And to determine the anti-inflammatory and anti- cancer mechanisms of the hybrid substances, studies of PGE2 production inhibition activity, cell cycle analysis meth[r]
Trang 1AND TRAINING SCIENCE AND TECHNOLOGY
GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY
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NGUYEN THI THUY HANG
“SYNTHESIS AND ANTIINFLAMMATORY, ANTIPROLIFERATIVE ACTIVITIES OF NEW COXIB–COMBRETASTATIN HYBRIDS’’
Scientific Field: Organic Chemistry
Trang 2Vietnam Academy of Science and Technology
Scientific Supervisors:
1 Assoc Prof Dr Ngo Quoc Anh
2 Assoc Prof Dr Vu Dinh Hoang
1st Reviewer:
2nd Reviewer:
3rd Reviewer:
The dissertation will be defended at Graduate University of Science And Technology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Cau Giay District, Ha Noi City At … hour… date… month … 2021
The dissertation can be found in National Library of Vietnam and the library
of Graduate University of Science And Technology, Vietnam Academy of Science and Technology
Trang 3INSTRODUCTION
1 The urgency of the thesis
Cancer is a group of diseases that involve disorganized cell division and cells that have the ability to invade other tissues by either growing directly into nearby tissue or moving to multiple locations different (metastatic) According
to the Global Cancer Organization GLOBOCAN 2018, there are currently more than 300,000 people living with cancer nationwide, there are 164,671 new cases, 114,871 people die from this disease Globally, there are about 23 million people infected, of which more than 14 million people are newly infected and 8 million 2 hundred thousand people die The World Health Organization (WHO) ranked Vietnam in the top 50 countries in the top 2 of the cancer map
Researching to find cancer treatment drugs with few side effects is one of the directions that is always interested in the scientific community Among current treatments, chemotherapy is a cancer treatment that uses one or more anticancer drugs - cytotoxic One of the anti-cancer drugs, used today in chemotherapy, influences the cell cycle to inhibit cancer cell growth and subsequently induce apoptosis (apoptosis)
Prospective treatment guidelines often recommend a combination of anticancer drugs acting on a variety of mechanisms Nowadays, research discovering drugs targeting multiple targets is gaining attention, with a desire to combine different molecular targets on a single chemical agent The advantages
of a hybrid molecule over a combination of many drugs can improve the technical limitations, side effects and resistance of a single target [1] One of the most important groups of anticancer agents are tubulin-binding molecules Although some drugs that target microtubule have been used in clinical
Trang 4practice, it is still necessary to look for new agents that can overcome the limitations of resistance and the undesirable side effects of these therapies current method [3]
2 Objectives of the dissertation
1 Structured design of coxib - combretastatin hybrid compounds
2 Synthesize coxib - combretastatin hybrid compounds
3 Screening for anti-cancer and anti-inflammatory activities of hybrid compounds
4 Identifying anti-inflammatory and anti-cancer mechanisms of hybrid compounds
5 Docking of a hybrid coxib - combretastatin compound with two purpose effect COX2 and tubulin
3 The main research contents of the thesis
- Research on coxib - combretastatin hybrid compounds synthesis
- Determination of the structure of coxib-combretastatin hybrid compounds
- Screening the activity of coxib - combretastatin hybrid substances
- Study on mechanism of action of some coxib - combretastatin hybrid substances
- Research docking of typical hybrid compounds with target effects of colchicine and COX2
Trang 5Figure 31: Simulation of coxib - combretastatin hybrid compound according to
set target
Trang 6DISSERTATION CONTENTS CHAPTER 1 LITERATURE REVIEW
General presentation of anticancer compounds by tubulin inhibitory mechanism, profile of tubulin mechanism The group of tubulin-based compounds has always been a topic of concern in the field of anti-cancer drug research Combretastatin compounds with rich biological activity have been used in the treatment of a number of cancers They are known for their cytotoxic activity by inhibiting tubulin polymerization at the colchicine site [ 2]
Up to now, these cancer treatment compounds with this mechanism are still being widely used and always a research direction that receives a lot of attention
Overview of combretastatin compounds, which belong to the class of
cis-stilbene, a rich source of pathogens in the search for new drugs, typical compounds such as resveratrol and combretastatin A-4 phosphate are currently
is clinically tested to treat Alzheimer's disease and cancer The recently isolated stilbene has been shown to have a diverse range of biological activities, including antioxidant, antibacterial, anti-malarial, cytotoxic, liver protective and anti-inflammatory properties Combretastatin A-4 (CA4) is also considered to
be a potential cytotoxic agent by strongly inhibiting microtubule polymerization
by binding to the binding point of colchicine on tubulin CA-4 is highly toxic
on many cancer cell models, making it a very interesting target structure
Overview of Pyrazole are pentagonal heterocyclics that form a group of compounds that are particularly useful in organic synthesis They are one of the most studied groups of compounds in the azol family Pyrazole is reported through the available literature and SAR shows that it is necessary for the design of selective COX2 inhibitors One of the most important and commonly used compounds in commercially applied pyrazole compounds is celecoxib, a
Trang 7substance known for its potent anti-inflammatory activity, which selectively
inhibits COX2 through its action Prostaglandins induce inflammation and pain
without effects on prostaglandins COX1 that have a protective effect on the
gastrointestinal tract Furthermore, Celecoxib inhibits the proliferation of
human breast cancer in vitro models such as MCF7 and MDAMB-231 Some
studies indicate that celecoxib and related compounds can induce cell cycle
arrest at G0 /G1 stage leading to apotosis cyclic apoptosis, inhibition of tumor
growth and prevents tumor angiogenesis in the absence of COX2
Thereby, it can be seen that combretastatin celecoxib hybrid compounds
are promising classes that are still new in terms of structural development as
well as bioactivity, contributing to the construction of new research projects
looking for different types anticancer drugs in the pharmaceutical industry
Trang 8CHAPTER 2 EXPERIMENTS 2.1 Materials and equipments
2.1.1 Materials
2.1.2 Equipments
2.2 Methods
2.2.1 Organic synthesis method
2.2.3 Biological activity test method
2.3 Synthesis of coxib- combrestatin hybrid compounds
2.3.1 Synthesis of ester derivatives of coxib - combretastatin hybrid ester hybrid
Figure 2.2 Ester derivative synthesis of coxib - combrestatin hybrids
compound
(i) Alkaline: t-BuOLi (3 mmol, 3 eq), refluxe, (ii) Ethyl chlorooxoacetate (1 mmol, 1 eq) (77), 5 ml THF; (iii) HCl (4 mmol); refluxe, 5 ml C2H5OH dry;
phenylhydrazin (1 mmol, 1 eq) (78)
Synthesized 20 hybrids esters of coxib - combretastatin ester form substances
Trang 9(a) 100 (1.0 mmol), 99 etyl trifluoroacetate (1,2 eq) and NaH (2,5 eq) in THF (5 mL), 6 h
(b), EtOH (5 mL), axit (1.0 eq); arylhydrazin hydrochloride 78 (1.0 mmol) is added
consecutively to the residue and restored for 6 hours Substance 102 was isolated by column
chromatography
2.3.3 Synthesis of acid derivatives of coxib - combretastatin hybrid
Figure 2.8 Synthesis of coxib - combretastatin hybrids
(i) Alkaline: t-BuOLi (3 mmol, 3 eq), refluxe, (ii) Ethyl chlorooxoacetate (1
mmol, 1 eq) (77), 5 ml THF; (iii) HCl (4 mmol); refluxe, 5 ml C2H5OH dry;
phenylhydrazin (1 mmol, 1 eq) (78) The product obtained after isolation
through column chromatography was dissolved in the solvent system THF /
MeOH / H2O = 3: 1: 1, then NaH (1,2 eq) was added to the mixture Carry out
the reaction in 3 hours to obtain compounds acid hybrids 103-122
successful synthesis of 20 acid hybridization of coxib – combretastatin hybrids
103-122
2.5 Biologically active testing of research compounds
The synthetic compounds were screened for anti-breast cancer activity MCF7,
colon cancer HT-29, hepatic carcinoma Hep-G2 and inhibited NO production
Trang 10CHAPTER 3 RESULTS AND DISCUSSIONS
The advantages of using a hybrid molecule over co-combination of multiple drugs at the same time may improve the limitations of adverse effects and resistance [107] Despite its outstanding activity, combretastatin still has many undesirable effects This is why the team aims to combine combretastatin, an anticancer compound, and celecoxib, a COX2-engineered anti-inflammatory agent, as derivatives for new hybrid compounds in hopes of finding new It has interesting biologically active properties such as the anticancer and anti-inflammatory properties of the parent substance and has less side effects
3.1 Design of the structure and biological activity of the hybrids
3.1.1 Design of hybrid molecular structure
In this study, we adopted the hybridization strategy to incorporate the important pharmacophoric groups of two original compounds celecoxib and CA-4 in a single molecule We utilized 1,2-diphenyl substituted pyrazole ring
of celecoxib as a scaffold to mimic the cis-1,2-diphenylethylene motif in CA-4
Replacement of the double bond with heterocyclic five membered rings was demonstrated to retain both cytotoxic and antitubulin activities of the compounds [108] Indeed, these cis-locked analogues provide several
advantages: preventing of isomerization from cis to trans; increasing specificity
of these drugs to cellular targets; and improving the therapeutic potential of these drugs The presence of the trimethoxybenzene moiety of CA-4 is also crucial to obtain relevant cytotoxic and antitubulin responses [76] We were particularly interested in maintaining the sulfonamid group or related bioisosteres of celecoxib which seems to result in its COX-2 selectivity [109] The COX-2 inhibitory effect, if any, will contribute to the overall anti-tumor activity of new molecules
Trang 11Figure 3.1 C Structure of celecoxib, combretastatin A4 and the coxib–
combretastatin hybrids
3.1.2 Design of hybrid molecular biology activities
The cytotoxic effects of these compounds against three human cancer cell lines HT-29, Hep-G2, MCF-7 as well as the inhibition of NO production using lipopolysaccharide (LPS)-activated murine macrophage RAW 264.7 cells were studied The role of NO in tumour biology is complex, because it has both facilitatory and inhibitory roles in cellular processes depending on the conditions, so NO inhibition is directly unrelated to the cytotoxicity But NO displays a variety of the same useful pharmacological properties as prostaglandins in the cardiovascular system including vasodilation, inhibition of platelet aggregation, modulation of platelet and leukocytes adherence to vessels [110] The use of selective COX-2 inhibitor celecoxib in chemoprevention of breast cancer and other malignancies has been limited by its adverse effects, in particular the risk of cardiovascular events principally connected to their ability
to reduce the production of prostacyclin PGI2 [111] Based on the observation that maintaining of NO production together with coxib-induced activities thus might help avoiding risk of cardiovascular toxicity, a strategy to design a multi-
Trang 12target drug by combining COX-2-selective inhibition with nitric oxide dependent activities has been initiated [112-114]
(NO)-In infammation, the activated immune cells such as macrophages secrete everal infammatory mediators such as proinfammatory cytokines, nitric oxide (NO) and prostaglandin E2 (PGE2) In our previous study, tested compounds and celecoxib were examined for their efects on nitric oxide (NO) production in LPS-activated murine macrophage RAW 264.7 cells 264.7 cells [115] Prostaglandin E2 is present in high concentrations in breast tumors and in metastatic cancers, in the absence of estrogen and progesterone receptors [116-117] Hence, the ability to inhibit production of PGE2 can be considered a good strategy in the design of anticancer agents However, the MCF7 breast cancer cells produced very low levels of PGE2, according to another report [109] Therefore, the active efficacy of the hybrid compounds was tested for the inflammatory response induced by LPS instead of by inhibiting PGE2 on MCF7 [118]
Therefore, cancer cell lines HT-29, Hep-G2, MCF-7 and inhibition of NO production were selected as initial tools to screen for anti-inflammatory activity
of the study agent class And to determine the inflammatory and cancer mechanisms of the hybrid substances, studies of PGE2 production inhibition activity, cell cycle analysis methods, apoptosis methods by nuclear staining cells with Hoechst 33342, study of apotosis-inducing activity by caspase-3 indicator, study of apoptosis induction by FITC-anexin V and PI stained cell photometric method Finally, substances selected and studied on biological mechanisms will be tested to interact with tubulin and COX2 targets
anti-by molecular docking method to confirm the biologic accuracy of the model in
vitro
3.2 Synthesis of coxib - combretastatin hybrids
The single-reaction method has been applied to prepare the 1H pyrazole-3-carboxylate ethyl oxalyl chlorid, 1,2-diarylethanon and ethyl
Trang 131,4,5-triaryl-oxalyl chloride arylhydrazine hydrochloride derivatives (Figure 2.1 , Figure 2.2, Figure 2.7, Figure 2.8) [109] Reaction of Claisen condensation between two components 1,2-diarylethanon and ethyl oxalyl chloride with alkaline agent using tert-BuOLi, obtained the lithium salt intermediate product of ethyl 2,4-dioxo-3,4-diarylbutanoate , the reaction is continued with arylhydrazine hydrochloride through the Knorr reaction catalyzed by hydrochloric acid to
produce triarylpyrazole-3-carboxylate (hybrids compound 79 to 98) and (hybrids compound 103 to 122) A cycle of celecoxib derivatization was also
applied (Figure 2.4, Figure 2.5) 3,4,5-trimethoxyphenyl -
1,1,1-trifluoro-2,4-butanedion (65, 102) obtained from Claisen condensation between
acetophenone and ethyl trifluoroacetate derivatives then reacts with sulfamidophenyl hydrazine halide salt to form 3,4,5-trimethoxyphenyl, a substance with structure similar to celecoxib
4-According to the reaction procedures described in Figure 2.1, Figure 2.2,
20 coxib-combretastatin hybrid compounds were successfully synthesized with efficiency from 64 to 83% (Table 3.1) 02 compounds containing CF3 group with difference rates are 95% respectively 78% (table 3.2) 20 acidic compounds of coxib - combretastatin hybrid with efficiency from 54 - 74% (Table 3.3); The substances are structurally determined by the methods of purification and nuclear magnetic resonance spectroscopy method NMR 1D, 2D
Of the hybrid compounds obtained, substances 79 to 98 and 102 to 122
were synthesized for the first time and never previously published And 01 compound has been published and also used as a comparator is celecoxib (65) which is known for many studies [78-79]
3.3 Biologically active coxib - combretastatin hybrids