Tmi π7_cιyrid-Lne PAF Antagonists This invention relates to specifically to σertain 4-su___etit__uted-l-(2-metfayliτnida_- >[4,5-c] yri -1- l)-benzene derivatives. The ccπpαunds are potent and selective antagonists of platelet activating factor having clinical utility in tte treatπιaτt of allergic and inflaπinatαry oαnditions in humans and animals.

Platelet activating factor (PAF, l-0-a_Uςyl-2-aoetyl-sn- is an ether phopholipid -whose structure was first elucidated in 1979. It is produced by, released frαn and interacts with many pt >-i-τfl--_maatαcy cells, platelets and the kidney. In addition to potent platelet aggregating activity, PAF e__d_ bits a wide Epectrum of biological activities elicited either directly αr via ths release of othsr powerful mediators such as thrrmbπxane A_ αr the leukotrienεs, ^• viiich make PAF _i_r_hibit_αrs of potential value in the treatment of a variety of conditions including allergic, inf___am_ratαry and hypersecretσry conditions such as asthma, arthritis, riiinitis, l_H_TDnchitis and urticaria, the treatment of cirαilatαry shock, gastric ulσeration, psoriasis and CTix_tiovasc_ular σσnditicns, including angina, 1_h_π_____bαsis and stroke.

In our European patent application no 0258033 v ^* e disclose a series of 2-subεtituted l,4- ihyd_ pyr_Ldi_r_e derivatives as PAF antagonists. In our later European patent application no 0310386 we disclose a further series of dihydropyridirie PAF antagonists %4ιerein the 2-position substitueπt includes in particular a 2-fflethyl-i_πιi_dazo[4,5-^ group. The present

T SHEET

iπvention provides further PAF antagonists having the forπula:

and their pharmaceutically acceptable salts, wherein K , R and

3 4

R are each H αr methyl, E is hydrogen, halo, C,-C ₄ aUkyl αr

C,-C. al-kαxy and n is an integer of from 1 to 3.

In the above definition, the term halo means fluoro, chloro, brαno αr ±odo. Alkyl and alkoxy groups of 3 or more carbon atoms may be straight αr branched-chain.

_1 2 3 In preferred embodiments of the invention R , R and R are

H, R is preferably chlorine and n is 1 or 2. When R is a branched alkyl or alkoxy group having 4 carbon atoms, the ccnpounds of the formula (I) may contain at least one asyπmetric centre and exist as a pair of enanticmers. Such isomers may be separable by physical methods, e.g. by fractional crystallisation αr chromatography of the parent corrpounds or of a suitable salt αr derivatives thereof. The invention includes all the enantiomers whether separated αr not.

The phaπnaceutically acceptable acid addition salts of the compounds of the formula (I) which form such salts are those

farmed from acids which form non-toxic acid addition salts, for example the hydrochlαride, hydrobrαnide, sulphate or hisulphate, phosphate or acid phosphate, acetate, citrate, fumerate, gluconate, lactate, maleate, succinate and tartrate salts.

The compound of forπula I may be obtained by cyclisation of a cαnpound of the formula:

—1 2 3 4 wherein IT ^" , R , R , R and n are as previously defined.

In a typical procedure the compound of formula II is simply heated in a high-hoiling organic solvent (for example toluene or xylene) at a temperature above 100°C for a period of several hours. The solvent is evaporated and the product recovered and purified by conventional procedures..

The intermediates of formula II are prepared by reaction of an amine of formul -:

with a benzoic acid or anhydride derivative of fαrπula-:

This reaction is achieved by either reacting the anhydride with the amine (III) in water or an aqueous organic solvent in the presence of a base (e.g. sodiτxn carbonate), or by reacting the benzoic acid with the amine using a diimide ∞up_l_ing reagent such as l-(3-d ^* j_αgtl ^* ylaminc)prcφyl)-3 iraide.

Appropriate reagents and conditions for these steps may be readily established by reference to standard text books and to the exaπples provided hereafter.

The activity of the ocπ_pounds of the invention is shown by their ability to inhibit the platelet aggregating activity of PAF in vitro. Testing is performed as follows:

Blood saπples are taken from either rabbit or man into 0.1

vol disodium et ylenediaπiine tetraacetic acid buffer and the saπples centrifuged for 15 minutes to obtain platelet rich plasma. ^~ ~bε plasma is further centrifuged to give a platelet pellet which is washed with a buffer solution (4 irtϊ KH-FO., 6ιriM Na_HP0., 100 mM NaCl, 0.1% glucose and 0.1% bovine serum albumin, pH 7.25) and finally resuspended in buffer solution to a concentration of 2 x

Q

10 platelets/ml. A saπple (0.5 ml) is pre-incubated with stirring for two minutes at 37 C in a Paton aggregometer, either with vehicle alone, αr with vehicle cxmtaiiiing the particular compound under test. PAF is added at a sufficient concentration to give a maxinun aggregating response in the absence of test

—8 —9 coπpound (10 to 10 molar), and the platelet aggregation is measured b following the increase in light transmission of the solution. The experiment is repeated in the presence of test ccπpound at a range of concentrations and the concentration of coπpound required to reduce the response to 50% of its maximm value in recorded as the IC ₅₀ value.

The activity of the ccnpounds of formula (I) is also demonstrated in vivo by their ability to protect mice from the lethal effect of an injection of PAF. A mixture of PAF (50 jgkg) and DL-propranolol (5 mg/kg) in 0.9% w/v sodium chloride is injected (0.2 ml) via a tail vein into mice. The compounds under test are either injected into the tail vein imnediately prior to the PAF/propranolol injection αr administered orally by gavage tvro hours earlier. The ccmpounds are tested at several doses in groups of 5 mice and the dose which reduces mortality to 50% is recorded as the H_> ₅ _ value.

For therapeutic use the compounds of the formula (I) will generally be administered in admixture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice. For exanple, they may be arfcni ig<-« ^** 'ττ ^p i orally in the form of tablets containing such excipients as starch αr lactose, αr in capsules αr ovules either alone αr in admixture with excipients, or in the form of elixirs or suspensions containing fla_vou_ring or colouring agents. They may be injected parenterally, far exanple, intravenously, iπtraπ scularly or subcutaneously. For parenteral administration, they are best used in the for of a sterile aqueous solution which may contain other substances, for exanple, enough salts or glucose to make the solution isotonic with blood.

Ear akninistration to man in the curative or prophylactic treatment of allergic branchial conditions and arthritis, oral dosages of the compounds will generally be in the range of from 2-1000 mg daily for an average adult patient (70 kg). Thus for a typical adult patient, individual tablets or capsules will ccxitain from 1 to 500 mg of active compound, in a suitable pharmaceutically acceptable vehicle or carrier. Dosages for intravenous ckάbiinistratiαn would typically be within the range 1 to 10 mg per single dose as requ red. For the treatment of allergic and bronchial, hyper-reactive conditions, inhalation via a nebuliser αr aerosol may be the preferred route of drug acininistration. Dose levels by this route would be within the range 0.1 to 50 mg per single dose as required. In practice the physician will determ ne the actual dosage which will be most suitable for an individual patient and it will vary with the age.

weight and response of the particular patient. The above dosages are exeπplary of the average case but there can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.

Thus in a further aspect the invention provides a pharmaceutical ccπposition ocnprising a coπpound of the formula (I), or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable diluent or carrier.

The invention also includes a ccπpound of the formula (I), or a pharmaceutically acceptable salt thereof, for use in medicine, in particular in the treatment of allergic, inflammatory and hypersecxetαry conditions in a human being.

The preparation of the intermediates of formula (II) and the compounds of formul (I) will now be more particularly .illustrated by reference to the following experimental Examples. The purity of coπpounds was routinely monitored by thin layer chromatography using Merck Kieselgel 60 F_ ₅₄ plates. TWϊuclear magnetic reasonance spectra were recorded using either a Nicolet QE-300 αr a Bruker AC-300 spectrometer and were in all cases consistent with the proposed structures. Chemical shifts are given in parts-per- ntillion dσwnfield from tetramethyl≤ilane using conventional abbreviations for designation of major peaks: s, singlet; d, doublet; t, triplet; m, nultiplet and br, broad.

EXflMP E 1 7-Chloro-3 _r 4-dihyd ^* ro-2-r4 2- ethyl_i^ pheπyl1-5H-l.4-benzodiazepin-5-one a) 4-Chloroisatoic anhydride (0.32 g, 1.6 πmol) was added to a solution of 2-aminι -4 ^/ -(2-methyli i azo[4,5-c3pryrid-l-yl)- acetophenone (0.57 g, 1.45 πmol) in water (6 ml). After stirring to "uniform consistency, a solution of sodiim -carbonate (0.19 g, 1.8 πmol) was added. After 1 hour, dichloromethane (12 ml) was adflpd and two phase stirring was resulted for 66 hours. The mixture was partitioned between saturated aqueous sodiim hydrogen carbonate and dLch_l_αrαnethane. The organic phase was separated, dried (MgSOu) and evaporated to an amorphous solid. Flash c_hromatography on silica, eluting with 15 then 25% methanol in ethyl acetate afforded 2-aminc^-5-chloro- i-{2-[4-(2-methylimida7.o- [4,5-c]pyrddr-l ^* ^l)pheα^l]-2-<3Xoetl l}benzamide as a solid (0.14 g, 23%), m.p. H6-118°C. ~ ^~ ϋ NMR (CD ₃ S0CD ₃ ): 2.52(3H,s), 4.79(2H,d,J 5.4Hz), 6.58hr exch (2H,s), 6.76(lH,d,J 8.9Hz), 7.21(lH,dd,J 8.9,2.2Hz), 7.28(lH,d,J 5.4Hz), 7.67(lH,d,J 2.2Hz), 7.82 arxi 8.28 (each 2H,d,J 8.4Hz), 8.35(lH,d,J 5.5Hz), 8.85 hr exch (lH,t,J 5.4Hz) and 8.94(lH,s). b) A suspension of the benzand.de from part (a) (0.11 g, 0.26 πmol) in xylene (5 ml, containing 2-3 drops ethanol to aid solubility) was heated to 140 C for 5 hours. The solvent was evaporated and the residue purified by flash chromatography on silica, eluting with 10 then 20% methanol in ethyl acetate to afford the title product as a pale-yellow amorphous solid (0.041 g, 40%), m.p. 225-227°C. Found: C,63.52; H,4.14; N,16.61. C ₂₂ ^ ₁₆ C1N ₅ 0, 0.75 B^O requires C,63.62; H,4.25; N,16.86%.

EXAMP E 2 7.8-Dicfalo_ro-3.4-dihydro-2-r4 ^/ -(2 phenyπ-5H-l.4-beπzodiazepin-5-one a) The procedure of Exanple 1 part (a) was followed but starting with 4,5-dichlαrαisatoic anhydride to yield 2-cin-Uio-4,5-dichlαro- N-{2-[4-(2-raet_t_y_I__u_-___da∞^ benzanri.de. hi MR (CDCl _j ): 2.63(3H,s), 5.00(2H,d,J 1.2Hz) , 5.69br exch. (2H,s), 6.83(lH,s), 7.18(2H,m), 7.62(3H,m), 8.33(2H,d,J 8.4Hz), 8.42(lH,d,J 5.5Hz), 9.11(lH,s). b) The above product was cyclised following the procedure of Exanple 1(b) to give the title product in 58% yield, m.p. 174-177°C. Found: C,58.08; H,3.41; N,14.94. requires C,58.16; H,3.77; N,15.40%.

EXAMPLE 3 7.9-Dic-Maro-3.4---dihydr^ pheπyl1-5K-l,4-benzodiazepin--5-αne a) A solution of 2-amino-4 '-(2-methylimidazo[4,5-c]pyrid-l-yl)- acetophencae hydrochloride (0.7 g, 1.8 πmol), 3,5-dichloro-2- aπ nαbenzoic acid (0.48 g, 2.3 πmol), (0.34 g, 2.4 nmol), HHaethylmαrphnline (1.61 ml, 16 πmol) and l-(3- dimRthylaminopropyl)-3-ethylcarhodii ide hydrochloride (0.89 g, 4.7 πmole) in dichloromethane (25 ml) was stirred at 25 C for 3 hours. All vαlatiles were evaporated and the residue was partitioned between ethyl acetate and water buffered to pH 5. The organic layer was separated, dried (MgS0 ₄ ) and evaporated. Flash chromatography of the residue on silica, eluting with 10%

methanol in ethyl acetate afforded 2-amino-3,5-dichloro-4f-{2-[4 -(2-methyliπ_idazo[4,5-c]pyrid-l-yl)phenyl]-2-αxoethyl}ben zamide as a pale-yellow gi (0.21 g, 26%). TI NMR (CDC ): 2.62(3H,s), 4.98(2H,d,J 5.7Hz), 6.10 br exch. (3H,m), 7.13(lH,d,J 5.7Hz), 7.39(lH,d,J 2Hz), 7.48(lH,d,J 2Hz), 7.57 and 8.30 (each 2H,d,J 8.4Hz), 8.43(lH,d,J 5.7Hz) and 9.07(lH,s). b) p-Toluenesu_lphonic acid hydrate (0.1 g, 0.5 nmol) was added to a re-fluxing solution of the ketoamine from part (a) (0.21 g, 0.47 nmol) in dichloromethane (14 ml). After 6 hours, the mixture was partitioned between dichlαromethane and saturated aqueous sodium hydrogen carbonate. The organic layer was dried (MgSO.) and evaporated to a yellow foam. Flash chromatography on silica, eluting with 5% methanol in ethyl acetate afforded the title product as an off white solid (0.065 g, 33%), m.p. 165-7°C. Found: C,58.92; H,3.85; N,14.57. 0.5 EtOAc requires C,58.91; H,4.12; N,14.31%.

FHEFARATIOW 2-Amino-4 ^, - ( 2-methyl i mi ήazoϊ .5-clpyrid-l-yl ) acetophenone hydrochlαride

(a) 4-(4--Acetylpheπyl)aπύno-3-nit_ιx^^ hydrochloride

A solution of 4-chlaK)-3-nitropyridine hydrochloride (9.75 g, 50 nmol) in ethanol (40 ml) was added to a slurry of p-aminoaceto- phenone (6.76 g, 50 nmol) in ethanol (25 ml), and the mixture was stirred at room temperature overnight. The mixture was chilled in ice, and the yellow solid filtered off and dried (10.1 g, 69%). m.p. 197-200°C.

(b) 4-(4-Acetyl|^τenyl)amino-3-aminopyridine hydrochloride (2.0 g,

78.8 nmol) was partitioned between aqueous sodium hydroxide and dichljαromethane ( 3 x 20 ml). The combined organic phases were ■ washed with water (20 ml) and concentrated under reduced pressure to give a solid. Ethanol (20 ml) was added, and the solution was hydrogenated over 5% palladitm on carbon (0.2 g) at 50 p.s.i. (3.4 bar) for 3.5 hours. The catalyst was filtered off, and the solvent removed under reduced pressure to give a brown solid, (1.8 g, ca 100%) which was used directly for the next stage without purification.

(c) 4-2-(Methyl_imidazor4.5-clpyrid-l-yl)acetophenone

A solution αf 4-(4-acet_yl|_heπyl)amiιx ^** ^3-a^ (68.0 g, 0.3 nmol) in acetic acid (204 ml) and acetic anhydride (204 ml) was heated at 95 C for 1.5 hours then cooled and concentrated under reduced pressure. The residue was dissolved in water (500 ml) and rendered basic by the addition of saturated aqueous aπmonia. The product was filtered off, washed with water (2 x 100

ml) and dried under -vacuum to give the title ccπpound, (61.0 g, 81%) as a brown solid, m.p. 155-156°C (from water).

(d) Ethyl 4 ^, -(2-^nethyliιaLdazor4.5-c1pyrid-l-yl)benzo^ A solution of 4-(2-^ιetl^l_i_nήdazo[4,5-c]pyrid--l-yl- acetophenone (17.5 g, 69.7 nmol) in dry tetxahydrofuran (175 ml) was added to a slurry of sodium hydride (3.68 g, 153 nmol) in a mixture of dry tetrahydrofuran (35 ml) and diethyl carbonate (24.7 g, 209 nmol) at reflux with stirring over 45 minutes. After a further 1 hour, the mixture was cooled, hexane (200 ml) was added, and the resulting precipitate was filtered off and washed with hexane (2 x 100 ml). The solid was suspended in ethyl acetate (200 ml) and acetic acid (10.2 g) was added. After stirring far 15 minutes, water (200 ml) was added, and the organic layer was separated. The aqueous phase was extracted with ethyl acetate (100 ml) and the combi ed organic solutions were washed with water (200 ml), dried (MgSO.) and concentrated to give the title product as a gun (17.3 g, 77%). Further purification by flash chrcπatography (eluting with ethyl acetate/methanol (7:1) gave the - title coπpound as a white solid, m.p. 11-112 C.

(e) Ethyl 4'-(2- ^** - ^* Met ^* hyl_-imidazc ^* pyriό>l-yl)

A solution of sodiim nitrite (3.3 g, 47 nmol) in water (40 ml) was added in drops to a solution of ethyl 4'-(2-methyl- i i azo[4,5-c3pyrid-l-yl)benzqylacetate (12.6 g, 39 nmol) in glacial acetic acid (45 ml) at 5°C. After 1.5 hours the mixture was partitioned between dichloromethane and brine. The organic layer was washed again with brine and then with saturated aqueous

sodii bicarbonate, dried (MgSO.) and evaporated to an oil which rapidly crystallised on addition of ether (9.61 g, 70%), (2:1 mixture of syn/anti isomers). M.p. 168-170°C

(f) Ethyl 2-acgtamido-4'-(2- ^* -g ^* tethy.Li d benzoylacetate

A solution of the product from e) above (6 g, 17 nmol) in acetic acid (33 ml) and acetic anhydride (9 ml) was hydrogenated over 5% palladium an carbon (1 g) at 50 p.s.i. (3.45 bar) at 30 C for 5 hours. The mixture was filtered through a filter pad, washing the cake with methanol and the filtrate was evaporated. The residue was chromatographed eluting with methanol and then 10% methanol in ethyl acetate to afford a colourless foam (6.1 g, 94%). M.p. 71-73°C. dihydroc oride

A solution of the product from f) above (1.2 g, 3.2 nmol) in 2M hydrochloric acid (30 ml) was heated at reflux for 3 hours. The solution was evaporated to dryness to yield the amine hydrochloride salt as a colourless foam (1.35 g), which was stored under vacuum.