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Synthesis of tricyclic quinazolinone-iminosugars as potential glycosidase inhibitors via a Mitsunobu reaction
Carbohydrate Research 478 (2019) 10–17
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Synthesis of tricyclic quinazolinone-iminosugars as potential glycosidase inhibitors via a Mitsunobu reaction
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Jiajing Sun, Yaqing Kang, Ligang Gao, Xin Lu, Huanhuan Ju, Xiaoliu Li∗∗, Hua Chen∗ Key Laboratory of Chemical Biology of Hebei Province, College of Chemistry and Environmental Science, Hebei University, Baoding, 071002, China
ARTICLE INFO
ABSTRACT
Keywords: Tricyclic iminosugars Quizanolinone Iodine-induced oxidative condensation Mitsunobu reaction Glycosidase inhibitory activity
A series of novel tricyclic quinazolinone-iminosugars 1 (a-c) were synthesized from the benzyl protected sugars through three steps. Firstly, the benzyl protected sugar (aldehyde) 5 reacted with o-aminobenzamide by the iodine-induced oxidative condensation to afford the corresponding aldo-quizanolinone 6. Secondly, through the intramolecular cyclization of the unprotected OH and the amide NH in 6, the tricyclic compounds 7 and 8 were constructed by the key Mitsunobu reaction. Finally, removal of the benzyl group gave the target tricyclic quinazolinone-iminosugars 1. The protocol was effective for the preparation of the tricyclic iminosugars in satisfactory yield. Interestingly, an unusual C-2 epimerization was observed with D-mannose and D-ribose compounds under the conditions of the Mitsunobu reaction that generated the products having the trans configuration at the C-2 and C-3 positions. Unfortunately, such tricyclic quinazolinone-iminosugars showed no inhibitory effects on the tested five glycosidases.
1. Introduction Glycosidases are the specific glycosidic bond hydrolases directly involved in the glycosylation on cell surface, and are also responsible for the glycosylation of the proteins or lipids with important biological activities [1]. The occurrence and development of many diseases, such as diabetes, cancers, viral infections, autoimmune diseases, are closely related to the abnormal expression of the glycosidases [2]. Therefore, the study of glycosidase inhibitors has become one of the hot topics in drug development [3]. As one of the most important classes of glycosidase inhibitor, iminosugars and their analogs are widely used for the treatment of carbohydrate-mediated diseases (compounds A-E, Fig. 1) [4]. They are the structural mimetics of the natural carbohydrates with endocyclic nitrogen atom, and can mimic the enzymatic transition state to inhibit glycosidase activity [5]. Generally, they can be easily recognized by glycosidases as the cationic intermediates adopting a half chair conformation under physiological conditions. Both structural features are becoming the key basis for the rational design of glycosidase inhibitors derived from iminosugars [6]. For examples, the amidine (compound F) and amidoxime (compound G) (Fig. 1) inhibitors could effectively inhibit both α- and β-glycosidases [7]. On the basis of the enzymatic transition state [8], a variety of bicyclic iminosugars fused to different nitrogen-containing
∗
heterocycles (compounds H-J, Fig. 1), which were called sp2-iminosugars [9–13] (the anomeric carbon having sp2-character), were rationally designed and displayed significant inhibitory activities against different glycosidases. The heterocycles included imidazole, trizaole, tetrazole, oxazole, and thiazole, etc. The increased potency of such sp2-iminosugars as glycosidase inhibitors suggested the preparation of other derivatives for the new carbohydrate-based drug discovery [14–16]. However, multicyclic (more than 3-fused rings) iminosugars [17–20] having sp2-character on the anomeric carbon have received much less attention possibly due to the difficulties of their synthesis. As a continuation of our studies on the bioactive multicyclic iminosugars [21–23], we would like to report the synthesis of novel tricyclic iminosugars fused 4-quinazolinone 1 (a-c) (Fig. 2) by a Mitsunobu reaction. As an important nitrogen-containing pharmacophore, 4(3H)-quinazolinone and its derivatives have attracted much attention due to their diverse biological activities, such as anti-cancer, anti-bacterial, anti-virus, anti-diabetes, and anti-glycosidase activities [24–26]. Such new tricyclic quinazolinone-iminosugars were thought to be capable of mimicking the enzymatic transition state of glycosidases due to the sp2-character of their anomeric carbons, and they were evaluated for their glycosidase inhibitory activity.
Corresponding author. Corresponding author. E-mail address: [email protected] (H. Chen).
∗∗
https://doi.org/10.1016/j.carres.2019.04.002 Received 16 February 2019; Received in revised form 9 April 2019; Accepted 14 April 2019 Available online 23 April 2019 0008-6215/ © 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. Examples of known iminosugars with effective glycosidase inhibitory activity. Fig. 2. The synthetic tricyclic quinazolinone-iminosugars 1 (a–c).
2. Results and discussion
induced oxidative condensation of D-mannose and o-aminobenzamides [28] to synthesize the corresponding tricyclic iminosugar fused quinazolinone 3. However, the reaction did not give the expected product under the same reaction conditions. Changing pyridine with sodium hydride (NaH) in DMF solution, the reaction gave an unexpected Cnucleoside [29] 4 in the good yield of 68% (Scheme 1), in which the intramolecular cyclization took place between 6-OH and 3-OH identified by its X-ray crystallographic structure (see supporting information) [30]. The reason that compound 4 is formed in preference to compound 3 is possibly that cyclization to form five-membered ring is much more favoured than cyclization to form a seven-membered ring.
2.1. Synthesis of the tricyclic gluco-iminosugars fused quinazolinone Sallam et al. [27] had reported that treatment of D-arabino-tetritol1-yl-benzimidazole with p-toluene sulfonyl chloride (TsCl) in pyridine solution could afford an interesting tricyclic benzimidazole fused iminosugar, which was achieved by the intramolecular SN2 nucleophilic substitution between the primary tosyl intermediate and the 1-nitrogen atom of the benzimidazole moiety (Scheme 1). Therefore, we attempted to use manno-quinazolinone 2 that was directly prepared by the iodine-
Scheme 1. Synthesis of an unexpected C-nucleoside 4. 11
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Scheme 2. The retrosynthesis route for the tricyclic quinazolinone-iminosugar.
As an alternative method, Mitsunobu reaction was widely used to construct nitrogen-containing heterocycles [31–33]. Cheon et al. [34] had reported the intramolecular cyclization of quinazolinone to afford mackinazolinone by Mitsunobu reaction (Scheme 2). Inspired by this positive result, we conceived that our compound 1 could be prepared via a Mitsunobu reaction on the benzyl protected aldo-quinazolinone 6 (Scheme 2). We initiated our synthesis from the benzyl protected aldo-quinazolinone 6 that could be easily prepared by the iodine-induced oxidative condensation of benzyl protected sugar aldehyde 5 and o-aminobenzamides [28]. Taking the synthesis of the tricyclic gluco-iminosugar 6a as example (Scheme 3), the available benzyl protected glucose (5a) reacted with o-aminobenzamide to afford the corresponding gluco-quinazolinone (6a) in yield of 67% according to the above mentioned method. Then, Mitsunobu reaction was successfully performed to realize the cyclization of the unprotected 5-OH and the amide NH in 6a in the presence of the DIAD and PPh3. Generally the Mitsunobu reaction generates the inversion product [35–37]. However, in this case, the reaction provided a pair of diastereoisomers, the C-5 configuration
inversion product 7a and the C-5 configuration retention one 8a, in total good yields of 66% although the reaction conditions were not optimized. The inversion product 7a was the predominant one and the ratio of 7a: 8a was 1.9:1. We conceived that the Mitsunobu reaction resulted in retention possibly due to neighbouring group participation [38] and double inversion (Scheme 4). The neighbouring group is most likely the 6-O-benzyl. Attack by the C-6 oxygen on C-5 with loss of Ph3PO would result in the (O-benzyl) C5eC6 oxiran (epoxy derivative). This, being analogous to a protonated epoxide, would be rapidly ringopened by the amide nitrogen attacking at C-5 (a favoured 6-exo-tet process) resulting in retention of configuration at C-5. Presumably, oxiran ring formation is competitive with nucleophilic attack by the amide nitrogen. Finally, removal of the benzyl group from 7a in CF3SO3H was carried out at −80 °C to give the target tricyclic glucoiminosugar in low yield of 22%. The temperature had important effect on the reaction. The reaction could not afford the target product under higher temperature, such as −40 °C, which rapidly cause the reaction complex. An alternative attempt to remove the benzyl group was in Pd (OH)2/C and THF/HCl under hydrogen atmosphere, however, under
Scheme 3. Synthesis of the tricyclic gluco-iminosugars fused quinazolinone 1a and 1b. Reagents and conditions: (a) o-aminobenzamide, I2 (1.9 equiv.), 60 °C, 24 h, 67%; (b) DIAD, PPh3, THF, ice bath, 20 h, total yield 66%; (c) CF3SO3H, CH2Cl2, -80 °C-rt, 8 h, 22%. 12
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Scheme 4. The proposed mechanism for the formation of the retention product 8a.
the conditions the substrate was degraded and the reaction ended up with a complex mixture. Thus, the three step reactions could proceed effectively for the preparation of the tricyclic iminosugars. Under the same conditions, the benzyl protected mannose 5b was used to synthesize the tricyclic manno-iminosugars (Scheme 5). The corresponding manno-quinazolinone 6b was successfully obtained. However, the subsequent Mitsunobu reaction did not yield the expected compounds 7b and 8b, it generated 7a and 8a instead, in which the configuration of C-2 was inverted.
reaction conditions, however, with the above results in hand, we conceived that the superiority of the trans configuration at C-2 and C-3 might be one of the potential reasons for the inversion of C-2. After removal of the benzyl in CF3SO3H, the tricyclic arabino-iminosugars fused quinazolinone 1c-1 and 1c-2 were obtained in the yields of 52% and 23%, respectively (Scheme 6). 2.3. Structure characterization of the compounds The structures of 6c-1 and 6c-2 were determined by their 2D NMR spectra (see supporting information). Taking the 6c-1 as example, after its C and H signals were assigned by the 1H, 1H COSY and HSQC spectra, the position of the unprotected hydroxyl group was determined at C-4 due to the absent JC–H correlation between C-4/BneCH2eH in the HMBC spectrum, while the cross peaks of C-2, C-3, and C-5 and the CH2eH on Bn were observed, respectively. The unprotected hydroxyl group at the C-5 in 6c-2 was also deduced from the HMBC spectrum in which there was no JC–H correlation between C-5/BneCH2eH. The structures of the tricyclic iminosugars were determined by the HMBC and NOESY spectra (Fig. 3). In the HMBC spectra, both the key JC–H couplings of C-1/5-H and C-7(C]O)/5-H in 1a and 1b, C-1/4-H and C-6(C]O)/4-H in 1c-1 and C-1/5-H and C-6(C]O)/5-H in 1c-2 determined the structure of the new ring formed by Mitsunobu reactions, respectively. The observed cross peak between 3-H and 5-H in the NOESY spectrum of 1b suggested that it was the C-5 configuration retention product in the Mitsunobu reaction. The absent relevant signals of 3-H and 5-H in 1a and 2-H and 4-H in 1c-1 implied that they were the inversion products.
2.2. Synthesis of the tricyclic arabino-iminosugars fused quinazolinone To obtain diverse tricyclic iminosugars, both D-arabinose and D-ribose with different configurations on C-2 were employed to synthesize their corresponding tricyclic iminosugars. The preparation of the benzyl protected furan-type arabinose 5c-1 was always accompanied by the pyrantype one 5c-2 (Scheme 6). After their benzyl protected arabino-quinazolinones 6c-1 and 6c-2 were obtained, they were used in the next Mitsunobu reaction to successfully yield the expected C-4 configuration inversion product 7c-1 (the retention one was not found) and 7c-2, respectively (Scheme 6). In the synthesis of the benzyl protected ribo-quinazolinone, both compounds 6d-1 and 6d-2 were also yielded (Scheme 7). As expected, the same phenomenon was observed that 7c-1 and 7c-2 were produced when the mixture of 6d-1 and 6d-2 was employed as the reactant in Mitsunobu reaction. Using 6d-1 solely, the reaction could yield 7C-1. Both 7c-1 and 7c-2 are the products with trans configuration at C-2 and C-3, in accordance with the results from 7a and 8a. It was difficult to explain why the C-2 epimerization occurred in the Mitsunobu
Scheme 5. Synthesis of the tricyclic gluco-iminosugars from D-mannose. Reagents and conditions: (a) o-aminobenzamide, I2 (0.5 equiv.), 60 °C, 24 h, 69%; (b) DIAD, PPh3, THF, ice bath, 20 h, total yield 42%. 13
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Scheme 6. Synthesis of the tricyclic arabino-iminosugar fused quinazolinone 1c-1 and 1c-2. Reagents and conditions: (a) o-aminobenzamide, I2 (0.9 equiv.), 60 °C, 24 h, 64%; (b) DIAD, PPh3, THF, ice bath, 20 h, yield 65% for 7c-1, 60% for 7c-2; (c) CF3SO3H, CH2Cl2, -80 °C-rt, 8 h, yield 52% for 1c-1, 23% for 1c-2.
2.4. The glycosidases inhibitory activities of the compounds
iminosugars were conceived to have a functional anomeric-like position, (a sp [3] carbon atom), capable of bearing anomeric substituents [14,16], in the compounds here reported this is not the case which probably compromises recognition by the enzyme. In conclusion, a series of novel tricyclic quinazolinone-iminosugars 1 (a-c) were constructed through the intramolecular cyclization of NH and OH by the key Mitsunobu reaction, providing an effective protocol for the preparation of the tricyclic iminosugars. Interestingly, the C-2
The glycosidases inhibitory activities of the four tricyclic quinazolinone-iminosugars 1 (a-c) were examined on hydrolytic reactions of α-/β-glucosidase, α-/β-galactosidase, α-mannosidase, respectively. Unfortunately, these four tricyclic iminosugars exhibited no inhibitory effects on the tested five glycosidases on the concentrations of 100 μmol/L and 10 μmol/L (see supporting information). Whereas sp2-
Scheme 7. Synthesis of the tricyclic arabino-iminosugars from D-ribose. Reagents and conditions: (a) o-aminobenzamide, I2 (0.9 equiv.), 60 °C, 24 h, total yield 59%; (b) DIAD, PPh3, THF, ice bath, 20 h, total yield 51%. 14
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Fig. 3. Key HMBC (C→H) and NOESY (H↔H) correlations of the tricyclic iminosugars.
epimers, such as glucose and mannose (or arabinose and ribose), would generate the same products having the trans configuration at C-2 and C3 in the ring closure. However, such compounds showed no inhibitory effects on the tested five glycosidases.
3.2.2. Synthesis of the benzyl protected aldo-quizanolione 6 A mixture of benzyl protected D-glucose 5a (3.8 g, 7.0 mmol) and oaminobenzamide (1.4 g, 1.5 equiv.) in 100 mL toluene was stirred at 60 °C under N2 atmosphere for 15 min, then iodine (3.4 g, 1.9 equiv.) was added into the solution. The mixture was stirred for 24 h till the reaction completed. After the solution cooled down, toluene was evaporated under vacuum to afford the crude product. The residue was purified using flash column chromatography (petroleum ether - ethyl acetate V/V = 2:1) to afford the benzyl protected gluco-quinazolinone 6a. Under the similar conditions, the corresponding aldo-quinazolinones 6b, 6c-1, 6c-2 and 6d-1 were obtained in satisfactory yields using different benzyl protected sugars, respectively. 2-((1S,2R,3R,4S)-1,2,3,5-tetrakis(benzyloxy)-4-hydroxypentyl) quinazolin-4(3H)-one (6a): Yellow oil, yield 67%; 1H NMR (600 MHz, CDCl3), δ: 9.73 (s, 1H), 8.14 (d, J = 7.8 Hz, 1H), 7.63 (t, J = 8.4 Hz, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.36 (t, J = 7.8 Hz, 1H), 7.23–7.14 (m, 16H), 6.94 (d, J = 4.2 Hz, 3H), 6.91–6.88 (m, 1H), 4.734 (d, J = 3.6 Hz, 1H), 4.66 (d, J = 11.4 Hz, 1H), 4.58 (d, J = 11.4 Hz, 1H), 4.51–4.40 (m, 4H), 4.32 (d, J = 11.4 Hz, 1H), 3.98 (dd, J = 5.4, 3.6 Hz, 1H), 3.91 (s, 1H), 3.83 (t, J = 6.6 Hz,1H), 3.59 (dd, J = 10.2, 3.6 Hz, 1H), 3.52 (dd, J = 10.2, 5.4 Hz, 1H), 2.90 (s, 1H); 13C NMR (150 MHz, CDCl3), δ: 161.4, 154.4, 148.5, 138.1, 138.1, 136.9, 136.4, 134.4, 128.6, 128.5, 128.5, 128.4, 128.3, 128.1, 127.9, 127.8, 127.8, 127.7, 127.3, 126.8, 126.6, 121.8, 81.1, 79.7, 77.7, 75.0, 74.2, 73.4, 71.5, 71.1; HR-ESI-MS: calcd for C41H40N2O6Na ([M+Na]+), 679.2784, found: 679.2786. 2-((1R,2R,3R,4R)-1,2,3,5-tetrakis(benzyloxy)-4-hydroxypentyl) quinazolin-4(3H)-one (6b): Yellow oil, yield 69%; 1H NMR (600 MHz, CDCl3), δ: 9.92 (s, 1H), 8.12 (d, J = 7.8 Hz, 1H), 7.59 (t, J = 7.2 Hz, 1H), 7.54 (d, J = 7.8 Hz, 1H), 7.35 (t, J = 7.2 Hz, 1H), 7.22–7.19 (m, 10H), 7.10–7.08 (m, 8H), 6.97 (t, J = 6.0 Hz, 2H), 4.78 (d, J = 4.8 Hz, 1H), 4.65 (d, J = 11.4 Hz, 1H), 4.59 (d, J = 11.4 Hz, 1H), 4.48–4.39 (m, 6H), 4.34 (d, J = 11.4 Hz, 1H), 4.21 (t, J = 4.8 Hz, 1H), 3.92 (s, 1H), 3.79 (dd, J = 7.8, 5.4 Hz, 1H), 3.60 (d, J = 3.6 Hz, 2H), 3.45 (d, J = 6.6 Hz, 1H); 13C NMR (150 MHz, CDCl3), δ: 161.4, 154.1, 148.1, 138.0, 137.7, 137.6, 136.7, 134.5, 128.6, 128.5, 128.4, 128.3, 128.2, 128.0, 127.8, 127.5, 127.3, 126.9, 126.6, 121.8, 81.9, 79.7, 78.7, 75.6, 74.4, 73.4, 72.4, 71.1, 70.6; MS(ESI); HR-ESI-MS: calcd for C41H40N2O6Na ([M+Na]+), 679.2784, found: 679.2787. 2-((1R,2R,3R)-1,2,4-tris(benzyloxy)-3-hydroxybutyl)quinazolin-4(3H)-one (6c-1): Yellow oil, yield 64%; 1H NMR (400 MHz, CDCl3), δ: 9.67 (s, 1H), 8.24 (d, J = 8.0 Hz, 1H), 7.71 (t, J = 7.2 Hz, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.44 (t, J = 7.2 Hz, 1H), 7.31–7.24 (m, 10H), 7.02–6.94 (m, 5H), 4.83 (s, 1H), 4.63–4.89 (m, 2H), 4.33 (d, J = 11.2 Hz, 1H), 4.11 (d, J = 11.2 Hz, 1H), 4.05 (s, 1H), 3.99 (d, J = 8.0 Hz, 1H), 3.69 (d, J = 2.0 Hz, 2H); 13C NMR (100 MHz, CDCl3), δ: 161.2, 154.9, 137.6, 136.6, 136.3, 134.5, 128.7, 128.6, 128.6, 128.5, 128.2, 128.1, 128.1, 127.9, 127.2, 126.8, 126.7, 121.7, 80.7, 78.9, 74.5, 74.0, 73.6, 70.4, 69.7; HR-ESI-MS: calcd for C33H32N2O5Na ([M +Na]+), 559.2209, found: 559.2213. 2-((1R,2S,3R)-1,2,3-tris(benzyloxy)-4-hydroxybutyl)quinazolin-4(3H)-one (6c-2): Yellow oil, yield 64%; 1H NMR (400 MHz,
3. Experimental 3.1. General methods 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 NMR, and 13C NMR spectra were measured on a RT-NMR Bruker AVANCE 600 (600 MHz) and AVANCE 400 (400 MHz) NMR spectrometers 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 densities for examining glycosidases inhibitation were measured on a BioRad Model 3550 microplate spectrophotometer, respectively. Thin-layer chromatography (TLC) was performed on precoated plates (Qingdao GF254) with detection by UV light or with phosphomolybdic acid in EtOH/H2O followed by heating. Column chromatography was performed using SiO2 (Qingdao 300–400 mesh). Five glycosidases: αglucosidase (Aspergillus niger), β-glucosidase (almonds), α-galatosidase (coffee beans), β-galatosidase (Escherichia coli), and α-Mannosidase (Jack beans), and their corresponding substrates, p-nitrophenyl glycopyranosides, were purchased from Sigma Chemical Co. 3.2. Experimental procedures 3.2.1. Synthesis of the unexpected C-nucleoside 4 A solution of manno-quinazolinone 2 (0.9 g, 3.0 mmol) in 10 mL DMF was dropwise added into a mixture of 60% NaH (0.4 g, 3.3 equiv.) in 5 mL THF at ice bath. After the solution was stirred for 30 min, the ice bath was withdrawn. Then, the solution of TsCl (0.7 g, 1.2 equiv.) in THF was dropwise added into the reaction solution. The mixture was stirred for 7 h at room temperature till the reaction completed. The mixture was quenched with 10 mL H2O. The solvent was evaporated under vacuum to afford the crude product. The residue was purified using flash column chromatography (ethyl acetate - methanol - water V/V/V = 250:14:11) to afford the unexpected C-nucleoside 4. 2-((R)-((2S,3R,4R)-3,4-dihydroxytetrahydrofuran-2-yl) (hydroxy)methyl)quinazolin-4(3H)-one (4): White solid, yield 68%, m. p. 180.8–182.0 °C, [α] 25D -68.4 (c 0.04, H2O); 1H NMR (600 MHz, DMSO‑d6), δ: 8.10 (d, J = 7.8 Hz, 1H), 7.80 (t, J = 7.2 Hz, 1H), 7.63 (d, J = 8.4 Hz, 1H), 7.50 (t, J = 7.2 Hz, 1H), 5.14 (s, 1H), 4.82 (s, 1H), 3.86 (d, J = 7.8 Hz, 1H), 3.67 (dd, J = 5.4, 1.8 Hz, 1H), 3.58 (dd, J = 10.8, 4.2 Hz, 1H), 3.51–3.39 (m, 5H); 13C NMR (150 MHz, CD3OD), δ: 161.5, 153.2, 148.2, 134.5, 126.8, 126.6, 125.9, 121.5, 73.0, 70.4, 62.9, 60.6, 52.9; HR-ESI-MS: calcd for C13H14N2O5Na ([M+Na]+), 301.0800, found: 301.0806. 15
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CDCl3), δ: 9.59 (s, 1H), 8.24 (d, J = 7.6 Hz, 1H), 7.73 (t, J = 7.2 Hz, 1H), 7.61 (d, J = 8.0 Hz, 1H), 7.46 (t, J = 7.2 Hz, 1H), 7.30–7.22 (m, 10H), 7.09–6.97 (m, 5H), 4.74 (d, J = 2.0 Hz, 1H), 4.56–4.51 (m, 3H), 4.39–4.23 (m, 3H), 4.13–4.11 (m, 1H), 4.02–3.97 (m, 1H), 3.83–3.81 (m, 2H); 13C NMR (100 MHz, CDCl3), δ: 161.1, 154.7, 148.5, 137.8, 136.6, 136.2, 134.5, 128.7, 128.7, 128.5, 128.4, 128.3, 127.9, 127.6, 127.2, 126.8, 126.7, 121.8, 79.4, 79.2, 78.5, 75.0, 73.4, 71.6, 59.3; HRESI-MS: calcd for C33H32N2O5Na ([M+Na]+), 559.2209, found: 559.2208. 2-((1S,2R,3R)-1,2,4-tris(benzyloxy)-3-hydroxybutyl)quinazolin-4(3H)-one (6d-1): Yellow oil, yield 59%; 1H NMR (600 MHz, CDCl3), δ: 9.93 (s, 1H), 8.24 (d, J = 7.8 Hz, 1H), 7.73 (t, J = 8.4 Hz, 1H), 7.67 (d, J = 8.4 Hz, 1H), 7.46 (t, J = 7.8 Hz, 1H), 7.34–7.24 (m, 13H), 7.17–7.16 (m, 2H), 4.91 (d, J = 2.4 Hz, 1H), 4.83 (d, J = 11.4 Hz, 1H), 4.65 (dd, J = 25.8, 11.4 Hz, 2H), 4.55–4.44 (m, 3H), 4.13 (d, J = 7.8 Hz, 1H), 3.81 (s, 2H), 3.57–3.51 (m, 2H); 13C NMR (150 MHz, CDCl3), δ: 161.4, 154.0, 148.1, 137.9, 137.6, 136.6, 134.6, 128.7, 128.4, 128.3, 128.1, 128.0, 127.9, 127.8, 127.2, 127.0, 126.6, 121.8, 80.8, 79.9, 74.7, 73.6, 73.2, 70.6, 70.1; HR-ESI-MS: calcd for C33H32N2O5Na ([M+Na]+), 559.2209, found: 559.2205.
137.7, 136.5, 136.2, 134.5, 128.8, 128.7, 128.6, 128.5, 128.3, 128.0, 128.0, 127.7, 127.1, 126.9, 126.7, 121.8, 79.3, 79.1, 78.4, 75.0, 73.4, 71.6, 59.2; HR-ESI-MS: calcd for C33H30N2O4Na ([M+Na]+), 541.2103, found: 541.2109. (6R,7R,8R)-6,7,8-tris(benzyloxy)-8,9-dihydro-6H-pyrido[2,1b]quinazolin-11(7H)-one (7c-2): Yellow oil, yield 60%; 1H NMR (600 MHz, CDCl3), δ: 8.28 (d, J = 7.8 Hz, 1H), 7.72 (t, J = 6.0 Hz, 2H), 7.48–7.45 (m, 1H), 7.35–7.24 (m, 15H), 4.93 (d, J = 11.4 Hz, 1H), 4.77 (d, J = 11.4 Hz, 1H), 4.73–4.61 (m, 5H), 4.39–4.34 (m, 2H), 4.07 (d, J = 5.0 Hz, 1H), 4.04–4.00 (m, 1H); 13C NMR (150 MHz, CDCl3), δ: 162.2, 151.2, 147.2, 137.8, 137.7, 137.6, 134.1, 128.5, 128.4, 127.9, 127.8, 127.8, 127.4, 127.0, 126.7, 120.9, 77.9, 74.4, 72.7, 72.6, 71.6, 71.1, 42.8; HR-ESI-MS: calcd for C33H30N2O4Na ([M+Na]+), 541.2103, found: 541.2108. 3.2.4. Synthesis of the tricyclic iminosugars 1 To a solution of 7a (0.5 g, 0.8 mmol) in 100 mL dried CH2Cl2 at −80 °C, trifloromethanesulfonic acid (CF3SO3H, 0.14 mL, 2.0 equiv.) was dropwise slowly added under N2 atmosphere. After the solution was stirred for 2 h at −80 °C, the temperature was improved to room temperature. The mixture was stirred at rt for 6 h until the reaction completed. K2CO3 Was added to the solution to neutralize CF3SO3H. After the solvent was evaporated under vacuum to afford the crude product, the residue was purified using flash column chromatography (dicholomethane - methanol V/V = 9:1) to afford compound 1a. Under the similar conditions, the corresponding compounds 1b, 1c1 and 1c-2 were obtained, respectively. (6S,7S,8R,9S)-6,7,8-trihydroxy-9-(hydroxymethyl)-8,9-dihydro-6H-pyrido[2,1-b]quinazolin-11(7H)-one (1a): White solid, yield 22%, m. p. 195.3–196.0 °C, [α] 25D +54.0 (c 0.04, H2O); 1H NMR (400 MHz, DMSO‑d6), δ: 8.12 (d, J = 8.0 Hz, 1H), 7.80 (t, J = 8.0 Hz, 1H), 7.65 (d, J = 8.0 Hz, 1H), 7.49 (t, J = 8.0 Hz, 1H), 4.79–4.74 (m, 1H), 4.27 (d, J = 6.8 Hz, 1H), 4.14 (dd, J = 10.4, 6.8 Hz, 1H), 3.90 (dd, J = 11.6, 2.4 Hz, 1H), 3.77(dd, J = 10.4, 6.0 Hz, 1H), 3.70 (dd, J = 11.2, 3.6 Hz, 1H); 13C NMR (100 MHz, DMSO‑d6), δ: 161.3, 157.5, 147.6, 134.8, 127.0, 126.8, 126.7, 120.6, 74.8, 72.4, 69.3, 56.8, 55.9; HR-ESI-MS: calcd for C13H14N2O5Na ([M+Na]+), 301.0800, found: 301.0803. (6S,7S,8R,9R)-6,7,8-trihydroxy-9-(hydroxymethyl)-8,9-dihydro-6H-pyrido[2,1-b]quinazolin-11(7H)-one (1b): White solid, yield 22%, m. p. 184.2–185.6 °C, [α] 25D +42.0 (c 0.04, H2O); 1H NMR (400 MHz, DMSO‑d6), δ: 8.15 (d, J = 7.2 Hz, 1H), 7.81 (t, J = 7.2 Hz, 1H), 7.67 (d, J = 8.0 Hz, 1H), 7.51 (t, J = 7.2 Hz, 1H), 5.58–4.77 (m, 6H), 4.26 (s, 1H), 4.16 (t, J = 6.8 Hz, 1H), 3.95 (d, J = 10.8 Hz, 2H), 3.70 (d, J = 7.2 Hz, 1H); 13C NMR (100 MHz, DMSO‑d6), δ: 161.3, 157.2, 147.4, 134.8, 127.0, 127.0, 126.8, 120.8, 70.4, 70.3, 67.7, 56.5, 56.0; HR-ESI-MS: calcd for C13H14N2O5Na ([M+Na]+), 301.0800, found: 301.0804. ((1S,2R,3R)-1,2,3,4-tetrahydroxybutyl)quinazolin-4(3H)-one (1c-1): White solid, yield 52%, m. p. 182.6–184.0 °C, [α] 25D +107.5 (c 0.04, H2O); 1H NMR (400 MHz, DMSO‑d6), δ: 8.14 (d, J = 7.6 Hz, 1H), 7.80 (t, J = 7.6 Hz, 1H), 7.68 (d, J = 8.0 Hz, 1H), 7.50 (t, J = 7.6 Hz, 1H), 6.12 (s, 1H), 5.90 (s, 1H), 5.01 (d, J = 8.4 Hz, 1H), 4.78 (s, 1H), 4.45 (d, J = 8.0 Hz, 1H), 4.30 (t, J = 8.4 Hz, 1H), 3.89 (s, 2H); 13C NMR (100 MHz, DMSO‑d6), δ: 160.1, 160.0, 149.2, 134.6, 127.4, 126.7, 126.3, 121.7, 76.2, 74.5, 59.5, 55.9; HR-ESI-MS: calcd for C12H12N2O4Na ([M+Na]+), 271.0695, found: 271.0694. (6R,7R,8R)-6,7,8-trihydroxy-8,9-dihydro-6H-pyrido[2,1-b]quinazolin-11(7H)-one (1c-2): White solid, yield 23%, m. p. 187.5–188.3 °C, [α] 25D +24.6 (c 0.04, H2O); 1H NMR (400 MHz, CD3OD) δ: 8.16 (d, J = 8.4 Hz, 1H), 7.78 (t, J = 7.2 Hz, 1H), 7.69 (d, J = 8.0 Hz, 1H), 7.49 (t, J = 7.6 Hz, 1H), 4.74 (d, J = 6.4 Hz, 1H), 4.44–4.41 (m, 1H), 4.11–4.04 (m, 3H); 13C NMR (100 MHz, CD3OD) δ: 162.3, 155.0, 147.1, 134.3, 126.8, 126.3, 125.9, 119.8, 71.8, 70.8, 64.4, 45.7; HR-ESI-MS: calcd for C12H12N2O4Na ([M+Na]+), 271.0695, found: 271.0691.
3.2.3. Synthesis of the tricyclic iminosugars 7 and 8 by Mitsunobu reaction To a solution of 6a (1.8 g, 2.7 mmol) in 150 mL dried THF, triphenylphosphine (PPh3, 2.2 g, 3.0 equiv.) was added under N2 atmosphere at ice bath. After the solution was stirred for 30 min, diisopropyl azodicarboxylate (DIAD, 1.7 mL) was dropwise added into the solution at ice bath. The mixture was stirred for 20 h till the reaction completed. After 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 = 8:1) to afford compounds 7a and 8a. Under the similar conditions, the corresponding compounds 7c-1 and 7c-2 were obtained in satisfactory yields, respectively. (6S,7S,8R,9S)-6,7,8-tris(benzyloxy)-9-((benzyloxy)methyl)-8,9dihydro-6H-pyrido[2,1-b]quinazolin-11(7H)-one (7a): Yellow oil, yield 43%; 1H NMR (400 MHz, CDCl3), δ: 8.37 (d, J = 7.6 Hz, 1H), 7.82 (d, J = 3.6 Hz, 2H), 7.57–7.53 (m, 1H), 7.48 (d, J = 6.8 Hz, 2H), 7.43–7.37 (m, 13H), 7.25–7.18 (m, 5H), 5.59 (dd, J = 9.6, 5.2 Hz, 1H), 5.01 (d, J = 11.6 Hz, 1H), 4.90–4.75 (m, 5H), 4.68 (d, J = 4.0 Hz, 1H), 4.54 (s, 2H), 4.47 (dd, J = 9.2, 4.0 Hz, 1H), 4.18 (dd, J = 10.8, 5.2 Hz, 1H), 4.07 (dd, J = 10.4, 4.0 Hz, 1H), 3.93 (dd, J = 9.2, 5.6 Hz, 1H); 13C NMR (100 MHz, CDCl3), δ: 161.6, 152.4, 147.2, 138.1, 138.0, 138.0, 137.8, 134.4, 128.5, 128.4, 128.4, 128.2, 128.0, 127.9, 127.8, 127.7, 127.6, 127.6, 127.4, 127.2, 127.1, 120.8, 81.0, 81.0, 78.4, 73.4, 73.1, 73.0, 72.5, 66.8, 51.6; HR-ESI-MS: calcd for C41H38N2O5Na ([M +Na]+), 661.2678, found: 661.2682. (6S,7S,8R,9R)-6,7,8-tris(benzyloxy)-9-((benzyloxy)methyl)8,9-dihydro-6H-pyrido[2,1-b]quinazolin-11(7H)-one (8a): Yellow oil, yield 23%; 1H NMR (600 MHz, CDCl3), δ: 8.25 (d, J = 7.6 Hz, 1H), 7.80–7.53 (m, 2H), 7.51–7.47 (m, 1H), 7.44 (d, J = 6.8 Hz, 2H), 7.40–7.26 (m, 13H), 7.11 (d, J = 7.6 Hz, 1H), 7.05 (t, J = 7.6 Hz, 2H), 6.91 (d, J = 7.2 Hz, 2H), 5.27–5.25 (m, 1H), 4.95–4.70 (m, 8H), 4.56 (dd, J = 9.2, 6.8 Hz, 1H), 4.32 (d, J = 12.4 Hz, 1H), 4.20 (d, J = 12.4 Hz, 1H), 4.13 (d, J = 9.2 Hz, 1H); 13C NMR (100 MHz, CDCl3), δ: 161.9, 152.6, 150.2, 147.1, 138.5, 138.3, 138.0, 137.6, 134.3, 128.5, 128.4, 128.3, 128.2, 127.8, 127.8, 127.7, 127.6, 127.5, 127.4, 127.2, 127.0, 127.0, 121.2, 76.1, 74.6, 73.8, 73.0, 72.8, 72.3, 72.1, 64.7, 53.7; HR-ESI-MS: calcd for C41H38N2O5Na ([M+Na]+), 661.2678, found: 661.2680. (1S,2R,3R)-2,3-bis(benzyloxy)-1-((benzyloxy)methyl)-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (7c-1): Yellow oil, yield 65%; 1H NMR (400 MHz, CDCl3), δ: 8.24 (d, J = 7.6 Hz, 1H), 7.82–7.74 (m, 2H), 7.52–7.46 (m, 3H), 7.41–7.34 (m, 8H), 7.16–7.07 (m, 5H), 5.38–5.35 (m, 2H), 5.00 (d, J = 11.6 Hz, 1H), 4.79–4.69 (m, 3H), 4.47–4.37 (m, 3H), 4.01 (dd, J = 10.2, 2.8 Hz, 1H), 3.89 (d, J = 10.0 Hz, 1H); 13C NMR (100 MHz, CDCl3), δ: 161.1, 154.8, 148.4, 16
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3.2.5. Glycosidases inhibition The inhibitory activity of the synthesized compounds against five glycosidases: α-glucosidase (Aspergillus niger), β-glucosidase (almonds), α-galatosidase (coffee beans), β-galatosidase (Escherichia coli), and αMannosidase (Jack beans). The enzymes and their corresponding substrates, p-nitrophenyl glycopyranosides, were purchased from Sigma Chemical Co. All other commercial reagents were used as received. Taken the α-Glucosidase assay as example: α-Glucosidase assay was performed using PNPG (2 mmol/L) as substrate in pH = 5.5 citric acid - phosphate buffers (CPBs) at 37 °C. 10 μL of enzyme solution (50 u/mL in CPBs), 20 μL of inhibitor (6 mmol/L) and 20 μL of buffer were incubated for 10 min at 37 °C, and then 50 μL of PNPG was added. The mixture were incubated for 20 min. The reaction was quenched with 100 μL of sodium carbonate (1 mol/L) for 10 min. Absorbance readings were taken on a BioRad Model 3550 microplate spectrophotometer at 405 nm using distilled deionized water as a blank control.
[11] [12] [13] [14] [15] [16] [17] [18] [19] [20]
Acknowledgments
[21] [22]
The financial supports from the National Natural Science Foundation of China (NSFC) (21772031 and 21778013).
[23]
Appendix A. Supplementary data
[25]
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.carres.2019.04.002.
[26] [27] [28]
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Synthesis of tricyclic quinazolinone-iminosugars as potential glycosidase inhibitors via a Mitsunobu reaction
T
Jiajing Sun, Yaqing Kang, Ligang Gao, Xin Lu, Huanhuan Ju, Xiaoliu Li∗∗, Hua Chen∗ Key Laboratory of Chemical Biology of Hebei Province, College of Chemistry and Environmental Science, Hebei University, Baoding, 071002, China
ARTICLE INFO
ABSTRACT
Keywords: Tricyclic iminosugars Quizanolinone Iodine-induced oxidative condensation Mitsunobu reaction Glycosidase inhibitory activity
A series of novel tricyclic quinazolinone-iminosugars 1 (a-c) were synthesized from the benzyl protected sugars through three steps. Firstly, the benzyl protected sugar (aldehyde) 5 reacted with o-aminobenzamide by the iodine-induced oxidative condensation to afford the corresponding aldo-quizanolinone 6. Secondly, through the intramolecular cyclization of the unprotected OH and the amide NH in 6, the tricyclic compounds 7 and 8 were constructed by the key Mitsunobu reaction. Finally, removal of the benzyl group gave the target tricyclic quinazolinone-iminosugars 1. The protocol was effective for the preparation of the tricyclic iminosugars in satisfactory yield. Interestingly, an unusual C-2 epimerization was observed with D-mannose and D-ribose compounds under the conditions of the Mitsunobu reaction that generated the products having the trans configuration at the C-2 and C-3 positions. Unfortunately, such tricyclic quinazolinone-iminosugars showed no inhibitory effects on the tested five glycosidases.
1. Introduction Glycosidases are the specific glycosidic bond hydrolases directly involved in the glycosylation on cell surface, and are also responsible for the glycosylation of the proteins or lipids with important biological activities [1]. The occurrence and development of many diseases, such as diabetes, cancers, viral infections, autoimmune diseases, are closely related to the abnormal expression of the glycosidases [2]. Therefore, the study of glycosidase inhibitors has become one of the hot topics in drug development [3]. As one of the most important classes of glycosidase inhibitor, iminosugars and their analogs are widely used for the treatment of carbohydrate-mediated diseases (compounds A-E, Fig. 1) [4]. They are the structural mimetics of the natural carbohydrates with endocyclic nitrogen atom, and can mimic the enzymatic transition state to inhibit glycosidase activity [5]. Generally, they can be easily recognized by glycosidases as the cationic intermediates adopting a half chair conformation under physiological conditions. Both structural features are becoming the key basis for the rational design of glycosidase inhibitors derived from iminosugars [6]. For examples, the amidine (compound F) and amidoxime (compound G) (Fig. 1) inhibitors could effectively inhibit both α- and β-glycosidases [7]. On the basis of the enzymatic transition state [8], a variety of bicyclic iminosugars fused to different nitrogen-containing
∗
heterocycles (compounds H-J, Fig. 1), which were called sp2-iminosugars [9–13] (the anomeric carbon having sp2-character), were rationally designed and displayed significant inhibitory activities against different glycosidases. The heterocycles included imidazole, trizaole, tetrazole, oxazole, and thiazole, etc. The increased potency of such sp2-iminosugars as glycosidase inhibitors suggested the preparation of other derivatives for the new carbohydrate-based drug discovery [14–16]. However, multicyclic (more than 3-fused rings) iminosugars [17–20] having sp2-character on the anomeric carbon have received much less attention possibly due to the difficulties of their synthesis. As a continuation of our studies on the bioactive multicyclic iminosugars [21–23], we would like to report the synthesis of novel tricyclic iminosugars fused 4-quinazolinone 1 (a-c) (Fig. 2) by a Mitsunobu reaction. As an important nitrogen-containing pharmacophore, 4(3H)-quinazolinone and its derivatives have attracted much attention due to their diverse biological activities, such as anti-cancer, anti-bacterial, anti-virus, anti-diabetes, and anti-glycosidase activities [24–26]. Such new tricyclic quinazolinone-iminosugars were thought to be capable of mimicking the enzymatic transition state of glycosidases due to the sp2-character of their anomeric carbons, and they were evaluated for their glycosidase inhibitory activity.
Corresponding author. Corresponding author. E-mail address: [email protected] (H. Chen).
∗∗
https://doi.org/10.1016/j.carres.2019.04.002 Received 16 February 2019; Received in revised form 9 April 2019; Accepted 14 April 2019 Available online 23 April 2019 0008-6215/ © 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. Examples of known iminosugars with effective glycosidase inhibitory activity. Fig. 2. The synthetic tricyclic quinazolinone-iminosugars 1 (a–c).
2. Results and discussion
induced oxidative condensation of D-mannose and o-aminobenzamides [28] to synthesize the corresponding tricyclic iminosugar fused quinazolinone 3. However, the reaction did not give the expected product under the same reaction conditions. Changing pyridine with sodium hydride (NaH) in DMF solution, the reaction gave an unexpected Cnucleoside [29] 4 in the good yield of 68% (Scheme 1), in which the intramolecular cyclization took place between 6-OH and 3-OH identified by its X-ray crystallographic structure (see supporting information) [30]. The reason that compound 4 is formed in preference to compound 3 is possibly that cyclization to form five-membered ring is much more favoured than cyclization to form a seven-membered ring.
2.1. Synthesis of the tricyclic gluco-iminosugars fused quinazolinone Sallam et al. [27] had reported that treatment of D-arabino-tetritol1-yl-benzimidazole with p-toluene sulfonyl chloride (TsCl) in pyridine solution could afford an interesting tricyclic benzimidazole fused iminosugar, which was achieved by the intramolecular SN2 nucleophilic substitution between the primary tosyl intermediate and the 1-nitrogen atom of the benzimidazole moiety (Scheme 1). Therefore, we attempted to use manno-quinazolinone 2 that was directly prepared by the iodine-
Scheme 1. Synthesis of an unexpected C-nucleoside 4. 11
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Scheme 2. The retrosynthesis route for the tricyclic quinazolinone-iminosugar.
As an alternative method, Mitsunobu reaction was widely used to construct nitrogen-containing heterocycles [31–33]. Cheon et al. [34] had reported the intramolecular cyclization of quinazolinone to afford mackinazolinone by Mitsunobu reaction (Scheme 2). Inspired by this positive result, we conceived that our compound 1 could be prepared via a Mitsunobu reaction on the benzyl protected aldo-quinazolinone 6 (Scheme 2). We initiated our synthesis from the benzyl protected aldo-quinazolinone 6 that could be easily prepared by the iodine-induced oxidative condensation of benzyl protected sugar aldehyde 5 and o-aminobenzamides [28]. Taking the synthesis of the tricyclic gluco-iminosugar 6a as example (Scheme 3), the available benzyl protected glucose (5a) reacted with o-aminobenzamide to afford the corresponding gluco-quinazolinone (6a) in yield of 67% according to the above mentioned method. Then, Mitsunobu reaction was successfully performed to realize the cyclization of the unprotected 5-OH and the amide NH in 6a in the presence of the DIAD and PPh3. Generally the Mitsunobu reaction generates the inversion product [35–37]. However, in this case, the reaction provided a pair of diastereoisomers, the C-5 configuration
inversion product 7a and the C-5 configuration retention one 8a, in total good yields of 66% although the reaction conditions were not optimized. The inversion product 7a was the predominant one and the ratio of 7a: 8a was 1.9:1. We conceived that the Mitsunobu reaction resulted in retention possibly due to neighbouring group participation [38] and double inversion (Scheme 4). The neighbouring group is most likely the 6-O-benzyl. Attack by the C-6 oxygen on C-5 with loss of Ph3PO would result in the (O-benzyl) C5eC6 oxiran (epoxy derivative). This, being analogous to a protonated epoxide, would be rapidly ringopened by the amide nitrogen attacking at C-5 (a favoured 6-exo-tet process) resulting in retention of configuration at C-5. Presumably, oxiran ring formation is competitive with nucleophilic attack by the amide nitrogen. Finally, removal of the benzyl group from 7a in CF3SO3H was carried out at −80 °C to give the target tricyclic glucoiminosugar in low yield of 22%. The temperature had important effect on the reaction. The reaction could not afford the target product under higher temperature, such as −40 °C, which rapidly cause the reaction complex. An alternative attempt to remove the benzyl group was in Pd (OH)2/C and THF/HCl under hydrogen atmosphere, however, under
Scheme 3. Synthesis of the tricyclic gluco-iminosugars fused quinazolinone 1a and 1b. Reagents and conditions: (a) o-aminobenzamide, I2 (1.9 equiv.), 60 °C, 24 h, 67%; (b) DIAD, PPh3, THF, ice bath, 20 h, total yield 66%; (c) CF3SO3H, CH2Cl2, -80 °C-rt, 8 h, 22%. 12
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Scheme 4. The proposed mechanism for the formation of the retention product 8a.
the conditions the substrate was degraded and the reaction ended up with a complex mixture. Thus, the three step reactions could proceed effectively for the preparation of the tricyclic iminosugars. Under the same conditions, the benzyl protected mannose 5b was used to synthesize the tricyclic manno-iminosugars (Scheme 5). The corresponding manno-quinazolinone 6b was successfully obtained. However, the subsequent Mitsunobu reaction did not yield the expected compounds 7b and 8b, it generated 7a and 8a instead, in which the configuration of C-2 was inverted.
reaction conditions, however, with the above results in hand, we conceived that the superiority of the trans configuration at C-2 and C-3 might be one of the potential reasons for the inversion of C-2. After removal of the benzyl in CF3SO3H, the tricyclic arabino-iminosugars fused quinazolinone 1c-1 and 1c-2 were obtained in the yields of 52% and 23%, respectively (Scheme 6). 2.3. Structure characterization of the compounds The structures of 6c-1 and 6c-2 were determined by their 2D NMR spectra (see supporting information). Taking the 6c-1 as example, after its C and H signals were assigned by the 1H, 1H COSY and HSQC spectra, the position of the unprotected hydroxyl group was determined at C-4 due to the absent JC–H correlation between C-4/BneCH2eH in the HMBC spectrum, while the cross peaks of C-2, C-3, and C-5 and the CH2eH on Bn were observed, respectively. The unprotected hydroxyl group at the C-5 in 6c-2 was also deduced from the HMBC spectrum in which there was no JC–H correlation between C-5/BneCH2eH. The structures of the tricyclic iminosugars were determined by the HMBC and NOESY spectra (Fig. 3). In the HMBC spectra, both the key JC–H couplings of C-1/5-H and C-7(C]O)/5-H in 1a and 1b, C-1/4-H and C-6(C]O)/4-H in 1c-1 and C-1/5-H and C-6(C]O)/5-H in 1c-2 determined the structure of the new ring formed by Mitsunobu reactions, respectively. The observed cross peak between 3-H and 5-H in the NOESY spectrum of 1b suggested that it was the C-5 configuration retention product in the Mitsunobu reaction. The absent relevant signals of 3-H and 5-H in 1a and 2-H and 4-H in 1c-1 implied that they were the inversion products.
2.2. Synthesis of the tricyclic arabino-iminosugars fused quinazolinone To obtain diverse tricyclic iminosugars, both D-arabinose and D-ribose with different configurations on C-2 were employed to synthesize their corresponding tricyclic iminosugars. The preparation of the benzyl protected furan-type arabinose 5c-1 was always accompanied by the pyrantype one 5c-2 (Scheme 6). After their benzyl protected arabino-quinazolinones 6c-1 and 6c-2 were obtained, they were used in the next Mitsunobu reaction to successfully yield the expected C-4 configuration inversion product 7c-1 (the retention one was not found) and 7c-2, respectively (Scheme 6). In the synthesis of the benzyl protected ribo-quinazolinone, both compounds 6d-1 and 6d-2 were also yielded (Scheme 7). As expected, the same phenomenon was observed that 7c-1 and 7c-2 were produced when the mixture of 6d-1 and 6d-2 was employed as the reactant in Mitsunobu reaction. Using 6d-1 solely, the reaction could yield 7C-1. Both 7c-1 and 7c-2 are the products with trans configuration at C-2 and C-3, in accordance with the results from 7a and 8a. It was difficult to explain why the C-2 epimerization occurred in the Mitsunobu
Scheme 5. Synthesis of the tricyclic gluco-iminosugars from D-mannose. Reagents and conditions: (a) o-aminobenzamide, I2 (0.5 equiv.), 60 °C, 24 h, 69%; (b) DIAD, PPh3, THF, ice bath, 20 h, total yield 42%. 13
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Scheme 6. Synthesis of the tricyclic arabino-iminosugar fused quinazolinone 1c-1 and 1c-2. Reagents and conditions: (a) o-aminobenzamide, I2 (0.9 equiv.), 60 °C, 24 h, 64%; (b) DIAD, PPh3, THF, ice bath, 20 h, yield 65% for 7c-1, 60% for 7c-2; (c) CF3SO3H, CH2Cl2, -80 °C-rt, 8 h, yield 52% for 1c-1, 23% for 1c-2.
2.4. The glycosidases inhibitory activities of the compounds
iminosugars were conceived to have a functional anomeric-like position, (a sp [3] carbon atom), capable of bearing anomeric substituents [14,16], in the compounds here reported this is not the case which probably compromises recognition by the enzyme. In conclusion, a series of novel tricyclic quinazolinone-iminosugars 1 (a-c) were constructed through the intramolecular cyclization of NH and OH by the key Mitsunobu reaction, providing an effective protocol for the preparation of the tricyclic iminosugars. Interestingly, the C-2
The glycosidases inhibitory activities of the four tricyclic quinazolinone-iminosugars 1 (a-c) were examined on hydrolytic reactions of α-/β-glucosidase, α-/β-galactosidase, α-mannosidase, respectively. Unfortunately, these four tricyclic iminosugars exhibited no inhibitory effects on the tested five glycosidases on the concentrations of 100 μmol/L and 10 μmol/L (see supporting information). Whereas sp2-
Scheme 7. Synthesis of the tricyclic arabino-iminosugars from D-ribose. Reagents and conditions: (a) o-aminobenzamide, I2 (0.9 equiv.), 60 °C, 24 h, total yield 59%; (b) DIAD, PPh3, THF, ice bath, 20 h, total yield 51%. 14
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Fig. 3. Key HMBC (C→H) and NOESY (H↔H) correlations of the tricyclic iminosugars.
epimers, such as glucose and mannose (or arabinose and ribose), would generate the same products having the trans configuration at C-2 and C3 in the ring closure. However, such compounds showed no inhibitory effects on the tested five glycosidases.
3.2.2. Synthesis of the benzyl protected aldo-quizanolione 6 A mixture of benzyl protected D-glucose 5a (3.8 g, 7.0 mmol) and oaminobenzamide (1.4 g, 1.5 equiv.) in 100 mL toluene was stirred at 60 °C under N2 atmosphere for 15 min, then iodine (3.4 g, 1.9 equiv.) was added into the solution. The mixture was stirred for 24 h till the reaction completed. After the solution cooled down, toluene was evaporated under vacuum to afford the crude product. The residue was purified using flash column chromatography (petroleum ether - ethyl acetate V/V = 2:1) to afford the benzyl protected gluco-quinazolinone 6a. Under the similar conditions, the corresponding aldo-quinazolinones 6b, 6c-1, 6c-2 and 6d-1 were obtained in satisfactory yields using different benzyl protected sugars, respectively. 2-((1S,2R,3R,4S)-1,2,3,5-tetrakis(benzyloxy)-4-hydroxypentyl) quinazolin-4(3H)-one (6a): Yellow oil, yield 67%; 1H NMR (600 MHz, CDCl3), δ: 9.73 (s, 1H), 8.14 (d, J = 7.8 Hz, 1H), 7.63 (t, J = 8.4 Hz, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.36 (t, J = 7.8 Hz, 1H), 7.23–7.14 (m, 16H), 6.94 (d, J = 4.2 Hz, 3H), 6.91–6.88 (m, 1H), 4.734 (d, J = 3.6 Hz, 1H), 4.66 (d, J = 11.4 Hz, 1H), 4.58 (d, J = 11.4 Hz, 1H), 4.51–4.40 (m, 4H), 4.32 (d, J = 11.4 Hz, 1H), 3.98 (dd, J = 5.4, 3.6 Hz, 1H), 3.91 (s, 1H), 3.83 (t, J = 6.6 Hz,1H), 3.59 (dd, J = 10.2, 3.6 Hz, 1H), 3.52 (dd, J = 10.2, 5.4 Hz, 1H), 2.90 (s, 1H); 13C NMR (150 MHz, CDCl3), δ: 161.4, 154.4, 148.5, 138.1, 138.1, 136.9, 136.4, 134.4, 128.6, 128.5, 128.5, 128.4, 128.3, 128.1, 127.9, 127.8, 127.8, 127.7, 127.3, 126.8, 126.6, 121.8, 81.1, 79.7, 77.7, 75.0, 74.2, 73.4, 71.5, 71.1; HR-ESI-MS: calcd for C41H40N2O6Na ([M+Na]+), 679.2784, found: 679.2786. 2-((1R,2R,3R,4R)-1,2,3,5-tetrakis(benzyloxy)-4-hydroxypentyl) quinazolin-4(3H)-one (6b): Yellow oil, yield 69%; 1H NMR (600 MHz, CDCl3), δ: 9.92 (s, 1H), 8.12 (d, J = 7.8 Hz, 1H), 7.59 (t, J = 7.2 Hz, 1H), 7.54 (d, J = 7.8 Hz, 1H), 7.35 (t, J = 7.2 Hz, 1H), 7.22–7.19 (m, 10H), 7.10–7.08 (m, 8H), 6.97 (t, J = 6.0 Hz, 2H), 4.78 (d, J = 4.8 Hz, 1H), 4.65 (d, J = 11.4 Hz, 1H), 4.59 (d, J = 11.4 Hz, 1H), 4.48–4.39 (m, 6H), 4.34 (d, J = 11.4 Hz, 1H), 4.21 (t, J = 4.8 Hz, 1H), 3.92 (s, 1H), 3.79 (dd, J = 7.8, 5.4 Hz, 1H), 3.60 (d, J = 3.6 Hz, 2H), 3.45 (d, J = 6.6 Hz, 1H); 13C NMR (150 MHz, CDCl3), δ: 161.4, 154.1, 148.1, 138.0, 137.7, 137.6, 136.7, 134.5, 128.6, 128.5, 128.4, 128.3, 128.2, 128.0, 127.8, 127.5, 127.3, 126.9, 126.6, 121.8, 81.9, 79.7, 78.7, 75.6, 74.4, 73.4, 72.4, 71.1, 70.6; MS(ESI); HR-ESI-MS: calcd for C41H40N2O6Na ([M+Na]+), 679.2784, found: 679.2787. 2-((1R,2R,3R)-1,2,4-tris(benzyloxy)-3-hydroxybutyl)quinazolin-4(3H)-one (6c-1): Yellow oil, yield 64%; 1H NMR (400 MHz, CDCl3), δ: 9.67 (s, 1H), 8.24 (d, J = 8.0 Hz, 1H), 7.71 (t, J = 7.2 Hz, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.44 (t, J = 7.2 Hz, 1H), 7.31–7.24 (m, 10H), 7.02–6.94 (m, 5H), 4.83 (s, 1H), 4.63–4.89 (m, 2H), 4.33 (d, J = 11.2 Hz, 1H), 4.11 (d, J = 11.2 Hz, 1H), 4.05 (s, 1H), 3.99 (d, J = 8.0 Hz, 1H), 3.69 (d, J = 2.0 Hz, 2H); 13C NMR (100 MHz, CDCl3), δ: 161.2, 154.9, 137.6, 136.6, 136.3, 134.5, 128.7, 128.6, 128.6, 128.5, 128.2, 128.1, 128.1, 127.9, 127.2, 126.8, 126.7, 121.7, 80.7, 78.9, 74.5, 74.0, 73.6, 70.4, 69.7; HR-ESI-MS: calcd for C33H32N2O5Na ([M +Na]+), 559.2209, found: 559.2213. 2-((1R,2S,3R)-1,2,3-tris(benzyloxy)-4-hydroxybutyl)quinazolin-4(3H)-one (6c-2): Yellow oil, yield 64%; 1H NMR (400 MHz,
3. Experimental 3.1. General methods 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 NMR, and 13C NMR spectra were measured on a RT-NMR Bruker AVANCE 600 (600 MHz) and AVANCE 400 (400 MHz) NMR spectrometers 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 densities for examining glycosidases inhibitation were measured on a BioRad Model 3550 microplate spectrophotometer, respectively. Thin-layer chromatography (TLC) was performed on precoated plates (Qingdao GF254) with detection by UV light or with phosphomolybdic acid in EtOH/H2O followed by heating. Column chromatography was performed using SiO2 (Qingdao 300–400 mesh). Five glycosidases: αglucosidase (Aspergillus niger), β-glucosidase (almonds), α-galatosidase (coffee beans), β-galatosidase (Escherichia coli), and α-Mannosidase (Jack beans), and their corresponding substrates, p-nitrophenyl glycopyranosides, were purchased from Sigma Chemical Co. 3.2. Experimental procedures 3.2.1. Synthesis of the unexpected C-nucleoside 4 A solution of manno-quinazolinone 2 (0.9 g, 3.0 mmol) in 10 mL DMF was dropwise added into a mixture of 60% NaH (0.4 g, 3.3 equiv.) in 5 mL THF at ice bath. After the solution was stirred for 30 min, the ice bath was withdrawn. Then, the solution of TsCl (0.7 g, 1.2 equiv.) in THF was dropwise added into the reaction solution. The mixture was stirred for 7 h at room temperature till the reaction completed. The mixture was quenched with 10 mL H2O. The solvent was evaporated under vacuum to afford the crude product. The residue was purified using flash column chromatography (ethyl acetate - methanol - water V/V/V = 250:14:11) to afford the unexpected C-nucleoside 4. 2-((R)-((2S,3R,4R)-3,4-dihydroxytetrahydrofuran-2-yl) (hydroxy)methyl)quinazolin-4(3H)-one (4): White solid, yield 68%, m. p. 180.8–182.0 °C, [α] 25D -68.4 (c 0.04, H2O); 1H NMR (600 MHz, DMSO‑d6), δ: 8.10 (d, J = 7.8 Hz, 1H), 7.80 (t, J = 7.2 Hz, 1H), 7.63 (d, J = 8.4 Hz, 1H), 7.50 (t, J = 7.2 Hz, 1H), 5.14 (s, 1H), 4.82 (s, 1H), 3.86 (d, J = 7.8 Hz, 1H), 3.67 (dd, J = 5.4, 1.8 Hz, 1H), 3.58 (dd, J = 10.8, 4.2 Hz, 1H), 3.51–3.39 (m, 5H); 13C NMR (150 MHz, CD3OD), δ: 161.5, 153.2, 148.2, 134.5, 126.8, 126.6, 125.9, 121.5, 73.0, 70.4, 62.9, 60.6, 52.9; HR-ESI-MS: calcd for C13H14N2O5Na ([M+Na]+), 301.0800, found: 301.0806. 15
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CDCl3), δ: 9.59 (s, 1H), 8.24 (d, J = 7.6 Hz, 1H), 7.73 (t, J = 7.2 Hz, 1H), 7.61 (d, J = 8.0 Hz, 1H), 7.46 (t, J = 7.2 Hz, 1H), 7.30–7.22 (m, 10H), 7.09–6.97 (m, 5H), 4.74 (d, J = 2.0 Hz, 1H), 4.56–4.51 (m, 3H), 4.39–4.23 (m, 3H), 4.13–4.11 (m, 1H), 4.02–3.97 (m, 1H), 3.83–3.81 (m, 2H); 13C NMR (100 MHz, CDCl3), δ: 161.1, 154.7, 148.5, 137.8, 136.6, 136.2, 134.5, 128.7, 128.7, 128.5, 128.4, 128.3, 127.9, 127.6, 127.2, 126.8, 126.7, 121.8, 79.4, 79.2, 78.5, 75.0, 73.4, 71.6, 59.3; HRESI-MS: calcd for C33H32N2O5Na ([M+Na]+), 559.2209, found: 559.2208. 2-((1S,2R,3R)-1,2,4-tris(benzyloxy)-3-hydroxybutyl)quinazolin-4(3H)-one (6d-1): Yellow oil, yield 59%; 1H NMR (600 MHz, CDCl3), δ: 9.93 (s, 1H), 8.24 (d, J = 7.8 Hz, 1H), 7.73 (t, J = 8.4 Hz, 1H), 7.67 (d, J = 8.4 Hz, 1H), 7.46 (t, J = 7.8 Hz, 1H), 7.34–7.24 (m, 13H), 7.17–7.16 (m, 2H), 4.91 (d, J = 2.4 Hz, 1H), 4.83 (d, J = 11.4 Hz, 1H), 4.65 (dd, J = 25.8, 11.4 Hz, 2H), 4.55–4.44 (m, 3H), 4.13 (d, J = 7.8 Hz, 1H), 3.81 (s, 2H), 3.57–3.51 (m, 2H); 13C NMR (150 MHz, CDCl3), δ: 161.4, 154.0, 148.1, 137.9, 137.6, 136.6, 134.6, 128.7, 128.4, 128.3, 128.1, 128.0, 127.9, 127.8, 127.2, 127.0, 126.6, 121.8, 80.8, 79.9, 74.7, 73.6, 73.2, 70.6, 70.1; HR-ESI-MS: calcd for C33H32N2O5Na ([M+Na]+), 559.2209, found: 559.2205.
137.7, 136.5, 136.2, 134.5, 128.8, 128.7, 128.6, 128.5, 128.3, 128.0, 128.0, 127.7, 127.1, 126.9, 126.7, 121.8, 79.3, 79.1, 78.4, 75.0, 73.4, 71.6, 59.2; HR-ESI-MS: calcd for C33H30N2O4Na ([M+Na]+), 541.2103, found: 541.2109. (6R,7R,8R)-6,7,8-tris(benzyloxy)-8,9-dihydro-6H-pyrido[2,1b]quinazolin-11(7H)-one (7c-2): Yellow oil, yield 60%; 1H NMR (600 MHz, CDCl3), δ: 8.28 (d, J = 7.8 Hz, 1H), 7.72 (t, J = 6.0 Hz, 2H), 7.48–7.45 (m, 1H), 7.35–7.24 (m, 15H), 4.93 (d, J = 11.4 Hz, 1H), 4.77 (d, J = 11.4 Hz, 1H), 4.73–4.61 (m, 5H), 4.39–4.34 (m, 2H), 4.07 (d, J = 5.0 Hz, 1H), 4.04–4.00 (m, 1H); 13C NMR (150 MHz, CDCl3), δ: 162.2, 151.2, 147.2, 137.8, 137.7, 137.6, 134.1, 128.5, 128.4, 127.9, 127.8, 127.8, 127.4, 127.0, 126.7, 120.9, 77.9, 74.4, 72.7, 72.6, 71.6, 71.1, 42.8; HR-ESI-MS: calcd for C33H30N2O4Na ([M+Na]+), 541.2103, found: 541.2108. 3.2.4. Synthesis of the tricyclic iminosugars 1 To a solution of 7a (0.5 g, 0.8 mmol) in 100 mL dried CH2Cl2 at −80 °C, trifloromethanesulfonic acid (CF3SO3H, 0.14 mL, 2.0 equiv.) was dropwise slowly added under N2 atmosphere. After the solution was stirred for 2 h at −80 °C, the temperature was improved to room temperature. The mixture was stirred at rt for 6 h until the reaction completed. K2CO3 Was added to the solution to neutralize CF3SO3H. After the solvent was evaporated under vacuum to afford the crude product, the residue was purified using flash column chromatography (dicholomethane - methanol V/V = 9:1) to afford compound 1a. Under the similar conditions, the corresponding compounds 1b, 1c1 and 1c-2 were obtained, respectively. (6S,7S,8R,9S)-6,7,8-trihydroxy-9-(hydroxymethyl)-8,9-dihydro-6H-pyrido[2,1-b]quinazolin-11(7H)-one (1a): White solid, yield 22%, m. p. 195.3–196.0 °C, [α] 25D +54.0 (c 0.04, H2O); 1H NMR (400 MHz, DMSO‑d6), δ: 8.12 (d, J = 8.0 Hz, 1H), 7.80 (t, J = 8.0 Hz, 1H), 7.65 (d, J = 8.0 Hz, 1H), 7.49 (t, J = 8.0 Hz, 1H), 4.79–4.74 (m, 1H), 4.27 (d, J = 6.8 Hz, 1H), 4.14 (dd, J = 10.4, 6.8 Hz, 1H), 3.90 (dd, J = 11.6, 2.4 Hz, 1H), 3.77(dd, J = 10.4, 6.0 Hz, 1H), 3.70 (dd, J = 11.2, 3.6 Hz, 1H); 13C NMR (100 MHz, DMSO‑d6), δ: 161.3, 157.5, 147.6, 134.8, 127.0, 126.8, 126.7, 120.6, 74.8, 72.4, 69.3, 56.8, 55.9; HR-ESI-MS: calcd for C13H14N2O5Na ([M+Na]+), 301.0800, found: 301.0803. (6S,7S,8R,9R)-6,7,8-trihydroxy-9-(hydroxymethyl)-8,9-dihydro-6H-pyrido[2,1-b]quinazolin-11(7H)-one (1b): White solid, yield 22%, m. p. 184.2–185.6 °C, [α] 25D +42.0 (c 0.04, H2O); 1H NMR (400 MHz, DMSO‑d6), δ: 8.15 (d, J = 7.2 Hz, 1H), 7.81 (t, J = 7.2 Hz, 1H), 7.67 (d, J = 8.0 Hz, 1H), 7.51 (t, J = 7.2 Hz, 1H), 5.58–4.77 (m, 6H), 4.26 (s, 1H), 4.16 (t, J = 6.8 Hz, 1H), 3.95 (d, J = 10.8 Hz, 2H), 3.70 (d, J = 7.2 Hz, 1H); 13C NMR (100 MHz, DMSO‑d6), δ: 161.3, 157.2, 147.4, 134.8, 127.0, 127.0, 126.8, 120.8, 70.4, 70.3, 67.7, 56.5, 56.0; HR-ESI-MS: calcd for C13H14N2O5Na ([M+Na]+), 301.0800, found: 301.0804. ((1S,2R,3R)-1,2,3,4-tetrahydroxybutyl)quinazolin-4(3H)-one (1c-1): White solid, yield 52%, m. p. 182.6–184.0 °C, [α] 25D +107.5 (c 0.04, H2O); 1H NMR (400 MHz, DMSO‑d6), δ: 8.14 (d, J = 7.6 Hz, 1H), 7.80 (t, J = 7.6 Hz, 1H), 7.68 (d, J = 8.0 Hz, 1H), 7.50 (t, J = 7.6 Hz, 1H), 6.12 (s, 1H), 5.90 (s, 1H), 5.01 (d, J = 8.4 Hz, 1H), 4.78 (s, 1H), 4.45 (d, J = 8.0 Hz, 1H), 4.30 (t, J = 8.4 Hz, 1H), 3.89 (s, 2H); 13C NMR (100 MHz, DMSO‑d6), δ: 160.1, 160.0, 149.2, 134.6, 127.4, 126.7, 126.3, 121.7, 76.2, 74.5, 59.5, 55.9; HR-ESI-MS: calcd for C12H12N2O4Na ([M+Na]+), 271.0695, found: 271.0694. (6R,7R,8R)-6,7,8-trihydroxy-8,9-dihydro-6H-pyrido[2,1-b]quinazolin-11(7H)-one (1c-2): White solid, yield 23%, m. p. 187.5–188.3 °C, [α] 25D +24.6 (c 0.04, H2O); 1H NMR (400 MHz, CD3OD) δ: 8.16 (d, J = 8.4 Hz, 1H), 7.78 (t, J = 7.2 Hz, 1H), 7.69 (d, J = 8.0 Hz, 1H), 7.49 (t, J = 7.6 Hz, 1H), 4.74 (d, J = 6.4 Hz, 1H), 4.44–4.41 (m, 1H), 4.11–4.04 (m, 3H); 13C NMR (100 MHz, CD3OD) δ: 162.3, 155.0, 147.1, 134.3, 126.8, 126.3, 125.9, 119.8, 71.8, 70.8, 64.4, 45.7; HR-ESI-MS: calcd for C12H12N2O4Na ([M+Na]+), 271.0695, found: 271.0691.
3.2.3. Synthesis of the tricyclic iminosugars 7 and 8 by Mitsunobu reaction To a solution of 6a (1.8 g, 2.7 mmol) in 150 mL dried THF, triphenylphosphine (PPh3, 2.2 g, 3.0 equiv.) was added under N2 atmosphere at ice bath. After the solution was stirred for 30 min, diisopropyl azodicarboxylate (DIAD, 1.7 mL) was dropwise added into the solution at ice bath. The mixture was stirred for 20 h till the reaction completed. After 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 = 8:1) to afford compounds 7a and 8a. Under the similar conditions, the corresponding compounds 7c-1 and 7c-2 were obtained in satisfactory yields, respectively. (6S,7S,8R,9S)-6,7,8-tris(benzyloxy)-9-((benzyloxy)methyl)-8,9dihydro-6H-pyrido[2,1-b]quinazolin-11(7H)-one (7a): Yellow oil, yield 43%; 1H NMR (400 MHz, CDCl3), δ: 8.37 (d, J = 7.6 Hz, 1H), 7.82 (d, J = 3.6 Hz, 2H), 7.57–7.53 (m, 1H), 7.48 (d, J = 6.8 Hz, 2H), 7.43–7.37 (m, 13H), 7.25–7.18 (m, 5H), 5.59 (dd, J = 9.6, 5.2 Hz, 1H), 5.01 (d, J = 11.6 Hz, 1H), 4.90–4.75 (m, 5H), 4.68 (d, J = 4.0 Hz, 1H), 4.54 (s, 2H), 4.47 (dd, J = 9.2, 4.0 Hz, 1H), 4.18 (dd, J = 10.8, 5.2 Hz, 1H), 4.07 (dd, J = 10.4, 4.0 Hz, 1H), 3.93 (dd, J = 9.2, 5.6 Hz, 1H); 13C NMR (100 MHz, CDCl3), δ: 161.6, 152.4, 147.2, 138.1, 138.0, 138.0, 137.8, 134.4, 128.5, 128.4, 128.4, 128.2, 128.0, 127.9, 127.8, 127.7, 127.6, 127.6, 127.4, 127.2, 127.1, 120.8, 81.0, 81.0, 78.4, 73.4, 73.1, 73.0, 72.5, 66.8, 51.6; HR-ESI-MS: calcd for C41H38N2O5Na ([M +Na]+), 661.2678, found: 661.2682. (6S,7S,8R,9R)-6,7,8-tris(benzyloxy)-9-((benzyloxy)methyl)8,9-dihydro-6H-pyrido[2,1-b]quinazolin-11(7H)-one (8a): Yellow oil, yield 23%; 1H NMR (600 MHz, CDCl3), δ: 8.25 (d, J = 7.6 Hz, 1H), 7.80–7.53 (m, 2H), 7.51–7.47 (m, 1H), 7.44 (d, J = 6.8 Hz, 2H), 7.40–7.26 (m, 13H), 7.11 (d, J = 7.6 Hz, 1H), 7.05 (t, J = 7.6 Hz, 2H), 6.91 (d, J = 7.2 Hz, 2H), 5.27–5.25 (m, 1H), 4.95–4.70 (m, 8H), 4.56 (dd, J = 9.2, 6.8 Hz, 1H), 4.32 (d, J = 12.4 Hz, 1H), 4.20 (d, J = 12.4 Hz, 1H), 4.13 (d, J = 9.2 Hz, 1H); 13C NMR (100 MHz, CDCl3), δ: 161.9, 152.6, 150.2, 147.1, 138.5, 138.3, 138.0, 137.6, 134.3, 128.5, 128.4, 128.3, 128.2, 127.8, 127.8, 127.7, 127.6, 127.5, 127.4, 127.2, 127.0, 127.0, 121.2, 76.1, 74.6, 73.8, 73.0, 72.8, 72.3, 72.1, 64.7, 53.7; HR-ESI-MS: calcd for C41H38N2O5Na ([M+Na]+), 661.2678, found: 661.2680. (1S,2R,3R)-2,3-bis(benzyloxy)-1-((benzyloxy)methyl)-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (7c-1): Yellow oil, yield 65%; 1H NMR (400 MHz, CDCl3), δ: 8.24 (d, J = 7.6 Hz, 1H), 7.82–7.74 (m, 2H), 7.52–7.46 (m, 3H), 7.41–7.34 (m, 8H), 7.16–7.07 (m, 5H), 5.38–5.35 (m, 2H), 5.00 (d, J = 11.6 Hz, 1H), 4.79–4.69 (m, 3H), 4.47–4.37 (m, 3H), 4.01 (dd, J = 10.2, 2.8 Hz, 1H), 3.89 (d, J = 10.0 Hz, 1H); 13C NMR (100 MHz, CDCl3), δ: 161.1, 154.8, 148.4, 16
Carbohydrate Research 478 (2019) 10–17
J. Sun, et al.
3.2.5. Glycosidases inhibition The inhibitory activity of the synthesized compounds against five glycosidases: α-glucosidase (Aspergillus niger), β-glucosidase (almonds), α-galatosidase (coffee beans), β-galatosidase (Escherichia coli), and αMannosidase (Jack beans). The enzymes and their corresponding substrates, p-nitrophenyl glycopyranosides, were purchased from Sigma Chemical Co. All other commercial reagents were used as received. Taken the α-Glucosidase assay as example: α-Glucosidase assay was performed using PNPG (2 mmol/L) as substrate in pH = 5.5 citric acid - phosphate buffers (CPBs) at 37 °C. 10 μL of enzyme solution (50 u/mL in CPBs), 20 μL of inhibitor (6 mmol/L) and 20 μL of buffer were incubated for 10 min at 37 °C, and then 50 μL of PNPG was added. The mixture were incubated for 20 min. The reaction was quenched with 100 μL of sodium carbonate (1 mol/L) for 10 min. Absorbance readings were taken on a BioRad Model 3550 microplate spectrophotometer at 405 nm using distilled deionized water as a blank control.
[11] [12] [13] [14] [15] [16] [17] [18] [19] [20]
Acknowledgments
[21] [22]
The financial supports from the National Natural Science Foundation of China (NSFC) (21772031 and 21778013).
[23]
Appendix A. Supplementary data
[25]
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.carres.2019.04.002.
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