CA2794066A1 - Galactoside inhibitor of galectins - Google Patents

Abstract

The present invention relates to a compound of the general formula (I):
(see formula I) The compound of formula (I) is suitable for treating pulmonary fibrosis, such as Idiopathic pulmonary fibrosis in a mammal.

Description

NOVEL GALACTOSIDE INHIBITOR OF GALECTINS
Technical field The present invention relates to novel compounds, the use of said compounds as medicament and for the manufacture of a medicament for the treatment of pulmonary fibrosis, such as Idiopathic pulmonary fibrosis in mammals. The invention also relates to pharmaceutical compositions com-prising said novel compounds.
Background Art Idiopathic pulmonary fibrosis (IPF) represents a massive worldwide health burden. It is a chronic condition of unknown etiology in which repeated acute lung injury causes progressive fibrosis resulting in destruction of lung architecture, deteriorating lung function with consequent respiratory failure and death. Although idiopathic pulmonary fibrosis (IPF) is the archetypal and most common cause of lung fibrosis, numerous respiratory diseases can pro-gress to pulmonary fibrosis, and this usually signifies a worse prognosis. The median time to death from diagnosis is 2.5 years and the incidence and prev-alence of IPF continues to rise. It remains one of the few respiratory condi-tions for which there are no effective therapies, and there are no reliable bio-markers to predict disease progression. The mechanisms resulting in pulmo-nary fibrosis are unclear but centre around aberrant wound healing as a con-sequence of repetitive epithelial injury from an as yet unknown cause. IPF is characterized by fibroblastic foci containing fibroblasts/ myofibroblasts which show increased activation response to fibrogenic cytokines such as trans-forming growth factor-al (TGF-a1). Given the non-responsiveness of many cases of IPF to current anti-inflammatory treatments the myofibroblasts within fibroblastic foci represent a potential novel therapeutic target.
The bleomycin model of pulmonary fibrosis is the best characterised rodent model and is the industry standard model. It causes oxidant-mediated DNA damage and induces initial lung inflammation followed by progressive fibrosis over 2 ¨ 4 weeks. When administered during the later phase of the

2 injury the anti-fibrotic potential of novel compounds can be assessed.
There is a big unmet need for drugs for treatment of Idiopathic pulmo-nary fibrosis.
Galectin inhibitors, in particular Gal-3 inhibitors have been described by the some of the present inventors in earlier published patent applications.
None of these galectin inhibitors have been tested in a bleomycin model.
Some of the prior art galectin inhibitors have the following general formulas HO OH
..&...\____ R11_ RI
RvO Rv 0 -,Z ORvii RH' µRiv as described in WO/2005/113568, and Rii-Y HO OH
N Nõ X, 'N HO RI
as described in WO/2005/113569, in which RI can be a D-galactose, and OH OH
_y_......Ø..\__x Z
RI' as described in WO/2010/126435.
Summary of the invention Galectin-3 is a 8-galactoside binding lectin that is highly expressed in fibrotic tissue of diverse etiologies. The present inventors have examined the role of galectin-3 in bleomycin and TGF-81-induced lung fibrosis in mice, and have established its relevance in human IPF. In particular it is shown that galectin-3 inhibition may represent a novel therapeutic strategy for treatment of

3 lung fibrosis. A novel compound has been tested and shown to be an inhibitor of galectin-3, in particular the compound of the present invention blocked TGF-P-induced p-catenin activation in vitro and attenuated the late stage pro-gression of lung fibrosis following bleomycin in vivo.
Accordingly, the present invention relates to a compound of the gen-eral formula (I):
HO OH
N I, OH
N
HO1-1C) N OH
N
N
F
(I).
In a further aspect the present invention relates to a pharmaceutical composition comprising the compound of formula (I) and optionally a pharma-ceutically acceptable additive, such as carrier or excipient.
The compound of formula (I) of the present invention is suitable for use in a method for treating pulmonary fibrosis, such as Idiopathic pulmonary fi-brosis in a mammal. Typically, such mammal is a human subject.
Moreover, in a still further aspect the present invention relates to a method for treatment of pulmonary fibrosis, such as Idiopathic pulmonary fi-brosis comprising administering to a mammal a therapeutically effective amount of the compound of formula (I).
In another aspect the present invention concerns a process of prepar-ing a compound of formula I comprising the step of reacting bis-(3-deoxy-3-azido-p-D-galactopyranosyl) sulfane with 3-fluorophenylacetylene and an amine, such as triethylamine, optionally in the presence of a catalyst, such as Cu(I), in a solvent, such as N,N-dimethylformamide (DMF), resulting in the compound of formula I.

4 Detailed description of the invention In a broad aspect the present invention relates to a compound of the general formula (I):

'NJ S 0 HO 1-1(31'1.14( N OH
N
N
F
0) The compound of formula (I) has the chemical name (IUPAC) bis (3-deoxy-3-(3-fluoropheny1-1H-1,2,3-triazol-1-y1)-(3-D-galactopyranosyl) sulfane, and as used herein is intended to cover the compound of formula (I) in any possible form, such as solid or liquid, a salt, a solvate, or in free form.
Typically, the compound of formula (I) is bis (3-deoxy-3-(3-fluoropheny1-1H-1,2,3-triazol-1-y1)-(3-D-galactopyranosyl) sulfane as the free form. In a further embodiment the compound of formula (I) is bis (3-deoxy-3-(3-fluoropheny1-1H-1,2,3-triazol-1-y1)43-D-galactopyranosyl) sulfane as the free form without any solvate, such as anhydrated.
In a still further embodiment the compound of formula (I) is useful for treating pulmonary fibrosis, and therefore is suitable for use as a medicament.
In a further aspect the present invention relates to a compound of for-mula (I) for use in a method for treating pulmonary fibrosis, such as Idiopathic pulmonary fibrosis in a mammal. Such a mammal is typically a human sub-ject, preferably a human subject diagnosed with IPF.
In a still further aspect the present invention relates to a method for treatment of pulmonary fibrosis, such as Idiopathic pulmonary fibrosis com-prising administering to a mammal a therapeutically effective amount of a compound of formula (I).

When the compounds and pharmaceutical compositions herein dis-closed are used for the above treatment, a therapeutically effective amount of at least one compound is administered to a mammal in need of said treat-ment.

5 The term "treatment" and "treating" as used herein means the man-agement and care of a patient for the purpose of combating a condition, such as a disease or a disorder. The term is intended to include the full spectrum of treatments for a given condition from which the patient is suffering, such as administration of the active compound to alleviate the symptoms or complica-tions, to delay the progression of the disease, disorder or condition, to allevi-ate or relief the symptoms and complications, and/or to cure or eliminate the disease, disorder or condition as well as to prevent the condition, wherein prevention is to be understood as the management and care of a patient for the purpose of combating the disease, condition, or disorder and includes the administration of the active compounds to prevent the onset of the symptoms or complications. The treatment may either be performed in an acute or in a chronic way. The patient to be treated is preferably a mammal; in particular a human being, but it may also include animals, such as dogs, cats, cows, sheep and pigs.
The term "a therapeutically effective amount" of a compound of formula (I) of the present invention as used herein means an amount sufficient to cure, alleviate or partially arrest the clinical manifestations of a given disease and its complications. An amount adequate to accomplish this is defined as "therapeutically effective amount". Effective amounts for each purpose will depend on the severity of the disease or injury as well as the weight and gen-eral state of the subject. It will be understood that determining an appropriate dosage may be achieved using routine experimentation, by constructing a matrix of values and testing different points in the matrix, which is all within the ordinary skills of a trained physician or veterinary.
In a still further aspect the present invention relates to a pharmaceuti-cal composition comprising the compound of formula (I) and optionally a pharmaceutically acceptable additive, such as a carrier or an excipient.

6 As used herein "pharmaceutically acceptable additive" is intended without limitation to include carriers, excipients, diluents, adjuvans, colour-ings, aroma, preservatives etc. that the skilled person would consider using when formulating a compound of the present invention in order to make a pharmaceutical composition.
The adjuvants, diluents, excipients and/or carriers that may be used in the composition of the invention must be pharmaceutically acceptable in the sense of being compatible with the compound of formula (I) and the other in-gredients of the pharmaceutical composition, and not deleterious to the re-cipient thereof. It is preferred that the compositions shall not contain any ma-terial that may cause an adverse reaction, such as an allergic reaction. The adjuvants, diluents, excipients and carriers that may be used in the pharma-ceutical composition of the invention are well known to a person within the art.
As mentioned above, the pharmaceutical compositions as herein dis-closed may, in addition to the compounds herein disclosed, further comprise at least one pharmaceutically acceptable adjuvant, diluent, excipient and/or carrier. In some embodiments, the pharmaceutical compositions comprise from 1 to 99 weight % of said at least one pharmaceutically acceptable adju-vant, diluent, excipient and/or carrier and from 1 to 99 weight % of a corn-pound as herein disclosed. The combined amount of the active ingredient and of the pharmaceutically acceptable adjuvant, diluent, excipient and/or carrier may not constitute more than 100 A by weight of the pharmaceutical compo-sition.
In some embodiments only one compound as herein disclosed is used for the purposes discussed above.
In some embodiments two or more of the compound as herein dis-closed are used in combination for the purposes discussed above.
The pharmaceutical composition according to the present invention comprising a compound of the invention may be adapted for oral, intrave-nous, topical, intraperitoneal, nasal, buccal, sublingual, or subcutaneous ad-ministration, or for administration via the respiratory tract in the form of, for example, an aerosol or an air-suspended fine powder. Therefore, the phar-maceutical composition of the present invention may be in the form of, for

7 example, tablets, capsules, powders, nanoparticles, crystals, amorphous substances, solutions, transdermal patches or suppositories.
Thus, in a still further aspect the present invention relates to a pharma-ceutical composition for intrapulmonary administration. Typically, such com-position is delivered by a nebulizer or inhaler, preferably a nebulizer.
The following characteristics are required for the delivery device: It should be able to provide a specific dose accurately and repeatedly. It should be able to provide 2 or more different dose levels, for instance through repeated dosing or by adjusting the dose provide to the patient. The device should ensure that the drug is delivered to the bronchiolar space or preferably to the bronchiolar and the alveolar space of the lung preferably uniformly over the lung tissue.
Hence, the device should generate aerosols or dry powder of an adequately small size to ensure this delivery.
Inhalation nebulizers deliver therapeutically effective amounts of phar-maceuticals by forming an aerosol which includes particles of a size that can easily be inhaled. The aerosol can be used, for example, by a patient within the bounds of an inhalation therapy, whereby the therapeutically effective pharmaceutical or drug reaches the patient's respiratory tract upon inhalation.
A variety of inhalation nebulizers are known. EP 0 170 715 Al uses a compressed gas flow to form an aerosol. A nozzle is arranged as an aerosol generator in an atomizer chamber of the inhalation nebulizer and has two suction ducts arranged adjacent a compressed-gas channel. When com-pressed air flows through the compressed-gas channel, the liquid to be nebu-lized is drawn in through the suction ducts from a liquid storage container.
EP 0 432 992 A discloses a nebulizer comprising an aerosol generator having a liquid storage container, a perforate mebrane and a vibrator. The vibrator is operable to vibrate the membrane such that it dispenses an aerosol from a liquid through holes provided in the membrane.
US 5.918.593 relates to ultrasonic nebulizers generating an aerosol by interaction between an amount of liquid and a piezo electric element. Droplets of various sizes are expelled from a surface of a liquid bulk when vibrational energy is transferred from the piezo element to the liquid. The droplets thus generated are filtered in an atomizer chamber since oversized droplets have

8 to be removed from the droplets expelled from the surface in order to gener-ate an aerosol for inhalation by a patient. This nebulizer is representative of continuously operating inhalation nebulizers, in which the aerosol generator produces an aerosol not only during inhalation but also while the patient ex-hales. The aerosol produced by the aerosol generator is actually inhaled by the patient only in the inhalation phase, while any aerosol produced at other times is lost.
Dry powder inhalers, such as metered dose medicament inhalers are well known for dispensing medicament to the lungs of a patient. Some previ-ous inhalers have comprised a pressurized aerosol dispensing container, wherein the aerosols contain gas propellants in which the powdered medica-ment is suspended. Upon actuation, the aerosol contents are expelled, through a metering valve, and into the lungs of the patient.
Several types of non-aerosol, breath actuated dry powder inhalers have therefore been provided. For example, U.S. Patent No. 5,503,144 to Baconõ shows a breath-actuated dry-powder inhaler. The device includes a dry powder reservoir for containing a dry powdered medicament, a metering chamber for removal of the powdered medicament from the reservoir in dis-crete amounts, and an air inlet for entraining the removed powdered medica-ment through a mouth piece upon patient inhalation.
US5458135 discloses a method and apparatus for producing an aero-solised dose of a medicament for subsequent inhalation by a patient. The method comprises first dispersing a preselected amount of the medicament in a predetermined volume of gas, usually air. The dispersion may be formed from a liquid or a dry powder. The method relies on flowing substantially the entire aerosolised dose into a chamber which is initially filled with air and open through a mouthpiece to the ambient. After the aerosolised medicament has been transferred to the chamber, the patient will inhale the entire dose in a single breath.
US 6 065 472 discloses a powder inhalation device comprising a hous-ing containing a pharmacologically active compound, a conduit with an outlet extending into the housing through which a user can inhale to create an air-flow through the conduit, a dosing unit for delivering a dose of the compound

9 to the conduit and baffles arranged within the said conduit to aid disintegra-tion of powder agglomerates entrained in said airflow.
Regardless of whether an aerosol or non-aerosol inhaler is used, it is of utmost importance that particles of the dispensed dry powder medicament be small enough to ensure the adequate penetration of the medicament into the bronchial region of a patient's lungs during inhalation. However, because the dry powder medicament is composed of very small particles, and often provided in a composition including a carrier such as lactose, non-defined agglomerates or aggregates of the medicament form at random prior to being dispensed. It has therefore been found preferably to provide breath-actuated dry powder inhalers with means for breaking down the agglomerates of me-dicament or medicament and carrier before inhalation of the medicament.
The pharmaceutical composition of the present invention may option-ally comprise two or more compounds of the present invention. The composi-tion may also be used together with other medicaments within the art for the treatment of related disorders.
The typical dosages of the compounds of the present invention vary within a wide range and depend on many factors, such as the route of ad-ministration, the requirement of the individual in need of treatment, the indi-vidual's body weight, age and general condition.
The compound of formula (I) may be prepared as described in the ex-perimental section below.
Accordingly, the present invention relates to a process of preparing a compound of formula I comprising the step of reacting bis-(3-deoxy-3-azido-p-D-galactopyranosyl) sulfane with 3-fluorophenylacetylene and an amine, such as triethylamine, optionally in the presence of a catalyst, such as Cu(I), in a solvent, such as N,N-dimethylformamide (DMF), resulting in the com-pound of formula I. In a particular embodiment, the present invention relates to a process of preparing a compound of formula I comprising the steps as described in the scheme 1 in the experimental section. Moreover, the present invention relates to a compound of formula (I) obtainable by the step of react-ing bis-(3-deoxy-3-azido-3-D-galactopyranosyl) sulfane with 3-fluorophenylacetylene and an amine, such as triethylamine, optionally in the presence of a catalyst, such as Cu(I), in a solvent, such as N,N-dimethylformamide (DMF), resulting in the compound of formula I, such as obtainable by the steps as described in the scheme 1 in the experimental sec-tion.
5 Further embodiments of the process of the present invention are de-scribed in the experimental section herein, and each individual process as well as each starting material constitutes embodiments that may form part of claims.
The above embodiments should be seen as referring to any one of the

10 aspects (such as 'method for treatment', 'pharmaceutical composition', 'com-pound for use as a medicament', or 'compound for use in a method') described herein as well as any one of the embodiments described herein unless it is specified that an embodiment relates to a certain aspect or aspects of the pre-sent invention.
All references, including publications, patent applications and patents, cited herein are hereby incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.
All headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way.
Any combination of the above-described elements in all possible varia-tions thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
The terms "a" and "an" and "the" and similar referents as used in the context of describing the invention are to be construed to cover both the sin-gular and the plural, unless otherwise indicated herein or clearly contradicted by context.
Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless other-wise indicated herein, and each separate value is in-corporated into the specification as if it were individually recited herein.
Un-less otherwise stated, all exact values provided herein are representative of corresponding approximate values (e.g., all exact exemplary values provided

11 with respect to a particular factor or measurement can be considered to also pro-vide a corresponding approximate measurement, modified by "about,"
where appropriate).
All methods described herein can be performed in any suitable order unless other-wise indicated herein or otherwise clearly contradicted by con-text.
The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise indi-cated. No language in the specification should be construed as indicating any element is essential to the practice of the invention unless as much is explic-itly stated.
The citation and incorporation of patent documents herein is done for convenience only and does not reflect any view of the validity, patentability and/or enforceability of such patent documents.
The description herein of any aspect or embodiment of the invention using terms such as "comprising", "having", "including" or "containing" with reference to an element or elements is intended to provide support for a simi-lar aspect or embodiment of the invention that "consists of', "consists essen-tially of', or "substantially comprises" that particular element or elements, un-less otherwise stated or clearly contradicted by context (e.g., a composition described herein as comprising a particular element should be understood as also describing a composition consisting of that element, unless otherwise stated or clearly contradicted by context).
This invention includes all modifications and equivalents of the subject matter recited in the aspects or claims presented herein to the maximum ex-tent permitted by applicable law.
The present invention is further illustrated by the following examples which, however, are not to be construed as limiting the scope of protection.
The features disclosed in the foregoing description and in the following exam-ples may, both separately and in any combination thereof, be material for re-alizing the invention in diverse forms thereof.

12 Experimental Synthesis of bis (3-deoxy-3-(3-fluoropheny1-1H-1,2,3-triazol-1-y1)-13-D-galactopyranosyl) sulfane.
General Methods.
Melting points were recorded on a Kofler apparatus (Reichert) and are uncorrected. Proton nuclear magnetic resonance (1H) spectra were recorded on a Bruker DRX 400 (400 MHz) or a Bruker ARX 300 (300 MHz) spectrome-ter; multiplicities are quoted as singlet (s), doublet (d), doublet of doublets (dd), triplet (t), apparent triplet (at) or apparent triplet of doublets (atd). Carbon nuclear magnetic resonance (13C) spectra were recorded on a Bruker DRX
400 (100.6 MHz) spectrometer. Spectra were assigned using COSY, HMQC
and DEPT experiments. All chemical shifts are quoted on the 6-scale in parts per million (ppm). Low- and high-resolution (FAB-HRMS) fast atom bom-bardment mass spectra were recorded using a JEOL SX-120 instrument and low- and high- resolution (ES-HRMS) were recorded on a Micromass Q-TOF
instrument. Optical rotations were measured on a Perkin-Elmer 341 polarime-ter with a path length of 1 dm; concentrations are given in g per 100 mL. Thin layer chromatography (TLC) was carried out on Merck Kieselgel sheets, pre-coated with 60F254 silica. Plates were developed using 10% sulfuric acid.
Flash column chromatography was carried out on silica (Matrex, 60A, 35-70pm, Grace Amicon). Acetonitrile was distilled from calcium hydride and stored over 4A molecular sieves. DMF was distilled from 4A molecular sieves and stored over 4A molecular sieves.
Bis (3-deoxy-3-(3-fl uorophenyl-1 H-1,2,3-triazol-1 -yI)-p-D-galactopyranosyl) sulfane (TD139) was prepared in accordance with the reaction scheme 1 below:

13 Ph PhPh Ph Bu4N+NO2-iTf20 DMF
Tf20 0 pyridine 0 60% over 2 ci pyridine -() 0 -20 C o steps when 0 -20 C 0 .....\___ "AcCI ,...&\.Ø....\________)._ nii1:1_30._ HO SPh Tf0 SPh SPh 0,00.=\.....\__--SPh OH OAc OAc OAc OH OTf (1) (2) (3) (4) Ph Bu4N+N3-DMF
.\(:) AcOH HOOH Ac20 Ac0 OAc 59% over pyridine 0 (80%) 2 steps". 60 C \--0 ---11"
N3...&.. _SPh ....&04__ NA0.07,,,,..\_.-SPh N3 SPh OAc OAc OAc (6) (7) (5) Br2 CH2Cl2 Ac0 OAc 68% over ---S- MeCN
3 steps __.&\..Ø,. Et3N Ac0 OAc N3 50-60% Ac0 Ac0 over 2 steps ....&\6Ø.._\___ Br ----0"-(8) Ac0 OAc ONH
Thiourea a0Me MeCN
reflux HO Me0H (10) 75% NbAc OH
Ac0 OAc ......&4...\___ N S

OAc ) F
r.-NH2 (11) N3 OH
(9) H2N Br 3-fluorophenylacetylene e Cut + HO , triuetthylamine DMF
OH
76%
¨ \ 0 HO
NJ' HO
HO
F
/ \NO
N H
TD139 041, N
(Scheme 1) Compound (1) (cf. reaction scheme above) is commercial from Carbo-synth Limited 8 & 9 Old Station Business Park - Compton - Berkshire - RG20 6NE ¨ UK or synthesized in three near-quantitative steps from D-galactose,

14 (cf e.g. Li, Z. and Gildersleeve, J. C. J. Am. Chem. Soc. 2006, 128, 11612-11619) Phenyl 2-0-acetyl-4,6-0-benzylidene-1-thio-3-0-trifluoromethanesulfonyl-p-D-galactopyranoside (2) Compound 1 (10.5 g, 29.2 mmol) was dissolved in dried pyridine (4.73 mL , 58.4 mmol) and dried CH2Cl2 (132 mL ). The reaction mixture was cooled, under stirring, until -20 C (Ice and NaCI bath 3:1). Slowly and under N, atmosphere, Tf20 (5.68 mL, 33.6 mmol) was added. The reaction mixture was monitored by TLC (heptane:Et0Ac, 1:1 and toluene:acetone, 10:1).
When the reaction was complete, AcCI (2.29 ml , 32.1 mmol) was added and keeping stirring, the temperature was increased to room temperature. This mixture was monitored by TLC too (heptane:Et0Ac, 1:1 and toluene:acetone, 10:1). When it was complete, it was quenched with CH2Cl2 and washed with 5 % HCI, NaHCO3 (saturated ¨ hereafter sat) and NaCI (sat). The organic layer was dried over MgSO4, filtered and concentrated under reduced pressure.
Phenyl 2-0-acetyl-4,6-0-benzyliden-1-thio-p-D-gulopyranoside (3) Tetrabutylammonium nitrite (25.3 g, 87.7 mmol) was added to a solu-tion of compound 2 (15.6 g, 29.2 mmol) in DMF (110 mL ) and was kept stir-ring, under N2 atmosphere, at 50 C. (The reaction started being purple and turned garnet). The reaction was monitored by TLC (heptane:Et0Ac, 1:1 and toluene:acetone, 10:1) and quenched with CH2Cl2. The mixture was washed with 5 % HCI, NaHCO, (sat) and NaCI (sat). The organic layer was dried over MgSO4, filtered and concentrated under reduced pressure followed by purifi-cation by flash chromatography (Eluent heptane:Et0Ac, 1:1 and hep-tane:Et0Ac, 1:2) and recrystallized from a mixture of Et0Ac and Heptane (1:3). 1F1 NMR in CDCI, 67.60-7.57 (m, 2H, Ar), 7.43-7.40 (m, 2H, Ar), 7.37-7.34 (m, 3H, Ar), 7.29-7.25 (m, 3H, Ar), 5.50 (s, 1H, PhCH), 5.15 (d, 1H, J=10.29 Hz, H-1), 5.10 (dd, 1H, J=10.27 Hz, 2.85 Hz, H-2), 4.36 (dd, 1H, J=
12.49 Hz,1.4 Hz, H-6), 4.18 (br s, 1H, H-3), 4.08 (dd, 1H, J= 3.59 Hz, 1.04 Hz, H-6), 4.03 (dd, 1H, J= 12.53 Hz, 1.75 Hz, H-4), 3.88 (s, 2H, H-5 + OH), 2.12 ( s, 3H, OAc).
Phenyl 2-0-acetyl-4,6-0-benzylidene-1-thio-3-0-trifluoromethanesulfonyl-p-D-gulopyranoside (4) 5 Compound 3(1.00 g, 2.48 mmol) was dissolved in dried CH2Cl2 (12.5 mL) and dried pyridine (0.40 mL , 4.96 mmol). The reaction mixture was cooled, under stirring, until -20 C (Ice and NaCI bath 3:1). Slowly and under N2 atmosphere, Tf20 (0.48 mL, 2.85 mmol) was added. The reaction mixture was monitored by TLC (heptane:Et0Ac, 1:1 and toluene:acetone, 10:1) and 10 when it was complete, it was quenched with CH2Cl2 and washed with 5 %
HCI, NaHCO, (sat) and NaCI (sat). The organic layer was dried over MgSO4, filtered and concentrated under reduced pressure until being dry.
Phenyl 2-0-acetyl-3-azido-4,6-0-benzylidene-3-deoxy-1-thio-p-D-

15 galactopyranoside (5) Tetrabutylammonium azide (2.12 g, 7.44 mmol) was added carefully to a solution of compound 4 (1.3256 g, 2.48 mmol) in DMF (10 mL) and was kept stirring, under N2 atmosphere, at 50 C. The reaction was monitored by TLC (E:H, 1:1) and concentrated under reduced pressure followed by purifi-cation by flash chromatography (Eluent heptane:Et0Ac, 2:1 and hep-tane:Et0Ac, 1:1).1H NMR in CDCI, 6 7.61-7.58 (m, 2H, Ar), 7.44-7.41 (m, 2H, Ar), 7.39-7.36 (m, 3H, Ar), 7.30-7.24 (m, 3H, Ar), 5.59 (s, 1H, PhCH), 5.35 (t, 1H, J= 9.95 Hz, H-2), 4.73 (d, 1H, J= 9.63 Hz, H-1), 4.44 (dd, 1H, J= 6.24 Hz, 1.60 Hz, H-6), 4.35-4.34 (dd, 1H, J= 3.33 Hz, 0.88 Hz, H-4), 4.11 (dd, 1H, J=
12.48 Hz, 1.67 Hz, H-6), 3.57 (d, 1H, J= 1.15 Hz, H-5), 3.44 (dd, 1H, J= 10.21 Hz, 3.29 Hz, H-3), 2.17 (s, 3H, OAc).
Phenyl 2-0-acetyl-3-azido-3-deoxy-1-thio-13-D-galactopyranoside (6) Compound 5 (470 mg, 1.1 mmol) was dissolved in 80% acetic acid (75 mL) and the mixture was heated at 60 C. The reaction was monitored by TLC (heptane:Et0Ac, 1:1). When the reaction was complete, the mixture was concentrated under reduced pressure and heating.

16 Phenyl 2,4,6-tri-O-acetyl-3-azido-3-deoxy-1-thio-p-D-galactopyranoside (7) Acetic anhydride (30 mL) was added to a solution of compound 6 (373 mg, 1.1mmol) in dry pyridine (30 mL). The reaction was monitored by TLC
(heptane:Et0Ac, 1:1) and when it was complete, it was concentrated under reduced pressure. 1H NMR in CDCI3 6 7.54-7.51 (m, 2H, Ar), 7.35-7.30 (m, 3H, Ar), 5.46 (dd, 1H, H-4), 5.23 (t, 1H, H-2), 4.73 (d, 1H, H-1), 4.15 (d, 2H, H-6, H-6), 3.94 (dt, 1H, H-5), 3.68 (dd, 1H, H-3), 2.18 (s, 3H, OAc), 2.15 (s, 3H, OAc), 2.06 (s, 3H, OAc).
2,4,6-tri-O-acety1-3-azido-3-deoxy-a-D-galactopyranosyl bromide (8) Compound 7 (237.4 mg, 560 pmol) was dissolved in dry CH2Cl2 (2 mL), and bromine (32 pl, 620 pmol) was added. The reaction was monitored by TLC (heptane:Et0Ac, 1:1). When the reaction was complete, a small amount of cyclopentene was added to the reaction mixture to remove the rests of Br2.
The mixture was concentrated under reduced pressure and purified by quick Flash chromatography (Eluent: 500mL heptane:Et0Ac, 2:1).
2,4,6-tri-O-acetyl-3-azido-3-deoxy-a-D-galactopyranose-1-isothiouronium bromide (9) The sensitive bromide 8 (70.6 mg, 180 pmol) was immediately dis-solved in dry acetonitrile (1.7 mL) and refluxed with thiourea (13.7 mg, 180 pmol) under N2 for 4 hours. The reaction was monitored by TLC (hep-tane:Et0Ac, 1:1) and when it was complete, the mixture was cooled.
Bis-(2,4,6-tri-O-acetyl-3-azido-3-deoxy-p-D-galactopyranosyl)-sulfane (10) The sensitive bromide 8 (77.0 mg, 196 pmol) and Eth (60 pl, 430 pmol) was added to the last mixture (9). The reaction was monitored by TLC
(heptane:Et0Ac, 1:1). When it was complete, the reaction mixture was con-centrated under reduced pressure and without heating. The residue was pun-

17 tied by flash chromatography (Eluent: heptane:Et0Ac, 1:1).1H NMR in CDCI3 6 5.50 (dd, 2H, H-4,), 5.23 (t, 2H, H-2, H-2'), 4.83 (d, 2H, H-1, H-1'), 4.15 (dd, 4H, H-6, H-6, H-6', H-6'), 3.89 (dt, 2H, H-5, H-5'), 3.70 (dd, 2H, H-3, H-3'), 2.19 (s, 6H, 20Ac), 2.15 (s, 6H, 20Ac), 2.18 (s, 6H, 20Ac).
Bis-(3-azido-3-deoxy-13-D-galactopyranosyl)-sulfane (11) Compound 10 (160 mg, 0.00024 mol) was dissolved in dry Me0H (2.6 mL) and dry CH2Cl2 (1.6 mL), and Na0Me (1M, 24 pL, 24 pmol) was added.
The reaction was monitored by TLC (heptane:Et0Ac 1:1 and D:M 5:1). When the reaction was complete, the mixture was neutralized with Duolite C436 until pH 7, filtered and washed with Me0H. The filtered solution was concen-trated under reduced pressure. The residue was purified by flash chromatog-raphy (Eluent: CH2C12:Me0H, 5:1) to give pure 11(74.1 mg, 75%). 1H NMR
in CDCI3 6 4.72 (d, 2H, J=9.7 Hz, H-1, H-1'), 3.95 (br s, 2H, H-4, H-4'), 3.84 (t, 2H, J= 9.8 Hz, H-2, H-2'), 3.74 (dd, 2H, J= 11.47 Hz, 7.23 Hz, H-6, H-6'), 3.64 (dd, 2H, J= 11.48 Hz, 4.72 Hz, H-6, H-6'), 3.60-3.55 (ddd, 2H, 7.15 Hz, 4.67 Hz, 0.93 Hz, H-5, H-5'), 3.36 (dd, 2H, J= 10 Hz, 3.05 Hz, H-3, H-3').
Bis-{3-deoxy-344-(3-fluoropheny1)-1H-1,2,3-triazol-1-y1]-13-D-galactopyranosyl} sulfane (Named TD139) TD139 was synthesized at ambient temperature by Cu(I)-catalyzed cycloaddition between bis-(3-azido-3-deoxy-13-D-galactopyranosyl)-sulfane (11) and 3-fluorophenylacetylene (3 eq.) with Cu(I) (0.2 eq), triethylamine (2 eq.) in N,N-dimethylformamide (DMF, 100 mUmmol sulfane). The reaction was monitored with tic until complete, concentrated and first purified by flash chromatography (Eluent: CH2C12:Me0H, 8:1), followed by final purification by preparative hplc to give TD139 in 76% yield as a white amorphous solid. 1H-NMR (CD30D, 400 MHz) 8 8.59 (s, 2H, triazole-H), 7.63 (br d, 2H, 7.6 Hz, Ar-H), 7.57 (br d, 2H, 8.4 Hz, Ar-H), 7.41 (dt, 2H, 6,0 and 8.0 Hz, Ar-H), 7.05 (br dt, 2H, 2.4 and 6.4 Hz, Ar-H), 4.93 (dd, 2H, 2,4 and 10.4 Hz, H3), 4.92 (d, 2H, 10.4 Hz, H1), 4.84 (2H, 10.4 Hz, H2), 4.18 (d, 2H, 2.4 Hz, H4), 3.92 (dd, 2H, 4.2 and 7.6 Hz, H5), 3.84 (dd, 2H, 7.6 and 11.4 Hz, H6), 3.73 (dd, 2H, 4.2 and

18 11.4 Hz, H6); FAB-HRMS m/z calcd for C28H30F2N6Na08S (M+Na+), 671.1712; found, 671.1705.
Model of bleomycin-induced lung fibrosis Female C57/BI6 mice (10-14 weeks old) were anaesthetized with halo-thane, and bleomycin or saline was administered intratracheally (33 pg in 50 pl of saline) and lungs were harvested on day 26. TD139 was instilled into the lungs of mice on days 18, 20, 22 and 24 of bleomycin induced lung injury.
Fibrosis was assessed by histological score of collagen stained lung sections and by total collagen content by Sircol assay.
Mice were treated with bleomycin (bleo) or saline (control) and bleo-mycin treated mice were treated with 200 mg/kg pirfenidone twice daily on days 18-24. TD139 was administered intratracheally on days 18, 20, 22 and 24. Lungs were harvested on day 26.
Figure 1 shows (A) Total lung collagen measured by Sircol assay; (B) Fibrosis score; and (C) Inflammatory score. Results represent the mean and SEM (A) or box and whiskers (median, interquartile range, minimum to maxi-mum, B and C) of n=8 mice per group (n=7 bleo). ***P<0.005, **P<0.01, *P<0.05. Figure 1E) Beta-catenin activation in vivo was assessed by scoring sections of bleomycin treated mouse lung (control and 10 ug TD139 treated) stained with an anti-active beta catenin.
Effect on alveolar epithelial cells Primary alveolar epithelial cells from WT mice were plated and treated with TGF-131 in the presence or absence of 10 1.1M TD139. Figure 1D) Cells were lysed and analyzed for active p-catenin, total p-catenin and 13-actin by western blot.
In conclusion TD139 is a galectin-3 inhibitor and blocked TGF-I3-induced catenin activation in vitro and bleomycin induced lung fibrosis in vivo and is believed to represent a novel therapeutic strategy for treatment of lung fibro-

19 sis in mammals, in particular humans.
Drug treatment Mice were divided into the following groups:
Group Induction Treatment Dose Dosing days Administration 1 Control Vehicle N/A
18, 20, 2 Bleomycin Vehicle 22 Intratracheal and 24 3 Bleomycin TD139 10 ug 18,20, 22 Intratracheal and 24 4 Bleomycin TD139 3 ug 18, 20, 22 Intratracheal and 24 5 Bleomycin TD139 1 ug 18, 20, 22 Intratracheal and 24 , 6 Bleomycin TD139 0.1 ug 18,20 22 Intratracheal and 24 200 bid, from 7 oral Bleomycin Pirfenidone mg/kg day 18 Immunohistochemistry Paraffin-embedded sections of mouse tissue were stained with Mas-son's trichrome and haemotoxylin and eosin (H&E) as per manufacturer's instructions. Sections were processed for immunohistochemistry and the fol-lowing primary antibodies used: mouse anti-active (ABC) beta-catenin (Milli-pore) and sections visualized and quantified.
Determination of lung fibrosis and inflammation Histological lung inflammation and fibrosis score were carried out in Masson's trichronne stained sections. Inflammation (peribronchiolar, perivas-cular, and alveolar wall thickness) scored in > 5 random fields at magnifica-tion X630 using the following system (peribronchiolar and perivascular, 1 = no cells, 2 = <20 cells, 3 = 20 ¨ 100 cells, 4 = > 100 cells; alveolar wall thick-ness, 1 = no cells, 2 = 2 ¨ 3 cells thick, 3 = 4 ¨ 5 cells thick, 4 = > 5 cells thick). The combined inflammatory score was the sum of these scores. Fi-brosis score was evaluated as the area of the section positively stained for collagen (1 = none, 2 = <10%, 3 = <50%, 4 => 50%). Only fields where the majority of the field was composed of alveoli were scored.

Determination of lung collagen by sircol assay Collagen content in the left lung lobe was determined by sircol assay as per manufacturer's instructions. The left lobe was minced in 5 ml of 3 5 mg/ml pepsin in 0.5 M acetic acid and incubated with shaking at 4oC for 24 h.
Cleared lung extract (0.2 ml) was incubated with 0.8 ml sircol reagent for 1 h at room temperature and precipitated collagen centrifuged at 10,000g for 5 min at 4oC. Pellets solubilised in 1 ml 1 M NaOH and absorbance measured at 570 nm alongside collagen standards.
Primary Type ll alveolar epithelial cell isolation Treated and control mouse type II lung alveolar epithelial cells (AECs) were extracted following a standard method. Briefly, 1 ml of 50 U/m1 dispase (BD Biosciences) was administered intratracheally into perfused lungs fol-lowed by instillation of 0.5 ml of 1% low melting point agarose. The agarose within the upper airways was allowed to set on ice for 2 minutes and the lungs were placed in 4 ml 50 U/ml dispase for 45 min at room temperature. The lung lobes minus the upper airways were then dispersed in DMEM containing 50 pg/ml DNAse I (Sigma-Aldrich, UK). The cell suspension was passed through a 100-pm cell strainer and the cells washed in DMEM followed by resuspension in DMEM containing 10% FCS. The cell suspension was plated onto tissue culture plastic for 1 h to allow any contaminated fibroblasts and macrophages to adhere. Non-adherent epithelial cells were counted and cul-tured for 2 days on tissue culture plastic or cover-slips pre-coated with 5 pg/ml collagen (AMS Biotechnology) and 10 pg/m1 fibronectin (Sigma-Aldrich), Cells were washed three times in PBS before treatment. Epithelial cells were either incubated in DMEM containing 10% FCS, 50 Wm! penicillin, 50 pg/ml streptomycin and 5 pg/ml L-glutamine or transferred to complete mouse media (DMEM/F-12 containing 0.25% BSA, 10 nM hydrocortisone, 5 pg/ml Insulin-Transferrin-Sodium-Selenite (ITS) and supplemented with 0.1 mg/ml sodium succinate, 75 pg/ml succinic acid and 1.8 pg/m1 choline bitar-trate).

Western Blotting Cells were lysed in 25 mM HEPES pH 7.4, 0.3 M NaCI, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5% triton X-100, 0.5 mM dithiothreitol, 1 mM sodium or-thovanadate and protease inhibitors (Boehringer Mannheim, Sussex, UK; pre-pared as per manufacturers instructions). Lysates equilibrated for protein using Pierce BCA protein assay reagent (Pierce) and resolved on 12% SDS-PAGE gels. Western blot analysis undertaken using the following primary antibodies; rabbit anti beta-catenin, (BD Biosciences), rabbit polyclonal anti-beta-actin antibody (Sigma, UK), mouse anti-active (ABC) beta-catenin (Milli-pore).

Claims (16)

WE CLAIM:

1. A compound of the general formula (i):

2. The compound of claim 1 selected from bis (3-deoxy-3-(3-fluorophenyl-1H-1,2,3-triazol-1-yl)-.beta.-D-galactopyranosyl) sulfane as the free form.

3. The compound according to any one of claims 1-2, for use as a me-dicament.

4. A pharmaceutical composition comprising the compound of any one of claims 1-3 and optionally a pharmaceutically acceptable additive, such as a carrier or an excipient.

5. The pharmaceutical composition of claim 4 wherein the composition is administered by the pulmonary route.

6. The compound of any one of the claims 1-3 for use in a method for treating pulmonary fibrosis, such as Idiopathic pulmonary fibrosis in a mam-mal.

7. The compound of claim 6, wherein the compound is administered by the pulmonary route.

8. The compound of claim 6 or 7 wherein said mammal is a human subject.

9. A method for treatment of pulmonary fibrosis, such as Idiopathic pulmonary fibrosis comprising administering to a mammal a therapeutically effective amount of the compound of any one of claims 1-3.

10. The method of claim 9, wherein the compound of any one of claims 1-3 is administered by the pulmonary route.

11. A process of preparing a compound of formula I comprising a step of reacting bis-(3-deoxy-3-azido-.beta.-D-galactopyranosyl) sulfane with 3-fluorophenylacetylene and an amine in a solvent, resulting in the compound of formula I.

12. The process of claim 11 wherein the amine is triethylamine, a cata-lyst is present, such as Cu(l), and the solvent is an organic solvent, such as N,N-dimethylformamide (DMF).

13. A nebulizer device for pulmonary administration comprising a com-pound of any one of claims 1-3.

14. The nebulizer device of claim 13, wherein the compound is bis (3-deoxy-3-(3-fluorophenyl-1H-1,2,3-triazol-1-yl)-.beta.-D-galactopyranosyl) sulfane as the free form.

15. A dry powder device for pulmonary administration comprising a compound of any one of claims 1-3.

16. The dry powder device of claim 15, wherein the compound is bis (3-deoxy-3-(3-fluorophenyl-1H-1,2,3-triazol-1-yl)-.beta.-D-galactopyranosyl) sul-fane as the free form.