jurnal internasional Synthesis of a New Pyridine Pharmaceutical Intermediate inPotential Anti-cancer of HDACI

2016 3rd International Conference on Engineering Technology and Application (ICETA 2016)

ISBN: 978-1-60595-383-0

Synthesis of a New Pyridine Pharmaceutical Intermediate inPotential Anti-cancer of HDACI

Xiaopan Zhang & Zhiyi Yao*

College of Chemical and Environmental Engineering, Shanghai Institute of Technology, Shanghai, China

ABSTRACT: In order to obtain better anti-cancer activity of HDACI, new HDCIs analog and a pyridine pharmaceutical intermediate were designed by comparing the molecule of FK228 and Largazole and introducing the pyridine intermediate to the cyclic tetrapeptide pharmacophore. The synthetic route of HDCIs analog and the pyridine intermediate were designed. The HDCIs analog and the pyridine intermediate were all synthesized, and the optimum conditions of synthesizing the pyridine intermediate were got by using parallel test. The pyridine intermediate can be used to manufacture new potential HDACIs by forming new structure of the polypeptide. Due to the special structure of the pyridine intermediate, it can also be widely used in other medical field. Through our work we have designed synthetic routes of the pyridine intermediate and discussed the optimum conditions. And we provide a new synthesis method of cyclopeptide HDCIs.

Keywords: FK228 analogue; HDACIs; pyridine; tetrapeptide

1 INTRODUCTION

Pyridine compound is a high value-added fine chemical product. Its chemically active can participate in a variety of chemical reactions. Pyridine compounds chlorinated, fluoride and ammonia oxidation can be prepared for a variety of intermediates in the pharmaceutical synthesis and fine chemical industries that have been widely used. Pyridine compounds are currently used in medicine, pesticides, spices, feed additives, adhesives and synthetic materials. Such compounds are being used to develop the most widely used range of fine chemical products global manufactures pyridine compounds of $50 billion in annual sales. In the field of medicine, pyridine can be used to produce cephalexin, prednisone dexamethasone acetate, sulfa sulfuric acid piperazine, hydrocortisone, idoxuridine, progesterone, norfloxacin, vitamin A, D2 or D3, the fourth generation of cephalosporins, and more than 40 kinds of synthetic materials of commonly used medicines.

For a long time, cancer has been considered to be the result of a wide variety of genetic and genomic alterations, such as amplifications, translocations, deletions and point mutations [1]. But recent research showed cancer development is not only related to the genetic changes but also involves epigenetic changes.
Epigenetics is concerned with the inheritance of information based on gene-expression levels. The main epigenetic modifications in humans are DNA methylation and posttranslational histone modifications.

2 MATERIALS AND METHODS

Histones are small basic proteins that are rich in amino acids, lysine and arginine [2]. Histone acetyltransferase (HAT) and histone deacetylase (HDAC) can make histone N-terminal amino acid residue be acetylated or deacetylated in vivo, both enzymes work together to determine histone acetylation levels, and they also regulate expression of gene and some other cellular processes, and studies have shown that acetylation and deacetylation process and the occurrence of cancer are closely linked. Therefore, histone deacetylase (HDACs) are targets for anticancer drugs. Histone deacetylase inhibitor (HDACi) has become a kind of hot areas of research. Histone deacetylase inhibitor can improve the level of p21 and other genes expression by increasing cell level of histone acetylation. The process of tumor cells proliferation has showed a very good inhibitory effect, and the inhibitor can in-duce tumor cell differentiation and/or the occurrence of apoptosis, so it has the effect of inhibiting tumor [5,6]. Romidepsin (Istodax), a cyclic peptide, has been recently approved as an HDAC inhibitor against CTCL, indicating that cyclic peptides are pharmacophores of interest in this field. Histone deacetylases (HDACs) are promising targets for such a novel NB therapy. HDACs catalyze the removal of acetul groups from the lysine residues of proteins including the core nucleosomal histones. Histone tails contain 40 lysine residues, which are acetylated by HATs. Acetylation induces a conformational change within chromation, allowing the transcriptional machinery access to DNA thus promoting gene expression. HDACs repress the gene expression by deacetulating the lysine tails, allowing the positively charged lysines to be tightly bound to the negatively charged DNA and denying the transcriptional machinery access to genes, thereby repressing gene expression. Thus, these post- translational modifications play a key role in directing gene expression, and can create a phenotype that is unrelated to changes in DNA. Histone deacetylases play a prominent role in the regulation of gene transcription by histone deacetylation. They are involved in the remodeling of chromatin, and consequently, in the regulation of gene expression. There is growing evidence that perturbation of epigenetic balances promotes the initiation and progression of cancer. HDAC inhibitors have been found to have therapeutic properties in many different human tumor cell lines, including those derived from the bladder, breast, prostate, lung, ovary and colon among others, indicating that the inhibition of HDAC activity may be a viable strategy for the treatment of cancers. HDAC inhibitors can be divided into short-chain fatty acids, hydroxamic acids, cyclopeptide HDCIs, benzamides, electrophilic ketones, trithiocarbonate and other categories; however, in this article we focus on the cyclic peptides. Cyclic peptides were the most complex in all the HDACIs. Surface recognition structure of macrocyclic inhibitors given the complex structure, but also to ensure the inhibitor with the enzyme molecule interactions more fully, this may be one of the reasons to ensure cyclic peptide inhibitor activity. Like most of other inhibitors, the structure of cyclic peptide HDACIs is divided into three domains [7], which are surface recognition domain, linker domain and Zinc binding domain. FK228 (formerly named FR901228), also known as depsipeptide, is produced by Chromobacterium violaceum and shows potent in vivo antitumor activity. FK228 received FDA approval in 2009 for the treatment of cutaneous T-cell lymphoma. The naturally occurring FK-228 displays impressive anticancer and anti-angiogenesis activities through the HDAC inhibition, and it is now approved as a cancer drug. FK228 is a bicyclic depsipeptide with intramolecular disulfide bridge, which is reductively activated after uptake into the cells generating the sulfhydryl groups. Inspired by the stability of FK228 due to its prodrug nature [12], several HDAC inhibitors with the disulfide bond have been proposed with more simplified structure for the ease of synthetic study. In the past decade, a considerable number of synthetic analogs have been reported as cyclic-tetrapeptide HDAC inhibitors. Main nucleus of 4-aniline quinazoline and hydroxylamine part of histone acetyl off enzyme (HDAC) inhibitor fragments are connected. By this way, they developed an EGFR inhibitors, ErbB2 and HDAC targets, the acetylation of EGFR phosphorylation and HDAC has certain inhibitory effect. Predecessors by the way of connecting hydroxylamine fragments at site-6,7 of 4-aminoquinazolinone obtained good active compound such as compound h1and h2, therefore, we imagined that if at site-4 ' of 4 - aniline quinazoline was substituted by different hydroxyl oxime acid group to get some new compounds. Largazole is a natural cyclic peptide which can selectively inhibit Class I histone deacetylase and show remarkable selectivity between transformed and non-transformed cells. The design of these analogs has been done either by changing the amino acids sequence in the cyclic tetrapeptide framework to achieve high affinity of the inhibitors with the enzyme surface or by the introduction of different type of functional groups as zinc ligands to achieve a strong binding affinity to the enzyme’s active site. The functional groups introduced are mostly full of electron. And more functional groups have been introduced to the non-peptide HDAC inhibitors. In order to develop HDAC inhibitors with various functional groups, we replaced the epoxyketone moiety of chlamydocin scaffold with a pyridine ring. As the first part of this paper, we have designed and synthesized 2 cyclic peptide compounds as HDACi based on the structures of Largazole and FK228. We tried to derive novel artificial amino acids and a pyridine ring to introduce them into the chlamydocin scaffold [9].








Figure 1. Structural regions of cyclopeptide HDCIs.


By comparing the structures of FK228 and Largazole, we designed and synthesized other compounds. The cyclic tetrapeptide structure in surface recognition domain was changed by introducing an amino acid containing a pyridine ring for a fuller contact with the surface and enzymes. The pyridine intermediate has a similar structure with amino acid, and it can be widely used in the medical field, especially as cyclic peptide intermediate fragment deacetylase inhibitors as FK228 analogues. Details are shown as follows in Figure 1.1169 Since the molecules have a cyclic tetrapeptide structure, we split the molecule into three different fragments and then they are connected end to end by a peptide bond or ester bond [14, 15]. Through the retrosynthesis the molecule can be split into three different parts as Figure 2 shows by breaking the amide bond and one ester bond. The zinc domain and the linker can be synthesized by fragment 1. The cyclic peptide was divided into valine and fragment 3. So the amino acid containing the pyridinering becomes the key intermediate in the total synthesis of molecule DCE-3529415.
Details are shown as follows in Figure 3.

Figure 2. Retrosynthesis of DCE-3529415.

Figure 3. Design of compound structure.

The main challenges in the synthesis include the asymmetric construction of the hydroxyl mercapto heptenoic acid unit, the 16-membered cyclic depsipeptide ring itself (that is, four amino acids and hydroxyl mercapto heptenoic acid), and the intramolecular oxidative coupling of the thiol moieties to produce the stable prodrug form of the molecule. Using this method, Wen et al. utilized a lactamization as an alternative route to cyclization in 2008. Yurek-George et al. [13-16] examined the importance of several features of the romidepsin structure. To fragment 3, as the former through breaking the amide bond, the molecule can be divided to 2 parts, one is threonine. The hydroxyl of threonine can react with MsCl and then be removed by DABCO, so the double bond can form [14]. Details are shown as follows in Figure 4.The other one is an amino acids analogue with pyridine ring. The key to the amino acids analogue is to introduce synthetic carboxyl and amino functional groups. So we choose the 2, 6-dimethylpyridine as the raw material. Reacting with KMnO4 the one methyl of 2,6-dimethylpyridine can be oxidized to carboxyl and the other methyl can stay the same [17]. Then the methyl can be transferred into benzyl reacting with NBS by radical reaction which is the material of Gabriel reaction in the next step changing the benzyl to amino.

Details are shown as follows in Figure 5.

O

NH

HO

O

N

NH

Boc

3

NH2

HO

O

+ N

HO NH2

O

N

HO Br

O

N

Figure 4. Retrosynthesis of fragment 3 of DCE-3529415.

Figure 5. Synthesis of fragment 3 of DCE-3529415.

3 EXPERIMENTAL SECTION

All solvents were A.R. grade and purchased from Shanghai Chemical Reagent Company. Melting points were determined on a WPS-2Acapillary apparatus and were uncorrected. The 1HNMR and spectra were recorded on a Bruker Avance III 500 NMR spectrometer using TMS as an internal standard and chemical shifts were given in ppm with tetramethylsilane (TMS). The HRMS spectra were acquired on a Solari X-70FT-MS apparatus. TLC was carried out on silica gel plates 1170 (GF254) with visualization of components by UV light (254nm) or exposure to I2. Column chromatography was carried out on silica gel (300–400 mesh).
Compound 19 (21.43g, 0.2mol) dissolved in 1000mL H2O (not completely dissolve and not affect the reaction) was added KMnO4 47.41g. The solid was added in for ten times, and the interval between two times is 30min. After adding solid raising the temperature to 70°C, reflux for 5h. After the material was completely disappeared, the reaction solution was cooled to room temperature, filtered. The filtrate was evaporated to dryness, and then 200mL HCl (70%) was added to the solid dissolved. Extract the solution with ethanol, the solution was evaporated to be dryness and recrystallization, giving product (17.3g).
Compound 19’ (15g, 0.11mol) was dissolved in 500mL methanol; thionyl chloride was dropped into

methanol at 0°C. After the dropwise addition the reaction apparatus was brought to room temperature, reacting for 5 hours. Methanol was removed by rotary evaporation to give a white solid. The solid was dissolved in 200mL of sodium hydroxide solution at a concentration of 1mol/L. The pH value was adjusted to 9. Ethyl acetate was added to the solution and then was extracted. The organic phase was collected and dried. The solvent was evaporated under reduced pressure to give the product 19’’ (10g).
Compound 19’’ (10g, 0.066mol) was dissolved in 60mL CCl4, NBS 14.1g (0.079mol) was added under stirring. AIBN was added for 3 times about 5g (0.03mol). After the addition reflux at 80°C for 6 hours, add a certain amount of water and stir for 15min, the water is separated from CCl4 then dried. The CCl4 was removed under reduced pressure to give the crude product. The crude product was recrystallized to give pure product (6g).

Compound 20 (6g, 26.20mmol) dissolved in DMF (100mL) was added with potassium phthalimide

(5.82g, 31.44mmol), and reaction was carried out at room temperature for 5h. TLC showed the reaction was completed and concentrated under reduced pressure. NaOH solution was added with 0.2mol/L of washing. Water was added, extracted with ethyl acetate, dried over anhydrous sodium sulfate and finally concentrated to give compound 21 (6.42 g, 83%).

Compound 21 (8g, 20.26mmol) dissolved in methanol (1/4, 200mL) was added with hydrazine hydrate compound (2.9mL, 50mmol) and refluxed overnight. After the reaction was completed, it was concentrated under reduced pressure obtained compound 22 (2.59g,77%) by column chromatography.

At 0 °C, the compound 22 (1.7g, 10.24mmol) in THF was added with DIPEA (2.0mL) and Boc2O

(2.79g), and reacted at room temperature overnight. TLC showed the reaction was complete, concentrated under reduced pressure, water was added, with ethyl ester extraction, dried over anhydrous sodium sulfate, and concentrated by column chromatography to obtain the product compound 23 (2.3g, 86%)

The compound 23 (2.0g, 7.93mmol) and H-Thr-Ome (1.6g, 9.52mmol) in DCM (40mL) was added PyBop (4.13g, 7.93mmol) and DIPEA (5.3mL), Reaction was carried out at room temperature overnight, quenched with saturated ammonium chloride and extracted with dichloromethane, washed with water and saturated brine, and concentrated, dried over anhydrous sodium sulfate, and concentrated by column chromatography to give compound 6 (1.48 g, 51%).

At 0 °C, under the protection of Ar gas, the compound 24 (1.0 g, 2.72mmol) in DCM (10mL) were sequentially added DMAP (16.6 mg), TEA (1.13 mL), MsCl (0.63 mL), Reaction was carried out at room temperature for 2 hours, quenched with saturated ammonium chloride, extracted with dichloromethane, washed with water and saturated brine, concentrated, dried over anhydrous sodium sulfate, and concentrated to give a pale yellow compound 980.8mg. At 0 °C, the pale yellow compound (900mg, 2.02mmol) in DCM (10mL) was added with DABCO (2.27g), and reaction was carried out at room temperature 2h, quenched with saturated ammonium chloride, extracted with dichloromethane, washed with water and saturated brine, concentrated, dried over anhydrous sodium

sulfate, and concentrated by column chromatography to give compound 25 (620.1mg, 95%).

Compound 25 (600mg, 1.72mmol) dissolved in methanol was added with LiOH (123.5mg,). Reaction

was carried out at room temperature for 10 hours. After completion of the reaction, the solution was

filtered to remove LiOH and concentrated to give compound 3 (547.6mg, 95%).

6-Methyl-2-pyridinecarboxylic acid

1HNMR(501MHz,CDCl3)δ13.17(s,1H),8.20(d,J=7.4Hz,1H),8.11(d,J=7.6Hz,1H),7.78(D,J=8.3Hz,1H),2.17(s,3H)ESI-HRMS calcd for C7H7NO2 [M + H]+,138.06

6-Methylpicolinic acid methyl ester

1HNMR(501MHz,CDCl3)δ8.20(d,J=7.4Hz,1H),8.13(d,J=7.8Hz,1H),7.77(D,J=8.3Hz,1H),3.23(s,3H),2.50(s,3H)ESI-HRMS calcd for C8H9NO2 [M + H]+,152.07

6-(Bromomethyl)picolinic acid methyl ester

1HNMR(501MHz,CDCl3)δ8.20(d,J=7.4Hz,1H),8.13(d,J=7.8Hz,1H),7.77(D,J=8.3Hz,1H),5.3(s,2H),3.3(s,3H)ESI-HRMS calcd for C8H8BrNO2[M+H]+, 229. 98

2-Pyridinecarboxylicacid,6-[(1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl)methyl]-,methyl ester
1HNMR(501MHz,CDCl3)δ8.83(d,J=7.6Hz,1H),8.27(d,J=12.3Hz,2H),8.10(d,J=7.6Hz,1H),7.74(d,J=8.9Hz,1H),7.11(d,J=8.9Hz,1H),5.75(s,3H),3.90(s,3H)ESI-HRMS calcd for C16H16N2O4[M + H]+,297.09

2-Pyridinecarboxylicacid, 6-(aminomethyl)-, methyl ester

1HNMR(501MHz,CDCl3)δ8.42(d,J=8.8Hz,1H),8.35(d,J=8.3Hz,1H),7.87(d,J=6.3Hz,1H),4.76(s,2H),3.58(s,3H) ESI-HRMS calcd for C8H10N2O2[M + H]+,167.08

6-[[(tert-Butoxycarbonyl)amino]methyl]pyridine-2-1171carboxylic acid

1HNMR(501MHz,CDCl3)δ12.1(s,1H),8.52(d,J=8.3Hz,1H),8.35(d,J=8.6Hz,1H),7.99(d,J=6.9Hz,1H),4.55(s,2H),1.33(s,9H) ESI-HRMS calcd for C12H16N2O4 [M+ H]+,253.12

Threnine,N-[[6-[[[(1,1-dimethylethoxy)carbonyl]amino]methyl]-2-pyridinyl]carbonyl]-, methyl ester

1HNMR(501MHz,CDCl3)δ8.55(d,J=8.7Hz,1H),8.50(d,J=8.7Hz,1H),8.01(d,J=9.7Hz,1H),7.81(m,2H),5.1(d,J=6.1Hz,1H), 4.7(d, J=6.1Hz,1H),4.4(s,2H), 3.3(s,3H),2.3(m,1H),1.3(s,9H)ESI-HRMS calcd for C17H25N3O6[M + H]+,368.18

2-Butenoicacid,2-[[[6-[[[(1,1-dimethylethoxy)carbonyl]amino]methyl]-2-pyridinyl]carbonyl]amino]-,

methyl ester

1HNMR(501MHz,CDCl3)δ8.12(d,J=7.4Hz,1H),7.85(s,1H),7.83(d,J=8.7Hz,1H),7.02(s,1H),4.48(d,J=5.6Hz,2H),1.89(s,3H),1.57(s,3H),1.43(d,J=29.6Hz,9H)ESI-HRMS calcd for C17H23N3O5[M + H]+,350.17

2-Butenoicacid,2-[[[6-[[[(1,1-dimethylethoxy)carbonyl]amino]methyl]-2-pyridinyl]carbonyl]amino]

1HNMR(501MHz,CDCl3)δ9.50(d,J=72.4Hz,1H),8.12(d,J=7.4Hz,1H),7.85(s,1H),7.43(d,J=50.9Hz,1H),7.02(s,1H),4.48(d,J=22.2Hz,2H),1.89(s,3H),1.43(d,J=29.6Hz,9H). ESI-HRMS calcd for C16H21N3O5[M+H]+,336.16

4 RESULTS AND CONCLUSION

During the process for preparing compound 19’, we found that the equivalent of KMnO4 has a greater impact on the yield of the product. So we carried out experiments at different equivalents, and the results are shown in Table 1.

Table 1 showed the yield of 19’ declining with theincreasing equivalent of KMnO4. However, in the actual process, the reaction time is shortened with the increase of KMnO4 equivalents. Balance based on efficiency and productivity, the 1.5 equiv. of KMnO4 at 80°C is the optimum condition.

During the process for preparing compound 20, we found that the equivalent of NBS have a greater impact on the yield of the product. So we carried out experiments at different equivalents, the results are shown as follows. The chart showed the yield of 19’ declining with the increasing equivalent of NBS. Because, the dibromide increases very quickly with the increasing equivalent of NBS, the 1.2 equiv. of NBS was chosen to be the optimum condition.

Data in Table 1 showed the equivalent of KMnO4 are the key to control yield of 6-Methyl-2-Prydinecarboxylic acid. With the increasing equivalent of KMnO4, more and more 6-Methyl-2-Pyridinecar boxylic acid are transfered into 2,6-pyridinedicarboxylic acid. With the increasing equivalent of NBS, more and more 6-Methylpicolinic acid methyl ester r are transfered into 2-pyri-dinecarboxylic acid, 6-(dibromomethyl)-methyl ester and 2-pyridinecarboxylic acid,6-(trbromomethyl)- methyl ester. Accoding to Figure 7, the A point which is 1.2 equivalent of NBS was chosen to be the optimum condition.

In summary, by means of comparing the structures of FK228 and Largazole, we designed and synthesized several compounds. The cyclic tetrapeptide structure in surface recognization domain was changed by introducing an amino acid containing a pyridine ring for a fuller contact with the surface and enzymes. Through the retrosynthesis analysis, a pyridine pharmaceutical intermediate was designed and synthesized. The pyridine intermediate has a similar structure with amino acid, it can be widely used in the medical field, especially as cyclic peptide intermediate fragment deacetylase inhibitors as FK228 analogues. The key intermediate fragment for the entire molecule

cyclic tetrapeptide structure plays an important role in the surface recognization domain. Through continuous improvement and exploration of the conditions, we

Table 1. Equivalent of KMnO4 effect on yield.

No. Material(g) Equivalent(KMnO4) Product(19’g) Yield(%)

1 11.3 1.1 10.3 71

2 11.3 1.3 10.5 72

3 11.7 1.5 9.7 67

4 11.3 1.7 8.1 56

5 11.1 1.9 6.6 46

6 11.3 2.1 5.0 35

Table 2. Equivalent of NBS effect on yield.

No. Material(g) Equivalent(NBS) Product(g) Yield(%)

1 10.1 1.0 5.5 36

2 10.0 1.1 6.3 41

3 10.1 1.2 6.7 44

4 10.0 1.3 4.1 27

5 10.0 1.4 4.1 27

6

7

10.0

10.0

1.5

1.6

5.0

4.5

33

30

1172

found more optimal conditions. And this has important implications for the synthesis of analogues

of FK228, and the development of new drugs is also important.

ACKNOWLEDGMENTS

This work was supported by Shanghai Institute of Technology.

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(ISTN)

KIMIA MEDISINAL

Dosen Pembimbing : Lia Puspitasari, S.Farm, M.Si,Apt.

Mahasiswa

Nama : Suyatno

NIM   : 17330730

Kelas A

Synthesis of a New Pyridine Pharmaceutical Intermediate in

Potential Anti-cancer of HDACI

Abstrak : Untuk mendapatkan aktivitas anti kanker yang lebih baik dari HDACI, analog HDACI baru dan dari sedian farmasi piridin intermediate dirancang dengan membandingkan molekuk FK228 dan Largazole dan mengenalkan piridin dengan farmakofor tetrapeptida siklik. Jalur sintesis analog HDCI dan piridin mulai dirancang. HDCIs analog dan intermediate piridin semuanya disintetis, pada kondisi optimum untuk mensintesis piridin intermediate didapat dengan menggunakan uji paralel. Piridin intermediate dapat digunakan untuk memproduksi HDACIs potensi baru dengan membentuk struktur baru polipeptida. Karena struktur khusus piridin intermediate, juga dapat digunakan secara luas dibidang medis lainya. Melalui pekerjaan kami, kami telah merancang rute sintetik piridin intermediate dan membahas yang optimum kondisi. Dan kami menyediakan metode sintesis baru dari HDCIs siklopeptidin.

Latar Belakang

Senyawa piridin adalah produk kimia halus bernilai tambah tinggi. Piridin dapat berpatisiasi dalam berbagai reaksi kimia. Senya piridin terklorinasi,oksidasi fluorida dan amonia dapat disiapkan untuk berbagai zat antara dalam sintesis farmasi dan industri kimia. Senyawa piridin saat ini digunakan dalam pengobatan, pestisida, rempah-rempah, aditif pakan, perekat dan bahan sintetis. Dibidang kedokteran, piridin dapat digunakan untuk memproduksi cephalexin, prednisone, dexamethason acetat, sulfo sulfur sulfat, pipozet, hydrokortison, idoksuridin, progesteron, norfloksasin, vitamin A, D2 atau D3, generasi keempat sefalosporin, dan lebih dari 40 jenis bahan sintetis obat yang biasa digunakan.

Untuk waktu yang lama, kanker telah dianggap sebagai hasil dari beragam peubahan genetik dan genomik, seperti amplifikasi, translokasi, delesi dan mutasi titik. Namun penelitian terbaru menunjukan perkembangan kanker tidak hanya terkait dengan perubahan genetik namun juga melibatkan perubahan epigenetik.

Epigenetika berkaitan dengan pewarisan informasi berdasarkan tingkat ekspresi gen.

Modifikasi epigenetik utama pada manusia adalah metilasi DNA dan modifikasi histone posttranslasional.

Metode Penelitian yang Digunakan

Histones adalah protein dasar kecil yang kaya akan asam amino, lisin dan arginin. Histone acetyltransferase (HAT) dan histone deacetylase (HDAC) dapat membuat residu asam amino n-terminal histon menjadi asetilasi atau deasetilasi invivo, kedua enzim bekerja sama untuk menentukan tingkat asetilasi histon, mereka juga mengatur ekspresi gen dan beberapa proses seluler lainya, penelitian telah menunjukkan bahwa proses asetilasi dan deasetilasi dan terjadinya kanker terkait erat. Oleh karena itu, histone deacetylase (HDACs) menjadi target obat antikanker.

Histone deacetylase inhibitor (HDACi) telah menjadi semacam daerah penelitian yang panas. Histone deacetylase inhibitior dapat memperbaiki tingkat p21 dan ekspresi gen lainya dengan meningkatkan tingkat aset asetone histon. Proses proliferasi sel tumor telah menunjukkan efek penghambatan yang sangat baik, dan inhibitor dapat menyebabkan diferensiasi sel tumor dan atau terjadinya apoptosis, sehingga memiliki efek menghambat tumor.

Romidepsin (Istodax), peptida siklik, baru-baru ini disetujui sebagai penghambat HDAC melawan CTCL, yang menunjukan bahwa peptida siklik adalah farmakope yang menarik dalam bidang ini. Histone deacetylase (HDACs) merupakan target yang menjanjikan untuk terapi NB yang baru. HDAC mengkatalisis penghapusan gugus asetat dari residu lisin protein termasuk hambatan inti nukleosom.

 Ekor histone mengandung 40 residu lisin, yang dilarutkan dengan HAT. Asetilasi menginduksi perubahan konformasi dalam kromasi, memungkinkan akses mesin trankripsi ke DNA sehingga mendorong ekspresi gen. HDACs menekan ekspresi gen dengan deasetilasi ekor lisin, yang memungkinkan lisin bermuatan positif terikat erat dengan DNA bermuatan negatif dan menolak akses mesim transkripsional terhadap gen, sehingga menepis eksprsi gen. Dengan demikian, modifikasi pasca translasi ini memainkan peran kunci dalam mengarahkan ekspressi gen, dan dapat menciptakan fenotipe yang tidak terkait dengan perubahan DNA. Histone deacetylases memainkan peran penting dalam pengaturan transkripsi gen denga deasetilasi histone. Mereka terlibat dalam remodeling kromatin, dan akibatnya, dalam regulasi ekspresi gen. Ada bukti yang berkembang epigenetik mendorong inisiasi dan perkembangan kanker. Penghambat HDAC telah ditemukan memiliki sifat terapeutik pada banyak sel tumor manusia yang berbeda termasuk yang berasal dari kandung kemih, payudarah, prostat, paru-paru, ovarium dan usus besar antara lain, menunjukan bahwa penghambatan aktivitas HDAC dapat menjadi strategi yang tepat untuk pengobatan kanker.

Penghambat HDAC dapat dibagi menjadi asam lemak rantai pendek, asam hidroksamat, HDACls siklopeptida, benzamida, keton elektrofilik, tritokarbonat dan kategori lainnya; Namun, dalam artikel ini kita fokus pada peptida siklik.

Peptida siklik adalah yang paling komplek di semua HDACI. Struktur pengenalan permukaan penghambat macrocyclic mengingat struktur kompleks, tetapi juga untuk memastikan penghambat inteaksi enzim secara lebih penuh, ini mungkin salah satu alasan untuk memastikan aktivitas inhibitor peptida siklik. Seperti kebanyakan penghambat lainya, struktur HDACI peptida siklik dibagi menjadi tiga domain, yaitu domain pengenalan permukaan, domain linker dan domain pengikat seng. FK228 (dahulu bernama FR901228), juga dikenal sebagai depsipeptide, diproduksi oleh Chromobacterium violaceum dan menunjukkan aktivitas antitumor in vivo yang manjur. FK228 menerima persetujuan FDA pada tahun 2009 untuk pengobatan limfoma sel T kutaneous. FK-228 yang terjadi secara alami menampilkan aktivitas antikanker dan anti-angiogenesis yang mengesankan melalui penghambat HDAC, dan sekarang disetujui sebagai obat kanker.

FK228 adalah depsipeptida bicyclic dengan jembatan disulfida intramolekul, yang diaktifkan secara reduksi setelah dilakukan pengambilan ke dalam sel yang menghasilkan gugus sulfhidril. Terinspirasi oleh stabilitas FK228 karena produg nature, beberapa penghambat HDAC dengan ikatan disulfida telah diusulkan dengan struktur yang lebih sederhana untuk memudahkan studi sintetis.

Dalam dekade terakhir, sejumlah besar analog sintetis telah dilaporkan sebagai inhibitor HDAC siklik-tetrapeptida. Inti inti dari 4-anilin quinazoline dan bagian hidroksilamina fragmen inhibitor enzim histone acetyl off enzyme (HDAC) dihubungkan. Dengan cara ini, mereka mengembangkan inhibitor EGFR, target ErbB2 dan HDAC, asetilasi fosforilasi EGFR dan HDAC memiliki efek penghambat tertentu. Pendahulunya dengan cara menghubungkan fragmen hidroksilamina di tempat-6,7 dari 4-aminoquinazolinone memperoleh senyawa aktif yang baik seperti senyawa h1 dan h2, oleh karena itu, kita membayangkan bahwa jika di tempat-4 ‘anina 4-anilin diambil dari hidroksil oksimena yang berbeda kelompok asam untuk mendapatkan beberapa senyawa baru.

Largazole adalah peptida siklik alami yang secara selektif dapat menghambat deasetilase histone Kelas I dan menunjukkan selektivitas yang luar biasa antara sel yang berubah dan tidak berubah. Perencanaan analog ini telah dilakukan baik dengan mengubah urutan asam amino dalam kerangka tetrapeptida siklik untuk mencapai afinitas tinggi inhibitor dengan permukaan enzim atau dengan pengenalan berbagai jenis gugus fungsional sebagai ligan seng untuk mencapai afinita ikatan yang kuat, kesitus aktif enzim. Kelompok fungsional yang diperkenalkan sebagai besar penuh dengan elektron. Dan kelompok yang lebih fungsional telah diperkenalkan pada inhibitor HDAC non-peptida. Untuk mengembangkan penghambat HDAC dengan berbagai kelompok fungsional, kami mengganti bagian epoxyketone perancah chlamydocin dengan cicin piridin. Sebagai bagian pertama dari jurnal ini, kami telah merancang dan mensintesis 2 senyawa siklik peptida sebagai HDACi berdasarkan struktur Largazole dan FK228. Kami mencoba untuk mendapatkan asam amino buatan baru dan cincin piridin untuk mengenalkannya ke perancah chlamydocin.

Dengan membandingkan struktur FK228 dan Largazole, kami merancang dan mensintesis senyawa lainnya. Struktur tetrapeptida siklik dalam domain pengenalan permukaan diubah dengan memasukan asam amino yang mengandung cincin piridin untuk kontak lebih penuh dengan permukaan dan enzim. Piridin intermediate memiliki struktur yang sama dengan asam amino, dan dapat digunakan secara luas di bidang medis, terutama sebagai penghambat deacetylase fragmen siklik peptida sementara sebagai analog FK228. Rincian ditunjukkan seperti Figure 1 karena molekul memiliki struktur tetrapeptida siklik, kita membagi molekul menjadi tiga fragmen yang berbeda dan kemudian terhubung ujungnya dengan ikatan peptida atau ikatan ester. Melalui retrosynthesis, molekul dapat dibagi menjadi tiga bagian yang berbeda seperti di tunjukkan pada figure 2 dengan memecah ikatan amida dan satu ikatan ester.

Domain seng dan linker dapat disintesis dengan fragmen 1. Cincin menjadi kunci antara sintesis total molekul DCE-3529415

Rincian ditunjukan pada Figure 3. Desain struktur majemuk.

  

Tentang utama dalam sintesis meliputi kontruksi asimetris unit asam heptenoat hidroksil merkapto, cincin depsipeptida siklik beranggota 16 itu sendiri (yaitu empat asam amino dan asam hidroksil merkapto heptenoat), dan kopling oksidatif intramolekul dari bagian tiol untuk menghasilkan bentuk prodrug stabil dari molekul. Dengan menggunakan metode ini, Wen et al. memanfaatkan laktamisasi sebagai jalur alternatif untuk siklisasi pada tahun 2008. Yurek-George et al. meneliti pentingnya beberapa ciri struktur romidepsin. Untuk fragmen 3, sebagai bekas ikatan amida, molekul dapat di bagi menjadi 2 bagian, satu adalah treonin. Hidroksil dari treonin dapat bereaksi dengan MsCI dan kemudian dikeluarkan oleh DABCO, sehingga ikatan rangkap dapat terbentuk.

Rincian ditunjukan pada Figure 4.

 

  

Yang lainnya adalah asam amino yang analog dengan cincin piridin. Kunci untuk asam amino analog adalah mengenalkan gugus fungsi karboksil dan amino sintetis. Jadi kita memilih 2,6-dimetilpiridin sebagai bahan baku. Bereaksi dengan KMnO4 satu metil 2,6-dimetilpiridin dapat dioksidasi menjadi karboksil dan metil lainya bisa tetap sama. Kemudian metil dapat di pindahkan ke benzil yang bereaksi dengan NBS melalui reaksi radikal yang merupakan bahan reaksi Gabriel pada langkah selajutnya untuk mengubah benzil menjadi amino.

Rincian ditunjukkan pada Figure 5.

Hasil dan Analisis

Mereka membandingkan struktur FK228 dan Largazole, dan merancang mensintesis beberapa senyawa. Struktur tetrapeptida siklik di domain pengenalan permukaan diubah dengan memperkenalkan asam amino yang mengandung cincin piridin untuk kontak lebih penuh dengan permukaan dan enzim

Melalui analisis Retrosynthesis intermediate farmasi piridin dirancang dan disintesis. Piridin intermediate memiliki struktur yang sama dengan asam amino, dapat digunakan secara luas di bidang medis, terutama sebagi penghambat deakeetylase fragmen siklik peptida sementara sebagai analog FK228.

Saran karena memiliki struktur yang sama dengan asam amino dan dapat digunakan secara luas di bidang medis dan ini memiliki implikasi penting untuk sintesis analog dari FK228 dan pengembangan obat-obat baru.