TGFβ inhibitors and prodrugs

Abstract

Disclosed are a compound of formula (I) and two prodrugs thereof having improved TGFβ signaling pathway inhibitory activity, improved therapeutic efficacy and improved toxicity characteristics. Also disclosed are compositions comprising the TGFβ signaling pathway inhibitor and its prodrug. In addition, the present invention discloses a compound of formula (I) and its prodrug for use in a method for treating a disease responsive to TGFβ signaling pathway inhibition.

Description

Technical Field

[0001] The present invention relates to novel TGFβ signaling pathway inhibitors with improved inhibitory activity, improved therapeutic capacity and improved toxicity characteristics, and prodrugs thereof (including vanilla acetone-derived fragments or carbamate fragments), which trap TGFβ signaling inhibition activity, but when the prodrug is hydrolyzed, it is able to deliver a more active TGFβ signaling pathway inhibitor in vivo. Compositions comprising the TGFβ signaling pathway inhibitor and its prodrug are also disclosed. In addition, the present invention discloses the TGFβ signaling pathway inhibitor and its prodrug, which are used in methods for treating diseases responsive to TGFβ signaling inhibition. Background Art

[0002] Transforming growth factor β isoforms TGFβ1, TGFβ2 and TGFβ3 are small secreted homodimeric signaling proteins. They are only found in vertebrates and are required for the normal development of various organs and tissues and for homeostasis. Most, if not all, of the activities that regulate cell fate and the function of the TGFβ signaling pathway are mediated by specific receptor complexes that assemble after ligand binding and contain TGFβ type II receptors and type I receptors.

[0003] TGFβ receptors (TGFβRs) include TGFβ type I receptors and TGFβ type II receptors. TGFβ type I receptors include seven activin receptor-like kinases: ALK1 (ACVRL1), ALK2 (ACVR1), ALK3 (BMPR1A), ALK4 (ACVR1B), ALK5 (TGFBR1), ALK6 (BMPR1B) and ALK7 (ACVR1C), and TGFβ type II receptors include TGFBR2, BMPR2, ACVR2A and ACVR2B. TGFβ ligands use ALK5 / TGFBR1 and TGFBR2 for signaling across the cell membrane and are therefore targets for specific inhibition of the TGFβ pathway. ALK5 in particular has been used as a target when using small molecules, but because the receptors ALK4 and ALK5 are closely related, inhibitor specificity (for either of them) is a known problem. ALK4 / ACVR1B mediates signaling of additional molecules from the TGFβ superfamily, including Activin A, GDF1, GDF11, and Nodal. However, similar to ALK5 signaling, ALK4 activity has been implicated in tissue fibrosis (Jin. et al., Journal of Mecinal Chemistry 2014, 57:4213-4238; Sugiyama M et al., Gastroentenology 1998, 114(3):550-558; Matsuse T et al., Am. J. Respir. Cell Mol. Biol. 1995, 13(1):17-24; Pawlowski, J. E et al., J. Clin. Invest. 1997, 100(3):639-648; Hübner, G. et al., Dev. Biol. 1996, 173(2):490-498; Munz, B. et al., EMBO J. 1999, 18(19):5205-5215; Wankell, M. et al., EMBO J. 2001, 20(19): 5361-5372. Therefore, dual inhibition of ALK4 / ALK5 may enhance the therapeutic efficacy of TGFβ pathway inhibitors in fibrotic diseases.

[0004] TGFβ signaling has been implicated in a variety of pathologies, including genetic disorders (including Camurati-Engelmann disease, Marfan syndrome, muscular dystrophy and Fanconi Anemia), obesity, diabetes, hematological disease, cardiovascular disease, skin diseases such as psoriasis and fibrotic diseases, and plays a key role in wound healing or scarring, for example, in burn wounds.

[0005] TGFβ signaling also plays a key role in cancer progression. Although TGFβ signaling potentially affects all the different cell types in a tumor in complex and disparate ways, the overall effect appears to strongly promote tumor growth, invasion, and metastasis, particularly in the late stages of the disease. Therefore, inhibition of the TGFβ signaling pathway is strongly clinically relevant, and the development and testing of effective agents targeting this pathway is actively underway. Clinical trials are ongoing for a variety of cancer types, including blood cancers such as multiple myeloma and myelodysplastic syndrome; brain cancers such as glioblastoma; soft cancers such as Ewing's sarcoma and malignant pleural mesothelioma; and solid tumors, including breast cancer, gastric and gastroesophageal cancer, colorectal cancer, pancreatic cancer, liver cancer, lung cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, or melanoma; as well as metastases associated with the above-mentioned cancer types.

[0006] The documents Li et al. (J. Med. Chem. 2008, 51(7): 2302-2306), WO02094833 A1 and WO2014072517 A1 describe a wide range of dihydropyrrolopyrazole derivatives useful as inhibitors of the TGFβ signaling pathway.

[0007] Recently, it has been shown that TGFβ signaling inhibition activates anti-tumor immune responses and enhances other types of immunotherapy, making it possible to become a component of combined immunotherapy for the treatment of different types of cancer in the near future (Batlle and Massagué. Immunity. 2019, 50 (4): 924-940). It has been shown that inhibition of interstitial TGFβ signaling releases the immune system against tumor cells, and this therapeutic effect can also cure metastatic disease (i.e., the main cause of death in cancer patients) (Tauriello et al. Nature. 2018, 554 (7693): 538-543.; Mariathasan et al. Nature. 2018, 554 (7693): 544-548).

[0008] Although the TGFβ pathway has been widely recognized as a potential therapeutic target, the clinical use of the TGFβ pathway is hampered by significant toxicity issues. The development of a variety of small molecule inhibitors and antibodies against pathway components has been abandoned due to their extensive toxicity in preclinical models and patients (Garber, K. (2009). JNCI Journal of the National Cancer Institute, 101 (24), 1664–1667.). The small molecule LY2157299 (galunisertib) is a TGFβ pathway inhibitor that has been extensively tested in cancer patients. Ganicitinib is a relatively weak inhibitor of TGFβ receptor I (ALK5) (Herbertz S et al., Drug Design, Development and Therapy, 2015, 9:4479-4499) that has entered clinical trials, initially for brain cancer, and has been tested in a variety of other cancer types over the past 10 years (Herbertz S et al., Drug Design, Development and Therapy, 2015, 9:4479-4499). Given its associated toxicities in preclinical models, including oral cardiovascular, gastrointestinal, immune, bone / cartilage, reproductive, and renal toxicities (Stauber et al., J Clin Pract 2014, 4(3): 1-10), both the dose and frequency of administration of ganicitinib were limited to a maximum of 300 mg / day in humans on a 14-day on-14-day off regimen (Herbertz S et al., Drug Design, Development and Therapy, 2015, 9: 4479-4499). However, despite extensive testing in clinical trials, the therapeutic effects of ganicitinib reported to date in cancer patients using this dosage regimen are modest or nonexistent (Herbertz S et al.; 2015; Melisi et al., British Journal of Cancer, 2018, 119(10): 1208–1214,; Kelley RK et al., Clin Transl Gastroenterol. 2019 Jul; 10(7): e00056.; Brandes, A. et al., Neuro-Oncology, 18(8): 1146–1156.). Therefore, inhibitors of the TGFβ signaling pathway that exhibit lower toxicity profiles and enhanced activity at therapeutic doses represent an important clinical need.

[0009] More generally, many effective drugs are known to provide many adverse effects or are toxic to healthy tissues and cells. Other drugs can only be delivered locally to avoid the systemic effects of drugs. Prodrugs are compounds derived from active agents or drugs (parent compounds) that have been modified to regulate properties such as solubility, absorption, distribution, metabolism, excretion and toxicity. Therefore, prodrugs are such compounds that include parent compounds connected to fragments that reduce or block activity, while regulating one or more properties such as solubility, absorption, distribution, metabolism, excretion and toxicity. In this sense, Yoon et al. (Molecular Cancer Therapeutics, 2003, 2 (11): 1171-1181) reported the use of ester-linked 4-piperidinyl piperidine groups as prodrugs of active metabolites 7-ethyl-10-hydroxycamptothecin. In addition, Reiter et al. (Green Chem., 2013, 15 (5): 1373-1381) reported the enzymatic cleavage of lignin β-4 aryl ether bonds using LigF (β-etherase) enzymes.

[0010] Therefore, one object of the present invention is to develop more effective inhibitors of the TGFβ signaling pathway that exhibit improved toxicity profiles and enhanced therapeutic effects.

[0011] Additionally, it is an object of the present invention to develop prodrugs that can be used to more effectively deliver drugs in target tissues or organs, thereby avoiding adverse or undesirable systemic effects: producing highly effective local doses without toxicity to the rest of the body. Summary of the invention

[0012] The present invention relates to a compound of formula (I), or a pharmaceutically acceptable salt or pharmaceutically acceptable solvate thereof:

[0013]

[0014] The present invention also relates to a pharmaceutical composition comprising an effective amount of a compound of formula I, or a pharmaceutically acceptable salt or pharmaceutically acceptable solvate thereof, and / or at least one pharmaceutically acceptable excipient or carrier, and optionally, at least one other active ingredient.

[0015] In addition, the present invention relates to a compound of formula I or a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof, or a pharmaceutical composition comprising the compound of formula I or a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof, for use as a medicament, in particular for treating diseases responsive to TGFβ pathway inhibitors.

[0016] The present invention also relates to a prodrug of a compound of formula I, wherein the prodrug is a prodrug of formula (II) or a pharmaceutically acceptable salt or pharmaceutically acceptable solvate thereof:

[0017]

[0018] in

[0019] - R1 is selected from the group consisting of H, alkyl, cycloalkyl, haloalkyl, hydroxyalkyl, aminoalkyl, alkanoylamino, alkoxy, aryl, alkylaryl, arylalkyl, amino, N-alkylamino and N,N-dialkylamino; and

[0020] - R2 and R3 are each independently selected from the group consisting of H, alkyl and haloalkyl.

[0021] The present invention also relates to a prodrug of a compound of formula I, wherein the prodrug has formula (III), or a pharmaceutically acceptable salt or pharmaceutically acceptable solvate thereof:

[0022]

[0023] Furthermore, the present invention relates to a prodrug of a compound of formula I, wherein the prodrug has formula (IV), or a pharmaceutically acceptable salt or pharmaceutically acceptable solvate thereof:

[0024]

[0025] In addition, the present invention also relates to a pharmaceutical composition, which comprises an effective amount of a prodrug of a compound of formula I, which is selected from a prodrug of formula II, a prodrug of formula III, or a prodrug of formula IV, or a pharmaceutically acceptable salt or pharmaceutically acceptable solvate thereof, and at least one pharmaceutically acceptable excipient or carrier.

[0026] The present invention also relates to a prodrug of a compound of formula I, which is selected from a prodrug of formula II, a prodrug of formula III, or a prodrug of formula IV, or a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof, or a pharmaceutical composition comprising the prodrug, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate, for use as a medicament, in particular for treating diseases responsive to TGFβ pathway inhibitors. In particular, for a disease responsive to inhibition of the TGFβ signaling pathway, the disease is selected from the group consisting of: cancer, scleroderma, psoriasis, anemia, sarcopenia, Alzheimer's disease, Marfan syndrome, aneurysm, pulmonary hypertension, osteogenesis imperfecta, idiopathic pulmonary fibrosis, liver fibrosis, cirrhosis, hepatic steatosis, hypertrophic cardiomyopathy, myelofibrosis, neurofibromatosis type I, fibrotic kidney disease, focal segmental glomerulosclerosis, The invention relates to the treatment of various diseases, including cataract, glomerulosclerosis, radiation-induced fibrosis, skin fibrosis in systemic sclerosis, diffuse systemic sclerosis, scarring, corneal primary pterygium, fibrosis, uterine leiomyoma, obesity, diabetes, microangiopathy in diabetic retinopathy and nephropathy, and more particularly for the treatment of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 : IC obtained in a cell-based assay using a CAGA-luciferase reporter gene 50 Value. Compounds of formula I Compound of formula III and a compound of formula IV Bioluminescence at different doses (y-axis). Calculated IC 50 Values ​​are shown in the legend.

[0028] Figure 2 : Photographs of liver metastasis sections stained for phosphorylated SMAD2 by immunohistochemistry: Arrows point to stained nuclei, indicating active TGFβ signaling in controls (vehicle), but not in mice treated with a compound of Formula I, a compound of Formula IV, or ganicitinib. Ganicitinib, a compound of Formula I, and a compound of Formula IV were administered at equimolar doses (5x) over three days.

[0029] Figure 3 : Mouse liver metastasis initiation assay, as a readout of TGFβ inhibition and related anticancer efficacy in mice with immune competence implanted in mouse tumor organoids (Mouse Tumor Organoid, MTO). Relative to controls treated with empty vehicle, after treatment with a compound of Formula I or ganisitinib at 0.3x, 1x, 3x, and 9x molar equivalents of a standard mouse dose of 80 mg / kg bid ganisitinib (which is converted to 2 mg / mouse, assuming an average weight of 25 g), the number of liver metastases (LiM) was measured (y axis). The dose of 0.3x ganisitinib (0.6 mg / mouse bid) (nt) was not tested.

[0030] Figure 4 : Mouse liver metastasis initiation assay as a readout for TGFβ inhibition and associated anticancer efficacy. Normalized bioluminescent signals quantified by in vivo imaging using luciferase-expressing MTOs implanted in immunocompetent mice showed tumor growth and rejection between days 3 and 14 following treatment with different doses (0.3x, 1x, and 3x molar equivalents of the standard mouse ganicitinib dose of 80 mg / kg bid) of the compound of Formula I and ganicitinib.

[0031] Figure 5 : In vitro cytotoxicity assay of compounds of formula I (relative to ganicitinib) in HEK293T cells. DMSO was used as a control.

[0032] Figure 6: In vitro fluorescence assay of the enzymatic cleavage of the β-ketoether moiety of 4-methylumbelliferyl vanillyl ethyl ketone (a model compound for prodrugs of formula II and in particular prodrugs of formula III) using purified Lig F. The graph shows fluorescence versus time.

[0033] Figure 7 : In vitro fluorescence assay using HEK293T cells stably expressing LigF for cellular enzymatic cleavage of the β-ketoether moiety of 4-methylumbelliferyl vanillone (MUAV), a model compound for prodrugs of Formula II and in particular prodrugs of Formula III. The figure shows the fluorescence of MUAV exposed to different concentrations of LigF relative to time. Three concentrations of MUAV substrate are indicated: 10 μM (solid squares), 30 μM (triangles), and 100 μM (solid circles). Controls using 100 μM MUAV but without LigF are shown with open circles.

[0034] Figure 8 : HPLC-MS experiments of purified LigF cleaving the β-ether bond of the prodrug of formula VII to give 4-OHT. (A) Negative control. (B) Time = 0. (C) Time = 1 hour. (D) Time = 3 hours. (E) Time = 44 hours.

[0035] Fig. 9 : Quantification of the conversion of the prodrug of Formula VII (shown as isomers E and Z) to 4-OHT (both isomers Z and E are shown) measured by mass spectrometry; as relative abundance (y-axis) as a percentage of the total at different time points (x-axis).

[0036] Fig.10 : Using cells that are sensitive or insensitive to 4-OHT (carrying or not carrying the ubiquitous Cre-E RT2 Co-culture assay of mouse intestinal tumor organoids expressing β-etherase with MEFs containing 4-OHT protein and treated with a compound of Formula VII or a 4-OHT therapeutic agent. The fraction of recombinant cells measured by FACS analysis is shown.

[0037] Fig.11 : TCF4-ER in cells treated with 4-OHT, prodrug VII at 1 μM and 10 μM, or DMSO as a control T2 Normalized gene expression of fusion proteins.

[0038] Fig.12 : Immunohistochemical staining of EGFP in mouse liver sections (the mouse liver expresses Ub-Cre-ER T2 and mTmG reporter gene cassette), with negative control (oil, Fig.12 A) or 1 μmol ( Fig.12B) or 5 μmol ( Fig.12 C) is treated with a compound of formula VII.

[0039] Fig.13 : Assay for mouse liver metastasis formation as a readout of TGFβ signaling inhibition and related anticancer efficacy. MTO was implanted into C57BL / 6 mice and treated with the indicated compound two days after inoculation of tumor cells. The therapeutic efficacy of the indicated compound was evaluated by measuring its ability to inhibit liver metastasis formation. Mice were treated with a compound of Formula I, a compound of Formula IV, Compound 338 or ganicitinib at the following molar equivalent doses: 9x, 3x, 1x, 0.3x. Control mice were treated with an empty vehicle. The number of liver metastases (LiM) was measured after treatment (y axis). All mice were treated from day 2 to day 14 after inoculation of tumor cells, and metastases were counted 4 weeks after MTO inoculation. The results are pooled from three independent experiments, represented by the shape of the data points (square, triangle or circle). The P value relative to the pooled control is expressed as: *: <0.05; **: <0.001; ***: <0.0001. Their values ​​are: Gal 1x: 0.72; Gal 3x: 0.0107; Gal 9x: 0.0078; (IV) 1x: 0.075; (I) 0.3x: 0.026; (I) 1x: 0.0002; (I) 3x: 0.0004; (I) 9x: 0.035; 338 1x: 0.72; Mann-Whitney two-tailed test.

[0040] Fig.14 : The therapeutic effect of the compound in mice with obvious metastatic disease. MTO was implanted into mice with immune competence, and the mice were treated on the 16th day after inoculation when metastatic disease was obvious. Mice were treated with ALK5 inhibitors alone or in combination with antibodies against the checkpoint molecule PD-1. The therapeutic efficacy of the indicated compound was evaluated by measuring its ability to reduce the number of established liver metastases. The indicated compound was given at an equivalent 1x molar dose. For antibody administration, mice were treated by intraperitoneal injection of 200 μg every 3 days. Control mice were treated with gavage vehicle and 200 μg IgG2a isotype control antibody. From the 16th day to the 24th day, mice were treated. The number of liver metastases (LiM, y-axis) was measured at the endpoint. The results are pooled from two independent experiments, represented by the shape of the data points (square or circle). The P value relative to the pooled control is expressed as: *: <0.05; **: <0.001; ***: <0.0001. Their values ​​are: Gal 1x: 0.58; (I) 1x: 0.048; aPD-1: 0.0068; Gal+aPD-1: 0.18; (I)+aPD1: 0.0001; Mann-Whitney two-tailed test.

[0041] Fig.15 : Therapeutic effect of compounds in mice with overt metastatic disease. MTOs were implanted into immunocompetent mice and mice were treated on day 16 post-inoculation when hepatic metastatic disease was overt. Therapeutic efficacy was assessed by measuring the percentage of mice surviving after treatment with the indicated compound, which was given at an equivalent 1x molar dose. Control mice were treated with gavage vehicle. All mice were treated from day 16 to day 24. Data from Fig.14 Same experiment as shown. P values ​​relative to pooled controls are: (I): 0.0018; Gal: 0.67; Log-rank test.

[0042] Fig.16 : MTO was implanted into immunocompetent mice and mice were treated on day 16 post-inoculation when metastatic disease was evident. The therapeutic efficacy of the indicated compound was assessed by measuring the survival percentage after treatment with an anti-PD-1 antibody at 1x equimolar dose (200 μg every 3 days) or with a combination immunotherapy with an anti-PD-1 antibody plus a compound of Formula I or with nicitinib. Control mice were treated with gavage vehicle and 200 μg of IgG2a isotype control antibody. Mice were treated from day 16 to day 24. Data from Fig.14 Same experiment as shown. P values ​​relative to pooled controls are: (I) + aPD-1: <0.0001; aPD-1: 0.0048; Gal + aPD1: 0.43; log-rank test.

[0043] Fig.17 :MTO was implanted into C57BL / 6 mice, and treatment with the indicated compound was started two days after inoculation of tumor cells. The therapeutic efficacy of the indicated compound was evaluated by measuring its ability to inhibit the formation of liver metastases. Mice were treated with a compound of Formula I, compound 338 or compound 337 at 1x. Control mice were treated with an empty vehicle. The number of liver metastases (LiM) was measured after treatment (y axis). All mice were treated from the 2nd day to the 18th day after inoculation of tumor cells. The number of liver metastases was counted 21 days after MTO inoculation. Ns is non-significant, and * indicates <0.05 by the Mann-Whitney two-tailed test.

[0044] Description of the invention

[0045] The present invention relates to a compound of formula (I), or a pharmaceutically acceptable salt, or a pharmaceutically acceptable solvate, polymorph or co-crystal thereof:

[0046]

[0047] In particular, the present invention relates to a compound of formula I, or a pharmaceutically acceptable salt or pharmaceutically acceptable solvate thereof.

[0048] The present invention also relates to a pharmaceutical composition comprising an effective amount of a compound of formula I, or a pharmaceutically acceptable salt, or a pharmaceutically acceptable solvate, or a polymorph, or a co-crystal thereof, and at least one pharmaceutically acceptable excipient.

[0049] The present invention relates to a compound of formula I, or a pharmaceutical composition comprising the compound, which is used as a medicament, in particular for treating a disease that responds to inhibition of the TGFβ pathway or inhibition of TGFβ signaling. For the purposes of the present invention, the term "inhibitor of the TGFβ pathway" is equivalent to the term "inhibitor of the TGFβ signaling pathway" and the term "TGFβ inhibitor", and these three terms are used indiscriminately throughout the specification. For this reason, for the purposes of the present invention, the term "disease that responds to inhibition of the TGFβ pathway" is therefore equivalent to the term "disease that responds to inhibition of the TGFβ signaling pathway".

[0050] The compound of formula I is an inhibitor of the TGFβ pathway. The inhibitor of the TGFβ signaling pathway can inhibit one or more TGFβ signaling pathway receptors selected from the group consisting of: ALK1, ALK2, ALK3, ALK4, ALK5, ALK6, ALK7, TGFBR2, BMPR2, ACVR2A and ACVR2B. Therefore, the present invention also relates to a compound of formula I for use in treating a disease responsive to inhibition of one or more TGFβ signaling pathway receptors, wherein the TGFβ signaling pathway receptor is a TGFβ signaling pathway type I receptor or a TGFβ signaling pathway type II receptor, and is selected from the group consisting of: ALK1, ALK2, ALK3, ALK4, ALK5, ALK6, ALK7, TGFBR2, BMPR2, ACVR2A and ACVR2B. Therefore, the present invention relates to a compound of formula I, which is used as an inhibitor of one or more TGFβ signaling pathway receptors selected from the group consisting of: ALK1, ALK2, ALK3, ALK4, ALK5, ALK6, ALK7, TGFBR2, BMPR2, ACVR2A and ACVR2B.

[0051] The results contained in Table 1 of Example 2 show that the compound of Formula I has a surprisingly higher (8.6-fold) binding affinity to ALK5 compared to ganicitinib. This comparison has been taken into account using the same technology (KINOMEscan TM) based on the public data (Jonathan M et al., Oncotarget, 2018, 9(6): 6659-6677). In addition, the compound of Formula I has about 2.5 times the ability to inhibit ALK5 kinase enzymatic activity and TGFβ signaling in cultured cells as ganicitinib, as shown in Table 2 and Figure 1 In addition, selective determination (KINOMEscan TM Technology) shows that the compound of formula I is characterized by a selectivity of more than three hundred times for ALK5 relative to ALK1, ALK2 and ALK3, 100 times for TGFBR2, and 5 times and 8 times for ACVR2B and ALK6, respectively, as described in Example 2 (Table 1). In addition, compared with ganicitinib, the compound of formula I has greater selectivity for all tested proteins.

[0052] Thus, the structural differences of the compounds of formula I from the prior art compounds surprisingly lead to strongly improved TGFβ signaling inhibition capacity, which is associated with better ALK5 inhibition. The compounds of formula I are also more selective than the prior art compounds in terms of binding affinity for ALK5. On the other hand, the compounds of formula I have passed toxicity screening, including screening for genotoxicity, protein perturbations strongly associated with adverse drug reactions (SafetyScreen 44). TM , as described in Example 2) and in vivo (mouse) toxicity (Example 4), thereby demonstrating that compounds of Formula I provide an improved toxicity profile compared to ganicitinib, potentially obviating the need for intermittent treatment.

[0053] Compounds of formula I can be synthesized from para-aniline with a masked or protected phenol in the para position. In this sense, the synthesis of compounds of formula I can use different routes, as can be seen in the retrosynthetic analysis of Scheme 1:

[0054] Scheme I

[0055]

[0056] Example 1 includes all synthesis details corresponding to the preparation of compounds of formula I according to preferred synthesis routes (a) and (d), but synthesis routes (b) and (c) are also suitable routes to obtain compounds of formula I according to the invention.

[0057] Option II: Route (a)

[0058]

[0059] Route (a) comprises the synthesis of compounds of formula I starting from protected p-hydroxyaniline, wherein the protected phenol is deprotected only at the end. In a first step, 6-methoxy-4-methylquinoline is obtained via a Doebner-Miller reaction in the presence of a Lewis acid catalyst. The Doebner-Miller reaction is well known in the art ( FW Chem. Rev. 1944,35,153).

[0060] In the second step, acylation of 6-methoxy-4-methylquinoline is carried out by adding methyl 6-methylpicolinate in the presence of a strong base (pKa of the conjugate acid is higher than 17) and appropriate conditions.

[0061] Finally, the conversion of the ketone intermediate to the methyl-protected compound of Formula I is carried out by first using 1-aminopyrrolidin-2-one and a base (such as dimethylpyridine) under Dean-Stark conditions to obtain the hydrazone intermediate, which is then cyclized using appropriate conditions to obtain the methyl-protected compound of Formula I after dehydration under Dean-Stark conditions.

[0062] The methyl group can be removed by refluxing HBr in AcOH, but other common deprotection conditions such as BBr 3 , BCl 3 , TMSCl / NaI or BF 3 / RSH can also be used.

[0063] Option III: Route (b)

[0064]

[0065] Route (b) comprises a first step identical to that of route (a), wherein a modified Dobner-Müller reaction of p-nitroaniline is carried out. The second step also comprises acylation of 6-nitro-4-methylquinoline by addition of methyl 6-picolinate in the presence of a base. Subsequent steps to complete the formation of the hydrazone and cyclization are carried out in an identical manner to route (a). Reduction of the nitro derivative is carried out by any known method (H2 / Pd / C; Sn / HCl, etc.), and the amino group is converted to phenol by hydrolysis of the diazonium salt.

[0066] Option IV: Route (c)

[0067]

[0068] Route (c) includes a first step equivalent to route (a), wherein p-fluoroaniline and an oxidant (such as chloranil) are used to perform a modified Dobner-Miller reaction. The second step also includes the addition of 6-methylpicolinate in the presence of a strong base to perform the acylation of 6-fluoro-4-methylquinoline. The subsequent steps of completing the formation of the hydrazone and the cyclization are performed in an equivalent manner to route (a). The conversion of the fluoro derivative to phenol can be performed in a variety of ways, including transition metal catalysis, or using acetoxy hydroxamic acid, as described by Fier and Maloney (Org. Lett. 2016, 18, 2244).

[0069] Option V: Route (d)

[0070]

[0071] Option V (continued): Route (d)

[0072]

[0073] Route (d) includes a first step equivalent to route (a), wherein air is used as an oxidant to carry out a modified Dobner-Müller reaction of p-bromoaniline. The second step also includes acylation of 6-bromo-4-methylquinoline by adding 6-methylpicolinate in the presence of a strong base. The subsequent steps of completing the formation of hydrazone and cyclization are carried out in a manner equivalent to route (a). Subsequently, the compound of formula I can be obtained using Miyaura borylation (T.Ishiyama, M.Murata, N.Miyaura, J.Org.Chem.1995,60:7508–7510), wherein the aryl bromide is converted into the corresponding boric acid ester via a Pd-catalyzed cross-coupling reaction with a diboronyl ester such as bis(pinacol)diboron (B2Pin2). The coupling is carried out via a Pd(0) / Pd(II) mechanism, so any Pd(0) source can carry out the reaction. In a preferred embodiment, [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (Pd(DPPF)Cl2) is a suitable reagent. Without any further purification, the boric acid ester can be oxidized with hydrogen peroxide and, after subsequent hydrolysis in alkaline medium, the compound of formula I is obtained.

[0074] The present invention also relates to prodrugs. Prodrugs are compounds derived from active agents or drugs (parent compounds) that have been modified to regulate properties such as solubility, absorption, distribution, metabolism, excretion and toxicity. Therefore, prodrugs are compounds that include a parent compound connected to a fragment that reduces or blocks activity while regulating one or more properties such as solubility, absorption, distribution, metabolism, excretion and toxicity.

[0075] In particular, the present invention relates to prodrugs of compounds of formula I.

[0076] One embodiment of the present invention relates to a prodrug of a compound of formula I, wherein the prodrug has formula II, or a pharmaceutically acceptable salt, or a pharmaceutically acceptable solvate, or a polymorph, or a cocrystal thereof:

[0077]

[0078] in

[0079] - R1 is selected from the group consisting of H, alkyl, cycloalkyl, haloalkyl, hydroxyalkyl, aminoalkyl, alkanoylamino, alkoxy, aryl, alkylaryl, arylalkyl, amino, N-alkylamino and N,N-dialkylamino; and

[0080] - R2 and R3 are each independently selected from the group consisting of H, alkyl and haloalkyl.

[0081] One embodiment relates to a prodrug of a compound of formula I, wherein the prodrug has formula II, or a pharmaceutically acceptable salt or pharmaceutically acceptable solvate thereof, wherein R1 is selected from the group consisting of H, alkyl, cycloalkyl, haloalkyl, hydroxyalkyl, aminoalkyl, alkanoylamino, alkoxy, aryl, alkylaryl, arylalkyl, amino, N-alkylamino and N,N-dialkylamino; R2 is methyl and R3 is H. In another embodiment, R1 is selected from the group consisting of H, alkyl, haloalkyl, aminoalkyl and hydroxyalkyl; R2 is methyl and R3 is H.

[0082] In a preferred embodiment of the invention, R1 is H, R2 is methyl and R3 is H, and the prodrug of the compound of formula I is a prodrug of formula (III), or a pharmaceutically acceptable salt, or a pharmaceutically acceptable solvate, or a polymorph, or a cocrystal thereof:

[0083]

[0084] Preferably, the prodrug of the compound of formula I is a prodrug of formula (III), or a pharmaceutically acceptable salt or pharmaceutically acceptable solvate thereof. As illustrated in Example 1, the prodrug of formula III is obtained by alkylating the phenolic group of the compound of formula I with a vanilla acetone halogenated derivative having a protected vanilla acetone phenolic group, followed by deprotection of the vanilla acetone moiety.

[0085] In another preferred embodiment, the prodrug of the compound of formula I is a prodrug of formula (IV), or a pharmaceutically acceptable salt, or pharmaceutically acceptable solvate, or polymorph, or cocrystal thereof:

[0086]

[0087] Preferably, the prodrug of the compound of formula I is a prodrug of formula (IV), or a pharmaceutically acceptable salt or pharmaceutically acceptable solvate thereof. As illustrated in Example 1, the prodrug of formula IV is obtained by reacting the compound of formula I with 4-piperidinylpiperidine-1-carbonyl chloride.

[0088] The term "comprising" should be understood as including a set of features (A, B, C), but it does not exclude embodiments in which other features are also included, as long as they do not make the embodiment infeasible. In addition, for the purposes of the present invention, the term "comprising" also covers embodiments in which only the features (A, B, C) are included, in which case it can be replaced by the term "consisting of..."

[0089] The term "pharmaceutically acceptable salt" refers to any pharmaceutically acceptable salt which, upon administration to a patient, is capable of providing (directly or indirectly) a compound as described herein.

[0090] Such salt is preferably an acid addition salt or a base addition salt. The example of acid addition salt includes inorganic acid addition salt, such as, for example, hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, phosphate, and organic acid addition salt, such as, for example, acetate, trifluoroacetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, mesylate and p-toluenesulfonate. The example of base addition salt includes inorganic salt, such as, for example, sodium salt, potassium salt, calcium salt and ammonium salt, and organic alkali salt, such as, for example, ethylenediamine salt, ethanolamine salt, N, N-dialkylenethanolamine salt (N, N-dialkylenethanolamine), triethanolamine salt and basic amino acid salt. However, it should be understood that non-pharmaceutical salts also fall within the scope of the present invention, because they can be used to prepare pharmaceutical salts. The procedure for salt formation is conventional in the art.

[0091] In one embodiment of the invention, the pharmaceutically acceptable salt of the compound of formula I is a base addition salt or an acid addition salt. In a preferred embodiment, the pharmaceutically acceptable salt of the compound of formula I is selected from the group consisting of sodium salts, potassium salts, calcium salts, ammonium salts, alkylammonium salts, dialkylammonium salts, trialkylammonium salts, ethanolamine salts, N,N-dialkyleneethanolamine salts and amino acid salts.

[0092] In another preferred embodiment, the pharmaceutically acceptable salt of the compound of formula I is selected from the group consisting of hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, nitrate, phosphate, hydrogen phosphate, acetate, trifluoroacetate, maleate, fumarate, citrate, lactate, oxalate, succinate, gluconate, tartrate, maleate, mandelate, methanesulfonate and p-toluenesulfonate.

[0093] In one embodiment of the invention, the pharmaceutically acceptable salts of the prodrugs of formula II and in particular the prodrugs of formula III are acid addition salts. In a preferred embodiment, the pharmaceutically acceptable salts of the compounds of formula II and in particular the prodrugs of formula III are selected from the group consisting of hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, nitrate, phosphate, hydrogen phosphate, acetate, trifluoroacetate, maleate, fumarate, citrate, lactate, oxalate, succinate, gluconate, tartrate, maleate, mandelate, mesylate and p-toluenesulfonate.

[0094] In one embodiment of the invention, the pharmaceutically acceptable salt of the compound of formula IV is a base addition salt or an acid addition salt. In a preferred embodiment, the pharmaceutically acceptable salt of the compound of formula IV is selected from the group consisting of hydrochloride, hydrobromide, hydroiodide, sulfate, phosphate, acetate, trifluoroacetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, mesylate and p-toluenesulfonate. Most preferably, the pharmaceutically acceptable salt of the compound of formula IV is hydrochloride.

[0095] In another preferred embodiment, the pharmaceutically acceptable salt of the compound of formula IV is selected from the group consisting of sodium salt, potassium salt, calcium salt, ammonium salt, alkylammonium salt, dialkylammonium salt, trialkylammonium salt, ethanolamine salt, N,N-dialkyleneethanolamine salt and amino acid salt.

[0096] The term "solvate" according to the present invention is understood to mean any form of the active compound according to the present invention, wherein the compound is bound to another molecule (usually a polar solvent) by a non-covalent bond, which includes in particular hydrates and alcoholates. In one embodiment of the present invention, the pharmaceutically acceptable solvate is an alcoholate. In one embodiment of the present invention, the pharmaceutically acceptable solvate is a hydrate. In one embodiment of the present invention, the compound of formula I, the prodrug of formula II, the prodrug of formula III or the prodrug of formula IV is a monohydrate.

[0097] The term "polymorph" refers to a crystalline form having the same chemical composition but having different spatial arrangements of molecules, atoms and / or ions forming the crystal. Thus, the same compound of Formula I or a pharmaceutically acceptable salt or pharmaceutically acceptable solvate thereof may include different crystalline polymorphs, depending on the spatial arrangements of the atoms or ions forming the crystalline form.

[0098] The compounds of formula I are TGFβ signaling pathway inhibitors with improved inhibitory potency and low toxicity profile, as shown in Examples 2 to 4 and Examples 8 to 10.

[0099] Therefore, one embodiment relates to a compound of formula I, or a pharmaceutically acceptable salt, or a pharmaceutically acceptable solvate, or a polymorph, or a cocrystal thereof, for use in treating a disease responsive to inhibition of the TGFβ signaling pathway. Preferably, it relates to a compound of formula I, or a pharmaceutically acceptable salt, or a pharmaceutically acceptable solvate thereof, for use in treating a disease responsive to inhibition of the TGFβ pathway.

[0100] After cleavage of the fragment linked to the agent or drug, the prodrug is converted to the parent compound. This can be done by a variety of enzymes.

[0101] Upon metabolism, the prodrugs of the compounds of formula I release the compounds of formula I and are thus locally administered to the tumor, thereby preventing in this way systemic pathway inhibition, which would have adverse effects if continuous inhibition of the TGFβ signaling pathway were performed. Therefore, the prodrugs of the compounds of formula I can also be used to prevent and / or treat diseases that respond to inhibition of the TGFβ pathway.

[0102] As in Figure 1 As seen in Table 2, the prodrugs of the compound of Formula I, i.e., the prodrugs of Formula II, Formula III, and Formula IV, have reduced TGFβ signaling inhibition ability in vitro, but the prodrugs of the compound of Formula I, i.e., the prodrugs of Formula II, Formula III, and Formula IV, delivered in vivo are prodrugs that are metabolized in vivo to release the active compound of Formula I.

[0103] Indeed, the 4-piperidinylpiperidine group of the prodrug of Formula IV provides water solubility and is cleaved via hydrolysis by in vivo carboxylesterases expressed, for example, in hepatocytes and intestinal cells.

[0104] Thus, when the prodrug of formula IV is administered, the compound of formula I is released upon hydrolysis by carboxylesterases in hepatocytes and intestinal cells. The major advantage is that it allows the administration of high local doses as well as TGFβ signaling inhibition with reduced systemic toxicity.

[0105] On the other hand, the vanillone-derived fragments of the prodrugs of formula II and especially the prodrugs of formula III can be oxidized and cleaved by enzymes such as the cytochrome P450 family of enzymes (primarily found in the liver), which hydrolyze the ester bond, thereby delivering the compound of formula I. On the other hand, the compound of formula I can also be obtained from its prodrug of formula II using specific β-etherases from non-mammalian sources, such as the bacterial Sphingobium sp. strain SYK-6 enzymes LigE and LigF, or the fungal Dichomitus squalens Ds-GST1, which can be administered in combination with the prodrugs of formula II, and preferably in combination with the prodrugs of formula III.

[0106] Thus, one embodiment relates to a prodrug of formula II for use in treating a disease responsive to inhibition of the TGFβ pathway, wherein a β-etherase is administered with, after or before the compound of formula II. A preferred embodiment relates to a prodrug of formula III for use in treating a disease responsive to inhibition of the TGFβ pathway, wherein a β-etherase is administered with or after the compound of formula III. Example 6 shows how LigF, a β-etherase, can hydrolyze the ether bond, thereby cleaving the vanilla ethyl ketone fragment, in a model experiment performed with the vanilla ethyl ketone ether of 4-methylumbelliferone and with the vanilla ethyl ketone ether of 4-hydroxytamoxifen.

[0107] In a particular embodiment, the β-etherase is administered in cells expressing the β-etherase as a cell therapy, such as adoptive T cell transfer or dendritic cell vaccination. Alternatively, in another particular embodiment, the β-etherase is administered coupled to a tumor or matrix specific antibody, optionally with glutathione. Certain tissues such as the lung lining contain high extracellular levels of glutathione, and therefore do not require administration of glutathione.

[0108] The prodrugs of Formula II, Formula III and Formula IV, or pharmaceutically acceptable salts or solvates thereof, have greatly reduced activity as TGFβ signaling pathway inhibitors, allowing them to be administered with little or no effect on the TGFβ pathway unless the β-ether bond is cleaved.

[0109] When the prodrug of Formula II or Formula III or a pharmaceutically acceptable salt or solvate thereof is administered, the compound of Formula I is released in the target tumor stroma following local administration of β-etherase. The major advantage is that it allows for administration of high local doses with reduced systemic toxicity as well as TGFβ signaling pathway inhibition.

[0110] On the other hand, the prodrug of the compound of Formula I (having Formula IV), or a pharmaceutically acceptable salt thereof, also provides reduced TGFβ signaling inhibitory activity in vitro, and when administered in vivo, is hydrolyzed in vivo by the enzymatic action of carboxyl esterases expressed in various body tissue cells such as hepatocytes and intestinal cells and produces the compound of Formula I.

[0111] It was then found that in in vivo experiments, the compound of formula I was effective at much lower doses (in fact, as low as one-tenth) compared to the effective dose of ganicitinib. In addition, at effective doses, the compound of formula I was not toxic (either grossly in vivo or pathologically on tissue sections), while ganicitinib at mouse-related doses was indeed toxic (see Examples 2 to 4). As in Figure 1As seen in Table 2, the prodrugs of the compound of Formula I, such as the prodrugs of Formula III and the prodrugs of Formula IV, have up to one-tenth lower TGFβ signaling inhibitory ability in vitro, indicating that the TGFβ signaling inhibitory ability of the compound of Formula I is successfully trapped or blocked.

[0112] Figure 2 The high TGFβ signaling inhibitory activity of the compound of Formula I is shown, while the prodrug of Formula IV, which does not show significant TGFβ signaling inhibitory activity in vitro, provides high TGFβ signaling pathway inhibitory activity in vivo, because the prodrug is hydrolyzed in vivo to deliver the active compound of Formula I.

[0113] Thus, one embodiment of the present invention relates to a prodrug of a compound of formula I, wherein the prodrug is a prodrug of formula II, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate or polymorph or cocrystal thereof, for use as a medicament, in particular for the treatment of diseases responsive to TGFβ signaling. Preferably, the prodrug is a prodrug of formula III. A preferred embodiment relates to a compound of formula III, or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate thereof, for use as a medicament, in particular for the treatment of diseases responsive to inhibition of the TGFβ signaling pathway.

[0114] Another embodiment relates to the use of a compound of formula I, or a pharmaceutically acceptable salt, or pharmaceutically acceptable solvate, or polymorph or cocrystal thereof, or a composition comprising said compound of formula I, in the preparation of a medicament for treating a disease responsive to inhibition of the TGFβ signaling pathway.

[0115] A further embodiment is directed to a pharmaceutical composition comprising a compound of formula I, or a pharmaceutically acceptable salt, or pharmaceutically acceptable solvate, or polymorph or cocrystal thereof, and at least one pharmaceutically acceptable excipient, and optionally one additional active ingredient or therapeutic agent.

[0116] For purposes of the present invention, the term "active ingredient" or "therapeutic agent" refers to another compound or therapy that improves or enhances the therapeutic effect of a compound of the present invention or a prodrug thereof.

[0117] Another embodiment of the present invention relates to a prodrug of a compound of formula I, wherein the prodrug is a prodrug of formula IV, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate or a polymorph or a cocrystal thereof, for use as a medicament, in particular for the treatment of diseases responsive to inhibition of the TGFβ signaling pathway. Preferably, the compound of formula IV, or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate thereof, is for use as a medicament, in particular for the treatment of diseases responsive to inhibition of the TGFβ signaling pathway.

[0118] An alternative embodiment of the present invention relates to the use of the compound of formula I or its prodrug (i.e., the prodrug of formula II, formula III and formula IV) or its pharmaceutically acceptable salt or solvate as a medicament, alone or formulated as a composition (especially a pharmaceutical composition), wherein the composition comprises at least the compound of formula I or its prodrug (i.e., the prodrug of formula II, formula III and formula IV) in combination with at least one other active compound or therapeutic agent having additive or synergistic biological activity.

[0119] In this sense, one embodiment of the present invention relates to a compound of formula I or a prodrug thereof (i.e., a prodrug of formula II, formula III and formula IV) or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising at least one of said compounds, for use in combination with other cancer targeted therapies or with chemotherapy to prevent and / or treat diseases responsive to inhibition of the TGFβ signaling pathway.

[0120] For the purposes of the present invention, the term "chemotherapy" refers to any treatment that acts to stop the growth of cancer cells, either by killing the cells or by stopping them from dividing.

[0121] Therefore, one embodiment of the present invention relates to a compound of Formula I or its prodrug (i.e., prodrug of Formula II, Formula III and Formula IV) or its pharmaceutically acceptable salt or solvate, or a pharmaceutical composition comprising at least one of the compounds, for use with chemotherapy or in combination with chemotherapeutic agents. In particular, in combination with a chemotherapeutic agent selected from the group consisting of the following: a platinum-based antitumor agent, an anti-mitotic chemotherapeutic agent, a poly (ADP-ribose) polymerase (PARP) inhibitor, a type I topoisomerase inhibitor, a type II topoisomerase inhibitor, an epothilone, a cycloskeletal disruptor, an alkylating agent, an epothilone, a histone deacetylase inhibitor, a kinase inhibitor, an antifolate, a kinase inhibitor, a peptide antibiotic, a retinoid, a vinca alkaloid, and a thymidylate synthase inhibitor. Preferably, the chemotherapeutic agent is selected from the group consisting of cyclophosphamide, ifosfamide, busulfan, temozolomide, mechlorethamine, chlorambucil, melphalan, dacarbazine, daunorubicin, doxorubicin, daunorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, paclitaxel, docetaxel, abraxane, taxotere, re), epothilone, vorinostat, romidepsin, irinotecan, topotecan, camptothecin, exatecan, lurtotecan, etoposide, teniposide, tafluposide, bortezomib, erlotinib, gefitinib, imatinib, vemurafenib, vismodegib, azacitadine, azathioprine, capecitabine,cytarabine, cladribine, fludarabine, doxifluridine, fluorouracil, gemcitabine, hydroxyurea, mercaptopurine, methotrexate, pemetrexed, tioguanine, bleomycin, actinomycin, carboplatin, cisplatin, oxaliplatin, tretinoin, alitretinoin, bexarotene, vinblastine, vincristine, vindesine, and vinorelbine. ,

[0122] For the purposes of the present invention, the term "cancer targeted therapy" refers to any type of therapy that targets specific genes, proteins, or tissue environments that contribute to cancer growth and survival. Therefore, one embodiment of the present invention relates to a compound of formula I or its prodrug (i.e., prodrugs of formula II, formula III, and formula IV) or a pharmaceutically acceptable salt or solvate thereof, or a composition comprising at least one of the compounds, which is used to prevent and / or treat diseases in response to TGFβ signaling pathway inhibition in combination with cancer targeted therapy, preferably, the cancer targeted therapy is selected from the group consisting of: hormone therapy, signal transduction inhibitors, gene expression regulators, apoptosis inducers, angiogenesis inhibitors, immunotherapeutics, monoclonal antibodies for delivering toxic molecules, cancer therapy, and gene therapy.

[0123] Therefore, one embodiment of the present invention relates to a compound of formula I or a prodrug thereof (i.e., a prodrug of formula II, formula III and formula IV) or a pharmaceutically acceptable salt or solvate thereof, or a composition comprising at least one of said compounds, for use in combination with immunotherapy to prevent and / or treat diseases responsive to inhibition of the TGFβ signaling pathway.

[0124] For the purposes of the present invention, the term "immunotherapy" refers to an immunotherapeutic agent, compound or composition that activates the immune system to kill cancer cells. More preferably, the immunotherapy or immunotherapeutic agent is an immune checkpoint inhibitor and even more preferably an anti-PD-1 or PDL1 inhibitor.

[0125] In this sense, another embodiment of the present invention relates to a compound of formula I or a prodrug thereof (i.e., a prodrug of formula II, formula III and formula IV) or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising at least one of the compounds, for use in combination with an immunotherapeutic agent (preferably, an immune checkpoint inhibitor; more preferably, an anti-PD1 or PDL1 inhibitor; still more preferably, an anti-PD1 antibody) to prevent and / or treat a disease that responds to inhibition of the TGFβ signaling pathway.

[0126] Programmed cell death protein 1 (PD-1) is a transmembrane protein expressed on T cells that acts as an immune checkpoint that negatively regulates T cell activation and causes downregulation of the immune system. Upon administration, anti-PD-1 monoclonal antibodies bind to and inhibit PD-1 and its downstream signaling pathways, thereby restoring immune function by activating T cells and cell-mediated immune responses against tumor cells.

[0127] In a preferred embodiment, the at least one other active compound or therapeutic agent is selected from the group consisting of: oncolytic viruses that inhibit PD1, and antibodies against PD-1 or PDL1, bispecific monoclonal antibodies that inhibit PD-1 or PDL1, small molecules that inhibit PD-1 or PDL-1, proteins that antagonize PD-1, and vaccines targeting PD-1.

[0128] In a preferred embodiment, the at least one additional active compound or therapeutic agent is selected from the group consisting of nivolumab, pembrolizumab, cemiplimab, camrelizumab, sintilimab, toripalimab, tislelizumab, atezolizumab, avelumab, durvalumab, KN-035, CK-301, AUNP12, CA-170, BMS-986189, MDX-1105, MEDI0680, LY3300054, LY-3434172, APL-502, bintrafusp alfa, CS-1001, SHR-1316, BGBA-333, CX-072, GEN-1046, GS-4224, IO-103, KD-005, KLA-167, KN-046 , lazertinib, STIA-1014, WP-1066, ADG-104, BCD-135, BCD-135, FAZ-053, FPT-155, FS-118, H LX-20, IBI-318, INBRX-105, INCB-86550, JS-003, lodapolimab, LP-002, MCLA-145, MSB-2311, RG-6084, SHR-1701, SL-279252, STIA-1015, LYN-00102, ACE-05, AP-201, AP-505, AVA-004, AVA-02 1. BBI-801, BH-2996h, BH-29XX, CA-327, CDX-527, CTX-8371, DPDL-1E, DSP-105, DSP-106, Gensci-04 7, HS-636, IBI-322, IBI-323, IKT-201, IMM-2505, JBI-426, KD-033, KY-1043, ND-021, PH-790, PMC-1 22. PRS-344, SNA-02, STT-01, TJL-14B, TJL-1C4, TJL-1D5, TJL-1H3, TJL-1I7, Toca-521, VXM-10, Y-1 11. ABM-101, ABP-160, AVA-027, AVA-040, DB-002, DB-003, IMC-2101, IMC-2102, IMM-2510, TE-7212,TXB-4BC3, YBL-007, YBL-008, ALN-PDL, BH-2941, BMS-936559, CBA-0710, GXP-2, HTI-1316, IGEM-P, IMM-25, IMM-2502, IMM-2503, IMM-2504, IO-104, KD-036, KY-1003, KY-1055, MSB-002, PMC-305, STIA-100X, STIA-1010, STIA-1011, STIA-1012, STIB-010X, LY-3415244, AK-103, AK-104, AK-105, AK-1 06, dostarlimab, HLX-10, prolgolimab, spartalizumab, APL-501, balstilimab, BAT-1306, BI-754091, cetrelimab, GLS-010, MGA-012, pidilizumab, SCTI-10A, AMG-404, BCD-217, budigalimab, CC-90006, CS-1003, F-520, HAB-21, HX-009, IBI-31 8. JTX-4014, LY-3434172, LZM-009, MEDI-5752, MGD-013, MGD-019, ONO-4685, RO-7121661, sasanlimab, sulituzumab, Sym-021, XmAb-20717 , -8371, GNR-051, HEISCOIII-003, IBI-319, KEY-Vaxx, LD-01, LD-10, MD-402, mDX-400, OT-2, PC-101, PEGMP-7, PH-762, PRS-332, PT-001, RO-7247669, SG-001, TSR-075, AT-16201, DB-004, IKT-202, IMT-200, KF-082, LBL-006, SSI-361, YBL-006, JY-034, TRS-007, AMP-224, AUR-012, BGB-108, BH-2941,BLSM-101, CX-188, ENUM-244C8, ENUM-388D4, IMM-1802, MEDI-0680, SNA-01, STIA-1110 and Sym-016. ,

[0129] Alternatively, the composition can be formulated with at least one inert ingredient as a carrier or excipient (such as: a co-solvent, a surfactant, an oil, a wetting agent, a lubricant, a preservative, a stabilizer and an antioxidant). Any pharmacologically acceptable buffer can be used, such as TRIS or a phosphate buffer.

[0130] Another embodiment relates to a pharmaceutical composition comprising a prodrug of a compound of Formula I (the prodrug has Formula II or Formula III), or a pharmaceutically acceptable salt, or a pharmaceutically acceptable solvate, or a polymorph or cocrystal thereof, and at least one pharmaceutically acceptable excipient, and optionally one additional active ingredient. Yet another embodiment relates to a pharmaceutical composition comprising a prodrug of a compound of Formula I (the prodrug has Formula IV), or a pharmaceutically acceptable salt, or a pharmaceutically acceptable solvate, or a polymorph or cocrystal thereof, and at least one pharmaceutically acceptable excipient, and optionally one additional active ingredient.

[0131] Another embodiment relates to the use of a prodrug of a compound of formula I, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate, or a polymorph or cocrystal, or a composition comprising a prodrug of a compound of formula I in the preparation of a medicament for treating a disease responsive to inhibition of TGFβ signaling. A preferred embodiment relates to the use of a prodrug of formula II, more preferably a prodrug of formula III, or a prodrug of formula IV in the preparation of a medicament for treating a disease responsive to inhibition of TGFβ signaling.

[0132] Another embodiment of the present invention is directed to a method for treating a disease responsive to inhibition of TGFβ signaling, the method comprising administering to a subject in need thereof an effective amount of a compound of formula I, or a pharmaceutically acceptable salt, or a pharmaceutically acceptable solvate, or a polymorph or cocrystal thereof, or a composition comprising the compound of formula I.

[0133] Another embodiment of the present invention relates to a method for treating a disease responsive to inhibition of TGFβ signaling, the method comprising administering to a subject in need thereof an effective amount of a prodrug of a compound of formula I, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate, or a polymorph or cocrystal, or a composition comprising a prodrug of a compound of formula I. Preferably, the prodrug of the compound of formula I is a prodrug of formula II or formula III or formula IV. A preferred embodiment relates to a method for treating a disease responsive to inhibition of TGFβ signaling, the method comprising administering to a subject in need thereof an effective amount of a prodrug of formula II or formula III or formula IV, or a pharmaceutically acceptable solvate thereof, or a polymorph or cocrystal, or a composition comprising the prodrug.

[0134] In one embodiment of the invention, the disease responsive to inhibition of the TGFβ signaling pathway is selected from the group consisting of cancer, scleroderma, psoriasis, anemia, sarcopenia, Alzheimer's disease, Marfan syndrome, aneurysm, pulmonary hypertension, osteogenesis imperfecta, idiopathic pulmonary fibrosis, liver fibrosis, liver cirrhosis, liver steatosis, hypertrophic cardiomyopathy, myelofibrosis, neurofibromatosis type I, fibrosing nephropathy, focal segmental glomerulosclerosis, radiation-induced fibrosis, skin fibrosis in systemic sclerosis, diffuse systemic sclerosis, scarring, primary pterygium of the cornea, fibrosis, uterine leiomyoma, obesity, diabetes, diabetic retinopathy and microangiopathy in nephropathy. In a particular embodiment, scarring is pathological skin scarring, cutaneous scarring or corneal scarring.

[0135] In a particular embodiment, the disease responsive to inhibition of the TGF[beta] signaling pathway also includes arterial restenosis.

[0136] In cancer progression, TGFβ signaling has been implicated in cell proliferation, angiogenesis, epithelial to mesenchymal transition, immune infiltration and regulation, metastatic spread, and drug resistance. As mentioned previously, the overall effects of the TGFβ signaling pathway appear to strongly promote tumor growth, invasion, and metastasis.

[0137] In a preferred embodiment, the disease responsive to inhibition of the TGFβ signaling pathway is cancer. In a preferred embodiment, the cancer is a solid tumor. In another preferred embodiment, the cancer is not a solid cancer. In an additional preferred embodiment, the cancer is a pediatric cancer.

[0138] As in Figure 3As seen in Figure 3, the compound of Formula I provides improved ability to block liver metastasis formation compared to the reference compound ganicitinib, even at one-third of the equimolar dose of ganicitinib.

[0139] Fig.13 It is shown how the compound of formula I exhibits a huge improvement in therapeutic activity and blocks the formation of liver metastases to a much greater extent compared to ganicitinib and, unexpectedly, also compared to the reference compound 4-[2-(methyl-pyridin-2-yl)5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-quinolin-7-ol (reference compound 338) disclosed in the prior art:

[0140]

[0141] As you can see, Fig.13 The results shown in are highly unexpected because reference compound 338 is a structural isomer of the compound of formula I having a phenolic hydroxyl group at the 7-position of the quinoline moiety, whereas the compound of formula I is characterized in that the phenolic hydroxyl group is at the 6-position of the quinoline moiety.

[0142] In this sense, the compounds of Formula I have an inhibitory effect on the enzymatic activity of ALK5 (as IC 50 Measurements) also confirmed that the compounds of the present invention have unexpectedly high potency compared to prior art compounds 337, 338 and ganicitinib.

[0143] The structure of 4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinolin-7-ol (reference compound 337) differs from that of the compound of Formula I in that reference compound 337 does not include a methyl group at the 2-position of the pyridine moiety and is characterized in that the phenolic hydroxyl group is at the 7-position of the quinoline moiety rather than at the 6-position of the quinoline moiety as in the compound of Formula I.

[0144]

[0145] As can be seen in Table 2 appearing in Example 2, the results show that, unexpectedly, the compound of formula (I) is 2.1 times more potent than reference compound 338 and 1.8 times more potent than reference compound 337, recognizing that the compounds differ structurally only in the position of the phenolic hydroxyl group and the presence or absence of a methyl group in the pyridine moiety.

[0146] Additional in vivo assays compared the therapeutic effects of the compound of Formula I with compounds 337 and 338. Fig.17It was shown that the compound of formula I exhibited a great improvement in therapeutic activity and blocked liver metastasis formation to a much greater extent compared to compound 338, confirming that Fig.13 The results shown in . The experiment also confirmed that in these in vivo transfer assays, the compound of formula (I) showed enhanced therapeutic activity compared to compound 337. Therefore, the present invention shows that the type of substitution and its position in the parent core structure surprisingly provide the unexpectedly improved efficacy shown by the compound of formula (I). In addition, Li et al. (J. Med. Chem. 2008, 51 (7): 2302-2306) disclose that electron-deficient and relatively small groups can be tolerated at the 6-position, which teaches that in contrast to the introduction of electron-donating groups such as hydroxyl groups as features at the 6-position in the compound of formula (I), when compared to the prior art structural isomer 338 characterized by the hydroxyl group at the 7-position instead, the compound of formula (I) instead leads to surprisingly strong efficacy.

[0147] on the other hand, Fig.13 It is also shown that the prodrug of Formula IV has up to one-tenth the TGFβ signaling pathway inhibitory ability in vitro compared to the compound of Formula I, but provides similar therapeutic activity in an in vivo metastasis assay. Therefore, although the prodrug of Formula III and the prodrug of Formula IV have up to one-tenth the TGFβ signaling inhibitory ability in in vitro assays compared to the compound of Formula I, they exhibit similar therapeutic activity in vivo. This result supports that the TGFβ signaling inhibitory ability of the prodrugs of Formula III and Formula IV is trapped or blocked in vitro, but is released in vivo.

[0148] In another preferred embodiment, the cancer is a metastatic cancer. Preferably, the metastatic cancer is overt metastasis or overt metastatic disease.

[0149] In this sense, Fig.14 It is shown how the compound of formula I exerts a very substantially improved activity in an animal model of overt metastatic disease compared to the prior art compound ganotinib when administered at equimolar doses. In addition, Fig.15 It was confirmed that administration of the compound of formula I also resulted in a higher survival rate in the animal model of overt metastatic disease.

[0150] On the other hand, the compound of formula I can be administered in combination with other therapies. As mentioned previously, in one embodiment, the compound of formula I, or a prodrug thereof having formula II, formula III, or formula IV, or a pharmaceutically acceptable salt, or a pharmaceutically acceptable solvate, or a polymorph or cocrystal thereof, can be used with immunotherapy, for example, with immunotherapy directed to immune checkpoints.

[0151] Therefore, a preferred embodiment relates to a pharmaceutical composition comprising a compound of formula I, or a prodrug thereof having formula II, formula III, or formula IV, or a pharmaceutically acceptable salt, or a pharmaceutically acceptable solvate, or a polymorph or cocrystal thereof, and an immune checkpoint inhibitor, more preferably, an anti-PD1 or PDL1 antibody. To this end, Fig.14 and 16 It was shown how combination therapy with a compound of Formula I and an anti-PD-1 antibody provided a significant increase in survival and reduced the number of liver metastases in an animal model of overt metastatic disease.

[0152] In a preferred embodiment, the disease responsive to inhibition of the TGFβ signaling pathway is a cancer selected from the group consisting of: hematologic cancer; B-cell or T-cell leukemia, non-Hodgkin lymphoma, non-Hodgkin lymphoma B-cell or T-cell types, Burkitt lymphoma, Hodgkin lymphoma, leukemias, lymphoma B-cell or T-cell types, multiple myeloma, brain cancer, cancer of glial lineage of the central nervous system, system)(glioma), gliobastoma, sarcomas, fibrosarcoma, malignant fibroushistiocytoma, Ewing's sarcoma, osteosarcoma, malignant pleural mesothelioma, breast cancer, breast cancer resistant to anti-HER2 therapy, breast carcinoma, breast adenocarcinoma, metastatic breast cancer, gastric and gastroesophageal cancer, gastric carcinoma, gastricadenocarcinoma, colorectal cancer, colon carcinoma, colon adenocarcinoma adenocarcinoma, rectum cancer, colorectalcarcinoma,metastatic colon cancer, pancreatic cancer, pancreas carcinoma, pancreatic adenocarcinoma, renal cell carcinoma, clear cell renal cell carcinoma, liver cancer, liver metastatic cancer, metastatic disease, lung cancer, lung carcinoma, lung adenocarcinoma, non-small-cell lung cancer, small-cell lung cancer, ovarian cancer, ovarian carcinoma, ovarian adenocarcinoma, ovarian carcinoma, endometrial carcinoma, endometrial stromal sarcoma, carcinoma of the uterine cervix, thyroid carcinoma, metastasizing papillary thyroid carcinoma, follicular thyroid carcinoma, bladder carcinoma, urine bladder carcinoma, transitional cell carcinoma of the urinary bladder, prostate cancer, prostate carcinoma, neuroendocrine cancers, squamous cell carcinoma, osteosarcoma, rhabdomyosarcoma, embryonal cancers, neuroblastoma,Medulloblastoma, retinoblastoma, nephroblastoma, hepatoblastoma, melanoma and skin cancer.

[0153] In a preferred embodiment, the disease responsive to inhibition of the TGF[beta] signaling pathway is a metastatic disease.

[0154] Preferably, the blood cancer is leukemia, multiple myeloma or myelodysplastic syndrome.Preferably, the brain cancer is glioblastoma.

[0155] In a preferred embodiment, the solid tumor is selected from the group consisting of: breast cancer, gastric and gastroesophageal cancer, colorectal cancer, pancreatic cancer, liver cancer, liver metastases, metastatic disease, lung cancer, ovarian cancer, prostate cancer, neuroendocrine cancer. Preferably, the skin cancer is melanoma.

[0156] In a preferred embodiment, the disease responsive to inhibition of the TGFβ signaling pathway is a cancer selected from the group consisting of leukemia, multiple myeloma, myelodysplastic syndrome, glioblastoma, Ewing sarcoma, osteosarcoma, malignant pleural mesothelioma, breast cancer, gastric and gastroesophageal cancer, colorectal cancer, pancreatic cancer, liver cancer, liver metastases, metastatic disease, lung cancer, ovarian cancer, prostate cancer, neuroendocrine cancer and melanoma.

[0157] In addition, for the prodrug of formula II containing a fragment derived from vanilla acetone, the present invention also discloses other prodrugs derived from vanilla acetone. Therefore, the vanilla acetone derived fragment (AcV) of the prodrug of formula II:

[0158]

[0159] It can also be used to obtain prodrugs of other drugs than the compounds of formula I, wherein the drug must contain one hydroxyl group. In this sense, the drug containing one hydroxyl group having the structure R-OH can react with the compound AcV-X containing the vanilla acetone fragment to form the ether bond of the prodrug AcV-OR of formula V, as seen in Scheme VI:

[0160]

[0161] Solution VI

[0162] When the β-ketoether bond is hydrolyzed in the presence of β-etherase or by liver enzymes such as cytochrome P450, a drug R-OH containing one hydroxyl group is released. Examples of drugs containing one hydroxyl group with formula R-OH are, but are not limited to, 4-hydroxytamoxifen or 7-ethyl-10-hydroxy-camptothecin (SN-38, CAS No: 86639-52-3, an active metabolite of irinotecan).

[0163] Example 6 shows how LigF, a β-etherase, can hydrolyze the ether bond, thereby cleaving the vanilla ethyl ketone fragment, in model experiments with the vanilla ethyl ketone ether of 4-methylumbelliferone and with the vanilla ethyl ketone ether of 4-hydroxytamoxifen.

[0164] In this sense, the present invention also generally relates to a prodrug of formula V of a drug containing one hydroxyl group having the formula R-OH, wherein the prodrug comprises a vanillyl acetonide-derived fragment:

[0165]

[0166] in

[0167] -R-OH is a drug containing a hydroxyl group;

[0168] - R1 is selected from the group consisting of H, alkyl, cycloalkyl, haloalkyl, hydroxyalkyl, aminoalkyl, alkanoylamino, alkoxy, aryl, alkylaryl, arylalkyl, amino, N-alkylamino and N,N-dialkylamino; and

[0169] - R2 and R3 are each independently selected from the group consisting of H, alkyl and haloalkyl.

[0170] In one embodiment, R1 is selected from the group consisting of H, alkyl, cycloalkyl, haloalkyl, hydroxyalkyl, aminoalkyl, alkanoylamino, alkoxy, aryl, alkylaryl, arylalkyl, amino, N-alkylamino and N,N-dialkylamino; R2 is methyl and R3 is H. In another embodiment, R1 is selected from the group consisting of H, alkyl, haloalkyl, aminoalkyl and hydroxyalkyl; R2 is methyl and R3 is H.

[0171] One embodiment is directed to a pharmaceutical composition comprising an effective amount of a prodrug of Formula V, and at least one pharmaceutically acceptable excipient.

[0172] One embodiment of the present invention relates to a prodrug of 4-hydroxytamoxifen of formula VI:

[0173]

[0174] in

[0175] - R1 is selected from the group consisting of H, alkyl, cycloalkyl, haloalkyl, hydroxyalkyl, aminoalkyl, alkanoylamino, alkoxy, aryl, alkylaryl, arylalkyl, amino, N-alkylamino and N,N-dialkylamino; and

[0176] - R2 and R3 are each independently selected from the group consisting of H, alkyl and haloalkyl.

[0177] One embodiment relates to a prodrug of 4-hydroxytamoxifen of formula VI, wherein R1 is selected from the group consisting of H, alkyl, cycloalkyl, haloalkyl, hydroxyalkyl, aminoalkyl, alkanoylamino, alkoxy, aryl, alkylaryl, arylalkyl, amino, N-alkylamino and N,N-dialkylamino; R2 is methyl and R3 is H. In another embodiment, R1 is selected from the group consisting of H, alkyl, haloalkyl, aminoalkyl and hydroxyalkyl; R2 is methyl and R3 is H.

[0178] Although one isomer is depicted, both isomers Z and E and mixtures thereof are included in the prodrugs of Formula VI. In one embodiment, the prodrug of Formula VI is the Z-isomer. In another embodiment, the prodrug of Formula VI is the E-isomer. In another embodiment, the prodrug of Formula VI is a mixture of the Z-isomer and the E-isomer. In particular, the mixture includes a mixture of the two isomers in a ratio of 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 99:1, or any other ratio.

[0179] In a preferred embodiment, R1 is H, R2 is methyl and R3 is H, and the prodrug of 4-hydroxytamoxifen of formula VI is a prodrug of formula VII:

[0180]

[0181] Although one isomer is depicted, both isomers Z and E and mixtures thereof are included in the prodrugs of Formula VII. In one embodiment, the prodrug of Formula VII is the Z-isomer. In another embodiment, the prodrug of Formula VII is the E-isomer. In another embodiment, the prodrug of Formula VII is a mixture of the Z-isomer and the E-isomer. In particular, the mixture includes a mixture of the two isomers in a ratio of 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 99:1 or any other ratio.

[0182] One embodiment is directed to a pharmaceutical composition comprising an effective amount of a prodrug of Formula VI or Formula VII, and at least one pharmaceutically acceptable excipient.

[0183] Compared to 4-hydroxytamoxifen (4-OHT), the prodrugs of Formula VI or Formula VII are much less active.

[0184] 4-Hydroxytamoxifen is an estrogen receptor (ER) antagonist used in the treatment of cancer (preferably but not exclusively for the prevention and / or treatment of breast cancer). In addition, it is an ER mutant (especially ER T2 variants), which can be used to trigger fusion proteins such as Cre-ER T and Cre-ER T2 (T = tamoxifen-inducible) activation, as described in patent EP1692936B1, the main activity of the fusion protein is in the nucleus.

[0185] In particular, Cre-ER T The fusion protein comprises at least a portion of a nuclear estrogen receptor having ligand binding activity. The ligand binding activity of one of the nuclear receptor or its fragments has at least one mutation selected from the following: (1) a glycine to arginine mutation at position 521 of the human nuclear estrogen receptor sequence or a natural variant of the sequence (G521 R); (2) a glycine to valine mutation at position 400 of the human nuclear estrogen receptor sequence or a natural variant of the sequence (G400V); and (3) a (methionine-leucine) to (alanine-alanine) mutation at positions 543-544 of the human nuclear estrogen receptor sequence or a natural variant of the sequence (M543A / L544A mutation).

[0186] As described in patent EP1692936B1, Cre-ER T2 It is a triple mutant protein G400V / M543A / L544A and Cre-ERT fusion protein, which carries the G521 R mutation.

[0187] In the absence of 4-OHT, the fusion protein is sequestered in the cytoplasm and is thus inactive; T2 After binding, this causes the fusion protein to move to the nucleus. T2 With Cre( Able to restructure ( C an R ecombine)) protein fusion, and when utilizing specific genetic regions flanked by Cre recognition sites (loxP sites), this allows the use of 4-OHT to activate or delete genes in cells at specific locations. In this sense, the prodrug can be cleaved locally only around the cell encoding the specific enzyme, thereby releasing 4-hydroxytamoxifen (4-OHT), as seen in Example 6.

[0188] As described in Example 5, the prodrug of formula VII was synthesized starting from the known compound 4,4'-(1-phenylbut-1-ene-1,2-diyl)diol (Compound 5-43) and vanillyl acetone. In this sense, Scheme VII describes the synthetic route used:

[0189] Scheme VII

[0190]

[0191] In this sense, the synthesis starts from compound 5-43 (CAS No. 91221-46-4) (a dihydroxy precursor of tamoxifen) from 4,4'-dihydroxybenzophenone and acetophenone. First, the hydroxyl group corresponding to the position characterized by the NN-dimethylaminoethyl ether group of 4-hydroxytamoxifen is converted into an ether with glycidol. The diol is then protected with 2,2-dimethoxypropane to couple the fragment derived from vanilla acetone by alkylation in the second hydroxyl group. Other steps include deprotecting the diol group, then oxidizing it to an aldehyde, and then converting it to an amine via reductive amination.

[0192] The prodrug of formula VII is inactive, but can release 4-OHT in the presence of β-etherase. It is able to activate or delete genes in cells at specific locations (such as where Cre-ER is used). T2 enzymes as shown in Example 6), or act as anticancer agents, thereby antagonizing wild-type estrogen receptors.

[0193] In this way, 4-hydroxytamoxifen is released only in cells encoding a β-etherase such as LigF, which is encoded in the DNA of tumor cells as described in Example 6. The enzymatic activity of LigF will release 4-hydroxytamoxifen from the prodrug of Formula VI or the prodrug of Formula VII.

[0194] Alternatively, the prodrug of Formula VI or the prodrug of Formula VII may be cleaved (perhaps with lower efficiency than LigF) by enzymes such as the cytochrome P450 enzyme family (primarily present in the liver). Example 7 describes a mouse treatment in which the liver was found to actively cleave the prodrug and undergo genetic recombination in a dose-dependent manner, suggesting that this prodrug strategy can be used to specifically target the liver using any compound of Formula V (including vanilla acetonide β-ether and in particular the prodrug of Formula VI). Genetic recombination in peripheral tissues was lower when compared to the liver, suggesting the possibility of targeting the liver.

[0195] In this sense, the prodrugs of Formula VI or the prodrugs of Formula VII can be used as research tools in biomedicine, genetics, developmental biology, cell biology, stem cell biology or related fields.

[0196] Thus, this article describes a method for expressing Cre-ER in the presence of 4-hydroxytamoxifen in a group of T2 A method for performing recombination of one or more genes or one or more target sequences in the mouse in cells of a mouse expressing a fusion protein, wherein the one or more genes or sequences belong to the natural genome of the mouse, the method comprising:

[0197] a) providing a mouse, the mouse comprising one or more recognition sites for a Cre recombinase protein, the recognition sites being inserted into the target DNA sequence located in one or more chromosomes of the mouse genome; and a Cre-ER that has substantially no recombinase activity in the absence of 4-hydroxytamoxifen or in the presence of natural estrogen. T2 Fusion protein, wherein the recombinase activity is induced by a small amount of 4-hydroxytamoxifen;

[0198] b) contacting the mouse with a prodrug of formula VI;

[0199] c) when recombination is required, contacting at least a portion of the cells of the mouse with β-etherase to react with the compound of formula VI, thereby releasing 4-hydroxytamoxifen and inducing the Cre-ER T2 recombinase activity of the fusion protein and obtaining recombination of the one or more genes or target sequences in a localized specific region where the beta-etherase is present, which is the source of the released 4-hydroxytamoxifen; and

[0200] However, in the absence of 4-hydroxytamoxifen or in the presence of natural estrogen, expression of Cre-ER T2 Substantially no recombination occurs in said cell containing the fusion protein.

[0201] In one embodiment, the β-etherase is LigF and the prodrug of Formula VI has Formula VII. For purposes of the present invention, a Cre-ER having substantially no recombinase activity in the absence of 4-hydroxytamoxifen or in the presence of natural estrogen T2 The fusion protein means that the recombinase activity of the fusion protein is less than 10%, or less than 5%, or even less than 1%. In a similar manner, in the absence of 4-hydroxytamoxifen or in the presence of natural estrogen, the expression of Cre-ER T2 Essentially no recombination occurs in cells containing the fusion protein, which also means that recombination occurs at less than 10%, 5% or even 1% of the situation in the absence of 4-hydroxytamoxifen or in the presence of natural estrogen.

[0202] Another embodiment of the present invention relates to a prodrug of formula VI or formula VII, or a pharmaceutical composition thereof, which is used as a drug. Another embodiment relates to a prodrug of formula VI, preferably a prodrug of formula VII, which is used to treat cancer. Preferably, the cancer is selected from the group consisting of: blood cancer; B-cell or T-cell leukemia, non-Hodgkin's lymphoma, B-cell or T-cell non-Hodgkin's lymphoma, Burkitt's lymphoma, Hodgkin's lymphoma, leukemia, B-cell or T-cell lymphoma, multiple myeloma, brain cancer, cancer of the glial lineage of the central nervous system (glioma), sarcoma, fibrosarcoma, malignant fibrous histiocytoma, Ewing's sarcoma, osteosarcoma, malignant pleural mesothelioma, breast cancer, breast cancer for anti-HER2 therapy, breast cancer, breast adenocarcinoma, gastric and gastroesophageal cancer, gastric cancer, gastric adenocarcinoma, colorectal cancer, colon cancer, colon adenocarcinoma, rectal cancer, colon Rectal cancer, metastatic colon cancer, pancreatic cancer, pancreatic cancer, pancreatic adenocarcinoma, renal cell carcinoma, clear cell renal cell carcinoma, liver cancer, lung cancer, lung adenocarcinoma, non-small cell lung cancer, small cell lung cancer, ovarian cancer, ovarian cancer, ovarian adenocarcinoma, ovarian cancer, endometrial cancer, endometrial stromal sarcoma, cervical cancer, thyroid cancer, metastatic papillary thyroid cancer, follicular thyroid cancer, bladder cancer, urinary bladder cancer, urinary bladder transitional cell carcinoma, prostate cancer, prostate cancer, neuroendocrine cancer, squamous cell carcinoma, osteosarcoma, rhabdomyosarcoma, embryonal carcinoma, neuroblastoma, medulloblastoma, retinoblastoma, Wilms tumor, hepatoblastoma, melanoma, and skin cancer.

[0203] Preferably, the blood cancer is leukemia, multiple myeloma or myelodysplastic syndrome.Preferably, the brain cancer is glioblastoma.

[0204] In a preferred embodiment, the solid tumor is selected from the group consisting of breast cancer, gastric and gastroesophageal cancer, colorectal cancer, pancreatic cancer, liver cancer, lung cancer, ovarian cancer, prostate cancer, neuroendocrine cancer.

[0205] Preferably, the skin cancer is melanoma.

[0206] Another embodiment of the present invention relates to a prodrug of 7-ethyl-10-hydroxy-camptothecin of formula VIII:

[0207]

[0208] in

[0209] - R1 is selected from the group consisting of H, alkyl, cycloalkyl, haloalkyl, hydroxyalkyl, aminoalkyl, alkanoylamino, alkoxy, aryl, alkylaryl, arylalkyl, amino, N-alkylamino and N,N-dialkylamino; and

[0210] - R2 and R3 are each independently selected from the group consisting of H, alkyl and haloalkyl.

[0211] One embodiment relates to a prodrug of 7-ethyl-10-hydroxy-camptothecin of formula VIII, wherein R1 is selected from the group consisting of H, alkyl, cycloalkyl, haloalkyl, hydroxyalkyl, aminoalkyl, alkanoylamino, alkoxy, aryl, alkylaryl, arylalkyl, amino, N-alkylamino and N,N-dialkylamino; R2 is methyl and R3 is H. In another embodiment, R1 is selected from the group consisting of H, alkyl, haloalkyl, aminoalkyl and hydroxyalkyl; R2 is methyl and R3 is H.

[0212] In a preferred embodiment of the prodrug of formula VIII, R1 is H, R2 is methyl and R3 is H, and the prodrug of 7-ethyl-10-hydroxy-camptothecin is a prodrug of formula IX:

[0213]

[0214] One embodiment is directed to a pharmaceutical composition comprising an effective amount of a prodrug of Formula VIII or a prodrug of Formula IX, and at least one pharmaceutically acceptable excipient.

[0215] One embodiment described relates to a prodrug of 7-ethyl-10-hydroxy-camptothecin of formula VIII or formula IX, or a pharmaceutical composition thereof, for use as a medicament, in particular for the treatment of cancer. Preferably, the cancer is selected from the group consisting of: blood cancer; B-cell or T-cell leukemia, non-Hodgkin's lymphoma, B-cell or T-cell non-Hodgkin's lymphoma, Burkitt's lymphoma, Hodgkin's lymphoma, leukemia, B-cell or T-cell lymphoma, multiple myeloma, brain cancer, cancer of the glial lineage of the central nervous system (glioma), sarcoma, fibrosarcoma, malignant fibrous histiocytoma, Ewing's sarcoma, osteosarcoma, malignant pleural mesothelioma, breast cancer, breast cancer on anti-HER2 therapy, breast cancer, breast adenocarcinoma, gastric and gastroesophageal cancer, gastric cancer, gastric adenocarcinoma, colorectal cancer, colon cancer, colon adenocarcinoma, rectal cancer, colon Rectal cancer, metastatic colon cancer, pancreatic cancer, pancreatic cancer, pancreatic adenocarcinoma, renal cell carcinoma, clear cell renal cell carcinoma, liver cancer, lung cancer, lung adenocarcinoma, non-small cell lung cancer, small cell lung cancer, ovarian cancer, ovarian cancer, ovarian adenocarcinoma, ovarian cancer, endometrial cancer, endometrial stromal sarcoma, cervical cancer, thyroid cancer, metastatic papillary thyroid cancer, follicular thyroid cancer, bladder cancer, urinary bladder cancer, urinary bladder transitional cell carcinoma, prostate cancer, prostate cancer, neuroendocrine cancer, squamous cell carcinoma, osteosarcoma, rhabdomyosarcoma, embryonal carcinoma, neuroblastoma, medulloblastoma, retinoblastoma, Wilms tumor, hepatoblastoma, melanoma, and skin cancer.

[0216] Preferably, the blood cancer is leukemia, multiple myeloma or myelodysplastic syndrome.Preferably, the brain cancer is glioblastoma.

[0217] In a preferred embodiment, the solid tumor is selected from the group consisting of breast cancer, gastric and gastroesophageal cancer, colorectal cancer, pancreatic cancer, liver cancer, lung cancer, ovarian cancer, prostate cancer, neuroendocrine cancer.

[0218] Preferably, the skin cancer is melanoma.

[0219] The vanilla acetone derived fragments of the prodrugs of formula V and in particular the prodrugs of formulas VI, VII, VIII and IX can be oxidized and cleaved by liver enzymes such as cytochrome P450 enzymes, which hydrolyze the ester bond, thereby delivering the drug R-OH containing one hydroxyl group, which is 4-hydroxytamoxifen in the case of the prodrugs of formulas VI and VII, and 7-ethyl-10-hydroxy-camptothecin in the case of the prodrugs of formulas VIII and IX. On the other hand, the ether bond can also be hydrolyzed by specific β-etherases from non-mammalian sources, such as the bacterial Sphingobium sp. strain SYK-6 enzymes LigE and LigF, or the fungal Dichomitus squalens Ds-GST1, which can be administered in combination with the prodrugs of formula V, and preferably in combination with the prodrugs of formulas VI, VII, VIII or IX.

[0220] In a particular embodiment, the β-etherase is administered in cells expressing the β-etherase as a cell therapy, such as adoptive T cell transfer or dendritic cell vaccination. Alternatively, in another particular embodiment, the β-etherase is administered coupled to a tumor or matrix specific antibody, optionally with glutathione. Certain tissues such as the lung lining contain high extracellular levels of glutathione, and therefore do not require administration of glutathione.

[0221] For the purposes of the present invention, the term "active compound" refers to the chemical entity or active ingredient that exerts a therapeutic effect when administered to humans or animals.

[0222] Typical compositions comprise the compound of the present invention together with at least one pharmaceutical excipient, for example, the excipient can be a carrier or a diluent. Such compositions can be in the form of capsules, sachets, paper or other containers. In preparing compositions, conventional techniques for preparing pharmaceutical compositions can be used. For example, the target compound will usually be mixed with a carrier, or diluted by a carrier, or enclosed in a carrier that can be in the form of an ampoule, capsule, sachets, paper or other container. When a carrier is used as a diluent, it can be a solid, semisolid or liquid material that serves as a vehicle, excipient or medium for the active compound. The target compound can be adsorbed on a granular solid container, for example in a sachet. Some examples of suitable carriers are water, saline solution, alcohol, polyethylene glycol, polyhydroxyethoxylated castor oil, peanut oil, olive oil, lactose, terra alba, sucrose, cyclodextrin, amylose, magnesium stearate, talc, gelatin, agar, pectin, gum arabic, stearic acid or cellulose lower alkyl ethers, silicic acid, fatty acids, fatty acid amines, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, polyoxyethylene, hydroxymethylcellulose and polyvinylpyrrolidone. Similarly, carriers or diluents can include any sustained-release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or in combination with wax. Preparations can also include wetting agents, emulsifiers and suspending agents, preservatives, sweeteners or flavorings. By adopting procedures well known in the art, preparations of the present invention can be formulated to provide a quick, continuous or delayed release of active ingredients after being applied to a patient.

[0223] The pharmaceutical compositions can be sterilized and, if desired, mixed with auxiliary agents, emulsifiers, salts for influencing the osmotic pressure, buffers and / or coloring substances etc. which do not deleteriously react with the active compounds.

[0224] A preferred embodiment of the present invention relates to the route of administration, which can be any route that effectively delivers the target compound to the appropriate or desired site of action, such as oral, buccal, nasal, topical, pulmonary, transdermal or parenteral, for example, rectal, subcutaneous, intravenous, intraurethral, ​​intramuscular, intranasal, eye drops or ointment.

[0225] For nasal administration, the formulation may contain the target compound dissolved or suspended in a liquid carrier, particularly an aqueous carrier, for aerosol application. The carrier may contain additives such as solubilizers, e.g., propylene glycol, surfactants, absorption enhancers such as lecithin (phosphatidylcholine) or cyclodextrins, or preservatives such as parabens.

[0226] To prepare a topical formulation, the target compound is placed in a dermatological vehicle as known in the art. The amount of the target compound to be applied in the topical formulation and the concentration of the compound depend on the selected vehicle, delivery system or device, the patient's clinical condition, the side effects and stability of the compound in the formulation. Therefore, based on the clinical experience of the patient in question or similar patients, the doctor uses an appropriate formulation containing an appropriate concentration of the target compound and selects the amount of the formulation to be applied.

[0227] For ophthalmic use, the subject compounds are formulated into solutions, suspensions and ointments suitable for application to the eye. The concentrations are generally as discussed above for topical preparations.

[0228] For oral administration, solid or fluid unit dosage forms can be prepared. In order to prepare solid compositions such as tablets, the target compound is mixed into the preparation with conventional ingredients such as talc, magnesium stearate, dicalcium phosphate, magnesium aluminum silicate, calcium sulfate, starch, lactose, gum arabic, methylcellulose and materials similar in function to pharmaceutical diluents or carriers.

[0229] Capsules are prepared by mixing the target compound with an inert pharmaceutical diluent and filling the mixture into a hard gelatin capsule of appropriate size. Soft gelatin capsules are prepared by mechanically encapsulating the slurry of the target compound with acceptable vegetable oils, light liquid paraffin or other inert oils. Fluid unit dosage forms such as syrups, elixirs and suspensions for oral administration can be prepared. Water-soluble forms can be dissolved in aqueous vehicles with sugar, aromatic flavorings and preservatives to form syrups. Elixirs are prepared by using a hydroalcohol (e.g., ethanol) vehicle with suitable sweeteners such as sugar and saccharin together with aromatic flavorings. Suspensions can be prepared by suspending agents such as gum arabic, tragacanth, methylcellulose, etc. using aqueous vehicles.

[0230] Suitable formulations for parenteral use will be apparent to those of ordinary skill, such as using suitable injectable solutions or suspensions. The sterile formulation is suitable for a variety of topical or parenteral routes, including intradermal, intramuscular, intravascular and subcutaneous.

[0231] In addition to the target compound, the composition may also contain a pharmaceutically acceptable non-toxic carrier or diluent, which includes vehicles commonly used to form pharmaceutical compositions for animal or human administration, depending on the desired formulation and delivery mode. The diluent is selected so as not to unduly affect the biological activity of the combination.

[0232] Examples of such diluents, which are particularly useful for injectable preparations, are water, various saline solutions, organic or inorganic salt solutions, Ringer's solution, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or preparation may include additives such as other carriers; adjuvants; or non-toxic, non-therapeutic, non-immunogenic stabilizers, etc.

[0233] In addition, excipients may be included in the formulation. Examples include cosolvents, surfactants, oils, wetting agents, lubricants, preservatives, stabilizers, and antioxidants. Any pharmacologically acceptable buffer may be used, such as tris or phosphate buffers. Effective amounts of diluents, additives, and excipients are those amounts that are effective in obtaining a pharmaceutical formulation in terms of solubility or biological activity.

[0234] The target compound can be incorporated into microspheres. The target compound can be loaded into albumin microspheres, from which such microspheres can be recovered in dry powder form for nasal administration. Other materials suitable for preparing microspheres include agar, alginate, chitosan, starch, hydroxyethyl starch, albumin, agarose, dextran, hyaluronic acid, gelatin, collagen and casein. Microspheres can be produced by various methods known to those skilled in the art such as spray drying or emulsification.

[0235] For example, albumin microspheres can be prepared by adding rabbit serum albumin in a phosphate buffer to olive oil under stirring to produce a water-in-oil emulsion. A glutaraldehyde solution is then added to the emulsion, and the emulsion is stirred to crosslink the albumin. The microspheres can then be separated by centrifugation, the oil removed, and the microspheres washed, for example, with petroleum ether followed by ethanol. Finally, the microspheres can be sieved and collected and dried by filtration.

[0236] Starch microspheres can be prepared in the following manner: under stirring, a warm starch aqueous solution such as a potato starch aqueous solution is added to a heated solution of polyethylene glycol in water to form an emulsion. When a two-phase system (wherein the starch solution is used as the internal phase) has been formed, the mixture is cooled to room temperature under continuous stirring, and the internal phase is subsequently converted into gel particles. These particles are then filtered out at room temperature and made into a slurry in a solvent such as ethanol, and then the particles are filtered out again and placed in air to dry. Microspheres can be hardened by well-known cross-linking procedures such as heat treatment or by using a chemical cross-linking agent. Suitable reagents include dialdehydes (including glyoxal, malondialdehyde, succinicaldehyde (succinicaldehyde), adipaldehyde, glutaraldehyde and phthalaldehyde), diketones such as diacetyl, epichlorohydrin, polyphosphates and borates. Dialdehydes are used to cross-link proteins (such as albumin) by interaction with amino groups, and diketones form Schiff bases (Schiff bases) with amino groups. Epichlorohydrin utilizes nucleophilic groups such as amino or hydroxyl groups to activate compounds as epoxide derivatives.

[0237] Another preferred embodiment of the present invention is a dosage regimen. The term "unit dosage form" refers to a physically discrete unit suitable as a single dose for a subject, such as a mammalian subject (e.g., a human, dog, cat, and rodent), each unit containing a predetermined amount of active material calculated to produce the desired drug effect in combination with a desired drug diluent, carrier, or vehicle. The specifications of the unit dosage form used in the present invention are determined by and depend on the following: (a) the unique characteristics of the active material and the specific effect to be achieved and (b) the inherent limitations in the field of formulating such active materials for use in humans and animals. Examples of unit dosage forms are tablets, capsules, pills, powder packets, wafers, suppositories, granules, cachets, teaspoons, tablespoons, drops, ampoules, vials, aerosols with metered discharge, any of the aforementioned separation multiples, and other forms as described herein. The composition can be included in a kit, which can contain one or more unit dosage forms of the composition and instructions for use for treating one or more disorders described herein.

[0238] Slow or extended release delivery systems, including various biopolymers (biologically based systems), systems using liposomes, colloids, resins, and any of other polymer delivery systems or compartmentalized reservoirs, can be used with the compositions described herein to provide a continuous or long-term source of therapeutic compounds. Such slow release systems are suitable for formulations delivered via topical, intraocular, oral, and parenteral routes.

[0239] An effective amount of the target compound is used in treatment. The dosage of the compound used according to the present invention varies depending on the compound and the condition being treated, such as the age, weight and clinical condition of the recipient patient. Other factors include: the route of administration, the patient, the patient's medical history, the severity of the disease process and the efficacy of the specific compound. The dosage should be sufficient to alleviate the symptoms or signs of the disease being treated without causing unacceptable toxicity to the patient. Typically, the effective amount of the compound is to provide subjective relief of symptoms or an objectively identifiable improvement noted by a clinician or other qualified observer. Example

[0240] The embodiments of the present invention described below are intended to illustrate some of the disclosed embodiments without limiting their scope. In addition, reference examples are included, which relate to the embodiments disclosed in this specification but are not included in the scope of the claims. All experiments using mouse models were approved by the Animal Care and Use Committee of Barcelona Science Park (CEEA-PCB) and the Government of Catalonia. Mice were kept in a facility with a 12-hour light-dark cycle without specific pathogens (SPF) and fed with a standard diet and water (without restriction). The animal welfare of all mice was closely monitored.

[0241] General information regarding compound administration in mouse studies: The molecular weights of ganicitinib (LY2157299 monohydrate), compounds of formula I and IV, and compounds 338 and 337 are: 387.4; 342.4; 536.7; 342.4; 328.4, respectively. Therefore, the corresponding molecular weight ratios are: 1:0.88:1.39:0.88:0.85. All mouse treatments described in the following figures and in the examples were calculated using the 1x dose for ganicitinib as a reference, which corresponds to 160 mg / kg / day divided into 2 oral administrations (morning and evening), i.e. 80 mg / kg bid. Thus, the 1x molar equivalent dose of the compound of formula I is 70.8 mg / kg bid, the 1x molar equivalent dose of the compound of formula IV is 111.2 mg / kg bid, the 1x molar equivalent dose of compound 338 is 70.8 mg / kg bid and the 1x molar equivalent dose of compound 337 is 68.0 mg / kg b.id.

[0242] Prior art Compounds 337 (CAS No. 476474-11-0) and 338 (CAS No. 476477-67-5) were prepared according to patent WO2005094833 (both 338 and 337) and J. Med Chem. 2008, 51, 2302 (only 337 is described).

[0243] Example 1: Synthesis of compounds of formula I, III and IV:

[0244] Unless otherwise stated, all reactions were performed under an inert atmosphere (N2). Anhydrous THF, DCM, and diethyl ether were obtained from Innovative Technology Inc. Puresolv Solvent Purification System (SPS). Other anhydrous solvents were purchased and used without any further purification or drying.

[0245] All experiments were monitored by analytical thin layer chromatography (TLC) using silica gel TLC-aluminum plates (Merck 60 F254). The results were visualized under UV light (254 or 365 nm) and displayed using KMnO4 staining when necessary.

[0246] Unless otherwise stated, flash column chromatography was performed at (Teledyne Isco) or on a Puriflash 430 (Interchim). The mobile phase was a gradient of hexane / ethyl acetate or a gradient of dichloromethane / methanol. In some cases where there was a basic nitrogen on the molecule to be purified, the column was preconditioned with a 2.5% triethylamine solution in hexane.

[0247] Samples for NMR were dissolved in CDCl3, CD3OD, DMSO or D2O.

[0248] 1 H and 13 C spectra were referenced to residual solvent peaks and / or tetramethylsilane. Coupling constants are measured in Hertz (Hz). All IR spectra were performed on NaCl disks using the thin film method and recorded on a Thermo Nicolet Nexus Ft-IR Fourier transform spectrometer.

[0249] 1.1. Synthesis of compounds of formula I from p-bromoaniline - route (d):

[0250] The compound of formula I is synthesized from p-bromoaniline, including a final step in which the bromine is converted to phenol:

[0251]

[0252] 6-Bromo-4-methylquinoline

[0253]

[0254] To a stirred solution of 4-bromoaniline (20 g, 117 mmol) and chloranil (34.7 g, 141 mmol, 1.2 eq) in ethanol (200 mL, 10 vol) was added 37% HCl (29.3 mL, 351 mmol, 3 eq) over five minutes at room temperature. After stirring at room temperature for five minutes, the reaction mixture was heated to 75°C, where methyl vinyl ketone (15.45 mL, 190.5 mmol, 1.5 eq) diluted in ethanol (15 mL, 1 vol) was added to the reaction mixture over thirty minutes. After four hours, no starting material was observed by TLC. The reaction was cooled to 60°C, where it was diluted with THF (165 mL, 11 vol), stirred at 60°C for one hour, and then allowed to cool to room temperature overnight. The suspension was filtered through a sintered Buchner funnel. The residue was rinsed with THF (2 x 45 mL, 6 volumes total) and then dried in a vacuum oven at 50 °C overnight to afford the title compound (21.1 g, 81%) as a brown solid.

[0255] 1 H NMR(400MHz, CDCl3)δ8.78(d,J=4.4Hz,1H),8.16–8.14(m,1H),7.97(d,J=9.0Hz,1 H), 7.77 (dd, J=9.0, 2.2Hz, 1H), 7.25 (dd, J=4.4, 0.9Hz, 1H), 2.68 (d, J=0.9Hz, 3H).

[0256] 13 C NMR (101MHz, DMSO) δ145.76,138.20,135.96,133.04,129.21,127.79,125.12,123.25,122.18,19.24.

[0257] 6-Bromo-4-(2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)quinoline

[0258]

[0259] To a stirred solution of 6-bromo-4-methylquinoline (3.70 g, 14.31 mmol) in THF (60 mL) was added 1 M NaHMDS (57 mL, 57 mmol, 4 eq) at -78 °C. The reaction mixture was stirred for 3 hours, followed by the addition of methyl 6-methylpicolinate (2.60 g, 17.17 mmol, 1.2 eq) diluted in THF (7.4 mL, 2 volumes) dropwise over 30 minutes. The reaction was allowed to warm to room temperature overnight, then cooled to 0 °C, and brought to pH 1 with 3 M HCl. Allowed to warm to room temperature over an hour, then brought to pH 8 with solid NaHCO. The mixture was diluted with water (50 mL) and extracted with EtOAc (1 x 100 mL), followed by CHCl (1 x 100 mL). The combined organic extracts were dried (MgSO4), evaporated and the residue recrystallized from MeOH (30 mL, 8 vol) to give 2-(6-bromoquinolin-4-yl)-1-(6-methylpyridin-2-yl)ethan-1-one (2.57 g, 53%) as an off-white solid.

[0260] 1 H NMR (400MHz, cdcl3) δ8.84(d,J=4.4Hz,1H),8.36(d,J=2.1Hz,1H),7.97(d,J=9.0Hz,1H),7.86(d,J=7.7Hz,0H),7.75 (dd, J=9.0, 2.2Hz, 1H), 7.73 (t, J=7.7Hz, 1H), 7.43 (d, J=4.4Hz, 1H), 7.38 (d, J=8.6Hz, 1H), 4.96 (s, 1H), 2.70 (s, 2H).

[0261] 13 C NMR(101MHz,cdcl3)δ197.87,158.43,152.01,150.47,147.24,141.30,137.39, 132.77,131.95,129.36,127.58,127.09,124.09,120.89,119.76,40.53,24.60.

[0262] 6-Bromo-4-(2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)quinoline

[0263]

[0264] To a stirred solution of 2-(6-bromoquinoline-4-yl)-1-(6-methylpyridin-2-yl)ethan-1-one (2.57 g, 7.53 mmol) in DMF (15.5 mL, 6 volumes), toluene (26 mL, 10 volumes) and 2,6-lutidine (2.6 mL, 1 volume) at room temperature was added 1-aminopyrrolidin-2-one tosylate (2.05 g, 7.53 mmol, 1 eq) and stirred at room temperature for 5 minutes. The reaction was heated to reflux under Dean-Stark conditions until most of the starting material had been consumed (by TLC). The reaction mixture was cooled to room temperature, CsCO (4.17 g, 12.8 mmol, 1.7 eq) was charged thereto and heated back to reflux. The disappearance of the intermediate of monitoring reaction, and once all consumed, just distill toluene until reaction mixture reaches 145 ℃, then cool to room temperature, distribute between water (30mL) and EtOAc (30mL), and separate each phase.Aqueous phase is extracted with EtOAc (2x20mL), the organic extract merging is washed with 5%LiCl aqueous solution (2x20mL) and with organic layer drying (MgSO ), evaporate, and residue is carried out chromatographic analysis (0-10%MeOH, in DCM), obtain title compound (2.05g, 67%), be brown solid.

[0265] 1 H NMR (400MHz, cdcl3) δ8.85 (d, J = 4.4Hz, 1H), 7.98 (d, J = 8.9Hz, 1H), 7.92 (d, J = 2.2Hz, 1H), 7.71 (dd, J = 8.9, 2.2Hz, 1H), 7.35 (t, J = 7.7Hz, 1H), 7.31 (d, J = 4.4Hz, 1H), 7.11 (d, J = 7.8Hz, 1H), 6.92 (d, J = 7.6Hz, 1H), 4.37 (t, J = 7.2Hz, 2H), 2.90–2.84 (m, 2H), 2.75–2.66 (m, 2H), 2.24 (s, 3H).

[0266] 13 C NMR (101MHz, cdcl3) δ158.22, 153.60, 153.24, 151.25, 150.22, 147.11, 146.66, 140.88, 136.29 ,132.50,131.34,128.72,128.66,123.03,121.77,120.20,118.86,48.33,25.98,24.19,23.15.

[0267] UPLC-MS: 407m / z[M+H]

[0268] Compound of formula I: 4-(2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)quinolin-6-ol

[0269]

[0270] To a flame-dried flask under N2 was added 6-bromo-4-(2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)quinoline (200 mg, 0.493 mmol), Pd(dppf)Cl2 (36 mgs, 0.050 mmol, 0.1 eq), KOAc (145 mgs, 1.48 mmol, 3 eq) and bis(pinacolato)diboron (137 mgs, 0.542 mmol, 1.1 eq). Degassed dioxane (5 mL) was then added to the reaction flask and heated to 100°C for 2 hours, at which time the reaction was cooled to room temperature and diluted with 1 M NaOH (4.93 mL, 4.93 mmol, 10 eq) and 30% H2O2 (46.6 μL, 0.542 mmol, 1.1 eq) was added dropwise. After an additional 2 hours at room temperature, the reaction was quenched with 1.6 M sodium sulfite (1 mM, 625 μL). After stirring for 30 minutes, the reaction mixture was checked for the presence of peroxide by diatomaceous earth. The mixture was filtered through a pad of CHCl and brought to pH 8 using 37% HCl. The mixture was diluted with water (5 mL) and extracted with EtOAc (3 × 5 mL) and chloroform (1 × 5 mL). The combined organic extracts were dried (MgSO ), evaporated, and the residue was chromatographed (0-100% EtOAc in hexane) to give title compound I (130 mgs, 77%).

[0271] 1 H NMR (400MHz, DMSO) δ9.66 (s, 1H), 8.58 (d, J = 4.4Hz, 1H), 7.86 (d, J = 9.0Hz, 1H), 7.56 (t, J = 7.7Hz, 1H), 7.48 (d, J = 7.8Hz, 1H), 7.29–7. 16 (m, 2H), 6.96 (d, J = 7.5Hz, 1H), 6.91 (d, J = 2.7Hz, 1H), 4.27 (t, J = 7.2Hz, 2H), 2.79 (t, J = 6.5Hz, 2H), 2.69–2.55 (m, 2H), 1.89 (s, 3H).

[0272] 13C NMR(101MHz,DMSO)δ156.49,155.08,152.01,151.70,146.55,146.24,143.18,139.37,136 .48,130.60,128.71,122.47,121.16,117.73,109.89,106.98,47.86,25.53,23.41,22.50.

[0273] IR(Thin Film):3412,2949,2833,1650,1618,1508,1236,1010cm -1

[0274] 1.2. Synthesis of compounds of formula I from p-methoxyaniline - Route (a):

[0275] 6-Methoxy-4-methylquinoline

[0276]

[0277] To a stirred solution of p-anisidine (5 g, 40.6 mmol) and ZnCl2 (719 mg, 5.3 mmol, 0.13 eq) in ethanol (20 mL) was added FeCl3 (17.45 g, 107.6 mmol, 2.65 eq) in portions at room temperature over 30 min. The reaction mixture was stirred for 30 min and heated to 75°C for two hours while methyl vinyl ketone (3.38 mL, 40.6 mmol, 1 eq) was added. It was then cooled to room temperature and poured into NaOH (1 M, 220 mL) at 0°C. The mixture was extracted with EtOAc (3 x 40 mL) and the combined organic extracts were dried (MgSO4), evaporated, and the residue was chromatographed (0-100% EtOAc in hexanes) to give the title compound (2.88 g, 41%) as a brown oil.

[0278] 1 H NMR (400MHz; CDCl3) δ8.65(d,J=4.4Hz,1H),8.02(d,J=9.2Hz,1H),7.37(dd,J=9.2, 2.8Hz, 1H), 7.21 (d, J = 4.4Hz, 1H), 7.19 (d, J = 2.8Hz, 1H), 3.96 (s, 3H), 2.67 (s, 3H).

[0279] 2-(6-Methoxyquinolin-4-yl)-1-(6-methylpyridin-2-yl)ethan-1-one

[0280]

[0281] To a stirred solution of 6-methoxy-4-methylquinoline (2.5 g, 14.4 mmol) in THF (80 mL) was added LDA (33.19 mmol, 2.3 equiv) at -78 ° C. When methyl 6-methylpicolinate (3.27 g, 21.6 mmol, 1.5 equiv) was added dropwise to the reaction mixture over 30 minutes, the reaction mixture was stirred for 30 minutes. The reaction was placed at -78 ° C and allowed to warm to room temperature overnight. It was then quenched with saturated NH4Cl (20 mL), diluted with EtOAc (40 mL), and the phases were separated. The aqueous phase was extracted with chloroform (2x50 mL), the combined organic extracts were dried (MgSO4), evaporated, and the residue was subjected to chromatography (0-100% EtOAc in hexane) to obtain the title compound (1.91 g, 51%) as a brown oil;

[0282] 6-Methoxy-4-(2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)quinoline

[0283]

[0284] 1-aminopyrrolidin-2-one tosylate (1.49 g, 5.47 mmol, 1 equivalent) was added to a stirred solution of 2-(6-methoxyquinoline-4-yl)-1-(6-methylpyridin-2-yl) ethyl-1-one (1.60 g, 5.47 mmol) in DMF (9.6 mL, 6 volumes), toluene (16 mL, 10 volumes) and 2,6-lutidine (1.6 mL, 1 volume) at room temperature, and stirred at room temperature for 5 minutes. The reaction was heated to reflux under Dean-Stark conditions until most of the starting material had been consumed (by TLC). The reaction mixture was cooled to room temperature, CsCO (3.029 g, 9.299 mmol, 1.7 equivalents) was loaded therein, left to stand for 5 minutes, and then heated back to reflux. The disappearance of the intermediate of the reaction was monitored, and once all consumed, the reaction was cooled to room temperature and distributed between water (50 mL) and EtOAc (50 mL). The aqueous phase was extracted (2x25mL), the combined organic extracts were washed with 5% LiCl aqueous solution (2x50mL), and the organic layer was dried (MgSO4), evaporated, and the residue was subjected to chromatography (0-10% MeOH in DCM). The fraction containing the product was dissolved in a small amount of CHCl3, and diluted slowly with hexane until the mixture appeared slightly turbid. The flask was placed in a refrigerator for 48 hours to form crystals (1.28g, 67%) as a white solid;

[0285] 1H NMR(400MHz,cdcl3)δ8.74(d,J=4.4Hz,1H),7.98(d,J=9.2Hz,1H),7.35–7.20(m,4H),6.96–6.85(m,3H),4.37(t,J=7.2Hz,2H),3.54(s,3H),2.91(br s,2H),2.75–2.65(m,2H),2.38(s,3H).

[0286] 13 C NMR(101MHz,cdcl3)δ158.62,157.67,153.65,151.83,147.66,146.69,144.89,139.90,136.43,13 1.17,128.25,122.41,122.22,121.93,119.50,110.50,104.00,55.52,48.46,26.19,24.58,23.32.

[0287] HRMS (ESI): m / z [M+H] + for C 22 H 20 Calculated value of N4O: 356.1637; experimental value: 356.1639.

[0288] Compound of formula I: 4-(2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)quinolin-6-ol

[0289]

[0290] To a stirred solution of 6-methoxy-4-(2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)quinoline (432 mg, 1.21 mmol) in acetic acid (5 mL) was added HBr (5 mL, 44.45 mmol, 36.7 equiv) at room temperature. The reaction mixture was stirred at 120 °C for 48 hours, then cooled to room temperature and the solvent was removed in vacuo. The residue was partitioned between water (5 mL) and EtOAc (10 mL) and brought to pH 8 with 40% NaOH, the phases were separated, and the aqueous phase was extracted with EtOAc (3x10 mL). The combined organic extracts were dried (MgSO4), evaporated, and the residue was suspended in refluxing toluene for one hour, then cooled to room temperature within one hour. The solid was filtered and dried in vacuo to give the title compound (328 mg, 79%) as an off-white solid;

[0291] 1H NMR (400MHz, DMSO) δ9.66 (s, 1H), 8.58 (d, J = 4.4Hz, 1H), 7.86 (d, J = 9.0Hz, 1H), 7.56 (t, J = 7.7Hz, 1H), 7.48 (d, J = 7.8Hz, 1H), 7.29–7. 16(m,2H),6.96(d,J=7.5Hz,1H),6.91(d,J=2.7Hz,1H),4.27(t,J=7.2Hz,2H),2.79(t,J=6.5Hz,2H),2.69–2.55(m,2H),1.89(s,3H).

[0292] 13 C NMR(101MHz,DMSO)δ156.49,155.08,152.01,151.70,146.55,146.24,143.18,139.37,136 .48,130.60,128.71,122.47,121.16,117.73,109.89,106.98,47.86,25.53,23.41,22.50.

[0293] IR(Thin Film):3412,2949,2833,1650,1618,1508,1236,1010cm -1

[0294] HRMS (ESI): m / z [M+H] + for C 21 H 18 Calculated value of N4O: 342.1481; experimental value: 342.1480.

[0295] 1.3. Synthesis of prodrug of formula III:

[0296] 1-(4-Hydroxy-3-methoxyphenyl)-2-((4-(2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)quinolin-6-yl)oxy)ethan-1-one

[0297] The compound of formula I, i.e. 4-(2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole-3-yl)quinoline-6-ol (42 mg, 0.12 mmol) and CsCO (134 mg, 0.41 mmol) were dissolved in 1.5 mL of anhydrous DMF and heated at 60 ° C for 1.5 hours under N atmosphere. Then a solution of 4-(2-bromoacetyl)-2-methoxyphenyl pivalate in 0.5 mL of anhydrous DMF was added to the bright green suspension via cannula. The resulting mixture was stirred at 60 ° C for 2 hours. The reaction was then cooled to room temperature, diluted with water (15 mL) and quenched with 0.5 mL of a 1M solution of HCl. The resulting water layer was extracted with EtOAc (3x20 mL), the organic layer was dried with anhydrous MgSO, and the solvent was removed in vacuo. The crude product is purified by column chromatography (0 to 20% MeOH in DCM) to obtain a brown oil, which is used in the LiOH.H in 1.5mL MeOH o (13mg, 0.31mmol) is treated for 1 hour. The reaction is quenched with 1M HCl (0.3mL), diluted with water and extracted with EtOAc (3x20mL). Anhydrous MgSO for the organic layer is dried, the solvent is removed in a vacuum, and the crude product is subjected to chromatographic analysis (0 to 20% MeOH in EtOAc), 51.9mg (82%) of the title compound is obtained.

[0298] 1 H NMR (400MHz, CDCl3) δ8.73(d,J=4.6Hz,1H),8.11(d,J=9.2Hz,1H),7.50(d,J=1.9Hz,1H),7.47–7.43(m,2H),7.32–7.26(m,2H),7.06 –6.95(m,3H),6.89(d,J=7.6Hz,1H),4.94(bs,2H),4.27(t,J=7.3Hz,2H),3.93(s,3H),2.82(bs,2H),2.61(bs,2H),2.24(s,3H)ppm.

[0299] 13 C NMR (101MHz, CDCl3) δ192.3,158.5,156.3,153.4,152.0,151.6,147.5,147.2,146.9,144.2,141.1,136.5,130.7,1 28.3,127.1,123.5,122.7,122.6,122.0,119.2,114.5,110.2,110.1,105.7,70.4,56.2,48.4,26.0,24.3,23.2ppm.

[0300] IR (thin film): 3074, 2958, 2925, 1686, 1620, 1589, 1509, 1428, 1274, 1225, 1198, 1088cm -1 .

[0301] HRMS (ESI): m / z [M+H] + for C 30 H 27 Calculated value of N4O4: 507.2027; experimental value: 507.2016.

[0302] Mp:162-163℃.

[0303] 1.4. Synthesis of prodrug of formula IV:

[0304] 4-(2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)quinolin-6-yl[1,4'-bipiperidinyl]-1'-carboxylate

[0305] A solution of the compound of formula I synthesized in 1.1. (47 mg, 0.14 mmol) in 3 mL of CHCl3 was prepared and transferred to a flask containing Schlenk flask of activated molecular sieve. The solution was stirred at room temperature for 1 hour. Then [1,4'-bipiperidine]-1'-carbonyl chloride (32mg, 0.14mmol, 1 equivalent), Et3N (23μL, 0.17mmol, 1.2 equivalents) and 4-dimethylaminopyridine crystals in 1mL of CHCl3 were added dropwise. The resulting mixture was stirred overnight. Certain starting materials were detected by TLC on the second day, and therefore [1,4'-bipiperidine]-1'-carbonyl chloride (16mg, 0.07mmol, 0.5 equivalents), Et3N (12μL, 0.08mmol, 0.6 equivalents) and 4-dimethylaminopyridine crystals were added to the reaction mixture in 0.5mL of CHCl3 Another solution, and it was stirred overnight. The reaction was quenched by adding 20mL of H2O. The aqueous layer was extracted with EtOAc (3 x 20 mL), dried over MgSO4 and concentrated to give the crude product, which was purified by column chromatography (eluting with a gradient of 10-20% MeOH in DCM) to afford 64 mg (87%) of the desired product as an orange oil.

[0306] 1H NMR (400MHz, DMSO) δ8.80(d,J=4.4Hz,1H),8.01(d,J=9.1Hz,1H),7.56(dd,J=24.0,7.5H z,2H),7.47(dd,J=9.1,2.6Hz,1H),7.38(d,J=4.4Hz,1H),7.28(d,J=2.6Hz,1H),6.95(d ,J=7.8Hz,1H),4.27(t,J=7.2Hz,2H),4.01(dd,J=37.1,14.3Hz,2H),3.65–3.14(m,1H), 2.81(d,J=16.6Hz,4H),2.61(m,3H),1.82(s,3H),1.74(d,J=10.6Hz,3H),1.44(m,10H).

[0307] 13 C NMR (101MHz, DMSO) δ156.59,152.55,151.92,151.57,149.18,148.60,147.08,145.62,141.10,136.57,13 0.20,127.54,124.70,122.63,121.25,117.67,116.92,109.27,61.31,49.59,47.95,25.53,23.29,22.41.

[0308] UPLC-MS: 537.8 (M+H)

[0309] 1.5. Synthesis of the hydrochloride salt of the prodrug of formula IV:

[0310] 1-(1-(((4-(2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)quinolin-6-yl)oxy)carbonyl)piperidin-4-yl)piperidin-1-ium chloride

[0311] The compound of formula IV (65 mg, 0.12 mmol) was dissolved in DCM (1.5 mL) and treated with 0.3 mL (1.21 mmol) of a 2M solution of HCl in dioxane for 2 hours. The solvent was then removed to give a light orange solid (70 mg, 100%).

[0312] 1H NMR (400MHz, methanol-d4) δ9.18 (d, J = 5.6Hz, 1H), 8.35 (d, J = 9.2Hz, 1H), 8.12 (d, J = 5.6Hz, 1H), 8 .06(t,J=7.9Hz,1H),8.02–7.93(m,2H),7.74(d,J=7.9Hz,1H),7.28(d,J=7.9Hz,1H), 4.56– 4.39 (m, 3H), 4.31–4.28 (m, 1H), 3.59–3.56 (m, 2H), 3.52–3.45 (m, 2H), 3.19–2.95 (m, 6H), 2 .88(s, 3H), 2.85–2.81(m, 1H), 2.25–2.20(m, 2H), 2.01–1.85(m, 7H), 1.60–1.50(m, 1H)ppm.

[0313] 13 C NMR (101 MHz, methanol-d4) δ 157.0, 153.3, 153.2, 152.2, 151.0, 147.2, 146.1, 145.5, 144.9, 137.62, 131.4, 129.3, 128.0, 124.6, 124.5, 124.1, 119.5, 111.0, 66.9, 64.6, 51.4, 50.4, 44.3, 44.0, 27.5, 27.2, 27.1, 24.5, 24.2, 22.9, 20.1, 15.5 ppm.

[0314] IR(Thin Film):3411,3048,2933,2857,1716,1589,1420,1195,1020,804cm -1 .

[0315] HRMS (ESI): m / z [M+H] + for C 32 H 37 Calculated value of N6O2: 537.2973; experimental value: 537.2968.

[0316] Mp: 214-215℃.

[0317] Example 2: In vitro TGFβ inhibitor activity

[0318] 2.1 Molecular affinity and selectivity determination

[0319] By using KINOMEscan from Eurofins TMThe affinity of the compounds of Formula I for the ALK family was determined using the ELISA technique, which was measured as the Kd (dissociation constant). The selectivity towards ALK1 (ACVRL1), ALK2 (ACVR1), ALK3 (BMPR1A), ALK4 (ACVR1B), ALK6 (BMPR1B), ACVR2B and TGFβR2 was determined.

[0320] The KINOMEscan screening platform employs an active site-directed competition binding assay to quantitatively measure the interaction between a test compound and a kinase. The KINOMEscan assay does not require ATP and thus reports true thermodynamic interaction affinities.

[0321] The assay is performed by combining three components: DNA-tagged kinase; immobilized ligand; and test compound. The ability of the test compound to compete with the immobilized ligand is measured via quantitative PCR of the DNA tag.

[0322] The binding reaction was assembled by combining kinase, ligand affinity beads and test compounds in 1x binding buffer (20% SeaBlock, 0.17x PBS, 0.05% Tween 20, 6mM DTT). The test compound was prepared as a 111X stock solution in 100% DMSO. Kd was determined using an 11-point 3-fold compound dilution series, with three DMSO control points at the highest concentration of 30 μM. All compounds used for Kd measurement were distributed by acoustic transmission (non-contact distribution) in 100% DMSO. The compound was then diluted directly into the assay so that the final concentration of DMSO was 0.9%. All reactions were performed on polypropylene 384-well plates. The final volume of each was 0.02 ml. The assay plate was incubated at room temperature for 1 hour under shaking, and the affinity beads were washed with wash buffer (1x PBS, 0.05% Tween 20). The beads were then resuspended in elution buffer (1xPBS, 0.05% Tween 20, 0.5 μM non-biotinylated affinity ligand) and incubated for 30 minutes at room temperature with shaking. The kinase concentration in the eluate was measured by qPCR.

[0323] Compounds that bind to the kinase active site and directly (sterically) or indirectly (allosterically) prevent the kinase from binding to the immobilized ligand will reduce the amount of kinase captured on the solid support. In contrast, test molecules that do not bind to the kinase have no effect on the amount of kinase captured on the solid support. The dissociation constant (Kd) for the test compound-kinase interaction is calculated by measuring the amount of kinase captured on the solid support as a function of the test compound concentration.

[0324] As can be seen in Table 1 below, the compound of Formula I is 8.6 times more potent than ganicitinib in terms of ALK5 affinity. Using the same technique, ganicitinib published data (Jonathan M et al., Oncotarget, 2018, 9(6): 6659-6677) were considered for comparison.

[0325] Table 1.KINOMEscan TM Affinity data

[0326]

[0327] The enzyme activity inhibition (as IC ) of the compounds of formula I, the prodrugs of formula II and IV of the present invention and the prior art compounds 337, 338 and ganicitinib on ALK5 was also determined by using the Kinase Profiler service from Eurofins. 50 measured).

[0328] Briefly, human ALK5 was incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA, 1 mM MnCl2, 2 mg / mL casein, 10 mM Mg acetate, and [γ-33P-ATP] (specific activity approximately 500 cpm / pmol, desired concentration). The reaction was initiated by adding the MgATP mixture. After incubation for 40 minutes at room temperature, the reaction was terminated by adding 3% phosphoric acid solution. 10 μL of the reaction was then spotted onto a P30 filter pad and washed three times in 75 mM phosphoric acid for 5 minutes and once in methanol before drying and scintillation counting.

[0329] Compounds were tested using a 9-point curve of half-log serial dilutions. The highest concentration tested was 10 μM. Test compounds were prepared in 100% DMSO.

[0330] The results shown in Table 2 indicate that the compound of formula I is 2.5 times more potent than ganicitinib. The results for the prodrugs of formula III and IV of the present invention and prior art compounds 337, 338 are also shown in the same table below:

[0331] Table 2. Kinase Profiler TM Enzyme activity data

[0332]

[0333] As can be seen in Table 2, the results show that, unexpectedly, the compound of formula (I) is 2.1 times more potent than reference compound 338, which differs in structure only in the position of the phenolic hydroxyl group. In addition, a similar result was shown in comparison with reference compound 337, i.e., the compound of formula (I) is 1.8 times more potent than the compound, which differs in structure only in the position of the phenolic hydroxyl group and the presence or absence of a methyl group in the pyridine moiety.

[0334] As can be seen, the prior art has identified families of compounds that provide activity as TGFβ pathway inhibitors, however, surprisingly, the present invention demonstrates that the type of substitution and their position within the core structure are critical in providing the unexpectedly improved potency exhibited by the compounds of formula (I).

[0335] Furthermore, Li et al. (J. Med. Chem. 2008, 51(7):2302-2306) disclosed that electron-deficient and relatively small groups can be tolerated at the 6-position, which teaches the opposite of introducing an electron-donating group such as a hydroxyl group as a feature at the 6-position in compounds of formula (I).

[0336] In terms of prodrugs, the compound of formula I was 4.7 and 12.9 times more potent than the prodrugs of the compound of formula (I) having formulas (III) and (IV), respectively, highlighting that TGFβ inhibitory activity was blocked in vitro.

[0337] The selectivity of the compound of formula I was also measured against a panel of 97 proteins using the scanEDGE (KINOMEscan, Eurofins) assay. The selectivity profile of the compound of formula was good.

[0338] In addition, by using SafetyScreen44 from Eurofins TMServices are used for safety assessment. The targets are all strongly associated with adverse drug reactions in vivo, as mentioned in the publication Reducing safety-related drug attrition: the use of in vitro pharmacological profiling (Bowes J et al., Nature Review Drug Discovery 2012, 11 (12): 909-922). This group represents a reasonable first step in preliminary safety assessments because they provide early identification of potential off-target interactions to optimize safety margins. The safety group includes both enzyme assays and binding assays. For all proteins tested, the inhibition percentages of the compounds of Formula I at 10 μM were less than 50%. Considering the binding affinity of the compound for ALK5 (20 nM), the selectivity ratio for proteins in the SafetyScreen44 group is greater than 500 times.

[0339] In vitro luciferase assay

[0340] HEK293T cells were purchased from ATCC and cultured in DMEM (Life Technologies) supplemented with L-glutamine and 10% fetal bovine serum at 37°C and 5% CO2. Cells were seeded in 24-well plates and transfected with plasmids encoding 12xCAGA-Firefly_Luc and Tk-Renilla_Luc (75ng and 10ng per well, respectively) using polyethyleneimine (Polysciences) as a transfection reagent. After 7 hours, the culture medium was replaced with starvation medium (DMEM + 0.05% FBS). The next day, ganicitinib, a compound of formula I, and a prodrug (a compound of formula III and a compound of formula IV) (from a 10mM stock in DMSO, diluted 10 3 Up to 10 8 fold) (as indicated) and 5ng / ml recombinant human TGFB1 (Peprotech). After 16 hours, luciferase activity was measured using a dual luciferase assay kit (Promega): the culture medium was aspirated and the cells were lysed in 200μl passive lysis buffer (kit) for 20 minutes. Bioluminescence was measured in a Berthold Lumat LB6507 luminometer (18μl reagent, 10 seconds measurement).

[0341] When TGβ binds to the receptor, phosphorylation of TGFBR1 occurs, which in turn phosphorylates SMAD2 / 3, which then forms a complex with SMAD4 and enters the nucleus. The SMAD2 / 3-SMAD4 complex then binds to the promoter (or TGF-β response element) and binds to various cofactors, allowing transcription of downstream genes or luciferase reporter genes.

[0342] When the luciferase substrate (luciferin) is added to the cell lysate, the emission of light due to the production of pSMAD2 / 3–SMAD4 would suggest an active TGF-β pathway, whereas the absence or little observed light would suggest that the pathway is inhibited by the inhibitor. To normalize the results, the Stop& The firefly luciferase / substrate was quenched and the Renilla transfection control was activated, and the light generated was again measured by a luminometer and used to normalize the data.

[0343] Figure 1 It shows how the TGFβ signaling inhibitory potency of the compound of formula I is more than 2 times higher than that of ganicitinib, i.e., the IC of ganicitinib was found to be 50 The value is 359 nM and the IC of the compound of formula I 50 The value is 165 nM, while the prodrugs of the compound of Formula I (compounds of Formula III and compounds of Formula IV) have up to one-eighth lower TGFβ signaling inhibitory ability than the compound of Formula I. This means that due to the specific structural features of the compound of Formula I, its TGFβ signaling inhibitory ability is strong, and in addition, the modifications made to obtain the prodrugs (compounds of Formula III and compounds of Formula IV) successfully entrap or block the TGFβ signaling inhibitory activity.

[0344] Example 3: In vivo studies of TGFβ inhibitors

[0345] Mouse tumor organoids (MTOs) were derived from C57BL6 / J strain compound transgenic mice (conditional mutations in Apc, Kras, Trp53, and Tgfbr2, and carrying Lgr5-EGFP / CreER T2), and cultured as described in Tauriello et al. 2018 Nature 554(7693):538-543.doi:10.1038 / nature25492. Organoids are tumor epithelial cells grown in a 3D matrix in which they adopt an organotypic structure, typically as a spheroid with an inner cavity. In this way, the cells maintain relatively normal polarity (which means that the outer surface, inner / luminal surface, and cell-cell contacts are individually specialized), which is more physiological than conventional 2D cell culture. In addition, these organoids are able to recapitulate the complex tumor architecture of human cancers, including the recruitment of a rich and tumorigenic (i.e., TGFβ-rich) matrix; a system for transplanting human-like tumors and studying liver metastases has not been described before.

[0346] For in vivo anticancer efficacy and TGFβ inhibitor studies, C57BL / 6J mice were purchased from Janvier at 6 weeks of age and injected at 7-8 weeks. MTO was trypsinized (digested the matrix and broke the organoids into single cells), counted, and 300,000 cells were injected into the spleen in the form of HBSS (Hank's balanced salt solution, Lonza), thereby delivering the cells directly to the portal vein connecting the intestine to the liver. MTO129 (Tauriello et al. Nature. 2018) was used in all experiments described in the present invention.

[0347] Treatment with compounds (compounds of Formula I and ganicitinib) was performed by gavage (tube feeding) of 4 to 120 mg / ml suspension in 1% sodium carboxymethylcellulose (Sigma), 0.4% sodium dodecyl sulfate (Sigma), 0.085% polyvinyl pyrrolidone, 0.05% antifoam A (Sigma) in Milli-Q water. Mice were treated with 150 μl twice a day for 14 days or until endpoint.

[0348] The effects of treatment on biomarkers were assessed in mice where treatment was initiated two weeks after MTO transplantation and continued for three days before sacrifice. 4 μm tissue sections were stained by immunohistochemistry with an antibody specific for phosphorylated SMAD2 (Cell Signaling, 3108) using procedures well known in the art. Figure 2 Shown are nuclei (marked with arrows) that are positive for phospho-SMAD2 in control treated animals (vehicle), but absent in mice treated with ganicitinib or with a compound of Formula I or a prodrug thereof (compound of Formula IV).

[0349] Figure 3Liver tumor counts (LiM) of the compound of Formula I and ganicitinib at different doses (0.3x, 1x, 3x and 9x the molar equivalent of the usual mouse dose of ganicitinib at 80 mg / kg bid, which converts to 2 mg / mouse when assuming an average weight of 25 g) are shown. In mice, especially in mouse metastatic colorectal cancer, ganicitinib is ineffective at the standard 1x dose of 160 mg / Kg / day; in the above-mentioned MTO paper (Tauriello et al. Nature et al. 2018), we need to use doses up to 1600 mg / kg / day (10x) to effectively treat metastatic cancer. However, the dose may cause toxicity (cardiotoxicity, muscle tissue and cartilage defects, bone development, and altered inflammatory responses in the skin and intestines). In patients, the treatment dose is reduced to 80 to 150 mgb.id in a 14-day medication-14-day withdrawal regimen. As Figure 3 As shown in , the compound of Formula I caused significantly lower amounts of liver tumors when compared to ganicitibi.

[0350] MTO expressing a luciferase reporter gene were measured by bioluminescence imaging using retro-orbital injections of 50 μl of 15 mg / ml D-luciferin potassium salt (Resem BV). Photons per second from regions across the thorax / abdomen were quantified and normalized to measurements on the day of injection. Figure 4 The ability of the compound of formula I and ganicitinib to inhibit metastatic tumor growth at different doses (0.3x, 1x, 3x and 9x the mouse dose of 80 mg / kg bid (i.e., 160 mg / kg / day)) during a 12-day treatment period (day 3 to day 14) is shown. Figure 4 As seen in Figure 2, the 1x dose (160 mg / kg / day mouse dose) was ineffective for ganicitinib and had to be increased to a 3x dose (corresponding to 480 mg / kg / day) to be most effective (the 9x dose of 1440 mg / kg / day was highly effective, as shown in Figure 2). Figure 3 ), while the compound of Formula I was effective at a molar equivalent of 0.3x dose (corresponding to 42.5 mg / Kg / day; this is after correction for the molecular weight ratio of Formula I to ganicitinib monohydrate of 0.88:1, since the molecular weight of the compound of Formula I is 342.4 and the molecular weight of ganicitinib is 387.4), showing surprisingly and strongly improved in vivo anti-tumor activity.

[0351] Example 4: Toxicity Assessment

[0352] For safety assessments on proteins often associated with toxicity, see the SafetyScreen44 assay in Example 2.1.

[0353] To evaluate the toxicity of the compounds of Formula I in vitro, HEK293T cells were seeded in 96-well plates and treated with the indicated compounds for 24 hours. DMSO was used as a control. The compounds of Formula I or ganicitinib were added at different concentrations ranging from 1 to 100 μM. Cell viability was assessed by XTT assay (Biological Industries) according to the manufacturer's instructions and incubated for 4 hours before measurement. The cell viability at 100 μM was 64%, and the cell viability at 30 μM was 93% ( Figure 5 ), which indicates extremely low toxicity at physiological levels, especially when considering the IC of the compound of Formula I for inhibition of ALK5 50 When the value is in the nM range.

[0354] To evaluate in vivo toxicity, mice were inoculated with MTO129 and Figure 3 As described in, different molar equivalent doses of ganicitinib and compounds of formula I were treated within two weeks. The number of cured mice (i.e., mice without metastasis at the end of the experiment) was scored. The intestines, skin samples, hearts, thoracic bones, and limbs of these mice were fixed in phosphate-buffered 10% formalin (Sigma). The soft tissues were embedded in paraffin blocks, sliced, and stained with hematoxylin and eosin. The bones were first decalcified for 2 weeks and then embedded, sliced, and stained. An experienced pathologist analyzed the tissue for histopathological abnormalities. It was found that the obvious pectus excavatum phenotype (collapse within the sternum) was associated with defects in the sternocostal cartilage region (cartilage present between the sternum and the ribs). In addition, for defects in the hypertrophic zone, the cartilage ossification zone at the end of the long bones was analyzed. No pathological abnormalities were observed in the analysis of the skin and intestine using 1x doses of ganicitinib or compounds of formula I.

[0355] Table 3: Comparison of efficacy and toxicity of ganicitinib (Gal) and compounds of formula I. Toxicity was scored for each number of mice tested, combined and reported as a percentage; nd: not determined

[0356]

[0357] Table 3

[0358] Ganicitinib was effective (>50% efficacy) starting at 3x dose equivalents (480 mg / kg / day) and was toxic (>50% toxicity) at 9x (1440 mg / kg / day). The compound of Formula I was effective at 0.3x dose equivalents (42.5 mg / kg / day) and was also toxic (>50% toxicity) at 9x dose equivalents (1272 mg / kg / day).

[0359] Based on this, the therapeutic index (ratio between toxic dose / effective dose) of the compound of formula I compared to ganicitinib can be calculated.

[0360] Ganicitinib therapeutic index = 9 / 3 = 3. Compound of formula I therapeutic index = 9 / 0.3 = 30. The results show that the compound of formula I provides at least a 10-fold improvement compared to Ganicitinib.

[0361] Example 5: Synthesis of prodrug of formula VII:

[0362] In order to couple the vanilla acetonide fragment to 4-hydroxytamoxifen, the halogenated vanilla acetonide fragment was first synthesized starting from commercial vanilla acetonide.

[0363] 1-(4-(Benzyloxy)-3-methoxyphenyl)ethan-1-one

[0364]

[0365] To a stirred solution of vanilla acetone (6 g, 36.1 mmol) in DMF (120 mL) was added K2CO3 (7.48 g, 54.15 mmol, 1.5 eq.) at room temperature, followed by dropwise addition of benzyl bromide (4.66 mL, 38.99 mmol, 1.08 eq.). The reaction mixture was stirred at 40 °C for 14 hours, then cooled to room temperature, where it was poured into a 1 L water / ice mixture and stirred for 10 minutes. The suspension was then filtered using a Büchner funnel under vacuum, the solid was washed with water (3x70 mL) at 0 °C, and then left in a Büchner funnel under vacuum for one hour. The solid was then transferred to a round-bottom flask and dried to constant weight on a high vacuum line to give 8.99 g (97% yield) of the title compound (C. Miesch, T. Emrick, J. Colloid Interface Sci. 2014, 425, 152–8).

[0366] 1 H NMR (400 MHz, CHLOROFORM-d) δ 7.55 (d, J = 2.0 Hz, 1H), 7.50 (dd, J = 8.4, 2.0 Hz, 1H), 7.43 (dd, J = 8.2, 1.4 Hz, 2H), 7.38 (m, 2H), 7.34–7.29 (m, 1H), 6.91–6.87 (m, 1H), 5.23 (s, 2H), 3.94 (s, 3H), 2.55 (s, 3H).

[0367] 1-(4-(Benzyloxy)-3-methoxyphenyl)-2-bromoethan-1-one (5-8a)

[0368]

[0369] To a stirred solution of the previously obtained 1-(4-(benzyloxy)-3-methoxyphenyl)ethan-1-one (7.86 g, 30.69 mmol) in EtOH (150 mL) was added bromine (4.90 g, 30.69 mmol, 1 eq) at 60 °C. The reaction mixture was stirred for 6 hours and then allowed to slowly cool to room temperature overnight. In the morning, the reaction mixture was cooled to 0 °C where it was kept stirring for one hour. It was then filtered through a glass Buchner funnel, washed with cold EtOH (2 x 30 mL) and then dried on a high vacuum line. This gave the title compound (7.25 g, 71%) as a white solid (neat, filtered through 1 1H NMR).

[0370] 1 H NMR (400MHz, cdcl3) δ7.58–7.49(m,2H),7.46–7.29(m,5H),6.92(d,J=8.4Hz,1H),5.25(s,2H),4.39(s,2H),3.95(s,3H).

[0371] The synthesis of the prodrug of formula VII starting from 4,4'-(2-phenylbut-1-ene-1,1-diyl)diphenol (CAS No. 4120-45-0) can be as shown in Scheme VIII:

[0372] Scheme VIII

[0373]

[0374] 3-(4-(1-(4-hydroxyphenyl)-2-phenylbut-1-en-1-yl)phenoxy)propane-1,2-diol, (5-46)

[0375]

[0376] In a sealed flask, NEt in EtOH (24mL) is added to the solution of diol 5-43 (1.6g, 5.06mmol) (0.706mL, 5.06mmol, 1 equivalent) and glycidol (0.301mL, 4.55mmol, 0.9 equivalent), and heated until 80 degrees overnight. In the morning, the reaction mixture is cooled to room temperature and the solvent is removed in a vacuum. Residue is distributed between EtOAc (40mL) and saturated ammonium chloride (40mL), then organic layer MgSO4 is dried and concentrated to dryness in a vacuum. Residue obtains title compound by flash column chromatography purification, is viscous colorless oil (735mg, 37.2% yield).

[0377] 1 H NMR(400MHz,cd3od)δ7.19–7.06(m,1H),7.04(d,J=8.4Hz,1H),6.96(t,J=5.7Hz,1H),6.82–6.74(m,1H),6.67(d,J=8.5Hz,1H) ,6.62–6.55(m,1H),6.42(d,J=8.5Hz,1H),4.13–3.80(m,1H),3.79–3.53(m,1H),2.49(p,J=7.5Hz,1H),0.92(t,J=7.4Hz,1H).

[0378] 13 C NMR(101MHz,cd3od)δ159.14,158.29,157.25,156.36,144.11,144.10,1 41.99,141.80,139.74,139.71,137.87,137.50,136.34,135.98,133.02, 133.00,131.57,130.89,130.87,128.84,126.94,115.84,115.18,115.09 ,114.35,71.84,71.76,70.34,70.08,64.21,64.14,29.89,29.85,13.94. **Due to the fact that the double bond is a 1:1 mixture of cis and trans isomers, and the fact that the glycidol used is a racemate, there are more carbons than expected. This is caused by diastereomers.**

[0379] 4-(1-(4-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)phenyl)-2-phenylbut-1-en-1-yl)phenol, (5-49)

[0380]

[0381] Under N2, a solution of 5-46 (106 mg, 0.271 mmol) was added to a flask and dissolved in acetone (3 mL) at room temperature, wherein p-TSA (7.7 mg, 0.040 mmol, 0.15 equivalent) and 2,2-dimethoxypropane (66 μL, 542 mmol, 2 equivalents) were added at 50 degrees, and it was reacted for 5 hours. After all starting materials disappeared by TLC, the reaction mixture was cooled to room temperature, wherein it was diluted with 10% NaHCO3 (2 mL) and EtOAc (6 mL), each phase was separated, and the aqueous layer was extracted with EtOAc (2x6 mL), the organic layers were merged and dried with MgSO4, and then concentrated in a vacuum. The residue was then purified by silica to obtain the title compound (102 mgs, 87% yield), which was a colorless oil.

[0382] 1 H NMR(400MHz,cdcl3)δ7.19–7.04(m,7H),6.92–6.83(m,1H),6.82–6.69(m,3H),6.56–6.52(m,1H),6.48–6.44(m,1 H), 4.96 (s, 0.5H), 4.70 (s, 0.5H), 4.54–3.77 (m, 5H), 2.52–2.42 (m, 2H), 1.49–1.34 (m, 6H), 0.92 (t, J = 7.4Hz, 3H).

[0383] 13 C NMR (101MHz, cdcl3) δ157.20,156.37,154.22,153.41,142.53,141.20,141.16,137. 63,137.61,136.69,136.33,136.23,135.89,132.09,131.92,130.76,130.59,129.6 7,127.83,127.81,125.92,114.93,114.23,114.04,113.29,109.79,109.69,74.03,73.98,68.71,68.42,66.89,66.80,29.01,29.00,26.79,26.73,25.37,25.34,13.59.

[0384] **Due to the fact that the double bond is a 1:1 mixture of cis and trans isomers, and the fact that the glycidol used is a racemate, there are more carbons than expected. This is caused by diastereomers.

[0385] IR(Thin Film):3400,2920,2851,1722,1673,1598,1496,1273cm -1

[0386] HRMS (ESI): m / z (M+H) for C 28 H 30 Calculated value of O4: 430.2144; experimental value: 430.2143.

[0387] 1-(4-(Benzyloxy)-3-methoxyphenyl)-2-(4-(1-(4-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)phenyl)-2-phenylbut-1-en-1-yl)phenoxy)ethan-1-one, (5-50)

[0388]

[0389] To 5-49 (500mg, 1.16mmol, 1 equivalent) in the stirring solution of acetone (15mL), add K2CO3 (321mg, 2.32mmol, 2 equivalents), then add α bromoketone 5-8a (582mg, 1.74mmol, 1.5 equivalents), wherein it is stirred at room temperature overnight. The next morning, the solvent is removed in a vacuum, wherein then it is diluted with EtOAc (50mL) and water (30mL). Separate each phase, and water layer is extracted with EtOAc (2x20mL), organic layer is merged and MgSO4 is used for drying, then concentrated in a vacuum. Then residue is passed through silica purification, obtains title compound (737mg, 92.7% yield), is colorless oil.

[0390] 1 H NMR (400MHz, cdcl3) δ7.61–7.27(m,7H),7.19–6.84(m,10H),6.78–6.71(m,2H),6.59–6.50(m,2H),5.30–5.05(m,4H),4.53–4.34(m, 1H),4.22–4.02(m,1H),3.99–3.86(m,5jH),3.85–3.74(m,1H),2.46(q,J=7.5Hz,2H),1.49–1.34(m,6H),0.91(td,J=7.3,1.0Hz,3H).

[0391] 13C NMR(101MHz,cdcl3)δ193.25,193.20,157.39,156.98,156.55,156.15,153.18,153.05,149.91,149.82,142.62,142.58,141.53,141.48, 137.66,137.23,136.76,136.73,136.26,136.23,132.10,132.07,13 0.78,130.75,129.80,128.86,128.84,128.32,128.30,128.13,128.1 2,127.98,127.31,127.30,126.09,122.75,122.68,114.53,114.20,113.79,113.45,112.38,112.32,110.94,110.91,109.88,109.78,74.17,74.12,70.98,70.95,70.91,70.78,68.89,68.60,67.06,66.97,56.27,56.23,29.19,29.14,26.98,26.89,25.52,25.50,13.74,13.73.

[0392] **Due to the fact that the double bond is a 1:1 mixture of cis and trans isomers, and the fact that the glycidol used is a racemate, there are more carbons than expected. This is caused by diastereomers.**

[0393] UPLC-MS: 685.9 (M+H)

[0394] 1-(4-(Benzyloxy)-3-methoxyphenyl)-2-(4-(1-(4-(2,3-dihydroxypropoxy)phenyl)-2-phenylbut-1-en-1-yl)phenoxy)ethan-1-one, (5-51)

[0395]

[0396] The stirred solution of above-mentioned 5-50 (634mg, 1.09mmol) is dissolved in 6:1THF: water (20mL), wherein 2M HCl (2mL) is added at room temperature. Follow the reaction by TLC, and after all SM consumption, use 10%NaHCO (10mL) quencher. The reaction mixture is diluted with EtOAc and each phase is separated, and aqueous phase is extracted with EtOAc (5mL) and chloroform (5mL). By the organic extract MgSO4 merged, dry, then concentrate in a vacuum. Then residue is purified by silica (MeOH / DCM), obtain title compound (527mg, 89% yield), is colorless oil.

[0397] 1 H NMR(400MHz,cdcl3)δ7.61–7.30(m,7H),7.19–7.05(m,7H),6.95–6.85(m,3H),6.79–6.72(m,2H),6.5 9–6.51(m,2H),5.26–5.02(m,4H),4.23–3.53(m,8H),2.50–2.40(m,2H),0.91(td,J=7.3,1.1Hz,3H).

[0398] 2-(4-(1-(4-(2-(4-(benzyloxy)-3-methoxyphenyl)-2-oxoethoxy)phenyl)-2-phenylbut-1-en-1-yl)phenoxy)acetaldehyde, (5-52)

[0399]

[0400] To a stirred solution of the above-mentioned 5-51 (250 mg, 0.388 mmol) in DCM (7 mL) was added NaIO4 (249 mgs, 1.16 mmol, 3 equivalents) in water (2 mL) at room temperature. The reaction was stirred vigorously for 3 hours, wherein most of the starting material was consumed. It was diluted with saline (4 mL), and then each phase was separated. The organic phase was dried (MgSO4), and the solvent was removed in vacuo, and then purified by flash column chromatography to obtain the title compound as a colorless oil (155 mgs, 63% yield).

[0401] 1H NMR(400MHz,cdcl3)δ9.88(br s,0.5H),9.77(br s,0.5H),7.62–7.28(m,7H),7.20–7.05(m,7H),6.94–6.84(m,3H),6.81–6.70(m,2H),6.60–6.49( m,2H),5.33–4.98(m,4H),3.93(d,J=14.5Hz,4H),2.51–2.38(m,2H),0.92(dt,J=8.5,4.2Hz,3H).

[0402] 1-(4-(Benzyloxy)-3-methoxyphenyl)-2-(4-(1-(4-(2-(dimethylamino)ethoxy)phenyl)-2-phenylbut-1-en-1-yl)phenoxy)ethan-1-one (5-54)

[0403]

[0404] To a stirred solution of 5-52 (143 mg, 0.233 mmol, 1 eq.) in THF (5 mL) was added a 2M solution of dimethylamine in THF (0.175 mL, 0.350 mmol, 1.5 eq.) at room temperature under N2, followed by the addition of Na(AcO)3BH (75 mg, 0.700 mmol, 3 eq.) in portions. The reaction was allowed to react for 5 hours, at which time no SM was seen by TLC. The reaction mixture was then diluted with brine (3 mL) and EtOAc (10 mL), and the phases were separated after stirring for 15 minutes. The aqueous phase was extracted with EtOAc (2x10 mL) and chloroform (1x10 mL), the organic extracts were combined, dried (MgSO4), and the solvent was removed in vacuo. The residue was purified by flash column chromatography (DCM / MeOH-0-15%) to give the title compound as a viscous oil (93 mgs, 62% yield).

[0405] 1H NMR(400MHz,cdcl3)δ7.62–7.55(m,1H),7.55–7.48(m,1H),7.46–7.28(m,5H),7.21–7.06(m, 7H),6.94–6.81(m,3H),6.78–6.71(m,2H),6.59–6.45(m,2H),5.24(s,1H),5.22(s,2H),5.06( s, 1H), 4.26 (t, J = 5.1Hz, 1H), 4.11–4.07 (m, 1H), 3.95 (s, 1.5H), 3.91 (s, 1.5H), 3.11 (dt, J = 3 6.9, 4.3Hz, 2H), 2.63 (s, 3H), 2.57 (s, 3H), 2.46 (q, J = 6.7Hz, 2H), 0.91 (td, J = 7.4, 1.8Hz, 3H).

[0406] 2-(4-(1-(4-(2-(dimethylamino)ethoxy)phenyl)-2-phenylbut-1-en-1-yl)phenoxy)-1-(4-hydroxy-3-methoxyphenyl)ethan-1-one, (prodrug of formula VII)

[0407]

[0408] Pd / C (9 mg) was added to a stirred solution of 5-54 (94 mg, 0.146 mmol) in degassed 10% MeOH / EtOAc (12 mL) at room temperature. In three cycles using H2 balloons and vacuum, N2 atmosphere was replaced with H2 atmosphere. The reaction was tracked by TLC, and when SM was consumed, the atmosphere was replaced with N2 and purged for 5 minutes. The reaction mixture was filtered through diatomaceous earth and the solvent was removed in vacuo. The residue was purified by flash column chromatography (DCM / MeOH-gradient) to obtain the title compound, which was a viscous oil (74 mg, 92% yield).

[0409] 1H NMR(400MHz,cdcl3)δ7.65–7.58(m,1H),7.55–7.50(m,1H),7.17–7.05(m,7H) ,7.00–6.86(m,3H),6.79–6.71(m,2H),6.59–6.51(m,2H),5.23(s,1H),5.07( s,1H),4.14(t,J=5.6Hz,1H),4.01–3.93(m,4H),2.86(t,J=5.6Hz,1H),2.77( t,J=5.4Hz,1H),2.50–2.40(m,5H),2.38(s,3H),0.91(td,J=7.4,2.0Hz,3H).

[0410] Example 6: LigF enzyme assay

[0411] 6.1 Synthesis of 4-methylumbelliferyl vanilla acetone

[0412] 4-Methylumbelliferone (4-MU) derivatives are known compounds. The synthesis of MUAV can be illustrated as follows:

[0413]

[0414] 7-(2-(4-(Benzyloxy)-3-methoxyphenyl)-2-oxoethoxy)-4-methyl-2H-chromen-2-one, (5-4b)

[0415]

[0416] To a stirred solution of 4-methylumbelliferone (400 mg, 2.27 mmol) in acetone (30 mL) was added 1-(4-(benzyloxy)-3-methoxyphenyl)-2-bromoethyl-1-one (5-8a, synthesized in Example 5) (967 mg, 2.72 mmol, 1.2 equivalents) at 25 ° C. The reaction mixture was stirred for 16 hours, then filtered, and the solid residue was washed with acetone (2x30 mL). The solvent was then removed under reduced pressure, then suspended in toluene and heated until a solution was formed. The flask was then cooled to room temperature within 90 minutes, where it was filtered, washed with cold toluene (10 mL), and dried on a high vacuum line. This gave the title compound as a white solid (801 mg, 82%).

[0417] 1H NMR (400MHz, cdcl3) δ7.57–7.30(m,8H),6.94(d,J=4.5Hz,2H),6.78(d,J=2.5Hz,1H),6.14(d,J=1.2Hz,1H),5.31(s,2H),5.26(s,2H),3.95(s,3H).

[0418] 7-(2-(4-Hydroxy-3-methoxyphenyl)-2-oxoethoxy)-4-methyl-2H-chromen-2-one, (5-4a)

[0419]

[0420] At room temperature, 5-4b (500 mg, 1.16 mmol) and 10% Pd / C (25 mg, 5% wrt relative to the mass of sm) in degassed 10% MeOH / EtOAc (10 mL) were added to a pressure reactor. 1 bar (g) of hydrogen was loaded into the reactor, and the reaction was tracked by TLC using a suitable needle sampling that could withstand pressure. After 4 hours, the starting material had been consumed. Hydrogen was released from the reactor and replaced with nitrogen via 3 vacuum-nitrogen cycles. The reaction mixture was diluted with CHCl3 (20 mL) and filtered through diatomaceous earth. The filtrate was concentrated under reduced pressure. The crude material was subjected to chromatographic analysis (0-10% MeOH: DCM) to obtain the title compound 5-4a (307 mg, 78%) as a white solid.

[0421] 1 H NMR (400MHz, chloroform-d) δ7.59–7.55(m,2H),7.51(d,J=8.8Hz,1H),7.00(d,J=8.1Hz,1H),6.94(dd,J=8.8,2.6Hz,1H),6.79(d,J=2.6Hz,1H),6.18(br s, 1H), 6.14 (d, J = 1.2Hz, 1H), 5.33 (s, 2H), 3.97 (s, 3H), 2.39 (d, J = 1.2Hz, 3H).

[0422] 6.2 In vitro enzymatic fluorescence assay

[0423] In order to study the kinetics of cleavage of the ether bond in the prodrugs of Formula II and Formula V in general (including the compound of Formula III, i.e., the prodrug of the compound of Formula I; the compound of Formula VII, i.e., the prodrug of 4-hydroxytamoxifen; and the compound of Formula IX, i.e., the prodrug of SN-38), and to clarify the selectivity of the enzyme for the recognition fragment, the fluorescent compound fluorescent 4-methylumbelliferone was used as a model compound.

[0424]

[0425] To test the enzymatic activity of LigF, pure LigF protein was expressed by the protein expression core facility within IRB (Barcelona) by inserting His-tagged LigF DNA into the pOPINF vector and expressed in Escherichia coli (E. coli) BL21 (DE3) Rosetta lysS cells. After expression and lysis, the protein was purified by using a nickel column followed by gel filtration. It is noteworthy that the His tag was not removed from the protein after purification.

[0426] For in vitro fluorescence assay, purified LigF was diluted to 15 μg / ml in 20 mM Tris-HCl (pH 7.5), and 1 mM glutathione and 30 μM 4-methylumbelliferyl vanilla acetonide (MUAV) model compound were added.

[0427] Reactions were performed in 96-well solid polystyrene plates (Corning) and measured at 360 excitation, 485 emission, and 100 sensitivity using a top well plate reader (BioTek FL600 Fluorometer).

[0428] Due to the rapid rate of production of 4-methylumbelliferone (4-MU) caused by the cleavage of the β-ether bond of compound MUAV, the protocol described by Masai (E. Masai et al., J. Bacteriol. 2003, 185, 1768–75) was modified by reducing the concentration of LigF enzyme. Using this optimized protocol, compound MUAV was incubated at room temperature in the presence of buffer glutathione (GSH) and LigF, and the cleavage was tracked by observing the production of 4-MU using fluorescence. Appropriate controls (including wells without GSH, LigF, and MUAV) showed that the ether bond was not broken down under the assay conditions and that enzyme activity was required. The enzyme LigF cleaved the product MUAV in approximately 30-40 minutes ( Figure 6 ).

[0429] For the assay in cell culture, cells were stably transduced to express LigF by lentiviral infection using the pLenti PGK puro vector and packaging plasmids pCAG-RTR2, pCAG-VSVG, and pCAG-KGP1R. After selection with 1 μg / ml puromycin (InvivoGen), infected cells were seeded into 24-well plates and 4-methylumbelliferyl vanilla acetone (MUAV) was added. At the time point after adding various concentrations of MUAV (i.e., 10, 30, and 100 μM), the culture medium was taken for measurement of 4-MU fluorescence.

[0430] Figure 7The results showed that the ether bond of MUAV was indeed cleaved by cell-expressed LigF, as indicated by a dose-dependent increase in fluorescence. No cleavage was observed in the following control experiment: HEK293T cells expressing empty vector (instead of LigF) treated with 100 μM. These experiments indicate that LigF is active in mammalian cells and that no other enzymes capable of cleaving the β-ether bond are present, at least in the cells tested.

[0431] 6.3. In vitro LigF assay for prodrugs of Formula VII

[0432] For further testing in an in vitro enzymatic assay with LigF and vanillyl acetonide-derived prodrugs, the compound of Formula VII, a prodrug of 4-hydroxytamoxifen, was used.

[0433] Analogously to the in vitro assay in 5.2., the compound of formula VII was diluted from a 30 mM stock in DMSO to 50 μM in 20 mM Tris-HCl (pH 7.5), 1 mM glutathione and 15 μg / ml Lig F were added. However, the reaction mixture was incubated at 37°C and the readout was different: HPLC-MS / MS was used. The reaction samples, as well as the control samples without enzyme and Z-4-OHT (4-hydroxytamoxifen) as a standard were diluted 2:3 in MeOH and 2 μl was injected onto a BioBasic C18 column (5 μm, 2.1×150 mm, Thermo; using a Thermo EC Finnigan Mod Micro AS autosampler and Thermo EC Quarternary pump, Finnigan Mod. Surveyor MS chromatograph) using a water-MeOH gradient (60-90% MeOH in 30 minutes; 90-100% in 5 minutes; 100 μl / min flow rate). LC-MS coupling was performed using an Advion Triversa Nanomate (Advion BioSciences, Ithaca, NY, USA) as a nano-ESI source for nano-electrospray by chip technology. Nanomate is attached to LTQ-FT Ultra mass spectrometer and runs under the spray voltage of 1.7kV and the delivery pressure of 0.5psi in positive mode.In LTQ-FT Ultra (Thermo Scientif) mass spectrometer, 40V capillary voltage and 120V sleeve lens voltage are used for analysis.MS / MS is operated in data dependent acquisition (DDA) mode and selected reaction monitoring (SRM) mode.In FT, obtain inspection MS scan, wherein resolution (defined under 400m / z) is set to 100,000.In each scan, the maximum six of the strongest ions are fragmented and detected in the linear ion trap.For the inspection scan, the ion count target value is 1,000,000, and for the MS / MS scan, the ion count target value is 50,000.The target ion selected for MS / MS has been dynamically excluded for 30s.For SRM, the maximum injection amount is set to 400ms and an average of 2 micro scans.

[0434] Data were expressed as XIC (Extracted Ion Chromatogram) peak areas of parallel reaction monitoring (PRM) transitions using QuanBrowser (Xcalibur software 2.0 SR2).

[0435] LigF is able to cleave the β-ether bond of the prodrug of formula VII connecting the recognition fragment (vanillyl acetone) and the 4-OHT core, thereby producing 4-OHT. Note that in control experiments, when all other components except LigF are present, the molecule remains intact, indicating extended stability at body temperature. Unexpectedly, the E-isomer of the prodrug of formula VII cleaves at a faster rate than the Z isomer, resulting in the specific release of the isomer (Z)-4-OHT. (The fact that the E-isomer of the prodrug of formula VII cleaves to obtain (Z)-4-OHT is caused by the Cahn-Ingold-Prelog priority rule.)

[0436] Figure 8 HPLC-MS / MS experiments showing cleavage of the beta ether bond of the prodrug of Formula VII by LigF to give 4-OHT. (A) Negative control. (B) Time = 0. (C) Time = 1 hour. (D) Time = 3 hours. (E) Time = 44 hours.

[0437] Cleavage of the prodrug of Formula VII reaches a 50% plateau after approximately 30 hours and reaches halfway to the plateau after 3 hours, which we envision as sufficient rates to be a useful in vivo tool, such as Fig. 9 As seen in.

[0438] 6.4 Cell co-culture assay:

[0439] These assays were performed to assess whether this specific LigF enzymatic reaction for compounds of Formula VII can occur in a cellular setting, including whether this prodrug can cross the cell membrane and whether, after cleavage by LigF, 4-OHT can diffuse to neighboring cells and activate mutant ER T2 In this example, the fusion proteins are Cre-ER T2 and the active portion of the transcription factor (NTCF4-ER T2 )-.

[0440] For the first experiment, in which genetic recombination in cells adjacent to cells expressing LigF was confirmed, mouse embryonic fibroblasts (MEFs) were used. The mouse model carries the recombination reporter gene mTmG (JAX Catalog No.: 007676). This system expresses membrane-bound tdTomato (tandem dimer tomato; mT stands for membrane Tomato), which is a red fluorescent protein. After recombination with activated Cre enzyme, the coding sequence for mT between the loxP sites and the stop codon are excised. This in turn leads to the expression of membrane-bound enhanced green fluorescent protein (EGFP; mG). Effectively, this means that in the presence of Cre enzyme or in the Cre-ER activated by 4-OHT,T2 In the presence of the fusion protein, the cells ceased their tdTomato production and continued to express EGFP; this genetic conversion can be efficiently quantified by flow cytometric analysis. T2 Fusion protein mouse hybrid (UbC-Cre-ER T2 ; Jax Catalog No.: 008085). From double transgenic mice and from mice without Cre-ER T2 In the reporter strain of , MEFs were harvested from 14-day-old embryos. Briefly, 14 days after plug formation, pregnant females were sacrificed, the uterus was removed, and the embryos were dissected: the head and fetal liver were discarded. The remainder was minced and trypsinized and grown in 10-cm dishes with DMEM supplemented as above.

[0441] MEF Reconstitution: MTO129 cells transduced with pLenti PGK-LigF(puro) or with an equivalent empty vector were trypsinized and single cells were added with UbC-Cre-ER T2 On; mTmG (or control only mTmG) MEF grown in 12-well plates. The next day, Z-4-OHT or the prodrug of formula VII was added at a concentration in the range of 1000 to 1nM. Two days later, the cells were trypsinized and analyzed by flow cytometry for TdTomato / EGFP fluorescence (Gallios, Beckman Coulter). The fluorescence on living cells was analyzed in 2 dimensions and scored as a percentage in 4 quadrants. EGFP-positive quadrants (Q2 and Q4, with or without TdTomato) were considered to be recombined, and the scores in the overall population were calculated. Data are the mean values ​​(+SD) from two independent experiments.

[0442] Fig.10 The results of the assay are shown, where the bar graph below UVCre-mTmG shows the expression of Cre-ER T2 The results obtained with MEFs containing the recombinant reporter cassette (mTmG) are shown in the figure below, while the bar graph below Con-mTmG shows the lack of Cre-ER T2 Results of MEFs expressing WT and WT were used as control.

[0443] As in Fig.10 As seen in the Cre-ER T2 Regardless of expression, there was a dose-dependent genetic recombination by the addition of 4-OHT. When the prodrug of Formula VII was added, this achieved a pattern of genetic recombination in MEFs that was very similar to 4-OHT alone in MEFs expressing LigF etherase. T2In MEFs of 100 μg / mL, there was only some recombination at the highest dose, which could be explained by a small amount of impurities or intrinsically reduced activity. This suggests that the prodrug of Formula VII is largely inactive, but is cleaved in MTO129 expressing Lig-F co-cultured with MEFs, and this reaction is sufficient to allow the released 4-OHT to diffuse to MEFs and affect genetic recombination.

[0444] For which ER T2 The second experiment, in which the fusion product was a transcription factor, used LS174T-NTCF4-ER in co-culture with HEK293T cells expressing Lig-F. T2 (LS-NE) cells (as described in Whissell et al., 2014, Nature Cell Biology, 16, 695-707. Original LS174T cells were purchased from ATCC). These LS-NE cells were cultured in DMEM (Life Technologies) supplemented with L-glutamine and 10% fetal bovine serum at 37°C and 5% CO2.

[0445] NTCF4 represents the N-terminus of the transcription factor TCF4. This construct has a dominant negative function, shutting down signaling in the Wnt pathway (Wnt-OFF), which is important in development and (intestinal) stem cell regulation. In addition, most colorectal cancer (CRC) cells are addicted to Wnt signaling, so that its inhibition will lead to a strong response in the gene expression of pro-tumorigenic stem cell genes (down, inhibition) and cell differentiation genes (up, activation). T2 The fusion of NTCF4-ER keeps this construct outside the nucleus and is therefore inactive. T2 In the modified CRC cell line LS174T, addition of 4-OHT resulted in the described changes in endogenous gene expression.

[0446] HEK293T cells transduced with pLenti PGK-LigF(puro) or with an equivalent empty vector were co-transduced with LS174T-NTCF4-ER T2(LS-NE) cells were co-plated in 6-well plates at a ratio of 1 / 10. Co-cultures were treated with DMSO, 1 μM Z-4-OHT or a prodrug of Formula VII at 1 or 10 μM. After 16 hours, cells were lysed in trizol for RNA extraction (PureLinkRNA Mini Kit, Life Technologies) and cDNA preparation (High Capacity cDNA RT kit, Applied Biosysytems). On the StepOnePlus real-time PCR system (Applied Biosystems), Taqman probes (Applied Biosystems) were used to analyze stem cell genes ASCL2 (inhibited by 4-OHT-mediated Wnt-OFF; Hs_00270888_S1), differentiation genes KRT20 (activated by 4-OHT-mediated Wnt-OFF; Hs_00300643_m1) and housekeeping genes PPIA (Hs_99999904_m1). Fig.11 The data in have been normalized for housekeeping genes and DMSO controls. The data are the mean (+SD) of technical replicates from a single experiment. While in the presence of HEK293T negative for LigF, only 4-OHT was able to induce a transcriptional switch (activating gene KRT20 up, and repressing gene ASCL2 down), in the presence of HEK293T-LigF, the prodrug of Formula VII achieved the same response at 10 μM.

[0447] Example 7: In vivo studies of 4-OHT prodrugs of formula VII

[0448] Use UbCre-ER as described in Example 6 T2 ; mTmG mice, we treated these animals with the prodrug of Formula VII at 1 or 5 μmol (approximately 0.6 or 3 mg, respectively) or with vehicle control (oil). Five days after treatment, the mice were sacrificed and the livers were fixed overnight in 10% phosphate buffered formalin (sigma) and embedded in paraffin, similar to the description in Example 3 ( Figure 1 ). Sections were cut out and stained for EGFP using immunohistochemistry. Fig.12 The results shown in show that the enzyme present in mouse liver (but not in our cultured cells, see Example 6) is able to cleave the prodrug of formula VII, leading to a dose-dependent activation of gene recombination and EGFP expression.

[0449] Example 8: Additional studies of TGFβ signaling inhibition in vivo using cancer models.

[0450] Additional in vivo studies were conducted to further evaluate the efficacy of the compounds of formula I of the present invention and their prodrugs of formula IV relative to the prior art reference compounds ganicitinib and reference compound 338. Mouse tumor organoids (MTO129) were developed as shown in Example 3. For in vivo anticancer efficacy and TGFβ signaling inhibition studies, C57BL / 6J mice were purchased from Janvier at 6 weeks of age and injected at 7-8 weeks. In order to transplant MTO129 into the liver, the cells were trypsinized (digesting the matrix and breaking the organoids into single cells), counted, and 300,000 cells were injected into the spleen in the form of HBSS (Hank's Balanced Salt Solution, Lonza), thereby delivering the cells directly to the portal vein connecting the intestine to the liver.

[0451] The compound (compound of formula I, compound of formula IV, ganicitinib or compound 338) was prepared as a suspension in a vehicle solution consisting of 1% sodium carboxymethylcellulose (Sigma), 0.4% sodium dodecyl sulfate (Sigma), 0.085% polyvinyl pyrrolidone, 0.05% antifoam A (Sigma) in milli-Q water. Treatment with the compound was performed by gavage (tube feeding), with 150 μl of the compound suspension treated twice a day. Control mice were treated with 150 μl of vehicle.

[0452] exist Fig.13 In the experiment shown, the efficacy of the indicated compound in inhibiting metastasis formation was evaluated. Mice were treated with the indicated compound starting two days after MTO129 transplantation and continued for two weeks. Mice were killed 4 weeks after transplantation and liver metastasis (LiM) was counted by naked eye on fixed livers. Figure 3 Liver tumor counts (LiM) of the compound of Formula I and ganicitinib at different molar equivalent doses (9x, 3x, 1x, 0.3x) as described in the legend are shown. The compound of Formula I at a 1x dose (70.8 mg / Kg bid) provided significantly improved activity in preventing metastasis formation when compared to the reference compound 338 (the closest compound to the compound of Formula I in structure) at the same molar dose (70.8 mg / Kg bid).

[0453] also, Fig.13 It is shown that the compound of formula I is still effective in preventing liver metastasis at a 0.3x dose (21.25 mg / Kg bid), and this therapeutic effect is comparable to a 3x dose of ganicitinib. In addition, the activity of the compound of formula I is significantly superior to that of reference compound 338, which is highly unexpected because reference compound 338 is a structural isomer of the compound of formula I.

[0454] The prodrug of the compound of Formula I (Formula IV) more potently reduced the number of liver metastases in this assay compared to ganicitinib.

[0455] in addition, Fig.14 and Fig.15 The experiments shown evaluate the therapeutic efficacy of the compound of Formula I against overt metastatic disease compared to the prior art compound, ganicitinib, both at 1x equimolar doses (70.8 mg / kg bid for the compound of Formula I and 80 mg / Kg bid for ganicitinib). Fig.13 The protocol for vaccination of mice with MTO129 was described, but here treatment with the compound was started on day 16 after vaccination. At this time point, the mice had already developed large metastases. Treatment was terminated on day 24. Results ( Fig.14 ) showed that, when compared to an equimolar dose of ganicitinib, the compound of Formula I provided improved efficacy in reducing the number of liver metastases in this experimental setting. On the other hand, Fig.15 Survival of mice with overt metastatic disease after completion of treatment with the compound is shown. In these experiments, the vast majority (>90%) of control mice died from liver metastatic disease. Mice treated with the compound of formula I had a higher survival rate than mice treated with ganicitinib. Fig.13 , Fig.14 and Fig.15 The experiments shown in illustrate that the compound of formula (I) has unexpectedly enhanced therapeutic efficacy compared to the chemically related compound ganicitinib and reference compound 338 (which is in fact a conformational isomer of the compound of formula (I)).

[0456] Example 9: In vivo combination therapy assay utilizing PD1 / PDL1 checkpoint immunotherapy.

[0457] In vivo studies were performed to evaluate the activity of the compound of Formula I relative to the prior art reference compound plus nicitinib, alone or in combination with PD1 / PDL1 immune checkpoint inhibitors such as anti-PD-1 antibody treatment, in mice with overt metastatic disease.

[0458] As shown in Example 3, mouse tumor organoids (MTO129) were developed. C57BL / 6J mice were purchased from Janvier at 6 weeks of age and injected at 7-8 weeks. In order to transplant MTO129 into the liver, the cells were trypsinized (digesting the matrix and breaking the organoids into single cells), counted, and 300,000 cells were injected into the spleen in the form of HBSS (Hank's balanced salt solution, Lonza), thereby delivering the cells directly to the portal vein connecting the intestine to the liver. The compound (compound of Formula I or ganicitinib) was prepared as a suspension in a vehicle solution consisting of 1% sodium carboxymethylcellulose (Sigma), 0.4% sodium dodecyl sulfate (Sigma), 0.085% polyvinyl pyrrolidone, and 0.05% defoamer A (Sigma) in milli-Q water. Treatment with compounds was performed by gavage (tube feeding), and mice were treated twice daily with 150 μl of compound suspension in vehicle. Mice were treated with 1x equimolar doses of each compound, corresponding to 80 mg / k bid of ganicitinib or 70.8 mg / kg b.id of the compound of Formula I. 200 μg of anti-PD1 (clone RMPI-14; Leinco Technologies) was administered once every 3 days by intraperitoneal injection. Control mice were given gavage vehicle and IgG2a isotype control antibody.

[0459] exist Fig.14 and Fig.16 In the experiment shown, treatment with the compound and the indicated antibody was started on day 16 after inoculation with MTO129. At this time point, the mice had developed large liver metastases. Treatment was terminated on day 24. Fig.14 Liver tumor counts (LiM) after treatment with a compound of Formula I alone, both at 1x equimolar doses (80 mg / k bid of ganicitinib or 70.8 mg / kg bid of a compound of Formula I), and liver tumor counts (LiM) after treatment with an anti-PD-1 antibody or a combination of an anti-PD-1 antibody plus a compound of Formula I or ganicitinib are shown compared to the prior art compound ganicitinib. Again, the results indicate that the compound of Formula I provides improved activity at equimolar doses as a monotherapy. In addition, the combination therapy of an anti-PD-1 antibody and a compound of Formula I provides improved activity compared to the combination of an anti-PD-1 antibody and ganicitinib. Significantly, the majority of mice treated with a compound of Formula I in combination with a PD-1 antibody therapy remained metastasis-free at the experimental time point. Fig.16The survival rate of mice with overt metastatic disease after the end of treatment is shown. In these experiments, the vast majority (>90%) of control mice died from liver metastatic disease. In contrast, 80% of mice treated with a combination of a compound of Formula 1 and an anti-PD1 antibody survived after treatment. The survival rate of mice treated with a combination of a compound of Formula I and an anti-PD1 antibody was much higher than that of mice treated with ganishitinib plus an anti-PD1 antibody (80% vs. 20%).

[0460] Example 10: In vivo therapeutic efficacy of the compound of formula I and reference compounds 338 and 337

[0461] In another experiment ( Fig.17 ), we compared the in vivo therapeutic activity of compounds of formula I with related compounds 338 and compound 337. As shown in Example 3, mouse tumor organoids (MTO129) were developed. C57BL / 6J mice were purchased from Janvier at 6 weeks of age and injected at 7-8 weeks. In order to transplant MTO129 into the liver, the cells were trypsinized (digesting the matrix and breaking the organoids into single cells), counted, and 200,000 cells were injected into the spleen in the form of HBSS (Hank's balanced salt solution, Lonza), thereby delivering the cells directly to the portal vein connecting the intestine to the liver. Mice were treated with 1x equivalent molar doses of each compound (70.8 mg / kg bid of compound of formula I, 70.8 mg / kg bid of compound 338 or 68.0 mg / kg bid of compound 337). All mice were treated from day 2 to day 18 after inoculation of tumor cells. The number of liver metastases was counted 21 days after MTO inoculation. Fig.17 ) demonstrated that the compound of Formula I had enhanced potency in blocking metastasis formation compared to Compound 338 and Compound 337.

Claims

1. A compound of formula (I), or a pharmaceutically acceptable salt thereof: (I)。 2. Use of the compound of formula I or its pharmaceutically acceptable salt for preparing a drug: (I) The medicament is for preventing and / or treating a disease responsive to a TGFβ pathway inhibitor, wherein the disease is cancer.

3. The use according to claim 2, wherein the drug is administered in combination with chemotherapy, cancer targeted therapy, immunotherapy or immune checkpoint inhibitors.

4. The method of claim 2 , wherein the disease responsive to a TGFβ pathway inhibitor is a cancer selected from the group consisting of: hematological cancer; B-cell or T-cell leukemia, non-Hodgkin's lymphoma, non-Hodgkin's lymphoma of the B-cell or T-cell type, Burkitt's lymphoma, Hodgkin's lymphoma, leukemia, lymphoma of the B-cell or T-cell type, multiple myeloma, brain cancer, cancer of the glial lineage of the central nervous system (glioma), sarcoma, fibrosarcoma, malignant fibrous histiocytoma, Ewing's sarcoma, osteosarcoma, malignant pleural mesothelioma, breast cancer, breast cancer on anti-HER2 therapy, breast cancer, breast adenocarcinoma, gastric and gastroesophageal cancer, gastric cancer, gastric adenocarcinoma, colorectal cancer, colon carcinoma, colon adenocarcinoma, rectal cancer, colorectal cancer, metastatic colon cancer, pancreatic cancer, pancreatic cancer, pancreatic adenocarcinoma, renal cell carcinoma, clear cell renal cell carcinoma, liver cancer, liver metastases, metastatic disease, lung cancer, lung adenocarcinoma, non-small cell lung cancer, small cell lung cancer, ovarian cancer, ovarian cancer, ovarian adenocarcinoma, ovarian cancer, endometrial cancer, endometrial stromal sarcoma, cervical cancer, thyroid cancer, metastatic papillary thyroid cancer, follicular thyroid cancer, bladder cancer, urinary bladder cancer, urinary bladder transitional cell carcinoma, prostate cancer, prostate cancer, neuroendocrine cancer, squamous cell carcinoma, osteosarcoma, rhabdomyosarcoma, embryonal carcinoma, neuroblastoma, medulloblastoma, retinoblastoma, Wilms tumor, hepatoblastoma, melanoma, and skin cancer.

5. The use according to claim 4, wherein the cancer is a metastatic cancer.

6. Use of the compound of formula I or its pharmaceutically acceptable salt for preparing a drug: (I) The medicament is used to prevent cancer metastasis.

7. A pharmaceutical composition comprising an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof: (I) and at least one pharmaceutically acceptable excipient or carrier.

8. The pharmaceutical composition according to claim 7, further comprising at least another active ingredient, wherein the at least another active ingredient is an immunotherapeutic agent or a chemotherapeutic agent.

9. A prodrug of a compound of formula I, (I) The prodrug has formula (III), or a pharmaceutically acceptable salt thereof: (III).

10. A prodrug of a compound of formula (I), (I) Wherein the prodrug has formula (IV), or a pharmaceutically acceptable salt thereof: (IV).

11. Use of a prodrug of a compound of formula (I) according to any one of claims 9 or 10 for the preparation of a medicament for the prevention and / or treatment of a disease responsive to a TGFβ pathway inhibitor, wherein the disease is cancer.

12. The use according to claim 11, wherein the drug is administered in combination with chemotherapy, cancer targeted therapy, immunotherapy or immune checkpoint inhibitors.

13. The method of claim 11 , wherein the disease is a cancer selected from the group consisting of: hematological cancer; B-cell or T-cell leukemia, non-Hodgkin's lymphoma, non-Hodgkin's lymphoma of the B-cell or T-cell type, Burkitt's lymphoma, Hodgkin's lymphoma, leukemia, lymphoma of the B-cell or T-cell type, multiple myeloma, brain cancer, cancer of the glial lineage of the central nervous system (glioma), sarcoma, fibrosarcoma, malignant fibrous histiocytoma, Ewing's sarcoma, osteosarcoma, malignant pleural mesothelioma, breast cancer, breast cancer on anti-HER2 therapy, breast cancer, breast adenocarcinoma, gastric and gastroesophageal cancer, gastric cancer, gastric adenocarcinoma, colorectal cancer, colon cancer, colon adenocarcinoma carcinoma, rectal cancer, colorectal cancer, metastatic colon cancer, pancreatic cancer, pancreatic cancer, pancreatic adenocarcinoma, renal cell carcinoma, clear cell renal cell carcinoma, liver cancer, liver metastases, metastatic disease, lung cancer, lung adenocarcinoma, non-small cell lung cancer, small cell lung cancer, ovarian cancer, ovarian cancer, ovarian adenocarcinoma, ovarian cancer, endometrial cancer, endometrial stromal sarcoma, cervical cancer, thyroid cancer, metastatic papillary thyroid cancer, follicular thyroid cancer, bladder cancer, urinary bladder cancer, urinary bladder transitional cell carcinoma, prostate cancer, prostate cancer, neuroendocrine cancer, squamous cell carcinoma, osteosarcoma, rhabdomyosarcoma, embryonal carcinoma, neuroblastoma, medulloblastoma, retinoblastoma, Wilms tumor, hepatoblastoma, melanoma, and skin cancer.

14. The use according to claim 13, wherein the cancer is a metastatic cancer.

15. Use of a prodrug of a compound of formula (I) according to any one of claims 9 or 10 for the preparation of a medicament for preventing cancer metastasis.

16. A pharmaceutical composition comprising an effective amount of a prodrug of a compound of formula (I) according to any one of claims 9 or 10 or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient or carrier.

17. The pharmaceutical composition according to claim 16, further comprising at least another active ingredient, wherein the at least another active ingredient is an immunotherapeutic agent or a chemotherapeutic agent.

Images