(R)-4-Acetyl-10-(2-chloro-1,3-thiazol-4-yl)-5,11,13-trihydroxy-2,12-dimethyl-8-oxatricyclo[7.4.0.0^2,7]trideca-1(13),4,6,9,11-pentaen-3-one

Abstract

A novel usnic acid derivative with 2-chlorothiazole substituent was obtained by a three-step synthesis from usnic acid. The structure of the product was proved by a set of physical methods, including ¹H, ¹³C, HRMS, HSQC, HMBC and IR spectroscopy.

1. Introduction

Studying the properties of natural plant metabolites has been one of the main activities of scientists since the birth of chemistry as a science. At present, this section of science has not only not lost its significance but has also acquired new facets and possibilities. Thus, the use of plant metabolites as starting compounds for the creation of new biologically active agents has become one of the main areas of modern medicinal chemistry. One of the most interesting plant metabolites is usnic acid 1, first isolated from lichens more than a century and a half ago [1,2]. Usnic acid 1 is produced in a number of lichens, such as Cladonia (Cladoniaceae), Usnea (Usneaceae), Lecanora (Lecanoraceae), Ramalina (Ramalinaceae), Evernia, Parmelia (Parmeliaceae), Alectoria (Alectoriaceae), etc. It exhibits a wide range of biological activities, including antibacterial, antiviral, anticancer, anti-inflammatory and analgesic [2,3,4,5,6,7,8].

Semisynthetic derivatives of usnic acid are promising candidates for medicinal products. One of the most promising directions of modification is formation of a thiazole ring by reaction of 14-bromousnic derivative 2 with certain thioamide derivatives (Scheme 1). There are some derivatives described in the literature which have the substituent in the second position of the thiazole ring; these include alkyl, aminoalkyl and hydrazonoalkyl derivatives. Moreover, aminothiazole derivatives exhibit antimycobacterial properties, and hydrazonothiazole derivatives exhibit inhibitory properties with respect to several DNA repair enzymes—tyrosyl-DNA phosphodiesterases 1 and 2 (TDP1 and TDP2) and poly(ADP-ribose)polymerase 1 (PARP1)—and antiviral properties with respect to the SARS-CoV-2 strain line [9,10].

In the present work, synthesis of a novel thiazole derivative of usnic acid with chlorine as substituent in the second position of the thiazole ring from usnic acid was carried out in three steps.

2. Results and Discussion

To obtain thiazoles, in particular their 2-chloro- or 2-hydroxy derivatives, the method of cyclization of α-thiocyanate ketones by the action of mineral acids is used [11]. This method was chosen to obtain the 2-chlorothiazole derivative of (+)-usnic acid (Scheme 2).

Compound 4 was prepared as described in [12], starting from (+)-usnic acid through intermediate 14-bromousnic acid 2, which was converted into thiocyanate by reaction with sodium thiocyanate in acetone. The reaction proceeds according to the theory of hard and soft (Lewis) acids and bases (HSAB). Substitution of the bromine atom occurs using an atom of sulfur, as a soft base. Consequently, no side processes occur at this stage. Compound 4 was isolated in 91% yield after column chromatography.

At the final stage of the synthesis of compound 5, gaseous hydrogen chloride was passed through a solution of substance 4 in tetrahydrofuran. Chlorothiazole 5 was obtained with a yield of 64% and HPLC purity of 96.7%. The decrease in yield was due to the difficulty of chromatographic purification of the compound from the decomposition products of THF under reaction conditions.

The structure of compound 5 was confirmed based on NMR (¹H, ¹³C) spectroscopy (Figure 1 and Supplementary Materials (Figures S1 and S2)). No methylene proton signal was observed in the ¹H NMR spectrum. At the same time there was a new signal at 7.67 ppm, which is typical for the thiazole proton. A change in the signal of one of the acidic protons was observed. The OH⁷ group signal of the new compound shifted from 12.26 to 11.42, indicating modification of a neighboring acetyl group. One of the key changes in the ¹³C NMR spectrum was the absence of one of the carbonyl carbon atoms and the appearance of three new thiazole signals at 115.6, 147.2 and 150.9. According to HMBC data (Supplementary Materials (Figure S4)) cross-peaks between thiazole proton and all thiazole carbon atoms were detected. A cross-peak between the H¹⁴ proton and the C⁶ benzene carbon atom also was observed. The position of the C⁶ signal of the carbon atom was confirmed by observed interaction of this atom with the hydrogen atoms of the methyl group of the benzene ring, as well as the hydroxyl group OH⁷. This confirmed the formation of the thiazole ring and its position relative to the benzene ring and to the usnic acid core. HRMS data confirming the molecular formula of the new compound and also the presence of the chlorine atom are presented in the Supplementary Materials (Figure S6).

IR spectral analysis of compound 5 confirmed two distinct carbonyl stretching vibrations at 1620 and 1683 cm⁻¹, corresponding to the carbonyl groups in the usnic core. Also observed was an absorption band at 1064 cm⁻¹, corresponding to the C-Cl group. The detailed IR absorption bands are presented in the Supplementary Materials (Figure S5).

3. Materials and Methods

Analytical and spectral studies were conducted at the Chemical Service Center for the collective use of the Siberian Branch of the Russian Academy of Science.

The ¹H and ¹³C spectra for solutions of the compound in CDCl₃ were recorded on a Bruker AV-400 spectrometer (Bruker Corporation, Karlsruhe, Germany; operating frequencies 400.13 MHz for ¹H, 100.61 MHz for ¹³C). The residual signals of the solvent were used as references (δ_H 7.24, δ_C 76.90 for CDCl₃). Mass spectra (ionizing-electron energy 70 eV) were recorded on a DFS Thermo Scientific high-resolution mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Electron impact ionization was used for measurement of mass spectra. Specific rotation was determined on a PolAAr 3005 polarimeter (Optical Activity Ltd., Huntingdon, UK) and was expressed in (deg × mL) × (g × dm)⁻¹, whereas concentrations of solutions were shown in g × (100 × mL)⁻¹. Thin-layer chromatography was performed on TLC Silica gel 60F₂₅₄ (Merck KGaA, Darmstadt, Germany). IR spectra were measured on a Bruker Vector 22 IR (Bruker Corporation, Billerica, MA, USA) spectrophotometer. Melting points were determined on an Electrothermal IA 9100 (Electrothermal, Chelmsford, UK) apparatus (1 °C/min). Synthetic starting materials and reagents were acquired from Reachem (Moscow, Russia). (+)-Usnic acid was obtained from Zhejiang Yixin Pharmaceutical Co., Ltd., (Lanxi, China). All chemicals were used as described unless otherwise noted. The atom numbers in the compound provided for the assignment of signals in the NMR spectra corresponded to the traditional numeration for usnic acid.

3.1. Usnic Acid Bromination

Synthesis of 14-bromousnic acid 2 was performed by reaction of usnic acid 1 with solution Br₂ in dioxane as described in [12]. Detailed information about the synthesis protocol can be found in Supplementary Information.

3.2. Synthesis of Thiocyanate Derivative of Usnic Acid 4

Synthesis of compound 4 was performed by reaction of 14-bromousnic acid 2 with sodium thiocyanate as described in [12]. Detailed information about the synthesis protocol can be found in Supplementary Information.

3.3. Synthesis of 2-Chlorothiazole Derivative of Usnic Acid 5

Compound 4 (500 mg, 1.25 mmol) was dissolved in 60 mL of THF. Gaseous HCl was bubbled through the reaction mixture. To achieve this, 280 mL of concentrated hydrochloric acid was added to a 500 mL flask equipped with a trap (a 250 mL flask and a Drexel attachment) to avoid suction of the reaction mixture into the flask with hydrochloric acid. During the reaction the color of the reaction mixture changed from yellow to red. An hour later, the passage of HCl was stopped. The reaction mixture was diluted with water, saturated solution of NaHCO₃ was added to neutral pH, and extraction was then performed with chloroform. After that, the reaction mixture was evaporated and purified by column chromatography on silica gel (eluent—gradient of methanol in dry methylene chloride (from 0% to 0.5% upward)). As result a yellow powdery substance was isolated.

(R)-4-acetyl-10-(2-chloro-1,3-thiazol-4-yl)-5,11,13-trihydroxy-2,12-dimethyl-8-oxatricyclo[7.4.0.0²,⁷]trideca-1(13),4,6,9,11-pentaen-3-one (5).

Yellow amorphous powder. Yield: 335 mg, 64%. M. p. 114.7–116.0 °C. ¹H NMR (CDCl₃, δ): 1.72 (3H, s, H-15), 2.15 (3H, s, H-10), 5.90 (1H, s, H-4), 10.32 (1H, s, OH-9), 11.42 (1H, s, OH-7), 18.79 (1H, s, OH-3). ¹³C NMR (CDCl₃, δ): 8.21 (C-10), 27.70 (C-12), 32.02 (C-15), 59.28 (C-9b), 96.36 (C-6), 97.63 (C-4), 103.49 (C-9a), 105.15 (C-2), 109.38 (C-8), 115.64 (C-14), 147.14 (C-13 or C-16), 150.89 (C-13 or C-16), 151.46 (C-5a), 152.10 (C-9), 156.47 (C-7), 180.08 (C-4a), 191.55 (C-3) 197.92 (C-1), 201.35 (C-11). [α]_D^23,7 +308 (c 0.1160, CHCl₃). IR (cm⁻¹): 3600–3300, 3144, 2925, 2854, 1683, 1620, 1533, 1461, 1346, 1330, 1305, 1180, 1128, 1095, 1064, 1031, 958, 941, 829, 765, 740, 692, 539. HRMS: m/z 419.0235 [M]+ (calcd for (C₁₉H₁₄O₆N₁³²S₁³⁵Cl₁)^+∙: 419.0225).

3.4. HPLC Analysis

HPLC analysis was carried out using an HPLC-UV system (Agilent 1100, Agilent Technologies Inc., Santa Clara, CA, USA) with a Zorbax RX C18 column (150 × 4.6 mm with 5.0 µm particle size; Agilent Technologies Inc., Santa Clara, CA, USA). The column was thermostatically controlled at 30 °C. Gradient elution was used for separation of the peak of the main compound from other peaks, as follows: from 60%(B) to 100%(B) for 20 min, 100%(B) for 5 min, where solvent (A) was trifluoroacetic acid 0.05% and solvent (B) was 100% acetonitrile (for HPLC, gradient grade, ≥99.9%; Sigma-Aldrich, Saint-Quentin-Fallavier, France). The flow rate was 1.0 mL/min. The elution volume was 8 μL. Peaks were detected using a wavelength of 255 nm. Peaks from solvents were not included in the purity calculations

4. Conclusions

In this work, a new usnic acid derivative containing 2-chlorothiazole fragment was obtained. This compound was synthesized by reaction of a 14-thiocyanate derivative of usnic acid with hydrogen chloride, accompanied by intramolecular cyclization.