Pyrimidine: a review on anticancer activity with key emphasis on SAR

Cancer is a global health challenge, it impacts the quality of life and its treatment is associated with several side effects. Resistance of the cancer cells to the existing drugs has led to search for novel anticancer agents. Pyrimidine, a privileged scaffold, is part of living organisms and plays vital role in various biological procedures as well as in cancer pathogenesis. Due to resemblance in structure with the nucleotide base pair of DNA and RNA, it is recognized as valuable compound in the treatment of cancer. Many novel pyrimidine derivatives have been designed and developed for their anticancer activity in the last few years. The present review aims to focus on the structure activity relationship (SAR) of pyrimidine derivatives as anticancer agent from the last decade. This review intends to assist in the development of more potent and efficacious anticancer drugs with pyrimidine scaffold.

moderate to highly active against the A549 (IC 50 = 12.05 ± 0.45 μM), HTC-116 (IC 50 = 1.31 ± 0.41 μM), and MCF-7 (IC 50 = 20.53 ± 6.13 μM) cell lines and exhibited potent aurora kinase inhibition against both aurora A and B kinase. Apoptosis was induced due to upregulation of Bax and downregulation in Bcl-xl. The SAR studies suggests that the benzene ring when replaced with cyclohexyl group gave better activity and replacement of NH in urea with CH 2 lead to decrease in activity [5]. It was established further that the blockade of G2-M phase of cell cycle occurred by accumulation of the contents at S phase due to decrease in mitochondrial membrane potential making 2,4-diaminopyrimidine derivatives potential anticancer agents. 3a (IC 50 = 2.14 to 5.52 μM) and 3b (IC 50 = 1.98 to 4.27 μM) were most potent against the PC-3, A549, MCF-7, and HCT-116 cancer cell lines due to the variation of substitution on aromatic ring and terminal aniline on the pyrimidine moiety [6]. The induction of apoptosis was observed for cancer cell line K562 by novel anilino substituted pyrimidine sulfonamides. Cell viability was tested through MTT and tunnel assay. Compounds 4(a-c) demonstrated a promising activity with IC 50 range = 5.6 to 12.3 μM [7]. Figure 1 depicts the chemical structure of 2,4-disubstituted pyrimidine derivatives.
Trisubstituted pyrimidines 2, 5, 6-Trisubstituted pyrimidines A new series of 2,4-diaminopyrimidines were reported as potent and selective aurora A kinase inhibitors. The best potency of the compounds was elucidated against HeLa, A-549, HCT-8, and Hep-G2 cells compared with the VX-680 as positive control by MTT assay. Compound 7 exhibited highest cytotoxicity with an IC 50 = 0.5-4.0 μm as well as led to cell cycle arrest in HeLa cells at G2/M phase [10].

2, 4, 5-Trisubstituted pyrimidines
New 5-alkyl pyrimidine derivatives, alkyl Nmethoxymethyl pyrimidine derivatives, and 5,6-dihydrofuro [2,3-d]pyrimidines were reported with cytostatic activities using 5-FU as the positive control. 5-chloroethyl-2,6 dichloro pyrimidine 8 (IC 50 = 0.8 ± 0.2 μM) exerted cytostatic effect on HCT-116 cancer cell line which led to cell cycle arrest at G2/M phase due to DNA damage. The SAR suggests that the presence of two aromatic and an aliphatic chlorine atom linked to the pyrimidine ring gave the compound with maximum potential [11]. Furthermore, 2-arylaminopyrimidine derivative bearing a 2amino-N-methylbenzamide at C4 and chlorine at C5 positions were designed as a potent inhibitor of c-Met in cellular and enzymatic assays. C2 benzazepinone were found to be the most potent c-Met inhibitors, 9 (IC 50 = 10 nM) being the best analog. Incorporation of fluorine at C3 position of aminobenzamide moiety led to selectivity for c-Met kinase [12].

2, 4, 6-Trisubstituted pyrimidines
Recently, in a study of synthesis and anticancer activity of trisubstituted pyrimidines and their N-alkyl derivatives was studied via ELISA, BRU, and MTT assay and 10 posed exceptional activity. They were tested against A549, Hep3B, HT29 FL, MCF-7, and HeLa cell lines with the IC 50 range from 2 to10 μm/ml [13]. Moreover, anthranillic acid ester moiety-linked 2,4,6-trisubstituted pyrimidines were tested for cytotoxic activty. The compounds were screened against U-937, CEM-13, MDA-MB-231, DU-145, and BT-474 cancer cell lines by conventional MTT assays. 11a and 11b were known to be the most potent in the series and also as CDK9 inhibitors. The SAR studies reveal that the major activity is due to the (E)-styryl moiety at C-6 position, methyl group at R 2 position, and the presence of methylanthranilate moiety with an EDG at C-4 lead to better activity ( Fig. 4) [14].
Pyrimidines and triazolopyrimidines as antiproliferative agents exhibited COX-1/2 inhibitory potential. Compound 12 (IC 50 range = 8.68 ± 0.2 to 36.56 ± 0.9 μg/ml) displayed in vitro activity against cancer cell lines HepG-2, MCF-7, CaCo-2, and A549 alongside COX-2 inhibition using 5-FU as the reference drug [15]. Previously in a study, combretastatin bridged pyrimidine derivatives were tested for antitumor activity against the MCF-7 and A549 using MTT assay. 13a (IC 50 = 4.67 μM; 3.38 μM) and 13b (IC 50 = 0.63 μM; 3.71 μM) were concluded to have the best potential. 13a induced apoptosis by ROS-regulated intrinsic apoptotic pathway; they were non-toxic to harmful cells and were more potent inhibitors than cholchicne in the tunnel assay. The SAR demonstrated that the R 2 and R 3 substituted rings affected the activity, EWG such as 2,4-dichlorosubstitution on the rings manifested good activity, and interchange of amine, methyl with hydrogen in R 1 position of the pyrimidine ring displayed no activity. Replacement of rings with napthyl gave less activity and no substitution in any of the three rings also depicted activity [16].
In a library of N-trisubstituted pyrimidine scaffold, compound 14 (IC 50 = 12.2 nM) elucidated the best activity in the inhibition of U937 cell line. It caused the inhibition by inducing polyploidy (4N, 8N, and 16N) in the cancer cells by inducing defects in both chromosome formation and spindle formation. The SAR studies are depicted in Fig. 5 [17].
In a continued study, a series of pyrimidinebenzimidazole compound 15 with an IC 50 = 1.06 to 12.89 μM, was tested against the MGC-803, SMMC-7721, EC-9706, and MCF-7 cell lines antiproliferative activity. The cell cycle came to rest at G2/M phase by the active compound accompanied by an increase in apoptotic cell death of MGC-803 [18]. Additionally, in the previous year, the novel thiazolopyrimidine derivatives were studied against the human cancer cell lines and primary CLL cells. 16 displayed excellent anticancer activity against the cell lines and led to cell death by apoptosis as it inhibited the CDK enzyme [19].
Evaluation for pharmacological activity was conducted for some novel 1, 2, 4-triazole containing pyrimidine derivatives. 17(a-b) were found to have effective activity on HOP-92 as evaluated using the MTT assay [20]. In an earlier work, MTT assay was conducted to determine cytotoxic study of the series novel pyrimidine derivatives 18a (% inhibition at 50 μg/mL = 43.62) containing 4chlorophenyl substitution on 6th position of pyrimidine nucleus and 18b (% inhibition at 50 μg/mL = 39.52) containing thiophene ring, showed the most potency. The SAR revealed that the most of the activity was due to the substitution of mono or di chlorine at the R1 position of the phenyl ring [21]. The in vitro anticancer activity of novel pyrimidine derivatives was reported in which it was concluded that 19 showed the best activity against the whole panel of 60 cancer cell lines (especially lung cancer cell lines). The cell lines were tested at different concentration of the compounds, and the SAR studies revealed that the EDG groups like NH 2 and EWG like Cl and C=O at orthoand paraposition of the ring is highly influential for the activity (Fig. 6) [22].
Tetrasubstituted pyrimidine derivatives 2,4,5,6-Tetrasubstituted pyrimidines A study reported ERα and VEGFR-2 ligands in the form of 2, 4-disubstituted pyrimidine derivatives and tested them against the MCF-7 cancer cell lines. 20 had ERα binding affinity (IC 50 = 1.64 μM) and inhibition activity against VEGFR-2 (IC 50 = 0.085 μM). It acted by suppressing the progesterone inhibition mRNA and in vivo angiogenesis inhibition in CAM assay. Suppression of cell migration, apoptosis, and transduction reticence of Raf-1/MAPK/ERK in MCF-7 cells were also reported. SAR studies showed that the hydrogen bonding interaction at the head is significant for increase in ERα binding affinity (Fig. 7) [23].
Pyrimidine substituted with 1, 2, 3-triazole-urea was screened for oncogenic activity against MGC-803, B16-F10, EC-109, and MCF-7 cancer cell lines. Compounds 23a, 23b, and 23c were reported to exhibit potent activity against B16-F10 with an IC 50 = 32 nM, 35 nM, and 42 nM respectively. The SAR revealed that the electronic effect on the phenyl ring affects the activity, an EDG group, gives more activity and at R 1 the presence of 4methyl and 4-methoxyl attributes better activity than 2methyl, 3-methyl, and 2-methoxyl substitutions [26]. Additionally, anticancer activity of pyrimidine-thiourea derivatives was studied. The derivatives inhibited histone LSD1 which was over expressed in many tumor cells. Compound 24 (IC 50 = 0.65 ± 0.12 μM) was most potent against gastric cancer cell line and it inhibited cell migration and invasion with tumor suppressing and antimetastasis functions. Amino, thio, and urea groups are essential for the LSD-1 inhibition. Thio urea instead of urea offered better activity and subsequently the proprgyl and trimethoxy phenyl group increases anticancer activity ( Fig. 9) [27].
1, 2, 3-Triazole substituted pyrimidines were studied for anticancer activity against the MGC-803, EC-109,    A library of pyrimidine substituted with polymethoxy chalcones and thiazolopyrimidine for anticancer potential was reported. The activity was tested against 60 cell cancer line panel. 27 exhibited significant inhibition of tumor growth being highly efficient with cytotoxic and cytostatic attribute. SAR illustrated in Fig. 11 [30].

Pyrazolo [1, 5-a]pyrimidines
In a recent report, some novel fused pyrazolopyrimidine derivatives were studied for anticancer activity as well as COX-2 inhibition against a 60 cancer cell line panel. Compound 29 was potent in case of both the studies. It was selective towards COX due to the presence of 5-amino-1-oxo-substituted-pyrazole-4-  SAR studies revealed that the order of antitumor activity was 4 methyl phenyl> 4 chloro phenyl> phenyl derivative against the cell lines and chlorine atom at 2 position was more active than 3 and 4 positions [33]. In another study by the same group, antitumor activities of pyrazolo [1, 5-a] pyrimidines was screened against HepG-2 and MCF-7 using MTT assay. 31a (IC 50 =63.2 ± 5.9 μg/mL) was reported have the best potential against MCF-7 carcinoma cells and 31b (IC 50 =70.3 ±4.1 μg/mL) against HepG2 carcinoma cells. SAR suggested that substitutions with bulky groups like methoxy and bromo gave significant antitumor activity ( Fig. 13) [34]. A new series of diamide substituted pyrazolo [1, 5-a] pyrimidine derivatives were reported. 32(a-c) were active against HeLa cell lines where IC 50 value of each was less than 10 μM better than the marketed drug cisplatin. MTT assay was conducted to evaluate the cytotoxicity of the compounds. SAR discussed in Fig. 14 [35]. Chalcone-linked pyrazolo [1, 5-a] pyrimidines were reported as potential anticancer agents. 33 (IC 50 = 2.6 μM) was most potent against the MDA-MB231 cancer cell line as screened by MTT assay. The compound caused increase in the expression of apoptosis-inducing proteins like p53, p21, and Bax, and decreased proapoptotic proteins like Bcl-2 and procaspase-9, while stopping the cell cycle at sub G1 phase. The activity was mainly due to C-5 substitution phenylprop-2-en-1-one and C-7 phenyl ring [36].
The antiproliferative activity of the pyrazolo [1, 5-a] pyrimidine derivatives bearing nitrogen mustard moiety were tested for antitumor activity against cell lines A549, SH-SY5Y, HepG2, MCF-7, and DU145 via MTT assay. 34 inhibited the cell growth at the G1 phase of the cell cycle by inducing apoptosis in all the five cancer cell lines with the IC 50 range = 0.2 to 8.3 μM. The compound was tested against human HepG2, HCC tumor xenograft in nude mice, and gave better potency than positive control drug sorafenib and cyclophosphamide also being less toxic to normal human cells. N-mustard pharmacophore at C-7 and other substituent at C-5 of the pyrrazolo-pyrimidine derivatives exhibited potent in vitro cytotoxicity but when N-mustard pharmacophore is attached at C-5 and various aniline moieties at C-7 the compound formed is found to be ineffective [37]. In a consecutive study a regioselective synthesis of pyrazolo [1, 5-a] pyrimidine derivatives was conducted in presence of KHSO4(aq) assisted by ultrasound. The  In vitro cytotoxicity of pyrazolo [1,5-a] pyrimidine against cell lines HCT-116, A549, HepG2, and MCF-7 was reported. 36a and 36b have the best cytotoxicity and cancer cell growth inhibitory properties compared to the standard drug DOX. Order of activity in substituted pyrazolo [1,5-a] pyrimidines was phenyl groups> 4-chlorophenyl >4-methylphenyl group [39]. Moreover, some novel pyrazolo [1,5-a]pyrimidines were reported as the inhibitors of CDK9 which is often linked to cancer. The study used PIK-75 as a positive control and found out that the synthesized compound 37 (IC 50 = 203-> 1000 nM) is a better lead compound compared to PIK-75 due to lack of structural liabilities. The compound was also effective against FLT3 and MV4; 11 (IC 50 = 0.177 and 219 μM respectively) cancer cell lines ( Fig. 16) [40].
The synthesis of 2-aminobenzothizole conjugate linked with pyrazole [1,5-a] pyrimidines were tested for antitumor activity against cell lines namely A549, DU-145, MCF-7, ACHN, and HeLa via MTT assay. 38a (IC 50 range =1.94-3.46 μM) and 38b (IC 50 range = 2.01-7.07 μM) showed the best activity. Apoptosis was induced in caspase-3-dependent manner along with the arrest of cell cycle at G2/M and lowering of CDK1expression. SAR analysis suggests that substitution on the phenyl ring of aminobenzothiazole group does not show any promising effect on the activity whereas substitution on the 7-phenyl ring of pyrazolo [1,5-a]pyrimidine group is significant for the activity [41]. Interestingly, pim kinase inhibitors in the form of novel pyrazolo [1,5-a]pyrimidines were reported and it was concluded that compound 39 is a potential lead with low picomolar potency on the three isoforms of pim kinase. This particular enzyme is a significant target for cancer therapeutics but is hard to inhibit due to high affinity for ATP [42]. Novel 3-phenylpyrazolopyrimidine-1,2,3-triazole conjugates were studied for Src kinase inhibition and anticancer activity against SK-Ov-3, MDA-MB231, and HT-29 cell lines. 40a and 40c inhibited the enzyme with the   was the most efficacious and as per reports on CHO cell line it is non-toxic to normal human cells [45]. In a subsequent study, compound 43 showed the best anticancer activity against enzymes CDK2/cyclin E Abl kinases with significant activity against the K-562 and MCF-7 cell lines. Better CDK2 inhibition was observed with compounds with thiophenethyl group at C-6 and monosubstituted aniline at C-4 positions compared to thiopentane at C-6 and disubstituted aniline at C-4 position. Significant enzyme inhibitory activity was reported for compounds with 2-chloro, 3-nitro, and 4-methythio aniline at C-4 [46]. Synthesis as well as SAR studies of 6′-fluorocyclopentenyl pyrimidine derivatives was carried out along with assay against six human cancer cell lines including HCT-116, SNU-638, A-549, PC-3, SK-Hep-1, and MDA-MB-231 via SRB assay. 44a (IC 50 =1.10-2.17 μM) and 44b (IC 50 = 2.14-15.3 μM) exhibited good activity. The anticancer activity was attributed to arrest of sadenosylhomocysteine hydrolase which led to inhibition of histone methyltransferase. N6-amino group was essential for anticancer activity whereas deamination, introduction of bulky alkyl group or amino group at C2 position results loss of activity [47]. In simultaneous study, 4-aminopyrazolo [3,4-d] pyrimidines were reported as the potent inhibitors of both IGFIR/Src and as well as an anticancer agent with minimum toxicity to normal cells. 45 inhibited the kinase enzymes efficiently as well as induced apoptosis in the cancer cell lines MCF-7 and A549 with the IC 50 range = 9.7 to 15 μM. In vivo toxicity of the compound was evaluated on xenograft tumors and mutant Kras-driven lung tumorigenesis [48].
Pyrazolo [3,4-d] pyrimidines were reported as transmembrane RET inhibitors. 46 inhibited the RET phosphorylation and downstream signaling in BaF3/CCDC6-RET cells being a potent RET inhibitor in the various biochemical assays conducted. It had an IC 50 = 61 nM which makes it 6-folds more potent than the second best compound which was moderately efficacious against BaF3/CCDC6-RET cells with IC 50 = 433 nM in the cellular assays. Flexible side chain on the isoxazoline moiety like hydroxymethyl turned out to be most potent RET kinase inhibitor. Extending the carbon chain, capping the free hydroxyl group or addition of esteric substituent/alkyl substituents bearing a terminal pyrrolyl/morpholinyl moieties led to decrease in activity [49]. In a parallel study, in vivo and in vitro anticancer activity of pyrazolo [3,4-d]pyrimidines, 47(a-b) were reported in the form of prodrug. The compounds were evaluated against U-87 cancer cell line (in vitro), against Src and Abl kinase enzyme and also in vivo [50]. A series of novel N-4 substituted benzylidene acetohydrazide pyrazolo [3,4-d] pyrimidine was assessed for in vitro cytotoxicity against MCF-7, A549, and HT-29 cell lines. The compounds exhibited potent antitumor activity with 48 being the most potent. Molecular docking studies against the EGFR-TK revealed the compound was most effective inhibitor with docking score of − 28.8 Kcal/mol [51]. Pyrazolo [3,4-d] pyrimidine-3-carbonitriles were evaluated for activity against Hep 2 cancer cell line using MTT assay. Compound 49a (IC 50 =36.9 μM) and compound 49b (IC 50 = 21.3 μM) were found to be more active compared to the standard anticancer drug 5-FU (IC 50 = 41.5 μM). The presence of NH-C-S moiety and N-tosyl group in the pyrazolo-pyrimidine may be accounted for the activity [52].
Pyrazolo [3,4-d]pyrimidinones derivatives 50 (a-e) containing 1,2,3-triazole displayed good anticancer activity against the MCF-7 and HCT-116 cell lines using MTT assay. At the concentration of 100 μM, the inhibition percent of the cancer cell growth by these compounds is of the range 43-75% with tamoxifen as positive control. The major anticancer activity in this series of compounds is observed due to the presence of triazole group linked to the pyrazolo-pyrimidine ring through the methylene spacer linker [53]. Previously, novel pyrazolo[3,4-d]pyrimidine derivatives having anticancer potential were evaluated using MTT assay. 51 was tested against a panel of various cancer cell lines had the best potential against the A549 cells with IC 50 = 2.49 μM which is comparatively better than the positive control DOX [54].
In a study on cross-docking simulation of pyrazolo-[3, 4-d]pyrimidine on Bcr-Abl T315l mutant, 52a and 52b exhibited the best activity. 4-Bromo atom in paraposition of the N1 side chain of phenyl ring was responsible for interaction with the T315l mutant. The antitumor activity was tested in vivo using a mouse model xenograft [55]. Interestingly, a series of pyrazolo [3,4-d] pyrimidine was formed via substituting different polar moieties in C4 and C-6 positions of a promising anti leukemia lead. The compounds exhibited Src/Abl inhibitory activity and potent antiproliferative activity in leukemia cell lines (KU-812, MEG-01, and K-562). 53a and 53b showed good in vitro ADME properties as well as active in hypoxic leukemia cells [56].
Pyrazolo [3,4-d] pyrimidines with benzylidene hydrazinyl group were tested for their cytotoxic activity against MCF-7 cell line. 1-Phenyl substituted derivatives exhibited better activity compared to 1-(4-methoxyphenyl) derivatives compound 54 (IC 50 = 7.5nM) was the most potent [57]. One pot synthesis of pyrazolo [3, 4-d] pyrimidine derivatives along with their antiproliferative activity was reported. 55 (a-d) showed the most potency due to the presence of p-Me-Ph, p-Cl-Ph, or p-OMe-Ph group at C-3 position and phenyl or 2-quinolinyl groups at N-1 [58]. The green synthesis of pyrazolo [3,4]-pyrimidine-thiones was carried out and 56 (a-b) were most potent with IC 50 = 66 and 35 mg/ml against EAC cell lines ( Fig. 17) [59]. Compound 57 was reported to be most active among the series of substituted pyrazolo [3, 4-d] pyrimidines with the in vitro evaluation against MCF-7 cancer cell lines. The level of hydrogen peroxide and activity of superoxide dismutase were noticed to increase significantly whereas catalase activity and glutathione peroxidase levels were lowered. SAR is illustrated in Fig. 18 [60].

Pyrazolo [4,3-d]-pyrimidine
Pyrazolo [4, 3-d] pyrimidines with substitutions at 3, 5, 7 positions were reported as the inhibitors of CDK and also tested for in vivo and in vitro anticancer activity. 58 was the most potent, inhibited CDK 2, 5 and 9 (IC 50 = 0.002 μM) as well as exhibits both activities, and was better than the positive control CR8. When tested against 60 panels of cancer cancer cell lines, apoptosis was induced due to activation of caspases, dephosphorylation of CDK substrates, downregulation of XIAP, and MCL-1 and cleavage of PARP-1. In vivo activity was evaluated against various xenograft models. It was suggested that the activity was due to the modification of 2aminoethylthio group [61]. Previously, pyrazolo [4,3-d] pyrimidine a bioisoester of roscovitine was evaluated for the dual property of both CDK inhibition and antiproliferative activity. 59 was the bioisoster responsible for both the activities of CDK inhibition being the primary one. The anticancer activity of the compound was tested against a cancer cell line panel of 60 (IC 50 range = 3.6 ± 0.3 to 11 ± 1.8 μM) [62].
In vitro activity of substituted pyrazolo[4,3-d]-pyrimidine derivatives were reported against MCF-7, HT-29, and HepG2 tumor cell lines. Tetrazole ring fused to pyrazole [4,3-d]-pyrimidine 60 was most potent against all the cell lines with IC 50 range = 0.13 ± 0.03 to 0.36 ± 0.04 μmol/L respectively. The presence of tetrazole moiety fused to the pyrazolo-pyrimidine ring and also the nature of the substituted groups at the aryl ringlinked carbohydrazide with the pyrazole unit led to increased cytotoxic activity [63]. In a simultaneous study, a series of 5- pyrimidine derivatives were reported and it was concluded that compound 61 was the most potent inhibitor of both CDK2 (IC 50 = 21n M) and CDK5 (IC 50 = 35 n M). The activity was mainly due to the presence of hydroxyalkylamines at the 5 position of the pyrazolo-pyrimidine ring. The cell cycle was arrested at S and G2/M phase which resulted in induction of apoptosis [64].
In study selective inhibitors of CDK2, CDK5, and aurora A kinase with in vitro anti-angiogenic activity were reported. 62 was the most potent in the series and was known to inhibit all the three kinase enzymes as well as cause the downregulation of cyclins A and B, the dephosphorylation of histone H3 at Ser10, and the induction of mitochondrial apoptosis in the HCT-116 cancer cell line. It also reduced cell migration in human endothelial cells. The anti-angiogenic property of the  compound is linked to CDK5 inhibition [65]. The microwave assisted synthesis of 1H-pyrazolo [4,3-d] pyrimidin-7(6H)-ones and their anticancer activity were reported. The compounds were tested against HeLa, CAKI-I, PC-3, MiaPaca-2, and A549 human cancer cell lines by MTT assay. 63 was the most potent as it exhibits anticancer activity against all cell lines and the IC 50 range = 14 to 38 μM, with apoptosis mechanism along with mTOR inhibition at nM potency ( Fig. 19) [66].  Fig. 20 [67]. Previously, some Src tyrosine kinase family selective novel pyrrolo [2, 3-d] pyrimidine derivatives were reported. The inhibitory activity was tested against the Fyn, Lyn, Hck, and c-Src members of the Src kinase family. 65 was known to show the most potent activity Fyn, Lyn, and c-Src though it was non selective and not show activity against the Hck. PP2 and CGP77675 were used as positive control [68]. A novel series of pyrrolopyrimidine and pyrrolo-pyridine compounds bearing pyridazinone moiety were studied as c-Met kinase inhibitors, evaluated for in vitro and in silico molecular docking activity against the A549, HepG2, PC-3, and MCF-7 cancer cell lines. 66a was more active than 66b showed excellent activity against the cell lines with the IC 50 (μM) range 3.62 ± 1.24 to 9.61 ± 0.95 and 2.73 ± 0.98 to 3.77 ± 1.24 respectively. The compound induced apoptosis in HePG2 cell line. The compounds were screened against 4 tyrosine kinase (Slp-3, VEGRF-2, c-Kit, and EGFR). EWG on the aryl group in the pyridazinone moiety makes the compound increasingly active. The pyridine derivatives containing pyridazinone moiety are better than the pyrimidine moieties comparatively and, thus, a pyridine derivative was selected as c-Met kinase inhibitor [69].
A series of novel sulfonamide bearing pyrrolopyrimidine derivatives were screened against the MCF-7 cancer cell line. 67a and 67b (IC 50 = 8.30 and 8.39 μM) exhibited comparable activity to the positive control DOX. For the study of the mechanism of action, a molecular docking study on the compounds was conducted for the Src kinase enzyme and it was suggested that the inhibition of this enzyme is the reason for the anticancer potential of the compounds (Fig. 21) [70].  [71].
In a study based on the cytotoxicity of 1, 2, 3-triazoles and 1, 2, 4-triazolo fused with [1, 5-a] pyrimidines in MCF-7 cancer cell line. 69 showed better activities compared to other compounds in the series. VEGF and its receptor VEGFR are essential for the neovasculature; it is also associated with the progression of various malignancies which make this a significant target for the cancer treatment. The effect of the compounds on the MCF-7 cancer cell line was tested with the help of immunoblotting assay. 1, 2, 3-triazoles cause the inhibition of VEGFR1 giving a lead to evaluate the effect of EFT of    (Fig. 24) [76].

Other triazolo pyrimidine derivatives
A group of novel pyrimidine-2, 4-dione-1,2,3-triazole and furo[2,3-d]pyrimidine2-one-1,2,3-triazole hybrids derivatives were reported. The evaluation was carried out with a vast range of cell line and 5-FU was used as positive control. 74 exhibited the best cytostatic activity against HepG2 and HeLa cells with an IC 50 = 2.67 μM and 6.51 μM respectively. In terms of mechanism, cytostatic effect was attributed to its property of inhibition of Wee-1 kinase and finishing of sphingolipid signaling mediated by acid sphingosine kinase 1 and ceramidase. The compound was nonmitochondrial toxic agent (Fig. 24) [77].

Imidazolo pyrimidine derivatives
Recently, antiproliferative activity of N-9-and N-7-1,2,3 triazole analogs of 2,6-di-substituted purines was reported. They were tested against HCT-1, THP-1, IMR-32, and A-549 cancer cell lines. 75 was the most potent against the THP-1 and A-549 cell lines with the IC 50 = 0.08 and 0.4 μM respectively. The activity was mainly due to C6 position substitution with amines like aminoethnol and benzyl amine, and C2 position was substituted with cyclic secondary amines like piperidine and pyrolidine [78]. Earlier, the anticancer activity of newly synthesized imidazo[1,2-a]pyrimidine mannich bases was studied. In a series of 29 compounds 76(a-c) were found to be the most potent giving similar activity to DOX. The compounds were evaluated against the human lung, pancreatic, cervical, and breast adenocarcinoma cell lines. The compound 76a inhibited the growth of all the four cancer cell lines. The two series of compounds were evaluated by the SRB assay. From the SAR analysis, it was concluded that the main activity of the compounds is due to the C-2 and C-3 substitutions [79].

Pyrido[2,3-d]pyrimidine derivatives
In a parallel study HepG-2 and HCT-116 cancer cell lines were screened by novel pyrido[2,3-d]pyrimidine derivatives and 80(a-d) were found to be most active (Fig. 26) [83]. Pyrido  The derivatives with a SeH group in the 4th and 2nd positions usually have better activity than the analogs with selenoalkyl group [87]. In later researches, cancer cell lines HeLa, A549, PANC 1, and MDA MB-231 at < 10 μM concentration were used to test the anticancer activity of new triazole/isoxazole functionalized 7-(trifluoromethyl) pyrido [2,3-d]pyrimidine derivatives. 85a against PANC1 and 85b against A549 had the best activity better compared to positive control nocodazole. The high anticancer activity of the compound was due to the presence of ethyl chain at C-2 position and the long alkyl/ perfluoroalkyl chain at R on triazole ring. Many structures showed activity regardless of the substitutions on the C-2 position or the triazole ring [88]. Interestingly, some pyrido[2,3-d]pyrimidine derivatives inhibiting EGFR were reported for elucidating potency against A549, MCF-7, BT-474, MDA-MB-231, and SK-BR-3. 86a and 86b were found to have potent inhibition activity against the EGFR tyrosine kinase with the IC 50 = 2.97 nM and 3.58 nM respectively and also showed good anti-proliferative activity against the SK-BR-3 cells IC 50 = 3.10 (86a) and 5.87 μM (86b). Similar binding pattern to the EGFR was observed standard drug gefitinib (Fig. 28) [89].

Pyrido[3,4-d]pyrimidines
Potential anticancer agents in the form of new pyrido [3, 4-d]pyrimidine derivatives were evaluated against the panel of 60 cancer cell lines resulting to be selective towards the renal and breast cancer. 88a had good activity and selectivity towards both breast and renal cancer cell lines. 88b had selective for breast cancer cell line due to the expression of certain kind of kinase enzymes namely, PDGFR, ErbB-1, HER2/Neu (ErbB-2, over expressed in 15-20% of breast cancer), EGFR, and VEGFR [91].  50 (μM) range = 5.22 to 93.2 ± 0.14 with 5-FU was used as control. 89a and 89c inhibited topoisomerase I (TopI) activity, which was comparable to camptothecin thus making it a significant target for the anticancer activity. The compounds were also tested on the normal cell line HUVEC and had an IC 50 more than 80 μM making it non-toxic for the normal cells [92]. Subsequently, substituted pyrido pyrimidines derivatives were evaluated for against cancer cell lines, PC-3, HepG-2, MCF-7, HCT-116, and A-549. 90 showed the best activity against the cell lines with the IC 50 = 0.5 to 7 μM with DOX as positive control. 90 also exhibited promising activity against three kinases CDK4/cyclin D1, EGFR, and PDGFR β, at two different concentrations of 100 μM and 50 μM at a mono measurement. In further assessment of the compound, molecular docking studies were conducted to check the binding of the compound with EGFR and CDK4/cyclin D1 kinases. Overall, the potency of the compound was credited to remarkable kinase inhibitory activity (Fig. 30) [93].

Thieno pyrimidine derivatives Thieno[2,3-d]pyrimidines
Novel 2,3-disubstituted-4-oxo-5,6,7,8-tetrahydrobenzo [4,5]thieno[2,3-d]pyrimidines were studied against breast and liver cancer cell lines with DOX a positive control. 91 showed the best activity in the series with the IC 50 = 0.19 μM (breast cancer cell line). The SAR is illustrated in Fig. 31 [94]. Some novel thieno[2,3-d]pyrimidines with aminophosphonate were studied anticancer activity against the HepG2, MGC-803, and EC109 by MTT assay. 92 was the most potent with the inhibitory percent of 91.2 and 94.4 at 50 μg/ml concentration specifically against the HepG2 and MGC-803 respectively. SAR is depicted in Fig. 32 [95]. Antitumor activity of some 3-ethyl-2-mercaptothieno [2,3-d]pyrimidin-4(3H)-ones was reported and screened against HT-29, MDA-MB231, HeLa, and HepG2 as well as Lep-3. 93a was active against the HepG2 cell line with the (IC 50 = 0.99 μM), 93b was potent against the HeLa cell line (IC 50 = 0.83 μM), and 93c with the (IC 50 = 0.001 μM) against the HT-29 cell lines. In the theinopyrimidine derivative linked to the thiadiazole, activity was due to the presence of oxygen 4th position of the theinopyrimidine ring [96]. A series of thieno [2,3-d]pyrimidine derivatives were reported to initiate apoptosis and EGFR/HER2 arrest. Targeting a tyrosine kinase-related entity makes a drug important in cancer treatment regime. The compounds were tested in vitro for the inhibition of the EGFR and those which passed the test were further evaluated for the mutant EGFR and HER2 kinase inhibition. MCF-7, HCT-116, A431, and HepG2 cell lines were used to screen compound 94 (IC 50 range = The major mechanism of action in the whole process was that the active compounds upregulated the expression of p-53, downregulated the expression of caspase-3, led to the cell cycle arrest, and further apoptosis leading to cancer cell destruction. These compounds increased the p-53 expression by 3-4-folds compared to the 2folds of DOX [98]. Thieno [2,3-d] pyrimidine derivatives were active against 60 human cancer cell line panel by the primary  anticancer assay as a result 96 was found to have the best activity. Although the compound 96 exhibited a wide spectrum of activity, it was not selective towards any particular group of human cancer cell lines [99]. Evaluation for in vitro antiproliferative activity of new thieno [2, 3-d] pyrimidine and thiophene derivatives was conducted and 97(a-e) were the best compounds, with the IC 50 (μM) = 4.30 ± 0.3 to 23.57 ± 1.8 when tested against five cancer cell lines namely, HepG-2, Hep-2, MCF-7, PC-3, and HeLa by the standard MTT assay. 97d was efficient than the positive control DOX. The abovementioned compounds were also tested for DNA binding and enzyme inhibition activity, the order of inhibition: DNA polymerase> thymidylate synthase >tyrosine kinase [100]. In a parallel study, some novel thieno [2, 3-d] pyrimidine derivatives were tested for antitumor activity against the human breast and cervical cancer cell line. 98b was highly active with the IC 50 = 18.87 ± 0.2 μg/ml, and the 98a was active against cervical cancer cell line with the IC 50 =40 ± 1.7 μg/ml both better than the positive control, Paclitaxel [101].
Recently, urea derivative based thieno [2,3-d]pyrimidine-based were screened for in vitro antitumor activity against the tamoxifen sensitive and resistant breast cancer cell lines. 99 a sorafenib analog and a theinopyrimidine-based urea derivative inhibited VEGF R2 tyrosine kinase activity up to 65% and also showed anticancer activity against its parental MCF7 BC cells, LCC2 and TAM-resistant. The compound was also sensitive to the LCC2 due to the upregulation of key enzymes for apoptosis, LDH in media downregulated the mRNA expression of Ki-67, survivin, and Akt and reduced levels of ROS and glucose uptake led to cell death of proteins like caspases 8, 9, 3, p53, Bax/Bcl-2 ratio [102]. Some novel thieno [2, 3-d] pyrimidines were evaluated in 2012 and compounds 100(a-b) showed the best antitumor activity against the HCT-116 with the IC 50 = 15.92 and 25.85 μM and 19 to 2.26 times more efficacious than imatinib (IC 50 = 34.40 μM). SAR suggested that N-ethyl substitution imparted comparable activity to the positive control whereas N-phenyl or Nsubstituted phenyl gave moderate activity in case of thienopyrimidine derivatives (Fig. 33) [103].
A series of thieno [2, 3-d] pyrimidine derivatives were reported and evaluated for the in vitro activity against  tested for growth inhibition and 103c exhibited selectivity towards MDAMB-453 and MCF-7 cell and arrested cell cycle at G0/G1 phase in MCF-7 cells. This potential was attributed to the presence of cinnamide moiety and trifluoromethyl group. 103(d-e) was selective towards A549 and MDA-MB-453cell lines, respectively, with ZSTK474 as positive control [106]. Previously, the varied substitution of groups at the R position of the thieno [2, 3-d] pyrimidine derivatives led to properties like c-Met and VEGFR-2 inhibition which is an important target Recently, condensed thieno [2, 3-d] pyrimidines were evaluated for their cytotoxic potential against A431 and H9c2 cancer cell lines through MTT assay. Most of the synthesized compounds except 105 had 85% cell death in both rat and H9c2 cell lines. The presence of ethyl, methyl, and ethyl-fused five-membered rings in the structure showed better activity in A431 than in H9c2 cells and in case of the six-membered structures, methyl-substituted cyclohexyl compounds inhibited A431 cell growth, although dimethyl substituted thiophene derivatives displayed better activity in H9c2 cells. Nitromethyl, cyclohexyl, and dimethyl substitution lowered activity compared to chloro substitution fused with seven membered rings and the methyl-substituted structures had better potency than the aryl substituted structures [108].
Anticancer activity, apoptosis-inducing ability, and cell cycle profiling of hexahydrocyclooctathieno [2, 3-d] pyrimidines was reported using DOX as positive control. 106(a-b) were tested for antiproliferative activity against 56 cancer cell lines and 106b was potent against a broad range of cell lines. 106a and 106b led to the induction of S-phase and G2/M-cell cycle arrest in MCF7 and HCT116 cell lines with a significant increase in the pre-G phase cell population in a time-dependent way; 106b also increased cleaved caspase-3 as a marker of apoptosis   [109]. A series of new 2-pyridyl hexahydrocycloocta [4,5]thieno [2, 3-d] pyrimidine derivatives were evaluated with DOX as positive control. 107 (a-e) manifested the most potent activity due to the substitution of 2-pyridyl group at C-2, amino group at C-4 increases activity, and chlorine group at C-4 leading to an inactive compound and the bulkiness of the amino at C-4 effects the activity [110].

Thieno [3, 2-d] pyrimidine
N-substituted thieno [3, 2-d] pyrimidine and thiophene derivatives were studied for the antitumor activity and 108(a-b) had more potency than standard drug DOX in MTT assay against the cancer cell lines of liver, colon, and lung. According to the SAR the pyrrolo-pyrimidine moiety and triazolopyrimidine moiety at the C-3 position of the theinopyrimidine ring and EWG at the paraposition of the phenyl ring contributed to the high activity, on the contrary an EDG at the same position gives moderate activity [111]. Screening of antitumor activity of 4-morpholinothieno [3, 2-d]-pyrimidine derivatives bearing aryl methyl hydrazine moiety were reported. The activity was mainly due to heteroaryl methylene hydrazinyl moiety and various substitutions on the benzene ring when screened against H460, HT-29, and MDA-MB-231 cell lines. 109 bearing 3,4-methylenedioxy phenyl group was most efficacious against all the three abovementioned cell lines with the IC 50 = 0.003 μM, 0.42 μM, and 0.74 μM respectively with more selectivity towards lung cancer cell line and was 1.6-to 290-folds more active than GDC0941 [112]. Diaryl urea moiety containing thieno [3,2-d] pyrimidine derivatives were tested for their antitumor activity by MTT assay using sorafenib and GDC-0941 (positive control). 110 was tested against various tumor cell lines gave the most potent activity against the MDA-MB-231, MKN-45, HT-29, and H460 and cell lines with the IC 50 = 0.23 μM, 0.18 μM, 0.058 μM, and 0.081 μM respectively. The SAR of the study concluded that the compounds with the mono halogen group on the phenyl ring were more efficient than the one's with methyl group or double groups and presence of chlorine atom at the 6,7-position of thieno [3,2-d]pyrimidine moiety lead to detrimental activity but had good selectivity towards HT-29 and H460 cell lines. In the enzymatic evaluation, it was found that the compound was active against the PI3Kα kinase (IC 50 = 0.13 μM). The apoptosis in the cancer cell was induced by the compound in concentration dependent manner with the increase in protein expression of Clv-PARP [113]. Diaryl semicarbazone linked to thieno [3,2-d] pyrimidine derivatives as potential antitumor agent were studied in the same year. The compound also showed promising activity against the PI3Kα better than the standard reference GDC-0941 indicating that the compound will make this particular kinase enzyme a potential target in future. By the help of the western blot method, it was determined that the compound can be used for induction of apoptosis in a concentration dependent manner [114].
Recently, the biological activity of the new series of novel thieno[3,2-d]pyrimidine derivatives were reported along with the test for the fak inhibition. These compounds were designed in a way that it mimics the conformation of the diaminopyrimidine moiety. 112 most potently inhibited the enzyme with the IC 50 (28.2 = n M) and also acted against the proliferation in the U-87MG, A-549, and MDA-MB231 cancer cell lines with the IC 50 = 0.16, 0.27 and 0.19 μM respectively and was less toxic to HK2 cell line. It arrested the cell cycle at G0/G1 phase in the MDA-MB231 cancer cell line, also preventing the migration and induced apoptosis [115]. HDAC was inhibited by novel thieno [3, 2-d] pyrimidines and for their anticancer activity was evaluated on three cell lines including, MCF-7, HeLa, and HCT-116 by using MTT assay. The HDAC inhibition in the compound was studied using fluorimetric assay. 113 inhibited HDAC potently with the IC 50 = 0.38 μM by inducing apoptosis in HCT-116 cell line and cell cycle arrest at G2/M phase with SAHA as positive control [116]. In the SAR study, it has been summarized that substitution at C-2 position highly affects the potency and presence of methylene between electronegative moieties and pyrimidin-4-one ring, Cl, and arylamino increases the activity. Arylcarbothioyl derivative did not affect the activity, whereas the 2-nitrophenyl group increased the activity by 2.89 folds. Molecular docking was used to study the binding and affinity of the compound towards the CDK2 receptor [118].

Other thieno pyrimidine derivatives
In silico method was used to determine the anticancer activity of new annelated thieno [2, 3-e ] [1][2][3] triazolo [1, 5-a] pyrimidines, using 60 human cancer cell lines. Compounds 116(a-b) were the most potent and showed high antiproliferative activity at sub micromolecular concentration with low toxicity to normal cells and highly potent towards cancer cells. The basic chain histamine or methylpiperazine was important for potency enhancement of the activity and the isosters pyridothieno were less effective than the benzothieno bicycle [119]. Using the unusual dimroth rearrangement in an in silico research study conducted simultaneously led to synthesis of annelated thieno [3,2-d] [1,2,3]triazolo[1,5-a]pyrimidines. 117a (angular isomers) showed excellent anticancer activity against the 60 cancer cell line panel at sub nM concentration. Compound 117b (linear isomer) displayed good antiproliferative activity. In the preliminary in vivo test of compound 117b, it showed high potency (Fig. 36) [120].

Thiazolopyrimidine derivatives Thiazolo [1, 2-a] pyrimidine
3-carboxylate derivatives of benzo [4,5]thiazolo [1, 2-a] pyrimidines were reported and 118(a-b) displayed potency against MDA-MB-231 and MCF-7 cancer cell lines. Compound 118a showed activity against both with the IC 50 = 0.58 and 1.58 μM respectively, and 118b had the best activity against MDA-MB-231 with the IC 50 = 5.01 μM. The activity was imparted to the MCF-7 and MDA-MB231 due condensation of methyl piperazine and piperidine with carboxylic acid at 3′-position of the benzothiazopyrimidine ring. Condensation of β-alanine at the third position of benzothiazopyrimidine ring led to increase in chain length subsequently decreasing the activity compared to piperidine and methylpiperazine moieties [121].

Thiazolo [3,2-a]pyrimidines
Recently, thiazolo [3, 2-a] pyrimidine derivatives were tested for anticancer activity against MCF-7, HeLa, A549, and SKNSH by MTT assay. 119a with IC 50 = 2.2 ± 0.6 μM against A549 and compound 119b with IC 50 = 5.6 ± 0.4 μM against HeLa exhibited better effects than positive control DOX. The presence of methoxyphenyl groups and naphthyl on thiazolopyrimidine demonstrated enhanced activity [122]. Novel thiazolo [3, 2-a] pyrimidines were screened for in vitro antiproliferative The different activity between the compounds was due to variation of substitutions in the phenyl group of the molecule and the presence of sulfur and nitrogen atom subsequently enhanced the activity. Thiopyrimidine moieties fused to N-methylpipredine ring boosted the activity [125].

Thiazolo [5, 4-d] pyrimidines
Apoptosis-inducing and antiproliferative activity of thiazolo [5,4-d]pyrimidines were reported using the SRB assay against cancer cell lines A549, A431, T98G, T47D, PC-3, HL-60, NCI-H322, and MIAPaCa-2. 123 showed the induction of apoptosis in A549 cells at the concentration of 10 μM determined by the high sub -G1 population. In the western blotting assay, the compound demarcated cleavage in PARP-1 and lead to procaspase-3 inhibited apoptosis [126]. The study of some thiazolo [5,4-d]pyrimidine derivatives were carried out by the  drug repurposing strategy and evaluated against three cancer cell lines. 124 showed the best anti-cancer activity with the IC 50 = 1.03 and 38.95 μM against the MGC803 and GES1 cell lines respectively, with efficient selectivity between cancer cell and normal cells. The compound also inhibited the formation and migration of MGC803 cells and also led to apoptosis by expression of some changes in the apoptosis concerned proteins by upregulation of Bax and caspase-3/9 and downregulation of Bcl-2, confirmed by the western blot assay [127]. The antiproliferative activity of novel thiazolo [5,4d]pyrimidines were evaluated. 125 exhibited efficacious selectivity between the human cells and cancer cells which was confirmed as it had IC 50 = 1.22 μM against HGC-27 cell line and low toxicity towards the GES-1 cells. The induction of apoptosis was due to upregulated expression of Bax, downregulation of Bcl-2, and cleavage in caspased-3/9. It also prevented the migration and formation of new HGC-27 cell proving it as an efficacious lead for future anticancer drugs [128].

Other thiazolopyrimidine derivatives
Novel 1-thia-4-azaspiro [4.5] decane and their derived thiazolopyrimidines were studied for activity against the HepG-2, PC-3 and HCT116 cancer cell lines. 126(a-b) showed good anticancer activity against the HCT116 cell line with the IC 50 range = 92.2 to 120.2 μM and showed some activity against the HepG-2 cell line. The MTT assay was conducted for the cytotoxicity with compound dosage of 100 ppm and DOX was used as positive control (Fig. 39) [129].

Oxazolo-pyrimidine derivatives
Recently, work on the anticancer activity of some 4-(Oxazolo [5,4-d]pyrimidine derivatives linked with 1,2,4oxadiazole was reported. Compounds were tested against the human breast, lung, colon, and ovarian cancer cell line using the MTT assay with etoposide as the positive control. 127a showed activity against all the cancer cell lines with the IC 50 range = 0.046 to 0.12 μM and 127b showed activity against cell lines namely breast, lung, and ovarian cancer with the IC 50 range = 0.012 to 0.63 μM. In the SAR studies, it was concluded that the compound with an EDG had the most potent activity and compounds with 3,5-dimethoxy, 4-methoxy group depicted low activity. Compounds with one nitro group had moderate activity whereas the ones with bromo di nitro group had good activity. A weak EDG group with the compound showed considerable activity [130].
New series of oxazolo [5,4-d]pyrimidines were reported as efficacious VEGFR-2 inhibitors. The compounds were tested against HUVEC and VEGFR-2 with 128 being the most potent in the series with the IC 50 = 0.33 μM for VEGFR-2 and IC 50 = 0.29 μM for HUVEC.
Compounds of this series were also moderate inhibitors of EGFR and sunitib was used as positive control [131]. In a parallel study, a newly synthesized oxazolo [5,4d]pyrimidine derivative, 129 was reported. HUVEC was inhibited in a dose dependent manner with the IC 50 = 9.30 ± 1.24 μM. Reported in other in vitro and ex vivo studies, it also inhibited new micro vessels sprouting from rat aortic ring. The migration and chemotactic invasion was stopped along with the downstream signaling of VEGFR-2 and downregulation of phosphorylation of PI3K, ERK1/2, and p38 MAPK (Fig. 40) [132].

Quinazoline containing derivatives
Anticancer activity of 2,3-disubsituted quinazoline was reported and the compounds were tested using MTT assay against MCF-7 cancer cell line with amphotericin B as positive control. 130(a-c) exhibited anticancer with an IC 50 (μM) = 6, 6, and 4 respectively [133]. New quinazoline derivatives were being studied for anticancer activity against the MDA-MB231 and HeLa cancer cell lines, using MTT assay, while using gefitinib as positive control. 131(a-c) had the lowest (IC 50 = 1.85 to 2.81 μM) in relation to gefitinib (IC 50 = 4.3 and 28.3 μM) against MDA-MB231and HeLa cells, respectively. They were deemed as most potent because they acted through EGFR-TK pathway. The addition of heteroalkyl atom led to decrease in cytotoxicity. The NO 2 , CN, phthalimido, and morpholino groups alongside the N-benzyl group and the substitution at R 2 position affected the cytotoxic activity to a great extent [134].
2-furano-4(3H)-quinazolinones, diamides (open ring quinazolines), and quinoxalines were evaluated against OVCAR-4 and NCI-H522 cancer cell lines and 132 was found to be the most efficient compound. The potency was due to the blocking of hydrophobic pocket of tyrosine kinase because of 2-chloro benzylideneamine group at 3-position of quinazoline. The presence of OH and benzoic acid at N3 of the ring lowered the activity whereas 2-chloro benzylideneamine group increased activity [135].
Novel amine substituted quinozoline-linked benzimidazole compounds were tested for antitumor activity. 135 elucidated the best activity against the prostate and colon cancer cell lines. In the SAR studies, it was revealed that the substituents at C-2 and C-4 position of the quinozoline ring affected the activity of the compounds and with benzimidazole at the C-4 position it also possessed growth inhibition property [138]. A regioisomeric series of quinazoline and benimidazole hybrids were studied as anticancer agents and screened against a panel of 60 cancer cell lines. 136 was most potent of all, better than 5-FU (positive control) [139].
A series of quinozoline derivatives were reported in a simultaneous study for their activity against human tumor cell lines derived from 9 different sources using the SRB assay. 137 showed antiproliferative potency against all the cell lines due to the presence of the phenylvinylic substituent at 2 position. Other compounds with methylene, trichlorophenoxymethylene, and benzyl displayed moderate activity against specific cell lines (Fig. 41) [140].

Miscellaneous fused ring pyrimidine derivative
Some novel pyrido-thieno-pyrimidine derivatives were responsible for the induction of phosphorylation and acetylation which led to the activation of p53 in the colorectal cancer cells. 138 was responsible for the p53mediated activity which led to activation of downstream genes like p51 and PUMA (which resulted towards delay in growth, cell cycle stoppage at G1, cell senesce, and cell death). Controlled interaction between p53 and MDM2 led reduced degradation of p53 and due to these attributes, 138 was considered a potential anticancer agent [141]. Pyrimidooxadiazine and triazolopyrimidooxadiazine derivatives were reported as anticancer agents. 139(a-b) elucidated the best potency against the A549, MCF7, and HepG2 cancer cell lines because of the change in substitutions at triazole moiety fused to pyrimido[4,5e][1,3,4]oxadiazine heterocyclic ring and C-7 position. Compounds with fused triazole ring were more active compared to the chlorine or pyrrolidine moiety at the C-7 position. Induction of cell death by apoptosis was caused by various cellular modifications such as biochemical changes like protein cleavage, protein crosslinking, DNA fragmentation, and phagocytic recognition [142].
Recently, a novel series of thiazolopyrimidine hydrobromides and triazolopyrimidines which act as topoisomerase II inhibitors was reported. The compounds were screened against 60 human cancer cell lines by the MTT colorimetric assay. Results elucidated 140 as the most significant inhibitor against the A498 (IC 50 = 3.5 μM) cell line. The cell cycle was arrested at the G2/M phase leading to inhibition of cell proliferation and induced apoptotic activity. 140 also elucidated potent topoisomerase II inhibitory activity (IC 50 =2.89 μM) versus DOX (IC 50 = 2.67 μM) which was used as a positive control [143].
Fused pyrimidine hybrids were tested using MTT assay against the B-16, A-549, MCF-7, ACHN, and COLO-205 cancer cell lines. 141a (IC 50 = 9.5 μM) and 141b (IC 50 = 7.7 μM) showed the best activity against A-549 cancer cell line due to induction of apoptosis at the G0/G1 phase of the cell cycle. SAR suggested that 4chloro and 4-bromo pyrimidine hybrid have 5-6 times more activity against the A-549 whereas 2-3 times more activity against the other abovementioned cell lines than the parent compound [144]. Novel azacalix [2]aren e [2] pyrimidines were screened against the MCF-7 cancer cell line. 142(a-b) were the best with anticancer activity with the IC 50 = 0.58 and 1.82 μM respectively. Phenyl ring containing pyrrolidine group shows good ctytotoxic activity against A549, MCF7, and SHSY5Y cell lines but not against L02 cells. Compounds methyl and methoxy substituent on phenyl ring instead of no substitutions shows decreased activity. Other substitutions on the phenyl ring gave moderate activity whereas CF 3 group on the phenyl ring showed good activity [145].
Novel anilino pyrimidine derivatives were evaluated in 2018 and compound 145 (IC 50 = 0.5 μM) depicted the most potent anticancer activity against A549 cell line mainly due to the presence of the linkage via ester moiety. SAR is illustrated in Fig. 43 [148].

Conclusions
Pyrimidine is a significant scaffold due to its presence in naturally occurring nucleotides and is being explored extensively for its anticancer profile. The present manuscript has discussed the anticancer potential of substituted pyrimidine at various positions as well as pyrimidine fused with other heterocyclic ring. Substitutions at the C-2, C-4, and C-6 position of the pyrimidine core has a great influence on the antitumor activity especially a thio or amino group at C-2 and substituted phenyl group at C-4. Pyrimidine fused with five member rings like pyrazolo, pyrrolo, triazolo, imidazole, oxazolo, thiazolo, and thieno exhibited more distinct anticancer activity compared to the six member rings like pyrido and quinazoline. Pyrimidine analogs act as anticancer agent through diverse mechanism of action including, kinase (erbB2, raf, CDK, Src etc.) enzyme inhibition, cell cycle arrest, activation of oncogenes, reduction of mitochondrial membrane potential, increase of ROS, and induction of apoptosis by upregulation of apoptotic and downregulation of anti-apoptotic proteins. The current manuscript can be valuable to scientists and researchers around the globe to optimize and select specific targets for the development of potent lead molecules as anticancer agents in future.