Design, synthesis and In Vitro evaluation on glucosamine-6P synthase of aromatic analogs of 2-Aminohexitols-6P

ARTICLE

Design, synthesis and In Vitro evaluation on glucosamine-6P synthase of aromatic analogs of 2-Aminohexitols-6P

Danielle F. Dias^I; Céline Roux^II; Philippe Durand^II; Bogdan Iorga^II; Marie A. Badet-Denisot^II; Bernard Badet^II; Ricardo J. AlvesI, ^*

^IFaculdade de Farmácia, Universidade Federal de Minas Gerais, 31970-201 Belo Horizonte-MG, Brazil

^IIInstitut de Chimie des Substances Naturelles-CNRS, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette, France

ABSTRACT

The aminosugars are very important structural components of bacterial and fungi cell walls. Glucosamine-6-phosphate synthase (GlmS), which catalyses the first step of the aminosugar biosynthetic pathway i.e. the formation of D-glucosamine-6-phosphate from D-fructose-6-phosphate, is therefore an interesting target in the fight against microorganisms. In this work is described the synthesis of aromatic analogs of 2-amino-2-deoxy-D-glucitol-6-phosphate (ADGP) and its epimer 2-amino-2-deoxy-D-manitol-6-phosphate (ADMP), two important inhibitors of GlmS. The aromatic analogs displayed modest inhibitory activity against GlmS, with IC₅₀ in the mmol L^-1 range.

Keywords: glucosamine synthase, inhibitors, aminohexitols derivatives

RESUMO

A glicosamina-6-fosfato sintase (GlmS) catalisa a formação de glicosamina-6-fosfato a partir de frutose-6-fosfato. Esta é a primeira etapa da via biossintética de aminoaçúcares, componentes estruturais importantes da parede celular de fungos e bactérias. Este trabalho descreve o planejamento, a síntese e a avaliação da atividade inibitória da GlmS de dois novos compostos aromáticos enantioméricos, análogos de 2-amino-2-desoxi-D-glucitol-6-fosfato (ADGP) e de seu epímero 2-amino-2-desoxi-D-manitol-6-fosfato (ADMP), dois importantes inibidores da GlmS. Os análogos aromáticos apresentaram atividade inibitória modesta, na faixa de mmol L^-1.

Introduction

Systemic fungi infections remain a problem in modern chemotherapy. Current therapeutic arsenal does not fulfill all medical needs. The increasing number of immunocompromised patients, due to anticancer chemotherapy, organ transplantation and AIDS, along with drug resistance, makes things more complicated.^1,2 Thus the search for new antifungal agents is mandatory. Glucosamine-6-phosphate synthase (GlmS) catalyses the transformation of D-fructose-6-phosphate (Fru-6P) into D-glucosamine-6-phosphate (GlcN-6P). This is, in turn, converted into UDP-GlcNAc, the nucleotide form of N-acetylglucosamine that is the building block for the biosynthesis of chitin.^3,4 The enzyme has been validated as a new target for the development of new antifungal agents.^4,5 GlmS has two catalytic domains, namely, a glutaminase and an isomerase domains. The glutaminase domain is responsible for the hydrolysis of glutamine and generation of ammonia and glutamate. The ammonia released travels across a hydrophobic tunnel to reach the isomerase domain, where it is transferred to enzyme-bound Fru-6P.^6,7

Several inhibitors of both domains have been reported.^8-11 Inhibitors of the glutaminase domain are L-glutamine mimics whereas inhibitors of the isomerase domain have been trying to mimic the putative transition state intermediates.⁴ Therefore 2-amino-2-deoxy-D-glucitol-6-phosphate (ADGP) and 2-amino-2-deoxy-D-manitol-6-phosphate (ADMP) are, along with D-arabinose-oxime-6-phosphate, the most potent inhibitors described thus far.^9,10 Nevertheless, these compounds possess weak in vitro antifungal activity which was attributed to low cell penetration due to the high hydrophilic character of these compounds.¹² Lipophilic derivatives of ADGP were prepared and some of them did display enhanced in vitro activity,¹¹ reinforcing the importance of hydrophilic/ lipophilic balance for good activity.

The replacement of carbohydrate moieties of bioactive compounds by an aromatic ring has been a useful strategy for the obtention of active analogs.¹³ Besides increasing the lipophilic character, the ring introduces conformational restriction, which might eventually enhances activity by selecting the bioactive conformation. The 6-phosphate and 2-amino groups of ADGP have been suggested as crucial for its enzyme inhibitory potency.¹¹ Substitution of the C3-C6 backbone chain by a meta phenyloxy ring linking the two groups was envisaged as one possibility for aromatic introduction. This was supported by molecular modeling (docking) which points out a similar distance between C2 and O6 atoms in the two molecules (5.3 Å for ADGP vs 5.7 Å for 2), and the good fitting for some of the aromatic molecule conformers with the crystallographic structure of ADGP in the active site of GlmS (E. coli) (Figure 1).

With this in mind we planned the synthesis of compounds 1 and 2, according to the retrosynthetic analysis shown in Figure 2.

The synthesis of 1 and 2 were then carried out according to the synthetic route shown in Figure 3.

Results and Discussion

Initially, the commercially available protected serine 3 was reacted with LiBH₄ in THF/CH₃OH at -10 ºC to give the alcohol 4.¹⁴ Reaction of 4 with 3-benzoyloxyphenol under Mitsunobu conditions¹⁵ at 80 °C afforded the corresponding ether 5. Removal of the benzoyl protecting group was achieved upon treatment with CH₃ONa in CH₃OH at 0 °C to afford 6 in high yield. Phosphorylation with dibenzylphosphite (HPO(BnO)₂), in the presence of NaOH and CBr₄,¹⁶ gave the aromatic phosphate 7 in 90% yield. Hydrogenolysis of the benzyl phosphate (H₂, Pd-C, Parr hydrogenator, 40 psi) and subsequent removal of N-Boc and dimethyl acetal groups with TFA¹⁷ followed by preparative HPLC purification gave the target compounds 1S or 2R, in 19% and 30% overall yield, respectively. Inhibitory assay against GlmS from E. coli was performed according to published procedures.¹⁸ Results are given in Table 1.

As shown in Table 1, compounds 1 and 2 were poor inhibitors of GlmS, displaying IC₅₀ in the millimolar range. Although the replacement of the hydroxyl groups in ADGP and ADMP by an aromatic ring can provide higher lipophilicity to the resulting analogs, as predict by ACD software calculations of Clog D pH 7 (-6.5 for ADGP and ADMP vs -4.6 for 1 and 2), it is deleterious for their interaction with the enzyme active site.

Conclusions

In conclusion, we reported the design, synthesis and evaluation of two chiral aromatic analogs of the GlmS inhibitors ADGP and ADMP. Although the target compounds displayed weak inhibitory activity, they represent a new class of Glms inhibitors whose activity can be improved by further modification. This is in progress and will be reported in due time.

Experimental

General procedures

The reactions were monitored by thin-layer chromatography (TLC) performed on silica gel 60 F254 (Merck®). Melting points were determined with a Microquímica MQAPF 301 apparatus and are uncorrected. Flash chromatography was performed on silica gel 230-600 mesh (Merck®). NMR spectrum was recorded on a Bruker AC300 or AC500 spectrometer at ambient temperature or under heating at 363K. Chemical shifts (δ) were reported in ppm and standardized to the residual undeuterated solvents as the reference peaks and coupling constants (J) in Hz. The products 1S and 2R were purified on Atlantis® Prep dC₁₈ OBD preparative column (19 mm × 150 mm, 5 μm) using an HPLC apparatus (Waters) equipped with a photodiode array detector. Gradient elution was employed using solvent A (10 mmol L^-1 NH₄OAc), solvent B (CH₃CN); gradient: 0-7 min 100% A; 7-9 min linear change to A-B (40:60); 9-30 min, isocratic elution with 60% B (column rinsing); 30-50 min 100% A (column equilibration). The flow rate was kept at 17 mL min^-1 and the sample injection volume was 0.5 mL. The alcohol 4 was synthesized following standard procedure.^14,19

Synthesis of (R)-tert-butyl 4-((3-(benzoyloxy)phenoxy)methyl)-2,2-dimethyl-oxazolidine-3-carboxylate 5R

Diethyl azodicarboxylate (0.20 mL, 1.20 mmol) was added to a mixture of 3-benzoyloxyphenol (0.25 g, 1.20 mmol), alcohol 4R (0.20 g, 1.00 mmol) and PPh₃ (0.31 g, 1.20 mmol) in toluene (8 mL) at room temperature under an argon atmosphere and the reaction mixture was stirred for 40 h at 80 °C. The solution was cooled to room temperature and was washed with 1 mol L^-1 NaOH solution (20 mL) and then H₂O (2 × 20 mL). The organic phase was dried over MgSO₄, filtered and evaporated under reduced pressure. The residue was purified by flash chromatography on silica gel (n-hexane/EtOAc 9:1) to give the desired ether 5R (0.23 g, 67%). [α]_D²⁰ -49.0 (c 1.0, CHCl₃). HRMS (ESI+) calculated for C₂₄H₂₉NO₆Na (M+Na), 450.1893. Found: 450.1898. ¹H NMR (500 MHz, DMSO-d6, 363 K) δ 1.44 (s, 9H, ^tBu); 1.48 and 1.54 (2s, 6H, ⁱPr); 3.96-4.18 (m, 5H); 6.88-6.94 (m, 3H, CH-aromatics); 7.36 (t, 1H, ²J 7.75 Hz, CH-aromatic); 7.60 (t, 2H, ²J 7 Hz; mCH-benzoyl group); 7.73 (t, 1H, ²J 7.5 Hz, pCH-benzoyl group); 8.13 (d, 2H, ²J 7.5 Hz, oCH-benzoyl group). ¹³C NMR (125 MHz, DMSO-d6, 363K) δ 23.32 (CH₃-ⁱPr); 26.17 (CH₃-ⁱPr); 27.56 (CH₃-^tBu); 55.15 (CH); 64.47 (CH₂); 67.0 (CH₂); 79.09 (C-^tBu); 92.81 (C-ⁱPr); 108.15 (C-aromatic); 112.09 (CH-aromatic); 113.74 (CH-aromatic); 128.24 (mCH-benzoyl group); 128.80 (C-benzoyl group); 129.10 (oCH-benzoyl group); 129.37 (mCH-aromatic); 133.19 (pCH-benzoyl group); 150.86 (C=O NBoc); 151.32 (C-ipso); 158.89 (C-ipso); 163.83 (C=O benzoyl group).

Synthesis of (S)-tert-butyl 4-((3-(benzoyloxy)phenoxy)methyl)-2,2-dimethyl-oxazolidine-3-carboxylate 5S

The S enantiomer was prepared following the same procedure described above to prepare 5R (0.21 g, 0.9 mmol) to give 5S (0.29 g, 76%). [α]_D²⁰ 49.2 (c 1.05, CHCl₃). HRMS (ESI+) calculated for C₂₄H₂₉NO₆Na (M+Na), 450.1893. Found: 450.1892. ¹H NMR and ¹³C NMR spectra, as for 5R.

Synthesis of (R)-tert-butyl 4-((3-hydroxyphenoxy)methyl)-2,2-dimethyloxazolidine-3-carboxylate 6R

To a solution of 5R (0.19 g, 0.44 mmol) in dry CH₃OH (3 mL), was added CH₃ONa (24 mg, 0.44 mmol) at 0 °C. After 30 min. the solvent was removed under reduced pressure. The residue was purified by flash chromatography on silica gel (n-hexane/ AcOEt 8:2) to give pure 6R as a white solid, (0.14 g; 95%). mp 110.6-111.6 °C. [α]_D²⁰ -34.5 (c 1.0, CHCl₃). HRMS (ESI+) calculated for C₁₇H₂₅NO₅Na (M+Na), 346.1631. Found: 346.1634. ¹H NMR (500 MHz, DMSO-d6, 363K) δ 1.45 (s, 9H, CH₃-^tBu); 1.47 (s, 3H, CH₃-ⁱPr); 1.53 (s, 3H, CH₃-ⁱPr); 3.87 (t, 1H, ²J 9.4Hz, CH₂); 3.93 (d, 1H, ²J 8.5Hz, CH₂); 4.00 - 4.02 (m, 1H, CH₂); 4.05 (t, 1H, ²J 9.4Hz, CH₂); 4.13 (br, s, 1H, CH); 6.38 - 6.42 (m, 3H, CH-aromatics); 7.04 (t, 1H, ²J 8 Hz, mCH-aromatic); 8.98 (br, s, 1H, OH). ¹³C NMR (125 MHz, DMSO-d6, 363 K) δ 23.37 (CH₃-ⁱPr); 26.17 (CH₃-ⁱPr); 27.59 (CH₃-^tBu); 55.30 (CH); 64.51 (CH₂); 66.52 (CH₂); 79.02 (C-^tBu); 92.76 (C-ⁱPr); 102.08 (CH-aromatic); 105.04 (CH-aromatic); 108.04 (CH-aromatic); 129.16 (CH-aromatic); 150.86 (C=O NBoc); 158.16 (C-ipso phenolic); 159.22 (C-ipso).

Synthesis of (S)-tert-butyl 4-((3-hydroxyphenoxy)methyl)-2,2-dimethyloxazolidine-3-carboxylate 6S

The S enantiomer was prepared following the same procedure described above to prepare 6R (0.18 g, 0.42 mmol) to give pure phenol 6S (0.12 g, 85%). mp 110.7-111.6 °C. [α]_D²⁰ 36.0 (c 1.02, CHCl₃). HRMS (ESI+) calculated for C₁₇H₂₅NO₅Na (M+Na), 346.1631. Found: 346.1636. ¹H NMR and ¹³C NMR spectra, as for 6R.

Synthesis of (R)-tert-butyl 4-((3-(bis(benzyloxy)phosphoryloxy)phenoxy)methyl)-2,2-dimethyloxazolidine-3-carboxylate 7R

A solution of dibenzylphosphite (0.12 g, 0.46 mmol) and phenol 6R (0.10 g, 0.31 mmol) in CH₂Cl₂ (2 mL) was added to an ice-cold two-phase system consisting of CBr₄ (0.053 g, 0.16 mmol), 30% (m/v) aqueous NaOH (100 μL), H₂0 (1 mL) and CH₂Cl₂ (1 mL). The system was stirring for 30 min at room temperature. CH₂Cl₂ (10 mL) was added and the organic layer was separated, washed with H₂O (3 × 5 mL), dried over MgSO₄ and filtrated. Concentration followed by flash chromatography on silica gel of the crude material afforded the product 7R (0.17 g, 89% yield) as colorless oil. [α]_D²⁰ -37.0 (c 1.03, CHCl₃). HRMS (ESI+) calculated for C₃₁H₃₈NO₈PNa (M+Na), 606.2233. Found: 606.2230. ¹H NMR (500 MHz, DMSO-d6, 363 K) δ 1.44 (br, s, 9H, CH₃-^tBu); 1.47 (s, 3H, CH₃-ⁱPr); 1.53 (s, 3H, CH₃-ⁱPr); 3.90-4.14 (m, 5H); 5.15-5.17 (m, 4H, CH₂-benzyl group); 6.77-6.64 (m, 3H, CH-aromatics); 7.26 (t, 1H, ²J 8.5 Hz; CH-aromatic); 7.36 (br, s, 10H, CH-benzyl group). ¹³C NMR (75 MHz, CDCl₃) δ 23.30; 24.50; 27.00; 27.80 (CH₃-ⁱPr); 28.60; 28.70 (CH₃-^tBu); 56.00; 56.20 (CH); 65.40; 65.6 (CH₂); 66.50; 67.10 (CH₂); 70.10; 70.20 (CH₂-benzyl group); 80.50; 80.80 (C-^tBu); 93.80; 94.30 (C-ⁱPr); 107.20; 107.30 (CH-aromatic); 111.40; 111.7 (CH-aromatic); 112.7; 112.80 (CH-aromatic); 128.20; 128.80 (CH-aromatic benzyl group); 130.30 (CH-aromatic); 135.60; 135.70 (C-ipso benzyl group); 151.60; 151.90 (C-aromatic and C=O NBoc), 159.70 (C-ipso). ³¹P NMR (121.5 MHz, CDCl₃, external 85% H₃PO₄) δ -6.38 and -6.43.

Synthesis of (S)-tert-butyl 4-((3-(bis(benzyloxy)phosphoryloxy)phenoxy)methyl)-2,2-dimethyloxazolidine-3-carboxylate 7S

The S enantiomer was prepared following the same procedure described above to prepare 7R (0.12 g, 0.37 mmol) to give 7S (0.15g, 76% yield). [α]_D²⁰ 37.4 (c 1.01, CHCl₃). HRMS (ESI+) calculated for C₃₁H₃₈NO₈PNa (M+Na), 606.2233. Found: 606.2228. ¹H NMR and ¹³C NMR spectra, as for 7R.

Synthesis of 3-(2-(S)-amino-3-hydroxypropoxy)phenyl dihydrogenphosphate 1

A solution of dibenzylphosphate 7R (0.16 g, 0.27 mmol) in THF (2 mL) was hydrogenolyzed under hydrogen pressure (40 psi) in presence of 10% activated Pd-C (40 mg) at room temperature for 1h. The reaction mixture was filtered and the THF evaporated under vacuum. TFA (1 mL) was then added to a solution of the residue in CH₃OH (2 mL). The reaction mixture was stirred at room temperature for 3h. TFA was eliminated by co-distillation with CH₂Cl₂ and CH₃OH. The product 1S was purified by preparative RP-HPLC and obtained as an amorphous white solid (35 mg, 48% yield). mp 168.0-168.7 °C. [α]_D²⁰ 7.2 (c 0.5, H₂O). HRMS (ESI+) calculated for C₉H₁₄NO₆PNa (M+Na), 286.0456. Found: 286.0455. ¹H NMR (500 MHz, D₂O) δ 3.79-3.84 (m, 1H, CH); 3.92 (dd, 1H, J_gem 12.3 Hz, ²J 6.6 Hz, CH₂); 3.99 (dd, 1H, J_gem 12.2 Hz, ²J 4.7 Hz, CH₂); 4.26 (dd, 1H, J_gem 10.5 HZ, ²J 6.6 Hz, CH₂); 4.36 (dd, 1H, J_gem 10.5 Hz, ²J 3.8 Hz, CH₂); 6.77 (dd, 1H, ²J 8.2 Hz, ³J 2.2 Hz, CH-aromatic); 6.89 (d, 1H, ²J 8.3 Hz, CH-aromatic); 6.95 (s, 1H, CH-aromatic); 7.32 (t, 1H, ²J 8.1 Hz, CH-aromatic). ¹³C NMR (125 MHz, D₂O) δ 52.18 (CH); 58.74 (CH₂); 65.01 (CH₂); 107.15 (d, ³J 4.6 Hz, CH-aromatic); 108.81 (CH-aromatic); 113.96 (d, ³J 4.6 Hz, CH-aromatic); 129.96 (CH-aromatic); 154.87 (d, ²J 6.4 Hz, C-ipso); 158.31 (C-ipso). ³¹P NMR (202.5 MHz, D₂O, external 85% H₃PO₄) δ -0.273.

Synthesis of 3-(2-(R)-amino-3-hydroxypropoxy)phenyl dihydrogenphosphate 2

The R enantiomer was prepared following the same procedure using 7S (0.13g, 0.22 mmol) to give the desired phosphate 2R (39 mg; 68% yield). mp 163.9-165.0 °C. [α]_D²⁰ -7.5 (c 0.5, H₂O). HRMS (ESI+) calculated for C₉H₁₄NO₆PNa (M+Na), 286.0456. Found: 286.0451. ¹H NMR and ¹³C NMR spectra, as for 1.

Determination of enzyme activity

GlmS was purified to homogeneity using a protocol adapted from the procedure first reported by Badet and co-workers.²⁰ GlmS activity was spectrophotometrically assayed using the modified microplate Morgan-Elson method.¹⁸ The assay mixture contained, in a final volume of 100 μL for each well of the microplate, 100 mmol L^-1 phosphate buffer, pH 7.2, 50 mmol L^-1 KCl, 1 mmol L^-1 EDTA, 0.4 mmol L^-1 D-Fru-6P (a concentration close to the K_m value was chosen), and 4 mmol L^-1 Gln (saturating concentration). Inhibitors were used at the following concentrations: ADGP at 0-0.5 mmol L^-1, 1 or 2 at 0-7 mmol L^-1. Following preincubation at 37 °C for 10 min, the enzymatic reaction was initiated by the addition of 0.06 unit of GlmS. Precisely 30 min later (initial rate conditions), the reaction was stopped by the addition of 10 μL of a 1.5% (v/v) acetic anhydride solution in acetone (prepared daily) followed by 50 μL of a 0.2 mol L^-1 borate solution. After being heated to 80 °C for 30 min, the microplate (containing the various reaction mixtures) was centrifuged before addition of 130 μL of Ehrlich reagent (10% (m/v) solution of p-dimethyl-aminobenzaldehyde in acetic acid/concentrated HCl mixture (87/13; v/v) to each well. Then, the amount of GlcN-6P formed was measured from the absorbance of the Ehrlich adduct at 585 nm (ε value was determined daily from a GlcN-6P standard solution). The initial rate of formation of Glc-N6P as a function of the inhibitor concentration allowed the determination of the IC₅₀ value (inhibition that gives an initial rate equal to 50% of the rate in the absence of inhibitor).

Molecular modeling

The tridimensional structures of compounds 1 and 2 were constructed using Corina v3.44. Docking studies were performed on the dimer structure of GlmS (PDB code 2J6H)²¹ with Gold 4.0,²² using the default parameters and the GoldScore scoring function. A number of five water molecules (W166, W251, W254, W256, W257), which are conserved in all GlmS structures, were considered in the docking procedure. Ligand 2-deoxy-2-amino-D-glucitol-6-phosphate (ADGP) from the structure 1MOS ²³ was used as reference in the docking analysis. Raytraced image was produced with UCSF Chimera²⁴ and POV-Ray.²⁵

Acknowledgments

The authors thank the CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico - Brasil), CAPES (Coordenação de Aperfeiçoamento Pessoal de Nível Superior - Brasil), FAPEMIG (Fundação de Amparo à Pesquisa do Estado de Minas Gerais - Brasil), CNRS (Centre National de la Recherche Scientifique - France) and ICSN (Insitut de Chimie des Substances Naturelles) for grant and fellowships.

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Received: July 1, 2009

Web Release Date: December 21, 2009

Publication Dates

• Publication in this collection
  21 May 2010
• Date of issue
  2010

History

• Accepted
  21 Dec 2009
• Received
  01 July 2009