WO2008056149A1

WO2008056149A1 - Quinazoline derivatives and pharmaceutical compositions containing them

Info

Publication number: WO2008056149A1
Application number: PCT/GB2007/004264
Authority: WO
Inventors: Surinder Chana
Original assignee: Arrow Therapeutics Limited
Priority date: 2006-11-09
Filing date: 2007-11-08
Publication date: 2008-05-15

Abstract

Compounds of formula (I) are found to be active in inhibiting replication of flaviviridae viruses, R is C1-C4 alkyl or C1-C4 alkoxy.

Description

QUINAZOLINE DERIVATIVES AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM

The present invention relates to specific quinazoline derivatives which are useful in treating or preventing a flaviviridae infection and which have enhanced permeability, absorption and bioavailability.

Viruses of the family flaviviridae are small, icosahedral, enveloped viruses that contain a positive-sense RNA genome. The family consists of three genera, flavivirus, pestivirus and hepacivirus. Many of the flaviviridae viruses are important human pathogens. Indeed, the hepacivirus genus includes the hepatitis C virus. Compounds active against flaviviridae infections are therefore of potential significant therapeutic benefit.

A pre-requisite of orally bioavailable drugs is that they are absorbed from the gastrointestinal tract. Compounds exhibiting higher rate constants for absorption are inherently more likely to achieve complete absorption, earlier achievement of therapeutic plasma concentrations and higher overall bioavailability for a given dose level. Mandin Darby Canine Kidney (MDCK) cells are a common model for studying drug transport as they differentiate into columnar epithelium and form tight junctions when cultured on semi-permeable membranes. They can therefore be used to determine the absorption potential of a drug. This absorption potential can be expressed as an apparent permeability rate constant, Papp, with units of 1 x 1OE^"6 cm/sec.

WO 2005/105761 discloses lipophilic 6-heteroaryl and 6-aryl quinazolines. Many such compounds have low Papp values (e.g. Papp < 5), which can translate into poor exposure and variable pharmacokinetic profiles in animal studies. However, surprisingly, the introduction of an appropriate substituent ortho to the anilino-quinazoline nitrogen atom in specific 6-thiazolyl quinazoline compounds consistently increases the Papp value 7-fold or higher and hence the compounds are likely to be more extensively and/or more rapidly absorbed.

Therefore, it is one object of the invention to provide compounds with a higher rate of permeability. It is another object of the invention to provide compounds with greater absorption. Permeability is only one of a number of factors contributing to bioavailability and unexpectedly the ortho substituted compounds of the invention also have higher bioavailability than many of the other compounds disclosed in WO2005/105761. Therefore it is a further object of the invention to provide compounds of superior bioavailability.

The present invention therefore provides a quinazoline derivative of formula (I), or a pharmaceutically acceptable salt thereof,

wherein R represents C₁-C₄ alkyl or C₁-C₄ alkoxy. Preferably, R represents a methyl, methoxy or ethoxy substituent.

Particularly preferred compounds of formula (I) are therefore: (2-methyl-4-morpholin-4-yl-phenyl)-(6-thiazol-2-yl-quinazolin-4-yl)-amine; (2-methoxy-4-morpholin-4-yl-phenyl)-(6-thiazol-2-yl-quinazolin-4-yl)-amine; (2-ethoxy-4-morpholin-4-yl-phenyl)-(6-thiazol-2-yl-quinzolin-4-yl)-amine; and pharmaceutically acceptable salts thereof.

As used herein, a pharmaceutically acceptable salt is a salt with a pharmaceutically acceptable acid or base. Pharmaceutically acceptable acids include both inorganic acids such as hydrochloric, sulphuric, phosphoric, diphosphoric, hydrobromic or nitric acid and organic acids such as citric, fumaric, maleic, malic, ascorbic, succinic, tartaric, benzoic, acetic, methanesulphonic, ethanesulphonic, benzenesulphonic oτp- toluenesulphonic acid. Pharmaceutically acceptable bases include alkali metal (e.g. sodium or potassium) and alkali earth metal (e.g. calcium or magnesium) hydroxides and organic bases such as alkyl amines, aralkyl amines and heterocyclic amines.

The compounds of the invention can be prepared according to the reaction schemes set out in WO 2005/105761, which is incorporated herein by reference. They can also be prepared by the following processes. Scheme 1 - R = CI-CU alkyl

Scheme 2-R= Ci-C₄ alkoxy NaH/THF/(Ci-Ci alkyl)-!

H₂/Pd/C/DMF

The starting materials in the above reaction schemes are known compounds, or can be prepared by analogy with known methods.

The compounds of the present invention are therapeutically useful. The present invention therefore provides a quinazoline derivative of the formula (I), as defined above, or a pharmaceutically acceptable salt thereof, for use in treating the human or animal body.

Also provided is a pharmaceutical composition comprising a quinazoline derivative of the formula (I), as defined above, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or diluent.

Said pharmaceutical composition typically contains up to 85 wt% of a compound of the invention. More typically, it contains up to 50 wt% of a compound of the invention.

Preferred pharmaceutical compositions are sterile and pyrogen free.

As explained above, the compounds of the invention are active against a flaviviridae infection. The present invention therefore provides the use of a quinazoline derivative of the formula (I), as defined above, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in treating or preventing a flaviviridae infection.

Also provided is a method for treating a patient suffering from or susceptible to a flaviviridae infection, which method comprises administering to said patient an effective amount of a quinazoline derivative of formula (I) or a pharmaceutically acceptable salt thereof. The flaviviridae family contains three genera. These are hepacivirus, fiavivirus and pestivirus. The compounds of the invention are active in treating or preventing a hepacivirus infection, a fiavivirus infection or a pestivirus infection.

Typical pestivirus infections which can be treated with the compounds of the invention include bovine viral diarrhea virus, classical swine fever virus and border disease virus. Typical fiavivirus infections which can be treated with the compounds of the invention include yellow fever virus, dengue fever virus, Japanese encephalitis virus and tick borne encephalitis virus.

Typical hepacivirus infections that can be treated with the compounds of the invention include hepatitis C virus. Compounds of the present invention are especially active against hepatitis C.

Typically, said fiavivirus is therefore hepatitis C virus. The compounds of the invention may be administered in a variety of dosage forms. Thus, they can be administered orally, for example as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules. The compounds of the invention may also be administered parenterally, whether subcutaneously, intravenously, intramuscularly, intrasternally, transdermally or by infusion techniques. The compounds may also be administered as suppositories.

The compounds of the invention are typically formulated for administration with a pharmaceutically acceptable carrier or diluent. For example, solid oral forms may contain, together with the active compound, diluents, e.g. lactose, dextrose, saccharose, cellulose,o corn starch or potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents; e.g. starches, arabic gums, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone; disaggregating agents, e.g. starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; sweeteners; wetting agents, such as lecithin, polysorbates, laurylsulphates; and,s in general, non toxic and pharmacologically inactive substances used in pharmaceutical formulations. Such pharmaceutical preparations may be manufactured in known manner, for example, by means of mixing, granulating, tableting, sugar coating, or film coating processes.

Liquid dispersions for oral administration may be syrups, emulsions and suspensions.o The syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.

Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. The suspension or solutions for intramuscular injections may contain, together with the₅ active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.

Solutions for injection or infusion may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions. o Compounds of the present invention may be used in conjunction with known anti- viral agents. Preferred known anti- viral agents in this regard are interferon and ribavirin, and derivatives thereof, which are known for the treatment of hepatitis C (Clinical Microbiology Reviews, 2000, 67-82). The said medicament therefore typically further comprises interferon or a derivative thereof and/or ribavirin or a derivative thereof. Further, the present invention provides a pharmaceutical composition comprising:

(a) a quinazoline derivative of the formula (I), as defined above, or a pharmaceutically acceptable salt thereof;

(b) interferon or a derivative thereof and/or ribavirin or a derivative thereof; and

(c) a pharmaceutically acceptable carrier or diluent. Also provided is a product comprising:

(a) a quinazoline derivative of the formula (I), as defined above, or a pharmaceutically acceptable salt thereof; and

(b) interferon or a derivative thereof and/or ribavirin or a derivative thereof, for separate, simultaneous or sequential use in the treatment of the human or animal body.

A preferred interferon derivative is PEG-interferon. A preferred ribavirin derivative is viramidine.

A therapeutically effective amount of a compound of the invention is administered to a patient. A typical dose is from about 0.01 to 100 mg per kg of body weight, according to the activity of the specific compound, the age, weight and conditions of the subject to be treated, the type and severity of the disease and the frequency and route of administration. Preferably, daily dosage levels are from 0.05 to 16 mg per kg of body weight, more preferably, from 0.05 to 1.25 mg per kg of body weight.

The following Examples illustrate the invention.

EXAMPLES

Thin layer chromatography (TLC) was carried out on Si 6OG coated plastic plates with uv254 indicator (Polygram). All NMR spectra were obtained at 250MHz in dβ- DMSO unless stated otherwise.

LC-MS CONDITIONS

Samples were run on a MicroMass ZMD, using electrospray with simultaneous positive - negative ion detection. Column: Synergi Hydro-RP, 30 x 4.6 mm LD, 4 μm.

Gradient: 95:5 to 5:95 v/v H₂O/CH₃CN + 0.05% formic acid over 4.0 min, hold 3 min, return to 95:5 v/v H₂O/CH₃CN + 0.05% formic acid over 0.2 min and hold at 95:5 v/v H₂O/CH₃CN + 0.05% formic acid over 3 min. Detection: PDA 250 - 340 nm. Flow rate: 1.5 ml/min.

Abbreviations:

DCM dichloromethane DMA N,N'-dimethylacetamide

DMF N,N'-dimethylformamide

DMSO dimethylsulfoxide rt retention time

RT room temperature

Intermediate 1: 2-Amino-5-iodobenzonitrile

Prepared by the method of A. Rosowsky and H. Chen, J. Org. Chem., 2001, 66, 7522- 7526.

¹H NMR (CDCl₃) δ 7.64 (IH, s), 7.55 (IH, dd, J 8.5, 2.5Hz), 6.53 (IH, d, J 8.5Hz), 4.66 (2H, br s); LC-MS rt 2.42 min, m/z 243 ES^".

Intermediate 2: N'-(2-Cyano-4-iodo-phenyl)-N,N-dimethyl-formamidine

A solution of 2-amino-5-iodobenzonitrile (50 g, 0.2 mol) in DMF-DMA (2.5 eq, 68 ml) was heated to 120 ⁰C for 2h. The excess DMF-DMA was removed by concentration to leave the title compound as a viscous brown oil (61 g, 100%) which solidified on standing at 4⁰C to a brown solid.

¹HNMR(CDCl₃) δ 7.79 (IH, d, J 1.9Hz), 7.65 (IH, dd, J 1.9, 8.5Hz), 7.57 (IH, s), 6.70 (IH, d, J 8.2Hz), 3.08 (6H, s); LC-MS rt 2.1 min, m/z 300 ES⁺.

Intermediate 3: N'-(2-Cyano-4-thiazol-2-yl-phenyl)-N,N-dimethyl-formamidine A 50 ml round bottomed flask was charged with intermediate 2 (6 g), 2-tributylstannylthiazole (8.25 g), lithium chloride (4 g), and bistriphenylphosphine palladium dichloride (0.7 g) in toluene (100 ml) and heated to 120 °C for 24h. The cooled reaction mixture was concentrated and purified by column chromatography on silica gel with gradient elution of DCM : 2.5% methanol - DCM to give the title compound (4 g, 78%).

¹H NMR (de-DMSO) δ 8.12 (2H, m), 8.05 (IH, dd, J = 8.85Hz, 2.5Hz), 7.91 (IH, d, J = 3.16Hz), 7.77 (IH, d, J = 3.16Hz), 7.32 (IH, d, J = 8.85Hz), 3.13 (3H, s), 3.05 (3H, s); LC- MS rt 2.12 min, m/z 258 ES .

Intermediate 4: 4-(3-Methyl-4-nitrophenyl)morpholine

A 150 ml RB flask was charged with 4-fluoro-2-methyl-l -nitrobenzene (5 g), morpholine (3 g), triethylamine (6 g) and anhydrous acetonitrile (50 ml). The mixture was heated at 90 °C for 18h, cooled, and concentrated to give a yellow solid. This solid was triturated with water (200 ml), filtered and washed with further water. Drying in vacuo gave the title compound as a yellow solid (4 g, 59%).

¹HNMR (d₆-DMSO) δ 8.01 (IH, d, J = 10.1 IHz), 6.93 (2H, m), 3.73 ( 4H, t, J = 5.05Hz)), 3.37 (4H, t, J = 5.05Hz), 2.56 (3H, s); LC-MS rt 2.44 min, (M+H)⁺ 223.

Intermediate 5: 2-Methyl-4-morpholin-4-yl-phenylamine

4-(3-Methyl-4-nitrophenyl)morpholine (2 g) was hydrogenated at RT and pressure over 10% Pd/C (0.2 g) in DMF (40 ml) for 24h. The mixture was filtered through celite and the filtrate concentrated to give the title compound as a red-brown solid (1.7 g).

¹H NMR (d₆-DMSO) δ 6.61 (IH, s), 6.55 (2H, s), 4.36 (2H, b), 3.69 (4H, t, J = 5.05Hz), 2.87 (4H, t, J = 5.05), 2.04 (3H, s).

Intermediate 6: 4-(3-Methoxy-4-nitro-phenyl)-morpholine

5-Morpholino-2-nitrophenol (Ig) was added portionwise over 5 min to a suspension of sodium hydride (0.2 g) in dry DMF (15 ml) at RT. After 15 min, methyl iodide (0.7 g) was added and the mixture left to stir overnight. Water (5 ml) was added and the mixture was concentrated. The residue was triturated with water, filtered and the filter cake washed with water, 2N NaOH and water before being dried in vacuo to give the title compound as a yellow solid (1 g).

¹H NMR (de-DMSO) δ 7.94 (IH, d, J = 9.5Hz), 6.64 (IH, dd, 8.85, 2.5Hz), 6.59 (IH, d, J = 2.57Hz), 3.96 (3H, s), 3.77 (4H, t, J = 5.05Hz) , 3.46 (4H, t, J = 5.05Hz); LC-MS rt 2.22 min, (M+H)⁺ 239.

Intermediate 7: 4-(3-Ethoxy-4-nitro-phenyl)-morpholine

The title compound (0.55 g) was prepared by the method of Intermediate 6, but using ethyl iodide (0.76 g).

¹H NMR (de-DMSO) δ 7.92 (IH, d, J = 9.5Hz), 6.63 (IH, dd, J = 9.50Hz, 2.53Hz), 6.59

(IH, s), 4.23 (2H, q, J = 6.95 Hz), 3.76 (4H, t, 5.06Hz), 3.42 (4H, t, J = 5.06Hz), 1.39 (3H, t, J = 6.95Hz); LC-MS. rt 2.40 min, (M+H)⁺ 253.

Intermediate 8: 2-Methoxy-4-morpholin-4-yl-phenylamine

Intermediate 6 (0.5 g) was hydrogenated at RT and pressure over 10% Pd/C (0.06 g) in DMF (15 ml) for 18h. The mixture was filtered through celite and the filtrate concentrated to give the title compound as a purple oil which solidified on standing (0.4 g).

¹H NMR (d₆-DMSO) δ 6.54 (IH, d, J = 8.20 Hz), 6.51 (IH, d, J = 2.53Hz), 6.30 (IH, dd, J = 8.85Hz, 2.53Hz), 4.25 (2H, b), 3.75 (3H, s), 3.71 (4H, t, J = 5.0Hz), 2.92 (4H, t, 5.0Hz);

Intermediate 9: 2-Ethoxy-4-morpholin-4-yl-phenylamine

Intermediate 7 (0.5 g) was hydrogenated at RT and pressure over 10% Pd/C (0.06 g) in DMF (15 ml) for 18h. The mixture was filtered through celite and the filtrate concentrated to give the title compound as a purple oil (0.4 g)

¹H NMR (d₆-DMSO) δ 6.55 (IH, d, J = 8.20Hz), 6.50 (IH, d, J = 1.90Hz), 6.30 (IH, dd, J

= 8.85, 2.5Hz), 4.23 (2H, b), 3.99 (2H, q, J = 6.95Hz), 3.71 (4H, t, J = 5.05Hz), 2.91 (4H, t, J = 4.4Hz), 1.33 (3H, t, J = 6.55Hz); LC-MS rt 0.8 min, (M+H)⁺ 223.

Example 1 (2-Methyl-4-morpholin-4-yl-phenyl)-(6-thiazol-2-yl-quinazolin-4-yl)- amine Intermediate 3 (1.5 g) and Intermediate 5 (1.24 g) were combined in acetic acid (15 ml) and heated to 120 °C for 3h. The cooled mixture was concentrated in vacuo and purified by column chromatography on silica with gradient elution 2.5% MeOH / DCM to 5% MeOH / DCM. This gave the title compound as a yellow solid (1.2 g, 50%). 1H NMR (d₆-DMSO) δ 10.06 (IH, s), 9.12 (IH, s), 8.49 (IH, d, J = 1.90Hz), 8.45 (IH, s),

8.07 (IH, d, J = 3.16Hz), 7.94 (IH, d, J = 3.16Hz), 7.90 (IH, d, J = 8.85Hz), 7.21 (IH, d, J = 8.85Hz), 6.97 (IH, s), 6.91 (IH, dd, J = 8.85Hz, 2.53Hz), 3.83 (4H, b), 3.19 (4H, b), 2.21 (3H, s); LC-MS rt 2.28 min, (M + H)⁺ 404, (M -H)⁺ 402.

Example 2 (N-(2-Methoxy-4-morpholinophenyl)-6-(thiazol-2-yl)quinazolin-4-amine Intermediate 3 (100 mg) and Intermediate 8 (89 mg) were combined in acetic acid (4 ml) and heated at 120 °C for 3h. Workup as for Example 1 gave the title compound (90 mg).

¹HNMR (d_δ-DMSO) δ 9.60 (IH, s), 8.86 (IH, s), 8.24 (IH, d, J = 1.90Hz), 8.21 (IH, s),

7.82 (IH, d, J= 3.16Hz), 7.70 (IH, d, J = 3.16Hz), 7.63 (IH, d, J = 8.85Hz), 7.05 (IH, d, J = 8.85Hz), 6.51 (IH, s), 6.38 (IH, dd, J = 8.85Hz, 2.53Hz), 3.58 (7H, b), 3.0 (4H, b); LC- MS rt 2.17 min, (M + H)⁺ 420, (M-H)⁺ 418.

Example 3 : (N-(2-Ethoxy-4-morpholinophenyl)-6-(thiazol-2-yl)quinazolin-4-amine

Intermediate 3 (100 mg) and Intermediate 9 (93 mg) were combined in acetic acid (4 ml) and heated at 120 °C for 3h. Workup as for Example 1 gave the title compound (90 mg).

¹HNMR ^₆-DMSO) δ 9.67 (IH, s), 8.95 (IH, s), 8.35 (IH, s), 8.32 (IH, d, J= 1.86Hz),

7.94 (IH, d, J = 3.16Hz), 7.81 (IH, d, J = 3.16Hz), 7.75 (IH, d, J = 8.85Hz), 7.22 (IH, d, J = 8.85Hz), 6.62 (IH, s), 6.49 (IH, dd, J = 8.85, 1.89Hz), 3.98 (2H, q, J = 6.95Hz), 3.70 (4H, b), 3.09 (4H, b), 1.09 (3H, t, J = 6.95Hz); LC-MS rt 2.37 min, (M+H)⁺434, (M-H)⁺ 432. Activity Example 1

Cells used:

HCV replicon cells Huh 9B (ReBlikon), containing the firefly luciferase - ubiquitin - neomycin phosphotransferase fusion protein and EMCV-IRES driven HCV polyprotein with cell culture adaptive mutations.

Cell culture conditions:

Cells were cultured at 37 °C in a 5% CO₂ environment and split twice a week on seeding at 2 x 10E6 cells/flask on day 1 and 1 x 10E6 3 days later. Some 0.25 mg/ml G418 was added to the culture medium (125 μl per 25ml) but not to the assay medium.

The culture medium consisted of DMEM with 4500 g/1 glucose and glutamax (Gibco 61965-026) supplemented with 1 x non-essential amino acids, penicillin (100 IU/ml) / streptomycin (100 μg/ml), FCS (10%, 50 ml) and 1 mg/ml G418 (Invitrogen cat no 10131-027) and 10% foetal calf serum.

Assay procedure:

A flask of cells was trypsinised and a cell count carried out. Cells were diluted to 100,000 cells/ml and 100 μl of this used to seed one opaque white 96-well plate (for the replicon assay) and one flat-bottomed clear plate (for the tox assay) for every seven compounds to be tested for IC50. Wells G12 and H12 were left empty in the clear plate as the blank. Plates were then incubated at 37 ⁰C in a 5% CO₂ environment for 24h.

On the following day compound dilutions are made up in medium at twice their desired final concentration in a clear round bottomed plate. AU dilutions have a final DMSO concentration of 1%.

Once the dilution plate had been made up, controls and compounds were transferred to the assay plate (containing the cells) at 100 μl /well in duplicate plates.

Exception: in the white (replicon) plate, no compound was added to wells Al and A2 and 100 μl of 1% DMSO was added to these instead. In the clear (Tox) plate, wells E12 & F12 only contained the DMSO control. Plates were then incubated at 37 ⁰C with 5% CO₂ for 72h.

At the end of the incubation time, the cells in the white plate were harvested by washing with 200 μl/ well of warm (37 °C) PBS and lysed with 20 μl cell culture lysis buffer (Promega). After 5 min incubation at RT, luciferin solution was added to the luciferase assay buffer (LARB at 200 μl per 10 ml LARB). The M injector of the microplate luminometer (Lmax, Molecular Devices) was primed with 4 x 300 μl injections. Plates were inserted into the luminometer and 100 μl luciferase assay reagent was added by the injector on the luminometer. The signal was measured using a 1 second delay followed by a 4 second measurement programme. The IC50, the concentration of the drug required for reducing the replicon level by 50% in relation to the untreated cell control value, can be calculated from the plot of the percentage reduction of the luciferase activity vs. drug concentration.

The clear plate was stained with 100 μl 0.5% methylene blue in 50% ethanol at RT for Ih, followed by solvation of the absorbed methylene blue in 100 μl per well of 1% lauroylsarcosine. Absorbance of the plate was measured on a microplate spectrophotometer (Molecular Devices) and the absorbance for each concentration of compound expressed as a proportion of the relative DMSO control. The TD50, the concentration of drug required to reduce the total cell area by 50% relative to the DMSO controls can be calculated by plotting the absorbance at 620 nm vs drug concentration.

Table 1

Activity Example 2

Permeability

The in vitro apical to basolateral permeability of the compounds was tested in a permeability assay using an MDCK cell line (ATCC Catalogue number: CCL-34, Passage number: 55, Lot number: 3563161). The integrity of the monolayers used in the assay is then checked with a Lucifer Yellow rejection assay.

Cells were grown in 75 cm² tissue culture flasks in an atmosphere of 5% (v/v) CO₂ in a 37 °C humidified incubator. Cells were passaged at 90% confluence by rinsing the flask with PBS, followed by a two-minute incubation with Versene, followed by a five to ten minute incubation with trypsin-EDTA. Detached cells were washed from the flask with growth medium. Cell number was estimated by counting using a heamocytometer slide. Cell suspensions were then diluted accordingly in growth medium. At day 0, cells were seeded in growth medium at 1 x 10⁵ cells/cm² in a 24- well plate containing tissue culture treated inserts, and allowed to grow for 48h at 5% (v/v) CO₂, 37 ⁰C in a humidified incubator. Growth medium was then renewed and cells allowed to grow a further 24h. On day 3, compounds were diluted to 10 μM in HBSS. Cells were washed three times with 500 μl HBSS and the inserts were transferred to the 24-well plate containing the 500 μl/well fresh HBSS. Each compound dilution (500 μl) was then added in the inserts containing the cells, in duplicate, alongside a mix of control compounds (atenolol, dexamethasone and propranolol). Final compound concentrations in the assay was 10 μM ; final DMSO concentration was 1% (v/v). Cells were incubated for 2h at 5% (v/v) CO₂, 37 ⁰C in a humidified incubator. The reaction was stopped by removing the inserts from the plate. Then, a 200 μl sample was taken from the inserts, and diluted 1 :2 with acetonitrile + 0.05% (v/v) formic acid, directly into the LC-MS analysis plate. Then, 500 μl acetonitrile + 0.05% (v/v) formic acid were added to the wells containing the samples from the permeability assay, to improve the recovery of compounds. The plate was left at room temperature for a further 15 min before sampling for LC-MS analysis. Lucifer Yellow was diluted to 100 μM in HBSS and a 24 well-plate was filled with

500 μl/well HBSS. The inserts recovered from the permeability assay were washed three times with 500 μl HBSS and were transferred to the 24-well plate containing the fresh HBSS. The Lucifer Yellow solution (500 μl) was then added in all the inserts, to check the monolayer integrity. Cells were incubated for 2h at 5% (v/v) CO₂, 37 ⁰C in a humidified incubator. The reaction was stopped by removing the inserts from the plate. Then, a 100 μl sample was taken from the wells, dispensed in a black 96-well plate alongside the 100 μM Lucifer Yellow solution and HBSS as a blank. The fluorescence was estimated by reading RPU at 530 nm (excitation 430nm).

The apparent permeability coefficient (Papp) was calculated using the following formula:

_ receiver Volume (ml) Cf (nM)

Papp= — — x — -

Area (cm )x Time (sec) Ci (nM)

using the Volume of the receiver (here basolateral compartment, 500 μl), the Area of the filter membrane (0.6 cm²), the Time of total experiment (7200 sec), Cf the final drug concentration recovered (obtained after LC-MS analysis of the samples), and Ci the initial drug concentration (10⁴nM).

For each experiment, compounds were rank ordered alongside control compounds to determine their permeability.

The Lucifer Yellow rejection was calculated using the following formula:

using the fluorescence of the starting 100 μM solution, and where the receiver is the basolateral side (samples from the wells).

The % rejection is considered very good if between 98 and 100% and good if between 96 and 98%. A rejection below 96% suggests that the monolayers were likely compromised during the assay.

Bioavailability Rats were dosed orally at 5 mg/kg of compound as a suspension in 1% carboxymethyl cellulose (CMC) by gavage. For intravenous administration the compounds were formulated in a mixture of DMSO and saline at 1 mg/mL with a dose volume of 1 mL/kg giving a 1 mg/kg dose. Blood samples were taken over 24h; the plasma was separated by centrifugation and then extracted using protein precipitation. The compounds were measured using a highly specific LC-MS/MS analytical method and the data used to determine the area under the plasma concentration time profile, (AUCo_-∞). Bioavailability was established by comparison of the dose normalised systemic exposure of the compounds following oral administration to that obtained from the intravenous route.

Table 2 below shows the respective bioavailability figures for Example 1 and an analogue lacking the substitution ortho to the anilino-quinazoline nitrogen atom.

Table 2

Claims

1. A compound which is a quinazoline derivative of formula (I), or a pharmaceutically acceptable salt thereof,

R is Ci-C₄ alkyl or Ci-C₄ alkoxy.

2. A compound according to claim 1 , wherein R is methyl, methoxy or ethoxy.

3. (2-Methyl-4-morpholin-4-yl-phenyl)-(6-thiazol-2-yl-quinazolin-4-yl)-amine, or a pharmaceutically acceptable salt thereof.

4. (2-Methoxy-4-morpholin-4-yl-phenyl)-(6-thiazol-2-yl-quinazolin-4-yl)-amine, or a pharmaceutically acceptable salt thereof.

5. (2-Ethoxy-4-morpholin-4-yl-phenyl)-(6-thiazol-2-yl-quinazolin-4-yl)-amme, or a pharmaceutically acceptable salt thereof.

6. A quinazoline derivative as defined in any one of the preceding claims, or a pharmaceutically acceptable salt thereof, for use in treating the human or animal body.

7. A pharmaceutical composition which comprises a quinazoline derivative as defined in any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or diluent.

8. Use of a quinazoline derivative as defined in any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in treating or preventing a flaviviridae infection.

9. Use according to claim 8, wherein the flaviviridae infection is a pestivirus infection.

10. Use according to claim 9, wherein the pestivirus infection is an infection by a bovine viral diarrhea virus, classical swine fever virus or border disease virus.

11. Use according to claim 8, wherein the flaviviridae infection is a flavivirus infection.

12. Use according to claim 11, wherein the flavivirus infection is an infection by a yellow fever virus, dengue fever virus, Japanese encephalitis virus or tick borne encephalitis virus. s

13. Use according to claim 8,wherein the flaviviridae infection is a hepacivirus infection.

14. Use according to claim 13, wherein the hepacivirus infection is an infection by a hepatitis C virus. o

15. Use according to claim 14, wherein the medicament further comprises (a) interferon or a derivative thereof and/or (b) ribavirin or a derivative thereof.

16. Use according to claim 15 wherein the interferon derivative is PEG-interferon and/or the ribavirin derivative is viramidine. 5

17. A product containing:

(a) a quinazoline derivative as defined in any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof; and

(b) interferon, or an interferon derivative as defined in claim 15 or 16 and/oro ribavirin or a ribavirin derivative as defined in claim 15 or 16; for simultaneous, separate or sequential use in the treatment of the human or animal body.

18. A method of alleviating or reducing the incidence of a flaviviridae infection, as defined in any one of claims 8 to 14, in a patient, which method comprises administering to said patient an effective amount of a quinazoline derivative as defined in any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof.