First total synthesis of a novel amide alkaloid derived from Aconitum taipeicum and its anticancer activity

Article abstract of DOI:10.1080/14786419.2017.1340283

A concise total synthesis of a naturally occurring 3-isopropyl-tetrahydropyrrolo[1, 2-a]pyrimidine-2, 4(1H, 3H)-dione (ITPD) isolated from Aconitum taipeicum with a three-step approach was depicted in this study for the first time. Two key intermediates, diethyl isopropylmalonate (2) and pyrrolidin-2-amine (3), being synthsesised separately from initial diethyl malonate (4) and 3, 4-dihydro-2H-pyrrol-5-amine (5), were utilised to obtain the compound entitled ITPD. ITPD showed a promising anticancer activity in vitro on SMMC-7721 cell lines. Flow cytometry and cell cycle analysis revealed that ITPD could induce apoptosis and cell cycle arrest in S phase. The occurrence of apoptosis possibly attributed to the mechanism that ITPD could mediate the mitochondrial pathway through activating caspase-3/9 and increasing the ratio of Bax/Bcl-2 to finally trigger cell apoptosis and DNA damage. Collectively, the possibility to produce sufficient quantity of synthetic ITPD provided the base for further bio-evaluation in vivo and in vitro. The bioactive assay suggested that it may be a potential candidate for further chemical optimisation and use in cancer therapy.

Full text of DOI:10.1080/14786419.2017.1340283

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
First total synthesis of a novel amide alkaloid
derived from Aconitum taipeicum and its
anticancer activity
Xinxin Zhang, Dandan Li, Xuanji Xue, Yan Zhang, Jie Zhang, Chen Huang,
Zengjun Guo & Nigatu Tadesse
To cite this article: Xinxin Zhang, Dandan Li, Xuanji Xue, Yan Zhang, Jie Zhang, Chen
Huang, Zengjun Guo & Nigatu Tadesse (2017): First total synthesis of a novel amide alkaloid
derived from Aconitum taipeicum and its anticancer activity, Natural Product Research, DOI:
10.1080/14786419.2017.1340283
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Date: 20 June 2017, At: 20:49

Natural Product research, 2017
https://doi.org/10.1080/14786419.2017.1340283
First total synthesis of a novel amide alkaloid derived from
Aconitum taipeicum and its anticancer activity
Xinxin Zhanga, Dandan Lia, Xuanji Xuea, Yan Zhanga,c, Jie Zhanga, Chen Huangb,
Zengjun Guoa and Nigatu Tadessed
aschool of Pharmacy, health science center, Xi’an Jiaotong university, Xi’an, P.r. china; bdepartment of
c
Genetics and Molecular Biology, health science center, Xi’an Jiaotong university, Xi’an, P.r. china; school
of Pharmacy, Xi’an Medical university, Xi’an, P.r. china; dschool of International education, Xi’an Jiaotong
university, Xi’an, china
ABSTRACT
ARTICLE HISTORY
received 29 March 2017
accepted 13 May 2017
A concise total synthesis of a naturally occurring 3-isopropyl-
tetrahydropyrrolo[1, 2-a]pyrimidine-2, 4(1H, 3H)-dione (ITPD)
isolated from Aconitum taipeicum with a three-step approach was
depicted in this study for the first time. Two key intermediates, diethyl
isopropylmalonate (2) and pyrrolidin-2-amine (3), being synthsesised
separately from initial diethyl malonate (4) and 3, 4-dihydro-2H-
pyrrol-5-amine (5), were utilised to obtain the compound entitled
ITPD. ITPD showed a promising anticancer activity in vitro on SMMC-
7721 cell lines. Flow cytometry and cell cycle analysis revealed that
ITPD could induce apoptosis and cell cycle arrest in S phase. The
occurrence of apoptosis possibly attributed to the mechanism that
ITPD could mediate the mitochondrial pathway through activating
caspase-3/9 and increasing the ratio of Bax/Bcl-2 to finally trigger cell
apoptosis and DNA damage. Collectively, the possibility to produce
sufficient quantity of synthetic ITPD provided the base for further
bio-evaluation in vivo and in vitro. The bioactive assay suggested that
it may be a potential candidate for further chemical optimisation and
use in cancer therapy.
KEYWORDS
total synthesis; ItPd;
Aconitum taipeicum;
anticancer; apoptosis
1. Introduction
Alkaloids, a large group of naturally occurring compounds, are widely distributed in the
plant kingdom and reveal a broad spectrum of remarkably biological activities such as
CONTACT Zengjun Guo
guozj@mail.xjtu.edu.cn
supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2017.1340283.
© 2017 Informa uK limited, trading as taylor & Francis Group

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X. ZHANG ET AL.
antimicrobial, antifungal, anti-inflammatory effects (Tavares Lde et al. 2014; Li et al. 2017;
Shi et al. 2017), especially anticancer property (Zhao et al. 2016). Particularly, camptothecin
and leurocristine are capable of confronting a variety of tumours and being developed into
new drugs with enormous efforts (Liang et al. 2017). Aconitum taipeicum Hand. -Mzt. (A.
taipeicum) belonging to Ranunculaceae family is a perennial herb and uniquely distributed
in the Southern Shaanxi Province of China (The Flora Committee of Chinese Academy of
Sciences 1979). The Traditional Medicine Principle describes that A. taipeicum exhibits expel-
ling wind and promoting blood circulation, relieving spasm and pain, anaesthetising and
combating toxicity and modulating immunity (Shaanxi Chinese Herbal Medicine 1971). The
phytochemical studies have demonstrated the presence of various compounds in this plant
(Wang and Fang 1982; He et al. 2008; Xu et al. 2008, 2012) and the alkaloidal constituents
are probably responsible for the pharmacological efficacies for example antioxidant and
anticancer (Xu and Guo 2008).
A novel amides alkaloid i.e. 3-isopropyl-tetrahydropyrrolo[1, 2-a]pyrimidine-2,4 (1H,
3H)-dione (ITPD) in Figure S1 was recently isolated from the roots of A. taipeicum which
exhibited significant anti-leukaemia activity in a prior screening research (Xu et al. 2008) and
antileishmanial effect due to its diterpenoid alkaloids constituents (Shyaula et al. 2016).
Enough amount of ITPD could guarantee further pharmacological evaluations. However,
relatively a limited quantity was finally obtained from A. taipeicum after undergoing an
exhaustive procedure by conventional separation. Considering the potential activity of ITPD
and due to our interests towards the naturally occurring bioactive alkaloid’s constituents, a
first efficient and concise total synthesis of the compound ITPD was synthesised via a novel
methodology involving three steps with intense efforts. This promising inspection could
make it accessible for comprehensive pharmacological evaluation of ITPD in subsequent
researches. Meanwhile, the underlying mechanism of this alkaloid against SMMC-7721 was
preliminarily investigated in this current study.
2. Results and discussion
The retrosynthetic analysis of the preparation of ITPD (1) was carried out according to the
method depicted in Scheme 1 which commercially available diethyl malonate (4) and 3,
4-dihydro-2H-pyrrol-5-amine (5) were chosen as the precursor materials in the three-step
approach. Accordingly, one key intermediate diethyl isopropylmalonate (2) was obtained
when 4 reacted with isopropyl bromide through alkylation of sodium enolate by refluxing
for 8 h with 81.46% yield. Based on the different partition coefficients of 2 and 4, the resultant
colourless and transparency liquid product was further separated by preparative thin-layer
chromatography (200–300 mesh) with silica gel and petroleum ether-ethyl acetate at ratio
20:1 as the solid and mobile phase, respectively. Conversely, the conventional distillation
method under reduced pressure merely provided a 37.62% yield of 2. Therefore, the former
one was utilised as a purification method. Another crucial intermediate pyrrolidin-2-amine
(3) in this synthesis route was accomplished after the reduction of 5 in the presence of
sodium borohydride via refluxing for 5 h with a yield of 32.30% assisted by ethanol and its
sodium salt. With the aim of combining 2 and 3, they were refluxed together for 15 h to
finally lead to the production of ITPD in yield of 31.08% via asymmetric double amidation
reaction. The filtrate was concentrated as yellow solid after reaction. Then the water layer
was collected and concentrated to attain the product when crude residues were dissolved

NATURAL PRODUCT RESEARCH
3
Scheme 1. retrosynthetic approach and total synthesis of the naturally occurring compound of ItPd.
in water, which was extracted with ethyl acetate and n-butanol, successively. With the pur-
pose of improving its purity, ITPD was dissolved with trichloromethane and placed overnight
in 4 °C refrigerator which later precipitated as a white solid. Its purity was determined by
HPLC and shown in Figure S2.
The reaction procedure and the purity of the compounds were continuously monitored
online through thin-layer chromatography (TLC). The structures of the intermediates 2 and
1
3 including the final product 1 were unambiguously elaborated and characterised by H
NMR, 13C NMR and HR-MS spectrometric analysis. The recorded spectra of the synthesised
ITPD closely resembled the natural product that was previously isolated from A. taipeicum
in our laboratory. The NMR spectra of ITPD were depicted in Figure S3.
The anticancer activity of this synthetic ITPD was investigated by MTT assay against SMMC-
7721, HCT116 and RKO human cell lines. The result demonstrated that ITPD revealed satis-
factory activity confronting SMMC-7721 with IC50 at 37.85 2.94 μM after comparison with
5-FU as the positive drug with IC50 at 19.73 1.30 μM. On the contrary, no promising effects
were observable for HCT116 and RKO. Therefore, SMMC-7721 was chosen to evaluate the
underlying mechanism of ITPD in vitro. Annexin V-FITC/PI assay was performed to quantita-
tively determine the rate of apoptosis. As shown in Figure S4, the negative cells appeared
in the control group after annexin V-FITC and PI staining when SMMC-7721 were treated
with ITPD at the concentration of 10 and 20 μM for 24 and 48 h, respectively. Accompanying
with augmentation of doses and extension of periods, the cancer cells presented an increase

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X. ZHANG ET AL.
proportion of annexin V-positive cells, suggesting that they were at an early stage of apop-
tosis after exposure to ITPD treatment. Meanwhile, the increase in double positive staining
cells proved that they were at the late stage of apoptosis or necrosis. Altogether, the afore-ob-
tained findings provided credible evidences that ITPD exhibited anti-cancer effect via induc-
ing apoptosis of SMMC-7721.
As shown in Figure S5, compared to the control group, ITPD, especially at 20 μM, caused
a remarkable increase in accumulation of S phase cells in a dose-dependent manner, while
the cell quantity in G1 and G2 phase was obviously reduced. It implied that ITPD could
capture the S phase of cell cycle and then caused inhibition of cell proliferation, resulting in
SMMC-7721 damage.
The expression of the pro-apoptotic protein Bax and the anti-apoptotic protein Bcl-2 is
crucial determinants of the apoptotic response mediated by many agents. A high Bax/Bcl-2
ratio is clearly correlated with increased apoptotic sensitivity to anticancer reagents. The
results in Figure S6 showed that the expression of Bax increased, whereas Bcl-2 decreased,
which accounted for the obviously increased ratio of Bax/Bcl-2. The resultant activation of
the caspase cascades including caspase-3 and caspase-9 was further analysed and both
increased in a dose-dependent manner after treatment with ITPD. Thus, ITPD could induce
apoptosis and DNA damage against SMMC-7721 cells through activation of mitochondria
pathways.
3. Experimental section
The experimental section is available online in supplementary material.
4. Conclusions
In summary, a facile and efficient total synthesis of ITPD was described using a three-step
approach involving the formation of 2 and 3 and following their asymmetric double ami-
dation reaction. The target compound exhibited potentially anti-cancer effect against SMMC-
7721 after activity-guided screening assay and the underlying mechanism was probably
attributed to mitochondria signalling pathways mediating the apoptotic process and DNA
damage. This successful synthesis guaranteed the enough quantity of ITPD for comprehen-
sive bio-evaluation in subsequent researches and ITPD may be a potential candidate for
further chemical optimisation and use in cancer therapy.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This study was financially supported by the National Natural Science Foundation of China [grant
number 81603267], [grant number 81172957]; the Fundamental Research Funds for the Central
Universities [grant number 121]; the China Postdoctoral Science Foundation [First-class, grant num-
ber 2016M590958]; the Scientific Research Supporting Project for New Teacher of Xi’an Jiaotong
University [grant number 1191320023]; the Natural Science Foundation of Shaanxi Province [grant
number 2015JQ2048]; the Shaanxi Health and Family Planning Commission [grant number 2016D089].

NATURAL PRODUCT RESEARCH
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