Purine derivatives with heterocyclic moieties and related analogues as new antitumour agents

For Review Only PURINE DERIVATIVES WITH HETEROCYCLIC MOIETIES AND RELATED ANALOGUES AS NEW ANTITUMOUR AGENTS Journal: Future Medicinal Chemistry Manuscript ID FMC-2018-0291.R3 Manuscript Type: Research Article Keywords: quinoline, antiproliferative activity, apoptosis Note: The following files were submitted by the author for peer review, but cannot be converted to PDF. You must view these files (e.g. movies) online. Figure 1.cdx Figure 2.cdx Figure 3.cdx Figure 4.cdx Figure 5.cdx Compound 4c at 30 uM-1.mp4 https://mc04.manuscriptcentral.com/fs-fmc Future Medicinal Chemistry For Review Only 1 Abstract Aim: Identification of new antiproliferative compounds. Methodology: Four series of compounds were synthesized by the Mitsunobu reaction. Their antiproliferative activity was studied against several cancer cells and a non-cancerous fibroblast cell line. Their apoptotic activity was analyzed using a caspase 3/7 fluorescence assay. Results & Conclusion: 9-Alkylated-6-halogenated and 2,6-dihalogenated purines show remarkable inhibition of tumour cell proliferation, with the dichloro derivatives being the most potent of all the series. The most promising compound, tetrahydroquinoline 4c, exhibits significant antiproliferative activity against the cancer cells tested, while displaying a 19-fold lower potency against non-cancerous fibroblasts, a key feature that indicates potential selectivity against cancer cells. This compound produces a high percentage of apoptosis (58%) after 24h treatment in the human breast cancer MCF-7 cells. Graphical abstract O SN N N N N Y X N N N N Cl Cl Ts 4c2a-c Isosteric replacement in fragment A A B Promising tetrahydroquinoline linked to 2,6-dichloropurine for future drug development Keywords: benzoxazine, quinoline, pyridoxazine, Mitsunobu, antiproliferative activity, apoptosis. Page 1 of 66 https://mc04.manuscriptcentral.com/fs-fmc Future Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Review Only 2 Introduction Cancer is one of the major public health problems in the world with 17.5 million cases worldwide and 8.7 million deaths in 2015 [1]. An increase of 13.1 million cancer deaths has been estimated in 2030, according to the World Health Organization [2]. Although in recent years there have been important advances in the understanding of the underlying mechanisms leading to many cancers, some of them are still very difficult to treat and remain unmet clinical needs. Multi-drug resistance and systemic toxicity are some of the main drawbacks limiting the efficacy of current cancer treatments [3]. To tackle these limitations, the design of more effective and safer drugs is required. It is known that many drugs activate apoptosis as a mechanism for their antitumour activity. Apoptosis is a cellular natural process whereby cells induce their own death in response to several biochemical signals that are typically triggered by an irreparable damage to DNA. It is a fundamental process of protection and maintenance of homeostasis. Generally, a group of cysteine aspartyl proteases known as caspases are responsible for the initiation (e.g. caspases 2, 8, 9 and 10) and effecting (e.g. caspases 3, 6 and 7) of apoptosis [4]. The development of drugs that induce apoptosis is an area of great activity for the discovery of new anticancer therapies [5]. Our research group has previously published several compounds that effectively induce caspase-mediated apoptosis, including a series of benzofused six-membered rings linked to purines through a methylene group. Substituted 9-(2,3-dihydro-1,4-benzo[b][1,4]oxathiin-3-ylmethyl)-9H-purines (1ac) [6] and their isomers 9-(2,3-dihydro-1,4-benzo[b][1,4]oxathiin-2-ylmethyl)-9Hpurines (2a-c) [7] (Figure 1) showed interesting antiproliferative activities and high apoptosis levels on the human breast adenocarcinoma MCF-7 cells. In this article, we have introduced several structural modifications on the scaffold 2 to explore a new chemical space (Figure 1). A bioisosteric replacement was made by changing the sulfur atom of the 6-membered ring by a nitrogen one, obtaining the 2H-1,4-benzoxazine derivatives 3 (series A). For Page 2 of 66 https://mc04.manuscriptcentral.com/fs-fmc Future Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Review Only 3 synthetic reasons, in order to carry out the Mitsunobu reaction, the nitrogen atom in the heterocycle was converted into the tosylsulfonamide group. This moiety was maintained in the structure as the removal of the oand p-nosyl group in a series of benzoxapine derivatives proved to be deleterious for the antiproliferative activity in a previous work [8] (see Figure S.1. in Supplementary Information). Additionally, the toxicity described for the NO2 group [9, 10] led us to replace it by the p-CH3 on the benzene ring (tosyl group). The influence of the distance between the heterocyclic ring and the purine was also evaluated by introducing a linker of two carbon atoms. Electron-withdrawing groups (Cl, Br, di-Cl) were used as substituents at positions 2 and 6 of the purine ring since they conferred good antiproliferative properties in previously reported derivatives 1 and 2 [6, 7]. Additionally, we decided to explore the introduction of a trifluoromethyl group (CF3), a common substituent used in medicinal chemistry. This group offers not only high lipophilicity but also increased electron density, and is found in approved drugs such as efavirenz (for HIV disease) [11] and desoxyepothilone b (anticancer activity) [12]. Finally taking 3 as a reference scaffold, the following modifications were also introduced: (a) Elimination of the oxygen atom in the heterocycle and shortening of the side chain (derivatives 4, series B); (b) change of the benzene ring for a pyridine moiety and linking the (purine-9yl)ethyl fragment through the position 3 of the [1,4]oxazine (compounds 5, series C); (c) aperture of the heterocycle to obtain open analogues, in which the tosyl group and the substituted purine moieties have been maintained (derivatives 6 and 7, series D). Page 3 of 66 https://mc04.manuscriptcentral.com/fs-fmc Future Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Review Only 4 O SNN N NX Y O S N N NN X Y 1 2 1a, X = Br, Y = H 1b, X = Cl, Y = H 1c, X = Y = Cl 2a, X = Br, Y = H 2b, X = Cl, Y = H 2c, X = Y = Cl N N Ts N NN X Y NN N N Y X O O R N Ts O O a) b) c) 4a-d 3a-d 6a-d, R = Me 7a-d, R = Et 5b-c series B series A series C series D X = Br, Cl, CF3; Y = H, Cl X = Br, Cl, CF3; Y = H, Cl X = Cl; Y = H, Cl X = Br, Cl, CF3; Y = H, Cl O N Ts NN N N X Y N O N Ts NN N N X Y Figure 1. Benzo-fused six-membered rings linked to purines described by our research group (1 – 2) and chemical structure of the novel compounds objective of this article (3 – 7). Materials and methods Chemistry The determination of melting points was made in open capillaries employing an Electrothermal 1A 6301 instrument and they are unrectified. Elemental analyses were designated by the element symbols, the results were not over 0.4% of the postulated values and they were carried out in a Thermo Scientific Flash 2000 analyzer. Silica (0.035-0.070 mm), 60 Å used for flash chromatography was manufactured by Acros Organic. NMR spectra were carried out on a Varian Direct Drive 400 spectrometer operating at 400 MHz for 1H and 101 MHz for Page 4 of 66 https://mc04.manuscriptcentral.com/fs-fmc Future Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Review Only 5 13C, on a Varian Direct Drive 500 spectrometer operating at 500 MHz for 1H and 126 MHz for 13C at room temperature (rt) in all cases. Chemicals shifts () are reported in ppm and are referenced to the residual solvent peak. A VG AutoSpec Q high-resolution apparatus (Fision Instrument) was used to perform the high-resolution mass spectroscopy (HRMS). Anhydrous reactions were carried out under argon atmosphere. Reagents were purchased from SigmaAldrich (now Merck) and Acros Organics (part of Thermo Fisher Scientific). Synthesis and characterization of the intermediate derivatives are presented in the Supplementary Information. Characterization of 6-bromopurines 3a, 4a, 6a, and 7a, are described in the Supplementary Information. Synthesis and characterization of 6-trifluoromethylpurines 3d, 4d, 6d and 7d are reported in the Supplementary Information. General synthetic procedure of halo and dihalopurine derivatives The tosylated derivatives 9, 11, 16 or 18 (0.31 mmol), Ph3P (164.43 mg, 0.63 mmol) and the adequate halo or dihalopurine (0.34 mmol) were purged with 3 cycles of vacuum-argon exchanges in a Schlenck line previously anhydrified. Anhydrous THF was added (2 mL) under argon atmosphere and the reaction mixture was cooled to -20°C. After that, DIAD (124 µl, 0.63 mmol) was added dropwise and stirred from -20°C to rt. After 36 h the solvent was evaporated and the residue was purified by flash chromatography (EtOAc/hexane, 2:1). Characterization of 6-chloropurines 3b, 4b, 5b, 6b and 7b. 2-(2-(6-Chloro-9H-purine-9-yl)ethyl)-4-tosyl-3,4-dihydro-2Hbenzo[b][1,4]oxazine (3b) White solid (92 mg, 0.195 mmol), yield 63%, mp: 189 - 190°C; 1H NMR (400 MHz, CDCl3):  (ppm) 8.75 (s, 1H, Hpurine), 7.96 (s, 1H, Hpurine), 7.79 (dd, J1 = 8.3 Hz, J2 = 1.5 Hz, 1H, Hbenz), 7.34 (d, J = 8.1 Hz, 2H, 2xHtosyl), 7.16 – 7.00 (m, 3H, 2xHtosyl,Hbenz), 7.01 – 6.88 (m, 1H, Hbenz), 6.78 (dd, J1 = 8.2 Hz, J2 = 1.5 Hz, 1H, Hbenz), 4.52 – 4.39 (m, 2H, CH2N), 4.20 (dd, J1 = 14.1 Hz, J2 = 1.9 Hz, 1H, Hoxazine), 3.38 – 3.27 (m, 1H, Hoxazine), 3.21 (dd, J1 = 14.1 Hz, J2 = 9.7 Hz, 1H, Page 5 of 66 https://mc04.manuscriptcentral.com/fs-fmc Future Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Review Only 6 Hoxazine), 2.39 (s, 3H, CH3tosyl), 2.35 – 2.18 (m, 1H, CH2CH2N), 2.10 – 1.93 (m, 1H, CH2CH2N); 13C NMR (101 MHz, CDCl3):  (ppm) 152.0 (CHpurine), 151.7 (Cpurine), 151.1 (Cpurine), 146.0 (Cbenz), 145.2 (Cpurine), 144.6 (Ctosyl), 135.3 (Ctosyl), 131.6 (Cpurine), 129.7 (2xCHtosyl), 126.9 (2xCHtosyl), 126.3 (CHbenz), 124.3 (CHbenz), 123.5 (Cbenz), 121.4 (CHbenz), 117.2 (CHbenz), 68.3 (CHoxazine), 48.2 (CH2oxazine), 40.3 (CH2N), 31.7 (CH2CH2N), 21.6 (CH3tosyl); HRMS (ESI-TOF) (m/z) calcd. for C22H21N5O3SCl (M + H)+ 470.1054, found 470.1055. Anal. Calc. for C22H20N5O3SCl: C, 56.23; H, 4.29; N, 14.90. Found: C, 56.22; H, 4.31; N, 14.88. 3-((6-Chloro-9H-purine-9-yl)methyl)-1-tosyl-1,2,3,4-tetrahydroquinoline (4b) White solid (85 mg, 0.186 mmol), yield 60%, mp: 202 - 203°C; 1H NMR (500 MHz, CDCl3):  (ppm) 8.74 (s, 1H, Hpurine), 8.04 (s, 1H, Hpurine), 7.73 (dd, J1 = 8.3 Hz, J2 = 11.2 Hz, 1H, Hbenz), 7.39 (d, J = 8.1 Hz, 2H, 2xHtosyl), 7.23 – 7.17 (m, 1H, Hbenz), 7.12 (d, J = 8.0 Hz, 2H, 2xHtosyl), 7.10 – 7.05 (m, 1H, Hbenz), 6.98 (dd, J1 = 7.6 Hz, J2 = 1.5 Hz, 1H, Hbenz), 4.25 (dd, J1 = 14.2 Hz, J2 = 7.2 Hz, 1H, CH2N), 4.15 (dd, J1 = 14.2 Hz, J2 = 6.7 Hz, 1H, CH2N), 4.01 (dd, J1 = 13.5 Hz, J2 = 4.1 Hz, 1H, Hpyr), 3.45 (dd, J1 = 13.5 Hz, J2 = 8.9 Hz, 1H, Hpyr), 2.59 (dd, J1 = 16.0 Hz, J2 = 5.3 Hz, 1H, Hpyr), 2.42 – 2.37 (m, 1H, Hpyr), 2.36 (s, 3H, CH3tosyl), 2.30 (dd, J1 = 15.9 Hz, J2 = 9.1 Hz, 1H, Hpyr); 13C NMR (101 MHz, CDCl3):  (ppm) 152.0 (CHpurine), 151.8 (Cpurine), 151.3 (Cpurine), 145.2 (CHpurine), 144.0 (Ctosyl), 136.3 (Cbenz), 136.2 (Ctosyl), 131.6 (Cpurine), 129.7 (2xCHtosyl), 129.3 (CHbenz), 127.3 (Cbenz), 127.2 (CHbenz), 126.8 (2xCHtosyl), 125.2 (CHbenz), 124.0 (CHbenz), 48.5 (CH2pyr), 46.7 (CH2N), 32.8 (CHpyr), 30.7 (CH2pyr), 21.5 (CH3tosyl); HRMS (ESI-TOF) (m/z) calcd. for C22H21N5O2SCl (M + H)+ 454.1104, found 454.1098. Anal. calc. for C22H20N5O2SCl: C, 58.21; H, 4.44; N, 15.43. Found: C, 58.19; H, 4.46; N, 15.45. 3-(2-(6-Chloro-9H-purine-9-yl)ethyl)-4-tosyl-3,4-dihydro-2H-pyrido[3,2b][1,4]oxazine (5b) Yellowish solid (80 mg, 0.171 mmol), yield 55%, mp: 180 - 181°C; 1H NMR (500 MHz, CDCl3):  (ppm) 8.73 (s, 1H, Hpurine), 8.30 (s, 1H, Hpurine), 8.02 (dd, J1 = 4.7 Hz, J2 =1.5 Hz, 1H, Hbenz), 7.84 (d, J = 8.1 Hz, 2H, 2xHtosyl), 7.25 (d, J = 8.0 Hz, Page 6 of 66 https://mc04.manuscriptcentral.com/fs-fmc Future Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Review Only 7 2H, 2xHtosyl), 7.15 (dd, J1 = 8.0 Hz, J2 = 1.5 Hz, 1H, Hbenz), 6.96 (dd, J1 = 8.0 Hz, J2 = 4.6 Hz, 1H, Hbenz), 4.80 – 4.75 (m, 1H, Hoxazine), 4.61 – 4.43 (m, 2H, CH2N), 4.18 (dd, J1 = 11.4 Hz, J2 = 1.4 Hz, 1H, Hoxazine), 3.77 (dd, J1 = 11.4 Hz, J2 = 2.3 Hz, 1H, Hoxazine), 2.49 – 2.39 (m, 1H, CH2CH2N), 2.38 (s, 3H, CH3tosyl), 2.20 – 2.09 (m, 1H, CH2CH2N); 13C NMR (126 MHz, CDCl3):  (ppm) 151.8 (Cpurine), 151.7 (CHpurine), 151.0 (Cpurine), 146.0 (CHpurine), 144.4 (Ctosyl), 140.9 (CHbenz), 140.3 (Cbenz), 137.1 (Cbenz), 136.2 (Ctosyl), 131.8 (Cpurine), 129.4 (2xCHtosyl), 128.2 (2xCHtosyl), 124.7 (CHbenz), 120.4 (CHbenz), 67.0 (CH2pyr), 51.2 (Cpyr), 41.6 (CH2N), 30.8 (CH2CH2N), 21.5 (CH3tosyl); HRMS (ESI-TOF) (m/z) calcd. for C21H20N6O3SCl (M + H)+ 471.1006, found 471.0981. Anal. calc. for C21H19ClN6O3S: C, 53.56; H, 4.07; N, 17.85. Found: C, 53.54; H, 4.09; N, 17.88. Methyl-2-(N-tert-butoxycarbonyl)-N-tosylamino)-3-(6-chloro-9H-purine-9yl)propanoate (6b) White solid (110 mg, 0.217 mmol), yield 70%, mp: 164 - 165°C; 1H NMR (500 MHz, CDCl3):  (ppm) 8.67 (s, 1H, Hpurine), 8.20 (s, 1H, Hpurine), 7.59 (d, J = 8.1 Hz, 2H, 2xHtosyl), 7.18 (d, J = 8.1 Hz, 2H, 2xHtosyl), 5.58 (dd, J1 = 9.4 Hz, J2 = 4.9 Hz, 1H, Hprop), 5.05 (dd, J1 = 14.7 Hz, J2 = 4.9 Hz, 1H, Hprop), 4.95 (dd, J1 = 14.7 Hz, J2 = 9.4 Hz, 1H, Hprop), 3.83 (s, 3H, OCH3), 2.41 (s, 3H, CH3tosyl), 1.34 (s, 9H, 3xCH3); 13C NMR (126 MHz, CDCl3):  (ppm) 167.8 (COOMe), 152.0 (Cpurine), 151.9 (CHpurine), 150.9 (Cpurine), 149.8 (COOBut), 145.5 (CHpurine), 144.8 (Ctosyl), 135.6 (Ctosyl), 131.3 (Cpurine), 129.0 (2xCHtosyl), 128.1 (2xCHtosyl), 86.1 (C(CH3)3), 58.1 (CHprop), 53.0 (OCH3), 43.4 (CH2prop), 27.6 (C(CH3)3), 21.5 (CH3tosyl); HRMS (ESI-TOF) (m/z) calcd. For C21H25N5O6SCl (M + H)+ 510.1214, found 510.1215. Anal. calc. for C21H24N5O6SCl: C, 49.46; H, 4.74; N, 13.73. Found: C, 49.48; H, 4.74; N, 13.71. Ethyl-2-(N-tert-butoxycarbonyl)-N-tosylamino)-3-(6-chloro-9H-purine-9yl)propanoate (7b) White solid (146 mg, 0.279 mmol), yield 90%, mp: 175 - 176°C; 1H NMR (400 MHz, CDCl3):  (ppm) 8.71 (s, 1H, Hpurine), 8.18 (s, 1H, Hpurine), 7.62 (d, J = 8.2 Hz, 2H, 2xHtosyl), 7.19 (d, J = 8.2 Hz, 2H, 2xHtosyl), 5.55 (dd, J1 = 9.2, J2 = 5.0 Hz, 1H, Hprop), 5.03 – 4.97 (m, 2H, Hprop), 4.27 (q, J = 7.2 Hz, 2H, OCH2CH3), Page 7 of 66 https://mc04.manuscriptcentral.com/fs-fmc Future Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Review Only 8 2.43 (s, 3H, CH3tosyl), 1.33 (s, 9H, 3xCH3), 1.26 (t, J = 7.3 Hz, 3H, OCH2CH3); 13C NMR (101 MHz, CDCl3):  (ppm) 167.3 (COOEt), 152.1 (Cpurine), 151.8 (CHpurine), 150.9 (Cpurine), 149.8 (COOBut), 145.4 (CHpurine), 144.7 (Ctosyl), 135.7 (Ctosyl), 131.4 (Cpurine), 128.9 (2xCHtosyl), 128.0 (2xCHtosyl), 85.9 (C(CH3)3), 62.3 (OCH2CH3), 58.1 (CHprop), 43.3 (CH2prop), 27.6 (C(CH3)3), 21.4 (CH3tosyl), 13.8 (OCH2CH3); HRMS (ESI-TOF) (m/z) calcd. forC22H27N5O6SCl (M + H)+ 524.1371, found 524.1370. Anal. Calc. for C22H26N5O6SCl: C, 50.43; H, 5.00; N, 13.37. Found: C, 50.41; H, 5.03; N, 13.35. Characterization of 2,6-dihalopurines 3c, 4c, 5c, 6c and 7c. 2-(2-(2,6-Dichloro-9H-purine-9-yl)ethyl)-4-tosyl-3,4-dihydro-2Hbenzo[b][1,4]oxazine (3c) White solid (91 mg, 0.221 mmol), yield 68%, mp: 195-196°C; 1H NMR (400 MHz, CDCl3):  (ppm) 7.96 (s, 1H, Hpurine), 7.77 (dd, J1 = 8.3 Hz, J2 = 1.6 Hz, 1H, Hpurine), 7.40 (d, J = 8.2 Hz, 2H, 2×Htosyl), 7.16 – 7.02 (m, 3H, 2×Htosyl, Hbenz), 7.01 – 6.88 (m, 1H, Hbenz), 6.76 (dd, J1 = 8.2 Hz, J2 = 1.6 Hz, 1H, Hbenz), 4.46 - 4.41 (m, 2H, CH2N), 4.19 (dd, J1 = 14.2 Hz, J2 = 2.3 Hz, 1H, Hoxazine), 3.42 – 3.29 (m, 1H, Hoxazine), 3.28 – 3.13 (m, 1H, Hoxazine), 2.39 (s, 3H, CH3tosyl), 2.30 – 2.14 (m, 1H, CH2CH2N), 2.09 – 1.97 (m, 1H, CH2CH2N ); 13C NMR (101 MHz, CDCl3): (ppm) 153.2 (2×Cpurine), 151.9 (CPurine) 146.1 (CPurine), 146.0 (Cbenz), 144.8 (Ctosyl), 135.5 (Ctosyl), 130.9 (Cpurine), 129.9 (2×CHtosyl), 127.1 (2×CHtosyl), 126.4 (CHbenz), 124.3 (CHbenz), 123.7 (Cbenz),, 121.6, (CHbenz), 117.4 (CHbenz), 68.7 (CHoxazine), 48.3 (CH2oxazine), 40.6 (CH2N), 31.9 (CH2CH2N), 21.8 (CH3tosyl); HRMS (ESI-TOF) (m/z) calcd. for C22H20N5O3SCl2 (M + H)+ 504,0586, found 504,0636; Anal. Calc. for C22H19N5O3SCl2: C, 52.39; H, 3.80; N, 13.89. Found: C, 52.37; H, 3.82; N, 13.91. 3-((2,6-Dichloro-9H-purine-9-yl)methyl)-1-tosyl-1,2,3,4-tetrahydroquinoline (4c) White solid (102 mg, 0.232 mmol), yield 75%, mp: 207-208°C; 1H NMR (500 MHz, CDCl3):  (ppm) 8.07 (s, 1H, Hpurine), 7.69 (dd, J1 = 8.3 Hz, J2 =1.1 Hz, 1H, Hbenz), 7.38 (d, J = 8.0 Hz, 2H, 2×Htosyl), 7.21 – 7.12 (m, 3H, Hbenz, 2×Htosyl), 7.08 – 7.03 (m, 1H, Hbenz), 6.97 (dd, J1 = 7.6 Hz, J2 = 1.6 Hz, 1H, Hbenz), 4.20 (dd, J1 Page 8 of 66 https://mc04.manuscriptcentral.com/fs-fmc Future Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Review Only 15 nowadays in the top-20 of the most deadly ones, therefore the identification of new treatments is required. We first analysed the antiproliferative properties of the 18 synthesized compounds against MCF-7 cell line. Those structures that exhibited an EC50 value lower than 50 μM were further investigated in A-375 and HCT-116 cells. The results of this assessment are shown in Table 1. 5-Fluorouracil (5-FU) has been used as a control for the breast and colon carcinoma cell lines. Today this drug and its derivatives such as capecitabine, are widely used in different solid tumours despite its high toxicity and side effects [28, 29]. In general, 6-halo, 2,6-dihalo and 6-trifluoromethyl groups on the 9-alkylated purine show a notable inhibition on cell proliferation, with the dichloro analogue being the most interesting in each of the series and tumour cell lines. This data are in agreement with the results previously published by our research group where the highest antiproliferative activity were obtained with electronwithdrawing groups in the purine ring [7], specifically derivatives bearing two chloro atoms at 2 and 6 positions. Accordingly, the most active compounds in MCF-7 cell line are the 9-alkylated 2,6-disubstituted purines with two chlorine atoms, with 4c being the most potent inhibitor (EC50 = 4.26 ± 0.15 μM), only slightly less active than the positive control 5-FU. Compounds 4c-d (within series B), which feature a tetrahydroquinoline heterocycle, exhibited higher activity than their isosteric benzoxazines 3c-d (series A). The open structures included in series D (6a-d and 7a-d) mediated no or low inhibition of cell proliferation. Only the most lipophilic derivative, 7c (R = Et; X = Cl, Y = Cl), showed some level of activity. This might be due to a reduced capacity of the open structures to penetrate the cell membrane. Regarding the A-375 cell line, derivatives with a tetrahydroquinoline (4c) and pyridoxazine (5c) heterocycle show similar inhibition values (EC50 = 5.54 ± 0.64 and 5.66 ± 0.65 μM, respectively). In this cell line there is not much difference of activity between the 6-chloro (3b) and 2,6-dichloro (3c) derivatives from series A. Page 15 of 66 https://mc04.manuscriptcentral.com/fs-fmc Future Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Review Only 16 The best inhibition values were obtained in the carcinoma colon line HCT-116 where the 2,6-dicloro derivatives 3c (7.06 ± 0.80 µM), 4c (2.80 ± 0.31 µM) and 5c (3.13 ± 0.35 µM) mediated high activity. 4c, the most potent derivative of the series, showed an activity comparable to the reference compound 5-FU. Interestingly, in this tumour cell line the open derivative with a methyl carboxylate moiety (6c) shows a better inhibition than the one with an ethyl carboxylate group, unlike the other two cell lines. Generally, in all derivatives it is observed that the oxygen atom of the benzoxazine ring (derivatives 3) is not essential for the antitumour activity since when it was eliminated (derivatives 4) compounds show better inhibition values. Furthermore, if we compare derivatives 3 and 5, the substitution of the benzene ring by its isosteric pyridine, as well as the change of the side chain position in the condensate system, have led to an improvement in the activity. Finally, the presence of a heterocyclic system is very important for the antitumour inhibition since compounds are less active when it is not present (open derivatives 6 and 7). Table 1. In vitro anticancer activity of final compounds against MCF-7, A-375 and HCT-116 tumour cells. Comp X Y R Cancer cell lines MCF-7 EC50 (μM)a A-375 EC50 (μM)a HCT-116 EC50 (μM)a 3a Br H - 27.45 ± 0.18 26.11 ± 3.01 17.30 ± 2.67 3b Cl H - 37.63 ± 4.85 13.32 ± 0.43 17.54 ± 0.86 3c Cl Cl - 13.00 ± 0.11 10.85 ± 1.40 7.06 ± 0.80 3d CF3 H - 33.61 ± 3.62 34.59 ± 2.80 56.06 ± 2.66 4a Br H - > 100 - - 4b Cl H - > 100 - - 4c Cl Cl - 4.26 ± 0.15 5.54 ± 0.64 2.80 ± 0.31 4d CF3 H - 21.95 ± 1.7 61.57 ± 0.67 60.14 ± 5.35 5b Cl H - > 100 - - 5c Cl Cl - 11.56 ± 0.28 5.66 ± 0.65 3.13 ± 0.35 6a Br H Me > 100 - - 6b Cl H Me > 100 - - Page 16 of 66 https://mc04.manuscriptcentral.com/fs-fmc Future Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Review Only 17 6c Cl Cl Me 45.96 ± 5.69 20.48 ± 2.40 22.20 ± 1.57 6d CF3 H Me > 100 - - 7a Br H Et > 100 - - 7b Cl H Et > 100 - - 7c Cl Cl Et 26.15 ± 3.22 17.62 ± 1.90 38.32 ± 2.01 7d CF3 H Et > 100 - - 5-FU 1.5 ± 0.31 - 2.40 ± 0.62 aCell viability was measured after 5 days treatment using the PrestoBlueTM reagent. Experiments were conducted in triplicate. Data are the mean ± SD of 3 independent determinations. To analyze if the observed growth inhibition was due to an apoptotic effect, NucView™ 488 caspase-3 substrate was used to assess the rate of caspase3/7 mediated apoptosis in MCF-7 cells once treated with derivatives 3c, 4c, 4d and 5c. This fluorescent probe contains a peptide sequence (DEVD) that after cleaved by caspase-3/7 activity releases a DNA-binding dye which stains the cell nucleus bright green. As shown in Figure 6, image-based measurement of caspase 3/7 activity demonstrated significant levels of apoptotic cell death in a time-dependent manner in comparison with the control DMSO. Figure 6. (A) Percentage of apoptotic cells after treatment with compounds 3c, 4c, 4d and 5c (30 µM) for 12h (blue), 24 h (light green) and 48h (dark green). (B) Representative images of MCF-7 cells stained using caspase 3/7-detecting reagent after 24 h treatment with compound 4c (30 µM). The most interesting compound was the tetrahydroquinoline 4c that induces 58% apoptosis after 24 h treatment at 30 µM. This structure also displayed strong apoptotic activity at lower concentrations (see Figure S.2. of the Page 17 of 66 https://mc04.manuscriptcentral.com/fs-fmc Future Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Review Only 18 Supplementary Information). On the contrary, compound 5c (which showed moderate activity in MCF-7 with an EC50 value of 11.56 ± 0.28 μM) did not produce an important increase in apoptotic cells (18% at 48 h). This fact could indicate a different mechanism of action (e.g. cell cycle inhibitor, cytotoxic, etc.) for this pyridoxazine derivative. In addition, the change of the 2,6-dichloropurine moiety (4c) to 6-trifluoromethylpurine (4d) has produced a significant decrease of apoptosis (from 58 to 25% after 24h) as we observed with the antiproliferative activity. The benzoxazine 3c has shown moderate apoptosis after 48h of treatment (45%). Furthermore, as a preliminary assessment of the safety profile of compound 4c, a cell proliferation study was performed in non-tumour cells (RFP TERT immortalized fibroblasts). As shown in Figure 7, treatment of non-cancerous fibroblasts with compound 4c resulted in a significantly lower antiproliferative activity, with a reduction of activity of up to 19-fold respect to the HCT-116 tumour cell line, indicating promising preferential activity towards cancerous cells. Figure 7. Semilog dose-response curves and EC50 values for compound 4c against RFP TERT immortalized fibroblasts, A-375, MCF-7 and HCT-116 cells. Error bars: ± SD from n = 3. Page 18 of 66 https://mc04.manuscriptcentral.com/fs-fmc Future Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Review Only 19 Conclusion In an attempt to find new antitumour agents, eighteen purine molecules linked to different heterocycles (benzoxazine, quinoline and pyridoxazine) and open analogues (methyl and ethyl propanoate derivatives) were synthesized. We evaluated their antiproliferative activity against three cancerous cell lines (MCF7, A-375 and HCT-116) and the induction of apoptosis in MCF-7. In this study we have shown the importance of the substitution in the purine rings since compounds with a 2,6-dichloropurine moiety are consistently more active than other members of the series. In addition, compounds with the complete heterocyclic systems have been more active than open analogues, highlighting the compound with the quinoline ring. In addition, an increase in the distance between the purine and the heterocycle leads to a decrease of antiproliferative activity. 4c is the most active compound in the cancer cell lines tested and an apoptotic inducer through activation of caspases 3/7, with a low cytotoxicity in non-cancerous cells. In conclusion, this derivative can be considered as a promising antiproliferative lead compound for future optimization campaigns. Future prespective One of the main hallmarks of cancer is the ability of malignant cells to evade programed cell death. Therefore, the development of chemical structures able to effectively induce apoptosis represents an interesting approach in the finding of new anticancer treatments. In the search for improved therapies, chemical moieties such as purines and related heterocycles are privileged structures in medicinal chemistry. This work reports an interesting derivative 4c (3-((2,6dichloro-9H-purine-9-yl)methyl)-1-tosyl-1,2,3,4-tetrahydroquinoline) that was easily prepared and had a promising activity against three cancer cells lines. Moreover, this compound induces apoptosis through activation of caspases, and shows a good safety profile. This quinoline could be an interesting starting point for further structural optimization to obtain new promising antitumour agents. Page 19 of 66 https://mc04.manuscriptcentral.com/fs-fmc Future Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Review Only 20 Executive summary Purine derivatives linked to heterocycles as a promising scaffold • 18 Novel purine compounds incorporating different heterocycles and their open analogues were designed, synthesized and evaluated for their antiproliferative activity. • In the 4 series of derivatives the most interesting molecules have a moiety of 2,6-dichloropurine. Antiproliferative and apoptosis activity • Compound 4c with a quinoline ring shows the lowest EC50 values against MCF-7 (4.26 μM), A-375 (5.54 μM) and HCT-116 (2.80 μM). • 4c induced apoptosis by activation of caspases 3/7 in a time-dependent manner. It shows a pronounced selectivity against cancer cells. • 4c is therefore a leading structure for future anticancer drug development due to its straightforward synthesis and relevant bioactivity. Supplementary data See online the supplementary data with the synthesis and characterization of intermediate compounds, 6-bromo and 6-trifluoromethylpurines derivatives and a two-day time-lapse motion picture of MCF-7 cell proliferation under treatment with 30 μM of 4c with Nucview 488 (apoptosis fluorescent marker). Financial & competing interest disclosure This paper was supported financially by the Junta de Andalucía (project no. CS2016.1). The authors have no other relevant affiliations or financial involvement with any organization or entity with financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript. Page 20 of 66 https://mc04.manuscriptcentral.com/fs-fmc Future Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Review Only 21 References Papers of special note have been highlighted as: • of interest; •• of considerable interest • Importance of cancer in today's society 1. Fitzmaurice C, Allen C, Barber RM et al. Global, Regional, and National Cancer Incidence, Mortality, Years of Life Lost, Years Lived With Disability, and Disability-Adjusted Life-years for 32 Cancer Groups, 1990 to 2015. JAMA Oncol. 3(4), 524–548 (2017). 2. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2018. CA Cancer J. Clin. 68, 7–30 (2018). 3. Singla H, Kalra S, Kheterpal P et al. Role of genomic alterations in HER2 positive breast carcinoma: focus on susceptibility and trastuzumab-therapy. Curr. Cancer Drug Target 17, 344–356 (2017). •• Caspases as apoptosis regulators, an attractive strategy in cancer therapy 4. Shalini S, Dorstyn L, Dawar S, Kumar S. Old, new and emerging functions of caspases. Cell Death Differ. 22, 526-539 (2015). 5. Bai L, Wang S. Targeting apoptosis pathways for new cancer therapeutics. Annu Rev Med. 65, 139-155 (2014). •• Interesting purine derivatives linked to heterocycles previously published 6. Díaz‐Gavilán M, Conejo‐García A, Cruz‐López O et al. Synthesis and Anticancer Activity of (RS)‐9‐(2,3‐Dihydro‐1,4‐Benzoxathiin‐3‐ylmethyl)‐9H‐Purines. ChemMedChem. 3, 127-135 (2008). 7. Conejo-García A, García-Rubiño ME, Marchal J et al. Synthesis and anticancer activity of (RS)-9-(2,3-dihydro-1,4-benzoxaheteroin-2-ylmethyl)-9Hpurine. Eur. J. Med. Chem. 46, 3795-380 (2011). 8. Díaz-Gavilán M, Gómez-Vidal JA, Rodríguez-Serrano F et al. Anticancer Activity of (1,2,3,5-Tetrahydro-4,1-Benzoxazepine-3-yl)-Pyrimidines and - Page 21 of 66 https://mc04.manuscriptcentral.com/fs-fmc Future Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Review Only 22 Purines against the MCF-7 Cell Line: Preliminary cDNA Microarray Studies. Bioorg. Med. Chem. Lett. 18, 1457-1460 (2008). Nitro group toxicity 9. Kovacic P, Somanathan R. Nitroaromatic compounds: Environmental toxicity, carcinogenicity, mutagenicity, therapy and mechanism. J Appl Toxicol. 34, 810824 (2014). 10. Purohit V, Basu AK. Mutagenicity of nitroaromatic compounds. Chem. Res. Toxicol. 13, 673–692 (2000). The trifluoromethyl group in medicinal chemistry 11. Chen J, Akhtari FS, Wagner M J et al. Pharmacogenetic Analysis of the Model-Based Pharmacokinetics of Five Anti-HIV Drugs: How Does This Influence the Effect of Aging?. Clin Transl Sci 11, 226–236 (2018). 12. Rivkin, A, Biswas K, Chou T, Danishefsky SJ. On the Introduction of a Trifluoromethyl Substituent in the Epothilone Setting: Chemical Issues Related to Ring Forming Olefin Metathesis and Earliest Biological Findings. Org. Lett. 4(23), 4081-4084 (2002). Biological assays procedure 13. Munro J, Steeghs K, Morrison V, Ireland H, Parkinson EK. Human fibroblast replicative senescence can occur in the absence of extensive cell division and short telomeres. Oncogene, 20 (27), 3541-3552 (2001). 14. Rubio-Ruiz B, Weiss JT, Unciti-Broceta A. Efficient Palladium-Triggered Release of Vorinostat from a Bioorthogonal Precursor. J. Med. Chem. 59 (21), 9974-9980 (2016). 15. Fraser C, Dawson JC, Dowling R et al. Rapid Discovery and StructureActivity Relationships of Pyrazolopyrimidines That Potently Suppress Breast Cancer Cell Growth via SRC Kinase Inhibition with Exceptional Selectivity over ABL Kinase. J. Med. Chem. 59 (10), 4697-4710 (2016). Chemical procedures for syntheses Page 22 of 66 https://mc04.manuscriptcentral.com/fs-fmc Future Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Review Only 23 16. Mayer S, Guillaumet G, Merour JY. Regioselective formylation of ethyl 3,4dihydro-2H-1,4-benzoxazine-2-carboxylate or 2-acetate derivatives. Heterocycles, 55(10), 1873-1888 (2001). 17. Véliz EA, Stephens OM, Beal PA. Synthesis and analysis of RNA containing 6-trifluoromethylpurine ribonucleoside. Org. Lett. 3(19), 2969–2972 (2001). 18. Ferrabosch iP, Ciceri S, Grisenti P. Chemoenzymatic synthesis of the enantiomerically pure 1,2,3,4-tetrahydroquinoline moiety of the antithrombotic (21R)- and (21S)-argatroban. Tetrahedron: Asymmetry 24, 1142-1147 (2013). 19. PCT Int. Appl. WO 2016008411 (2016) 20. Provoost OY, Hazelwood AJ, Harrity J. P. Pd-Catalysed [3 + 3] annelations in the stereoselective synthesis of indolizidines. Beilstein J. Org. Chem. 3 (8), 113 (2007). 21. Van Dijk M, Postma TM, Rijkers, DTS, Liskamp RMJ, Nostrum CF, Hennink WE. Synthesis and characterization of tailorable biodegradable thermoresponsive methacryloylamide polymers based on L-serine and Lthreonine alkyl esters. Polymer 51(12), 2479–2485 (2010). 22. Takahashi K, Fukushima K, Tsubuki M, Honda T.The formal synthesis of lucentamycin A: Construction of cis-2,3-disubstituted pyrrolidine core by application of SmI2-DMPU system. Tetrahedron Lett. 59, 1435-1437 (2018). 23. Shields SWJ, Manthorpe JM. Efficient, scalable and economical preparation of tris(deuterium)- and 13C-labelled N-methyl-N-nitroso-p-toluenesulfonamide (Diazald®) and their conversion to labelled diazomethane. J. Label. Compd. Radiopharm. 57 (12), 674–679 (2014). 24. Myers MC, Wang, Iera JA, Bang J, Hara T, Saito S, Zambetti GP, Appella DH. A new family of small molecules to probe the reactivation of mutant P53. J. Am. Chem. Soc. 127(17), 6152–6153 (2005). • Experimental cell models in drug discovery 25. Lee AV, Oesterreich S, Davidson NE. MCF-7 cells--changing the course of breast cancer research and care for 45 years. J Natl Cancer Inst. 107(7): djv073 (2015). 26. Datta A, Dey S, Das P, Kayum Alam SK, Roychoudhury, S. Transcriptome profiling identifies genes and pathways deregulated upon floxuridine treatment Page 23 of 66 https://mc04.manuscriptcentral.com/fs-fmc Future Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Review Only 24 in colorectal cancer cells harboring GOF mutant p53. Genomics Data 8, 47–51 (2016). 27. Rozzo C, Sanna D, Garribba E et al. Antitumoral effect of vanadium compounds in malignant melanoma cell lines. J. Inorg. Biochem. 174, 14–24 (2017). • Reported the pharmacological importance of a lead compound such as 5-FU 28. Xiaoyan P, Chen W, Fang W et al. Development of 5-Fluorouracil Derivatives as Anticancer Agents. Curr. Med. Chem. 18, 4538-4556 (2011). 29. Walther R, Rautio J, Zelikin A N. Prodrugs in medicinal chemistry and enzyme prodrug therapies. Adv. Drug Deliv. Rev. 118, 65-77 (2017). Page 24 of 66 https://mc04.manuscriptcentral.com/fs-fmc Future Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Review Only 4 To a solution of 12 (950 mg, 3.59 mmol) in anhydrous THF (36 mL) at 0ºC, LiAlH4 1M (4.5 mL, 4.5 mmol) was added dropwise and stirred for 1h. After this time, ethyl acetate (3 mL) and sodium potassium tartrate (3 mL) were added. The resulting precipitate was filtered over celite. The filtrated solvent was extracted with ethyl acetate. The organic layer was dried over Na2SO4, filtered, and evaporated. The residue was purified by flash chromatography (EtOAc/hexane, 5:1). Yellow solid (455 mg, 2.51 mmol), yield 70%, mp: 90 - 91°C; 1H NMR (500 MHz, CDCl3):  (ppm) 7.57 (dt, J1 = 5.1 Hz, J2 = 1.4 Hz, 1H, Hpyr), 6.93 (dt, J1 = 7.8 Hz, J2 = 1.3 Hz, 1H,Hpyr), 6.49 (ddd, J1 = 7.8 Hz, J2 = 5.0 Hz, J3 = 1.1 Hz, 1H, Hpyr), 6.14 (bs, 1H, OH), 5.12 (bs, 1H, NH), 4.15 (ddd, J1 = 10.3 Hz, J2 = 2.8 Hz, J3 = 1.3 Hz, 1H, Hoxazine), 3.99 – 3.95 (m, 1H, CH2OH), 3.88 – 3.85 (m, 1H, CH2OH), 3.82 (ddd, J1 = 10.3 Hz, J2 = 7.6 Hz, J3 = 1.3 Hz, 1H, Hoxazine), 3.78 – 3.74 (m, 1H, Hoxazine), 1.78 – 1.61 (m, 2H, CH2CH2OH); 13C NMR (126 MHz, CDCl3):  (ppm) 147.0 (Cpyr), 139.1 (Cpyr), 139.0 (CHpyr), 121.7 (CHpyr), 113.2 (CHpyr), 68.7 (CH2oxazine), 60.1 (CH2OH), 49.2 (CHoxazine), 33.3 (CH2CH2OH); HRMS (ESI-TOF) (m/z) calcd. for C9H13N2O2 (M + H)+ 181.0977, found 181.0965. 3-((((tert-Butyldimethyl)silyl)oxy)ethyl)-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazine (14) To a suspension of DMAP (14 mg, 0.11 mmol), Et3N (427µL, 2,22 mmol), 13 (200 mg, 1.11 mmol) in anhydrous DCM (5 mL), TBDMSCl (250 mg, 1,66 mmol) dissolved in anhydrous DCM (1 mL) was added under argon atmosphere and stirred at rt. After 12h the reaction mixture was poured into aq. AcOH (5%) and extracted with DCM. The organic layer was dried over Na2SO4, filtered, and evaporated. The residue was purified by flash chromatography (EtOAc/hexane, 1:5). Yellow oil (278 mg, 0.94 mmol), yield 86%; 1H NMR (400 MHz, CDCl3):  (ppm) 7.66 (d, J = 5.0 Hz, 1H, Hpyr), 6.94 (d, J = 7.7 Hz, 1H, Hpyr), 6.52 (dd, J1 = 7.7 Hz, J2 = 4.9 Hz, 1H, Hpyr), 5.32 (bs, 1H, NH), 4.21 (dd, J1 = 10.7 Hz, J2 = 2.9 Hz, 1H, Hoxazine), 3.87 (dd, J1 = 10.6 Hz, J2 = 7.0 Hz, 1H, Hoxazine), 3.84 – 3.77 (m, 2H, CH2O), 3.77 – 3.70 (m, 1H, Hoxazine), 1.76 – 1.65 (m, 2H, CH2CH2O), 0.91 (s, 9H, (CH3)3), 0.07 (s, 6H, (CH3)2Si); 13C NMR (101 MHz, CDCl3):  (ppm) 147.5 (Cpyr), 140.5 (CHpyr), 139.6 (Cpyr), 122.1 (CHpyr), 114.2 (CHpyr), 69.3 (CH2oxazine), 60.7 (CH2O), 48.6 (CHoxazine), 35.3 (CH2CH2O), 26.4 ((CH3)3), 18.7 (C(CH3)3), -4.9 (CH3Si), -5.0 (CH3Si); HRMS (ESI-TOF) (m/z) calcd. for C15H27N2O2Si (M + H)+ 295.1842, found 295.1863. 3-(2-((tert-Butyldimethylsilyl)oxy)ethyl)-4-tosyl-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazine (15) Page 31 of 66 https://mc04.manuscriptcentral.com/fs-fmc Future Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Review Only 5 To a suspension of DMAP (73.30 mg, 0.6 mmol), Et3N (812µL, 5.82 mmol) and 14 (857 mg, 2.91 mmol) in dry DCM (10 mL) under argon atmosphere, p-toluenesulfonyl chloride was added (1.165 g, 6.11 mmol) at 0°C and stirred at rt. After 36h the reaction mixture was poured into fresh water. The precipitate was filtered off and the solvent was purified by flash chromatography (EtOAc/hexane, 1:10). Yellow sirup (391 mg, 0.87 mmol), yield 30%; 1H NMR (400 MHz, CDCl3):  (ppm) 8.01 (d, J = 8.3 Hz, 2H, Htosyl), 7.94 (dd, J1 = 4.7 Hz, J2 = 1.6 Hz, 1H, Hpyr), 7.28 (d, J = 5.9 Hz, 2H, Htosyl), 7.12 (dd, J1 = 8.0 Hz, J2 = 1.6 Hz, 1H, Hpyr), 6.90 (dd, J1 = 8.0 Hz, J2 = 4.7 Hz, 1H, Hpyr), 5.01 - 4.96 (m, 1H, Hoxazine), 4.37 (dd, J1 = 11.1 Hz, J2 = 1.6 Hz, 1H, Hoxazine), 4.02 (dd, J1 = 11.1, J2 = 2.5 Hz, 1H, Hoxazine), 3.72 – 3.62 (m, 2H, CH2O), 2.40 (s, 3H, CH3tosyl), 1.73 (q, J = 6.4 Hz, 2H, CH2CH2O) 0.90 (s, 9H, (CH3)3), 0.04 (s, 3H, (CH3)2Si), 0.05 (s, 3H, (CH3)2Si); 13C NMR (101 MHz, CDCl3):  (ppm) 143.4 (Ctosyl), 140.7 (Cpyr), 140.1 (CHpyr), 138.0 (Cpyr), 137.9 (Ctosyl), 128.9 (2xCHtosyl), 128.3 (2xCHtosyl), 123.9 (CHpyr), 119.6 (CHpyr), 67.4 (CH2oxazine), 59.4 (CH2O), 50.9 (CHoxazine), 33.3 (CH2CH2O), 25.7 ((CH3)3), 21.4 (CH3tosyl), 18.0 (C(CH3)3), -5.5 (CH3Si), -5.7 (CH3Si); HRMS (ESI-TOF) (m/z) calcd. for C22H33N2O4SiS (M + H)+ 449.1930, found 449.1964. 2-(4-Tosyl-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazine-3-yl)etanol (16) The silyl derivative 15 (200 mg, 0.44 mmol) was added to a mixture of THF/H2O (1:1) (6.6 mL) and glacial acetic acid (16 mL) was added. After stirred for 22h the mixture was poured into sat. NaHCO3 (25 ml). The aqueous fractions were extracted with EtOAc. The organic layers were combined, dried over Na2SO4, filtered and evaporated. The residue was purified by flash chromatography (EtOAc/hexane, 1:2). Colourless oil (74 mg, 0.22 mmol), yield 50 %; 1H NMR (400 MHz, CDCl3):  (ppm) 7.97 (dd, J1 = 4.7 Hz, J2 = 1.6 Hz, 1H, Hpyr), 7.93 (d, J = 8.3 Hz, 2H, Htosyl), 7.26 (d, J = 8.1 Hz, 2H, Htosyl), 7.14 (dd, J1 = 8.1 Hz, J2 = 1.5 Hz, 1H, Hpyr), 6.92 (dd, J1 = 8.1 Hz, J2 = 4.7 Hz, 1H, Hpyr), 4.95 (m, 1H, Hoxazine), 4.27 (dd, J1 = 11.2 Hz, J2 = 1.5 Hz, 1H, Hoxazine), 3.86 (dd, J1 = 11.2 Hz, J2 = 2.5 Hz, 1H, Hoxazine), 3.84 – 3.79 (m, 1H, CH2OH), 3.74 – 3.68 (m, 1H, CH2OH), 2.38 (s, 3H, CH3tosyl), 1.89 - 1.71 (m, 2H, CH2CH2OH); 13C NMR (101 MHz, CDCl3):  (ppm) 144.0 (Ctosyl), 140.5 (Cpyr), 140.2 (CHpyr), 137.4 (Cpyr), 136.7 (Ctosyl), 129.2 (2xCHtosyl), 128.2 (2xCHtosyl), 124.5 (CHpyr), 119.8 (CHpyr), 67.4 (CH2oxazine), 58.3 (CH2OH), 50.8 (CHoxazine), 33.4 (CH2CH2OH), 21.7 (CH3tosyl); HRMS (ESI-TOF) (m/z) calcd. for C16H19N2O4S (M + H)+ 335.0987, found 335.0878. Methyl/Ethyl 2-(N-(tert-butoxycarbonyl)-N-tosylamino)-3-hydroxypropanoate (18a,b) Page 32 of 66 https://mc04.manuscriptcentral.com/fs-fmc Future Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Review Only 6 To a suspension of DMAP (11.4 mg, 0.1 mmol), Et3N (156.3 µl, 1.12 mmol) and 17a,b (360 mg, 0.95 mmol) in anhydrous DCM (3 ml) under argon atmosphere, (Boc)2O was added (244 mg, 1.12 mmol), dissolved in anhydrous DCM (3 ml) and stirred at rt. After 2h the reaction mixture was poured into aq. AcOH (5%) and extracted with DCM. The organic layer was dried over Na2SO4, filtered, and evaporated. The crude obtained without isolation was added to a mixture of THF/H2O (1:1) (10 ml) and glacial acetic acid was added (40 ml). After stirring for 22h the mixture reaction was poured into brine and sat. NaHCO3. The aqueous fraction was extracted with EtOAc, and the organic layer was dried, filtered and evaporated. The residue was purified by flash chromatography (EtOAc/hexane, 1:7). (18a): White solid (262 mg, 0.846 mmol), yield 89%, mp: 102 - 103°C;1H NMR (500 MHz, CDCl3):  (ppm) 7.93 – 7.90 (m, 2H, Htosyl), 7.34 – 7.30 (m, 2H, Htosyl), 5.22 (t, J = 6.5 Hz, 1H, Hprop), 4.31 (dd, J1 = 11.6 Hz, J2 = 6.4 Hz, 1H, Hprop), 3.96 (dd, J1 = 11.6 Hz, J2 = 6.5 Hz, 1H, Hprop), 3.76 (s, 3H, OCH3), 2.45 (s, 3H, CH3tosyl), 1.31 (s, 9H, 3xCH3); 13C NMR (126 MHz, CDCl3):  (ppm) 169.6 (COOMe), 150.2 (COOBut), 144.4 (Ctosyl), 136.5 (Ctosyl), 129.1 (2xCHtosyl), 128.3 (2xCHtosyl), 85.4 (C(CH3)3), 61.8 (CH2prop), 59.9 (CHprop), 52.4 (OCH3), 27.7 (C(CH3)3), 21.5 (CH3tosyl); HRMS (ESI-TOF) (m/z) calcd. for C16H24NO7S (M + H)+ 374.1273, found 374.1278. (18b): White solid (282 mg, 0.874 mmol), yield 92%, mp: 119 - 120°C; 1H NMR (500 MHz, CDCl3):  (ppm) 7.94 – 7.90 (m, 2H, Htosyl), 7.33 – 7.30 (m, 2H, Htosyl), 5.19 (t, J = 6.5 Hz, 1H, Hprop), 4.32 (dd, J1 = 11.5 Hz, J2 = 6.6 Hz, 1H, Hprop), 4.21 (q, J = 7.1 Hz, 2H, OCH2CH3), 3.95 (dd, J1 = 11.5 Hz, J2 = 6.4 Hz, 1H, Hprop), 2.45 (s, 3H, CH3tosyl), 1.31 (s, 9H, 3xCH3), 1.23 (t, J = 7.1 Hz, 3H, OCH2CH3); 13C NMR (126 MHz, CDCl3):  (ppm) 169.7 (COOEt), 150.6 (COOBut), 144.9 (Ctosyl), 137.1 (Ctosyl), 129.5 (2xCHtosyl), 128.8 (2xCHtosyl), 85.9 (C(CH3)3), 62.3 (OCH2CH3), 62.2 (CH2prop), 60.3 (CHprop), 28.2 (C(CH3)3), 22.1 (CH3tosyl), 14.4 (OCH2CH3); HRMS (ESI-TOF) (m/z) calcd. for C17H26NO7S (M + H)+ 388.1430, found 388.1444. Synthesis and characterization of 6-bromopurines 3a, 4a, 6a, and 7a For the preparation of these compounds the general synthetic procedure of halopurine derivatives is followed. 2-(2-(6-Bromo-9H-purine-9-yl)ethyl)-4-tosyl-3,4-dihydro-2H-benzo[b][1,4]oxazine (3a) Yellow solid (120 mg, 0.232 mmol), yield 75%, mp: 192 - 193°C; 1H NMR (500 MHz, CDCl3):  (ppm) 8.69 (s, 1H, Hpurine), 7.98 (s, 1H, Hpurine), 7.77 (dd, J1 = 8.3 Hz, J2 = 1.6 Page 33 of 66 https://mc04.manuscriptcentral.com/fs-fmc Future Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Review Only 7 Hz, 1H, Hbenz), 7.44 – 7.31 (m, 2H, Htosyl), 7.12 – 7.01 (m, 3H, 2xHtosyl, Hbenz), 6.93 (ddd, J1 = 8.8 Hz, J2 =7.3 Hz, J3 =1.5 Hz, 1H, Hbenz), 6.77 (dd, J1 = 8.2 Hz, J2 = 1.5 Hz, 1H, Hbenz), 4.54 – 4.38 (m, 2H, CH2N), 4.19 (dd, J1 = 14.2 Hz, J2 = 2.3 Hz, 1H, Hoxazine), 3.36 – 3.32 (m, 1H, Hoxazine), 3.21 (dd, J1 = 14.2 Hz, J2 = 9.7 Hz, 1H, Hoxazine), 2.39 (s, 3H, CH3tosyl), 2.30 – 2.26 (m, 1H, CH2CH2N), 2.10 – 1.96 (m, 1H, CH2CH2N); 13C NMR (126 MHz, CDCl3):  (ppm) 151.8 (CHpurine), 150.4 (Cpurine), 145.9 (Cbenz), 145.0 (CHpurine), 144.5 (Ctosyl), 143.1 (Cpurine), 135.2 (Ctosyl), 134.1 (Cpurine), 129.6 (2xCHtosyl), 126.8 (2xCHtosyl), 126.2 (CHbenz), 124.1 (CHbenz), 123.4 (Cbenz), 121.3 (CHbenz), 117.2 (CHbenz), 68.3 (CHoxazine), 48.1 (CH2oxazine), 40.3 (CH2N), 31.7 (CH2CH2N), 21.6 (CH3tosyl); HRMS (ESI-TOF) (m/z) calcd. for C22H21N5O3SBr (M + H)+ 514.0548, found 514.0565. Anal. calc. for C22H20N5O3SBr: C, 51.37; H, 3.92; N, 13.61. Found: C, 51.35; H, 3.90; N, 13.63. 3-((6-Bromo-9H-purine-9-yl)methyl)-1-tosyl-1,2,3,4-tetrahydroquinoline (4a) White solid (115 mg, 0.23 mmol), yield 73%, mp: 204 - 205°C; 1H NMR (400 MHz, DMSO-d6):  (ppm) 8.76 (s, 1H, Hpurine), 8.70 (s, 1H, Hpurine), 7.61 (d, J = 8.3 Hz, 1H, Hbenz), 7.25 (d, J = 7.9 Hz, 2H, 2xHtosyl), 7.21 – 7.12 (m, 3H, 2xHtosyl, Hbenz), 7.07 (d, J = 4.5 Hz, 2H, 2xHbenz), 4.26 (dd, J1 = 8.0 Hz, J2 = 7.6 Hz, 2H, CH2N), 4.05 (dd, J1 = 13.5, J2 = 4.0 Hz, 1H, Hpyr), 3.35 (dd, J1 = 9.4 Hz, J2 = 4.3 Hz, 2H, 2xHpyr) 2.55 (dd, J1 = 16.5 Hz, J2 = 5.3 Hz, 1H, Hpyr), 2.30 (s, 3H, CH3tosyl), 2.15 – 2.04 (m, 1H, Hpyr); 13C NMR (101 MHz, DMSO-d6):  (ppm) 151.8 (CHpurine), 150.9 (Cpurine), 147.6 (CHpurine), 144.0 (Ctosyl), 142.0 (Cpurine), 136.0 (Cbenz), 135.7 (Ctosyl), 133.6 (Cpurine), 129.9 (2xCHtosyl), 129.7 (CHbenz), 128.8 (Cbenz), 126.7 (CHbenz), 126.6 (2xCHtosyl), 125.1 (CHbenz), 123.5 (CHbenz), 48.5 (CH2pyr), 46.1 (CH2N), 32.6 (CHpyr), 30.3 (CH2pyr), 21.1 (CH3tosyl); HRMS (ESI-TOF) (m/z) calcd. for C22H21N5O2SBr (M + H)+ 498.0599, found 498.0619. Anal. calc. for C22H20N5O2SBr: C, 53.02; H, 4.04; N, 14.05. Found: C, 53.05; H, 4.01; N, 14.02. Methyl-2-(N-tert-butoxycarbonyl)-N-tosylamino)-3-(6-bromo-9H-purine-9-yl)propanoate (6a) White solid (139 mg, 0.251 mmol), yield 81%; mp: 160 - 161°C; 1H NMR (500 MHz, CDCl3):  (ppm) 8.56 (d, J = 6.4 Hz, 1H, Hpurine), 8.19 (s, 1H, Hpurine), 7.53 (d, J = 8.0 Hz, 2H, 2xHtosyl), 7.14 (d, J = 8.0 Hz, 2H, 2xHtosyl), 5.54 (dd, J1 = 9.4 Hz, J2 = 4.9 Hz, 1H, Hprop), 4.98 – 4.93 (m, 2H, Hprop), 3.78 (s, 3H, OCH3), 2.36 (s, 3H, CH3tosyl), 1.30 (s, 9H, 3xCH3); 13C NMR (126 MHz, CDCl3):  (ppm) 168.0 (COOMe), 151.9 (Cpurine), 151.0 (CHpurine), 149.9 (COOBut), 145.6 (CHpurine), 144.9 (Ctosyl), 143.0 (Cpurine), 135.7 (Ctosyl), 134.1 (Cpurine), 129.1(2xCHtosyl), 128.1 (2xCHtosyl), 86.2 (C(CH3)3), 58.2 (CHprop), 53.1 Page 34 of 66 https://mc04.manuscriptcentral.com/fs-fmc Future Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Review Only 8 (OCH3), 43.5 (CH2prop), 27.8 (C(CH3)3), 21.7 (CH3tosyl); HRMS (ESI-TOF) (m/z) calcd. for C21H25N5O6SBr (M + H)+ 554.0709, found 554.0728. Anal. calc. for C21H24N5O6SBr: C, 45.49; H, 4.36; N, 12.63. Found: C, 45.51; H, 4.34; N, 12.61. Ethyl-2-(N-tert-butoxycarbonyl)-N-tosylamino)-3-(6-bromo-9H-purine-9-yl)propanoate (7a) White solid (140 mg, 0.248 mmol), yield 80%, mp: 171 - 172°C; 1H NMR (500 MHz, CDCl3):  (ppm) 8.63 (s, 1H, Hpurine), 8.19 (s, 1H, Hpurine), 7.60 (d, J = 8.1 Hz, 2H, 2xHtosyl), 7.19 (d, J = 8.1 Hz, 2H, 2xHtosyl), 5.55 (dd, J1 = 9.3 Hz, J2 = 5.0 Hz, 1H, Hprop), 5.04 (dd, J1 = 14.7 Hz, J2 = 5.0 Hz, 1H, Hprop), 4.95 (dd, J1 = 14.7 Hz, J2 = 9.3 Hz, 1H, Hprop), 4.28 (q, J = 7.4 Hz, 2H, OCH2CH3), 2.41 (s, 3H, CH3tosyl), 1.33 (s, 9H, 3xCH3), 1.27 (t, J = 7.4 Hz, 3H, OCH2CH3); 13C NMR (126 MHz, CDCl3):  (ppm) 167.6 (COOEt), 152.1 (CHpurine), 151.1 (Cpurine), 150.1 (COOBut), 145.6 (CHpurine), 145.0 (Ctosyl), 143.3 (Cpurine), 136.0 (Ctosyl), 134.3 (Cpurine), 129.2 (2xCHtosyl), 128.3 (2xCHtosyl), 86.3 (C(CH3)3), 62.6 (OCH2CH3), 58.4 (CHprop), 43.7 (CH2prop), 27.9 (C(CH3)3), 21.8 (CH3tosyl), 14.1 (OCH2CH3); HRMS (ESI-TOF) (m/z) calcd. for C22H27N5O6SBr (M + H)+ 568.0865, found 568.0867; Anal. calc. for C22H26N5O6SBr: C, 46.49; H, 4.61; N, 12.32. Found: C, 46.51; H, 4.60; N, 12.34. Synthesis and characterization of 6-trifluoromethylpurines 3d, 4d, 6d and 7d A mixture of FSO2CF2CO2Me (MFSDA, 32µl, 0.25 mmol), CuI (32 mg, 0.17 mmol), HMPA (30.5 µl, 0.175 mmol) and the appropriate bromopurines (3a, 4a, 6a and 7a) (0.14 mmol) in anhydrous DMF was stirred for 13h at 70°C. After this time, the reaction was cooled, dissolved in EtOAc/hexane (7:3), washed with sat. aq. NH4Cl, sat. aq. NaHCO3, water and brine, dried, filtered and the solvent was evaporated off. The residue was purified by flash chromatography using EtOAc/hexane as eluent. 2-(2-(6-Trifluromethyl-9H-purine-9-yl)ethyl)-4-tosyl-3,4-dihydro-2H-benzo[b][1,4]oxazine (3d) (EtOAc/hexane, 1:1), white solid, (47 mg, 0.094 mmol), yield 67%, mp: 178 - 179°C; 1H NMR (500 MHz, CDCl3):  (ppm) 9.08 (s, 1H, CHpurine), 8.13 (s, 1H, CHpurine), 7.76 (dd, J1 = 8.3 Hz, J2= 1.6 Hz, 1H, CHbenz), 7.46 – 7.35 (m, 2H, 2xCHtosyl), 7.10 (d, J = 8.0 Hz, 2H, 2xCHtosyl), 7.08 – 7.03 (m, 1H, CHpurine), 6.95 – 6.90 (m, 1H, CHpurine), 6.74 (dd, J1 = 8.2 Hz, J2= 1.5 Hz, 1H, CHpurine), 4.55 – 4.49 (m, 2H, CH2CH2N), 4.20 (dd, J1 = 14.2 Hz, J2 = 2.4 Hz, 1H, CH2oxazine), 3.46 – 3.42 (m, 1H, CHoxazine), 3.23 (dd, J1 = 14.3 Hz, J2 = Page 35 of 66 https://mc04.manuscriptcentral.com/fs-fmc Future Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Review Only 9 9.6 Hz, 1H,CH2oxazine), 2.37 (s, 3H, CH3tosyl), 2.30 – 2.25 (m, 1H, CH2CH2N), 2.13 – 2.05 (m, 1H, CH2CH2N); 13C NMR (126 MHz, CDCl3):  (ppm) 153.6 (Cpurine), 152.4 (Cpurine),151.8 (CHpurine), 145.8 (Cbenz), 145.2 (q, J = 37.1 Hz, Cpurine),144.6 (Ctosyl), 135.3 (Ctosyl), 129.9 (Cpurine), 129.7 (2xCHtosyl), 126.8 (2xCHtosyl), 126.2 (CHbenz), 124.0, (CHbenz), 123.4 (Cbenz), 121.4 (CHbenz), 120.69 (q, J = 274.9 Hz, CF3), 117.1 (CHbenz), 68.6 (CHoxazine), 48.1 (CH2oxazine), 40.1 (CH2CH2N), 31.8 (CH2CH2N), 21.4 (CH3tosyl); HRMS (ESI-TOF) (m/z) calcd. for C23H21N5O3SF3 (M + H)+ 504.1317, found 504.1345. Anal. Calc. for C23H20N5O3SF3: C, 54.87; H, 4.00; N, 13.91. Found: C, 54.86; H, 4.03; N, 13.94. 3-((6-Trifluoromethyl-9H-purine-9-yl)methyl)-1-tosyl-1,2,3,4-tetrahydroquinoline (4d) (EtOAc/hexane, 1:1), yellowish solid, (41 mg, 0.105 mmol), yield 75%, mp: 195 - 196°C;1H NMR (500 MHz, CDCl3):  (ppm) 9.08 (s, 1H, CHpurine), 8.21 (s, 1H, CHpurine), 7.72 (d, J = 8.3 Hz, 1H), CHbenz, 7.40 (d, J = 7.9 Hz, 2H, 2xCHtosyl), 7.20 (t, J = 7.8 Hz, 1H, CHbenz), 7.16 – 7.09 (m, 2H, 2xCHtosyl), 7.07 (d, J = 7.3 Hz, 1H, CHbenz), 6.99 (d, J = 7.6 Hz, 1H, CHbenz), 4.32 (dd, J1= 14.3 Hz, J2 = 6.6 Hz, 1H, CH2N), 4.21 (dd, J1 = 14.4 Hz, J2 = 6.4 Hz, 1H, CH2N), 4.02 (dd, J1 = 13.5 Hz, J2 = 3.7 Hz, 1H, CH2pyr), 3.48 (dd, J1 = 13.5 Hz, J2 = 8.5 Hz, 1H, CH2pyr), 2.61 (dd, J1 = 15.9 Hz, J2 = 5.1 Hz, 1H. CH2pyr), 2.49 – 2.38 (m, 1H, CHpyr), 2.35 (s, 3H, CH3tosyl), 2.31 (d, J = 8.6 Hz, 1H, CH2pyr); 13C NMR (126 MHz, CDCl3):  (ppm) 154.2 (Cpurine), 152.4 (CHpurine), 147.9 (CHpurine), 145.8 (q, J = 37.7 Hz, Cpurine),144.4 (Ctosyl), 136.7 (Cbenz), 136.6 (Ctosyl), 130.4 (Cpurine), 130.1 (2xCHtosyl), 129.8 (CHbenz), 127.6 (CHbenz), 127.5 (Cbenz), 127.2 (2xCHtosyl), 125.6 (CHbenz), 124.3 (CHbenz), 120.8 (q, J = 275.9 Hz, CF3), 48.9 (CH2pyr), 47.0 (CH2N), 33.2 (CHpyr), 31.1 (CH2pyr), 21.9 (CH3tosyl); HRMS (ESI-TOF) (m/z) calcd. for C23H21N5O2SF3 (M + H)+ 488.1368, found 488.1415. Anal. Calc. for C23H20N5O2SF3: C, 56.67; H, 4.14; N, 14.37. Found: C, 56.65; H, 4.15; N, 14.40. Methyl-2-(N-tert-butoxycarbonyl)-N-tosylamino)-3-(6-trifluoromethyl-9H-purine-9yl)propanoate (6d) (EtOAc/hexane, 1:1), white solid, (41 mg, 0.075 mmol), yield 50%, mp: 156 - 157°C; 1H NMR (500 MHz, CDCl3):  (ppm)  9.03 (s, 1H, CHpurine), 8.36 (s, 1H, CHpurine), 7.63 (d, J = 8.1 Hz, 2H, 2xCHtosyl), 7.19 (d, J = 8.1 Hz, 2H, 2xCHtosyl), 5.61 (dd, J1 = 9.2 Hz, J2 = 4.9 Hz, 1H, Hprop), 5.12 (dd, J1 = 14.7 Hz, J2 = 4.8 Hz, 1H, Hprop), 5.01 (dd, J1 = 14.7 Hz, J2 = 9.2 Hz, 1H, Hprop), 3.85 (s, 3H, OCH3), 2.40 (s, 3H, CH3tosyl), 1.28 (s, 9H, 3xCH3); 13C NMR (126 MHz, CDCl3):  (ppm) 168.1 (COOMe), 154.3 (Cpurine), 152.1 (CHpurine), 150.1 (COOBut), 148.0 (CHpurine), 145.3 (Ctosyl), 145.1 (q, J = 37.2 Hz, Cpurine), 135.9 Page 36 of 66 https://mc04.manuscriptcentral.com/fs-fmc Future Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Review Only 10 (Ctosyl), 130.1 (Cpurine), 129.3 (2xCHtosyl), 128.4 (2xCHtosyl), 120.6 (q, J = 275.9 Hz, CF3),86.5 (C(CH3)3), 58.1 (CHprop), 53.3 (OCH3), 43.7 (CH2prop), 27.8 (C(CH3)3), 21.7 (CH3tosyl); HRMS (ESI-TOF) (m/z) calcd. for C22H25N5O6SF3 (M + H)+ 544.1478, found 544.1480. Anal. calc. for C22H24N5O6SF3: C, 48.62; H, 4.45; N, 12.89. Found: C, 48.63; H, 4.42; N, 12.91. Ethyl-2-(N-tert-butoxycarbonyl)-N-tosylamino)-3-(6-trifluoromethyl-9H-purine-9yl)propanoate (7d) (EtOAc/hexane, 1:1), white solid, (47 mg, 0.084 mmol), yield 61%, mp: 160 - 161°C; 1H NMR (400 MHz, CDCl3):  (ppm) 9.03 (s, 1H, CHpurine), 8.36 (s, 1H, CHpurine), 7.65 (d, J = 8.2 Hz, 2H, 2xCHtosyl), 7.21 (t, J = 8.2 Hz, 2H, 2xCHtosyl), 5.57 (dd, J1 = 9.0 Hz, J2 = 5.1 Hz, 1H, Hprop), 5.07 – 5.03 (m, 2H, Hprop), 4.29 (q, J = 7.1 Hz, 2H, OCH2CH3), 2.41 (d, J = 8.9 Hz, 3H, CH3tosyl), 1.30 (s, 9H, 3xCH3), 1.27 (t, J = 7.2 Hz, 3H, OCH2CH3); 13C NMR (101 MHz, CDCl3):  (ppm) 167.9 (COOEt), 154.7 (Cpurine), 152.4 (CHpurine), 150.4 (COOBut), 148.4 (CHpurine), 145.2 (q, J = 37.4 Hz, Cpurine), 144.9 (Ctosyl), 136.3 (Ctosyl), 130.4 (Cpurine), 129.5 (2xCHtosyl), 128.7 (2xCHtosyl), 120.6(q, J = 275.9 Hz, CF3), 86.7 (C(CH3)3), 63.0 (OCH2CH3), 58.5 (CHprop), 43.9 (CH2prop), 28.2 (C(CH3)3), 22.0 (CH3tosyl), 14.4 (OCH2CH3); HRMS (ESI-TOF) (m/z) calcd. for C23H27N5O6SF3 (M + H)+ 558.1634, found 558.1634. Anal. calc. for C23H26N5O6SF3: C, 49.55; H, 4.70; N, 12.56. Found: C, 49.57; H, 4.69; N, 12.56. Figure S.1. Chemical structure of benzoxazepine derivatives [Díaz-Gavilán, M. et al. Bioorg. Med. Chem. Lett. 18, 1457-1460 (2008)] O N R1 N N N N R2 aR1= SO2-C6H4-oNO2, R2= SPh bR1= SO2-C6H4-pNO2, R2= SPh cR1= H, R2= SPh Figure S.1. Benzoxazepine derivatives previously published by our group. Page 37 of 66 https://mc04.manuscriptcentral.com/fs-fmc Future Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Review Only 11 Figure S.2. Apoptosis after treatment with 4c at lower concentrations Figure S.2. Percentage of apoptotic cells after treatment with compound 4c at 3, 10 and 30 µM. Supplementary video: two-day time-lapse motion picture of MCF-7 cell proliferation under treatment with 30 μM of 4c with Nucview488 (apoptosis fluorescent marker) 1H and 13C-RMN spectra of intermediate derivatives Page 38 of 66 https://mc04.manuscriptcentral.com/fs-fmc Future Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Review Only 12 1H NMR 2-(3,4-Dihydro-2H-1,4-benzo[b][1,4]oxazine-2-yl)ethanol (8) 2.02.53.03.54.04.55.05.56.06.57.0 f1 (ppm) 13C NMR 20253035404550556065707580859095100105110115120125130135140145150155 f1 (ppm) CARBON_01 Page 39 of 66 https://mc04.manuscriptcentral.com/fs-fmc Future Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Review Only 13 1H NMR (3,4-Dihydro-4-tosyl-2H-1,4-benzo[b][1,4]oxazine-2-yl)ethanol (9) 1.82.02.22.42.62.83.03.23.43.63.84.04.24.44.64.85.05.25.45.65.86.06.26.46.66.87.07.27.47.67.8 f1 (ppm) proton STANDARD FLUORINE PARAMETERS 13C NMR 1520253035404550556065707580859095100105110115120125130135140145150155 f1 (ppm) carbono Page 40 of 66 https://mc04.manuscriptcentral.com/fs-fmc Future Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Review Only 20 1H NMR Methyl 2-(N-(tert-butoxycarbonyl)-N-tosylamino)-3-hydroxypropanoate (18a) 1.21.41.61.82.02.22.42.62.83.03.23.43.63.84.04.24.44.64.85.05.25.45.65.86.06.26.46.66.87.07.27.47.67.88.0 f1 (ppm) 13C NMR 2030405060708090100110120130140150160170 f1 (ppm) CARBON_01 Page 47 of 66 https://mc04.manuscriptcentral.com/fs-fmc Future Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Review Only 21 1H NMR Ethyl 2-(N-(tert-butoxycarbonyl)-N-tosylamino)-3-hydroxypropanoate (18b) 1.21.41.61.82.02.22.42.62.83.03.23.43.63.84.04.24.44.64.85.05.25.45.65.86.06.26.46.66.87.07.27.47.67.88.0 f1 (ppm) PROTON_01 13C NMR 102030405060708090100110120130140150160170 f1 (ppm) CARBON_01 Page 48 of 66 https://mc04.manuscriptcentral.com/fs-fmc Future Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Review Only 22 1H and 13C-RMN spectra of 6-halo and 2,6-dihalopurines 1H NMR 2-(2-(6-Bromo-9H-purine-9-yl)ethyl)-4-tosyl-3,4-dihydro-2Hbenzo[b][1,4]oxazine (3a) 2.02.22.42.62.83.03.23.43.63.84.04.24.44.64.85.05.25.45.65.86.06.26.46.66.87.07.27.47.67.88.08.28.48.68.8 f1 (ppm) PROTON_01 13C NMR 20253035404550556065707580859095100105110115120125130135140145150 f1 (ppm) CARBON_01 Page 49 of 66 https://mc04.manuscriptcentral.com/fs-fmc Future Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Review Only 23 1H NMR 2-(2-(6-Chloro-9H-purine-9-yl)ethyl)-4-tosyl-3,4-dihydro-2Hbenzo[b][1,4]oxazine (3b) 2.02.53.03.54.04.55.05.56.06.57.07.58.08.59.0 f1 (ppm) proton 13C NMR 20253035404550556065707580859095100105110115120125130135140145150155 f1 (ppm) carbono Page 50 of 66 https://mc04.manuscriptcentral.com/fs-fmc Future Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Review Only 24 1H NMR 2-(2-(2,6-Dichloro-9H-purine-9-yl)ethyl)-4-tosyl-3,4-dihydro-2Hbenzo[b][1,4]oxazine (3c) 2.02.22.42.62.83.03.23.43.63.84.04.24.44.64.85.05.25.45.65.86.06.26.46.66.87.07.27.47.67.88.0 f1 (ppm) proton 13C NMR 20253035404550556065707580859095100105110115120125130135140145150155 f1 (ppm) carbono Page 51 of 66 https://mc04.manuscriptcentral.com/fs-fmc Future Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Review Only 25 1H NMR 3-((6-Bromo-9H-purine-9-yl)methyl)-1-tosyl-1,2,3,4-tetrahydroquinoline (4a) 2.02.53.03.54.04.55.05.56.06.57.07.58.08.59.0 f1 (ppm) proton 13C NMR 20253035404550556065707580859095100105110115120125130135140145150 f1 (ppm) carbono Page 52 of 66 https://mc04.manuscriptcentral.com/fs-fmc Future Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Review Only 26 1H NMR 3-((6-Chloro-9H-purine-9-yl)methyl)-1-tosyl-1,2,3,4-tetrahydroquinoline (4b) 2.53.03.54.04.55.05.56.06.57.07.58.08.59.0 f1 (ppm) PROTON_01 13C NMR 253035404550556065707580859095100105110115120125130135140145150 f1 (ppm) carbono Page 53 of 66 https://mc04.manuscriptcentral.com/fs-fmc Future Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Review Only 27 1H NMR 3-((2,6-Dichloro-9H-purine-9-yl)methyl)-1-tosyl-1,2,3,4-tetrahydroquinoline (4c) 2.42.62.83.03.23.43.63.84.04.24.44.64.85.05.25.45.65.86.06.26.46.66.87.07.27.47.67.88.0 f1 (ppm) PROTON_01 13C NMR 1520253035404550556065707580859095100105110115120125130135140145150 f1 (ppm) CARBON_01 Page 54 of 66 https://mc04.manuscriptcentral.com/fs-fmc Future Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Review Only 28 1H NMR 3-(2-(6-Chloro-9H-purine-9-yl)ethyl)-4-tosyl-3,4-dihydro-2H-pyrido[3,2b][1,4]oxazine (5b) 2.02.53.03.54.04.55.05.56.06.57.07.58.08.5 f1 (ppm) 13C NMR Page 55 of 66 https://mc04.manuscriptcentral.com/fs-fmc Future Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Review Only 29 1H NMR 3-(2-(2,6-Dichloro-9H-purine-9-yl)ethyl)-4-tosyl-3,4-dihydro-2H-pyrido[3,2b][1,4]oxazine (5c) 2.53.03.54.04.55.05.56.06.57.07.58.08.5 f1 (ppm) 13C NMR Page 56 of 66 https://mc04.manuscriptcentral.com/fs-fmc Future Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Review Only 36 1H and 13C-RMN spectra of 6-trifluoromethylpurines 1H NMR 2-(2-(6-Trifluromethyl-9H-purine-9-yl)ethyl)-4-tosyl-3,4-dihydro-2Hbenzo[b][1,4]oxazine (3d) 2.02.53.03.54.04.55.05.56.06.57.07.58.08.59.0 f1 (ppm) PROTON_01 13C NMR 20253035404550556065707580859095100105110115120125130135140145150155 f1 (ppm) CARBON_01 Page 63 of 66 https://mc04.manuscriptcentral.com/fs-fmc Future Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Review Only 37 1H NMR 3-((6-Trifluoromethyl-9H-purine-9-yl)methyl)-1-tosyl-1,2,3,4tetrahydroquinoline (4d) 2.53.03.54.04.55.05.56.06.57.07.58.08.59.0 f1 (ppm) 13C NMR 253035404550556065707580859095100105110115120125130135140145150155 f1 (ppm) CARBON_01 Page 64 of 66 https://mc04.manuscriptcentral.com/fs-fmc Future Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Review Only 38 1H NMR Methyl-2-(N-tert-butoxycarbonyl)-N-tosylamino)-3-(6-trifluoromethyl-9H-purine9-yl)propanoate(6d) 1.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.0 f1 (ppm) 13C NMR 102030405060708090100110120130140150160170 f1 (ppm) CARBON_01 Page 65 of 66 https://mc04.manuscriptcentral.com/fs-fmc Future Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Review Only 39 1H NMR Ethyl-2-(N-tert-butoxycarbonyl)-N-tosylamino)-3-(6-trifluoromethyl-9H-purine9-yl)propanoate(7d) 1.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.0 f1 (ppm) 13C NMR 2030405060708090100110120130140150160170 f1 (ppm) carbono Page 66 of 66 https://mc04.manuscriptcentral.com/fs-fmc Future Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60