Synthesis of sildenafil analogues from anacardic acid and their phosphodiesterase-5 inhibition

Article abstract of DOI:10.1021/jf0258050

Anacardic acid (6-pentadecylsalicylic acid), a major component of cashew nut shell liquid, consists of a heterogeneous mixture of monoenes, dienes, and trienes. The enes mixture of anacardic acid was hydrogenated to a saturated compound. Using saturated a

Full text of DOI:10.1021/jf0258050

J. Agric. Food Chem. 2002, 50, 7709−7713 7709
Synthesis of Sildenafil Analogues from Anacardic Acid and
Their Phosphodiesterase-5 Inhibition
R. PARAMASHIVAPPA, P. PHANI KUMAR, P. V. SUBBA RAO, AND
A. SRINIVASA RAO*
Vittal Mallya Scientific Research Foundation, P.O. Box 406, K. R. Road, Bangalore-560 004, India
Anacardic acid (6-pentadecylsalicylic acid), a major component of cashew nut shell liquid, consists
of a heterogeneous mixture of monoenes, dienes, and trienes. The enes mixture of anacardic acid
was hydrogenated to a saturated compound. Using saturated anacardic acid as a starting material,
analogues of sildenafil [a potent phosphodiesterase-5 (PDE5) inhibitor and an orally active drug for
the treatment of erectile dysfunction] were synthesized, to observe the effect of the pentadecyl side
chain on PDE5 inhibition. The synthesized compounds were characterized by spectral studies and
tested for PDE5 inhibition, and the results were compared with those obtained with sildenafil.
KEYWORDS: Anacardic acid; cashew nut shell liquid; sildenafil; PDE5 inhibitor; erectile dysfunction
INTRODUCTION
Phosphodiesterases (PDEs) are the key enzymes in the
regulation of smooth muscle tone and play a very important
physiological role by regulating the intracellular level of cyclic
nucleotides. They are classified into five isoenzymes (1, 2),
PDE1, PDE2, PDE3, PDE4, and PDE5, according to their
specificity toward the hydrolysis of cyclic 3′,5′-adenosine
monophosphate (cAMP) and cyclic 3′,5′-guanosine monophos-
phate (cGMP). PDEs catalyze the hydrolysis of cyclic nucle-
otides [3′,5′-guanosine monophosphate (cGMP) or adenosine
monophosphate (cAMP)] to the corresponding 5′-nucleotides.
Among phosphodiesterase isoenzymes, PDE5 plays a very
important role in male “erectile dysfunction” by inhibiting the
hydrolysis of cGMP (3).
Erectile dysfunction is a fairly common medical problem (4),
affecting about 40% of men over the age of 40 and 70% of
men over the age of 70. It is also estimated that the number of
men who have this condition will more than double in the next
25 years, ultimately affecting more than 330 million men
worldwide (5). Sildenafil (Figure 1) is a potent PDE5 inhibitor
and an orally active drug in the treatment of erectile dysfunction
(6, 7), but it is associated with several side effects (8).
Anacardic acid (6-pentadecylsalicylic acid), a major compo-
nent of cashew nut shell liquid (CNSL), is obtained by solvent
extraction of cashew nut shells. It exists as a heterogeneous
mixture of monoenes, dienes, and trienes along with toxic
phenols (9). The presence of a long alkyl chain in anacardic
acid is attributed to a variety of biological activities, such as
antibacterial activity (10, 11), antimicrobial activity (12),
prostaglandin synthase inhibition (13), and tyrosinase (14) and
lipoxygenase inhibition (15). To explore the potential of
anacardic acid, it was extensively derivatized to drug analogues
Figure 1. Structure of sildenafil (18).
by several researchers (16, 17). By considering biological and
industrial application of CNSL constituents, we have developed
a novel method for comprehensive isolation of all major
phenolic constituents of CNSL (18). To make use of abundantly
available anacardic acid for pharmaceutical application, our
group has been working on the synthesis of drug analogues,
and recently we reported the synthesis of 1,4-dihydropyridine
derivatives as T-type calcium channel blockers (19).
In continuation of our ongoing effort to make a druglike
candidate, we have designed and synthesized analogues of
sildenafil from anacardic acid instead of salicylic acid. The
presence of a long alkyl chain in these compounds may enhance
the fat solubility, which in turn may increase the bioavailability
of the resultant compound. Hence, to determine the effect of
the pentadecyl side chain on PDE5 inhibition, synthesis of
sildenafil analogues from anacardic acid was attempted, and
our preliminary results are described in this communication.
MATERIALS AND METHODS
Chemicals and Solvents. 2-Pentanone, diethyl oxalate, N-meth-
ylpiperazine, and dicyclohexylcarbodiimide were purchased from
Aldrich Chemical Co. Inc. (Milwaukee, WI). Hydrazine hydrate,
triethylamine, ferric chloride, potassium tert-butoxide, ammonium
* To whom correspondence should be addressed. Tel.: +91-80-6611664.
Fax: +91-80-6612806. E-mail: asr@vmsrf.org, alapati61@hotmail.com.
10.1021/jf0258050 CCC: $22.00 © 2002 American Chemical Society
Published on Web 11/20/2002

7710 J. Agric. Food Chem., Vol. 50, No. 26, 2002
Paramashivappa et al.
hydroxide, and chlorosulfonic acid were purchased from Merck (India).
Radioactive cGMP (specific activity, 40.0 Ci/mol) and cold cGMP were
purchased from Amersham Pharmacia Biotech Ltd. (UK). MgCl2,
EDTA, and DEAE cellulose anion-exchange resin were purchased from
Sigma Chemicals (St. Louis, MO). Alkaline phosphatase (specific
activity, 3143 u/mg) was purchased from Genei (Bangalore, India).
Instrumentation. 1H and 13C NMR spectra were recorded in CDCl3
at 200 MHz on a Bruker A G spectrometer, and chemical shifts are
reported in parts per million (δ) downfield from tetramethylsilane
(Me4Si) as internal standard. IR spectra were recorded with a Galaxy
series FT-IR, and mass spectra were taken using GC-MS (Hewlett-
Packard HP 5890/Shimadzu QP 5050A) and electrospray mass
spectrometry (quadrupole mass spectrometer, Quattro II). Phosphodi-
esterase assay was performed using a liquid scintillation counter (LKB,
Wallac Beta, Bruker). Melting points are uncorrected and were
determined with a melting point apparatus (Arco Steels Pvt Ltd.).
Elemental analyses were performed on a Carlo Erba Strumentazones
elemental analyzer (MOD-1106).
Preparation of Saturated Anacardic Acid (1). To a solution of
an ene mixture of anacardic acid (500 g, 1300 mmol) in methanol (2.0
L) was added 5% palladium-carbon (25 g), and then hydrogen gas
was passed through the solution at 2.5 kg/cm2 until consumption of
hydrogen gas ceased (4-5 h). The reaction mass was filtered over a
Celite bed and washed with methanol (100 mL). The filtrate was
concentrated under reduced pressure to give an off-white solid (490
g), which on recrystallization from hexane yielded 475 g (95%) of the
title compound. Mp: 89-90 °C [lit. (18) mp 90 °C].
Preparation of Ethyl 2-Ethoxy-6-pentadecylbenzoate (2). To a
stirred solution of anacardic acid 1 (50 g, 143 mmol) in acetone (300
mL) was added anhydrous powdered potassium carbonate (80 g, 580
mmol). Diethyl sulfate (44.25 g, 290 mmol) was added in portions for
about 10 min at room temperature. After the addition was complete,
the solution was heated to reflux temperature on a water bath and
maintained for 3 h. The solution was cooled to room temperature and
then concentrated under reduced pressure. Distilled water (200 mL)
was added to the reaction mixture, which was then extracted with ethyl
acetate (200 mL). The organic layer was washed with distilled water
(2 × 200 mL), dried over anhydrous sodium sulfate, and concentrated
to yield the title compound (48 g, 82%). Mp: 34 °C. IR (KBr): 3010,
2920, 2863, 1730, 1588, 1463, 1260, 1185, 1109, 1070, 742 cm-1. GC-
MS: m/z (relative intensity) 404 (M+, 23), 362 (55), 330 (20), 287
(16), 199 (12), 161 (100), 134 (30), 43 (60).
by TLC using a chloroform-methanol (9:1) solvent system. After the
reaction was complete, distilled water (50 mL) was added. The organic
layer was washed with 5% hydrochloric acid (100 mL), followed by
distilled water (100 mL). The dichloromethane layer was dried over
anhydrous sodium sulfate and then concentrated under reduced pressure
to yield a crude mass. The solid was recrystallized from hexane to
give an off-white solid (6.5 g, 72%). Mp: 145-148 °C. IR (KBr):
3527, 3484, 3383, 2962, 2922, 1688, 1606,1512, 1462, 1387, 1300,
1250, 1192, 1142, 1092, 1056, 870 cm-1 1H NMR (200 MHz,
.
CDCl3): δ 0.84-0.90 (3 H, t, CH3, J ) 6.8 Hz), 0.93-1.011 (3 H, t,
CH3, J ) 7.4 Hz), 1.24 (24 H, bs, (CH2)12), 1.35-1.42 (3 H, t,
OCH2CH3), 1.58 (2 H, m, CH2), 1.64-1.75 (2 H, m, CH2), 2.53-2.61
(2 H, t, ArCH2, J ) 8.0 Hz), 2.66-2.70 (2 H, t, J ) 8.1 Hz, PyCH2),
4.06-4.17 (2 H, q, OCH2, J ) 7.0 Hz), 4.08 (3 H, s, N-CH3), 5.6 (2
H, bs, NH2), 6.78-6.82 (1 H, d, Ar H, J ) 8.2 Hz), 6.88-6.92 (1 H,
d, Ar H, J ) 7.2 Hz), 7.27-7.35 (1 H, t, Ar H, J ) 8.1 Hz), 7.6 (1 H,
bs, NH). ES-MS: m/z 541.5 (M + 1).
Preparation of 4-[2-Ethoxy-5-(chlorosulfonyl)-6-pentadecylbenz-
amido]-1-methyl-3-n-propylpyrazole-5-carboxamide (11). To the
chlorosulfonic acid (42 g, 2790 mmol) was added compound 9 (6 g,
11.4 mmol) while maintaining the temperature at 0-5 °C. The reaction
was allowed to proceed at 5 °C until TLC analysis indicated the absence
of starting material. After the reaction was complete, it was quenched
on ice. The precipitated milky solid was filtered, washed thoroughly
with distilled water (200 mL), and dried under suction to yield an off-
white solid. The resultant solid was redissolved in dichloromethane
and washed with distilled water (50 mL), and the organic layer was
dried over anhydrous sodium sulfate and concentrated to yield the title
compound as a milky white solid (6 g, 84%). Mp: 116-118 °C. IR
(KBr): 3525, 3483, 3382, 2960, 2924, 1686, 1606,1622, 1480, 1380,
1320, 1280, 1190, 1140, 1092, 1050, 875 cm-1
.
Preparation of 4-[2-Ethoxy-5-(4-methylpiperazin-1-ylsulfonyl)-
6-pentadecylbenzamido]-1-methyl-3-n-propylpyrazole-5-carboxam-
ide (13). To a stirred solution of compound 11 (6 g, 9.3 mmol) in
distilled water (20 mL) was added N-methylpiperazine (1.4 g, 14 mmol)
while maintaining the temperature at 5 °C. The reaction was allowed
to proceed for 2 h, and the precipitated solid was filtered, washed
thoroughly with distilled water, and dried under vacuum. The crude
mass was purified on a silica gel column using chloroform-methanol
to yield a yellowish-brown solid (2.4 g, 36%). Mp: 65-70 °C. IR
(KBr): 3449, 3306, 2960, 1688, 1579, 1464, 1288, 1152 cm-1. 1H NMR
(200 MHz, CDCl3): δ 0.84-0.90 (3 H, t, CH3, J ) 6.7 Hz), 0.95-
1.031 (3 H, t, CH3, J ) 7.4 Hz), 1.23 (24 H, bs, (CH2)12), 1.40-1.47
(3 H, t, OCH2CH3, J ) 7.0 Hz), 1.67-1.78 (2 × 2 H, m, CH2, CH2),
2.26 (3 H, s, N-CH3), 2.44 (4 H, ds t, (CH2)2), 2.53-2.61 (2 H, t,
ArCH2, J ) 8.1 Hz), 2.97 (2 H, ds t, PyCH2), 3.1 (4 H, ds t, (CH2)2),
4.05 (3 H, s, N-CH3), 4.13-4.23 (2 H, q, OCH2, J ) 6.9 Hz), 5.82 (2
H, bs, NH2), 6.84-6.88 (1 H, d, Ar H, J ) 9.1 Hz), 7.27 (1 H, s, NH),
7.88-7.93 (1 H, d, Ar H, J ) 9.0 Hz). ES-MS: m/z 703.4 (M + 1).
Preparation of 5-[2-Ethoxy-5-(4-methylpiperazin-1-ylsulfonyl)-
6-pentadecylphenyl]-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-
one (15). To a stirred solution of compound 13 (1.2 g, 1.7 mmol) in
dry xylene (20 mL) was added dicyclohexylcarbodiimide (DCC) (2 g,
9.6 mmol). The solution was heated to reflux until the reaction was
complete (38 h). The progress of the reaction was monitored by TLC
analysis using chloroform-methanol (9:1) as the mobile phase. The
reaction mixture was concentrated to remove the solvent under reduced
pressure. Distilled water (25 mL) was added to the residue and extracted
with ethyl acetate (25 mL). The organic layer was dried over anhydrous
sodium sulfate, concentrated to dryness, and then purified by column
chromatography on silica gel by eluting with chloroform-methanol
(1-5%) of increasing polarity. The resultant compound was recrystal-
lized in ethyl alcohol to give the title compound (0.6 g, 51.7%). Mp:
154-156 °C. IR (KBr): 3370, 3308, 2942, 2859, 1663, 1602, 1456,
1336, 1289, 1155 cm-1. 1H NMR (200 MHz, CDCl3): δ 0.84-0.90 (3
H, t, CH3, J ) 6.7 Hz), 0.97-1.04 (3 H, t, CH3, J ) 7.4 Hz), 1.24 (24
H, bs, (CH2)12), 1.32-1.39 (3 H, t, OCH2CH3, J ) 7.0 Hz), 1.61 (2 H,
s, CH2), 1.73-1.80 (2 H, m, CH2), 2.27 (3 H, s, N-CH3), 2.43 (4 H,
ds t, (CH2)2), 2.84-2.88 (2 H, t, ArCH2, J ) 8.0 Hz), 2.91-2.95 (2 H,
ds t, PyCH2), 3.17 (4 H, ds t, (CH2)2), 4.03-4.10 (2 H, q, J ) 7.0 Hz,
OCH2), 4.11 (3 H, s, N-CH3), 6.72-6.82 (1 H, d, Ar H, J ) 9.0 Hz),
Preparation of 2-Ethoxy-6-pentadecylbenzoic Acid (4). To a
stirred solution of compound 2 (10 g, 240 mmol) in dimethyl sulfoxide
(40 mL) was added potassium tert-butoxide (10 g, 890 mmol) in
portions. The solution was heated to 70 °C on a water bath for 2 h,
and the progress of the reaction was monitored by TLC using a hexane-
ethyl acetate (8:2) solvent system. The reaction mass was cooled to 10
°C, poured into ice water, and then acidified with 5% dilute hydrochloric
acid. The precipitated solid was filtered and washed thoroughly with
distilled water, and the crude mass was recrystallized in hexane (50
mL) to yield an off-white solid (7.5 g, 80%). Mp: 58-60 °C. IR
(KBr): 2920, 2851, 1708, 1649, 1591, 1419, 1388, 1305, 1246, 1121,
1076, 800 cm-1. GC-MS: m/z (relative intensity) 390 (M+, 14), 359
(18), 245 (5), 189 (15), 175 (100), 134 (10), 43 (12) (for methyl ester).
Preparation of 2-Ethoxy-6-pentadecylbenzoyl Chloride (6). To
a stirred solution of compound 4 (6.5 g, 16 mmol) in hexane (60 mL)
were added thionyl chloride (2.5 g, 21 mmol) and N,N-dimethylform-
amide (0.5 mL). The reaction mixture was heated to reflux for 1 h.
After the reaction was complete, the solvent was evaporated under
reduced pressure to yield the desired acid chloride, which was
redissolved in dichloromethane (50 mL) and used for the next step
without further isolation. Mp: 46-48 °C. IR: 2920, 2850, 1792, 1586,
1470, 1270, 1248, 846, 648 cm-1
.
Preparation of 4-(2-Ethoxy-6-pentadecylbenzamido)-1-methyl-
3-n-propylpyrazole-5-carboxamide (9). To the cold solution of
carboxamide 8 (3 g, 16 mmol) containing triethylamine (4.35 g, 43
mmol) in dichloromethane (50 mL) was added acid chloride 6 (6.5 g,
16.4 mmol) while maintaining the temperature at 0 °C. The reaction
mixture was kept at 0-5 °C for 1 h and then brought to room
temperature (28-30 °C). The progress of the reaction was monitored

Sildenafil Analogues from Anacardic Acid
J. Agric. Food Chem., Vol. 50, No. 26, 2002 7711
7.93-7.98 (1 H, s, J ) 8.9 Hz), 9.52 (1 H, s, NH, D2O exchangeable).
13C NMR (300 MHz, CDCl3): δ 14.05, 14.43, 14.45, 21.7, 22.6, 24.6,
24.2, 25.2, 29.2, 29.6, 29.7, 30.7, 31.88, 32.0, 32.8, 39.59, 44.9, 45.5,
50.08, 54.1, 64.1, 108.2, 120.1, 127.6, 130.3, 130.7, 133.0, 143.2, 144.7,
157.9, 159.2, 164.2, 170.27. ES-MS: m/z 685.45 (M + 1). Anal.
Found: C, 64.97; H, 8.86; N, 12.30. Calcd for C37H60N6O4S: C, 64.87;
H, 8.82; N, 12.26.
12 (4 g, 6.4 mmol) was condensed with N-methylpiperazine (0.9 g,
8.9 mmol) to yield an off-white solid (2.6 g, 59%). Mp: 80-85 °C.
IR (KBr): 3450, 3310, 2960, 1688, 1579, 1464, 1288, 1152 cm-1. 1H
NMR (200 MHz, CDCl3): δ 0.84-0.90 (3 H, t, CH3, J ) 6.8 Hz),
0.94-1.01 (3 H, t, CH3, J ) 7.2 Hz), 1.23 (24 H, bs, (CH2)12), 1.63 (2
H, m, CH2), 1.72-1.80 (2 H, m, CH2), 2.25 (3 H, s, N-CH3), 2.42 (4
H, ds t, (CH2)2), 2.54-2.62 (2 H, t, ArCH2, J ) 8.0 Hz), 2.92-2.97 (2
H, ds t, PyCH2), 3.14 (4 H, ds t, (CH2)2), 3.86, (3H, s, OCH3), 4.12 (3
H, s, N-CH3), 5.84 (2 H, bs, NH2), 6.82-6.84 (1 H, d, Ar H, J ) 8.9
Hz), 7.25 (1 H, bs, NH), 7.86-7.90 (1 H, d, Ar H, J ) 9.0 Hz).
Preparation of 5-[2-Methoxy-5-(4-methylpiperazin-1-ylsulfonyl)-
6-pentadecylphenyl]-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-
one (16). In a procedure similar to that for the synthesis of 15,
compound 14 (2 g, 2.9 mmol) was cyclized with dicyclohexylcarbo-
diimide (3 g, 14.5 mmol) to give the title compound (0.5 g, 25%).
Mp: 150-152 °C. IR (KBr): 3370, 3308, 2942, 2859, 1663, 1602,
1456, 1336, 1155 cm-1. 1H NMR (200 MHz, CDCl3): δ 0.84-0.90 (3
H, t, CH3, J ) 6.8 Hz), 0.97-1.04 (3 H, t, CH3, J ) 7.4 Hz), 1.25 (24
H, bs, (CH2)12), 1.63 (2 H, m, CH2), 1.72-1.83 (2 H, m, CH2), 2.27 (3
H, s, N-CH3), 2.43 (4 H, ds t, (CH2)2), 2.83-2.87 (2 H, t, ArCH2, J
) 8.0 Hz), 2.91-2.95 (2 H, ds t, PyCH2), 3.18 (4 H, ds t, (CH2)2),
3.83 (3 H, s, OCH3), 4.11 (3 H, s, N-CH3), 6.80-6.85 (1 H, d, Ar H,
J ) 9.0 Hz), 7.96-8.01 (1 H, d, Ar H, J ) 9.0 Hz), 9.48 (1 H, s, NH,
D2O exchangeable). GC-MS: m/z (relative intensity) 670 (M+, 6), 462
(10), 373 (13), 319 (10), 232 (8), 146 (100), 135 (29), 104 (10), 70
(23). Anal. Found: C, 64.68; H, 8.81; N, 12.45. Calcd for C36H58N6O4S:
C, 64.44; H, 8.71; N, 12.52.
Isolation of PDE5 Isoenzyme. Freshly dissected goat penile tissue
was chopped into small pieces using a sterilized knife and homogenized
in a buffer containing 0.2 mM Tris-hydrochloride, 0.2 mM EDTA, and
0.25 mM mercaptoethanol. The homogenate was filtered through
cheesecloth and centrifuged at 8 k/20′/4 °C in a Beckman centrifuge.
The supernatant was filtered through cheesecloth, partially purified by
DEAE-Sepharose column chromatography (20), and frozen at -20 °C.
This sample was used in each phosphodiesterase inhibition assay.
Phosphodiesterase5 Inhibition Assay. Synthesized compounds were
tested for PDE5 inhibition by the method of Kincaid and Manganiello
(21); sildenafil was used as an active control in the PDE5 inhibition
assay.
Preparation of 5-(2-Ethoxy-6-pentadecylphenyl)-1-methyl-3-n-
propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (17). To a
solution of compound 9 (0.5 g, 0.92 mmol) in dry xylene (10 mL) was
added dicyclohexylcarbodiimide (1 g, 4.8 mmol). The reaction mixture
was heated to reflux until the reaction was complete (38 h). After the
reaction was complete, the reaction mixture was concentrated under
reduced pressure. Distilled water (10 mL) was added to the residue
and extracted with ethyl acetate (20 mL). The organic layer was dried
over anhydrous sodium sulfate, concentrated under reduced pressure
to yield a brown residue, and then purified by column chromatography
on silica gel by eluting with chloroform-methanol (1-5%). The
resultant solid was recrystallized in ethyl alcohol (10 mL) to give the
1
colorless title compound (0.3 g, 62%). Mp: 126-127 °C. H NMR
(200 MHz, CDCl3): δ 0.84-0.9 (3 H, t, CH3, J ) 6.6 Hz), 0.96-1.03
(3 H, t, CH3, J ) 7.4 Hz), 1.24 (24 H, bs, (CH2)12), 1.28-1.35 (3 H,
t, OCH2CH3, J ) 7.0 Hz), 1.60 (2 H, bs, CH2), 1.72-1.84 (2 H, m,
CH2), 2.61-2.69 (2 H, t, ArCH2, J ) 8.1 Hz), 2.85-2.93 (2 H, t,
PyCH2, J ) 8.1 Hz), 3.92-4.03 (2 H, q, J ) 7.0 Hz, OCH2), 4.10 (3
H, s, N-CH3) 6.68-6.72 (1 H, d, Ar H, J ) 7.9 Hz), 6.79-6.82 (1 H,
d, Ar H, J ) 7.9 Hz), 7.16-7.26 (1 H, t, Ar H, J ) 8.0 Hz), 9.37 (1
H, s, NH, D2O exchangeable).
Preparation of Methyl 2-Methoxy-6-pentadecylbenzoate (3). In
a procedure similar to that for the synthesis of 2, anacardic acid 1 (50
g, 143 mmol) was treated with dimethyl sulfate (36.2 g, 280 mmol)
and anhydrous potassium carbonate (80 g, 580 mmol) in acetone to
yield the title compound (51 g, 94.4%). Mp: 37-38 °C. IR (KBr):
3006, 2922, 2853, 1732, 1588, 1465, 1268, 1185, 1109, 1071, 752 cm-1
.
GC-MS: m/z (relative intensity) 376 (M+, 35), 345 (46), 317 (3), 273
(2), 204 (9), 161 (100), 121 (15), 91 (10).
Preparation of 2-Methoxy-6-pentadecylbenzoic Acid (5). In a
procedure similar to that for the synthesis of 4, compound 3 (20 g, 53
mmol) was hydrolyzed in dimethyl sulfoxide (80 mL) with potassium
tert-butoxide (20 g, 178 mmol) to give an off-white solid (14 g, 73%).
Mp: 78-79 °C. IR (KBr): 2920, 2851, 1708, 1649, 1591, 1419, 1388,
1305, 246, 1121, 1076, 800 cm-1. GC-MS: m/z (relative intensity)
376 (M+, 35), 345 (46), 317 (3), 273 (2), 204 (9), 161 (100), 121 (15),
91 (10) for methyl ester.
RESULTS AND DISCUSSION
The ene mixture of anacardic acid isolated from cashew nut
shell liquid was hydrogenated to saturated anacardic acid 1 (18)
(saturated anacardic acid is generally termed as tetrahydroana-
cardic acid or anacardic acid). During the synthesis of sildenafil
analogues from anacardic acid, different methods were attempted
for selective O-alkylation of anacardic acid with a variety of
alkylating agents, such as methyl iodide, ethyl iodide, dimethyl
sulfate, and diethyl sulfate. In all of these experiments, alkoxy
ester formation was observed. Further hydrolysis of alkoxy ester
2/3 to acid 4/5 was not achieved with either sodium hydroxide
or hydrochloric acid/sulfuric acid, probably due to steric
hindrance. Finally, it was achieved with potassium tert-butoxide
in anhydrous DMSO, a method described for the hydrolysis of
sterically hindered ester (22). Acid chloride 6 was prepared by
heating a mixture of alkoxyanacardic acid 4 and thionyl chloride
in the presence of a catalytic amount of N,N-dimethylformamide
(Figure 2). 4-Amino-1-methyl-3-n-propylpyrazole-5-carboxa-
mide was prepared according to the reported procedure (23).
Compound 9 was prepared by condensation of amine 8 with
acid chloride 6 in dichloromethane in the presence of triethyl-
amine as acid scavenger. This was further reacted with chloro-
sulfonic acid at 5 °C to give the corresponding chlorosulfonated
compound 11, which was condensed with N-methylpiperazine
to yield compound 13. Cyclization of 13 was not effected by
either the method reported for the preparation of sildenafil (22,
23) or the reaction with potassium tert-butoxide in t-BuOH/
Preparation of 2-Methoxy-6-pentadecylbenzoyl Chloride (7). In
a procedure similar to that for the synthesis of 6, compound 5 (5.0 g,
13.8 mmol) in hexane (50 mL) was treated with thionyl chloride (2 g,
16.8 mmol) to give an acid chloride, which was taken up for the next
step. Mp: 52-54 °C. IR (KBr): 2919, 2850, 1794, 1585, 1472, 1271,
1247, 1078, 847, 645 cm-1
.
Preparation of 4-(2-Methoxy-6-pentadecylbenzamido)-1-methyl-
3-n-propylpyrazole-5-carboxamide (10). In a procedure similar to that
for the synthesis of 9, compound 8 (2.6 g, 14 mmol) was condensed
with 7 to yield an off-white solid (5.2 g, 75%). Mp: 150-152 °C. 1H
NMR (200 MHz, CDCl3): δ 0.84-0.90 (3 H, t, CH3, J ) 6.8 Hz),
0.94-1.01 (3 H, t, CH3, J ) 7.4 Hz), 1.24 (24 H, bs, (CH2)12), 1.64 (2
H, bs, CH2), 1.67-1.75 (2 H, m, CH2), 2.54-2.61 (2 H, t, ArCH2, J )
8.0 Hz), 2.66-2.73 (2 H, t, PyCH2, J ) 8.1 Hz), 3.85 (3 H, s, OCH3),
4.08 (3 H, s, NCH3), 5.73 (2 H, bs, NH2), 6.79-6.83 (1 H, d, Ar H, J
) 8.2 Hz), 6.89-6.93 (1 H, d, Ar H, J ) 7.8 Hz), 7.29-7.39 (1 H, t,
Ar H, J ) 8.0 Hz), 7.63 (1 H, bs, NH, D2O exchangeable).
Preparation of 4-[2-Methoxy-5-(chlorosulfonyl)-6-pentadecyl-
benzamido]-1-methyl-3-n-propylpyrazole-5-carboxamide (12). In a
procedure similar to that for the synthesis of 11, compound 10 (4.5 g,
8.5 mmol) was treated with chlorosulfonic acid to yield an off-white
solid (4 g, 75%). Mp: 158-160 °C. IR (KBr): 3524, 3482, 3380, 2966,
2921, 1685, 1602, 1627, 1485, 1382, 1322, 1280, 1195, 1144, 1090,
1050, 874 cm-1
.
Preparation of 4-[2-Methoxy-5-(4-methylpiperazin-1-ylsulfonyl)-
6-pentadecylbenzamido]-1-methyl-3-n-propylpyrazole-5-carboxam-
ide (14). In a procedure similar to that for the synthesis of 13, compound

7712 J. Agric. Food Chem., Vol. 50, No. 26, 2002
Paramashivappa et al.
while that on the propyl chain on pyrazolopyrimidone appeared
as a multiplet at δ 1.73-1.80. Benzylic protons are seen as a
triplet at δ 2.84-2.88, and methylene protons on the pyrazole
ring also appeared as a triplet at δ 2.91-2.95. The four
methylene groups of the N-methylpiperazine ring appeared as
two broad singlets (distorted triplets) at δ 2.43 and 3.17. The
methylene protons of the ethoxy group are seen as a quartet at
δ 4.03-4.1. The N-methyl protons on pyrazolopyrimidone and
the piperazine ring appeared as singlets at δ 4.11 and 2.27,
respectively. Two aromatic protons on the phenyl ring appeared
as doublets at δ 6.72-6.82 and 7.93-7.98, and the NH proton
on the pyrazolopyrimidone ring appeared as a singlet at δ 9.52,
as compared to δ 10.5 for sildenafil, which was readily
exchangeable with D2O. The IR spectrum bands at 3370 (N-H),
3036 (Ar C-H), and 1663 cm-1 (CdO) were similar to bands
observed for sildenafil. Also, electrospray mass spectrometry
showed an M + 1 peak, similar to that observed for sildenafil.
Figure 2. Synthesis of 2-alkoxy-6-pentadecylbenzoyl chloride (6/7).
DMSO/DMF at reflux temperature. The reaction with phos-
phoric acid or with phosphorous pentoxide yielded an undesired
product. Finally, cyclization was achieved with dicyclohexyl-
carbodiimide in xylene to yield sildenafil analogue 15 (Figure
3). Sildenafil 18 was synthesized by the reported method (23,
24) and characterized by spectral studies. This sample was used
as a reference sample for comparison and for PDE5 inhibition
assay.
Sildenafil analogues (Figure 4) synthesized from anacardic
acid were evaluated on partially purified PDE5 enzyme. The
enzyme was isolated by a method similar to that reported by
Burns et al. (20) from the penile tissue of a goat. PDE5 inhibition
was studied by the method of Kincaid and Manganiello (21),
and the results are summarized in Table 1.
1
13
Compounds 15 and 16, which have a pentadecyl side chain
on the phenyl ring, showed an IC50 of 145 and 125 µM,
respectively, whereas the reference molecule sildenafil 18 has
shown 38 µM under similar experimental conditions. Compound
17, an analogue of 15 without the N-methylpiperazinesulfona-
mide moiety, inhibited the PDE5 enzyme with an IC50 of 160
µM.
The sildenafil analogues 15 and 16, synthesized from ana-
cardic acid, have shown less potency compared to sildenafil
18. Hence, introduction of a pentadecyl side chain may alter
The H and C NMR spectra of analogue 15 were similar
to those of sildenafil, except for a few additional peaks due to
1
the pentadecyl side chain. In the H NMR spectra of 15, the
triplet at δ 0.84-0.90 is assigned to the terminal methyl of the
pentadecyl side chain and that at δ 0.97-1.04 to methyl protons
of propyl chain, while another triplet at δ 1.32-1.39 is assigned
to the methyl group of the ethoxy group. The broad singlet at
δ 1.24 is attributed to 12-methylene groups of the C15 alkyl
side chain. The methylene group adjacent to the benzylic group
in the C15 side chain appeared as a distorted multiplet at δ 1.61,
Figure 3. Synthesis of sildenafil analogues (15, 16, and 17).

Sildenafil Analogues from Anacardic Acid
J. Agric. Food Chem., Vol. 50, No. 26, 2002 7713
(6) Terrett, N. K.; Bell, A. K.; Brown, D.; Ellis, P. Sildenafil (Viagra)
a potent and selective inhibitor of type-5 cyclic GMP phos-
phodiesterase with utility for the treatment of male erectile
dysfunction. Bioorg. Med. Chem. Lett. 1996, 6, 1819-1824.
(7) Boolell, M.; Allen, M. J.; Ballard, S. A.; Gepi-Attee, S.;
Muirhead, G. J.; Naylor, A. M.; Osterloh, I. H.; Gingell, C.
Sildenafil: an orally active type-5 cyclic GMP specific phos-
phodiesterase inhibitor for treatment of penile erectile dysfunc-
tion. Int. J. Impotence Res. 1996, 8, 47-52.
(8) Beavo, J. A. Cyclic nucleotide phosphodiesterases functional
implication of multiple isoforms. Physiol. ReV. 1995, 75, 725-
748.
(9) Tyman, J. H. P. Non-isoprenoid long chain phenols. Chem. Soc.
ReV. 1975, 8, 499-537.
(10) Gellerman, J. L.; Walsh, N. J.; Werner, N. K.; Schlenk, H.
Antimicrobial effects of anacardic acids. Can. J. Microbiol. 1969,
15, 1219-1223.
(11) Muroi, H.; Kubo, I. Bactericidal activity of anacardic acids
against Streptococcus mutant and their potentiation. J. Agric.
Food Chem. 1993, 41, 1780-1783.
Figure 4. Structure of sildenafil analogues.
Table 1. Results of PDE Inhibition of Sildenafil Analogues
5
compound
R
R
IC50 (µM)
2
15
16
17a
18
CH CH
C H
15 31
C H
C H
15 31
H
145
125
160
38
2
3
CH
3
15 31
CH CH
2
3
CH CH
2
3
(12) Kubo I.; Muroi, H.; Himejima, M. Structure antibacterial
relationship of anacardic acids. J. Agric. Food Chem. 1993, 41,
1016-1019.
a N-Methylpiperazinesulfonamide moiety is absent.
(13) Grazzini, R.; Hesk, D.; Heiminger, E.; Mumma, R. O.; Hilden-
brandt, G.; Reddy, C. C.; Cox-Foster, D.; Medlford, J.; Craig,
R. Inhibition of lipoxygenase and prostaglandin endoperoxide
synthase by anacardic acids. Biochem. Biophys. Res Commun.
1991, 176, 775-780.
(14) Kubo, I.; Kinst-Hori, I.; Yokawa, Y. Tyrosinase inihibitors from
Anacardium occidentale fruits. J. Nat. Prod. 1994, 57, 545-
551.
the orientation of the sildenafil molecule at the site of action,
resulting in a decrease in the PDE5 inhibition. Compound 17 is
less potent compared to 15, indicating the essentiality of the
N-methylpiperazinesulfonamide group in the molecule, and this
observation is consistent with the SAR reported on sildenafil
(6).
(15) Shobha, S. V.; Ramadoss, C. S.; Ravindranath, B. Inhibition of
lipoxygenase by anacardic acids, cardols and cardanols. J. Nat.
Prod. 1994, 57, 1755-1757.
(16) Gulati, A. S.; Subba Rao, B. C. S. Drug analogues from phenolic
constituents of cashew nut shell liquid. Indian J. Chem. 1964,
2, 337-339.
(17) ElSholy, M. A.; Adawadkar, P. D.; Benigni, D. A.; Watson, E.
S.; Little, T. L., Jr. Analogues of poison ivy urushiol, synthesis
and biological activity of di-substituted alkylbenzene. J. Med.
Chem. 1986, 29, 606-611.
(18) Paramashivappa, R.; Phanikumar, P.; Vithayathil, P. J.; Srinivasa
Rao, A. Novel method for isolation of major phenolic constitu-
ents from cashew (Anacardium occidentale. L.) nut shell liquid.
J. Agric. Food Chem. 2001, 49, 2548-2551.
(19) Phani Kumar, P.; Stotz, S. C.; Paramashivappa, R.; Beedle, A.
M.; Zamponi, G. W.; Srinivasa Rao, A. Synthesis and evaluation
of new class of nifedipine analogues with T-type calcium channel
blocking activity. Mol. Pharmacol. 2002, 61, 649-658.
(20) Burns F.; Rodger, I. W.; Pyne, N. J. The catalytic subunit of
protein kinase A triggers activation of the type V cyclic GMP
specific phosphodiesterase from guinea-pig lung. Biochem. J.
1992, 283, 487-491.
SAFETY PRECAUTIONS
Use protective apron, hand gloves, goggles, and air respirator
during the preparation of compounds 6, 7, 11, and 12. Special
care must be taken to avoid the inhalation of vapors of thionyl
chloride and chlorosulfonic acid.
ACKNOWLEDGMENT
We thank Dr. C. S. Ramadoss, Prof. P. J. Vithayathil, and Dr.
Ganesh for their valuable guidance and encouragement. We also
acknowledge Dr. M. Srinivas and Ms Uma Maheswari for
technical assistance and helpful discussions during biological
assay. P.P.K. thanks the Council of Scientific and Industrial
Research (India) for a senior research fellowship.
Supporting Information Available: 1H NMR spectra of 9,
1
13, 16, and 17 and comparative H NMR spectra of 15 and
sildenafil 18; 13C NMR spectrum of 15; and mass spectra of 9,
13, and 15. This material is available free of charge via the
Internet at http://pubs.acs.org.
(21) Kincaid, R. L.; Manganiello, V. C. Assay of cyclic nucleotide
phosphodiesterase using radiolabelled and fluorescent substrate.
Methods Enzymol 1988, 159, 457-491.
LITERATURE CITED
(22) Chang, F. C.; Wood, N. F. Reaction potassium tertiary butoxide
in DMSO III, Hydrolysis of hindered carboxylic acid esters.
Tetrahedron Lett. 1964, 2969-2973.
(23) Bell, A. S.; Brown, D. Terrett, N. K. Process for preparation of
sildenafil. European Patent 0463756 B1, 1995.
(1) Beavo, J. A.; Reifsnyder, D. M. Primary sequence of cyclic
nucleotide phosphodiesterase isoenzyme and design of selective
inhibitor. Trends Pharmacol. Sci. 1990, 11, 150-155.
(2) Nicholeson, C. A.; Challis, J. R. A.; Shadid, M. Differential
modulation of tissue function and therapeutic potential of
selective inhibitor of cyclic nucleotide phosphodiesterase iso-
enzyme. Pharmacol. Sci. 1991, 12, 18-27.
(24) Dunn, P. J.; Wood. A. S. Richard, H. J. Pyrazolo pyrimidinone
as antianginal agents. European Patent 0812845 A1, 1997.
(3) Andersson, K.-E.; Wagner, G. Physiology of penile erection.
Physiol. ReV. 1995, 75, 191-236.
(4) Jeffecoate, W. J. The investigation of impotence. Br. J. Urol.
1991, 68, 449-453.
(5) Goldstein, I. Male sexual circuitry. Sci. Am. 2000, 283, 56-61.
Received for review July 10, 2002. Revised manuscript received October
15, 2002. Accepted October 16, 2002.
JF0258050