Synthesis and antibacterial evaluation of 3-Farnesyl-2-hydroxybenzoic acid from Piper multiplinervium

Article abstract of DOI:10.1016/j.fitote.2014.01.005

3-Farnesyl-2-hydroxybenzoic acid is an antibacterial agent isolated from the leaves of Piper multiplinervium. This compound has activity against both Gram positive and Gram negative bacteria including Escherichia coli, Staphylococcus aureus and Helicobact

Full text of DOI:10.1016/j.fitote.2014.01.005

Fitoterapia 93 (2014) 189–193
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Synthesis and antibacterial evaluation of
3-Farnesyl-2-hydroxybenzoic acid from Piper multiplinervium
b
b
Ibrahim Malami a, , Simon Gibbons , John P. Malkinson
⁎
a
Department of Pharmacognosy and Ethnopharmacy, Usmanu Danfodiyo University, Sokoto, P.M.B 2346 Sokoto, Nigeria
Department of Pharmaceutical and Biological Chemistry, UCL School of Pharmacy, 29-39 Brunswick Square, London WC1N 1AX, United Kingdom
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 15 April 2013
Accepted in revised form 31 December 2013
Available online 18 January 2014
3-Farnesyl-2-hydroxybenzoic acid is an antibacterial agent isolated from the leaves of Piper
multiplinervium. This compound has activity against both Gram positive and Gram negative
bacteria including Escherichia coli, Staphylococcus aureus and Helicobacter pylori. This research
aimed to synthesize a natural antibacterial compound and its analogs. The synthesis of 3-Farnesyl-
2-hydroxybenzoic acid consists of three steps: straightforward synthesis involving protection of
phenolic hydroxyl group, coupling of suitable isoprenyl chain to the protected aromatic ring at
ortho position followed by carboxylation with concomitant deprotection to give the derivatives
of the salicylic acid. All the three prenylated compounds synthesized were found to exhibit
spectrum of activity against S. aureus (ATCC) having MIC: 5.84 × 10−3, 41.46 × 10−2 and
6.19 × 10−1 μmol/ml respectively. The compounds also displayed activity against resistance
strain of S. aureus (SA1119B) having MIC: 5.84 × 10−3, 7.29 × 10−3 and 3.09 × 10−1 μmol/ml
respectively. This synthesis has been achieved and accomplished with the confirmation of it
structure to that of the original natural product, thus producing the first synthesis of the natural
product and providing the first synthesis of its analogs with 3-Farnesyl-2-hydroxybenzoic acid
having biological activity higher than that of the original natural product.
Keywords:
3-Farnesyl-2-hydroxybenzoic acid
Prenylated salicylic acid
Piper multiplinervium
Staphylococcus aureus
Antibacterial
© 2014 Elsevier B.V. All rights reserved.
1. Introduction
ones [1]. However, mithicillin-resistance S. aureus (MRSA) is
resistant to all β-lactam antibiotics [2] and thus making the
Resistance to antibiotics by bacterial strains especially multi-
drug resistance has become a major concern in public health all
over the world. Widespread use of antibacterial agents in
hospitals, food production, and veterinary treatments among
others has resulted in rapid emergence of antibiotics resistance.
The search for new antibacterial agents, particularly those which
are effective against multi-drug resistant pathogens has become
a major global focus and subject of research in almost all research
institutions. It is certain that most of the bacterial strains
(e.g. Staphylococcus aureus) found in most research institutions
are resistant to antibiotic. S. aureus is one of the most important
pathogenic bacteria, causing a wide range of infections from local
and usually superficial infections to extreme severe systematic
antibiotic treatment option limited. Therefore, the widespread
of multidrug resistant bacterial strains necessitates the discov-
ery of new class of antibacterial and compounds that inhibit
these resistance mechanisms [3].
Medicinal plants are used for centuries as remedies for
human diseases and offer the richest biosources of drugs or
traditional medical system, modern medicines, neutraceuticals,
food supplements, folk medicines, pharmaceuticals, intermedi-
ate and chemical entities for synthetic drugs [4]. Among the
estimated 250,000–500,000 plant species [5], Piper species are
estimated to contain more than 4000 species widely distributed
in the tropical and subtropical region of the world, its species
are chemically very rich and contained major classes of com-
pounds [6,7] of which among the classes of compounds include
prenylated benzoic acid derivatives [8].
3-Farnesyl-2-hydroxybenzoic acid (1) is an antibacterial
compound isolated and characterized from the leaves of Piper
⁎
Corresponding author. Tel.: +234 8166923199.
E-mail address: keepibinformed@yahoo.co.uk (I. Malami).
0367-326X/$ – see front matter © 2014 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.fitote.2014.01.005

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I. Malami et al. / Fitoterapia 93 (2014) 189–193
multiplinervium. The plant is traditionally used by the local
Kuna Indians of Panama for the treatment of stomach ache.
Compound 1 was found to have broad spectrum of activity
against both Gram positive and Gram negative bacteria includ-
ing Escherichia coli, S. aureus and Helicobacter pylori [9]. 1 is
composed of C22H31O3 with a molecular weight of 342.2273
and possessed a remarkable structure that the farnesyl group is
found at the ortho position to hydroxyl group of phenol.
a Finnigan MassLab Navigator quadruple instrument using
electrospray (ES) visualization.
2.1. Synthesis of 3-Farnesyl-2-hydroxybenzoic acid (1) and its
analogs
All the reaction procedures in this experiment were
followed according to the procedure reported [12] as outlined
in Fig. 1. Sodium hydride (1.5 Eq.) was suspended in
tetrahydrofuran (40 mL) and allowed to cool to 0 °C under
argon atmosphere. To the NaH suspension, 4 dissolved in dry
THF was added dropwise and allowed to stir at r.t for 2 h. To
the reaction mixture, Chloromethyl methyl ether (3 Eq.) was
added dropwise and allowed to stir for 2 h. The reaction
mixture was concentrated in a rotary evaporator and the
residue partitioned between ether and 0.1 M NaOH. The
aqueous layer was extracted again with ether and the organic
layers were then combined, washed with 0.1 M NaOH and
with brine respectively. The organic layers were dried
over MgSO4, filtered and evaporated to dryness in a rotary
evaporator. The organic oil obtained was then purified by
column chromatography. 5 (3.38 mmol) was dissolved in
ether (5 mL) and allowed to cool to −78 °C. To the reaction
mixture, t-BuLi (2.2 Eq) was added dropwise over 5 min and
allowed to stir maintaining the temperature at −78 °C for
over 1 h. To the reaction mixture, Farnesyl, geranyl or
dimethylallyl bromide was added dropwise over 2 min and
the reaction mixture was allowed to stir at r.t over 1 h. The
reaction mixture was then diluted with ether and quenched
with saturated Na2CO3 (5 mL). The aqueous layer was ex-
tracted with ether and the organic layers was then combined
and washed with brine. The organic extract was dried
over MgSO4, filtered and evaporated to dryness in a rotary
evaporator. The crude product obtained was purified by
flash chromatography and further purified by TLC. 6, 7, or 8
was dissolved in THF and allowed to cool to −78 °C. To the
reaction mixture, t-BuLi (2.2 Eq.) was added dropwise over
5 min and allowed to stir maintaining the temperature at −
78 °C for over 1.5 h. To the reaction mixture, CO2(g) was
bubbled directly via a column of drierite and allowed to stir
at −78 °C for over 1.5 h. HCl was added to the reaction
mixture at r.t and allowed to stir overnight. The resulting
reaction mixture was extracted twice with ether, washed
with brine and dried over MgSO4. The organic filtrate obtained
was evaporated to dryness in a rotary evaporator and purified
by preparative TLC.
Phytochemical investigations previously carried out on
different Piper species revealed to contain several interesting
prenylated benzoic acid with interesting biological activities
and have been described to contain structurally similar com-
pounds [10,11]. Despite the frequent isolation of these interest-
ing compounds, there is less report available on the antibacterial
activity from Piper species. In view of this, the present studies
aimed to synthesize a natural antibacterial compound 1 and its
analogs and evaluate them against bacteria. This compound
is given much interest because of the increasing demand on
antibacterial agents against multi-drug resistant bacteria.
2. Materials and methods
All the chemical reactions were conducted inside the
fume cupboard under an inert gas atmosphere of either argon
or nitrogen using well oven-dried glassware. THF and ether
were allowed to dry distilled over sodium before use. Most
reagents and equipments used were commercially available
and are obtained from Sigma Aldrich while CO2 was provided
by passing through a column of Drierite. All the reactions
were monitored by TLC (Silica gel 60F254), and spotted under
UV and the major spot identified by spraying with PMA
in EtOH. The synthesized compounds and their intermedi-
ates were purified by chromatography and analyzed by
NMR spectroscopy recorded on Bruker Avance spectrometer
(1H 400 MHz, 13C 100 MHz in CDCl3, δ 7.26 as internal
reference). The spectral data were recorded in ppm (δ),
coupling constant in Hertz, NMR pick patterns are in broad
(br), singlets (s), doublets (d), double-doublet (dd), triplets
(t) and multiplets (m). Mass spectroscopy was recorded on
1.5 Eq. NaH, THF
R =
+
O
Cl
Br
Br
Br
Br
O
5
O
OH
4
R =
R =
THF, t-BuLi
2.2 Eq. t-BuLi
Ether, 1.2 Eq. R
CO2
HOOC
R
Br
R
OH
O
O
Fig. 1. Synthesis of 3-Farnesyl-2-hydroxybenzoic acid and it analogs in three steps.

I. Malami et al. / Fitoterapia 93 (2014) 189–193
191
2.2. Assessment of antibacterial activity
3.65 (s, 3H), 5.09 (s, 2H), 5.10 (m, 2H), 5.31 (m, 1H), 6.93
(t, J = 8.0 Hz, 1H), 7.13 (dd, J = 7.6 Hz, 1H), 7.39 (dd,
J = 7.6 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ: 16.39,
16.52, 18.04, 26.05, 26.84, 27.08, 29.10, 40.03, 58.18,
100.14, 117.71, 121.68, 121.72, 122.32, 124.35, 124.69,
124.72, 125.94, 129.42, 129.89, 131.41, 131.65, 135.52
S. aureus (ATCC) and S. aureus (SA1119B) were used in the
assessment of antibacterial assay. The bacterial strains used
were obtained from the American Type Culture Collection
(ATCC). All the compounds were determined in duplicate
by minimum inhibitory concentration (MIC) values using
micro-broth dilution method [13] and Norfloxacin was used
as positive control. The microplate was incubated overnight
at 37 °C and the MIC values were determined as the lowest
concentration of the compounds that inhibits visible growth
of the bacterial strains.
2-bromo-6-geranyl-1-methoxymethoxybenzene (10)
10 was obtained as (0.167 g, 14%); Rf 0.59(9:1 hexane/
EtOAc), 1H NMR (400 MHz, CDCl3) δ: 1.60(s, 3H), 1.68(s,
3H), 1.70(s, 3H), 2.06–2.13 (m, 4H), 3.46(d, J = 7.12 Hz,
2H), 3.65(s, 3H), 5.09(s, 2H), 5.10(m, 1H), 5.30(m, J =
7.2 Hz, 1H), 6.93(t, J = 7.8 Hz, 1H), 7.13(dd, J = 7.6 Hz,
1H), 7.40(dd, J = 7.9 Hz, 1H). 13C NMR (100 MHz, CDCl3)
δ: 16.5, 18.1, 26.1, 40.0, 26.9, 29.1, 58.2, 100.1, 124.5, 122.4,
125.9, 129.4, 131.4, 131.9, 117.7, 153.0, 137.4, 137.8.
3. Result and discussion
3.1. Chemical synthesis
The basic principles of the reactions are highlighted in Fig. 1.
The spectral data of the synthesized compounds and their
intermediates are given below:
2-bromo-6-dimethlyallyl-1-methoxymethoxybenzene (11)
11 was obtained as (0.120 g, 12.4%), Rf 0.34 (10% EtAOc/
Hexane) 1H NMR (400 MHz, CDCl3) δ: 1.71(s, 3H), 1.75 (s,
3H3.45 (d, 2H), 3.65 (s, 3H), 5.09 (s, 2H), 5.30 (m, 1H),
6.93 (t, J = 8.0 Hz, 1H), 7.12 (dd, J = 6.8 Hz, 1H), 7.39
(dd, J = 7.9 Hz 1H). 13C NMR (100 MHz, CDCl3) δ: 18.21,
26.08, 29.32, 58.20, 100.17, 117.75, 122.45, 125.96, 129.48,
131.45, 133.68, 137.82, 152.97.
1, 3-dibromo-2-methoxymethoxybenzene (5)
Colorless oil, (12.05 g, 88%); Rf 0.34 (9:1 Hexane/EtOAc),
1H NMR (400 MHz, CDCl3) δ: 3.73(s, 3H), 5.18(s, 2H),
6.88(t, J = 8.0 Hz, 1H), 7.52(d, J = 8.0 Hz, 2H). 13C NMR
(100 MHz, CDCl3) δ: 58.5, 99.6, 188.6, 125.5, 132.9, 151.9.
2-bromo-6-Farnesyl-1-methoxymethoxybenzene (9)
Compound 9 was obtained as (0.45 g, 21%); Rf 0.54 (19:1,
hexane/EtOAc), 1H NMR (400 MHz, CDCl3) δ: 1.60 (s, 6H),
1.68 (s, 3H), 1.78 (s, 3H), 1.95–2.14 (m, 8H), 3.47 (d, 2H),
3-Farnesyl-2-hydroxybenzoic acid (1)
1 was afforded in 0.033 g, 10.7%; Rf 0.4 (3:1:2 drops
CHCl3/MeOH/AcOH), 1H NMR (400 MHz, CDCl3) δ: 1.61
(s, 6H), 1.67 (s, 3H), 1.71 (s, 3H), 1.96–2.15 (m, 8H), 3.39
(d, 2H), 5.11 (m, 2H), 5.33 (br m, 1H), 6.86 (br t, J =
7.52 Hz, 1H), 7.39 (dd, J = 7.12 Hz, 1H), 7.79 (dd, J =
7.76 Hz, 1H), 10.67 (br, 1H). 13C NMR (100 MHz, CDCl3)
δ: 16.40, 16.46, 18.03, 26.03, 26.88, 27.10, 27.99, 40.10,
40.99, 111.01, 119.37, 120.79, 124.46, 124.74, 128.79,
130.69, 131.63, 135.47, 136.77, 138.20, 160.70, 175.43.
Electrospray (ES) m/z calculated for C22H31O3 (M + K)+
381.24, found 342.14 and (M + H)− 341.41, found 342.42.
HOOC
OH
1
HOOC
OH
2
HOOC
OH
3
3-Geranyl-2-hydroxybenzoic acid (2)
2 was obtained as pale yellow oil in 0.029 g, 22.1%; Rf 0.38
(3:1:2 drops CHCl3/MeOH/AcOH), 1H NMR (400 MHz,
CDCl3) δ: 1.60 (s, 3H), 1.68 (s, 3H), 1.70 (s, 3H), 2.13–2.05
(m, 4H), 3.37 (d, J = 7.0 Hz, 2H), 5.11 (t, J = 6.08 Hz, 1H),
5.33 (br t, J = 6.8 Hz, 1H), 6.82 (br, 1H), 7.35 (d, J = 7.0 Hz,
1H), 7.75 (br d, 1H). Electrospray (ES) m/z calculated for
C17H22O3 (M + H)− 273.36, found 274.368.
Br
O
O
6
Br
O
O
7
3-Dimethylallyl-2-hydroxybenzoic acid (3)
5 was obtained as amorphous solid in 0.0203 g, 23.3%; Rf
0.6 (3:1 CHCl3/MeOH), 1H NMR (400 MHz, CDCl3) δ: 1.72
(s, 3H), 1.76 (s, 3H), 3.37 (d, 2H), 5.32 (br t, J = 7.16 Hz,
1H), 6.84 (br t, J = 7.2 Hz, 1H), 7.36 (dd, J = 7.3 Hz, 1H),
7.78 (br d, 1H). Electrospray (ES) m/z calculated for
C12H14O3 (M + H)− 205.36, found 206.878.
Br
O
O
8
Fig. 2. Structure of 3-Farnesyl-2-hydroxybenzoic acid and it derivatives.

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I. Malami et al. / Fitoterapia 93 (2014) 189–193
The synthesis of prenylated salicylic acid commenced
geranylated or farnesylated have been reported to inhibit
NorA MDR pump of S. aureus effluxes. Apart from prenylated
benzoic acid derivative reported with antibacterial activity
from P. multiplinervium [9], only few plant polyphenolic
compounds found as prenylated, geranylated or farnesylated
have been reported to show antibacterial activity or inhibition
of NorA MDR pump of S. aureus effluxes. Iinuma et. al [17]
reported the antibacterial activity of rubraxanthone isolated
from the bark of Garcinia dioica Dl. with activity against
both methicillin-resistant S. aureus and methicillin-sensitive
S. aureus. Rubraxanthone showed greater activity than
that of the antibiotic Vancomycin against staphylococcal
strains having MIC values ranging from 0.31 to 1.25 μg/ml. In
with the synthesis of the starting material 2,6-dibromo MOM
protected phenol (5) in larger scale from commercially readily
available 2,6-bromophenol (4). Compound 5 was successfully
protected using Chloromethyl methyl ether (MOMCl) to afford
the MOM ether in high yield (12.05 g, 88%) as shown in Fig. 1.
The 1H NMR of 5 conformed to the spectral data previously
reported [12] in the presence of two aromatic proton signals at
δ 6.88(t, J = 8.0 Hz, 1H) and 7.52(d, J = 8.0 Hz, 2H), two
singlets at δ: 3.73(s, 3H) and 5.18(s, 2H) which was assigned to
CH3 and CH2 of MOM respectively.
The common method widely used for the prenylation of
aromatic compounds is via halogen–metal exchange [14].
The desired compounds 9, 10 and 11, were successfully afforded
in 21%, 14% and 12.4% yield respectively.
However, the remaining bromide at the ortho position
to hydroxyl group of phenol is subsequently exchanged by
bromine–lithium exchange and trapped with carbon dioxide at
−78 °C. The addition of acidic workup (6 M HCl) protonated
CO2 and cleaved the MOM ether in situ at room temperature
[15] to afford the desired compounds 1, 2, and 3 in 10%, 23.3%
and 22.1% yield respectively. The 1H NMR of 1 conformed to the
previously reported spectral data [9].
another report,
2 prenylated derivatives of benzoic acid
4-hydroxy-(3′,7′-dimethyl-1′-oxo-octa-E-2′-6′-dienyl) benzoic
acid and 4-hydroxy-(3′,7′-dimethyl-1′-oxo-octa-2′-Z-6′-dienyl)
benzoic acid isolated from Piper gaudichaudianum showed
antibacterial activity against S. aureus and Baccilus subtilis having
MIC of 31.25 μg/mL and 12.5 μg/mL for S. aureus, respectively,
and 7.81 μg/ml and 6.25 μg/ml for B. subtilis, respectively [2].
Prenylated flavonoids with activity against methicillin-resistant
S. aureus have also been reported in Delia scandens val.
Paucifolia [18]. One of the prenylated flavonoids isolated from
the root of this plant is 2(S)-5′-(-1‴,1‴-dimethylallyl)-8-(3″,3″-
dimethylallyl)-2′,4′,5,7-tetrahydroxyflavanone. This compound
displayed spectrum of activity against both resistant and
sensitive strains of S. aureus having MIC value of 1.56 μg/ml. It
is noteworthy to suggest that the isoprenyl side chain on the
salicylic acid of these compounds plays a crucial role in the
antibacterial activity.
The literature survey revealed that there is no report so
far on compounds 2 and 3 and their antibacterial properties.
Therefore, this research reports the synthesis and antibacte-
rial evaluation of two new compounds 1 and 2. The present
studies showed that the antibacterial activity is higher in
longer isoprenyl side chain on the salicylic acid than those of
the shorter isoprenyl side chain. The results show that the
longer the side chain the greater the biological activity, which
might result from an enhanced ability to cross the bacterial
lipid membrane.
3.2. Antibacterial assessment
All the compounds 1, 2, and 3 synthesized were eval-
uated against both S. aureus (ATCC) and resistance S. aureus
(SA1119B) as presented in Table 1. All the compounds showed
activity against both S. aureus (ATCC) and the SA1119B type
S. aureus using a standard antibacterial drug (Norfloxacin) as
positive control. Compound 1 (MIC: 5.84 × 10−3 μmol/ml)
and 2 (MIC: 1.46 × 10−2 μmol/ml) exhibit higher activity
against S. aureus (ATCC) with 1 being the most active while
compound 3 (MIC: 6.19 × 10−1 μmol/ml) has the lowest
activity. All the three compounds displayed more activity
against SA1119B type than that evaluated on ATCC type.
Both compounds 1 and 2 displayed activity with MIC value at
5.84 × 10−3 and 7.29 × 10−3 μmol/ml much higher than
that of Norfloxacin (MIC: 1.57 × 10−3 μmol/ml) while 3 has
the minimum activity with MIC value at 3.09 × 10−1 μmol/ml.
Compound 1, however, displayed higher activity against
S. aureus (ATCC) at MIC: 5.84 × 10−3 μmol/ml in compari-
son to that reported natural product 1 (MIC: 12.5 μg/ml) [9].
Bacterial resistance modulators are compounds which
potentiate the activity of an antibiotic against a resistant strain.
These compounds may target a resistance mechanism such as
the inhibition of multidrug resistance (MDR), e.g. inhibition of
the NorA MDR pump of S. aureus effluxes [3,16]. However,
few plant secondary metabolites found as prenylated,
4. Conclusion
The search for potential antibacterial agent especially
against multi-drug resistance bacteria led to the synthesis of
prenylated salicylic acid isolated from P. multiplinervium.
However, the present research studies have been a success in
the synthesis of natural 3-Farnesyl-2-hydroxybenzoic acid.
Additionally, two new prenylated compounds 2 and 3 have
been successfully synthesized. All compounds synthesized
has been achieved and accomplished with the confirmation
of its structure to that of the original natural product, thus
producing the first synthesis of the natural product and
providing the first synthesis of its analogs. All the three
prenylated compounds displayed spectrum of activity against
both S. aureus (ATCC) and S. aureus (SA1119B).
Table 1
Antibacterial activity of compounds 1, 2, and 3.
Compound
S. aureus (ATTC)
MIC (μmol/ml)
S. aureus (SA1119B)
MIC (μmol/ml)
1
2
3
5.84 × 10−3
1.46 × 10−2
6.19 × 10−1
1.57 × 10−3
5.84 × 10−3
7.29 × 10−3
3.09 × 10−1
1.00 × 10−1
5. Conflict on interest
Norfloxacin
MIC: minimal inhibitory concentration (μmol/ml).
Authors declare that there are no conflict of interest.

I. Malami et al. / Fitoterapia 93 (2014) 189–193
193
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Acknowledgments
This research project was carried out in part fulfillment of
the requirements for the Master in Pharmacognosy degree,
UCL School of Pharmacy.
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