Allosteric AKT inhibitors for the treatment of hereditary hemorrhagic telangiectasia

Abstract

The present application provides a compound of Formula (I) or a pharmaceutically acceptable salt thereof for use in the treatment of hereditary hemorrhagic telangiectasia (HHT).

Description

Technical Field

[0001] This invention relates to allosteric AKT inhibitors and their use in the treatment of hereditary hemorrhagic telangiectasia (HHT). Background Technology

[0002] Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant genetic disorder affecting approximately one in 5,000-8,000 people worldwide. Clinical symptoms include arteriovenous malformations (AVMs) in the lungs, brain, and liver, which consist of direct connections between arteries and veins without the involvement of capillaries. If left untreated, AVMs can lead to life-threatening complications. Individuals with HHT may also develop small arteriovenous malformations called telangiectasia in the nose, mouth, and gastrointestinal tract. These fragile blood vessels are prone to rupture and bleeding, leading to recurrent anemia after severe and frequent bleeding episodes (Dupuis-Girod et al., (2010), "Hereditary hemorrhagic telangiectasia: Frommolecular biology to patient care", Journal of Thrombosis and Haemostasis. https: / / doi.org / 10.1111 / j.1538-7836.2010.03860.x; Iriarte et al., (2019), "PI3K (Phosphatidylinositol 3-Kinase) Activation and Endothelial Cell Proliferation in Patients with Hemorrhagic Hereditary Telangiectasia Type 1", Cells. https: / / doi.org / 10.3390 / cells8090971).

[0003] Most mutations discovered to date are found in two genes. HHT1 is caused by mutations in the ENG (endoglin) gene, while HHT2 is caused by mutations in the ACVRL1 (activin receptor-like kinase 1, ALK1) gene. Both are receptors for transforming growth factor-β (TGF-β) / bone morphogenetic protein (BMP), which is primarily expressed in endothelial cells. The mutations represent null alleles, suggesting that haploid deficiency is the underlying cause of HHT. Little is known about how mutations in ENG or ACVRL1 (ALK1) lead to pathological angiogenesis.

[0004] The identification of pathogenic gene mutations and the establishment of animal models have revealed that reduced transforming growth factor-β (TGF-β) / bone morphogenetic protein (BMP) signaling and increased vascular endothelial growth factor (VEGF) signaling activity in endothelial cells are the causes of vascular malformation development in hemorrhagic thrombosis (HHT). Disruptions to these key pathways lead to endothelial cell activation, causing parietal cells to detach from the endothelium (Galaris et al., (2019), "Pericytes in Hereditary Hemorrhagic Telangiectasia", Advances in Experimental Medicine and Biology. https: / / doi.org / 10.1007 / 978-3-030-16908-4_10). This initial instability results in inadequate vascular response to angiogenesis triggers, leading to excessive vascular growth and the formation of vascular abnormalities, making the vessel prone to bleeding.

[0005] Recently, a series of studies from mouse models and in vitro experiments have revealed the link between the PI3K / AKT signaling pathway and the increased endothelial cell activation observed in HHT. The loss of ALK1 signaling in endothelial cells occurs simultaneously with the increase in PI3K / AKT signaling induced by PTEN reduction (Jin et al., (2017), "Endoglin prevents vascular malformation by regulating flow-induced cell migration and specification through VEGFR2 signalling", Nature Cell Biology, 19(6), 639–652. https: / / doi.org / 10.1038 / ncb3534; Ola et al., (2016), "PI3 kinase inhibition improves vascular malformations in mouse models of hereditary haemorrhagic telangiectasia", Nature Communications, 7. https: / / doi.org / 10.1038 / ncomms13650; Ola et al. (2018), "SMAD4 Prevents Flow Induced Arteriovenous Malformations by Inhibiting Casein Kinase 2", Circulation, 138(21), 2379–2394). This observation was confirmed in skin capillary dilatation biopsies of HHT1 and HHT2 patients, where increased endothelial cell proliferation was associated with increased downstream genes of the PI3K / AKT pathway (Alsina-Sanchiset al., (2018), "ALK1 loss results in vascular hyperplasia in mice and humans through PI3K activation", Arteriosclerosis, Thrombosis, and Vascular Biology. https: / / doi.org / 10.1161 / ATVBAHA.118.310760).

[0006] Targeting Pi3K / AKT / mTOR signaling offers a therapeutic strategy for treating pathological angiogenesis observed in HHT. However, safe and effective therapeutic agents remain unknown. For example, the widely used pan-Pi3K inhibitor Wortmannin can inhibit AVM formation in ALK1 or SMAD4 iKO retinal mouse models (Ola et al., (2016), (2018), ibid.), but not in endothelial glycoprotein-induced knockout (Eng-iKO) models (Jin et al., (2017), ibid.). Similarly, the typical mTOR inhibitor sirolimus cannot block AVM formation in Eng-iKO models (Ruiz et al., (2019), "Sirolimus plusnintedanib treats vascular pathology in HHT mouse models", BioRxiv CellBiology. https: / / doi.org / 10.1101 / 739144).

[0007] Therefore, there is a need to provide effective HHT treatments with acceptable safety profiles. Summary of the Invention

[0008] According to a first aspect of the invention, a compound of formula (I) is provided: Or a pharmaceutically acceptable salt thereof, used for the treatment of hereditary hemorrhagic telangiectasia (HHT) in subjects, wherein: Each X is independently either O or S; R 1 Selected from hydrogen and C1-C 10 Alkyl groups, wherein each C1-C 10 Alkyl groups are optionally surrounded by one or more elements selected from halogens, -CN, and -OR. 7 Or substituents of 3- to 6-membered cycloalkyl groups; R 2 and R 3 Each is independently selected from hydrogen and C1-C 10 Alkyl; or R 2 and R 3 Together with the nitrogen atoms they are attached to, they form 3- to 6-membered heterocycles; R 4 and R 5 Each is independently selected from C1-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl; or R 4 and R 5Together with the carbon atoms to which they are attached, they form 3- to 6-membered cycloalkyl rings or 3- to 6-membered heterocycles, which are optionally connected by one or more rings selected from C1-C2. 10 Alkyl, -CN, -OR 7 Halogen, -COR 7 CO2R 7 CONR 7 2 and -NR 7 Substitution of 2 by substituents; R 6 It is a 5- to 7-membered aryl ring or a 5- to 7-membered heteroaryl ring, optionally composed of one or more elements selected from C1-C2. 10 Alkyl, -CN, -OR 7 Halogen, -COR 7 CO2R 7 CONR 7 2 and -NR 7 Substitution of 2; and Each R 7 Independently selected from hydrogen and C1-C 10 alkyl.

[0009] In some embodiments, the compound of formula (I) has the following structure: .

[0010] In some implementation schemes, R 6 It is a phenyl group substituted with one or more substituents selected from C1-C1. 10 Alkyl, -CN, -OR 7 Halogen, -COR 7 CO2R 7 CONR 7 2 and -NR 7 2. In some embodiments, the one or more substituents are selected from C1-C6 alkyl groups, -CN, -OH, and halogens. In other embodiments, R 6 It is an unsubstituted phenyl group.

[0011] In some implementation schemes, R 6 It is a 5- to 7-membered heteroaryl ring substituted with one or more substituents selected from C1-C2. 10 Alkyl, -CN, -OR 7 Halogen, -COR 7 CO2R 7 CONR 7 2 and -NR 7 2. In some embodiments, the one or more substituents are selected from C1-C6 alkyl groups, -CN, -OH, and halogens. In other embodiments, R6 It is an unsubstituted 5- to 7-membered heteroaryl ring. In some embodiments, the heteroaryl ring is a 6-membered heteroaryl ring. In some embodiments, the heteroaryl ring contains at least one nitrogen atom. In some embodiments, the heteroaryl ring contains at least one oxygen atom. In some embodiments, the heteroaryl ring contains at least one sulfur atom. For example, the heteroaryl ring may be pyridine, pyran, thioran, pyrrole, furan, or thiophene.

[0012] In some implementation schemes, R 4 and R 5 Together with the carbon atoms they are attached to, they form unsubstituted 3- to 6-membered cycloalkyl rings or heterocycles. In other embodiments, R 4 and R 5 Together with the carbon atoms they are attached to, they form unsubstituted 3- to 6-membered cycloalkyl rings or heterocycles, which are selected from one or more C1-C1 carbon atoms. 10 Alkyl, -CN, -OR 7 Halogen, -COR 7 CO2R 7 CONR 7 2 and -NR 7 Substituent substitution of 2. For example, the cycloalkyl ring may be a cyclopropyl ring, a cyclobutyl ring, a cyclopentyl ring, or a cyclohexyl ring. In some embodiments, the heterocycle contains at least one nitrogen atom. In some embodiments, the heterocycle contains at least one oxygen atom. In some embodiments, the heterocycle contains at least one sulfur atom. For example, the heterocycle may be piperidine, tetrahydropyran, thiane, pyrrolidine, tetrahydrofuran, or tetrahydrothiophene.

[0013] In some implementation schemes, R 4 and R 5 Together with the carbon atoms to which they are attached, they form an unsubstituted cyclopropyl ring, cyclobutyl ring, cyclopentyl ring, or cyclohexyl ring. In other embodiments, the cyclopropyl ring, cyclobutyl ring, cyclopentyl ring, or cyclohexyl ring is substituted by one or more substituents selected from C1-C1. 10 Alkyl, -CN, -OR 7 Halogen, -COR 7 CO2R 7 CONR 7 2 and -NR 7 2. In some embodiments, the one or more substituents are selected from C1-C6 alkyl, -CN, -OH, and halogens. In some embodiments, R 4 and R 5Together with the carbon atoms to which they are attached, they form an unsubstituted cyclobutyl ring. In other embodiments, the cyclobutyl ring is substituted by one or more substituents selected from C1-C1. 10 Alkyl, -CN, -OR 7 Halogen, -COR 7 CO2R 7 CONR 7 2 and -NR 7 2. In some embodiments, the one or more substituents are selected from C1-C6 alkyl groups, -CN, -OH, and halogens.

[0014] In some implementations, each X is O.

[0015] In some implementation schemes, R 1 It is unsubstituted C1-C 10 Alkyl, unsubstituted C1-C6 alkyl, or unsubstituted C1-C4 alkyl. In some embodiments, R 1 It is methyl, ethyl, propyl, butyl, pentyl, or hexyl. In some embodiments, R 1 It is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl. In some embodiments, R 1 It is methyl or ethyl. In some embodiments, R 1 It is a C1-C substituted with one or more substituents selected from halogens, -CN, -OH or 3- to 6-membered cycloalkyl groups. 10 Alkyl, C1-C6 alkyl, or C1-C4 alkyl. In some embodiments, R 1 It is -(CH2) n CN, where n is 1, 2, 3, 4 or 5.

[0016] In some implementation schemes, R 2 and R 3 Each is independently selected from hydrogen and C1-C 10 Alkyl group. In some embodiments, R 2 and R 3 Each is independently selected from hydrogen and C1-C6 alkyl groups. In some embodiments, R 2 and R 3 Each is independently selected from hydrogen, methyl, and ethyl. In some embodiments, R 2 and R 3 Each is hydrogen.

[0017] In some embodiments, the compound of formula (I) is: Or a pharmaceutically acceptable salt thereof, wherein R 8Selected from -OH, -CN, halogens and C1-C6 alkyl groups.

[0018] In some embodiments, the compound of formula (I) is: Or its pharmaceutically acceptable salt.

[0019] In some embodiments, the pharmaceutically acceptable salt is a tartrate, methanesulfonate, or phosphate. In some embodiments, the pharmaceutically acceptable salt is a tartrate. In some such embodiments, the tartrate is an L-tartrate.

[0020] In some embodiments, the compound is formulated for oral administration to the subject.

[0021] In some embodiments, the subject is a human being. In some embodiments, the subject is an adult. In some such embodiments, the compound of formula (I) is administered to the subject at a dose of 20 mg to 75 mg QD. In other such embodiments, the compound of formula (I) is administered to the subject at a dose of 10 mg to 50 mg QD, 20 mg to 40 mg QD, or 20 mg to 30 mg QD.

[0022] In some embodiments, treatment of HHT includes reducing the frequency, duration, or intensity of HHT-related bleeding. In some such embodiments, the HHT-related bleeding is gastrointestinal (GI) bleeding. In some embodiments, the HHT-related bleeding is nasal bleeding.

[0023] In some implementations, the treatment of HHT includes increasing the subject's hemoglobin levels.

[0024] In some embodiments, the HHT treatment includes reducing the number of capillary dilatations in the subject. In some embodiments, the HHT treatment includes reducing the size of capillary dilatations in the subject. In some embodiments, the capillary dilatations are cutaneous capillary dilatations. In some embodiments, the capillary dilatations are nasal capillary dilatations. In some embodiments, the capillary dilatations are oral capillary dilatations. In some embodiments, the capillary dilatations are gastrointestinal capillary dilatations.

[0025] In some embodiments, the HHT treatment includes reducing the number and / or size of arteriovenous malformations (AVMs) in the subject. In some embodiments, the HHT treatment includes preventing the formation of AVMs in the subject. In some such embodiments, the AVM is a pulmonary AVM. In some embodiments, the AVM is a brain (cerebral) AVM. In some embodiments, the AVM is a visceral AVM.

[0026] In some embodiments, the treatment of HHT includes reducing heart failure and pulmonary hypertension (PAH). In some embodiments, the treatment of HHT includes preventing heart failure and pulmonary hypertension (PAH).

[0027] In some embodiments, the HHT treatment includes preventing right-to-left shunting induced by pulmonary AVMs in the subject. In some embodiments, the HHT treatment includes reducing the degree of right-to-left shunting induced by pulmonary AVMs in the subject.

[0028] In some embodiments, treatment of HHT includes reducing the subject's need for iron supplementation, such as reducing the number of iron infusions required by the subject. In some embodiments, treatment of HHT includes reducing the number of blood transfusions required by the subject.

[0029] In some implementations, the treatment of HHT includes reducing hepatic blood flow.

[0030] In some implementations, the HHT treatment reduces the subject's need for a liver transplant.

[0031] In some implementations, treatment of HHT includes reducing the frequency and / or severity of additional symptoms of HHT. Such additional symptoms may include difficulty breathing, migraines, fatigue, neurological events, and embolic events.

[0032] The present invention also provides the use of compounds of formula (I) as defined in any of the above embodiments or pharmaceutically acceptable salts thereof in the preparation of medicaments for the treatment of hereditary hemorrhagic telangiectasia (HHT).

[0033] The present invention also provides a method for treating hereditary hemorrhagic telangiectasia (HHT) in a subject in need, the method comprising administering to the subject an effective amount of a compound of formula (I) as defined in any of the above embodiments or a pharmaceutically acceptable salt thereof.

[0034] In some embodiments, the method of treating HHT results in a reduction in the frequency, duration, or intensity of HHT-related bleeding. In some such embodiments, the HHT-related bleeding is gastrointestinal (GI) bleeding. In some embodiments, the HHT-related bleeding is nasal bleeding.

[0035] In some implementations, the method of treating HHT results in an increase in the subject's hemoglobin levels.

[0036] In some embodiments, the method of treating HHT results in a reduction in the number of dilated capillaries in the subject. In some embodiments, the method of treating HHT results in a reduction in the size of dilated capillaries in the subject. In some embodiments, the dilated capillaries are cutaneous capillaries. In some embodiments, the dilated capillaries are nasal capillaries. In some embodiments, the dilated capillaries are oral capillaries. In some embodiments, the dilated capillaries are gastrointestinal capillaries.

[0037] In some embodiments, the method of treating HHT results in a reduction in the number of arteriovenous malformations (AVMs) in the subject. In some embodiments, the method of treating HHT results in a reduction in the size of the arteriovenous malformations (AVMs) in the subject. In some embodiments, the method prevents the formation of AVMs in the subject. In some such embodiments, the arteriovenous malformation is a pulmonary AVM. In some embodiments, the arteriovenous malformation is a cerebral AVM. In some embodiments, the arteriovenous malformation is a visceral AVM.

[0038] In some implementations, the method of treating HHT results in an improvement in the subject's quality of life.

[0039] In some embodiments, the method of treating HHT results in a reduction of heart failure and pulmonary hypertension (PAH). In some embodiments, the method of treating HHT prevents heart failure and pulmonary hypertension (PAH).

[0040] In some embodiments, the method of treating HHT prevents right-to-left shunting induced by pulmonary AVMs in the subject. In some embodiments, the method of treating HHT results in a reduction in the degree of right-to-left shunting induced by pulmonary AVMs in the subject.

[0041] In some embodiments, the method of treating HHT results in a reduction in the subject's need for iron supplementation, such as a reduction in the number of iron infusions required by the subject. In some embodiments, the method of treating HHT results in a reduction in the number of blood transfusions required by the subject.

[0042] In some implementations, the method of treating HHT results in a reduction in hepatic blood flow.

[0043] In some implementations, the method of treating HHT reduces the need for liver transplantation in the subject.

[0044] In some implementations, the methods for treating HHT result in a reduction in the frequency and / or severity of additional HHT symptoms. Such additional symptoms may include difficulty breathing, migraines, fatigue, neurological events, and embolic events.

[0045] In some implementations, the method of treating HHT leads to a comprehensive improvement in the subject's quality of life. Attached Figure Description

[0046] Figure 1A The overall structure of compound 6 combined with AKT2 is shown. Figure 1B The structure of the complex was shown, the allosteric bag was magnified, and the interactions of the amino acids were revealed. Figure 1C This is a schematic diagram of amino acid interactions.

[0047] Figure 1D The structure of the free base of VAD044 complexed with AKT2 is shown (magnified allosteric bag and amino acid interactions are shown). Figure 1E This is a schematic diagram of amino acid interactions.

[0048] Figure 2A It is a tree diagram showing the relative sequence homology of different kinases (where kinases on the same branch and located close to each other have a high level of sequence homology; while those on different branches have significantly different sequence homology). For example... Figure 2A As shown, AKT1, AKT2, and AKT3 exhibit a very high level of sequence homology. PDK1 is located on the same main branch as AKT1 / 2 / 3 (labeled "AGC"), indicating a relatively high level of sequence homology with AKT1 / 2 / 3; while p38α is located on a completely different main branch (labeled "CMGC"), indicating a lower level of sequence homology between AKT1 / 2 / 3 and p38α. Figure 2B The diagram shows VAD044 (6-(4-(1-amino-3-hydroxycyclobutyl)phenyl)-1-ethyl-7-phenyl-1H-pyrido[2,3-b][1,4]). The percentage of inhibition of these five selected kinases (AKT1, AKT2, AKT3, PDK1 and p38α) by azinon-2(3H)-one L-tartrate.

[0049] Figure 3A and Figure 3BThe study demonstrated the safety and potential toxicity of perifosine. Figure 3A It showed the mortality rate of young mice. Figure 3B Shows the difference in animal weight on day 4 (P4) and day 7 (P7) after birth. All error bars represent SEM. **** p<0.0001 This is the result of a one-way ANOVA and Dunnet post-hoc test, comparing the mean of each group with a control group that received only the injectable vehicle. "ns" = not significant.

[0050] Figures 4A-4D The study showed that perifoxine has poor anti-angiogenic properties in mice. Figure 4A The image shows isolectin-B4 stained endothelial cells in the retinal vessels of wild-type mice at P7, following intraperitoneal injection at P4 with the carrier alone or with 5 mg / kg, 10 mg / kg, 25 mg / kg, 50 mg / kg, or 100 mg / kg. The vascular network is shown at a higher magnification at the bottom. Figure 4B This shows a quantitative representation of radial expansion. Figure 4C The surface area of ​​isolectin-B4 positive cells was shown, and Figure 4D The number of branches for each field of view is displayed. All error bars represent SEM. * p<0.05 and** p<0.01 This is the result of a one-way ANOVA and Dunnet's post-hoc test, which compares the mean of each group with the control group treated only with the carrier. "ns" = insignificant.

[0051] Figures 5A-5F This demonstrates the effectiveness of perifoxine in preventing AVM formation in the Eng-iKO mouse model. Figure 5A This is a diagram illustrating the injection protocol for perifoxine. Figure 5B This image shows a confocal image demonstrating the effect of perifoxane on the Eng-iKO retina stained with isolectin-B4 to visualize endothelial cells. The bottom image shows a vascular network at higher magnification. Figure 5C The number of AV shunts per mouse is shown. Figure 5D This shows a quantitative representation of radial expansion. Figure 5E The area showing positive for isolectin-B4, and Figure 5F The number of branches for each field of view is shown. All error bars represent SEM. *p<0.05 is the result of one-way ANOVA and Dunnet post-hoc test comparing the mean of each group to the Eng-iKO group. “ns” = not significant.

[0052] Figure 6A and Figure 6BThe safety and potential toxicity of Uprosertib were demonstrated. Figure 6A It showed the mortality rate of young mice. Figure 6B The difference in animal body weight at day 4 (P4) and day 7 (P7) after birth is shown. All error bars represent SEM. *p<0.05 is the result of one-way ANOVA and Dunnet post-hoc test comparing the mean of each group to the control group injected with the vector alone. “ns” = not significant.

[0053] Figures 7A-7D Uprosertib was shown to have anti-angiogenic properties in mice. Figure 7A The image shows isolectin-B4 stained endothelial cells in the retinal vessels of wild-type mice at P7, following intraperitoneal injection of Uprosertib at P4 alone or with doses of 2.5 mg / kg, 5 mg / kg, 10 mg / kg, or 25 mg / kg. The vascular network is shown at a higher magnification at the bottom. Figure 7B This shows a quantitative representation of radial expansion. Figure 7C The surface area of ​​isolectin-B4 positive cells was shown. Figure 7D The number of branches for each field of view is shown. All error bars represent SEM. *p<0.0, **p<0.01, **p<0.001, and ****p<0.0001 are the results of one-way ANOVA and Dunnet post-hoc tests comparing the mean of each group with the control group treated alone with the carrier. “ns” = insignificant.

[0054] Figure 8A This is a diagram illustrating the Uprosertib injection protocol. Figure 8B The lethality of juvenile mice was shown. P2 EngFlox / flox;cdh5-CreERT2 animals were injected with tamoxifen (50 μg) to induce near-complete gene knockout in newborn mice, followed by intraperitoneal injections of 0 mg / kg or 5 mg / kg body weight of Uprosertib at P3 and P5. The same volume of the vector was injected as a control.

[0055] Figure 9A This is a diagram illustrating the injection protocol for VAD044. Figure 9BThe lethality of juvenile mice was shown. Near-complete gene knockout was induced in newborn mice by injection of tamoxifen (50 μg) into EngFlox / flox;cdh5-CreERT2 animals at P2, followed by intraperitoneal injections of VAD044 at P3 and P5 at 0 mg / kg, 2.5 mg / kg, 5 mg / kg, or 10 mg / kg body weight. The same volume of the vector was injected as a control. Mice were then sacrificed at P7, and their retinas were immunofluorescently stained as described (Lebrin et al., 2010).

[0056] Figures 10A-10E This demonstrates the effectiveness of VAD044 in preventing AVM formation in the Eng-iKO mouse model. Figure 10A Confocal images showing the effect of VAD044 on the Eng-iKO retina stained with isolectin-B4 to visualize endothelial cells are displayed. Figure 10B The number of AV shunts per mouse is shown. Figure 10C This shows a quantitative representation of radial expansion. Figure 10D The area showing positive for isolectin-B4 was observed. Figure 10E The number of branch points for each field of view is shown. All error bars represent SEM. *p<0.05, **p<0.01, and ***p<0.001 are the results of one-way ANOVA and Dunnet post-hoc tests comparing the mean of each group to the Eng-iKO group. “ns” = not significant.

[0057] Figures 11A-11D A method is shown for isolating and characterizing primary mouse endothelial cells and Cre recombinase from the lungs of Eng-iKO mice by adenovirus infection to induce deletion of the Eng gene (and subsequently the Eng protein) in the cells.

[0058] Figure 12 The signaling of AKT and SMAD in normal endothelial cells (ECs) and Eng-deficient endothelial cells is shown.

[0059] Figure 13A This study demonstrates the effect of Cre adenovirus infection on endothelial glycoprotein expression in normal ECs and Eng-iKO ECs. Figure 13B The IC50 of VAD-044 in mouse lung endothelial cells with normal Eng expression levels and in mouse lung endothelial cells with complete loss of Eng expression are shown. Figure 13C p42 / p44 phosphorylation was shown and analyzed as a selective control. Detailed Implementation

[0060] As mentioned above, targeting the Pi3K / AKT / mTOR signaling pathway could provide a treatment strategy for HHT. However, safe and effective therapeutic agents remain unknown.

[0061] AKT is a serine / threonine protein kinase with three isoforms: AKT1, AKT2, and AKT3. AKT1 plays a crucial role in endothelial cells by regulating important downstream effectors such as eNOS, Ang2, and FOXO, which promote several aspects of angiogenesis signaling, such as shoot elongation and vascular remodeling (Lee et al., (2014), "Endothelial Akt1 mediates angiogenesis by phosphorylating multiple angiogenic substrates", Proceedings of the National Academy of Sciences of the United States of America. https: / / doi.org / 10.1073 / pnas.1408472111). Several studies have shown that sustained endothelial AKT activation leads to increased vessel size and systemic edema caused by chronic vascular permeability (Phunget et al., (2006), "Pathological angiogenesis is induced by sustained Akt signaling and inhibited by rapamycin", Cancer Cell. https: / / doi.org / 10.1016 / j.ccr.2006.07.003). Similarly, uncontrolled activation of AKT1 in endothelial cells induces vascular malformations in vivo (Perry et al., (2007), "AKT1 overexpression in endothelial cells leads to the development of cutaneous vascular malformations in vivo", Archives of Dermatology. https: / / doi.org / 10.1001 / archderm.143.4.50). Therefore, AKT may be a key molecular target controlling the pathological angiogenesis and vascular malformations observed in HHT.

[0062] The inventors tested different types of AKT inhibitors and evaluated their toxicity profiles, their effects on normal angiogenesis, and their ability to prevent the development of vascular malformations in an HHT mouse model. The results are discussed in detail in the examples below.

[0063] In summary, the inventors have surprisingly discovered that a specific allosteric AKT inhibitor having the following formula (I) can prevent the development of vascular malformations in an Eng-iKO mouse model of type 1 HHT (HHT1). The safety / toxicity profiles of this compound (and two other comparative AKT inhibitors) were obtained, as discussed in more detail in the examples below, and the effects of each compound on day 7 (P7) after birth were determined using a neoretinal angiogenesis model. C57BL / 6J The minimum effective dose for inhibiting angiogenesis in wild-type mice was determined. The effects of each compound on angiogenesis were also determined. Eng-iKO Minimum effective dose for preventing the development of arteriovenous malformations (AVMs) in mice.

[0064] The inventors unexpectedly discovered that the allosteric AKT inhibitor of formula (I) can treat and / or prevent HHT-related vascular malformations at surprisingly low doses (about 10 times lower than the doses commonly used in oncology) while exhibiting acceptable safety.

[0065] Therefore, in a first aspect, the present invention provides a compound of formula (I): Or a pharmaceutically acceptable salt thereof, which is used to treat HHT in subjects, wherein: Each X is independently either O or S; R 1 Selected from hydrogen and C1-C 10 Alkyl groups, wherein each C1-C 10 Alkyl groups are optionally surrounded by one or more elements selected from halogens, -CN, and -OR. 7 Or substituents of 3- to 6-membered cycloalkyl groups; R 2 and R 3 Each is independently selected from hydrogen and C1-C 10 Alkyl; or R 2 and R 3 Together with the nitrogen atoms they are attached to, they form 3- to 6-membered heterocycles; R 4 and R 5 Each is independently selected from C1-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl; or R 4 and R 5 Together with the carbon atoms to which they are attached, they form 3- to 6-membered cycloalkyl rings or 3- to 6-membered heterocycles, which are optionally connected by one or more rings selected from C1-C2.10 Alkyl, -CN, -OR 7 Halogen, -COR 7 CO2R 7 CONR 7 2 and -NR 7 Substitution of 2 by substituents; R 6 It is a 5- to 7-membered aryl ring or a 5- to 7-membered heteroaryl ring, optionally composed of one or more elements selected from C1-C2. 10 Alkyl, -CN, -OR 7 Halogen, -COR 7 CO2R 7 CONR 7 2 and -NR 7 Substitution of 2; and Each R 7 Independently selected from hydrogen and C1-C 10 alkyl.

[0066] In some embodiments, the compound of formula (I) has the following structure: .

[0067] In some implementation schemes, R 6 It is a phenyl group substituted with one or more substituents selected from C1-C1. 10 Alkyl, -CN, -OR 7 Halogen, -COR 7 CO2R 7 CONR 7 2 and -NR 7 2. In some embodiments, the one or more substituents are selected from C1-C6 alkyl groups, -CN, -OH, and halogens. In other embodiments, R 6 It is an unsubstituted phenyl group.

[0068] In some implementation schemes, R 6 It is a 5- to 7-membered heteroaryl ring substituted with one or more substituents selected from C1-C2. 10 Alkyl, -CN, -OR 7 Halogen, -COR 7 CO2R 7 CONR 7 2 and -NR 7 2. In some embodiments, the one or more substituents are selected from C1-C6 alkyl groups, -CN, -OH, and halogens. In other embodiments, R 6It is an unsubstituted 5- to 7-membered heteroaryl ring. In some embodiments, the heteroaryl ring is a 6-membered heteroaryl ring. In some embodiments, the heteroaryl ring contains at least one nitrogen atom. In some embodiments, the heteroaryl ring contains at least one oxygen atom. In some embodiments, the heteroaryl ring contains at least one sulfur atom. For example, the heteroaryl ring may be pyridine, pyran, thioran, pyrrole, furan, or thiophene.

[0069] In some implementation schemes, R 4 and R 5 Together with the carbon atoms they are attached to, they form unsubstituted 3- to 6-membered cycloalkyl rings or heterocycles. In other embodiments, R 4 and R 5 Together with the carbon atoms they are attached to, they form unsubstituted 3- to 6-membered cycloalkyl rings or heterocycles, which are selected from one or more C1-C1 carbon atoms. 10 Alkyl, -CN, -OR 7 Halogen, -COR 7 CO2R 7 CONR 7 2 and -NR 7 Substituents of 2. For example, the cycloalkyl ring may be a cyclopropyl ring, a cyclobutyl ring, a cyclopentyl ring, or a cyclohexyl ring. In some embodiments, the heterocyclic ring contains at least one nitrogen atom. In some embodiments, the heterocyclic ring contains at least one oxygen atom. In some embodiments, the heterocyclic ring contains at least one sulfur atom. For example, the heterocyclic ring may be piperidine, tetrahydropyran, thiane, pyrrolidine, tetrahydrofuran, or tetrahydrothiophene.

[0070] In some implementation schemes, R 4 and R 5 Together with the carbon atoms to which they are attached, they form an unsubstituted cyclopropyl ring, cyclobutyl ring, cyclopentyl ring, or cyclohexyl ring. In other embodiments, the cyclopropyl ring, cyclobutyl ring, cyclopentyl ring, or cyclohexyl ring is substituted by one or more substituents selected from C1-C1. 10 Alkyl, -CN, -OR 7 Halogen, -COR 7 CO2R 7 CONR 7 2 and -NR 7 2. In some embodiments, the one or more substituents are selected from C1-C6 alkyl, -CN, -OH, and halogens. In some embodiments, R 4 and R 5 Together with the carbon atoms to which they are attached, they form an unsubstituted cyclobutyl ring. In other embodiments, the cyclobutyl ring is substituted by one or more substituents selected from C1-C1.10 Alkyl, -CN, -OR 7 Halogen, -COR 7 CO2R 7 CONR 7 2 and -NR 7 2. In some embodiments, the one or more substituents are selected from C1-C6 alkyl groups, -CN, -OH, and halogens.

[0071] In some implementation schemes, each X is O.

[0072] In some implementation schemes, R 1 It is unsubstituted C1-C 10 Alkyl, unsubstituted C1-C6 alkyl, or unsubstituted C1-C4 alkyl. In some embodiments, R 1 It is methyl, ethyl, propyl, butyl, pentyl, or hexyl. In some embodiments, R 1 It is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl. In some embodiments, R 1 It is methyl or ethyl. In some embodiments, R 1 It is a C1-C substituted with one or more substituents selected from halogens, -CN, -OH or 3- to 6-membered cycloalkyl groups. 10 Alkyl, C1-C6 alkyl, or C1-C4 alkyl. In some embodiments, R 1 It is -(CH2) n CN, where n is 1, 2, 3, 4 or 5.

[0073] In some implementation schemes, R 2 and R 3 Each is independently selected from hydrogen and C1-C 10 Alkyl group. In some embodiments, R 2 and R 3 Each is independently selected from hydrogen and C1-C6 alkyl groups. In some embodiments, R 2 and R 3 Each is independently selected from hydrogen, methyl, and ethyl. In some embodiments, R 2 and R 3 Each is hydrogen.

[0074] In some embodiments, the compound of formula (I) is: Or a pharmaceutically acceptable salt thereof, wherein R 8 Selected from -OH, -CN, halogens and C1-C6 alkyl groups.

[0075] In some embodiments, the compound of formula (I) is: Or its pharmaceutically acceptable salt.

[0076] In some embodiments, the pharmaceutically acceptable salt is a tartrate, methanesulfonate, or phosphate. In some embodiments, the pharmaceutically acceptable salt is a tartrate. In some such embodiments, the tartrate is an L-tartrate.

[0077] In some embodiments, the compound is formulated for oral administration to the subject.

[0078] In some embodiments, treatment of HHT includes reducing the frequency, duration, or intensity of HHT-related bleeding. In some such embodiments, the HHT-related bleeding is gastrointestinal (GI) bleeding. In some embodiments, the HHT-related bleeding is nasal bleeding.

[0079] In some implementations, the treatment of HHT includes increasing the subject's hemoglobin levels.

[0080] In some embodiments, the HHT treatment includes reducing the number of capillary dilatations in the subject. In some embodiments, the HHT treatment includes reducing the size of capillary dilatations in the subject. In some embodiments, the capillary dilatations are cutaneous capillary dilatations. In some embodiments, the capillary dilatations are nasal capillary dilatations. In some embodiments, the capillary dilatations are oral capillary dilatations. In some embodiments, the capillary dilatations are gastrointestinal capillary dilatations.

[0081] In some embodiments, the HHT treatment includes reducing the number of arteriovenous malformations (AVMs) in the subject. In some embodiments, the HHT treatment includes reducing the size of the AVMs in the subject. In some embodiments, the HHT treatment includes preventing the formation of AVMs in the subject. In some such embodiments, the AVM is a pulmonary AVM. In some embodiments, the AVM is a brain (cerebral) AVM. In some embodiments, the AVM is a visceral AVM.

[0082] In some embodiments, the treatment of HHT includes reducing heart failure and pulmonary hypertension (PAH). In some embodiments, the treatment of HHT includes preventing heart failure and pulmonary hypertension (PAH).

[0083] In some embodiments, the HHT treatment includes preventing right-to-left shunting induced by pulmonary AVMs in the subject. In some embodiments, the HHT treatment includes reducing the degree of right-to-left shunting induced by pulmonary AVMs in the subject.

[0084] In some embodiments, treatment of HHT includes reducing the subject's need for iron supplementation, such as reducing the number of iron infusions required by the subject. In some embodiments, treatment of HHT includes reducing the number of blood transfusions required by the subject.

[0085] In some implementations, the treatment of HHT includes reducing hepatic blood flow.

[0086] In some implementations, the HHT treatment reduces the subject's need for a liver transplant.

[0087] In some implementations, treatment of HHT includes reducing the frequency and / or severity of additional symptoms of HHT. Such additional symptoms may include difficulty breathing, migraines, fatigue, neurological events, and embolic events.

[0088] The present invention also provides a method for treating hereditary hemorrhagic telangiectasia (HHT) in a subject in need, the method comprising administering to the subject an effective amount of a compound of formula (I) as defined in any of the above embodiments or a pharmaceutically acceptable salt thereof.

[0089] In some embodiments, the method of treating HHT results in a reduction in the frequency, duration, or intensity of HHT-related bleeding. In some such embodiments, the HHT-related bleeding is gastrointestinal (GI) bleeding. In some embodiments, the HHT-related bleeding is nasal bleeding.

[0090] In some implementations, the method of treating HHT results in an increase in the subject's hemoglobin levels.

[0091] In some embodiments, the method of treating HHT results in a reduction in the number of dilated capillaries in the subject. In some embodiments, the method of treating HHT results in a reduction in the size of dilated capillaries in the subject. In some embodiments, the dilated capillaries are cutaneous capillaries. In some embodiments, the dilated capillaries are nasal capillaries. In some embodiments, the dilated capillaries are oral capillaries. In some embodiments, the dilated capillaries are gastrointestinal capillaries.

[0092] In some embodiments, the method of treating HHT results in a reduction in the number of arteriovenous malformations (AVMs) in the subject. In some embodiments, the method of treating HHT results in a reduction in the size of the arteriovenous malformations (AVMs) in the subject. In some embodiments, the method prevents the formation of AVMs in the subject. In some such embodiments, the arteriovenous malformation is a pulmonary AVM. In some embodiments, the arteriovenous malformation is a cerebral AVM. In some embodiments, the arteriovenous malformation is a visceral AVM.

[0093] In some implementations, the method of treating HHT results in an improvement in the subject's quality of life.

[0094] In some embodiments, the method of treating HHT results in a reduction of heart failure and pulmonary hypertension (PAH). In some embodiments, the method of treating HHT prevents heart failure and pulmonary hypertension (PAH).

[0095] In some embodiments, the method of treating HHT prevents right-to-left shunting induced by pulmonary AVMs in the subject. In some embodiments, the method of treating HHT results in a reduction in the degree of right-to-left shunting induced by pulmonary AVMs in the subject.

[0096] In some embodiments, the method of treating HHT results in a reduction in the subject's need for iron supplementation, such as a reduction in the number of iron infusions required by the subject. In some embodiments, the method of treating HHT results in a reduction in the number of blood transfusions required by the subject.

[0097] In some implementations, the method of treating HHT results in a reduction in hepatic blood flow.

[0098] In some implementations, the method of treating HHT reduces the need for liver transplantation in the subject.

[0099] In some implementations, the methods for treating HHT result in a reduction in the frequency and / or severity of additional HHT symptoms. Such additional symptoms may include difficulty breathing, migraines, fatigue, neurological events, and embolic events.

[0100] In some implementations, the method of treating HHT leads to a comprehensive improvement in the subject's quality of life.

[0101] Definitions and abbreviations As used in this article, the term "allosteric AKT inhibitor" refers to a substance that inhibits AKT activity by binding to AKT at a site other than the enzyme's active site.

[0102] As used herein, the term "halogen" refers to -F, -Cl, -Br, and -I. Abbreviations: Table 1: Abbreviations Example Example 1: 6-(4-(1-amino-3-hydroxycyclobutyl)phenyl)-1-ethyl-7-phenyl-1H-pyrido[2,3- b][1,4] Synthesis of azinon-2(3H)-one 6-(4-(1-amino-3-hydroxycyclobutyl)phenyl)-1-ethyl-7-phenyl-1H-pyrido[2,3-b][1,4] was synthesized according to the scheme described in WO2011077098 (see Examples 97, 113 and 139 for details, reproduced below) Azine-2(3H)-one (referred to in this paper as "VAD044 free base"): 6-(4-((1s,3s)-1-amino-3-hydroxycyclobutyl)phenyl)-1-ethyl-7-phenyl-1H-pyrido[2,3- b][1,4] Synthesis of azinon-2(3H)-one: Example 139 from WO2011077098 Step 1: ((1s,3s)-1-(4-(1-ethyl-2-oxo-7-phenyl-2,3-dihydro-1H-pyrido[2,3-b]) [1,4] Azine-6-yl)phenyl)-3-hydroxycyclobutyl)carbamate tert-butyl Add 1,4-dioxanone to a 15 mL reaction tube Cesium carbonate (204 mg, 0.625 mmol), 6-bromo-1-ethyl-7-phenyl-1H-pyrido[2,3-b][1,4] in a mixture of alkane (2.3 ml) and water (0.8 ml) Azine-2(3H)-one* (50 mg, 0.150 mmol), ((1s,3s)-3-hydroxy-1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)phenyl)cyclobutyl)carbamate tert-butyl ester** (49 mg, 0.125 mmol) were added to give a colorless solution. The mixture was degassed by purging with nitrogen for 15 min, followed by the addition of [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II)dichloromethane adduct (20 mg, 0.025 mmol) and further degassed for 5 min. The reaction mixture was heated to 50 °C under a nitrogen atmosphere and maintained for one hour, then cooled to room temperature, diluted with water (5 ml), and extracted into ethyl acetate (3 × 5 ml). The combined organic phases were dried over Na₂SO₄, filtered, and concentrated under reduced pressure to dryness. The residue was purified by Biotage chromatography (cyclohexane:ethyl acetate, gradient elution from 90:10 to 0:100) to give the target product (45 mg, 70% yield) as an off-white solid. 1 ¹H-NMR (500MHz, CDCl₃) δ 7.29–7.35 (5H, m), 7.28 (1H, s), 7.18–7.24 (4H, m), 4.96 (1H, br s), 4.88 (2H, s), 4.05 (1H, br s), 4.01 (2H, q), 2.98 (2H, br s), 2.75 (2H, br s), 1.20–1.51 (9H, br m), 1.32 (3H, t). LCMS (Method D) RT = 1.25 min, M+H + = 516.20. Step 2: 6-(4-((1s,3s)-1-amino-3-hydroxycyclobutyl)phenyl)-1-ethyl-7-phenyl-1H-pyridyl [2,3-b][1,4] Azine-2(3H)-one ((1s,3s)-1-(4-(1-ethyl-2-oxo-7-phenyl-2,3-dihydro-1H-pyrido[2,3-b][1,4]) (45 mg, 0.087 mmol) tert-butyl carbamate (6-yl)phenyl)-3-hydroxycyclobutyl)carbamate was dissolved in TFA (1 mL) and stirred for 30 seconds. The solution was immediately concentrated under reduced pressure to dryness. The residue was dissolved in diethyl ether (~3 mL) and concentrated under reduced pressure to dryness, repeated three times. The residue was then slurried in diethyl ether (3 mL) and the supernatant was removed by pipetting after sedimentation. This process was repeated three times. The remaining solvent was removed by freeze-drying overnight to give the target compound (33 mg, 71% yield) as a creamy white solid. 1H-NMR (500 MHz, MeOD) δ 7.55 (1H, s), 7.39–7.42 (4H, m), 7.27–7.31 (3H, m), 7.20–7.24 (2H, m), 4.93 (2H, s), 4.01–4.11 (3H, m), 3.03–3.11 (2H, m), 2.42–2.50 (2H, m), 1.28 (3H, t). LCMS (Method D) RT = 0.74 min, M+H + = 416.20. * 6-Bromo-1-ethyl-7-phenyl-1H-pyrido[2,3-b][1,4] Synthesis of azinon-2(3H)-one: from Example 113 (steps 1-4) of WO2011077098 and page 222 (step 5): Step 1: 2-((5-bromo-3-nitropyridin-2-yl)oxy)ethyl acetate Within 30 minutes, towards 1, 4-2 Add ethyl glycolate (12.56 ml, 133 mmol) dropwise to a suspension of sodium hydride (5.31 g, 133 mmol) in alkyl (250 ml), ensuring the temperature is maintained below 30°C. Stir the resulting thick suspension at room temperature for 15 minutes. In another 1 L round-bottom flask, add 1,4-di(2,3 ... 5-Bromo-2-chloro-3-nitropyridine (21 g, 88 mmol) was added to alkyl (150 ml) to give a brown solution. A suspension of sodium hydride and ethyl glycolate was added dropwise at 0 °C over 30 minutes. The resulting reaction mixture was heated to 80 °C overnight.

[0103] The reaction mixture was concentrated under reduced pressure, and the crude residue was purified by Biotage silica gel chromatography (ethyl acetate in a solution of n-hexane with a gradient of 0% to 10%) to give the title compound (1.8 g, 44%). 1 H NMR (500 MHz, CDCl3) δ8.48 (1H, s), 8.42 (1H, s), 5.07 (2H, s), 4.28-4.24 (2H, q), 1.31-1.28 (3H,t). Step 2: 2-((3-nitro-5-phenylpyridin-2-yl)oxy)ethyl acetate In a 1 L round-bottom flask, cesium fluoride (45.6 g, 300 mmol), ethyl 2-(5-bromo-3-nitropyridin-2-yloxy)acetate (18.33 g, 60.1 mmol), phenylboronic acid (10.99 g, 90 mmol), and triphenylphosphine (4.73 g, 18.02 mmol) in 300 mL of 1,2-dimethoxyethane were added to give a yellow solution. The reaction mixture was degassed by purging with nitrogen for 30 min. Palladium(II) acetate (2.023 g, 9.01 mmol) was added, and the mixture was heated to 75 °C overnight under a nitrogen atmosphere. The reaction mixture was cooled to room temperature and concentrated under reduced pressure to dryness to give a brown solid. It was redissolved in dichloromethane, filtered, and concentrated under reduced pressure to dryness to give a brown solid. The crude residue was purified by Biotage chromatography (ethyl acetate in a 5% to 60% hexane solution) to give the title compound (6.9 g, 38%). 1H NMR (500MHz, CDCI3) δ 8.58 (1H, s), 8.56 (1H, s), 7.59-7.52 (2H, m), 7.48-7.46 (2H,m), 7.45-7.43 (1H, m), 5.13 (2H, s), 4.30-4.26 (2H, q), 1.33-1.30 (3H, t). Step 3: 7-Phenylaceto[2,3-b][1,4] Azine-2(3H)-one Ethyl 2-(3-nitro-5-phenylpyridin-2-yloxy)acetate (4.6 g, 15.22 mmol) in 37% hydrochloric acid (40 mL) was added to a 500 mL round-bottom flask to give a yellow suspension. The mixture was cooled to 0-5 °C, and tin powder (9.94 g, 84 mmol) was added in portions. This addition process has been confirmed to be exothermic. Care should be taken during addition. The mixture was then stirred at room temperature for another 30 minutes until all foaming ceased. The reaction mixture was heated to 80 °C under a nitrogen atmosphere for 3 hours. The reaction mixture was cooled to room temperature and diluted with water (800 mL). The white precipitate was separated by filtration, washed with water (100 mL), and dried to give a white solid. The solid was azeotropically reacted with toluene (3 × 30 mL) to give a white solid, the title compound (2.6 g, 77%). 1 H NMR (500 MHz, (CD3)2SO) δ 10.41 (1H, s), 8.10 (1H, s), 7.59 (2H, d), 7.49-7.42 (2H, t), 7.39-7-38 (1H, d), 4.83 (2H, s). Step 4: 6-Bromo-7-phenyl-1H-pyrido[2,3-b][1,4] Azine-2(3H)-one The 10 ml microwave-safe vial contains N-bromosuccinimide (78.6 mg, 0.441 mmol) and 7-phenyl-1H-pyrido[2,3-b][1,4] from dimethylformamide (1 ml). Azine-2(3H)-one (50 mg, 0.221 mmol). The reaction mixture was heated to 80 °C for 30 min under microwave irradiation. The reaction mixture was cooled to room temperature and diluted with ethyl acetate (10 ml). The organic solution was washed with water (2 × 10 ml) and brine (2 × 10 ml). The organic phase was dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude residue was purified by Biotage chromatography (methanol in a 0% to 5% dichloromethane solution gradient) to give the title compound (61 mg, 90%) as a yellow solid. 1 H NMR (500 MHz, CD3OD) δ 7.48-7.32 (5H, m), 7.12 (1 H, s), 4.82 (2H, s). Step 5: 6-Bromo-1-ethyl-7-phenyl-1H-pyrido[2,3-b][1,4] Azine-2(3H)-one Add potassium carbonate (408 mg, 2.95 mmol) and 6-bromo-7-phenyl-1H-pyrido[2,3-b][1,4] from anhydrous N,N-dimethylformamide (1 mL) to a 15 mL reaction tube. A azinon-2(3H)-one (300 mg, 0.983 mmol) and iodoethane (0.095 mL, 1.180 mmol) were reacted to give a brown suspension. The mixture was stirred at 50 °C under a nitrogen atmosphere for 60 min. The reaction mixture was diluted with saturated sodium bicarbonate solution (5 mL) and extracted with ethyl acetate (3 × 5 mL). The combined organic phases were washed with 50:50 water: saline solution (3 × 5 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure to dryness to give a brown solid. Purification was performed by Biotage chromatography (25 g silica gel column, cyclohexane:ethyl acetate, gradient elution from 90:10 to 20:80) to give the title compound as a beige solid (160 mg, 48.8% yield). 1 ¹H NMR (500 MHz, CDCI₃) δ 7.58–7.37 (5H, m), 7.21 (1H, s), 4.86 (2H, s), 3.96 (2H, q), 1.27 (3H, t). LCMS (Method D) RT 1.293 min, M+1 = 334. **((1s,3s)-3-hydroxy-1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)benzene Synthesis of tert-butyl(cyclobutyl)carbamate (Example 97 from WO2011077098): In a 40 mL reaction tube, tert-butyl (1s,3s)-1-(4-bromophenyl)-3-hydroxycyclobutylcarbamate*** (0.25 g, 0.731 mmol) from anhydrous tetrahydrofuran (14 mL) was added to obtain a colorless solution. The solution was degassed by purging with nitrogen for 20 minutes, followed by the addition of [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II)dichloromethane adduct (60 mg, 0.073 mmol). After purging with nitrogen for another 15 minutes, potassium acetate (143 mg, 1.461 mmol) and bis(pinacol)diboron (223 mg, 0.877 mmol) were added. The reaction mixture was heated to reflux overnight, then concentrated under reduced pressure to dryness, and purified by Biotage chromatography (cyclohexane:ethyl acetate, gradient elution from 88:12 to 0:100) to give the target product (240 mg, 84% yield) as a colorless oil (which solidified upon standing). 1 H-NMR (500 MHz, CDCI3) δ 7.71 (2H, d),7.44 (2H, d), 4.15 (1H, br s), 2.87-2.98 (2H, m), 2.27-2.44 (2H, m), 1.22-1.49 (21H, br m). (The composition of *** is described in WO2009148887 and WO2009148916) Example 2: In vitro pharmacodynamics In biochemical kinase analysis, 6-(4-(1-amino-3-hydroxycyclobutyl)phenyl)-1-ethyl-7-phenyl-1H-pyrido[2,3-b][1,4] Azine-2(3H)-one L-tartrate (VAD044) was found to effectively and selectively inhibit the activity of full-length AKT1 and AKT2, with an IC50 value of [missing information]. 50 The values ​​were 125 nM and 95 nM, respectively (compared to >500 nM for AKT3). However, all three mutant forms of the AKT isoform containing the inactive pleckstrin homology (PH) domain were not suppressed by VAD044. Furthermore, compared with ATP... K m Compared to the experiments conducted, incubation of VAD044 with increased concentrations of ATP resulted in an increase in the IC50 response. 50 The value had no effect. This confirms that the binding mode of VAD044 is allosteric and independent of ATP concentration.

[0104] By combining AKT2 with an approximate analogue of the free base of VAD044 (compound 6, below) (see...) Figure 1A The high-resolution (2.32 Å) eutectic structure obtained verifies the hypothesized VAD044 allosteric bonding mode. like Figure 1B As shown, compound 6 is bound in an allosteric binding bag approximately 10 Å from the hinge region at the interface between the N-lobes and C-lobes of the PH domain and the kinase domain.

[0105] Example 3: Kinase selectivity, and an overview of receptors and ion channels VAD044 is a potent allosteric inhibitor of AKT1 and AKT2. The selectivity window of VAD044 against target-independent protein kinases, G protein-coupled receptors, and ion channels was tested. A favorable selectivity window indicates good safety at the expected pharmacological concentrations effective in patients.

[0106] Kinase selectivity When tested on a panel of 450 kinases, VAD044 showed high selectivity for AKT1 and AKT2 at 10 μM (achieving 89% inhibition of AKT1 and 95% inhibition of AKT2, as shown in the figure). Figure 2B (As shown). Notably, only two other kinases, p38α and PDK1, achieved inhibition of over 75% (84% and 77% inhibition, respectively, as shown). Figure 2B As shown). Only 72% AKT3 suppression was achieved (see...). Figure 2B ).

[0107] Further analysis of the 10-point titration curve confirmed the IC50 values ​​of p38α and PDK1. 50 Greater than 10 μM.

[0108] In addition, VAD044 is compatible with ICs for AKT1 and AKT2. 50 Values ​​(respectively IC) 50 = 125 nM and IC 50 = 95 nM) was found to be much lower than its IC for the AKT3 subtype. 50 Value (VAD044 EC for AKT3 subtype) 50 (Value greater than 500 nM).

[0109] Therefore, the above results indicate that VAD044 is an effective and selective inhibitor of AKT1 and AKT2.

[0110] Overview of receptors and ion channels When tested at 10 μM, VAD044 did not inhibit cardiac ion channels Nav1.5, Cav1.2, Kv4.3, KChIP2, Kv1.5, KCNQ1, minK, KiR2.1, and HCN4 by less than 25% of their activity, and demonstrated excellent selectivity across a panel of 76 receptors and ion channels (Millipore Drug Discovery Safety and Responsibility Panel). Notably, at 10 μM, only three G protein-coupled receptors (GPCRs) showed inhibition exceeding 50%, namely LPA1, motilin, and P2Y1 receptors (showing mean percentage inhibition values ​​of 57.4%, 50.0%, and 50.6%, respectively), and only one GPCR showed an increase in agonistic activity exceeding 20% ​​(i.e., GPR14, with a 29.5% increase observed at 10 μM). These results are shown in Table 2 below: Table 2: Results of the Millipore Drug Discovery Safety and Responsibility Suite The above results indicate that VAD044 does not interfere with the tested receptors and ion channels.

[0111] Therefore, the in vitro enzyme profile of VAD044 can be summarized as follows: -VAD044 effectively and selectively inhibits AKT1 and AKT2 (IC50 = 125 nM and 95 nM, respectively), with a window at least 5 times larger than that of the AKT3 isotype (IC50 > 500 nM). -VAD044 does not interfere with a wide range of tested kinases, receptors, and ion channels.

[0112] Example 4: Safety / toxicity of three AKT inhibitor compounds in Eng-iKO mice; anti-angiogenic properties Research on sex and the ability to prevent AVM formation. Materials / Methods mice The pregnant female C57Bl / 6J mice were provided by the Janvier Laboratory. EngFlox / flox Mice (Mahmoud etal., (2010), "Pathogenesis of arteriovenous malformations in the absence ofendoglin", Circulation Research. https: / / doi.org / 10.1161 / CIRCRESAHA.109.211037) and Cdh5(PAC)-CreERT2 (12) Hybridization to produce endothelial-specific induction Eng knock remove The animal facilities and procedures complied with Dutch government guidelines and European Parliament Directive 2010 / 63 / EU. All efforts were made to minimize the number of animals used and their suffering. The Animal Welfare Institution Committee of Leiden University Medical Center (Project No. AVD1160020171628) approved all protocols. Male and female mice were used in the first week after birth to study retinal angiogenesis.

[0113] AKT inhibition in vivo Safety / toxicity characteristics and determination of the minimum effective dose for inhibiting angiogenesis: Wild-type C57Bl / 6J juvenile mice were injected once at P4 with perifoxine at doses of 5 mg, 10 mg, 25 mg, 50 mg, or 100 mg / kg body weight; or uprosertib at doses of 0 mg, 2.5 mg, 5 mg, 10 mg, 25 mg, or 50 mg / kg body weight; or VAD044 at doses of 2.5 mg, 5 mg, and 10 mg / kg body weight. The juvenile mice were then sacrificed at P7. The retina was treated with immunofluorescence staining as described above (Lebrin et al., (2010), "Thalidomide stimulates vessel maturation and reduces epistaxis in individuals with hereditary hemorrhagictelangiectasia", Nature Medicine. https: / / doi.org / 10.1038 / nm.2131).

[0114] Determination of the minimum effective dose for preventing AVM formation in Eng-iKO mice: Tamoxifen (50 μg) was used on P2 EngFlox / flox; cdh5-CreERT2 Injections were administered to induce near-complete gene knockout in newborn mice, followed by intraperitoneal injections (IP) at P3 and P5 with 0 mg, 25 mg, or 50 mg per kg body weight, or IP injections at P3, P4, P5, and P6 with 10 mg per kg body weight. Mice injected with the same volume of the vector served as control animals. Mice were then euthanized at P7, and their retinas were treated with immunofluorescence staining as described above (Lebrin et al., ibid.).

[0115] Tamoxifen (50 mg) for P2 EngFlox / flox; cdh5-CreERT2Injections were administered to induce near-complete gene knockout in newborn mice, followed by a single intraperitoneal injection (IP) at P3 with 0, 1, 2.5, or 5 mg of Uprosertib per kg body weight. Mice injected with the same volume of the vector served as control animals. Mice were then euthanized at P7, and their retinas were treated with immunofluorescence staining as described above (Lebrin et al., ibid.).

[0116] Tamoxifen (50 mg) for P2 EngFlox / flox; cdh5-CreERT2 Injections were performed to induce near-complete gene knockout in newborn mice, followed by intraperitoneal injections of VAD044 at P3 and P5 at doses of 0, 2.5, 5, or 10 mg / kg body weight. Mice injected with the same volume of the vector served as control animals. Mice were then euthanized at P7, and their retinas were treated with immunofluorescence staining as described above (Lebrin et al., ibid.).

[0117] retinal vascular network analysis The quantification of the number of AV shunts per mouse for the retinal vascular network was performed manually. Radial dilation, vessel length, and branching points of the optic nerve in at least four visual fields of each retina were analyzed using ImageJ software (National Institutes of Health). Examples include: Lebrin et al., (2010), “Thalidomide stimulates vessel maturation and reduces epistaxis in individuals with hereditary hemorrhagic telangiectasia”, Nature Med, 16(4), 420-8; Gkatzis et al., (2016), “Interaction Between ALK1 Signaling and Connexin40 in the Development of Arteriovenous Malformations”, Arterioscler Thromb Vasc Biol., 36(4), 707-17; and Thalgott et al., (2018), “Decreased Expression of Vascular Endothelial Growth Factor Receptor 1 Contributes to the Pathogenesis of Hereditary Hemorrhagic Telangiectasia Type 2”, Circulation, 138(23). Described in 2698-2712. Customized macros from our own lab allow for automated image processing using the same processing and quantization parameters. In short, image brightness is enhanced by modifying pixel extrema, depending on the specific image histogram content. A Gaussian filter is applied to reduce homogeneous intensity. The filter order depends on the image resolution. Next, a threshold step size is calculated based on global thresholding techniques such as Otsu's or Li's methods, producing a binary image. A neutralizing filter is then applied to eliminate potential local irregularities that could lead to erroneous branch point detection. Vessel density is quantized from the binary image (calculated as the ratio of vein-related pixels to the total number of pixels in the image). For other parameters, the image skeleton is first calculated using ImageJ, and then the total vessel length is quantized using ImageJ's Measure Skeleton Length plugin. The number of branch points is extracted from ImageJ's AnalyzeSkeleton plugin.All results were transferred to Microsoft Office Excel and automatically rearranged using a lab-customized program. Visual management was implemented to ensure no outliers were detected, as all images generated at each processing step were saved.

[0118] Statistical analysis was performed using Prism 7 software (GraphPad). For most experiments, one-way ANOVA was used for multiple comparisons. Results are expressed as mean ± SEM. For post-hoc pairwise comparisons, the Dunnett test was used. *P<0.05、**P <0.01、***P<0.001 or ****P<0.0001 The value indicates statistical significance. The inventors had previously observed approximately 15% intragroup variation in parameters associated with changes in vascular structure, and therefore expected a difference of approximately 20% to 30% between control mice and mutant mice. Therefore, a group of eight mice under each condition was expected to show statistical significance.

[0119] Isolation, culture and stimulation of mouse endothelial cells Surgical resection Engflox / floxLungs of young mice were flushed in ice-cold DMEM and endothelial cells were isolated, as described in Galaris et al., (2021) “In vitro Three-Dimensional Sprouting Assay of Angiogenesis using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing” J Vis Exp (171). In summary, tissues were minced with scissors, digested in DMEM-3 mg / ml collagenase A (10103586001, Roche) at 37°C for 15 min, and then filtered through a 70-μm filter. The cell suspension was centrifuged at 200 g for 5 min, and CD45+ cells were removed using Dynabeads sheep anti-rat IgG (11035, Invitrogen) coated with rat anti-mouse CD45 antibody (550539, BD Pharmigen). Endothelial cells were sorted using Dynabeads sheep anti-rat IgG (11035, Invitrogen) coated with rat anti-mouse PECAM1 antibody (550274, BD Pharmigen). After washing five times with DMEM-0.1% BSA, cells were seeded in 6-well plates. Lung and liver ECs were maintained for 2 to 3 passages in endothelial cell growth medium 2 (C-22011, PromoCell) supplemented with fetal bovine serum, human epidermal growth factor, basic fibroblast growth factor, insulin-like growth factor, human vascular endothelial growth factor-165, ascorbic acid, heparin, and hydrocortisone (C-39211, SupplementPack ECGM2, PromoCell).

[0120] ECs were seeded in six-well plates and allowed to grow to 90% confluence. Cells were then washed with PBS and serum-starved for 6 hours. Cells were treated with 0.05 mM, 0.1 mM, 0.25 mM, 0.5 mM, 1.0 mM, 5.0 mM, or 10.0 mM VAD044 in endothelial cell growth medium 2 (C-22011, PromoCell) for 1 hour, followed by stimulation with VEGF-A (25 ng / ml) for 30 minutes. Cells were washed with cold PBS and then lysed in RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), 0.5% deoxycholate) containing protease and phosphatase inhibitors (PPC1010, Sigma-Aldrich).

[0121] Adenovirus infection ECs of the lungs or liver were infected with an adenovirus expressing Cre recombinase (SL100707, SignaGen Laboratories) at a multiplicity of infection (moi) of 500 for 6 hours, washed with PBS, and then cultured in endothelial cell growth medium 2 for 12 to 60 hours before use.

[0122] Immunofluorescence staining The eyes were fixed in 4% paraformaldehyde (PFA) in PBS at room temperature for 10 minutes. The retina was dissected and fixed overnight in 4% PFA in PBS at 4°C, followed by immunostaining. The retina was permeated overnight in PBS, 1% BSA, and 0.5% Triton X-100 at 4°C, rinsed in PBS, and washed twice in PBlec (PBS, pH 6.8, 1% Triton-X-100, 0.1 mM CaCl2, 0.1 mM MgCl2, 0.1 mM MnCl2), and then biotinylated in PBlec. Griffonia simplicifoliaThe samples were incubated overnight at 4°C in lectin (iso-lectin B4) (B-1205, Vector Laboratories, 1:50). After washing five times in PBS, the samples were incubated for 2 hours at 4°C with streptavidin Cy-3 (PA43001, Sigma-Aldrich, 1:100) diluted with PBS, 0.5% BSA and 0.25% X-100, and FITC-conjugated α-SMA (clone 1A4) (F3777, Sigma-Aldrich). After washing, flat whole-mount retina were laid on Dako medium (S3023, DAKO). High-resolution three-dimensional renderings of the whole-mount retina were obtained using a laser scanning microscope SP5 or SP8 (Leica).

[0123] Western blotting and quantification of proteins The samples were boiled for 10 minutes, and the proteins were separated on a 10% acrylamide gel and transferred to a nitrocellulose membrane. The membrane was then blocked with 5% BSA or 5% milk powder / Tris-buffered saline / Tween 20 and incubated with the following primary antibodies: rabbit antiphosphorylated Akt1 (Ser473) (44-621G, Invitrogen), rabbit anti-Akt1 (2938S, Cell Signaling), rabbit antiphosphorylated Smad1 / 5 (Ser463 / 465) (9516S, Cell Signaling), rabbit anti-Smad 1 (6944S, Cell Signaling), mouse antiphosphorylated p44 / 42 MAPK (Thr202 / Tyr204) (E10) (9106S, Cell Signaling), and rabbit p44 / 42 MAPK (Erk1 / 2) (9102S, Cell Signaling). Signaling, goat anti-endothelial glycoprotein (AF1097, R&D systems), and mouse anti-β-actin (A5441, Sigma-Aldrich) were used for detection. HRP anti-rabbit IgG or HRP anti-mouse IgG (W4011 or W4021, Promega, respectively) or HRP anti-goat IgG (HAF017, R&D systems) were used for detection, followed by scanning on a BIO-RAD ChemiDoc imager. Images were acquired within the linear range, and protein and protein phosphorylation levels were measured using ImageJ software. For all blots, background was subtracted and the results were normalized.

[0124] Pharmacokinetic (PK) characteristics of VAD044 in newborn mice VAD044 was prepared in sterile PBS and administered intraperitoneally (ip) to newborn mice of P3 and P5 at a dose concentration of 2.5 mg / kg and a dose volume of 10 µl / g. Terminal blood samples were collected from each mouse under isoflurane anesthesia at 24 and 48 hours post-injection for plasma preparation. Four newborn mice were used at each time point in this study. Blood was collected into EDTA-coated microtubes (Sartsed) for plasma preparation. The blood was centrifuged at 2700 × g for 10 min, and approximately 30 to 40 µl of plasma was collected and aliquoted into polypropylene tubes (two aliquots per animal). Plasma samples were stored at -80°C until bioanalysis. VAD044 compound levels in plasma samples were determined by LC-MS / MS. The 24-hour and 48-hour VAD044 plasma concentrations allowed for the calculation of Cavg using the following formula: AUC0 - inf / (number of 48-hour intervals).

[0125] Human Dosage Simulation Human dose simulation analysis was performed using a nonlinear mixed-effects modeling approach with MONOLIX (version 2019R1) properly installed. R (version 3.5.3) was used for preprocessing and postprocessing of data and model outputs. All raw PK data generated from the mouse, rat, and canine species used to build the model are summarized in Table 3, which provides an overview of the number of animals and sample sizes included in the overall analysis.

[0126] Table 3 Summary of available concentration observations for VAD044 The PK parameters for the human model were determined based on the allometric scaling of clearance and volume parameters with body weight. Assumptions must be made for bioavailability and absorption. The absorption rate kabs was derived from canine values ​​using allometric scaling of body weight with an exponent of -0.25. This exponent has previously been observed in other parameters associated with the same physical units as kabs, 1 / time, see Dawson TH. Allometric relations and scaling laws for the cardiovascular system of mammals. Systems. 2014 Jun;2(2):168-85. The data in the current analysis do not provide bioavailability values ​​for humans. Therefore, reasonable assumptions must be made, and 50% human bioavailability is considered a sufficiently conservative estimate.

[0127] As described below, the blood exposure of 2.5 mg / kg of VAD044 free base in neonatal mouse plasma, as determined by LC / MS / MS bioanalysis, corresponds to a Cavg of 22.9 ng / ml (or 55.1 nM) over a 48-hour dosing interval. Based on PK models, this exposure is expected to be achieved in humans with a single fixed-dose QD of 20 to 40 mg (VAD044 free base). This dose was calculated for an adult weighing 70 kg, and the expected bioavailability of VAD044 is 50% to 75%.

[0128] Compounds evaluated Three compounds with different AKT kinase inhibitory properties were tested: (i) Perifosine (comparative compound): an alkylphospholipid AKT inhibitor. Unlike ATP-binding kinase inhibitors, perifosine targets the plek substrate protein homology (PH) domain of AKT, thereby preventing its translocation to the plasma membrane (Gradziel et al., (2014), "Cytotoxic amphiphiles and phosphoinositides bindto two discrete sites on the Akt1 PH domain", Biochemistry, https: / / doi.org / 10.1021 / bi401720v; Kondapaka et al., (2003), "Perifosine, a novel alkylphospholipid, inhibits protein kinase B activation", Molecular CancerTherapeutics; Ríos-Marco et al., (2017), "Alkylphospholipids: An update onmolecular mechanisms and clinical relevance", In Biochimica et BiophysicaActa - Biomembranes, https: / / doi.org / 10.1016 / j.bbamem.2017.02.016 (ii) Uprosertib (comparative compound): an ATP-competitive inhibitor of AKT1, AKT2, and AKT3. It may also inhibit members of the PKC family, including PRKACA, PRKACB, and cGMP-dependent protein kinases PRKG1 and ROCK kinase (Dumble et al., (2014), "Discovery of novel AKT inhibitors with enhanced anti-tumoreffects in combination with the MEK inhibitor", PLoS ONE. https: / / doi.org / 10.1371 / journal.pone.0100880) (iii) VAD044 (the compound of this invention): a novel AKT1 / 2 allosteric inhibitor with excellent selectivity for other kinases. As described above, VAD044 is the L-tartrate salt of the following compound (referred to as "VAD044 free base"): IUPAC Name: 6-(4-(1-amino-3-hydroxycyclobutyl)phenyl)-1-ethyl-7-phenyl-1H-pyrido[2,3-b][1,4] Azine-2(3H)-one Perifosin Overview toxicity No adverse reactions were observed in neonatal mice receiving a single injection of perifoxine at P4 at doses ranging from 5 mg / kg to 25 mg / kg. Administration of 50 mg / kg perifoxine delayed weight gain, while administration of 100 mg / kg perifoxine induced significant mortality in pups (see [link to relevant documentation]). Figure 3A and Figure 3B Therefore, when compared with drugs such as thalidomide, the safety profile of perifoxine is satisfactory. Thalidomide has been previously tested in newborn mice and has been reported to reduce the severity and frequency of nosebleeds in patients with HHT (Lebrin et al., ibid.).

[0129] Anti-angiogenic properties in C57Bl / 6J mice A single injection of a low dose of perifoxine (dose range 5 mg / kg to 10 mg / kg) at P4 did not affect angiogenesis (see [link]). Figures 4A-4DOnly high doses of perifoxine, ranging from 25 mg / kg to 100 mg / kg, resulted in slight changes in the retinal vascular bed, with a slight decrease in the number of branch points and vascular area (see [link to original text]). Figures 4A-4D Perifoxine at 100 mg / kg also resulted in reduced radial vasodilation and exhibited mild endothelial cell anti-migration properties (see [link]). Figures 4A-4B In summary, perifoxine exhibited weak anti-angiogenic properties.

[0130] Ability to prevent AVM formation in Eng-iKO mice For P2 Eng-iKO Mice were injected with 50 μg tamoxifen. Then at P3 and P5 (25 mg or 50 mg per kg body weight, ip) Figure 5A Or administer periporphine intraperitoneally to mice daily (10 mg / kg body weight, ip) from P3 to P6. Retinal analysis was performed at P7. Figure 5A Litter mice injected with the vector alone or with tamoxifen and the vector alone were used as controls. As expected, in P2... Eng Conditional absence leads to the formation of retinal AVMs ( Figure 5B ) and moderately reduced angiogenesis ( Figure 5D ). The tested dosage and injection regimen ( Figures 5A-5F Under these conditions, perifoxine cannot prevent [the spread of disease]. Eng-iKO Formation of AVM in mice ( Figure 5C ), and cannot inhibit angiogenesis ( Figures 5D-5E In addition, daily intraperitoneal administration of 25 mg / kg of perifoxine resulted in the death of young mice (data not shown).

[0131] Uprosertib Overview toxicity Following a single injection of Uprosertib (at concentrations ranging from 10 mg to 50 mg per kg body weight) at P4, the survival rate and body weight of wild-type mice observed at P4 through P7 demonstrate the high toxicity of Uprosertib. (See [link to relevant documentation]). Figures 6A-6B . Found to be Eng-iKO Administering doses of 1 to 5 mg per kg of body weight to mice will cause the death of most young mice.

[0132] Anti-angiogenic properties in C57Bl / 6J mice Even with a single administration of low doses (5 mg / kg to 25 mg / kg, ip) at P4, Uprosertib inhibited angiogenesis, accompanied by a slight decrease in radial dilation. Figure 7B ); and vascular area ( Figure 7C) and number of branch points ( Figure 7D The significant reduction in endothelial cells indicates their anti-proliferative effect. These results suggest that Uprosertib is an effective inhibitor of angiogenesis in the mouse retina.

[0133] Ability to prevent AVM formation in Eng-iKO mice Eng-iKO mice were injected twice with Uprosertib at P3 and P5. Figure 8A However, it was found that two injections of Uprosertib led to the death of most young mice. Figure 8B This hindered further investigation into the ability of Uprosertib to prevent AVM formation in mice at the test dose.

[0134] VAD044 Overview In previous preclinical studies of tumor xenograft models, compound VAD044 has demonstrated good safety, therefore no additional safety / toxicity studies were conducted.

[0135] Ability to prevent AVM formation in Eng-iKO mice The tested concentration was comparable to the concentration of Uprosertib used. Figure 9A At these concentrations, when VAD044 is used on newborns at concentrations ranging from 1.25 mg to 10 mg per kg of body weight... Eng-iKO No adverse reactions were observed in mice after two injections (at P3 and P5). Figure 9B At concentrations of 2.5 mg·kg⁻¹ and above, VAD044 effectively inhibited… Eng-iKO Formation of AV shunt in mice ( Figures 10A-10B A dose of 1.25 mg / kg was found to be ineffective in preventing the formation of AV shunts. Figures 10A-10B VAD044 also normalized the density of the retinal vascular network in Eng-iKO mice. Figure 10D At the same time, it has little effect on angiogenesis, slightly reducing... Eng-iKO radial dilatation of vascular network ( Figure 10C ) and the number of branch points ( Figure 10E The blood exposure of VAD044 free base at a dose of 2.5 mg·kg⁻¹ in neonatal mouse plasma, as determined by LC / MS / MS bioanalysis, corresponds to 22.9 ng / ml (or 55.1 nM) Cavg over a 48-hour dosing interval.

[0136] The loss of endothelial glycoprotein in primary mouse endothelial cells preferentially induces AKT activation. Primary endothelial cells were isolated from the lungs of Engflox / flox pups using collagenase I-based enzymatic digestion, followed by cell sorting with PECAM1-coated microbeads. Figure 11A CD45-coated microbeads are used to deplete CD45+ / PECAM1+ immune cell populations ( Figure 11A The isolated CD45- / PECAM1+ cell population resembled an endothelial cell population and could be cultured for at least three generations. Platelet endothelial cell adhesion molecule 1 (PECAM1) and vascular endothelial cadherin (VE-cadherin) staining confirmed the endothelial cell identity. Figure 11B ).

[0137] Primary endothelial cell cultures were infected with a recombinant adenovirus encoding Cre recombinase using the Cre-Lox system to excise the Eng gene. Adenovirus infection was highly efficient and had no effect on cell viability. Almost all cells underwent gene recombination within the first 10 hours. Since endothelial glycoprotein is a relatively stable protein with an estimated half-life of approximately 17 hours, homogeneous endothelial cell cultures were obtained within the first 60 hours post-infection, as endothelial glycoprotein levels decreased from normal and by half to complete loss, as shown by staining and Western blot analysis. Figures 11C-11D ).

[0138] We then investigated the activation of Akt and Smad1 signaling pathways in lung endothelial cells lacking Eng protein expression. Cells were treated with VEGF, TGF-β1, and BMP9 for 30 minutes at different time points following infection, corresponding to different endothelial glycoprotein expression levels. As previously published in Jakobsson's lab (Jin et al., 2017–ibid.), we were able to demonstrate that decreased endothelial glycoprotein expression was associated with increased Akt phosphorylation. Figure 12 Most interestingly, AKT phosphorylation was more sensitive to reductions in Eng protein, as a 50% reduction in Eng protein induced an increase in AKT phosphorylation compared to endothelial cells expressing normal levels of endothelial glycoproteins. Figure 12 ), while Smad1 phosphorylation remained unchanged ( Figure 12 This result indicates that halved endothelial glycoprotein expression induced increased VEGF response and increased AKT signaling (increasing proliferation and migration, leading to vascular malformations), while classic BMP / SMAD signaling remained normal.

[0139] Measurement of VAD044 IC50 in control and Eng-iKO mouse endothelial cells The effects of VAD044 on primary lung endothelial cells isolated from Eng-iKO mice were then tested. As expected, Cre adenovirus infection resulted in complete loss of endothelial glycoprotein expression at 60 hours post-infection. Figure 13A ECs from the control group with normal Eng expression levels were highly sensitive to VAD044, with an IC50 of 55 nM, while the IC50 of VAD044 in lung mouse endothelial cells exhibiting complete loss of Eng expression was 93 nM. Figure 13B When the Eng protein is completely knocked out, the increase in AKT activation (approximately 2 to 4-fold) can explain this approximately 2-fold IC50 shift. Figure 12 Meanwhile, analysis of p42 / p44 phosphorylation as a selective control confirmed that VAD044 had no effect on the MAPK pathway. Figure 13C ).

[0140] Results / Discussion Uprosertib is a competitive AKT inhibitor that binds to the ATP kinase-binding pockets of AKT1, AKT2, and AKT3 isoforms. This compound is a typical low-molecular-weight scaffold with potent pan-ATP kinase pocket inhibition (Dumble et al., 2014). However, there is high homology between the ATP-binding pockets of AKT and protein kinase A (PKA), protein kinase C (PKC), or protein kinase G (PKG). Therefore, Uprosertib exhibits strong inhibition of PKA, PKG, PKC, and ROCK kinases. This “off-target” kinase activity is often associated with increased side effects (Rodon et al., (2013), "Development of PI3K inhibitors: Lessons learned from early clinicaltrials", Nature Reviews Clinical Oncology, https: / / doi.org / 10.1038 / nrclinonc.2013.10).

[0141] In contrast, allosteric inhibitors appear to have higher specificity for AKT. For example, VAD044 is a novel AKT allosteric inhibitor that offers the highest selectivity among such inhibitors, exhibiting potent activity against both AKT1 and AKT2. US9221838B2 Perifoxine is an unusual allosteric inhibitor that acts on the plek substrate protein homologous domain of AKT, thereby preventing its transport to the plasma membrane required for activation.

[0142] VAD044 exhibits comparable biochemical and cellular potency to Uprosertib in inhibiting AKT kinase. For example, based on similar biochemical kinase activity analyses, Uprosertib and VAD044 show similar IC50 values ​​for AKT1. 50The values ​​were 180 nM and 125 nM, respectively (see Pachl, et al (2013). “Characterization of a chemical affinity probe targeting Akt kinases”, Journal of Proteome Research, https: / / doi.org / 10.1021 / pr400455j; and Example 3 of VAD044 above). Similarly, both molecules showed comparable AKT kinase inhibition IC50 values ​​in Pi3Kα or PTEN mutant cell lines (e.g., MCF7, BT474, or LnCAP). 50 The range of activity for Uprosertib was 34 nM to 143 nM, and for VAD044 it was 50 nM to 130 nM. In contrast, perifoxine was less potent than VAD044 and Uprosertib, with inhibitory activity against similar cell lines of AKT in the μM range (Gradziel et al., 2014; Kondapaka et al., 2003; Ríos-Marco et al., 2017 – ibid.).

[0143] The above results indicate that it is impossible to determine a therapeutic dose window for both perifoxine and uprosertib that simultaneously possess acceptable safety and the desired inhibitory potency. For perifoxine, even at high doses expected to completely inhibit AKT kinase, its effects on normal angiogenesis and AVM shunts are only modest. At the highest tolerable doses (25 mg / kg and 50 mg / kg), no significant effect on shunt formation was observed in the HHT1 mouse model. A trend toward efficacy was observed when 10 mg / kg was administered daily, but this did not reach statistical significance. In contrast, uprosertib demonstrated potent anti-angiogenic properties after a single injection at P3. (5 mg / kg) -1 The dosage was well tolerated and was selected for efficacy studies. However, when administered twice at P3 and P6, 5 mg / kg... -1The dosage resulted in high levels of mortality in young mice, thus ruling out any further evaluation of its efficacy in preventing AVM shunt formation. This is likely due to the off-target inhibition of PKA, PKG, and ROCK kinases by Uprosertib, which may be involved in the development of young mice from P3 to P7 (Shi et al., (2011), "Rho-kinase in development and heart failure: Insights from genetic models", Pediatric Cardiology. https: / / doi.org / 10.1007 / s00246-011-9920-0).

[0144] At approximately 5 mg / kg -1 At the recommended dose, two injections of VAD044 were well tolerated, and no fatalities were observed. At 2.5 mg / kg... -1 At this dose, VAD044 completely prevented the formation of AVM shunts. Surprisingly, this dose had almost no effect on normal angiogenesis while completely and effectively preventing shunt formation.

[0145] These results indicate that VAD044 may be able to treat vascular defects observed in HHT by inhibiting the AKT pathway. This also suggests that inhibiting a portion of the pathway may be sufficient to treat the vascular defects observed in HHT. First, the results confirm that Eng deficiency in endothelial cells preferentially overactivates AKT signaling (2 to 4-fold), while SMAD signaling remains normal. The minimum effective dose of 2.5 mg·kg⁻¹ determined in the above results corresponds to a blood exposure of 22.9 ng / ml (or 55.1 nM) of the free base Cavg of VAD044. This concentration is low, equivalent to the IC50 of VAD044 inhibiting AKT in normal endothelial cells and the IC30–IC40 of VAD044 inhibiting AKT in Eng protein-deficient endothelial cells. Surprisingly, the minimum effective dose of VAD044 in the HHT model is significantly lower than in tumor indications. Although the IC50 of VAD044 in vitro is similar (approximately 50 nM) in MCF7 tumor cells, the minimum effective dose in vivo in a tumor xenograft model is 30 mg / kg. -1 This is approximately 10 times higher than in the HHT1 mouse model described above. Based on different PK models, this effective concentration (equivalent to 22.9 mg / mL Cavg) is expected to be achievable in humans with a single fixed-dose QD of 20 mg to 40 mg (VAD044 free base). This dose was calculated for an adult weighing 70 kg, and the expected bioavailability of VAD044 is 50% to 75%.

[0146] In summary, these results indicate that blood vessels and endothelial cells carrying the HHT gene deletion are highly sensitive to AKT inhibition. Therefore, when this pathway is overactivated, low doses of VAD044 can effectively control vascular malformations, possibly due to the inhibition of BMP9 signaling observed in HHT, or possibly due to the acquisition of functional mutations, as observed in overgrowth syndromes or venous malformations caused by somatic mutations in PI3Kα or TIE2 (Castillo et al., (2016), "Phosphoinositide 3-kinase: a new kid on the block in vascularanomalies", Journal of Pathology (Vol. 240, Issue 4, pp. 387–396). John Wiley and Sons Ltd. https: / / doi.org / 10.1002 / path.4802).

Claims

1. A compound of formula (I): Use of its pharmaceutically acceptable salt or in the preparation of a medicament for treating hereditary hemorrhagic telangiectasia (HHT) in a subject, wherein: Each X is O; R 1 Selected from hydrogen and C1-C 10 alkyl; R 2 and R 3 Each is hydrogen; R 6 It is a benzene ring; and R 8 It is -OH.

2. The use according to claim 1, wherein R 1 It is a C1-C6 alkyl group.

3. The use according to claim 2, wherein R 1 It is methyl or ethyl.

4. The use according to claim 1, wherein the compound of formula (I) is: Or its pharmaceutically acceptable salt.

5. The use according to any one of claims 1-4, wherein the pharmaceutically acceptable salt is a tartrate, a methanesulfonate, or a phosphate.

6. The use according to claim 5, wherein the pharmaceutically acceptable salt is a tartrate.

7. The use according to claim 6, wherein the tartrate is an L-tartrate.

8. The use according to claim 5, wherein the compound is formulated for oral administration to the subject.

9. The use according to claim 5, wherein the subject is a human.

10. The use according to claim 9, wherein the subject is an adult.

11. The use according to claim 10, wherein the compound of formula (I) is administered to the subject at a dose of 20 mg to 75 mg QD.

12. The use according to claim 10, wherein the compound of formula (I) is administered to the subject at a dose of 10 mg to 50 mg QD.

13. The use according to claim 12, wherein the compound of formula (I) is administered to the subject at a dose of 20 mg to 40 mg QD.

14. The use according to claim 13, wherein the compound of formula (I) is administered to the subject at a dose of 20 mg to 30 mg QD.

15. The use according to claim 5, wherein the treatment of HHT includes reducing the frequency, duration, or intensity of HHT-related bleeding.

16. The use according to claim 15, wherein the HHT-related bleeding is gastrointestinal (GI) bleeding.

17. The use according to claim 15, wherein the HHT-related bleeding is nasal bleeding.

18. The use according to claim 5, wherein the treatment of HHT includes increasing the hemoglobin level of the subject.

19. The use according to claim 5, wherein the treatment of HHT includes reducing the number of capillary dilatations in the subject.

20. The use according to claim 19, wherein the capillary dilation is dermal capillary dilation.

21. The use according to claim 19, wherein the capillary dilation is nasal capillary dilation.

22. The use according to claim 19, wherein the capillary dilation is oral capillary dilation.

23. The use according to claim 19, wherein the capillary dilation is gastrointestinal capillary dilation.

24. The use according to claim 5, wherein the treatment of HHT comprises reducing the size of capillary dilation in the subject.

25. The use according to claim 24, wherein the capillary dilation is dermal capillary dilation.

26. The use according to claim 24, wherein the capillary dilation is nasal capillary dilation.

27. The use according to claim 24, wherein the capillary dilation is oral capillary dilation.

28. The use according to claim 24, wherein the capillary dilation is gastrointestinal capillary dilation.

29. The use according to claim 5, wherein the treatment of HHT includes reducing the number of arteriovenous malformations (AVMs) in the subject.

30. The use according to claim 29, wherein the arteriovenous malformation is a cerebral AVM or a visceral AVM.

31. The use according to claim 5, wherein the treatment of HHT includes reducing the size of the arteriovenous malformation (AVM) in the subject.

32. The use according to claim 31, wherein the arteriovenous malformation is a cerebral AVM or a visceral AVM.

33. The use according to claim 5, wherein the treatment of HHT includes prevention of the formation of arteriovenous malformation (AVM) in the subject.

34. The use according to claim 33, wherein the arteriovenous malformation is a cerebral AVM or a visceral AVM.

35. The use according to claim 30, 32 or 34, wherein the visceral AVM is a pulmonary AVM.

36. The use according to claim 5, wherein the treatment of said HHT includes reducing heart failure and pulmonary hypertension (PAH).

37. The use according to claim 5, wherein the treatment of said HHT includes prevention of heart failure and pulmonary hypertension (PAH).

38. The use according to claim 5, wherein the treatment of HHT includes prevention of right-to-left shunting induced by pulmonary AVM in the subject.

39. The use according to claim 5, wherein the treatment of HHT includes reducing the degree of right-to-left shunting induced by pulmonary AVM in the subject.

40. The use according to claim 5, wherein the treatment of HHT includes reducing the subject's need for iron supplementation.

41. The use according to claim 5, wherein the treatment of HHT includes reducing the number of blood transfusions required by the subject.

42. The use according to claim 5, wherein the treatment of HHT includes reducing hepatic blood flow.

43. The use according to claim 5, wherein the HHT treatment reduces the subject's need for a liver transplant.

44. The use according to claim 5, wherein the treatment of HHT includes reducing the frequency and / or severity of additional symptoms of HHT.

45. The use according to claim 44, wherein the additional symptoms are dyspnea, fatigue, neurological events, and / or embolic events.

46. ​​The use according to claim 45, wherein the neural event is a migraine.

Images