WO2008152142A1 - 2 -aminoquinazoline derivatives and their therapeutic uses - Google Patents

Abstract

A compound of formula (I) wherein X is NH2or OH and W is -C(O)-O- or -O-C(O)-; use thereof as a medicament, in particular for the treatment of inflammation, autoimmune disorder, immunogenic rejection reactions and cancer and for non-surgical abortion.

Description

2 -AMINOQUINAZOLINE DERIVATIVES AND THEIR THERAPEUTIC USES

Field of the invention

The present invention relates to derivatives of phenyl-2,4-diaminoquinazoline-6-carboxylate and 2,4-diaminoquinazoline-6-yl-benzoate and salts thereof, useful for the treatment of diseases/conditions that can be therapeutically treated by immuno -modulating or cytostatic compounds.

Background to the invention Inflammatory bowel disease (IBD) is a general term that includes both ulcerative colitis and Crohn's disease, disorders of unknown ethiology that result in inflammation in the gastrointestinal tract.

Ulcerative colitis is an inflammatory disease of the large intestine. Ulcers develop in the inner lining, or mucosa, of the colon or rectum, often resulting in diarrhoea, blood, and pus. Crohn's disease is an inflammation that extends into the deeper layers of the intestinal wall. It is found most often in the ileum and the first part of the large intestine, known as the ileocecal region.

Ulcerative colitis and Crohn's disease share many symptoms, although they also differ in im- portant ways. Both are chronic diseases characterized by frequent relapses and remissions, and symptoms usually appear in young adults. The most common symptom of both ulcerative colitis and Crohn's disease is diarrhoea. Constipation may develop during active flare-ups of both Crohn's disease and ulcerative colitis. Cramps can occur from intestinal contractions caused by inflammation. Fever, fatigue and loss of appetite are often present. Neurologic or psychiatric symptoms may be early signs of Crohn's disease when accompanied by gastrointestinal problems.

Drugs presently on the market cannot cure IBD, but some are effective in reducing the inflammation and accompanying symptoms in up to 80% of patients. Many such drugs are available, including corticosteroids, aspirin-like medications, and drugs that suppress the immune system. The primary goal of drug therapy is to put acute flares into remission and/or prevent relapse. Mesalazine is the common name of the compound 5 -aminosalicylic acid or 5- ASA, which inhibits substances in the immune system that cause inflammation. For very active IBD that does not respond to standard treatments, immunosuppressant drugs are now being used for long-term treatment. All of these drugs suppress actions of the immune system and thereby its inflammatory response that causes ulcerative colitis and Crohn's disease. The two most common immunosuppressant used for IBD are azathioprine and mer- captopurine. Other immunosuppressants investigated for IBD and showing promising results in promoting remission include cyclosporine and methotrexate. Metronidazole is an antibiotic used for infections caused by anaerobic bacteria and is useful for people with Crohn's disease. Other antibiotics used for Crohn's disease include trimethoprim (TMP) /sulfamethoxazole, ciprofloxacin, and tetracycline.

Of some promise is a genetically engineered antibody that acts against tumour necrosis factor (TNF), a major factor in the inflammatory process that causes IBD. Recent results show a reduction in disease activity and improving symptoms in both Crohn's disease and ulcerative colitis. A similar drug, cA2, is also showing promising results against Crohn's disease.

Asthma is a chronic lung disease and causes breathing problems. Asthma medicines keep the air tubes in the lungs open. There are two groups of asthma medicines: bronchodilators and anti-inflammatory active agents. Inhaled corticosteroids are important in therapy.

Psoriasis is a common condition affecting the skin. It causes red, scaly patches. In addition it can affect the joints, nails and eyes. Although the exact cause is unknown, psoriasis is believed to be related to faulty signals sent by the body's immune system. It has a genetic component that makes certain people more likely to develop it. Treatments include moisturising creams and ointments, oils for the bath, creams, ointments, lotions and shampoos based on tar, vitamin D, salicylic acid, sunshine, stronger medications, e.g. methotrexate, and mild steroid creams and ointments, used for short periods, for psoriasis affecting the face or body folds.

The AIDS epidemic, cancer chemotherapy and organ transplantation have significantly in- creased the number of patients with impaired immune systems who are suffering from severe opportunistic infections including pneumonia caused by the fungus Pneumocystis carinii. P. carinii pneumonia (PCP), which is a serious disease with high prevalence, constitutes the major cause of death in patients with AIDS. Current treatment of the disease with trimethoprim (TMP):

a nonclassical inhibitor of dihydro folate reductase (DHFR), in combination with a sulfonamide is still the standard therapy for PCP.

Inhaled aerosolised/nebulised pentamidine is used for prophylaxis of PCP. When applied systematically pentamidine exhibits a considerable toxicity. Trimetrexate (TMX):

and piritrexim (PTX) :

two lipophilic agents originally developed against cancer, are now used in clinic as second- line therapy (structures below). Although TMX and PTX are both potent inhibitors of DHFR from P. carinii, they are not selective and inhibit the mammalian enzyme even more efficiently. The clinical use of TMX and PTX is therefore limited due to their systemic host toxicity and require an expensive co-therapy with the rescue agent leucovorin (5-formyl- tetrahydrofolate). Leucovorin, a classical folate cofactor for one-carbon metabolism, is taken up via active transport only by mammalian cells and thereby reverses toxicity associated with the lipophilic DHFR inhibitors. Today considerable research efforts are devoted to the identification of more selective and potent DHFR inhibitors with the overall goal to improve therapy and to minimise the adverse effects. Rheumatoid arthritis is another inflammatory condition, the signs and symptoms of which include: pain and swelling in the smaller joints of hands and feet, overall aching or stiffness of the joints and muscles, especially after sleep or after periods of rest, loss of motion of the affected joints, loss of strength in muscles attached to the affected joints, fatigue, which can be severe during a flare-up, low-grade fever, deformity of the joints as time goes on. Medications for rheumatoid arthritis can relieve its symptoms. Nonsteroidal anti- inflammatory drugs can slow or halt its progression. Treatment with NSAIDs can, however, lead to such side effects as indigestion and stomach bleeding, as well as damage to the liver and kidneys, ringing in the ears (tinnitus), fluid retention, and high blood pressure. COX-2 inhibitors, which is a new class of NSAIDs may be less damaging to the stomach, but may have higher incidents of other side-effects than conventional NSAIDs.

Corticosteroids reduce inflammation and slow joint damage. Disease-modifying antirheumatic drugs (DMARDs) are another group of drugs prescribed. Some commonly used DMARDs include hydroxychloroquine sulfate (Plaquenil), gold compounds (Ridaura, Solga- nal), sulfasalazine (Azulfidine) and minocycline (Minocin). Other forms of DMARDs include immunosuppressants and TNF blockers. Some of the commonly used immunosuppressants include methotrexate, leflunomide, azathioprine, cyclosporine and cyclophosphamide. These medications can have potentially serious side effects such as increased susceptibility to infec- tion and disease.

Sulfonamides are structural analogues of p-aminobenzoic acid. They interfere with the early stages of folic acid synthesis by competitive inhibition of dihydropteroic acid synthetase, which condenses p-aminobenzoic acid with dihydropteroic acid. The sulfonamide may also be erroneously incorporated into the folic acid molecule to produce an inactive product. Bacterial cells synthesize folic acid, whereas mammalian cells use the preformed dietary vitamin, and this is the basis of the selective antibacterial action of sulfonamides.

Diaminopyrimidines, like trimethoprim and the antimalarial compound, pyrimethamine, act at a later stage on the same pathway by inhibiting dihydro folate reductase, the enzyme that generates the active product, tetrahydro folate, from dihydrofolate. The affinity of trimethoprim for DHFR of bacteria is several orders of magnitude higher than the affinity for the mammalian enzyme; similarly pyrimethamine has a very high affinity for the DHFR of malaria parasites. Because sulfonamides and diaminopyrimidines act on the same metabolic pathway, they exhibit a strongly synergic interaction, at least in vitro. However, because tetrahydro folate is reoxidized to dihydro folate during the biosynthesis of thymidylic acid, diaminopyrimidines rapidly trap the vitamin in the unusable dihydro folate form.

Sulfonamides, in contrast, cut off the supply of dihydro folate and act rather slowly because the folate pool becomes depleted only after several cell divisions. For this reason, if there is sufficient diaminopyrimidine present to halt tetrahydro folate regeneration completely, the sulfonamide does not have an opportunity to contribute to the antibacterial action.

In Med. Res. Rev., 2000, 20, 58-101, the "soft drug" concept is discussed. According to the authors of said paper, the soft drug design represents an approach aimed to design safer drugs with an increased therapeutic index by integrating metabolism considerations into the drug design process. Soft drugs are therapeutic agents that undergo metabolism to inactive metabolite after exerting their therapeutic effect.

Certain ester analogues of the above DHFR inhibitors as well as methotrexate analogs have been published both in the academic and patent literature, in particular by Hallberg et al. (Chem. Pharm. Bull, 1998,46, 591-601, J. Med. Chem. 2001, 44, 2391-2402, J. Med. Chem., 2000,43, 3852-3861, WO2004020418 and WO2004020417). In J. Med. Chem. 2001, 44, 2391-2402 the authors describe TMX and PTX ester analogs which act as soft drugs. Said ester compounds have a three-atom ester linker, (viz. a 3-membered chain containing an ester functionality) linking the two cyclic parts of the molecule, and in the paper it is stated that a compound having a two-atom linker will be too metabolically instable. WO2004020418 dis- closes methotrexate analogs of the general formula

wherein m and n are independently selected from 0-5 and W is

wherein the "@" signs denote the points of attachment to the rest of the molecule.

While theoretically n and m in the formula of WO2004020418 may both be 0 so as to provide a two-atom ester linker, no example of such a compound is provided and in fact it is stated that n preferably is 3, i.e. the linking group is an at least 5-atom linker. The present inventors however have tested and found that the the exemplified three atom linker disclosed in WO2004020418 as well as two atom linker analogs of methotrexate are surprisingly metab- olically stable and therefore will not act as soft drugs.

WO2004020417, on the other hand, discloses a compound of the general formula

i.e. having two moieties A and B connected by a 3 atom ester linker, wherein A and B are independently selected from various moieties including quinazolin (A) and substituted phenyl (B). However, once again three atom- linker is not enough metabolically labile for acting as a soft drug.

Summary of the invention

It has now been discovered that certain active analogues of known lipophilic DHFR structures tend to be deactivated by a fast hydro lytic metabolism in vivo. These new compounds, comprising a 2-atom linker ester in the middle region, are more easily metabolised than the corresponding 3-atom linker esters. The compounds show good response in a DSS induced colitis model in rat but surprisingly, in view of the structurally similarity and the positive in- vivo results, they are very poor DHFR inhibitors. The compounds of the invention will in general be administered near the site of action following the criteria for the soft drug concept (Med. Res. Rev., 2000, 20, 58-101). The invention thus provides a new entry to efficient and safe treatment of diseases that can be therapeutically treated by immuno -modulating or cytostatic effective compounds, in particular DHFR inhibitors, applied either topically, orally or par- enterally, or cancer forms being sensitive to methotrexate. IBD, i. e. ulcerative colitis and Crohn's disease, is a further indication that can be treated, and some other are colorectal cancer, cancer of the urinary bladder, the lung and other cancer types that may be reached from the "outside" of the body; psoriasis, PCP, other fungal (vaginal and others), protozoal and bacterial (pulmonary infections, urinary tract infections and others) infections, non-surgical abortions (intrauterin administration), asthma, or other serious pulmonary diseases, rheumatoid arthritis (other inflammatory conditions). The inventive compounds can also be used to prevent transplant rejection following liver transplantation or intestine transplantation. As a short-lived duration of exposure is sufficient, systemic treatment of e. g., rheumatoid arthritis or other inflammatory conditions, is possible as well. The compounds of the invention can also be used for treating nephritis, e. g. IgA nephritis.

Thus, according to a first aspect, the present invention provides compounds of the formula I:

(I) wherein

X is selected from NH₂ and OH;

W is selected from -C(O)-O- and -O-C(O)-;

each R¹ is independently selected from R², OR², SR², N(R²R³), C(O)R², OC(O)R², NNHH--CC((OO))--RR²²,, NNHHCC((OO))OORR²² oorr ttwwoo RR¹¹ aattttaacchheedd ttoo aaddjjaacceenntt aattoommss ooff ttlhe phenyl ring are linked together so as to form a 5-6 membered ring fused to the phenyl ring;

R² and R³ are independently selected from H and C1-C6 alkyl, optionally substituted by one or several several moieties selected from halogen and nitro;

as well as a pharmaceutically acceptable salt thereof.

According to a further aspect, the invention provides a pharmaceutical composition comprising a compound of formula (I) as defined herein or a pharmaceutically acceptable salt thereof. According to still a further aspect, the present invention provides a compound of formula (I) as defined herein or a pharmaceutically acceptable salt thereof for use in the treatment of inflammation.

According to still a further aspect, the present invention provides a compound of formula (I) as defined herein or a pharmaceutically acceptable salt thereof for use in the treatment of diseases which can be therapeutically treated by immunomodulating or cytostatic compounds, in particular dihydrofolate reductase inhibitors, applied either topically, orally, rectally, or par- enterally, or cancer forms being sensitive to methotrexate, inflammatory bowel disease i. e. ulcerative colitis and Crohn's disease, asthma, Pneumocystis carinii pneumonia (PCP), psoriasis, inflammations caused by bacteria, fungi, protozoa, rheumatoid arthritis as well as other inflammatory conditions, colorectal cancer, cancer of the urinary bladder, the skin, the lung and other cancer types that may be reached from the outside of the body, non- surgical abortions (intrauterin administration), or for preventing immunogenic rejection reactions in liver or intestine transplantations.

According to still a further aspect, the present invention provides the use of a compound of formula (I) as defined herein or a pharmaceutically acceptable salt thereof in the manufacturing of a medicament for the treatment of inflammation and/or diseases that can be therapeuti- cally treated by immunomodulating or cytostatic compounds, in particular dihydrofolate reductase inhibitors, either applied topically, orally, rectally, or parenterally, or cancer forms being sensitive to methotrexate, inflammatory bowel disease i. e. ulcerative colitis and Crohn's disease, asthma and other serious pulmonary diseases, Pneumocystis carinii pneumonia (PCP), psoriasis, inflammations caused by bacteria, fungi, protozoa, rheumatoid arthritis as well as other inflammatory conditions, colorectal cancer, cancer of the urinary bladder, the skin, the lung and other cancer types that may be reached from the outside of the body, nonsurgical abortions (intrauterin administration), or for preventing immunogenic rejection reactions in liver or intestine transplantations.

According to one aspect, a method of treatment of a mammalian subject in need thereof is provided, by administering a compound of formula (I) as defined herein or a pharmaceutically acceptable salt thereof. Brief description of the drawings

Fig. IA-C shows bar charts representing (A) length, (B) weight and (C) % affected mucosal area of colon of mice in the DSS model at completion of test period.

Fig. 2A-E shows bar charts representing (A) concentration of white blood cells (WBC), (B) concentration of red blood cells (RBC), (C) concentration of haemoglobin (HGB), (D) hema- tocrite (HCT), and (E) platelet count (PLT) in blood from mice in the DSS model at completion of test period.

Fig. 3 is a bar chart showing % weight change of mice in the DSS model during days 5-14 of the treatment period. Fig. 4 is a bar chart showing the relative remaining LC/PDA peak areas for inventive compound No. 31 in 0 and 60 min incubations, with and without cofactors. Fig. 5 is a bar chart showing the relative remaining LC/PDA peak areas for inventive Compound No. 32 in 0 and 60 min, with and without cofactors. Fig. 6 is a bar chart showing LC/MS peak areas for the metabolites of inventive Compound No. 31 in 0 and 60 min human liver S9 fraction incubations, with and without cofactors.

Fig. 7 is a bar chart showing LC/MS peak areas for the metabolites of inventive Compound No. 31 in 0 and 60 min rat liver S9 fraction incubations, with and without cofactors. Fig. 8. shows the suggested structures for detected metabolites of inventive Compound No. 31. Fig. 9 shows the suggested structures for detected metabolites of inventive Compound No. 32.

Detailed description of the invention

In the compounds of formula (I) according to the invention

X is selected from NH₂ and OH;

W is selected from -C(O)-O- and -O-C(O)-; each R¹ is independently selected from R², OR², SR², N(R²R³), C(O)R², OC(O)R², NH-C(O)-R², NHC(O)OR² and/or two R¹ attached to adjacent atoms of the phenyl ring may be linked together so as to form a 5-6 membered ring fused to the phenyl ring;

R² and R³ are independently selected from H and C1-C6 alkyl, optionally substituted by one or several several moieties selected from halogen and nitro.

By "(R¹^" in formula (I) is meant five moieties attached to the phenyl ring, which moieties are denoted R¹ and which moieties may be different from each other or the same. For example R¹ may be all H, or else one, two, three or four of R¹ may be H, while the other ones are independently selected from substituents as defined herein above. In one embodiment, at least one R¹ is different from H.

Any alkyl moiety according to the invention may, at each occurence in the compound, be in- dependently selected from branched or unbranched C1-C6 alkyl, e.g. C1-C4 alkyl or C1-C3 alkyl, e.g. methyl, ethyl, propyl and butyl.

The number of halogen and/or nitro substituents on any alkyl moiety may range from none to several, e.g. 3 or 4. For example, in an alkyl moiety all hydrogens may be replaced by halo- gens.

In one embodiment, any alkyl is selected from methyl and ethyl.

As used herein the term "halogen" or "halo" means a fluorine, chlorine, bromine or iodine, e.g. F and Cl, in particular F and Cl.

In one embodiment, any R¹ is independently selected from H, C1-C6 alkyl, C1-C6 alkoxy; halogen; C1-C6 alkyl-C(O)-; C1-C6 alkyl-C(O)O-; C1-C6 alkyl-C(O)NH-; C1-C6 alkyl-S-; C1-C6 alkyl-S-; C1-C6 alkyl-NH-; C1-C6 alkyl-OC(O)NH-; and (C1-C6 alkyl)₂N- ; wherein any alkyl moiety optionally is substituted with halogen or NO₂;.

In one embodiment, any R¹ is independently selected from H, C1-C6 alkyl, optionally substituted with halogen or NO₂; C1-C6 alkoxy; halogen; C1-C6 alkyl-C(O)-; C1-C6 alkyl-C(O)O-; C1-C6 alkyl-C(O)NH-; C1-C6 alkyl-S-; C1-C6 alkyl-S- wherein the alkyl optionally is substituted with halogen; C1-C6 alkyl-NH-; C1-C6 alkyl-OC(O)NH-; and (C1-C6 alkyl)₂N-.

In one embodiment, any R¹ is independently selected from H, CH₃-, CH₃O-, F, CH₃CH₂-, CH₃C(O)-, CH₃C(O)O-, CH₃C(O)NH-, CH₃S-, CF₃-, Cl, CF₃S-, CH₃CH2O-, NO₂CH₂-, CH₃NH-, CH₃CH₂OC(O)NH- and (CH₃)₂N-.

In one embodiment, two R¹ attached to adjacent atoms of the phenyl are linked together so as to form a 5-6 membered ring fused to the phenyl. This 5-6 membered ring may be carbocyclic or heterocyclic and contain one or several heteroatoms selected from N, O and S. For example the ring may be 5-6 membered, e.g. 5-membered, and contain oxygen atoms, e.g. 2 oxygen atoms. Thus, in one embodiment, two adjacent R¹ form together a methylene dioxy radical - 0-CH₂-O-, giving a fϊve-membered heterocyclic ring fused to the phenyl ring of the com- pound of formula (I) .

In one embodiment of the invention, in a compound of formula (I), X is NH₂. In another embodiment of a compound of formula (I), X is OH.

It should be understood that when X is OH, due to amide-oxime tautomerism a compound of formula (I) may be present as an equilibrium mixture of the oxime form and the corresponding amide. This rapid tautomeric equilibrium may be represented as:

oxime form amide form.

Therefore, it should be understood that any reference to a compound of formula (I) where X is OH also includes the corresponding carbonyl-containing form of the compound, and vice versa.

In one embodiment of the invention, in a compound of formula (I), W is -C(O)-O-. In another embodiment of a compound of formula (I), W is -O-C(O)-. In one embodiment of the invention, a compound of formula (I) is provided, wherein the moiety

is selected from

In one embodiment of the invention, a compound of formula (I) is provided, wherein the moiety

is selected from

wherein the * denotes the point of attachment to the moiety W in formula (I).

In one embodiment a compound is provided, selected from: 3,4-dimethoxyphenyl-2,4-diaminoquinazoline-6-carboxylate; 2,4-dimethoxyphenyl-2,4-diaminoquinazoline-6-carboxylate; l,3-benzodioxol-5-yl-2,4-diaminoquinazoline-6-carboxylate;

2,6-dimethoxyphenyl-2,4-diaminoquinazoline-6-carboxylate;

2,3-dimethoxyphenyl-2,4-diaminoquinazoline-6-carboxylate;

4-fluoro-2-methoxyphenyl-2,4-diaminoquinazoline-6-carboxylate; 4-ethyl-2-methoxyphenyl-2,4-diaminoquinazoline-6-carboxylate;

4-acetyl-2-methoxyphenyl-2,4-diaminoquinazoline-6-carboxylate;

2,4-diaminoquinazolin-6-yl-4-(acetyloxy)-3,5-dimethoxybenzoate;

3-(acetylamino)phenyl-2,4-diaminoquinazoline-6-carboxylate;

4-(methylthio)phenyl-2,4-diaminoquinazoline-6-carboxylate; 3,4,5-trifluorophenyl-2,4-diaminoquinazoline-6-carboxylate;

4-(acetylamino)phenyl-2,4-diaminoquinazoline-6-carboxylate;

3,5-bis(trifluoromethyl)phenyl-2,4-diaminoquinazoline-6-carboxylate;

3,5-dichlorophenyl-2,4-diaminoquinazoline-6-carboxylate;

4-[(trifluoromethyl)thio]phenyl-2,4-diaminoquinazoline-6-carboxylate; 2,4-diaminoquinazolin-6-yl-3,5-dimethoxy-4-methylbenzoate;

2,4-diaminoquinazolin-6-yl-3,4,5-triethoxybenzoate;

2,4-diaminoquinazolin-6-yl-2-methoxybenzoate;

2,4-diaminoquinazolin-6-yl-2-methoxy-5-methylbenzoate;

2,4-diaminoquinazolin-6-yl-3-(methylthio)benzoate; 2,4-diaminoquinazolin-6-yl-2,5-bis(trifluoromethyl)benzoate;

2,4-diaminoquinazolin-6-yl-2-methoxy-5-(nitromethyl)benzoate;

2,4-diaminoquinazolin-6-yl-2-(methylamino)benzoate;

2,4-diaminoquinazolin-6-yl-2-[(ethoxycarbonyl)amino]-4,5-dimethoxybenzoate

2,4-diaminoquinazolin-6-yl-2-(methylthio)benzoate; 2,4-diaminoquinazolin-6-yl-5-fluoro-2-(trifluoromethyl)benzoate;

3-(dimethylamino)phenyl-2,4-diaminoquinazoline-6-carboxylate;

2,4-diaminoquinazolin-6-yl-4-amino-2-methoxybenzoate;

5-amino-2-methoxyphenyl-2,4-diaminoquinazoline-6-carboxylate;

3,4,5-trimethoxyphenyl-2,4-diaminoquinazoline-6-carboxylate; 2,4-diaminoquinazolin-6-yl-2,5-dimethoxybenzoate;

3,5-dichlorophenyl-2-amino-4-oxo-3,4-dihydroquinazoline-6-carboxylate; l,3-benzodioxol-5-yl-2-amino-4-oxo-3,4-dihydroquinazoline-6-carboxylate;

3-(acetylamino)phenyl-2-amino-4-oxo-3,4-dihydroquinazoline-6-carboxylate;

4-(methylthio)phenyl-2-amino-4-oxo-3,4-dihydroquinazoline-6-carboxylate; and 3,4,5-trimethoxyphenyl-2-amino-4-oxo-3,4-dihydroquinazoline-6-carboxylate, or a pharmaceutically acceptable salt thereof.

The compounds of the present invention may be prepared e.g. by methods as outlined herein below.

Thus, in the embodiment where X in formula (I) is NH2 and W is -C(O)-O-, the inventive compounds may be prepared by a method as schematically represented in Reaction Scheme 1.

Reaction Scheme 1

In this reaction sequence, in step 1, the acid function of 3-cyano-4-fluro benzoic acid (a) is protected, e.g. as an ester function. The protective ester group may be achieved by reacting compound a with an alcohol ROH, e.g methanol, suitably in the presence of a coupling agent, such as thionyl chloride. Compound b is cyclized using guanidine carbonate (step 2), and the acid protecting group is then removed, e.g by alkaline hydrolysis (step 3), to give 2,4- diaminoquinazoline-6-carboxylic acid (d). Finally, in step 4 compound d is esterified using an appropriately substituted or unsubstituted phenol giving compound e of the invention. In another embodiment, where X in formula (I) is NH2 and W is -O-C(O)-, the inventive compounds may be prepared by a method as schematically represented in Reaction Scheme 2.

ion

Reaction Scheme 2

In this reaction sequence, in step 1, the ether function of commercially available 2-fluoro-5- methoxybenzonitrile (f) is cleaved to yield 2-fluoro-5-hydroxy benzonitrile (g), e.g by reac- tion with BBr₃. In step 2, the alcohol function is protected, e.g. by benzylation using a benzyl halogenide in the presence of a base, such as sodium hydride (NaH), giving 5-(benzyloxy)-2- fluorobenzonitrile (h). Step 3 is a cyclization using guanidine carbonate, to provide 6- (benzyloxy)quinazoline-2,4-diamine (i). By deprotection of the alcohol function (step 4) 2,4- diaminoquinazolin-6-ol (j) is obtained, which in step 5 is esterifϊed using an appropriately substituted or unsubstituted benzoic acid, to provide compound (k) according to the invention.

In an embodiment where X in formula (I) is OH and W is -C(O)-O-, the inventive compounds may be prepared by a method as schematically represented in Reaction Scheme 3. protection of

m

Reaction Scheme 3

In this reaction sequence, in step 1, the methyl groups of commercially available m- nitroxylene (1) are oxidized to carboxylic groups, using a suitable oxidant, e.g. KMnO₄, giving 4-nitro-isophthalic acid (m). Next, both carboxylic groups of compound m are protected, e.g. by esterification (step 2) to give 4-nitro-isophthalic acid diester (n). This esterification may be achieved e.g. using a suitable alcohol, such as methanol, optionally in the presence of a cou- pling agent, such as thionyl chloride. In step 3, the nitro group of compound n is reduced to a primary amine group, e.g. by use of H₂ over Pd/C, giving 4-amino-isophthalic acid diester (o). Cyclization of compound o using guanidine carbonate gives of 2-amino-4-oxo-3,4-dihydro- quinazoline-6-carboxylic acid ester (p). Deprotection, (step 5), e.g. by alkaline hydrolysis, of the remaining carboxylic acid function to provide 2-amino-4-oxo-3,4-dihydro-quinazoline-6- carboxylic acid (q) and reaction thereof (step 6) with an appropriately substituted or unsubsti- tuted phenol provide compound r according to the invention.

In an embodiment where X in formula (I) is OH and W is -O-C(O)-, the inventive compounds may be prepared by a method as schematically represented in Reaction Scheme 4. thyl

OH

w

Reaction Scheme 4

In this reaction sequence, in step 1, the hydroxy group of 3-methyl-4-nitrophenol (s) is protected, e.g. by esterification using an acid chloride, such as acetyl choride. In step 2, the methyl group of compound t is oxidized to a carboxylic group, using a suitable oxidant, e.g. KMnO₄, compound u. Next, the carboxylic group of compound u is protected (step 3), e.g. by esterification to give compound v, e.g. by use of an alcohol, such as methanol, in the presence of a coupling agent, such as thionyl chloride. In step 4, the nitro group of compound v is reduced to a primary amine group, e.g. by use of H₂ over Pd/C, giving compound w. Cyclization (step 5) of compound w using guanidine carbonate gives compound x. Deprotection, (step 6), of the hydroxy function of compound x to provide 2-amino-6-hydroxyquinazolin-4(3H)-one (y) and reaction of compound y (step 7) with an appropriately substituted or unsubstituted benzoic acid will provide compound z according to the invention.

Although certain of the inventive compounds may show decreased potency against pathogen or cellular DHFR relative to current inhibitors, they may exhibit better in- vivo effect and, importantly, a more rapid metabolism to inactive metabolites in vivo. Accordingly, the compounds of the invention have utility in the treatment of diseases which can be therapeutically treated by immuno- modulating or cytostatic compounds, either applied topically, orally, rec- tally, or parenterally, or cancer forms being sensitive to methotrexate. Another utility are the treatments of IBD, i. e. ulcerative colitis and Crohn's disease. Asthma, Pneumocystis carinii pneumonia (PCP), psoriasis, rheumatoid arthritis (other inflammatory conditions), colorectal cancer, cancer of the urinary bladder, the lung and other cancer types that may be reached from the "outside" of the body, inflammatory conditions caused by bacterial, fungal (vaginal and others) or protozoal infections, non-surgical abortions (intrauterin administration), liver transplantation, or other serious pulmonary diseases may be treated, especially in immunocompromised individuals.

Compounds of formula I and/or their pharmaceutically acceptable salts have valuable pharmacological properties, making them useful for the treatment of inflammation such as in- flammations related to the production of nitric oxide, inflammation related to increased amounts (upregulated amounts) of inducible nitric oxide synthase, inflammation related to activation of transcriptional activators, inflammation related to nuclear factor kappa beta, inflammation related to macrophages, neutrophils, monocytes, keratinocytes, fibroblasts, melanocytes, pigment cells and endothelial cells, inflammation related to increased produc- tionand/or release of inflammatory cytokines, such as e. g. interleukins, in particular inter- leukin 1 (IL-I), interleukin 6 (IL-6) and tumor necrosis factor a (TNF-a).

In the present specification, increased production refers to increased formation, increased release, or increased amount of an endogenous compound locally, regionally or systemically in a patient compared to the amount of said endogenous compound in a healthy individual. In the present specification, upregulated refers to an increased activity or amount of the compound compared with that in a healthy individual. In particular, positive treatment effects or preventive effects may be seen in conditions where inflammation or an inflammatory- like condition is caused by or being associated with one or more of the following: allergy, hypersensitivity, bacterial infection, viral infection, inflammation caused by toxic agent, fever, autoimmune disease, radiation damage by any source in- eluding UV-radiation, X-ray radiation, γ-radiation, α- or β- particles, sun burns, elevated temperature or mechanical injury. Moreover, inflammation due to hypoxia, which is optionally followed by reoxygenation of the hypoxic area, is typically followed by severe inflammation, which condition may be positively affected by treatment with a compound of the invention. In very specific embodiments of the invention, a compound of the invention may be adminis- tered for the prevention or therapeutic treatment of inflammatory diseases of the skin (including the dermis and epidermis) of any origin, including skin diseases having an inflammatory component. Specific examples of this embodiment of the invention include treatment of contact dermatitis of the skin, sunburns of the skin, burns of any cause, and inflammation of the skin caused by chemical agents, psoriasis, vasculitis, pyoderma gangrenosum, discoid lupus erythematosus, eczema, pustulosis palmo-plantaris, and phemphigus vulgaris.

According to one aspect of the invention a compound of formula I or a pharmacologically acceptable salt thereof is used for the treatment of an inflammatory disease in the abdomen, including an abdominal disease having an inflammatory component. Specific examples of the treatment of such a disease with a compound of the invention are gastritis, including one of unknown origin, gastritis perniciosa (atrophic gastritis), ulcerous colitis (colitis ulcerosa), morbus Crohn, systemic sclerosis, ulcus duodeni, coeliac disease, oesophagitis and ulcus ven- triculi.

The free bases of formula I may be converted to their therapeutically active acid addition salts, which form an additional aspect of the invention. Appropriate pharmaceutically acceptable salts of the compounds of general Formula I include salts of organic acids, especially carboxylic acids, including but not limited to acetate, trifluoroacetate, lactate, gluconate, citrate, tartrate, maleate, malate, pantothenate, isethionate, adipate, alginate, aspartate, benzoate, butyrate, digluconate, cyclopentanate, glucoheptanate, glycerophosphate, oxalate, heptanoate, hexanoate, fumarate, nicotinate, palmoate, pectinate, 3-phenylpropionate, picrate, pivalate, proprionate, lactobionate, pivolate, camphorate, undecanoate and succinate, organic sulphonic acids such as methanesulphonate, ethanesulphonate, 2-hydroxyethane sulphonate, camphor- sulphonate, 2-napthalenesulphonate, benzenesulphonate, p-chlorobenzenesulphonate and p- toluenesulphonate; and inorganic acids such as hydrochloride, hydrobromide, hydroiodide, sulphate, bisulphate, hemisulphate, thiocyanate, persulphate, phosphoric and sulphonic acids. Hydrochloric acid salts are convenient. The salt may also be hydrated to various degrees, e. g. mono-, di- or tri-hydrates.

Conversely, the salt form may be converted into the free base form by treatment with alkali.

The compounds of the invention are particularly suited to topical administration, such as pulmonary, dermally, optically, vaginally, nasally, transdermally but may also be administered orally, rectally, or parenterally, for instance orally in a bioadhesive composition to adhere to the gastro-intestinal tract or parenterally, intramuscularly, intraperitoneally, intravenously or epidurally. The compounds may be administered alone, for instance in a capsule, but will generally be administered in conjunction with a pharmaceutically acceptable carrier or diluent. The invention extends to methods for preparing a pharmaceutical composition comprising bringing a compound of formula I or its pharmaceutically acceptable salt in conjunction or association with a pharmaceutically acceptable carrier, adjuvant, excipient or vehicle.

The term topical herein means any application on the outside of the body but also applies to the topical administration on the mucous membranes of the gastro-intestinal tract, such as by means of a mucoadhesive composition adhering to e. g. the intestines where it serves its therapeutical effect.

Oral formulations are conveniently prepared in unit dosage form, such as capsules or tablets, employing conventional carriers or binders such as magnesium stearate, chalk, starch, lactose, wax, gum or gelatine. Liposomes or synthetic or natural polymers such as HPMC or PVP may be used to afford a sustained release formulation. Alternatively the formulation may be presented as a nasal or eye drop, syrup, gel or cream comprising a solution, suspension, emulsion, oil-in-water or water-in-oil preparation in conventional vehicles such as water, saline, ethanol, vegetable oil or glycerine, optionally with flavouring agent and/or preservative and/or emulsifier. Any formulation may contain 0.5 to 99.5% by weight of the therapeutically active compound.

Examples

The following examples are intended to illustrate but not to limit the scope of the invention. Example 1

3,4,5-trimethoxyphenyl-2,4-diaminoquinazoline-6-carboxylate (Compound No. 31)

STEP-I: To a solution of 3-cyano 4-fluro benzoic acid (2 g, 0.012 mol) in dry MeOH (20 ml) was added thionyl chloride (1.2 ml, 0.018 mol) at 0 ⁰C and heated to reflux for 3 hours. The reaction mixture was concentrated under vacuum to afford the step-1 product (2 g, 95 %) as white solid. This was used as such for the next step. Rf = 0.5 (CHCI3: MeOH, 9:1).

STEP-2: To a solution of step-1 product (2 g, 0.011 mol) in dry (dimethyl formamide) DMF (40 ml) was added guanidine carbonate (4 g, 0.022 mol) and heated to 100 ⁰C for 3 hours. The reaction mixture was cooled to RT and added to ice cold water (200 ml). The solid precipitated was filtered and dried to afford the step-2 product (2 g, 86 %) as a yellow solid. Rf = 0.3 (CHCl₃: MeOH, 8:2).

STEP-3: To a solution of step-2 product (1 g, 0.0045 mol) in 1 :1 THF:water (30 ml) was added sodium hydroxide (0.56 g, 0.013 mol) and heated to reflux at 70 ⁰C for 4 hours. After completion of the reaction, the reaction mixture was cooled to room temperature (RT) and concentrated under vacuum. The residue was neutralized with 1.5 N HCl (50 ml). The solid precipitated was filtered and dried to afford the step-3 product (0.7 g, 77 %) as white solid.

STEP-4: To a solution of step-3 product (0.5 g, 0.0024 mol) in dry DMF (20 ml) were added l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) (0.9 g, 0.0048 mol), 1-hydroxybenzotriazole (HOBt) (0.11 g, 0.00072 mol), triethyl amine (1 ml, 0.0072 mol), and 3,4,5-trimethoxy phenol (0.45 g, 0.0024 mol). The reaction mixture was stirred overnight at RT. The reaction mixture was added to ice cold water (100 ml) and the solid precipitated was filtered and dried. The crude material was purified by silica gel column chromatography using 10 % MeOH in CHCI3 as an eluent to afford the final product, Compound No. 31, (0.15 g, 18 %) as a white powder. R_f = 0.3 (CHCl₃: MeOH, 9:1).

Example 2

2,4-diaminoquinazolin-6-yl-2,5-dimethoxybenzoate (Compound No. 32)

Guanidine carbonate Step-3

STEP-I: To a solution of 2-fluoro-5-methoxy benzonitrile (10 g, 0.066 mol) in dry di- chloromethane (DCM) (200 ml) was added BBr₃ (16 ml, 0.0092 mol) at 0 ⁰C and stirred at RT for two days. Reaction mixture was quenched with 10 % sodium bicarbonate (50 ml). The aqueous layer was extracted with DCM (2 X 200 ml). The combined organic layers were dried over anhydrous sodium sulphate and concentrated to afford step-1 product (5.5 g, 61 %). Rf= 0.4 (CHCl₃: MeOH, 9:1).

STEP-2: To a suspension of sodium hydride (2 g, 0.048 mol) in dry DMF (50 ml) at 0 ⁰C was added a solution of step-1 product (5.5 g, 0.040 mol) in dry DMF (50 ml) and stirred for

15 minutes. To this was added benzyl bromide (5 ml, 0.040 mol) in a drop-wise manner. The reaction mixture was allowed to stir at RT for 6 hours under N₂ atmosphere. The reaction mixture was quenched with ice-cold water (100 ml). The solid precipitated was filtered off and dried to afford the step-2 product (8 g, 88 %) as white solid. Rf = 0.5 (petroleum ether: EtOAc, 7:3). STEP-3: To a solution of step-2 (8 g, 0.035 mol) in dry DMF (100 ml) was added guanidine carbonate (12.6 g, 0.070 mol) and heated to 145 ⁰C for 10 hours under N₂. The reaction mixture was cooled to RT and poured into ice cold water (250 ml). The solid precipitated was filtered and dried. The crude material was purified by silica gel column chromatography using 80 % EtoAc in petroleum ether as an eluent to afford the step-3 product (3 g, 32 %) as a pale yellow solid. R_f = 0.35 (CHCl₃: MeoH, 8:2).

STEP-4: To a solution of Step-3 product (3 g, 0.0112 mol) in dry methanol (200 ml) was added palladium on carbon (0.5 g) followed by hydrogenation under balloon pressure for 8 hours. The reaction mixture was filtered through celite and washed thoroughly using MeOH. The combined filtrate and washings were concentrated to afford the step-4 product (1.5 g, 78%) as pale yellow solid. R_f= 0.2 (CHCl₃: MeOH, 8:2).

STEP-5: To a solution of step-4 compound (0.5 g, 0.0028 mol) in dry DMF (30 ml) was added EDCI (1 g, 0.0056 mol), HOBt (0.12 g, 0.00084 mol), triethyl amine (1.2 ml, 0.0084 mol), and 2,5 dimethoxy benzoic acid (0.52 g, 0.0028 mol). The reaction mixture was stirred overnight at RT under N₂. After completion of the reaction, water (20 ml) was added to the reaction mixture and extraction with ethyl acetate (2 X 50 ml) was effected. The combined organic layer was washed with water (2 X 50 ml), dried over anhydrous sodium sulphate and concentrated. The crude material was purified by silica gel column chromatography using 8 %

MeOH in CHCl₃ as an eluent to afford the final product, Compound No. 32, (150 mg, 15%) as an off-white solid. R_f = 0.3 (CHC13: MeoH, 8:2).

Example 3 3,5-dichlorophenyl-2-amino-4-oxo-3,4-dihydroquinazoline-6-carboxylate

Pd/C

Step-3 H2

4-Nitro-isophthalic acid: To an emulsion of m-nitroxylene (10 g, 0.065 mol) in water (500 ml) was added KMnO₄ (104.2 g, 0.65 mol) at RT and heated at 85 ⁰C for 2 h. The solid MnO₂ that settled was filtered off. The filtrate was neutralized with 1.5N HCl (50 ml) and extracted with EtOAc (2x100 ml). The organic layer was washed with water and brine solution, dried over anhydrous Na₂SO₄ and concentrated to obtain a white solid 2. Yield: 19.6 % (2.75 g).

4-Nitro-isophthalic acid dimethyl ester: To a stirred solution of 4-nitro-isophthalic acid (3.4 g, 0.016 mol) in dry methanol (35 ml) was added thionyl chloride (5.8 ml 0.08 mol). The solution was refluxed for 1O h. On removing methanol, the solid was obtained and was washed with water to remove excess thionyl chloride. The crude solid was dissolved in EtOAc and washed with water two times. The organic layer was dried over anhydrous Na₂SO₄ and concentrated to result in 3 as a white solid. Yield: 70.5 % (2.7 g).

4-Amino-isophthalic acid dimethyl ester: To a suspension of 4-nitro-isophthalic acid dimethyl ester (3.5 g, 0.014 mol) in dry methanol (200 ml) was added Pd/C (7 g, 50 % w/w) in an autoclave. The suspension was stirred at RT under 3 kg H₂ pressure. The solution was filtered through a celite bed and the filtrate concentrated to afford 4. Yield: 63.7 % (1.95 g).

3,5-dichlorophenyl-2-amino-4-oxo-3,4-dihydroquinazoline-6-carboxylate (or 2-Amino-4- oxo-3,4-dihydro-quinazoline-6-carboxylic acid methyl ester): The diester 4-Amino- isophthalic acid dimethyl ester (2.4 g, 0.011 mol) was added to a mixture containing dimethyl sulfone (12 g) and chloroformamidine hydrochloride (2.64 g 0.022 mol). The reaction content was heated at 150 ⁰C for 2h and cooled to RT. The solid mass was added with water (50 ml) and stirred for 30 min. On adding aqueous ammonia to this suspension, the solid was precipitated out and filtered off. The solid was washed with water and dried to obtain 5 as off- white solid. Yield: 85 % (2.1 g)

2-Amino-4-oxo-3,4-dihydro-quinazoline-6-carboxylic acid: A suspension of 2-Amino-4- oxo-3,4-dihydro-quinazoline-6-carboxylic acid methyl ester (1.3 g, 0.006 mol) in NaOH

/water (1 :1) was refluxed at 75⁰C for 12 h. The reaction mixture was rotoevaporated and residue neutralized withl.5N HCl. The resultant precipitate was filtered to obtain 6 as white solid. Yield: 70 % (0.85 g).

2-Amino-4-oxo-3,4-dihydro-quinazoline-6-carboxylic acid 3,5-dichloro-phenyl ester: To a suspension of 2-Amino-4-oxo-3,4-dihydro-quinazoline-6-carboxylic acid (100 mg, 0.49 mmol) in DMF (20 ml) was added EDCLHCl (190 mg, 0.97 mmol), HOBT (20 mg, 0.1 mmol), phenol (80 mg, 0.48 mmol) and triethylamine (0.1 ml). The reaction was stirred at RT for two days. It was poured onto ice and extracted with ethyl acetate (25 ml). The organic layer was washed with water (2x100 ml) and brine, dried over Na₂SO₄ and filtered. The filtrate was concentrated and the solid recrystallized using methanol to afford 7 as a pale yellow solid. Yield: 15 % (25 mg). Additionally, the compounds represented in table 1 were synthesized using the above described methods.

Table 1

Cmp. Structural formula Name

No.

3 ,4-dimethoxyphenyl-2,4- diaminoquinazo line-6- carboxylate

2,4-dimethoxyphenyl-2,4- diaminoquinazo line-6- carboxylate

1 ,3-benzodioxol-5-yl-2,4- diaminoquinazo line-6- carboxylate

2,6-dimethoxyphenyl-2,4- diaminoquinazo line-6- carboxylate

2,3-dimethoxyphenyl-2,4- diaminoquinazo line-6- carboxylate

4-fluoro-2-methoxyphenyl-

2,4-diaminoquinazoline-6- carboxylate

3,5-bis(trifluoromethyl)- phenyl-2,4-diamino- quinazo line-6-carboxylate

3,5-dichlorophenyl-2,4- diaminoquinazo line-6- carboxylate;

4- [(trifluoromethyl)thio] - phenyl-2,4-diamino- quinazo line-6-carboxylate

2,4-diaminoquinazolin-6-yl-

3,5-dimethoxy-4-methyl- benzoate

2,4-diaminoquinazolin-6-yl- 3,4,5-triethoxybenzoate;

2,4-diaminoquinazolin-6-yl- 2-methoxybenzoate

2,4-diaminoquinazolin-6-yl- 2-methoxy-5 -methylbenzoate

2,4-diaminoquinazolin-6-yl- 3 -(methylthio)benzoate;

2,4-diaminoquinazolin-6-yl- 2 , 5 -bis(trifluoromethy I)- benzoate;

2,4-diaminoquinazolin-6-yl- 2-methoxy-5 -(nitromethyl)- benzoate

2,4-diaminoquinazolin-6-yl- 2-(methylamino)benzoate

2,4-diaminoquinazolin-6-yl- 2- [(ethoxycarbonyl)amino] - 4,5-dimethoxybenzoate

2,4-diaminoquinazolin-6-yl- 2-(methylthio)benzoate

2,4-diaminoquinazolin-6-yl- 5 -fluoro-2-(trifluoromethyl)- benzoate

3 -(dimethylamino)phenyl- 2,4-diaminoquinazoline-6- carboxylate

2,4-diaminoquinazolin-6-yl- 4-amino-2-methoxybenzoate

5-amino-2-methoxyphenyl-

2,4-diaminoquinazoline-6- carboxylate

3,4,5-trimethoxyphenyl-2,4- diaminoquinazo line-6- carboxylate

2,4-diaminoquinazolin-6-yl- 2,5-dimethoxybenzoate

3,5-dichlorophenyl-2-amino- 4-0X0-3, 4-dihydro- quinazo line-6-carboxylate

Herein below, biological tests performed with compounds of the invention are described.

Biological tests Compounds of the invention were tested in a number of biological tests, in vivo and in vitro.

1. IN VIVO TEST OF DSS-INDUCED MURINE COLITIS

The main objective was to study the effects of drugs to alleviate dextran sodium sulphate (DSS) induced colitis in mice. DSS-induced colitis, is one of the most widely used animal models for inflammatory bowel disease (IBD).

Fifty-one 7 weeks old female Balb/c mice were used in the study. The animals were treated with dextran sodium sulphate dissolved in water in concentrations (2 - 3.5%) during a 29 days period where after groups of animals were treated for 5.5 days by rectal instillation of Compound No. 31 and Compound No. 32, in 100 or 300 μmolar concentrations. NMP (N- methylpyrrolidone) 10% in a solution of 2.0% hydroxypropyl methyl cellulose (HPMC) in 2 % glycerol (aq.) for tonicity, the vehicle, was used as control item. The DSS treatment was discontinued at day 32.

On commencement of the DSS treatment the animals were weighed once daily and checked for change in food intake, activity etc as signs of a change in general health status. On completion of the study the animals were anaesthetised and blood was obtained for hae- matology. The colons were excised and the wet weight and length thereof were determined before placing them in 4% buffered formalin for fixation and subsequent histopathological determination of lesions and infiltration of inflammatory cells.

The treatment with DSS in a concentration of 3.5% which started at day 24 produced a drastic increase in number of animals showing blood in faeces. At this time body weights started to decrease. Some animals decreased rapidly in weight and were found moribund and were thus euthanized. A fair number of animals did nevertheless survive the whole experimental period.

1.1 Test Item The active substances were weighed and dissolved in N-methylpyrrolidon (NMP) and were thereafter diluted in a polymer solution consisting of 2% hydroxypropyl methyl cellulose (HPMC; Hypromellos) in 2% glycerol (aq). The final concentration of NMP was 10%. The solutions were prepared in 20 ml glass vials with Al-shrink caps. All vials were kept at room temperature protected from light until use and between administrations.

1.2 Control Item

The control item consisted of NMP (N-methylpyrrolidone) 10% in a solution of 2.0% hydroxypropyl methyl cellulose (HPMC) in 2 % glycerol (aq.) for tonicity.

1.3 Animals

Fifty-one 7 weeks old female Balb/c mice were purchased and allowed an acclimatisation period of a minimum of 7 days prior to the commencement of the experiment. The study was performed in two series, with about half of the animals per each series (se division below). The experiment was approved by the regional animal experimental ethics committee in Uppsala (C207/6).

The animals were treated with vehicle, Compound No. 31 and Compound No. 32, rectally twice daily for 5.5 days starting on day 29 of the DSS treatment. The instillation of drugs was performed in conscious animals. A volume of 100 μl of drug solution or vehicle was delivered into the colon through a 1.2 x 38 mm Teflon gavage tubing with silicone tip (AgnThos, 5202) by the rectal approach after lubrication. The tip of the tubing was inserted about 30 mm into the colon as measured from anus.

The animals were allocated to treatment groups as shown in table 2.

Table 2

Note: n = number of animals

On commencement of the DSS treatment the animals were weighed once daily during the treatment period.

The animals were checked daily for change in food intake, activity etc as signs of a change in general health status.

On completion of the study the animals were anaesthetised with Isofluran one hour after drug administration at day 6 and as much blood as possible was obtained through the orbital plexus and collected in K /EDTA tubes. The blood samples were analysed for concentration of white blood cells (WBC), concentration of red blood cells (RBC), haemoglobin, hematocrite, and platelet count (Fig. T). The remaining blood was centrifuged to obtain plasma. The plasma was frozen at -80°C and stored until shipped to the sponsor for bioanalyses. The animals were euthanized at day 34 after start of DSS treatment. Colons were excised from ampulla coli and as close to the pelvic bone as possible dorsal to the bladder and the wet weight and length was determined before the sample was placed in 4% buffered formalin for fixation. Notes were taken about the macroscopic appearance of the colon epithelia at ne- cropsy including % mucosal area affected. Since the lengths of colons differed the colons were divided as follows to obtain, as far as possible, sections from comparable regions: Two pieces were cut one centimetre from each ends, the remaining part was then cut in two halves and these two pieces were in turn cut in two halves. This procedure provided 5 cut surfaces from which sections were taken for histopathological determination of lesions and infiltration of inflammatory cells.

1.4 Results

Animal weights

Animals treated with Compound No. 31, high dose were the sole group of animals that had gained weight at day 34 in comparison to controls (Fig. 3).

Health status

At start of the treatment 2% DSS was mixed in the drinking water. It was anticipated that this would result in a decrease in body weight and signs of colitis such as diarrhoea and blood in faeces. However, after 13 days of treatment no such signs were observed, therefore the DSS concentration was increased to 2.5 %. During 2.5% DSS treatment a few animals were observed to have some blood in faeces but without weight reductions. At day 19 the DSS concentration was increased to 3.0% again without any remarkable changes in health status or weights. After increasing the DSS concentration to 3.5% at day 24 a drastic increase in num- ber of animals that showed blood in faeces were noted, and the body weights started to decrease.

Necropsy

At necropsy the weight, length and % affected area of colons were recorded (Fig. 1). Animals treated with Compound No. 31 had generally longer colons and less affected epithelium.

Notes from necropsy show that the most obvious sign of disease was bleedings from the epithelium. No other macroscopic lesions were observed. Pathology

Dissected colons were sectioned transversally at 5 levels and scored for destruction and infiltration of inflammatory cells. The destruction scores were 0 - morphologically normal, 1 - focal destruction of the epithelial surface and/or focal destruction of crypts, 2 - zonal destruction of the epithelial surface and/or zonal loss of crypts, 3 - diffuse ulceration of mucosa involving the submucosa and/or diffuse loss of crypts. The infiltration scores were 0 - no infiltration of inflammatory cells, 1 - infiltration of lamina propria, subepithelially or in crypts, 2 - infiltration down to muscularis mucosae, 3 - severe and extensive infiltrations down to submucosa and /or engagement of tunica muscularis.

The median of destruction and infiltration scores respectively were calculated for each animal. Group-wise scores are shown in table 3. These scores are further summarized in table 4. Animals treated with, Compound No. 31 high dose and Compound No. 32 low dose had the lowest scores on destruction of colon epithelium and infiltration of inflammatory cells.

Table 3

Table 4

Group Destruction Infiltration

Vehicle Median 1.0 1.0 n 3.0 3.0

Comp. No. 31 Low dose Median 1.0 1.0 n 5.0 5.0

Comp. No. 31 High dose Median 0.0 0.5 n 4.0 4.0

Comp. No. 32 Low dose Median 0.5 0.5 n 4.0 4.0

Comp. No. 32 High dose Median 1.0 2.0 n 5.0 5.0

Total Median 1.0 1.0 n 21 21

Note: n = number of animals.

1.5 Discussion

It is evident that the DSS treatment affected the general health status of the animals shown mostly as a reduction in weight but also as blood in faeces. Animals treated with the high dose of Compound No. 31 were the only ones that at day 34 had gained weight in comparison to controls. This observation along with the findings that animals treated with Compound No. 31 had the longest dissected colons and the less affected area, less destruction of colon epithelium accompanied with less infiltration of inflammatory cells indicates that Compound No. 31 produced some protective effects against the DSS induced adverse effects on colon. The low dose of Compound No. 32 seems also to have had some protective actions similar to Compound No. 31, but less pronounced.

1.6 Conclusion

Although few animals were included in the different treatment groups or survived throughout the whole experiment it seems as the high dose of Compound No. 31 and the low dose of Compound No. 32 produced protection against the deleterious effects of DSS on colon and improved the general health status of animals shown as an increase in weights. 2. IN-VITRO DHFR INHIBTION

2.1 Principle of the assay

The DHFR enzyme is involved in the reduction of dihydrofolate (DHF) to tetrahydro folate (THF). In this process, NADPH is converted into NADP. The activity of DHFR enzyme is measured with its ability to effect the conversion of NADPH to NADP. This reduction reaction is being monitored kinetically in a spectrophotometer at the wave length of 340 nm for 3 min.

2.2 Preparation of compounds Vehicle control : DMSO 0.1% (final concentration)

100% DMSO diluted in IX assay buffer to make concentration to 10 % 10 μL of 10% DMSO was added in 1 mL reaction to give a final concentration of 0.1%.

Preparation of test compound: About 1-2 mg of the test compounds were weighed in a sterile microcentrifuge tube, IOOX stock were prepared and 10 μL was added into the 1 mL reaction. Stock concentration (100 mM) of compounds was prepared with 100% DMSO as described in table 5.

2.3 Methodology The assay was performed with human DHFR expressed and purified from E.coli expression system.

Short protocol: IX assay buffer containing 25 μL of DHFR enzyme (1.5 x 10^" units) and appropriate concentration of inhibitor was pre-incubated for 3 min at room temperature. Then, the whole mixture was transferred to 1 mL quartz cuvette (10 mm path length). The reaction was initiated by adding NADPH (60 μm) and Dihydro folic acid (50 μm). The cuvette was mixed by inversion and the reading was started immediately at 340 nm (22-25⁰C) in spectrophotometer. Decrease in absorbance was monitored for 3 min in kinetic mode. Decreased ab- sorbance measured from the initial absorbance and the specific activity of the enzyme is cal- culated using the following formula.

(ΔOD/min. inhibitor-(ΔOD/min. Blank) x d Specific activity (Units/mg) =

12.3 xV x mg protein/mL Where:

ΔOD/min. blank: Decreased absorbance with blank ΔOD/min. inhibitor: Decreased absorbance with inhibitor 12.3: Excitation coefficient (ε, mM^"1 cm^"1) for the DHFR reaction at 340 nm V: Enzyme volume in mL used in the assay d: Dilution factor of the enzyme sample mg protein/mL: Enzyme concentration/mL Units/mg: Specific activity on μM/min/mg protein

Unit definition: One unit will convert 1.0 μM of DHF to THF in 1 min (pH 7.5) at 22⁰C (equivalent to the conversion of NADPH to NADP).

Reaction protocol

The final volume (1 mL) in the reaction mixture was made as per the table 5.

Table 5

Results

Inventive compounds were tested for their inhibition potential against the human rDHFR enzyme activity.

The percentage inhibitions of the tested compounds are presented in table 6. Table 6

3. IN-VITRO PK STUDIES

3.1 Biological materials and incubations

Liver samples Human liver samples were obtained from transplantation donors. The Ethics Committee of the Medical Faculty has approved the collection of surplus human tissues for research purposes. For most studies, a pool of five liver microsomal preparations (HL20 through HL24), which have been extensively characterized to be used for the primary screening (sufficient model activities, no known polymorphisms, expected effects of model inhibitors, quantitation of CYPs by Western blotting), were employed. Rat liver samples were from storaged livers of male Sprague-Dawley rats, which had been obtained and used for other experiments than the current one. Livers were leftover tissues, which were collected on the basis of a permission of Animal Experiment Committee of the Experimental Animal Centre of the University of OuIu

Normal- looking liver tissue was homogenized in four volumes of ice-cold 0.1 mM phosphate buffer, pH 7.4. S9 fraction was prepared by centrifuging the liver homogenate for 30 min at 10 000 rpm and collecting the supernatant.

Incubation conditions

The study compounds were incubated with liver S9-fractions in the presence of appropriate cofactors. The basic incubation mixture consisted of 100 μl S9-fraction, substrate in DMSO and 1 mM NADPH. The substrate concentration used was 100 μM. Two parallel incubates, one with cofactors and one without, were employed. Each reaction mixture was preincubated for 2 minutes at +37 °C in a shaking incubator block (Eppendorf Thermomixer 5436, Hamburg, Germany). Reaction was started by addition of co factor mixture. Samples of 200 μl volume were taken from incubates after incubation at +37 °C for 0 and 60 min, and put into vials containing 100 μl of ice-cold acetonitrile. The tubes were stored at -18 °C until analysis. The incubates were transferred to Novamass Analytical in ice. 3.2 Analytical methods

Sample preparation

The incubation samples were thawed at room temperature (RT), shaken and centrifuged for 10 min at 12 100 x g (Eppendorf Mini Spin, Eppendorf AG, Hamburg, Germany) and pipetted to Total Recovery vials (Waters Corporation, Milford, Massachusetts, USA) to wait for an autosampler run.

HPLC conditions A Waters Alliance 2695 chromatographic system (Waters Corp., Milford, MA, USA) with autosampler, vacuum degasser and column oven was used. The analytical column used for all compounds was a Waters Xbridge ShieldRP18, 2.1x50 mm, 3.5 μm together with Phenome- nex Luna Cl 8 precolumn, 4.0^χ2.0 mm, 3.0 μm (Phenomenex, Torrance, California, USA). The eluents were (A) 0.1% acetic acid in water (pH 3.2) and (B) acetonitrile. A linear gradient elution from 5% B to 60% B in 6 minutes was applied, following equilibration with initial conditions. The flow rate was 0.3 ml/min and the column oven temperature was 3O⁰C. The flow was split post-column with an Acurate Post-Column Stream Splitter (LC Packings, Amsterdam, The Netherlands) with a ratio of 1 :3 to MS and Waters 2996 photo-diode-array- (PDA)-detector, respectively.

LC/ESI/MS conditions

LC/MS data were recorded with a Micromass LCT time-of- flight (TOF) high resolution mass spectrometer (Micromass Ltd., Manchester, England) equipped with a LockSpray electros- pray ionization source. A positive ionization mode of electrospray was used with cone voltage of 30V. The mass range of m/z 140 - 800 was acquired. The mass spectrometer and HPLC system were operated under Micromass MassLynx 4.0 software. Complete LC/MS parameters of the final method are presented in Appendix I. Before LC/MS runs, the instrument was tuned with 1 μg/ml solution of substrate in order to maximally produce molecular ions using a syringe pump (Harvard Apparatus, Holliston, USA) with a flow rate of 6 μL/min. Leucine enkephalin was used as a lock mass compound ([M+H]⁺ m/z 556.2771) for accurate mass measurements and was delivered into LockSpray probe with syringe pump to obtain ion count of about 160 per spectrum. No quantification was performed. The disappearances of study substrates were determined by comparing the LC/PDA peak area in appropriate 0 min sample (without NADPH) to peak area of corresponding metabolized sample at wavelengths of 283 nm and 324 nm for Compounds No. 31 and 32, respectively. The relative abundances of individual metabolites in metabolic profiles were determined by ESI/MS peak areas of molecular ion of a particular metabolite, assuming their responses to be directly comparable. The area of that metabolite was then compared to the combined area of all metabolites found in that same incubation.

3.3 Results And Discussion

The disappearance of the substrates

The remaining LC/PDA peak areas (relative to 0 min incubation samples, %) detected for the substrates are shown in the Figs. 4 and 5. As can be seen, very similar disappearance was observed in human and rat cells, and also with and without co factors for both of the compounds. This suggests that the disappearance is not cofactor dependent and thus not due to oxidative metabolism, but rather due chemical degradation or binding into the walls of the incubation vessel or the proteins present in the incubations.

Identification of the metabolites/degradation products

The LC/MS data obtained from the detected metabolites is shown in the table 7. The suggested structures for the detected metabolites are shown in the Figs. 8 and 9.

Three metabolites were detected for Compound No. 31. The main metabolites, Ml and M2, were formed via hydrolysis of the ester bond of the substrate. No accurate mass data was obtained for M2 due to low its m/z value. The minor metabolite, M3, was identified to be formed via demethylation, hydroxylation and addition Of H₂O. The abundance of M3 was too low for obtaining adequate fragmentation data, and thus biotransformation site in the substrate was not elucidated.

Also for Compound No. 32 three metabolites were detected. All of the metabolites were tentatively identified to be formed via opening of the ester bond (hydrolysis) in the substrate molecule. No accurate mass measurement was obtained for the metabolites of Compound No. 32 due to m/z values that were under calibration range. Table 7 LC/ESI/TOF MS data obtained from the substrates and their metabolites.

Comp RT* m/z identification meas. calc. accurate accurate mass (Da) mass (Da)

Compound 5.3 371.1 [M+H]^τ 371.1352 371.1355

No. 31 min

Ml 1.5 205.1 [M+H]⁺ 205.0731 205.0725 min

M2 5.3 185.1 [M+H]⁺ 185. 0814 min 170.1 [M+H-CH₃]⁺ 170. 0579

153.1 [M+H-CH₃OH]⁴ 153. 0551

M2 4.4 391.1 [M+H]⁺ 391 .1277 391. 1253 min

Compound 5.5 341.1 [M+H]⁺ 341 .1250 341. 1250

No. 32 mmimn

Ml 11..44 117777..11 Γ [MM++HHI]⁺⁴ 177.0776 min

M2 5.4 165.1 [M+H]⁴ 165.0551 min

M3 5.9 165.1 [M+H]⁴ 165.0551 min

* out of calibration range

3.4 Metabolic profiles

The LC/MS peak areas for the detected metabolites in different incubations are shown in Figs. 6 and 7. The detected metabolites were formed almost equally with and without cofac- tors, being thus not cofactor-dependent, and also suggesting these compounds not to be formed by P450-mediated metabolism, but rather due to liver esterases or by chemical degradation. The main metabolites for Compound No. 31 were Ml and M2, having 64% and 35% of the total combined metabolite peak area, respectively. The main metabolite for Compound No. 32 was Ml, having almost 50% of the total combined metabolite peak area, while the M2 and M3 had both about 25% of the total combined metabolite peak area.

Claims

Claims

1. A compound of formula (I)

(I)

wherein

X is selected from NH₂ and OH;

W is selected from -C(O)-O- and -O-C(O)-;

each R¹ is independently selected from R², -OR², -SR², -N(R²R³), -C(O)R², -OC(O)R², -NH-C(O)-R², -NHC(O)OR² and/or two R¹ are attached to adjacent atoms of the phenyl linked together so as to form a 5-6 membered ring fused to the phenyl ring;

R² and R³ are independently selected from H and C1-C6 alkyl, optionally substituted by one or several several moieties selected from halogen and nitro; or a pharmaceutically acceptable salt thereof.

2. A compound according to claim 1, wherein X is NH2.

3. A compound according to claim 1, wherein X is OH.

4. A compound according to any one of the claims 1-3, wherein W is -C(O)-O-.

5. A compound according to any one of the claims 1-3, wherein W is -0-C(O)- .

6. A compound according to any one of the claims 1-5, wherein R² and R³ are independently selected from H and C1-C3 alkyl, optionally substituted by one or several moieties selected from halogen and nitro.

5 7. A compound according to any one of the claims 1-6, wherein each R¹ is independently selected from H, CH₃-, CH₃O-, F, CH₃CH₂-, CH₃C(O)-, CH₃C(O)O-, CH₃C(O)NH-, CH₃S-, CF₃-, Cl, CF₃S-, CH₃CH2O-, NO₂CH₂-, CH₃NH-, CH₃CH₂OC(O)NH-, (CH₃)₂N- and/or two adjacent R¹ forming together a methylene dioxy radical -0-CH₂-O-. 0

8. A compound according to claim 1, selected from

3,4-dimethoxyphenyl-2,4-diaminoquinazoline-6-carboxylate;

2,4-dimethoxyphenyl-2,4-diaminoquinazoline-6-carboxylate; l,3-benzodioxol-5-yl-2,4-diaminoquinazoline-6-carboxylate;

2,6-dimethoxyphenyl-2,4-diaminoquinazoline-6-carboxylate; 5 2,3-dimethoxyphenyl-2,4-diaminoquinazoline-6-carboxylate;

4-fluoro-2-methoxyphenyl-2,4-diaminoquinazoline-6-carboxylate;

4-ethyl-2-methoxyphenyl-2,4-diaminoquinazoline-6-carboxylate;

4-acetyl-2-methoxyphenyl-2,4-diaminoquinazoline-6-carboxylate;

2,4-diaminoquinazolin-6-yl-4-(acetyloxy)-3,5-dimethoxybenzoate; 0 3-(acetylamino)phenyl-2,4-diaminoquinazoline-6-carboxylate;

4-(methylthio)phenyl-2,4-diaminoquinazoline-6-carboxylate;

3,4,5-trifluorophenyl-2,4-diaminoquinazoline-6-carboxylate;

4-(acetylamino)phenyl-2,4-diaminoquinazoline-6-carboxylate;

3,5-bis(trifluoromethyl)phenyl-2,4-diaminoquinazoline-6-carboxylate; 5 3,5-dichlorophenyl-2,4-diaminoquinazoline-6-carboxylate;

4-[(trifluoromethyl)thio]phenyl-2,4-diaminoquinazoline-6-carboxylate;

2,4-diaminoquinazolin-6-yl-3,5-dimethoxy-4-methylbenzoate;

2,4-diaminoquinazolin-6-yl-3,4,5-triethoxybenzoate;

2,4-diaminoquinazolin-6-yl-2-methoxybenzoate; O 2,4-diaminoquinazo lin-6-yl-2-methoxy-5 -methylbenzoate;

2,4-diaminoquinazolin-6-yl-3-(methylthio)benzoate;

2,4-diaminoquinazo lin-6-yl-2,5-bis(trifluoromethyl)benzoate;

2,4-diaminoquinazo lin-6-yl-2-methoxy-5-(nitromethyl)benzoate;

2,4-diaminoquinazo lin-6-yl-2-(methylamino)benzoate; 2,4-diaminoquinazolin-6-yl-2-[(ethoxycarbonyl)amino]-4,5-dimethoxybenzoate

2,4-diaminoquinazolin-6-yl-2-(methylthio)benzoate;

2,4-diaminoquinazolin-6-yl-5-fluoro-2-(trifluoromethyl)benzoate;

3-(dimethylamino)phenyl-2,4-diaminoquinazoline-6-carboxylate; 2,4-diaminoquinazolin-6-yl-4-amino-2-methoxybenzoate;

5-amino-2-methoxyphenyl-2,4-diaminoquinazoline-6-carboxylate;

3,4,5-trimethoxyphenyl-2,4-diaminoquinazoline-6-carboxylate;

2,4-diaminoquinazolin-6-yl-2,5-dimethoxybenzoate;

3,5-dichlorophenyl-2-amino-4-oxo-3,4-dihydroquinazoline-6-carboxylate; l,3-benzodioxol-5-yl-2-amino-4-oxo-3,4-dihydroquinazoline-6-carboxylate;

3-(acetylamino)phenyl-2-amino-4-oxo-3,4-dihydroquinazoline-6-carboxylate;

4-(methylthio)phenyl-2-amino-4-oxo-3,4-dihydroquinazoline-6-carboxylate; and

3,4,5-trimethoxyphenyl-2-amino-4-oxo-3,4-dihydroquinazoline-6-carboxylate, or a pharmaceutically acceptable salt thereof.

9. A compound according to any one of the claims 1 to 8, or a pharmaceutically acceptable salt thereof, for use as a medicament.

10. A pharmaceutical composition comprising a compound according to any one of the claims 1 to 8, or a pharmaceutically acceptable salt thereof, together with one or more carriers, adjuvants or excipients.

11. Use of a compound according to any one of the claims 1-8, or a pharmaceutically acceptable salt thereof, in the manufacturing of a medicament for the treatment of a disorder se- lected from inflammation, autoimmune disorder, immunogenic rejection reactions and cancer and for non-surgical abortion.

12. Use according to claim 11, where the disorder is selected from inflammatory bowel disease i. e. ulcerative colitis and Crohn's disease, asthma, Pneumocystis carinii pneumonia (PCP), psoriasis, inflammations caused by bacteria, fungi, protozoa, rheumatoid arthritis, colorectal cancer, cancer of the urinary bladder, the skin, or the lung; or an immunogenic rejection reaction in connection to liver or intestine transplantation.

13. A compound according to any one of the claims 1-8, or a pharmaceutically acceptable salt thereof, for the treatment of a disorder selected from inflammation, autoimmune disorder, immunogenic rejection reactions and cancer and for non- surgical abortion.

14. A compound according to claim 13, where the disorder is selected from inflammatory bowel disease i. e. ulcerative colitis and Crohn's disease, asthma, Pneumocystis carinii pneumonia (PCP), psoriasis, inflammations caused by bacteria, fungi, protozoa, rheumatoid arthritis, colorectal cancer, cancer of the urinary bladder, the skin, or the lung; or an immunogenic rejection reaction in connection to liver or intestine transplantation.

14. A method for the treatment of a mammal suffering from a disorder selected from inflammation, autoimmune disorder, cancer and susceptibility to develop an immunogenic rejection reaction or to provoke a non-surgical abortion in said mammal, by administering to said mammal a therapeutically effective amount of a compound according to any one of the claims 1-8.

15. A method according to claim 14, wherein the disorder is selected from inflammatory bowel disease i. e. ulcerative colitis and Crohn's disease, asthma, Pneumocystis carinii pneumonia (PCP), psoriasis, inflammations caused by bacteria, fungi, protozoa, rheumatoid arthri- tis, colorectal cancer, cancer of the urinary bladder, the skin, or the lung; or an immunogenic rejection reaction in connection to liver or intestine transplantations.