(Lidorestat) and Congeners as Highly Potent and ... - ACS Publications

J. Med. Chem. 2005, 48, 3141-3152

3141

Discovery of 3-[(4,5,7-Trifluorobenzothiazol-2-yl)methyl]indole-N-acetic Acid (Lidorestat) and Congeners as Highly Potent and Selective Inhibitors of Aldose Reductase for Treatment of Chronic Diabetic Complications Michael C. Van Zandt,*,† Michael L. Jones,† David E. Gunn,† Leo S. Geraci,† J. Howard Jones,† Diane R. Sawicki,† Janet Sredy,† Jorge L. Jacot,† A. Thomas DiCioccio,† Tatiana Petrova,‡ Andre Mitschler,‡ and Alberto D. Podjarny‡ The Institute for Diabetes Discovery, LLC, 23 Business Park Drive, Branford, Connecticut 06405, and Departement de Biologie et Genomique Structurales, UMR 7104 du CNRS, IGBMC, 1 rue Laurent Fries, B.P. 163, 67404 Illkirch Cedex, France Received September 30, 2004

Recent efforts to identify treatments for chronic diabetic complications have resulted in the discovery of a novel series of highly potent and selective 3-[(benzothiazol-2-yl)methyl]indoleN-alkanoic acid aldose reductase inhibitors. The lead candidate, 3-[(4,5,7-trifluorobenzothiazol2-yl)methyl]indole-N-acetic acid (lidorestat, 9) inhibits aldose reductase with an IC50 of 5 nM, while being 5400 times less active against aldehyde reductase, a related enzyme involved in the detoxification of reactive aldehydes. It lowers nerve and lens sorbitol levels with ED50’s of 1.9 and 4.5 mg/kg/d po, respectively, in the 5-day STZ-induced diabetic rat model. In a 3-month diabetic intervention model (1 month of diabetes followed by 2 months of drug treatment at 5 mg/kg/d po), it normalizes polyols and reduces the motor nerve conduction velocity deficit by 59% relative to diabetic controls. It has a favorable pharmacokinetic profile (F, 82%; t1/2, 5.6 h; Vd, 0.694 L/kg) with good drug penetration in target tissues (Cmax in sciatic nerve and eye are 2.36 and 1.45 µg equiv/g, respectively, when dosed with [14C]lidorestat at 10 mg/kg po). Introduction Diabetes mellitus and its disabling complications, which include blindness, renal failure, neuropathy, limb amputation, myocardial infarction, and stroke, affect some 17 million people in the United States with an estimated cost of over 130 billion dollars annually.1 Through various clinical studies, including the Diabetes Control and Complications Trial (DCCT) and the United Kingdom Prospective Diabetes Study (UKPDS), the development and progression of these complications in type 1 and type 2 patients have been clearly linked to elevated blood glucose levels.2 Although glucose is preferentially metabolized through the glycolytic pathway, during conditions of hyperglycemia, as observed in diabetes mellitus, elevated blood glucose levels saturate the normal pathways of glucose metabolism and a dramatic increase in flux through the polyol pathway results (Scheme 1). Glucose entering the polyol pathway is reduced to sorbitol by aldose reductase (ALR2) and NADPH. Sorbitol is subsequently oxidized to fructose by sorbitol dehydrogenase and NAD.+ This increased flux through the polyol pathway results in a reduced ratio of NADPH to NADP+ and an increased ratio of NADH to NAD.+ These changes, which alter the reduction potential of the cell, are collectively termed oxidative stress. The impact of oxidative stress has been clearly demonstrated.3 It is linked to depleted intracellular levels of reduced glutathione, increased nonenzymatic glycation, and activation of protein kinase C. In addition to sorbitol and fructose,4 myo-inositol is also an important sugar that is effected by flux through * To whom correspondence should be addressed. Fax: (203) 3154002. Tel: (203) 315-5951. E-mail: michael.vanzandt@ ipd-discovery.com. † The Institute for Diabetes Discovery. ‡ Departement de Biologie et Genomique Structurales.

Scheme 1

the polyol pathway.5 As glucose and sorbitol levels are increased, myoinositol levels are decreased. This decrease results in reduced production of diacylglycerol, diminished Na+-K+-ATPase activity, and eventually a reduced nerve conduction velocity.6 In addition to oxidative stress, accumulation of sorbitol in certain cells results in osmotic stress that can lead to cell swelling and eventually cell death.7 Inhibitors of aldose reductase can prevent these metabolic and biochemical changes, which have been linked to the pathogenesis of diabetic complications.8 Further supporting the role of ALR2 in the development of diabetic complications, certain allelic variants of the hALR2 gene associated with overexpression of aldose reductase are well-correlated with increased risk of developing diabetic complications.9 Similar conclusions can be drawn from the ALR2 knockout mouse. Even when severely diabetic, these animals are protected against decline in motor nerve conduction velocity.10 As a result, a high ALR2 level is now considered an important risk factor for chronic diabetic complications, including neuropathy, retinopathy, and nephropathy.11 During the past 25 years at least 14 different potent and highly efficacious aldose reductase inhibitors (ARIs) have been identified and advanced through phase II clinical development (Figure 1).12 Despite this tremen-

10.1021/jm0492094 CCC: $30.25 © 2005 American Chemical Society Published on Web 03/30/2005

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Figure 1. Inhibitors of aldose reductase advancing to phase II clinical development.

Scheme 3a

Figure 2. Aldose reductase inhibitors with 4,5,7-trifluorobenzothiazole side chain.

Scheme 2a

a Reagents: (a) morpholine, K CO , DMSO, 80 °C (97%); (b) 2 3 phenol, K2CO3, DMSO, 80 °C (63%); (c) dimethylformamide dimethyl acetal, pyrrolidine, 100 °C; (d) H2, Pd-C, ethanol, 55 psi (31%, 2 steps); (e) analogous to method set forth in Scheme 2.

a Reagents: (a) Ac O, pyridine, 120 °C (82%); (b) P S , benzene 2 4 10 (88%); (c) NaH, toluene (96%); (d) 30% aqueous NaOH, ethylene glycol, 125 °C (73%); (e) ethyl bromoacetate, NaH, acetonitrile (49%); (f) trifluoroethanol, cat. BHT, 80 °C (59%); (g) aqueous NaOH, ethanol (98%).

dous effort, none are currently marketed for worldwide use. Although epalrestat is currently available in Japan, and fidarestat and AS-3201 are in late-stage clinical development, many of these candidates failed to gain acceptance due to an inadequate therapeutic index.13 In some cases this toxicity may have resulted from a lack of selectivity relative to aldehyde reductase (ALR1). The physiological importance of ALR1 has been clearly

demonstrated.14 It converts highly reactive 2-oxoaldehydes such as 3-deoxyglucosone and methyl glyoxal to their corresponding nonreactive alcohols.15 These aldehydes, which are involved in the formation of various protein cross-links and other advanced glycation end products (AGEs), are formed as degradation products from glucose and fructose.16 During conditions of prolonged hyperglycemia, high concentrations of these aldehydes are formed, resulting in the development of chronic diabetic complications and accelerated aging.17 Our work in this area has focused primarily on identifying new carboxylic acid-based templates with significantly improved oral efficacy and selectivity relative to aldehyde reductase. In this paper we report our work on a novel series of indole-N-acetic acids, including our clinical candidate, 3-[(4,5,7-trifluorobenzothiazol-2yl)methyl]indole-N-acetic acid (lidorestat), compound 9.

Potent and Selective Inhibitors of Aldose Reductase

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9 3143

Table 1. Physical and in Vitro Properties of Indoleacetic Acid ARIs

no.

substituents

mp, °C

empirical formula

analysis

hALR2a (nM)

HALRb (nM)

17 18 19 20 21 22 23 24 25 26 27 9 28 29 30 31 32 33 34 35 36 16 37 15 38 39 40 41 42 43 44 45 46 47

5′-CF3 5-CH3, 5′-CF3 5-Cl, 5′-CF3 6-Br, 5′-CF3 6′-Cl 4′-F 5′-F 6′-F 7′-F 5′-F, 6′-F 4′-F, 6′-F 4′-F, 5′-F, 7′-F 2-CH3, 4′-F, 5′-F, 7′-F 2-Ph, 4′-F, 5′-F, 7′-F 4-Cl, 4′-F, 5′-F, 7′-F 5-Cl, 4′-F, 5′-F, 7′-F 5-F, 4′-F, 5′-F, 7′-F 5-Br, 4′-F, 5′-F, 7′-F 5-CH3, 4′-F, 5′-F, 7′-F 5-OCH3, 4′-F, 5′-F, 7′-F 5-OBn, 4′-F, 5′-F, 7′-F 5-OPh, 4′-F, 5′-F, 7′-F 5-Ph, 4′-F, 5′-F, 7′-F 5-morpholino, 4′-F, 5′-F, 7′-F 6-F, 4′-F, 5′-F, 7′-F 6-Cl, 4′-F, 5′-F, 7′-F 6-CH3, 4′-F, 5′-F, 7′-F 6-OCH3, 4′-F, 5′-F, 7′-F 6-Ph, 4′-F, 5′-F, 7′-F 6-morpholino, 4′-F, 5′-F, 7′-F 7-F, 4′-F, 5′-F, 7′-F 7-Cl, 4′-F, 5′-F, 7′-F 7-Br, 4′-F, 5′-F, 7′-F 7-CH3, 4′-F, 5′-F, 7′-F

233-234 248-249 238-240 265-267 158-160 170-172 208 203 181-183 193-195 216-218 177-178 178-180 238-239 203-206 188-189 165-168 143-144 131-133 165-167 165-168 128-130 156-159 119-121 200-203 194-195 211-213 118-120 156-159 178-180 194-196 228-230 228-230 216-218

C19H13F3N2O2S C20H15F3N2O2S C19H12ClF3N2O2S C19H12F3N2O2SBr C18H13ClN2O2S C18H13FN2O2S C18H13FN2O2S C18H13FN2O2S C18H13FN2O2S C18H12F2N2O2S C18H12F2N2O2S C18H11F3N2O2S C19H13F3N2O2S C24H15F3N2O2S C18H10ClF3N2O2S C18H10ClF3N2O2S C18H10F4N2O2S C18H10BrF3N2O2S C19H13F3N2O2S C19H13F3N2O3S C25H17ClF3N2O3S C24H15F3N2O3S C24H15F3N2O2S C22H18F3N3O3S H2O C18H10F4N3O2S C18H10F3N2O2SCl C19H13F3N2O2S C19H13F3N2O3S·H2O C24H15F3N2O2S C22H18F3N3O3S C28H10F4N2O2S·H2O C18H10F3N2O2SCl C18H10F3N2O2SBr C19H13F3N2O2S

C,H,N,S C,H,N,S NDc C,H,N,S C,H,N,S C,H,N,S C,H,N,S C,H,N,S C,H,N,S C,H,N,S C,H,N,S C,H,N,S C,H,N,S C,H,N,S C,H,N,S C,H,N,S C,H,N,S C,H,N,S C,H,N,S ND C,H,N,S C,H,N,S C,H,N,S C,H,N,S ND C,H,N,S C,H,N,S C,H,N,S C,H,N,S C,H,N,S C,H,N,S C,H,N,S C,H,N,S C,H,N,S

99 100 130 52 660 34 9 1 000 55 560 690 5 8 100 11 10 11 13 8 8 12 30 53 8 7 8 10 5 25 15 7 9 14 6

5 600 10 200 4 900 3 400 2 100 11 000 6 400 4 500 18 000 13 000 5 100 27 000 13 000 16 000 14 000 11 000 21 000 4 700 34 000 21 000 4 800 11 000 10 000 41 000 7 500 2 700 13 000 12 000 6 100 44 000 7 000 19 000 18 000 21 000

a

Recombinant human aldose reductase. b Recombinant human aldehyde reductase. c Not determined.

Inhibitor Design and Synthesis As we began work on our indole-based ARIs, we were particularly intrigued by a series of inhibitors containing a 4′,5′,7-trifluorobenzothiazole side chain (Figure 2). Although these compounds do not appear to have been developed clinically, published data suggests that the side chain provides significant improvements in in vitro and in vivo activity relative to the 5-trifluoromethylbenzothiazole or 2-fluoro-4-bromobenzyl used in previously developed ARIs.18 In our efforts to identify and develop a new ARI with the potency, selectivity, and safety required to proceed through clinical development, we found that the same side chain was optimal in our indole-N-acetic acid based series. The structure-activity relationship for the series is described in the Results and Discussion. The target compounds were synthesized using methods illustrated in Schemes 2 and 3. The synthesis of 3-[(4,5,7-trifluorobenzothiazol-2-yl)methyl]indole-Nacetic acid (9) is described in Scheme 2. Here, tetrafluoroaniline 1 is acylated with acetic anhydride in pyridine at 120 °C followed by treatment with P4S10 in benzene to give thioamide 3 in good yield. Subsequent cyclization of the thioamide using sodium hydride provides 2methylbenzothiazole 4. Hydrolysis of the heterocyclic ring with aqueous sodium hydroxide followed by acidi-

fication with 2 N HCl gives the 2-aminothiophenol as the hydrochloride salt (5). Although prepared in high yield, this intermediate is highly unstable and completely dimerizes within 24 h. This oxidation can be easily prevented, however, by addition of a free radical inhibitor. With the addition of a catalytic amount of BHT (a few crystals) this key intermediate is stable on the benchtop for several months. Indole-3-acetonitrile-N-acetic acid ethyl ester 7 was prepared from indole-3-acetonitrile (6) by alkylation with ethyl bromoacetate and sodium hydride. Substituted indole examples where the 3-acetonitrile substituent was not commercially available were conveniently prepared from the corresponding indole via the gramine intermediate using classical procedures.19 Condensation of 2-aminothiophenol 5 with indole 3-acetonitrile 7 in 2,2,2-trifluoroethanol provided benzothiazole 8 in 59% yield. Initial results using ethanol as solvent were considerably less efficient due to partial esterification of the cyano group. Finally, hydrolysis with aqueous NaOH gives the target compound (9) in 29% overall yield (six linear steps). Compounds 15 and 16, where the 5′-position on the indole ring is substituted with a morpholino or phenoxy group, were prepared using the chemistry illustrated in Scheme 3. 4-Nitro-3-methylfluorobenzene 10 is treated

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Table 2. Physical and in Vitro Properties of Indoleacetic Acid ARIs

no.

A

B

mp, °C

empirical formula

analysis

hALR2a (nM)

hALR1b (nM)

9 48 49 50 51

-CH2-(CO)-CH2CH2-CH2-CH2-

-CH2-CH2-CH2-CH2CH2-CH(CH3)-

177-178 >250 198-199 200-201 176-177

C18H11F3N2O2S C18H9F3N2O3S C19H13F3N2O3S C19H13F3N2O3S C19H13F3N2O2S·H2O

C,H,N,S C,H,N,S C,H,N,S C,H,N,S C,H,N,S

5 1 600 8 17 163

27 000 NDc 11 000 88 000 6 500

a

Recombinant human aldose reductase. b Recombinant human aldehyde reductase. c Not determined.

with morpholine or phenol with K2CO3 in DMSO to provide substituted products 11 and 12, respectively. Subsequent treatment with dimethylformaldehyde dimethyl acetal followed by hydrogenation provides the corresponding indole intermediates 13 and 14. Once formed, the substituted indole intermediates were converted to the target compounds using chemistry outlined in Scheme 2. Results and Discussion The major objective of this program has been to identify highly potent, selective, and efficacious aldose reductase inhibitors for treatment of chronic diabetic complications. All compounds in the program were initially tested for potency against human aldose reductase (hALR2) and selectivity relative to human aldehyde reductase (hALR1). The results from these experiments are listed in Tables 1 and 2. Selected compounds with significant in vitro activity against hALR2 were also tested in the streptozotocin (STZ) induced diabetic rat model. X-ray crystallography was used throughout the program. The structures provided a clear understanding of the important interactions that make up the enzymeinhibitor complexes. This information was used to focus efforts on the specific fragments of the inhibitor template that provided the greatest opportunities for optimization of potency and selectivity relative to aldehyde reductase. The complexes were obtained using hALR2 expressed in Escherichia coli and crystallized with the oxidized form of the coenzyme β-NADP+ at pH 5 and 277 K. Initial results with 9 provided a precise picture of the inhibitor-enzyme complex (1.04 Å resolution). As illustrated in Figure 3, the inhibitor is oriented in the active site of hALR2 in a manner such that the hydrophilic carboxylate head forms tight hydrogen bonds with the OH of Tyr48 (2.72 Å), the NE2 of His110 (2.69 Å), the O7N of NADP+ (3.41 Å), and the NE1 of Trp111 (2.95 Å). These interactions anchor the inhibitor deep within the enzyme active site. As this class of inhibitors binds to the hALR2 active site, a conformational change occurs, opening a pocket localized between Trp111 and Leu300.20 The specific opening of the pocket varies to accommodate each inhibitor, producing an “induced fit.” Since the residues lining this pocket are not conserved in aldehyde reductase, the interactions are specific for ALR2.21 As a result,

Figure 3. Compound 9 bound to ALR2. Enzyme-inhibitor complex of 9 bound in the active site of human aldose reductase. Contacts shorter than 3.5 A are indicated. Note short contacts of the inhibitor carboxylic acid head with residues Tyr48, His110, and Trp111 and the perfect π-stacking interaction of the benzothiazole side chain with the indole moiety of Trp111.

inhibitors with binding interactions in this “specificity pocket” are generally highly selective for aldose reductase. The specific interactions within the specificity pocket for 9 include a nearly perfect π-stacking interaction of the benzothiazole side chain with the indole moiety of Trp111 and H-bonding interactions between the amide N-H of Leu300 and the benzothiazole N (3.42 Å) and the 4′-fluorine (3.38 Å). Within this series, the indole, benzothiazole, and carboxylic acid moieties were kept constant while the various substituents and linkers were optimized. Since selectivity relative to aldehyde reductase was a critical program objective, initial efforts focused on optimization of the benzothiazole ring system.22 On the basis of work published for zopolrestat, SG-210, and GP-1447, various examples with fluorine or trifluoromethyl substituents were evaluated. The first examples with interesting in vitro activity were those with the 5′-trifluoromethyl substituent (17-20). This side chain provides modest in vitro activity with IC50’s of approximately 100 nM. In addition to the CF3 substituent, fluorine substituents



in various positions around the benzothiazole ring provided an interesting structure-activity relationship. As seen in examples 22-25, introduction of a fluorine substituent in the 4′-, 5′-, and 7′-positions (22, 23, and 25, respectively) provides excellent in vitro activity, with the 5′-position being most important, giving an IC50 of 9 nM. Unlike the 4′-, 5′-, and 7′-positions, compound 24, with the 6′-fluoro substituent, is relatively inactive, with an IC50 of 1000 nM. This same trend is seen in the difluoro-substituted analogues. When the 6′-fluoro substituent is added to the 5′-fluoro (23) and 4′-fluoro (22) analogues, giving the corresponding difluoro compounds 26 and 27, the in vitro activity is decreased by about 10-fold (IC50’s > 500 nM). Conversely, when the 4′-, 5′-, and 7′-fluorine substituents are combined, the binding affinity is enhanced, giving example 9 with an IC50 of 5 nM. This structure-activity relationship is consistent with the well-defined interactions between the benzothiazole moiety and the specificity pocket. Introducing the 6′-fluorine into the crystal structure of 9 requires a short contact (about 2 Å) with the OG1 of Thr113. This unfavorable steric interaction results in the loss of binding affinity. In addition to the potency of these compounds, the selectivity relative to aldehyde reductase is also additive. The 4′-, 5′-, and 7′-fluorine-substituted examples (22, 23, and 25) have selectivity ratios (hALR1/hALR2) of 710, 320, and 330, respectively, but when the substituents are combined together, the selectivity ratio for compound 9 becomes 5400. In contrast to the strong substitution effects seen on the benzothiazole, substituents on the indole framework have little effect. This is due primarily to the orientation of the inhibitors in the active site. While the carboxylate and benzothiazole side chains reach deep within the active site, the space around the indole core is relatively open. While the 4′,5′,7′-trifluorobenzothiazole is kept constant, introduction of a methyl at the 2-position gives 28, which has an IC50 of 8 nM. Even with the large phenyl substituent in the 2-position (29), the IC50 only increases to 100 nM. Examples with halogens or methyl in the 4- or 5-position have IC50’s of about 10 nM (30, 4-cloro; 31, 5-cloro; 32, 5-floro; 33, 5-bromo; 34; 5-methyl). Other alkoxy substituents in the 5-position, including methoxy (35), benzyloxy (36), and phenoxy (16), have IC50’s of 12, 30, and 53 nM, respectively. The 5-phenyl example (37) is somewhat less active, with an IC50 of 53 nM, while the 5-morpholino is quite interesting, with an IC50 of 8 nM and selectively ratio of >5000 (15). Similar results are observed for the corresponding examples with substituents in the 6- and 7-positions (38-47). In addition to the benzothiazole and indole portions of the inhibitor template, some modifications of the linkers were also investigated. Oxidation of the indolebenzothiazole linker A to a carbonyl (48) results in a significant loss in activity (IC50 ) 1600 nM), while the homologue (A ) -CH2CH2-, 49) remains very active, with an IC50 of 8 nM. The loss of activity for example 48 is expected on the basis of the increased bond angle required for the sp2 hybridized carbon atom. In this configuration the substituted benzothiazole and carboxylate are unable to interact with their respective

Table 3. Activity of Lidorestat (9) Relative to Other Clinically Developed ARIsa % inhibn of sorbitol accum (10 mg/kg/d)c

IC50 group

hALR2a

hALR1b

nerve

lens

tolrestat zopolrestat zenarestat lidorestat (9)

13 9 8 5

1 940 38 600 9 900 27 000

27 80 64 100

7 9 2 77

a Recombinant human aldose reductase. b Recombinant human aldehyde reductase. c Percent improvement of drug treated group relative to difference between nondiabetic and diabetic control groups.

Table 4. Nerve Function Assesment with Lidorestat (9) in 1-month Diabetic Prevention Modela group

dose (mg/kg/d)

MNCV

% imp.b

nondiabetic control diabetic control lidorestat (9) lidorestat (9)

5 10

47.73 ( 41.15 ( 0.43 44.77 ( 0.69* 46.25 ( 0.94*

55 78

1.08c

a One month of diabetes following STZ injection. b Percent improvement of drug-treated group relative to the difference between nondiabetic and diabetic control groups. c All values are mean ( SEM. *p < 0.05 as compared to diabetic control group.

pockets at the same time. In contrast, the homologue is able to retain these binding interactions by simply moving the indole moiety slightly. It is interesting to note that the same homologue in the zopolrestat series is about 100-fold less active.23 This likely results from a reduced flexibility around the phthalazine ring. Homologation of the methylene connecting the indole moiety to the carboxylic acid (B ) -CH2CH2-, 50) is also quite active, with an IC50 of 17 nM. In addition to its good potency, this example is also quite selective, with a selectivity ratio of over 5000. Introduction of a methyl substituent to linker B (51) results a significant loss of activity (IC50 ) 163 nM). The reduced activity for this example arises from a steric interaction with Trp20. As the optimization program was developing, compounds with good in vitro activity were selected for further evaluation in vivo. In general, the streptozotocin (STZ)-induced diabetic rat model was used to evaluate the in vivo efficacy of compounds from this series. Sorbitol concentrations in nerve and lens tissues were used as the primary biochemical endpoints in the initial 5-day screening assay. In this model, where sorbitol levels in the sciatic nerve and lens are increased 10 and 20-fold in the diabetic controls, lidorestat is highly efficacious, normalizing this polyol with an ED50 of 1.9 and 4.5 mg/kg/d, respectively. When compared to other clinically developed ARIs, lidorestat is one of the most potent, selective, and efficacious (Table 3). With these excellent results, lidorestat was subsequently tested in more long-term studies to better define its pharmacodynamic effects. Dose-response prevention and lowdose intervention study designs were employed in which the animals either received drug 4 days after STZinduction of diabetes (prevention) or were diabetic for 28 days prior to receiving daily drug treatment for the two following months (intervention). In the prevention study, after one-month of STZinduced diabetes, the untreated diabetic animals dem-

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Table 5. Biochemical and Nerve Function Assessment with Lidorestat (9) in 3-Month Diabetic Intervention Modela

group

sorbitol (nmol/mg of dry wt)

fructose (nmol/mg of dry wt)

myo-inositol (nmol/mg of dry wt)

MNCV

nondiabetic control diabetic control 9 at 5.0 mg/kg/d

0.24 ( 0.02b 4.84 ( 0.69* BQLc **

2.16 ( 0.11 20.10 ( 1.53* 1.62 ( 0.05**

9.90 ( 0.27 6.89 ( 0.18* 9.26 ( 0.26**

51.00 ( 0.79 44.58 ( 0.49 48.39 ( 0.42

% improvementd

100

100

79

59

One month of diabetes followed by 2 months of drug treatment. b All values are mean ( SEM. *p < 0.05 as compared to nondiabetic control group. **p < 0.05 as compared to untreated diabetic control group. c Below quantifiable level. d Percent improvement of drugtreated group relative to the difference between nondiabetic and diabetic control groups. a

Figure 4. Slit-lamp assessment of cataract formation with lidorestat. Slit-lamp evaluation of cataract formation in rat lens after 3 months of diabetes. Image A, nondiabetic control; image B, diabetic control; and image C, diabetic with lidorestat at 25 mg/kg/d po. Results indicate that the drug-treated group is indistinguishable from the nondiabetic control group.

onstrated a significant loss in motor nerve conduction velocity (MNCV, 6.58 m/s relative to the control group). Lidorestat, administered at 5 and 10 mg/kg/d, substantially reduced the MNCV decline, resulting in an improvement of 3.62 and 5.10 m/s, respectively (Table 4). This improvement corresponds to a 55% and 78% recovery of the MNCV-deficit relative to the untreated diabetic rats. In the intervention study, at three-months, untreated diabetic rats had a decline in MNCV of 6.42 m/s compared to the nondiabetic control group (Table 5). Diabetic animals treated for the last 2 months with lidorestat lost only 2.61 m/s in MNCV. This represents a 59% improvement relative to the diabetic control group. In addition to MNCV, in the intervention study, nerves were removed and analyzed for polyol and sugar content. As expected, the concentrations (nmol/mg/dry wt) of glucose, sorbitol, and fructose were significantly increased and that of myo-inositol significantly decreased in the sciatic nerve of untreated diabetic rats relative to the nondiabetic control group. Treatment with lidorestat (5 mg/kg/d) in the intervention study completely eliminated the accumulation of sorbitol and fructose and corrected myo-inositol concentrations (79%) without altering nerve glucose levels. In the eye, activation of the polyol pathway and corresponding increase of sorbitol concentration in the lens epithelium is an early initiating event in cataractogenesis.24 This increase in lens sorbitol level has also been shown to correlate with the rate of diabetic cataract formation.25 Once formed inside the cell, sorbitol only slowly transverses the cell membrane, resulting in accumulation inside the cell. This results in intracellular hypertonicity, followed by an influx of water, which leads to cellular edema. Continued intracellular hydration leads to vacole formation and loss of membrane integrity, leading to irreversible lens opacification. Treatment with lidorestat exhibited a dosedependent prevention of cataract formation in STZ-

Table 6. Triglyceride Lowering with Lidorestat

group control nondiabetic diabetic 9-treated 4.8 mg/kg/d 1.9 mg/kg/d 1.4 mg/kg/d 0.95 mg/kg/d 0.48 mg/kg/d

glucose (mg/dL)

plasma triglycerides (mg/dL)

% red.a

72 ( 2b 439 ( 12**

81 ( 18 349 ( 78**

-

417 ( 12** 440 ( 16** 427 ( 26** 430 ( 13** 432 ( 16**

189 ( 34* 218 ( 27** 242 ( 58* 261 ( 81* 301 ( 102

60 49 40 33 18

a Percent reduction of plasma triglycerides in drug-treated group relative to difference between nondiabetic and diabetic controls. b All values are means ( SEM . **p < 0.01 as compared to nondiabetic control group. *p < 0.05 as compared to nondiabetic control group.

induced diabetic rats (5-25 mg/kg/d admixed in diet). After 3 months, untreated diabetic control animals had full nuclear cataracts formed while groups treated with 5 and 10 mg/kg/d exhibited only diffuse cloudiness and loss of transparency. The 25 mg/kg/d group suffered no loss of clarity and was indistinguishable from the nondiabetic control group. Figure 4 provides a slit-lamp evaluation of the rat lens at 3 months for the control STZ-induced diabetic rat verses the same animals treated with lidorestat at 25 mg/kg/d. In addition to polyols, MNCV, and cararact formation, lidorestat was also evaluated for its effect on plasma triglyceride levels. In the 15-day STZ-induced diabetic rat model, where blood glucose and plasma triglyceride levels are increased 5.7- and 5.4-fold relative to the nondiabetic controls (Table 6), treatment with lidorestat at doses of 0.48-4.8 mg/kg/d resulted in a significant dose-related reduction in triglyceride concentration (1860%). These data suggests that in addition to its direct beneficial effect on the peripheral nerve and lens, lidorestat has the added advantage of lowering the



Table 7. Pharmacokinetics of [14C]Lidorestat in Rata

a

group

dose (mg/kg/d)

Tmax (h)

Cmax (µg equiv/ mL)

t1/2 (h)

AUC0-24h (µg equiv/ h/mL)

Vd (L/kg)

ClT (mL/ min/kg)

iv, fasted po, fasted po, nonfasted

10 6 6

0.08 3.50 5.00

24.0 4.18 6.09

5.04 5.61 5.18

97.1 43.5 72.8

0.694 -

1.70 2.24 1.37

Study measures unchanged drug in plasma.

elevated levels of triglycerides that are commonly seen in people with diabetes. A pharmacokinetic evaluation of [14C]lidorestat was conducted in rat using both intravenous and oral dosing (Table 7). Following intravenous dosing with 10 mg/kg/d in fasted animals, [14C]lidorestast has a terminal elimination half-life (t1/2) of 5.04 h with a volume of distribution and total body clearance of 0.694 L/kg and 1.70 mL/ min/kg, respectively. The oral bioavailability was 82% with a Cmax of 4.18 µg equiv/mL and tmax of 3.50 h. The t1/2 following oral administration was determined to be 5.61 h. In a separate tissue distribution study with [14C]lidorestat also dosed orally at 10 mg/kg in male rats, the Cmax in sciatic nerve and eye were 2.36 and 1.45 µg equiv/g, respectively. Unlike many other carboxylic acid based aldose reductase inhibitors,4 the favorable pharmacokinetic profile of lidorestat provides excellent penetration in the hard-to-reach tissues where complication occur (nerve and lens). The details of this tissue distribution and pharmacokinetic study will be published separately. Lidorestat has proceeded through early development into a phase II clinical trial. Preliminary results in man demonstrate complete normalization of sural nerve sorbitol levels at all doses tested (25-400 mg/d). Measured sorbitol levels after 60 days were 0.00-0.09 nM/ mg of dry weight compared to 2.5-8.0 nM/mg of dry weight that would be expected from untreated diabetic patients.26 Although these results are preliminary, it is clear lidorestat is highly efficacious in man and will likely require a dose significantly less than 25 mg/d. In conclusion, a novel series of 3-[(benzothiazol-2-yl)methyl]indole-N-acetic acid derivatives has been identified and optimized as highly potent and selective inhibitors of aldose reductase. In vivo evaluation of 3-[(4,5,7-trifluorobenzothiazol-2-yl)methyl]indole-N-acetic acid (lidorestat), compound 9, in the STZ-induced diabetic rat model resulted in normalization of nerve sugars (sorbitol, fructose, and myo-inositol), improvement of MNCV, reduction of triglycerides, and prevention of diabetic cataracts. Continued development of these compounds may result in an important new treatment for chronic diabetic complications. Experimental Section Chemistry. General Methods. Melting points were determined in open capillary tubes on a Thomas-Hoover apparatus and are uncorrected. Proton nuclear magnetic resonance (1H NMR) spectra were determined on a Varian Gemini 2000 (300 MHz) instrument. Chemical shifts are provided in parts per million (ppm) downfield from tetramethylsilane (internal standard) with coupling constants in hertz (Hz). Multiplicity is indicated by the following abbreviations: singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), broad (br). Mass spectra were recorded on a Perkin-Emer API 100. Elemental analyses (C, H, N, S) were performed by Atlantic Microlab, Inc. (Norcross, Georgia) and are within (0.4% of

theory unless otherwise noted. Products from all reactions were purified by either flash column chromatography or medium-pressure liquid chromatography using silica gel 60 (230-400 mesh Kieselgel 60, EM Reagents) unless otherwise indicated. Thin-layer chromatography using glass-backed silica plates containing a fluorescent indicator (0.25 mm, Whatman, Merck) was used to monitor reactions. The chromatograms were visualized using ultraviolet illumination, exposure to iodine vapors, or dipping in an aqueous potassium permanganate solution. All starting materials were used without further purification. All reactions were carried out under an atmosphere of dried nitrogen. General Procedures for the Synthesis of 3-(Benzothiazol-2-yl)methylindole-N-alkanoic Acid ARIs. 3-(Benzothiazol-2-yl)methylindole-N-alkanoic acids 9 and 15-51 (Tables 1 and 2) were prepared from the desired 2-fluoroaniline or corresponding commercially available substituted 2-aminothiophenol and the indole moiety as outlined in Scheme 2 via the general experimental methods described for 9 below. The desired indole moiety used in the synthesis was prepared by the method outlined in Schemes 3 or purchased commercially. 3-(4,5,7-Trifluorobenzothiazol-2-yl)methylindole-Nacetic Acid (9). Step 1: Synthesis of 2,3,5,6-Tetrafluoroacetanilide (2). A solution of 2,3,5,6-tetrofluoroaniline (200 g, 1.21 mol) in anhydrous pyridine (103 mL, 1.27 mol) was treated with acetic anhydride (120 mL, 1.27 mol) and heated to 120 °C for 2 h. After cooling to room temperature, the solution was poured into ice-cold water (500 mL). The resulting precipitate was filtered, dissolved in ethyl acetate, dried over MgSO4, filtered, and concentrated. The solid material was washed with heptane (200 mL) and dried to give 2,3,5,6tetrafluoroacetanilide as a white crystalline solid (206 g, 82%): mp 136-137 °C; Rf 0.48 (50% ethyl acetate in heptane); 1 H NMR (DMSO-d6, 300 MHz) δ 10.10 (s, 1 H), 7.87-7.74 (m, 1 H), 2.09 (s, 3 H). Anal. (C8H5F4NO) C, H, N. Step 2: 2,3,5,6-Tetrafluorothioacetanilide (3). A flamedried, four-necked, 5000 mL round-bottomed flask was charged with phosphorus pentasulfide (198 g, 0.45 mol) and diluted with anhydrous benzene (3000 mL, 0.34 M). 2,3,5,6-Tetrafluoroacetanilide (185 g, 0.89 mol) was added in one portion and the bright yellow suspension was heated to a gentle reflux for 3 h. The solution was cooled to 0 °C and filtered. The insoluble material was washed with ether (2 × 250 mL) and the combined filtrate was extracted with 10% aqueous NaOH (750 mL, 500 mL). After cooling the aqueous layer to 0 °C, it was carefully acidified with concentrated HCl (pH 2-3). The precipitated product was collected by filtration and washed with water (500 mL). The yellow-orange material was dissolved in ethyl acetate (1000 mL), dried over MgSO4 and activated charcoal (3 g), filtered through a short pad of silica (50 g), and concentrated. The resulting solid was triturated with heptane (500 mL) and filtered to give 2,3,5,6-tetrafluorothioacetanilide (174.9 g, 88%): mp 103-104 °C; Rf 0.67 (50% ethyl acetate in heptane); 1H NMR (DMSO-d6, 300 MHz) δ 11.20 (s, 1 H), 8.00-7.88 (m, 1 H), 2.66 (s, 3 H). Anal. (C8H5F4NS) C, H, N, S. Step 3: 4,5,7-Trifluoro-2-methylbenzothiazole (4). A flame-dried, 5000 mL round-bottomed flask equipped with overhead stirrer was charged with sodium hydride (15.9 g, 0.66 mol) and diluted with anhydrous toluene (3000 mL, 0.2 M). The suspension was cooled to 0 °C, and treated with 2,3,5,6tetrafluorothioacetanilide (134 g, 0.60 mol) in one portion. The solution was warmed to room temperature over 1 h and then

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heated to a gentle reflux. After 30 min, dimethylformamide (400 mL) was carefully added and the mixture was stirred for an additional 2 h. The solution was cooled to 0 °C and added to ice-water (2000 mL). The solution was extracted with ethyl acetate (1500 mL) and washed with saturated aqueous NaCl (1000 mL). The organic layer was concentrated to dryness, diluted with heptane, and successively washed with water (300 mL) and saturated aqueous NaCl (1000 mL). The organic layer was dried over MgSO4, filtered, and concentrated to give 4,5,7trifluoro-2-methylbenzothiazole (116.8 g, 96%) as a light brown solid: mp 91-92 °C; Rf 0.56 (30% ethyl acetate in heptane); 1 H NMR (DMSO-d6, 300 MHz) δ 7.76-7.67 (m, 1 H), 2.87 (s, 3 H). Anal. (C8H4F3NS) C, H, N, S. Step 4: 2-Amino-3,4,6-trifluorothiophenol Hydrochloride (5). A solution of 4,5,7-trifluoro-2-methylbenzothiazole (25.0 g, 123 mmol) in ethylene glycol (310 mL, 0.4 M) and 30% aqueous NaOH (310 mL, 0.4 M) was degassed using a nitrogen stream then heated to a gentle reflux (125 °C) for 3 h. The solution was cooled to 0 °C and acidified to pH 3-4 using concentrated HCl (appox. 200 mL). The solution was extracted with ether (750 mL) and washed with water (200 mL). The organic layer was dried over Na2SO4, filtered, and treated with 2,2-di-tert-butyl-4-methylphenol (0.135 g, 0.5 mol %). After concentrating to dryness, the crude product was dissolved in anhydrous methanol (200 mL) and treated with an HCl solution in 1,4-dioxane (37 mL, 4 N, 148 mmol). The resulting mixture was concentrated to dryness, triturated with isopropyl ether (100 mL), and filtered to give 2-amino-3,4,6-trifluorothiophenol hydrochloride (19.3 g, 73%) as a light brown solid that was used without further purification: mp 121-124 C; Rf 0.43 (30% ethyl acetate in heptane). Anal. (C6H5ClF3NS) C, H, N, S. Step 5: 3-Cyanomethylindole-N-acetic Acid, Ethyl Ester (7). Under an atmosphere of nitrogen, a solution of 3-indolyl acetonitrile (25.0 g, 160 mmol) in dry acetonitrile (530 mL, 0.3 M) was treated with sodium hydride (95%, 4.2 g, 168 mmol) and stirred for 30 min. Ethyl bromoacetate (21.3 mL, 192 mmol) was added in a dropwise manner over 10 min and the solution was stirred at room temperature for 16 h. After concentrating under reduced pressure, the resulting residue was dissolved in ethyl acetate and washed with saturated aqueous NaCl. The organic extracts were dried over MgSO4, filtered, and concentrated. The crude product was recrystallized from heptane and ethyl acetate to give the target compound as a white crystalline solid (19 g, 49%): mp 98-99 °C; Rf 0.29 (30% ethyl acetate in heptane); 1H NMR (DMSOd6, 300 MHz) δ 7.59 (dd, J1 ) 7.8 Hz, J2 ) 0.6 Hz, 1 H), 7.40 (dd, J1 ) 8.1 Hz, J2 ) 0.6 Hz, 1 H), 7.36 (s, 1 H), 7.18 (b t, J ) 7.2 Hz, 1 H), 7.10 (b t, J ) 7.2 Hz, 1 H), 5.12 (s, 2 H), 4.14 (q, J ) 7.2 Hz, 2 H), 4.06, (s, 2 H), 1.20 (t, J ) 7.2 Hz, 3 H); LRMS calcd for C14H14N2O2 242.3, found 243.0 (M + 1)+. Anal. (C14H14N2O2) C, H, N. Step 6: 3-(4,5,7-Trifluorobenzothiazol-2-yl)methylindole-N-acetic Acid, Ethyl Ester (8). Under a nitrogen atmosphere, a solution of 3-acetonitrileindole-N-acetic acid, ethyl ester (11.0 g, 45.4 mmol) and 2,6-di-tert-butyl-4-methylphenol (BHT, 10 mg) in anhydrous trifluoroethanol (90 mL, 0.5 M) was treated with 2-amino-3,4,6-trifluorothiophenol hydrochloride (12.7 g, 59.0 mmol) and heated to a gentle reflux for 16 h. After cooling to room temperature, the solution was concentrated under reduced pressure, diluted with ethyl acetate, and washed with 2 N HCl and saturated aqueous NaCl. The organic layer was dried over MgSO4, filtered, and concentrated. Purification by MPLC (10-50% ethyl acetate in heptane, 23 mL/min, 150 min) to give 3-(4,5,7-trifluorobenzothiazol-2-yl)methylindole-N-acetic acid ethyl ester (10.8 g, 59%) as a white crystalline solid: mp 110-111 °C; Rf 0.41 (30% ethyl acetate in heptane); 1H NMR (DMSO-d6, 300 MHz) δ 7.74-7.66 (m, 1 H), 7.54 (d, J ) 7.8 Hz, 1 H), 7.46 (s, 1 H), 7.40 (d, J ) 8.1 Hz, 1 H), 7.15 (br t, J ) 6.9 Hz, 1 H), 7.04 (br t, J ) 7.8 Hz, 1 H), 5.14, s, 2 H), 4.66 (s, 2 H), 4.14 (q, J ) 7.2 Hz, 3 H); LRMS calcd for C20H15F3N2O2S 404.4, found 405.0 (M + 1)+. Anal. (C20H15F3N2O2S) C, H, N, S.

Step 7: 3-(4,5,7-Trifluorobenzothiazol-2-yl)methylindole-N-acetic Acid (9). A solution of 3-(4,5,7-trifluorobenzothiazol-2-yl)methylindole-N-acetic acid ethyl ester (5.91 g, 14.6 mmol) in 1,2-dimethoxyethane (73 mL, 0.2 M) was cooled to 0 °C and treated with aqueous NaOH (1.25 N, 58 mL, 73.1 mmol) in a dropwise manner over 15 min. After the addition was complete, the solution was stirred for an additional 30 min, acidified to pH 3 with 2 N HCl, and concentrated under reduced pressure. The residue was dissolved in ethyl acetate (200 mL) and washed with saturated aqueous NaCl (30 mL). The organic extract was dried over Na2SO4, filtered, and concentrated. The resulting material was stirred as a suspension in heptane, filtered, and dried to give 3-(4,5,7-trifluorobenzothiazol-2-yl)methylindole-N-acetic acid (5.38 g, 98%) as a pale yellow solid: mp 177-178 °C; Rf 0.44 (20% methanol in dichloromethane); 1H NMR (DMSO-d6, 300 MHz) δ 7.747.65 (m, 1 H), 7.53 (d, J ) 7.5 Hz, 1 H), 7.46 (s, 1 H), 7.40 (d, J ) 8.1 Hz, 1 H), 7.15 (b t, J ) 6.9 Hz, 1 H), 7.03 (b t, J ) 7.2 Hz, 1 H), 5.03 (s, 2 H), 4.65 (s, 2 H); LRMS calcd for C18H11F3N2O2S 376.4, found 375.0 (M - 1)-. Anal. (C18H11F3N2O2S) C, H, N, S. Examples with a phenyl substituent coupled to the 5- and 6-positions of the indole ring (compounds 37 and 42, respectively) were prepared from the corresponding bromides via a Suzuki coupling.27 The following example is provided to illustrate the general method. Preparation of 6-Phenylindole. A solution of 6-bromoindole (2.0 g, 10.20 mmol) in anhydrous toluene (20 mL) under a nitrogen atmosphere was treated with Pd[P(Ph3)]4 (10 mol %). After stirring for 30 min, a solution of phenylboronic acid (1.87 g, 15.30 mmol) in anhydrous ethanol (10 mL) and saturated aqueous NaHCO3 (6 mL) was added and the biphasic mixture was heated to reflux for 24 h. After cooling to room temperature, the mixture was added to a saturated aqueous NaCl solution and extracted with ethyl acetate (2×). The combined organic layers were dried over MgSO4, filtered, and concentrated in vacuo. The resulting residue was purified by flash column chromatography (50% dichloromethane in heptane) to give the desired material as a white powder (900 mg, 45%): 1H NMR (DMSO-d6, 300 MHz) δ 11.15 (br s, 1H), 7.587.66 (m, 4H), 7.41-7.47 (m, 2H), 7.36 (m, 1H), 7.26-7.31 (m, 2H), 6.42 (m, 1H). The 5-morpholino and 5-phenoxy examples (15 and 16) were prepared from 4-fluoro-2-methylnitrobenzene 10 as illustrated in Scheme 3 and described in detail below. Preparation of 5-Morpholino-2-nitrotoluene (11). A solution of 5-fluoro-2-nitrotoluene (5.11 g, 32.9 mmol), morpholine (4.31 mL, 49.4 mmol), and K2CO3 (6.83 g, 49.4 mmol) in anhydrous DMSO (80 mL) was heated to 80 °C for 24 h. After cooling to room temperature, deionized water was added and the resulting mixture was extracted with ethyl acetate (3 × 50 mL). The organic layer was washed with saturated aqueous NaCl (100 mL), dried over MgSO4, filtered, and concentrated. The remaining solid was triturated in heptane (200 mL) and filtered to give the desired material (7.10 g, 97%) as a yellow powder: Rf 0.40 (25% ethyl acetate in heptane): 1 H NMR (DMSO-d6, 300 MHz) δ 7.96 (d, J ) 9.9 Hz, 1 H), 8.85-8.88 (m, 2 H), 3.70 (t, J ) 5.0 Hz, 4 H), 3.35 (t, J ) 5.0 Hz, 4 H), 2.53 (s, 3 H). Preparation of 5-Morpholinoindole (13). Under an atmosphere of nitrogen, a solution of 5-morpholinyl-2-nitrotoluene (7.0 g, 31.5 mmol) in DMF (100 mL) was treated with dimethylformamide dimethyl acetal (4.81 mL, 36.2 mmol) and pyrrolidine (2.62 mL, 31.5 mL), and heated to 100 °C for 12 h. After cooling to room temperature, the mixture was concentrated to give the crude enamine as a brick-red solid. The intermediate enamine was dissolved in ethyl acetate (200 mL) and added to a precharged Parr bottle containing 10% Pd/C (600 mg) in ethyl acetate (40 mL). The mixture was hydrogenated on a Parr shaker at 55 psi for 2.5 h. After the hydrogenation was complete, the reaction mixture was filtered through a Celite plug and washed with ethyl acetate. The resulting filtrate was concentrated and purified by flash column chromatography (50% heptane in ethyl acetate) to give



2.0 g (31% over 2 steps) of the desired indole as a off-white powder: Rf 0.30 (10% methanol in chloroform); 1H NMR (DMSO-d6, 300 MHz) δ 10.77 (br s, 1 H), 7.24 (s, 1 H), 7.187.20 (m, 1 H), 6.97 (d, J ) 1.8 Hz, 1 H), 6.81 (dd, J1 ) 8.7 Hz, J2 ) 2.1 Hz, 1 H), 6.25 (dd, J1 ) 3.0 Hz, J2 ) 1.8 Hz, 1 H), 3.7 (t, J ) 4.50 Hz, 4 H), 2.96 (t, J ) 4.50 Hz, 4 H). Preparation of 5-Phenoxy-2-nitrotoluene (12). A solution of phenol (12.16 g, 0.129 mol) in anhydrous DMSO was treated with K2CO3 (17.88 g, 0.129 mol) and stirred at room temperature for 15 min. 5-Fluoro-2-nitrotoluene (13.38 g, 0.086 mol) was added and the resultant mixture was heated to 80 °C for 12 h. After cooling to room temperature, the mixture was poured into deionized water (100 mL) and extracted with ethyl acetate (2 × 100 mL). The combined organic layers were washed with a saturated aqueous NaCl, dried over MgSO4, filtered, and concentrated. The resulting residue was purified by flash column chromatography (0-10% ethyl acetate in heptane) to give 5-phenoxy-2-nitrotoluene as a yellow crystalline solid (12.50 g, 63%). Rf 0.60 (15% ethyl acetate in heptane); 1 H NMR (DMSO-d6, 300 MHz) δ 8.05 (d, J ) 9.0 Hz, 1 H), 7.44-7.47 (m, 2 H), 7.23-7.29 (m, 1 H), 7.12-7.16 (m, 2 H), 7.04 (d, J ) 2.7 Hz, 1 H), 6.90 (dd, J1 ) 9.0 Hz, J2 ) 2.7 Hz, 1 H), 2.51 (s, 3 H). Preparation of 5-Phenoxyindole (14). A solution of 5-phenoxy-2-nitrotoluene (10.03 g, 0.0428 mol) in anhydrous DMF was treated with N,N-dimethylformamide dimethyl diacetal (6.73 mL, 0.0508 mol) and pyrrolidine (3.63 mL, 0.0438 mol) and heated to 110 °C for 2.5 h. After cooling to room temperature, the mixture was diluted with ethyl acetate (500 mL) and washed successively with deionized water (500 mL) and saturated aqueous NaCl. The remaining organic layer was dried over MgSO4, filtered, and concentrated. The crude intermediate was dissolved in glacial acetic acid (250 mL) and warmed to 85 °C. Zinc ribbon (24.62 g, 0.377 mol) was added to the solution over 30 min and the mixture was heated to reflux for 4 h. After cooling to room temperature, the mixture was neutralized with saturated aqueous NaHCO3 and extracted with diethyl ether (3 × 300 mL). The combined organic layers were washed with saturated aqueous NaCl, dried over MgSO4, filtered, and concentrated. The resulting residue was purified by flash column chromatography (0-30% ethyl acetate in heptane) to give the 5-phenoxyindole as a white crystalline powder (3.1 g, 34%, 2 steps): Rf 0.50 (30% ethyl acetate in heptane); 1H NMR (DMSO-d6, 300 MHz) δ 11.12 (br s, 1 H), 7.48 (s, 1 H), 7.30-7.38 (m, 1 H), 7.25-7.29 (m, 2 H), 7.17 (d, J ) 2.7 Hz, 1 H), 6.89-7.02 (m, 1 H), 6.86-6.88 (m, 2 H), 6.80 (dd, J1 ) 8.7 Hz, J2 ) 2.4 Hz, 1 H), 6.37 (m, 1 H). Pharmacology. Enzyme Preparation. Recombinant human aldose reductase (hALR2) with a His6-tag was overexpressed in E. coli using a T7-based expression system and purified using a metal affinity column.28 The enzyme was precipitated in 3.5 M ammonium sulfate, 10 mM Tris-HCl buffer, and 1 mM DTT. The enzyme was centrifuged at 10 000 rpm for 10 min at 4 °C and the pellet was resuspended to an approximate concentration of 10-15 mg of protein/mL in (NH4)2SO4 in sodium phosphate, pH 6.6. Protein content was determined using Coomassie Protein Assay reagent (Pierce, Rockford, IL). Aliquots were made and the enzyme was stored at -40 °C. Recombinant human aldehyde reductase (hALR1), supplied by Dr. K. Gabbay (Baylor College of Medicine, Houston, TX), was prepared as previously described.29 The enzyme was stored in 5 mM sodium phosphate, pH 7.4, and 0.1 mM EDTA at 4 °C. Protein content was determined using Coomassie Protein Assay reagent (Pierce, Rockford, IL). Enzymatic Assays. The method to examine the inhibition of aldose reductase (ALR2) was similar to those methods used by numerous other laboratories.30 Each assay is done in triplicate. Enzymatic activity was measured by monitoring the disappearance rate of NADPH. Compound or solvent is placed in the microplate well to which buffer is added and mixed for 1 min. Enzyme is added to the well and incubated at 37 °C and shaken for 10 min. DL-Glyceraldehyde and NADPH are

added to initiate the reaction. The microplate is placed in the plate reader for 30 min at 340 nm and read at 2 min intervals. The reaction contents in a final volume of 300 µL are 6.6% w/v (NH4)2SO4, 33 mM NaH2PO4 (pH 6.6), 0.11 mM NADPH, 4.7 mM DL-glyceraldehyde, 0.59 µg of enzyme, 1% DMSO, and compound. Percent inhibition is calculated on the basis of the enzyme activity in the presence or absence of compound. The IC50 is calculated using SAS release 6.10 and a four-parameter logistic regression of data. The inhibition of aldehyde reductase (ALR1) was assessed using a standard spectrophotometric assay.31 The procedure is similar to the method described above for determining ALR2 inhibition. The reaction contents in a final volume of 300 µL are 33 mM NaH2PO4 (pH 6.6), 0.11 mM NADPH, 4.7 mM DL-glyceraldehyde, 0.28 µg of enzyme, 1% DMSO, and compound. STZ-Induced Diabetic Rat Model (General). Male Sprague-Dawley rats (Harlan, Indianapolis, IN), weighing 150 g, are allowed to acclimate for 1 week and placed on a standard diet (7012, Harlan Teklad certified LM-485 mouse/ rat, Madison, WI). Water is supplied ad libitum. After a 24 h fast, 30 mg/kg STZ (S01230 Sigma, St. Louis, MO) prepared in 0.03 M citrate buffer, pH 4.5, is administered ip. Control animals receive citrate buffer. Four days after receiving STZ, tail blood glucose is measured using a One Touch II blood glucose meter (Lifescan, Inc., Johnson & Johnson, Milpitas, CA). Diabetic rats whose blood glucose levels are g300 mg/dL are randomized according to blood glucose levels into diabetic-control and diabetic-treatment groups. Five-Day STZ-Induced Diabetic Rat Screening Model. Using the STZ-induced diabetic rat model (as described above), each study includes nondiabetic controls (n ) 5), diabetic controls (n ) 10), and diabetic rats for each drug treated group (n ) 7). Beginning on day 4 after induction of diabetes with STZ, the compound, suspended in a vehicle of 2% Tween 80 in saline, is administered po consecutively for 5 days. Nondiabetic and diabetic controls receive vehicle during this period. After the initial dose of compound, animals continue to receive the standard diet for the remainder of the study. After receiving the final dose of compound, the food is removed. Four hours after the final dose is administered, the rats are euthanized by asphyxiation with CO2 and sciatic nerves and lens are removed, weighed, frozen on porcelain plates on dry ice, and stored at -80 °C for sorbitol analysis. Tissue Sorbitol Analysis. Sciatic nerve sorbitol levels are measured by a standard enzymatic method that uses sorbitol dehydrogenase.32 A D-sorbitol kit purchased from Boehringer Mannheim (670 057 Indianapolis, IN) was adapted to a microplate format. Tissues are homogenized in ice-cold 0.2% perchloric acid in methanol using a DUALL tissue grinder and centrifuged at 10 000 rpm for 10 min at 4 °C. A sampling of the supernatant is transferred to a microplate. A reaction mixture combining buffer, diaphorase, NAD+, and iodonitrotetrazolium chloride (INT) is added to each sample followed by an initial absorbance reading at 490 nm. Sorbitol dehydrogenase (SDH) is added to initiate the reaction and the plate is mixed for 90 s on an orbital shaker. Plates are incubated at room temperature in the dark for 30 min and the final absorbance reading at 490 nm is made. The reaction contents in a final volume of 255 µL at pH 8.7 are 14 mM potassium phosphate, 94 mM triethanolamine, 0.94% Triton X-100, 0.16 U diaphorase, 1.66 mM NAD, 0.034 mM INT, and 0.82 U SDH. Sciatic nerve sorbitol content is calculated as nmol of sorbitol/mg of wet tissue weight by taking the difference in the final and initial absorbance readings and comparing to a sorbitol standard curve. Percent inhibition is calculated by comparing treatment groups to both the nondiabetic and diabetic control groups within the study as described by the equation: (t - n)/(d - n) where n ) nondiabetic control, d ) diabetic control, and t ) drug-treated diabetic control. The ED50 was calculated using Sigma plot, version 4.0, using a Hill slope four-parameter logistic equation.

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Motor Nerve Conduction Velocity (MNCV). Using the STZ-induced diabetic rat model (as described above), each study includes nondiabetic controls, diabetic controls, and diabetic rats for each drug treated group (prevention study, n ) 10 per group; intervention study, n ) 12 per group). MNCV was measured in the sciatic/tibialis-interosseous system by the method of Sharma.33 The rats are anesthetized with 4% isoflurane in oxygen (1 L/min) for induction, 2.5% for controls, and 2% for diabetics. After the initial anesthetization, flow is reduced to 0.5 L/min for maintenance. Near-nerve/subcutaneous temperature was recorded and maintained at 37 °C using a wire thermister and thermometer (YSI Tele-thermometer, Model 4000A, Yellow Springs Instrument Co. Inc.), a Heat Therapy System (Baxter K-MOD 100 pump and Duo Therm pad, Baxter Healthcare Corp, Deerfield, IL), and a source of radiant heat. The left sciatic/tibial nerve was stimulated at the sciatic notch and Achilles tendon by square wave pulses (duration ) 0.1 m/second, intensity ) 20 V) and compound muscle action potentials were recorded from the interosseus muscle using needle electrodes (Sapphire II, 4ME electrophysiological system, Teca Corp, Pleasantville, NY). An average of 10 action potentials traces was recorded for each stimulation point. The latencies to inflection (takeoff) of the compound muscle action potential were measured for each stimulation point, and the nerve length was measured after dissection. MNCV ) nerve length (mm)/[sciatic notch latency - Achilles tendon latency (m/s)]. When the measurements were completed, rats were euthanized and both sciatic nerves were removed, weighed, and frozen for polyol determination by capillary gas chromatography. Data from all groups were compared using one-way analysis of variance and the Tukey post-test (Graph Pad Prism, version 2). Cataract Assessment by Slit-Lamp Biomicroscopy. Using the STZ-induced diabetic rat model (as described above), each study includes nondiabetic controls, diabetic controls, and diabetic rats for each drug treated group (n ) 8 per group). Assessment of cataract progression was conducted by slit-lamp biomicroscopy (American Optical Slit-lamp biomicroscopemodel 11665, SN AP5813) in a dark room. Two drops of 1% mydriacyl (Alcon Inc., Ft. Worth, TX) were administered to cause full dilation of the pupil. Rats were maintained anestheized by an inhalation system, which provided continuously delivered isoflurane (Ohmeda Isotec 3, BOC Health Care, Yorkshire, England). While under anesthesia, rats were positioned in front of the slit-lamp on an adjustable height platform. In vivo photodocumentation was conducted by using a reversal-coupling ring for reverse mounting of a 28 mm f/2.8 lens on a camera body (Pentax). The camera was placed on a minitripod with the camera lens maintained at the largest aperture. Color reversal film (AGFAchrome RSX 50) was used to capture the images. Triglyceride Determination.34 The assay used to demonstrate the compounds ability to lower elevated serum triglyceride levels is described as follows. Using the STZinduced diabetic rat model (as described above), the study includes a nondiabetic control group (n ) 5), an untreated diabetic control group (n ) 7), and drug treated diabetic group (n ) 7). The daily dosages are administered by gavage as a single dose of the test compound in 2% Tween 90 in saline each day. The nondiabetic and diabetic control groups are administered vehicle. After the final dose, all groups of animals are fasted for 4 h and anesthetized with CO2, and blood is collected by cardiac puncture into EDTA tubes. The plasma is separated from the red blood cells by centrifugation. Plasma triglyceride levels are quantitated on an automated COBAS chemistry system utilizing the Roche reagent for triglycerides (Cat. #44119).35 Plasma samples are diluted with 1:1 saline and run in quadruplicate. Data are given as mean ( SEM. Statistical comparisons between groups employed a one-tailed t-test and demonstrated statistical significance with p > 0.01. Pharmacokinetics and Tissue Distribution. In an unpaired study, 12 male Sprague Dawley rats received a 10 mg/

Van Zandt et al. kg dose of [14C]lidorestat, by either the intravenous or oral route (n ) 6 per group). Blood was drawn at 0.083 (iv only), 0.25, 0.5, 1, 2, 4, 7, 12, and 24 h following each dose via a surgically implanted jugular vein cannula. The corresponding plasma was harvested and analyzed for total radioactivity and unchanged drug using a scintillation counter and LC/MS/MS. Pharmacokinetic parameters were calculated using modelindependent pharmacokinetics. Whole-body autoradiography was used to determine drug concentrations in specific tissues. X-ray Crystallography. Overproduction and Purification of Recombinant Human ALR2. The ORF of the human ALR2 gene (Accession GenBank/embl Data Bank Number J05017) was amplified by PCR from its cDNA and cloned into the T7 RNA polymerase-based vector pET15b (Novagen). Expression of the (His)6-human ALR2 in the E. coli strain BL21(DE3) (Novagen) is induced by IPTG (Euromedex) during a 3 h culture at 37 °C. The pellet from a 4 L culture was disrupted by sonication and centrifuged at 4 °C. The supernatant was applied on a Talon metal affinity column (Clontech). After thrombin cleavage of the hexahistidine extension, the detagged protein was then loaded on a DEAE Sephadex A-50 column (Pharmacia) and eluted with a NaCl gradient. Crystallization Trials and Preliminary Crystallographic Analysis. All trials were carried out in Linbro 24well tissue culture plates (Flow Laboratories) using the hanging-drop vapor diffusion method. The enzyme was dialyzed against 50 mM ammonium citrate, pH 5.0. The drops of a final volume of 12 mL contained 15 mg/mL of human ALR2 with 2 equiv of NADP+ (Sigma) and 5% of PEG 6000; they were equilibrated against a well containing 120 mM ammonium citrate, pH 5.0, 20% PEG 6000 and the same concentration of NADP+ as above. Crystals were grown at 4 °C and reached the size of 0.5 × 0.4 × 0.4 mm. Soaking with the Inhibitor. Crystals of the ALR2inhibitor complex were obtained by soaking of the native crystals with a solution containing 2 mg/mL of inhibitor dissolved in the mother liquor containing 120 mM ammonium citrate, pH 5.0, and 25% of PEG 6000. The soaking time was 3 weeks. Data Collection and Refinement. The diffraction analysis was performed using a rotating anode laboratory source and an image plate, and the data were treated with the HKL package.36 The inhibitor-soaked crystals diffracted to 1.04 Å and were isomorphous to the native crystals. The native structure was used as a model for the molecular replacement; after a rigid body minimization, the inhibitor was placed in the electron density shown by a Fourier difference map. The model of the complex was used as starting point of the refinement, which included a rigid-body refinement step, a slow-cooling step, a Powell minimization, and a temperaturefactor refinement. Water molecules were then placed in a difference map and minimization was performed as above without putting restraints on the water molecules; bulk solvent correction was also applied. This was followed by anisotropic refinement and H-atom addition. The programs O,37 X-PLOR,38 and SHELXL39 were used for model building and refinement. Diffraction experiments were conducted on one crystal at the X-ray beam of the undulator line W32 at LURE. The atomic coordinates and structure factors of the hALR2NADP+-9 (lidorestat) complex have been deposited in the Protein Data Bank, accession code 1Z3N.

Acknowledgment. This work was supported, in part, by the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Sante´ et de la Recherche Me´dicale, and the Hoˆpital Universitaire de Strasbourg (H.U.S.). We thank the personnel of the LURE for their help in data collection. Supporting Information Available: 1H NMR spectra and combustion analysis. This material is available free of charge via the Internet at http://pubs.acs.org.



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JM0492094

(Lidorestat) and Congeners as Highly Potent and ... - ACS Publications

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