Synthesis of New Heteroaryl Substituted Morpholine ...

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RESEARCH ARTICLE

Synthesis of New Heteroaryl Substituted Morpholine Tagged Triazines and Evaluation of their Cytotoxic Activity Prasad Kasturia,b, Sujatha Surarapua, Srinivas Uppalanchia, Jaya Shree Anireddyb,*, Hasitha Shilpa Anantarajuc, Shubham Dwivedic, Perumal Yogeeswaric and Krishna S. Ethiraja a

Department of Medicinal Chemistry, GVK Biosciences Pvt. Ltd, Plot.No.28 A, IDA, Nacharam, Hyderabad 500076, Telangana State, India; bCentre for Chemical Sciences & Technology, Institute of Science and Technology, JNTUH, Kukatpally, Hyderabad 500085, Telangana State, India; cBiology Division, Drug Discovery Research Laboratory, Department of Pharmacy, Birla Institute of Technology & Science-Pilani, Hyderabad Campus, Hyderabad 500078, Telangana State, India

A R T I C L E H I S T O R Y

Abstract: Background: In the present study, new triazine derivatives 3, 4, 5, 6, 8 and 10 were synthesized starting from readily available cyanuric chloride 1 via nucleophilic displacement with morpholine followed by Suzuki or Stille coupling reactions and then the thermal displacement of chlorine atom with diverse substituted amines.

Received: March 04, 2017 Revised: May 10, 2017 Accepted: May 26, 2017

Method: All synthesized compounds were screened for their cytotoxic activity against HT-29, MDA-MB-231, and HEK293 cell lines.

DOI: 10.2174/1570180814666170605115335

Results and Conclusion: Compounds 6a (IC50 (µM): 0.32 for HT-29 and 2.92 for MDA-MB-231) and 8c (IC50 (µM): 1.40 for HT-29 and 1.60 for MDA-MB-231) have been identified and compared with Doxorubicin and ZSTK474 as the reference standards.

Keywords: Cyanuric chloride, cytotoxic activity, stille coupling, suzuki reaction and triazines. 1. INTRODUCTION Cancer is one of the leading causes of human death exceeded only by cardiovascular diseases [1]. Every year, tens of millions of people are diagnosed with cancer around the world and more than half of the patients eventually, die from it. There are three major groups of anticancer drugs: 1) Cytotoxic drugs the largest and these include alkylating agents, [2] antimetabolites, [3] antitumor antibodies, plant alkaloids and miscellaneous cytotoxic drugs, 2) Hormones [4] and hormone antagonists, 3) Immuno modulators. Traditional anticancer drugs have disadvantages like lack of specificity, toxicity, drug resistance and a lot of side effects. Hence, the development of new anticancer agents and more effective treatment strategies are of immense importance [5]. Although, several kinase inhibitors, like PI3K/mTOR, [6-8] have been discovered recently but still there is strong demand for the discovery of improved cytotoxic agents. Triazine derivatives have attracted organic chemists due to their various biological and chemotherapeutic importance. *Address correspondence to this author at the Centre for Chemical Sciences & Technology, Institute of Science and Technology, Faculty of Chemistry, JNTUH University, P.O. Box: 500085, Hyderabad, Telangana State, India; Tel: +0-040-2315-6128; Fax: +0-040-2305-8729; E-mail: [email protected] 1570-1808/17 $58.00+.00

1,3,5-triazine derivatives containing various amino groups at position 2, 4 or 6 such as Tretamine, Furazil and Dioxadet are in the clinical practice as anticancer drugs [9-15] (Fig. 1). Structural modifications in benzimidazole hybrids by the replacement of ethyleneimino moiety with either dialkylamino, alkoxy, alkylaryloxy or hydroxy groups led to the discovery of novel chemotherapeutic agents [16-21]. Moreover, numerous 2,4-diamino-1,3,5-triazines possess various biological activity. Several literature reports describe their potential as cardiotonic, [22] neuroleptic, [23] nootropic, [24] anti-mycobacterial, [25] tuberculostatic, [26] anti-HIV, [27] antiviral, [28] anti-microbial agents, [29, 30] histamine H4 receptor ligands, [31] human carbonic anhydrases II, IX and XII, [32] inhibitors of phosphoinositide 3kinase, [33] FAK inhibitors with anti-angiogenic activity [34]. Literature survey revealed that triazines with aryl groups have been studied [35, 36]. However, triazines substituted with heteroaryl groups like furan, thiophene and indole etc. have not been explored. In this context, we envisaged that different heteroaryl substituted morpholine tagged triazine derivatives may act as potential anticancer agents. We commenced our synthetic strategy towards a series of morpholine tagged heteroaryl substituted triazine derivatives (Schemes 1-4). Our key focus was to synthesize indole, furan & thio©2017 Bentham Science Publishers

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Kasturi et al.

2. RESULTS AND DISCUSSION Synthetic strategies to make heteroaryl substituted morpholine tagged triazines are depicted in Schemes 1-4. Controlled nucleophilic substitution of one of the chlorine atoms of cyanuric chloride 1 with one equivalent of morpholine in the presence of diisopropylethylamine at -78 oC furnished exclusively the dichloro-mono-morpholine triazine 2 product in 92% yield (Scheme 1). The addition of two equivalent of morpholine at 0 °C afforded the di-morpholine-monochloro triazine 9 as the major product in 95% yield (Scheme 4).

Fig. (1). Potent anticancer compounds possessing triazine core.

Suzuki coupling of dichloro-mono-morpholine triazine 2 with furan, indole & methyl indole boronic acids37 furnished the corresponding bis-heteroarylated morpholine triazines 3a, 3c, 3e and the monochloro-heteroaryl morpholine triazines 3b, 3d and 3f in approximately 1:2 ratio, respectively. Bis-heteroaryl and monochloro heteroaryl morpholine products were separated by silica gel column purification. Corresponding thiophene analogues 3g and 3h were synthesized using Stille conditions. Bis-thiophene triazine 3g and monochloro-thiophene triazine 3h formed in 1:2 ratio were easily separable by silica gel column purification (Scheme 1).

phene substituted triazines and screen their cytotoxic activity against MDA-MB-231, HT-29 and HEK293 cell lines. To compare the cytotoxic activity, Doxorubicin and ZSTK474 anticancer compounds were used as the reference standards.

Second nucleophilic displacement of chlorine atom in monochloro heteroaryl triazines with various amines resulted in three series of compounds 4, 5 & 6, respectively (Scheme 2) in moderate to good yields, ~59-85%. The cytotoxic activity of thiophene analogues was found very encouraging & hence

Synthesis of New Heteroaryl Substituted Morpholine Tagged Triazines

more number of compounds in this series was made. In addition to nucleophilic displacement (6a-6m), two more aryl substituted triazines 6n and 6o were made under Suzuki reaction conditions (Scheme 2). 1-Cyclopropylethanamine substituted triazine 8c showed promising activity, hence 8a8h were synthesized by coupling of 7 with corresponding amines or boronic acids or stannane reagents in good yields (Scheme 3). To compare the activity of synthesized compounds with ZSTK474, dimorpholine tagged s-triazine compounds 10a-

Letters in Drug Design & Discovery, 2017, Vol. 14, No. 0

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10e were prepared by reacting 9 with corresponding boronic acids or stannane under standard Suzuki and Stille reaction conditions to yield products in good yields (Scheme 4). Six series of heteroaryl-triazine derivatives have been synthesized (3a-3h, 4a-4d, 5a-5e, 6a-6o, 8a-8h and 10a10e) (Schemes 1-4) to explore the effect of furan, indole and thiophene groups on the cytotoxic activity against two types of human cancer cell lines, breast (MDA-MB-231) and colon cancer (HT-29) and human normal cell line (HEK293) using Doxorubicin and ZSTK474 as standards.


Kasturi et al.

Compounds with a percentage of inhibition greater than 70% at 25 µM concentration were shortlisted and IC50 were calculated using MTT assay method38 in a concentrationdependent manner. It is evident from the bioactivity assay results that most of the test compounds have shown significant cytotoxic activity on HT-29 & MDA-MB-231 cell lines with a degree of variation (Table 1). Out of 45 compounds made, 11 compounds have been shown moderate (IC50 of 7.4 µM) to excellent (IC50 of 0.32 µM) cytotoxic activity. Compounds such as 3f, 3h, 6a, 8c, 8f and 10a showed activity only on MDA-MB-231 cell line. Out of these, compound 3h was found as the most promising against MDATable 1.

a

MB-231 with an IC50 of 0.26±0.12µM. Similarly, compounds 6a, 8b, 8c and 8e showed activity against HT-29 cell lines and among these compounds 6a and 8e were found to be more potent with IC50 of 0.32±0.26µM, 0.33±0.11µM, respectively. The order of toxicity over HT-29 cell lines is 6a > 8e > ZSTK474 and remaining compounds showed moderate activity on both the cell lines. Morpholine tagged difuranyl triazine 3a and its corresponding monochloro-furan triazine 3b have shown negligible or no cytotoxic activity. Nucleophilic displacement of chlorine in 3b with various amines resulted 4a-d and 8c (Schemes 2, 3). Compounds 4a, 4b and 4c have witnessed

In vitro cytotoxicity of heteroaryl substituted morpholine tagged 1, 3, 5-triazine derivatives against HT-29 and MDA-MB231 cancer cell lines.

Compound No.

HT-29 (µM)

MDA- MB-231 (µM)

HEK 293 Viability in Percentage

Compound No.

HT-29 (µM)

MDA- MB231 (µM)

HEK 293 Viability in Percentage

3d

8.56±0.12

11.35±0.02

84.63768

6j

32.3±0.12

7.47±0.21

69.74381

79.18841

6l

43.21±1.13

7.50±0. 1

62.36482

a

3e

18.64±0.63

ND

3f

7.71±0.22

2.69±0.12

76.52174

6m

12.7±0.25

3.74±0. 5

92.65486

3g

5.53±0.05

6.16±0.25

82.36546

6n

10.5±0.16

16.6±0.11

84.48368

3h

21.7±0.2

0.26±0.12

76.78261

8b

1.92±0.02

55.7±1.12

78.25917

4d

3.37±0.11

17.74±1.02

69.56522

8c

1.40±0.13

1.608±0.12

83.69472

6a

0.32±0.26

2.92±0.05

87.69824

8d

5.64±0.18

NDa

80.25497

6b

4.13±0.27

17.81±0.95

74.43478

8e

0.33±0.11

14.6±0.01

76.68724

6c

12.31±0.1

4.5±0.20

80.11594

8f

7.5±0.16

1.3±0.10

88.93247

6e

52.9±0.2

12.98±0.24

75.47826

10a

3.82±0.16

1.47±0.14

93.68762

6f

11.41±0.13

52.8±0.02

86.49275

10c

22.6±0.12

3.6±0.11

85.55912

6h

31.5±0.3

5.8±0.24

91.94203

10d

12.5±0.14

6.4±0.17

84.69275

6i

28.5±0.02

5.3±0.25

62.65483

ZSTK474

0.9±0.01

1.2±0.02

96.68827

Doxorubicin

0.30±0.02

1.0±0.01

Not developed. All values with a standard deviation of ±0.55 and were analysed using Graph Pad Prism software. Higher cell viability against HEK-293 cells represent that compounds are nontoxic at 25 µM.



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Fig. (2). Promising compounds with cytotoxic activities identified.

no cytotoxic potency against both the cell lines. In 4 series, among all aliphatic amines in furan coupled triazines, 2methylazitidine derivative 4d has showed considerable activity. Surprisingly, a significant increase in the cytotoxic activity was encountered against both the cell lines by replacing the cyclic amine in 4a-4d series with 1- cyclopropylethanamine in furan coupled triazine 8c (IC50 (µM): 1.40 for HT29 and 1.60 for MDA-MB-231) and identified as one of the most promising compound. Replacing the furan ring with N-methyl indole or indole groups, twelve compounds were made to evaluate and compare the cytotoxic potency. Both the Bis-N-methyl indole analogue 3c, Bis-N-methyl indole analogue 3e analogues has shown negligible potency and a moderate activity was observed in the case of both the corresponding chloro derivatives 3d and 3f. The indole substituted triazines 3e, 3f and 10c exhibited moderate potency against both the cell lines. A dramatic increase in the cytotoxic potency was observed with indole-1-cyclopropylethanamine triazine 8e against HT29 (IC50 of 0.33) and which is comparable to Doxorubicin and is more active than ZSTK474, corresponding N-methyl indole-1-cyclopropylethanamine triazine 8d also showed good activity. Remaining all indole coupled compounds showed no cytotoxic activity. Encouraging results were observed by replacing furan or indole group with the thiophene functionality. Hence we extended the work, fifteen thiophene derivatives were made in this series. The di-thiophene substituted triazine 3g has shown moderate potency. An excellent anti-proliferative (IC50 of 0.26 µM) activity was displayed by the key intermediate 3h which is more potent than the standards against MDA-MB-231 cell-lines. The key intermediate 3h was used to make 6a-6m using various amines and 6n and 6o with the corresponding aryl boronic acids. 2-Methylazetidine

derivative 6a has exhibited very good activity against HT-29 cell lines which is better than ZSTK474 and comparable with Doxorubicin. The closest analogue of ZSTK474, 6m has shown lower potency than both the standards. Corresponding 4hydroxyphenyl derivative 6n displayed significant effect on both the cell lines whereas 4-nitrophenyl derivative 6o showed no cytotoxic activity on both the cell lines. Moderate (IC50 of 7.4 µM) to excellent (IC50 of 0.26) activity were shown by the thiophene derivatives. To compare the cytotoxic activity with ZSTK474, five di-morpholine tagged chloro triazines were coupled with heteroaryl or aryl groups to synthesize 10a-10e. Lower but considerable potency was observed in the case of thiophene (10a) & 4-hydroxy derivatives (10d) when compared to ZSTK474. Finally, it is clearly observed that among all aliphatic amines, 1-cyclopropylethanamine having triazine derivatives showed promising activity. Thiophene attached morpholine tagged triazine derivatives showed promising activity when compared to furan or indole attached derivatives. The order of toxicity over MDA-MB-231, HT-29 and HEK293 cell lines are thiophene > furan > indole > N-methyl indole. Among all aromatic derivatives, 4-hydroxy phenyl derivatives showed considerable activity. The activity data is tabulated in Table 1. Promising compounds 3f, 3h, 6a, 8c, 8f and 10a (Fig. 2) were identified against MDA-MB-231, HT-29 cell lines. All active compounds were further tested for their cytotoxicity effect on human normal cell line and were found to be safe in-vitro with a lesser percentage of toxicity at 25 µM. The differential activity among the cell lines may be due to the structure-activity relationship of these molecules.


EXPERIMENTAL Chemistry Melting points were determined in capillaries, recorded on Buchi Melting Point B-540 and are uncorrected. IR data was recorded on Perkin Elmer Spectrum 100. 1H and 13 CNMR spectra were recorded on Varian 400 MHz and 300 MHz spectrometer, using CDCl3 and DMSO-d6 as solvents. Chemical shifts are given in ppm with TMS as an internal reference. J values are given in Hertz. Reactions were monitored by thin layer chromatography (TLC) coated with Silica Gel. Column chromatography was performed with 100-200 mesh silica. 4-(4,6-Dichloro-1,3,5-triazin-2-yl)morpholine (2) Cyanuric chloride 1 (20.0 g, 0.10 mol) was dissolved in dichloromethane (200 mL) at -78 °C, followed by the addition of diisopropylethylamine (17.93 mL, 0.10 mol). The reaction mixture was stirred for 5 minutes at -78 °C. Morpholine (9.61 mL, 0.10 mol) was added drop-wise to the reaction mixture for 10 min and stirred for 30 min at -78 °C. The resulting white precipitate was filtered, washed with water (100 mL) followed by diethyl ether (50 mL) and dried to afford 23.5 g (92.15%) of 4-(4,6-dichloro-1,3,5-triazin-2yl)morpholine 2 as white solid. 1H NMR (400 MHz, DMSOd6) δ 3.76 (t, J = 5.2 Hz, 4H), 3.66 (t, J = 5.2 Hz, 4H); 13C NMR (100 MHz, DMSO-d6) δ 169.17, 149.95, 65.49, 44.29; LC-MS (ESI) m/z Calcd. for C7H8Cl2N4O: 234.01, found: 235.27 [M+H] + General Procedure for Compounds 3a-f, 6n & 6o, 8c-h and 10b-e Chloro derivative (1 mol), arylboronic acid (1 mol) and Cs2CO3 (2 mol) were taken in 1,4-dioxane-water (10 v, 3:1), the reaction mixture was degassed with argon for 10 minutes, then Pd(PPh3)4 (0.05 mol) was added. The reaction mixture was heated to 100 °C, stirred for 16 h. Then the reaction mixture was cooled to room temperature, diluted with water (50 v) and extracted with EtOAc (2 x 50 v). Combined organic layers were dried over anhydrous sodium sulphate, evaporated the solvent in vacuo. Crude products were purified by column chromatography. (Note: 3a, 3c and 3e (diheteroarylated products) were isolated in the preparation of 3b, 3d and 3f) 4-(4,6-Di(furan-3-yl)-1,3,5-triazin-2-yl)morpholine (3a) Yield: 32 %; MR: 148-151 °C; 1H NMR (300 MHz , CDCl3) δ 8.29 (d, J = 0.6 Hz, 2H), 7.49 (t, J = 1.8 Hz, 2H), 7.03 (d, J = 1.5 Hz, 2H), 3.97 (t, J = 5.1 Hz, 4H), 3.79 (t, J = 5.4 Hz, 4H); 13C NMR (100 MHz, CDCl3) δ 167.61, 164.54, 146.25, 143.67, 126.28, 109.48, 66.77, 43.53; LC-MS (ESI) m/z Calcd. for C15H14N4O3: 298.11, found: 299.21 [M+H]+ 4-(4-Chloro-6-(furan-3-yl)-1,3,5-triazin-2-yl)morpholine (3b): Yield: 63%; MR: 120-123 °C; 1H NMR (400 MHz, CDCl3) δ 8.28 (s, 1H), 7.47 (d, J = 2.0 Hz, 1H), 6.97 (d, J = 1.6 Hz, 1H), 3.98-3.95 (m, 2H), 3.90-3.88 (m, 2H), 3.80-3.75 (m, 4H); 13C NMR (100 MHz, CDCl3) δ 170.58, 168.67,

Kasturi et al.

164.51, 147.33, 144.00, 124.99, 109.35, 66.55, 44.04 & 43.91; LC-MS (ESI) m/z Calcd. for C11H11ClN4O2: 266.06, found: 266.88 [M+H]+ 4-(4,6-Bis(1-methyl-1H-indol-5-yl)-1,3,5-triazin-2-yl) morpholine (3c) Yield: 26 %; MR: 165-169 °C; 1H NMR (400 MHz, CDCl3) δ 8.97 (d, J = 0.6 Hz, 2H), 8.54 (dd, J = 8.4 and 1.2 Hz, 2H), 7.39 (d, J = 8.8 Hz, 2H), 7.09 (d, J = 2.8 Hz, 2H), 6.64 (d, J = 3.2 Hz, 2H), 4.14 (t, J = 5.2 Hz, 4H), 3.87-3.81 (m, 10H); 13C NMR (100 MHz, CDCl3) δ 171.69, 165.17, 139.01, 129.66, 128.50, 128.42, 122.52, 122.38, 108.75, 102.53, 66.98, 43.73, 33.01; LC-MS (ESI) m/z Calcd. for C25H24N6O: 424.20, found: 425.28 [M+H]+ 4-(4-Chloro-6-(1-methyl-1H-indol-5-yl)-1,3,5-triazin-2-yl) morpholine (3d) Yield: 60%; MR: 159-162 °C; 1H NMR (400 MHz, CDCl3) δ 8.77 (d, J = 1.2 Hz, 1H), 8.30 (dd, J = 8.8 and 1.6 Hz, 1H), 7.34 (d, J = 8.8 Hz, 1H), 7.09 (d, J = 2.8 Hz, 1H), 6.59 (d, J = 2.8 Hz, 1H), 4.08 (br s, 2H), 3.91 (br s, 2H), 3.83 (s, 3H), 3.80-3.77 (m, 4H); 13C NMR (100 MHz, CDCl3) δ 173.29, 170.65, 164.77, 139.48, 130.12, 128.42, 126.19, 123.32, 122.52, 108.98, 102.82, 66.66, 44.09 & 43.95, 33.01; LC-MS (ESI) m/z Calcd. for C16H16ClN5O: 329.10, found: 330.05 [M+H] + 4-(4,6-Di(1H-indol-5-yl)-1,3,5-triazin-2-yl)morpholine (3e) Yield: 28 %; MR: 301-304 °C; IR (KBr, cm-1) 3374 (NH); 1H NMR (400 MHz, CDCl3) δ 9.00 (s, 2H, NH), 8.52 (d, J = 8.7 Hz, 2H), 8.27 (s, 2H), 7.49 (d, J = 8.7 Hz, 2H), 7.28 (s, 2H), 6.72 (s, 2H), 4.14 (t, J = 4.5 Hz, 4H), 3.86 (t, J = 4.8 Hz, 4H); LC-MS (ESI) m/z Calcd. for C23H20N6O : 396.17, found: 397.63 [M+H]+ 4-(4-Chloro-6-(1H-indol-5-yl)-1,3,5-triazin-2-yl)morpholine (3f) Yield: 57%; MR: 208-212 °C; IR (KBr, cm-1) 3368 (NH); 1H NMR (400 MHz, CDCl3) δ 8.79 (s, 1H, NH), 8.29 (d, J = 1.6 Hz, 1H), 8.27 (d, J = 1.6 Hz, 1H), 7.43 (d, J = 8.8 Hz, 1H), 7.25 (s, 1H), 6.66 (s, 1H), 4.08 (br s, 2H), 3.92 (br s, 2H), 3.80-3.78 (m, 4H); 13C NMR (100 MHz, CDCl3) δ 173.19, 170.64, 164.70, 138.65, 127.86, 126.76, 125.37, 123.13, 122.97, 110.88, 104.19, 66.64, 44.07 & 43.90; LCMS (ESI) m/z Calcd. for C15H14ClN5O: 315.09, found: 315.90 [M+H] + 4-(4-Morpholino-6-(thiophen-2-yl)-1,3,5-triazin-2-yl)phenol (6n) Yield: 67%; MR: 231-234 °C; IR (KBr, cm-1) 3431 (OH); 1H NMR (400 MHz, DMSO-d6) δ 10.16 (s, 1H, OH), 8.33 (d, J = 8.8 Hz, 2H), 8.12 (dd, J = 3.6 and 1.2 Hz, 1H), 7.85 (dd, J = 5.2 and 1.6 Hz, 1H), 7.25 (dd, J = 4.8 and 4.0 Hz, 1H), 6.91 (d, J = 8.8 Hz, 2H), 3.93 (br s, 4H), 3.81-3.65 (m, 4H); 13C NMR (100 MHz, DMSO-d6) δ 169.78, 166.42, 163.99, 161.40, 142.01, 132.00, 130.43, 130.20, 128.41, 126.38, 115.27, 65.87, 43.15; LC-MS (ESI) m/z Calcd. for C17H16N4O2S: 340.10, found: 341.23 [M+H]+


4-(4-(4-Nitrophenyl)-6-(thiophen-2-yl)-1,3,5-triazin-2yl)morpholine (6o) Yield: 61%; MR: 211-215 °C; IR (KBr, cm-1) 1557 (NO2); 1H NMR (400 MHz, DMSO-d6) δ 8.68 (d, J = 8.8 Hz, 2H), 8.38 (d, J = 8.8 Hz, 2H), 8.20 (dd, J = 3.6 and 1.2 Hz, 1H), 7.92 (dd, J = 4.8 and 1.6 Hz, 1H), 7.28 (dd, J = 4.8 and 4.0 Hz, 1H), 4.05-3.90 (m, 4H), 3.79-3.70 (m, 4H); 13C NMR (100 MHz, DMSO-d6) δ 168.36, 166.93, 163.95, 149.69, 141.63, 141.25, 132.85, 131.24, 129.41, 128.63, 123.70, 65.80, 43.44. N-(1-Cyclopropylethyl)-4-(furan-3-yl)-6-morpholino-1,3,5triazin-2-amine (8c) -1

Yield: 69%; MR: 77-80 °C; IR (KBr, cm ) 3425 (NH); H NMR (400 MHz, CDCl3) δ 8.11 (s, 1H), 7.40 (t, J = 1.6 Hz, 1H), 6.90 (s, 1H), 5.17 (s, 1H, NH), 3.81 (br s, 4H), 3.70 (t, J = 4.8 Hz, 4H), 3.51 (s, 1H), 1.24 (d, J = 6.4 Hz, 3H), 0.90-0.84 (m, 1H), 0.51-0.25 (m, 3H), 0.24-0.20 (m, 1H); 13 C NMR (100 MHz, CDCl3) δ 167.03, 165.46, 164.95, 145.38, 143.34, 126.53, 109.47, 66.83, 50.49, 43.51, 20.15, 17.58, 3.33, 2.80; LC-MS (ESI) m/z Calcd. for C16H21N5O2: 315.17, found: 316.23 [M+H]+ 1

N-(1-Cyclopropylethyl)-4-(1-methyl-1H-indol-5-yl)-6morpholino-1,3,5-triazin-2-amine (8d) Yield: 67%; MR: 89-92 °C; IR (KBr, cm-1) 3414 (NH); H NMR (300 MHz, DMSO-d6) δ 8.58 (s, 1H), 8.16 (d, J = 8.7 Hz, 1H), 7.43 (d, J = 9.0 Hz, 1H), 7.37 (d, J = 2.8 Hz, 1H), 7.32-7.13 (m, 1H, NH), 6.54 (s, 1H), 3.81-3.66 (m, 11H), 3.56-3.51 (m, 1H), 1.24 (t, J = 7.5 Hz, 3H), 1.05-0.89 (m, 1H), 0.39-0.19 (m, 4H); 13C NMR (75 MHz, CDCl3) δ 171.49, 165.75, 165.25, 138.75, 129.50, 128.85, 128.35, 128.27, 121.99, 108.58, 102.35, 66.94, 50.51, 43.59, 32.97, 20.26, 17.69, 3.37, 2.83; LC-MS (ESI) m/z Calcd. for C21H26N6O: 378.22, found: 379.0 [M+H]+ 1

N-(1-Cyclopropylethyl)-4-(1H-indol-5-yl)-6-morpholino1,3,5-triazin-2-amine (8e) Yield: 62%; MR: 118-121 °C; IR (KBr, cm-1) 3414 (NH, br); 1H NMR (300 MHz, DMSO-d6) δ 11.26 (s, 1H, NH), 8.58 (s, 1H), 8.11 (d, J = 8.4 Hz, 1H), 7.42-7.28 (m, 3H), 6.53 (s, 1H), 3.80-3.78 (m, 4H), 3.67-3.65 (m, 4H), 3.543.52 (m, 1H), 1.22 (d, J = 7.2 Hz, 3H), 1.00-0.97 (m, 1H), 0.37-0.19 (m, 4H); 13C NMR (75 MHz, CDCl3) δ 171.44, 165.73, 165.24, 137.90, 128.86, 127.75, 124.77, 122.52, 121.78, 110.45, 103.89, 66.93, 50.52, 43.06, 20.19, 17.68, 3.37, 2.83; LC-MS (ESI) m/z Calcd. for C20H24N6O: 364.20, found: 365.21 [M+H]+ 4-(4-(1-Cyclopropylethylamino)-6-morpholino-1,3,5triazin-2-yl)phenol (8f) Yield: 69%; MR: 238-242 °C; IR (KBr, cm-1) 3342 (NH), 3076 (OH); 1H NMR (400 MHz, DMSO-d6) δ 9.91 (br s, 1H, OH), 8.15 (d, J = 8.3 Hz, 2H), 7.31-7.07 (m, 1H, NH), 6.82 (d, J = 7.8 Hz, 2H), 3.90-3.58 (m, 8H), 3.56-3.44 (m, 1H), 1.21 (t, J = 5.9 Hz, 3H), 1.04-0.91 (m, 1H), 0.49-0.12 (m, 4H); 13C NMR (100 MHz, DMSO-d6) δ 169.03, 165.16 & 165.07, 164.62 & 164.54, 160.47 & 160.35, 129.72 & 129. 62, 127.69, 114.79, 65.98, 49.57 & 49.49, 43.13, 20.43 &


7

20.17, 17.47 & 17.31, 3.29 & 3.19, 2.74 & 2.65; LC-MS (ESI) m/z Calcd. for C18H23N5O2: 341.19, found: 342.30 [M+H]+ 4-(4-Aminophenyl)-N-(1-cyclopropylethyl)-6-morpholino1,3,5-triazin-2-amine (8g) Yield: 64%; MR: 85-90 °C; IR (KBr, cm-1) 3426 (NH), 3405 (NH2); 1H NMR (400 MHz, CDCl3) δ 8.17 (d, J = 7.6 Hz, 2H), 6.68 (d, J = 8.8 Hz, 2H), 5.30 (br s, 2H, NH2), 3.873.73 (m, 8H), 3.58-3.50 (m, 1H), 1.28-1.26 (m, 3H), 0.940.86 (m, 1H), 0.53-0.12 (m, 4H); LC-MS (ESI) m/z Calcd. for C18H24N6O: 340.20, found: 341.35 [M+H]+ N-(1-Cyclopropylethyl)-4-morpholino-6-(4-nitrophenyl)1,3,5-triazin-2-amine (8h) Yield: 64%; MR: 182-184 °C; IR (KBr, cm-1) 3416 (NH), 1545 (NO2); 1H NMR (400 MHz, DMSO-d6) δ 8.50 (d, J = 8.4 Hz, 2H), 8.34-8.30 (m, 2H), 7.69-7.50 (m, 1H, NH), 3.96-3.60 (m, 8H), 3.58-3.44 (m, 1H), 1.31-1.15 (m, 3H), 1.08-0.92 (m, 1H), 0.52-0.20 (m, 4H); 13C NMR (100 MHz, DMSO-d6) δ 167.59 & 167.49, 165.15 & 164.95, 164.53 & 164.38, 149.17 & 149.09, 143.01, 128.98 & 128.86, 123.40, 65.90, 49.75, 43.21, 20.35 & 20.03, 17.37 & 17.21, 3.32 & 3.19, 2.77 & 2.65; LC-MS (ESI) m/z Calcd. for C18H22N6O3: 370.18, found: 371.28 [M+H]+ 4,4'-(6-(Furan-3-yl)-1,3,5-triazine-2,4-diyl)dimorpholine (10b) Yield: 77%; MR: 116-119 °C; 1H NMR (300 MHz, CDCl3) δ 8.16 (s, 1H), 7.44 (t, J = 1.6 Hz, 1H), 6.94 (d, J = 1.5 Hz, 1H), 3.85 (br s, 8H), 3.78-3.71 (m, 8H); LC-MS (ESI) m/z Calcd. for C15H19N5O3: 317.15, found: 318.23 [M+H]+ 4,4'-(6-(1H-Indol-5-yl)-1,3,5-triazine-2,4-diyl)dimorpholine (10c) Yield: 72%; IR (KBr, cm-1) 3447 (NH); 1H NMR (400 MHz, DMSO-d6) δ 11.30 (s, 1H, NH), 8.65 (s, 1H), 8.17 (d, J = 8.8 Hz, 1H), 7.44-7.39 (m, 2H), 6.55 (s, 1H), 3.84 (br s, 8H), 3.67 (br s, 8H); 13C NMR (100 MHz, DMSO-d6) δ 170.48, 164.64, 138.05, 127.54, 127.37, 126.37, 121.44, 121.07, 110.79, 102.34, 66.05, 43.26; LC-MS (ESI) m/z Calcd. for C19H22N6O2: 366.12, found: 367.17 [M+H]+ 4-(4,6-dimorpholino-1,3,5-triazin-2-yl)phenol (10d) Yield: 69% ; IR (KBr, cm-1) 3747 (OH), 3271, 2857, 1539; 1H NMR (DMSO-d6, 400 MHz) δ = 9.96 (s, 1H), 8.19 (d, 2H, J = 8.8 Hz), 6.82 (d, 2H, J = 8.8 Hz), 4.16 - 3.46 (m, 16H); 13C NMR (DMSO-d6, 400 MHz) δ = 169.14, 164.53, 160.64, 129.92, 127.39, 114.87, 65.98, 43.20; LC-MS m/z Calcd. for C17H21N5O3: 343.16, Found 344.32 [M+H]+ 4,4'-(6-(4-Nitrophenyl)-1,3,5-triazine-2,4-diyl)dimorpholine (10e) Yield: 64%; MR: 227-230 °C; IR (KBr, cm-1) 1559 (NO2); 1H NMR (300 MHz, CDCl3) δ 8.55 (d, J = 7.7 Hz, 2H), 8.28 (d, J = 8.1 Hz, 2H), 3.93 (br s, 8H), 3.79 (br s, 8H); 13C NMR (300 MHz, CDCl3) δ 168.46, 165.09, 149.62, 143.42, 129.19, 123.23, 66.80, 43.70; LC-MS (ESI) m/z Calcd. for C17H20N6O4: 372.15, found: 373.36 [M+H]+


General Procedure for Compounds 3g, 3h, 8b and 10a Chloro derivative (1 mol), tributyl(thiophen-2yl)stannane (1 mol) and LiCl (1 mol) were taken in 1,4dioxane (10 v), the reaction mixture was degassed with argon for 10 minutes, then Pd(PPh3)4 (0.05 mol) was added. The reaction mixture was heated to 100 °C, stirred for 16 h. Then the reaction mixture was cooled to room temperature, diluted with water (50 v) and extracted with EtOAc (2 x 50 v). Combined organic layers were dried over anhydrous sodium sulphate, evaporated the solvent in vacuo. Crude products were purified by column chromatography. (Note: 3g (diheteroarylated product) was isolated in the preparation of 3h) 4-(4,6-Di(thiophen-2-yl)-1,3,5-triazin-2-yl)morpholine (3g) 1

Yield: 28 %; MR: 264-267 °C; H NMR (300 MHz, CDCl3) δ 8.12 (dd, J = 3.6 and 1.2 Hz, 2H), 7.52 (dd, J = 5.1 and 0.9 Hz, 2H), 7.15 (t, J = 4.8 Hz, 2H), 3.98 (t, J = 4.2 Hz, 4H), 3.79 (t, J = 5.1 Hz, 4H); 13C NMR (75 MHz, CDCl3): δ 167.25, 164.33, 142.41, 131.00, 130.48, 128.02, 66.75, 43.59; LC-MS (ESI) m/z Calcd. for C15H14N4OS2: 330.06, found: 331.11 [M+H]+ 4-(4-Chloro-6-(thiophen-2-yl)-1,3,5-triazin-2yl)morpholine (3h) Yield: 65%; MR: 142-145 °C; 1H NMR (300 MHz, CDCl3) δ 8.08 (dd, J = 3.6 and 1.2 Hz, 1H), 7.57 (dd, J = 5.1 and 1.2 Hz, 1H), 7.15 (t, J = 4.8 Hz, 1H), 3.99-3.96 (m, 2H), 3.91-3.88 (m, 2H), 3.80-3.78 (m, 4H); 13C NMR (100 MHz, CDCl3) δ 170.50, 168.15, 164.31, 140.52, 132.36, 131.85, 128.27, 66.51, 44.05 & 43.92; LC-MS (ESI) m/z Calcd. for C11H11ClN4OS: 282.03, found: 283.08 [M+H]+

Kasturi et al.

were dried over anhydrous sodium sulphate, evaporated the solvent in vacuo. Crude products were purified by column chromatography. 4-(4-(Furan-3-yl)-6-(5-methylhexahydropyrrolo[3,4c]pyrrol-2(1H)-yl)-1,3,5-triazin-2-yl)morpholine (4a) Yield: 72%; MR: 253-255 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.33 (d, J = 1.6 Hz, 1H), 7.74 (s, 1H), 6.92 (d, J = 1.6 Hz, 1H), 3.76 (br s, 6H), 3.63 (br s, 6H), 3.02 (br s, 4H), 3.60 (br s, 3H), 1.25 (s, 2H); LC-MS (ESI) m/z Calcd.for C18H24N6O2: 356.20, found: 357.20 [M+H]+ N,N-Diethyl-1-(4-(furan-3-yl)-6-morpholino-1,3,5-triazin2-yl)piperidin-4-amine (4b) Yield: 82%; MR: 192-195 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.38 (s, 1H), 7.75 (d, J = 1.6 Hz, 1H), 6.95 (d, J = 1.2 Hz, 1H), 4.96-4.82 (m, 2H), 3.77 (s, 4H), 3.65-3.63 (m, 5H), 3.28-3.22 (m, 2H), 3.11-3.06 (m, 2H), 2.94-2.88 (m, 2H), 2.05 (d, J = 11.2 Hz, 2H), 1.59-1.57 (m, 2H), 1.23 (t, J = 6.8 Hz, 6H); 13C NMR (75 MHz, CDCl3) δ 167.11, 164.91, 164.49, 145.53, 143.38, 126.55, 109.45, 66.75, 60.59, 44.79, 43.49, 41.70, 26.16, 9.95; LC-MS (ESI) m/z Calcd. for C20H30N6O2: 386.24, found: 387.24 [M+H]+ 4-(4-(Furan-3-yl)-6-(2-methylpyrrolidin-1-yl)-1,3,5-triazin2-yl)morpholine (4c) Yield: 74%; MR: 131-134 °C; 1H NMR (300 MHz, CDCl3) δ 8.15 (d, J = 1.5 Hz, 1H), 7.42 (t, J = 1.5 Hz, 1H), 6.95 (d, J = 1.8 Hz, 1H), 4.40-4.38 (m, 1H), 3.85-3.83 (m, 4H), 3.75-3.73 (m, 4H), 3.66-3.60 (m, 2H), 2.04-1.60 (m, 4H), 1.27 (s, 3H); LC-MS (ESI) m/z Calcd. for C16H21N5O2: 315.17, found: 316.23 [M+H]+

N-(1-Cyclopropylethyl)-4-morpholino-6-(thiophen-2-yl)1,3,5-triazin-2-amine (8b)

4-(4-(Furan-3-yl)-6-(2-methylazetidin-1-yl)-1,3,5-triazin-2yl)morpholine (4d)

Yield: 81%; MR: 104-107 °C; IR (KBr, cm-1) 3433 (NH); 1H NMR (400 MHz, DMSO-d6) δ 7.86 (d, J = 3.6 Hz, 1H), 7.71 (d, J = 4.8 Hz, 1H), 7.47 (d, J = 8.4 Hz, 1H, NH), 7.15 (t, J = 4.8 Hz, 1H), 3.73 (br s, 4H), 3.64-3.63 (m, 4H), 3.58-3.47 (m, 1H), 1.19 (d, J = 6.0 Hz, 3H), 0.97-0.94 (m, 1H), 0.42-0.14 (m, 4H); LC-MS (ESI) m/z Calcd. for C16H21N5OS: 331.05, found: 332.30 [M+H]+

Yield: 79%; MR: 103-107 °C; 1H NMR (300 MHz, CDCl3) δ 8.15 (d, J = 0.9 Hz, 1H), 7.42 (t, J = 1.8 Hz, 1H), 6.93 (d, J = 1.5 Hz, 1H), 4.53-4.50 (m, 1H), 4.07-3.98 (m, 2H), 3.83-3.82 (m, 4H), 3.74-3.71 (m, 4H), 2.46-2.40 (m, 1H), 1.98-1.95 (m, 1H), 1.54 (s, 3H); LC-MS (ESI) m/z Calcd. for C15H19N5O2: 301.15, found: 302.24 [M+H]+

4,4'-(6-(Thiophen-2-yl)-1,3,5-triazine-2,4diyl)dimorpholine (10a) 1

Yield: 72%; MR: 191-194 °C; H NMR (400 MHz, CDCl3) δ 7.95 (dd, J = 4.0 and 1.2 Hz, 1H), 7.44 (dd, J = 4.8 and 1.2 Hz, 1H), 7.12 (dd, J = 5.2 and 3.6 Hz, 1H), 3.89 (br s, 8H), 3.80-3.73 (m, 8H); 13C NMR (100 MHz, CDCl3) δ 166.64, 164.81, 143.34, 129.93, 129.28, 127.71, 66.82, 43.55; LC-MS (ESI) m/z Calcd. for C15H19N5O2S: 333.13, found: 334.30 [M+H]+ General Procedure for Compounds 4a-d, 5a-e and 6a-l Chloro derivative (1 mol), amine (1 mol) and K2CO3 (2 mol) were taken in DMF (10 v). The reaction mixture was heated to 100 °C, stirred for 16 h. Then the reaction mixture was cooled to room temperature, diluted with water (50 v) extracted with EtOAC (2 x 50 v). Combined organic layers

4-(4-(1H-Indol-5-yl)-6-(5-methylhexahydropyrrolo[3,4c]pyrrol-2(1H)-yl)-1,3,5-triazin-2-yl)morpholine (5a) Yield: 67%; MR: 186-190 °C; IR (KBr, cm-1) 3245 (NH); 1H NMR (400 MHz, DMSO-d6) δ 11.26 (s, 1H, NH), 8.63 (s, 1H), 8.15 (dd, J = 8.0 and 1.2 Hz, 1H), 7.42-7.38 (m, 2H), 6.54 (s, 1H), 3.82 (br s, 6H), 3.66 (br s, 6H), 2.88 (s, 2H), 2.50 (s, 2H), 2.21 (s, 3H), 1.25 (s, 2H); 13C NMR (75 MHz, CDCl3) δ 170.92, 165.09, 163.96, 137.89, 129.51, 127.72, 124.73, 122.60, 121.79, 110.35, 103.78, 66.96, 62.89, 51.46, 43.59, 42.23, 41.79; LC-MS (ESI) m/z Calcd. for C22H27N7O: 405.23, found: 406.27 [M+H]+ 1-(4-(1H-Indol-5-yl)-6-morpholino-1,3,5-triazin-2-yl)-N,Ndiethylpiperidin-4-amine (5b) Yield: 67%; MR: 173-175 °C; IR (KBr, cm-1) 3232 (NH); 1H NMR (400 MHz, DMSO-d6) δ 11.30 (s, 1H, NH), 8.63 (s, 1H), 8.15 (d, J = 8.4 Hz, 1H) 7.44-7.39 (m, 2H),


6.55 (s, 1H), 5.07-4.84 (m, 2H), 3.83 (s, 4H), 3.67 (s, 4H), 3.63 (br s, 1H), 3.16-2.93 (m, 4H), 2.98-2.75 (m, 2H), 1.771.74 (m, 2H), 1.61 (s, 2H), 1.23 (t, J = 6.8 Hz, 6H); 13C NMR (75 MHz, CDCl3) δ 171.43, 165.29, 164.80, 138.04, 128.97, 127.71, 125.06, 122.39, 121.81, 110.57, 103.56, 66.86, 59.91, 44.28, 43.62, 42.19, 26.69, 11.02; LC-MS (ESI) m/z Calcd. for C24H33N7O: 435.27, found: 436.28 [M+H] + 4-(4-(1H-Indol-5-yl)-6-(2-methylazetidin-1-yl)-1,3,5triazin-2-yl)morpholine (5c) Yield: 66%; MR: 116-119 °C; IR (KBr, cm-1) 3414 (NH); 1H NMR (300 MHz, DMSO-d6) δ 11.27 (s, 1H, NH), 8.59 (s, 1H), 8.12 (dd, J = 8.4 and 1.5 Hz, 1H), 7.43-7.38 (m, 2H), 6.55 (s, 1H), 4.51 (s, 1H), 4.10-3.89 (2H, m), 3.84-3.81 (m, 4H), 3.67-3.66 (m, 4H), 2.43-2.42 (m, 1H), 1.93 (m, 1H), 1.53 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 170.68, 166.35, 165.05, 137.89, 129.46, 127.72, 124.68, 122.65, 121.83, 110.35, 103.89, 66.96, 58.35, 46.45, 43.56, 24.63, 21.63; LC-MS (ESI) m/z Calcd. for C19H22N6O: 350.19, found: 351.21 [M+H]+ tert-Butyl 1-(4-(1H-indol-5-yl)-6-morpholino-1,3,5-triazin2-yl)pyrrolidin-3-ylcarbamate (5d) Yield: 62%; MR: 201-206 °C; IR (KBr, cm-1) 3401 (NH), 3314 (NH), 1694 (CO); 1H NMR (400 MHz, CDCl3) δ 8.77 (s, 1H, NH), 8.31 (d, J = 8.8 Hz, 1H), 8.23 (s, 1H), 7.41 (d, J = 8.8 Hz, 1H), 7.23 (s, 1H), 6.64 (s, 1H), 3.94-3.68 (m, 12H), 3.53-3.48 (m, 1H), 2.27-2.24 (m, 1H), 1.97-1.94 (m, 1H), 1.45 (s, 9H); LC-MS (ESI) m/z Calcd. for C24H31N7O3: 465.25, found: 466.43 [M+H]+ 1-(4-(1H-Indol-5-yl)-6-morpholino-1,3,5-triazin-2yl)pyrrolidin-3-amine (5e) Yield: 61%; MR: chared above 250 °C; IR (KBr, cm-1) 3428 (NH2) and 3241 (NH); 1H NMR (400 MHz, D2O) δ 8.43 (s, 1H), 7.89 (d, J = 8.4 Hz, 1H), 7.66 (d, J = 8.8 Hz, 1H), 7.55 (d, J = 3.2 Hz, 1H), 6.75 (d, J = 2.8 Hz, 1H), 4.083.98 (m, 5H), 3.90-3.87 (m, 6H), 3.78-3.74 (m, 2H), 2.602.54 (m, 1H), 2.34-2.31 (m, 1H); LC-MS (ESI) m/z Calcd. for C19H23N7O: 365.20, found: 366.31 [M+H]+ 4-(4-(2-Methylazetidin-1-yl)-6-(thiophen-2-yl)-1,3,5-triazin2-yl)morpholine (6a) Yield: 76%; (liquid); 1H NMR (300 MHz, CDCl3) δ 7.94 (dd, J = 3.9 and 1.2 Hz, 1H), 7.42 (dd, J = 4.8 and 1.2 Hz, 1H), 7.09-7.06 (m, 1H), 4.55-4.52 (m, 1H), 4.13-4.07 (m, 2H), 3.85 (br s, 4H), 3.75-3.72 (m, 4H), 2.47-2.41 (m, 1H), 1.98-1.95 (m, 1H), 1.54 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 166.40, 165.82, 164.64, 143.60, 129.67, 129.11, 127.61, 66.85, 58.40, 46.38, 43.49, 24.56, 21.38; LC-MS (ESI) m/z Calcd. for C15H19N5OS: 317.13, found: 318.19 [M+H]+ 4-(4-(2-Methylpyrrolidin-1-yl)-6-(thiophen-2-yl)-1,3,5triazin-2-yl)morpholine (6b) Yield: 74%; MR: not recorded (liquid); 1H NMR (300 MHz, CDCl3) δ 7.95 (dd, J = 3.6 and 1.2 Hz, 1H), 7.41 (dd, J = 5.1 and 1.2 Hz, 1H), 7.10-7.07 (m, 1H), 4.42-4.38 (m, 1H), 3.87-3.85 (m, 4H), 3.76-3.73 (m, 4H), 3.69-3.56 (m, 2H),


9

2.08-1.68 (m, 4H), 1.32-1.26 (m, 3H); LC-MS (ESI) m/z Calcd. for C16H21N5OS: 331.15, found: 332.10 [M+H]+ 4-(4-(2-Methylpiperidin-1-yl)-6-(thiophen-2-yl)-1,3,5triazin-2-yl)morpholine (6c) Yield: 73%; MR: 83-86 °C; 1H NMR (300 MHz, DMSOd6) δ 7.91 (dd, J = 3.3 and 0.9 Hz, 1H), 7.72 (dd, J = 5.1 and 1.2 Hz, 1H), 7.16 (dd, J = 4.8 and 3.6 Hz, 1H), 4.61-4.47 (m, 1H), 3.86-3.50 (m, 8H), 3.02-2.68 (m, 2H), 1.78-1.45 (m, 6H), 1.16 (d, J = 6.9, 3H); 13C NMR (75 MHz, DMSO-d6) δ 165.77, 164.34, 163.74, 142.87, 130.71, 129.26, 127.94, 65.92, 44.79, 43.16, 37.53, 29.70, 25.28, 18.69 & 18.51, 14.71; LC-MS (ESI) m/z Calcd. for C17H23N5OS: 345.16, found: 346.27 [M+H]+ 4-(4-(4-Methylpiperazin-1-yl)-6-(thiophen-2-yl)-1,3,5triazin-2-yl)morpholine (6d) Yield: 81%; MR: 135-138 °C; 1H NMR (300 MHz, DMSO-d6) δ 7.93 (dd, J = 3.6 and 1.2 Hz, 1H), 7.74 (dd, J = 5.0 and 1.2 Hz, 1H), 7.16 (dd, J = 4.8 and 4.0 Hz, 1H), 3.76 (br s, 8H), 3.68-3.60 (m, 4H), 2.40-2.26 (m, 4H), 2.21 (s, 3H); 13C NMR (75 MHz, DMSO-d6) δ 165.84, 164.14, 164.06, 142.61, 130.91, 129.48, 128.00, 65.92, 54.31, 45.73, 43.18, 42.59; LC-MS (ESI) m/z Calcd. for C16H22N6OS: 346.16, found: 347.28 [M+H]+ 4-(4-(Piperazin-1-yl)-6-(thiophen-2-yl)-1,3,5-triazin-2yl)morpholine (6e) Yield: 85%; MR: 221-224 °C; IR (KBr, cm-1) 3409 (NH); 1H NMR (400 MHz, DMSO-d6) δ 8.89 (br s, 1H, NH), 8.03 - 7.89 (m, 1H), 7.78 (dd, J = 6.4 and 1.2 Hz, 1H), 7.19 (dd, J = 4.8 and 4.0 Hz, 1H), 4.00 (br s, 4H), 3.79 (br s, 4H), 3.66 (br s, 4H), 3.18 (br s, 4H); 13C NMR (75MHz, DMSOd6) δ 166.09, 164.18, 164.08, 142.58, 131.41, 129.94, 128.17, 65.94, 43.24, 43.21, 42.39; LC-MS (ESI) m/z Calcd. for C15H20N6OS: 332.14, found: 333.16 [M+H]+ tert-Butyl 4-(4-morpholino-6-(thiophen-2-yl)-1,3,5-triazin2-yl)piperazine-1-carboxylate (6f) Yield: 82%; MR: 180-185 °C; IR (KBr, cm-1) 1629 (CO); H NMR (400 MHz, DMSO-d6) δ 7.96 (dd, J = 3.6 and 0.9 Hz, 1H), 7.44 (dd, J = 5.1 and 1.2 Hz, 1H), 7.10 (dd, J = 4.8 and 3.6 Hz, 1H), 3.86 (br s, 8H), 3.79-3.71 (m, 4H), 3.533.44 (m, 4H), 1.49 (s, 9H); 13C NMR (300 MHz, CDCl3) δ 166.70, 164.87, 164.52, 154.77, 143.37, 129.94, 129.31, 127.71, 80.01, 66.83, 43.59, 43.28, 42.97, 28.42; LC-MS (ESI) m/z Calcd. for C20H28N6O3S: 432.19, found: 433.41 [M+H] + 1

4-(4-(5-Methylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-6(thiophen-2-yl)-1,3,5-triazin-2-yl)morpholine (6g) Yield: 69%; MR: 114-117 °C; 1H NMR (300 MHz, CDCl3) δ 7.95 (dd, J = 3.6 and 1.2 Hz, 1H), 7.43 (dd, J = 5.1 and 1.2 Hz, 1H), 7.09 (dd, J = 4.8 and 3.6 Hz, 1H), 3.95-3.82 (m, 4H), 3.80-3.66 (m, 8H), 3.14-2.96 (m, 4H), 2.52 (d, J = 4.8 Hz, 2H), 2.47 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 166.37, 164.60, 163.73, 143.46, 129.76, 129.15, 127.66, 66.84, 62.22, 50.91, 43.50, 41.90, 41.43; LC-MS (ESI) m/z Calcd. for C18H24N6OS: 372.17, found: 373.30 [M+H]+


4-(4-Morpholino-6-(thiophen-2-yl)-1,3,5-triazin-2yl)hexahydro-2H-furo[3,2-b]pyrrole (6h) Yield: 72%; MR: 84-87 °C; 1H NMR (300 MHz, CDCl3) δ 7.96 (d, J = 2.8 Hz, 1H), 7.43 (d, J = 4.8 Hz, 1H), 7.09 (t, J = 3.9 Hz, 1H), 4.60-4.57 (m, 1H), 4.28-4.17 (m, 1H), 4.103.97 (m, 1H), 3.94-3.80 (m, 6H), 3.79-3.70 (m, 4H), 3.563.34 (m, 1H), 2.44-1.88 (m, 4H); 13C NMR (75 MHz, CDCl3) δ 166.49 & 166.07, 164.73 & 164.46, 162.97, 143.67 & 143.49, 129.76, 129.12 & 129.06, 127.65, 82.74, 68.43, 66.84, 62.78 & 62.52, 45.19 & 45.02, 43.51, 34.45 & 33.96, 31.78 & 31.61; LC-MS (ESI) m/z Calcd. for C17H21N5O2S: 359.14, found: 360.26 [M+H]+ 2-(4-Morpholino-6-(thiophen-2-yl)-1,3,5-triazin-2ylamino)ethanol (6i) Yield: 59%; MR: 185-188 °C; IR (KBr, cm-1) 3413 (OH), 3402 (NH); 1H NMR (400 MHz, DMSO-d6) δ 7.91-7.86 (m, 1H), 7.74-7.70 (m, 1H), 7.18-7.14 (m, 1H), 4.78-4.58 (m, 1H, NH), 3.77 (br s, 4H), 3.66 (d, J = 4.9 Hz, 4H), 3.56-3.47 (m, 2H), 3.44-3.33 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ 165.94 & 165.69, 165.57 & 165.43, 164.27 & 164.12, 142.70, 130.86 & 130.61, 129.35 & 129.07, 127.95, 65.93, 59.89 & 59.47, 43.12, 42.84; LC-MS (ESI) m/z Calcd. for C13H17N5O2S: 307.11, found: 308.21 [M+H]+ 4-Morpholino-6-(thiophen-2-yl)-N-(3,3,3-trifluoropropyl)1,3,5-triazin-2-amine (6j) Yield: 68%; MR: 168-171 °C; IR (KBr, cm-1) 3319 (NH); 1H NMR (300 MHz, DMSO-d6) δ 7.92-7.88 (m, 1H), 7.75-7.66 (m, 1H), 7.18-7.15 (m, 1H), 3.78 (br s, 4H), 3.683.65 (br s, 4H), 3.56-3.43 (m, 2H), 2.60-2.50 (m, 2H); 13C NMR (75 MHz, DMSO-d6) δ 165.86, 165.34 & 165.23, 164.17, 142.60 & 142.50, 130.81 & 130.11, 129.44 & 129.25, 128.01, 124.99, 65.89, 43.13, 33.62, 32.42 & 32.07; LC-MS (ESI) m/z Calcd. for C14H16F3N5OS: 359.10, found: 360.26 [M+H] + N1,N1-Dimethyl-N2-(4-morpholino-6-(thiophen-2-yl)1,3,5-triazin-2-yl)ethane-1,2-diamine (6k) Yield: 62%; 1H NMR (400 MHz, CDCl3) δ 7.94 (br s, 1H), 7.44 (d, J = 4.4 Hz, 1H), 7.10 (t, J = 4.0 Hz, 1H), 3.87 (br s, 4H), 3.74 (br s, 4H), 3.49 (br s, 2H), 2.52 (br s, 2H), 2.29 (s, 6H); LC-MS (ESI) m/z Calcd. for C15H22N6OS: 334.16, found: 335.24 [M+H]+ N,N-Dimethyl-4-morpholino-6-(thiophen-2-yl)-1,3,5triazin-2-amine (6l) 1

Yield: 64%; MR: 121-124 °C; H NMR (300 MHz, CDCl3) δ 7.97 (dd, J = 3.9 and 1.2 Hz, 1H), 7.43 (dd, J = 5.1 and 1.2 Hz, 1H), 7.10 (dd, J = 4.8 and 3.6 Hz, 1H), 3.95-3.81 (m, 4H), 3.79-3.71 (m, 4H), 3.34-3.06 (m, 6H); 13C NMR (75 MHz, CDCl3) δ 166.32, 165.35, 164.81, 143.73, 129.62, 129.00, 127.64, 66.88, 43.54, 36.03; LC-MS (ESI) m/z Calcd. for C13H17N5OS: 291.12, found: 292.27 [M+H]+ General Procedure for Compounds 6m, 8a and 10e (ZSTK474) 2-(Difluoromethyl)-1H-benzo[d]imidazole (1 mol), chloro derivative (1 mol) and K2CO3 (2 mol) were taken in

Kasturi et al.

DMSO (10 v). The reaction mixture was heated to 130 °C, stirred for 4 h. Then the reaction mixture was cooled to room temperature, diluted with water (50 v) extracted with EtOAC (2 x 50 v), evaporated the solvent under reduced pressure to get the crude compound. Crude products were purified by column chromatography. 4-(4-(2-(Difluoromethyl)-1H-benzo[d]imidazol-1-yl)-6(thiophen-2-yl)-1,3,5-triazin-2-yl)morpholine (6m) Yield: 65%; MR: 232-236 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.52 (d, J = 8.4 Hz, 1H), 8.19 (dd, J = 3.6 and 1.2 Hz, 1H), 7.98 (dd, J = 5.2 and 1.2 Hz, 1H), 7.96-7.69 (m, 2H), 7.56 (t, J = 8.8 Hz, 1H), 7.47 (t, J = 7.6 Hz, 1H), 7.32 (dd, J = 4.2 and 3.6 Hz, 1H), 3.98 (br s, 2H), 3.89 (br s, 2H), 3.77 (br s, 4H); 13C NMR (100 MHz, DMSO-d6) δ 167.30, 164.08, 161.46, 146.03 (t), 141.38, 140.48, 133.55, 132.75, 131.76, 128.89, 126.15, 124.68, 120.80, 116.14, 108.58 (t), 65.74, 43.87; LC-MS (ESI) m/z Calcd. for C19H16F2N6OS: 414.11, found: 415.24 [M+H]+ N-(1-Cyclopropylethyl)-4-(2-(difluoromethyl)-1H-benzo[d] imidazol-1-yl)-6-morpholino-1,3,5-triazin-2-amine (8a) Yield: 74%; MR: 181-183 °C; IR (KBr, cm-1) 3340 (NH); 1H NMR (400 MHz, DMSO-d6) δ 8.58 (d, J = 7.8 Hz, 1H), 8.11-7.63 (m, 3H), 7.54-7.37 (m, 2H), 3.77 (br s, 4H), 3.69 (br s, 4H), 3.60-3.50 (m, 1H), 1.25 (d, J = 7.2 Hz, 3H), 1.03-0.99 (m, 1H), 0.49-0.23 (m, 4H); 13C NMR (100 MHz, DMSO-d6) δ 164.98, 164.57 & 164.29, 161.36 & 161.19, 146.02 (t), 141.29, 133.04 & 132.93, 125.72 & 125.56, 124.21, 120.64 & 120.47, 116.72 & 115.84, 108.69 (t), 65.77, 50.09 & 49.85, 43.51, 20.37 & 20.01, 17.21 & 17.01, 3.48, 2.88 & 2.72; LC-MS (ESI) m/z Calcd. for C20H23F2N7O: 415.19, found: 416.33 [M+H]+ 4,4'-(6-(2-(Difluoromethyl)-1H-benzo[d]imidazol-1-yl)1,3,5-triazine-2,4-diyl)dimorpholine (10e) (ZSTK474) Yield: 73%; MR: 215-218 °C; 1H NMR (300 MHz, CDCl3) δ 8.33 (d, J = 7.8 Hz, 1H), 7.89 (d, J = 7.2 Hz, 1H), 7.74-7.26 (m, 3H), 3.89 (br s, 8H), 3.79 (br s, 8H); 13C NMR (75 MHz, CDCl3) δ 164.96, 161.97, 145.89 (t), 141.92, 133.52, 125.75, 124.37, 121.31, 115.80, 108.38 (t), 66.61, 43.98; LC-MS (ESI) m/z Calcd. for C19H21F2N7O2: 417.17, found: 418.35 [M+H]+ 4-Chloro-N-(1-cyclopropylethyl)-6-morpholino-1,3,5triazin-2-amine (7) K2CO3 (2 mol) was added to a solution of 1cyclopropylethanamine (1 mol) in ACN (10 v) after 5 minutes, Intermediate 2 (1 mol) was added at 0 °C, slowly warmed to 50 °C, stirred for 3 h. The reaction mixture was diluted with water (50 v) extracted with EtOAC (2 x 50 v), evaporated the solvent in vacuo. Crude product was purified by column chromatography to afford 4-chloro-N-(1cyclopropylethyl)-6-morpholino-1,3,5-triazin-2-amine (7) as white solid. Yield: 88%; IR (KBr, cm-1) 3331(NH); 1H NMR (400 MHz, DMSO-d6) δ 8.09-7.67 (m, 1H, NH), 3.64-3.60 (m, 8H), 3.45-3.37 (m, 1H), 1.16 (d, J = 6.8 Hz, 3H), 1.010.81 (m, 1H), 0.48-0.11 (m, 4H); 13C NMR (100 MHz, DMSO-d6) δ 168.71 & 168.12, 164.62 & 164.41, 164.02 & 163.77, 65.71, 49.97 & 49.91, 43.37, 20.21 & 19.85, 17.06


& 17.00, 3.19 & 3.05, 2.74 & 2.56; LC-MS (ESI) m/z Calcd. for C12H18ClN5O: 283.12, found: 284.20 [M+H]+ 4,4'-(6-Chloro-1,3,5-triazine-2,4-diyl)dimorpholine (9) Cyanuric chloride 1 (10.0 g, 0.054 mol) was dissolved in dichloromethane (150 mL) at 0 °C, followed by the addition of diisopropylethylamine (17.93 mL, 0.108 mol). The reaction mixture was stirred for 5 minutes at 0 °C. Morpholine (9.50 mL, 0.108 mol) was added drop-wise to the reaction mixture for 10 min, stirred for 30 min at 0 °C. The resulting white precipitate was filtered, washed with water (100 mL) followed by diethylether (50 mL) and dried to afford 14.71 g (95.02%) of 4,4'-(6-chloro-1,3,5-triazine-2,4-diyl)dimorpholine (9) as white solid. Yield: 95%; 1H NMR (400 MHz, CDCl3) δ 3.80-3.68 (m, 16H); 13C NMR (75MHz, CDCl3) δ 169.65, 164.47, 66.57, 43.83; LC-MS (ESI) m/z Calcd. for C11H16ClN5O2: 285.10, found: 286.09 [M+H]+ BIOLOGY Cytotoxicity Assay Cell Lines and Cell Culture The cell lines MDA-MB-231 (human breast adenocarcinoma cell line), HT29 (human colorectal adenocarcinoma cell line) and HEK293 (human embryonic kidney normal cell line) were obtained from the National Centre for Cellular Sciences (NCCS), Pune, India. Cells were cultured either in RPMI1640 (MDA-MB-231, HEK293) or DMEM (HT-29) media, supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1 mM NaHCO3, 2 mM-glutamine, 100 units/mL penicillin and 100 µg/mL streptomycin. All cell lines were maintained in culture at 37° C in an atmosphere of 5% CO2. Test Concentrations Initially, stock solutions of each test substances were prepared in 100% Dimethyl Sulfoxide (DMSO, Sigma Chemical Co., St. Louis, MO) with a final concentration of 10mM. Further dilutions were made with culture medium to obtain the experimental stock concentration of 100-0.01µM. Exactly 100µl of each diluent was added to 100µl of cell suspension (total assay volume of 200µl) and incubated for 48h at 37 °C in 5% CO2.


11

The rate of color formation was measured at 570 nm in a spectrophotometer (Spectra MAX Plus; Molecular Devices; supported by SOFTmax PRO-5.4). The percent inhibition of cell viability was determined with reference to the control values (without test compound). The data were normalised with the lowest and highest values and subjected to nonlinear regression analysis to obtain log (concentration) Vs normalised expression (variable slope) using graph pad prism 6. The GI50 (inhibition of cell growth) concentrations were further obtained using the Graph pad prism 6. CONCLUSION In conclusion, we have synthesized a series of new heteroaryl substituted morpholine tagged triazines and evaluated their cytotoxic activity. Compounds 3f, 3h, 6a, 8c, 8f and 10a showed significant cytotoxic activity against MDA-MB-231, HT-29 cell lines. Among all these compounds 6a and 8c are active against both the cell lines and are considered to be more potent. Based on the present results, it is warranted that these derivatives can be further evaluated on other cancer cell lines. The promising active compounds (3f, 3h, 6a, 8c, 8f and 10a) will be tested for PI3K / mTOR inhibition activities. SAR studies by further structural modification of these active derivatives may lead to a prospective anticancer candidate molecule and further work in this area is in progress. ETHICS APPROVAL PARTICIPATE

AND

CONSENT

TO

Not applicable. HUMAN AND ANIMAL RIGHTS No Animals/Humans were used for studies that are base of this research. CONSENT FOR PUBLICATION Not applicable. CONFLICT OF INTEREST The authors declare no conflict of interest, financial or otherwise.

Cytotoxicity Cytotoxicity was measured using the MTT [3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] assay, according to the method of Johan van Meerloo (2011). Briefly, the cells (5 x 103) were seeded in each well containing 0.1 mL of medium in 96 well plates. After overnight incubation at 37 °C in 5% CO2, the cells were treated with 100µL of different test concentrations of test compounds (0.01 to 100µM) at identical conditions with three replicates each. The final test concentrations were equivalent to 0.01 µM to 100µM. The cell viability was assessed after 48h, by adding 10µL of MTT (5 mg/mL) per well. The plates were incubated at 37 °C for additional 2-4 hours. The medium was discarded and the formazan violet crystals, which were colored for the viable cells, were dissolved in 100 µL of DMSO.

ACKNOWLEDGEMENTS Authors wish to sincerely thank GVK Biosciences Pvt. Ltd., for the Higher Education Program, financial support and encouragement. Help from the analytical department for all spectral analysis is highly appreciated. We are thankful to Dr. Sudhir Kumar Singh for his immense support and motivation. We are also thankful to JNTUH, Hyderabad and BITS-Pilani, Hyderabad. SUPPLEMENTARY MATERIAL Supplementary material is available on the publisher’s web site along with the published article.


Kasturi et al.

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