Summary of chemistry dotoral thesis: Study on chemical constituents, cytotoxic and antiinflammatory activities of the sponges Rhabdastrella providentiae and Xestospongia muta living in the

Objectives of the thesis: Determine the chemical constituents of two marine sponge species Rhabdastrella providentiae and Xestospongia muta living in the sea area of Central Vietnam. Evaluate cytotoxic and anti-inflammatory activities of isolated compounds to seek for active compounds for further studies to develop healthy care products for the community.

GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY

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STUDY ON CHEMICAL CONSTITUENTS, CYTOTOXIC AND ANTI-INFLAMMATORY ACTIVITIES OF THE

SPONGES Rhabdastrella providentiae AND Xestospongia muta

LIVING IN THE SEA AREA OF CENTRAL VIETNAM

Major: Organic chemistry Code: 9.44.01.14

SUMMARY OF CHEMISTRY DOTORAL THESIS

Ha Noi - 2019

This thesis was completed at: Graduate university of Science and

Technology - Vietnam Academy of Science and Technology

The thesis can be found in:

- The Library of Graduate University of Science and Technology, Vietnam Academy of Science and Technology

- National Library of Vietnam

INTRODUCTION

1 The urgency of the thesis

In recent years, with the fast development of advanced techniques, a large number of natural compounds have been actively isolated and evaluated for biological activities Many drugs from marine species have been available on the market by major pharmaceutical companies in the world such as: Cytarabine, Halaven, Ziconotide, Vidarabine, Trabectedin

Vietnam has the advantage of a long coastline with more than 3,260

km from North to South and many coastal islands, notably Truong Sa and Hoang Sa archipelagoes located in the middle of East Sea Such geographical conditions have brought many advantages and potentials for rich natural resources, creating an extreme diversity and abundant marine ecosystem The research showed that there were about 160 marine sponge species distributed mainly in coastal and offshore islands Among them, there were only 20 species which have been studied on the chemical

composition and biological activity Particularly, the sponge Rhabdastrella providentiae has not been studied in Vietnam and over the world The sponge Xestospongia muta has not been studied in Vietnam

The study and survey of chemical constituents and biological activity of marine species are topic of interest for scientists in the world In Vietnam, there were some studies on chemical constituents and biological activity of marine species published in prestigious international magazines but not many Therefore, the task of studying the chemical composition and biological activities of marine life in our country is very important From

that point, the topic "Study on chemical constituents, cytotoxic and

anti-inflammatory activities of the sponges Rhabdastrella providentiae and Xestospongia muta living in the sea area of Central Vietnam" was

chosen

Objectives of the thesis:

+ Determine the chemical constituents of two marine sponge species

Rhabdastrella providentiae and Xestospongia muta living in the sea area of

Central Vietnam

+ Evaluate cytotoxic and anti-inflammatory activities of isolated compounds to seek for active compounds for further studies to develop healthy care products for the community

The content of the thesis includes:

1 Isolate compounds from two species of marine sponges

Rhabdastrella providentiae and Xestospongia muta living in the sea area of

Central Vietnam by chromatographic methods:

2 Determine the chemical structure of compounds isolated by physical and chemical methods

3 Evaluate in vitro cancer cell toxicity activity of isolated

compounds;

4 Evaluate in vitro anti-inflammatory activity of isolated

compounds

2 The objectives of the thesis

Study on chemical constituents of two marine sponge species named

Rhabdastrella providentiae and Xestospongia muta

Evaluation of cytotoxic and anti-imflammatory biological activities of

isolated compounds to seek for potential compounds for further researches

3 The main contents of the thesis

Isolation of compounds from the sponges Rhabdastrella providentiae and Xestospongia muta living in the sea area of Central Vietnam

Determination of chemical structures of isolated compounds

Evaluation of cytotoxic and anti-imflammatory biological activies of

isolated compounds to provide scientific evidences for applied researches

1.2 The study of sponges of the genus Rhabdastrella

1.3 The study of sponges of the genus Xestospongia

CHAPTER 2: EXPERIMENT AND RESULTS

2.1 Sponge materials

Sample of Rhabdastrella providentiae (Dendy, 1916) was collected at Con Co, Quang Tri, Vietnam

Sample of Xestospongia muta (Schmidt, 1870) was collected at Vinh

Moc, Quang Tri, Vietnam

2.2 Methods

2.2.1 Methods for isolation of secondary metabolites

2.2.2 Methods for determination of chemical structure of compounds

2.2.3 Cytotoxic assay

2.3 Isolation of compounds

2.3.1 Isolation of compounds from R providentiae

Figure 2.1 Isolation of compounds from R providentiae

2.3.2 Isolation of compounds from X muta

Figure 2.2 Isolation of compounds from X muta

2.4 Physical and spectroscopic data of compounds

2.5 Results on activity of compounds

2.5.1 Results on anti-inflammatory activity of compounds from R providentiae

Table 2.1 The results of evaluation inhibited NO production activity in BV2 cells

2.5.2 Results on anti-inflammatory activity of compounds from X muta

Table 2.2 I nhibited NO production activity in BV2 cells of compounds XM11

2.5.3 Results on cytotocic activity of compounds from R providentiae

Table 2.3 Cytotocic activity of compounds RP1-RP18

Positive control: Ellipticine

2.5.4 Results on cytotocic activity of compounds from X muta

Table 2.4 Cytotocic activity of compounds XM1-XM11

Evaluation of anti-cancer mechanism of XM1 against MCF-7 - human breast cancer cell line: XM1 triggers cell apoptosis by altering the

expression level of related proteins in human breast cancer cells MCF-7 At the same time, this compound also affects MCF-7 human breast cancer cell

cycle in G2/M phase

CHAPTER 3: DISCUSSIONS 3.1 Determination of chemical structure of compounds from R

providentiae

3.1.12 Compound RP12: rhabdaprovidine G (new compound)

Figure 3.30 Chemical structure of compound RP12 and RP10

Compound RP12 is a light yellow amorphous powder The

molecular formula of compound RP12 was determined to be C30H46O4 by

the exhibition of a quasi-molecular ion peak at m/z 493.3289 [M+Na]+

(calcd for C30H46O4Na: 493.3294) in the HR-ESI-MS The 1H-NMR

spectrum of RP12 contained signals corresponding to seven methyl groups

at δH 0.95, 1.05, 1.09, 1.49, 1.61, 1.68, 1.71, two olefinic protons at δH 5.10, 5.81, and three oxygenated methines at δH 4.15, 4.24, 4.70 ppm The 13C-

NMR of RP12 contained signals corresponding to 30 carbon atoms that

were preliminary assigned, according to HSQC data, as one carbonyl carbon at δC 219.9; four olefinic carbons at δC 121.8, 124.1, 131.6, and 141.9; and three oxygenated methines at δC 68.1, 76.3, 77.0 The NMR

spectral data of RP12 were compared with the corresponding data of compound RP10 (its structure was elucidated) and found to match from

carbons C-1 to C-8, and different from carbon C-9, suggesting that the ring

C (5C) of RP12 was changed This suggestion was further confirmed by

HMBC and COSY correlations at rings A and B (Figure 3.32)

Table 3.12 NMR spectral data of RP12 and reference compound

The COSY cross peaks H-9 (δH 1.90)/ H-11 (δH 4.24)/ H-12 (δH

4.15)/ H-13 (δH 1.71) and HMBC correlation between H-30 (δH1.49) and

C-13 (δC 54.9) established five-membered ring C in RP12 The chemical

shifts of C-11 (δC 77.0), C-12 (δC 76.3), and C-13 (δC 54.9) characterized for

two oxygenated methines 11 and C-12) and one aliphatic methine 13) Other connections of carbon backbone were established by consecutive analysis of HMBC and COSY spectra, including HMBC correlations between terminal methyl protons H-26 (δH 1.68)/ H-27 (δH 1.61) and C-25 (δC 131.6)/ C-24 (δC 124.1), COSY cross peaks H-24 (δH 5.10)/ H-23 (δH

(C-2.08, 2.14)/ H-22 (δH 2.06, 2.11), HMBC correlations between H-21 (δH1.71) and C-22 (δC 39.8)/ C-20 (δC 141.9)/ C-17 (δC 121.8), COSY cross peaks H-17 (δH 5.81)/ H-16 (δH 4.70)/ H-15 (δH 1.13, 2.26), HMBC correlations between H-18 (δH 2.65, 2.76) and C-15 (δC 36.4)/ C-14 (δC

54.0)/ C-13 (δC 54.9) In addition, an HMBC correlation between H-16 (δH

4.70) and C-12 (δC 76.3) indicated an ether bridge between C-16 and C-12 Also, the presence of an epoxide ring at C-14/C-18 was conclusively

confirmed by the molecular formula of RP12 (C30H46O4), chemical shift of the methylene carbon C-18 (δC 49.8), the presence of a non-protonated carbon C-14 (δC 54.0), the J-coupling constant of geminal protons H-18

(4.5 Hz), and by comparison with previously published data The

stereochemistry of compound RP12 was then proposed by analysis of

NOESY and CD spectra An E-configuration of double bond C-17/C-20

was confirmed by a NOESY correlation between H-16 (δH 4.70) and H-21 (δH 1.71) NOESY correlations between H-19 (δH 0.95) and H-9 (δH 1.90)/ H-29 (δH 1.05), H-5 (δH 2.48) and H-28 (δH 1.09)/ H-30 (δH 1.49) indicated

a trans-syn-trans junction between rings A-C (Figure 3.32) This means that C-29, C-19, and H-9 are located on the same side, assuming a β-

configuration Therefore, the NOESY correlations H-19 (δH 0.95)/ H-11 (δH 4.24), H-9 (δH 1.90)/ H-15ax (δH 2.26), H-15ax/ H-16 (δH 4.70)

indicated β-configurations for H-11 and H-16 Additional NOESY

correlations H-17 (δH 5.81)/ H-12 (δH 4.15), and H-12/H-13 (δH 1.71)

suggested an α-configuration of H-12 and H-13 The epoxide ring at 14/C-18 adopted an α-configuration based on the NOESY correlation

C-between H-30 (δH 1.49) and H-18 (δH 2.76) Finally, an absolute structure of

RP12 was deduced by the similarity of experimental CD spectrum of RP12

in comparison with that of calculated CD spectrum for enantiomer RP12a

(5R,8S,9S,10S,11S,12S,13S,14S,16S, Figure 3.33) Consequently, structure

of compound RP12 was established and named as rhabdaprovidine G

Figure 3.31 Chemical structure and important HMBC, COSY and NOESY

correlations of compound RP12 3.1.19 Total compounds isolated from R providentiae RP1-RP18

18 compounds (RP1-RP18) were isolated from the marine sponge

Rhabdastrella providentiae These include: 12 new compounds

(RP1-RP12) named: rhabdastrellins G-K (RP1-RP5), rhabdaprovidines A-G (RP6-RP12) and 6 known compounds: jaspolide C (RP13), globostelletin

C (RP14), globostelletin D (RP15), jaspiferin A (RP16), mollisolactone A (RP17), gibepyrone F (RP18) (Figure 3.50) Besides, some of these

isolated compounds demonstrate some specically structural characteristics such as:

- The new compound RP12 has a complex structure with 8 chiral carbons

and an epoxy ring closure forming a 5-rings system was first found in isomalabaricane frame compounds In this study, we have determined the

absolute configuration of RP12 through NOESY and CD spectrum

analyses

- The new compound RP11 with a special three-ring stereotype is arranged

trans-syn-cis instead of trans-syn-trans often found in marine sponge

Rhabdastrella species The absolute configuration of -RP11 is also

determined through NOESY and CD spectra

- The new compound RP3 has a γ-lactone ring closure in the vascular

fraction is rarely found in isomalabaricane compounds The absolute configuaration of two chiral carbons at the side chain of this compound are also determined through analysis of δC values, NOESY and CD spectral interactions

- In previous publications on the isomalabaricane skeleton, the 13E/13Z

isomers are often reported as a mixed form (as globostelletin C and

globostelletin D) In this study, new compounds RP4/ RP5, RP6/ RP7 and

two known compounds RP14/RP15 are 13E/13Z isomer pairs, however,

they were separated one by one with high purity

Figure 3.50 Chemical structure of compounds RP1-RP18

- The isomalabaricane skeleton compounds are specialized secondary

substances synthesized by marine species like Stelletta, Jaspis, Geodia and Rhabdastrella (of the family Astrophorida) Therefore, the detection of new

isomalabaricane compounds (RP1-RP12) can be considered as marker

compounds to identify marine species of Rhabdastrella The special

structure of compounds RP11 and RP12 can be used as an indicator for

identification of R providentiae species.

3.2 Determination of chemical structure of compounds from X muta 3.2.1 Compound XM1: araguspongine C

Figure 3.51 Chemical structure of compound XM1

The compound XM1 was obtained in the form of a white, amorphous powder TLC analysis showed that XM1 reacted positively with

Dragendorff's solution suggesting for an alkaloid compound Compound

XM1 was shown to have a chemical formula similar to that of

araguspongine C, C28H50O4N2, which was deduced from a cluster of quasi

molecular ion peaks in the HR-ESI-MS at m/z 479.3845 [M+H]+ (Calcd for [C28H51O4N2]+, 479.3843), 501.3656 [M+Na]+ (Calcd for [C28H50O4N2Na]+, 501.3663), and 477.3701 [M−H]ˉ (Calcd for [C28H49O4N2]−, 477.3692) The chemical structure of araguspongine C was presented to form by symmetric cycloaddition of a pair of 9-hydroxy-1-oxaquinolizidine moieties through C6 linear chains The 1H NMR and 13C- NMR spectral data of XM1

indicated that two these compounds share the same macrocyclic

bis-1-oxaquinolizidine skeleton Interestingly, only 14 carbon signals were observed in the 13C NMR spectrum of XM1, which also suggested the

symmetricity of its chemical structure Hence, detailed chemical structure

of XM1 was elucidated on a half of its molecule HMBC correlations from

H-10 (δH 4.10) to C-4 (δC 53.2) and C-6 (δC 45.5), and their chemical shifts, demonstrated connections of C-10, C-4, and C-6 via nitrogen atom to form

a quinolizidine backbone Moreover, replacement by an oxygen atom at C-1

of the quinolizidine structure of XM1 was confirmed by the presence of

oxygenated carbon signals of C-10 (δC 91.0) and C-2 (δC 77.6) and by HMBC correlation between H-10 and C-2 In addition, a singlet signal of

H-10 and HMBC correlations from H-10 to the non-protonated C-9 (δC

72.0), and methylene carbons C-8 (δC 33.8)/C-11 (δC 41.3) indicated the presence of a hydroxy group and linear carbon chain at C-9 It was hypothesized that C6-linear chain arises from the C-9 Thus, HMBC correlation from H-2 to C-16′ and COSY cross peak of H-2/H-16′ indicated

head-to-tail cyclization of two half molecules of XM1 by connections

C-2/C-16′: C-16/C-2′ Due to the presence of a system with two fused membered rings and three chiral carbons, stereochemistry of XM1 was

six-studied by NOESY and conformational analyses It was assumed that both six-membered rings were in a stable chair forms Thus, as in decalin, cis- or

trans-fused conformations should be acquired for a 1-oxaquinolizidine

structure (Figure 3.53)

Table 3.19 NMR spectral data of XM1 and reference compound

C # δ C d δ C b,d δ C a,b δ H a,c (mult., J = Hz)