Synthetic Communicationsw, 37: 3509–3518, 2007 Copyright # Taylor & Francis Group, LLC ISSN 0039-7911 print/1532-2432 online DOI: 10.1080/00397910701555725

Chemoenzymatic Synthesis and Biological Evaluation of (2)-Conduramine C-4 Ana Bellomo Faculty of Chemistry, Department of Organic Chemistry, University of the Republic, Montevideo, Uruguay

Cecilia Giacomini and Beatriz Brena Faculty of Chemistry, Department of Bioscience, University of the Republic, Montevideo, Uruguay

Gustavo Seoane and David Gonzalez Faculty of Chemistry, Department of Organic Chemistry, University of the Republic, Montevideo, Uruguay

Abstract: Previously unreported (2)-conduramine C-4 was synthesized in six steps from a bacterial bromobenzene metabolite in 23% overall yield. The chemoenzymatic route involved toluene dioxygenase dihydroxylation, b-epoxidation, epoxide ringopening, Staudinger reduction, radical debromination, and Amberlite- catalyzed hydrolysis. (2)-Conduramine C-4 and other related compounds synthesised were assayed for galactosidase-activity inhibition against b-D -galactoside galactohidrolase isolated from Aspergillus oryzae. Keywords: chemoenzymatic, conduramine, glycosidase inhibitors, toluene dioxygenase

INTRODUCTION The cyclitols are a diverse class of compounds that share a polyhydroxylated cycloalkane ring as a common structural motif. The major subclasses among

Received in the USA February 23, 2007 Address correspondence to David Gonzalez, Faculty of Chemistry, Department of Organic Chemistry, University of the Republic, C.C. 1157, Gral. Flores 2124, Montevideo, Uruguay. E-mail: [email protected] 3509

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the cyclitols are inositols, conduritols, and their deoxygenated analogs. The chemoenzymatic approach to several of these cyclitols and cyclitol analogs via whole-cell oxidation of aromatics by toluene dioxygenase has been extensively explored by Hudlicky,[1 – 6] Carless,[7 – 9] Nicolosi,[10 – 14] and ourselves.[15,16] As a result, syntheses of six of the nine possible inositol isomers have been disclosed.[15,17 – 20] Conduritols (5-cyclohexen-1,2,3,4-tetrols) are a prominent class of compounds useful for the preparation of inositol derivatives.[21,22] In view of the presence of four stereogenic centers, conduritols exist as 10 stereoisomers: four enantiomeric pairs and two meso-forms (Fig. 1). Conduramines and other deoxygenated congeners of conduritols arise in even greater structural diversity (32 isomers) because of their decreased number of symmetry planes and axes. Interest in conduritols and conduramines has expanded in recent years for various reasons.[23 – 28] Conduritols and conduramines inhibit certain glycosidases,[29 – 31] they are important building blocks for the synthesis of more complex natural products,[32] and the densely functionalised nature of these compounds (four stereogenic centers on a cyclohexene ring) is synthetically challenging. Conduramines are purely synthetic aminocyclohexentriols formally derived from conduritols. Some conduramines have significant glycosidaseinhibitory activity;[33] they are well-known synthetic precursors of amino- and diaminocyclitols, many of which are the aglycon portions of the therapeutically useful aminoglycoside antibiotics. In addition, conduramines have been utilized as intermediates in the preparation of azasugars,[34] aminosugars, sphingosines,[32] and narcissus alkaloids.[35] Given the importance of conduramines as synthetic building blocks, significant effort has been devoted to the development of useful preparative routes to these compounds[36,37] and their derivatives.[38] Hudlicky et al. have devised an enantiomerically controlled approach to

Figure 1.

Structures of all conduritol isomers.

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several conduramines from a single, optically pure 1-halo-cis-2,3-dihidrobenzene-2,3-diol.[23] As a part of our program for the synthesis of cyclitol conjugates and glycosides to be tested as glycosidase inhibitors and insect repellents,[39] we needed to prepare a series of known and unknown cyclitols. We have recently published the synthesis of phenylthioconduritol F,[16] and in continuation with our studies, we report the preparation of the previously unknown (2)-conduramine C-4 (8) in an optically pure form.

RESULTS AND DISCUSSION Whole-cell fermentation of bromobenzene with Pseudomonas putida 39/D furnished the chiral cis-cyclohexenedienediol 1 in an isolated yield of 1.5 g of optically pure metabolite per liter of cell-free broth. After the chirality transfer through the biocatalytic step, the homochiral cis-cyclohexadienediol was subjected to a precisely defined sequence of stereospecific chemical transformations. The absolute stereochemistry of the next quiral centers introduced after the enzymatic oxidation was controlled by the syn-directing effect of the free diol or by the anti-directing effect of the rigid acetonide protective group. cis-Diol 1 was protected as the corresponding acetonide 2, which reacted with N-bromosuccinimide in the presence of water to render a mixture of bromohydrins 3a and 3b.[40] These products resulted from the hydrolytic ring opening at the allylic carbon of two stereochemically different bromonium intermediates. Treatment of the crude mixture of bromohydrins with sodium hydroxide promoted the formation of epoxides 4a and 4b,[41,42] which were isolated after column chromatography in a relative ratio of 7:1. Azido alcohol 5 was prepared in high yield by ring opening of epoxide 4a with NaN3 in an aqueous/organic media (Scheme 1).[43] To generate the desired conduramine, two different routes starting from azide 5 were tested (Scheme 2). We first attempted a classical two-step

Scheme 1.

Synthesis of homochiral azide 5 from bromobenzene.

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Scheme 2.

Synthesis of (2)-conduramine C-4 from bromoazide 5.

sequence for the consecutive reduction of both functional groups. Staudingertype reduction of the azide group[44] rendered the aminoalcohol 6 in reasonable yield. This operation was followed by radical dehalogenation using SnBu3H as the hydrogen donor and azobiscyclohexancarbonitrile (ABCC) as initiator to furnish amine 7. The overall yield for both steps was 56% of isolated conduramine derivative 7. Because LiAlH4 is a known azide group reducer and debrominating agent, we decided to use it in an alternative onestep procedure for the complete reduction of key intermediate 5. Our results showed that the reaction of 5 with LiAlH4 in THF did furnished 7, albeit in poorer yield (31%). Derivative 7 is a very hygroscopic and unstable compound that repeatedly failed elemental analysis. Therefore, for synthetic purposes, the material was carried to the next step with only minimal purification. Intermediate 7 was subjected to Amberlite resin – catalyzed hydrolysis to render enantiopure (2)-conduramine C-4 as the only product in high yield. This last step was conducted inside a chromatography column loaded with Amberlite in its acidic form and in a mass ratio of 20:1 relative to the crude amine 7. In that sense, this last reaction overlapped with the purification procedure for the previous step, which therefore become redundant and unnecessary from a purely synthetic perspective. Synthetic (2)-conduramine C-4 is a new compound; consequently its optical rotation has not been previously reported. Nevertheless, the recorded value –166 (c 0.3, MeOH) was compared to the reported number for the known enantiomer (þ)-conduramine C-4 þ155 (c 0.7, MeOH) synthesized by Nicolosi et al. by lipase-catalyzed desymmetrisation.[13] The compound was included in a small library of compounds structurally related to conduritols and assayed for galactosidase-activity inhibition against b-D -galactoside galactohidrolase (EC 3.2.1.23) isolated from Aspergillus

(2)-Conduramine C-4 Table 1. Entry 1 2 3 4 5 6

3513 Comparison of inhibitory activities Compound

Inhibition activity (%)

8 9 10 11 12 13

— 13 17 — 3 —

oryzae. Neither (2)-conduramine C-4 nor its brominated analog 11 exhibited activity at a concentration range of 0.069 mM to 2.76 mM (Table 1). The deprotected derivatives of other intermediates in the synthesis were also tested (Fig. 2). The bromoazidotriol 10 afforded from the hydrolysis of bromoazide 5 showed the highest activity: 17% inhibition at a concentration of 10 mM. Its debrominated analog 12 was much less active, and a similar effect was observed for the diasteromeric bromoazidotriol 9. Finally, compound 13, which lacks the vinyl bromine atom and presents the opposite configuration at C-1 and C-6, was completely inactive. These results could suggest that the concomitant presence of an azido group and a bromide atom is required to obtain high inhibition degrees and that the presence of three contiguous cis-hydroxyl groups is important for bioactivity. Furthermore, these results are useful to determine the structure –activity relation for these inhibitors and to design alternative strategies to improve their potency. Several other conduritol-like compounds as well as conduritol conjugates have been prepared in our laboratory and are being evaluated as glycosidase inhibitors. The synthetic strategy and the biological testing results for all libraries will be reported in due course.

Figure 2. Compounds tested for galactosidase-activity inhibition.

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EXPERIMENTAL General All nonhydrolytic reactions were carried out under a nitrogen atmosphere, with standard techniques for the exclusion of moisture. All solvents were distilled prior to use. Melting points were determined on a Leitz Microscope Heating Stage model 350 apparatus and are uncorrected. Optical rotations were measured on a Perkin-Elmer 241 polarimeter using a 7-mL cell. Infrared spectra were recorded on a Matheson Excalibur spectrometer. Mass spectra were recorded on a Shimadzu GS-MS QP 1100 EX instrument using the electron impact mode. Nuclear magnetic resonance spectra were recorded on Bruker Avance DPX-400 instrument with Me4Si as the internal standard and chloroform-d as solvent unless otherwise indicated. Chemical shifts are reported in parts per million(ppm). Elemental analyses were performed in a Fisons EA 1108 CHNS-O analyzer or submitted to Atlantic Microlabs, Northcross, GA, USA. Analytical thin-layer chromatography(TLC) was performed on silica-gel 60F-254 plates and visualized with UV light (254 nm) and/or anisaldehyde-H2SO4-AcOH as detecting agent. Flash-column chromatography was performed in silica gel (Kieselgel 60, EM Reagents, 230–400 mesh). Compound 5 has been previously reported;[43] therefore, some spectral data are omitted here. Compound 7 was so unstable that we could not obtain acceptable CHN results in spite of using spectroscopically pure samples and performing the analysis four times on this compound using different operators in different machines.

(1S,2R,3S,6R)-6-Amino-4-bromocyclohexene-2,3-isopropylidendioxy1-ol (6) Azide 5 was dissolved in tetrahydrofuran (15 mL) containing 1 equivalent of acetic acid. Triphenylphosphine (0.73 g, 2.78 mmol) was added under N2. The reaction was stirred for 24 h at rt followed by addition of water (25 eq.) and stirred for a further 24 h at rt. The reaction mixture was concentrated at reduced pressure, and the yellow residue was subjected to flash chromatography on silica, eluting first with EtOAc (to remove triphenylphosphine and triphenylphosphine oxide) and then a gradient of 10:90–30:70 MeOH–EtOAc to afford the amino compound 6. Yield: 78%; white needles; mp 87–898C; [a]19 D ¼ 298 (c 0.73, CH2Cl2). Anal. calcd. for C11H18BrNO5 (amine 6 acetate): C, 41.06; H, 5.76; N, 4.52. Found: C, 40.76; H, 5.60; N, 4.32. IR (KBr): 3539–2575 b, 1589, 1231, 1059, 864 cm21; MS (m/z, %): 263 (Mþ, 0.10), 190 (5), 165 (83), 109 (38), 101 (100), 80 (19), 55 (54), 43 (37); 1H RMN (CDCl3): d ¼ 1.39 (s, 6H), 3.67 (bs, 2H, H-3 and H-6), 4.44 (bs, 3H), 4.48 (d, 1H, H-1), 4.60 (d, 1H, J23 5.0 Hz, H-2), 6.03 (s, 1H, H-5); 13C NMR (CDCl3): d ¼ 26.9, 27.8, 51.9, 72.5, 77.8, 78.8, 111.1, 124.2, 132.3.

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(1S,2R,3R,6R)-6-Amino-cyclohex-4-ene-2,3-isopropylidendioxy1-ol (7) Bu3SnH (0.42 g, 1.44 mmol) was added to a mixture of azobiscyclohexancarbonitrile (0.09 g, 0.36 mmol) and the amino compound 5 in dry tetrahydrofuran (15 mL). The reaction mixture was refluxed for 4 h. Concentration at reduced pressure and purification of the oily residue by flash chromatography (AcOEt – MeOH 70:30) furnished the pure product 7. Yield: 75%; white solid; mp 99– 1008C; [a]20 D ¼ 276 (c 0.51, CH2Cl2); IR (KBr): 3450– 2882 b, 1237, 1086, 1036, 882 cm21; MS (m/z, %): 170 (Mþ-CH3, 5), 149 (4), 110 (11), 98 (21), 85 (100), 55 (42), 43 (29); 1H NMR (CDCl3): d ¼ 1.39 (s, 6H), 2.14 (bs, 3H), 3.40 (d, 1H, J16 8.1 Hz, H-1), 3.50 (bd, 1H, H-6), 4.51 (bs, 1H, H-2), 4.63 (d, 1H, J32 2.6 Hz, H-3), 5.64 (m, 2H, H-4 and H-5); 13C NMR (CDCl3): d ¼ 26.9, 27.9, 50.2, 74.4, 75.3, 76.3, 110.1, 126.8, 132.6. An alternative procedure for 7 is as follows: To a solution of azide 5 (0.30 g, 1.05 mmol) in dry tetrahydrofuran (10 mL), LiAlH4 (0.05 g, 1.31 mmol) was added. The resulting mixture was stirred at rt for 36 h. The reaction was quenched with water and treated with 20% aqueous sodium hydroxide (1 mL). The layers were separated, and the organic phase was dried (anh. MgSO4), filtered, and concentrated under reduced pressure to provide the crude amine (7), which was purified by flash chromatography as described above. Yield: 31%.

(1S,2R,3R,6R)-6-Amino-cyclohex-4-ene-1,2,3-triol (Conduramine C-4, 8) Compound 7 was dissolved in methanol and applied to a Dowex-50 (Hþ form) column, which was eluted with 2 N NH4OH (20 mL). The alkaline eluate was concentrated at 508C (20 Torr) to afford (2)-8. Yield: 98%; white solid; mp 82–848C (d); [a]20 D ¼ 2160 (c 0.30, MeOH), Anal. calcd. for C6H11NO3: C, 49.63; H, 7.65; N, 9.65. Found: C, 49.54; H, 8.01; N 9.44, IR (KBr) 3385– 3279 b, 1613, 1305, 1050, 1024, 847, 690 cm21; MS (m/z, %) 128 (Mþ 221, 0.25), 110 (3), 85 (100), 68 (6), 56 (12), 42 (5); 1H NMR (D2O): d ¼ 3.44 (m, 1H, H-2), 3.49 (m, 1H, H-3), 4.04 (s, 1H, H-1), 4.36 (s, 1H, H-6), 5.56 (d, 1H, J54 10.3 Hz, H-5), 5.59 (d, 1H, J45 10.3 Hz, H-4); 13C NMR (D2O): d ¼ 50.2, 68.6, 72.8, 75.0, 128.6, 130.0.

ACKNOWLEDGMENT The authors are grateful to IFS (Grants F/3078-1 and F/3078-2) and CONICYT (FCE 8068) for financial support of this work. A. B. is grateful to PEDECIBA and DINACYT for a doctoral fellowship. The authors

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acknowledge Jorge Adum for carrying out the fermentation process and Horacio Pezzaroglo for recording nuclear magnetic resonance (NMR) spectra.

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