Design and synthesis of novel pyrazolo[1,5-a]pyrimidine derivatives bearing nitrogen mustard moiety and evaluation of their antitumor activity in vitro and in vivo

Design and synthesis of novel pyrazolo[1,5-a]pyrimidine derivatives bearing nitrogen mustard moiety and evaluation of their antitumor activity in vitro and in vivo

European Journal of Medicinal Chemistry 119 (2016) 183e196 Contents lists available at ScienceDirect European Journal of Medicinal Chemistry journal...

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European Journal of Medicinal Chemistry 119 (2016) 183e196

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Research paper

Design and synthesis of novel pyrazolo[1,5-a]pyrimidine derivatives bearing nitrogen mustard moiety and evaluation of their antitumor activity in vitro and in vivo Mingxia Zhao a, Hongyu Ren a, Jin Chang a, Diqin Zhang a, Yating Yang a, Yong He a, b, Chuanmin Qi a, *, Huabei Zhang a a b

Key Laboratory of Radiopharmaceuticals, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, PR China Experimental Chemistry Center, Beijing Normal University, Beijing 100875, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 October 2014 Received in revised form 27 April 2016 Accepted 28 April 2016 Available online 30 April 2016

A series of novel pyrazolo[1,5-a]pyrimidine derivatives bearing nitrogen mustard moiety were designed, synthesized and evaluated for their antiproliferative activities against five human cancer cell lines (A549, SH-SY5Y, HepG2, MCF-7 and DU145) in vitro. Among these compounds, 13b exhibited potent inhibitory effect on the proliferation of the five tumor cells and was able to inhibit cell cycle arrest at G1 phase and induce cell apoptosis. In HepG2 HCC xenograft compound 13b was selected for evaluating the antitumor activity in vivo which exhibited significant cancer growth inhibition with low host toxicity in vivo. © 2016 Elsevier Masson SAS. All rights reserved.

Keywords: Pyrazolo[1,5-a]pyrimidine Nitrogen mustard derivatives Anti-tumor Cell cycle Apoptosis Xenograft mice model

1. Introduction It is widely known that cancer is one leading cause of death in developed countries that may be induced by a plethora of both external and internal factors [1]. Among the anticancer drugs, Nitrogen mustard bifunctional DNA-alkylating agents which were widely used in clinical could cause DNA damage by interrupting DNA biosynthesis and in doing so caused DNA damage [2]. Many of these drugs, such as Melphalan, Chlorambucil, Cyclophosphamide and Bendamustine and so on, have revolutionized the treatment of cancers. However, these derivatives have many drawbacks, including a low specificity to target the DNA of tumor cells, an eventual loss in activity due to cellular DNA repair mechanisms, a

Abbreviations: MTT, (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; A549, human alveolar adenocarcinoma cell line; SH-SY5Y, human neuroblastoma cell line; HepG2, human liver carcinoma cell line; MCF-7, human breast cancer cell line; DU145, human prostate cancer cell lines; GES-1, human gastric epithelial cell line; FBS, Fetal Bovine Serum; PBS, Phosphate Buffer solution; PI, propidium iodide; RNase A, Ribonuclease A. * Corresponding author. E-mail address: [email protected] (C. Qi). http://dx.doi.org/10.1016/j.ejmech.2016.04.068 0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

high chemical reactivity and induced bone-marrow toxicity [3e6]. To conquer these problems of nitrogen mustards, one of the effective strategies is to link the alkylating pharmacophore with DNA-affinic molecules. To this end, numerous affinity tumor heterocyclic have been designed as carriers to transport the alkylating pharmacophore into the tumor site [7e11]. Emerging evidence shows that connecting DNA-affinic molecules to alkylating agents could improve therapeutic efficacy than the corresponding untargeted alkylating agents. Cyclin-dependent protein kinases (CDKs) are serine/threonine kinases which are central to the appropriate regulation of cell proliferation, apoptosis, and gene expression [12e14]. Most human cancers have abnormalities in some component of CDK activity, hence, synthetic inhibitors of CDK activity present as a logical approach in the development of new cancer therapies. Pragmatically, most efforts have focused on using CDK2 as a template to achieve the absolute selectivity, given its ready applicability to protein structure guided drug design [15]. Pyrazolo[1,5-a]pyrimidine derivatives are widely used as inhibitors of Cyclin-dependent kinase 2 [16e19], and are also found may demonstrate antitumor effect in various cancer cell lines [20e22] and other biologically

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activities [23e27]. Herein, we choose pyrazolo[1,5-a]pyrimidine derivatives as the carrier to selectively transport the alkylating pharmacophore into the tumor site. We coupled the aniline Nmustard residue with the pyrazolo[1,5-a]pyrimidine derivatives to afford 43 new compounds. The biological results showed that compounds 4-13a and 4-13b exhibited promising antitumor activity against five human tumor cells in vitro. In addition, cell cycle analysis and cellular apoptosis experiment indicated the excellent therapeutic efficacy of compound 13b at the cell level. To evaluate the toxicity and in vivo targeting therapeutic efficacy of compound 13b, we conducted the acute toxicity test and in vivo therapeutic experiment. 2. Results and discussion 2.1. Chemistry The synthetic pathway for target compounds 4-15a, 4-15b and 18a-s was illustrated in Schemes 1 and 2. 5-chloromethyl-7-chloro3-cyanopyrazolo[1,5-a]pyrimidine (1) and N1,N1-bis(2-chloroethyl) benzene diamine (2a-b) were prepared by following the literature methods [28e30]. Treatment of compound 1 with N1,N1-bis(2-chloroethyl)benzene diamine (2a-b) in ethanol under reflux at nitrogen atmosphere for 2e3 h gave compounds 3a-b in good yields. Compounds 3a-b were then converted into target compounds 4-15a and 4-15b by nucleophilic substitution reaction using different amines. Compounds 16a-s were obtained in high yield using the method similar to compounds 3a-b. The synthesis of the key intermediates 17a-s was achieved from compounds 16a-s with diethanolamine in the presence potassium carbonate in N,N-Dimethylformamide at 40  C for 2e4 h. Chlorination of the intermediates 17a-s with thionyl chloride in dichloromethane at room temperature overnight yielded the target compounds 18a-s. 2.2. Pharmacology 2.2.1. MTT assay Using Sorafenib and Melphalan as the positive control, the cytotoxic activities of all target compounds 4-15a, 4-15b and 18a-s have been evaluated against the A549, SH-SY5Y, HepG-2, MCF-7, DU145 cell lines using the MTT assay [31,32]. The results expressed as IC50 values are shown in Table 1. Compounds 4-13a and 4-13b showed excellent cytotoxic activity against different cancer cells, in which compounds 4-13b and 13a were more potent than Sorafenib and Melphalan against one or more cell lines. For example, compounds 4-5b, 7-9b 11-13b, 9a and 13a showed stronger anti-proliferative activities against HepG-2 cell line. Compounds 4-5b, 7-10b and 13a-b exhibited more promising activities against SH-SY5Y cell line. For A549 cell line, compound 9b showed the strongest activity with the IC50 values of 5.122 uM. The IC50 values of compounds 4b and 13b against MCF-7 cell line were 6.538 and 6.138 uM, respectively. DU145 cells were negligibly affected by most of the compounds, with only the compounds 9b and 13b inducing a significant effect. However, compounds 14-15a and 14-15b were totally ineffective against all cancer cell lines. Analysis of compounds 4-13a and 4-13b revealed that the nitrogen mustard group in the para position of the benzene ring contributes more to enhancement of potency than that in the meta position. The anti-proliferative activities of derivatives 18a-s, in which the nitrogen mustard pharmacophore was in the 5-position of the pyrazolo[1,5-a]pyrimidine ring, were almost ineffective against all the five cancer cell lines. The IC50 values of the most promising compound 13b were 6.023, 0.217, 6.318, 8.317 and 6.82 uM against the A549, SH-SY5Y, HepG-2, MCF-7, DU145 cell lines, respectively,

indicating that this compound was more active than Sorafenib and Melphalan. Human gastric epithelial cell line (GES-1) was treated with the potent compounds (those showing the higher cytotoxicity) and the clinical drugs Sorafenib and Melphalan to evaluate the cytotoxicity to normal cells. The result is shown in Table 2. Cytotoxicity of 4b, 8b, 9b and 13b against GES-1 was comparable to that of Sorafenib. Compounds 5b, 6b, 10b and 12b possessed smaller cytotoxicity than Sorafenib. Base on the above results, compound 13b was selected for further study in vitro and in vivo.

2.2.2. Cell cycle inhibition To investigate the effect of compound 13b on cell cycle progression, cellular DNA content was measured by flow cytometry. Human liver carcinoma HepG2 cells were treated with compound 13b at the concentrations of 1 and 3 uM for 24 h. Thereafter, the cells were collected and washed with ice-cold PBS twice, then fixed in 70% ice-cold ethanol at least for 24 h. After that, the cells were resuspended in ice-cold PBS into which RNase A was added. This solution was maintained for about 30 min. The cells were stained with propidium iodide (PI) and analyzed with a flow cytometer. The percentages of HepG2 cells in G1, S, and G2/M phases were calculated and the results were presented in Fig. 1. Cell cycle analysis showed that compound 13b accumulated the cells at G1 phase after treatment with HepG2 for 24 h.

2.2.3. Cell apoptosis An Annexin V-FITC&PI Apoptosis Detection Kit was used to evaluate the efficacy of compound 13b on HepG2 tumor cell line. The result is shown in Fig. 2. The upper right section represents the cells which were in the late apoptotic or necrotic cells, and the lower right section represents the early or middle apoptosis. Compared with the control, compound 13b increased the apoptosis percentage of HepG2 whatever the late apoptotic or the early apoptotic cells after 24 h. The results confirmed that compound 13b could induce cell apoptosis.

2.2.4. In vivo therapeutic activity Based on the in vitro cytotoxicity, Compound 13b was selected for evaluating the antitumor therapeutic efficacy in vivo. The HepG2 HCC xenograft models were generated by injecting HepG2 cell in the left flank of each female nude mouse. Compound 13b was administered via intraperitoneal injection every day for 15 times at the dose of 10 mg/kg and 20 mg/kg body weight. Positive control Sorafenib, Cyclophosphamide and negative control vehicle were administered in the same way as compound 13b. The result is shown in Fig. 3. Compound 13b showed significant tumor growth inhibition (43.88% and 58.26%, respectively) in comparison to negative control at the doses of 10 mg/kg and 20 mg/kg. The inhibition of compound 13b was a slight lower than the positive control Sorafenib (47.90%), but higher than cyclophosphamide (42.11%) at 10 mg/kg body weight. When the dosage of compound 13b was 20 mg/kg, the inhibition was better than the positive control Sorafenib (54.93%) and Cyclophosphamide (51.48%) at the same dose level. On the other hand, compound 13b induced a 7.15% body-weight change at 10 mg/kg dose level during the treatment that similar to Cyclophosphamide (7.04%). Although compound 13b induced a 9.21% body-weight change at 20 mg/kg body weight, it was better than the positive control Cyclophosphamide (10.63%). The result is shown in Fig. 4. The body-weight of mice was readily recovered after cession of treatment. Therefore, we could conclude that compound 13b has an effective therapy in vivo for HepG2 HCC tumors and relatively low toxicity to the hosts.

M. Zhao et al. / European Journal of Medicinal Chemistry 119 (2016) 183e196

Scheme 1. Synthesis of pyrazolo[1,5-a]pyrimidine nitrogen mustard derivatives 4a-15a and 4b-15b.

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Scheme 2. Synthesis of pyrazolo[1,5-a]pyrimidine nitrogen mustard derivatives 18a-s.

3. Conclusion We have synthesized a series of pyrazolo[1,5-a]pyrimidine nitrogen mustard derivatives for antitumor evaluation. We demonstrated that most of these derivatives in which the N-mustard pharmacophore was attached at the C-7 and different substituents were introduced to the C-5 moiety of the pyrazolo[1,5-a]pyrimidines exhibited potent cytotoxicity in vitro. However, when the Nmustard pharmacophore was attached at the C-5 and different aniline moieties were introduced to the C-7, the derivatives were almost ineffective. Among them, Compound 13b possessed antiproliferative activity with IC50 values of 0.22e8.32 mM against 5 diverse human cancer cell lines, and was selected as candidate for further research. In the present studies we show that compound 13b could inhibit cell cycle arrest at G1 phase and induce cell early apoptosis. On the basis of above studies, compound 13b was selected for evaluating their antitumor activity against human HepG2 HCC tumor xenograft in nude mice. The tumor weight of the compound 13b treated mice increased much more slowly than that of the mice group treated with saline with the tumor inhibition rate 58.26% at dose levels of 20 mg/kg body weight. The result is slightly better than the positive control drug Sorafenib and

Cyclophosphamide. The body-weight of mice was recovered after stopping treatment showed that compound 13b was less toxic to the host. With the description above, compound 13b has potential as a promising anticancer agent. 4. Experimental section 4.1. General methods and materials All solvents and reagents were commercially available and used without further purification. Melting points were taken on a RY-1 apparatus and uncorrected. IR spectra were recorded on a Nicolet-AVATAR 360 FT-IR spectrometer (KBr pellets). 1H NMR and 13 C NMR spectra were performed on a Brucker spectrometer (400 MHz and 100 MHz) in DMSO-d6 or CDCl3, and chemical shifts (d values) were reported in ppm (d) relative to TMS. Mass spectra were obtained using a Brucker Apex IV FTM instrument. The human cell lines SH-SY5Y (neuroblastoma cells), HepG-2 (liver cancer cells), A549 (lung cancer cells), DU145 (prostate cancer cells), MCF-7 (breast cancer cells) and GES-1 (human gastric epithelial cell line) were obtained from Cell Resource Center, Chinese Academy of Medicinal Sciences (CAMS). Female nude mice

M. Zhao et al. / European Journal of Medicinal Chemistry 119 (2016) 183e196 Table 1 The cell cytotoxicity of newly synthesized pyrazolo[1,5-a]pyrimidine nitrogen mustard derivatives. Compd

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40  C for 2e3 h. The mixture was poured into 10 mL cold water and the solid was filtered, dried and recrystallized from methanol to give the title compounds 4-15a (4-15b).

IC50(uM)

4a 4b 5a 5b 6a 6b 7b 7a 8a 8b 9a 9b 10a 10b 11a 11b 12a 12b 13a 13b 14a 14b 15a 15b 18a 18b 18c 18d 18e 18f 18g 18h 18i 18j 18k 18l 18m 18n 18o 18p 18q 18r 18s Sorafenib Melphalan

SH-SY5Y

HepG2

A549

MCF-7

DU145

40.75 8.68 15.74 9.17 25.85 19.21 6.71 60.96 15.27 7.42 15.35 7.86 22.73 8.40 36.69 13.66 30.72 11.52 9.36 6.02 >100 74.08 >100 >100 >100 50.65 49.11 >100 64.43 >100 >100 >100 79.90 >100 >100 >100 >100 >100 >100 >100 >100 >100 77.93 27.71 >100

33.59 3.16 14.42 5.54 33.83 23.56 5.51 33.39 17.64 2.92 9.22 4.01 16.64 10.71 29.72 9.11 18.41 8.54 9.07 0.21 88.13 74.21 >100 >100 64.25 39.28 37.21 55.50 52.11 67.17 56.61 49.66 62.14 44.17 >100 >100 >100 >100 59.38 >100 >100 89.44 38.96 8.42 18.26

39.78 6.22 17.47 9.84 35.923 18.30 13.60 >100 19.53 8.90 12.67 5.122 16.49 14.78 35.83 20.58 27.94 12.80 13.21 6.13 >100 >100 >100 >100 >100 79.54 61.36 >100 85.40 >100 >100 >100 >100 >100 >100 >100 >100 >100 97.35 >100 >100 >100 75.04 19.54 43.78

54.64 6.53 37.46 12.41 >100 82.91 11.34 >100 41.10 12.19 25.16 12.36 23.94 13.79 70.38 20.41 41.05 16.20 25.06 8.31 >100 >100 >100 >100 77.45 33.03 28.30 33.04 26.40 >100 23.01 41.03 25.82 33.99 >100 >100 99.20 84.40 >100 >100 75.08 76.75 37.95 11.34 40.59

59.28 18.88 35.38 27.17 55.82 43.37 10.66 >100 50.54 23.56 26.98 7.67 44.05 21.71 >100 20.91 33.36 26.75 22.38 6.82 >100 >100 >100 >100 84.43 42.88 33.11 95.07 37.33 >100 43.47 >100 34.96 83.14 >100 >100 >100 >100 >100 >100 >100 27.36 76.09 24.91 >100

Table 2 IC50 of the potent compounds against GES-1. Cell line

GES-1

Compd

4b

5b

6b

8b

9b

10b

12b

13b

Sorafenib

IC50(uM)

10.97

13.68

17.54

8.29

11.12

13.49

17.67

9.54

10.68

were purchased from the Animal Center of Peking University (Beijing, China) for the in vivo research. Animal experiments were carried out in compliance with the Animal Management Rules of the Ministry of Health of the People's Republic of China (document no. 55, 2001) and in the guidelines for the Care and Use of Laboratory Animals of Beijing Normal University. 4.2. Synthesis 4.2.1. General procedure to synthesize compounds 4-15a and 4-15b Compound 3a (3b) (0.5 mmol, 96 mg) was mixed with corresponding amines (mmol) and K2CO3 in 2 mL of DMF and stirred at

4.2.2. 5-(Dimethylamino)-methyl-7-(4-bis(2-chloroethyl)amino) phenylamino)-3-cyanopyrazolo[1,5-a] pyrimidine (4a) Yield: 83.9%. mp: 125e126  C. 1H NMR (400 MHz, CDCl3): d 8.19 (s, 1H, eCH), 7.94 (s, 1H, eNH), 7.28 (t, J ¼ 8.00 Hz, 1H, ArH), 6.83 (s, 1H, eCH), 6.68 (d, J ¼ 8.24 Hz, 1H, ArH), 6.57 (d, J ¼ 6.48 Hz, 2H, ArH), 3.70 (t, J ¼ 6.80 Hz, 4H, eCH2CH2Cl), 3.61 (t, J ¼ 6.36 Hz, 4H, eCH2CH2Cl), 3.49 (s, 2H, eCH2), 2.22 (s, 6H, eCH3). 13C NMR (100 MHz, CDCl3): d 165.11, 150.43, 147.64, 146.29, 145.75, 137.15, 131.04, 113.68, 112.41, 110.38, 107.13, 88.75, 80.80, 65.56, 53.35, 45.77, 40.40. MS (ESIþ) m/z: 433.9 [MþH]þ. IR (KBr pellet, cm1): 3356, 2947, 2769, 2222, 1624, 1531, 1508, 1450, 1261, 1163, 744. 4.2.3. 5-(Dimethylamino)-methyl-7-(3-bis(2-chloroethyl)amino) phenylamino)-3-cyanopyrazolo[1,5-a] pyrimidine (4b) Yield: 70.1%. mp: 83e84  C. 1H NMR (400 MHz, CDCl3): d 8.26 (s, 1H, eCH), 7.89 (s, 1H, eNH), 7.24 (d, J ¼ 9.24 Hz, 2H, ArH), 6.77 (d, J ¼ 8.92 Hz, 2H, ArH), 6.59 (s, 1H, eCH), 3.79 (t, J ¼ 7.08 Hz, 4H, eCH2CH2Cl), 3.68 (t, J ¼ 6.68 Hz, 4H, eCH2CH2Cl), 3.55 (s, 2H, eCH2), 2.28 (s, 6H, eCH3). 13C NMR (100 MHz, CDCl3): d 164.84, 150.35, 146.59, 146.37, 145.52, 126.53, 125.01, 113.66, 112.89, 88.09, 80.92, 65.73, 53.49, 45.76, 40.30. MS (ESIþ) m/z: 432.1 [MþH]þ. IR (KBr pellet, cm1): 3348, 2821, 2224, 1620, 1585, 1518, 1354, 1255, 1185, 819. 4.2.4. 5-(Pyrrolidin-1-ly)methyl-7-(4-bis(2-chloroethyl)amino) phenylamino)-3-cyanopyrazolo[1,5-a] pyrimidine (5a) Yield: 61.5%. mp: 72e73  C. 1H NMR (400 MHz, CDCl3): d 8.27 (s, 1H, eCH), 8.05 (s, 1H, eNH), 7.35 (t, J ¼ 8.12 Hz, 1H, ArH), 6.88 (s, 1H, eCH), 6.75 (d, J ¼ 7.16 Hz, 1H, ArH), 6.63e6.66 (m, 2H, ArH), 3.78 (t, J ¼ 8.44 Hz, 4H, eCH2CH2Cl), 3.78 (s, 2H, eCH2), 3.67 (t, J ¼ 6.68 Hz, 4H, eCH2CH2Cl), 2.58e2.62 (m, 4H, eCH2), 1.78e1.81 (m, 4H, eCH2). 13C NMR (100 MHz, CDCl3): d 165.50, 150.39, 147.66, 146.34, 145.49, 137.18, 131.10, 113.61, 112.33, 110.37, 88.70, 81.06, 62.06, 54.37, 53.40, 40.32, 23.76. MS (ESIþ) m/z: 458.1 [MþH]þ. IR (KBr pellet, cm1): 3253, 2960, 2974, 2224, 1624, 1558, 1500, 1361, 1172, 758. 4.2.5. 5-(Pyrrolidin-1-ly)methyl-7-(3-bis(2-chloroethyl)amino) phenylamino)-3-cyanopyrazolo[1,5-a] pyrimidine (5b) Yield: 74.2%. mp: 72e74  C. 1H NMR (400 MHz, CDCl3): d 8.25 (s, 1H, eCH), 7.89 (s, 1H, eNH), 7.23 (d, J ¼ 8.96 Hz, 2H, ArH), 6.77 (d, J ¼ 8.96 Hz, 2H, ArH), 6.56 (s, 1H, eCH), 3.79 (t, J ¼ 7.08 Hz, 4H, eCH2CH2Cl), 3.74 (s, 2H, eCH2), 3.68 (t, J ¼ 6.68 Hz, 4H, eCH2CH2Cl), 2.59 (s, 4H, eCH2), 1.78 (s, 4H, eCH2). 13C NMR (100 MHz, CDCl3): d 164.92, 150.39, 146.58, 146.37, 145.51, 126.54, 125.04, 113.76, 112.65, 88.31, 80.73, 62.06, 54.32, 53.46, 40.36, 23.67. MS (ESIþ) m/ z: 458.1 [MþH]þ. IR (KBr pellet, cm1): 3367, 2960, 2224, 1618, 1587, 1518, 1356, 1280, 1172, 817. 4.2.6. 5-(4-Methylpiperidin-1-yl)methyl-7-(4-bis(2-chloroethyl) amino)phenylamino)-3- cyanopyrazolo[1,5-a]pyrimidine (6a) Yield: 82.6%. mp: 72e74  C. 1H NMR (400 MHz, CDCl3): d 8.26 (s, 1H, eCH), 7.36 (t, J ¼ 7.92 Hz, 1H, ArH), 6.95 (s, 1H, eCH), 6.77 (d, J ¼ 8.60 Hz, 1H, ArH), 6.65 (d, J ¼ 8.40 Hz, 2H, ArH), 3.78 (t, J ¼ 7.00 Hz, 4H, eCH2CH2Cl), 3.68 (t, J ¼ 6.48 Hz, 4H, eCH2CH2Cl), 3.61 (s, 2H, eCH2), 2.83 (d, J ¼ 11.52 Hz, 2H, eCH2), 2.11 (t, J ¼ 11.52 Hz, 2H, eCH2), 1.61 (d, J ¼ 12.56 Hz, 2H, eCH2), 1.24e1.42 (m, 1H, eCH), 1.15e1.24 (m, 2H, eCH2), 0.89 (s, 3H, eCH3). 13C NMR (100 MHz, CDCl3): d 165.60, 150.44, 147.64, 146.27, 145.43, 137.28, 137.28, 131.01, 113.65, 112.14, 110.30, 106.88, 88.70, 80.92, 64.56, 54.23, 53.39, 40.37, 34.38, 30.43, 21.89. MS (ESIþ) m/z: 488.0

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Fig. 1. Cell cycle inhibition in HepG2 cell line by treating with compound 13b for 24 h.

Fig. 2. Effects of compound 13b on cell apoptosis of HepG2 for 24 h.

[MþH]þ. IR (KBr pellet, cm1): 3348, 2922, 2224, 1624, 1568, 1535, 1444, 1315, 1172, 758. 4.2.7. 5-(4-Methylpiperidin-1-yl)methyl-7-(3-bis(2-chloroethyl) amino)phenylamino)-3- cyanopyrazolo [1,5-a]pyrimidine (6b) Yield: 60.7%. mp: 151e152  C. 1H NMR (400 MHz, CDCl3): d 8.25

(s, 1H, eCH), 7.24 (d, J ¼ 8.82 Hz, 1H, ArH), 6.78 (d, J ¼ 8.96 Hz, 1H, ArH), 6.66 (s, 1H, eCH), 3.80 (t, J ¼ 7.00 Hz, 4H, eCH2CH2Cl), 3.69 (t, J ¼ 6.68 Hz, 4H, eCH2CH2Cl), 3.59 (s, 2H, eCH2), 2.80e2.96 (m, 2H, eCH2), 2.09 (t, J ¼ 11.48 Hz, 2H, eCH2), 1.61 (d, J ¼ 12.04 Hz, 2H, eCH2), 1.34e1.39 (m, 1H, eCH), 1.13e1.22 (m, 2H, eCH2), 0.88 (s, 3H, eCH3). 13C NMR (100 MHz, CDCl3): d 165.23, 150.47, 146.51, 146.36,

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189

Fig. 3. The average tumor size changes of 13b in nude mice bearing HepG2 HCC tumors in mice (ip injection, n ¼ 5), the p values for the treated versus the untreated group from day 12 to 26 is *p < 0.05.

Fig. 4. The average body weight changes of the bearing HCC mice during the process of therapy with 13b, the p values for the treated versus the untreated group from day 12 to 26 is **p < 0.01.

145.43, 126.42, 125.19, 113.76, 112.86, 88.24, 80.70, 64.43, 54.14, 53.50, 40.32, 34.39, 30.45, 21.92. MS (ESIþ) m/z: 487.8 [MþH]þ. IR (KBr pellet, cm1): 3354, 2922, 2224, 1620, 1585, 1517, 1313, 1253, 817. 4.2.8. 5-Morpholinomethyl-7-(4-bis(2-chloroethyl)amino) phenylamino)-3-cyanopyrazolo[1,5-a] pyrimidine (7a) Yield: 76.5%. mp: 176e178  C. 1H NMR (400 MHz, CDCl3): d 8.28 (s, 1H, eCH), 8.09 (s, 1H, eNH), 7.37 (t, J ¼ 8.04 Hz, 1H, ArH), 6.91 (s, 1H, eCH), 6.76 (d, J ¼ 7.68 Hz, 1H, ArH), 6.64e6.68 (m, 2H, ArH), 3.79 (t, J ¼ 6.92 Hz, 4H, eCH2CH2Cl), 3.66e3.71(m, 8H, eCH2CH2Cl and eCH2), 3.65 (s, 2H, eCH2), 2.54 (s, 4H, eCH2). 13C NMR (100 MHz, CDCl3): d 164.22, 150.42, 147.74, 146.38, 145.52, 137.13, 131.07, 113.48, 112.21, 110.47, 106.98, 88.62, 81.25, 66.94, 64.52, 53.76, 53.36, 40.32. MS (ESIþ) m/z: 474.0 [MþH]þ. IR (KBr pellet,

cm1): 3248, 2989, 2224, 1620, 1566, 1502, 1170, 1111, 750.

4.2.9. 5-Morpholinomethyl-7-(3-bis(2-chloroethyl)amino) phenylamino)-3-cyanopyrazolo[1,5-a] pyrimidine (7b) Yield: 81.3%. mp: 191e192  C. 1H NMR (400 MHz, CDCl3): d 8.19 (s, 1H, eCH), 7.84 (s, 1H, eNH), 7.17 (d, J ¼ 8.92 Hz, 2H, ArH), 6.72 (d, J ¼ 8.84 Hz, 2H, ArH), 6.53 (s, 1H, eCH), 3.74 (t, J ¼ 6.88 Hz, 4H, eCH2CH2Cl), 3.62e3.64(m, 8H, eCH2CH2Cl and eCH2), 3.55 (s, 2H, eCH2), 2.46 (s, 4H, eCH2). 13C NMR (100 MHz, CDCl3): d 162.82, 149.42, 145.54, 145.44, 144.58, 125.44, 123.97, 112.59, 111.86, 87.14, 80.04, 65.91, 63.42, 52.71, 52.47, 39.31. MS (ESIþ) m/z: 473.8 [MþH]þ. IR (KBr pellet, cm1): 3346, 2852, 2224, 1618, 1587, 1517, 1315, 1263, 1112, 819.

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4.2.10. 5-(4-Methylpiperazin-1-yl)methyl-7-(4-bis(2-chloroethyl) amino)phenylamino)-3- cyanopyrazolo[1,5-a]pyrimidine (8a) Yield: 72.8%. mp: 74e75  C. 1H NMR (400 MHz, CDCl3): d 8.27 (s, 1H, eCH), 8.07 (s, 1H, eNH), 7.37 (t, J ¼ 8.08 Hz, 1H, ArH), 6.91 (s, 1H, eCH), 6.76 (d, J ¼ 7.64 Hz, 1H, ArH), 6.63e6.68 (m, 2H, ArH), 3.79 (t, J ¼ 7.00 Hz, 4H, eCH2CH2Cl), 3.68 (t, J ¼ 6.52 Hz, 4H, eCH2CH2Cl), 3.65 (s, 2H, eCH2), 2.58 (brs, 4H, eCH2), 2.43 (brs, 4H, eCH2), 2.28 (s, 3H, eCH3). 13C NMR (100 MHz, CDCl3): d 164.69, 150.41, 147.67, 146.32, 145.49, 137.18, 131.04, 113.55, 112.22, 110.40, 106.95, 88.69, 81.08, 64.04, 55.07, 53.38, 53.29, 40.36. MS (ESIþ) m/z: 486.9 [MþH]þ. IR (KBr pellet, cm1): 3356, 2799, 2224, 1624, 1551, 1535, 1456, 1315, 1168, 758. 4.2.11. 5-(4-Methylpiperazin-1-yl)methyl-7-(3-bis(2-chloroethyl) amino)phenylamino)-3- cyanopyrazolo[1,5-a]pyrimidine (8b) Yield: 82.6%. mp: 78e80  C. 1H NMR (400 MHz, CDCl3): d 8.25 (s, 1H, eCH), 7.91 (s, 1H, eNH), 7.24 (d, J ¼ 8.08 Hz, 2H, ArH), 6.79 (d, J ¼ 8.48 Hz, 2H, ArH), 6.60 (s, 1H, eCH), 3.80 (t, J ¼ 6.48 Hz, 4H, eCH2CH2Cl), 3.69 (t, J ¼ 6.16 Hz, 4H, eCH2CH2Cl), 3.63 (s, 2H, eCH2), 2.57 (brs, 4H, eCH2), 2.43 (brs, 4H, eCH2), 2.28 (s, 3H, eCH3). 13C NMR (100 MHz, CDCl3): d 164.25, 150.45, 146.58, 146.40, 145.50, 126.50, 125.06, 113.66, 112.87, 88.27, 80.83, 63.87, 55.06, 53.47, 53.17, 46.02, 40.32. MS (ESIþ) m/z: 487.1 [MþH]þ. IR (KBr pellet, cm1): 3348, 2800, 2224, 1620, 1568, 1517, 1456, 1251, 1010, 815. 4.2.12. 5-(4-(Dimethylamino)piperidin-1-yl)methyl-7-(4-bis(2chloroethyl)amino)phenylamino)-3- cyanopyrazolo[1,5-a] pyrimidine (9a) Yield: 84.8%. mp: 82e83  C. 1H NMR (400 MHz, CDCl3): d 8.27 (s, 1H, eCH), 8.03 (s, 1H, eNH), 7.35 (t, J ¼ 8.04 Hz, 1H, ArH), 6.95 (s, 1H, eCH), 6.76 (d, J ¼ 7.88 Hz, 1H, ArH), 6.61e6.66 (m, 2H, ArH), 3.77 (t, J ¼ 7.04 Hz, 4H, eCH2CH2Cl), 3.67 (t, J ¼ 6.48 Hz, 4H, eCH2CH2Cl), 3.63 (s, 2H, eCH2), 2.91 (d, J ¼ 11.80 Hz, 2H, eCH2), 2.27 (s, 6H, eCH3), 2.03e2.16 (m, 3H, eCH2 and eCH), 1.82 (d, J ¼ 12.12 Hz, 2H, eCH2), 1.47e1.50 (m, 2H, eCH2). 13C NMR (100 MHz, CDCl3): d 165.33, 150.41, 147.68, 146.31, 145.48, 137.16, 131.07, 113.54, 112.15, 110.37, 106.85, 88.51, 81.07, 64.14, 61.99, 53.38, 53.31, 41.96, 40.26, 28.90. MS (ESIþ) m/z: 515.1 [MþH]þ. IR (KBr pellet, cm1): 3277, 2225, 1616, 1591, 1517, 1411, 1246, 1159, 748.

[MþH]þ. IR (KBr pellet, cm1): 3348, 2810, 2224, 1624, 1568, 1502, 1313, 1166, 1016, 758. 4.2.15. 5-(4-Ethylpiperazin-1-yl)methyl-7-(3-bis(2-chloroethyl) amino)phenylamino)-3- cyanopyrazolo[1,5-a]pyrimidine (10b) Yield: 78.8%. mp: 106e108  C. 1H NMR (400 MHz, CDCl3): d 8.25 (s, 1H, eCH), 7.92 (s, 1H, eNH), 7.24 (d, J ¼ 9.00 Hz, 2H, ArH), 6.78 (d, J ¼ 9.00 Hz, 2H, ArH), 6.60 (s, 1H, eCH), 3.80 (t, J ¼ 7.20 Hz, 4H, eCH2CH2Cl), 3.69 (t, J ¼ 6.92 Hz, 4H, eCH2CH2Cl), 3.63 (s, 2H, eCH2), 2.38e2.58 (m, 10H, eCH2), 1.08 (t, J ¼ 7.20 Hz, 3H, eCH3). 13C NMR (100 MHz, CDCl3): d 164.15, 150.48, 146.65, 146.35, 145.50, 126.53, 125.05, 113.73, 112.86, 88.39, 80.60, 63.82, 53.44, 53.14, 52.70, 52.24, 40.37, 11.91. MS (ESIþ) m/z: 501.1[MþH]þ. IR (KBr pellet, cm1): 3356, 2814, 2222, 1622, 1585, 1518, 1313, 1238, 1172, 819. 4.2.16. 5-(4-Hydroxypiperidin-1-yl)methyl-7-(4-bis(2-chloroethyl) amino)phenylamino)-3- cyanopyrazolo[1,5-a]pyrimidine (11a) Yield: 72.1%. mp: 84e85  C. 1H NMR (400 MHz, CDCl3): d 8.01 (s, 1H, eCH), 7.38 (s, 1H, eNH), 7.34 (t, J ¼ 8.12 Hz, 1H, ArH), 6.94 (s, 1H, eCH), 6.77 (d, J ¼ 8.80 Hz, 1H, ArH), 6.63e6.67 (m, 2H, ArH), 3.78 (t, J ¼ 7.24 Hz, 4H, eCH2CH2Cl), 3.67e3.73 (m, 5H, eCH2CH2Cl and eCH), 3.64 (s, 2H, eCH2), 2.77e2.82 (m, 2H, eCH2), 2.25e2.31 (m, 2H, eCH2), 1.86e1.91 (m, 2H, eCH2), 1.54e1.61 (m, 2H, eCH2). 13C NMR (100 MHz, CDCl3): d 165.07, 150.45, 147.67, 146.32, 145.66, 137.17, 131.01, 113.64, 112.19, 110.40, 107.04, 88.70, 80.81, 67.50, 63.93, 53.32, 51.43, 40.38, 34.41. MS (ESIþ) m/z: 488.0 [MþH]þ. IR (KBr pellet, cm1): 3361, 2968, 2222, 1612, 1584, 1514, 1415, 1240, 1176, 815. 4.2.17. 5-(4-Hydroxypiperidin-1-yl)methyl-7-(3-bis(2-chloroethyl) amino)phenylamino)-3- cyanopyrazolo[1,5-a]pyrimidine (11b) Yield: 53.7%. mp: 88e89  C. 1H NMR (400 MHz, CDCl3): d 8.19 (s, 1H, eCH), 7.84 (s, 1H, eNH), 7.17 (d, J ¼ 8.12 Hz, 2H, ArH), 6.71 (d, J ¼ 8.64 Hz, 2H, ArH), 6.57 (s, 1H, eCH), 3.73 (t, J ¼ 6.68 Hz, 4H, eCH2CH2Cl), 3.61e3.64 (m, 5H, eCH2CH2Cl and eCH), 3.54 (s, 2H, eCH2), 2.71 (d, J ¼ 11.00 Hz, 2H, eCH2), 2.19 (t, J ¼ 10.12 Hz, 2H, eCH2), 1.81 (d, J ¼ 9.88 Hz, 2H, eCH2), 1.45e1.55 (m, 2H, eCH2). MS (ESIþ) m/z: 488.1 [MþH]þ. IR (KBr pellet, cm1): 3354, 2927, 2223, 1618, 1587, 1517, 1064, 817.

4.2.13. 5-(4-(Dimethylamino)piperidin-1-yl)methyl-7-(3-bis(2chloroethyl)amino)phenylamino)-3- cyanopyrazolo[1,5-a] pyrimidine (9b) Yield: 95.8%. mp: 90e91  C. 1H NMR (400 MHz, CDCl3): d 8.25 (s, 1H, eCH), 7.89 (s, 1H, eNH), 7.24 (d, J ¼ 8.88 Hz, 2H, ArH), 6.77 (d, J ¼ 8.92 Hz, 2H, ArH), 6.67 (s, 1H, eCH), 3.79 (t, J ¼ 7.00 Hz, 4H, eCH2CH2Cl), 3.69 (t, J ¼ 7.00 Hz, 4H, eCH2CH2Cl), 3.59 (s, 2H, eCH2), 2.90 (d, J ¼ 11.52 Hz, 4H, eCH2), 2.27 (s, 6H, eCH3), 2.03e2.15 (m, 3H, eCH2 and eCH), 1.83 (d, J ¼ 11.68 Hz, 4H, eCH2), 1.41e1.50 (m, 2H, eCH2). 13C NMR (100 MHz, CDCl3): d 164.99, 150.41, 146.49, 146.32, 145.48, 126.32, 125.06, 113.69, 112.80, 87.99, 80.74, 64.04, 61.96, 53.47, 53.20, 41.93, 40.30, 28.85. MS (ESIþ) m/z: 515.1 [MþH]þ. IR (KBr pellet, cm1): 3491, 2941, 2224, 1622, 1568, 1535, 1313, 1168, 750.

4.2.18. 5-(4-(Hydroxymethyl)piperidin-1-yl)methyl-7-(4-bis(2chloroethyl)amino)phenylamino)-3- cyanopyrazolo[1,5-a] pyrimidine (12a) Yield: 82.4%. mp: 92e93  C. 1H NMR (400 MHz, CDCl3): d 8.27 (s, 1H, eCH), 8.02 (s, 1H, eNH), 7.36 (t, J ¼ 7.92 Hz, 1H, ArH), 6.93 (s, 1H, eCH), 6.76 (d, J ¼ 7.28 Hz, 1H, ArH), 6.64e6.67 (m, 2H, ArH), 3.78 (t, J ¼ 7.00 Hz, 4H, eCH2CH2Cl), 3.68 (t, J ¼ 6.92 Hz, 4H, eCH2CH2Cl), 3.63 (s, 2H, eCH2), 3.50 (d, J ¼ 6.24 Hz, 2H, eCH2), 2.89 (d, J ¼ 11.68 Hz, 2H, eCH2), 2.14 (t, J ¼ 11.52 Hz, 2H, eCH2), 1.32e1.73 (m, 5H, eCH2 and eCH). 13C NMR (100 MHz, CDCl3): d 165.26, 150.44, 147.67, 146.30, 145.55, 137.23, 130.00, 113.64, 112.17, 110.36, 106.96, 88.79, 80.85, 67.59, 64.48, 53.77, 53.35, 40.41, 38.21, 28.83. MS (ESIþ) m/z: 502.0 [MþH]þ. IR (KBr pellet, cm1): 3356, 2922, 2224, 1624, 1568, 1534, 1313, 1170, 1039, 758.

4.2.14. 5-(4-Ethylpiperazin-1-yl)methyl-7-(4-bis(2-chloroethyl) amino)phenylamino)-3- cyanopyrazolo [1,5-a]pyrimidine (10a) Yield: 68.3%. mp: 76e78  C. 1H NMR (400 MHz, CDCl3): d 8.27 (s, 1H, eCH), 8.08 (s, 1H, eNH), 7.36 (t, J ¼ 8.00 Hz, 1H, ArH), 6.91 (s, 1H, eCH), 6.76 (d, J ¼ 7.56 Hz, 1H, ArH), 6.64e6.67 (m, 2H, ArH), 3.79 (t, J ¼ 7.08 Hz, 4H, eCH2CH2Cl), 3.68 (t, J ¼ 6.84 Hz, 4H, eCH2CH2Cl), 3.66 (s, 2H, eCH2), 2.38e2.59 (m, 10H, eCH2), 1.08 (t, J ¼ 7.20 Hz, 3H, eCH3). 13C NMR (100 MHz, CDCl3): d 164.69, 150.42, 147.64, 146.31, 145.47, 137.19, 131.02, 113.58, 112.18, 110.13, 106.91, 88.71, 81.02, 64.07, 53.37, 53.31, 52.78, 52.26, 40.36, 12.01. MS (ESIþ) m/z: 501.1

4.2.19. 5-(4-(Hydroxymethyl)piperidin-1-yl)methyl-7-(4-bis(2chloroethyl)amino)phenylamino)-3- cyanopyrazolo[1,5-a] pyrimidine (12b) Yield: 79.6%. mp: 72e73  C. 1H NMR (400 MHz, CDCl3): d 8.25 (s, 1H, eCH), 7.93 (s, 1H, eNH), 7.24 (d, J ¼ 8.84 Hz, 2H, ArH), 6.78 (d, J ¼ 8.92 Hz, 2H, ArH), 6.64 (s, 1H, eCH), 3.80 (t, J ¼ 6.68 Hz, 4H, eCH2CH2Cl), 3.69 (t, J ¼ 7.08 Hz, 4H, eCH2CH2Cl), 3.60 (s, 2H, eCH2), 3.50 (d, J ¼ 6.24 Hz, 2H, eCH2), 2.88 (d, J ¼ 11.32 Hz, 2H, eCH2), 2.12 (t, J ¼ 11.52 Hz, 2H, eCH2), 1.70 (d, J ¼ 12.28 Hz, 2H, eCH2), 1.48e1.55 (m, 1H, eCH), 1.23e1.30 (m, 1H, eCH2). 13C NMR

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(100 MHz, CDCl3): d 164.91, 150.46, 146.55, 146.37, 145.47, 126.39, 125.11, 113.75, 112.87, 88.30, 80.63, 67.63, 64.34, 53.67, 53.46, 40.39, 38.20, 28.81. MS (ESIþ) m/z: 502.0 [MþH]þ. IR (KBr pellet, cm1): 3354, 2920, 2224, 1620, 1587, 1518, 1357, 1313, 1039, 817. 4.2.20. 5-(4-(Piperidin-1-yl)piperidin-1-yl)methyl-7-(4-bis(2chloroethyl)amino)phenylamino)-3- cyanopyrazolo[1,5-a] pyrimidine (13a) Yield: 94.5%. mp: 77e78  C. 1H NMR (400 MHz, CDCl3): d 8.06 (s, 1H, eCH), 7.39 (s, 1H, eNH), 7.35 (t, J ¼ 8.04 Hz, 1H, ArH), 6.95 (s, 1H, eCH), 6.75 (d, J ¼ 7.48 Hz, 1H, ArH), 6.64e6.67 (m, 2H, ArH), 4.14 (q, J ¼ 7.12 Hz, 2H, eCH2), 3.78 (t, J ¼ 6.92 Hz, 4H, eCH2CH2Cl), 3.68 (t, J ¼ 6.96 Hz, 4H, eCH2CH2Cl), 3.63 (s, 2H, eCH2), 2.87 (d, J ¼ 11.64 Hz, 2H, eCH2), 2.28e2.33 (m, 1H, eCH), 2.15e2.26 (m, 2H, eCH2), 1.90 (d, J ¼ 11.00 Hz, 2H, eCH2), 1.62e1.75 (m, 2H, eCH2), 1.26 (t, J ¼ 7.12 Hz, 2H, eCH3). 13C NMR (100 MHz, CDCl3): d 175.09, 165.20, 150.39, 147.72, 146.32, 145.46, 137.14, 131.12, 113.49, 112.13, 110.44, 106.78, 88.43, 81.19, 64.41, 60.35, 53.35, 53.25, 40.79, 40.27, 28.38, 14.21. MS (ESIþ) m/z: 544.0 [MþH]þ. IR (KBr pellet, cm1): 3357, 2939, 2225, 1726, 1624, 1568, 1535, 1500, 1317, 1170, 758. 4.2.21. 5-(4-(Piperidin-1-yl)piperidin-1-yl)methyl-7-(4-bis(2chloroethyl)amino)phenylamino)-3- cyanopyrazolo[1,5-a] pyrimidine (13b) Yield: 95.3%. mp: 80e82  C. 1H NMR (400 MHz, CDCl3): d 8.18 (s, 1H, eCH), 7.82 (s, 1H, eNH), 7.16 (d, J ¼ 8.96 Hz, 2H, ArH), 6.72 (d, J ¼ 9.04 Hz, 2H, ArH), 6.58 (s, 1H, eCH), 4.06 (q, J ¼ 7.12 Hz, 2H, eCH2), 3.73 (t, J ¼ 7.16 Hz, 4H, eCH2CH2Cl), 3.62 (t, J ¼ 6.84 Hz, 4H, eCH2CH2Cl), 3.53 (s, 2H, eCH2), 2.78 (d, J ¼ 11.68 Hz, 2H, eCH2), 2.22e2.26 (m, 1H, eCH), 2.18e2.21 (m, 2H, eCH2), 2.12e2.21 (m, 4H, eCH2), 1.66 (t, J ¼ 8.24 Hz, 3H, eCH3). 13C NMR (100 MHz, CDCl3): d 174.19, 163.82, 149.41, 145.53, 145.36, 144.51, 125.35, 123.97, 112.68, 111.81, 86.97, 79.82, 63.32, 59.36, 52.49, 52.16, 39.79, 39.32, 27.32, 13.22. MS (ESIþ) m/z: 543.9 [MþH]þ. IR (KBr pellet, cm1): 3354, 2947, 2224, 1626, 1618, 1585, 1518, 1315, 1186, 819. 4.2.22. 5-(Ethoxycarbonyl-piperidinyl)methyl-7-(4-bis(2chloroethyl)amino)phenylamino)-3- cyanopyrazolo [1,5-a] pyrimidine (14a) Yield: 62.9%. mp: 76e78  C. 1H NMR (400 MHz, CDCl3): d 8.29 (s, 1H, eCH), 8.11 (s, 1H, eNH), 7.36 (t, J ¼ 8.12 Hz, 1H, ArH), 6.94 (s, 1H, eCH), 6.76 (d, J ¼ 7.76 Hz, 1H, ArH), 6.61e6.67 (m, 2H, ArH), 3.77 (t, J ¼ 6.96 Hz, 4H, eCH2CH2Cl), 3.68 (t, J ¼ 6.76 Hz, 4H, eCH2CH2Cl), 3.62 (s, 2H, eCH2), 2.91 (d, J ¼ 11.68 Hz, 2H, eCH2), 2.49 (s, 4H, eCH2), 2.09e2.22 (m, 3H, eCH2 and eCH), 1.81 (d, J ¼ 12.08 Hz, 2H, eCH2), 1.43e1.61 (m, 8H, eCH2). 13C NMR (100 MHz, CDCl3): d 165.40, 150.39, 147.68, 146.31, 145.45, 137.17, 131.10, 113.53, 112.17, 110.38, 106.81, 88.52, 81.11, 64.20, 62.28, 53.73, 53.39, 50.42, 40.26, 28.35, 26.32, 24.73. MS (ESIþ) m/z: 554.9 [MþH]þ. IR (KBr pellet, cm1): 3354, 2933, 2796, 2224, 1623, 1562, 1535, 1313, 1170, 1111, 758. 4.2.23. 5-(Ethoxycarbonyl-piperidinyl)methyl-7-(3-bis(2chloroethyl)amino)phenylamino)-3- cyanopyrazolo[1,5-a] pyrimidine (14b) Yield: 76.7%. mp: 68e70  C. 1H NMR (400 MHz, CDCl3): d 8.25 (s, 1H, eCH), 7.88 (s, 1H, eNH), 7.23 (d, J ¼ 8.96 Hz, 2H, ArH), 6.78 (d, J ¼ 9.04 Hz, 2H, ArH), 6.66 (s, 1H, eCH), 3.80 (t, J ¼ 7.16 Hz, 4H, eCH2CH2Cl), 3.69 (t, J ¼ 6.60 Hz, 4H, eCH2CH2Cl), 3.56 (s, 2H, eCH2), 2.90 (d, J ¼ 11.68 Hz, 2H, eCH2), 2.46 (s, 4H, eCH2), 2.07e2.19 (m, 3H, eCH2 and eCH), 1.82 (d, J ¼ 12.68 Hz, 2H, eCH2), 1.52e1.61 (m, 8H, eCH2). 13C NMR (100 MHz, CDCl3): d 165.08, 150.41, 146.49, 146.33, 145.50, 126.34, 125.07, 113.66, 112.82, 87.96, 80.78, 64.09, 62.22, 53.61, 53.50, 50.44, 40.27, 28.37, 26.31, 24.70. MS (ESIþ) m/z: 555.2 [MþH]þ. IR (KBr pellet, cm1): 3354, 2933, 2798, 2224, 1618,

191

1566, 1518, 1355, 1111, 817. 4.2.24. 5-(4-Phenylpiperidin-1-yl)methyl-7-(4-bis(2-chloroethyl) amino)phenylamino)-3- cyanopyrazolo [1,5-a]pyrimidine (15a) Yield: 94.1%. mp: 75e76  C. 1H NMR (400 MHz, CDCl3): d 8.31 (s, 1H, eCH), 7.19e7.39 (m, 6H, ArH), 6.98 (s, 1H, eCH), 6.76 (d, J ¼ 7.36 Hz, 1H, ArH), 6.65e6.68 (m, 2H, ArH), 3.78 (t, J ¼ 7.08 Hz, 4H, eCH2CH2Cl), 3.67 (t, J ¼ 6.88 Hz, 4H, eCH2CH2Cl), 3.65 (s, 2H, eCH2), 2.99 (d, J ¼ 11.48 Hz, 2H, eCH2), 2.49 (t, J ¼ 7.64 Hz, 1H, eCH), 2.28 (t, J ¼ 11.68 Hz, 2H, eCH2), 1.84 (d, J ¼ 11.28 Hz, 2H, eCH2), 1.70e1.79 (m, 2H, eCH2). 13C NMR (100 MHz, CDCl3): d 165.29, 150.46, 147.73, 146.35, 146.16, 145.51, 137.26, 131.08, 128.42, 126.77, 126.19, 113.59, 112.24, 110.40, 106.93, 88.71, 81.12, 64.54, 54.59, 53.39, 42.14, 40.31, 33.47. MS (ESIþ) m/z: 549.9 [MþH]þ. IR (KBr pellet, cm1): 3346, 2933, 2798, 2224, 1622, 1568, 1535, 1500, 1359, 1170, 758. 4.2.25. 5-(4-Phenylpiperidin-1-yl)methyl-7-(3-bis(2-chloroethyl) amino)phenylamino)-3- cyanopyrazolo[1,5-a]pyrimidine (15b) Yield: 94.8%. mp: 86e87  C. 1H NMR (400 MHz, CDCl3): d 8.26 (s, 1H, eCH), 7.88 (s, 1H, eNH), 7.19e7.33 (m, 7H, ArH), 6.78 (d, J ¼ 8.96 Hz, 2H, ArH), 6.69 (s, 1H, eCH), 3.79 (t, J ¼ 7.36 Hz, 4H, eCH2CH2Cl), 3.68 (t, J ¼ 6.68 Hz, 4H, eCH2CH2Cl), 3.66 (s, 2H, eCH2), 2.99 (d, J ¼ 11.40 Hz, 2H, eCH2), 2.49e2.56 (m, 1H, eCH), 2.26 (t, J ¼ 11.44 Hz, 2H, eCH2), 1.84 (d, J ¼ 10.76 Hz, 2H, eCH2), 1.71e1.78 (m, 2H, eCH2). 13C NMR (100 MHz, CDCl3): d 164.94, 150.50, 146.56, 146.40, 146.21, 145.51, 128.45, 126.73, 126.46, 126.19, 125.15, 113.72, 112.86, 88.20, 80.83, 64.35, 54.47, 53.52, 42.14, 40.33, 33.46. MS (ESIþ) m/z: 548.1 [MþH]þ. IR (KBr pellet, cm1): 3346, 2933, 2796, 2224, 1620, 1585, 1517, 1490, 1313, 1178, 750. 4.2.25.1. General procedure to synthesize compounds 17a-s. NH(CH2CH2OH)2 (6 mmol, 0.13 g) was dissolved in DMF (5 mL), then K2CO3 (6 mmol, 0.83 g) was added to this solution and stirred for 20 min at room temperature. Thereafter, Compound 16a-s (3 mmol) was added into the solution and stirred at 40  C for 2e3 h. The reaction mixture was cooled and mixed with water and the solid was filtered. The solid obtained was washed with water and ethanol to give the intermediate compounds 17a-s. 4.2.26. 5-(Bis(2-hydroxyethyl)amino)methyl-7-(4fluorophenylamino)-3-cyanopyrazolo[1,5-a] pyrimidine (17a) Yield: 77.1%. mp: 160e161  C. 1H NMR (400 MHz, DMSO-d6): d 10.37 (s, 1H, eNH), 8.72 (s, 1H, eCH), 7.51 (d, J ¼ 7.36 Hz, 2H, ArH), 7.31 (d, J ¼ 8.76 Hz, 2H, ArH), 6.74 (s, 1H, eCH), 4.38 (t, J ¼ 5.28 Hz, 2H, eOH), 3.75 (s, 2H, eCH2), 3.39e3.44 (m, 4H, eCH2), 2.60 (t, J ¼ 6.20 Hz, 2H, eCH2). 13C NMR (100 MHz, DMSO-d6): d166.18, 161.18, 158.76, 150.63, 146.39, 132.89, 126.74, 126.65, 116.27, 116.04, 114.18, 88.25, 78.55, 60.86, 59.15, 56.77. MS (ESIþ) m/z: 372.2 [MþH]þ. IR (KBr pellet, cm1): 3421, 2879, 2225, 1622, 1571, 1514, 1490, 1313, 1217, 1076, 835, 542. 4.2.27. 5-(Bis(2-hydroxyethyl)amino)methyl-7-(4chlorophenylamino)-3-cyanopyrazolo[1,5-a] pyrimidine (17b) Yield: 87.5%. mp: 158e160  C. 1H NMR (400 MHz, DMSO-d6): d 10.45 (s, 1H, eNH), 8.73 (s, 1H, eCH), 7.52 (s, 4H, ArH), 6.88 (s, 1H, eCH), 4.41 (t, J ¼ 5.20 Hz, 2H, eOH), 3.77 (s, 2H, eCH2), 3.41e3.46 (m, 4H, eCH2), 2.61 (t, J ¼ 6.12 Hz, 4H, eCH2). 13C NMR (100 MHz, DMSO-d6): d166.25, 150.63, 146.51, 145.80, 135.74, 132.18, 129.27, 125.70, 114.12, 88.66, 78.71, 60.82, 59.10, 56.75. MS (ESIþ) m/z: 388.7 [MþH]þ. IR (KBr pellet, cm1): 3417, 2926, 2796, 2227, 1624, 1597, 1535, 1487, 1313, 1170, 827.

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4.2.28. 5-(Bis(2-hydroxyethyl)amino)methyl-7-(4bromophenylamino)-3-cyanopyrazolo[1,5-a] pyrimidine (17c) Yield: 87.4%. mp: 160e161  C. 1H NMR (400 MHz, DMSO-d6): d 10.45 (s, 1H, eNH), 8.73 (s, 1H, eCH), 7.62 (d, J ¼ 8.72 Hz, 2H, ArH), 7.46 (d, J ¼ 8.76 Hz, 2H, ArH), 6.90 (s, 1H, eCH), 4.42 (t, J ¼ 5.12 Hz, 2H, eOH), 3.78 (s, 2H, eCH2), 3.42e3.46 (m, 4H, eCH2), 2.62 (t, J ¼ 6.08 Hz, 4H, eCH2). 13C NMR (100 MHz, DMSO-d6): d166.26, 150.63, 146.50, 145.67, 136.19, 132.18, 129.27, 125.93, 125.69, 118.00, 114.11, 88.71, 78.73, 60.82, 59.09, 56.74. MS (ESIþ) m/z: 432.9 [MþH]þ. IR (KBr pellet, cm1): 3317, 2879, 2227, 16,222, 1562, 1535, 1485, 1290, 1076, 819, 534. 4.2.29. 5-(Bis(2-hydroxyethyl)amino)methyl-7-(4-(trifluoromethyl) phenylamino)-3-cyanopyrazolo [1,5-a]pyrimidine (17d) Yield: 88.1%. mp: 146e147  C. 1H NMR (400 MHz, DMSO-d6): d 10.68 (s, 1H, eNH), 8.76 (s, 1H, eCH), 7.81 (d, J ¼ 8.68 Hz, 2H, ArH), 7.74 (d, J ¼ 8.56 Hz, 2H, ArH), 7.10 (s, 1H, eCH), 4.44 (t, J ¼ 4.96 Hz, 2H, eOH), 3.81 (s, 2H, eCH2), 3.44e3.48 (m, 4H, eCH2), 2.64 (t, J ¼ 6.00 Hz, 4H, eCH2). 13C NMR (100 MHz, DMSO-d6): d166.62, 150.66, 146.54, 145.17, 140.95, 126.47, 126.43, 125.48, 125.13, 123.27, 114.03, 89.45, 78.95, 60.84, 59.05, 56.73. MS (ESIþ) m/z: 421.2 [MþH]þ. IR (KBr pellet, cm1): 3365, 2945, 2827, 2229, 1627, 1566, 1541, 1413, 1325, 1116, 761. 4.2.30. 5-(Bis(2-hydroxyethyl)amino)methyl-7-(4(ethoxycarbonyl)phenylamino)-3-cyanopyrazolo [1,5-a]pyrimidine (17e) Yield: 65.6%. mp: 136e138  C. 1H NMR (400 MHz, DMSO-d6): d 10.62 (s, 1H, eNH), 8.75 (s, 1H, eCH), 8.02 (d, J ¼ 8.44 Hz, 2H, ArH), 7.65 (d, J ¼ 8.72 Hz, 2H, ArH), 7.12 (s, 1H, eCH), 4.43 (t, J ¼ 5.28 Hz, 2H, eOH), 4.32 (q, J ¼ 7.04 Hz, 2H, eCH2), 3.80 (s, 2H, eCH2), 3.43e3.47 (m, 4H, eCH2), 2.63 (t, J ¼ 6.00 Hz, 4H, eCH2), 2.1.34 (t, J ¼ 7.08 Hz, 3H, eCH3). 13C NMR (100 MHz, DMSO-d6): d166.60, 165.08, 150.67, 146.49, 145.00, 141.57, 130.39, 126.20, 122.47, 114.01, 89.63, 78.98, 60.88, 60.64, 59.12, 56.79. MS (ESIþ) m/z: 425.1 [MþH]þ. IR (KBr pellet, cm1): 3439, 2821, 2231, 1716, 1626, 1595, 1562, 1537, 1276, 1176, 1022, 756. 4.2.31. 5-(Bis(2-hydroxyethyl)amino)methyl-7-(3fluorophenylamino)-3-cyanopyrazolo[1,5-a] pyrimidine (17f) Yield: 85.9%. mp: 154e155  C. 1H NMR (400 MHz, DMSO-d6): d 10.49 (s, 1H, eNH), 8.74 (s, 1H, eCH), 8.03 (m, 1H, ArH), 7.36 (t, J ¼ 8.80 Hz, 2H, ArH), 7.13 (t, J ¼ 8.76 Hz, 1H, ArH), 6.95 (s, 1H, eCH), 4.39 (s, 2H, eOH), 3.79 (s, 2H, eCH2), 3.44 (t, J ¼ 6.12 Hz, 4H, eCH2), 2.63 (t, J ¼ 6.00 Hz, 4H, eCH2). 13C NMR (100 MHz, DMSO-d6): d166.42, 163.56, 150.61, 146.48, 145.61, 138.71, 119.58, 114.08, 112.56, 110.99, 88.98, 78.81, 60.89, 59.22, 56.86. MS (ESIþ) m/z: 371.5 [MþH]þ. IR (KBr pellet, cm1): 3388, 2953, 2231, 1625, 1577, 1533, 1492, 1259, 1143, 756. 4.2.32. 5-(Bis(2-hydroxyethyl)amino)methyl-7-(3chlorophenylamino)-3-cyanopyrazolo[1,5-a] pyrimidine (17g) Yield: 93.8%. mp: 177e178  C. 1H NMR (400 MHz, DMSO-d6): d 10.48 (s, 1H, eNH), 8.73 (s, 1H, eCH), 7.55 (s, 1H, eArH), 7.48 (d, J ¼ 5.08 Hz, 1H, ArH), 7.34e7.37 (m, 2H, ArH), 6.89 (s, 1H, eCH), 4.38 (t, J ¼ 4.84 Hz, 2H, eOH), 3.77 (s, 2H, eCH2), 3.41e3.46 (m, 4H, eCH2), 2.62 (t, J ¼ 6.20 Hz, 4H, eCH2). 13C NMR (100 MHz, DMSOd6): d166.40, 150.66, 146.53, 145.73, 138.54, 133.58, 130.91, 125.63, 123.69, 122.33, 114.12, 88.89, 78.74, 60.90, 59.28, 56.89. MS (ESIþ) m/z: 388.8 [MþH]þ. IR (KBr pellet, cm1): 3350, 2953, 2816, 2231, 1624, 1589, 1566, 1479, 1315, 1080, 798. 4.2.33. 5-(Bis(2-hydroxyethyl)amino)methyl-7-(3bromophenylamino)-3-cyanopyrazolo[1,5-a] pyrimidine (17h) Yield: 90.1%. mp: 178e179  C. 1H NMR (400 MHz, DMSO-d6):

d 10.47 (s, 1H, eNH), 8.74 (s, 1H, eCH), 7.69 (s, 1H, ArH), 7.51 (q, J ¼ 7.92 Hz, 2H, ArH), 7.42 (t, J ¼ 7.92 Hz, 1H, ArH), 6.89 (s, 1H, eCH), 4.38 (s, 2H, eOH), 3.78 (s, 2H, eCH2), 3.44 (s, 4H, eCH2), 2.62 (t, J ¼ 6.20 Hz, 4H, eCH2). 13C NMR (100 MHz, DMSO-d6): d166.40, 150.64, 146.54, 145.71, 138.58, 131.17, 128.56, 122.74, 121.89, 114.11, 88.87, 78.77, 60.91, 59.30, 56.90. MS (ESIþ) m/z: 432.9 [MþH]þ. IR (KBr pellet, cm1): 3271, 2949, 2829, 2231, 1625, 1587, 1541, 1477, 1315, 1180, 796. 4.2.34. 5-(Bis(2-hydroxyethyl)amino)methyl-7-(m-toluidino)-3cyanopyrazolo[1,5-a]pyrimidine (17i) Yield: 61.7%. mp: 136e137  C. 1H NMR (400 MHz, DMSO-d6): d 10.26 (s, 1H, eNH), 8.71 (s, 1H, eCH), 7.36 (t, J ¼ 7.72 Hz, 1H, ArH), 7.28 (t, J ¼ 8.36 Hz, 2H, ArH), 7.13 (d, J ¼ 7.32 Hz, 1H, ArH), 6.81 (s, 1H, eCH), 4.39 (t, J ¼ 5.24 Hz, 2H, eOH), 3.76 (s, 2H, eCH2), 3.40e3.45 (m, 4H, eCH2), 2.61 (t, J ¼ 6.20 Hz, 4H, eCH2), 2.36 (s, 3H, eCH3). 13C NMR (100 MHz, DMSO-d6): d166.08, 150.67, 146.48, 146.14, 138.93, 136.51, 129.21, 126.79, 124.60, 121.36, 114.20, 88.45, 78.55, 60.96, 59.30, 56.88, 20.87. MS (ESIþ) m/z: 367.9 [MþH]þ. IR (KBr pellet, cm1): 3348, 2938, 2786, 2227, 1625, 1579, 1537, 1492, 1315, 1078, 798. 4.2.35. 5-(Bis(2-hydroxyethyl)amino)methyl-7-(3ethynylphenylamino)-3-cyanopyrazolo[1,5-a] pyrimidine (17j) Yield: 93.9%. mp: 176e178  C. 1H NMR (400 MHz, DMSO-d6): d 10.41 (s, 1H, eNH), 8.72 (s, 1H, eCH), 7.55 (d, J ¼ 8.68 Hz, 2H, ArH), 7.48 (t, J ¼ 7.64 Hz, 1H, ArH), 7.39 (d, J ¼ 7.60 Hz, 1H, ArH), 6.83 (s, 1H, eCH), 4.37 (t, J ¼ 5.32 Hz, 2H, -OH), 4.27 (s, 1H, eCH), 3.77 (s, 2H, eCH2), 3.40e3.45 (m, 4H, eCH2), 2.61 (t, J ¼ 6.20 Hz, 4H, eCH2). 13C NMR (100 MHz, DMSO-d6): d166.24, 150.67, 146.50, 145.92, 137.24, 129.79, 129.13, 127.08, 124.61, 122.86, 114.15, 88.69, 82.65, 81.64, 78.68, 60.88, 59.28, 56.88. MS (ESIþ) m/z: 377.13 [MþH]þ. IR (KBr pellet, cm1): 3278, 2821, 2227, 1624, 1571, 1539, 1487, 1082, 805. 4.2.36. 5-(Bis(2-hydroxyethyl)amino)methyl-7-(2chlorophenylamino)-3-cyanopyrazolo[1,5-a] pyrimidine (17k) Yield: 80.8%. mp: 163e164  C. 1H NMR (400 MHz, DMSO-d6): d 10.35 (s, 1H, eNH), 8.74 (s, 1H, eCH), 7.68 (d, J ¼ 7.40 Hz, 1H, ArH), 7.57 (d, J ¼ 7.72 Hz, 1H, ArH), 7.44e7.53 (m, 2H, ArH), 6.23 (s, 1H, eCH), 4.33 (s, 2H, eOH), 3.74 (s, 2H, eCH2), 3.37 (s, 4H, eCH2), 2.57 (t, J ¼ 6.32 Hz, 4H, eCH2). 13C NMR (100 MHz, DMSO-d6): d166.04, 150.49, 146.79, 146.48, 133.46, 130.99, 130.38, 129.41, 129.33, 128.47, 114.13, 88.66, 78.60, 60.75, 59.21, 56.78. MS (ESIþ) m/z: 388.9 [MþH]þ. IR (KBr pellet, cm1): 3307, 2808, 2229, 1624, 15,997, 1564, 1539, 1471, 1080, 763. 4.2.37. 5-(Bis(2-hydroxyethyl)amino)methyl-7-(2bromophenylamino)-3-cyanopyrazolo[1,5-a] pyrimidine (17l) Yield: 81.6%. mp: 172e173  C. 1H NMR (400 MHz, DMSO-d6): d 10.34 (s, 1H, eNH), 8.74 (s, 1H, eCH), 7.84 (d, J ¼ 7.92 Hz, 1H, ArH), 7.52e7.59 (m, 2H, ArH), 7.39 (t, J ¼ 8.24 Hz, 1H, ArH), 6.19 (s, 1H, eCH), 4.32 (s, 2H, eOH), 3.74 (s, 2H, eCH2), 3.34e3.38 (m, 4H, eCH2), 2.57 (t, J ¼ 6.32 Hz, 4H, eCH2). 13C NMR (100 MHz, DMSOd6): d165.91, 150.48, 146.79, 146.51, 134.88, 129.68, 129.59, 129.11, 121.81, 114.12, 88.71, 78.61, 60.71, 59.19, 56.76. MS (ESIþ) m/z: 432.9 [MþH]þ. IR (KBr pellet, cm1): 3300, 2808, 2229, 1622, 1593, 1537, 1471, 1313, 1078, 763. 4.2.38. 5-(Bis(2-hydroxyethyl)amino)methyl-7-(o-toluidino)-3cyanopyrazolo[1,5-a]pyrimidine (17m) Yield: 65.6%. mp: 116e117  C. 1H NMR (400 MHz, CDCl3): d 8.28 (s, 1H, eNH), 7.93 (s, 1H, eCH), 7.33e7.39 (m, 4H, ArH), 6.24 (s, 1H, eCH), 3.83 (s, 2H, eCH2), 3.60 (t, J ¼ 5.00 Hz, 4H, eCH2), 2.82 (t, J ¼ 4.96 Hz, 4H, eCH2), 2.33 (s, 3H, eCH3). 13C NMR (100 MHz, DMSO-d6): d165.58, 150.40, 146.56, 146.24, 134.23, 133.41, 128.29,

M. Zhao et al. / European Journal of Medicinal Chemistry 119 (2016) 183e196

127.52, 125.92, 113.15, 87.97, 81.29, 60.26, 59.79, 57.46, 17.64. MS (ESIþ) m/z: 367.0 [MþH]þ. IR (KBr pellet, cm1): 3292, 2873, 2229, 1622, 1575, 1537, 1479, 1309, 1078, 754. 4.2.39. 5-(Bis(2-hydroxyethyl)amino)methyl-7-(3,5difluorophenylamino)-3-cyanopyrazolo[1,5-a] pyrimidine (17n) Yield: 79.6%. mp: 177e178  C. 1H NMR (400 MHz, DMSO-d6): d 10.58 (s, 1H, eNH), 8.75 (s, 1H, eCH), 7.25 (d, J ¼ 8.52 Hz, 2H, ArH), 7.13 (t, J ¼ 9.32 Hz, 1H, ArH), 7.06 (s, 1H, eCH), 4.41 (s, 2H, eOH), 3.81 (s, 2H, eCH2), 3.45 (s, 4H, eCH2), 2.64 (t, J ¼ 6.12 Hz, 4H, eCH2). 13 C NMR (100 MHz, DMSO-d6): d166.67, 163.78, 161.49, 150.56, 146.52, 146.52, 145.11, 139.88, 113.97, 106.67, 106.39, 100.77, 88.73, 79.01, 60.84, 59.22, 56.94. MS (ESIþ) m/z: 389.2 [MþH]þ. IR (KBr pellet, cm1): 3350, 2850, 2231, 1614, 1583, 1537, 1473, 1313, 1145, 727. 4.2.40. 5-(Bis(2-hydroxyethyl)amino)methyl-7-(3,5dimethylphenylamino)-3-cyanopyrazolo[1,5-a] pyrimidine (17o) Yield: 82.4%. mp: 116e117  C. 1H NMR (400 MHz, DMSO-d6): d 10.24 (s, 1H, eNH), 8.69 (s, 1H, eCH), 7.03 (s, 2H, ArH), 6.95 (s, 1H, ArH), 6.79 (s, 1H, eCH), 4.38 (s, J ¼ 5.32 Hz, 2H, eOH), 3.75 (s, 2H, eCH2), 3.40e3.45 (m, 4H, eCH2), 2.61 (t, J ¼ 6.28 Hz, 4H, eCH2), 2.31 (s, 6H, eCH3). 13C NMR (100 MHz, DMSO-d6): d165.96, 150.72, 146.41, 146.21, 138.66, 136.65, 127.52, 121.77, 114.25, 88.48, 78.44, 61.02, 59.39, 56.92, 20.79. MS (ESIþ) m/z: 381.7 [MþH]þ. IR (KBr pellet, cm1): 3481, 2945, 2222, 1624, 1578, 1531, 1417, 1319, 1043, 688. 4.2.41. 5-(Bis(2-hydroxyethyl)amino)methyl-7-(3,5dimethoxyphenylamino)-3-cyanopyrazolo[1,5-a] pyrimidine (17p) Yield: 83.9%. mp: 140e142  C. 1H NMR (400 MHz, DMSO-d6): d 10.29 (s, 1H, eNH), 8.72 (s, 1H, eCH), 6.90 (s, 1H, ArH), 6.68 (s, 2H, ArH), 6.43 (s, 1H, eCH), 4.37 (s, J ¼ 5.00 Hz, 2H, eOH), 3.77 (s, 8H, eCH2 and eCH3), 3.40e3.45 (m, 4H, eCH2), 2.63 (t, J ¼ 6.16 Hz, 4H, eCH2). 13C NMR (100 MHz, DMSO-d6): d166.09, 160.89, 150.59, 146.46, 145.84, 138.41, 114.16, 102.05, 98.23, 89.25, 78.69, 60.85, 59.29, 57.00, 55.33. MS (ESIþ) m/z: 413.5 [MþH]þ. IR (KBr pellet, cm1): 3356, 2939, 2224, 1614, 1575, 1535, 1458, 1311, 1155, 680. 4.2.42. 5-(Bis(2-hydroxyethyl)amino)methyl-7-(4-fluoro-3(trifluoromethyl)phenylamino)-3- cyanopyrazolo [1,5-a]pyrimidine (17q) Yield: 81.9%. mp: 194e195  C. 1H NMR (400 MHz, DMSO-d6): d 8.02 (s, 1H, eCH), 7.29 (t, J ¼ 9.08 Hz, 1H, ArH), 7.13e7.17 (m, 1H, ArH), 7.08e7.10 (m, 1H, ArH), 5.84 (s, 1H, eCH), 4.39 (s, 2H, eOH), 3.41 (m, 6H, eCH2), 2.56 (t, J ¼ 6.40 Hz, 4H, eCH2). 13C NMR (100 MHz, DMSO-d6): d160.11, 153.37, 149.78, 148.86, 143.61, 127.87, 121.54, 119.76, 117.43, 117.23, 116.30, 87.54, 74.75, 60.47, 59.26, 56.92. MS (ESIþ) m/z: 439.1 [MþH]þ. IR (KBr pellet, cm1): 3343, 2903, 2222, 1626, 1580, 1518, 1313, 1076, 748. 4.2.43. 5-(Bis(2-hydroxyethyl)amino)methyl-7-(4-fluoro-3chlorophenylamino)-3-cyanopyrazolo [1,5-a]pyrimidine (17r) Yield: 96.5%. mp: 207e208  C. 1H NMR (400 MHz, DMSO-d6): d 8.04 (s, 1H, eCH), 7.22 (t, J ¼ 9.00 Hz, 1H, ArH), 6.96e6.99 (m, 1H, ArH), 6.83e6.86 (m, 1H, ArH), 5.87 (s, 1H, eCH), 4.40 (t, J ¼ 5.24 Hz, 2H, eOH), 3.37e3.42 (m, 6H, eCH2), 2.57 (t, J ¼ 6.44 Hz, 4H, eCH2). 13 C NMR (100 MHz, DMSO-d6): d160.08, 153.30, 150.54, 149.41, 148.99, 143.61, 123.33, 122.25, 118.97, 116.78, 116.28, 88.81, 74.77, 60.49, 59.29, 56.95. MS (ESIþ) m/z: 405.0 [MþH]þ. IR (KBr pellet, cm1): 3446, 2879, 2210, 1608, 1552, 1483, 1444, 1190, 704. 4.2.44. 5-(Bis(2-hydroxyethyl)amino)methyl-7-(4-fluoro-3methylphenylamino)-3-cyanopyrazolo [1,5-a]pyrimidine (17s) Yield: 86.3%. mp: 142e143  C. 1H NMR (400 MHz, DMSO-d6):

193

d 10.25 (s, 1H, eNH), 8.69 (s, 1H, eCH), 7.37 (d, J ¼ 6.84 Hz, 1H, ArH),

7.21e7.31 (m, 2H, ArH), 6.72 (s, 1H, eCH), 4.38 (t, J ¼ 5.20 Hz, 2H, eOH), 3.74 (s, 2H, eCH2), 3.39e3.44 (m, 4H, eCH2), 2.60 (t, J ¼ 6.20 Hz, 4H, eCH2), 2.27 (s, 3H, eCH3). 13C NMR (100 MHz, DMSO-d6): d166.06, 159.73, 157.32, 150.66, 146.45, 132.68, 127.65, 125.31, 123.95, 115.60, 114.23, 88.26, 78.45, 60.92, 59.25, 56.82, 14.09. MS (ESIþ) m/z: 385.8 [MþH]þ. IR (KBr pellet, cm1): 3383, 2924, 2226, 1624, 1577, 1535, 1498, 1313, 1076. 4.2.44.1. General procedure to synthesize compounds 18a-s. A solution of compounds 17a-s (1.7 mmol) and SOCl2 (3.5 mmol) in CH2Cl2 (5 mL) was stirred at room temperature overnight. The solution was poured into water and extracted with CHCl2. The organic phase was washed with water, then dried over anhydrous sodium sulfate, filtered and evaporated. The resulting solid obtained was purified by silica column chromatography with ethyl acetate and petrol as the eluent (v:v). 4.2.45. 5-(Bis(2-chloroethyl)amino)methyl-7-(4fluorophenylamino)-3-cyanopyrazolo[1,5-a] pyrimidine (18a) Yield: 16.8%. mp: 142e143  C. 1H NMR (400 MHz, CDCl3): d 8.28 (s, 1H, -CH), 8.04 (s, 1H, eNH), 7.40 (d, J ¼ 8.96 Hz, 2H, ArH), 7.20 (d, J ¼ 8.20 Hz, 2H, ArH), 6.98 (s, 1H, eCH), 3.89 (s, 2H, eCH2), 3.51 (t, J ¼ 6.32 Hz, 4H, eCH2CH2Cl), 2.98 (t, J ¼ 6.28 Hz, 4H, eCH2CH2Cl). 13 C NMR (100 MHz, CDCl3): d 165.68, 162.58, 160.12, 150.33, 146.48, 146.20, 131.24, 126.57, 117.07, 113.41, 88.11, 81.12, 60.90, 56.60, 42.08. MS (ESIþ) m/z: 407.1 [MþH]þ. IR (KBr pellet, cm1): 3354, 3097, 2819, 2224, 1620, 1579, 1571, 1512, 1319, 1155, 759. 4.2.46. 5-(Bis(2-chloroethyl)amino)methyl-7-(4chlorophenylamino)-3-cyanopyrazolo[1,5-a] pyrimidine (18b) Yield: 19.6%. mp: 122e123  C. 1H NMR (400 MHz, CDCl3): d 8.29 (s, 1H, eCH), 8.09 (s, 1H, eNH), 7.46 (d, J ¼ 8.76 Hz, 2H, ArH), 7.36 (d, J ¼ 8.68 Hz, 2H, ArH), 7.10 (s, 1H, eCH), 3.91 (s, 2H, eCH2), 3.54 (t, J ¼ 6.28 Hz, 4H, eCH2CH2Cl), 3.00 (t, J ¼ 6.28 Hz, 4H, eCH2CH2Cl). MS (ESIþ) m/z: 424.6 [MþH]þ. IR (KBr pellet, cm1): 3342, 2837, 2226, 1623, 1593, 1562, 1483, 1315, 1097, 805. 4.2.47. 5-(Bis(2-chloroethyl)amino)methyl-7-(4bromophenylamino)-3-cyanopyrazolo[1,5-a] pyrimidine (18c) Yield: 27.3%. mp: 100e101  C. 1H NMR (400 MHz, CDCl3): d 8.28 (s, 1H, eCH), 8.10 (s, 1H, eNH), 7.61 (d, J ¼ 8.72 Hz, 2H, ArH), 7.30 (d, J ¼ 8.72 Hz, 2H, ArH), 7.11 (s, 1H, eCH), 3.91 (s, 2H, eCH2), 3.54 (t, J ¼ 6.28 Hz, 4H, eCH2CH2Cl), 3.00 (t, J ¼ 6.24 Hz, 4H, eCH2CH2Cl). 13 C NMR (100 MHz, CDCl3): d 165.85, 150.28, 146.46, 145.32, 134.56, 133.11, 125.35, 120.23, 113.30, 88.32, 81.39, 60.96, 56.62, 42.14. MS (ESIþ) m/z: 468.9 [MþH]þ. IR (KBr pellet, cm1): 3363, 2926, 2222, 1627, 1562, 1531, 1481, 1319, 1126, 821. 4.2.48. 5-(Bis(2-chloroethyl)amino)methyl-7-(4-(trifluoromethyl) phenylamino)-3-cyanopyrazolo [1,5-a] pyrimidine (18d) Yield: 14.8%. mp: 90e92  C. 1H NMR (400 MHz, CDCl3): d 8.31 (s, 2H, eCH and eNH), 7.75 (d, J ¼ 8.48 Hz, 2H, ArH), 7.55 (d, J ¼ 8.44 Hz, 2H, ArH), 7.31 (s, 1H, eCH), 3.94 (s, 2H, eCH2), 3.56 (t, J ¼ 6.16 Hz, 4H, eCH2CH2Cl), 3.02 (t, J ¼ 6.12 Hz, 4H, eCH2CH2Cl). 13C NMR (100 MHz, CDCl3): d166.11, 150.28, 146.47, 138.99, 128.63, 127.24, 127.13, 125.05, 122.86, 113.22, 88.81, 81.55, 60.97, 56.64, 42.20. MS (ESIþ) m/z: 457.1 [MþH]þ. IR (KBr pellet, cm1): 3340, 2827, 2224, 1602, 1569, 1537, 1323, 1168, 750. 4.2.49. 5-(Bis(2-chloroethyl)amino)methyl-7-(4-(ethoxycarbonyl) phenylamino)-3-cyanopyrazolo [1,5-a]pyrimidine (18e) Yield: 17.8%. mp: 128e129  C. 1H NMR (400 MHz, CDCl3): d 8.32 (s, 1H, eCH), 8.30 (s, 1H, eNH), 8.16 (d, J ¼ 8.48 Hz, 2H, ArH), 7.48 (d, J ¼ 8.48 Hz, 2H, ArH), 7.33 (s, 1H, eCH), 4.41 (q, J ¼ 7.08 Hz, 2H,

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eCH2), 3.94 (s, 2H, eCH2), 3.56 (t, J ¼ 6.20 Hz, 4H, eCH2CH2Cl), 3.03 (t, J ¼ 6.20 Hz, 4H, eCH2CH2Cl), 1.43 (t, J ¼ 7.12 Hz, 2H, eCH3). 13C NMR (100 MHz, CDCl3): d 164.99, 164.58, 149.26, 145.43, 143.52, 138.79, 130.47, 127.29, 120.87, 112.22, 87.95, 80.61, 60.26, 59.97, 55.66, 41.15, 13.33. MS (ESIþ) m/z: 461.1 [MþH]þ. IR (KBr pellet, cm1): 3277, 2927, 2225, 1697, 1592, 1537, 1519, 1266, 1170, 767. 4.2.50. 5-(Bis(2-chloroethyl)amino)methyl-7-(3fluorophenylamino)-3-cyanopyrazolo[1,5-a] pyrimidine (18f) Yield: 32.4%. mp: 98e100  C. 1H NMR (400 MHz, CDCl3): d 8.29 (s, 1H, eCH), 8.17 (s, 1H, eNH), 8.45 (q, J ¼ 7.84 Hz, 1H, ArH), 7.15e7.22 (m, 3H, ArH), 7.05 (s, 1H, eCH), 3.92 (s, 2H, eCH2), 3.54 (t, J ¼ 6.16 Hz, 4H, eCH2CH2Cl), 3.01 (t, J ¼ 6.12 Hz, 4H, eCH2CH2Cl). 13C NMR (100 MHz, CDCl3): d 165.93, 164.50, 162.03, 150.30, 146.44, 145.25, 137.12, 131.23, 119.17, 113.95, 110.84, 88.59, 81.26, 60.88, 56.57, 42.11. MS (ESIþ) m/z: 407.1 [MþH]þ. IR (KBr pellet, cm1): 3325, 2814, 2224, 1629, 1577, 1496, 1313, 1149, 758. 4.2.51. 5-(Bis(2-chloroethyl)amino)methyl-7-(3chlorophenylamino)-3-cyanopyrazolo[1,5-a] pyrimidine (18g) Yield: 30.2%. mp: 119e120  C. 1H NMR (400 MHz, CDCl3): d 8.31 (s, 1H, eCH), 8.13 (s, 1H, eNH), 7.41e7.44 (m, 2H, ArH), 7.30e7.33 (m, 2H, ArH), 7.18 (s, 1H, eCH), 3.91 (s, 2H, eCH2), 3.54 (t, J ¼ 6.24 Hz, 4H, eCH2CH2Cl), 3.01 (t, J ¼ 6.24 Hz, 4H, eCH2CH2Cl). MS (ESIþ) m/z: 423.0 [MþH]þ. IR (KBr pellet, cm1): 3284, 2933, 2227, 1624, 1570, 1560, 1475, 1311, 883. 4.2.52. 5-(Bis(2-chloroethyl)amino)methyl-7-(3bromophenylamino)-3-cyanopyrazolo[1,5-a] pyrimidine (18h) Yield: 17.1%. mp: 86e88  C. 1H NMR (400 MHz, CDCl3): d 8.29 (s, 1H, eCH), 8.13 (s, 1H, eNH), 7.59 (s, 1H, ArH), 7.46e7.49 (m, 1H, ArH), 7.37 (s, 2H, ArH), 7.18 (s, 1H, eCH), 3.91 (s, 2H, eCH2), 3.54 (t, J ¼ 6.24 Hz, 4H, eCH2CH2Cl), 3.01 (t, J ¼ 6.24 Hz, 4H, eCH2CH2Cl). 13 C NMR (100 MHz, CDCl3): d 165.98, 150.27, 146.48, 145.21, 136.84, 131.22, 130.03, 126.57, 123.41, 122.21, 113.33, 88.47, 81.38, 60.95, 56.65, 42.16. MS (ESIþ) m/z: 468.9 [MþH]þ. IR (KBr pellet, cm1): 3294, 2812, 2218, 1606, 1535, 1477, 1427, 1307, 1174, 779.

13 C NMR (100 MHz, CDCl3): d 165.88, 150.30, 146.55, 145.04, 132.77, 130.61, 128.46, 128.10, 127.80, 124.70, 113.34, 88.62, 81.37, 60.91, 56.58, 42.15. MS (ESIþ) m/z: 423.0 [MþH]þ. IR (KBr pellet, cm1): 3298, 2845, 2228, 1625, 1555, 1543, 1328, 1128, 748.

4.2.56. 5-(Bis(2-chloroethyl)amino)methyl-7-(2bromophenylamino)-3-cyanopyrazolo[1,5-a] pyrimidine (18l) Yield: 21.4%. mp: 122e124  C. 1H NMR (400 MHz, CDCl3): d 8.32 (s, 1H, eCH), 8.30 (s, 1H, eNH), 7.74 (d, J ¼ 7.16 Hz, 1H, ArH), 7.66 (d, J ¼ 7.28 Hz, 1H, ArH), 7.45 (t, J ¼ 7.88 Hz, 1H, ArH), 7.23 (t, J ¼ 7.48 Hz, 1H, ArH), 7.09 (s, 1H, eCH), 3.91 (s, 2H, eCH2), 3.53 (t, J ¼ 6.24 Hz, 4H, eCH2CH2Cl), 3.00 (t, J ¼ 6.28 Hz, 4H, eCH2CH2Cl). MS (ESIþ) m/ z: 468.9 [MþH]þ. IR (KBr pellet, cm1): 3280, 2976, 2225, 1626, 1598, 1543, 1452, 1128, 746. 4.2.57. 5-(Bis(2-chloroethyl)amino)methyl-7-(o-toluidino)-3cyanopyrazolo[1,5-a]pyrimidine (18m) Yield: 21.6%. mp: 118e119  C. 1H NMR (400 MHz, CDCl3): d 8.29 (s, 1H, eCH), 7.89 (s, 1H, eNH), 7.32 (m, 4H, ArH), 6.69 (s, 1H, eCH), 3.87 (s, 2H, eCH2), 3.47 (t, J ¼ 6.40 Hz, 4H, eCH2CH2Cl), 2.96 (t, J ¼ 6.40 Hz, 4H, eCH2CH2Cl), 2.33 (s, 2H, eCH3). 13C NMR (100 MHz, CDCl3): d 165.51, 150.40, 146.53, 146.41, 134.16, 131.64, 128.18, 127.45, 126.04, 113.52, 88.23, 80.97, 60.78, 56.52, 41.94, 17.66. MS (ESIþ) m/z: 403.1 [MþH]þ. IR (KBr pellet, cm1): 3292, 2816, 2224, 1622, 1577, 1533, 1312, 750. 4.2.58. 5-(Bis(2-chloroethyl)amino)methyl-7-(3,5difluorophenylamino)-3-cyanopyrazolo[1,5-a] pyrimidine (18n) Yield: 16.9%. mp: 122e123  C. 1H NMR (400 MHz, CDCl3): d 8.29 (s, 1H, eCH), 8.19 (s, 1H, eNH), 7.32 (s, 1H, CH), 6.99 (d, 2H, J ¼ 5.72 Hz, ArH), 6.78 (t, J ¼ 8.68 Hz, 1H, eArH), 3.94 (s, 2H, eCH2), 3.56 (t, J ¼ 6.12 Hz, 4H, eCH2CH2Cl), 3.03 (t, J ¼ 6.12 Hz, 4H, eCH2CH2Cl). 13C NMR (100 MHz, CDCl3): d 166.20, 165.00, 161.92, 150.19, 146.48, 144.52, 137.88, 113.10, 106.46, 106.18, 88.90, 81.75, 60.97, 56.67, 42.10. MS (ESIþ) m/z: 425.1 [MþH]þ. IR (KBr pellet, cm1): 3327, 2816, 2229, 1614, 1573, 1545, 1481, 1307, 1118, 839.

4.2.53. 5-(Bis(2-chloroethyl)amino)methyl-7-(m-toluidino)-3cyanopyrazolo[1,5-a]pyrimidine (18i) Yield: 32.2%. mp: 104e105  C. 1H NMR (400 MHz, CDCl3): d 8.28 (s, 1H, eCH), 8.12 (s, 1H, eNH), 7.37 (t, J ¼ 7.76 Hz, 1H, ArH), 7.15e7.26 (m, 3H, ArH), 7.09 (s, 1H, eCH), 3.89 (s, 2H, eCH2), 3.53 (t, J ¼ 6.36 Hz, 4H, eCH2CH2Cl), 3.01 (t, J ¼ 6.28 Hz, 4H, eCH2CH2Cl), 2.42 (s, 3H, eCH3). MS (ESIþ) m/z: 403.1 [MþH]þ. IR (KBr pellet, cm1): 3313, 2802, 2222, 1624, 1577, 1533, 1480, 1323, 1122, 750.

4.2.59. 5-(Bis(2-chloroethyl)amino)methyl-7-(3,5dimethylphenylamino)-3-cyanopyrazolo[1,5-a] pyrimidine (18o) Yield: 16.6%. mp: 131e132  C. 1H NMR (400 MHz, CDCl3): d 8.27 (s, 1H, eCH), 8.06 (s, 1H, eNH), 7.09 (s, 1H, ArH), 7.10 (s, 2H, ArH), 6.97 (s, 1H, eCH), 3.89 (s, 2H, eCH2), 3.53 (t, J ¼ 6.24 Hz, 4H, eCH2CH2Cl), 2.99 (t, J ¼ 6.24 Hz, 4H, eCH2CH2Cl), 2.37 (s, 6H, eCH3). 13 C NMR (100 MHz, CDCl3): d 164.42, 149.34, 145.34, 144.78, 138.89, 134.22, 127.71, 120.25, 112.53, 87.44, 79.99, 59.89, 55.66, 41.10, 20.28. MS (ESIþ) m/z: 417.1 [MþH]þ. IR (KBr pellet, cm1): 3346, 2918, 2222, 1624, 1581, 1537, 1118, 835.

4.2.54. 5-(Bis(2-chloroethyl)amino)methyl-7-(3ethynylphenylamino)-3-cyanopyrazolo[1,5-a] pyrimidine (18j) Yield: 28.6%. mp: 121e122  C. 1H NMR (400 MHz, CDCl3): d 8.21 (s, 1H, eCH), 8.06 (s, 1H, eNH), 7.47 (s, 1H, ArH), 7.31e7.39 (m, 3H, ArH), 7.06 (s, 1H, eCH), 3.84 (s, 2H, eCH2), 3.46 (t, J ¼ 6.32 Hz, 4H, eCH2CH2Cl), 3.09 (s, 1H, eCH), 2.93 (t, J ¼ 6.36 Hz, 4H, eCH2CH2Cl). 13 C NMR (100 MHz, CDCl3): d 165.78, 150.30, 146.48, 145.46, 135.59, 130.60, 130.05, 127.05, 124.11, 113.34, 88.42, 82.15, 81.33, 78.91, 60.81, 56.61, 41.93. MS (ESIþ) m/z: 413.1 [MþH]þ. IR (KBr pellet, cm1): 3285, 2933, 2222, 1624, 1589, 1572, 1535, 1122.

4.2.60. 5-(Bis(2-chloroethyl)amino)methyl-7-(3,5dimethoxyphenylamino)-3-cyanopyrazolo[1,5-a] pyrimidine (18p) Yield: 18.7%. mp: 161e162  C. 1H NMR (400 MHz, CDCl3): d 8.21 (s, 1H, eCH), 7.99 (s, 1H, eNH), 7.04 (s, 1H, ArH), 6.45 (s, 2H, ArH), 6.36 (s, 1H, eCH), 3.83 (s, 2H, eCH2), 3.76 (s, 6H, eOCH3), 3.46 (t, J ¼ 6.32 Hz, 4H, eCH2CH2Cl), 2.94 (t, J ¼ 6.32 Hz, 4H, eCH2CH2Cl). MS (ESIþ) m/z: 449.1 [MþH]þ. IR (KBr pellet, cm1): 3323, 2927, 2222, 1627, 1575, 1537, 1458, 1311, 1149, 752.

4.2.55. 5-(Bis(2-chloroethyl)amino)methyl-7-(2chlorophenylamino)-3-cyanopyrazolo[1,5-a] pyrimidine (18k) Yield: 23.6%. mp: 149e150  C. 1H NMR (400 MHz, CDCl3): d 8.31 (s, 1H, eCH and eNH), 7.65 (d, J ¼ 8.00 Hz, 1H, ArH), 7.56 (d, J ¼ 8.00 Hz, 1H, ArH), 7.41 (t, J ¼ 7.52 Hz, 1H, ArH), 7.29 (t, J ¼ 7.92 Hz, 1H, ArH), 7.11 (s, 1H, eCH), 3.92 (s, 2H, eCH2), 3.53 (t, J ¼ 6.24 Hz, 4H, eCH2CH2Cl), 3.01 (t, J ¼ 6.24 Hz, 4H, eCH2CH2Cl).

4.2.61. 5-(Bis(2-chloroethyl)amino)methyl-7-(4-fluoro-3(trifluoromethyl)phenylamino)-3- cyanopyrazolo[1,5-a]pyrimidine (18q) Yield: 13.7%. mp: 119e120  C. 1H NMR (400 MHz, CDCl3): d 8.29 (s, 1H, eCH), 8.09 (s, 1H, eNH), 7.63e7.66 (m, 2H, ArH), 7.35 (t, J ¼ 8.84 Hz, 1H, ArH), 7.07 (s, 1H, eCH), 3.91 (s, 2H, eCH2), 3.52 (t, J ¼ 6.08 Hz, 4H, eCH2CH2Cl), 2.99 (t, J ¼ 6.08 Hz, 4H, eCH2CH2Cl). 13 C NMR (100 MHz, CDCl3): d 166.12, 150.24, 146.58, 145.65, 131.68,

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130.06, 129.98, 123.53, 118.90, 113.17, 88.19, 81.55, 61.00, 56.72, 42.18. MS (ESIþ) m/z: 475.0 [MþH]þ. IR (KBr pellet, cm1): 3356, 2816, 2226, 1620, 1573, 1510, 1489, 1323, 1141. 4.2.62. 5-(Bis(2-chloroethyl)amino)methyl-7-(4-fluoro-3chlorophenylamino)-3-cyanopyrazolo [1,5-a]pyrimidine (18r) Yield: 17.8%. mp: 126e128  C. 1H NMR (400 MHz, CDCl3): d 8.29 (s, 1H, eCH), 8.03 (s, 1H, eNH), 7.51 (d, J ¼ 6.12 Hz, 1H, ArH), 7.29e7.33 (m, 2H, ArH), 7.09 (s, 1H, eCH), 3.91 (s, 2H, eCH2), 3.53 (t, J ¼ 6.16 Hz, 4H, eCH2CH2Cl), 2.99 (t, J ¼ 6.16 Hz, 4H, eCH2CH2Cl). 13C NMR (100 MHz, CDCl3): d 165.06, 157.03, 154.54, 149.25, 144.66, 125.70, 123.28, 121.36, 116.90, 112.33, 87.26, 80.30, 59.96, 55.64, 41.21. MS (ESIþ) m/z: 443.0 [MþH]þ. IR (KBr pellet, cm1): 3346, 2933, 2224, 1625, 1568, 1506, 1489, 1213, 1126, 750. 4.2.63. 5-(Bis(2-chloroethyl)amino)methyl-7-(4-fluoro-3methylphenylamino)-3-cyanopyrazolo[1,5-a] pyrimidine (18s) Yield: 37.8%. mp: 126e127  C. 1H NMR (400 MHz, CDCl3): d 8.21 (s, 1H, eCH), 7.92 (s, 1H, eNH), 7.16 (s, 1H, ArH), 7.11 (t, J ¼ 8.52 Hz, 1H, ArH), 7.04 (t, J ¼ 8.76 Hz, 1H, ArH), 6.92 (s, 1H, eCH), 3.81 (s, 2H, eCH2), 3.45 (t, J ¼ 6.28 Hz, 4H, eCH2CH2Cl), 2.91 (t, J ¼ 6.28 Hz, 4H, eCH2CH2Cl), 2.26 (s, 3H, eCH3). 13C NMR (100 MHz, CDCl3): d 164.68, 160.13, 157.68, 149.31, 145.45, 129.81, 126.65, 125.91, 122.53, 115.27, 112.47, 87.13, 80.05, 59.93, 55.63, 41.13, 13.67. MS (ESIþ) m/z: 421.1 [MþH]þ. IR (KBr pellet, cm1): 3315, 2825, 2222, 1618, 1579, 1535, 1487, 1217, 1180, 813.

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with 2 uM, 4 uM and 6 uM of compound 13b for 24 h. The cells were washed twice with ice-cold PBS and resuspended in 300 uL 1  Binding Buffer. Annexin V-FITC (5 uL) was then added into each sample and incubated in the dark for 15 min and then propidium iodide (5 uL) was added and stained for 5 min at room temperature in darkness. After adding 200 uL 1  Binding Buffer, the cellular apoptosis was analyzed by flow cytometry. 4.6. Tumor xenografts HepG-2 tumor cells (5  106) were injected subcutaneously in not more than 0.2 mL volume into the left flank of the animals. The animals were randomized divided into seven groups (n ¼ 5) and treated with the following three different samples via i.p. injection: (A) saline; (B) compound 13b (10 mg/kg); (C) compound 13b (20 mg/kg); (D) cyclophosphamide (10 mg/kg); (E) cyclophosphamide (20 mg/kg); (F) sorafenib (10 mg/kg); (G) sorafenib (20 mg/ kg). The tumor dimension and body weight of each mouse were recorded once every other day and volumes were calculated using the formula 1/2 [length (mm)] [width (mm)]2 when tumors had reached a volume of 130e160 mm3. The density of the tumor is equal to water. Each mouse underwent once daily peritoneal injections for 15 days. Relative body weight was calculated using the formula W/W0, in which W and W0 are the body weight of mouse on the day of measurement and on the initial day, respectively. Acknowledgments

4.3. Cytotoxic activities against tumor cells assay The cytotoxic activities of compounds 4-15a, 4-15b and 18a-s were evaluated against A549, SH-SY5Y, HepG-2, MCF-7, DU145 and GES-1 cell lines using the standard MTT assay in vitro, with Sorafenib and Melphalan used as the positive control. The cancer cell lines were cultured in DMEM or RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS). Approximate 2  103 cells, suspended in DMEM or RPMI 1640 medium, were plated onto each well of a 96-well plate and incubated in 5% CO2 at 37  C for 24 h. The title compounds tested at the indicated final concentrations (range from 0.1 uM to 1000 uM) were added to the culture medium and the cell cultures were continued for 72 h. Each concentration was tested in 5 wells. Fresh MTT (10 uL/well) was added to each well and incubated with cells at 37  C for 4 h. Then cell lysates were added into each well (100 uL/well) and incubated overnight. The absorbency was measured at 570 nm (for absorbance of MTT formazan) and 650 nm (for the reference wavelength) on an automated microplate spectrophotometer (The Wallac 1420 VICTOR3™). The results expressed as IC50 (inhibitory concentration 50%) were calculated using GraphPad Prism software (San Diego, CA). 4.4. Flow cytometry cell cycle analysis Cells were treated with compound 13b at 1 and 3 uM for 24 h and then were trypsinized, washed in ice-cold PBS, and fixed in icecold 70% alcohol at least 24 h. Thereafter, cells were resuspended in 0.4 mL PBS, stained with 30 mL RNase A (1 mg/ml) and 50 mL PI (500ug/ml). The staining process lasted 30 min at 4  C in darkness. The stained cells were then analyzed using flow cytometry and the cycle distribution was quantified. 4.5. Cell apoptosis assay To detect the apoptotic ratio of compound 13b-treated HepG2 tumor cells, the cells were planted at a density of 2  105 per well in 6-well plate and incubated for 24 h. Then the cells were treated

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