Synthesis and Glycosidase Inhibition of Broussonetine M and Its Analogues

Cross-metathesis (CM) and Keck asymmetric allylation, which allows access to defined stereochemistry of a remote side chain hydroxyl group, are the key steps in a versatile synthesis of broussonetine M (3) from the d-arabinose-derived cyclic nitrone 14. By a similar strategy, ent-broussonetine M (ent-3) and six other stereoisomers have been synthesized, respectively, starting from l-arabino-nitrone (ent-14), l-lyxo-nitrone (ent-3-epi-14), and l-xylo-nitrone (2-epi-14) in five steps, in 26%–31% overall yield. The natural product broussonetine M (3) and 10’-epi-3 were potent inhibitors of β-glucosidase (IC50 = 6.3 μM and 0.8 μM, respectively) and β-galactosidase (IC50 = 2.3 μM and 0.2 μM, respectively); while their enantiomers, ent-3 and ent-10’-epi-3, were selective and potent inhibitors of rice α-glucosidase (IC50 = 1.2 μM and 1.3 μM, respectively) and rat intestinal maltase (IC50 = 0.29 μM and 18 μM, respectively). Both the configuration of the polyhydroxylated pyrrolidine ring and C-10’ hydroxyl on the alkyl side chain affect the specificity and potency of glycosidase inhibition.

According to our previous study on the synthesis and glycosidase inhibition of broussonetines [39,40], enantiomers of the above broussonetines would probably demonstrate a similar inhibitory profile by analogy with LAB (ent-1) and l-DMDP (ent-2), and as such [22,26,41,42], these compounds may have potential in the treatment of type II diabetes, cancers, and viral infections [43][44][45][46]. Hence, in this work, broussonetine M (3) was selected as the research objective; synthesis and glycosidase inhibition of the natural product and its analogues, including l-enantiomers and pyrrolidine core stereoisomers, were finished, aiming for a better understanding of structure-activity relationship (SAR) of this interesting class of pyrrolidine iminosugars. Though the first synthesis of broussonetine M (3) was accomplished with d-serine as the starting material [47], we have shown that most broussonetines can be efficiently constructed via a general synthetic strategy employing sugar-derived cyclic nitrones [39,40]. The pyrrolidine core of this class of iminosugars can be derived from cyclic sugar nitrones [44,[48][49][50] with the corresponding stereochemistry in the hydroxylated pyrrolidine ring, while the various side chains could be installed via cross-metathesis (CM) reactions [46,47,51,52]. This general strategy is capable of synthesizing a number of natural broussonetines, as well as a variety of broussonetine analogues for SAR study; it has been successful in the synthesis of broussonetine I (7), J 2 (10), and W (11) [39,40]. Therefore, this strategy was applied in the construction of broussonetine M (3) and its analogues.

Synthesis of Broussonetine M
Our retrosynthesis for broussonetine M (3) is presented in Scheme 1. The precursor 12 of broussonetine M was obtained by the CM reaction between the pyrrolidine 13 and the alcohol 15. The pyrrolidine 13 was conveniently prepared from d-arabinose-derived cyclic nitrone 14 [53][54][55][56][57]. The alcohol 15, which contains one stereocenter, was synthesized through asymmetric Keck allylation of aldehyde 16. In this synthetic route, only the stereocenter in alcohol 15 is constructed by virtue of an asymmetric reaction; of the four stereocenters on the pyrrolidine ring, three were determined by sugar-derived cyclic starting nitrone 14 and the fourth was formed by the high diastereoselectivity of organometallic addition to the nitrone 14. For the synthesis of broussonetine M (3), addition of Grignard reagent 18, prepared from 8-bromo-1-octene, to d-arabino-nitrone (14) afforded the hydroxylamine 19 in high yield and excellent diastereoselectivity; none of the other diastereomers were formed (Scheme 2) [54]. Due to its chemical instability, the hydroxylamine 19 was directly used in the next step of reaction without further purification. Successive zinc reduction and N-Cbz protection of the crude hydroxylamine 19 provided the pyrrolidine 13, as the required CM reaction precursor, in 64% overall yield in three steps. The configuration of the newly formed C-5 chiral center was determined as R by nuclear Overhauser effects experiment on 19, with the observation of the strong correlation between H-5 and H-6a, H-6b (Scheme 2). The key step in the synthesis of the chiral alkyl alcohol 15 was the asymmetric Keck allylation (Scheme 3) [58]. Treatment of butyl glycol 20 with BnBr/NaH/TBAI in DMF-THF gave the mono-O-benzylated alcohol 21 in 91% yield. Swern oxidation of alcohol 21 afforded aldehyde 16 (89%), which on enantioselective Keck allylation using (S)-BINOL produced alcohol 15 (93% ee) [59]. Using (R)-BINOL as the ligand, alcohol ent-15 was also prepared by the same method (See the Supplementary Materials for HPLC analysis of compounds 15 and ent-15). In this case, the free hydroxyl group of the alcohol 15 was tolerated for the CM reaction with no need for O-protection. CM reaction of pyrrolidine 13 and alcohol 15 promoted by Grubbs II catalyst produced olefin 12 as an inseparable Z/E mixture in moderate yield (43%). Pd/C-catalyzed hydrogenation of compound 12 in acidic methanol afforded the target product broussonetine M (3) in quantitative yield (Scheme 4). Thus, broussonetine M (3) was synthesized in five linear steps starting from d-arabino-nitrone (14) in 28% total yield. The 1 H-and 13 C-NMR spectra and the specific rotation of the synthetic broussonetine M (3) were all consistent with those reported for the synthetic broussonetine M (3) [47], but had some differences with those of natural products [36] (See the Supplementary Materials for comparison of NMR data). Since the structure and configuration of product 3 were ensured by all the synthetic materials and procedures, as in the work of Alberto Marco et al. [47], the NMR spectra differences may be explained by minute pH variation or metal impurities [60,61].

Synthesis of Analogues of Broussonetine M
Glycosidase inhibition by pyrrolidine iminosugars are among those that are most difficult to predict [16]; minor modification of the iminosugar can lead to a distinct change of the inhibition profile. In order to explore the preliminary structure-activity relationship of this type of iminosugar, seven analogues of broussonetine M (3), including its l-enantiomer and other six stereoisomers, were then prepared. By the same strategy as that for pyrrolidine 13, synthesis of ent-13, ent-3-epi-13, and 2-epi-13 were accomplished from the corresponding sugar-derived cyclic nitrones ent-14, ent-3-epi-14, and 2-epi-14. The configurations of the newly constructed chiral centers in these compounds were all confirmed by NOE experiments. With these pyrrolidines in hand, CM reaction with alcohol 15, and subsequent hydrogenation, provided the target products, i.e., ent-10'-epi-3, ent-3,10'-di-epi-3, and 2-epi-3, of which, the C10'-hydroxyls retained S configuration as that of the natural product (Table 1).
In order to evaluate the influence of the C10'-hydroxyl on glycosidase inhibition, the C10'-epimers of broussonetine M (3), including 10'-epi-3, ent-3, ent-3-epi-3, and 2,10'-di-epi-3, were also synthesized from the above four pyrrolidines and alcohol ent-15 by the same strategy. a Total yield in 3 steps starting from cyclic nitrones to the corresponding pyrrolidine. b Total yield in 2 steps starting from pyrrolidine cores to broussonetine M or its analogues.
Therefore, configurations of both the pyrrolidine ring and C-10' have significant influences on glycosidase inhibition of broussonetine M and its analogs. In detail, comparison of the inhibitory profiles of ent-broussonetine M (ent-3) and ent-10'-epi-3 with those of broussonetine M (3) and 10'-epi-3 showed high similarity to those of DAB and LAB derivatives (or DMDP and l-DMDP derivatives), of which we have previously reported opposite inhibitions toward αand β-glycosidases for enantiomers [16,18,42]. In contrast, inhibitory activities of other broussonetine analogues with l-altro-DMDP and d-gluco-DMDP pyrrolidine cores are more difficult to predict, but the presence of the 13-carbon chains basically narrowed down the inhibitory profiles.
The structure-activity relationship uncovered in this work would be helpful in the research and development of new glycosidase inhibitors.

General Methods
NMR spectra was recorded at 300 MHz, 400 MHz, or 500 MHz ( 1 H-NMR) and 75 MHz, 100 MHz, or 125 MHz ( 13 C-NMR) in CDCl 3 (with TMS as internal standard), C 5 D 5 N, or CD 3 OD (with solvent signal as internal standard). High-resolution mass spectra (HRMS) were performed on a LTQ/FT linear ion trap mass spectrometer. All reagents were used as received without any further purification or prepared, as described in the literature. CH 2 Cl 2 was freshly distilled from CaH 2 . Tetrahydrofuran was distilled from sodium benzophenone. TLC plates were visualized by ultraviolet light or by treatment with a spray of Pancaldi reagent ((NH 4 ) 6 MoO 4 , Ce(SO 4 ) 2 , H 2 SO 4 , H 2 O) or a 0.5% solution of KMnO 4 in acetone. Column chromatography was performed on a flash column chromatography with silica gel (200-300 mesh). Polarimetry was determined using an Optical Activity AA-10R polarimeter with concentrations (c) given in grams per 100 mL. IR data were measured as films on KBr plates and are given only when relevant functions are present. Chiral HPLC analyses were performed on an Agilen 1100 Series using a Daicel Chiralpak (OD-H) column with hexanes/i-PrOH as the eluent.

Material and Methods for the Enzyme Inhibition Assay
With rat intestinal maltase as an exception, other enzymes were purchased from Sigma-Aldrich Chemical Co. (St. Louis, Mo. USA). Brush border membranes prepared from rat small intestine according to the method of Kessler et al. [63] were assayed at pH 6.8 for rat intestinal maltase using maltose. The released d-glucose was determined colorimetrically using the Glucose CII-test Wako (Wako Pure Chemical Ind.; Osaka, Japan). Other glycosidase activities were determined using an appropriate p-nitrophenyl glycoside as substrate in a buffer solution at the optimal pH value of each enzyme. The reaction was stopped by adding 400 mM Na 2 CO 3 . The released p-nitrophenol was measured spectrometrically at 400 nm [16].

Synthesis of 4-(benzyloxy)butanal (16)
A solution of DMSO (8.74 mL, 0.26 mol) in dry CH 2 Cl 2 (20 mL) was added dropwise to a solution of (COCl) 2 (24.57 mL, 0.29 mol) in dry CH 2 Cl 2 (100 mL) at -78 • C. The mixture was stirred for 5 min. A solution of 4-(benzyloxy)butan-1-ol 21 (43.3 g, 0.24 mol) in dry CH 2 Cl 2 (50 mL) was then added dropwise while the temperature was kept below −65 • C. After 15 min, NEt 3 (166.94 mL, 1.2 mol) was added dropwise. After stirring for 10 min at −78 • C, the reaction mixture was allowed to warm to room temperature and diluted with CH 2 Cl 2 (200 mL). The organic layer was washed with brine (2 × 100 mL). The combined organic extracts were dried over MgSO 4 , filtered, and concentrated under reduced pressure. Purification by flash chromatography on silica gel (petroleum ether/EtOAc = 10/1) afforded 4-(benzyloxy)butanal 16 (39.8 g, 93% yield) as a yellow oil. 1  Under Ar atmosphere, (S)-BINOL (52 mg, 0.2 mmol) and Ti(O i Pr) 4 (45 mg, 0.2 mmol) were added to a solution of dried 4 Å molecular sieves (2.2 g) in CH 2 Cl 2 (20 mL). The reaction mixture was heated at reflux for 1 h, and then allowed to cool to room temperature. A solution of aldehyde 16 (356 mg, 2 mmol) in CH 2 Cl 2 (15 mL) was added to the reaction mixture. After stirring for 0.5 h at room temperature, the solution was cooled to −78 • C and allyltributyltin (993 mg, 3 mmol) was added dropwise. The reaction mixture was stirred for an additional 20 min at −78 • C, then kept at −20 • C. After 12 h, the reaction mixture was filtered through a pad of celite into a 200 mL flask that contained a stirred sat. aq. NaHCO 3 solution (50 mL); the resulting reaction mixture was stirred for 1 h. Then, the layers were separated and the aqueous layer was extracted with CH 2 Cl 2 (3 × 50 mL). The combined organic extracts were dried with MgSO 4 ; subsequent removal of all volatiles under reduced pressure and column chromatography of the residue on silica gel (petroleum ether/EtOAc = 20/1) afforded homoallylic alcohol 15 (729.7 mg, 83% yield) as colorless oil.  19, ent-19, ent-3-epi-19, and 2-epi-19, with 19 as an Example Part of the solution of 8-bromo-1-octene (573.3 mg, 3.0 mmol) in THF (2 mL) was quickly added via syringe to a stirred solution of Mg (1.16 g, 5.0 mmol) and I 2 (cat.) in THF (5 mL) under Ar atmosphere. The mixture was heated until the color disappeared; then, the remaining 8-bromo-1-octene was added dropwise. After the addition was completed, the resulting reaction mixture was heated to reflux for 1 h and then was allowed to cool to room temperature. The prepared Grignard reagent was added slowly to a solution of d-arabino-nitrone (14) (417.5 mg, 1.0 mmol) in THF (10 mL) via syringe at 0 • C under Ar atmosphere. The reaction mixture was stirred for 0.5 h; then sat. aq. NH 4 Cl was added to quench the reaction. The organic layer was separated and the aqueous layer was extracted with EtOAc (3 × 20 mL). The combined organic phases were dried over MgSO 4 and filtered; the solvent was removed under reduced pressure to give the crude product hydroxylamine 19, which was used without further purification because of its instability. The sample for structure characterization was purified by flash column chromatography on silica gel (petroleum ether/EtOAc = 5/1) as a colorless syrup.
Data for (2R,3R,4R,5R)-3,4-bis(benzyloxy)-2-((benzyloxy)methyl)-1-hydroxyl-5-(oct-7-en-1-yl)pyrrolidine  Zinc powder (653.8 mg, 10 mmol) was added to a suspension of Cu(OAc) 2 (18.2 mg, 0.1 mmol) in AcOH (10 mL) and the reaction mixture was stirred for 0.5 h. Then, a solution of the crude hydroxylamine 19 in AcOH (5 mL) was added and the reaction mixture was stirred for 10 h. The solid was removed by filtration and all volatiles were removed under reduced pressure. The residue was dissolved in EtOAc (20 mL) and sat. aq. NaHCO 3 was added to neutralize the solution. The resulting precipitate was removed by filtration and the organic layers were collected; the aqueous layer was extracted with EtOAc (3 × 10 mL). The combined organic layer was dried over MgSO 4 , filtered, and concentrated under reduced pressure to give the crude amine, which was used in the next step without further purification. NaHCO 3 (252.0 mg, 3.0 mmol) and CbzCl (255.9 mg, 1.5 mmol) were added slowly to a stirred solution of the crude amine in methanol (10 mL) and the reaction mixture was stirred at room temperature for 6 h. Then sat. aq. NaHCO 3 (20 mL) was added to quench the reaction and EtOAc (20 mL) was added. The organic layer was separated and the aqueous layer was extracted by EtOAc (3 × 10 mL). The combined organic layers were dried over MgSO 4 , filtered, and the solvent was removed under reduced pressure. Purification by flash chromatography on silica gel (petroleum ether/EtOAc = 15/1) afforded the carbamate 13 as light yellow syrup (423 mg, yield: 64% for 3 steps).

Conflicts of Interest:
The authors declare no conflict of interest.