Patent Application
20240294465
The present invention relates to new cinnamic acid amides which may be used for treatment of fibrosis and neoplasia and to cinnamic acid amides for use in the treatment of fibrosis, neoplasia, arthrolithiasis, familiar mediterranean fever and pericarditis. Further, the invention relates to a pharmaceutical composition comprising said cinnamic acid amides and to a screening essay for identifying compounds suitable for the treatment of fibrosis.
Fibrotic diseases affect nearly every tissue in the body, account for over 45% of all deaths in the industrialized world, and progressive forms of the disease rapidly lead to organ dysfunction, organ failure and ultimately death (1-3). Due to its ubiquitous existence and high mortality, fibrosis, or “scarring”, has become a high medical need for novel drug discovery strategies (3, 4). However, effective antifibrotic therapeutics are missing in the clinics. The lack of antifibrotic therapies and its concomitant high medical need is best exemplified by idiopathic pulmonary fibrosis (IPF), which is a rapidly progressive and fatal fibrotic disorder. Patients with this common form of interstitial fibrotic lung disease face a median survival time of 3-5 years (5-7). Currently, only two approved anti-fibrotic drugs for IPF are on the market, Pirfenidone and Nintedanib, however, both substances partially slow down the rate in lung function decline but do not stop disease progression (8-10). Therefore, new therapeutic strategies and approaches are urgently required. In fibrotic pathogenesis repetitive and constant injury leads to a sustained and self-perpetuating activation of fibroblasts, leading to their transdifferentiation into synthetic and highly contractile α-smoothmuscle-actin (αSMA)-expressing myofibroblasts, that massively deposit extracellular matrix (ECM), which stiffens the lung and destroys normal lung architecture (3, 6, 11, 12). The matrisome of fibrotic ECM was shown to harbor a disease- and progression specific signature of fibrillar collagens (types I, Ill, and V), proteoglycans, fibronectin, glycosaminoglycans, matrix-Gla protein, and microfibrillar-associated proteins (11, 13-16).
Of all pro-fibrotic signals reported, multifunctional TGFP1 is the most intensively studied and central player in various fibrotic diseases capable of triggering transdifferentiation of fibroblasts into myofibroblasts (17-21). TGFP1 binds to its TGFβ1-receptor and downstream signaling occurs by post translational modifications of cytoplasmic members of the SMAD family, which act as transcription factors in the cell nucleus, regulating the expression of common profibrotic genes, including ECM proteins (22-25). Plasminogen activator inhibitor-1 (PAI-1) is an essential downstream target of the TGFβ1 pathway, suppresses the fibrinolytic system and is considered as a therapeutic target option for fibrosis (26). Additionally, in IPF profibrotic IL8 was recently found to be secreted by a special fibrogenic mesenchymal progenitor cell population with autocrine effects on proliferation and motility, as well as paracrine effects on macrophage recruitment (27).
Tranilast is known as a mast cell degranulation inhibitor developed by Kissei Pharmaceuticals and was already approved 1982 in Japan and South Korea for the treatment of bronchial asthma, keloid and hypertrophic scars. The drug appears to work by inhibiting the release of histamine from mast cells but its molecular target(s) remain unknown. Even though the antifibrotic properties of Tranilast have also been reported in the prior art, its potency is very low (IC50˜150 μM) and would require high-dose administration in humans, which reportedly causes liver toxicity. So far, medicinal chemistry optimization efforts failed to significantly improve the antifibrotic activity of Tranilast (28).
Thus, there is a general need for antifibrotic drugs and assays for identification of suitable antifibrotic drugs. Tranilast may be a suitable lead compound for further medicinal chemistry optimization.
The Invention is directed to a compound for use in the treatment of fibrosis and neoplasia, preferably a fibrosis or neoplasia located in the heart, the lung, the renal tract, the liver, in the skin, in the pleura and retroperitoneum, more preferably the fibrosis is selected from pleural fibrosis, retroperitoneal fibrosis, atrial fibrillation, myocardial interstitial fibrosis, idiopathic pulmonary fibrosis (IPF), interstitial lung diseases, chronic kidney disease, non-alcoholic fat liver disease, skin scars, keloids, tumor-associated desmoplastic reaction wherein said compound is a compound according to formula (I)
—(CH2)u(C2)alkynyl;
X is selected from the group consisting of O, —NH— or S;
preferably H or benzyl, most preferably H;
The invention is further related to a compound according to formula (II)
—O(CH2)u(C3-C10)aryl,
preferably H or benzyl, most preferably H;
In a further embodiment, the invention is related to a pharmaceutical composition comprising the compounds as defined above.
In a further embodiment, the invention is directed to the compounds as defined above and the pharmaceutical composition for use in medicine, in particular for use in the treatment of fibrosis and neoplasia, preferably a fibrosis or neoplasia located in the heart, the lung, the renal tract, the liver, in the skin, in the pleura and retroperitoneum, more preferably the fibrosis is selected from pleural fibrosis, retroperitoneal fibrosis, atrial fibrillation, myocardial interstitial fibrosis, idiopathic pulmonary fibrosis (IPF), interstitial lung diseases, chronic kidney disease, non-alcoholic fat liver disease, skin scars, keloids, tumour-associated desmoplastic reaction.
In a further embodiment, the invention is directed to the compounds as defined above and the pharmaceutical composition for use in medicine, in particular for use in the treatment of inflammatory diseases, such as arthrolithiasis, familiar mediterranean fever and pericarditis.
Moreover, the invention is directed to a screening assay, comprising the steps
In dose-response relationship studies, applying the assay of the present invention, the inventive compounds proved to be >100 fold more potent compared to Tranilast in inhibiting ECM deposition (see
For the inventive assay, it has been found that immunostaining according to step b) prior to fixation according to step c) identifies exclusively the extracellular deposited proteins, and does not lead to “false-positive” hits due to staining of intracellular ECM precursor proteins. The assay can be used for the quantification of deposited ECM of any adherent cells that produce ECM (primary patient derived, primary animal derived, human cell lines, animal cell lines), derived from any organ (healthy or diseased) or from various animal species and/or animal disease model. In one embodiment patient derived human primary cells (for example lung fibroblasts from IPF patients) are used. This generates efficacy and potency data with the highest clinical relevance possible in vitro, especially when compared to assays that use immortalized cell lines or cells from different animal species.
The solution of the present invention is described in the following, exemplified in the appended examples, illustrated in the Figures and reflected in the claims.
The term “alkyl” refers to a monoradical of a saturated straight or branched hydrocarbon. Preferably, the alkyl group comprises from 1 to 10 carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 1 to 8 carbon atoms, such as 1 to 6 or 1 to 4 carbon atoms. Exemplary alkyl groups include methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, tert-butyl, n-pentyl, iso-pentyl, sec-pentyl, neo-pentyl, 1,2-dimethyl-propyl, iso-amyl, n-hexyl, iso-hexyl, sec-hexyl, n-heptyl, iso-heptyl, n-octyl, 2-ethyl-hexyl, n-nonyl, n-decyl, and the like.
The term “cycloalkyl” represents cyclic non-aromatic versions of “alkyl” and “alkenyl” with preferably 3 to 10 carbon atoms, i.e., 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, more preferably 3 to 8 carbon atoms, even more preferably 3 to 7 carbon atoms. Exemplary cycloalkyl groups include cyclopropyl, cyclopropenyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, cyclononyl, cyclononenyl, cylcodecyl. Preferred examples of cycloalkyl include (C3-C8)-cycloalkyl, in particular cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl.
In an alkyl group or cycloalkyl group, one or more hydrogen atoms may be replaced by a halogen atom, such as Cl, Br, F, preferably F.
The term “alkenyl” refers to a monoradical of an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond. Generally, the maximal number of carbon-carbon double bonds in the alkenyl group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkenyl group by 2 and, if the number of carbon atoms in the alkenyl group is uneven, rounding the result of the division down to the next integer. For example, for an alkenyl group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenyl group has 1 to 4, i.e., 1, 2, 3, or 4, carbon-carbon double bonds. Preferably, the alkenyl group comprises from 2 to 10 carbon atoms, i.e., 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, more preferably 2 to 8 carbon atoms, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in a preferred embodiment, the alkenyl group comprises from 2 to 10 carbon atoms and 1, 2, 3, 4, or 5 carbon-carbon double bonds, more preferably it comprises 2 to 8 carbon atoms and 1, 2, 3, or 4 carbon-carbon double bonds, such as 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon double bonds or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds. The carbon-carbon double bond(s) may be in cis (Z) or trans (E) configuration. Exemplary alkenyl groups include vinyl, 1-propenyl, 2-propenyl (i.e., allyl), 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl, 6-nonenyl, 7-nonenyl, 8-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl, 4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, 9-decenyl, and the like. If an alkenyl group is attached to a nitrogen atom, the double bond cannot be alpha to the nitrogen atom.
The term “alkenylene” refers to a diradical of an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond. Generally, the maximal number of carbon-carbon double bonds in the alkenylene group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkenylene group by 2 and, if the number of carbon atoms in the alkenylene group is uneven, rounding the result of the division down to the next integer. For example, for an alkenylene group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenylene group has 1 to 4, i.e., 1, 2, 3, or 4, carbon-carbon double bonds. Preferably, the alkenylene group comprises from 2 to 10 carbon atoms, i.e., 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, more preferably 2 to 8 carbon atoms, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in a preferred embodiment, the alkenylene group comprises from 2 to 10 carbon atoms and 1, 2, 3, 4, or 5 carbon-carbon double bonds, more preferably it comprises 2 to 8 carbon atoms and 1, 2, 3, or 4 carbon-carbon double bonds, such as 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon double bonds or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds. The carbon-carbon double bond(s) may be in cis (Z) or trans (E) configuration. Exemplary alkenylene groups include ethen-1,2-diyl, vinyliden, 1-propen-1,2-diyl, 1-propen-1,3-diyl, 1-propen-2,3-diyl, allyliden, 1-buten-1,2-diyl, 1-buten-1,3-diyl, 1-buten-1,4-diyl, 1-buten-2,3-diyl, 1-buten-2,4-diyl, 1-buten-3,4-diyl, 2-buten-1,2-diyl, 2-buten-1,3-diyl, 2-buten-1,4-diyl, 2-buten-2,3-diyl, 2-buten-2,4-diyl, 2-buten-3,4-diyl, and the like. If an alkenylene group is attached to a nitrogen atom, the double bond cannot be alpha to the nitrogen atom.
The term “alkynyl” refers to a monoradical of an unsaturated straight or branched hydrocarbon having at least one carbon-carbon triple bond. Generally, the maximal number of carbon-carbon triple bonds in the alkynyl group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkynyl group by 2 and, if the number of carbon atoms in the alkynyl group is uneven, rounding the result of the division down to the next integer. For example, for an alkynyl group having 9 carbon atoms, the maximum number of carbon-carbon triple bonds is 4. Preferably, the alkynyl group has 1 to 4, i.e., 1, 2, 3, or 4, more preferably 1 or 2 carbon-carbon triple bonds. Preferably, the alkynyl group comprises from 2 to 10 carbon atoms, i.e., 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, more preferably 2 to 8 carbon atoms, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in a preferred embodiment, the alkynyl group comprises from 2 to 10 carbon atoms and 1, 2, 3, 4, or 5 (preferably 1, 2, or 3) carbon-carbon triple bonds, more preferably it comprises 2 to 8 carbon atoms and 1, 2, 3, or 4 (preferably 1 or 2) carbon-carbon triple bonds, such as 2 to 6 carbon atoms and 1, 2 or 3 carbon-carbon triple bonds or 2 to 4 carbon atoms and 1 or 2 carbon-carbon triple bonds. Exemplary alkynyl groups include ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 1-heptynyl, 2-heptynyl, 3-heptynyl, 4-heptynyl, 5-heptynyl, 6-heptynyl, 1-octynyl, 2-octynyl, 3-octynyl, 4-octynyl, 5-octynyl, 6-octynyl, 7-octynyl, 1-nonylyl, 2-nonynyl, 3-nonynyl, 4-nonynyl, 5-nonynyl, 6-nonynyl, 7-nonynyl, 8-nonynyl, 1-decynyl, 2-decynyl, 3-decynyl, 4-decynyl, 5-decynyl, 6-decynyl, 7-decynyl, 8-decynyl, 9-decynyl, and the like. If an alkynyl group is attached to a nitrogen atom, the triple bond cannot be alpha to the nitrogen atom.
The term “heterocyclyl” means a cycloalkyl group as defined above in which from 1, 2, 3, or 4 carbon atoms in the cycloalkyl group are replaced by heteroatoms of O, S, or N. Preferably, in each ring of the heterocyclyl group the maximum number of 0 atoms is 1, the maximum number of S atoms is 1, and the maximum total number of O and S atoms is 2. The term “heterocyclyl” is also meant to encompass partially or completely hydrogenated forms (such as dihydro, tetrahydro or perhydro forms) of the above-mentioned heteroaryl groups. Exemplary heterocyclyl groups include morpholino, isochromanyl, chromanyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl, piperazinyl, indolinyl, isoindolinyl, di- and tetrahydrofuranyl, di- and tetrahydrothienyl, di- and tetrahydrooxazolyl, di- and tetrahydroisoxazolyl, di- and tetrahydrooxadiazolyl (1,2,5- and 1,2,3-), dihydropyrrolyl, dihydroimidazolyl, dihydropyrazolyl, di- and tetrahydrotriazolyl (1,2,3- and 1,2,4-), di- and tetrahydrothiazolyl, di- and tetrahydrothiazolyl, di- and tetrahydrothiadiazolyl (1,2,3- and 1,2,5-), di- and tetrahydropyridyl, di- and tetrahydropyrimidinyl, di- and tetrahydropyrazinyl, di- and tetrahydrotriazinyl (1,2,3-, 1,2,4-, and 1,3,5-), di- and tetrahydrobenzofuranyl (1- and 2-), di- and tetrahydroindolyl, di- and tetrahydroisoindolyl, di- and tetrahydrobenzothienyl (1- and 2), di- and tetrahydro-1H-indazolyl, di- and tetrahydrobenzimidazolyl, di- and tetrahydrobenzoxazolyl, di- and tetrahydroindoxazinyl, di- and tetrahydrobenzisoxazolyl, di- and tetrahydrobenzothiazolyl, di- and tetrahydrobenzisothiazolyl, di- and tetrahydrobenzotriazolyl, di- and tetrahydroquinolinyl, di- and tetrahydroisoquinolinyl, di- and tetrahydrobenzodiazinyl, di- and tetrahydroquinoxalinyl, di- and tetrahydroquinazolinyl, di- and tetrahydrobenzotriazinyl (1,2,3- and 1,2,4-), di- and tetrahydropyridazinyl, di- and tetrahydrophenoxazinyl, di- and tetrahydrothiazolopyridinyl (such as 4,5,6-7-tetrahydro[1,3]thiazolo[5,4-c]pyridinyl or 4,5,6-7-tetrahydro[1,3]thiazolo[4,5-c]pyridinyl, e.g., 4,5,6-7-tetrahydro[1,3]thiazolo[5,4-c]pyridin-2-yl or 4,5,6-7-tetrahydro[1,3]thiazolo[4,5-c]pyridin-2-yl), di- and tetrahydropyrrolothiazolyl (such as 5,6-dihydro-4H-pyrrolo[3,4-d][1,3]thiazolyl), di- and tetrahydrophenothiazinyl, di- and tetrahydroisobenzofuranyl, di- and tetrahydrochromenyl, di- and tetrahydroxanthenyl, di- and tetrahydrophenoxathiinyl, di- and tetrahydropyrrolizinyl, di- and tetrahydroindolizinyl, di- and tetrahydroindazolyl, di- and tetrahydropurinyl, di- and tetrahydroquinolizinyl, di- and tetrahydrophthalazinyl, di- and tetrahydronaphthyridinyl (1,5-, 1,6-, 1,7-, 1,8-, and 2,6-), di- and tetrahydrocinnolinyl, di- and tetrahydropteridinyl, di- and tetrahydrocarbazolyl, di- and tetrahydrophenanthridinyl, di- and tetrahydroacridinyl, di- and tetrahydroperimidinyl, di- and tetrahydrophenanthrolinyl (1,7-, 1,8-, 1,10-, 3,8-, and 4,7-), di- and tetrahydrophenazinyl, di- and tetrahydrooxazolopyridinyl, di- and tetrahydroisoxazolopyridinyl, di- and tetrahydropyrrolooxazolyl, and di- and tetrahydropyrrolopyrrolyl. Exemplary 5- or 6-membered heterocyclyl groups include morpholino, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl, piperazinyl, di- and tetrahydrofuranyl, di- and tetrahydrothienyl, di- and tetrahydrooxazolyl, di- and tetrahydroisoxazolyl, di- and tetrahydrooxadiazolyl (1,2,5- and 1,2,3-), dihydropyrrolyl, dihydroimidazolyl, dihydropyrazolyl, di- and tetrahydrotriazolyl (1,2,3- and 1,2,4-), di- and tetrahydrothiazolyl, di- and tetrahydroisothiazolyl, di- and tetrahydrothiadiazolyl (1,2,3- and 1,2,5-), di- and tetrahydropyridyl, di- and tetrahydropyrimidinyl, di- and tetrahydropyrazinyl, di- and tetrahydrotriazinyl (1,2,3-, 1,2,4-, and 1,3,5-), and di- and tetrahydropyridazinyl.
The term “aryl” refers to a monoradical of an aromatic cyclic hydrocarbon. Preferably, the aryl group contains 3 to 10 (e.g., 5 to 10, such as 5, 6, or 10) carbon atoms which can be arranged in one ring (e.g., phenyl) or two or more condensed rings (e.g., naphthyl). Exemplary aryl groups include cyclopropenylium, cyclopentadienyl, phenyl, indenyl, naphthyl, azulenyl, fluorenyl, anthryl, and phenanthryl. Preferably, “aryl” refers to a monocyclic ring containing 6 carbon atoms or an aromatic bicyclic ring system containing 10 carbon atoms. Preferred examples are phenyl and naphthyl.
The term “heteroaryl” means an aryl group as defined above in which one or more carbon atoms in the aryl group are replaced by heteroatoms of O, S, or N. Preferably, heteroaryl refers to a five or six-membered aromatic monocyclic ring wherein 1, 2, or 3 carbon atoms are replaced by the same or different heteroatoms of O, N, or S. Alternatively, it means an aromatic bicyclic or tricyclic ring system wherein 1, 2, 3, 4, or 5 carbon atoms are replaced with the same or different heteroatoms of O, N, or S. Preferably, in each ring of the heteroaryl group the maximum number of O atoms is 1, the maximum number of S atoms is 1, and the maximum total number of O and S atoms is 2. Exemplary heteroaryl groups include furanyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl (1,2,5- and 1,2,3-), pyrrolyl, imidazolyl, pyrazolyl, triazolyl (1,2,3- and 1,2,4-), tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl (1,2,3- and 1,2,5-), pyridyl, pyrimidinyl, pyrazinyl, triazinyl (1,2,3-, 1,2,4-, and 1,3,5-), benzofuranyl (1- and 2-), indolyl, isoindolyl, benzothienyl (1- and 2-), 1H-indazolyl, benzimidazolyl, benzoxazolyl, indoxazinyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzotriazolyl, quinolinyl, isoquinolinyl, benzodiazinyl, quinoxalinyl, quinazolinyl, benzotriazinyl (1,2,3- and 1,2,4-benzotriazinyl), pyridazinyl, phenoxazinyl, thiazolopyridinyl, pyrrolothiazolyl, phenothiazinyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxathiinyl, pyrrolizinyl, indolizinyl, indazolyl, purinyl, quinolizinyl, phthalazinyl, naphthyridinyl (1,5-, 1,6-, 1,7-, 1,8-, and 2,6-), cinnolinyl, pteridinyl, carbazolyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl (1,7-, 1,8-, 1,10-, 3,8-, and 4,7-), phenazinyl, oxazolopyridinyl, isoxazolopyridinyl, pyrrolooxazolyl, and pyrrolopyrrolyl. Exemplary 5- or 6-memered heteroaryl groups include furanyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl (1,2,5- and 1,2,3-), pyrrolyl, imidazolyl, pyrazolyl, triazolyl (1,2,3- and 1,2,4-), thiazolyl, isothiazolyl, thiadiazolyl (1,2,3- and 1,2,5-), pyridyl, pyrimidinyl, pyrazinyl, triazinyl (1,2,3-, 1,2,4-, and 1,3,5-), and pyridazinyl.
The term “azido” means N3.
In an alkyl group, cycloalkyl group, heterocyclyl, alkenyl group, alkenylene group, aryl group, or heteroaryl group, one or more hydrogen atoms may be replaced by a halogen atom, such as Cl, Br, F, preferably F, —OH, —NH2, —NHC(O)CH3, —CN, —N3, —COOH, and/or —C(O)NH.
The invention comprises a compound according to formula (I)
—(CH2).(C2)alkynyl.
Preferably, for
u is 1 and (C1-C6)alkyl is methyl.
u is 0 to 6.
R2 to R5 are independently selected from the group consisting of H, —OR12, —(C1-C10)alkyl, halogen, cyano, isocyano, cyanato, isocyanato, thiocyanato, isothiocyanato, azido, —(C2-C10)alkenyl, —(C2-C10)alkynyl, —(C3-C10)cycloalkyl, —(C3-C10)heterocyclyl, —(C3-C10)aryl, —(C3-C10)heteroaryl, —CHZ2, —CZ3—CH2Z, —OCHZ2, —OCZ3, —OCH2Z—N(R13)(R14), —N(R16)(OR16), —S(O)0-2R′7, —S(O)1-2OR18, —OS(O)1-2R19, —OS(O)1-2OR20, —S(O)1-2N(R21)(R22), —OS(O)1-2N(R23)(R24), —N(R25)S(O)1-2R26, —NR27S(O)1-2OR28, —NR29S(O)1-2N(R30)(R31), —C(═X)R32, —C(═X)XR33, —XC(═X)R34, and —XC(═X)XR35, —OR36, —O(CH2)v(C3-C10)aryl, —O(CH2)v(C3-C10)cycloalkyl, —O(CH2)v(C2)alkynyl.
R6 is selected from the group consisting of H, —(C1-C10)alkyl, and —(CH2)1-5(C3-C10)cycloalkyl; wherein —(C1-C10)alkyl, benzyl and —(CH2)1-5(C3-C10)cycloalkyl optionally are further substituted with at least one substituent selected from the group consisting of Halogen, preferably F.
R7 to R11 are independently selected from the group consisting of H, —OR12, —SR12, —(C1-C10)alkyl, halogen, —(C1-C10)alkylO(C1-C10)alkyl, cyano, isocyano, cyanato, isocyanato, thiocyanato, isothiocyanato, azido, —(C2-C10)alkenyl, —(C2-C10)alkynyl, —O(C2-C10)alkynyl, —(C3-C10)cycloalkyl, —(C3-C10)heterocyclyl, —(C3-C10)aryl, —(C3-C10)heteroaryl, —(CH2)CHZ2, —CZ3—CH2Z, —OCHZ2, —OCZ3, —OCH2Z, —N(R13)(R14), —N(R15)(OR16), —S(O)0-2R17, —S(O)1-2OR18, —OS(O)1-2R19, —OS(O)1-2OR20, —S(O)1-2N(R21)(R22), —OS(O)1-2N(R23)(R24), —N(R25)S(O)1-2R26, —NR27S(O)1-2OR28, —NR29S(O)1-2N(R30)(R31), —C(═X)R32, —C(═X)XR33, —XC(═X)R34, and —XC(═X)XR35, —O(CH2)v(C3-C10)cycloakyl, —O(CH2)v(C1-C10)alkyl and —O(CH2)v(C3-C10)aryl.
Two adjacent rests of R1 to R5 and R7 to R11, preferably R8 to R10 optionally may form a ring attached to the underlying aromatic ring of formula (I) according to formula (III) to (XI)
J1 to J4 are independently selected from C or N, preferably J1 to J4 are C;
R12 to R36 are independently selected from the group consisting of H, —(C1-C10)alkyl, —(C2-C10)alkenyl, —(C2-C10)alkynyl, —(C3-C10)cycloalkyl, —(C3-C10)heterocyclyl, —(C3-C10)aryl, —(C3-C10)heteroaryl.
R1 to R11, independently selected from the group consisting of, —(C1-C10)alkyl, —(C2-C10)alkenyl, —(C2-C10)alkynyl, —(C3-C10)cycloalkyl, —(C3-C10)heterocyclyl, —(C3-C10)aryl, —O(CH2)v(C3-C10)cycloalkyl, —O(CH2)v(C1-C10)alkyl and —O(CH2)(C3-C10)aryl and R12 to R35 optionally are further substituted with at least one substituent selected from the group consisting of OR12, —(C1-C10)alkyl, halogen, cyano, isocyano, cyanato, isocyanato, thiocyanato, isothiocyanato, azido, —(C2-C10)alkenyl, —(C2-C10)alkynyl, —(C3-C10)cycloalkyl, —(C3-C10)heterocyclyl, —(C3-C10)aryl, —(C3-C10)heteroaryl, —CHZ2, —CZ3—CH2Z, —OCHZ2, —OCZ3, —OCH2Z, —N(R13)(R14), —N(R15)(OR16), —NHC(O)(C1-C10)alkyl, —S(O)0-2R17, —S(O)1-2OR18, —OS(O)1-2R19, —OS(O)1-2OR20, —S(O)1-2N(R21)(R22), —OS(O)1-2N(R23)(R24), —N(R25)S(O)12R26, —NR27S(O)1-2OR28, —NR29S(O)2N(R30)(R31), —C(═X)R32, —C(═X)XR33, —XC(═X)R34, and —XC(═X)XR35, —OR36, and —O(CH2)v(C3-C10)aryl.
v is 0 to 5;
Z is halogen;
X is selected from the group consisting of O, —NH— or S;
A is selected from the group consisting of
preferably H or benzyl, most preferably H;
With the proviso that Rs is not —COOH.
In one embodiment,
In another embodiment, R2 to R5 are independently selected from the group consisting of H, —OR12, —(C1-C10)alkyl, halogen, cyano, isocyano, cyanato, isocyanato, thiocyanato, isothiocyanato, azido, —(C2-C10)alkenyl, —(C2-C10)alkynyl, —(C3-C10)cycloalkyl, —(C3-C10)heterocyclyl, —(C3-C10)aryl, —(C3-C10)heteroaryl, —CHZ2, —CZ3—CH2Z, —OCHZ2, —OCZ3, —OCH2Z, —N(R13)(R14), —N(R15)(OR16), —C(═X)R32, —C(═X)XR33, —XC(═X)R34, and —XC(═X)XR35, —O(CH2)v(C3-C10)aryl, —O(CH2)v(C3-C10)cycloalkyl, —O(CH2)v(C2)alkynyl.
In another embodiment, R7 to R11 are independently selected from the group consisting of H, —SR12, —OR12, —(C1-C10)alkyl, halogen, cyano, isocyano, cyanato, isocyanato, thiocyanato, isothiocyanato, azido, —(C2-C10)alkenyl, —(C2-C10)alkynyl, —O(C2-C10)alkynyl, —(C3-C10)cycloalkyl, —(C3-C10)heterocyclyl, —(C3-C10)aryl, —(C3-C10)heteroaryl, —(CH2)vCHZ2, —CZ3—CH2Z, —OCHZ2, —OCZ3, —OCH2Z, —N(R13)(R14), —N(R15)(OR16), —C(═X)R32, —C(═X)XR33, —XC(═X)R34, and —XC(═X)XR35, —O(CH2)v(C3-C10)cycloakyl, —O(CH2)v(C1-C10)alkyl and —O(CH2)v(C3-C10)aryl,
In a further embodiment,
If an R12 is substituted with a further substituent such as —OR12 then the specific R12 selected from the group as specified above may be different in the underlying R12 and in its substituent —OR12.
In a further embodiment, R9 is selected from the group consisting of —OR12, halogen, —O(C2-C10)alkynyl, —CZ3, —OCHZ2, —OCZ3, —OCH2Z, —O(CH2)v(C3-C10)cycloakyl, —O(CH2)(C1-C10)alkyl and —O(CH2)v(C3-C10)aryl, wherein R9 selected from the group consisting of —O(C1-C10)alkyl, —OCH2Z, —O(CH2)(C3-C10)cycloakyl, —O(CH2)v(C1-C10)alkyl and —O(CH2)u(C3-C10)aryl optionally is further substituted with at least one substituent selected from the group consisting of Halogen, —OH, —NH2, —NHC(O)CH3, —CN, —N3, and —COOH, —C(O)NH2.
Further, the invention comprises a compound according to formula (II)
R1 to R4 and R6, R7, R9 and R10 are independently selected from the group consisting of H, —(C1-C10)alkyl, —SR12, halogen, azido, cyano, —O(C1-C10)alkyl, —(CH2)u(C3-C10)aryl, —O(CH2)u(C3-C10)cycloalkyl, —(CH2)u(C3-C10)cycloalkyl, —(C2-C10)alkenyl,
R5 is —(C1-C10)alkyl, —(C5—C)heteroaryl, —(C3-C10)aryl, —(CH2)u(C3-C10)cycloalkyl, preferably —(C4—C)alkyl, or benzyl, most preferably —(C4)alkyl.
R8 is H, —O(C1-C10)alkyl, —O(CH2)u(C3-C14)aryl, —O(CH2)u(C3-C10)cycloalkyl, —O(C3-C10)cycloalkyl, or, —O(C2-C10)alkenyl, preferably —OCH3.
u is 0 to 6.
R is H, (C1-C6)alkyl, cyano, —(C3-C10)cycloalkyl, benzyl or part of a ring wherein R is connected with R7 or R11 by
preferably H or benzyl, most preferably H.
R11 is H, —(C1-C10)alkyl, and —(CH2)1-5(C3-C10)cycloalkyl.
R1 to R11, independently selected from the group consisting of, —(C1-C10)alkyl, —O(C1-C10)alkyl, —(C2-C10)alkenyl, —(C2-C10)alkynyl, —(C3-C10)cycloalkyl, —(CH2)u(C3-C14)cycloalkyl, —(C3-C10)aryl, and —(CH2)u(C3-C10)aryl and R12 to R35 optionally are further substituted with at least one substituent selected from the group consisting of Halogen, —OH, —NH2, —NHC(O)CH3, —CN, —N3, and —COOH, —C(O)NH2.
R12 is —(C1-C10)alkyl, preferably —(C3-C5)alkyl, more preferably —(C4)alkyl.
Two adjacent rests of R8 to R10 optionally may form a ring attached to the underlying aromatic ring of formula (II) according to
Het is selected from O.
G is selected from CH, NH.
R38 is independently selected from the group consisting of H, —(C1-C10)alkyl.
J1 to J4 are independently selected from C or N, preferably J1 to J4 are C.
Wherein if any one of J1 to J4 is N, the corresponding R1 to R4 attached to the respective J1 to J4 which is (are) N is absent.
In one embodiment one of J1 to J4 is N. In another embodiment two of J1 to J4 are N.
With the proviso that
In a further embodiment, if J, to J4 are C the following compounds are not or are also not comprised by the compounds according to formula (II):
In respect to the compounds according to formula (I) or (II), the invention further comprises the following embodiments i) to xxiv):
preferably according to formula (III)
In respect to the compounds according to formula (II), the invention further comprises the following embodiment(s):
and/or
A selection of compounds within the scope of the present invention is listed in the following Table:
The invention is further directed to the compound according to formula (I), (II) or according to table 1 for use in medicine.
The invention is further directed to the compound according to formula (I), (II) or according to table 1 for use in the treatment of fibrosis and neoplasia, preferably a fibrosis or neoplasia located in the heart, the lung, the renal tract, the liver, in the skin, in the pleura and retroperitoneum, more preferably the fibrosis is selected from pleural fibrosis, retroperitoneal fibrosis, atrial fibrillation, myocardial interstitial fibrosis, idiopathic pulmonary fibrosis (IPF), interstitial lung diseases, chronic kidney disease, non-alcoholic fat liver disease, skin scars, keloids, tumour-associated desmoplastic reaction.
The invention is further directed to the compound according to formula (I), (II) or according to table 1 use in the treatment of inflammatory diseases, such as arthrolithiasis, familiar mediterranean fever and pericarditis.
Compounds of the invention which contain a basic functionality may form salts with a variety of inorganic or organic acids. Exemplary inorganic and organic acids/bases as well as exemplary acid/base addition salts of the compounds of the present invention are given in the definition of “pharmaceutically acceptable salt” in the section “Pharmaceutical composition”, below. The compounds of the invention which contain an acidic functionality may form salts with a variety of inorganic or organic bases. The compounds of the invention which contain both basic and acidic functionalities may be converted into either base or acid addition salt. The neutral forms of the compounds of the invention may be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner.
Many compounds of the present invention are commercially available. Compounds which are not commercially available are in general obtainable as follows and specific examples for the preparation of compounds of the present invention are described in the example part.
As shown in Scheme 1, the compounds of the present invention may be synthesized by an amide formation between a respective aromatic acid and an aniline.
Several reagents and methods for amide formation from acids and amins are known.
Examplary Methods and/or Reagents are:
Moreover, the invention is directed to a pharmaceutical composition comprising the compound as described above and at least one carrier.
“Pharmaceutical composition” refers to one or more active ingredients, and one or more inert ingredients that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the pharmaceutical compositions of the present invention encompass any composition made by admixing a compound of the present invention and a pharmaceutically acceptable carrier.
“Carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, including but not limited to peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered orally. Saline and aqueous dextrose are preferred carriers when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions are preferably employed as liquid carriers for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositions will contain a therapeutically effective amount of the therapeutic, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.
A composition of the present invention can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for the preparation of such formulations are generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
To administer a compound of the invention by certain routes of administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation. For example, the compound may be administered to an individual in an appropriate carrier, for example, liposomes, or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes (Strejan et al., J. Neuroimmunol. 7: 27 (1984)).
Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.
Pharmaceutical compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration.
Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the individuals to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
Examples of pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulphate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
For the therapeutic/pharmaceutical formulations, compositions of the present invention include those suitable for enteral administration (such as oral or rectal) or parenteral administration (such as nasal, topical (including vaginal, buccal and sublingual)). The compositions may conveniently be presented in unit dosage form and may be prepared by any methods known in the art of pharmacy. The amount of active ingredient (in particular, the amount of a compound of the present invention) which can be combined with a carrier material to produce a pharmaceutical composition (such as a single dosage form) will vary depending upon the individual being treated, and the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect.
Generally, out of 100% (for the pharmaceutical formulations/compositions), the amount of active ingredient (in particular, the amount of the compound of the present invention, optionally together with other therapeutically active agents, if present in the pharmaceutical formulations/compositions) will range from about 0.01% to about 99%, preferably from about 0.1% to about 70%, most preferably from about 1% to about 30%, wherein the reminder is preferably composed of the one or more pharmaceutically acceptable excipients.
The amount of active ingredient, e.g., a compound of the invention, in a unit dosage form and/or when administered to an individual or used in therapy, may range from about 0.1 mg to about 1000 mg (for example, from about 1 mg to about 500 mg, such as from about 10 mg to about 200 mg) per unit, administration or therapy. In certain embodiments, a suitable amount of such active ingredient may be calculated using the mass or body surface area of the individual, including amounts of between about 1 mg/Kg and 10 mg/Kg (such as between about 2 mg/Kg and 5 mg/Kg), or between about 1 mg/m2 and about 400 mg/m2 (such as between about 3 mg/M2 and about 350 mg/m2 or between about 10 mg/m2 and about 200 mg/m2).
Compositions of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate. Dosage forms for the topical or transdermal administration of compositions of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
The expressions “enteral administration” and “administered enterally” as used herein mean that the drug administered is taken up by the stomach and/or the intestine.
Examples of enteral administration include oral and rectal administration. The expressions “parenteral administration” and “administered parenterally” as used herein mean modes of administration other than enteral administration, usually by injection or topical application, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraosseous, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, intracerebral, intracerebroventricular, subarachnoid, intraspinal, epidural and intrasternal administration (such as by injection and/or infusion) as well as topical administration (e.g., epicutaneous, inhalational, or through mucous membranes (such as buccal, sublingual or vaginal)).
Examples of suitable aqueous and non-aqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, pH buffering agents, and dispersing agents. Prevention of the presence of microorganisms may be ensured both by sterilization procedures, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art (cf., e.g., Remington, “The Science and Practice of Pharmacy” edited by Allen, Loyd V., Jr., 22nd edition, Pharmaceutical Sciences, September 2012; Ansel et al., “Pharmaceutical Dosage Forms and Drug Delivery Systems”, 7th edition, Lippincott Williams & Wilkins Publishers, 1999.).
Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start with doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
In general, a suitable daily dose of a composition of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. It is preferred that administration be oral, intravenous, intramuscular, intraperitoneal, or subcutaneous, preferably administered proximal to the site of the target. If desired, the effective daily dose of a pharmaceutical composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation/composition.
In one embodiment, the compounds or compositions of the invention may be administered by infusion, preferably slow continuous infusion over a long period, such as more than 24 hours, in order to reduce toxic side effects. The administration may also be performed by continuous infusion over a period of from 2 to 24 hours, such as of from 2 to 12 hours. Such regimen may be repeated one or more times as necessary, for example, after 6 months or 12 months.
In yet another embodiment, the compounds or compositions of the invention are administered by maintenance therapy, such as, e.g., once a week for a period of 6 months or more.
For oral administration, the pharmaceutical composition of the invention can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutical acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone, hydroxypropyl methylcellulose), fillers (e.g., lactose, microcrystalline cellulose, calcium hydrogen phosphate), lubricants (e.g., magnesium stearate, talc, silica), disintegrants (e.g., potato starch, sodium starch glycolate), or wetting agents (e.g., sodium lauryl sulphate). Liquid preparations for oral administration can be in the form of, for example, solutions, syrups, or suspensions, or can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparation can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol, syrup, cellulose derivatives, hydrogenated edible fats), emulsifying agents (e.g., lecithin, acacia), non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, fractionated vegetable oils), preservatives (e.g., methyl or propyl-p-hydroxycarbonates, sorbic acids). The preparations can also contain buffer salts, flavouring, coloring and sweetening agents as deemed appropriate. Preparations for oral administration can be suitably formulated to give controlled release of the pharmaceutical composition of the invention.
The pharmaceutical composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
For administration by inhalation, the pharmaceutical composition of the invention is conveniently delivered in the form of an aerosol spray presentation from a pressurised pack or a nebulizer, with the use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, nitrogen, or other suitable gas). In the case of a pressurised aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatine, for use in an inhaler or insufflator can be formulated containing a powder mix of the pharmaceutical composition of the invention and a suitable powder base such as lactose or starch.
The pharmaceutical composition of the invention can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. Formulations for injection can be presented in units dosage form (e.g., in phial, in multi-dose container), and with an added preservative. The pharmaceutical composition of the invention can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing, or dispersing agents. Alternatively, the agent can be in powder form for constitution with a suitable vehicle (e.g., sterile pyrogen-free water) before use. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition can also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilised powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.
Therapeutic/pharmaceutical compositions can be administered with medical devices known in the art. For example, in a preferred embodiment, a therapeutic/pharmaceutical composition of the invention can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or U.S. Pat. No. 4,596,556. Examples of well-known implants and modules useful in the present invention include those described in: U.S. Pat. No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Pat. No. 4,486,194, which discloses a therapeutic device for administering medicants through the skin; U.S. Pat. No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Pat. No. 4,475,196, which discloses an osmotic drug delivery system.
Many other such implants, delivery systems, and modules are known to those skilled in the art. In certain embodiments, the compounds of the invention can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the compounds of the invention cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, and thus enhance targeted drug delivery (see, e.g., V. V. Ranade (1989) J. Clin. Pharmacol. 29: 685). Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153: 1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357: 140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39: 180); and surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233: 134).
In one embodiment of the invention, the compounds of the invention are formulated in liposomes. In a more preferred embodiment, the liposomes include a targeting moiety. In a most preferred embodiment, the compounds in the liposomes are delivered by bolus injection to a site proximal to the desired area. Such liposome-based composition should be fluid to the extent that easy syringability exists, should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi.
A “therapeutically effective dosage” for therapy/treatment can be measured by objective responses which can either be complete or partial. A complete response (CR) is defined as no clinical, radiological or other evidence of a condition, disorder or disease. A partial response (PR) results from a reduction in disease of greater than 50%. Median time to progression is a measure that characterizes the durability of the objective response.
A “therapeutically effective dosage” for therapy/treatment can also be measured by its ability to stabilize the progression of a condition, disorder or disease. The ability of a compound to inhibit, reduce or ameliorate non-apoptotic regulated cell-death and/or to reduce oxidative stress can be evaluated in appropriate animal model systems as such as one or more of those set fourth below. Alternatively, these properties of a compound of the present invention can be evaluated by examining the ability of the compound using in vitro assays known to the skilled practitioner such as one or more of those set fourth below. A therapeutically effective amount of a compound of the present invention can cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the condition, disorder or disease or the symptoms of the condition, disorder or disease or the predisposition toward the condition, disorder or disease in an individual. One of ordinary skill in the art would be able to determine such amounts based on such factors as the individual's size, the severity of the individual's symptoms, and the particular composition or route of administration selected.
An injectable composition should be sterile and fluid to the extent that the composition is deliverable by syringe. In addition to water, the carrier can be an isotonic buffered saline solution, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
The pharmaceutical composition of the invention can also, if desired, be presented in a pack, or dispenser device which can contain one or more unit dosage forms containing the said agent. The pack can for example comprise metal or plastic foil, such as blister pack. The pack or dispenser device can be accompanied with instruction for administration.
The pharmaceutical composition of the invention can be administered as sole active agent or can be administered in combination with other therapeutically and/or cosmetically active agents.
Further, the invention is directed to a screening assay, comprising the steps: a) culturing adherent cells which deposit at least one protein in the presence of at least one test compound; b) staining of at least one protein deposited by the adherent cells; c) fixation of adherent cells and the at least one protein; d) microscopic detection of a signal of the at least one stained deposited protein; e) data analysis of signals detected in step d) comprising quantification of the amount of the at least one protein deposited in the presence of the at least one test compound. wherein step b) is carried out before step c).
In step a), cells are cultured in an adherent cell culture. The cells may be cultured in an adherent cell culture applying any conditions known to the person skilled in the art suitable to culture the respective cell type. Optionally, the cells are starved before application in step a), preferably for 5 to 48 h, more preferably 10 to 30 h, most preferably for 20 to 26 h.
Preferably, the adherent cells are primary cells. In one embodiment, the adherent cells are primary patient derived human cells most preferably human lung fibroblasts. In another embodiment the cells are primary animal derived cells or any adherent immortalized cells.
Preferably, the at least one protein in step a) is an extracellular matrix protein.
More preferably, the at least one extracellular matrix protein is selected from the group consisting of collagen type 5, collagen type 1, and fibulin 1. In a further embodiment more than one protein is deposited. For example at least two, at least three or at least four proteins are deposited.
Preferably, step a) is carried out for at least 24 h, more preferably 60 to 90 hours, particular preferred 65 to 80 hours, most preferably for 70 to 75 hours.
The at least one test compound is preferably a compound which is expected to inhibit deposition of the at least one protein. It is the purpose of the assay to identify potential compounds which inhibit the deposition of the at least one protein and to quantify the extend of the inhibition by the respected test compound. Optionally, the at least one test compound in step a) is a small molecule; and/or oligonucleotides, peptides, proteins, protacs, anticalins, antibodies, or CRISPRs.
Optionally, in step a) at least one growth factor, preferably at least one Transforming Growth Factor β (TGF β), more preferably, TGF β1 is present.
In one embodiment, staining in step b), comprises the binding of at least one antibody to the at least one protein or at least one probe binding to the at least one protein.
Optionally, the antibody or probe comprises at least one detectable label that is directly conjugated to the antibody and wherein optionally the detectable label has fluorescence property, preferably the detectable lable is a fluorophore selected from AlexaFluor 488, AlexaFluor 555, AlexaFluor 637; AlexaFluor 647, AlexaFluor 568, AlexaFluor 568 and/or Qdots.
In a further embodiment, staining in step b) comprises the binding of at least one first antibody (FA) to at least one protein and the subsequent binding of at least one first antibody (FA) with at least one secondary antibody (SA), wherein the at least one second antibody (SA) comprises at least one detectable label that is conjugated to the antibody and wherein optionally the detectable label has fluorescence property, preferably the detectable label is a fluorophore selected from AlexaFluor 488, AlexaFluor 555, AlexaFluor 637; AlexaFluor 647, AlexaFluor 568, AlexaFluor 568, and/or Qdot.
Optionally, in step b) at least one further co-staining is present and selected from the group consisting of cell-nuclei staining, live-dead staining, myofibroblast markers (e.g. αSMA-staining), apoptosis markers (e.g. Caspase3/7 staining).
In step c) fixation may be carried out with any reagents which are suitable for the purpose and known to the person skilled in the art. Respective conditions are known in the art.
Examplary conditions used in certain embodiments of the inventions are 4% PFA for 30 min at 37° C. or 100% methanol for 2 min at −20° C. Staining in step b) is carried out before fixation in step b).
Preferably, in step d) 2D, 3D or 4D imaging is carried out. More preferably, step d) is carried out with a conventional or confocal imaging apparatus.
Data analysis in step e) may comprise using a machine learning model, such as neural networks.
(E)-3-(4-hydroxyphenyl)acrylic acid (120 mg, 0.73 mmol) was dissolved in 2 mL of dry DMF. The solution was cooled in an ice bath and 2-butoxyaniline (0.87 mmol, 1.2 eq) was added followed by a solution of benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (1.1 mmol, 1.5 eq) in 2 mL of dichloromethane. Subsequently, triethylamine (1.46 mmol, 2 eq) was added. The mixture was stirred at 0° C. for 30 min and then at room temperature overnight. The mixture was diluted by dichloromethane and washed with 1M aqueous hydrochloric acid, saturated sodium carbonate and brine. The organic layer was dried over magnesium sulphate and concentrated in vacuo. Purification by silica gel chromatography yielded (E)-N-(2-butoxyphenyl)-3-(4-hydroxyphenyl)acrylamide (87 mg).
Diisopropyl azodicarboxylate (2 eq) was added over a period of 10 min at 0° C. to a solution of (E)-N-(2-butoxyphenyl)-3-(4-hydroxyphenyl)acrylamide (1 eq.), cyclopropylmethanol (2 eq.), and triphenyl phosphine (2 eq.) in absolute tetrahydrofurane (15 mL). After 30 minutes the cooling bath was removed, and the solution was stirred at room temperature for 16 h. The solvent was concentrated in vacuo, and the remaining residue was purified by flash chromatography to yield (E)-N-(2-butoxyphenyl)-3-(4-(cyclopropylmethoxy)phenyl)acrylamide in 77% yield.
1H-NMR (CDCl3, 400 MHz) 0.36 (2H, q, J=5.11 Hz), 0.64-0.69 (2H, m), 1.03 (3H, t, J=7.40 Hz), 1.25-1.31 (1H, m), 1.54 (2H, sextet, J=7.47 Hz), 1.86 (2H, quintet, J=7.06 Hz), 3.83 (2H, d, J=6.92 Hz), 4.07 (3H, t, J=6.60 Hz), 6.42 (2H, d, J=15.45 Hz), 6.87-6.91 (3H, m), 6.98 (1H, dt, J=1.40, 7.66 Hz), 7.03 (1H, dt, J=1.73, 7.69 Hz), 7.50 (2H, d, J=8.72 Hz), 7.69 (1H, d, J=15.45 Hz), 7.92 (1H, s), 8.51 (1H, d, J=6.52 Hz). 13C-NMR (CDCl3, 101 MHz) 3.36, 10.30, 14.04, 19.48, 31.37, 68.55, 73.00, 111.01, 114.99, 118.91, 120.01, 121.17, 123.70, 127.47, 128.26, 129.68, 141.73, 147.38, 160.66, 164.16. HRMS (ESI): m/z [M+Na]+ calcd for C23H27NO3: 388.1889, Found: 388.1894.
Concentrated sulfuric acid (0.67 mL) was added to a solution of (E)-3-(4-hydroxyphenyl)acrylic acid (0.8 g) in methanol (40 mL). The solution was heated to reflux for 5 h, cooled to room temperature and then quenched by addition of saturated aqueous sodium bicarbonate solution. The aqueous phase was extracted with ethyl acetate and the combined organic fractions were washed with water, brine, dried over magnesium sulphate and concentrated in vacuo providing methyl (E)-3-(4-hydroxyphenyl)acrylate (800 mg), which was sufficiently pure for further conversion. 1H-NMR (CDCl3 400 MHz) 3.81 (3H, s), 6.02 (1H, s), 6.30 (1H, d, J=15.93 Hz), 6.86 (2H, d, J=8.60 Hz), 7.42 (2H, d, J=8.56 Hz), 7.64 (1H, d, J=15.97 Hz). HLM-01-046, yielding 90%
A dry flask was charged with NaH (1.2 equiv) under argon. Dry dimethylsulfoxide (4 mL) was added to the reaction flask and stirred at 0° C. for 15 min. A solution of Methyl (E)-3-(4-(2,2,2-trifluoroethoxy)phenyl)acrylate (890 mg, 5 mmol, 1 equiv) in DMSO (2 mL) was slowly added to the suspension over 10 min. The reaction mixture was allowed to stir at 0° C. for 30 min. 2,2,2-trifluoroethyl iodide (1.5 mL, 15 mmol, 3 equiv) was added to the reaction flask. The reaction was then stirred at 80° C. for 24 h. After completion of the reaction, it was quenched by addition of water and extracted with ethyl acetate. The organic phase was evaporated to dryness and purified by silica gel chromatography.
To a solution of methyl (E)-3-(4-(2,2,2-trifluoroethoxy)phenyl)acrylate (1 equiv.) in methanol (4 mL) was added potassium carbonate (5 equiv.) dissolved in water (4 mL). The reaction mixture was refluxed for 3 h after which methanol was removed under reduced pressure. The solution was then cooled to 0° C. and acidified to pH 2 by addition of hydrochloric acid (1M). The mixture was extracted with diethyl ether and the combined organic layer was washed with brine, dried with sodium sulphate, and evaporated in vacuo to give the product as white solid in 90% yield.
(E)-N-(2-butoxyphenyl)-3-(4-(2,2,2-trifluoroethoxy)phenyl)acrylamide was prepared from (E)-3-(4-(2,2,2-trifluoroethoxy)phenyl)acrylic acid and 2-butoxyaniline in a similar fashion as described for (E)-N-(2-butoxyphenyl)-3-(4-hydroxyphenyl)acrylamide (Example 1, step a). The product was obtained in 63% yield.
1H-NMR (CDCl3 400 MHz) 1.03 (3H, t, J=7.40 Hz), 1.54 (2H, sextet, J=7.47 Hz), 1.86 (2H, quintet, J=7.06 Hz), 4.07 (2H, t, J=6.60 Hz), 4.38 (2H, q, J=8.04 Hz), 6.46 (1H, d, J=15.49 Hz), 6.89 (2H, dd, J=1.04, 7.96 Hz), 6.94-7.00 (3H, m), 7.04 (1H, dt, J=1.57, 7.70 Hz), 7.53 (2H, d, J=8.68 Hz), 7.69 (1H, d, J=15.49 Hz), 7.95 (1H, s), 8.52 (1H, d, J=6.48 Hz). 13C-NMR (CDCl3) 14.03, 19.47, 31.36, 65.82 (1C, q, J=35.83 Hz), 68.57, 111.04, 115.28, 120.05, 120.23, 121.17, 123.31 (1C, q, J=277.95 Hz), 123.87, 128.13, 129.40, 129.77, 141.08, 147.41, 158.62, 163.80. HRMS (ESI): m/z [M+Na]+ calcd for C21H22F3NO3: 416.1449, Found: 416.1445.
A suspension of (E)-3-(4-methoxyphenyl)acrylic acid (150 mg, 0.84 mmol, 1 eq.) in dry dichloromethane (3 mL) was treated with oxalyl chloride (1.68 mmol, 2 eq.) and a catalytic amount of dimethyl formamide at 0° C. under argon. After 5 min, the solution was allowed to warm and stirred at ambient temperature for one hour. Subsequently the solvent was removed under reduced pressure to give the acid chloride as a yellow solid. A solution of the acid chloride in dry dichloromethane (4 mL) was added to a solution of 2-phenoxyaniline (1.01 mmol, 1.2 eq) and triethyl amine (1.01 mmol 1.2 eq) in dichloromethane (3 mL) at 0° C. The suspension was stirred at room temperature for 16 h and was quenched by addition of water. The mixture was diluted with dichloromethane and washed with aqueous ammonium chloride, saturated sodium bicarbonate, then dried over magnesium sulphate and concentrated in vacuo. The crude product was purified by silica gel chromatography to give 139 mg of (E)-3-(4-methoxyphenyl)-N-(2-phenoxyphenyl)acrylamide in 48% yield (139 mg).
1H-NMR (CDCl3 400 MHz) 3.82 (1H, s), 6.42 (1H, d, J=15.45 Hz), 6.86 (1H, dd, J=1.30, 8.14 Hz), 6.89 (2H, d, J=8.76 Hz), 7.01 (1H, dt, J=1.53, 7.79 Hz), 7.06 (2H, dd, J=0.96, 8.60 Hz), 7.13-7.18 (2H, m), 7.35-7.39 (2H, m), 7.47 (2H, d, J=8.76 Hz), 7.69 (1H, d, J=15.49 Hz), 7.93 (1H, s), 8.62 (1H, d, J=7.92 Hz). 13C-NMR (CDCl3): 55.45, 114.38, 117.70, 118.65, 118.88, 121.04, 123.97, 124.08, 124.18, 127.43, 129.72, 130.11, 130.24, 142.11, 145.78, 156.55, 161.23, 164.36. HRMS (ESI): m/z [M+Na]+ calcd for C22H19NO3: 368.1263, Found: 368.1264.
To a solution of 3-nitropyridin-4-ol (1.0 g, 7.13 mmol) and triphenyl phosphine (2.8 g, 10.71 mmol, 1.5 eq.) in anhydrous tetrahydrofurane (10 mL) was added diisopropyl azodicarboxylate (2.16 g, 10.71 mmol, 1.5 eq) in tetrahydrofurane (2.5 mL) and 1-butanol (794 mg, 10.71 mmol, 1.5 eq.) in tetrahydrofurane (2.5 mL) at 0° C. simultaneously. The temperature of the reaction mixture was raised slowly to room temperature and stirring continued at room temperature overnight. At the end of this period diluted with ethyl acetate (25 mL) and washed with water (2×50 mL), dried by Na2SO4, filtered and the solvent was evaporated. The residue was purified by silica column (dichloromethane/methanol=50/1) to afford 4-butoxy-3-nitropyridine as light yellow solid (400 mg).
1H-NMR (CDCl3 400 MHz) 0.99 (3H, t, J=7.36 Hz), 1.40 (2H, q, J=7.55 Hz), 1.84 (2H, m, J=7.47 Hz), 3.94 (2H, t, J=7.32 Hz), 6.68 (1H, d, J=7.68 Hz), 7.32 (1H, q, J=3.37 Hz), 8.53 (1H, d, J=2.36 Hz).
A suspension of 4-butoxy-3-nitropyridine (386 mg, 2.0 mmol) in ethanol (9.2 ml) and acetic acid (0.51 ml) was heated at 60° C. After adding of iron powder (678 mg, 12.0 mmol, 6.0 eq) and iron (III) chloride hexahydrate (55 mg), the mixture was stirred under reflux for 18 hours. After cooling to room temperature, the mixture was diluted with ethyl acetate (100 ml) and filtered through Celite. The filtrate and washings were combined and washed by water. The organic phase was dried by MgSO4 and then concentrated in vacuum to afford 4-butoxypyridin-3-amine as a light yellow solid (310 mg). The crude product can be used in next step without further purification.
A suspension of (E)-3-(4-methoxyphenyl)but-2-enoic acid (1.4 mmol, 250 mg) in dry dichloromethane (5 mL) was treated with oxalyl chloride (1.5 eq.) and a catalytic amount of dimethyl formamide (2 drops) at 0° C. under argon atmosphere. After 5 min, the solution was allowed to warm at room temperature and allowed to stir at ambient temperature for 1 h. The solvent was removed under reduced pressure to give the acid chloride as a yellow solid. A solution of the acid chloride in extra dried dichloromethane (7 mL) was added to a solution of 4-butoxypyridin-3-amine (300 mg, 1.2 eq.) and triethylamine (1.2 eq.) in dichloromethane (5 mL) at 0° C. The suspension was stirring at ambient temperature for 4 h and was quenched by water. The mixture was diluted by dichloromethane and washed by aqueous ammonium chloride, saturated sodium bicarbonate, and then dried by MgSO4. After concentrated in vacuum, the residue was purified by a silica gel column (dichloromethane/methanol 15:1) to afford (E)-N-(4-butoxypyridin-3-yl)-3-(4-methoxyphenyl)acrylamide as a white solid (530 mg).
1H-NMR (DMSO-d6, 400 MHz) 0.90 (3H, t, J=7.36 Hz), 1.27 (2H, sextet, J=7.48 Hz), 1.69 (2H, quintet, J=7.30 Hz), 3.79 (3H, s), 3.95 (2H, t, J=7.06 Hz), 6.25 (1H, d, J=7.28 Hz), 6.99 (2H, d, J=8.76 Hz), 7.20 (1H, d, J=15.65 Hz), 7.46 (1H, d, J=15.61 Hz), 7.59 (2H, d, J=8.76 Hz), 7.71 (1H, dd, J=2.26, 7.30 Hz), 8.89 (1H, d, J=2.24 Hz), 9.27 (1H, s). 13C-NMR (DMSO-d6, 100 MHz) 13.41, 18.86, 32.44, 55.28, 56.25, 112.89, 114.37, 119.69, 127.45, 127.66, 129.44, 129.51, 138.60, 140.00, 160.56, 164.27, 169.02.
HRMS (ESI): Found 349.1527. Calc. 349.1528, [M+Na]+, M=C19H22N2O3
To a solution of 2-nitrophenol in acetonitrile, anhydrous potassium carbonate was added. The mixture was stirred for 30 minutes at room temperature. Catalytic amount of potassium iodide and 1-(bromomethyl)-4-fluorobenzene were added. The reaction was stirred at 75° C. for 8 h. Water (125 mL) was added to the reaction mixture and the reaction mixture was extracted with ethyl acetate. The combined organic layer was washed with brine and dried over anhydrous sodium sulphate. The solvent was evaporated under reduced pressure and the residue was purified by flash chromatography.
1H-NMR (CDCl3): 5.19 (2H, s), 7.08 (4H, m, J=4.53 Hz), 7.44 (2H, q, J=4.65 Hz), 7.51 (1H, m, J=4.36 Hz), 7.86 (1H, q, J=3.21 Hz).
To a solution of 1-((4-fluorobenzyl)oxy)-2-nitrobenzene (1 eq.) in methanol (7.5 mL) and water (7.5 mL) was added ammonium chloride (10 eq.) and iron powder (5 eq.). The reaction mixture was allowed to stir at 90° C. for 16 h. The mixture was filtered, and water (50 mL) was added to the filtrate. The filtrate was extracted with ethyl acetate and the combined organic layer was extracted with brine, dried over magnesium sulphate and concentrated in vacuo to give the crude 2-((4-Fluorobenzyl)oxy)aniline (80% yield), which was used without purification for further conversion.
1H-NMR (CDCl3): 1H-NMR (CDCl3, 400 MHz) 3.84 (3H, s), 5.11 (2H, s), 6.36 (1H, d, J=15.45 Hz), 6.90 (2H, d, J=8.72 Hz), 6.93-6.96 (1H, m), 7.00-7.04 (2H, m), 7.12 (2H, t, J=8.62 Hz), 7.42 (1H, dd, J=5.40, 8.44 Hz), 7.48 (2H, d, J=8.68 Hz), 7.67 (1H, d, J=15.45 Hz), 7.87 (1H, s), 8.54 (1H, s). 13C-NMR (CDCl3): 55.48, 70.44, 111.72, 114.40, 115.89 (d, J=21.60 Hz), 119.61 (d, J=157.91 Hz), 121.86, 123.77, 127.48, 128.42, 129.69, 129.78, 132.34 (d, J=3.27 Hz), 141.85, 147.13, 161.22, 161.62, 164.07, 164.19.
(E)-3-(4-methoxyphenyl)acrylic acid and 2-((4-fluorobenzyl)oxy)aniline were reacted in a similar fashion as described for the synthesis of (E)-3-(4-methoxyphenyl)-N-(2-phenoxyphenyl)acrylamide (Example 3) to yield (E)-N-(2-((4-fluorobenzyl)oxy)phenyl)-3-(4-methoxyphenyl)acrylamide in 57% yield.
1H-NMR (DMSO-d6, 400 MHz): 3.84 (3H, s), 5.11 (2H, s), 6.36 (1H, d, J=15.45 Hz), 6.90 (2H, d, J=8.72 Hz), 6.95 (1H, m, J=2.37 Hz), 7.03 (2H, t, J=3.96 Hz), 7.12 (2H, t, J=8.62 Hz), 7.42 (2H, q, J=4.61 Hz), 7.48 (2H, d, J=8.68 Hz), 7.67 (1H, d, J=15.45 Hz), 7.87 (1H, s), 8.54 (1H, s). 13C-NMR (DMSO-d6, 100 MHz): δ5.48, 70.44, 111.72, 114.40, 115.89 (d, J=21.60 Hz), 119.61 (d, J=157.91 Hz), 121.86, 123.77, 127.48, 128.42, 129.69, 129.78, 132.34 (d, J=3.27 Hz), 141.85, 147.13, 161.22, 161.62, 164.07, 164.19. HRMS (ESI): Found 400.1310 [M+Na]+ calc. 400.1325.
A mixture of 2.2 g (11.8 mmol) 2,2-difluoro-benzo[1,3]dioxole-5-carbaldehyde, 2.71 g (26.0 mmol) malonic acid, 0.2 g (2.4 mmol) piperidine and 9 ml pyridine was kept at reflux temperature until carbon dioxide development ceased (3 h). After cooling to room temperature the reaction mixture was poured onto 100 g of ice and 30 ml of 6N HCl. The precipitate was isolated, washed with water and dried to afford the desired product HLM1319 (2.53 g, 11.1 mmol, yielding 94%) as white solid, which was used in next step without further purification.
By following the synthesis procedure of Example 3 ((E)-3-(4-methoxyphenyl)-N-(2-phenoxyphenyl)acrylamide), (E)-3-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(2-methoxyphenyl)acrylamide was obtained as brown solid (400 mg, 1.2 mmol, yielding 68%) from (E)-3-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)acrylic acid (400 mg, 1.75 mmol) after purification by silica gel chromatography (PE/EA=8/1).
1H-NMR (CDCl3 400 MHz): 1.03 (3H, t, J=7.40 Hz), 1.53 (2H, sextet, J=7.47 Hz), 1.86 (2H, quintet, J=7.06 Hz), 4.08 (2H, t, J=6.62 Hz), 6.46 (1H, d, J=15.41 Hz), 6.90 (1H, dd, J=0.96, 8.04 Hz), 6.99 (1H, dt, J=1.24, 7.76 Hz), 7.04 (1H, dd, J=1.36, 7.80 Hz), 7.08 (1H, d, J=8.16 Hz), 7.26-7.30 (2H, m), 7.69 (1H, d, J=15.45 Hz), 7.95 (1H, s), 8.51 (1H, d, J=6.84 Hz). 13C-NMR (CDCl3, 100 MHz): 13.92, 19.35, 31.30, 36.55, 68.17, 107.86, 109.85, 111.04, 120.09, 121.16, 121.67, 124.08, 125.07, 127.93, 131.39, 131.76 (1C, t, J=256.34 Hz), 140.69, 144.46, 144.81, 147.41, 163.22. HRMS (ESI): Found: 398.1181 [M+Na] calc. 398.1180.
The following examples, listed in the Table 2 below, were obtained using similar methods as used for synthesis of Examples 1-6.
1H-NMR (CDCl3)
13C-NMR (CDCl3)
(Azidomethyl)benzene (77 mg, 0.58 mmol) and (E)-N-(2-butoxyphenyl)-3-(4-(pent-4-yn-1-yloxy)phenyl)acrylamide (220 mg, 0.58 mmol) (1.00 equiv.) were dissolved in dichloromethane. To this solution, a freshly prepared solution of copper (11) sulphate pentahydrate (12 mg, 0.047 mmol, 0.08 equiv.) and sodium ascorbate (23 mg, 0.12 mmol, 0.20 equiv.) in water was added, while the mixture was stirred vigorously. The reaction mixture was allowed to stir for 2 h at room temperature. After the reaction was complete, the reaction mixture was extracted with dichloromethane and the organic phase was dried over magnesium sulphate. After the organic layer was evaporated to dryness, the residue was purified by silica gel chromatography (PE/EA=1/1) to afford (E)-3-(4-(3-(1-benzyl-1H-1,2,3-triazol-4-yl)propoxy)phenyl)-N-(2-butoxyphenyl)acrylamide (153 mg, 0.3 mmol, 51%) as white solid. 1H-NMR (CDCl3 400 MHz): 1.03 (3H, t, J=7.40 Hz), 1.54 (2H, sextet, J=7.47 Hz), 1.86 (2H, quintet, J=7.02 Hz), 2.17 (2H, quintet, J=6.80 Hz), 2.90 (2H, t, J=7.46 Hz), 4.02 (2H, t, J=6.16 Hz), 4.08 (2H, t, J=6.60 Hz), 5.49 (2H, s), 6.41 (1H, d, J=15.45 Hz), 6.85-6.90 (3H, m), 6.98 (1H, dt, J=1.15, 7.65 Hz), 7.03 (1H, dt, J=1.61, 7.72 Hz), 7.21-7.25 (3H, m), 7.35-7.37 (3H, m), 7.48 (2H, d, J=8.60 Hz), 7.68 (1H, d, J=15.45 Hz), 7.92 (1H, s), 8.52 (1H, d, J=6.16 Hz). 13C-NMR (CDCl3): 14.04, 19.47, 22.25, 28.84, 31.35, 54.15, 67.05, 68.54, 111.01, 114.88, 118.94, 119.99, 121.01, 121.15, 123.70, 127.50, 128.11, 128.24, 128.78, 129.19, 129.66, 134.95 (1C, s), 141.67, 147.36, 147.65, 160.53, 164.12. HRMS (ESI): Found 360.1572, calc. 360.1576, [M+Na]+, M=C31H34N4O3
(E)-N-(2-butoxyphenyl)-3-(3,4-dimethoxyphenyl)acrylamide (example 84, 1 eq.) was added to a suspension of sodium hydride (1.4 eq) in dry THF (8 mL). The mixture was allowed to stir for 5 min until hydrogen evolution ceased. Methyl iodide (1.4 eq.) was added and stirring was continued for 16 hr. The mixture was poured into ether and washed with brine, dried over magnesium sulphate and evaporated in vacuo to dryness. Purification of the crude material by flash chromatography afforded (E)-N-(2-butoxyphenyl)-3-(3,4-dimethoxyphenyl)-N-methylacrylamide in 76% yield.
1H-NMR (400 MHz, CDCl3) 0.89 (3H, t, J=7.40 Hz), 1.42 (2H, sextet, J=7.46 Hz), 1.71 (2H, quintet, J=7.04 Hz), 3.30 (3H, s), 3.80 (3H, s), 3.86 (3H, s), 3.98 (2H, t, J=6.22 Hz), 6.16 (1H, d, J=15.49 Hz), 6.75-6.78 (2H, m), 6.91 (1H, d, J=8.24 Hz), 6.95-6.99 (2H, m), 7.19 (1H, dd, J=1.38, 8.14 Hz), 7.32 (1H, dt, J=1.32, 7.82 Hz), 7.59 (1H, d, J=15.45 Hz). 13C-NMR (CDCl3, 100 MHz) 13.87, 19.32, 31.27, 36.39, 55.96, 56.02, 68.11, 110.58, 111.14, 112.88, 117.00, 120.75, 121.35, 128.64, 129.32, 129.72, 132.29, 141.12, 149.01, 150.34, 154.97, 167.00. HRMS (ESI): m/z [M+Na]+ calcd for C22H27NO4: 392.1838, Found: 392.1823.
The following compounds, as listed in Table 3 below can be prepared in a similar manner as described for Example 67.
1H-NMR
13C-NMR
To a solution of p-anisaldehyde (32 g, 0.24 mol) and ethyl-(2-dimethoxyphosphinyl)-2-propanoate (58.8 g, 0.28 mol) in toluene (160 mL) was added NaOtBu (33.6 g, 0.35 mol) at 0° C. under N2, atmosphere over the course of 30 minutes. The reaction mixture was warmed to room temperature and stirred for 15 min. After completion of the reaction, the reaction mixture was neutralised with 10% aq. HCl The toluene layer was separated, washed with water, dried over sodium sulphate and concentrated in vacuo. The residue was purified by silica gel chromatography to give 22.7 g ethyl (E)-3-(4-methoxyphenyl)-2-methylacrylate.
1H-NMR (CDCl3): 1.35 (3H, t, J=7.12 Hz), 2.13 (3H, d, J=1.20 Hz), 3.84 (3H, s), 4.26 (2H, q, J=7.12 Hz), 6.92 (2H, d, J=8.72 Hz), 7.39 (2H, d, J=8.64 Hz), 7.64 (1H, s).
A 50 mL round-bottom flask was charged with ethyl (E)-3-(4-methoxyphenyl)-2-methylacrylate (10 mmol, 1 eq.), LiOH·H2O (50 mmol, 5 eq.) in a mixture of tetrahydrofurane/water (1:1, 0.25 M) The reaction flask was heated to 80° C. and stirred for 3 h. After cooling to room temperature, the mixture was extracted with diethyl ether. The aqueous phase was acidified by addition of 2N hydrochloric acid and extracted with ethyl acetate. The combined organic layers were dried over magnesium sulphate and subsequently evaporated to dryness (18.6 g).
By following the synthesis procedure of Example 3 ((E)-3-(4-methoxyphenyl)-N-(2-phenoxyphenyl)acrylamide), (E)-N-(2-butoxyphenyl)-3-(4-methoxyphenyl)-2-methylacrylamide was obtained in 92% yield, employing (E)-3-(4-methoxyphenyl)-2-methylacrylic acid and 2.butoxyaniline. 1H-NMR (CDCl3). 1.00 (1H, t, J=7.40 Hz), 1.54 (2H, sextet, J=7.50 Hz), 1.80-1.87 (2H, m), 2.24 (3H, d, J=1.32 Hz), 3.85 (3H, s), 4.07 (2H, t, J=6.38 Hz), 6.89 (1H, dd, J=1.56, 7.88 Hz), 6.93-6.95 (2H, m), 6.99 (1H, dt, J=1.60, 7.70 Hz), 7.04 (1H, dt, J=1.88, 7.67 Hz), 7.37 (2H, d, J=8.68 Hz), 7.47 (1H, s), 8.41 (1H, s), 8.50 (1H, dd, J=1.82, 7.82 Hz). 13C-NMR (CDCl3): 13.99, 14.33, 19.50, 31.45, 55.45, 68.36, 110.90, 114.01, 119.75, 121.22, 123.65, 128.26, 128.75, 130.71, 131.17, 134.60, 147.61, 159.54, 167.39. The examples listed in the following Table 4 were obtained in a similar fashion as described for Example 78.
1H-NMR (CDCl3)
13C-NMR (CDCl3)
To a solution of the methyl (E)-3-(4-hydroxyphenyl)acrylate (2.8 mmol, 500 mg, 1.0 equiv.) and propargyl bromide (3.4 mmol, 401 mg, 1.2 equiv.) in acetone (5.6 mL), K2CO3 (3.4 mmol, 465 mg, 1.2 equiv.) was added and the mixture was stirred at 60° C. for 24 h. The reaction mixture was cooled down to room temperature, filtered to remove the solid, and the volatiles removed under reduced pressure. The crude product was purified by column chromatography on silica gel chromatography to afford methyl (E)-3-(4-(prop-2-yn-1-yloxy)phenyl)acrylate (573 mg, 94%).
1H-NMR (CDCl3 400 MHz): 2.54 (1H, t, J=2.38 Hz), 3.79 (3H, s), 4.72 (2H, d, J=2.40 Hz), 6.32 (1H, d, J=15.97 Hz), 6.98 (1H, d, J=8.76 Hz), 7.49 (2H, d, J=8.76 Hz), 7.65 (1H, d, J=15.97 Hz).
13C-NMR (CDCla 101 Hz): 51.76, 55.94, 76.09, 78.16, 115.36, 115.95, 128.09, 129.79, 144.43, 159.33, 167.81.
Methyl (E)-3-(4-(prop-2-yn-1-yloxy)phenyl)acrylate (573 mg, 2.7 mmol) was dissolved in methanol (10 ml). 1M sodium hydroxide aqueous solution (10 mL) was added. The reaction mixture was stirred at room temperature for 2 hours. The resultant was acidified (pH=3) with 2N HCl, and the aqueous layer was extracted with ethyl acetate. The combined organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give (E)-3-(4-(prop-2-yn-1-yloxy)phenyl)acrylic acid 510 mg (yield: 95%, white solid), which was used in next step without purification.
Added (E)-3-(4-(prop-2-yn-1-yloxy)phenyl)acrylic acid (293 mg, 1 mmol), DIPEA (1 mmol, 174 μL) and HATU (418 mg, 1.1 mmol) in 10 mL DCM at 0° C. and stirred for 30 min. Then 2-aminophenol (2 mmol, 218 mg) was added and stirring was continued overnight. After quenching by 1 M HCl, it was extracted by ethyl acetate and the combined organic phase was dried with Na2SO4. After being concentrated in vacuum, the residue was purified by silica column to afford (E)-N-(2-hydroxyphenyl)-3-(4-(prop-2-yn-1-yloxy)phenyl)acrylamide (270 mg, 92%)
1H-NMR (DMSO 400 MHz): 3.61 (1H, t, J=2.14 Hz), 4.86 (2H, d, J=2.20 Hz), 6.80 (1H, t, J=7.58 Hz), 6.89 (1H, d, J=6.84 Hz), 6.96 (1H, t, J=7.52 Hz), 7.00-7.06 (3H, m), 7.53 (1H, d, J=15.61 Hz), 7.60 (2H, d, J=8.64 Hz), 7.92 (1H, d, J=7.72 Hz), 9.44 (1H, s), 9.99 (1H, s).
HRMS (ESI): Found: 292.0976 [M−H]− calc. 290.0974.
To a mixture of (E)-N-(2-hydroxyphenyl)-3-(4-(prop-2-yn-1-yloxy)phenyl)acrylamide (0.27 mmol, 80 mg), triphenyl phosphine (1.2 eq, 0.33 mmol, 86 mg), (3-methyl-3H-diazirin-3-yl)ethanol [prepared according to ref. 42] (1.5 eq, 0.41 mmol, 41 mg) in THF (3 ml), diethyl azodicarboxylate (1.2 eq, 0.33 mmol, 51 μL) was added at 0° C. After the solution was stirred overnight at room temperature, the solvent was removed in vacuum, and the residue was purified by silica column to afford (E)-N-(2-(2-(3-methyl-3H-diazirin-3-yl)ethoxy)phenyl)-3-(4-(prop-2-yn-1-yloxy)phenyl)acrylamide (HLM-01-543, 37 mg, 36%).
1H-NMR (CDCl3, 400 MHz): 1.14 (3H, s), 1.91 (2H, t, J=5.82 Hz), 2.55 (1H, t, J=2.40 Hz), 4.04 (2H, t, J=5.82 Hz), 4.73 (2H, d, J=2.40 Hz), 6.66 (1H, d, J=15.53 Hz), 6.84-6.87 (1H, m), 6.98-7.03 (4H, m), 7.55 (2H, d, J=8.72 Hz), 7.74 (1H, d, J=15.53 Hz), 8.38 (1H, s), 8.56 (2H, d, J=3.92 Hz).
13C-NMR (CDCl3 101 MHz): 19.58, 24.76, 34.36, 55.95, 64.19, 76.03, 78.27, 111.19, 115.35, 119.76, 120.18, 121.98, 123.60, 128.57, 128.65, 129.63, 141.37, 146.88, 158.97, 164.38.
HRMS (ESI): Found: 398.1476 [M−H]− calc. 398.1481.
By following the synthesis procedure of (E)-N-(2-(2-(3-methyl-3H-diazirin-3-yl)ethoxy)phenyl)-3-(4-(prop-2-yn-1-yloxy)phenyl)acrylamide (HLM-01-543), (E)-N-(2-(2-(3-methyl-3H-diazirin-3-yl)ethoxy)phenyl)-3-(4-(pent-4-yn-1-yloxy)phenyl)acrylamide (HLM-02-544) was obtained as white solid (51 mg, 53%)
1H-NMR (CDCl3, 400 MHz). 1.04 (3H, s), 1.81 (2H, t, J=5.82 Hz), 1.87-1.95 (3H, m), 2.32 (1H, dt, J=2.65, 6.95 Hz), 3.94 (2H, t, J=5.82 Hz), 4.01 (2H, t, J=6.10 Hz), 6.54 (1H, d, J=15.53 Hz), 6.75-6.77 (1H, m), 6.82 (1H, d, J=8.76 Hz), 6.91-6.94 (2H, m), 7.43 (2H, d, J=8.64 Hz), 7.64 (1H, d, J=15.53 Hz), 8.26 (1H, s), 8.46 (1H, s).
13C-NMR (CDCl1 101 MHz). 15.29, 19.60, 24.75, 28.20, 34.38, 64.19, 66.36, 69.14, 83.44, 111.18, 114.97, 119.24, 120.20, 122.00, 123.56, 127.75, 128.71, 129.70, 141.60, 146.87, 160.49, 164.51.
HRMS (ESI): Found: 426.1791 [M−H]− calc. 426.1794.
Primary human lung fibroblasts (phLFs) were isolated by outgrowth from human lung tissue derived from lung explants or tumor-free areas of lung resections as previously described (29, 30). Cells were cultured in Dulbecco's Modified Eagle Medium F-12 with 20% (v/v) special processed fetal bovine serum (PAN Biotech, Cat. No. and 100 International Units Penicillin per mL and 100 μg per mL Streptomycin. Medium was changed every 2-3 days and cells were passaged at 80-90% confluency in a ratio of 1:5 or 1:6. Cells were used for experiments until passage 7. For ECM deposition drug screening, 0.5-1×106 cells were expanded from passage 1 to passage 5, each time in a ratio of 1:6. More than 100×106 cells were trypsinized at passage 5 and cryopreserved in 90% (v/v) fetal bovine serum and 10% (v/v) dimethyl sulfoxide. Cells were frozen slowly by using Mr. Frosty (ThermoFisher Scientific) freezing containers. For reseeding phLFs were thawed in a water bath at 37° C. and the cells were washed with culture medium, prior to plating. After reaching confluency in passage 6, cells were used for the ECM deposition assays. Primary human dermal fibroblast (Cat #DF-F) were purchased from ZenBio Inc. and cultured according to the manufacturer's instructions.
phLFs were cultured in DMEM F-12 medium with 20% fetal bovine serum (FBS) and antibiotic supplement as mentioned above. Cells were seeded with 6000 cells/well in 384-well CellCarrier plates (Perkin Elmer, Cat #6007550). Following overnight incubation, cells were starved in serum-reduced medium (1% FBS with 0.1 mM 2-phosphoascorbate (Sigma, Cat #49752)) for 24 h. Afterwards, cells were treated with TGFβ1 (1 ng/ml) or vehicle, and additionally small molecules or appropriate vehicle controls were added. After 72 h of incubation, medium was changed for starving medium with 1 μg/mL AlexaFluor-488 fluorophore conjugated anti-collagen-type-5 antibodies (SantaCruz, Clone C-5, Cat #sc-166155 AF488), 0.66 μg/mL AlexaFluor-555 fluorophore conjugated anti-collagen-type-1 antibodies (Rockland, Cat #600-401-103-0.1), and 1 μg/mL AlexaFluor-637 fluorophore conjugated anti-fibulin 1 antibodies (SantaCruz, Clone C-5, Cat #sc-25281 AF647) and 1 μg/mL Hoechst H33342 (Sigma). Fluorescenceconjugation of the collagen type 1 antibody was done by using the AlexaFluor-555 Protein Labeling Kit (Invitrogen, Cat #A20174) according to manufacturer's instructions. Labeling efficacy was controlled by photometrical means.
Following four hours of incubation, cells were washed three times with PBS and fixed with paraformaldehyde (PFA). For automated liquid handling in 384 well plates, an INTEGRA Assist Plus (INTEGRA, Zizers, Switzerland) equipped with an INTEGRA Viaflo 1I pipette (INTEGRA, Zizers, Switzerland, Cat #4642), 125 μL GripTips™ pipette tips (INTEGRA, Zizers, Switzerland, Cat #6464) and sterile reagent reservoirs (INTEGRA, Cat #4311) were applied. All automated pipetting steps with the INTEGRA Assist Plus were performed at 9.5 μL/s in order to ensure proper integrity and attachment of the deposited ECM to the culturing surface within the wells of the 384-well plates. During cell seeding the automated liquid handling was performed at 89.3 μL/s. Removal of liquids from the well-plates was done by manually inverting the plates.
Following fixation, the automated imaging was achieved by using aconfocal laser scanning microscope (LSM710, Zeiss) with automated focus detection for three-dimensional image acquisition (1024 px×1024 px×9 px which equals a dimension of 1417 μm×1417 μm×16 μm). For post-acquisition analysis images were imported into IMARIS software (Bitplane) and volume detection or alternatively quantification of the mean fluorescence intensity, as well as Hoechst-stained cell nuclei were automatically counted by using Imaris' spot detection algorithm.
PCLS were prepared as described before (31, 32). Shortly, PCLS were prepared from tumor-free peri-tumor tissue. The lung tissue was inflated with 3% agarose solution and solidified at 4° C. Tissue blocks were cut in μm thick PCLS using a vibration microtome Hyrax V50 (Zeiss). PCLS were cultured in DMEM F-12 medium and treated with a profibrotic cocktail, as described before (31), or vehicle, as well as with small molecules or vehicles for 7 days. After culturing and treatments, supernatants were harvested. PCLS were 500 washed in PBS and protein was extracted as previously described (33). Briefly, PCLS were pooled in an Eppendorf tube and lysed in 500 μl ice-cold RIPA buffer (50 mM Tris-CI pH 7.4, 150 mM NaCl, 1% NP40, 0.25% Na-deoxycholate) containing 1× Roche complete mini protease inhibitor cocktail (Roche, Cat. #11697498001). After an incubation of 2 hours rotating at 4° C., the lung slices were removed from the lysates and the protein content was measured.
Viability/Cytotoxicity Assay Kit for Animal Live and Dead Cells was obtained from Biotium, Cat. No. 3002). CellEvent™ Caspase 3/7 Green Detection Reagent was acquired from Invitrogen, Cat. No. C10423. For MTT-assays, Thiazolyl Blue Tetrazolium was bought from SigmaAldrich (M5655-1G). All these kits and assays were used according to the manufacturer's instructions.
For immunofluorescence microscopy the following antibodies were used: monoclonal mouse anti-collagen type 5 (1 mg/mL) from Sigma Aldrich (Cat #sc-166155), monoclonal mouse anti-collagen type 5 AlexaFluor-488 conjugate from Sigma Aldrich (Cat #sc-166-155AF488), polyclonal rabbit anti-collagen type 1 from Rockland (Cat #600-401-103-0.5), monoclonal mouse anti-fibulin 1 from SantaCruz (Cat #sc-25281), monoclonal mouse anti-fibulin 1 AlexaFluor-647 conjugate (1 mg/mL) from SantaCruz (Cat #sc-25281AF647), and polyclonal rabbit anti-fibronectin (1 mg/mL) from SantaCruz (Cat #sc-9068). Hoecst-33342 was obtained from Sigma (Cat #B2261). The following secondary antibodies were used: AF488 donkey-anti-mouse Ab (Invitrogen, Cat. #A21202), AF568 donkey-anti-mouse Ab (Invitrogen, Cat. #A11004), and AF568 donkey-anti-mouse Ab (Invitrogen, Cat. #A11011). For immunofluorescence stainings of actin-stress fibers Alexa Fluor 568 Phalloidin (Invitrogen, A12380) was used. 4′, 6-diamidino-2-phenylindole (DAPI) was acquired from Sigma-Aldrich (Cat #D9564).
For standard immunofluorescence staining, 5000 phLFs were seeded into 96 well imaging plates with a flat bottom (Cat #353376, BD Biosciences). After incubation, cells were fixed with either 4% PFA for 30 min at 37° C. or 100% methanol for 2 min at −20° C. If needed, phLFs were permeabilized with 0.25% (v/v) Triton X-100 in PBS for 15 min. After washing with 100 μL of PBS blocking was done by incubation with 5% (w/v) BSA in PBS for one hour. Primary antibodies were diluted in 1% bovine serum albumin (BSA, Sigma) in PBS, incubated for 16 hours at 4° C. and subsequently washed three times with PBS for 20 minutes each. Secondary antibodies were diluted in 1% bovine serum albumin (BSA, Sigma) in PBS, incubated for one hour at room temperature and subsequently washed three times with PBS for 20 minutes each. 4% paraformaldehyde in phosphate buffered saline (w/v) was prepared from paraformaldehyde from Sigma (Cat #15,812-7). Bovine serum albumin was obtained from Sigma (Cat #A3059). Triton X-100 was obtained from AppliChem (Cat #A1388).
Confocal time-lapse microscopy was implemented on an LSM710 system (Carl Zeiss) containing an inverted AxioObserver.Z1 stand equipped with phase-contrast and epi-illumination optics and operated by ZEN2009 software (Carl Zeiss). The following objectives were used for imaging: EC Plan-Neofluar 20×/0.8 NA (Carl Zeiss), LD C-Apochromat 40x/1.1 NA water objective lens (Carl Zeiss) and LCI PLN-NEOF DICIII 63×/1.30 NA water objective lens (Carl Zeiss). For 4D imaging the cells were kept in an incubation chamber (Carl Zeiss) under standard cultivation conditions (37° C. and 5% CO2). Thickness of single confocal layers within the z-stacks was set according to optimized values suggested by the ZEN2009 software. The confocal data sets were either maximum intensity projected in the ZEN2009 software (Carl Zeiss) and/or imported into Imaris 9.0.0-9.3.1 software (Bitplane) for analysis.
Cells were scraped off the plastic dish directly into 200 μl ice-cold RIPA buffer containing 1× Roche complete mini protease inhibitor cocktail. After incubating the samples for 30 minutes on ice, insoluble material was removed by centrifugation at 14.000 g for 15 minutes at 4° C. and the supernatant was further processed. Samples were mixed with 50 mM Tris-HCl, pH 6.8, 100 mM DTT, 2% SDS, 1% bromphenol blue, and 10% glycerol, and proteins were separated using standard SDS-10% PAGE. For immunoblotting, proteins were transferred to PVDF (Millipore (Billerica, MA, (USA)), 0.45 μm or 0.2 μm) membranes, which were blocked with 5% milk in TBST (0.1% Tween 20 in TBS) and incubated with primary, followed by HRP-conjugated secondary antibodies over night at 4° C. and at room temperature for 1 hour, respectively. For immunoblotting the following primary antibodies were used: monoclonal mouse anticollagen type 5 (1 mg/mL) from Sigma Aldrich (Cat #sc-166155), polyclonal rabbit anti-collagen type 3 (1 mg/mL) from Rockland (Cat #600-401-105), polyclonal rabbit anti-collagen type 1 (1 mg/mL) from Rockland (Cat #600-401-103-0.5), monoclonal mouse anti-fibulin 1 (1 mg/mL) from SantaCruz (Cat #sc-25281), polyclonal rabbit anti-fibronectin (1 mg/mL) from SantaCruz (Cat #sc-9068), and monoclonal mouse anti-p-actin-peroxidase (AC-15, Sigma, 1:10000). Goat anti-rabbit and goat anti-mouse IgG conjugated to horseradish peroxidase (Cell Signaling, 1:10000) were applied as secondary antibodies. CXCL/IL-8 concentrations were determined using Human IL-8/CXCL8 DuoSet ELISA (DY208-05) according to the manufacturer's protocol.
mRNA Isolation, cDNA Synthesis and qRT-PCR
RNA extraction from cultured phLFs was performed using the PeqGold RNA kit (Peqlab) according to the manufacturer's instruction. The concentration of the isolated RNA was assessed spectrophotometrically at a wavelength of 260 nm (NanoDrop 1000). cDNA was synthesized with the GeneAMP PCR kit (Applied Biosystems (Foster City, CA, USA)) utilizing random hexamers using 1 μg of isolated RNA for one 301 reaction. Denaturation was performed in an Eppendorf Mastercycler with the following settings: 302 303 lid=45° C., 70° C. for 10 minutes and 4° C. for 5 minutes. Reverse transcription was performed in an Eppendorf Mastercycler with the following settings: lid=105° C., 20° C. for 10 minutes, 42° C. for 60 minutes and 99° C. for 5 minutes. qRT-PCR reactions were performed in triplicates with SYBR Green I Master in a LightCycler@ 48011 (Roche (Risch, Switzerland)) with standard conditions: 95° C. for 5 min followed by 45 cycles of 95° C. for 5 s (denaturation), 59° C. for 5 s (annealing) and 72° C. for 20 s (elongation). Target genes were normalized to HPRT expression.
Total RNA was isolated PEQGold Total RNA Kit (PeqLab) according to the manufacturer's instructions including gDNA elimination. The Agilent 2100 Bioanalyzer was used to assess RNA quality and RNA with RIN>7 was used for microarray analysis. Total RNA (150 ng) was amplified using the WT PLUS Reagent Kit (Thermo Fisher Scientific Inc., Waltham, USA). Amplified cDNA was hybridized on Human ClariomS arrays (Thermo Fisher Scientific). Staining and scanning (GeneChip Scanner 3000 7G) was done according to manufacturer's instructions. Transcriptome Analysis Console (TAC; version 4.0.0.25; Thermo Fisher Scientific) was used for quality control and to obtain annotated normalized SST-RMA gene-level data. Statistical analyses were performed by utilizing the statistical programming environment R (R Development Core Team Ref1). Genewise testing for differential expression was done employing the paired limma t-test and Benjamini-Hochberg multiple testing correction (FDR<10%). To reduce background, gene sets were filtered using DABG p-values<0.05 in at least one sample per pair and in at least two of three pairs per analysis. Heatmaps were generated using GraphPad Prism v7, The regulation pattern clustering (RPC) was based on uniform manifold approximation and projection (UMAP) (35). mRNA abundancies from the microarray data were normalized (as seen as an example in FIG. F) and abundancies of all four different conditions summarized in a linear vector (
The KERAS high-level API (https github.com/fchciet/keras/) with TensorFlow implementation was used to train Convolutional Neural Network (CNN) on a complex image detection and classification task. The CNN design (
tiles with a 3/4 tile overlap (
Each data tile T was rotated by θE {0°, 90°, 180°, 270° }, representing different spatial orientations of the ECM (
tiles were saved from each original image leading to a significant augmentation of data (100-fold for np=512, 676-fold for np=256 and 3364-fold for np=128). The convolutional neural network as shown in
not overlapping np×np sized tiles (
The image clustering chosen was performed using the UMAP (Uniform Manifold Approximation and Projection), a widely used manifold learning technique for dimension reduction. UMAP is constructed from a theoretical framework based in Riemannian geometry and algebraic topology (35). Each m×m dimensional image pixel matrix (m=1024) is flattened as a linear vector (
6000 cells/well phLFs were seeded in 384-well CellCarrier plates. Following overnight incubation, cells were starved in serum-reduced medium (1% FBS) for 24 h.
Afterwards, cells were treated with TGFβ1 (1 ng/ml) and different compounds. After 48 h, cells were fixed with 100% ice-cold methanol. Cells were stained for DAPI and αSMA antibody conjugated to Cy3 (Cat. No. C6198-2ML, Sigma). For automated liquid handling in 384 well plates, an INTEGRA Assist Plus was used. Following fixation, the automated imaging was achieved by using a confocal laser scanning microscope (LSM710, Zeiss) with automated focus detection for three-dimensional image acquisition (ECM Deposition Assay). Images were analyzed by measuring the mean fluorescence intensity (MFI) of the αSMA signal in Zen Blue v2.5 (Zeiss).
In a 96-well imaging plate 50 μl 3D collagen gels were casted per well as described before (30), and 20.000 phLFs per well were seeded on top. Cells were treated with 1 ng/mL TGFβ1 and/or example 84. After 72 h cells were fixed with 4% paraformaldehyde. Collagen gels were imaged using an Axiolmager2 (Zeiss) and the gel diameter was determined using Zen Blue v2.5 (Zeiss).
Each 10 μg of protein extract was digested using a modified FASP protocol (36, 37). Briefly, proteins were reduced and alkylated using dithiothreitol and iodoacetamide, and diluted to 4 M urea prior to centrifugation on a 30 kDa filter device (Sartorius). After several washing steps using 8 M urea and 50 mM ammoniumbicarbonate, proteins were digested on the filter by Lys-C and trypsin overnight. Generated peptides were eluted by centrifugation, acidified with TFA and stored at −20° C. Samples were measured on a QExactive HF-X mass spectrometer (Thermo scientific) online coupled to an Ultimate 3000 nano-RSLC (Dionex). Tryptic peptides were automatically loaded on a trap column (300 μm inner diameter (ID)×5 mm, Acclaim PepMap100 C18, 5 μm, 100 Å, LC Packings) prior to C18 reversed phase chromatography on the analytical column (nanoEase MZ HSS T3 Column, 100 Å, 1.8 μm, 75 μm×250 mm, Waters) at 250 nl/min flow rate in a 95 minutes non-linear acetonitrile gradient from 3 to 40% in 0.1% formic acid. Profile precursor spectra from 300 to 1500 m/z were recorded at 60000 resolution with an automatic gain control (AGC) target of 3e6 and a maximum injection time of 30 ms. Subsequently TOP15 fragment spectra of charges 2 to 7 were recorded at 15000 resolution with an AGC target of 1e5, a maximum injection time of 50 ms, an isolation window of 1.6 m/z, a normalized collision energy of 28 and a dynamic exclusion of 30 seconds. Generated raw files were analyzed using Progenesis QI for proteomics (version 4.1, Nonlinear Dynamics, part of Waters) for label-free quantification as described (38, 39). Features of charges 2-7 were used and all MSMS spectra were exported as mgf file. Peptide search was performed using Mascot search engine (version 2.6.2) against the Swissprot human protein database (20237 sequences, 11451954 residues).
Search settings were: 10 ppm precursor tolerance, 0.02 Da fragment tolerance, one missed cleavage allowed, carbamidomethyl on cysteine as fixed modification, deamidation of glutamine and asparagine allowed as variable modification, as well as oxidation of methionine. Applying the percolator algorithm (40) resulted in a peptide false discovery rate (FDR) of 0.46%. Search results were reimported in the Progenesis Q1 software. Proteins were quantified by summing up the abundances of all unique peptides per protein after normalization to identified GAPDH and ACTB peptides. Resulting protein abundances were used for calculation of fold-changes between conditions and repeated-measures ANOVAs within the Progenesis QI software. Proteomics expression data is provided as Table S4.
Smurf2 siRNA-Mediated Silencing
phLFs were reverse transfected with 2 nM or 10 nM Silencer® Pre-designed Smurf2 siRNA (Cat #: AM16708, Ambion, ThermoFisher Scientific, Carlsbad, USA) or 10 nM scrambled Silencer® Negative control No. 1 siRNA (AM4611, Ambion, ThermoFisher Scientific, Carlsbad, USA) in Lipofectamine® RNAiMax transfection reagent (13778-150, ThermoFisher Scientific, Carlsbad, 130 USA) as indicated followed by 1 ng/ml TGFβ1 treatment for 48 h if not indicated differently.
Experimental outline: Primary human lung IPF-fibroblasts (phLFs), which were derived from 3 different idiopathic-pulmonary-fibrosis (IPF) patients (n=3), were cultured in DMEM F-12 medium with 20% fetal bovine serum (FBS) and antibiotic supplements. Cells were seeded with 6000 cells/well in 384-well CellCarrier plates. After overnight incubation, cells were starved in serum-reduced medium (1% FBS with 0.1 mM 2-phosphoascorbate for 24 h). Then, phLFs were treated with TGFβ1 (1 ng/ml) or vehicle, and incubated together with synthesized N23Ps (10 μM) or appropriate vehicle controls. After 72 h of incubation, medium was changed for starvation medium with 1 μg/mL AlexaFluor-637 fluorophore conjugated anti-fibulin 1 antibodies (SantaCruz, Clone C-5, Cat #sc-25281 AF647) and 1 μg/mL Hoechst H33342 (Sigma). Following four hours of incubation, cells were washed three times with PBS and fixed with paraformaldehyde (PFA). Following fixation, the automated imaging was achieved by using a confocal laser scanning microscope (LSM710, Zeiss) with automated focus detection for three-dimensional image acquisition (1024 px×1024 px×9 px which equals a dimension of 1417 μm×1417 μm×16 μm). For post-acquisition analysis images were imported into IMARIS software (Bitplane) and quantified for the mean fluorescence intensity, as well as Hoechst-stained cell nuclei were automatically counted by using Imaris' spot detection algorithm.
Assessment of ECM inhibition I: Mean fluorescence intensity (MFI) values represented the degree of fibulin-1 ECM deposition in three different IPF-phLFs (n=3). The inhibitory activity of the tested N23P compounds was graded, whereas we defined a compound as “inactive” if all TGFβ1-induced ECM deposition was preserved (=0% inhibition by the compound). If no increase in TGFβ1-induced ECM deposition was detected, the compound was rated as active (100% inhibition by the compound). Based on this definition three different classes of inhibitory activities have been created: (+++) compounds which displayed >90% inhibition, (++) compounds which displayed 60-90% inhibition, and (+) compounds which displayed 20-60% inhibition of TGFβ1-induced ECM deposition. Compounds which showed an inhibition <20% were classified as inactive. Tranilast exhibits no inhibition at 10 μM.
Assessment of ECM inhibition It: Mean fluorescence intensity (MFI) values represented the degree of fibulin-1 ECM deposition in one or two different IPF-phLFs (n=1-2). The inhibitory activity of the tested N23P compounds was graded, whereas we defined a compound as “inactive” if all TGFβ1-induced ECM deposition was preserved (=0% inhibition by the compound). If no increase in TGFβ1-induced ECM deposition was detected, the compound was rated as active (100% inhibition by the compound). Based on this definition three different classes of inhibitory activities have been created: (+++) compounds which displayed more than 90% inhibition, (++) compounds which displayed 60-90% inhibition, and (+) compounds which displayed 20-60% inhibition of TGFβ1-induced ECM deposition. Compounds which showed an inhibition <20% were classified as inactive.
| Number | Date | Country | Kind |
|---|---|---|---|
| 21178481.4 | Jun 2021 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/065773 | 6/9/2022 | WO |
