NZ714519B2 - THIENO[3,2-d]PYRIMIDINES DERIVATIVES FOR THE TREATMENT OF VIRAL INFECTIONS - Google Patents
THIENO[3,2-d]PYRIMIDINES DERIVATIVES FOR THE TREATMENT OF VIRAL INFECTIONS Download PDFInfo
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- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 8
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Abstract
This invention relates to thieno[3,2-d]pyrimidine derivatives of formula (I), processes for their preparation, pharmaceutical compositions, and their use in treating viral infections.
Description
THIENO[3,2-d]PYRIMIDINES DERIVATIVES FOR THE TREATMENT OF VIRAL INFECTIONS This invention relates to thieno[3,2-d]pyrimidine derivatives, processes for their preparation, pharmaceutical itions, and their use in treating viral infections.
The present invention relates to the use of thieno[3,2-d]pyrimidine derivatives in the treatment of viral ions, immune or inflammatory disorders, whereby the modulation, or agonism, of ike-receptors (TLRs) is involved. Toll-Like Receptors are primary transmembrane proteins terized by an extracellular |eucine rich domain and a cytoplasmic extension that contains a conserved . The innate immune system can recognize pathogen-associated lar 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 mulatory molecules on phagocytes. This leads to the modulation ofT 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 identified in humans and mice together, and equivalent forms of many of these have been found in other mammalian species. However, equivalents of certain TLR found in humans are not present in all mammals. For example, a gene coding for a protein 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 . 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 immunity.
For reviews on TLRs see the following journal es. nn, J.A., Nature, 426, p33-38, 2003; Akira, 8., Takeda, K., and Kaisho, T., Annual Rev. Immunology, 21, p335-376, 2003; Ulevitch, R. J., Nature Reviews: Immunology, 4, 20, 2004.
Compounds indicating activity on Toll-Like receptors have been previously described such as purine derivatives in , adenine tives in WO 98/01448 and WO 21, and pyrimidines in .
However, there exists a strong need for novel Toll-Like receptor modulators having preferred selectivity, higher potency, and an improved safety profile ed to the compounds of the prior art.
In accordance with the present ion a compound of formula (I) is provided R3 (I) or a pharmaceutically acceptable salt, tautomer(s), -isomeric form, solvate or polymorph thereof, wherein R1 is selected from hydrogen, n, -CH3 or -CF3, R2 is selected from hydrogen, halogen, 01-6 alkyl or 03-6 cycloalkyl, R3 is 01-8 alkyl optionally substituted by one or more substituents independently selected from aryl, aryloxy, halogen, hydroxyl, alkylamino, dialkylamino, C1_6alkenyl, 01.6 alkoxy, carboxylic acid, carboxylic ester, carboxylic amide, nitrile, sulfonamide, sulfamide, acyl sulfonamide, or R3 is an alkylaryl optionally substituted by one or more substituents independently selected from aryl, aryloxy, halogen, alkylamino, dialkylamino, 01.6 alkyl, C1_6alkenyl, 01.6 , carboxylic acid, carboxylic ester, carboxylic amide, nitrile, amide, ide, or acyl amide.
The compounds of formula (I) and their pharmaceutically acceptable salts, tautomer(s), stereo-isomeric forms, e or polymorph thereof have activity as pharmaceuticals, in particular as modulators of Toll-Like Receptors 7 and 8 (especially TLR 8).
In a further aspect the present ion provides a pharmaceutical ition comprising a compound of formula (I) or a pharmaceutically acceptable salt, tautomer, stereo-isomeric form, solvate or polymorph thereof together with one or more pharmaceutically acceptable excipients, diluents or carriers.
Furthermore a compound of formula (I) or a pharmaceutically acceptable salt, solvate, tautomer, stereo-isomeric form or polymorph thereof according to the current ion, or a pharmaceutical composition comprising said compound of formu|a(|) or a ceutically acceptable salt, solvate, tautomer, stereo-isomeric form or polymorph thereof can be used as a medicament.
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 ition sing said nd of a (I) or a pharmaceutically acceptable salt, solvate, tautomer, stereo-isomeric form or polymorph thereof can be used accordingly in the treatment of a er in which the modulation of TLR7 and /or TLR8 is involved preferably TLR8.
The term "(C1-8)-alkyl" and "(C1-6)-alkyl" refers to a straight-chain or branched-chain ted aliphatic hydrocarbon containing the specified number of carbon atoms.
The term "halogen" refers to fluorine, chlorine, bromine or iodine.
The term "alkylaryl" refers to a straight-chain or branched-chain saturated aliphatic hydrocarbon containing the specified number of carbon atoms substituted by an aryl wherein "aryl" is defined as below.
The term "alkenyl" refers to an alkyl 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 (carbon and hydrogen chain) group singular bonded to oxygen like for instance a methoxy group or ethoxy group.
The term "aryl" means an aromatic ring structure optionally comprising one or two atoms selected from N, O and S, in particular from N and O. Said aromatic ring ure may have 5, 6 or 7 ring atoms. In ular, said aromatic ring structure may have or 6 ring atoms.
The term "aryloxy" refers to an aromatic ring structure. Said aromatic group is singularly bonded to oxygen.
As used herein, any al 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, S) around one or more atoms, contemplates each possible 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 isomer 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.
Diastereomers (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 enantiomers, diastereomers, racemates, E isomers, Z isomers, cis isomers, trans isomers and mixtures thereof, whenever chemically possible.
The meaning of all those terms, i.e. enantiomers, diastereomers, racemates, E isomers, Z isomers, cis isomers, trans isomers and es thereof are known to the skilled person.
The absolute configuration is specified ing to the Cahn-lngold-Prelog system. The configuration at an asymmetric atom is specified by either R or 8. ed isomers 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 (-) ing on the direction in which they rotate plane zed 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 nd 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 Formula (I) is for instance ied as E, this means that the compound is substantially free of the Z isomer; when a compound of Formula (I) is for ce specified as cis, this means that the compound is substantially free of the trans isomer.
Pharmaceutically acceptable salts of the compounds of formula (I) include the acid addition and base salts thereof. 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 ated 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 t molecules, for example, ethanol.
WO 14815 The term "polymorph" refers to the ability of the nd of the invention to exist in more than one form or crystal structure.
The compounds of the present invention may be stered as crystalline or amorphous products. They may be obtained for example as solid plugs, powders, 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 invention 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 factors such as the particular mode of administration, the effect of the ent on solubility and stability, and the nature of the dosage form.
The compounds of the present invention or any up 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 ion, an effective amount of the particular compound, optionally in on salt form, as the active ingredient is combined in intimate admixture with a pharmaceutically acceptable r, which carrier may take a wide variety of forms depending on the form of preparation desired for administration.
These pharmaceutical compositions are bly in unitary dosage form suitable, for example, for oral, rectal, or percutaneous administration. For example, in preparing the itions in oral dosage form, any of the usual pharmaceutical media may be employed 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, egrating agents and the like in the case of powders, pills, capsules, and tablets. Because of their ease in administration, s and capsules represent the most advantageous oral dosage unit forms, in which case solid pharmaceutical carriers are obviously ed. Also included are solid form preparations 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 wetting agent, optionally combined with suitable additives of any nature in minor proportions, which additives do not introduce a icant deleterious effect on the skin. Said additives may facilitate the administration to the skin and/or may be helpful for preparing the desired compositions. 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 formulations employed in the art for stration via this way. Thus, in general the compounds of the present invention may be administered to the lungs in the form of a solution, a suspension or a dry powder.
It is especially advantageous to formulate the aforementioned pharmaceutical itions in unit dosage form for ease of administration and uniformity of . Unit dosage form as used herein refers to physically discrete units suitable as unitary dosages, each unit ning a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the ed pharmaceutical carrier. Examples of such unit dosage forms are tablets (including scored or coated tablets), capsules, pills, powder packets, wafers, suppositories, injectable ons or suspensions and the like, and segregated multiples thereof.
Those of skill in the treatment of infectious es will be able to determine the effective amount from the test results presented after. In general 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 als throughout the day.
Said ses 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 ion being treated, the age, weight and general physical condition of the particular 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. 2014/066219 Pre aration of com ounds offormula | N=C—NH2 H R3\NH // ML» R \SI\N +|3M 3 R2 A NH2 R \N EtOH,ref|ux DMF,rt \' A O N NH2 N NH2 0 R1 R1 The preparation of compounds of type I are described in the literature (Synthetic Communications, 9(8), , 1979; Synthetic Communications, 32(16), 2565-2568; 2002). 3-aminothiophenecarboxylates are mixed with cyanamide in a polar solvent (e.g. ethanol) ning acid (e.g. HCI) to form intermediates II with heat as bed in the literature (Synthesis, (9), p1428, 2010). Intermediate II in polar, aprotic solvent can be mixed with BOP or PyBOP in combination with a base (e.g. DBU) and the amine to lead to the formation of final products (III) at room temperature. Alternatively, the alcohol in intermediates of type II can be converted to chlorine using described methods and chlorinating agents, such as POCI3, often with heat and in the presence of solvent, and optionally with base. After isolation, the 4-chloro intermediate can then be used to form products of type III by heating with the amine in base and polar solvent (e.g. acetonitrile).
Preparation of 1 m H OH H2N NH 8 \ S \ . N \ A DBU, BOP m \ A N NH2 N NH2 DMF, rt B 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 for 16 hours at room temperature. LC-MS showed conversion to product. The crude reaction mixture was purified by preparatory HPLC (RP SunFire Prep C18 OBD-10 um, 30 x 150 mm, mobile phase 0.25% aq. ammonium carbonate, to itrile). The best fractions were pooled and the solvents were removed under reduced pressure to afford a white solid, 1. LC-MS m/z = 237 (M+H).
Table 1. Compounds of formula (I) and corresponding analytical data. Compounds were prepared according to the methods described in the experimental section.
LC , LC-MS Mass # STRUCTURE 1H NMR Rt (min) Found (M+H) 1H NMR (400 MHz, DMSO-ds) 6 ppm 0.91 (t, J=7.4 Hz, 3 H), 1.33 (dq, J=14.9, 7.4 Hz, 2 H), 1.49 - A, 0.8 237 1.61 (m, 2 H), 3.35 (s, 3 H), 3.36 Mm, \N381H2 - 3.42 (m, 2 H), 5.74 (s, 2 H), 6.69 (s, 1 H), 7.03 (t, J=5.5 Hz, 1 H) 1H NMR (400 MHz, DMSO-ds) 6 — ppm 0.91 (t, J=7.4 Hz, 3 H), 1.26 2 s / N 237 - 1.42 (m, 2 H), 1.48 - 1.62 (m, 2 B, 1-52 MNH \NJNHZ H), 2.17(d,J=1.1 Hz,3 H), 3.37- 3.46 (m, 2 H), 5.83 (s, 2 H), 7.14 (s, 1 H), 7.43 (d, J=1.1 Hz,1 H) 1H NMR (400 MHz, DMSO-ds) 6 ppm 0.90 (t, J=7.4 Hz, 3 H), 1.27 3 s / - 1.35 (m, 2 H), 1.36 (s, 9 H), B, 1.83 279 X 1.47 - 1.60 (m, 2 H), 3.35 - 3.43 MM, \N H2 (m, 2 H), 5.72 (s, 2 H), 6.73 (s, 1 H), 7.04 (t, J=5.5 Hz, 1 H) M 1H NMR (400 MHz, DMSO-ds) 6 ts / x, ppm 0.85 (br. s., 3 H), 1.17-1.39 W (m, 4 H), 1.43 - 1.56 (m, 1 H), m 1.65 (br. s., 1 H), 3.39 - 3.54 (m, A, 0.70 267 ",1 2 H), 4.26 (d, J=4.4 Hz, 1 H), 4.65 (br. s., 1 H), 5.75 (s, 2 H), 6.84 (d, J=8.4 Hz, 1 H), 6.95 (d, J=5.3 Hz, 1 H), 7.81 (d, J=5.3 Hz, 1 H) 1H NMR (400 MHz, DMSO-ds) 6 «M ppm 0.78 - 0.94 (m, 3 H), 1.16 - 3 / 1%,,2 1.41 , 1.45- 1.69 (m,2 0" 3366 MN" H), 3.47 - 3.53 (m, 1 H), 4.30 - A, 0.63 253 4.47 (m, 2 H), 7.18 - 7.28 (m, 1 H), 7.77 (br. s., 2 H), 8.18 (d, J=5.3 Hz, 1 H), 8.92 (d, J=8.4 Hz, 1 H), 13.26 (br.s., 1 H) WO 14815 LC Method, LC-MS Mass STRUCTURE 1HNMR Rt (min) Found (M+H) 1H NMR (400 MHz, DMSO-ds) 6 ppm 0.84 (br. s., 3 H), 1.19 - 1.39 (m, 4 H), 1.42 - 1.57 (m, 1 H), 1.65 (br. s., 1 H), 3.37 - 3.55 (m, 4070 267 2 H), 3.71 - 4.21 (m, 1 H), 4.28 (d, J=4.6 Hz, 1 H), 5.97 (br. s., 2 H), 6.97 (d, J=5.3 Hz, 1 H), 7.05 (d, J=8.4 Hz, 1 H), 7.84 (d, J=5.3 Hz, 1 H) 1H NMR (400 MHz, CHLOROFORM-d) 6 ppm 0.98 (t, J=7.4 Hz, 3 H), 1.39 - 1.51 (m, 2 H), 1.61 - 1.69 (m, 2 H), 1.74 (s, 8071 223 1 H), 3.59 (td, J=7.2, 5.7 Hz, 2 H), 4.71 (br. s., 2 H), 7.11 (d, J=5.3 Hz, 1 H), 7.56 (d, J=5.3 Hz, 1 H) 1H NMR (400 MHz, DMSO-ds) 6 ppm 0.81 - 0.93 (m, 3 H), 1.20 - 1.40 (m, 4 H), 1.52 - 1.65 (m, 2 H), 1.74 (q, J=6.6 Hz, 2 H), 3.40 - 4076 281 3.50 (m, 2 H), 4.38 - 4.52 (m, 2 H), 7.22 (d, J=5.5 Hz, 1 H), 7.63 - 7.82 (m, 2 H), 8.18 (d, J=5.5 Hz, 1 H), 8.82 (d, J=8.4 Hz, 1 H) 1H NMR (400 MHz, DMSO-ds) 6 ppm 0.89 (t, J=7.3 Hz, 3 H), 1.16 (d, J=6.6 Hz, 3 H), 1.26 - 1.38 (m, 2 H), 1.39 - 1.51 (m, 1 H), 1.53 - 4082 237 1.64 (m, 1 H), 4.28 - 4.39 (m, 1 H), 5.77 (s, 2 H), 6.95 (d, J=5.3 Hz, 1 H), 7.01 (d, J=8.4 Hz, 1 H), 7.81 (d, J=5.3 Hz, 1 H) 1H NMR (400 MHz, DMSO-ds) 6 ppm 0.84 - 0.98 (m, 3 H), 1.27 - 1.51 (m, 2 H), 1.57 - 1.70 (m, 1 H), 1.80 - 1.98 (m, 1 H), 3.69 (s, 4076 281 3 H), 4.76 - 4.92 (m, 1 H), 7.27 (d, J=5.3 Hz, 1 H), 7.89 (br. s., 2 H), 8.26 (d, J=5.3 Hz, 1 H), 9.47 (d, J=7.3 Hz, 1 H) LC Method, LC-MS Mass STRUCTURE 1HNMR Rt (min) Found (M+H) 9%M2M N 1H NMR (400 MHz, DMSO-ds) 6 W ppm 0.87 (t, J=6.9 Hz, 3 H), 1.25 11 - 1.37 (m, 4 H), 1.57 (br. s., 2 H), A084 237 3.39 - 3.44 (m, 2 H), 5.80 (s, 2 H), 6.95 (d, J=5.3 Hz, 1 H), 7.25 (s, 1 H), 7.80 (d, J=5.3 Hz, 1 H) 1H NMR (400 MHz, DMSO-ds) 6 ppm 0.88 (t, J=7.3 Hz, 3 H), 1.21 - 1.43 (m, 2 H), 1.50 (dtd, J=13.5, 9.0, 9.0, 5.0 Hz, 1 H), 1.57 - 1.89 12 A061 253.1 (m, 1 H), 3.38 - 3.53 (m, 2 H), 4.29 (d, J=4.6 Hz, 1 H), 4.82 (br. s., 1 H), 5.80 (s, 2 H), 6.87 (d, J=8.4 Hz, 1 H), 6.96 (d, J=5.3 Hz, 1 H), 7.82 (d, J=5.3 Hz, 1 H) 1H NMR (400 MHz, CHLOROFORM-d) 6 ppm 0.95 (t, J=7.3 Hz, 3 H), 1.32 - 1.50 (m, OH 5/ 2 H), 1.51 - 1.71 (m, 2 H), 2.31 13 (d, J=1.1 Hz, 3 H), 3.34 (s, 1 H), A067 267.1 SNH N H2 3.67 (dd, J=11.0, 6.4 Hz, 1 H), 3.83 (dd, , 3.3 Hz, 1 H), 4.19 - 4.38 (m, 1 H), 4.77 (d, J=7.3 Hz, 1 H), 4.87 (s, 2 H), 7.19 (d,J=1.1 Hz, 1 H) 1H NMR (400 MHz, DMSO-ds) 6 ppm 0.80 - 0.94 (m, 3 H), 1.20 - 1.39 (m, 4 H), 1.49 - 14 1.64 (m, 2 H), 2.17 (cl, 8169 251.0 J=1.1 Hz, 3 H), 3.36 - 3.43 (m, 2 H), 5.82 (s, 2 H), 7.15 (t, J=5.5 Hz, 1 H), 7.43 (cl, J=1.1 Hz, 1 H) 1H NMR (400 MHz, CHLOROFORM-d) 6 ppm 0.96 (t, J=7.4 Hz, 3 H), 1.22 - 1.33 (m, 1 H), 1.35 - 1.52 (m, 1 H), 1.74 - 1.86 (m, 1 H), 1.87 - 2.01 (m, 1 5102 295.2 H), 2.33 (d, J=1.1 Hz, 3 H), 3.76 (s, 3 H), 4.75 (br. s., 2 H), 4.97 (td, J=7.5, 5.6 Hz, 1 H), 5.10 (d, J=7.7 Hz, 1 H), 7.22 (d, J=1.1 Hz, 1 H) WO 14815 LC Method LC-MS Mass # STRUCTURE 1H NMR ' Rt (min) Found (M+H) 1H NMR (400 MHz, CHLOROFORM-d) 5 ppm 0.92 (t, J=7.4 Hz, 3 H), 1.26 - 1.50 (m, 3 H), 1.51 - 1.66 (m, 2 H), 1.68 - 1.79 (m, 1 H), 1.86 - 2.03 (m, 1 H), 2.32 (d, J=1.1 Hz, 3 H), 3.45 - 3.68 (m, 2 H), 4.41 (ddd, J=11.1, .4, 2.9 Hz, 1 H), 4.52 (d, J=8.8 Hz, 1 H), 4.97 (s, 2 H), 7.20 (d, J=1.1 Hz, 1 H) 1H NMR (400 MHz, DMSO-d6) d ppm 0.85 (t, J=6.5 Hz, 3 H), 1.11 - 1.35 (m, 4 H), 1.38 - 1.56 (m, 1 H), 1.57 - 1.74 (m, 1 H), 2.18 (d, J=0.9 Hz, 3 H), 3.39 - 3.55 (m, 2 H), 4.19 - 4.35 (m, 1 H), 4.66 (br. s., 1 H), 5.79 (s, 2 H), 6.75 (d, J=8.4 Hz, 1 H), 7.44 (d, J=1.1 Hz, 1 H) 1H NMR (400 MHz, DMSO-ds) 6 ppm 0.76 - 0.91 (m, 3 H), 1.16 - 1.36 (m, 4 H), 1.44 - 1.58 (m, 2 H), 1.59 - 1.79 (m, 2 H), 2.17 (d, J=1.1 Hz, 3 H), 3.38 - 3.49 (m, 2 H), 4.34 (d, J=7.5 Hz, 1 H), 4.40 (t, J=5.4 Hz, 1 H), 5.83 (s, 2 H), 6.87 (d, J=8.6 Hz, 1 H), 7.44 (d, J=1.1 Hz, 1 H) 1H NMR (400 MHz, DMSO-ds) 6 ppm 0.88 (t, J=7.3 Hz, 3 H), 1.18 (d, J=6.5 Hz, 3 H), 1.23 - 1.38 (m, 2 H), 1.40 - 1.69 (m, 2 H), 2.58 (s, 3 H), 4.25 - 4.45 (m, 1 H), 6.93 (s, 1 H), 7.50 (br. s., 2 H), 8.50 (br.s.,1 H) WO 14815 LC Method, LC-MS Mass # STRUCTURE 1H NMR Rt (min) Found (M+H) 1H NMR (400 MHz, DMSO-ds) ppm 0.85 (t, J=6.5 Hz, 3 H), 1.17 - 1.40 (m, 4 H), 1.43-1.71 (m,2 H), 2.59 (s, 3 H), 3.45 - 3.50 (m, 2 H), 4.20 - 4.41 (m, 1 H), 6.99 (d, J=0.8 Hz, 1 H), 7.66 (br. s., 2 H), 8.71 (d, J=8.5 Hz,1 H), 13.00 (br. s., 1 H) 1H NMR (400 MHz, DMSO-ds) 6 ppm 0.85 (t, J=6.7 Hz, 3 H), 1.08 - 1.38 (m, 4 H), 1.43-1.71 (m,2 H), 2.52 (br. s., 2 H), 2.57 (m, J=1.0 Hz, 3 H), 4.29 (d, J=5.0 Hz, 1 H), 4.82 (br. s., 1 H), 6.92 (d, J=1.3 Hz, 1 H), 7.20 (br. s., 2 H), 8.22 (br. s., 1 H) 1H NMR (400 MHz, DMSO-ds) 6 ppm 0.85 (t, J=6.8 Hz, 3 H), 1.16 - 1.35 (m, 4 H), 1.40 (s, 9 H), 1.51 (dd, J=9.0, 4.8 Hz, 1 H), 1.64 (d, J=6.0 Hz, 1 H), 3.47 (br. s., 2 H), 4.33 (d, J=4.8 Hz, 1 H), 4.83 (br. s., 1 H), 7.00 (s, 1 H), 7.67 (br. s., 2 H), 8.66 (d, J=8.5 Hz, 1 H) General information: the LC measurement was performed using an Acquity UPLC (Waters) system comprising a binary pump, a sample organizer, a column heater (set at 55 0C), a array detector (DAD) and a column as specified in the respective methods below.
Flow from the column was split to a MS spectrometer. The MS detector was configured with an ospray ionization source. Mass spectra were ed 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 Methods.
Flow Mobile phase (lemin)l Temp (°C) A: 10mM Waters : CH3COONH4 in From 95% A to BEH C18 95% H20 + 5% 5% A in 1.3 min, (1.7pm, 2.1 CH3CNB: held for 0.7 min. x 50mm) CH3CN A: 10mM From 100% A to CH3COONH4 in 5% A in 2.10 min, 95% H20 + 5% to 0% A in 0.90 CH3CN min, to 5% A in B: CH3CN 0.5min 100% Afor 1min s Agilent: TC- ACES/334391 A’o to 40% A in 4min, C18 (5pm, to15%Ain 2.1x50mm) CF39OOH 0'05" 2.5min, to 100% In CH3CN A In 2min. . 90% Afor A' CF3000H 0.1% Agilent: TC- 0.8min, to 20% A in water, B: C18 (5pm, in 3.7min, held CFgCOOH 0.05% 2.1x50mm) for 3min, back to in CH3CN 90% A in 2min.
A: 10mM CT3COONH4 Ln.
From 950/ Ato 0 Waters : 5% A in 1.3 min, 90" H20 +105 BEH C18 held for 0.2 min, , CHsCN to 95% A in 2.1*50mm) 0.2 min held for 31 WW 0.1 min.
Biolo ical Activit of com ounds of formula | Description of ical Assays Assessment of TLR7 and TLR8 activity The ability of compounds to activate human TLR7 and/or TLR8 was assessed in a cellular er assay using HEK293 cells transiently transfected with a TLR7 or TLR8 expression vector and NFKB-IUC reporter uct.
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 % 002 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 microliters 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 |uciferase activity was determined by adding 15 uL of Steady Lite Plus ate (Perkin Elmer) to each well and readout performed on a ViewLux ultraHTS microplate imager (Perkin Elmer). Dose response curves were generated from measurements performed in quadruplicates. Lowest effective concentrations (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 compound with 40 uL per well of cells transfected with the CMV-TLR7 construct alone (1.25 x 105 cells/mL), in 384-well plates. Cell ity was measured after 6 hours tion at 37°C, % 002 by adding 15 uL of ATP lite (Perkin Elmer) per well and reading on a x ultraHTS microplate imager (Perkin Elmer). Data was reported as 0050.
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 uc er construct alone (1.25 x 105 cells/mL). Six hours after incubation at 37°C, 5% 002, the |uciferase activity was ined by adding 15 ul of Steady Lite Plus substrate (Perkin Elmer) to each well and readout med on a ViewLux ultraHTS late 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 GAAACT is highly responsive to the STAT1-STAT2- |RF9 ription factor, activated upon binding of IFN-l to their receptor IFNAR (Clontech, PT3372—5W). The plasmid plSRE-Luc from Clontech (ref. 631913) ns 5 copies of this ISRE element, followed by the firefly |uciferase ORF. A HEK293 cell line stably transfected with plSRE-Luc (HEK-lSREluc) was ished to profile the conditioned PBMC cell e media.
Briefly, PBMCs were prepared from buffy coats of at least two donors using a standard Ficoll centrifugation protocol. lsolated PBMCs were resuspended in RPMI medium supplemented with 10% human AB serum and 2 x 105 cells/well were dispensed into 384- well plates ning nds (70 uL total volume). After overnight incubation, 10 uL of supernatant was transferred to 384-well plates containing 5 x 103 HEK-lSREluc cells/well in uL (plated the day before). Following 24 hours of incubation, tion 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 Elmer). The stimulating activity of each compound on the HEK-lSREluc cells was reported as LEC value, defined as the compound concentration applied 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 interferon or-2a (Roferon-A) was used as a standard l compound.
Table 2. Biological Activity of compounds of a (I).
Human TLR 7 Human TLR 8 HEK-ISRE (LEC) uM (LEC) uM luc (LEC) ["9fo \immmo —x—xo M001 VN01 O 01 _\ '_x 12 07 33 25 61 27 m- 21 39 VN01 N _\ _\ N 0." MN .07 00 —x «a VN01 co 01 .01 N —x 03 9° \l o co 2014/066219 Human TLR 7 Human TLR 8 HEK-ISRE (LEC) uM (LEC) uM luc (LEC) All compounds showed no activity (LEC >25 uM) in the HEK 293 NF-kB counterscreen assay described above.
Claims (11)
1. A compound of formula (I) or a pharmaceutically acceptable salt, er(s), stereo-isomeric form, solvate or polymorph thereof, 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 C1-8 alkyl optionally substituted by one or more substituents independently selected from aryl, aryloxy, halogen, hydroxyl, alkylamino, dialkylamino, C 1-6 alkenyl, C1-6 alkoxy, carboxylic acid, carboxylic ester, carboxylic amide, nitrile, sulfonamide, sulfamide, acyl sulfonamide, or R3 is an alkylaryl optionally substituted by one or more substituents independently selected from aryl, aryloxy, halogen, mino, dialkylamino, C1-6 alkyl, C1-6 alkenyl, C1-6 alkoxy, carboxylic acid, carboxylic ester, carboxylic amide, e, sulfonamide, sulfamide, or acyl sulfonamide.
2. A compound, pharmaceutically able salt, tautomer, stereo-isomeric form, solvate or polymorph thereof, according to claim 1, which is selected from nds #1-23: # STRUCTURE # URE # URE # URE # URE # STRUCTURE S NH
3. A compound according to claim 1 wherein R1 and R2 are both hydrogen and wherein R3 is C1-8 alkyl substituted by hydroxyl.
4. A compound ing to claim 2 having the structure
5. A pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt, tautomer(s), stereo-isomeric forms , solvate or polymorph thereof ing to claim 1 together with one or more pharmaceutically acceptable ents, diluents or carriers.
6. A compound of formula (I) or a pharmaceutically acceptable salt, tautomer(s), stereo-isomeric forms, solvate or polymorph thereof according to claim 1 or a pharmaceutical composition according to claim 5 for use as a medicament.
7. A compound of formula (I) or a pharmaceutically acceptable salt, er(s), stereo-isomeric forms , e or polymorph thereof according to claim 1, or a pharmaceutical composition ing to claim 5 for use in the treatment of a disorder in which the modulation of TLR7 and/or TLR8 is involved.
8. A compound of formula (I) or a pharmaceutically acceptable salt, tautomer(s), stereo-isomeric forms, e or polymorph f according to claim 1, or a pharmaceutical ition according to claim 5 for use in the treatment of a disorder in which the modulation of TLR8 is involved.
9. Use of the compound of formula (I) or a pharmaceutically acceptable salt, tautomer(s), stereoisomeric forms, solvate or polymorph thereof according to claim 1, or a pharmaceutical composition according to claim 5, in the manufacture of a medicament for the treatment of a disorder in which the modulation of TLR7 and/or TLR8 is involved.
10. Use according to claim 9, wherein the treatment of the disorder es the modulation of TLR8.
11. The compound according to claim 1, substantially as described with reference to any one of the Examples thereof.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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EP13178534 | 2013-07-30 | ||
PCT/EP2014/066219 WO2015014815A1 (en) | 2013-07-30 | 2014-07-29 | THIENO[3,2-d]PYRIMIDINES DERIVATIVES FOR THE TREATMENT OF VIRAL INFECTIONS |
Publications (2)
Publication Number | Publication Date |
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NZ714519A NZ714519A (en) | 2021-07-30 |
NZ714519B2 true NZ714519B2 (en) | 2021-11-02 |
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