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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: [email protected]

Materials and Methods (2-9)

Biological Evaluation & Molecular docking studies (10-12)

Spectral Copies (13-51)

HPLC data-(52-68)

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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

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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

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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,

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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.

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(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,


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,


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,


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%


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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,


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,



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,



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,


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,


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%


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

https://www.rcsb.org/

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

15

IR and 1H-NMR spectra of compound 5

16

13C-NMR and MS spectra of compound 5

18


20


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

26

13C-NMR and MS spectra of compound 10b

27

IR and 1H-NMR spectra of compound 10c

29

13C-NMR and MS spectra of compound 10c

30

IR and 1H-NMR spectra of compound 10d

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

36

IR and 1H-NMR spectra of compound 10f

38

13C-NMR and MS spectra of compound 10f

39

IR and 1H-NMR spectra of compound 10g

41

13C-NMR and MS spectra of compound 10g

42

IR and 1H-NMR spectra of compound 10h

44

13C-NMR and MS spectra of compound 10h

45

IR and 1H-NMR spectra of compound 10i

47

13C-NMR and MS spectra of compound 10i

48

IR and 1H-NMR spectra of compound 11a

50

13C-NMR and MS spectra of compound 11a

52

IR and 1H-NMR spectra of compound 11b

54

13C-NMR and MS spectra of compound 11b

55

IR and 1H-NMR spectra of compound 11c

57

13C-NMR and MS spectra of compound 11c

58

IR and 1H-NMR spectra of compound 11d

60

13C-NMR and MS spectra of compound 11d

61

IR and 1H-NMR spectra of compound 11e

63

13C-NMR and MS spectra of compound 11e

64

IR and 1H-NMR spectra of compound 11f

66

13C-NMR and MS spectra of compound 11f

67

IR and 1H-NMR spectra of compound 11g

69

13C-NMR and MS spectra of compound 11g

71


73


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

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