Optimized protocol for multigram preparation of emodin anthrone, a precursor in the hypericin synthesis

Article abstract of DOI:10.1080/14786419.2018.1457661

Emodin reduction to emodin anthrone comprise one of three process steps involved in the hypericin synthesis, a powerful natural photosensitiser found in plants of the genus Hypericum. In this communication, an optimized protocol was established for emodin reduction enabling an efficient multigram preparation of emodin anthrone. A screening of reducing agent (SnCl₂·2H₂O and HCl_conc) under different reaction times was employed in micro-scale and monitored by electronic absorption spectroscopy technique. Data showed lower yields of emodin anthrone when some experimental conditions previously described in the literature were reproduce. However, using the optimized protocol for the emodin reduction these yields were overcoming, and a gram-scale supply experiment was reproducible for the preparation of 10 grams of emodin anthrone with excellent yield.

Full text of DOI:10.1080/14786419.2018.1457661

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
Optimized protocol for multigram preparation of
emodin anthrone, a precursor in the hypericin
synthesis
Renato Sonchini Gonçalves, Gabriel Batista César, Patrícia Magalhães
Barbosa, Noboru Hioka, Celso Vataru Nakamura, Marcos Luciano Bruschi &
Wilker Caetano
To cite this article: Renato Sonchini Gonçalves, Gabriel Batista César, Patrícia Magalhães
Barbosa, Noboru Hioka, Celso Vataru Nakamura, Marcos Luciano Bruschi & Wilker Caetano
(2018): Optimized protocol for multigram preparation of emodin anthrone, a precursor in the
hypericin synthesis, Natural Product Research, DOI: 10.1080/14786419.2018.1457661
To link to this article: https://doi.org/10.1080/14786419.2018.1457661
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Natural Product research, 2018
ꢁꢁpꢂ://ꢃꢄi.ꢄꢅg/10.1080/14786419.2018.1457661
ꢀ
SHORT COMMUNICATION
Optimized protocol for multigram preparation of emodin
anthrone, a precursor in the hypericin synthesis
a
a
a
Renato Sonchini Gonçalves , Gabriel Batista César , Patrícia Magalhães Barbosa ,
a
b
b
Noboru Hioka , Celso Vataru Nakamura , Marcos Luciano Bruschi and
a
Wilker Caetano
ꢆ
b
dꢇpꢆꢅꢁmꢇnꢁ ꢄf cꢀꢇmiꢂꢁꢅy, sꢁꢆꢁꢇ univꢇꢅꢂiꢁy ꢄf Mꢆꢅingá, Mꢆꢅingá, Bꢅꢆziꢈ; dꢇpꢆꢅꢁmꢇnꢁ ꢄf Pꢀꢆꢅmꢆꢉy, sꢁꢆꢁꢇ
univꢇꢅꢂiꢁy ꢄf Mꢆꢅingá, Mꢆꢅingá, Bꢅꢆziꢈ
ABSTRACT
ARTICLE HISTORY
Emodin reduction to emodin anthrone comprise one of three
process steps involved in the hypericin synthesis, a powerful natural
photosensitiser found in plants of the genus Hypericum. In this
communication, an optimized protocol was established for emodin
reduction enabling an efficient multigram preparation of emodin
rꢇꢉꢇivꢇꢃ 25 oꢉꢁꢄbꢇꢅ 2017
aꢉꢉꢇpꢁꢇꢃ 18 Mꢆꢅꢉꢀ 2018
KEYWORDS
hypꢇꢅiꢉin; ꢇmꢄꢃin; ꢇmꢄꢃin
ꢆnꢁꢀꢅꢄnꢇ; ꢁin(II) ꢉꢀꢈꢄꢅiꢃꢇ
ꢃiꢀyꢃꢅꢆꢁꢇ; ꢅꢇꢃꢊꢉꢁiꢄn
ꢅꢇꢆꢉꢁiꢄn; ꢇꢈꢇꢉꢁꢅꢄniꢉ
ꢆbꢂꢄꢅpꢁiꢄn ꢂpꢇꢉꢁꢅꢄꢂꢉꢄpy;
ꢄpꢁimizꢇꢃ pꢅꢄꢁꢄꢉꢄꢈ
anthrone. A screening of reducing agent (SnCl ·2H O and HClconc)
2
2
under different reaction times was employed in micro-scale and
monitored by electronic absorption spectroscopy technique. Data
showed lower yields of emodin anthrone when some experimental
conditions previously described in the literature were reproduce.
However, using the optimized protocol for the emodin reduction
these yields were overcoming, and a gram-scale supply experiment
was reproducible for the preparation of 10 grams of emodin anthrone
with excellent yield.
CONTACT rꢇnꢆꢁꢄ sꢄnꢉꢀini Gꢄnçꢆꢈvꢇꢂ
ꢅꢂgꢄnꢉꢆꢈvꢇꢂ2@ꢊꢇm.bꢅ
sꢊppꢈꢇmꢇnꢁꢆꢈ ꢃꢆꢁꢆ fꢄꢅ ꢁꢀiꢂ ꢆꢅꢁiꢉꢈꢇ ꢉꢆn bꢇ ꢆꢉꢉꢇꢂꢂꢇꢃ ꢆꢁ ꢀꢁꢁpꢂ://ꢃꢄi.ꢄꢅg/10.1080/14786419.2018.1457661.
©
2018 Infꢄꢅmꢆ uK limiꢁꢇꢃ, ꢁꢅꢆꢃing ꢆꢂ tꢆyꢈꢄꢅ & Fꢅꢆnꢉiꢂ Gꢅꢄꢊp

2
R. S. GONÇALVES ET AL.
1
. Introduction
Emodin is a naturally occurring anthraquinone derivative found in a variety of medicinal
plant species. Although emodin has been widely studied due its broad spectrum of phar-
macological activities, it serves as a precursor to access the emodin anthrone in the prepa-
ration of naphtodianthrones, comprising mainly the chemical synthesis and biosynthesis of
hypericin and its derivative fagopyrin (Falk 1999; Abd-Alla et al. 2009; Zdunić et al. 2011;
Sytar et al. 2013; Huang et al. 2014; Dong et al. 2016; Braumann et al. 2017). During last years,
a number of antimicrobial and anticancer effects of hypericin and fagopyrine have been
discovered, justifying the importance of developing a methodology for the preparation of
its precursor (Wada et al. 2002; Karioti and Bilia 2010; Sytar et al. 2016; Montanha et al. 2017).
Therefore, in order to overcome the main difficulty concerning the hypericin isolation in
a pure state direct from plant material, in the last decade a number of synthetic routes have
been proposed, standing out one simplified three process steps starting from emodin reduc-
tion reaction (Rodewald et al. 1977; Mazur et al. 1989). However, a large number of divergence
in the experimental conditions, mainly related to initial emodin reduction step can be found
in the previous works (Falk et al. 1993, 1995, 1998; Altmann et al. 1998; Motoyoshiya et al.
2007; Tobia et al. 2012).
Moreover, some experimental conditions reproduced by us led to very lower yields of
emodin anthrone, and considering the expensive commercial cost of emodin (30 mg ~ USD
70) an increase in the final production cost of hypericin is unavoidable hindering its pro-
2
duction on large scale. In this present communication, we show that the yield of the emodin
anthrone and consequently the overall yield of hypericin synthesis provided by previous
works can be significantly improved if the emodin reduction step is performed under optimal
experimental condition set here (Scheme 1).
2
. Results and discussion
Initially a significant amount of emodin was accessed with high purity degree (Figure S1) by
reproduce of the optimized protocol for the isolation of anthraquinones from Rhamnus
Frangula L. reported by us in a previously communication (Gonçalves et al. 2017). To an initial
−2
−1
emodin solution (5.0 × 10 mol·L ) in acetic acid, tin(II) chloride dihydrate (SnCl ·2H O) and
2
2
concentrated hydrogen chloride were employed as reducing agents in agreement with the
reported works (Mazur et al. 1989; Falk et al. 1993). Thus, we performed a screening of
the molar amounts of reducing agents and of reaction time (Table S1), with monitoring of
the reaction progress by electronic absorption technique. In the first screening (entries 1–3),
Scheme 1. opꢁimizꢇꢃ ꢇxpꢇꢅimꢇnꢁꢆꢈ ꢉꢄnꢃiꢁiꢄn fꢄꢅ ꢁꢀꢇ ꢇmꢄꢃin ꢅꢇꢃꢊꢉꢁiꢄn ꢅꢇꢆꢉꢁiꢄn.

NATURAL PRODUCT RESEARCH
3
no significant change was observed in the intensity of emodin electronic absorption band
at λmax = 450 nm, even to a prolonged reaction time of 6 h. Next, we set the same molar
amount of the reducing agents (entry 4), described by Falk et al. (1993), however the emodin
anthrone yield was much lower than that of reported reaction. For an added 50-fold molar
excess of hydrogen chloride, an improvement of 61% in the emodin reduction was achieved
for a reaction time of 3 h (entry 5). At this stage, it becomes evident that a large excess of
HClconc should be used to promote an effective reduction of emodin with a significant reduc
ing of the reaction time.
-
For our delight, the optimal experimental condition was set for the combination of 3.2
and 100.0 equivalents of SnCl ·2H O and HClconc, respectively resulting in a very efficient
2
2
conversion of emodin to emodin anthrone with 87% yield (Figures S2 and S3), and with an
expressive reduction of the reaction time for 30 min (entry 6). The UV-vis monitoring show
that in the first five minutes of reaction, the emodin absorption band (λmax = 450 nm)
decrease rapidly from 0.4 to approximately 0.1, accompanied of an increase of 0.2 in the
emodin anthrone absorption band at λmax = 356 nm, which reached a maximum value of
0.7 for a reaction time of 30 min (Figure S4(A)). In this way, the next experiments were per-
formed out in order to demonstrate the effect of some extreme experimental conditions,
reported previously. For instance, Motoyoshiya et al. described the use of a large excess of
reducing agents (10.0 and 2.505 M equivalent of SnCl ·2H O and HClconc, respectively) and
2
2
−1
a prolonged reaction time of 24 h for the reduction of an 0.048 mol·L initial emodin solution
Motoyoshiya et al. 2007). For comparison purposes, the optimal molar amounts of reducing
agents was maintained, and the emodin reducing was evaluated with reaction times of 3,
and 6 h (entries 7–9). However, for 3 h of reaction, the maximum intensity of emodin
(
5
anthrone absorption band show an expressive decrease of 25% (0.7 to 0.5) (Figure S4(B)).
Moreover, the decrease of absorption intensity become even more pronounced for reaction
times of 5 and 6 h, leading to a dramatic decrease of 42 and 58%, respectively. Interestingly,
at this reaction stage (6 h), the reaction proceeded with a yellow (optimal condition) to green
1
color change (Figure S5). Analyzing the H NMR spectrum of crude product, the presence of
additional aromatic and aliphatic hydrogen signals were observed (Figure S6), probably, due
the correspondent formation of over-reduced products, justifying this abrupt color change.
Finally, a gram-scale supply experiment was performed in order to evaluate the repro-
ducibility of the optimal experimental condition, initially accessed in micro-scale (entry 6).
−1
However, 10.0 g of emodin (0.1 mol·L ) was reduced to emodin anthrone with excellent
yield after a reaction time of ca. 90 min, proving the efficiency of the set optimized param-
eters. Furthermore, the optimal protocol provided here can be extended to other anthraqui-
nones derivatives.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico [grant
number 308142/2014‐4].

4
R. S. GONÇALVES ET AL.
ORCID
Renato Sonchini Gonçalves
http://orcid.org/0000-0003-2701-6101
References
Abd-Alla HI, Shaaban M, Shaaban KA, Abu-Gabal NS, Shalaby NMM, Laatsch H. 2009. New bioactive
compounds from Aloe hijazensis. Nat Prod Res. 23(11):1035–1049.
Altmann R, Falk H, Gruber HJ. 1998. Synthesis and properties of ionophore conjugated hypericin
derivatives. Monatshefte fur Chemie. 129(3):235–244.
Braumann C, Koplin G, Geier C, Höhn P, Pohlenz J, Dubiel W, Rogalla S. 2017. Dose-dependent role of
novel agents emodin and BTB14431 in colonic cancer treatment in rats. Acta Chir Belg. 117(6):376–
384.
Dong X, Fu J, Yin X, Cao S, Li X, Lin L, Ni J. 2016. Emodin: a review of its pharmacology, toxicity and
pharmacokinetics. Phyther Res. 30(8):1207–1218.
Falk H. 1999. From the photosensitizer hypericin to the photoreceptor stentorin – the chemistry of
phenanthroperylene quinones. Angew Chemie. 38(21):3116–3136.
Falk H, Meyer J, Oberreiter M. 1993. A convenient semisynthetic route to hypericin. Monatshefte für
Chemie Chem Mon. 124(3):339–341.
Falk H, Sarhan AEWAO, Tran HTN, Altmann R. 1998. Synthesis and properties of hypericins substituted
with acidic and basic residues: hypericin tetrasulfonic acid - a water soluble hypericin derivative.
Monatshefte fur Chemie. 129(3):309–318.
Falk H, Vaisburg AF, Amer AM. 1995. On the synthesis of ω-appended hypericin derivatives. Monatshefte
für Chemie Chem Mon. 126(8–9):993–1000.
Gonçalves RS, Silva EL, Hioka N, Nakamura CV, Bruschi ML, Caetano W. 2017. An optimized protocol for
anthraquinones isolation from Rhamnus frangula L. Nat Prod Res. 32(3):366–369.
Huang LF, Wang ZH, Chen SL. 2014. Hypericin: chemical synthesis and biosynthesis. Chin J Nat Med.
12(2):81–88.
Karioti A, Bilia AR. 2010. Hypericins as potential leads for new therapeutics. Int J Mol Sci. 11(2):562–594.
Mazur Y, Bock H, Lavie D. 1989. Preparation oh hypericin. Patent, CA 2 029 993.
Montanha MC, Silva LL, Pangoni FBB, Cesar GB, Gonçalves RS, Caetano W, Hioka N, Tominaga TT,
Consolaro MEL, Diniz A, Kimura E. 2017. Response surface method optimization of a novel Hypericin
formulation in P123 micelles for colorectal cancer and antimicrobial photodynamic therapy.
J Photochem Photobiol B Biol. 170:247–255.
Motoyoshiya J, Masue Y, Nishi Y, Aoyama H. 2007. Synthesis of hypericin via emodin anthrone derived
from a twofold Diels-Alder reaction of 1,4-benzoquinone. Nat Prod Commun. 2(1):67–70.
Rodewald G, Arnold R, Griesler J, Steglich W. 1977. Synthesis of hypericin and related meso-naphtho-
dianthrones by alkaline dimerization of hydroxyan- thraquinones. Angew Chem. 16(1):46–47.
Sytar O, Brestic M, Rai M. 2013. Possible ways of fagopyrin biosynthesis and production in buckwheat
plants. Fitoterapia. 84:72–79.
Sytar O, Švedienė J, Ložienė K, Paškevičius A, Kosyan A, Taran N. 2016. Antifungal properties of hypericin,
hypericin tetrasulphonic acid and fagopyrin on pathogenic fungi and spoilage yeasts. Pharm Biol.
54(12):3121–3125.
Tobia AJ, Cabana BE, Vadlaplatla V, Connolly RH. 2012. Methods for preparing hypericin. Patent,
US20120245392A1.
Wada A, Sakaeda T, Takara K, Hirai M, Kimura T, Ohmoto N, Zhou J, Nakamura T, Kobayashi H, Okamura
N, et al. 2002. Effects of St John’s Wort and hypericin on cytotoxicity of anticancer drugs. Drug Metab
Pharmacokinet. 17(5):467–474.
Zdunić G, Gođevac D, Šavikin K, Novaković M, Milosavljević S, Petrović S. 2011. Isolation and identification
of phenolic compounds from Hypericum richeri Vill. and their antioxidant capacity. Nat Prod Res.
25(3):175–187.