Synthesis of novel L-rhamnose derived acyclic C-nucleosides with substituted 1,2,3-triazole core as potent sodium-glucose co-transporter (SGLT) inhibitors Siddamal Reddy Putapatri a , Abhinav Kanwal b , Sanjay K. Banerjee b,c,⇑ , Srinivas Kantevari a,c,⇑ a Organic Chemistry Division-II (CPC Division), CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India b Medicinal Chemistry and Pharmacology Division, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India c Academy of Scientific and Innovative Research, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India article info Article history: Received 4 November 2013 Revised 12 January 2014 Accepted 30 January 2014 Available online 6 February 2014 Keywords: Triazole Rhamnose Diabetes Click chemistry SGLT2 inhibitors abstract Sodium-glucose co-transporter (SGLT) inhibitors are a novel class of therapeutic agents for the treatment of type 2 diabetes by preventing renal glucose reabsorption. In our efforts to identify novel inhibitors of SGLT, we synthesized a series of L-rhamnose derived acyclic C-nucleosides with 1,2,3-triazole core. The key b-ketoester building block 4 prepared from L-rhamnose in five steps, was reacted with various aryl azides to produce the respective 1,2,3-triazole derivatives in excellent yields. Deprotection of acetonide group gave the desired acyclic C-nucleosides 7a–o. All the new compounds were screened for their sodium-glucose co-transporters (SGLT1 and SGLT2) inhibition activity using recently developed cell- based nonradioactive fluorescence glucose uptake assay. Among them, 7m with IC 50 : 125.9 nM emerged as the most potent SGLT2 inhibitor. On the other hand compound 7d exhibited best selectivity for inhi- bition of SGLT2 (IC 50 : 149.1 nM) over SGLT1 (IC 50 : 693.2 nM). The results presented here demonstrated the utility of acyclic C-nucleosides as novel SGLT inhibitors for future investigations. Ó 2014 Elsevier Ltd. All rights reserved. In recent years, inhibition of sodium-glucose co-transporters (SGLTs) was considered as one of the therapeutic options to reduce blood glucose level independent of insulin. 1 Of several subtypes identified in the SGLT family, sodium-glucose co-transporters 2 (SGLT2) expressed in the early convoluted segment (S1) of the prox- imal tubule is responsible for 90% of the renal glucose reabsorption. 2 Remaining 10% of renal glucose reabsorption occurs through SGLT1 is expressed in the proximal tubule (S3). Other tissues like intestine, heart and trachea also express SGLT1. Thus, enhancing glucose excretion into urine by selectively inhibiting SGLT2 should lead to effective control of type 2 diabetes mellitus (T2DM). 3 Over the past 10 years, a series of O-glucosides and C-glucosides has been re- ported as SGLT2 inhibitors (Fig. 1). 4 T-1095 5 is the first structural derivative of Phlorizin, 6 a natural non selective SGLT inhibitor. Sergliflozin 7a and remogliflozin 7b,c are other representatives of the O-glucoside class of SGLT2 inhibitors. However, these two synthetic aryl O-glucosides were abandoned during clinical trials due to their poor selectivity/ efficacy and inherent metabolic instability to gas- trointestinal b-glucosidases. 8 Meanwhile, dapagliflozin, 9 followed by canagliflozin 10 and empagliflozin 11 have emerged as C-aryl glu- coside class of SGLT2 inhibitors. These three were found to exhibit more potent hSGLT2 inhibition in vitro and gastrointestinal stability in vivo than O-glucosides. 9–11 However, recent clinical results indi- cated that none of these SGLT2 inhibitors could exert more than 50% renal glucose reabsorption in humans. 12 Thus the quest for develop- ing novel small molecule carbohydrate mimics as inhibitors of SGLT2 is higher than ever before. 13 Besides this, C-glucosides with triazole, indole, benzisothiazole and thiophene aglycon were also investigated as inhibitors of SGLT2 (Fig. 2). 14 From all these reports it is evident that development of SGLT inhibitors is focused mainly at modifying aglycon unit of C-aryl glu- coside leaving the sugar moiety untouched. Departing from this ongoing trend, we asked ourselves whether acyclic C-nucleosides can inhibit SGLT activity. In our efforts to find new and potent acy- clic C-nucleosides 15 as novel SGLT inhibitors, we herein present our results on the synthesis and evaluation of the first examples of L- rhamnose derived acyclic C-nucleosides with substituted 1,2,3-tri- azole core as potential sodium-glucose co-transporter inhibitors. Here, L-rhamnose, a naturally occurring, easily available 6-deoxy- sugar having no known role in mammalian metabolism and toxic- ity, 16a was considered as carbohydrate mimic replacing D-glucose for the development of new antidiabetic agents. The choice of http://dx.doi.org/10.1016/j.bmcl.2014.01.077 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved. ⇑ Corresponding authors. Tel.: +91 4027191438; fax: +91 4027198933. E-mail addresses: [email protected](S.K. Banerjee), [email protected], [email protected](S. Kantevari). Bioorganic & Medicinal Chemistry Letters 24 (2014) 1528–1531 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Siddamal Reddy Putapatri a, Abhinav Kanwal b, Sanjay K. Banerjee b,c,⇑, Srinivas Kantevari a,c,⇑a Organic Chemistry Division-II (CPC Division), CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, Indiab Medicinal Chemistry and Pharmacology Division, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, Indiac Academy of Scientific and Innovative Research, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India
a r t i c l e i n f o
Article history:Received 4 November 2013Revised 12 January 2014Accepted 30 January 2014Available online 6 February 2014
Sodium-glucose co-transporter (SGLT) inhibitors are a novel class of therapeutic agents for the treatmentof type 2 diabetes by preventing renal glucose reabsorption. In our efforts to identify novel inhibitors ofSGLT, we synthesized a series of L-rhamnose derived acyclic C-nucleosides with 1,2,3-triazole core. Thekey b-ketoester building block 4 prepared from L-rhamnose in five steps, was reacted with various arylazides to produce the respective 1,2,3-triazole derivatives in excellent yields. Deprotection of acetonidegroup gave the desired acyclic C-nucleosides 7a–o. All the new compounds were screened for theirsodium-glucose co-transporters (SGLT1 and SGLT2) inhibition activity using recently developed cell-based nonradioactive fluorescence glucose uptake assay. Among them, 7m with IC50: 125.9 nM emergedas the most potent SGLT2 inhibitor. On the other hand compound 7d exhibited best selectivity for inhi-bition of SGLT2 (IC50: 149.1 nM) over SGLT1 (IC50: 693.2 nM). The results presented here demonstratedthe utility of acyclic C-nucleosides as novel SGLT inhibitors for future investigations.
� 2014 Elsevier Ltd. All rights reserved.
In recent years, inhibition of sodium-glucose co-transporters(SGLTs) was considered as one of the therapeutic options to reduceblood glucose level independent of insulin.1 Of several subtypesidentified in the SGLT family, sodium-glucose co-transporters 2(SGLT2) expressed in the early convoluted segment (S1) of the prox-imal tubule is responsible for 90% of the renal glucose reabsorption.2
Remaining 10% of renal glucose reabsorption occurs through SGLT1is expressed in the proximal tubule (S3). Other tissues like intestine,heart and trachea also express SGLT1. Thus, enhancing glucoseexcretion into urine by selectively inhibiting SGLT2 should lead toeffective control of type 2 diabetes mellitus (T2DM).3 Over the past10 years, a series of O-glucosides and C-glucosides has been re-ported as SGLT2 inhibitors (Fig. 1).4 T-10955 is the first structuralderivative of Phlorizin,6 a natural non selective SGLT inhibitor.Sergliflozin7a and remogliflozin7b,c are other representatives of theO-glucoside class of SGLT2 inhibitors. However, these two syntheticaryl O-glucosides were abandoned during clinical trials due to theirpoor selectivity/ efficacy and inherent metabolic instability to gas-trointestinal b-glucosidases.8 Meanwhile, dapagliflozin,9 followed
by canagliflozin10 and empagliflozin11 have emerged as C-aryl glu-coside class of SGLT2 inhibitors. These three were found to exhibitmore potent hSGLT2 inhibition in vitro and gastrointestinal stabilityin vivo than O-glucosides.9–11 However, recent clinical results indi-cated that none of these SGLT2 inhibitors could exert more than 50%renal glucose reabsorption in humans.12 Thus the quest for develop-ing novel small molecule carbohydrate mimics as inhibitors ofSGLT2 is higher than ever before.13 Besides this, C-glucosides withtriazole, indole, benzisothiazole and thiophene aglycon were alsoinvestigated as inhibitors of SGLT2 (Fig. 2).14
From all these reports it is evident that development of SGLTinhibitors is focused mainly at modifying aglycon unit of C-aryl glu-coside leaving the sugar moiety untouched. Departing from thisongoing trend, we asked ourselves whether acyclic C-nucleosidescan inhibit SGLT activity. In our efforts to find new and potent acy-clic C-nucleosides15 as novel SGLT inhibitors, we herein present ourresults on the synthesis and evaluation of the first examples of L-rhamnose derived acyclic C-nucleosides with substituted 1,2,3-tri-azole core as potential sodium-glucose co-transporter inhibitors.Here, L-rhamnose, a naturally occurring, easily available 6-deoxy-sugar having no known role in mammalian metabolism and toxic-ity,16a was considered as carbohydrate mimic replacing D-glucosefor the development of new antidiabetic agents. The choice of
Scheme 1. Synthesis of L-rhamnose derived b-ketoester building block 4. Reactionconditions: (i) NaBH4, methanol, rt; (ii) dimethoxypropane, acetone, pTSA, rt; (iii)35% aq AcOH; (iv) NaIO4, methanol/water (1:1); (v) N2CHCOOEt, dichloromethane,SnCl2�2H2O.
O
OHHO OH
HO
Dapagliflozin
Cl OEt
O O
OH
O
Sergliflozin
O
O
O
O
OHHO OH
HO
S
F
Canagliflozin
O
OH
(b)
O O
OHHO OH
HO
OHHO
O
OH
Phlorizin
O O
OHHO OH
O
T-1095
O
O
H3C OH
O
O(a)
HO OH
HO
Cl O
O
Empagliflozin
OHHO
O O
OH
O
Remogliflozin
O
O
HO OH
NN
O
Figure 1. Examples of some (a) O-glucoside and (b) C-glucoside SGLT2 inhibitors.
O
OHHO OH
HO
Cl
O O
OH
HO
O
OHHO OH
HOO
OHHO OH
HO
S
NF
NNN
CN
NS
HO OH
HN
I III
II IV
Figure 2. Representative C-glucosides with triazole(I), indole(II), benzisothiaz-ole(III), and thiophene(IV) aglycons as SGLT2 inhibitors.
S. R. Putapatri et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1528–1531 1529
6-deoxy L-mannose (L-rhamnose) is also due to recent discovery of6-deoxydapagliflozin as a more active SGLT2 inhibitor (IC50 = 0.67 -nM against human SGLT2 (hSGLT2) vs 1.16 nM for dapagliflozin.16b
Biological evaluation of fifteen new synthetic analogs revealed twocompounds 7m with IC50: 125.9 nM emerged as the most potentSGLT2 inhibitor and 7d exhibited best selectivity for inhibition ofSGLT2 (IC50: 149.1 nM) over SGLT1(IC50: 693.2 nM).
Synthetic approach for the preparation of L-rhamnose derivedacyclic C-nucleosides with substituted 1,2,3-triazole core 7a–oare presented in schemes 1 and 2. The key b-ketoester buildingblock [ethyl 3-((4R,5S)-5-((S)-1-hydroxyethyl)-2,2-dimethyl-1,3-dioxolan-4-yl)-3-oxopropanoate] 4 required for library generationis synthesized from commercially available L-rhamnose (Scheme 1).L-Rhamnitol obtained after NaBH4 reduction,15 was protected byreacting with 2,2-dimethoxy propane. Selective deprotection ofacetonide in 35% aqueous acetic acid gave triol 2 in 85% yield.Oxidative cleavage of 2 with NaIO4 in methanol: water producedreactive aldehyde 3 in excellent yield (95%). Aldehyde 3 was used
as such without any further purification and characterization. Fi-nally, the required key b-ketoester building block 4 was obtainedin 80% yields after reacting 3 with ethyl diazoacetate at room tem-perature in dichloromethane. Compounds 1, 2 and 4 were fullycharacterized by their IR, NMR and mass spectral data.17 On theother hand, the required azides 5a–n were also prepared fromthe respective amines by following the reported procedures,18
and were characterized by their spectral data correlating withliterature.
Click chemistry originally described by Huisgen and later devel-oped by Sharpless, has become a ubiquitous chemical tool withapplications in nearly all areas of modern chemistry, includingdrug discovery.19 Besides original Cu(I)-catalyzed azide–alkynecycloaddition, the variants like inverse electron demand Diels–Al-der reaction, and other types of bio-orthogonal click ligations havegained importance for the synthesis of bioactive molecules andradiopharmaceuticals.20 One such variant of click reaction is organ-ocatalytic 3+2 cycloaddition of b-ketoesters with aryl azides toproduce multi-substituted 1,2,3-triazoles.21
Having L-rhamnose derived b-ketoester 4 in hand, we employed3+2 cycloaddition reaction with aryl azides in the presence of anorganocatalyst. After series of experiments with varying reactionparameters and organocatalysts (proline, pyrrolidine), cycloaddi-tion of b-ketoester 4 with aryl azides 5a–n (Scheme 2) were bestachieved using pyrrolidine as organocatalyst.21 For example, 3+2cycloaddition of b-ketoester 4 with phenyl azide 5a in the presenceof pyrrolidine (10 mol %) in DMSO at 60 �C for 14 h produced ethyl5-((4S,5S)-5-((S)-1-hydroxyethyl)-2,2-dimethyl-1,3-dioxolan-4-yl)-1-phenyl-1H-1,2,3-triazole-4-carboxylate (6a) in 81% yield. Use ofproline as catalyst resulted 6a in lower yield (45%). All the azides5a–o reacted well with b-ketoester 4 to give the respective 1,4,5-tri-substituted 1,2,3-triazole derivatives 6a–o in excellent yields(Scheme 2). Compound 6a–o was fully characterized by IR, 1HNMR, 13C NMR, mass spectral (HRMS) analysis.22 Finally deprotec-tion of 1,4,5-trisubstituted 1,2,3-triazole derivatives 6a–o in dichlo-romethane using trifluoroacetic acid/water (1:1) gave the desiredacyclic C-nucleosides 7a–o in very good (71–85%) yields. All the fi-nal products 7a–o were purified over silica gel column chromatog-raphy and were fully characterized by IR, 1H NMR, 13C NMR, electronspray ionization (ESI), and high-resolution mass spectral (HRMS)analysis.22
The in vitro inhibitory potential of all fifteen new acyclicC-nucleosides 7a–o with aryl substituent’s appended to nitrogen on1,2,3-triazole core was assessed using recently developed23 cell-based nonradioactive fluorescent glucose uptake assay for functionalscreening of sodium-glucose co-transporters SGLT1 and SGLT2 deter-mined at excitation/emission maxima of 465/540 nm. SGLT1 & SGLT2transfected Human Embryonic Kidney (HEK293) cells were propa-gated at 37 �C in 5% CO2 in Dulbecco’s minimal essential medium(DMEM) supplemented with 1.0% of penicillin-streptomycin and
Table 1Evaluation of SGLT1 and SGLT 2 activity for 7a–o
Scheme 2. Synthesis of acyclic C-nucleosides 7a–o via organocatalytic variants of click reaction.
1530 S. R. Putapatri et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1528–1531
10% heat inactivated fetal bovine serum (FBS). The cells were culturedin a 90 mm dish in DMEM with 10% FBS until 70–80% confluences ob-tained for further use for screening SGLT1 & SGLT2 inhibition activity.To measure SGLT1 and SGLT2-mediated glucose uptake,23b transfec-ted stable HEK cells were plated separately at 1 � 104/well in 96-wellplate; all culture medium was removed from each well and replacedwith 100 ll of culture medium with newly synthesized acyclic C-nucleosides 7a–o at five different concentrations ranging from 50 to1000 nM (i.e. 50, 100, 250, 500 and 1000 nM). After half an hour, afluorescently-labeled 2-deoxyglucose analog, 2-NBDG (2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose) was added tothe plates and incubated for 30 min. After washing three times, cellswere then lysed and fluorescence of aliquots from the lysate was mea-sured at excitation/emission maxima of �465/540nm. Phlorizin, anatural non-specific SGLT1 and SGLT2 inhibitor was used as a refer-ence compound in this fluorescent based in vitro activity evaluationsystem. The IC50 values (concentration to inhibit 50% D-glucose up-take in cells) of new acyclic C-nucleosides 7a–o were determinedfrom the glucose uptake inhibition curves with reference to phlorizin.IC50 values obtained for synthetic compounds 7a–o are depicted inTable 1. All fifteen new compounds 7a–o showed SGLT1 and SGLT2inhibition activity with IC50 ranging from 125.9 to 693.2 nM (Table 1).Thirteen new analogs, except 7k and 7n exhibited SGLT2 inhibition atlower concentrations (IC50 ranging from 125 to 227 nM) than SGLT1.Among all the derivatives, p-nitro phenyl analogue 7m is the most po-tent SGLT2 inhibitor with IC50: 125.9 nM. Other analogs 7a(134.7 nM), 7b (163.2 nM), 7d (149.1 nM), 7e (146.9 nM), 7h(150.0 nM), and 7i (151.8 nM) also exhibited potency for SGLT2 inhi-bition at concentrations in comparable range. Compound 7n is non-specific analogue similar to reference drug and inhibited both SGLT1and SGLT2 at concentration of 434 and 458 nM respectively. Com-pound 7k is the only compound in this series inhibited SGLT1 (IC50:256 nM) at slightly lower concentration than SGLT2 (IC50: 367 nM).We next correlated selectivity index of all the compounds 7a–o forSGLT2 over SGLT1 inhibitory activities (Table 1). Unfortunately, mostof the acyclic C-nucleoside SGLT2 inhibitors 7a–o tested showedmoderate selectivity index. Among all analogs 7a–o, one compoundethyl 1-(2,5-dimethoxyphenyl)-5-((1S,2S,3S)-1,2,3-trihydroxybu-tyl)-1H-1,2,3-triazole-4-carboxylate 7d (IC50: 149 nM) inhibitedSGLT2 five times more selectively over SGLT1. Although the selectiv-ity of new compounds for SGLT2 over SGLT1 is not high, these results
identified first examples of acyclic C-nucleosides 7a–o as inhibitorsfor SGLT1 and SGLT2.
In conclusion, the present study led to the identification of thefirst examples of a series of new L-rhamnose derived acyclic C-nucleosides 7a–o bearing substituted 1,2,3-triazole core as potentsodium-glucose co-transporter (SGLT) inhibitors. The key b-keto-ester building block 4 was reacted with various aryl azides to give6a–o. Deprotection of acetonide group in 6a–o produced therespective 1,2,3-triazole derivatives 7a–o in excellent yields. Allthe new compounds 7a–o were evaluated for sodium-glucose co-transporters SGLT1 and SGLT2inhibitory activity using recentlydeveloped cell-based nonradioactive fluorescence glucose uptakeassay. Among all, two compounds 7m with IC50: 125.9 nMemerged as the most potent SGLT2 inhibitor and 7d exhibited bestselectivity for inhibition of SGLT2 (IC50: 149.1 nM) over SGLT1(IC50: 693.2 nM). It is worth mentioning that recently developedcell-based nonradioactive fluorescence glucose uptake assay24 isused for the first time in evaluating compounds as SGLT1/ SGLT2inhibitors. Over all the results presented here demonstrated the
S. R. Putapatri et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1528–1531 1531
utility of acyclic C-nucleosides as novel SGLT inhibitors for futureinvestigations in development of drugs to treat T2DM.
Acknowledgments
Authors are thankful to Dr. M. Lakshmikantam, Director and Dr.V.J. Rao, Head, CPC Division, IICT, Hyderabad for their continuoussupport, encouragement and assistance through CSIR 12th FYPNetwork (ORIGIN, CSC0108; DENOVA, CSC0205; SmiLE, CSC0110) & MLP 0002 projects. S.K.B. is thankful to Department ofBiotechnology (DBT) for providing Ramalingaswami Fellowship.S.K., (BT/Indo-Aus/07/06/2013) and S.K.B. (BT/PR13768/MED/30/300/2010) thanks DBT for grants. S.R.P. & A.K. (SRF) are thankfulto CSIR for financial assistance.
Supplementary data
Supplementary data (Experimental methods, 1H, 13C NMR andHRMS spectral data of all new compounds 2, 4, 6a–o and 7a–o andcopies of 1H, 13C NMR and HRMS spectra for all the new compounds2, 4, 6a–o and 7a–o.) associated with this article can be found, in theonline version, at http://dx.doi.org/10.1016/j.bmcl.2014.01.077.
References and notes
1. (a) Kanwal, A.; Banerjee, S. K. Pharm. Pat. Anal. 2013, 2, 77; (b) Hinnen, D. J.Cardiovas. Nurs. 2013, 28, 157; (c) Mather, A.; Pollock, C. Nat. Rev. Nephrol. 2010,6, 307; (d) Wright, E. M.; Loo, D. D. F.; Hirayama, B. A. Physiol. Rev. 2011, 91,733.
2. (a) Deshmukh, A. B.; Patel, M. C.; Mishra, B. Ren. Fail. 2013, 35, 566; (b) Raskin,P. Diabetes Metab. Res. Rev. 2013, 5, 347.
3. (a) Ghosh, R. K.; Ghosh, S. M.; Chawla, S.; Jasdanwala, S. A. J. Clin. Pharmacol.2012, 52, 457; (b) Washburn, W. N. Expert Opin. Ther. Pat. 2012, 22, 483.
4. (a) Vasilakou, D.; Karagiannis, T.; Athanasiadou, E.; Mainou, M.; Liakos, A.;Bekiari, E.; Sarigianni, M.; Matthews, D. R.; Tsapas, A. Ann. Intern. Med. 2013,159, 262; (b) Liu, J.; Lee, T.W. SGLT2 Inhibitors for Type 2 Diabetes, Macor, J. E.(Ed.), Book Series: Annual Reports in Medicinal Chemistry, 2011, 46, 103.; (c)Pfister, M.; Whaley, J. M.; Zhang, L.; List, J. F. Clin. Pharmacol. Ther. 2011, 89,621; (d) Ho, L.-T.; Kulkarni, S. S.; Lee, J.-C. Curr. Top. Med. Chem. 2011, 11, 1476.
5. Chao, E. C.; Henry, R. R. Nat. Rev. Drug Disc. 2010, 9, 551.6. Hung, H.-Y.; Qian, K.; Morris-Natschke, S. L.; Hsu, C.-S.; Lee, K.-H. Nat. Prod. Rep.
Fujikura, H.; Isaji, M. J. Pharmacol. Exp. Ther. 2007, 320, 323; (b) Dobbins, R. L.;O’Connor-Semmes, R.; Kapur, A.; Kapitza, C.; Golor, G.; Mikoshiba, I.; Tao, W.;Hussey, E. K. Diabetes Obes. Metab. 2012, 14, 15; (c) Fujimori, Y.; Katsuno, K.;Nakashima, I.; Ishikawa-Takemura, Y.; Fujikura, H.; Isaji, M. J. Pharmacol. Exp.Ther. 2008, 327, 268.
8. (a) Isaji, M. Kidney Int. 2011, 79, S14; (b) Hardman, T. C.; Dubrey, S. W. DiabetesTher. 2011, 2, 133; (c) Colca, J. R. Expert Opin. Invest. Drugs 2009, 18, 1243.
9. Ohtake, Y.; Sato, T.; Kobayashi, T.; Nishimoto, M.; Taka, N.; Takano, K.;Yamamoto, K.; Ohmori, M.; Yamaguchi, M.; Takami, K.; Yeu, S. Y.; Ahn, K. H.;Matsuoka, H.; Morikawa, K.; Suzuki, M.; Hagita, H.; Ozawa, K.; Yamaguchi, K.;Kato, M.; Ikeda, S. J. Med. Chem. 2012, 55, 7828.
10. Lamos, E. M.; Younk, L. M.; Davis, S. N. Expert Opin. Drug Metab. Toxicol. 2013, 9,763.
11. Grempler, R.; Thomas, L.; Eckhardt, M.; Himmelsbach, F.; Sauer, A.; Sharp, D. E.;Bakker, R. A.; Mark, M.; Klein, T.; Eickelmann, P. Diabetes Obes. Metab. 2012, 14,83.
12. (a) Liu, J. J.; Lee, T.; DeFronzo, R. A. Diabetes 2012, 61, 2199; (b) Sheridan, C. Nat.Biotechnol. 2012, 30, 899.
13. Robinson, R. P.; Mascitti, V.; Boustany-Kari, C. M.; Carr, C. L.; Foley, P. M.;Kimoto, E.; Leininger, M. T.; Lowe, A.; Klenotic, M. K.; Macdonald, J. I.; Maguire,R. J.; Masterson, V. M.; Maurer, T. S.; Miao, Z.; Patel, J. D.; Préville, C.; Reese, M.R.; She, L.; Steppan, C. M.; Thuma, B. A.; Zhu, T. Bioorg. Med. Chem. Lett. 2010,20, 1569.
14. (a) Yamamoto, Y.; Kawanishi, E.; Koga, Y.; Sakamaki, S.; Sakamoto, T.; Ueta, K.;Matsushita, Y.; Kuriyama, C.; Tsuda-Tsukimoto, M.; Nomura, S. Bioorg. Med.Chem. Lett. 2013, 23, 5641; (b) Koga, Y.; Sakamaki, S.; Hongu, M.; Kawanishi, E.;Sakamoto, T.; Yamamoto, Y.; Kimata, H.; Nakayama, K.; Kuriyama, C.;Matsushita, Y.; Ueta, K.; Tsuda-Tsukimoto, M.; Nomura, S. Bioorg. Med. Chem.2013, 21, 5561; (c) Li, L. T.; Zhou, L. F.; Li, Y. J.; Huang, J.; Liu, R. H.; Wang, B.;Wang, P. Bioorg. Med. Chem. Lett. 2012, 22, 642; (d) Zhou, H.; Danger, D. P.;Dock, S. T.; Hawley, L.; Roller, S. G.; Smith, C. D.; Handlon, A. L. ACS Med. Chem.Lett. 2010, 1, 19.
15. Kantevari, S.; Putapatri, S. R. Synlett 2010, 2251.16. (a) Ghoneim, A. A. Chem. Cent. J. 2011, 5, 1; (b) Zhang, L.; Wang, Y.; Xu, H.; Shi,
17. Ethyl 3-((4R,5S)-5-((S)-1-hydroxyethyl)-2,2-dimethyl-1,3-dioxolan-4-yl)-3-oxopropanoate (4): Compound 3 (4 g, 22.9 mmol) in dichloromethane(50 mL), ethyl diazoacetate (3.1 g, 27.5 mmol) and SnCl2�2H2O (100 mg) wasadded at 0 �C and stirred at rt for 7 h. The reaction was quenched with 1 N HCl(20 mL) and extracted with dichloromethane (2 � 30 mL). The combinedorganic extracts were washed with brine, dried over anhydrous Na2SO4,concentrated under reduced pressure. The crude residue was purified bycolumn chromatography over silica gel eluted with ethyl acetate/hexane(2.5:7.5) to afford 4 (4.70 g, 80%) as syrupy liquid. Yield: 80%; ½a�29
18. Karine, B.; Adam, D. M.; John, E. M. Org. Lett. 2007, 9, 1807.19. For recent reviews, see: (a) Thirumurugan, P.; Matosiuk, D.; Jozwiak, K. Chem.
Rev. 2013, 113, 4905; (b) Grammel, M.; Hang, H. C. Nat. Chem. Biol. 2013, 9, 475.20. (a) Zeng, D.; Zeglis, B. M.; Lewis, J. S.; Anderson, C. J. J. Nucl. Med. 2013, 54, 829;
(b) Kolb, H. C.; Sharpless, K. B. Drug Discovery Today 2003, 8, 1128.21. Wang, L.; Peng, S.; Danence, L. J. T.; Gao, Y.; Wang, J. Chem. Eur. J. 2012, 18,
6088.22. General procedure for amine-catalyzed Huisgen [3+2] cycloaddition 6(a–o): To a
solution of aryl azide 5 (1 equiv) and b-ketoester 4 (1.2 equiv) in DMSO (1 mL)pyrrolidine was added (0.1 equiv) and stirred at 60 �C for 14 h. The reactionmixture was diluted with ethyl acetate (20 mL), washed with water, dried overanhydrous Na2SO4 and concentrated under reduced pressure. The cruderesidue thus obtained was purified over silica gel column eluted withgradient mixture of ethyl acetate/hexane to give triazoles 6a–o as paleyellow syrupy liquids. Ethyl 5-((4S,5S)-5-((S)-1-hydroxyethyl)-2,2-dimethyl-1,3-dioxolan-4-yl)-1-phenyl-1H-1,2,3-triazole-4-carboxylate(6a). Yield: 81%;½a�29
D 33.5 (c 1.0, CHCl3). 1H NMR (300 MHz, CDCl3) d 7.59–7.49 (m, 5H), 5.71 (d,J = 7.9 Hz, 1H), 4.52 (q, J = 7.1 Hz, 2H), 3.93–3.88 (m,2H), 3.10 (br s, 1H), 1.48 (t,J = 7.1 Hz, 3H), 1.32 (s, 3H), 1.15 (d, J = 6.0 Hz, 3H), 0.87 (s, 3H). IR (neat) 3447,2983, 2933, 1725, 1377, 1219, 1066, 767 cm�1. MS (ESI) m/z 362[M+H]+; HR-MS (ESI) calcd for C18H24N3O5 [M+H]+: 362.0817, found: 362.0803. A typicalprocedure for the preparation of 7a–o: To a solution of 6 (0.5 mmol) indichloromethane (1 mL) trifluroacetic acid: water (1:1, 4 mL) was added at0 �C. After 2 h of stirring at rt, the reaction mixture was neutralized withsaturated NaHCO3 and extracted with ethyl acetate (2 � 15 mL). The combinedorganic extracts were washed with brine, dried over anhydrous Na2SO4,concentrated under reduced pressure. Crude residue thus obtained waspurified over silica gel column eluted with ethyl acetate/hexane (1:1)afforded 7a–o. (4S,5R,6S,7R)-7-Methyl-3-phenyl-4,5,6,7-tetrahydro-[1,2,3]triazole [1,5-a]pyridine-4,5,6-triol (7a). Yield: 90%; ½a�25
23. (a) Chang, H. C.; Yang, S. F.; Huang, C. C.; Lin, T. S.; Liang, P. H.; Lin, C. J.; Hsu, L.C. Mol. BioSyst. 2013, 9, 2010; (b) Kanwal, A.; Singh, S. P.; Grover, P.; Banerjee, S.K. Anal. Biochem. 2012, 429, 70.
24. Cell culture: Human embryonic kidney (HEK293) cells were purchased fromATCC, USA and made two stable cell lines after expressing SGLT1 & SGLT2,respectively. Previously, we searched the selectivity of these cell lines forSGLT1 and SGLT2 inhibition study. Glucose uptake by these cell lines was onlyinhibited by SGLT inhibitors but not by any other GLUTs inhibitors. SGLT1 &SGLT2 transfected HEK cell lines were propagated at 37 �C in 5% CO2 inDulbecco’s minimal essential medium (DMEM) supplemented with 1.0% ofpenicillin-streptomycin and 10% heat inactivated fetal bovine serum (FBS). Thecells were cultured in a 90 mm dish in DMEM with 10% FBS until 70–80%confluency was obtained for further use for SGLT1 and SGLT2 inhibitionactivity. SGLT1 inhibition assay: SGLT1 transfected stable HEK cells were platedat 1 � 104/well in 96-well plate and used at sub confluence after 24 h pre-incubation. For measuring SGLT1-mediated glucose uptake, all culture mediawas removed from each well and replaced with 100 ll of culture medium withnewly synthesized molecules at different concentrations (50, 100, 250, 500 and1000 nm). After half an hour, fluorescent 2-deoxy-glucose (2-NBDG) wasadded to the plates and incubated at 37 �C with 5% CO2 for a period of 30 min.23b Cells were lysed with 50 ll of 0.1 N NaOH and fluorescence of aliquots fromthe lysate was measured at excitation/emission maxima of �465/540 nm.Phlorizin (non-Specific SGLT1 inhibitor) was used as a standard for this study.SGLT2 inhibition assay: SGLT2 transfected stable HEK cells were plated at1 � 104/well in 96-well plate and used at sub confluence after 24 h pre-incubation. For SGLT2-mediated glucose uptake, all culture medium wasremoved from each well and replaced with 100 ll of culture medium withnewly synthesized molecules at different concentrations (50, 100, 250, 500 and1000 nm). After half an hour, florescent glucose (2-NBDG) was added to theplates and incubated at 37 �C with 5% CO2 for a period of 30 min. Cells werethen lysed with 50 ll of 0.1 N NaOH and fluorescence of aliquots from thelysate was measured at excitation/emission maxima of �465/540 nm.Phlorizin (non-specific SGLT2 inhibitor) was used as a standard for the study.
