Meroterpene-like compounds derived from β-caryophyllene as potent α-glucosidase inhibitors

Article abstract of DOI:10.1039/c8ob02687d

Meroterpenoids isolated from guava (Psidium guajava) and Rhodomyrtus tomentosa possess special skeletons which incorporate terpenoids with phloroglucinol derivatives. Most of these meroterpenoids showed high cytotoxicity against cancer cell lines. However

Full text of DOI:10.1039/c8ob02687d

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Meroterpene-like compounds derived from
β-caryophyllene as potent α-glucosidase
inhibitors†
Cite this: Org. Biomol. Chem., 2018,
16, 9454
a
Shuang-Jiang Ma,‡a Jie Yu, ‡a Da-Wei Yan,a Da-Cheng Wang,a Jin-Ming Gao
*
a,b
and Qiang Zhang
*
Meroterpenoids isolated from guava (Psidium guajava) and Rhodomyrtus tomentosa possess special skel-
etons which incorporate terpenoids with phloroglucinol derivatives. Most of these meroterpenoids
showed high cytotoxicity against cancer cell lines. However, their chemical diversity is very limited.
Herein, we employed a biomimetic hetero-cycloaddition starting from ortho-quinone methides and an
abundant natural product, β-caryophyllene, to generate meroterpene-like compounds. Considering that
the source plant has hyperglycemic functions, α-glucosidase was selected as a target for bioassay. Nine
compounds were screened out for promising activities (IC50 < 15 μM), which were better than the positive
controls genistein and acarbose. The best inhibitor 12 (IC50 2.73 μM) possesses two caryophyllene moi-
eties. They represented a new type of skeleton possessing activities against α-glucosidase. The kinetic
study exhibited that these inhibitors belong to a non-competitive type. All these inhibitors may provide an
opportunity to develop a new class of antidiabetic agents.
Received 30th October 2018,
Accepted 26th November 2018
DOI: 10.1039/c8ob02687d
rsc.li/obc
arising from phloroglucinols (Scheme 1a and b).13–23 Some of
the meroterpene metabolites, such as the main meroterpene
Introduction
The chemical diversity of natural products (NPs) and their component (guajadial) from guava leaves (Fig. 1a), displayed
derivatives provides a huge contribution for developing novel
drugs or lead compounds. However, the difficulty in obtaining
novel scaffolds has recently declined in pharmaceutical
research due to elaborate isolation procedures or lengthy path-
ways of total synthesis.1,2 Thus, it is urgently required to
improve the diversity of NPs and their derivatives. In this
regard, diversity-oriented synthesis (DOS) has emerged
recently as an efficient strategy for constructing complex and
diverse molecules from simple precursors.3,4 Easily accessible
NPs as starting scaffolds are particularly effective for obtaining
chemically diverse libraries that are useful in drug discovery.5–8
This strategy can avoid too many reaction steps for building
complex scaffolds.6,9–12
Meroterpenoids isolated from guava (Psidium guajava) and
Rhodomyrtus tomentosa are a typical group of meroterpenoids
aShaanxi Key Laboratory of Natural Products & Chemical Biology,
College of Chemistry & Pharmacy, Northwest A&F University, Yangling 712100,
PR China. E-mail: zhangq@nwsuaf.edu.cn, jinminggao@nwsuaf.edu.cn
bState Key Laboratory of Medicinal Chemical Biology, Nankai University,
Tianjin 300071, People’s Republic of China
†Electronic supplementary information (ESI) available: 1H and 13C NMR and
HRMS spectra of products. CCDC 1838685–1838687. For ESI and crystallo-
graphic data in CIF or other electronic format see DOI: 10.1039/c8ob02687d
‡These authors contributed equally to this article.
Scheme 1 Typical meroterpene skeletons from Psidium guajava and
Rhodomyrtus tomentosa. (a) Representative meroterpenoid structures;
(b) total synthesis pathway; (c) the epimerization of guajadial.
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Fig. 1 X-ray ORTEP views of compounds 11, 17 and 27 (CCDC No. 1838685–1838687†).
high cytotoxicities against cancer cell lines via inhibiting the Besides the natural β-caryophyllene (4, Scheme 2c), two other
Top1 enzyme (IC50 160 nM).14 Inspired by the hyperglycemic derivatives (5 and 6) were synthesized (Scheme 2d) and
function of guava leaves in traditional Chinese medicine, we employed as sesquiterpenoid building blocks to improve the
envisioned that the typical meroterpenes also have similar diversity of the products.
bioactivities. However, the compound guajadial is not so
stable and tends to epimerize into another NP, psidial A
(Scheme 1c).24
Results and discussion
Synthesis of the meroterpene-like compounds
In synthetic chemistry, these products were totally syn-
thesized by a one-pot procedure from ortho-quinone methides
(o-QM) and terpenoids (Scheme 1b).25,26 o-QMs are highly The pre-QM building blocks 1 and 2 were synthesized accord-
useful building blocks for the total synthesis of many natural ing to the reported procedures.29–31 The derivatives of caryo-
products (NPs). Their inherent reactivity can be used in cas- phyllene 5 and 6 were synthesized from caryophyllene epoxide
caded reactions for constructing complex scaffolds.27 We whose terminal vinyl was cleared by NaIO4/RuCl3 and the
selected three natural precursors of o-QM (pre-QM, epoxide group was reduced to vinyl by Zn powder (Scheme 2d).
Scheme 2a), which can also be regarded as privileged struc- Due to the steric effect, the hydroxyl group in compound 6 is
tures due to the high bioactivities of their derivatives. The pre- β-oriented (R), which was also indicated by the X-ray analysis
QMs 1 and 2 commonly exist in these natural meroterpenes. of its derivative 27 (Fig. 1).
QM 3 is an essential moiety of the highly bioactive compound
Every pre-QM was combined with every caryophyllene
lapachol which showed potential against many targets, derivative respectively to afford 24 products, which can be
especially human cancer cells DU145 (IC50 64 nM).28 These classified into 5 different scaffolds (Scheme 3). Despite the
pre-QMs can transform into o-QMs in the cascaded one-pot total synthesis of these types of meroterpenes having been
procedure to construct meroterpene-like products. For the reported, obtaining enantioselective products is still difficult.
example of 3 (Scheme 2b), once compound 3 reacts with paraf- On the other hand, the stereoisomers would enhance the
ormaldehyde, the o-QM intermediate will generate immedi- diversity of products and help understand the relationship
ately, which is followed by cycloaddition or Michael addition25 between the skeletons and the bioactivities. To recognize the
on the vinyl of terpenoids to yield meroterpene scaffolds. absolute configurations (ACs) of these newly formed chiral
Scheme 2 (a) Selected o-QM precursors; (b) formation and reactivity of o-QM; (c) structures of β-caryophyllene (4) and its derivatives (5 and 6); (d)
synthesis of the caryophyllene derivatives 5 and 6.
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Scheme 3 Diversity-oriented synthesis of natural-like meroterpenes.
carbons (C-4 and C-5), we carried out NOESY, X-ray diffraction centers near chromophores, such as C-4 and C-5 in these com-
analysis, and ECD calculations. pounds, can yield obvious CEs around their λmax. The chiral
Since the chiralities of C-1 and C-9 remained unchanged in centers at C-1 and C-9 are far away from the chromophores;
the reactions (Scheme 3), they can be selected as inside refer- thus they only give the CE at around 208 nm. Comprising
ence groups for determining ACs. For all the compounds these experimental ECD spectra with theoretical ECD curves,
except for 11 and 12, NOESY correlations can be recognized the consistency of the whole spectra (from 190 to 400 nm)
from H3-12 or H-5 to H-1 or H-9, which indicate the ACs of the allowed us to confirm their ACs as shown therein.
chiralities of C-4 and C-5. All these products possess chromo- Furthermore, X-ray diffraction of compound 17 also verified
phores close to the newly formed chiral carbons (C-4 and C-5); the whole configurations (Cu Kα, Flack coefficient 0.02).
thus we also employed ECD spectra associated with quantum
When the pre-QM 1 was combined with the caryophyllene
calculations to verify their ACs. As typical examples, we derivative 5, more complex skeletons (11 and 12) were gener-
selected compounds 9, 10, 17, 18, 23 and 24 to analyze their ated containing 2 caryophyllene substructures. The planar
theoretical CD spectra since these compounds possess the structure and configuration of compound 11 were verified by
same sesquiterpenoid moiety. Due to different UV absorptions X-ray diffraction (Cu Kα, Flack coefficient 0.02, Fig. 1), which
arising from the incorporated chromophores, these six com- possesses (4R,5S,4′R,5′S)-stereochemistry. Product 12 showed
pounds showed obvious Cotton effects at 307, 223, and much-closer 1H NMR and 13C NMR spectra data to those of
394 nm on the CD spectra, respectively (Fig. 2). The calculated compound 11, but they have different retention times (tR) on
theoretical spectra indicated that the (4R,5S)-products of pre- an ordinary HPLC C18 column, which indicated that the two
QMs 1 and 3 have negative CE at 307 nm and 394 nm respect- compounds are diastereoisomers. The NOESY correlations
ively, while (4S,5R)-products have positive CE. However, the shown in Fig. 3a indicated that H3C-12, H-9, H-5′ and H-9′ are
products synthesized from pre-QM 2 showed different negative all on the same α-side of the caryophyllene macrocycle. In
CEs at 223 nm. It should be noted that these spectra showed addition, the cycloaddition mechanism indicates that a trans
almost mirror-image CD curves. In general, only those chirality double bond will yield trans-chiral centers (C-4/C-5 and C-4′/
Fig. 2 Experimental and calculated ECD spectra of 9, 10, 17, 18, 23 and 24. See Fig. S1 in the ESI† for the other ECD spectra.
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Fig. 3 (a) Selected NOESY correlations of 12; (b) experimental and calculated ECD spectra of 12.
C-5′). Thus, the configuration of 12 was determined as 50 μM concentrations. The compounds listed in the table
(4R,5S,4′S,5′R). Furthermore, the consistent CEs between the showed a high inhibition rate (more than 50%) at 25 μM level;
experimental and calculated spectra (Fig. 3b) confirmed the thus, they were evaluated further for IC50 values. Except com-
correct configuration assignment.
pound 22, they all showed higher inhibition than the positive
control genistein and the drug acarbose (Table 1). In particu-
lar, the polycyclic compounds 11, 12 and 29 showed potent
activity against α-glucosidase with IC50 values ranging from 2.7
to 5.4 μM.
Evaluation of the α-glucosidase inhibiting effect of the
natural-like compounds
α-Glucosidase is an important enzyme catalyzing carbohydrate
digestion.32 Inhibiting this enzyme will postpone glucose
absorption, and then lower postprandial blood glucose.33,34
α-Glucosidase inhibitors such as acarbose, miglitol, and vogli-
bose are being utilized as oral antidiabetic drugs. To screen
out lead compounds from the synthetic products, all the com-
pounds were primarily bioassayed on this enzyme at 25 and
In the primary screening, the building blocks 1–6 have not
been found to have any inhibition on the α-glucosidase
enzyme. Only when these pre-QMs (1–3) were combined with
terpenoid moieties 4–6 to form meroterpenoid-like products,
obvious inhibition was detected. These indicated that both the
pre-QMs and terpenoid moieties are essential for bioactivity.
Although the pre-QM moiety in the meroterpenoid skeletons
provides binding sites for hydrogen bonds, the terpenoid moi-
eties are also essential due to their hydrophobic interaction
with the target enzyme. In particular, compounds 11 and 12
possess more large hydrophobic substructures, which contrib-
ute to their high activities. The chiralities of C-4 and C-5 may
have limited influence on the bioactivities, such as pairs of
compounds 11/12 and 25/26, which showed similar
bioactivities.
Table 1 IC50 against α-glucosidase
IC50 (μM)
IC50 (μM)
11
12
13
21
22
25
5.42 0.71
2.73 0.13
9.47 0.64
8.82 0.48
13.95 1.47
11.92 0.30
26
28
29
G
6.57 0.66
9.24 0.50
3.32 0.25
22.64 3.03
>50
A
To explore the interaction mechanism of the typical pro-
ducts 12 and 21 with high activities, the enzyme kinetic
studies were carried out using the Lineweaver–Burk plot ana-
G, genistein, A, acarbose.
Fig. 4 Lineweaver–Burk plot analysis of the kinetics of α-glucosidase inhibition exerted by compounds 12 (a) and 21 (b).
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lysis.35,36 α-Glucosidase was treated with pNPG at various con- GUI tool. Column chromatography was performed on silica gel
centrations (0.3–1.5 mM) in the absence or presence of 12 and (90–150 μm) and Chromatorex C18 gel (40–75 μm). GF254 plates
21 at five different concentrations. As shown in Fig. 4, 12 and were used for thin-layer chromatography (TLC). HPLC analysis
21 showed
a
noncompetitive type of inhibition against and preparations were performed on
a
Waters 1525
α-glucosidase. Replotting the slope and Y-intercept values instrument.
taken from each line in the primary Lineweaver–Burk plot
Preparation of meroterpenoid-like products 7–30
(see the ESI†) allowed extrapolation of the inhibition constants
Ki,free (a measure of the affinity to the free enzyme) and Ki,bound To a solution of 1.1 mmol pre-QM in 1,4-dioxane (3.0 ml) was
(a measure of the affinity to the complex enzyme–substrate). added paraformaldehyde (774 mg) and 3 eq. β-caryophyllene
The Ki,free and Ki,bound values of 12 were 1.98 and 5.24 μM, (or its derivative). After being stirred under reflux for 24 h, the
whereas the values of compound 21 were 7.06 and 3.02 μM.
solvent was removed from the reaction mixture under vacuum.
The crude products were separated on a silica gel column
(PE–EtOAc from 50 : 1 to 10 : 1) and a semi-HPLC C18 column
(gradient MECN 80–100%) repeatedly to yield pure products.
The spectra data of compounds 9, 11, 17 and 25 were selected
Conclusions
In conclusion, we have reported the construction of a set of as representative examples listed as below. For the full list of
meroterpenoid-like compounds starting from caryophyllene all the synthetic products, please find them in the ESI.†
and natural essential moieties. All 24 products represent the
Compound 9. Colorless oil (53 mg, 12%); [α]2D0 = −47.0
creation of five different frameworks prepared in a biomimetic (c 0.05 in CH3CN); UV λmax(MeCN)/nm (log ε) 277 (4.51), 344
1
reaction, thereby providing the candidates to identify poten- (3.43); H NMR (400 MHz, CDCl3) δ ppm 1.00 (s, 3 H), 1.02 (s,
tially new chemotypes. Due to the limited stereoselectivity of 3 H), 1.24 (s, 3 H), 1.45 (dd, J = 10.96, 7.43 Hz, 1 H), 1.55–1.66
the construction reaction, all the ACs of the products were (m, 1 H), 1.73–1.95 (m, 4 H), 2.01–2.24 (m, 4 H), 2.25–2.34 (m,
determined unambiguously by ECD calculations or X-ray diffr- 1 H), 2.46 (dt, J = 13.69, 5.67 Hz, 1 H), 2.72 (dd, J = 16.63,
action analysis. Furthermore, the commercial availability of 4.89 Hz, 1 H), 2.77–2.86 (m, 1 H), 3.08–3.18 (m, 1 H), 10.00 (s,
the starting materials allowed us to scale up reactions and 1 H), 10.14 (s, 1 H), 13.23 (s, 1 H), 13.39 (s, 1 H); 13C NMR
obtain sufficient amounts of compounds for further investi- (100 MHz, CDCl3) δ ppm 21.2, 21.9, 22.7, 23.6, 29.6, 29.6, 31.6,
gations. Eight of the products, 11, 12, 13, 21, 25, 26, 28 and 29, 34.6, 35.0, 37.3, 41.3, 46.0, 50.7, 83.9, 100.2, 103.7, 103.9,
showed potential activities against α-glucosidase, which could 162.6, 168.1, 168.4, 191.6, 191.6, 212.7; HRESIMS m/z [M + H]+
be considered as promising lead compounds for developing calcd for C23H29O6 401.1964, found 401.1968.
new antidiabetic drugs. Furthermore, our findings have
Compound 11. Colorless oil (78 mg, 12%); [α]2D0 = −78.0
demonstrated that combining the abundant terpenoids with (c 0.05 in CH3CN); UV λmax(MeCN)/nm (log ε) 304(4.31); 1H
natural essential substructures will conveniently expand the NMR (400 MHz, CDCl3) δ ppm 0.99 (s, 3 H), 1.01 (s, 3 H), 1.05
chemical space of NPs and generate diverse skeletons covering (s, 6 H), 1.15 (s, 6 H), 1.44 (dt, J = 11.0, 8.2 Hz, 2 H), 1.50–1.63
potential lead compounds for further development.
(m, 2 H), 1.63–1.75 (m, 2 H), 1.76–1.93 (m, 5 H), 1.93–2.00 (m,
1 H), 2.00–2.10 (m, 3 H), 2.10–2.17 (m, 2 H), 2.17–2.30 (m,
4 H), 2.38–2.49 (m, 2 H), 2.53 (dd, J = 16.6, 5.3 Hz, 1 H),
2.72–2.80 (m, 2 H), 2.83–2.92 (m, 1 H), 3.04–3.20 (m, 2 H),
12.46 (s, 1 H); 13C NMR (100 MHz, CDCl3) δ ppm 20.8, 21.3,
21.7, 22.1, 22.7, 22.8, 24.0, 25.2, 29.7, 29.7, 29.8, 30.2, 31.5,
Experimental
General procedure
NMR spectra were recorded in CDCl3 using an AVANCE III 31.8, 34.5, 34.7, 35.2, 35.4, 37.4, 37.4, 41.3, 41.3, 45.4, 46.7,
spectrometer (400 MHz) and an AVANCE III spectrometer 50.7, 50.7, 80.6, 81.9, 100.0, 100.0, 100.1, 154.7, 159.0, 160.6,
(500 MHz). TMS was used as an internal reference for chemical 191.4, 213.1, 213.4; HR ESI-MS m/z [M + H]+ calcd for
shifts (in ppm). Coupling constants, J, are reported in hertz C37H51O6 591.3686, found 591.3689.
(Hz). Melting points were determined using a MEL-TEMP
Compounds 17. White solid (229 mg, 52%), [α]2D0 = +27.4
1101D apparatus and are uncorrected. ESI-MS spectra were (c 0.05 in CH3CN); UV λmax(MeCN)/nm (log ε) 261(4.12); 1H
recorded on a Thermo Fisher Scientific LTQ Fleet instrument. NMR (400 MHz, CDCl3) δ ppm 0.99 (s, 3 H), 1.01 (s, 3 H), 1.14
HR ESIMS data were obtained by using an AB Sciex Triple (s, 3 H), 1.31 (s, 3 H), 1.32 (s, 3 H), 1.34 (s, 3 H), 1.36 (s, 3 H),
TOF 4600 system. Optical rotations were measured on a 1.44 (dd, J = 11.0, 7.4 Hz, 1 H), 1.51–1.62 (m, 1 H), 1.62–1.70
PerkinElmer 341 polarimeter with a thermally jacketed 5 cm (m, 1 H), 1.74 (dtd, J = 11.7, 5.9, 5.9, 3.1 Hz, 2 H), 1.78–1.89
cell at approximately 20 °C, and concentrations (c) are given in (m, 2 H), 2.00–2.08 (m, 1 H), 2.09–2.16 (m, 1 H), 2.17–2.28 (m,
g per 100 ml. ECD spectra were obtained on a Chirascan CD 2 H), 2.43 (dt, J = 13.7, 5.9 Hz, 1 H), 2.54 (dd, J = 17.0, 5.3 Hz,
spectrometer. Crystallographic data were collected on a Bruker 1 H), 2.72–2.83 (m, 1 H), 3.08–3.19 (m, 1 H); 13C NMR
Smart Apex CCD area detector diffractometer with graphite- (100 MHz, CDCl3,) δ ppm 20.7, 21.9, 22.5, 23.8, 24.6, 25.2,
monochromated Cu Kα radiation (λ = 1.54184 Å) at 293(2) K 25.3, 25.4, 29.1, 29.4, 31.8, 34.6, 35.5, 36.9, 41.4, 46.1, 47.4,
using the ω-scan technique. Crystal structures were solved and 50.7, 54.8, 83.0, 106.7, 169.8, 197.4, 212.9, 213.5; HR ESI-MS
refined by SHELXT and SHELXS37 associated with Olex2 as a m/z [M + H]+ calcd for C25H37O4 401.2692, found 401.2696.
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Compound 25. Rufous solid (21 mg, 5%), [α]2D0 = −44.6 Y-intercepts of these reciprocal plots were also replotted
(c 0.05 in CH3CN); UV λmax(MeCN)/nm (log ε) 257(4.30); against the inhibitor concentration, respectively. Data analysis
1H NMR (500 MHz, CDCl3) δ ppm 0.99 (s, 3 H), 1.04 (s, 3 H), was performed using Prism software.
1.28 (s, 3 H), 1.46 (dd, J = 11.0, 7.6 Hz, 1 H), 1.62–1.71 (m,
1 H), 1.75–1.97 (m, 4 H), 2.06 (dd, J = 17.3, 11.0 Hz, 1 H), 2.15
(t, J = 10.2 Hz, 1 H), 2.19–2.27 (m, 2 H), 2.31–2.40 (m, 1 H), Conflicts of interest
2.43–2.51 (m, 1 H), 2.71–2.85 (m, 2 H), 3.12–3.20 (m, 1 H), 7.52
There are no conflicts to declare.
(t, J = 7.6 Hz, 1 H), 7.65 (td, J = 7.7, 1.3 Hz, 1 H), 7.76 (d, J =
7.9 Hz, 1 H), 8.04–8.09 (m, 1 H); 13C NMR (125 MHz, CDCl3)
δ ppm 21.3, 21.8, 22.7, 24.6, 29.4, 29.6, 31.7, 34.7, 35.3, 37.3,
Acknowledgements
41.2, 45.8, 50.7, 84.9, 112.6, 123.8, 128.7, 130.2, 130.8, 132.2,
134.8, 161.4, 178.0, 179.7, 212.6; HR ESI-MS m/z [M + H]+ calcd
This work is financially supported by the Natural Science and
Basic Research Plan of Shaanxi Province (No. 2016JM2002) and
for C25H29O4 393.2066, found 393.2065.
the State Key Laboratory of Medicinal Chemical Biology
(Nankai University, No. 201502003).
ECD calculation method
All the conformers of every calculated compound were
searched by Conflex using the MMFF94s force field.38,39
Further optimization were performed at the B3LYP/6-31+G(d,p)
Notes and references
level in Gaussian 09 package.40 The theoretical CD
spectra were calculated by cam-B3LYP/TZVP and summated
in SpecDis41 according to their Boltzmann-calculated
distributions.
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α-Glucosidase inhibitory assay
The α-glucosidase inhibitory assay of the synthesized com-
pounds was performed as reported in the literature.36 Briefly,
α-glucosidase was purchased from Sigma (EC 3.2.1.20), which
was isolated from Saccharomyces cerevisiae. The enzyme was
dissolved in 200 μL of 10 mM phosphate buffer (pH 6.80) and
incubated in the presence of 12 μL of the test compound in
DMSO at 37 °C for 5 min. The reaction was started by the
addition of 36 μL of 4-nitrophenyl α-D-glucopyranoside
(p-NPG) and maintained under 37 °C for 40 min. The amount
of released 4-nitrophenol was measured as the absorbance at
400 nm. The assay was performed with 5 or 6 different concen-
trations around the IC50 values, approximately estimated in
previous experiments. In each set of experiments, the assay
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