ars.els-cdn.com · web viewall chemicals were purchased from lancaster (alfa aesar, johnson matthey...
Post on 19-Sep-2020
1 Views
Preview:
TRANSCRIPT
1
Rational design, molecular docking and synthesis of novel homopiperazine linked
imidazo[1,2-a]pyrimidine derivatives as potent cytotoxic and antimicrobial agents
Manohar Mantipallya, Madhusudhana Reddy Gangireddya, Rambabu Gundla*a, Vishnu Nayak
Badavathb, Santhosh Reddy Mandhaa, Venkatanarayana Chowdary Maddipatia a Department of Chemistry, School of Technology, GITAM University, Hyderabad-
502102,Telangana, India.b Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Pathumwan,
Bangkok-10330, Thailand.
* Corresponding author E-mail address: rambabu.gundla@gitam.edu
Materials and Methods (2-9)
Biological Evaluation & Molecular docking studies (10-12)
Spectral Copies (13-51)
HPLC data-(52-68)
2
1. Materials and Methods
General Method for Synthesis
All chemicals were purchased from Lancaster (Alfa Aesar, Johnson Matthey Co, Ward
Hill, MA, USA), Combi-blocks (USA) and Spectrochem Pvt Ltd (Mumbai, India). Reactions
were monitored by TLC, performed on silica gel glass plates containing 60 F-254 and
visualization on TLC was achieved by UV light. 1H NMR spectra were recorded on Avance (300
& 400 MHz); Bruker, Fallanden, Switzerland instruments. Chemical shifts were reported in δ
values downfield from internal TMS standard. Spectral patterns were designated as s, singlet; d,
doublet; dd, double doublet; t, triplet; bs, broad singlet; m, multiplet. ESI spectra were recorded
on Micro mass, Quattro LC using ESI+ software with capillary voltage of 3.98 kV and ESI mode
positive ion trap detector. IR spectra were recorded on a FT-IR spectrometer and only major
peaks are reported in cm-1.
Synthesis of tert-butyl 4-(2-aminopyrimidin-4-yl)-1,4-diazepane-1-carboxylate (3)
To a solution of 4-chloropyrimidin-2-amine 1 (1 mmol), N,N’-diisopropylethyl amine (10
mL) was added tert-butyl 1,4-diazepane-1-carboxylate 2 (1.1 mmol) and the resulting mixture
was heated to 120 ºC for 24 h under inert condition. The mixture was cooled to room
temperature and concentrated under reduced pressure. The crude residue was dissolved in water
and extracted with EtOAc (3 × 50 mL). The combined organic layers were dried over anhydrous
Na2SO4, filtered and concentrated under reduced pressure to obtain tert-butyl 4-(2-
aminopyrimidin-4-yl)-1,4-diazepane-1-carboxylate 3 (85%) as an off-white solid. LC-MS (APCI
+ ESI) m/z: 294 [M + H] +; 1H-NMR (400 MHz, DMSO-d6): δ 7.71 (d, J = 5.6 Hz, 1H), 5.91 (bs,
3H), 3.64–3.40 (m, 6H), 3.26–3.21 (m, 2H), 1.77–1.68 (m, 2H), 1.31 (d, 9H).
Synthesis of tert-butyl 4-(2-(4-fluorophenyl)imidazo[1,2-a]pyrimidin-7-yl)-1,4-diazepane-1-
carboxylate (5)
To a solution of tert-butyl 1,4-diazepane-1-carboxylate 3 (1 mmol), 2-bromo-1-(4-
fluorophenyl)ethanone 4 (1.1 mmol) in EtOH (15 mL) was added NaHCO3 (1.1 mmol) and the
resulting mixture was heated to 80 ºC for 4 h. The reaction mixture was cooled to room
temperature and concentrated under reduced pressure, whereupon the residue was partitioned
between EtOAc and saturated aqueous NaHCO3 solution (200 mL:50 mL). The layers were
3
separated and the EtOAc layer dried over anhydrous Na2SO4, filtered and concentrated under
reduced pressure to obtain tert-butyl 4-(2-(4-fluorophenyl)imidazo[1,2-a]pyrimidin-7-yl)-1,4-
diazepane-1-carboxylate 5 (82%) as an off-white solid. mp: 192-196 °C; IR (KBr): ν 2870, 1740,
1446, 1367, 1226, 788, 692 cm-1; LCMS (ESI+APCI): m/z = 412 [M + H]+; 1H-NMR (400 MHz,
DMSO-d6): δ 8.50 (t, J = 6.4 Hz, 1H), 7.91–7.87 (m, 2H), 7.84 (s, 1H), 7.24–7.20 (m, 2H), 6.68
(d, J = 7.6 Hz, 1H), 3.80 (bs, 2H), 3.70 (bs, 2H), 3.59 (bs, 1H), 3.52–3.49 (m, 1H), 3.32–3.25 (m,
2H), 1.85 (bs, 1H), 1.77 (bs, 1H), 1.24 (d, 9H); 13C-NMR (75 MHz, DMSO-d6): δ 162.96,
159.75,155.32, 154.26, 148.95,141.38, 134.60, 130.76, 126.72, 115.38, 115.10, 104.66, 97.13,
47.76, 47.57, 46.79, 45.54, 44.96, 30.55, 27.78; Anal. Calcd for C22H26FN5O2: C, 64.22; H, 6.37;
N, 17.02. Found: C, 64.31; H, 6.39; N, 17.08.
Synthesis of 7-(1,4-diazepan-1-yl)-2-(4-fluorophenyl)imidazo[1,2-a]pyrimidine (6)
To a solution of tert-butyl 4-(2-(4-fluorophenyl)imidazo[1,2-a]pyrimidin-7-yl)-1,4-
diazepane-1-carboxylate 5(1 mmol) in CH2Cl2 (10 mL) was added a solution of HCl (4.0 M in
1,4-dioxane, 5 mL) and the reaction mixture was stirred at room temperature for 4 h. The solid
obtained in the reaction was collected by filtration under vacuum, washed with CH2Cl2 (50 mL).
The solid was then suspended in water (50 mL) and basified with saturated aqueous NaHCO3
solution (50 mL), whereupon an off-white solid precipitated. The solid was collected by filtration
under vacuum, washed with water and dried to provide 7-(1,4-diazepan-1-yl)-2-(4-
fluorophenyl)imidazo[1,2-a]pyrimidine 6 (95%). mp: 130-135 °C; IR (KBr): ν 1641, 1549, 1462,
1366, 1221, 840, 689 cm-1; 1H-NMR (400 MHz, DMSO-d6): δ 8.50–8.45 (m, 1H), 7.91–7.82 (m,
3H), 7.22 (t, J = 8.8 Hz, 2H), 6.70–6.62 (m, 1H), 3.80–3.71 (m, 4H), 3.54 (t, J = 5.6 Hz, 1H),
3.32 (t, J = 5.2 Hz, 1H), 2.86 (t, J = 5.2 Hz, 1H), 2.67 (t, J = 5.6 Hz, 1H), 1.82–1.74 (m, 2H); 13C-NMR (75 MHz, DMSO-d6): δ 162.97, 159.75, 155.76, 149.15, 141.26, 134.72, 130.83,
126.81, 115.40, 115.11, 104.60, 97.15, 48.08, 47.14, 45.58, 44.99, 28.78; LCMS (ESI+APCI):
m/z = 312 [M + H]+; Anal. Calcd for C17H18FN5: C, 65.58; H, 5.83; N, 22.49. Found: C, 65.63; H,
5.86; N, 22.55.
General procedure for the synthesis of compounds (10a-i, 11a-g, 12)
To a mixture of 7-(1,4-diazepan-1-yl)-2-(4-fluorophenyl)imidazo[1,2-a]pyrimidine 6 (1
mmol) in CH2Cl2 was added Et3N (2 mmol), followed by corresponding acid chloride or sulfonyl
4
chloride or alkyl halide (1.1 mmol) at room temperature and the mixture was allowed to stir for 3
h. The mixture was diluted with water (50 mL), extracted with CH2Cl2 (2 × 50 mL) and the
combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated under
reduced pressure. The residue was purified by column chromatography over silica gel [100–200
mesh; CH3OH:CH2Cl2 (1:9)]. Fractions containing the product were concentrated under vacuum
to obtain title compounds (10a-i, 11a-g, 12).
Methyl 4-(2-(4-fluorophenyl)imidazo[1,2-a]pyrimidin-7-yl)-1,4-diazepane-1-carboxylate (10a).
Yield: 75%; off-white solid; mp: 171-174 °C; IR (KBr): ν 2940, 1740, 1696, 1639, 1480, 1227,
775 cm-1; 1H-NMR (400 MHz, DMSO-d6): δ 8.50 (d, J = 7.6 Hz, 1H), 7.91–7.87 (m, 2H), 7.84
(s, 1H), 7.24–7.20 (m, 2H), 6.69 (d, J = 8.0 Hz, 1H), 3.82 (bs, 2H), 3.73 (bs, 2H), 3.58–3.50 (m,
5H), 3.37–3.33 (m, 2H), 1.82 (bs, 2H); 13C-NMR (75 MHz, DMSO-d6): δ 174.82, 163.01,
159.78, 155.44, 148.87, 141.35, 134.76, 130.71, 126.85, 115.42, 115.14, 104.80, 97.04, 48.23,
47.28, 46.08, 45.14, 44.23, 29.08; LC-MS (APCI + ESI) m/z: 370 [M + H] +; Anal. Calcd for
C19H20FN5O2: C, 61.78; H, 5.46; N, 18.96. Found: C, 61.63; H, 5.47; N, 18.91; HPLC: >99%
(AUC), tR= 6.26 min.
1-(4-(2-(4-Fluorophenyl)imidazo[1,2-a]pyrimidin-7-yl)-1,4-diazepan-1-yl)ethanone (10b).
Yield: 68%; off-white solid; mp: 98-103 °C ; IR (KBr): ν 2983, 1737, 1633, 1454, 1367, 1213,
739 cm-1; 1H-NMR (400 MHz, DMSO-d6): δ 8.52–8.49 (m, 1H), 7.92–7.88 (m, 2H), 7.85 (d, J =
3.2 Hz, 1H), 7.25–7.20 (m, 2H), 6.72–6.68 (m, 1H), 3.89 (bs, 1H), 3.77–3.62 (m, 5H), 3.45–3.37
(m, 2H), 1.99–1.87 (m, 4H), 1.77 (t, J = 5.2 Hz, 1H ); 13C-NMR (100 MHz, DMSO-d6): δ
169.34, 162.68, 160.26, 155.47, 149.02, 141.50, 134.95, 130.81, 126.91, 115.46, 115.24, 104.92,
97.05, 47.42, 46.73, 44.67, 44.06, 26.31, 20.92; LC-MS (APCI + ESI) m/z: 354 [M + H] +; Anal.
Calcd for C19H20FN5O: C, 64.57; H, 5.70; N, 19.82. Found: C, 64.47; H, 5.67; N, 19.76; HPLC:
>99% (AUC), tR= 6.37 min.
4-(2-(4-Fluorophenyl)imidazo[1,2-a]pyrimidin-7-yl)-N,N-dimethyl-1,4-diazepane-1-
carboxamide (10c). Yield:73%; off-white solid; mp: 130-135 °C ; IR (KBr): ν 3009, 2961, 1740,
1634, 1488, 1356, 1218, 734 cm-1; 1H-NMR (400 MHz, DMSO-d6): δ 8.49 (d, J = 8.0 Hz, 1H),
7.91–7.87 (m, 2H), 7.84 (s, 1H), 7.22 (t, J = 9.2 Hz, 2H), 6.69 (d, J = 7.6 Hz, 1H), 3.85 (bs, 1H),
3.74 (bs, 2H), 3.45 (t, J = 5.6 Hz, 2H), 3.25 (t, J = 5.6 Hz, 2H), 2.65 (s, 6H), 1.89 (t, J = 5.2 Hz,
2H); 13C-NMR (75 MHz, DMSO-d6): δ 164.07, 162.98, 159.76, 155.42, 148.96, 141.27, 134.70,
5
130.71, 126.72, 115.41, 115.12, 104.75, 97.05, 48.12, 47.87, 46.96, 46.41, 38.57, 26.53; LC-MS
(APCI + ESI) m/z: 383 [M + H] +; Anal. Calcd for C20H23FN6O: C, 62.81; H, 6.06; N, 21.98.
Found: C, 63.02; H, 6.09; N, 21.93; HPLC: >99% (AUC), tR= 6.28 min.
Cyclopropyl(4-(2-(4-fluorophenyl)imidazo[1,2-a]pyrimidin-7-yl)-1,4-diazepan-1-yl)methanone
(10d). Yield:72%; off-white solid; mp: 203-208 °C ; IR (KBr): ν 2944, 1740, 1617, 1484, 1364,
1218, 734 cm-1; 1H-NMR (300 MHz, DMSO-d6): δ 8.50 (d, J = 7.8 Hz, 1H), 7.92–7.85 (m, 3H),
7.22 (t, J = 8.7 Hz, 2H), 6.69 (d, J = 7.5 Hz, 1H), 3.92 (bs, 2H), 3.76–3.62 (m, 5H), 3.42 (t, J =
5.7 Hz, 1H), 1.95–1.84 (m, 2H), 1.76 (bs, 1H), 0.67 (d, J = 6.3 Hz, 2H), 0.47 (bs, 2H); 13C-NMR
(75 MHz, DMSO-d6): δ 172.09, 171.66, 163.00, 155.40, 148.95, 141.34, 134.78, 130.72, 126.75,
115.42, 115.14, 104.78, 97.10, 48.12, 47.55, 45.31, 44.64, 26.29, 10.38, 6.96, 6.61; LC-MS
(APCI + ESI) m/z: 380 [M + H] +; Anal. Calcd for C21H22FN5O: C, 66.47; H, 5.84; F; N, 18.46.
Found: C, 66.38; H, 5.85; N, 18.39; HPLC: >99% (AUC), tR= 6.26 min.
Cyclohexyl(4-(2-(4-fluorophenyl)imidazo[1,2-a]pyrimidin-7-yl)-1,4-diazepan-1-yl)methanone
(10e). Yield:82%; off-white solid; mp: 108-112 °C ; IR (KBr): ν 3008, 2932, 1740, 1636, 1452,
1366, 1218, 738 cm-1; 1H-NMR (300 MHz, DMSO-d6): δ 8.50 (d, J = 6.9 Hz, 1H), 7.91–7.83 (m,
3H), 7.22 (t, J = 9.0 Hz, 2H), 6.68 (d, J = 7.8 Hz, 1H), 3.90 (bs, 1H), 3.74–3.63 (m, 5H), 3.48
(bs, 1H), 3.40 (bs, 1H), 1.88 (bs, 1H), 1.72–1.48 (m, 5H), 1.26–1.10 (m, 7H); 13C-NMR (100
MHz, DMSO-d6): δ 174.93, 162.72, 160.29, 155.49, 148.94, 141.48, 134.86, 130.83, 126.95,
115.48, 115.27, 104.90, 97.13, 47.38, 46.19, 45.25, 44.32, 29.44, 29.17, 26.93, 25.44, 25.09,
24.94, 24.71; LC-MS (APCI + ESI) m/z: 422 [M + H] +; Anal. Calcd for C24H28FN5O: C, 68.39;
H, 6.70; N, 16.61. Found: C, 68.28; H, 6.72; N, 16.56; HPLC: 97.5% (AUC), tR= 6.70 min.
(4-Fluorophenyl)(4-(2-(4-fluorophenyl)imidazo[1,2-a]pyrimidin-7-yl)-1,4-diazepan-1-
yl)methanone (10f).Yield:85%; off-white solid; mp: 180-184 °C ; IR (KBr): ν 3006, 2945, 1740,
1634, 1457, 1367, 1220, 844, 759 cm-1; 1H-NMR (400 MHz, DMSO-d6): δ 8.53–8.51 (m, 1H),
7.89–7.86 (m, 3H), 7.38 (bs, 1H), 7.25–7.21 (m, 3H), 7.07 (bs, 2H), 6.75–6.38 (m, 1H), 3.91 (bs,
1H), 3.82 (bs, 4H), 3.60 (bs, 2H), 3.55 (bs, 1H), 1.92 (bs, 1H), 1.68 (bs, 1H); 13C-NMR (100
MHz, DMSO-d6): δ 169.41, 162.70, 160.28, 155.27, 148.84, 141.51, 134.96, 132.82, 132.10,
130.82, 128.60, 126.94, 115.46, 115.25, 115.05, 104.89, 97.01, 48.02, 46.76, 45.50, 44.05, 26.93,
24.79; LC-MS (APCI + ESI) m/z: 434 [M + H] +; Anal. Calcd for C24H21F2N5O: C, 66.50; H,
4.88; N, 16.16. Found: C, 66.38; H, 4.92; N, 16.23; HPLC: 96.9% (AUC), tR= 6.44 min.
6
(4-Chlorophenyl)(4-(2-(4-fluorophenyl)imidazo[1,2-a]pyrimidin-7-yl)-1,4-diazepan-1-
yl)methanone (10g).Yield:83%; off-white solid; mp: 222-226 °C ; IR (KBr): ν 3006, 2944, 1740,
1621, 1459, 1367, 1217, 840, 748 cm-1; 1H-NMR (300 MHz, DMSO-d6): δ 8.53–8.51 (m, 1H),
7.91–7.87 (m, 3H), 7.48 (d, J = 7.8 Hz, 1H), 7.34 (d, J = 7.5 Hz, 1H), 7.23 (t, J = 8.7 Hz, 3H),
6.99 (bs, 1H), 6.75–6.64 (m, 1H), 3.91 (bs, 1H), 3.81 (bs, 4H), 3.60–3.54 (m, 3H), 1.91 (bs, 1H),
1.68 (bs, 1H); 13C-NMR (75 MHz, DMSO-d6): δ 169.15, 162.99, 159.77, 155.14, 148.91, 141.49,
134.89, 133.49, 130.71, 128.38, 127.89, 126.82, 115.36, 115.08, 104.79, 97.10, 96.89, 47.83,
45.30, 43.91, 24.68; LC-MS (APCI + ESI) m/z: 450 [M + H] +; Anal. Calcd for C24H21ClFN5O:
C, 64.07; H, 4.70; N, 15.57. Found: C, 63.95; H, 4.68; N, 15.52; HPLC: 96.8% (AUC), tR= 6.54
min.
(4-(2-(4-Fluorophenyl)imidazo[1,2-a]pyrimidin-7-yl)-1,4-diazepan-1-yl)(thiophen-2-
yl)methanone (10h). Yield:79%; off-white solid; mp: 190-193 °C; IR (KBr): ν 3003, 2947, 1741,
1633, 1454, 1365, 1219, 842, 697 cm-1; 1H-NMR (300 MHz, DMSO-d6): δ 8.51–8.47 (m, 1H),
7.92–7.87 (m, 3H), 7.27–7.21 (m, 3H), 6.85 (bs, 1H), 6.79–6.68 (m, 2H), 3.91–3.68 (m, 8H),
3.56 (t, J = 5.6 Hz, 1H), 3.43 (t, J = 5.4 Hz, 1H), 1.80 (d, J = 4.8 Hz, 2H); 13C-NMR (75 MHz,
DMSO-d6): δ 169.03, 163.08, 159.85, 155.59, 148.68, 140.51, 137.26, 134.94, 126.82, 126.21,
125.97, 124.82, 115.50, 115.21, 104.97, 97.43, 97.16, 46.67, 45.79, 45.26, 44.41, 33.58, 26.43;
LC-MS (APCI + ESI) m/z: 436 [M + H] +; Anal. Calcd for C23H22FN5OS: C, 63.43; H, 5.09; N,
16.08; S, 7.36. Found: C, 63.55; H, 5.12; N, 16.16; S, 7.42; HPLC: 95.9% (AUC), tR= 6.17 min.
1-(4-(2-(4-Fluorophenyl)imidazo[1,2-a]pyrimidin-7-yl)-1,4-diazepan-1-yl)-2-phenylethanone
(10i). Yield:71%; off-white solid; mp: 100-105 °C ; IR (KBr): ν 2932, 1740, 1634, 1483, 1453,
1365, 1217, 729 cm-1; 1H-NMR (400 MHz, DMSO-d6): δ 8.48 (d, J = 7.6 Hz, 1H), 7.93–7.89 (m,
2H), 7.86 (s, 1H), 7.25–7.21 (m, 2H), 7.13–7.10 (m, 4H), 7.04–7.02 (m, 1H), 6.64 (d, J = 7.6 Hz,
1H), 3.80 (bs, 2H), 3.71–3.63 (m, 6H), 3.52 (t, J = 5.6 Hz, 1H), 3.44 (t, J = 5.2 Hz, 1H), 1.78 (bs,
2H) ; 13C-NMR (100 MHz, DMSO-d6): δ 169.97, 162.69, 160.27, 155.50, 148.99, 141.46,
135.88, 134.78, 130.82, 128.83, 128.06, 126.94, 126.19, 115.47, 115.26, 104.89, 97.21, 46.79,
45.70, 45.22, 44.28, 26.47, 24.95; LC-MS (APCI + ESI) m/z: 430 [M + H] +; Anal. Calcd for
C25H24FN5O: C, 69.91; H, 5.63; N, 16.31. Found: C, 70.06; H, 5.65; N, 16.34; HPLC: 97.2%
(AUC), tR= 6.47 min.
7
2-(4-Fluorophenyl)-7-(4-(methylsulfonyl)-1,4-diazepan-1-yl)imidazo[1,2-a]pyrimidine (11a).
Yield:71%; off-white solid; mp: 200-205 °C ; IR (KBr): ν 3007, 2965, 1638, 1461, 1369, 1323,
1211, 1145, 786 cm-1; 1H-NMR (400 MHz, DMSO-d6): δ 8.53 (d, J = 7.6 Hz, 1H), 7.91–7.88 (m,
2H), 7.87 (s, 1H), 7.23 (t, J = 8.8 Hz, 2H), 6.73 (d, J = 7.6 Hz, 1H), 3.88 (bs, 2H), 3.80 (bs, 2H),
3.47 (t, J = 5.2 Hz, 2H), 3.26 (t, J = 5.6 Hz, 2H), 2.86 (s, 3H), 1.91–1.85 (m, 2H); 13C-NMR (75
MHz, DMSO-d6): δ 163.05, 155.55, 148.77, 141.06, 134.95, 130.47, 126.89, 126.78, 115.45,
115.17, 104.91, 97.18, 48.32, 47.19, 46.55, 46.30, 36.84, 27.51; LC-MS (APCI + ESI) m/z: 390
[M + H] +; Anal. Calcd for C18H20FN5O2S: C, 55.51; H, 5.18; N, 17.98; S, 8.23. Found: C, 55.58;
H, 5.21; N, 18.03; S, 8.25; HPLC: 94.0% (AUC), tR= 6.31 min.
4-(2-(4-Fluorophenyl)imidazo[1,2-a]pyrimidin-7-yl)-N,N-dimethyl-1,4-diazepane-1-sulfonamide
(11b). Yield:81%; off-white solid; mp: 155-160 °C ; IR (KBr): ν 3058, 2921, 1644, 1455, 1344,
1213, 1145, 715 cm-1; 1H-NMR (300 MHz, DMSO-d6): δ 8.52 (d, J = 7.5 Hz, 1H), 7.92–7.87 (m,
2H), 7.85 (s, 1H), 7.22 (t, J = 9.0 Hz, 2H), 6.71 (d, J = 7.8 Hz, 1H), 3.85 (bs, 2H), 3.78 (bs, 2H),
3.49 (t, J = 5.1 Hz, 2H), 3.35 (bs, 2H), 2.63 (s, 6H), 1.86 (t, J = 5.1 Hz, 2H); 13C-NMR (75 MHz,
DMSO-d6): δ 159.77, 155.45, 148.85, 141.25, 134.83, 130.61, 126.85, 126.75, 115.41, 115.13,
104.80, 97.11, 48.31, 47.77, 47.27, 46.35, 37.46, 27.42; LC-MS (APCI + ESI) m/z: 419 [M + H] +; Anal. Calcd for C19H23FN6O2S: C, 54.53; H, 5.54; N, 20.08; S, 7.66. Found: C, 54.58; H, 5.55;
N, 20.09; S, 7.69; HPLC: >99% (AUC), tR= 6.52 min.
7-(4-(Cyclopropylsulfonyl)-1,4-diazepan-1-yl)-2-(4-fluorophenyl)imidazo[1,2-a]pyrimidine
(11c). Yield:79%; off-white solid; mp: 211-215 °C ; IR (KBr): ν 3032, 2947, 1638, 1455, 1327,
1220, 1147, 718 cm-1; 1H-NMR (400 MHz, DMSO-d6): δ 8.52 (d, J = 7.6 Hz, 1H), 7.91–7.88 (m,
2H), 7.85 (s, 1H), 7.22 (t, J = 8.8 Hz, 2H), 6.73 (d, J = 8.0 Hz, 1H), 3.88 (bs, 2H), 3.80 (bs, 2H),
3.52 (t, J = 5.6 Hz, 2H), 3.23 (t, J = 6.0 Hz, 2H), 2.60–2.55 (m, 1H), 1.92–1.86 (m, 2H), 0.90–
0.87 (m, 4H); 13C-NMR (75 MHz, DMSO-d6): δ 159.77, 155.48, 148.87, 141.36, 134.87, 130.68,
126.85, 126.74, 115.41, 115.13, 104.81, 97.05, 48.43, 47.53, 46.77, 46.20, 27.89, 27.69, 4.17;
LC-MS (APCI + ESI) m/z: 416 [M + H] +; Anal. Calcd for C20H22FN5O2S: C, 57.82; H, 5.34; N,
16.86; S, 7.72. Found: C, 57.84; H, 5.37; N, 16.90; S, 7.76; HPLC: 98.5% (AUC), tR= 6.42 min.
2-(4-Fluorophenyl)-7-(4-((4-methoxyphenyl)sulfonyl)-1,4-diazepan-1-yl)imidazo[1,2-
a]pyrimidine (11d). Yield:78%; off-white solid; mp: 160-165 °C ; IR (KBr): ν 2945, 2846, 1641,
1458, 1332, 1216, 1152, 838, 599 cm-1; 1H-NMR (300 MHz, DMSO-d6): δ 8.46 (d, J = 7.5 Hz,
8
1H), 7.92–7.87 (m, 2H), 7.84 (s, 1H), 7.64 (d, J = 8.7 Hz, 2H), 7.22 (t, J = 8.7 Hz, 2H), 6.95 (d, J
= 9.0 Hz, 2H), 6.61 (d, J = 7.5 Hz, 1H), 3.84 (bs, 2H), 3.73 (bs, 5H), 3.40 (bs, 2H), 3.25 (bs, 2H),
1.82 (bs, 2H); 13C-NMR (75 MHz, DMSO-d6): δ 162.14, 159.77, 155.28, 148.79, 141.37, 134.73,
130.73, 130.40, 128.63, 126.85, 126.74, 115.39, 115.11, 114.19, 104.77, 96.96, 55.39, 47.66,
47.00, 46.48, 46.35, 27.44; LC-MS (APCI + ESI) m/z: 482 [M + H] +; Anal. Calcd for
C24H24FN5O3S: C, 59.86; H, 5.02; N, 14.54; S, 6.66. Found: C, 59.81; H, 5.05; N, 14.49; S, 6.69;
HPLC: >99% (AUC), tR= 6.59 min.
2-(4-Fluorophenyl)-7-(4-((4-methyl-3-nitrophenyl)sulfonyl)-1,4-diazepan-1-yl)imidazo[1,2-
a]pyrimidine (11e). Yield:74%; off-white solid; mp: 163-168 °C ; IR (KBr): ν 2932, 2865, 1641,
1533, 1487, 1342, 1215, 1161, 842, 697 cm-1; 1H-NMR (400 MHz, DMSO-d6): δ 8.43 (d, J = 7.6
Hz, 1H), 8.22 (d, J = 2.0 Hz, 1H) 7.91–7.88 (m, 3H), 7.84 (s, 1H), 7.45 (d, J = 8.4 Hz, 1H), 7.23
(t, J = 8.8 Hz, 2H), 6.59 (d, J = 7.6 Hz, 1H), 3.84 (bs, 2H), 3.71 (bs, 2H), 3.51 (t, J = 5.6 Hz,
2H), 3.42 (bs, 2H), 2.41 (s, 3H), 1.83 (bs, 2H); 13C-NMR (75 MHz, DMSO-d6): δ 159.79,
155.11, 148.64, 148.41, 141.35, 138.06, 137.45, 134.67, 133.90, 130.66, 130.42, 126.88, 126.77,
122.45, 115.41, 115.12, 104.84, 96.84, 47.30, 46.67, 46.50, 27.44, 19.35; LC-MS (APCI + ESI)
m/z: 511 [M + H] +; Anal. Calcd for C24H23FN6O4S: C, 56.46; H, 4.54; N, 16.46; S, 6.28. Found:
C, 56.41; H, 4.51; N, 16.41; S, 6.26; HPLC: 97.5% (AUC), tR= 6.70 min.
7-(4-((2,4-Difluorophenyl)sulfonyl)-1,4-diazepan-1-yl)-2-(4-fluorophenyl)imidazo[1,2-
a]pyrimidine (11f). Yield:76%; off-white solid; mp: 178-182 °C ; IR (KBr): ν 2859, 1641, 1485,
1339, 1218, 1158, 845, 737 cm-1; 1H-NMR (300 MHz, DMSO-d6): δ 8.49 (d, J = 7.5 Hz, 1H),
7.92–7.80 (m, 4H), 7.43–7.35 (m, 1H), 7.26–7.16 (m, 3H), 6.67 (d, J = 7.8 Hz, 1H), 3.88 (bs,
2H), 3.76 (bs, 2H), 3.51 (bs, 2H), 3.37 (bs, 2H), 1.87 (bs, 2H); 13C-NMR (75 MHz, DMSO-d6):
δ 163.00, 159.78, 155.27, 148.74, 141.33, 134.79, 132.17, 132.03, 130.66, 126.87, 126.77,
115.40, 115.11, 112.29, 112.00, 105.93, 104.79, 97.00, 47.92, 46.79, 46.55, 46.34, 27.52; LC-
MS (APCI + ESI) m/z: 488 [M + H] +; Anal. Calcd for C23H20F3N5O2S: C, 56.67; H, 4.14; N,
14.37; S, 6.58. Found: C, 56.70; H, 4.19; N, 14.41; S, 6.60; HPLC: 96.8% (AUC), tR= 6.66 min.
4-((4-(2-(4-Fluorophenyl)imidazo[1,2-a]pyrimidin-7-yl)-1,4-diazepan-1-yl)sulfonyl)benzonitrile
(11g). Yield:83%; off-white solid; mp: 163-168 °C ; IR (KBr): ν 2926, 2857, 2234, 1641, 1487,
1338, 1217, 1157, 696 cm-1; 1H-NMR (300 MHz, DMSO-d6): δ 8.45 (d, J = 7.5 Hz, 1H), 7.92–
7.83 (m, 7H), 7.22 (t, J = 8.7 Hz, 2H), 6.60 (d, J = 7.8 Hz, 1H), 3.86 (bs, 2H), 3.71 (bs, 2H), 3.50
9
(bs, 2H), 3.38 (bs, 2H), 1.83 (bs, 2H); 13C-NMR (75 MHz, DMSO-d6): δ 159.80, 155.13, 148.66,
142.99, 141.44, 134.77, 133.15, 130.72, 127.21, 126.93, 126.82, 117.36, 115.39, 115.11, 114.81,
104.88, 96.89, 47.51, 46.75, 46.51, 27.48; LC-MS (APCI + ESI) m/z: 477 [M + H] +; Anal. Calcd
for C24H21FN6O2S: C, 60.49; H, 4.44; N, 17.64; S, 6.73. Found: C, 60.55; H, 4.48; N, 17.65; S,
6.74; HPLC: 98.7% (AUC), tR= 6.67 min.
(4-(2-(4-Fluorophenyl)imidazo[1,2-a]pyrimidin-7-yl)-1,4-diazepan-1-yl)(3-
methoxyphenyl)methanone (12). Yield:78%; off-white solid; mp: 120-125 °C ; IR (KBr): ν 3057,
2989, 2944, 1645, 1448, 1258, 1212, 784 cm-1; 1H-NMR (400 MHz, DMSO-d6): δ 8.47 (d, J =
7.5 Hz, 1H), 7.91–7.87 (m, 2H), 7.84 (s, 1H), 7.25–7.19 (m, 3H), 6.85 (bs, 2H), 6.78 (d, J = 7.6
Hz, 1H), 6.65 (d, J = 7.6 Hz, 1H), 3.75–3.70 (m, 7H), 3.60 (s, 2H), 2.72 (bs, 2H), 2.55 (bs, 2H),
1.87 (s, 2H); 13C-NMR (75 MHz, DMSO-d6): δ 162.95, 159.13, 155.84, 149.02, 141.28, 140.73,
134.52, 130.79, 129.03, 126.80, 126.69, 120.48, 115.38, 115.09, 113.68, 112.17, 104.63, 97.09,
60.91, 54.75, 54.41, 53.59, 46.19, 26.87; LC-MS (APCI + ESI) m/z: 432 [M + H] +; Anal. Calcd
for C25H26FN5O: C, 69.59; H, 6.07; N, 16.23. Found: C, 69.73; H, 6.11; N, 16.26; HPLC: 95.9%
(AUC), tR= 6.17 min.
10
2. Biological Evaluation
2a. Cytotoxicity assay
The effect of synthesized compounds on the growth of cancer cell lines was evaluated
according to the procedure adopted by the National Cancer Institute for in vitro anticancer drug
screening that uses the protein-binding dye sulforhodamine B to estimate cell growth. Briefly,
cells in their log phase of growth were harvested, counted and seeded (104 cells/ well in 100 mL
medium) in 96-well microtitre plates. After 24 h of incubation at 37 °C and 5% CO2 to allow cell
attachment, cultures were treated with varying concentrations (0.1e100 mM) of test samples
made with 1:10 serial dilutions. Four replicate wells were set up for each experimental condition.
Test samples were left in contact with the cells for 48 h under same conditions. Thereafter cells
were fixed with 50% chilled TCA and kept at 4 °C for 1 h, washed and air-dried. Cells were
stained with sulforhodamine B dye. The adsorbed dye was dissolved in tris-buffer and the plates
were gently shaken for 10 min on a mechanical shaker. The optical density (OD) was recorded
on ELISA reader at 540 nm. The cell growth was calculated by subtracting mean OD value of
the respective blank from the mean OD value of experimental set. Percentage of growth in the
presence of test material was calculated considering the growth in the absence of any test
material as 100% and the results are reported in terms of IC50 values. VP-16 was taken as
positive control.
2b. In vitro antimicrobial activity
All the newly synthesized compounds were evaluated by the agar cup diffusion technique
using 1 mg/mL solution of synthesized compound in DMSO. The test organisms utilized were
Bacillus subtilis (MTCC-441) and Bacillus megaterium (MTCC-428) as examples of gram
positive bacteria and Escherichia coli (MTCC-443) and Pseudomonas Spp. (2496) as examples
of gram negative bacteria. Antifungal activity was screened against fungal strain, Candida
albicans (MTCC-227). Inocculated müller Hinton agar for bacteria and yeast peptone dextrose
agar for fungi was poured onto the sterilized petri dishes (25–30 mL: each petri dish). The
poured material was allowed to set (40 min) and thereafter the cups (8 mm diameter) was made
by punching into the agar surface with a sterile cork borer and scooping out the punched part of
11
the agar. The test solution (200 µL) was added into the cups with the help of a micro pipette. The
plates were incubated at 37 °C and the results were recorded for antibacterial activity after 24 h
and for antifungal activity after 72 h. A control using DMSO without the test compound was
included for each organism. Ampicillin and gentamicin were used as standard antibacterial
agents, whereas amphotericin B was used as antifungal reference drugs. Each experiment was
carried out in triplicate and the results were recorded as the average diameter of inhibition zones
of bacterial or fungal growth in mm. The minimal inhibitory concentration (MIC, µg/mL)
determination method of the biologically active compounds (Table 2) was applied using
different series of dilutions against Gram-positive, Gram-negative bacteria and fungi.
3. Molecular docking studies
3a. Protein preparation
The crystal structure of receptors has been extracted from the website of
https://www.rcsb.org (PDB ID: 3E22). Protein was preprocessed by adding the polar hydrogen
only, filling the missing chains, loops along with capping the terminals. Hydrogen bonds were
optimized and co-crystallized water was deleted. Energy minimization was done with using
OPLS3 force field; pH was maintained at 7 ± 0.2 during the preparation of whole protein
preparation.
3b. Receptor grid generation
Grid generation at the active site of the receptor was done using Glide module, which
facilitates Grid box along with vanderwaals radius for scaling factor (1.0) and partial charge cut-
off (0.25) without any force. It implied the binding site by picking the pre-docked ligand. The
site of the grid box was on the centroid of pre-dock ligand. The grid box spacing was kept at 20
Å before running the grid file.
3c. Ligand preparation and docking
Ligand preparation of for the docking was carried out by LigPrep wizard and started
developing the ligands by minimizing using OPLS3 force field; pH was kept constant at 7 ± 0.2
through Epik. Stereoisomers were generated for each ligand. Standard precision of flexible
molecular docking was started by keeping the rest of the parameters by default. Molecular
12
docking analyses were employed out using Maestro (Schrödinger) and Marvin Sketch
(Chemaxon) was used for sketching of ligand.
13
1H-NMR and MS spectra of compound 3
14
15
IR and 1H-NMR spectra of compound 5
16
13C-NMR and MS spectra of compound 5
17
18
IR and 1H-NMR spectra of compound 6
19
20
13C-NMR and MS spectra of compound 6
21
22
IR and 1H-NMR spectra of compound 10a
23
13C-NMR and MS spectra of compound 10a
24
IR and 1H-NMR spectra of compound 10b
25
26
13C-NMR and MS spectra of compound 10b
27
IR and 1H-NMR spectra of compound 10c
28
29
13C-NMR and MS spectra of compound 10c
30
IR and 1H-NMR spectra of compound 10d
31
32
13C-NMR and MS spectra of compound 10d
33
IR and 1H-NMR spectra of compound 10e
34
13C-NMR and MS spectra of compound 10e
35
36
IR and 1H-NMR spectra of compound 10f
37
38
13C-NMR and MS spectra of compound 10f
39
IR and 1H-NMR spectra of compound 10g
40
41
13C-NMR and MS spectra of compound 10g
42
IR and 1H-NMR spectra of compound 10h
43
44
13C-NMR and MS spectra of compound 10h
45
IR and 1H-NMR spectra of compound 10i
46
47
13C-NMR and MS spectra of compound 10i
48
IR and 1H-NMR spectra of compound 11a
49
50
13C-NMR and MS spectra of compound 11a
51
52
IR and 1H-NMR spectra of compound 11b
53
54
13C-NMR and MS spectra of compound 11b
55
IR and 1H-NMR spectra of compound 11c
56
57
13C-NMR and MS spectra of compound 11c
58
IR and 1H-NMR spectra of compound 11d
59
60
13C-NMR and MS spectra of compound 11d
61
IR and 1H-NMR spectra of compound 11e
62
63
13C-NMR and MS spectra of compound 11e
64
IR and 1H-NMR spectra of compound 11f
65
66
13C-NMR and MS spectra of compound 11f
67
IR and 1H-NMR spectra of compound 11g
68
69
13C-NMR and MS spectra of compound 11g
70
71
IR and 1H-NMR spectra of compound 12
72
73
13C-NMR and MS spectra of compound 12
74
HPLC spectra of compound 10a
75
HPLC spectra of compound 10b
76
HPLC spectra of compound 10c
77
HPLC spectra of compound 10d
78
HPLC spectra of compound 10e
79
HPLC spectra of compound 10f
80
HPLC spectra of compound 10g
81
HPLC spectra of compound 10h
82
HPLC spectra of compound 10i
83
HPLC spectra of compound 11a
84
HPLC spectra of compound 11b
85
HPLC spectra of compound 11c
86
HPLC spectra of compound 11d
87
HPLC spectra of compound 11e
88
HPLC spectra of compound 11f
89
HPLC spectra of compound 11g
90
HPLC spectra of compound 12
top related