Semisynthesis of Antitrypanosomal p-Quinone Analog Possesing the Komaroviquinone Pharmacophore

Abstract

A p-quinone analog having the komaroviquinone pharmacophore fused with a more conformationally flexible cycloheptane ring, was semisynthesized from natural demethlsalvicanol isolated from Perovskia abrotanoides via four steps in 26% overall yield. The IC₅₀ for the antitrypanosomal activity of the analog was 0.55 µM.

Introduction

Human African trypanosomiasis (HAT), also known as sleeping sickness, is caused by the protozoan parasite Trypanosoma brucei rhodesiense (the causative parasite of γ-HAT) or T. b. gambiense (the causative parasite of γ-HAT), and is a major threat to communities in the sub-Saharan areas of Africa.^1,2) In the case of γ-HAT, the parasite invades the central nervous system, causing behavioral changes including coma (sleeping sickness) and eventually death if left untreated. All drugs currently used to treat HAT have low efficacy and produce undesirable side effects³⁾ with the exception of fexinidazole, which was recently approved by U. S. Food and Drug Administration (FDA) as the first oral treatment for γ-HAT.⁴⁾ Apparently, there is a need for more effective drugs that are safe, cheap, and easy to administer. Some natural products have potent antitrypanosomal activity.^5–9) One of them, komaroviquinone (2) from Dracocephalum komarovi, an icetexane diterpene having p-quinone komaroviquinone pharmacophore fused with a 8-oxabicyclo[3.2.1]octane core to form a rigid structure, exerts a trypanocidal effect on the epimastigote of T. cruzi, the causative parasite of American trypanosomiasis.^10,11) In this study, we designed analogue 3 in which the p-quinone pharmacophore was fused with a more conformationally flexible cycloheptane ring, semisynthesized 3 from natural demethlsalvicanol (1),^12–18) and evaluated the in vitro antitrypanosomal activity of 3.

Results and Discussion

We prepared p-quinone analog 3 having the 2-isopropyl-3-methoxy-1,4-benzoquinone pharmacophore and exhibiting antitrypanosomal activity (Fig. 1) from demethylsalvicanol (1), an icetexane diterpene from Perovskia abrotanoides.^12–14)

Fig. 1. Structures of Demethylsalvicanol (1), Komaroviquinone (2), and Prepared Analog 3

The synthetic sequence for the preparation of analog 3 from compound 1 is shown in Chart 1. Kita and colleagues reported efficient preparation of p-quinones in water via oxidative demethylation of phenol ethers using [bis(trifluoroacetoxy)iodo]benzene (PIFA).¹⁹⁾ Therefore, the direct oxidation of demethylsalvicanol (1) was first attempted by using PIFA in a mixture of water and methanol. This reaction produced not p-quinone 5 but o-quinone 4¹²⁾ in 40% yield. Then, the oxidation of compound 1 after functionalization at 14-position was studied. The phenolic hydroxy groups in compound 1 were methylated with methyl iodide and potassium carbonate to give corresponding dimethoxy derivative 6 in 65% yield.²⁰⁾ However, the oxidation of 6 by using PIFA in a mixture of water and methanol did not give compound 3. Mild bromination of compound 6 with N-bromosuccinimide (NBS) in N,N-dimethylformamide (DMF) afforded 14-Br compound 7 in 94% yield.¹²⁾ In the presence of copper(I) iodide, bromide 7 was treated with sodium methoxide in DMF to give 11,12,14-trimethoxy derivative 8²¹⁾ in 88% yield. The oxidation of trimethoxy compound 8 was carried out under several reaction conditions, as shown in Table 1.

Chart 1. Semisynthesis of Target Quinone (3)

i) PIFA/H₂O–MeOH (40:1)/r.t.; ii) MeI/K₂CO₃/dry Me₂CO/reflux; iii) NSB/DMF/r.t.; iv) CuI/NaOMe/dry DMF/90–120 °C.

Table 1. Oxidation of Trimethoxy Compound 8 under Several Reaction Conditions

Entry	Reagents (equiv.)	Sovlents	Yield (%) of 3
1	PIFA (1.5)	H2O : MeOH = 40 : 1	21
2	PIFA (1.5)	H2O : MeOH = 1 : 2	No reaction
3	PIFA (4.0)	H2O : MeOH = 40 : 1	10
4	Fremy’s salt (4.0)	H2O : MeOH = 1 : 10	No reaction
5	CANa) (4.0)	MeOH	trace
6	Ag2O (4.0)/6N HNO3	1,4-Dioxane	48

a) Cerium (IV) ammonium nitrate (CAN).

The oxidation of 8 with PIFA in water:methanol (40 : 1) gave compound 3 in 21% yield (entry 1), and the oxidation with silver(I) oxide in a mixture of 6 N nitric acid and 1,4-dioxane gave target compound 3 in 48% yield (entry 6). The structure of compound 3 was confirmed by analysis of its ¹H, ¹³C, and two dimensional (2D) NMR data (See Supplementary Materials). Thus, p-quinone analog 3 having the komaroviquinone pharmacophore could be semisynthesized from demethylsalvicanol (1) via four steps in 26% overall yield. Recently, total synthesis of compound 3 has been reported by Yang et al.²²⁾

p-Quinone 3 and 11,12,14-trimethoxy compound 8 were subjected to assay for in vitro antitrypanosomal activity against T. b. brucei GUTat3.1 strain. Therapeutic drugs pentamidine, suramin, and eflornithine were used as positive control. The average IC₅₀ values for the antitrypanosomal activity against GUTat3.1 strain and the cytotoxicity toward MRC-5 cells of the assayed compounds are presented in Table 2, along with the selectivity indexes (cytotoxicity IC₅₀/antitrypanosomal activity IC₅₀).^23,24)

Table 2. IC₅₀ Values of in vitro Antitrypanosomal Activity (T. b. brucei GUTat 3.1) and of Cytotoxicity (MRC-5 Cells) of Compounds 3 and 8, and of the Currently Used Drugs

Compound	IC50 (µM)		Selectivity index (SI)b)
	Antitrypanosomal activitiy	Cytotoxicity
3	0.55	33.0	60.0
8	9.88	122.0	12.3
Pentamidine	0.0018	5.9	3333
Suramina)	1.20	>77	>64
Eflornithinea)	12.4	>549	>44

a) Ref. 5. b) Cytotoxicity IC₅₀/Antitrypanosomal activity IC_50.

The IC₅₀ of p-quinone analog 3 having the komaroviquinone pharmacophore was lower than that of trimethoxy analog 8. The in vivo antitrypanosomal activity of 3 was assessed in T. b. brucei S427-infected mice at the dose of 50 mg/kg × 4 in the intraperitoneal (i.p.) route as previously reported.⁶⁾ Unfortunately, compound 3 was inactive.

Conclusion

In conclusion, the designed analogue 3 in which the p-quinone pharmacophore was fused with a more conformationally flexible cycloheptane ring, was semisynthesized from natural demethlsalvicanol (1) via four steps in 26% overall yield. The p-quinone substructure is significant for compounds to show antitrypanosomal activity and increase selectivity index. Further studies including the optimization of 3 to create derivatives having antitrypanosomal activity in animal models are in progress.

Experimental

General Procedures

Melting points were determined on a Yanaco MP-3 apparatus and recorded uncorrected. IR spectra were recorded on a JASCO FT/IR 620 spectrophotometer, optical rotation was measured on a JASCO DIP-360 automatic digital polarimeter, and mass spectra were recorded on a Micromass LCT spectrometer. NMR spectra were recorded in CDCl₃ on Bruker AM-400 and DRX-500 spectrometers at 300 K.

Materials

Demethylsalvicanol (1) was isolated from P. abrotanoides and purified as described in our previous paper,^12,23) and its absolute configuration was determined as depicted as Fig. 1.¹²⁾

Experimental Procedures

Oxidation of Demethylsalvicanol (1) with PIFA

A mixture of demethylsalvicanol (1) (10 mg, 0.031 mmol) and PIFA (18 mg, 0.043 mmol) in MeOH (2.5 µL) and H₂O (100 µL) was stirred at room temperature for 24 h. Then, the reaction mixture was diluted with H₂O (10 mL), and the aqueous solution was extracted with AcOEt (3 × 10 mL). The combined organic layer was washed with sat. NaCl (3 × 15 mL), dried over MgSO₄, filtered, and evaporated in vacuo to give an oily residue that was subjected to medium pressure liquid chromatography (MPLC) (SiO₂, hexane : AcOEt = 20 : 1). The product, o-quinone 4 (3.9 mg, 40%), was obtained as a pale red amorphous solid, whose spectral data were identical with those reported previously.¹²⁾

Methylation of Demethylsalvicanol (1) with Methyl Iodide

Demethylsalvicanol (1) (50 mg, 0.16 mmol) was dissolved in dry Me₂CO (2.0 mL), and the solution was treated with K₂CO₃ (640 mg, 3.1 mmol) and methyl iodide (MeI) (0.19 mL, 3.1 mmol). After stirring at 65 °C for 5 h under Ar atmosphere, the reaction mixture was diluted with H₂O (20 mL), and the aqueous solution was extracted with AcOEt (3 × 10 mL). The combined organic layer was washed with sat. NaCl (3 × 15 mL), dried over MgSO₄, filtered, and evaporated in vacuo to give an oily residue. MPLC (SiO₂, hexane : AcOEt = 49 : 1) of the residue gave 11-O-methylsalvicanol (6) (36 mg, 65%) as a colorless amorphous solid, whose spectral data were identical with those reported previously.^15–18)

Bromination of 11-O-Methylsalvicanol (6) with N-Bromosuccinimide (NBS) in DMF

A solution of compound 6 (35 mg, 0.10 mmol) and NBS (20 mg, 0.11 mmol) in dry DMF (2.0 mL) was stirred at room temperature for 20 h. The reaction mixture was poured into ice-cooled H₂O (20 mL), and the aqueous phase was extracted with AcOEt (15 mL × 3). The combined organic layer was washed with sat. NaCl (15 mL × 3), dried over MgSO₄, filtered, and evaporated in vacuo to give an oily residue, which was subjected to MPLC (SiO₂, hexane : AcOEt = 49 : 1). A colorless amorphous solid (41 mg, 94%) was obtained, and its spectral data showed that it was known bromide 7.¹²⁾

Methoxylation of Compound 7

To a solution of CuI (32 mg, 6.3 mmol) in dry DMF (2 mL) was added 4.17 M sodium methoxide solution in dry MeOH (1.5 mL, 6.3 mmol) under Ar atmosphere, and to this, compound 7 (18 mg, 0.040 mmol) in dry DMF (2 mL) was added at 90 °C. The reaction mixture was stirred at 120 °C for 3.5 h. After the reaction mixture was cooled to room temperature, it was poured into H₂O (20 mL) and the aqueous phase was extracted with AcOEt (3 × 20 mL). The combined organic layer was washed with sat. NaCl solution (3 × 15 mL), dried over MgSO₄, filtered, and evaporated in vacuo to give an oily residue. MPLC (SiO₂, hexane : AcOEt = 49 : 1) of the residue gave compound 8²¹⁾ (14 mg, 88%) as a colorless amorphous solid. Compound 8: mp 47–50 °C (MeOH); [α]_D²⁵: +25.2° (c 0.19, CHCl₃); ¹H-NMR (CDCl₃, 400 MHz) δ: 3.84 (3H, s, 12-OCH₃), 3.76 (3H, s, 11-OCH₃), 3.65 (3H, s, 14-OCH₃), 3.42 (1H, sept, J = 7.1 Hz, H-15), 3.32 (1H, dd, J = 14.4, 7.8 Hz, H-7), 3.24 (1H, d, J = 14.0 Hz, H-20), 2.50 (1H, d, J = 14.0 Hz, H-20), 2.26 (1H, dd, J = 14.4, 11.4 Hz, H-7), 2.05 (1H, m, H-6), 1.87 (1H, ddd, J = 13.4, 3.2, 3.2 Hz, H-2), 1.83 (1H, m, H-1), 1.55 (1H, ddd, J = 14.1, 14.1, 3.6 Hz, H-1), 1.48 (1H, m, H-2), 1.44 (1H, m, H-3), 1.34 (3H, d, J = 7.1 Hz, H-16), 1.34 (1H, m, overlapped, H-5), 1.32 (3H, d, J = 7.1 Hz, H-17), 1.27 (1H, m, H-3), 1.18 (1H, m, H-6), 0.93 (3H, s, H-18), 0.89 (3H, sH-19); ¹³C-NMR (CDCl₃, 100 MHz) δ: 150.84 (s, C-12), 150.77 (s, C-14), 149.3 (s, C-11), 133.4 (s, C-13), 132.5 (s, C-8), 128.9 (s, C-9), 70.6 (s, C-10), 62.3 (q, 14-OCH₃), 60.4 (q, 11-OCH₃), 60.3 (q, 12-OCH₃), 58.4 (d, C-5), 42.4 (t, C-3), 42.1 (t, C-1), 41.5 (t, C-20), 34.3 (s, C-4), 32.2 (q, C-18), 27.2 (t, C-7), 26.0 (d, C-15), 23.7 (t, C-6), 22.3 (q, C-19), 22.1 (q, C-16), 21.6 (q, C-17), 18.8 (t, C-2); IR (film) cm⁻¹: 3468 (OH), 2938, 1454, 1413, 1340, 1247, 1121, 1079, 1040, 965, 844, 758 cm⁻¹; high resolution (HR)-MS (electrospray ionization (ESI)): Calcd for C₂₃H₃₆O₄Na (M + Na⁺): 399.2511. Found: 399.2518.

Oxidation of Compound 8 with PIFA

A mixture of compound 8 (14 mg, 0.036 mmol), PIFA (19 mg, 0.043 mmol), MeOH (20 µL), and H₂O (800 µL) was stirred at room temperature for 4 h. Then, the reaction mixture was diluted with H₂O (10 mL), and the aqueous solution was extracted with AcOEt (3 × 10 mL). The combined organic layer was washed with sat. NaCl (3 × 15 mL), dried over MgSO₄, filtered, and evaporated in vacuo to give an oily residue. The residue was then subjected to MPLC (SiO₂, hexane : AcOEt = 20 : 1) to give a pale yellow oil, which was identified as p-quinone 3 (2.6 mg, 21%).

Oxidation of Compound 8 with Ag₂O in 6 M HNO₃

To a mixture of compound 8 (43 mg, 0.11 mmol) and Ag₂O (56 mg, 0.45 mmol) in 1,4-dioxane (1.5 mL) was added 6 M HNO₃ (0.2 mL). After stirring at room temperature for 1 h, the reaction mixture was diluted with H₂O (10 mL), and the aqueous solution was extracted with AcOEt (3 × 10 mL). The combined organic layer was dried over MgSO₄, filtered, and evaporated in vacuo to give an oily residue, which was subjected to MPLC (SiO₂, hexane:AcOEt = 20 : 1) to give p-quinone 3 (19 mg, 48%) as a pale yellow oil. p-Quinone 3: [α]_D²⁵: +63.3° (c = 0.13, CHCl₃); IR (film) cm⁻¹: ν_max 3502 (OH), 1647, 1610 (C=O); HR-MS (ESI): Calcd for C₂₁H₃₀O₄Na (M + Na⁺): 369.2041. Found: 369.2042. ¹H- and ¹³C-NMR spectral data are shown in Supplementary Materials.

Acknowledgments

This work was supported by JSPS KAKENHI Grant Number 22590019 and a Grant from the Drugs for Neglected Diseases initiative (DNDi).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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