Total synthesis of cytotoxic metabolite (±)-desmethyldiaportinol from Ampelomyces sp.

Article abstract of DOI:10.1080/14786419.2013.866111

A concise total synthesis of (±)-desmethyldiaportinol isolated from Ampelomyces sp. is described. Microwave-assisted cyclocondensation of 3,5-dimethoxyhomopthalic acid with 3,4-dibromobutanoyl chloride afforded the 3-(2,3-dibromopropyl)-6, 8-dimethoxyisocoumarin in 2-3 min as the pivotal step. The 3,4-dibromobutanoyl chloride was itself synthesised from 3-butenoic acid via bromination in carbon tetrachloride at room temperature to yield 3,4-dibromobutanoic acid followed by reaction with thionyl chloride. The replacement of bromo-by hydroxyl substituent was achieved under mild conditions involving the refluxing in a mixture of acetone and water to provide (±)-3-(2,3-dihydroxypropyl)-6,8-dimethoxyisocoumarin which on complete demethylation furnished the title natural product.

Full text of DOI:10.1080/14786419.2013.866111

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Total synthesis of cytotoxic metabolite
(
± )-desmethyldiaportinol from
Ampelomyces sp.
a
a
Aamer Saeed & Muhammad Qasim
a
Department of Chemistry, Quaid-I-Azam University,
5320Islamabad, Pakistan
4
Published online: 04 Dec 2013.
To cite this article: Aamer Saeed & Muhammad Qasim (2014) Total synthesis of cytotoxic
metabolite ( ± )-desmethyldiaportinol from Ampelomyces sp., Natural Product Research: Formerly
Natural Product Letters, 28:3, 185-190, DOI: 10.1080/14786419.2013.866111
To link to this article: http://dx.doi.org/10.1080/14786419.2013.866111
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Natural Product Research, 2014
Vol. 28, No. 3, 185–190, http://dx.doi.org/10.1080/14786419.2013.866111
Total synthesis of cytotoxic metabolite (6)-desmethyldiaportinol from
Ampelomyces sp.
Aamer Saeed* and Muhammad Qasim
Department of Chemistry, Quaid-I-Azam University, 45320 Islamabad, Pakistan
(
Received 19 August 2013; final version received 31 October 2013)
A concise total synthesis of (^)-desmethyldiaportinol isolated from Ampelomyces
sp. is described. Microwave-assisted cyclocondensation of 3,5-dimethoxyhomopthalic
acid with 3,4-dibromobutanoyl chloride afforded the 3-(2,3-dibromopropyl)-6,
8-dimethoxyisocoumarin in 2–3 min as the pivotal step. The 3,4-dibromobutanoyl
chloride was itself synthesised from 3-butenoic acid via bromination in carbon
tetrachloride at room temperature to yield 3,4-dibromobutanoic acid followed by
reaction with thionyl chloride. The replacement of bromo- by hydroxyl substituent was
achieved under mild conditions involving the refluxing in a mixture of acetone and
water to provide (^)-3-(2,3-dihydroxypropyl)-6,8-dimethoxyisocoumarin which on
complete demethylation furnished the title natural product.
Keywords: desmethyldiaportinol; isocoumarin; Ampelomyces sp
1
. Introduction
Isocoumarins (1H-2-benzopyran-1-ones) are the positional isomers of coumarins with an
inverted lactone moiety. More than 300 isocoumarins have been isolated so far from a wide
range of natural sources (microbes, plants, insects and marine organisms) (Kutschera et al. 2003;
Engelmeier et al. 2004). Several biological activities have been reported for these metabolites
including cytotoxic, antimicrobial, antiallergic, algicidal, gastroprotective, protease inhibitor
¨
and antifungal (Krohn et al. 2001; Ozcan et al. 2007; Guimaraes et al. 2008; Oliveira et al. 2009)
as well as plant growth regulatory activities (Kuramata et al. 2007). In addition, nephratoxic,
protease inhibitor, antifungal, cytotoxic, immunomodulatory, antiallergic and antimalarial
activities have also been attributed to this class of natural products (Krohn et al. 2001; Di Stasi
et al. 2004; Kostova et al. 2005; Devienne et al. 2007; Zhang et al. 2008). Most of the natural
isocoumarins posses a 3-alkyl chain (C –C ) or a 3-(un) substituted phenyl ring and
1
17
6
,8-dioxygenation pattern due to their typical biosynthetic origin (Hill 1986).
Fungal endophytes have been proved to be a source of new and biologically active natural
products for specific medicinal and agrochemical uses (Strobel 2003). Thus, Proksch and
coworkers isolated a phenolic isocoumarin metabolite from the ethyl acetate extract of fungal
endophyte Ampelomyces sp. obtained from the flowers of Urospermum picroides. The structure
of was established unambiguously by spectroscopic techniques as 3-(2,3-dihydroxypropyl)-6,8-
dihydroxy-1H-isochromen-1-one which displayed potent in vitro cytotoxic activity against
L5178Y cells of mouse (Aly et al. 2008). The determination of configuration at C-3 proved it to
be the R-enantiomer (Figure 1).
*
Corresponding author. Email: aamersaeed@yahoo.com
q 2013 Taylor & Francis

1
86
A. Saeed and M. Qasim
HO
OH
O
OH
(1)
Figure 1. (^)-3-(2,3-Dihydroxypropyl)-6,8-dihydroxyisocoumarin.
OH
O
As a continuance of our focus on the synthesis and bioevaluation of this important class of
secondary metabolites (Saeed 2003, 2004, 2013; Saeed & Ehsan 2005; Saeed & Ashraf 2008;
Saeed et al. 2011), herein we report a simple and efficient total synthesis of the title compound as
a racemic mixture. The synthesis not only confirms the structural assignment but also makes it
accessible for comprehensive evaluation of its bioactivity.
2
. Results and discussion
An efficient synthesis of (^)-3-(2,3-dihydroxypropyl)-6,8-dihydroxy-1H-isochromen-1-one (1)
was carried out according to the synthetic route revealed in Schemes 1 and 2. Accordingly,
3
-butenoic acid (2) was treated with bromine in carbon tetrachloride at room temperature to
provide (^)-3,4-dibromobutanoic acid (3) which was converted to 3,4-dibromobutanoyl
chloride on treatment with thionyl chloride as a viscous yellow oil which was used in the
following step. The completion of the reaction and purity of the compound were checked by
analytical thin layer chromatography (TLC) where the spot characteristic of acid chlorides was
observed (Scheme 1).
The key intermediate 3,5-dimethoxyhomophthalic acid (4) was prepared starting from 3,5-
dihydroxybenzoic acid according to the procedure reported earlier (Saeed et al. 2003).
Cyclocondensation of 3,4-dibromobutanoyl chloride with 4 afforded 3-(2,3-dibromopropyl)-6,8-
dimethoxyisocoumarin (5) under microwave irradiation in 2–3 min in high yield, while the same
reaction takes 4–8 h under the conventional heating method. Isocoumarin (6) was separated by
preparative thin layer chromatography (PTLC) using silica gel and petroleum ether:ethyl acetate
(2:1) as an eluent and further purified by recrystallisation from methanol. In the FT-IR spectrum, the
d-lactonic carbonyl stretching appeared as a strong absorption at 1724 cm , besides those at
21
2
1
21
126 cm for (CvCZH) and 1570 cm for the aromatic moiety. Isocoumarin 5 exhibited the
3
0
characteristic singletforH-4 olefinic protonat d6.57, the triplet for H-1 at d2.46 (J ¼ 7.5 Hz) andthe
characteristic carbon signals at d 159.3 C-3, 103.5forC-4 and 168.1forlactonic carbon, respectively.
The replacement of bromo- substituents in 5 with hydroxyls was achieved by refluxing with a
mixture of acetone and water in the absence of any base to provide 3-(2,3-dihydroxypropyl)-6,8-
1
dimethoxyisocoumarin (6). In the H NMR spectrum, the singlets at d 3.6, 4.39 ppm and 3.95
confirmed the presence of hydroxy and methoxy protons respectively and the double doublets at
1
,2
1,3
d 2.1 and 2.3 with J ¼ 9.0 Hz, J ¼ 12.0 Hz those of the methylenic protons. The signals at d
1
3
1
65.6 (C-1), 157.2 (C-3), 104.9 (C-4) and 56.17 (2-OMe) were observed in the C NMR
spectrum.
Complete demethylation of (^)-3-(2,3-dihydroxypropyl)-6,8-dimethoxyisocoumarin (6)
was achieved by refluxing with hydrobromic acid in glacial acetic acid (Zou et al. 2008) to afford
O
Br
O
Br
O
(i)
H
(ii)
H
Br
Br
OH
OH
Cl
(2)
(3)
Scheme 1. Synthesis of (^)-3,4-dibromobutanoyl chloride.

Natural Product Research
187
Br
O
O
OH
OH
Br
H
H
Br
MeO
Cl
MeO
(i)
(ii)
O
Br
OMe
O
(5)
OMe
O
(4)
OH
OH
H
H
MeO
HO
(iii)
O
OH
6)
Scheme 2. Synthesis of (^)-desmethyldiaportinol.
O
OH
OMe
O
OH
O
(
(±)-(1)
(
stretching appeared at 3421 cm and in the mass spectrum, base peak was observed at m/z 206.
^)-desmethyldiaportinol (1) (Scheme 2). In the FT-IR spectrum, the characteristic (OZH)
1
2
1
In the H NMR spectrum of isocoumarin (1), besides the disappearance of C-6 and C-8
methoxyls, a characteristic broad singlet for ZOH protons appeared at d 4.84 ppm. Furthermore,
0
H-3 proton appeared as a multiplet at d 5.63 and a characteristic downfield double doublets were
1
3
detected for H-4 proton at d 3.42 and 3.28 ppm. In the C NMR spectrum, carbonyl carbon
CvO) was observed at d 169.6 and an upfield signal for C-3 appeared at d 88.4 ppm. In
(
addition, two downfield carbons C-6 and C-8 were observed at d 160.7 and 160.7 ppm,
respectively. The product was further characterised by the close agreement of its physicospectral
data with that of the natural isocoumarin.
3
3
. Experimental
.1. General methods
All chemicals used in the synthesis were purchased from Sigma-Aldrich and were used as such.
Thin layer chromatography was used to monitor the progress of the reactions. All of the
compounds were purified over silica gel column. Solvents were distilled before use for
purification purposes. Melting points were recorded using a digital Gallenkamp (SANYO,
1
13
Loughborough, UK) model MPD BM 3.5 apparatus and are uncorrected. H NMR and C NMR
spectra were recorded using Bruker AM-300 spectrophotometer (Billerica, Middlesex
Massachusetts, USA) at 300 MHz and 75.5 MHz, respectively, using TMS as an internal
standard. The chemical shift values are recorded on d scale and the coupling constants (J) are in
Hz. FT-IR spectra were recorded using Bio Rad Excalibur FTS 3000 MX spectrophotometer
(Madison, WI, USA). R values are reported in the following solvent system: petroleum ether
f
and ethyl acetate (2:1).
3
3
.1.1. (^)-3,4-Dibromobutanoic acid (3)
-Butenoic acid (2) (6 ml, 0.0025 mol) was dissolved in dry CCl (35 ml) and treated drop wise
4
with bromine (4.5 ml) and the reaction mixture was stirred at room temperature for 3 h. On
completion (TLC control), the reaction mixture was rotary evaporated and then poured into
water. Extracted with ethyl acetate, followed by separation, drying over anhydrous sodium

1
88
A. Saeed and M. Qasim
sulphate and rotary evaporation yielded (^)-3,4-dibromobutanoic acid as a viscous brown oil.
2
Yield 72%. R 0.35. IR (neat) n (cm ); 1722 (CvO acid), 875 (–Br). MS (70 eV) m/z (%):
1
f
þz
2
C, 19.54%; H, 2.46%.
45.87, 243.87 [M] ; elemental analysis: found: C, 19.49%; H, 2.51%; calcd. for C H Br O :
2 2
4
6
3
.1.2. (^)-3,4-Dibromobutanoyl chloride
(^)-3,4-Dibromobutanoic acid (3) (2 ml, 0.0011 mol) was refluxed with thionyl chloride (1 ml)
for 2 h to yield (^)-3,4-dibromobutanoyl chloride as a viscous yellow oil which was used in the
following step.
3
.1.3. (^)-3-(2,3-Dibromopropyl)-6,8-dimethoxyisocoumarin (5)
A homogeneous mixture of 3,5-dimethoxyhomothalic acid (4) (1.9 g, 0.0012 mol) and (^)-3,4-
dibromobutanoyl chloride (0.0024 mol) was irradiated for 2–3 min in an alumina bath inside a
microwave oven with intermittent cooling. The progress of the reaction was followed by TLC
examination using hexane ethyl acetate (2:1). On completion, the reaction mixture was dissolved
in ethyl acetate and purified by PTLC using petroleum ether and ethyl acetate (2:1) to afford (5)
2
1
as a brown solid. Yield 85%. R 0.32. m.p. 118–1208C. IR (neat) n (cm ); 1724 (CvO ester),
f
1
3
126 (CvCZH), 835 (CZBr). H NMR (CDCl ): d 6.6–7.30 (2H, Ar–H), 6.56 (1H, s,
3
1
,2
CvCZH), 3.95 (6H, s, OMe), 3.6 (3H, m, ZCH ZBr), 1.9, 2.0 (2H, dd, J ¼ 8 Hz,
2
1
,3
13
J ¼ 11 Hz, CH ). C NMR (CDCl ): d 165.6 (C-1), 163.4 (C-6), 160.4 (C-8), 157.2 (C-3),
2
3
0
1
3
43.9 (C-9), 112.3 (C-10), 104.9 (C-4), 102.3 (C-5), 100.7 (C-7), 56.17 (2-OMe), 44.45 (C-3 ),
þz
0
0
0.0 (C-4 ) 26.1 (C-5 ). MS (70 eV) m/z (%): 405.92, 403.93 [M] ; elemental analysis: found:
C, 41.39%; H, 3.51%; calcd. for C H Br O : C, 41.41%; H, 3.48%.
2 4
1
4
14
3
3
.1.4. (^)-3-(2,3-Dihydroxypropyl)-6,8-dimethoxyisocoumarin (6)
-(2,3-Dibromopropyl)-6,8-dimethoxyisocoumarin (5) (0.18 g, 0.00032 mol) was refluxed with
an acetone–water mixture (1:5) for 3 h. On completion, the reaction mixture was poured into
water and extracted with ethyl acetate. The organic layer was washed with 5% sodium
bicarbonate and then dried over anhydrous sodium sulphate and rotary evaporated to leave a
solid which was recrystallised from methanol to afford isocoumarin (6) as a light yellow solid.
2
1
Yield 70%. R 0.22. m.p. 130–1338C. IR (neat) n (cm ); 1722 (CvO ester), 3145 (CvCZH),
f
1
H NMR (CDCl ): d 6.6–7.30 (2H, ArZH), 6.57 (1H, s, CvCZH), 4.39 (3H, m, CH -OH), 3.95
2
3
1
,2
1,3
13
(
(
(
(
6H, s, ZOMe), 3.6 (2H, s, ZOH), 2.1, 2.3 (2H, dd, J ¼ 9 Hz, J ¼ 12 Hz, CH ). C NMR
2
CDCl ): d 165.6 (C-1), 163.4 (C-6), 160.4 (C-8), 157.2 (C-3), 143.9 (C-9), 112.3 (C-10), 104.9
3
0
0
0
C-4), 102.3 (C-5), 100.7 (C-7), 56.17 (2-OMe), 44.45 (C-3 ), 57.2 (C-4 ) 67.1 (C-5 ). MS
þz
70 eV) m/z (%): 280.1 [M] ; elemental analysis: found: C, 60.09%; H, 5.77%; calcd. for
C H O : C, 59.99%; H, 5.75;%.
14 16 6
3
.1.5. (^)-Desmethyldiaportinol (1)
3
-(2,3-Dihydroxypropyl)-6,8-dimethoxyisocoumarin (6) (0.1 g, 0.000034 mol) was refluxed
with 33% hydrobromic acid in glacial acetic acid (6 ml) for 2 h. The reaction mixture was poured
into ice 50 g and then solid sodium carbonate was added to manage to pH 6. The compound was
extracted with ethyl acetate and dried and rotary evaporated to give a solid recrystallised from
methanol to afford racemic desmethyldiaportinol (1) as an yellowish powder. Yield 68%. R
f
2
.22. m.p. 1788C. IR (neat) n (cm ); 1722 (CvO ester), 3145 (CvCZH), 3455 (ZOH). H
1
1
0
NMR (CDCl ): d 6.6–7.30 (2H, Ar–H), 6.57 (1H, s, CvCZH), 5.2 (2-OHZAr), 4.39 (3H, m,
3
1
,2
1,3
ZCH ZOH), 3.95 (6H, s, ZOMe), 3.6 (2H, s, OH), 1.9, 2.0 (2H, dd, J ¼ 9.0 Hz, J ¼ 12 Hz,
2

Natural Product Research
189
1
CH2). C NMR (CDCl ): d 165.6 (C-1), 165.4 (C-6), 163.4 (C-8), 157.2 (C-3), 143.9 (C-9),
3
3
0
0
1
6
12.3 (C-10), 104.9 (C-4), 102.3 (C-5), 100.7 (C-7), 56.17 (2-OMe), 44.45 (C-3 ), 57.2 (C-4 )
þz
0
7.1 (C-5 ). MS (70 eV) m/z (%): 252 [M] ; elemental analysis: found: C, 57.21%; H, 4.78%;
calcd. for C H O : C, 57.14%; H, 4.80%.
12 12 6
4
. Conclusions
The first total non-enantioselective synthesis of recently isolated natural cytotoxic metabolite
desmethyldiaportinol was achieved. The pivotal steps include the condensation of acid chloride
with homopthalic acid under microwave irradiation, which drastically reduced the reaction time
and improved the yield, and successful substitution of bromo- with hydroxyls under mild
conditions using acetone and water in the absence of any base to avoid ring opening.
Supplementary material
Supplementary material relating to this article is available online, alongside Figures S1 and S2.
References
Aly AH, Ebel RE, Wray V, Muller WEG, Kozytska S, Hentschel U, Proksch P, Ebel R. 2008. Bioactive metabolites from
the endophytic fungus Ampelomyces sp. isolated from the medicinal plant Urospermum picroides.
Phytochemistry. 69:1716–1725.
Devienne KF, Calgaro-Helena AF, Dorta DJ, Prado, Leda MR, Raddi MSG, Vilegas W, Uyemura SA, Santos AC, Curti
C. 2007. Antioxidant activity of isocoumarins isolated from Paepalanthus bromelioides on mitochondria.
Phytochemistry. 68(7):1075–1080.
Di Stasi LC, Camuesco D, Nieto A, Vilegas W, Zarzuelo A, Galvez J. 2004. Intestinal anti-inflammatory activity of
Paepalantine, an isocoumarin isolated from capitula of Paepalanthus bromelioides, in the trinitrobenzenesul-
phonic acid model of rat colitis. Planta Med. 70:315–330.
Engelmeier D, Hadacek F, Hofer O, Kutschera GL, Nagl M, Wurz G, Greger H. 2004. Antifungal 3-butylisocoumarins
from Asteraceae–Anthemideae. J Nat Prod. 67:19–25.
Guimaraes KL, Souza-Filho JD, Mares-Guia TR, Braga FC. 2008. Dihydroisocoumarin from Xyris pterygoblephara
active against dermatophyte fungi. Phytochemistry. 69:439–444.
Hill RA. 1986. Isocoumarins. Developments since 1950. Fortschritte der Chemie Organischer Naturst Fortschr. 49:1–78.
Kostova I. 2005. Synthetic and natural coumarins as cytotoxic agents. Curr Med Chem. 5:29–46.
Krohn K, Florke U, Rao MS, Steingrover K, Aust H-J, Draeger S, Schulz B. 2001. Metabolites from fungi 15. New
isocoumarins from an endophytic fungus isolated from the canadian thistle Cirsium arvense. Nat Prod Lett.
15:353–361.
Kuramata M, Fujioka S, Shimada A, Kawano T, Kimura Y. 2007. Citrinolactones A, B and C, and Sclerotinin C, plant
growth regulators from Penicillium citrinum. Biosci Biotechnol Biochem. 71(2):499–503.
Kutschera GL, Engelmeier D, Hadacek AW, Greger H, Hofer O. 2003. Synthesis of side chain substituted 3-
butylisocoumarins and absolute configuration of natural isocoumarins from Artemisia dracunculus. Monatsh
Chem. 134:1195–1206.
Oliveira CM, Silva GH, Regasini LO, Zanardi LM, Evangelista AH, Young MCM, Bolzani VS, Ara u´ jo AR. 2009.
Bioactive metabolites produced by Penicillium sp. 1 and sp. 2, two endophytes associated with Alibertia
macrophyllan (Rubiaceae). Z Naturforsch C. 64:824–830.
¨
Ozcan S, Sahin E, Balci M. 2007. The synthesis of unusual isocoumarin derivatives: the chemistry of homophthalic acid.
Tetrahedron Lett. 48:151–2154.
Saeed A. 2003. Stereoselective synthesis of (3R)-3,4-dihydro-6,8-dimethoxy-3-undecyl-1H-[2]-benzopyran-1-one and
derivatives, metabolites from Ononis natrix. Helv Chim Acta. 86:377–383.
0
0
0
Saeed A. 2004. Synthesis of 6,8-dihydroxy-3-(2 -acetyl-3 ,5 -dihydroxyphenyl)methylisocoumarin related to Feralolide.
Nat Prod Res. 18:373–378.
Saeed, A. 2013. An efficient total synthesis of ruprechstyril from Ruprechtia tangarana. Nat Prod Res. 27(13):1153–
1158.
Saeed A, Ashraf Z. 2008. Synthesis of some 3-aryl-1H-isochromene-1-thiones. J Heterocycl Chem. 45:679–682.
Saeed A, Ashraf Z, Rafique H. 2011. Total synthesis and cytotoxic activity of stellatin. J Asian Nat Prod Res. 13:97–104.
Saeed A, Ehsan S. 2005. Synthesis of (^)-Kigelin. J Braz Chem Soc. 16(4):739–741.

1
90
A. Saeed and M. Qasim
Saeed A, Rama NH, Irfan M. 2003. An efficient synthesis of 3,5-dimethoxyhomophthalic acid, a key intermediate for
synthesis of natural isocoumarins. J Heterocycl Chem. 40:519–522.
Strobel GA. 2003. Endophytes as sources of bioactive products. Microbes Infection. 5:535–544.
Zhang W, Krohn K, Draeger S, Schulz B. 2008. Bioactive isocoumarins isolated from the endophytic fungus
Microdochium bolleyi. J Nat Prod. 71:1078–1081.
Zou CL, Ji H, Xie GB, Chen DL, Wang FP. 2008. An effective O-demethylation of some C19-diterpenoid alkaloids with
HBr–glacial acetic acid. J Asian Nat Prod Res. 10:1063–1067.