Docsa, Tibor

Gergely, Pál

Juhász, László

Somsák, László

Varga, Gergely

Bioorganic & Medicinal Chemistry Letters

DEA University of Debrecen Electronic Archive

Our reference: BMCL 20030 P-authorquery-v11AUTHOR QUERY FORMJournal: BMCL Please e-mail or fax your responses and any corrections to:Article Number: 20030E-mail: corrections.essd@elsevier.sps.co.inFax: +31 2048 52799Dear Author,Please check your proof carefully and mark all corrections at the appropriate place in the proof (e.g., by using on-screen annotation in theﬁle) or compile them in a separate list. Note: if you opt to annotate the ﬁle with software other than Adobe Reader then please also highlPDFightthe appropriate place in the PDF ﬁle. To ensure fast publication of your paper please return your corrections within 48 hours.For correction or revision of any artwork, please consult http://www.elsevier.com/artworkinstructions.Any queries or remarks that have arisen during the processing of your manuscript are listed below and highlighted by ﬂags in the proof. Clickon the ‘Q’ link to go to the location in the proof.Location inarticleQuery / Remark: click on the Q link to goPlease insert your reply or correction at the corresponding line in the proofQ1 Please confirm that given names and surnames have been identified correctly.Q2 Please check the e-mail addresses, and correct if necessary.Thank you for your assistance.Please check this box if you have nocorrections to make to the PDF fileGraphical abstractBMCL 20030 No. of Pages 1, Model 5G28 January 2013pp xxx–xxxSynthesis of tartaric acid analogues of FR258900 and their evaluation as glycogen phosphorylase inhibitorsGergely Varga, Tibor Docsa, Pál Gergely, László Juhász *, László Somsák *HOOC(S)(R) OO(E)O(E)HOOcore unitHOOC(S)(S)COOHOHHOHOOC(R)(R)COOHOHHOHOOC(R)(S)COOHOHHOD-tartaric acid L-tartaric acid meso-tartaric acidor or(E) COOH R1 = R2 = H: cinnamic acidcore unitreplaced byCOOH OHFR258900R2R1R1 = H; R2 = OH: p-coumaric acidR1 = OCH3; R2 = OH: ferulic acidKi = 5.47 µM (against G1P) tartaric acids Ki = 3.36 -109 µM (against G1P)K = 0.2 - 0.46 µM (against AMP) esterified by K = 2.0 - 26.4 µM (against AMP)1i iFR258900 and their evaluationJuhász a,⇑, László Somsák a,⇑Debrecen, Egyetem tér 1, PO Box 10, H-4010 Debrecen, Hungaryf Debrecen, Egyetem tér 1, H-4032 Debrecen, Hungary20coumaroylated, and -feruloylated D-, L- and meso-tartaric acids were synthesizedral product FR258900, a glycogen phosphorylase (GP) inhibitor with in vivo anti-. The new compounds inhibited rabbit muscle GP in the low micromolar range,ric d2.0 ed.is-ss40 -snnleicnd50g60dItfeyy5--y70oa,,Q2QBioorganic & Medicinal Chemistry Letters xxx (2013) xxx–xxxle at SciVerse ScienceDirectcinal Chemistry Lettersw.elsevier .com/ locate/bmclBMCL 20030 No. of Pages 5, Model 5G29 January 2013Synthesis of tartaric acid analogues ofas glycogen phosphorylase inhibitorsGergely Varga a, Tibor Docsa b, Pál Gergely b, LászlóaDepartment of Organic Chemistry, Faculty of Science and Technology, University ofbDepartment of Medical Chemistry, Medical and Health Science Centre, University oa r t i c l e i n f oArticle history:Received 4 December 2012Revised 10 January 2013Accepted 12 January 2013Available online xxxxKeywords:FR258900Tartaric acid derivativesDiabetesGlycogen phosphorylaseInhibitora b s t r a c tDi-O-cinnamoylated, -p-as analogues of the natuhyperglycaemic activityand bound to the allosteand had Ki values of(non-competitive).The number of patients suffering from diabetes mellitus (DM)dramatically increasing. In 2011 the international diabetes federation project (IDF) indicated that the number of diabetic patientwas more than 360 million worldwide.1 In 2001 this number wapredicted to be reached in 2030 only.2 More than 90% of the diagnosed cases belong to type 2 or non-insulin dependent diabetemellitus (T2DM or NIDDM) characterized by peripheral insuliresistance, elevated hepatic glucose production, and defects ipancreatic insulin secretion.3 Although several drugs are in clinicause for symptomatic treatment of T2DM,4,5 these therapies arinadequate for 30–40% of the patients.6Among several investigational ﬁelds to diminish hepatglucose output in T2DM, glycogen phosphorylase (GP) as a mairegulatory enzyme of glycogen metabolism has become a validatetarget.21Contents lists availabBioorganic & Medijournal homepage: wwProtein crystallographic studies have shown that endogenousand synthetic modulators can bind to six major sites in GP: thecatalytic, the inhibitor, the allosteric (or AMP-binding), the glyco-gen storage, and the new allosteric sites,7 as well as the newlydiscovered benzimidazole site.8 A number of GP inhibitors for thedifferent binding sites have been disclosed and several of themhave considerable in vivo effects towards normalizing bloodglucose and liver glycogen levels.9,100960-894X/$ - see front matter  2013 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: juhasz.laszlo@science.unideb.hu (L. Juhász), somsak.laszlo@science.unideb.hu, somsak@tigris.unideb.hu (L. Somsák).Please cite this article in press as: Varga, G.; et al. Bioorg. Med. Chemsite of the enzyme. The best inhibitor was 2,3-di-O-feruloylmeso-tartaric acilM against AMP (competitive) and 3.36 lM against glucose-1-phosphat 2013 Published by Elsevier LtIn recent years, the interaction of GP and glycogen targetinsubunit (GL) of protein phosphatase 1 (PP1) has been identiﬁeas a novel molecular target for the treatment of T2DM.11–13was demonstrated in an in vivo mouse model that disruption oGL–GP interaction resulted in an increased glycogen synthasactivity and the mice had improved glucose tolerance.14 X-racrystallography showed that the GL–GP interaction took place bbinding the C-terminal region of GL to the allosteric site of GP.1Thus, occupation of this site may prevent GL–GP interaction, thereby enhancing glycogen synthesis which, on the other hand, diminishes hepatic glucose production. This effect could be achieved bmodulators that bind to the allosteric site of GP.Several molecules of high structural diversity were reported tbind to the allosteric site of GP, such as derivatives of acyl uredihydropyridine dicarboxylic acid, pentanedioic acid, phthalic acidN,N0-diaryl-urea, and pentacyclic triterpenoids.9,16HOOCCOOHOOOOHHOOFR2589001core unitIC50 = 2.5 μM (human liver GP)17Ki = 0.46 μM (rabbit muscle GP)18. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2013.01.042FR258900, a bis-O-(p-coumaroylated)2,3-dihydroxypentane-dioic acid derivative 1, was isolated from the fermentation brothof fungi No. 138354.17 The compound was shown to inhibit glyco-80 gen phosphorylases and to bind to the allosteric site of GP.18 Com-pound 1 stimulated glycogen synthesis in primary rat hepatocytes,and investigations on glucagon-induced hyperglycemia in C57BL/6mice suggested that 1 could suppress hepatic glucose output invivo.19On the basis of the above information and as a continuation ofour research on the design and synthesis of small molecule inhib-itors of GP we envisaged the preparation of structural analogues of1. Since the synthesis of 2,3-dihydroxy-pentanedioic acid is un-known in the literature, the core unit was planned to be replaced90 by easily available tartaric acid, also allowing to study the inﬂu-ence of conﬁgurational isomerism on the biological activity. Fur-thermore, the substitution pattern of the aromatic rings was alsomodiﬁed.A similar derivative to our target compounds is chicoric acid20(2) with immunostimulator and HIV-1 integrase inhibitor activi-ties.21OOCOOHHOOCOOHOHOOHOH2Crucial points for both the syntheses of chicoric acid20–23 and100 the preparation of our target compounds are the protections ofphenolic OH and COOH groups. Acylation of unprotected tartaricacids with carbonylcaffeoyl chloride is feasible, but this carbonatetype protection can be used only for caffeic acid.20 Benzylic protec-tion of both functionalities seemed very attractive, however, forthe removal an equimolar amount of Pd(OAc)2 for each protectivegroup was necessary rendering this method extremely expen-sive.23 Orthogonal ester type protection (acetyl for OH and tert-bu-tyl for COOH22 or methoxycarbonyl for OH and diphenylmethyl forCOOH21) were also applied in the syntheses of 2 and their ana-110 logues. Our comparative preliminary experiments showed thatintroduction of the latter pair of protecting groups was more efﬁ-cient 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) bydiphenyldiazomethane (DPDAM) generated in situ from benzophe-none-hydrazone (6) by oxidation with activated MnO2 in CH2Cl224(Scheme 1). DPM esters 7–9 were isolated in excellent yields aswhite crystals and used further without any puriﬁcation.Next, acid-chlorides 15–17 were prepared from commercially120 available cinnamic (10), p-coumaric (11), and ferulic acids (12),respectively (Scheme 2), whereby phenolic OH groups of 11 and12 were protected as methyl-carbonates 13 and 14, respectively.Thionyl-chloride treatment of carboxylic acids 10, 13, and 14 gaveacid-chlorides 15–17 which were used for acylations without fur-ther puriﬁcation.Acylations of 7–9 were carried out in dry toluene using2.2 equiv of acid-chlorides 15–17 and 2.2 equiv dry pyridine asbase (Scheme 3). The fully protected 18–25 were isolated by col-umn chromatography in acceptable yields.130Subsequent deprotections following the suggested protocol21(removal of methoxycarbonyl groups with Na2CO3/aq THF andcleavage of DPM esters with 70% aq AcOH) caused in our hands to-tal decomposition of the molecules, irrespective of the order of thedeprotection steps.Therefore, a new protocol for the cleavage of protecting groupsin 18–25was developed. The DPM esters could be cleaved by usingdrﬁc(P1404)usisosustrmbitsute150inagap(psudrthbode160ofp-urMnO2 - MgSO4dry CH2Cl2rt.Ph2CNNH26a then b10 - 14 15 - 17or bR1R2COOH R1R2COOR3a: ClCOOCH3 in 50% aq. NaOH at 0 °C; b: SOCl2, reflux, 6 h.R1 R2 R3 Yield (%) Abbreviation10 H H Cinn-OH11 H OH Coum-OH12 OCH3 OH Feru-OH13 H OCOOCH3 OH 84 4-MC-Coum-OH14 OCH3 OCOOCH3 OH 88 4-MC-Feru-OH15 H H Cl 100* 4-MC-Cinn-Cl16 H OCOOCH3 Cl 100* 4-MC-Coum-Cl17 OCH3 OCOOCH3 Cl 100* 4-MC-Feru-Cl* Conversion of the starting material.Scheme 2. Preparation of acid chlorides 15–17.2 G. Varga et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxxBMCL 20030 No. of Pages 5, Model 5G29 January 2013PlPh2CN2dry CH2Cl2rt.3 - 5 7 - 9DPMOOCOHCOODPMHOCOOHHOHOOCOHStarting 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 onScheme 1. Preparation of DPM esters 7–9.ease cite this article in press as: Varga, G.; et al. Bioorg. Med. Chem. Letty anisole–TFA reagent in dry CH2Cl2 at room temperature.25 Puri-ation of the crude products by column chromatographyhCH3:AcOH 3:1) gave 26–33 in good to excellent yields (Scheme. Hydrolysis of the methoxycarbonyl esters was achieved bying an aq solution of NH3 in MeOH and the products 34–39werelated in good to excellent yields. Reversing of the sequence re-lted in decomposition of the molecules in the ﬁrst step. Theucture of the molecules was identiﬁed by NMR and MSeasurements.The synthesized derivatives were evaluated as inhibitors of rab-muscle glycogen phosphorylase b (rmGPb), and the results aremmarized in Table 1. For comparison FR258900 (1) was alsosted under our conditions. Compound 1 proved a competitivehibitor against AMP and the obtained Ki of 0.2 lM showed a goodreement with the literature value. When tested against G1P, 1peared as a non-competitive inhibitor with a Ki of 5.47 lMlease, see Fig. 1 in the Supplementary data for details of the mea-rements and plots of the data). Similar conclusions could beawn from the kinetic studies of compounds 32–39, as well,ereby indicating that in general the tartaric acid derivativesund to the same site as FR258900 (1).As non-competitive inhibitors against G1P, the cinnamoylrivatives 32 and 33, lacking the 4-OH substituents characteristicthe natural product 1, proved practically inefﬁcient. In thecoumaroyl (34–36) and feruloyl (37–39) series the meso-conﬁg-ed compounds 36 and 39 proved most efﬁcient. The latter dem-strated that introduction of an additional substituent in the. (2013), http://dx.doi.org/10.1016/j.bmcl.2013.01.042170R3thloridpyroluens wG. Varga et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx 3BMCL 20030 No. of Pages 5, Model 5G29 January 201325 eq. CF3COOH7 eq. dry anisoledry CH2Cl218 - 25DPMOOCOCOODPMO RRHOOCOCOOHORto 32, 3acid-cdrydry t7 - 9DPMOOCOHCOODPMHOStarting compound R78 Cinn7894-MC-Coum7894-MC-FeruScheme 3. Acylatioaromatic rings (3-CH3O) was very advantageous and 39 provedequipotent with 1.As competitive inhibitors against AMP, beside the meso-conﬁg-ured 36 and 39, the L-conﬁgured 38 proved most efﬁcient. Never-theless, the efﬁciency of the inhibitors lagged behind that of 1.The presence of the 3-CH3O moieties in 39 rendered this com-pound the best inhibitor under these test conditions, as well.In order to determine the molecular basis of the efﬁciency ofthese synthetic compounds, preparation of further analogues as26 - 31Products AR Starting compound(Configuration) Products ACinn 18 (D) -19 (L) -20 (D) 264-MC-Coum 21 (L) 2722 (meso) 2823 (D) 294-MC-Feru 24 (L) 3025 (meso) 31Scheme 4. Cleavage oTable 1Inhibition (Ki [lM]) of rabbit muscle glycogen phosphorylase b by the syntheticcompoundsCompound (conﬁguration) G1P dependence AMP dependence1 (La) 5.47 0.200.461832 (D) 800b —33 (L) No inh. —34 (D) 109 26.435 (L) 300b —36 (meso) 71.4 5.6837 (D) 29.1 19.038 (L) 28.5 2.6839 (meso) 3.36 2.0a Conﬁguration of the natural product corresponds to that of L-tartaric acid.b Calculated from the IC50 value by using a web-based tool.26Please cite this article in press as: Varga, G.; et al. Bioorg. Med. Chem25% NH3 solutionOHOOCCOOHOOOR2R1R2R1MeOH32 - 39o 34-39Products Be (15 - 17)idinene, r.t.18 - 25DPMOOCOCOODPMO RRProduct (Configuration) Yield (%)18 (D)19 (L)635220 (D)21 (L)22 (meso)54488323 (D)24 (L)25 (meso)485043ith acid chlorides 15–17.well as molecular dockings and X-ray crystallographic studies arein progress, and will be reported in due course.In conclusion, three series of D-, L- and meso-tartaric acids di-O-acylated by cinnamic, p-coumaric, and ferulic acids were preparedas synthetic analogues of FR258900, a natural product withglycogen phosphorylase inhibitory and in vivo antihyperglycemicactivities. The syntheses were characterized by using methoxycar-180bonyl (MC) and diphenylmethyl (DPM) esters to protect phenolicOH and COOH groups, respectively. New methods were appliedfor the removal of these protective groups (anisole–TFA for thecleavage of DPM, aq NH3–MeOH to hydrolyse MC). Some of thenew compounds proved inhibitors of rabbit muscle glycogen phos-phorylase b (competitive inhibition against AMP, non-competitiveagainst G1P) in the low micromolar range. It was demonstrated bythis work that simple synthetic compounds can bind to rmGPbsimilarly to the natural product, thereby opening up a new wayfor further studies of allosteric site inhibitors. The synthetic avail-190ability of these analogs offers obvious advantages over FR258900.AcknowledgmentsThis work was supported by NKTH-OTKA (CK-77712), TÁMOP4.2.1/B-09/1/KONV-2010-0007 and TÁMOP-4.2.2./B-10/1-2010-0024 projects co-ﬁnanced by the European Union and theEuropean Social Fund, as well as János Bolyai Research Scholarships(to L.J. and T.D.) of the Hungarian Academy of Sciences. AstellasYield (%) Products B R1 R2 Yield (%)- 32 H H 83- 33 H H 8688 34 H OH 8582 35 H OH 8982 36 H OH 3085 37 CH3O OH 8179 38 CH3O OH 6992 39 CH3O OH 88f the protective groups.. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2013.01.042Pharma Inc., Japan is gratefully thanked for kind provision of asample of FR258900.Supplementary data200 Supplementary data (kinetics of FR258900 inhibition of rmGPb)associated with this article can be found, in the online version, athttp://dx.doi.org/10.1016/j.bmcl.2013.01.042.References and notes1. Whiting, D. R.; Guariguata, L.; Weil, C.; Shaw, J. Diabetes Res. Clin. Pract. 2011,94, 311.2. Treadway, J. L.; Mendys, P.; Hoover, D. J. Expert Opin. Invest. Drugs 2001, 10, 439.3. Hengesh, E. J. In Principles of Medicinal Chemistry; Foye, W. O., Lemke, T. L.,Williams, D. A., Eds.; Williams & Wilkins: Baltimore, 1995; p 581.4. Israili, Z. H. Am. J. Ther. 2011, 18, 117.210 5. Somsák, L.; Bokor, É.; Czifrák, K.; Juhász, L.; Tóth, M. In Topics in the Prevention,Treatment and Complications of Type 2 Diabetes; Zimering, M. B., Ed.; InTechOpen Access Publisher: Rijeka, 2011; p 103.6. Wagman, A. S.; Nuss, J. M. Curr. Pharm. Des. 2001, 7, 417.7. 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Z.; Mudunuri, U.; Stephens, R.; Lebeda, F. J. Nucleic Acids Res. 2009, 37,W441.4 G. Varga et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxxBMCL 20030 No. of Pages 5, Model 5G29 January 2013Please 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

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

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

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