First total synthesis of prasinic acid and its anticancer activity

Article abstract of DOI:10.1016/j.bmcl.2012.08.116

The first total synthesis of prasinic acid is being reported along with its biological evaluation. The ten step synthesis involved readily available and cheap starting materials and can easily be transposed to large scale manufacturing. The crucial steps of the synthesis included the formation of two different aromatic units (7 and 9) and their coupling reaction. The synthetic prasinic acid exhibited moderate antitumor activity (IC₅₀ 4.3-9.1 μM) in different lines of cancer cells.

Full text of DOI:10.1016/j.bmcl.2012.08.116

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6608–6610
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Bioorganic & Medicinal Chemistry Letters
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First total synthesis of prasinic acid and its anticancer activity
Narayan Chakor, Ganesh Patil, Diana Writer, Giridharan Periyasamy, Rajiv Sharma,
⇑
Abhijit Roychowdhury, Prabhu Dutt Mishra
Department of natural products chemistry, Piramal Healthcare Limited, 1 Nirlon Complex, Goregaon (E), Mumbai 400 063, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 July 2012
Revised 28 August 2012
Accepted 30 August 2012
Available online 13 September 2012
The first total synthesis of prasinic acid is being reported along with its biological evaluation. The ten step
synthesis involved readily available and cheap starting materials and can easily be transposed to large
scale manufacturing. The crucial steps of the synthesis included the formation of two different aromatic
units (7 and 9) and their coupling reaction. The synthetic prasinic acid exhibited moderate antitumor
activity (IC50 4.3–9.1 lM) in different lines of cancer cells.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Natural Products
Prasinic Acid
Anticancer
Total synthesis
Our own anti-cancer screening program led to the discovery of
the biological activity of prasinic acid (Fig. 1). It was isolated and
characterized from fungus Sporothrix sp. Prasinic acid (1) and two
unique diphenyl ethers methoxymicareic acid and micareic acid
were first isolated in 1984 by Elix et.al.1 from Lichen Micareaprasi-
na Fr. These diphenyl ethers are classified as depsides and are
primarily known for their antibiotic,2 anti-HIV,3 antioxidant,4
anti-proliferative activities5 and anti-inflammatory6 activity. These
types of compounds are characterized by the presence of two or
three carboxylic groups held together by ester linkages. Since the
prasinic acid isolated in our laboratory exhibited moderate anti-
cancer activity it was decided to proceed with a total synthesis
of prasinic acid to further evaluate its biological potential. Thus
in this paper we report the first total synthesis of prasinic acid
along with its anti-cancer activity.
The structure of prasinic acid consists of two repeating units of
2-heptyl-4, 6-dihydroxybenzoic acid. The retrosynthetic strategy
involved the synthesis of 2-heptyl-4, 6-dihydroxybenzoic acid
and its subsequent coupling. We envisaged that starting from tri-
hydroxy benzoic acid, 5-heptyl-7-hydroxy-2,2-dimethyl-4H-ben-
zo[d][1,3]dioxin-4-one (6) can be synthesized using a modified
version of the literature procedure, similar to a synthetic strategy
exemplified by Dushin and Danishefsky.7 Following literature pro-
cedures trihydroxybenoic acid upon treatment with trifluoroacetic
acid and trifluoroacetic anhydride in acetone yielded benzodioxin
type compound (2) in 48% yield. Mitsunobu reaction using benzyl
alcohol, triphenyl phosphine, diisopropylazodicarboxylate (DIAD)
in tetrahydrofuran afforded desired benzyl protected compound
(3) in 62% yield. This was further converted to the aryl triflate (4)
using triflic anhydride in pyridine in excellent yields (90%). Cross
coupling of the triflate (4) with 1-heptyne in presence of triphen-
ylphosphine palladium (II) chloride in DMF:TEA yielded the cross
coupled product (5) in good yields (85%). Reduction of the triple
bond and debenzylation of the phenolic hydroxyl was achieved
in one step using 10% Palladium hydroxide on carbon using EtOH
as solvent in a Parr shaker hydrogenator apparatus to yield the de-
sired compound (6) in quantitative yield. Following literature pro-
cedure, our original synthetic design involved coupling of acid 11
with the phenolic hydroxyl of compound 6 to yield the desired es-
ter linkage and subsequently prasinic acid. In order to get acid 11,
compound 5 was treated with 48% KOH in DMSO at 60 °C. Eventu-
ally, acid 11was found to be prone for decarboxylation under
hydrolysis as well as in EDC/DMAP assisted esterification reaction
(Scheme 1).
Thus we revisited the retrosynthetic strategy and planed
protection of hydroxyl moieties to reduce the chances of
COOH
OH
O
O
OH
OH
Figure 1. Structure of prasinic acid.
⇑
Corresponding author. Tel.: +91 22 30818811; fax: +91 22 30818036.
E-mail address: prabhu.mishra@piramal.com (P.D. Mishra).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.116

N. Chakor et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6608–6610
6609
O
O
O
O
O
O
COOH
OH
TFAA,TFA,
Acetone
HO
O
HO
O
HO
BnOH, PPh3,
TfO
O
Tf2O, Py.
90 %
DIAD, THF
62 %
48 %
OBn
OH
4
OH
OBn
3
2
1-Heptyene,
(PPh3)2PdCl2
85 %
DMF:TEA
C5H11
O
OBn
OH
O
O
O
O
O
C5H11
C7H15
O
BnOLi
i)Pd(OH)2, H2
DCM
47 %
EtOH
99 %
OH
OH
OBn
7
6
5
60 oC,
30 min.
48% KOH
DMSO
O
OH
OH
C5H11
OBn
11
Scheme 1.
decarboxylation as a side reaction. Thus treatment of compound 6
with freshly prepared lithium salt of benzyl alcohol (prepared
in situ by using benzyl alcohol and lithium hexamethyldisilazide
ꢀ78 °C in DCM) in THF/DCM under reflux conditions afforded the
required intermediate 7 in 47% yield. The hydroxyls were further
protected using excess K2CO3 and BnBr in acetone to yield benzyl
ether (8) in excellent yields (83%). The carboxylic acid was depro-
tected under basic conditions to afford the desired coupling
precursor (9) in reasonable yields (68%). The phenol (7) and the
carboxylic acid (9) were coupled using EDC/DMAP protocol in
DCM to afford the desired compound (10) as the major product.
A global debenzylation of compound 10 under hydrogenation
C5H11
O
OBn
OH
C5H11
O
OBn
OBn
O
OH
OBn
48 % KOH,
DMSO
K2CO 3, BnBr,
Acetone
C5H11
83 %
83 %
OH
OBn
OBn
7
8
9
7, EDC. HCl
DMAP, DCM
67 %
O
OBn
OH
C5H11
C5H11
O
OH
OH
H2,Pd(OH)2
MeOH
C5H11
O
O
C5H11
O
O
99 %
OBn
10
OH
OBn
OH
Prasinic Acid (1)
Scheme 2.
Table 1
Anti-proliferative activity for synthetic prasinic acid
Sample
ACHN (renal
cancer)
Panc1 (pancreatic
cancer)
Calu1 (lung
cancer)
H460 (non small cell lung
cancer)
HCT116 (colon
cancer)
MCF10A (normal breast
epithelium cells)
Prasinic acid IC50
8.2 0.63
8.5 0.74
4.3 0.32
9.1 0.86
7.8 0.57
>30
(lM)

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N. Chakor et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6608–6610
4. Reynertson, K. A.; Wallace, A. M.; Adachi, S.; Gil, R. R.; Yang, H.; Basile, M. J.;
D’Armiento, J.; Weinstein, B.; Kennelly, E. J. J. Nat. Prod. 2006, 69, 1228.
5. Kumar, K. C. S.; Müller, K. J. Nat. Prod. 1999, 62, 821.
6. Kumar, K. C. S.; Müller, K. J. Eur. J. Med. Chem. 2000, 35, 405.
7. Biological evaluation of the Prasinic acid
condition using Pd(OH)2 in methanol led to prasinic acid in excel-
lent yields (99%). The total synthesis of prasinic acid has been
achieved in ten steps using commercially cheap starting materials
with an overall yield of 9.26% (Scheme 2).
Prasinic acid was evaluated across a panel of cell lines for its
anti-cancer activity. IC50 was determined across different cancer
cells with eight different concentrations of prasinic acid for 48 h
(table 1). The result revealed that, prasinic acid showed concentra-
tion dependent antitumor activity with the IC50 range of
Maintenance of the cell lines
Human tumour cell lines Panc-1 (Pancreatic Cancer), HCT 116 (Colorectal
Cancer), ACHN (Renal Cell Carcinoma), Calu-1 (Lung Carcinoma), were grown in
Minimal Essential Media with Eagle’s Basal Salts (MEM – EBS) obtained from
AMIMED (BioConcept
- Switzerland). H460 (Small Cell Lung Cancer) were
cultured in RPMI 1640 (AMIMED, BioConept, Switzerland). All tumour cell lines
were supplemented with 10% Foetal Bovine Serum (FBS) (GIBCO), 1% Penicillin/
Streptomycin (Sigma) and 1% Anti-Anti (GIBCO) and grown in T-175 tissue
culture flasks (Nunc). MCF-10A non-tumourigenic cell line was cultured in
Mammary Epithelial Basal Medium (MEBM) with all standard additions (Lonza,
Catalog. No. CC-3150). All cells were grown at 37 °C with 5% CO2. Cells were
passaged at 80–90% confluence. Adherent cells were trypsinised using Trypsin-
EDTA (Sigma) and maintained. All cell lines were purchased from ATCC
(Rockville, MD, USA)
4.3–9.1
l
M in different cancer cells, while in normal breast epithe-
M at 48 h. This data
lium cells (MCF10A) it showed an IC50 of >30
l
also predicts partial selectivity of this compound towards highly
proliferating cancer cells and thus might lead to an acceptable
therapeutic window.
In conclusion we have reported for the first time a total synthe-
sis of prasinic acid in ten steps. The successful coupling of the two
aromatic moieties (7 and 9) achieved with proper choice of the
protecting groups. The natural product is of biological significance
as exhibited by its anti-cancer activity across various cell lines. The
total synthesis will also lead to the initiation of a medicinal chem-
istry project around prasinic acid to improve activity and impart
drug-likeliness to this natural product.
Sample preparation
Prasinic acid is weighed and dissolved in the required amount of DMSO to give a
required stock solution of 20 mM.Eight different concentrations of prasinic acid
were prepared by serial dilution of stock solution finally resulting in
(compared to the test concentration) 200-fold higher concentration. Prasinic
acid was tested at 0.01, 0.03, 0.1, 0.3, 1, 3, 10 and 30
evaluated in triplicate.
a
lM. Each concentration was
Method for IC50 determination
A
modified Propidium Iodide assay was used to assess the effect of the
compound of formula (1) on the growth of the human tumor cell lines and was
designed as in reference, Anti-cancer Drugs, 6, 522–532, (1995).
Cells were plated in 96-well flat-bottom microtiter plates at a cell density of
Acknowledgments
4000 cells/well. After
exponential growth, the compound of formula (1) was applied at
concentrations in triplicates and treatment continued for 2 days. After 2 days
of treatment, cells were next washed with 200 l phosphate buffer solution
(PBS) to remove dead cells, then 200 of solution containing g/ml
a 24 h recovery period to allow the cells to resume
8
We are indebted to the analytical facility at Piramal Healthcare
Ltd. for recording spectral data of all the compounds.
l
l
l
a
7 l
propidium iodide (PI) and 0.1%(v/v) Triton X-100 were added to the wells. After
an incubation period of 1–2 h at room temperature, fluorescence (FU) was
measured using the Cytofluor 4000 microplate reader (excitation k = 530 nm,
emission k = 620 nm) to quantify the amount of attached viable cells.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.08.
116.
ðReading of Vehicle controlꢀReading of Treated cellsÞ
Percent Cytotoxicity¼
Reading of Vehicle Control
ꢁ100
References and notes
1. Elix, John A.; Jones, Alan J.; Lajide, Labunmi; Coppins, Brian. J.; James, P. W. Aust.
J. Chem. 1984, 37, 2349.
2. Nielsen, J.; Halfdan, P.; Frisvad, J. C. Phytochemistry 1999, 50, 263.
3. Neamati, N.; Hong, H.; Muzumder, A.; Wang, S.; Sunder, S.; Nicklaus, M. C.;
Milne, G. W. A; Proksa, B.; Pommier, Y. J. Med. Chem. 1997, 40, 942.
If Graph Pad Prism could not calculate reliable IC50 values by non-
linear regression, the IC50 value was estimated by visual inspection
of the concentration-effect curve.