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cyclization and their HIV-RT inhibitory activity
Carbohydrate Research 456 (2018) 45e52
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Synthesis of tetracyclic azasugars fused benzo[e][1,3]thiazin-4-one by the tandem Staudinger/aza-Wittig/cyclization and their HIV-RT inhibitory activity Jie Shao, Mo Zhu, Ligang Gao, Hua Chen**, Xiaoliu Li* Key Laboratory of Chemical Biology of Hebei Province, College of Chemistry and Environmental Science, Hebei University, Baoding 071002, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 November 2017 Received in revised form 12 December 2017 Accepted 12 December 2017 Available online 15 December 2017
Azasugar aldehydes 6a and 6b containing azido groups were prepared from D-mannose. Three novel tetracyclic azasugars fused benzo[e][1,3]thiazin-4-one 9a-1, 9a-2 and 9a-3 were conveniently synthesized from 6a by the tandem intramolecular Staudinger/aza-Wittig/cyclization reaction under microwave radiation. Two unexpected elimination compounds 8b-1 and 8b-2 were achieved as the main products from 6b in the same processes. The newly synthesized azasugars were examined for their HIV reverse transcriptase (RT) inhibitory activities. The results showed that all the tested compounds could effectively inhibit RT activity. Among them, compound 8b-1 with the protective group (isopropylidene group) was the best one with the IC50 value of 0.76 mM. The structure activity relationship analysis suggested that improvement of the molecular hydrophilicity might be beneficial for their anti-HIV RT activities. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Tetracyclic azasugar Benzthiazinan-4-one Staudinger/aza-Wittig/cyclization Anti-HIV RT activity
1. Introduction Iminosugars or azasugars, which exhibit effective inhibition against carbohydrate-processing enzymes, have been paid much attention due to their potential clinical applications as anti-HIV, anti-diabetic, and anti-cancer agents and immunomodulators in past decades [1e4]. The bicyclic azasugars fused different azoles (A and B, Fig. 1) [5e7] were synthesized and evaluated for their biological activities. The promising activities of the azasugars demanded to prepare their novel derivatives to meet the evergrowing requirements for the new drug discovery [8e10]. However, multicyclic (more than 3-fused rings) azasugars have received much less attention [11e13]. The tetracyclic azasugars were so far scarcely reported for their synthesis and biological activities. By using the microwave-assisted Staudinger/aza-Wittig/cyclization reaction, we have conveniently synthesized a series of novel bi- and tri-cyclic thiazolidin-4-one- and benzothiazin-4-one-fused azasugars (C-E, Fig. 1), which exhibit strong HIV reverse transcriptase (HIV-RT) inhibitory activity [14e18]. It was well known that most HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) generally showed butterfly-like conformation when they
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (H. Chen), [email protected] (X. Li). https://doi.org/10.1016/j.carres.2017.12.005 0008-6215/© 2017 Elsevier Ltd. All rights reserved.
were binding with HIV-RT [19e21]. Taking account this dominant conformation as determinant for anti-HIV RT activity, recently, the tetracyclic azasugar fused benzo[e][1,3]thiazin-4-one F [22] (Fig. 1) was rationally designed and was synthesized via the threecomponents condensation of the azasugar aldehyde, amino acid ester, and 2-mercaptobenzoic acid, then followed by the intramolecular cyclo-amidation reaction. Compound F was found to possess significant anti-HIV-RT activity with the IC50 value of 0.82 mM possibly due to its constrained butterfly-like conformation, just as the known NNRTIs G (MEN 10979) and H (Fig. 1) did [23,24]. However, the redundant synthesis of F limited its further application for the very low yield. As a continuation of our research, we would like herein to report the synthesis of novel tetracyclic azasugars fused benzo[e][1,3]thiazin-4-one by the tandem Staudinger/ Aza-Wittig/cyclization as an alternative protocol. Such newly synthetic azasugars were evaluated for their HIV-RT inhibitory activities in order to further investigate the structure-activity relationship (SAR). 2. Results and discussion 2.1. Synthesis of the azasugar aldehydes 6a and 6b containing azido groups The requisite azasugar aldehydes 6a and 6b were prepared from
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Fig. 1. The structures of some bi/tri/tetracyclic azasugars A-F and conformationally constrained NNRTIs G and H.
bismesylated (Ms) compound 1, which was derived from Dmannose according to the previous procedures (Scheme 1) [25]. Compound 1 reacted with S-1-amino-2-propanol to give compound 2a by the amination cyclization in 130 C. Then, the hydroxyl in the side chain was changed with the azido group to generate the azidosugar 4a by the nucleophilic substitution via the tosylated (Ts) intermediate 3a. The isopropylidene group was selectively removed in 80% CH3COOH to afford compound 5a. The vicinal diols in 5 were oxidated into the aldehyde group by sodium periodate to achieve compound 6a bearing with both the aldehyde and azido groups. Following the same procedures, the bisfunctional compound 6b was obtained when 2-aminoethanol was used. We attempted to synthesize 4b from compound 1 and 2-azido ethylamine to simplify the preparation of 6b, however, the reactions were failed to produce 4b in different basic conditions (TEA,
NaHCO3 or NaOH). It should be noted that compound 6 was unstable in its preparation process and should be used directly for the next step after simple work-up. 2.2. Synthesis of the tetracyclic iminosugars fused benzo[e][1,3] thiazin-4-one The key reaction for the construction of the tetracyclic azasugars fused benzthiazinan-4-one was the intramolecular Staudinger/azaWittig/cyclization reaction using 6 and thiosalicylic acid 7 as the starting materials (Scheme 2 and Table 1). The tandem reaction was performed under microwave radiation following the reported procedures [14e18]. 6a Reacted with 7a (entry 1, Table 1) stereospecifically afforded the single tetracyclic product 8a-1 in middle yield of 40.5%, while the reaction of 6a and 7b (entry 2) generated a
Scheme 1. The synthesis of the sugar aldehydes containing azido group 6a and 6b. Reagents and conditions: (i) S-1-amino-2-propanol or 2-aminoethanol, 130 C; (ii) p-TsCl, TEA, CH2Cl2, ice bath; (iii) NaN3, DMF, 90 C; (iv) 80% AcOH, 50 C (v) sodium periodate, 80% ethanol, rt.
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Scheme 2. The synthesis of the tetracyclic azasugars fused benzthiazinan-4-one 8 and 9 by the microwave-assisted Staudinger/aza-Wittig/cyclization reaction.
Table 1 The synthesis of the tetracyclic azasugars 8a and 8b by the microwave-assisted one-pot tandem Staudinger/aza-Wittig/cyclization reactions. Sugar aldehydes
6ac
6bc
a
Mercaptan acids
Entry
Products
Total Yields (%)a
Ratio of 9R:9Sb
9R
9S
1
8a-1
N. D.d
40.5
1:0
2
8a-2
8a-3
37.6e
3:1
3
8b-1f
42.3
4
8b-2f
40.7
Isolated yield. Determined by 1H NMR. c 6a (or 6b) And Ph3P were firstly stirred in 3 mL dry toluene for 10 min at M. W. 80 C, then thiosalicylic acid 7a (or 7b) was added accompanied with DCC (1.2 equiv.) and DMAP (0.2 equiv.) for another 20 min stirring at M. W. 80 C. The ratio of reactants: 6a (1 mmol): Ph3P: thiosalicylic acid ¼ 1: 1.5: 1.2, and the reaction was performed in 10 mL sealed tube. d Not detected. e The total yields of 8a-2, and 8a-3. f Elimination product. b
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pair of inseparable diastereoisomers 8a-2 and 8a-3 with the stereoselectivity ratio of 3:1. It was possible that the hindrance effect of R-methyl adjacent to the imine mainly induced the formation of the compounds 8a-1 and 8a-2 with 9R configuration as the dominant products (Scheme 3). However, the reaction of 6b and 7a (or 7b) gave the unexpected elimination products 8b-1 and 8b-2, respectively (Scheme 2, entry 3 and 4 in Table 1). It was likely that the reaction didn't performed according to the general procedures as shown in Scheme 3. It was difficult for us to explain why the elimination happened. The hypothesized formation mechanism of 8b-1 and 8b-2 was shown in Scheme 4. The amide derivative might be the key intermediate achieved by the intramolecular Schimdt-Boyer reaction [26], which generally provided an alternative and effective way for the preparation of amide via the condensation of the aldehyde and azido in the acidic condition [27,28]. Deprotection of the isopropylidene group in 90% CF3COOH at room temperature, the target tetracyclic azasugars fused benzthiazinan-4-one 9a-1, 9a-2 and 9a-3 were obtained in good yields (Scheme 2). However, compound 9b couldn't be prepared from 8b under the same conditions, possibly due to the instability of 8b in acidic environment. The structures of all the newly synthesized tetracyclic iminosugars 9a-1, 9a-2 and 9a-3 were determined by their 1H, 13C NMR, and HRMS (ESI) spectra. The typical coupling constants of 9-H and 1-H indicated that both compounds 9a-1 and 9a-2 with big J1,9 values (10.2 Hz) should have trans-relationship between 1-H and 9-H, while the corresponding diastereomer 9a-3 is of cis form (J1,9 value of 4.2 Hz). Thus, the absolute configuration
of C-9 in compounds 9a-1 and 9a-2 could be determined to be of (9R), which was consistent with their X-ray crystallographic data (Fig. 2) [29]. The other (9a-3) was confirmed to be of (9S). The structure of compound 8b-1 was determined by its 1H, 13C NMR, HSQC, HMBC and HRMS (ESI) spectra. Using HRESIMS, its molecular formula was deduced to be C17H18N2O3S, two H atoms less than those in the expected compound (not obtained, Fig. 3). This observation was identified with the absent signals in the 1H NMR spectrum, suggesting that there is a new double bond in 8b-1. The peaks of 135.7 ppm and 96.5 ppm in 13C NMR were determined as two quaternary carbons of the double bond by the analysis of HSQC spectrum, and they were assigned on C-1 and C-9, respectively, by the observation of the key JCeH couplings between C-1/2H (dH 5.11 ppm), 3-H (dH 4.82 ppm), 4-H (dH 3.38e3.31 ppm) and 6H (dH 3.43 ppm), C-9/2-H and 7-H (dH 4.73 ppm), and C-17 (dC 163.0 ppm)/7-H (dH 4.73 ppm and 3.38e3.31 ppm) in the HMBC spectrum (Fig. 3). Both the analytical and spectral data of 8b-1 are in agreement with the proposed structure. Compound 8b-2 has very similar 1H and 13C NMR spectra with those of 8b-1. 2.3. HIV-RT inhibition assay HIV-1 reverse transcriptase (RT) inhibitory activity was preliminarily evaluated with the tetracyclic azasugars 9a-1, 9a-2 and 9a-3 and the elimination products 8b-1 and 8b-2 (Scheme 1) by determining their percentage inhibition of HIV-RT activity in HIV1-RT kit by comparison with AZT [30]. The results are shown in Table 2. It could be seen that all the tested compounds showed
Scheme 3. The formation mechanism of the 4-benzthiazinanone-azasugar hybrids 8a-1 and 8a-2.
Scheme 4. Hypothesized formation mechanism of the compounds 8b-1 and 8b-2.
Fig. 2. X-Ray crystographical structures of 9a-1 and 9a-2.
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Fig. 3. Key HMBC (C/H) correlations of 8b-1.
Table 2 In vitro HIV-1-RT kit assay for the tetracyclic azasugars. Compds
IC50 (mM) (HIV-RT kit assay)
Compds
IC50 (mM) (HIV-RT kit assay)
9a-1 9a-2 9a-3
1.96 ± 0.05 5.94 ± 0.96 1.97 ± 0.82
8b-1 8b-2 AZT
0.76 ± 0.14 1.32 ± 0.48 14.95 ± 3.20
densities for examining HIV-RT inhibition were measured on a BioRad Model 3550 microplate spectrophotometer. Thin-layer chromatography (TLC) was performed on precoated plates (Qingdao GF254) with detection with phosphomolybdic acid in EtOH/H2O followed by heating. Column chromatography was performed using SiO2 (Qingdao 300-400 mesh). 3.2. Experimental procedures
significant HIV-RT inhibitory activities, better than that of positive control AZT. Especially, compound 8b-1 showed a more significant HIV-RT inhibitory activity with the IC50 value of 0.76 mM, which indicated that 8b-1 might be better accommodated into the HIV-1 RT binding site, a hydrophobic and flexible binding pocket [21]. The inhibitory activity of the 8b (bearing with isopropylidene group) is much higher than that of 9a, suggesting that the improvement of the molecular hydrophilicity might be favorable to their HIV-RT inhibitory activities. Moreover, 9a exhibit significant anti HIV-RT activities, implying that the naked hydroxyl groups were also tolerant in the binding with HIV-1 RT. In conclusion, novel tetracyclic azasugars fused benzthiazinan4-one 9a-1, 9a-2 and 9a-3 were conveniently synthesized from 6a by the microwave-assisted Staudinger/aza- Wittig/cyclization reaction. Two elimination compounds 8b-1 and 8b-2 were achieved from 6b in the same process as the unexpected products. All the newly synthesized azasugars showed significant HIV-RT inhibitory activities. Among them, compound 8b-1 with the protective group (isopropylidene group) was the best one with the IC50 value of RT inhibitory activity of 0.76 mM. The structure activity relationship (SAR) analysis indicated that the improvement of the molecular hydrophilicity might be favorable to their anti-HIV-RT inhibitory activities.
3. Experimental 3.1. General methods All microwave-assisted reactions were carried out on a CEM Discover S-Class Synthesizer (CEM Co. Ltd. USA). Melting points were measured on an SGW® X-4 micro melting point apparatus and were uncorrected. Optical rotations were determined on an SGW®1 automatic polarimeter. 1H and 13C NMR spectra were measured on a RT-NMR Bruker AVANCE 600 (600 MHz), NMR spectrometer using tetramethylsilane (Me4Si) as an internal standard. Mass Spectra (MS) and High Resolution Mass Spectra (HRMS) were carried out on a FTICR-MS (Ionspec 7.0T) mass spectrometer with electrospray ionization (ESI). X-Ray crystallographic measurements were made on a Bruker SMART CCD Diffractometer. The optical
3.2.1. Synthesis of compounds 2a and 2b A mixture of bismesylated sugar 1 [25] (150 mg, 0.4 mmol) and S-1-amino-2-propanol (20.0 equiv.) was stirred at 130 C under N2 atmosphere for 4-5 h. After the reaction completed (monitored by TLC), water (30 mL) was poured into the solution. The solution was extracted with CH2Cl2 (30 mL 3). The organic solvent was washed with the saturated brine (20 mL 1), dried with MgSO4 for 5 h, then evaporated under vacuum to afford the crude product. The residue was purified using flash column chromatography (petroleum ether - ethyl acetate V/V ¼ 2:1) to afford compound 2a. Under the same conditions, compound 2b was obtained using 2aminoethanol as reactant. 2-((3aS,4S,6aR)-4-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2,2dimethyldihydro-3aH-[1,3]dioxolo[4,5-c]pyrrol-5(4H)-yl) ethanol (2a): yellow oil, yield 90%, [a] 25D -12.7 (c 0.052, CH3OH); 1 H NMR (600 MHz, CDCl3), dH (ppm): 4.62 (dd, J ¼ 10.6, 6.2 Hz, 1H, CH), 4.30 (dd, J ¼ 6.5, 3.3 Hz, 1H, CH), 4.12e4.03 (m, 2H, CH2), 3.85e3.80 (m, 1H, CH), 3.43 (dd, J ¼ 10.0, 3.9 Hz, 1H, CH), 3.34e3.22 (m, 2H, CH2), 3.13e3.05 (m, 2H, CH2), 2.77 (dd, J ¼ 10.8, 4.1 Hz, 1H, CH), 1.49 (s, 3H, CH3), 1.43 (s, 3H, CH3), 1.31 (3H, CH3), 1.29 (s, 3H, CH3), 0.97 (d, J ¼ 6.0 Hz, 3H, CH3); 13C NMR (150 MHz, CDCl3), dC (ppm): 112.8, 109.6, 81.8, 78.9, 76.9, 68.7, 66.2, 64.4, 55.6, 50.0, 27.0, 26.5, 25.1, 24.9, 10.3; MS (ESI), m/z: 324.4 ([ M þ Na ]þ). 2-((3aS,4S,6aR)-4-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2,2dimethyldihydro-3aH-[1,3]dioxolo[4,5-c]pyrrol-5(4H)-yl) ethanol (2b): yellow oil, yield 90%, [a] -37.9 (c 0.038, CH3OH); 1H NMR (600 MHz, CD3OD), dH (ppm): 4.65 (dd, J ¼ 10.3, 6.1 Hz, 1H, CH), 4.35 (dd, J ¼ 6.6, 3.8 Hz, 1H, CH), 4.14 (q, J ¼ 6.9 Hz, 1H, CH), 4.09e4.05 (m, 1H, CH), 3.82e3.77 (m, 1H, CH), 3.61e3.56 (m, 2H, CH2), 3.39 (dd, J ¼ 10.9, 5.8 Hz, 1H, CH), 3.24 (dt, J ¼ 13.0, 6.5 Hz, 1H, CH), 2.82 (dd, J ¼ 7.0, 3.7 Hz, 1H, CH), 2.70e2.59 (m, 2H, CH2), 1.47 (s, 3H, CH3), 1.39 (s, 3H, CH3), 1.33 (s, 3H, CH3), 1.29 (s, 3H, CH3); 13C NMR (150 MHz, CD3OD), dC (ppm): 114.0, 110.7, 83.4, 80.4, 78.4, 73.5, 67.5, 61.5, 60.5, 58.5, 27.5, 26.9, 25.6, 25.0; MS (ESI), m/z: 310.4 ([ M þ Na ]þ). 3.2.2. Synthesis of compounds 4a and 4b A mixture of 2a (150 mg, 0.5 mmol) and TEA (1.5 equiv.) was dissolved in 5 mL dry CH2Cl2 in 25 mL flask. The solution of p-TsCl
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J. Shao et al. / Carbohydrate Research 456 (2018) 45e52
(1.5 equiv.) in dry CH2Cl2 (5 mL) was slowly dropped into the flask under N2 atmosphere. The mixture was stirred at ice bath for 12 h. After the reaction completed (monitored by TLC), water (3 mL) was poured into the solution to quench the reaction, and the solution was extracted with CH2Cl2 (30 mL 3). The organic solvent was washed with the saturated brine (20 mL 1), dried with MgSO4 for 5 h, then evaporated under vacuum to afford the crude product 3a (87% yield). To a solution of 3a (150 mg, 0.3 mmol) in DMF (5 mL) added NaN3 (64 mg, 3.0 equiv.). The solution was stirred at 90 C under N2 atmosphere for 5-6 h. After the reaction completed (monitored by TLC), water (3 mL) was poured into the solution to quench the reaction, and the solution was extracted with ethyl acetate (10 mL 3). The organic solvent was washed with the saturated brine (15 mL 1), dried with MgSO4 for 5 h, then evaporated under vacuum to afford the crude product. The residue was purified using flash column chromatography (petroleum ether - ethyl acetate V/ V ¼ 8:1) to afford the compound 4a. Under the same conditions, compound 4b was obtained. (3aS,4S,6aR)-5-((R)-2-azidopropyl)-4-((R)-2,2-dimethyl-1,3dioxolan-4-yl)-2,2-dimethyltetrahydro-3aH-[1,3]dioxolo[4,5-c] pyrrole (4a, rotamer): yellow oil, yield 69%, [a] -5.6 (c 0.018, CH3OH); 1H NMR (600 MHz, CDCl3), dH (ppm): 4.61 (dd, J ¼ 6.1, 3.4 Hz, 1H, CH), 4.33e4.28 (m, 1H,CH), 4.16 (d, J ¼ 6.7 Hz, 0.4H), 4.09e4.01 (m, 1.7H, CH2), 3.81 (dt, J ¼ 15.3, 7.4 Hz, 1H, CH), 3.53 (dd, J ¼ 13.1, 6.6 Hz, 0.7H, CH), 3.36 (dd, J ¼ 11.0, 5.6 Hz, 0.6H, CH), 3.22 (dd, J ¼ 12.7, 8.9 Hz, 0.4H), 3.12 (dd, J ¼ 9.8, 6.3 Hz, 1H), 3.02e2.93 (m, 0.4H, CH), 2.90 (dd, J ¼ 6.4, 3.3 Hz, 1.4H, CH), 2.75e2.67 (m, 0.6H, CH2), 2.41 (d, J ¼ 14.6 Hz, 1H), 1.49 (s, 3H, CH3), 1.42 (d, J ¼ 12.8 Hz, 3H, CH3), 1.34 (d, J ¼ 6.1 Hz, 3H, CH3), 1.29 (d, J ¼ 5.4 Hz, 3H, CH3), 1.16 (d, J ¼ 6.6 Hz, 2H, CH2), 1.01 (d, J ¼ 6.5 Hz, 1H, CH); 13C NMR (150 MHz, CD3OD), dC (ppm): 112.9, 112.7, 109.5, 109.4, 82.3, 81.2, 79.4, 78.1, 72.0, 68.0, 66.5, 66.3, 61.1, 60.5, 57.8, 54.4, 53.2, 50.4, 27.3, 27.2, 26.5, 25.4, 25.2, 25.1, 24.8, 17.5, 10.6; MS (ESI), m/z: 349.4 ([ M þ Na ]þ). (3aS,4S,6aR)-5-(2-azidoethyl)-4-((R)-2,2-dimethyl-1,3dioxolan-4-yl)-2,2-dimethyltetrahydro-3aH-[1,3]dioxolo[4,5-c] pyrrole (4b): yellow oil, yield 69%, [a] 25D -16.2 (c 0.028, CH3OH); 1 H NMR (600 MHz, CD3OD), dH (ppm): 4.72 (dd, J ¼ 10.3, 6.2 Hz, 1H, CH), 4.44 (dd, J ¼ 6.6, 3.6 Hz, 1H, CH), 4.22 (dd, J ¼ 13.9, 6.8 Hz, 1H, CH), 4.14 (dd, J ¼ 8.2, 6.5 Hz, 1H, CH), 3.87 (t, J ¼ 7.9 Hz, 1H, CH), 3.46 (dd, J ¼ 10.7, 5.8 Hz, 1H, CH), 3.41 (dd, J ¼ 9.3, 5.2 Hz, 1H, CH), 3.38e3.33 (m, 2H, CH2), 2.92 (dd, J ¼ 6.7, 3.7 Hz, 1H, CH), 2.86 (dt, J ¼ 13.5, 6.5 Hz, 1H, CH), 2.73e2.69 (m, 1H, CH), 1.55 (s, 3H, CH3), 1.47 (s, 3H, CH3), 1.41 (s, 3H, CH3), 1.36 (s, 3H, CH3); 13C NMR (150 MHz, CD3OD), dC (ppm): 114.0, 110.7, 83.5, 80.3, 78.6, 73.0, 67.5, 60.3, 55.2, 50.9, 27.5, 26.9, 25.7, 25.0. MS (ESI), m/z: 335.4 ([ M þ Na ]þ). 3.2.3. Synthesis of compounds 5a and 5b A mixture of 4a (150 mg, 0.5 mmol) and 80% acetic acid was stirred at 50 C under N2 atmosphere for 40 h. After the reaction completed (monitored by TLC), the solvent was evaporated under vacuum to afford the crude product. The residue was purified using flash column chromatography (petroleum ether - ethyl acetate V/ V ¼ 1:1) to afford the compound 5a. Under the same conditions, compound 5b was obtained. (R)-1-((3aS,4S,6aR)-5-((R)-2-azidopropyl)-2,2dimethyltetrahydro-3aH-[1,3]dioxolo[4,5-c]pyrrol-4-yl)ethane1,2-diol (5a, rotamer): yellow oil, yield 70%, [a]eq \o(\s\up 6(25),\s\do 2(D)) -6.7 (c 0.036, CH3OH); 1H NMR (600 MHz, CDCl3), dH (ppm): 4.72 (dd, J ¼ 13.0, 3.4 Hz, 1H), 4.52 (d, J ¼ 5.8 Hz, 0.6H), 4.48 (d, J ¼ 5.9 Hz, 0.4H), 3.82 (dd, J ¼ 11.7, 3.4 Hz, 1H), 3.65 (dt, J ¼ 11.7, 4.2 Hz, 1H), 3.51 (dd, J ¼ 11.2, 5.3 Hz, 1.5H), 3.33e3.23 (m, 3H), 3.20e3.09 (m, 2.52H), 3.03 (dd, J ¼ 12.9, 5.1 Hz, 0.5H), 2.91 (dd,
J ¼ 13.6, 8.6 Hz, 0.7H), 1.48 (s, 3H), 1.29 (s, 3H), 1.23 (d, J ¼ 6.6 Hz, 2H), 1.15 (d, J ¼ 6.3 Hz, 1H); 13C NMR (150 MHz, CD3OD), dC (ppm): 111.9, 111.6, 83.8, 83.5, 82.6, 81.1, 73.2, 70.3, 69.5, 68.6, 64.0, 63.9, 63.6, 59.9, 58.3, 56.4, 51.4, 26.5, 26.4, 24.0, 23.3, 17.1, 14.8; MS (ESI), m/z: 309.3 ([ M þ Na ]þ. (R)-1-((3aS,4S,6aR)-5-(2-azidoethyl)-2,2dimethyltetrahydro-3aH-[1,3]dioxolo[4,5-c]pyrrol-4-yl)ethane1,2-diol (5b): yellow oil, yield 70%, [a]eq \o(\s\up 6(25),\s\do 2(D)) -10.0 (c 0.028, CH3OH); 1H NMR (600 MHz, CD3OD), dH (ppm): 4.89e4.86 (m, 1H, CH), 4.81 (d, J ¼ 6.0 Hz, 1H, CH), 4.89e4.86 (m, 1H), 4.81 (d, J ¼ 6.0 Hz, 1H), 3.87 (dd, J ¼ 11.3, 4.4 Hz, 1H, CH), 3.76 (dd, J ¼ 11.3, 5.5 Hz, 1H, CH), 3.67 (d, J ¼ 5.1 Hz, 1H, CH), 3.54 (dd, J ¼ 12.6, 5.3 Hz, 1H, CH), 3.48 (dd, J ¼ 12.0, 6.2 Hz, 1H, CH), 3.42 (dd, J ¼ 12.6, 4.9 Hz, 1H, CH), 3.33 (dt, J ¼ 18.6, 5.2 Hz, 2H, CH2), 3.24e3.19 (m, 1H, CH), 3.06 (d, J ¼ 12.6 Hz, 1H, CH), 1.63 (s, 3H, CH3), 1.46 (s, 3H, CH3); 13C NMR (150 MHz, CD3OD), dC (ppm): 112.9, 85.2, 83.0, 74.1, 72.5, 64.7, 60.2, 58.2, 52.2, 27.2, 24.2; MS (ESI), m/z: 295.3 ([ M þ Na ]þ. 3.2.4. Synthesis of compounds 8a-1, 8a-2, 8a-3, 8b-1 and 8b-2 To a solution of 5a (150 mg, 0.5 mmol) in 80% ethanol (10 mL) added NaIO4 (168 mg, 1.5 equiv.). The solution was stirred at room temperature under N2 atmosphere for 3 h. After the reaction completed (monitored by TLC), the organic solvent was evaporated under vacuum below 30 C, and the residue was extracted with CH2Cl2 (10 mL 3). The organic solvent was washed with the saturated brine (15 mL 1), and evaporated under vacuum below 30 C to afford the crude product 6a (yield 70%). A mixture of sugar aldehyde 6a (100 mg, 0.4 mmol), and PPh3 (154 mg, 1.5 equiv.) was dissolved in 3 mL anhydrous toluene and was stirred in sealed tube at 80 C under microwave irradiation in a CEM Discover S-Class Synthesizer for 10 min. After cooling, 2mercaptobenzoic acid 7a (1.2 equiv.), DCC (1.2 equiv.) and DMAP (0.2 equiv.) were added and the mixture was stirred at 80 C in sealed tube by irradiating microwave for another 20 min reaction. After the reaction finished, solid K2CO3 was added to the solution to neutralize 2-mercaptobenzoic acid and then was removed with DCU by filtration. Subsequently, the solution was extracted with CH2Cl2 (10 mL 3), then, the organic layer was combined and dried with anhydrous MgSO4 for 5h. The solvent was evaporated under reduced pressure to get a crude product that was purified using flash column chromatography (petroleum ether - ethyl acetate V/ V ¼ 4:1) to afford the compound 8a-1. Under the same conditions, the mixture of two inseparable diastereoisomers 8a-2 and 8a-3 was obtained using 5-methyl-2mercaptobenzoic acid 7b as the starting material. The elimination products 8b-2 and 8b-3 were obtained using 6b as the starting material. (3aR,7R,14aR,14bR,14cS)-2,2,7-trimethyl-3a,4,6,7,14b,14c-hexahydro-[1,3]dioxolo[400 ,5'':3′,4']pyrrolo[2′,1':3,4]pyrazino[2,1-b] benzo[e][1,3]thiazin-9(14aH)-one (8a-1): yellow solid, mp. 179.8 C, yield 40.5%, [a] 25D þ66.7 (c 0.012, CH3OH), 1H NMR (600 MHz, CD3OD), dH (ppm): 8.02 (d, J ¼ 7.7 Hz, 1H, Ar-H), 7.45 (t, J ¼ 7.6 Hz, 1H, Ar-H), 7.29 (t, J ¼ 8.0 Hz, 2H, Ar-H), 5.01 (d, J ¼ 10.2 Hz, 1H, CH), 4.78 (dd, J ¼ 12.5, 6.5 Hz, 1H, CH), 4.75e4.71 (m, 1H, CH), 4.42 (t, J ¼ 6.7 Hz, 1H, CH), 3.45 (dd, J ¼ 9.1, 6.5 Hz, 1H, CH), 2.93 (d, J ¼ 11.3 Hz, 1H, CH), 2.56 (dd, J ¼ 11.3, 3.8 Hz, 1H, CH), 2.40e2.34 (m, 2H, CH2), 1.49 (s, 3H, CH3), 1.34 (s, 3H, CH3), 1.30 (d, J ¼ 6.7 Hz, 3H, CH3); 13C NMR 0 MHz, CDCl3), dC (ppm): 164.3, 133.6, 132.1, 130.6, 128.7, 126.5, 126.1, 115.0, 83.0, 77.7, 71.4, 59.7, 58.4, 55.5, 48.3, 27.2, 25.3, 16.6; HR-ESI-MS: calcd for C18H22N2O3SNa ([MþNa]þ), 369.1249, found: 369.1246. (3aR,14 cS)-2,2-dimethyl-3a,4,6,7-tetrahydro-[1,3]dioxolo [400 ,5'':3′,4']pyrrolo[2′,1':3,4]pyrazino[2,1-b]benzo[e][1,3]thiazin-9(14 cH)-one (8b-1): yellow oil, yield 42.3%, [a] 25D -140.0 (c
J. Shao et al. / Carbohydrate Research 456 (2018) 45e52
0.01, CH3OH); 1H NMR (600 MHz, CD3OD), dH (ppm): 7.97 (d, J ¼ 7.0 Hz, 1H, Ar-H), 7.38 (s, 1H, Ar-H), 7.23e7.18 (m, 2H, Ar-H), 5.11 (d, J ¼ 6.3 Hz, 1H, CH), 4.82 (t, J ¼ 5.4 Hz, 1H, CH), 4.73 (dt, J ¼ 12.9, 3.0 Hz, 1H, CH), 3.43 (dt, J ¼ 11.7, 2.7 Hz, 1H, CH), 3.38e3.32 (m, 2H, CH2), 3.09 (dd, J ¼ 10.3, 4.9 Hz, 1H, CH), 2.96e2.90 (m, 1H, CH), 1.44 (s, 3H, CH3), 1.37 (s, 3H, CH3); 13C NMR (150 MHz, CD3OD), dC (ppm): 163.0, 138.7, 135.7, 133.7, 131.2, 129.1, 126.8, 126.4, 113.7, 96.5, 80.4, 79.0, 57.6, 46.0, 39.5, 27.3, 25.4; HR-ESI-MS: calcd for C17H18N2O3S ([M]þ), 330.1038, found: 330.1041. (3aR,14 cS)-2,2,11-trimethyl-3a,4,6,7-tetrahydro-[1,3]dioxolo [400 ,5'':3′,4']pyrrolo[2′,1':3,4]pyrazino[2,1-b]benzo[e][1,3]thiazin-9(14 cH)-one (8b-2): yellow oil, yield 40.7%, [a] 25D -230.0 (c 0.008, CH3OH); 1H NMR (600 MHz, CD3OD), dH (ppm): 7.81 (s, 1H, Ar-H), 7.24 (d, J ¼ 7.8 Hz, 1H, Ar-H), 7.10 (d, J ¼ 7.9 Hz, 1H, Ar-H), 5.12 (d, J ¼ 6.3 Hz, 1H, CH), 4.85e4.81 (m, 1H, CH), 4.75 (dt, J ¼ 12.8, 2.8 Hz, 1H, CH), 3.45 (d, J ¼ 11.7 Hz, 1H, CH), 3.38 (dd, J ¼ 16.1, 6.6 Hz, 2H, CH2), 3.10 (dd, J ¼ 10.3, 4.9 Hz, 1H, CH), 2.95 (td, J ¼ 11.2, 3.5 Hz, 1H, CH), 2.33 (s, 3H, CH3), 2.10 (s, 3H, CH3), 1.46 (s, 3H, CH3), 1.38 (s, 3H, CH3); 13C NMR (150 MHz, CD3OD), dC (ppm): 163.1, 137.0, 135.4, 134.5, 131.5, 128.9, 126.4, 113.7, 97.0, 80.4, 79.1, 57.7, 46.1, 39.6, 27.3, 25.9, 21.0; HR-ESI-MS: calcd for C18H20N2O3S ([M]þ), 344.1195, found: 344.1198. 3.2.5. Synthesis of compounds 9a-1, 9a-2 and 9a-3 The compound 8a-1 (100 mg, 0.3 mmol) was dissolved in 5 mL 90% CF3COOH. The mixture was stirred at ice bath for 3 h. After the reaction completed (monitored by TLC), solid NaHCO3 was added to the solution to neutralize the acid, and the solution extracted with water (3 mL) and CH2Cl2 (10 mL 3). The organic solvent was washed with the saturated brine (15 mL 1), dried with MgSO4 for 5 h, then evaporated under vacuum to afford the crude product. The residue was purified using flash column chromatography (petroleum ether - ethyl acetate V/V ¼ 4:1) to afford the compound 9a-1. Under the same conditions, compounds 9a-2 and 9a-3 were obtained using the mixture of 8a-2 and 8a-3 as the reactant. (1S,2R,6R,13aR,13bR)-1,2-dihydroxy-6-methyl-1,2,3,5,6,13bhexahydrobenzo[e]pyrrolo[2′,1':3,4]pyrazino[2,1-b][1,3]thiazin8(13aH)-one (9a-1): yellow solid, mp. 186.7 C, yield 50.2%, [a] þ116.5 (c 0.046, CH3OH); 1H NMR (600 MHz, CD3OD), dH (ppm): 8.13 (d, J ¼ 7.8 Hz, 1H, Ar-H), 7.36 (d, J ¼ 7.5 Hz, 1H, Ar-H), 7.24 (t, J ¼ 8.2 Hz, 2H, Ar-H), 4.92 (d, J ¼ 10.0 Hz, 1H, CH), 4.88e4.83 (m, 1H, CH), 4.31 (d, J ¼ 6.7 Hz, 1H, CH), 3.88 (s, 1H, CH), 3.50 (dd, J ¼ 9.5, 6.7 Hz, 1H, CH), 2.85 (d, J ¼ 11.0 Hz, 1H, CH), 2.54 (dd, J ¼ 11.1, 3.8 Hz, 1H, CH), 2.35 (dd, J ¼ 9.8, 7.5 Hz, 1H, CH), 2.26 (dd, J ¼ 9.5, 6.0 Hz, 1H, CH), 1.30 (d, J ¼ 6.7 Hz, 3H, CH3); 13C NMR (150 MHz, CDCl3), dC (ppm): 164.1, 133.2, 132.1, 130.7, 128.6, 126.5, 126.2, 73.4, 71.1, 67.6, 61.0, 58.7, 55.5, 48.6, 16.6; HR-ESI-MS: calcd for C15H18N2O3SNa ([MþNa]þ), 329.0936, found: 329.0934. (1S,2R,6R,13aR,13bR)-1,2-dihydroxy-6,10-dimethyl1,2,3,5,6,13b-hexahydrobenzo[e]pyrrolo[2′,1':3,4]pyrazino[2,1b][1,3]thiazin-8(13aH)-one (9a-2): yellow solid, mp. 71.2 C, yield 15.07%, [a] þ112.5 (c 0.016, CH3OH); 1H NMR (600 MHz, CD3OD), dH (ppm): 7.83 (s, 1H, Ar-H), 7.26 (dd, J ¼ 7.9, 1.5 Hz, 1H, Ar-H), 7.17 (d, J ¼ 7.9 Hz, 1H, Ar-H), 4.96 (d, J ¼ 10.1 Hz, 1H, CH), 4.74e4.70 (m, 1H, CH), 4.15 (q, J ¼ 6.8 Hz, 1H, CH), 3.81 (t, J ¼ 7.3 Hz, 1H, CH), 3.40 (dd, J ¼ 9.2, 6.7 Hz, 1H, CH), 2.94e2.90 (m, 1H, CH), 2.48 (dd, J ¼ 11.2, 3.9 Hz, 1H, CH), 2.34 (s, 3H, CH3), 2.26 (dd, J ¼ 10.0, 7.2 Hz, 1H, CH), 2.21 (dd, J ¼ 9.2, 6.6 Hz, 1H, CH), 1.29 (d, J ¼ 6.7 Hz, 3H, CH3); 13C NMR (150 MHz, CDCl3), d(ppm): 164.5, 136.2, 133.1, 131.0, 129.7, 128.4, 73.3, 71.1, 67.6, 61.0, 58.8, 55.5, 48.6, 21.0, 16.6; HR-ESI-MS: calcd for C16H20N2O3SNa ([MþNa]þ), 343.1092, found: 343.1088. (1S,2R,6R,13aS,13bR)-1,2-dihydroxy-6,10-dimethyl1,2,3,5,6,13b-hexahydrobenzo[e]pyrrolo[2′,1':3,4]pyrazino[2,1b][1,3]thiazin-8(13aH)-one (9a-3): yellow solid, mp. 159.8 C, yield 6.01%, [a] -60.0 (c 0.002, CH3OH); 1H NMR (600 MHz, CD3OD), dH
51
(ppm): 7.77 (s, 1H, Ar-H), 7.32 (d, J ¼ 8.0 Hz, 1H, Ar-H), 7.27 (s, 1H), 5.40 (d, J ¼ 4.4 Hz, 1H, Ar-H), 4.15 (d, J ¼ 4.5 Hz, 1H, CH), 4.02 (s, 1H), 3.84 (dd, J ¼ 13.4, 4.8 Hz, 1H, CH), 3.63 (dd, J ¼ 10.2, 6.3 Hz, 1H, CH), 3.38 (dd, J ¼ 13.4, 7.6 Hz, 1H, CH), 3.09 (dd, J ¼ 8.1, 4.4 Hz, 1H, CH), 2.68 (dd, J ¼ 12.1, 6.2 Hz, 1H, CH), 2.36 (s, 3H, CH3), 2.32 (dd, J ¼ 10.3, 4.3 Hz, 1H, CH), 1.15 (d, J ¼ 6.2 Hz, 3H, CH3); 13C NMR (150 MHz, CD3OD), dC (ppm): 169.4, 137.4, 134.0, 131.9, 130.7, 129.9, 128.8, 73.8, 69.6, 69.2, 66.5, 59.7, 57.3, 46.3, 20.9, 18.9; HR-ESI-MS: calcd for C16H20N2O3SNa ([MþNa]þ), 343.1092, found: 343.1095. 3.3. In vitro HIV-RT kit assay The HIV-RT inhibition assay was performed by using an RT assay kit (Roche), and the procedure for assaying RT inhibition was performed as described in the kit protocol. Briefly, the reaction mixture consists of template/primer complex, 20 -deoxy-nucleotide- 50 - triphosphates (dNTPs) and reverse transcriptase (RT) enzyme in the lysis buffer with or without inhibitors. After 1 h incubation at 37 C the reaction mixture was transferred to streptavidine-coated microtitre plate (MTP). The biotin labeled dNTPs that are incorporated in the template due to activity of RT were bound to streptavidine. The unbound dNTPs were washed using wash buffer and antidigoxigenin-peroxidase (DIG-POD) was added in MTP. The DIG-labeled dNTPs incorporated in the template was bound to antiDIG-POD antibody. The unbound anti-DIG-POD was washed and the peroxide substrate (ABST) was added to the MTP. A colored reaction product was produced during the cleavage of the substrate catalyses by a peroxide enzyme. The absorbance of the sample was determined at OD 405 nM using microtiter plate ELISA reader. The resulting color intensity is directly proportional to the actual RT activity. The percentage inhibitory activity of RT inhibitors was calculated by comparing to a sample that does not contain an inhibitor. The percentage inhibition was calculated by formula as given below: % Inhibition ¼ 100- [(OD 405 nm with inhibitor/OD 405 nm without inhibitor) 100]. Acknowledgments The financial supports from the National Natural Science Foundation of China (NSFC) (21372060, 21772031), Hebei Province Natural Science Fund for Distinguished Young Scholars (B2015201005). Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.carres.2017.12.005. References [1] P. Compain, O.R. Martin (Eds.), Iminosugars: from Synthesis to Therapeutic Applications, Wiley, Chichester, 2007. [2] N. Asano, H. Hashimoto, Azaglycomimetics: synthesis and chemical biology, in: B.O. Fraser-Reid, K. Tatsuta, J. Thiem (Eds.), Glycoscience: Chemistry and Chemical Biology, Springer-Verlag, Berlin, 2008, pp. 1887e1911. [3] G.N. Wang, G. Reinkensmeier, S.W. Zhang, J. Zhou, L.R. Zhang, L.H. Zhang, et al., J. Med. Chem. 52 (2009) 3146e3149. [4] Y.X. Li, K. Kinami, Y. Hirokami, A. Kato, J.K. Su, Y.M. Jia, et al., Org. Biomol. Chem. 14 (2016) 2249e2263. [5] R. Lahiri, A.A. Ansariw, Y.D. Vankar, Chem. Soc. Rev. 42 (2013) 5102e5118. n, M.I. García-Moreno, E. Nanba, K. Higaki, A.L. Concia, [6] T. Mena-Barraga s, et al., Eur. J. Med. Chem. 121 (2016) 880e891. P. Clape Lope €ck, O. z, Å. Tobiesen, J.G. Fern ~ os, M.O. Sydnes, Org. [7] E. Lindba andez-Bolan Biomol. Chem. 15 (2017) 8709e8712. [8] A. Stütz (Ed.), Iminosugars as Glycosidase Inhibitors: Nojirimycin and beyond, Wiley-VCH, Weinheim, 1999. [9] R. Zelli, J.F. Longevial, P. Dumy, A. Marra, New J. Chem. 39 (2015) 5050e5074. [10] S.G. Gouin, Chem. Eur J. 20 (2014) 11616e11628. [11] L.D.S. Yadav, C. Awasthi, Carbohydr. Res. 345 (2010) 318e323.
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[12] S. Senthilkumar, S.S. Prasad, A. Das, S. Baskaran, Chem. Eur J. 21 (2015) 15914e15918. [13] Y.D. Vankar, T. Linker, Eur. J. Org Chem. (2015) 7633e7642. [14] X.L. Li, Z.B. Qin, T.Y. Yang, H.Z. Zhang, S.N. Wei, C.X. Li, et al., Bioorg. Med. Chem. Lett 22 (2012) 2712e2716. [15] H. Chen, T.Y. Yang, S.N. Wei, H.Z. Zhang, R. Li, Z.B. Qin, et al., Bioorg. Med. Chem. Lett 22 (2012) 7041e7044. [16] H. Chen, R. Li, Z.Y. Liu, S.N. Wei, H.Z. Zhang, X.L. Li, Carbohydr. Res. 365 (2013) 1e8. [17] H. Chen, L. Hao, M. Zhu, T.Y. Yang, S.N. Wei, Z.B. Qin, et al., Bioorg. Med. Chem. Lett 24 (2014) 3426e3429. [18] Y.H. Hou, S.K. Xing, J. Shao, Z.Q. Yin, L. Hao, T.Y. Yang, et al., Carbohydr. Res. 429 (2016) 105e112. [19] P. Zhan, X.Y. Liu, Z.Y. Li, C. Pannecouque, E. De Clercq, Curr. Med. Chem. 16 (2009) 3903e3917. [20] D.W. Kang, Z.J. Fang, Z.Y. Li, B.S. Huang, H. Zhang, X.Y. Lu, et al., J. Med. Chem. 59 (2016) 7991e8001. [21] P. Zhan, X.W. Chen, D.Y. Li, Z.J. Fang, E. De Clercq, X.Y. Liu, Med. Res. Rev. 33 (S1) (2013) E1eE72.
[22] Z.Q. Yin, M. Zhu, S.N. Wei, J. Shao, Y.H. Hou, H. Chen, et al., Bioorg. Med. Chem. Lett 26 (2016) 1738e1741. [23] D. Bellarosa, G. Antonelli, F. Bambacioni, D. Giannotti, G. Viti, R. Nannicini, et al., Antivir. Res. 30 (1996) 109e124. [24] R. Silvestri, M. Artico, B. Bruno, S. Massa, E. Novellino, G. Greco, et al., Antivir. Chem. Chemother. 9 (1998) 139e148. [25] X.L. Li, S.N. Wei, H.B. Zhang, P.Z. Zhang, W. Wang, H. Chen, Chin. J. Org. Chem. 32 (2012) 1708e1717. [26] H. Chen, R. Li, F. Gao, X.L. Li, Tetrahedron Lett. 53 (2012) 7147e7149. [27] S. Lang, J.A. Murphy, Chem. Soc. Rev. 2 (2006) 146e156. , J. Am. Chem. Soc. 134 (2012) [28] R.Z. Liu, O. Gutierrez, D.J. Tantillo, J. Aube 6528e6531. [29] CCDC-1584727 (for 9a-1) and CCDC-1584728 (for 9a-2) contain the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc. cam.ac.uk/data_ request/cif. [30] Reverse Transcriptase Assay, Colorimetric Kit, Roche Diagnostics GmbH, Roche Applied Science, Sandhofer Strasse 116, D-68305 Mannheim, Germany.
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Synthesis of tetracyclic azasugars fused benzo[e][1,3]thiazin-4-one by the tandem Staudinger/aza-Wittig/cyclization and their HIV-RT inhibitory activity Jie Shao, Mo Zhu, Ligang Gao, Hua Chen**, Xiaoliu Li* Key Laboratory of Chemical Biology of Hebei Province, College of Chemistry and Environmental Science, Hebei University, Baoding 071002, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 November 2017 Received in revised form 12 December 2017 Accepted 12 December 2017 Available online 15 December 2017
Azasugar aldehydes 6a and 6b containing azido groups were prepared from D-mannose. Three novel tetracyclic azasugars fused benzo[e][1,3]thiazin-4-one 9a-1, 9a-2 and 9a-3 were conveniently synthesized from 6a by the tandem intramolecular Staudinger/aza-Wittig/cyclization reaction under microwave radiation. Two unexpected elimination compounds 8b-1 and 8b-2 were achieved as the main products from 6b in the same processes. The newly synthesized azasugars were examined for their HIV reverse transcriptase (RT) inhibitory activities. The results showed that all the tested compounds could effectively inhibit RT activity. Among them, compound 8b-1 with the protective group (isopropylidene group) was the best one with the IC50 value of 0.76 mM. The structure activity relationship analysis suggested that improvement of the molecular hydrophilicity might be beneficial for their anti-HIV RT activities. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Tetracyclic azasugar Benzthiazinan-4-one Staudinger/aza-Wittig/cyclization Anti-HIV RT activity
1. Introduction Iminosugars or azasugars, which exhibit effective inhibition against carbohydrate-processing enzymes, have been paid much attention due to their potential clinical applications as anti-HIV, anti-diabetic, and anti-cancer agents and immunomodulators in past decades [1e4]. The bicyclic azasugars fused different azoles (A and B, Fig. 1) [5e7] were synthesized and evaluated for their biological activities. The promising activities of the azasugars demanded to prepare their novel derivatives to meet the evergrowing requirements for the new drug discovery [8e10]. However, multicyclic (more than 3-fused rings) azasugars have received much less attention [11e13]. The tetracyclic azasugars were so far scarcely reported for their synthesis and biological activities. By using the microwave-assisted Staudinger/aza-Wittig/cyclization reaction, we have conveniently synthesized a series of novel bi- and tri-cyclic thiazolidin-4-one- and benzothiazin-4-one-fused azasugars (C-E, Fig. 1), which exhibit strong HIV reverse transcriptase (HIV-RT) inhibitory activity [14e18]. It was well known that most HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) generally showed butterfly-like conformation when they
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (H. Chen), [email protected] (X. Li). https://doi.org/10.1016/j.carres.2017.12.005 0008-6215/© 2017 Elsevier Ltd. All rights reserved.
were binding with HIV-RT [19e21]. Taking account this dominant conformation as determinant for anti-HIV RT activity, recently, the tetracyclic azasugar fused benzo[e][1,3]thiazin-4-one F [22] (Fig. 1) was rationally designed and was synthesized via the threecomponents condensation of the azasugar aldehyde, amino acid ester, and 2-mercaptobenzoic acid, then followed by the intramolecular cyclo-amidation reaction. Compound F was found to possess significant anti-HIV-RT activity with the IC50 value of 0.82 mM possibly due to its constrained butterfly-like conformation, just as the known NNRTIs G (MEN 10979) and H (Fig. 1) did [23,24]. However, the redundant synthesis of F limited its further application for the very low yield. As a continuation of our research, we would like herein to report the synthesis of novel tetracyclic azasugars fused benzo[e][1,3]thiazin-4-one by the tandem Staudinger/ Aza-Wittig/cyclization as an alternative protocol. Such newly synthetic azasugars were evaluated for their HIV-RT inhibitory activities in order to further investigate the structure-activity relationship (SAR). 2. Results and discussion 2.1. Synthesis of the azasugar aldehydes 6a and 6b containing azido groups The requisite azasugar aldehydes 6a and 6b were prepared from
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Fig. 1. The structures of some bi/tri/tetracyclic azasugars A-F and conformationally constrained NNRTIs G and H.
bismesylated (Ms) compound 1, which was derived from Dmannose according to the previous procedures (Scheme 1) [25]. Compound 1 reacted with S-1-amino-2-propanol to give compound 2a by the amination cyclization in 130 C. Then, the hydroxyl in the side chain was changed with the azido group to generate the azidosugar 4a by the nucleophilic substitution via the tosylated (Ts) intermediate 3a. The isopropylidene group was selectively removed in 80% CH3COOH to afford compound 5a. The vicinal diols in 5 were oxidated into the aldehyde group by sodium periodate to achieve compound 6a bearing with both the aldehyde and azido groups. Following the same procedures, the bisfunctional compound 6b was obtained when 2-aminoethanol was used. We attempted to synthesize 4b from compound 1 and 2-azido ethylamine to simplify the preparation of 6b, however, the reactions were failed to produce 4b in different basic conditions (TEA,
NaHCO3 or NaOH). It should be noted that compound 6 was unstable in its preparation process and should be used directly for the next step after simple work-up. 2.2. Synthesis of the tetracyclic iminosugars fused benzo[e][1,3] thiazin-4-one The key reaction for the construction of the tetracyclic azasugars fused benzthiazinan-4-one was the intramolecular Staudinger/azaWittig/cyclization reaction using 6 and thiosalicylic acid 7 as the starting materials (Scheme 2 and Table 1). The tandem reaction was performed under microwave radiation following the reported procedures [14e18]. 6a Reacted with 7a (entry 1, Table 1) stereospecifically afforded the single tetracyclic product 8a-1 in middle yield of 40.5%, while the reaction of 6a and 7b (entry 2) generated a
Scheme 1. The synthesis of the sugar aldehydes containing azido group 6a and 6b. Reagents and conditions: (i) S-1-amino-2-propanol or 2-aminoethanol, 130 C; (ii) p-TsCl, TEA, CH2Cl2, ice bath; (iii) NaN3, DMF, 90 C; (iv) 80% AcOH, 50 C (v) sodium periodate, 80% ethanol, rt.
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Scheme 2. The synthesis of the tetracyclic azasugars fused benzthiazinan-4-one 8 and 9 by the microwave-assisted Staudinger/aza-Wittig/cyclization reaction.
Table 1 The synthesis of the tetracyclic azasugars 8a and 8b by the microwave-assisted one-pot tandem Staudinger/aza-Wittig/cyclization reactions. Sugar aldehydes
6ac
6bc
a
Mercaptan acids
Entry
Products
Total Yields (%)a
Ratio of 9R:9Sb
9R
9S
1
8a-1
N. D.d
40.5
1:0
2
8a-2
8a-3
37.6e
3:1
3
8b-1f
42.3
4
8b-2f
40.7
Isolated yield. Determined by 1H NMR. c 6a (or 6b) And Ph3P were firstly stirred in 3 mL dry toluene for 10 min at M. W. 80 C, then thiosalicylic acid 7a (or 7b) was added accompanied with DCC (1.2 equiv.) and DMAP (0.2 equiv.) for another 20 min stirring at M. W. 80 C. The ratio of reactants: 6a (1 mmol): Ph3P: thiosalicylic acid ¼ 1: 1.5: 1.2, and the reaction was performed in 10 mL sealed tube. d Not detected. e The total yields of 8a-2, and 8a-3. f Elimination product. b
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pair of inseparable diastereoisomers 8a-2 and 8a-3 with the stereoselectivity ratio of 3:1. It was possible that the hindrance effect of R-methyl adjacent to the imine mainly induced the formation of the compounds 8a-1 and 8a-2 with 9R configuration as the dominant products (Scheme 3). However, the reaction of 6b and 7a (or 7b) gave the unexpected elimination products 8b-1 and 8b-2, respectively (Scheme 2, entry 3 and 4 in Table 1). It was likely that the reaction didn't performed according to the general procedures as shown in Scheme 3. It was difficult for us to explain why the elimination happened. The hypothesized formation mechanism of 8b-1 and 8b-2 was shown in Scheme 4. The amide derivative might be the key intermediate achieved by the intramolecular Schimdt-Boyer reaction [26], which generally provided an alternative and effective way for the preparation of amide via the condensation of the aldehyde and azido in the acidic condition [27,28]. Deprotection of the isopropylidene group in 90% CF3COOH at room temperature, the target tetracyclic azasugars fused benzthiazinan-4-one 9a-1, 9a-2 and 9a-3 were obtained in good yields (Scheme 2). However, compound 9b couldn't be prepared from 8b under the same conditions, possibly due to the instability of 8b in acidic environment. The structures of all the newly synthesized tetracyclic iminosugars 9a-1, 9a-2 and 9a-3 were determined by their 1H, 13C NMR, and HRMS (ESI) spectra. The typical coupling constants of 9-H and 1-H indicated that both compounds 9a-1 and 9a-2 with big J1,9 values (10.2 Hz) should have trans-relationship between 1-H and 9-H, while the corresponding diastereomer 9a-3 is of cis form (J1,9 value of 4.2 Hz). Thus, the absolute configuration
of C-9 in compounds 9a-1 and 9a-2 could be determined to be of (9R), which was consistent with their X-ray crystallographic data (Fig. 2) [29]. The other (9a-3) was confirmed to be of (9S). The structure of compound 8b-1 was determined by its 1H, 13C NMR, HSQC, HMBC and HRMS (ESI) spectra. Using HRESIMS, its molecular formula was deduced to be C17H18N2O3S, two H atoms less than those in the expected compound (not obtained, Fig. 3). This observation was identified with the absent signals in the 1H NMR spectrum, suggesting that there is a new double bond in 8b-1. The peaks of 135.7 ppm and 96.5 ppm in 13C NMR were determined as two quaternary carbons of the double bond by the analysis of HSQC spectrum, and they were assigned on C-1 and C-9, respectively, by the observation of the key JCeH couplings between C-1/2H (dH 5.11 ppm), 3-H (dH 4.82 ppm), 4-H (dH 3.38e3.31 ppm) and 6H (dH 3.43 ppm), C-9/2-H and 7-H (dH 4.73 ppm), and C-17 (dC 163.0 ppm)/7-H (dH 4.73 ppm and 3.38e3.31 ppm) in the HMBC spectrum (Fig. 3). Both the analytical and spectral data of 8b-1 are in agreement with the proposed structure. Compound 8b-2 has very similar 1H and 13C NMR spectra with those of 8b-1. 2.3. HIV-RT inhibition assay HIV-1 reverse transcriptase (RT) inhibitory activity was preliminarily evaluated with the tetracyclic azasugars 9a-1, 9a-2 and 9a-3 and the elimination products 8b-1 and 8b-2 (Scheme 1) by determining their percentage inhibition of HIV-RT activity in HIV1-RT kit by comparison with AZT [30]. The results are shown in Table 2. It could be seen that all the tested compounds showed
Scheme 3. The formation mechanism of the 4-benzthiazinanone-azasugar hybrids 8a-1 and 8a-2.
Scheme 4. Hypothesized formation mechanism of the compounds 8b-1 and 8b-2.
Fig. 2. X-Ray crystographical structures of 9a-1 and 9a-2.
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49
Fig. 3. Key HMBC (C/H) correlations of 8b-1.
Table 2 In vitro HIV-1-RT kit assay for the tetracyclic azasugars. Compds
IC50 (mM) (HIV-RT kit assay)
Compds
IC50 (mM) (HIV-RT kit assay)
9a-1 9a-2 9a-3
1.96 ± 0.05 5.94 ± 0.96 1.97 ± 0.82
8b-1 8b-2 AZT
0.76 ± 0.14 1.32 ± 0.48 14.95 ± 3.20
densities for examining HIV-RT inhibition were measured on a BioRad Model 3550 microplate spectrophotometer. Thin-layer chromatography (TLC) was performed on precoated plates (Qingdao GF254) with detection with phosphomolybdic acid in EtOH/H2O followed by heating. Column chromatography was performed using SiO2 (Qingdao 300-400 mesh). 3.2. Experimental procedures
significant HIV-RT inhibitory activities, better than that of positive control AZT. Especially, compound 8b-1 showed a more significant HIV-RT inhibitory activity with the IC50 value of 0.76 mM, which indicated that 8b-1 might be better accommodated into the HIV-1 RT binding site, a hydrophobic and flexible binding pocket [21]. The inhibitory activity of the 8b (bearing with isopropylidene group) is much higher than that of 9a, suggesting that the improvement of the molecular hydrophilicity might be favorable to their HIV-RT inhibitory activities. Moreover, 9a exhibit significant anti HIV-RT activities, implying that the naked hydroxyl groups were also tolerant in the binding with HIV-1 RT. In conclusion, novel tetracyclic azasugars fused benzthiazinan4-one 9a-1, 9a-2 and 9a-3 were conveniently synthesized from 6a by the microwave-assisted Staudinger/aza- Wittig/cyclization reaction. Two elimination compounds 8b-1 and 8b-2 were achieved from 6b in the same process as the unexpected products. All the newly synthesized azasugars showed significant HIV-RT inhibitory activities. Among them, compound 8b-1 with the protective group (isopropylidene group) was the best one with the IC50 value of RT inhibitory activity of 0.76 mM. The structure activity relationship (SAR) analysis indicated that the improvement of the molecular hydrophilicity might be favorable to their anti-HIV-RT inhibitory activities.
3. Experimental 3.1. General methods All microwave-assisted reactions were carried out on a CEM Discover S-Class Synthesizer (CEM Co. Ltd. USA). Melting points were measured on an SGW® X-4 micro melting point apparatus and were uncorrected. Optical rotations were determined on an SGW®1 automatic polarimeter. 1H and 13C NMR spectra were measured on a RT-NMR Bruker AVANCE 600 (600 MHz), NMR spectrometer using tetramethylsilane (Me4Si) as an internal standard. Mass Spectra (MS) and High Resolution Mass Spectra (HRMS) were carried out on a FTICR-MS (Ionspec 7.0T) mass spectrometer with electrospray ionization (ESI). X-Ray crystallographic measurements were made on a Bruker SMART CCD Diffractometer. The optical
3.2.1. Synthesis of compounds 2a and 2b A mixture of bismesylated sugar 1 [25] (150 mg, 0.4 mmol) and S-1-amino-2-propanol (20.0 equiv.) was stirred at 130 C under N2 atmosphere for 4-5 h. After the reaction completed (monitored by TLC), water (30 mL) was poured into the solution. The solution was extracted with CH2Cl2 (30 mL 3). The organic solvent was washed with the saturated brine (20 mL 1), dried with MgSO4 for 5 h, then evaporated under vacuum to afford the crude product. The residue was purified using flash column chromatography (petroleum ether - ethyl acetate V/V ¼ 2:1) to afford compound 2a. Under the same conditions, compound 2b was obtained using 2aminoethanol as reactant. 2-((3aS,4S,6aR)-4-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2,2dimethyldihydro-3aH-[1,3]dioxolo[4,5-c]pyrrol-5(4H)-yl) ethanol (2a): yellow oil, yield 90%, [a] 25D -12.7 (c 0.052, CH3OH); 1 H NMR (600 MHz, CDCl3), dH (ppm): 4.62 (dd, J ¼ 10.6, 6.2 Hz, 1H, CH), 4.30 (dd, J ¼ 6.5, 3.3 Hz, 1H, CH), 4.12e4.03 (m, 2H, CH2), 3.85e3.80 (m, 1H, CH), 3.43 (dd, J ¼ 10.0, 3.9 Hz, 1H, CH), 3.34e3.22 (m, 2H, CH2), 3.13e3.05 (m, 2H, CH2), 2.77 (dd, J ¼ 10.8, 4.1 Hz, 1H, CH), 1.49 (s, 3H, CH3), 1.43 (s, 3H, CH3), 1.31 (3H, CH3), 1.29 (s, 3H, CH3), 0.97 (d, J ¼ 6.0 Hz, 3H, CH3); 13C NMR (150 MHz, CDCl3), dC (ppm): 112.8, 109.6, 81.8, 78.9, 76.9, 68.7, 66.2, 64.4, 55.6, 50.0, 27.0, 26.5, 25.1, 24.9, 10.3; MS (ESI), m/z: 324.4 ([ M þ Na ]þ). 2-((3aS,4S,6aR)-4-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2,2dimethyldihydro-3aH-[1,3]dioxolo[4,5-c]pyrrol-5(4H)-yl) ethanol (2b): yellow oil, yield 90%, [a] -37.9 (c 0.038, CH3OH); 1H NMR (600 MHz, CD3OD), dH (ppm): 4.65 (dd, J ¼ 10.3, 6.1 Hz, 1H, CH), 4.35 (dd, J ¼ 6.6, 3.8 Hz, 1H, CH), 4.14 (q, J ¼ 6.9 Hz, 1H, CH), 4.09e4.05 (m, 1H, CH), 3.82e3.77 (m, 1H, CH), 3.61e3.56 (m, 2H, CH2), 3.39 (dd, J ¼ 10.9, 5.8 Hz, 1H, CH), 3.24 (dt, J ¼ 13.0, 6.5 Hz, 1H, CH), 2.82 (dd, J ¼ 7.0, 3.7 Hz, 1H, CH), 2.70e2.59 (m, 2H, CH2), 1.47 (s, 3H, CH3), 1.39 (s, 3H, CH3), 1.33 (s, 3H, CH3), 1.29 (s, 3H, CH3); 13C NMR (150 MHz, CD3OD), dC (ppm): 114.0, 110.7, 83.4, 80.4, 78.4, 73.5, 67.5, 61.5, 60.5, 58.5, 27.5, 26.9, 25.6, 25.0; MS (ESI), m/z: 310.4 ([ M þ Na ]þ). 3.2.2. Synthesis of compounds 4a and 4b A mixture of 2a (150 mg, 0.5 mmol) and TEA (1.5 equiv.) was dissolved in 5 mL dry CH2Cl2 in 25 mL flask. The solution of p-TsCl
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J. Shao et al. / Carbohydrate Research 456 (2018) 45e52
(1.5 equiv.) in dry CH2Cl2 (5 mL) was slowly dropped into the flask under N2 atmosphere. The mixture was stirred at ice bath for 12 h. After the reaction completed (monitored by TLC), water (3 mL) was poured into the solution to quench the reaction, and the solution was extracted with CH2Cl2 (30 mL 3). The organic solvent was washed with the saturated brine (20 mL 1), dried with MgSO4 for 5 h, then evaporated under vacuum to afford the crude product 3a (87% yield). To a solution of 3a (150 mg, 0.3 mmol) in DMF (5 mL) added NaN3 (64 mg, 3.0 equiv.). The solution was stirred at 90 C under N2 atmosphere for 5-6 h. After the reaction completed (monitored by TLC), water (3 mL) was poured into the solution to quench the reaction, and the solution was extracted with ethyl acetate (10 mL 3). The organic solvent was washed with the saturated brine (15 mL 1), dried with MgSO4 for 5 h, then evaporated under vacuum to afford the crude product. The residue was purified using flash column chromatography (petroleum ether - ethyl acetate V/ V ¼ 8:1) to afford the compound 4a. Under the same conditions, compound 4b was obtained. (3aS,4S,6aR)-5-((R)-2-azidopropyl)-4-((R)-2,2-dimethyl-1,3dioxolan-4-yl)-2,2-dimethyltetrahydro-3aH-[1,3]dioxolo[4,5-c] pyrrole (4a, rotamer): yellow oil, yield 69%, [a] -5.6 (c 0.018, CH3OH); 1H NMR (600 MHz, CDCl3), dH (ppm): 4.61 (dd, J ¼ 6.1, 3.4 Hz, 1H, CH), 4.33e4.28 (m, 1H,CH), 4.16 (d, J ¼ 6.7 Hz, 0.4H), 4.09e4.01 (m, 1.7H, CH2), 3.81 (dt, J ¼ 15.3, 7.4 Hz, 1H, CH), 3.53 (dd, J ¼ 13.1, 6.6 Hz, 0.7H, CH), 3.36 (dd, J ¼ 11.0, 5.6 Hz, 0.6H, CH), 3.22 (dd, J ¼ 12.7, 8.9 Hz, 0.4H), 3.12 (dd, J ¼ 9.8, 6.3 Hz, 1H), 3.02e2.93 (m, 0.4H, CH), 2.90 (dd, J ¼ 6.4, 3.3 Hz, 1.4H, CH), 2.75e2.67 (m, 0.6H, CH2), 2.41 (d, J ¼ 14.6 Hz, 1H), 1.49 (s, 3H, CH3), 1.42 (d, J ¼ 12.8 Hz, 3H, CH3), 1.34 (d, J ¼ 6.1 Hz, 3H, CH3), 1.29 (d, J ¼ 5.4 Hz, 3H, CH3), 1.16 (d, J ¼ 6.6 Hz, 2H, CH2), 1.01 (d, J ¼ 6.5 Hz, 1H, CH); 13C NMR (150 MHz, CD3OD), dC (ppm): 112.9, 112.7, 109.5, 109.4, 82.3, 81.2, 79.4, 78.1, 72.0, 68.0, 66.5, 66.3, 61.1, 60.5, 57.8, 54.4, 53.2, 50.4, 27.3, 27.2, 26.5, 25.4, 25.2, 25.1, 24.8, 17.5, 10.6; MS (ESI), m/z: 349.4 ([ M þ Na ]þ). (3aS,4S,6aR)-5-(2-azidoethyl)-4-((R)-2,2-dimethyl-1,3dioxolan-4-yl)-2,2-dimethyltetrahydro-3aH-[1,3]dioxolo[4,5-c] pyrrole (4b): yellow oil, yield 69%, [a] 25D -16.2 (c 0.028, CH3OH); 1 H NMR (600 MHz, CD3OD), dH (ppm): 4.72 (dd, J ¼ 10.3, 6.2 Hz, 1H, CH), 4.44 (dd, J ¼ 6.6, 3.6 Hz, 1H, CH), 4.22 (dd, J ¼ 13.9, 6.8 Hz, 1H, CH), 4.14 (dd, J ¼ 8.2, 6.5 Hz, 1H, CH), 3.87 (t, J ¼ 7.9 Hz, 1H, CH), 3.46 (dd, J ¼ 10.7, 5.8 Hz, 1H, CH), 3.41 (dd, J ¼ 9.3, 5.2 Hz, 1H, CH), 3.38e3.33 (m, 2H, CH2), 2.92 (dd, J ¼ 6.7, 3.7 Hz, 1H, CH), 2.86 (dt, J ¼ 13.5, 6.5 Hz, 1H, CH), 2.73e2.69 (m, 1H, CH), 1.55 (s, 3H, CH3), 1.47 (s, 3H, CH3), 1.41 (s, 3H, CH3), 1.36 (s, 3H, CH3); 13C NMR (150 MHz, CD3OD), dC (ppm): 114.0, 110.7, 83.5, 80.3, 78.6, 73.0, 67.5, 60.3, 55.2, 50.9, 27.5, 26.9, 25.7, 25.0. MS (ESI), m/z: 335.4 ([ M þ Na ]þ). 3.2.3. Synthesis of compounds 5a and 5b A mixture of 4a (150 mg, 0.5 mmol) and 80% acetic acid was stirred at 50 C under N2 atmosphere for 40 h. After the reaction completed (monitored by TLC), the solvent was evaporated under vacuum to afford the crude product. The residue was purified using flash column chromatography (petroleum ether - ethyl acetate V/ V ¼ 1:1) to afford the compound 5a. Under the same conditions, compound 5b was obtained. (R)-1-((3aS,4S,6aR)-5-((R)-2-azidopropyl)-2,2dimethyltetrahydro-3aH-[1,3]dioxolo[4,5-c]pyrrol-4-yl)ethane1,2-diol (5a, rotamer): yellow oil, yield 70%, [a]eq \o(\s\up 6(25),\s\do 2(D)) -6.7 (c 0.036, CH3OH); 1H NMR (600 MHz, CDCl3), dH (ppm): 4.72 (dd, J ¼ 13.0, 3.4 Hz, 1H), 4.52 (d, J ¼ 5.8 Hz, 0.6H), 4.48 (d, J ¼ 5.9 Hz, 0.4H), 3.82 (dd, J ¼ 11.7, 3.4 Hz, 1H), 3.65 (dt, J ¼ 11.7, 4.2 Hz, 1H), 3.51 (dd, J ¼ 11.2, 5.3 Hz, 1.5H), 3.33e3.23 (m, 3H), 3.20e3.09 (m, 2.52H), 3.03 (dd, J ¼ 12.9, 5.1 Hz, 0.5H), 2.91 (dd,
J ¼ 13.6, 8.6 Hz, 0.7H), 1.48 (s, 3H), 1.29 (s, 3H), 1.23 (d, J ¼ 6.6 Hz, 2H), 1.15 (d, J ¼ 6.3 Hz, 1H); 13C NMR (150 MHz, CD3OD), dC (ppm): 111.9, 111.6, 83.8, 83.5, 82.6, 81.1, 73.2, 70.3, 69.5, 68.6, 64.0, 63.9, 63.6, 59.9, 58.3, 56.4, 51.4, 26.5, 26.4, 24.0, 23.3, 17.1, 14.8; MS (ESI), m/z: 309.3 ([ M þ Na ]þ. (R)-1-((3aS,4S,6aR)-5-(2-azidoethyl)-2,2dimethyltetrahydro-3aH-[1,3]dioxolo[4,5-c]pyrrol-4-yl)ethane1,2-diol (5b): yellow oil, yield 70%, [a]eq \o(\s\up 6(25),\s\do 2(D)) -10.0 (c 0.028, CH3OH); 1H NMR (600 MHz, CD3OD), dH (ppm): 4.89e4.86 (m, 1H, CH), 4.81 (d, J ¼ 6.0 Hz, 1H, CH), 4.89e4.86 (m, 1H), 4.81 (d, J ¼ 6.0 Hz, 1H), 3.87 (dd, J ¼ 11.3, 4.4 Hz, 1H, CH), 3.76 (dd, J ¼ 11.3, 5.5 Hz, 1H, CH), 3.67 (d, J ¼ 5.1 Hz, 1H, CH), 3.54 (dd, J ¼ 12.6, 5.3 Hz, 1H, CH), 3.48 (dd, J ¼ 12.0, 6.2 Hz, 1H, CH), 3.42 (dd, J ¼ 12.6, 4.9 Hz, 1H, CH), 3.33 (dt, J ¼ 18.6, 5.2 Hz, 2H, CH2), 3.24e3.19 (m, 1H, CH), 3.06 (d, J ¼ 12.6 Hz, 1H, CH), 1.63 (s, 3H, CH3), 1.46 (s, 3H, CH3); 13C NMR (150 MHz, CD3OD), dC (ppm): 112.9, 85.2, 83.0, 74.1, 72.5, 64.7, 60.2, 58.2, 52.2, 27.2, 24.2; MS (ESI), m/z: 295.3 ([ M þ Na ]þ. 3.2.4. Synthesis of compounds 8a-1, 8a-2, 8a-3, 8b-1 and 8b-2 To a solution of 5a (150 mg, 0.5 mmol) in 80% ethanol (10 mL) added NaIO4 (168 mg, 1.5 equiv.). The solution was stirred at room temperature under N2 atmosphere for 3 h. After the reaction completed (monitored by TLC), the organic solvent was evaporated under vacuum below 30 C, and the residue was extracted with CH2Cl2 (10 mL 3). The organic solvent was washed with the saturated brine (15 mL 1), and evaporated under vacuum below 30 C to afford the crude product 6a (yield 70%). A mixture of sugar aldehyde 6a (100 mg, 0.4 mmol), and PPh3 (154 mg, 1.5 equiv.) was dissolved in 3 mL anhydrous toluene and was stirred in sealed tube at 80 C under microwave irradiation in a CEM Discover S-Class Synthesizer for 10 min. After cooling, 2mercaptobenzoic acid 7a (1.2 equiv.), DCC (1.2 equiv.) and DMAP (0.2 equiv.) were added and the mixture was stirred at 80 C in sealed tube by irradiating microwave for another 20 min reaction. After the reaction finished, solid K2CO3 was added to the solution to neutralize 2-mercaptobenzoic acid and then was removed with DCU by filtration. Subsequently, the solution was extracted with CH2Cl2 (10 mL 3), then, the organic layer was combined and dried with anhydrous MgSO4 for 5h. The solvent was evaporated under reduced pressure to get a crude product that was purified using flash column chromatography (petroleum ether - ethyl acetate V/ V ¼ 4:1) to afford the compound 8a-1. Under the same conditions, the mixture of two inseparable diastereoisomers 8a-2 and 8a-3 was obtained using 5-methyl-2mercaptobenzoic acid 7b as the starting material. The elimination products 8b-2 and 8b-3 were obtained using 6b as the starting material. (3aR,7R,14aR,14bR,14cS)-2,2,7-trimethyl-3a,4,6,7,14b,14c-hexahydro-[1,3]dioxolo[400 ,5'':3′,4']pyrrolo[2′,1':3,4]pyrazino[2,1-b] benzo[e][1,3]thiazin-9(14aH)-one (8a-1): yellow solid, mp. 179.8 C, yield 40.5%, [a] 25D þ66.7 (c 0.012, CH3OH), 1H NMR (600 MHz, CD3OD), dH (ppm): 8.02 (d, J ¼ 7.7 Hz, 1H, Ar-H), 7.45 (t, J ¼ 7.6 Hz, 1H, Ar-H), 7.29 (t, J ¼ 8.0 Hz, 2H, Ar-H), 5.01 (d, J ¼ 10.2 Hz, 1H, CH), 4.78 (dd, J ¼ 12.5, 6.5 Hz, 1H, CH), 4.75e4.71 (m, 1H, CH), 4.42 (t, J ¼ 6.7 Hz, 1H, CH), 3.45 (dd, J ¼ 9.1, 6.5 Hz, 1H, CH), 2.93 (d, J ¼ 11.3 Hz, 1H, CH), 2.56 (dd, J ¼ 11.3, 3.8 Hz, 1H, CH), 2.40e2.34 (m, 2H, CH2), 1.49 (s, 3H, CH3), 1.34 (s, 3H, CH3), 1.30 (d, J ¼ 6.7 Hz, 3H, CH3); 13C NMR 0 MHz, CDCl3), dC (ppm): 164.3, 133.6, 132.1, 130.6, 128.7, 126.5, 126.1, 115.0, 83.0, 77.7, 71.4, 59.7, 58.4, 55.5, 48.3, 27.2, 25.3, 16.6; HR-ESI-MS: calcd for C18H22N2O3SNa ([MþNa]þ), 369.1249, found: 369.1246. (3aR,14 cS)-2,2-dimethyl-3a,4,6,7-tetrahydro-[1,3]dioxolo [400 ,5'':3′,4']pyrrolo[2′,1':3,4]pyrazino[2,1-b]benzo[e][1,3]thiazin-9(14 cH)-one (8b-1): yellow oil, yield 42.3%, [a] 25D -140.0 (c
J. Shao et al. / Carbohydrate Research 456 (2018) 45e52
0.01, CH3OH); 1H NMR (600 MHz, CD3OD), dH (ppm): 7.97 (d, J ¼ 7.0 Hz, 1H, Ar-H), 7.38 (s, 1H, Ar-H), 7.23e7.18 (m, 2H, Ar-H), 5.11 (d, J ¼ 6.3 Hz, 1H, CH), 4.82 (t, J ¼ 5.4 Hz, 1H, CH), 4.73 (dt, J ¼ 12.9, 3.0 Hz, 1H, CH), 3.43 (dt, J ¼ 11.7, 2.7 Hz, 1H, CH), 3.38e3.32 (m, 2H, CH2), 3.09 (dd, J ¼ 10.3, 4.9 Hz, 1H, CH), 2.96e2.90 (m, 1H, CH), 1.44 (s, 3H, CH3), 1.37 (s, 3H, CH3); 13C NMR (150 MHz, CD3OD), dC (ppm): 163.0, 138.7, 135.7, 133.7, 131.2, 129.1, 126.8, 126.4, 113.7, 96.5, 80.4, 79.0, 57.6, 46.0, 39.5, 27.3, 25.4; HR-ESI-MS: calcd for C17H18N2O3S ([M]þ), 330.1038, found: 330.1041. (3aR,14 cS)-2,2,11-trimethyl-3a,4,6,7-tetrahydro-[1,3]dioxolo [400 ,5'':3′,4']pyrrolo[2′,1':3,4]pyrazino[2,1-b]benzo[e][1,3]thiazin-9(14 cH)-one (8b-2): yellow oil, yield 40.7%, [a] 25D -230.0 (c 0.008, CH3OH); 1H NMR (600 MHz, CD3OD), dH (ppm): 7.81 (s, 1H, Ar-H), 7.24 (d, J ¼ 7.8 Hz, 1H, Ar-H), 7.10 (d, J ¼ 7.9 Hz, 1H, Ar-H), 5.12 (d, J ¼ 6.3 Hz, 1H, CH), 4.85e4.81 (m, 1H, CH), 4.75 (dt, J ¼ 12.8, 2.8 Hz, 1H, CH), 3.45 (d, J ¼ 11.7 Hz, 1H, CH), 3.38 (dd, J ¼ 16.1, 6.6 Hz, 2H, CH2), 3.10 (dd, J ¼ 10.3, 4.9 Hz, 1H, CH), 2.95 (td, J ¼ 11.2, 3.5 Hz, 1H, CH), 2.33 (s, 3H, CH3), 2.10 (s, 3H, CH3), 1.46 (s, 3H, CH3), 1.38 (s, 3H, CH3); 13C NMR (150 MHz, CD3OD), dC (ppm): 163.1, 137.0, 135.4, 134.5, 131.5, 128.9, 126.4, 113.7, 97.0, 80.4, 79.1, 57.7, 46.1, 39.6, 27.3, 25.9, 21.0; HR-ESI-MS: calcd for C18H20N2O3S ([M]þ), 344.1195, found: 344.1198. 3.2.5. Synthesis of compounds 9a-1, 9a-2 and 9a-3 The compound 8a-1 (100 mg, 0.3 mmol) was dissolved in 5 mL 90% CF3COOH. The mixture was stirred at ice bath for 3 h. After the reaction completed (monitored by TLC), solid NaHCO3 was added to the solution to neutralize the acid, and the solution extracted with water (3 mL) and CH2Cl2 (10 mL 3). The organic solvent was washed with the saturated brine (15 mL 1), dried with MgSO4 for 5 h, then evaporated under vacuum to afford the crude product. The residue was purified using flash column chromatography (petroleum ether - ethyl acetate V/V ¼ 4:1) to afford the compound 9a-1. Under the same conditions, compounds 9a-2 and 9a-3 were obtained using the mixture of 8a-2 and 8a-3 as the reactant. (1S,2R,6R,13aR,13bR)-1,2-dihydroxy-6-methyl-1,2,3,5,6,13bhexahydrobenzo[e]pyrrolo[2′,1':3,4]pyrazino[2,1-b][1,3]thiazin8(13aH)-one (9a-1): yellow solid, mp. 186.7 C, yield 50.2%, [a] þ116.5 (c 0.046, CH3OH); 1H NMR (600 MHz, CD3OD), dH (ppm): 8.13 (d, J ¼ 7.8 Hz, 1H, Ar-H), 7.36 (d, J ¼ 7.5 Hz, 1H, Ar-H), 7.24 (t, J ¼ 8.2 Hz, 2H, Ar-H), 4.92 (d, J ¼ 10.0 Hz, 1H, CH), 4.88e4.83 (m, 1H, CH), 4.31 (d, J ¼ 6.7 Hz, 1H, CH), 3.88 (s, 1H, CH), 3.50 (dd, J ¼ 9.5, 6.7 Hz, 1H, CH), 2.85 (d, J ¼ 11.0 Hz, 1H, CH), 2.54 (dd, J ¼ 11.1, 3.8 Hz, 1H, CH), 2.35 (dd, J ¼ 9.8, 7.5 Hz, 1H, CH), 2.26 (dd, J ¼ 9.5, 6.0 Hz, 1H, CH), 1.30 (d, J ¼ 6.7 Hz, 3H, CH3); 13C NMR (150 MHz, CDCl3), dC (ppm): 164.1, 133.2, 132.1, 130.7, 128.6, 126.5, 126.2, 73.4, 71.1, 67.6, 61.0, 58.7, 55.5, 48.6, 16.6; HR-ESI-MS: calcd for C15H18N2O3SNa ([MþNa]þ), 329.0936, found: 329.0934. (1S,2R,6R,13aR,13bR)-1,2-dihydroxy-6,10-dimethyl1,2,3,5,6,13b-hexahydrobenzo[e]pyrrolo[2′,1':3,4]pyrazino[2,1b][1,3]thiazin-8(13aH)-one (9a-2): yellow solid, mp. 71.2 C, yield 15.07%, [a] þ112.5 (c 0.016, CH3OH); 1H NMR (600 MHz, CD3OD), dH (ppm): 7.83 (s, 1H, Ar-H), 7.26 (dd, J ¼ 7.9, 1.5 Hz, 1H, Ar-H), 7.17 (d, J ¼ 7.9 Hz, 1H, Ar-H), 4.96 (d, J ¼ 10.1 Hz, 1H, CH), 4.74e4.70 (m, 1H, CH), 4.15 (q, J ¼ 6.8 Hz, 1H, CH), 3.81 (t, J ¼ 7.3 Hz, 1H, CH), 3.40 (dd, J ¼ 9.2, 6.7 Hz, 1H, CH), 2.94e2.90 (m, 1H, CH), 2.48 (dd, J ¼ 11.2, 3.9 Hz, 1H, CH), 2.34 (s, 3H, CH3), 2.26 (dd, J ¼ 10.0, 7.2 Hz, 1H, CH), 2.21 (dd, J ¼ 9.2, 6.6 Hz, 1H, CH), 1.29 (d, J ¼ 6.7 Hz, 3H, CH3); 13C NMR (150 MHz, CDCl3), d(ppm): 164.5, 136.2, 133.1, 131.0, 129.7, 128.4, 73.3, 71.1, 67.6, 61.0, 58.8, 55.5, 48.6, 21.0, 16.6; HR-ESI-MS: calcd for C16H20N2O3SNa ([MþNa]þ), 343.1092, found: 343.1088. (1S,2R,6R,13aS,13bR)-1,2-dihydroxy-6,10-dimethyl1,2,3,5,6,13b-hexahydrobenzo[e]pyrrolo[2′,1':3,4]pyrazino[2,1b][1,3]thiazin-8(13aH)-one (9a-3): yellow solid, mp. 159.8 C, yield 6.01%, [a] -60.0 (c 0.002, CH3OH); 1H NMR (600 MHz, CD3OD), dH
51
(ppm): 7.77 (s, 1H, Ar-H), 7.32 (d, J ¼ 8.0 Hz, 1H, Ar-H), 7.27 (s, 1H), 5.40 (d, J ¼ 4.4 Hz, 1H, Ar-H), 4.15 (d, J ¼ 4.5 Hz, 1H, CH), 4.02 (s, 1H), 3.84 (dd, J ¼ 13.4, 4.8 Hz, 1H, CH), 3.63 (dd, J ¼ 10.2, 6.3 Hz, 1H, CH), 3.38 (dd, J ¼ 13.4, 7.6 Hz, 1H, CH), 3.09 (dd, J ¼ 8.1, 4.4 Hz, 1H, CH), 2.68 (dd, J ¼ 12.1, 6.2 Hz, 1H, CH), 2.36 (s, 3H, CH3), 2.32 (dd, J ¼ 10.3, 4.3 Hz, 1H, CH), 1.15 (d, J ¼ 6.2 Hz, 3H, CH3); 13C NMR (150 MHz, CD3OD), dC (ppm): 169.4, 137.4, 134.0, 131.9, 130.7, 129.9, 128.8, 73.8, 69.6, 69.2, 66.5, 59.7, 57.3, 46.3, 20.9, 18.9; HR-ESI-MS: calcd for C16H20N2O3SNa ([MþNa]þ), 343.1092, found: 343.1095. 3.3. In vitro HIV-RT kit assay The HIV-RT inhibition assay was performed by using an RT assay kit (Roche), and the procedure for assaying RT inhibition was performed as described in the kit protocol. Briefly, the reaction mixture consists of template/primer complex, 20 -deoxy-nucleotide- 50 - triphosphates (dNTPs) and reverse transcriptase (RT) enzyme in the lysis buffer with or without inhibitors. After 1 h incubation at 37 C the reaction mixture was transferred to streptavidine-coated microtitre plate (MTP). The biotin labeled dNTPs that are incorporated in the template due to activity of RT were bound to streptavidine. The unbound dNTPs were washed using wash buffer and antidigoxigenin-peroxidase (DIG-POD) was added in MTP. The DIG-labeled dNTPs incorporated in the template was bound to antiDIG-POD antibody. The unbound anti-DIG-POD was washed and the peroxide substrate (ABST) was added to the MTP. A colored reaction product was produced during the cleavage of the substrate catalyses by a peroxide enzyme. The absorbance of the sample was determined at OD 405 nM using microtiter plate ELISA reader. The resulting color intensity is directly proportional to the actual RT activity. The percentage inhibitory activity of RT inhibitors was calculated by comparing to a sample that does not contain an inhibitor. The percentage inhibition was calculated by formula as given below: % Inhibition ¼ 100- [(OD 405 nm with inhibitor/OD 405 nm without inhibitor) 100]. Acknowledgments The financial supports from the National Natural Science Foundation of China (NSFC) (21372060, 21772031), Hebei Province Natural Science Fund for Distinguished Young Scholars (B2015201005). Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.carres.2017.12.005. References [1] P. Compain, O.R. Martin (Eds.), Iminosugars: from Synthesis to Therapeutic Applications, Wiley, Chichester, 2007. [2] N. Asano, H. Hashimoto, Azaglycomimetics: synthesis and chemical biology, in: B.O. Fraser-Reid, K. Tatsuta, J. Thiem (Eds.), Glycoscience: Chemistry and Chemical Biology, Springer-Verlag, Berlin, 2008, pp. 1887e1911. [3] G.N. Wang, G. Reinkensmeier, S.W. Zhang, J. Zhou, L.R. Zhang, L.H. Zhang, et al., J. Med. Chem. 52 (2009) 3146e3149. [4] Y.X. Li, K. Kinami, Y. Hirokami, A. Kato, J.K. Su, Y.M. Jia, et al., Org. Biomol. Chem. 14 (2016) 2249e2263. [5] R. Lahiri, A.A. Ansariw, Y.D. Vankar, Chem. Soc. Rev. 42 (2013) 5102e5118. n, M.I. García-Moreno, E. Nanba, K. Higaki, A.L. Concia, [6] T. Mena-Barraga s, et al., Eur. J. Med. Chem. 121 (2016) 880e891. P. Clape Lope €ck, O. z, Å. Tobiesen, J.G. Fern ~ os, M.O. Sydnes, Org. [7] E. Lindba andez-Bolan Biomol. Chem. 15 (2017) 8709e8712. [8] A. Stütz (Ed.), Iminosugars as Glycosidase Inhibitors: Nojirimycin and beyond, Wiley-VCH, Weinheim, 1999. [9] R. Zelli, J.F. Longevial, P. Dumy, A. Marra, New J. Chem. 39 (2015) 5050e5074. [10] S.G. Gouin, Chem. Eur J. 20 (2014) 11616e11628. [11] L.D.S. Yadav, C. Awasthi, Carbohydr. Res. 345 (2010) 318e323.
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