Synthesis of Homodrimane Sesquiterpenoids Bearing 1,3-Benzothiazole Unit and Their Antimicrobial Activity Evaluation

Abstract

Based on some homodrimane carboxylic acids and their acyl chlorides, a series of fourteen 2-homodrimenyl-1,3-benzothiazoles, N-homodrimenoyl-2-amino-1,3-benzothiazoles, 4′-methyl-homodrimenoyl anilides and 4′-methyl-homodrimenthioyl anilides were synthesized and their biological activities were evaluated on five species of fungi (Aspergillus niger, Fusarium solani, Penicillium chrysogenum, P. frequentans, and Alternaria alternata) and two strains of bacteria (Bacillus sp. and Pseudomonas aeruginosa). The synthesis involved the decarboxylative cyclization, condensation and thionation of the said acids, anhydrides or their derivatives with 2-aminothiophenol, 2-aminobenzothiazole, p-toluidine and Lawesson’s reagent. As a result, together with the desired compounds, some unexpected products 8, 25, and 27 were obtained, and the structures and mechanisms for their formation have been proposed. Compounds 4, 9, and 25 showed higher antifungal and antibacterial activity compared to the standards caspofungin (MIC = 1.5 μg/mL) and kanamycin (MIC = 3.0 μg/mL), while compound 8 had comparable activities. In addition, compounds 6, 17, and 27 showed selective antifungal activity at MIC = 2.0, 0.25, and 1.0 μg/mL, respectively.

1. Introduction

Natural labdane-type diterpenes isolated from terrestrial plants and marine sources are still interesting objects of study due to a wide range of their biological activities [1]. Some of them are obtained from sources in sufficient amounts to be used as precursors for the synthesis of natural analogs, special purpose compounds, or pharmaceutical agents with remarkable properties [2].

The chemistry of 1,3-benzothiazole and its 2-substituted derivatives has become a separate area of research due to a high degree of structural diversity, which generates a wide variety of their applications or pharmacological activities [3,4,5].

A wide variety of reagents and methods which lead to 2-substituted 1,3-benzothiazoles are known. One of the most requested methods of their synthesis involves the cyclocondensation of aromatic aldehydes or others carbonyl compounds such as carboxylic acids, esters, acyl halides, etc., with o-aminophenol [6,7] or its disulfides [8]. Frequently, for the conversion of the resulting amides into the corresponding thioamides, Lawesson’s reagent is used, but the course of reactions and their yields strongly depend on the structure of substrates [9].

The synthesis of terpeno-heterocyclic hybrid compounds with a cumulative biological potential is a new direction of organic chemistry that has emerged in the last decade. Research in this field has been successful, a large number of molecular hybrids containing both terpene and diazine [10,11], 1,2,4-triazole and carbazole [12,13], azaheterocyclic [14,15], hydrazinecarbothioamide and 1,2,4-triazole [16], 1,3,4-oxadiazole and 1,3,4-thiadiazole [17], thiosemicarbazone and 1,3-thiazole [18] units were reported, many of which showed excellent antifungal and/or antibacterial activity.

In continuation of our work aimed at the preparation of hybrid terpeno-heterocyclic compounds, herein, we report the result of the synthesis of novel homodrimane sesquiterpenoids bearing 2-substituted 1,3-benzothiazole, N-substituted 2-amino-1,3-benzothiazole and N-substituted p-toluidine units and their antimicrobial properties evaluation.

2. Results and Discussion

2.1. Synthesis and Characterization

According to the synthesis strategy of the desired compounds, at first, the intermediate carboxylic acids were obtained from commercial (+)-sclareolide (1). It was converted to methoxyester 2 in two steps, with an overall yield of 25%, applying the known procedure [19], followed by the saponification into acid 3 in 89% yield. Starting from sclareolide (1) carboxylic acids 5 and 7 were obtained in five and six steps, with overall yields of 81% and 62%, respectively [13,20] (Scheme 1).

Further, the one-pot decarboxylative cyclization reactions of acids 3, 5, and 7 with 2-aminothiophenol promoted by triphenylphosphine and triethylamine [21] were performed under reflux for 4 h, which after silica gel chromatography afforded 2-homodrimenyl-1,3-benzothiazoles 4, 6, 8, and 9, in the yields as depicted in Scheme 1.

The structures of intermediary compounds as well as final products were confirmed by ¹H, ¹³C, ¹⁵N, and 2D NMR spectroscopy and HRMS analysis. The formation of the desired hybrid compounds 4, 6, 8, and 9 was proved, first of all, with the presence of signals attributed to aromatic protons from a common 2-substituted-1,3-benzothiazole unit in a range of 7.32–7.96 ppm. In addition, some individual signals such as a singlet corresponding to protons of C₇-bonded methoxy group at 3.39 ppm, or doublet of doublets of C₆- and C₇-bonded protons at 5.88 and 5.95 ppm confirmed the presence of a terpene unit. Those structures were fully confirmed by the carbon spectral data.

It should be noted that, in the case of acid 7, surprisingly, in addition to the desired compound 9, obtained with a yield of only 5%, the compound 8 with an unexpected structure was afforded as a major reaction product, in 27% total yield. The rearrangement of the carbon skeleton of compound 8 was confirmed by a shift of some signals in the ¹H NMR spectrum compared to the starting acid 7, e.g., by singlet signals of the C₈- and C₉-bonded methyl groups at 0.92 and 1.05 ppm and the appearance of new multiplet signals of the C₈-bonded proton at 1.54–1.56 ppm. The ¹³C NMR spectra confirmed this by signals of the C₈ (34.5 ppm) and C₉ (42.1 ppm), those at 130.8 and 139.4 ppm being attributed to C₁₀ and C₅, respectively.

The NMR data of compound 8 have been assigned on the basis of their 1D (¹H, ¹³C, DEPT-135°) and 2D homo- (¹H/¹³C HSQC, ¹H/¹³C HMBC and ¹H/¹H COSY-45°) correlation spectra. An analysis of the ¹H, ¹³C, ¹H/¹H COSY and ¹H/¹³C HSQC NMR spectra suggested the presence of two isolated spin systems: CH₂CH₂CH₂ (C₁ to C₃) and CH₂CH₂CH (C₆ to C₈) (Figure 1). The rearranged carbon framework of compound 8 was indisputable according to a detailed analysis of its ¹H/¹³C HMBC spectrum. Thus, the observed correlations of H2-C₂ with two sp² hybridized carbons (C₅, δ_C 139.4 and C₁₀, δ_C 130.8) were indicative of the Δ^5,10 double bond localization, which was also supported by the correlations of H3-C₁₈/C₅, H3-C₉/C₁₀ and H2-C₁₁/C₁₀. The migration of H3-C₂₀ methyl from the C₁₀ to C₉ position was ascertained by the H3-C₂₀/C₁₀, H3-C₂₀/C₈ and H3-C₂₀/C₉ and the H3-C₂₀/C₁₁ cross-peaks in the HMBC spectrum.

The rearrangement of 2-homodrimenyl-1,3-benzothiazole 8 carbon skeleton can be explained by the following reaction pathway (Scheme 2). The reaction started with formation of triphenylphosphonium chloride as product of Ph₃P and CCl₄ interaction, followed by its condensation with carboxylic acid which led to acylphosphonium intermediate 10. Next, the nucleophilic attack of the amino group to the carbonyl gave the intermediate amide 11 and triphenylphosphine oxide. Then, the nucleophilic attack of the deprotonated sulfur atom led to the unstable cyclic intermediate 12. Next, the formation of compound 8 was a result of the elimination reaction, which led to the desired 2-homodrimenyl-1,3-benzothiazole 9 that by protonation gave carbocation 13. The latter suffered a rearrangement of the carbon skeleton as a result of the C₁₀-bonded methyl group migration to C₉, followed by both C₅ deprotonation and C₅-C₁₀ double bond formation.

Next, a series of new N-homodrimenoyl-2-amino-1,3-benzothiazoles were prepared, starting from the intermediate carboxylic acids 3, 5, 7, and 20, via their acyl chlorides 14, 16, 18, and 21, generated in situ in 25–65% yields. It should be mentioned that the acid 20 was obtained from the commercially available (+)-sclareolide (1) in 6 steps, with an overall yield of 60%, according to the known method [11]. The desired N-substituted 2-amino-1,3-benzothiazoles 15, 17, 19, and 22 were obtained with yields between 40–84% by acylation of 2-amino-1,3-benzothiazole with the mentioned sesquiterpene acyl chlorides under the mentioned conditions (Scheme 3).

According to the NMR spectra, the hybrids involved both heterocyclic and terpene units, and their accurate masses were confirmed by a high-resolution mass spectrometry (HRMS) analysis. All proton spectra of compounds 15, 17, 19, and 22, include the signals of aromatic protons in a range of 7.30–7.82 ppm, together with the signals specific for terpene unit such as singlets of C₈-bonded methyl groups at 1.69–1.82 ppm and C₇-bonded methoxy group at 3.40 ppm, C₆- and C₇-bonded protons at 3.50, 5.93, and 5.94 ppm, singlets of C₁₇-exomethylene group at 4.39 and 4.73 ppm, and broad singlets of amidic protons in a range of 9.73–11.26 ppm. The structures of the reported N-substituted 2-amino-1,3-benzothiazoles were additionally confirmed by the ¹³C NMR spectra.

Then, effort was devoted to prepare 2-substituted 6-methyl-benzothiazoles starting from the carboxylic acids 3 and 5, as well as, from acyl chlorides 14, 16, 18 using p-toluidine. The one-pot condensation of homodrimane acyl chlorides 14, 16, and 18, generated in situ from acids 3, 5 and 7 (see Scheme 3) with p-toluidine, yielded amides 23, 26, and 28 in 50–54% yields (Scheme 4).

An attempt to perform the direct amidation of acids 3 and 5 with p-toluidine in the presence of N,N′-dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (4-DMAP) gave better results, because the yields of amides 26 and 28 increased up to 76% and 94%, respectively (Scheme 4). Together with the signals of protons from terpene units, the proton spectra of amides 23, 26, and 28 contained the singlets of C_4′-bonded methyl in a range of 2.30–2.32 ppm and doublets of aromatic protons from 7.11 ppm to 7.37 ppm, and a broad singlet of the amidic proton at 7.53–7.70 ppm. The structures of the mentioned compounds were fully confirmed by the ¹³C NMR spectra.

After that, amides 23, 26, and 28 were submitted to the thionation reaction using Lawesson’s reagent (LR) in toluene [22]. In the case of amide 23, a reaction occurred, and thioamide 24 was obtained in 50% yield (Scheme 4). Its structure was confirmed by the NMR and HRMS analyses. In the ¹H and ¹³C NMR spectra, down field shifts of the amidic proton to 9.03 ppm and of C₁₂ to 200.9 ppm compared to the initial amide 23 were observed.

Heterocyclization of thioamide 24 performed in the presence of potassium ferricyanide under basic conditions (30% NaOH) [22] did not lead to the desired 2-substituted 6-methyl-benzothiazole and gave an unexpected compound 25 in 41% yield (Scheme 4). Its structure was elucidated based on the NMR and HRMS spectra. Comparing with the initial thioamide 24, its ¹H NMR spectra did not contain the signals of an amidic proton and of one of C₁₁-bonded proton, but the singlets of a C₈-bonded methyl group and one of C₁₁-H were shifted to 1.72 and 6.19 ppm, respectively. The same is true of the carbon spectrum, where some signals are strongly shifted, e.g., C₈ (62.9 ppm), C₁₁ (123.6 ppm), C₉ (170.4 ppm), and C₁₂ (176.6 ppm).

The analysis of the structure of compound 25 by ¹H, ¹³C, ¹H/¹H COSY and ¹H/¹³C HSQC NMR spectra suggested the presence of two isolated spin systems: CH₂CH₂CH₂ (C₁ to C₃) and CHCH₂CH₂ (C₅ to C₇) (Figure 2). In the ¹H/¹³C HMBC spectrum correlations of H-C₁₁ with quaternary carbons (C₉, δ_C 170.4 and C₁₂, δ_C 176.6) were observed, which was also supported by the correlations of H-C₁₁/C₈. In addition, the correlation of H3-C₁₇/C₁₂ confirms the formation of the 5-membered heterocycle.

In the NOESY spectrum of compound 25, there is no NOE correlation between C₁₁-H and C_2′-H from the aromatic ring (Figure 2) that clearly indicates the E-configuration for C₁₂ = N- double bond.

The thionation of amide 26 under the same conditions occurred and gave an unexpected cyclic thioamide 27 in 52% yield. The formation of the N-C₈ bond was confirmed by the absence of an amidic proton signal, a shift of the singlet signal of C₈-CH₃ to 1.56 ppm and by the appearance of the C₉-H doublet at 1.88 ppm. The upfield chemical shift of C₈ and C₉ atoms to 59.2 and 58.2 ppm and a downfield shifted signal of C₁₂ (175.9 ppm) in the ¹³C NMR spectra also confirmed the structure of compound 27.

Structural analysis of compound 27 by ¹H, ¹³C, ¹H/¹H COSY and ¹H/¹³C HSQC NMR spectra suggested the presence of two isolated spin systems: CH₂CH₂CH₂ (C₁ to C₃) and CH₂CHCH (C₅ to C₇) (Figure 3). The rearranged carbon framework of compound 27 was indisputable by a detailed analysis of its ¹H/¹³C HMBC spectrum. Thus, the observed correlations of H-C₅ with two sp² hybridized carbons (C₆, δ_C 126.8 and C₇, δ_C 130.6) were indicative of the Δ^6,7 double bond localization, which was also supported by the correlations of H3-C₁₇/C₇. The relative configuration at C₁₇ was deduced from the absence of H3-C₁₇/H3-C₁₀ NOESY correlation. The position of N has been confirmed by the ¹H/¹⁵N HMBC spectra and was supported by the correlations of H2-C₁₁/N and H-C_2′/N cross-peaks (Figure 3).

However, in the case of amide 28, from the thionation reaction mixture, the same amides 26 and 27 were isolated in 37% and 21% yields, respectively (Scheme 4). Their spectral data were in accordance with those obtained earlier.

Returning to the compound 25, it can be said that its formation in place of the desired benzothiazole is due to the reaction conditions and the molecular structure of thioamide 24 which permits the existence of a tautomeric thioketo-enothiol 24<—>29 system. In the basic medium, the tautomer 29 easily generated enothiolate which due to the activation by hexacyanofferate ions attacked the C₈-C₉ double bond and generated the intermediate carbocation 30 (Scheme 5). In such a way, the formation the new C₈-S bond occurred simultaneously with the one C₁₁-proton elimination giving compound 25.

The formation of compound 27 can be explained by a sequence of transformations depicted in Scheme 6. In this case, the Lawesson’s reagent played a double role. The first role is to interact with axial the C₇–bonded methoxy group of the amide 28, stimulating its elimination and generating the carbocation 32 which by deprotonation offered amide 26 (see Scheme 4 and Scheme 6). On the other hand, the thionation with Lawesson’s reagent gave an intermediate carbocation 33 which suffered a cyclization followed by deprotonation into cyclic thioamide 27. Note that there are several resonance structures, but from our point of view, intermediates 32 and 33 are more stable.

2.2. Antimicrobial Activity

All synthesized compounds were subjected to preliminary screening for their in vitro antifungal and antibacterial activities [23] against pure cultures of fungal species Aspergillus niger, Fusarium solani, Penicillium chrysogenum, Penicillium frequentans, and Alternaria alternata and both Gram-positive Bacillus sp. and Gram-negative Pseudomonas aeruginosa bacteria strains. The obtained minimum inhibitory concentration (MIC) values revealed that compounds 4 and 17 possessed a high nonselective antifungal (MIC 0.094 and 0.25 µg/mL, respectively) activity (

Table 1. In vitro antifungal and antibacterial activities of compounds 4, 6, 8, 9, 15, 17, 19, 22–28.

Entry	Compound	MIC (μg/mL)
		Aspergillus niger	Fusarium solani	Penicillium chrysogenum	Penicillium frequentans	Alternaria alternata	Bacillus sp.	Pseudomonas aeruginosa
1	4	0.094 *	0.094 *	0.094 *	0.094 *	0.094 *	0.75 *	0.75 *
2	6	2 **	2 **	2 **	2 **	2 **	32 ***	32 ***
3	8	1.5	1.5	1.5	1.5	1.5	2.75	2.75
4	9	1	1	1	1	1	2.5	2.5
5	15	32 *4	32 *4	32 *4	32 *4	32 *4	192 *	192 *
6	17	0.25 *5	0.25 *5	0.25 *5	0.25 *5	0.25 *5	3 *6	3 *6
7	19	>32	>32	>32	>32	>32	>256	>256
8	22	>32	>32	>32	>32	>32	>256	>256
9	23	>32	>32	>32	>32	>32	>256	>256
10	24	16 *7	16 *7	16 *7	16 *7	16 *7	128 *8	128 *8
11	25	0.95	0.95	0.95	0.95	0.95	1.5	1.5
12	26	>32	>32	>32	>32	>32	>256	>256
13	27	1 *9	1 *9	1 *9	1 *9	1 *9	24 *10	24 *10
14	28	>32	>32	>32	>32	>32	>256	>256
15	Caspofungin	1.5	1.5	1.5	1.5	1.5	-	-
16	Kanamycin	-	-	-	-	-	3	3

Standard deviation (mean of three measurements ± SD). * SD ± 0.002 μg/mL; ** SD ± 0.05 μg/mL; *** SD ± 0.028 μg/mL; *⁴ SD ± 0.001 μg/mL; *⁵ SD ± 0.019 μg/mL; *⁶ SD ± 0.012 μg/mL; *⁷ SD ± 0.09 μg/mL; *⁸ SD ± 0.027 μg/mL; *⁹ SD ± 0.085 μg/mL; *¹⁰ SD ± 0.060 μg/mL.

, entries 1 and 6) in comparison with caspofungin. Moreover, compounds 6, 8, 9, 25, and 27 possessed a promising antifungal activity (Table 1, entries 2–4, 11 and 13) at MIC in a range from 0.95 to 2 µg/mL, vs. the same standard. At the same time, compounds 4 and 25 possessed high nonselective antibacterial (MIC 0.75 and 1.5 µg/mL, respectively) activities (Table 1, entries 1 and 11) relative to the standard kanamycin. Compounds 8, 9, and 17 possessed a moderate antibacterial activity (Table 1, entries 3, 4, and 11). As to compounds 15, 19, 22, 23, 24, 26, and 28, they were biologically inactive.

In conclusion, it can be mentioned that the greatest antimicrobial activity was presented by homodrimane sesquiterpenoids bearing benzothiazole units, as well as those containing the NCS fragment, rigidly bound in space with the involvement of another ring, which sterically creates a stable bond similar to that in benzothiazole.

3. Materials and Methods

3.1. Synthesis and Characterization

The IR spectra were recorded on a Spectrum 100 FT-IR spectrometer (Perkin-Elmer, Shelton, CT, USA) using an ATR technique. The ¹H, ¹³C, and ¹⁵N NMR (400, 100, and 40 MHz, respectively) and COSY, ¹H–¹³C HSQC, ¹H–¹³C HMBC, DEPT, and ¹H–¹⁵N HSQC, ¹H–¹⁵N HMBC spectra were acquired on a Bruker Avance DRX 400 spectrometer (Bruker BioSpin, Rheinstetten, Germany) in CDCl₃ (NMR spectra for all the compounds are available online, see the Supplementary Materials). The ¹H NMR chemical shifts were reported relative to the residual solvent protons as internal standards (7.26 ppm). The solvent carbon atoms served as internal standard for the ¹³C NMR spectra (77.0 ppm). The ¹⁵N NMR spectra were obtained using MeNO₂ (380.5 ppm) and urea (73.4 ppm) as internal standards. Optical rotations measurements were performed on a Jasco DIP-370 polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA) with a 10 cm microcell. Melting points were determined on a Boetius (VEB Analytik, DDR) hot stage apparatus and were not uncorrected. The progress of reactions and purity of products were examined by TLC on Merck silica gel 60 plates, eluent CH₂Cl₂ or a mixture of CH₂Cl₂–MeOH, 99:1; 49:1. Visualization was achieved by the treatment with conc. H₂SO₄ and heating at 80 °C or using an UV lamp (254 or 365 nm). All solvents were purified and dried by standard techniques prior to use.

Compound 3: Compound 2 (294 mg, 1 mmol) was dissolved in EtOH (10 mL) and solid KOH (615 mg, 11 mmol) was added. The resulting mixture was heated at 50 °C for 3 h, and then, 2/3 of alcohol was distilled under reduced pressure on a rotary evaporator. The residue was diluted with H₂O (20 mL), acidified with 40% HCl (20 mL) and extracted with Et₂O (3 × 20 mL). The organic layer was washed with H₂O (30 mL), dried over anhydrous Na₂SO₄ and concentrated, giving compound 3 (249 mg, 89% yield) as a colorless oil. ${\left[\alpha \right]}_{\mathrm{D}}^{20}$ +50.6 (c 2.4, CHCl₃). IR spectrum, ν, cm⁻¹: 736, 1070, 1376, 1459, 1626, 1704, 2927. ¹H NMR (400 MHz, CDCl₃) δ 0.82 (3H, s, 10-CH₃), 0.87 (3H, s, 4-CH₃), 0.88 (3H, s, 4-CH₃), 1.09–1.15 (2H, m, CH₂), 1.16–1.19 (1H, m, CH₂), 1.35–1.58 (5H, m, H-5, 2CH₂), 1.64 (3H, s, 8-CH₃), 1.91–1.94 (1H, m, CH₂), 3.01 (2H, d, J = 7.0 Hz, H-11), 3.32 (3H, s, 7-CH₃), 3.43 (1H, d, J = 2.6 Hz, H-7). ¹³C NMR (100 MHz, CDCl₃) δ 17.7 (C-17), 17.9 (C-20), 18.7 (C-2), 21.5 (C-18), 22.5 (C-6), 32.7 (C-19), 32.8 (C-4), 33.0 (C-11), 35.9 (C-1), 39.3 (C-10), 41.1 (C-3), 45.7 (7-OCH₃), 56.4 (C-5), 79.4 (C-7), 130.0 (C-8), 139.1 (C-9), 171.9 (C-12). HRMS (ESI) calculated for C₁₇H₂₈O₃ [M + H]⁺, 280.4035. Found: 280.4127.

Compounds 4, 6, 8, and 9 (General method).

To an ice bath-cooled solution of Ph₃P (786 mg, 3 mmol) and Et₃N (0.16 mL, 1.2 mmol) dissolved in CCl₄ (7 mL), one of the acids 3 (280 mg, 1 mmol), 5 (248 mg, 1 mmol) or 7 (250 mg, 1 mmol) was added. After 10 min of stirring, the solution of 2-aminothiophenol (150 mg, 1.2 mmol) dissolved in CCl₄ (3 mL) was added and the reaction mixture was refluxed under stirring for 4 h. The solvents were removed under a reduced pressure on a rotary evaporator to dryness and crude reaction products were subjected to silica gel flash chromatography (CH₂Cl₂).

Compound 4. (180 mg, 49%), colorless oil. ${\left[\alpha \right]}_{\mathrm{D}}^{20}$ 78.3 (c 0.6, CHCl₃). IR spectrum, ν, cm⁻¹: 729, 758, 1080, 1373, 1437, 1456, 1509, 1707, 1759, 2926. ¹H NMR (400 MHz, CDCl₃) δ 0.86 (3H, s, 10-CH₃), 0.93 (3H, s, 4-CH₃), 0.99 (3H, s, 4-CH₃), 1.09–1.16 (2H, m, CH₂), 1.23–1.27 (1H, m, CH₂), 1.35–1.40 (2H, m, CH₂), 1.51–1.55 (3H, m, H-5, CH₂), 1.83 (3H, s, 8-CH₃), 2.01–2.05 (1H, m, CH₂), 3.43 (3H, s, 7-CH₃), 3.51 (1H, t, J = 2.7 Hz, 7-CH), 3.88 (2H, t, J = 17.4 Hz, H-11), 7.31 (1H, dt, J = 7.5, 1.0 Hz, H-6′), 7.43 (1H, dt, J = 7.7, 1.0 Hz, H-7′), 7.78 (1H, d, J = 7.8 Hz, H-5′), 7.93 (1H, d, J = 8.0 Hz, C-8′). ¹³C NMR (100 MHz, CDCl₃) δ 18.6 (C-20 and C-17), 18.7 (C-2), 21.7 (C-18), 22.7 (C-6), 32.8 (C-19), 32.8 (C-11), 32.9 (C-4), 36.0 (C-1), 39.9 (C-10), 41.2 (C-3), 46.0 (7-OCH₃), 56.9 (C-5), 79.3 (C-7), 121.5 (C-5′), 122.3 (C-8′), 124.4 (C-6′), 125.7 (C-7′), 131.1 (C-8), 135.1 (C-9), 142.5 (C-4′), 153.4 (C-9′), 173.6 (C-2′). ¹⁵N NMR (400 MHz, CDCl₃) δ 301. HRMS (ESI) calculated for C₂₃H₃₁NOS [M + H]⁺, 369.5667. Found: 369.5693.

Compound 6. (185 mg, 55%), yellow oil. ${\left[\alpha \right]}_{\mathrm{D}}^{20}$ −260.94 (c 0.59, CHCl₃). IR spectrum, ν, cm⁻¹: 729, 757, 1369, 1456, 1508, 1726, 2924. ¹H NMR (400 MHz, CDCl₃) δ 0.88 (3H, s, 10-CH₃), 0.95 (3H, s, 4-CH₃), 0.96 (3H, s, 4-CH₃), 1.10–1.82 (6H, m, 3CH₂), 1.86 (3H, s, 8-CH₃), 2.15 (1H, t, J = 2.9 Hz, H-5), 3.85 (1H, d, J = 16.7 Hz, H-11), 3.96 (1H, d, J = 16.7 Hz, H-11), 5.88 (1H, dd, J = 9.5 Hz, J = 2.5 Hz, H-6), 5.95 (1H, dd, J = 9.5, 3.0 Hz, H-7); 7.33 (1H, ddd, J = 7.5, 7.2 Hz, J = 1.1 Hz, H-6′), 7.44 (1H, ddd, J = 8.1, 7.2, 1.2 Hz, H-7′), 7.82 (1H, dm, J = 8.3 Hz, H-5′), 7.96 (1H, dm, J = 8.1 Hz, H-8′). ¹³C NMR (100 MHz, CDCl₃) δ 15.5 (C-20), 18.5 (C-8), 18.8 (C-2), 22.7 (C-18), 32.1 (C-11), 32.3 (C-19), 32.9 (C-4), 35.1 (C-1), 39.2 (C-10), 40.8 (C-3), 52.9 (C-5), 121.4 (C-5′), 122.4 (C-8′), 124.5 (C-6′), 125.7 (C-7′), 128.7 (C-6), 129.1 (C-7), 135.4 (C-8), 139.9 (C-9), 139.9 (C-9′), 153.6 (C-4′), 174.0 (C-2′). ¹⁵N NMR (400 MHz, CDCl₃) δ 303. HRMS (ESI) calculated for C₂₂H₂₇NS [M + H]⁺, 337.1864. Found: 337.1949.

Compound 8. (92 mg, 27%), mp 58–59 °C, ${\left[\alpha \right]}_{\mathrm{D}}^{20}$ 7.63 (c 3.2, CHCl₃). IR spectrum, ν, cm⁻¹: 737, 763, 1122, 1311, 1377, 1433, 1454, 1505, 1713, 2920, 3059. ¹H NMR (400 MHz, CDCl₃) δ 0.92 (3H, d, J = 6.8 Hz, 8-CH₃), 1.01 (3H, s, 4-CH₃), 1.02 (3H, s, 4-CH₃), 1.05 (3H, s, 9-CH₃), 1.30–1.40 (2H, m, CH₂), 1.46–1.63 (5H, m, H-8, 2CH₂), 1.87–2.04 (2H, m, H-6), 2.17–2.22 (2H, m, CH₂), 3.27 (2H, d, J = 3.9 Hz, H-11), 7.32 (1H, td, J = 7.8, 1.2 Hz, H-6′), 7.42 (1H, td, J = 7.0, 1.2 Hz, H-7′), 7.83 (1H, dm, J = 8.0 Hz, H-5′), 7.96 (1H, d, J = 8.0 Hz, H-8′). ¹³C NMR (100 MHz, CDCl₃) δ 16.2 (C-17), 20.1 (C-2), 21.4 (C-20), 24.9 (C-6), 26.6 (C-1), 27.0 (C-7), 27.8 (C-18), 28.3 (C-19), 34.5 (C-8); 34.7 (C-4), 39.6 (C-3), 42.1 (C-11, C-9), 121.3 (C-5′), 122.5 (C-8′), 124.5 (C-6′), 125.6 (C-7′), 130.8 (C-10), 135.8 (C-9′), 139.4 (C-5), 152.1 (C-4′), 169.5 (C-2′). ¹⁵N NMR (400 MHz, CDCl₃) δ 305. HRMS (ESI) calculated for C₂₂H₂₉NS [M + H]⁺, 339.2021. Found: 339.2106.

Compound 9. (17 mg, 5%), yellow oil. ${\left[\alpha \right]}_{\mathrm{D}}^{20}$ +28.62 (c 0.2, CHCl₃). IR spectrum, ν, cm⁻¹: 729, 757, 1014, 1124, 1146, 1293, 1376, 1435, 1456, 1506, 1577, 1694, 2865, 2926. ¹H NMR (400 MHz, CDCl₃) δ 0.84 (3H, s, 4-CH₃), 0.90 (3H, s, 4-CH₃), 1.02 (3H, s, 10-CH₃), 1.05–1.11 (2H, m, CH₂), 1.25–1.40 (3H, m, H-5, CH₂), 1.45–1.60 (4H, m, 2CH₂), 1.70 (3H, s, 8-CH₃), 2.09–2.21 (2H, m, H-7), 3.83 (1H, d, J = 17.2 Hz, H-11), 3.90 (1H, d, J = 17.2 Hz, H-11), 7.32 (1H, ddd, J = 7.9 Hz, J = 7.3, 1.1 Hz, H-6′), 7.43 (1H, ddd, J = 8.2, 7.3, 1.3 Hz, H-7′), 7.81 (1H, ddd, J = 7.9, 1.3, 0.5 Hz, H-5′), 7.94 (1H, d, J = 8.2 Hz, H-8′). ¹³C NMR (100 MHz, CDCl₃) δ 18.8 (C-6), 18.9 (C-2), 20.3 (C-20), 20.7 (C-17), 21.6 (C-18), 32.8 (C-11), 33.2 (C-19), 33.3 (C-4); 33.6 (C-7), 36.6 (C-1), 39.1 (C-10), 41.6 (C-3), 51.8 (C-5), 121.3 (C-5′), 122.4 (C-8′), 124.3 (C-6′), 125.6 (C-7′), 131.2 (C-8), 135.4 (C-9′), 137.7 (C-9), 153.6 (C-4′), 175.4 (C-2′). ¹⁵N NMR (400 MHz, CDCl₃) δ 299. HRMS (ESI) calculated for C₂₂H₂₉NS [M + H]⁺, 339.2021. Found: 339.2107.

Compounds 15, 17, 19, and 22 (General method).

The solution of one of the acids 3 (280 mg, 1 mmol), 5 (248 mg, 1 mmol), 7 (250 mg, 1 mmol) or 20 (250 mg, 1 mmol) dissolved in anhydrous C₆H₆ (5 mL) was treated with a solution of (COCl)₂ (0.95 mL, 11 mmol) dissolved in C₆H₆ (2.5 mL). The reaction mixture was stirred at room temperature for 1 h and subsequently refluxed for 1 h. The C₆H₆ and excess of (COCl)₂ were removed at a reduced pressure on a rotary evaporator. Next, 2-aminobenzothiazole (225 mg, 1.5 mmol) was added to the solution of an acyl chloride 14, 16, 18 or 21 in CH₂Cl₂ (10 mL), and the resulting mixtures were stirred at r.t. for 3 h, then refluxed for 4–10 h. After cooling, the precipitates were filtered off, washed with CH₂Cl₂, and the filtrates were concentrated to dryness at a reduced pressure on a rotary evaporator. The crude reaction products were purified by silica gel flash chromatography (1 → 2% MeOH-CH₂Cl₂).

Compound 15. (164 mg, 40%), white crystals, mp 93–94 °C. ${\left[\alpha \right]}_{\mathrm{D}}^{20}$ 99.96 (c 1.4, CHCl₃). IR spectrum, ν, cm⁻¹: 728, 755, 1076, 1341, 1264, 1441, 1538, 1600, 1697, 2926, 3182. ¹H NMR (400 MHz, CDCl₃) δ 0.85 (3H, s, 10-CH₃), 0.91 (3H, s, 4-CH₃), 0.93 (3H, s, 4-CH₃); 1.09–1.20 (2H, m, CH₂), 1.37–1.58 (5H, m, H-5, 2CH₂), 1.62–1.66 (1H, m, CH₂), 1.77 (3H, s, 8-CH₃), 2.01 (1H, d, J = 13.0 Hz, CH₂), 3.22 (1H, d, J = 17.4 Hz, H-11), 3.32 (1H, d, J = 17.4 Hz, H-11), 3.40 (3H, s, 7-CH₃), 3.50 (1H, d, J = 5.6 Hz, H-7), 7.30 (1H, dt, J = 7.5, 0.9 Hz, H-6′), 7.42 (1H, dt, J = 7.6, 1.0 Hz, H-7′), 7.77 (1H, d, J = 8.0 Hz, H-5′), 7.80 (1H, d, J = 7.7 Hz, H-8′), 9.91 (1H, br.s, NH). ¹³C NMR (100 MHz, CDCl₃) δ 18.2 (C-20), 18.3 (C-17), 18.7 (C-2), 21.6 (C-18), 22.4 (C-6), 32.7 (C-19), 32.8 (C-4), 35.7 (C-11), 35.8 (C-1), 39.6 (C-10), 40.9 (C-3), 45.5 (7-CH₃), 56.8 (C-5), 78.9 (C-7), 120.8 (C-5′), 121.3 (C-8′), 123.9 (C-6′), 126.2 (C-7′), 132.1 (C-4′), 132.7 (C-8), 138.9 (C-9), 148.1 (C-9′), 158.2 (C-2′), 169.6 (C = O). ¹⁵N NMR (400 MHz, CDCl₃) δ 140, 259. HRMS (ESI) calculated for C₂₄H₃₂N₂O₂S [M-H]^-, 412.2184. Found: 412.2112.

Compound 17. (23 mg, 52%), white crystals, mp 83–84 °C. ${\left[\alpha \right]}_{\mathrm{D}}^{20}$ −172.5 (c 0.1, CHCl₃). IR spectrum, ν, cm⁻¹: 728, 755, 908, 1147, 1267, 1343, 1442, 1536, 1599, 1702, 2925, 3178. ¹H NMR (400 MHz, CDCl₃) δ 0.84 (3H, s, 10-CH₃), 0.96 (6H, s, 4-CH₃, 4-CH₃), 1.15–1.80 (6H, m, 3CH₂), 1.83 (3H, s, 8-CH₃), 2.10 (1H, t, J = 2.2 Hz, H-5), 3.21 (1H, d, J = 17.3 Hz, H-11), 3.42 (1H, d, J = 17.0 Hz, H-11), 5.93 (1H, d, J = 1.8 Hz, H-6), 5.94 (1H, d, J = 2.2 Hz, H-6), 7.31 (1H, dt, J = 1.0, 0.8 Hz, H-6′), 7.44 (1H, dt, J = 1.2, 1.0 Hz, H-7′), 7.75 (1H, d, J = 8.2 Hz, H-5′), 7.82 (1H, dd, J = 7.87, 0.47 Hz, H-8′), 9.74 (1H, br.s, NH). ¹³C NMR (100 MHz, CDCl₃) δ 15.1 (C-20), 18. (C-17), 18.7 (C-2), 22.8 (C-18), 32.2 (C-19), 33.0 (C-4), 35.0 (C-1), 35.3 (C-11), 39.1 (C-10), 40.6 (C-3), 52.9 (C-5), 120.7 (C-5′), 121.4 (C-8′), 124.0 (C-6′), 126.3 (C-7′), 128.8 (C-6), 129.6 (C-7), 130.9 (C-9), 132.1 (C-4′), 135.7 (C-8), 148.0 (C-9′), 157.9 (C-2′), 169.9 (C-12). ¹⁵N NMR (400 MHz, CDCl₃) δ 139, 254. HRMS (ESI) calculated for C₂₃H₂₈N₂OS [M + H]⁺, 380.1922. Found: 380.2011.

Compound 19. (171 mg, 45%), white crystals, mp 84–85 °C. ${\left[\alpha \right]}_{\mathrm{D}}^{20}$ +102.5 (c 2.0, CHCl₃). IR spectrum, ν, cm⁻¹: 727, 755, 883, 907, 975, 1018, 1152, 1267, 1334, 1379, 1443, 1533, 1600, 1704, 1773, 2927, 3175, 3365. ¹H NMR (400 MHz, CDCl₃) δ 0.80 (3H, s, 4-CH₃), 0.84 (3H, s, 4-CH₃), 0.88 (3H, s, 10-CH₃), 0.95–1.18 (4H, m, 2CH₂), 1.27 (1H, dd, J = 12.6 Hz, J = 1.9 Hz, H-5), 1.33–1.57 (4H, m, 2CH₂), 1.67 (3H, s, 8-CH₃), 2.09 (1H, dd, J = 18.0 Hz, J = 6.4 Hz, H-7), 2.27 (1H, ddd, J = 18.4 Hz, J = 11.2 Hz, J = 7.4 Hz, H-7), 3.12 (1H, d, J = 17.6 Hz, H-11), 3.33 (1H, d, J = 17.6 Hz, H-11), 7.30 (1H, ddd, J = 8.0 Hz, J = 7.3 Hz, J = 1.0 Hz, H-7′), 7.42 (1H, ddd, J = 8.0 Hz, J = 7.3 Hz, J = 1.2 Hz, H-6′), 7.79 (1H, d, J = 8.0 Hz, H-5′), 7.81 (1H, ddd, J = 8.0 Hz, J = 1.2 Hz, J = 0.6 Hz, H-8′), 9.73 (1H, br.s., NH). ¹³C NMR (100 MHz, CDCl₃) δ 18.7 (C-6), 18.8 (C-2), 19.7 (C-20), 20.4 (C-17), 21.6 (C-18), 33.1 (C-19), 33.3 (C-4), 33.5 (C-7), 35.9 (C-11), 36.3 (C-1), 38.8 (C-10), 41.3 (C-3), 51.5 (C-5), 120.7 (C-5′), 121.5 (C-8′), 124.0 (C-7′), 126.3 (C-6′), 132.1 (C-9′), 133.0 (C-9), 134.2 (C-8), 148.0 (C-4′), 158.4 (C-2′), 170.4 (C-12). ¹⁵N NMR (400 MHz, CDCl₃) δ 137, 257. HRMS (ESI) calculated for C₂₃H₃₀N₂OS [M + H]⁺, 382.2079. Found: 382.2165.

Compound 22. (320 mg, 84%), white crystals, mp 99–100 °C. ${\left[\alpha \right]}_{\mathrm{D}}^{20}$ −34.2 (c 2.0, CHCl₃). IR spectrum, ν, cm⁻¹: 750, 884, 998, 1018, 1135, 1157, 1215, 1270, 1292, 1324, 1332, 1365, 1383, 1443, 1457, 1542, 1599, 1644, 1697, 2932, 3063, 3178. ¹H NMR (400 MHz, CDCl₃) δ 0.46 (3H, s, 10-CH₃), 0.76 (3H, s, 4-CH₃), 0.84 (3H, s, 4-CH₃), 0.99–1.18 (3H, m, H-5, CH₂), 1.25 (1H, dd, J = 13.0, 4.4 Hz, H-6), 1.30–1.54 (4H, m, 2CH₂), 1.68 (1H, dm, J = 13.0 Hz, H-6), 2.05 (1H, td, J = 13.0, 5.1 Hz, H-7), 2.33 (1H, ddd, J = 13.0, 4.0, 2.1 Hz, H-7), 2.45 (1H, dd, J = 11.2, 10.3 Hz, H-9), 2.47 (1H, dd, J = 6.2, 10.3 Hz, H-11), 2.63 (1H, dd, J = 26.2, 11.2 Hz, H-11), 4.39 (1H, s, 8-CH₂), 4.73 (1H, s, 8-CH₂), 7.33 (1H, ddd, J = 8.1, 7.1, 1.0 Hz, H-7′), 7.44 (1H, ddd, J = 8.2, 7.1, Hz, 1.1 Hz, H-6′), 7.81 (1H, dd, J = 8.2, 1.0 Hz, H-5′), 7.84 (1H, dd, J = 8.1, 1.1 Hz, H-8′), 11.26 (1H, br.s., NH). ¹³C NMR (100 MHz, CDCl₃) δ 14.3 (C-20), 19.2 (C-2); 21.6 (C-18), 23.9 (C-6), 32.8 (C-11), 33.4 (C-4), 33.5 (C-19), 37.5 (C-7), 38.9 (C-1), 39.0 (C-10), 41.8 (C-3), 52.2 (C-9), 55.0 (C-5), 106.5 (8-CH₂), 120.6 (C-5′), 121.7 (C-8′), 123.9 (C-7′), 126.3 (C-6′), 132.1 (C-9′), 147.8 (C-4′), 148.7 (C-8), 159.6 (C-2′), 171.8 (C-12). ¹⁵N NMR (400 MHz, CDCl₃) δ 251 (C = N). HRMS (ESI) calculated for C₂₃H₃₀N₂OS [M + H]⁺, 382.2079. Found: 382.2166.

Compounds 23, 26, and 28. (Typical procedure).

Method 1. The solution of one of the acids 3 (280 mg, 1 mmol), 5 (248 mg, 1 mmol) or 7 (250 mg, 1 mmol) dissolved in anhydrous C₆H₆ (5 mL) was treated with a solution of (COCl)₂ (0.95 mL, 11 mmol) dissolved in C₆H₆ (2.5 mL). The reaction mixture was stirred at room temperature for 1 h, and additionally refluxed for 1 h. The C₆H₆ and excess of (COCl)₂ were removed at reduced pressure on a rotary evaporator. Next, p-toluidine (160 mg, 1.5 mmol) was added to the solutions of acyl chlorides 14, 16 or 18 obtained in situ in CH₂Cl₂ (10 mL), and the resulting mixtures were stirred at r.t. for 5 h, and refluxed for 10–12 h. After cooling, the precipitate was filtered off, washed with CH₂Cl₂, and the filtrate was concentrated to dryness on a rotary evaporator. The crude reaction products were purified by silica gel flash chromatography (1→2% MeOH-CH₂Cl₂) to give products 23, 26, and 28.

Method 2. A solution of DCC (412 mg, 2 mmol), 4-DMAP (244 mg, 2 mmol), p-toluidine (214 mg, 2 mmol) and an acid 3 (280 mg, 1 mmol) or 5 (248 mg, 1 mmol) dissolved in CH₂Cl₂ (8 mL) was stirred for 10 h at room temperature. After the reaction period, the mixture was filtered, and the solvent was removed under a reduced pressure on a rotary evaporator to give the crude product which was purified by silica gel flash chromatography (CH₂Cl₂) to give compounds 26 and 28.

Compound 23. (183 mg, 54%), white crystals, mp 152–153 °C. ${\left[\alpha \right]}_{\mathrm{D}}^{20}$ +132.64 (c 1.0, CHCl₃). IR spectrum, ν, cm⁻¹: 733, 818, 908, 1171, 1248, 1346, 1405, 1458, 1516, 1603, 1661, 2867, 2927, 3293. ¹H NMR (400 MHz, CDCl₃) σσ 0.85 (3H, s, 4-CH₃), 0.92 (3H, s, 4-CH₃), 0.99 (3H, s, 10-CH₃), 1.04–1.10 (2H, m, CH₂), 1.17 (1H, dd, J = 12.6, 1.9 Hz, H-5), 1.40–1.62 (4H, m, 2CH₂), 1.68 (3H, s, 8-CH₃), 1.71–1.82 (2H, m, CH₂), 2.08–2.26 (2H, m, H-7), 2.31 (3H, s, 4′-CH₃), 3.04 (1H, d, J = 17.5 Hz, H-11), 3.22 (1H, d, J = 17.5 Hz, H-11), 7.12 (2H, d, J = 8.2 Hz, H-3′ and H-5′), 7.35 (2H, d, J = 8.3 Hz, H-2′ and H-6′), 7.53 (1H, br.s, NH). ¹³C NMR (100 MHz, CDCl₃) δ 18.8 (C-6), 18.9 (C-2), 20.1 (C-20), 20.3 (C-17), 21.7 (C-18), 29.9 (C-7′), 33.3 (C-19), 33.4 (C-4), 33.7 (C-7), 36δ.3 (C-1), 37.2 (C-11), 39.0 (C-10), 41.6 (C-3), 52.4 (C-5), 119.9 (C-2′ and C-6′), 129.5 (C-3′ and C-5′), 132.1 (C-8), 133.9 (C-4′), 135.3 (C-1′), 136.4 (C-9), 169.6 (C-12). ¹⁵N NMR (400 MHz, CDCl₃) δ 127. HRMS (ESI) calculated for C₂₃H₃₃NO [M + H]⁺, 339.2562. Found: 339.2649.

Compound 26. (256 mg, 76%), white crystals, mp 69–70 °C, ${\left[\alpha \right]}_{\mathrm{D}}^{20}$ −155.56 (c 0.69, CHCl₃). IR spectrum, ν, cm⁻¹: 817, 1177, 1607, 1245, 1351, 1454, 1513, 1542, 1653, 2927, 3292. ¹H NMR (400 MHz, CDCl₃) δ 0.87 (3H, s, 10-CH₃), 0.97 (3H, s, 4-CH₃), 0.99 (3H, s, 4-CH₃), 1.10–1.62 (6H, m, 3CH₂), 1.83 (3H, s, 8-CH₃), 2.04 (1H, t, J = 2.5 Hz, H-5), 2.32 (3H, s, 4′-CH₃), 3.08 (1H, d, J = 16.9 Hz, H-11), 3.31 (1H, d, J = 16.9 Hz, H-11),5.92 (1H, dd, J = 9.4, 2.4 Hz, H-6), 5.97 (1H, dd, J = 9.5, 2.7 Hz, H-7), 7.14 (2H, d, J = 8.2 Hz, H-2′ and H-6′), 7.37 (2H, d, J = 8.4 Hz, H-3′ and H-5′), 7.66 (1H, s, NH). ¹³C NMR (100 MHz, CDCl₃) δ 15.0 (C-17), 18.4 (C-20), 18.7 (C-2); 20.8 (C-7′); 22.7 (C-18), 32.4 (C-19), 33.0 (C-4), 34.8 (C-1), 36.5 (C-11), 39.1 (C-10), 40.8 (C-3), 53.6 (C-5), 119.9 (C-2′ and C-6′), 128.9 (C-6), 129.4 (C-7), 129.7 (C-3′and C-5′), 129.9 (C-8), 135.1 (C-9), 138.1 (C-1′), 169.0 (C-12). ¹⁵N NMR (400 MHz, CDCl₃) δ 125. HRMS (ESI) calculated for C₂₃H₃₁NO [M + H]⁺, 337.2406. Found: 337.2492.

Compound 28. (346 mg, 94%), white solid, mp 187–188 °C, ${\left[\alpha \right]}_{\mathrm{D}}^{20}$ +70.2 (c 0.39, CHCl₃). IR spectrum, ν, cm⁻¹: 816, 1086, 1243, 1310, 1448, 1536, 1571, 1624, 2928, 3322. ¹H NMR (400 MHz, CDCl₃) δ 0.87 (3H, s, 10-CH₃), 0.93 (3H, s, 4-CH₃), 0.95 (3H, s, 4-CH₃), 1.10–1.19 (2H, m, CH₂), 1.39–1.60 (5H, m, H-5 and 2CH₂), 1.78 (3H, s, 8-CH₃), 2.00–2.04 (2H, m, CH₂), 2.30 (3H, s, 4′-CH₃), 3.05 (1H, d, J = 17.6 Hz, H-11), 3.22 (1H, d, J = 17.6 Hz, H-11), 3.39 (3H, s, 7-CH₃), 3.48 (1H, d, J = 2.6 Hz, H-7), 7.11 (2H, d, J = 8.2 Hz, H-2′ and H-6′), 7.35 (2H, d, J = 8.4 Hz, H-3′ and H-5′), 7.70 (1H, s, NH). ¹³C NMR (100 MHz, CDCl₃) δ 18.2 (C-20), 18.4 (C-2), 18.6 (C-17), 20.8 (C-7′), 21.6 (C-18), 22.4 (C-6), 32.8 (C-19), 32.9 (C-4), 35.4 (C-1), 37.0 (C-11), 39.7 (C-10), 41.2 (C-3), 45.9 (7-OCH₃), 56.8 (C-5), 79.0 (C-7), 120.1 (C-2′ and C-6′), 129.3 (C-3′ and 5′), 132.0 (C-8), 133.3 (C-4′), 135.1 (C-9), 140.8 (C-1′), 168.6 (C-12). ¹⁵N NMR (400 MHz, CDCl₃) δ 128. HRMS (ESI) calculated for C₂₄H₃₅NO₂ [M-31]⁺, 369.2668. Found: 338.2492.

Compounds 24 and 27. (Typical procedure)

To a solution of one of the amides 23 (339 mg, 1 mmol), 26 (337 mg, 1 mmol) or 28 (369 mg, 1 mmol) dissolved in toluene (8 mL), Lawesson’s reagent (203 mg, 0.5 mmol) was added and the reaction mixture was refluxed for 48–50 h. Then, the mixture was filtered, and the solvent was removed under a reduced pressure on a rotary evaporator to afford the crude reaction product, which was purified by silica gel flash column chromatography (1% MeOH-CH₂Cl₂).

Compound 24. (177 mg, 50%), white solid, mp 104–105 °C. ${\left[\alpha \right]}_{\mathrm{D}}^{20}$ +45.63 (c 0.5, CHCl₃). IR spectrum, ν, cm⁻¹: 730, 826, 852, 908, 998, 1056, 1066, 1267, 1395, 1406, 1453, 1516, 1599, 2052, 2214, 2972, 2987, 3147, 3246. ¹H NMR (400 MHz, CDCl₃) δ 0.86 (3H, s, 4-CH₃), 0.91 (3H, s, 4-CH₃), 1.00 (3H, s, 10-CH₃), 1.03–1.14 (2H, m, CH₂), 1.15 (1H, dd, J = 12.6, 2.0 Hz, H-5), 1.34–1.59 (4H, m, 2CH₂), 1.67 (3H, s, 8-CH₃), 1.72–1.88 (2H, m, CH₂), 2.11–2.23 (2H, m, H-7), 2.36 (3H, s, 4′-CH₃), 3.71 (2H, s, H-11), 7.22 (2H, d, J = 8.4 Hz, H-3′ and H-5′), 7.50 (2H, d, J = 8.4 Hz, H-2′ and H-6′), 9.03 (1H, s, NH). ¹³C NMR (100 MHz, CDCl₃) δ 18.7 (C-2), 18.8 (C-6), 20.1 (C-20), 20.2 (C-17), 21.1 (4′-CH₃), 21.6 (C-18), 33.2 (C-19), 33.4 (C-4), 33.6 (C-7), 36.2 (C-1), 39.2 (C-10), 41.6 (C-3), 47.8 (C-11), 52.5 (C-5), 123.8 (C-2′ and C-6′), 129.5 (C-3′ and C-5′), 134.2 (C-8), 136.2 (C-4′), 136.8 (C-9), 136.9 (C-1′), 200.9 (C = S). HRMS (ESI) calculated for C₂₃H₃₃NS [M + H]⁺, 355.2334. Found: 355.2419.

Compound 27. (176 mg, 52%), white solid, mp 103–105 °C. ${\left[\alpha \right]}_{\mathrm{D}}^{20}$ +155.73 (c 0.83, CHCl₃). IR spectrum, ν, cm⁻¹: 817, 1033, 1097, 1365, 1454, 1502, 1600, 1625, 2917. ¹H NMR (400 MHz, CDCl₃) δ 0.82 (3H, s, 4-CH₃), 0.87 (3H, s, 4-CH₃), 0.92 (3H, s, 10-CH₃), 1.08–1.40 (2H, m, CH₂), 1.46–1.55 (2H, m, CH₂), 1.56 (3H, s, 8-CH₃), 1.64–1.72 (2H, m, CH₂), 1.88 (1H, d, J = 8.2 Hz, H-9), 2.23 (3H, s, H-7′), 3.07 (1H, d, J = 17.5 Hz, H-11), 3.19 (1H, dd, J = 17.5, 8.2 Hz, H-11), 5.62 (1H, dd, J = 10.1, 2.1 Hz, H-6), 5.66 (1H, dd, J = 10.1, 1.2 Hz, H-7), 6.75 (2H, d, J = 8.0 Hz, H-2′ and H-6′), 7.11 (2H, d, J = 8.0 Hz, H-3′ and H-5′). ¹³C NMR (100 MHz, CDCl₃) δ 14.5 (C-20), 18.2 (C-2), 20.9 (C-7′), 21.6 (C-18), 32.5 (C-4), 33.9 (C-19), 33.9 (C-17), 37.6 (C-1), 37.7 (C-10), 40.7 (C-11), 40.9 (C-3), 51.9 (C-5), 58.2 (C-9), 59.2 (C-8), 119.9 (C-2′ and C-6′), 126.8 (C-6), 129.6 (C-3′ and C-5′), 130.6 (C-7), 133.4 (C-4′), 149.8 (C-1′), 175.9 (C-12). ¹⁵N NMR (400 MHz, CDCl₃) δ 293. HRMS (ESI) calculated for C₂₃H₃₁NS [M + H]⁺, 353.5677. Found: 353.5748.

Compound 25. To a solution of carbothioamide 24 (355 mg, 1 mmol) dissolved in EtOH (9 mL), 30% NaOH (1 mL, 7.9 mmol) was added. The mixture was diluted with EtOH (20 mL) to give 10% NaOH. Portions of this mixture were added to a stirred solution of K₃[Fe(CN)₆] (1.3 g, 3.9 mmol) in H₂O (2 mL) at 85 °C. The resultant mixture was further heated at 85 °C for 5 h and filtered to isolate a light yellow solid (720 mg) K₄[Fe(CN)₆]·3H₂O. Then, the solvent was removed in vacuo from the filtrate. To the residue, H₂O (20 mL) was added and the obtained mixture was extracted with CH₂Cl₂ (2 × 30 mL). The combined extracts were washed with H₂O (2 × 20 mL), dried over MgSO₄, and the solvent was removed to afford an orange oil. The crude reaction product was purified by flash column chromatography (SiO₂, elution CH₂Cl₂) to give compound 25.

Compound 25. (145 mg, 41%), yellow oil. ${\left[\alpha \right]}_{\mathrm{D}}^{20}$ −92.68 (c 2.0, CHCl₃). IR spectrum, ν, cm⁻¹: 730, 824, 851, 909, 1004, 1127, 1147, 1169, 1201, 1249, 1306, 1379, 1451, 1504, 1594, 1618, 2868, 2927. ¹H NMR (400 MHz, CDCl₃) δ 0.89 (3H, s, 4-CH₃), 0.91 (3H, s, 4-CH₃), 1.07 (1H, dd, J = 12.5 Hz, J = 2.5 Hz, H-5), 1.19–1.22 (1H, m, CH₂), 1.23 (3H, s, 10-CH₃), 1.42–1.50 (2H, m, CH₂), 1.58–1.65 (2H, m, CH₂), 1.72 (3H, s, 8-CH₃), 1.78–1.95 (4H, m, 2CH₂), 2.22–2.27 (1H, m, CH₂), 2.33 (3H, s, 4′-CH₃), 6.19 (1H, s, H-11), 6.98 (2H, d, J = 8.0 Hz, H-3′ and H-5′), 7.14 (2H, d, J = 8.0 Hz, H-2′ and H-6′). ¹³C NMR (100 MHz, CDCl₃) δ 18.6 (C-6); 19.6 (C-20), 19.9 (C-2), 21.0 (C-7′), 21.6 (C-18), 29.4 (C-17), 33.3 (C-19), 34.0 (C-4), 39.0 (C-1), 41.2 (C-10), 41.7 (C-3), 43.2 (C-7), 55.2 (C-5), 62.9 (C-8), 120.5 (C-2′ and C-6′), 123.6 (C-11), 129.6 (C-3′ and C-5′), 134.0 (C-4′), 148.9 (C-1′), 170.4 (C-9), 176.6 (C-12). ¹⁵N NMR (400 MHz, CDCl₃) δ 191. HRMS (ESI) calculated for C₂₃H₃₁NS [M + H]⁺, 353.5677. Found: 353.5725.

3.2. Antifungal and Antibacterial Activity Assay

Pure cultures of the fungi Aspergillus niger, Fusarium, Penicillium chrysogenum, Penicillium frequentans, and Alternaria alternata and bacteria Pseudomonas aeruginosa and Bacillus sp. were obtained from the American Type Culture Collection (ATCC). Suspensions of microorganisms in DMSO were prepared according to direct colony method and serial dilution procedure. Then, the final concentration of the stock inoculum was 1·10⁻⁴ μg/mL. Both antifungal and antibacterial activity assay were performed by applying a mixture of a microorganism suspension and a solution of the target compound in a ratio 1:1 to Petri dishes with a solid medium—Merck Sabouraud agar or agar-agar. The DMSO did not have any inhibitory effect on the tested organisms.

4. Conclusions

A series of 14 novel hybrid terpeno-heterocyclic compounds containing homodrimane and 2-substituted 1,3-thiadiazole, N-substituted 2-amino-1,3-benzothiazole and N-p-toluidyl units were designed, synthesized, and assessed as antimicrobial agents. Several of them showed higher antifungal and antibacterial activities than reference drugs.