1,3-Dihydroimidazole-2-Thione Derivatives for Use in the Treatment of Pulmonary Arterial Hypertension and Lung Injury

Abstract

The present invention relates to compounds of formula I:

##STR00001##

for use in treating pulmonary arterial hypertension and associated conditions, where R.sub.1, R.sub.2 and R.sub.3 are the same or different and signify hydrogens, halogens, alkyl, alkylaryl, alkyloxy, hydroxy, nitro, amino, alkylcarbonylamino, alkylamino or dialkylamino group; R.sub.4 signifies hydrogen, alkyl, -alkylaryl or -alkylheteroaryl; X signifies CH.sub.2, oxygen atom or sulphur atom; n is 1, 2 or 3, with the proviso that when n is 1, X is not CH.sub.2; and the individual (R)- and (S)-enantiomers or mixtures of enantiomers and pharmaceutically acceptable salts thereof.

Claims

1. A pharmaceutical composition comprising a compound of formula I ##STR00009## wherein, R.sub.1, R.sub.2 and R.sub.3 are the same or different and signify hydrogen, halogen, alkyl, alkylaryl, alkyloxy, hydroxy, nitro, amino, alkylcarbonylamino, alkylamino or dialkylamino group; R.sub.4 signifies hydrogen, alkyl, -alkylaryl or -alkylheteroaryl; X signifies CH.sub.2, oxygen atom or sulphur atom; n is 1, 2 or 3, with the proviso that when n is 1, X is not CH.sub.2; and the individual (R)- and (S)-enantiomers or mixtures of enantiomers and pharmaceutically acceptable salts thereof, in combination with at least one prostacyclin analogue.

2. The pharmaceutical composition according to claim 1, wherein the compound of formula I has the formula IA ##STR00010## wherein, R.sub.1, R.sub.2 and R.sub.3 are the same or different and signify hydrogen, halogen, alkyl, alkylaryl, alkyloxy, hydroxy, nitro, amino, alkylcarbonylamino, alkylamino or dialkylamino group; R.sub.4 signifies hydrogen, alkyl or -alkylaryl group; X signifies CH.sub.2, oxygen atom or sulphur atom; and n is 1, 2 or 3, with the proviso that when n is 1, X is not CH.sub.2.

3. The pharmaceutical composition according to claim 1, wherein the compound of formula I has the formula IB ##STR00011## wherein R.sub.1, R.sub.2 and R.sub.3 are the same or different and signify hydrogen, halogen, alkyl, nitro, amino, alkylcarbonylamino, alkylamino or dialkylamino group; R.sub.4 signifies -alkyl-aryl or -alkyl-heteroaryl; X signifies CH.sub.2, oxygen atom or sulphur atom; and n is 2 or 3.

4. The pharmaceutical composition according to claim 1, wherein the compound of formula I is selected from the group consisting of: (S)-5-(2-aminoethyl)-1-(1,2,3,4-tetrahydronaphthalen-2-yl)-1,3-dihydroimidazole-2-thione; (S)-5-(2-aminoethyl)-1-(5,7-difluoro-1,2,3,4-tetrahydronaphthalen-2-yl)-1,3-dihydroimidazole-2-thione; (R)-5-(2-aminoethyl)-1-chroman-3-yl-1,3-dihydroimidazole-2-thione; (R)-5-(2-aminoethyl)-1-(6-hydroxychroman-3-yl)-1,3-dihydroimidazole-2-thione; (R)-5-(2-aminoethyl)-1-(8-hydroxychroman-3-yl)-1,3-dihydroimidazole-2-thione; (R)-5-(2-aminoethyl)-1-(6-methoxychroman-3-yl)-1,3-dihydroimidazole-2-thione; (R)-5-(2-aminoethyl)-1-(8-methoxychroman-3-yl)-1,3-dihydroimidazole-2-thione; (R)-5-(2-aminoethyl)-1-(6-fluorochroman-3-yl)-1,3-dihydroimidazole-2-thione; (R)-5-(2-aminoethyl)-1-(8-fluorochroman-3-yl)-1,3-dihydroimidazole-2-thione; (R)-5-(2-aminoethyl)-1-(6,7-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione; (R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione; (S)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione; (R)-5-(2-aminoethyl)-1-(6,7,8-trifluorochroman-3-yl)-1,3-dihydroimidazole-2-thione; (R)-5-(2-aminoethyl)-1-(6-chloro-8-methoxychroman-3-yl)-1,3-dihydroimidazole-2-thione; (R)-5-(2-aminoethyl)-1-(6-methoxy-8-chlorochroman-3-yl)-1,3-dihydroimidazole-2-thione; (R)-5-(2-aminoethyl)-1-(6-nitrochroman-3-yl)-1,3-dihydroimidazole-2-thione; (R)-5-(2-aminoethyl)-1-(8-nitrochroman-3-yl)-1,3-dihydroimidazole-2-thione; (R)-5-(2-aminoethyl)-1-[6-(acetylamino)chroman-3-yl]-1,3-dihydroimidazole-2-thione; (R)-5-aminomethyl-1-chroman-3-yl-1,3-dihydroimidazole-2-thione; (R)-5-aminomethyl-1-(6-hydroxychroman-3-yl)-1,3-dihydroimidazole-2-thione; (R)-5-(2-aminoethyl)-1-(6-hydroxy-7-benzylchroman-3-yl)-1,3-dihydroimidazole-2-thione; (R)-5-aminomethyl-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione; (R)-5-(3-aminopropyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione; (S)-5-(3-aminopropyl)-1-(5,7-difluoro-1,2,3,4-tetrahydronaphthalen-2-yl)-1,3-dihydroimidazole-2-thione; (R,S)-5-(2-aminoethyl)-1-(6-hydroxythiochroman-3-yl)-1,3-dihydroimidazole-2-thione; (R,S)-5-(2-aminoethyl)-1-(6-methoxythiochroman-3-yl)-1,3-dihydroimidazole-2-thione; (R)-5-(2-benzylaminoethyl)-1-(6-methoxychroman-3-yl)-1,3-dihydroimidazole7-2-thione; (R)-5-(2-benzylaminoethyl)-1-(6-hydroxychroman-3-yl)-1,3-dihydroimidazole-2-thione; (R)-1-(6-hydroxychroman-3-yl)-5-(2-methylaminoethyl)-1,3-dihydroimidazole-2-thione; (R)-1-(6,8-difluorochroman-3-yl)-5-(2-methylaminoethyl)-1,3-dihydroimidazole-2-thione; (R)-1-chroman-3-yl-5-(2-methylaminoethyl)-1,3-dihydroimidazole-2-thione; and (R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thione.

5. The pharmaceutical composition according to claim 1, wherein the compound of formula I is (R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione hydrochloride.

6. The pharmaceutical composition according to claim 1, wherein the compound of formula I is (R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thione.

7. The pharmaceutical composition according to claim 6, wherein the dose of (R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thione is from 10 mg/day to 50 mg/day.

8. The pharmaceutical composition according to claim 1, wherein the prostacyclin analogue is selected from the group consisting of epoprostenol, iloprost, treprostinil and beraprost.

9. The pharmaceutical composition according to claim 1, wherein the prostacyclin analogue is epoprostenol.

10. The pharmaceutical composition according to claim 1, wherein the prostacyclin analogue is iloprost.

11. The pharmaceutical composition according to claim 1, wherein the prostacyclin analogue is treprostinil.

12. The pharmaceutical composition according to claim 1, wherein the prostacyclin analogue is beraprost.

13. The pharmaceutical composition according to claim 1, wherein the composition is in the form of a single daily dosage.

14. The pharmaceutical composition according to claim 1, further comprising one or more inert pharmaceutically acceptable carriers.

15. The pharmaceutical composition according to claim 1 in unit dosage form.

16. The pharmaceutical composition according to claim 1, wherein the compound of formula I is (R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thione and the prostacyclin analogue is selected from the group consisting of epoprostenol, iloprost, treprostinil and beraprost.

17. A method of treating pulmonary arterial hypertension comprising administering a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, in combination with at least one prostacyclin analogue, to a subject in need thereof.

18. The method according to claim 17, wherein the compound of formula I is (R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione hydrochloride.

19. The method according to claim 17, wherein (R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thione.

20. The method according to claim 17, wherein the prostacyclin analogue is selected from the group consisting of epoprostenol, iloprost, treprostinil and beraprost.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0123] Reference is now made to the accompanying Figures, in which:

[0124] FIG. 1represents a schematic of the study described below.

[0125] FIG. 2Kaplan-Meyer survival curves from the day of MCT administration from an initial n=20 of rats in each group of animals.

[0126] FIG. 3Kaplan-Meyer survival curves from the day of MCT administration from an initial n=20 of rats in each group of animals, except the MCT+Bos300 (n=10).

[0127] FIG. 4Ratios of heart weight over bodyweight (BW) (FIG. 4a) and Fulton index (RV/LF+S) (FIG. 4b) from MCT (.circle-solid.), MCT+Bos300 (.square-solid.), MCT+Bos100 (.box-tangle-solidup.), MCT+Compound B (.Math.), MCT+Compound B+Bos100 (.diamond-solid.), SHAM () and SHAM+Compound B () treated rats at 28 days after MCT or vehicle (Sham) administration. Data represents meanssem of n=4-14. Significantly different from MCT (*P<0.05) or from Sham and Sham+Compound B (# P<0.05).

[0128] FIG. 5Ratios of right ventricle (RV) (FIG. 5a) and left ventricle plus septum (LV/S) weight over bodyweight (BW) (FIG. 5b) from MCT (.circle-solid.), MCT+Bos300 (.square-solid.), MCT+Bos100 (.box-tangle-solidup.), MCT+Compound B (.Math.), MCT+Compound B+Bos100 (.diamond-solid.), SHAM () and SHAM+Compound B () treated rats at 28 days after MCT or vehicle (Sham) administration. Data represents meanssem of n=4-14. Significantly different from MCT (*P<0.05) or from Sham and Sham+Compound B (# P<0.05).

MATERIALS AND METHODS

In Vivo Studies

[0129] One of the most-used animal models is the Monocrotaline (MCT) lung injury model of Pulmonary Arterial Hypertension. Administration of MCT, that is metabolized in the liver to the reactive metabolite dehydromonocrotaline, results in a syndrome characterized by acute lung injury, interstitial pulmonary fibrosis, necrotizing pulmonary arteritis, pulmonary hypertension, right ventricular (RV) hypertrophy, myocarditis and hepatic venoocclusive disease.

Experimental Procedure

The Monocrotaline (MCT) Lung Injury Model of Pulmonary Arterial Hypertension Set Up

[0130] The protocol to use is a standard one (Curr. Protoc. Pharmacol. 46:5.56.1-5.56.11(2009) John Wiley & Sons Inc). Rats, of 6-7 weeks old (150-200 g) are administered MCT 60 mg/kg subcutaneously, and the pathology usually develops in the course of initial 3 to 4 weeks with the animals dying within the next 2 weeks (5.sup.th-6.sup.th week). A decrease of 50% in food and water consumption is thought to be indicative of pathological status severe enough for testing.

Experimental Design

[0131] Two studies were conducted separately and a combination analysis performed on the two studies. Each study consisted in the scheme represented in FIG. 1.

[0132] Compound B refers to (R)-5-(2-benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thione (i.e. compound (R)-B as shown above). The structure is as follows:

##STR00008##

[0133] The treatment groups for each study were as follows.

TABLE-US-00001 Group Treatment n G1 MCT + Vehicle 10 G2 MCT + Bosentan 300 mg/kg 10 G3 MCT + Bosentan 100 mg/kg 10 G4 MCT + Compound B 30 mg/kg 10 G5 MCT + Compound B 30 mg/kg + 10 Bosentan 100 mg/kg G6 Vehicle SHAM 6 G7 Compound B 30 mg/kg SHAM 6

[0134] Compound B was obtained from BialPortela & Ca., S.A. Monocrotaline was obtained from Sequoia Research Products Limited. Bosentan (CAS number 157212-55-0) was obtained from APICHEM.

[0135] Abbreviations:

[0136] a. Bos=Bosentan

[0137] b. Bos100=Bosentan 100 mg/kg

[0138] c. Bos300=Bosentan 300 mg/kg

[0139] d. LV+S=Left ventricle+septum

[0140] e. MCT=Monocrotaline

[0141] f. RV=Right ventricle

[0142] The experimental design of both studies was the same, with animals randomized in seven groups, five groups of 10 animals each (MCT, MCT+Bos300, MCT+Bos100, MCT+Compound B, MCT+Compound B+Bos100) and 2 groups of 6 animals each (SHAM and SHAM+Compound B).

[0143] The experimental details are described in more detail below; briefly, compounds were given to rats mixed with food starting, Compound B (30 mg/kg/day) for groups MCT+Compound B and SHAM+Compound B, bosentan (300 mg/kg/day) for group MCT+Bos300, bosentan (100 mg/kg/day) for group MCT+Bos100, Compound B+bosentan (30+100 mg/kg/day) for group MCT+Compound B+Bos100, and no compound for groups MCT and SHAM. Animals from groups MCT were administered MCT and animals from groups SHAM were administered vehicle. Twenty eight days later, surviving animals were sacrificed, tissues collected and weighed. During the entire experimental period food, water consumption and animal weight were monitored. The ratios organ weight to body weight (BW) were calculated to evaluate organ hypertrophy. The index of RV/LV+S was calculated as an index of RV hypertrophy. In each study an n=10 animals was used for each MCT treatment group, and an n=6 was used for the groups not given MCT. The animals from MCT+Bos300 group from Study 1 were not considered for the combined survival analysis since these unexpectedly presented a higher mortality effect as compared to the MCT group. The surviving animals from this group were nevertheless considered for the cardiac remodelling analysis.

[0144] Administration volumes: 3 ml/kg for subcutaneous administration.

[0145] Storage conditions: The substances will be stored according to conditions defined in the respective certificate of analysis.

Data Acquisition and Analysis

[0146] Raw data acquisition was performed with Gilson UniPoint System Software version 5.11. All data analysis was performed using Prism 5 for Windows software, version 5.02 (GraphPad Software Inc., San Diego, Calif.).

Statistical Analysis

[0147] All data is expressed as meansem (Standard Error of the Mean) unless otherwise indicated. Differences between treatment groups were analysed by one-way ANOVA and post-hoc Newman-Keuls multiple comparison test. P<0.05 was considered statistically significant.

[0148] Survival curves were derived by the product limit method of Kaplan and Meier, and compared using the logrank (Mantel Cox) test.

Study 1Further Details

[0149] Animals were given powdered food for an adaptation period of 3-5 days before treatment start. Compounds were given to rats mixed with food (powdered diet) starting on day 2. Forty-eight hours later, on day zero animals from groups GI through G5 were administered MCT subcutaneously (60 mg/kg) while remaining groups (G6 and G7) were given vehicle instead of MCT. Treatment was prolonged for 28 days. Food, water consumption and animal weight were monitored over the entire duration of the experiment.

[0150] On day 28 surviving animals were sacrificed and the following tissues/organs collected and weighed: liver, kidney, lung, heart and spleen. Femurs were removed and measured. Heart was dissected into right ventricle, left ventricle+septum and these were also weighed. The ratio organ to body weight (BW) was calculated as well as the ratio organ weight to femur length. The index of RV/LV+S is an index of RV hypertrophy. Catecholamines were evaluated only on right ventricle and left ventricle+septum.

[0151] Six weeks old male Wistar rats (151-187 g) were obtained from Harlan (Spain). Rats were kept 5 per cage, under controlled environmental conditions (12 hr light/dark cycle and room temperature 221 C.).

[0152] All animal groups were fed powdered food, MCT (G1, n=10) and SHAM (G6, n=6) were given normal food and all other groups were given food mixed with the compounds: MCT-Bos300 (G2, n=10) received food with 300 mg/kg/day bosentan, MCT-Bos100 (G3, n=10) received food with 100 mg/kg/day bosentan, MCT-1058 (G4, n=10) received food with 30 mg/kg/day BIA 5-1058, MCT-1058-Bos100 (G5, n=10) received food with 100 mg/kg/day bosentan plus 30 mg/kg/day BIA 5-1058 and SHAM-1058 (G7, n=6) received food with 30 mg/kg/day BIA 5-1058.

[0153] Forty eight hours later animals groups 1 through 5 were administered MCT (60 mg/kg) and groups 6 and 7 were administered vehicle subcutaneously in a volume of 3 ml/kg.

[0154] MCT was prepared by dissolving at 300 mg/kg in 1M HCl, neutralizing with 1 M NaOH and diluting to 20 mg/ml with sterile water.

[0155] Water and food consumption for each cage and weights of the individual animals were measured twice a week.

[0156] Animals were kept under treatment for 28 days after MCT administration. The animals that survived were, at that time, anaesthetized with pentobarbital 60 mg/kg administered intraperitoneally, and subjected to tissue and organ collection.

[0157] Whole spleen, whole liver, left kidney and lungs were removed, cleaned and weighed. Heart was removed, cleaned from atria and vascular tissue and weighed. Right ventricle was then dissected from the rest of the heart (left ventricle+septum) and both were weighed and put in tubes containing 0.2 M PCA. Femurs were removed, cleaned and length measured with a calliper.

[0158] The ratios organ weight to body weight and organ weight to femur length were calculated as well as the ratio right ventricular weight to left ventricular+septum weight.

[0159] Tissues were left in 0.2 M PCA (perchloric acid) for 24 h in the dark at 4 C. and were then frozen at 80 C. Catecholamines were measured in right ventricle and left ventricle+septum.

Catecholamines Quantification

[0160] Reagents and Materials: All reagents were obtained from Sigma-Aldrich. SPE columns Sep-Pak Vac Alumina A cartridge 1 cc/100 mg 50-300 im 100/box were obtained from Waters.

[0161] Tissues: Frozen tissues in 0.2 M PCA were thawed, the liquid phase removed, and filtered through 0.22 m Spin-X filters (Corning Costar) by centrifugation in microfuge for 10 minutes at 5000 rpm, 4 C. Noradrenaline and dopamine were quantified in filtrates by high-pressure liquid chromatography with electrochemical detection (HPLC-ED).

[0162] Test system: Gilson HPLC-ED 142

[0163] Test Method: Chromatographic conditions were: [0164] Flow rate: 1 ml/min [0165] Analytical Column: Spheri-5 RP18 5 m, 4.6250 mm, Perkin Elmer serial #28918, lot#07H8-1318806 [0166] Temperature: Ambient [0167] Injection volume: 50 l [0168] Mobile phase: 0.15 mM EDTA, 0.1M Sodium acetate, 0.1M Citric acid monohydrate, 1 mM Octyl sulphate, 1.0 mM Dibutylamine, 10% Methanol, pH 3.5 with PCA [0169] Detector set at: Mode Amperometric [0170] 0.75 V potential [0171] 2 nA sensitivity

Study 2Further Details

[0172] Animals were given powdered food for an adaptation period of 3-5 days before treatment start. Compounds were given to rats mixed with food (powdered diet) starting on day 2. Forty-eight hours later, on day zero animals from groups GI through G5 were administered MCT subcutaneously (60 mg/kg) while remaining groups (G6 and G7) were given vehicle instead of MCT. Animal weight, food and water consumption were evaluated twice a week and animals were regularly observed for any disease signs. Mortality was registered and on day 28 surviving animals were sacrificed, selected organs weighed and femurs removed and measured.

[0173] Organs weighed were: liver, kidney, lung, heart and spleen. Heart was dissected into right ventricle, left ventricle+septum and these were also weighed. The ratio organ to body weight (BW) was calculated as well as the ratio organ weight to femur length. The index of RV/LV+S is an index of RV hypertrophy.

[0174] Rats were housed in groups of 5 in macrolon cages on wood litter with free access to powder chow diet (Code 113-SAFE, 89290 Augy, France) and tap water. The animal house was maintained in a 12-hour light/dark cycle (0700 to 1900 hours) in a controlled ambient temperature of 221 C.

[0175] Treatment with compound in food started on day 2: for groups G4 and G7 BIA 5-1058 (30 mg/kg/day), for group G2 bosentan (300 mg/kg/day), for group 3 bosentan (100 mg/kg/day), for group 5 BIA 5-1058+bosentan (30+100 mg/kg/day). The remaining animals (groups GI and G6) were given normal rat chow food. Forty-eight hours later, on day zero, animals from groups GI through G5 were administered MCT subcutaneously (60 mg/kg/3 ml). Groups G6 and G7 were given vehicle instead of MCT. Treatment was prolonged for 4 weeks.

[0176] MCT was dissolved at 300 mg/kg in 1 N HCl, neutralized with 1 N NaOH and diluted to 20 mg/ml with sterile water.

[0177] Food, water consumption and animal weight was monitored over the all experiment. Water and food consumption was measured for each cage twice a week. The weights of the individual animals were determined twice a week.

[0178] Animals were kept under treatment for 28 days after MCT administration. The animals that survived were, at that time, anaesthetized with pentobarbital 60 mg/kg administered intraperitoneally, and subjected to tissue and organ collection.

[0179] Whole spleen, whole liver, both kidneys and lungs were removed, cleaned and weighed. Heart was removed, cleaned from atria and vascular tissue and weighed. Right ventricle was then dissected from the rest of the heart (left ventricle+septum) and both were weighed. Femurs were removed, cleaned and length measured with a calliper.

[0180] The ratios organ weight to body weight and organ weight to femur length were calculated as well as the ratio right ventricular weight to left ventricular+septum weight.

Irwin Test

[0181] The Irwin test (Irwin S. Psycopharmacologia 1968:13; 222-57) is a systematic observational procedure for assessing and scoring the behavioral and physiological state of rodents. Animals are observed according to a standardised observation battery in order to detect neurobehavioural, neurovegetative or psychotropic signs or neurotoxic effects. A total of 30 parameters are scored using a standardized procedure based on that described by Irwin, 1968. These parameters are distributed as followed: 16 items for the behavioral profile, 9 items for the neurological profile and 5 items for the autonomic profile as represented in Table 1 below:

TABLE-US-00002 TABLE 1 Behavioural Spontaneous activity locomotor activity spatial locomotion stereotyped behaviour writhing Motor-affective responses transfer arousal (appearance) touch response Provoked biting vocalisation easy of handling/passivity Sensoro-motor visual placing reflex responses pain response (tail pinch) pain response (toe pinch) startle response Posture straub tail abnormal limb position abnormal body carriage (posture) Neurologic Muscle tone body tone abdominal tonus limb muscle tonus grip strength Equillibrium and gait righting reflex abnormal gait catalepsy CNS excitability tremors/twitches/jerks convulsions Autonomic Eyes ptosis Secretions lacrimation salivation General hypothermia piloerection

Results

[0182] Survival curves (see FIGS. 2 and 3) clearly show that the combination of Compound B (30 mg/kg/day) plus bosentan (100 mg/kg/day) significantly (P<0.05) increased the number of animals that survived the 28 days observing period when compared to the MCT group. A slight increase in survival was also observed for the MCT+Compound B group translated by a shift to the right in the survival curve. No clear benefit was observed with either bosentan treatments, 300 or 100 mg/kg/day as compared to the MCT group.

[0183] Thus, the use of Compound B alone resulted in greater survival rates compared to the use of bosentan alone. Further, the combined use of Compound B and bosentan resulted in a synergistic effect on survival rates.

[0184] No animals died on the SHAM groups.

[0185] As shown in FIGS. 4a, 4b, 5a and 5b, Compound B alone or in combination with bosentan significantly decreased the heart, RV and Fulton Index (where the Fulton Index=the ratio of right ventricle weight to left ventricle plus septum weight, i.e. RV/LV+S) as compared to the MCT group; the magnitude of the decrease was higher with the combination Compound B+Bos100 than with the other treatments. Bosentan 300 mg/kg/day significantly decreased the RV and Fulton Index as compared to the MCT group.

[0186] All groups administered with MCT had significantly increased heart, RV, LV+S and Fulton Index as compared to SHAM and SHAM+Compound B groups.

[0187] Regarding cardiac remodeling a marked and statistically significant decrease in right ventricular hypertrophy was observed in the MCT+Compound B+Bos100 group as compared to the MCT group, similarly to what observed in the MCT+Bos300 group. A significant decreased right ventricular hypertrophy was also observed in the MCT+Bos100 and MCT+Compound B groups.

[0188] Thus, Compound B, whether used alone or in combination with bosentan, exhibits beneficial effects in relation to lung injury, particularly pulmonary arterial hypertension.

SYNTHETIC EXAMPLES

Example 1

(R)-5-aminomethyl-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione Hydrochloride

[0189] A stirred mixture of (R)-6,8-difluorochroman-3-ylamine hydrochloride (0.22 g, 1.0 mmol), [3-(tert-butyldimethylsilanyloxy)-2-oxopropyl]carbamic acid tert-butyl ester (0.33 g, 1.1 mmol), potassium thiocyanate (0.11 g, 1.1 mmol) and acetic acid (0.3 mL, 5.0 mmol) in ethyl acetate (3 mL) was refluxed for 2 hours, cooled to room temperature, then washed by sodium bicarbonate solution, dried over anhydrous magnesium sulphate and evaporated in vacuo. The residue was purified by the column chromatography over silica gel using ethyl acetate-petroleum ether mixture as eluent. The resulting oil (0.23 g) was dissolved in ethyl acetate (2 ml), whereupon 2M HCl solution in ethyl acetate was added (2 mL, 4 mmol) and the mixture was stirred for 2 hours at room temperature. The precipitate was removed by filtration and washed with ethyl acetate to give crystals of m.p. 192 C. (decomp.).

Example 2

(R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thione

[0190] To (R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione (2.36 g, 7.58 mmol) and benzaldehyde (0.85 ml, 8.34 mmol) in a mixture of methanol (15 ml), and dichloromethane (15 ml) sodium cyanoborohydride (0.67 g, 10.66 mmol) was added at 20-25 C. in portions. The mixture was stirred for 64 h, quenched with IN HCl (12 ml) with stirring followed by 3N NaOH (12 ml). The mixture was extracted with DCM (100 ml), the organic phase was washed with brine (50 ml), dried (MgSO.sub.4) and evaporated to dryness. The residue was purified on a silica gel column with ethyl acetate and a mixture of ethyl acetate with methanol (9:1) as eluents. Fractions containing the product were collected, evaporated under reduced pressure to approx 20 ml then cooled on ice. The precipitate was collected, washed with ethyl acetate-petroleum ether (1:1) mixture, dried on air. Yield was 1.25 g (41%), the product having a mp 188-90 C. (2-propanol-DCM).

[0191] It will be appreciated that the invention may be modified within the scope of the appended claims.