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N. Sunduru et al. / European Journal of Medicinal Chemistry 101 (2015) 595e603
mixture was stirred for 12 h at room temperature under inert at-
mosphere. The reaction mixture was diluted with water, extracted
with EtOAc, dried over Na2SO4 and concentrated. The crude residue
was dissolved in EtOAc and triturated with heptanes. The precipi-
tate formed was filtered and dried under vacuum to afford com-
pound 13.
4.16.1. (E)-3-(4-((E)-2-(3-Fluoro-2-hydroxybenzylidene)
hydrazinecarbonyl)phenyl)-N-(4-(N-(5-methylisoxazol-3-yl)
sulfamoyl)phenyl)acrylamide (15a)
Yield: 34%; ESIMS m/z calcd for C27H22FN5O6S [M þ H]þ, 564.14;
found 563.94; 1H NMR (400 MHz, (CD3)2SO):
d 12.16 (bs, 1H), 11.51
(bs, 1H), 11.17 (bs, 1H), 10.53 (s, 1H), 8.68 (s, 1H), 8.02 (d, 2H,
J ¼ 7.00 Hz), 7.89e7.79 (m, 6H), 7.71 (d,1H, J ¼ 15.64 Hz), 7.38 (d,1H,
J ¼ 7.76 Hz), 7.27 (t, 1H, J ¼ 9.46 Hz), 6.98e6.90 (m, 2H), 6.10 (s, 1H),
4.13.1. tert-Butyl 2-(4-bromobenzoyl)hydrazinecarboxylate (13)
Yield: 45%; ESIMS m/z calcd for C12H15BrN2O3 [M þ H]þ, 315.03;
2.30 (s, 3H). 13C (100 MHz, (CD3)2SO):
d 170.76, 164.27, 162.74,
found 314.99.; 1H NMR (400 MHz, (CD3)2SO):
d
10.28 (bs, 1H), 8.94
158.04, 152.17, 150.57, 148.20, 145.83, 145.74, 143.85, 140.50, 138.43,
133.91, 133.85, 128.88, 128.61, 128.45, 125.36, 124.20, 121.65, 119.74,
119.69, 119.58, 118.11, 117.99, 95.86, 12.53; HRMS (ES): m/z calcd for
(bs, 1H), 7.79 (d, 2H, J ¼ 8.32 Hz), 7.71 (dd, 2H, J ¼ 6.72, 1.88 Hz), 1.43
(s, 9H). 13C (100 MHz, (CD3)2SO):
129.92, 126.04, 79.74, 28.55.
d 165.61, 155.88, 132.10, 132.00,
C
27H22FN5O6S [M þ Na]þ, 586.1167; found 586.1164.
4.14. Procedure for the synthesis of compound 14a
4.16.2. (E)-3-(4-((E)-2-(3,5-Dichloro-2-hydroxybenzylidene)
hydrazinecarbonyl)phenyl)-N-(4-(N-(5-methylisoxazol-3-yl)
sulfamoyl)phenyl)acrylamide (15b)
To a stirred solution of Compound 13 (2.53 mmol) and com-
pound 12 (3.04 mmol) in dry DMF (10 mL), was added TEA
(3.80 mmol), Pd(OAc)2 (0.25 mmol) followed by diphenylphos-
phinopropane (0.25 mmol) under nitrogen. The mixture was
heated to 100 ꢀC and continued stirring for 24 h under nitrogen
atmosphere. The reaction mixture was cooled down to room tem-
perature, diluted with water and extracted with EtOAc. The organic
layer was dried over Na2SO4 and concentrated. The crude mixture
was suspended in EtOAc and sonicated for 15 min. The precipitate
formed was filtered, washed with hot EtOAc and dried under vac-
uum to obtain compound 14a.
Yield: 39%; ESIMS m/z calcd for C27H21Cl2N5O6S [M þ H]þ,
614.07; found 613.81; 1H NMR (600 MHz, (CD3)2SO):
d 12.58 (s, 1H),
12.49 (s, 1H), 11.36 (s, 1H), 10.73 (s, 1H), 8.60 (s, 1H), 8.03 (d, 2H,
J ¼ 6.90 Hz), 7.90 (d, 2H, J ¼ 7.92 Hz), 7.85e7.81 (m, 4H), 7.72e7.62
(m, 3H), 6.97 (d, 1H, J ¼ 15.42 Hz), 6.15 (s, 1H), 2.30 (s, 3H). 13C
(150 MHz, (CD3)2SO):
d 170.77, 164.25, 162.85, 158.02, 152.75,
147.62, 143.86, 140.44, 138.62, 133.82, 133.47, 130.80, 128.97, 128.90,
128.61, 128.45, 124.31, 123.43, 121.97, 121.25, 119.58, 95.86, 12.54;
HRMS (ES): m/z calcd for C27H21Cl2N5O6S [M þ Na]þ, 636.0482;
found 636.0477.
4.14.1. (E)-tert-Butyl 2-(4-(3-((4-(N-(5-methylisoxazol-3-yl)
sulfamoyl)phenyl)amino)-3-oxoprop-1-en-1-yl)benzoyl)
hydrazinecarboxylate (14a)
4.16.3. (E)-3-(4-((E)-2-(4-(Diethylamino)-2-hydroxybenzylidene)
hydrazinecarbonyl)phenyl)-N-(4-(N-(5-methylisoxazol-3-yl)
sulfamoyl)phenyl)acrylamide (15c)
Yield: 29%; ESIMS m/z calcd for C25H27N5O7S [MꢃH]þ, 540.16;
Yield: 54%; ESIMS m/z calcd for C31H32N6O6S [M þ H]þ, 617.22;
found 540.01; 1H NMR (400 MHz, (CD3)2SO):
d
11.35 (bs, 1H), 10.66
found 616.96; 1H NMR (400 MHz, (CD3)2SO):
d 11.89 (bs, 1H), 11.36
(bs, 1H), 10.27 (bs, 1H), 8.95 (bs, 1H), 7.93e7.81 (m, 6H), 7.75 (d, 2H,
J ¼ 8.40 Hz), 7.68 (d, 1H, J ¼ 15.72 Hz), 6.93 (d, 1H, J ¼ 15.80 Hz), 6.12
(bs, 2H), 10.72 (s, 1H), 8.45 (s, 1H), 7.99 (d, 2H, J ¼ 8.32 Hz), 7.90 (d,
2H, J ¼ 9.00 Hz), 7.85e7.78 (m, 4H), 7.70 (d, 1H, J ¼ 15.68 Hz),
7.23e7.21 (m, 1H), 6.96 (d, 1H, J ¼ 15.76 Hz), 6.30e6.28 (m, 1H), 6.14
(s, 1H), 2.29 (s, 3H), 1.44 (s, 9H). 13C (100 MHz, (CD3)2SO):
d 170.54,
165.87, 164.28, 158.43, 155.92, 143.69, 140.53, 138.10, 134.25, 133.91,
128.54, 128.33, 124.05, 119.53, 95.93, 79.73, 28.56, 12.52.
(s, 1H), 3.40e3.37 (m, 4H), 2.30 (s, 3H), 1.11 (t, 6H, J ¼ 6.98 Hz). 13
C
(100 MHz, (CD3)2SO):
d 170.76, 164.33, 162.24, 158.03, 143.93,
140.51, 138.12, 134.31, 133.78, 132.12, 128.73, 128.60, 128.37, 124.07,
4.15. Procedure for the synthesis of compound 14b
119.56, 95.86, 44.61, 12.89, 12.53; HRMS (ES): m/z calcd for
C
31H32N6O6S [M þ Na]þ, 639.1996; found 639.1989.
To the suspension of compound 14a (0.73 mmol) in MeOH
(4 mL) at 0 ꢀC, was added 4 M HCl in 1,4-dioxane solution (8 mL)
drop wise under nitrogen. The resulting mixture was stirred for
overnight at ambient temperature under nitrogen. The precipitate
formed was filtered and dried under vacuum under nitrogen to
afford compound 14b.
4.16.4. (E)-3-(4-((E)-2-(5-Bromo-2-hydroxybenzylidene)
hydrazinecarbonyl)phenyl)-N-(4-(N-(5-methylisoxazol-3-yl)
sulfamoyl)phenyl)acrylamide (15d)
Yield: 46%; ESIMS m/z calcd for C27H22BrN5O6S [M þ H]þ,
624.05; found 623.64; 1H NMR (600 MHz, (CD3)2SO):
d 12.25 (bs,
1H), 11.35 (s, 1H), 11.26 (bs, 1H), 10.72 (bs, 1H), 8.64 (s, 1H), 8.02 (d,
2H, J ¼ 7.44 Hz), 7.90 (d, 2H, J ¼ 8.10 Hz), 7.85e7.80 (m, 5H), 7.70 (d,
1H, J ¼ 15.66 Hz), 7.44 (d,1H, J ¼ 7.62 Hz), 6.98e6.91 (m, 2H), 6.14 (s,
4.15.1. (E)-3-(4-(Hydrazinecarbonyl)phenyl)-N-(4-(N-(5-
methylisoxazol-3-yl)sulfamoyl)phenyl)acrylamide
hydrochloride (14b)
1H), 2.30 (s, 3H). 13C (100 MHz, (CD3)2SO):
d 170.77, 164.28, 162.77,
Yield: 90%; ESIMS m/z calcd for C20H20ClN5O5S [M þ HeHCl]þ,
158.02, 156.90, 146.20, 143.87, 140.51, 138.34, 134.12, 134.08, 133.81,
130.81, 128.88, 128.61, 128.40, 124.16, 121.82, 119.57, 119.17, 110.94,
95.86, 12.54; HRMS (ES): m/z calcd for C27H22BrN5O6S [M þ Na]þ,
646.0366; found 646.0350.
442.12; found 441.98; 1H NMR (400 MHz, (CD3OD)):
d 7.94 (d, 2H,
J ¼ 8.40 Hz), 7.87 (m, 4H), 7.79e7.73 (m, 3H), 6.91 (d, 1H,
J ¼ 15.72 Hz), 6.14 (s, 1H), 2.32 (s, 3H). 13C (100 MHz, (CD3)2SO):
d
170.75, 165.79, 164.27, 158.02, 143.95, 140.11, 139.11, 133.77, 131.71,
128.91, 128.56, 128.48, 124.75, 119.55, 95.86, 12.53.
5. Biological assay methods
4.16. General procedure for the synthesis of compounds 15aed
The synthesized compounds (4aed, 5aed, 11aed and 15aed)
were tested for growth inhibition of C. trachomatis and C. pneu-
moniae as previously described [12]. HeLa 229 cells (CCL-2.1, ATCC,
Manassas, VA) were infected independently with C. trachomatis L2
serovar (VR-902B, ATCC) and C. pneumoniae strain T45 [26]. The
synthesized compounds were dissolved in DMSO and two-fold
dilution series were used for the biological testing in 1% final
A mixture of compound 14b (0.20 mmol) and respective sali-
cylaldehyde (0.21 mmol) in EtOH (5 mL) was heated to 70 ꢀC for 4 h.
The precipitate formed was filtered when the reaction is hot,
washed with hot EtOH (2 ꢁ 5 mL) and dried under vacuum to
obtain the compounds 15aed in respective yields.