 1 M HCl, and 18 precipitated as a
white solid, which was collected by filtration and dried
1
(0.059 g, 77%): mp 236–237 ꢂC. H NMR (DMSO-d6):
dH 3.73 (s, 2H, C6H5CH2), 7.21–7.36 (m, 5H, C6H5),
7.71–7.79 (m, 3H, H2, H5, H6), 8.05* (br s, 4H,
N@C(NH2)2), 10.0* (s, 1H, NHCOCH2C6H5), 10.73*
(br, COOH). MS (ES+) m/z 313 ([M+H]+, 100), 335
([M+Na]+, 52). HR-ES-MS (m/z) [M+H]+ calcd for
C16H17N4O3: 313.1295. Found: 313.1307.
4.2.10. 3-Amino-4-phenylacetylaminobenzoic acid (19). A
mixture of 16 (20 mg, 0.070 mmol), NaOH 0.5 M (1 mL)
and THF (0.5 mL) was stirred at room temperature for
16 h. The resulting suspension was filtered, the filtrate
diluted with 2 mL of water and the pH adjusted to ꢀ7 with
1 M HCl. The precipitate thus formed was collected by fil-
tration and dried, giving 19 as a fine white powder
4.2.13. Methyl 4-benzylmercapto-3-guanidino-benzoate
hydrochloride (23). A mixture of 22 (0.7 g, 2.6 mmol),
cyanamide (2.7 g, 64 mmol) and concd HCl (0.33 mL)
in EtOAc (20 mL) was refluxed for 2 h. The reaction mix-
ture was cooled to room temperature and a solid crystal-
lised. This solid was filtered, washed with EtOAc and
1
(0.011 g, 58%): mp 168–170 ꢂC. H NMR (DMSO-d6):

J. Neres et al. / Bioorg. Med. Chem. 15 (2007) 2106–2119
2117
dried, yielding 23 as a white solid (0.8 g, 88%): mp 245–
246 ꢂC (recryst from EtOH). H NMR (DMSO-d6): dH
(1.74 g, 5.9 mmol) and DCC (1.1 g, 5.5 mmol) and the
mixture stirred for a few minutes on the ice bath and then
at room temperature for 16 h. The suspended solid was
removed by filtration and the filtrate evaporated in vacuo.
The resulting residue was suspended in EtOAc (50 mL)
and the insoluble material filtered out (solid A). The fil-
trate was chromatographed over a silica-gel column,
starting with CH2Cl2 with an increasing gradient of
CH2Cl2: EtOAc (up to 30% EtOAc). Fractions contain-
ing the product were combined with solid A and solvent
removed in vacuo. Successive recrystallisations of the res-
idue from EtOAc and EtOH yielded 26 as a white solid
1
3.85 (s, 3H, OCH3), 4.36 (s, 2H, C6H5CH2S), 7.26–7.48
(m, 9H, decreased to 5H after D2O shake, C6H5CH2S,
NH@C(NH2)2), 7.62 (d, J = 8.4 Hz, 1H, H5), 7.73 (d,
J = 1.8 Hz, 1H, H2), 7.90 (dd, J = 1.8 and 8.4 Hz, 1H,
H6), 9.66* (br s, 1H, NH@C(NH2)2). 13C NMR
(DMSO-d6): dC 34.8 (C6H5CH2S), 52.2 (OCH3), 126.5
(C2), 126.8 (C1), 127.4 (C40), 128.5 (C20 and C60),
128.89–128.93 (C5, C6, and C30 and C50), 131.2 (C4),
135.8 (C10), 143.5 (C3), 156.4 (NH@C(NH2)2), 165.3
(COOCH3). MS (ES+) m/z 316 ([M]+, 100), (ESꢁ) m/z
1
350
([MꢁH+Cl]ꢁ,
100).
Anal.
Calcd
for
(0.65 g, 33%): mp 193–195 ꢂC. H NMR (DMSO-d6):
C16H18N3O2SCl: C, 54.6; H, 5.2; N, 11.9; S, 9.1; Cl,
10.1. Found: C, 54.7; H, 5.1; N, 12.0; S, 8.9; Cl, 10.4.
dH 3.83–3.88 (br s, 5H, OCH3 and NHCOCH2NHFmoc),
4.24–4.33 (m, 5H, C6H5CH2S; CH and CH2 Fmoc), 7.20–
7.35 (m, 7H, C6H5CH2S and 2 Car H Fmoc), 7.42 (t,
J = 7.3 Hz, 2H, 2 Car H Fmoc), 7.54 (d, J = 8.3 Hz, 1H,
H5), 7.69–7.74 (m, 3H, H6 and 2 Car H Fmoc), 7.79*
(app t, 1H, NHCOCH2NHFmoc), 7.90 (d, J = 7.4 Hz,
2H, 2 Car H Fmoc), 8.11 (s, 1H, H2), 9.47* (s, 1H,
NHCOCH2NHFmoc). 13C NMR (DMSO-d6): dC 36.1
(C6H5CH2S), 43.9 (NHCOCH2NHFmoc), 46.5 (CH
Fmoc), 52.0 (OCH3), 65.8 (CH2 Fmoc), 120.0 (CH
Fmoc), 124.8 (C2), 125.1 (CH Fmoc), 125.9 (C6), 127.0
(CH Fmoc), 127.2 (C40), 127.5 (CH Fmoc), 128.3 (C20
and C50), 128.6 (C5), 128.9 (C30 and C50), 135.22,
135.24, 136.2, 136.6 (C1, C3, C4 and C10), 140.6 and
143.7 (2 · Cquat Fmoc), 156.5 (CO Fmoc), 165.6
(COOCH3), 168.4 (NHCOCH2NHFmoc). MS (ES+) m/
z 575 ([M+Na]+, 100); (ESꢁ) m/z 551 ([MꢁH]ꢁ, 100).
HR-ES-MS (m/z) [M+Na]+ calcd for C32H28N2O5SNa:
575.1611. Found: 575.1607.
4.2.14. 4-Benzylmercapto-3-guanidino-benzoic acid (24).
A solution of 23 (0.3 g, 0.85 mmol) in THF (1 mL) was
added to 1 M NaOH (4 mL) and the mixture heated to
70 ꢂC for 1 h. The solvent was evaporated, and the res-
idue obtained re-dissolved in water (3 mL) and the pH
adjusted to 6–7 with 1 M HCl. A solid slowly came
out of solution, which was filtered and dried, yielding
24 as a white solid, (0.22 g, 87%): mp >260 ꢂC (recryst
1
from EtOH/water). H NMR (DMSO-d6): dH 4.23 (s,
2H, C6H5CH2S), 7.21–7.41 (m, 6H, H5 and C6H5CH2S),
7.62 (d, J = 1.5 Hz, 1H, H2), 7.76 (dd, J = 1.5 and
8.1 Hz, 1H, H6), 8.00* (br s, 4H, NH@C(NH2)2),
11.36* (br s, 1H, NH@C(NH2)2). 13C NMR (DMSO-
d6): dC 35.6 (C6H5CH2S), 126.9 (C2), 127.2 (C40),
128.4 (C20 and C60), 128.7 (C5 and C6), 128.9 (C30
and C50), 131.7 (C1), 136.5, 137.2, 137.3 (C3, C4, C10)
156.4 (NH@C(NH2)2), 169.5 (COOH). MS (ES+) m/z
302 ([M+H]+, 100), (ESꢁ) m/z 300 ([MꢁH]ꢁ, 100). Anal.
Calcd for C15H15N3O2SÆH2O: C, 56.4; H, 5.4; N, 13.2; S,
10.0. Found: C, 56.3; H, 5.0; N, 13.2; S, 9.8.
4.2.17. 3-[(Aminoacetyl)amino]-4-benzylmercapto-benzo-
ic acid (27). A solution of 26 (0.125 g, 0.23 mmol) in
EtOAc (5 mL) and diethylamine (5 mL) was refluxed
for 1 h and the resulting solution evaporated to dryness
in vacuo. The resulting residue was suspended in MeOH
(30 mL) and filtered. The filtrate was evaporated to dry-
ness and the residue dissolved in THF (2 mL) and 1 M
NaOH (4 mL), and refluxed for 1 h. The mixture was fil-
tered and the pH adjusted to ꢀ7 with 1 M HCl. The sol-
id formed was collected by filtration and dried, giving 27
as a white solid (0.026 g, 36%): mp colour change to
dark brown at 210 ꢂC; melted at 240–241 ꢂC. 1H
NMR (DMSO-d6, TFA): dH 3.81–3.83 (br q, 2H,
NHCOCH2NH3þ), 4.33 (s, 2H, C6H5CH2S), 7.25–7.43
(m, 5H, C6H5CH2S), 7.56 (d, J = 8.4 Hz, 1H, H5) 7.76
(dd, J = 1.7 and 8.4, 1H, H6), 7.94 (dþ, J = 1.6 Hz, 1H,
H2), 8.17* (br s, 3H, NHCOCH2NH3 ), 10.07* (s, 1H,
4.2.15. Synthesis of 3-amino-4-benzylmercapto-benzoic
acid (25). A mixture of 22 (0.42 g, 1.54 mmol), 1 M
NaOH (3 mL) and MeOH (0.5 mL) was stirred at
40 ꢂC for 8 h. The volume of the resulting solution
was reduced to one third and the pH adjusted to 4 with
1 M HCl. The solution was extracted with EtOAc
(30 mL) and the organic phase dried over anhydrous
Na2SO4 and evaporated in vacuo, giving 25 as an off-
white solid (0.35 g, 88%): mp 155–157 ꢂC (recryst from
MeOH). 1H NMR (CDCl3): dH 4.10 (s, 2H,
SCH2NC6H5), 5.45* (s, 2H, NH2), 4.44 (2H,
J = 5.6 Hz, C6H5CH2NH), 7.03 (dd, J = 1.8 and
8.0 Hz, 1H, H6), 7.17–7.29 (m, 6H, C6H5CH2S and
H5), 7.31 (d, J = 1.8 Hz, 1H, H2), 12.71* (br s, 1H,
COOH). 13C NMR (DMSO-d6): dC 36.4 (SCH2NC6H5)
114.6 and 116.9 (C1 and C6), 122.0 (C4), 126.9 (C40),
128.2 (C20 and C60), 128.8 (C30 and C50), 130.1 (C1),
131.2 (C5), 137.4 (C10), 147.7 (C3), 167.4 (COOH).
MS (ESꢁ) m/z 258 ([MꢁH]ꢁ, 100). Anal. Calcd for
C14H13NO2S: C, 64.8; H, 5.1; N, 5.4; S, 12.4. Found:
C, 64.7; H, 5.0; N, 5.3; S, 12.2.
NHCOCH2NH þ). 13C NMR (DMSO-d6): dC 35.9
3
(C6H5CH2S), 40.9 (NHCOC H2 NH3þ), 126.9, 127.4
and 127.6 (C2, C5, C6, C40), 128.2 (C20 and C60),
129.2 (C30 and C50), 134.0, 136.4 and 138.5 (C3, C4,
C10), 165.9 (NHCOCH2NH3þ), 166.8 (COOH). MS
(ESꢁ) m/z 315 ([MꢁH]ꢁ, 100). HR-ES-MS (m/z)
[MꢁH]ꢁ calcd for C16H15N2O3S: 315.0802. Found:
315.0809.
4.2.16. Methyl 4-benzylmercapto-3-[2-(9H-fluoren-9-
ylmethoxycarbonylamino)-acetylamino] benzoate (26).
To a solution of 22 (1 g, 3.7 mmol) in anhydrous DMF
(5 mL) cooled in an ice bath were added Fmoc-Gly-OH
4.3. Expression and purification of recombinant TcTS
The protocol for expression and purification of recombi-
nant TcTS was adapted from Buschiazzo et al.46

2118
J. Neres et al. / Bioorg. Med. Chem. 15 (2007) 2106–2119
Competent E. coli BL21(DE3)pLysS (Promega, South-
ampton, UK) cells were transformed with a plasmid con-
taining the TcTS gene (pTrcTS611/247) and grown
overnight in LB broth containing 100 lg/mL ampicillin
at 37 ꢂC. The culture was diluted 1:50 with TB broth con-
taining 100 lg/mL ampicillin and incubation continued
under the same conditions to A600 ꢀ 1.2. The tempera-
ture was adjusted to 18 ꢂC and bacteria induced to
over-express recombinant TcTS by adding IPTG
(1 mM). Induction was maintained for 14–16 h, after
which cells were harvested and frozen. After one thaw/
freeze cycle, lysis was achieved in the presence of
20 mM Tris–HCl, pH 8.5, 30 mM NaCl, 0.5% Triton
X-100, 5 lg/mL DNAse I, protease inhibitor cocktail
and lysozyme produced by the cells, followed by sonica-
tion (6 · 30 s pulses of ultrasound). The lysate was centri-
fuged at 16,000 rpm for 45 min at 4 ꢂC and the
supernatant subjected to iminodiacetic metal affinity
chromatography (HisTrap FF 1 mL), on an AKTA-
FPLC system (GE Healthcare, Little Chalfont, UK),
after adjustment of the NaCl concentration to 0.5 M
and pH to 8.5. After applying the lysate, the column
was washed with buffer containing 20 mM Tris–HCl,
pH 8.5, 0.5 M NaCl and 10 mM imidazole, and the pro-
tein eluted with a linear gradient of imidazole (10–
500 mM) in the same buffer. Fractions containing the
protein were desalted (HiPrep 26/10 desalting column)
and further purification was achieved by FPLC anionic
exchange (MonoQ 5/50 GL) applying a linear elution gra-
dient of NaCl (0–0.5 M). Purified protein was quantified
using the Bradford method, using BSA as a standard.48
sion wavelengths of 365 and 442 nm, respectively. Inhi-
bition type was assessed by analysing the patterns of
three diagnostic classes of plot: 1/vo versus 1/[So] for var-
ious [I]; 1/vo versus [I] for various [So]; [So]/vo versus [I]
at various [So]. Values of Ki and K0i for mixed inhibition
were determined by direct weighted (1=v2o for weighting)
least squares non-linear regression analysis of the raw
data using the appropriate equation using the Grafit
program,34 viz., for mixed inhibition, v ¼ V max½Soꢂ=
ð½Soꢂð1 þ ½Iꢂ=Ki0Þ þ Kmð1 þ ½1ꢂ=KiÞ. Values of Vmax and
Km were obtained by least squares non-linear regression
analysis using Grafit.
Acknowledgments
We are grateful to Professor A. Carlos C. Frasch and
´
Laura Ratier (Instituto de Investigaciones Biotecnolog-
icas, Universidad Nacional de San Martın, Argentina)
´
for general advice and to the Portuguese Foundation
for Science and Technology (F.C.T.) for financial
support.
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