Semi-synthetic analogs of pinitol as potential inhibitors of TNF-α cytokine expression in human neutrophils

Article abstract of DOI:10.1016/j.bmcl.2009.02.050

Semi-synthetic analogs of pinitol were subjected to screening by determining TNF-α expression in human neutrophils using flowcytometry. Among the tested compounds, three derivatives displayed more than 50% inhibition of TNF-α cytokine secretion in LPS ind

Full text of DOI:10.1016/j.bmcl.2009.02.050

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1939–1943
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Semi-synthetic analogs of pinitol as potential inhibitors of TNF-
a cytokine
expression in human neutrophils
*
Khurshid A. Bhat, Bhahwal A. Shah, Kuldeep K. Gupta, Anjali Pandey, Sarang Bani, Subhash C. Taneja
Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu 180001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Semi-synthetic analogs of pinitol were subjected to screening by determining TNF-
a
expression in
Received 2 December 2008
Revised 22 January 2009
Accepted 12 February 2009
Available online 20 February 2009
human neutrophils using flowcytometry. Among the tested compounds, three derivatives displayed more
than 50% inhibition of TNF- cytokine secretion in LPS induced stimulated neutrophils and can be con-
sidered as potent anti-inflammatory moieties.
a
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Pinitol
Lipase
Acylates
Diacetonide
TNF-a expression
Flowcytometry
Inositols and their derivatives have attracted considerable
Pinitol is one of the major constituents and mainly being iso-
attention due to their innumerable roles in living organisms,1
and diverse biological activities.2 They also serve as starting mate-
rial in the synthesis of natural products.3 The major naturally
occurring inositol is myo-inositol. Other naturally occurring iso-
mers (occur in low concentrations) are scyllo-, chiro-, muco-, and
lated from Bougainvillea spectabilis,6 Pinus lambertiana7 and soya-
bean (Glycine max).8 There is also a widespread presence of
D-
pinitol in Alfalfa and legumes.9 Pinitol appears to mimic the effects
of insulin by acting downstream in the insulin signaling pathway.10
Bougainvillea spectabilis is used as a traditional medicine for the
treatment of diabetes in Asia and West Indies.6 Pinitol has been
shown to lower the blood glucose concentration in diabetic rats
and in normal rats given glucose.11 Its hypoglycemic and anti-dia-
betic activities are attributed to insulin like effects in normal and
alloxan induced diabetic albino mice.12 The insulin is able to up-
neo-inositol. Pinitol is 3-O-methyl ether of
known as Mateziol or Sennitol or Cathrtomannitol (Fig. 1). The con-
figuration of
-chiro-inositol has been determined by Posternak,4
D-chiro-inositol also
D
although the position of the methyl group in pinitol was estab-
lished by Arthur, et al.5 in 1952, assigning its structure as
3-O-methyl-chiro-inositol.
regulate the expression of the TNF-a gene in macrophages derived
from the THP-1 cell,13 and TNF-
a is involved in insulin resistance
through the inhibitory effect on insulin sensitivity.14
OMe
Pinitol derivatives (e.g., aminocyclitols) are reported to exhibit
glycosidase inhibitory activities,15 thus adding to their biological
importance, since glycosidase inhibitors are the potential thera-
HO
HO
OH
OH
peutic agents.16 Azole nucleoside analogs of
D-pinitol have recently
been reported as potent antitumor agents.17 Pilot studies have
indicated that co-administration of creatine (a natural nutrient
found in animal foods, claimed to be an effective nutritional ergo-
genic aid to enhance sports related activities and physical perfor-
OH
mance18) with low doses of
D-pinitol help in augmenting whole
body creatine stores by non-caloric means.19 The pinitol has shown
its potential as anti-inflammatory agent,20,21 and a remedy for fe-
ver and respiratory disorders.22 Pinitol has been demonstrated to
decrease the lipid laden foam cell formation in THP-1 derived
Figure 1.
* Corresponding author. Tel.: +91 191 2569111; fax +91 191 2569333.
E-mail address: sc_taneja@yahoo.co.in (S.C. Taneja).
human macrophages23 and inhibit TNF-
a
production.24 From the
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.02.050

1940
K. A. Bhat et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1939–1943
literature survey it is apparent that pinitol and its derivatives are
associated with a wide range of bioactivities, for example, anti-dia-
betic, anti-inflammatory, anticancer, stamina enhancing. There-
reaction mixture comprising the products was separated by col-
umn chromatography and characterized as -1,2,3,5-tetra-O-acet-
yl-4-O-methyl-chiro-inositol (2) and -1,2,4,5-tetra-O-acetyl-3-O-
D
D
fore,
inhibiting TNF-
D
-pinitol analogs were subjected to study their role in
cytokine expression in LPS activated human neu-
methyl-chiro-inositol (3) on the basis of 1H NMR, 13C NMR,
1H–1H COSY and 1H–13C COSY spectral analysis (Scheme 1).
Various acyl analogs of pinitol (new as well as reported ones)
were also synthesized for bioactivity screening (Schemes 2–4).26
a
trophils, as they play essential roles in host defence through their
ability to clear bacterial infections. The literature scan revealed
that there are no reports of the preparation of selectively acylated
and randomly/multi-acylated derivatives of pinitol. We therefore
envisaged synthesizing various acylated derivatives of pinitol. In
this Letter, we report the preparation of selectively acylated pinitol
derivatives through (a) biotransformation, and (b) chemical ap-
Preparation of 1
O-butanoyl-4-O-methyl-chiro-inositol (6b),
propanoyl-chiro-inositol (6c) and -3-O-iso-butanoyl-4-O-
D
-3-O-acetyl-4-O-methyl-chiro-inositol (6a), 1
D-3-
1D
-3-O-methyl-4-O-
1D
methyl-chiro-inositol (6d) was achieved in a three step reaction
process (Scheme 2). The vicinal syn hydroxyl functions were first
protected through diacetonide formation followed by esterification
of the remaining hydroxyl group by reacting it with an acid anhy-
dride in presence of pyridine as a base to produce 5 in almost
quantitative yield. Deprotection of isopropylidene acetals was
achieved by a method using HClO4–SiO2.27 Therefore, HClO4–SiO2
was prepared according to the standard procedure and used for
the deprotection of isopropylidene acetals in acetonitrile. Catalytic
amount of the reagent (0.035 mmol/mmol of diacetonide) was suf-
ficient to cleave the diacetonide in half an hour to furnish the prod-
ucts 6 (a–d) in 90–92% yield.
Alkoxy derivatives 7a–c were synthesized from a common
diacetonide intermediate 4, by performing standard alkylation
reactions, respectively, in presence of a base. Removal of the aceto-
nide groups under acidic conditions described as above yielded the
desired derivatives 8a–c, respectively (Scheme 3; Joanne B. H.
2004).28
proaches and their TNF-a inhibitory studies.
Enzymes are known to be the versatile catalysts for the kinetic
resolution and desymmetrization of prostereogenic substrates. A
variety of lipases of different origins are known to be useful in reg-
ioselective transesterification as well as hydrolysis reactions. Since
it was almost impossible to get selectively acylated derivatives of
pinitol through chemical transformation in one step reaction, it
was envisaged that transesterification of pinitol would be an alter-
native process. Our attempts for the preparation of regioselectively
acylated derivatives through transesterification of pinitol turned
futile, though different solvent combinations, and temperature
conditions were tried. Therefore, we proceeded through selective
hydrolysis route for the preparation of desired compounds. In this
approach, regioselective lipase catalyzed hydrolysis of penta-acy-
lates of pinitol was studied. A number of lipases from institutional
microbial repository and commercial sources were screened for
the selective hydrolysis of penta-acetates, penta-butyrates and
penta-benzoates of pinitol. Using the commercial lipase CAL-A+B,
monodeacylated products 2 and 3 in the ratio of 48:52 were ob-
3-Keto derivative 10 was prepared by oxidation of diacetonide, 4
(822 mg, 3.0 mmol) with pyridinium chlorochromate (PCC, 1.290 g,
6.0 mmol)indichloromethane (DCM)at 0 °C(5 h). Ontheotherhand
diacetonide 4 was converted into 3-chloro analog 9 with thionyl
chloride in excess of pyridine in 45% yield (Scheme 4).
tained by regioselective hydrolysis of D-1,2,4,5,6-penta-O-acetyl-
3-O-methyl-chiro-inositol 1 with an overall yield of 72%.25 The
OAc
OAc
OAc
OH
OAc
OH
OAc
CAL(A+B)
MeO
AcO
MeO
AcO
MeO
AcO
+
pH-7.0
AcO
AcO
AcO
OAc
3
OAc
2
1
D-1, 2, 3, 5-tetra-O-acetyl-4-O- D-1, 2,4, 5-tetra-O-acetyl-3-O-
methyl chiro-inositol
methyl chiro-inositol
D-1,2,4,5,,6-penta-O-acetyl-4-O-
methyl-chiro-inositol
Scheme 1.
OH
OH
O
O
O
O
O
O
MeO
HO
(i)
(ii)
MeO
RO
MeO
HO
HO
O
O
OH
Pinitol
4
OH
OH
5
(iii)
MeO
RO
R = Acetyl-,
butanoyl,
propanoyl,
5a, 6a
5b, 6b
5c, 6c
HO
6
OH
iso- butanoyl-, 5d, 6d
Scheme 2. Reagents and conditions: (i) p-TSA, 2,2-dimethoxy propane, dry acetone; (ii) acid anhydride, pyridine; (iii) HClO4–SiO2/CH3CN.

K. A. Bhat et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1939–1943
1941
OH
O
O
O
OH
OH
O
O
(i)
MeO
HO
(ii)
MeO
HO
MeO
RO
HO
O
O
Pinitol
O
(iii)
4
OH
OH
7
MeO
RO
HO
OH
R = All, 7a, 8a
Me, 7b, 8b
Et, 7c, 8c
8
Scheme 3. Reagents and conditions: (i) p-TSA, 2,2-dimethoxy propane, dry acetone; (ii) allyl bromide/methyliodide/ethyliodide, NaH, DMF; (iii) TFA/H2O (1:1).
O
O
O
O
O
O
O
O
O
MeO
MeO
HO
MeO
(i)
(ii)
Cl
O
O
O
O
10
4
9
Scheme 4. Reagents and conditions: (i) PCC, DCM; (ii) SOCl2, Pyridine.
From 1H NMR studies including coupling constants, it was
established that chlorination occurred with the retention of
configuration. The J-value of the double doublet for the proton at
C-3 position in 4 is almost the same as that of the proton at C-4
ity to clear bacterial infections. Their inappropriate activation con-
tributes to a variety of inflammatory diseases, from the acute
respiratory distress syndrome to asthma, chronic obstructive pul-
monary disease and rheumatoid arthritis. TNF-a cytokine was cho-
position in 1
chiro-inositol (9). Penta-acyl derivatives of pinitol, that is,
4,5,6-penta-O-acetyl-3-O-methyl-chiro-inositol (11), 1 -3-O-meth-
yl-1,2,4,5,6-penta-O-propanoyl-chiro-inositol (12), -1,2,4,5,6-pen-
ta-O-butanoyl-3-O-methyl-chiro-inositol (13), and -1,2,4,5,
6-penta-O-benzoyl-3-O-methyl-chiro-inositol (14) were prepared
from pinitol (5 mmol), and respective alkanoic anhydride (25 mmol)
in presence of dimethyl amino pyridine (DMAP, 10–15 mg) by known
methods.
D
-3-chloro-1,2:5,6-di-O-isopropylidene-4-O-methyl-
sen as the target for anti-inflammatory activity screening because
of the fact that it is a pro-inflammatory cytokine and plays a major
role in the pathogenesis of septic shock induced by LPS (Lipopoly-
saccharide) endotoxin injection.29 LPS is an important triggering
factor for in vivo systemic inflammatory response.30 LPS activates
neutrophils via engagement of TLR4 (Toll-like receptor 4),31 result-
ing in the induction of a characteristic proinflammatory phenotype
and prolongation of cell lifespan.32 In brief, neutrophils extraction
was performed by taking human blood and centrifuging it at 250g
for 20 min. Two layers were formed, the platelet rich upper layer
was discarded and the lower layer centrifuged at 1800g. Again
three layers were formed: the platelet poor upper layer was dis-
carded, the middle layer which appeared to be the buffy coat layer
D-1,2,
D
D
1D
Flowcytometric studies were carried out to determine the anti-
inflammatory effect of pinitol (Schemes 2–5) and its derivatives on
TNF-a cytokine expression in LPS activated human neutrophils.
Neutrophils play essential roles in host defence through their abil-
OH
OH
OR
OR
MeO
HO
MeO
RO
(i)
HO
RO
OR
OH
R= COCH3
R=COCH2CH3
11
12
R=COCH2CH2CH3 13
R=COPh 14
Scheme 5. Reagents and conditions: (i) acid anhydride, pyridine.

1942
K. A. Bhat et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1939–1943
was taken and the lower layer which was RBC rich layer was also
discarded. Middle layer was taken and layered with histopaque
and incubated for 10 min. The samples were centrifuged at 700 g.
After centrifugation two layers were observed, upper layer was ta-
ken and Facs Lysing solution added for the lysis of any traces of
RBC. The samples were washed with PBS (200 g) for 10 min. The
are considered to have potent TNF-
found to be efficacious in suppressing intracellular TNF-
the dose of 1.0 g/ml when compared to the LPS control. Therefore,
these compounds can be considered as lead molecules for detailed
investigations of their anti-inflammatory potential both in vitro
and in vivo.
a
inhibitory activity. It was
a
level at
l
intracellular TNF-
of neutrophils. Neutrophils separation was carried out as described
a
level was then estimated in gated population
Partial information about SAR has also been obtained from
these studies, though no definite relationship could be established.
In general, the molecules protected with ester or acetonide groups
(A new method for detecting TNF-
a secreting cells using direct-
immunofluoresence surface membrane staining).33 Cells were
stimulated with LPS and incubated with test compounds for 3 h,
in CO2 incubator. For the permeabilisation of the cells Permeablis-
ing sol. (1X) (BD-Biosciences) was added and incubated for 10 min.
displayed much higher TNF-a inhibition than the corresponding
unprotected compounds. Furthermore, the dialkylated molecules,
for example, 8a–c exhibited higher activity than their acylated
counterparts (e.g., 6a–c). In case of alkylated analogs, for example,
6a–c, respectively, the bioactivity improved with the increase in
the length of side chain; however, the effect is reversed in case
of 5a–c, where the activity decreased with increase in the length
of side chain. The oxo derivative (10) was more active than the
chloro derivative (9), the reason possibly may be attributed to
the presence of oxo functionality which could be involved in the
interaction (through hydrogen bonding) at the receptor site. Out
of all semi-synthetic analogs, the peracylated derivatives, with
longer alkyl chain were found to be more potent than those with
The cells were then labeled with conjugated anti-mouse TNF-
a
monoclonal antibody (BD-Biosciences). Incubation of the cells
was carried out for 30 min in dark. After washing with PBS (Phos-
phate buffered saline, Sigma Chemicals, USA) the samples were ac-
quired directly on BD-LSR Flowcytometer (BD-LSR, Beckton–
Dickinson Biosciences, CA, USA). A fluorescence trigger was set
on the PE (FL1) parameter of the gated neutrophil populations
(10,000 events). Fluorescence compensation, data analysis, and
data presentation was performed using Cell Quest Pro software
(Beckton–Dickinson Biosciences, CA, USA).
smaller alkyl chain. Much higher TNF-a inhibition displayed by
Most of the semi-synthetic analogs displayed a range of TNF-
expression inhibition from low to high. Pinitol displayed average
inhibition of the expression of TNF- in the range of 30.03% using
flowcytometry. From the results given in Table 1, (flowcytometric
studies) it is clear that analogs 10 (1 -1,2: 5,6-di-O-isopropyli-
dene-3-oxo-4-O-methyl-chiro-inositol), 13 (1 -1,2,4,5,6-penta-O-
butanoyl-3-O-methyl-chiro-inositol), and 14 (1 -1,2,4,5,6-penta-
O-benzoyl-3-O-methyl-chiro-inositol) displayed maximum inhibi-
tory effect on TNF- cytokine secretion in murine isolated neutro-
phils in response to LPS stimulant. Neutrophils were in vitro
treated with 1.0 g/ml concentration of 10, 13, and 14 showing
50.57%, 52.47%, and 51.71% TNF- suppression respectively. The
a
compound 3 in comparison to 2 can not be explained though it
may be attributed to stereochemical factors (position of hydroxyl
groups), given that the molecule is having 6 chiral centers. It ap-
pears that lipophilicity played an important role and improved
the inhibitory activity, indicating that alkylated part of the mole-
a
D
D
cule probably interacts with the binding site of TNF-a receptor.
D
In conclusion 23 semi-synthetic analogs of pinitol were pre-
pared using chemo and chemo-enzymatic methodologies. Some
interesting structure activity relationships (SAR) could be drawn
from the data. However, the three analogs viz. 10, 13, and 14 dis-
a
l
played significant TNF-a inhibition activity, which is almost one
a
log higher than the known standard rolipram, and just about dou-
ble as compared to parent molecule pinitol. The identified lead
molecules are being studied to ascertain their potential as chemo-
therapeutic anti-inflammatory agents.
compounds showing more than 50% inhibition of TNF-a expression
Table 1
Effect of different pinitol derivatives on intracellular TNF-
activated neutrophils
a
expression by LPS
Activity (%)
References and notes
S.No.
Sample
Concn (
lg/ml)
Mean S.E.
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LPS Control
Pinitol
2
3
4
5a
5b
5c
5d
6a
6b
6c
6d
7a
7b
7c
8a
8b
8c
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
10
2.63 0.01
1.84 0.04
2.60 0.08
1.75 0.04
2.19 0.11
1.51 0.02
1.59 0.03
1.71 0.02
2.19 0.15
2.57 0.04
2.41 0.08
2.08 0.01
2.07 0.11
1.71 0.07
1.68 0.04
1.62 0.04
1.85 0.05
1.60 0.02
1.67 0.02
1.64 0.05
1.30 0.03
1.75 0.04
1.82 0.09
1.25 0.02
1.27 0.06
1.24 0.07
—
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
30.03 ;
1.14 ;
33.46 ;
13.09 ;
42.58 ;
39.54 ;
32.14 ;
20.65 ;
2.28 ;
8.36 ;
20.91 ;
17.85;
34.98 ;
33.33 ;
38.40 ;
29.65 ;
39.16 ;
36.50 ;
37.64 ;
50.57 ;
33.46 ;
30.79 ;
52.47 ;
51.71 ;
52.85 ;
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9
10
11
12
13
14
Rolipram
Samples showing TNF alpha suppression around 50% are considered to be active.
%;: indicates suppression of TNF- alpha expression; No. of observations = 3.

K. A. Bhat et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1939–1943
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242(11), 213(10), 210(46), 200(42), 188(34), 182(68),169(29), 168(100),
158(45). Anal. Calcd for C15H22O10: C, 49.72; H, 6.12. Found C, 49.83; H, 6.01.
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(500 MHz, CDCl3): d 1.98 (3H, s), 2.01 (6H, s), 2.07 (3H, s), 3.37 (3H, s), 3.57 (1H,
t, J = 10.8 Hz), 4.10 (1H, s), 5.26 (1H, d, J = 9.0 Hz), 5.34 (1H, s), 5.39 (1H, t,
J = 9.7 Hz), 5.46 (1H, t, J = 3.9 Hz); 13C NMR (50 MHz, CDCl3): d 21.4, 60.3, 68.1,
69.0, 70.0, 70.5, 71.4, 79.2, 168.1, 168.5, 169.2, 170.2; MS: M+ÀH2O = 345(2),
289(8), 259(6), 242(13), 213(14), 210(40), 200(35), 188(37), 182(60), 169(38),
168(100), 158(35). Anal. Calcd for C15H22O1O: C, 49.72; H, 6.12. Found C, 49.67;
H, 6.23.
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J. Med. Food 2007, 10, 594.
26. Preparation of
1D-3-O-butanoyl-4-O-methyl-chiro-inositol (6b): Freshly
27
prepared HClO4–SiO2
(70 mg) were added to
a solution of 1D-3-O-
butanoyl-1,2: 5,6-di-O-isopropylidene-4-O-methyl-chiro-inositol (1.0 mmol)
in acetonitrile (5 ml) and the reaction mixture was stirred at room
temperature for 45 min. After the completion of reaction, the reaction
mixture was filtered with ethylacetate through Celite bed and concentrated
to give the title compound. The sample was further purified by flash
chromatography with ethyl acetate:toluene (70:30) as the eluent to give 6b
in 90% yield. Compound 6b: IR (KBr): 3385, 2965, 2935, 1728, 1383, 1192,
1089, 670; 1H NMR (200 MHz, D2O): d 0.98 (3H, d, J = 7.4 Hz), 1.68 (2H, m), 2.38
(2H, t, J = 7.2 Hz), 3.31 (1H, t, J = 1.5 Hz), 3.51 (3H, s), 3.81–3.92 (4H, m), 4.85
(4H, bs), 5.15 (1H, t, J = 10.0 Hz); 13C NMR (50 MHz, CDCl3): d 14.0, 18.5, 36.5,
60.0, 69.6, 69.9, 71.7, 71.9, 74.6, 81.2, 170.0; ESI-MS (m/z) = 287 [M+Na]+; Anal.
Calcd for C11H20O7: C, 49.99; H, 7.63. Found C, 50.10; H, 7.51.
27. Agnihotri, G.; Misra, A. K. Tetrahedron Lett. 2006, 47, 3653.
28. Hart, J. B.; Kroger, L.; Falshaw, A.; Falshaw, R.; Farkas, E.; Thiem, J.; Win, A. L.
Carbohydr. Res. 2004, 339, 1857.
24. Bauer, J.; Berkenbosch, F.; Van-Dam, A. M.; Dijkstra, C. D. J. Neuroimmunol.
1993, 48, 13.
25. Enzymatic Preparation of
and -1,2,4,5-tetra-O-acetyl-3-O-methyl-chiro-inositol (3):
-1,2,4,5,6-penta-O-acetyl-3-O-methyl-chiro-inositol (1) (808 mg, 2 mmol),
D
-1,2,3,5-tetra-O-acetyl-4-O-methyl-chiro-inositol (2)
D
A
suspension of
D
CAL (A+B) in powder form (600 mg) and sodium phosphate buffer (0.1 M, pH
7.0, 5 ml) was stirred at 30 1 °C, maintaining pH 7.0 by the addition of 0.01 M
NaOH solution. The course of reaction was monitored by TLC. After the
completion of reaction the contents were centrifuged at 10,000 rpm. The clear
solution and the centrifuged cell pellet were extracted separately with
ethylacetate (3 Â 25 ml). The organic layers were combined and washed with
water, dried and concentrated under reduced pressure. The resulting crude
mixture was separated by column chromatography over silica gel to furnish
29. Remick, D. G.; Strieter, R. M.; Eskandari, M. K.; Nguyen, D. T.; Genord, M. A.;
Raiford, C. L. Am. J. Path. 1990, 136, 49.
D-1,2,3,5-tetra-O-acetyl-4-O-methyl-chiro-inositol (2) (272 mg, 0.75 mmol) as
30. Bone, R. C. Chest 1991, 100, 802.
a
semi solid and -1,2,4,5-tetra-O-acetyl-3-O-methyl-chiro-inositol (3)
D
31. Sabroe, I.; Read, R. C.; Whyte, M. K. B.; Dockrell, D. H.; Vogel, S. N.; Dower, S. K.
J. Immunol. 2003, 171, 1630.
32. Lee, A.; Whyte, M. K. B.; Haslett, C. J. Leukoc. Biol. 1993, 54, 283.
33. Clara, B.; Arancha, R. C.; Andres, G. M.; Atanasio, P.; Julie, A.; Alberto, O. J.
Immun. Meth. 2002, 264, 77.
(250 mg, 0.69 mmol) as a semisolid, respectively.
Compound 2: IR (KBr): 3479, 2938, 1752, 1372, 1227, 1078, 1046, 983; 1H NMR
(500 MHz, CDCl3): d 2.04 (3H, s), 2.13 (3H, s), 2.16 (6H, s), 3.47 (3H, s), 3.56 (1H,
m), 3.99 (1H, d, J = 10.0 Hz), 5.16 (2H, m), 5.30 (1H, d, J = 6.2 Hz), 5.35 (1H, s);