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[1,2,4]Triazino[4,3- a ] b e n z i m i d a z o l e A c e t i c A c i d D e r i v a t i v e s : A N e w C l a s s o f

S e l e c t i v e A l d o s e R e d u c t a s e I n h i b i t o r s

Federico Da Settimo,*,† Giampaolo Primofiore,† Antonio Da Settimo,† Concettina La Motta,† Sabrina Taliani,†

Fra ncesca Simorini,† Ettore Novellino,‡ Giovan ni Greco,‡ Antonio Lavecchia,‡ and Enr ico Boldrini§

Dipartimento di Scienze Farmaceutiche, Universita di Pisa, Via Bonanno 6, 56126 Pisa, Italy, Dipartimento di Chimica

Farm aceutica e T ossicologica, Un iversita di Na poli “Federico II”, Via Domenico Montesano, 49, 80131 N apoli, Italy, an d

Farmigea S .p.A., Via Carm ignani 2, 56127, Pisa, Italy

Received May 8, 2001

Acetic a cid deriva tives of [1,2,4]tria zino[4,3-a]benzimidazole (TBI) were synt hesized an d test edin vitro and in vivo as a novel class of aldose reductase (ALR2) inhibitors. Compound 3, (10-benzyl[1,2,4]triazino[4,3- a]benzimidazol-3,4(10 H )-dion-2-yl)acetic acid, displayed the highestinhibitory activity (IC50 ) 0.36 µM) and was found to be effective in preventing cataractdevelopment in severely galactosemic ra ts wh en a dministered as a n eyedrop solution. All thecompounds investigated were selective for ALR2, since none of them inhibited appreciablyaldehyde reductase, sorbitol dehydrogenase, or glutathione reductase. The activity of 3 wa slowered by inserting various substituents on the pendant phenyl ring, by shifting the aceticacid moiety from t he 2 to th e 3 position of th e TBI nu cleus, or by cleaving t he TBI system toyield benzimidazolylidenehydrazines as open-cha in an alogues. A th ree-dimensional model of hu ma n ALR2 was built, ta king into account t he conform at iona l cha nges induced by th e bindingof inhibitors such as zopolrestat, to simulate the docking of 3 into the enzyme active site. Thetheoretical binding mode of 3 was fully consistent with th e stru ctu re-activity relationships inth e TBI series a nd will guide th e design of novel ALR2 inhibitors.

I n t r o d u c t i o n

Despite r ecent advan ces in t he chemistry a nd m olec-ular pha rma cology of ant idiabetic drugs, diabetes stillremains a life-threatening disease. Tissues capable of insulin-independent glucose uptake develop structur aland functional damage (retinopathy, nephropathy, cata-ract, keratopath y, neuropathy, and a ngiopathy) in morethan 50% of diabetics. A strict glycemic control prevents

or at least delays the onset of such complications.However, because close control is difficult to maintainand becau se th ere is a par allel risk of severe h ypogly-cemia a nd obesity in inten sive insulin-treated patients,considerable efforts have been made to find novel,effective antidiabetic a gents acting by mechanismsindependent of the control of blood glucose.1-3

Several experimental data have revealed a l inkbetween glucose metabolism via the polyol pathwayan d long-term diabetic complicat ions. Aldose redu ctase(alditol/NADP + oxidoredu ctas e, EC 1.1.1.21, ALR2), thefirst enzyme of the pathway, catalyzes the NADPH-depend ent redu ction of glucose to sorbitol.2,4 Therefore,inhibit ion of ALR2 has become an attractive thera-peutic stra tegy, an d a large nu mber of ALR2 inhibitors(ARIs) have been identified to dat e. Curr ently kn ownARIs can be divided into t hr ee classes: (i) acetic acidderivatives, including a lrestat in, tolrestat, zopolresta t,and epalresta t; (ii) cyclic imides s uch as sorbinil; (iii)flavonoids, whose prototype is quercetin (Chart 1).5

Stru ctura l requirement s for ALR2 inhibitory activity area pla na r cyclic moiety an d an acidic function ionized atthe ALR2 active site.6-9

One of the most important requirements of clinicallyuseful ARIs is ALR2 selectivity over closely relatedenzymes such as aldehyde reductase (ALR1), sorbitoldehydrogenase (SD), and glutathione reductase (GR).ALR1 belongs to the aldo-keto reductase family likeALR2 and exhibits the highest homology in structure(51% sequence identity) an d activity with ALR2.10 SDis the second enzyme in the polyol pathway, and i ts

* To whom correspondence should be addressed. Ph one: 39 50500209. Fa x: 39 50 40517. E-ma il: fsettim o@farm .unipi.it.

† Univers ita di Pisa .‡ Univer sita “Feder ico II” di N apoli.§ Far migea S.p.A.

Char t 1. Well-Known ALR2 Inhibitors

4359 J. Med. Chem. 2001, 44 , 4359-4369

10.1021/jm0109210 CCC: $20.00 © 2001 American Chemical SocietyPublished on Web 11/09/2001



inhibition increases sorbitol levels, thus reducing theeffects of ALR2 inh ibition.11 GR ma inta ins p hysiologicallevels of glutathione, which in turn prevents glycosyl-ation of cellular protein and protects against oxidativestress. The int ra cellular accumu lation of oxidized glu-tathione, result ing from inhibit ion of GR, leads to astru ctura l modification of ALR2 tha t r educes its sensi-tivity to ARI action.12,13

In addition to problems r elated to selectivity a monghomologous enzymes, ARI-based therapy suffers frommany l imitations due to pharmacokinetic problems,reversibility of diabetic neur opathy, adverse reactions,and poor reproducibility of clinical measurements.14

Only epalrestat is on t he J apanese mar ket nowadays.For these reasons, there is still a need to identify anddevelop clinically effective an d well-tolerat ed ARIs.

This paper describes th e synth esis and the biologicalevaluation of novel ARIs featuring an acetic acid residueat the 2 or 3 posit ion of the [1,2,4]triazino[4,3-a]-benzimida zole (TBI) nucleus (compoun ds 1-11 or 23 -

25 , respectively). The TBI system p roved to be a suita blescaffold for s evera l compounds recent ly described by usas selective l igands at the benzodiazepine15 a n d A1

adenosine16 receptors. An overlay of molecular modelsof 3 and 25 on t he ALR2-boun d conform at ion of zopol-restat 6 gave support to our project because it showed asat isfactory mat ch of th e common acetic acid chain, th ephth alazine/TBI systems a nd the benzothiazole/phenylrings (Figure 1). To better delineate the structure-

activity rela tionsh ips (SARs) of th is new class of ARIs,the benzimidazolylidenehydrazine (BIH) derivatives12 -22 , intermediates in th e synthesis of 1-11 , werelikewise tested.

Compound 3, the most active ALR2 inhibitor amongthose examined, and i ts i-propyl ester 73 were alsoinvestigat ed in vivo for th eir ability to preven t cata ra ctdevelopment in galactosemic r ats. Finally, dockingsimulations of 3 into th e ALR2 active site were car riedout to rationalize the SARs observed and to guide,perspectively, th e design of new an alogues.

C h e m i s t r y

The title compounds 1-11 were synthesized as out-lined in Scheme 1. The substituted 1-alkyl-2-chloro-benzimidazoles 27 -36 (Table 1) were pr epar ed in goodyields from the commer cially a vailable 2-chlorobenz-imidazole 26 by reaction with the appropriate alkylhalide in t he pr esence of sodium h ydride.17 Treatmentof 26 -36 with hydrazine hydrate18 resul ted in thehydrazino derivatives 37 -47 (Table 1), which werecyclized to the corresponding triazinobenzimidazoles48 -58 (Table 2) by refluxing with an equimolar am ountof diethyl oxalate in ethanolic solution.19 Reaction of

48-

58 with eth yl bromoacetat e in refluxing acetone, inthe pr esence of an hydrous potassium carbonate,11 gavethe ester derivatives 59 -69 (Table 3). These werehydrolyzed by aqueous sodium hydroxide to give, afteracidification, the dicarboxylic acids 12 -22 (Table 4),which underwent thermal cyclization to the triazino-benzimidazole acetic acids 1-11 (Table 5). Altern at ively,the t arget acids 1 -11 could be obtained by hydrolysisof compounds 59 -69 with concentrated hydrochloricacid at 100 °C.

The regioisomer ic acetic acids 23-25 were preparedin accordance with th e synth etic rout e shown in Scheme2. Treatment of the hydrazino derivatives 37 , 38 , and40 with diethyl a cetylenedicar boxylate in refluxing

methanol20

afforded the ethyl esters 70 -72 (Table 6),which by hydrolysis with concentrated hydrochloric acidyielded the desired acids 23 -25 (Table 6).

To improve the ocular bioavailability of carboxylic acid3, the more lipophilic i-propyl ester 73 was also pre-pared. Thus, a mixture of compound 51 with i-propylbromoacetate was refluxed in acetone solution in thepresence of anh ydrous potassium car bonat e to give th etarget ester 73 (see Experimenta l Section).

B i o c h e m i s t r y a n d P h a r m a c o l o g y

Compounds 1-25 were evaluated for their in vitroinhibitory activity against ALR2 as well as againstALR1, SD, and GR. Primary in vitro screening was

F i g u r e 1 . Overlay of 3 (red) and 25 (blue) on the experimen -tally determined ALR2-bound conformat ion of zopolrestat(yellow). The conformat ions of the TBI derivatives wereselected and aligned as descr ibed in the ComputationalChemistry part of the Experimental Section.

S c h e m e 1

4360 J ou rn al of M ed icin al Ch em istry, 2001, V ol. 44, N o. 25 Da S ettim o et al.



condu cted on a water -soluble enzymatic extract pu rifiedfrom rat lenses.21 -24 IC50 values were determined bylinear regression analysis of the log of the concentra-tion -response curve. Compounds 1-25 were also as-

sayed for their abil i ty to inhibit SD25 and two otherenzymes not involved in the polyol pathway, namely,ALR126 and GR.27 Sorbinil,28 tolrestat,29,30 and querce-t in 31 were used as th e reference standards.

Compound 3 and i t s i-propyl ester 73 were investi-gated in vivo for their abil ity to prevent cataractdevelopment in severely galactosemic r ats.28 Theireffectiveness was evalua ted with r espect t o tolrestat asa highly potent reversible inhibitor of lenticular ALR2when topical ly administered to rats fed with 50%

galactose diet.30

R e s u l t s a n d D i s c u s s i o n

B i o l o g i c a l E v a l u a t i o n . Table 7 lists the inhibitoryactivities against ALR2 of compounds 1-25 expressedas IC50 values. Several compounds (6, 7, 9, 10 , 14 , 19 ,20 , and 25 ) exhibited I C50 values in th e low micromolarra nge. The TBI derivative 3 tur ned out th e most potentinhibitor of the series, with an IC 50 value (0.36 µM)similar to that of sorbinil (0.65 µM) and 7-fold worsethan that of tol restat . The act ivi ty was lowered byinserting various substituents on the phenyl ring of 3

to yield 4-10 , as well as r eplacing the benzyl group of 3 with much less l ipophi l ic subst i tuents such as a

T a b l e 1 . Physical Pr operties of Benzimidazole Derivatives 30 , 31 , 34 -36 , 38 , 39 , 41 -47

no. R1 R2 yield (%) recryst solv m p (°C) formula a

30 Cl CH 2C6H 4-4-CH 3 85 pet roleum et her 60-80 119-120 C15H 13ClN2

31 Cl CH 2C6H 4-4-OCH 3 85 AcOE t 96-97 C15H 13ClN2O34 Cl CH 2C6H 4-4-CF 3 64 pet roleum et her 60-80 84-85 C15H 10ClF 3N2

35 Cl CH 2C6H 3-3,4-F2 74 pet roleum et her 60-80 88-89 C14H 9ClF 2N2

36 Cl CH 2C6H 3-2-F -4-Br 97 EtOH 106-107 C14H 9BrClFN 2

38 NHNH 2 CH 3 91 H 2O 148-151 C8H 10N4

39 NHNH 2 CH 2CH 2CH 3 99 H 2O 108-111 C10H 14N4

41 NHNH 2 CH 2C6H 4-4-CH 3 56 H 2O 86 -89 C15H 16N4

42 NHNH 2 CH 2C6H 4-4-OCH 3 96 toluene 83-86 C15H 16N4O43 NHNH 2 CH 2C6H 4-4-Cl 70 H 2O 193-198 (dec) C14H 13ClN4

44 NHNH 2 CH 2C6H 4-4-F 67 H 2O 138-151 (dec) C14H 13FN4

45 NHNH 2 CH 2C6H 4-4-CF 3 88 H 2O 156-160 C15H 13F 3N4

46 NHNH 2 CH 2C6H 3-3,4-F2 90 i-Pr OH 108-111 C14H 12F 2N 4

47 NHNH 2 CH 2C6H 3-2-F -4-Br 85 EtOH 116-120 C14H 12BrFN 4

a Elemental analyses for C, H, N were within (0.4% of the calculated values.

T a b l e 2 . Physical Properties of 10-Alkyl[1,2,4]triazino[4,3- a]benzimidazol-3,4(10 H )-dioneDerivatives 49 -58

no. Ryield(%)

recrystsolv m p (°C) for mu la a

49 CH 3 53 DMF >300 C10H 8N4O2

50 CH 2CH 2CH 3 66 DMF 294-2 95 C12H 12N4O2

51 CH 2C6H 5 74 DMF >300 C16H 12N4O2

52 CH 2C6H 4-4-CH 3 62 DMF >300 C17H 14N4O2

53 CH 2C6H 4-4-OCH 3 46 DMF >300 C17H 14N4O3

54 CH 2C6H 4-4-Cl 42 DMF >300 C16H 11ClN 4O2

55 CH 2C6H 4-4-F 33 DMF 278-2 80 C16H 11FN4O2

56 CH 2C6H 4-4-CF 3 31 DMF 296-2 98 C17H 11F 3N4O2

57 CH 2C6H 3-3,4-F2 81 DMF 210-2 12 C16H 10F 2N4O2

58 CH 2C6H 3-2-F -4-Br 54 DMF 278-2 80 C16H 10BrFN4O2

a Elemental analyses for C, H, N were within (0.4% of thecalculated values.

T a b l e 3 . Physical Properties of Ethyl(10-Alkyl[1,2,4]triazino[4,3- a]benzimidazol-3,4(10 H )-dion-2-yl)acetateDerivatives 59 -69

no. Ryield(%)

recrystsolv m p (°C) for mu la a

59 CH 3 74 Et OH 208-2 09 C14H 14N4O4

60 CH 2CH 2CH 3 90 Et OH 168-1 69 C16H 18N4O4

61 CH 2C6H 5 57 Et OH 211-2 13 C20H 18N4O4

62 CH 2C6H 4-4-CH 3 70 Et OH 195-1 96 C21H 20N4O4

63 CH 2C6H 4-4-OCH 3 90 Et OH 177-1 78 C21H 20N4O5

64 CH 2C6H 4-4-Cl 73 E t OH 218-2 20 C20H 17ClN 4O4

65 CH 2C6H 4-4-F 71 AcOEt 222-2 23 C20H 17FN 4O4

66 CH 2C6H 4-4-CF 3 63 Et OH 243-2 46 C21H 17F3N4O4

67 CH 2C6H 3-3,4-F2 75 Et OH 182-1 83 C20H 16F2N4O4

68 CH 2C6H 3-2-F -4-Br 59 E tOH 206-2 09 C20H 16BrFN4O4

69 CH 2COOEt 39 Et OH 202-2 03 C17H 18N4O4

a Elemental analyses for C, H, N were within (0.4% of thecalculated values.

T a b l e 4 . Physical Properties of (1-Substituted-3-oxalo-1 H ,3 H -benzimidazol-2-ylidenehydrazino)aceticAcids 12 -22

no. Ryield(%)

recrystsolv

cycliz tempa

(°C) formula b

12 CH 3 62 H 2O 231-235 C12H 12N4O5

13 CH 2CH 2CH 3 95 H 2O 167-168 C14H 16N4O5

14 CH 2C6H 5 85 H 2O 238-250 C18H 16N4O5

15 CH 2C6H 4-4-CH3 64 H 2O 205-215 C19H 18N4O5

16 CH 2C6H 4-4-OCH 3 61 MeOH 193-196 C19H 18N4O6

17 CH 2C6H 4-4-Cl 57 AcOEt 197-200 C18H 15ClN 4O5

18 CH 2C6H 4-4-F 44 H 2O 200-202 C18H 15FN 4O5

19 CH 2C6H 4-4-CF3 4 1 a ce t on e 2 05-206 C19H 15F3N4O5

20 CH 2C6H 3-3,4-F2 76 AcOE t 215-216 C18H 14F2N4O5

21 CH 2C6H 3-2-F -4-Br 70 AcOE t 185-190 C18H 14BrFN4O5

22 CH 2COOH 77 H 2O 178-185 C13H 12N4O7

a Temperat ure at which compounds cyclize to triazinobenzimi-dazole derivatives 1-11 . b Elemental a nalyses for C, H, N werewithin (0.4% of the calculat ed values.

A ld ose R ed uctase In hibitors J ou rn al of M ed icin al Ch em istry, 2001, V ol. 44, N o. 25 4361



methyl (1), an n -propyl (2), or a n a cetic acid group (11 ).Taken together, these data suggest that a small-sizehydrophobic pocket within the enzyme active site isavailable to th e benzyl moiety of 3. The BIH derivat ives12 -22 were genera lly less active than th eir corr espond-ing closed-chain analogues TBIs 1-11 , th e only excep-t i on t o t h i s t rend be i ng t he pa i r 19 / 8. The higherflexibility of BIHs compared with their TBI counterpartsma y explain th e difference in activity observed betweenthe two series.

The shift of the acetic acid moiety from position 2 to

position 3 of the TBI system, to give compounds 23-

25 , was tolerated (compare 1 vs 24 ) or detrimental(compare 3 vs 25). Evidently, the TBI-2-acetic acidsfeature the best spatial relationship among the pha r-macophoric groups.

Finally, compounds 1 -25 were all found to be selec-tive inhibitors of ALR2 becau se n one of them showedany appr eciable inhibitory property toward ALR1, SD,and GR.

P h a r m a c o l o g i c a l E v a l u a t i o n o f C o m p o u n d s 3

a n d 7 3 . Compound 3 was administered as an eyedropsolution in the precorneal region to investigate its invivo ability to prevent cat ar act developmen t in sever elygalactosemic rats. Topical administra tion can in pr in-

ciple achieve significant drug levels in the lens withnegligible effects on other tissues, thus avoiding bio-availability and/or met abolism-relat ed pr oblems associ-ated with systemic administ rat ion.30 I t h a s b ee nreported32 -34 that esters of acid drugs display a bettercorneal permeability and ocular bioavailability by virtueof their higher lipophilicity. Considering tha t i-propylesters have shown good permeabili ty in the cornealtissue,32 the i-propyl ester 73 was prepared and testedin vivo as a pr odrug of compound 3. The pharmacologi-cal data ar e report ed in Table 8. After 21 da ys of a 50%

galactose diet , 90% of th e animals treated only withvehicle developed n uclear catara ct. Those t reat ed with3% ophtha lmic solution of 3 and 73 and w i t h 1%ophth almic solut ion of tolrest at wer e protected by 50%,60%, an d 53%, respectively, whereas no nu clear cata-racts were detected in rats that were administered a3% ophth almic solution of tolrestat . The similar fairlyhigh potency displayed by 3 and its prodrug 73 suggeststhat 3 is already char acterized by an optimal hydro-philic-l ipophilic balance to permeate the cornea andthat 73 is quickly hydrolyzed to the active acid bycorneal esterases.

Molecular Modeling. To rat iona lize th e SARs of theTBI derivatives at t he m olecular level and to perspec-

T a b l e 5 . Physical Properties of (10-Substituted[1,2,4]triazino[4,3-a]benzimidazol-3,4(10 H )-dion-2-yl)acetic Acids 1-11

no. R yielda (%) recryst solv m p (°C) formula b

1 CH 3 38 (39) MeOH >300 C12H 10N4O4

2 CH 2CH 2CH 3 75 (65) E tOH 199-200 C14H 14N4O4

3 CH 2C6H 5 68 (63) E tOH 252-255 (dec) C18H 14N4O4

4 CH 2C6H 4-4-CH3 73 (70) E tOH 258-262 (dec) C19H 16N4O45 CH 2C6H 4-4-OCH 3 61 (60) E tOH 258-261 C19H 16N4O5

6 CH 2C6H 4-4-Cl 85 (88) EtOH 255-257 (dec) C18H 13ClN4O4

7 CH 2C6H 4-4-F 63 (60) EtOH 250-253 (dec) C18H 13FN4O4

8 CH 2C6H 4-4-CF 3 46 (48) MeOH 240-246 (dec) C19H 13F 3N4O4

9 CH 2C6H 3-3,4-F2 71 (65) E tOH 250-256 (dec) C18H 12F 2N4O4

10 CH 2C6H 3-2-F -4-Br 92 (90) EtOH 262-266 (dec) C18H 12BrFN 4O4

11 CH 2COOH 53 (54) E tOH 238-244 (dec) C13H 10N4O6

a Thermal cyclization yield (method A). In parentheses ester hydrolysis yield (method B). b Element al an alyses for C, H, N were within(0.4% of the calculated values.

T a b l e 6 . Physical Properties of ([1,2,4]Triazino[4,3-a]benzimidazol-4(10 H )-on-3-yl)acetic Acid Derivat ives 23 -25 a nd 70 -72

no. R1 R2 yield (%) recryst solv mp (°C) form ula a

70 CH 2CH 3 H 75 toluene 237-238 (dec) C13H 12N4O3

71 CH 2CH 3 CH 3 86 MeOH 195-198 (dec) C14H 14N4O3

72 CH 2CH 3 CH 2C6H 5 85 E tOH 161-164 C20H 18N4O3

23 H H 95 AcOH >300 C11H 8N4O3

24 H CH 3 90 AcOH 230-231 C12H 10N4O3

25 H CH 2C6H 5 92 AcOH 180-182 C18H 14N4O3

a Elemental analyses for C, H, N were within (0.4% of the calculated values.

S c h e m e 2




tively guide the design of novel ARIs, we developed amodel of compound 3 bound to ALR2. The results of these studies are summ arized here, whereas details aregiven in the Computat ional Chemist ry part of theExperimenta l Section.

Crystal structures of ALR2 complexed with zopol-restat ,6 tolrestat,7 sorbinil,7 and alrestatin 8 clearly showthat inhibitors bind at the catalytic si te and inducesignificant conformational changes in a loop (residues121-135) and a short segment (residues 298-303) toopen an d fill a contiguous h ydrophobic pocket. This so-called specificity pocket, closed in the absence of the

inhibitor, is a key determ inant for selectivity, since ithosts hydrophobic moiet ies of ARIs that are moreeffective aga inst ALR2 tha n ALR1. Alth ough th e thr ee-dimensional structure of th e huma n ALR2/NADP+ / zopolrestat complex has been determined by Wilson etal.,6 only the corr esponding coordina tes of the CR atomshave been filed in th e Brookha ven Protein Data Bank 35

(PDB entry code 1MAR). To take into account theconformational switching undergone by ALR2 upon ARIbinding, we built a m odel of hu ma n ALR2 sta rt ing fromthe crystal str uctures of the hum an ALR2 holoenzyme(PDB ent ry code 1ADS)36 an d th e porcine ALR2/NADP + / tolrestat complex (PDB entry code 1AH3).7 Briefly,fragments 121-135 and 298-303 of human ALR2(closed specificity pocket) were replaced with those of porcine ALR2 (open specificity pocket) and nonidenticalside chains were m uta ted into those of the hum an t ypeusing the SYBYL/MUTATE command. The resultingprotein model was geometry-optimized by molecularmecha nics calcula tions u sing th e AMBER force field.37,38

Although the inhibit ion assays on our compoundswere conducted on rat ALR2, the use of a model of human ALR2 for docking studies is justified by thefollowing facts: (i) the crystal structure of rat ALR2 isunknown; ( i i ) the human and rat sequences of thisenzyme are characterized by 81% of identity and 88%of homology;39 (iii) all active-site residues, includingthose of the specificity pocket, are largely conservedacross the ALR2 isoforms so far sequenced.

Representative energetically stable conformations of 3 were sought for docking calculations. For this purpose,we used the SYBYL/SEARCH module to scan thetorsion angles τ 1, τ 2, τ 3, and τ 4 defined in Figur e 2. Low-energy conformations (those within 2 kcal/mol of the

global minimum conformation) were then clusteredinto 16 families thr ough th e SYBYL/SPREADSHEET/ FAMILY routine. The lowest energy member of eachfamily was submitted to docking using the automatedDOCK softwar e p ackage.40 -43 This program estimatesthe l ikelihood of each generated binding mode by ascoring function based on a molecular mechanics forcefield. The geometry of the top scoring ALR2/NADP + / 3complex was refined by extensive energy minimizationand molecular dynamics simulations in a solvatedsystem at room temperature. Interestingly, the bestdocking conformation of 3 yielded the most satisfactoryoverlay on th e ALR2-bound conforma tion of zopolresta t(Figure 1).

T a b l e 7 . ALR2 In hibition Data of Acid Derivatives 1-25 a

a All compounds did not inhibit ALR1, SD, and GR up to aconcentration of 10-3 M. b IC 50 (95% CL) values represent the

concentra tion required to produce 50% enzyme inhibition.

T a b l e 8 . Effect of Treatment with Ophthalmic Solution of 3,73 , and Tolrestat on Development of Nuclear Cataract inSeverely Galactosemic Rat s

rats with nuclear cataract (%)

day of t r ea t m en t con t r ol

3(3%)

73(3%)

tolrestat(1%)

tolrestat(3%)

11 13 0 0 0 012 25 0 10 0 013 25 0 10 0 014 25 10 10 23 0

15 25 10 10 32 016 31 10 10 32 017 50 10 10 32 018 50 40 40 43 019 75 40 40 43 020 88 50 40 47 021 90 50 40 47 0




The main features of the final docking model are

schemat ical ly represented in Figure 2. The three-dimensional str ucture of the ALR2/NADP + / 3 complexis shown in Figure 3, where only the amino acids locatedwithin 5 Å of the inhibitor ar e displayed.

Throughout the entire m olecular dynamics simula-tion, the carboxylate oxygens of 3 were anchored intothe so-called anionic binding si te via a network of hydrogen bonds involving Tyr48, His110, an d Trp111side chains. This suggests that the carboxylate groupis an essential requirement for the inhibitory activityof 3 and its TBI derivatives. A further hydrogen bondbetween the carbonyl oxygen at the 4 position of the TBIsystem and the N 1 hydrogen of Trp20 wa s a lso foundto contr ibute t o complex sta bilization, a lthough it was

observed to be frequently cleaved dur ing the simu lation,giving an avera ge distan ce longer th an tha t of an idealhydrogen bond.

A hydrophobic cavity ma de u p of the Trp20, Trp79,Phe122, and Trp219 side chains hosts the TBI nu cleusof the inhibitor. The Ph e122 side chain mak es conta ctwith th e fused benzene an d th e benzyl moiety of 3 viaa π -stacking a nd a T-sha ped inter action, r espectively.

The phenyl r ing of 3 fits into the specificity pocket(the same one th at hosts th e zopolresta t benzothiazolemoiety)6 made up of four aromatic residues (Trp79,Trp111, Ph e115, Phe122), two alipha tic residues (Val130,Leu300), and five polar residues (Cys80, Thr113, Cys298,

Ser302, Cys303). This specificity pocket does not appearto tolerate any further steric bulk, consistent with thedet rimental effects exerted by subst i tuents on thephenyl ring of 3. Our m odel is not contr adicted by thefavora ble effect of the tr ifluoromethyl group featu redby the ben zothiazole moiety of zopolrest at as well as bythe fused-benzene system of tolrestat , both occupyingthe specificity pocket. In fact, the phenyl ring of 3 isnot exactly coincident in spa ce with either the benzo-thiazole or the fused-benzene moieties of zopolrestat andtolrestat (Figure 4). From a methodological point of view, it is wort h n oting tha t th e phar macophore-based(Figure 1) and the docking-based (Figure 4) alignmentsof 3 on zopolresta t are fairly similar.

C o n c l u s i o n s

We have presented acetic acid derivatives of the[1,2,4]triazino[4,3-a]benzimidazole (TBI) nucleus as anovel class of selective ARIs. Compound 3, the mostpotent derivative of the series, exhibited an ALR2inhibitory activity (IC50 ) 0.36 µM) similar to tha t of sorbin il (0.65 µM) and proved to be effective in pr event-ing catar act development in severely galactosemic ra tswhen administered as an eyedrop solution. Docking

simulations of 3 into a model of the ALR2 binding sitewere performed to explain SARs and to guide, perspec-tively, the design of new an alogues.

E x p e r im e n t a l S e c t i o n

1 . C h e m i s t r y . Melting points were determined using aReichert Kofler hot-stage appara tus and are uncorrected.Infrared spectra were recorded with a PYE/UNICAM Infracordmodel PU 9516 spectrophotometer in Nujol mulls. Routinenuclear magnetic resonance spectra were recorded in DMSO-d 6 solution on a Varian CFT 20 spectrometer operating at 80MHz, using tetramethylsilane (TMS) as the internal standard.Mass spectra were obtained on a Hewlett-Packard 5988 Aspectrometer using a direct injection probe and an electronbeam energy of 70 eV. Evaporations were made in vacuo

(rotary evaporat or). Analytical TLC was carried out on Merck0.2 mm precoated silica gel aluminum sheets (60 F-254).Elemental analyses were performed by our analytical labora-tory, and th e results a greed with theoretical values to within(0.4%.

The alkyl halides 4-methylbenzyl chloride, 4-methoxybenzylchloride, 4-(tr ifluorometh yl)benzyl chloride, 3,4-difluorobenzylbromide, an d 4-bromo-2-fluorobenzyl bromide used to obtaincompounds 30 , 31 , 34 -36 , resp ectively, an d 2-chlorobenzimi-dazole, 26 , were from Sigma-Aldrich. The following compoundswere prepared in accordance with reported procedures: 2-chloro-1-methylbenzimidazole, 27 , mp 114-116 °C (lit.44 mp 107-

113.5 °C); 2-chloro-1-n -propylbenzimidazole, 28 , bp 140-143°C, 0.5 mmHg (lit.44 bp 110.5-111.5 °C, 0.3 mmH g); 1-benzyl-2-chlorobenzimidazole, 29 , mp 108-109 °C (lit.44 mp 108-111°C); 1-(4-chlorobenzyl)-2-chloroben zimida zole, 32 , mp 73-75

°C (lit.45

mp 73-75 °C); 2 -chloro-1-(4-fluoroben zyl)benzimida -zole, 33 , m p 7 4-79 °C (lit.46 m p 75-76 °C); 2-hydrazino-benzimidazole, 37 , mp 148-151 °C (lit.18 mp 146-147 °C);1-benzyl-2-hydrazinobenzimidazole, 40 , m p 134-135 °C(lit.18 mp 140-145 °C); [1,2,4]tria zino[4,3-a]benzimidazol-3,4-(10 H )-dione 48 , mp >300 °C (lit.19 mp 360 °C).

G e n e r a l P r o c e d u r e f o r t h e S y n t h e s i s o f 1 -A lk y l -2 -c h l o r o b e n z i m i d a z o l e s 3 0 , 3 1, 3 4-36 . Sodium hydride (1.2mmol, 50% dispersion in mineral oil) was added portionwise,under a nitrogen atmosphere, to an ice-cooled solution of 2-chlorobenzimidazole (26 , 0.153 g, 1 mmol) in 5 m L of fresh lydisti l led DMF. Once hydrogen evolution had ceased, theappropriate a lkyl halide (1.2 mm ol) was a dded dropwise andthe reaction mixture was maintained under stirring at roomtemperature until the disappearance of the starting material(2-18 h, TLC ana lysis). The solution was t hen slowly poured

onto crushed ice, and the solid precipita te was collected,washed with water, and recrystallized. Yields, recrystallizationsolvents, and melting points of the products are reported inTable 1. Spectra l dat a for 30 , which is representat ive of thetitle compounds, are listed below.

2-Chloro-1-(4-methylbenzyl)benzimidazole, 30. IR , νcm -1: 1450, 1360, 720. 1H NMR, δ: 2.25 (s, 3H, CH3), 5.44 (s,2H, CH 2), 7.10-7.49 (m, 8H, ArH). MS, m / e: 256 [M+], 105,base.

G e n e r a l P r o c e d u r e f o r t h e S y n t h e s i s o f 1 -A lk y l -2 -h y d r a z i n o b e n z i m i d a z o l e s 3 8 , 39 , 4 1-47 . The appropriate1-alkyl-2-chlorobenzimidazole (27 , 28 , 30 -36 , 1 mmol) washeated at 160 °C in a Pyrex capped tube with 0.1 mL of hydrazine hydrate for 5 h. After the mixture was cooled, awhite solid separa ted, which was collected an d recrysta llized.Yields, recrystallization solvents, a nd melting points of the

F i g u r e 2 . Scheme of the m ain int eractions observed in th emolecular dyna mics simula tion of th e ALR2/NADP+ / 3 complex.Mean values of intermolecular hydrogen bond dista nces andof their standard deviations are given. The values of the torsionangles τ 1, τ 2, τ 3, and τ 4 define t he conformat ion of th e inhibitor.




products are reported in Table 1. Spectral data for 38 , whichis representa tive of the t itle compounds, ar e listed below.

1 -M e t h y l -2 -h y d r a z i n o b e n z i m i d a z o l e , 3 8 . IR , ν cm-1

:3320, 3225, 1550, 720. 1H NMR, δ: 3.48 (s, 3H, CH3), 6.91-

7.30 (m, 4H, ArH ). MS, m / e: 162 [M+], base.General Procedure for the Synthesis of 10-Alkyl[1,2,4]-

triazino[4,3- a ]benzimidazol-3,4(10 H )-dione s 49 -58 . A so-lution of t he appropriate 1-alkyl-2-hydrazinobenzimidazole(38 -47 , 1 mmol) and diet hyl oxalate (0.16 mL, 1.2 mmol) in 5mL of absolute e thanol was heated under ref lux until thedisappeara nce of the st art ing mater ial (3-20 h, TLC analysis).After th e mixtu re was cooled, a yellow solid separ at ed, whichwas collected an d r ecrystallized. Yields, recrystallization sol-vents, and m elting points of the pr oducts ar e reported in Table2. Spectral data for 49 , which is representative of the titlecompounds, are listed below.

10-Methyl[1,2,4]triazino[4,3- a ]benzimidazol-3,4(10 H )-d i o n e , 4 9 . IR , ν cm -1: 3150, 1700, 1625, 1450. 1H NMR, δ:

3.44 (s, 3H, CH 3), 7.13-8.13 (m, 4H, ArH), 12.11 (s, 1H, NH ,exch with D2O). MS, m / e: 216 [M+], 131, base.

G e n e r a l P r o c e d u r e f o r t h e S y n t h e s i s o f E t h y l ( 1 0 -Alkyl[1,2,4]triazino[4,3- a ]benzimidazol-3,4(10 H )-dion-2-y l ) a c e t a t e s 5 9-69 . A suspension of 48 -58 (1 mmol), ethylbromoacetate (0.13 mL, 1.2 mmol), and anhydrous potassiumcarbonat e (0.166 g, 1.2 mmol) in 5 m L of acetone was heat edunder r eflux until the disappeara nce of the start ing material(7-24 h, TLC analysis). After the mixture was cooled, the solidprecipitate was collected, washed with water to remove the

inorganic salts, and recrystallized. Yields, recrystallizationsolvents, and melting points of the products are reported inTable 3. Spectra l dat a for 59 , which is representat ive of thetitle compounds, are listed below.

Ethyl (10-Methyl[1,2,4]triazino[4,3- a ]benzimidazol-3,4-(1 0 H )-dion -2-yl)ace tate, 59. IR , ν cm -1: 1740, 1700, 1620,1200. 1H NMR, δ: 1.23 (t, 3H, CH 2C H 3), 3.44 (s, 3H, CH 3),4.17 (q, 2H, C H 2CH 3), 4.69 (s, 2H, NCH 2), 7.13-8.12 (m, 4H,ArH). MS, m / e: 302 [M+], 201, base.

G e n e r a l P r o c e d u r e f o r t h e S y n t h e s i s o f ( 1 -A lk y l -3 -o x a l o - 1 H ,3 H - b e n z i m i d a z o l - 2 - y l i d e n e h y d r a z i n o ) a c e t i cAcids 12-22 . A suspension of the a ppropriate ester deriva-tive (59 -69 , 1 mmol) in 3 mL of 3% NaOH was left understirring at room temperature until a solution was achieved(1-48 h). The solution was then filtered and acidified withconcentrated HCl under ice-cooling. The white solid precipitate

was collected, washed with water, and recrystallized. Yields,recrystallization solvents, and cyclization temperatures toproducts 1-11 are r eported in Table 4. Spectra l data for 12 ,which is representative of the t i t le compounds, are l istedbelow.

(1-Methyl-3-oxalo-1 H ,3 H - b e n z i m i d a z o l - 2 - y l i d e n e h y -drazino)acetic Acid, 12. IR , ν cm -1: 3250, 3150, 1710, 1600,1580, 1260. 1H NMR, δ: 3.78 (s, 3H, CH 3), 4.51 (s, 2H, CH 2),7.39-7.63 (m, 4H, ArH ). MS, m / e: 77, base.

G e n e r a l P r o c e d u r e f o r t h e S y n t h e s i s o f ( 10 -Al k y l-[1,2,4]triazino[4,3- a ] b e n z i m i d a z o l - 3 , 4 ( 1 0 H )-dion-2-yl)-a c e t i c A c i d s 1-11. Method A. The dicarboxylic derivatives12 -22 (1 mmol) were heated at t he appropriate temperat ure(see Table 4) unt il t hermal cyclization occurred. Yields, re-crystallization solvent s, and meltin g point s of the pr oducts arereported in Table 5.

F i g u r e 3 . Compound 3 docked int o the ALR2 active site. Only amin o acids located with in 5 Å of th e inhibit or are d isplayed forthe sake of clarity.

F i g u r e 4 . Overlay of the putative bioactive conformation of 3 (red) on the experimentally determined ALR2-bound con-formations of zopolrestat (yellow) and tolrestat (green).




M e t h o d B . A suspension of the ester derivative (59 -69 , 1mmol) in 1 mL of concentr ated H Cl was heated, un der stirr ingat 100 °C, until hydrolysis was complete (12 -72 h , TLCana lysis). After cooling, the r eaction mixtur e was dilut ed withwater and t he yellow solid tha t precipitat ed was collected an drecrystallized from the appropriate solvent. Spectral data for1, which is represent ative of the t itle compounds, are listedbelow.

(10-Methyl[1,2,4]triazino[4,3- a ]benzimidazol-3,4(10 H )-dion-2-yl)acetic Acid, 1. IR , ν cm -1: 3400, 1720, 1590, 1200,900. 1H NMR, δ: 3.45 (s, 3H, CH 3), 4.60 (s, 2H , CH 2), 7.16-

8.12 (m, 4H, ArH ). MS, m / e: 274 [M+], 77, base.G e n e r a l P r o c e d u r e f o r t h e S y n t h e s i s o f E t h y l ( 1 0 -

Alkyl[1,2,4]triazino[4,3- a ]benzimidazol-4(10 H )-on-3-yl)-a c e t a t e s 7 0-72 . A solution of the suitable 2-hydrazinobenz-imidazole 37 , 38 , and 40 (1 mmol) an d dieth yl acetylenedicar-boxylate (0.19 mL, 1.2 mmol) in 5 mL of metha nol was h eatedunder r eflux until the disappeara nce of the start ing material(8-48 h, TLC analysis). After the mixture was cooled, theyellow solid precipitat e was collected an d r ecrystallized. Yields,recrystallization solvents, and melting points of the productsa r e r e por te d in Ta ble 6 . S pect r a l da ta for 72 , which i srepresentat ive of the t itle compounds, ar e listed below.

E t h y l ( 1 0 -B e n z y l [ 1 ,2 ,4 ] t r i a z i n o [ 4 ,3 - a ] b e n z i m i d a z o l -4(10 H )-on-3-yl)acetate, 72. IR , ν cm -1: 1720, 1660, 1560. 1HNMR, δ: 1.19 (t, 3H, CH2C H 3), 3.98 (s, 2H, CH 2), 4.11 (q, 2H,C H 2CH 3), 5.18 (s, 2H, CH 2Ar), 7.37-8.41 (m, 9H, ArH). MS,m / e: 362 [M+], 91, base.

G e n e r a l P r o c e d u r e f o r t h e S y n t h e s i s o f ( 1 0 -A lk y l -[1,2,4]triazino[4,3- a ]benzimidazol-4(10 H )-on-3-yl)aceticAcids 23-25 . A suspension of the appropriat e ester derivative(70 -72 , 1 mmol) in 1 mL of concentrated HCl was heatedunder stirring a t 100 °C until hydrolysis was complete (2-4h, TLC analysis). After cooling, the reaction mixture wasdiluted with wa ter a nd th e solid precipitat e was collected andrecrystallized. Yields, recrystallization solvents, and meltingpoints of the pr oducts a re reported in Table 6. Spectra l datafor 24 , which is representative of the title compounds, arelisted below.

(10-Methyl[1,2,4]triazino[4,3- a ]benzimidazol-4(10 H )-on-3-yl)acetic Acid, 24. IR , ν cm -1: 1650, 1640, 1550. 1HNMR, δ: 3.83 (s, 3H, CH3), 3.86 (s, 2H, CH 2), 7.30-8.37 (m,

4H, ArH). MS,m

/ e

: 118, base.i -Propyl (10-Benzyl[1,2,4]triazino[4,3- a ]benzimidazol-3,4(10 H )-dion -2-yl)ace tate, 73. Compound 73 was obtainedfollowing a procedure analogous to that employed for theprepar ation of compounds 59 -69 using i-propyl bromoacetat einstead of ethyl bromoacetate. After recrystallization fromi-propyl alcohol, pur e 73 was obtained with a 61% yield. Mp:246-247 °C. IR, ν cm -1: 1740, 1700, 1600, 1180. 1H NMR, δ:1.17 (s, 3H, CH 3), 1.25 (s, 3H , CH 3), 3.09 (m, 1H, CH ), 4.66 (s,2H, NCH 2), 5.17 (s, 2H, CH 2Ar), 7.33-8.18 (m, 9H, ArH). MS,m / e: 392 [M+], 91, base. Anal. Ca lcd for C 21H 20N4O4.

2. Biology. 2.1. Materials an d Metho ds. Aldose reductase(ALR2) and aldehyde reductase (ALR1) were obtained fromSpra gue Dawley albino rats , 120-140 g body weight , suppliedby Har lan Nossan, I ta ly. In ordered to minimize cross-contamination between ALR2 and ALR1 in the enzyme

preparation, ra t lens, in which ALR2 is the predominantenzyme, and kidney, where ALR1 shows the highest concen-trat ion, were used for isolation of ALR2 an d ALR1, respec-tively.

Glutathione reductase (GR) type IV from baker’s yeast(100-300 U/mg), sorbitol dehydr ogenase (SD) from sh eep liver(10 U/mg of protein), pyridine coenzymes, D,L-glyceraldehyde,glutathione disulfide, sodium D-glucuronate, sorbitol, andquercetin were from Sigma Chemical Co. Sorbinil was a giftfrom Pfizer, Groton, CT. Tolrestat was extracted from LorestatRecordati, Italy. All other chemicals were of reagent grade.

2 .2 . E n z y m e P r e p a r a ti o n . 2 .2 .1 . Al d o s e R e d u c t a s e(ALR2). A purified rat lens extract was prepared in accordancewith the m e thod of Ha ym a n a nd Kinoshi ta 47 with slightmodifications. Lenses were qu ickly removed from norma l killedrats and homogenized (Glas-Potter) in three volumes of cold,

deionized water. The homogenate was centrifuged at 12 000r p m a t 0-4 °C for 30 min. Saturated ammonium sulfatesolution was added t o the supern ata nt fraction to form a 40%solution, which was stirred for 30 min at 0 -4 °C and thencentrifuged at 12 000 rpm for 15 min. Following this sameprocedure, the recovered superna tan t was su bsequently frac-tionated with sat ura ted amm onium sulfate solution, using firsta 50% and then a 75% of salt saturation. The precipita terecovered from the 75% saturated fraction, containing ALR2activity, was redissolved in 0.05 M NaCl and dialyzed over-night in 0.05 M NaCl. The dialyzed mater ial was used for the

enzymatic assay.2.2.2. Aldehyde Reductase (ALR1). Rat kidney ALR1 was

prepared in accordance with a previously reported meth od.26

Kidneys were quickly removed from normal killed rats andhomogenized (Glas-Potter) in t hr ee volumes of 10 mM sodiumphosphate buffer, pH ) 7.2, containing 0.25 M sucrose, 2.0mM EDTA dipotassium salt, and 2.5 mM β-mercaptoethanol.The homogenate was centrifuged at 12 000 rpm at 0 -4 °C for30 m in , a nd the supe r na ta nt wa s subje c te d to a 40 -75 %amm onium su lfate fractionat ion, following th e sam e procedurepreviously described for ALR2. The pr ecipita te obta ined fromthe 75% ammonium sulfate saturation, containing ALR1activity, was redissolved in 50 volumes of 10 mM sodiumphosphate buffer, pH ) 7.2, containing 2.0 mM EDTA dipo-ta ss ium sa l t a nd 2 . 0 m M β-mercaptoethanol and dialyzedovernight u sing th e same buffer. The dialyzed mat erial wasused in the enzymatic assay.

2 .3 . E n z y m a t i c A s s a y s . The activity of the four testenzymes was determined spectrophotometrically by monitoringthe change in absorbance at 340 nm, which is due to theoxidation of NADPH or the reduction of NAD+ catalyzed byALR2, ALR1, and GR or SD, respectively. The change inpyridine coenzyme concentration per minute was determinedusing a Beckma n DU-64 kinet ics software pr ogram (Solf PackTM m odule).

ALR2 activity was assayed at 30 °C in a reaction m ixturecontaining 0.25 mL of 10 mM D,L-glyceraldehyde, 0.25 mL of 0.104 mM NADPH, 0.25 mL of 0.1 M sodium phosphate buffer(pH ) 6.2), 0.1 mL of enzyme extr act, an d 0.15 mL of deionizedwater in a total volume of 1 mL. All the a bove reagent s, exceptD,L-glyceraldehyde, were incubated at 30 °C for 10 min; t hesubstrate was then added to star t the reaction, which wasmonitored for 5 min. En zyme activity was calibrated bydiluting the enzymatic solution in order t o obtain a n a veragereaction rate of 0.011 ( 0.0010 AU/min for the sample.

ALR1 activity was determined at 37 °C in a reaction mixturecont ainin g 0.25 mL of 20 mM sodium D-glucurona te, 0.25 mLof 0.12 mM NADPH , 0.25 mL of dialyzed en zymatic solution,and 0.25 mL of 0.1 M sodium phosphate buffer (pH ) 7.2) ina total volume of 1 mL. The enzyme activity was calibratedby diluting th e dialyzed enzymatic solut ion in order to obtainan average reaction rate of 0.015 ( 0.0010 AU/min for thesample.

SD activity25 was determined at 37 °C in a reaction mixturecontaining 0.25 mL of 10 mM sorbitol, 0.25 mL of 0.47 mMNAD+, 0.25 mL of 3.75 mU/mL enzymatic solution, 0.25 mLof 100 mM Tris-HCl buffer (pH ) 8) in a total volume of 1

mL. All the reagents were incubated at 37 °C for 1 min, afterwhich the reaction was m onitored for 3 min.GR activity27 w as d et e rm in ed a t 3 7 ° C i n a m ixt u r e

cont ainin g 0.25 mL of 1 mM glut at hione disulfide, 0.25 mL of 0.36 mM NADPH, 0.25 mL of 4.5 mU/mL en zymatic solution,0.25 mL of 0.125 sodium phospha te bu ffer (pH ) 7.4) supple-mented with 6.3 mM EDTA potassium salt, in a total volumeof 1 mL.

2 .4 . E n z y m a t i c I n h i b i t i o n . The inhibitory activity of thenewly synthesized compounds against ALR2, ALR1, SD, andGR was assayed by adding 0.1 mL of the inhibitor solution tothe reaction mixture described above. All the inhibitors weredissolved in water , an d the solubility was facil ita ted byadjustment to a favorable pH. After complete dissolution, thepH was readjusted to 7. To correct for the nonenzymaticoxidation of NADPH or reduction of NAD+ a n d f o r t h e




absorption by the compounds tested, a reference blank con-taining all th e above assay components except th e substra tewas pr epared. The inh ibitory effect of the new derivat ives wasroutinely estimated at a concentration of 10-5 M. Thosecompounds found to be active were t ested at additionalconcentrations between 10-5 and 10-8 M. The determinat ionof the IC50 values was performed by using linear regressionanalysis of the log of the dose -response curve, which wasgenerated using at least four concentrations of inhibitor ,causing an inhibition between 20% and 80% with two repli-cates at each concentration. The 95% confidence limits (95%

CL) were calculated from t values for n - 2, where n is thetotal num ber of determinat ions.

3 . P h a r m a c o l o g y . 3 . 1. M a t e r i a ls a n d M e t h o d s . Experi-ments were carr ied out u sing Sprague Dawley albino rats , 45-

55 g body weight, supplied by Har lan-Nossan, It aly. Animalcare and treatment conformed to the ARVO resolution on theuse of animals in ophthalmic and vision research. The galac-tose diet consisted of a pulverized mixt ur e of 50% D-galactoseand 50% TRM (Harlan Teckland, U.K.) and laboratory chow,and the contr ol diet consisted of normal TRM. Both contr oland experiment al rat s had a ccess to food and wat er ad libitum.

3.2. Preven tion of Cataract Developmen t. Animals wererandomly divided into groups of equal average body weightwith 15 rat s per group. The test compounds 3, 73 , and tolrestatwere administ ered four t imes daily as eyedrops of appr opriateconcentr ations. The vehicle in wh ich ARIs were contain ed wasadminist ered with th e same dose regimen to the contr ol group,which was given access to the gala ctose diet, and t o the groupfed with norma l diet, which was included to record t he as pectof normal lenses. Groups treat ed with t he test ed compoundswere predosed 1 day before switching their diet to galactose-containing chow.

Lenses were examined using slit-lamp microscopy, afterdila ting the pupils with 1% atropina, Farmigea, I ta ly, toestablish their st atu s of integrity.

Nuclear cataracts, which appeared as a pronounced centralopacity readily visible as a white spot, were considered. Thenum ber of animals that a tta ined this stage was recorded, andthe ability of the test compounds to prevent cataract develop-ment was assessed on the basis of comparison with galac-tosemic rat s t reated only with the vehicle.

4 . C o m p u t a t i o n a l C h e m i s t r y . Molecular modeling andgraphics manipulations were performed using the SYBYL 48

and MIDAS49 software packages, running them on a SiliconGraphics R10000 workstation. Model building and conforma-tional a nalysis of compound 3 were accomplished with theTRIPOS force field50 available within SYBYL. Point chargesof this inhibitor were calculated using the semiempir icalquant um mechan ics AM1 method51 implemented in the MO-PAC program.52 Energy minimizations and molecular dynam-ics simulations of the ALR2/NADP + an d ALR2/NADP + / 3complexes were realized by employing the AMBER pro-gram,37,38 selectin g th e all-atom Corn ell et a l. force field.53

4.1. Building a Model of Huma n ALR2. It is known fromcrystallographic6,7 and modeling studies11,54 that the bindingof inhibit ors to th e ALR2 active site indu ces fairly significantconformational changes in a loop (residues 121-135) and a

short segment (residues 298-303). These chan ges are a ssoci-ated with the opening of the so-called specificity pocket, whichis completely closed in th e absen ce of the in hibitor. The X-raystr uctur e of th e hum an ALR2/NADP+ /zopolrestat complex hasbeen published by Wilson et al.6 but could not be used in th ismodeling study because only the coordinates of the corre-sponding CR atoms ar e filed in the Brookhaven Protein DataBank 35 (PDB entry code 1MAR). A model of human ALR2 wasbuilt start ing from the crystal stru ctur es of the hum an ALR2holoenzyme (PDB en tr y code 1ADS)36 and the porcine ALR2/ NADP + /tolrestat complex (PDB entry code 1AH3).7 A super-position of the proteins about their CR atoms yielded a root-mean -square (rms) deviation of 0.83 Å. Such a close str uctur alsimilarity is not surprising because the two ALR2 isoformsshare more th an 84% sequence identity, the only differencesbeing confined to the loop 121-135 and the segment 298-

303. The backbone of the above-listed residues in the humanALR2 holoenzyme (closed specificity pocket) was repla ced withthat of porcine ALR2/NADP + /tolres ta t complex (open specific-ity pocket). All the nonidentical side chains were then mut atedinto those of the hu man type th rough th e SYBYL/MUTATEcomman d. The Asn129 side chain atoms, not included in th ecrystal structure of human ALR2 owing to disorder , weregenerated using th e AMBER internal coordinate database.With t he exception of His110, all His residues were treat edas neutral species with the hydrogen assigned to the N δ1.His110 was modeled in its N 2 ta ut omeric form on th e basis of

the crystallographic evidence that the carboxylate group of zopolrestat is salt-linked to the N 2 hydrogen of His110.9,55-57

Recent modeling studies of substrates in the ALR2 bindingsite suggest that His110 is not protonated.58 Asp and Gluresidues were assumed to be negatively charged, while Argan d Lys were positively char ged. Hydrogen at oms were addedto the protein through the SYBYL/BIOPOLYMER module.

To refine the resulting model, we adopted the followingenergy minimization protocol. Stretches 121 -135 and 298-

303 were locally minimized through 200 steps of steepestdescent followed by 300 step s of conjuga te gra dient, wh ile therest of the system was kept fixed. Next, th e whole protein wa sminimized in vacuo using a distance-dependent dielectricfunction ( ) 4r ) and const rain ing the position of th e backboneat oms with a ha rm onic force consta nt of 10 kcal/mol Å2. Duringth is step, NADP+ was a llowed to move. En ergy minimizat ionswere realized by setting a nonbonded cutoff of 8 Å and anenergy gra dient of 0.01 kcal/mol as t he convergence criter ion.In all AMBER calculations, the atomic charges for NADP +

were calculat ed by an electr ostat ic potent ial fit 59 to a STO-3Gab initio wave function.

4 .2 . Mo d e l B u i l d i n g a n d C o n f o r m a t i o n a l A n a l y s i s o f C o m p ou n d 3 . A molecular model of compound 3 was con-stru cted using standar d bond lengths a nd bond angles of theSYBYL fragment library. Th e carboxylate group was takenas dissociated. Atom-center ed par tial char ges were calculatedusing the AM1 Hamiltonian 51 as implemented in MOPAC52

(CHARGE ) -1; keywords, PREC, GNORM ) 0.01, EF,MMOK). Geometry optimizations were realized with theSYBYL/MAXIMIN2 minim izer by a pplying t he BFGS (Broy-den, Fletcher, Goldfarb, and Sha nn on) algorithm 60 and settingan r ms gra dient of the forces acting on each at om of 0.05 kcal/ mol Å as the convergence criterion.

Representa tive energetically stable conformat ions of 3 weresought for a pharmacophore-based superposition on the ALR2-bound conformation of zopolrestat, as well as for dockingcalculations. A conformational analysis on 3 was carried outusing the SYBYL/SEARCH module. The torsional anglesdefined in Figure 2 were scanned by 20° increments through-out 0-340° (τ 2 a nd τ 3) or 0-160° (τ 1 a nd τ 4). A van der Wa alsscaling factor of 0.75 was applied t o “soften ” ster ic conta cts t otake into account the lack of relaxation in the rigid rotamers.The number of output conformations to be examined wasreduced by setting the “energy window” (energy differencebetween the generated conformation and the current mini-mum) to 5.0 kcal/mol. The resulting conformations wereclassified in t he gr id spa ce of torsional a ngles int o 16 “families”,using the FAMILY option of the SYBYL/MOLECULARSPREADSHEET routine.

The lowest energy conformation of each family was super-imposed on th e ALR2-bound geomet ry of zopolrest at 6 by fitt ingthe carboxylate carbons, the triazinobenzimidazole N1 andN10 on the phthalazine N2 and N3, respectively. The confor-mation of 3 affording the best overlay on zopolrestat (Figure1) was defined by t he following va lues of torsion an gles: τ 1 )

135°, τ 2 ) 106°, τ 3 ) -115°, and τ 4 ) 39°. Compoun d 25 wa smodeled and aligned on zopolrestat , following a similarprocedure. The conforma tion of 25 i l lustrated in Figure 1featur es th e following values of torsion an gles (defined a na lo-gously to th ose of 3): τ 1 ) 59°, τ 2 ) 63°, τ 3 ) -118°, an d τ 4 )

44°.4.3. Docking of Compoun d 3 into the ALR2 Active S ite.

Docking calculations were performed using t he DOCK suite




of programs.40 -43 DOCK describes th e binding site of the ta rgetprotein as a cluster of spheres filling the binding cavity toconform to its shape. Docking is achieved by treating theprotein and the l igand as r igid entit ies and searching formatches between interatomic and intersphere distances. Tota ke int o account th e flexibility of the ligand, a set of differentgeometr ies of th e ligand, represen ta tive of its conformat ionalchances, can be individually docked. Several binding modesare gener at ed for each ligand conforma tion, with each r eceiv-ing a score dictated by the steric and electrostatic energies

calculat ed by a molecular mechan ics force field.40 -43

The refined model of the ALR2/NADP + complex, withouthydrogen atoms, was used to create the solvent-accessiblemolecular surface in accordance with the Connolly algo-r ithm.61,62 The pr ogr a m S P HGEN wa s use d to ge ner a tespheres th at fill the enzyme active site. The target ar ea wascovered by a man ually edited cluster ma de up of 34 individualspheres. Each sphere was assigned a close contact limitingdistance of 1.3 and 1.8 Å for the polar and nonpolar atoms,resp ectively. A cutoff dista nce of 4.5 Å for “good cont acts ” withthe protein was used.61,62 The contact and force field gridsgenerated using t he DISTMAP a nd CHE MGRID modules inDOCK were used to score the different orientations of theligand bound t o the enzyme. DOCK was run in th e SINGLEmode option by investigat ing all th e possible binding orienta -tions.

The reliability of DOCK and t he validity of th e assu mpt ionsconcerning the modeled enzyme structure were confirmedby attempting the redocking of the crystal structures of zopolrestat and tolrestat. The most stable docking modelsidentified by DOCK closely reproduced the two experimentallydetermined binding m odes.

Each of the above representative 16 low-energy conforma-tions of 3 was a t this point docked into t he ALR2 active site.Interestingly, the top scoring value was achieved by theconformation yielding the best overlay on the ALR2-boundconformation of zopolrestat (Figure 1). Out of the 1305orientations of this conformation, 43 were within 5 kcal/molof th e best orienta tion based on t he scoring fun ction. Insp ectionof the docked structures revealed that the best and many of the top-scoring orientations placed the negatively chargedcarboxylate group of 3 into th e a nionic binding site of ALR2lined by residues Tyr48, His110, and Trp111 to make anetwork of hydrogen bonds. From t his cluster of stru ctures,we selected one (the top-scoring one with a force field score of -43.6 kcal/mol) in which t he benzyl group was locat ed in t hespecificity pocket occupied by the benzothiazole moiety of zopolrestat in th e crystal st ructure.

The param eters of 3 were set consistently with the Cornellet al. force field;53 missing bond and angle parameters wereassigned on the basis of analogy with known parameters inthe databa se and calibrated t o reproduce th e AM1 optimizedgeometry. The complex was solvated by th e a ddition of 217TIP3P water molecules63 within 20 Å of the inhibitor . Thewater molecules alone were minimized (20 000 cycles or 0.1kcal/mol rms d eviation in ener gy) an d equilibrat ed for 5 ps in

a consta nt temperat ure (300 K) bath. The entire system wasthen subjected to SANDER energy minimization (<0.01 kcal/ mol rms deviation) followed by a 200 ps MD run. During thesimulation the positional constraints on the protein backbonewere gradually r educed from 5 to 0.1 kcal Å-2 mol-1. TheSHAKE option was used to constrain bonds involving hydro-gen. A 1 fs time step wa s used a long with a n onbonded cutoff of 8 Å at 1 a t m of constant pressure. The temperature wasmainta ined at 300 K using the Berendsen algorithm 64 with acoupling constant of 0.2 ps. Four snapsh ots, extracted every25 ps from th e last 100 ps MD simulat ion, proved to be verysimilar in term s of rms deviation. An a verage structure wascalculated from the last 100 ps trajectory, and the energy wasminimized using t he steepest descent an d conjugate gradientmethods available within the SANDER module of AMBER.

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