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Synthesis, method optimization, anticancer activity of 2,3,7-trisubstituted Quinazoline derivatives and targeting EGFR-tyrosine kinase by rational approach
*Corresponding author. Tel.: +91 9417563874; fax: +91 1881263655 mnoolvi@yahoo.co.uk (Malleshappa N. Noolvi), mallesh7301@rediffmail.com (Malleshappa N. Noolvi),
-
Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer-review under responsibility of King Saud University.
Available online 1 January 2011
Abstract
A novel 3-(substituted benzylideneamino)-7-chloro-2-phenyl quinazoline-4(3H)-one (7–27) has been synthesized and characterised by IR, 1H NMR, 13C NMR spectroscopy, and elemental analysis. We changed the methodology for the synthesis of 3-amino 7-chloro-2-phenyl quinazolin-4(3H)-one 6 to fusion reaction at 250 °C, instead of using solvent, to avoid the problem of ring opening, which is commonly observed while synthesizing quinazolines from benzoxazinone. NCI selected, 7-chloro-3-{[(4-chlorophenyl) methylidene] amino}-2-phenylquinazolin-4(3H)-one 12, with GI50 value of −5.59 M, TGI value of −5.12 M, and LC50 value of −4.40 M showed remarkable activity against CNS SNB-75 Cancer cell line. Rational approach and QSAR techniques enabled the understanding of the pharmacophoric requirement for 2,3,7-tri substituted quinazoline derivatives to inhibit EGFR-tyrosine kinase as antitumor agents and could be used as an excellent framework in this field that may lead to discovery of potent anti tumor agent.
Keywords
Synthesis 3-(benzylideneamino)-7-chloro-2-phenyl quinazoline-4(3H)-one
Fusion
QSAR
Rational design
Anticancer activity
1 Introduction
Protein tyrosine kinases are enzymes involved in many cellular processes such as cell proliferation, metabolism, survival, and apoptosis. Several protein tyrosine kinases are known to be activated in cancer cells and to drive tumor growth and progression. Blocking tyrosine kinase activity therefore represents a rational approach to cancer therapy. Protein kinases (PTKs) catalyze the phosphorylation of tyrosine and serine/threonine residues in various proteins involved in the regulation of all functions (Jordan et al., 2000). They can be broadly classified as receptor such as EGFR, or non-receptor kinases. Inappropriate or uncontrolled activation of many of these kinases, by over-expression, constitutive activation, or mutation, has been shown to result in uncontrolled cell growth (Blume-Jensen and Hunter, 2001). Overexpression of these receptors was found in a number of cancers (e.g., breast, ovarian, colon, and prostate), their expression levels often correlate with vascularity, and is associated with poor prognosis in patients (Slichenmeyer et al., 2001; Fricker, 2006). Inhibitors of the EGFR PTK are therefore expected to have great therapeutic potential in the treatment of malignant and nonmalignant epithelial diseases. Drug discovery efforts have targeted this aberrant kinase activity in cancer, asthma, psoriasis, and inflammation (Cohen, 2002). Recent advances in the identification of erbB family kinase inhibitors have created hope for the modulation of uncontrolled cell growth in cancer therapy for solid tumors (Gschwind et al., 2004)
This strongly suggests that these targets represent drug intervention opportunities due to pivotal role in governing cellular proliferation, survival, and metastasis. A great number of different structural classes of tyrosine kinase inhibitors have been reported and reviewed (Adams, 2001; Yarden and Sliwkowski, 2001; Dumas, 2001). The most promising small molecule selective EGFR-TK inhibitors include quinazolines. Chart 1 includes some examples that are currently approved drugs or in clinical trials (Fricker, 2006).
EGFR-tyrosine kinase inhibitors.
In view of previous rational and in continuation of our research for newer anti-cancer agents (Manjula et al., 2009; Badiger et al., 2006) in the present study new series of 2,3,7-tri substituted quinazoline have been optimized and synthesized (Fig. 1). Quinazoline ring opening is a common phenomenon in the synthesis of quinazoline from benzoxazinone. We changed the methodology for the synthesis of 3-amino 7-chloro-2-phenyl quinazolin-4(3H)-one 6 to fusion reaction at 250 °C, instead of using solvent, to avoid the problem of ring opening, which is commonly observed while synthesizing quinazolines from benzoxazinone. By using this new methodology the preparation of 3-amino 7-chloro-2-phenyl quinazolin-4(3H)-one 6 is simple, time-saving and eco-friendly process without solvent and also avoid the problem of ring opening. Thus, it is a step towards green chemistry.
Reported and proposed antitumor quinazoline derivatives.
2 Rational and design
In recent years, quinazolines have emerged as a versatile template for inhibition of a diverse range of receptor tyrosine kinases. The most widely studied of these is the epidermal growth factor receptor (EGFR), with the small-molecule inhibitor gefitinib being the first agent from this class to be approved for the treatment of Non-Small Cell Lung Cancer refractory to prior chemotherapeutic intervention (Peter et al., 2006; Ranson, 2004). Subsequent research aimed at further exploration of the SAR of this novel template has led to discovery of highly selective compounds that target EGFR. These compounds act via competing with ATP for binding at the catalytic domain of tyrosine kinase. Later on, a great structural variety of compounds of structurally diverse classes have proved to be highly potent and selective ATP-competitive inhibitors. The ATP binding site has the following features; Adenine region – contains two key hydrogen bonds formed by the interaction of N-1 and N-6 amino group of the adenine ring. Many potent inhibitors use one of these hydrogen bonds. Sugar region – a hydrophilic region, except a few e.g., EGFR. Hydrophobic pocket – though not used by ATP but plays an important role in inhibitor selectivity. Hydrophobic channels – it is not used by ATP and may be exploited for inhibitor specificity. Phosphate binding region – this is used for improving inhibitor selectivity (Fig. 2) (Fabbro et al., 2002). In this study, we present a new sub-family of compounds containing 2,3,7-tri substituted quinazoline core as EGFR inhibitors. Our strategy is directed toward designing a variety of ligands with diverse chemical properties hypothesizing that the potency of these molecules might be enhanced by adding alternative binding group such as phenyl ring at position 2-, imines at position 3-, and chloro group at position 7- of the quinazoline ring. In this way, such substitution pattern could target different regions of the ATP-binding site of the protein kinase domain to create differentially selective molecules. The design of our ligands was done based on previous quantitative structure–activity relationship (QSAR) of 4-anilinoquinazolines as EGFR inhibitor (Noolvi and Patel, 2010, Noolvi et al., 2011). We introduced larger moiety at 3 position of the quinazoline such as substituted arylidene moiety in a fashion similar to lapatinib which binds in the ATP-binding cleft, so that the bulky group could be oriented deep in the back of the ATP binding site and makes predominantly hydrophobic interactions with the protein mimicking the 3′-chloro-4′-[(3-fluorobenzyl)oxy]aniline group of lapatinib (Fig. 3).
Model of the ATP-binding site of protein kinases. ATP is depicted in red; Sug1, Hyp1, and Hyc1 are residues lining the sugar region, hydrophobic pocket (Hyp), hydrophobic channel (Huc), and hinge region (Hin), respectively.
Proposed hypothetical model of the 2,3,7-trisubstituted quinazoline bound to ATP binding site of EGFR-protein tyrosine kinase.
3 Materials and methods
3.1 Chemistry
4-Chloro 2-amino benzoic acid 1 reaction with benzoyl chloride 2 yielded 7-chloro 2-phenyl-4H-3, 1-benzoxazin-4 one 3 by N-acylation via dehydrative cyclization mechanism. Subsequently which was refluxed with hydrazine hydrate in dry pyridine in an attempt to obtain 3-amino 7-chloro-2-phenyl quinazolin-4(3H)-one, but we got mixture of 3-amino 7-chloro-2-phenyl quinazolin-4(3H)-one 5 and ring opened N-(5-chloro-2(hydrazine carbonyl) phenyl) benzamide 4. Later we tried the same reaction at 250 °C by fusion without any solvent and this time we got the desired product 3-amino 7-chloro-2-phenyl quinazolin-4(3H)-one 6 without any ring opened structure (diamides). Fusion is more convenient and time saving method since it takes only 0.5 h, where conventional takes 3.0 h for the reaction. On the other hand ring opening is a common phenomenon in the synthesis of quinazoline from benzoxazinone. This problem can be avoided by fusing benzoxazinone at high temperature resulting in the synthesis of desired quinazoline otherwise one will get mixture of quinazoline and ring opened quinazoline (diamides), as shown mechanistically in Chart 2. Condensation of 6 with different substituted aromatic aldehydes gave corresponding arylidene derivatives of quinazoline 7–27 in glacial acetic acid (Scheme 1).
Reaction mechanism and ring opening phenomenon in the synthesis of target compound 7–27 (Scheme 1).
Synthetic route for the synthesis of quinazoline derivatives (7–27).
3.2 Anticancer screening at NIH, Bethesda, Maryland, USA
The tumor growth inhibition properties of compounds 12 with the NCI codes NSC D-753447/1 selected among 7–27 synthesized compounds (Scheme 1) by the National Cancer Institute (NCI), USA, were screened on human tumor cell lines at the NIH, Bethesda, Maryland, USA, under the drug discovery program of the NCI, for one and five dose anti-cancer assay.
The screening is a two-stage process, beginning with the evaluation of all compounds against the 60 cell lines at a single dose of 10 μM. The output from the single dose screen is reported as a mean graph and is available for analysis by the COMPARE program. Compounds which exhibit significant growth inhibition are evaluated against the 60 cell panel at five concentration levels. The human tumor cell lines of the cancer screening panel are grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM l-glutamine. For a typical screening experiment, cells are inoculated into 96-well microtiter plates in 100 μl at plating densities ranging from 5000 to 40,000 cells/well depending on the doubling time of individual cell lines. After cell inoculation, the microtiter plates are incubated at 37 °C, 5% CO2, 95% air, and 100% relative humidity for 24 h prior to addition of experimental drugs.
After 24 h, two plates of each cell line are fixed in situ with TCA, to represent a measurement of the cell population for each cell line at the time of drug addition (Tz). Experimental drugs are solubilized in dimethyl sulfoxide at 400-fold the desired final maximum test concentration and stored frozen prior to use. At the time of drug addition, an aliquot of frozen concentrate is thawed and diluted to twice the desired final maximum test concentration with complete medium containing 50 μg/ml gentamicin. Additional four, 10-fold or ½ log serial dilutions are made to provide a total of five drug concentrations plus control. Aliquots of 100 μl of these different drug dilutions are added to the appropriate microtiter wells already containing 100 μl of medium, resulting in the required final drug concentrations.
Following drug addition, the plates are incubated for an additional 48 h at 37 °C, 5% CO2, 95% air, and 100% relative humidity. For adherent cells, the assay is terminated by the addition of cold TCA. Cells are fixed in situ by the gentle addition of 50 μl of cold 50% (w/v) TCA (final concentration, 10% TCA) and incubated for 60 minutes at 4 °C. The supernatant is discarded, and the plates are washed five times with tap water and air dried. Sulforhodamine B (SRB) solution (100 μl) at 0.4% (w/v) in 1% acetic acid is added to each well, and plates are incubated for 10 minutes at room temperature. After staining, unbound dye is removed by washing five times with 1% acetic acid and the plates are air dried. Bound stain is subsequently solubilized with 10 mM trizma base, and the absorbance is read on an automated plate reader at a wavelength of 515 nm. For suspension cells, the methodology is the same except that the assay is terminated by fixing settled cells at the bottom of the wells by gently adding 50 μl of 80% TCA (final concentration, 16% TCA). Using the seven absorbance measurements[time zero, (Tz), control growth, (C), and test growth in the presence of drug at the five concentration levels (Ti)], the percentage growth is calculated at each of the drug concentrations levels. Percentage growth inhibition is calculated as:
Three dose–response parameters are calculated for each experimental agent. Growth inhibition of 50% (GI50) is calculated from[(Ti − Tz)/(C − Tz)] × 100 = 50, which is the drug concentration resulting in a 50% reduction in the net protein increase (as measured by SRB staining) in control cells during the drug incubation. The drug concentration resulting in total growth inhibition (TGI) is calculated from Ti = Tz. The LC50 (concentration of drug resulting in a 50% reduction in the measured protein at the end of the drug treatment as compared to that at the beginning) indicating a net loss of cells following treatment is calculated from[(Ti − Tz)/Tz] × 100 = −50. Values are calculated for each of these three parameters if the level of activity is reached; however, if the effect is not reached or is exceeded, the value for that parameter is expressed as greater or less than the maximum or minimum concentration tested (Alley et al., 1988; Grever et al., 1992; Boyd and Paul,1995).
4 Result and discussion
In the present work, 2,3,7-trisubstituted quinazolines were prepared according to reaction Scheme 1. The spectral data of 7–27 showed IR band at 1634–1678 cm−1 due to stretching vibration of C⚌O group of the quinazoline moiety and absorption band at 1372–1436 cm−1 is due to the C–N stretching vibration. C–Cl stretching vibration is appeared at 721–768. Further 1H NMR of compounds 7–27 showed presence of a singlet between δ 8.77 and 9.34 ppm indicate the formation of imine (>HC⚌N–). Peak at δ 7.10–8.30 ppm showed presence of aromatic protons. Synthesis of 7–27 compounds was also confirmed by 13C NMR data, peak at around δ 168.23 ppm has confirmed formation imine (>HC⚌N–).
The tumor growth inhibition properties of compounds 12 with the NCI codes NSC D-753447/1 selected among 7–27 synthesized compounds (Scheme 1) by the National Cancer Institute (NCI), USA, were screened on human tumor cell lines at the NIH, Bethesda, Maryland, USA, under the drug discovery program of the NCI, for one and five dose anti-cancer assay.
The tested quinazoline derivative showed a distinctive pattern of selectivity. With regard to sensitivity against individual cell lines (Fig. 4), compound 12 showed remarkably lowest cell growth promotion against Renal A-498 cancer cell line of −43.73, apart from this it also exhibited broad spectrum cell growth inhibition against Non-Small Cell Lung Cancer NCI-H522 (cell growth promotion 20.09%, inhibition 79.91%), Colon cancer HCT-116 (cell growth promotion 38.26%, inhibition 61.74%), CNS cancer U-251 (cell growth promotion 37.22%, inhibition 62.78%), and Ovarian OVCAR-8 cell line (cell growth promotion 33.54%, inhibition 66.46%) at concentration of 10−5 M in one dose primary assay.
One dose mean graph of compound 12 at 10−5 M concentration.
The same compound was further screened for 5-log dose range as it has shown prominent cell growth inhibition at 10−5 M concentration against verity of cell lines. Three response parameters, median growth inhibition (GI50), total growth inhibition (TGI), and median lethal growth inhibition (LC50) were calculated for each cell line (Skehan et al., 1990). It showed remarkable activity against CNS SNB-75 cancer cell line with GI50 value of −5.59 and TGI value of −5.12 apart from this it also exhibited broad spectrum cell growth inhibition against: Non-Small Cell Lung Cancer Lines; HOP-62 (GI50 value −5.13 and TGI value −4.51), NCI-H226 (GI50 value −5.40 and TGI value −4.70), NCI-H23 (GI50 value −5.08 and TGI value −4.23), NCI-H460 (GI50 value −5.51 and TGI −5.08), and NCI-H522 (GI50 value −5.56 &and TGI value −5.19), Colon HCT-116 cancer cell lines (GI50 value of −5.50 and TGI value −4.85), CNS cancer cell lines; SNB-19 (GI50 value −5.24 and TGI value −4.39), SF-539 (GI50 value −5.43 and TGI value −4.73), and U251 (GI50 value −5.36 and TGI value −4.38), Melanoma UACC-62 cancer cell line (GI50 value −5.15 and TGI value −4.65), Ovarian cancer cell lines; IGROV1 (GI50 value −5.31 and TGI value −4.81), OVCAR 3 (GI50 value −5.56 and TGI value −5.19), and SK-OV-3 (GI50 value −5.11 and TGI value −4.40), Renal cancer cell lines; 786-0 (GI50 value −5.37 and TGI value −4.29), A498 (GI50 value −5.07 and TGI value −4.10), RXF-393 (GI50 value −4.79 and TGI value −4.14), TK-10 (GI50 value −4.96 and TGI value −4.16), and UO-31 (GI50 value −5.40 and TGI value −4.07), Breast BT-549 cancer cell line with GI50 value of −5.42 and TGI value of −4.71. It was also found to be active at median lethal concentration against CNS SNB-75 (LC50 value −4.40), Melanoma UACC-62 (LC50 value −4.21), and Ovarian OVCAR 3 cancer cell line (LC50 value −4.47) at 5-log dose range as shown in Tables 1 and 2. The highest activity of this compound might be because of its structural resemblance with lapatinib since it contain 4-chloro benzylideneamine group at C-3 position of quinazoline which suppose to block the hydrophobic pocket of tyrosine kinase (Fig. 3).
NSC: D-753447/1
Experiment ID: 1009 NS13
Units: Molar
COMI: SIH6 (96135)
Strain reagent: SRB Dual Pass
SSPL: 0XXS
Parent cell line
Time
Mean optical densities
Percent growth
GI50
TGI
LC50
Zero
Control
−8.0
−7.0
−6.0
−5.0
−4.0
−8.0
−7.0
−6.0
−5.0
−4.0
Leukemia
CCRFCEM
0.356
1.727
1.680
1.633
1.696
1.628
1.559
97
93
98
93
88
>1.00 E−4
>1.00 E−4
>1.00 E−4
MOLT-4
0.439
1.652
1.712
1.741
1.718
1.628
1.520
105
107
105
98
89
>1.00 E−4
>1.00 E−4
>1.00 E−4
RPMI-8226
0.795
2.712
2.696
2.643
2.631
2.033
1.772
99
96
96
65
51
>1.00 E−4
>1.00 E−4
>1.00 E−4
SR
0.240
1.406
1.428
1.455
1.447
0.743
0.547
102
104
104
43
26
7.70 E−6
>1.00 E−4
>1.00 E−4
Non-Small Cell Lung Cancer
A549/ATCC
0.253
1.319
1.262
1.286
1.293
0.664
0.699
95
97
98
39
42
6.39 E−6
>1.00 E−4
>1.00 E−4
EKVX
0.720
1.673
1.653
1.674
1.611
1.288
1.158
98
100
94
60
46
5.05 E−5
>1.00 E−4
>1.00 E−4
HOP-62
0.421
1.062
1.027
1.005
1.004
0.703
0.226
94
91
91
44
−46
7.44 E−6
3.06 E−5
>1.00 E−4
HOP-92
1.396
2.139
1.989
1.969
1.916
1.721
1.468
80
77
70
44
10
5.77 E−6
>1.00 E−4
>1.00 E−4
NCI-H226
0.630
1.349
1.341
1.347
1.353
0.743
0.397
99
100
101
16
−37
3.94 E−6
1.98 E−5
>1.00 E−4
NCI-H23
0.514
1.382
1.406
1.388
1.432
0.909
0.444
103
101
106
45
−14
8.40 E−6
5.88 E−5
>1.00 E−4
NCI-H322M
0.715
1.092
1.171
1.117
1.152
0.807
0.725
121
106
116
24
3
5.23 E−6
>1.00 E−4
>1.00 E−4
NCI-H460
0.154
1.363
1.434
1.455
1.455
0.141
0.148
106
108
108
−9
−4
3.13 E−6
8.41 E−6
>1.00 E−4
NCI-H522
0.607
1.456
1.487
1.552
1.545
0.449
0.447
104
111
110
−26
−21
2.77 E−6
6.45 E−6
>1.00 E−4
Colon cancer
COLO 205
0.266
1.128
1.184
1.205
1.201
1.116
1.068
107
109
109
99
93
>1.00 E−4
>1.00 E−4
>1.00 E−4
HCC-2998
0.474
1.476
1.465
1.511
1.541
1.095
1.027
99
103
106
62
55
>1.00 E−4
>1.00 E−4
>1.00 E−4
HCT-116
0.171
1.540
1.590
1.536
1.486
0.230
0.130
104
100
96
4
−24
3.18 E−6
1.42 E−5
>1.00 E−4
HCT-15
0.193
1.375
1.367
1.331
1.311
1.073
0.924
99
96
95
74
62
>1.00 E−4
>1.00 E−4
>1.00 E−4
HT-29
0.155
0.883
0.941
0.960
0.995
0.783
0.729
108
111
115
86
79
>1.00 E−4
>1.00 E−4
>1.00 E−4
KM 12
0.415
1.793
1.855
1.848
1.826
1.480
1.333
104
104
102
77
67
>1.00 E−4
>1.00 E−4
>1.00 E−4
SW-620
0.124
0.842
0.854
0.886
0.871
0.629
0.455
102
106
104
70
46
6.90 E−5
>1.00 E−4
>1.00 E−4
CNS cancer
SF-268
0.346
1.231
1.271
1.264
1.230
0.395
0.360
104
104
100
6
9
3.38 E−6
>1.00 E−4
>1.00 E−4
SF-295
0.701
1.779
1.730
1.697
1.717
1.242
0.895
95
92
94
50
18
1.01 E−5
>1.00 E−4
>1.00 E−4
SF-539
0.492
1.757
1.742
1.711
1.690
0.707
0.269
99
96
95
17
−45
3.76 E−6
1.87 E−5
>1.00 E−4
SNB-19
0.417
1.200
1.200
1.226
1.256
0.666
0.334
100
103
107
32
−20
5.73 E−6
4.12 E−5
>1.00 E−4
SNB-75
0.662
1.136
1.049
1.108
1.108
0.574
0.172
82
94
94
−13
−74
2.57 E−6
7.51 E−6
4.02 E−5
U251
0.279
1.391
1.388
1.364
1.378
0.531
0.240
100
98
99
23
−14
4.37 E−6
4.12 E−5
>1.00 E−4
Melanoma
LOX IMVI
0.141
0.945
0.912
0.939
0.838
0.582
0.429
96
99
87
55
36
1.78 E−5
>1.00 E−4
>1.00 E−4
M14
0.284
1.199
1.199
1.164
1.236
1.291
0.865
100
96
104
110
63
>1.00 E−4
>1.00 E−4
>1.00 E−4
MDA-MB-435
0.340
1.243
1.221
1.237
1.257
0.981
0.971
98
99
101
71
70
>1.00 E−4
>1.00 E−4
>1.00 E−4
SK-MEL-2
0.821
2.277
2.389
2.405
2.415
1.322
0.849
108
109
110
34
9
6.20 E−6
>1.00 E−4
>1.00 E−4
SK-MEL-28
0.485
1.120
1.157
1.144
1.216
1.034
0.797
106
104
115
86
49
9.47 E−5
>1.00 E−4
>1.00 E−4
SK-MEL-5
0.316
1.746
1.763
1.736
1.689
1.062
0.749
101
99
96
52
30
1.25 E−5
>1.00 E−4
>1.00 E−4
UACC-257
0.466
1.075
1.065
1.047
1.051
0.876
0.800
98
95
96
67
55
>1.00 E−4
>1.00 E−4
>1.00 E−4
UACC-62
0.598
1.775
1.770
1.823
1.807
1.076
0.152
100
104
103
41
−75
7.05 E−6
2.25 E−5
6.12 E−5
Ovarian cancer
IGROV1
0.499
1.085
1.175
1.184
1.203
0.532
0.377
115
117
120
6
−24
4.10 E−6
1.54 E−5
>1.00 E−4
OVCAR3
0.282
0.859
0.917
0.918
0.907
0.212
0.077
110
110
108
−25
−73
2.74 E−6
6.51 E−6
3.36 E−5
OVCAR4
0.468
0.864
0.875
0.901
0.908
0.370
0.345
103
109
111
−21
−26
2.90 E−6
6.93 E−6
>1.00 E−4
OVCAR5
0.433
1.386
1.343
1.318
1.334
1.142
0.932
95
93
95
74
52
>1.00 E−4
>1.00 E−4
>1.00 E−4
OVCAR8
0.253
1.037
1.078
1.054
1.007
0.337
0.267
105
102
96
11
2
3.47 E−6
>1.00 E−4
>1.00 E−4
NCI-RES
0.442
1.482
1.559
1.533
1.496
0.636
0.518
107
105
101
19
7
4.18 E−6
>1.00 E−4
>1.00 E−4
SK-OV-3
0.579
1.289
1.307
1.289
1.313
0.886
0.412
103
100
103
43
−29
7.70 E−6
3.97 E−5
>1.00 E−4
Renal cancer
786-0
0.582
1.943
1.856
1.893
1.781
0.955
0.518
94
96
88
27
−11
4.25 E−6
5.15 E−5
>1.00 E−4
A498
0.798
1.312
1.305
1.340
1.315
1.035
0.758
98
105
100
46
−5
8.47 E−6
7.98 E−5
>1.00 E−4
ACHN
0.213
1.255
1.303
1.297
1.235
0.626
0.401
105
104
98
40
18
6.63 E−6
>1.00 E−4
>1.00 E−4
CAKI-1
0.570
1.525
1.565
1.510
1.367
1.057
1.184
104
98
83
51
64
>1.00 E−4
>1.00 E−4
>1.00 E−4
RXF-393
0.464
1.040
1.037
1.072
1.032
0.844
0.413
99
106
99
66
−11
1.61 E−5
7.20 E−5
>1.00 E−4
SN12C
0.431
1.502
1.562
1.588
1.540
1.009
0.686
106
108
104
54
24
1.35 E−5
>1.00 E−4
>1.00 E−4
TK-10
0.544
1.369
1.391
1.398
1.447
0.979
0.490
103
104
109
53
−10
1.11 E−5
6.92 E−5
>1.00 E−4
UO-31
0.679
1.233
1.184
1.164
1.200
0.792
0.669
91
87
94
20
−2
3.95 E−6
8.50 E−5
>1.00 E−4
Prostate cancer
PC-3
0.413
1.309
1.284
1.300
1.265
0.619
0.534
97
99
95
23
14
4.22 E−6
>1.00 E−4
>1.00 E−4
DU-145
0.298
1.204
1.243
1.214
1.194
0.765
0.566
104
101
99
51
30
1.17 E−5
>1.00 E−4
>1.00 E−4
Breast cancer
MCF7
0.288
1.086
1.176
1.076
1.106
0.607
0.417
111
99
102
40
16
>1.00 E−4
>1.00 E−4
>1.00 E−4
MDA-MB-231
0.503
1.182
1.203
1.195
1.207
0.925
0.684
103
102
104
62
27
>1.00 E−4
>1.00 E−4
>1.00 E−4
HS-578T
0.636
1.257
1.272
1.291
1.271
0.956
0.733
102
105
102
51
16
>1.00 E−4
>1.00 E−4
>1.00 E−4
BT-549
0.866
1.733
1.743
1.689
1.729
0.994
0.555
101
95
100
15
−36
>1.00 E−4
>1.00 E−4
>1.00 E−4
T-47D
0.575
1.094
.055
1.088
1.071
0.647
0.610
93
99
96
14
7
>1.00 E−4
>1.00 E−4
>1.00 E−4
MDA-MB-468
0.462
1.057
1.036
1.097
1.069
0.896
0.720
96
105
102
73
43
>1.00 E−4
>1.00 E−4
>1.00 E−4
NSC: D-753447/1
Experiment ID: 1009 NS13
Units: Molar
COMI: SIH6 (96135)
Strain reagent: SRB Dual Pass
SSPL: 0XXS
Cell Lines
log10 GI50
log10 TGI
log10 LC50
Leukemia
CCRFCEM
>−4.00
>−4.00
>−4.00
MOLT-4
>−4.00
>−4.00
>−4.00
RPMI-8226
>−4.00
>−4.00
>−4.00
SR
−5.11
>−4.00
>−4.00
Non-Small Cell Lung Cancer
A549/ATCC
−5.19
>−4.00
>−4.00
EKVX
−4.30
>−4.00
>−4.00
HOP-62
−5.13
−4.51
>−4.00
HOP-92
−5.24
>−4.00
>−4.00
NCI-H226
−5.40
−4.70
>−4.00
NCI-H23
−5.08
−4.23
>−4.00
NCI-H322M
−5.28
>−4.00
>−4.00
NCI-H460
−5.51
−5.08
>−4.00
NCI-H522
−5.56
−5.19
>−4.00
Colon cancer
COLO 205
>−4.00
>−4.00
>−4.00
HCC-2998
>−4.00
>−4.00
>−4.00
HCT-116
−5.50
−4.85
>−4.00
HCT-15
>−4.00
>−4.00
>−4.00
HT-29
>−4.00
>−4.00
>−4.00
KM 12
>−4.00
>−4.00
>−4.00
SW-620
−4.16
>−4.00
>−4.00
CNS cancer
SF-268
−5.47
>−4.00
>−4.00
SF-295
−4.99
>−4.00
>−4.00
SF-539
−5.43
−4.73
>−4.00
SNB-19
−5.24
−4.39
>−4.00
SNB-75
−5.59
−5.12
−4.40
U251
−5.36
−4.38
>−4.00
Melanoma
LOXIMVI
−4.75
>−4.00
>−4.00
M14
>−4.00
>−4.00
>−4.00
MDA-MB-435
>−4.00
>−4.00
>−4.00
SK-MEL-2
−5.21
>−4.00
>−4.00
SK-MEL-28
−4.02
>−4.00
>−4.00
SK-MEL-5
−4.90
>−4.00
>−4.00
UACC-257
>−4.00
>−4.00
>−4.00
UACC-62
−5.15
−4.65
−4.21
Ovarian cancer
IGROV1
−5.31
−4.81
>−4.00
OVCAR3
−5.56
−5.19
−4.47
OVCAR4
−5.54
>−4.00
>−4.00
OVCAR5
>−4.00
>−4.00
>−4.00
OVCAR8
−5.46
>−4.00
>−4.00
NCI-RES
−5.38
>−4.00
>−4.00
SK-OV-3
−5.11
−4.40
>−4.00
Renal cancer
786-0
−5.37
−4.29
>−4.00
A498
−5.07
−4.10
>−4.00
ACHN
−5.18
>−4.00
>−4.00
CAKI-1
>−4.00
>−4.00
>−4.00
RXF-393
−4.79
−4.14
>−4.00
SN12C
−4.87
>−4.00
>−4.00
TK-10
−4.96
−4.16
>−4.00
UO-31
−5.40
−4.07
>−4.00
Prostate cancer
PC-3
−5.37
>−4.00
>−4.00
DU-145
−4.93
>−4.00
>−4.00
Breast cancer
MCF7
−5.16
>−4.00
>−4.00
MDA-MB-231
−4.66
>−4.00
>−4.00
HS-578T
−4.96
>−4.00
>−4.00
BT-549
−5.42
−4.71
>−4.00
T-47D
−5.44
>−4.00
>−4.00
MDA-MB-468
−4.23
>−4.00
>−4.00
5 Conclusion
A novel 3-(substituted benzylideneamino)-7-chloro-2-phenyl quinazoline-4(3H)-one (7–27) has been synthesized. We changed the methodology for the synthesis of 3-amino 7-chloro-2-phenyl quinazolin-4(3H)-one 6 to fusion reaction at 250 °C, instead of using solvent, to avoid the problem of ring opening, which is commonly observed while synthesizing quinazolines from benzoxazinone. Fusion is more convenient and time saving method since it takes only 0.5 h, where conventional takes 3.0 h for the reaction. By using this new methodology the preparation of 3-amino 7-chloro-2-phenyl quinazolin-4(3H)-one 6 is simple, time-saving and eco-friendly process without solvent and also avoid the problem of ring opening. NCI selected, 7-chloro-3-{[(4-chlorophenyl) methylidene] amino}-2-phenylquinazolin-4(3H)-one 12, with GI50 value of −5.59 M, TGI value of −5.12 M, and LC50 value of −4.40 M showed remarkable activity against CNS SNB-75 cancer cell line. Rational approach and QSAR techniques enabled the understanding of the pharmacophoric requirement for quinazoline derivatives. The overall outcome of this model revealed that: (i) the quinazoline ring is satisfactory backbone for antitumor activity, (ii) the presence of hydrophobic group at 3 position of quinazoline enhances the activity. These preliminary encouraging results of biological screening of the tested compounds could offer an excellent framework in this field that may lead to discovery of potent antitumor agent.
6 Experimental
All chemicals and solvents were supplied by Merck, S.D. Fine Chemical Limited, Mumbai. All the solvents were distilled and dried before use. The reactions were monitored with the help of thin-layer chromatography using pre-coated aluminum sheets with GF254 silica gel, 0.2 mm layer thickness (E. Merck). Melting points of the synthesized compounds were recorded on the Veego (VMP-MP) melting point apparatus. IR spectrum was acquired on a Shimadzu Infra Red Spectrometer, (model FTIR-8400S). Both 1H NMR (DMSO) spectra of the synthesized compounds were performed with Bruker Avance-II 400 NMR Spectrometer operating at 400 MHz in SAIF, Punjab University (Chandigarh). Chemical shifts were measured relative to internal standard TMS (δ: 0). 13C NMR spectra were recorded at 67.8 MHz on the same instrument with internal TMS (δ: 0, DMSO). Chemical shifts are reported in δ scale (ppm). Elemental analyses were performed at the Microanalytical Laboratory of the SAIF, Punjab University.
6.1 Synthesis of 7-chloro 2-phenyl-benzo[d][1,3] oxazine-4-one (3)
4-Chloro 2-amino benzoic acid 1 (0.1 mole) was dissolved in minimum volume of dry pyridine (30 ml) by shaking. To this solution benzoyl chloride 2 (0.2 mole) taken in dry pyridine (30 ml), was added slowly with constant stirring. When the addition was completed (the operation of addition required half an hour), the resultant solution was subjected to vigorous stirring for one hour mechanically subsequently, it was left as such for one hour at room temperature and treated with a solution of sodium bicarbonate (10%). Addition of sodium bicarbonate solution was continued till the effervescence due to the evolution of carbon-dioxide ceased. The separated solid was allowed to settle down and filtered off. It was washed with cold water repeatedly till there was no smell of pyridine and unreacted benzoyl chloride. The crude benzo-oxazine was dried in vacuum overnight and recrystallization from diluted ethanol afforded analytically pure sample of 2-phenyl-benzo[d][1,3] oxazin-4-one 3 as white crystalline mass. Yield 82%; mp 195–198 °C. IR (KBr, νmax, cm−1): 3062 (C–H), 1745 (C⚌O), 1510 (C⚌N), 1480 (C⚌C), 1364 (C–N), 771 (C–Cl). 1H NMR (DMSO-d6) δ ppm: 7.60–8.21 (m, 8H, Ar-H). 13C NMR (DMSO-d6) δ ppm: 164.2, 158.4, 156.2, 140.4, 136.2, 134.6, 130.8, 128.6, 128.4, 126.6, 124.4, 114.4. Anal. Calcd for C14H8ClNO2: C, 65.26; H, 3.13; N, 5.44. Found: C, 65.42; H, 3.04; N, 5.65%.
6.2 Synthesis of isomers of N-(5-chloro-2-(hydrazine carbonyl) phenyl) benzamide (4) and 3-amino 7-chloro-2-phenyl quinazolin-4(3H)-one (5)
A mixture of 7-chloro 2-phenyl-benzo[d][1,3] oxazine-4-one 3 (0.01 mol) and hydrazine hydrate (0.01 mol) in dry pyridine (50 ml) was heated under reflux for 3 h. Subsequently, mixture was poured into water (containing few drops of HCl) solid thus separated was filtered, washed repeatedly with water. It was dried and crystallized from ethanol. Isomers were separated by column chromatography using benzene: acetone in 7:3 ratio.
6.2.1 N-(5-Chloro-2-(hydrazine carbonyl) phenyl) benzamide (4)
This compound was prepared, purified, and separated as per the above mentioned procedure: yield 35%; mp 215–217 °C. IR (KBr, νmax, cm−1): 3212 (s), 1560 (b) (NH), 3057 (C–H), 1665 (C⚌O), 1484 (C⚌C), 748 (C–Cl). 1H NMR (DMSO-d6) δ ppm: 6.21 (s, 2H, NH2), 7.14–8.60 (m, 8H, Ar-H), 10.19 (s, 1H, –NHCO–), 9.12 (s, 1H, –CONH–). 13C NMR (DMSO-d6) δ ppm: 168.6, 168.2, 145.6, 142.7, 136.6, 132.1, 130.3, 128.8, 126.8, 122.4, 120.5, 118.4. Anal. Calcd for C14H12ClN3O2: C, 58.04; H, 4.17; N, 14.50. Found: C, 58.18; H, 4.42; N, 14.81%.
6.2.2 3-Amino 7-chloro-2-phenyl quinazolin-4(3H)-one (5)
This compound was prepared, purified and separated as per the above mentioned procedure: yield 41%; mp 206–209 °C. IR (KBr, νmax, cm−1): 3270 (s), 1582 (b) (NH2), 3078 (CH), 1666 (C⚌O), 1564 (C⚌N), 1491 (C⚌C), 1371 (C–N), 762 (C–Cl). 1H NMR (DMSO-d6) δ ppm: 5.25 (s, 2H, NH2), 7.14–8.18 (m, 8H, Ar-H). 13C NMR (DMSO-d6) δ ppm: 164.8, 158.4, 154.6, 138.6, 132.6, 132.6, 130.8, 130.6, 128.6, 127.6, 120.8. Anal. Calcd for C14H10ClN3O: C, 61.89; H, 3.71; N, 15.47. Found: C, 61.66; H, 3.78; N, 15.68%.
6.2.3 Optimized method for the synthesis of 3-amino 7-chloro-2-phenyl quinazolin-4(3H)-one (6)
A mixture of 7-chloro 2-phenyl-benzo[d][1,3] oxazine-4-one 3 and hydrazine hydrate was fused together at 250 °C in an oil bath for 0.5 h. The mixture was cooled and methanol was added to the mixture. The separated solid was collected by filtration, washed with methanol, dried, and crystallized from ethanol. Yield 68%; mp 206–209 °C. IR (KBr, νmax, cm−1): 3270 (s), 1582 (b) (NH2), 3078 (CH), 1666 (C⚌O), 1564 (C⚌N), 1491 (C⚌C), 1371 (C–N), 762 (C–Cl). 1H NMR (DMSO-d6) δ ppm: 5.25 (s, 2H, NH2), 7.14–8.18 (m, 8H, Ar-H). 13C NMR (DMSO-d6) δ ppm: 164.8, 158.4, 154.6, 138.6, 132.6, 132.6, 130.8, 130.6, 128.6, 127.6, 120.8. Anal. Calcd for C14H10ClN3O: C, 61.89; H, 3.71; N, 15.47. Found: C, 61.46; H, 3.85; N, 15.66%.
6.3 General procedure for the synthesis of 3-(substituted benzylideneamino)-7-chloro-2-phenyl quinazoline-4(3H)-one (7–27)
A mixture of 3-amino-7-chloro-2-phenyl-3H-quinazoline-4-one 6 and different aldehydes in equimolar quantities in glacial acetic acid was heated under reflux for six hours. The solution was cooled and poured carefully into crushed ice. A solid separated out which was allowed to settle down. It was filtered off, washed successively with water and dried. The solid thus obtained was on recrystallization from diluted ethanol afforded white crystalline solid mass.
6.3.1 3-(Anthracene-9-yl methyleneamino)-7-chloro-2-phenyl quinazoline-4(3H)-one (7)
This compound was prepared and purified as per the above mentioned procedure: yield 62%; mp 288–291 °C. IR (KBr, νmax, cm−1): 3052 (CH), 1650 (C⚌O), 1545 (C⚌N), 1495 (C⚌C), 1384 (C–N), 752 (C–Cl). 1H NMR (DMSO-d6) δ ppm: 6.54–8.28 (m, 17H, Ar-H), 8.77 (s, 1H, N⚌CH). 13C NMR (DMSO-d6) δ ppm: 170.5, 168.7, 156.6, 154.5, 146.8, 140.8, 132.8, 131.2, 131.1, 129.9, 129.8, 129.2, 129.0, 125.6, 124.6, 123.8, 122.2, 118.0. Anal. Calcd for C29H18ClN3O: C, 75.73; H, 3.94; N, 9.14. Found: C, 75.64; H, 3.64; N, 9.06%.
6.3.2 3-(Benzylideneamino)-7-chloro-2-phenyl quinazoline-4(3H)-one (8)
This compound was prepared and purified as per the above mentioned procedure: yield 68%; mp 234–236 °C. IR (KBr, νmax, cm−1): 3089 (CH), 1654 (C⚌O), 1552 (C⚌N), 1488 (C⚌C), 1386 (C–N), 724 (C–Cl). 1H NMR (DMSO-d6) δ ppm: 7.41–8.28 (m, 13H, Ar-H), 9.29 (s, 1H, N⚌CH). 13CNMR (DMSO-d6) δ ppm: 170.7, 168.6, 160.4, 156.4, 154.2, 140.4, 135.7, 132.0, 131.2, 131.1, 130.6, 129.8, 129.2, 125.6, 122.6, 118.0. Anal. Calcd for C21H14ClN3O: C, 70.10; H, 3.92; N, 11.68. Found: C, 70.24; H, 3.68; N, 11.51%.
6.3.3 3-(3-Bromo benzylideneamino)-7-chloro-2-phenyl quinazoline-4(3H)-one (9)
This compound was prepared and purified as per the above mentioned procedure: yield 71%; mp 248–251 °C. IR (KBr, νmax, cm−1): 3065 (CH), 1634 (C⚌O), 1556 (C⚌N), 1494 (C⚌C), 1389 (C–N), 735 (C–Cl), 685 (C–Br). 1H NMR (DMSO-d6) δ ppm: 7.05–8.36 (m, 12H, Ar-H), 8.98 (s, 1H, N⚌CH). 13C NMR (DMSO-d6) δ ppm: 170.7, 168.6, 156.2, 154.6, 152.5, 140.2, 136.9, 134.9, 132.7, 131.2, 131.1, 130.2, 129.8, 129.2, 125.1, 123.7, 122.4, 118.0. Anal. Calcd for C21H13BrClN3O: C, 57.49; H, 2.99; N, 9.58. Found: C, 57.36; H, 2.91; N, 9.45%.
6.3.4 3-(4-Cyano benzylideneamino)-7-chloro-2-phenyl quinazoline-4(3H)-one (10)
This compound was prepared and purified as per the above mentioned procedure: yield 82%; mp 222–225 °C. IR (KBr, νmax, cm−1): 3071 (CH), 2245 (cyano), 1645 (C⚌O), 1534 (C⚌N), 1488 (C⚌C), 1392 (C–N), 765 (C–Cl). 1H NMR (DMSO-d6) δ ppm: 7.41–8.28 (m, 12H, Ar-H), 9.29 (s, 1H, N⚌CH). 13C NMR (DMSO-d6) δ ppm: 170.7, 168.3, 160.8, 156.4, 154.5, 141.2, 140.2, 135.3, 131.2, 131.1, 129.8, 125.4, 126.3, 122.6, 118.0, 116.6, 114.6. Anal. Calcd for C22H13ClN4O: C, 68.67; H, 3.41; N, 14.56. Found: C, 68.75; H, 3.24; N, 14.39%.
6.3.5 3-(2-Chloro benzylideneamino)-7-chloro-2-phenyl quinazoline-4(3H)-one (11)
This compound was prepared and purified as per the above mentioned procedure: yield 83%; mp 211–214 °C. IR (KBr, νmax, cm−1): 3058 (CH), 1647 (C⚌O), 1536 (C⚌N), 1490 (C⚌C), 1421 (C–N), 756 (C–Cl). 1H NMR (DMSO-d6) δ ppm: 7.12–8.28 (m, 12H, Ar-H), 9.12 (s, 1H, N⚌CH). 13C NMR (DMSO-d6) δ ppm: 170.8, 168.4, 156.3, 154.2, 146.6, 140.1, 136.7, 135.9, 132.4, 131.1, 129.8, 129.2, 125.4, 125.2, 126.9, 122.4, 118.0. Anal. Calcd for C21H13Cl2N3O: C, 63.98; H, 3.32; N, 10.66. Found: C, 63.88; H, 3.27; N, 10.63%.
6.3.6 3-(4-Chloro benzylideneamino)-7-chloro-2-phenyl quinazoline-4(3H)-one (12)
This compound was prepared and purified as per the above mentioned procedure: yield 63%; mp 228–230 °C. IR (KBr, νmax, cm−1): 3052 (CH), 1657 (C⚌O), 1530 (C⚌N), 1495 (C⚌C), 1412 (C–N), 725 (C–Cl). 1H NMR (DMSO-d6) δ ppm: 7.34–8.61 (m, 12H, Ar-H), 9.23 (s, 1H, N⚌CH). 13C NMR (DMSO-d6) δ ppm: 170.6, 168.3, 160.4, 156.7, 154.5, 140.1, 136.6, 132.8, 131.6, 131.2, 131.1, 129.9, 129.8, 129.2, 125.4, 122.4, 118.0. Anal. Calcd for: C21H13Cl2N3O C, 63.98; H, 3.32; N, 10.66. Found: C, 63.82; H, 3.36; N, 10.58%.
6.3.7 3-(4-Dimethylamino benzylideneamino)-7-chloro-2-phenyl quinazoline-4(3H)-one (13)
This compound was prepared and purified as per the above mentioned procedure: yield 52%; mp 218–220 °C. IR (KBr, νmax, cm−1): 3059 (CH), 1659 (C⚌O), 1538 (C⚌N), 1478 (C⚌C), 1423 (C–N), 734 (C–Cl). 1H NMR (DMSO-d6) δ ppm: 2.48 (s, 6H, CH3), 7.39–8.39 (m, 12H, Ar-H), 9.34 (s, 1H, N⚌CH). 13C NMR (DMSO-d6) δ ppm: 170.8, 168.3, 160.8, 156.6, 156.1, 154.2, 140.1, 131.2, 131.1, 129.8, 129.3, 129.2, 125.4, 123.6, 122.4, 118.0, 114.8, 41.6. Anal. Calcd for C23H19ClN4O: C, 68.57; H, 4.75; N, 13.91. Found: C, 68.52; H, 4.78; N, 13.63%.
6.3.8 3-(3-Methoxy 4-hydroxy benzylideneamino)-7-chloro-2-phenyl quinazoline-4(3H)-one (14)
This compound was prepared and purified as per the above mentioned procedure: yield 78%; mp 260–263 °C. IR (KBr, νmax, cm−1): 3410 (OH), 3058 (CH), 1678 (C⚌O), 1562 (C⚌N), 1485 (C⚌C), 1435 (C–N), 710 (C–Cl). 1H NMR (DMSO-d6) δ ppm: 3.94 (s, 3H, OCH3), 7.51–8.34 (m, 11H, Ar-H), 8.90 (s, 1H, N⚌CH), 10.12 (s, 1H, OH). 13C NMR (DMSO-d6) δ ppm: 170.8, 168.7, 156.2, 154.5, 153.8, 152.4, 150.1, 140.8, 131.9, 131.2, 131.1, 129.8, 129.2, 125.4, 122.8, 122.3, 118.0, 116.6, 114.6. Anal. Calcd for C22H16ClN3O3: C, 65.11; H, 3.97; N, 10.35. Found: C, 65.18; H, 3.63; N, 10.21%.
6.3.9 3-(4-Methoxy benzylideneamino)-7-chloro-2-phenyl quinazoline-4(3H)-one (15)
This compound was prepared and purified as per the above mentioned procedure: yield 62%; mp 251–254 °C. IR (KBr, νmax, cm−1): 3055 (CH), 1664 (C⚌O), 1540 (C⚌N), 1489 (C⚌C), 1414 (C–N), 714 (C–Cl). 1H NMR (DMSO-d6) δ ppm: 3.96 (s, 3H, OCH3), 7.10–8.26 (m, 12H, Ar-H), 9.11 (s, 1H, N⚌CH). 13C NMR (DMSO-d6) δ ppm: 170.4, 168.2, 164.1, 160.4, 156.2, 154.5, 140.8, 131.2, 131.1, 129.8, 129.2, 125.4, 126.0, 122.4, 118.0, 116.4, 54.4. Anal. Calcd for C22H16ClN3O2: C, 67.78; H, 4.14; N, 10.78. Found: C, 67.81; H, 4.05; N, 10.67%.
6.3.10 3-(2-Hydroxy benzylideneamino)-7-chloro-2-phenyl quinazoline-4(3H)-one (16)
This compound was prepared and purified as per the above mentioned procedure: yield 69%; mp 210–213 °C. IR (KBr, νmax, cm−1): 3390 (OH), 3088 (CH), 1656 (C⚌O), 1648 (C⚌C), 1510 (C⚌N), 1498 (C⚌C), 1445 (C–N), 724 (C–Cl). 1H NMR (DMSO-d6) δ ppm: 7.56–8.30 (m, 12H, Ar-H), 9.10 (s, 1H, N⚌CH), 11.99 (s, 1H, OH). 13C NMR (DMSO-d6) δ ppm: 170.4, 168.3, 160.6, 156.1, 154.2, 146.1, 140.4, 134.4, 131.2, 131.1, 129.8, 129.2, 125.5, 125.4, 122.2, 121.1, 118.0, 114.4, 112.4. Anal. Calcd for C21H14ClN3O2: C, 67.12; H, 3.75; N, 11.18. Found: C, 67.32; H, 3.72; N, 11.05%.
6.3.11 3-(4-Hydroxy benzylideneamino)-7-chloro-2-phenyl quinazoline-4(3H)-one (17)
This compound was prepared and purified as per the above mentioned procedure: yield 72%; mp 244–247 °C. IR (KBr, νmax, cm−1): 3418 (OH), 3079 (CH), 1644 (C⚌O), 1524 (C⚌N), 1478 (C⚌C), 1405 (C–N), 768 (C–Cl) cm−1. 1H NMR (DMSO-d6) δ ppm: 7.16–8.70 (m, 12H, Ar-H), 9.16 (s, 1H, N⚌CH), 11.99 (s, 1H, OH). 13C NMR (DMSO-d6) δ ppm: 170.4, 168.6, 164.8, 160.3, 156.6, 154.2, 140.1, 131.6, 131.2, 131.1, 129.8, 129.2, 125.4, 126.5, 122.4, 118.0, 114.2. Anal. Calcd for C21H14ClN3O2: C, 67.12; H, 3.75; N, 11.18. Found: C, 67.04; H, 3.67; N, 11.26%.
6.3.12 3-(2,5-Dimethoxy benzylideneamino)-7-chloro-2-phenyl quinazoline-4(3H)-one (18)
This compound was prepared and purified as per the above mentioned procedure: yield 78%; mp 251–254 °C. IR (KBr, νmax, cm−1): 3071 (CH), 1646 (C⚌O), 1522 (C⚌N), 1491 (C⚌C), 1428 (C-N), 745 (C–Cl). 1H NMR (DMSO-d6) δ ppm: 3.96 (s, 6H, OCH3), 7.10–8.64 (m, 11H, Ar-H), 8.88 (s, 1H, N⚌CH). 13C NMR (DMSO-d6) δ ppm: 170.4, 168.3, 156.6, 156.1, 154.4, 150.9, 146.2, 140.2, 131.2, 131.1, 129.8, 129.2, 125.4, 122.6, 118.0, 116.9, 116.6, 114.6, 112.4, 54.2. Anal. Calcd for C23H18ClN3O3: C, 65.79; H, 4.32; N, 10.01. Found: C, 65.98; H, 4.21; N, 10.14%.
6.3.13 3-(4-Methyl benzylideneamino)-7-chloro-2-phenyl quinazoline-4(3H)-one (19)
This compound was prepared and purified as per the above mentioned procedure: yield 62%; mp 223–226 °C. IR (KBr, νmax, cm−1): 3078 (CH), 1662 (C⚌O), 1512 (C⚌N), 1498 (C⚌C), 1434 (C–N), 724 (C–Cl). 1H NMR (DMSO-d6) δ ppm: 2.48 (s, 3H, CH3), 7.12–8.87 (m, 12H, Ar-H), 8.90 (s, 1H, N⚌CH). 13C NMR (DMSO-d6) δ ppm: 170.8, 168.3, 160.2, 156.4, 154.2, 142.7, 140.2, 131.7, 131.2, 131.1, 130.5, 129.8, 129.2, 125.4, 126.1, 122.4, 118.0, 22.4. Anal. Calcd for C22H16ClN3O: C, 70.68; H, 4.31; N, 11.24. Found: C, 70.66; H, 4.12; N, 11.65%.
6.3.14 3-(3-Nitro benzylideneamino)-7-chloro-2-phenyl quinazoline-4(3H)-one (20)
This compound was prepared and purified as per the above mentioned procedure: yield 70%; mp 246–250 °C. IR (KBr, νmax, cm−1): 3076 (CH), 1648 (C⚌O), 1518 (C⚌N), 1545, 1354 (NO2), 1496 (C⚌C), 1421 (C–N), 752 (C–Cl). 1H NMR (DMSO-d6) δ ppm: 7.18–8.33 (m, 12H, Ar-H), 9.28 (s, 1H, N⚌CH). 13C NMR (DMSO-d6) δ ppm: 170.1, 169.2, 168.3, 156.4, 154.2, 150.4, 140.2, 136.8, 132.4, 131.2, 131.1, 130.2, 129.8, 129.2, 125.4, 123.6, 122.1, 118.0. Anal. Calcd for C21H13ClN4O3: C, 62.31; H, 3.24; N, 13.84. Found: C, 62.16; H,3.32; N, 13.63%.
6.3.15 3-(4-Nitro benzylideneamino)-7-chloro-2-phenyl quinazoline-4(3H)-one (21)
This compound was prepared and purified as per the above mentioned procedure: yield 71%; mp 232–234 °C. IR (KBr, νmax, cm−1): 3072 (CH), 1676 (C⚌O), 1528 (C⚌N), 1554, 1348 (NO2), 1486 (C⚌C), 1428 (C–N), 760 (C–Cl). 1H NMR (DMSO-d6) δ ppm: 7.65–8.53 (m, 12H, Ar-H), 9.45 (s, 1H, N⚌CH). 13C NMR (DMSO-d6) δ ppm: 170.8, 169.4, 168.3, 160.2, 156.4, 154.1, 144.7, 140.2, 131.2, 130.1, 129.8, 129.2, 125.7, 125.4, 122.6, 121.1, 118.0. Anal. Calcd for C21H13ClN4O3: C, 62.31; H, 3.24; N, 13.84. Found: C, 62.29; H, 3.21; N, 13.88%.
6.3.16 3-(2-Nitro benzylideneamino)-7-chloro-2-phenyl quinazoline-4(3H)-one (22)
This compound was prepared and purified as per the above mentioned procedure: yield 62%; mp 262–265 °C. IR (KBr, νmax, cm−1): 3064 (CH), 1676 (C⚌O), 1510 (C⚌N), 1561, 1352 (NO2), 1484 (C⚌C), 1436 (C–N), 736 (C–Cl). 1H NMR (DMSO-d6) δ ppm: 7.65–8.43 (m, 12H, Ar-H), 9.12 (s, 1H, N⚌CH). 13C NMR (DMSO-d6) δ ppm: 170.3, 168.8, 168.4, 156.3, 154.1, 146.1, 140.8, 136.8, 132.8, 130.2, 130.2, 130.1, 130, 129.8, 129.2, 125.4, 124.6, 122.6, 121.1, 118.0. Anal. Calcd for C21H13ClN4O3: C, 62.31; H, 3.24; N, 13.84. Found: C, 62.38; H, 3.16; N, 13.98%.
6.3.17 3-(4-Bromo benzylideneamino)-7-chloro-2-phenyl quinazoline-4(3H)-one (23)
This compound was prepared and purified as per the above mentioned procedure: yield 68%; mp 268–270 °C. IR (KBr, νmax, cm−1): 3068 (CH), 1656 (C⚌O), 1538 (C⚌N), 1486 (C⚌C), 1398 (C–N), 764 (C–Cl), 671 (C–Br). 1H NMR (DMSO-d6) δ ppm: 7.16–8.54 (m, 12H, Ar-H), 9.35 (s, 1H, N⚌CH). 13C NMR (DMSO-d6) δ ppm: 170.6, 168.4, 160.5, 156.3, 154.2, 140.8, 134.7, 132.7, 131.2, 131.1, 129.8, 129.5, 129.2, 125.4, 124.4, 122.4, 118.0. Anal. Calcd for C21H13BrClN3O: C, 57.49; H, 2.99; N, 9.58. Found: C, 57.64; H, 3.08; N, 9.45%.
6.3.18 3-(4-Fluro benzylideneamino)-7-chloro-2-phenyl quinazoline-4(3H)-one (24)
This compound was prepared and purified as per the above mentioned procedure: yield 58%; mp 224–226 °C. IR (KBr, νmax, cm−1): 3064 (CH), 1658 (C⚌O), 1544 (C⚌N), 1492 (C⚌C), 1368 (C–N), 1214 (C–Fl), 728 (C–Cl). 1H NMR (DMSO-d6) δ ppm: 7.55–8.64 (m, 12H, Ar-H), 9.14 (s, 1H, N⚌CH). 13C NMR (DMSO-d6) δ ppm: 170.0, 168.7, 165.3, 160.6, 156.5, 154.2, 140.2, 131.8, 131.2, 131.1, 130.2, 129.8, 129.2, 125.4, 122.6, 118.0, 114.8. Anal. Calcd for C21H13ClFN3O: C, 66.76; H, 3.47; N, 11.12. Found: C, 66.64; H, 3.62; N, 11.02%.
6.3.19 3-(3,4-Dihydroxy benzylideneamino)-7-chloro-2-phenyl quinazoline-4(3H)-one (25)
This compound was prepared and purified as per the above mentioned procedure: yield 52%; mp 236–241 °C. IR (KBr, νmax, cm−1): 3428 (OH), 3057 (CH), 1639 (C⚌O), 1515 (C⚌C), 1556 (C⚌N), 1494 (C⚌C), 1388 (C–N), 732 (C–Cl). 1H NMR (DMSO-d6) δ ppm: 7.16-8.51 (m, 11H, Ar-H), 8.91 (s, 1H, N⚌CH), 9.63 (s, 2H, OH). 13C NMR (DMSO-d6) δ ppm: 170.4, 168.6, 156.4, 154.4, 152.4, 150.8, 148.2, 140.6, 132.3, 131.2, 131.1, 129.8, 129.2, 125.4, 123.5, 122.5, 118.0, 116.2, 114.2. Anal. Calcd for C21H14ClN3O3: C, 64.37; H, 3.60; N, 10.72. Found: C, 64.56; H, 3.32; N, 10.62%.
6.3.20 3-(3-Hydroxy benzylideneamino)-7-chloro-2-phenyl quinazoline-4(3H)-one (26)
This compound was prepared and purified as per the above mentioned procedure: yield 48%; mp 214–218 °C. IR (KBr, νmax, cm−1): 3412 (OH), 3061 (CH), 1638 (C⚌O), 1532 (C⚌C), 1565 (C⚌N), 1489 (C⚌C), 1372 (C–N), 721 (C–Cl). 1H NMR (DMSO-d6) δ ppm: 7.56–8.58 (m, 12H, Ar-H), 8.96 (s, 1H, N⚌CH), 9.32 (s, 1H, OH). 13C NMR (DMSO-d6) δ ppm: 170.4, 168.4, 159.4, 156.2, 154.1, 152.2, 140.7, 131.2, 131.1, 129.8, 129.2, 125.4. Anal. Calcd for C21H14ClN3O2: C, 67.12; H, 3.75; N, 11.18. Found: C, 67.15; H, 3.89; N, 11.24%.
6.3.21 3-(3-Phenyl allyllidene amino)-7-chloro-2-phenyl quinazoline-4(3H)-one (27)
This compound was prepared and purified as per the above mentioned procedure: yield 71%; mp 283–285 °C. IR (KBr) νmax 3066 (CH), 2110 (C⚌C), 1651 (C⚌O), 1514 (C⚌N), 1482 (C⚌C), 1394 (C–N), 764 (C–Cl). 1H NMR (DMSO-d6) δ ppm: 6.95–9.32 (a set of signals, 16H, Ar-H, olefinic CH⚌CH and N⚌CH). 13C NMR (DMSO-d6) δ ppm: 170.6, 168.4, 156.2, 154.2, 140.6, 138.6, 136.2, 134.1, 131.2, 131.1, 129.8, 129.6, 129.5, 129.2, 125.9, 125.4, 122.4, 118.0. Anal. Calcd for C23H16ClN3O: C, 71.59; H, 4.18; N, 10.89. Found: C, 71.68; H, 4.31; N, 10.61%.
Acknowledgements
The authors thank Director General, Department of Science and Technology, New Delhi for funding the project (Grant No. SR/FT/LS-0024/2008), Chairman, Captain M.P. Singh and Sardar Sangat Singh Longia, Secretary ASBASJSM College of Pharmacy for providing the necessary facilities.
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