NO770992L - PROCEDURES FOR THE PREPARATION OF 2-NITROINDANDION DERIVATIVES - Google Patents

Description

The invention relates to certain 2-nitroindanedione derivatives, pharmaceutical preparations comprising such compounds, and methods for their preparation.

It is well known that some types of cells are activated by certain antibody/antigen combinations and release substances such as

t mediates the allergic response. It has been reported that SRS-A

(the slow-reacting substance for anaphylaxis) plays an important role in the development of allergic and asthmatic phenomena.

The 2-nitroindan-1,3-dione derivatives according to the invention not only inhibit the action of SRS-A, but they also prevent the release of the mediators from activated cells. They are therefore useful in the prophylaxis and treatment of allergic diseases. Examples of such diseases include bronchial asthma, rhinitis and allergic eczema.

Accordingly, the invention provides a compound of formula (I) and pharmaceutically acceptable salts thereof: where R, and R 2 are the same or different and represent hydrogen, lower alkyl, lower alkenyl, lower alkynyl or lower alkoxy, or R 2 and R 2 , when on adjacent carbon atoms, represents an alkylene group containing 3-5 carbon atoms or a 1,4-buta-1,3-dienylene group, and A represents a group of formula (II):

where. X is a bond or an oxygen atom and Q represents a linear alkylene group containing 1-9 carbon atoms, one methylene group within the group Q, which is not a methylene group covalently bound to an ether oxygen, is optionally substituted

. with hydroxyl, and , R4, R1., R^ and R^ each represent hydrogen, halogen, nitro, hydroxy, cyano, carboxyl, amino, lower

alkyl, lower a.lkenyl, lower alkynyl, lower alkoxy, lower alkoxycarbonyl, lower acyl, lower acyloxy, mono- and di-lower alkylamino, mono- and di-lower acylamino, phenyl, lower alkyl-phenyl, phenoxycarbonyl, benzyloxycarbonyl, or .any two of R^ to R^, when on adjacent carbon atoms', together represent an alkylene group containing 3-5 carbon atoms, or a 1,4-buta-1,3-dienylene group.

As used here, the terms lower alkyl, lower alkenyl and lower alkynyl, lower alkoxy and lower acyl mean such groups containing from 1-6 carbon atoms.

The term 2-nitroindane-1,3-dione core here means a group of formula (III):

and the positions referred to by numbers in the 2-nitrb-indan-1,3-dione are as indicated above. When reference is made here to "the phenyl ring in the substituent A", this means the phenyl ring shown in formula (II) bearing the s-substituents, R 1 , R 4 , R 5 , R 1 and R 7 and the positions referred to by number in the phenyl ring in substituent A is as indicated below:

At least one, but not more than 4 of R-. to R₂ suitably represents hydrogen, and where these substituents are other than hydrogen, it is preferred that they are different.

Examples of suitable lower alkyl substituents falling within the definitions of R₁ to R₂ are methyl, ethyl, n- and iso-propyl, n-, iso and t-butyl.

Examples of suitable lower alkenyl groups represented by 'R^-R^ are 2-propenyl; 2- and 3-butenyl; 2-, 3- and 4-pentenyl;

2-, 3-, 4- and .5-hexenyl; 1- and 2,-methyl-2-propenyl; 1-, 2- and 3-methyl-2- and 3-butenyl; 1-, 2-, 3- and 4-methyl-2, 3- and 4-pentenyl;

1- and 2-ethyl-2-propenyl; 1-, 2- and 3-ethyl-2- and 3-butenyl.

Examples of suitable lower alkynyl groups represented by R^-R^ are 2-propynyl; 2- and 3-butynyl; 2-, 3- and 4-pentynyl.;

2-, 3-, 4- and 5- hexynyl; 1- and 2-methyl-2-propynyl; 1-, 2- and

3-methyl-2- and 3-butynyl; 1-, 2-, 3- and 4-methyl-2-, 3- and 4-pentynyl; 1- and 2-ethyl-2-propynyl; 1-, 2- and 3-ethyl-2- and 3-butynyl.

Examples of suitable lower alkoxy substituents falling within the definitions for R^ to R^ are methoxy, ethoxy, n-

and iso-propoxy, n, iso- and t-butoxy. Examples of suitable lower acyl groups included within the definition of R^ to R^ are acetyl, propionyl, n- and iso-butyryl. Examples of lower alkoxycarbonyl substituents included within the definition for R 1 to R 2

is methoxycarbonyl, ethoxycarbonyl, n- and iso-propoxycarbonyl,

n- and iso-butoxycarbonyl. Examples of lower acyloxy groups falling within the definition for R^ to R^ are acetoxy, propypoxy, butyryloxy and pentanoyloxy. These previously mentioned examples of lower alkyl and lower acyl groups are also suitable as examples of the lower alkyl and lower acyl groups contained in mono- and di-lower alkyl and lower acylamino substituents which are included within the definition for R^ to R^ . The suitable halogen atoms which. are included within the definition for R^ to R^ are fluorine, chlorine and bromine. The most suitable halogen is fluorine.

The alkyl, alkoxy, alkenyl, alkynyl, acyl, acyloxy and alkoxycarbonyl groups represented by R-^ to R^. is preferably unbranched. If a highly substituted compound of. formula (I) kr eves, it will be understood that the substituents must be chosen with regard to steric compatibility. For example, if two or three of the substituents are groups with complex stereochemistry, e.g. strongly branched lower alkyl, lower alkoxy or similar groups, then these will not occupy adjacent positions.

Examples of linear alkylene groups represented by Q are methylene, ethylene, propylene, butylene, pentylene, hexylene or heptylene. The optional hydroxyl group can occupy any position on any of such alkylene groups other than the methylene bound to an ether- oxygen. It will be understood in this context that if a methylene within the group Q is substituted with hydroxyl, then the thus substituted methylene is asymmetric.

It should therefore be understood that when the compounds according to the invention are referred to herein, mixtures of enantiomers as well as pure enantiomers are included.

Within the general framework of compounds according to the invention there are certain more suitable subgroups of compounds, depending on the nature of the substituents R 1 to R 2 . thus a first, more suitable subgroup consists of the compounds of formula (I), where R^, R^, Rg, Rg and R^ are as previously defined, and R^ and R^.

is hydrogen or lower alkyl. More conveniently, only one of R^ or R^ is hydrogen while the other is a lower alkyl group which preferably occupies a position close to both the substituent A and the ring junction.

Another, more suitable group of compounds that fall within the general scope of the invention are those in which the phenyl ring in the substituent A does not carry more than two polar groups.

This class of compounds has formula (I), and R 1 and R 2 are as previously defined, R 2 is hydrogen, lower alkyl, lower alkoxy, lower acyl, lower acyloxy, lower alkoxycarbonyl, phenyl, cyano, carboxyl, halogen, nitro . lower

alkyl group, or any two of R^, R^ and R^-, when they are

on adjacent carbon atoms, represents an alkylene group containing 3-5 carbon atoms, or a 1,4-buta-1,3-dienylene group, and Rg and are both hydrogen.

A third suitable group of compounds falling within the scope of the invention are those where the phenyl ring in substituent A carries a single lower alkyl substituent, i.e. that R 3 is lower alkyl and R 4 to R 4 are hydrogen. Particularly suitable lower alkyl groups are methyl, ethyl and n-propyl. Most advantageously, this lower alkyl group occupies position 2' in the phenyl ring.

A fourth suitable group of compounds that fall within the scope of the invention are those where the phenyl ring in substituent A carries a single fluorine atom, i.e. that R3 is fluorine and R4 to R? all are hydrogen. Most suitable, the fluorine substituent occupies position 4' in the phenyl ring.

A fifth group of compounds falling within the scope of the invention are those where R^ is a fluorine atom at position 4' in the phenyl ring in substituent A, R^, a lower alkyl group, preferably "propyl, at position 2', and to R^ is hydrogen atoms.

A particularly suitable combination of groups for incorporation into the phenyl ring in substituent A and which transfers a favorable activity level to the indanedion consists of one polar group, e.g. lower acyl, one hydrophilic group, e.g. hydroxyl, and one hydrophobic group, e.g. lower alkyl. It is particularly suitable in such a combination that the hydroxyl group R^ occupies position 3', the lower acyl group R4 occupies position 4', and the lower alkyl group R1- occupies position 2" in the phenyl ring and that R^ and R^ both represent hydrogen. In this combinasorf<r>R4 preferably acetyl and R^preferably n-propyl.

In the compounds according to the invention, R^ and R- 2*°9R3R7i^ke need to be other than hydrogen. That is to say, neither the phenyl ring in substituent A nor the indane dione core is necessarily substituted. A group of compounds of this type that fall within the scope of the invention bear only substituents in the indane ion nucleus and have the general formula (I), where R^ and R2 are as previously defined with respect to formula (I), and R^ to R_, are all hydrogen. A further group of compounds of this type only bear substituents within the phenyl ring of substituent A and have formula (I), where R^ and R^ are both hydrogen and R^ to R^ are as previously defined. Another group of compounds that fall within the scope of the invention carry substituents that are neither in the indahdione nucleus nor in the phenyl ring of substituent A. These compounds have the general formula (i) where R, to R^ all represent hydrogen. Compounds of this type which bearing few substituents are preferred, as they are easy to synthesize. The substituent AO is shown occupying position 5 in the 2-nitroindan-1,3-dione core. The substituents R^ and R^ may occupy any of the remaining positions in the nucleus, but one preferably occupies position 4. Within the general framework of compounds according to the invention, X represents oxygen or a covalent bond. Among the compounds of formula (I) where X is a covalent bond, those compounds where Q is methylene or ethylene have useful oral absorption properties. Where X is oxygen, Q is preferably methylene, but is more suitable ethylene, propylene, butylene, pentylene or hexylene, eventu-. or substituted with hydroxyl, a particularly suitable group being 2-hydroxypropylene. Examples of special compounds that fall within the scope of the invention are: 5-(2-[4-fluorophenyljetoxy)-2-nitroindan-1,3-dione. 5-(3-[4-Acetylphenyl Jpropoxy)-2-nitroindan-1,3-dione. 2-nitro-5-(3-phenoxypropoxy)indan-1,3-dione. -2-nitro-5-(2-phenoxyethoxy)indan-1,3-dione. ;2-nitro-5-(5-phenoxypentoxy)indan-1,3-dione. ;5-(4-chlorophenylmethoxy)-2-nitroindan-1,3-dione. ;2-nitro-5-(3-[2-n-propylphenoxy]-propoxy)-indan-1,3-dione. 2-Nitro-5-(2-hydroxy-3-[2-n-propylphenoxy]-propoxy)-indan-1,3-dione. ;5-(3-[4-acetyl-3-hydroxy-2-n-propylphenoxy]-2-hydroxypropoxy)-2-nitroindan-1,3-dione. ;5-(3-[4-acetyl-3-hydroxy-2-n-propylphenoxy]-propoxy)-2-nitro-indan-1,3-dione. 5-(3-(4-acetyl-3-hydroxy-2-n-propylphenoxy)-2-hydroxy-propoxy)-2-nitro-4-n-propylindan-1,3-dione. Examples of suitable salts of the compounds of formula (I) are the alkali metal salts, especially of sodium and potassium; the alkaline earth metal salts, e.g. of.calcium and magnesium; the aluminum salts and salts with organic bases, e.g. amines or amino compounds, e.g. N-methyl-D-glucamine. If the substituent in formula (II) contains amino groups, then examples of suitable acid addition salts include the acetate, the tartrate, the hydrochloride and the hydrobromide. For administration purposes, the compounds according to the invention can be presented in different dosage forms. According to the invention, a pharmaceutical product is also provided which has SRS-A inhibitory activity, comprising a compound of formula (i) as defined above, or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier. ;, Examples and preferred values of , R^ and A are as previously discussed. The preparations according to the invention can be presented as a microfine powder for snorting (in such a case the particles of the active compound often have diameters of less than 50 microns, preferably less than 10 microns) or in the form of an aerosol or a solution for a mist former. The preparations may also be presented together with a sterile, liquid carrier for injection, or in an ointment, cream, lotion or solution for topical application, or as suppositories. Compounds of formula (I) which are active when given orally can be formulated in the form of syrups, tablets, capsules, pills and the like. The preparations are preferably in unit-dose form or in a form in which the patient can administer a single dose to themselves. For example, if the preparation is in the form of a tablet, pill, lozenge, powder, or various kinds of lozenges, any suitable pharmaceutical carrier can be used for the preparation of solid preparations, e.g. magnesium stearate, starch, lactose, glucose, rice flour and lime. The preparation may also be in the form of a digestible capsule (e.g. of gelatin) containing the compound, or in the form of a syrup, a liquid solution or suspension. Suitable liquid pharmaceutical carriers include ethyl alcohol, glycerol, saline and water together with flavoring or coloring agents to form syrups. A suitable dosage unit may contain from 0.1 to 500 mg of active ingredient. The effective dose of compound (i) depends on the particular compound used, but is generally in the range from 0.01 mg/kg/day to 100 mg/kg body weight per day. ;Where appropriate, small amounts of anti-asthmatics and bronchodilators, e.g. sympathomimetic amines, e.g. isoprenaline, isoetarine, salbutamol, phenylephrine and ephedrine; xantinderiVater, e.g. theophylline and aminophylline; corticosteroids, e.g. prednisolone and adrenal simulants, e.g. ACTH, is included. It is common practice for the preparations to be accompanied by written or printed instructions for use in the medical be- . action in question, in this case as an anti-allergy pot for the treatment of e.g. asthma, hay fever or rhinitis. Compounds of formula (I) can be prepared by a booth-catalyzed rearrangement of the corresponding nitromethylene phthalide derivative. Accordingly, the invention provides a method for producing a compound of formula (I) or a pharmaceutically acceptable salt thereof, the method comprising reacting a compound of formula (IV) and (IVa) or a mixture thereof:;ir ; ; where., R^ and A are as defined for formula (I), with a tertiary base, in the presence of an aprotic solvent and then, if desired, converting the product thus formed into a pharmaceutically acceptable salt. By tertiary base is meant a tertiary amine or a hetero-. ;cyclic aromatic amine..;Suitable tertiary bases include pyridine, picoline and ;tri-lower alkylamines, e.g. triethylamine, which is preferred. ;Suitable aprotic solvents include halogenated hydrocarbons, e.g., chloroform and carbon tetrachloride, and other ;conventional inert solvents, e.g. lower alkyl ethers, dioxane and tetrahydrofuran. Alternatively, the solvent may be the base itself. The reaction is preferably carried out at room temperature. Compounds of formula (i) can also be prepared by nitration of the corresponding indane ion derivatives. Accordingly, the invention also provides a process for the preparation of compounds of formula (I), and the method comprises react a compound of formula (V) : ; where R and A are as defined above with respect to formula (I), with a nitrating agent. The nitration can be carried out using any conventional nitrating agent, e.g. : (I) Acetic and concentrated nitric acid; (II) fuming nitric acid in chloroform and/or ether. The temperature at which the reaction is carried out depends on the nitration reagent used, with the aim of avoiding nitration of the phenyl ring on the side chain (II): ; it is most preferable to use a temperature below 25°C .. ;and one reaction time of up to 90 minutes.;The nitrating agent is preferably fuming nitric acid in ether, and the reaction is preferably carried out in a temperature range from -5° to +5° C, preferably wood. 0°C, for between 15 and 90 minutes. The intermediates of formula (IV) and (IVa) are prepared by coupling a suitable halide of formula (VI) with a dimethyl hydroxyphthalate (VII) using a suitable base, e.g. potassium carbonate, and converting the resulting ester (VIII) to the anhydride (IX). This anhydride is then converted with nitromethane and base to the 2-nitroacetylbenzoic acid derivative (X), which is then dehydrated. ;This is illustrated in form 1 below. ; The compounds of formulas (X) (IV) and (IVa) prepared in this sequence will be in isomeric mixture as shown. This mixture can be used without separation, as both isomers give the same 2-nitro-indan-1,3-dione. In the event that the halide AY contains active groups, e.g. hydroxyl or acetyl which may interfere with the coupling reaction, these are possibly protected with standard protecting groups. ;In the event that the substituent A contains groups that are prone to acylation, the dehydration reaction is carried out with a mild dehydration reagent, e.g. NN'-dicyclohexyl carbodiimide. A further suitable method for preparing the intermediates (IV) (IVa) is to connect the aralkyl halide AY to a hydroxynitromethylene phthalide: ; by a method analogous to that used for the preparation of the diester (VIII). This method is also particularly well suited in the event that the substituent A contains groups which are prone to acylation. The intermediate (v) can be prepared by condensation of the diester (VIII) with ethyl acetate and a strong base. If the substituent A in the diester (VIII) contains active groups, e.g. hydroxyl and/or acetyl which would interfere with the condensation reaction, these are optionally protected by means of conventional protecting groups which are removed after the indan-1,3-dione nucleus has been formed. The diester of formula (VII) and the intermediates of formulas (IV) and (IVa) in which the alkylene chain Q in the substituent A includes a hydroxyl group can be prepared by coupling either the hydroxydimethyl phthalate (V) or the hydroxynitromethylenephthalides (IX), with a epoxide of formula: ; where n . is an integer from 1 to 6.;This coupling reaction is carried out in a polar aprotic solvent, e.g. dimethylformamide (DMF) in the presence of a strong base, e.g. sodium hydride. If the epoxide contains any activated substituents, e.g. acetyl and/or hydroxyl which can interfere in the coupling reaction, these are optionally protected with standard protecting groups. The following examples illustrate the preparation and properties of compounds within the framework of the invention. ;Example 1 Dimethyl-4-(3-phenoxypropoxy) phthalate ;A mixture of dimethyl-4-hydroxyphthalate (2.1 g, 0.01 mol), l-bromo-3-phenoxypropane (2.15 g, 0.01 mol) and anhydrous potassium carbonate (1.52 g, 0.015 mol) in 30 mL of dry butanone were stirred at reflux overnight and the cooled mixture filtered. Evaporation in vacuo gave the title compound in quantitative yield.^max (film) 1730 , 1605 cm-1; X (CDCl 3 ) 7.77 (2H, quintet, J 6.5Hz); 6.16 (3H,s); 6.11 (3H, s); 5.86 (2H, t, J ; 6.5Hz); 5.78 (2H, t, J 6.7Hz); 3.20-2.55 (7H, m); 2.19 (1H, d, j8.5Hz); (found: 65.98% C; 5.89% H; CigH2o05 required:.. 66 , 27% C ; ;.5.85% H). in a similar manner the compounds of Table I were prepared. ; Example 5; 4-(3-phenoxypropoxy). phthalic acid; A stirred mixture of dimethyl 4-(3-phenoxypropoxy)-phthalate (50 g, 0.145 mol), 2.5N sodium hydroxide (570 mL) and methanol (130 mL) was heated overnight at 80°C. The clear solution was diluted with water and acidified so that the diacid was obtained in the form of a white, crystalline solid with m.p. 155-159°c in 90% yield. Recrystallization from water/ethanol gave a material with m.p. 155-157°. (Found: 64.55% C; 5.36% H; c^7<H>i505 required: 64.55% C; 5.10% H). The compounds listed in Table II were prepared in a similar manner. ; Example 9: 4-(3-phenoxypropoxy)phthalic anhydride; 4-(3-phenoxypropoxy)phthalic acid (41 g, 0.134 mol) was refluxed for 30 minutes with excess acetic anhydride, and the product was evaporated to dryness in vacuo. Recrystallization of the resulting white solid from ethyl acetate gave a material with m.p. 96-98°. (Found: 68.48% C; 4.75% H; C17H14O5 required: 68.45% C; 4.73% H). The compounds in Table III were prepared in a similar manner. ; Example 13; 3-nitromethylene-5 and/or 6-(3-phenoxypropoxy)-phthalide; To a stirred suspension of 4-(3-phenoxypropoxy)-phthalic anhydride (13.6 g, 0.0472 mol) in 1 liter of dry ether at 0°, nitromethane (6.16 g, 0.102 mol) was added followed by a cold ethanolic solution of sodium ethoxyde (from Na [1.06 g, 0.0472 mol] and ethanol [24 mL]). The resulting pink suspension was stirred for 6 hours at 0° and 250 ml of water was added. The water phase was separated off, acidified and extracted with ether. Evaporation of the dried (MgSO 4 ) ether extract in vacuo gave a yellow oil which was refluxed for 2 hours with excess acetic anhydride to effect dehydration of the intermediate nitroacetylbenzoic acids. evaporation of the excess of anhydride gave an oil which was chromatographed on silica gel by elution with chloroform, and the yellow fraction crystallized by trituration with ethanol. Recrystallization from ethanol gave 2.1 g (12%) of mixed phthalides with m.p. 115-119°. ;^. max (minor) 1800, 1655, 1600 cm<-1>. (Found-:63.39% C; ;4.71% H; 3 .65% N; C18H15N06 requires: 63.34% .C; 4.43% H; 4.10% N. ;Compounds 14 and 15 in Table IV was prepared in a similar manner. The increased yield of compound 16 was obtained by dropwise addition of base over 2 hours to a mixture containing 1/3 of the amount of anhydride. This was the preferred method. ; Example 17; 2-nitro-5-(3-phenoxypropoxy)indan-1,3-dione; A solution of the mixed 3-nitromethylene-5- and/or 6-(3-phenoxypropoxy)phthalides (1.026 g, 0.003 mol) ) in 30 ml of ethanol-free chloroform was treated with 0.42 ml of dry triethylamine and the red solution stirred overnight. The solvent was removed in vacuo and the product partitioned between water and ether. Evaporation of the aqueous phase gave 0.889 g of the triethylamine salt of the title compound as a red oil. ;\<>>max (CHC;13) 2980, 1700, 1640, 1600 cm"<1>; T (CDCl3) , ;8.72 (9H, t, J 7.2Hz)7.75 (2H, quintet , J 6Hz); .26(8H, m); 1.92 (2H, broad exchangeable); A sample was converted to the sodium salt which had m.p. 218-220° (dec.). (Found: ;57.06% C; 4.30% H; 3.76% N; C-^gH-j^NNaO^ .i^O requires: 56.70% C; 4 , 23% H; 3 .67% N).. By the same procedure, the compounds in table V prepared. ; . Example 21;5-(3-phenoxypropoxy)indan-1,3-dione;A solution of dimethyl 4-(3-phenoxypropoxy)phthalate (13.6 g, 0.0395 mol) in 12 ml of ethyl acetate was added to a 50 % dispersion of sodium hydride in 2.54 g of mineral oil and the mixture heated at reflux for 4 hours. After cooling, the mixture was triturated with 50% ethanol, ether and the yellow solid filtered off and added to a warm (70-80°C) 10% solution of hydrochloric acid (150 ml). After. 2 minutes a yellow oily solid separated out, which was isolated by decantation and taken up in acetone from which it crystallized by concentration to give 2.533 g (22%) of material with m.p. 91-94°C , S max (minor) . 1740, 1710, 1605 cm<-1>; ;T(MSO) 7.78 (2H quintet; J 6.4Hz); 6.68 (2H, s); 5.83 (2H, t, J ;* 6.4Hz); 5.62 (2H, tr J 6.4Hz); 3.21-2.44 (7H, m); 2.12 (1H, m).

(Found: 72.97% C; 5.61% H; <C>18H16°4 requires: 72.96% C; 5.44%H).

Example 22

2-nitro-5-(3-phenoxypropoxy)-indan-1,3-dione

A suspension of 5-(3-phenoxypropoxy)-indan-1,3-dione (0.296 g, 0.001 mol) was suspended in 10 mL dry ether and cooled to 0°C. The stirred mixture was treated with 1 ml fuming nitric acid added dropwise over 15 minutes, and stirred in the ice bath for 1 hour. The mixture was then stirred at room temperature for an additional 1 1/2 hours. During this time, all the solid dissolved, leaving a deep yellow solution. 20 ml of dilute hydrochloric acid was added and the ether evaporated in vacuo. A yellow gum formed, and this solidified by scraping to give 0.25 g of a yellow solid with m.p. 77-79°c (cleaves), yield 73%.

(Found: 60.71% C; 4.58% H; 3.66% N; C1QH15N06.0.75H20 required: 60.92% C; .4.69% H; 3.95%N).

BIOLOGICAL DATA

SRS-A-antagonist activity

The compounds have been evaluated as direct antagonists of slow-reacting substance in anaphylaxis (SRS-A) by investigation using the isolated ileum from guinea pigs.

SRS-A rat was obtained from the peritoneal cavity 1 rat after passive peritoneal anaphylaxis by a method based on R.P. Orange, D. J. Stechschulte and K. F. Austen, j. Immunology, 105 1087 (1970) as described by B.A. Spicer, J.W. Ross and

H. Smith, Clin.exp.Immunol. 1975, 21_, 419. The sensitizing serum containing reaginic antibody was produced in rats as described by B.A. Spicer, et al. ibid. 2 ml of a 1 in 5 dilution of the sensitizing serum was injected ad peritoneally into recipient rats, and after 2 hours 5 ml of Thyroid solution containing 0.4 mg/ml ovalbumin (Sigma grade III) and 50 pg/ml heparin was injected ad the same way. 5 minutes after the injection, the rats were stunned and bled and the peritoneal fluids collected in polycarbonate tubes on ice. After centrifugation at 150Gi for 5 minutes, the supernatant was combined, heated in a boiling water bath for 5 minutes, cooled and stored at -20°C. The combined peritoneal fluids contained SRS-A and were used in the antagonism studies. The SRS-A studies were performed on isolated strips of -7 guinea pig ileum in Tyrode's solution containing atropine 5 x 10 m and mepyramine 10 µ m as described by W. E. Brocklehurst, J. Physiology, 151, 416 (1960).

The activity of the antagonists was determined by their ability to reduce submaximal. responses induced by SRS-Å. The antagonists were added to a 4 ml bath in a 0.1 ml volume in aqueous solution 1/2 minute before the addition of SRS-A and were present during induced contraction. Two or three concentrations of antagonists were applied and percent inhibition of the SRS-A response plotted against the bath concentration of the antagonist. The line of best fit was drawn and the concentration causing 50% inhibition ICcj0 read graphically.

Passive cutaneous anaphylaxis

Serum containing heat-labile homocytotropic antibody was cultured in rats to crystallized ovalbumin XOA by the method of Mota (I.Mota, Immunology, _7,681 (1964)) using Bordettela pertussis vaccine as an adjuvant.

Passive cutaneous anaphylaxis (PCA') was performed by a method

which is based on Ovary and Bier (A.Ovary and 0,G.Bier, Proe. Soc.

Exp. Biol. Med 81, 584, (1952)), modified by Goose and Blair..

Male Wistar rats weighing 250-300 g were given 0.1 ral

of each of six two-fold serial dilutions of pooled anti-

serum in 0.9% saline injected intradermally at various sites on their shaved backs. 72 hours later, the animals were given an intra-venous injection of 0.3 ml of a 1% solution of ovalbumin in an isotonic solution of saline buffered with 0.5 M Sørensen buffer (PBS) pH 7.2, mixed, with 0.2 ml of a 5% solution of Pontamine Sky Blue (6BX CI. 24410, Raymond A. Lamb, London) in isotonic saline. The rats were killed after 20 minutes and the diameter of the blue, swollen areas at the antisto.ff injection sites was measured at the outer surface of the skin. The starting dilution of the serum was adjusted so that there was no response after injection at the injection site for the highest dilution and maximal response at the lowest dilutions. Typically, six-fold serial dilutions of the serum from 1/4 to 1/128 were used.

Compounds were tested for ability to reduce the diameter of the raised areas at the intradermal sites which in control animals produced a less than maximal response. Each dose of the compound was administered intravenously to 6 rats in isotonic saline, adjusted to pH 7 with sodium bicarbonate if necessary (2 ml/kg body weight) just before intravenous injection with ovalbumin. Control groups of 6 animals were given the same volume of vehicle fluid at the same time.

The results were calculated as follows: % inhibition of PCA=100(1 - a/b) where a = the sum of the diameters of the raised sites produced in the test animal at the sites of antibody dilutions used in control groups, and b = mean -

the sum of the diameters of the swollen sites produced in the control group of animals at the antibody sites where at least 5 out of 6 of the animals

gave less than maximum response. A typical variation in the control group of animals was SEN-6%.

The dose of compound required to inhibit the PCA response by 50% was obtained from the log dose-response curve.

BIOLOGICAL RESULTS

Claims (12)

1. Process for the preparation of a compound of formula (T) and pharmaceutically acceptable salts thereof: where and R^ are the same or different and represent hydrogen, lower alkyl, lower alkenyl, lower alkynyl or lower alkoxy, or R^ and R^, when on adjacent carbon atoms, represent an alkylene group containing 3-5 carbon atoms or a 1 ,4-buta-1,3-dienylene group, and A represents a group of formula (II): where X is a bond or an oxygen atom and Q represents a linear alkylene group containing 1-9 carbon atoms, one methylene group within the group Q, which is not a methylene group covalently bound to an ether oxygen, is optionally substituted with hydroxyl, and R^ , R^ , R,., R^ and R_, each represent hydrogen, halogen, nitro , hydroxy , cyano , carboxyl , amin.o , lower alkyl, lower alkenyl, lower alkynyl, lower alkoxy, lower alkoxycarbonyl, lower acyl, lower acyloxy, mono- and di-lower alkylamino, mono- and di-lower acylamino, phenyl, lower alkyl-phenyl, phenoxycarbonyl, benzyloxycarbonyl, or any two of R^ to R^, when on adjacent carbon atoms, together represent an alkylene group containing 3-5 carbon atoms, or a 1,4-buta-1,3-dienylene group, characterized by reaction of a compound of formula (IV), (IVa) or a mixture thereof: wherein R 1 , R 2 and A are as defined with respect to formula (I); with a tertiary base, in the presence of an aprotic solvent, and then, if desired, conversion of the product thus formed, to a pharmaceutically acceptable salt. ir 2. Method as stated in claim 1, characterized in that pyridine, picoline or a tri-lower alkylamine is used as the tertiary base. 3. Method as stated in claim 2, characterized in that triethylamine is used as tertiary base. 4. Process for producing a compound of formula (I) as stated in claim 1, characterized by reacting a compound of formula (V): with a nitrating agent and then, if desired, salt formation of the thus prepared compound of formula (I). 5. Method as stated in claim 4, characterized in that fuming nitric acid in chloroform and/or ether is used as nitrating agent. 6..Process as stated in claim 4, character, i-, in that x is oxygen and Q represents an alkylene group containing up to 8 carbon atoms. 7. Method as stated in claim 4, characterized in that X is a covalent bond and Q is an alkylene group containing 2-8 carbon atoms'. 8. Process as stated in claim 6 or 7, characterized in that R, and R^ are hydrogen, lower alkyl or lower alkenyl; and R 1 , R 1 and R 1 are hydrogen or lower alkyl, lower alkoxy, lower alkylcarbonyl, lower alkylcarbonyloxy, lower alkoxycarbonyl, phenyl, halogen, nitro or hydroxyl; or any two of R 1 , R 4 or R 1 . on adjacent carbon atoms together represent a 1,3-buta-1,4-dienylene group or a C 1 -C 2 -alkylene group, and R 2 and R 2 both is hydrogen. 9. Method as set forth in any of claims 1 to 8, characterized in that R^ is a lower alkyl group at position 2' of the phenyl ring in substituent A, and R^ is hydrogen. 10. A reaction method as set forth in any of claims 1 to 8, characterized in that R^ is a hydroxyl group at position 3', R^ is a lower acyl group at -position 4', and R,, is a lower alkyl group in position 2' of the phenyl ring in substituent A. 11. Method as stated in any one of claims 1 to 10, characterized in that R 1 is hydrogen and R 2 is a lower alkyl group. 12. Method as stated in any one of claims 1 to 11, characterized in that X is oxygen and Q is 2-hydroxypropylene.