WO2020115009A1 - LPAAT-β INHIBITORS FOR TREATMENT OF CANCER - Google Patents

Abstract

The present invention relates to a compound of formula (1), wherein R1 and R4 are selected from halogen, small hydrocarbon or alkoxy moieties, R2 is a short alkyl moiety, and X is selected from -S-, -O-, -N(R5)-, wherein R5 is selected from H or a short alkyl moiety. Furthermore, the compound of formula (1) may be used as a medicament, particularly in the treatment of cancer. The invention also relates to a method of inhibiting lysophosphatidic acid acyltransferase β (LPAAT-β).

Description

LPAAT-b inhibitors for treatment of cancer

Background of the invention

Angiogenesis, i.e. the formation of new blood vessels, is a key biological process during embryogenesis and wound healing. Angiogenesis inhibition is an important target to address diabetic retinopathy and cancer, in particular by interfering with the vascular endothelial growth factor receptor 2 (VEGFR-2), but also with other receptor tyrosine kinases such as Axl for which several inhibitors have been investigated.

Less studied targets of angiogenesis inhibition are transferases such as lysophosphatidic acid acyltransferase b (LPAAT-b). A known LPAAT-b inhibitor (compound 43) has been shown to inhibit ERK and AKT pathways in several cancer models,

This compound is characterized by a p-bromo substituted aniline moiety and a substituted phenyl moiety.

Further known LPAAT-b inhibitors include a m-substituted analog (compound 52) and a similar compound sharing the same core structure (compound 57),

Compounds with an amended core structure (compound 44) were so far only known to inhibit other targets such as the adenosine receptor 2a, which is a G-protein coupled receptor associated with the synthesis of intracellular cAMP,

Surprisingly, compounds characterized by a substituted benzofuran moiety instead of a phenyl moiety and a specifically substituted aniline moiety were found to be potent inhibitors of LPAAT-b as well expanding the rather limited pharmacology of LPAAT-b inhibitors.

Based on the above-mentioned state of the art, the objective of the present invention is to provide new compounds for treatment of cancer, in particular by inhibition of lysophosphatidic acid acyl transferase b (LPAAT-b). This objective is attained by the subject- matter of the independent claims of the present specification.

Terms and definitions

A Ci-C₆ alkyl in the context of the present specification signifies a saturated linear or branched hydrocarbon having 1 , 2, 3, 4, 5 or 6 carbon atoms, wherein one carbon-carbon bond may be unsaturated. Non-limiting examples for a CrC₆ alkyl include the examples given for CrC₄ alkyl below, and additionally 3-methylbut-2-enyl, 2-methylbut-3-enyl, 3- methylbut-3-enyl, n-pentyl, 2-methylbutyl, 3-methylbutyl, 1 ,1-dimethylpropyl, 1 ,2- dimethylpropyl, 1 ,2-dimethylpropyl, pent-4-inyl, 3-methyl-2-pentyl, and 4-methyl-2-pentyl.

Where used in the context of chemical formulae, the following abbreviations may be used: Me is methyl CH₃, Et is ethyl -CH₂CH₃, Pr is propyl -(CH₂)2CH₃ (n-propyl, n-pr) or -CH(CH₃)₂ (iso-propyl, i-pr), Bu is butyl -C₄H₉, -(CH₂)₃CH₃, -CHCH₃CH₂CH₃, -CH₂CH(CH₃)₂ or -C(CH₃)₃.

The term halo alkyl refers to an alkyl according to the above definition that is modified by one or several halogen atoms selected (independently) from F, Cl, Br, I, particularly F. Further non-limiting examples of halo-substituted alkyl include -CH₂F, -CHF₂, -(CH₂)₂F, -(CHF)₂H, - (CHF)₂F, -C₂F_S, -(CH₂)₃F, -(CHF)₃H, -(CHF)₃F, -C₃F_7I -(CH₂)₄F, -(CHF)₄H, -(CHF)₄F and -

C F₉.

The term aryl in the context of the present specification signifies a cyclic aromatic C₅-Ci₀ hydrocarbon that may comprise a heteroatom (e.g. N, O, S). Examples of aryl include, without being restricted to, phenyl and naphthyl, and any heteroaryl. A heteroaryl is an aryl that comprises one or several nitrogen, oxygen and/or sulphur atoms. Examples for heteroaryl include, without being restricted to, pyrrole, thiophene, furan, imidazole, pyrazole, thiazole, oxazole, pyridine, pyrimidine, thiazin, quinoline, benzofuran and indole. An aryl or a heteroaryl in the context of the specification additionally may be substituted by one or more alkyl groups.

As used herein, the term treating or treatment of any disease or disorder (e.g. cancer) refers in one embodiment, to ameliorating the disease or disorder (e.g. slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment "treating" or "treatment" refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient. In yet another embodiment, "treating" or "treatment" refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. Methods for assessing treatment and/or prevention of disease are generally known in the art, unless specifically described herein below.

The skilled person is aware that any specifically mentioned drug may be present as a pharmaceutically acceptable salt of said drug. Pharmaceutically acceptable salts comprise the ionized drug and an oppositely charged counterion. Non-limiting examples of pharmaceutically acceptable anionic salt forms include acetate, benzoate, besylate, bitatrate, bromide, carbonate, chloride, citrate, edetate, edisylate, embonate, estolate, fumarate, gluceptate, gluconate, hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl bromide, methyl sulfate, mucate, napsylate, nitrate, pamoate, phosphate, diphosphate, salicylate, disalicylate, stearate, succinate, sulfate, tartrate, tosylate, triethiodide and valerate. Non-limiting examples of pharmaceutically acceptable cationic salt forms include aluminium, benzathine, calcium, ethylene diamine, lysine, magnesium, meglumine, potassium, procaine, sodium, tromethamine and zinc.

Dosage forms may be for enteral administration, such as nasal, buccal, rectal, transdermal or oral administration, or as an inhalation form or suppository. Alternatively, parenteral administration may be used, such as subcutaneous, intravenous, intrahepatic or intramuscular injection forms. Optionally, a pharmaceutically acceptable carrier and/or excipient may be present.

Wherever alternatives for single separable features are laid out herein as“embodiments”, it is to be understood that such alternatives may be combined freely to form discrete embodiments of the invention disclosed herein.

The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope.

A first aspect of the invention relates to a compound comprising the general formula (1 )

wherein

R¹ is selected from -I, -Br, -Cl, C-_|-C₆-alkyl, C₃-C_4.cycloalkyl, CrC₆-haloalkyl, -OR^3a, -SR^3a, and wherein R^3a is selected from CrC₆-alkyl

R² is selected from CrC₆-alkyl, and

R⁴ is selected from -I, -Br, -Cl, -F, CrC₆-alkyl, C₃-C₆-cycloalkyl, CrC₆-haloalkyl, C₆-aryl, - OR^3b, -SR^3b, and wherein R^3b is selected from CrC₆-alkyl, and with m being 0, 1 , 2, 3 or 4, and

X is selected from -S-, -0-, -N(R⁵)-, wherein R⁵ is selected from H, CrC₃-alkyl, particularly wherein R⁵ is methyl

with the proviso that the compound is not 6-(3-Methyl-2-benzofuranyl)-/V²-(4-ethylphenyl)- 1 , 3, 5-triazine-2, 4-diamine, 6-(3-Methyl-2-benzofuranyl)-/V²-(4-methylphenyl)-1 ,3,5-triazine- 2, 4-diamine, 6-(3-Methyl-2-benzofuranyl)-A/²-(4-chlorophenyl)-1 , 3, 5-triazine-2, 4-diamine, 6- (3-Methyl-2-benzofuranyl)-/V²-(4-ethoxyphenyl)-1 , 3, 5-triazine-2, 4-diamine.

6-(3-Methyl-2-benzofuranyl)-/V²-(4-ethylphenyl)-1 , 3, 5-triazine-2, 4-diamine is a compound of formula 1 with R¹ being ethyl, R² being methyl, m being 0 and X being -0-. Thus, the combination R¹ = C₂-alkyl, R² = Ci-alkyl, m = 0 and X = -O- is excluded from the scope of the first aspect.

6-(3-Methyl-2-benzofuranyl)-/V²-(4-methylphenyl)-1 , 3, 5-triazine-2, 4-diamine is a compound of formula 1 with R¹ being methyl, R² being methyl, m being 0 and X being -0-. Thus, the combination R¹ = Cralkyl, R² = Cralkyl, m = 0 and X = -O- is excluded from the scope of the first aspect.

6-(3-Methyl-2-benzofuranyl)-/V²-(4-chlorophenyl)-1 , 3, 5-triazine-2, 4-diamine is a compound of formula 1 with R¹ being -Cl, R² being methyl, m being 0 and X being -0-. Thus, the combination R¹ = -Cl, R² = Cralkyl, m = 0 and X = -O- is excluded from the scope of the first aspect.

6-(3-Methyl-2-benzofuranyl)-A/²-(4-ethoxyphenyl)-1 ,3, 5-triazine-2, 4-diamine is a compound of formula 1 with R¹ being -O-ethyl, R² being methyl, m being 0 and X being -0-. Thus, the combination R¹ = -0-R^3a with R^3a being C -alkyl, R² = Ci-alkyl, m = 0 and X = -O- is excluded from the scope of the first aspect.

In certain embodiments, R¹ is selected from -Br, -Cl, CrC₃-alkyl, C _.cycloalkyl, CrC₃- haloalkyl and -OR^3a, in particular -Br, -Cl, methyl, ethyl, iso-propyl, -CF₃, and -OR^3a.

In certain embodiments, R¹ is selected from Br, -Cl, ethyl, -CF₃ and -OMe, in particular from - Cl, ethyl, and -OMe.

In certain embodiments, R² is selected from Ci-C -alkyl.

In certain embodiments, is selected from ethyl and methyl, in particular ethyl.

In certain embodiments, R⁴ is selected from -F, -Br, -Cl, CrC₃-alkyl and -OR^3b, in particular - F, -Cl, methyl and -OR^3b.

In certain embodiments, R⁴ is selected from -F and methyl.

In certain embodiments, m is selected from 0, 1 or 2.

In certain embodiments, R^3a is selected from CrC₃-alkyl, in particular ethyl and methyl, more particular methyl.

In certain embodiments, R^3b is selected from Ci-C₄-alkyl.

Inhibition of LPAAT-b seems to require a small, hydrophobic scaffold at the indene-like moiety that is not a hydrogen bond donor such as benzofuran or methyl-indole.

In certain embodiments, X is selected from -O- and -N(Me)-, more particularly X is -O-.

In certain embodiments, the compound of the invention comprises the formula 2

wherein,

R⁶, R⁷ and R⁸ independently from each other are selected from -H, -F, -Cl, methyl and -OR^3b, in particular from -F and methyl, wherein in particular

o one of R⁶, R⁷ and R⁸ is selected from - -F, -Cl, methyl and -OR^3b, in particular from -F and methyl, and the others are H or

o R⁶ and R⁸ are selected independently from each other from -F, -Cl, methyl and -OR^3b, in particular from -F and methyl, more particularly -F, and R⁷ is H, or o R⁶, R⁷ and R⁸ are H,

wherein R¹ and R² have the same meaning as defined above.

In certain embodiments, the compound according to the first aspect of the invention comprises a salt of the compound of formula 1 , in particular a physiological acceptable salts of the compound of formula 1.

The embodiments and effects described for the first aspect of the invention also apply for all further aspects described below.

According to a second aspect, the invention comprises a compound comprising the general formula (1 ) for use in the treatment of a disease,

(1 ).

- wherein R¹ is selected from -I, -Br, -Cl, C-_|-C₆-alkyl, C₃-C_4.cycloalkyl, Ci-C₆-haloalkyl, - O ^3a, -SR^3a, and wherein R^3a is selected from CrC₆-alkyl

R² is selected from Ci-C₆-alkyl, and

R⁴ is selected from -I, -Br, -Cl, -F, Ci-C₆-alkyl, C₃-C₆-cycloalkyl, CrCe-haloalkyl, C₆- aryl, -OR^3b, -SR^3b, and wherein R^3b is selected from Ci-C₆-alkyl, and with m being 0, 1 , 2, 3 or 4, and

X is selected from -S-, -0-, -N(R⁵)-, wherein R⁵ is selected from H, Ci-C₃-alkyl, particularly wherein R⁵ is methyl.

The compound of the second aspect of the invention may be used as a medicament, in particular a medicament for use in the treatment of cancer.

The invention further comprises a method for treating or preventing a disease, comprising administrating a compound according to the second aspect of the invention to a patient in need thereof, in particular in a pharmaceutically effective amount, more particularly wherein said disease is cancer.

The invention also comprises a pharmaceutical composition comprising a compound of formula 1 according to the second aspect of the invention in combination with a pharmaceutical acceptable carrier or diluent. According to a third aspect, the invention comprises a compound comprising the general formula (1 ) for use in the treatment of a cancer,

wherein R¹ is selected from -I, -Br, -Cl, C-_|-C₆-alkyl, C₃-C₄_cycloalkyl, Ci-C₆-haloalkyl, - OR^3a, -SR^3a, and wherein R^3a is selected from Ci-C₆-alkyl

R² is selected from C-_|-C₆-alkyl, and

R⁴ is selected from -I, -Br, -Cl, -F, C-_|-C₆-alkyl, C₃-C_6.cycloalkyl, C-_|-C₆-haloalkyl, C₆- aryl, -OR^3b, -SR^3b, and wherein R^3b is selected from CrC₆-alkyl, and with m being 0, 1 , 2, 3 or 4, and

X is selected from -S-, -0-, -N(R⁵)-, wherein R⁵ is selected from H, Ci-C₃-alkyl, particularly wherein R⁵ is methyl.

According to a fourth aspect, the invention relates to a method for reducing the activity of lysophosphatidic acid acyltransferase b (LPAAT-b) comprising contacting LPAAT-b with a compound or salt thereof, or in combination with a pharmaceutical acceptable carrier or diluent, in an amount effective to reduce the activity of LPAAT-b, wherein the compound comprises the general formula (1 ),

wherein R¹ is selected from -I, -Br, -Cl, C-_|-C₆-alkyl, C₃-C_4.cycloalkyl, Ci-C₆-haloalkyl, - OR^3a, -SR^3a, and wherein R^3a is selected from CrC₆-alkyl

R² is selected from CrC₆-alkyl, and

R⁴ is selected from -I, -Br, -Cl, -F, CrC₆-alkyl, C₃-C₆-cycloalkyl, CrC₆-haloalkyl, C₆- aryl, -OR^3b, -SR^3b, and wherein R^3b is selected from CrC₆-alkyl, and with m being 0, 1 , 2, 3 or 4, and X is selected from -S-, -0-, -N(R⁵)-, wherein R⁵ is selected from H, CrC₃-alkyl, particularly wherein R⁵ is methyl.

The compound as according to the fourth aspect of the invention may be used as an inhibitor, in particular used as an LPAAT-b Inhibitor.

In certain embodiments, LPAAT-b resides in a mammal, particularly in a mammal.

The invention further relates to a method for inhibiting the proliferation of a cell in which the activity of lysophosphatidic acid acyltransferase b (LPAAT-b) is required for the proliferation of the cell comprising contacting the cell with a compound of formula 1 according to the fourth aspect of the invention or salt thereof, or in combination with a pharmaceutical acceptable carrier or diluent, in an amount effective to inhibit the proliferation of the cell, wherein in particular the cell resides in an animal, particularly in a mammal.

Furthermore, the invention relates to a method for treating a cancer in which lysophosphatidic acid acyltransferase b (LPAAT-b) activity is associated comprising administering to an animal in need, a compound of formula 1 according to the fourth aspect of the invention or salt thereof, or in combination with a pharmaceutically acceptable carrier or diluent, in an amount effective to treat the cancer, wherein in particular the animal is a mammal, more particularly a human.

Examples

An angiogenesis inhibitor with potent cytotoxicity

The Triazine 1 (Compound 1 in Table 1 ) disrupted cellular network formation in the low nanomolar range, and induced strong underdevelopment of zebrafishes at 500 nM under conditions where receptor tyrosine kinase inhibitors show an effect on neovascularization in the mM range after 24 h (Figure 3). Activity required at least a methyl substituent on the furan ring and a para-substituent on the aniline ring (Table 1 ).

Table 1 : Activity test of compounds according to the invention on co-cultured human umbilical cord endothelial cells (HUVEC) and pulmonary-derived vascular smooth muscle cells (PA-vSMC). Compounds were initially tested at 1 , 3 and 10 mM. IC₅₀ value was determined when the compound showed an activity at 1 pM. When nothing is specified, R is hydrogen. The co-cultured cells were incubated with the compound solution for 72 h at 5 % C0₂ and 37 °C.

2 Cl Me 0.005

8 F Me 3

LR-C3 Me Me 0.040

LR-C4 OEt Me 0.37

LR-C6 H Me 3

LR-C10 F Me 10

LR-C12 Me Me 10

LR-C14 Me Me 10

LR-C23 CF^ Me 10

LR-C34 did Me

LR-C3S Me Me Me

LR C 7 Cl M M

To further elucidate the structure-activity relationship (SAR) of this compound series, the inventors synthesized additional triazines with this substitution pattern. Activity screening for cytotoxicity on HeLa cells revealed three additional strongly cytotoxic triazines bearing an ethyl substituent on the furan ring and an ethyl (12), methoxy (14) or chloro (16) substituent on the aniline ring (Table 2).

Table 2. Cytotoxicity of triazines on HeLa cells. ^[a]

[a] aHeLa cells (10 000 cells/well) were incubated with various concentrations of compounds for 96 h at 37 °C and 5% C0₂. Viability of HeLa cells was determined after the addition of WST-8 working solution and phenazine ethosulfate. Final absorbance was measured at 450 nm. Results were normalized to the control values.

No. R¹ R² ICso ± SD (nM)

1 Et Me 1 1 1 ± 12

2 Cl Me 252 ± 7 10

3 OMe Me 190 ± 3

4 /Pr Me 814 ± 20

9 Br Me 264 ± 5

10 CF₃ Me 263 ± 6

12 Et Et 51 ± 1

13 Et Bu 780 ± 30 _{1 5}

14 OMe Et 12.4 ± 0.2

16 Cl Et 140 ± 8

17 Br Et 69 ± 13

18 CF₃ Et 110 ± 56 The structural resemblance of hit 1 and analogs to known triazine type kinase inhibitors suggested that angiogenesis inhibition and cytotoxicity might result from inhibition of one or more kinases. To check this hypothesis whole kinome profiling was performed with 1 and analog 14 at 10 mM, which should reveal any possible interaction with kinases. However, both compounds turned out almost completely inactive. The original triazine 1 only gave a weak inhibition signal with JAK1 , PIP5K2C and TYK2. In the case of 14, three kinases gave submicromolar iC₅₀ values, namely JAK1 , EPHB6 and GAK. Given the high intracellular ATP concentration however these values were far too weak to explain the observed nanomolar activity on cells (Table 3). Table 3. Kinome scan with triazines 1 and 14^[a]

No. Kinase % Ctrl at 10 mM K_D (nM)

1 JAK1 (JH2domain-pseudopocket) 4.5 n.d.

thePIP5K2C 16 n.d.

TYK2 (JH2domain-pseudopocket) 31 n.d.

14 JAK1 (pseudokinase) 4.2 380

EPHB6 5.3 860

PIKFYVE 6 3100

GRK7 6 > 60 000

TAK1 7.2 > 60 000

IRAK3 12 3500

GAK 15 490

TYK2 (JH2 domain pseudokinase) 15 1800

[a] The kinome scan was performed for binding of the ATP binding pockets on 442 kinases by DiscoveRx in San Diego. The tested concentration was 10 mM.

The source literature associated with ChEMBL compounds 43, 52 and 57 as LPAAT-b inhibitors reported that these compounds were cytotoxic at levels comparable to the inventors’ triazines. The inventors synthesized a pure sample of 43 following the published procedure, and indeed observed strong cytotoxicity in cells to the reported level. To confirm that LPAAT-b was a target of the inventive triazines, the inventors measured their activity on LPAAT-b directly by expressing this protein in oocytes and measuring its catalytic activity by quantifying the free coenzyme A (CoA-SH) released by catalyzed acyl transfer from palmitoleyl-CoA colorimetrically using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB).The measurement was performed with triazines 1 and 16 and gave iC₅₀ values in the low nanomolar range compatible with their observed effects on cells, and even stronger than the LPAAT-b inhibition measured with the reference inhibitor 43, which was similar to the reported values for this compound (Figure 1 ).

To gain a closer insight into the biological effects of triazines according to the invention, the inventors evaluated cell proliferation of the most potent triazines 1 , 12, 14 and 16 in comparison with the known LPAAT- b inhibitor 43 by subjecting MEF (non-cancerous cells) and SK-OV-3 (ovarian cancer cells), as well as CHO and HEK 293 cells to increasing concentrations of the respective compounds. The expression of LPAAT-b was confirmed on the mRNA level by reverse transcription PCR (Figure 2a) as well as on the protein level by western blot staining (Figure 2b). Proliferation in all cell lines was inhibited at similar levels (Figure 2c, Table 2). The inventors were further interested in comparing the effect of their most effective compound 14 with the known inhibitor 43 on the cell cycle of MEF and SK-OV- 3 cells at concentrations that inhibited growth in the proliferation assay. Strikingly, both compounds triggered a G2 arrest in both cell lines (Figure 2d). Due to the shorter cell cycle duration of MEF cells, the observed effect was more dramatic in this cell line. Both compounds had very similar effects on the cell cycle, suggesting that they engage in a similar mechanism. Considering that the reference compound 43 has been shown to inhibit ERK and AKT pathways in several cancer models, the inventors also observed the effect of the two compounds on the activity of the RAF-MEK-ERK and the PI3’K-AKT pathways by measuring levels of ERK and AKT phosphorylation respectively (Figure 2e). As control compounds, the inventors used PD- 325901 (a potent MEK inhibitor) and GDC-0941 (a pan class I PI3’K inhibitor) to target the RAF-MEK-ERK and the PI3’K-AKT pathways respectively. Whereas the inventors could not observe an effect on ERK phosphorylation at the used doses, AKT phosphorylation was significantly reduced by 25% with by both compounds. These data replicate results from earlier publications using 43, and further solidifies the hypothesis that the inventors’ triazines (e.g. 14) and 43 act on the same pathway. The assignment of LPAAT-b as their target, supported by the biochemical assay discussed above, is also consistent with the results of LPAAT-b deletion by siRNA on cells.

Table 4. IC50 ± SD (nM) values for growth inhibition. ^[al

1 12 14 16 43

CHO 63±9 32±5 23±5 102±32 94±25

HEK 293 25±7 19±5 1 1±1 51±10 37±15

MEF 48±19 20±4 23±3 62±11 42±23

SK-OV-3 79±43 58±18 18± 5 179±139 92±83

[a] CHO, MEF, SK-OV-3 and HEK 293 cells were subjected to increasing doses of 1 , 12, 14, 16 and 43. Dose response was measured by crystal violet nuclei staining, data from Figure 2c.

Further LPAA T-b inhibitors

Table 5 shows the cytotoxicity of further triazines on HeLa cells. HeLa cells (10 000 cells/well) were incubated with various concentrations of compounds for 96 h at 37 °C and 5% C0₂. Viability of HeLa cells was determined after the addition of WST-8 working solution and phenazine ethosulfate. Final absorbance was measured at 450 nm. Results were normalized to the control values. Table 5: Cytotoxicity of triazines with more than two substituents

Compound R¹ R² R⁶ R⁷ R⁸

GG291 OMe Me F H F 0.065

GG287 Br Me F H F 0.120

GG050 Et Me F H H 0.150

GG081 Et Me H H Me 0.150

GG051 Et Me H F H 0.200

GG400 OMe Me H Me H 0.200

GG399 Et Me H Me H 0.210

GG058 Et Me H OBu H 0.500

GG039 Et Me H Cl H 0.500

GG023 Et Me H OMe H 0.625

Materials and methods

All reagents were purchased from commercial sources and were used without further purification. Flash chromatography purifications were performed with silica Gel 60 (Fluka,

0.040-0.063 nm, 230-400 mesh ASTM). Low resolution mass spectra were obtained by electron spray ionization (ESI-MS) in the positive mode on a Thermo Scientific LCQ Fleet.

High resolution mass spectra were obtained by electron spray ionization (HR-ESi-MS) in the positive mode recorded on a Thermo Scientific LTQ Orbitrap XL. 1 H and 13C-NMR spectra were measured on a Bruker Avance 300 spectrometer (at 300 MHz and 75 MHz, respectively) or on a Bruker Avance II 400 spectrometer (at 400 MHz and 101 MHz, respectively). 1 H and 13C chemical shifts are quoted relative to solvent signals, and resonance multiplicities are reported as s (singlet), d (doublet), t (triplet), q (quartet), p (pentet), and m (multiplet); br = broad peak. Compound purities were assessed by analytical reversed phase HPLC (RP-HPLC) at a detection wavelength of 214 nm. The purity of tested compounds was > 87 %, unless otherwise stated. Analytical RP-HPLC was performed on a Dionex Ultimate 3000 RSLC System (DAD-3000 RS Photodiode Array Detector) using a Dionex Acclaim RSLC 120 column (C18, 3.0 x 50 mm, particle size 2.2 pm, 120 A pore size) and a flow rate of 1.2 mL min-1. Data were recorded and processed with Dionex Chromeleon Management System (version 6.8) and Xcalibur (version 2.2, Thermo Scientific). Eluents for analytical RP-HPLC were as follows: (A) milliQ-deionized water containing 0.05 % TFA, and (D) HPLC-grade acetonitrile/milliQ-deionized water (9:1 ) containing 0.05 % TFA. Conditions for analytical RP-HPLC were as follows: in 2.2 min from 100% A to 100% D, then staying on 100% D (method A), in 2.2 min from 50% A/50% D to 100% D, then staying on 100% D

(method B), in 7.5 min from 100% A to 100% D, then staying on 100% D (method C). Melting point were recorded on a BQchi B-545 apparatus and are un corrected.

The general synthesis of the compounds with X being -O- is depicted in scheme 1.

1 - 18 Scheme 1 : Synthesis of triazines 1. Conditions: a) N-cyanoguanidine, HCI, 2-BuOH, 100°C, 24 h. b) CICH₂C0₂Me, NaOMe, MeOH, 25°C, 2 h, then HCI, H₂0, 25°C, 15 h. c) K₂C0₃, DMF, 155°C, 2 h.

Step A (general procedure A):

A mixture of aniline (1 eq) and N-cyanoguanidine (1.1 eq) in HCI (cone.) and i-PrOH or 2- BuOH was stirred at 105 °C for 24 hours. After allowing the mixture to cool to room temperature, the residue was washed with i-PrOH and dried in vacuo to afford the biguanide hydrochloride salts.

Step B

Finely powdered biguanide salt (1 eq) was added to a solution of methyl chloroacetate (1 eq) and sodium methoxide (1.2 eq, 0.5 M) in MeOH at 0 °C. The reaction was stirred for 3 hours at room temperature before the addition of HCI (88 pl_ cone HCI in 250 pl_ H₂0 per mmol). After stirring for 12-18 hours at room temperature, water was added and the precipitate filtered off and dried in vacuo.

Step C ( general procedure C)

A mixture of triazine (1 eq), hydroxyphenone (1 eq) and anhydrous K₂C0₃ in dimethylformamide (1 M) was heated to 155 °C for 3 hours. After cooling down, cold water was added and the precipitate filtered, washed with water and dried in vacuo. Where necessary, purification by silica gel chromatography delivered the benzofuran product as a white solid.

Compounds according to the invention with X being - S- are synthesised according to the general procedure depicted in Scheme 2 (Ar refers to benzothiophene) followed by the general procedure A (Scheme 1 ) and the general procedure D (Scheme 3).

Scheme 2. A solution of carboxylic acid (1 eq) and sulphuric acid (cone. 0.3 mL per mmol acid) in methanol (0.25 M) were heated to 70°C for 5 hours. After removing 2/3 of the solvent, the aqueous phase was washed with ether (3 *) and the combined organic layers washed with water and brine, dried with sodium sulfate and concentrated in vacuo. The methyl ester was isolated by column chromatography when required. O

Scheme 3: A mixture of biguanine (1.3 eq) and methyl ester (1 eq) was heated to 75°C in a sodium methoxide (0.4-0.5 M) solution for 18 hours. The solvent was removed in vacuo and the crude mixture purified by HPLC or precipitation in DMF/H₂0.

Compounds according to the invention with X being - N(R⁵)- are synthesised according to the general procedure A (Scheme 1 ) followed by the general procedure D (Scheme 3), wherein the respective N methyl carboxy indole are purchased or produced according to scheme 2.

6-( 3-Meth yl-2-benzofuranyl) -N²- (4-eth ylphen vD- 1 , 3, 5-triazine-2, 4-diamine ( 1 )

Following general procedure C, using triazine 30 (132 mg,

to 15:85). RP-UPLC: t_R = 2.02 min (method A); Mp: 228-229 °C; ¹H NMR (300 MHz, DMSO- d₆) d 9.49 (1 H, br. s, NHAr). 6-( 3-Methyl-2-benzofuranvi)-N²-(4-isopropylphenvi)-1,3, 5-triazine-2, 4-diamine (4)

Following general procedure C, using triazine 33 (69 mg,

to 15:85). RP-UPLC: t_R = 1 .24 min (method B); Mp: 187-188 °C; ¹ H NMR (300 MHz, DMSO- d₆) d 9.52 (1 H, br. s, NHAr). 6-( 3-Butyl-2-benzofuranyl)-N²-(4-ethylphenyl)-1 , 3.5-triazine-2, 4-diamine (13)

15:85). RP-UPLC: t_R = 2.36 min (method A); Mp: 237-238 °C; ¹H NMR (300 MHz, DMSO-d₆) <5 9.80 (1 H, br. s, NH_Ar). 6-( 3-Eth yl-2-benzofuranyl) -N²- ( 4-trifluorometh ylphen vD- 1 , 3, 5-triazine-2, 4-diamine (18)

Following general procedure C, using triazine 39 (38 mg,

50:50 to 15:85). RP-UPLC: t_R = 5.13 min (method C); Mp: 180-181 °C; ¹H NMR (300 MHz, CDCIa) d 7.79 (2H, d, J = 8.5 Hz, H_aniiine).

6-(4-Fluoro-3-methyl-2-benzofuranyl)-N²-(4-methoxyDhenyl)-1,3, 5-triazine-2, 4-diamine

(GG291)

F ll i l d C i t i i GG030

(100%A - 100%D, 5 min); Mp: 206-207 °C; HRMS calculated for C^H^OaNs: m/z 366.1361, m/z found 366.1363.

6-(4-Fluoro-3-methyl-2-benzofuranyl)-N²-(4-bromoDhenyl)-1,3,5-triazine-2,4-diamine

(GG287)

. _R .

(100%A - 100%D, 5 min); Mp: 239-240 °C; HRMS calculated for Ci₈H₁₄ON₅ ⁷⁹BrF: m/z 414.0360, m/z found 414.0364.

6-(4-Fluoro-3-methyl-2-benzofuranyl)-N²-(4-ethylphenyl)-1, 3, 5-triazine-2, 4-diamine (GG050)

soae as a rown so a er precp a on. - : _R = 1.30 min (50%A/50%D - 100%D, 5 min); Mp: 183-184 °C; HRMS calculated for C20H-19ON5F: m/z 376.1768, m/z found 376.1760. 6-( 3, 7-Dimethyl-2-benzofuranyl)-N²-(4-ethylphenyl)-1 , 3, 5-triazine-2, 4-diamine ( GG081)

F ll i l d C i ti i GG007 66

isolated as a white solid after purifiacation by HPLC (gradient, A/D 50:50 to 100%D in 30 min, t_R = 65% D). RP-UPLC: t_R = 2.24 min (100%A - 100%D, 5 min); Mp: 258-259 °C; HRMS calculated for C₂₁H₂₂ON₅: m/z 360.1819, m/z found 360.1810.

6-(6-Butyloxy-3-methyl-2-benzofuranyl)-N -(4-ethylphenyl)-1,3,5-triazine-2,4-diamine

(GG058)

(gradient, hexane/ethyl acetate, 50:50 to 5:85). RP-UPLC: t_R = 2.47 min (100%A - 100%D, 5 min); Mp: 140 °C (decomp.); HRMS calculated for C₂₄H₂₈O₂N₅: m/z 418.2238, m/z found 418.2240. 6-( 6- Chloro-3-meth yl-2-benzofuran yl) -N²- (4-eth ylphen vD-1 , 3, 5-triazine-2, 4-diamine ( GG039)

t_R = 2.44 min (100%A - 100%D, 5 min); Mp: 216-217 °C; HRMS calculated for C₂₀H₁₉ON₅ ³⁵CI: m/z 380.1273, m/z found 380.1262.

6-(6-Methoxy-3-methyl-2-benzofuranyl)-N²-(4-ethylphenyl)-1 ,3.5-triazine-2, 4-diamine

(GG023)

50:50 to 15:85). RP-UPLC: t_R = 2.23 min (100%A - 100%D, 5 min); Mp: 213-214 °C; HRMS calculated for C₂₁H₂₂O₂N₅: m/z 376.1768, m/z found 376.1760.

EC-vSMC co-culture Angiogenesis Assay

Co-cultures were seeded in 96-well plates with 12Ό00 human umbilical cord endothelial cells (HUVEC) and 50Ό00 pulmonary artery-derived vascular smooth muscle cells (PA-vSMC) per well. Cells were allowed to attach for 3 h (network formation) or 72 h (disintegration of formed network) before compound addition. 100 pl_ of EGM-2 was pipetted to each well of another 96-well plate. 1 pL of compound solution was added and mixed with the medium. These were carefully transferred to the pre-seed ed co-cultures. The co-cultured cells were incubated with the compound solution (200 m l/well) for 72 hours at 5% C0₂ and 37 °C. Imaging was performed with a BD Pathway 855 Bioimager with a 10x objective. Images were acquired as 2x2 montages with a GFP LP515 filter with 0.11 s exposure time. Human Iliac Vein Endothelial Cells (HIVEC) was trans-ducted with a GFP-construct and showed stable expression of GFP. Image analysis was performed with Attovisoin v1.6.2 using the tube formation algorithm.

Cell culture

HeLa cells were obtained from the European Collection of Animal Cell Cultures or the American Type Culture Collection and were cultured according to the supplier's instructions. Cells were seeded in 96-well plates and incubated with inhibitors for 96 h. Cell viability was assessed using Dojindo Cell Counting Kit-8 to measure WST-8 (2-(2-methoxy-4- nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium) conversion.

Cloning of LPAA T into the pMJB08 Expression Vector

Optimized for Eukaryotes DNA from LPAAT-beta (UniProtKB entry: 015120, GeneWiki: AGPAT2) was produced by GeneArt® and was cloned from a carrier construct of GeneArt® into the pMJB08[35] expression vector without tags. The vectors were digested with the restriction enzymes BamHI and Hindlll, and ligated into the pMJB08 vector. The DNA construct was verified by sequencing. [36] Expression of recombinant LPAAT-b using this construct results in a protein without tags. The calculated molecular masses of recombinant LPAAT-b based on the amino acid sequence are -30.9 kDa (full-length).

Protein Expression in Xenopus laevis Oocytes

Ovarian tissue was surgically removed from the frogs and treated with 3 mg/mL of collagenase A (Roche) for 2 h in Modified Barth's Medium (MBM: 88 mM NaCI, 1 mM KCI, 2.4 mM NaHCOa, 0.82 mM MgS0₄, 0.66 mM NaN0₃, 0.75 mM CaCI₂, 10 mM HEPES-NaOH (pH 7.4)) but without CaCI₂ and supplemented with P/S antibiotics (GIBCO™ Penicillin- Steptomycin liquid, Invitrogen). This buffer is isoosmolar relative to the X. laevis plasma (-200 mOsm). Subsequently, defolliculated stage V-VI oocytes were isolated, transferred into plates containing MBM medium supplemented with P/S antibiotics and maintained at 18 °C prior to injection. Oocytes were injected with 40 ng of cDNA-derived cRNA (in-vitro transcription with the m MESSAGE m MACHINE® T7 kit, Ambion) and maintained for 3 days at 18 °C in MBM supplemented with P/S antibiotics. Isolation of egg yolk-depleted LPAAT-b from oocytes

LPAAT-b from oocytes.1 Ό00 oocytes were homogenized in 10 mL of ice-cold 25 mM HEPES-HCI (pH 7.5), 1 mM EDTA, 100 mM NaCI supplemented with the Complete® protease inhibitor cocktail (Roche). To remove cell debris, nuclei and pigments, the homogenate was subjected to a low spin centrifugation (1 ,500 g; 2 min; 4 °C) and the supernatant was saved for enzyme assay.

LPAAT-b enzymatic activity

LPAAT-b activity was measured by formation of PA (200 mM of 18:1-CoA, 200 mM sn-1-18:1 lysoPA, 500 pM DTNB, 25 mM HEPES-HCL, pH 7.5, 1 mM EDTA, 100 mM NaCI and various concentrations of inhibitors) during a 3-min incubation period at room temperature in flat-bootom, MicroWell 96-well polystyrene plates (TPP, Switzerland, Trasadingen). Product formation was monitored by the change in absorbance at 405 nm over 3 min on a Specta Max 250 Microplate Reader from Molecular Devices. IC₅₀ values were calculated using a nonlinear regression of the software GraphPad Prism.

Kinome Scan

Kinase profiling was performed for binding of the ATP binding pockets on 442 kinases by DiscoveRx in San Diego. The tested concentration was 10 pM in DMSO.

Evaluation of iCm

MEF, SK-OV-3 (AKR-225; LuBioscience), CHO and HEK-293 cells were cultivated in Dulbecco’s Modified Eagle Medium (DMEM) (D5796-6X500ML; Sigma) complemented with 10% fetal calf serum (FCS) (A3160801 ; Life Technologies), 2 mM L-Glutamine (25030123; LuBioscience), MEM Non-essential amino acids (NEAA) 1 : 100 (5-13K00-H; Bioconcept), Penicillin 100 U/ml and Streptomycin 0.1 mg/ml (P0781-100ML; Sigma-Aldrich). In order to assure HEK-293 cell adherence to the wells, plates were coated with with Poly-D-lysine hydrobromide from (P6407-5MG ; Sigma-Aldrich). We incubated plates with 50 pL/ well of an 0.1 mg/ mL solution of polylysine diluted in sterile water. Then plates were washed two times with 100 pL of sterile water and allowed to dry for 10 min before seeding the cells. All other cell types were seeded without coating. Cells were seeded in 96-Well plates at a density of 10% in 100 pL medium, treated with decreasing doses of 1 , 12, 14, 16 and 43 and fixed with neutrally buffered 10% formalin solution (HT501128; Sigma-Aldrich) at the time the DMSO (D4540-1 L; Sigma-Aldrich) controls reached a confluency of 100%. Fixation was carried out as follows: Without removing the 100 pL of cell culture medium, 100 pL of the formalin solution was added to each well. Fixation was allowed to continue for 10 min at room temperature (RT). After fixation, the plates were washed 2 times with 100 pL PBS (137 mM NaCI (S9888; Sigma-Aldrich); 2.7 mM KCI (P391 1 ; Sigma-Aldrich); 18 mM KH₂P0₄ (P9791 ; Sigma-Aldrich)_; 100 mM Na₂HP0 (S5136; Sigma-Aldrich)). After washing, cells were stained with crystal violet. To that end, 100 mI_ of a crystal violet solution containing 0.2% crystal violet (C3886; Sigma-Aldrich) and 2% EtOH (G0152; Dr. Grogg Chemie AG) in H₂0. Fixed cells were stained for 10 min at RT. After staining, the plates were washed by plunging in a water bath, until all the surplus ink was washed out. Then, plates were dried overnight. The dried crystal violet in the plates was solubilized with 100 mί 1 % SDS (05030; Sigma-Aldrich) solution by shaking gently for 30 min at RT and the optical density at 500 nm (OD₅₅₀) was measured. Values are displayed as the ratio of the OD₅₅₀ of the treated cells to the OD₅₅₀ of the respective DMSO control.

Western Blots

To evaluate the effect of 43 and 14 on the activity of the PI3’K-AKT and the RAF-MEK-ERK pathways, levels of AKT phosphorylation (pAKT) and ERK phosphorylation (pERK) were measured. To that end, SK-OV-3 cells were seeded at subconfluent densities in complete DMEM (see above) in 10 cm cell culture petri dishes. When cells had grown to 70-80% confluency 2-3 days after seeding, they were starved over night with DMEM containing 0.5% FBS. After the overnight starvation, cells were treated with the corresponding drugs for one hour. Drug concentrations were accordingly: PD-325901 : 100 nM (Abmole Bioscience); GDC-0941 , 1 mM (Abmole Bioscience); 43: 260 nM; 14: 70 nM. Concentrations of 14 and 43 were chosen to be growth inhibitory. For protein isolation, cells were washed twice with ice cold PBS, recovered by scraping and pelleted by centrifugation at 2300 g 5 min at 4 °C. Then, pellets were lysed in RIPA buffer (20 mM TRIS base (T1503; Sigma-Aldrich) pH 8; 150 mM NaCI; 1 % Triton-X100 (T8787; Sigma-Aldrich); 0.1 % SDS; 0.5% deoxycholate Na⁺ (D6750; Sigma-Aldrich)) complemented with Halt™ Inhibitor Cocktail 1 :100 (1 183411 1 ; Pierce, ThermoFischer Scientific). For lysing, pellets were resuspended by pipetting up and down, followed by short vortexing and incubation at 4 °C for 30 min. Lysates were cleared by 30 min of centrifugation at full speed and 4 °C. Lysates were mixed with 5x sample buffer (0.294 M sucrose (84097; Sigma-Aldrich), 2% SDS, 1 mM EDTA (E9884; Sigma-Aldrich), 60 mM TRIS pH 8.8, 0.05% Bromophenol blue (114391 ; Sigma-Aldrich), and 26 mM Dithiothreitol (43819; Sigma-Aldrich)). For denaturation, samples were heated at 95 °C for 5 min. For western blotting, protein extracts were either loaded separately (for quantifications) or pooled (for figure pictures) for each treatment condition. Proteins were run on TGX precast 4-20% gels (456-1096; BioRad, Switzerland), transferred onto Trans-Blot transfer pack nitrocellulose (170-4158; BioRad, Switzerland). Western blots were probed with the following antibodies and concentrations: Primary antibodies: ERK1/2 (1 :5000, Cell Signaling, 9107); pERK1/2 (1 :2000, Cell Signaling, 4370); pan-AKT (1 :2000, Cell Signaling, 2920); pAKT- Ser473 (1 :2000, Cell Signaling, 4060); ACTIN (1 :4000), Sigma, A5316); LPAAT-b (1 :500, Biorbyt, orb5859). Secondary antibodies, Li-Cor Biosciences (Bad Homburg Germany): IRDye 680RD Goat anti-Mouse IgG (H + L) (1 :10O00, 926-68071 ), IRDye 800CW Goat anti- Rabbit IgG (H + L) (1 :10O00, 926-32210), IRDye 680RD Goat anti Rabbit IgG (H+L) (1 :10Ό00 926-68071 ); IRDye 800CW Goat anti mouse IgG (H+L) (1 :10000 926-32210). Blots were scanned using a Li-Cor ODYSSEY Sa fluorescent western blot scanner and quantified with the ODYSSEY Image Studio software. Ratios in the graph are depicted as the ratio of the brightness of the phosphorylated band (pERK; pAKT) band to the total (tERK; pan-AKT) band, normalized to control conditions.

Cell Cycle Analysis

To investigate the effect of the compounds on the cell cycle MEF and SK-OV-3 cells were seeded in 10 cm dishes and left to proliferate for 2, respectively 3 days until they reached a confluency of 80% before the experiment was started. Then Cells were treated with either DMSO, 14 or 43 for 24 h. MEF cells were treated with 260 nM 43 and 200 nM 14, SK-OV-3 cells were treated with 260 nM 43 and 70 nM 14. These doses of drugs were selected to be fully growth inhibitory. After treatment, the supernatant was removed and centrifuged for 5 min at 300 g to pellet detached cells, carefully discard supernatant. Cells on plates were detached with 3 ml trypsin-EDTA 5% (25300054; Thermo Fisher) for 5 min at 37 °C. The detached cells in trypsin were taken up in 7 mL complete DMEM medium and added to the pellet of the detached cells. Cells were centrifuged for 5 min at 300 g at room temperature and resuspended in 10 mL PBS for counting. After counting, cells were pelleted once more to the above conditions and resuspended in PBS for washing. After washing, cells were pelleted for 5 min at 300 g and 4 °C, then resuspended in ice cold PBS at a concentration of 3.33 million cells/mL (or 0.3 mL of PBS per one million cells). For fixation, -20°C cold EtOH is added to the cells drop wise while vortexing, to reach a final concentration of 1 million cells/mL (or 0.7 mL EtOH per one million cells). Cells were then stored at 4 °C overnight, after they were kept at -20 °C before analysis. For analysis, 1 mL of the fixed cells are centrifuged for 10 min at 1000 g and 4 °C. After centrifugation, the supernatant is decanted and the pellet washed with 1 mL of ice cold PBS. The cells were pelleted by centrifugation for 10 min at 1000 g and 4 °C. Then pellets were resuspended in 250 pL ice cold PBS and RNAse A (R4642; Sigma-Aldrich) was added at a final concentration of 0.2-0-5 mg/mL and the solution was incubated for 1 h at 37 °C for RNA digestion. For staining of DNA propidium iodide (P4864; Sigma-Aldrich) was added at a final concentration of 40 pg/mL incubated for 10 min at room temperature in the dark. Samples were scanned with a Becton Dickinson FACScan FACS machine. Numbers are depicted as percentage of total counts of a certain cell cycle state. Quantitative PCR

After reaching a confluency of around 80-90%, cells were washed two times with cold PBS and detached from culture flasks by scraping. For cell pelleting, they were centrifuged at 4 °C for 5 min at 1000 g and pellets were snap frozen and stored at - 80 °C for later RNA isolation. Total RNA extraction was performed using standard a phenol-chloroform protocol. Phenol was substituted by QIAzol Lysis Reagent from Qiagen and the chloroform by chlorophorm- isoamyl alcohol mixture (25668-100ML; Sigma-Aldrich). RNA was precipitated with isopropanol (1096341000; Merck) and the obtained pellet was washed with 75% ethanol (diluted absolute alcohol, 02860-4X2.5L; Sigma-Aldrich). The pure RNA pellet was resuspended in 70 pL of treated water with DEPC (D5758-50ML; Sigma-Aldrich). Reverse transcription was performed in two steps using 500 ng of total RNA. First a 14 pL mix containing, 0.5 pL of Oligo(dT)12-18 Primer (0.5 pg / pL, 18418-012; Invitrogen), 1 pL of dNTPS mix (10 mM solution from set of dATP, dCTP, dGTP, dTTP 25 pmole each, U1420; Promega), 500 ng of total RNA and DE PC-treated water was heated for 5 min at 65 °C. Second step, the inventors added 6 pL mix containing DEPC-treated water, 40 U of reverse transcriptase (Superscript™ II Reverse Transcriptase, 10Ό00 U, 18064-014; Invitrogen), 4 pL of FSB (5x first-strand buffer, supplied with the enzyme, Invitrogen) and 1 pL of DTT (100 mM DTT, supplied with the enzyme, Invitrogen). Tubes were incubated for 1 hour at 42 °C for cDNA synthesis. The reaction was inactivated by incubation at 70 °C for 15 min. Real time qPCR was performed using the GoTaq® qPCR Master Mix (A6002; Promega). A single tube contained 10 pL of GoTaq® qPCR Master Mix, 5 pL of DEPC-treated water, 1 pL of primers mix (10 pM of forward and reverse primers) and 4 pL of cDNA product (diluted 5 times with DEPC-treated water). For control, GAPDH was used. Following primers were used: forward mouse Agpat2 : CT CAAAGT GT GG AT CT ACCCAG , reverse mouse Agpat2: GCACTT GT ACCTT GATT GTT CC (size product: 190 bp), forward mouse Gapdh: T GCCCCCAT GTTT GT GAT G , reverse mouse Gapdh T GT GGT CAT GAGCCCTT CC (size product: 151 bp), forward human AGPAT2: GT GGGCCT CAT CAT GT ACCTC, reverse human AGPAT2: CGATGGGCACGTTCTCCC (product size: 1 18 bp), forward human GAPDH. AAT CCCAT CACCAT CTT CCA, reverse human GAPDH. T GG ACT CCACG ACGT ACT CA (product size: 82 bp). Mouse primers were used on MEF and CHO cell lines and human primers were used on SK-OV-3 and HEK cell lines.

Brief description of the figures

Fig. 1 shows activity of triazines 1 , 16 and 43 on LPAAT-b by quantifying the release of free CoA-SH in presence of DTNB monitored by the change in absorbance at 405 nm over 3 min at room temperature. Fig. 2 (a) shows reverse transcription PCR, demonstrating that all the used cell lines contain RNA transcripts for LPAAT-b. GAPDH was used for control (b) Western blot confirming the expression of LPAAT-b protein in all cell lines used b -ACT IN was used as a loading control (c). CHO, MEF, SK-OV-3 and HEK 293 cells respectively were subjected to increasing doses of triazines 1 , 12, 14, 16 and 43. (d) Effect of 43 and 14 on the cell cycle given as percentage of cells in G1 , S or G2 phases. Doses of drugs were selected to be growth inhibitory. For MEF cells: 43: 260 nM; 14: 200 nM. For SK-OV-3 cells: 43: 260 nM; 14: 70 nM. (e). Effect of 43 and 14 on the activity of the MAPK- pathway (ERK phosphorylation) and the PI3’K-AKT pathway (AKT phosphorylation) of SK-OV-3 cells. The same doses were used as in (d).

Fig. 3 shows aminotriazine 1 as an angiogenesis inhibitor. Zebra fish assay, i.

Control ii. zebrafish treated with Sorafenib for 24 h at 0.5 mM. iii. zebrafish treated with Sorafenib for 24 h at 1 mM. iv. zebrafish treated with Sorafenib for 24 h at 5 pM. v. zebrafish treated with 1 for 24 h at 500 nM.

Claims

Claims

1. A compound comprising the general formula (1 ),

OR^3a, -SR^3a, and wherein R^3a is selected from Ci-C₆-alkyl

R² is selected from C-_|-C₆-alkyl, and

R⁴ is selected from -I, -Br, -Cl, -F, CrC₆-alkyl, C₃-C₆-cycloalkyl, CrC₆-haloalkyl, C₆- aryl, -OR^3b, -SR^3b, and wherein R^3b is selected from CrC₆-alkyl, and with m being 0, 1 , 2, 3 or 4, and

X is selected from -S-, -0-, -N(R⁵)-, wherein R⁵ is selected from H, CrC -alkyl, particularly wherein R⁵ is methyl,

with the proviso that the compound is not 6-(3-Methyl-2-benzofuranyl)-/V²-(4- ethylphenyl)-1 , 3, 5-triazine-2, 4-diamine, 6-(3-Methyl-2-benzofuranyl)-A/²-(4- methylphenyl)-1 , 3, 5-triazine-2, 4-diamine, 6-(3-Methyl-2-benzofuranyl)-/V²-(4- chlorophenyl)-1 , 3, 5-triazine-2, 4-diamine, 6-(3-Methyl-2-benzofuranyl)-/V²-(4- ethoxyphenyl)-1 , 3, 5-triazine-2, 4-diamine.

2. The compound according to claim 1 , wherein R¹ is selected from -Br, -Cl, CrC₃-alkyl, C_3.cycloalkyl, CrC₃-haloalkyl and -OR^3a, in particular -Br, -Cl, methyl, ethyl, iso propyl, -CF₃, and -OR^3a.

3. The compound according to any one of the previous claims, wherein R¹ is selected from Br, -Cl, ethyl, -CF₃ and -OMe, in particular from -Cl, ethyl, and -OMe.

4. The compound according to any one of the previous claims, wherein R² is selected from Ci-C -alkyl.

5. The compound according to any one of the previous claims, wherein R² is selected from ethyl and methyl, in particular ethyl.

6. The compound according to claim 1 , wherein R⁴ is selected from -F, -Br, -Cl, CrC₃- alkyl and -OR^3b, in particular -F, -Cl, methyl and -OR^3b.

7. The compound according to any one of the previous claims, wherein R⁴ is selected from -F and methyl.

8. The compound according to any one of the previous claims, wherein m is selected from 0, 1 or 2.

9. The compound according to any one of the previous claims, wherein R^3a is selected from Ci-C₃-alkyl, in particular ethyl and methyl, more particular methyl.

10. The compound according to any one of the previous claims, wherein R^3b is selected from Ci-C₄-alkyl.

11. The compound according to any one of the previous claims, wherein X is selected from -O- and -N(Me)-, more particularly X is -O-.

12. The compound according to any one of the previous claims, comprising the formula 2

wherein,

R⁶, R⁷ and R⁸ independently from each other are selected from -H, -F, -Cl, methyl and -OR^3b, in particular from -F and methyl, wherein in particular

o one of R⁶, R⁷ and R⁸ is selected from - -F, -Cl, methyl and -OR^3b, in particular from -F and methyl, and the others are H or

o R⁶ and R⁸ are selected independently from each other from -F, -Cl, methyl and -OR^3b, in particular from -F and methyl, more particularly -F, and R⁷ is H, or o R⁶, R⁷ and R⁸ are H,

wherein R¹ and R² have the same meaning as defined above.

13. A compound comprising the general formula (1 ) for use in the treatment of a disease,

wherein R¹ is selected from -I, -Br, -Cl, C-_|-C₆-alkyl, C₃-C_4.cycloalkyl, C-_|-C₆-haloalkyl, - OR^3a, -SR^3a, and wherein R^3a is selected from Ci-C₆-alkyl

R² is selected from CrC₆-alkyl, and

R⁴ is selected from -I, -Br, -Cl, -F, CrC₆-alkyl, C₃-C₆-cycloalkyl, Ci-C₆-haloalkyl, C₆- aryl, -OR^3b, -SR^3b, and wherein R^3b is selected from Ci-C₆-alkyl, and with m being 0, 1 , 2, 3 or 4, and

X is selected from -S-, -0-, -N(R⁵)-, wherein R⁵ is selected from H, Ci-C₃-alkyl, particularly wherein R⁵ is methyl.

14. A compound comprising the general formula (1 ) for use in the treatment of cancer,

wherein R¹ is selected from -I, -Br, -Cl, Ci-C₆-alkyl, C₃-C_4.cycloalkyl, C-_|-C₆-haloalkyl, - OR^3a, -SR^3a, and wherein R^3a is selected from Ci-C₆-alkyl

R² is selected from Ci-C₆-alkyl, and

R⁴ is selected from -I, -Br, -Cl, -F, Ci-C₆-alkyl, C₃-C₆-cycloalkyl, CrC₆-haloalkyl, C₆- aryl, -OR^3b, -SR^3b, and wherein R^3b is selected from Ci-C₆-alkyl, and with m being 0, 1 , 2, 3 or 4, and

X is selected from -S-, -0-, -N(R⁵)-, wherein R⁵ is selected from H, Ci-C₃-alkyl, particularly wherein R⁵ is methyl.

15. A method for reducing the activity of lysophosphatidic acid acyltransferase b (LPAAT- b) comprising contacting LPAAT-b with a compound or salt thereof, or in combination with a pharmaceutical acceptable carrier or diluent, in an amount effective to reduce the activity of LPAAT-b, wherein the compound comprises the general formula (1 ),

wherein R¹ is selected from -I, -Br, -Cl, C-_|-C₆-alkyl, C₃-C_4.cycloalkyl, Ci-C₆-haloalkyl, - OR^3a, -SR^3a, and wherein R^3a is selected from Ci-C₆-alkyl

R² is selected from CrC₆-alkyl, and

R⁴ is selected from -I, -Br, -Cl, -F, CrC₆-alkyl, C₃-C₆-cycloalkyl, Ci-C₆-haloalkyl, C₆- aryl, -OR^3b, -SR^3b, and wherein R^3b is selected from Ci-C₆-alkyl, and with m being 0, 1 , 2, 3 or 4, and

X is selected from -S-, -0-, -N(R⁵)-, wherein R⁵ is selected from H, Ci-C₃-alkyl, particularly wherein R⁵ is methyl.