Send Orders of Reprints at
[email protected] Medicinal Chemistry, 2013, 9, 77-83
77
Novel 2-hydrazino-pyrimidin-4(3H)-one Derivatives with Pseudofunctional- Similarity to Siderophores as Potential Antimycobacterial Agents Ranjeet Bairwaa, Nilesh R. Tawaria, Amruta Alatea, Swapnil Anama, Mariam S. Degania* Muktikant Rayb and Ramakrishna M. G. Rajanb a
Institute of Chemical Technology, Nathalal Parikh Marg, Matunga, Mumbai-400019, India
b
Radiation Medicine Center, Bhabha Atomic Research Centre, Tata Memorial Hospital Annex, Parel, Mumbai-400012 Abstract: Siderophores are small molecules produced by bacteria under iron-scarcity conditions faced by bacteria inside host. Sideophores bind iron with high affinity (Kd < 10-25 M) and are required for iron transport into the bacterial cell. Small molecules interfering with siderophore functioning can be promising anti-mycobacterial agents. Several molecules with hydrazone as a structural feature are known to have metal chelating property. This prompted us to investigate the metal chelating ability of 2-hydrazino-pyrimidin-4(3H)-one derivatives. In this light, a library of 22 novel molecules with 2hydrazino-pyrimidin-4(3H)-one moiety was synthesized and the compounds were evaluated against M. tuberculosis under iron-limiting and iron-rich conditions. Interestingly, several molecules showed promising (MIC: 7 fold active against Mtb under iron-limiting
Novel 2-hydrazino-pyrimidin-4(3H)-one Derivatives with Pseudo-functional-
Medicinal Chemistry, 2013, Vol. 9, No. 1
79
Table 1. Antimycobacterial Evaluation of 2-hydrazino-pyrimidin-4(3H)-one Derivatives Under Iron-Limiting (GAST-D) and IronRich (GAST-D-Fe) Conditions O R4
HN R1
N
N H
N
R3
R2 Structure R1
R2
R3
R4
MIC GAST-D (M)
Code
MIC GAST-DFe (M)
*Ratio
CC50 (M)
4a
-Ph
-Ph
-CH3
-H
15.51
62.04
4
483.00
4b
-Ph
-Ph
-Ph
-H
20.49
81.97
4
327.49
4c
-Ph
-Ph
-CH3
-CH2Ph
116.75
253.81
2
291.53
4d
-Ph
-Ph
-CH3
-(CH2)2 CH3
109.29
273.22
3
360.82
4e
1H-1,2,4triazol-1-yl
2,4di-FPh
-CH3
-H
88.77
355.07
4
347.51
4f
1H-1,2,4triazol-1-yl
2,4di-FPh
-Ph
-H
162.16
324.32
2
311.75
4g
1H-1,2,4triazol-1-yl
2,4di-FPh
-CH3
-CH2Ph
289.60
373.68
1
273.29
1H-1,2,4triazol-1-yl
2,4di-FPh
-CH3
-(CH2)2 CH3
167.82
335.63
2
264.68
4i
1H-1,2,4triazol-1-yl
2,4di-FPh
-CH3
-(CH2)3 CH3
275.97
389.61
1
331.33
4j
-Ph
4-ClPh
-CH3
-H
11.48
91.88
8
457.51
4k
-Ph
2,4di-OHPh
-CH3
-H
6.48
50.25
8
454.90
4l
-Ph
-CH2Ph
-CH3
-H
32.84
65.69
2
439.74
4m
-Ph
-(CH2)2 –4Cl-Ph
-CH3
-H
4.38
35.05
8
373.46
4n
-Ph
-(CH2)2 –triOCH 3-Ph
-CH3
-H
13.99
55.93
4
286.73
4o
-Ph
-(CH2)2 –4Cl-Ph
-Ph
-H
7.56
120.90
16
333.40
4p
-4ClPh
4-ClPh
-CH3
-H
16.84
134.72
8
375.09
4q
-Ph
-CH2 CH 3
-CH3
-H
42.45
339.62
8
632.81
4r
-CH3
-CH2CH (CH3) 2
-CH3
-H
41.39
82.79
2
742.27
-CH3
-CH2CH (CH3) 2
-Ph
-H
39.61
79.23
2
523.98
4h
4s
4t
2-OHPh
-CH3
-CH3
-H
10.01
80.08
8
441.38
4u
2,4di-OHPh
-CH3
-CH3
-H
4.56
72.99
16
445.26
4v
3-OHPh
-CH3
-CH3
-H
9.23
38.76
2
534.30
500
INH
-
*Ratio = MIC GAST-D-Fe/MIC GAST-D
condition than iron-rich condition out of which two compounds 4o and 4u stood out in the group with 16 fold higher potency under iron-limiting condition. The compounds were also evaluated for their cytotoxicity in VERO cell line to estimate their safety profile. Calculated CC50 values are given in Table 1. Interestingly, the most potent compound 4m exhibited good safety profile with selectivity index ~ 85.
5. CONCLUSION In conclusion a novel scaffold, incorporating 2hydrazino-pyrimidin-4(3H)-one core was envisaged as potential part of molecule which could act as pseudosiderophore-mimics. A library of 22 compounds was synthesized and the molecules were evaluated against M. tuberculosis under iron-limiting conditions which mimic the iron-
80 Medicinal Chemistry, 2013, Vol. 9, No. 1
scarcity condition that bacteria encounter in host and ironrich condition for comparison. Interestingly, several of the compounds showed promising selective activity under ironlimiting condition. Of particular interest, compound, 4m, showed 4.38 M, activity under iron-limiting media, with 16 fold selectivity to that of under iron-rich media. Furthermore, compound 4m was found to be non-toxic at 100 M. Thus this study provides several promising lead molecules which could be used for further development against tuberculosis. 6. EXPERIMENTAL 6.1. Chemistry All melting points (m.p.) were recorded on Thermomik Compbell electronics, having oil-heating system and were uncorrected. The microwave reactions were carried out using CEM Focused Microwave System in monomode, Model Discover. Analytical thin-layer chromatography (TLC) was carried out on precoated plates SiO2 (silica gel 60, F 254, Merck). SiO2 (Silica gel 420, Merck) was used for column chromatography using CombiFlash® RETRIEVE® system. FTIR spectra were recorded on “Buck scientific infrared spectroscopy M500 spectrophotometer” using KBr pellets. All the NMR spectra were recorded on FT-NMR JEOL 60 MHz or JEOL AL 300 MHz spectrometer with CDCl3 as solvent using tetramethyl silane (TMS) as internal reference: s = singlet, bs = broad singlet, d = doublet, d = double doublet, t = triplet, m = multiplet. The mass spectra were recorded on a Waters Q/TOF Micromass spectrometer or on API 3000 LCMS. 6.1.1. Synthesis of 2-hydrazino-pyrimidin-4(3H)-one A mixture of ketone 1 (16.48 mmol), semicarbazide hydrochloride 2 (16.48 mmol) and -ketoester 3 (16.48 mmol) were suspended in 30 mL absolute ethanol. This reaction mixture was subjected to microwave irradiation with power of 60 Watt, for 10 min (target temperature 100°C). After 10 min triethylamine (8.24mmol) was added in and reaction mixture was subjected to microwave irradiation at the power of 100 Watt, for 15-20 min (target temperature 100°C). Most of the starting material consumed after this time, the reaction mixture was then concentrated under reduced pressure and partitioned between water (50 mL) and EtOAc (50 mL). The organic layer was separated, and the aqueous layer was extracted further with EtOAc (2 25 mL). The combined organic layers were washed with brine (75 mL), dried (NaSO4), and concentrated in vacuo. Column purification using flash chromatography (SiO2, 5 g, 15 % EtOAc/hexanes) of the crude product yielded pure 2hydrazino-pyrimidin-4(3H)-one derivatives.
Bairwa et al.
CDCl3): ppm 9.20 (s, 1H, NH), 7.57-7.27 (m, 10H, ArH), 5.79 (s, 1H, pyrimidone-H), 2.20 (s, 3H, CH3), 1.72 (bs, 1H, NH); 13C NMR (300 MHz, CDCl3): ppm 165.67, 162.63, 152.14, 151.41, 136.28, 131.54, 131.52, 131.21, 130.20, 130.15, 130.12, 129.90, 129.85, 128.49, 128.45, 127.68, 104.16, 23.73; MS: 305 [M+1]+. 2-(2-(diphenylmethylene)hydrazinyl)-6-phenylpyrimidin4(3H)-one (4b): White crystalline solid; Yield: 66%; m.p.: 278 ºC; IR (KBr): Vmax cm-1 3331, 3154, 2937,1674, 1565, 1492, 1435, 1120, 1015, 805, 699; 1H NMR (300 MHz, CDCl3): ppm 9.78 (s, 1H, NH), 7.95-7.31 (m, 15H, Ar-H), 6.35 (s, 1H, pyrimidone-H), 1.86 (bs, 1H, NH); 13C NMR (300 MHz, CDCl3): ppm 163.70, 151.91, 151.80, 136.91, 130.35, 130.22, 130.11, 129.90, 129.86, 128.94, 128.84, 128.72, 128.60, 128.46, 128.20, 128.07, 127.63, 127.56, 127.52, 126.88, 126.81, 126.78, 101.54; MS: 367 [M+1]+. 5-benzyl-2-(2-(diphenylmethylene)hydrazinyl)-6methylpyrimidin-4(3H)-one (4c): Pale yellow solid; Yield: 43.8%; m.p.: 250 ºC; IR (KBr): Vmax cm-1 3351, 3024, 2916, 1640, 1601, 1575, 1492, 1442, 1117, 772; 1H NMR (300 MHz, CDCl3): ppm 9.61(s, 1H, NH), 7.54-7.16 (m, 15H, Ar-H); 3.86 (s, 2H, CH2 -Ar), 2.14 (s, 3H, CH3), 1.78 (bs, 1H, NH); 13C NMR (300 MHz, CDCl3): ppm 162.47, 162.04, 151.53, 149.27, 140.23, 136.32, 131.57, 130.31, 130.12, 130.05, 129.92, 129.88, 128.71, 128.49, 128.43, 128.49, 128.43, 128.35, 128.19, 127.63, 127.57, 127.50, 127.49, 125.9, 114.933, 104.35, 30.73, 21.72; MS: m/z 395 [M+1]+, 396 [M+2]+. 2-(2-(diphenylmethylene)hydrazinyl)-6-methyl-5propylpyrimidin-4(3H)-one (4d): Pale yellow solid; Yield: 39 %; m.p.: 202ºC; IR (KBr): Vmax cm-1 3320, 3060, 2916, 1654, 1555, 1384, 1166, 958; 1H NMR (300 MHz, CDCl3): ppm 8.99 (bs, 1H, NH), 7.55-7.26 (m, 10H Ar-H), 2.46-2.43 (t, 2H, CH2), 2.14 (s, 3H, CH3), 1.7-1.45 (m, 3H, CH2-, NH), 0.95-0.91(t, 3H, CH2CH3); 13C NMR (300 MHz, CDCl3): ppm 166.25, 164.77, 162.31, 161.22, 160.25, 151.08, 148.81, 136.43, 131.67, 130.32, 130.13, 129.90, 128.89, 128.53, 128.47, 127.47, 116.46, 31.09, 25.24, 22.77, 14.04; MS: m/z 347 [M+1] +.
Representative example: 2-(2-(diphenylmethylene)hydrazinyl)-6-methylpyrimidin-4(3H)-one (4a):
2-(2-(1-(2,4-difluorophenyl)-2-(1H-1,2,4-triazol-1yl)ethylidene)hydrazinyl)-6-methyl pyrimidin-4(3H)-one (4e): Brown solid; Yield: 39.64 %; m.p.: 248 ºC; IR (KBr): Vmax cm-1 3351, 3120, 2967, 2801, 1642, 1572, 1509, 1422, 1271, 1140, 1017, 708; 1H NMR (300 MHz, CDCl3): ppm 9.71(bs, 1H, NH), 8.63 (s, 1H, triazole-H); 8.39(s, 1H, triazole-H), 7.86-7.71 (m, 3H, Ar-H,); 6.14 (S, 1H, pyrimidoneH), 3.28 (s, 2H, CH2-triazole), 2.57 (bs, 1H, NH), 2.15 (s, 3H, CH3); 13C NMR (300 MHz, CDCl3): ppm 161.03, 161.00, 160.52, 151.67, 151.67, 150.24, 143.67, 131.161, 110.64, 110.59, 110.52, 103.15, 45.49, 18.21. MS: 346 [M+1]+, 347[M+2]+.
The ethanolic solution (30 mL) of benzophenone (2g, 10.98 mmol), aminoguanide hydrochloride (1.20 g, 10.98 mmol) and ethylacetoacetate (1.43g, 10.98 mmol) triethyl amine (0.55 g, 5.49 mmol) was subjected to microwave irradiation as described in general procedure. The workup followed by column purification yielded pure white solid. Yield: 50%; m.p.: 262ºC; IR (KBr): Vmax cm-1 3140, 3046, 2916, 2874, 1634, 1151, 1132, 826; 1H NMR (300 MHz,
2-(2-(1-(2,4-difluorophenyl)-2-(1H-1,2,4-triazol-1yl)ethylidene)hydrazinyl)-6-phenyl pyrimidin-4(3H)-one (4f): Brown solid; Yield: 37.5%; m.p.: 262 ºC; IR (KBr): Vmax cm-1 3139, 3047, 2957, 2889, 1633, 1511, 1235, 1170, 1132, 826; 1H NMR (300 MHz, CDCl3): ppm 8.40 (s, 1H, triazole-H), 8.04(bs, 2H, triazole-H and NH), 7.99-6.88 (m, 8H, Ar-H), 5.73 (s, 1H, pyrimidone-H), 3.27 (s, 2H, CH2 triazole), 2.59 (bs, 1H, -NH); 13C NMR (300 MHz, CDCl3):
Novel 2-hydrazino-pyrimidin-4(3H)-one Derivatives with Pseudo-functional-
ppm 170.02, 163.49, 160.18, 156.84, 151.43, 151.29, 143.40, 141.61, 139.46, 128.11, 127.82, 126.80, 126.28, 121.00, 118.16, 118.06, 110.95, 110.70, 110.36, 55.23. MS: 408 [M+1]+, 409 [M+2]+. 5-benzyl-2-(2-(1-(2,4-difluorophenyl)-2-(1H-1,2,4-triazol-1-yl)ethylidene)hydrazinyl)-6-methylpyrimidin-4(3H)one (4g): Light brown solid; Yield: 48.75 %; m.p.: 238 ºC; IR (KBr): Vmax cm-1 3352, 3180, 1654, 1604, 1534, 1402, 1245, 1146, 1017, 973; 1H NMR (300 MHz, CDCl3): ppm 8.2 (s, 1H, triazole-H); 7.9 (s, 1H, triazole-H), 7.59-6.86 (m, 6H, Ar-H and NH,); 5.72 (s, 1H, pyrimidone-H), 4.72 (s, 2H, CH2-triazole), 2.90 (s, 3H, CH3), 2.59(bs, 1H, NH); 13C NMR (300 MHz, CDCl3): ppm 163.75, 160.51, 160.48, 151.67, 151.63, 143.27, 141.85, 139.33, 128.98, 128.81, 126.70, 126.70, 121.12, 120.97, 118.19, 111.10, 111.07, 110.76, 46.48, 31.46, 17.65; MS: 436 [M+1]+, 437 [M+2]+. 2-(2-(1-(2,4-difluorophenyl)-2-(1H-1,2,4-triazol-1-yl) ethylidene)hydrazinyl)-6-methyl-5-propylpyrimidin-4(3H)-one (4h): Dark brown solid; Yield: 43.2 %; m.p.: 211 ºC; IR (KBr): Vmax cm-1 3339, 3046, 2887, 1635, 1511, 1430, 1235, 1132, 1016, 827; 1H NMR (300 MHz, CDCl3): ppm 8.28 (s, 1H, triazole-H), 7.91 (s, 1H, triazole-H), 7.87-6.26 (m, 4H, Ar-H and NH), 5.61(s, 1H, pyrimidone-H), 3.51(s, 2H, CH2-triazole), 2.34 (t, 2H, CH2), 2.18 (s, 3H, CH3), 1.461.39 (m, 5H, CH2-CH2-CH3 and NH), 0.96-0.92 (t, 3H, CH3); MS: 388 [M+1]+, 389 [M+2]+. 5-butyl-2-(2-(1-(2,4-difluorophenyl)-2-(1H-1,2,4-triazol1-yl)ethylidene)hydrazinyl)-6-methylpyrimidin-4(3H)-one (4i): Brown solid; Yield: 48.32%; m.p.: 205 ºC; IR (KBr): Vmax cm-1 3266, 3123, 1645, 1597, 1559, 1500, 1434, 1278, 1136, 966; 1H NMR (300 MHz, CDCl3): ppm 8.19 (s, 1H, triazole-H), 7.96 (s, 1H, triazole-H), 7.61 (m, 4H, Ar-H and NH), 4.73 (s, 2H, CH2-triazole), 2.48 (t, 2H, CH2), 2.14 (s, 3H, CH3), 1.70 (bs, 1H, NH), 1.467-1.37 (m, 2H, CH2-CH3), 0.953-0.91 (t, 3H, CH3); MS: 402 [M+1]+, 403 [M+2]+. 2-(2-((4-chlorophenyl)(phenyl)methylene)hydrazinyl)-6methylpyrimidin-4(3H)-one (4j): Red crystaline solid; Yield:62 %; m.p.:231 ºC; IR (KBr): Vmax cm-1 3414, 3300, 3057, 1638, 1596, 1508, 1136, 1012, 965; 1H NMR (300 MHz, CDCl3): ppm 9.14(bs, 1H, NH), 7.56-7.27 (m, 10H Ar-H), 5.80 (s, 1H, pyrimidone-H), 2.14(s, 3H, CH3); MS: 339 [M+1]+, 340 [M+2]+. 2-(2-((2,4-dihydroxyphenyl)(phenyl)methylene)hydrazinyl)-6-methylpyrimidin-4(3H)-one (4k): Dark brown solid powder; Yield: 49.7 %; m.p.:185 ºC; IR (KBr): Vmax cm-1 3443, 3398 2928, 1679, 1586, 1252; 1H NMR (300 MHz, CDCl3): ppm 9.67 (bs, 1H, NH), 7.98-6.1(m, 10H, Ar-H), 5.52 (s,1H, pyrimidone-H), 3.34 (bs, 2H, 2xOH), 2.11(s, 3H, CH3); MS: 337 [M+1]+. 2-(2-(1,2-diphenylethylidene)hydrazinyl)-6-methylpyrimidin-4(3H)-one (4l): White solid; Yield: 60.47 %; m.p.:205 ºC; IR (KBr): Vmax cm-1 3433, 3365, 2915, 1663, 1443, 1337, 1125; 1H NMR (300 MHz, CDCl3): ppm 9.9 (bs, 1H, NH), 7.75-7.16 (m, 11H, Ar-H), 5.42 (s, 1H, pyrimidone-H), 4.35(s, 2H, CH2), 2.71(bs, 1H, NH), 2.11 (s, 3H, CH3); MS: 319 [M+1]+. 2-{2-[3-(4-chlorophenyl)-1-phenylpropylidene]hydrazino}-6-methylpyrimidin-4(3H)-one (4m): White crystalline
Medicinal Chemistry, 2013, Vol. 9, No. 1
81
solid; Yield: 54%; m.p.:181 ºC; IR (KBr): Vmax cm-1 3355, 3055, 2920, 1640, 1575, 1491, 1442, 1117, 772, 701; 1H NMR (300 MHz, CDCl3): ppm 9.61(bs, 1H, NH),7.677.47(dd, 4H, 4-Cl-phenyl-H,) 7.41 -7.09(m, 5H, Ar-H), 5.65 (s, 1H, pyrimidone-H), 3.2(s, 1H, NH), 3.09-3.03(t, 2H, ArCH2), 2.1(s, 2H, Ar-CH2-CH2), 2.19 (s, 3H, CH3); 13C NMR (300 MHz, CDCl3): ppm 186.47, 153.46, 151.46, 138.7, 136.3, 132.4, 129.9, 129.7, 129.6, 128.7, 128.6, 128.5, 128.3, 126.7, 126.5, 126.3, 102.8, 31.5, 29, 22.9; MS: 367 [M+1]+, 368 [M+2]+. 6-methyl-2-(2-(1-phenyl-3-(3,4,5-trimethoxyphenyl)propylidene)hydrazinyl) pyrimidin -4(3H)-one (4n): Yellow solid; Yield: 44.6%; m.p.:241 ºC; IR (KBr): Vmax cm-1 3310, 3176, 2958, 2864, 1669, 1583, 1443, 1294, 1121, 1056, 769; 1 H NMR (300 MHz, CDCl3): ppm 8.21(bs, 1H, NH), 7.926.25 (m, 6H, Ar-H), 5.77 (s, 1H, pyrimidone-H), 3.88 (s, 9H, 3xOCH3), 2.56-2.45 (t, 2H Ar-CH2-CH2), 1.89-1.62 (t, 2H Ar-CH2-CH2), 2.15 (s, 3H, CH3), MS: 423 [M+1]+, 424 [M+2]+. 2-{-2-[3-(4-chlorophenyl)-1-phenylpropylidene]hydrazino}-6-phenylpyrimidin-4(3H)-one (4o): Pale yellow crystalline solid; Yield: 45.0%; m.p.: 212ºC; IR (KBr): Vmax cm-1 3138, 3040, 2919, 2874, 1634, 1511, 1428, 1133, 1016, 826; 1 H NMR (300 MHz, CDCl3): ppm 7.8-7.701 (dd, 4H, 4-Clphenyl-H), 7.44-7.15 (m, 11H, Ar-H and NH), 6.25 (s, 1H, pyrimidone-H), 3.04-2.99 (t, 2H, Ar-CH2-), 2.92-2.85 (t, 2H, Ar-CH2-CH2), 1.73 (bs, 1H, NH); 13C NMR (300 MHz, CDCl3): ppm 163.4, 152.3, 151.8, 138.3, 136.8, 135.9, 132.8, 130.49 130.42, 130, 129.6, 129.5, 129, 128.9, 128.78, 128.72, 128.6, 128.58, 126.88, 126.33, 126.29, 101.41, 31.4, 28.7; MS: 429 [M+1]+, 430 [M+2]+. 2-(2-(bis(4-chlorophenyl)methylene)hydrazinyl)-6methylpyrimidin-4(3H)-one (4p): Red solid; Yield: 36.00%; m.p.:252 ºC; IR (KBr): Vmax cm-1 3434, 2921, 1657, 1619, 1486, 1H NMR (300 MHz, CDCl3): ppm 9.9 (bs, 1H, NH); 7.43-7.16 (m, 9H, Ar-H), 5.42 (s, 1H, pyrimidone-H), 2.11 (s, 3H, CH3); MS: 374 [M+1]+. 4-methyl-6-(2-(1-phenylpropylidene)hydrazinyl)pyridin2(1H)-one (4q): Yield: 55.0%; m.p.:143 ºC; IR (KBr): Vmax cm-1 3058, 1649, 1598, 1492, 1237, 1083; 1H NMR (300 MHz, CDCl3): ppm 7.77-7.26 (m, 6H, Ar-H and NH), 5.77 (s, 1H, pyrimidone-H), 2.83-2.59 (q, 2H, N=C-CH2), 2.19 (s, 3H, -COCH3), 1.77 (bs, 1H, -NH), 1.34-1.09 (t, 3H, CH3); 13 C NMR (300 MHz, CDCl3): 166, 162, 151.5, 143, 137.2, 131, 128.9, 128.9, 128.5, 128.5, 127.7, 103.0, 29.9, 22.4, 14.1; MS: 256 [M+1]+ 4-methyl-6-(2-(5-methylhexan-3-ylidene)hydrazinyl)pyridin-2(1H)-one (4r): Yield: 60.0%; m.p.: 130 ºC; IR (KBr): Vmax cm-1 3250, 3014, 2956, 1667, 1584, 1437, 1336, 1054; 1 H NMR (300 MHz, CDCl3): ppm 9.38 (bs, 1H, NH), 5.73 (s, 1H, pyrimidone-H), 2.24 (s, 3H, -CH3), 2.16-1.89- (m, 5H, -CH-CH2-C-CH2, NH,), 0.95-0.87 (m, 9H, 3xCH3); 13C NMR (300 MHz, CDCl3): ppm 166.0, 160.7, 152.4, 147.0, 128.1, 105.6, 43.5, 26.2, 25.4, 25.0, 22.4, 14.3, MS: 236 [M+1]+ 6-(2-(5-methylhexan-3-ylidene)hydrazinyl)-4-phenylpyridin-2(1H)-one (4s): White solid; Yield: 52.0%; m.p.: 184 ºC; IR (KBr): Vmax cm-1 3130, 2947, 1681, 1579, 1470, 1415, 1123, 999; 1H NMR (300 MHz, CDCl3): ppm 8.99
82 Medicinal Chemistry, 2013, Vol. 9, No. 1
(bs, 1H, NH), 7.84-7.27 (m, 5H, Ar-H), 6.35 (s, 1H, pyrimidone-H), 2.13-1.87 (m, 6H, -CH-CH2-C-CH2 and NH), 0.970.88 (m, 9H, 3xCH3); 13C NMR (300 MHz, CDCl3): ppm 165, 161.5, 157.1, 143.5, 141.1, 129.2, 129.2, 128.3, 128.3, 126.5, 114.2, 104.2, 42.0, 27.7, 25.4, 25.2, 20.4, 14.8; MS: 298 [M+1]+ 6-(2-(1-(2-hydroxyphenyl)ethylidene)hydrazinyl)-4methylpyridin-2(1H)-one (4t): Dark brown solid; Yield: 52.6%; m.p.: 165 ºC; IR (KBr): Vmax cm-1 3424, 3382, 3300, 3014, 1649, 1445, 1367, 1254, 1162, 964; 1H NMR (300 MHz, CDCl3): ppm 10.25 (bs, 1H, NH), 7.25-6.39 (m, 6H, Ar-H), 5.49 (s, 1H, pyrimidone-H), 3.24 (s, 3H, N=C-CH3), 2.03 (s, 3H, -CH3); MS: 259 [M+1]+ 6-(2-(1-(2,5-dihydroxyphenyl)ethylidene)hydrazinyl)-4methylpyridin-2(1H)-one (4u): Yield: 52.6%; m.p.: 201 ºC; IR (KBr): Vmax cm-1 3400, 3191, 2916, 1659, 1629, 1571, 1315, 1210; 1H NMR (300 MHz, CDCl3): ppm 8.13 (bs, 1H, NH), 7.99-7.93 (m, 2H, Ar-H), 7.39 (s, 1H, Ar-H), 5.49 (s, 1H, pyrimidone-H), 3.4 (bs, 2H, 2xOH), 3.21 (s, 3H, N=C-CH3), 2.03 (s, 3H, -CH3); MS: 274 [M+1]+ 6-(2-(1-(3-hydroxyphenyl)ethylidene)hydrazinyl)-4methylpyridin-2(1H)-one (4v): Yield: 52.6%; m.p.: 198ºC; IR (KBr): Vmax cm-1 3407, 3234, 3092, 1644, 1508, 1452, 1245, 1122, 976; 1H NMR (300 MHz, CDCl3): ppm 8.13 (bs, 1H, NH), 7.99-7.93 (m, 2H, Ar-H), 7.39 (s, 1H, Ar-H), 5.49 (s, 1H, pyrimidone-H), 3.4 (bs, 2H, 2xOH), 3.21 (s, 3H, -N=C-CH3), 2.03 (s, 3H, -CH3); MS: 258.13 [M+1]+
Bairwa et al.
MIC was determined visual inspection by the dye color changed from blue to pink. 6.2.2. Cytotoxicity The compounds were further examined for toxicity in a mammalian VERO cell line (C1008). Cells were seeded in 96-well microtitre plates (Falcon Becton Dickinson, Franklin Lakes, NJ) at a density of 1105 cells/well and incubated at 37 °C in 5% CO2 and observed for morphological changes at 24, 48 and 72 h. Following 72 h of incubation at 37 ºC in 5% CO2, viability was assessed on the basis of cellular conversion of (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) into a formazan product [28]. The concentration required for 50% inhibition (CC50 value) was determined by reduced the optical density values (OD570) with respect to non-treated cells by 50%. CONFLICT OF INTEREST The author(s) confirm that this article content has no conflicts of interest. ACKNOWLEDGEMENTS Ranjeet Bairwa, Amruta Alate, Swapnil Anam are thankful to University Grand Commission (UGC), India and Nilesh R. Tawari is thankful to Department of Biotechnology (DBT), India for financial support. This work was supported by DBT grant no. BT/PR7858/Med/14/1142/2006.
6.2. Biology
REFERENCES
6.2.1. Antitubercular Activity
[1]
All synthesized compounds (4a-v) were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mg/mL and were screened against M. tuberculosis H37Rv.
[2]
Preparation of mycobaterial cell culture: M. tuberculosis (H37Rv) was grown in Youman’s media till mid log phase. The pellicle was homogenized in Middlebrook 7H9 broth by vortexing along with glass beads in a sealed tube. The suspension was allowed to stand for 15 min and then the supernatant from the top was removed to a fresh tube and OD540 adjusted to 0.1 ( 107 cells/ml).
[3] [4] [5] [6]
Media preparation and siderophore production: Irondeficient media GAST-D and iron-rich media GAST-D-Fe were prepared according to reported procedure [11, 13]. Mid log phase cells of M. tuberculosis (H37Rv) were grown on GAST-D media for 15 days at 37°C without shaking in order to induce siderophore production [11, 13]. Anti-mycobacterial evaluation: Compounds were tested against M. tuberculosis H37Rv under iron-limiting and iron rich conditions in order to determine their minimum inhibitory concentration (MIC) in the two fold broth microdilution assay [32-36]. Homogenous mycobacterial culture suspension from GAST-D media was seeded in microtitre plates at density of 105 cells per well along with serially diluted compound to a final volume of 200 l in GAST-D media for iron-deficient conditions and GAST-D-Fe media for ironrich conditions [11, 13]. The control received equivalent amount of DMSO. The plates were incubated at 37 C for 8 days. Resazurin dye (0.02%) was added incubated for 48 hr.
[7]
[8] [9]
WHO Report 2008 Global tuberculosis control - surveillance, planning, financing http://www.who.int/tb/publications/global_report/2008/summary/en/index.html Dye, C.; Scheele, S.; Dolin, P. Pathania, V.; Raviglione, M. C. for the WHO Global Surveillance and Monitoring Project (1999) Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country. J. Am. Med. Assoc. 1999, 282, 677-686. Murray, J.F. Tuberculosis and HIV infection: a global perspective. Respiration 1998, 65, 335-342. B. R. Bloom, Eds. Tuberculosis: Pathogenesis, Protection, and Control, ASM Press: Washington DC, 1994. Zignol, M.; Hosseini, M. S.; Wright, A.; Weezenbeek, C. L.; Nunn, P.; Watt, C. J.; Williams, B. G.; Dye, C. Global incidence of multidrug-resistant tuberculosis. J. Infect. Dis. 2006, 194, 479-485. Cole, S. T.; Brosch, R.; Parkhill, J.; Garnier. T.; Churcher. C.; Harris. D.; Gordon, S. V.; Eiglmeier, K.; Gas, S.; Barry, C. E.; III.; Tekaia, F.; Badcock, K.; Basham, D.; Brown, D.; Chillingworth, T.; Connor, R.; Davies, R.; Devlin, K.; Feltwell, T.; Gentles, S.; Hamlin, N.; Holroyd, S.; Hornsby, T.; Jagels, K.; Krogh, A.; McLean, J.; Moule, S.; Murphy, L.; Oliver, K.; Osborne, J.; Quail, M. A.; Rajandream, M.-A.; Rogers, J.; Rutter, S.; Seeger, K.; Skelton, J.; Squares, R.; Squares, S.; Sulston, J. E.; Taylor, K.; Whitehead, S.; Barrell, B. G. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 1998, 393, 537-544. Fleischmann, R .D.; Alland, D.; Eisen, J. A.; Carpenter, L.; White, O.; Peterson J. DeBoy, R.; Dodson, R.; Gwinn, M.; Haft, D.; Hickey, E.; Kolonay, J. F.; Nelson, W. C.; Umayam, L. A.; Ermolaeva, M.; Salzberg, S. L.; Delcher, A.; Utterback, T.; Weidman, J.; Khouri, H.; Gill, J.; Mikula, A.; Bishai, W.; Jacobs, W. R.; Jr.; Venter, J. C.; Fraser, C. M. Whole-genome comparison of Mycobacterium tuberculosis clinical and laboratory strains. J. Bacteriol. 2002, 184, 5479-5490. Raymond, K. N.; Dertz, E. A.; Kim, S. S. Enterobactin: an archetype for microbial iron transport. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 3584-3588. Ratledge, C.; Dover, L. G. Iron metabolism in pathogenic bacteria. Annu. Rev. Microbiol. 2000, 54, 881-941.
Novel 2-hydrazino-pyrimidin-4(3H)-one Derivatives with Pseudo-functional[10]
[11]
[12] [13]
[14]
[15]
[16]
[17]
[18]
[19]
[20] [21]
[22] [23]
[24]
Miethke, M.; Marahiel, M. A. Siderophore-based iron acquisition and pathogen control. Microbiol. Mol. Biol. Rev. 2007, 71, 413451. De Voss, J. J.; Rutter, K.; Schroeder, B. G.; Su, H.; Zhu, Y.; Barry, C. E., III The salicylate-derived mycobactin siderophores of Mycobacterium tuberculosis are essential for growth in macrophages. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 1252-1257. Rodriguez, G. M.; Smith, I. Siderophore-based iron acquisition and pathogen control. Microbiol. J. Bacteriol. 2006, 188, 424-451. Ferreras, J. A.; Ryu, J. S.; Di Lello, F.; Tan, D. S.; Quadri, L. E. Small-molecule inhibition of siderophore biosynthesis in Mycobacterium tuberculosis and Yersinia pestis. Nat. Chem. Biol. 2005, 1, 29-32. Somu, R. V.; Boshoff, H.; Qiao, C.; Bennett, E. M.; Barry, C. E. III; Aldrich, C. C. Rationally designed nucleoside antibiotics that inhibit siderophore biosynthesis of Mycobacterium tuberculosis. J. Med. Chem. 2006, 49, 31-34. Neres, J.; Labello, N. P.; Somu, R. V.; Boshoff, H. I.; Wilson, D. J.; Vannada, J.; Chen, L.; Barry, C. E. III; Bennett, E. M.; Aldrich, C. C. Inhibition of siderophore biosynthesis in Mycobacterium tuberculosis with nucleoside bisubstrate analogues: Structure activity relationships of the nucleobase domain of 5'-O-[N(Salicyl)sulfamoyl]adenosine. J. Med. Chem. 2008, 51, 5349-5370. Tawari, N.R.; Degani, M. S. Predictive models for nucleoside bisubstrate analogs as inhibitors of siderophore biosynthesis in Mycobacterium tuberculosis: Pharmacophore mapping and chemometric QSAR study. Mol. Div. 2011, 15, 435-444. Stirrett, K. L.; Ferreras, J. A.; Jayaprakash, V.; Sinha, B. N.; Renc, T.; Quadri, L. E. N. Small molecules with structural similarities to siderophores as novel antimicrobials against Mycobacterium tuberculosis and Yersinia pestis. Bioorg. Med. Chem. Lett. 2008, 18, 2662-2668. Hu, J.; Miller, M. Total Synthesis of a mycobactin S: A siderophore and growth promoter of Mycobacterium Smegmatis, and determination of its growth inhibitory activity against Mycobacterium tuberculosis. J. Am. Chem. Soc. 1997, 119, 3462-3468. Xu, Y.; Miller, M. Total syntheses of mycobactin analogues as potent antimycobacterial agents using a minimal protecting group strategy. J. Org. Chem. 1998, 63, 4314-4322. Cocco, M. T.; Congiu, C.; Onnis, V.; Pusceddu, M. C.; Schivo, M. L.; De Logu, A. Synthesis and antimycobacterial activity of some isonicotinoylhydrazones. Eur. J. Med. Chem. 1999, 34, 10711076. da Silva Lourenço, M.; C.; Ferreira, M. de L.; de Souza, M. V. N. M.; Peralta, A.; Vasconcelos, T. R. A. Synthesis and antimycobacterial activity of (E)-N'-(monosubstituted-benzylidene) isonicotinohydrazide derivatives. Eur. J. Med. Chem. 2008, 43, 1344-1347. Sastry, P. S. S. J.; Lonibala, R. K.; Rao, T. R. Synthesis and structural studies on 3d metal complexes of furfuralisonicotinoyl hydrazone, Synth. React. Inorg. Met.-Org. Chem. 1995, 25, 1261-1278. Narang, K. K.; Singh, M. K.; Goyle, M. R.; Bhuvaneshwari, K. Synthesis, physico-chemical studies and biological activity of glyoxalmonoisonicotinoyl hydrazone complexes with some bivalent transition metal ions and oxdcations. Synth. React. Inorg. Met.Org. Chem. 1991, 21, 1569-1585. Tsintsadze, G.V.; Kurtanidze, R.S.; Mdivani, M.A. Coordination compounds of iron(II) and iron(III) with pyridine carboxylic acid
Recieved: September 06, 2012
Medicinal Chemistry, 2013, Vol. 9, No. 1
[25]
[26] [27]
[28]
[29]
[30]
[31] [32]
[33]
[34]
[35]
[36]
[37]
83
hydrazides and derivatives of isonicotinic acid hydrazide. Prol. Sovren. Bioneorg. Khim. Mater. Vyzed. Ses. Novsibirik 1984, Chem. Abstr. 1987, 107, 16751j. Dubois, J-E.; Fakhrayan, H.; Doucet, J-P.; Chahine, J-M. E. H. Kinetic and thermodynamic study of complex formation between Iron(II) and pyridoxal isonicotinoylhydrazone and other synthetic chelating agents. Inorg. Chem. 1992, 31, 853-859. Edward, J. T.; Gauthier, M.; Chubb, F.L.; Ponka, P. Synthesis of new acylhydrazones as iron-chelating compounds. J. Chem. Eng. Data 1988, 33, 538-540. Degani, M. S.; Bag, S.; Bairwa, R.; Tawari N. R.; Queener. S. F. Novel 2-hydrazino-pyrimidin-4(3h)-one derivatives as potential dihydrofolate reductase inhibitors. J. Heterocyclic Chem. 2010, 47, 558-563. Tawari, N. R.; Degani, M. S. Pharmacophore mapping and electronic feature analysis for a series of nitroaromatic compounds with antitubercular activity. J Comput Chem 2010, 31, 739-751. Bairwa, R.; Kakwani, M.; Tawari, N. R.; Lalchandani, J.; Ray, M. K.; Rajan, M. G. R.; Degani, M. S. Novel molecular hybrids of cinnamic acids and guanylhydrazones as potential antitubercular agents. Bioorg. Med. Chem. Lett. 2010, 20, 1623-1625. M. J. Cooper, R. Hull, M. Wardleworth. Synthesis of some 1,2,4triazoles and 1,2,4-triazolines by reaction of oxamidrazone condensation products with acetic anhydride. J. Chem. Soc. Perkin. Trans. 1 1975, 1433-1437. Bag, S.; Vaze V. V.; Degani, M. S. Microwave assisted benzoin condensation using thiamine as catalyst. J. Chem. Res. 2006, 4, 267-269. Zampieri, D.; Mamolo, M. G.; Vio, L.; Banfi, E.; Scialino, G.; Fermeglia, M.; Ferronec, M.; Priclc, S. Synthesis, antifungal and antimycobacterial activities of new bis-imidazole derivatives, and prediction of their binding to P45014DM by molecular docking and MM/PBSA method. Bioorg. Med. Chem. 2007, 15, 7444-7458. Prado, S.; Janin, Y. L.; Saint-Joanis, B.; Brodin, P.; Michel, S.; Koch, M.; Cole, S. T.; Tillequinc, F.; Bost, P-E. Synthesis and antimycobacterial evaluation of benzofurobenzopyran analogues. Bioorg. Med. Chem. 2007, 15, 2177-2186. Gasse, C.; Douguet, D.; Huteau, V.; Marchal, G.; MunierLehmanna, H.; Pochet, S. Substituted benzyl-pyrimidines targeting thymidine monophosphate kinase of Mycobacterium tuberculosis: Synthesis and in vitro anti-mycobacterial activity. Bioorg. Med. Chem. 2008, 16, 6075-6085. Figueiredo, R.; Moiteiro, C.; Medeiros, M. A.; da Silva, P. A.; Ramos, D.; Spies, F.; Ribeiro, M. O.; Lourenço, M. C. S.; Júnior, I. N.; Gaspar, M. M.; Cruz, M. E. M.; Curto, M. J. M.; Franzblau, S. G.; Orozco, H.; Aguilar, D.; Hernandez-Pando, R.; Costa, M. C. Synthesis and evaluation of rifabutin analogs against Mycobacterium avium and H37Rv, MDR and NRP Mycobacterium tuberculosis. Bioorg. Med. Chem. 2009, 17, 503-511. Palomino, J. C.; Martin, A.; Camacho, M.; Guerra, H.; Swings, J.; Portaels, F. Resazurin microtiter assay plate: simple and inexpensive method for detection of drug resistance in Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 2002, 46, 2720-2722. Freshney, R. L. Culture of animal cells: A Manual of Basic Techniques, 4th edition, Willy-LISS, 2000.
Revised: October 02, 2012
Accepted: October 04, 2012
