GB2327675A

GB2327675A - Nicotinic acid amide derivatives

Info

Publication number: GB2327675A
Application number: GB9816099A
Authority: GB
Inventors: Akiharu Kajiwara; Satoshi Nagato; Joanna Elizabeth Brashaw; Mary Louise Morgan; James Martin Staddon; Lee Laurence Rubin
Original assignee: Eisai Co Ltd
Current assignee: Eisai Co Ltd
Priority date: 1997-07-23
Filing date: 1998-07-23
Publication date: 1999-02-03
Anticipated expiration: 2018-07-23
Also published as: JP3987645B2; GB2327675A8; GB9715584D0; JPH11147874A; GB9816099D0; GB2327675B

Abstract

A nicotinic acid amide derivative of formula (I), pharmacologically acceptable salts and pharmaceutical compositions thereof: wherein R 1 is H or lower alkyl; R 2 is optionally substituted pyridinyl, pyridinemethyl, N-benzylpiperidinyl, isoquinolyl or benzyl; R 3 is optionally substituted cyclopentyl, cyclohexyl, cyclooctyl, norbornyl, adamantyl, piperidyl, pyridyl, isoquinolyl or azabicyclooctyl wherein the ring system is attached to the nicotinamide ring by means of an oxygen or NH group and R 4 is H or lower alkoxy. The compounds are useful for the prevention and treatment of stroke, brain edema after stroke and a variety of allergic and inflammatory diseases, e.g. asthma, chronic bronchitis, shock, rheumatoid arthritis, reperfusion injury, encephalomyelitis and multiple sclerosis. The compounds are prepared from intermediates wherein R 3 is chlorine which in turn are prepared from reaction of the corresponding acid with HNR 1 R 2 .

Description

NICOTINAMIDE DERIVATIVES AND THEIR USE AS MEDICAMENTS The present invention relates to a novel nicotinamide derivative which is useful for the prevention, treatment or amelioration of stroke, brain edema after stroke and a variety of allergic and inflammatory diseases such as asthma, chronic bronchitis, shock, rheumatoid arthritis, reperfusion injury, encephalomyelitis and multiple sclerosis.

Recently, patients suffering from allergic or inflammatory diseases are increasing markedly. These diseases severe symptoms and are intractable and can be recurrent.

There are no therapeutic drug having high effectiveness and safety. Therefore, a therapeutic drug having high efficacy and safety in the clinical field has been strongly desired.

Cyclic 3',5'-adenosine monophosphate (hereinafter abbreviated as cAMP) is a well known second messenger that mediates the functional responses of cells to hormones, autocoids, neurotransmitters and drugs, Sutherland et al Pharmacol Rev 12 265 (1996). The cellular levels of cAMP are regulated by mechanisms which control its synthesis and breakdown. The breakdown of cAMP is controlled by a family of phosphodiesterases (hereinafter abbreviated to PDE) (Beavo et al TiPS 11, 1150, 1990). It has been shown that PDE IV plays a main role to regulate cAMP concentrations in airway smooth muscle and inflammatory cells, Dent et al British J Pharmacology 90, 163, (1990). and inhibition of PDE IV can lead to prevent inflammatory mediator release, Verghese et al J Mol Cell Cardiol 12 (Suppl II), S61 (1989). Thus, compounds that inhibit PDE IV would be useful for the treatment of inflammatory disease.

In the meantime, brain capillary endothelial cells (hereinafter abbreviated as ECs) form the blood-brain barrier (hereinafter abbreviated as BBB), which is usually spoken of as having an essential role in maintaining the normal extracellular environment of the central nervous system (hereinafter abbreviated as CNS). The BBB is a real molecular barrier, permitting only small hydrophobic molecules, a limited set of specifically transported nutrients (glucose and certain amino acids), and a restricted number of specifically transcytosed macromolecules, such as transferrin, entry into the brain. Two separate properties of brain capillary ECs account for the limited molecular transport: their low rates of fluid-phase endocytosis (and correspondingly low rates of transcellular flux) and their coupling by high electrical resistance tight junctions (which severely limit paracellular flux).

For some time, the BBB has been known to be clinically important. When tight junctions are grossly disrupted, as sometimes happens following stroke, the resulting entry of proteins and ions into the brain leads to edema because of the associated influx of water. Even an apparently normal BBB can be breached, as occurs with the entry of certain types of lymphocytes and metastatic cells into the brain; such conditions are associated with serious disease (multiple sclerosis and metastatic brain tumour. for example).

In the case of cultured brain ECs, addition of a membrane permeant cyclic AMP derivative has been shown to decrease tight junction permeability and to produce a reorganization of the actin cytoskeleton, reducing the number of stress fibres, thereby yielding a well defined cortical actin belt. It follows that inhibition of cyclic AMP phosphodiesterase (hereinafter abbreviated as PDE) activity could produce a similar effect. This is indeed the case as inhibitors of PDE IV, such as rolipram, do decrease tight junction permeability.

In the case of stroke, PDE IV inhibitors would be useful agents to decrease the vasogenic edema that severely complicates this condition. By reversing the increased permeability of the tight junctions of the brain endothelial cells, the entry of ions and protein into the brain would be prevented and the CNS environment would return to normal. Vasogenic edema would be prevented.

In multiple sclerosis, activated T cells bind to the brain endothelium and transmigrate to enter the CNS. If CNS antigen is encountered, the cells remain to trigger an inflammatory cascade, ultimately resulting in demyelination. Transmigration of activated T cells across brain endothelial cells can be inhibited in vitro by PDE IV inhibitors. The mechanism of inhibition may be due to blockade of T cell-initiated, endothelial signalling processes that are necessary for transmigration. It follows that PDE IV inhibitors could be agents to block activated T cell entry into the brain in multiple sclerosis, thereby providing a potential therapy.

In those diseases, cyclic AMP Phosphodiesterase (hereinafter, abbreviated as PDE) plays a important role. For example, it is disclosed that Rolipram, a selective type IV PDE inhibitor, is a potential anti multiple sclerosis drug [Nature Medicine, 1(3),244248,1995.]. Furthermore, it is disclosed also that PDE inhibitors can prevent experimental allergic encephalomyelitis [Proc.Natl,Acad,Sci,USA,92(4),3601- 3605,1995.]. Since Rolipram is an antidepressant, adverse reactions such as sleepiness, lowering of concentration or reflex movement ability are unavoidable.

Regarding the foregoing problems, the present inventors have proceeded with extensive research. As a result, it has been found that a novel nicotinamide derivative represented by the formula (I) has excellent efficacy and safety.

According to a first aspect of the present invention there is provided a compound of general formula (I):

wherein R1 represents a hydrogen atom or a lower alkyl group; R2 represents a group selected from the following group:

wherein X represents a halogen atom; or R1 and R2 can form a 4-methylpiperazinyl group together which may be substituted; R3 represents a group selected from the following group;

R4 represents a hydrogen atom or a lower alkyl group.

The present invention offers a potential anti allergic and inflammatory drug, especially for asthma, chronic bronchitis, shock, rheumatoid arthritis, reperfusion injury, encephalomyelitis and multiple sclerosis.

With respect to the above definition of the above formulas, particular examples of the halogen atom include chlorine atom, fluorine atom, bromine atom and iodine atom, among which chlorine atom is preferable. Particular examples of the lower alkyl group include alkyl groups having 1 to 6 carbon atoms, such as methyl group, ethyl group, npropyl 0group, i-propyl group, n-butyl group, i-butyl group, t-butyl group, pentyl group and hexyl group.

More specific examples of the nicotinamide derivative represented by the above formula (I) according to the present invention include the following compounds, though the nicotinamide derivative is not limited to them; ( 1 ) N-(4-pyridyl)-2-cyclopentyloxynicotinicamide, (2) N-(4-pyridyl)-2-exonorbornyloxynicotinic amide, (3) N-(4-pyridyl)-2-(4-fluorophenyloxy) nicotinic amine, (4) N-(4-pyridyl)-2-(3-hydroxycyclohexyloxy) nicotinic amide, (5) N-(4-pyridyl)-2-(2-exonorbornylamino) nicotinic amide, (6) N-(4-pyridyl)-2-cyclooctylaminonicotinic amide, (7) N-(4-pyridyl)-2-adamantylaminonicotinic amide, (8) N-(4-pyridyl)-2-exonorbomyloxy-6-methyl nicotinic amide, (9) N-(3-pyridyl)-2-exonorbomyloxynicotinic amide, (10) N-(3,5-dichloro-4-pyridyl)-2-(2-exonorbomyloxy) nicotinic amide, (11) N-(5-isoquinolynyl)-2-(2-exonorbornyloxy) nicotinic amide, ( 12) N-(4-picolyl)-2-(4-fluorophenyloxy) nicotinic amide, (13) N-(4-picolyl)-2-(3-nitrophenylamino) nicotinic amide, (14) N-(3-picolyl)-2-(2-Exonorbomyloxy) nicotinic amide, (15) N-(3-picolyl)-2-(3,4-dimethoxyphenyloxy) nicotinic amide and; (16) N-( 1 Benzyl-4-piperidyl)-2-(2-exonorbornyloxy) nicotinic amide.

The present invention provides a method for treating a variety of allergic and inflammatory diseases such as asthma, chronic bronchitis, shock, rheumatoid arthritis, reperfusion injury, encephalomyelitis and multiple sclerosis, etc. accompanied by PDE IV activity by administering to a human patient a pharmacologically effective amount of a compound according to general formula (I) above for inhibiting the PDE IV activity. In other words, there is provided the use of a compound or a pharmacologically acceptable salt thereof according to the present invention for the making of a medicament for treating or ameliorating a disease against which phosphodiesterase antagonsim is efficacious.

The invention further provides a therapeutic composition which comprises a pharmacologically effective amount of a compound according to general formula (I) above and a pharmacologically acceptable carrier.

Specifically, the compounds of general formula (I) of the present invention may be effective for treatment, prevention, remission, improvement, etc. of a variety of allergic and inflammatory diseases such as asthma, chronic bronchitis, shock, rheumatoid arthritis, reperfusion injury, encephalomyelitis and multiple sclerosis.

Where a compound of general formula (I) of the present invention is used as a pharmaceutical agent in the treatment or amelioration for these diseases, it may be orally or parenterally administered. In general, it is parenterally administered in the form of injections, such as intravenous, subcutaneous, and intramuscular injections, suppositories, or sublingual tablets. The dose will remarkably vary depending upon the symptom; age, sex, weight, and sensitivity of patients; method of administration; time and intervals of administration and properties, dispensing, and kind of pharmaceutical preparations; kind of effective ingredients, etc., so that there is no particular limitation with respect to the dose. Normally the compound may be administered in a dose of about 0.1 to 1000 mg, preferably 0.5 to 500 mg, more preferably 1 to 100 mg, per day per adult, ordinarily in one to four portions.

In preparing injections, the effective ingredient may be blended, if necessary, with a pH modifier, a buffer, a suspending agent, a solubilizing agent, a stabilizer, a tonicity agent, a preservative, etc., followed by preparation of an intravenous, subcutaneous, or intramuscular injection according to an ordinary method. In this case, if necessary, these preparations may be lyophilized according to an ordinary method.

Examples of the suspending agents include methylcellulose, Polysorbate 80, hydroxyethylcellulose, acacia, powdered tragacanth, sodium carboxymethylcellulose, and polyoxyethylene sorbitan monolaurate.

Examples of the solubility agent include polyoxyethylene hydrogenated castor oil, Polysorbate 80, nicotinamide, polyoxyethylene sorbitan monolaurate, Macrogol, and an ethyl ester of castor oil fatty acid.

Examples of the stabilizer include sodium sulfite, sodium metasulfite and ether, and examples of the preservative include methyl p-hydroxybenzoate, ethyl phydroxybenzoate, sorbic acid, phenol, cresol, and chlorocresol.

According to a further aspect of the present invention there is provided a process for the preparation of compound of general formula (II),

comprising derivatising an optionally protected compound of general formula (III),

and optionally thereafter converting the compound of general formula (II) so formed into another compound of general formula (II), in which Rl to R4 have the same meaning as defined in accordance with the first aspect of the present invention.

In this process, the derivativisation of the compound of general formula (m) to form a compound of general formula (II) may be carried out by treatment with: (a) a chlorinating agent (b) a mixed acid anhydride forming agent or, (c) 1,3-dicyclohexylcarbodiimide (DCC), and a primary or secondary amine of general formula (IV), HNR'R2 (IV) wherein R' and R2 have the same meaning as defined in accordance with the first aspect of the present invention.

The chlorinating agent may conveniently be thionyl chloride, sulfuryl chloride, oxalyl chloride, phosphorus pentachloride, phosphorous trichloride or phosphorous oxychloride. The mixed acid anhydride forming agent may be methyl chloroformate or ethyl chloroformate.

The compound of general formula (II) prepared by a process in accordance with this aspect of the present invention may be further treated with an alcohol or an amine to give a compound of general formula (I).

The present invention also extends to a compound of general formula (II), in which R' to R4 have the same meaning as defined in claim 1, The present invention also extends to a compound of general formula (II), in which RX to R4 have the same meaning as defined in claim 1,

with the proviso that N-(4-pyridyl)-2-chloronicotinic amide, N-(3-pyridyl)-2chloronicotinic amide and N-(2-pyridyl)-2-chloronicotinic amide are excluded.

More specific examples of such compounds include, (1) N-(4-pyridyl)-2-chloro-6-methyl nicotinic amine, (2) N-(3 ,5-dichloro4-pyridyl)-2-chloronicotinic amide, (3) N-(5-isoquinolyl)-2-chloronicotinic amine, (4) N-(4-picolyl)-2-chloronicotinic amine, (5) N-(3-picolyl)-2-chloronicotinic amide and; (6) N-(N-benzylpiperidin4-yl)-2-chloronicotinic amine.

Preferred features of the second and subsequent features are as for the first aspect mutatis mutandis.

The invention will now be described by way of example with reference to the accompanying Examples which are provided for the purposes of illustration and are not to be construed as being limiting on the present invention. Reference is made in the Examples to a number of Figures in which: FIGURE 1 shows coronal sections of rat brain illustrating division of right (R) and left (L) hemispheres into six regions for measurement of tissue Evans Blue content.

A - level anterior to infarct (bregma + 2.7mm); B - level of infarct (bregma - 0.3mm).

FIGURE 2 shows the effect of the compound of example 2 (5 hour infusion) on BBB disruption.

Evans Blue was extracted from 6 areas of brain 5 hours after MCAo as described in the test. The compound was administered after onset of occlusion. Data are expressed as mean + SE. The compound (n=5) vs. vehicle (n=5) *p < 0.05 (Student T-test).

FIGURE 3 shows the effect of the compound of example 2 (48 hour infusion) on BBB disruption.

Evan Blue was extracted from 6 areas of the brain 48 hours after MCAo as described in the test. The compound was administered after onset of occlusion. Data are expressed as mean z SE. The compound (n=6) vs vehicle (n=7) * p < 0.05 (Student T-test).

Preparative Examples (1) Synthetic route

0 0 0 1)SOCI2 RH 1OH 1NR1R2 < NR N:R2l R4 N Cl 2) NHR1R2 R4 NCI R4 NR3 "intemediate" (I) (wherein R1 to R4 have the same meaning as defined above.) 2-Chloronicotinic acid or its 6-substituted derivative was treated with; 1 ) chlorinating agent (e.g., thionyl chloride, sulfuryl chloride, oxalyl chloride, phosphorous pentachloride, phosphorous trichloride, phosphorous oxychloride) or 2) mixed acid anhydride forming agent (e.g., methyl chloroformate, ethyl choloroformate) or 3)1 ,3-dicyclohexylcarobodiimide (DCC); and primary or secondary amine to afford intermediate amide compounds, and this was treated with alcohol or amine to give the nicotinamide derivative (I).

(2) Synthesis of intermediates PreDarative Example 1 (intermediate 1) N-(4-pyridyl)-2-chloronicotinic amide

The mixture of 2-chloronicotinic acid (lSg,0.095mo1) and thionyl chloride(l50ml) was heated at 600C for 8 hours followed by evaporation of excess thionyl chloride to give crude acid chloride. This was washed with ether and dried and then dissolved in 1 some of dichloromethane. To the solution of 4-aminopyridine (1 0g,0.1mol) and triethylamine (20ml) in 200ml of dichloromethane was added the solution of above acid chloride in dichloromethane at 0 C for 30 minutes and stirred for 10 hours at room temperature (hereinafter abbreviated as RT).

The reaction mixture was poured onto water and extracted with dichloromethane twice, combined organic layer was washed with brine and dried over MgSQ4. The dichloromethane was evaporated to dryness to afford 20g of the titled compound.

'H-NMR(CDCl3); 6(ppm) 8.82 (brs,lH), 8.52(m,3H), 8.1 4(d-d,1 H,J=7Hz,2Hz), 7.58 (d,2H,J=8Hz).

Preparative Example 2 (intermediate 2) N-(4-pyridyl)-2-chloro-6-methyl nicotinic amide

2-Chloro-6-methylnicotinic acid and 4-aminopyridine were reacted according to the synthesis of (1) to give the titled compound. lH-NMR(CDCl3); #(ppm) 8.58(d,2H,J=7Hz), 8.52(brs, 1 H), 8.14(d,2H,J=7Hz), 7.60(d,2H,J=7Hz), 7.29(d,2H,J=7Hz), 2.60(s,3H).

Preparative Example 3 (intermediate 3) N-(3-pyridyl)-2-chloronicotinic amide

2-Chloronicotinic acid and 3-aminopyridine were reacted according to the synthesis of (1) to give the titled compound.

Preparative Example 4 (intermediate 4) N-(3 ,5-dichloro-4-pyridyl)-2-chloronicotinic amide

2-Chloronicotinic acid and 3,5-dichloro-4-aminopyridine were reacted according to the synthesis of (1) to give the titled compound.

Preparative Example 5 (intermediate 5) N-(5-isoquinolyl)-2-chloronicotinic amide

2-Chloronicotinic acid and 5-aminoisoquinoline were reacted according to the synthesis of (1) to give the titled compound.

Preparative Example 6 (intermediate 6) N-(4-picolyl)-2-chloronicotinic amide

2-Chloronicotinic acid and 4-picolylamine were reacted according to the synthesis of (1) to give the titled compound.

Preparative Example 7 (intermediate 7) N-(3-picolyl)-2-chloronicotinic amide

2-Chloronicotinic acid and 3-picolylamine were reacted according to the synthesis of (1) to give the titled compound.

Preparative Example 8 (intermediate 8) N-(N-benzylpiperidin-4-yl)-2-chloronicotinic amide

2-Chloronicotinic acid and 1 -benzyl-4-aminopiperizine were reacted according to the synthesis of (1) to give the titled compound.

EXAMPLES Example 1 N-(4-pyridyl)-2-cyclopentyloxynicotinicamide

To the suspension of 50% of sodium hydride (4.3g,0,09mol) in dimethylformamide was added cyclopentanol (7.74g,0,09mol) at RT and stirred for 1 hour and then the intermediate (1) was added and reacted at 110-120"C for 4 hours. The reaction mixture was poured onto ice-water and extracted with ethyl acetate(twice) and the organic layer was washed with water, brine and dried over MgS04. The organic phase was evaporated to dryness and the residue was purified on 200g of silica gel column chromatography (2% ethanol-dichloromethane) to afford 9.0g of the titled compound.

1H-NMR(D20) ; o(ppm) 8.50(d,2H,J=7Hz), 8.20(m,2H), 8.03(d,2H,J=7Hz), 7.10(dd,lH,J=7Hz,7Hz), 5 .40(m, 1 H), 1.5-2.0 (m,8H).

Example 2 N-(4-pyridyl)-2-exonorbornyloxynicotinic amide

Exonorborneol and intermediate (1) were reacted according to the procedure of example (1) to afford the titled compound.

H-NMR(CDCl3) ; #(ppm) 10.30(brs,1H), 8.52(m,3H), 8.30(d-d,1H,J=7Hz,1Hz), 7.5 8(d,2H,J=7Hz), 7.06(d-d,1H,J=7Hz,7Hz), 5.1 8(d, 1 H,J=5Hz), 2.60(m, 1 H), 2.42(m,1H), 1.1-2.1(m,8H), Example 3 N-(4-pyridyl)-2-(4-fluorophenyloxy) nicotinic amide

4-Fluorophenol and intermediate (1) were reacted according to the procedure of example (1) to afford the titled compound.

'H-NMR (HCl salt in D20) ; #(ppm) 8.57(d,2H,J=7Hz), 8.25(d-d,lH,J= 7Hz,lHz), 8.10(m,3H), 7.30(d-d,1H,J=7Hz,7Hz), 7.15(d,4H,J=7Hz).

Example 4 N-(4-pyridyl)-2-(3 -hydroxycyclohexyloxy) nicotinic amide

1,3-Cyclohexanediol and intermediate(l) were reacted according to the procedure of example (1) to afford the titled compound.

'H-NMR(CDCl3) ; #(ppm) 10.35(brs,1H), 8.56(m,3H), 8.30(d-d,lH, J=7Hz,lHz), 7.68(d,2H,J=7Hz), 7.12(d-d,1H,J=7Hz,7Hz), 5.48(m,1H), 4.00(brs,1H), 1.42.4(m,8H).

Example 5 N-(4-pyridyl)-2-(2-exonorbornylamino) nicotinic amide

To the solution of exo-2-norbornyl amine (710mg,6.4mM) and intermediate (l) (1.5g,6.4mM) in 15ml of dimethylformamide was added copper(II) acetate (58mg,0.32mM) and n-ethylmorphorine(0.8ml) and heated to 1100C for 15 hours. The reaction mixture was poured onto ice-water and extracted with ethyl acetate(three times) and the organic layer was washed with brine, dried over MgSO4 and evaporated to dryness. The residue was purified on silica gel column chromatography (3% ethanol-dichloromethane) to give 1.1 g of the titled compound.

H-NMR (CDCl3); #(ppm) 8.30(d,2H,J=7Hz), 7.94(brs,2H), 7.70(d-d,1H,J=7Hz,1Hz), 7.50(brs,2H), 6.51(d-d,1H,J=7Hz,7Hz), 3 .80(m, 1 H), 2.30(brs,2H), 1.1-2.0(m,8H).

Example 6 N-(4-pyridyl)-2-cyclooctylaminonicotinic amide

Cyclooctylamine and intermediate (1) was reacted according to the procedure of example (5) to afford the titled compound.

H-NMR(CDCl3); #(ppm) 8.50(d,2H,J=7Hz), 8.28(m,1H), 8,12(brs,1H), 8 .08(d,J= 10Hz), 7.70(d, 1 H,J=7Hz), 7.52(d,2H,J=7Hz), 6.50(d-d,1H,J=7Hz,7Hz), 4.30 (m,lH), 1.4-2.1(m,14H).

Example 7 N-(4-pyridyl)-2-adamantylaminonicotinic amide

Adamantylamine and intermediate (1) was reacted according to the procedure of example (5) to afford the titled compound.

H-NMR(CDCl3); #(ppm) 8.50(d,2H,J=7Hz), 8.22(m,1H), 8.13(brs,1H), 7.90(brs,1H), 7.72(d-d, I H,J=7Hz, 1Hz), 7.53(d,2H,J=7Hz), 6.48(d-d, 1 H,J=7Hz,7Hz), 1.62.3(m,15H).

Example 8 N-(4-pyridyl)-2-exonorbornyloxy-6-methyl nicotinic amide

Exonorborneol and intermediate (1) was reacted according to the procedure of example (5) to afford the titled compound.

H-NMR(CDCl3); #(ppm) 10.33(brs,1H), 8.55(d,2H,J=7Hz), 8.42(d,1H,J=7Hz), 7.59(d,2H,J=7Hz), 6.92(d, 1 H,J=7Hz), 5.21 (m, 1 H), 2.50(s,3H), 1 .2-2.6(m, 1 OH).

Example 9 N-(3-pyridyl)-2-exonorbornyloxynicotinic amide

Exonorborneol and intermediate (3) was reacted according to the procedure of example (5) to afford the titled compound.

'H-NMR(HCI salt in D20); #(ppm) 9.29(s,lH), 8.43(d,1H,J=7Hz), 8.28(m,1H), 8.12(m,2H), 7.88(d-d,1H,J=7Hz), 7.01(d-d,1H,J=7Hz,7Hz), 4.76(m,1H), 2.38(m,1H), 2.20(m,1H), 1.0-1.8(m,8H).

Example 10 N-(3,5 -di chloro-4-pyridyl)-2 -(2-exonorbomyloxy) nicotinic amide

2-Exonorborneol and intermediate (4) was reacted according to the procedure of example (5) to afford the titled compound.

'H-NMR (CDCl3); #(ppm) 9.92(brs,1H), 8.60(m,1H), 8.57(s,2H), 8.36(m,1H), 7.08(dd, 1 H,J=7Hz, 1Hz), 5 .20(m, 1 H), 1 .0-2.6(m, 1 OH).

Example 11 N-(5-isoquinolynyl)-2-(2-exonorbornyloxy) nicotinic amide

2-Exonorborneol and intermediate (5) was reacted according to the procedure of example (5) to afford the titled compound.

H-NMR(D6-DMSO); #(ppm) 10.65(s,1H), 9.95(s,1H), 8.78(d,1H,J=7Hz), 8.51 (d, 1 H,J=7Hz), 8.40(m,3H), 8.1 0(m,2H), 7.1 8(d-d, 1 H,J=7Hz,7Hz), 5.02(d,1H,J=5Hz), 1.1-2.5(m,10H).

Example 12 N-(4-picolyl)-2-(4-fluorophenyloxy) nicotinic amide

4-Fluoropheol and intermediate (6) was reacted according to the procedure of example (5) to afford the titled compound.

H-NMR(CDCl3); #(ppm) 8.55(m,2H), 7.43(d,1H,J=7Hz), 7.2-7.4(m,3H), 7.04(m,2H), 6.80(t,2H,J=8Hz), 6.20(t, H,J=7Hz), 5.04(s,2H).

Example 13 N-(4-picolyl)-2-(3-nitrophenylamino) nicotinic amide

3-Nitroaniline and intermediate (6) was reacted according to the procedure of example (5) to afford the titled compound.

'H-NMR(CDCl3); #(ppm) 9.08(t,1H,J=5Hz), 8.87(m,1H), 8.55(d,2H, J=7Hz), 8.39(m,1H), 8.16(d-d,1H,J=7Hz,1Hz), 7.88(d,1H,J=7Hz), 7.78(d,1H,J=7Hz), 7.40(t, lH,J=7Hz), 7.28(d,2H,J=7Hz), 6.86(d-d,1H,J-7Hz, 1Hz), 4.58(d,2H,J=5Hz).

Example 14 N-(3 -picolyl)-2-(2-Exonorbornyloxy) nicotinic amide

2-Exonorborneol and intermediate (6) was reacted according to the procedure of example (5) to afford the titled compound.

'H-NMR(CDCl3); 6(ppm) 8.62(brs,1H), 8.55(m,1H), 8 .50(d-d, 1 H,J=7Hz, 1Hz), 8.40(m,1H), 8.25(m,1H), 7.70(d-d, 1 H,J=7Hz, 1Hz), 7.28(d-d,lH,J=7Hz,7Hz), 7.03(dd,1H,J=7Hz,7Hz), 5.03(m,1H),4,65(d,2H,J=5Hz), 3.78(m,1H), 1.0-2.4(m,10H).

Example 15 N-(3-picolyl)-2-(3,4-dimethoxyphenyloxy) nicotinic amide

3,4-Dimethoxyphenol and intermediate (6) was reacted according to the procedure of example (5) to afford the titled compound.

'H-NMR (HCl salt in D2O); #(ppm) 8.70(s,lH), 8.60(d,lH,J=7Hz), 8.45(d,lH,J=8Hz), 8,19 (d-d,lH, J=7Hz, 1Hz), 8.1 2(m, 1 H), 7.90(d-d,1H,J=7Hz), 7,22(dd, 1 H,J=7Hz,7Hz), 6.96(d,1H,J=Hz), 6,76(s,1H), 6.62(m,1H), 4.68(s,2H), 3 .76(s,3H), 3.68(s,3H).

Example 16 N-(1-Benzyl-4-piperidyl)-2-(2-exonorbornyloxy) nicotinic amide

2-Exonorborneol and intermediate (8) was reacted according to the procedure of example (5) to afford the titled compound.

'H-NMR (CDCl3); #(ppm) 8.46(d-d,1H,J=7Hz,1Hz), 8 .20(m, 1 H), 8.08 (d, I H,J-7Hz), 7.2-7.3 (m,5H), 6.98(d-d,1H,J=7Hz,7Hz), 5.08(m,1H), 4.00(m,1H), 3.53(s,2H), 2.72.9(m,2H), 1.1-2.5(m,16H).

PHARMACOLOGICAL EXPERIMENTS (1) In vitro studv: Effects on transcellular resistance (TER) of pig brain endothelial cell cultures 1) Materials The compound of example 2 was used as a representative of the present invention.

Rolipram was used as a positive control, and two compounds disclosed in the example 4 and 8 ofUS-4,861,891 (EP-357,316), close to the present invention structure, were used as controls.

Control 1 Control 2

2) Method Microvessel isolation and pig brain endothelial cell (hereinafter abbreviated as PBEC) culture; Essentially as described in Rubin et al. 1991') for bovine brain endothelial cells. Pig cortex is homogenized and microvessel fragments collected by filtration. Vessel fragments are cultured for 6 days, passaged and PBEC plated on collagen-treated polycarbonate Transwells. After 4 days the medium is changed to 50% astrocyteconditioned medium (ACM), 50% defined medium [(hereinafter abbreviated as DMEM) with 10pLg/ml transferrin, 100CLM putrescine and 30nM sodium selenite], using a total volume of 1 ml, 2501l1 in upper chamber, 750111 in lower chamber.

Transcellular electrical resistance (hereinafter abbreviated as TER) across the PBEC monolayer is determined using an EVOM resistance system (World Precision Instruments, Hertfordshire, UK). Resistance is corrected for resistance across an empty filter and expressed as Ohms x cm2 (Qcm2). Electrical resistance across the monolayer exceeds 100 Ohms x cm2 (#cm before addition of test compounds.

Astrocyte-conditioned medium Cultures of astrocytes that are over 95% pure are prepared from l day old Sprague Dawley rat cortex, essentially as described by Lillien and coworkers (1988)2).

Conditioned medium is collected every 2 days from confl

2); Lillien, L. E., Sendtner, M., Rohrer, H., Hughes, S. M., and Raff, M. C. (1988).

Type-2 astrocyte development in rat brain cultures is initiated by a CNTF-like protein produced by type-l astrocytes. Neuron, 1,485-94.

2) Results Results are shown in table 1.

(TER shown as % of 0 hour reading, mean# standard deviation for triplicate Transwells) Table 1

TER,% Compound I Content at 1.5h I at 2.5h i at 24h Rolipram 10 M 257#29 237#36 118#16 Control 1 1x104nM 292#4 291#13 188#9 Control 1 1x10 nM 239#12 232#24 124#6 Control 1 1x10nM 190#25 173#8 91#5 Control 1 1x10nM 134#10 111#3 63#3 Control 2 1x104nM 253#5 246#15 149#9 Control 2 1x10 nM 184#32 159#14 80#1 Control 2 1x10nM 142#7 123#4 75#11 Control 2 1x10nM 128#7 108#2 62#2 Example 2 1x104nM 281#16 304#14 120#10 Example 2 1x10 nM 260#27 257#15 130#5 Example 2 1x10nM 173#23 151#15 99#1 Example 2 1x10nM 123+6 98i2 65+1 (2) In vitro study: c-AMP Content (Control=1 .00) 1) Materials - The following tested compounds correspond to the examples.

2) Method Brain endothelial cells were cultured on 96 well plates. After attainment of confluence, culture medium was replaced with 50 l of fresh medium and the cultures were incubated for a further 2 hours at 37cC. Compounds were diluted in pH equilibrated medium at 370C to give twice the desired final concentration. Then, 50 1 was added in triplicate to the cultures for 2 hours. In order to extract cellular cAMP, the medium was then rapidly replaced with ice-cold O.lM-HCI. After 30 minutes at 40C, the extract was assayed following acetylation by radioimmuno-scintillation proximity assay (Amersham RIA-SPA: RPA 538).

3) Results - Results are shown in table 2.

Table 2

Compound | cyclic AMP content I 1 M 10mM Solubility in water Control 1 insol.

Control 2 1.32 1.93 5.61 1 insol.

Example 1 1.92 2.90 4.85 ' sol.

Example 2 2.63 5.68 | 6.93 sol.

Example3 1.26 1.75 | 3.19 sol.

Example 5 1.33 1.33 1 1.50 sol.

Example 6 1.04 1.14 1.33 sol.

Example7 1.03 1.11 0.81 a insol.

Example 9 1.37 1.71 2.38 ' sol.

Example 10 1.27 1.33 1.50 1 sol.

Example 12 1.07 1.55 1.94 sol.

Example 13 1.39 1.22 1.50 sol.

Example 14 1.02 0.95 1.30 sol.

Example 15 0.86 1.62 1.41 sol.

Example 16 1.08 1.16 1.54 sol.

Most of the present invention compounds are soluble in water, whereas Control 1 and 2 are insoluble. This physical property is very advantageous for the treatment of cerebrovascular diseases, because water soluble materials can be formulated easily and stably as an injection form and can pass through BBB.

(3) In vivo studv: Effect of the present invention compound on BBB integrity in the middle cerebral arterv occlusion model in rats 1) Material The compound of example 2 was used as a representative of the present invention.

2) Method Male Sprague-Dawley rats (270-320g) were anesthetized with halothane and subjected to 120 min of temporary MCAo by retrograde insertion of an intraluminal nylon suture') coated with poly-L-Iysine2) through the extemal carotid artery into the internal carotid artery and MCA. Temperature probes were inserted in the rectum and the left temporalis muscles. Heating lamps were used to maintain rectal and temporalis muscle temperatures at 37 to 38 "C. In all rats, polyethylene catheters were introduced into the right femoral artery and vein for blood pressure recording, blood sampling, for Evans Blue and drug infusion. Mean arterial blood pressure (hereinafter abbreviated as MABP), plasma glucose and blood gases were continuously measured during the operation.

The neurological status was evaluated during occlusion (60 min) in all groups; 3 and 5 hours after MCAo in A and B groups; 24 and 48 hours in C and D groups. A grading scale of 0-12 was used to assess the effects of occlusion (normal score -0; maximal score -12; Table 3).

I); Zea Longa, E.L., Einstein, P.R., Carlson, S. and Cummins, R., Reversible middle cerebral artery occlusion without craniectomy in rats, Stroke, 20 (1989) 84-91.

2); Belayev, L., Alonso, O.F., Busto, R., Zhao, W. and Ginsberg, M.D., Middle cerebral artery occlusion in the rat by intraluminal suture: neurological and pathological evaluation of an improved model, Stroke, 27 (1996)1616-1623.

Table 3 Neurological evaluation of rats with MCAo

Item I Normal Score I Deficit Postual Reflex ("Hang Test")* | I | 2 Placing Test** (performed on each side) Visual Placing Forward 0 2 Sideways 0 2 Tactile Placing Dorsal Surface of Paw 0 2 Lateral Surface of Paw 0 2 Proprioceptive Placing 0 2 Total Score 0 12 * Postural Reflex ("Hang Test") ** Placing Test O-no observable deficit O-complete immediate placing 1-limb flexion during hang test l-incomplete and / or delayed placing ( < 2sec.) 2-deficit on lateral push 2-absence ofplacing MCAo, middle cerebral artery occlusion 3) Drug infusion Tested compound (example 2 in saline, 1 mg/kg, i.v.) or vehicle (0.9% saline) was administered by infusion after the onset of MCAo (Table 4).

Table 4 Experimental Groups

Group n Procedure MCAo Evans Blue Sacrificed Treatment Dose Route (min) injection A (Vehicle) 5 MCAo 120 3h 5h 0-5h 2mg/Kg i.v.

Saline-5h B (Example 2) 5 MCAo 120 3h 5h 0-5h 1mg/Kg i.v.

1mg/Kg-5h C l ' Vehicle 9 MCAo 120 46h 48h 0-48h 1mg/Kg i.v.

Saline-48h D I (Example 2) 8 MCAo 120 46h 48h 0-48h 1mg/Kg i.v.

1mg/Kg-48h Four animal groups were studied: Groups A and B were treated by infusion of vehicle or drug over 5 hours, and Groups C and D were treated by infusion of vehicle or drug over 48 hours.

4) Evaluation of BBB integrity The integrity of the BBB was investigated using Evans-Blue extravasation, according to Uyama et al.3). Animals were divided into groups as listed in Table 4. Evans Blue (EB, 2%, in saline, 4 ml/Kg) was injected intravenously at 3 h after the onset of MCAo in Groups A and B; and at 46 h in Groups C and D. The chest was subsequently opened under halothane anesthesia 2 h later. Rats were perfused with saline through the left ventricle at 110 mm Hg pressure until colorless perfusion fluid was obtained from the right atrium.

After decapitation, the brain was blocked into 2 segments that included the levels bregma +2.7 and -0.3 mm. Coronal blocks were next divided into right and left hemispheres and were cut into six regions for local measurement of EB dye (Fig. 1).

Samples were weighed and placed in 50% trichloroacetic acid solution. Following homogenization and centrifugation, the extracted dye was diluted with ethanol (1:3), and its fluorescence was determined (excitation at 620nm and emission at 680nm) with a Perkin-Elmer LS-5B Luminescence spectrometer.

Calculations were based on external standards in the same solvent (100-500 ng/ml).

The tissue content of EB was quantified from a linear standard curve derived from known amounts of the dye and was expressed per gram of tissue.

3); Uyama, O., Okamura, N., Yanase, M., Narita, M., Kawabata, K. and Sugita, M., Quantitative evaluation of vascular permeability in the gerbil brain after transient ischemia using Evan's blue fluorescence, J Cereb. Blood Flow Metab., 8 (1988) 282284.

5) Results Rectal and cranial (temporalis muscle) temperatures, MABP, blood gases and plasma glucose in the 27 animals of this study showed no significant differences between groups (Tables 5-9).

Table 10 summarises the neurological outcome after MCAo. The neurological scores at 3 h and 5 h after MCAo were significantly better in the compound 2 treated group (1 mg/kg, 5-hour infusion) than in the vehicle-treated group (mean j SE; 6.2 + 0.5 vs. 8.4 + 0.2; 5.8 t 0.6 vs. 8.4 + 0.2, respectively; A vs B, p < 0.003).

The compound 2 also significantly improved the neurological score at 60 min, 24 h and 48 h in the compound 2 treated group (1 mg/kg. 48-hours infusion) compared to the vehicle group (mean j SE; 7.3 j 0.5 vs. 9.0 j 0; 4.7 j 0.4 vs. 6.7 j 0.4; 4.5 j 0.3 vs. 7.0 + 0.4 respectively; C vs D. p < 0.004).

The effect of the compound 2 on BBB integrity after MCAo is shown in Table 11.

The compound 2 (1 mg/kg, 5 hours infusion) significantly decreased in the dye extravasation (Fig. 2) into the cortex (mean j SE; 9.3 + 2.9 vs. 24.3 + 5.8 llg/g. sample 3, p=0.05), striatum (26.4 + 3.1 vs. 47.3 + 4.7 pg/g, sample 5. p= 0.01) and right hemisphere (41.2 + 5.4 vs. 82.4 + 9.2,uglg, p= 0.005) compared to the vehicle-treated group. Total EB from whole brain was also significantly decreased in the compound 2 treated rats compared with the vehicle group (63.9 + 10.5 vs. 111.8 + 12.9 llg/g; p=0.02).

The compound 2 (1 mg/Kg, 48 hours infusion) also significantly decreased dye extravasation (Fig. 3) into the cortex (mean + SE; 7.4 + 2.5 vs. 29.0 + 8.3 pg/g, sample 3, p= 0.05). striatum (17.2 + 2.2 vs. 50.8 + 12.1 g/g. sample 5, p= 0.03) and right hemisphere (30.7 + 4.0 vs. 93.2 + 18 llg/g, p=O.O1) compared to the vehicle-treated group. Total EB from whole brain was also significantly decreased in the compound 2 treated rats compared with the vehicle group (44.8 + 6,2 vs. 118.9 + 19.6 g/g; p=0.01).

Four animals died in our study (two in group C and two in group D). None died in Groups A or B (vehicle and the compound 2 treated rats).

Table 5 Head Temperature Chanoes in Rats

Groups No.of Before During MCAo After MCAo animal MCAo (min) (min) (h) 15 0 15 120 2.15 3 5 24 48 6 10303 37.2 37.1 37.1 37.0 37.1 37.2 37.1 A 6 11042 37.3 37.2 37.1 37.3 37.2 37.4 37.2 (Vehicle) 6 11074 37.2 37.2 37.1 37.1 37.2 37.2 37.3 Saline-Sh 701313 37.0 37.0 37.1 37.0 37.1 37.0 37.0 7 02061 37.2 37.1 37.2 37.3 37.1 37.0 37.2 Average 37.2 37.1 37.1 37.1 37.1 37.2 37.2 S.D. 0.11 0.08 0.04 0.15 0.05 0.17 0.11 S.E. | 0.05 0.04 0.02 0.07 0.02 0.07 0.03 6 11261 37.2 37.2 37.1 37.3 37.4 37.1 37.0 B 7 01092 37.3 37.2 37.0 37.2 37.1 37.0 37.0 (Example2) 7 01103 37.0 37.0 37.1 37.1 37.1 37.1 37.2 1mg/Kg-5h 7 01173 37.1 37.0 37.0 37.0 37.0 37.1 37.2 7 01232 37.2 37.1 37.2 37.0 37.0 37.1 37.0 Average 37.2 37.1 37.1 37.1 37.1 37.1 37.1 S.D. 0.11 0.10 0.08 0.13 0.16 0.04 0.11 S.E. 0.05 0.04 0.04 0.06 0.07 0.02 0.05 6 11063 37.2 37.1 37.0 37.2 37.1 37.3 37.0 6 11111 37.3 37.2 37.1 37.1 37.2 37.1 37.2 7 01283 37.2 37.4 37.3 37.0 37.0 37.4 37.0 C 7 01284 37.2 37.1 37.0 37.0 37.1 37.9 37.9 (Vehicle) 7 02031 37.3 37.2 37.1 37.0 37.1 died Saline-48 7 02042 37.0 37.0 37.0 37.1 37.0 37.1 7 02043 37.0 37.1 37.0 37.1 37.0 died 7 02052 37.2 37.1 37.2 37.3 37.1 37.2 37.0 7 02053 37.1 37.1 37.0 37.1 37.2 37.1 37.5 Average 37.2 37.1 37.1 37.1 37.1 37.3 37.2 S.D. 0.11 0.11 0.11 0.11 0.07 0.3 0.3 S.E. 0.04 0.04 0.04 0.04 0.02 0.1 0.1 611054 37.3 37.1 1 37.2 37.4 37.3 37.1 6 11123 37.2 37.1 37.2 37.3 37.3 37.0 37.8 7 11124 37.0 37.1 37.2 37.1 37.0 died D 6 11253 37.3 37.2 37.1 37.2 37.3 36.3 died (Example 2) 7 01143 37.3 37.2 37.1 37.0 37.0 37.0 37.0 1mg/Kg-48 7 01215 37.1 37.1 37.1 37.2 37.1 38.3 37.0 7 01272 37.3 37.2 37.2 37.0 37.0 36.4 37.0 7 01273 37.2 37.2 37.2 37.0 37.0 37.3 37.0 Average 37.2 37.2 37.2 37.2 37.1 37.1 37.2 S.D. 0.11 0.05 0.05 0.15 0.15 0.7 0.3 S.E. 0.04 0.02 0.02 0.05 0.05 0.3 0.1 T-test(Avs.B) 0.78 0.74 0.37 0.83 0.80 T-test(Cvs.D) 0.41 0.90 0.06 0.34 0.66 0.48 0.63 Table 6 Rectal Temperature Changes in Rats

Groups No. of Before During MCAo After MCAo Animal MCAo (min) (min) (h) 15 0 15 60 120 2.15 3 5 24 48 6 10303 37.4 37.3 37.4 37.3 37.2 37.3 37.4 37.4 A 6 11042 37.5 37.4 37.3 37.4 37.5 37.4 37.5 37.4 (Vehicle) 6 11074 37.5 37.4 37.3 37.4 37.3 37.4 37.3 37.5 Saline-5h 7 01313 37.2 37.1 37.3 37.3 37.2 37.3 37.0 37.2 7 02061 37.4 37.3 37.3 37.4 37.5 37.2 37.3 37.4 Average 37.4 37.3 37.3 37.4 37.3 37.3 37.3 37.4 S.D. 0.12 0.12 0.04 0.05 0.15 0.08 0.19 0.11 S.E. 0.05 0.05 0.02 0.02 0.07 0.04 0.08 0.05 6 11261 37.5 37.4 37.3 37.4 37.5 37.5 37.3 37.2 B 7 01092 37.5 37.4 37.1 37.3 37.4 37.3 37.1 37.1 (Example2) 7 01103 37.2 37.1 37.3 37.4 37.3 37.2 37.1 37.4 1mg(Kg-5h 7 01173 37.3 37.1 37.2 37.2 37.1 37.2 37.3 37.4 7 01232 37.4 37.3 37.4 37.3 37.1 37.2 37.3 37.0 Average 37.4 37.3 37.3 37.3 37.3 37.3 37.2 37.2 S.D. 0.13 0.15 0.11 0.08 0.18 0.13 0.11 0.18 S.E. 0.06 0.07 0.05 0.04 0.08 0.06 0.05 0.08 6 11063 37.4 37.3 37.2 37.1 37.4 37.3 37.5 37.2 6 11111 37.5 37.4 37.3 37.4 37.3 37.4 37.4 37.5 7 01283 37.4 37.5 37.5 37.3 37.1 37.2 37.7 37.2 C 7 01284 37.4 37.3 37.2 37.1 37.2 37.3 38.2 38.4 7 02031 37.5 37.4 37.3 37.2 37.2 37.3 died (Vehicle) 7 02042 37.1 37.2 37.3 37.0 37.2 37.3 37.2 37.3 Saline-48h 7 02043 37.2 37.3 37.1 37.0 37.2 37.1 died 7 02052 37.4 37.3 37.4 37.3 37.5 37.4 37.5 37.0 7 02053 37.4 37.3 37.2 37.2 37.4 37.3 37.4 37.3 38.0 Average 37.4 37.3 37.3 37.2 37.3 37.3 37.5 37.5 S.D. 0.13 0.09 0.12 0.16 0.12 0.10 0.33 0.50 S.E. 0.04 0.03 0.04 0.05 0.04 0.03 0.13 0.19 6 11054 37.5 37.3 37.4 37.3 37.6 37.5 37.5 37.3 6 11123 37.4 37.3 37.3 37.5 37.4 37.5 37.1 38.1 D 7 11124 37.2 37.3 37.4 37.4 37.3 37.2 died (Example 2) 6 11253 37.5 37.4 37.3 37.5 37.4 37.5 36.5 died 1mg/Kg-48h 7 01143 37.5 37.4 37.3 37.3 37.0 37.1 37.2 37.0 7 01215 37.3 37.2 37.3 37.2 37.4 37.3 38.6 37.0 7 01272 37.5 37.4 37.4 37.3 37.1 37.1 36.7 37.9 7 01273 37.4 37.3 37.4 37.2 37.0 37.2 37.6 37.2 Average 37.4 37.3 37.4 37.3 37.3 37.3 37.3 37.3 S.D. 0.11 0.07 0.05 0.12 0.22 0.18 0.69 0.43 S.E. 0.04 0.03 0.02 0.04 0.08 0.06 0.26 0.17 T-test(Avs.B) 0.81 0.66 0.31 0.40 0.58 0.58 T-test(Cvs.D) 0.46 0.83 0.14 0.06 0.92 1.00 0.45 0.37 Table 7 Arterial Blood Pressure in Rats

Groups No. of 15min MCAo Occlusion After animal Before (min) MCAo MCAo MCAo O | IS 1 120 2.15h 6 10303 110 110 120 100 90 A 6 11042 120 130 115 | 105 105 (Vehicle) 6 11074 80 90 110 70 70 Saline-5h 7 01313 105 125 110 80 85 7 02061 100 130 110 90 80 Average 103.0 117.0 113.0 89.0 86.0 S.D. 14.8 17.2 4.5 14.3 12.9 S.E. 6.6 | 7.7 . 2.0 6.4 i 5.8 611261 130 120 125 110 , 105 B 701092 115 120 120 90 70 (Example2) 701103 130 130 120 105 105 1mg/Kg-5h 701173 100 110 120 80 80 701232 80 110 110 80 90 Average 111.0 118.0 119.0 93.0 90.0 S.D. 21.3 8.4 5.5 14.0 15.4 S.E. 9.5 3.7 2.4 ~ 6.2 6.9 6 11063 90 110 110 110 90 611111 110 130 130 120 110 7 01283 | 90 | 120 | 110 | 100 | 90 C 701284 100 130 105 80 95 (Vehicle) 7 02031 90 100 85 80 90 Saline-48h 7 02042 90 120 120 90 90 7 02043 90 90 90 80 80 7 02052 85 90 105 80 80 7 02053 85 120 110 110 85 Average 92.2 112.2 107.2 94.4 90.0 S.D. 7.9 15.6 13.7 15.9 9.0 S.E. 2.6 5.2 4.6 5.3 3.0 6 11054 120 120 110 100 90 6 11123 80 85 90 90 80 7 11124 95 100 110 70 70 D 6 11253 100 110 80 80 75 (Example 2) 7 01143 110 130 140 90 90 1mg/Kg-48h 7 01215 90 110 100 90 80 7 01272 90 100 110 80 80 7 01273 80 110 100 80 90 Average 95.6 108.1 105.0 85.0 81.9 S.D. 14.0 13.6 17.7 9.3 7.5 S.E. 4.9 4.8 6.3 3.3 2.7 T-test(Avs.B) 0.51 0.91 0.09 0.67 0.67 T-test(Cvs.D) 0.54 0.58 0.78 0.16 0.06 Table 8 Arterial Blood Gates in Rats

Groups No. of 15min Before MCAo During MCAo (15min) animal pH pO2 pCO2 pH pO2 pCO2 6 10303 7.41 95.6 38.4 7.42 105.3 38.3 A 6 11042 7.41 108.5 46.6 7.43 101.6 47.5 (Vehicle) 6 11074 7.42 92.5 38.7 7.40 98.6 40.5 Saline-5h 7 01313 7.30 116.7 7.30 116.6 7 02061 7.46 98.9 37.2 7.43 82.7 40.4 Average 7.40 102.4 40.2 7.40 101.0 41.7 S.D. 0.06 10.0 4.3 0.06 12.3 4.0 S.E. 0.03 4.5 2.1 0.02 5.5 2.0 6 11261 7.42 128.9 38.9 7.41 118.1 38.3 B 7 01092 7.43 94.7 40.3 7.40 106.3 39.0 (Example 2) 7 01103 7.37 109.7 39.9 7.40 99.3 37.5 1mg/Kg-5h 7 01173 7.40 124.4 41.2 7.43 125.8 38.9 7 01232 7.43 101.2 36.2 7.42 108.8 40.3 Average 7.41 111.8 39.3 7.41 111.7 38.8 S.D. 0.03 14.7 1.9 0.01 10.4 1.0 S.E. 0.01 6.6 0.9 0.01 4.6 0.5 6 11063 7.37 120.6 40.5 7.39 92.4 37.1 6 11111 7.41 91.9 41.5 7.41 96.4 40.4 Table 9 Arterial Glucose in Rats

Groups No. of 15min During animal Before MCAo MCAo (15min) 6 10303 95 84 A 6 11042 122 105 (Vehicle) 6 11074 131 137 Saline-5h 7 01313 98 112 7 02061 146 111 Average 118.4 109.7 S.D. 21.8 19.1 S.E. 9.8 8.5 6 11261 130 115 B 701092 139 168 (Example2) 701103 171 138 1mg/Kg-5h 7 01173 111 141 7 01232 130 144 Average 136.2 141.2 S.D. 22.0 18.9 S.E. 9.8 8.4 611063 121 118 611111 138 112 701283 103 100 C 701284 108 114 (Vehicle) 702031 125 144 Saline-48h 702042 104 110 702043 106 113 702052 94 110 7 02053 96 114 Average 110.6 115.0 S.D. 14.5 11.9 S.E. 4.8 4.0 6 11054 133 101 611123 99 114 D 7 11124 96 117 (Example 2) 6 11253 121 111 1mg/Kg-48h 7 01143 200 139 701215 108 134 701272 120 129 701273 154 128 Average 128.9 121.6 S.D. 34.3 12.9 S.E. 12.1 | 4.6 T-test(A vs.B) 0.23 0.09 T-test (C vs. D) | | 0.16 1 0.29 Table 10 Neurological Outcome Following lrOmin MCAo in Rats

Groups No. of MCAo Score Score Score After MCAo animal (min) before during MCAo MCAo 15min 60min 3h 5h 24h 48h 6 10303 120 0 9 8 8 A 611042 120 0 8 8 8 (Vehicle) 6 11074 120 0 9 9 9 Saline-5h 7 01313 120 0 9 9 9 7 02061 120 0 8 8 8 Average 8.6 8.4 8.4 S.D. 0.5 0.5 0.5 S.E. 0.2 0.2 0.2 6 11261 120 0 6 6 5 B 7 01092 120 0 8 6 6 (Example2) 7 01103 120 0 8 8 8 1mg/Kg-5h 7 01173 120 0 6 6 5 7 01232 120 0 9 5 5 Average 7.4 6.2 5.8 S.D. 1.3 1.1 1.3 S.E. 0.6 0.5 0.6 6 11063 120 0 9 8 9 6 11111 120 0 9 7 7 7 01283 120 0 9 5 6 C 7 01284 120 0 9 7 7 (Vehicle) 7 02031 120 0 9 died Saline-48h 7 02042 120 0 9 6 6 7 02043 120 0 9 died 7 02052 120 0 9 8 8 7 02053 120 0 9 6 6 Average 9 6.7 7.0S.D. 0 1.1 1.2 S.D. 0 1.1 1.2 S.E. 0 0.4 0.4 6 11054 120 0 9 5 6 6 11123 120 0 6 4 4 7 11124 120 0 5 died D 6 11253 120 0 8 6 died (Example 2) 7 01143 120 0 8 6 5 1mg/Kg-48h 7 01215 120 0 8 4 4 7 01272 120 0 8 4 4 7 01273 120 0 6 4 4 Average 7.3 4.7 4.5 S.D. 1.4 1.0 0.8 S.E. 0.5 0.4 0.3 T-test(Avs.B) 0.10 0.004 0.003 T-test(Cvs.D) 0.002 0.004 0.001 Table 11 Tissue Evans Blue Content

Groups No. of Evans Blue Hemispheres Total animal ( g/g tissue) EB ( g/g tissue) EB Samp. Samp. Samp. Samp. Samp. Samp. right left g/g 1 2 3 4 5 6 6 10303 11.13 7.87 25.48 10.49 32.41 13.81 69.0 32.2 101.2 A 6 11042 9.62 7.99 16.81 9.76 50.28 13.55 76.7 31.3 108.0 (Vehicle) 6 11074 18.69 10.57 48.28 15.74 52.85 16.27 117.8 42.6 160.4 Saline-5h 7 01313 7.59 5.53 18.03 6.24 41.61 4.26 67.2 16.0 83.3 7 02061 7.18 9.11 14.82 7.52 59.27 8.1 81.3 24.7 106.0 Average 10.8 8.2 24.3 10.0 47.3 11.2 82.4 29.4 111.8 S.D. 4.7 1.9 12.9 3.7 10.5 4.9 20.6 9.8 28.9 S.E. 2.09 0.83 5.79 1.64 4.67 2.19 9.21 4.39 12.92 6 11261 8.03 9.68 17.3 13.5 37.16 16.38 62.5 39.6 102.1 B 7 01092 1.76 2.28 12.07 5.77 22.13 14.11 36.0 22.2 58.1 (Example2) 7 01103 6.89 7.94 11.8 9.44 19.03 13.53 37.7 30.9 68.6 1mg/Kg-5h 7 01173 6.32 2.52 1.42 8.36 28.9 0.7 36.6 1.6 48.2 7 01232 4.02 2.4 4.07 3.47 24.91 3.76 33.0 9.6 42.6 Average 5.4 5.0 9.3 8.1 26.4 9.7 41.2 22.8 63.9 S.D. 2.5 3.6 6.5 3.8 7.0 7.0 12.1 12.7 23.5 S.E. 1.12 1.59 2.89 1.70 3.14 3.12 5.39 5.69 10.51 6 11063 20.54 12.67 13.12 9.4 24.85 10.27 58.5 32.3 90.9 6 11111 15.42 7.24 14.48 6.38 21.43 5.82 51.3 19.4 70.8 C 7 01283 6.08 5.02 65.85 9.03 81.97 8.16 153.9 22.2 176.1 (Vehicle) 7 01284 14.59 13.11 54.38 8.9 68.97 12.91 137.9 34.9 172.9 Saline-48h 7 02042 11.9 7.2 12.32 5.8 35.61 5.47 59.8 18.5 78.3 7 02052 10.89 8.22 17.23 6.03 23.49 5.14 51.6 19.4 71.0 7 02053 14.52 7.41 25.69 11.86 99.04 13.72 139.3 33.0 172.2 Average 13.4 8.7 29.0 8.2 50.8 8.8 93.2 25.7 118.9 S.D. 4.5 3.0 22.0 2.2 32.0 3.6 47.6 7.4 51.8 S.E. 1.69 1.14 8.30 0.84 12.09 1.35 18.00 2.78 19.56 6 11054 9.42 8.55 9.73 9.64 15.81 12.12 35.0 30.3 65.3 D 6 11123 8.04 5.07 18.91 5.12 14.97 0.23 41.9 10.4 52.3 (Example 2) 7 01143 1.99 1.23 4.43 0.83 15.36 3.99 21.8 6.1 27.8 1mg/Kg-48h 7 01215 3.04 2.21 2.25 2.63 20.68 3.71 26.0 8.6 34.5 7 01272 8.76 6.2 5.67 5.55 26.01 3.93 40.4 15.7 56.1 7 01273 5.24 6.49 3.22 2.74 10.43 4.33 18.9 13.6 32.5 Average 6.1 5.0 7.4 4.4 17.2 4.7 30.7 14.1 44.8 S.D. 3.1 2.8 6.2 3.1 5.4 3.9 9.8 8.7 15.2 S.E. 1.28 1.13 2.54 1.26 2.20 1.60 4.00 3.53 6.19 T-teat(Avs.B) 0.05 0.11 0.05 0.46 0.01 0.70 0.005 0.39 0.02 T-test(Cvs.D) 0.01 0.04 0.04 0.03 0.03 0.08 0.01 0.02 0.01

Claims

CLAIMS 1. A compound of general formula (I) or a pharmacologically acceptable salt thereof:

wherein R1 represents a hydrogen atom or a lower alkyl group; R2 represents a group selected from the following:

wherein X represents a halogen atom; or R1 and R2 can form a 4-methylpiperazinyl group together which may be substituted; R3 represents a group selected from the following group;

R4 represents a hydrogen atom or a lower alkoxy group.
2. A compound or a pharmacologically acceptable salt thereof as claimed in claim 1, which is a compound selected from: ( 1 ) N-(4-pyridyl)-2-cyclopentyloxynicotinicamide, (2) N-(4-pyridyl)-2-exonorbornyloxynicotinic amide, (3) N-(4-pyridyl)-2-(4-fluorophenyloxy) nicotinic amide, (4) N-(4-pyridyl)-2-(3-hydroxycyclohexyloxy) nicotinic amide, (5) N-(4-pyridyl)-2-(2-exonorbornylamino) nicotinic amide, (6) N-(4-pyridyl)-2-cyclooctylaminonicotinic amide, (7) N-(4-pyridyl)-2-adamantylaminonicotinic amide, (8) N-(4-pyridyl)-2-exonorbomyloxy-6-methyl nicotinic amide, (9) N-(3-pyridyl)-2-exonorbornyloxynicotinic amid, (10) N-(3 ,5 -dichloro-4-pyridyl)-2-(2-exonorbornyloxy) nicotinic amide, (11) N-(5-isoquinolynyl)-2-(2-exonorbornyloxy) nicotinic amide, (12) N-(4-picolyl)-2-(4-fluorophenyloxy) nicotinic amide, (13) N-(4-picolyl)-2-(3-nitrophenylamino) nicotinic amide, (14) N-(3-picolyl)-2-(2-Exonorbornyloxy) nicotinic amide, (15) N-(3-picolyl)-2-(3,4-dimethoxyphenyloxy) nicotinic amide and; (16) N-(1-Benzyl-4-piperidyl)-2-(2-exonorbornyloxy) nicotinic amide.
3. A pharmaceutical composition comprising a therapeutically or ameliorative effective amount of a compound or a pharmacologically acceptable salt thereof as defined in claim 1 and a pharmacologically acceptable vehicle.
4. The use of a compound or a pharmacologically acceptable salt thereof as defined in claim 1 for the making of a medicament for treating or ameliorating a disease against which phosphodiesterase antagonism is efficacious.
5. A method for treating or ameliorating a disease which comprises administering a pharmaceutically effective amount of a compound or a pharmacologically acceptable salt thereof as defined in claim 1 to a patient suffering from a disease against which phosphodiesterase antagonism is efficacious.
6. A method for treating or ameliorating a disease which comprises administering a pharmaceutically effective amount of a compound or a pharmacologically acceptable salt thereof as defined in claim 1 to a patient suffering from a variety of allergic and inflammatory diseases such as asthma, chronic bronchitis, shock, rheumatoid arthritis, stroke, reperfusion injury, encephalomyelitis and multiple sclerosis.
7. A process for preparation of a compound of general formula (II),

comprising derivatising an optionally protected compound of general formula (m),

and optionally thereafter converting the compound of general formula (11) so formed into another compound of general formula (II), in which Rl to R4 have the same meaning as defined in claim 1.
8. A process as claimed in claim 7, in which the derivativisation of the compound of general formula (Ill) to form a compound of general formula (II) is treatment with: (a) a chlorinating agent (b) a mixed acid anhydride forming agent or, (c) 1,3-dicyclohexylcarbodiimide (DCC), and a primary or secondary amine of general formula (IV), HNRtR2 (IV) (wherein R' and R2 have the same meaning as defined in claim 1).
9. A process as claimed in claim 8, in which the chlorinating agent is thionyl chloride, sulfuryl chloride, oxalyl chloride, phosphorus pentachloride, phosphorous trichloride or phosphorous oxychloride.
10. A process as claimed in claim 8, in which the mixed acid anhydride forming agent is methyl chloroformate or ethyl chloroformate.
11. A process as claimed in any one of claims 8 to 10, in which the compound of general formula (II) is further treated with an alcohol or an amine to give a compound of general formula (I).
12. A compound of general formula (II), in which Rl to R4 have the same meaning as defined in claim 1,

with the proviso that N-(4-pyridyl)-2-chloronicotinic amide, N-(3-pyridyl)-2chloronicotinic amide and N-(2-pyridyl)-2-chloronicotinic amide are excluded.
13. A compound as claimed in claim 12 which is, (1) N-(4-pyridyl)-2-chloro-6-methyl nicotinic amide, (2) N-(3,5-dichloro-4-pyridyl)-2-chloronicotinic amide, (3) N-(5-isoquinolyl)-2-chloronicotinic amide, (4) N-(4-picolyl)-2-chloronicotinic arnide, (5) N-(3-picolyl)-2-chloronicotinic amide and; (6) N-(N-benzylpiperidin-4-yl)-2-chloronicotinic amid.