Vanadyl−Thiazolidinedione Combination Agents for Diabetes Therapy

Cohen, N., Halberstam, M., Shlimovich, P., Chang, C. J., Shamoon, H., and Rossetti, L. (1995) Oral vanadyl sulfate improves hepatic and peripheral ins...
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Bioconjugate Chem. 2003, 14, 212−221

Vanadyl-Thiazolidinedione Combination Agents for Diabetes Therapy Tim Storr,† Devin Mitchell,†,‡ Pe´ter Buglyo´,†,§ Katherine H. Thompson,† Violet G. Yuen,| John H. McNeill,*,| and Chris Orvig*,† Medicinal Inorganic Chemistry Group, Department of Chemistry, University of British Columbia, Vancouver, BC, V6T 1Z1, Canada, and Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada. Received September 16, 2002; Revised Manuscript Received October 9, 2002

A series of vanadium compounds, chelated by ligands containing a thiazolidinedione moiety as an additional insulin-enhancing component, were produced in this study to create potentially synergistic compounds. A set of four bifunctional ligand precursors were synthesized: (()-5-{4-[(5-hydroxy-4oxo-4H-pyran-2-ylmethyl)amino]benzyl}thiazolidine-2,4-dione (HL1), (()-5-{4-[(5-hydroxy-1-methyl4-oxo-1,4-dihydro-pyridin-2-ylmethyl)amino]benzyl}thiazolidine-2,4-dione (HL2), 5-[4-(5-hydroxy-4-oxo4H-pyran-2-ylmethoxy)benzylidene]thiazolidine-2,4-dione (HL3), and (()-5-[4-(5-hydroxy-4-oxo-4Hpyran-2-ylmethoxy)benzyl]thiazolidine-2,4-dione (HL4), each containing a metal chelating portion as well as a thiazolidinedione moiety. From this set of ligand precursors, air-stable VO(L1)2, VO(L3)2, and VO(L4)2 were prepared. The four ligand precursors and three complexes were tested for insulinenhancing potential in STZ-diabetic rats and compared to rosiglitazone and BMOV, respectively. Both the ligand precursors HL1 and HL3 showed enhanced activity compared with that of rosiglitazone. The complex VO(L3)2 showed the most efficacious hypoglycemic effects in this study; however, neither additive nor synergistic effects were observed using this acute animal model.

INTRODUCTION

Diabetes mellitus includes a heterogeneous group of diseases which have become an important health concern in our society, affecting 1 in 20 persons in industrialized nations. The disease is characterized by hyperglycemia, alterations in carbohydrate and lipid metabolism, and vascular and neurological complications. It can be roughly divided into two classes: type 1, insulin-dependent diabetes mellitus (IDDM), and type 2, non-insulindependent diabetes mellitus (NIDDM). Treatment may involve daily subcutaneous injections of insulin (most common for type 1), diet and exercise, administration of one or more of the currently available hypoglycemic agents (type 2), or combination therapy. Numerous therapies designed to supplement or enhance insulin action have been developed to aid in the treatment of NIDDM. Combined with diet and exercise, these regimens have proven to be fairly effective, but none are ideal. Thus, there is a need to find effective, orally active drugs that mimic or enhance the properties of insulin. Vanadium was first demonstrated to have insulin-like properties in vitro in 1979 (1), in skeletal muscle and adipose tissue, and in vivo in 1985 (2). Since this time there has been a great deal of interest in determining the biological role of vanadium, including its mechanism of action, and in the development of new vanadium * Corresponding authors. E-mails: [email protected] and [email protected]. † Department of Chemistry, University of British Columbia. ‡ Present address: Department of Chemistry, University of Victoria, Victoria, BC, V8W 3V6, Canada. § Present address: Department of Inorganic and Analytical Chemistry, University of Debrecen, H-4010 Debrecen, P.O. Box 21, Hungary. | Faculty of Pharmaceutical Sciences, University of British Columbia.

compounds as alternatives to conventional diabetes therapy. The mechanism of vanadium’s in vivo effects has been the subject of much debate (3-8), most probably due to the multiplicity of its effects (5, 9). Current evidence points to a site (or sites) of action downstream from the insulin receptor (10) in the insulin signaling cascade. The therapeutic value of inorganic vanadium, in the form of vanadate ([VO4]3-) or vanadyl ([VO]2+), as an orally active agent against diabetes has been well documented (5, 11). Poor absorption from the gastrointestinal tract (GI) into the bloodstream, as well as a narrowness of the window of optimal effectiveness in vivo, limits the utility of administering vanadium to diabetic patients in this form. However, vanadium complexes such as bis(maltolato)oxovanadium(IV), commonly known by its acronym BMOV (12, 13) (Chart 1), have shown increased efficacy over inorganic vanadium in STZ-diabetic rat studies (14). BEOV (the ethyl analog) has recently completed phase 1 human clinical trials. Complexation of vanadyl ([VO]2+) with the approved food additive maltol improved GI (gastrointestinal) absorption, thereby decreasing the vanadium dose required for effective glucose lowering (14, 15). Numerous small ligands have been subsequently used in this manner to increase the potency of the vanadium center (15, 16). In an effort to further increase the efficacy of the drug candidates, the coupling of vanadium with a ligand that contains an insulin-action-enhancing drug has been explored (17). The coupling of vanadyl with a series of biguanides failed to produce additive or synergistic effects; however, this was most likely due to the large dosage differences required for these two classes of drugs (17-19). In an advance on this approach herein, we have chelated the vanadium center to a series of thiazolidinedione-containing ligands; dosage differences be-

10.1021/bc025606m CCC: $25.00 © 2003 American Chemical Society Published on Web 11/16/2002

Vanadyl−Thiazolidinedione Combination Agents Chart 1. Bis(maltolato)oxovanadium(IV) (BMOV, R ) CH3), bis(ethylmaltolato)oxovanadium(IV) (BEOV, R ) C2H5)

Chart 2

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significant antidiabetic activity (16). The planar ring system of kojic acid was also well suited for interacting with the binding domain of the PPARγ receptor (26). It was hypothesized that kojic acid would be the most versatile binding moiety, as the planar ring system is also capable of chelating vanadium. The concept of combining vanadium and a thiazolidinedione-containing ligand to form a compound that exhibits additive or synergistic effects is quite appealing. The preparation and characterization of a series of such bifunctional ligand precursors and their associated oxovanadium(IV) complexes are presented herein. Results from preliminary acute STZ-diabetic rat studies of four ligand precursors and three of the corresponding complexes are also reported and compared to rosiglitazone and BMOV, respectively. RESULTS AND DISCUSSION

Chart 3

tween oxovanadium(IV) compounds and representative thiazolidinediones are small (18, 20). The thiazolidinediones are named for the five-membered heteronuclear ring common to all compounds of this class (Chart 2). Of the thiazolidinedione compounds, ciglitazone, troglitazone, englitazone, pioglitazone, and rosiglitazone have been clinically examined as potential antidiabetic compounds (21). Both pioglitazone and rosiglitazone are available on the market for the treatment of type 2 diabetes. Thiazolidinediones act by indirectly enhancing peripheral insulin sensitivity, thereby lowering the levels of both glucose and insulin (22). Thiazolidinediones are known to activate the peroxisome proliferator-activated receptor gamma (PPARγ). PPARγ regulates numerous metabolic pathways involving lipoprotein lipase, glucose transporters and insulin signaling pathways (23). Nonetheless, binding affinity of the thiazolidinediones for PPARγ does not always correlate with euglycemic activity (24). A major benefit of the thiazolidinediones is that unlike sulfonylurea derivatives, R-glucosidase inhibitors, or insulin, they influence insulin resistance. Compounds which improve insulin resistance enable the continued treatment of NIDDM patients without inducing hypoglycemia (21). To form stable neutral complexes with the vanadyl ion, bifunctional ligands were developed, tethering the active portion of the thiazolidinedione molecule to a suitable chelator. Kojic acid 1 (Chart 3) was chosen for this purpose because, like maltol, it forms water soluble, neutraly charged complexes with appropriate metal ions (25). Simple kojic acid complexes with VO2+ have shown

Ligand Preparation. All four ligand precursors HL1, HL2, HL3, and HL4 were prepared by various routes from kojic acid 1. Kojic acid 1 was first protected at the ring hydroxyl using the method of Thomas and Marxer (27) to afford the benzyl protected compound 2. This compound was further elaborated to synthesize the four distinct ligand precursors. An effort was made to preserve the active portion of the thiazolidinediones (the 5-(4substituted-benzyl)thiazolidine-2,4-dione moiety) in these bifunctional ligands (Chart 2). For the amino-tethered ligand (Scheme 1), 5-benzyloxy2-hydroxymethyl-pyran-4-one 2 was reacted with methanesulfonyl chloride to afford the mesylate 3. Compound 3 was then coupled with (()-5-(4-amino-benzyl)thiazolidine-2,4-dione 6, which was synthesized by a more direct route than that available in the literature (28), to afford 7. To synthesize 6, 4-nitrobenzaldehyde 4 was coupled with thiazolidine-2,4-dione using reported conditions (29) to afford 5, which was then reduced using NaBH4 in the presence of a cobalt/dimethylglyoxime catalyst formed in situ (30). Using this method, both the nitro group and the double bond were reduced simultaneously. Benzyl group removal from the coupled product 7 in strong acid afforded the amino-tethered thiazolidinedione ligand HL1. Synthesis of the pyridinone-type ligand (Scheme 2) proceeded in much the same manner as with HL1. A pyridinone-type chelator was examined because N-substitution into a pyrone ring has been shown to increase metal binding affinity (31, 32). Benzyl-protected kojic acid 2 was reacted with methylamine to form the pyridinone 8 in an improved yield over that reported (33). Compound 8 was then converted to the chloride 9 with thionyl chloride in good yield. The coupling step proceeded to give the protected compound 10 from which the benzyl group was removed to afford the pyridinone-type ligand precursor HL2. The unsaturated ether-tethered ligand (Scheme 3) HL3 was synthesized using the mesylate 3 as the starting material. Compound 3 was coupled with 4-hydroxybenzaldehyde to afford the aldehyde 11, which then underwent a Knoevenagel condensation with thiazolidine-2,4dione to yield compound 12. The original intent at this point was to reduce the C-C double bond connecting the thiazolidinedione ring. Unfortunately we were not able to effect this transition selectively, despite employing a variety of conditions, because of the many other unsaturation points in the molecule. Because previous work (34, 35) had shown that the activity of certain thiazolidinediones with an unsaturation point in an identical

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Scheme 1. The Synthesis of HL1: (A) Thiazolidine-2,4-dione, Piperidine, Benzoic Acid, Toluene, 55%; (B) CoCl2, Dimethylglyoxime, NaBH4, H2O, 71%; (C) Na, Benzyl Chloride, CH3OH, 55%; (D) Triethylamine, Methanesulfonyl Chloride, CH2Cl2; (E) K2CO3, CH3CN, 69%.; (F) HCl, CH3COOH, 70%

Scheme 2. The Synthesis of HL2: (A) Methylamine, EtOH/H2O, 77%; (B) SOCl2, CH2Cl2, 96%; (C) Triethylamine, DMF, 88%; (D) HCl, CH3COOH, 73%

Scheme 3. The Synthesis of HL3: (A) 4-Hydroxybenzaldehyde, K2CO3, DMF, 64%; (B) Thiazolidine-2,4-dione, Piperidine, Benzoic Acid, Toluene, 57%; (C) HCl, CH3COOH, 67%

location exhibited equal or more impressive activity than their saturated analogues, we decided to continue by deprotecting 12 to form the unsaturated ether-tethered ligand HL3. The ether-tethered ligand (Scheme 4) HL4 was synthesized in a manner similar to that employed for HL1. In this case 5-benzyloxy-2-chloromethyl-pyran-4-one 13 was used in the coupling step because the mesylate 3 did not afford any coupled product. The slower reactivity of the chloride derivative presumably minimized sidereactions allowing the coupling reaction to take place. Compound 13 was coupled with 5-(4-hydroxy-benzyl)thiazolidine-2,4-dione (28) 16, which was synthesized by a Knoevenagel condensation of 4-hydroxybenzaldehyde 14 with thiazolidine-2,4-dione to form 15, and then subsequent reduction. The coupled product 17 was then deprotected to afford the ether-tethered ligand HL4. The low yield of the coupling step here is most likely because of the similar nucleophilicities of the thiazolidinedione

nitrogen and the ring hydroxyl oxygen of compound 16. Indeed, both coupling products were isolated, with the O-alkylation product occurring in a higher proportion. The saturated ligand precursors HL1, HL2, and HL4 are presumed to exist as a mixture of enantiomers with the stereogenic center located at C-5 of the thiazolidinedione ring. No attempt was made to synthesize or isolate selectively one enantiomer because this center is prone to rapid racemization at physiological pH (36). Interestingly, it has been reported that the (S)-(-)enantiomer of rosiglitazone 5 was responsible for the binding affinity to PPARγ with a t1/2 for racemization of 3 h at pH 7.2(36). Vanadyl-Thiazolidinedione Complexes. The neutral oxovanadium(IV)-thiazolidinedione complexes were prepared (Scheme 5) by refluxing vanadyl sulfate and two equivalents of the appropriate ligand precursor in mildly acidic aqueous medium (pH ∼5). The precipitated products were isolated and washed with water and

Vanadyl−Thiazolidinedione Combination Agents

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Scheme 4. The Synthesis of HL4: (A) Thiazolidine-2,4-dione, Piperidine, Benzoic Acid, Toluene, 98%; (B) CoCl2, Dimethylglyoxime, NaBH4, H2O, 64%; (C) SOCl2, CH2Cl2, 60%; (D) NaH, DMF, 35%; (E) HCl, CH3COOH, 93%

Scheme 5. The Synthesis of VO(L1)2 (83%), VO(L3)2 (93%), and VO(L4)2 (88%)a

a

VOSO4, H2O.

diethyl ether to afford, after drying in vacuo light brown, gray, and brown solids, characterized as VO(L1)2, VO(L3)2, and VO(L4)2, respectively. Unfortunately, the extremely low solubility of the pyridinone-type ligand HL2 precluded complexation to vanadium under a variety of conditions. Characteristic stretching frequencies of the VdO bond in oxovanadium(IV) complexes generally occur in the region 930-1030 cm-1(37). The complexes VO(L1)2, VO(L3)2, and VO(L4)2 exhibit VdO stretching frequencies of 982, 985, and 975 cm-1, respectively. The corresponding bis(kojato)oxovanadium(IV) (VO(ka)2) complex exhibits a similar VdO stretch at 980 cm-1 (16). Elemental analyses of the three complexes were consistent with the calculated values. All exhibited one molecule of residual water which could not be eliminated with prolonged drying. The mass spectra (+ ESIMS) support the VOL2 formulation showing parent ion M + 1 peaks ([HVOL2]+), as well as expected fragmentation patterns. All complexes prepared were paramagnetic in the solid state. Room-temperature paramagnetic susceptibilities were obtained. By correcting for the diamagnetic susceptibilities of the ligands using Pascal’s constants, the effective magnetic moments of the complexes were calculated. With an electronic configuration of [Ar]3d1, vanadium(IV) has one unpaired electron for which the spin-only formula predicts a magnetic moment of 1.73 µB. The experimental values are in the range 1.69-1.75 µB for the three vanadyl complexes. Electron paramagnetic resonance spectroscopic data for the three complexes showed the characteristic eight-line

Table 1. Spin Hamiltonian Parameters for VO(L)2; L ) L1, L3, L4, Ma, Ka complex

gisoa

Aisob

VO(L1)2 (MeOH:CH2Cl2 1:1) VO(L3)2 (MeOH:CH2Cl2 1:1) VO(L4)2 (MeOH:CH2Cl2 1:1) VO(ma)2 (BMOV) (CHCl3)c VO(ka)2 (CHCl3)d

1.967 1.967 1.967 1.963 1.963

104.0 108.0 105.0 103.7 104.5

a Error, (0.001. b Units for the vanadium hyperfine coupling are ( 0.1 × 10-4 cm-1. c Ref 12. d Ref 16.

pattern expected for V (IV). The three vanadiumthiazolidinedione complexes exhibited identical g-values and very similar isotropic vanadium nuclear hyperfine couplings (Table 1). In addition, the experimental values compare very well with those reported for BMOV (12) and VO(ka)2 (16) (Table 1). Dissolution of each vanadiumthiazolidinedione complex in MeOH:CH2Cl2 (1:1) most probably results in solvated MeOH species. Possible sites for solvent coordination are trans (T) or cis (C) to the oxoO, yielding six possible species in solution (38). X-ray crystal-structure analysis would give a much more detailed understanding of the coordination environment; however, many crystallization attempts did not produce the required single crystals. In Vivo Results. Animal studies were completed in four sets of trials the first three of which used, in each trial, a diabetic control, a ligand precursor, the associated vanadyl complex of the ligand precursor, and BMOV as a representative standard. The fourth trial used a diabetic control, a ligand precursor, rosiglitazone, and BMOV. The percent decrease in blood glucose was

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Table 2. Glucose Lowering of Four Thiazolidinedione Ligand Precursors and Three of Their Corresponding Complexes Compared with BMOV, and Rosiglitazone % decrease in blood glucosea,b compound HL1 VO(L1)2 HL2 HL3 VO(L3)2 HL4 VO(L4)2 Rosiglitazone BMOV (average)c

12 h

24 h

48 h

6.4 ( 2.0 3.9 ( 11.6 10.9 ( 5.7 54.5 ( 2.4 59.2 ( 3.3 3.6 ( 2.9 21.5 ( 6.7 6.4 ( 4.7 41.9 ( 6.0

19.9 ( 1.9 25.9 ( 11.0 5.6 ( 3.3 49.6 ( 3.5 57.5 ( 4.6 7.3 ( 1.8 28.8 ( 10.3 9.4 ( 5.1 57.1 ( 6.2

27.2 ( 5.3 24.5 ( 10.6 2.0 ( 2.3 27.5 ( 4.0 47.6 ( 6.0 0.9 ( 2.8 3.3 ( 2.5 10.2 ( 6.3 52.3 ( 5.3

a Glucose lowering of test compounds in STZ-diabetic rats was calculated as in eq 1 (21). Each value represents the mean ( SEM (n ) 10). b Dose: 0.1 mmol/kg. c Combination of four trials (n ) 26).

Figure 1. Hypoglycemic activity (% plasma glucose lowering) of four thiazolidinedione ligand precursors and three of their corresponding complexes compared with BMOV and rosiglitazone.

calculated for each compound utilizing a common method (21, 39), and the results are displayed in Table 2. A visual representation of the data is presented in Figure 1. The BMOV results are a combination of the four trials. The results for the amine tethered ligand precursor HL1 and the associated complex VO(L1)2 suggest that these compounds may be taken up more slowly because there was no observed glucose lowering at 12 h. Positive effects were evident, however, at the 24 and 48 h time points, with both compounds exhibiting half of the in vivo activity of BMOV in acute testing. The pyridinone-type ligand precursor HL2 showed minimal glucose lowering at 12 h, but the effect was not sustained. The very low solubility of this compound leading to a lack of absorption in vivo was most probably responsible for the observed response. This low solubility precluded complexation with vanadium as well. The unsaturated ether-tethered ligand precursor HL3 and complex VO(L3)2 showed the most efficacious hypoglycemic effects in this study. Both compounds exhibited enhanced glucose lowering compared to BMOV at the 12 h time point. Whereas the glucose lowering effect of the ligand was lessened from 12 to 48 h, the effect of VO(L3)2 on blood glucose levels was sustained, similar to BMOV. On the basis of these promising results, we

tested the saturated analogues HL4 and VO(L4)2. The ligand precursor did not show any glucose lowering, while the complex exhibited glucose-lowering at 12 and 24 h but the effect was not sustained to 48 h. The results for VO(L4)2 contrast that for VO(L1)2 in that the former complex was faster acting but the positive effects were not sustained. These results could be explained by quicker uptake of VO(L4)2 followed by faster metabolism and/or excretion. Both the ligand precursors HL1 and HL3 were more proficient in lowering plasma glucose levels in this testing protocol when compared with rosiglitazone (Avandia), a marketed PPARγ agonist. While these results are preliminary, the increased activity of HL1 and HL3 compared to that of rosiglitazone is very encouraging. HL2 showed minimal hypoglycemic activity at 12 h, but the effect was not sustained. The very low solubility of this compound most probably hampered its activity. HL3 lowered plasma glucose much more effectively than rosiglitazone, a significant finding that warrants further attention. It has been reported that unsaturated thiazolidinediones show lesser fold transactivation of PPARR and PPARγ than their saturated analogues (29, 40). This suggests that HL3 and other unsaturated thiazolidinediones exhibit their antidiabetic effects through other, yet to be determined, mechanisms. Interestingly the saturated analogue HL4 did not show any activity in this testing protocol. This lack of activity could have been due to poor pharmacokinetics as the solubility of this ligand precursor was satisfactory. Further testing in a more specific animal model of type 2 diabetes, such as db/db mice, is planned to elucidate more clearly the potential of these structurally novel thiazolidinediones, especially HL3 (34, 35). Studies of the combined effects of treatment with rosiglitazone and BMOV on glucose-lowering in STZdiabetic rats have shown possible additivity (41). Neither additive nor synergistic effects were observed in this study; however, this could be due to the limited amount of information available from this short-term testing protocol. With these preliminary results in hand, it is clear that VO(L3)2 is comparable to BMOV in lowering plasma glucose levels in STZ diabetic rats. Again, further testing would be prudent in a specific model of type 2 diabetes whereby effects on other important parameters such as plasma triglycerides and insulin could be determined (39). CONCLUDING REMARKS

To design vanadyl-thiazolidinedione complexes was appealing because of the potential for associative or synergistic effects via complexation of vanadium to thiazolidinedione-containing compounds. There is considerable evidence for the orally effective glucose-lowering properties of V(V) and V(IV), and a number of thiazolidinediones are either in late-stage clinical trials or are available for the treatment of type 2 diabetes. In preliminary animal investigations with this class of compounds the ligand precursors HL1 and HL3 showed enhanced activity in lowering blood glucose compared with that of rosiglitazone. The complex VO(L3)2 exhibited comparable activity to BMOV and had a more prolonged treatment effect than the ligand alone. EXPERIMENTAL PROCEDURES

Chemicals and Instrumentation. All solvents and chemicals (Fisher, Aldrich) were reagent-grade and used without further purification unless otherwise specified.

Vanadyl−Thiazolidinedione Combination Agents

Rosiglitazone was a gift from Kinetek Pharmaceuticals Inc. Water was deionized (Barnstead D9802 and D9804 cartridges) and distilled (Corning MP-1 Megapure Still) before use. Melting points were measured on a Mel-Temp apparatus and are uncorrected. The ligands and complexes synthesized were characterized by infrared (IR) spectroscopy, mass spectrometry, elemental analysis, magnetic susceptibility, and 1H NMR, where appropriate. Infrared spectra were recorded as KBr disks in the range 4000-400 cm-1 on a Galaxy Series FTIR spectrometer. Mass spectra (+ ion) were obtained with a Kratos MS 50 (electron-impact ionization, EI), a Kratos MS 80 (desorption chemical ionization, DCI), or a Macromass LCT (electrospray ionization, ESI) instrument. Room-temperature (293 K) magnetic susceptibilities were measured on a Johnson Matthey balance, using Hg[Co(NCS)4] as the susceptibility standard; diamagnetic corrections were estimated using Pascal’s constants (42). C, H, and N analyses were performed either in this department by Mr. Peter Borda (Carlo Erba analytical instrumentation) or by Delta Microanalytical Services. 1H NMR spectra of samples were recorded on either a Bruker AM-300 or an AX-400 instrument at 300.13 or 400.13 MHz, respectively. Room-temperature X-band EPR spectra were recorded on a Bruker ECS106 EPR spectrometer in 20µL quartz capillaries. Computer simulations of the isotropic EPR spectra were performed using Bruker’s WINEPR/SIMFONIA package. Synthesis of Bifunctional Ligands. 5-Benzyloxy-2hydroxymethyl-pyran-4-one (2). This compound was prepared in a manner similar to that in the literature (27). To a solution of sodium (2.3 g, 100 mmol) in dry MeOH (200 mL) was added kojic acid 1 (14.2 g, 100 mmol) in portions. To this solution was added benzyl chloride (14 mL, 100 mmol) dropwise, and the mixture was heated to reflux for 5 h, cooled to room temperature, and then poured into H2O (1 L). The precipitate was collected and recrystallized from a minimum of hot EtOH (80 mL) to afford 12.7 g (55%) of the title compound 7 as colorless crystals; mp 120-122 °C. 1H NMR (CD3OD, 300 MHz): δ 8.00 (s, 1H), 7.38 (m, 5H), 6.50 (s, 1H), 5.01 (s, 2H), 4.39 (s, 2H). EIMS m/z (relative intensity) ) 232 (M+, 60), 91 (100). Anal. Calcd. (found) for C13H12O4: C, 67.23 (67.42); H, 5.21 (5.26). Methanesulfonic Acid 5-Benzyloxy-4-oxo-4H-pyran-2ylmethyl Ester (3). To a suspension of 5-benzyloxy-2hydroxymethyl-pyran-4-one 2 (0.303 g, 1.3 mmol) in dry CH2Cl2 (10 mL) was added triethylamine (0.36 mL, 2.6 mmol). The suspension was then cooled in an ice bath and methansulfonyl chloride (0.12 mL, 1.6 mmol) was added dropwise. The mixture was stirred for 0.5 h, quenched with saturated NaHCO3 (15 mL), and then extracted with CH2Cl2 (2 × 15 mL); the combined organic extracts were dried over anhydrous Na2SO4, and then concentrated. The slightly colored oil 3 was used without further purification. 1H NMR (CDCl3, 300 MHz): δ 7.57 (s, 1H), 7.38 (m, 5H), 6.55 (s, 1H), 5.08 (s, 2H), 4.95 (s, 2H), 3.08 (s, 3H). 5-(4-Nitro-benzylidene)thiazolidine-2,4-dione (5). To a solution of 4-nitrobenzaldehyde 4 (5.00 g, 33.0 mmol) and thiazolidine-2,4-dione (3.87 g, 33.0 mmol) in toluene (200 mL) was added benzoic acid (0.68 g, 5.5 mmol) and piperidine (0.55 mL, 5.5 mmol). The resulting mixture was refluxed for 2 h with the continuous removal of water using a Dean-Stark water separator. The reaction was then cooled to room temperature and the resulting precipitate was collected and washed with CH2Cl2 and Et2O to yield 4.50 g (55%) of compound 5 as a pale yellow solid; mp 250 °C. 1H NMR (DMSO-d6, 300 MHz): δ 8.35

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(d, J ) 8.6 Hz, 2H), 7.91 (s, 1H), 7.87 (d, J ) 8.6 Hz, 2H). EIMS m/z (relative intensity) ) 250 (M+, 35), 179 (100), 89 (70). Anal. Calcd. (found) for C10H6N2O4S: C, 48.00 (48.00); H, 2.42 (2.46); N, 11.20 (10.99). (()-5-(4-Amino-benzyl)thiazolidine-2,4-dione (6). This compound was prepared by a more direct method than that reported in the literature (28). CoCl2‚6H2O (0.30 g, 1.3 mmol) and dimethylglyoxime (0.56 g, 4.9 mmol) were added to H2O (300 mL). After addition of NaOH (3 mL, 1 M), the brown solution was cooled to 0 °C in an ice bath and NaBH4 (7.90 g, 209 mmol) was added in portions. 5-(4-Nitro-benzylidene)thiazolidine-2,4-dione 5 (5.22 g, 20.9 mmol) was then added slowly as a slurry in THF (50 mL). The reaction mixture was stirred at roomtemperature overnight. The pH was then adjusted to 9 with CH3COOH, and the mixture was extracted with THF (3 × 200 mL). The combined organic extracts were dried over Na2SO4 and concentrated. The residue was chromatographed over silica gel using a mixture of MeOH and CH2Cl2 (5:95) as the eluent to afford 3.31 g (71%) of the title compound 6 as a pale yellow solid; mp 152-153 °C. 1H NMR (DMSO-d6, 300 MHz): δ 6.89 (d, J ) 8.3 Hz, 2H), 6.49 (d, J ) 8.3 Hz, 2H), 4.65 (dd, J ) 9.3, 4.4 Hz, 1H), 3.20 (dd, J ) 14.4, 4.4 Hz), 2.91 (dd, J ) 14.4, 9.3 Hz). EIMS m/z (relative intensity) ) 222 (M+, 60), 106 (100). Anal. Calcd. (found) for C10H10N2O2S: C, 54.04 (54.26); H, 4.53 (4.53); N, 12.60 (12.45); S, 14.42 (14.26). (()-5-{4-[(5-Benzyloxy-4-oxo-4H-pyran-2-ylmethyl)amino]benzyl}thiazolidine-2,4-dione (7). Methanesulfonic acid 5-benzyloxy-4-oxo-4H-pyran-2-ylmethyl ester 3 (2.67 g, 9.05 mmol) and (()-5-(4-amino-benzyl)thiazolidine-2,4dione 6 (2.00 g, 9.0 mmol) were dissolved in dry CH3CN (50 mL). K2CO3 (2.50 g, 18 mmol) was then added as a solid, and the resulting mixture was heated at 50 °C for 24 h with stirring. The solvent was then removed and the residue partitioned between CH2Cl2 (50 mL) and saturated NaHCO3 (30 mL). The mixture was extracted with CH2Cl2 (2 × 50 mL); the organic extracts were dried over Na2SO4 and concentrated. The crude product was chromatographed on silica gel using a mixture of MeOH and CH2Cl2 (3:97) as the eluent to afford 2.73 g (69%) of the title compound 7 as a pale yellow solid; mp 57-59 °C. 1H NMR (acetone-d6, 300 MHz): δ 7.86 (s, 1H), 7.37 (m, 5H), 6.95 (d, J ) 8.3 Hz, 2H), 6.59 (d, J ) 8.3 Hz, 2H), 6.21 (s, 1H), 5.02 (s, 2H), 4.82 (dd, J ) 8.4, 4.1 Hz, 1H), 4.57 (s, 1H), 3.33 (dd, J ) 14.1, 4.1 Hz, 1H), 3.09 (dd, J ) 14.1, 8.4 Hz, 1H). EIMS m/z (relative intensity) ) 436 (M+,10), 196 (20), 106 (100), 91 (55). Anal. Calcd. (found) for C23H20N2O5S: C, 63.29 (63.39); H, 4.62 (4.59); N, 6.42 (6.43). (()-5-{4-[(5-Hydroxy-4-oxo-4H-pyran-2-ylmethyl)amino]benzyl}thiazolidine-2,4-dione (HL1). 5-{4-[(5-Benzyloxy4-oxo-4H-pyran-2-ylmethyl)amino]benzyl}thiazolidine2,4-dione 7 (3.75 g, 8.6 mmol) was dissolved in a mixture of concentrated HCl (20 mL) and CH3COOH (40 mL) and heated at 70 °C with stirring for 48 h. The yellow solution was rotary evaporated to dryness and then H2O (3 mL) was added and the pH adjusted to 5. The precipitate was isolated and washed with a minimum of cold H2O to afford, after drying, 2.71 g (70%) of the title compound as a pale yellow solid; mp 146 °C. 1H NMR (D2O, 300 MHz): δ 7.85 (s, 1H), 7.29 (d, J ) 8.5 Hz, 2H), 7.22 (d, J ) 8.5 Hz, 2H), 5.76 (s, 1H), 4.58 (dd, J ) 6.4, 4.8 Hz, 1H), 4.5 (s, 2H), 3.76 (dd, J ) 15.5, 6.4 Hz, 1H), 3.63 (dd, J ) 15.5, 4.8 Hz, 1H). EIMS m/z (relative intensity) ) 346 (M+, 4), 106 (100). Anal. Calcd. (found) for C16H14N2O5S‚0.5H2O: C, 54.08 (54.21); H, 4.25 (4.06); N, 7.88 (7.74).

218 Bioconjugate Chem., Vol. 14, No. 1, 2003

5-Benzyloxy-2-hydroxymethyl-1-methyl-1H-pyridin-4one (33) (8). This compound was synthesized in a higher yield than that reported in the literature (33). 5-Benzyloxy-2-hydroxymethyl-pyran-4-one 2 (0.99 g, 4.27 mmol) was suspended in a mixture of EtOH and H2O (10 mL/ 10 mL), and methylamine (0.56 mL, 40% w/w, 6.42 mmol) was added. The mixture was heated to reflux for 6 h and then cooled to r.t., at which time the product crystallized from the solution. The solid was washed with Et2O and dried in vacuo to afford 0.81 g (77%) of the title compound 8 as an off-white solid; mp 218-220 °C. 1H NMR (CD3OD, 300 MHz): δ 7.60 (s, 1H), 7.48 (d, J ) 7.2 Hz, 2H), 7.37 (m, 3H), 6.61 (s, 1H), 5.10 (s, 2H), 4.55 (s, 2H), 3.77 (s, 3H). DCIMS m/z ) 245 (M+, 100), 168 (15), 139 (85), 91 (74). Anal. Calcd. (found) for C14H15N O3: C, 68.56 (68.42); H, 6.16 (6.22); N, 5.71 (5.81). 5-Benzyloxy-2-chloromethyl-1-methyl-1H-pyridin-4one Hydrochloride Salt (9). 5-Benzyloxy-2-hydroxymethyl-1-methyl-1H-pyridin-4-one 8 (2.10 g, 8.57 mmol) was added with stirring to form a suspension in dry CH2Cl2 (20 mL). Thionyl chloride (5.0 mL, 68.9 mmol) was then added dropwise. The resulting light yellow solution was stirred overnight under Ar, and then the solvent was removed under reduced pressure. Ethyl acetate (15 mL) was added, and the triturated solid was collected and washed with hexanes to afford 2.45 g (96%) of the title compound 9 as a white solid; mp 170-172 °C. 1H NMR (DMSO-d6, 300 MHz): δ 8.62 (s, 1H), 7.40 (m, 6H), 5.17 (s, 2H), 5.04 (s, 2H). DCIMS m/z ) 263 (M+, 16), 228 (20), 157 (30), 91 (100). Anal. Calcd. (found) for C14H14ClNO2: C, 56.00 (56.00); H, 5.00 (5.04); N, 4.67 (4.72). (()-5-{4-[(5-Benzyloxy-1-methyl-4-oxo-1,4-dihydro-pyridin-2-ylmethyl)amino]benzyl}thiazolidine-2,4-dione (10). (()-5-(4-Amino-benzyl)thiazolidine-2,4-dione 6 (1.55 g, 6.98 mmol) and 5-benzyloxy-2-chloromethyl-1-methyl-1Hpyridin-4-one hydrochloride salt 9 (2.08 g, 6.98 mmol) were dissolved in DMF (50 mL). Triethylamine (3 mL, 21 mmol) was added dropwise, and the resulting mixture was stirred for 22 h. Water (60 mL) was added and the resulting mixture extracted with CH2Cl2 (2 × 70 mL). The combined organic extracts were dried over Na2SO4, filtered, and then evacuated. The crude product was chromatographed on silica gel eluting with MeOH:CH2Cl2 (5:95) to afford 2.65 g (88%) of the title compound 10 as a pale yellow solid; mp 85-87 °C. 1H NMR (CDCl3, 300 MHz): δ 7.32 (m, 5H), 6.85 (s, 1H), 6.84 (d, J ) 8.3 Hz, 2H), 6.43 (d, J ) 8.3 Hz, 2H), 6.12 (s, 1H), 5.08 (s, 2H), 4.44 (dd, J ) 8.0, 3.9 Hz, 1H), 4.42 (s, 2H), 3.35 (s, 3H), 3.20 (dd, J ) 14.2, 3.9 Hz, 1H), 3.06 (dd, J ) 14.2, 8.0 Hz, 1H); EIMS m/z ) 449 (M+, 45), 344(10), 238(35), 106(80), 91(100). Anal. Calcd. (found) for C24H23N3 O4S‚ HCl: C, 59.32 (59.52); H, 4.98 (4.90); N, 8.65 (8.49). (()-5-{4-[(5-Hydroxy-1-methyl-4-oxo-1,4-dihydro-pyridin-2-ylmethyl)amino]benzyl}thiazolidine-2,4-dione (HL2). The title compound HL2 (1.50 g, 73%) was prepared as a light yellow solid from (()-5-{4-[(5-benzyloxy-1-methyl4-oxo-1,4-dihydro-pyridin-2-ylmethyl)amino]benzyl}thiazolidine-2,4-dione 10 (2.60 g, 5.80 mmol) by a procedure analogous to that described for HL1; mp 108-110 °C. 1H NMR (DMSO-d6, 300 MHz): δ 7.58 (s, 1H), 6.94 (d, J ) 8.1 Hz, 2H), 6.58 (d, J ) 8.1 Hz, 2H), 5.99 (s, 1H), 5.06 (dd, J ) 8.9, 4.2 Hz, 1H), 4.69 (s, 2H), 3.69 (s, 3H), 3.34 (dd, J ) 14.1, 4.2 Hz, 1H), 3.04 (dd, J ) 14.1, 8.9 Hz, 1H). EIMS m/z ) 359 (M+, 30), 254 (60), 181 (30), 106 (100). Anal. Calcd. (found) for C17H17N3O4S‚0.5H2O: C, 55.42 (55.19); H, 4.92 (4.75); N, 11.41 (11.28). 4-(5-Benzyloxy-4-oxo-4H-pyran-2-ylmethoxy)benzaldehyde (11). Methanesulfonic acid 5-benzyloxy-4-oxo-4Hpyran-2-ylmethyl ester 3 (5.37 g, 17.3 mmol) was dis-

Storr et al.

solved in DMF (150 mL) and 4-hydroxybenzaldehyde 14 (2.11 g, 17.3 mmol) was added. To this stirred solution K2CO3 (7.18 g, 52 mmol) was added and the mixture was heated at 60 °C overnight. Water (250 mL) was then added and the mixture extracted with CH2Cl2 (3 × 200 mL). The combined organic extracts were washed with brine (50 mL), dried over Na2SO4, and concentrated. The crude material was washed with acetone to afford 3.72 g (64%) of 11 as a white solid; mp 155 °C. 1H NMR (acetone-d6, 300 MHz): δ 9.93 (s, 1H), 8.04 (s, 1H), 7.92 (d, J ) 8.5 Hz, 2H), 7.40 (m, 5H), 7.25 (d, J ) 8.5 Hz, 2H), 6.54 (s, 1H), 5.15, (s, 2H), 5.07 (s, 2H). EIMS m/z (relative intensity) ) 336 (M+, 4), 214 (10), 91 (100). Anal. Calcd. (found) for C20H16O5: C, 71.42 (71.59); H, 4.79 (4.72). 5-[4-(5-Benzyloxy-4-oxo-4H-pyran-2-ylmethoxy)benzylidene]thiazolidine-2,4-dione (12). The title compound 12 (1.50 g, 57%) was prepared as a light orange solid from 4-(5-benzyloxy-4-oxo-4H-pyran-2-ylmethoxy)benzaldehyde (2.11 g, 6.28 mmol), thiazolidine-2,4-dione (0.74 g, 6.28 mmol), benzoic acid (0.15 g, 1.25 mmol), and piperidine (0.12 mL, 1.25 mmol) by a procedure analogous to that described for 5; mp 253-254 °C. 1H NMR (DMSOd6, 300 MHz): δ 12.52 (s, 1H), 8.27 (s, 1H), 7.75 (s, 1H), 7.58 (d, J ) 8.6 Hz, 2H), 7.39 (m, 5H), 7.20 (d, J ) 8.6 Hz, 2H), 6.56 (s, 1H), 5.10 (s, 2H), 4.94 (s, 2H). EIMS m/z (relative intensity) ) 435 (M+, 7), 91 (100). Anal. Calcd. (found) for C23H17NO6S: C, 63.44 (63.16); H, 3.93 (4.06); N, 3.22 (3.21). 5-[4-(5-Hydroxy-4-oxo-4H-pyran-2-ylmethoxy)benzylidene]thiazolidine-2,4-dione (HL3). The title compound HL3 (1.60 g, 67%) was prepared as a light beige solid from 5-[4-(5-benzyloxy-4-oxo-4H-pyran-2-ylmethoxy)benzylidene]thiazolidine-2,4-dione 12 (3.00 g, 6.90 mmol) by a procedure analogous to that described for HL1; mp 275-276 °C. 1H NMR (DMSO-d6, 300 MHz): δ 12.54 (s, 1H), 9.26 (s, 1H), 8.12 (s, 1H), 7.76 (s, 1H), 7.59 (d, J ) 8.8 Hz, 2H), 7.20 (d, J ) 8.8 Hz, 2H), 6.57 (s, 1H), 5.10 (s, 2H). DCIMS m/z (relative intensity) ) 346 (M+, 100), 221 (40), 203 (35), 150 (60), 125 (100). Anal. Calcd. (found) for C16H11NO6S: C, 55.65 (55.23); H, 3.21 (3.19); N, 4.06 (4.03). 5-Benzyloxy-2-chloromethyl-pyran-4-one (13). The title compound 13 (2.01 g, 62%) was prepared as a white solid from 5-benzyloxy-2-hydroxymethyl-pyran-4-one (3.00 g, 12.9 mmol) by a procedure analogous to that described for 9; mp 109-110 °C. 1H NMR (CDCl3, 300 MHz): δ 7.58 (s, 1H), 7.38 (m, 5H), 6.56 (s, 1H), 5.09 (s, 2H), 4.29 (s, 2H). EIMS m/z (relative intensity) ) 250 (M+, 7), 126 (10), 108 (28), 91 (100). Anal. Calcd. (found) for C13H11ClO3: C, 62.29 (62.41); H, 4.42 (4.41). 5-(4-Hydroxy-benzylidene)thiazolidine-2,4-dione (15). The title compound (17.69 g, 98%) was prepared as a bright yellow solid from 4-hydroxybenzaldehyde 14 (10.00 g, 81.9 mmol), thiazolidine-2,4-dione (9.58 g, 81.9 mmol), benzoic acid (1.51 g, 12.3 mmol), and piperidine (1.2 mL, 12.1 mmol) by a procedure analogous to that described for 5; mp 291-292 °C. 1H NMR (CD3OD, 300 MHz): δ 7.72 (s, 1H), 7.43 (d, J ) 8.6 Hz, 2H), 6.90 (d, J ) 8.6 Hz, 2H). EIMS m/z (relative intensity) ) 221 (M+, 40), 151 (10), 150 (100), 121 (17), 75 (11). Anal. Calcd. (found) for C10H7NO3S: C, 54.29 (54.69); H, 3.19 (3.30); N, 6.33 (6.44). (()-5-(4-Hydroxy-benzyl)thiazolidine-2,4-dione(28) (16). CoCl2‚6H2O (0.02 g, 0.08 mmol) and dimethylglyoxime (0.04 g, 0.3 mmol) were added to H2O (80 mL). After the addition of NaOH (1 M, 1 mL), the brown solution was cooled to 0 °C in an ice bath and NaBH4 (1.20 g, 31.7 mmol) was added in portions. 5-(4-Hydroxy-benzylidene)-

Vanadyl−Thiazolidinedione Combination Agents

thiazolidine-2,4-dione 15 (1.00 g, 4.5 mmol) was then added slowly as a solid over 15 min, and then the reaction mixture was stirred at room-temperature overnight. The pH was adjusted to 6 with CH3COOH, and then the mixture was extracted with CHCl3 (3 × 60 mL). The combined organic extracts were dried over Na2SO4, filtered, and decolorized with charcoal (refluxed for 5 min and filtered). Evaporation of the solvents afforded 0.64 g (64%) of the title compound 16 as a white solid; mp 156-157 °C. 1H NMR (CD3OD, 300 MHz): δ 7.06 (d, J ) 8.6 Hz, 2H), 6.71 (d, J ) 8.6 Hz, 2H), 4.65 (dd, J ) 9.2, 3.9 Hz, 1H), 3.34 (dd, J ) 14.2, 3.9 Hz, 1H), 3.03 (dd, J ) 14.2, 9.2 Hz, 1H). EIMS m/z (relative intensity) ) 223 (M+, 35), 107 (100), 91 (4), 77, (13). Anal. Calcd. (found) for C10H9O3S: C, 53.80 (53.76); H, 4.06 (3.95); N, 6.27 (6.15). (()-5-[4-(5-Benzyloxy-4-oxo-4H-pyran-2-ylmethoxy)benzyl]thiazolidine-2,4-dione (17). Sodium hydride (0.40 g, 16.7 mmol) was added to DMF (45 mL) cooled in an ice bath. To this stirring mixture (()-5-(4-hydroxy-benzyl)thiazolidine-2,4-dione 16 (1.70 g, 7.6 mmol) was added as a solid in portions causing considerable gas evolution. After 0.5 h 5-benzyloxy-2-chloromethyl-pyran-4-one 13 (2.00 g, 8.0 mmol) was added dropwise as a solution in DMF (30 mL). The resulting mixture was warmed to room temperature and stirred for 18 h. Saturated NaHCO3 (125 mL) was then added and the mixture was extracted with CH2Cl2 (3 × 125 mL). The combined organic portions were washed with brine (100 mL), dried over Na2SO4, and then the solvent was evaporated. The crude product was chromatographed on silica gel eluting with MeOH:CH2Cl2 (1:99) to afford 1.20 g (35%) of 17 as a pale yellow solid; mp 165-166 °C. 1H NMR (CDCl3, 400 MHz): δ 8.71 (s, 1H), 7.57 (s, 1H), 7.40 (m, 5H), 7.16 (d, J ) 8.6 Hz, 2H), 6.86 (d, J ) 8.6 Hz, 2H), 5.07 (s, 2H), 4.80 (s, 2H), 4.47 (dd, J ) 9.2, 3.9 Hz, 1H), 3.42 (dd, J ) 14.2, 3.9 Hz, 1H), 3.11 (dd, J ) 14.2, 9.2 Hz, 1H). EIMS m/z (relative intensity) ) 437 (M+, 3), 214 (10), 107 (53), 91 (100). Anal. Calcd. (found) for C23H19NO6S: C, 63.15 (63.09); H, 4.38 (4.40); N, 3.20 (3.32). (()-5-[4-(5-Hydroxy-4-oxo-4H-pyran-2-ylmethoxy)benzyl]thiazolidine-2,4-dione (HL4). The title compound HL4 (0.50 g, 93%) was prepared as a white solid from (()-5[4-(5-benzyloxy-4-oxo-4H-pyran-2-ylmethoxy)benzyl]thiazolidine-2,4-dione 17 (0.68 g, 1.6 mmol) by a procedure analogous to that described for HL1; mp 174 °C. 1H NMR (CD3OD, 300 MHz): δ 8.04 (s, 1H), 7.24 (d, J ) 8.6 Hz, 2H), 6.98 (d, J ) 8.6 Hz, 2H), 6.60 (s, 1H), 4.99 (s, 2H), 4.73 (dd, J ) 8.9, 4.2 Hz, 1H), 3.42 (dd, J ) 14.2, 4.2 Hz, 1H), 3.11 (dd, J ) 14.2, 8.9 Hz, 1H); EIMS m/z (relative intensity) ) 347 (M+, 10), 231 (70), 125 (100), 107 (63). Anal. Calcd. (found) for C16H13NO6S: C, 55.33 (54.87); H, 3.77 (3.75); N, 4.03 (4.03). Synthesis of Vanadyl-Thiazolidinedione Complexes. Bis((()-5-{4-[(5-alkoxy-4-oxo-4H-pyran-2-ylmethyl)amino]benzyl}thiazolidine-2,4-dionato)oxovanadium(IV) Hydrate (VO(L1)2‚H2O). HL1 (0.91 g, 2.64 mmol) was dissolved in H2O (15 mL) and VOSO4‚5H2O (0.28 g, 1.12 mmol) was added. The pH was adjusted to 5 with 1 M NaOH and the mixture was refluxed under Ar for 22 h. The reaction mixture was filtered hot and the precipitate was washed with H2O to afford, after drying in vacuo, 0.71 g (83% based on V) of the title compound as a brown solid; mp 175-176 °C. IR (cm-1, KBr disk): 3500-3000 (νN-H, νC-H); 1752, 1681 (νCdO thiazolidinedione); 1610, 1563, 1516, 1475 (pyrone νCdO, ring νCdC); 982 (νVdO). ESIMS m/z (relative intensity) ) 758 ([HVO(L1)2]+,(M+1)+, 6), 740 (16), 444 (15), 412 (60), 347 (100). The room-temperature solid-state magnetic moment was 1.75

Bioconjugate Chem., Vol. 14, No. 1, 2003 219

µB. Anal.Calcd. (found) for C32H26N4O11S2V‚H2O: C, 49.55 (49.25); H, 3.64 (3.54); N, 7.22 (6.94). Bis((()-5-[4-(5-alkoxy-4-oxo-4H-pyran-2-ylmethoxy)benzylidene]thiazolidine-2,4-dionato)oxovanadium(IV) Hydrate (VO(L3)2‚H2O). The title compound VO(L3)2‚H2O (1.00 g, 88%) was prepared as a gray solid from HL3 (1.03 g, 2.98 mmol) and VOSO4‚5H2O (0.38 g, 1.60 mmol) by a procedure analogous to that described for VO(L1)2; mp 189-192 °C. IR (cm-1, KBr disk): 3500-3000 (νN-H, νC-H); 1745, 1679 (νCdO thiazolidinedione); 1596, 1562, 1510, 1472 (pyrone νCdO, ring νCdC); 985 (νVdO). ESIMS m/z ) 756 ([HVO(L3)2]+,(M+1)+, 9), 685 (4), 593 (25), 458 (100), 443 (56), 368 (20). The room-temperature solidstate magnetic moment was 1.72 µB. Anal. Calcd. (found) for C32H20N2O13S2V‚H2O: C, 49.68 (50.01); H, 2.87 (2.67); N, 3.62 (3.71). Bis((()-5-[4-(5-alkoxy-4-oxo-4H-pyran-2-ylmethoxy)benzyl]thiazolidine-2,4-dionato)oxovanadium(IV) Hydrate (VO(L4)2‚H2O). The title compound VO(L4)2‚H2O (0.51 g, 93%) was prepared as a brown solid from HL4 (0.71 g, 2.05 mmol) and VOSO4‚5H2O (0.29 g, 1.21 mmol) by a procedure analogous to that described for VO(L1)2; mp 174 °C. IR (cm-1, KBr disk): 3500-3000 (νN-H, νC-H); 1750, 1692 (νCdO thiazolidinedione); 1610, 1563, 1510, 1476 (pyrone νCdO, ring νCdC); 975 (νVdO). ESIMS m/z ) 760 ([HVO(L4)2]+, (M+1)+, 50), 445 (100), 413 (49), 370 (10). The room-temperature solid-state magnetic moment was 1.69 µB. Anal. Calcd. (found) for C32H24N2O13S2V‚ H2O: C, 49.43 (48.98); H, 3.37 (3.25); N, 3.60 (3.52). Glucose-Lowering Studies. Male Wistar rats weighing 190-210 g (Animal Care Unit, UBC) were housed two per cage (polycarbonate) on a 12 h light:dark cycle. Animals were allowed ad libitum access to food (Purina Rat Chow #5001) and water, and were cared for in accordance with the principles and guidelines of the Canadian Council for Animal Care. Animals were allowed to acclimatize for a period of 7 days, and then diabetes was induced by a single tail vein injection of streptozotocin (STZ) (60 mg kg-1 in 0.9% NaCl) under light halothane anesthesia. Diabetes was confirmed 3 days after STZ-injection by tail-vein blood glucose determination (Ames Glucometer II and Glucostix), with blood levels over 13 mM being accepted as diabetic. One week after STZ-injection, animals were divided into treatment groups: carboxymethylcellulose (CMC) alone, thiazolidinedione compound, congeneric vanadium complex, or BMOV. All drugs were administered as suspensions in 1% CMC. Animals were not fasted prior to drug administration. Blood, 50 µL, was collected for glucose analysis immediately prior to drug administration and at selected times up to 72 h after drug administration. Blood was collected from the tail vein into heparinized capillary tubes and centrifuged (10 000g × 15 min), and plasma was collected for immediate determination of glucose using Boehringer Mannheim kits (glucose oxidase method). The hypoglycemic activity of the test compounds was calculated as shown in eq 1 (21, 39):

Hypoglycemic activity (%) )

(

)

PGc - PGT × 100% PGc (1)

PGC is plasma glucose in control mice, and PGT is plasma glucose in the mice treated with test compounds. Values are presented as means ( SEM at 12, 24, and 48 h. At all time points, the animals were observed for signs of toxicity (e.g. diarrhea). Intraperitoneal (i.p.) Administration in STZDiabetic Rats. Studies were done on 4 separate occa-

220 Bioconjugate Chem., Vol. 14, No. 1, 2003

sions, in each study four groups were examined CMC, BMOV, ligand, and vanadyl complex. For VO(L1)2, the animals were divided into CMC (n ) 5), BMOV (n ) 5), HL1 (n ) 10), VO(L1)2 (n ) 10); for HL2, CMC (n ) 5), BMOV (n ) 6), HL2 (n ) 10), rosiglitazone (n ) 10); for VO(L3)2, CMC (n ) 6), BMOV (n ) 10), HL3 (n ) 10), VO(L3)2 (n ) 10); and for VO(L4)2, (n ) 5), BMOV (n ) 5), HL4 (n ) 10), VO(L4)2 (n ) 10). All drug candidates were administered by i.p. injection in a volume of 2.5 mLkg-1 at a dose of 0.1 mmol kg-1. The control groups (CMC) received an equivalent volume of 1% CMC alone. There was no significant effect of drug administration on body weight over the treatment period in any of the trials. ACKNOWLEDGMENT

The authors gratefully acknowledge the Canadian Institutes of Health Research (CHIR) for an operating grant and Kinetek Pharmaceuticals Inc. for personnel support. We also thank Dr. B.D. Liboiron for his assistance with the EPR spectra. T.S. acknowledges NSERC (Natural Sciences and Engineering Research Council) of Canada for a postgraduate fellowship, and P.B. acknowledges a NATO Science Fellowship. LITERATURE CITED (1) Tolman, E. L., Barris, E., Burns, M., Pansini, A., and Partridge, R. (1979) Effects of vanadium on glucose metabolism in vitro. Life Sci. 25, 1159-1164. (2) Heyliger, C. E., Tahiliani, A. G., and McNeill, J. H. (1985) Effect of vanadate on elevated blood glucose and depressed cardiac performance of diabetic rats. Science 227, 1474-1477. (3) Li, J., Elberg, G., Crans, D. C., and Shechter, Y. (1996) Evidence for the distinct vanadyl(+4)-dependent activating system for manifesting insulin-like effects. Biochemistry 35, 8314-8318. (4) Fantus, I. G., Ahmad, F., and Deragon, G. (1994) Vanadate augments insulin-stimulated insulin receptor kinase activity and prolongs insulin action in rat adipocytes. Evidence for transduction of amplitude of signaling into duration of response. Diabetes 43, 375-383. (5) Shechter, Y., Meyerovitch, J., Farfel, Z., Sack, J., Bruck, R., BarMeir, S., Amir, S., Degani, H., and Karlish, S. J. D. (1990) In Vanadium in Biological Systems (N. D. Chasteen, Ed.), pp 129, Kluwer, Dordrecht, The Netherlands. (6) Shechter, Y., Li, J., Meyerovitch, J., Gefel, D., Bruck, R., Elberg, G., Miller, D. S., and Shisheva, A. (1995) Insulin-like actions of vanadate are mediated in an insulin-receptorindependent manner via nonreceptor protein tyrosine kinases and protein phosphotyrosine phosphatases. Mol. Cell. Biochem. 153, 39-47. (7) Shisheva, A., and Shechter, Y. (1993) Mechanism of pervanadate stimulation and potentiation of insulin-activated glucose transport in rat adipocytes: dissociation from vanadate effect. Endocrinology 133, 1562-1568. (8) Meyerovitch, J., Farfel, Z., Sack, J., and Shechter, Y. (1987) Oral administration of vanadate normalizes blood glucose levels in streptozotocin-treated rats. Characterization and mode of action. J. Biol. Chem. 262, 6658-6662. (9) Nechay, B. R. (1984) Mechanisms of action of vanadium. Annu. Rev. Pharmacol. Toxicol. 24, 501-524. (10) Tsiani, E., and Fantus, I. G. (1997) Vanadium compounds. Biological actions and potential as pharmacological agents. Trends Endocrinol. Metab. 8, 51-58. (11) Thompson, K. H., Yuen, V. G., McNeill, J. H., and Orvig, C. (1998) Chemical and pharmacological studies of a new class of antidiabetic vanadium complexes. In Chemistry, Biochemistry and Therapeutic Applications, ACS Symposium Series 711, pp 329, American Chemical Society, New York. (12) Caravan, P., Gelmini, L., Glover, N., Herring, F. G., Li, H., McNeill, J. H., Rettig, S. J., Setyawati, I. A., Shuter, E., Sun, Y., Tracey, A. S., Yuen, V. G., and Orvig, C. (1995)

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