Synthesis of competitive inhibitors of phospholipase A2 (PLA2)

Article abstract of DOI:10.1080/10426500214877

The synthetic routes for preparation of several phospholipid analogues such as lithium alkyl-3-alkoxy allylvinyl-sn-glycerol 5 and 6 and lithium trans-2-amidomethyl phosphate 7 and lithium cholesterol ester phosphate 8 containing ether, amide, and phospha

Full text of DOI:10.1080/10426500214877

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Phosphorus, Sulfur, and Silicon and the Related
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Synthesis of Competitive Inhibitors of Phospholipase A
2 (PLA 2 )
Nosrat O. Mahmoodi a
a University of Guilan , Rasht, Iran
Published online: 27 Oct 2010.
To cite this article: Nosrat O. Mahmoodi (2002) Synthesis of Competitive Inhibitors of Phospholipase A 2 (PLA 2 ), Phosphorus,
Sulfur, and Silicon and the Related Elements, 177:12, 2887-2893, DOI: 10.1080/10426500214877
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Phosphorus, Sulfur and Silicon, 2002, Vol. 177:2887–2893
C
Copyright 2002 Taylor & Francis
1042-6507/02 $12.00 + .00
DOI: 10.1080/10426500290146938
SYNTHESIS OF COMPETITIVE INHIBITORS
OF PHOSPHOLIPASE A2 (PLA2)
Nosrat O. Mahmoodi
University of Guilan, Rasht, Iran
(Received January 9, 2002; accepted April 2, 2002)
The synthetic routes for preparation of several phospholipid ana-
logues such as lithium alkyl-3-alkoxy allylvinyl-sn-glycerol 5 and 6
and lithium trans-2-amidomethyl phosphate 7 and lithium cholesterol
ester phosphate 8 containing ether, amide, and phosphate ester moieties
are described.
Keywords: Inhibitor; phospholipase A2 (PLA2); phospholipid
INTRODUCTION
This article focuses on the synthesis of inhibitor for the class of 14-KDa
4
secreted phospholipase A2 (PLA2)1 which catalyzes the hydrolysis
of the sn-2-acyl chain of naturally occurring phospholipids 1 at the 2-
position of the glycerol backbone into a 1-acyllysophospholipids 2 and
fatty acids; it is one of the best examples of heterogeneous interfacial
10
catalysts in biological systems (see Figure 1).5
The venom of poisonous snakes contains this class of enzymes known
as phospholipases, enzymes that cause the breakdown of phospho-
lipids. Venom of the Indian cobra (Nag naja), which kills several thou-
sand people each year, and the eastern diamondback rattlesnake (Cro-
talus adamanteus) both among other things contain phospholipase A2.
Lysolecithin, the phospholipid breakdown product of this reaction, acts
as a detergent and dissolves the membranes of red blood cells.11
Various forms of PLA2 are distributed widely, and they are abun-
dant in pancreatic digestive fluid, venoms, and inflammatory exudates.
Recently, several phospholipid analogues12 (e.g., 3 and 4)13 containing
The author is grateful to Professor Mahendra K. Jain and Department of Chemistry
and Biochemistry at the University of Delaware, Newark, Delaware, USA, and the re-
search committee of Guilan University for their support to this study.
Address correspondence to Nosrat O. Mahmoodi, Laboratory of Organic Synthe-
sis, Faculty of Chemistry, University of Guilan, PO Box 1914, Rasht, Iran. E-mail:
mahmoodi@cd.gu.ac.ir
2887

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N. O. Mahmoodi
FIGURE 1
a phosphonate or amide function in place of the ester at the 2-position
of the glycerol backbone were synthesized (Figure 2).14,15
These compounds were shown as inhibitors of the phospholipase A2
enzyme. As an extension to the study of 3 and 4, compounds 5–8 con-
taining phosphate and amide moieties were synthesized (Figure 3).
RESULTS AND DISCUSSION
The basic procedure for the synthesis of the lithium glycerol-sn-2-
phosphoesters 5, 6 is outlined in Scheme 1, and for lithium phospho-
dimethyl ester amide 7 and lithium cholesterol phosphate ester 8 are
18
outlined in (Schemes 2 and 3) respectively.16
In all cases the isolated yields were over 50%, however no attempt
was made to optimize the synthetic procedures. The structure of the
intermediates and the final products were consistent with their Bruker
250 MHz 1H NMR (CDCl3). The mass spectra were run on Nicolet FT-
2000. All reagents and chromatographic silica gel (60 A, 230–400 mash)
were from Aldrich. TLC plates were from Analtech.
EXPERIMENTAL SECTION
Preparation of 1-Allyloxy-3-octyloxy-propan-2-ol (12a)
and 1-Allyloxy-3-octadecyloxy-propan-2-ol (12b)
To a solution of 20 mmol vinyl allyl ether epoxide 9 and under stream of
nitrogen was added 22 mmol alcohol 10 or 11 in dry distilled methylene
chloride (30 ml) in round bottom flask were added 5 drops of BF3 in di-
ethyl ether (Fluka, distilled). The solvent was removed and the residue
FIGURE 2

Synthesis of Competitive Inhibitors of Phospholipase A
2889
2
FIGURE 3
SCHEME 1
SCHEME 2
SCHEME 3

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N. O. Mahmoodi
was pumped. The crude material was taken on column of silicagel,
compounds 12a and 12b were eluted with mixture of EtOAc: ligroin
(7:93 v/v), and latter with EtOAc: ligroin (20:80 v/v), and finally with
CHCl3: ether (90:10 v/v) 55 fractions were recovered, fractions 37–52
were pure product. MS for 12a = m/z 244 (M+), for 12b = m/z 384 (M+).
Preparation of Phosphoric Acid 1-Allyloxymethyl-
2-octadecyloxy-ethyl Ester Dimethyl Ester (14) (the Same
Procedure Could be Applied for Preparation of (13))
To a three-neck round bottom flask equipped with an addition funnel
and drying tube, at 0–6 C (ice bath) was added 0.34 ml (3.6 mmol,
1.2 eq) of POCl3 (Aldrich freshly distilled). To this from addition funnel
was added dropwise solution of 1.16 g (3 mmol) secondary alcohol
12 (12a = 1-Allyloxy-3-octyloxy-propan-2-ol or 12b = 1-Allyloxy-3-
octadecyloxy-propan-2-ol and 1 ml (2.4 eq, 7.2 mmol, d = 0.726) three
ethyl amine (TEA) in 15 ml THF dropwise added, the temperature was
kept at 0 C, and allowed to react for 30 min. After completion of addi-
tion, the reaction mixture was allowed to warm up to r.t. for 48 h, them
heated to 60 C for 30 min. The TLC was run, and to this was added
10 ml brine and the resulting solution extracted with 2 15 ml hexane.
The hexane layer was combined and dried over MgSO4. After removal
of solvent, the weight of residue was 1.07 g; TLC showed only one spot.
MS for 13 = m/z 352 (M+), for 14 m/z = 492 (M+).
Preparation of Lithium Salt of (5) and (6)
To a 25 ml round-bottom flask was added 1.07 g (2.17 mmol) phos-
phodimethyl ester and 0.246 g (2.82 mmol, 1.3 eq) LiBr and 10 ml
reagent grade acetone. The reaction was heated to reflux overnight.
A white solid precipitated on cooling to 20 C, was filtered and washed
with cold acetone; if no solid precipitated, the mixture was concen-
trated and the residue was chromatographed on silicagel. 0.9 g of crude
compound was taken on 25 g silicagel column. The solvent used was
MeOH:CHCl3:NH4OH (10:88:2), fractions 27–32 (each collection 10 ml)
gave a white crystal weighing 0.6 g.
1H NMR compound 5: 0.8 ppm (t, 3H, J = 7 Hz), 1.3 ppm (s, 12H),
1.48 ppm (b, 1H), 3.6 ppm (m, 7H), 4.1 ppm (d, 3H, J = 7 Hz), 4.4 ppm
(b, 1H), 5.1–5.3 ppm (dd, 2H, J = 25), 5.9 ppm (m, 1H), MS: m/z 344
(M+), Anal. Calcd for C15H30O6PLi: C, 52.33, H, 8.78 Found, C, 52.51,
H, 8.61.
1H NMR of compound 6: 0.9 ppm (t, 5H, J = 7 Hz), 1.21 ppm
(s, 13H), 1.55 ppm (m, 2H), 3.57 ppm (m, 20H), 4 ppm (d, 9H, J = 13 Hz),
5.16–5.36 ppm (dd, 2H, J = 25 Hz), 5.8–6 ppm (m, 1H), MS: m/z

Synthesis of Competitive Inhibitors of Phospholipase A
2891
2
484 (M+). Anal. Calcd. for C25H50O6PLi: C, 61.97, H, 10.40, Found,
C, 61.89; H, 10.51.
Preparation of Decanoic Acid (Trans-2-hydroxy-
cyclohexyl)-amide (16)
To a 100 ml round-bottom flask and under stream of nitrogen was added
1g (6.59 mmol) of mainly trans-2-aminocyclohexanol hydrochloride. To
this was added 10 ml water and 3.7 ml (4 eq, 26.36 mmol) of three ethyl
amine. The solution was stirred, to this was added a solution of and
1.3 eq (0.9 eq, 5.9 mmol) of decanoyl chloride (98%, d = 0.919) in 10 ml
THF. The solution was stirred at r.t. overnight.
The reaction was monitored by TLC, after completion the crude prod-
uct was recrystallized from water and MeOH. The weight of pure prod-
uct after two days of vacuum drying was 1.8 g.
1H NMR (250 MHz): 0.94 ppm (t, 3H, J = 7 Hz), 1.17 ppm (s, 16H),
1.58–1.8 ppm (m, 5H), 1.8–2.2 ppm (m, 2H), 2.18–2.4 (m, 1H), 3.2–3.4
(m, 2H), 3.58–3.8 (b, 1H, NH), 5.4 (b, 1H, OH). MS: m/z 269 (M+).
Preparation of Trans-Phosphoric Acid-2-decanoylamino-
cyclohexyl Ester Dimethyl Ester (17)
To a three-neck round-bottom flask equipped with a dropping fun-
nel and CaCl2 tube at 0 C was added 0.7 ml (7.4 mmol, 1.2 eq) of
POCl3 (Aldrich, freshly distilled). To this was added solution of 1.65 g
(6.12 mmol) trans-amidocyclohexanol in 15 ml THF (dry and freshly dis-
tilled). The solution was cooled to 0 C and 2.5 ml (17.76 mmol 2.8 eq)
three ethylamine added dropwise over 1 h, and the solution allowed
to stay at 0 C for 4 h. The progress of reaction was monitored
by TLC. After completion of reaction excess of MeOH and 1.75 ml
(12.43 mmol) three ethylamine was added. The reaction mixture was al-
lowed to warm to r.t. and then heated to 60 C for 30 min. The reaction
was then cooled to r.t. to this was added 10 ml brine and the mixture
extracted with hexane. The hexane extract was dried and taken on col-
umn of silica gel. The weight of pure 17 was 0.56 g. TLC showed one
spot. 1H NMR (250 MHz): 0.95 ppm (t, 3H J = 7 Hz), 1.18 ppm (s, 16H),
1.59–1.8 ppm (m, 5H), 1.8–2.3 ppm (m, 2H), 2.18–2.4 (m, 1H), 3.2–3.4
ppm (m, 2H), 3.68 ppm (d, 6H), 5.4 ppm (b, 1H, OH), MS: m/z 377 (M+).
Preparation of Lithium Salt of Trans-Phosphoric Acid
2-Decanoylamino-cyclohexyl Ester Methyl Ester (7)
To a 30 ml round-bottom flask was added 0.58 g (1.53 mmol) of phos-
phodimethyl ester amide and 0.16 g (1.2 eq, 1.85 mmol) LiBr and 10 ml

2892
N. O. Mahmoodi
acetone (reagent grade). The flask was equipped with CaCl2 tube and
heated to reflux for 6 h. After this time the solution changed to cloudy.
The solution was cooled to 20 C and was filtered. The filtrate was
washed with acetone. The TLC showed only one spot. The weight of
final compound was 0.28 g. 1H NMR (250 MHz): 0.94 ppm (t, 3H,
J = 7 Hz), 1.18 ppm (s, 16H), 1.6–1.8 ppm (m, 5H), 1.8–2.25 ppm
(m, 2H), 2.18–2.4 (m, 1H), 3.2–3.4 ppm (m, 2H), 3.63 ppm (d, 3H), 5.2
(b, 1H, OH), MS: m/z 369 (M+). Anal. Calcd. C, 55.25; H, 9.01, Found
C, 55.51; H, 9.14.
Preparation of 3-Hexadecyloxy-propan-1-ol Ether (21)
To a 250 ml three-neck round-bottom flask was added 9.2 g (120 mmol,
3 eq) of 1,3-propanediol and 50 ml tert-amylalcohol 99% and 7.01 g
(60 mmol, 95%) potassium tert-butoxide. The flask was equipped with
reflux condenser and addition funnel. The reaction run under CaCl2,
and heated to reflux. To this from addition funnel was added a solution
of 12.2 g (12.21 ml, d = 0.999) 1-bromohexadecane in 50 ml THF drop-
wise over period of 3 h. The reaction mixture was stirred for 50 h. The
reaction mixture was cooled to r.t. and poured into a 50 ml H2O and
the water phase was acidified with 10% HCl. To the mixture was added
100 ml hexane and the organic phase was separated and to the aqouse
phase was extracted with additional 100 ml hexane. The organic layers
were combined and dried over MgSO4. The solvent was removed. The
weight of residue after vacuum drying was 10 g. The crude compound
was recrystallized from panthane. The yield of pure product went 58%.
1H NMR: 0.88 ppm (t, 3H, J = 7 Hz), 1.28 ppm (s, 28H), 1.8–2 (pentate,
2H, J = 7 Hz), 2.23 ppm (b, 1H), 3.4 ppm (t, 2H, J = 7 Hz), 3.6 (t, 2H,
J = 7 Hz), 3.8 (t, 2H, J = 7 Hz).
1H NMR of proton decoupling of 21: 0.85 ppm (t, 3H, J = 7 Hz), 1.3
ppm (s, 28H), 3.4 ppm (t, 3H), 3.63 ppm (s, 2H), 3.93 ppm (s, 2H). MS:
m/z 300 (M+).
Preparation of (8), Lithium Salt of Phosphoric Acid
17(1,5-Dimethyl-hexyl)-10, 13-Dimethyl-
2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-
1H-cyclopenta[a]phenanthren-3-yl]
2-Hexadecyloxy-ethylester
A three-neck round-bottom flask was equipped with addition funnel
and CaCl2 drying tube. To the addition funnel was added 1.9 g (6.32
mmol) of hexadecanol ether 21 (purified by recrystalization), and 20 ml
THF dry over CaH2, NaOH) and 1.33 ml (1.5 eq, 9.4 mmol) three ethyl

Synthesis of Competitive Inhibitors of Phospholipase A
2893
2
amine. This solution was added to the 0 C solution of 0.65 ml POCl3
(redistilled, 7 mmol) dropwise over period of 45 min and allowed to stay
at this condition for 210 min. The process of reaction was monitored by
TLC. After completion of reaction to this was added solution of 2.56 g
(1.2 eq, 6.95 mmol) of cholesterol (5,6-cholesten-3-ol) 22 in 25 ml dry
THF over 30 min at the same temperature. The reaction allowed to
stir while warming to r.t. overnight. Next day the reaction was stopped
and to this was added 10 ml NaOH 10% and 10 ml H2O. The reaction
mixture was heated to 60 C for 2 min, and extracted with CH2Cl2.
The CH2Cl2 layer was separated. 1H NMR of 8: 0.67 ppm (s, 3H),
0.87 ppm (m, 19H), 1–1.6 ppm (m, 46H), 1.6–2.18 ppm (b, 1H). (m, 6H),
2.2–2.4 ppm (m, 4H), 3.36–3.48 ppm (tt, 3H, J = 7 Hz), 3.93 ppm (bd,
2H). MS: m/z 754 (M+), Exact mass: Calcd. 754.6216; Foud, 754.6211.
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