Compounds for the treatment of conditions modulated by TGF-beta and bmp

Abstract

Compounds for use in the Treatment of Conditions modulated by Transforming Growth Factor β (TGF-β) and Bone Morphogenic Protein (BMP) Signalling Pathways This invention relates to compounds for use in various methods of treatment, along with the methods themselves. The compounds are useful for the treatment of conditions caused by dysregulated TGF-β and / or bone morphogenic protein (BMP) signalling pathways. The compounds are useful for the treatment of pulmonary arterial hypertension (PAH) and TGF-β and BMP-associated conditions.

Description

COMPOUNDS FOR THE TREATMENT OF CONDITIONS MODULATED BY TGF-BETA AND BMP

[0001] This invention relates to compounds for use in various methods of treatment,5 along with the methods themselves. The compounds are useful for the treatment of conditions caused by dysregulated TGF-p and / or bone morphogenic protein (BMP) signalling pathways. The compounds are useful for the treatment of pulmonary arterial hypertension (PAH) as well as conditions associated with TGF-p and BMP signalling pathways.BACKGROUND

[0002] Subversion of transforming growth factor-p (TGF-P) signalling has been implicated in cancer, fibrosis, autoimmune and cardiovascular disorders. On such cardiovascular disorder is Pulmonary Arterial Hypertension (PAH), a devastating cardiovascular disease caused by narrowing of the small blood vessels in the lungs. Such15 blood vessel narrowing and other vascular remodelling is caused by excessive proliferation and apoptosis resistance of the pulmonary vasculature which are attributable to the pathogenesis of this fatal disease. Heterozygous germline mutations in the bone morphogenetic protein type II receptor (BMPR2) gene, a member of the TGF-p superfamily receptors underlie the majority of the familial and heritable forms of the20 disease. Currently, there is no cure for PAH. Agents that either inhibit hyperactive TGFp or promote bone morphogenetic protein (BMP) signalling elicit beneficial effects in PAH animal models.

[0003] Transforming growth factor-p (TGF-P) superfamily comprises many cytokines including TGF-ps, bone morphogenetic proteins (BMPs), activins, inhibins and growth and25 differentiation factors. TGF-ps and BMPs like other members of this superfamily have crucial roles in multiple processes including differentiation, proliferation, migration and apoptosis for development and maintenance of tissue homeostasis. Both TGF-p and BMP transduce signals through respective type I and type II receptors which in turn phosphorylate cytoplasmic SMAD2 / 3 (for TGF-p) or SMAD1 / 5 (for BMP) proteins.30 Phosphorylated SMADs in complex with common partner SMAD4 translocate to the nucleus, acting as transcription factor complexes to express their target genes namely Pai- 1 (TGF-p signalling) and Id-1 (BMP signalling). Both TGF- and BMP can also activate SMAD independent non-canonical pathways involving p38 MAPK, c-jun N-terminal kinase and ERK1 / 2.

[0004] Heterozygous germline mutations in the bone morphogenetic protein type II receptor (BMPR2) gene, a member of the TGF-p superfamily receptors, are linked to the onset of the majority (>80%) of heritable pulmonary arterial hypertension (HPAH) and 10- 40% of idiopathic (IPAH) forms of PAH. Mutations have been identified in multiple genes within this superfamily including activin receptor-like kinase type I (ALK1), endoglin (ENG) (8), BMPR1B (9), BMP9 and TGFBRII and SMAD 1, 4 and 9 in PAH patients. Additionally, mutations have also been observed in non- TGF-p superfamily genes including TBX4, EIF2KA4, ATP13A3, AQP1 and S0X17 genes although they are an infrequent cause of the disease.

[0005] PAH is a devastating cardiovascular condition where the mean pulmonary arterial pressure (mPAP) is recorded above 20 mmHg accompanied by pulmonary artery wedge pressure (PAWP) < 15 mmHg and pulmonary vascular resistance > 3 Wood units (Wil). PAH is incurable and characterised by luminal obliteration of the small peripheral pulmonary arteries attributed to uncontrolled hypertrophy and hyperplasia. The pathological features observed in PAH patients include deposition of extracellular matrix, perivascular infiltration of inflammatory immune cells and the formation of plexiform lesions leading to substantial vascular remodelling. Current maintenance therapies for PAH are expensive and combination therapy could potentially reach £200,000-£300,000 per year per patient without the care cost. Additionally, the current therapies were established prior to the fact that PAH has substantial genetic elements and hence there is a need to identify novel therapeutic intervention prior to or following onset of the disease. As such, embodiments of the present invention seek to provide compounds and methods for the treatment or prevention of PAH. Optionally, wherein the compound or method for treatment or prevention targets a mutant member of the TGF-p superfamily receptors.

[0006] TGF-p signalling has been found to be overactivated in PAH patients and in experimental animal models. The levels of TGF-p ligands, phosphorylated SMAD2 and 3 proteins, Pai-1 transcripts have been found to be elevated in lungs of both PAH rats and PAH patients. It has been demonstrated that BMPR2 mutations have a deleterious effect on the balance between the BMP and TGF-p pathways, resulting in a dysregulated pro- proliferative response that is mediated via the TGF-P-TAK1-MAPK dependent pathway. It has been demonstrated that inhibition of this overactive TGF-p pathway by established drugs such as beraprost sodium (BPS) and treprostinil and other small molecule TGFp inhibitors including SD208, IN-1233 and SB525334, prevent PAH disease progression in animal models. These observations suggest that targetting the overactive TGF-p signalling pathway may offer new therapeutic options for PAH. As such, it is an object of certainembodiments of the present invention to provide compounds or methods that inhibit the TGF-p signalling pathway.

[0007] The BMP pathway has been established as a viable target for the treatment of PAH. Beneficial effects in PAH animal models have been achieved by the promotion of BMP signalling by small molecule agents including prostacyclin analogues, PDE5 inhibitors, FK506, chloroquine, biological agents such as BMP9 ligand, and BMPR2 gene therapy (46, 47). As such, it is an object of certain embodiments of the present invention to promote the BMP pathway, in particular to promote BMP signalling.

[0008] Dysfunctional BMP-mediated signalling contributes to the disease pathogenesis and targeting the attenuated BMPRII-SMAD1 / 5 / 8 axis using chemical and biological agents showed beneficial effects in multiple PAH models. Stimulation of endothelial cells (ECs) derived from PAH patients with BMP9 ligand inhibits endothelial cell apoptosis while administration of this biological agent prevents and reverses the disease in PAH animal models. FK506 (tacrolimus), a FDA approved small molecule drug has also been found to potentiate SMAD1 / 5-dependent signalling and showed beneficial effects in PAH animal models. Prostacyclin analogues including beraprost sodium, iloprost and treprostinil have rescued the BMP / SMAD1 / 5 / id1 axis in pulmonary arterial smooth muscle cells (PASMCs) derived from PAH patients and reversed the progression of established PAH in MCT- induced rat models. Furthermore, administration of chloroquine, an anti-malarial quinoline agent, has reduced mean pulmonary artery pressure (mPAP) in the monocrotaline-induced PAH model by promoting the expression of BMPR-II protein, inhibiting autophagy and proliferation of PASMCs.

[0009] Compounds of the present invention aim to promote BMP signalling. The compounds of the present invention may promote BMP signalling by promotion of phosphorylation of SMAD1 / 5 proteins. In addition to their pro-BMP effects, certain compounds of the present invention additionally or alternatively aim to have an anti-TGF-p effect. The anti-TGF-p effect of the present invention is mediated through the inhibition of phosphorylation of SMAD3 protein. Furthermore, compounds of the present invention promote apoptosis and inhibit excessive proliferation of pulmonary arterial hypertension- pulmonary arterial smooth muscle cells and mouse pulmonary arterial smooth muscle cells harbouring a pathogenic BMPR2 mutation. The balance between BMP and TGF-p pathways may be restored by certain compounds of the invention providing a novel treatment strategy not only for PAH but also for other BMP and TGFp-associated disorders.BRIEF SUMMARY OF THE DISCLOSURE

[0010] In accordance with the present inventions there is provided compounds for use in various methods of treatment. The methods of treatment are also contemplated by the present invention. All methods of treatment contemplated by the present invention may comprise the step of administering a therapeutic amount of a compound of the present invention. The present invention contemplates both the treatment, prevention and amelioration of a disease state. Treatment includes controlling symptoms of the disease state and does not necessarily include reversal or curing the disease state. However, the present invention does also contemplate the reversal of a disease state. The methods of treatment will be discussed in detail below. The compounds for use in the methods of treatment have a structure according to Formula (I):wherein:R1, R2, R3, R4, R5and R6are each independently selected from: H, halo, C1-6 alkyl, C1-6 haloalkyl, C6-io aryl, -OR7, -SO2R7, -C(O)OR7, -C(O)NR7R8, -NO2and -CN; andR7and R8are each independently selected from: H, C1-6 alkyl, phenyl and toluenyl.

[0011] In embodiments R1, R2, R3, R4, and R5are each independently selected from: H, halo, C1-6 alkyl, Ci-6haloalkyl, -OR7, -SO2R7, -C(O)OR7, -C(O)NR7R8, -NO2and -CN.

[0012] In embodiments R1, R2, R3, R4, and R5are each independently selected from: H, halo, C1-6 alkyl, Ci-6haloalkyl, -OR7, -SO2R7, -C(O)NR7R8, -NO2and -CN.

[0013] In embodiments R1, R2, R3, R4, and R5are each independently selected from: H, halo, -OR7, and -NO2.

[0014] In embodiments R1, R2, R3, R4, and R5are each independently selected from: H, halo, and -NO2.

[0015] In embodiments R1, R2, R3, R4, and R5are each independently selected from: H, chloro, bromo, fluoro, and -NO2.

[0016] In embodiments R6is H, -OR7, or phenyl.

[0017] In embodiments R6is H or -OR7.

[0018] In embodiments R7is H or Ci-6 alkyl. In embodiments R7is H or methyl.

[0019] In embodiments R8is H or C1-6 alkyl. In embodiments R8is H or methyl.

[0020] In embodiments R1, R2, R3, R4, and R5are each independently selected from: H, halo, C1-6 alkyl, Ci-6haloalkyl, -OR7, -SO2R7, -C(O)OR7, -C(O)NR7R8, -NO2and -ON.;R6is H, -OR7or phenyl; andR7is H or methyl.

[0021] In embodiments R1, R2, R3, R4, and R5are each independently selected from: H, halo, and -NO2;R6is H or -OR7; andR7is H.

[0022] In embodiments R1is selected from: H, halo, C1-6 alkyl, and -OR7. In embodiments R1is H, methyl, -OMe, bromo or fluoro. In embodiments R1is H, bromo or fluoro.

[0023] In embodiments R2is selected from: H, halo, C1-6 alkyl, and -OR7. In embodiments R2is H, bromo, methyl, or -OMe. In embodiments R2is H.

[0024] In embodiments R3is selected from: H, halo, C1-6 haloalkyl, -OR7, -SO2R7, - C(O)NR7R8, -NO2 and -ON. In embodiments R3is H, -OMe, fluoro, bromo, -CF3, -SO2Me, - C(O)NH2, -NO2, or -ON. In embodiments R3is H, bromo or -NO2.

[0025] In embodiments R4is selected from: H or halo. In embodiments R4is H or chloro.

[0026] In embodiments R5is selected from: H, halo, C1-6 alkyl, -OR7, -NO2 In embodiments R5is H, chloro, methyl, -OMe, or -NO2. In embodiments R5is H or -NO2.

[0027] In embodiments R6is H, C1-6 alkyl, -OR7, or Ce- aryl. In embodiments R6is H, methyl, OH, or phenyl. In embodiments R6is H or OH.

[0028] In embodiments the compound for use in the methods of the invention is one of the following compounds:

[0029] In an aspect of the invention there is provided a compound disclosed herein for use in a method of treatment of a condition caused by dysregulated TGF-p and / or bone morphogenic protein (BMP) signalling pathways.

[0030] In an aspect of the invention there is provided a compound disclosed herein for use in a method of treatment of a condition modulated by overexpression of TGF-p.

[0031] In a further aspect of the invention there is provided a compound disclosed herein for use in a method of treatment of a condition modulated by under expression of bone morphogenic protein (BMP).

[0032] In embodiments of the present invention the compound disclosed herein is for use in a method of treatment of a condition modulated by overexpression of TGF-p and under expression of bone morphogenic protein (BMP).

[0033] In an aspect of the invention there is provided a compound disclosed above for use in treating pulmonary hypertension, for example pulmonary arterial hypertension (PAH). In embodiments the pulmonary arterial hypertension is caused by a mutation in the bone morphogenic protein receptor of a patient suffering from pulmonary arterial hypertension. In certain embodiments the mutation is BMPR2-R899X+ / -. As such, the present invention contemplates a method of treating pulmonary arterial hypertension in patients possessing a mutation in the bone morphogenic protein receptor, as well as compounds for use in such a method.

[0034] In an aspect of the invention the compounds are for use in promoting apoptosis of cells within a human or animal tissue. In certain embodiments, the compounds for use in promoting apoptosis of cells is within human cardiovascular tissue, for example smooth muscle cells, fibroblasts or endothelial cells. The smooth muscle cells, fibroblasts or endothelial cells are optionally pulmonary arterial cells. In certain embodiments the promotion of apoptosis is of lung tissue cells or pulmonary arterial tissue cells. For example, the cells may be pulmonary arterial smooth muscle cells.

[0035] In certain embodiments, of any aspect of the invention, the treatment of pulmonary arterial hypertension comprises promoting apoptosis.

[0036] In an aspect of the invention there is provided a method of promoting apoptosis of cells within a human or animal tissue, the method comprising administering a therapeutic amount of the compound of the invention. In certain embodiments, the method of promoting apoptosis of cells is within human cardiovascular tissue, for example smooth muscle cells, fibroblasts or endothelial cells. The smooth muscle cells, fibroblasts or endothelial cells are optionally pulmonary arterial cells. In certain embodiments the promotion of apoptosis is of lung tissue cells or pulmonary arterial tissue cells. For example, the cells may be pulmonary arterial smooth muscle cells.

[0037] In an aspect of the invention there is provided a method of reducing the rate of proliferation of cells (optionally under normal or hypoxic conditions) within cardiovascular tissue, for example smooth muscle cells, fibroblasts or endothelial cells wherein the method comprises administering a therapeutically relevant amount of a compound of thepresent invention to a patient. The cells are optionally pulmonary arterial cells. In embodiments, the method is for use in a patient suffering from pulmonary arterial hypertension. In certain embodiments the promotion of apoptosis is of lung tissue cells or pulmonary arterial tissue cells. For example, the cells may be pulmonary arterial smooth muscle cells.

[0038] In an aspect the compounds of the present invention are for use in the reversal of vascular remodelling in patients, optionally wherein those patients are suffering from PAH. Furthermore, the present invention contemplates the method of reversing the vascular remodelling in patients, wherein the method comprises administering a therapeutically relevant amount of a compound of the present invention to a patient.BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:Figure 1A shows a BMP responsive reporter assay in cells transfected with reporter plasmids.Figure 1B shows a BMP responsive reporter assay in cells transfected with reporter plasmids and a plasmid encoding the wild type BMPRII.Figure 2 shows a BMP responsive reporter assay in HEK293T cells transfected with reporter plasmids.Figure 3 shows a BMP responsive reporter assay in HEK293T cells in the presence of BMPRII receptor at a range of concentrations.Figure 4 shows the results of a BMP responsive assay in HEK 293T cells transfected with the BMP-responsive reporter promoted with BMP9 ligand.Figure 5 shows a Western Blot analysis of phosphorylation of SMAD1 / 5 proteins.Figure 6 shows the results of a SMAD-binding element (SBE) responsive secreted alkaline phosphatase (SEAP) assay.Figure 7 shows the results of an TGF-p responsive SBE-Luc reporter assay utilising HEK 293T cells overexpressing TGFBRII receptor.Figure 8A-D show the effect of compounds of the invention on Pai-1 transcripts.Figure 9 shows the effect of compounds of the invention on phosphorylation level of SMAD3 (p-SMAD3) protein.Figure 10 shows the effect on Pai-1 expression in PASMCs derived from transgenic mice harbouring a pathogenic BMPR2 mutation (bmpr2R899X+ / -)Figures 11 A-D show the effect on expression of Pai-1.Figure 12 shows the effect on SMAD3 phosphorylation.Figure 13A-D shows the effect on Pai-1 expression levels in pulmonary arterial hypertension - pulmonary arterial smooth muscle cells (PAH-PASMCs) isolated from explanted lung samples of a PAH patient harbouring the pathogenic BMPR2 mutation (p. R899X).Figure 14 shows a western blot of phosphorylation of SMAD3 in PAH-PASMCs.Figure 15 shows the rate of apoptosis in a caspase Gio 3 / 7 assay in mouse PASMCs.Figure 16 shows the rate of apoptosis in a caspase Gio 3 / 7 assay in human PASMCs.Figure 17 shows the relative proliferation rate of mouse PASMCs.Figure 18 shows the relative proliferation rate of mouse PASMCs stimulated with TGF-pi .Figure 19 shows the relative proliferation rate of human PASMCs.Figure 20 shows the relative proliferation rate of human PASMCs stimulated with TGF-pi .Figure 21 shows TGF-p responsive SBE-Luc reporter assay utilising HEK 293T cells overexpressing TGFBRII receptor.DETAILED DESCRIPTION

[0040] The compounds of the present invention have a beneficial effect on both BMP and TGF-p pathways in PAH. The compounds of the present invention have the dual effect of activating the diminished BMPRII-SMAD1 / 5 / ID1 axis while downregulating overactive TGF-p signalling. As such, the compounds of the invention effect both pathways that have been shown to be abnormal in patients suffering from PAH. Additionally, compounds of the invention inhibit excessive proliferation and induce apoptosis in pulmonary arterial hypertension - pulmonary arterial smooth muscle cells (PAH-PASMCs). Thus, the compounds of the invention may be able to prevent PAH through correction of the dysregulated BMP and TGF-p pathways and reverse damage to tissue caused by irregular cell growth resulting from PAH. This is the first time that compounds have been identified with both BMP and TGF-p signalling modulatory effects.

[0041] Compounds of the present invention were initially screened for their BMP activity by a BMP responsive reporter assay. Compounds were then scrutinised in a cellular modeland the compounds of the present invention were found to have pro-BMP effects in HEK293T cells stimulated with BMP9 ligand and overexpressed with BMPRII receptor. Furthermore, in these cells, the compounds of the invention increased the expression of Id1 transcripts, a recognized target of BMP signalling pathway. Expression of Id1 was also increased in cells stimulated with BMP9 ligand and was further enhanced in cells treated with compounds of the invention.

[0042] Single halogen substitution is a preferred embodiment of the present invention because compounds with this functionality provide a more pronounced and persistent BMP promoting effect in cells. Notably, while most of the analogues tested enhanced expression of Id1 transcripts in HEK 293T cells, in mouse PASMCs harbouring a pathogenic BMPR2 mutation, only the 8-chloro- analogue increased expression.

[0043] In addition, the compounds of the present invention upregulate phosphorylation of SMAD1 / 5 protein in HEK293T cells. This suggests that compounds of the invention elevate the BMPRII / SMAD1 / 5 / / d7 axis in these cells.

[0044] In addition to the pro-BMP effects, the compounds of the present invention possess anti-TGF-p effects. Firstly, cryptolepine analogues inhibited SBE-SEAP reporter activity in mouse embryonic fibroblasts (mFB-F1) stimulated with TGF-pi ligand and SBE- Luc reporter activity in HEK293T cells overexpressed with TGFBR2 receptor. Secondly, compounds of the invention downregulated the expression of Pai-1 transcript, a recognised target of TGF-p signalling in mouse fibroblasts as well as in both mouse and human PASMCs. Thirdly, in these PAH relevant cell types, phosphorylation of SMAD3 protein was inhibited following treatment by compounds of the present invention. Notably, the compound of the invention substituted with a bromo group and a nitro group potently inhibited the TGF-p signalling pathway. This suggests that the compounds of the invention may inhibit the TGF-p target genes by reducing the phosphorylation of SMAD3 protein.

[0045] Vascular remodelling observed in PAH lungs is caused by a combination of excessive proliferation and attenuation of apoptosis. Therefore, the compounds of the present invention are able to rescue these cellular defects. Treatment of both human and mouse PASMCs harbouring the BMPR2 mutation with compounds of the present invention significantly upregulated caspase 3 / 7 activities. Additionally, compounds of the present invention also inhibited excessive proliferation of both human and mouse mutant PASMCs in the presence or absence of TGF-pi ligand stimulation. The anti-proliferative and pro- apoptotic activities of the compounds of the present invention may aid to reverse vascular remodelling in PAH.

[0046] EXAMPLES

[0047] Compounds of the present invention can be produced following a representative procedure shown in Wright et. al., J. Med. Chem. 2001 , 44, 3187-3194, which is hereby incorporated by reference.

[0048] Methods:

[0049] The BMP and TGFp signalling modulating effects of cryptolepine and its analogues were tested through reporter assays, gene expression studies and western blot analyses. To determine the anti-proliferative and pro-apoptotic effects of the compounds, MTS and Caspase-Glo®3 / 7 assays were employed respectively. Finally, the comet assay was performed to examine whether the cryptolepines possess any cytotoxic effects.

[0050] The cryptolepines exert anti-TGFp effects through the inhibition of phosphorylation of SMAD3 and p38MAPK proteins. These compounds also promote BMP signalling by increasing the phosphorylation of SMAD1 / 5 proteins. Furthermore, these compounds inhibit excessive cell proliferation, induce apoptosis, and protect human lymphocytes from oxidative damage.

[0051] Compounds

[0052] Cryptolepine and its 2-bromo-, 2, 7-dibromo- and 11 -chloro- analogues were synthesised as reported in Defaux J, Sala M, Formosa X, Galdeano C, Taylor MC, Alobaid WA, et al. Huprines as a new family of dual acting trypanocidal-antiplasmodial agents. Bioorg Med Chem. 2011;19(5):1702-7, which is incorporated herein by reference. The 2- bromo-7-nitro-, 8-chloro- and derivatives 3-fluoro-7, 9-dinitrocryptolepine were prepared as described as were 3-fluoro- and 3-fluoro7, 9-dinitrocryptolepine and 5- / V- hydroxyethanequindoline. All compounds were prepared as their hydrochloride salts and dissolved in DMSO.

[0053] Cell Culture, transfection, drug treatment and enzymatic assays

[0054] Cell culture, transfection and enzymatic assays were carried out as described in Nasim MT, Ghouri A, Patel B, James V, Rudarakanchana N, Morrell NW, et al.Stoichiometric imbalance in the receptor complex contributes to dysfunctional BMPR-II mediated signalling in pulmonary arterial hypertension. Hum Mol Genet.2008;17(11):1683-94, which is incorporated herein by reference. Human Embryonic Kidney cells (HEK 293T) (ATCC) and mouse fibroblast cells (mFB-F11) (University of Stanford, USA) were used for BMP or TGFp responsive reporter assays and secreted alkaline phosphatase assays (SEAP) respectively. Dulbecco’s modified Eagle medium (DMEM) containing 10% foetal calf serum (FCS, Sigma-Aldrich), 1% penicillin streptomycin (P / S) and 1% glutamax (both from Invitrogen) or DMEM with phenol red containing 10% FCS and 1% P / S were used as growth medium for the cell lines. Wild typehuman and mouse pulmonary arterial smooth muscle cells (PASMCs) (bmpr2 + / +) were purchased from Lonza, UK. Transgenic knock-in (bmpr2 R899X+ / -) mouse PASMCs and PAH-patient derived PASMCs (PAH-PASMCs) were also used in the present study. These primary cells harbour a pathogenic mutation (p. R899X) in the BMPR2 gene and were gifted by Professor Nicholas Morrell, University of Cambridge, UK. In addition, primary PAH-PASMCs (bmpr2 R899X+ / -) were immortalized by the expression of human telomerase gene (hTERT) in Dr. Nasim’s laboratory (Kanaganti et al., unpublished data) and used in the present study. 20% FCS and 1% P / S containing DM EM with phenol red media was used as the growth media for these cells. Wild type human pulmonary arterial endothelial cells (HPAEC) were gifted by Professor Timothy Palmer, University of Hull, UK. EGM-2 Bulletkit medium (Lonza, UK) was prepared according to manufacturer’s direction by adding all the growth supplements provided and used for growing HPAEC cells.Primary PASMCs and HPAECs were used for no more than 12 or 9 passages respectively to conduct the scheduled experiments.

[0055] BMP Responsive Reporter Assay

[0056] BMP responsive reporter assays were carried out as described elsewhere Nasim MT, Ghouri A, Patel B, James V, Rudarakanchana N, Morrell NW, et al. Stoichiometric imbalance in the receptor complex contributes to dysfunctional BMPR-II mediated signalling in pulmonary arterial hypertension. Hum Mol Genet. 2008;17(11):1683-94, which is incorporated herein by reference. HEK 293T cells were transfected in a 96 well-half area microtiter plate with plasmid containing BMP responsive element inserted at the promoter region of the luciferase reporter plasmid (3GC2-Lux). In addition, pJ7 LacZ plasmid which encodes the bacterial lacZ gene was also transfected as an internal control. To study the TGF-p pathway inhibitory properties of the experimental compounds a previously established SBE-Luc reporter assay was utilized using SBE-Luc reporter plasmid constructed by fusing a SMAD binding element (SBE) to a luciferase reporter gene. Additionally, plasmids encoding wild type BMPRII and TGFBRII receptors also were used wherever appropriate. HEK293T cells were transfected with plasmid DNAs using Gene Jammer transfection reagent (Stratagene, Agilent Technologies, USA) according to the manufacturer’s protocol. An appropriate volume of Gene Jammer reagent was diluted with serum-free DM EM media, plasmid DNA was added to this mixture and incubated for 30 minutes. The transfection mixture was added to each well and cells were incubated in a humidified incubator at 37°C, 5% CO2 overnight. 0.1% FCS, 1% P / S and 1% glutamax containing DM EM was used to prepare the appropriate concentrations of compounds. For ligand stimulation and treatment, ligands were first diluted with the latter medium at appropriate concentrations and further used for dilution of compounds to the desiredconcentrations. After treatment, cells were returned to the humidified incubator at 37°C, 5% CO2. Cells were stimulated with ligand in the presence or absence of compounds for 16 hours in reporter assays and 1 hour for RNA isolation / protein extraction.

[0057] Cell extracts were prepared by using reporter lysis buffer (Promega, UK). To determine luciferase and p-galactosidase activities, Dual-Light Reporter Assay system (Applied Biosystems, Carlsbad, CA) was employed, using an ORION-II Plate Luminometer (Berthold, Bad Wildbad, Germany) according to the manufacturer’s protocols.

[0058] SEAP Assay

[0059] Mouse embryonic fibroblast cells (mFB-F11), null for TgfblfTgfb ') that are stably transfected with a TGF-p responsive SMAD-binding element fused to the secreted alkaline phosphatase reporter gene (SBE-SEAP) were employed for the SEAP assay. This assay provides the convenience of directly correlating the magnitude of TGF-p ligand / receptor stimulation with the luminescence produced. mFB-F11 cells were seeded at an appropriate number optimized for this assay. On the following day, cells were treated with compounds in the presence or absence TGF-pi ligand (1ng / ml) and returned to the incubator at 37°C, 5% CO2 for 16 hours incubation. SEAP activity of the cells was measured following manufacturer’s protocol. Briefly, plates were centrifuged, cell supernatants were collected, diluted with dilution buffer in 1 :4 ratios and incubated at 65°C for 30 minutes. Samples were then transferred to a 96-well NulconTM Delta surface plate. Inactivation buffer was added to each well in 1 :1 ratio and incubated for 5 minutes at RT. SEAP substrate was prepared following manufacturer’s protocol, added in a 1 :1 ratio to the samples and incubated for 10 minutes at RT. SEAP activity was measured by Orion II Microplate Luminometer for 1 second.

[0060] RNA extraction, reverse transcription, and quantitative polymerase chain reaction (qPCR)

[0061] RNA extraction, cDNA synthesis and QPCR experiments were carried out using established protocols. Briefly, total RNA was extracted using PureLink RNA kit (Invitrogen) and then reverse transcribed using random primers and MMLV Reverse Transcriptase (Promega) following the manufacturer’s instructions. Expression levels of selected genes were quantified using TaqMan Gene Expression Assay (Applied Biosystems). Reactions were amplified on a StepOnePlusTM Real-Time PCR System (Applied Biosystems). Relative expression of target mRNA was normalized to GAPDH or B2M using the AACT method and expressed as fold change relative to the relevant control.

[0062] Western blotting analysis

[0063] Western blots were carried out using established protocols. For extraction of protein from mFB-F11, PASMCs and HPAECs, cells were lysed with protein extraction buffer containing reporter lysis buffer (Promega, UK), Laemmli buffer, protease inhibitors (Roche, UK), and phosphatase inhibitors. For HEK 293T cells, cell lysate was prepared by freeze and thaw method. Equal volumes of protein samples in sample buffer were loaded onto 12% SDS / PAGE gels and subjected to electrophoresis followed by western blotting. Membranes were incubated with Phospho-SMAD1 / 5 (Ser463 / 465) (41D10), SMAD1 (D59D7) XP®, SMAD2 / 3 and Phospho-SMAD3 (Ser423 / 425) (C25A9) antibodies (all from Cell Signalling Technologies). HRP-linked Anti-rabbit IgG (Cell signalling Technologies) was used as secondary antibody. The protein amounts loaded were normalized according to the p-actin signal, using a p-actin antibody (Cell signalling Technologies).

[0064] Bands were visualised using ECL Plus western blotting detection reagents (GE Healthcare) in a ChemiDoc™ Imager (Bio-Rad). The relative intensity of the immunoreactive bands was determined by densitometry using Image J software.

[0065] Proliferation assay

[0066] The relative rate of proliferation was determined as previously described. Briefly, 0.2X105PASMCs were seeded in a clear 96 well plate and incubated for 24 hours at 37 °C, 5% CO2. Subsequently, cell culture medium was removed from the wells and the cells were treated with selected compounds in 0.1% FBS media in the presence or absence of TGF-pi ligand (10ng / ml) and the 96 well plate was then returned to the incubator. After 72 hours’ incubation, cell proliferation was measured using CellTiter 96® AQueous One Solution Cell Proliferation Assay kit following manufacturer’s protocol. Briefly, MTS reagent was prepared by mixing with 0.1%FBS containing media in a 1 :5 ratio. Cell treatment medium was removed and the reagent mixture was added to the wells and incubated at 37 °C, 5% CO2. Absorbance was measured at 490nm using an iMark™ Microplate Absorbance Reader (Bio-Rad) after two hours.

[0067] Apoptosis assay

[0068] The relative rate of proliferation was determined as previously described. Briefly, 1X104PASMCs were seeded in Nucleon™ Delta Surface 96-well white plate and incubated at 37°C, 5% CO2for 24 hours. Following incubation, the cell culture medium was removed and compounds containing 0.1% FBS medium were added to each well and returned to the incubator. After 24 hours incubation, activation of caspase 3 / 7 in the cells was quantified using the Caspase-Gio® 3 / 7 assay kit (Promega, UK) following manufacturer’s protocol. In brief, caspase reagent was prepared by mixing the Caspase- Glo buffer and Caspase-Gio substrate. This reagent was mixed with 0.1% FBS containingmedium in a 1:1 ratio. Medium was removed and the mixture was added to the cells. Cells were incubated at RT and luminescence activity was measured after one hour using Orion II microplate luminometer.

[0069] Comet assay

[0070] For this assay, consent was obtained from 10 healthy volunteers. Collected whole blood samples were stored at -80°C after diluting with Roswell Park Memorial Institute- 1640 (RPMI-1640) medium in a ratio of 1 :1 plus 10% DMSO.

[0071] To run the Comet assay, 10OpI of whole blood samples were incubated for 30 minutes at 37°C with cryptolepines, dissolved in DMSO at concentrations of 1, 10 and 100pM in RPMI-1640 medium in the presence of 75mM of H2O2. The negative control consisted of a mixture of lymphocytes and RPMI while the vehicle control was a mixture of lymphocytes and DMSO. The positive control was a mixture of lymphocytes and 75mM H2O2. The samples were then incubated at 37°C for 30 mins. Following incubation, the samples were centrifuged at 3000 rpm for 5 minutes. The supernatant was removed and 10OpI of 0.5% low melting agarose mixed with 40pl of the pellet. The mixture was spread on the frosted slide, coverslip added and incubated on ice for 5 minutes. After removal of coverslips, the slides were incubated overnight in lysis solution at 4-8°C. Following electrophoresis and washing with neutralising buffer, the slides were stained with ethidium bromide and scored (100 cells / slide / 200 cells from each dose) using a fluorescent microscope at 20 X magnification with CCD camera. Komet 6 software and Kinetic Imaging (Andor Technology Ltd, Belfast) were used for automated scoring of Olive tail moment and % Tail DNA.

[0072] Statistical analysis

[0073] All data were analysed using Graphpad Prism 6.0 software. Graphs were constructed with bars for means and have error bars representing Standard error of Mean (SEM). Statistical significance was determined by student’s t-test for comparing two groups or one-way ANOVA and a Tukey’s Post Hoc test for analysis of more than two groups. Significance was set to p<0.05 (*), p<0.01 (**) and p<0.001 (***).

[0074] Example 1

[0075] Modulation of BMP signalling in human embryonic kidney cells (HEK293T)

[0076] A cell-based BMP responsive luciferase assay was used to investigate whether or not the BMP signalling pathway was modulated by the compounds disclosed herein. The BMP responsive luciferase assay utilises a BMP responsive element inserted in the promoter region of the luciferase gene (3GC2-Lux). Another reporter expressing the bacterial lacZ gene was used as an internal standard. HEK 293T cells were transfectedwith reporter plasmids together with the BMPRII receptor followed by stimulation with BMP9 ligand. BMP9 was included as studies suggest therapeutic potential of this ligand in the context of PAH.

[0077] The validity of the protocol was established by monitoring the BMP response in the presence of overexpression of either BMPRII or BMP9 ligand stimulation or combination of both. In all cases, the BMP responsive reporter activity was increased compared with the untreated control cells.

[0078] The pro-BMP effect of compounds of the invention was investigated by the following procedure. HEK 293T cells were transfected with either i) the reporter plasmids only, or ii) reporter plasmids and BMPRII receptor. Cells were treated with quinoline compounds including, chloroquine diphosphate, mefloquine hydrochloride, quinine dihydrochloride, a non-quinoline atovaquone and the indoloquinoline anti-malarial, cryptolepine. In both conditions (i) and (ii), quinoline mefloquine hydrochloride and indoloquinoline cryptolepine significantly enhanced both basal and BMPRII-mediated reporter activation, as shown in Figures 1A and 1B. However, the other quinoline chloroquine diphosphate and quinine dihydrochloride and non-quinoline atovaquone, failed to induce any discernible reporter activity compared with the untreated control cells (Figures 1A and 1 B).

[0079] Example 2

[0080] Potentiation of BMPRII-mediated signalling pathway using cryptolepine and halogenated analogues in HEK293T cells

[0081] Compounds 1 to 9 (Table 1) were investigated for their effects in HEK293T cells using the BMP responsive reporter assay described in Example 1. As shown in Figure 2, the parent compound cryptolepine, 2-bromo, 2,7-dibromo, 2-fluoro and 3-fluoro compounds were able to enhance the reporter activity more than 6-fold at 10pM fold as compared with the untreated control. The 2-bromo- and 3-fluoro- analogues compounds significantly increased luciferase activity in cells treated with both 1 and 10 pM concentrations while the parent compound cryptolepine and its 2-fluoro- analogue showed significant activity only at 10pM concentration. This data demonstrates that certain tested compounds have a promoting effect on BMP.

[0082] Table ! Structures of tested compounds.

[0083] Example 3

[0084] The effects of the five analogues which promoted BMP signalling were tested in the presence of BMPRII receptor at a range of concentrations. HEK293T cells overexpressing BMPRII receptor were treated with analogues at 0.1 , 1 and 10 pM concentrations (except for 8-chlorocryptolepine, tested at 1 and 10 pM only). All compounds tested promoted BMPRII-mediated reporter activity (Figure 3). These findings suggested that cryptolepine, 2-bromo, 8-chloro, 2,7-dibromo, 2-fluoro and 3- fluorocryptolepines promote BMP signalling and this effect is at least partly mediated through the BMPRII receptor.

[0085] Example 4

[0086] Next, to determine whether these compounds were able to increase the reporter activity in the presence of a BMP ligand, HEK 293T cells transfected with the BMP- responsive reporter were treated with these compounds together with the BMP9 ligand. BMP9 ligand was selected as the ligand both prevented and reversed the PAH in animal models. All analogues tested were able to increase the ligand stimulated signalling activity predominantly at 10pM concentration (Figure 4). Notably, cryptolepine at both 1pM and 10pM concentrations as well as 2-bromocryptolepine at 1pM concentration increased the luciferase activity (Figure 4).

[0087] Example 5

[0088] The mechanism of action of compounds of the invention was investigated. The goal was to determine whether the promotion of BMP signalling mediated by compounds of the invention occurred through the SMAD1 / 5-dependent pathway. HEK 293T cells overexpressing the BMPRII receptor for 24 hours were treated with compounds overnight. An elevated level of phosphorylation of SMAD1 / 5 proteins was observed upon treatment with cryptolepine, 2,7-dibromo-, 8-chloro- and 3-fluorocryptolepine despite the fact that overexpression of BMPRII receptor did not lead to a noticeable increase in SMAD1 / 5 phosphorylation compared with un-transfected control cells (Figure 5). These observations suggested that the compounds of the invention might promote BMP9-BMPRII-SMAD1 / 5- mediated signalling pathway.

[0089] Example 6

[0090] Overactivation of TGF-p signalling contributes to the pathogenesis of PAH and curbing this pathway with agents such as prostacyclins or ALK5 inhibitors elicit therapeutic benefits. To determine whether TGF-p signalling pathway can be modulated by compounds of the invention, we first employed SMAD-binding element (SBE) responsive secreted alkaline phosphatase (SEAP) assay. A time course experiment was carried out for optimizing the duration of stimulation with TGF-pi ligand. The mFB-F11 cells were stimulated with TGF-pi ligand (1ng / ml) at various time points starting from 0 minute to 20 hours and the resultant SEAP activity was measured. A sharp rise in luminescence activity was observed in the stimulated cells from 4 hours up to 20 hours, whilst no observable change in the SEAP activity was determined in unstimulated cells (Additional file 1: Fig. S4A). Next, to determine the inhibitory effect of the compounds of the invnetion, mFB-F11 cells were stimulated with TGF-pi ligand in the presence of the compounds for 16 hours. All compounds significantly inhibited the TGF-pi stimulated SEAP reporter activity except for 3-fluoro-7,9-dinitrocryptolepine (Figure 6). These findings indicate that compounds of the invention modulate the TGF-pi -mediated signalling pathway.

[0091] Example 7

[0092] Whether the compounds of the invention mediated inhibition of TGF-p signalling through the Type II receptor, TGFBRII, was investigated. A previously established TGF-p responsive SBE-Luc reporter assay was employed (Ogo T, Chowdhury HM, Yang J, Long L, Li X, Torres Cleuren YN, et al. Inhibition of overactive transforming growth factor-beta signaling by prostacyclin analogs in pulmonary arterial hypertension. Am J Respir Cell Mol Biol. 2013;48(6):733-41). Both overexpression of TGFBRII or TGF-pi stimulation significantly enhanced the reporter activity (p<0.001), whilst a recognized TGF-p type I receptor (ALK5) inhibitor, SD208, inhibited the activity, supporting the validity of the assay system.

[0093] HEK 293T cells overexpressing TGFBRII receptor were treated with cryptolepine, 2-bromo-7-nitro, 2-fluoro and 3-fluorocryptolepine at three concentrations (0.1 , 1 and 10pM) and their reporter activity determined. The 2-bromo-7-nitro analogue showed anti- TGF-p effects both in SEAP assay and SBE-Luc reporter assays (Figure 7). The parent cryptolepine showed inhibitory effects at 0.1 pM and 1 M whilst the 2-fluoro compound showed diminished luciferase activity at 10pM (Figure 7). These findings suggest that cryptolepine analogues can exert anti-TGF-p activities.

[0094] Example 8

[0095] Since heightened expression of Pai-1, a TGF-p target gene and phosphorylation of SMAD2 / 3 had been identified in various animal models and plexiform lesions of I PAH patients, the compounds were tested to determine whether they inhibited expression of Pai-1 transcripts and the phosphorylation of SMAD3 protein.

[0096] The effect of cryptolepine and its halogenated analogues on Pai-1 expression were investigated. In mouse fibroblast cells (mFB-F11), the parent compound cryptolepine, its analogues 2-bromo-7-nitro, 2-fluoro and 3-fluorocryptolepine significantly lowered Pai-1 expression at 10pM (Figure 8A, B, C and D) whilst 2-fluorocryptolepine reduced the expression at both 1 M concentrations also (Figure 8C).

[0097] Example 9

[0098] Following identification of inhibition of Pai-1 expression in mouse fibroblast cells, the phosphorylation level of SMAD3 (p-SMAD3) protein was determined in mFB-F11 cells following stimulation with TGF-pi and in the presence of cryptolepine, 2-bromo-7- nitrocryptolepine, 2-fluorocryptolepine and 3-fluorocryptolepine (Figure 9). As expected, TGF-pi stimulation upregulated the phospho-SMAD3 protein. With treatments, SMAD3 phosphorylation was reduced in cells treated with cryptolepine (10pM), 2-bromo-7-nitro (1 M and 10pM) and 2-fluorocryptolepine (1 M) (Figure 9). Interestingly, 3- fluorocryptolepine did not have any discernible effect on SMAD3 phosphorylation (Figure 9).

[0099] Example 10

[0100] Inhibition of Pai-1 expression and SMAD3 phosphorylation in mouse and human PASMCs harbouring a pathogenic BMPR2 mutation using cryptolepine compounds

[0101] The inhibitory effect of compounds of the invention on Pai-1 expression was further investigated in PASMCs derived from transgenic mice harbouring a pathogenic BMPR2 mutation (bmpr2R899X+ / -). Elevated expression of Pai-1 and a diminished level of BMPR2 transcripts were observed in untreated mutant cells compared to the wild type which is consistent with PAH. The compounds of the present invention reduced expressionof Pai-1 transcripts in mutant mouse PASMCs compared with the untreated control (Figure 10).

[0102] Example 11

[0103] Following the finding of the reduced expression of Pai-1 in the absence of any promoter, the effect of TGF-pi ligand stimulation was investigated. Pai-1 expression increased more than 4-fold in both wild type and mutant PASMCs under TGF-pi ligand stimulation. Treatment of the TGF-pi ligand stimulated PASMCs with cryptolepine, 2- fluoro-, 3-fluoro-, and 2-bromo-7-nitrocryptolepine analogues significantly downregulated expression of Pai-1 (Figure 11A-D). In these cells, TGF-pi ligand also stimulated SMAD3 phosphorylation, which was reduced in cells treated with cryptolepine, 2-bromo-7-nitro, 2- fluoro and 3-fluorocryptolepine (Figure 12).

[0104] Example 12

[0105] Having established the inhibitory effects of compounds in mouse PASMCs, their effects in human PASMCs were determined. The PAH-PASMCs isolated from explanted lung samples of a PAH patient harbouring the pathogenic BMPR2 mutation (p. R899X) exhibited a reduced level of the BMPR2 transcripts compared with the wild type cells. A 15-20 fold increase in Pai-1 expression in response to the TGF-pi ligand stimulation was observed in mutant cells and all cryptolepine analogues significantly downregulated Pai-1 expression levels (Figures 13A-D). Discernible reduction in Pai-1 expression was also observed in wild type cells for 2-fluoro and 3-fluoro compounds (Figure 13C, D).

[0106] Example 13

[0107] Following ligand stimulation, phosphorylation of SMAD3 was further elevated in PAH-PASMCs whilst TGF-pi stimulation only slightly increased this protein in the wild type cells (Figure 14). The 2-bromo-7-nitro, 2-fluoro and 3-fluoro analogues strongly downregulated SMAD3 phosphorylation in both wild type and mutant cells. However, the parent compound cryptolepine did not show any discernible effect on phospho-SMAD3 protein in both cell types (Figure 14).

[0108] Example 14

[0109] It has been demonstrated that mutations in the BMPR2 gene alter the response of PASMCs towards TGF-p and BMP ligands conferring resistance to apoptosis. Apoptosis is a significant pathology with PAH; thus, agents inducing pro-apoptotic activity might have therapeutic benefits. Therefore, pro-apoptotic effects of the compounds of the invention were tested.

[0110] A well-established Caspase-Gio 3 / 7 assay (Promega, UK) was employed to quantify the rate of apoptosis in mouse and human PASMCs. Higher caspase 3 / 7 activity in the mutant human PASMCs was observed when treated with a recognized caspase 3 / 7 activator staurosporine, (0.2pM) for 16 hours compared to untreated cells which established the validity of the assay. Consistent with previous observations, it was found that the level of caspase 3 / 7 activities was lower in mutant cells compared with wild-type.

[0111] Example 14A

[0112] Pro-apoptotic effects of compounds of the invention were determined in mouse PASMCs. Cryptolepine, 2-bromo-7-nitro- and 3-fluorocryptolepine induced apoptosis in both wild type and mutant cells (Fig. 15). 2-fluorocryptolepine increased caspase 3 / 7 activity in wild type but not in mutant mouse cells whilst 8-chlorocryptolepine failed to exert any discernible pro-apoptotic effect in either cell type (Figure 15).

[0113] Example 14B

[0114] In human PASMCs, cryptolepine and 3-fluorocryptolepine elicited a significant apoptotic response in both wild-type and PAH-PASMCs. Interestingly, the 2-bromo-7-nitro- analogue showed no effect in wild-type cells whilst significantly increased caspase 3 / 7 activity in mutant cells (Figure 16). However, 8-chloro and 2-fluoro compounds failed to exert pro-apoptotic effect in both type of cells (Figure 16).

[0115] Example 15

[0116] Thickening of the pulmonary arterial vessels observed in PAH lungs is conferred by the uncontrolled proliferation of vascular cells which is further enhanced by aberrant pro-proliferative action of TGF-pi ligand. As compounds eliciting anti-proliferative effects might have therapeutic potential, we determined the effects of cryptolepine and its analogues on mutant cell proliferation. Stimulation of cells with TGF-pi ligand significantly enhanced proliferation rates of mutant PASMCs when investigated in both mouse and human models.

[0117] Example 15A

[0118] Mouse PASMCs were treated with compounds of the invention in the presence or absence of TGF-pi ligand and relative rates of proliferation determined after 72 hours. In the absence of TGF-pi ligand, cryptolepine, 2-bromo-7-nitro-, 2-fluoro- and 3- fluorocryptolepine significantly reduced proliferation rates in unstimulated and TGF-pi stimulated cells (Figure 17) while 8-chlorocryptolepine exerted significant anti-proliferative effect in wild type but not in mutant cells (Figure 17). On the other hand, TGF-pi stimulation elevated the proliferation of mutant mouse cells more than 2-fold compared to the unstimulated PASMCs (Figure 18). Cryptolepine, 2-bromo-7-nitro-, 2-fluoro- and 3-fluoro-cryptolepine significantly downregulated the ligand-induced excessive proliferation in mutant cells. 8-chlorocryptolepine failed to inhibit ligand induced excessive proliferation (Fig. 18).

[0119] Example 15B

[0120] Anti-proliferative activity of cryptolepines was also tested in human PAH- PASMCs. Significant attenuation of proliferation was observed when these cells were treated with cryptolepine, 2-bromo-7-nitro-, 2- fluoro- and 3-fluorocryptolepine (Fig. 19). However, as with mouse PASMCs, 8-chlorocryptolepine failed to show any discernible inhibition (Figure 19). TGF-pi ligand stimulation significantly enhanced the proliferation of mutant human PASMCs but not of the wild type cells (Fig. 20). Cryptolepine, 2-bromo-7- nitro and 2- fluorocryptolepine inhibited TGF-pi induced excessive proliferation in PAH- PASMCs (Fig. 20). The 3-fluoro and 8-chlorocryptolepine exerted a cell specific response. These compounds showed no effect in wild-type cells whilst it inhibited aberrant proliferation in mutant PASMCs (Fig. 20).

[0121] Cryptolepine analogues show antioxidant effects on human lymphocytes

[0122] Persistent DNA damage has been found in blood cells of PAH patients (43) and reduced level of BMPRII may contribute to DNA damage (44). Hence, we investigated whether cryptolepine could provide protection from oxidative damage. The Comet assay was carried out on whole blood samples (n=10) derived from healthy subjects as a measure of oxidative stress induced DNA damage. The samples were treated with cryptolepine in the presence of an oxidative agent (H2O2, positive control) and effects on DNA damage were compared with samples treated with H2O2 alone. A significant increase in DNA damage was detected in the positive control (p-value <0.01) for both Olive tail moment (OTM) (Fig. 9C) and Tail DNA (TDNA)% (Fig. 9D) compared with untreated samples or vehicle control indicating the validity of the assay system. The parent cryptolepine, 2-fluoro and 3-fluoro compounds significantly reduced the DNA damage induced by H2O2 indicating the antioxidant effects of cryptolepine analogues at the concentrations tested.

[0123] Conclusion

[0124] In this study, we have provided extensive evidence to suggest that cryptolepine, a traditional antimalarial indoloquinoline agent, and its derivatives reinstate the equilibrium between the dysregulated BMP and TGF-p signalling, elicit pro-apoptotic and antiproliferative responses which, in turn, may reverse the vascular remodelling observed in PAH. Furthermore, cryptolepines may confer an antioxidant activity in PAH lungs. Additional studies including bioavailability, microsomal stability, half-life and elimination arerequired to test the beneficial effects of indoloquinoline compounds in PAH animal models prior to testing them in Phase l-lll clinical trials. We believe that natural compounds like indoloquinoline agents may provide a translational opportunity for the development of a novel treatment strategy not only for PAH but also for other BMP and TGFp-associated disorders.

[0125] Example 16

[0126] The Synthesis of Compound 16

[0127] To a solution of N,N,N-triethyl-2-hydroxy-1-(4-methylbenzenesulfonyl)-2,3- dihydro-1 H-indol-3-aminium bromide (1.99 g, 4.24 mmol) in EtOAc (40 mL) was added 4- bromo-N-methylaniline (862.7 mg, 4.66 mmol, 580.0 pL, 1.1 equiv.) and triethylamine (900.24 mg, 8.9 mmol, 1.24 mL, 2.1 equiv.). The reaction mixture was refluxed overnight. After completion of the reaction (monitored by LCMS), the resulting mixture was diluted with water (70 mL) and extracted with EtOAc (3 x 40 mL). The organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure to obtain 3-[(4-bromophenyl)(methyl)amino]-1-(4-methylbenzenesulfonyl)-2,3-dihydro-1 H-indol-2-ol (1.9 g, 4.01 mmol, 94.7% yield).

[0128] To a solution of 3-[(4-bromophenyl)(methyl)amino]-1-(4-methylbenzenesulfonyl)- 2,3-dihydro-1 H-indol-2-ol (1.9 g, 4.01 mmol) in EtOAc (50 mL) was added ethoxyethane; trifluoroborane (2.85 g, 20.03 mmol, 2.53 mL, 5.0 equiv.). The reaction mixture was warmed to 50 °C and stirred for 6 hours. After completion of the reaction (monitored by LCMS), the resulting mixture was diluted with an aqueous solution of NaHCCh and extracted with EtOAc (3 x 50 mL). The organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography to obtain N-(4-bromophenyl)-N-methyl-1-(4-methylbenzenesulfonyl)-1 H-indol-3-amine (850.0 mg, 94.0% purity, 1.75 mmol, 43.8% yield).

[0129] Phosphoroyl trichloride (133.25 mg, 877.33 pmol, 80.0 pL, 2.0 equiv.) was slowly added dropwise in dry DMF (2 mL) at -16 °C, and the mixture was stirred for 0.5 h. Then N-(4-bromophenyl)-N-methyl-1-(4-methylbenzenesulfonyl)-1 H-indol-3-amine (199.63 mg, 438.4 pmol) dissolved in DMF (1 mL) was added dropwise at 0 °C. The reaction mixture was warmed to room temperature and stirred for 1 hours. After completion of the reaction, the resulting mixture was diluted with ice water and filtered. The filter cake was dried under reduced pressure to obtain 2-bromo-5-methyl-10-(4-methylbenzenesulfonyl)-10H- indolo[3,2-b]quinolin-5-ium chloride (250.0 mg, 88.0% purity, 438.4 pmol, 100% yield). (If no precipitate formed, H2O was concentrated under reduced pressure, and the compound with DMF was used in the next step)Compound 16

[0130] To a solution of 2-bromo-5-methyl-10-(4-methylbenzenesulfonyl)-10H-indolo[3,2- b]quinolin-5-ium chloride (250.0 mg, 498.18 pmol) in DMF (3 mL) was added dimethylamine hydrochloride (80.73 mg, 996.22 pmol, 2.0 equiv.). The reaction mixture was warmed to 150 °C and stirred for 1.5 hours. The resulting mixture was cooled to room temperature and filtered. The obtained precipitate was washed with CH3CN (10 mL) and MTBE (10 mL). The filter cake was mixed with an aqueous solution of K2CO3 (10 mL) and the mixture was stirred for 10 min and filtered. The solid was washed with H2O (10 mL), dried under reduced pressure and purified by HPLC to obtain 2-bromo-5-methyl-10H- indolo[3,2-b]quinolin-5-ium-10-ide (Compound 16) (21.2 mg, 95.0% purity, 64.72 pmol, 13% yield). 1H (500 MHz, DMSO-d6) 8.90 (1 H, s), 8.68 (1 H, s), 8.48 (2H, apparent d, J 9.8 Hz), 7.96 (1H, dd, J 8.8, 3.8 Hz), 7.65 (1H, d, J 9.8 Hz), 7.53 (1H, t, J 7.5 Hz), 7.05 (1 H, t, J 7.5 Hz), 4.95 (3H, s).

[0131] Example 17

[0132] The Synthesis of Compound 17

[0133] To a solution of N,N,N-triethyl-2-hydroxy-1-(4-methylbenzenesulfonyl)-2,3- dihydro-1 H-indol-3-aminium bromide (1.99 g, 4.24 mmol) in EtOAc (40 mL) was added 3- bromo-N-methylaniline (862.7 mg, 4.66 mmol, 590.0 pL, 1.1 equiv.) and triethylamine (900.24 mg, 8.9 mmol, 1.24 mL, 2.1 equiv.). The reaction mixture was refluxed overnight. After completion of the reaction (monitored by LCMS), the resulting mixture was diluted with water (70 mL) and extracted with EtOAc (3 x 40 mL). The organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure to obtain 3-[(3-bromophenyl)(methyl)amino]-1-(4-methylbenzenesulfonyl)-2,3-dihydro-1 H-indol-2-ol (2.0 g, 84.0% purity, 3.55 mmol, 83.7% yield).

[0134] To a solution of 3-[(3-bromophenyl)(methyl)amino]-1-(4-methylbenzenesulfonyl)- 2,3-dihydro-1 H-indol-2-ol (2.0 g, 4.22 mmol) in EtOAc (50 mL) was added ethoxyethane; trifluoroborane (3.0 g, 21.13 mmol, 2.67 mL, 5.0 equiv.). The reaction mixture was warmed to 50 °C and stirred for 6 hours. After completion of the reaction (monitored by LCMS), the resulting mixture was diluted with an aqueous solution of NaHCOs and extracted with EtOAc (3 x 50 mL). The organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography to obtain N-(3-bromophenyl)-N-methyl-1-(4-methylbenzenesulfonyl)-1 H-indol-3-amine (1.1 g, 92.0% purity, 2.22 mmol, 52.6% yield).

[0135] Phosphoroyl trichloride (400.1 mg, 2.63 mmol, 240.0 pL, 2.0 equiv.) was slowly added dropwise in dry DMF (2 mL) at -16 °C and the mixture was stirred for 0.5 h. Then N- (3-bromophenyl)-N-methyl-1-(4-methylbenzenesulfonyl)-1 H-indol-3-amine (600.0 mg, 1.32 mmol) dissolved in DMF (1.0 mL) was added dropwise at 0 °C. The reaction mixture was warmed to room temperature and stirred for 1 hours. After completion of the reaction, the resulting mixture was diluted with ice water and filtered. The precipitate was dried under reduced pressure to obtain 3-bromo-5-methyl-10-(4-methylbenzenesulfonyl)-10H- indolo[3,2-b]quinolin-5-ium chloride (700.0 mg, 74.0% purity, 1.03 mmol, 78.4% yield). (If no precipitate formed, H2O was concentrated under reduced pressure, and the compound with DMF was used in the next step)Compound 17

[0136] To a solution of 3-bromo-5-methyl-10-(4-methylbenzenesulfonyl)-10H-indolo[3,2- b]quinolin-5-ium chloride (700.0 mg, 1.39 mmol) in DMF (3 mL) was added dimethylamine hydrochloride (226.04 mg, 2.79 mmol). The reaction mixture was warmed to 150 °C and stirred for 1.5 hours. The resulting mixture was cooled to room temperature and filtered. The obtained precipitate was washed with CH3CN (10 mL) and MTBE (10 mL). The filter cake was mixed with an aqueous solution of K2CO3 (10 mL) and the mixture was stirred for 10 min and filtered. The solid was washed with H2O (10 mL), dried under reduced pressure and purified by HPLC to obtain 3-bromo-5-methyl-10H-indolo[3,2-b]quinolin-5- ium-10-ide (Compound 17) (40.0 mg, 91.0% purity, 116.98 pmol, 8.4% yield). 1 H (500 MHz, DMSO-d6) 8.93 (1 H, s), 8.76 (1 H, s), 8.49 (1 H, d, J 8.7 Hz), 8.34 (1 H, d, J 8.7 Hz), 7.81 (1 H, d, J 8.7 Hz), 7.64 (1 H, d, J 8.7 Hz), 7.52 (1 H, t, J 6.2 Hz), 7.02 (1 H, t, J 6.2 Hz), 4.90 (3H, s).

[0137] Example 18

[0138] The Synthesis of Compound 18

[0139] To a solution of N,N,N-triethyl-2-hydroxy-1-(4-methylbenzenesulfonyl)-2,3- dihydro-1 H-indol-3-aminium bromide (2.0 g, 4.26 mmol) in EtOAc (50 mL) was added N- ethylaniline (567.34 mg, 4.69 mmol, 590.0 pL, 1.1 equiv.) and triethylamine (904.49 mg, 8.94 mmol). The reaction mixture was refluxed overnight. After completion of the reaction (monitored by LCMS), the resulting mixture was diluted with water (70 mL) and extractedwith EtOAc (3 x 40 mL). The organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure to obtain 3-[ethyl(phenyl)amino]-1-(4- methylbenzenesulfonyl)-2,3-dihydro-1 H-indol-2-ol (1.7 g, 92.0% purity, 3.83 mmol, 89.9% yield).

[0140] To a solution of 3-[ethyl(phenyl)amino]-1-(4-methylbenzenesulfonyl)-2,3-dihydro- 1 H-indol-2-ol (1.7 g, 4.16 mmol) in EtOAc (50 mL) was added ethoxyethane; trifluoroborane (2.96 g, 20.81 mmol). The reaction mixture was warmed to 50 °C and stirred for 6 hours. After completion of the reaction (monitored by LCMS), the resulting mixture was diluted with an aqueous solution of NaHCOs and extracted with EtOAc (3 x 50 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography to obtain N-ethyl-1-(4- methylbenzenesulfonyl)-N-phenyl-1 H-indol-3-amine (900.0 mg, 91.0% purity, 2.1 mmol, 50.4% yield).

[0141] Phosphoroyl trichloride (351.05 mg, 2.31 mmol, 210.0 pL, 2.0 equiv.) was slowly added dropwise in dry DMF (2 mL) at -16 °C and the mixture was stirred for 0.5 h. Then N- ethyl-1-(4-methylbenzenesulfonyl)-N-phenyl-1 H-indol-3-amine (451.31 mg, 1.16 mmol) dissolved in DMF (1.0 mL) was added dropwise at 0 °C. The reaction mixture was warmed to room temperature and stirred for 1 hour. After completion of the reaction, the resulting mixture was diluted with ice water and filtered. The filter cake was dried under reduced pressure to obtain 5-ethyl-10-(4-methylbenzenesulfonyl)-10H-indolo[3,2-b]quinolin-5-ium chloride (500.0 mg, 94.0% purity, 1.08 mmol, 93.1% yield). (If no precipitate formed, H2O was concentrated under reduced pressure, and the compound with DMF was used in the next step)Compound 18

[0142] To a solution of 5-ethyl-10-(4-methylbenzenesulfonyl)-10H-indolo[3,2-b]quinolin-5- ium chloride (500.0 mg, 1.14 mmol) in DMF ( mL) was added dimethylamine hydrochloride (185.56 mg, 2.29 mmol). The reaction was warmed to 150 °C and stirred for 1.5 hours.The resulting mixture was cooled to room temperature and filtered. The obtained precipitate was washed with CH3CN (10 mL) and MTBE (10 mL). The filter cake was mixed with an aqueous solution of K2CO3 (10 mL) and the mixture was stirred for 10 min and filtered. The solid was washed with H2O (10 mL) and dried under reduced pressure toobtain 5-ethyl-10H-indolo[3,2-b]quinolin-5-ium-10-ide (Compound 18) (71.0 mg, 95.0% purity, 273.85 pmol, 23.9% yield).

[0143] Example 19

[0144] The Synthesis of Compound 19

[0145] To a solution of N,N,N-triethyl-2-hydroxy-1-(4-methylbenzenesulfonyl)-2,3- dihydro-1 H-indol-3-aminium bromide (2.01 g, 4.27 mmol) in EtOAc (40 mL) was added N,4-dimethylaniline (543.42 mg, 4.49 mmol, 560.0 pL, 1.05 equiv.) and triethylamine (907.5 mg, 8.97 mmol, 1.25 mL, 2.1 equiv.). The reaction mixture was refluxed overnight. After completion of the reaction (monitored by LCMS), the resulting mixture was diluted with water (70 mL) and extracted with EtOAc (3 x 40 mL). The organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure to obtain 3-[methyl(4- methylphenyl)amino]-1-(4-methylbenzenesulfonyl)-2,3-dihydro-1 H-indol-2-ol (2.0 g, 58.0% purity, 2.84 mmol, 66.4% yield).

[0146] To a solution of 3-[methyl(4-methylphenyl)amino]-1-(4-methylbenzenesulfonyl)- 2,3-dihydro-1 H-indol-2-ol (2.0 g, 4.9 mmol) in EtOAc (50 mL) was added ethoxyethane; trifluoroborane (3.48 g, 24.48 mmol, 3.09 mL, 5.0 equiv.). The reaction mixture was warmed to 50 °C and stirred for 6 hours. After completion of the reaction (monitored by LCMS), the resulting mixture was diluted with an aqueous solution of NaHCCh and extracted with EtOAc (3 x 50 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography to obtain N-methyl-1- (4-methylbenzenesulfonyl)-N-(4-methylphenyl)-1 H-indol-3-amine (700.0 mg, 95.0% purity, 1.7 mmol, 34.8% yield).

[0147] Phosphoroyl trichloride (272.5 mg, 1.79 mmol, 170.0 pL, 2.0 equiv.) was slowly added dropwise in dry DMF (3 mL) at -16 °C and the mixture was stirred for 0.5 h. Then N- methyl-1-(4-methylbenzenesulfonyl)-N-(4-methylphenyl)-1 H-indol-3-amine (350.0 mg, 896.29 pmol) dissolved in DMF (1 mL) was added dropwise at 0 °C. The reaction mixture was warmed to room temperature and stirred for 1 hour. After completion of the reaction, the resulting mixture was diluted with ice water and filtered. The filter cake was dried under reduced pressure to obtain 2,5-dimethyl-10-(4-methylbenzenesulfonyl)-10H-indolo[3,2- b]quinolin-5-ium chloride (350.0 mg, 91.0% purity, 728.91 pmol, 81.3% yield). (If no precipitate formed, H2O was concentrated under reduced pressure, and the compound with DMF was used in the next step)Compound 19

[0148] To a solution of 2,5-dimethyl-10-(4-methylbenzenesulfonyl)-10H-indolo[3,2- b]quinolin-5-ium chloride (360.0 mg, 823.89 pmol) in DMF (3 mL) was added dimethylamine hydrochloride (133.57 mg, 1.65 mmol). The reaction mixture was warmed to 150 °C and stirred for 1.5 hours. The resulting mixture was cooled to room temperature and filtered. The obtained precipitate was washed with CH3CN (10 mL) and MTBE (10 mL). The filter cake was mixed with an aqueous solution of K2CO3 (10 mL) and the mixture was stirred for 10 min and filtered. The solid was washed with H2O (10 mL), dried under reduced pressure and purified by HPLC to obtain 2,5-dimethyl-10H-indolo[3,2- b]quinolin-5-ium-10-ide (Compound 19) (47.1 mg, 95.0% purity, 181.66 pmol, 22% yield). 1 H (500 MHz, DMSO-d6) 8.84 (1 H, s), 8.48 (1 H, d, J 8.8 Hz), 8.43 (1 H, d, J 8.8 Hz), 8.14 (1 H, s), 7.75 (1 H, d, J 7.5 Hz), 7.63 (1 H, d, J 7.5 Hz), 7.53 (1 H, t, J 7.5 Hz), 7.07 (1H, t, J 7.5 Hz), 4.89 (3H, s), 2.55 (3H, s).

[0149] Example 20

[0150] The Synthesis of Compound 20

[0151] To a solution of N,N,N-triethyl-2-hydroxy-1-(4-methylbenzenesulfonyl)-2,3- dihydro-1 H-indol-3-aminium bromide (2.0 g, 4.26 mmol) in EtOAc (40 mL) was added 4- methoxy-N-methylaniline (613.37 mg, 4.47 mmol, 560.0 pL, 1.05 equiv.) and triethylamine (904.91 mg, 8.95 mmol, 1.25 mL, 2.1 equiv.). The reaction mixture was refluxed overnight. After completion of the reaction (monitored by LCMS), the resulting mixture was diluted with water (70 mL) and extracted with EtOAc (3 x 40 mL). The organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure to obtain 3-[(4-methoxyphenyl)(methyl)amino]-1-(4-methylbenzenesulfonyl)-2,3-dihydro-1 H-indol-2- ol (1.7 g, 90.0% purity, 3.6 mmol, 84.6% yield).

[0152] To a solution of 3-[(4-methoxyphenyl)(methyl)amino]-1-(4- methylbenzenesulfonyl)-2,3-dihydro-1 H-indol-2-ol (1.7 g, 4.01 mmol) in EtOAc (50 mL) was added ethoxyethane; trifluoroborane (2.85 g, 20.03 mmol, 2.53 mL, 5.0 equiv.). The reaction mixture was warmed to 50 °C and stirred for 6 hours. After completion of the reaction (monitored by LCMS), the resulting mixture was diluted with an aqueous solution of NaHCOs and extracted with EtOAc (3 x 50 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatographyto obtain N-(4-methoxyphenyl)-N-methyl-1-(4-methylbenzenesulfonyl)-1 H-indol-3-amine (1.05 g, 95.0% purity, 2.45 mmol, 61.2% yield).

[0153] Phosphoroyl trichloride (560.29 mg, 3.69 mmol, 340.0 pL, 3.0 equiv.) was slowly added dropwise in dry DMF (3 mL) at -16 °C and the mixture was stirred for 0.5 h. Then N- (4-methoxyphenyl)-N-methyl-1-(4-methylbenzenesulfonyl)-1 H-indol-3-amine (500.0 mg, 1.23 mmol) dissolved in DMF (1 mL) was added dropwise at 0 °C. The reaction mixture was warmed to room temperature and stirred for 1 hour. After completion of the reaction, the resulting mixture was diluted with ice water and filtered. The filter cake was dried under reduced pressure to obtain 2-methoxy-5-methyl-10-(4-methylbenzenesulfonyl)-10H- indolo[3,2-b]quinolin-5-ium chloride (500.0 mg, 1.1 mmol, 89.8% yield). (If no precipitate formed, H2O was concentrated under reduced pressure, and the compound with DMF was used in the next step)Compound 20

[0154] To a solution of 2-methoxy-5-methyl-10-(4-methylbenzenesulfonyl)-10H- indolo[3,2-b]quinolin-5-ium chloride (500.0 mg, 1.1 mmol) in DMF (3 mL) was added dimethylamine hydrochloride (179.18 mg, 2.21 mmol). The reaction mixture was warmed to 150 °C and stirred for 1.5 hours. The resulting mixture was cooled to room temperature and filtered. The obtained precipitate was washed with CH3CN (10 mL) and MTBE (10 mL). The filter cake was mixed with an aqueous solution of K2CO3 (10 mL) and the mixture was stirred for 10 min and filtered. The solid was washed with H2O (10 mL), dried under reduced pressure and purified by HPLC to obtain 2-methoxy-5-methyl-10H- indolo[3,2-b]quinolin-5-ium-10-ide (Compound 20) (200.0 mg, 95.0% purity, 724.35 pmol, 65.5% yield). 1 H (500 MHz, DMSO-d6) 8.77 (1 H, s), 8.44 (2H, apparent d, J 8.7), 7.79 (1 H, d, J 4.3), 7.60 (1H, d, J 7.6), 7.50 (1 H, dd, J 4.3, 8.7), 7.48 (1 H, t, J 7.6), 6.99 (1 H, t, J 7.8), 4.89 (3H, s), 3.95 (3H, s).

[0155] Example 21

[0156] The Synthesis of Compound 21

[0157] To a solution of 5-nitro-1 H-indole (5.0 g, 30.86 mmol) in DMF (100 mL) was added sodium hydride (1.48 g, 60.0% purity, 37.03 mmol) at 0 °C under argon atmosphere. The mixture was stirred for 30 min at 0 °C and then 4-methylbenzene-1- sulfonyl chloride (6.45 g, 33.94 mmol) was added. The reaction mixture was warmed to room temperature and stirred overnight. After that, a solution of NH4CI was slowly addedand the resulting mixture was extracted with EtOAc (3 x 100 mL). The organic layer was washed with brine (3 x 50 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to obtain 1-(4-methylbenzenesulfonyl)-5-nitro-1 H-indole (8.5 g, 90.0% purity, 24.18 mmol, 78.4% yield).

[0158] To a solution of 1-(4-methylbenzenesulfonyl)-5-nitro-1 H-indole (8.26 g,26.11 mmol) in a mixture of acetone / water (100 / 10 mL) was added 1 -bromopyrrolidine-2, 5- dione (5.08 g, 28.72 mmol) at 0 °C. The reaction mixture was warmed to ambient temperature and stirred overnight. After completion of the reaction (monitored by TLC), triethylamine (2.9 g, 28.72 mmol, 4.0 mL, 1.1 equiv.) was added at 0 °C and the resulting mixture was stirred for 1 h and concentrated under reduced pressure. The residue was diluted with water (100 mL) and washed with EtOAc (3 x 50 mL), and the aqueous layer was concentrated under reduced pressure to obtain N,N,N-triethyl-2-hydroxy-1-(4- methylbenzenesulfonyl)-5-nitro-2,3-dihydro-1 H-indol-3-aminium bromide (5.6 g, 92.0% purity, 10.01 mmol, 38.4% yield).

[0159] To a solution of N,N,N-triethyl-2-hydroxy-1-(4-methylbenzenesulfonyl)-5-nitro-2,3- dihydro-1 H-indol-3-aminium bromide (4.0 g, 7.78 mmol) in EtOH (60 mL) was added N- methylaniline (915.75 mg, 8.55 mmol) and triethylamine (1.65 g, 16.33 mmol). The reaction mixture was refluxed overnight. After completion of the reaction (monitored by LCMS), the resulting mixture was diluted with water (100 mL) and extracted with EtOAc (3 x 70 mL). The organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography to obtain 3-[methyl(phenyl)amino]-1-(4-methylbenzenesulfonyl)-5-nitro-2,3-dihydro-1 H-indol-2-ol (500.0 mg, 1.14 mmol, 14.6% yield).

[0160] To a solution of 3-[methyl(phenyl)amino]-1-(4-methylbenzenesulfonyl)-5-nitro-2,3- dihydro-1 H-indol-2-ol (600.0 mg, 1.37 mmol) in EtOAc (100 mL) was added ethoxyethane; trifluoroborane (969.17 mg, 6.82 mmol, 860.0 pL, 5.0 equiv.). The reaction mixture was warmed to 50 °C and stirred for 6 hours. After completion of the reaction (monitored by LCMS), the resulting mixture was diluted with an aqueous solution of NaHCCh and extracted with EtOAc (3 x 50 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography to obtain N-methyl-1- (4-methylbenzenesulfonyl)-5-nitro-N-phenyl-1 H-indol-3-amine (150.0 mg, 66.0% purity, 234.89 pmol, 17.2% yield).

[0161] Phosphoroyl trichloride (108.57 mg, 714.86 pmol, 70.0 pL, 2.0 equiv.) was slowly added dropwise in dry DMF (1 mL) at -16 °C and the mixture was stirred for 0.5 h. Then N- methyl-1-(4-methylbenzenesulfonyl)-5-nitro-N-phenyl-1 H-indol-3-amine (150.42 mg, 356.9 pmol) dissolved in DMF (1 mL) was added dropwise at 0 °C. The reaction mixturewas warmed to room temperature and stirred for 1 hour. After completion of the reaction, the resulting mixture was diluted with ice water and filtered. The filter cake was dried under reduced pressure to obtain 5-methyl-10-(4-methylbenzenesulfonyl)-7-nitro-10H-indolo[3,2- b]quinolin-5-ium chloride (170.0 mg, 46.0% purity, 167.12 pmol, 46.8% yield). (If no precipitate formed, H2O was concentrated under reduced pressure, and the compound with DMF was used in the next step)Compound 21

[0162] To a solution of 5-methyl-10-(4-methylbenzenesulfonyl)-7-nitro-10H-indolo[3,2- b]quinolin-5-ium chloride (180.0 mg, 384.68 pmol) in DMF (3 mL) was added dimethylamine hydrochloride (62.54 mg, 771.79 pmol). The reaction mixture was warmed to 150 °C and stirred for 1.5 hours. The resulting mixture was cooled to room temperature and filtered. The obtained precipitate was washed with CH3CN (10 mL) and MTBE (10 mL). The filter cake was mixed with an aqueous solution of K2CO3 (10 mL) and the mixture was stirred for 10 min and filtered. The solid was washed with H2O (10 mL), dried under reduced pressure and purified by HPLC to obtain 5-methyl-7-nitro-10H-indolo[3,2- b]quinolin-5-ium-10-ide (Compound 21) (36.4 mg, 95.0% purity, 124.71 pmol, 32.3% yield). 1 H (500 MHz, DMSO-d6) 9.46 (1 H, s), 9.25 (1 H, s), 8.70 (1 H, d, J 9.0 Hz), 8.52 (1 H, d, J 7.8 Hz), 8.40 (1 H, d, J 9.0 Hz), 8.08 (1 H, t, J 7.8 Hz), 7.86 (1 H, t, J 7.1 Hz), 7.76 (1 H, d, J 9.0 Hz), 5.05 (3H, s).

[0163] Example 22

[0164] The Synthesis of Compound 22

[0165] To a solution of 1 H-indole-5-carbonitrile (4.0 g, 28.16 mmol) in DMF (100 mL) was added sodium hydride (1.58 g, 60.0% purity, 39.42 mmol) at 0 °C under argon atmosphere. The mixture was stirred for 30 min at 0 °C and then 4-methylbenzene-1- sulfonyl chloride (5.88 g, 30.98 mmol) was added. The reaction mixture was warmed to room temperature and stirred overnight. After that, a solution of NH4CI was slowly added and the resulting mixture was extracted with EtOAc (3 x 100 mL). The organic layer was washed with brine (3 x 50 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to obtain 1-(4-methylbenzenesulfonyl)-1 H-indole-5-carbonitrile (8.0 g, 90.0% purity, 24.3 mmol, 86.3% yield).

[0166] To a solution of 1-(4-methylbenzenesulfonyl)-1 H-indole-5-carbonitrile (8.01 g, 27.04 mmol) in a mixture of acetone / water (100 / 10 mL) was added 1 -bromopyrrolidine-2, 5- dione (5.26 g, 29.74 mmol) at 0 °C. The reaction mixture was warmed to ambient temperature and stirred overnight. After completion of the reaction (monitored by TLC), triethylamine (3.01 g, 29.74 mmol, 4.14 mL, 1.1 equiv.) was added at 0 °C and the resulting mixture was stirred for 1 h and concentrated under reduced pressure. The residue was diluted with water (100 mL) and washed with EtOAc (3 x 50 mL), and the aqueous layer was concentrated under reduced pressure to obtain 5-cyano-N,N,N-triethyl- 2-hydroxy-1-(4-methylbenzenesulfonyl)-2,3-dihydro-1 H-indol-3-aminium bromide (8.0 g, 80.0% purity, 12.94 mmol, 47.9% yield).

[0167] To a solution of 5-cyano-N,N,N-triethyl-2-hydroxy-1-(4-methylbenzenesulfonyl)-2.3-dihydro-1 H-indol-3-aminium bromide (4.0 g, 8.09 mmol) in EtOAc (50 mL) was added N-methylaniline (952.92 mg, 8.9 mmol, 960.0 pL, 1.1 equiv.) and triethylamine (1.8 g, 17.8 mmol, 2.48 mL, 2.2 equiv.). The reaction mixture was refluxed overnight. After completion of the reaction (monitored by LCMS), the resulting mixture was diluted with water (70 mL) and extracted with EtOAc (3 x 50 mL). The organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure to obtain 2-hydroxy-3-[methyl(phenyl)amino]-1- (4-methylbenzenesulfonyl)-2,3-dihydro-1 H-indole-5-carbonitrile (4.0 g, 73.0% purity, 6.96 mmol, 86% yield).

[0168] To a solution of 2-hydroxy-3-[methyl(phenyl)amino]-1-(4-methylbenzenesulfonyl)-2.3-dihydro-1 H-indole-5-carbonitrile (3.9 g, 9.3 mmol) in EtOAc (80 mL) was added ethoxyethane; trifluoroborane (6.6 g, 46.48 mmol, 5.87 mL, 5.0 equiv.). The reaction mixture was warmed to 50 °C and stirred for 6 hours. After completion of the reaction (monitored by LCMS), the resulting mixture was diluted with an aqueous solution of NaHCO3 and extracted with EtOAc (3 x 50 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography to obtain 3-[methyl(phenyl)amino]-1-(4-methylbenzenesulfonyl)-1 H-indole-5-carbonitrile (500.0 mg, 1.25 mmol, 13.4% yield).

[0169] Phosphoroyl trichloride (230.3 mg, 1.52 mmol, 140.0 pL, 2.0 equiv.) was slowly added dropwise in dry DMF (2 mL) at -16 °C and the mixture was stirred for 0.5 h. Then 3- [methyl(phenyl)amino]-1-(4-methylbenzenesulfonyl)-1 H-indole-5-carbonitrile (304.74 mg, 759.04 pmol) dissolved in DMF (1.0 mL) was added dropwise at 0 °C. The reaction mixture was warmed to room temperature and stirred for 1 hour. After completion of the reaction, the resulting mixture was diluted with ice water and filtered. The filter cake was dried under reduced pressure to obtain 7-cyano-5-methyl-10-(4-methylbenzenesulfonyl)- 10H-indolo[3,2-b]quinolin-5-ium chloride (340.0 mg, 759.04 pmol, 100% yield). (If noprecipitate formed, H2O was concentrated under reduced pressure, and the compound with DMF was used in the next step)Compound 22

[0170] To a solution of 7-cyano-5-methyl-10-(4-methylbenzenesulfonyl)-10H-indolo[3,2- b]quinolin-5-ium chloride (340.0 mg, 759.04 pmol) in DMF (3 mL) was added dimethylamine hydrochloride (122.83 mg, 1.52 mmol). The reaction was warmed to 150 °C and stirred for 1.5 hours. The resulting mixture was cooled to room temperature and filtered. The obtained precipitate was washed with CH3CN (10 mL) and MTBE (10 mL). The filter cake was mixed with an aqueous solution of K2CO3 (10 mL) and the mixture was stirred for 10 min and filtered. The solid was washed with H2O (10 mL), dried under reduced pressure and purified by HPLC to obtain 7-cyano-5-methyl-10H-indolo[3,2- b]quinolin-5-ium-10-ide (Compound 22) (70.0 mg, 95.0% purity, 258.46 pmol, 34.1% yield). 1 H (500 MHz, DMSO-d6) 9.08 (2H, apparent d, J -10.6 Hz), 8.62 (1 H, d 9.1 Hz), 8.45 (1 H, d, J 9.1 Hz), 7.95 (1 H, t, J 9.1 Hz), 7.74 (1H, t, J 9.1 Hz), 7.68 (2H, s), 4.94 (3H, s).

[0171] Example 23

[0172] The Synthesis of Compound 23

[0173] To a solution of N,N,N-triethyl-2-hydroxy-1-(4-methylbenzenesulfonyl)-2,3- dihydro-1 H-indol-3-aminium bromide (2.0 g, 4.26 mmol) in EtOAc (50 mL) was added N- benzylaniline (858.17 mg, 4.69 mmol, 810.0 pL, 1.1 equiv.) and triethylamine (861.68 mg, 8.52 mmol, 1.19 mL, 2.0 equiv.). The reaction mixture was refluxed overnight. After completion of the reaction (monitored by LCMS), the resulting mixture was diluted with water (70 mL) and extracted with EtOAc (3 x 50 mL). The organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure to obtain 3-[benzyl(phenyl)amino]-1-(4-methylbenzenesulfonyl)-2,3-dihydro-1 H-indol-2-ol (2.4 g, 70.0% purity, 3.57 mmol, 83.8% yield).

[0174] To a solution of 3-[benzyl(phenyl)amino]-1-(4-methylbenzenesulfonyl)-2,3- dihydro-1 H-indol-2-ol (2.4 g, 5.1 mmol) in EtOAc (50 mL) was added ethoxyethane; trifluoroborane (3.62 g, 25.5 mmol, 3.22 mL, 5.0 equiv.). The reaction mixture was warmed to 50 °C and stirred for 6 hours. After completion of the reaction (monitored by LCMS), the resulting mixture was diluted with an aqueous solution of NaHCOs and extracted withEtOAc (3 x 50 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography to obtain N-benzyl-1-(4- methylbenzenesulfonyl)-N-phenyl-1 H-indol-3-amine (1.05 g, 90.0% purity, 2.09 mmol, 40.9% yield).

[0175] Phosphoroyl trichloride (402.95 mg, 2.65 mmol, 2.0 equiv.) was slowly added dropwise in dry DMF (2 mL) at -16 °C and the mixture was stirred for 0.5 h. Then N-benzyl- 1-(4-methylbenzenesulfonyl)-N-phenyl-1 H-indol-3-amine (600.0 mg, 1.33 mmol) dissolved in DMF (1.0 mL) was added dropwise at 0 °C. The reaction mixture was warmed to room temperature and stirred for 1 hour. After completion of the reaction, the resulting mixture was diluted with ice water and filtered. The filter cake was dried under reduced pressure to obtain 5-benzyl-10-(4-methylbenzenesulfonyl)-10H-indolo[3,2-b]quinolin-5-ium chloride (320.0 mg, 98.0% purity, 628.43 pmol, 47.4% yield).Compound 23

[0176] To a solution of 5-benzyl-10-(4-methylbenzenesulfonyl)-10H-indolo[3,2-b]quinolin- 5-ium chloride (320.0 mg, 641.25 pmol) in DMF (3 mL) was added dimethylamine hydrochloride (104.06 mg, 1.28 mmol). The reaction mixture was warmed to 150 °C and stirred for 1.5 hours. The resulting mixture was cooled to room temperature and purified without work up by HPLC (system H2O / MeOH / 0.1% NH4OH) to obtain 5-benzyl-10H- indolo[3,2-b]quinolin-5-ium-10-ide (Compound 23) (26.9 mg, 95.0% purity, 82.87 pmol, 12.9% yield). 1 H (500 MHz, DMSO-d6) 9.07 (1 H, s), 8.45 (1 H, d, J 8.3 Hz), 8.29 (1 H, d, J 9.1 Hz), 8.03 (1H, d, J 9.1 Hz), 7.82 (1 H, t, J 7.6 Hz), 7.74-7.63 (2H, m), 7.46 (1H, t 7.6 Hz), 7.32-7.22 (3H, m), 7.09 (2H, d, J 7.6 Hz), 6.88 (1H, t, J 7.6 Hz), 6.68 (2H, s).

[0177] Example 24

[0178] Further analogues Compound 16 (CR095), Compound 17 (CR107) and Compound 18 (CR244) were tested for their effect on TGFp signalling and compared against the parent cryptoleptine compound (CR). This was investigated using SMAD- responsive reporter assay as set out in Example 7. HEK293T cells were overexpressed with TGFBRII receptor together with SBE-Luc and pJ7-LacZ reporter plasmids. pJ7-LacZ reporter plasmid encodes p-gal gene and used here as an internal standard. 24 hours after transfection, cells were treated with cryptolepine compounds as well as with analoguesand the luc-gal activities were determined. The values of untreated cells were set as 100 and compared with cells treated with cryptolepine analogues. The parent cryptolepine compound as well as the analogues inhibited reporter activity with the exception of CR095, which failed to inhibit the reporter activity. These data suggest that the newly synthesized cryptolepine compounds inhibit TGF p signalling. Relative luciferase-p-galactosidase activity is presented in Figure 21 as the mean with error bars representing the Standard Error of Mean (SEM). (ns-0.39, ****P< 000. n=4-6).

[0179] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0180] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0181] The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

Claims

CLAIMS1 . A compound for use in a method of treatment, the compound being selected from a compound according to Formula (I):wherein:R1, R2, R3, R4, R5and R6are each independently selected from: H, halo, C1-6 alkyl, C1-6 haloalkyl, C6-io aryl, -OR7, -SO2R7, -C(O)OR7, -C(O)NR7R8, -NO2and -CN; andR7and R8are each independently selected from: H, C1-6 alkyl, phenyl and toluenyl.

2. The compound for use of claim 1 , wherein R1, R2, R3, R4, and R5are each independently selected from: H, halo, C1-6 alkyl, C1-6 haloalkyl, -OR7, -SO2R7, -C(O)OR7, - C(O)NR7R8, -NO2and -CN.

3. The compound for use of claim 1 , wherein R1, R2, R3, R4, and R5are each independently selected from: H, halo, -OR7, and -NO2.

4. The compound for use of claim 1 , wherein R6is H, -OR7, or phenyl.

5. The compound for use of claim 1 , wherein R7is H or C1-6 alkyl.

6. The compound for use of claim 1 , wherein R1, R2, R3, R4, and R5are each independently selected from: H, halo, C1-6 alkyl, C1-6 haloalkyl, -OR7, -SO2R7, -C(O)OR7, - C(O)NR7R8, -NO2and -CN.;R6is H, -OR7or phenyl; andR7is H or methyl.

7. The compound for use of claim 1 wherein the compound is selected from:

8. The compound for use of claims 1 to 7, wherein the method of treatment is the treatment of a condition caused by dysregulated TGF-p and / or bone morphogenic protein (BMP) signalling pathways.

9. The compound for use of claims 1 to 7, wherein the method of treatment is the treatment of a condition modulated by overexpression of TGF-p.

10. The compound for use of claims 1 to 7, wherein the method of treatment is the treatment of a condition modulated by under expression of bone morphogenic protein (BMP).

11. The compound for use of claims 1 to 7, wherein the method of treatment is the treatment of a condition modulated by overexpression of TGF-p and under expression of bone morphogenic protein (BMP).

12. The compound for use of claims 1 to 7, wherein the compound is for use in treating pulmonary hypertension, for example pulmonary arterial hypertension (PAH).

13. The compound for use of claim 12, wherein the pulmonary hypertension is pulmonary arterial hypertension, optionally wherein the pulmonary arterial hypertension is caused by a mutation in the bone morphogenic protein receptor of a patient suffering from pulmonary arterial hypertension.

14. The compound for use of claims 1 to 7, wherein the compound is for use in promoting apoptosis of cells within a human or animal tissue.

15. The compound for use of claims 1 to 7, wherein the compound is for use in the reversal of vascular remodelling in patients.

16. A method of treatment of a condition modulated by overexpression of TGF-p, wherein the method comprises administering a therapeutically effective amount of a compound selected from a compound according to Formula (I):wherein:R1, R2, R3, R4, R5and R6are each independently selected from: H, halo, C1-6 alkyl, C1-6 haloalkyl, C6-io aryl, -OR7, -SO2R7, -C(O)OR7, -C(O)NR7R8, -NO2and -CN; andR7and R8are each independently selected from: H, C1-6 alkyl, phenyl and toluenyl.

17. The method of treatment of claim 16, wherein R1, R2, R3, R4, and R5are each independently selected from: H, halo, C1-6 alkyl, C1-6 haloalkyl, -OR7, -SO2R7, -C(O)OR7, - C(O)NR7R8, -NO2and -CN.

18. The method of treatment of claim 16, wherein R1, R2, R3, R4, and R5are each independently selected from: H, halo, -OR7, and -NO2.

19. The method of treatment of claim 16, wherein R6is H, -OR7, or phenyl.

20. The method of treatment of any one of claims 16 to 19, wherein R7is H or C1-6 alkyl.21 . The method of treatment of claim 16, wherein R1, R2, R3, R4, and R5are each independently selected from: H, halo, C1-6 alkyl, C1-6 haloalkyl, -OR7, -SO2R7, -C(O)OR7, - C(O)NR7R8, -NO2and -CN.;R6is H, -OR7or phenyl; andR7is H or methyl.

22. The method of treatment of claim 16, wherein the compound is selected from:

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