WO2005092908A1

WO2005092908A1 - Base addition salts of monosaccharides derivatives as inhibitors of airway hyper reactivity and airway inflammation

Info

Publication number: WO2005092908A1
Application number: PCT/IB2004/000800
Authority: WO
Inventors: Jag Mohan Khanna; Gyan Chand Yadav; Abhijit Ray; Naruganahalli Siddarangaih Krishna
Original assignee: Ranbaxy Laboratories Limited
Priority date: 2004-03-18
Filing date: 2004-03-18
Publication date: 2005-10-06

Abstract

The present invention relates to the use of base addition salts of Formula I for treating or preventing airway hyper reactivity and airway inflammation in a mammal. It in turn may also prove a potential benefit in the treatment of Chronic Obstructive Pulmonary Disease (COPD).

Description

BASE ADDITION SALTS OF MONOSACCHARIDES DERIVATIVES AS INHIBITORS OF AIRWAY HYPER REACTIVITY AND AIRWAY INFLAMMATION

Field of the Invention

The present invention relates to the use of base addition salts of 2,3-O-isopropylidene- l-O-substituted-5,6-dideoxy-5-N-[4-(2-hydroxy-2-oxoethyl)-phenylaminocarbonyl] amino-/3- L-gulofuranoside of Formula I

FORMULA I

for treating or preventing airway hyper reactivity and airway inflammation in a mammal. It in turn may also prove a potential benefit in the treatment of Chronic Obstructive Pulmonary Disease (COPD).

This invention also relates to the use of pharmaceutical composition of compound of Formula I for treating or preventing airway hyper reactivity and airway inflammation in a mammal, using the base addition salts of Formula I.

Further this invention also relates to a process for the synthesis of base addition salts of Formula I. Background of Invention Cell adhesion is a process by which cells associate with each other, migrate towards a specific target or localize within the extra-cellular matrix. These interactions are mediated by specialized molecules called cell adhesion molecules (CAM). CAMs have been demonstrated to participate in various cell-cell, cell-extracellular matrix, and platelet interactions. They influence the adhesion of leukocytes to the vascular endothelium, their transendothelial migration, retention at extravascular sites and activation of T cells and eosinophils. These processes are central to the pathogenesis of inflammatory and autoimmune diseases. Therefore, adhesion molecules are considered as potential targets to treat such disorders.

CAMs can be classified into three groups - integrins, selectins and the immunoglobulin superfamily. Out of these, integrins are key mediators in the adhesive interactions between hemopoietic cells and their microenvironment. They include of alpha- beta heterodimers and integrate signals from outside of the cells to inside and vice versa. Integrins can be classified on the basis of the alpha and beta subunits they contain. For example, beta-1 subfamily contains beta-1 subunit non-covalently linked to one of the 10 different alpha subunits.

The alpha-4 beta-1 integrin, also known as NLA4 (very late activation antigen 4), is a member of beta 1 integrin family and consists of alpha-4 and beta-1 subunits. NLA₄ interacts with two specific ligands - the vascular cell adhesion molecule (NCAM-1) and the the CSl region of fibronectin. Adhesion mediated by NLA4 is central to the process of transendothelial migration of leukocytes. Ligation of NLA4 is followed by gross rearrangement of the cytoskeleton leading to flattening of cells along the blood vessel wall followed by expression of specific molecules which digest the endothelial cell wall and diapedesis. Once in the exti'aluminal region, the interactions of NLA4 with extracellular fibronectin play a crucial role in migration to the site of inflammation, T cell proliferation, expression of cytokines and inflammatory mediators. In addition, NLA4 ligation provides costimulatory signal to the leukocytes resulting in enhanced immunoreactiviτy. Therefore, it is expected that NLA4 antagonists would ameliorate the immune response through twofold actions - inhibition of T cell recruitment at the site of inflammation and inhibition of costimulatory activation of immune cells.

In this respect, inhibitors of NLA4 interactions have demonstrated beneficial therapeutic effects in several animal models of inflammatory, and allergic diseases including sheep allergic asthma, experimental allergic encephomyelitis, contact hypersensitivity and inflammatory bowel.

Region of CS 1 moiety of fibronectin involved in the interaction with NLA4 was identified as the tripeptide Leu-Asp-Nal. also known as LDN. Taking a lead from this, several peptides containing the LDN sequence were synthesised which have shown to inhibit the in vivo interaction of NLA4 to its ligands.

Despite these advances, there remains a need for small and specific inhibitors of NLA4 dependent cell adhesion molecules. Ideally such inhibitors should be water soluble with oral efficacy. Such compounds would provide useful agents for treatment, prevention or suppression of various inflammatory pathologies mediated by NLA4 binding.

2,3-O-isopropylidene-l-O-substituted-5,6-dideoxy-5-Ν-[4-(2-hydroxy-2-oxoethyl) phenylaminocarbonyl]amino-(3, L-gulofuranoside are among compounds described in PCT application WO 00/42053, and US Patent No. 6,329,344. These compounds have potential as cell adhesion inhibitors and for the prevention of bronchial asthma, rheumatoid arthritis, multiple sclerosis, type I diabetes, psoriasis, allograft rejections and cell adhesion mediated pathologies including inflammatory and autoimmune disorders.

Summary of the Invention Herein is provided base addition salts of 2,3-O-isopropylidene-l-O-substituted-5,6- dideoxy-5-N-[4-(2-hydroxy-2-oxoethyl)ρhenyl aminocarbonyl] amino-β, L-gulofuranoside of Formula II (shown as Formula I) which are useful for treating or preventing airway hyper reactivity and airway inflammation in a mammal. The base addition salts of compounds of Formula I,

FORMULA I

have demonstrated protective effect in experimental models of airway hyper reactivity and airway inflammation. Standard assays have been performed on compounds of the present invention to ascertain their efficacy on airway reactivity, and airway inflammation. The efficacy of compounds of the present invention in the experimental models of airway reactivity and inflammation may indicate potential benefit in the treatment of Chronic Obstructive Pulmonary Disease (COPD).

Thus it has been found that the novel alkaline salts of compounds of Formula II (shown by the compounds of Formula I), have shown "Maximum Inhibitory Effect" superior to the corresponding free acid form, i.e., compound of Formula II,

FORMULA II

wherein in the Formula I, n is 1,2 or 4 and Aⁿ is Li , Na , K , Mg , Ca² or Ti . The salts of Formula II are easier to handle than the free acid form in the manufacture of pharmaceutical dosage form. A particular group of salts of Formula II are those wherein Aⁿ⁺ is Li ⁺, Na⁺, K⁺, Mg²⁺ , Ti ²⁺ and Ca²⁺. Further particular salts are Na⁺ and Mg²⁺, for example the Na⁺ salt. In the Formulae I and II, R is C₁ to ₅ alkyl, alkene, alkyne (straight chain or branched), aryl, substituted aryl or alkylaryl. The present invention also includes within its scope use of prodrugs of the compounds of Formula I for the inhibition and prevention of cell-adhesion mediated inflammatory and autoimmune disorders. In general, such prodrugs will be functional derivatives of these compounds which readily get converted in vivo into the defined compounds. Conventional procedures for the selection and preparation of suitable prodrugs are known. The invention also includes the use of enantiomers, diastereomers, N-oxides as well as metabolites for the inhibition and prevention of cell-adhesion mediated inflammatory and autoimmune disorders. The disclosed compounds of Formula I have five stereo chemical centers, the absolute configurations of which can be IS, 2S, 3S, 4R and 5S. In some particular embodiments, the stereo chemical configuration at the position 5, located outside the ring, can be 'S'.

The invention further includes the use of pharmaceutical compositions for the inhibition and prevention of cell-adhesion mediated inflammatory and autoimmune disorders comprising the compound of Formula I or prodrugs, metabolites, enantiomers, diastereomers, N-oxides, pharmaceutically acceptable solvents or polymorphs in combination with pharmaceutically acceptable carrier and optionally included excipient.

Another object of the present invention is to provide a process for the preparation of base addition salts of Formula I.

Other objects and advantages of the invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by the practice of the invention. The objects and the advantages of the invention may be realized and obtained by means of the mechanisms and combinations pointed out in the appended claims. Detailed Description of the Invention

The novel salts of Formula II, i.e., compound of Formula I

FORMULA I

are prepared by reacting compound of Formula II, as shown in Scheme I

SCHEME-I

FORMULA II

Base

FORMULA 1 with a base capable of releasing a cation Aⁿ⁺ wherein Aⁿ is as defined above to give a compound of the Formula I which is then isolated by methods known in the prior art.

The key starting compound of Formula II can be prepared, for example, by following processes described in PCT application No. WO 00/42053.

Examples of bases capable of releasing the cation Aⁿ⁺ and examples of reaction conditions are given below.

(a) Compounds of Formula I, wherein A is Li, Na, K are prepared by treating the free acid of Formula II with LiOH, NaOH or KOH in an aqueous or non-aqueous medium; wherein aqueous medium can be for example, aqueous ethanol, aqueous methanol, aqueous acetone or mixtures thereto and non-aqueous medium can be for example, ethylacetate-methanol, ethylacetate-ethanol, ethylacetate-isopropylalcohol, diethylether-methanol, diethylether-ethanol, diethylether-isopropylalcohol, or mixtures thereof, also particular mixtures of aqueos and non aqueos media can also be employed. Or

(b) by treating with LiORi NaOR₁ or KOR_t, wherein

is an alkyl group containing 1-4 carbon atoms, in a non aqueous medium, such as alcohol, for example, alcohols can be methanol, ethanol, propanol or isopropyl alcohol.

(c) Compounds of Formula I (wherein A is Mg, Ca or Ti) are prepared by treating compound of Formula II with Mg (ORι)₂, Ca (OR ₂ or Ti(ORι) wherein K_\ is an alkyl group containing 1-4 carbon atoms, in a non-aqueous solvent such as alcohol, for example, alcohols can be methanol, ethanol, propanol and isopropyl alcohol.

(d) A compound of Formula I may be converted to another salt of the same Formula by exchanging the cation. For example, Na⁺ as a counter ion may be exchanged for Ca²⁺ or Mg²⁺. Illustrative examples of the radical Ri are CH₃, C₂H_5j n-C₃H₇, n-C₄H₉, i-C₄H₉, sec- C₄H₉ and ter-C H . The examples mentioned below demonstrate general syntheses and specific preparations of particular compounds. In the above preparations, mixtures of aqueos and non aqueos media can be used. The examples are given to illustrate the details of invention and should not be constrained to limit the scope of the present invention.

Example 1; Preparation of 2,3-O-isopropylidene-l-O-dodecyl-5,6-dideoxy-5-N-{|^"4-(2- methoxy -2-oxoethv phenyllamino carbonyl amino}-3-L-gulofuranoside

Step 1 : 2,3-O-isopropylidene-l-O-dodecyl-6- eoxy-5-p-tosyl-3-L-gulofuranoside

2,3-O-isopropylidene-l-O-dodecyl-6-deoxy-|3-L-gulofuranoside (prepared according to the method reported in US Patent No, 5,360,794) (6.0 gm) was dissolved in pyridine (5 ml) and cooled to 0-5 °C. To this, was added p-toluenesulfonyl chloride (5.3 g) portionwise with stirring. After about 7 hours, solvent was removed under vacuum to get the residue which was extracted with ethyl acetate, washed with saturated sodium bicarbonate solution and brine. The ethyl acetate layer was dried over anhydrous sodium sulphate and the solvent was evaporated in vacuo to get an oily product which was purified by column chromatography using ethylacetate-hexane (5:95) as eluent to afford the title compound in 82% yield.

The compounds prepared similarly using the appropriate starting material were as follows in comparable yields:

2,3-O-isopropylidene-l-O-butyl-6-deoxy-5-p-tosyl- jS-L-gulofuranoside 2,3-O-isopropylidene-l-O-hexyl-6-deoxy-5-p-tosyl- -L-gulofuranoside 2,3-O-isopropylidene-l-O-heptyl-6-deoxy-5-p-tosyl-jS-L-gulofuranoside 2,3-O~isopropylidene-l-O-decyl-6-deoxy-5-p-tosyl-j8-L-gulofuranoside

Step 2 : 2,3-O-isopropylidene-l-O-dodecyl-5,6-dideoxy-5-azido-/3-L-gulofuranoside

A mixture of the compound obtained from step 1(9.0 g), sodium azide (9.0 g), and DMF (50 ml) was heated at 100 °C for about 48 hours. The solvent was removed under vacuum and the residue obtained was dissolved in ethylacetate. It was washed with water (2 x 50 ml) the organic layer was dried over anhydrous sodium sulphate and the solvent was removed under vacuum. The crude material was purified by column chromatography by eluting with a mixture of 2% ethyl acetate in hexane to get an oily product in 42% yield. The compounds prepared similarly from the products of Step 1 were as follows in comparable yields:

2,3-O-isopropylidene-l-O-butyl-5,6-dideoxy-5-azido-/3-L-gulofuranoside 2,3-O-isopropylidene-l-O-hexyl-5,6-dideoxy-5-azido-/3-L-gulofuranoside 2,3-O-isopropylidene-l-O-heptyl-5,6-dideoxy-5-azido-jS-L-gulofuranoside 2,3-O-isopropylidene-l-O-decyl-5,6-dideoxy-5-azido -/3-L-gulofuranoside

Step 3 : 2,3-O-isopropylidene-l-O-dodecyl-5,6-dideoxy-5-amino-(3-L-gulofuranoside 0 To a suspension of lithium aluminium hydride (2.0 gm) in dry THF (50 mL) at 0-5 C, was added a solution of the above compound obtained from step 2(3.0 gm in 10 mL of THF) dropwise. Once the addition was over, the reaction mixture was further stirred at room temperature for about 2 hours. The excess of LAH was decomposed by addition of ice-water mixture. The reaction mixture was then filtered through celite, washed with THF and the solvent was evaporated in vacuo. The residue was dissolved in ethyl acetate, washed with water and brine. The solvent was dried over anhydrous sodium sulphate and the solvent was removed under reduced pressure. The crude product was purified by column chromatography using ethylacetate-hexane (50:50) as an eluent to yield pure product in 61% yield and over 98% purity by HPLC.

The compounds prepared similarly from the products of Step 2 were as follows in comparable yields: 2,3-O-isopropylidene- 1 -O-butyl-5,6-dideoxy-5-amino-/3-L-gulofuranoside 2,3-O-isopropylidene-l-O-hexyl-5,6-dideoxy-5-amino-/3-L-gulofuranoside 2,3-O-isopropylidene-l-O-heptyl-5,6-dideoxy-5-amino-|S-L-gulofuranoside 2,3-O-isopropylidene-l-O-decyl-5,6-dideoxy-5-amino- -L-gulofuranoside

Step 4 : 2,3-O-isopropylidene-l-O-dodecyl-5,6-dideoxy-5-N-{[4-(2-methoxy-2-oxo ethyl)phenyl] amino carbonyl amino}~/?-L-gulofuraιιoside

To a cold (0-5°C) solution of the amine obtained from step 3 (0.5 g) in dry methylene chloride, was added the methyl ester of isocyanate-4-phenyl acetic acid (250 mg) dissolved in dry methylene chloride at 0-5°C and the reaction was stirred at same temperature for about 3 hours. Excess of methylene chloride was added to it, washed with water and brine. The methylene chloride layer was dried over anhydrous sodium sulphate and the solvent was removed under vacuum to get an oily crude product. This product was purified by column chromatography over silica (230-400 mesh) and eluted with ethyl acetate-hexane (20:80) mixture to get a pure white solid in 80% yield and over 98% purity by HPLC.

The compounds prepared similarly from the products of Step 3 were as follows in comparable yields:

2,3-O-isopropylidene-l-O-butyl-5,6-dideoxy-5-N-{[4-(2-methoxy-2- oxoethyl)phenyl]aminocarbonyl amino} - -L-gulofuranoside 2,3 -O-isopropylidene- 1 -O-hexyl-5 ,6-dideoxy-5-N- { [4-(2-methoxy-2- oxoethyl)phenyl]aminocarbonyl amino} -β-L-gulofuranoside 2,3-O-isopropylidene-l-O-heptyl-5,6-dideoxy-5-N-{[4-(2-methoxy-2- oxoethyl)phenyl] aminocarbonyl amino} - -L-gulofuranoside 2,3-O-isopropylidene-l-O-decyl-5,6-dideoxy-5-N-{[4-(2-methoxy-2- oxoethyl)phenyl] aminocarbonyl amino} -|3-L-gulofuranoside

Example 2; Preparation of 2,3,O-isopropyridene-l-O-dodecyl-5,6-dideoxy-5-N-{r4-(2- hvdroxy-2-oxoethyl phenvHamino carbonyl aminoj -ff-L-gulofuranoside

A mixture of ester (0.3 g) obtained in step 4 of Example 1 and aqueous sodium hydroxide (IN, 10 ml) was heated at 50°C for about two hours. The reaction mixture was cooled to 0-5°C, acidified to pH3 with 3N HCl, a white solid separated out was filtered, which became oil on being at room temperature. This anhydrous sodium sulphate and the solvent was removed in vacuum to get a crude oily product. The crude product was purified by flash column chromatography by eluting with ethyl acetate hexane mixture (35:65) to get a low melting solid in 75% yield.

Example 3: Preparation of sodium salt of dodecyl 2,3-O-isopropylidene-5,6-dideoxy5-N- 4- (2-hvdroxy-2-oxoethy phenylaminocarbonyl amino-i8-L-gulofuranoside. To a solution of dodecyl 2,3 ,-O-isopropylidene-5,6-dideoxy-5-N-[4-(2-hydroxy-2- oxoethyl)phenylaminocarbonyl]amino-j8-L-gulofuranoside (54.8 g, 0.1 mole) in ethylalcohol (274 ml) was added sodium ethoxide (6.8 g, 0.1 mole, 1 equivalent) at room temperature and stirred for 1 hour to get a clear solution. Ethyl alcohol was removed under reduced pressure on rotary evaporator at 40-45°C. A foaming solid was obtained which was dried at 40-45°C under high vacuum for 8-10 hours. The foaming hygroscopic solid thus obtained was powdered under controlled humidity as off white hygroscopic solid, yield 57g, sodium content = 3.8-4.2% w/w (Purity by HPLC=99.64%). The optical rotation of this compound ([o;]²⁵ o) is ⁺19 - ⁺22° (C = 2.0 in methanol).

¹HNMR (300 MHz), DMSO d₆) δ: 0.83-0.87 (3H, t), 1.10-1.12 (3H, d), 1.23-1.52 (21H, bs), 1.38 (3H, s), 1.46-1.48 (2H, m), 3.17 (2H, s), 3.29-3.35 (1H, s), 3.54-3.60 (1H, m), 3.76-3.80 (1H, t), 3.80-4.0 (1H, m), 4.49-4.51 (1H, d), 4.70-4.72 (1H, m), 4.89 (1H, s), 6.73-6.75 (1H, d), 7.02-7.05 (2H, d), 7.23-7.26 (2H, d), 8.90 (1H, s)

Mass: m/z = 571.2 (MH⁺), 549.3 (MH⁺ of acid) IR (DCM Film) Cm^-1: 3348, 2925, 2854, 1656, 1600.

Example 4; Preparation of potassium salt of dodecyl 2,3-O-isopropylidene-5,6-dideoxy-5-N- [4-(2-hvdroxy-2-oxoethyl)phenylaminoearbonyllamino-ig-L-gulofuranoside.

To a solution of dodecyl 2,3-O-isopropylidene-5,6-dideoxy-5-N-[4-(2-hydroxy-2- oxoethyl)phenylaminocarbonyl]amino-i3-L-gulofuranoside (16.44g, 0.03 moles) in methanol (85 ml) was added potassium hydroxide (1 mol. eq.) at room temperature and stirred for 1 hour to get a clear solution. Methanol was removed under reduced pressure at 40-45°C on rotary evaporator. The foaming solid thus obtained was dried at 40-45°C under high vacuum for 8-10 hours. It was then powdered under controlled humidity to afford off white hygroscopic powder (yield 17g, potassium content = 6.3-6.9% w/w).

¹HNMR (300 MHz), DMSO d₆) δ: 0.83-0.85 (3H, t), 1.08-1.11 (3H, d), 1.23-1.25 (21H, bs), 1.38 (3H, s), 1.46-1.47 (2H, m), 3.07 (2H, s), 3.33-3.62 (2H, m), 3.77-3.82 (1H, m), 3.9-4.0 (1H, m), 4.48-4.50 (1H, d), 4.69-4.72 (1H, m), 4.87 (1H, s), 6.99-7.01 (2H, d), 7.21-7.24 (3H,m), 9.30 (1H, s)

Mass: m z = 549.3 (MH⁺ of acid) IR (DCM film) cm^"1: 3361.8, 2925.1, 2854.5, 1657.3 Example 5; Preparation of calcium salt of dodecyl 2,3-O-isopropylidene-5,6-dideoxy-5-N-[4- ^-hydroxy^-oxoethy^phenylaminocarbonyllamino-g-L-gulofuranoside. To a solution of compound prepared in Example 3 (l.lg, 1.93 mmol) in water (11 ml) calcium chloride (0.214, 1.93 mmol, 1 mol. Eqv.) was added and stirred at room temperature for about lhour. Calcium salt was separated out as white solid. It was filtered, washed with water and dried under vacuum at 40-50°C (yield lg, calcium content = 3.52% w/w).

¹HNMR (300 MHz), DMSO d₆) δ: 0.83-0.87 (3H, t), 1.10-1.20 (3H, d), 1.21-1.25 (21H, bs), 1.37 (3H, s), 1.48 (2H, bs), 3.22 (2H, s), 3.34-3.59 (2H, m), 3.74-3.78 (IH, m), 3.94-4.02 (IH, m), 4.49-4.51 (IH, d), 4.69-4.72 (IH, m), 4.89 (IH, s), 6.52-6.55 (IH, d), 7.05-7.08 (2H, d), 7.24-7.27 (2H, d), 8.74 (IH, s)

Mass: m/z = 549.2 (MH+ of acid)

IR (film, DCM) = 3344.7, 2926.8, 2855, 1652.3

Example 6: Preparation of magnesium salt of dodecyl 2,3-O-isopropylidene-5,6-dideoxy-5-N- r4-(2-hvdroxy-2-oxoethyl phenylaminocarbonyl1amino-^-L-gulofuranoside. Magnesium turnings (0.328g, 0.014 mole, 0.5 mol. Eqv.) were added to 60 ml of methyl alcohol and it was heated at 50-60°C for about 1 hour. A hazy solution thus obtained was added to a solution of dodecyl 2,3-O-isopropylidene-5,6-dideoxy-5-N-[4-(2-hydroxy-2- oxoemyl)phenylaminocarbonyl]amino- ?-L-gulofuranoside in methanol (15g, 0.027 moles in 60 ml of methanol). The reaction mixture so obtained was stirred for about 2 hours at 50- 60°C. It was then filtered and methanol was removed from the filtrate on rotary evaporator at 50-55°C to afford off-white solid, which was dried under vacuum for 2 hours (Yield 15 g, Mg content = 2.04-2.26% w/w).

¹HNMR (DMSO d₆ , 300 MHz) δ: 0.83-0.87 (3H, t), 1.11-1.14 (3H, d), 1.23-1.25 (21H, bs), 1.37 (3H, s), 1.46-1.48 (2H, m), 3.17 (2H, s), 3.35-3.41 (2H, m), 3.54-3.58 (IH, m), 3.76-3.77 (IH, m), 3.94-4.0 (IH, m), 4.50-4.52 (IH, d), 4.70-4.72 (IH, m), 4.90 (IH, s) 6.15 (IH, bs), 7.05-7.08 (2H, d), 7.23-7.26 (2H, d), 8.40 (IH, s)

Mass: m/z = 549.2 (MH⁺ of acid)

IR (DCM, film): 3359.3, 2925.4, 2854.4, 1651.4 PHARMACOLOGICAL ACTIVITY

The compounds disclosed herein have demonstrated protective effects in experimental models of airway hyperreactivity and airway inflammation. Standard assays have been performed on compounds of the present invention to ascertain their efficacy on airway reactivity, and airway inflammation. These include :

1. Ovalbumin - induced airway reactivity and airway inflammation in guinea pig.

2. LPS - induced airway reactivity and airway inflammation in rat.

Enhanced airway reactivity and airway inflammation are a common feature of respiratory diseases like bronchial asthma and chronic obstructive pulmonary disease (COPD) (O'Byrne and Postma; Am. J. Respir. Crit. Care Med. 159, S41, 1999; Rutgers et al., Clin. Exp. Aller., 31_, 193, 2001). Guinea pigs sensitised and challenged with ovalbumin demonstrate intense airway reactivity to histamine with accompanying influx of eosinophil into the airways, a model representative of bronchial asthma (O'Byrne and Postma; Am. J. Respir. Crit. Care Med. 159, S41, 1999). By contrast, following LPS exposure rats develop airway reactivity with accompanying neutrophil influx into the airways, a condition simulating COPD (Wheeldon et al., Lab. Anim. 26, pg.29, 1992). Thus efficacy of compounds in above described models of airway reactivity and inflammation may indicate potential benefit in the treatment of asthma and COPD.

Guinea pig airway reactivity and inflammation

1. Sensitisation of guinea pigs

Guinea pigs were sensitised on days 0, 7 and 14 with 50 μg ovalbumin and 10 mg aluminium hydroxide injected intraperitoneally. On days 19 and 20 guinea pigs were exposed to 0.1% w/v ovalbumin or PBS for 10 min. On day 21 guinea pigs were exposed to 1% ovalbumin for 30 min. One group of guinea pigs were exposed intraperitoneal ovalbumin as described but were challenged with phosphate buffered saline (PBS) and served as control. Mepyramine 5 mg/kg, i.p., was administered to each guinea pig 30 min before ovalbumin/ PBS challenge.

2. Administration of compounds

Treatment with any of the following: TRIS salt of compound of Example 2; the compound of Example 3; montelukast; dexamethasone; or vehicle once daily, was started from day 18 onwards and continued for 5 days {i.e. on days 18, 19, 20, 21, 22). Ovalbumin / PBS challenges were performed 2 hours after different drug treatment.

3. Airway Reactivity Measurement : Histamine dose response

On day 22, 2 hours after drug treatment or vehicle administration, animals were transferred to a whole body plethysmograph (Buxco Electronics, USA) to study the airway reactivity as described in (Harnelmann et al., Am. J. Respir. Crit. Care Med., 156, 766, 1997 and Chong et al. ; J. Pharmacol. Toxicol. Methods, 39, 163, 1998). Animals were allowed to acclimatise in the body box and the basal PenH value (an index of airway resistance) was recorded using Biosystem XA software, (Buxco Electronics, USA). This was followed by successive challenges, each of 2 min duration, with PBS (vehicle for histamine) or histamine (0.1, 0.3, 0.6, 1, 1.5, 3, 6 and 10 mg/ml). Recording was stopped at once airway resistance increased 3 fold over PBS response.

4. Bronchoalveolar lavage (BAD After histamine dose response study, animals were sacrificed using thiopentone sodium (150 mg/kg/i.p.). Trachea was cannulated and BAL was performed using Hank's

Buffer salt solution (HBSS) (5 ml x 10 times). Lavage fractions were pooled and centrifuged at 30O0 rpm for 5 min, at 4°C. Pellet was collected and resuspended in 1-ml HBSS. Total leukocyte count was performed in the resuspended sample by using hemocytometer. A smear was prepared using the resuspended BAL fluid on a glass slide and was left to dry. Dried slides were kept on a staining tray and flooded with Leishmann's stain for 1 min. The stain was then diluted in the ratio of 1 :2 with buffer (pH 7) and left for 10 min. Slides were washed with tap water and left to dry and then differential leukocyte counts was done. 5. Data analysis: i. Airway Reactivity PC200 computation

PenH values in each guinea pig were obtained in the presence of PBS and different doses of histamine. PenH, at any chosen dose of histamine was, expressed as percent of PBS response. The PenH values thus calculated and the corresponding histamine dose were fed into Graph Pad Prism software (Graphpad Software Inc.,USA) and using a nonlinear regression analysis PC200 (dose of histamine increasing PenH 3 folds of PBS value) values were computed.

EDgn computation PC200 values obtained in the presence of different doses of test compounds were expressed as percent protection using the formula given below. ED₅₀ value was obtained by linear regression analysis of concentration and percent protection data.

PC200TEST - PC200 OVA % Protection = X 100 PC200 MAX - PC200 OVA

Where,

PC200_TEST ⁼ PC200 value obtained in the presence of a given dose of test compound PC200 _MA_X ⁼ Maximum PC200 value obtained in the presence of test compound

PC200 _OV_A ⁼ PC200 value obtained in the absence of test compound

ii. Airway Inflammation

Eosinophil count in bronchoalveolar lavage fluid was expressed as percent of total leukocyte count. Inhibition of eosinophil influx into airway was computed using the following formula: EOSQVA - EOSTEST % Inhibition = X 100 EOSQVA - EOSCON Where, EOS_OVA - Percentage of eosinophil in untreated ovalbumin challenged group EOS_TEST — Percentage of eosinophil in group treated with a given dose of test compound EOS_CON = Percentage of eosinophil in group not challenged with ovalbumin ED₅₀ dose for inhibition of eosinophil influx was calculated by linear regression analysis of concentration and percent protection data. Table 1 shows the effect of Tris salt of compound of Example 2, Example 3 , montelukast and dexamethasone on airway reactivity in guinea pig

(1) Effect observed at 10 mg/kg; (2) Effect observed at 0.1 mg/kg TRIS implies [tris (hydroxymethylaminomethane)]

Table 2 shows the effect of Compound of Example 3, TRIS salt of compound of Example 2, montelukast and dexamethasone on airway inflammation in guinea pig

(^l) Effect observed at 10 mg/kg; Compared to Montelukast, our compounds of Examples 2 and 3 exhibit better anti- inflammatory effect. The dose of Montelukast (0.1 g/ g) that exhibits comparable efficacy to our compounds on airway reactivity, inhibits airway inflammation marginally (35%). The reported anti-inflammatory effect of Montelukast happens at 10 mg/Kg dose that is 100 times more effective dose for protection of airway reactivity. Our compounds exhibit comparable airway reactivity and anti-inflammatory effect to Dexamethasone. Rat airway reactivity and inflammation

1. Administration of compounds

Animals were dosed orally every day of five days with vehicle, Compound of Example 3, TRIS salt of compound of Example 2, montelukast and dexamethasone.

2. Sensitisation of rats

On the day of experiment, animals were exposed to LPS (100 μg/ml) for 40 min (Toward and Broadley, Bri. J. Pharmacol., 131, 271, 2000). One group of vehicle treated rats were exposed to phosphate buffered saline for 40 min. Two hours after LPS/PBS exposure, animals were placed inside a whole body plethysmograph and exposed to acetylcholine (1, 3, 6, 12, 24 and 48 mg/ml) aerosol for 2 min. Between each challenge a gap of 7 min was allowed. In order to minimise movement artefacts during experiment, animals were sedated by intraperitoneal injection of diazepam (10 mg/kg, intraperitoneally) in 3% Tween® 80. 3. Bronchoalveolar lavage (BAL) Twenty four hours after acetylcholine dose response study, animals were sacrificed using thiopentone sodium (150 mg/kg/i.p.). Trachea was cannulated and BAL was performed using Hank's Buffer salt solution (HBSS) (5 ml x 10 times). Lavage fractions were pooled and centrifuged at 3000 rpm for 5 min, at 4°C. Pellet was collected and resuspended in 1-ml HBSS. Total leukocyte count was performed in the resuspended sample by using hemocytometer. A smear was prepared using the resuspended BAL fluid on a glass slide and was left to dry. Dried slides were kept on a staining tray and flooded with Leishmann's stain for 1 min. The stain was then diluted in the ratio of 1:2 with buffer (pH 7) and left for 10 min. Slides were washed with tap water and left to dry and then differential leukocyte counts were done. 4. Data analysis:

PenH values in each rat were obtained in the presence of PBS and different doses of acetylcholine. PenH, at any chosen dose of acetylcholine was, expressed as percent of PBS response. Total neutrophil were expressed as million cells/ml of BAL. Protective effect on airway reactivity and inflammation were calculated using the following formula : Respons^β ps - ResponseχEsτ % Inhibition = X 100 Response_Lps- ResponsecoN Where,

Response ps = PenH (% Initial) or airway inflammation (Total neutrophil count) in LPS challenged rats in the absence of drug treatment Responsej_EST - PenH (% Initial) or airway inflammation (Total neutrophil count) in LPS challenged rats treated with a given dose of test compound Responseco_N ⁼ PenH (% Initial) or airway inflammation (Total neutrophil count) in PBS challenged rats Table 3 shows the maximum inhibitory effects of TRIS salt of compound of Example 2, Example 3 and dexamethasone on airway reactivity and airway inflammation in rat

(1) Effect observed at 0.1 mg/kg dose; (2) Effect observed at 10 mg/kg dose;. The compounds of our invention were tested in the experimental animal models and it exhibited better anti-inflammatory effect (% inhibition of inflammatory cell influx) in both guinea pig and rat. While the present invention has been described in terms of its specific embodiments, certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the present invention.

Claims

We Claim: 1. A method for treating or preventing airway hyper reactivity and airway inflammation in a mammal comprising administering to the said mammal a therapeutically effective amount of a base addition salt of 2,3-O-isopropylidene-l-O-substituted-5,6-dideoxy- 5-N-[4-(2-hydroxy-2-oxoethyl)-phenylaminocarbonyl]amino-/3-L-gulofuranoside of Formula I,

FORMULA I its pharmaceutically acceptable solvates, enantiomers, diasteromers, N-oxides, polymorphs, prodrugs or metabolites wherein R is C₁ to C₁₅ alkyl, alkene, alkyne (straight chain or branched), aryl, substitued aryl or akylaryl; n is selected from integers 1, 2 and 4;

Aⁿ⁺ is selected from a group comprising of Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺ and Ti⁴⁺'

2. A method for treating or preventing chronic obstructive pulmonary disease (COPD) in a mammal comprising administering to the said mammal a therapeutically effective amount of a base addition salt of 2,

3-O-isopropylidene-l-O-substituted-5,6-dideoxy- 5-N-[4-(2-hydroxy-2-oxoethyl)-phenylaminocarbonyl]amino- -L-gulofuranoside of Formula I ,

FORMULA I its pharmaceutically acceptable solvates, enantiomers, diasteromers, N-oxides, polymorphs, prodrugs or metabolites wherein

R is C₁ to C₁₅ alkyl, alkene, alkyne (straight chain or branched), aryl, substitued aryl or akylaryl; n is selected from integers 1, 2 and 4;

Aⁿ⁺ is selected from a group comprising of Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺ and Ti⁴⁺' 3. The method according to any one of the claims 1 and 2 wherein Aⁿ⁺ is Na⁺.

4. The method according to any one of the claims 1 and 2 wherein Aⁿ⁺ is K⁺.

5. The method according to any one of the claims 1 and 2 wherein Aⁿ⁺ is Mg²⁺.

6. The method according to any one of the claims 1 and 2 wherein Aⁿ⁺ is Ca²⁺.

7. The method according to any one of the claims 1 and 2 wherein the base addition salt is a sodium salt of 2,3-O-isopropylidene-l-O-substituted-5,6-dideoxy-5-N-[4-(2- hydroxy-2-oxoethyl)-phenylaminocarbonyl]amino-j8-L-gulofuranoside.

8. The method according to any one of the claims 1 and 2 wherein the base addition salt is a potassium salt of 2,3-O-isopropylidene-l-O-substituted-5,6-dideoxy-5-N-[4- (2-hydroxy-2-oxoethyl)-phenylaminocarbonyl]amino-j8-L-gulofuranoside.

9. The method according to any one of the claims 1 and 2 wherein the base addition salt is a calcium salt of 2,3-O-isopropylidene-l-O-substituted-5,6-dideoxy-5-N-[4-(2- hydroxy-2-oxoethyl)-phenylaminocarbonyl]amino-ι(3-L-gulofuranoside.

10. The method according to any one of the claims 1 and 2 wherein the base addition salt is a magnesium salt of 2,3-O-isopropylidene-l-O-substituted-5,6-dideoxy-5-N- [4-(2-hydroxy-2-oxoethyl)-phenylaminocarbonyl]amino-j8-L-gulofuranoside.

11. A method for treating or preventing airway hyper reactivity and airway inflammation in a mammal comprising administering to the said mammal a therapeutically effective amount of a pharmaceutical compoisition comprising a base addition salt of 2,3-0- isopropylidene-l-O-substituted-5,6-dideoxy-5-N-[4-(2-hydroxy-2-oxoethyl)- phenylaminocarbonyl]amino-|3-L-gulofuranoside of Formula I,

FORMULA I its pharmaceutically acceptable solvates, enantiomers, diasteromers, N-oxides, polymorphs, prodrugs or metabolites wherein R is Ci to Cι₅ alkyl, alkene, alkyne (straight chain or branched), aryl, substitued aryl or akylaryl; n is selected from integers 1, 2 and 4; Aⁿ⁺ is selected from a group comprising of Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺ and Ti⁴⁺ and a pharmaceutically acceptable carrie.

12. A method for treating or preventing chronic obstructive pulmonary disease (COPD) in a mammal comprising administering to the said mammal a therapeutically effective amount of a pharmaceutical composition comprising a base addition salt of 2,3-0- isopropylidene-l-O-substituted-5,6-dideoxy-5-N-[4-(2-hydroxy-2-oxoethyl)- phenylaminocarbonyl]amino-j8-L-gulofuranoside of Formula I ,

FORMULA I its pharmaceutically acceptable solvates, enantiomers, diasteromers, N-oxides, polymorphs, prodrugs or metabolites wherein R is Ci to Ci₅ alkyl, alkene, alkyne (straight chain or branched), aryl, substitued aryl or akylaryl; n is selected from integers 1, 2 and 4;

Aⁿ is selected from a group comprising of Li , Na , K , Mg2+ , Ca ~2+ and T ιi-4+ and a pharmaceutically acceptable carrier.

13. The method according to any one of the claims 11 and 12 wherein Aⁿ⁺ is Na⁺.

14. The method according to any one of the claims 11 and 12 wherein .Aⁿ⁺ is K .

15. The method according to any one of the claims 11 and 12 wherein A , nⁿ+^τ is Mg²⁺.

16. The method according to any one of the claims 11 and 12 wherein Aⁿ⁺ is Ca²⁺.

17. The method according to any one of the claims 11 and 12 wherein the base addition salt is a sodium salt of 2,3-O-isopropylidene-l-O-substituted-5,6-dideoxy-5-N-[4-(2- hydroxy-2-oxoethyl)-phenylaminocarbonyl]amino-j8-L-gulofuranoside.

18. The method according to any one of the claims 11 and 12 wherein the base addition salt is a potassium salt of 2,3-O-isopropylidene-l-O-substituted-5,6-dideoxy-5-N-[4- (2-hydroxy-2-oxoethyl)-phenylaminocarbonyl]amino-^-L-gulofuranoside.

19. The method according to any one of the claims 11 and 12 wherein the base addition salt is a calcium salt of 2,3-0-isopropylidene-l-0-substituted-5,6-dideoxy-5-N-[4-(2- hydroxy-2-oxoethyl)-phenylaminocarbonyl]amino-(8-L-gulofuranoside.

20. The method according to any one of the claims 11 and 12 wherein the base addition salt is a magnesium salt of 2,3 -O-isopropylidene- l-O-substituted-5,6-dideoxy-5-N- [4-(2-hydroxy-2-oxoethyl)-phenylaminocarbonyl]amino-_]8-L-gulofuranoside.

21. A process for preparing a base addition salt of 2,3 -O-isopropylidene- 1 -O- substituted-5,6-dideoxy-5-N-[4-(2-hydroxy-2-oxoethyl)-phenylaminocarbonyl] amino-jS-L- gulofuranoside of Formula I

FORMULA I and its pharmaceutically acceptable solvates, enantiomers, diasteromers, N-oxides, polymorphs, prodrugs or metabolites wherein R is Ci to Cis alkyl, alkene, alkyne (straight chain or branched), aryl, substitued aryl or akylaryl; n is selected from integers 1, 2 and 4; Aⁿ⁺ is selected from a group comprising Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺ and Ti⁴⁺ ; which comprises reacting a compound of Formula II

FORMULA II with a base capable of releasing a cation Aⁿ⁺.

22. The process of claim 21 wherein Aⁿ⁺ is Na .

23. The process of claim 21 wherein Aⁿ⁺ is K⁺.

24. The process of claim 21 wherein Aⁿ⁺ Mg²⁺. The process of claim 21 wherein Aⁿ⁺ is Ca⁺.

25. The process of claim 21 wherein a compound of Formula I is prepared by treating the free acid of Formula II with LiOH, NaOH, or KOH in an aqueous or non-aqueous medium.

26. The process of claim 26 wherein the aqueous medium is selected from the group comprising of aqueous methanol, aqueous ethanol and aqueous acetone.

27. The process of claim 26 wherein the non-aqueous medium is selected from the group comprising of ethylacetate-methanol, ethylacetate-ethanol, ethylacetate-isopropyl alcohol, diethylether-methanol, diethylether-ethanol, and diethylether-isopropyl alcohol.

28. The process of claim 21 wherein a compound of Formula I is prepared by treating the free acid of Formula II with LiORi , NaORi or KORi wherein Ri is an allcyl group containing 1-4 carbon atoms, in a non-aqueous medium.

29. The process according to claim 29 wherein the non-aqueous medium is alcohol.

30. The process of claim 30 wherein the alcohol is selected from the group comprising of methanol, ethanol, propanol and isopropyl alcohol.

31. The process claim 21 wherein a compound of Formula I (when A = Mg, Ca or Ti) is prepared by reacting a compound of Formula II with Mg (ORι)₂ , Ca (ORι)₂ or Ti (ORι)₄ wherein Ri is an alkyl group containing 1-4 carbon atoms, in a non-aqueous solvent.

32. The process according to claim 32 wherein the the non-aqueous solvent is an alcohol.

33. The process of claim 33 wherein the alcohol is selected from the group comprising of methanol, ethanol, propanol and isopropyl alcohol.

34. The process of claim 21 wherein a compound of Formula I may be converted to another salt of the same Formula by exchanging the cation