This invention relates to the substantially pure 5R enantiomers of the known glitazone drug class, such as the known pharmaceutical products pioglitazone and rosiglitazone, in which the hydrogen at position 5 of the thiazolidinone ring which characterises the glitazones is replaced by deuterium and to the use of these compounds for pulmonary administration by inhalation, for treatment of inflammatory respiratory diseases.

Background to the invention

A broad spectrum of respiratory diseases and disorders has been recognized, many of which have overlapping and interacting etiologies. Two of the most widespread and prevalent of these diseases are chronic obstructive pulmonary disorder (COPD) and asthma. Respiratory diseases have a significant inflammatory component. For example, current therapy for COPD and severe asthma focuses mainly on the reduction of symptoms using short and long acting bronchodilators either as monotherapies or combinations of long acting β ₂ agonist bronchodilators with inhaled corticosteroids (ICS). The disappointing anti-inflammatory data for ICS either alone or in combination with β ₂ agonists has intensified the search for an effective antiinflammatory drug for COPD. COPD is clearly a chronic inflammatory disorder that involves complex interactions between cells of the innate and acquired immune response both in the lung and potentially also systemically. One hypothesis under intense investigation is whether novel, demonstrably anti-inflammatory agents can halt or slow function decline characteristic of COPD. Reducing the frequency and severity of exacerbations has become an increasingly important target for COPD therapy as the prognosis for patients following exacerbations is poor. Antiinflammatory therapy in COPD, and in asthma, is expected to reduce the frequency and severity of exacerbations. It is also desirable that decline in lung function and quality of life are also improved with treatment.

Hence, new treatments for inflammatory respiratory diseases, including asthma, chronic obstructive pulmonary disease (COPD), allergic airway syndrome, bronchitis, cystic fibrosis and emphysema, are constantly sought.

Peroxisome Proliferation Receptor gamma receptor (PPARγ) agonists are a class of drug which increase sensitivity to glucose in diabetic patients. Physiological activation of PPARγ is believed to increase the sensitivity of peripheral tissues to insulin, thus facilitating the clearance of glucose from the blood and producing the desired anti-diabetic effect.

Many PPARγ agonists are known from the patent and other literature, but currently only two are approved for clinical use in diabetes; Rosiglϊtazone and Pioglitazone. See Campbell IW, Curr MoI Med. 2005 May; 5(3):349-63. Both of these compounds are thiazolidinediones ("TZDs" or "glitazones"), and are in practice administered by the oral route for systemic delivery.

In addition to its effect on glucose metabolism, a variety of reports have been published which demonstrate that rosiglitazone also exerts anti-inflammatory effects. For instance, (i) rosiglitazone has been reported to exert effects in diabetic patients consistent with an anti-inflammatory effect (Haffner et al, Circulation. 2002 Aug 6; 106(6):679-84, Marx et al, Arterioscler Thromb Vase Biol. 2003 Feb 1 ;23(2):283-8); (ii) Rosiglitazone has been reported to exert anti-inflammatory effects in a range of animal models of inflammation, including: carageenan-induced paw oedema (Cuzzocrea et al, Eur J Pharmacol. 2004 Jan 1 ;483(1 ):79-93), TNBS-induced colitis (Desreumanux et al, J Exp Med. 2001 Apr 2;193(7):827-38, Sanchez-Hidalgo et al, Biochem Pharmacol. 2005 Jun 15;69(12): 1733-44), experimental encephalomyelitis (Feinstein et al, Ann Neurol. 2002 Jun;51(6):694-702) collagen-induced (Cuzzocrea et al, Arthritis Rheum. 2003 Dec;48(12):3544-56) and adjuvant-induced arthritis (Shiojiri et al, Eur J Pharmacol. 2002 Ju1 19;448(2-3):231-8), carageenan-induced pleurisy (Cuzzocrea et al, Eur J Pharmacol. 2004 Jan 1 ;483(1 ):79-93), ovalbumin- induced lung inflammation (Lee et al, FASEB J. 2005 Jun; 19(8): 1033-5) and LPS- induced lung tissue neutrophilia (Birrell et al, Eur Respir J. 2004 Jul;24(1): 18-23) and (iii) rosiglitazone has been reported to exert anti-inflammatory effects in isolated cells, including ϊNOS expression in murine macrophages (Reddy et al, Am J Physiol Lung Cell MoI Physiol. 2004 Mar;286(3):L613-9), TNFα-induced MMP-9 activity in human bronchial epithelial cells (Hetzel et al, Thorax. 2003 Sep;58(9):778-83), human airway smooth muscle cell proliferation (Ward et al, Br J Pharmacol. 2004 Feb;141(3):517-25) and MMP-9 release by neutrophils (WO 0062766). PPARγ agonists have also been shown to be effective in models of pulmonary fibrosis (Milam et al., Am. J. Physiol. Lung Cell. MoI. Physiol, 2008, 294(5):L891-901 ) and pulmonary arterial hypertension (Crossno et al., Am. J. Physiol. Lung Cell. MoI. Physiol, 2007, 292(4):L885-897). Based on observations of anti-inflammatory activity in cells relevant to the lung, the utility of other PPARγ agonists has been suggested for the treatment of inflammatory respiratory disorders including asthma, COPD, cystic fibrosis and pulmonary fibrosis. See WO0053601 , WO0213812 and WO0062766. These suggestions include administration by both the systemic oral and pulmonary inhalation routes.

Unfortunately, PPARγ agonists also have unwanted cardiovascular effects, including haemodilution, peripheral and pulmonary oedema and congestive heart failure

(CHF). These effects are also believed to result from activation of PPARγ. In particular, a significant effort has been devoted to investigating the hypothesis that

PPARγ agonists disturb the normal maintenance of fluid balance via binding to the

PPARγ receptor in the kidney. See Guan et al, Nat Med. 2005;11 (8):861=6 and Zhang et al, Proc Natl Acad Sci USA. 2005 28;102(26):9406-11. Treatment with

PPARγ agonists by the oral route for systemic delivery is also associated with an unwanted increase in body weight.

COPD patients are known to be at a higher risk than other clinical populations from congestive heart failure (CHF) (Curkendali et al, Ann Epidemiol, 2006; 16: 63-70,

Padeletti M et al, lnt J Cardiol. 2008;125(2):209-15) and so it is important that systemic activation of the PPARγ receptors is kept to a minimum in these patients to avoid increasing the likelihood of CHF being observed. Administering respiratory drugs by the inhaled route is one approach to target the lung with an anti- inflammatory agent whilst keeping systemic exposure of the drug low reducing the likelihood of systemic activity and observation of side effects.

Pioglitazone has structural formula (I)

and can be named as 5-{4-[2-(5-ethylpyridin-2-yl)ethoxy]benzyl}-1 ,3-thiazolidine-2,4- dione. The carbon atom in the 5-position of the thiazolidine-dione ring of pioglitazone, indicated by an arrow in formula (I) above, is asymmetric, so pioglitazone has two enantiomers, the 5R and 5S enantiomers.

Rosiglitazone has the structural formula (H) and can be named as 5-(4-{2-[methyI (pyridin-2-yI)amino]ethoxy]benzyl}-1 ,3-thiazolidine-2,4-dione. The carbon atom in the 5-position of the thiazolidine=dione ring of rosigϋtazone, indicated by an arrow in formula (II) below, is also asymmetric, so rosigϋtazone also has two enantiomers, the 5R and 5S enantiomers.

The 5S enantiomer of rosiglitazone has a higher binding affinity for the PPARγ receptor than the 5R enantiomer (30nm vs 2μM, Parks et al, 1998, Bioorg Med Chem Lett. 8(24):3657-8). For another member of the glitazone class, Rivogiitazone the 5S enantiomer also has higher receptor binding affinity than the 5R enantiomer (see page 13 of WO2007100027).

In practice, pioglitazone and rosiglitazone are administered for treatment of diabetes as a mixture of 5R and 5S enantiomers (a 1 :1 racemic mixture) by the oral route for systemic delivery. The individual enantiomers of these compounds, and the glitazone family generally, are known to equilibrate rapidly in vivo after oral administration ( see for example J. CHn. Pharmacol. 2007, 47, 323-33; Rapid Commun. Mass Spectrum. 2005, 19, 1125-9; J. Chromatography, 835 (2006), 40-46; Biopharmaceutics and Drug Disposition 1997, 18 (4), 305-24; Chem. Pharm. Bull 1984, 32, (11 ) 4460-65; T. J. Med. Chem. 1991 , 34, 319-25) so there is no difference in practice between oral administration of either substantially pure isomer and oral administration of the racemic mixture. Specifically in relation to pioglitazone, it has been stated in a submission to the Federal Drug Administration (FDA) that there was no difference in activity following oral administration of the racemate and the individual enantiomers in a rodent diabetes model (wwwida.gov/medwatch/SAFETY/2007/Sep Pl/Actoplus MeLELM): "(Pioglitazone) contains one asymmetric carbon, and the compound is synthesized and used as the racemic mixture. The two enantiomers of pioglitazone interconvert in vivo. No differences were found in the pharmacologic activity between the two enantiomers".

The effects of pulmonary inhalation of rosiglitazone or piogϋtazone (or indeed any other glitazone) in either racemic or single enantiomer form do not appear to have been studied, ft appears nothing has been published concerning the potential equilibration of the 5R and 5S enantiomers of either compound, or any other glitazone, when contacted directly with lung tissue.

The glitazone class of PPARγ agonists as a whole is characterised by the presence in the molecule of a thiazolidin-2,4-dione radical (A), often as part of a (thiazolidin- 2,4,dione-5-yI)methylphenyl radical (B):

and the ring carbon atom indicated by the arrow is numbered as the 5-position of the thiazolidinone ring. The term "glitazone" as used herein refers to a PPARγ agonist compound whose structure includes a thiazolidin-2,4-dione radical (A), or a (thiazolidin-2,4,dione ^» 5-yl)methylphenyl radical (B):

Besides the approved and marketed rosiglitazone and pioglitazone, there is a multitude of glitazones known from the patent and scientific literature. Known examples include the following:

Ciglitazone Englitazone Darglitazone

Edaglitazone MK-0767 Rivoglitazone

Trogiitazone Netoglitazone Baiaglitazone

Lobeglitazone Inoiitazone

In any organic biologically active compounds, one or more protons can be in principle be replaced by the hydrogen isotope deuterium. Many studies have shown that for a given biologically active compound, deuteration usually changes one or more pharmacodynamic properties of the compound, for example absorption, distribution, metabolism and execretion (ADME) properties. For example amphetamines are more readily transported into the brain in the deuterated form (Wenzel, 1989); halogenated anaesthetics, such as selvoflurane, when deuterated are no longer oxidized to toxic forms within the body (Baker et a/., 1993), and the deuterated forms of certain antimicrobial compounds [long-chain fatty acids (Abrahamson et a/., 1982) and D- fluorophenylalanine (Merck and Co., 1977)] are not oxidized by the affected microorganism arid hence are toxic for longer times.

Clark et al. J. Med. Chem. 1991 , 34, 319-325 made the racemic 5-deuterated glitazone of formula (III)

and reported that loss of deuterium occurred within minutes under physiological conditions. However, it appears that the effects on ADME of substitution of deuterium for the 5-proton of the 5R glitazone enantiomers has not been studied.

Brief summary of the invention

This invention is based on the finding that in an animal model of treatment of inflammatory respiratory disease by inhalation, the 5R-enantiomer of a glitazone, for example pioglitazone and rosiglitazone, in which the hydrogen at position 5 of the thiazolidinone ring is replaced by deuterium is active, whereas the corresponding 5S enantiomer is essentially inactive. This finding leads to the conclusion that inhaled pulmonary administration of the 5-deuterated, 5R giitazone enantiomer allows the anti-inflammatory effect of the giitazone to be achieved more efficiently than by similar administration of the racemate, with all the concomitant reduced side effect benefits of lower systemic exposure than oral administration.

The invention also includes a particularly efficient method for the resolution of racemic 5-deuterated pioglitazone-chϊral resolving agent salts.

Brief description of the figures

Figure 1 is a bar graph (mean ±SD) that illustrates the effect of intranasal administration to laboratory mice with vehicle (0.2% tween 80 in saline), 5S-5- deuterated pioglitazone (5S-D-Pio) (1 or 3 μg/kg) or 5R-5-deuterated pioglitazone (5R-D-Pio) (1 or 3 μg/kg) on the number of BAL cells induced by tobacco smoke for 4 days examined 24 hours post the finaf exposure.

Figure 2 is a bar graph (mean ±SD) that illustrates the effect of intranasal administration to laboratory mice with vehicle (0.2% tween 80 in saline), 5S-5- deuterated pioglitazone (5S-D-Pio) (1 or 3 μg/kg) or 5R-5-deuterated pioglitazone (5S-D-Pio) (1 or 3 μg/kg) on the number of BAL neutrophils induced by tobacco smoke for 4 days examined 24 hours post the final exposure.

Figure 3 is a bar graph (mean ±SD) that illustrates the effect of intranasal administration to laboratory mice with vehicle (0.2% tween 80 in saline), 5S-5- deuterated rosiglitazone (5S-D-Rosi) (3 or 10 μg/kg), 5R-5-deuterated rosiglitazone (5R-D-Rosi) (3 or 10 μg/kg) or racemic 5-deuterated rosiglitazone (D-Rosi) (10 μg/kg) on the number of BAL cells induced by tobacco smoke for 4 days examined 24 hours post the final exposure.

Figure 4 is a bar graph (mean ±SD) that illustrates the effect of intranasal administration to laboratory mice with vehicle (0.2% tween 80 in saline), 5S-5- deuterated rosiglitazone (5S-D-Pio) (3 or 10 μg/kg) or 5R-5-deuterated rosigiitazone (SR-D-Pio) (3 or 10 μg/kg) on the number of BAL neutrophils induced by tobacco smoke for 4 days examined 24 hours post the final exposure.

Figure 5 is a line graph that illustrates the amount of 5R and 5S Pioglϊtazone detected in the blood of rats following i.t. administration of 5R protonated Pioglitazone (30 μg/kg) up to 8 hrs post administration (protonated = H).

Figure 6 is a line graph that illustrates the amount of 5R and 5S 5-deuterated Pioglitazone detected in the blood of rats following i.t. administration of 5R-5- deuterated Pioglitazone (30 μg/kg) up to 8 hrs post administration (deuterated = D).

Figure 7 is a line graph that illustrates the amount of 5S protonated Pioglitazone and 5S 5-deuterated Pioglitazone detected in the blood of rats following i.t. administration of 5R protonated Pioglitazone or 5R 5-deuterated Pioglitazone (30 μg/kg) respectively up to 8 hrs post administration (protonated = H, deuterated = D).

Detailed description of the invention

As used herein, the term "glitazone" has the meaning ascribed to it above, i.e., a PPARγ agonist compound whose structure includes a thiazolidin-2,4-dione radical (A), or a (thiazolidin-2,4,dione-5-yi)methylphenyl radical (B):

As used herein, the terms "pioglitazone", "pioglitazone component", "5H-pioglitazone" and "5-protonated pioglitazone" mean the compound of formula (I) above.

As used herein, the terms "5R-pioglitazone", "5R-5H-pioglitazone" and "5R-5- protonated pioglitazone" mean the 5R enantiomer of the compound of formula (1) above.

As used herein, the terms "5S-pioglitazone", "5S-5H-pioglitazone" and "5S-5- protonated pioglitazone" mean the 5S enantiomer of the compound of formula (I) above.

As used herein the terms "5-deuterated pioglitazone" and 5- ² H pioglitazone" mean 5- {4-[2-(5-ethylpyridin-2-yl)ethoxy]-benzyl}-(5- ² H)-1 ,3-thiazolidϊnβ-2,4-dione of formula (IV) below.

As used herein, the terms "5R-5-deuterated pϊoglitazone", "5R-S- ² H pioglitazone" mean the 5R-enantiorner of 5-deuterated pioglitazone.

As used herein, the terms "5S-5-deuterated pioglitazone" and "5S-S- ² H pioglitazone" mean the 5S-enantiorner of 5-deuterated pioglitazone.

As used herein, the terms "rosiglitazone", "rosiglitazone component", "5H- rosiglitazone" and "5-protonated rosiglitazone" mean the compound of formula (II) above.

As used herein, the terms "5R-rosiglitazone", "5R-5H-rosiglitazone" and "5R-5- protonated rosiglitazone" mean the 5R enantiomer of the compound of formula (H) above.

As used herein, the terms "5S-rosigiitazone", "5S-5H-rosiglitazone" and "5S-5- protonated rosiglitazone" mean the 5S enantiomer of the compound of formula (II) above.

As used herein the terms "5-deuterated rosiglitazone" and 5- ² H rosigiitazone" mean 5-{4-[2-(5-ethylpyridin-2-yl)ethoxy]-benzyi}-(5- ² H)-1 ,3-thiazolidine-2,4-dione of formula (V) below.

As used herein, the terms "5R-5-deuterated rosiglitazone" and "5R-S- ² H rosiglitazone" mean the 5R-enantiomer of 5-deuterated rosiglitazone.

As used herein, the terms "5S-5-deuterated rosiglitazone" and "5S-S- ² H rosiglitazone" mean the 5S-enantiomer of 5-deuterated rosiglitazone. As used herein, the symbols D and ² H are interchangeable and refer to the deuterium isotope of hydrogen.

As used herein, the term "enantiomeric excess" or its abbreviation "e.e." is defined as the percentage:

((R-S)/(R+S)) x 100 percent

where R and S are the respective weight fractions of the R and S enantiomers in a sample. Thus for a glitazone sample containing 95% by weight of the 5R enantiomer and 5% of the 5S enantiomer, the enantiomeric excess of R over S enantiomer is ((95-5)/95+5)) x 100 = 90%.

In one aspect, the present invention provides a composition of matter comprising a 5R-5-deuterated glitazone or a salt thereof, in which composition the 5R-5 deuterated glitazone component includes less than 5% by weight in total of 5R-5-protonated glitazone, 5S-5-protonated glitazone and 5S=5-deuterated glitazone. Preferably, the glitazone component includes less than 3%, more preferably less than 1%, by weight in total of 5R-5-protonated glitazone, 5S-5-protonated glitazone and 5S-5-deuterated glitazone.

In one preferred aspect, the composition of matter of the invention is a pharmaceutical composition adapted for pulmonary administration by inhalation. The pharmaceutical composition may additionally comprise a bronchodiiator drug and/or another anti-inflammatory drug.

The invention also provides the use of a composition of matter comprising a 5R-5- deuterated glitazone or a salt thereof, in which composition the 5R-5 deuterated glitazone component includes less than 5% by weight in total of 5R-5-protonated glitazone, 5S-5-protonated glitazone and 5S-5-deuterated glitazone, for the treatment of inflammatory respiratory disease by pulmonary administration by inhalation, and the use of such composition of matter in the preparation of a medicament for the treatment of inflammatory respiratory disease by pulmonary administration by inhalation. The invention also provides a method of treatment of inflammatory respiratory disease comprising pulmonary administration by inhalation to a subject of an effective amount of a composition of matter comprising a 5R-5-deuterated glitazone or a salt thereof, in which composition the glitazone component includes less than 5% by weight in total of 5R-5-protonated glitazone, 5S-5-protonated glitazone and 5S-5-deuterated glitazone.

The method of the invention may also include the step wherein the subject is additionally administered an amount of a bronchodilator drug and/or another anti- inflammatory drug wherein the amount of 5R-5-deuterated giitazone, or salt thereof and the amount of the conventional respiratory treatment agent together comprises a therapeutically effective amount.

The invention also includes a kit for treatment of respiratory disorders in a subject, the kit comprising one dosage form comprising 5R-5-deuterated glitazone or a salt thereof, in which composition the glitazone component includes less than 5% by weight in tota! of 5R-5-protonated glitazone, 5S-5-protonated glitazone and 5S-5- deuterated glitazone.

In all aspects of the invention, the 5R-5-deuterated glitazone component may be inhaled via the nose or the mouth. Preferably it is inhaled via the mouth.

In all aspects of the invention, the 5R-5-deuterated glitazone component may, in particular, be 5R-5-deuterated pioglitazone or 5R-5-deuterated rosiglitazone.

As stated, the 5R-5-deuterated glitazone component can be in the form of a pharmaceutically acceptable salt. The term "pharmaceutically acceptable salt" refers to salts prepared from pharmaceutically acceptable inorganic and organic acids and bases.

Pharmaceutically acceptable inorganic bases include metallic ions. More preferred metallic ions include, but are not limited to, appropriate alkali metal salts, alkaline earth metal salts and other physiological acceptable metal ions. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc, and the like and in their usual valences. Exemplary salts include aluminum, calcium, lithium, magnesium, potassium, sodium and zinc. Particularly preferred are the ammonium, calcium, magnesium, potassium, and sodium salts.

Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, including in part, trimethylamine, dϊethylamine, N, N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamϊne, ethylenediarnine, meglumine (N-methylglucamϊne) and procaine; substituted amines including naturally occurring substituted amines; cyclic amines; quaternary ammonium cations; and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,N-dibenzyIethyIenediamine, diethylamine, 2- diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N- ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropySamine, lysine, mβthylglucamϊne, rnorpholinβ, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like.

Illustrative pharmaceutically acceptable acid addition salts of the compounds of the present invention can be prepared from the following acids, including, without limitation formic, acetic, propionic, benzoic, succinic, glycolic, gluconic, lactic, maleic, maiic, tartaric, (-)-dibenzoyl-L-tartaric, citric, nitric, ascorbic, glucuronic, fumaric, pyruvic, aspartic, glutamic, hydrochloric, deuterochloric, hydrobromic, hydroiodic, isocitric, trifluoroacetic, pamoic, anthranilic, mesylic, napadisyiate, oxalacetic, oleic, stearic, salicylic, p-hydroxybenzoic, nicotinic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, phosphoric, phosphonic, ethanesulfonic, benzenesulfonic, pantothenic, toluenesulfonϊc, 2-hydroxyethanesulfonic, sulfanilic, sulfuric, salicylic, cyclohexylaminosulfonic, algenic, β-hydroxybutyric, galactaric and galacturonic acids. Exemplary pharmaceutically acceptable salts include the salts of hydrochloric acid, hydrobromic acid, (-)-dibenzoyl-L-tartaric acid and deuterochloric acid.

Compositions of the invention are useful for treatment of inflammatory respiratory disorders, for example asthma (mild, moderate or severe), e.g., bronchial, allergic, intrinsic, extrinsic, exercise-induced, drug-induced (including aspirin and NSAID- induced) and dust-induced asthma, steroid resistant asthma, smoking asthmatics, bronchitis including infectious and eosinophilic bronchitis, chronic obstructive pulmonary disease (COPD), cystic fibrosis, pulmonary fibrosis including cryptogenic fibrosing alveolitis, idiopathic pulmonary fibrosis, idiopathic interstitial pneumonias, fibrosis complicating antineoplastic therapy and chronic infection, including tubercuSosϊs and aspergillosis and other fungal infections; complications of lung transplantation; vasculitic and thrombotic disorders of the lung vasculature, and pulmonary hypertension (including pulmonary arterial hypertension); antitussive activity including treatment of chronic cough associated with inflammatory and secretory conditions of the airways, and iatrogenic cough; acute and chronic rhinitis including rhinitis medicamentosa, and vasomotor rhinitis; perennial and seasonal allergic rhinitis including rhinitis nervosa (hay fever); nasal polyposis; acute viral infection including the common cold, and infection due to respiratory syncytia! virus, influenza, coronavirus (including SARS) and adenovirus, pulmonary edema, pulmonary embolism, pneumonia, pulmonary sarcoidosis, silicosis, farmer's lung and related diseases; hypersensitivity pneumonitis, respiratory failure, acute respiratory distress syndrome, emphysema, chronic bronchitis, tuberculosis, and lung cancer. In particular, the methods and compositions of the present invention encompass the prevention and treatment of the respiratory disorder, COPD.

As used herein, the term "chronic obstructive pulmonary disease" or "COPD" refers to a set of physiological symptoms including chronic bronchitis, chronic cough, expectoration, exertional dyspnea and a significant, progressive reduction in airflow that may or may not be partly reversible. Emphysema may also be present in the lungs. COPD is a disease characterized by a progressive airflow limitation caused by an abnormal inflammatory reaction to the chronic inhalation of particles.

In subjects with the disorder, poor gas exchange in the lungs leads to decreased oxygen levels in the blood, increased levels of carbon dioxide and shortness of breath. Chronic airflow obstruction in COPD is complicated by the loss of lung elasticity resulting from enzymatic destruction of the lung parenchyma. Rather than a single pathologic condition, COPD is an umbrella term encompassing chronic obstructive bronchitis and emphysema.

Compositions suitable for administration by inhalation via the mouth or the nose are known, and may include carriers and/or diluents that are known for use in such compositions. The composition may contain 0.01-99% by weight of the 5R-5- deuterated glitazone component. Preferably, a unit dose comprises the 5R-5- deuterated glitazone component in an amount of 1 μg to 15 mg. The most suitable dosage level may be determined by any suitable method known to one skilled in the art. It will be understood, however, that the specific amount for any particular patient will depend upon a variety of factors, including the activity of the specific compound that is used, the age, body weight, diet, general health and sex of the patient, time of administration, the route of administration, the rate of excretion, the use of any other drugs, and the severity of the disease undergoing treatment. Optimum dosages will be determined by clinical trial, as is required in the art.

Compositions of the invention may be used in combination with other therapeutic agents that are used in the treatment/prevention/suppression or amelioration of the diseases or conditions for which present compounds are useful. Such other therapeutic agents may be administered, by a route and in an amount commonly used therefore, contemporaneously or sequentially with the 5R-5-deuterated glitazone component. When a compound of the invention is used contemporaneously with one or more other therapeutic agents, a pharmaceutical composition containing such other therapeutic agents in addition to the glitazone component is preferred. Accordingly, the pharmaceutical compositions of the present invention include those that also contain one or more other active ingredients, in addition to the 5R-5-deuterated glitazone component.

Suitable therapeutic agents for a combination therapy with the 5R-5-deuterated glitazone compositions of the invention include one or more other therapeutic agents selected from anti-inflammatory agents, bronchodilators, mucolytic agents, antitussive agents, leukotriene inhibitors, and antibiotics.

Suitable therapeutic agents for a combination therapy with the 5R-5-deuterated glitazone compositions of the invention include: (1 ) a (1 ) a steroid drug such as a corticosteroid, for example beclomethasone, (e.g., as the mono or the dipropionate ester), flunisolide, fluticasone (e.g., as the propionate or furoate ester), ciclesonide, mometasone (e.g., as the furoate ester), mometasone desonide, rofleponide, hydrocortisone, prednisone, prednisolone, methyl prednisolone, naflocort, deflazacort, halopredone acetate, fluocinolone acetonide, fluocinonide, clocortolone, tipredane, prednicarbate, alclometasone dipropionate, halometasone, rϊmexolonβ, deprodone propionate, triamcinolone, betamethasone, fludrocortisone, desoxycorticosterone, etiprendnol dicloacetate and the like. Steroid drugs can additionally include steroids in clinical or pre-clinical development for respiratory diseases such as GW-685698, GW-799943, GSK 870086, QAE397, NCX=I 010, NCX-1020, NO-=dexamethasone, PL-2146, NS-126 (formerly ST-126) and compounds referred to in international patent applications WO0212265, WO0212266, WO02100879, WO03062259, WO03048181 and WO03042229. Steroid drugs can also additionally include next generation molecules in development with reduced side effect profiles such as selective glucocorticoid receptor agonists (SEGRAs), including ZK-216348 and compounds referred to in international patent applications WO-00032585, WO-QQQ21Q143, WQ-2Q05Q34939, WO-2005003098, WO-2005035518 and WO-2005035502 and functional equivalents and functional derivatives thereof; (2) a β2-adrenoreceptor agonist, such as albuterol, bambuterol, terbutaline, fenoterol, formoterol, formoterol fumarate, salmeterol, salmeterol xinafoate, arformoterol, arfomoterol tartrate, indacaterol (QAB-149), carmoterol, picumeterol, BI 1744 CL, GSK159797, GSK59790, GSK159802, GSK642444, GSK678007, GSK96108, clenbuterol, procaterol, bitolterol, and broxaterol,TA-2005 and also compounds of EP1440966, JP05025045, WO93/18007, WO99/64035, US2002/0055651 , US2Q05/Q133417, US2005/5159448, WO00/0751 14, WO01/42193, WO01 /83462, WO02/66422, WO02/70490, WO02/76933, WO03/24439, WO03/42160, WO03/42164, WO03/72539, WO03/91204, WO03/99764, WO04/16578, WO04/016601 , WO04/22547, WO04/32921 , WO04/33412, WO04/37768, WO04/37773, WO04/37807, WO0439762, WO04/39766, WO04/45618, WO04/46083, WO04/71388, WO04/80964, EP1460064, WO04/087142, WO04/89892, EP01477167, US2004/0242622, US2004/0229904, WO04/108675, WO04/108676, WO05/033121 , WO05/040103, WO05/044787, WO04/071388, WO05/058299, WO05/058867, WO05/065650, WO05/066140, WO05/070908, WO05/092840, WO05/092841 , WO05/092860, WO05/092887, WO05/092861 , WO05/090288, WO05/092087, WO05/080324, WO05/080313, US20050182091 , US20050171147, WO05/092870, WO05/077361 , DE10258695, WO05/111002, WO05/111005, WO05/110990, US2005/0272769 WO05/110359, WO05/121065, US2006/0019991 , WO06/016245, WO06/014704, WO06/031556, WO06/032627, US2006/0106075, US2006/0106213, WO06/051373, WO06/056471 , WO08/096112, WO08/104790, WO08/096119, WO08/096112; (3) a leukotriene modulator, for example, montelukast or pranlukast; (4) anticholinergic agents, for example, selective muscarinic-3 (M3) receptor antagonists such as ipratropium bromide, tiotropium, tiotropium bromide (Spiriva®), glycopyrollate, NVA237, LAS34273, GSK656398, GSK233705, GSK 573719, LAS35201 , QAT370 and oxytropium bromide; (5) phosphodiesterase-IV (PDE-IV) inhibitors, for example, roflumilast or cilomilast; (6) an antitussive agent, such as codeine or dextramorphan; (7) a non-steroidal anti-inflammatory agent (NSAID), for example, ibuprofen or ketoprofen; (8) a mucolytic, for example, N acetyl cysteine or fudostein; (9) a expectorant/mucokinetic modulator, for example, ambroxol, hypertonic solutions (e.g., saline or mannitof) or surfactant; (10) a peptide mucolytic, for example, recombinant human deoxyribonoclease I (dornase-alfa and rhDNase) or helicidin; (11 ) antibiotics, for example, azithromycin, tobramycin and aztreonam; and (12) p38 MAP kinase inhibitors such as GSK 856553 and GSK 681323.

In one aspect, the invention provides for the use of inhaled administration of the 5R- 5-deuterated glitazone compositions of the invention in combination with other anti- inflammatory drugs and bronchodiiator drug combinations (i.e. triple combination product), including but not limited to salmeterol xinafoate/fluticasone propionate (Advair/Seretide®), formoterol fumarate/budesonide (Syrnbicort®), formoterol fumarate/mometasone furoate, formoterol fumarate/beclometasone dipropionate (Foster®), formoterol fumarate/fluticasone propionate (FlutϊForm®), Indacateroi/mometasone furoate, lndacaterol/QAE-397, GSK159797/GSK 685698, GSK159802/GSK 685698, GSK642444/GSK 685698, formoterol fumarate/ciclesonide, arformoterol tartrate/ciclesonide.

In another aspect, the invention provides for the use of inhaled administration of the 5R-5-deuterated glitazone compositions of the invention in combination with other bronchodiiator drug combinations, particularly B2 agonist/M3 antagonist combinations (i.e. triple combination product), including but not limited to salmeterol xinafoate/tiotropium bromide, formoterol fumarate/tiotropium bromide, BI 1744 CL/tiotropium bromide, indacaterol/NVA237, indacterol/QAT-370, formoterol/ LAS34273, GSK159797/GSK 573719, GSK159802/GSK 573719, GSK642444/GSK 573719, GSK159797/GSK 233705, GSK159802/GSK 233705, GSK642444/GSK 233705, and compounds which possess both B2 agonist and M3 antnagonist activity in the same molecule (dual functionality) such as GSK 961081.

Thus in another aspect, the invention provides a kit for treatment of respiratory disorders in a subject, the kit comprising one dosage form comprising a composition adapted for pulmonary administration by inhalation, which composition comprises a 5R-5-deuterated glitazone, particularly 5R-5-deuterated pioglitazone or 5R-5- deuterated rosiglitazone, and one or more pharmaceutically acceptable carriers and/or excipients, wherein the 5R-5-deuterated glitazone content of the composition consists of at least 95% by weight of the 5R-5-duterated glitazone and less than 5% by weight in total of 5R-5-protonated glitazone, 5S-5-protonated glitazone and 5S-5- deuterated glitazone and a second dosage form comprising another therapeutic agent, for example as discussed above, selected from anti-inflammatory agents, bronchodilators, mucolytic agents, antitussive agents, leukotriene inhibitors, and antibiotics.

For delivery by inhalation, the active compound is preferably in the form of microparticles. These may be prepared by a variety of techniques, including spray- drying, freeze-drying and micronisation. Following size reduction to produce microparticles, particle size distribution (PSD) of the compound is examined and generally described in the art by specifying d10, d50 and d90 values. The average particle size, i.e. the average equivalent diameter, is defined as the diameter where 50 mass-% (of the particles) of the powder have a larger equivalent diameter, and the other 50 mass-% have a smaller equivalent diameter. Hence the average particle size is denoted as equivalent d50. For inhaled use a d50 of less than 10 microns, preferably less than 5 microns is desired.

By way of example, a composition of the invention may be prepared as a suspension for delivery from a nebuliser or as an aerosol in a liquid propellant, for example for use in a pressurised metered dose inhaler (PMDI). Propellants suitable for use in a PMDI are known to the skilled person, and include CFC-12, HFA-134a, HFA-227, HCFC-22 (CCI ₂ F ₂ ) and HFA-152 (CH ₄ F ₂ and isobutane).

For delivery by inhalation, the active compound is preferably in the form of microparticles. They may be prepared by a variety of techniques, including spray- drying, freeze-drying and micronisation.

In a preferred embodiment of the invention, a composition of the invention is in dry powder form, for delivery using a dry powder inhaler (DPI). Many types of DPI are known.

Microparticles for delivery by inhalation may be formulated with excipients that aid delivery and release. For example, in a dry powder formulation, microparticles may be formulated with large carrier particles that aid flow from the DPI into the lung. Suitable carrier particles are known, and include lactose particles; they may have a mass median aerodynamic diameter of greater than 90 μm.

Aerosol generation can be carried out using, for example, pressure-driven jet atomizers or ultrasonic atomizers, preferably using propellant-driven metered aerosols or propellant-free administration of rnicronized active compounds from, for example, inhalation capsules or other "dry powder" delivery systems.

The compositions may be dosed as described depending on the inhaler system used. In addition to the active compounds, the administration forms may additionally contain excipients, such as, for example, propellents (e.g. Frϊgen in the case of metered aerosols), surface-active substances, emulsifiers, stabilizers, preservatives, flavorings, fillers (e.g. lactose in the case of powder inhalers) or, if appropriate, further active compounds.

For the purposes of inhalation, a large number of systems are available with which aerosols of optimum particle size can be generated and administered, using an inhalation technique which is appropriate for the patient. In addition to the use of adaptors (spacers, expanders) and pear-shaped containers (e.g. Nebulator®, Volumatic®), and automatic devices emitting a puffer spray (Autohaler®), for metered aerosols, in particular in the case of powder inhalers, a number of technical solutions are available (e.g. Diskhaler®, Rotadisk®, Turbohaler® or the inhalers for example as described EP-A-0505321 ).

Methods of preparation of Deuterated GIStazone Enantiomers

Deuferated glitazones can be prepared, using pioglitazone (I) by way of example, as shown in Scheme 1. A base (II) can be used to deprotonate the C-5 position of the glitazone. Suitable bases include, but are not restricted to, 1 ,5- diazabicyclo[4.3.0]non-5-ene and more preferably to sodium deuteroxide. Deuterated reagents (III), preferably deuterochloride can be used to incorporate the C-5 deuterium.

Scheme 1

Deuterated glitazones can be separated using chiral HPLC (see for example Methods 1 , 7 and 10 below). Chiral columns include CHIRALPAK AD, AD-H, AS-V, IA, IC, OD, OF, OG, OJ, OK, and OZ Preferred chiral columns for HPLC are CHIRALPAK AD-H and CHSRALPAK IA using elution with ethanol and varying portions of TFA, preferably O 05-0 2% TFA alternatively heptane, ethanof and acetic acid in varying proportions, preferably 30% heptane, 70% ethanol and 0 2% acetic acid

An alternative method of resolution, using deuterated pioglitazone as an example, is shown in Scheme 2 (see also Example 3, 4 and 5)

Scheme 2

Recent review articles on optical resolution methods E Fogassy et a/ , Tetrahedron asymmetry, 2008, 19, 519-536, S H Wilen, Topics in Stereochemistry, Wiley- lnterscience NY, 1972, 6, 107 Eds E L Ehel, N L Alhnger, P Newman, Optical resolution Procedures of Chiral Compounds 1-3, Resolution Information Center, NY, 1978-1984, J Jaques, S H Wilen, A Collett, Enantiomers Racemates and Resolutions, Wiley-lnterscience NY, 1991 , R A Scheldon, Chirotechnology, Marcel Dekker, NY, 1993, Optical Resolutions via diastereomeπc salt formation, CRC Press, 2002, Ed David Kozma

A preferred set of chiral acid resolving agents for use in the present invention includes (-)-Q,O'-dιbenzoyl-L-tartaπc acid (anhydrous), (-)-O,O'-dιbenzoy!-L-tartaπc acid monohydrate, (-)-dι-O-p-toluoyl-L-tartaπc acid, L-(+)-tartaπc acid and (-)-dι-p- anisolyl-L-tartaric acid The most preferred chiral acid resolving agent is (-)-O,Q'-dιbenzoyI-L-tartarιc acid

(anhydrous)

Chiral acid resolving agents can be used in different stoichiometrics to effect resolutions The above resolving agents can be used in a ratio of 1 part (I) to 10 parts of chiral acids (V), more preferably in a ratio of 1 part (I) to 5 parts of chiral acids (V), and most preferably in a ratio of 1 part (I) to 2 part of chiraf acids (V) Even more preferred is a ratio of 1 part (I) to 1 part of chirai acids (V)

A variety of solvents may be useful to form diastereomeric salts (Vl) with chiral acids (V) Preferred solvents will include ethyl acetate, dichloromethane tetrahydrofuran, 1 ,4-dιoxane, acetone, acetonitπle, alcohols such as MeOH, EtOH, IPA, 1-propanol, 1-butanol, 2-butanol, t-butanol, and deuterated forms of such alcohols such as IvIeOD, d ₄ -fvieOH, EtOD ₁ 1 ,2-dimethoxyethane, diethyl ether, dichloroethane, tert- butylmethyl ether, 2-butanone, toluene, cyclohexane, heptane, hexane, H2O, DIvIF, D ₂ O, petroleum ethers and CHCI ₃

Solvents may be used in combination with each other and preferred combinations include 10% HCI in H2θ, 10% H2O in DMF, 10% H2O in EtOH, 10% H2O in IPA, methanol or deuteromethanol and water or deuterium oxide in varying portions, methanol or deuteromethanol and water or deuterium oxide in varying portions with HCI or DCI ₁ CHCI ₃ and ethyl acetate in varying portions, EtOH or EtOD and water or deuterium oxide in varying portions, IPA and water in varying portions, 1-propanol and water in varying portions and CHCI ₃ and dioxane in varying portions

More preferred solvent combinations include CHCi ₃ and dioxane in varying portions, CHCI ₃ and ethyl acetate in varying portions, methanol and water in varying portions with HCl and methanol and water in varying portions

Commonly used chiral amine resolving agents (VII) include, without limitation, 1- phenylethyl amine, cinchonidine, cinchonine, quinidine, codeine, morphine, strychnine, brucine, quinine, ephedπne, ammo butanol, dehydroabiethylamine, 2- phenyl glycinol, 1 ,2-cyclohexyldιamιne, 1-naphthylethylamιne, 2-amιno-1- phenylpropane-1 ,3-dιol, 4-chloro-1-pheny!amme, N-(4-methoxybenzyl)-1-pheny!βthyl amine, fenchylamine, N-benzyl-1-phenylethyl amine, N-(4-dimethylammobenzyl)-1- phenylethy! amine, 3-amino-2,2-dimethyl-3-phenylpropan-1-ol, sparteine, proline, serine, phenylalanine, lysine, threonine, valine, histidine, alanine, glutamic acid and glutamine, arginine, homo-arginine, N-α-acetyl lysine, N-ε-acetyllysϊne and ornithine.

As an alternative method of resolution, deuterated glitazones can be also be prepared and then resolved in the same flask, as exemplified in example 4.

The compositions with which the invention is concerned are of high enantiomeric and deuterated purity. When the glitazone is pioglitazone, a method has been identified which efficiently enables the resolution of racemic pioglitazone to that high level of purity. This, in a preferred aspect of the invention, there is provided a method for the preparation of a salt of 5R-5-deuterated pioglitazone with a chiral acid resolving agent, containing less than 5% by weight in total of the corresponding salt of 5R-5- protonated piogiitazone, 5S-5-protonated pioglitazone and 5S-5-deuterated pioglitazone. which method comprises:

(a) forming a solution of a salt of racemic 5-deuterated pioglitazone with a chiral resolving agent which is an L-tartaric acid derivative, in an alcohol solvent, and adding H ₂ O or D ₂ O to the resultant solution, thereby precipitating 5R-5-deuterated pioglitazone-chiral resolving agent salt and recovering the precipitate; and

(b) forming a solution of the precipitate from step (a) in an alcohol solvent in the presence of an acid, and adding H ₂ O or D ₂ O to the resultant solution, thereby precipitating 5R-5-deuterated pioglitazone-chiral resolving agent salt, and recovering the precipitate.

Alcohol solvents have been discussed above, but a preferred alcohol for use in the pioglitazone resolution method is CH ₃ OH or CH ₃ OD.

The L-tartaric acid derivative resolving agent may be, for example, (-)-O,O'- dibenzoyl-L-tartaric acid (available as anhydrous or monohydrate), (-)-di-O-p-toluoyl- L-tartaric acid, L-(+)-tartaric acid or(-)-di-p-anisolyl-L-tartaric acid. The most preferred chiral acid resolving agent is (-)-O,O'-dibenzoyl-L-tartaric acid (preferably anhydrous). The acid whose presence is required in step (b) is preferably hydrochloric acid or deuteor hydrochloric acid, but other acids which may be used include sulphuric, hydrobromic, trifluoroacetic, citric, dibenzoyl tartatric, malic, maleic, ditoloyl and nitric acids, or deuterated forms of any of those acids. The presence of acid appears to be essential for the stability of the pioglitazone=chiral resolving agent salt in the solution.

The 5R-5-deuterated pϊoglitazone-chira! resolving agent salt prepared according to this aspect of the invention may be converted by salt exchange to a pharmaceutically acceptable salt of 5R-5-deuterated pioglitazone, such as the hydrochloride or the hydrobromide.

The following Examples illustrate the preparation of the 5R-5-deuterated pioglitazone or 5R-5-deuterated rosiglitazone enantiomers, and the biological results on which the present invention is based:

General Experimental Details

Abbreviations used in the experimental section: c = concentration, h - hour, H ₂ O = distilled water; D ₂ O = deuterium oxide; HPLC = high performance liquid chromatography; LCMS = liquid chromatography mass spectrometry; MeOH = methanol; MeOD = mono-Deuteromethanol; nM = nanometer; TFA = trifluoroacetic acid; DMSO = dimethyl sulphoxide; HCI = hydrogen chloride; DCI = deutero chloride; EtOH = ethanol; Et ₂ O - diethyl ether; min = minutes; RT = room temperature; Rt = retention time; e.e. = enantiomeric excess; d.e. = diastereomeric excess; MP-Carbonate = macroporous triethylammonium methyipolystyrene carbonate (0.5% inorganic antistatic agent); UV = ultraviolet; DBN = 1 ,5-diazabicyclo[4.3.0]non-5-ene; L-DBTA = (-)-dibenzoyl-L-tartaric acid monohydrate; NaOD = sodium deuteroxide; CH ₃ CN = acetonitrile

The nomenclature of structures was assigned using ACD Labs version 10.

NMR spectra were obtained on a Varian Unity Inova 400 spectrometer with a 5mm inverse detection triple resonance probe operating at 400MHz or on a Bruker Avance DRX 400 spectrometer with a 5mm inverse detection triple resonance TXI probe operating at 400MHz or on a Bruker Avance DPX 300 spectrometer with a standard 5rnm dual frequency probe operating at 300MHz. Shifts are given in ppm relative to tetramethylsilane. Optical rotations were measured using an AA-10R automatic polarimeter with 5x25 mm jacketed sample cell.

All solvents and commercial reagents were used as received.

The Liquid Chromatography Mass Spectroscopy (LC/MS) systems used: Method 1

CHIRALPAK IA (250 x 21 mm, 5 μm), elution with EtOH +0.05%TFA - flow rate 13 ml/min. Detection -In-line LJV detection set at 250 nM wavelength.

CHIRALPAK IA (250 x 4.6 mm, 5μM), elution with ethanol +0.05%TFA - flow rate 0.7 ml/min. Detection - In-line DAD set at 280 nM wavelength.

Waters Micromass ZQ2000 with a C18-reverse-phase column (100 * 3.0 mm Higgins Clipeus with 5 μm particle size), elution with A: water + 0.1 % formic acid; B: acetonitrile + 0.1 % formic acid. Gradient:

Gradient - Time flow rnL/min %A %B 0.00 1.0 95 5 1.00 1.0 95 5 15.00 1.0 5 95 20.00 1.0 5 95 22.00 1.0 95 5 25.00 1.0 95 5

Detection - MS, ELS ₁ UV (100 μl split to MS with in-line UV detector). MS ϊonisation method - Electrospray (positive ion)

Method 4

CHIRALCEL OD-RH (150 X 4.6 mm), elution with 90% MeOH + 10% H ₂ O - flow rate

0.5 ml/min. Detection - In-line UV detection set at 254 nM wavelength. CHIRALPAK IA (250 x 21 mm, 5 μm), elution with EtOH +0.2%TFA - flow rate 10 mL/min. Detection -In-line UV detection set at 220 nM wavelength.

Waters Platform LC Quadrupole mass spectrometer with a C18-reverse-phase column (30 * 4.6 mm Phenomenex Luna 3 μm particle size), elution with A: water + 0.1 % formic acid; B: acetonitrile + 0.1 % formic acid. Gradient:

Gradient - Time flow mL/min %A %B

0.00 2.0 95 5

0.50 2.0 95 5

4.50 2.0 5 95

5.50 2.0 5 95

6.00 2.0 95 5

Detection - MS, ELS, UV (200 μl split to MS with in-line UV detector). MS ionization method - Electrospray (positive and negative ion).

Chiral-AGP (150 x 4.0 mm, 5μM), elution with A: 86% 10 mM potassium dihydrogen phosphate buffer pH 7.0; B: 14% acetonitrile + 0.1% formic acid - flow rate 0.8 ml/min. Detection - In-line DAD set at 254 nM wavelength.

CHIRALPAK IA (250 x 21 mm, 5 μm), elution with heptane: EtOH: AcOH (30: 70: 0.2) - flow rate 18 mL/min. Detection -In-line UV detection set at 254 nM wavelength. Desired fractions were concentrated with a few drops of TFA.

CHIRALPAK IA (250 x 4.6 mm, 5 μm), elution with heptane: EtOH: AcOH (30: 70: 0.2) - flow rate 1.0 mL/min. Detection -In-line UV detection set at 254 nM wavelength.

Method 10 Waters Platform LC Quadrupole mass spectrometer with a Higgins CIipeus Smicron C18 column 100 x 3.0mm, maintained at 40 ⁰ C. Elution with A: Water 0.1 % Formic Acid ; B: Methanol 0.1% Formic Acid. Gradient:

Gradient - Time flow rnL/mϊn %A %B

0.00 1.0 85 15

1.0 1.0 85 15

13 1.0 5 95

20 1.0 5 95

22 1.0 85 15 25 1.0 85 15

Detection - MS, ELS ₁ UV (100μl split to MS with in-line UV DAD detector). MS ionisation method - Electrospray (positive/negative ion).

CHIRALPAK IA (250 x 4.6 mm, 5 μm), elution with EtOH +0.05%TFA: heptane: IPA (40: 30: 30%) - flow rate 0.7 ml/min. Detection -In-line UV detection set at 280 nM wavelength.

All reactions were carried out under an atmosphere of nitrogen unless specified otherwise. Racernic pioglϊtazone and rosiglitazone were used as free base.

(5S)-5-{4-[2-(5-Ethylpyridin-2-yl)ethoxy]benzyl}-(5- ² H)-1,3-thiazolidine-2,4'dione

a. (±)-5-{4-[2-(5-EthyIpyridIn-2-yI)ethoxy|-ben2yI}-(5- ² H)-1 ₅ 3-thsazoIidIne-2,4-

DBN (17.3 μL, 0.14 mmo!) was added to a suspension of (±)-5-{4-[2-(5-ethylpyιϊdin-2- yl)ethoxy]benzyl}-1 ,3-thiazolidine-2,4-dione (1.0 g, 2.81 mmol) in MeOD (150 mL) and refluxed overnight. The reaction was cooled to RT and the precipitate obtained was filtered and dried to afford the title compound (916 mg, 91%) as a white solid. ¹ H NMR (400 MHz, DMSQ-d ₆ ): δ 12.02-1192 (1 H, bs), 8.37=8.35 (1 H, d, J = 2.3 Hz), 7.59-7.54 (1 H, dd, J = 7.9, 2.4Hz), 7.28-7.25 (1 H, d, J = 7.9 Hz), 7.16-7.10 (2 H, d, J = 8.6 Hz), 6.88-6.84 (2 H, d, J = 8.6 Hz) ₁ 4.33=4.26 (2 H ₁ t, J = 6.8Hz), 3.30-3.26 (1 H ₁ d, J = 14.2 Hz ) 3.15-3.09 (2 H, t, J = 6.8 Hz), 3.06-3.00 (1 H ₁ d, J = 14.2 Hz), 2.63-2.55 (2 H, q, J = 7.6 Hz), 1.20-1.51 (3 H, t, J = 7.6

(+)-5-{4-[2-(5-Ethyipyridin-2-yI)ethoxy]ben2y!}-(5- ² H)-1,3-thIa2θS!dine-2,4-

-1.25 M HCI/MeOH (6.16 ml_, 7.7 mmol) was added to a suspension of example 1a

(916 mg, 2.57 mmol) in MeOD (20 ml_) and stirred at RT for 30 min. The solvent was concentrated in vacuo to afford the title compound (983 mg, 89%) as a white solid. ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 12.05-11.93 (1 H, bs), 8.72-8.67 (1 H, s), 8.39-8.32 (1 H, d, J = 8.1 Hz) ₁ 7.96-7.90 (1 H ₁ d, J = 8.3 Hz) ₁ 7.17-7.11 (2 H, d, J = 8.7 Hz), 6.89- 6.84 (2 H, d, J = 8.7 Hz), 4.41-4.34 (2 H, t, J = 6.3 Hz), 3.48-3.41 (2 H, t, J = 6.3 Hz), 3.31-3.25 (1 H, d, J = 14.2 Hz), 3.08-3.01 (1 H ₁ d, J = 14.2 Hz), 2.82-2.73 (2 H, q, J = 7.5 Hz), 1.26-1.20 (3 H, q, J = 7.5 Hz). Contains 1.1% (±)-5-{4-[2-(5-ethyIpyridin-2- yl)ethoxy]benzyl}-1 ,3-thiazolidine-2,4-dione hydrochloride.

c. (5S)-5-{4-[2-(5-Ethylpyridin-2-yl)ethoxy]ben2Eyl}-(5- ² H)-1,3-thiazolidine-2,4-

The title compound (121 mg) was prepared by preparative chiral HPLC purification (Method 1 ) using the product from example 1 b. e.e. (Method 2) > 99%, Rt 9.82 min.

d. (5S)-5~{4-ϊ2-f5-EthyIpyriciϊn-2-yI)etiioxy]ben2y!}-(5- ² H)->1,3-thIazoIidine-2 _! 4. ^-Carbonate (271 mg, 0.78 mmol) was added to a solution of example 1c (121 nig, 0.26 mmol) in MeOD (1.0 mL) and stirred at RT for 2 h. The suspended solid was decanted and the remaining resin washed with MeOD (3 X 2.0 mL). The suspension and washings were treated with -1.25 M HCI in MeOH (0.13 mL, 0.16 mmol) and stirred at RT for 30 min then concentrated in vacuo to afford the title compound (81 mg, 80%). LCMS (Method 3): Rt 6.12 mϊn, m/z 358 [M+Hf. [α] _D ²³ -100° (c 1.0, MeOH). e.e. (Method 4) 98.7%, Rt 15.84 min. ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 12.00 (1 H, s), 8.69 (1 H, d, J = 2.0 Hz), 8.31 (1 H, d, J = 8.2 Hz), 7.89 (1 H, d, J = 8.2 Hz) ₁ 7.19-7.11 (2 H, m), 6.90-6.83 (2 H, m), 4.37 (2 H, t, J = 6.2 Hz), 3.41 (2 H ₁ t, J = 6.2 Hz), 3.29 (1 H, d, J = 14.2 Hz), 3.06 (1 H, d, J = 14.2 Hz), 2.77 (2 H, q, J = 7.6 Hz), 1.24 (3 H, t, J = 7.6 Hz). Contains 1.6% (5S)-5-{4-[2-(5-ethyIpyridin-2- yl)ethoxy]benzyl}-1 ,3-thiazolidine-2,4-dione hydrochloride.

The title compound was prepared using a method analogous to that outlined in example 1c. e.e (Method 2) ≥ 99%, Rt 6.59 min.

b. (5/?)-5-{4-[2-(5-Ethylpyridin-2-yl)ethoxy]bβnzyl}-(5- ² H)-1,3-thiazolidinβ-2,4- dione hydrochloride

The title compound was prepared using a method analogous to that outlined in example 1d using the product from example 2a. LCIVIS (Method 3) Rt 6.13 min, m/z 358 [M-Cl ⁺ ]. [α] _D ²³ ÷108° (c 1.0, MeOH). e.e. (Method 4) 98.5%, Rt 14.14 min. Stereochemistry at C-5 was assigned (R) configuration by single crystal X-ray diffraction analysis. ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 11.99 (1 H, s), 8.68 (1 H, s), 8.29 (1 H, d, J = 8.2 Hz) ₁ 7.88 (1 H, d, J = 8.2 Hz), 7.18-7.11 (2 H, m), 6.90=6.83 (2 H, m), 4.36 (2 H, t, J = 6.2 Hz), 3.40 (2 H, t, J = 6.2 Hz), 3.29 (1 H, d, J = 14.2 Hz), 3.05 (1 H, d, J = 14.2 Hz), 2.76 (2 H, q, J = 7.6 Hz), 123 (3 H, f, J = 7.6 Hz). Contains 1.7% (5R)-5={4-[2-(5-ethylpyridin-2-yI)ethoxy]benzyI}-1 ,3-thiazolidine-2,4-dione hydrochloride.

a. (±)-5-{4-[2-(5-Ethylpyridin-2-yl)βthoxy]benzylH5- ² H)-1,3-thiazolidine-2,4-dione

To a suspension of (±)-5-{4-[2-(5-ethylpyridin=2-yl)ethoxy]-benzyI}-(5- ² H)-1 ,3- thiazolidine-2,4-dione (0.85 g, 2.4 mmol) in MeOH (25 tnL) was added dropwise HCI (37 wt%, 0.3 mL). The reaction was stirred at RT for 10 min and then concentrated in vacuo to give the title compound as a white solid (0.934 g, 99%). ¹ H NMR (400 MHz, DMSO-^ ₆ ): δ 12.01 (1 H, s), 8.70 (1 H, s), 8.34 (1 H, d, J = 8.2 Hz), 7.92 (1 H ₁ d, J = 8.2 Hz), 7.15 (2 H ₁ d, J = 8.4 Hz), 6.87 (2 H, d, J = 8.4 Hz), 4.37 (2 H ₁ t, J = 6.2 Hz), 3.43 (2 H, t, J = 6.3 Hz), 3.29 (2 H ₁ d, J = 14.2 Hz), 3.05 (1 H, d, J = 14.2 Hz), 2.77 (2 H, q, J = 7.6 Hz), 1.23 (3 H ₁ 1, J = 7.6 Hz).

b. (5/!)-5-{4-[2-(5-Ethylpyridin-2-yl)ethoxy]bβnzylH5- ² H)-1,3-thiazolidinβ-2,4- dione (-)-O,O'-dibenzoyl-L-tartrate

L-DBTA

To a solution of the product from example 3a (0.934 g, 2.37 mmol) in MeOD (20 mL) was added (-)-dibenzoyl-L-tartaric acid (0.85 g, 2.37 mmol). The reaction was stirred at room temperature for 5 min until a solution was obtained and then H ₂ O (15 mL) was added dropwise. The resulting suspension was stirred at RT for 20 hours. The granular, crystalline solid was collected by filtration and dried at 40 ^° C under vacuum to give title compound (0.467 g, 55%). (Method 2) 83.04%, Rt 10.54 min; 16.96%, Rt 14.03 ruin. 66.08% d.e. ¹ H (MMR (400 MHz, DMSQ-Cf ₆ ): δ 11.99 (1 H, m), 8.37 (1 H, m), 8.02 (4 H, m), 7.76=7.71 (2 H, m), 7.61-7.59 (5 H, m), 7.28 (1 H, dd, J = 7.9, 0.8 Hz), 7.13-7.12 (2 H, m), 6.87=6.85 (2 H, m), 5.87 (2 H, s), 4.30 (2 H, t, J = 6.7 Hz), 3.28 (1 H, d, J = 14.2 Hz). 3.14 (2 H, m), 3.08 (1H, m) 2.59 (2 H, q, J = 7.6 Hz), 1.18 (3 H, t, J = 7.6 Hz).

dϊone H-O,O ^! -dibenzoyI=L-tartrate

L-DBTA

To pre-mixed MeOH (4.63 ml.) and 1 M HCI (0.65 mL) was added the product from example 3b (0.467 g, 0.653 mmol) followed by dropwise addition of H ₂ O (3.97 mL) and the resulting suspension stirred at RT for 72 hours. The granular, crystalline solid was collected by filtration and dried at 40 ^° C under vacuum to give the title compound (0.307 g, 66%). (Method 7) 95.66%, Rt 10.11 min; 4.39% Rt 14.76 min. 91.3% d.e. LCMS (Method 6): Rt . 2.92 min, m/z 358 [M+Hf. ¹ H NMR (400 MHz, DMSO=d ₆ ): δ 1 1.93 (1 H, s), 8.32 (1 H, d, J = 2.3 Hz), 7.98-7.95 (4 H, m), 7.70=7.67 (2 H, m), 7.58- 7.51 (5 H, m), 7.23 (1 H, d, J = 7.9 Hz), 7.10=7.06 (2 H, m), 6.85-6.79 (2 H, m), 5.82 (2 H, s), 4.25 (2 H ₁ 1, J = 6.7 Hz), 3.22 (1 H, m), 3.08 (2 H, t, J = 6.7 Hz), 2.99 (1 H, d, J = 14.2 Hz), 2.54 (2 H, q, J = 7.6 Hz), 1.13 (3 H, t, J = 7.6 Hz). Contains 2.4% of (5R)-5-{4-[2-(5-ethylpyridin-2-yl)ethoxy]-benzyl}-1 ,3-thiazolidine-2,4-dione(-)-O,O ^! - dibenzoyl-L-tartrate.

d. (5/?)-5-{4-[2-(5-Ethylpyridin-2-yl)βthoxy]benzylH5- ² H)-1,3-thiazolidine-2,4- dione hydrochloride

The product from example 3c (0.3 g, 0.42 mmol) was dissolved in premixed MeOH (1.5 mL) and HCI 37 wt% (0.087 mL) at -45 ⁰ C (internal temperature). Ethyl acetate (4.5 mL) was added and the reaction allowed to cool with stirring over 1 h. The solid was collected by filtration, washed with ethyl acetate (0.5 mL) and dried for 18 h at 40 °C under vacuum to give title compound (0.128 g, 77.5%). (Method 7) 98.26%, Rt 15.00 min; 174%, Rf 16.82 min.; 96.52% e.e. LCMS (Method 6): Rt 2.89 min, m/z 358 [M+Hf. ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 11.99 (1 H, s), 8.69 (1 H, d, J = 2.0 Hz), 8.32 (1 H, d, J = 8.3 Hz), 7.90 (1 H, d, J = 8.3 Hz), 7.16=7.12 (2 H, m), 6.90-6.83 (2 H, rn), 4.37 (2 H, t, J = 6.2 Hz), 3.42 (2 H ₁ 1, J = 6.3 Hz), 3.28 (1 H, d, J = 14.2 Hz), 3.05 (1 H, d, J = 14.2 Hz), 2.77 (2 H, q, J = 7.6 Hz), 1.23 (3 H ₁ t, J = 7.6 Hz). Contains 2.6% of (5R)-5-{4-[2-(5-ethylpyridin-2-yI)ethoxy]-benzyϊ}-1 ,3-thiazoIϊdine- 2,4-dione hydrochloride.

L-DBTA

NaOD 40 wt% (0.6 ml) was added to a suspension of (±)-5-{4-[2-(5-ethylpyridin-2- yl)ethoxy]benzyl}-1 ,3-thiazolidine-2,4-dione (1.0 g, 2.81 mmol) in MeOD (25.0 mL) and then heated at 40 ⁰ C for 1 h. The reaction mixture was cooled to RT, diluted with D ₂ O (15 ml) and treated with DCI 35 wt% (0.8 mL) to reach pH 5. (-)-Dibenzoyl-L- tartaric acid (1.1 g, 3.07 mmol) was added to the suspension and the reaction heated to 35 ⁰ C. The resulting solution was cooled to RT, treated with D ₂ O (15 mL) and stirring continued at RT for 20 hours. The resulting precipitate was filtered and dried under vacuum at 40 ⁰ C to afford the title compound (0.442 g, 44%) as a white solid. (Method 2) 87.93%, Rt 10.92 min.; 12.07%, Rt 13.97 min.; 75.86% d.e. ¹ H NMR (400 MHz, DMSO-Cf ₆ ): δ 11.98 (1 H, s), 8.37 (1 H, d, J = 2.2 Hz), 8.04-8.00 (4 H ₁ m), 7.76-7.71 (2 H, m), 7.62-7.58 (5 H, m), 7.28 (1 H, d, J = 7.9 Hz), 7.14-7.12 (2 H ₁ m), 6.87-6.85 (2 H, m), 5.87 (2 H, s), 4.30 (2 H, t, J = 6.7 Hz), 3.28 (1 H, d, J = 14.2 Hz), 3.13 (2 H, t, J = 6.7 Hz), 3.04 (1 H, d, J = 14.2 Hz), 2.59 (2 H, q, J = 7.6 Hz), 1.18 (3 H, t, J = 7.6 Hz). Contains 1.3% of (5R)-5-{4-[2-(5-ethylpyridin-2-yl)ethoxy]- benzyl}-1 ,3-thiazolidine-2,4-dione.

Example 5 (5R)-5-{4-[2-(5-Ethylpyridin-2-yl)ethoxy]benzylH5- ² H)-1,3-thiazolidine-2,4-dionβ

NaOD 40 wt% (8 mL, 133 mmol) was added to a suspension of (±)-5-{4-[2-(5- ethylpyridin-2-yl)ethoxy]benzyl}-1 ,3-thiazolidine-2,4-dione (10.0 g, 28.1 mmol) in MeOD (250 mL) and heated at 40 ⁰ C with stirring for 1 hour. The reaction mixture was cooled to RT over 1 hour and the reaction mixture treated with DCI 35 wt% (11 mL) to reach pH 6-7. The resulting precipitate was filtered and dried under vacuum at 40 ⁰ C to afford the title compound (10.03 g, 100%) as a white solid. ¹ H NMR (400 MHz ₁ DMSO-d ₆ ): δ 11.99 (1 H ₁ s), 8.36 (1 H, d, J = 2.3 Hz) ₁ 7.57 (1 H, dd, J = 7.9, 2.3 Hz) ₁ 7.27 (1 H, d, J = 7.9 Hz) ₁ 7.13 (2 H, m), 6.87-6.84 (2 H, m), 4.30 (2 H ₁ t, J - 6.7 Hz), 3.29 (1 H ₁ d, J = 14.2 Hz), 3.16-3.12 (2 H, m), 3.03 (1 H ₁ d, J = 14.2 Hz), 2.59 (2 H ₁ q, J = 7.6 Hz) ₁ 1.18 (3 H ₁ t, J = 7.6 Hz). Contains 0.7% of (±)-5-{4-[2-(5- ethylpyridin-2-yl)ethoxy]-benzyl}-1 ,3-thiazolidine-2,4-dione.

b. (5/7)-5-{4-[2-(5-Ethylpyridin-2-yl)ethoxy]benzyl}-(5- ² H)-1,3-thiazolidine-2 _l 4. dione (-)-O,O'-dibenzoyS-L-tartrate

L-DBTA

A suspension of the product from example 5a (10.03 g, 28.1 mmol) in MeOD (200 mL) was treated with DCI 35 wt% (2.5 mL) to give a solution to which was added (-)- dibenzoyl-L-tartaric acid (11 g, 30.7 mmol). The reaction was stirred at room temperature for 5 min until a solution was obtained and then D ₂ O (180 mL) was added dropwise. The resulting suspension was stirred at RT for 20 hours. The granular, crystalline solid was collected by filtration and dried at 40 ^° C under vacuum to give the title compound (6.3 g, 62.7%). (Method 2) 83.02%, Rt 10.34 min.; 16.98%, Rt 14.98 min.; 66.04% d.e. ¹ H NMR (400 MHz ₁ DMSO-c/ ₆ ): δ 11.98 (1 H, s), 8.37 (1 H, d, J = 2.2 Hz) ₁ 8.04-7.99 (4 H, m), 7.76-7.71 (2 H ₁ m), 7.62-7.55 (5 H ₁ m), 7.28 (1 H ₁ d, J = 7.9 Hz), 7.15-7.08 (2 H, m), 6.89-6.82 (2 H, m), 5.87 (2 H ₁ s), 4.30 (2 H, t, J = 6.7 Hz), 3.28 (1 H ₁ d, J = 14.2 Hz), 3.13 (2 H ₁ t, J = 6.7 Hz), 3.04 (1 H, d, J = 14.2 Hz), 2.59 (2 H, q, J = 7.6 Hz), 1.18 (3 H ₁ 1, J = 7.6 Hz). Contains 0.6% of (5R)-5-{4-[2- (5-ethylpyridin-2-yl)ethoxy]-benzyl}-1 ,3-thiazolidine-2,4-dione (-)-O.O'-dibenzoyl-L- tartrate

L=DBTA

To pre-mixed MeOD (63 ml) and DCI 35% (0.73 ml) was added the product from example 5b (6.3 g, 8.81 mmol). The solution was treated dropwise with D ₂ O (63 ml). The resulting suspension was stirred at RT for 72 hours. The granular, crystalline solid was collected by filtration and dried at 40 ^° C under vacuum to give the title compound (3.90 g, 62%). (Method 7) 95.38%, Rt 16.02 min.; 4.62%, Rt 17.85 min.; 90.76% d.e. ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 1198 (1 H, s), 8.37 (1 H, d, J = 2.2 Hz), 8.03-7.98 (4 H, m), 7.77-7.70 (2 H, m), 7.64-7.56 (5 H, m), 7.28 (1 H, d, J = 7.9 Hz), 7.13 (2 H ₁ d, J = 8.4 Hz), 6.86 (2 H, d, J = 8.4 Hz), 5.87 (2 H, s), 4.30 (2 H, t, J = 6.7 Hz), 3.28 (1 H, d, J = 14.2 Hz), 3.13 (2 H, t, J = 6.7 Hz), 3.04 (1 H, d, J = 14.2 Hz), 2.59 (2 H, q, J = 7.6 Hz), 1.18 (3 H ₁ t, J = 7.6 Hz). Contains 0.7% of (5R)-5-{4-[2-(5- ethylpyridin-2-yl)ethoxy]-benzyl}-1 ,3-thiazolidine-2,4-dione.

d. (5R)-5-{4-[2-(5-Ethylpyridin-2-yl)βthoxy]benzyl}-(5- ² H)-1,3-thiazolidinβ-2,' dione deuterochloride

The product from example 5c (3.82 g, 5.34 mmol) was dissolved in premixed MeOD (19 ml) and DCI 35% (1.34 ml) at -45 °C (internal temperature). Ethyl acetate (57 ml) was added and the reaction mixture was allowed to cool with stirring over 1 h. The solid was collected by filtration, washed with ethyl acetate (30 ml_), and dried for 18 h at 40 ⁰ C under vacuum to give the title compound (1.75 g, 83%). (Method 7) 97.54%, Rt 16.27 min.; 2.46%, Rt 18.19 min.; 95.08% e.e. LCMS (Method 6): Rt 2.86 min, m/z 358 [M+Hf. ¹ H NMR (400 MHz, DMSO-c/ ₆ ): δ 12.01 (1 H, s), 8.71 (1 H, d, J = 2.0 Hz), 8.37 (1 H, d, J = 8.3 Hz), 7.94 (1 H, d, J = 8.3 Hz), 7.13-7.15 (2 H, m), 6.88-6.85 (2 H, m), 4.38 (2 H ₁ t, J = 6.22 Hz), 3.45 (2 H, t, J = 6.37 Hz), 3.29 (1 H, d, J = 142 Hz), 3.05 (1 H, d, J = 14.2 Hz), 2.78 (2 H, q, J = 7.6 Hz), 1.24 (3 H, t, J = 7.6 Hz). Contains 0.7% of (5R)-5-{4-=[2-(5-ethyipyridin-2-y!)ethoxy]-benzyI}-1 ,3- fhiazoIidine-=2,4-d!one.

The title compound was prepared from 5-{4-[(methyI-pyridin-2-yI-amino)-ethoxy]- benzyi}-}-thiazolidine-2,4-dione using the methods described in example 1a and 1 b. ¹ H NMR (400 MHz, DMSOd ₆ ): δ 3.04 (1 H, d, J = 14.8Hz), 3.25 (3H , s), 3.27 (1 H, d, J = 14.8Hz), 4.01-4.07 (2H, m), 4.17-4.24 (2H, m), 6.82 (2H, d, J = 4Hz), 6.89 (1H, t, J = 4Hz), 7.14 (2H, d, J = 4Hz), 7.26 (1 H , br s), 7.93 (1 H, m), 8.00 (1 H, m), 11.99 (1 H, br s).

The title compound (572mg) was isolated by preparative chiral HPLC purification (Method 8) using the product of example 6. e.e. (Method 10) ≥ 99%, Rt 11.12 min. (Method 9) 100%, Rt 11.12 min; 100% e.e. Contains 5.8% (5R)-5-{4-[(methyl- pyridin-2-yi-amino)-ethoxy]-benzyl}-}-(5)-thiazolidine-2,4-d ione trifluoroacetate.

Example 8 (5R)-5-{4-[(Methyl-pyridin-2-yl-amino)-ethoxy]-bβnzyl}-H5- ² H)-thiazolidine-2,4-

The title compound was prepared from the product of example 7 using the methods described in example 1d. (Method 9) 96.97%, Rt 11.71 min; 3.03%, Rt 27.6min; 93.9% e.e. Contains 6.5% (5R)-5-{4-[(methyI-pyridin-2-yl-amino)-ethoxy]-benzyI}-}- (5)-thiazoIidine-2,4=dione hydrochloride.

The product from example 8 (278mg) was suspended in CH ₃ CN (1 mL) containing cHCl (1eq) and warmed to 3O ⁰ C. A clear solution formed followed by the formation of a precipitate. The flask was allowed to cool and 5mL CH ₃ CN and then 3mL Et ₂ O was added over 0.5h. The solid was collected after stirring 45min, washed with 5mL Et ₂ CVCH ₃ CN (2:1 ) and dried at 35 ⁰ C for 18 under vacuum to give title compound (223mg). (Method 9) 97.19%, Rt 11.92 min; 2.8%, Rt 28.4min; 94.4% e.e. (Method 10) >99%, Rt 5.51 min. e.e. [α] _D ²³ +116° (c 1.0, MeOH). ¹ H NMR (400 MHz, DMSO- Cf ₆ ): δ 3.04 (1 H, d, J = 14.8Hz), 3.25 (3H, s), 3.27 (1 H, d, J = 14.8Hz), 4.01-4.07 (2H, m), 4.17-4.24 (2H, m), 4.85 (0.07H, dd, J = 4Hz, 4Hz), 6.82 (2H, d, J = 4Hz), 6.89 (1 H, t, J = 4Hz), 7.14 (2H, d, J = 4Hz), 7.26 (1 H, br s), 7.93 (1 H, m), 8.00 (1 H, m), 11.99 (1 H, br s). Contains -7.0% (5R)-5-{4-[(methyl-pyridin-2-yl-amino)-ethoxy]- benzyl}-}-(5)-thiazolidine-2,4-dione.

Example 10

(5S)-5-{4-[(Methyl-pyrIdϊn-2-yi-amino)-ethoxy]-ben2yI}-} -(5- ² H)-thiazo!Idine-2,4- εϋlone trifluoroacetate

The title compound (445mg) was isolated by preparative chiral HPLC purification (Method 8) using the the product of example 6. (Method 9) 1.79%, Rt 11.92 min; 98.2%, Rt 24.15min; 96.4% e.e. Contains 6.0% (5S)-5-{4-[(methyI-pyridin-2-yl= amino)-ethoxy]-benzyi}-}-(5)-thiazolidine-2,4-d)one trifluoroacetate.

The title compound was prepared from the product of example 10 using the method described in example 1d. (Method 9) 5.1%, Rt 11.62 min; 94.9%, Rt 25.37; 89.8% e.e. Contains 7.6% (5S)-5-{4-[(methyl-pyridin-2-yl-amino)-ethoxy]-benzyl}-}-(5) - thiazoIidine-2,4-dione hydrochloride.

^■ pyridin-2-yl-ami Ie mono

The product from example 11 (235mg) was crystallised as described in example 9 to give the title compound (170mg). (Method 10) >99%, Rt 2.6 min. (Method 9) 1.56%, Rt 11.96 min; 98.44%, Rt 26.42min; e.e. 96.9%. ¹ H NMR (400 MHz, DMSOd ₆ ): δ 3.04 (1 H, d, J = 14.8Hz), 3.25 (3H, s), 3.27 (1 H, d, J = 14.8Hz), 4.01-4.07 (2H, m), 4.17-4.24 (2H, m), 4.85 (-0.09H, dd, J = 4Hz, 4Hz) ₁ 6.82 (2H, d, J = 4Hz), 6.89 (1 H, t, J = 4Hz) ₁ 7.14 (2H, d, J = 4Hz), 7.26 (1 H, br s), 7.93 (1 H, m), 8.00 (1 H, m), 11.99 (1 H ₁ br s). Contains 9.0% (5S)-5-{4-[(methyl-pyridin-2-yl-amino)-ethoxy]-benzyl}-}-(5) - thiazo!idine-2,4-dione hydrochloride. In Example 3, step c, acid was included in the MeOH solvent for recrystallisation of the R-enantiomer as the chira! salt. The following comparative Example shows that omission of the acid results in recovery of less than 1 :1 stoichiometry of the salt as 5 an enantiomeric mixture rather than the required R enantϊomer of the chiral salt with > 90% d.e.:

J ₀ L-DBTA

To pre-mixed MeOD (2 mL) and D ₂ O (100 uL) was added (5R)-5-{4-[2-(5- ethyIpyridin-2-yl)ethoxy]benzyl}-(5- ² H)-1 ₎ 3-thiazolidine-2 _I 4-dione ( ^» )-O,O'-dibenzoyl-L- tartrate (0.1 g, d.e. 58.9%) followed by dropwϊse addition of D ₂ O (2 mL) and the resulting suspension stirred at RT for 66 hours. The solid was collected by filtration,

15 washed with MeOD-D ₂ O (1 :2, 2 mL) and dried at 40 ^° C under vacuum to give the title compound (71 mg). (Method 11 ) 93.67%, Rt 9.83 min; 6.33% Rt 12.95 min.; 87.34% d.e. ¹ H NMR (400 MHz, DMSQ-d ₆ ): δ 11.93 (1 H, s), 8.32 (1 H, d, J = 2.3 Hz), 7.98- 7.95 (3.2 H, m), 7.70-7.67 (1.67 H, m), 7.58-7.51 (4.3 H, m), 7.23 (1 H, d, J = 7.9 Hz), 7.10-7.06 (2 H, m), 6.85-6.79 (2 H, m), 5.82 (2 H, s), 4.25 (2 H, t, J - 6.7 Hz), 3.22 (1 0 H, m), 3.08 (2 H ₁ t, J = 6.7 Hz), 2.99 (1 H, d, J = 14.2 Hz), 2.54 (2 H, q, J = 7.6 Hz), 1.13 (3 H, t, J = 7.6 Hz). Base:Salt ratio = 1 :0.8. Contains 2.4% of (5R)-5-{4-[2-(5= ethylpyridin-2-yl)ethoxy]-benzyl}-1 ,3-thiazolidine-2,4-dione(-)-0 _> 0'-dibenzoyl-L- tartrate. 5 Comparative Example 14 in Example 13, MeOD-D ₂ O was used as solvent for recrystallisation of the R- enantiomer as the chiral salt. The following comparative Example is an example run in parallel to Example 13 and shows that addition of acid results in recovery of required R enantiomer of the chiral salt, in 1 :1 stoichiometry, with enhanced d.e.

30 compared to Example 13:

(5R)-5-{4-[2-(5-Ethylpyridin-2-yl)βthoxy]benzyl}-(5- ² H)-1,3-thiazolidine-2,4-dione (-)-O,O'-dibenzoyl-L-tartrate

L-DBTA

To pre-msxed MeOD (2 mL) and DCI (100 uL of 35% solution in D ₂ O) was added (5f?)-5-{4-[2-(5-ethyIpyπdιn-2-yI)ethoxy]benzyIH5- ² H)-1 ,3-thιazolid[ne-2,4=dιone (-)- 0,0'-dιbenzoyl-L-tartrate (0 1 g, d e 58 9%) to give a clear solution D ₂ O (2 mL) was added dropwise and the resulting suspension stirred at RT for 66 hours The granular solid was collected by filtration, washed with MeOD-D ₂ O (1 2, 2 mL) and dried at 40 ^° C under vacuum to give the title compound (23 mg) (Method 11 ) 96 38%, Rt 10 08 mm, 3 61% Rt 13 54 mm , 92 77% d e ¹ H NMR (400 MHz, DMSO-cfe) δ 11 93 (1 H, s), 8 32 (1 H, d, J = 2 3 Hz), 7 98-7 95 (4 H, m), 7 70-7 67 (2 H, m), 7 58-7 51 (5 H, m), 7 23 (1 H, d, J = 7 9 Hz), 7 10-7 06 (2 H, m), 6 85-6 79 (2 H, m), 5 82 (2 H, s), 4 25 (2 H, t, J = 6 7 Hz), 3 22 (1 H, m), 3 08 (2 H, t, J = 6 7 Hz), 2 99 (1 H, d, J = 14 2 Hz), 2 54 (2 H, q, J = 7 6 Hz), 1 13 (3 H, t, J = 7 6 Hz) Base Salt ratio = 1 1 06 Contains 2 4% of (5R)-5-{4-[2-(5-ethylpyrιdιn-2-yl)ethoxy]-benzyl}-1 ,3-thιazolιdιne- 2,4-dιone(-)-O,O'=dibenzoyI-L-tartrate

In Example 13, MeOD-D ₂ O was used as solvent for recrystallisation of the R- enantiomer as the chiral salt The following comparative Example shows that stirring in MeOD results in recovery of the free base as an enantiomeric mixture rather than the required R enantiomer of the chiral salt with enhanced d e

(5f?)-5-{4-[2-(5-Ethylpyridin-2-yl)ethoxy]bβnzyl}-(5- ² H)-1,3-thiazolidine-2,4'dionβ

To MeOD (2 mL) was added (5R)-5-{4-[2-(5-ethylpyπdιn-2-yl)ethoxy]benzyl}-(5- ² H)- 1 ,3-thiazolidine-2,4-dione (-)-0,0'-dibenzoyl-L-tartrate (0 1 g, d e 58 9%) to give a suspension which was stirred at RT for 66 hours The solid was collected by filtration, washed with MeOD-D ₂ O (1 2, 2 mL) and dried at 40 ^° C under vacuum to give the title compound (38 mg) (Method 11 ) 76 17%, Rt 9 59 mm, 23 82% Rt 13 36 mm ,

52 35% d e ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8 32 (1 H, d, J = 2 3 Hz), 7 58-7 51 (1 H, m), 7 23 (1 H, d, J = 7 9 Hz), 7 10-7 06 (2 H, m), 6 85-6 79 (2 H, m), 4 25 (2 H, t, J

= 6 7 Hz), 3 22 (1 H, m), 3 08 (2 H, t, J = 6 7 Hz), 2 99 (1 H, d, J = 14 2 Hz) ₁ 2 54 (2

H, q, J = 7 6 Hz), 1 13 (3 H, t, J = 7 6 Hz) Base Salt ratio = 1 0 Biological Results:

Pre-clinical mouse model of COPD inflammation - Tobacco smoke induced pulmonary inflammation.

Previous studies have established that the number of inflammatory cells recovered in the bronchoalveolar lavage (BAL) is significantly elevated 24 h following the fina! Tobacco Smoke (TS) exposure of 4 or 11 consecutive daily TS exposures, this time point was used in the study reported here.

Protocols for the exposure of mice to TS, obtaining bronchoalveolar lavage (BAL), preparation of cytospin slides for differential ceil counts are as outlined below.

Exposure of mice to TS daily for 4 or 11 consecutive days

In this exposure protocol, mice were exposed in groups of 5 in individual clear polycarbonate chambers (27 cm x 16 cm x 12 cm). The TS from the cigarettes was allowed to enter the exposure chambers at a flow rate of 100 ml/min. In order to minimise any potential problems caused by repeated exposure to a high level of TS (6 cigarettes), the exposure of the mice to TS was increased gradually over the exposure period to a maximum of 6 cigarettes. The exposure schedule used for 4 days was as follows: Day 1 : 4 cigarettes (approximately 32 min exposure)

Day 2: 4 cigarettes (approximately 32 msn exposure)

Day 3: 6 cigarettes (approximately 48 min exposure)

Day 4: 6 cigarettes (approximately 48 min exposure)

The exposure schedule used for 11 days exposure was as follows:

Day 1 : 2 cigarettes (approximately 16 min exposure)

Day 2: 3 cigarettes (approximately 24 min exposure)

Day 3: 4 cigarettes (approximately 32 min exposure) Day 4: 5 cigarettes (approximately 40 min exposure)

Day 5 to 11 : 6 cigarettes (approximately 48 min exposure)

A further group of mice were exposed to air on a daily basis for equivalent lengths of time as controls (no TS exposure).

Bronchoalveolar lavage (BAL) analysis Bronchoalveolar lavage was performed as follows: the trachea was cannulated using a Portex nylon intravenous cannula (pink luer fitting) shortened to approximately 8 mrn. Phosphate buffered saline (PBS) was used as the lavage fluid. A volume of 0.4 ml was gently instilled and withdrawn 3 times using a 1 ml syringe and then placed in an Eppendorf tube and kept on ice prior to subsequent determinations.

Cell counts:

Lavage fluid was separated from cells by centrifugation and the supernatant decanted and frozen for subsequent analysis. The cell pellet was re-suspended in a known volume of PBS and total eel! numbers calculated by counting a stained (Turks stain) aliquot under a microscope using a haemocytometer.

Differential cell counts were performed as follows:

The residual cell pellet was diluted to approximately 10 ⁵ cells per ml. A volume of 500 μl was placed in the funnel of a cytospin slide and centrifuged for 8 min at 800 rpm. The slide was air dried and stained using 'Kwik-DifP solutions (Shandon) as per the proprietary instructions. When dried and cover-sϋpped, differential cells were counted using light microscopy. Up to 400 cells were counted by unbiased operator using light microscopy. Cells were differentiated using standard morphometric techniques.

Drug Treatment

Rodents such as mice and rats are obligate nasal breathers thus oral delivery of test materials (such as therapeutic agents) for inhalation will not produce good lung exposure. As a consequence, delivery of therapeutic agents to the lungs in rodents is generally achieved by intra-nasal, intra-tracheal or inhalation by whole body aerosol exposure in a chamber.

The chamber method utilises large amounts of test material and is generally reserved for inhalation toxicology studies rather than pharmacological efficacy studies. Intra- tracheal administration is a very efficient delivery method as almost all of the test material is delivered to the lungs, but this is quite an invasive technique. For studies in the mouse particularly, it is also quite technically demanding as the diameter of the trachea is quite small. The intranasal route is less invasive than the intra-tracheal route and so is particularly suitable for repeat dosing studies such as the 4-11 day mouse model described below. Following intranasal administration ~50% of the dose administered is delivered to the lungs (Eyles JE, Williamson ED and Alpar HO. 1999, In! J Pharm, 189(1):75-9).

As a surrogate route for oral inhalation, mice were dosed intra-nasally with vehicle (0.2% tween 80 in saline), 5S=5-deuterated pioglitazone (as prepared in Example 1d) (3 μg/kg), 5S-5-deuterated pioglitazone (as prepared in Example 1d) (1 μg/kg), 5R-5- deuterated pioglitazone (as prepared in Example 2b) (3 μg/kg) or 5R- deuterated pioglitazone (as prepared in Example 2b) (1 μg/kg). The control group of mice received vehicle 1 hr prior to being exposed to air daily for a maximum of 50 minutes per day. Following 4 days of TS exposure, BAL was performed 24 h following the final TS exposure. All compounds were dosed as the HCI salt with doses corrected as base. The compounds were micronised prior to use in this study. The particle size distribution of the compounds achieved foilowing micronϊsation was suitable for inhalation.

In a second experiment, mice were dosed intra-nasally with vehicle (0.2% tween 80 in saline), 5S-5-deuterated rosiglitazone as prepared in Example 12 (3 μg/kg), 5S-5- deuterated rosiglitazone as prepared in Example 12 (10 μg/kg), 5R-5-deuterated rosiglitazone as prepared in Example 9 (3 μg/kg), 5R=5-deuterated rosiglitazone as prepared in Example 9 (10 μg/kg) or racemic 5-deuterated rosiglitazone (10 μg/kg). The control group of mice received vehicle 1 hr prior to being exposed to air daily for a maximum of 50 minutes per day. Following 4 days of TS exposure, BAL was performed 24 h following the final TS exposure. All compounds were dosed as the HCi salt with doses corrected as base. The compounds were micronised prior to use in this study. The particle size distribution of the compounds achieved following micronisation was suitable for inhalation.

Data management and statistical analysis All results are presented as individual data points for each animal and the mean value was calculated for each group. Since tests for normality were positive the data was subjected to a one way analysis of variance test (ANOVA), followed by a Bonferroni correction for multiple comparisons in order to test for significance between treatment groups. A "p" value of < 0.05 was considered to be statistically significant. Percentage inhibitions were automatically calculated within the Excel spreadsheets for the cell data using the formula below: .. , . .. ... . f Treatment group result - sham group result \ . _nn

% Inhibition = 1 - x 100 ^ TS vehicle group result - sham group result J

Inhibition data for other parameters were calculated manually using the above formula.

As illustrated in Figure 1 , there was a clear difference in activity between the two enantiomers of 5-deuterated pioglitazone on total cell BAL numbers following exposure to TS. The 5R enantiomer (e.e. 98.5%) of 5-deuterated pioglitazone significantly inhibited the BAL cell influx induced by TS at both 1 and 3 μg/kg when administered by the intranasal route, in contrast, the 5S-enantiomer (e.e. 98.7%) of 5-deuterated pioglitazone failed to inhibit the BAL cell inflammation at either dose examined.

Foliowing examination of the BAL cell cytospins, BAL neutrophil numbers were determined. In concert with the activity on total BAL cells, the 5R enantiomer of 5= deuterated pioglitazone significantly inhibited BAL neutrophil numbers induced by TS exposure at both doses whereas the 5S enantiomer of 5-deuterated pioglifazone was ineffective (Figure 2).

As iilustrated in Figure 3, there was a clear difference in activity between the two enantiomers of 5-deuterated Rosiglitazone on total cell BAL numbers following exposure to TS. The 5R enantiomer (e.e. 94.4%) of 5-deuterated rosiglitazone significantly inhibited the BAL cell influx induced by TS at both 3 and 10 μg/kg when administered by the intranasal route by more than 50% (66% with 3 μg/kg and 59% with 10 μg/kg). In contrast, the 5S-enantiomer (e.e. 96.9%) of 5-deuterated rosiglitazone failed to inhibit the BAL cell inflammation at 10 μg/kg and had a small effect at 3 μg/kg (24%).

Following examination of the BAL cell cytospins, BAL neutrophil numbers were determined. In concert with the activity on total BAL cells, the 5R enantiomer of 5- deuterated rosiglitazone significantly inhibited BAL neutrophil numbers induced by TS exposure at both doses whereas the 5S enantiomer of 5-deuterated rosiglitazone was ineffective (Figure 4). Racemic 5-deuterated rosiglϊtazone (which contains 50% 5R enantiomer of 5- deuterated rosϊglitazone) at the 10 μg/kg dose also significantly inhibited total and neutrophil BAL cells induced by TS.

Taken together, the results of the two studies identify the 5R enantiomer of both 5= deuterated Pioglitazone and 5-deuterated Rosiglitazone as possessing the anti- inflammatory activity required for inhibition of BAL cell influx whilst the 5S enantiomer does not.

Whilst the e.e. of the preparation of the 5R enantiomer of 5-deuterated Rosiglitazone (Example 9) was high (94.4%), it also contained some protonated 5R enantiomer. In total, the preparation contained 91% 5R enantiomer of 5-deuterated Rosiglitazone by weight. The preparation of the 5S enantiomer of 5-deuterated Rosiglϊtazone (Example 12) contained 90% 5S enantiomer by weight. As significant activity of the 5R enantiomer was observed at both doses in the TS model with minimal activity observed with the 5S enantiomer, this suggests that an optimal preparation of 5R enantiomer of 5-deuterated Rosiglitazone (>95% by weight, preferably >97% and most preferably >99% by weight) would show similar or even improved activity compared with the data presented herein. Therefore the data presented herein is indicative of what would be achieved with a preparation that contains by weight at least 95% 5R enantiomer of 5-deuterated Rosiglitazone.

Preparations of the 5S enantiomer or 5R enantiomer of 5-deuterated Pioglitazone contained >95% by weight of the appropriate 5-deuterated Pioglitazone enantiomer.

Example 17

Pharmacokinetic analysis of blood 5R and 5S pioglitazone following intra-tracheal administration of 5R-protonated or 5R-5-deuterated pioglitazone to male SD rats.

Male Sprague Dawley rats (n = 5/time point) were administered 5R-protonated or 5R- 5-deuterated pioglitazone at a nominal dose of 30μg/kg by the intra-tracheal (i.t.) route. Blood samples were taken at 0.25, 0.5, 1 , 2, 4, 8 and 24 hours following administration. Blood samples were thawed and kept on ice. The samples were vortex mixed thoroughly and a 40μL aliquot was transferred to an eppendorf tube containing 80μL ice cold 0.5% formic acid in acetonitrile, containing diazepam internal standard (at a concentration of 500 ng/mL).. Following vortex mixing and centrϊfugation (13,000 rpm, 4°C for 20 mϊns), a 20μL aliquot of supernatant was transferred to a weil of a 96-well midi-eppendorf plate and diluted by addition of 180 μl_ 0.5% formic acid in methanol. The plate was sealed and shaken on a plate shaker to ensure sample homogeneity. The extracts were assayed by chiral LC-MS/MS to determine the enantiomeric ratio. The samples were then assayed for total protonated pioglitazone or 5-deuterated pioglitazone by LC-MS/MS to allow determination of the amount of each enantiomer present.

As expected, rapid racemisation occurred in the blood following i.t. administration of 5R-protonated pϊogiitazone with both enantiomers present in equal amounts at around 1 hr (Figure 5). Published literature indicates that in rodents the S enantiomer for several glitazones predominates in the blood at equilibrium (R:S ratio of -40:60 rather than 50:50) and this was observed in this experiment where the amount of 5S-pioglitazone reached a peak at 4 hours and then declined as both enantiomers were cleared from the blood.

In contrast to protonated 5R-pioglitazone, significant racemisation did not occur in the blood following i.t. administration of 5R-5-deuterated pioglitazone. As illustrated in Figure 6, the amount of 5R-5-deuterated pioglitazone steady declined over 8 hours as it was cleared from the blood whilst the amount of 5S-5-deuterated pioglitazone remained low (<3 ng/ml) at all time points. Figure 7 illustrates this significant difference in the amount of 5S pioglitazone enantiomer present in the blood following i.t. administration of the respective 5R enantiomer by plotting the respective amounts of 5S enantiomer determined.

As the 5S enantiomer is believed to be largely responsible for the clinical systemic side effects of the glitazones these data suggest that inhalation of the 5R-5- deuterated glitazone enantiomer (such as 5R-5-deuterated pioglitazone) will lead to significantly lower amounts of the 5S enantiomer present systemically than would be produced following inhalation of the 5R protonated glitazone enantiomer or the racemate. in turn this would be predicted to result in an improved therapeutic index to patients due to reduced side effects, and allow higher dosages of the 5R-5- deuterated glitazone enantiomer to be administered compared with either the 5R protonated glitazone enantiomer or the glitazone racemate without increasing the systemic side effects mediated by the 5S enantiomer.