Design, Synthesis and Antitumor Evaluation of Novel

molecules Article

Design, Synthesis and Antitumor Evaluation of Novel Pyrazolopyrimidines and Pyrazoloquinazolines Mohamed El-Naggar 1 , Ashraf S. Hassan 2, * 1 2 3 4 5

*

ID

, Hanem M. Awad 3 and Mohamed F. Mady 4,5, *

ID

Chemistry Department, Faculty of Sciences, University of Sharjah, Sharjah 27272, UAE; [email protected] Organometallic and Organometalloid Chemistry Department, National Research Centre, Dokki, Cairo 12622, Egypt Department of Tanning Materials and Leather Technology, National Research Centre, Dokki, Cairo 12622, Egypt; [email protected] Department of Green Chemistry, National Research Centre, Dokki, Cairo 12622, Egypt Deaprtment of Chemistry, Bio Science and Environmental Technology, Faculty of Science and Technology, University of Stavanger, N-4036 Stavanger, Norway Correspondence: [email protected] (A.S.H.); [email protected] (M.F.M.); Tel.: +20-100-664-5444 (A.S.H.); +47-912-569-33 (M.F.M.)

Received: 2 May 2018; Accepted: 22 May 2018; Published: 23 May 2018







Abstract: A series of N-aryl-7-aryl-pyrazolo[1,5-a]pyrimidines 18a–u and N-aryl-pyrazolo[1,5-a] quinazolines 25a–c were designed and synthesized via the reaction of 5-aminopyrazoles 11a–c with enaminones 12a–g or 19, respectively. The new compounds were screened for their in vitro antitumor activity toward liver (HepG-2) and breast (MCF-7) human cancer cells using 3-[4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide MTT assay. From the results, it was found that all compounds showed dose-dependent cytotoxic activities against both HepG-2 and MCF-7 cells. Two compounds 18o and 18a were selected for further investigations. Cell cycle analysis of liver (HepG-2) cells treated with 18o and breast (MCF-7) cells treated with 18a showed cell cycle arrest at G2/M phase and pro-apoptotic activity as indicated by annexin V-FITC staining. Keywords: pyrazolopyrimidines; pyrazoloquinazolines; synthesis; antitumor activity; cell cycle analysis

1. Introduction Pyrazolo[1,5-a]pyrimidine ring 1 and its derivatives occupy a unique place in medicinal chemistry due to its various pharmacological activities [1–6] especially antitumor properties [7–9]. In 2006, Li et al. synthesized compound 2 which exhibited significant in vitro antitumor activity against Bel-7402 (liver) and HT-1080 (fibrosarcoma) cell lines [10]. In 2009, Ahmed et al., prepared compound 3 which was more effective and exhibited cytotoxicity against HCT116 (colon) and HeLa (cervix) cell lines [11]. In 2010, Abdel-Aziz and co-workers have described a facile synthesis of compound 4 which exhibited promising in vitro antitumor activity against CaCo-2 (colon) and BHK (normal fibroblast) cell lines [12]. Furthermore, we have reported the synthesis of compounds 5 and 6 in high yield by treating 5-aminopyrazole with 2-(2-chlorobenzylidene)malononitrile and ethyl acetoacetate, respectively, these compounds show good antitumor activities against HCT-116 and HepG2 cells [13,14] (Figure 1).

Molecules 2018, 23, 1249; doi:10.3390/molecules23061249

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In addition, derivatives have been reported as potent addition, pyrazolo[1,5-a]pyrimidine pyrazolo[1,5-a]pyrimidine derivatives potent enzymes enzymes inhibitors [15–17]. Mukaiyama et al., have prepared compound 7 which exhibited potent inhibitory al., have prepared compound 7 which exhibited potent inhibitory activity Kinase andand good CNSCNS penetration [18]. Very Kumar et al., have activity against againstc-Src c-Src Kinase good penetration [18].recently, Very recently, Kumar et prepared al., have pyrazolo[1,5-a]pyrimidine carboxamide 8 which showed good aurora kinase A and B activity [19] prepared pyrazolo[1,5-a]pyrimidine carboxamide 8 which showed good aurora kinase A and B (Figure 1). activity [19] (Figure 1). NC N

R1 N

N

O

R R2

PhHN

N N

N N

NHR Pyrazolo[1,5-a]pyrimidine

H2 N

N N

3

2 R1

R1

N R

N N

N

R1

R2 R

N N

CN

R= -NH-C6H4-4-OCH3 R1 = CONH-C6 H5

6

5 O

NH2 N

R

CH3

N N R2

R= -NH-C6H3-3,5-di-OCH3 R1 = 2-amino-2-methylpropylamino

7

CH3

OH

R= -NH-C6H4-4-OCH3 R1= CONH-C6H4-4-CH3 R2= 2-chlorophen-1-yl

4

N

N N

NH2

R2 R1= -N=N-(4-FC6H4) R2= 3-methylbenzo[d]thiazolo[3,2-a]imidazol-2-yl

R1

R= CONH-C6 H4 -4-Cl R1= naphth-2-yl

R= 3,5-bis(trifluoromethyl)phenyl NR1 R2 = piperdinyl

1

N

O

H N NH 2 N N N

R

R= -NH-CO-C6 H3 -3,5-di-F

8

Figure 1. 1. Structures of the the antitumor antitumor activity activity compounds compounds 1–6 1–6 and and enzymes enzymes inhibitors inhibitors 7–8. 7–8. Figure Structures of

In continuation of our research program [20–27] and following the potent biological activity In continuation of our research program [20–27] and following the potent biological results against MCF-7 and HepG2 carcinoma cells which were obtained from our synthesized activity results against MCF-7 and HepG2 carcinoma cells which were obtained from our compounds such as 7-(4-chlorophenyl)-2-(phenylamino)pyrazolo[1,5-a]pyrimidine (9, IC50 = 63.2 ± synthesized compounds such as 7-(4-chlorophenyl)-2-(phenylamino)pyrazolo[1,5-a]pyrimidine 5.9 µM) and 2-(phenylamino)-pyrazolo[1,5-a]quinazoline (10, IC50 = 77.6 ± 4.3 µM) compared to (9, IC50 = 63.2 ± 5.9 µM) and 2-(phenylamino)-pyrazolo[1,5-a]quinazoline (10, IC50 = 77.6 ± 4.3 µM) doxorubicin [28]. In this work, we have planned to modify the pyrazolo[1,5-a]pyrimidine 9 and compared to doxorubicin [28]. In this work, we have planned to modify the pyrazolo[1,5-a]pyrimidine pyrazolo[1,5-a]quinazoline 10 to obtain N-aryl-7-aryl-pyrazolo[1,5-a]pyrimidines 18a–u and 9 and pyrazolo[1,5-a]quinazoline 10 to obtain N-aryl-7-aryl-pyrazolo[1,5-a]pyrimidines 18a–u and N-aryl-pyrazolo[1,5-a]quinazolines 25a–c, respectively, incorporating different aryl groups (blue N-aryl-pyrazolo[1,5-a]quinazolines 25a–c, respectively, incorporating different aryl groups (blue and and green) into the structures to evaluate their in vitro antitumor activities against HepG-2 and green) into the structures to evaluate their in vitro antitumor activities against HepG-2 and MCF-7 MCF-7 human cells to find novel and potent antitumor compounds (Figure 2). human cells to find novel and potent antitumor compounds (Figure 2).

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Figure 2. 2. of novel N-aryl-pyrazolo[1,5-a]pyrimidines 18a–u and Figure DesignDesign of novel N-aryl-pyrazolo[1,5-a]pyrimidines 18a–u and N-aryl-pyrazolo[1,5-a] N-aryl-pyrazolo[1,5-a]quinazolines 25a–c-based amide linkages. quinazolines 25a–c-based amide linkages.

2. Results and Discussion 2. Results and Discussion 2.1. Chemistry 2.1. Chemistry The syntheses synthesesoftarget oftargetcompounds compounds 18a–u 25a–c are illustrated in Schemes andstarting 2. The The 18a–u andand 25a–c are illustrated in Schemes 1 and 2.1 The starting materials, 5-amino-N-aryl-1H-pyrazole-4-carboxamides 11a–c were synthesized according materials, 5-amino-N-aryl-1H-pyrazole-4-carboxamides 11a–c were synthesized according to our to ourwork [29]. previous [29]. 11a–c Reaction of compounds 11a–c with previous Reactionwork of compounds with 1-(aryl)-3-(dimethylamino)prop-2-en-1-ones 1-(aryl)-3-(dimethylamino)prop-2-en-1-ones 12a–g in glacial acetic acid furnished one 15a–u isolable 12a–g in glacial acetic acid furnished one isolable product 5-aryl-pyrazolo[1,5-a]pyrimidines or product 5-aryl-pyrazolo[1,5-a]pyrimidines 15a–u or 7-aryl-pyrazolo[1,5-a]pyrimidines 18a–u. As 7-aryl-pyrazolo[1,5-a]pyrimidines 18a–u. As depicted in Scheme 1, the final products were confirmed depicted in Scheme 1, the final products were confirmed by the spectral analysis. by the spectral analysis. 1 The 1H-NMR or 15c, 15c, characteristic characteristic two two The H-NMR spectrum spectrum (CDCl (CDCl33,, δδ ppm) ppm) exhibited, exhibited, in in each each case case 18c 18c or doublets of the pyrimidine protons at 6.87 (1H, H-6) and at 8.40 (1H, H-5) (each with J = 8.4 Hz) doublets of the pyrimidine protons at 6.87 (1H, H-6) and at 8.40 (1H, H-5) (each with J = 8.4 and Hz) respectively. The The 1313C-NMR C-NMR four four signals at 3.80, 3.91, 9.36 and 10.02 and signals at 3.80, 3.91, 9.36 and 10.02due duetoto2OCH 2OCH3 3 and and 2NH, 2NH, respectively. spectrum (CDCl (CDCl3,, δδ ppm), ppm), in in each each case case 18c 18c or or 15c, 15c, also also characterized characterized by by signals signals of of –OCH –OCH3,, –OCH –OCH3,, spectrum 3 3 3 3-pyrazolopyrimidine, C6-pyrazolopyrimidine, C5-pyrazolopyrimidine and C=O at 55.53, 55.60, C C3 -pyrazolopyrimidine, C6 -pyrazolopyrimidine, C5 -pyrazolopyrimidine and C=O at 55.53, 55.60, 87.45, 106.43, 106.43, 157.52 157.52 and and 163.21, 163.21, respectively. respectively. 87.45, 1H- and 13C-NMR spectra cannot differentiate between 15a–u and 18a–u, 1H-15N Although, 1 13 Although, H- and C-NMR spectra cannot differentiate between 15a–u and 18a–u, 1 H-15 N 1 1 15N HMBC spectrum of HMBC HMBC spectrum spectrum used used for for differentiating differentiating between betweenthe thetwo twoisomers. isomers.The TheHH-15 N HMBC spectrum thethe final product of of final productshows showsthe themost mostimportant importantcorrelated correlatedcoupling coupling between between the the proton proton H-5 H-5 of 1H, at 8.40 ppm) with N-4 of pyrimidine (15 15N, at 255 ppm) 22J (H-5, N-4) gave pyrazolopyrimidine ( 1 pyrazolopyrimidine ( H, at 8.40 ppm) with N-4 of pyrimidine ( N, at 255 ppm) J (H-5, N-4) gave absolute confirmation confirmation for for the the structure structure of of 18a–u 18a–u and and conclude conclude 15a–u 15a–u (cf. (cf. Supporting Supporting Information). absolute Information). Also,the of of 18a–u waswas supported by X-ray crystallography of similar products Also, thestructure structure 18a–u supported by X-ray crystallography of analogs similar and analogs and [12]. products [12].

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O Ar

HN Ar 1

O

NH2

N H N

NH

Ar 2

+

N

CH 3

CH3 12a-g

11a-c

-HN(CH3 )2 Glacial AcOH

O Ar

N H

N

Ar

O

N

HN Ar 1

O

Ar

NH2

HN

13a-u

H HO Ar 2 N

O N H

Ar 11a ; Ar = C 6H 5 11b ; Ar = 4-CH 3-C6 H 4 11c; Ar = 4-Cl-C 6H 4

N N

HN Ar1

14a-u

16a-u

O N H HN Ar 1

N

N H HN Ar 1

N

N

N N Ar 2 N HO

-H 2O

12a; Ar 2 = C 6H 5 12b; Ar 2= 4-CH 3-C6 H4 12c; Ar 2 = 4-CH 3 O-C 6H 4 12d; Ar 2= 4-Cl-C 6 H4 12e; Ar 2 = 4-Br-C6 H4 12f; Ar2 = 4-F-C6 H 4 12g; Ar 2= thiophen-2-yl

Ar2

O

H

17a-u

Ar 1= 4-CH 3O-C 6H 4

-H 2O

Ar

O

NH

N

Ar 1

Ar 2

Ar 2

H N

N H

Ar

O

N

N H

N

HN

N

Ar 2

Ar1 18a-u

15a-u

13-18 a b c d e f g h i j k

Ar C 6H 5 C 6H 5 C 6H 5 C 6H 5 C 6H 5 C 6H 5 C 6H 5 4-CH3-C6H4 4-CH3-C6H4 4-CH3-C6H4 4-CH3-C6H4

Ar2 C 6H5 4-CH3-C6H4 4-CH3O-C 6H4 4-Cl-C6H 4 4-Br-C6H4 4-F-C6H4 thiophen-2-yl C 6H5 4-CH3-C6H4 4-CH3O-C 6H4 4-Cl-C6H 4

13-18 l m n o p q r s t u

Ar 4-CH3-C6H4 4-CH3-C6H4 4-CH3-C6H4 4-Cl-C6H4 4-Cl-C6H4 4-Cl-C6H4 4-Cl-C6H4 4-Cl-C6H4 4-Cl-C6H4 4-Cl-C6H4

Ar2 4-Br-C6H4 4-F-C6H4 thiophen-2-yl C 6H5 4-CH3-C6H4 4-CH3O-C 6H4 4-Cl-C6H4 4-Br-C6H4 4-F-C6H4 thiophen-2-yl

Scheme 1. Synthesis of 7-aryl-pyrazolo[1,5-a]pyrimidines18a–u. Scheme 1. Synthesis of 7-aryl-pyrazolo[1,5-a]pyrimidines 18a–u.

In addition, N-aryl-2-(arylamino)-pyrazolo[1,5-a]quinazolines 25a–c were formed by the In addition, N-aryl-2-(arylamino)-pyrazolo[1,5-a]quinazolines 25a–c were formed by (19) the condensation of 11a–c with 2-((dimethylamino)methylene)-5,5-dimethylcyclohexane-1,3-dione condensation of 11a–c with 2-((dimethylamino)methylene)-5,5-dimethylcyclohexane-1,3-dione (19) in in a glacial AcOH (Scheme 2) while pyrazolo[1,5-a]quinazolines 22a–c were not formed. The spectral aanalysis glacial of AcOH (Scheme 2) while pyrazolo[1,5-a]quinazolines 22a–c were not formed. The spectral the products supported the structures of 25a–c. analysis of the products supported the structures of 25a–c.

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5 of 20 5 of 20 O

Ar

O

NH 2

N H N

HN Ar 1

NH

+

H 3C

O

H 3C

CH3 N CH 3

19

11a-c -HN(CH 3 )2 Glacial AcOH

Ar

O

NH 2 O

N H N

HN Ar 1

CH 3 Ar

CH3

N O

H3 C Ar

HO HN

O N H

N

HN Ar1

O

O

N

N H HN Ar1

21a-c

N

O

NH 23a-c

HN

O

N N HO H3 C

CH3

24a-c

11a; Ar = C 6H 5 11b; Ar = 4-CH 3-C6 H4 11c; Ar = 4-Cl-C6 H 4

-H2 O

H N

N H

CH3 Ar

O

O

HN Ar 1

20a-c

CH 3 CH 3

-H2 O

Ar 1= 4-CH3O-C6 H4 O

Ar HN

N

HN Ar 1

N

CH 3 CH 3

N O 22a-c

Ar 20-25 a b c

Ar C6H5 4-CH 3-C6H4 4-Cl-C6H 4

Ar 1 4-CH3O-C6H4 4-CH3O-C6H4 4-CH3O-C6H4

O

N

N H HN Ar1

N

O

N H3 C

CH3

25a-c

Scheme 2. Synthesis of N-aryl-2-(arylamino)-pyrazolo[1,5-a]quinazolines 25a–c. Scheme 2. Synthesis of N-aryl-2-(arylamino)-pyrazolo[1,5-a]quinazolines 25a–c.

The mass spectrum of 25b confirmed the molecular formula C27H27N5O3 (469.53) {MS (m/z, %): The mass spectrum of 25b confirmed the molecular formula C27 Hwas O3 (469.53) {MS (m/z, +, 93.88)}. 27 N5characterized 469 (M The 1H-NMR (CDCl 3, 400 MHz, δ ppm) spectrum of 25b by sharp + , 93.88)}. The 1 H-NMR (CDCl , 400 MHz, δ ppm) spectrum of 25b was characterized %): 469 (M signals of 2CH3, CH2 and CH2 groups of the3 dimedone at 1.19, 2.52 and 3.22, respectively. The OCH3 by sharp signals ofof2CH CH of the dimedone at 1.19, 2.52 and 3.22,8.90, respectively. 3 , CH2 and 2 groups group, H-5 proton quinazoline and 2NH protons appeared as singlet signals at 3.81, 9.41 and The OCH group, H-5 proton of quinazoline and 2NH protons appeared as singlet signals at 3.81, 3 9.72, respectively. The aromatic protons of 4-methoxyphenylamino ring appeared as two doublets at 8.90, 9.41 and 9.72, respectively. The aromatic protons of 4-methoxyphenylamino ring appeared 6.91 (2H) and 7.58 (2H) with the coupling constant J = 9.0 Hz and the four aromatic protons of as two doublets at 6.91 (2H) and as 7.58 (2H) with at the7.16 coupling J = (J 9.0= 8.3 Hz Hz). and Also, the four N-(4-methylphenyl) ring appeared two doublets (J = 8.2 constant Hz) and 7.54 the aromatic protons of N-(4-methylphenyl) ring appeared as two doublets at 7.16 (J = 8.2 Hz) and 13 C-NMR (CDCl3, 100 MHz, δ ppm) spectrum showed characteristic signals at 28.70 corresponding 7.54 (J = 8.3 Hz). Also, the 13 C-NMR (CDCl3 , 100 MHz, δ ppm) spectrum showed characteristic to 2CH 3, at 32.65 for a C8 (quinazoline), two signals at 37.52 and 50.99 corresponding to 2CH2 and signals at 28.70 corresponding to 2CH , at 32.65 for a C (quinazoline), two signals at 37.52 and 50.99 1 15 3 8 signal at 194.01 due to C=O (quinazoline). The H- N HMBC spectrum showed that the two most 1 H-15 N HMBC spectrum corresponding to 2CH and signal at 194.01 due to C=O (quinazoline). The 2 important correlated coupling which gave absolute and unique confirmation for the structure of showedthe that thewas twobetween most important correlated coupling which absolute 25a–c, first the proton H-5 of quinazoline (1H,gave at 8.90 ppm) and withunique N-4 ofconfirmation quinazoline 1 H, at 8.90 ppm) with for the structure of 25a–c, the first was between the proton H-5 of quinazoline ( 15 2 1 ( N, at 260 ppm) J (H-5, N-4) and the second was between the CH2 of quinazoline ( H, at 3.22 ppm) 2 J (H-5, N-4) and the second was between the CH of quinazoline N-4 ofN-10 quinazoline (15 N, at(15260 3J (H-9, N-10) (cf. Supporting Information) 2 (Figure 3). with of quinazoline N, ppm) at 216 ppm) If the compound 22b was obtained, its 1H-15N HMBC spectrum would have exhibited that correlated coupling between the proton H-9 of quinazoline with N-10 of quinazoline 2J (H-9, N-10) and correlated coupling between the CH2 of quinazoline with N-4 of quinazoline 3J (H-5, N-4), but,

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(1 H, at 3.22 ppm) with N-10 of quinazoline (15 N, at 216 ppm) 3 J (H-9, N-10) (cf. Supporting Information) (Figure 3). If the compound 22b was obtained, its 1 H-15 N HMBC spectrum would have exhibited that correlated coupling between the proton H-9 of quinazoline with N-10 of quinazoline 2 J (H-9, N-10) and Molecules 2018, 23, x FOR PEER REVIEW 6 of 20 correlated coupling between the CH2 of quinazoline with N-4 of quinazoline 3 J (H-5, N-4), but, these two correlated couplingcoupling were notwere detected in 1 H-15 HMBC spectrum (Figure 3).3). Furthermore, 1H15N HMBC these two correlated not detected inN spectrum (Figure Furthermore,X-ray diffraction of similar analogs added unequivocal evidence for the structures of 25a–c X-ray diffraction of similar analogs added unequivocal evidence for the structures of and 25a–cconfirmed and the reaction mechanism confirmed the reaction[30]. mechanism [30]. H3 C

H3 C H

O HN HN

4

N

3

NH

5

O

6

N 1

N

8

9

10

O

H

5

2

HN

N

22b

1

N

10

H 6

3

7

2

4

N

O 7

9

8

H 25b

OCH3

OCH3

Figure 3. Diagnostic correlations in the H- N HMBC spectrum for the two isomers 22b and 25b. 1

15

Figure 3. Diagnostic correlations in the 1 H-15 N HMBC spectrum for the two isomers 22b and 25b.

2.2. In Vitro Cytotoxic Activity

2.2. In Vitro Cytotoxic Activity

For evaluation of in vitro cytotoxic activity of compounds {5-aminopyrazoles 11a–c,

pyrazolo[1,5-a]pyrimidines 18a–ucytotoxic and pyrazolo[1,5-a]quinazolines 25a–c} {5-aminopyrazoles against liver (HepG-2)11a–c, For evaluation of in vitro activity of compounds ® was and breast (MCF-7) human carcinoma cell lines, MTT assay was used [31–33]. Doxorubicin pyrazolo[1,5-a]pyrimidines 18a–u and pyrazolo[1,5-a]quinazolines 25a–c} against liver (HepG-2) and ® used as a reference cytotoxic compound. The results were expressed as growth inhibitory breast (MCF-7) human carcinoma cell lines, MTT assay was used [31–33]. Doxorubicin was used as concentration (IC50) values (Table 1). a reference cytotoxic compound. The results were expressed as growth inhibitory concentration (IC50 ) From the results of in vitro cytotoxic activity, it was found that most of the prepared values (Table 1). compounds displayed comparable IC50 values against liver (HepG-2) and breast (MCF-7) cancer cell From the results of in vitro cytotoxic activity, it was found that most of the prepared compounds lines compared to positive control. displayedFor comparable IC50 values against (HepG-2) and did breast cell lines compared HepG-2 cancer cells, most of theliver tested compounds not(MCF-7) show anycancer significant difference to positive control. compared to the positive control. Only four compounds (11c, 18b, 18f and 18g) showed significant For HepG-2 cancer cells, most of the tested compounds not18d show significant difference in their activities. Compounds 18c (IC 50 = 75.9 ± 5.3did µM), (IC50any = 77.1 ± 4.2 µM),difference 18h (IC 50 = 73.2 ± 3.2 µM), 18j (IC 50 = 77.4 ± 2.9 µM), 18k (IC 50 = 74.0 ± 3.1 µM), 18l (IC 50 = 78.7 ± 5.1 µM), compared to the positive control. Only four compounds (11c, 18b, 18f and 18g) showed significant 50 = 72.2 ±activities. 3.8 µM), 18q (IC50 = 72.8 ±18c 3.9(IC µM), 18r (IC50 = 73.0 ± 1.9 µM), 18s (IC50 = 78.2 ± 3.2 18o (ICin difference their Compounds 50 = 75.9 ± 5.3 µM), 18d (IC50 = 77.1 ± 4.2 µM), µM), = 18t73.2 (IC50 78.7µM), ± 4.718j µM) and =25c (IC± 50 = 79.5 ± 4.8 µM) showed slightly higher activities than 18h (IC ±=3.2 (IC 77.4 2.9 µM), 18k (IC50 = 74.0 ± 3.1 µM), 18l (IC50 = 78.7 ± 50 50 doxorubicin (IC50 = 80.9 ± 2.1 µM). In addition, compound 18m (IC50 = 80.3 ± 3.9 µM) was almost 5.1 µM), 18o (IC50 = 72.2 ± 3.8 µM), 18q (IC50 = 72.8 ± 3.9 µM), 18r (IC50 = 73.0 ± 1.9 µM), 18s (IC50 equipotent as doxorubicin (IC50 = 80.9 ± 2.1 µM), while, compounds 11a (IC50 = 81.3 ± 4.1 µM), 18e = 78.2 ± 3.2 µM), 18t (IC50 = 78.7 ± 4.7 µM) and 25c (IC50 = 79.5 ± 4.8 µM) showed slightly higher (IC50 = 81.2 ± 5.5 µM), 18n (IC50 = 82.5 ± 5.7 µM) and 25b (IC50 = 81.9 ± 5.9 µM) displayed slightly less activities than doxorubicin (IC50 = 80.9 ± 2.1 µM). In addition, compound 18m (IC50 = 80.3 ± 3.9 µM) activities compared to doxorubicin (IC50 = 80.9 ± 2.1 µM). was almost as doxorubicin (IC50of =thecompounds 80.9 ± 2.1 µM), while,any compounds (IC50 = 81.3 In equipotent case of MCF-7 cell lines, none showed significant 11a differences ± 4.1compared µM), 18eto(IC = 81.2 ± 5.5 µM), 18n (IC = 82.5 ± 5.7 µM) and 25b (IC = 81.9 5.9 µM) 50 (IC50 = 63.1 ± 3.1 µM), 18b (IC50 =5064.9 ± 3.1± the50positive control. Compounds 18a µM), displayed less compared to doxorubicin ± 2.1 18c (ICslightly 50 = 64.3 ± 4.2 activities µM), 18j (IC 50 = 64.3 ± 3.1 µM), 11o (IC50(IC = 64.7 1.9 µM) andµM). 18u (IC50 = 64.5 ± 2.9 50 =± 80.9 65.6significant ± 4.2 µM). differences Whilst, compounds µM) showed slightly activities than doxorubicin (IC50 =any In case of MCF-7 cellhigher lines, none of thecompounds showed compared to 11a (IC50control. = 65.5 ±Compounds 4.3 µM), 18d (IC = 65.1 ± 2.8 µM), 18n (IC 50 = 65.9 ± 3.1 µM), 18q (IC 50 = 65.5 ± 50 2.1= 64.3 the positive 18a50(IC = 63.1 ± 3.1 µM), 18b (IC = 64.9 ± 3.1 µM), 18c (IC 50 50 µM) and 18r (IC 50 = 65.9 ± 2.6 µM) displayed equipotent as doxorubicin (IC50 = 65.6 ± 4.2 µM). ± 4.2 µM), 18j (IC50 = 64.3 ± 3.1 µM), 11o (IC50 = 64.7 ± 1.9 µM) and 18u (IC50 = 64.5 ± 2.9 µM) showed Whereas, compounds 18f (IC50 = 66.1 ± 2.9 µM), 18h (IC50 = 66.8 ± 2.6 µM), 18k (IC50 = 66.8 ± 3.9 µM), slightly higher activities than doxorubicin (IC50 = 65.6 ± 4.2 µM). Whilst, compounds 11a (IC50 = 65.5 ± 18l (IC50 = 66.7 ± 3.2 µM), 18m (IC50 = 66.2 ± 3.8 µM), 18s (IC50 = 66.8 ± 5.0 µM), 25b (IC50 = 66.2 ± 2.9 4.3 µM), 18d (IC50 = 65.1 ± 2.8 µM), 18n (IC50 = 65.9 ± 3.1 µM), 18q (IC50 = 65.5 ± 2.1 µM) and 18r (IC50 µM) and 25c (IC50 = 66.5 ± 3.1 µM) displayed slightly less activities. = 65.9 ± 2.6 µM) displayed equipotent as doxorubicin (IC50 = 65.6 ± 4.2 µM). Whereas, compounds 18f (IC50 = 66.1 ± 2.9 µM), 18h (IC50 = 66.8 ± 2.6 µM), 18k (IC50 = 66.8 ± 3.9 µM), 18l (IC50 = 66.7 ± 3.2 µM), 18m (IC50 = 66.2 ± 3.8 µM), 18s (IC50 = 66.8 ± 5.0 µM), 25b (IC50 = 66.2 ± 2.9 µM) and 25c (IC50 = 66.5 ± 3.1 µM) displayed slightly less activities.

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Table 1. The The IC IC50 50 (µM) Table 1. (µM) values values of of compounds compounds 11a–c, 11a–c, 18a–u 18a–u and and 25a–c 25a–c using using MTT MTT assay assay against against two two human carcinoma cell lines (HepG-2 and MCF-7). human carcinoma cell lines (HepG-2 and MCF-7). Ar

O

NH 2

NH

Ar

O NH

NH NH Ar 1

N 11a-c

Compounds Compounds 11a 11a 11b 11b 11c 11c 18a 18a 18b 18b 18c 18c 18d 18d 18e 18e 18f 18f 18g 18g 18h 18h 18i 18i 18j 18j 18k 18k 18l 18l 18m 18m 18n 18n 18o 18o 18p 18q 18p 18r 18q 18s 18r 18t 18s 18u 18t 25a 18u 25b 25a 25c Doxorubicin 25b 25c Doxorubicin

ArAr

NH Ar 1

Ar

N

N

N Ar 2

O

N

N H HN

O

N N

Ar 1

CH 3

25a-c

18a-u

Ar11 Ar

H3 C

Ar1Ar1

IC (µM) IC5050(µM) HepG-2 MCF-7 HepG-2 MCF-7

4-CH3O-C O-C6H H4 81.3 ± 4.1 C6H C65 H5 4-CH - 81.3 ± 4.1 3 6 4 3-C6H4 4-CH 3O-C6H4 86.2 ± 4.5 4-CH 4-CH3 -C6 H4 4-CH3 O-C6 H4 86.2 ± 4.5 6 H 4 4-CH 3 O-C 6 H 4 94.8 ± 6.5 4-Cl-C 4-Cl-C6 H4 4-CH3 O-C6 H4 94.8 ± 6.5 4-CH33O-C O-C66H44 C56H5 85.4 ± 5.1 C6H C65 H5 4-CH C6 H 85.4 ± 5.1 C65 H5 4-CH 4-CH H46H4 90.9 ± 6.5 C6H 4-CH33O-C O-C66H44 4-CH 90.9 ± 6.5 3 -C36-C C65 H5 4-CH 4-CH 75.9 ± 5.3 4-CH33O-C O-C66H4 4-CH 3O-C 75.9 ± 5.3 C6H 3 O-C 6 H46H4 C H 4-CH O-C H 4-Cl-C H 77.1 ± 4.2 6 5 3 6 4 6 4 4-CH3O-C6H4 4-Cl-C6H4 77.1 ± 4.2 C6H5 C65 H5 4-CH 4-Br-C 81.2 ± 5.5 6 H46H4 4-CH33O-C O-C66H H44 4-Br-C 81.2 ± 5.5 C6H C6 H5 4-CH3 O-C6 H4 4-F-C6 H4 92.8 ± 6.7 4-CH3O-C6H4 4-F-C6H4 92.8 ± 6.7 C6H5 C H 4-CH3 O-C6 H4 thiophen-2-yl 91.1 ± 6.4 4-CH3O-C6H4 thiophen-2-yl 91.1 ± 6.4 C6H65 5 4-CH3 -C6 H4 4-CH3 O-C6 H4 C6 H5 73.2 ± 3.2 3-C6H4 4-CH 3O-C6H4 C 6H5 73.2 ± 3.2 4-CH 4-CH3 -C6 H4 4-CH3 O-C6 H4 4-CH3 -C6 H4 83.3 ± 4.3 3-C6H4 4-CH3O-C O-C6H H4 4-CH 3-C6H4 83.3 ± 4.3 4-CH 4-CH -C H 4-CH 4-CH O-C H 77.4 ± 2.9 3 6 4 3 6 4 3 6 4 3-C6-C 4-CH33O-C O-C66H44 4-CH63O-C 77.4 ± 2.9 4-CH 4-CH 4-CH 4-Cl-C H4 6H4 74.0 ± 3.1 3 H46 H4 3-C H46 H4 4-CH33O-C O-C66H44 4-Cl-C 74.0 ± 3.1 4-CH 4-CH 4-CH 4-Br-C 78.7 ± 5.1 36-C 6 H46H4 4-CH 4-CH 4-F-C 80.3 ± 3.9 3-C H46 H4 4-CH33O-C O-C66H44 4-Br-C 78.7 ± 5.1 4-CH 36-C 6 H46H4 4-CH 4-CH thiophen-2-yl 82.5 ± 5.7 36-C 3-C H46 H4 4-CH33O-C O-C66H H4 4-F-C6H4 80.3 ± 3.9 4-CH 4-Cl-C 4-CH C6 H5 72.282.5 ± 3.8 * 6H 3-C6H 4 4 4-CH33O-C O-C66H H44 thiophen-2-yl ± 5.7 4-CH 4-Cl-C H 4-CH O-C H 4-CH -C H 87.8 ± 5.4 6 4 3 6 4 3 6 4 72.2 ± 3.8 * 4-CH3O-C6H4 C6H5 4-Cl-C6H4 4-Cl-C H 4-CH3 O-C6 H4 4-CH3 O-C6 H4 72.8 ± 3.9 4-CH3O-C6H4 4-CH3-C6H4 87.8 ± 5.4 4-Cl-C6H64 4 4-Cl-C6 H4 4-CH3 O-C6 H4 4-Cl-C6 H4 73.0 ± 1.9 4-CH3O-C6H4 4-CH3O-C6H4 72.8 ± 3.9 4-Cl-C6H4 4-Cl-C6 H4 4-CH3 O-C6 H4 4-Br-C6 H4 78.2 ± 3.2 6H4 4-CH 3O-C6H4 4-Cl-C 6H 4 73.0 ± 1.9 4-Cl-C 4-Cl-C6 H4 4-CH3 O-C6 H4 4-F-C6 H4 78.7 ± 4.7 6 H 4 4-CH 3 O-C 6 H 4 4-Br-C 6 H 4 78.2 ± 3.2 4-Cl-C 4-Cl-C6 H4 4-CH3 O-C6 H4 thiophen-2-yl 83.1 ± 5.1 6 H 4 4-CH 3 O-C 6 H 4 4-F-C 6 H 4 78.7 ± 4.7 4-Cl-C C6 H5 4-CH3 O-C6 4 87.9 ± 6.0 4-CH33O-C O-C66H44 thiophen-2-yl 83.1 ± 5.1 4-Cl-C 4-CH63H -C4 6 H4 4-CH 81.9 ± 5.9 4-Cl-C 4-CH - 79.5 ± 4.8 6H5 6 H4 4-CH33O-C O-C66H4 87.9 ± 6.0 C - 6H4 - 80.9 ± 2.1 4-CH3O-C 81.9 ± 5.9 4-CH3-C-6H4 4-CH3O-C6H4 79.5 ± 4.8 4-Cl-C6H4 * The most potent compound and selected for further experiments. 80.9 ± 2.1

65.5 ± ± 4.3 65.5 4.3 69.2 ± 3.9 69.2 ± 3.9 69.1 ± ± 3.7 69.1 3.7 ± 3.1 63.1 ± 3.1** 64.9 ± 3.1 ± 3.1 64.3 ± 4.2 ± 4.2 65.1 ± 2.8 65.1 ± 2.8 68.1 ± 4.0 68.1 ± 4.0 66.1 ± 2.9 66.1 ± 2.9 69.2 ± 3.2 69.2 ± 3.2 66.8 ± 2.6 66.8 ± 2.6 67.7 ± 2.7 67.7 ± ± 2.7 64.3 3.1 64.3 ± ± 3.1 66.8 3.9 66.8 ± ± 3.9 66.7 3.2 66.2 3.8 66.7 ± ± 3.2 65.9 3.1 66.2 ± ± 3.8 64.7 1.9 65.9 ± ± 3.1 67.1 ± 2.1 64.7 ± 1.9 65.5 ± 2.1 67.1 ± 2.1 65.9 ± 2.6 65.5 ± 2.1 66.8 ± 5.0 65.9 ± 2.6 67.0 ± 1.8 66.8 ± 5.0 64.5 ± 2.9 67.0 ± ± 1.8 68.9 4.2 64.5 ± ± 2.9 66.2 2.9 66.5 3.1 68.9 ± ± 4.2 65.6 4.2 66.2 ± ± 2.9 66.5 ± 3.1 65.6 ± 4.2

* The most potent compound and selected for further experiments.

2.3. Structure Activity Relationship (SAR) 2.3. Structure Relationship (SAR) activity of the synthesized compounds against liver (HepG2) From theActivity results of in vitrocytotoxic cell lines, that, (IC50 = 79.5 ± activity 4.8 µM) of >25b 5.9 µM) >25aagainst (IC50 =liver 87.9 From we thefound results of 25c in vitrocytotoxic the(IC synthesized 50 = 81.9 ± compounds ± 6.0 µM)cell in lines, the series of pyrazolo[1,5-a]quinazolines 25a–c, in (IC addition, (IC = 72.2 ± 3.8 µM) (HepG2) we found that, 25c (IC50 = 79.5 ± 4.8 µM) >25b 50 = 81.918o ± 5.9 µM) >25a (IC 50 = 87.9 50 >18h (IC50 in = 73.2 ± 3.2 µM) >18a (IC50 = 85.4 ± 5.1 µM); 18r (IC = 73.0 ± 18o 1.9 µM) 74.0 ± 6.0 µM) the series of pyrazolo[1,5-a]quinazolines 25a–c, in50addition, (IC50>18k = 72.2(IC ± 50 3.8=µM) ± 3.1(IC µM) = 77.1 ± 4.2 18s±(IC = 78.2 3.250 µM) 78.7 (IC ± 5.1 >18h 50 =>18d 73.2 (IC ± 3.2 >18a (ICµM); 50 = 85.4 5.150µM); 18r±(IC = 73.0>18l ± 1.9(IC µM) 50 = µM) 74.0 ±>18c 3.1 50 µM) 50 =>18k (IC = 75.9 ± 5.3 µM) and 18t (IC = 78.7 ± 4.7 µM) >18m (IC = 80.3 ± 3.92 µM) >18f (IC = 92.8 µM) (IC50 = 78.7 ± 5.1 µM) >18c (IC ± 50 >18d (IC50 = 77.1 ± 4.2 µM); 18s 50 (IC50 = 78.2 ± 3.2 µM) >18l50 5050 = 75.9± 6.7 µM) the18t series effect 4-Cl-C H4 5.3 µM) in and (IC50of= pyrazolo[1,5-a]pyrimidines 78.7 ± 4.7 µM) >18m (IC50 =18a–u. 80.3 ± This 3.92 was µM)concerning >18f (IC50 =the 92.8 ± 6.7ofµM) in 6the group of (chloride atom as electron withdrawing group) 4-CH3 -Cthe groupof (methyl series pyrazolo[1,5-a]pyrimidines 18a–u. This was and concerning 4-Cl-Cas 6Helectron 4 group 6 H4 effect releasing group) in the two series. Whence, the derivatives bearing Ar = 4-Cl-C H group (at position (chloride atom as electron withdrawing group) and 4-CH3-C6H4 group (methyl6 as4 electron releasing 3 in thein two were slightly active than bearing those bearing Ar = 64-CH than those group) theseries) two series. Whence,more the derivatives Ar = 4-Cl-C H4 group (at position 3 in the 3 -C6 H 4 group bearing Ar = C6 Hslightly group. two series) were more active than those bearing Ar = 4-CH 3 -C 6 H 4 group than those bearing 5 addition, Ar = In C6H 5 group. we observed that, there was a ranking in the order of rings bearing halogen atoms (Cl, Br F) in the series of 18a–u, where, (IC50 = in 77.1 4.2 µM) >18e bearing (IC50 = 81.2 ± 5.5 µM) In and addition, we observed that, there was18d a ranking the±order of rings halogen atoms (Cl, Br and F) in the series of 18a–u, where, 18d (IC50 = 77.1 ± 4.2 µM) >18e (IC50 = 81.2 ± 5.5 µM) >18f

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>18f (IC50 = 92.8 ± 6.7 µM); 18k (IC50 = 74.0 ± 3.1 µM) >18l (IC50 = 78.7 ± 5.1 µM) >18m (IC50 = 80.3 = 92.8 ± 6.7 (IC50 =±74.0 3.1 µM) = 78.7 5.1µM) µM) >18t >18m(IC (IC5050 == 80.3 3.94.7 µM) ± (IC 3.950µM) and 18rµM); (IC5018k = 73.0 1.9 ±µM) >18s>18l (IC50(IC=5078.2 ±±3.2 78.7±± µM). and 18r (IC 50 = 73.0 ± 1.9 µM) >18s (IC 50 = 78.2 ± 3.2 µM) >18t (IC 50 = 78.7 ± 4.7 µM). Therefore, the Therefore, the derivatives bearing Ar2 = 4-Cl-C6 H4 group (at position 7) > Ar2 = 4-Br-C6 H4 group > Ar2 derivatives bearing Ar2 = 4-Cl-C6H4 group (at position 7) > Ar2 = 4-Br-C6H4 group > Ar2 = 4-F-C6H4 = 4-F-C 6 H4 group. group. Moreover, the derivatives bearing Ar2 = 4-CH3 O-C6 H4 group (at position 7) > Ar2 = 4-CH3 -C6 H4 derivatives bearing Ar2 = 4-CH3O-C6H4 group (at position 7) > Ar2 = 4-CH3-C6H4 group, Moreover, where, 18cthe(IC 50 = 75.9 ± 5.3µM) >18b (IC50 = 90.9 ± 6.5 µM); 18j (IC50 = 77.4 ± 2.9µM) group, where, 18c (IC 50 = 75.9 ± 5.3µM) >18b (IC50 = 90.9 ± 6.5 µM); 18j (IC50 = 77.4 ± 2.9µM) >18i (IC50 = >18i (IC50 = 83.3 ± 4.3 µM) and 18q (IC50 = 72.8 ± 3.9 µM) >18p (IC50 = 87.8 ± 5.4 µM). Therefore, ± 4.3 µM) and 18q (IC50 = 72.8 ± 3.9 µM) >18p (IC50 = 87.8 ± 5.4 µM). Therefore, the replacement of the83.3 replacement of the 4-CH3 -C6 H4 group by 4-CH3 O-C6 H4 group was impacted and increased the the 4-CH3-C6H4 group by 4-CH3O-C6H4 group was impacted and increased the activity against liver activity against liver cancer. cancer. Furthermore, we observed that, the derivatives bearing phenyl group (at position 7) more active Furthermore, we observed that,the derivatives bearing phenyl group (at position 7) more active than those bearing thiophen-2-yl group, where,18a (IC50 = 85.4 ± 5.1 µM) >18g (IC50 = 91.1 ± 6.4 µM); than those bearing thiophen-2-yl group, where,18a (IC50 = 85.4 ± 5.1 µM) >18g (IC50 = 91.1 ± 6.4 µM); 18h (IC50 = 73.2 ± 3.2 µM) >18n (IC50 = 82.5 ± 5.7 µM) and 18o (IC50 = 72.2 ± 3.8 µM) >18u (IC50 = 83.1 18h (IC50 = 73.2 ± 3.2 µM) >18n (IC50 = 82.5 ± 5.7 µM) and 18o (IC50 = 72.2 ± 3.8 µM) >18u (IC50 = 83.1 ± ± 5.1 5.1 µM). µM). Therefore, Therefore,the theintroduction introduction thiophen-2-yl group in the series decreased the activity. of of thiophen-2-yl group in the series decreased the activity. A A brief relationship(SAR) (SAR)study study has been presented in Figure brief Structure-activity Structure-activity relationship has been presented in Figure 4. 4. 4-Chlorophenyl group were more active than 4-methylphenyl group and phenyl group

Ar

O

N

N H HN Ar 1

N

O

N H 3C

CH 3

Ar1 = 4-CH 3O-C 6H 4

4-Chlorophenyl group were more active than 4-methylphenyl group and phenyl group

Ar

O

N

NH NH

N

N Ar 2

Ar1

Ar 2 = C6 H 5 Ar 2 = 4-Cl-C 6H 4

Ar 1 = 4-CH3O-C6 H4

Ar 2 = 4-Br-C 6 H4 Ar 2 = 4-F-C6 H4

Ar = C6 H5

Ar

O NH

Ar = 4-CH 3-C6H4 Ar = 4-Cl-C 6 H4

4-Chlorophenyl group were more active than 4-bromophenyl group and 4-f lorophenyl group

N N

NH Ar1

N

Ar 2

Ar 1 = 4-CH3 O-C6 H4

4-Methoxyphenyl group were more active than 4-methylphenyl group Phenyl group were more active than thiophene-2-yl group

Figure 4. A brief Structure-activity relationship (SAR) study of 18a–u and 25a–c against liver Figure 4. A brief Structure-activity relationship (SAR) study of 18a–u and 25a–c against liver (HepG2) (HepG2) cell lines. cell lines.

2.4. Cell-Cycle Analysis and Apoptotic Changes 2.4. Cell-Cycle Analysis and Apoptotic Changes Cell cycle can be defined as cell reproduction via replication of the DNA followed by division of Cell cycleand can partitioning be defined as via replication of the DNA division of the nucleus of cell the reproduction cytoplasm to yield two daughter cells. Thisfollowed cell cyclebycomprises thefour nucleus and partitioning of the cytoplasm to yield two daughter cells. This cell cycle comprises four different phases. G1 phase occurs between nuclear division (M phase) and DNA synthesis (S different G1occurs phase between occurs between (M phase) and DNA synthesis phase); phase);phases. G2 phase S phasenuclear and M division phase. These gaps allow for the repair of(SDNA G2damage phase occurs betweenerrors S phase M phase. These gaps allow for theinrepair DNA damage and replication [34].and According to the cytotoxicity screening Table of 1, and because most of the compounds did not show statistical significant differences compared to the positive

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and replication errors [34]. According to the cytotoxicity screening in Table 1, and because most Molecules 2018, 23, x FOR PEER REVIEW 9 of 20 of the compounds did not show statistical significant differences compared to the positive control, two compounds (18o and 18a) selected forselected further experiments. The effect ofThe compounds control, two compounds (18o have and been 18a) have been for further experiments. effect of 18o and 18a after 24 h of treatment by propidium iodide on cell cycle progression, using the flow compounds 18o and 18a after 24 h of treatment by propidium iodide on cell cycle progression, using cytometry (Figure 5a), was investigated against HepG-2 and MCF-7, respectively. the flow cytometry (Figure 5a), was investigated against HepG-2 and MCF-7, respectively. Compound Compound 18o 18o induced induced significant significant alterations alterations in in the the cell-cycle cell-cycle phases phases of of HepG2 HepG2 cells cells when when compared to control. Interestingly, exposure of HepG2 cells to 18o induced a significant increase in compared to control. Interestingly, exposure of HepG2 cells to 18o induced a significant increasethe in percentage of cells pre-G1 and G2/M by 6.6 folds andfolds 1.7 folds, withfolds, a concurrent significant the percentage of atcells at pre-G1 and phases G2/M phases by 6.6 and 1.7 with a concurrent reduction the percentage cells at G0/G1 foldsby without anywithout significant in S phase significantinreduction in the of percentage of cellsbyat1.2 G0/G1 1.2 folds any changes significant changes compared to control, respectively. in S phase compared to control, respectively. Moreover, MCF-7 cancer cancer cells cellswith withcompound compound18a 18acaused causeda asignificant significant increase Moreover, treatment treatment of of MCF-7 increase in in pre-G1 and G2/M phases percent by 7.9 folds and 3.5 folds with a significant reduction in the pre-G1 and G2/M phases percent by 7.9 folds and 3.5 folds with a significant reduction in the percentage by1.4 1.4folds foldsand andslightly slightlyincrease increaseininS Sphases phasesbyby0.90.9 folds compared percentage of of cells cells at at G0/G1 G0/G1 by folds compared to to control, respectively (Figure5b). 5b).However, However,the thepositive positivecontrol control showed showed better better results. results. In In case control, respectively (Figure case of of HepG-2 HepG-2 cancer cancer cells, cells, Doxorubicin-induced Doxorubicin-induced aa significant significant increase increase in in the the percentage percentage of of cells cells at at pre-G1 pre-G1 and phases by by 2.2 2.2 folds folds and and G2/M G2/M phases and 1.6 1.6 folds, folds, with with aa significant significant reduction reduction in in the the percentage percentage of of cells cells at at G0/G1 and S phases by 1.14 and 1.46 folds compared to compound 18o. In addition, in case of MCF-7 G0/G1 and S phases by 1.14 and 1.46 folds compared to compound 18o. In addition, in case of MCF-7 cancer a significant increase in the of cells pre-G1 and G2/M cancercells, cells,Doxorubicin-induced Doxorubicin-induced a significant increase in percentage the percentage ofatcells at pre-G1 and phases by 1.7-folds and 1.4 folds, with a significant reduction in the percentage of cells at G0/G1 and G2/M phases by 1.7-folds and 1.4 folds, with a significant reduction in the percentage of cells at did not show any significant increase in S phases compared to compound 18o. From these results, G0/G1 and did not show any significant increase in S phases compared to compound 18o. From itthese can be concluded compounds 18o and 18a inhibit the 18a cell inhibit growththe through cell cycle arrestcell at results, it can that be concluded that compounds 18o and cell growth through G2/M phase,atwhich turn induces deathinduces by apoptosis. These are inThese agreement with cycle arrest G2/Minphase, which cell in turn cell death byresults apoptosis. results arethe in cytotoxicity screening results. agreement with the cytotoxicity screening results.

Figure5. 5. (a) (a) Effect Effect of of compound compound 18o 18o on on DNA-ploidy DNA-ploidy flow flow cytometric cytometric analysis analysis of of HepG-2 HepG-2 cancer cancer cells, cells, Figure the cells were treated with DMSO as control and with doxorubicin as a positive control, for 24 h. the cells were treated with DMSO as control and with doxorubicin as a positive control, for 24(b) h. Effect of of compound 18a ononDNA-ploidy cells, the the cells cells (b) Effect compound 18a DNA-ploidyflow flowcytometric cytometricanalysis analysisof of MCF-7 MCF-7 cancer cancer cells, weretreated treatedwith withDMSO DMSOas ascontrol controland andwith withdoxorubicin doxorubicinasasaapositive positivecontrol, control,for for24 24h.h. were

2.5. Annexin V-FITC Apoptosis Assay The apoptotic effect of compounds 18o and 18a was carried out using Annexin V-FITC/PI (AV/PI) dual staining assay (Figure 6). The results revealed that HepG2 and MCF-7cells, treated with compounds 18o and 18a, respectively, showed a significant increase in the percent of annexin

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2.5. Annexin V-FITC Apoptosis Assay The apoptotic effect of compounds 18o and 18a was carried out using Annexin V-FITC/PI (AV/PI) dual staining assay (Figure 6). The results revealed that HepG2 and MCF-7cells, treated Molecules 2018, 23, x FOR PEER REVIEW 10 of 20 with compounds 18o and 18a, respectively, showed a significant increase in the percent of annexin V-FITC cells (UR(UR & LR) 11.6byfolds 9.8 folds control, respectively. V-FITCpositive positiveapoptotic apoptotic cells & by LR) 11.6and folds and compared 9.8 folds to compared to control, However, doxorubicin showed 2.2 folds and 1.7 folds increases in apoptotic cells % compared respectively. However, doxorubicin showed 2.2 folds and 1.7 folds increases in apoptotic cells to % compounds 18ocompounds and 18a, respectively. results reveal These that theresults cytotoxicity of compounds compared to 18o and These 18a, respectively. revealactivities that the cytotoxicity 18o and 18a are due to their pro-apoptotic activity. activities of compounds 18opotent and 18a are due to their potent pro-apoptotic activity.

Figure 6.6.(a) (a)Effect Effectof of compound on percentage the percentage of annexin in Figure compound 18o18o on the of annexin V-FITCV-FITC positivepositive stainingstaining in HepG-2 HepG-2cells, cancer cells werewith treated withasDMSO asand control with doxorubicin as a positive cancer thecells, cells the were treated DMSO control withand doxorubicin as a positive control, control, for 24 h. (b)Effect of compound 18a on the percentage of annexin V-FITC positive staining in for 24 h. (b) Effect of compound 18a on the percentage of annexin V-FITC positive staining in MCF-7 MCF-7 cancer cells, the cells were treated with DMSO as control and with doxorubicin as a positive cancer cells, the cells were treated with DMSO as control and with doxorubicin as a positive control, control, for 24 h. for 24 h.

3. Materials Materials and and Methods Methods 3. 3.1. Chemistry Chemistry 3.1. All melting meltingpoints points were measured on a Gallenkamp melting point and apparatus and are All were measured on a Gallenkamp melting point apparatus are uncorrected. uncorrected. The IR spectra were recorded (KBr disk) on a 1650 FT-IR instrument (Perkin The IR spectra were recorded (KBr disk) on a 1650 FT-IR instrument (Perkin Elmer, Waltham, MA,Elmer, USA). 1H-NMR 1Waltham, 13 C-NMR MA,MHz) USA).and (400 (100 MHz) and 13 C-NMRwere (100recorded MHz) spectra were recorded on a H-NMR (400 MHz) spectra on a Varian spectrometer Varian spectrometer (Varian, Inc., Palo Alto, CA, USA) using DMSO-d 6 or CDCl 3 as solvent and TMS (Varian, Inc., Palo Alto, CA, USA) using DMSO-d6 or CDCl3 as solvent and TMS as an internal standard. as an internal standard. Chemical shifts are reported in ppm. Coupling constants are spectra expressed in Chemical shifts are reported in ppm. Coupling constants (J) are expressed in Hz. (J) Mass were Hz. Masson spectra wereMAT recorded on a Varian at MAT 112Elemental spectrometer at 70 were eV. Elemental analyses recorded a Varian 112 spectrometer 70 eV. analyses performed at the were performed at the Microanalytical Center, Cairo University, Egypt. The progress of the reactions Microanalytical Center, Cairo University, Egypt. The progress of the reactions was monitored by was monitored by thin-layer chromatography (TLC) using aluminum sheets coated with silica gel thin-layer chromatography (TLC) using aluminum sheets coated with silica gel F254 (Merck, Darmstadt, F254 (Merck, Darmstadt, Germany), viewing under a short-wavelength UV lamp effected detection. All evaporations were carried out under reduced pressure at 40 °C. Synthesis of 5-amino-3-(arylamino)-1H-pyrazole-4-carboxamides 11a–c. Compounds of this series were prepared according to the literature procedure. 5-Amino-3-(4-methoxyphenylamino)-N-phenyl-1H-pyrazole-4-carboxamide (11a). White crystals; m.p.

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Germany), viewing under a short-wavelength UV lamp effected detection. All evaporations were carried out under reduced pressure at 40 ◦ C. Synthesis of 5-amino-3-(arylamino)-1H-pyrazole-4-carboxamides 11a–c. Compounds of this series were prepared according to the literature procedure. 5-Amino-3-(4-methoxyphenylamino)-N-phenyl-1H-pyrazole-4-carboxamide (11a). White crystals; m.p. 175–177 ◦C [29]. 5-Amino-3-(4-methoxyphenylamino)-N-(4-methylphenyl)-1H-pyrazole-4-carboxamide (11b). White crystals; m.p. 198–200 ◦ C [29]. 5-Amino-3-(4-methoxyphenylamino)-N-(4-chlorophenyl)-1H-pyrazole-4-carboxamide (11c). White crystals; m.p. 190–192 ◦ C [29]. General Procedure for Synthesis of 7-aryl-2-(arylamino)pyrazolo[1,5-a]pyrimidine-3-carboxamides 18a–u. A mixture of compounds 11a–c (0.01 mol) with enaminones 12a–g {e.g., 3-(dimethylamino)-1phenylprop-2-en-1-one (12a), 3-(dimethylamino)-1-(4-methylphenyl)prop-2-en-1-one (12b), 3-(dimethylamino)-1-(4-methoxyphenyl)prop-2-en-1-one (12c), 1-(4-chlorophenyl)-3-(dimethyl-amino)prop -2-en-1-one (12d), 1-(4-bromophenyl)-3-(dimethylamino)prop-2-en-1-one (12e), 3-(dimethylamino)-1(4-fluorophenyl)prop-2-en-1-one (12f) or 3-(dimethylamino)-1-(thiophen-2-yl)prop-2-en-1-one (12g)} (0.01 mol) in glacial acetic acid (25 mL), the reaction mixture was refluxed for 1 h and then left to cool. The solid product was filtered off, washed with ethanol, dried and finally recrystallized from DMF/H2 O to afford the corresponding pyrazolo[1,5-a]pyrimidine derivatives 18a–u. 2-(4-Methoxyphenylamino)-N,7-diphenylpyrazolo[1,5-a]pyrimidine-3-carboxamide (18a). Yellow crystals, m.p. 218–220 ◦ C, yield (72%). IR (KBr) νmax /cm−1 3346 (NH), 1658 (C=O). 1 H-NMR (CDCl3 , 400 MHz, δ ppm): 3.80 (s, 3H, OCH3 ), 6.88 (d, 2H, J = 9.0 Hz,ArH), 6.96 (d, 1H, J = 4.8 Hz, pyrimidine), 7.12 (t, 1H, ArH), 7.36–7.42 (m, 5H, ArH), 7.62 (d, 2H, J = 9.0 Hz,ArH), 7.74 (d, 2H, J = 8.4 Hz,ArH), 8.11 (d, 2H, J = 8.3 Hz,ArH), 8.49 (d, 1H, J = 4.8 Hz, pyrimidine), 9.40 (s, 1H, NH), 10.05 (s, 1H, NH). 13 C-NMR (CDCl3 , 100 MHz, δ ppm): 55.7 (C, OCH3 ), 87.8 (C, C3 -pyrazolopyrimidine), 107.0 (C, C6 -pyrazolopyrimidine), 114.4, 119.2, 120.2, 123.7, 127.7, 129.1, 129.5, 129.6 (14C, Ar), 134.1 (C, C3a -pyrazolopyrimidine), 138.8, 142.4, 146.7 (3C, Ar), 147.9 (C, C7 -pyrazolopyrimidine), 149.6 (C, Ar), 154.5 (C, C2 -pyrazolopyrimidine), 157.8 (C, C5 -pyrazolopyrimidine), 163.3 (C=O). MS (m/z, %): 435 (M+ , 73.86). Anal. Calcd. (%) for C26 H21 N5 O2 (435.48): C, 71.71; H, 4.86; N, 16.08. Found: C, 71.80; H, 4.81; N, 16.00%. 2-(4-Methoxyphenylamino)-N-phenyl-7-(4-methylphenyl)-pyrazolo[1,5-a]pyrimidine-3-carboxamide (18b). Yellow crystals, m.p. 219–221 ◦ C, yield (77%). IR (KBr) νmax /cm−1 3337 (NH), 1658 (C=O). 1 H-NMR (CDCl3 , 400 MHz, δ ppm): 2.49 (s, 3H, CH3 ), 3.80 (s, 3H, OCH3 ), 6.87 (d, 2H, J = 8.9 Hz, ArH), 6.91 (d, 1H, J = 4.7 Hz, pyrimidine), 7.12 (t, 1H, ArH), 7.36 (d, 2H, J = 8.3 Hz, ArH), 7.38 (t, 2H, ArH), 7.60 (d, 2H, J = 8.9 Hz, ArH), 7.73 (d, 2H, J = 7.6 Hz, ArH), 8.08 (d, 2H, J = 8.1 Hz,ArH), 8.43 (d, 1H, J = 4.7 Hz, pyrimidine), 9.38 (s, 1H, NH), 10.01 (s, 1H, NH). 13 C-NMR (CDCl3 , 100 MHz, δ ppm): 21.8 (C, CH3 ), 55.7 (C, OCH3 ), 87.7 (C, C3 -pyrazolopyrimidine), 107.0 (C, C6 -pyrazolopyrimidine), 114.4, 119.1, 120.1, 123.7, 127.6, 129.1, 129.4, 129.6 (14C, Ar), 134.1 (C, C3a -pyrazolopyrimidine), 138.8, 142.3, 146.6 (3C, Ar), 147.8 (C, C7 -pyrazolopyrimidine), 149.6 (C, Ar), 154.4 (C, C2 -pyrazolopyrimidine), 157.7 (C, C5 -pyrazolopyrimidine), 163.3 (C=O). MS (m/z, %): 449 (M+ , 67.43). Anal. Calcd. (%) for C27 H23 N5 O2 (449.50): C, 72.14; H, 5.16; N, 15.58. Found: C, 72.10; H, 5.20; N, 15.60%. 7-(4-Methoxyphenyl)-2-(4-methoxyphenylamino)-N-phenylpyrazolo[1,5-a]pyrimidine-3-carboxamide (18c). Yellow crystals, m.p. 206–208 ◦ C, yield (76%). IR (KBr) νmax /cm−1 3340 (NH), 1646 (C=O). 1 H-NMR (CDCl3 , 400 MHz, δ ppm): 3.80 (s, 3H, OCH3 ), 3.91 (s, 3H, OCH3 ), 6.87 (d, 2H, J = 8.9 Hz, ArH), 6.89 (d, 1H, J = 4.8 Hz, pyrimidine), 7.05 (d, 2H, J = 8.8 Hz, ArH), 7.11 (t, 1H, ArH), 7.37 (t, 2H, ArH), 7.60 (d, 2H, J = 8.9 Hz, ArH), 7.72 (d, 2H, J = 7.6 Hz, ArH), 8.18 (d, 2H, J = 8.8 Hz, ArH), 8.40 (d, 1H, J = 4.8 Hz, pyrimidine), 9.36 (s, 1H, NH), 10.02 (s, 1H, NH). 13 C-NMR (CDCl3 , 100 MHz, δ ppm): 55.5 (C, OCH3 ), 55.6 (C, OCH3 ), 87.4 (C, C3 -pyrazolopyrimidine), 106.4 (C, C6 -pyrazolopyrimidine), 113.9,

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114.2, 119.0, 120.0, 122.4, 123.5, 128.9, 131.3 (14C, Ar), 134.0 (C, C3a -pyrazolopyrimidine), 138.6, 146.0 (2C, Ar), 147.7 (C, C7 -pyrazolopyrimidine), 149.3 (C, Ar), 154.3 (C, C2 -pyrazolopyrimidine), 157.5 (C, C5 -pyrazolopyrimidine), 162.2 (C, Ar), 163.2 (C=O). MS (m/z, %): 465 (M+ , 69.48). Anal. Calcd. (%) for C27 H23 N5 O3 (465.50): C, 69.66; H, 4.98; N, 15.04. Found: C, 69.70; H, 4.95; N, 15.00%. 7-(4-Chlorophenyl)-2-(4-methoxyphenylamino)-N-phenylpyrazolo[1,5-a]pyrimidine-3-carboxamide (18d). Yellow crystals, m.p. 252–253 ◦ C, yield (72%). IR (KBr) νmax /cm−1 3343 (NH), 1648 (C=O). 1 H-NMR (CDCl3 , 400 MHz, δ ppm): 3.81 (s, 3H, OCH3 ), 6.88 (d, 2H, J = 9.0 Hz, ArH), 6.94 (d, 1H, J = 4.7 Hz, pyrimidine), 7.13 (t, 1H, ArH), 7.39 (t, 2H, ArH), 7.58 (d, 4H, J = 8.8 Hz, ArH), 7.74 (d, 2H, J = 8.6 Hz, ArH), 8.15 (d, 2H, J = 8.7 Hz, ArH), 8.52 (d, 1H, J = 4.7 Hz, pyrimidine), 9.42 (s, 1H, NH), 9.99 (s, 1H, NH). 13 C-NMR (CDCl3 , 100 MHz, δ ppm): 55.7 (C, OCH3 ), 88.0 (C, C3 -pyrazolopyrimidine), 107.0 (C, C6 -pyrazolopyrimidine), 114.4, 119.2, 120.2, 123.8, 129.1, 129.1, 130.9, 131.8 (14C, Ar), 133.9 (C, C3a -pyrazolopyrimidine), 134.6, 138.0, 138.7 (3C, Ar), 145.3 (C, C7 -pyrazolopyrimidine), 149.7 (C, Ar), 154.6 (C, C2 -pyrazolopyrimidine), 157.9 (C, C5 -pyrazolopyrimidine), 163.2 (C=O). MS (m/z, %): 469 (M+ , 78.23). Anal. Calcd. (%) for C26 H20 ClN5 O2 (469.92): C, 66.45; H, 4.29; N, 14.90. Found: C, 66.40; H, 4.30; N, 14.95%. 7-(4-Bromophenyl)-2-(4-methoxyphenylamino)-N-phenylpyrazolo[1,5-a]pyrimidine-3-carboxamide (18e). Yellow crystals, m.p. 278–280 ◦ C, yield (69%). IR (KBr) νmax /cm−1 3365 (NH), 1650 (C=O). 1 H-NMR (DMSO-d6 , 400 MHz, δ ppm): 3.73 (s, 3H, OCH3 ), 6.93 (d, 2H, J = 9.0 Hz, ArH), 7.12 (t, 1H, ArH), 7.39 (t, 2H, ArH), 7.41 (d, 1H, J = 4.8 Hz, pyrimidine), 7.59 (d, 2H, J = 9.0 Hz, ArH), 7.73 (d, 2H, J = 7.6 Hz, ArH), 7.90 (d, 2H, J = 8.7 Hz, ArH), 8.20 (d, 2H, J = 8.7 Hz, ArH), 8.74 (d, 1H, J = 4.8 Hz, pyrimidine), 9.26 (s, 1H, NH), 10.03 (s, 1H, NH). 13 C-NMR (DMSO-d6 , 100 MHz, δ ppm): 55.7 (C, OCH3 ), 87.6 (C, C3 -pyrazolopyrimidine), 106.9 (C, C6 -pyrazolopyrimidine), 114.4, 119.1, 120.5, 123.3, 129.4, 129.8, 131.0, 131.6 (14C, Ar), 133.7 (C, C3a -pyrazolopyrimidine), 133.4, 136.1, 138.7 (3C, Ar), 145.2 (C, C7 -pyrazolopyrimidine), 149.5 (C, Ar), 154.8 (C, C2 -pyrazolopyrimidine), 157.1 (C, C5 -pyrazolopyrimidine), 163.7 (C=O). MS (m/z, %): 514 (M+ , 81.26). Anal. Calcd. (%) for C26 H20 BrN5 O2 (514.37): C, 60.71; H, 3.92; N, 13.62. Found: C, 60.65; H, 3.97; N, 13.65%. 7-(4-Fluorophenyl)-2-(4-methoxyphenylamino)-N-phenylpyrazolo[1,5-a]pyrimidine-3-carboxamide (18f). Yellow crystals, m.p. 237–239 ◦ C, yield (70%). IR (KBr) νmax /cm−1 3343 (NH), 1647 (C=O). 1 H-NMR (DMSO-d6 , 400 MHz, δ ppm): 3.73 (s, 3H, OCH3 ), 6.91 (d, 2H, J = 9.0 Hz, ArH), 7.11 (t, 1H, ArH), 7.37 (d, 1H, J = 4.9 Hz, pyrimidine), 7.39 (d, 2H, J = 7.6 Hz, ArH), 7.52 (t, 2H, ArH), 7.58 (d, 2H, J = 9.0 Hz, ArH), 7.71 (d, 2H, J = 8.6 Hz, ArH), 8.31 (d, 2H, J = 8.9 Hz, ArH), 8.71 (d, 1H, J = 4.8 Hz, pyrimidine), 9.23 (s, 1H, NH), 10.01 (s, 1H, NH). 13 C-NMR (DMSO-d6 , 100 MHz, δ ppm): 55.2 (C, OCH3 ), 86.7 (C, C3 -pyrazolopyrimidine), 108.3 (C, C6 -pyrazolopyrimidine), 114.3, 115.7, 115.9, 118.8, 119.4, 123.5, 126.4, 129.1 (14C, Ar), 132.4 (C, C3a -pyrazolopyrimidine), 133.3, 138.4 (2C, Ar), 145.0 (C, C7 -pyrazolopyrimidine), 147.1 (C, Ar), 151.1 (C, C2 -pyrazolopyrimidine), 154.1 (C, C5 -pyrazolopyrimidine), 156.6 (C, Ar), 162.2 (C=O). MS (m/z, %): 453 (M+ , 87.33). Anal. Calcd. (%) for C26 H20 FN5 O2 (453.47): C, 68.86; H, 4.45; N, 15.44. Found: C, 68.95; H, 4.40; N, 15.50%. 2-(4-Methoxyphenylamino)-N-phenyl-7-(thiophen-2-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (18g). Yellow crystals, m.p. 233–235 ◦ C, yield (71%). IR (KBr) νmax /cm−1 3356 (NH), 1652 (C=O). 1 H-NMR (DMSO-d6 , 400 MHz, δ ppm): 3.78 (s, 3H, OCH3 ), 7.03 (d, 2H, J = 8.4 Hz, ArH), 7.12 (t, 1H, ArH), 7.40 (t, 2H, ArH), 7.47 (t, 1H, J = 4.9 Hz, thiophene), 7.74 (d, 2H, J = 7.8 Hz, ArH), 7.84 (d, 2H, J = 8.5 Hz, ArH), 7.90 (d, 1H, J = 4.6 Hz, pyrimidine), 8.28 (d, 1H, J = 4.4 Hz, thiophene), 8.58 (d, 1H, J = 2.8 Hz, thiophene), 8.71 (d, 1H, J = 4.4 Hz, pyrimidine), 9.44 (s, 1H, NH), 10.07 (s, 1H, NH). 13 C-NMR (DMSO-d6 , 100 MHz, δ ppm): 55.7 (C, OCH3 ), 86.8 (C, C3 -pyrazolopyrimidine), 107.3 (C, C6 -pyrazolopyrimidine), 114.8, 119.2, 120.8, 126.3 (7C, Ar), 128.1, 129.8 (2C, thiophene), 130.1 (2C, Ar), 133.2 (C, thiophene), 133.9 (C, C3a -pyrazolopyrimidine), 134.5, 137.1 (2C, Ar), 139.4 (C, thiophene), 147.3 (C, Ar), 150.8 (C, C2 -pyrazolopyrimidine), 154.4 (C, C5 -pyrazolopyrimidine),

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156.3 (C, C7 -pyrazolopyrimidine), 162.9 (C=O). MS (m/z, %): 441 (M+ , 100). Anal. Calcd. (%) for C24 H19 N5 O2 S (441.50): C, 65.29; H, 4.34; N, 15.86. Found: C, 65.35; H, 4.30; N, 15.90%. 2-(4-Methoxyphenylamino)-7-phenyl-N-(4-methylphenyl)-pyrazolo[1,5-a]pyrimidine-3-carboxamide (18h). Yellow crystals, m.p. 251–253 ◦ C, yield (76%). IR (KBr) νmax /cm−1 3374 (NH), 1660 (C=O). 1 H-NMR (CDCl , 400 MHz, δ ppm): 2.35 (s, 3H, CH ), 3.80 (s, 3H, OCH ), 6.88 (d, 2H, 3 3 3 J = 9.0 Hz, ArH), 6.95 (d, 1H, J = 4.8 Hz, pyrimidine), 7.18 (d, 2H, J = 8.2 Hz, ArH), 7.40 (d, 2H, J = 8.2 Hz, ArH), 7.60–7.64 (m, 5H, ArH), 8.11 (d, 2H, J = 8.2 Hz, ArH), 8.48 (d, 1H, J = 4.8 Hz, pyrimidine), 9.42 (s, 1H, NH), 9.97 (s, 1H, NH).13 C-NMR (CDCl3 , 100 MHz, δ ppm): 21.0 (C, CH3 ), 55.7 (C, OCH3 ), 87.8 (C, C3 -pyrazolopyrimidine), 106.9 (C, C6 -pyrazolopyrimidine), 114.4, 119.1, 120.2, 127.7, 129.4, 129.6, 133.3 (14C, Ar), 134.2 (C, C3a -pyrazolopyrimidine), 136.2, 142.4, 146.6 (3C, Ar), 147.8 (C, C7 -pyrazolopyrimidine), 149.6 (C, Ar), 154.4 (C, C2 -pyrazolopyrimidine), 157.8 (C, C5 -pyrazolo-pyrimidine), 163.2 (C=O). MS (m/z, %): 449 (M+ , 92.11). Anal. Calcd. (%) for C27 H23 N5 O2 (449.50): C, 72.14; H, 5.16; N, 15.58. Found: C, 72.20; H, 5.11; N, 15.50%. 2-(4-Methoxyphenylamino)-N,7-di-(4-methylphenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (18i). Yellow crystals, m.p. 261 ◦ C, yield (74%). IR (KBr) νmax /cm−1 3293 (NH), 1642 (C=O). 1 H-NMR (CDCl3 , 400 MHz, δ ppm): 2.35 (s, 3H, CH3 ), 2.49 (s, 3H, CH3 ), 3.80 (s, 3H, OCH3 ), 6.87 (d, 2H, J = 9.0 Hz, ArH), 6.91 (d, 1H, J = 4.8 Hz, pyrimidine), 7.18 (d, 2H, J = 8.2 Hz, ArH), 7.37 (d, 2H, J = 8.0 Hz, ArH), 7.60 (d, 2H, J = 9.0 Hz, ArH), 7.61 (d, 2H, J = 8.5 Hz, ArH), 8.08 (d, 2H, J = 8.3 Hz, ArH), 8.43 (d, 1H, J = 4.8 Hz, pyrimidine), 9.40 (s, 1H, NH), 9.94 (s, 1H, NH). 13 C-NMR (CDCl3 , 100 MHz, δ ppm): 21.0 (C, CH3 ), 21.8 (C, CH3 ), 55.7 (C, OCH3 ), 87.7 (C, C3 -pyrazolopyrimidine), 106.9 (C, C6 -pyrazolopyrimidine), 114.4, 119.1, 120.1, 127.6, 129.4, 129.5, 129.6, 133.2 (14C, Ar), 134.1 (C, C3a -pyrazolopyrimidine), 136.2, 142.3, 146.5 (3C, Ar), 147.7 (C, C7 -pyrazolopyrimidine), 149.5 (C, Ar), 154.4 (C, C2 -pyrazolopyrimidine), 157.7 (C, C5 -pyrazolopyrimidine), 163.2 (C=O). MS (m/z, %): 463 (M+ , 100). Anal. Calcd. (%) for C28 H25 N5 O2 (463.53): C, 72.55; H, 5.44; N, 15.11. Found: C, 72.55; H, 5.44; N, 15.11%. 7-(4-Methoxyphenyl)-2-(4-methoxyphenylamino)-N-(4-methylphenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (18j). Yellow crystals, m.p. 244–245 ◦ C, yield (75%). IR (KBr) νmax /cm−1 3368 (NH), 1649 (C=O). 1 H-NMR (CDCl , 400 MHz, δ ppm): 2.35 (s, 3H, CH ), 3.80 (s, 3H, OCH ), 3.94 (s, 3H, OCH ), 6.88 3 3 3 3 (d, 2H, J = 9.0 Hz, ArH), 6.93 (d, 1H, J = 4.8 Hz, pyrimidine), 7.10 (d, 2H, J = 9.0 Hz, ArH), 7.18 (d, 2H, J = 8.2 Hz, ArH), 7.61–7.64 (m, 4H, ArH), 8.22 (d, 2H, J = 8.9 Hz, ArH), 8.45 (d, 1H, J = 4.8 Hz, pyrimidine), 9.42 (s, 1H, NH), 9.98 (s, 1H, NH). 13 C-NMR (CDCl3 , 100 MHz, δ ppm): 21.0 (C, CH3 ), 55.7 (C, OCH3 ), 55.7 (C, OCH3 ), 87.7 (C, C3 -pyrazolopyrimidine), 106.5 (C, C6 -pyrazolopyrimidine), 114.1, 114.4, 119.1, 120.2, 122.7, 129.6, 131.5, 133.2 (14C, Ar), 134.2 (C, C3a -pyrazolopyrimidine), 136.2, 146.2 (2C, Ar), 147.9 (C, C7 -pyrazolopyrimidine), 149.5 (C, Ar), 154.4 (C, C2 -pyrazolopyrimidine), 157.8 (C, C5 -pyrazolopyrimidine), 162.3 (C, Ar), 163.3 (C=O). MS (m/z, %): 479 (M+ , 92.77). Anal. Calcd. (%) for C28 H25 N5 O3 (479.53): C, 70.13; H, 5.25; N, 14.60. Found: C, 70.05; H, 5.30; N, 14.55%. 7-(4-Chlorophenyl)-2-(4-methoxyphenylamino)-N-(4-methylphenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (18k). Yellow crystals, m.p. 267–269 ◦ C, yield (71%). IR (KBr) νmax /cm−1 3315 (NH), 1662 (C=O). 1 H-NMR (CDCl , 400 MHz, δ ppm): 2.35 (s, 3H, CH ), 3.81 (s, 3H, OCH ), 6.88 (d, 2H, J = 9.0 Hz, 3 3 3 ArH), 6.93 (d, 1H, J = 4.7 Hz, pyrimidine), 7.19 (d, 2H, J = 8.2 Hz, ArH), 7.57–7.62 (m, 6H, ArH), 8.14 (d, 2H, J = 8.7 Hz, ArH), 8.50 (d, 1H, J = 4.7 Hz, pyrimidine), 9.43 (s, 1H, NH), 9.91 (s, 1H, NH). 13 C-NMR (CDCl , 100 MHz, δ ppm): 21.0 (C, CH ), 55.7 (C, OCH ), 88.0 (C, C -pyrazolopyrimidine), 3 3 3 3 107.0 (C, C6 -pyrazolopyrimidine), 114.4, 119.2, 120.2, 129.1, 129.6, 130.9, 131.7, 133.4 (14C, Ar), 134.1 (C, C3a -pyrazolopyrimidine), 134.4, 136.0, 137.9 (3C, Ar), 146.1 (C, C7 -pyrazolopyrimidine), 149.7 (C, Ar), 154.6 (C, C2 -pyrazolopyrimidine), 159.4 (C, C5 -pyrazolopyrimidine), 163.1 (C=O). MS (m/z, %): 483 (M+ , 87.08). Anal. Calcd. (%) for C27 H22 ClN5 O2 (483.95): C, 67.01; H, 4.58; N, 14.47. Found: C, 67.10; H, 4.50; N, 14.50%. 7-(4-Bromophenyl)-2-(4-methoxyphenylamino)-N-(4-methylphenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (18l). Yellow crystals, m.p. 278–279 ◦ C, yield (68%). IR (KBr) νmax /cm−1 3325 (NH), 1649 (C=O).

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1 H-NMR

(DMSO-d6 , 400 MHz, δ ppm): 2.30 (s, 3H, CH3 ), 3.74 (s, 3H, OCH3 ), 6.95 (d, 2H, J = 9.3 Hz, ArH), 7.20 (d, 2H, J = 8.6 Hz, ArH), 7.44 (d, 1H, J = 4.1 Hz, pyrimidine), 7.62 (d, 2H, J = 8.4 Hz, ArH), 7.63 (d, 2H, J = 7.8 Hz, ArH), 7.93 (d, 2H, J = 8.3 Hz, ArH), 8.22 (d, 2H, J = 8.3 Hz, ArH), 8.77 (d, 1H, J = 4.3 Hz, pyrimidine), 9.31 (s, 1H, NH), 9.98 (s, 1H, NH). 13 C-NMR (DMSO-d6 , 100 MHz, δ ppm): 21.0 (C, CH3 ), 55.7 (C, OCH3 ), 88.0 (C, C3 -pyrazolopyrimidine), 107.0 (C, C6 -pyrazolopyrimidine), 114.9, 119.2, 120.5, 125.2, 129.7, 129.4, 131.0, 133.5 (14C, Ar), 134.2 (C, C3a -pyrazolopyrimidine), 134.4, 135.9, 137.9 (3C, Ar), 146.0 (C, C7 -pyrazolopyrimidine), 149.7 (C, Ar), 154.5 (C, C2 -pyrazolopyrimidine), 159.3 (C, C5 -pyrazolopyrimidine), 163.1 (C=O). MS (m/z, %): 528 (M+ , 26.25). Anal. Calcd. (%) for C27 H22 BrN5 O2 (528.40): C, 61.37; H, 4.20; N, 13.25. Found: C, 61.45; H, 4.16; N, 13.30%. 7-(4-Fluorophenyl)-2-(4-methoxyphenylamino)-N-(4-methylphenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (18m). Yellow crystals, m.p. 255–257 ◦ C, yield (69%). IR (KBr) νmax /cm−1 3329 (NH), 1652 (C=O). 1 H-NMR (DMSO-d , 400 MHz, δ ppm): 2.30 (s, 3H, CH ), 3.73 (s, 3H, OCH ), 6.93 (d, 2H, J = 9.0 Hz, 6 3 3 ArH), 7.19 (d, 2H, J = 8.3 Hz, ArH), 7.39 (d, 1H, J = 4.8 Hz, pyrimidine), 7.54 (d, 2H, J = 8.9 Hz, ArH), 7.60 (d, 2H, J = 9.0 Hz, ArH), 7.62 (d, 2H, J = 8.4 Hz, ArH), 8.32 (d, 2H, J = 9.0 Hz, ArH), 8.74 (d, 1H, J = 4.8 Hz, pyrimidine), 9.27 (s, 1H, NH), 9.97 (s, 1H, NH). 13 C-NMR (DMSO-d6 , 100 MHz, δ ppm): 21.0 (C, CH3 ), 55.8 (C, OCH3 ), 87.9 (C, C3 -pyrazolopyrimidine), 107.0 (C, C6 -pyrazolopyrimidine), 114.6, 116.1, 119.5, 120.5, 129.5, 130.9, 131.7 (13C, Ar), 134.2 (C, C3a -pyrazolopyrimidine), 134.2, 136.1, 138.0 (3C, Ar), 146.2 (C, C7 -pyrazolopyrimidine), 149.6 (C, Ar), 154.3 (C, C2 -pyrazolopyrimidine), 159.0 (C, C5 -pyrazolopyrimidine), 160.1 (C, Ar), 162.9 (C=O). MS (m/z, %): 467 (M+ , 45.13). Anal. Calcd. (%) for C27 H22 FN5 O2 (467.49): C, 69.37; H, 4.74; N, 14.98. Found: C, 69.30; H, 4.80; N, 15.05%. 2-(4-Methoxyphenylamino)-7-(thiophen-2-yl)-N-(4-methylphenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (18n). Yellow crystals, m.p. 278–279 ◦ C, yield (70%). IR (KBr) νmax /cm−1 3345 (NH), 1652 (C=O). 1 H-NMR (DMSO-d6 , 400 MHz, δ ppm): 2.30 (s, 3H, CH3 ), 3.78 (s, 3H, OCH3 ), 7.03 (d, 2H, J = 8.9 Hz, ArH), 7.20 (d, 2H, J = 8.1 Hz, ArH), 7.47 (t, 1H, thiophene), 7.62 (d, 2H, J = 8.0 Hz, ArH), 7.83 (d, 2H, J = 8.6 Hz, ArH), 7.90 (d, 1H, J = 3.6 Hz, pyrimidine), 8.28 (d, 1H, J = 4.7 Hz, thiophene), 8.59 (d, 1H, J = 2.3 Hz, thiophene), 8.70 (d, 1H, J = 4.8 Hz, pyrimidine), 9.46 (s, 1H, NH), 10.00 (s, 1H, NH). 13 C-NMR (DMSO-d6 , 100 MHz, δ ppm): 21.0 (C, CH3 ), 55.7 (C, OCH3 ), 87.5 (C, C3 -pyrazolopyrimidine), 107.1 (C, C6 -pyrazolopyrimidine), 114.1, 119.5, 120.3 (6C, Ar), 127.6, 128.1, 129.8 (3C, thiophene), 130.0 (2C, Ar), 133.6 (C, C3a -pyrazolopyrimidine), 133.5, 134.5, 137.1 (3C, Ar), 139.8 (C, thiophene), 147.8 (C, Ar), 151.5 (C, C2 -pyrazolopyrimidine), 154.1 (C, C5 -pyrazolopyrimidine), 157.2 (C, C7 -pyrazolopyrimidine), 163.0 (C=O). MS (m/z, %): 455 (M+ , 65.71). Anal. Calcd. (%) for C25 H21 N5 O2 S (455.53): C, 65.92; H, 4.65; N, 15.37. Found: C, 66.00; H, 4.60; N, 15.31%. N-(4-Chlorophenyl)-2-(4-methoxyphenylamino)-7-phenylpyrazolo[1,5-a]pyrimidine-3-carboxamide (18o). Yellow crystals, m.p. 252–254 ◦ C, yield (73%). IR (KBr) νmax /cm−1 3336 (NH), 1650 (C=O). 1 H-NMR (DMSO-d6 , 400 MHz, δ ppm): 3.72 (s, 3H, OCH3 ), 6.90 (d, 2H, J = 9.0 Hz, ArH), 7.40 (d, 1H, J = 4.8 Hz, pyrimidine), 7.44 (d, 2H, J = 8.8 Hz, ArH), 7.61 (d, 2H, J = 9.0 Hz, ArH), 7.68–7.70 (m, 3H, ArH), 7.78 (d, 2H, J = 8.9 Hz, ArH), 8.23 (d, 2H, J = 7.2 Hz, ArH), 8.75 (d, 1H, J = 4.8 Hz, pyrimidine), 9.20 (s, 1H, NH), 10.11 (s, 1H, NH). 13 C-NMR (DMSO-d6 , 100 MHz, δ ppm): 55.7 (C, OCH3 ), 87.9 (C, C3 -pyrazolopyrimidine), 107.0 (C, C6 -pyrazolopyrimidine), 114.5, 119.1, 120.2, 124.0, 128.4, 129.1, 130.9, 131.8 (13C, Ar), 134.0 (C, C3a -pyrazolopyrimidine), 134.6, 135.9, 138.0, 138.7 (4C, Ar), 145.6 (C, C7 -pyrazolopyrimidine), 149.7 (C, Ar), 154.8 (C, C2 -pyrazolopyrimidine), 158.0 (C, C5 -pyrazolopyrimidine), 163.8 (C=O). MS (m/z, %): 469 (M+ , 29.83). Anal. Calcd. (%) for C26 H20 ClN5 O2 (469.92): C, 66.45; H, 4.29; N, 14.90. Found: C, 66.40; H, 4.35; N, 14.85%. N-(4-Chlorophenyl)-2-(4-methoxyphenylamino)-7-(4-methylphenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (18p). Yellow crystals, m.p. 261 ◦ C, yield (75%). IR (KBr) νmax /cm−1 3322 (NH), 1658 (C=O). 1 H-NMR (DMSO-d6 , 400 MHz, δ ppm): 2.32 (s, 3H, CH3 ), 3.74 (s, 3H, OCH3 ), 6.93 (d, 2H, J = 7.6 Hz, ArH), 7.42 (d, 1H, J = 4.8 Hz, pyrimidine), 7.45 (d, 2H, J = 7.7 Hz, ArH), 7.51 (d, 2H, J = 8.6 Hz, ArH), 7.64 (d, 2H, J = 7.8 Hz, ArH), 7.79 (d, 2H, J = 8.4 Hz, ArH), 8.19 (d, 2H, J = 7.5 Hz, ArH), 8.74 (d, 1H,

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J = 4.8 Hz, pyrimidine), 9.23 (s, 1H, NH), 10.14 (s, 1H, NH). 13 C-NMR (DMSO-d6 , 100 MHz, δ ppm): 21.0 (C, CH3 ), 55.7 (C, OCH3 ), 87.5 (C, C3 -pyrazolopyrimidine), 107.0 (C, C6 -pyrazolopyrimidine), 114.4, 119.3, 120.9, 129.0, 129.6, 131.0, 131.7, 133.4 (14C, Ar), 134.0 (C, C3a -pyrazolopyrimidine), 134.3, 136.0, 137.9 (3C, Ar), 146.1 (C, C7 -pyrazolopyrimidine), 149.7 (C, Ar), 154.5 (C, C2 -pyrazolopyrimidine), 159.4 (C, C5 -pyrazolopyrimidine), 163.1 (C=O). MS (m/z, %): 483 (M+ , 22.71). Anal. Calcd. (%) for C27 H22 ClN5 O2 (483.95): C, 67.01; H, 4.58; N, 14.47. Found: C, 67.10; H, 4.50; N, 14.55%. N-(4-Chlorophenyl)-7-(4-methoxyphenyl)-2-(4-methoxyphenylamino)pyrazolo[1,5-a]pyrimidine-3-carboxamide (18q). Yellow crystals, m.p. 266–267 ◦ C, yield (74%). IR (KBr) νmax /cm−1 3365 (NH), 1661 (C=O). 1 H-NMR (DMSO-d , 400 MHz, δ ppm): 3.74 (s, 3H, OCH ), 3.92 (s, 3H, OCH ), 6.95 (d, 2H, J = 8.8 Hz, 6 3 3 ArH), 7.24 (d, 2H, J = 8.7 Hz, ArH), 7.40 (d, 1H, J = 4.7 Hz, pyrimidine), 7.44 (d, 2H, J = 8.7 Hz, ArH), 7.65 (d, 2H, J = 8.8 Hz, ArH), 7.78 (d, 2H, J = 8.6 Hz, ArH), 8.31 (d, 2H, J = 8.6 Hz, ArH), 8.70 (d, 1H, J = 4.7 Hz, pyrimidine), 9.22 (s, 1H, NH), 10.15 (s, 1H, NH). 13 C-NMR (DMSO-d6 , 100 MHz, δ ppm): 55.6 (C, OCH3 ), 55.7 (C, OCH3 ), 87.8 (C, C3 -pyrazolopyrimidine), 106.4 (C, C6 -pyrazolopyrimidine), 114.2, 114.6, 119.0, 120.5, 122.8, 129.5, 131.6 (13C, Ar), 133.1 (C, C3a -pyrazolopyrimidine), 134.8, 135.0, 136.1 (3C, Ar), 147.9 (C, C7 -pyrazolopyrimidine), 149.4 (C, Ar), 154.5 (C, C2 -pyrazolopyrimidine), 157.8 (C, C5 -pyrazolopyrimidine), 161.5 (C, Ar), 163.2 (C=O). MS (m/z, %): 499 (M+ , 18.46). Anal. Calcd. (%) for C27 H22 ClN5 O3 (499.95): C, 64.86; H, 4.44; N, 14.01. Found: C, 64.95; H, 4.40; N, 14.05%. N,7-bis(4-Chlorophenyl)-2-(4-methoxyphenylamino)pyrazolo[1,5-a]pyrimidine-3-carboxamide (18r). Yellow crystals, m.p. 282–284 ◦ C, yield (70%). IR (KBr) νmax /cm−1 3317 (NH), 1653 (C=O). 1 H-NMR (DMSO-d6 , 400 MHz, δ ppm): 3.74 (s, 3H, OCH3 ), 6.94 (d, 2H, J = 8.8 Hz, ArH), 7.44 (d, 2H, J = 8.6 Hz, ArH), 7.45 (d, 1H, J = 3.8 Hz, pyrimidine), 7.61 (d, 2H, J = 8.8 Hz, ArH), 7.78 (d, 4H, J = 8.4 Hz, ArH), 8.29 (d, 2H, J = 8.6 Hz, ArH), 8.76 (d, 1H, J = 4.7 Hz, pyrimidine), 9.23 (s, 1H, NH), 10.10 (s, 1H, NH). 13 C-NMR (DMSO-d6 , 100 MHz, δ ppm): 55.8 (C, OCH3 ), 87.4 (C, C3 -pyrazolopyrimidine), 106.9 (C, C6 -pyrazolopyrimidine), 115.0, 119.3, 120.4, 129.1, 129.7, 129.9, 131.6 (13C, Ar), 133.1 (C, C3a -pyrazolopyrimidine), 133.8, 134.3, 136.0, 137.9 (4C, Ar), 146.0 (C, C7 -pyrazolopyrimidine), 149.8 (C, Ar), 154.5 (C, C2 -pyrazolopyrimidine), 159.4 (C, C5 -pyrazolopyrimidine), 162.9 (C=O). MS (m/z, %): 504 (M+ , 22.87). Anal. Calcd. (%) for C26 H19 Cl2 N5 O2 (504.37): C, 61.91; H, 3.80; N, 13.89. Found: C, 62.00; H, 3.75; N, 13.80%. 7-(4-Bromophenyl)-N-(4-chlorophenyl)-2-(4-methoxyphenylamino)pyrazolo[1,5-a]pyrimidine-3-carboxamide (18s). Yellow crystals, m.p. 275–277 ◦ C, yield (67%). IR (KBr) νmax /cm−1 3327 (NH), 1648 (C=O). 1 H-NMR (DMSO-d , 400 MHz, δ ppm): 3.74 (s, 3H, OCH ), 6.94 (d, 2H, J = 7.3 Hz, ArH), 7.44 6 3 (d, 2H, J = 7.2 Hz, ArH), 7.45 (d, 1H, J = 4.4 Hz, pyrimidine), 7.61 (d, 2H, J = 7.1 Hz, ArH), 7.79 (d, 2H, J = 7.9 Hz, ArH), 7.92 (d, 2H, J = 7.3 Hz, ArH), 8.21 (d, 2H, J = 7.8 Hz, ArH), 8.76 (d, 1H, J = 4.3 Hz, pyrimidine), 9.23 (s, 1H, NH), 10.11 (s, 1H, NH). 13 C-NMR (DMSO-d6 , 100 MHz, δ ppm): 55.7 (C, OCH3 ), 87.6 (C, C3 -pyrazolopyrimidine), 106.9 (C, C6 -pyrazolopyrimidine), 114.4, 119.2, 120.2, 123.2, 129.1, 129.6, 130.7, 131.4 (14C, Ar), 132.7 (C, C3a -pyrazolopyrimidine), 134.2, 136.0, 137.5 (3C, Ar), 146.0 (C, C7 -pyrazolopyrimidine), 149.8 (C, Ar), 154.5 (C, C2 -pyrazolopyrimidine), 159.5 (C, C5 -pyrazolopyrimidine), 163.2 (C=O). MS (m/z, %): 548 (M+ , 20.55). Anal. Calcd. (%) for C26 H19 BrClN5 O2 (548.82): C, 56.90; H, 3.49; N, 12.76. Found: C, 57.00; H, 3.40; N, 12.80%. N-(4-Chlorophenyl)-7-(4-fluorophenyl)-2-(4-methoxyphenylamino)pyrazolo[1,5-a]pyrimidine-3-carboxamide (18t). Yellow crystals, m.p. 251–252 ◦ C, yield (67%). IR (KBr) νmax /cm−1 3339 (NH), 1651 (C=O). 1 H-NMR (DMSO-d , 400 MHz, δ ppm): 3.74 (s, 3H, OCH ), 6.93 (d, 2H, J = 7.8 Hz, ArH), 7.44 6 3 (d, 2H, J = 8.7 Hz, ArH), 7.45 (d, 1H, J = 4.2 Hz, pyrimidine), 7.56 (d, 2H, J = 8.0 Hz, ArH), 7.62 (d, 2H, J = 8.0 Hz, ArH), 7.78 (d, 2H, J = 7.4 Hz, ArH), 8.33 (d, 2H, J = 8.1 Hz, ArH), 8.75 (d, 1H, J = 4.5 Hz, pyrimidine), 9.21 (s, 1H, NH), 10.10 (s, 1H, NH). 13 C-NMR (DMSO-d6 , 100 MHz, δ ppm): 55.8 (C, OCH3 ), 86.7 (C, C3 -pyrazolopyrimidine), 108.3 (C, C6 -pyrazolopyrimidine), 114.3, 115.9, 119.0, 119.4, 124.6, 128.1, 129.1 (13C, Ar), 132.3 (C, C3a -pyrazolopyrimidine), 133.2, 134.3, 138.4 (3C, Ar), 145.0 (C, C7 -pyrazolopyrimidine), 147.3 (C, Ar), 151.1 (C, C2 -pyrazolopyrimidine), 154.0

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(C, C5 -pyrazolopyrimidine), 156.6 (C, Ar), 162.3 (C=O). MS (m/z, %): 487 (M+ , 21.30). Anal. Calcd. (%) for C26 H19 ClFN5 O2 (487.91): C, 64.00; H, 3.93; N, 14.35. Found: C, 64.10; H, 4.00; N, 14.30%. N-(4-Chlorophenyl)-2-(4-methoxyphenylamino)-7-(thiophen-2-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (18u). Yellow crystals, m.p. 289–291 ◦ C, yield (71%). IR (KBr) νmax /cm−1 3293 (NH), 1644 (C=O). 1 H-NMR (DMSO-d , 400 MHz, δ ppm): 3.78 (s, 3H, OCH ), 7.04 (d, 2H, J = 9.0 Hz, ArH), 7.33 (d, 2H, 6 3 J = 9.2 Hz, ArH), 7.47 (t, 1H, thiophene), 7.79 (d, 2H, J = 9.1 Hz, ArH), 7.84 (d, 2H, J = 8.9 Hz, ArH), 7.91 (d, 1H, J = 4.8 Hz, pyrimidine), 8.29 (d, 1H, J = 5.1 Hz, thiophene), 8.60 (d, 1H, J = 2.9 Hz, thiophene), 8.71 (d, 1H, J = 5.7 Hz, pyrimidine), 9.39 (s, 1H, NH), 10.14 (s, 1H, NH). 13 C-NMR (DMSO-d6 , 100 MHz, δ ppm): 55.7 (C, OCH3 ), 87.8 (C, C3 -pyrazolopyrimidine), 107.1 (C, C6 -pyrazolopyrimidine), 114.0, 119.5, 120.4, (6C, Ar), 127.6, 128.2, 129.7 (3C, thiophene), 130.0 (2C, Ar), 133.1 (C, C3a -pyrazolopyrimidine), 133.6, 134.5, 136.9 (3C, Ar), 139.7 (C, thiophene), 148.0 (C, Ar), 151.4 (C, C2 -pyrazolopyrimidine), 154.2 (C, C5 -pyrazolopyrimidine), 157.2 (C, C7 -pyrazolopyrimidine), 162.9 (C=O). MS (m/z, %): 475 (M+ , 74.59). Anal. Calcd. (%) for C24 H18 ClN5 O2 S (475.95): C, 60.56; H, 3.81; N, 14.71. Found: C, 60.50; H, 3.90; N, 14.80%. General Procedure for Synthesis of N-aryl-2-(arylamino)-8,8-dimethyl-6-oxo-6,7,8,9-tetrahydropyrazolo[1,5-a] quinazoline-3-carboxamides 25a–c. A mixture of compounds 11a–c (0.01 mol) with 2-((dimethyl-amino) methylene)-5,5-dimethylcyclohexane-1,3-dione (19, 0.01 mol, 1.95 g) in glacial acetic acid (25 mL), the reaction mixture was refluxed for 1 h and then left to cool. The solid product was filtered off, washed with ethanol, dried and finally recrystallized from DMF/H2 O to afford the corresponding pyrazolo[1,5-a]quinazolines 25a–c. 2-(4-Methoxyphenylamino)-8,8-dimethyl-6-oxo-N-phenyl-6,7,8,9-tetrahydropyrazolo[1,5-a]quinazoline-3carboxamide (25a). Yellow crystals, m.p. 270–272 ◦ C, yield (73%). IR (KBr) νmax /cm−1 3302 (NH), 1655 (C=O). 1 H-NMR (CDCl3 , 400 MHz, δ ppm): 1.24 (s, 6H, 2CH3 ), 2.58 (s, 2H, CH2 ), 3.31 (s, 2H, CH2 ), 3.82 (s, 3H, OCH3 ), 6.95 (d, 2H, J = 9.0 Hz, ArH), 7.14 (t, 1H, ArH), 7.39 (t, 2H, ArH), 7.64 (d, 2H, J = 9.0 Hz, ArH), 7.71 (d, 2H, J = 7.5 Hz, ArH), 8.99 (s, 1H, quinazoline), 9.48 (s, 1H, NH), 9.88 (s, 1H, NH). 13 C-NMR (CDCl3 , 100 MHz, δ ppm): 28.7 (2C, 2CH3 ), 32.7 (C, C8 -quinazoline), 37.7 (C, CH2 ), 51.1 (C, CH2 ), 55.7 (C, OCH3 ), 90.9 (C, C3 -quinazoline), 113.8 (C, C5a -quinazoline), 114.5, 119.6, 120.3, 124.2, 129.2 (9C, Ar), 133.4 (C, C3a -quinazoline), 138.2, 147.1, 148.6 (3C, Ar), 151.5 (C, C2 -quinazoline), 155.0 (C, C5 -quinazoline), 159.1 (C=O), 162.6 (C, C9a -quinazoline), 193.9 (C=O). MS (m/z, %): 455 (M+ , 71.06). Anal. Calcd. (%) for C26 H25 N5 O3 (455.51): C, 68.56; H, 5.53; N, 15.37. Found: C, 68.50; H, 5.55; N, 15.40%. 2-(4-Methoxyphenylamino)-8,8-dimethyl-6-oxo-N-(4-methylphenyl)-6,7,8,9-tetrahydropyrazolo[1,5-a]quinazoline3-carboxamide (25b). Yellow crystals, m.p. 266–268 ◦ C, yield (77%). IR (KBr) νmax /cm−1 3316 (NH), 1659 (C=O). 1 H-NMR (CDCl3 , 400 MHz, δ ppm): 1.19 (s, 6H, 2CH3 ), 2.34 (s, 3H, CH3 ), 2.52 (s, 2H, CH2 ), 3.22 (s, 2H, CH2 ), 3.81 (s, 3H, OCH3 ), 6.91 (d, 2H, J = 9.0 Hz, ArH), 7.16 (d, 2H, J = 8.2 Hz, ArH), 7.54 (d, 2H, J = 8.3 Hz, ArH), 7.58 (d, 2H, J = 9.0 Hz, ArH), 8.90 (s, 1H, quinazoline), 9.41 (s, 1H, NH), 9.72 (s, 1H, NH). 13 C-NMR (CDCl3 , 100 MHz, δ ppm): 21.0 (C, CH3 ), 28.7 (2C, 2CH3 ), 32.6 (C, C8 -quinazoline), 37.5 (C, CH2 ), 50.9 (C, CH2 ), 55.6 (C, OCH3 ), 90.7 (C, C3 -quinazoline), 113.7 (C, C5a -quinazoline), 114.4, 119.4, 120.1, 129.6 (8C, Ar), 133.4 (C, C3a -quinazoline), 133.7, 135.7, 146.8, 148.5 (4C, Ar), 151.5 (C, C2 -quinazoline), 154.8 (C, C5 -quinazoline), 158.8 (C=O), 162.3 (C, C9a -quinazoline), 194.0 (C=O). MS (m/z, %): 469 (M+ , 93.88). Anal. Calcd. (%) for C27 H27 N5 O3 (469.53): C, 69.07; H, 5.80; N, 14.92. Found: C, 69.15; H, 5.75; N, 15.00%. N-(4-Chlorophenyl)-2-(4-methoxyphenylamino)-8,8-dimethyl-6-oxo-6,7,8,9-tetrahydropyrazolo[1,5-a]quinazoline3-carboxamide (25c). Yellow crystals, m.p. 291–293 ◦ C, yield (72%). IR (KBr) νmax /cm−1 3299 (NH), 1662 (C=O). 1 H-NMR (DMSO-d6 , 400 MHz, δ ppm): 1.16 (s, 6H, 2CH3 ), 2.59 (s, 2H, CH2 ), 3.36 (s, 2H, CH2 ), 3.76 (s, 3H, OCH3 ), 6.98 (d, 2H, J = 8.8 Hz, ArH), 7.45 (d, 2H, J = 8.6 Hz, ArH), 7.72 (d, 2H, J = 8.6 Hz, ArH), 7.76 (d, 2H, J = 8.8 Hz, ArH), 8.95 (s, 1H, quinazoline), 9.30 (s, 1H, NH), 10.01 (s, 1H, NH). 13 C-NMR (DMSO-d6 , 100 MHz, δ ppm): 28.7 (2C, 2CH3 ), 32.6 (C, C8 -quinazoline), 37.6 (C, CH2 ), 50.0 (C, CH2 ), 55.7

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(C, OCH3 ), 90.8 (C, C3 -quinazoline), 113.7 (C, C5a -quinazoline), 114.4, 119.3, 120.1, 129.3 (8C, Ar), 133.4 (C, C3a -quinazoline), 133.8, 136.3, 146.9, 148.1 (4C, Ar), 151.5 (C, C2 -quinazoline), 154.8 (C, C5 -quinazoline), 158.9 (C=O), 162.4 (C, C9a -quinazoline), 193.9 (C=O). MS (m/z, %): 489 (M+ , 63.07). Anal. Calcd. (%) for C26 H24 ClN5 O3 (489.95): C, 63.74; H, 4.94; N, 14.29. Found: C, 63.80; H, 5.00; N, 14.20%. 3.2. Biological Evaluation 3.2.1. In-Vitro Anticancer Activity Cell culture of HepG-2 (human liver carcinoma) and MCF-7 (human breast adenocarcinoma) cell lines were purchased from the American Type Culture Collection (Rockville, MD, USA) and maintained in DMEM medium which was supplemented with 10% heat-inactivated FBS (fetal bovine serum), 100 U/mL penicillin and 100 U/mL streptomycin. The cells were grown at 37 ◦ C in a humidified atmosphere of 5% CO2 . 3.2.2. MTT Cytotoxicity Assay The antitumor activity against HepG-2 and MCF-7 human cancer cell lines was estimated using the 3-[4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, which is based on the cleavage of the tetrazolium salt by mitochondrial dehydrogenases in viable cells [31–33]. Cells were dispensed in a 96 well sterile microplate (5 × 104 cells/well), and incubated at 37 ◦ C with series of different concentrations, in DMSO, of each tested compound or Doxorubicin® (positive control) for 48 h in a serum free medium prior to the MTT assay. After incubation, media were carefully removed, 40 µL of MTT (2.5 mg/mL) were added to each well and then incubated for an additional 4 h. The purple formazan dye crystals were solubilized by the addition of 200 µL of DMSO. The absorbance was measured at 590 nm using a SpectraMax® , Paradigm® Multi-Mode microplate reader. The relative cell viability was expressed as the mean percentage of viable cells compared to the untreated control cells. 3.2.3. Statistical Analysis All experiments were conducted in triplicate and repeated on three different days. All the values were represented as mean ± SD. IC50 s were determined by probit analysis using the SPSS software program (SPSS Inc., Chicago, IL, USA). 3.2.4. Cell Cycle Analysis and Apoptosis Detection Cell cycle analysis and apoptosis detection were carried out by flow cytometry [35]. Both HepG-2 and MCF-7 cells were seeded at 8 × 104 and incubated at 37 ◦ C, 5% CO2 overnight, after treatment with the tested compounds, for 24 h. Cell pellets were collected and centrifuged (300 g, 5 min). For cell cycle analysis, cell pellets were fixed with 70% ethanol on ice for 15 min and collected again. The collected pellets were incubated with propidium iodide (PI) staining solution (50 µg/mL PI, 0.1 mg/mL RNaseA, 0.05% Triton X-100) at room temperature for 1 h and analyzed by Gallios flow cytometer (Beckman Coulter, Brea, CA, USA). Apoptosis detection was performed by FITC Annexin-V/PI commercial kit (Becton Dickenson, Franklin Lakes, NJ, USA) following the manufacture protocol. The samples were analyzed by fluorescence-activated cell sorting (FACS) with a Gallios flow cytometer (Beckman Coulter) within 1 h after staining. Data were analyzed using Kaluza v. 1.2 (Beckman Coulter). 4. Conclusions A series of N-aryl-7-aryl-pyrazolo[1,5-a]pyrimidines 18a–u and N-aryl-pyrazolo[1,5-a] quinazolines 25a–c have been synthesized and investigated for their in vitroantitumor activity. All the investigated compounds showed dose-dependent cytotoxic activities against two cancer types (liver and breast cancer). The IC50 values of these compounds did not reveal statistical significant differences compared to the positive control (doxorubicin). Therefore, two compounds (18o and 18a) have been selected to study their cell cycle and apoptotic effect against HepG2 and

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MCF-7 cancer cell lines. Compounds 18o and 18ashowed slightly higher cytotoxicity compared to doxorubicin against HepG-2 cells (IC50 = 72.2 ± 3.8 vs. 80.9 ± 2.1 µM) and against MCF-7 cells (IC50 = 63.1 ± 3.1 vs. 65.6 ± 4.2µM), respectively. Cell cycle analysis of HepG-2 cells treated with 18o and MCF-7 cells treated with 18a revealed a significant G2/M phase arrest coupled with an increase in the percentage of cells in pre-G phase, which is indicative of apoptosis. The pro-apoptotic activity of 18a and 18o was inferred by the significant increase in the percentage of annexin V-FITC-positive apoptotic cells. Supplementary Materials: Spectra of compounds are available online. Author Contributions: A.S.H. formulated the research idea; M.E.-N., A.S.H. and M.F.M. carried out the experimental, interpreted the data and prepared the manuscript; H.M.A. performed the biological screening. All authors have read and approved the final manuscript. Conflicts of Interest: The authors declare no conflicts of interest.

References 1.

2.

3.

4. 5.

6.

7.

8.

9.

10. 11. 12.

13. 14.

Abd El Razik, H.A.; Abdel Wahab, A.E. Synthesis and Biological Evaluation of Some Novel Fused Pyrazolopyrimidines as Potential Anticancer and Antimicrobial Agents. Arch. Pharm. Chem. Life Sci. 2011, 11, 184–196. [CrossRef] [PubMed] Aggarwal, R.; Masan, E.; Kaushik, P.; Kaushik, D.; Sharma, C.; Aneja, K.R. Synthesis and biological evaluation of 7-trifluoromethylpyrazolo[1,5-a]pyrimidines as anti-inflammatory and antimicrobial agents. J. Fluor. Chem. 2014, 168, 16–24. [CrossRef] Kaping, S.; Kalita, U.; Sunn, M.; Singha, L.I.; Vishwakarma, J.N. A facile, regioselective synthesis of pyrazolo[1,5-a]pyrimidine analogs in the presence of KHSO4 in aqueous media assisted by ultrasound and their anti-inflammatory and anti-cancer activities. Monatsh. Chem. 2016, 147, 1257–1276. [CrossRef] Deshmukh, S.; Dingore, K.; Gaikwad, V.; Jachak, M. An efficient synthesis of pyrazolo[1,5-a]pyrimidines and evaluation of their antimicrobial activity. J. Chem. Sci. 2016, 128, 1459–1468. [CrossRef] El-Mekabaty, A.; Habib, O.M.O.; Moawad, E.B.; Hasel, A.M. Synthesis and Antioxidant Activity of New Pyrazolo[1,5-a]Pyrimidine Derivatives Incorporating a Thiazol-2-yldiazenyl Moiety. J. Heterocycl. Chem. 2016, 53, 1820–1826. [CrossRef] Hassan, A.S.; Masoud, D.M.; Sroor, F.M.; Askar, A.A. Synthesis and biological evaluation of pyrazolo[1,5-a]pyrimidine-3-carboxamide as antimicrobial agents. Med. Chem. Res. 2017, 26, 2909–2919. [CrossRef] Kumar, A.K.A.; Bodke, Y.D.; Lakra, P.S.; Sambasivam, G.; Bhat, K.G. Design, synthesis and anti-cancer evaluation of a novel series of pyrazolo[1,5-a]pyrimidine substituted diamide derivatives. Med. Chem. Res. 2017, 26, 714–744. [CrossRef] Rahmouni, A.; Souiei, S.; Belkacem, M.A.; Romdhane, A.; Bouajila, J.; Ben Jannet, H. Synthesis and biological evaluation of novel pyrazolopyrimidines derivatives as anticancer and anti-5-lipoxygenase agents. Bioorg. Chem. 2016, 66, 160–168. [CrossRef] [PubMed] Zhao, M.; Ren, H.; Chang, J.; Zhang, D.; Yang, Y.; He, Y.; Qi, C.; Zhang, H. Design andSynthesis of pyrazolo[1,5-a]pyrimidine derivativesbearing nitrogen mustard moiety and evaluation of their antitumor activity in vitro and in vivo. Eur. J. Med. Chem. 2016, 119, 183–196. [CrossRef] [PubMed] Li, J.; Zhao, Y.F.; Zhao, X.L.; Yuan, X.Y.; Gong, P. Synthesis and Anti-tumor Activities of Novel Pyrazolo[1,5-a]pyrimidines. Arch. Pharm. Chem. Life Sci. 2006, 339, 593–597. [CrossRef] [PubMed] Ahmed, O.M.; Mohamed, M.A.; Ahmed, R.R.; Ahmed, S.A. Synthesis and anti-tumor activities of some new pyridines and pyrazolo[1,5-a]pyrimidines. Eur. J. Med. Chem. 2009, 44, 3519–3523. [CrossRef] [PubMed] Abdel-Aziz, H.A.; Saleh, T.S.; El-Zahabi, H.S.A. Facile Synthesis and In-Vitro Antitumor Activity of Some Pyrazolo[3,4-b]pyridines and Pyrazolo[1,5-a]pyrimidines Linked to a Thiazolo[3,2-a]benzimidazoleMoiety. Arch. Pharm. Chem. Life Sci. 2010, 343, 24–30. [CrossRef] Hassan, A.S.; Mady, M.F.; Awad, H.M.; Hafez, T.S. Synthesis and antitumor activity of some new pyrazolo[1,5-a]pyrimidines. Chin. Chem. Lett. 2017, 28, 388–393. [CrossRef] Hassan, A.S.; Hafez, T.S.; Osman, S.A.M.; Ali, M.M. Synthesis and In Vitro cytotoxic activity of novel pyrazolo[1,5-a]pyrimidines and related Schiff bases. Turk. J. Chem. 2015, 39, 1102–1113. [CrossRef]

Molecules 2018, 23, 1249

15.

16.

17.

18.

19.

20. 21. 22.

23.

24.

25.

26. 27. 28. 29.

30.

31.

32.

19 of 20

Gopalsamy, A.; Ciszewski, G.; Hu, Y.; Lee, F.; Feldberg, L.; Frommer, E.; Kimb, S.; Collins, K.; Wojciechowicz, D.; Mallon, R. Identification of pyrazolo[1,5-a]pyrimidine-3-carboxylates as B-Raf kinase inhibitors. Bioorg. Med. Chem. Lett. 2009, 19, 2735–2738. [CrossRef] [PubMed] Mukaiyama, H.; Nishimura, T.; Kobayashi, S.; Komatsu, Y.; Kikuchi, S.; Ozawa, T.; Kamada, N.; Ohnota, H. Novel pyrazolo[1,5-a]pyrimidines as c-Src kinase inhibitors that reduce IKr channel blockade. Bioorg. Med. Chem. 2008, 16, 909–921. [CrossRef] [PubMed] Liu, Y.; Laufer, R.; Patel, N.K.; Ng, G.; Sampson, P.B.; Li, S.-W.; Lang, Y.; Feher, M.; Brokx, R.; Beletskaya, I.; et al. Discovery of Pyrazolo[1,5-a]pyrimidine TTK Inhibitors: CFI-402257 is a Potent, Selective, Bioavailable Anticancer Agent. Med. Chem. Lett. 2016, 7, 671–675. [CrossRef] [PubMed] Mukaiyama, H.; Nishimura, T.; Shiohara, H.; Kobayashi, S.; Komatsu, Y.; Kikuchi, S.; Tsuji, E.; Kamada, N.; Ohnota, H.; Kusama, H. Discovery of Novel 2-Anilinopyrazolo[1,5-a]pyrimidine Derivatives a c-Src Kinase Inhibitors for the Treatment of Acute Ischemic Stroke. Chem. Pharm. Bull. 2007, 55, 881–889. [CrossRef] [PubMed] Kumar, A.K.A.; Bodke, Y.D.; Sambasivam, G.; Lakra, P.S. Design, synthesis, and evaluation of novel hydrazide hydrochlorides of 6-aminopyrazolo[1,5-a]pyrimidine-3-carboxamides as potent Aurora kinase inhibitors. Monatsh. Chem. 2017, 148, 1767–1780. [CrossRef] Hassan, A.S.; Awad, H.M.; Magd-El-Din, A.A.; Hafez, T.S. Synthesis and in vitro antitumor evaluation of novel Schiff bases. Med. Chem. Res. 2018, 27, 915–927. [CrossRef] Hassan, A.S.; Hafez, T.S.; Ali, M.M.; Khatab, T.K. Design, synthesis and cytotoxic activity of some new pyrazolines bearing benzofuran and pyrazole moieties. Res. J. Pharm. Biol. Chem. Sci. 2016, 7, 417–429. Abd El-All, A.S.; Hassan, A.S.; Osman, S.A.; Yosef, H.A.A.; Abdel-Hady, W.H.; El-Hashash, M.A.; Atta-Allah, S.R.; Ali, M.M.; El Rashedy, A.A. Synthesis, characterization and biological evaluation of new fused triazine derivatives based on 6-methyl-3-thioxo-1,2,4-triazin-5-one. Acta Poloniae Pharm. Drug Res. 2016, 73, 79–92. Osman, S.A.; Mousa, H.A.; Yosef, H.A.A.; Hafez, T.S.; El-Sawy, A.A.; Abdallah, M.M.; Hassan, A.S. Synthesis, characterization and cytotoxicity of mixed ligand Mn(II), Co(II) and Ni(II) complexes. J. Serb. Chem. Soc. 2014, 79, 953–964. [CrossRef] Hafez, T.S.; Osman, S.A.; Yosef, H.A.A.; Abd El-All, A.S.; Hassan, A.S.; El-Sawy, A.A.; Abdallah, M.M.; Youns, M. Synthesis, structural elucidation and in vitro antitumor activities of some pyrazolopyrimidines and Schiff bases derived from 5-amino-3-(arylamino)-1H-pyrazole-4-carboxamides. Sci. Pharm. 2013, 81, 339–357. [CrossRef] [PubMed] Osman, S.A.; Yosef, H.A.A.; Hafez, T.S.; El-Sawy, A.A.; Mousa, H.A.; Hassan, A.S. Synthesis and antibacterial activity of some novel chalcones, pyrazoline and 3-cyanopyridine derivatives based on khellinone as well as Ni(II), Co(II) and Zn(II) complexes. Aust. J. Basic Appl. Sci. 2012, 6, 852–863. Elgemeie, G.H.; Elsayed, S.H.; Hassan, A.S. Design and synthesis of the first thiophene thioglycosides. Synth. Commun. 2009, 39, 1781–1792. [CrossRef] Elgemeie, G.H.; Elsayed, S.H.; Hassan, A.S. Direct route to a new class of acrylamide thioglycosides and their conversions to pyrazole derivatives. Synth. Commun. 2008, 38, 2700–2706. [CrossRef] Hassan, A.S.; Moustafa, G.O.; Awad, H.M. Synthesis and in vitro anticancer activity of pyrazolo[1,5-a] pyrimidines and pyrazolo[3,4-d][1,2,3]triazines. Synth. Commun. 2017, 47, 1963–1972. [CrossRef] Hassan, A.S.; Hafez, T.S.; Osman, S.A. Synthesis, characterization, and cytotoxicity of some new 5-aminopyrazole and pyrazolo[1,5-a]pyrimidine derivatives. Sci. Pharm. 2015, 83, 27–39. [CrossRef] [PubMed] Sadek, K.U.; Mekheimer, R.A.; Mohamed, T.M.; Moustafa, M.S.; Elnagdi, M.H. Regioselectivity in the multicomponent reaction of 5-aminopyrazoles, cyclic 1,3-diketones and dimethyl formamide dimethylacetal under controlled microwave heating. Beilstein J. Org. Chem. 2012, 8, 18–24. [CrossRef] [PubMed] Hamdy, N.A.; Anwar, M.M.; Abu-Zied, K.M.; Awad, H.M. Synthesis, tumor inhibitory and antioxidant activity of new polyfunctionally 2-substituted 5,6,7,8-tetrahydronaphthalene derivatives containing pyridine, thioxopyridine and pyrazolopyridine moieties. Acta Poloniae Pharm. Drug Res. 2013, 70, 987–1001. Awad, H.M.; Abd-Alla, H.I.; Mahmoud, K.H.; El-Toumy, S.A. In vitro anti-nitrosative, antioxidant, and cytotoxicity activities of plant flavonoids: A comparative study. Med. Chem. Res. 2014, 23, 3298–3307. [CrossRef]

Molecules 2018, 23, 1249

33.

34. 35.

20 of 20

Soliman, H.A.; Yousif, M.N.M.; Said, M.M.; Hassan, N.A.; Ali, M.M.; Awad, H.M.; Abdel-Megeid, F.M.E. Synthesis of novel 1,6-naphthyridines, pyrano[3,2-c]pyridines and pyrido[4,3-d]pyrimidines derived from 2,2,6,6-tetramethylpiperidin-4-one for in vitro anticancer and antioxidant evaluation. Der Pharma Chem. 2014, 6, 394–410. Massagué, J. G1 cell-cycle control and cancer. Nature 2004, 432, 298–306. [CrossRef] [PubMed] Thornton, T.M.; Rincon, M. Non-classical P38 map kinase functions: Cell cycle checkpoints and survival. Int. J. Biol. Sci. 2009, 5, 44–52. [CrossRef] [PubMed]

Sample Availability: Samples of the compounds are all available from the authors. © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).