Synthesis of tartaric acid analogues of FR258900 and their evaluation as glycogen phosphorylase inhibitors

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Please check this box if you have no corrections to make to the PDF file Graphical abstract pp xxx–xxxSynthesis of tartaric acid analogues of FR258900 and their evaluation as glycogen phosphorylase inhibitors Gergely Varga, Tibor Docsa, Pál Gergely, László Juhász * , László Somsák * HOOC (S) (R) COOH O O (E) O (E) OH HO O FR258900 core unit HOOC (S) (S) COOH OH HO HOOC (R) (R) COOH OH HO HOOC (R) (S) COOH OH HO D -tartaric acid L -tartaric acid meso-tartaric acid or or (E) COOH R 2 R 1 R 1 =R 2 = H: cinnamic acid R 1 =H;R 2 =OH:p-coumaric acid R 1 =OCH 3 ;R 2 =OH:ferulicacid K i =5.47µM (against G1P) K i =0.2-0.46µM (against AMP) core unit replaced by tartaric acids esterified by K i =3.36-109µM(againstG1P) K i =2.0-26.4µM(againstAMP) BMCL 20030 No. of Pages 1, Model 5G 28 January 2013 1 Synthesis of tartaric acid analogues of FR258900 and their evaluation as glycogen phosphorylase inhibitors Gergely Varga a ,Tibor Docsa b ,Pál Gergely b ,László Juhász a, ⇑ ,László Somsák a, ⇑ a Department of Organic Chemistry, Faculty of Science and Technology, University of Debrecen, Egyetem tér 1, PO Box 10, H-4010 Debrecen, Hungary b Department of Medical Chemistry, Medical and Health Science Centre, University of Debrecen, Egyetem tér 1, H-4032 Debrecen, Hungary article info Article history: Received 4 December 2012 Revised 10 January 2013 Accepted 12 January 2013 Available online xxxx Keywords: 20 FR258900 Tartaric acid derivatives Diabetes Glycogen phosphorylase Inhibitor abstract Di-O-cinnamoylated, -p-coumaroylated, and -feruloylated D-, L-and meso-tartaric acids were synthesized as analogues of the natural product FR258900, a glycogen phosphorylase (GP) inhibitor with in vivo antihyperglycaemic activity. The new compounds inhibited rabbit muscle GP in the low micromolar range, and bound to the allosteric site of the enzyme. The best inhibitor was 2,3-di-O-feruloyl meso-tartaric acid and had K i values of 2.0 l M against AMP (competitive) and 3.36 l M against glucose-1-phosphate (non-competitive). Ó2013 Published by Elsevier Ltd. The number of patients suffering from diabetes mellitus (DM) is dramatically increasing. In 2011 the international diabetes federation project (IDF) indicated that the number of diabetic patients was more than 360 million worldwide. 1 In 2001 this number was 40 predicted to be reached in 2030 only. 2 More than 90% of the diagnosed cases belong to type 2 or non-insulin dependent diabetes mellitus (T2DM or NIDDM) characterized by peripheral insulin resistance, elevated hepatic glucose production, and defects in pancreatic insulin secretion. 3 Although several drugs are in clinical use for symptomatic treatment of T2DM, 4,5 these therapies are inadequate for 30–40% of the patients. 6 Among several investigational fields to diminish hepatic glucose output in T2DM, glycogen phosphorylase (GP) as a main regulatory enzyme of glycogen metabolism has become a validated 50 target. 2 Protein crystallographic studies have shown that endogenous and synthetic modulators can bind to six major sites in GP: the catalytic, the inhibitor, the allosteric (or AMP-binding), the glycogen storage, and the new allosteric sites, 7 as well as the newly discovered benzimidazole site. 8 A number of GP inhibitors for the different binding sites have been disclosed and several of them have considerable in vivo effects towards normalizing blood glucose and liver glycogen levels. 9,10 In recent years, the interaction of GP and glycogen targeting 60 subunit (G L ) of protein phosphatase 1 (PP1) has been identified as a novel molecular target for the treatment of T2DM. 11–13 It was demonstrated in an in vivo mouse model that disruption of G L –GP interaction resulted in an increased glycogen synthase activity and the mice had improved glucose tolerance. 14 X-ray crystallography showed that the G L –GP interaction took place by binding the C-terminal region of G L to the allosteric site of GP. 15 Thus, occupation of this site may prevent G L –GP interaction, thereby enhancing glycogen synthesis which, on the other hand, diminishes hepatic glucose production. This effect could be achieved by 70 modulators that bind to the allosteric site of GP. Several molecules of high structural diversity were reported to bind to the allosteric site of GP, such as derivatives of acyl urea, dihydropyridine dicarboxylic acid, pentanedioic acid, phthalic acid, N,N 0 -diaryl-urea, and pentacyclic triterpenoids. 9,16 HOOC COOH O O OOH HO O FR258900 1 core unit IC50 = 2.5 μM (human liver GP)17 Ki= 0.46 μM (rabbit muscle GP)18 0960-894X/$ - see front matter Ó2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.bmcl.2013.01.042 ⇑ Corresponding authors. Tel.: +36 52 512900x22474; fax: +36 52 512744 (L.J.), tel.: +36 52 512900x22348; fax: +36 52 512744 (L.S.). E-mail addresses: [email protected] (L. Juhász), somsak.laszlo@ science.unideb.hu,[email protected] (L. Somsák). Q2 Q1 Bioorganic & Medicinal Chemistry Letters xxx (2013) xxx–xxx Contents lists available at SciVerse ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl BMCL 20030 No. of Pages 5, Model 5G 29 January 2013 Please cite this article in press as: Varga, G.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2013.01.042 FR258900, a bis-O-(p-coumaroylated)2,3-dihydroxypentanedioic acid derivative 1,was isolated from the fermentation broth of fungi No. 138354. 17 The compound was shown to inhibit glyco80 gen phosphorylases and to bind to the allosteric site of GP. 18 Compound 1stimulated glycogen synthesis in primary rat hepatocytes, and investigations on glucagon-induced hyperglycemia in C57BL/6 mice suggested that 1could suppress hepatic glucose output in vivo. 19 On the basis of the above information and as a continuation of our research on the design and synthesis of small molecule inhibitors of GP we envisaged the preparation of structural analogues of 1. Since the synthesis of 2,3-dihydroxy-pentanedioic acid is unknown in the literature, the core unit was planned to be replaced 90 by easily available tartaric acid, also allowing to study the influence of configurational isomerism on the biological activity. Furthermore, the substitution pattern of the aromatic rings was also modified. A similar derivative to our target compounds is chicoric acid 20 (2) with immunostimulator and HIV-1 integrase inhibitor activities. 21 O O COOH HOOC O O HO HO OH OH 2 Crucial points for both the syntheses of chicoric acid 20–23 and 100 the preparation of our target compounds are the protections of phenolic OH and COOH groups. Acylation of unprotected tartaric acids with carbonylcaffeoyl chloride is feasible, but this carbonate type protection can be used only for caffeic acid. 20 Benzylic protection of both functionalities seemed very attractive, however, for the removal an equimolar amount of Pd(OAc) 2 for each protective group was necessary rendering this method extremely expensive. 23 Orthogonal ester type protection (acetyl for OH and tert-butyl for COOH 22 or methoxycarbonyl for OH and diphenylmethyl for COOH 21 ) were also applied in the syntheses of 2and their ana110 logues. Our comparative preliminary experiments showed that introduction of the latter pair of protecting groups was more efficient and easier to reproduce than that of the benzylic protection. Thus, the COOH groups of D -, L -and meso-tartaric acids (3–5) were transformed into diphenylmethyl (DPM) esters (7–9)by diphenyldiazomethane (DPDAM) generated in situ from benzophenone-hydrazone (6) by oxidation with activated MnO 2 in CH 2 Cl 224 (Scheme 1). DPM esters 7–9were isolated in excellent yields as white crystals and used further without any purification. Next, acid-chlorides 15–17 were prepared from commercially 120 available cinnamic (10), p-coumaric (11), and ferulic acids (12), respectively (Scheme 2), whereby phenolic OH groups of 11 and 12 were protected as methyl-carbonates 13 and 14, respectively. Thionyl-chloride treatment of carboxylic acids 10,13, and 14 gave acid-chlorides 15–17 which were used for acylations without further purification. Acylations of 7–9 were carried out in dry toluene using 2.2 equiv of acid-chlorides 15–17 and 2.2 equiv dry pyridine as base (Scheme 3). The fully protected 18–25 were isolated by column chromatography in acceptable yields. 130 Subsequent deprotections following the suggested protocol 21 (removal of methoxycarbonyl groups with Na 2 CO 3 /aq THF and cleavage of DPM esters with 70% aq AcOH) caused in our hands total decomposition of the molecules, irrespective of the order of the deprotection steps. Therefore, a new protocol for the cleavage of protecting groups in 18–25 was developed. The DPM esters could be cleaved by using dry anisole–TFA reagent in dry CH 2 Cl 2 at room temperature. 25 Purification of the crude products by column chromatography (PhCH 3 :AcOH 3:1) gave 26–33 in good to excellent yields (Scheme 140 4). Hydrolysis of the methoxycarbonyl esters was achieved by using an aq solution of NH 3 in MeOH and the products 34–39 were isolated in good to excellent yields. Reversing of the sequence resulted in decomposition of the molecules in the first step. The structure of the molecules was identified by NMR and MS measurements. The synthesized derivatives were evaluated as inhibitors of rabbit muscle glycogen phosphorylase b (rmGPb), and the results are summarized in Table 1. For comparison FR258900 (1) was also tested under our conditions. Compound 1proved a competitive 150 inhibitor against AMP and the obtained K i of 0.2 l M showed a good agreement with the literature value. When tested against G1P, 1 appeared as a non-competitive inhibitor with a K i of 5.47 l M (please, see Fig. 1 in the Supplementary data for details of the measurements and plots of the data). Similar conclusions could be drawn from the kinetic studies of compounds 32–39, as well, thereby indicating that in general the tartaric acid derivatives bound to the same site as FR258900 (1). As non-competitive inhibitors against G1P, the cinnamoyl derivatives 32 and 33, lacking the 4-OH substituents characteristic 160 of the natural product 1, proved practically inefficient. In the p-coumaroyl (34–36) and feruloyl (37–39) series the meso-configured compounds 36 and 39 proved most efficient. The latter demonstrated that introduction of an additional substituent in the MnO2MgSO4 dry CH2Cl2 rt. Ph2CNNH2 6 Ph2CN2 dry CH2Cl2 rt. 3-5 7-9 DPMOOC OH COODPM HO COOH HO HOOC OH Starting compound Configuration Product Yield (%) 3 D or (2S,3S) 7 85 4 L or (2R,3R) 8 83 5 meso or (2R,3S) 9 82 Scheme 1. Preparation of DPM esters 7–9. a then b 10 - 14 15 - 17 or b R1 R2 COOH R1 R2 COOR3 a: ClCOOCH 3 in 50% aq. NaOH at 0 °C; b: SOCl 2 , reflux, 6 h. R 1 R 2 R 3 Yield (%) Abbreviation 10 H H Cinn-OH 11 HOH Coum-OH 12 OCH 3 OH Feru-OH 13 H OCOOCH 3 OH 84 4-MC-Coum-OH 14 OCH 3 OCOOCH 3 OH 88 4-MC-Feru-OH 15 H H Cl 100* 4-MC-Cinn-Cl 16 H OCOOCH 3 Cl 100* 4-MC-Coum-Cl 17 OCH 3 OCOOCH 3 Cl 100* 4-MC-Feru-Cl * Conversion of the starting material. Scheme 2. Preparation of acid chlorides 15–17. 2G. Varga et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx BMCL 20030 No. of Pages 5, Model 5G 29 January 2013 Please cite this article in press as: Varga, G.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2013.01.042 aromatic rings (3-CH 3 O) was very advantageous and 39 proved equipotent with 1. As competitive inhibitors against AMP, beside the meso-configured 36 and 39, the L -configured 38 proved most efficient. Nevertheless, the efficiency of the inhibitors lagged behind that of 1. The presence of the 3-CH 3 O moieties in 39 rendered this com170 pound the best inhibitor under these test conditions, as well. In order to determine the molecular basis of the efficiency of these synthetic compounds, preparation of further analogues as well as molecular dockings and X-ray crystallographic studies are in progress, and will be reported in due course. In conclusion, three series of D -, L -and meso-tartaric acids di-Oacylated by cinnamic, p-coumaric, and ferulic acids were prepared as synthetic analogues of FR258900, a natural product with glycogen phosphorylase inhibitory and in vivo antihyperglycemic activities. The syntheses were characterized by using methoxycar180 bonyl (MC) and diphenylmethyl (DPM) esters to protect phenolic OH and COOH groups, respectively. New methods were applied for the removal of these protective groups (anisole–TFA for the cleavage of DPM, aq NH 3 –MeOH to hydrolyse MC). Some of the new compounds proved inhibitors of rabbit muscle glycogen phosphorylase b (competitive inhibition against AMP, non-competitive against G1P) in the low micromolar range. It was demonstrated by this work that simple synthetic compounds can bind to rmGPb similarly to the natural product, thereby opening up a new way for further studies of allosteric site inhibitors. The synthetic avail190 ability of these analogs offers obvious advantages over FR258900. Acknowledgments This work was supported by NKTH-OTKA (CK-77712), TÁMOP 4.2.1/B-09/1/KONV-2010-0007 and TÁMOP-4.2.2./B-10/1-20100024 projects co-financed by the European Union and the European SocialFund, as well as János Bolyai Research Scholarships (to L.J. and T.D.) of the Hungarian Academy of Sciences. Astellas 25% NH3solution 25 eq. CF3COOH 7 eq. dry anisole dry CH2Cl2 O HOOC COOH O O O R2 R1 R2 R1 MeOH 32 - 39 18 - 25 26 - 31 Products A DPMOOC O COODPM O R R HOOC O COOH O R R to 32, 33 to 34-39 Products B RStarting compound (Configuration) Products A Yield (%) Products B R 1 R 2 Yield (%) Cinn 18 ( D )--32 H H 83 19 ( L )--33 H H 86 20 ( D )26 88 34 H OH 85 4-MC-Coum 21 ( L )27 82 35 HOH 89 22 (meso)28 82 36 HOH 30 23 ( D )29 85 37 CH 3 OOH 81 4-MC-Feru 24 ( L )30 79 38 CH 3 OOH 69 25 (meso)31 92 39 CH 3 OOH 88 Scheme 4. Cleavage of the protective groups. acid-chloride (15 - 17) dry pyridine dry toluene, r.t. 7-9 18 - 25 DPMOOC OH COODPM HO DPMOOC O COODPM O R R Starting compound R Product (Configuration) Yield (%) 7 8Cinn 18 ( D ) 19 ( L ) 63 52 7 8 9 4-MC-Coum 20 ( D ) 21 ( L ) 22 (meso) 54 48 83 7 8 9 4-MC-Feru 23 ( D ) 24 ( L ) 25 (meso) 48 50 43 Scheme 3. Acylations with acid chlorides 15–17. Table 1 Inhibition (K i [ l M]) of rabbit muscle glycogen phosphorylase b by the synthetic compounds Compound (configuration) G1P dependence AMP dependence 1( L a ) 5.47 0.20 0.46 18 32 ( D ) 800 b — 33 ( L ) No inh. — 34 ( D ) 109 26.4 35 ( L ) 300 b — 36 (meso) 71.4 5.68 37 ( D ) 29.1 19.0 38 ( L ) 28.5 2.68 39 (meso) 3.36 2.0 a Configuration of the natural product corresponds to that of L -tartaric acid. b Calculated from the IC 50 value by using a web-based tool. 26 G. Varga et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx 3 BMCL 20030 No. of Pages 5, Model 5G 29 January 2013 Please cite this article in press as: Varga, G.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2013.01.042 Pharma Inc., Japan is gratefully thanked for kind provision of a sample of FR258900. 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Lett. xxx (2013) xxx–xxx BMCL 20030 No. of Pages 5, Model 5G 29 January 2013 Please cite this article in press as: Varga, G.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2013.01.042