Alkoxyamino-substituted plastoquinone derivatives with anticancer activity.

Abstract

The invention relates specifically to the synthesis of plastoquinone derivatives, namely 5-(4-methoxyphenylamino)-2,3-dimethyl-1,4-benzoquinone (C1), 5-(3-ethoxyphenylamino)-2,3-dimethyl-1,4-benzoquinone (C2) and 5-(3,4-dimethoxyphenylamino)-2,3-dimethyl-1,4-benzoquinone (C3), for use as anticancer agents in the treatment of leukemia, as well as compositions containing these compounds and their use as anticancer / biological agents and other commercial applications.

Description

TARIFF Alkoxyamino-substituted plastoquinone derivatives with anticancer activity. Technical Area The invention relates to the synthesis of alkoxy-amino-substituted plastoquinone derivatives with anticancer activity for use in the treatment of leukemia. The invention specifically relates to methods for synthesizing plastoquinone derivatives, namely 5-(4-methoxyphenylamino)-2,3-dimethyl-1,4-benzoquinone, 5-(3-ethoxyphenylamino)-2,3-dimethyl-1,4-benzoquinone and 5-(3,4-dimethoxyphenylamino)-2,3-dimethyl-1,4-benzoquinone, for use as anticancer agents in the treatment of leukemia, and to compositions containing these compounds, as well as their use as anticancer / biological agents and other commercial applications. State of the Art A study conducted by the Turkish Statistical Institute in 2015 showed that in Türkiye, in 2014 and In 2016, it was determined that cancer-related deaths accounted for 20% of total deaths. This shows that one in five deaths is due to cancer (1). Leukemia is particularly important among cancer types because of the age group it affects. This type of cancer, which occurs most often in children under 15, is also frequently seen in adults aged 55. In a study conducted by the Turkish Ministry of Health in 2015, leukemia ranked first among cancers seen in children under 15 in Türkiye with a rate of 31.5% (2). Acute leukemias are subdivided into acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia (AML); chronic leukemias are subdivided into chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML) (3). In a study conducted in recent years, it was observed that ALL occurs approximately five times more often than AML and accounts for 78% of leukemias diagnosed in children (4).Studies published in the literature show that molecules with quinone skeletons exhibit activity against many cancer cells (5, 6, 7). For this reason, the design and synthesis of new quinone molecules with anticancer activity are becoming increasingly important. It is known from studies in the literature that highly active anticancer drugs such as adriamycin, daunorubicin, carminomsin, rubidazone, mitomycin C, streptonigrin, and lapacol contain quinone skeletons and act as free radical carriers by interacting with mammalian microsomes. Many studies investigating the mechanisms of action of these agents, which have mutagenic and carcinogenic effects in addition to their anticancer properties, have revealed that these structures are effective in DNA and RNA replication or in mitochondrial oxidative mechanisms.In this context, studies are being conducted on the mechanism of action of anthracycline antibiotics containing N-heterocyclic quinone and naphthoquinone anticancer agents in the current technique, and it is suggested that quinone anticancer agents act as pharmacological and site-specific free radicals by monitoring their intracellular movement (8). In a published article, novel thymoquinone derivatives with added gallate and fluorogallate pharmacophores were synthesized and characterized, and their effects against pancreatic cancer cell lines were tested. The synthesized molecules showed superior antiproliferative activity and excellent chemical selectivity against pancreatic cancer cells intracellularly (9). In another study conducted by our team in 2017, a number of novel alkoxy group-containing arylamine-sulfanyl naphthoquinone compounds were synthesized, and two of these molecules were selected by the National Cancer Institute (NCI) and tested against 60 human tumor cell lines from 9 neoplastic diseases. It was found that one of these molecules inhibited the growth of two colon cancer cells known as HCT116 and HCT15, while the other showed activity against leukemia cell lines such as K-562, MOLT-4, and SR.It has been determined that these compounds have a significant selectivity among blood cells because they do not affect peripheral blood mononuclear cells (PBMC) (10). Today, the fact that plastoquinone is a component of chloroplasts and plays a crucial role as an electron carrier in photosynthesis, the most vital metabolic process in plants, makes the synthesis of new plastoquinone analogs and the investigation of the biological properties of these molecules a popular topic. This compound is a molecule containing a 1,4-benzoquinone skeleton with two methyl groups at positions 2 and 3. In the current state of the technique, the antitumor activity of a plastoquinone derivative called SkQ was investigated and it was found that it inhibits the growth of human colon cancer HCT116 (11). In another study, a number of SkQ derivatives were synthesized and their antitumor activity was investigated and it was seen that they inhibited the growth of some tumor cells (12). Although there are studies in the literature on compounds with a quinone skeleton and their areas of use, derivatives that can show high activity against cancer cells and at the same time not affect normal peripheral blood mononuclear cells are few in number. Application EP1716119B1 provides for benzoquinone analogs containing ansamycins and their use in modulating and treating disorders associated with hyperproliferation such as cancer. The present invention provides analogs of benzoquinones containing ansamycins in which benzoquinone is reduced to a hydroquinone, trapped by a suitable acid reaction, preferably with acids that increase the stability and solubility of the resulting (17)-ammonium hydroquinone ansamycin analog in air. The application directly involves the use of plastoquinone derivatives as anticancer agents and the compounds in the invention also show high activity against leukemia. Consequently, due to the aforementioned drawbacks and shortcomings, the need for innovation in the relevant technical field has arisen. Purpose of the Invention The present invention relates to alkoxyamino-substituted plastoquinone derivatives with anticancer activity, which meet the aforementioned requirements and offer some additional advantages. The primary aim of this invention is to synthesize 5(4-methoxyphenylamino)-2,3-dimethyl-1,4-benzoquinone, 5-(3-ethoxyphenylamino)-2,3-dimethyl-1,4benzoquinone and 5-(3,4-dimethoxyphenylamino)-2,3-dimethyl-1,4-benzoquinone compounds with anticancer activity for use in the treatment of leukemia. The aim of this invention is to develop novel alkoxyamino-substituted plastoquinone derivatives with potential for use in biological applications and industrial settings. The aim of this invention is to synthesize plastoquinone derivatives that contain substituents such as aryl, amine, and alkoxy groups, known as activity-enhancing groups, in addition to the benzoquinone backbone in their structure. The aim of this invention is to synthesize plastoquinone derivatives that may exhibit high activity against three cancer types: K562 (Chronic Myeloid Leukemia), jurkat, and MT-2 (other leukemias). One aim of the invention is to explore the possibility of using the synthesized plastoquinone derivatives as a raw material for original anticancer drugs in the treatment of leukemia. To fulfill the purposes described above, the invention is a method for obtaining alkoxy-amino-substituted plastoquinone derivatives, including the synthesis of 2,3-dimethyl-1,4-benzoquinone from 2,3-dimethylbenzene-1,4-diol, for use as an anticancer agent in the treatment of leukemia; its characteristic feature is: The process involves the following steps: • mixing 2,3-dimethyl-1,4-benzoquinone and substituted aniline in absolute ethanol and heating, • cooling the resulting mixture to room temperature and evaporating the solvent, • extracting the resulting residue with dichloromethane and washing the organic phase with water, • drying, filtering, and concentrating the collected organic phase with calcium chloride, • purifying the compounds by column chromatography with suitable solvents until pure compounds are obtained. To fulfill the purposes described above, the invention is a method for obtaining an alkoxy-amino-substituted plastoquinone derivative for use as an anticancer agent in the treatment of leukemia, and its characteristic feature is; The process involves the following steps: • suspension of 2,3-dimethylhydroquinone and substituted aniline in methanol, • addition of aqueous solution of sodium iodate to the resulting mixture and stirring at room temperature, • evaporation of the solvent and extraction of the resulting residue with dichloromethane, • washing of the resulting organic phase with water, • drying and filtering of the organic solution with anhydrous calcium chloride and removal of the solvent under vacuum, • purification by column chromatography with suitable solvents until pure compounds are obtained. The structural and characteristic features and all the advantages of the invention are more clearly understood thanks to the detailed description given below, and therefore the evaluation should be made taking this detailed description into account. Detailed Description of the Invention The invention relates to the synthesis of plastoquinone derivatives, namely 5-(4-methoxyphenylamino)-2,3-dimethyl-1,4-benzoquinone (C1), 5-(3-ethoxyphenylamino)-2,3-dimethyl1,4-benzoquinone (C2) and 5-(3,4-dimethoxyphenylamino)-2,3-dimethyl-1,4-benzoquinone (C3), for use as anticancer agents in the treatment of leukemia. C1 C2 C3 In the synthesis of the plastoquinone derivatives subject to the invention, 2,3-dimethylbenzene-1,4-diol (A) and 2,3-dimethyl-1,4-benzoquinone (B) compounds are used as starting materials. 2,3-dimethylbenzene-1,4-diol (A) compound is purchased commercially, and 2,3-dimethyl-1,4-benzoquinone (B) compound is synthesized from 2,3-dimethylbenzene-1,4-diol (A) compound (13). Synthesis method of AJkoxy amino-substituted plastoquinone derivatives (C1, C2, C3); OH 0 <BrO., H.SO HjC^JL $ul 0 R- JL JIH A asotuye anilinler - jr vp :'T ~ HsC^f^ OH A Xdk.S-C ’ ^-l^Atoı- 0 B ûrefluks.--:: *at 0 R; C 1-3 __________________________________________________________________________________________________________________________________________________________________________________________________________ Substituted anilines. Na₂O₃ -1.0. Room conditions. 1-2 hours. Substitution Groups Ri r2 r3 R4 C1 HH OCH3 H C2 H OCH2CH3 HH C3 H OCH3 OCH3 H Synthesis of compounds C1 and C2; appropriate amount of substituted anilines (4.40 mmol, 1.2 equiv.) and 2,3-dimethyl-1,4-benzoquinone (B) (0.500 g, 3.67 mmol) is refluxed in absolute ethanol (25 mL) for 4–12 hours, stirring until the quinone compound is depleted. The reaction is observed under UV light until the spots of the starting materials disappear. The reaction mixture is cooled to room temperature. After evaporation of the solvent, the residue is dissolved in dichloromethane (3 x 50 mL) and the solution is washed successively with water (3 x 30 mL). The organic phase is collected, dried with calcium chloride, filtered, and concentrated. It is purified by column chromatography until pure compounds are obtained. In the method described in this invention, 4-methoxyaniline, as an aniline derivative, is used to obtain compound C1, and 3-ethoxyaniline is used to obtain compound C2. Synthesis of compound C3: In a round-bottomed flask, substituted aniline (4.00 mmol, 2 equiv.) and 2,3-dimethylhydroquinone (A) (0.276 g, 2.00 mmol) are suspended in methanol (12 mL). Sodium iodate (6.00 mmol, 3 equiv.) in water (12 mL) is added to this mixture and stirred at room temperature for 10-24 hours. The reaction is observed under UV light until the stains of the starting materials disappear. After evaporation of the solvent, the residue is extracted with dichloromethane (3 x 50 mL) and the combined organic phases are washed with water (3 x 50 mL). Finally, the organic solution is dried with anhydrous calcium chloride, filtered, and the solvent is evaporated under vacuum. The residue is purified by column chromatography on silica gel until pure compounds are obtained. In the method described in this invention, 3,4-dimethoxyaniline is used as an aniline derivative to obtain compound C3. Plastoquinone compounds with anticancer activity were synthesized according to the method in the literature (14, 15). The accuracy of the structures of the synthesized compounds was proven by FTIR, 1H NMR, 13C NMR and MS spectra. The colors and physical properties of the obtained compounds are given in Table T. Table-1: Physical properties of compounds C1, C2 and C3 obtained by the method described in this invention. Color Melting point (°C) Yield (%) C1 Purple 103-104 28 C2 Dark red 99-100 6 C3 Purple 151-153 62 Anticancer tests of alkoxyamino-substituted plastoquinone compounds (C1, C2, and C3); The in vitro anticancer activities of the synthesized (C1), (C2), and (C3) molecules were tested against three cancer cell lines, K 562 (Chronic Myeloid Leukemia), Jurkat, and MT-2 (other leukemias), using the MTT method. The effect of C1 and C3 against normal peripheral blood mononuclear cells (PBMC) was also observed. Normal peripheral blood mononuclear cells (PBMCs) are isolated from healthy blood using the Ficoll-Hypaque gradient method and incubated in RPMI 1640 medium containing 10% fetal bovine serum. The medium is supplemented with 89 pg / mL Streptomycin and incubated at 37 °C in an atmosphere containing 95% air and 5% CO2. Growing cells are transferred to 96-well cell culture dishes at a concentration of 6 x 10⁵ cells / mL and incubated for 24 hours, after which the C1, C2, and C3 compounds to be tested are added. Stock solutions of these compounds (in the range of 3-30 mM) are prepared in dimethyl sulfoxide (DMSO) and added to fresh culture medium. The final concentration of DMSO in the culture medium is 1%. The cellular reduction level of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is measured by making minor modifications, as previously described in the literature. The C1, C2, and C3 compounds to be tested are incubated with the cells for 24 hours at a final concentration of 3-300 μM. At the end of this period, MTT is added to the cells in the culture at a final concentration of 0.275 mg / mL and incubated for another 4 hours at 37 °C. The medium is removed, and formazan crystals are dissolved by adding 100 gL of DMSO to each cell and then measured using a microtiter plate spectrophotometer at 630 nm absorbance. The anticancer activities of the synthesized molecules (C1, C2, C3) mentioned in the invention are being investigated at a concentration of 10 μM. The synthesized 5-(4-methoxyphenylamino)-2,3-dimethyl-1,4-benzoquinone (C1), 5-(3-ethoxyphenylamino)-2,3-dimethyl-1,4-benzoquinone (C2) and 5-(3,4-dimethoxyphenylamino)- were selected as prototypes. IC50 values ​​were calculated by investigating the in vitro activities of 2,3-dimethyl-1,4-benzoquinone (C3) molecules at four different concentrations (100 μM, 30 μM, 10 μM, and 3 μM) against three different human cancer cell lines. Tests revealed that when growth percentage values ​​were examined, the cancer cell types most susceptible to plastoquinone derivatives C1, C2, and C3 were K562 and Jurkat cancer cells. Of these, C1 and C3 were found to be the most effective derivatives, particularly against the K562 cancer cell line, with IC50 values ​​of 9.66 and 8.91 μM, respectively. Furthermore, compound C2 was found to be more effective against the Jurkat cancer cell line than the reference compound, Imatinib, with an IC50 value of 8.95 μM. In summary, the IC50 values ​​of C1 and C3 against K562 cancer cells and C2 against Jurkat cancer cells were <10 μM. As the results show, at concentrations below 10 μM, these three compounds killed 50% of the specified cancer cells. Table 2 presents the in vitro antibacterial activity results of the C1-C3 molecules mentioned in the invention. Table 2: In vitro antibacterial activity results of C1-C3 molecules mentioned in the invention. Substitution Groups Cell Type (IC50, μM)3 R1 R2 R3 R4 K562b Jurkatb MT-2b C1 HH och3 H 9.66 12.31 22.75 11.93 53.96 13.81 C2 H OCH2CH3 HH 20.43 13.78 8.95 10.87 53.07 14.64 C3 H OCH3 och3 H 8.91 11.26 14.47 11.35 35.79 10.89 lmatinibc 7.47 12.22 9.49 12.46 22.09 11.76 According to the results, for these three compounds to be considered as anticancer drug candidates against the aforementioned cancer types, they must not kill normal cells. Therefore, the activities of these compounds against normal peripheral blood mononuclear cells (PBMCs) at concentrations of 10 μM were investigated at Kumamoto University in Japan, where collaborative studies were conducted. The cytotoxic effects of compounds C1, C2, and C3 against normal peripheral blood mononuclear cells (PBMCs) are shown in Table 3. These results showed that C1 and C3 were selective against K562 cancer cells, and C2 was selective against NCI-H23 Jurkat cancer cells. In other words, they inhibited cancer cells while showing no negative effect on healthy cells. Therefore, these compounds can be considered as potential anticancer agents. Table 3: Cytotoxicity and Selectivity Index (SI) of C1-C3 molecules mentioned in the invention. Substitution Groups Cell Type (IC50, μM) sib R1 R2 R3 R4 K562a PBMCa C1 HH och3 Η 9.66 12.31 72.68 16.51 7.52 C3 H OCH3 och3 Η 8.91 11.26 69.35 17.12 778 lmatinibc 7.47 12.22 39.81 14.38 5.33 In conclusion, in this invention, the compounds 5-(4-methoxyphenylamino)-2,3-dimethyl-1,4-benzoquinone (C1), 5-(3-ethoxyphenylamino)-2,3-dimethyl-1,4-benzoquinone (C2), and 5-(3,4-dimethoxyphenylamino)-2,3-dimethyl1,4-benzoquinone (C3) were synthesized and evaluated for their anticancer activity. All three compounds selected as prototypes showed significant inhibition against specific cancer cells, and since their SI values ​​were higher than those of the reference compound Imatinib, they can be identified as suitable compounds for the development of anticancer drugs.

Claims

CLAIMS 1. The invention relates to the synthesis of 2,3-dimethyl-1,4-benzoquinone (B) from 2,3-dimethylbenzene-1,4-diol (A) and the production of alkoxy-amino-substituted plastoquinone derivatives for use as an anticancer agent in the treatment of leukemia, including the following process steps: a. Mixing 2,3-dimethyl-1,4-benzoquinone (B) and substituted aniline in absolute ethanol and heating, b. Cooling the resulting mixture to room temperature and evaporating the solvent, c. Extracting the resulting residue with dichloromethane and washing the organic phase with water, d. Drying, filtering and concentrating the accumulated organic phase with calcium chloride, e. Purification by column chromatography with suitable solvents until pure compounds are obtained.

2. This is the method for obtaining the plastoquinone derivative according to claim 1, characterized by the fact that the substituted aniline mentioned in step a is 4-methoxyaniline to obtain compound C1 and 3-ethoxyaniline to obtain compound C2.

3. Compound C1 obtained by the method mentioned in either claim 1-2 is 5-(4-methoxyphenylamino)-2,3-dimethyl-1,4-benzoquinone.

4. Compound C2 obtained by the method mentioned in either claim 1-2 is 5-(3-ethoxyphenylamino)-2,3-dimethyl-1,4-benzoquinone.

5. The C1 compound mentioned in claim 3 is selective against K562 cancer cells.

6. The C2 compound mentioned in claim 4 is selective against Jurkat cancer cells.

7. The invention is a method for obtaining an alkoxy-amino-substituted plastoquinone derivative for use as an anticancer agent in the treatment of leukemia, and its characteristic features include the following process steps: a) suspension of 2,3-dimethylhydroquinone (A) and substituted aniline in methanol, b) addition of an aqueous solution of sodium iodate to the resulting mixture and stirring at room temperature, c) evaporation of the solvent and extraction of the resulting residue with dichloromethane, d) washing of the resulting organic phase with water, e) drying of the organic solution with anhydrous calcium chloride and filtering under vacuum, f) purification by column chromatography with suitable solvents until pure compounds are obtained.

8. This is the method for obtaining the plastoquinone derivative in accordance with claim 7, and its characteristic is that the substituted aniline mentioned in step a is converted to 3,4-dimethoxyaniline to obtain compound C3.

9. Compound C3 obtained by the method mentioned in either claim 7-8 is 5-(3,4-dimethoxyphenylamino)-2,3-dimethyl-1,4-benzoquinone.

10. Claim 9 states that compound C3 is selective against K562 cancer cells.