HK1217707B - Thieno[3,2-d]pyrimidines derivatives for the treatment of viral infections - Google Patents

Description

Thieno[3,2-d]pyrimidine derivatives for treating viral infections

The present invention relates to thieno[3,2-d]pyrimidine derivatives, processes for their preparation, pharmaceutical compositions, and their use in the treatment of viral infections.

The present invention relates to the use of thieno[3,2-d]pyrimidine derivatives in the treatment of viral infections, immune or inflammatory disorders, and thus to the regulation or stimulation of toll-like receptors (TLRs). Toll-like receptors are major transmembrane proteins characterized by an extracellular leucine-rich domain and a cytoplasmic extension containing a conserved region. The innate immune system can recognize pathogen-associated molecular patterns through these TLRs expressed on the cell surface of certain types of immune cells. Recognition of exogenous pathogens activates the production of cytokines and the upregulation of co-stimulatory molecules on phagocytes. This leads to the regulation of T cell behavior.

It is estimated that most mammalian species have between ten and fifteen types of Toll-like receptors. Thirteen TLRs (i.e., TLR1 to TLR13) have been identified in humans and mice, and many equivalents of these have been found in other mammalian species. However, equivalents of some TLRs found in humans are not present in all mammals. For example, a gene encoding a protein similar to TLR10 in humans exists in mice, but the gene appears to have been damaged by a retrovirus at a certain site in the past. On the other hand, mice express TLR 11, TLR 12, and TLR 13, none of which are present in humans. Other mammals may express TLRs not found in humans. Other non-mammalian species may have TLRs that are different from mammals, as demonstrated by TLR14 found in pufferfish. This can complicate the method of using experimental animals as models of human innate immunity.

For reviews of TLRs, see the following journal articles: Hoffmann, J.A., Nature, 426, 33-38, 2003; Akira, S., Takeda, K., and Kaisho, T., Annual Rev. Immunology, 21, 335-376, 2003; and Ulevitch, R.J., Nature Reviews: Immunology, 4, 512-520, 2004.

Compounds indicated to be active at Toll-like receptors have been previously described, for example purine derivatives in WO 2006/117670, adenine derivatives in WO 98/01448 and WO 99/28321, and pyrimidines in WO 2009/067081.

However, there is still a pressing need for novel toll-like receptor modulators with superior selectivity, higher potency and improved safety profile compared to prior art compounds.

According to the present invention, there is provided a compound having the chemical formula (I)

or a pharmaceutically acceptable salt, one or more tautomers, stereoisomeric forms, solvates or polymorphs thereof, wherein

R ₁ is selected from hydrogen, halogen, -CH ₃ or -CF ₃ ,

_R2 is selected from hydrogen, halogen, _C1-6 alkyl or _C3-6 cycloalkyl,

_R3 is _C1-8 alkyl optionally substituted with one or more substituents independently selected from the group consisting of aryl, aryloxy, halogen, hydroxy, alkylamino, dialkylamino, _C1-6 alkenyl, _C1-6 alkoxy, carboxylic acid, carboxylate, carboxylic acid amide, nitrile, sulfonamide, sulfamide, acylsulfonamide, or

R ₃ is alkylaryl optionally substituted with one or more substituents independently selected from the group consisting of aryl, aryloxy, halogen, alkylamino, dialkylamino, C _1-6 alkyl, C _1-6 alkenyl, C _1-6 alkoxy, carboxylic acid, carboxylate, carboxylic acid amide, nitrile, sulfonamide, sulfamide, or acylsulfonamide.

The compounds of formula (I) and pharmaceutically acceptable salts, one or more tautomers, stereoisomeric forms, solvates or polymorphs thereof are active as pharmaceuticals, particularly as modulators of Toll-like receptors 7 and 8 (especially TLR8).

In another aspect, the present invention provides a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt, tautomer, stereoisomeric form, solvate or polymorph thereof together with one or more pharmaceutically acceptable excipients, diluents or carriers.

In addition, the compound of the present invention having chemical formula (I) or its pharmaceutically acceptable salt, solvate, tautomer, stereoisomeric form or polymorph, or a pharmaceutical composition comprising the compound of the present invention having chemical formula (I) or its pharmaceutically acceptable salt, solvate, tautomer, stereoisomeric form or polymorph can be used as a medicament.

Another aspect of the present invention is that the compound of formula (I) or its pharmaceutically acceptable salt, solvate, tautomer, stereoisomeric form or polymorph, or the pharmaceutical composition comprising the compound of formula (I) or its pharmaceutically acceptable salt, solvate, tautomer, stereoisomeric form or polymorph can be used accordingly to treat conditions in which the regulation of TLR7 and/or TLR8, preferably TLR8, is involved.

The terms "(C _1-8 )-alkyl" and "(C _1-6 )-alkyl" refer to straight-chain, branched or cyclic saturated aliphatic hydrocarbons containing the indicated number of carbon atoms.

The term "halogen" refers to fluorine, chlorine, bromine or iodine.

The term "alkylaryl" refers to a straight or branched chain saturated aliphatic hydrocarbon containing the specified number of carbon atoms substituted with an aryl group, wherein "aryl" is defined below.

The term "alkenyl" refers to an alkyl group as defined above consisting of at least two carbon atoms and at least one carbon-carbon double bond.

The term "cycloalkyl" refers to a carbocyclic ring containing the specified number of carbon atoms.

The term "alkoxy" refers to an alkyl group (carbon and hydrogen chain) solely bonded to an oxygen, such as, for example, a methoxy group or an ethoxy group.

The term "aryl" means an aromatic ring structure which may optionally include one or two heteroatoms selected from N, O and S, in particular selected from N and O. The aromatic ring structure may have 5, 6 or 7 ring atoms. Specifically, the aromatic ring structure may have 5 or 6 ring atoms.

The term "aryloxy" refers to an aromatic ring structure wherein the aryl group is singly bonded to an oxygen.

As used herein, any chemical formula having bonds shown only as solid lines and not as solid or dashed wedges, or otherwise expressed as having a particular configuration (e.g., R, S) around one or more atoms, contemplates every possible stereoisomer, or mixture of two or more stereoisomers.

Above or below, the terms "stereoisomer", "stereoisomeric form" or "stereochemically isomeric form" are used interchangeably.

The present invention includes all stereoisomers of the compounds of the present invention as pure stereoisomers or as mixtures of two or more stereoisomers.

Enantiomers are stereoisomers that are non-superimposable mirror images of each other. A 1:1 mixture of a pair of enantiomers is a racemate or racemic mixture.

Diastereomers (or diastereomers) are stereoisomers that are not enantiomers, i.e., they are not mirror-image related. If the compound contains a double bond, these substituents may be in the E or Z configuration. If the compound contains at least two substituted non-aromatic ring groups, these substituents may be in the cis or trans configuration.

Therefore, the present invention includes enantiomers, diastereomers, racemates, E isomers, Z isomers, cis isomers, trans isomers and mixtures thereof, wherever chemically possible.

The meaning of all those terms (ie enantiomers, diastereomers, racemates, E isomers, Z isomers, cis isomers, trans isomers and mixtures thereof) is known to the skilled person.

Absolute configuration is assigned according to the Cahn-Ingold-Prelog system. Configuration at asymmetric atoms is specified by R or S. Resolved stereoisomers whose absolute configuration is unknown can be assigned by (+) or (-) based on the direction in which they rotate plane-polarized light. For example, resolved enantiomers whose absolute configuration is unknown can be assigned by (+) or (-) based on the direction in which they rotate plane-polarized light.

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

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

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

The term "polymorph" means that a compound of the present invention can exist in more than one form or crystal structure.

The compounds of the present invention can be administered as crystalline or amorphous products. These compounds can be obtained, for example, as solid plugs, powders, or films by methods such as precipitation, crystallization, freeze drying, spray drying, or evaporative drying. They can be administered alone or in combination with one or more other compounds of the present invention or in combination with one or more other drugs. Typically, they will be administered as a formulation combined with one or more pharmaceutically acceptable excipients. The term "excipient" is used herein to describe any ingredient other than one or more compounds of the present invention. The choice of excipient generally depends on factors such as the specific mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.

The compounds of the present invention or any subgroup thereof can be formulated into different pharmaceutical forms for administration purposes. As suitable compositions, all compositions commonly used for systemic administration of drugs can be cited. To prepare the pharmaceutical compositions of the present invention, an effective amount of a specific compound, optionally in the form of an addition salt, is combined as an active ingredient with a pharmaceutically acceptable carrier in a close mixture, which carrier can take a variety of forms depending on the desired formulation form. These pharmaceutical compositions are desirably in unit dosage forms suitable for, for example, oral, rectal or transdermal administration. For example, in the preparation of a composition in an oral dosage form, any common pharmaceutical medium can be used, such as water, glycols, oils, alcohols, etc. in the case of oral liquid preparations (such as suspensions, syrups, elixirs, emulsions and solutions); or solid carriers such as starch, sugar, kaolin, diluents, lubricants, binders, disintegrants, etc. in the case of powders, pills, capsules and tablets. Tablets and capsules represent the most advantageous oral unit dosage forms due to their ease of administration, in which case solid pharmaceutical carriers are obviously used. Also included are solid form preparations that can be converted into liquid form shortly before use. In compositions suitable for transdermal administration, the carrier may optionally include a penetration enhancer and/or a suitable wetting agent, optionally in combination with a small proportion of suitable additives of any nature which do not cause significant adverse effects on the skin. The additives may facilitate administration to the skin and/or may contribute to the preparation of the desired composition. These compositions may be administered in various ways, for example, as a transdermal patch, as a spot-on, or as an ointment. The compounds of the present invention may also be administered via inhalation or insufflation using methods and formulations employed in the art for administration in this manner. Thus, generally, the compounds of the present invention may be administered to the lungs in the form of a solution, suspension, or dry powder.

For ease of administration and uniformity of dosage, it is particularly advantageous to formulate the above-described pharmaceutical compositions in unit dosage form. As used herein, a unit dosage form refers to a physically discrete unit suitable as a unit dosage, each unit containing a predetermined amount of the active ingredient calculated to produce the desired therapeutic effect in combination with the required pharmaceutical carrier. Examples of such unit dosage forms are tablets (including scored or coated tablets), capsules, pills, powder packets, wafers, suppositories, injectable solutions or suspensions, and similar dosage forms, and segregated multiples thereof.

Those skilled in the art of treating infectious diseases will be able to determine an effective amount from the test results presented below. It is generally believed that the effective daily dose will 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. The desired dose can be appropriately administered as two, three, four or more sub-doses at appropriate intervals throughout the day. The sub-doses can be formulated into unit dosage forms, for example, containing 1 to 1000 mg, and specifically 5 to 200 mg, of the active ingredient per unit dosage form.

As is well known to those of ordinary skill in the art, the precise dosage and frequency of administration depend on the specific compound with chemical formula (I) used, the specific condition being treated, the severity of the condition being treated, the age, body weight and general physical condition of the specific patient, and other drugs that the individual can take. In addition, it is apparent that the effective amount can be reduced or increased, depending on the reaction of the subject being treated and/or depending on the assessment of the physician prescribing the compound of the present invention. Therefore, the scope of the effective amount mentioned above is merely a guide and is not intended to limit the scope or purposes of the present invention to any extent.

Preparation of compounds of formula (I)

Overall plan.

The preparation of compounds of type I is described in the literature (Synthetic Communications, 9(8), pp. 731-4, 1979; Synthetic Communications, 32(16), 2565-2568; 2002). As described in the literature (Synthesis, (9), p. 1428, 2010), 3-aminothiophene-2-carboxylate is mixed with cyanamide in a polar solvent (e.g., ethanol) containing an acid (e.g., HCl) under heating to form intermediate II. Intermediate II in a polar aprotic solvent can be mixed with BOP or PyBOP in combination with a base (e.g., DBU) and an amine at room temperature to form the final product (III). Alternatively, the alcohol in the intermediate of type II can be converted to chloride using the described method and a chlorinating agent (e.g., POCl ₃ ), typically under heating in the presence of a solvent and optionally in the presence of a base. After isolation, the 4-chloro intermediate can then be used to form Type III products by heating with a base and an amine in a polar solvent such as acetonitrile.

Preparation of 1

Into a 50 mL glass vial was placed B (500 mg, 2.76 mmol), anhydrous DMF (5 mL), DBU (1.26 g, 8.28 mmol), n-butylamine (605 mg, 8.3 mmol) and BOP (1.46 g, 3.31 mmol). The vial was sealed and shaken at room temperature for 16 hours. LC-MS showed conversion to product. The crude reaction mixture was purified by preparative HPLC (RP SunFire Prep C18 OBD-10 μm, 30×150 mm, mobile phase 0.25% aqueous ammonium carbonate to acetonitrile). The best fractions were pooled and the solvent removed under reduced pressure to provide a white solid, 1. LC-MS m/z=237 (M+H).

Table 1. Compounds of formula (I) and corresponding analytical data. The compounds were prepared according to the methods described in the experimental part.

Analytical methods

General information: LC measurements were performed using an Acquity UPLC (Waters) system comprising a binary pump, sample organizer, column heater (set to 55° C.), a diode array detector (DAD), and the column specified in the corresponding methods below. The flow from the column was diverted to the MS spectrometer. The MS detector was equipped with an electrospray ionization source. Mass spectra were acquired by scanning from 100 to 1000 in 0.18 seconds using a dwell time of 0.02 seconds. The capillary needle voltage was 3.5 kV and the source temperature was maintained at 140° C. Nitrogen was used as the nebulizer gas.

LC-MS method.

Biological activities of compounds of formula (I)

Bioassay Description

Assessment of TLR7 and TLR8 activity

The ability of these compounds to activate human TLR7 and/or TLR8 was assessed in a cellular reporter gene assay using HEK293 cells transiently transfected with TLR7 or TLR8 expression vectors and an NFκB-luc reporter gene construct.

In brief, HEK293 cells were grown in culture medium (DMEM supplemented with 10% FCS and 2 mM glutamine). For the transfection of cells in 15 cm culture dishes, cells were dispersed with trypsin-EDTA, and cells were transfected with a mixture of CMV-TLR7 or TLR8 plasmid (1700 ng), NFKB-luc plasmid (850 ng) and transfection reagent and incubated for 48 h at 37 ° C in a humidified 5% CO ₂ atmosphere. The transfected cells were then washed in PBS, separated with trypsin-EDTA and resuspended in culture medium to a density of 1.25 × 10 ⁵ cells/mL. 40 microliters of cells were then distributed to each well in a 384-well plate, where 200 nL of compound (in 100% DMSO) was already present. After incubation for 6 hours at 37°C, 5% _CO2 , luciferase activity was determined by adding 15 μL of Steady Lite Plus substrate (PerkinElmer) to each well and reading on a ViewLux ultraHTS microplate imager (PerkinElmer). Dose-response curves were generated from measurements performed in quadruplicate. The minimum effective concentration (LEC) value for each compound was determined, defined as the concentration that elicited an effect that exceeded the standard deviation of the assay by at least two-fold.

In a 384-well plate, similar dilution series of the compound and 40 μL of cells transfected with CMV-TLR7 constructs per well (1.25×10 ⁵ cells/mL) were used to determine compound toxicity in parallel. After incubation for 6 hours at 37°C, 5% CO ₂ , cell viability was measured by adding 15 μL of ATP lite (PerkinElmer) to each well and reading on a ViewLux ultraHTS microplate imager (PerkinElmer). Data were reported as CC ₅₀ .

In parallel, a similar dilution series of compound (200 nL of compound in 100% DMSO) and 40 μL of cells transfected with NFκB-luc reporter constructs per well (1.25 × ¹⁰ cells/mL) were _used . After incubation at 37°C, 5% CO for 6 hours, luciferase activity was determined by adding 15 μL of Steady Lite Plus substrate (PerkinElmer) to each well and reading on a ViewLux ultraHTS microplate imager (PerkinElmer). Counter screen data were reported as LEC.

Activation of ISRE promoter elements

The potentiality of these compounds for inducing IFN-I was also assessed by measuring the activation of the interferon-stimulated response element (ISRE) caused by the conditioned medium from PBMC. The ISRE element height with sequence GAAACTGAAACT is responsive to STAT1-STAT2-IRF9 transcription factors, which are activated when IFN-I is bound to their receptor IFNAR (Clontech, PT3372-5W). The plasmid pISRE-Luc from Clontech (reference number 631913) comprises 5 copies of this ISRE element, followed by firefly luciferase ORF. A HEK293 cell line of a stable transfection pISRE-Luc (HEK-ISREluc) was set up to analyze PBMC conditioned cell culture medium.

Briefly, PBMCs were prepared from buffy coats of at least two donors using a standard Ficoll centrifugation protocol. The isolated PBMCs were resuspended in RPMI medium supplemented with 10% human AB serum, and 2 × 10 ⁵ cells/well were distributed into 384-well plates containing the compound (70 μL total volume). After overnight incubation, 10 μL of supernatant was transferred to a 384-well plate containing 5 × 10 ³ HEK-ISREluc cells/well (30 μL) (plated the day before). After 24 hours of incubation, the activation of the ISRE element was measured by measuring luciferase activity (using 40 μL/well SteadyLitePlus substrate (PerkinElmer) and measured with a ViewLuxultraHTS microplate imager (PerkinElmer)). The stimulatory activity of each compound on HEK-ISREluc cells is reported as the LEC value, which is defined as the concentration of compound that results in luciferase activity exceeding the assay standard deviation by at least two times when applied to PBMC. The LEC further indicates the extent of ISRE activation when a defined amount of PBMC culture medium is transferred. Recombinant interferon α-2a (Roferon-A) is used as a standard control compound.

Table 2 Biological activities of compounds of formula (I).

All compounds showed no activity (LEC > 25 μM) in the HEK 293 NF-κB counter screen assay described above.

Claims (8)

1. A compound having the chemical formula (I) Or its pharmaceutically acceptable salt or stereoisomer, wherein _R1 is selected from hydrogen, halogen, _-CH3 , or _-CF3 . _R2 is selected from hydrogen, halogen, _C1-6 alkyl, or _C3-6 cycloalkyl. _R3 is a _C1-8 alkyl group optionally substituted with one or more substituents independently selected from the following: halogen, hydroxyl, alkylamino, dialkylamino, _C1-6 alkoxy, carboxylic acid, carboxylic acid ester, carboxylic acid amide, nitrile, sulfonamide, thioamide, acylsulfonamide. 2. The compound according to claim 1, wherein both _R1 and _R2 are hydrogen and wherein _R3 is a _C1-8 alkyl group substituted with a hydroxyl group. 3. The compound according to claim 2, having the following structure: . 4. A pharmaceutical composition comprising a compound having chemical formula (I) according to claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, together with one or more pharmaceutically acceptable excipients or carriers. 5. A pharmaceutical composition comprising a compound having chemical formula (I) according to claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, together with one or more pharmaceutically acceptable diluents. 6. Use of the compound having chemical formula (I) according to claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, or the pharmaceutical composition according to claim 4 or 5, in the preparation of an agent for treating viral infections. 7. Use of a compound having chemical formula (I) according to claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, or a pharmaceutical composition according to claim 4 or 5, in the preparation of a pharmaceutical agent for treating a condition in which regulation of TLR7 and/or TLR8 is involved. 8. The use according to claim 7, wherein the agent is used to treat a condition in which TLR8 regulation is involved.