Anomalous Products in the Halogenation Reactions of Vinca Alkaloids 1.949

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Current Organic Chemistry, 2016, 20, 2639-2646 RESEARCH ARTICLE

ISSN: 1385-2728 eISSN: 1875-5348

Impact Factor: Anomalous Products in the Halogenation Reactions of Vinca Alkaloids 1.949

BENTHAM SCIENCE

András Keglevich,1 László Hegeds,2 Lilla Péter,1 Judit Gyenese,1 Csaba Szántay, Jr.,3 Zsófia Dubrovay,3+ Miklós Dékány,3 Áron Szigetvári,3 Ana Martins,4++ József Molnár,4 Attila Hunyadi,5 Péter Keglevich1* and László Hazai1*

1Department of Organic Chemistry and Technology, University of Technology and Economics, Budapest, Hungary, H-1111 Budapest, Gellért tér 4. Hungary; 2MTA–BME Organic Chemical Technology Research Group, Hungarian Academy of Sciences, Department of Organic Chemistry and Technology, Budapest University of Technology and Economics, Budafoki út 8, H-1111 Budapest, Hungary; 3Spectroscopic Research Division, Gedeon Richter Plc., H-1475 Budapest 10, P. O. Box 27, Hungary; 4Department of Medical Microbiology and Immunobiology, University of Szeged, 6720 Szeged, Dóm tér 10., Hungary; 5Institute of Pharmacognosy, University of Szeged, H-6720 Szeged, Eötvös u. 6., Hungary

Abstract: Halogenation reactions of vindoline and 14,15-dihydrovindoline and its hydrochloric salt were investigated and the anomalous reductions were discussed. Performing the hydrogenation in the presence of chlorine-containing solvent, e.g. dichloromethane, hydrogenolysation reaction of chlorine also took place. In this case unexpected chlorinated product could be observed. Performing the hydrogenation reaction only in the presence of methanol, the expected reduced derivative was obtained. Upon bromination of vindoline with excess NBS, oxidation products with ring contraction and developing an oxygen bridge were isolated. The fluorination reactions of vinblastine using Selectfluor® and xenon difluoride as the fluorination reagents were unsuccessful because of the decomposition of the starting material. Reactions of vindoline with Selectfluor® a mixture of products were obtained. Using xenon difluoride as fluorination agent resulted in a pure quinoidal product containing the fluorine atom in the bridgehead carbon atom of the indole ring. The fluorination of catharan- A R T I C L E H I S T O R Y thine gave an anomalous indolenine type product. Received: February 22, 2016 Revised: May 24, 2016 Accepted: June 15, 2016 Dedicated to the memory of Professor Csaba Szántay who passed away on January 17, 2016.

DOI: 10.2174/1385272820666160617080202

Keywords: Vindoline, catharanthine, halogenation, Selectfluor®, xenon difluoride, quinoidal products.

1. INTRODUCTION plant in China, but it was likely an artefact because hydrochloric acid was used in the extraction [4]. The chemical space of organohalogen natural compounds com- prises of halogenated alkaloids in about 25% [1]. Brominated and The Vinca alkaloids vindoline (1) and catharanthine (2) are the chlorinated nitrogen heterocycles such as pyrrols, indoles, car- components of the famous antimitotic agents vinblastine (3) and bolines etc. can mostly be found in the marine environment [1]. In vincristine (4) which are present in the Madagascar periwinkle Ca- these compounds iodine or fluorine substituents rarely occur. Sev- tharanthus roseus (Scheme 1). Halogenation of vindoline (1) in eral halogenated alkaloids, mainly those with a bromo substitutent position 10 was carried out with N-chloro- and N-iodosuccinimide, isolated from marine intervebrates, exhibit cytotoxic protein kinase respectively [5]. The reaction with N-bromosuccinimide led to the inhibitory and antimicrobial activity [2]. From a hairy root culture corresponding 10-bromovindoline [6, 7]. Recently, derivatives of of Catharanthus roseus which contained exogenously added chlo- vinblastine (3) and vincristine (4), including iodo- and chloro sub- rinated or brominated tryptamine, halogenated indole alkaloids, e.g. stituted dimer alkaloids, were reviewed [8]. Halogenation of ca- chlorinated tabersonine could be obtained [3]. A similar chloro tharanthine gave indolenine-type derivatives halogenated in bridge- derivative was isolated from Alstonia yunnanensis, a medicinal head position 7 [9]. Catharanthine bearing two fluorine atoms in the methylene group of the ethyl substituent in position 19 was synthesized; however, its coupling with vindoline did not result in the *Address correspondence to these authors at the Department of Organic Chemistry expected fluorinated vinblastine [10]. 10’-Fluorovinblastine and and Technology, University of Technology and Economics, Budapest, Hungary, H-1111 Budapest, Gellért tér 4. Hungary; E-mails: [email protected], 10’-fluorovincristine with excellent antitumor activity were pre- [email protected] pared by Boger et al [11, 12]. In this case the fluoro atom was in- + Present address: XiMo Hungary Ltd., 1031 Budapest, Záhony u. 7., Hungary ++ Present address: Synthetic Systems Biology Unit, Institute of Biochemistry, Biologi- troduced in position 10 of catharanthine via 10-amino substituent cal Research Centre, 6726 Szeged, Temesvári krt. 62., Hungary following the coupling reaction with vindoline. Current Organic Chemistry

5 N 3 4 14 6 5 3 9 21 15 9 6 4 H 20 8 7 10 18 10 N 8 7 17 14 15 11 2 17 19 1 16 11 1 H CO 13 N OCOCH 13 N 2 16 21 20 3 12 3 H H OH 12 COOCH H 3 19 18 CH3 COOCH3 2 1 21' 4' 5' OH 18' N 20' 9' 6' 15' 19' 8' 10' 7' 3' 14' 5 11' 1' 16' 17' N 3 14 13' N 2' 4 12' H 9 6 21 15 20 3; R= CH3 H3COOC 18 10 8 7 4; R=CHO 17 11 19 1 2 16 13 H3CO 12 N OCOCH3 H OH R COOCH3

Scheme 1.

Cl H

N 14 N 14 N 14 15 15 15 10 10 H Pd/C aq. NaHCO 10 2, 3 EtOH, CH Cl 2 2 H3CO N OCOCH3 H3CO N OCOCH3 H CO N OCOCH H OH H OH 3 3 CH COOCH CH H OH 3 3 3 COOCH3 CH3 COOCH3 5 6 1

NBS, CH2Cl2 NBS, CH2Cl2 90%

N 14 N 14 N 14 15 10 15 15 Cl 10 Br Br 10

H3CO N OCOCH3 H3CO N OCOCH3 H3CO N OCOCH3 H OH 12 H OH H OH CH3 COOCH3 Br CH3 COOCH3 CH3 COOCH3

7 8 9

Scheme 2.

2. RESULTS AND DISSCUSSION Performing the hydrogenation in methanolic solution under pressure, only 14,15-dihydrovindoline base (6) formed [14]. The 2.1. Chemistry HCl was generated by the hydrogenolization of dichloromethane As a continuation of our work aimed at synthesizing Vinca al- resulting in the salt (5), which was supported with several samples kaloid derivatives with an antitumor activity [13], 14,15- in connection with hydrodehalogenation of halogenated hydrocar- dihydrovindoline (6) [14], saturated in positions 14 and 15, was bons using Pd catalyst [15-17]. In the course of NBS bromination prepared. Catalytic hydrogenation of vindoline (1) was achieved in of the base (6) the expected 10-bromo-14,15-dihydrovindoline (9) a mixture of ethanol-dichloromethane in the presence of Pd/C cata- [13] was obtained in 90% yield. In the bromination reaction of the lyst at room temperature under atmospheric pressure. The weight of hydrochloric salt of 14,15-dihydrovindoline (5), however, three the used catalyst had to be the same as of the substrate, otherwise products were isolated which could not be separated: 10-chloro- the reduction failed and was very slow, especially in larger quanti- 14,15-dihydrovindoline (7), 10,12-dibromo-14,15-dihydrovindoline ties (e.g. 5-10 g). In this case the product proved to be the 14,15- (8), and 10-bromo-14,15-dihydrovindoline (9) [13], as the major dihydrovindoline hydrochloric salt (5) and the base (6) was isolated product. Identification of the positions of halogen atoms in com- after washing with sodium hydrogen carbonate (Scheme 2). pounds 7 and 8 are discussed in Experimental. Chlorination can be Anomalous Products in the Halogenation Reactions Current Organic Chemistry, 2016, Vol. 20, No. 24 2641

N 14 N D 15 Br A OH NBS OH H CO N OCOCH CHCl H CO N OCOCH 3 H 3 3 3 H 3 CH3 COOCH3 Br CH3 COOCH3 1 10

CHCl3 NBS

H 22 23 H 14 O O

N O N O DD22' 23' Br Br A O + A O

H3CO N OCOCH3 H3CO N OCOCH3 H H CH3 COOCH3 CH3 COOCH3 11 12 Scheme 3.

N N F 10 XeF2 K2CO3 8 CH2Cl2 H3CO N OCOCH3 -40oC O N OCOCH3 H OH H OH CH3 COOCH3 10% CH3 COOCH3 1 13

SelectF CH3CN

N N F F 10 F 10 + 13 + 8

H3CO N OCOCH3 O N OCOCH3 H OH H OH CH3 COOCH3 CH3 COOCH3 14 15

Scheme 4. explained by NBS oxidation of the chloride ion to chlorine. This an aldehyde function formed and gave with ethyl alcohol (stabiliz- type of oxidation of halogen ions by N-haloimides to free halogen is ing the chloroform) the acetal 11, and the carboxylic acid (evolving mentioned by Filler [18]. Moreover, NBS mediated oxidation is from the aldehyde) resulted in the ethyl ester 12. Vindoline (and extensively known in the literature [18-21]. vinblastine) derivatives with a 5-membered D-ring were synthe- Nevertheless, directly introducing two bromo atoms into 14,15- sized by Boger and co-workers [24]. Nevertheless, NBS induced dihydrovindoline was unsuccessful [22]. In the course of bromina- ring contraction in the presence of alcohol is presented by Karimi tion reactions a complete transformation could not be observed et al. [25]. Similarly, in our case without alcohol no reaction could either in the reaction of 14,15-dihydrovindoline (6), or in the case be observed, moreover the two compounds (11, 12) could not be of 10-bromo-14,15-dihydrovindoline (9) [13]; only inseparable separated and thus could not be characterized in pure form, because complex mixtures were obtained. vinflunine, which was synthesized by Fahy et al. [26], exhibits a Therefore bromination of vindoline (1) was investigated high activity in the treatment of breast, lung and bladder cancers, (Scheme 3). Using two equivalents of NBS in chloroform reflux, the fluorination reactions of Vinca alkaloids are of increased interest. Therefore, the direct fluorination of vinblastine (3) was investi- instead of the expected dibromo derivative 10, the oxidation prod- ® ucts 11 and 12 were obtained in a 2:1 mixture. gated. The reaction of vinblastine (3) with Selectfluor and xenon difluoride, however, did not result in the expected fluoro deriva- An oxygen bridge developed between rings C and D of vindo- tives; from the reaction mixture only decomposition products could line. This reaction is one of the steps of the MnO oxidation of vin- 2 be obtained. doline which is a very complex process [23]. After ring contraction 2642 Current Organic Chemistry, 2016, Vol. 20, No. 24 Keglevich et al.

F H 10 N

N H 7 H H COOCH3 10 N SelectF, CH3CN, 16 19% N H or XeF2 COOCH H 3 CH2Cl2, K2CO3 F H 2 24% 7 N

N H COOCH3

17 Scheme 5.

Table 1. Results on the cytotoxicity effect of compound 13 alone, and of doxorubicin in the presence and absence of 50M of 13.

IC50 (M)

Doxorubicin 13 0M of 13 50M of 13

Mean SEM Mean SEM Mean SEM

L5178PAR >50 n.d. 0.25 0.03 0.11 0.03

L5178MDR >50 n.d. 1.92 0.25 0.36** 0.05

**p<0.01, unpaired t-test, n=3.

Fluorination of the monomer alkaloids, first vindoline (1) was pound 13, however, could effectively sensitize L5178B1 to doxoru- tried using Selectfluor® reagent in acetonitrile solution (Scheme 4). bicin (p<0.05), and, although in a statistically not significant man- In the product three fluorine-containing derivatives (13, 14, 15 in ner, the same tendency was observed in the sensitive mouse lym- 0.2:0.36:1 molar ratio, respectively) were detected. Besides the phoma cells; results are presented in Table 1. A lower, 25M con- expected 10-fluorovindoline (14), two quinoidal derivatives, 13 and centration of compound 13, however, failed to present the same 15 formed, however the compounds could not be separated. effect (not shown). Then the reaction with xenon difluoride was studied (Scheme Potential efflux pump inhibitory activity of compound 13 was 4). In this reaction quinone 13 could be isolated in 10% yield with- investigated through the accumulation of Rhodamine 123, a fluo- out other fluorinated derivatives. Hydrogen fluoride formed during rescent dye that is a well-known Pgp substrate. Compound 13 the reaction can work as a demethylating agent of the methoxy showed no inhibition of the efflux pump’s function. This, together group and moreover the oxidant properties of these fluorination with the results on the sensitive cell line, strongly suggests that a reagents are known [27, 28]. This would be the explanation of the mechanism other than Pgp inhibition should be responsible for the formation of quinoidal structures. The anticancer activity of com- sensitizing activity. Although the activity is not very strong and it is pound 13 was investigated (see below). observable only at a quite high dose, similar compounds might be The fluorination of catharanthine (2) was investigated by using of interest as chemosensitizers. Selectfluor® and xenon difluoride, respectively. Instead of the formation of the expected 10-fluoro derivative (16), similarly to the 3. EXPERIMENTAL chlorination reaction of catharanthine (2) [9], both of the reagents resulted electrophilic fluorination reactions [27, 28] and the indo- General lenine-type 7-fluorocatharanthine (17) was obtained in 19% and Melting points were measured on a VEB Analytik Dresden 24% yield (Scheme 5). PHMK-77/1328 apparatus and are uncorrected. IR spectra were recorded on Zeiss IR 75 and 80 instruments. NMR measurements 2.1. Biology were performed on a Varian 800 MHz NMR spectrometer equipped The antitumor potential of compound 13 was investigated on with a 1H{13C/15N} Triple Resonance 13C Enhanced Salt Tolerant two cancer cell line pairs, both pairs including a drug sensitive cell Cold Probe operating at 800 MHz for 1H and 201 MHz for 13C, and line and its multi-drug resistant (MDR) counterpart: L5178 mouse a Varian 500 MHz NMR spectrometer equipped with a 1H 13 15 13 lymphoma cell line and L5178B1 (transfected to express the human { C/ N} 5 mm PFG Triple Resonance C Enhanced Cold Probe 1 13 ABCB1 transporter commonly referred to as P-glycoprotein or Pgp) operating at 500 MHz for H and 125 MHz for C. Chemical shifts [29]; and two human breast cancer cell lines, MCF-7 and its sub- are given on the delta scale as parts per million (ppm) with 1 13 cell line obtained by adaptation to doxorubicin, MCF-7DOX that also tetramethylsilane (TMS) ( H) or dimethylsulfoxide-d6 ( C) as the overexpresses Pgp [30]. No toxic effect was observed on any of the internal standard (0.00 ppm and 39.5 ppm, respectively). 1H-1H, cell lines at a concentration as high as 50M. This dose of com- direct 1H-13C, and long-range 1H-13C scalar spin-spin connectivities

Anomalous Products in the Halogenation Reactions Current Organic Chemistry, 2016, Vol. 20, No. 24 2643 were established from 2D gDQFCOSY, zTOCSY, gHSQCAD, and molecule 8 implies that the two bromine atoms are at the positions gHMBCAD experiments, respectively. All pulse sequences were 10 and 12. applied by using the standard spectrometer software package. All MS: M+H=615. ESI-MS-MS (CID=35 %) (rel. int. %): 597(3); experiments were performed at 298 K. HRMS and MS analyses 555(73); 553(7); 523(2); 495(4); 344(7). were performed on an LTQ FT Ultra as well as an LTQ XL (Thermo Fisher Scientific, Bremen, Germany) system. The ioniza- Reaction of Vindoline (1) with Excess NBS tion method was ESI operated in positive ion mode. For the CID Vindoline (1) (150 mg, 0.33 mmol) in chloroform (6 mL) was experiment helium was used as the collision gas, and normalized refluxed after adding N-bromosuccinimide (117 mg, 0.66 mmol) for collision energy (expressed in percentage), which is a measure of 2 h. The organic phase was washed with 5% aqueous sodium hy- the amplitude of the resonance excitation RF voltage applied to the drogen carbonate (6 mL) and then with water (6 mL). After drying endcaps of the linear ion trap, was used to induce fragmentation. with magnesium sulfate the solvent was evaporated and after prepa- The protonated molecular ion peaks were fragmented by CID at a rative TLC (dichloromethane-methanol=10:1) of the residue, a 2:1 normalized collision energy of 35–45%. The samples were dis- mixture of compounds 11 and 12 was obtained. solved in methanol. Data acquisition and analysis were accom- Oxidation Product (11) plished with Xcalibur software version 2.0 (Thermo Fisher Scien- 1 tific). TLC was carried out using Kieselgel 60F254 (Merck) glass H NMR (799.7 MHz, DMSO-d6): 0.68 (3H, t, J=7.5 Hz, H3- plates. 18); 1.12 (3H, t, J=7.0 Hz), 1.16 (3H, t, J=7.0 Hz), H3-23, H3-23’; 1.39-1.46 (2H, buried m, H2-19); 1.88-1.94 (1H, buried m), 2.01- Bromination of 14,15-dihydrovindoline hydrochloride (5) 2.07 (1H, buried m), H2-6; 1.97 (3H, s, C(17)-OC(O)CH3); 2.71 The bromination reaction was carried out, yielding 10-bromo- (3H, s, N(1)-CH3); 2.91-3.00 (1H, buried m, Hx-5); 2.96 (1H, d, 14,15-dihydrovindoline (9) [13] as the major component of the J=7.9 Hz, H-3); 3.24-3.27 (1H, m, Hy-5); 3.46 (1H, s, H-2); 3.56- mixture of the products (7, 8, 9). With reference to our previous 3.70 (4H, buried m, H2-22, H2-22’); 3.72 (3H, s, C(16)-COOCH3); results [13], 10-chloro-14,15-dihydrovindoline (7) and 10,12- 3.76 (1H, s, H-21); 3.81 (3H, s, C(11)-OCH3); 4.18 (1H, ~s, H-15); dibromo-14,15-dihydrovindoline (8) were identified as the minor 4.24 (1H, d, J=7.9 Hz, H-14); 5.29 (1H, s, H-17); 6.34 (1H, s, H- 1 13 13 components on the basis of their most characteristic H and C 12); 7.29 (1H, s, H-9). C NMR (201.1 MHz, DMSO-d6): 9.4 (C- NMR assignments. 18); 15.2, 15.5 (C-23, C-23’); 20.6 (C(17)-OC(O)CH3); 21.5 (C- 19); 36.8 (N(1)-CH ); 46.4 (C-6); 51.6 (C-7); 52.0 (C(16)- 10-Chloro-14,15-dihydrovindoline (7) 3 COOCH3); 54.3 (C-5); 56.1 (C(11)-OCH3); 56.3 (C-20); 61.6, 62.7 1 H NMR (499.9 MHz, DMSO-d6): 2.57 (3H, s, N(1)-CH3); 3.80 (C-22, C-22’); 73.0 (C-17); 75.9 (C-3); 76.1 (C-21); 80.5 (C-2); 13 (3H, s, C(11)-OCH3); 6.45 (1H, s, H-12); 7.23 (1H, s, H-9). C 87.4 (C-16); 89.5 (C-15); 93.7 (C-12); 97.7 (C-10); 103.0 (C-14); NMR (125.7 MHz, DMSO-d6): 52.0 (C-7); 95.2 (C-12); 110.8 (C- 125.6 (C-9); 126.8 (C-8); 152.0 (C-13); 156.0 (C-11); 168.6 (C(16)-

10); 124.0 (C-9); 126.9 (C-8); 152.9 (C-13); 155.2 (C-11). COOCH3); 169.0 (C(17)-OC(O)CH3).

The structure of 7 was deduced from the loss of the characteris- HRMS: M+H=623.19671 (C29H40O8N2Br; delta=0.7 ppm). HR- 1 tic double doublet H-10 resonance in the H NMR spectrum. Other ESI-MS-MS (CID=35 %) (rel. int. %): 577(100). possible halogenation positions could be rejected. Instead of two Oxidation Product (12) singlets for H-9 and H-12, the 9-chloro-14,15-dihydrovindoline and 1 the 12-chloro derivative would have resulted in two aromatic H NMR (799.7 MHz, DMSO-d6): 0.64 (3H, t, J=7.3 Hz, H3- dublets with ca. 2 Hz and ca. 8 Hz J-coupling, respectively. 18); 1.12-1.18 (1H, buried m, Hx-19); 1.20 (3H, t, J=7.0 Hz, H3- MS: M+H=493. ESI-MS-MS (CID=35 %) (rel. int. %): 475(2); 23); 1.39-1.46 (1H, buried m, Hy-19); 1.92-2.01 (2H, buried m, H2- 433(100); 431(7); 401(5); 373(6); 222(14). 6); 1.97 (3H, s, C(17)-OC(O)CH3); 2.73 (3H, s, N(1)-CH3); 2.91- 3.00 (1H, buried m, Hx-5); 3.31-3.35 (1H, buried m, Hy-5); 3.44 10,12-Dibromo-14,15-dihydrovindoline (8) (1H, s, H-2); 3.72 (3H, s, C(16)-COOCH3); 3.81 (3H, s, C(11)- 1 H NMR (499.9 MHz, DMSO-d6): 0.37 (3H, t, J=7.5 Hz ,H- OCH3), 3.86 (1H, ~s, H-3); 3.92 (1H, s, H-21), 4.06-4.12 (2H, m, H -22); 4.44 (1H, ~s, H-15); 5.30 (1H, s, H-17); 6.34 (1H, s, H-12); 18); 0.72-0.81 (1H, m, Hx-19); 1.12-1.19 (1H, buried m, Hx-15); 2 7.38 (1H, s, H-9). 13C NMR (201.1 MHz, DMSO-d ): 9.1 (C-18); 1.22-1.27 (1H, buried m, Hy-15); 1.25-1.34 (1H, buried m, Hy-19); 6 14.1 (C-23); 20.6 (C(17)-OC(O)CH3); 20.8 (C-19); 36.6 (N(1)- 1.50-1.57 (1H, m, Heq-14); 1.68-1.79 (1H, m, Hax-14); 1.98 (3H, s, CH3); 45.2 (C-6); 51.5 (C-7); 52.1 C(16)-COOCH3); 54.7 (C-5); C(17)-OC(O)CH3); 1.95-2.04 (1H, buried m, Hax-3); 2.26-2.38 (2H, 56.1 (C(11)-OCH3); 56.5 (C-20); 60.4 (C-22); 72.4 (C-17); 73.6 (C- buried m, H2-6); 2.17 (1H, s, H-21); 2.47-2.59 (1H, buried m, Hx- 3); 74.6 (C-21); 79.3 (C-2); 87.6 (C-16); 90.5 (C-15); 93.6 (C-12); 5); 2.79 (3H, s, N(1)-CH3); 3.00-3.08 (1H, m, Heq-3); 3.09-3.16 97.9 (C-10); 125.1 (C-8); 125.9 (C-9); 151.9 (C-13); 155.9 (C-11); (1H, m, Hy-5); 3.67 (1H, s, H-2); 3.68 (3H; s, C(16)-COOCH3); 168.3 (C(16)-COOCH3); 169.0 (C(17)-OC(O)CH3); 171.3 (C-14). 3.72 (3H, s, C(11)-OCH3); 5.36 (1H, s, H-17); 7.53 (1H, s, H-9); 13 HRMS: M+H=593.15009 (C H O N Br; delta=1.3 ppm). HR- 9.29 (1H, s, C(16)-OH). C NMR(125.7 MHz, DMSO-d6): 7.7 (C- 27 34 8 2 ESI-MS-MS (CID=45 %) (rel. int. %): 565(43); 519(100); 431(3); 18); 20.7 (C(17)-OC(O)CH3); 22.3 (C-14); 29.4 (C-15); 32.9 (C- 402(2); 360(2). 19); 39.6 (C-20); 41.9 (N(1)-CH3); 43.2 (C-6); 50.7 (C-3); 51.6 (C- 5); 52.2 (C-7); 52.3 (C(16)-COOCH3); 60.3 (C(11)-OCH3); 71.2 Fluorination of Vindoline (1) with Xenon Difluoride (C-21); 75.8 (C-17); 77.8 (C-16); 85.2 (C-2); 100.9 (C-12); 106.3 To a solution of 200 mg (0.44 mmol) of vindoline (1) in di- (C-10); 126.3 (C-9); 135.0 (C-8); 151.8 (C-13); 154.0 (C-11); 169.8 chloromethane (6 mL) 76 mg (0.55 mmol) of potassium carbonate (C(17)-OC(O)CH3); 171.9 (C(16)-COOCH3). was added. Then 76 mg (0.44 mmol) of xenon difluoride dissolved 1 13 The intense HMBC cross peak at 7.53/52.2 ( H/ C coordinates in 16 mL dichloromethane was added to the mixture dropwise in in ppm) proves that the remaining aromatic proton and C-7 are argon atmosphere at -40 oC for 20 min. The mixture was stirred at - separated by no more than three bonds. The presence of H-9 in the 40 oC for 30 min, and distilled water (4 mL) was added slowly 2644 Current Organic Chemistry, 2016, Vol. 20, No. 24 Keglevich et al. while the temperature rised to rt. Then saturated sodium hydrogen MS: M+H=475. ESI-MS-MS (CID=35 %) (rel. int. %): carbonate solution (10 mL) was added and the mixture was ex- 457(11); 415(100); 397(2); 383(3); 355(1); 206(16); 180(2). 1 tracted with dichloromethane (3x 20 mL). The combined organic 15: H NMR (800 MHz, DMSO-d6): 0.50 (t, J=7.5 Hz, 3H, H3- phases were dried with magnesium sulfate and the solvent was 18); 1.28-1.33 (m, 1H, Hx-19); 1.61-1.67 (m, 1H, Hy-19); 1.99 (s, evaporated. After repeated preparative TLC (dichloromethane- 3H, OCOCH3); 2.19-2.32 (m, 2H, H2-6); 2.54-2.57 (m, 1H, Hx-5); methanol=19:1, then 15:1) 20 mg (10%) of pure product (13) was 2.47 (s, 1H, H-21); 2.78 (s, 3H, N-CH3); 2.79-2.83 (m, 1H, Hx-3); o obtained. Mp 115-117 C. TLC (dichloromethane-methanol=20:1); 3.20-3.23 (m, 1H, Hy-5); 3.46 (dd, J=16.7, 4.2 Hz, 1H, Hy-3); 3.73 -1 Rf=0.37. IR (KBr) 1742, 1650, 1596, 1236 cm . (s, 3H, COOCH3); 4.32 (s, 1H, H-2); 4.94 (s, 1H, H-17); 4.99 (d, 1 4 H NMR (799.7 MHz, DMSO-d6): 0.46 (3H, t, J=7.4 Hz, H3- J=10.1 Hz, 1H, H-15); 5.33 (d, JHF=5.9 Hz, 1H, H-12); 5.85 (ddd, 3 18); 1.33-1.38 (1H, m, Hx-19); 1.57-1.62 (1H, m, Hy-19); 1.98 (3H, J=10.1, 4.2; 1.5 Hz, 1H, H-14); 6.91 (dd, JHF=10.1, 1.4 Hz, 1H, H- 13 s, C(17)-OC(O)CH3); 2.34 (1H, ddd, J=14.4, 10.5, 9.1 Hz, Hx-6); 9). C NMR (201 MHz, DMSO-d6): 8.1 (C-18); 20.6 (C(17)- 2.38 (1H, s, H-21); 2.47-2.53 (1H, m, Hx-5); 2.57-2.62 (1H, m, Hy- OC(O)CH3); 29.5 (C-6); 31.6 (C-19); 33.8 (N(1)-CH3); 43.7 (C- 6); 2.74 (3H, s, N(1)-CH3); 2.77 (1H, ~d, J=16.0 Hz, Hx-3); 3.24 20); 50.5 (C-3), 52.7 (C-5); 53.1 (C(16)-COOCH3); 56.1 (C-7); 3 (1H, ~t, J=9.1 Hz, Hy-5); 3.47 (1H, dd, J=16.0, 4.3 Hz, Hy-3); 3.73 62.8 (C-21); 74.6 (C-17); 75.3 (C-2); 78.6 (C-16); 92.3 (d, JCF=2.4 1 3 (3H, s, C(16)-COOCH3); 4.27 (1H, s, H-2); 4.96 (1H, s, H-17); 4.98 Hz, C-12); 96.4 (dd, JCF=162.1 Hz, JCF=14.5 Hz, C-8); 107.0 (~t, 2 (1H, ~d, J=10.4 Hz, H-15); 5.21 (1H, dd, J=1.8, 1.4 Hz, H-12); JCF=17.8 Hz, C-9); 124.3 (C-14); 129.7 (C-15); 157.0 (dd, 3 1 3 2 5.84 (1H, ddd, J=10.4, 4.3, 1.6 Hz, H-14); 6.21 (1H, ddd, JHF=4.5 JCF=273 Hz, JCF=12.0 Hz, C-10); 163.6 (dd, JCF=19.5 Hz, 4 Hz, JHH=9.9, 1.4 Hz, H-10); 7.08 (1H, dd, J=9.9, 1.8 Hz, H-9); 8.24 JCF=2.2 Hz, C-13); 170.3 (C(17)-OC(O)CH3); 170.8 (C(16)- 13 2 4 19 13 (1H, s, C(16)-OH). C NMR (201.1 MHz, DMSO-d6): 8.1 (C-18); COOCH3); 175.3 (dd, JCF=24.0 Hz, JCF=5.0 Hz, C-11). F- C 3 20.6 (C(17)-OC(O)CH3); 29.1 (d, JCF=9.5 Hz, C-6); 31.2 (C-19); coupling constants belonging to C-6, C-7 and C-21 could not be 32.6 (N(1)-CH3); 43.3 (C-20); 50.3 (C-3); 51.4 (C-5); 52.5 (C(16)- unambiguously determined due to the overlap of carbon resonances 2 3 COOCH3); 55.0 (d, JCF=24.1 Hz, C-7); 62.2 (d, JCF=3.9 Hz, C- in the sample. 1 21); 74.3 (C-17); 74.8 (C-2); 77.9 (C-16); 93.39 (d, JCF=160.5 Hz, MS: M+H=479. ESI-MS-MS (CID=35 %) (rel. int. %): 2 C-8) 93.43 (C-12); 124.1 (C-14); 129.4 (C-15); 131.3 (d, JCF=15.4 461(35); 437(20); 419(100); 401(9); 399(18); 387(5); 359(2); 3 2 Hz, C-9); 135.1 (d, JCF=8.7 Hz, C-10); 163.1 (d, JCF=18.4 Hz, C- 339(4); 242(3); 192(12). 4 13); 183.7 (d, JCF=5.3 Hz, C-11); 170.2 (C(17)-OC(O)CH3); 170.8 (C(16)-COOCH3). 7-Fluorocatharanthine (17) HRMS: M+H=461.20771 (C24H30O6N2F; delta=-1.2 ppm). HR- a. With xenon-difluoride ESI-MS-MS (CID=45 %) (rel. int. %): 443(39); 441(41); 419(19); 401(100); 383(9); 381(18); 369(8); 363(2); 359(2); 341(2); 321(4); 100 mg (0.30 mmol) of catharanthine (2), obtained from 130 274(2); 242(2); 227(3). mg catharanthine sulfate, was dissolved in dichloromethane (4 mL), and under argon atmosphere at -40 oC 51 mg (0.37 mmol) of dry Fluorination of vindoline (1) with Selectfluor® potassium carbonate was added. Then 51 mg (0.30 mmol) of xenon difluoride dissolved in 10 mL dichloromethane was added to the 200 mg (0.44 mmol) of vindoline (1) was dissolved in acetoni- mixture dropwise in argon atmosphere at -40 oC for 20 min. The trile (4 mL), and in argon atmosphere 155 mg (0.44 mmol) of Se- mixture was stirred at -40 oC for 30 min and distilled water (2.5 ® lectfluor was added. The reaction mixture was stirred at rt for 2 h mL) was added slowly while the temperature rised to rt. Then satu- and was then poured into ice-water. After extraction with dichloro- rated sodium hydrogen carbonate solution (10 mL) was added and methane (3x30 mL) the mixture was washed with aqueous sodium the mixture was extracted with dichloromethane (3x 20 mL). The hydrogen carbonate solution (2x40 mL), dried with magnesium combined organic phases were dried with magnesium sulfate and sulfate and the solvent was evaporated. The crude product was puri- the solvent was evaporated. After preparative TLC (dichloro- fied with preparative TLC (dichloromethane-methanol=20:1), giv- methane-methanol=15:1) 25 mg (24%) of pure product (17) was ing a mixture of three products containing fluorine (14 and 13, 15 obtained. Mp. 96-98°C. TLC (dichloromethane-methanol); Rf quinoidal derivatives) which could not be separated. =0.66. IR (KBr) 2963, 1740, 1460, 1434, 1243, cm-1. 1 1 14: H NMR (799.7 MHz, DMSO-d6): 0.42 (3H, t, J=7.4 Hz, H NMR (499.9 MHz, DMSO-d6): 0.95 (3H, t, J=7.3 Hz, H3- H3-18); 0.93-0.98 (1H, m, Hx-19); 1.45-1.50 (1H, m, Hy-19); 1.94 18); 1.75 (1H, dd, J=13.1, 1.9 Hz, Hx-17); 1.88-1.97 (1H, m, Hx- (3H, s, C(17)-OC(O)CH3); 2.20-2.32 (2H, m, H2-6); 2.54-2.62 (1H, 19); 2.04-2.13 (1H, m, Hy-19); 2.16-2.26 (1H, m, Hx-6); 2.26-2.34 buried m, Hx-5); 2.57 (3H, s, N(1)-CH3); 2.66 (1H, s, H-21); 2.77- (1H, m, Hy-6); 2.56 (1H, dt, J=8.4, 2.4 Hz, Hx-3); 2.60-2.65 (1H, m, 2.84 (1H, m, Hx-3), 3.24-3.30 (1H, m, Hy-5); 3.32 (1H, s, H-2); H-14); 2.70 (1H, ~d, J=8.4 Hz, Hy-3); 2.78 (1H, dt, J=13.1, 3.0 Hz, 3.38-3.45 (1H, m, Hy-3); 3.66 (3H, s, C(16)-COOCH3); 3.80 (3H, s, Hy-17); 2.94-3.01 (1H, m, J=14.8 Hz, Hx-5); 3.48 (1H, ddd, C(11)-OCH3); 5.07-5.11 (1H, m, H-15); 5.19 (1H, s, H-17); 5.83 J=14.8, 12.2, 2.4 Hz, Hy-5); 3.53 (3H, s, C(16)-COOCH3); 4.15- 4 (1H, ddd, J=10.2, 4.9, 1.6 Hz, H-14); 6.49 (1H, d, JHF=7.2 Hz, H- 4.17 (1H, m, H-21); 5.81 (1H, ~d, J=6.7 Hz, H-15); 7.28-7.32 (1H, 3 13 12); 7.15 (1H, d, JHF=11.1 Hz, H-9); 8.79 (1H, s, C(16)-OH). C m, H-10); 7.40-7.44 (2H, m, H-11, H-12); 7.52-7.56 (1H, m, H-9). 13 NMR (201.1 MHz, DMSO-d6): 7.6 (C-18); 20.7 (C(17)- C NMR (125.7 MHz, DMSO-d6): 10.9 (C-18); 25.8 (C-19); 30.3 2 3 OC(O)CH3); 30.6 (C-19); 39.0 (N(1)-CH3); 42.4 (C-20); 43.6 (C- (C-14); 30.5 (d, JCF=26.3 Hz; C-6); 37.9 (C-17); 46.5 (d, JCF=1.9 3 6); 50.4 (C-3); 51.1 (C-5); 51.8 (C(16)-COOCH3); 52.4 (C-7); 56.0 Hz; C-5); 47.4 (C-3); 52.3 (C(16)-COOCH3); 57.1 (d, JCF=0.9 Hz; 4 1 (C(11)-OCH3); 66.0 (C-21); 75.8 (C-17); 78.6 (C-16); 82.8 (C-2); C-16); 58.7 (d, JCF=2.6 Hz; C-21); 104.1 (d, JCF=189 Hz; C-7); 2 4 96.1 (C-12); 110.4 (d, JCF=20.2 Hz, C-9); 124.1 (C-14); 124.5 (d, 120.5 (C-12); 122.6 (C-15); 122.9 (C-9); 127.0 (d, JCF=1.3 Hz; C- 3 1 5 2 JCF=7.2 Hz, C-8); 129.8 (C-15); 146.1 (d, JCF=235.0 Hz, C-10); 10); 131.1 (d, JCF=1.8 Hz; C-11); 135.8 (d, JCF=19.2 Hz; C-8); 2 3 147.6 (d, JCF=12.0 Hz, C-11); 148.7 (C-13); 170.1 (C(17)- 147.1 (C-20); 152.1 (d, JCF=6.2 Hz; C-13); 170.5 (C(16)- 2 OC(O)CH3); 171.6 (C(16)-COOCH3). COOCH3); 184.3 (d, JCF=16.9 Hz; C-2). Anomalous Products in the Halogenation Reactions Current Organic Chemistry, 2016, Vol. 20, No. 24 2645

HRMS: M+H=355.18118 (C21H24O2N2F; delta=-1.3 ppm). HR- CONFLICT OF INTEREST ESI-MS-MS (CID=45 %) (rel. int. %): 335(100); 323(7); 303(5); The author(s) confirm that this article content has no conflict of 293(4); 248(14); 228(12); 191(7); 171(17). interest. b. With Selectfluor® 106 mg (0.32 mmol) of catharanthine base (2) was dissolved in ACKNOWLEDGEMENTS ® acetonitrile (5 mL) and 112 mg (0.32 mmol) of Selectfluor was The authors are grateful to Gedeon Richter Plc for financial as- added under argon atmosphere After stirring for 4.5 h the solvent sistance. was evaporated and the crude product was purified with preparative TLC (dichloromethane-methanol=15:1), resulting in 16 mg (19%) REFERENCES of product (17), which was identical with the compound obtained in [1] Gribble, G.W. Occurrence of halogenated alkaloid. Alkaloids; San Diego, the fluorination reaction with xenon difluoride. 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