K. Suthagar et al. / Carbohydrate Research 457 (2018) 32e40
39
yellow waxy solid. [
a
]
[20] þ14.3 (c, 0.35 in CH3OH); ymax (neat)
120.5, 131.7 (2 ꢂ d, 4 ꢂ Ar(C)H), 137.0 (d, C-6), 137.7 (s, 2 ꢂ Ar(C)),
D
3268 (N-H), 1350 (s, S¼O), 1170 (s, S¼O) cmꢀ1
;
dH (400 MHz, D2O)
150.8 (s, C-2), 165.0 (s, C-4); HRMS (ESI) calculated for
79
1.76 (3H, s, 5-CH3), 2.55e2.75 (2H, m, H-2a’, H-2b’), 3.17e3.40 (2H,
m, H-5a’ and H-5b’), 4.28e4.36 (1H, m, H-40), 5.03e5.12 (1H, m, H-
30), 6.01 (1H, t, J1,2 7.0 Hz, H-10), 7.34 (1H, s, H-6); dC (100 MHz, D2O)
11.3 (q, 5-CH3), 35.2 (t, C-20), 40.6 (t, C-50), 79.7 (d, C-30), 79.8 (d, C-
40), 88.0 (d, C-10), 111.2 (s, C-5), 139.4 (d, C-6), 151.4 (s, C-2), 166.5 (s,
C-4); HRMS (ESI) calculated for C10H17N4O6S: 321.0863. Found
321.0873 (MHþ).
C H
BrN4O6S: 431.0281. Found 475.0281 (MHþ).
16 20
4.2.14. 50-N-[N-(4-Azidophenyl)sulfamoyl]amino]- 50-deoxy-
b-D-
thymidine 2k
General Procedure A, using sulfamoyl chloride 7d, and purifi-
cation by RP-HPLC (Luna C-18 column (Phenomenex); eluent: A
(0.05% TFA in H2O) and B MeCN; gradient: the sample was run at
1 mL/min with a gradient of 0e45% B; column oven: 40 ꢁC; detec-
tion: UV 210 nm and 280 nm), afforded sulfamide 2k (18 mg, 34%)
4.2.11. 50-Deoxy-50-N-[N-(3-trifluoromethyl-4-chlorophenyl)
sulfamoyl]amino]-b-D-thymidine 2h
as a light brown solid. m.p 133e135 ꢁC (EtOH/Et2O); [
a
]
D [20] þ10.4
General Procedure A, using sulfamoyl chloride 7a, and purifi-
cation by RP-HPLC (Luna C-18 column (Phenomenex); eluent: A
(0.05% TFA in H2O) and B MeCN; gradient: the sample was run at
1 mL/min with a gradient of 0e45% B; column oven: 40 ꢁC; detec-
tion: UV 210 nm and 280 nm), afforded sulfamide 2h (43 mg, 65%)
(c, 0.25 in CH3OH); ymax (neat) 3219 (N-H), 1331 (s, S¼O), 1148 (s,
S¼O) cmꢀ1
; dH (400 MHz, CD3OD) 1.86 (3H, s, 5-CH3), 2.19 (2H, at, J
6.3 Hz, H-2a’, H-2b’), 3.13e3.28 (2H, m, H-5a’ and H-5b’), 3.86 (1 H,
aq, J 4.7 Hz, H-40), 4.27 (1 H, aq, J 5.1 Hz, H-30), 6.11 (1H, t, J1,2 7.0 Hz,
H-10), 6.98, 7.23 (4H, 2 ꢂ d, J 8.6 Hz, 4 ꢂ Ar-H), 7.49 (1H, s, H-6); dC
(100 MHz, CD3OD) 10.9 (q, 5-CH3), 38.8 (t, C-20), 44.2 (t, C-50), 71.1
(d, C-30), 84.7 (d, C-40), 85.5 (d, C-10), 110.3 (s, C-5), 119.2, 121.0
(2 ꢂ d, 4 ꢂ Ar(C)H), 137.0 (d, C-6), 137.2 (s, 2 ꢂ Ar(C)), 150.8 (s, C-2),
165.0 (s, C-4); HRMS (ESI) calculated for C16H20N7O6S: 438.1190.
Found 438.1200 (MHþ).
as a pale yellow solid. m.p 105e107 ꢁC (EtOH/Et2O); [
a]
D [20] þ10.3
(c, 0.35 in CH3OH); ymax (neat) 3273 (N-H), 1370 (s, S¼O), 1165 (s,
S¼O) cmꢀ1
; dH (500 MHz, CD3OD) 1.86 (3H, d, J 1.0 Hz, 5-CH3),
2.18e2.23 (2H, m, H-2a’, H-2b’), 3.19 (1H, dd, J5a’,5b’ 14.6 Hz,
J4,5a’6.6 Hz, H-5a’), 3.28 (1H, dd, J5a’,5b’ 13.6 Hz, J4,5b’ 4.5 Hz, H-5b’),
3.85e3.87 (1 H, m, H-40), 4.25e4.27 (1 H, m, H-30), 6.11 (1H, t, J1,2
6.8 Hz, H-10), 7.41 (1H, dd, J 8.9 Hz, 2.7 Hz, Ar-H-6), 7.47 (1H, d, J
1.0 Hz, H-6), 7.49 (1H, d, J 8.9 Hz, Ar-H-5), 7.57 (1H, d, J 2.7 Hz, Ar-H-
2); dC (100 MHz, CD3OD) 10.9 (q, 5-CH3), 38.7 (t, C-20), 44.2 (t, C-50),
71.2 (d, C-30), 84.6 (d, C-40), 85.5 (d, C-10), 110.3 (s, C-5), 117.3, 122.7,
131.9 (d, q, Ar(C)H, CF3), 136.9 (d, C-6), 137.0, 138.0 (2 ꢂ s, 3 x Ar(C)),
150.8 (s, C-2), 164.9 (s, C-4); HRMS (ESI) calculated for
4.3. Measurement of TMPKmt enzymatic activity
The reaction medium (0.5 mL final volume) contained 50 mM
Tris-HCl pH 7.4, 50 mM KCl, 2 mM MgCl2, 0.2 mM NADH, 1 mM
phosphoenol pyruvate and 2 units each of lactate dehydrogenase,
pyruvate kinase and nucleoside diphosphate kinase. Activity was
determined using the coupled spectrophotometric assay at 334 nm
(0.5 mL final) in an Eppendorf ECOM 6122 photometer [38]. One
C
17H19ClF3N4O6S: 499.0660. Found 499.0677 (MHþ).
4.2.12. 50-N-[N-(4-Chlorophenyl)sulfamoyl]amino]- 50-deoxy-
b-D-
thymidine 2i
unit of enzyme activity corresponds to 1
mmole of the product
formed in 1 min at 30 ꢁC and pH 7.4. The concentration of ATP
(0.5 mM) and dTMP (0.05 or 0.5 mM) was kept constant, whereas
the concentrations of the sulfamide analogues were varied be-
tween 0.06 and 0.6 mM.
General Procedure A, using sulfamoyl chloride 7b, and purifi-
cation by RP-HPLC (Luna C-18 column (Phenomenex); eluent: A
(0.05% TFA in H2O) and B MeCN; gradient: the sample was run at
1 mL/min with a gradient of 0e45% B; column oven: 40 ꢁC; detec-
tion: UV 210 nm and 280 nm), afforded sulfamide 2i (26 mg, 49%) as
4.4. Measurement of anti-mycobacterial activity vs. M. smegmatis
a white solid. m.p 105e107 ꢁC (EtOH/Et2O); [
in CH3OH); ymax (neat) 3207 (N-H), 1328 (s, S¼O), 1150 (s, S¼O)
cmꢀ1
dH (400 MHz, CD3OD) 1.87 (3H, s, 5-CH3), 2.20 (2H, at, J
a
]
D [20] þ22.4 (c, 0.25
The anti-mycobacterial activities of sulfamides 2g-k were per-
formed using M. smegmatis. Test compounds and ethambutol (EB)
were prepared in DMSO at 40 mg/mL, and subsequent 2 fold serial
;
6.3 Hz, H-2a’, H-2b’), 3.13e3.28 (2H, m, H-5a’ and H-5b’), 3.85(1 H,
aq, J 4.7 Hz, H-40), 4.28 (1 H, aq, J 5.1 Hz, H-30), 6.12 (1H, t, J1,2 7.0 Hz,
H-10), 7.18, 7.26(4H, 2 ꢂ d, J 8.2 Hz, 4 ꢂ Ar-H), 7.50 (1H, s, H-6); dC
(100 MHz, CD3OD) 10.9 (q, 5-CH3), 38.8 (t, C-20), 44.2 (t, C-50), 71.2
(d, C-30), 84.7 (d, C-40), 85.5 (d, C-10), 110.3 (s, C-5), 120.3, 128.6
(2 ꢂ d, 4 ꢂ Ar(C)H), 137.0 (d, C-6), 137.2 (s, 2 ꢂ Ar(C)), 150.8 (s, C-2),
165.0 (s, C-4); HRMS (ESI) calculated for C16H20ClN4O6S: 431.0787.
Found 431.0788 (MHþ).
dilutions were performed in 100
in 96 well microplates, producing compound concentrations across
the plate of 1000, 500, 250, 125, 62, 31, 15, 7.5, and 3.75 g/mL.
ml of LB/T media and DMSO (1.25%)
m
Following the serial dilation each well had a constant concentration
of DMSO (2.5%). Approximately 4.5 ꢂ 106 cfu/mL of M. smegmatis was
added to each well, to give a total volume of 200 ml. Control wells
contained only bacteria with 2.5% DMSO in LB/T media. The plates
were incubated at 37 ꢁC for 18 h. After this time, 10
ml of Alamar Blue
4.2.13. 50-N-[N-(4-Bromophenyl)sulfamoyl]amino]- 50-deoxy-
b-D-
thymidine 2j
dye was added to all wells, and the plate was then incubated for a
further 5 h. The wells were then observed for a colour change (blue
to pink), and the MIC value was determined visually.
General Procedure A, using sulfamoyl chloride 7c, and purifi-
cation by RP-HPLC (Luna C-18 column (Phenomenex); eluent: A
(0.05% TFA in H2O) and B MeCN; gradient: the sample was run at
1 mL/min with a gradient of 0e45% B; column oven: 40 ꢁC; detec-
tion: UV 210 nm and 280 nm), afforded sulfamide 2j (24 mg, 41%) as
Acknowledgements
The authors thank the University of Canterbury (PhD Scholar-
ship to KS) and the Biomolecular Interaction Centre for financial
a white solid. m.p 115e117 ꢁC (EtOH/Et2O); [
a
]
D [20] þ15.2 (c, 0.25
in CH3OH); ymax (neat) 3208 (N-H), 1386 (s, S¼O), 1150 (s, S¼O)
cmꢀ1 dH (400 MHz, CD3OD) 1.87 (3H, s, 5-CH3), 2.18 (2H, at, J 6.3 Hz,
H-2a’, H-2b’), 3.14e3.28 (2H, m, H-5a’ and H-5b’), 3.85 (1 H, aq, J
5.1 Hz, H-40), 4.27 (1 H, aq, J 4.7 Hz, H-30), 6.11 (1H, t, J1,2 7.0 Hz, H-
10), 7.13 (2H, d, J 8.6 Hz, 2 ꢂ Ar-H), 7.40 (2H, d, J 9.0 Hz, 2 ꢂ Ar-H),
7.50 (1H, s, H-6); dC (100 MHz, CD3OD) 11.0 (q, 5-CH3), 38.8 (t, C-20),
44.2 (t, C-50), 71.2 (d, C-30), 84.7 (d, C-40), 85.5 (d, C-10),110.3 (s, C-5),
ꢀ ꢁ
support. Helene Munier-Lehmann was supported by INSERM, CNRS
and the Institut Pasteur.
References