key: cord-0855889-05fqdmei
authors: Chopade, Anil U.; Chopade, Manojkumar U.; Chanda, Bhanu M.; Sawaikar, Dilip D.; Sonawane, Kiran B.; Gurjar, Mukund K.
title: A synthesis of (±)-thia-calanolide A, its resolution and in vitro biological evaluation
date: 2016-11-30
journal: Arabian Journal of Chemistry
DOI: 10.1016/j.arabjc.2012.04.027
sha: a2c3f980ef40558ea9b4416a890d18b2dd7deb47
doc_id: 855889
cord_uid: 05fqdmei

Abstract A synthesis of (±)-thia-calanolide A 3 has been successfully accomplished starting from 3,5-dimethoxythiophenol 4, in six steps in an overall yield of 4.5%. The key reaction involved Friedel–Crafts tigloylation of 5,7-dihydroxy-4-n-propyl thiocoumarin 6 employing an appropriate solvent of CS2–PhNO2 in a ratio of 7:3. In its biological evaluation for anti-HIV activity, (±)-thia-calanolide A 3 demonstrated comparatively less activity with calanolide A and its synthetic analogue aza-calanolide. Further, (±)-3 has been resolved by chiral HPLC to (+) and (−)-3.

(+)-Calanolide A [(+)-1], non-nucleoside reverse transcriptase inhibitor (NNRTI) and few new coumarin derivatives isolated from Calophyllum langerum (Kashman et al., 1992) which showed promising activity towards human immunodeficiency virus (HIV-1) reverse transcriptase (Currens et al., 1996a,b) .

It is also active against many strains of Mycobacterium tuberculosis including those resistant to the standard antitubercular drugs (Xu et al., 2004) and is reported through molecular dynamics simulation of SARS-CoV to be a potent proteinase inhibitor (Lee et al., 2003) . Calanolide A is in clinical testing phase II as anti-HIV drug; (Buckheit et al., 2000; Cragg and Newman, 2003) interest therefore arose particularly in developing its analogues. So far, most of the analogues were either less potent or devoid of any anti-HIV activity (Galinis et al., 1996; Zhou et al., 1999; Zembower et al., 1997; Xu et al., 1998; Newman et al., 1998) ; however, aza-calanolide A [(±)-2] (Sharma et al., 2003) exhibited enhanced in vitro anti-HIV reverse transcriptase activity. These findings prompted us to undertake the total synthesis of thia-calanolide A 3 by replacing oxygen in ring B with a sulphur atom, which might exhibit better activity. Herein, we describe the first synthesis, optical resolution and in vitro biological evaluation of (±)thia-calanolide A 3 (Fig. 1 ).

Thiophenol 4, obtained from phloroglucinol (Wolfers et al., 1975) or from 3,5-dimethoxyaniline by diazotization followed by reaction with Na 2 S + S or NaSH (Allen and Mackay, 1943) was subjected to Pechmann condensation with ethylbutyrylacetate in presence of sulphuric acid (Sethna and Phadke, 1953) . However, it yielded the undesired 7-hydroxy-5-methoxy-2-propyl-4H-thiochromen-4-one. The required 5,7-dimethoxy-4-propyl-2H-thiochromen-2-one 5 was obtained by a modified Pechmann method where triflic acid was used instead of H 2 SO 4 . Demethylation with AlCl 3 yielded 5,7-dihydroxythiocoumarin 6. Friedel-Crafts acylation (Olah, 1964) of 6 with tigloyl chloride yielded a 3:1 mixture of 5,7-ditigloyloxy thiocoumarin 7a and 5-hydroxy-7-tigloyloxy thiocoumarin 7b instead of the required C-8 tigloyl coumarin 7 (Scheme 1).

In an alternate route (Sekino et al., 2004) thiocoumarin 5 was subjected to Friedel-Crafts reaction with tigloylchloride to yield 5-methoxy-7-hydroxy-8-tigloylthiacoumarin 8. Intramolecular oxo-Michael addition (IMA) of the latter furnished the chromanone 9. Subsequent demethylation of 9 with various reagents like BBr 3 /CH 2 Cl 2 at À70°C (Vickery et al., 1979) , BBr 3 -SMe 2 /CH 2 Cl 2 (Williard and Fryhle, 1980) , BF 3 -Et 2 O/CH 2 Cl 2 (Preston et al., 1983) , AlCl 3 /PhCl at 120°C (Chenera et al., 1993) , and MgI 2 /K 2 CO 3 /PhH (Sekino et al., 2004) under reflux for 15 days did not proceed satisfactorily to yield the expected demethylated product. With BBr 3 / CH 2 Cl 2 , compound 8 was regenerated, whereas the starting thiocoumarin 9 was recovered unchanged with other reagents (Scheme 2).

Successful tigloylation was achieved via Friedel-Crafts reaction from compound 6 employing an appropriate solvent in a ratio CS 2 -PhNO 2 (7:3). The product 7 thus formed was cyclized with K 2 CO 3 yielded the desired 8,9-dimethyldihydrothiopyranones 10 as an 1:1 epimeric mixture. The required 'C' ring was constructed from 10 by reaction with 3-chloro-3methyl-1-butyne to yield chromene-chromanone-thiacoumarins 11 and 12 in a 1.8:1 ratio and in an overall yield of 34% (Scheme 3).

Both the ketones 11 and 12 were separately reduced with NaBH 4 to afford (±)-3 and (±)-thia-calanolide C 14 in 44% and 71% yields, respectively. Reduction of 11 with NaBH 4 -CeCl 3 under Luche condition resulted in a 3:2 mixture of (±)-3 and (±)-thia-calanolide B (±)-13, respectively (Scheme 4).

For the first time, in the calanolide series, especially in the thia-calanolide A synthesis, application of HPLC method for determination and separation of small quantities of the intermediates in the total synthesis of (+)-thia-calanolide has been developed. Resolution of a mixture of cis and trans of 10 was found to be excellent using Bondapak silica porasil 30 cm column. Resolution of (±)-3 was achieved by chiral HPLC (Chiralcel (OD), 9:1 hexane-isopropanol, 1 mL/min) to afford the two isomers (+)-3 and (-)-3 having optical rotation [a] D + 54.3°(c 0.5, CHCl 3 ) and [a] D À49.8°(c 0.3, CHCl 3 ), respectively (Scheme 5).

Compound (±)-3 was evaluated for anti-HIV reverse transcriptase activity by following HIV-1 p24 antigen capture ELISA (Perkin Elmer NEN) as per manufacturer's protocol. Briefly, CEM cells were infected with HIV-1 NL4.3 isolate for 4 h in presence of Poltbrene in complete medium. The infected cells were kept for 7-10 days in culture before the supernatant was used for HIV-1 p24 antigen capture. The percentage inhibition of virus production was calculated based on p24 values of untreated control. (±)-3 was found to be 28% inhibitory as compared to synthetic (±)-1 which was found to exhibit 99.7% inhibition of virus production.

The first synthesis of (±)-thia-calanolide 3 has been successfully accomplished. It has also been resolved by chiral HPLC to the (+) and (À) enantiomers which have been fully characterized. Biological evaluation for anti-HIV activity revealed that 3 is not as achieved as the natural calanolide A and its synthetic analogue aza-calanolide A. The amphoteric and Reactions with moisture-sensitive chemicals were performed under nitrogen in flame-dried reaction flasks. Solvents were dried by standard methods. Column chromatography was carried out using 60-80 mesh and 230-400 mesh silica gel. Phloroglucinol, dimethylthiocarbamoyl chloride, trifluoromethanesulfonic acid, ethyl butyrylacetate, tiglic acid, 3chloro-3-methyl-1-butyne were purchased from Aldrich and were used without purification.

A mixture of 4 (0.540 g, 3.17 mmol) and ethylbutyrylacetate (0.527 g, 3.3 mmol) was added dropwise to cold (5°C) trifluoromethanesulfonic acid (0.55 mL, 6.34 mmol) over a 10 min period and the reaction mixture was warmed to 25°C and stirred for 17 h. After quenching with ice water (10 mL), the mixture was extracted with EtOAc (3 · 10 mL). The combined organic layer was washed with brine (2 · 10 mL), dried (Na 2 SO 4 ) and concentrated. The residue was subjected to chromatographic purification (EtOAc-pet-ether, 2:8) to give 5 as lemon yellow crystals (0.301 g, 36%). Mp 110-112°C, IR (CHCl 3 ) 3016, 2965, 1630, 1593 cm À1 ; 1 H NMR (300 MHz, CDCl 3 ) d 0.97 (t, J = 7.3 Hz, 3H), 1.58 (m, 2H), 2.90 (t, J = 7.3 Hz, 2H), 3.82 (s, 3H), 3.86 (s, 3H), 6.20 (s, 1H), 6.37 (d, J = 2.5 Hz, 1H), 6.49 (d, J = 2.5 Hz, 1H). 13 C NMR (75 MHz, CDCl 3 ) d 14. 1, 24.0, 41.8, 55.5, 55.6, 98.2, 101.8, 111.5, 122.7, 142.4, 156.9, 160.7, 160.8, 184.1. Anal . Calcd for C 14 H 16 O 3 S: C, 63.61; H, 6.10; S, 12.13. Found: C, 63.61; H, 6.21; S, 11.07.

To a stirred solution of 5 (1 g, 3.78 mmol) in chlorobenzene (10 mL) was added AlCl 3 (2.5 g, 1.89 mmol) in portions over a period of 15 min and the reaction mixture was heated to 100°C under stirring for 12 h. The mixture was then cooled to room temperature and poured on crushed ice (20 g) and stirred well for 30 min. The solid precipitated was filtered, washed with water (50 mL) till neutral, then washed with hexane (5 mL) and dried in vacuum to give 6 (0.849 g, 95%) as a dark brown solid. Mp 230-235°C (decomp.): IR (CHCl 3 ) 3159, 1600 cm À1 ; 1 H NMR (300 MHz, CDCl 3 + DMSO-d 6 ) d 0.95 (t, J = 7.3 Hz, 3H), 1.60 (m, 2H), 2.97 (t, J = 7.3 Hz, 2H), 6.04 (s, 1H), 6.39 (s, 2H), 9.70 (s, 1H) , 10.02 (s, 1H); 13 C NMR (75 MHz, DMSO-d 6 ) d 14. 0, 24.0, 40.7, 102.6, 104.4, 108.2, 120.3, 140.9, 158.1, 159.7, 159.9, 182.6; Anal. Calcd for C 12 H 12 O 3 S: C, 61.00; H, 5.12; S, 13.57. Found: C, 60.75; H, 4.98; S, 12.38. 4.1.3 . (E)-5,7-Dihydroxy-8-(2-methylbut-2-enoyl)-4-n-propyl-2H-thiochromen-2-one (7) A mixture of 6 (0.5 g, 2.11 mmol), AlCl 3 (1.4 g, 10.59 mmol) and CS 2 (7 mL) was heated at 50°C while stirring for 30 min PhNO 2 (2 mL) was added dropwise and stirred for additional 30 min to get a homogeneous mixture. Tigloyl chloride (0.262 g, 2.21 mmol) in PhNO 2 (1 mL) was then added dropwise and the reaction mixture was stirred at 50°C for 24 h. It was then cooled to room temperature and quenched with crushed ice and dil. HCl before extracting with EtOAc (3 · 30 mL). The combined organic layer was washed with brine (3 · 20 mL), dried (Na 2 SO 4 ) and concentrated. The residue obtained was purified by column chromatography on silica gel 4:6) 1, 102.1, 107.9, 117.4, 120.1, 137.9, 138.1, 142.4, 156.7, 158.4, 159.9, 181.6, 196.2; Anal. Calcd for C 17 H 18 O 4 S: C, 64.13; H, 5.70; S, 10.07. Found: C, 63.65; H, 5.58; S, 9dihydrothiopyrano[2, ]chromene-2,10-dione (10) To a solution of 7 (0.4 g, 1.25 mmol) in 2-butanone (5 mL), anhydrous K 2 CO 3 (0.520 g, 3.77 mmol) was added and the mixture was refluxed for 1.5 h. It was then cooled to room temperature, acidified (pH 2) with dil. HCl and extracted with EtOAc (3 · 20 mL). The combined organic layer was washed with brine (3 · 10 mL), dried (Na 2 SO 4 ) and concentrated. The residue was chromatographed on silica gel column (EtOAc-pet-ether, 3:7) to give a 1:1 mixture of isomeric chromanones 10 (0.360 g, 90%) as white solid, mp 243-245°C: IR (Nujol) 3130, 3020, 1621 cm À1 ; Anal. Calcd for C 17 H 18 O 4 S: C, 64.13; H, 5.70; S, 10.07. Found: C, 63.86; H, 5.72; S, 9.05. 4.1.5. 6 ,7-Dihydro-2H-1,5-dioxa-12-n-propyl-2,2,6,7tetramethyl-9-thia-triphenylene-8,10-dione (11 trans and 12 cis) To a stirred solution of 10 (0.5 g, 1.57 mmol) in a mixture containing 2-butanone: DMF (10 mL, 9:1), at room temperature, anhydrous K 2 CO 3 (0.65 g, 4.71 mmol), 3-chloro-3-methyl-1butyne (0.8 g, 7.85 mmol), n-Bu 4 NI (0.49 g, 1.22 mmol) were added successively. The reaction mixture was heated at 60°C for 1 h, and then anhydrous ZnCl 2 (0.278 g, 2.04 mmol) was added. The resultant mixture was further heated to 70°C for 20 h. It was cooled to room temperature and quenched with saturated aq. solution of NH 4 Cl (3 mL) and extracted with EtOAc (2 · 30 mL). The combined organic layer was washed with brine (20 mL), dried (Na 2 SO 4 ) and concentrated. The residue obtained was purified through column chromatography

CDCl 3 ) d 1.01 (t, J = 7.2 Hz, 3H), 1.22 (d, J = 6.9 Hz, 3H), 1.53 (d, J = 6.5 Hz, 3H), 1.58 (s, 6H), 1.67 (m, 2H), 2.60 (m, J = 6.8, 11.5 Hz, 1H), 2.98 (t

Hz, 3H), 1.43 (d, J = 5.8 Hz, 3H), 1.56 (s, 6H), 1.64 (m, 2H), 2.70-2.75 (m

S, 8.30. Found: C, 68.88

dioxa-8-hydroxy-12-n-propyl-2,2,6,7-tetramethyl-9-thia-triphenylene-10-one (3, 13) To a solution of (±)-11 (0.020 g, 0.052 mmol) in EtOH

CDCl 3 ) d 1.01 (t, J = 7.3 Hz, 3H), 1.12 (d, J = 7 Hz, 3H), 1.48 (d, J = 6.8 Hz, 3H), 1.52 (s, 6H), 1.64 (m, 2H), 2.05 (sext

S, 8.30. Found: C, 68.81

thia-calanolide B (±)-13 (0.003 g, 14%) as a white solid mp 156-158°C: IR

1.22 (d, J = 6.9 Hz, 3H), 1.41 (d, J = 6.3 Hz, 3H), 1.49 (s, 6H), 1.65 (m, 2H), 2.30 (m

To a solution of 12 (0.050 g, 0.13 mmol) in EtOH (5 mL) at 0°C was added NaBH 4 (0.009 g, 0.26 mmol) while stirring and the temperature was allowed to rise to 30°C and the mixture was stirred further for 1 h. After completion of reaction, ice water (1 mL) was added and the reaction mixture was extracted with EtOAc (3 · 5 mL). The combined organic layer was washed with brine (5 mL), dried (Na 2 SO 4 ) and concentrated. The residue obtained was purified through flash column chromatography on silica gel (230-400 mesh, EtOAc-petether, 2:8) to give (±)-14 (0.036 g, 71%) as a white solid

Friedel Crafts and Related Reactions

Organic Reactions

A.U.C. thank UGC-CSIR, New Delhi, for the award of a research fellowship. NCCS (Pune) is thanked for anti-HIV activity assays.