■βf BACKGROUND OF THE INVENTION

5 Field of the Invention

The present invention relates to novel 3-substituted-1-alkylamino-2-propanol derivatives having β-adrenergic blocking properties; to pharmaceutical compositions containing- same and methods of treatment

10 involving their use. Prior Art β-adrenergic blockers were first reported to be useful for the therapeutic treatment of glaucoma in 1967 [Phillips et al, Brit. J. Ophthal., 1967, 5J_, 222] . In

15 1978 timolol was approved for market use and since that time the drug has become very popular with ophthalmologists as an effective antiglaucoma agent. Recently, however, a vast number of serious cardiovascular, respiratory, CNS, and ocular side effects secondary to topical ocular timolol

20 administration has been reported [Ahmad, The Lancet, 1979, 2_, 1028; Buskirk, Ophthalmology, 1980, J37.' 447; Mishra et al, J. Anaesth. , 1983, _55_, 897; and Linkewich et al, Am. J. Hosp. Pharm. , 1981, 3JL' ⁶ "l • Currently, timolol is no longer the sole β-blocker used to treat glaucoma.

25 Befanolol, carteolol and metipranolol were introduced recently and a number of other newer β-adrenergic antagonists (e.g., L-bunolol , betaxolol, celiprolol, cetamolol, etc.) are currently under investigation as antiglaucoma agents.

30. It became desirable to design an antiglaucoma drug which could be delivered to the eye compartments in a sustained and controlled manner with minimal systemic absorption and/or no systemic side effects.

SUBSTITUTE SHEET

It was previously found that after topical application to the eye, esters of adrenalone but not adrenalone itself can be converted via a reduction-hydrolysis sequence to deliver adrenaline (epinephrine) at the iris-ciliary body, the desired site of action [Bodor et al, Exp. Eye. Res., 1984, J3_8_, 621] . Research was conducted to ascertain whether lipophilic ketones could also be reduced in the iris-ciliary body.

It was hypothesized that ketone precursors of β—blockers which are also β-hydroxylamines like adrenaline could then possibly be converted to the active β-blockers in the iris-ciliary body by a reductive process. Various attempts, however., to synthesize the ketones corresponding to a number of β-blockers (i.e., propranolol, timolol, carteolol, etc.) failed, due to the chemical instability of these β-amino-ke one esters.

It is an object of the present invention to provide novel hydrolytically sensitive precursors of the ketone precursors of the β-adrenergic blocking β-hydroxylamines which are readily converted to the active β-blockers in the iris-ciliary body by combined hydrolytic and reductive processes.

SUMMARY OF THE INVENTION

These and other objects are realized by the present invention which provides novel compounds having the formula: Y

I Ar - X - CH2 - C - CH2 - NHR (I)

Wherein: X is -0-, -CH2- or ; Y is =0 or a derivatized keto group, each of which is hydrolyzable or enzymatically convertible to a ketonic group,

R is substituted or unsubstituted alkyl having from 1 to 12 carbon atoms or substituted or unsubstituted aralkyl having from 7 to 20 carbon atoms,said substituents not adversely affecting the β-adrenergic

SUBSTITUTESHEET

blocking and other pharmaceutical properties of the compound and

Ar is the residue of a 1 -alkylamino- 2-propanol having a cyclic substituent at the 3-position thereof, said substituted propanol having- β-adrenergic blocking properties; or acid-addition salts thereof with pharmaceutically acceptable acids.

Another embodiment of the invention comprises a pharmaceutical composition in unit dosage form comprising a β-adrenergic blocking effective amount of one of the above described compounds or salts ^' and a pharmaceutically acceptable carrier therefor.

A further embodiment of the invention comprises a method for treating a human or non-human animal in need thereof comprising administering to the animal a 3-adrenergic blocking effective amount of one of the above described compounds or salts.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated on the discovery that the hydrolytically sensitive oxime-type and other labile ketone groups of the compounds of the invention would enable the delivery to and hydrolysis and reduction of the derivative at the iris-ciliary site of action to the active β-adrenergic blocking amino-alcohol.

The preferred compounds of the invention are those of the formula (I) above wherein is =0, =N-ORT , =N-NH ₂ ,

Wherein: R- _| and R may be the same or different and are H, alkyl having from 1 to 8 carbon atoms or aryl or aralkyl having from 6 to 15 carbon atoms;

SUBSTITUTE SHEET

R3 is R-) , -COOR- _] or

-CON(R-| ) ₂ , and

Ar and R have the meanings set forth above,

It will be understood, however, that Y may be any group or substituent which is readily hydrolyzed or enzymatically converted to a hydroxyl group at the intended site of action, preferably at the cornea or iris-ciliary body after administration of the parent compound to the animal undergoing treatment.

It is further preferred to employ those compounds of the above formula wherein R is a sterically hindered group such as the vertiary alkyls e.g., isopropyl, t-butyl, etc. Suitable aralkyl groups include benzyl, 3,4-dimethoxyρhenethyl, 1-phenethylethyl, etc.; it being understood that by the term, "aralkyl", is intended any hydrocarbyl group.

Ar may be any aromatic or heterocyclic residue of the known 3-aromatic or 3—heterocyclic substituted 1-alkylamino-2-proρanol β-blockers, e.g., those residues having the formulae:

SUBSTITUTE SHEET

CH,

SUBSTITUTE SHEET

- 6 -

Any pharmaceutically acceptable acid may be used to form the acid-addition salts of the invention, e.g., HCl, H2SO4, H3PO4, maleic, succinic, methanesulfonic, citric acids, etc.

The preferred compounds according to the present invention are those having the formulae in Table A.

ABLE A

Ar X - CH2 - Z - CH2 - NH - R

12 Bupranolol .U -cccn ₃ ) ₃

13 Celiprolol lm PX -CCCH ₃ ] ₃

H«CON(C ₂ H ₅ ) ₂

SUBSTITUTE SHEET

TABLE Λ (continued)

Ar - X - CH ₂ - Z - CH ₂ - NH - R

10 ια :-ιi8,ssι 12. cδπ -C(CH ₃ ) ₃ ϋH-,

17 IPS- 39 lr oτ_ιoι -CCCH ₃ ).

18 Labetolol Is _, ca ₂ — (o

19 cvobunolol IX ₃

20 Mepridolol - iϋ ₃ ) ₂

21 Mctlpranolol lv -CH(CH ₃ ) ₂

OCOCH3

22 Me aprolol l -CH(CH ₃ ) ₂

CHjCH-O-CHj

23 L-Moprolol in (or ³ - H(CH ₃ ) ₂

24 Nadolol i£ c°r -C(CH ₃ ) ₃

25 Diacetyl- laa aadoiol -C(CH ₃ ) ₃

26 Oxpireiiolol lbb

SUBSTITUTE SHEET

TΛBLF. Λ (continued)

AC - X - CH ₂ - 2 - CH ₂ - NH - R

- HCCH ₃ ) _Z

0 Z8 Piridolol ldd

0 29 I'ivaloyl lcc -CHCCH ₃ ) ₂

Pirldolol CO-C(CH ₃ ) ₃ HO-CH-CH ₇ NH—

0 3U Solalol Iff - H(CH ₃ ) ₂

0 31 Toliprolol teg -CH(CH ₃ ) _Z

CH-.

Particularly preferred are compounds le, lh, 11, lcc, Ik, lp, It, Iv, lbb, li and Is."

SUBSTITUTE SHEET

- 9 -

The following overall reaction scheme may be employed to prepare the products of the invention:

CH ₂ CH

Λr-OII A-CH ₂

NH ₂ 0H- HC 1

NO) I I Λr-O-CrUC-CrUCl

Λr-0CH ₂ CCH ₂ C1 <r ² δ ²

HHΠ. HΛ

. 5

SUBSTITUTE SHEET

The conventional reaction of Ar-OH and epichlorohydrin with a small amount of morpholine as catalyst affords a mixture of the chlorohydrin 1 and the epoxide 2; the latter being converted to 1 by treatment with cone- HC1. Oxidation of 1 by the Pfitzner-Moffat method [Pfitzner et al, 1965, J. Am. Chem. Soc. , 87, 5661 and 5670] yields the ketone 3. Subsequent reaction of 3 with hydroxylamine HCl gives the oxime 4, which is a mixture of the Z- and E-isomers. In the usual case the ratio is about 2:1, as determined by NMR [Silverstein, "Spectrometric identi ication of organic compounds", 1974, 3rd ed.: G. Clayton Bassler and Terence C. orril, New York, Wiley] .

The major product, 4 (Z-), can be isolated by recrystallization from benzene. Treatment of 4 with isopcopylamine in THF gives the oxime 5 essentially as the pure E-isomer, which can be converted to the HCl- salt 6. Alternately, the oxidation of the racemic 3-blocker OH Ar-0-CH ₂ -CK-CH2-NHR with DCC/(C0C1)2 at -20 ^* - 78 ^*

O II will result in the ketone Ar-0-CH2-C-CH2~NHR, which can be converted to 6 without isolation by adding H2 OH HCl.

The invention is illustrated by the following non-limiting examples wherein melting points were determined with a Fisher-Johns melting point apparatus and are uncorrected. The 90-MHz NMR spectra were taken on a

Varian EM390 NMR spectrometer. T C was performed on

0.25/mm Merk silica gel 60 F-254 glass plates.

SUBSTITUTE SHEET

EXAMPLE 1

The synthesis of the propanolone oxime (6a) is a typical example.

1-Isopropylamino-3-(l-naphthyl oxy)-2-propaπone oxime HCl propranolone oxime (6a)

3-Chloro-1-( 1-nap hyloxy)-2-propanol (la)

A mixture of 1-napthol (20g, 0.14 mole), ephichlorohydrin (51.3g , 0.55 mole), and morpholine (0.7 ml) was heated at 100-120°C for 7.5 h. Excess epichlorohydrin and morpholine were removed under reduced pressure, the residue was dissolved in chloroform and shaken with 10 ml of cone. HCl to convert (2a) to the chlorohydrin (1a). The organic layer was separated and washed with water, then with dil. NaHCθ3 and finally with water. It was dried over anhydrous MgS04 and concentrated to yield 29.8g (98%) of the crude product. This was used in the next step (oxidation) without purification.

Purification of a sample o -the crude (1a) was carried out by column chromatography (silica gel: Aldrich 100-200 mesh, 60 A _x 4W, eluent CHCI3). NMR (CDCI3) δ 8.15 (m, 1H), δ 7.75 (m, 1H) δ 7.5-7.2 (m, 4H), δ 6.7 (d, d _f J=7 Hz, J=1 Hz, 1H), δ 4.35-3.50 (m, 5H), δ 2.9 (d, J=6 Hz, 1H) . 3-Chloro-l-( 1-naph hyloxy)-2-propanone (3a)

To a solution of 1 ,3-dicyclohexylcarbodimide (DCC) (47.1g, 0.228 mole DMSO (36 ml), and pyridine (3.6 ml) in diethyl ether (300 ml) was added a solution of (1a) (18.Og, 76 mmole) in diethyl ether (36 ml). . To this solution was then added dropwise a solution of trifluoroacetic acid (1.8 ml) in diethyl ether under ice-water cooling, and the mixture was stirred at room temperature for 1 h and allowed to stand overnight. A solution of oxalic acid ( 18g ) in MeOH was added to the

SUBSTJTUT.- SHEET

reaction mixture in small portions, and the stirring was continued for 0.5 h. The dicyclohexylurea was filtered and washed with ether. The filtrate was washed with a 5% NaHC03 solution, then with water and dried over anhydrous MgSθ « From the filtrate 6.3g of the desired compound was recovered. The mother liquor was concentrated under reduced pressure and the residue was recrystallized from 2-propanol to yield additional 3.3g. The total yield was 9.6g (Y=56%). This product was used in the next step without further purification. -A pure sample was obtained by column chromatography (silica gel: Aldrich 100-200 mesh, 60 A x 7W, eluent CHC1 ₃ : hexane = 3.1). R CDCl3) δ 8.25 (m, 1H), δ 7.80 (m, 1H), δ 7.65 7.20 (m, 4H) , δ 6.75 (d, J=7 Hz, 1H), δ 4.83 (s, 2H), δ 4,43 (s, 2H). 3-Chloro-1-( 1-naphthyloxy)-2-propanone oxime (4a)

A mixture of (3a) (1.0g, 4.26 mmole) , hydroxylamine hydrochloride (0.36g, 5.1 mmole), and DMSO (10 ml) was heated at 40-60 ^* C for half an hour. Water (40 ml) was introduced and the solution was extracted with CHCI3. The organic layer was washed with water several times, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The crude yield was 1.1g (Y=100%). Further purification was carried out by column chromatography (silica gel: Aldrich 100-200 mesh, 60 A 30W, eluent: benzene:AcOE =4 : 1 ) . The product was a mixture of Z- and E- iso er (Z:E=2:1). This isomer mixture could be used in the next step. NMR (CDCI3 + DMSO-d6 (1 drop) δ 10.85 (s, 0.33H, -NOH of E-isomer), δ 10.75 (s, 0.67H, -NOH of Z-isomer) , δ 8.4-8.1 ( , 1H), δ 7.9-7.65 (m, 1H), δ 7.6-7.2 (m, 4H) , δ 6.95-6.7 (m, 1H), 55.2 (s, 1.33H, ^* OCH ₂ of Z-isomer), δ 4.9 (s, 0.67H, OCH of E-isomer), δ 4.45 (s, 0.67H, -CH ₂ Cl ₍ of E-isomer) δ 4.3 (s, T.33H, -CH ₂ C1 of Z-isomer).

The Z-isomer was isolated from the crude product by recrystallization from benzene. M.p. 162-163 ^* C.

SUBSTITUTE SHEET

1-IsoproρyIamino-3-( 1-naphthyloxy)-2-propanone oxime, Propranolone oxime (5a)

A mixture of (4a) (2.5g, 10 mmole), isopropylamine (6.0g, 8.7 ml, 100 mmole), and THF (50 ml) was heated at 50 ^* C for 1.5 h. The reaction mixture was concentrated under reduced pressure. To the residue was added dil. NaHC03 and the solution was extracted with ethyl acetate. After the organic extract was shaken with dil. HCl solution, the separated aqueous layer was made basic with dil. HCl solution, extracted with AcOEt, dried over anhydrous gS04 , and concentrated under ^' reduced pressure. The crude yield was 2.6g (Y=95%). The crude product was purified from a mixed solvent of isopropyl ether and hexane. The pure yield was 0.98g (Y=36%). M.p. 131.5-132.5 ^* C. This product was the E-isomer only. NMR(CDCl3) δ 8.30-8.20 (m, 1H, one of H of naphthalene), δ 6.90-6.80 (m, 1H, one of H of naphthalene), δ 5.16 (2, 2H, -OCH2-), δ 3.70 (s, 2H, -CH2N-) , δ 3.05-2.70 (m, 1H, N-CHO , 1.10. 1-Isopropylamino-3-( 1-naphthyloxy)-2-propanone oxime hydrochloride, Propranolone oxime Hydrochloride (6a)

To diethyl ether saturated with HCl gas was added a solution of propranolone oxime (5a) (0.30g) in diethyl ether. The mixture was stirred at room temperature for 0.5 h. The precipitated white crystals were filtered and dried in vacuo overnight. The yield was 0.32g (Y=94%). This product was essentially pure E-isomer. NMR (DMSO-d6) δ 12.00 (s, 1H, -NOH), δ 8.30-8.15 (ra, 1H, of naphthalene), δ 7.95-7.80 (m, 1H, of naphthalene), δ 7.65-7.30 (m, 4H, part of naphthalene), δ 7.05-6.95) (m, 1H, one of H of naphthalene), δ 5.15 (s, 211 , -OCH ), δ 3.96 (s, 2H, -CH ₂ N), δ 3.55-3.20 ( , 2H, NCH, -NH) , δ 1.27 (d, J=6 Hz, 60, -(CH ₃ )2). Anal. ⁽ C-| ₆ H2 ₀ θ2 ^N 2' ^HC1) C,H,N.

SHEE

EXAMPLE 2

l-( t-Butylamino)-3-[3-(4-morpholino-1 ,7., -thia izyloxy) ] - 2—prσpanone oxime oxalate ( iinolone oxime oxalate, (6b) 3-Chloro-1-3-(4-morpholino-l ,2 ,5-thiadiazyloxy)-2 pcopanol (lb) was synthesized according to the method given for (1a). Yield 83%; the crude compound was used in the next step. NMR (CDC1 ₃ ) δ 4.5 (d, J=5 Hz, 2H) , δ 4.2 (pentet, J=5 Hz, 1H), δ 3.8- 3.65 (m, 6H) , δ 3.55-3.40 (m, 411). The peak of C-OH cannot be identified. 3-Chloro-1- 3-(4-morpholino-1 ,2,5-thiadiazyloxy)-2- propanone (3b) was synthesized according to the method given for (3a). Yield: 65%. The crude product was used in the next step. Purification of the crude product (recrystallization from 2-propanol) yielded the pure sample. NMR (CDCI3) δ 5.22 (s, 2H) , δ 4.13 (s, 2H) , δ 3.75 (m, 4H, δ 3.50 (m, 4H).

3-Chloro-1-[3-(4-morpholino—1 ,2,5—thia iazyloxy)]- 2-propanone oxime (4b)

In a 500 ml round-bottomed flask were placed (3b) (13.6g, 49 mmole) and hydroxylamine hydrochloride

(5.1g, 73.4 mmole), in an ethanol-DMF mixed solvent (266 ml). The mixture was stirred at room temperature for 18 hours. The reaction mixture was poured into water (2L) and was extracted with ether. The organic extract was washed well with water, dried over anhydrous gS04 , and was concentrated in vacuo at 30 ^* C. The crude yield was 11.8g (Y=83%). The product was a mixture of Z- and E- isomers (Z: E=1.1: 1.0). This crude mixture can be used in the next step. NMR (CDCI3) δ 9.30 (broad s, Q .5U , -NOH of E-isomer), δ 9.10 (broad s, 0.5H -NOH of Z-isomer), δ 5.35 (s, 1H, 0-CH ₂ of Z-isomer) δ 5.10 (s, 1H, E-isomer), δ 4.30 (s, 1H, -CH ₂ -N of E-isomer), δ 4.17 (s, 1H, -CH ₂ -N of Z-isomer), δ 4.8-4.7 (m, 4H) δ 3.6-3.4 (m, 4H).

SUBSTITUTESHEET

1-( ter -Dutylamino)-3- [3 ( 4-mo phol no-1 ,2 ,5-t iad,iazyl- oxy)]-2-propanone oxime, timolone oxime (5b)

In a 250 ml round-bottomed flask fitted with a dropping funnel was placed (4b) (9.8g, 33.5 mmol) in THF (147 ml). The solution was cooled in an ice bath, and a solution of tert-butylamine (12.2g, 168 mmol) in THF (20 ml) was added through the dropping funnel during 10 minutes keeping the reaction temperature -5-0°C (pfl 6-7). The stirring was continued for 1 hour at the same temperature. The solvent was evaporated under reduced pressure at 25 ^β C. To the residue was added diluted HCl (2.8 ml cone. HCl) and it was extracted with ethyl acetate. To the aqueous layer separated was added a dil. NaHC03 solution ( aHCθ3 ^• 3.1g) at -5"C (pH <\» 6-7). It was extracted with ether to remove some impurities. Small amounts of NaHCθ3

(0.1-0.3g) were added to the aqueous layer to make it slightly basic, and the mixture was extracted with ether. This procedure was repeated 4 times (pH was- about 8) and another 3 times after raising the pH to about 9 with dil. NaOH solution. The organic extracts were combined, washed with water, dried over anhydrous MgSθ4, and concentrated in vacuo. Yield 3.1g (Y=28%). The crude product was triturated with isopropyl ether to yield 2.8g. This was recrystallized from isopropyl ether to yield 1.8g (Y=16%) of pure compound.

1-( ert-Butylamino)-3-[3-( 4-morpholino-1 ,2 ,5-thia iazyl- oxy) ]-2-propanone oxime oxalate, timolone oxime oxalate (βb)

In a 50 ml round bottomed flask was placed a solution of oxalic acid (0.20g, 2.22 mmole) in ether (10 ml). To this solution was added a solution of (5b) (0.43g, 1.3 mmole) in ether. The mixture was stirred at room temperature for 1 hour. The white non-hygroscopic crystals were filtered and dried in vacuo. Yield 0.53g (Y=97%). M.p. 165-166 ^* (dec:). NMR (CDCL ₃ ) δ 5.3 (S, 2H),

SUBSTITUTE SHEET

δ 3.9-3.7 ( , 4H), δ 3.6-3.4 (m, 6H) , δ 1.1 (s, 9H). Anal. (Cι ₈ H25θ7N ₅ ) , C,H,N.

EXAMPLE 3

5-13-(5—Bu yl)-2-hydroxylimiπo]-propoxy-3,4-dihyd ocarbo- styril hydrochloride; carteolone oxime HCl (6c) 5-(3-Chloro-2-hydroxy) ropoxy-3 ,4-dihydrocarbostyril ( 1 c)

In a 250 ml round-bottomed flask fitted with a reflux condenser were placed 5-hydroxycarbostyril (15.0g, 92 mmole), epichlocohydrin (34.2g, 0.37 mole), morpholine (1.5 ml), and dioxane (90 ml). The mixture was refluxed for 16 hours then it was concentrated in vacuo (20 mm/Hg) at 80-90 ^* C. To the residue was added 300 ml of 2N HCl, stirred for 15 minutes, then 0.8-1.0 L of ethylacetate was added and the mixture was stirred vigorously for 0.5 hour. The organic layer was separated, washed well with water, then with dil. aHCθ3 and was concentrated in vacuo. Yield: 19.9g (85%). NMR (DMSO-d6) S 10.3 (s, 1H -NH-) , δ 7.25-6.50 (m, 3U, Ph) , s 4.20-3.60 (m, 5H, OCH ₂ CHCH ₂ Cl) , 6 3.00-2.30 (m _r 4H, -CH ₂ CH ₂ CO-).

5-(3-Chloco-2-one)proρoxy-3 ,4- ihydrocarbos yril (3c) was synthesized according to the method described for (3a). NMR (DMSO-d6) δ 10.10 (s, 1H, -NH-) , δ 7.20-7.00 (m, 1H, Ph) , δ 6.65-6.50 ( , 2H, Ph) , δ 4.95 (s, 2H, 0CH ₂ ), δ 4.70 (s, 2H, CH ₂ Cl), δ 3.0-2.8 (m, 2H, -C-CH ₂ CO-), δ 2.5-2.3 (m, 2H, -CH ₂ -C-C0-).

5-(3-Chloro-2-hydroxyimino) Propoxy-3 ,4-dihydrocarbo¬ styril (4c) was synthesized similarly to the method given for (4b). NMR (DMSO-d6) δ 11.88 (s, 0.3H), E- of NOH), δ 11.80 (s, 0.7H, Z- of NOH) δ 10.08 (s, 1H, -NH-) , δ 7.30-6.50 (m, 3H, Ph), δ 4.93 (s, 1.4H, Z- of 0CH ₂ ) δ 4.73 (s, 0.6H, E- of OCH ₂ ), δ 4.3£ (s, 2H, Z & E- of CH ₂ Cl) , δ 3.00- δ 2.80 (m, 211, C-CH ₂ -CO- δ 2.65-2.40 (m, 2H, CH -C-CO) .

SUBSTITUTE SHEET

5-3-( tert-Butyl)-2-hydroxylimino propoxy-3- ,4- dihydrocarbostyril, carteolone oxime (5c)

In a 100 ml round-bottomed flask fitted with a dropping funnel were placed (4c) (2.0g, 7.45 mmole) and THF (70 ml). The solution was cooled to 0 ^β C and a solution of tert-butylamine (0.82g, 1.17 ml, 11.2 mmole) in THF was introduced through the dropping funnel. The mixture was stirred under cooling for 2 hours. To the reactive mixture was added a solution of oxalic acid (1.48g, 16.4 mmole) in THF. The precipitate was filtered, triturated with water (600-700 ml) by sticring well for 15 minutes, and it was filtered again. The filtrate was extracted with ethylacetate several times. The aqueous layer was cooled to 0 ^* C, basified with a dil. NaHC03 solution ( aIlCθ3 0.81g), and was immediately extracted with ethyl acetate. The extract was evaporated in vacuo (20 mHg) at 30°C. Yield: 0.63g (28%). Recrystallized from i-propanol, the product was E-isomer. M.p. 177-180°C (dec.) NMR (DMS0-d ₆ ) δ !1.PPM(S, 1H), δ 10.1 (S, 1H), δ 7.3-7.1 (m, 1H), δ 6.7-6.5 (m, 2H), δ 4.9 (S, 2H), δ 3.3 (S, 3H contain NH), δ 3.0-2.8 (m, 211), δ 2.6-2.3 (m, 2H), δ 1.05 (S, 9H).

5-3-( tert-Butyl)-2-hydroxylimino propoxy-3 ,4-dihydro¬ carbostyril Hydrochloride, carteolone oxime hydrochloride (6c)

The free base (5c) was converted to the hydrochloride salt (6c) in ether with HCl gas. M.p. 167-169 ^* C (dec). Anal. (C ₁₆ H ₂ 4θ ₃ Cl) C,H,N.

EXAMPLE 4

1-Isopropyla inp-3- [3-(4-morpholino-1 ,2,5-thiadiazyloxy)] - 2-propanone oxime (5d) in a 200 ml round-bottomed flask were placed

3-chloro-1- [3-(4-morpholino-1 ,2,5-thia iazyloxy) ]-2-

SUBSTITUTE SHEET

propanone oxime (4b) (3.53g, 12.1 mmole), isopropylamine (3.56g, 60.3 mmole), and THF (71 ml). The mixture was stirred at room temperture for 2.5 hours. The reaction mixture was concentrated in vacuo at room temperature. The residue was triturated with isopropyl ether and precipitated crystals were filtered with suction. The crystals were dissolved in dil. HCl solution. To the solution was added ether and NaIIC03 in small portions under vigorous stirring conditions. The organic layer was washed with water and dried over anhydrous MgSθ4, and concentrated in vacuo. Yield: 0.42g (Y=11.6%) from isopropyl ether.

1-Isopropylamino-3-[3-(4-morpholino-1 ,2 ,5-thiadiazyl- oxy) 3-2-propanone oxime Hydrochloride (6d) The oxime (4b) (0.25g) was dissolved in ether and ether saturated with HCl was introduced dropwise into the solution. The mixture was stirred for 10 minutes, filtered and dried in vacuo overnight. Yield: 89%. Anal. (C ₁₂ H ₂₂ N5θ ₃ S Cl) C,H,N.

Elemental Analyses

1 - 1-Isopropylamino-3-( 1 -Naphthyloxy) -2-propanone oxime , propranolone oxime hydrochloride ( 6a) , C _{1 6} H _{2 T} 0 ₂ N ₂ CL.

Calc : C, 62.23 ; H, 6.85 ; N, 9 .07 Found: C , 62.32; H, 6.89 ; N , 9.05.

2 - l-( -Butylamino-3-[3-(4-morpholino-1 ,2,5-thiadizyl- oxy)]-2-propanone oxime oxalate, timolone oxime oxalate (6b) , C ₁₅ H25θ7N ₅ S C ₁₃ H ₂ 3θ ₃ N5S- (COOH) ₂ .

Calc: C, 42.95; H, 6.01; N, 16.70 Found: C, 43.00; H, 6.04; N, 16.67.

SUBSTITUTE SHEET

3 - 5- [3-( t-Butyl)-2-hydroxylimino]-propoxy-3,4- dihydrocarbostyril hydrochloride; carteolone oxime HCL (6c), C16H24O3N3C .

Calc: ^' C, 56.22; H, 7.08; N, 12.30 Found: C, 56.10; H, 7.13; N, 12.21

4 - 1-Isopropylamino-3- [4-morpholino-1 ,2,5-thiadiazyl¬ oxy)]-2-propanone oxime hydrochloride (6d),

^C 12 ^H 22 ^N 5°3 ^{S L} - 3 H ₂ 0.

Calc: C, 40.27; H, 6.38; N, 19.57 Found: C, 40.54; H, 6.42; N, 19.46

The following non-limiting examples illustrate the pharmacological properties of the compounds of the invention.

EXAMPLE 5

Effect on the Intraocular Pressure (IOP) of Rabbits:

Adult male New Zealand albino rabbits weighing 2.5 - 3.5 kg were used. The animals were kept in individual cages with free access to food and water.

Intraocular pressure was measured using a.Digilab model 30R pneumatonometer. The pneuma onometer readings were checked at least twice a day using the Digilab calibration verifier. All measurements were obtained from unrestrained, unanesthetized rabbits. One drop of 0.5% propacaine (Ophthetic-Allergan Pharmaceuticals, Inc.) diluted 1:2 with saline was instilled in each eye immediately prior to IOP measurement. Drugs were administered as 1 or 2.5% solution in buffer pH 7.4 or in saline in both eyes of a group of at least four rabbits. Another group of at least three rabbits served as control and was administered the carrier only. IOP was recorded after 30 and 60 minutes and then after 2, 3, 4, 6 and 8 hours following the drug or carrier administration. Values are given as means i standard error (S.E.) of the mean.

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The significance of the change was determined using the student's t-test.

The animals were also observed for local action of the drugs on the eyes, e.g., irritation, congestion, redness, lacrimation, etc

The results are set forth in Tables 1 - 3.

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Table 1 Effect of 15 solutions of propranolol HCl.(la) and propranolone oxlme'HCl (6a ) on the IOP

(mm/Hg) of rabbits.

Propranolol Hcl (la) IS Propranolone oxime Hcl (6a ) (1 _* )

change ' change

Time after after after to ad i ni strati on Control Treated treatment Control Treated treatment c

CO I CO

H to

Zero 28.8 ± 0.33 30.8 ± 0.40 0.00 29.3 ± 0.50 27.8 ± 0.62 0.00 e 30 mi n. 32.0 •+ 0.44 28.0 ± 0.56 -9.10* 28.2 ± 0.65 26.9 ± 0.58 -3.23 60 mi n . 32.7 ± 0.26 27.6 ± 0.62 ^' 10.39* 29.1 + 0.60 24.0 ± 0.60 -11.51*

(n X ' . 2 hrs. 31.3 i 0.31 29.1 ± 0.38 5.52 27.7 ± 0.57 23.4 ± .-0.51 -15.82** m 3 hrs . 30.8 ± 0.32 27.4 ± 0.54 11.04* 26.3 ± 0.40 23.3 ± 0.35 -16.18**

H

4 hrs. 29.9 ± 0.61 28.2 ± 0.48 -8.44* 26.8 ± 0.33 22.2 i 0.42 -20.14** 6 hrs . 30.8 i 0.38 29.0 ± 0.45 -5.84 28.8 ± 0.52 ^" 25.8 ϊ 0.56 -7.08* 8 hrs. 30.7 ± 0.26 30.2 ± 0.43 -1.95 29.2 ± 0.51 27.9 ± 0.62 +0.50

. ^♦ Significant decrease 1n I.O.P. (P<0.05) **H1ghly significant decrease in I.O.P. (P<0.01 )

Table 2 Effect of 2.5X solutions of propranolol .HCl (la) and propranol.ne oxime HCl, (6a ) on the IOP (mm/Hg) of rabbits.

Propranolol 'HCl (la) (2.5Ϊ) Propranolone oxime'HCl (6a ) (2.55)

% change 5 change

Time after after after to Administration Control Treated treatment Control Treatment treatment c

CD ω Zero 26.6+0.56 25.4+0.56 0.00 25.8+0,55 26.0+0.48 0.00

H 30 min. 28.0±0.93 27.6i0.56 +8.66* 27.2+0.82 26.510.63 +1.92 ^• ιo 60 min. 26.6±0.51 27.9i0.60 +9.84* 26.2+0.63 23. 10.55 -10.00* to rπ

2 hrs. 24.6+0.19 25.710.55 + 1.18 26.3+0.71 22.6+0.43 -13,08* tn

3 hrs . 25.6i0.31 25.710.39 +1.18 26.710.66 21.210.28 -18.46* m m 4 hrs . 25.2±0.64 25.610.46 + 0.79 26,810.34 20.410.34 -21.54** 6 hrs. 26. 10.35 25.410.54 0.00 25.9+0.54 23.610.48 -9.23* 8 hrs. 26.010.52 25.210.68 -0.79 26.010.47 25.810.65 -0.77

*Sign1ficant change (P<0.05) **Hlghly significant change (P<0.01)

Table 3 Effect of lϊ of solutions of timolol maleate (lb) and timolone oxime oxalate (6b ) on the IOP (tim/Hg) of rabbits

Timolol maleate (lb) IS Timolone oxime oxlate (6b) 1*

to -Change 5 change c m Time after after after to admini stration Control Drug-treated treatment Control Drug-treated treatment

H

0 28.7210.54 29.1910.78 0.00 28.7210. 4 28.441.63 0.00 ω 30 min . 26.62+0.59 26.0011.23 -10.93* 26.62+0.59 27 .7510.68 -2.43 t

1 hr. 28.10+0.85 27.25+1.12 6.65 28.10+0.85 24.30+0.63 -14.55** m m 2 hr. 27.0210.92 27.2110.58 6.78 27.0210.92 24.0010.44 -15.61** H

3 hr . 25.95+0.44 25.00+0.87 14.35** 25.9510. 4 25.13+0.96 -11.64**

4 hr . 26.45+0.57 24.88+1 .00 14.77** 26.45+0.57 25.93+0.48 -5.31

5 hr. 27.0910.55 24.2511.18 16.92** 27.0910.55 26.8810.52 -5.49

6 hr. 27 .88+0.56 25.5810.85 12.37** 27.8810.56 28.7810.91 +1.20

8 hr. 27.6710.63 26.3310.40 9.80* 27.6710.63 26.3310.40 -7.42

^♦ Signi f icant change (P<0.05)

** Hi ghly si gni fi cant change (P <0.01 ) .

These results reveal that the ketoxime analogs of both propranolol and timolol display a certain degree of ocular hypotensive activity. Propranolone ketoxime (6a) has shown the highest activity at both tested concentration levels, 1 and 2.5%. This activity was much more pronounced and prolonged than that of propranolol itself administered at the same dose levels (Tables 1 and 2). In addition, the ketoxime (6a) was completely devoid of the ocular irritation which always accompanied propranolol administration at both dose levels. This irritant activity might have contributed to the reduced action of propranolol on the IOP at the 1% dose level and, also, might have completely masked its ocular hypotensive activity at the 2.5% dose level. Timolone ketoxime (6b) has also shown a significant ocular hypotensive activity which was faster in its onset and shorter in its duration than timolol (1b) itself (Table 3). On the other hand, the other ketoxime precursors, the ones for. the N-isopropyl analog (6d) of (6b) and (.6c) for carteolol, showed low activity at the dose levels used but showed some β-antagonist activity.

EXAMPLE 6 -

Effect on resting heart rate and on the isopreπaline- induced tachycardia in rats:

Male Sprague-Dawley rats weighing 150-250 g were used. Each animal was anesthetized with sodium pentobarbital (50 mg/kg) and the jugular vein was cannulated with PE50 tubing. This cannula was subcutaneously threaded around the neck and exteriorized dorsally. The cannula was filled with heparin solution (1000/μl) and sealed with a solid 22-gauge stylet. Animals were housed in individual stainless steel cages and at least 24 hours were allowed for recovery from the surgery. Food and water were provided ad libitum. On the day of

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the experiment, the heart rate of each rat was monitored with a plethsmograph and the data recorded on a Physioscribe II recorder. One hour was allowed as an equilibration period before any drugs were administered. Drugs were dissolved in normal saline as 0.3% solution and were administered intravenously at a dose of 6 mg/kg. The resting heart rate was then recorded after 1, 3, 5, 10 and 15 minutes following i.v. injection. Isoprenaline {Isoproterenol bitartarate) , was then administered subcutaneously at a dose of 50 μg/kg and the heart rate was recorded for 3, 5, 10, 15, 20, 30, 45 and 60 minutes after administration. A control group of seven animals was intravenously administered saline solution and was treated exactly in the same manner as the drug-treated groups. The significance of the difference between the effect of saline solution and the drugs under investigation on the resting heart rate and on isoprenaline tachycardia was analyzed using the student's "t" test. Values are given as mean ± S.E. of the mean. The results are depicted in Figure 1 which depicts the mean change in heart rate over time for ( □ ) propranolol HCl (1a), ( A ) timolol maleate (1b), ( t ) carteolol HCl (1c), ( p ) proprano¬ lone ketoxime HC1 (6a), ( V ) timolone ketoxime oxalate (6b), ( Δ ) N-isopropyl timolone ketoxime HCl (6d), ( B ) carteolone oxime HCl (6c) and (- - -) saline solution.

The results of this study revealed that most of the tested ketoximes exhibit a negative chronotropic action in rats. Again, the ketoximes of propranol (6a) and timolol (6b) have shown the highest activity in this test, whereas carteolone ketoxime' (6c) and the oxime (6d) were less active. It should be also noted that in this test carteolol (1c) itself has shown the lowest activity on the heart rate of rats.

suBST

EXAMPLE 7

Determination of 3-adrenergic antagonist activity.

In another set of experiments the effect of the oral administration of propranolol Hcl (1a) and propranolone oxime HC1 (6a) in doses of 25, 50 and 100 mg/kg on the resting heart rate and isoprenaline- tach cardia was evaluated in rats. Drugs were administered to groups of 5 rats using a stomach tube and the heart rate was re ^' corded for 1 hour. Then isoprenaline (50 μg/kg, s ^' .c) was administered and the heart rate was recorded after 3, 5, 10, 15, 20, 30, 45 and 60 minutes following administration. A control group of 5 rats was treated exactly in the same manner after the oral administration of the appropriate volume of saline solution.

The potential 3-adrenergic antagonist activity of the ketoxime precursors of propranolol, timolol and carteolol was assessed against isoprenaline-tachycardia using the parent compounds as obvious reference drugs. Results of this set of experiments were in agreement with the findings of the above studies, i.e., effect on the IOP and resting heart rate. Thus, the ketoxime precursors of propranolol (6a) and timolol (6b) were the most effective whereas (6c) and (6d) were the least active. See Figure 2.

These results indicate that at least two of the investigated ketoxime precursors (12a and 12b) have an antiglaucoma activity which is probably linked to their β-adrenergic antagonistic properties. Yet, whether these properties are due to an inherent intrinsic activity of the ketoximes themselves or are the result of their active biological conversion to their parent drugs needed to be verified. For this reason the in vivo disposition of the

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different ketoximes and their parent β-blockers in the different ocular tissues was studied in rabbits.

EXAMPLE 8

In vivo distribution - metabolism studies: A. In ocular tissues of rabbits:

Adult male New Zealand albino rabbits weighing 2.5-3.5 kg were used. Standard doses of 100 μl of 1% solution of the drugs in saline solution were administered topically to both eyes of each rabbit. After appropriate time intervals (30, 60 and 120 minues), the animals were sacrificed. Aqueous humor was obtained by making a sinqle puncture at the limbus using a 25 g x 5/8" needle attached to 1 c.c syringe. Then the cornea and the iris-ciliary body were isolated. The tissues were pooled and homogenized using a Tekmar SDT tissuemizer in ice cold perchloric acid (0.05 M) which contained 0.05% sodium etabisulfite as antioxidant. Samples were then rehomogenized in CH3OH to prepare 10% homogenates, transferred to micro-filters and centrifuged for 20 minutes at 10000 r/minute to precipitate proteins. Aqueous humor was analyzed as such without any further dilution. Aliquots of 5-20 μl of the 10% tissue homogenate samples were analyzed by HPLC. Quantitation was done by using a calibration curve obtained by the addition of known amounts of the compound to aqueous humor, iris-ciliary body or cornea obtained from a control rabbit after topical administration of saline solution. B. In rat's blood:

A group of seven adult male Sprague-Dawley rats weighing 150-250 g was used. Animals were intrajugularly injected with propranolone oxime (6a) at a dose of 6 mq/kg. After 1, 3, 5, 20, 40 and 60 minutes, one ml of blood was

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withdrawn from the jugular vein and dropped immediately into a tared tube containing 1 ml of ice-cold acetonitrile. The tubes were vigorously shaken, centrifuged, decanted and analyzed for propranolol (1a) and propranolone oxime (5a) by HPLC. Quantitation was done by using a calibration curve obtained by addition of known amounts of propranolol oxime HCl (6a) to blood obtained from a control rat pretreated with saline solution.

The results are set forth in Tables 4 and 5.

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Table 4 Tissue Concentration^ of propranolol (la) and propranolone oxime (5a) at various time Intervals following topical administration of propranolone oxime.HCl ( 6a ) (lϊ solution)

Concentration of propranolone oxime (5a) Concentration of Propranolol (la) (mcg/g tissue) (mcg/g tissue) to c cα to Tissue/Time 30 min. 60 min. 120 min. 30 min. 60 min. 120 min.

H

H t

H Cornea 23.75+4.91 16.4015.80 0.00+0.00 1.68+0.75 1.14+0.29 1.1410.22

VI

(J)

Iris-Ciliary 7.79+1.10 0.00+0.00 O.OO+O.OO 2.11+0.29 1.79+0.20 0.43+0.11 m m H

Aqueous 0.8210.09 0.8010.06 O.OOiO.OO 0.0410.02 0.7U0.11 - O.OOiO.OO Humor

^a Figures represent the mean 1 S.E. of the mean of at least 4 rabbits.

- 30 -

Table 5

Tissue Conceπtration ^a of propranolol at various timeinterval s following topical administration of propranolol .HCl (la)(ll solution ).

Concentration of propranolol (mcg/g tissue)

Tissue/Time 30 min. 60 min, 120 min.

Cornea 47.1015.57 14.54±2.97 0.00+0.00

Iris-ciliary 8.05±1.47 O.OO±O.OO 0.00+0.00- body

Aqueous 1.28+0.19 0.26±0.08 0.00+0.00 ^' humor

Figures represent the mean ± S.E. of the mean of four rabbits

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The results of this study show that propranolol (1a) could be detected in measurable concentrations in the different eye compartments for the first two hours following the topical administration of its ketoxime precursor (6a) at its effective ocular hypotensive dose level (1%) (Table 4). On the other hand, propranolol could not be detected in any of the tested eye tissues two hours after its ocular application (Table 5), and it had completely disappeared from the iris-ciliary body which is supposed to be the site of its ocular hypotensive action, one hour after administration. These results might explain the shorter duration of propranolol action on the IOP relative to that of its ketoxime precursor. In addition, these results might also suggest that the ocular hypotensive activity of the oxime is most probably due to its active conversion to propranolol in situ in the ocular tissues of rabbits. This is also supported by the finding that following the ophthalmic administration of the other ketoxime precursors (6b and 6c) of timolol and carteolol, respectively, at the low dose level used, the parent β-adrenergic antagonists in any of the eye compartments could not be found. This would suggest that either the ketone formed or the reduced form, the active β-blocker, is disposed of so fast that it cannot be detected, or that the ketones are not such good substrates for the reductase enzyme as the propanolone ketoxime.

Based on the previous observation of the necessity to convert adrenalone to lipophilic esters to be reduced, one could expect that the lipophilic propranolone is easily reduced, while the ketones derived from timolol and carteolol, being less lipophilic (heterocyclic ^• substitution of naphthalene), are not reduced that extensively. The N-isopropyl analog (6d) of timolol was synthesized and tested in order to assess the importance of the N-alkyl function. Propranolol contains an i-propyl

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group, like 6d, but 6d was still found inactive. The difference in the behavior of the 6a vs. 6b-d thus might be due to the difference in the Ar-group which appears to determine the substrate properties necessary to bind to the reductase enzyme. This hypothesis is supported by the relative HPLC retention times of the free bases 5a-5d which were 12.86, 7.22, 3.10, 6.10 for 5a, 5b, 5c and 5d, respectively, indicating that 5a is by far the most lipophilic It is believed that the compounds of the invention are converted to their parent β-blockers according to the following scheme A which shows the conversion of 5a to la. Similar conversions would follow scheme B. The hydrolytically sensitive precursors of the present invention comprise effective chemical delivery systems (CDS) for the β-blockers and intraocular pressure reducing agents.

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SCHEME A

to 5α α rπ m -I SCHEME B

N-OH 0 OH

H?0 II

;AΓ-CH ₂ -C- CH.-NHR * Ar-CH ^• C-CH ₂ -NHR - Ar-C-CH ₂ -NHR

The following example illustrates the biotransformation pathway of 6a after systemic administration.

EXAMPLE 9

ANALYTICAL METHOD

A high pressure liquid chromatography (HPLC) method was developed for .the assay of the β-blockers and their ketoxime analogs in biological fluids. The chromatographic analysis was performed on a system consisting of Beckman Model 112 solvent delivery system, Model 340 Injector, and Waters Model 481 variable wave length LC spectrophotometer. An ASI reverse phase chrompack C- _j g column, operated at ambient temperature, was used for all separations. The mobile phase used for separation of propranolol (1a) and propranolone oxime (5a) consisted of water, 1-heρtane sulfonic acid, 0.1M acetic acid, O.IM triethanolamine and methanol (90, 1g, 100, 799). With a flow rate of 1.5 ml/minute, the two compounds showed retention times of 2.44 and 3.21 minutes for propranolone oxime and propranolol, respectively. The mobile phase used for separation of carteolol (1c), carteolone oxime (5c), timolol (1b), timolone oxime (5b) and timolone ispropyl oxime (5d) consisted of water, 1-heptane sulfonic acid,

0.1M acetic acid, tetrahydrofuran, O.IM triethanolamine and methanol (430, 2, 40, 30, 100 and 398). With a flow rate of 1.5 ml/minute, the retention times for these compounds were 3.10, 3.54, 6.10, 7.22 and 9.15 minutes for carteolone oxime (5c), carteolol (1c), timolone isopropyl oxime (5d), timolone oxime (5b) and timolol (1b) , respectively.

The results are depicted in Figure 2 which is a plot of blood levels (μg/ml) vs. time of 5a after administration of 6a at a dose level of 6 mg/kg to rats.

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These studies revealed that the metabolic pathway of the oxime in the blood is quite different from that in ocular tissues. Thus, propranolol was not detected in rat's blood following the i.v. administration of the oxime and, instead, another more polar compound was detected. However, 5 minutes after injection even this compound had totally disappeared. The oxime itself appeared to have a very fast metabolism in blood (Fig. 2). The tl/2 in blood was equivalent to 7.64 + 0.55 minutes and one hour after i.v. administration the oxime had completely disappeared from the blood.

While these results would suggest that the propranolol formed in situ in the iris-ciliary body is responsible for the IOP reduction observed, the ketoxime itself might have intrinsic activity.

The compounds of the invention may be administered to animals in need thereof by instilling solutions thereof into the eye or via oral tablets, capsules, etc, or any other convenient route of administration at dosages of from about 0.001 to about 20 g/kg.

The compounds may be formulated with any conventional pharmaceutically acceptable carrier, such as those utilized for the parent amino-alcohol β-blockers. The research leading to the present invention was supported by NIH Grant R01 EY05800. The United States Government has certain rights in the present invention.

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