CA1209048A - Compositions of spiro-hydantoin compounds for inhibiting aldose reductase activity - Google Patents

Abstract

ABSTRACT

A pharmaceutical composition comprising:
(a) a compound of general formula:
wherein X and Y, independently, represent a group selected from H, F, Cl, Br, I, -CH3, -OCH3, -SCH3, -S(O)CH3 and -S(O2)CH3; and Z represents a group selected from H, F, Cl, Br, I, -CH3 and -OCH3; with the proviso: (i) that when X represents H and Z represents H or F, Y represents a group selected from Cl, Br, I, -CH3 and -OCH3; and (ii) when Y represents H and Z represents H or F, X
represents a group selected from Cl, Br, I, -CH3 and -OCH3; and (b) a pharmaceutical carrier.

Description

~2~9~4~
J
This is a divisional ~pplica-tion of copending ~pplication serial no. 425,866, filed April 14, 1983.

This invention relates to the method of inhibiting aldose reductase activity with hydantoin derivatives and new and useful hydantoin derivatives in medicinal chemistry. This invention particularly concerns the use of spiro-hydantoin compounds with a ~ubstituted and unsubstituted fluorene rin~ in the treatment of complications arising from diabetes mellitus such as cataracts and neuropathy.
~ urther, this invention relates to a process for the manufacture of spiro-~fluorofluoren-9,4'-imidazolidine)- 2',5'-diones and spiro-ldifluorofluoren-9,4'- imidazolidine)-

2'-,S'-diones. The process invention relates to the general preparation of spiro-(fluoren-9~4'- -i~ida~olidine)-2',5'-diones that are substituted in position 17 2, 3, or 4 with a fluoro group and~or spiro- (difluorofluoren-9-4 ' -imidazolidine)-2',5~-diones which are substituted in a combination of the 1, ~, 3, or 4 positions and in the S, 6, 7, or 8 positions with a ~luoro group. More particularly, the invention is concernea with the preparation of dl-spiro-~2~fluorofluoren-9,4'-imidazolidine)- 2',5'-dione and spiro-~2,7~difluorofluoren-9,4'-imidazolid~ne)-2,5-dione.
In diabete~ mellitus certain tissueC are sub~ect to exposure to high glucose levels. In the lens of the eye, nerves, kidney, retina and other tissues is the enzyme aldose reductaseO The latter is mainly responsible for the reduction of aldoses such as glucose to a polyol such as sorbitol and ~alactose to galactitol at the expense of NADPH. Accumulations of polyols such as sorbitol in diabetic subjects cause and~or are associated with diabetic complications including those of a lenticular, reninal, neuronal and renal nature.

These complications are generally kn~wn as diabetic cataract, retinopathy, neuropathy, and nephropat~y respectivel~ Elevated polyol such as sorbitol or galactitol in the eye lens lead to cataract and ~O5C O~
lens transparency. Elevated polyol such as sorbitol in the peripheral nerves interEeres with normal nervous function. Aldose reductase action on elevated gluc~se and/or galactose is also implicated in causin~
vasculature basement membrane thickening per se in ~he retinal capillaries and certain kidney tissues Aldose reductase inhibitors inhibit the activity of aldose reductase. Hence, aldose reductase inhibitors prevent the production of polyols which directly or indirec~ly lead to the f~rmation of cataracts, reduced nerve conduction~function, ~nd some ~iabetic vascula~ure pathophysiolo~y.
K. Sestanj et al. in the United States Patent No. 3~821,383 describes 1,3-dioxo-lH-benz-~d,33-isoquinoline-2~3H)-acetic acid and derivatives thereof as aldose reductase inhibitors~ Further, the search for effective antidiabetic agents ~o prevent ~r arrest chronic complications such as cataracts, neuropathy and retinopathy has revealed tha~ particu~ar spiro-hydantoin compounds are useful as aldos~ re~uctase inhibitors Sarges, United States Patent No. 4,117,230 describes a series o~ spiro-hydantoin compounds which include the 6-fluoro and 6,8-dichloro derivatives o~
spiro-chroman-imidazolidinediones.
Sarqes, United States Patent No~ 4,130,714 describes enhanced activity of specific dextro~atory spiro-hydantoin compounds such as d-spiro-[6-fluoro-chroman-4,4'-imidazolidine]-~ ione and d-spiro~[6'-fluoro-imidazolidine-4,4'-thiochroman~-2,5' dione in preventing chronic selected diabetic complications.

Sarges et al~, United States Patent N~s.
4,1Bl,728 and 4,181,729 de~cribe spiro~polycyclicimidazolidinedione derivatives and phenyl or phenoxy substituted spiro-imidazo~idinedione derivatives, respectively, which are useful to inhi~it the en~ymatic reduction of aldoses to prevent or reduce harmful and unwanted accumulations of polyols in the lens and nerves.
Methythio and methylsulfinyl (also called methylsulfoxyl) substituted spiro-chroman-imidazolidinediones ~hich are the subject o~ -~
U.S. paten~ 4,464,385 also are useful as aldose reductase inhibitors. With respect t~ sulphur containing derivatives in the instant inventio~ a methylsulfinyl derivative of spiro-Efluoren-9,4~-imidazolidine~-2',5'-dione may be biotransformed ln viv~
to the corresponding more active methylthio aerivative.
There can be an interconversion of the sulfoxide to ~he sulfide by thioredoxin-dependent reductases and/or related glutaredoxins. In the converse, the methylthio derivative can be oxidized by cytochrome containing oxidoreductases to the corresponding methylsulfinyl derivative. These interconversions can be stereospecific with respect to the chirality of ~he resulting sulfoxide. Other sulfoxiae or sul~iny~ dru~s such as Sulindac have been demonstrated to unaerg~ these biotransformations as a function of drug inter~ediary metabolism. Therefore it is believed tha~ the less in vitro active methylsulfinyl derivatives will exhibit greater than expected in vivo activity because o~ this intermediary metabolism.
In accordance with the present in~ention it has been found that compounds of the formulas:

Y~

x ~2~48 where x, y and z are hydrogen, halogen, rnethyl, meth~l-thio, methylsulfinyl, methoxyl, methylsuLfonyl and salts thereof with pharmacologically acceptable cations may be employed as aldose reductase inhibi-tors for the control and prevention of sorbitol levels in the scia-tic nerve and lens of diabetics, to reduce sorbitol levels in the lens o:E glucosemic subjects with resulting control of diabetic complications including diabetic cataracts, and to generally treat diabetic complications including diabetic cataract, neuropathy, retinopathy and nephropathy.
Further in accordance with the present invention it has been found that the novel compounds having the formulas:

NEI

~\ ~
~ ~

Il I .
.

where x is hydrogen, fluoro, methylthio, met~ylsulfinyl and methylsulfonyl and y is fluoro, methylthio, methyl-sulfinyl and methylsulfonyl and pharmaceutically acceptable salts thereof with pharmacologically acceptable cations may be employed as aldose reductase inhibitors fox the control and prevention of sorbitol levels in the sciatic nerve and lens of diabetics, and to reduce sorbitol levels in the lens of glucosemic subjects wi-th resulting control of diabetic complications including diabetic cataract, neuropathy, retinopathy, and nephropathy.

- mab/'~-~

~2~
rrhis application claims a pharmaceutical com-position comprising: (a) a compound of general formula:

~ ~ ~ ; NH>

'~
2: "

wherein X and Y, independently, represent a group selected from H, F, Cl, Br, I, -CH3, -OCH3, -SCH3, -StO)CE~3 and -Sto2)CH3; and Z represents a group selected from H, F, Cl, Br, I, -CH3 and -OCH3; with the proviso: ~i) that when X represents H and Z represents H or Fr Y represents a group selected from Cl, Br, I, -CH3 and -OCH3; and ~ii) when Y represents H and Z represents H or F, X
represents a group selected from Cl, Br, I, -CH3 and -OCH3; and (b) a pharmaceu-tical carrier.
The pharmaceutically acceptable metallic salts can be prepared from the corresponding unneutralized derivatives using conventional methods. Treatment of a derivative with an aqueous solution of the desired pharmaceutically acceptable metallic hydroxide or othex -- '1 cl mab/ ~

~2~ ~5~
metallic base and evaporation of the re~ulting solu~ion to dryness, usually under reduced pressure, will yield the salt. These compounds are uni~ue over the prior art in that they contain a tricylic ring system which is rigid and planar in conjunction wlth a heterocyclic ring The substantial increase in activity and potency of the aforedescribed tetracyclic spiro-hydantoin derivatives as seen over known compounds by in vitro and in vivo assays is unanticipated Prior art does not indicate such unanticipated increase in the activity and potency of such planar, rigid, tetracylic spiro~hydantoin derivatives. Further, the compounds are more active in vitro and in vivo than material found in the prior art and are anticipated to have char~ct~risti-s that will provide useful ~n v~vo hum~r activity and potency. Furthermore, several of the potent symmetrically substituted derivati~es do not require resolution as they are achiral.
It will be understood that these tetracylic spiro-hydantoin derivatives will contain an asymmetric center if the cubstitutio~ pattern on rin~s A an~ C are not symmetric. It is known in the art that ~or this class of molecules, a resolved compound is more p~tent than an unresolved compound. For example, a 2-fluoro substitution renders the molecule asymm2tric at the 9-position of the fluorene ring (the same carbon atom also may be designated to be at position 4' in the spiro hydantoin ring D). A 2,7 difluoro, 2,7-dibromor 3,6 difluoro etc., subctitu~ion pattern results in a symmetric molecule and ~ence no resolution is ~eeded for an increase in activity and potency~
It has also been found that tetcacyclic spiro-xanthene-imidazolidines such as spiro-~2,7-difluroxanthen-9,4'-imidazolidine)-2',5'-aione and spiro~(xanthen-9,4'-imidazolidine)-2'~5'-aione are unexpectedly less active as aldose reductase inhibitors and are not as potent as the correspondin~ tetracyclic fluorene compounds of the invention disclosed herein~

~2~

In accordance with the present process invention, a mono- or di-aminofluorene is reacted ~7ith fluoroboric acid and sodiurn nitrite to yield a mono- or di-diazonium fluoroborate salt wnich upon decomposltion yields a mono- or di-fluorofluorene. The mono- or di-fluoro~luorene is then oxidized to the ketone, a mono- or di-fluoro~luorenone, by oxygen under ~as~c conditions. Alternatively, this oxidation can be conducted with a more conventional oxidation meth~d such as ~otassium permanganate in pyridine This process may be illustrated as follows: - ~

~12 BF~

N~NO2 ~3 ~C-F,H or -NH2 Y=~,H orN2E

2, Tri t~n E~
Kl~nt::~

Z~jF

Z~
In accordance with this generalized scheme, 2-aminofluorene will yield 2-fluorofluorenone~ This ketone, in a specific example of this generalized process, will upon treatment with ammonium carbonate and potassium cyanide in an alcohol, alcohol-and-water or acetamide solvent in a pressure reactor at elevate~
temperature with time will yield dl-spiro-(2-fluorofluoren-9,4'-imidazolidine)-2',5'-dione. A

~ ~ 7.
specific example in the process may be illustrat~d 25 f ol 1 ow s ~

~F K~ ~-t~4)2C~3 . ,~

(~
Step 1 of the reaction is suitably carried out in aqueous solvent at cold temperature. The temperature is critical and should be controlled to less than 1~C.
but abo~7e O~C~ for maximum result. In step ~ ~ the process the decomposition of the diazonium fluoroborate salt should be accomplished in an inert high boilin~
solvent such as xylenes.
Conversion of the fluorofluorene t~ the ke~one in step 3 is accomplished by reacting a tetralkyl- or arylalkyl- trialkyl-ammonium hydroxide in an apr~tic basic solvent, such as anhydrous pyridine, with the fluorofluorene to facilitate oxidation by oxygen. The oxygen can be atmospheric oxygen, but better results are obtained by bubbling pure oxygen into the reactivn mixture, which excludes carbon dioxide, to generate ~he fluorofluorenone. The resulting ketone ~ro~uct can ~e pur;fied by distillation in vacuo. Likewise to step 1, the ratio of reactants in the formation of the ke~one is not critical as long as some excess ratio o~ equimolar amounts of the oxident is used. It is preferred t~
employ an excess of the tetralkyl- or arylalkyltrialkyl-ammonium hydroxide to facilitate a more rapid oxidation. The temperature during this exo~hermic oxidation is pre~erably held at or below the ~eflux temperature of pyridine~

~Z~9~

The ratio o~ reactants in step 4 o~ the reaction is important in the synthesis of the spiro-hydantoin product from the fluoro~luorenone.
Better results are ob~ained when the molar ratio or equivalents of potassium cyanide and ammonium carbonate are 1.5x to 3x and 2x to 5x in excess of the theoretical amount. The preferred solvent is absolute ethanol and the reaction is carried out at elevated temperatures under pressure. Other polar non-ketone or non-ester solvents such as acetamide or ethanol diluted with water can be employed. Purification can be accomplishe~ by diluting the reaction mixture with acid, pre~erably dilute hydrochloric acid, and collecting the c~u~e spiro-hydantoin precipitate. The base soluble and dimethylformamide soluble spiro-hydantoin can ~e puri~ied through a series of activated charcoal treatments, filtration and reprecipitation procedures~
Examples L, M, S and U are given for purposes of illustrating the process invention tExamples ~, M~ P.
Q, R, S, T and U also being given to illustrate the novel compositions of the invention) and such examples are not to be considered as limiting the inven~ion~
PREPAR~TION A
Spiro-(fluoren-9,4'-imidazolidine)-2',~'-dione has the formula:

o ~

Spiro-[fluoren-9,9'--imidazolidinel-2',5'-dione was prepared by the general method of W~ ScCown and .~ ~

~-9 -H. R. Henze as reported in J. AmPr. Chem. Soc 64, (1942) 689. Fluorenone (9 g, 50 mmol.), KCN (6~5 g, 10 mmol), ammonium carbonate (22.8 g) and 80-~ e~hanol (100 mL) were placed in a 200 cc stainless steel reaction vessel. The mixture was heated at 115-125~C. for 4~n hours, cooled and diluted with ice cold 10~ hydrochloric acid. The triturated solid was collected on the filter, washed with water and dried to yield 11.8 g {~5%) r m.p.326-329C. dec. Recrystallization from ethanol yielded needles, m.p. 350-353C. dec., m/e 250 ~or C15HlON202 ' PREPARATION B
Spiro-[2,7-dichlorofluoren-9,4'-imi~azolidlne~-2' ,5'~ dione has the formula '~

c~

This compound was prepared by the me~hod o~
Hsi-Lung Pan and T. L. Fletcher, J. Med. Chem lQ
(1967), 957-959. Spiro-[fluoren-9,4l-imidazol idine~ -2',5'-dione ~5 g, 20 mmol~, FeC13 (0~5 g) an~ acetic acid (350 mL) was stirred at 75C. while a s~luti~n C12 (3.6 y, 50 mmol) in acetic acid (60 mL1 was add~d in one portion. The mixture was stirred with heat fo~
twenty four hoùrsO After cooling water was aaded ana the product filtered. Recrystallization from ethan~l yave 0.9 g of product tl4~); m.p. 355-358DC. dec.;
r 15 8~12N2~
P~EPAXATIO~ C
dl-Spiro-[2-nitrofluoren-9,4'-imidazolidine}-~ o~4~
2 ' ~ 5 ' -dione ha s the formula N~ =

and may be prepared as ~ollows.
Powdered spiro-[fluoren-9,4'-imidazolidine~-2'~5'-dione (5.0 g, 20 mmol) was added ~o a stirred mixture of HN03 ~2 g, 22 mmol) and 60~ ~S04 ~100 mL) at 5~C. Th~n the mixt-.lre was allowed ~o cos~
room temperature and was stirred for twelve hours, ice water was added and the solid was filtered.
Recr~stallization from acetic acid gave a sampler m.p~
309-312~C. dec , m/e 295 for C15HgN304~ -PREPARATION D
Spiro [2,7-dinitrofluoren-~,4'-imidazolidin~l-2',5'-dione has the formula ~`~
~\
~ ~2 ..

It may be prepared according to ~si-Lun~ Pan and T. L. Fletcher, J. Med. Chem 10, (1967~ 957-g59.
Powdered spiro-[~I~oren-9~4~-imidozolidinel-2~r5~-dione ~6.0 ~) was added to a stirred mixture o~ HN03 ~10 g~

~2~
and 6C% H2SO4 (100 mL) at 50-60C. over a period o~
45 minutes. The suspension was stirred 3t the same temperature ~or 5 hours and cooled. Cold water was added and the solid was filtered. Several recr~stallizations from acetic acid gave a sample, m.p.
333-335C. dec., m/e 340 for C15H8N46 PREPA~ATION E
dl~Spiro-[2-bromofluoren-9,4'-imidazolidine~-2', 5'-dione has the formula tJ~ - D

Il . I
'~.D

It may be prepared according to ~ Pan and T. L. Fletcher, J. ~ed. Chem. 10, (1967) 957-959.
2-Bromofluorene (Trans World Che~icalst Inc.3 was oxidized by sodium dichromate to 2-bromofluo~enon~ The product, yellow needles, recryctalized from ethanol gave a m.p. 146-147~C. The ketone (2.6 g~ 10 mmol1, ~C~
~1.3 g, 20 mmol)~ ammonium carbonate t5.0 g~ an~ 80 ethanol l10O mL) were placed in a stainless steel reaction vessel. The mixture was heated at 115-125C~
for 4.0 hours~ The product was collected as i~
Preparation A and recrystallized from ethanol to yiPld white needles (2.7 g), m.p. 350-353C, m~e 328 for C15HgBrN202 .
PREPARATION F
Spiro-[2,7-dibromofluoren-9,4'-imi~az~lidine~-2',5'-dione has the formula Br ~U~

- D

`~~~ \ Zl r ~ 12-and may be prepared as follows.
To 2~7-dibromofluorene (1.5 ~r 4.6 mmol) in pyridine ~50 mL) was added potassium permanaganate (0 73 g, 4.6 mmol) and the mi~ture was stirred overnigh~, Alcohol tlO mL) was added and then water 100 ml and the mixture was extracted with ether tlOO mL, t~;ce). The combined extracts were washed with two portions of 5~
hydrochloric acid (20 mL each) and water ~50 ml~, The ether was evaporated to yield 1.~ g of the ketone needles from benzene, m/e 334 for the ketone, m.p.
196-l970C. for C13H6Br2 ~he keton~ tl,2 g, 3.6 mmol), KCN (.468, 7 mmol) and ammonium carbona~e (1.36 ~) in alcohol ~15 mL) were heated at 105~C~ overnigh~ in a stainless steel pressure reactor. The prod~ct was colle~ted as in Procedure A, the product having a m,p, 340C., m/e 406 for Cl5H8N2o2gr2.
PREPARATION G
.
dl-Spiro-[4-aminofluoren-9,4'-imidazDlidineJ-2',5 '- dione has the ~ormula and may be prepared as follows.
4-Amino-fluorenone (Pfaltz and ~auer~ I~c,) (~
g, 19 mmol), RCN (3.33 g, 51 mmol), ammonium carbonate ~9.9 g) in 95~ alcohol (50 mL) were heate~ in a glass pressure reactor at 105C. for ~4 hours, ~he cooled reaction mixture was poured into 400 ml cold water and adjusted to pH 5 with hydrochloric acid. The precipitate was filtered and redissolved in 5~ NaOH

pLessure reactor at 105C for 24 hours. The cooled reaction mix~ure was poured into 400 ml cold water and adjusted to pH 5 with hydrochloric acid. The precipitate was filtered and redis~olved in ~ N~O~
solution (100 mL) and refiltered throug~ a Celite pad.
Once again the pH was adjusted to an acid pH and the solid collected; recrystallization from hot methanol yielded a product m.p. 340-342C.; m/e 265 for PREPARA~ION H
dl Spiro-[2-aminofluoren-9,4'-imidazolidine]- -2',5'-dione has the formula ~1 ~ .
and may be prepared as follows.
2-Aminofluorenone ~Aldrich Chem}cal Co., Inc~
(1.95 g, 10 mmol), KCN (1.0 g, 15 mmol), ammonium carbonate (2.0 g) and 90~ ethanol (50 mL) ~ere hea~ed in a glass pressure reactvr at 105C for 24 hours~ The cooled reaction mixture was poured into 100 mL c~ld water and the pH was adjusted to 5 with 10~ hydrochl~r}c acid; the precipitate was filtered and redissolve~ in 5%
NaO~ (50 mL) and refiltered. Once again the p~ was adjusted to an acid p~ and solid collected.
Recrystallization from hot methanol yielded a proauct m p 310 314~C m/e~- 265 for C H N O
pP~PAR~TION I
Spiro-[2,7-diaminofluoren-9,4' imidazolidine]-2',5'-dione ha~ the formula ~, ", *trade n~ark ~J~

~ 9~

and may be prepared as follows.
Spiro-[2,7-dinitrofluoren-9,4'-i~idazolidine~-2',5'-dione (4.5 g, 13 mmol) was suspended in 1 liter reagent alcohol~ A stirred suspension o~ 85% hydrazine hydrate t25 mL) and Raney nickel ~1 g~ was added. The mixture was refluxed for two hours and filterea The filtrate was evaporated to yield a light brown solid The product was recrystallized from ethanol, m.p.
340-344C. dec~, m/e 230 for C15H12N42;
HRMS for C15H12N4O2: calc- 280-0960r obs-280~0951, error 0.9 mmu/3.2 ppm.
PREPARATION J
dl~Spiro-[2-iodofluoren-9,4'7 imidazolidine~-2',5'-dione has the formula ' .~
, and may be prepared as follows.
2-Iodofluorenone (Aldrich Chemical C~ ) ~4.0 g, 13 mmol), KCN (1.7 g, 30 mmol~, ammonium car~nate ~6.0 g) in 90~ ethanol (60 mL1 was placed in a stainless steel reaction vessel. The mixture was heated at 110-115C. for 12 hours~ The react;on vessel was cooled, and cold 10~ hydrochloric acid was ad~ed. The precipitate was collected by filtration~ The soli~ w~s dissolved in 5% sodi~m hydroxide and reprecipitated w;th cold 10~ hydrochloric ac;d. The collected solid was recrystallized from acetone and water to yield fine crystals, m.p. 358-359C~, m/e 376 for C15HgIT~202 ~

*trade- mar~

12qJ 90 ~15 13 mmol), KCN (1.7, 30 mmol), ammonium c~rbon~t~ ~6,~ g~
in 9~ ethan~l (60 mL) was placed in a stainless steel reaction vessel. The mixt~re was heated at 110-115C.
for 12 hours. The reaction vessel was co~ledt ~nd cold 10% hydrochloric acid was added. The precipitate was collected b.y filtration. The solid was dissolved in 5 sodium }Iydroxide and reprecipitated with cold 10 hydrochloric acid. The collected solid was recrystallized from acetone and water to ~ield fine crystals, m.p. 358-359C. t m/e~- 376 for C15HgIN202, --15P ~
PREPARATION K
dl-Spiro-~l-methylfluoren-9,4'-imidazolidineJ-2',5'-dione has the formula o `If '~ , ~ .

and may b~ prepared as follows~ -l~Methylfluorenone (2 g, 13 mmol) r RCN ~.7 g~
26 mmol), ammonium carbonate (5r l g) and 95~ ethan~l (50 mL) were placed in a glass pressure reactor ana heated overnight. The reaction mixture was diluted with ~OO mL
5% hydrochloric acid solution and ~he product was collected on a filter. The product was then dissolved in 5~ NaOH solution (150 mL); refiltered; reprecipitate~
with conc. hydrochloric acid. This product was recrystallized from hot methanol: yield 1.5 ~: m.p.
334-336C. dec.; m/e ' 264 for C16H12N2O2.
PREPARATION L
dl-Spiro(2~fluorofluoren-9,4'-imidazoli~ine) 2',5'-dione has the formula:
-.

)4~3 Fluoroboric acid (3000 ml, 48-50%~ i 5 diluted with water (1500 m~) and chilled. 2-Aminofluorene (Aldrich Chemical Co.) (543.7g, 3 mol3 in tetrahydrofuran (1500 ml) was portion wise a~ed with stirring to the chilled fluorboric acid solution ~45QO
mL). A pink brown solid formed during addition. The mixture was stirred until it became homogenous and then was chilled to approximately 5C. with a dry ice in an acetone bath~ A saturated aqueous solution of soaium nitrite (300 g with water to yield 600cc of solution~
was added dropwise while at 3-8C. After ~he addition, the mixture was stirred for one hour. The insoluble green product was collected by filtration an~ washe~
with 5% flouroboric acid (3 x 200 mL) r methanol (300 ~L~ 15% metl~anol ~n ether (5 x 2Q0 mL), e~her ~ ~ 20Q
mL) and then air dried overnight to yield the (dazonium salt (809g, 97~), m.p. 133-135 dec. The diazonium ~alt t800g) was suspended in xylenes (3000 mL) wi~h stirring. The mixture was heated to boiling~ ~s the temperature became 100C. nitrogen gas evol~ed an~
subsided as the temperature became 135C. T~e boiling mixture was filtered hot through a Celite (a diatomaceous filter aid) pad and the pad was washed with hot xylenes (3 x 200 mL~. The combined filtra~es were evaporated in vacuo with heat to dryness. Th~ residue .
was dissolved in boiling hexanes 13000 mL~ th~n filtere~
through a Celite*and Norite*(neutralized cha~coal) ~ilter pad with hot hexanes wash (400-600 mL~ The combined hexanes filtrates then were cooled w~th dry ice. The result;ng white precipitate was collected by filtration. The collected product was washeB with c~ld hexanes (200 mL), suction dried and oven dried at 5Q~C.
to yield 2-fluorofluorene (380g, 71.7%) with a m~p.
97.5-98.0~C. A second crop of product was c~llec~ea by concentrating the hexanes mother liquor, cooling and collecting the precipitate as before (total combined yield ~16g).

*tradc mark ~2~
2-Fluorofluorene (824.7g, g.48 mol~ was diss~lved in pyridine (4L) and stirred~ h 40% sol~tion of Triton B (100 m~ of benzyltrimethylammonium hydr~xide 40~ in pyridine according to a general procedure ~f U.
Sprinzak, J. Amer. Chem. Soc. 80 (1958) 5449) was added and oxygen was bubbled into the vigorously stirred solution. This exothermic reaction was run for 20 hours whereupon an additional portion of the Tri~n B solution (50 mL) was added with continued stirring and oxygen addition for an additional 24 hours. The resulting dark ~reen reaction mixture was treated with Rorite tlO0g~
neutralized charcoal~, stirred for 30'minutes an~
filtered through a Celite pad (diatomaceous ear~h ~llter aid). The filter pad was then washed with pyridine ~lL). The combined filtrates were concentrat~d to a small volume in vacuo with heat. A 5% solution o~
hydrochloric acid in water (lL) was added a~d the pyridine was azeotrophed in vacuo with heat, This process was repeated until a total of 6-8 ~ of~ wa~er were evaporated. The gxanular product was,filtered and washed with water (4L) and air dried. The yellow-orange product was melted and distilled through a large bvre short path distillation apparatus (bp 167~ C~; 1.5 ~m Hg) to yield into a chilled receiving flask the bright yellow 2-fluorofluorenone (6009) with a m.p. 113-115~C, and m/e 198 for C13H7FO.
2-Fluorof~uorenone (300g; 1~52 mol~ ammonium carbonate ~420g1 and potassium cyanide ~120g 7 1.84m~1 were suspended in absolute ethanol ~1.2L~ in a 2L
stainless steel Parr pressure reaction apparatus~ ~he sealed vessel was heated at 95-100C~ with mechanlcal stirring over 42-46 hours. The contents o~ the 2L
vessel was transferred into water (4L). The yellowish water mixture was made acidic by the slow aa~i~ion o~
concentrated hydrochloric acid (50~ m~) so as ~o be pH2~ The resulting precipitate was collected by filtration, washed with water ~4L) and suct;on dried.

*trade mar~
, ~

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The damp solid was partially solubilized wit~ l~J sodi~m hydroxide (l.SL) by stirring. Tne insolubles were removed by filtration through a Celite filter aid and neutralized charcoal pad to obtain a clear ~ol~tion The pad was washed wi~h lN sodium hydroxide (l.OL). The combined filtrates were acidified as before wi~h conc.
hydrochloric acid ~about 200 mL3 to pH2. The white precipitate was collected by ~iltration, washe~ with water (4L), suction dried, and washed with ether (4 X
300mL). The damp solid then was dried at 100~_ for 12 hours to yield dl-spiro-(2-fluorofluoren-9,4'- -imidazolidine)-2',5'-dione ~301 g, 74.3~ r 2B4g 0~ ~is product was dissolved in dimethylformami~e t~ mL) at 40~C. Norite ~40g, neutralized charcoal) was a~ded to the solutior. ~nd the mixture ~tirre~ at 45~_ for 40 min. The mixture was filtered through a Celite* pad and the filtrate was diluted with water ~2L). The solid was collected by filtration with water wash [500 m1~_ The damp solid was dissolved in lN sodium hydroxide ~ an~
treated with Darco G-6~ tFisher Scientific activated charcoal for chromatography) and stirred ~or 35 min at 30~C. The mixture was filtered through a care~lly prepared Celite pad with lN sodium hydroxide soluti~n wash (SOO mL) and water wash ~lL). The combine~
filtrates (2.5L) were neutralized with concentra~ed hydrochloxic acid to pH 6.5. The white pre~ipita~e was collected by filtration and was was~ed with water, air dried and oven dried at lOO~C. for 24 hrs to yiel~
(273g) of dl-spiro-~2~fluorofluoren-9,4'-imida2~1idine]-2~,5'-d;one D
A sample of the product, which was recrystallized rom ethanol, gave a m.p. 315C~ wi~h decomposition, HRMS analysis for C15HgFN202 calc. 268.0648, obs. 268.0659; error 1.1 ~muf4 ~ ppm, C15HgFN202r calc ~C 67.15 ~H 3.38 %~ 10.45 ~F
7.08, obs. %C 67.24 %H 3.56 ~N 10~42 ~F 7.22, obs. ~C

*trade mark , ~213~
--lg--67~17 %H 3.~2 %N 10.~1 ~F 6.98, obs. %C 67.29 %H 3.47 ~.J
10.50 ~ 7.27, obs. %C 67726 %H 3.35 ~N 10.41 ~F 7.03;
IR Spectrum: 3270 cm N-H stretch of II imide, 3170 cm 1; N-H stretch of imide, 3050 cm , Sp C-H stretch, 1775 cm 1, C=O stretch of imide, 1715 cm 1 C-O stretch of imide and amide, 1610, 1590, 1495 and 1455 cm 1 aromatic in-plane carbon stretching modes, 1422 cm 1 N-H in-plane bend of II cyclic amider 868, 830 and 752 cm 1 aromatic C-H out-of-plane deformationO and NMR Spectrum Assignments: delta 11.3 ppm: broad singlet, lH, imide proton; 8.7 ppm: br~aa singlet, lH~ amide proton; 7.9 ppm: multiplet, 2~, aromatic protons; 7.4 ppm: multiplet, 5H, aroma~ic protonsD
_REPARATION M
Spiro-[2,7-difluorofluoren ~4'-imidazolldine]-2',5'-dione has the formula:

~~ \;

can be prepared from 2,7-difluorofluorene which can be obtained from the Alfred Bader Library of Rare Chemicals~ Alternatively, 2,7-difluorofluorene may be prepared in accordance with the followin~ procedure~ To 27 7-diaminofluorene (Aldrich Chemical Co.) ~4.g~ g, 25.4 mmol) in tetrahydrofuran (90 mLl was added water (25 ml) and fluoroboric acid (48-50%, 50 m~). A thick paste of the fluoroborate salt formed~ While stirring and cooliny (5~C.), a saturated agueous solution ~f sodium nitrite (5 g~ was added dropwise while .
~2~

maintaining the temperature at 5-10C. After ad~ition, ~he mixture was stirred for ten minutes and filtered an~
washed with 5% fluoroboric acid, methanol and then ether. The dried product, the bis-diazonium salt, decomposed at 127C. The salt product was then mi~ed with boiling xylenes (50 mL) and heated for 30 minu~es yielding a d~rk tar material, 5.1 g. The tar was isolated and triturated with ether. The et~er was evaporated and the product, 2,7-difluoroflu~rener was recrystallized from ethanol, 3.9 g, m.p~ 80-82.5~C. T~e 2,7-difluorofluorene (2 g, 10 mmol) was dissolved in pyridine (30 mL) and potassium permanganate (1058 9) was added and stirred at room temperature overnight~ ~he reaction mixture was diluted with water and then acidi~ied with 5% HCl. The solid was filtered ana water washed~ The solid was suspended in 100 mL water and a solution of saturated sodium bisulfite and concen~ra~ed hydrochloric acid t7.27g NaHS03, 6.90g of conc. HCl~
was added and the mixture stirred for ~hirty mînute~_ The solid was collected by filtration and then dissolve in ether and refiltered. The ether extracte~ ketone product 2,7-difluorofluorenone (0.9 g3 yave mfe ~ ~16.
2,7-Difluorofluorenone ~43.2 g, 0 2 mol), ~C~
(16.93 g, 0.26 mol), ammonium carbonate (4~.59 y, 8.52 mol) and alcohol were reacted at gO-llO~C~ for 71 hours~ The workup on the hydantoin was in accordance with procedure L. Recrystallization o~ the acid ins~luble product from methanol and acetone was fo~lowed by a base solubili2ation and acid precipita~ion yield o~
sp;ro-[2,7-difluorofluoren-9,4'-imidazolidine3-2',~'-dione t32.8 g, 0.115 mol), m/e~- 286 for C15H8F2N202; m.p. 327-329~ dec.; and Calc~ ~C
62.94 ~H 2.82 ~F 13.28 %N 9.79, Obs. ~C 62~7R ~H 2.86 ~F
13.05 %~ 9.62.
PREPARATION N
dl-Spiro-[2-carboxyfluoren-9,4'-imidazolidine~

` ` ~z~Q~

,51_ dione has the formula C~ os~

and may be prepaxed as follows.
2~Carboxyfluorenone (2.5 g~ 2 mmo~ ~, ~N
(1.5 g, 23 mmol), ammonium carbonate (4.4 g~ and alcohol ~50 mL) were heated at 105~C. in a glass press~re reactor overnight. The reaction mixture was p~ured into 5% ~Cl (100 ~L) and the product filterea. The c~llected product was aissolved in 5% NaO~, refiltered and precipitated with acid, The filtered precipitate was washed with water and air dried havin~ a m.p_ 331 332 C. ec., C16 10 2 4 PREPARATION O
-dl-Spiro-[3-chlorofluoren-9,4'-imidazolidine~-2',5'-dione has the formula ~f and may be prepared as follows.
N-2-~3-chloro-9-oxofluorenyl)-trifluoroacetamide was prepared according to the ~ z~4~

procedure of H. Pan and T. L. ~letcher in J, Med. Chem, 7, ~1964) 31-38. This ketone !3.25 g~ 1~ mmol) was refluxed in 20% hydrochloric acid (100 mL~ for on~ hour and then cooled to 5C, The amine was then dia~otizea with sodium nitrite tl.0 g, 1.5 equivalents)_ Cold 50%
hypophosphorus acid (100 mL) then was slowly added. The mixture then was stored overnight at 5C~, then warmed to ambient temperature, diluted with water and ex~racted with benzene~ Chromato~raphy through alumina with ben2ene as an eluent yielded 3~chlorofluorenone ~1~6 ~). Yellow plates from ethanol had a m~p_ 156-158C, HRMS calc~ 214.0185 for C13H7ClO, obs, 214,0188, error 0.3 mmu/1.4 ppm.
The ketone (1,5 gr 7 mmol), KC~ (0~65 g, 10 mmol), ammonium carbonate (1.5 ~) and 90~ e~hanol t~O
ml) was placed in a stainless steel reaction vessel~
The mixture was heated at 110-115C. for 12 hours_ The reaction vessel was cooled and cold 10~ hyd~ochl~ric acid was added. The triturated solid wa~ collected by ~iltration, The resulting solid was dis~ol~d ;n 5 sodium hydroxide, filtered and the filtrate was acidified with concentrated hydrochloric acid, The precipitated solid was collected by filtra~ion ~na washed with cold water (1.1 ~) yielding a p~oduct wit~
m.p. 330C~ dec., HRMS calc. 284.0352 for C15HgClN2O2, obs. 284.0364, 1.1 mmu/4,2 ppm, PREPARATION P
dl-Sp;ro-[2-methylthiofluoren-~,4~-imi~azolidine]
- 2'~5~-dione has the formula CH -S~

~L2~ ?~
and may be prepa~ed as follows.
The ketone, 2-methylthiofluoren~ne, was prepared by suspending sodium h~dride (14.4g of ~he 50~
by weight mineral oil dispersion, 0.30 mol) in a~h~drous dimethylEormamide (400 mL)~ To this stirred sus~ension under an argon atmosphere was added methylmercap~an as a gas~ After the solution was saturated with methylmercaptan and the sodium hydride reacted, the yellow 2-fluorofluorenone (30g, 0.15 mol) was adaed in one portion. The resulting red solution was heated for ~our hours at 60-80~C~ Water (50 mL) was added and the solvent was removed with heat and reduced pre5sure~ The red residue was dissolved in ethyl acetate (500 ~ ) and extracted with O.lN sodium hydroxide (3 x 200 mL~_ These extractions were followed by washes with ~_lN
hydrochloric acid (200 mL) and ~l~ter ~3 x 200 ml~_ The yellow-orange ethyl acetate solution then was ~re~ted with activated charcoal and anhydrous sodium su~te.
After filtration the ethyl acetate solution was evaporated with heat and reduced pressure ~o yiel~ an oranoe solid which was triturated with ho, hexane~ ~00 mL) and then allowed to cool. The ketone was co~ected by filtration (31.1g, 91~3 r the product having a ~.p.
85C. (reported 84-85~C. by J.A. Parry and K.D. ~rren, J. Chem. Soc. 1965, 4049-4054); PMR (CDC13~ TMS)~
d~lta 2.4 (3H, methyl singlet); delta 6,95-7.7 t7~, aromatic multiplet); and m/e 226 for C14HloOS.
The ketone (22.6g, 0.10 mol), RCN ~13g, 0~20 mol), ammonium carbonate ~30g) and absolute etha~l (approx. 120 mL) were heated in a 200cc stainles5 steel pressure reactor for 15 hours at 105C. After c~oling the contents were poured with stirring onto ice [20~ cc}
and 5N hydrochloric acid ~100 mL). The resultin~ orange suspension then was collected by filtration onto sintered glass. Then lN sodium hydroxide (250 mL~ was filtered with stirring through the collecte~ solld. The bright orange filtrate then was precipitated w;th ~9~
-2~-concentrated hydrochloric acid and collected. ~he ~ir d~ied precipitate then was dissolved in hot dimethylformamide (100mLl. The solution was treate~
with Darco G-60 (activated charcoal sold by ~ischer Scientific Products for chromatography, 49) and fllterea with the aid of Celite Water (400 ml) and ice (100 cc) were added to the filtrate precipitating an off-white solid.
The collected precipitate was then redi~sol~ed in lN sodium hydroxide and treated as before with Darc~
G-60. After acidification the resultins white precipitate was collected and washed thoroughly with water and dried at 100C in a vacuum oven to yield dl-spiro- ~2-rnethylthiofluoren-9 ,4 ' -imidazoliaine] -2~ ,5'--dione (18.6g., 63%) with a m.p. 299-300DC. dec.; ~P~MS
for C16H12N2O2S c:alc. 2~6.0619, obs. ~96.0626, error 0.7 mmu/2.~ ppm; and PMR (DMSO-d6, TSP); delta 2.47 (3H, methyl); delta 7.0-8.8 (9H, aromatic and hydantoin multiplet); and elemental analysis~ calc ~C
64.85 %H 4.08 %N 9.45 %S 10~82. obs. ~C 64~89 ~E 4~12 ~N
9.44 ~S 10.95.
PREPARATION Q
Spiro-E2-(R,S)-methylsulfinylfluoren-9,~R,~
imida~olidine~~ 2',5'-dione has the formula C~3 5~
o ~~
,1 and the unresolved diastereomeric mixture o the four stereoisomers l(R,R),~R,S),(S,S~,(S,R) isomers¦ may ~e prepared as follows.

~trade mar};

The hydantoin product of procedure P(3.0g, 10 mmol) was suspended in 50% acetone and water (ln~ mL) and sodium metaperiodate (2.25g, 10.5 m~ol) was added.
This mixture was stirred at ambient temperature for 72 hours, whereupon 100 mL of lN hydrochloric aci~ were added and the suspension was filtered and washed thoroughly with water. The res~lting sclid was dissolved in lN NaOH (25mL), filtered and the filtrate acidified with concentrated hydrochloric acid.
~iltration and water washes yielded a white product (2.6g, with drying at 100~C.) with a m.p~ 284~286C~ --dec~; HRMS for C16H12N23S calc- 31~056~ obs_ 312.0565 error 0.3 mmu/l.Oppm; and PMR ~DMSO-d6~ TSP~:
delta 2.84 (3H, methyl singlet)l (9H, low ~iel~ ~romatic -and hydantoin multiplet).
PREPARATION R
dl-Spiro-~~methylsulfonylfluoren-9 imidazolidine)-2'5'-dione has the formula CH ~,--S~ ~ _O

and ~ay be prepared as follows.
The hydantoin product o~ procedure P t3.~g, 10 mmol) was suspended in 50~ acetone and water ~1~0 mL), and sodium metaperiodate (6.4~g, 30 mmol) was added.
This mixture was refluxed with stirring for 15 hours, whereupon 100 mL of lN hydrochloric acid and 100 cc of ice were added. The suspension was filtere~ and washed thorou~hly with water and air dried. Further dryin~ in a vacuum oven at 100~C. yielded a white fluffy product .

~Z~ 8 ~3.1 g) with ~ m.p. 309-311~C. dec.; H~S for C16~ll2N204S calc. 328.0518, obs~ 328.0527 error 0.9mmu/2.7ppm; and PMR (DMSO-d6, TSP~: delta 3.30 (3H, methyl singlet); ~9~I, low field aromatic and hydantoin multiplet) .
PREPARATION S
dl-Spiro-[l-fluorofluoren-9,4'-imidaz~lidine]-2',5'-dione has the formula ~,. ".
~ 1 and may be prepared as follows.
l-Fluorofluorenone was prepared from l-aminofluorenone (Pfaltz and Bauer, Inc~) ~y the procedure of T. L. Fletcher and ~l. J. Nam'~ung, Chemistry and Industry, Fehruary 11, 1961, pp. 179-18Q which yielded a ketone product m.p. 109-llO~C., m~e 19 l-Fluorofluorenone (3.96 9, 20 m~ol~, KCN (1~5 g, 30 mmol), ammonium carbonate (4.8 ~1 and 100% e~han~l tlOO ml) were reacted and a product (2.8 g~ collected as in Preparation L, the product having a m.p. 338-~41~C.
dec., HR~S for C15HgFN202 calc. 268.0648, obs~
268.0653 error 0.5 mmu/l.g ppm.
PREPARATIO~ T
dl-Spiro-[3-fluorofluoren-9/4'-imidazolidinel-2',5'-dione has the formula I~,J

2~9~48 and may be prepared as follows.

3-~luorofluorenone was prepared from 3-aminofluorenone by the procedure of T~ L. ~letcher and M. J. Namkung, Chemistry and Industry, E~ebruary 11, :L961, pp. 179-180 and T L. Fletcher et. al_, J. Orq.
Chem., 25, (1960) 1342 yielding the ketone proauct with p 129C. and m/e 198 for C13 7 prepar~tion of the spiro hydantoin was otherwise as in Preparation L, (yield 3.1 g) the resulting pro~uct wit~
a m.p. 328-332C. dec.,and HRMS for C15HgFN202 calc. 268.0648, obs. 268.0653, error D.5 mmu/1_9 ppm_ ~
PREPARATION U
dl-Spiro-[4--fluorofluoren-9,4'-imidaz~lidine~-2',5'-dione has the ~ormula F>~

and May be prepared as follows.

4-Flourofluorenone was prepare~ fro~
4~aminofluorenone (Pfaltz and Bauer, Inc.~ by t~e procedure of T~ L. Fletcher and M. J. Nzmkung, ¢hemistry and Industry, Feb. 11, 1961, pp. 179-180 yielding the ketone product with a m.p. 160-162C and m/e ' lg3 for C13H7FO. The preparation of the spiro hydantoin was otherwise as in Preparation ~ ~yield 3.0 q~ r the resulting product with a m.p. 330-333C. and ~RMS for C15HgE'N2O2 calc. 26~.0648, obs. 268.0660r error 1.2 mmu/A.5 ppm Preparations of dl-Spiro-(1,7-~1fluoFo fluoren-9,A'-imidazolidine)-2',5'-dione;

-2~-dl-spiro-(2~5-di~ rofluoren-9~4~-irnid~zolidine) 2',5t-dione, dl-spiro-(2,6-difluorofluoren-g,4--imidazolidine)-2',5~-dione; spiro-(2-fluoro-7-methYlthiofluoren-9,4'-imidazolidine)-2',5'-dione; and spiro-(2-~luoro-7-methylsulfinylfluoren-9,4'-imidazolidine)-2',5'-dione.
. _ . .
dl-Spiro- (1, 7-difluorofluoren-9,4'-imida zolidi ne) -2',5'-dione can be prepared from l-fluorofluorene_ dl-Spiro-(2,5-difluorofluoren-9,4'-imidazolidine~-~',5'-dione can be prepared from 4-fluorofluorene~
dl~Spiro-~2,6-difluorofluoren-9,4'-imidazolidine~-2',5'-dione can be prepared from 3-fluorofluorene. These fluorene precursors, l-fluorofluorene, 4-flu~rofluorene and 3-1uorofluorene can ~e prepared via the Schiemann reaction from the corresponding aminesr l-aminofluorene, 4-aminofluorene ~nd 3-aminofluorene, a~cor~ing to the procedure of T.L. Fletcher and M.L. Namkung, Chemistr~
and Industry, Februar~ 11, 1961, pp~ 179-180~ The resulting l-fluorofluorene, 4-fluorofluorene and 3-1uoro1uorene derivatives can be nitrated ;n the 7 po~ition using the ~eneral nitration method cited in . Synthesis, Coll. Vol 2, 447 (1943). The resulting nitro derivatives, 1-fluoro-7-nitrofluorene, 4-fluoro-7-nitrofluorene and 3-fluoro-7-nitro~luorene, can be reduced using the general reduction me~hod cited in Org~ Synthesis, Coll. Vol 5, 30 (1973~ t~ yiel~
7-amino-1-fluorofluorene, 2-amino-5-fluoro~luorene ~nd 2-amino-6-fluoro1uorene respectivelyO These amines employing the general procedure of T.L. Fletcher and M.L. ~amkung, Chemistry and Industry, ~ebruary llr 7961 pp. 179-180 can be transformed via the corresponaing diazonium tetrafluoroborate salts into lr7-difluorofluorene, 2,5-difluorofluorene and 2,6-difluorofluorene. These difluorofluorenes can be oxidized to the corresponding ketones using a general procedure o U. Sprinzak, J. Amer. Chem.__Soc., ~0 ~1958) or via procedures cited herein in Procedure ~ or ~ to ~z~

yield: 1,7~difluorofluorenone; 2,5-di~luoro~luorenone;
2,6-difluorofluorenone. As in procedues L an~ M these ketones can be converted into the corresponding spiro-hydantoins:
dl-spiro-(1,7-difluorofluoren-9,4'-imidazolidine) -2',5'-dione; dl-spiro-(2,5-difluorofluoren-9,4'-imidazolidine)-2',5'-dione; dl-spiro-~2~6-difluorofluoren-9,4'-imidazolidine)-2',51-dlone respectively.
dl-Spiro-~2-fluoro-7-methylthio~luoren-9,4'-imidazolidine)-2',5'-dione can be prepared fr~m 2,7-difluorofluorenone. The 2~7-difluoro~luorenone is reacted as in Procedure P except one equivalent of the sodium methylthiolate (prepared from sodium hydride and ~ethylmercaptan)is employed. The resul~ing 2-fluoro-7-me~hylthiofluorenone is synthetically transformed as in procedure L or P to the desired racemic spiro-~2-fluoro-7 methylthiofluoren-9,~'-imidazolidine)-2',5~-dione.
The diastereomeric mixture of four stereoisomers of spiro-(2-fluoro-7-methylsulfinylfluoren-9~4'-imidazolidine~-2',~'~dione can be prepared according to the Procedure Q ~ited herein whereby racemic spiro-(2-fluoro-7-methylthiofluoren-9,4~-imidazolidine)-2',~'~ di~ne is oxidized by sodium metaperiodate to yield the diastereomeric mixture ~f four stereo}somers o~
spiro-(2-fluoro-7-methylsulfinylfluoren-9,4'-imidazolidine)-2',5~-d;one.
Compounds A to U were tested for their ability to inhibit aldose reductase enzyme activity via the procedure of P.F. Rador, L.O. ~erola and ~.H ~inoshita as described in Documenta Ophthamologica, 1~ (1979) 117. The results are shown in Table I~ II and III.
TABLE I
IC*50-Human Aldose Reductase Inhibition ~ct;vity ~z~9~

COMPOUND IC50 ~Molar3 A (unsubstituted) 1,2 x 10 6 L (dl-2-Fluoro~ 9,~ x 10 M (2,7~ luoro) 5,2 x lD
Sarges Resolved ** 6~4 x 10 *ICso = Concentration of dru~ that inhibits 50% o~ the enzyme activity **Sarges Resolved Compound =
d-Spiro-(6~fluorochroman-4,4'-imidazolidine~-2',~'-dione, TABLE II --IC*50~Rat Aldose Reductase Inhibition Activity COMPOUND IC5~ ~Molar) L ldl-2-Fluoro) 1,5 x 10 M (2,7-Difluoro~ 4.4 x 10 8 P (dl-2-Methylthio) 4,2 x 10 ~
Q (dl-2-Methylsulfinyl) 1.7 x 10 6 R (dl-2-Methylsulfonyl) 5~ x 10 6 S (dl-l-Fluoro) . 5~ x 10 U (dl-4-Fluoro) 4.6 x 10 Sarges Racemic ** 4.3 x 10 Sarges Resolved *** 1.5 x 10 * ICso = Concentration of drug that inhibits ~0% of the enzyme activity.
** Sarges Racemic Compound =
dl-Spiro-(6-fluorochroman-4t4'-imidazolidine)-2',5'-dione.
*** Sarges Resolved Compound =
d-Spiro-(6-fluorochroman-4,41~imidazolindine3-2r,5'-dione.

Xt is noteworthy that Compounds L and M are as active or mGre active against human aldose reductase than rat aldose reductase and hence more selective against human aldose reductase than rat aldose reductase when compared to the Sarges resolved compoun~ These ~,Z~ 8 agents will exhibit more activity in humans than the Sarges resolved compound. The Sarges recolved compound shows only one fo~rth as much a~tivity against hum~n aldose reductase as compared to its activity against rat aldose reductase. It is apparent on analysis of ~oth in vitro and in vivo potency data described herein that compounds L and M will exhibit signiicantly greater relative potencies in humans against aldose reductase related diabetic complications. This i5 apparent from comparative studies cited herein on diabetic ana glactosemic rats and in vitro human enzyme inhibition studies. Both the racemic 2-fluo~o (L~ and 2,7-difluor~ -~M) deri~atives are 7X and 12X more acti~e against human aldose reductase than the Sarges resolved co~pound The Sarges resolved compound is found ~o be equiactive against rat aldose reductase to the racemic 2-fluoro ana half as active as the 2,7-difluoro derivative ln vitro, but b~th the 2-fluoro and 2,7-difluoro derivatiYes were found unexpectedly and significantly more ac~ive ln vivo than the Sarges compound in rat studies.
TABLE III
In vitro Aldose Reductase Tnhibiti~n:*
I~50 1~ 10-51o~6 10 7 10 ~ Species A 89 84 49 13 - Human ~ 65 30 12 - - Hu~an C 65 20 8 - - ~uman D 100 76 20 - - ~uman E 100 82 62 33 8 Ra~
- 94 76 55 12 Rat G 50 28 20 - - Human H ~3 31 0 - - Human I 50 25 10 - - ~uman J 94 88 77 39 0 Human K 96 85 47 6 - ~a~:
L 100 88 81 53 18 Human M - 81 71 64 3~ Human N B 9 54 0 - - Rat ~2~
* According to the procedllres o~ P.~ E';ador and M.E.
Sharpless, Biophysical Chemistry, 8 (1972) ~1-85; P~F.
Kador, L.O. Merola and ~.H. Kinoshita Docume~ta Ophthamolo~ica, 18 (1979~ 117.

In Vivo Evaluations A. The effect of dl-spiro-~2-fluoro-fluoren-~,4'-i~idazolidine)-2',5'-dione and spir~-[2,7~difluoro-fluoren-9,4 7 -imidazolidine)-2',5'-~ione on the prevention on retardation of cataracts in rats was studiea using the unresolved dl-spiro-(6-fluorochroman~4,4'-imidazolidine)-2~5'-dione based upon Sarges 4,117,230. The compounds ~ere formulated in ground Purina Rodent Laboratory C~w, Number 5001, containing 30% galactose.
Eighty-~our rats were divided int~ the following seven groups (six treated grou?~ ~aving different compound intake levels and an untreate~
galactose control) having diferent compoun~ intake levels as shown in Table IV~
T~BLE IV
Test Compound Dosages COMPOUND Mg INTAKE,~K~ BODYWE GHT/DAY
L (2-fluoro~ Racemic 4 Mg~Rg~day L (2-fluoro) Racemic 8 Mgf~g~y M ~2,7-difluoro) 4 Mg~g~day M l2,7-difluoro) 8 ~Sg/Kg/~y Sarges Racemic 4 ~Ig~K~day Sarges Racemic ~ Mg~y/day Galactose CONTROL ---The galactose control group ~t~el~e rats, twenty-four eyes) were fed ~round P~rina Rodent Laboratory Chow, No~ 5001 containing 30~ by weigh~

galactose~ The six test compound groups ~twelve rats, twenty-four eyes per group) wer~ fed the same 30%
galactose chow except that the chow was formulated to deliver mg/kgJday of the indicated test compound ~see Table IV).
It is known that young male rats fed a 30%
galactose diet (As previously described) will develop lenticular changes within several days which predicatively progress in time irreversibly to cataracts. Aldose reductase inhibitor compounds which are in vivo active will delay or prevent galac~ose induced cataracto~enesis. In this in vivo expe~iment, the test aldose reductase inhibitor compounds were formulated in the 30% galac~ose feed to evalua~e their _ vivo anticataract activity. The 168 ra~ eyes of the eighty-four rats were observed for the following conditions over a 32 day period.

Normal ~ormal lens, no vacuoles Time to begin Vacuoles just beginnin~ t~
cataract formation appear around lens perime~er Time to vacoule Vacuolization clearly visible on anterior lens sur~ace Time to Small opaque nflecks~ ~rossly snowflake visible in addition to ~acuoles Time to nuclear ~uclear cataract; interior cataract lens completely opaque Observations were made at days 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 3~ and 32_ All rat eyes in treatment L (2-fluoro) ~roups and M (2,7-difluoro) groups were asymptomatic at the ena of 32 days~ All rat eyes in the Sar~es yroups develop~d 9~4~3 -3~~
snowflake-like opacities without any indication of earlier vacuole formation~ The average time to snowflake-like opacities was 15.7 days for the Sarges Racemic 4 Mg/Kg group and 19.7 days for the Sarges Racemic 8 Mg~Kg ~roup. This difference i5 statis~ically ~p C.05) si~nificant. ~at eyes in the galatose control group followed the usual pattern of cataract formations. The average time to snowflake was 11.5 days. This was statistically (pC 05) significantly lower than the avera~e for either Sar~es group.
The "time to cataract~ is summarized in Table V_ TABLE V
Times to Cataract TreatmentXan~e (days) Mean (day) Standard Deviatlon Sarges Racemate 4 Mg/Ky/day 12 to 22 15~68 2~6B

Sarges Racemate 8 Mg/Kg/day 14 to 30 19.68 4,68 The "time to cataract" is on the average 4 days less or Sarges Racemic 4 Mg/Kg/day compound than for the Sarges Racemic 8 Mg/Kg/day compound. This difference is statistically significant at the 95% probability level~
Rat eyes in the galactose control gr~up followed the usual pattern of cataract formation~ The times to each type of cataract activity are summarized in Table VI.
TABLE VI
~

Times to Cataract for Galactose Control Stage ~ tdays) Mean (day) Standard De~iat~on Time to early 3 to 4 3~5B ~.50 vacoules Time to vacoules 4 to 10 6.00 2.00 Time to snow- 8 to 18 11 5~ ~.3 flake Time to nuclear16 to 32 24 04 6.52 catar~ct ~11 but 3 rat eyes (12.5%) developed nuclear cataracts by Day 32. The time to nuclear is estimate~ as 35 days for these three rats in the computation of the mean and standard deviation.
At Day 32, three rats were selected and sacrificed at random from the groups treatea with the racemic 2-fluoro derivative at 4 Mg/Kg/day, ~he racemic ~-fluoro derivative 8 Mg/~g/day, 2,1-difluor~ derivative 4 Mg/Kg/day~ 2,7-diEluoro derivative at ~ Mg~Ky~day ana the Sar~es Racemic compound at 4 and 8 Mg~Kg~day, and the dulcitol levels (galactitol and galatose3 in the lenses were measured.
The average galactitol levels for Sarges Racemic at 4 Mg/Kg/day and Sarges Racemic derivati~ at 8 Mq/K~/day were ~.238 and 9.107 respective7y, an~ are statistically (p~.05) significantly greater tha~ the averages for the racemic 2-fluoro derivative at 4 Mg/Kg/day and the racemic 2-fluoro derivat~ve at 8 Mg/Xg/day, 6.274 and 5.074 respectively, an~ the averages for 2,7-difluoro derivative at 4 and 8 Mg/Kg/day 2.560 and 1.399 respectively. The average galactitol levels for both doses of the 2,7-di~luoro derivative are statistically (p C 05~ less than the average for either dose of the racemic 2-~luoro compound. Both the racemic 2-fluoro and the 2,7-di1uoro derivatives inh;bit catarac~ development.
The 2,7-difluoro derivative is more poten~ than the 2-fluoro compound~ A dose response relationsh;p i5 observed between the 4 and the 8 mg racemic 2- luoro compound.

.

~z~ 8 B. In vivo dose response studies we~e conducted to test the in vivo activity of ~he followin~
compounds by intubation (per oral) in galac~ocemic rats ~n a 30~ galactose diet previously described-L* dl-Spiro-[2-1uorofluoren-9,4'-imidazoli~lne~-2',5'-dione M Spiro-~2,7-difluorofluoren-9,4'-imidazoli~ineJ-2',5'-dione; and Sarges dl-Spiro-[6-fluorochroman-4,4'-imidazolidi~e~-2,5'-dione.
* Compound L and Sarges Racemic are racemic.
Stock suspensions of the test compou~s ana all dilutions of the compound were formulated with 0 03~ by weight Tween 80.
_x~erimental nesi~n Healthy, Charles River CD (outbrea a~blno, Sprague-Dawley derived) male rats, aged 20-30 ~ysr without observable ocular defects and ranging ~n wei~ht from 35-45 grams, were acclimated to experimental conditions (in quarantine) ~or two days prior to beginning the study.
Rats were individually assigned to one o~
twenty-three groups according to a random numb~r scheduleO Two rats, belonging to the same drug-die~
treatment group, were housed per cage. Treatment a~
control groups consisted of six ~63 rats each ~ith n~
replacement in the event of death~ and were designa-~a as follows:
No. Drug Doses~ Mo.
Rats Dose Day (mg/kg~
Sarges Racemic A 6 40.000 1 14 Sarges Racemic B 6 12.600 1 14 Sarges Racemic C 6 4.000 1 14 Sarges Racemic D 6 1.260 1 14 Sarges Racemic E 6 0.400 1 ~
Sarges Racemic F 5 0.126 1 14 Sarges Racemic G 6 0.0~0 1 14 9~

2-fluoro (L)A* 640.000 1 14 2-fluoro (L~B* 612.600 1 14 2-fluoro (L)c* 64.~00 1 14 2-fluoro ~L)D* ~1.260 1 14 2 fluoro (L)E* 60.400 1 14 2-fluoro (L)~* 60.126 1 1 2-fluoro (L)G* 60.040 1 1~

Difluoro (M)A 640.000 1 14 Difluoro ~M~B 612.600 1 14 Difluoro (M)C 64.000 ~ 14 Difluoro (M)D 61~60 ~ 14 Difluoro tM)E 60.400 1 14 Dirluoro (M)F 60.126 1 14 Difluoro (~)G 60.040 1 14 Positive 6 0.000 1 14 Galactocemic Vehicle Control**
Positive 6 0.000 0 14 Galactosemic Control * Racemic ** This group was given an average dose of vehicle bas~
on body weight~
Lenses of all eyes were examined with a hand-held ophthalmoscope in the early stages o ca~arac~
development and a penlight for the stages~
snowflake-like, snow~lake, and nuclear cataract, ~ens grading occurred every day for the first ten study aays, and then every other day until study Day 14, The test was to determine the relative efficacy and potency o~ the dl-2-fluoro and 2,7-diflu~r~
compounds in terms of equal amounts of test drug per kg of body weight relative to Sarge's dl-spiro-[6-fluorochroman-4,4'-imida~olidine~-2',5l-dione, the positive anticata.ract control.

-3~-The relative potency determinativn was m3de ~t 7 drug levels over h~lf log dose intervals The response ~percent cataracts formed and/or ~he time to cataract formation~ was plotted against mg/kg ana/or moles/kg.
Assumptions on ~x~erimental Design The study was directed to the suppression and/or elimination of the initial stages of cataract development characterized by vacuoles or snowflake-like opacities only. It was assumed therefore that suppression and/or elimination of vacuoles o~ lens opacification hy the test drugs affects the transport, absorption and/or metabolism of galactose in such a manner as to prevent subsequent osmotic disturbances normally leading to tissue degeneration and advancea cataract. Advanced cataract defined as stages ~-4 o~
the Sipple classification was assumed to be ~f secondary importance to the etiology of cataract inhibition In the event where cataract stage ~her than vacuole formed, such as snowflake-like opacities, ~his cataract was counted similarly as vacuole in percent cataract formation~
Studies demonstrate that 90% of young ra~s ~50 gram) fed a 30~ galactose diet will develop ~acuole cataracts within 5-7 days. This study yielde~ ~his expected effect in the control groupsv Experimental Procedures All animals were fed Purina Laboratory Chow an~
water, ad libitum, for the two day acclimation perio All drugs were dosed orally by intubation using an animal feeding needle (Popper and Sons~ or 20 gauge, attached to a 1 or 3 ml syringe. Once the aosing time schedule was established, the schedule was followe~
throughout the study to compensate for any ~iurnal variation.
The animals were kept on a twel~e hour day/night cycle. The temperature and humidity were deterrnined each day at the scheduled dosing ~ime ~f 1:00 p.m~
The animals were weighed every other day ~nd adjustments to dosage amounts to compensate ~r weight gain were made each day prior to the dose time. All food was routinely withheld from all anima~ gr~ups for ~our hours (9:00 a.m.) prior to the dru~ dose and restored two hours (3~00 p~m.) post~dose. Otherwise sufficient amounts of chow or diet mixture were maintained in the food hopper to allow the animals to eat _ libitum. There were two days of drug dosag~ wit~
the animals remaining on Purina Laboratory Chow ~ays 1 and 2). The 30~ galactose diet mixture was intro~uced to the animals t~o hours post-dose on Day 3 which ~onstitut~d nay 1 of the study.
Ocular Examination and Grading All eyes were examined with a hand-hel~
ophthalmoscope and/or penlight at the times designa~ed in the Experimental Design section.
It has been found in previous studies ~ha~ -dilation of the eye with a mydriactic (i.e. Myariacyl) is not necessary for accurate grading, due ~ the semi-transparent nature of the rat iris~ In the e~ly stages (i.e.y grades ~ and ~) the lens was observed by reflecting the ophthalmoscope beam of the retina back through the lens and iris. Using this techni~ue, vacuoles appear as dar~, almost opaque patches. In the latter opacity stages (grades SL, S and ~) ~he lens were observed grossly with a penlight.
Each eye was g;ven one of the following grades:
~rade Description ~. .= .= _ - ~ormal lens, no vacuoles Vacuoles just beginning arouna lens perimeter Vacuoles cover greater than 1~3 ~ lens sur~ace 2~

S Lens completely opaque; small opaque flecks~
visible in lens N Nuclear cataract; white crys~alline material present in lens center SL Small opaque "flecks" visible wi~h no vacuole~
present ~snowflake-like) Statistical Analysis The percent of eyes with vacuoles ~c~taracts) and/or the time to vacuole formation were analyzed for each treatment/concentration group~ Since the ~ata were sufficient, the potency of the 2,7-difluoro an~ race.~c 2-fluoro compounds relative to the control were estimated by a probit analysis or other approprlate methodology.
Additionally, the incidence of non forMation (vacuoles, snowflake and nuclear cataract~ were determined at selected time points during the study and used to compare drugs and concentration. This was done in an effort to identify an optimal endpoin~ f~r this type of study.

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9~
-~3-~ = Percent eyes with cataracts fo~med.

Cataract is defined as distinct vacuoles or opacities which affect the transparency Qf the lens or the lens ability to transmit light without significan~ liyht scatter.

dl-Spiro-[6 fluorochroman-4,4'-imidazolidine]-2',5'-dione which is a Pfizer aldose reductase inhibitor.

** Racemic . The same data as in Table VII may ~e illustrated in a different form~t.
TABLE VIII
_ _ Percent of Rat Eye~ (n=12) ~ith at Least Vacuole~ Formed Day 6 Day 7 Day 8 Dav 9 Dav lODay 12 Day 1 Sarges Racemic 40.000 0.0% 0.0~ 0~0~ 0.0% 0-~ 0.0~ O.O~
12.600 0.0~ 0.0% .% -~ Q ~~ 0-3%
4 000 -~ 0.0~ 0.0~ 0.0~ 16~7~ so~%
1.260 0.0% 0.0% 8.3~ 16~7% 83~3~lO0.0~ ~O0.0 0,400 33.3~ 33.3% 50.0% 58.3% g3_3~83.3~ lOO~O~
0.126 33.3% 33.3~ 41.7% 7~.0% lO0~100.~ lOO.O~
0.0~0 0.0% 8.3~ 50.0~ 66~ 100_0%~O.Q~ lOO.O~

Racemic 2-fluoro (L) ~0.000 0.0% 0.0~ 0.0~ 0.0~ ~Q.O~n.0% 0.0 -12.600 0.0% 0.0% 0.0~ 0.0~ o.n~o~o~ o.~
4.000 0.0~ 0.0% 0.~% 0.~% ~.0~~.0~ 0~0 1.260 0.0% 0.0~ 0.0~ 0,0% ~ o_o~ o,~
0.400 0.0% ~.3~ 41.7% 41.7~ 66~3,3~ ~n~.o~
0.126 33.3% 33.3% 50.0~ 50~0~ 50.~~3,3~ ~3, 0.0~0 33.3% 50.0% 83.3~ ~3.3~ 100.0~100_~ lO0.0 ~ 2~ 4~

2,7-difluoro (M~
40,000 OOO~ 0.0% 0.0% 0.0% ~ 0~ 0.0~ 0.0 12.600 0.0~ 0.0% 0.0~ 0.0% ~.0~ ~.0~ D.0 4.000 0.0~ 0.0% 0.0% ~.0~ ~.0% ~.0% 0.0 1.260 0.0~ 0.0% 0.0% 0.0~ 0_0% 0.~% Q.
0.400 ~.0~ 0.0~ 0.0~ 0.0% 0_0~ 0.0% 0.0%
0.126 8.3~25.0%66.7~66.7% 66,7%66_7% 75.0%
0.0~0 50.0~66.7%83.3%83~3% 91_7~100.0% ~00.0%

Vehicle Control 33.3%50~0%100~0~100~0%100_0~1nO~0% 100.0%

Untreated Control 16.7% 58.3% 83.3~ 83,3% 83_3% ~00.0~ 100.0%

At observation day 12, the log rela~ive potencies for eyes with vacuoles or more ad~ance~
cataracts with their 95~ confidence limits-are:

., . ~...

4~
-~5-Log ~5'~ Confidence Pot~ncy I,imitc) 2-fluoro*
(L) relative to Sar~es Racemic 0 660~ (0.38~9 to 0.9346 2,7-difluoro t~) relat.ive to Sarges Racemic 1~1114 ~0.8501 to 1.3852 2,7-difluoro (M) relative to 2-fluoro*(L) 0.4473 ~0.1898 to ~.714~).

- The antilogs of the relative potencies with thei~ 95~ con~idence limits are: -Re~ative (95% Confldence Po~ency Limits) 2-fluoro*
(L) relative to Sarges Racemic 4~5~ ~2.45 ~o 8.60) 2,7-di~luoro ~M~ relative to Sarges Racemic 12~92 (7.08 ~ 24.2 2,7-difluoro (~) relative to 2-fluoro*(L) 2,B~ 55 to 5.18) * Racemic ~z~4~
-~6-C. Polyol Accumulation in Diabetic Rat Lens and Sciactic Nerves As a further test of the effic~cy of the racemic 2-fluoro and the 2,7-difluoro compound versus the Sarges Resolved compound, observation was made ~f the in vivo inhabition of polyol accumulation in the sciactic nerve and lens of rats with streptozotocin induced diabetic rats. The basis and techniques o~ the experiment are described in "Polyol Accumulation In Nervous Tissue Of Rats With Experimental ~iabete-c And-Galactosaemia", M. A. Steward, W. R. Sherm~n~ Mary ~_ Kurien, G~ I. Moonsammy and M. Wisgerhof~ ournal of Neuorchemistry, 14 (196~) 1057-1066.
In the test, eight male Sprague-Dawley rats weighing about 150 g were fasted overnight and given a~
injection o~ streptozotocin the next day The dose was ~-10 mg/100 g of animal weight in an acid citra~e buffer diluted in normal saline. The injection was given in or near the tail vein.
The respective aldose reductase inhibitors (the Sarges Resolved, racemic 2-fluoro, and 2,7-di~luoro compounds) were given to 4 of the rats at 4, 8 an~ 24 hours. The dose o~ 0.15 mg/100 g animal weight was given orally in abou. S ul of water. Each inhi~i~or was mixed in an erude suspension of water with a mortar and pestle. An aliauot of this mixture was ~hen ta~en, using an automatic pipet and fed to each animal.
On the day following the streptozotocin injection, the blood glucose of the ani~als W~5 determined using the glucose oxidase-peroxiaase method.
At 27 hours all the anima~s were sacrificed and the lences and sciatic nerves were removed, weighed and homogenized. These homogenates were ~hen analy7e~ for sorbitol content using the GLC method~ The experimental group (which was fed the inhibitor) was compared with the controls (not fed inhibitor) and the percentage o~
inhibition was determined. Each value represen~s 90D~

comparison of 4 values for control and 4 for experimental. The results of the test are ~hGwn in Table IX.
TABI,~ IX
In vivo Inhibition of Polyol ~ccum~lation in the Sciactic Nerve and Lens of Streptozotocin Induced Diabetic Rats mg/deciliter Percent Inhibition ~E~ Average Blood Glucose* ~erve Lens Sar~es Resolved** 298 + 17 77~ ~3%
~acemic ~-fluoro(L) 425 ~ 39 9~ 92%
2,7-difluoro ~M) 349 + 32 90-95% 95 * Eight animals were used for each study, 4 contr~l, 4 experimental.
**d-Spiro-16-fluorochroman 4,4'-imidazolidin~}-2~,5'-dione D. Nerve Conduction Study In Diabetic Rats The purpose is to evaluate the efficacy o~ the racemic 2-fluoro compound lL), an aldose re~uctase inhibitor, for inhibiting cataractogenesis and ner~e conduction deficit in diabetic rats.
Methods an~ Materials Diabetes was induced in male albino rat~ by ~he intravenous injection of 50 mg/kg streptozot~cin, The diabetic state was verified by measurin~ t~e bloo~
glucose level in each animal before and after in]ection. Diabetic animals were acsiynea randomly to three groups as follows. (l) vehicle trea~ea control;
(2) ~ mg/kg the racemic 2-fluoro compound SL~; ana ~3) 16 mg/kg Sarges Racemic. A fourth group consisted of non-diabetic and otherwise normal rats whic~ remained untreated. Treatments were given onc~ daily by oral lavage~ and c~ntinued through the day prior ~o sacrifice of the animal.
Each animal was we;ghed on a weekly b2sis.
Both lenses of each animal were examine~ by ophthalmoscope periodically and any cataractous chan~e~

were graded and noted. At the end of 15 weeks, ~-ach aniMal was anesthetized usiny pentobarbital and ~he nerve conduction velocities of the sciatic nerve were measured. After an animal was sacrificed, the sciatic nerve and other tissues and organs were taken, w~ighed, and frozen for future biochemical analyses Results Cataractous changes were graded on a scale of 0 to 4; 0 denoting a normal looking lens with no c~an~es and 4 denoting opacification involving the whole lens~
The lenses o~ those rats in the untreated, norma~
control group exhibited no apparent chanyes ana remained optically clear. Of the 32 lenses (16 rats) present at the end of 15 weeks in the vehicle-treated diabe~ic rats, 27 had a score of 4, 3 sco ed 3, and 2 scored 2 on cataractous changes. Thus, every lens ex~ibite~ ~aximal or near maximal opacity. Racemic 2-fluoro compo~nd (L~
was effective in preventin~ opacification of ~he lens in diabetic rats. Of 30 lenses (15 rats) in the Sa~es Racemic-treated group, all appeared normal. Thus, both co~pounds were effective for preventing cataracts; in diabetic rats at 8mg/kg of the racemic ~-~luoro compoun~
and 16mg/kg of the Sar~es Racemic compound~
The record of electrical activity from t~
sciatic nerve in response to electrical stimulat~ion ~f the nerve exhibited two inflections and two peaks;, probably representing different nerve fiber groups~ The effect of trea~ment on each of the four events was determined, and all are considered, although treatment effects appear to be greatest on the first inflection~
By comparison to the normal, untreated controls, the conduction velocities determined for the vehicle-treated, diabetic rats were as follows: first inflection, 83% of normal; first peak, 82% of no~mal;
second inflectiont 80.6% of normal; and, second peak, 76.4~ of normal Thus~ the impairment of nerve conduction, as manifest by a reduction in velocity of ~`:

~2~Q~
, _~,9_ conduction, ranged frorn 17% to 2'.6% and i~ attributa~l~
to the diabetic state of these animals~ In those ~a~
(diabetic) treated with the r~ce.mic ~-fluoro comp~und l~), the nerve conduction velocities as a percent of normal were as follows: first inElectio~. 93_3~; first peak, 8~.2~; second inflection, 87.4%; and s~con~ pea~, 85.0%. In these animals, then, the impair.~ent ran~e~
from 6.7% to 15~, representing an improvement by treatment o~ 34% to 61%. In those diabetic r~ts trea~e~
with Sarges Racemic, the nerve conduc~ion ~elocitie~ as a percent of normal were as follows: first inflec~ionr 88.6~; first peak, 83.9%; second inflection~ ~2.4~;
second pea~, 79.1~. This represents an impalrmen~
ranging from 11~4% to 20.9%, indicatin~ an improve~ent cver controls of 9% to 33%. Therefore, ~ot~ the riacemic 2-fluoro compound (L) and Sarges Racemic produce s~me beneficial effect to improve nerve conduction ~e~city and return it towards normal. .~owever, t~e racemlc 2-fluoro compound (L), at one-half the doce o~ Sa~ge~
Racemic, allows a greater improvement. S~a.istical analysis indicates that only the improvemen. by th~
racemic 2-fluoro compound (L) is significan'~ by comparison to the untreated diab~tic ra.~

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A pharmaceutical composition comprising:
(a) a compound of general formula:
(III) wherein X and Y, independently, represent a group selected from H; F, Cl, Br, I, -CH3, -OCH3, -SCH3, -S(O)CH3 and -S(O2)CH3; and Z represents a group selected from H, F, Cl, Br, I, -CH3 and -OCH3;
with the proviso: (i) that when X represents H and Z
represents H or F, Y represents a group selected from Cl, Br, I, -CH3 and -OCH3; and (ii) when Y represents H and Z represents H or F, X represents a group selected from Cl, Br, I, -CH3 and -OCH3; and (b) a pharmaceutical carrier.