Discovery of novel inhibitors targeting the Mycobacterium tuberculosis O-acetylserine sulfhydrylase (CysK1) using virtual high-throughput screening

Article abstract of DOI:10.1016/j.bmcl.2013.01.031

Cysteine biosynthesis in Mycobacterium tuberculosis (MTB) is crucial for this pathogen to combat oxidative stress and for long term survival in the host. Hence inhibition of this pathway is attractive for developing novel drugs against tuberculosis. In the present study, the crystal structure of the mycobacterial enzyme O-acetylserine sulfhydrylase CysK1 bound to an oligopeptide inhibitor was used as a framework for virtual screening of the BITS-Pilani in-house database to identify new scaffolds as CysK1 inhibitors. Thirty compounds were synthesized and evaluated in vitro for their ability to inhibit CysK1, activity against M. tuberculosis and cytotoxicity as steps towards the derivation of structure-activity relationships (SAR) and lead optimization. Compound 8-nitro-4-(2-(trifluoromethyl)phenyl)-4,4a-dihydro-2H-pyrimido[5,4-e] thiazolo[3,2-a]pyrimidine-2,5(3H)-dione (4n) emerged as the most promising lead with an IC₅₀ of 17.7 μM for purified CysK1 and MIC of 7.6 μM for M. tuberculosis, with little or no cytotoxicity (>50 μM).

Full text of DOI:10.1016/j.bmcl.2013.01.031

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1182–1186
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of novel inhibitors targeting the Mycobacterium
tuberculosis O-acetylserine sulfhydrylase (CysK1) using virtual
high-throughput screening
Variam Ullas Jean kumar a, Ömer Poyraz b, Shalini Saxena a, Robert Schnell b, Perumal Yogeeswari a,
Gunter Schneider b, , Dharmarajan Sriram a,
⇑
⇑
a Department of Pharmacy, Birla Institute of Technology & Science-Pilani, Hyderabad Campus, Shameerpet, R.R. District, Hyderabad 500078, Andhra Pradesh, India
b Department of Medical Biochemistry & Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
Cysteine biosynthesis in Mycobacterium tuberculosis (MTB) is crucial for this pathogen to combat oxida-
tive stress and for long term survival in the host. Hence inhibition of this pathway is attractive for devel-
oping novel drugs against tuberculosis. In the present study, the crystal structure of the mycobacterial
enzyme O-acetylserine sulfhydrylase CysK1 bound to an oligopeptide inhibitor was used as a framework
for virtual screening of the BITS-Pilani in-house database to identify new scaffolds as CysK1 inhibitors.
Thirty compounds were synthesized and evaluated in vitro for their ability to inhibit CysK1, activity
against M. tuberculosis and cytotoxicity as steps towards the derivation of structure–activity relationships
(SAR) and lead optimization. Compound 8-nitro-4-(2-(trifluoromethyl)phenyl)-4,4a-dihydro-2H-pyrimi-
do[5,4-e]thiazolo[3,2-a]pyrimidine-2,5(3H)-dione (4n) emerged as the most promising lead with an IC50
Received 30 October 2012
Revised 7 January 2013
Accepted 9 January 2013
Available online 16 January 2013
Keywords:
O-Acetylserine sulfhydrylase
CysK1
Tuberculosis
of 17.7
lM for purified CysK1 and MIC of 7.6
l
M for M. tuberculosis, with little or no cytotoxicity
(>50 M).
l
Ó 2013 Elsevier Ltd. All rights reserved.
Mycothiol, the major low molecular weight thiol used by myco-
bacteria to maintain their redox balance, contains a cysteine-de-
rived sulfhydryl moiety.1 Thus, the first line of defense of the
pathogen against free radicals, and hence its long term survival
within granulomas, is directly linked to the availability of cysteine.
Not surprisingly, gene expression and proteome analysis reveal
that genes from the cysteine biosynthetic pathway and sulfur
metabolism are up-regulated in dormancy models2,3 of Mycobacte-
rium tuberculosis. These metabolic processes therefore appear to
provide potential targets for novel anti-tuberculosis agents, specif-
ically directed toward the pathogen in its persistent phase.4 The
essential role of cysteine biosynthesis in this pathogen was further
emphasized by high density mutagenesis screens showing that
mutants carrying transposon insertions in genes related to this
pathway are attenuated in macrophages and in a mouse MTB mod-
el.5,6 cysD, cysN, cysH, sirA, genes from the sulphate reduction (APS/
PAPS-) pathway producing the sulphide used for cysteine biosyn-
thesis, and also cysE encoding the serine–acetyl transferase were
shown to be indispensible for growth.7 Cysteine biosynthesis is
attractive as target for new antibacterials also because this path-
way is absent in humans.8 One of the enzymes of this pathway,
the pyridoxalphosphate-dependent O-acetylserine sulfhydrylase
CysK1 (Rv2334), catalyzes the incorporation of sulfide into O-acet-
ylserine, yielding
that CysK1 is competitively inhibited by the four-residue peptide
DFSI with a Ki of 5 M and determined the crystal structure of
L
-cysteine.9 In our previous work we have shown
l
the CysK1:DFSI complex to 2.1 Å resolution (PDB ID 2Q3C). The
peptide inhibitor binds to the active site of CysK1 through direct
hydrogen bonds and hydrophobic interactions.10
A high-throughput virtual screening of our in-house (BITS-
Pilani) database was carried out using Glide XP (extra precision)
docking and we identified 8-nitro-2-thioxo-4-(2-nitrophenyl)-
4,4a-dihydro-2H-pyrimido[5,4-e]thiazolo[3,2-a]pyrimidin-5(3H)-
one (BITS-12, 5c) as a potential inhibitor in the DFSI site interacting
with amino acid residues Gly-222, Phe-145 and Ser-72 with a
docking score of À6.84, while the DFSI docking score was À8.34
(Fig. 1). Compound 5c (BITS-12) showed an IC50 of 30.3 lM when
tested for CysK1 inhibitory activity. To investigate the potential
of this lead, we synthesized a series of substituted nitro-5H-thiaz-
olo [3,2-a] pyrimidinones to screen against CysK1 enzyme activity,
for establishing structure–activity relationship (SAR), and to evalu-
ate antimycobacterial activity and cytotoxicity.
To date, to the best of our knowledge, the synthesis of substi-
tuted nitro-5H-thiazolo[3,2-a]pyrimidinone derivatives has been
described scarcely in the literature and further synthetic chemis-
try work was initiated in our lab to design a highly efficient
⇑
Corresponding authors.
E-mail addresses: Gunter.Schneider@ki.se (G. Schneider), drdsriram@yahoo.com
(D. Sriram).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.01.031

V. U. Jean kumar et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1182–1186
1183
S
N
NH
NO2
N
O
2N
S
N
O
BITS-12 (5c)
IC50 = 30.3 µM
Phe145
Ser72
Gly222
HIE218
Docked pose of
compound 5c
LIGPLOT of
compound 5c
Figure 1. Docking pose of BITS-12 (5c) identified by virtual high throughput screening (vHTS).
chemical reaction sequence which should provide a maximum of
structural complexity and biological diversity in a minimum
number of steps. A library was designed with the goal of obtain-
ing a lead series with tractable SAR and potencies better than the
previously identified virtual screening hit (BITS-12, 5c). In the
present protocol we report a simple and highly efficient micro-
wave-assisted synthesis of substituted 8-nitro-4-(aryl)-4,4a-dihy-
dro-2H-pyrimido[5,4-e]thiazolo[3,2-a]pyrimidine-2,5(3H)-dione
(4a–4o) and 8-nitro-4-(aryl)-2-thioxo-4,4a-dihydro-2H-pyrimi-
do[5,4-e]thiazolo[3,2-a]pyrimidin-5(3H)-one (5a–5o) derivatives
as potential antitubercular agents. The synthetic pathway used
to achieve the target compounds has been delineated in Figure 2.
Tadakazu et al. has previously reported the synthesis and reac-
tions of thiadiazolo/thiazolopyrimidine derivatives by condensa-
tion of 2-amino[1,3,4]thiadiazole/2-aminothiazole and ethyl
cyanoacetate in sodium ethoxide and ethanol.11 Our initial efforts
to extend this work to 5-nitro-2-aminothiazole derivatives re-
sulted in a very poor yield of the cyclised product, probably
due to the low nucleophilicity and poor solubility of 5-nitro-2-
aminothiazole compared to the previously reported derivatives.
In light of these findings, it was decided to perform a trial under
microwave irradiation. The effort not only improved the yield
dramatically but also reduced the reaction time to 8–10 min
compared to the conventional heating protocol. The so obtained
5-imino-2-nitro-5H-thiazolo[3,2-a]pyrimidin-7(6H)-one was then
hydrolysed by refluxing with 6 N HCl to give the corresponding
2-nitro-5H-thiazolo[3,2-a]pyrimidine-5,7(6H)-dione scaffold,12
which was further condensed with various substituted benzalde-
hydes, urea or thiourea in presence of catalytic amount of trieth-
ylamine in a microwave-assisted, one pot protocol to give the
titled compound in good yield.13–15 The microwave reaction
was carried out in Biotage intiator, the absorption level was kept
high with a pre-stirring of 30 s and stirring rate at 600 rpm. Ini-
tially the power was kept zero, followed by a gradual increase,
the reaction proceeded at a constant power of 320 W with tem-
perature 120 °C and 5-bar pressure. A library of 30 compounds
has been synthesised as summarized in Table 1, and as is evident,
the presence of electron withdrawing/electron donating substitu-
tions did not seem to have any significant limitation on the over-
all reactivity giving the desired product in quantitative yields,
thus validating the scope of the present protocol. Both analytical
and spectral data (1H NMR, 13C NMR, and mass spectra) of all
the synthesized compounds were in full agreement with the
proposed structures. The 1H NMR spectrum of all the final com-
pounds (4a–o, 5a–o) showed a doublet in the range of 3.47–3.53
and a doublet in the range 4.43–4.47 corresponding to the two
terminal protons of the pyrimidinone nucleas & not nucleas,
whereas the proton of the thiazole ring resonated in the range
X
R
NH
HN
O
O2N
N
S
O
O
c
a
b
N
S
N
NH2
O
N
N
N
N
S
N
O2N
O2N
X=O,S
S
O2N
1
2
3
4a-o, 5a-o
a. Ethylcyanoacetate, Na/C2H5OH, MWI; b. 6N HCl, reflux; c. (sub)Benzaldehyde, TEA, urea/thiourea, MWI
Figure 2. General synthesis procedure.

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V. U. Jean kumar et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1182–1186
Table 1
Physical constants and biological activity of compounds
X
NH
N
R
N
O
O2N
N
S
4a-o, 5a-o
Compd
X
R
Yield (%)
mp (°C)
Rel. activity at 100
l
M [IC50 in
l
M]
MICa
(l
M)
Cytotoxicityb (% inhibition)
25 50
l
M
lM
4a
5a
4b
5b
O
S
O
S
O
S
O
S
O
S
O
S
O
S
O
S
O
S
O
S
O
S
O
S
O
S
O
S
O
S
Phenyl
Phenyl
2-Hydroxyphenyl
2-Hydroxyphenyl
2-Nitrophenyl
2-Nitrophenyl
4-Nitrophenyl
4-Nitrophenyl
3-Bromophenyl
3-Bromophenyl
4-Bromophenyl
4-Bromophenyl
4-Fluorophenyl
86
84
88
79
91
83
94
79
73
66
73
76
68
81
63
68
89
92
81
74
65
63
74
68
83
96
91
88
72
84
135–138
215–218
178–180
188–190
210–212
254–256
195–197
178–179
172–174
194–195
180–181
241–242
213–215
261–263
188–190
255–257
168–170
230–232
143–144
266–268
199–201
199–201
210–211
140–142
240–242
281–283
124–126
159–161
131–133
243–345
84.1
90.4
107.4
68.8
93.0
11.3 [30.3]
54.7
70.6
60.1
39.9
89.1
65.6
100.2
60.0
93.9
9.1 [22.7]
80.0
102.5
92.7
100.4
102.7
15.9 [17.7]
57.7
106.2
57.8
>145.6
69.6
69.6
33.3
128.8
30.9
128.8
61.8
14.8
7.1
29.6
28.5
17.3
16.6
133.9
32.1
66.2
15.9
32.4
15.5
70.0
33.5
70.0
67.0
121.3
58.4
7.6
1.6
0
0
0.1
0
1.0
2.6
1.64
0
6.0
6.1
2.5
5.4
3.8
25.3
0
12.0
0
8.7
9.4
6.6
2.8
13.5
11.2
6.5
10.7
43.4
7.3
14.6
0
14.6
4.9
26.8
20.4
18.8
0
14.7
0
30.7
13.1
52.2
0
4c
5c (BITS-12)
4d
5d
4e
5e
4f
5f
4g
5g
4h
5h
4i
5i
4j
5j
4k
5k
4l
5l
4m
5m
4n
5n
4o
5o
DFSI
Isoniazid
Ethambutol
4-Fluorophenyl
30.6
4-Methoxyphenyl
4-Methoxyphenyl
4-Chlorophenyl
0
0.2
0
7.4
0
8.0
6.0
3.6
0
0
0
21.3
5.7
27.7
0
4-Chlorophenyl
4-Dimethylaminophenyl
4-Dimethylaminophenyl
2-Methylphenyl
2-Methylphenyl
4-Methylphenyl
4-Methylphenyl
2,6-Dichlorophenyl
2,6-Dichlorophenyl
2-Trifluromethylphenyl
2-Trifluromethylphenyl
4-Hydroxy-3-methoxyphenyl
4-Hydroxy-3-methoxyphenyl
113.2
13.8 [17.7]
89.4
90.5
89.2
[7.4]
NT
NT
7.3
128.4
123.3
NT
0.72
4.71
0
1.35
NT
NT
NT
NT
NT
NT
a
Minimum inhibitory concentration against M. tuberculosis.
Cytotoxicity against HEK 293T, NT indicates not tested.
b
of 7.21–7.23 ppm as a singlet. The structures were further
confirmed by their 13C NMR, and mass spectra and also the
elemental analysis as summarized in the experimental section.15
Thirty substituted nitro-5H-thiazolo[3,2-a]pyrimidinone deriv-
atives were screened against CysK1 enzyme activity based on
methods reported recently.10 The O-acetylserine-sulfhydrylase
activity assay was based on the spectrophotometric determina-
method16 adapted to multi-well format. In a first screening
round the novel compounds were evaluated at 100 M concen-
tration to identify the inhibitors in the derivative-set.15 The rel-
ative enzyme activity at 100 compound concentration
(Table 1) was used as a filter to score the compounds and select
the strongest inhibitors for further studies. Compounds inhibit-
ing CysK1 activity P50% were retested in dose–response in more
detail (Fig. 3).
l
lM
tion of the reaction product L-cysteine by the acid-ninhydrin
Figure 3. Dose–response curves of 5h (left) and 4n (right) for inhibition of CysK1.

V. U. Jean kumar et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1182–1186
1185
Phe145
Gly222
HIE218
Docked pose of compound 4n
LIGPLOT of compound 4n
Figure 4. Docked pose of compound 4n in the active site of CysK1.
Three compounds (4n, 5h and 5k) that showed at least 50%
phenyl)-4,4a-dihydro-2H-pyrimido[5,4-e]thiazolo[3,2-a]pyrimidin-
5(3H)-one (BITS-12, 5c) and further synthesis of analogues yielded
8-nitro-4-(2-(trifluoromethyl)phenyl)-4,4a-dihydro-2H-pyrimido
[5,4-e]thiazolo[3,2-a]pyrimidine-2,5(3H)-dione (4n) being more
active than the initial hit 5c.
inhibition at 100
and 17.7 M, respectively, and thus are improved when compared
to the initial hit 5c (BITS-12) whose IC50 was 30.3 M (Table 1).
lM concentration gave IC50 values 17.7, 22.7,
l
l
All the new nitro-5H-thiazolo[3,2-a]pyrimidinone derivatives
were screened for their in vitro anti-tubercular activity against
Mycobacterium tuberculosis H37Rv (ATCC27294) using an agar dilu-
tion method17 with compound concentrations ranging from 50 to
Acknowledgements
This research was supported by the Department of Biotechnogy
(DBT), Government of India and the Swedish Governmental Agency
for Innovation Systems (VINNOVA).
0.78 lg/mL in duplicates. The minimum inhibitory concentration
(MIC), defined as the minimum concentration of compound re-
quired to completely inhibit bacterial growth, was determined
for each compound. Isoniazid and ethambutol were used as refer-
ence compounds for comparison (Table 1). All the nitro-5H-thiaz-
olo[3,2-a]pyrimidinone derivatives showed in vitro activity
References and notes
1. Bornemann, C.; Jardine, M. A.; Spies, H. S. C.; Steenkamp, D. J. Biochem. J. 1997,
325, 623.
2. Schnappinger, D.; Ehrt, S.; Voskuil, M. I.; Liu, Y.; Mangan, J. A.; Monahan, I. M.;
Dolganov, G.; Efron, B.; Butche, P. D.; Nathan, C.; Schoolnik, G. K. J. Exp. Med.
2003, 198, 693.
against M. tuberculosis with MICs ranging from 7.1 to 145.6
Three compounds (5e, 4n and 5n) inhibited M. tuberculosis with
a MIC of less than 10 M and compound 5e [8-nitro-2-thioxo-
lM.
l
3. Hampshire, T.; Soneji, S.; Bacon, J.; James, B. W.; Hinds, J.; Laing, K.; Stabler, R.
A.; Marsh, P. D.; Butcher, P. D. Tuberculosis 2004, 84, 228.
4-(3-bromophenyl)-4,4a-dihydro-2H-pyrimido[5,4-e]thiazolo
[3,2-a]pyrimidin-5(3H)-one] emerged as the more potent antimy-
cobacterial molecule among the synthesized derivatives with a
4. Hatzios, S. K.; Bertozzi, C. R. PLoS Pathog. 2011, 7, e1002036.
5. Sassetti, C. M.; Rubin, E. J. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 12989.
6. Senaratne, R. H.; De Silva, A. D.; Williams, S. J.; Mougous, J. D.; Reader, J. R.;
Zhang, T.; Chan, S.; Sidders, B.; Lee, D. H.; Chan, J.; Bertozzi, C. R.; Riley, L. W.
Mol. Microbiol. 2006, 59, 1744.
7. Sassetti, C. M.; Boyd, R. H.; Rubin, E. J. Mol. Microbiol. 2003, 48, 77.
8. Bhave, D. P.; Muse, W. B., III; Carroll, K. S. Infect. Disord. Drug Targets 2007, 7,
140.
9. Schnell, R.; Schneider, G. Biochem. Biophys. Res. Commun. 2010, 396, 33.
10. Schnell, R.; Oehlmann, W.; Singh, M.; Schneider, G. J. Biol. Chem. 2007, 282,
23473.
11. Tadakazu, T. J. Heterocycl. Chem. 1991, 489.
12. Alain, L.; Jean-Pierre, F. Tetrahedron Lett. 1978, 3469.
MIC of 7.1 lM. All these compounds were less potent than stan-
dard first line anti-tubercular drugs. In general 2-thioxo derivatives
[5a–5o] were found to be more potent than 2-oxo derivatives
[4a–4o]. Substitutions at position 4 of the aryl ring with various
electron withdrawing groups such as bromo, trifluoromethyl
moieties, showed good activity, whereas substitution with electron
donating groups like hydroxyl, methyl, methoxyl moieties in
general resulted in less active compounds.
All compounds were also tested for in vitro cytotoxicity against
13. Sharma, R. L.; Daljeet, K.; Jasbir, S.; Surinder, K.; Poonam, G.; Shallu, G.;
Bhavneet, K.; Anand, S. J. Heterocycl. Chem. 2008, 1775.
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Chem. 1993, 460.
HEK293T cells at 25 and 50 lM by the (4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) assay.18 Table 1 summa-
rizes the percentage inhibition of cell growth. Most of the
15. Experimental section: Synthesis of 5-imino-2-nitro-5H-thiazolo[3,2-a]pyrimidin-
7(6H)-one (Step 1): To a freshly prepared solution of sodium ethoxide (0.47 g,
6.89 mmol) in ethanol (6 ml) were added, 2-amino-5-nitrothiazole (1 g,
6.89 mmol) and ethyl cyanoacetate (0.78 g, 6.89 mmol). The reaction mixture
was irradiated in microwave digestor at an intensity of 320 W with 30 s/cycle.
The number of cycle in turn depended on the completion of the reaction,
monitored by TLC and LCMS for completion. The reaction mixture was then
evaporated; hot water was added to completely dissolve the residue and
further neutralized with saturated sodium bicarbonate solution until pH 7. The
aqueous layer was extracted with dichloromethane (2 Â 15 ml). The combined
organic layer was then washed with brine (1 Â 15 ml) dried over anhydrous
sodium sulphate and evaporated in vacuo. The crude residue was then
recrystallised from ethanol to give the desired product as buff coloured solid.
(1.3 g, 89%).
compounds were not cytotoxic up to 50 lM and compounds with
a 2-thioxo group showed more toxicity than their corresponding
2-oxo analogues.
Based on the CysK1 inhibition, activity against M.tuberculosis and
cytotoxicity data, the most active compound in this series was found
to be compound 8-nitro-4-(2-(trifluoromethyl)phenyl)-4,4a-dihy-
dro-2H-pyrimido[5,4-e]thiazolo[3,2-a]pyrimidine-2,5(3H)-dione
(4n) with an IC50 of 17.7
lM (CysK1) and MIC of 7.6
lM (antimyco-
bacterial) and no cytotoxicity (>50
lM). Compound 4n was docked
into the DFSI pocket and was found to interact with a docking score
of À6.42 and the interacting residues were Gly-222, Phe-145 and
Hie-218 (histidine in epsilon state) (Fig. 4). In conclusion, our
present study identified an initial lead 8-nitro-2-thioxo-4-(2-nitro-
Synthesis of 2-nitro-5H-thiazolo[3,2-a]pyrimidine-5,7(6H)-dione (Step 2): The
above obtained 5-imino-2-nitro-5H-thiazolo[3,2-a]pyrimidin-7(6H)-one (1 g,
4.7 mmol) was added lot wise to 6 N HCl (5 ml), and the reaction mixture was
refluxed for 2 h (monitored by TLC and LCMS for completion). The reaction

1186
V. U. Jean kumar et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1182–1186
mixture was then poured into ice cold water, filtered, washed with water to
give titled product. (0.757 g, 75%).
J = 7.1 Hz), 4.47 (d, 1H, J = 7.0 Hz), 7.16–7.39 (m, 4H, 3Ar-H & 1Th-H), 7.86 (d,
1H, J = 8.1 Hz, Ar-H) 13C NMR (75 MHz, CDCl3): dc. 196.3, 164.7, 164, 162.9,
142.7, 138.1, 130.9, 137.1, 126, 125.7, 124.1, 123.8, 113.8, 48.9, 37.9. 19F NMR
(282 MHz, CDCl3): d À59.38 (3F, –CF3). EI-MS m/z 411.05 (M+). Anal. Calcd for
Step 3: Library synthesis: To the above obtained product (0.25 g, 1.17 mmol) in
acetonitrile (3 ml) were added, the corresponding aldehyde (for benzaldehyde
(0.124 g, 1.17 mmol)), urea/thiourea (for urea 0.07 g, 1.17 mmol) and
triethylamine (0.012 g, 0.117 mmol). The reaction mixture was irradiated in
microwave digestor at an intensity of 320 W with 30 s/cycle. The number of
cycle in turn depended on the completion of the reaction, monitored by TLC
and LCMS for completion. The reaction mixture was poured into ice cold water,
filtered, washed with water, dried and further recrystallised from ethanol–
ethyl acetate mixture (7:3 v/v) to give titled product in good yield as
summarized in Table 1. (a) 8-Nitro-2-thioxo-4-(2-nitrophenyl)-4,4a-dihydro-
2H-pyrimido[5,4-e]thiazolo[3,2-a]pyrimidin-5(3H)-one (5c): Yellowish orange
solid; mp 254–256 °C; Yield (83.67%); 1H NMR (300 MHz, CDCl3): dH 3.49 (d,
1H, J = 7.1 Hz), 4.43 (d, 1H, J = 7.0 Hz), 7.11–7.53 (m, 4H, 3Ar-H & 1Th-H), 8.09
(d, 1H, J = 8.1 Hz, Ar-H). 13C NMR (75 MHz, CDCl3): dc 197.6, 183.3, 164.1, 162.9,
148.1, 143, 138.3, 134.5, 126.7, 126.3, 124.7, 113.7, 46.7, 43.3. EI-MS m/z
404.03 (M+). Anal. Calcd for C14H8N6O5S2: C, 41.58; H, 1.99; N, 20.78. Found:
C, 41.63; H, 2.01; N, 20.71. (b) 8-Nitro-2-thioxo-4-(4-methoxyphenyl)-4,
4a-dihydro-2H-pyrimido[5,4-e]thiazolo[3,2-a]pyrimidin-5(3H)-one (5h): Brown
solid; mp 255–257 °C; Yield (68.01%); 1H NMR (300 MHz, CDCl3):dH 3.51 (d,
1H, J = 7.2 Hz), 3.87 (s, 3H, –OCH3), 4.45 (d, 1H, J = 7.1 Hz), 6.89–7.3 (m, 5H,
4Ar-H & 1Th-H). 13C NMR (75 MHz, CDCl3): dc 197.3, 186.1, 164.3, 163.1,
155.4, 142.7, 136.4, 127.0, 114, 113.8, 60.1, 51.7, 47.3. EI-MS m/z 389.07 (M+).
Anal. Calcd for C15H11N5O4S2: C, 46.27; H, 2.85; N, 17.98. Found: C, 46.23;
H, 2.81; N, 18.03. (c) 8-Nitro-2-thioxo-4-(o-tolyl)-4,4a-dihydro-2H-pyrimido
[5,4-e]thiazolo[3,2-a]pyrimidin-5(3H)-one (5k): Brown solid; mp 199–201 °C;
Yield (63.26%); 1H NMR (300 MHz, CDCl3): dH 2.41 (s, 3H, –CH3), 3.51
(d, 1H, J = 7.1 Hz), 4.46 (d, 1H, J = 7.0 Hz), 7.01–7.36 (m, 5H, 4Ar-H & 1Th-H).
13C NMR (75 MHz, CDCl3): 197.5, 186, 164.6, 163.2, 143.1, 140.3, 135.7,
127.9, 127.6, 124.8, 124.6, 113.7, 47.8, 45.8, 21.6. EI-MS m/z 373.01 (M+). Anal.
Calcd for C15H11N5O3S2: C, 48.25; H, 2.97; N, 18.76 Found: C, 48.19; H, 2.93; N,
18.72. (d) 8-Nitro-4-(2-(trifluoromethyl)phenyl)-4,4a-dihydro-2H-pyrimido
[5,4-e]thiazolo[3,2-a]pyrimidine-2,5(3H)-dione (4n): Yellowish brown solid;
mp 124–126 °C; Yield (91%); 1H NMR (300 MHz, CDCl3): dH. 3.53 (d, 1H,
C
15H8F3N5O4S: C, 42.15; H, 1.89; N, 16.39. Found: C, 42.21; H, 1.87; N, 16.35.
Enzymatic assay of CysK1: 1
well plates. Then, a total of 44
l
L of compounds was first transferred into 96-
L of assay mixture (MOPS [pH 7.0], O-acetyl-L-
l
serine, CysK1) was added to each well and incubated for 5 min at room
temperature to reach equilibrium. Positive controls (100% inhibition)
contained the assay mixture with the inhibitory DFSI tetrapeptide (final
concentration: 100 lM), negative controls (0% inhibition) did not contain any
inhibitory compounds. The reactions were started by the addition of sodium-
sulphide to 1 mM concentration, and incubated for 20 min. At the end of the
reaction time, reactions were stopped with 10
concentration: 16.6%) followed by addition of 60
l
L of trichloroacetic acid (final
of acid-ninhydrine
l
L
reagent and heating at 96 °C for 10 min in a 96-well PCR heating block. After
cooling reactions were transferred into new 96-well plates, diluted 1:1 in
ethanol (95%) and measured at 560 nm for their optical density in a BioTek
Synergy HT-1 plate reader. Each reaction was carried out in triplicate.
Determination of dose–response curves: Serial 1:3 compound dilutions were
first prepared in 50% DMSO in 100
and transferred into a 96-deep well block. Then, 890
added (final concentrations: 100 mM MOPS [pH 7.0], 2 mM O-acetyl-
1.3 g CysK1) and incubated for 5 min at room temperature. Reactions were
then started by addition of 10 of sodium-sulphide solution (final
concentration: 1 mM). At various time intervals, 100 aliquots were
removed and assayed for -cysteine formation as described above. The
l
L (final DMSO concentration in assay: 5%)
L assay mixture was
-serine,
l
L
l
l
L
l
L
L
specific enzyme activities were plotted against compound concentration and
IC50 values derived from sigmoid curves fit (GraphPad Prism).
16. Gaitonde, M. K. Biochem. J. 1967, 104, 627.
17. National Committee for Clinical Laboratory Standards Antimycobacterial
susceptibility testing for Mycobacterium tuberculosis Proposed standard M24-T;
National Committee for Clinical Laboratory Standards: Villanova, PA, 1995.
18. Gerlier, D.; Thomasset, N. Immunol. Methods 1986, 94, 57.