Stereoselective synthesis of C18-guggultetrol and C 18-phytosphingosine analogues from d-fructose

Article abstract of DOI:10.1016/j.carres.2012.07.016

A series of C₁₈-guggultetrol stereo isomers and C ₁₈-phytosphingosine regio/stereo isomers were synthesised in a stereoselective fashion involving metal mediated fragmentation, stereoselective reduction, 1,4 O→O silyl migration, and Grubbs' cross metathesis as key steps. d-Fructose was used as a raw material for the preparation of all the analogues. The isophytosphingosine derivatives were evaluated against their 5-LOX (5-lipoxigenase) inhibitory activity.

Full text of DOI:10.1016/j.carres.2012.07.016

Carbohydrate Research 360 (2012) 40–46
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Stereoselective synthesis of C18-guggultetrol and C18-phytosphingosine
analogues from D-fructose
Perali Ramu Sridhar a, , Mandava Suresh a, Patteti Venu Kumar a, Kalapati Seshadri a,
⇑
Chunduri Venkata Rao b
a School of Chemistry, University of Hyderabad, Hyderabad, India
b Department of Chemistry, Sri Venkateswara University, Tirupati, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 May 2012
Received in revised form 25 July 2012
Accepted 26 July 2012
Available online 4 August 2012
A series of C18-guggultetrol stereo isomers and C18-phytosphingosine regio/stereo isomers were synthes-
ised in a stereoselective fashion involving metal mediated fragmentation, stereoselective reduction, 1,4
O?O silyl migration, and Grubbs’ cross metathesis as key steps. D-Fructose was used as a raw material
for the preparation of all the analogues. The isophytosphingosine derivatives were evaluated against their
5-LOX (5-lipoxigenase) inhibitory activity.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
D-Fructose
C
18-Guggultetrol
Phytosphingosine
Isophytosphingosine
5-Lipoxigenase
1. Introduction
phytosphingosines as well as their stereoisomers.8 Interestingly,
the regioisomers of these amino-triols, where the amine is posi-
tioned on the C-1, C-3, or C-4 carbon are very less. To the best of
our knowledge, there is only one recent report in which the amine
was placed on the C-4 carbon and it was called isophytosphingo-
sine.9 The stereoisomers of this isophytosphingosine showed
excellent cytotoxic activity in B16 murine melanoma cells, in some
Guggultetrols1 are aliphatic long chain 1,2,3,4-tetrols isolated
from the gum resin of Commiphora mukul, a small tree of the family
Burseraceae, endemic to the Hindustan peninsula. Two major com-
pounds in guggultetrols series identified are
D-xylo-C18-guggulte-
-xylo-C20-guggultetrol (40%) (Fig. 1). The crude
trol (ꢀ50%) and
D
resin, called guggulu in Sanskrit, has been used especially for the
treatment of rheumatoid arthritis and lipid disorders in Ayurveda,
the traditional system of ancient Indian medicine.2 Along with
cases better activity than the D-ribo-C18-phytosphingosine itself. As
part of our research program aimed at developing enantioselective
methods for the synthesis of bioactive molecules starting from
commercially inexpensive carbohydrate raw materials,10 herein
we report the synthesis of guggultetrol epimers as well as the ste-
reo and regioisomers of phytosphingolipids from the commercially
inexpensive ketose, D-fructose. Further, the synthetic analogues of
phytosphingolipids were evaluated against their 5-LOX (5-lipoxi-
genase) inhibitory activity.
guggultetrols two other steroids namely
Z and E-pregna-
4,17(20)-diene-3,16-diones (Z- and E-guggulsterones) were also
isolated. However the recent studies of randomized, placebo-con-
trolled clinical trials revealed that guggulsterones do not improve
the serum cholesterol levels in adults with hypercholesterolemia.3
On the other hand, very few reports are available in the literature
for the synthesis of guggultetrols4 and their isomers5 and no bio-
logical studies were reported on these fatty alcohols. Since guggul-
tetrols are structurally similar to the naturally occurring
phytosphingolipids (Fig. 1), which are known to play multiple bio-
logical activities,6 these are also expected to exhibit interesting
biological properties.
2. Results and discussion
The key intermediates 2 and 3, for the preparation of guggulte-
trol epimers as well as phytosphingosine analogues, were synthe-
sized on a large scale from commercially available inexpensive raw
Recent work on the biological importance of D-ribo-C18-phyto-
sphingosine7 made it an attractive target to the synthetic commu-
nity. As a result a number of methods were published to prepare
material D-fructose 1 in a series of eight steps (2 in 27% and 3 in
22% overall yield) involving only three column chromatographic
purifications.10 Cross metathesis of alcohols 2 and 3 with tetra-
dec-1-ene using Grubbs II generation catalyst provided the alke-
nols 410a and 510b which were further hydrogenated with 10%
⇑
Corresponding author. Tel.: +91 40 66794823; fax: +91 40 23012460.
E-mail address: prssc@uohyd.ernet.in (P. Ramu Sridhar).
0008-6215/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2012.07.016

P. Ramu Sridhar et al. / Carbohydrate Research 360 (2012) 40–46
41
OH
OH
nucleophilic substitution with phthalimide at the primary carbon
center.11 Cross-metathesis of olefins 12 and 13 with tetradec-1-
ene provided the protected 5,6-unsaturated phytosphingosine ana-
logues 14 and 15. Pd/C/H2 mediated hydrogenation of the double
bond provided compounds 16 and 17 in good yield. A one-pot
deprotection of acid-sensitive TBDMS and acetonide protective
groups, by using 80% aqueous CH3COOH at 50 °C provided the
phthalimide protected regioisomeric phytosphingosine analogues
18 and 19 in excellent yield. Finally deprotection of phthalimide
achieved by stirring the reaction mixture in H2NNH2ꢁH2O/MeOH
gave phytosphingosine analogues 20 and 21 as white solids
(Scheme 2).
Further we also planned to synthesize 5,6-unsaturated isomeric
phytosphingosine derivatives. Thus, the deprotection of alcohol
protective groups in olefins 14 and 15 with AcOH at 50 °C fur-
nished 5,6-unsaturated triols 22 and 24. Final the removal of
phthalimide protective group using NH2NH2ꢁH2O/MeOH provided
the 5,6-unsaturated isomeric phytosphingosine derivatives 23
and 25 respectively in good yield (Scheme 3).
NH2 OH
HO
HO
n
n
OH
OH
n = 13 D-xylo-C18-guggultetrol
n = 15 -xylo-C20-guggultetrol
n = 13 C18-phytosphingolipid
n = 15 C20-phytosphingolipid
D
Figure 1. General structure of guggultetrols and phytosphingolipids.
Pd/C under hydrogen atmosphere to give alcohols 610a and 710a
respectively in good yield. A one-pot deprotection of TBDMS and
acetonide protective groups using p-TsOH/MeOH followed by
20% aqueous HCl provided D-ribo-C18-guggultetrol 8 and L-arabi-
no-C18-guggultetrol 9 respectively, in excellent yield as single
enantiomers. On the other hand, global deprotection of TBDMS
and acetonide protecting groups of alkenols 4 and 5 under similar
reaction conditions mentioned above furnished unsaturated gugg-
ultetrol analogues 10 and 11 (Scheme 1).
The synthesis of phytosphingosine analogues was envisaged
again starting from the key intermediate enols 2 and 3. Thus, the
reaction of 2 and 3 with Ph3P/DIAD/phthalimide in dry THF under
Mitsunobu reaction conditions provided enols 1211 and 1311
respectively, via a one-pot 1,4 O?O silyl migration followed by
3. In vitro 5-lipoxigenase (5-LOX) enzyme assay
It has been shown that 5-LOX and its metabolites, leukotrienes
(LTs) and 5-hydroxyeicosatetraenoic acid (5-HETE) are important
OH
O
R1
O
R2
OH
OH
Ref. 10a
8 steps
HO
TBDMSO
O
OH
1
2
2
R = H, R = OH (27%)
1
3 R1 = OH, R2 = H (22%)
D-Fructose
tetradec-1-ene,
Grubbs' II
CH2Cl2, reflux
R2
R2
O
R1
R1
O
10% Pd/C/H2
EtOAc
C12H25
C12H25
TBDMSO
O
TBDMSO
O
1
2
1
2
6
7
4
5
R = H, R = OH (87%)
R = H, R = OH (60%)
1
2
1
2
R = OH, R = H (85%)
R = OH, R = H (57%)
i) p-TsOH/MeOH
ii) 20% aq HCl
i) p-TsOH/MeOH
ii) 20% aq HCl
OH
OH
OH
OH
HO
HO
C12H25
C12H25
OH
OH
D-ribo-C18-guggultetrol 8 (79%)
5,6-dehydro-
D-ribo-C18-guggultetrol 10 (80%)
Or
Or
OH
OH
OH
OH
HO
HO
C12H25
C12H25
OH
OH
5,6-dehydro-
L-arabino-C18-guggultetrol 11 (77%)
L-arabino-C18-guggultetrol 9 (82%)
Scheme 1. Stereoselective synthesis of guggultetrol analogues.

42
P. Ramu Sridhar et al. / Carbohydrate Research 360 (2012) 40–46
R1
O
R1
R2
R2
O
Ph3P, DIAD
phthalimide
THF, 0-25°C
TBDMSO
O
PhthN
O
1
2
12 R1 = H, R2 = OTBDMS (75%)
2
3
R1 = H, R = OH
2
13 R1 = OTBDMS, R2 = H (75%)
R = OH, R = H
tetradec-1-ene,
Grubbs' II
CH2Cl2, reflux
R1
O
R1
R2
O
R2
10% Pd/C/H2
MeOH
C12H25
C12H25
PhthN
O
PhthN
O
16 R1 = H, R2 = OTBDMS (95%)
14 R1 = H, R2 = OTBDMS (88%)
17 R1 = OTBDMS, R2 = H (91%)
15 R1 = OTBDMS, R2 = H (87%)
OH OH
AcOH, 50°C
H2N
C12H25
OH
R1
OH
R2
20 (78%)
Or
NH2NH2.H2O
MeOH
C12H25
OH
OH
PhthN
OH
H2N
1
2
C12H25
18
19
R = H, R = OH (85%)
1
2
OH
21 (85%)
R = OH, R = H (90%)
Scheme 2. Stereoselective synthesis of isomeric phytosphingosine analogues.
OH OH
OH OH
NH2NH2.H2O
MeOH, 71%
AcOH
H2N
14
15
C12H25
C12H25
50°C
81%
PhthN
PhthN
OH
OH
22
23
OH OH
OH OH
NH2NH2.H2O
MeOH, 75%
AcOH
H2N
C12H25
C12H25
50°C
86%
OH
OH
24
25
Scheme 3. Synthesis of 5,6-unsaturated isomeric phytosphingosine analogues.
pro-inflammatory mediators participating in various forms of
acute and sub acute inflammation as well as potential survival fac-
tors for prostate cancer cells and the inhibition of 5-LOX triggered
massive apoptosis. Thus, the development of 5-LOX inhibitors
gains much importance in order to reduce the side effects caused
by the present drugs in the market. For example, COX-2 inhibition
by celecoxib (a selective COX-2 inhibitor, drug present in the mar-
ket) in cancer cell lines was shown to increase the formation of 5-
HETE, which is having tumor cell proliferative property.12 In our
studies the isophytosphingosine analogues 20, 21, 23, and 25 were
tested in vitro for their inhibitory properties against 5-LOX enzyme
Table 1
IC50 values of the isophytosphingosine derivatives against 5-LOX enzyme inhibition
Compound
5-LOX
IC50 (lM)
20
23
>100
46
21
50
25
NDGA
0.96
1.8
NDGA: nordihydroguaiaretic acid.

P. Ramu Sridhar et al. / Carbohydrate Research 360 (2012) 40–46
43
using nordihydroguaiaretic acid (NDGA) as a standard 5-LOX
inhibitor (IC50 = 1.8
M).13 Out of the 4 molecules tested com-
pound 21 and 23 showed moderate inhibition and the compound
25 exhibited excellent inhibitory activity with IC50 = 0.96
(Table 1). Comparing the activities of compounds 20 and 23, pos-
sessing -ribo configuration and compounds 21 and 25, possessing
-arabino configuration, indicates that incorporation of unsatura-
5.3. (2R,3R,4R)-Octadecane-1,2,3,4-tetraol (9)
l
Compound 9 was synthesized following the procedure de-
scribed for compound 8: mp: 87–90 °C. Yield 82%. Rf (5% MeOH/
lM
CH2Cl2) 0.41; ½a D25
ꢃ
+5 (c 1 in MeOH). IR (KBr, cmꢄ1): mmax 3391,
D
3292, 2920, 2849, 1469, 1323, 1298, 1076; 1H NMR (400 MHz,
CD3OD): d 3.89 (td, 1H, J = 2.0, 6.4 Hz), 3.62–3.60 (m, 3H), 3.33
(d, 1H, J = 2.0 Hz), 1.78–1.73 (m, 1H), 1.57–1.55 (m, 1H), 1.38–
1.29 (br m, 24H), 0.90 (t, 3H, J = 6.4 Hz). 13C NMR (100 MHz,
CD3OD): d 74.6, 74.1, 72.2, 64.7, 33.5, 33.0, 30.8, 30.7, 30.6, 30.5,
30.4, 30.3, 23.7, 14.4. Low-resolution MS (ESI): m/z: 319 (M+1)+.
Anal. Calcd for C18H38O4: C, 67.88; H, 12.03. Found: C, 67.75; H,
12.09.
L
tion at 5 position increases the 5-LOX inhibitory activity.
4. Conclusion
In conclusion, an efficient method for the stereoselective syn-
thesis of
their 5,6-unsaturated counterparts as well as regioisomers of phy-
tosphingosines were developed starting from -fructose. This is the
D-ribo-C18-guggultetrol, L-arabino-C18-guggultetrol, and
5.4. (2S,3R,4R,E)-Octadec-5-ene-1,2,3,4-tetraol (10)
D
first report on the synthesis of the isophytosphingosine analogue
in which the amine was placed on an achiral carbon. Further all
the phytosphingosine regioisomers were evaluated for their
in vitro 5-LOX inhibitory activity. Synthesis of other regio-isomers
of phytosphingosine family and the evaluation of their bioactivities
were under progress.z
To a stirred solution of 4 (60 mg, 0.12 mmol) in methanol (2 mL)
was added p-toluenesulfonic acid (36.6 mg, 0.19 mmol). The resul-
tant mixture was stirred at room temperature for 2 h. Then 20% aq
HCl (2 mL) was added to the mixture and stirring continued for an-
other 2 h. Removal of the solvents under reduced pressure pro-
vided a viscous residue. To the residue was added EtOAc (10 mL)
and water (5 mL). The organic layer was separated and the aqueous
layer was extracted with EtOAc (3 ꢂ 20 mL). The combined organic
layers were dried over anhydrous Na2SO4 and concentrated. Col-
umn chromatography of the crude product using CH2Cl2/MeOH
(95:5) provided compound 10 (32.0 mg, 80%) as a colorless solid.
5. Experimental
5.1. General
Mp: 69–72 °C. Rf (5% MeOH/CH2Cl2) 0.46; ½a D25
ꢃ
+1 (c 0.8 in MeOH).
All the reactions were carried out under inert atmosphere with
dry solvents under anhydrous conditions unless otherwise men-
tioned. TLC was run on Silica Gel 60 F254 (Merck) and the spots
were detected by staining with H2SO4 in methanol (5%, V/V) or
phosphomolybdic acid in ethanol (5%, W/V) and heat. Silica-gel
(100–200 mesh) was used as a stationary phase for column chro-
matography. NMR spectra were recorded at 25 °C on a Bruker
AvanceIII 400 (400 MHz for 1H and 100 MHz for 13C) or 500
(500 MHz for 1H and 125 MHz for 13C) instrument with CDCl3 or
CD3OD as solvent and residual CHCl3 (dH 7.26 ppm) or CH3OH (dH
3.31 ppm) as internal standard for 1H and CDCl3 (dC 77.0 ppm) or
CD3OD (dC 49.0 ppm) as internal standard for 13C. Chemical shifts
are given in d (ppm) and coupling constants (J) in Hz. IR spectra
were recorded on JASCO FT/IR-5300. Elemental analyses were re-
corded on a Thermo Finnigan Flash EA 1112 analyzer. Mass spectra
were recorded on Shimadzu-LCMS-2010A mass spectrometer.
IR (KBr, cmꢄ1): mmax 3339, 2920, 2849, 1649, 1518, 1466, 1325,
1228, 1051; 1H NMR (400 MHz, CD3OD): d 5.74 (dt, 1H, J = 6.8,
15.2 Hz), 5.58 (dd, 1H, J = 7.6, 15.6 Hz), 4.19 (dd, 1H, J = 4.0,
7.6 Hz), 3.76 (dd, 1H, J = 2.4, 10.8 Hz), 3.61 (dd, 1H, J = 5.6,
11.2 Hz), 3.56–3.53 (m, 2H), 2.10–2.03 (m, 2H), 1.43–1.38 (m,
2H), 1.33–1.29 (br m, 18H), 0.90 (t, 3H, J = 6.4 Hz); 13C NMR
(100 MHz, CD3OD): d 135.1, 129.9, 75.9, 75.1, 74.0, 64.7, 33.6,
33.0, 30.8, 30.7, 30.6, 30.5, 30.4, 30.3, 23.7, 14.4; Low-resolution
MS (ESI): m/z: 317 (M+1)+; Anal. Calcd for C18H36O4: C, 68.31; H,
11.47. Found: C, 68.21; H, 11.36.
5.5. (2R,3R,4R,E)-Octadec-5-ene-1,2,3,4-tetraol (11)
Compound 11 was synthesized following the procedure de-
scribed for compound 10: Yield 77%. Rf (5% MeOH/CH2Cl2): 0.39;
½
a 2D5 +4 (c 0.6 in MeOH). IR (KBr, cmꢄ1): mmax 3447, 2918, 2836,
ꢃ
1685, 1523, 1466, 1325, 1024; 1H NMR (400 MHz, CD3OD): d
5.74 (dt, 1H, J = 6.4, 15.6 Hz), 5.59 (dd, 1H, J = 6.8, 15.6 Hz), 4.09
(t, 1H, J = 7.2 Hz), 3.85 (td, 1H, J = 2.8, 6.8 Hz), 3.61 (dd, 2H,
J = 3.2, 5.6 Hz), 3.41 (dd, 1H, J = 2.4, 5.6 Hz), 2.10–2.05 (m, 2H),
1.43–1.38 (m, 2H), 1.33–1.29 (br m, 18H), 0.90 (t, 3H, J = 6.4 Hz);
13C NMR (100 MHz, CD3OD): d 134.3, 131.4, 74.6, 74.1, 72.2, 64.7,
33.5, 33.0, 30.8, 30.7, 30.6, 30.5, 30.4, 30.3, 23.7, 14.4; Low-resolu-
tion MS (ESI): m/z: 317 (M+1)+; Anal. Calcd for C18H36O4: C, 68.31;
H, 11.47. Found: C, 68.21; H, 11.55.
5.2. (2S,3R,4R)-Octadecane-1,2,3,4-tetraol (8)
To a stirred solution of 6 (60 mg, 0.12 mmol) in methanol (2 mL)
was added p-toluenesulfonic acid (36.4 mg, 0.19 mmol). The resul-
tant mixture was stirred at room temperature for 2 h. Then 20% aq
HCl (1.5 mL) was added to the mixture and stirred for another 2 h.
Removal of the solvents under reduced pressure provided a viscous
residue. To the residue was added EtOAc (20 mL) and water
(10 mL). The organic layer was separated and the aqueous layer
was extracted with EtOAc (3 ꢂ 20 mL). The combined organic lay-
ers were dried over anhydrous Na2SO4 and concentrated. Column
chromatography of the crude product using CH2Cl2/MeOH (95:5)
provided compound 8 (31 mg, 79%) as a colorless solid. Mp: 97–
5.6. 2-((S)-2-(tert-Butyldimethylsilyloxy)-2-((4R,5S)-2,2-
dimethyl-5-((E)-tetradec-1-enyl)-1,3-dioxolan-4-yl)ethyl)iso-
indoline-1,3-dione (14)
99 °C. Rf (5% MeOH/CH2Cl2): 0.47; ½a D25
ꢃ
+9 (c 0.9 in MeOH). IR
(KBr, cmꢄ1): mmax 3418, 3292, 2918, 2851, 1471, 1419, 1331,
1269, 1101; 1H NMR (400 MHz, CD3OD): d 3.77 (dd, 1H, J = 2.4,
10.8 Hz), 3.69–3.59 (m, 3H), 3.46 (t, 1H, J = 6.4 Hz), 1.71–1.65 (m,
1H), 1.57 (br s, 1H), 1.32–1.29 (br m, 24H), 0.90 (t, 3H,
J = 6.0 Hz); 13C NMR (100 MHz, CD3OD): d 76.0, 74.4, 74.0, 64.6,
33.4, 33.1, 30.9, 30.8, 30.5, 23.7, 14.4; Low-resolution MS (ESI):
m/z: 319 (M+1)+; Anal. Calcd for C18H38O4: C, 67.88; H, 12.03.
Found: C, 67.71; H, 12.11.
Compound 12 (275 mg, 0.63 mmol) and 1-tetradecene (0.50 g,
2.5 mmol) were dissolved in CH2Cl2 (25 mL) at room temperature.
Grubbs II generation catalyst (4 mol %) was added to the solution
and then the reaction mixture was refluxed under nitrogen for
24 h. After cooling the reaction mixture was concentrated and
purified by column chromatography with EtOAc:hexane (6:94) to
afford compound 14 (305 mg, 88%) as a colorless liquid. Rf (10%
EtOAc/hexane): 0.65. ½a D25
ꢃ
ꢄ30 (c 2 in CHCl3). IR (neat, cmꢄ1): mmax

44
P. Ramu Sridhar et al. / Carbohydrate Research 360 (2012) 40–46
3474, 2928, 2854, 1774, 1718, 1614, 1467, 1396; 1H NMR
(400 MHz, CDCl3): 7.82 (d, 2H, J = 3.2 Hz), 7.70 (d, 2H,
2H, J = 3.2, 5.6 Hz), 7.72 (dd, 2H, J = 3.2, 5.6 Hz), 4.17 (td, 1H,
J = 3.6, 8.4 Hz), 4.13–4.07 (m, 1H), 3.96 (dd, 1H, J = 5.2, 7.6 Hz),
3.84 (dd, 1H, J = 8.4, 13.6 Hz), 3.59 (dd, 1H, J = 3.6, 14.0 Hz), 1.64–
1.61 (m, 2H), 1.46 (s, 3H), 1.33 (s, 3H), 1.25 (br s, 24H), 0.87 (t,
3H, J = 6.4 Hz), 0.74 (s, 9H), 0.04 (s, 3H), ꢄ0.15 (s, 3H); 13C NMR
(100 MHz, CDCl3): d 168.2, 133.9, 132.0, 123.1, 107.8, 79.5, 77.1,
69.0, 40.9, 31.9, 29.8, 29.6, 29.5, 29.3, 28.2, 25.9, 25.8, 25.5, 22.6,
18.0, 14.1, ꢄ4.4. ꢄ4.9; Low-resolution MS (ESI): m/z: 603 (M+1)+;
Anal. Calcd for C35H59NO5Si: C, 69.84; H, 9.88; N, 2.33. Found: C,
69.92; H, 9.81; N, 2.29.
d
J = 2.0 Hz), 5.81–5.79 (br m, 2H), 4.64 (t, 1H, J = 12.8 Hz), 4.20 (t,
1H, J = 6.0 Hz), 4.10 (dd, 1H, J = 4.0, 6.8 Hz), 3.89 (dd, 1H, J = 6.8,
13.6 Hz), 3.74 (dd, 1H, J = 6.4, 14.0 Hz), 2.13–2.10 (m, 2H), 1.42–
1.38 (m, 2H), 1.33 (s, 3H), 1.26–1.23 (br m, 21H), 0.86 (t, 3H,
J = 5.6 Hz), 0.83 (s, 9H), 0.01 (s, 3H), ꢄ0.06 (s, 3H); 13C NMR
(100 MHz, CDCl3): d 168.3, 136.7, 133.8, 132.2, 126.2, 123.1,
108.0, 80.3, 79.1, 68.6, 41.3, 32.4, 31.9, 29.7, 29.6, 29.5, 29.3,
29.0, 27.0, 25.8, 22.7, 17.8, 14.1, ꢄ4.5, ꢄ4.6. Low-resolution MS
(ESI): m/z: 599 (M)+; Anal. Calcd for C35H57NO5Si: C, 70.07; H,
9.58; N, 2.33. Found: C, 70.21; H, 9.51; N, 2.45.
5.10. 2-((2S,3R,4R)-2,3,4-Trihydroxyoctadecyl)isoindoline-1,3-
dione (18)
5.7. 2-((R)-2-(tert-Butyldimethylsilyloxy)-2-((4R,5S)-2,2-
dimethyl-5-((E)-tetradec-1-enyl)-1,3-dioxolan-4-yl)ethyl)iso-
indoline-1,3-dione (15)
A solution of compound 16 (90 mg, 0.14 mmol) in 80% aqueous
acetic acid (5 mL) was stirred at 50 °C for 15 h. After completion of
the reaction (by TLC) the reaction mixture was cooled to room tem-
perature and the solvent was removed under reduced pressure. The
residual acetic acid was co-evaporated with toluene to give a white
solid. Flash column chromatography of this residue with
EtOAc:hexane (1:1) provided compound 18 (56.6 mg, 85%) as a
Compound 15 was synthesized following the procedure de-
scribed for compound 14: Yield 87%. Rf (10% EtOAc/hexane):
0.60. ½a 2D5
ꢃ
+10 (c 1 in CHCl3). IR (neat, cmꢄ1): mmax 3477, 2926,
2854, 1774, 1720, 1618, 1467, 1394; 1H NMR (400 MHz, CDCl3):
d 7.83 (dd, 2H, J = 3.2, 5.6 Hz), 7.71 (dd, 2H, J = 3.2, 5.6 Hz), 5.73
(dt, 1H, J = 6.4, 15.2 Hz), 5.62 (dd, 1H, J = 8.8, 15.6 Hz), 4.56 (dd,
1H, J = 6.0, 8.8 Hz), 4.15–4.10 (m, 1H), 4.03 (dd, 1H, J = 6.0,
8.0 Hz), 3.78–3.68 (m, 2H), 2.12–2.07 (m, 2H), 1.49 (s, 3H), 1.40–
1.38 (m, 2H), 1.33 (s, 3H), 1.25–1.23 (br m, 18H), 0.87 (t, 3H,
J = 6.0 Hz), 0.75 (s, 9H), 0.06 (s, 3H), ꢄ0.12 (s, 3H); 13C NMR
(100 MHz, CDCl3): d 168.3, 137.0, 133.9, 132.1, 125.8, 123.1,
108.1, 80.1, 78.6, 69.1, 40.1, 32.3, 31.9, 29.7, 29.6, 29.5, 29.3,
28.8, 28.0, 25.6, 25.3, 22.7, 18.0, 14.1, ꢄ4.4, ꢄ4.9; Low-resolution
MS (ESI): m/z: 600 (M)+; Anal. Calcd for C35H57NO5Si: C, 70.07; H,
9.58; N, 2.33. Found: C, 70.16; H, 9.49; N, 2.41.
white foam. Rf (50% EtOAc/hexane): 0.61. ½a D25
ꢄ1.2 (c 0.6 in CHCl3).
ꢃ
IR (KBr, cmꢄ1):
mmax 3485, 3312, 3302, 2916, 2849, 1780, 1707, 1467,
1392; 1H NMR (400 MHz, CDCl3): d 7.85 (dd, 2H, J = 3.2, 5.6 Hz), 7.72
(dd, 2H, J = 2.8, 5.6 Hz), 4.11–3.98 (m, 3H), 3.82–3.77 (m, 1H), 3.39
(br s, 1H), 3.27 (br s, 2H), 2.52 (br s, 1H), 1.77–1.68 (m, 1H), 1.53–
1.45 (m, 2H), 1.30–1.26 (br m 23H), 0.88 (t, 3H, J = 6.8 Hz); 13C
NMR (100 MHz, CDCl3): d 169.5, 134.2, 131.9, 123.5, 73.9, 73.8,
72.9, 41.0, 32.8, 31.8, 29.6, 29.5, 29.3, 25.4, 22.6, 14.0; Low-resolu-
tion MS (ESI): m/z: 448 (M+1)+; Anal. Calcd for C26H41NO5: C,
69.77; H, 9.23; N, 3.13. Found: C, 69.71; H, 9.15; N, 3.18.
5.11. 2-((2R,3R,4R)-2,3,4-Trihydroxyoctadecyl)isoindoline-1,3-
5.8. 2-((S)-2-(tert-Butyldimethylsilyloxy)-2-((4R,5S)-2,2-
dimethyl-5-tetradecyl-1,3-dioxolan-4-yl)ethyl)iso-indoline-1,3-
dione (16)
dione (19)
Compound 19 was synthesized following the procedure de-
scribed for compound 18: Yield 90%. Rf (50% EtOAc/hexane):
To a stirred solution of compound 14 (110 mg, 0.18 mmol) in
MeOH (6 mL) was added 10% palladium on charcoal (30 mg). Then
the mixture was degassed and stirred under hydrogen atmosphere
at room temperature overnight. After completion of the reaction
(by TLC) the suspension was filtered through a pad of celite and
concentrated. The crude product was purified by column chroma-
tography using hexane:EtOAc (19:1) to afford compound 16
(104 mg, 95%) as a colorless liquid. Rf (10% EtOAc/hexane): 0.68;
0.56. ½a 2D5
ꢃ
+1 (c 0.4 in CHCl3). IR (KBr, cmꢄ1): mmax 3435, 3306,
2918, 2849, 1778, 1714, 1467, 1361; 1H NMR (400 MHz, CDCl3):
d 7.86 (dd, 2H, J = 3.2, 5.6 Hz), 7.73 (dd, 2H, J = 2.8, 5.6 Hz), 4.19–
4.17 (m, 1H), 4.00 (dd, 1H, J = 7.6, 14.4), 3.87 (dd, 1H, J = 4.8,
14.0), 3.78–3.77 (m, 1H), 3.39 (t, 1H, J = 5.2 Hz), 3.28 (d, 1H,
J = 4.8 Hz), 3.01 (d, 1H, J = 7.2 Hz), 2.36 (d, 1H, J = 5.6 Hz), 1.62–
1.45 (m, 3H), 1.31–1.26 (br m, 23H), 0.88 (t, 3H, J = 6.4 Hz); 13C
NMR (100 MHz, CDCl3): d 169.0, 134.2, 132.0, 123.5, 74.2, 72.9,
69.8, 41.6, 33.6, 31.9, 29.7, 29.6, 29.5, 29.3, 25.9, 22.6, 14.0; Low-
resolution MS (ESI): m/z: 448 (M+1)+; Anal. Calcd for C26H41NO5:
C, 69.77; H, 9.23; N, 3.13. Found: C, 69.85; H, 9.16; N, 3.21.
½
a 2D5
ꢃ
ꢄ10 (c 1 in CHCl3). IR (KBr, cmꢄ1): mmax 2922, 2849, 1776,
1720, 1604, 1468, 1398; 1H NMR (400 MHz, CDCl3): d 7.82 (dd,
2H, J = 3.2, 5.6 Hz), 7.69 (dd, 2H, J = 3.2, 5.6 Hz), 4.21 (dd, 1H,
J = 6.8, 12.8 Hz), 4.14–4.09 (m, 1H), 4.03 (t, 1H, J = 6.0 Hz), 3.91
(dd, 1H, J = 6.8, 13.6 Hz), 3.74 (dd, 1H, J = 6.4, 13.6 Hz), 1.61–1.53
(m, 2H), 1.29–1.23 (br m, 24H), 1.18 (s, 3H), 1.15 (s, 3H), 0.86 (t,
3H, J = 6.4 Hz), 0.82 (s, 9H), 0.06 (s, 3H), ꢄ0.01 (s, 3H); 13C NMR
(100 MHz, CDCl3): d 168.5, 133.8, 132.3, 123.0, 107.9, 80.5, 78.0,
67.8, 42.3, 31.9, 29.8, 29.7, 29.6, 29.5, 29.4, 27.5, 26.2, 25.7, 25.2,
22.7, 17.9, 14.1, ꢄ3.9. ꢄ4.6; Low-resolution MS (ESI): m/z: 603
(M+1)+; Anal. Calcd for C35H59NO5Si: C, 69.84; H, 9.88; N, 2.33.
Found: C, 69.71; H, 9.81; N, 2.43.
5.12. (2S,3R,4R)-1-Aminooctadecane-2,3,4-triol (20)
To a stirred solution of compound 18 (50 mg, 0.11 mmol) in dry
CH3OH (3 mL) was added hydrazine hydrate (11.2 mg, 0.22 mmol)
at room temperature. The resulting mixture was stirred overnight.
After completion of the reaction the solvent was evaporated under
reduced pressure. The obtained residue was purified by flash col-
umn chromatography using CHCl3:MeOH:NH4OH (80:20:0.5) to
give compound 20 (27.5 mg, 78%) as a colorless solid. Mp: 90–
5.9. 2-((R)-2-(tert-Butyldimethylsilyloxy)-2-((4R,5S)-2,2-
dimethyl-5-tetradecyl-1,3-dioxolan-4-yl)-ethyl)iso-indoline-
1,3-dione (17)
93 °C. Rf (CHCl3/MeOH/NH4OH 80:20:0.5): 0.24. ½a D25
ꢃ
+13 (c 0.5 in
CHCl3). IR (KBr, cmꢄ1): mmax 3425, 2918, 2851, 1469, 1396; 1H
NMR (400 MHz, CD3OD):
d 3.64–3.58 (m, 2H), 3.38 (t, 1H,
J = 6.4 Hz), 2.90 (dd, 1H, J = 3.6, 13.2 Hz), 2.72 (dd, 1H, J = 7.2,
13.2 Hz), 1.72–1.66 (m, 1H), 1.57–1.54 (m, 1H), 1.29 (br s, 24H),
0.90 (t, 3H, J = 6.8 Hz); 13C NMR (100 MHz, CD3OD): d 76.7, 74.5,
74.0, 44.8, 33.6, 33.08 30.9, 30.8, 30.7, 30.5, 26.7, 23.7, 14.5;
Low-resolution MS (ESI): m/z: 318 (M+1)+; Anal. Calcd for
Compound 17 was synthesized following the procedure de-
scribed for compound 16: Yield 91%. Rf (5% EtOAc/hexane): 0.64.
½
a 2D5 +13 (c 0.7 in CHCl3). IR (neat, cmꢄ1): mmax 2926, 2854, 1776,
ꢃ
1720, 1614, 1467, 1392; 1H NMR (400 MHz, CDCl3): d 7.85 (dd,

P. Ramu Sridhar et al. / Carbohydrate Research 360 (2012) 40–46
45
C18H39NO3: C, 68.09; H, 12.38; N, 4.41. Found: C, 68.19; H, 12.28;
N, 4.51.
EtOAc/hexane): 0.53. ½a D25
ꢃ
+2 (c 1.7 in CHCl3). IR (KBr, cmꢄ1): mmax
3489, 2922, 2856, 1776, 1714, 1618, 1466; 1H NMR (400 MHz,
CDCl3): d 7.85 (dd, 2H, J = 3.2, 5.2 Hz), 7.73 (dd, 2H, J = 3.2,
5.6 Hz), 5.79 (dt, 1H, J = 6.8, 15.2 Hz), 5.52 (dd, 1H, J = 6.8,
15.6 Hz), 4.29 (t, 1H, J = 6.0), 4.18–4.15 (m, 1H), 4.00 (dd, 1H,
J = 8.0, 14.4 Hz), 3.83 (dd, 1H, J = 4.4, 14.0 Hz), 3.46 (dd, 1H,
J = 1.6, 5.2 Hz), 2.07–2.02 (m, 2H), 1.37–1.30 (m, 2H), 1.23 (br s,
18H), 0.87 (t, 3H, J = 6.8 Hz); 13C NMR (100 MHz, CDCl3): d 169.0,
134.9, 134.2, 131.9, 128.2, 123.5, 74.8, 72.9, 69.5, 41.5, 32.3, 31.9,
29.6, 29.5, 29.3, 29.2, 29.0, 22.8, 14.1; Low-resolution MS (ESI):
m/z: 445 (M)+; Anal. Calcd for C26H39NO5: C, 70.08; H, 8.82; N,
3.14. Found: C, 70.21; H, 8.75; N, 3.21.
5.13. (2R,3R,4R)-1-Aminooctadecane-2,3,4-triol (21)
Compound 21 was synthesized by following the procedure de-
scribed for compound 20: mp: 86–89 °C. Yield 85%. Rf (CHCl3/
MeOH/NH4OH, 80:20:0.5): 0.26. ½a D25
ꢃ
+6 (c 0.9 in CHCl3). IR (KBr,
cmꢄ1): mmax 3356, 2918, 2851, 1467, 1342; 1H NMR (400 MHz,
CD3OD): d 3.87 (ddd, 1H, J = 2.0, 4.4, 8.0 Hz), 3.58 (td, 1H, J = 2.8,
8.0 Hz), 3.22 (dd, 1H, J = 2.0, 8.0 Hz), 2.85 (dd, 1H, J = 8.0,
12.8 Hz), 2.77 (dd, 1H, J = 4.4, 13.2 Hz), 1.79–1.73 (m, 1H), 1.59–
1.55 (m, 1H), 1.32–1.26 (br m, 24H), 0.90 (t, 3H, J = 6.8 Hz); 13C
NMR (100 MHz, CD3OD): d 76.0, 72.6, 71.7, 45.5, 34.7, 33.0, 30.9,
30.8, 30.7, 30.5, 26.6, 23.7, 14.4; Low-resolution MS (ESI): m/z:
318 (M+1)+; Anal. Calcd for C18H39NO3: C, 68.09; H, 12.38; N,
4.41. Found: C, 68.21; H, 12.29; N, 4.38.
5.17. (2R,3R,4R,E)-1-Aminooctadec-5-ene-2,3,4-triol (25)
Compound 25 was synthesized following the procedure de-
scribed for compound 23: mp: 73–76 °C. Yield 75%. Rf (CHCl3/
MeOH/NH4OH 80:20:0.5): 0.25. ½a D25
ꢃ
+8 (c 0.4 in MeOH). IR (KBr,
5.14. 2-((2S,3R,4R,E)-2,3,4-Trihydroxyoctadec-5-
enyl)isoindoline-1,3-dione (22)
cmꢄ1): mmax 3385, 2920, 2851, 1626, 1467, 1340; 1H NMR
(400 MHz, CD3OD): d 5.74 (dt, 1H, J = 6.4, 15.6 Hz), 5.58 (dd, 1H,
J = 6.8, 15.2 Hz), 4.06 (t, 1H, J = 6.8 Hz), 3.82–3.78 (m, 1H), 3.28
(d, 1H, J = 2.4), 2.82–2.73 (m, 1H), 2.10–2.05 (m, 2H), 1.42–1.38
(m, 2H), 1.29 (br s, 18H), 0.90 (t, 3H, J = 6.4 Hz); 13C NMR
(100 MHz, CD3OD): d 134.4, 131.5, 75.8, 74.1, 72.3, 45.5, 33.5,
33.0, 30.8, 30.7, 30.6, 30.5, 30.4, 30.3, 23.7, 14.4; Low-resolution
MS (ESI): m/z: 316 (M+1)+; Anal. Calcd for C18H37NO3: C, 68.53;
H, 11.82; N, 4.44. Found: C, 68.45; H, 11.75; N, 4.38.
A solution of compound 14 (100 mg, 0.16 mmol) in 80% aqueous
acetic acid (8 mL) was stirred at 50 °C for 15 h. After completion of
the reaction (by TLC) the reaction mixture was cooled to room tem-
perature and the solvent was removed under reduced pressure. The
residual acetic acid was co-evaporated with toluene to give a white
solid. Flash column chromatography of this residue with
EtOAc:hexane (1:1) provided compound 22 (63.7 mg, 86%) as a
white foam. Rf (40% EtOAc/hexane): 0.58. ½a D25
ꢃ
+2 (c 1 in CHCl3).
5.18. In vitro 5-LOX inhibitory assay
IR (KBr, cmꢄ1): mmax 3479, 2918, 2852, 1776, 1714, 1618, 1464,
1388; 1H NMR (400 MHz, CDCl3,): d 7.85 (dd, 2H, J = 3.2, 5.6 Hz),
7.73 (dd, 2H, J = 3.2, 5.6 Hz), 5.81 (dt, 1H, J = 6.4, 13.6 Hz), 5.57
(dd, 1H, J = 7.2, 15.8 Hz), 4.26 (t, 1H, J = 6.0), 4.13–4.06 (m, 1H),
3.99 (dd, 1H, J = 5.2, 14.8 Hz), 3.93–3.89 (m, 1H), 3.45–3.41 (m,
1H), 3.27 (d, 1H, J = 4.0 Hz), 3.16 (d, 1H, J = 4.0 Hz), 2.08–2.03 (m,
2H), 1.38–1.33 (m, 2H), 1.26–1.24 (br m, 18H), 0.88 (t, 3H,
J = 6.8 Hz); 13C NMR (100 MHz, CDCl3): d 169.5, 135.8, 134.3,
131.8, 127.9, 123.6, 74.7, 73.4, 72.6, 41.0, 32.3, 31.9, 29.6, 29.5,
29.4, 29.3, 29.2, 29.0, 22.6, 14.0; Low-resolution MS (ESI): m/z:
446 (M+1)+; Anal. Calcd for C26H39NO5: C, 70.08; H, 8.82; N, 3.14.
Found: C, 70.15; H, 8.79; N, 3.22.
5-LOX from potato tubers was purified and assayed as per the
reported protocol.13 Enzyme activity was measured using polar
graphic method with a Clark’s oxygen electrode on Strathkelvin
Instruments, model 782, RC-300. Typical reaction mixture con-
tained 50–100 lL of enzyme and 10 lL of substrate [AA-133 lM
(final concentration)] 2 mL 100 mM phosphate buffer pH 6.3 and
the final volume made upto 3 mL with double distilled water. Since
LOXs are oxygen-consuming enzymes, the concentration of oxygen
decrease in the reaction mixture was taken as a measure of enzyme
activity. Reaction was allowed to proceed at 25 °C and the maxi-
mum slope generated was taken for calculating enzyme activity.
The activity was expressed as units/mg protein, where one unit
5.15. (2S,3R,4R,E)-1-Aminooctadec-5-ene-2,3,4-triol (23)
is defined as one
lmole of oxygen consumed per minute.
To a stirred solution of compound 22 (100 mg, 0.22 mmol) in dry
CH3OH (3 mL) was added hydrazine hydrate (22.4 mg, 0.45 mmol)
at 25 °C. The resulting mixture was stirred overnight. After comple-
tion of the reaction the solvent was evaporated under reduced pres-
sure. The obtained residue was purified by flash column
chromatography using CHCl3:MeOH:NH4OH (80:20:0.5) to give
compound 23 (50 mg, 71%) as a colorless solid. Mp: 76–79 °C. Rf
(CHCl3/MeOH/NH4OH 80:20:0.5): 0.18. IR (KBr, cmꢄ1): mmax 3418,
2918, 2851, 1614, 1467; 1H NMR (400 MHz, CD3OD): d 5.75 (dt,
1H, J = 6.4, 15.6 Hz), 5.58 (dd, 1H, J = 7.2, 15.2 Hz), 4.14 (dd, 1H,
J = 5.2, 7.2 Hz), 3.60–3.55 (m, 1H), 3.47 (dd, 1H, J = 5.2, 7.2 Hz),
2.95 (dd, 1H, J = 3.6, 13.2 Hz), 2.77 (dd, 1H, J = 7.2, 13.2 Hz), 2.10–
2.05 (m, 2H), 1.42–1.38 (m, 2H), 1.29 (br s 18H), 0.90 (t, 3H,
J = 6.8 Hz); 13C NMR (100 MHz, CD3OD): d 135.1, 130.0, 76.9, 75.0,
73.0, 44.6, 33.6, 33.1, 30.8, 30.7, 30.6, 30.5, 30.4, 23.7, 14.4; Low-res-
olution MS (ESI): m/z: 316 (M+1)+; Anal. Calcd for C18H37NO3: C,
68.53; H, 11.82; N, 4.44. Found: C, 68.45; H, 11.75; N, 4.51.
Acknowledgment
This work was supported by the Department of Science and
Technology (DST) FAST track project Grant No. SR/FTP/SC-64/2007.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.carres.2012.07.
016.
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