" 0,200 0,000 Ct Ploo 1100 Patent Application Publication Oct

111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111 US 20200323798Al (19) U nited States (12) Patent Application Publication (lO) Pub. No.: US 2020/0323798 Al BOTELLA CUBELLS et al. (43) Pub. Date: Oct. 15, 2020

(54) COMPOUNDS FOR TREATING VON (30) Foreign Application Priority Data HIPPEL-LINDAU DISEASE

Aug. 7, 2017 (ES) 000000000000000000000000000000000 P201731019 (71) Applicants:CONSEJO SUPERIOR DE INVESTIGACIONES CIENTÍFICAS, Publication Classification Madrid (ES); ALIANZA ESPAÑOLA DE FAMILIAS DE VON (51) Int. Cl. HIPPEL-LINDAU-VHL, Olías del A61K 311138 (2006.01) Rey (ES) A61P 35104 (2006.01) (52) U.S. Cl.

(72) Inventors: Luisa María BOTELLA CUBELLS, CPC 000000000000 A61K 311138 (2013.01); A61P 35104 Madrid (ES); Virginia ALBIÑANA (2018.01) DÍAZ, Madrid (ES); Karina VILLAR GOMEZ-DE LAS HERAS, Olías del (57) ABSTRACT Rey (ES) The invention relates to the use of a selective B2 adrenergic (21) Appl. No.: 16/636,959 receptor antagonist for treating and preventing a tumor in a patient with von Hippel-Lindau syndrome. In particular, the (22) PCT Filed: Aug. 6, 2018 invention relates to the use of an alkanolamine derivative or a pharmaceutically acceptable acid-addition salt thereof for (86) PCTNo.: PCT/EP2018/071220 treating and preventing a hemangioblastoma in VHL syn § 371 (c)(l), drome patients. (2) Date: Feb. 6, 2020 Specification includes a Sequence Listing.

HB11cib 1,200 > +-' 1,000 ..e ro 0,800 ·> QJ > 0,600 +-' -ro 0,400 QJ " 0,200 0,000 Ct PlOO 1100 Patent Application Publication Oct. 15, 2020 Sheet 1 of 14 US 2020/0323798 Al

HB11cib 1,200 > +J 1,000 ..0 ro 0,800 > Q) > 0,600 +J ro 0,400 Q) 0:::: 0,200

0,000 Ct P100 1100 FIG.1

FIG.2 Patent Application Publication Oct. 15, 2020 Sheet 2 of 14 US 2020/0323798 Al

.... u

N o

SlaAal UO!SSaJdxa "NHW JO saSue4J PIO:I

SlaAal UO!SSaJdxa "NHW <( JO saSu e4J PIO:I Patent Application Publication Oct. 15, 2020 Sheet 3 of 14 US 2020/0323798 Al

o o .-1 en c. "'ta u .... u

SlaAal UO!SSaJdxa "NH W .e ~o saSuelp PIO:I ¡..: -z o -u

.... u

SlaAal UO!SSaJdxa "NHW ~o saSuelp PIO:I Patent Application Publication Oct. 15, 2020 Sheet 4 of 14 US 2020/0323798 Al

FIG. 4 Patent Application Publication Oct. 15, 2020 Sheet 5 of 14 US 2020/0323798 Al

e Control PlOO J!M 1100 J!M

FIG. 4 (CONT.) Patent Application Publication Oct. 15, 2020 Sheet 6 of 14 US 2020/0323798 Al

A CONTROL PlOO ~M 1100 ~M

B 100

!: 90 ·,¡:¡o 80 tU lo. 70 + Control b.O ·e 60 --- 1100 50 ...... , PlOO Q) 40 u 30 -'$.. 20 - 10 o Oh Gh (time) FIG. 5 Patent Application Publication Oct. 15, 2020 Sheet 7 of 14 US 2020/0323798 Al

A Control

B 100

~ ~ o 80 ....,~ (1J 60 e: > ...., 40 ·~ e: e(1J 20 o Control 1100 11M PlOO 11M FIG. 6 Patent Application Publication Oct. 15, 2020 Sheet 8 of 14 US 2020/0323798 Al

U') .t:: e: 25 :S QJ > ·,¡:; 20 ca -QJ ' QJ U') 15 ca ' ...... QJ ·o 10 _.:S

Ct P50 P1 00 150 1100 DFO DFO DFO DFO DFO +P50 +P1 00 +150 +11 00

Normoxia Hypoxia

FIG. 7 Patent Application Publication Oct. 15, 2020 Sheet 9 of 14 US 2020/0323798 Al

HIF-Iuc promoter activation 8.0E+05 • ··· ···· · · ··· ." ······ ···.···...... ····"···· ...... ·· ...... ··· ...... 1 Normoxia mDFO

6.0E+05 ...... "......

5.0E+05 • ·· · ....". · ::J ...J 4.0E+05 1···· ···•· ···· ...... ······ · a:::

DMSO ICI-118,551 Propanolol Atenolol

Fig.8 Patent Application Publication Oct. 15, 2020 Sheet 10 of 14 US 2020/0323798 Al

---ICI Propranolol

A 12o~------

1oo a-~~------ -~ so+------~~------ ~ 60 +------"'ltr---"~--- :c >"' 40

20+------~~-

o+------~~------~ o 50 100 150 200 [uM)

B 120 100 -~ 80 ~ 60 Jl >"' 40 20 o o 50 100 150 200 [uM]

Fig. 9 Patent Application Publication Oct. 15, 2020 Sheet 11 of 14 US 2020/0323798 Al

e 120 ~------

100~------ ...... ~so +-~~~~------~------

-~ 60 +-----..,;:;~::------ :0 5 40+------~~~~~~--~

20 +------=~ ··

o+-----~----~~----~------~ O 50 100 150 200 [uM}

D 120 ~------

100~------ -~ so+-~~~------ ~ 60 +------~------ :c >"' 40

20·~------~~------

o+=====~====~======~==~~ O 50 100 150 200 [uM}

Fig. 9 (cont.} Patent Application Publication Oct. 15, 2020 Sheet 12 of 14 US 2020/0323798 Al

E 120 100 -"#. 80 ~ 60 -:S ra > 40 20 o o 50 100 150 200 [uM}

Fig. 9 (cont} Patent Application Publication Oct. 15, 2020 Sheet 13 of 14 US 2020/0323798 Al

1000 -Vehiele * 900 ----·ICI-118,551 800 * ;:¡"" 700 E - -Propranolol .§. 600 cu § 500 g ... 400 o E ~ 300 200

100

o 10 20 30 40 50 60 Days after injection

Fig.10 Patent Application Publication Oct. 15, 2020 Sheet 14 of 14 US 2020/0323798 Al

Cell viability -t-HMEC 120 _,._ HUVEC 100 ...... HB

- 80 ';/. -f" 60 .e 5 40 20

o 50 100 150 200 250 ICI [uM]

Fig.11 US 2020/0323798 Al Oct. 15, 2020 1

COMPOUNDS FOR TREATING VON tomas, these disappeared completely between 3 and 6 HIPPEL-LINDAU DISEASE months of treatment. Low blood pressure was recorded as a side effect (Albiñana et al., 2017) Therefore, the lack of FIELD OF THE INVENTION therapies for recurrent disease means there is an urgent requirement for effective drugs with reduced side effects for [0001] The invention relates to the field of therapeutics VHL patients, especially those that halt the progression of and prevention, more specifically to the treatment and pre tumors and delay surgical treatment. vention ofvon Hippel-Lindau disease. BRIEF DESCRIPTION OF THE INVENTION BACKGROUND OF THE INVENTION [0002] Von Hippel-Lindau (VHL) disease is arare type of [0009] The inventors of the present invention have sur prisingly found that ICI 118,551 decreases viability of cancer with an incidence of 1 per 36,000 individuals in the general population. VHL is an autosomal dominantly inher hemangioblastoma cells from VHL patients (FIGS. 1 and 2) and inhibits hemangiosphere formation from VHL patients ited genetic disorder. The disease was first described sepa (FIG. Additionally, the inventors have shown that ICI rately by von Hippel in 1911 and by Lindau in 1926. 3). 118,551 inhibits cell migration and angiogenesis ofendothe [0003] The clinical manifestations include multiple benign lial cells (FIGS. 3 and 4) and that inhibits HIF activity and malignant tumors that appear throughout the lifespan of induced by hypoxia. the patient: retina! hemangioblastoma, CNS hemangioblas [0010] Thus, the invention relates toa selective antagonist toma, clear cell renal cell carcinoma (CCRCC), pheochro of the ~ -adrenergic receptor for use in the treatment andlor mocytoma, pancreatic islet tumor, endolymphatic sac 2 tumors and cysts in testes and broad ligament. prevention of von Hippel-Lindau disease. [0004] Tumor cells from VHL have lost the von Hippel DETAILED DESCRIPTION OF THE FIGURES Lindau protein function, which leads to a constitutive expression of hypoxia inducible factor (HIF), even in nor [0011] FIG. l. ICI 118,551 and Propranolol decrease moxic conditions. viability of hemangioblastoma cells from VHL patients. [0005] HIF is responsible for activation of genes involved Propranolol, (P) is a non-selective beta blocker binding beta in angiogenesis, metabolism and apoptosis that promote 1 and 2 adrenergic receptors. ICI 118,551 (I), is a selective adaptation and survival under low 0 2 conditions (hypoxia), beta blocker binding only to receptor beta 2. Both beta such as might be found in ischemic tissues and in most solid blockers act by decreasing viability of hemangioblastoma tumors. primary cell cultures up to 40%-50% at 100 ¡.tM for 72 hours. [0006] In normoxia, the rapid degradation of HIF is con FIG. 1 shows the decrease in viability in primary cell culture trolled by p VHL binding to hydroxylated residues of HIF-a. HB11cib. Results are representative of 8 hemangioblastoma Under hypoxic conditions, PHD cannot hydroxylate HIPa primary cell cultures treated in the same way. Each measure subunits that then escape ubiquitin mediated proteolysis, comes from triplicates, and quantified by a luminescent kit allowing HIF ato accumulate, translocate to the nucleus, and detecting ATP (Cell titer Glo, from Promega). bind to HIF~. This heterodimer is able to bind specific DNA [0012] FIG. 2. ICI 118,551 acts as propranolol decreasing sequences ("hypoxia responsive elements" or HRE) to actí viability in hemangioblastoma cells from VHL. A. Heman vate transcription of more than 200 target genes. Main genes gioblastoma cell primary culture (patient sample HB18), regulated by pVHL are involved in tumor development, without (Control) and after 4 days of treatment with ICI angiogenesis (vascular endothelial growth factor [VEGF] 118,551 at 50 y 100 ¡.tM. After treatment with ICI 118,551 and platelet derivative growth factor [PDGF]), cell prolif 100 ¡.tM we can see empty spaces in the plate, compared with eration or survival (transforming growth factor [TGFa]), control, which is confluent after 4 days of culture, starting regulation of glucose uptake and metabolism (glucose trans with the same number of cells. B. Comparison of 4-day porter Glut-1) and erythropoiesis (EPO). Other target genes treatment with Propranolol (P) and ICI 118,551 (I) at the relevant to tumor biology are involved in extracellular same concentration 100 ¡.tM of hemangioblastoma cell pri matrix formation (matrix metalloproteinase 1 [MMP1 ]), mary culture (patient sample HB4). chemotaxis (stromal cell derived factor 1 [SDF1] and its [0013] FIG. 3. ICI 118,551 treatment induces cell apop receptor CXC chemokine receptor 4 [CXCR4], epidennal tosis by stimulating proapoptotic genes Bax and Caspase 9 growth factor receptor [EGFR], Hepatocyte growth factor A. Relative mRNA expression levels of BAX and CASP9 receptor [HGFR encoded by MET]), pH regulation (car (genes involved in apoptosis) detected by RT-qPCR in HB26 bonic anhydrases IX and XII [CA9 and CA12]), and cell hemangioblastoma cell culture, after 72 h of 100 ¡.tM ICI cycle (cyclin Dl [CCNDl]). 118,551 (1100). B. Relative mRNAexpression levels ofBax [0007] Thus far, therapeutic options for VHL patients are and Casp9 (genes involved in apoptosis) detected by RT derived from surgery. The systemic therapy used for meta qPCR in HB11 hemangioblastoma cell culture, after 72 h of static cancers has shown limited response in VHL pancreatic 100 ¡.tM ICI 118,551. Both genes, Bax and Casp 9 are and renal tumors, while CNS tumors do not respond at all. increased afteriCI 118,551 treatment, therefore, ICI 118,551 [0008] VHL Spanish Alliance conducted a clinical tria! is inducing apoptosis in these cells. with 7 VHL patients for 12 months, from December 2014 to [0014] FIG. 4. Propanolo! and ICI 118,551 are antitumoral April 2016. Patients were treated for retina hemangioblas agents, inhibiting hemangiosphere formation from VHL tomas with 120 mg of propranolol a day (a suboptimal do se, patient cells. A. Round sphere-like structures with a well for many patients under 2 mg/Kg body weight/day). As a defined border appear as aggregates from of VHL cell result, tumors did not grow in any case during the clinical tumors. Two different hemangioblastomas were used for tria! and in sorne cases visual acuity increased. More inter these experiments HB2 and HB3. However, when the cells estingly, in 2 cases with exudates from the hemangioblas are cultured for 7 days in the presence of 100 ¡.tM of either, US 2020/0323798 Al Oct. 15, 2020 2

Propranolol (P) or ICI 118,551 (I), the spheres appear antagonist) and propranolol (a non-specific beta-adrenergic disaggregated and the observed groups are irregular with receptors type 1 and 2 antagonist) decrease, in a dose few cells. Photos were taken the last day of treatment B. The dependent manner and with higher specificity, the viability same as in panel A but with other two hemangioblastomas of the VHL-/- cells. ATP content was measured using the from VHL patients. C. The same as in panels A and B but CellTiter-Glo® Assay from Promega. A: HUVEC cellline, with HB23 from a VHL patient. B: HMEC cell line, C: Hemangioblastoma primary culture [0015] FIG. 5. ICI 118,551 and propranolol inhibit cell 18, D: Hemangioblastoma primary culture 14, E: ccRCC migration of endothelial ce lis. A. Functional in vitro tests for Vhl-/- 786-0 cellline. Data shown represen! the mean+/ angiogenesis include "wound-healing" or plate-scratch, to SEM (n=3). follow the migration capacity of endothelial cells after short [0020] FIG. 10. Xenografts of 786-0 Vhl-/- human times. When a confiuent monolayer is disrupted with the tip ccRCC cells in mice. Male 7-8 weeks old NOD scid gamma of a micropipette to create a discontinuity, endothelial cells (NSG) mice were injected in the dorsal fiank with a single normally migrate to cover the discontinuity "wound the cell suspension of 106 786-0 cells. When tumor size reached healing" in a short time, depending on the size of the 100 mm3 volume, mi ce were randomly divided in 3 groups, "wound" but normally, less than 8 hours. This test measures of 9/10 mice each. Two groups were daily treated intraperi the migration by taking pictures at different times from the toneally with 10 mg/Kg body propranolol or ICI-118,551, moment the wound is created. Only the final picture after 6 respectively and a third group was injected with the solvent. hours is shown. The migrated distance is quantified, and in Tmnor size was measured by a caliper every 2-3 days and the following graph the evolution is shown. ICI 118,551 and volmnes were calculated following the formula: shortesex propranolol delay cell migration, hence they are antiangio largestx0.52. Mice were sacrificed when tmnor volmne genic. B. Migrated distance by untreated and propranolol/ average of the control group reached an end point estab ICI 118,551 treated-cells after 6 hours of making a scratch lished based on ethical procedures. Arrow marks the begin on the confiuent !ayer of HUVECs (Human Umbilical Vein ning of the treatment. Endothelial Cells ). [0021] FIG. 11. ICI 118.551 affects differentially Vhl-/ [0016] FIG. 6. ICI 118,551 and propranolol inhibit cell cells and normal endothelial cells HUVECs and HMECs. angiogenesis of endothelial cells. Tubulogenesis is another Primary culture of Haemangioblastoma from VHL, normal in vitro test for angiogenesis. Endothelial cells cultured on primary endothelial HUVECs, and the non tumoral microen matrigel have the property of building cell tube-like struc dothelial cell line HMEC-1 were cultured in the absence or tures, which mimic the "vessels" structure. A. Propranolol presence of different concentrations ofiCI 118.551 (50-250 and ICI 118,551 at 100 ¡.tM significantly inhibit the tubulo ¡.tM) to measure viability. According to viability curves genesis. B. The number of closed cells (density network) is Haemangioblastoma (HB) cells show only 20% ofviability quantified and averaged from 5 different fields in each case. at 100 ¡.tM, while HUVEC or HMEC-1 non tumoral cells Bar histograms represent the mean±SD. show 80% and 55% respectively decrease in viability at 100 [0017] FIG. 7. Propranolol and ICI 118,551 inhibit HIF ¡.tM in both cultures, mediated stimulation. The bar histograms show the relative luciferase activity depending on HIF -1 (hypoxia) in stable DETAILED DESCRIPTION OF THE transfectants of Hela cells for the reporter HRE9x-luc, that INVENTION contains 9 tandem repetitions of the hypoxia responsive [0022] The invention relates to a selective antagonist of sequence element bound by HIF -1 fused with luciferase the ~ 2 -adrenergic receptor for use in the treatment andlor reporter protein. Ct stands for control cells (normoxia and prevention of von Hippel-Lindau disease. hypoxia). Deferroxamin (DFO) at 100 ¡.tMolar produces [0023] Altematively, the invention relates to the use of a chemical hypoxia in treated cells. Treatment with Propa selective antagonist of the ~ 2 -adrenergic receptor in the nolol or with ICI 118,551 at 50 or 100 ¡.tMolar (PSO, P100, manufacture of a medicament for the treatment and/or ISO, IlOO, respectively, for 24 h), inhibits the hypoxia prevention of von Hippel-Lindau disease. stimulation induced by DFO. Remarkably, the effect of ICI [0024] Altematively, the invention relates to a method of 118,551 is clearly superior to propranolol at equal dose. treatment and/or prevention of von Hippel-Lindau disease Both DFO and drug treatment were simultaneously admin comprising administering to said patient a therapeutically istrated. effective amount of a selective antagonist of the ~ 2 -adren [0018] FIG. 8. Propranolol and ICI 118.551 downregulate ergic receptor. significantly HIF -dependen! transcription in HeLa cells but [0025] The term "~ 2 -adrenergic receptor" or "~2AR", as not Atenolol. Luciferase assays were performed in HRE used herein, refers to a class A of G protein-coupled recep luc-stably transfected HeLa cells under hypoxic conditions tors (GPCR) that responds to diffusible hormones and neu and treatment with Propranolol, atenolol and ICI118.551. rotransmitters and resides predominantly in smooth muscles. Propranolol and ICI (100 ¡.tM) prevented hypoxia stimula There are two main groups of adrenergic receptors, a and 13, tion in HeLa cells, as shown by the decrease in luciferase with severa! subtypes: activity, by inhibiting the activation of hypoxia elements [0026] a receptors have the subtypes a 1 (a Gq coupled (HRE) by HIF. Atenolol results were less significan! than the receptor) and a 2 (a G; coupled receptor). other ones (Propranolol and ICI). [0027] ~ receptors have the subtypes ~ 1 , ~ 2 and ~ 3 . All [0019] FIG. 9. ICI-118551 Shows antitumoral specific three are linked to Gs proteins, which in turnare linked activity in VHL-/- cells. Viability (ATP content) of severa! to adenylate cyclase. Agonist binding to these receptors cells (primary cultured and lines) exposed to different mM causes a rise in the intracellular concentration of the concentrations ofiCI-118551 or Propranolol, for 72 hours. second messenger cAMP. Control wells did not contain ICI-118551 or Propranolol. [0028] Agonist binding to the ~ 2 -adrenergic receptor ICI-118551 (a specific beta-adrenergic receptors type 2 results in smooth muscle relaxation. US 2020/0323798 Al Oct. 15, 2020 3

[0029] The term "~ 2 -adrenergic receptor antagonist", as [0036] The term "alkyl group", as used herein, refers to used herein, refers toa compound that binds a ~ 2 -adrenergic acyclic straight and branched groups derivable from alkanes, receptor and lacks any substantial ability to activate the and having the formula - CnH2n+ 1 by removal of a hydro receptor itself. The term "~ 2 -adrenergic receptor antagonist" gen atom. includes both neutral antagonists and inverse agonists. A [0037] The term "halogen", as used herein, refers to an "neutral antagonist" is a compound that blocks the action of atom selected from fluorine, chlorine, bromine and iodine. the agonist but has no effect on intrinsic or spontaneous [0038] R1 may be, for example, isopropyl or t-butyl. In a receptor activity. An "inverse agonist" is able to both block particular embodiment, R 1 is isopropyl. the action of the agonist at the receptor and attenuate the [0039] R2 may be, for example, methyl or ethyl. In a constitutive activity of the receptor. The term "antagonist" particular embodiment, R2 is methyl. also includes competitive antagonists, which are drugs that [0040] R3 may be, for example, hydrogen, chlorine, bro bind to the same site as the naturalligand; noncompetitive mine, methyl or ethyl. In a particular embodiment, R3 is antagonists which bind to a different site on the receptor than methyl. the natural ligand; reversible antagonists which bind and [0041] In a particular embodiment n is l. unbind the receptor at rates determined by receptor-ligand [0042] In a particular embodiment, R1 is isopropyl and R2 kinetics; and irreversible antagonists which bind perma is methyl. In a particular embodiment, R1 is isopropyl and nently to the receptor either by forming a covalent bond to R3 is methyl. In a particular embodiment, R1 is isopropyl the active site or just by binding so tightly that the rate of and n is l. In a particular embodiment, R2 is methyl and R3 dissociation is effectively zero. is methyl. In a particular embodiment, R2 is methyl and n is [0030] The term "selective ~ -adrenergic receptor antago 2 l. In a particular embodiment, R3 is methyl and n is l. nist", as used herein, means an antagonist which is selective [0043] In a particular embodiment, R1 is isopropyl and R2 for ~ -adrenergic receptors over ~ -adrenergic receptors. In 2 1 and R3 are methyl. In another particular embodiment, R1 is a particular embodiment, a selective ~ 2 -adrenergic receptor 2 antagonist exhibits at least 10-fold greater potency in bind isopropyl, R is methyl and n is l. In another particular embodiment, R1 is isopropyl, R3 is methyl and n is l. In a ing to ~ - than to ~ -adrenergic receptors, i.e. have a ~ /~ 2 1 2 1 2 3 l. selectivity ratio of at least 1O. More preferably, the selective particular embodiment, R and R are methyl and n is [0044] In a more particular embodiment, R 1 is isopropyl, ~ 2 receptor antagonist will have a ~i~ 1 selectivity ratio ofat R2 and/or R3 are methyl and n is l. least 50. The affinity of various active agents for ~ 2 - and [0045] In an even more particular embodiment, the ~ 1-adrenergic receptors can be determined by evaluating alkanolamine derivative has the formula JI: tissues containing a majority of ~ 2 receptors (e.g., rabbit ciliary process, rat liver, cat choroid plexus or lung), tissues containing a majority of ~ 1 receptors (e.g., cat and guinea pig heart), and tissues containing a mixture (e.g. guinea pig Formula 11 trachea). The methods of determining relative binding selec tivity for these different types of tissues are extensively disclosed in O'Donnell and Wanstall, Naunyn-Schmiede berg's Arch. Pharmaco., 308, 183-190 (1979), Nathanson, Science. 204, 843-844 (1979), Nathanson, Life Sciences, 26, 1793-1799 (1980), Minneman et al., Mol. Pharmacol., 15, 21-33 (1979a), and Minneman et al., Joumal ofPharmacol [0046] This compound of formula JI is al so known as ICI ogy and Experimental Therapeutics, 211, 502-508 (1979). 118,551 and its chemical name is erythro-D,L-1(methylin [0031] A significant number of compounds having selec den-4-yloxy)-3-isopropylaminobutan-2-ol. ICI 118,551 has tive ~ -adrenergic antagonist activity suitable for use in this 2 a ~ /~ selectivity ratio of at least 50, as determined and invention are known. In a particular embodiment, the selec 2 1 reported in Life Sciences, 27,671 (1980) and Bilsky et al., J. tive ~ 2 -adrenergic receptor antagonist is the alkanolamine Cardiovasc. Pharmacol., 5, 430-437 (1983). derivative of formula I [0047] It will be observed that the alkanolamine derivative of formula I possesses two asymmetric carbon atoms, namely those of the - CHOH- group and the ---CHR2 Formula! group, and that it can therefore exist in two racemic diaste reoisomeric forms , the threo and erythro forms, and four optically-active forms, those being the ( +) and (-) isomers of each of the racemic forms. It is to be understood that this invention encompasses any one of these isomeric forms which possess a selective ~ 2 -adrenergic receptor antagonis tic activity as defined above, it being a matter of common general knowledge how any particular isomer may be iso lated and how any selective ~ 2 -adrenergic receptor blocking [0032] wherein R1 is an alkyl group ofup to 6 carbon activity it may possess may be measured. atoms which is branched at the a-carbon atom, [0048] It is to be understood that in general an optical [0033] wherein R2 is an alkyl ofup to 3 carbon atoms, isomer which has the {S)-absolute configuration of the 3 [0034] wherein R is hydrogen, an halogen or an alkyl ---CHOH- group is more active as a P2 adrenergic blocking of up to three carbon atoms and agent than the corresponding isomer which has the {R) [0035] wherein n is 1 or 2, absolute configuration. It is also known that in general the or a pharmaceutically acceptable acid addition salt thereof. erythro-isomer is more P2 -selective than the corresponding US 2020/0323798 Al Oct. 15, 2020 4

threo-isomer, but that both threo- and erythro isomers ofthe [0061] The term "treatment", as used herein, refers to any compounds of the present invention possess the required process, action, application, therapy, or the like, wherein a selectivity. subject (or patient), including a human being, is provided [0049] The term "pharmaceutically acceptable acid-addi medica! aid with the object of improving the subject's tion salt" refers to any acid-addition salt, which, upon condition, directly or indirectly, or slowing the progression administration to the recipient is capable of providing (di of a condition or disorder in the subject, or ameliorating at rectly or indirectly) a compound as described herein. Pref least one symptom of the disease or disorder under treat erably, as used herein, the term "pharmaceutically accept ment. able salt" means approved by a regulatory agency of the [0062] The term "prevention", as used herein, refers to the Federal ora state govemment or listed in the U.S. Pharma administration of a compound of the invention in an initial copeia or other generally recognized pharmacopeia for use or early stage of the disease, or to also prevent its onset. in animals, and more particularly in humans. The prepara [0063] The term "von Hippel-Lindau disease" or "VHL tion of salts can be carried out by methods known in the art. disease" or "von Hippel-Lindau disease" or "VHL disease" Illustrative non-limitative examples of pharmaceutically or "VHL syndrome" or "von Hippel-Lindau syndrome", as acceptable acid-addition salt ofthe alkanolamine derivative used herein, refers to a rare disease caused by a mutation in offormula I is, for example, a salt derived from an inorganic the von Hippel-Lindau (VHL) tumor suppressor leading to acid, for example a hydrochloride, hydrobromide, phosphate an absence ofVHL protein orto an aberrant non-functional or sulphate, or a salt derived from an organic acid, for VHL protein. More than 370 inherited mutations in the VHL example an oxalate, lactate, tartrate, acetate, salicylate, gene have been identified in people with von Hippel-Lindau citrate, benzoate, ~-naphthoate , adipate or 1,1-methylene disease (http://www.umd.be/VHLI). VHL gene mutations bis(2-hydroxy-3-naphthoate), or a salt derived from an associated with this condition either prevent the production acidic synthetic resin, for example a sulphonated polysty of any VHL protein or lead to the production of an abnormal rene resin. In a particular embodiment, the pharmaceutically version of the protein. VHL di sease is characterized by the acceptable acid-addition salt is hydrochloride. In a more formation of multiple benign and malignant tumors and particular embodiment, the selective antagonist of the ~ 2 fluid-filled sacs (cysts) in many different parts of the body, adrenergic receptor antagonist is the hydrochloride salt of including: retina! hemangioblastoma, CNS hemangioblas the compound of formula II. toma, clear cell renal cell carcinoma (CCRCC), pheochro [0050] In another particular embodiment, the selective mocytoma, pancreatic islet tumor, endolymphatic sac ~ 2 -adrenergic receptor antagonist is selected from a list tumors and cysts in testes and broad ligament. comprising the following compounds: [0064] The term "patient" or "subject", as used herein, [0051] Butoxamine refers to any animal, preferably a mammal and includes, but [0052] The chemical name for butoxamine is DL-erythro is not limited to, domestic and farm animals, primates and humans, for example, human beings, non-human primates, a-(2,5-dimethoxyphenyl)-~-t-butyl aminopropanol hydro cows, horses, pigs, sheep, goats, dogs, cats, or rodents. In a chloride. Determination ofthe Beta2 selectivity ofbutoxam ine is reported in O'Donnell and Wanstall, Naunyn preferred embodiment, the subject is a human being of any Schmiedeberg's Arch. Pharmaco., 308, 183-190 (1979), age or race. In the present invention, the patient suffers from von Hippel-Lindau disease. which reports a ~ 2 /~ 1 selectivity ratio of at least 17. [0065] The term "patient with von Hippel-Lindau dis [0053] H35/25 ease", as used herein, means that the patient has been [0054] The chemical name for H35/25 is 1-(4'-methylphe diagnosed with the VHL disease. VHL disease can be nyl)-b 2,2-I-isopropylaminopropanol. diagnosed according with the following diagnostic criteria [0055] Prenalterol (Frantzen et al, Von Hippel-Lindau Syndrome, GeneR [0056] Prenalterol is a compound of formula eviews®): [0066] For an individual with no family history ofVHL disease, VHL disease is diagnosed if the patient pres ents two or more characteristic lesions: [0067] Two or more hemangioblastomas of the retina, spine, or brain ora single hemangioblastoma in association with a visceral manifestation (e.g. , multiple kidney or pancreatic cysts). [0068] Renal cell carcinoma. [0069] Adrenal or extra-adrenal pheochromocy [0057] The selective ~ 2 -adrenergic receptor antagonistic tomas. activity is described by Johansson and Waldeck, J. Pharm. [0070] Less commonly, endolymphatic sac tumors, Pharmacol., 1988, 32(9), 659-660. papillary cystadenomas of the epididymis or broad [0058] Various 4- and 5-[2-hydroxy-3-(isopropy ligament, or neuroendocrine tumors ofthe pancreas. lamino )propoxy ]benzimidazoles as described by [0071] For an individual with a positive history ofVHL Crooks et al, J. Med. Chem., 22(2), 210-214 (1979). disease, VHL disease is diagnosed if one or more ofthe [0059] 1-(t-butyl-amino-3-ol-2-propyl)oximino-9 fluo following disease manifestations is present: rene, as described by Imbs et al, Br. J. Pharmacol. [0072] Retina! angioma 60(3), 357-362 (1977). [0073] Spinal or cerebellar hemangioblastoma [0060] Various 2-(alpha-hydroxyarylmethyl)-3,3-dim [0074] Adrenal or extra-adrenal pheochromocytoma ethylaziridines as described by Jain et al, J. Med. [0075] Renal cell carcinoma Chem., 21(1), 68-72 (1978). [0076] Multiple renal and pancreatic cysts US 2020/0323798 Al Oct. 15, 2020 5

[0077] In any case where a heterozygous germline VHL cinoma (CCRCC), pheochromocytoma, pancreatic islet pathogenic variant is identified by molecular testing. tumor, endolymphatic sac tumors and cysts in testes and [0078] In a particular embodiment, the VHL disease broad ligament. occurs with the appearance of one or more tumors. [0084] The term "hemangioblastoma", as used herein, refers to a benign, highly vascular tumor that can occur in [0079] In a particular embodiment, the invention relates to the central nervous system (brain or spinal cord) and in the a selective antagonist of the ~ -adrenergic receptor for use 2 retina. In a more particular embodiment, the tumor is in the treatment andlor prevention of a tumor in a patient hemangioblastoma. In an even more particular embodiment, with von Hippel-Lindau disease. the tumor is a retina! hemangioblastoma ora central nervous [0080] The term "tumor" or "cancer", as used herein, system hemangioblastoma. refers to a broad group of diseases involving unregulated [0085] The term "clear cell renal cell carcinoma" or cell growth and which are also referred to as malignant "ccRCC", as used herein, refers to a renal cortical tumor neoplasms. The tenn is usually applied to a disease charac typically characterized by malignant epithelial cells with terized by uncontrolled cell division (or by an increase of clear cytoplasm and a compact-alveolar or acinar growth survival or apoptosis resistance) and by the ability of said pattem with arborizing vasculature. cells to invade other neighboring tissues (invasion) and [0086] The term "pheochromocytoma" or "PCC", as used spread to other areas of the body where the cells are not herein refers to a neuroendrocrine tumor of the medulla of normally located (metastasis) through the lymphatic and the adrenal glands, or extra-adrenal chromaffin tissue that blood vessels, circulate through the bloodstream, and then filed to involute after birth, that secretes high amounts of invade normal tissues elsewhere in the body. In a particular catecholamines, mostly norepinephrine and, to a lesser embodiment, the cancer appears as a benign tumor, i.e. extent, epinephrine. tumors that cannot spread by invasion or metastasis, i.e., [0087] The term "endolymphatic sac tumor", as used they only grow locally. In another particular embodiment, herein, refers to a papillary epithelial neoplasm arising the cancer appears as a malign tumor, i.e. a tumor that is within the endolymphatic sac or endolymphatic duct. capable of spreading by invasion and metastasis. [0088] The term "cyst", as used herein, refers to a cluster [0081] Illustrative non-limitative examples of tumors are of cells that have grouped together to form a sac. The hemangioblastoma, pheochromocytoma, endolymphatic sac distinguishing aspect of a cyst is that the cells forming the tumor, lymphocytic cancer, acute myeloid leukemia, alveo "shell" of such a sac are distinctly abnormal (in both lar rhabdomyosarcoma, borre cancer, brain cancer, breast appearance and behavior) when compared with all surround cancer, cancer of the anus, anal canal, or anorectum, cancer ing cells for that given location. It may contain air, fluids, or of the eye, cancer of the intrahepatic bile duct, cancer of the semi-solid material. In a particular embodiment, the cysts joints, cancer of the neck, gallbladder, or pleura, cancer of affect the testes or the broad ligament. the nose, nasal cavity, or middle ear, cancer of the vulva, [0089] For its administration to the patient, the selective ~ 2 chronic lymphocytic leukemia, chronic myeloid cancer, cer adrenergic receptor antagonist of the invention, preferably vical cancer, glioma, Hodgkin lymphoma, hypopharynx the alkanolamine derivative of formula I, more preferably cancer, kidney cancer, larynx cancer, liver cancer, lung the alkanolamine derivative of formula II, or a pharmaceu cancer, malignant mesothelioma, melanoma, multiple tically acceptable acid-addition salt thereof, will be formu myeloma, nasopharynx cancer, non-Hodgkin lymphoma, lated in a pharmaceutical composition. ovarian cancer, cyst in the broad ligamentum, peritoneum, [0090] The term "pharmaceutical composition", as used omentmn, and mesentery cancer, pharynx cancer, prostate herein, refers to a composition comprising a therapeutically cancer, rectal cancer, renal cancer, including renal cell effective amount of the selective ~ 2 adrenergic receptor carcinoma, skin cancer, soft tissue cancer, testicular cancer, antagonist of the invention, preferably the alkanolamine cysts in testes, thyroid cancer, ureter cancer, urinary bladder derivative of formula I, more preferably the alkanolamine cancer, and digestive tract cancer such as, e.g., esophageal derivative of fonnula II, or a pharmaceutically acceptable cancer, gastric cancer, pancreatic cancer, including pancre acid-addition salt thereof, and at least a pharmaceutically atic islet tumor, stomach cancer, small intestine cancer, acceptable excipient or carrier. gastrointestinal carcinoid tumor, cancer of the oral cavity, [0091] The term "therapeutically effective amount", as colorectal cancer, and hepatobiliary cancer. used herein, refers to the sufficient amount ofthe compound [0082] In a particular embodiment, the tumor is not a to provide the desired effect and will generally be deter pancreatic tumor. The term "pancreatic tumor", as used mined by, among other causes, the characteristics of the herein, includes both exocrine and endocrine tumors. Exo compound itself and the therapeutic effect to be achieved. It crine tumors in pancreas include adenocarcinoma, acinar will also depend on the subject to be treated, the severity of cell carcinoma, intraductal papillary-mucinous neoplasm the disease suffered by said subject, the chosen dosage form, (IPMN), mucinous cystadenocarcinoma among others. administration route, etc. Endocrine tumors in the pancreas, also called "islet cell [0092] For this reason, the doses mentioned in this inven tumors" include gastrinoma (Zollinger-Ellison syndrome ), tion must be considered only as guides for the person skilled glucagoma, insulinoma, somastostatinoma, VIPoma in the art, who must adjust the doses depending on the (Vemer-Morrison syndrome), nonfunctional islet cell tumor aforementioned variables. and multiple endrocrine neoplasia type-1 (MENl) among [0093] Even though individual needs vary, determination others. In a more particular embodiment, the tumor is notan of optimal ranges for therapeutically effective amounts of exocrine pancreatic tumor. In an even more particular the compounds for use according to the invention belongs to embodiment, the tumor is not a pancreatic adenocarcinoma. the common experience of those experts in the art. In [0083] In another particular embodiment, the tumor is general, the dosage needed to pro vide an effective treatment, selected from hemangioblastoma, clear cell renal cell car which can be adjusted by one expert in the art, will vary US 2020/0323798 Al Oct. 15, 2020 6

depending on age, health, fitness, sex, diet, weight, degree of animal dose (AD) in mg/kg can be converted to human alteration ofthe receptor, frequency oftreatment, nature and equivalent dose (HED) in mg/kg using the following for condition of the injury, nature and extent of impairment or mula: illness, medica! condition of the subject, route of adminis tration, pharmacological considerations such as activity, Animal K, efficacy, pharmacokinetic and toxicology pro file of the par HED(mg/kg) =AD(mg/kg) X -- ticular compound used, if using a system drug delivery, and Human K, if the compound is administered as part of a combination of drugs. The amount of the compound for use according to the wherein the K, for each species is shown in Table 1 (data invention that is therapeutically effective in the prevention extracted from Reagan-Shaw S. et al. "Dos e translation from ancl/or treatment of ischemia/reperfusion injury in a subject animal to human studies revisitecl'. FASEB J 2008, 22(3): can be determined by conventional clinical techniques (see, 659-661 ). for example, The Physician's Desk Reference, Medica! Economics Company, Inc., Oradell, N J, 1995, and Drug TABLE 1 Facts and Comparisons, Inc., St. Louis, Mo., 1993). K factor for conversion of AD to HED [0094] In a particular embodiment, the therapeutically effective amount produces the amelioration of one or more Species K, factor symptoms ofVHL disease. In a particular embodiment, the Human Adult 37 selective ~ adrenergic receptor antagonist, preferably the 2 Child 25 alkanolamine derivative of formula I, more preferably the Baboon 20 alkanolamine derivative of formula II, or the pharmaceuti Dog 20 cally acceptable acid-addition salt thereof, is administered at Monkey 12 a dose from about 0.2 mglkg/day to about 5 mg/kg/day, Rabbit 12 Guinea pig preferably from about 0.5 mg/kg/day, about 0.7 mg/kg/day, Rat about 1 mg/kglday, about 1.5 mglkg/day about 1.7 mg/kg/ Hamster day, about 1.9 mglkg/day, about 2.1 mg/kg/day, about 2.2 Mouse mg/kg/day, about 2.5 mg/kg/day, about 2.7 mg/kg/day, about 2.9 mglkg/day, about 3.1 mglkg/day, about 3.3 mglkg/day, to [0097] Thus, the experiments with doses of 10 mg/kg in about 4.9 mg/kg/day, about 4.8 mglkglday, about 4.7 mg/kg/ mice correspond to general do ses in humans of 0.8 mg/kg. day, about 4.6 mglkg/day, about 4.5 mg/kg/day. In a more [0098] In another particular embodiment, the selective ~ 2 particular embodiment, the selective ~ 2 adrenergic receptor antagonist, preferably the alkanolamine derivative of for adrenergic receptor antagonist, preferably the alkanolamine mula I, more preferably the alkanolamine derivative of derivative of formula I, more preferably the alkanolamine derivative offormula II, or the pharmaceutically acceptable formula II, or the pharmaceutically acceptable acid-addition salt thereof, is administered at a dose between 3.3 mg/kg acid-addition salt thereof, is administered in humans, at a dose wherein each administration ranges from 0.2 mglm2 to body/day and 4.5 mg/kg body/day. In another more particu 2 5 mglm . preferably from about 0.2 mglkg/day, about 0.25 lar embodiment, the selective ~ adrenergic receptor antago 2 mg/kg/day, about 0.3 mg/kg/day, about 0.35 mg/kg/day, nist, preferably the alkanolamine derivative of formula I, about 0.4 mg/kg/day, about 0.45 mg/kg/day, about 0.50 more preferably the alkanolamine derivative of formula II, or the pharmaceutically acceptable acid-addition salt mg/kg/day, about 0.55 mg/kglday, about 0.6 mg/kg/day, about 0.65 mg/kglday, about 0.7 mg/kg/day, about 0.75 thereof, is administered at a dose between 0.5 mg/kg body/ day and 1 mg/kg body/day. In an even more particular mg/kg/day, about 0.8 mg/kg/day, about 0.85 mg/kg/day, about 0.90 mg/kglday, about 0.95 mg/kg/day, about 1 embodiment, the selective ~ adrenergic receptor antagonist, 2 mg/kg/day, about 1.2 mg/kg/day, about 1.4 mg/kg/day, about preferably the alkanolamine derivative of formula I, more preferably the alkanolamine derivative of formula II, or the 1.6 mglkglday, about 1.8 mg/kg/day, about 2 mg/kg/day about 2.2 mg/kg/day, about 2.4 mglkg/day, about 2.6 mg/kg/ pharmaceutically acceptable acid-addition salt thereof, is administered at a dose of 0.81 mg/kg body/day. day, about 2.8 mg/kg/day, about 3 mg/kglday, about 3.2 mg/kg/day, to about 3.4 mg/kglday, about 3.6 mg/kg/day, [0095] Doses of the compounds of the invention may be about 3.8 mg/kg/day, about 4 mg/kg/day, about 4.5 mg/kg/ expressed either in mg of the antagonist per kg of body day, about 5 mg/kg/day. In a more preferred embodiment, weight or in mg of the antagonist per square meter of body the selective ~2 adrenergic receptor antagonist, preferably surface. The skilled person knows how to determine the do se the alkanolamine derivative of formula I, more preferably for a particular animal, in particular the dose for human the alkanolamine derivative of formula II, or the pharma beings, from the doses experimentally assayed in mice. For ceutically acceptable acid-addition salt thereof, is adminis example, the article from Reagan-Shaw S. et al. (Reagan tered at a dose between 0.7 mg/kg body/day and 1 mglkg Shaw S. et al. "Dose translation from animal to human body/day. In a still more preferred embodiment, the selective studies revisited". FASEB J 2008, 22(3):659-661) provides ~2 adrenergic receptor antagonist, preferably the the standard conversion factors used to convert mg/kg to alkanolamine derivative of formula I, more preferably the 2 mg/m . alkanolamine derivative of formula II, or the pharmaceuti 2 cally acceptable acid-addition salt thereof, is administered at Dose (mg/kg)xK, ~Dose (mg!m ) a dose of 0.81 mglkg body/day. (0096] The article also explains that this conversion is the (0099] In another particular embodiment, the selective ~ 2 basis for converting do se in a first animal species to do se in adrenergic receptor antagonist, preferably the alkanolamine a second animal species (allometric dose translation). Thus, derivative of formula I, more preferably the alkanolamine US 2020/0323798 Al Oct. 15, 2020 7

derivative of formula II, or the pharmaceutically acceptable alkanolamine derivative of formula II, or pharmaceutically acid-addition salt thereof, is administered daily preferably 1 acceptable acid-addition salt thereof, is administered orally time a day, 2 times a day, 3 times a day. In a more preferred or intravenously or intravitreal. In a more preferred embodi embodiment, it is administered 1 time a day. In another ment, the selective B2 adrenergic receptor antagonist of the particular embodiment, the selective B2 adrenergic receptor invention, preferably the alkanolamine derivative offormula antagonist, preferably the alkanolamine derivative of for I, more preferably the alkanolamine deriva tive offormula II, mula I, more preferably the alkanolamine derivative of or pharmaceutically acceptable acid-addition salt thereof, is formula II, or the pharmaceutically acceptable acid-addition administered intraperitoneally. salt thereof, is administered during 1O days, 15 days, 20 [0102] The invention is described below by way of the days, 25 days, 1 month, 2 months, 3 months, 4 months, 5 following examples, which are to be seen as merely illus months, 6 months, 7 months, 8 months, 9 months, 1O trative and not limitative of the scope of the invention. months, 11 months, 12 months or more than 12 months, preferably during 25 days. Examples [0100] The terms "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier", refer to any com Methods pound or combination of compounds that is essentially non-toxic to the subject at the dosage and concentration Cell Culture employed, and is compatible with the other components of [0103] HeLa 9XHRE cells were stably transfected with a a pharmaceutical composition. Thus, an excipient is an HRE!uc reporter carrying nine copies in tandem of the inactive substance formulated alongside the active ingredi hypoxia responsive element (HRE) followed by luciferase ent (i.e., the selective B2 adrenergic receptor antagonist, gene, and were cultured in DMEM (Dulbecco's Modified preferably the alkanolamine derivative of formula I, more Eagle Medium, Gibco, Grand Island, N.Y., USA) supple preferably the alkanolamine derivative of formula II, or the mented with 10% fetal bovine serum (FBS; Gibco). To pharmaceutically acceptable acid-addition salt thereof) of a induce hypoxic conditions, HeLa cells were cultured with pharmaceutical composition, for the purpose of bulking-up 100 ¡.tM desferrioxamine (DFO) (chemical hypoxia). When compositions that contain said active ingredients. Bulking required, HeLa cells were treated with Propranolol (non up allows convenient and accurate dispensation of a drug selective beta blocker which binds both Beta 1 and Beta 2 substance when producing a dosage form. Excipients also receptors), ICI 118.551 (specific of Beta 2) and Atenolol can serve various therapeutic enhancing purposes, such as (specific for Beta 1) (100 ¡.tM). Primary cultures of CNS facilitating compound (drug) absorption or solubility, or hemangioblastoma were obtained according to the previ other pharmacokinetic considerations. Excipients can also ously described by Albiñana et al., Orphanet J Rare Dis be useful in the manufacturing process, to aid in the handling eases, 2015, 10: 118. When required, primary cultures of of the active substance concemed such as by facilitating CNS hemangioblastoma were treated with propranolol and powder flowability or non-stick properties, in addition to ICI (100 ¡.tM). aiding in vitro stability such as prevention of denaturation over the expected shelf life. The selection of appropriate Real-Time RT-PCR excipients depends upon the rout of administration and the dosage form, as well as the active ingredient and other [0104] Total cellular RNA was extracted from hemangio factors. An excipient can be a non-toxic salid, semisolid or blastoma cells using a Nucleo Spin RNA kit (Macherey liquid filler, diluent, encapsulating material or formulation Nagel, Düren, Germany). One microgram oftotal RNA was auxiliary of any conventional type. Illustrative, non-limita reverse-transcribed in a final volume of 20 ¡.ti with the First tive, examples of excipients or carriers include water, salt Strand cDNA Synthesis Kit (Rache, Mannheim, Germany) (saline) solutions, alcohol, dextrose, vegetable oils, polyeth using random primers. SYBR Green PCR system (BioRad, ylene glycols, gelatin, lactase, amylose, magnesium stearate, Hercules, Calif., USA) was used to carry out real-time PCR tale, surfactants, silicic acid, viscous para:ffin, perfume oil, with an iQ5 system. Primers used for qPCR were: monoglycerides and diglycerides of fatty acids, fatty acid esters petroetrals, hydroxymethyl cellulose, polyvinylpyr TABLE 2 rolidone and the like. Primers used for gPCR [0101] The selective B2 adrenergic receptor antagonist of the invention, preferably the alkanolamine derivative of GENE Forward Reverse formula I, more preferably the alkanolamine derivative of 18S 5'-CTCAACACGGGAA 5'-CGCTCCACCAACT formula II, or a pharmaceutically acceptable acid-addition ACCTCAC-3' AAGAACG-3' . salt thereof, may be administered by any suitable adminis tration route, such as, but not limited to, parenteral, oral, BAX 5'-CACTCCCGCCACA 5'-CAAGACCAGGGTG topical, nasal, rectal, intravitreal route. In a particular AAGAT-3' GTTGG-3' embodiment, the selective B2 adrenergic receptor antagonist CASP9 5'-CCCAAGCTCTTTT 5'-TTACTGCCAGGGG of the invention, preferably the alkanolamine derivative of TCATCCA-3' ACTCGT-3' formula I, more preferably the alkanolamine derivative of formula II, or a pharmaceutically acceptable acid-addition salt thereof, is administered intraperitoneally, intravenously, Luminescent Cell Viability Assay subcutaneously, intradermically, intramuscularly or intrav itreal. In a preferred embodiment, the selective B2 adrenergic [0105] The viability of hemangioblastoma and HeLa receptor antagonist of the invention, preferably the 9XHRE cells was measured with a CellTiter-Glo Lumines alkanolamine derivative of formula I, more preferably the cent Cell Viability Assay (Promega, Madison, Wis., USA). US 2020/0323798 Al Oct. 15, 2020 8

This is a homogeneous method to determine the number of Results viable cells in culture based on quantitation of the ATP [0110] ICI 118,551 Decreases Viability ofHemangioblas present, which signals the presence of metabolically active toma and Inhibits Hemangiosphere Formation from VHL cells. A total of 10,000 cells were plated in 96-well plates Patients and cultured with propranolol/ICI118,551 in 100 ¡.ti of [0111] Results shown in examples demonstrate that ICI medium. After treatment, plates were equilibrated to room 118,551 , a selective beta 2 blocker, not only mirrors the temperature for 30 min befare the addition of 100 ¡.ti of Cell results obtained with propranolol (non-selective beta Titer-Glo reagent (Lysis buffer, Ultra-Glo Recombinant blocker which binds beta 1 and beta 2 receptors) (see Luciferase, Luciferine and Mg2+ ). Luminescence was mea Albiñana et al., Orphanet J of are diseases. 10:118 (2015)) sured using a Glomax Multidetection System (Promega). but al so shows superior results to propranolol. In particular, for the case of VHL-tumor derived cells, ICI 118,551 Wound Healing and Tube Formation Assay decreases viability by an increase in cell apoptosis, inhibits [0106] In vitro-scratched wounds were created by scrap hemangiosphere formation, which is a specific property of ing confiuent HlNEC-1 monolayers in P-24 plate wells tumors containing undifferentiated stem cells, and is an with sterile disposable pipet tips. The remaining cells were antiangiogenic agent through the inhibition of HIF-stimu washed with PBS and incubated with EGM-2 (Lanza) in the lated transcription of its target genes. absence or presence of propranolol/ICI 118,551 100 ¡.tM for [0112] Propranolol and ICI 118,551 decrease viability of up to 6-8 h. For tu be formation assays, HlNECs were plated hemangioblastoma cells from a primary culture derived as befare but on Matrigel plate ((BD Biosciences, Bedford, from a VHL tumor by at least 55-60% compared to the same Mass., USA) and incubated at 37° C., without and with untreated cells (FIG. 1). In most cases the effect of ICI propranolol/ICI 118,551 treatrnent (100 ¡.tM). Tube forma 118,551 is superior to propranolol, when both are used at tion was monitored for up to 8 hours. 100 ¡.tM, after 72 hours of treatment. The decrease of viability ranges from 60 to 40% depending of different Hemangiospheres Formation and Culture tumors, patients and time of culture. [0113] FIG. 2 presents the remaining cells in cultures after [0107] VHL-derived hemangioblastoma cells were grown 72 hours oftreatment with P (propranolol100 ¡.tM) and ICI in suspension in phenol red free DMEM:F-12 medium with 118,551 50 ¡.tM (HB 18) and 100 ¡.tM in (HB 4), compared GlutaMAX, supplemented with B27, 20 ng/ml EGF, 20 with Control (untreated cells of different hemangioblasto ng/ml bFGF and 1% penicillin/streptomycin at 37° C. in 5% mas from 2 different patients ). There is a significant reduc C02. Single cell suspensions were plated at a density of tion in the number of cells of up to 30-40% with respect to 50.000 cells/ml in 75 cm2 ultralow attachment fiasks (Com the untreated ones. The decrease is due to apoptosis as ing). Cultures were untreated or treated with propranolol/ICI shown for ICI 118,551 in FIG. 3. FIG. 3 shows the amount 118,551 at the shown concentrations. of mRNA from two proapoptotic genes, Bax and the execu tor protease Caspase 9, which is significantly upregulated Cell Viability Measure after cell treatment with ICI 118,551 at 100 ¡.tM. [01 08] Endothelial cell !ines (HlNECs and HMECs), [0114] One characteristic oftumoral cells in culture is the human ccRCC Vhl-/- 786-0 cellline, and primary cultures property of forming organized spheres around a nucleus of hemangioblastomas from patients (Hb14 and 18) were undifferentiated stem cells, these spheres have a well defined seeded in triplicate onto a 96-well plate (2x103 per well). border surrounded by an ordered interface with the medium. The next day, cells were treated with vehicle or various mM Antitumoral agents inhibit or dismpt the formation of the concentrations ofpropranolol or ICI-118,551 for 72 hours. spheres by disaggregation of the cells. FIG. 4 is showing Cell viability (cell proliferation, ATP content) was deter hemangiosphere cultures from different hemangioblastomas mined using the CellTiter-Glo Luminescent Cell Viability without treatment and upon 100 ¡.tM of either Propranolol or Assay kit (Promega) according to the supplier's instructions. ICI 118,551 treatment for 7 days in these particular cases Cell Titer-Glo reagent (Lysis buffer, Ultra-Glo Recombinant shown in this FIGS. 4 and 5. As shown in 5 different tumor Luciferase, Luciferine and Mg2+). Celllysis was induced in cultures, hemangiospheres are dismpted by beta-blockers an orbital shaker for 2 min, and then plates were incubated treatment, being especially conspicuous the case ofheman at room temperature for 1O min to stabilize the luminescent gioblastoma 4 (FIG. 4B) and 23 (FIG. 4C) after 100 ¡.tM ICI signa!. Luminescence was measured using a GlomaxMulti 118,551. detection System (Promega) Readings were normalized to [0115] Angiogenesis is the property endothelial cells have control (100%). to form ves seis from preexisting ones. For angiogenesis it is Xenografts of 786-0 Human ccRCC Cells in Mice necessary migration of endothelial cells (involving dismp [0109] Male 7-8 weeks old NOD scid gamma (NSG) mice tion on cell matrix ), and tubulogenesis or formation of new were injected in the dorsal fiank with a single cell suspension tubes (vessels). These properties may be studied in vitro by of 106 786-0 cells. When tumor size reached 100 mm3 the test ofwound healing (FIG. 5), and matrigel tubulogen volume, mice were randomly divided in 3 groups, of 9/10 esis (FIG. 6). mice each. Two groups were daily treated intraperitoneally [0116] If confiuent monolayers of endothelial cells are with 10 mg/Kg body propranolol or ICI-118,551, respec dismpted by scratching their surface, the endothelial cells tively anda third group was injected with the solvent. Tumor tend to migrate and fill the discontinuity. This "so-called" size was measured by a caliper every 2-3 days and volumes wound healing test may be followed over time to monitor the were calculated following the formula: shortest 2xlargestx migration of cells. FIG. 5A shows how ICI 118,551 and 0.52. Mice were sacrificed when tumor volume average of propranolol at 100 ¡.tM delay the elasure of the discontinuity the control group reached an end point established according as compared to untreated cells (control). The migration of to ethical procedures untreated cells is faster as quantified in the graph ofFIG. 5B, US 2020/0323798 Al Oct. 15, 2020 9

while both ICI 118,551 and propranolol are significantly therefore, the therapeutic effect ofiCI118,551 a specific beta interfering with the migration process. 2 blocker, for the von Hippel Lindau disease is explained by [0117] FIG. 6A is showing the characteristic network of a decrease of stimulation ofthe HIF gene program. Thus, ICI tubules imitating the capillary network formed by endothe 118,551 mimicks the effects ofpropranolol interfering with lial cells in matrigel after 3 hours. However, if cells are the hypoxia pathway, mainly through the blockade of the pretreated with ICI 118,551 or Propranolol (100 ¡.tM), the beta 2 adrenergic receptors. number of closed structures is dramatically decreased. The ICI 118,551 is Preferentially Targeting Vhl-/- Tumoral average quantification of closed cells in five different fields Cells, Versus Non Tumoral Cells as HUVECs and HMECs is shown in FIG. 6B. It is clear that propranolol and ICI 118,551 are functioning as inhibitors of angiogenesis. [0120] When Vhl-/- cells, primary cultures ofhemangio blastoma cells from VHL patients, including the Vhl-/ [0118] Angiogenesis is largely dependent of HIF tran renal carcinoma cellline 786-0 are treated at different doses scription program, triggered in the presence of hypoxic from O to 250 ¡.tM of Ser. No. 10/118,551 , they are more condition, oras in the case ofVon Hippel Lindau haeman sensitive than non tumoral cells like HUVECs (Human gioblastoma cells, dueto the lack ofVHL protein in charge umbilical vein endothelial cell) and HMECs (human micro oftargeting HIF protein for its proteasome processing under vasculature endothelial cells ). While LD50 for VHL heman normoxic conditions in wild type ce lis. A special peculiarity gioblastomas is between 50 and 100 ¡.tM ICI118,551, in ofhemangioblastoma cells, and clear cell kidney carcinoma 786-0 is 100 ¡.tM., in the case of HUVEC and HMEC is cells, is that they constitutively express HIF protein, which around 150 ¡.tM. This is very important in arder to selectively is active, and is translocated to nucleus where it may bind target the Vhl-/- cells atan ICI118,551 do se below the toxic and activates its gene targets such as: VEGF, PDGF, EPO, range for non Vhl-/- cells. See FIG. 9. Thus, ICI 118,551 is endoglin, severa! metalloproteases, carbonic anhydrase, preferentially targeting Vhl-/- tumoral cells, versus non Glut-1 and so on. To test that propranolol and ICI 118,551 tumoral cells. are inhibitors of HIF transcriptional activity a system of ICI 118,551 and Propranolol Act Decreasing the Tumor luciferase reporter under the control of nine copies in Growth of a ccRCC Vhl-/-786-0 Xenograft in NSG Mice, tandem of the hypoxia responsive element (HRE) followed an In Vivo Model by luciferase was used. HeLa 9XHRE cells were stably [0121] NSG mice treated with propranolol or ICI118,551 transfected with a HRE!uc reporter. To induce hypoxic (Beta-blockers) inhibits the tumor growth, as shown from conditions, HeLa cells were cultured with 100 ¡.tM desfer significant differences found between the size ofthe tumors, rioxamine (DFO) (chemical hypoxia). These hypoxic cells between 50-60 days after the cell inoculation (FIG. 10). were either untreated or treated with 100 ¡.tM of either There were no significant differences between propranolol Propranolol or ICI 118,551 , and the luciferase activity and ICI118,551 treatments, while these treatments showed a measured by luminometry. FIG. 7 shows how luciferase significant decrease around 30% of the size tumor versus expression is triggered under hypoxic conditions, and how vehicle treated mi ce. There were no toxic or adverse effects this upregulation is dampened by Propranolol and ICI 118, observed during the time ofthe treatment. Thus, ICI118,551 551. ICI 118,551 is clearly superior to Propranolol in acts decreasing the tumor growth of a ccRCC Vhl-/- 786-0 abolishing completely the luciferase activity induced by xenografts in an in vivo model of NSG mi ce. hypoxia, therefore ICI 118,551 is targeting the transcrip tional activity of HIF, which is reduced practically to "0". ICI 118.551 Affects Differentially Vhl-/- Cells and Normal Propranolol and ICI 118,551 are Interfering with the Endothelial Cells HUVECs and HMECs. Hypoxia Pathway, Mainly Through the Blockade ofthe Beta [0122] Primary culture ofHaemangioblastoma from VHL, 2 Adrenergic Receptors normal primary endothelial HUVECs, and the non tumoral [0119] When HeLa cells harbouring the HRE-luciferase microendothelial cell line HMEC-1 were cultured in the hypoxia reporter are treated with DFO (deferroxiamine), absence or presence of different concentrations of ICI 118. HIF is translocated to the nuclei, and binds the HRE 551 (50-250 ¡.tM) to measure viability. According to viability (Hypoxia Responsive Elements) fused to Luciferase curves Haemangioblastoma (HB) cells show only 20% of reporter, and the stimulation ofhypoxia target maybe quan viability at 100 ¡.tM, while HUVEC or HMEC-1 non tumoral tified by luminescence. However, the hypoxia stimulation is cells show 80% and 55% respectively decrease in viability extremely reduced in the presence of ICI118,551 and pro at 100 ¡.tM in both cultures, pranolol at 100 ¡.tM. As both are Beta 2 adrenergic receptor (0123] Propranolol and ICI 118,551 have a double way of blockers, atenolol, a specific beta 1 adrenergic blocker was action: on one hand, they are promoting apoptosis and in this also used. As shown in FIG. 8, atenolol, hardly reduced the way stopping growth and causing cell death in a pro hypoxia stimulation. Accordingly, it seems that ICI118,551 grammed way. On the other hand, they are abrogating the is mimicking propranolol targeting HIF-inducible pathway, transcriptional activation of HIF inducible program, and the action is carried out by blocking beta 2 type receptor. decreasing in this way the expression oftarget genes such as VHL derived tumors, have a constitutive HIF expression, VEGF, EPO, endoglin, metalloproteases and so on.

SEQUENCE LISTING

<160 > NUMBER OF SEQ ID NOS: 6

<210> SEQ ID NO 1 <211> LENGTH : 20 <212> TYPE: DNA US 2020/0323798 Al Oct. 15, 2020 10

-continued

<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: lBS qPRC forward primer

<400 > SEQUENCE: 1

ctcaacacgg gaaacctcac 20

<2 10 > SEQ ID NO 2 <2 11 > LENGTH: 20 <2 12 > TYPE : DNA <213> ORGANISM: Artificial Sequence <220 > FEATURE : <223> OTHER INFORMATION: lBS qPRC reverse primer

<400 > SEQUENCE: 2

cgctccacca actaagaacg 20

<210> SEQ ID NO 3 <211 > LENGTH : lB <212> TYPE: DNA <2 13 > ORGANISM: Artificial Sequence <22 0 > FEATURE : <223> OTHER INFORMATION: BAX qPCR forward primer

<400> SEQUENCE:

cactcccgcc acaaagat lB

<2 10 > SEQ ID NO 4 <2 11 > LENGTH : lB <212> TYPE: DNA <2 13 > ORGANISM: Artificial Sequence <220> FEATURE: <223 > OTHER INFORMATION : BAX qPCR reverse primer

<400> SEQUENCE: 4

caagaccagg gtggttgg lB

<210 > SEQ ID NO 5 <211> LENGTH: 20 <212 > TYPE : DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION : CASP9 qPCR forward primer

<4 00 > SEQUENCE : 5

cccaagct ct ttttcatcca 20

<210> SEQ ID NO 6 <2 11 > LENGTH: 19 <2 12 > TYPE : DNA <213> ORGANISM: Artificial Sequence <220 > FEATURE: <223> OTHER INFORMATION: CASP9 qPCR reverse primer

<400> SEQUENCE:

ttactgccag gggactcgt 19 US 2020/0323798 Al Oct. 15, 2020 11

l. A method oftreatment and/or prevention ofvon Hippel 8. The method according to claim 7, wherein the Lindau disease in a patient comprising administering to said alkanolamine derivative has the formula patient a therapeutically effective amount of a selective antagonist of the B2 -adrenergic receptor. 2. The method according to claim wherein the antagonist is an alkanolamine deriva tive of formula

9. The method according to claim 2, wherein the phar maceutically acceptable acid-addition salt is hydrochloride. 10. The method according to claim 1, wherein the alkanolamine derivative or pharmaceutically acceptable acid-addition salt thereof is administered at a dose between 0.2 mg/Kg body/day and 5 mg/kg body/day. wherein R 1 is an alkyl group of up to 6 carbon atoms which is branched at the a-carbon atom, 11. The method according to claim 10, wherein the wherein R2 is an alkyl of up to 3 carbon atoms, alkanolamine derivative or pharmaceutically acceptable wherein R3 is hydrogen, a halogen or an alkyl of up to acid-addition salt thereof is administered at a dose between three carbon atoms and wherein n is 1 or 2, 0.5 mg/Kg body/day and 1 mg/kg body/day. or a pharmaceutically acceptable acid-addition salt 12. The method according to claim 11 , wherein the thereof. alkanolamine derivative or pharmaceutically acceptable 3. The method according to claim 1, wherein the VHL acid-addition salt thereof is administered at a dose of 0.8 disease occurs with the appearance of one or more tumors. mg/kg body/day. 4. The method according to claim 1, wherein the tumor is 13. The method according to claim 1, wherein the a hemangioblastoma. alkanolamine derivative or pharmaceutically acceptable 5. The method according to claim 4, wherein the heman acid-addition salt thereof is administered intraperitoneally. gioblastoma is a retina! hemangioblastoma or a central nervous system hemangioblastoma. 14. The method according to claim 1, wherein the alkanolamine derivative or pharmaceutically acceptable 6. The method according to claim 1, wherein n is l. 1 acid-addition salt thereof is administered daily for 25 days. 7. The method according to claim 1, wherein R is 2 3 isopropyl, R is methyl and/or R is methyl. * * * * *