NZ709769B2 - 2-aminopyrimidine derivatives for the treatment of viral infections - Google Patents

Abstract

The invention relates to 2-aminopyrimidine derivatives of formula (I) and pharmaceutical compositions, as TLR7 and/or TLR8 agonists for use in treating viral infections.

Description

-AMINOPYRIMIDINE DERIVATIVES FOR THE TREATMENT OF VIRAL IONS This invention s to opyrimidine derivatives, processes for their preparation, pharmaceutical compositions, and their use in treating viral infections.

The present invention s to the use of 2-aminopyrimidine derivatives in the treatment of viral infections, immune or inflammatory ers, whereby the modulation, or agonism, of toll-like-receptors (TLRs) is involved. Toll-Like Receptors are primary transmembrane proteins characterized by an extracellular leucine rich domain and a cytoplasmic extension that ns a conserved region. The innate immune system can recognize pathogen- associated molecular patterns via these TLRs expressed on the cell surface of certain types of immune cells. Recognition of foreign pathogens activates the production of cytokines and upregulation of co-stimulatory molecules on phagocytes. This leads to the modulation of T cell behaviour.

It has been estimated that most mammalian species have between ten and fifteen types of Toll—like receptors. Thirteen TLRs (named TLR1 to TLR13) have been fied in humans and mice together, and equivalent forms of many of these have been found in other mammalian s. However, equivalents of certain TLR found in humans are not present in all mammals. For example, a gene coding for a n analogous to TLR10 in humans is present in mice, but appears to have been damaged at some point in the past by a retrovirus.

On the other hand, mice express TLRs 11, 12, and 13, none of which are represented in humans. Other mammals may express TLRs which are not found in humans. Other non-mammalian species may have TLRs distinct from mammals, as demonstrated by TLR14, which is found in the Takifugu pufferfish. This may complicate the process of using experimental animals as models of human innate ty.

For s on TLRs see the following journal articles. Hoffmann, J.A., Nature, 426, p33—38, 2003; Akira, S., Takeda, K., and Kaisho, T., Annual Rev.

Immunology, 21, p335-376, 2003; Ulevitch, R. J., Nature Reviews: logy, 4, p512-520, 2004. nds indicating activity on ike receptors have been previously bed such as purine derivatives in WO 17670, adenine derivatives in WO 98/01448 and WO 99/28321, and pyrimidines in .

However, there exists a strong need for novel Toll-Like receptor modulators having preferred selectivity, higher potency, higher metabolic ity, and an improved safety profile ed to the compounds of the prior art.

In accordance with the present invention a compound of formula (I) is provided or a pharmaceutically acceptable salt, tautomer(s), stereo-isomeric forms, solvate or polymorph thereof, wherein X represents S, S=O or O=S=O, R1 is hydrogen, (C1-6)-alkyl, (C1-6)-alkoxy or aryl, R2 is (C1-3)-alkyl or (C3-6)-cycloalkyl and n = 1 or 2.

In a particular aspect of the invention, there is provided a compound of formula (I) or a pharmaceutically able salt, tautomer(s), stereo-isomeric forms, solvate or polymorph thereof, wherein X represents S, S=O or O=S=O, R1 is hydrogen, straight-chain or branched-chain (C1-6)-alkyl, (C1-6)-alkoxy or aryl, R2 is straight-chain or branched-chain (C1-3)-alkyl or (C3-6)-cycloalkyl and n = 1 or 2.

[FOLLOWED BY PAGE 2a] - 2a - The compounds of a (I) and their pharmaceutically acceptable salts, tautomer(s), stereo-isomeric forms, solvate or polymorph thereof have activity as pharmaceuticals, in particular as modulators of Toll-Like Receptors (especially TLR7 and/or TLR8 activity).

In a further aspect the present ion provides a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt, tautomer, -isomeric form, e or polymorph thereof together with one or more pharmaceutically acceptable excipients, diluents or rs.

Furthermore a compound of formula (I) or a pharmaceutically acceptable salt, solvate, tautomer, stereo-isomeric form or polymorph f according to the current invention, or a pharmaceutical composition comprising said compound of formula(I) or a pharmaceutically acceptable salt, solvate, tautomer, stereo-isomeric form or polymorph thereof can be used as a medicament.

[FOLLOWED BY PAGE 3] Another aspect of the invention is that a compound of formula (I) or its pharmaceutically acceptable salt, solvate, tautomer, stereo-isomeric form or polymorph thereof, or said pharmaceutical composition comprising said nd of formula (I) or a pharmaceutically acceptable salt, solvate, tautomer, stereo—isomeric form or rph thereof can be used accordingly in the treatment of a disorder where the modulation of TLR’s, more ically TLR7 and /or TLR8, is involved.

The term "(C1_6)-alkyl" or "(C1_3)-alkyl refers to a ht-chain, branched-chain or cyclic saturated aliphatic hydrocarbon containing the specified number of carbon atoms.

The term "aryl" means an aromatic ring structure optionally comprising one or two heteroatoms selected from N, O and S, in particular from N and O. Said aromatic ring ure may have 4, 5, 6 or 7 ring atoms. In particular, said aromatic ring structure may have 5 or 6 ring atoms.

The term "(C1_6)-alkoxy refers to an alkyl n and hydrogen chain) group singular bonded to oxygen like for instance a methoxy group or ethoxy group.

The term "(Cs-e)-cycloalkyl" means refers to a carbocyclic ring containing the specified number of carbon atoms.

As used herein, any chemical formula with bonds shown only as solid lines and not as solid wedged or hashed wedged bonds, or otherwise indicated as having a particular configuration (e.g. R, 8) around one or more atoms, contemplates each le stereoisomer, or mixture of two or more stereoisomers.

The terms "stereoisomers", "stereoisomeric forms" or "stereochemically isomeric forms" hereinbefore or hereinafter are used interchangeably.

The invention includes all stereoisomers of the compounds of the invention either as a pure stereoisomer or as a mixture of two or more stereoisomers.

Enantiomers are stereoisomers that are perimposable mirror images of each other. A 1:1 mixture of a pair of enantiomers is a racemate or c mixture. reomers (or diastereoisomers) are stereoisomers that are not enantiomers, i.e. they are not related as mirror images. If a compound contains a double bond, the substituents may be in the E or the Z configuration. If a compound contains an at least disubstituted non-aromatic cyclic group, the substituents may be in the cis or trans configuration.

Therefore, the invention includes omers, diastereomers, racemates, E isomers, Z isomers, cis isomers, trans isomers and es f, whenever chemically possible.

The meaning of all those terms, i.e. enantiomers, diastereomers, racemates, E isomers, Z isomers, cis isomers, trans s and mixtures thereof are known to the skilled person.

The absolute configuration is specified according to the Cahn-lngold-Prelog system. The configuration at an asymmetric atom is specified by either R or 8.

Resolved stereoisomers whose absolute configuration is not known can be designated by (+) or (-) depending on the direction in which they rotate plane polarized light. For instance, resolved enantiomers whose absolute configuration is not known can be designated by (+) or (-) depending on the direction in which they rotate plane polarized light.

When a specific stereoisomer is identified, this means that said stereoisomer is substantially free, i.e. associated with less than 50%, preferably less than 20%, more preferably less than 10%, even more preferably less than 5%, in particular less than 2% and most preferably less than 1%, of the other stereoisomers. Thus, when a compound of Formula (I) is for instance specified as (R), this means that the compound is substantially free of the (S) isomer; when a compound of a (I) is for instance specified as E, this means that the compound is substantially free of the Z isomer; when a compound of a (I) is for instance ied as cis, this means that the compound is ntially free of the trans isomer.

Pharmaceutically acceptable salts of the compounds of formula (l) include the acid addition and base salts f. Suitable acid addition salts are formed from acids which form non-toxic salts. Suitable base salts are formed from bases which form non-toxic salts.

The compounds of the invention may also exist in unsolvated and solvated forms. The term "solvate" is used herein to describe a molecular complex comprising the compound of the invention and one or more pharmaceutically acceptable solvent molecules, for example, ethanol.

The term orph" refers to the ability of the compound of the invention to exist in more than one form or crystal structure.

The nds of the present invention may be administered as crystalline or ous products. They may be ed for example as solid plugs, s, or films by methods such as precipitation, crystallization, freeze drying, spray drying, or evaporative drying. They may be administered alone or in combination with one or more other compounds of the ion or in combination with one or more other drugs. Generally, they will be administered as a formulation in association with one or more pharmaceutically acceptable excipients. The term "excipient" is used herein to describe any ingredient other than the compound(s) of the invention. The choice of excipient depends largely on s such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.

The compounds of the present ion or any subgroup thereof may be formulated into various pharmaceutical forms for administration purposes. As appropriate itions there may be cited all compositions usually employed for systemically administering drugs. To prepare the pharmaceutical compositions of this invention, an effective amount of the particular nd, optionally in addition salt form, as the active ingredient is combined in intimate admixture with a pharmaceutically able carrier, which carrier may take a wide variety of forms depending on the form of preparation desired for administration. These pharmaceutical itions are desirably in unitary dosage form suitable, for example, for oral, rectal, or percutaneous administration. For example, in preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be ed such as, for example, water, glycols, oils, ls and the like in the case of oral liquid preparations such as suspensions, syrups, elixirs, emulsions, and solutions; or solid carriers such as starches, sugars, kaolin, diluents, lubricants, binders, disintegrating agents and the like in the case of powders, pills, capsules, and tablets.

Because of their ease in stration, tablets and capsules represent the most advantageous oral dosage unit forms, in which case solid pharmaceutical carriers are obviously employed. Also included are solid form ations that can be converted, y before use, to liquid forms. In the compositions suitable for percutaneous administration, the carrier optionally comprises a penetration enhancing agent and/or a suitable g agent, optionally combined with suitable additives of any nature in minor proportions, which additives do not introduce a significant deleterious effect on the skin. Said additives may facilitate the administration to the skin and/or may be helpful for preparing the desired itions. These compositions may be administered in various ways, e.g., as a transdermal patch, as a spot-on, as an ointment.

The compounds of the present invention may also be administered via inhalation or insufflation by means of methods and ations employed in the art for administration via this way. Thus, in general the compounds of the present ion may be administered to the lungs in the form of a on, a suspension or a dry powder.

It is ally advantageous to formulate the aforementioned pharmaceutical compositions in unit dosage form for ease of stration and uniformity of dosage. Unit dosage form as used herein refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ient calculated to produce the desired therapeutic effect in association with the required pharmaceutical r. Examples of such unit dosage forms are tablets (including scored or coated tablets), capsules, pills, powder packets, wafers, suppositories, injectable solutions or sions and the like, and segregated multiples thereof.

Those of skill in the treatment of infectious diseases will be able to determine the effective amount from the test results presented hereinafter. In l it is contemplated that an effective daily amount would be from 0.01 mg/kg to 50 mg/kg body weight, more preferably from 0.1 mg/kg to 10 mg/kg body weight. It may be appropriate to administer the required dose as two, three, four or more sub-doses at appropriate intervals throughout the day. Said sub-doses may be formulated as unit dosage forms, for example, containing 1 to 1000 mg, and in particular 5 to 200 mg of active ingredient per unit dosage form.

The exact dosage and frequency of administration depends on the particular compound of formula (I) used, the particular condition being treated, the severity of the condition being treated, the age, weight and general physical condition of the ular patient as well as other medication the individual may be taking, as is well known to those skilled in the art. Furthermore, it is evident that the effective amount may be lowered or increased depending on the response of the treated subject and/or depending on the evaluation of the physician prescribing the compounds of the instant invention. The effective amount ranges mentioned above are therefore only guidelines and are not intended to limit the scope or use of the invention to any extent.

Pre aration of com ounds ula | l scheme. Compound A was prepared according to procedures described in W02008147697 and W02009067081.

MsCI, Et3N 3N3 /\(S) ‘ \ /\(S€| HO/\\" \ NH DCM,OOC MSO NH 3 DMF, 70 0C NH BOC Boc BOC A B c dioxane/HCI HN N NH2 —> \ /\ S ‘ NH HCI —> ’ CH2C|2, rt 2 (S L K2C03 ,EtOH MW, 120 0C, 0.5 h S E Oxone H20, DMF | | O \ N Na|O4 O I A N water, EtOH, rt | A HI?! N NH2 Hg N NH2 (3) (S) 40 "O ? I \ Experimental Section MsCI, Et3N (8) (3) HO/\\v. /\ "- \ NH MSO DCM, 0 0C [NH BOC 300 A B Triethylamine (10.5 9, 103.75 mmol, 2.4 eq.) was added to the solution of A (10 g, 43.23 mmol, 1 eq.) in CH2C|2 (200 mL) at 0°C. Methanesulfonyl chloride (6.4 g, 55.87 mmol, 1.3 eq.) was added dropwise to the solution and stirred 1.5 hours at 0°C. CHZCI2 (500 mL) was added. The solution was washed with aq. NaH003, brine, and dried over Na2804, the solids were removed by filtration and the solvent of the filtrate was removed under reduced re to give B. Used as such without further purification.

/\(S) —>. \ /\(3) Boo Boo B C A mixture of B (12 g, 38.782 mmol, 1 eq.) and sodium thoxide (4.08 g, 58.17 mmol, 1.5 eq.) in DMF (60 mL) was stirred overnight at 70°C. The solids were removed by filtration and the solvents of the te were removed under reduced pressure. The crude was dissolved in ethyl acetate, washed with water, brine, dried over Na2804‘ the solids were removed by filtration and the solvents of the filtrate were removed under reduced pressure. The crude was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate from 40/1 to 3/1) to afford C. 1H NMR (400 MHz, chloroform-d) 8 ppm 0.70 — 0.85 (m, 5 H), 1.15 — 1.49 (m, 13 H), 1.49 — 1.61 (m, 1 H), 1.61 — 1.80 (m, 1 H), 2.05 (s, 3 H), 2.38 - 2.50 (m, 2 H), 3.51 (br. s.,1 H), 4.25 (br. s., 1 H) e/HCI ‘3) —> ‘8) \s/\"\. \ ". l}lH CH20I2, rt s/\‘ NH2'HCI c D HCI/dioxane (47 mL, 187.43 mmol, 10 eq.) was added drop wise to a stirred solution of C (4.9 g, 18.74 mmol, 1 eq.) in CH2CI2 at 0°C, and stirred for 1 hour at 25°C. The solution was concentrated under reduced pressure to give D.

Used as such in the next step.

O o \N \ l A 3QN CI N NH2 HN N NH2 \S/\\"‘ NH2_HC| .,l K2C03 ,EtOH MW, 120 0c, 0.5 h ’L D (0.75 g, 3.79 mmol, 1 eq.), 2-aminochloromethoxypyrimidine (0.908 g, .69 mmol, 1.5 eq.) and K2C03 (1.57 g 11.38 mmol, 3 eq.) were mixed in ethanol (20 mL). The mixture was d at 120°C in the microwave for 30 minutes. The solvent was removed under reduced pressure. The crude was purified by preparative silica thin layer chromatography (eluent: : CH3OH = 20:1) to afford E. 1H NMR (400 MHz, chloroform—d) 6 ppm 1.05 — 1.15 (m, 3 H), 1.40 - 1.60 (m, 4 H), 1.95 (m, 2H), 2.15 (m, 1 H), 2.30 (d, 3 H), 2.70 (t, 1 H), 2.90 (t, 1 H), 3.55 (m, 1 H), 4.50 (m, 1 H), 3.95 (s, 3 H), 6.20 (d, 1H), 6.60 (br. s., 2 H), 7.45 (s, 1 H) O O A Oxone Hl§| NANH 2 2 ., H20, DMF L (3) Oxone (6.959 g, 11.32 mmol, 3 eq.) was added to a solution of E (1.45 g, 3.773 mmol, 1 eq.) in DMF (100 mL) and water (100 mL). The mixture was stirred for 12 hours at 20°C. The solids were removed by filtration and the filtrate was basified to pH=8 with saturated, aq. Na2C03 solution. The resultant mixture was concentrated under reduced pressure. The residue was purified by preparative erformance liquid tography (column: gemini 150 x mm x 50m, C18, mobile phase: CH3CN/water (0.05% HCI), Gradient: 2-32% CchN, 0-8min, flow rate: 30mL /min). The best fractions were pooled and concentrated under reduced pressure to afford 1.

LC-MS 3.88 min 1H NMR (400 MHz, methanol-d4) 8 ppm 0.92 (t, J=6.9 Hz, 3 H), 1.21 - 1.50 (m, 4 H), 1.69 (q, J=7.1 Hz, 2 H), 1.95 - 2.28 (m, 2 H), 2.98 (s, 3 H), 3.09 - 3.22 (m, 2 H), 3.87 (s, 3 H), 4.37 - 4.55 (m, 1 H), 7.26 (s, 1 H) labile protons not observed.

(I) | m o / m/ HN N NH2 Nam—,4 HN N NH2 (3)., (s ’I Hzo-EtOH, rt "I, A on of E (40 mg, 0.14 mmol, 1 eq.) in ethanol (40 mL) was treated with a solution of NalO4 (0.2 g, 1 mmol, 7.5 eq.) in water (10 mL), and then stirred at room temperature overnight. The solution was trated under vacuum.

The residue was diluted with water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over NaZSO4, the solids were removed via filtration, and the solvent of the filtrate was removed under reduced pressure. The crude was ed by preparative high-performance liquid tography (C18 column, eluent: CH3CN, H20 from 3/97 to 33/67, 0.05% HCI). The desired fractions were collected and concentrated under vacuum to afford 2.

LC-MS 3.78 min 1H NMR (400 MHz, methanol-d4) 8 ppm 0.90 (t, J=6.8 Hz, 3 H), 1.19 - 1.49 (m, 4 H), 1.67 (d, J=6.5 Hz, 2 H), 1.91 - 2.15 (m, 2 H), 2.63 (br. s., 3 H), 2.69 - 2.96 (m, 2 H), 3.85 (s, 3 H), 4.46 (br. s., 1 H), 7.25 (s, 1 H) labile protons not observed.

WO 28189 LC-MS Analytical .

Column YMC-PACK ODS—AQ, 50X2.0mm, A:H20 (0.1%TFA) onitrile (0.05%TFA) TIME(min) A% 8% 0 100 0 Mobile Phase 1 100 0 40 60 7.5 40 60 8 100 0 Flow Rate 0.8mL/min Wavelenoth UV 220nm Column 50°C Temoerture MS oolarit oositive LCMS Agilent 1100 Biolo icalActivit ofcom ounds offormula | Description of Biological Assays Assessment of TLR7 and TLR8 activity The ability of nds to activate human TLR7 and/or TLR8 was assessed in a cellular reporter assay using HEK293 cells transiently transfected with a TLR7 or TLR8 expression vector and NFKB-IUC reporter construct.

Briefly, HEK293 cells were grown in culture medium (DMEM supplemented with 10% FCS and 2 mM Glutamine). For transfection of cells in 15 cm dishes, cells were detached with Trypsin-EDTA, transfected with a mix of CMV-TLR7 or TLR8 plasmid (1700 ng), NFKB-IUC plasmid (850 ng) and a transfection reagent and incubated for 48 h at 37°C in a humidified 5% C02 atmosphere.

Transfected cells were then washed in PBS, detached with Trypsin-EDTA and resuspended in medium to a density of 1.25 x 105 mL. Forty iters of cells were then dispensed into each well in 384-well plates, where 200 nL of compound in 100% DMSO was already present. Following 6 hours incubation at 37°C, 5% 002, the luciferase activity was ined by adding 15 uL of Steady Lite Plus substrate (Perkin Elmer) to each well and t performed on a ViewLux TS microplate imager (Perkin Elmer). Dose response curves were ted from measurements med in quadruplicates.

Lowest effective trations (LEC) values, defined as the concentration that induces an effect which is at least two fold above the standard deviation of the assay, were determined for each compound.

Compound toxicity was determined in parallel using a similar dilution series of nd with 40 uL per well of cells transfected with the CMV—TLR7 construct alone (1.25 x 105 cells/mL), in ll plates. Cell viability was measured after 6 hours incubation at 37°C, 5% C02 by adding 15 uL of ATP lite (Perkin Elmer) per well and reading on a ViewLux ultraHTS microplate imager (Perkin Elmer). Data was ed as CC50.

In parallel, a similar dilution series of compound was used (200 nL of compound in 100% DMSO) with 40 uL per well of cells transfected with NFKB- Iuc reporter construct alone (1.25 x 105 cells/mL). Six hours after tion at 37°C, 5% C02, the Iuciferase activity was determined by adding 15 ul of Steady Lite Plus substrate (Perkin Elmer) to each well and readout performed on a ViewLux ultraHTS microplate imager (Perkin Elmer). Counterscreen data is reported as LEC.

Activation of ISRE promoter elements The potential of compounds to induce IFN-l was also evaluated by measuring the activation of interferon—stimulated responsive elements (ISRE) by conditioned media from PBMC. The ISRE element of sequence GAAACTGAAACT is highly responsive to the STAT1-STAT2-IRF9 transcription factor, activated upon binding of lFN-l to their receptor IFNAR (Clontech, PT3372-5W). The plasmid Luc from Clontech (ref. 631913) contains 5 copies of this ISRE t, followed by the firefly Iuciferase ORF. A HEK293 cell line stably transfected with plSRE-Luc (HEK-lSREluc) was established to profile the conditioned PBMC cell culture media. y, PBMCs were prepared from buffy coats of at least two donors using a standard Ficoll centrifugation protocol. Isolated PBMCs were resuspended in RPMI medium supplemented with 10% human AB serum and 2 x 105 cells/well were dispensed into 384-well plates containing compounds (70 uL total -1 3- volume). After overnight tion, 10 uL of supernatant was transferred to 384-well plates containing 5 x 103 HEK-lSREluc well in 30 uL (plated the day before). Following 24 hours of incubation, activation of the ISRE elements was measured by assaying luciferase activity using 40 uL/well Steady Lite Plus substrate (Perkin Elmer) and measured with ViewLux ultraHTS microplate imager (Perkin . The stimulating activity of each compound on the HEK- lSREluc cells was reported as LEC value, defined as the compound concentration d to the PBMCs resulting in a luciferase activity at least two fold above the standard deviation of the assay. The LEC in turn indicates the degree of ISRE activation on transfer of a defined amount of PBMC culture medium. Recombinant eron d-2a (Roferon-A) was used as a standard control compound.

TABLE I: BIOLOGICAL ACTIVITY.

Human TLR 7 Human TLR 8 HEK-ISRE luc (LEC) uM (LEC) uM (LEC) uM NA = not available. All compounds showed no toxicity up to the highest tested tration. All compounds showed no activity (LEC >25 (M) in the HEK 293 NF—kB counterscreen assay described above.

Claims (7)

Claims 1.

1. A compound of formula (I) or a pharmaceutically acceptable salt, tautomer(s), stereo-isomeric forms, solvate or polymorph thereof, wherein X represents S, S=O or O=S=O, R1 is hydrogen, straight-chain or branched-chain (C1-6)-alkyl, (C1-6)-alkoxy or aryl, R2 is straight-chain or branched-chain (C1-3)-alkyl or (C3-6)-cycloalkyl and n = 1 or 2.

2. A pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt, tautomer(s), stereo-isomeric forms, solvate or polymorph f according to claim 1 together with one or more pharmaceutically acceptable excipients, diluents or carriers.

3. A compound of formula (I) or a pharmaceutically acceptable salt, tautomer(s), stereo-isomeric forms, solvate or rph thereof according to claim 1 or a pharmaceutical composition according to claim 2 for use as a medicament.

4. A compound of a (I) or a pharmaceutically acceptable salt, tautomer(s), stereo-isomeric forms, solvate or polymorph thereof according to claim 1, or a pharmaceutical ition according to claim 2 for use in the ent of a disorder in which the modulation of TLR7 and/or TLR8 is involved.

5. The use of a compound of formula (I) or a ceutically able salt, tautomer(s), stereo-isomeric forms, solvate or polymorph thereof ing to claim 1, or a pharmaceutical composition according to claim 2 for the cture of a medicament for the treatment of a disorder in which the modulation of TLR7 and/or TLR8 is involved.

6. The compound of claim 1 selected from the group consisting of: HN N NH2 O , , and .

7. The nd of claim 1, substantially as herein described with reference to the Experimental Section. 31993972v.1