WO2021167554A1

WO2021167554A1 - Use of synthesized compounds as an inhibitor of ef2-kinase enzyme

Info

Publication number: WO2021167554A1
Application number: PCT/TR2020/050204
Authority: WO
Inventors: Mehmet AY; Bulent Ozpolat; Ferah COMERT ONDER; Tugba TASKIN TOK; Esen Bellur Atici; Bekir Karliga; Hakan KANDEMIR; Ali Cagir; Nurettin Sahiner; Gizem TATAR
Original assignee: Canakkale Onsekiz Mart Universitesi Rektorlugu
Priority date: 2020-02-18
Filing date: 2020-03-16
Publication date: 2021-08-26

Abstract

The invention is related to the use of synthesized compounds, as an inhibitor of the EF2-Kinase enzyme which is active in breast, pancreas, brain, ovarian, lung, skin (melanoma) and blood cancers. The aim of this invention is to make use of synthesized compounds as an inhibitor of the EF2-Kinase enzyme.

Description

USE OF SYNTHESIZED COMPOUNDS AS AN INHIBITOR OF EF2-

KINASE ENZYME

Technical Field of the Invention The invention is related to the use of the compounds known in the art. These could use as inhibitors of EF2-Kinase enzyme that is active in breast, pancreas, brain, ovarian, lung, skin (melanoma) and blood cancers.

Prior Art

Specific and potent inhibitors suitable for the EF2-Kinase enzyme are not commercially available. Although several molecules have been published as inhibitors of EF2-Kinase, these are neither specific nor potent, i.e. they are inhibited by many other molecules and kinases or they do not act against the EF2- Kinase enzyme at low concentrations. Due to this reason, there is room for discovering effective molecules. [1] In the international patent document numbered WO2017201241A1 which is a part of the prior art, the process for preparing (S)-N-(5-((R)-2-(2,5- difluorophenyl)pyrrolidine-l-yl)pyrazolo [1,5-a] pyrimidine-3 -yl) -3- hydroxypyrrolidine- 1 -carboxamide or a salt thereof by reacting phenyl(5-((R)-2- (2,5- difluorophenyl)pyrrolidine-l-yl)-3,3a-dihydropyrazolo[l,5-a]pyrimidin-3- yl)carbamate or a similar derivative with (S)-pyrrolidine-3-ol is mentioned. The necessary processes for the preparation of phenyl (5-((R)-2-(2,5-difluorophenyl) pyrrolidine-l-yl)-3,3a-dihydropyrazo [1,5-a] pyrimidine-3-yl) carbamate are described. In the international patent document numbered W02007079164A2 which is a part of the prior art, the compounds that inhibit protein kinases, the compositions containing the compounds and the methods of treating diseases using the compounds are mentioned. NH125 that is an imidazole derivative has been published as an inhibitor of eEF2K [2], but the subsequent publications have set forth that this molecule does not inhibit eEF2K, and on the contrary that it increases the phosphorylation of eEF2. [3, 4]

A-484954 that is a pyrido-pyrimidine carboxamide derivative has been published as an inhibitor of eEF2K [5], but they exhibit very weak effects on cancer cells, and they have inhibited the cells at very high doses, such as 75 micromolar (pmol/L).

Due to these reasons, the need for the synthesis of novel compounds that inhibit the EF2-Kinase enzyme and that are effective, specific and potent (effective at low concentrations) has risen.

Aim of the invention

The aim of this invention is to make use of synthesized compounds as an inhibitor of the EF2-Kinase enzyme.

Detailed Description of the Invention Small molecules (Preliminary trial compounds 1-5 and 2A, 2B, 2C, 2D, 2K, 2L, 2M, 2N) that can be effective in the treatment of breast and pancreatic cancers by inhibiting EF2K enzyme are designed and synthesized. It has been observed that these synthesized compounds inhibit cell growth in the cells of breast cancer and pancreatic cancer. Synthesizing of preliminary trial compounds:

(1) and 7-hydroxy-4-methyl- (3, starting compound) mixed for 10 hours at 165°C and for

12 hours at room temperature, respectively, in a medium that contains 85% of H₃PO₄, and the reactions were successfully carried out with a yield of 65% and 85%. [6]

was synthesized by boiling under reflux for 10 hours in a medium that contains piperidine and ethanol. The compound of methyl

y y was synthesized with a yield of 71% as a result of mixing at room temperature overnight in the presence of K₂CO₃ and acetone. Compounds of

and 4-chloro-2-

were synthesized, respectively, with yields of 90% and 80% by mixing at room temperature for 24 hours in a medium that contains Et₃N and CH₂CI₂.

In the invention, first of all, the preliminary trial compounds whose general structure is expressed as a compound of the formula (A) are synthesized.

Yield: 50%; melting point: 238-240 °C; melting point literature: 244-245 °C. [7]

Yield: 33%; melting point: 249-252 °C; melting point literature: 246-249 °C.

Yield: 71%; melting point: 153-155 °C.

Yield: 90%; melting point: 103-105 °C; melting point literature: 103-105 °C. [10]

Yield: 80%; melting point: 163-165 °C.

Compounds of 3-acetylcoumarin and coumarinyl chalcone derivatives (2 A, 2B, 2C, 2D, 2K) were synthesized by Knoevenagel condensation reaction.

Scheme 1. General reaction scheme of the derivative compounds (2A, 2B, 2C, 2D, 2K)

General Method for 2A, 2D, 2K:

To the mixture of 3-acetylcoumarin (1 equivalent) and substituted benzaldehyde (1.96 equivalent) in ethanol, piperidine (5-10 drops) was added. The mixture was boiled under reflux. The precipitated solids were collected by filtration. They were washed with ethanol. [11, 12] They were purified by column chromatography.

2B and 2C; were synthesized by means of the condensation method in which n- butanol is used instead of ethanol as solvent. The compound of formula (A) was used in the synthesis of 2A, 2B, 2C, 2D, 2K, 2L, 2M, 2N compounds of the invention.

Yield: 90%; melting point: 98-100 °C; melting point literature: 100 °C. [14]

Yield: 45%; melting point: > 250 °C. 3094, 3052, 3030, 1715,

1666, 1608-1491, 1317. ¹³C-NMR (75 MHz, DMSO): 187.87, 159.00,

155.09, 147.88, 142.43, 139.05, 134.98, 131.11, 129.80, 127.86, 126.50, 126.44,

125.60, 118.93, 116.02.

2D. 3-((E)-3-(2-Hydroxyphenyl)acryloyl)-2H-chromene-2-one.

Yield: 75%; melting point: 278-280 °C; melting point literature: [17]. 281-283 °C.

Yield: 70%; melting point: > 250 °C melting point, literature: 245.5-245.7 °C [18].

7.63 (s, 1H), 7.59-7.56 (d, 1H), 7.46-7.44 (m, 2H), 7.41 (d, 1H), 7.39 (s, 1H), 7.31-7.30 (d, 1H), 7.29-7.28 (d, 1H), 3.78 (s, 3H).

Carboxamide derivatives

The aliphatic amine derivatives comprising various functional groups were used in the synthesis of derivative compounds in the 3 -position of the coumarin ring.

The exchange reactions from ester to amide groups were successfully carried out between the compound of 3-ethoxycarbonyl coumarin [19] whose synthesis was carried out with a yield of 90% by Knoevenagel condensation, and commercial amines including l-(2-aminoethyl)pyrrolidine, l-(2-aminoethyl)piperidine, 4-(2- aminoethyl)morpholine and l-(2-aminoethyl)piperazine. [20] (Scheme 2)

Scheme 2. General reaction scheme of the derivative compounds 2L, 2M, 2N

General method: 3-Ethoxycarbonyl coumarin derivative (1 equivalent) was dissolved in ethanol, aliphatic amine derivatives (1.1 equivalent) were added and the mixture was boiled under reflux for 24 hours. The solids that are formed were collected by filtration. [21]

They were purified from the silica column and/or recrystallized from ethanol.

Yield: 61%; melting point: 127-130 °C.

3323, 3108, 3045, 2968- 2749, 1693, 1702, 1655, 1610.

NH), 8.89 (s, 1H), 7.69-7.34 (Ar-H), 3.60 (d, 2H), 2.71 (t, 2H), 2.57 (t, 4H), 1,80 (m, 4H).

161.72, 154.09, 148.08, 138.63, 134.46, 129.85, 122.16, 119.09, 116.55, 106.81, 95.97, 54.72, 54.34, 23.54. LC- MS spectrum LC-MS (ESI) [M+l] = 287.

Yield: 41%; melting point: 125-128 °C. FT-IR

3336, 3046, 2951-2678, 1698, 1654, 1600, 1568.

R (400 MHz, CDCb) d (ppm) 9.12 (s, -NH), 8.87 (s, 1H), 7.67-7.33 (Ar-H), 3.73 (t, 2H), 3.55 (d, 2H), 2.54 (t, 2H), 2.43 (t, 1H),

1.60 (m, 1H). ¹³C-NMR (100 MHz, CDCb) d (ppm) 161.36, 154.49, 148.40, 134.44, 129.57, 125.01, 119.14, 116.98, 67.11, 56.81, 53.62, 36.84.

Yield: 55%; melting point: 138-141 °C. FT-IR 3324, 3040, 2969-2744,

1742, 1694, 1653, 1605-1541. (400 MHz, CDC1₃)

9.14 (s, -

NH), 8.68 (t, 1H), 7.69-7.34 (Ar-H), 3,73 (t, 2H), 3,57 (d, 2H), 2.59 (t, 2H), 2.51 (t, 2H). ¹³C-NMR (100 MHz, 161.36, 154.49, 148.40, 134.44,

129.57, 125.01, 119.14, 116.98, 67.11, 56.81, 53.62, 36.84. LC-MS spectrum LC- MS (ESI) [M+1] = 303.

The compound 2 and the derivatives thereof (2A, 2B, 2C, 2D, 2K, 2L, 2M, 2N) have been determined in vitro at expected sub-micromolar (less than 1 micromolar) concentrations (for example 0.1 micromolar or 100 nanomolar doses) in the drugs that are intended to be sent to the clinic, they inhibit EF2-kinase above 90% in breast cancer cells (MDA-MB-231) and pancreatic cancer (PANC1) cells which both are highly aggressive. The compound 2 was subjected to pre clinic studies in in vivo animal tests and it has been observed that it inhibits EF2- kinase when it is applied by systemic intravenous injection to tail veins of mice. In this section of computational chemistry and molecular modeling, first of all, the quantum identifier parameters (energy of formation, dipole

moment, hardness, chemical potential, and electrophilicity index.... etc.) for each intended compound on the basis of the method of density functional theory (DFT) at atomic and electronic levels under the quantum mechanics were directly and indirectly calculated, the correlation of structure and of activity were studied quantitatively. In this section of the conducted study, the determination of desired and undesired characteristics and the reasons behind them and how to eliminate them or how to increase the activity can be designated electrochemically.

With this stage, it has been assured that the pre-experimental stages are conducted more deliberately and rationally. Nowadays, there are great numbers of quantum chemical identifiers that identify lipophilic, electronic and steric effects of the molecule involved in numerous studies. [22] Although there are numerous quantum chemical identifiers, the parameters used in this study in the most basic and the most common biochemical activity and the physical definitions thereof have been defined and used in our previous studies [23, 24] Finding the candidate compounds for clinical trials that are able to be used in cancer treatment by improving small molecules that inhibit the EF2K enzyme which is active in human cancers, will provide new specific molecules to very expensive cancer drugs.

Since it stops the tumor from growing, the inhibition of the EF2K enzyme in cancer cells is effective, besides breast and pancreas cancers, also in ovarian cancer which is lethal and incurable, brain tumor, melanoma, lung and prostate cancers, and some blood cancers.

Since the EF2K enzyme is, in other species, genetically similar to the one in humans (homology), it probably plays an important role in animal tumors and it is possible to be applied to the veterinary field for the treatment of pets (dogs, cats).

It is also possible to use the EF2K inhibitor described above, in heart diseases (atherosclerosis), depression and brain degenerative diseases such as Alzheimer's which is the disease of our era, all of which are caused by the increased activity of EF2K. The effects of the discovered compounds against other types of cancer and/or other diseases can be investigated and the area of use and of treatment thereof can be enlarged. REFERENCES

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Claims

1. The invention is related to the use of a compound having the general structure of the formula (A) as an inhibitor of EF2 Kinase (EF2K),

characterized in that;

R is selected from any one of the formulas (I), (II), (III), (IV), (V), (VI), (VII), (VIII) or (IX)

and Rl, R2, R3, R4, and R5 are H.

2. The invention is related to the use of a compound having the general structure of the formula (A) as an inhibitor of EF2 Kinase (EF2K),

characterized in that R is formula (X),

Rl, Rl, R2, R4, and R5 are H.

3. The invention is related to the use of a compound having the general structure of formula (A) as an inhibitor of EF2 Kinase (EF2K),

characterized in that; R4 is selected from any one of the formulas (XI), (XII) or (XIII)

R1 is -CH3 and

R, R2, R3, and R5 are H.