(-)-Tarchonanthuslactone exerts a blood glucose-increasing effect in experimental type 2 diabetes mellitus

Article abstract of DOI:10.3390/molecules20035038

A number of studies have proposed an anti-diabetic effect for tarchonanthuslactone based on its structural similarity with caffeic acid, a compound known for its blood glucose-reducing properties. However, the actual effect of tarchonanthuslactone on bloo

Full text of DOI:10.3390/molecules20035038

Molecules 2015, 20, 5038-5049; doi:10.3390/molecules20035038
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Communication
(−)-Tarchonanthuslactone Exerts a Blood Glucose-Increasing
Effect in Experimental Type 2 Diabetes Mellitus
Gabriela F. P. de Souza 1,†, Luiz F. T. Novaes 2,†, Carolina M. Avila 2, Lucas F. R. Nascimento 1,
Licio A. Velloso 1 and Ronaldo A. Pilli 2,*
1
Laboratory of Cell Signaling, University of Campinas, UNICAMP, C.P. 6154,
CEP 13084-761 Campinas, São Paulo, Brazil; E-Mails: hbee_quimica@yahoo.com.br (G.F.P.S.);
lribeiro_bio@yahoo.com.br (L.F.R.N.); lavelloso.unicamp@gmail.com (L.A.V.)
2
Institute of Chemistry, University of Campinas, UNICAMP, C.P. 6154, CEP 13084-971 Campinas,
São Paulo, Brazil; E-Mails: luiztnovaes@gmail.com (L.F.T.N.); santanacma@gmail.com (C.M.A.)
†
These authors contributed equally to this work.
* Author to whom correspondence should be addressed; E-Mail: pilli@iqm.unicamp.br;
Tel.: +55-19-35213422; Fax: +55-19-35213023.
Academic Editor: Derek J. McPhee
Received: 8 December 2014 / Accepted: 6 March 2015 / Published: 19 March 2015
Abstract: A number of studies have proposed an anti-diabetic effect for
tarchonanthuslactone based on its structural similarity with caffeic acid, a compound known
for its blood glucose-reducing properties. However, the actual effect of tarchonanthuslactone
on blood glucose level has never been tested. Here, we report that, in opposition to the
common sense, tarchonanthuslactone has a glucose-increasing effect in a mouse model of
obesity and type 2 diabetes mellitus. The effect is acute and non-cumulative and is present
only in diabetic mice. In lean, glucose-tolerant mice, despite a slight increase in blood
glucose levels, the effect was not significant.
Keywords: tarchonanthuslactone; caffeic acid; diabetes

Molecules 2015, 20
5039
1. Introduction
Type 2 diabetes mellitus (T2DM) is a metabolic disease defined by the presence of chronic
hyperglycemia due to a simultaneous development of insulin resistance and a relative defect of the
pancreatic islet to secrete insulin [1]. It is currently known that obesity-related metabolic inflammation
plays an important role both in the installation of insulin resistance and damaging of the pancreatic
islets [1]. Therefore, it is expected that therapeutic methods aimed at reducing inflammation may impact
positively on the control of glucose homeostasis [2]. Currently, there is only one report of a clinical trial
showing the metabolic benefits of reducing inflammation in diabetes using salsalate, which is derived
from the bark of the willow tree [3]. However, additional studies, using different animal models [4] have
evaluated other natural compounds for their potential as therapy for this disease [5–7].
Tarchonanthuslactone (1), an ester of dihydrocaffeic acid (2), was isolated in 1979 from the leaves of
the tree Tarchonanthus trilobus [8]. The genus Tarchonanthus is found in southern Africa and is used
in folk medicine. Traditional health practitioners have used T. camphorate for diabetes treatment;
moreover, its anti-inflammatory and cytotoxic activities have been reported by van de Venter and
coworkers [9]. Tarchonanthuslactone (1) has a privileged structure, presenting the usually bioactive
α,β-unsaturated δ-lactone motif [10–13], which makes it a good target for new asymmetric synthetic
approaches [14–18]. Interestingly, Hsu and coworkers [19] have reported that caffeic acid (3) has an
antidiabetic effect and, because of the structural similarity between 1 and 3, a number of reports have,
thereafter, assigned a putative antidiabetic effect to 1 as well [14–18,20–22]. Here, we employed a mouse
model of diet-induced diabetes to evaluate the effect of 1 and related compounds (Figure 1) on blood
glucose levels. Such compounds may have effects on other metabolic parameters, however, we have
focused on blood glucose levels.
O
O
O
O
OH
OH
OH
OH
OH
OH
O
O
1
3
HO
HO
1
3
2
2
3
1
2
O
O
O
OH
OH
O
OH
O
O
4
5
Figure 1. Chemical structures of compounds 1–5.
2. Results and Discussion
Swiss mice belong to an outbread strain related to the diabetes prone Akr mouse [23]. Upon feeding
on a high-fat diet (31% fat from lard) Swiss mice rapidly develop obesity accompained by insulin
resistance and hyperglycemia [24]. In the present study, six-week old male Swiss mice were fed for eight
weeks on a high-fat diet and then employed in the experiments.
Compound 3, in the same dose as previously reported [19] was employed as a positive control.
Six-hour fasting diabetic mice (median fasting blood glucose levels = 200 mg/dL) were randomly
divided into three groups treated, by an intraperitoneal (ip) injection, with a single dose of either 1

Molecules 2015, 20
5040
(3.0 mg/kg), 2 (3.0 mg/kg) or 3 (3.0 mg/kg) and blood glucose levels were determined over the following
90 min. As depicted in Figure 2A,B, both 2 and 3 exerted a blood glucose-reducing effect, which was
significant 30 min after the ip injection of the compounds but resulted in no significant reduction of the
area under the glucose curve during the 90 min evaluation. Conversely, 1 exerted an unexpected blood
glucose-increasing effect, which was significant at 90 min but resulted in no significant change in the
area under the glucose curve during the 90 min evaluation. The effect of 3 obtained in our experiments
matches the results reported previously [19], as maximal effect was obtained as early as 30 min after the
injection of the compound. Since 2 presents similar glucose-reducing effect as 3, we hypothesized that
the presence of the double bond at C2-C3 (Figure 1), which is the only structural difference between 2
and 3, would be involved in this biological effect. In order to test this hypothesis, we synthetized an
analogue of 1 that possesses the C2-C3 double bond (4) and determined its effect on blood
glucose levels.
Figure 2. (A) Effect of a single intraperitoneal dose of 1, 2 and 3 (3.0 mg/kg) on the blood
glucose levels and (B) on area under the blood glucose response curve (AUC). Blood glucose
levels and the AUC was calculated and expressed as percentage of saline treated group
(mean ± SEM). * p < 0.05 vs. control.
As depicted in Figure 3A,B, in diabetic Swiss mice, 4 (3.0 mg/kg, ip) resulted in an even more potent
glucose-increasing effect than 1. Blood glucose levels were significantly higher than control at 30 and
60 min, leading to a significantly higher area under the glucose curve during the 90 min evaluation
period. Thus, we propose that the presence/abscence of the C2-C3 double bond plays no role in either
the glucose-reducing effect of 3 or the glucose-increasing effect of 1.
Next, we hypothesized that the alcohol part of esther 1 could be responsible for the glucose-increasing
effect produced by this compound. In order to test this hypothesis, diabetic Swiss mice were treated with the
synthetic intermediate 5 (3.0 mg/kg, ip) and glucose levels were assessed. As depicted in Figure 3A,B, 5 had
no effect on blood glucose levels in diabetic Swiss mice. Thus, we propose that neither the
presence/absence of the C2-C3 double bond nor the alcohol part of 1 and 4 are required, per se, for the
glucose regulatory effects of the compounds, rather, the whole molecules are required for the
glucose-increasing effect.

Molecules 2015, 20
5041
Figure 3. (A) Effect of a single intraperitoneal dose of 4 and 5 (3.0 mg/kg) on the blood
glucose levels and (B) on area under the blood glucose response curve (AUC). Blood glucose
levels and the AUC was calculated and expressed as percentage of saline treated group
(mean ± SEM). * p < 0.05 vs. control.
In addition, we evaluated the long-term and cumulative effect of 1 on blood glucose levels. For that,
diabetic Swiss mice were treated three times a week, for four weeks with 1 (3.0 mg/kg per dose, ip).
Two days after the last dose of the compound, the mice were submitted to a 6-hour fasting and blood
glucose levels were determined. As depicted in Figure 4A, the prolonged treatment with 1 resulted in no
significant change in fasting blood glucose level, suggesting that its blood-glucose increasing effect is
acute and non-cumulative.
Figure 4. (A) Effect of 1 on fasting glycemia of chronically treated diabetic mice expressed
as percentage of saline treated group; and (B) effect of a single intraperitoneal dose of 1
(3.0 mg/kg) on the blood glucose levels and (C) area under the blood glucose response curve
(AUC) of lean treated mice expressed as percentage of lean saline treated group
(mean ± SEM).
Finally, lean, non-diabetic Swiss mice, fed on chow (containing 4% fat), were accutely treated with
1 (3.0 mg/kg, ip) and blood glucose levels were determined over a 90 min time-frame. The median
six-hour fasting glucose levels was 120 mg/dL and as depicted in Figure 4B, despite a slight increase in
glucose levels at 60 min, 1 produced no statistically significant change in blood levels in lean mice.
In summary, 1 has no anti-diabetic effect as previously suggested [14–18]. In fact, despite its
structural similarities with 2 and 3, both of which capable of transiently reducing the blood glucose levels
of diabetic animals, the accute treatment of diabetic mice with 1 results in a transient increase in blood

Molecules 2015, 20
5042
glucose. Of note, the glucose-increasing effect of 1 is not due to particular features of the molecule, such
as double bond between carbons 2 and 3 or the alcohol part of the molecule, but rather, to the whole
molecule. The present study highlights how subtle structural modifications in chemical compounds can
affect profoundly and unexpectedly its biological activity. The results described herein has potential
impact on the design of more potent anti-diabetic compounds.
3. Experimental Section
3.1. Chemistry
3.1.1. General Procedures
Starting materials and reagents were obtained from commercial sources and used as received unless
otherwise specified. Dichloromethane was treated with calcium hydride and distilled before use.
Tetrahydrofuran was treated with metallic sodium and benzophenone and distilled before use.
Anhydrous reactions were carried out with continuous stirring under atmosphere of dry nitrogen.
Progress of the reactions was monitored by thin-layer chromatography (TLC) analysis (silica gel 60 F254
1
13
on aluminum plates, Merck, Darmstadt, Hesse, Germany). H-NMR and C-NMR were recorded on
Bruker 250, 400, 500 or 600 (Rheinstetten, Baden-Württemberg, Germany), the chemical shifts (δ) were
reported in parts per million (ppm) relative to deuterated solvent as the internal standard (CDCl3
7.26 ppm, 77.00 ppm, acetone-d6 2.05 ppm, 29.92 ppm, methanol-d4 3.31 ppm, 49.15 ppm), coupling
constants (J) are in hertz (Hz). Peaks multiplicities are reported as follows: singlet (s), doublet (d), triplet
(t), quartet (q), sextet (sext), multiplet (m), broad singlet (br s). Mass spectra were recorded on a Waters
Xevo Q-Tof apparatus operating in electrospray mode (ES). Infrared spectra with Fourier transform
(FT-IR) were recorded on a Therm Scientific Nicolet iS5, the principal absorptions are listed in cm−1.
The values of optical rotation were measured at 25 °C in a polarimeter Perkin-Elmer 341, with sodium
lamp, the measure is described as follow {[α]D (c = g/100 mL), solvent}.
3.1.2. Experimental Procedures
(R)-Butane-1,3-diol (ii). To a suspension of LiAlH4 (200 mg, 5.27 mmol) in anhydrous THF (9.2 mL)
at 0 °C, was added PHB (i, 602 mg), then the temperature increased until rt. The reaction was stirred for
90 min at this temperature, and for 5 h under reflux, and at rt again overnight. The reaction was cooled
at 0 °C and was added with intervals 0.2 mL of water, 0.2 mL of aqueous solution of NaOH 10% and
0.6 mL of water. The reaction was filtered and the solid was washed with THF (3 × 25 mL), the organic
phase was concentrated and the product was purified by column chromatography (SiO2, CHCl3/MeOH
95:5) to afford the corresponding diol ii (595 mg, 94%). Colorless oil. Rf 0.67 (SiO2, CHCl3/MeOH
90:10). 1H-NMR δ (CDCl3, 250 MHz): 1.15 (d, J = 6.2 Hz, 3H), 1.61 (q, J = 5.5 Hz), 3.62–3.82 (m, 2H),
3.90–4.02 (m, 3H). 13C-NMR δ (CDCl3, 62.9 MHz): 23.6, 40.2, 60.8, 67.3. [α]D (c = 1.0, CHCl3): −21.
Lit. [25]: [α]D (c = 1.66, CHCl3): −30.1.
(R)-2,2,3,3,5,9,9,10,10-Nonamethyl-4,8-dioxa-3,9-disilaundecane (iii). To a solution of the diol ii (1.00 g,
11.1 mmol, 1 eq.) in anhydrous CH2Cl2 (44 mL), was added imidazole (2.27 g, 33.3 mmol, 3 eq.) and
TBSCl (4.18 g, 27.7 mmol, 2.5 eq.), the reaction was stirred for 6 h, and were added to it, 75 mL of

Molecules 2015, 20
5043
water and 120 mL of EtOAc, the aqueous phase was extracted once with 50 mL of EtOAc, the organic
phases were grouped, dried (Na2SO4), filtered and concentrated, the crude was purified by column
chromatography (SiO2, hexanes/EtOAc 98:2) to afford iii (3.45 g, 98%). Colorless oil. Rf 0.51 (SiO2,
hexanes/EtOAc 98:2). 1H-NMR δ (CDCl3, 250 MHz): 0.04 (s, 6H), 0.05 (s, 6H), 0.89 (s, 18H), 1.14 (d,
J = 6.2 Hz, 3H), 1.50–1.75 (m, 2H), 3.67 (t, J = 6.5 Hz, 2H), 3.97 (sext, J = 6.1 Hz, 1H). 13C-NMR δ
(CDCl3, 62.9 MHz): −5.1 (2C), −4.6, −4.2, 18.3, 18.4, 24.2, 26.1 (3C), 26.1 (3C), 43.0, 60.2, 65.7. [α]D
(c = 1.0, CHCl3): −20. IR (NaCl, cm−1): 1255, 1384, 1472, 2858, 2886, 2930, 2956. HRMS:
[C16H38O2Si2+H]+ calculated 319.2489, observed 319.2471.
(R)-3-((tert-Butyldimethylsilyl)oxy)butan-1-ol (iv). To a solution of iii (2.54 g, 7.97 mmol, 1 eq.) in
anhydrous THF (30 mL) at 0 °C, was added a solution composed by pyridine (20 mL) and HF•pyridine
(8.8 mL) in THF (50 mL). The reaction was stirred at rt for 2.5 h. The reaction was neutralized with
solution of NaHCO3 (150 mL) and it was stirred for other 10 min. The reaction contents were diluted
with 150 mL of EtOAc and the aqueous phase was extracted with EtOAc (2 × 50 mL). The organic
phases were combined, dried (MgSO4) and concentrated, the product was purified by column
chromatography (SiO2, hexanes/EtOAc 75:25) to afford iv (895 mg, 55%). Colorless oil. Rf 0.40 (SiO2,
hexanes/EtOAc 80:20). 1H-NMR δ (CDCl3, 250 MHz): 0.06 (s, 3H), 0.06 (s, 3H), 0.87 (s, 9H), 1.17 (d,
J = 6.2 Hz, 3H), 1.54–1.82 (m, 2H), 2.72 (br s, 1H), 3.63–3.85 (m, 2H), 4.00–4.14 (m, 1H). 13C-NMR δ
(CDCl3, 62.9 MHz): −4.9, −4.3, 18.1, 23.6, 25.9 (3C), 40.7, 60.5, 68.3. [α]D (c = 1.0, CHCl3): −25.
Lit. [26]: [α]D (c = 0.41, CHCl3): −17.8.
(R)-3-((tert-Butyldimethylsilyl)oxy)butanal (v). In a dried 100 mL round bottom flask, was added a
solution of freshly distilled oxalyl chloride (0.74 mL, 8.59 mmol, 1.5 eq) dissolved in anhydrous CH2Cl2
(15 mL). The solution was cooled at −78 °C. After this, was added dropwise anhydrous DMSO (1.3 mL,
18.3 mmol, 3.2 eq.). The reaction stayed under stirring for 15 min at −78 °C, then the alcohol iv (1.17 g,
5.73 mmol, 1.0 eq.) dissolved in anhydrous CH2Cl2 (10 mL) was added to the mixture via cannula.
The reaction was stirred for other period of 15 min, and freshly distilled triethylamine (3.9 mL, 28 mmol,
5.0 eq.) was added. After 30 min of stirring, the reaction contents were diluted in 20 mL of Et2O and
15 mL of aqueous solution of NH4Cl. The aqueous phase was extracted with Et2O (3 × 15 mL) and the
organic phases were grouped, washed with brine, dried (MgSO4), filtered and concentrated. The product
was purified by column chromatography (SiO2, hexanes/EtOAc 95:5) to afford v (1.00 g, 86%).
Colorless oil. Rf 0.57 (SiO2, hexanes/EtOAc 90:10). 1H-NMR δ (CDCl3, 250 MHz): 0.02 (s, 3H), 0.03
(s, 3H), 0.82 (s, 9H), 1.19 (d, J = 6.2 Hz, 3H), 2.35–2.60 (m, 2H), 4.31 (sext, J = 6.1 Hz, 1H), 9.74 (t,
J = 2.3 Hz, 1H). 13C-NMR δ (CDCl3, 62.9 MHz): −4.9, −4.3, 18.0, 24.2, 25.8 (3C), 53.1, 64.6, 202.0.
[α]D (c = 1.0, CHCl3): −15. Lit. [27]: ent-compound [α]D (c = 1.0, CHCl3): +14.
(4S,6R)-6-((tert-Butyldimethylsilyl)oxy)hept-1-en-4-ol (vi). In a 50 mL round bottom flask with a
magnetic stir bar were added powdered activated molecular sieves 4 Å (2.5 g). After the addition of the
molecular sieves the flask was heated under flow of N2. After this anhydrous CH2Cl2 (10 mL), S-BINOL
(286 mg, 1.00 mmol, 0.2 eq.), TFA (1 μL) and Ti(O-iPr)4 (150 μL) were added to the flask. The mixture
was refluxed for 2 h, when it was observed a change of color from dark red to brown. After this period,
the bath of oil was removed and temperature decrease until rt and the aldehyde v (986 mg, 4.87 mmol,

Molecules 2015, 20
5044
1.0 eq.) dissolved in anhydrous CH2Cl2 (10 mL) was added. The mixture was maintained under stirring
for 15 min. The reaction was placed in a bath at −78 °C and allyltributylstannane (2.34 mL, 7.4 mmol,
1.5 eq.) was added slowly to the mixture. The reaction was maintained under stirring at −30 °C for
4 days. After this period, brine (40 mL) was added to the reaction and the temperature raised until rt.
After 1 h the mixture was filtered. The aqueous phase was extracted with CH2Cl2 (3 × 50 mL). The
organic phases were grouped, dried (Na2SO4), filtered and concentrated. The product was purified by
column chromatography (SiO2, hexanes/EtOAc 95:5) to afford vi (524 mg, 44%) and aldehyde v
(275 mg, 28%) was recovered. Colorless oil. Rf 0.34 (SiO2, hexanes/EtOAc 90:10). d.r. 12:1 (syn/anti).
1H-NMR δ (CDCl3, 250 MHz): 0.07 (s, 3H), 0.08 (s, 3H), 0.86 (s, 9H), 1.15 (d, J = 6.2 Hz, 3H),
1.48–1.57 (m, 2H), 2.11–2.22 (m, 2H), 3.26 (br s, 1H), 3.73–3.85 (m, 1H), 3.97–4.10 (m, 1H),
5.00–5.11 (m, 2H), 5.80 (ddt, J = 16.9, 10.2, 7.1 Hz, 1H). 13C-NMR δ (CDCl3, 62.9 MHz): −4.7, −3.8,
18.0, 24.6, 25.9 (3C), 42.1, 45.3, 69.8, 70.6, 117.3, 135.0. [α]D (c = 1.0, CHCl3): −30. Lit. [28]:
ent-compound [α]D,literature (c = 0.76, CHCl3): +32.8.
(4S,6R)-6-((tert-Butyldimethylsilyl)oxy)hept-1-en-4-yl acrylate (vii). To a solution of alcohol vi (2.00 g,
1.0 eq.) in anhydrous CH2Cl2 (41 mL) at 0 °C, was added freshly distilled triethylamine (2.3 mL,
2.0 eq.), followed by slowly addition of acryloyl chloride (1.0 mL, 1.5 eq.). The reaction was stirred for
4 h at rt. After this period, brine (20 mL) and aqueous solution of Rochelle salt (20 mL) were added. The
aqueous phase was extracted with EtOAc (3 × 50 mL). The organic phases were grouped, dried
(Na2SO4), filtered and concentrated. The product was purified by column chromatography (SiO2,
hexanes/EtOAc 95:5) to afford vii (1.80 g, 75%). Colorless oil. Rf 0.30 (SiO2, hexanes/EtOAc 99:1).
1H-NMR δ (CDCl3, 600 MHz): 0.04 (s, 3H), 0.05 (s, 3H), 0.88 (s, 9H), 1.15 (d, J = 6.0 Hz, 3H),
1.63–1.68 (m, 1H), 1.82–1.88 (m, 1H), 2.30–2.43 (m, 2H), 3.85 (sext, J = 6.2 Hz, 1H), 5.04–5.11 (m,
3H), 5.71–5.79 (m, 1H), 5.80 (dd, J = 10.4, 1.3 Hz, 1H), 6.09 (dd, J = 17.3, 10.5 Hz, 1H), 6.38 (dd,
J = 17.3, 1.3 Hz, 1H). 13C-NMR δ (CDCl3, 151 MHz): −4.7, −4.3, 18.2, 23.6, 26.0 (3C), 39.0, 43.7, 65.7,
71.1, 118.0, 128.9, 130.5, 133.5, 165.7. [α]D (c = 1.0, CHCl3): +13. IR (NaCl, cm−1): 1194, 1406, 1472,
1727, 2858, 2896, 2930, 2958. HRMS: [C16H30O3Si+H]+ calculated 299.2043, observed 299.2107.
(S)-6-((R)-2-((tert-butyldimethylsilyl)oxy)propyl)-5,6-dihydro-2H-pyran-2-one (viii). To a solution of
the acrylate vii (1969 mg, 6.6 mmol, 1.0 eq.) in CH2Cl2 (540 mL) at temperature of 40–45 °C, Grubbs
catalyst 1st generation (543 mg, 0.66 mmol, 0.1 eq.) was added. The mixture was refluxed for 4 h. After
this time the solvent was evaporated. The product was purified by column chromatography (SiO2,
hexanes/EtOAc 95:5) to afford viii (1.40 g, 80%). Brown oil. Rf 0.34 (SiO2, hexanes/EtOAc 80:20).
1H-NMR δ (CDCl3, 500 MHz): 0.00 (s, 3H), 0.02 (s, 3H), 0.82 (s, 9H), 1.15 (d, J = 6.1 Hz, 3H),
1.64–1.71 (m, 1H), 1.96–2.03 (m, 1H), 2.26–2.41 (m, 2H), 4.03 (sext, J = 6.1 Hz, 1H), 4.49–4.56 (m,
1H), 5.93–5.98 (m, 1H), 6.84 (ddd, J = 9.8, 5.8, 2.7 Hz, 1H). 13C-NMR δ (CDCl3, 126 MHz): −4.8, −4.3,
18.0, 23.4, 25.8 (3C), 29.7, 44.2, 64.9, 75.5, 121.4, 145.1, 164.4. [α]D (c = 1.0, CHCl3): −91. Lit. [29]:
[α]D (c = 0.84, CHCl3): −92.6.
(S)-6-((R)-2-Hydroxypropyl)-5,6-dihydro-2H-pyran-2-one (5). To a solution of viii (210 mg, 0.8 mmol,
1 eq.) in anhydrous THF (10 mL) at 0 °C, was added a solution composed by pyridine (1.3 mL) and
HF•pyridine (0.6 mL) in THF (13 mL). The reaction was stirred at rt for 3 days. The reaction was

Molecules 2015, 20
5045
neutralized with solution of NaHCO3 (30 mL). The reaction contents were diluted with 30 mL of ethyl
acetate and the aqueous phase was extracted with EtOAc (3 × 30 mL). The product was purified by
column chromatography (SiO2, EtOAc) to afford 5 (121 mg, quantitative yield). Colorless oil. Rf 0.40
(SiO2, EtOAc). 1H-NMR δ (CDCl3, 500 MHz): 1.20 (d, J = 6.3 Hz, 3H), 1.67–1.74 (m, 1H), 1.92–2.00
(m, 1H), 2.30–2.44 (m, 2H), 2.72 (br s, 1H), 3.96–4.05 (m, 1H), 4.55–4.63 (m, 1H), 5.95 (dd, J = 9.9,
1.7 Hz, 1H), 6.87 (ddd, J = 9.3, 5.8, 2.6 Hz, 1H). 13C-NMR δ (CDCl3, 126 MHz): 23.7, 29.4, 43.5, 64.9,
76.8, 121.0, 145.7, 164.4. [α]D (c = 1.0, CHCl3): −128. Lit. [18]: [α]D (c = 0.17, CHCl3): −115.5.
(E)-3-(3,4-bis((tert-Butyldimethylsilyl)oxy)phenyl)acrylic acid (ix). Freshly distilled diisopropylethylamine
(2.91 mL, 16.7 mmol, 5.0 eq) and tert-butyldimethylsilyl chloride (2.09 g, 13.9 mmol, 5.0 eq) were
added to a suspension of caffeic acid (500 mg, 2.78 mmol, 1 eq) in anhydrous CH2Cl2 (3.4 mL) at
25 °C, and the mixture became a solution, it was stirred at 25 °C for 14 h. The mixture was diluted with
EtOAc (15 mL), extracted with water (5 mL), aqueous solution of HCl 1 M (2 × 10 mL) and brine
(10 mL), then the organic phase was dried (MgSO4) and concentrated to obtain an yellow oil. This oil
was dissolved in THF (4 mL) and were added solid K2CO3 (400 mg) and water (0.7 mL), the reaction
was stirred for 2 h. Upon completion the reaction contents were diluted with ethyl acetate (15 mL),
extracted with water (10 mL), aqueous solution of HCl 1 M (10 mL) and brine (10 mL), then the organic
phase was dried (MgSO4) and concentrated. The solid was submitted to vacuo (10 mbar) at 60 °C for
4 h, to obtain ix (1.078 g, 95%). Pale yellow solid, mp 157–160 °C. Rf 0.40 (SiO2, hexanes/EtOAc
1
75:25), H-NMR δ (CDCl3, 250 MHz): 0.22 (s, 6H), 0.23 (s, 6H), 0.99 (s, 9H), 1.00 (s, 9H), 6.24 (d,
J = 15.8 Hz, 1H), 6.81–6.87 (m, 1H), 7.01–7.08 (m, 2H), 7.67 (d, J = 16.0 Hz, 1H), 13C-NMR δ (CDCl3,
62.9 MHz): −4.0 (2C), −3.9 (2C), 18.6, 18.6, 26.0 (6C), 115.0, 120.8, 121.3, 122.9, 127.8, 147.2, 147.4,
150.1, 173.2.
3-(3,4-Bis((tert-butyldimethylsilyl)oxy)phenyl)propanoic acid (x). 40 mg of Pd/C (5% m/m) were added
to a solution of acid ix (0.92 mmol) in ethyl acetate (5 mL). The heterogeneous mixture was stirred under
hydrogen atmosphere for 3 h. The mixture was filtered through celite, and the solvent was removed
under vacuo to afford the acid x (373 mg, 99%). pale yellow solid. mp 88–89 °C. Rf 0.54 (SiO2,
hexanes/EtOAc 75:25). 1H-NMR δ (CDCl3, 250 MHz): 0.18 (s, 12H), 0.98 (s, 18H), 2.62 (t, J = 7.5 Hz,
2H), 2.84 (t, J = 7.8 Hz, 2H), 6.60–6.78 (m, 3H). 13C-NMR δ (CDCl3, 62.9 MHz): −3.95 (4C), 18.6 (2C),
26.1 (6C), 30.1, 36.1, 121.2 (2C), 121.3, 133.4, 145.4, 146.8, 179.7.
(−)-Tarchonanthuslactone (1). To a solution of EDC•HCl (143 mg, 0.75 mmol, 2 eq.) and DMAP
(50 mg, 0.38 mmol, 1 eq.) in anhydrous CH2Cl2 (5 mL) were added a solution of acid x (308 mg,
0.75 mmol, 2 eq.) and alcohol 5 (60 mg, 0.31 mmol, 1 eq.) in anhydrous CH2Cl2 (5 mL) at 25 °C, and
the mixture was stirred at 25 °C for 1 day. Upon completion, the reaction contents were diluted in EtOAc
(90 mL), and extracted with solution of HCl 0.5 M (40 mL). The organic phase was washed with aqueous
solution of saturated NaHCO3 (30 mL), dried (MgSO4), and concentrated. The crude was dissolved in
anhydrous THF (10 mL), and benzoic acid (231 mg, 1.9 mmol, 5 eq.) and solution of TBAF 1 M in THF
(1.9 mL, 1.9 mmol, 5 eq.) were added at 0 °C. The reaction was stirred at this temperature for 30 min,
then aqueous solution of saturated NaHCO3 (30 mL) was added, and the aqueous phase was extracted
with EtOAc (2 × 60 mL). The organic phases were grouped, washed with brine (30 mL) and dried

Molecules 2015, 20
5046
(MgSO4). The product was purified by column chromatography (SiO2, hexanes/EtOAc 60:40 to 30:70)
1
to afford 1 (71 mg, 58%). Viscous colorless oil. Rf 0.50 (SiO2, hexanes/EtOAc 30:70). H-NMR δ
(CDCl3, 500 MHz): 1.22 (d, J = 6.3 Hz, 3H), 1.73 (ddd, J = 14.5, 6.9, 4.3 Hz, 1H), 2.06 (ddd, J = 14.6,
8.4, 6.4 Hz, 1H), 2.11–2.31 (m, 2H), 2.58 (t, J = 7.5 Hz, 2H), 2.80 (t, J = 7.2 Hz, 2H), 4.16–4.24 (m,
1H), 5.01–5.08 (m, 1H), 5.98 (dd, 9.8, 1.8 Hz, 1H), 6.55 (dd, J = 8.1, 1.8 Hz, 1H), 6.71 (d, J = 1.8 Hz,
1H), 6.73 (d, J = 7.9 Hz, 1H), 6.78–6.85 (m, 1H). 13C-NMR δ (CDCl3, 126 MHz): 20.4, 29.1, 30.3, 36.1,
40.8, 67.4, 75.4, 115.4, 115.5, 120.3, 120.8, 132.7, 142.6, 144.1, 146.0, 165.5, 173.1. [α]D (c = 1.0,
CHCl3): −75. Lit. [16]: [α]D (c = 0.6, CHCl3): −76.
(R)-1-((S)-6-oxo-3,6-Dihydro-2H-pyran-2-yl)propan-2-yl (E)-3-(3,4-dihydroxyphenyl)acrylate (4). To
a solution of EDC•HCl (143 mg, 0.75 mmol, 2 eq.) and DMAP (50 mg, 0.38 mmol, 1 eq.) in anhydrous
CH2Cl2 (5 mL) were added a solution of acid ix (310mg, 0.75 mmol, 2 eq.) and alcohol 5 (60 mg,
0.31 mmol, 1 eq.) in anhydrous CH2Cl2 (5 mL) at 25 °C, and the mixture was stirred at 25 °C for 1 day.
Upon completion, the reaction contents were diluted in EtOAc (90 mL), and extracted with solution of
HCl 0.5 M (40 mL). The organic phase was washed with aqueous solution of saturated NaHCO3
(30 mL), dried (MgSO4), and concentrated. The crude was dissolved in anhydrous THF (10 mL), and
benzoic acid (231 mg, 1.9 mmol, 5 eq.) and solution of TBAF 1 M in THF (1.9 mL, 1.9 mmol, 5 eq.)
were added at 0 °C. The reaction was stirred at this temperature for 60 min, then aqueous solution of
saturated NaHCO3 (30 mL) was added, and the aqueous phase was extracted with EtOAc (2 × 60 mL).
The organic phases were grouped, washed with brine (30 mL) and dried (MgSO4). The product was
purified by column chromatography (SiO2, hexanes/EtOAc 60:40 to 30:70) to afford 4 (61 mg, 50%).
1
Clear yellow oil. Rf 0.50 (SiO2, hexanes/EtOAc 30:70). H-NMR δ (methanol-d4, 250 MHz): 1.35 (d,
J = 6.3 Hz, 3H), 1.88–2.01 (m, 1H), 2.15–2.26 (m, 1H), 2.29–2.58 (m, 2H), 4.54–4.65 (m, 1H), 5.21 (st,
J = 9.8 Hz, 1H), 5.96 (ddd, J = 9.8, 2.4, 0.8 Hz, 1H), 6.24 (d, J = 15.8 Hz, 1H), 6.78 (d, J = 8.2 Hz, 1H),
6.94 (dd, J = 8.2, 1.9 Hz, 1H), 6.97–7.03 (m, 1H), 7.04 (d, J = 2.0 Hz, 1H), 7,54 (d, J = 15.9 Hz, 1H).
13C-NMR δ (methanol-d4, 62.9 MHz): 20.5, 30.3, 41.9, 68.8, 77.1, 115.3, 115.5, 116.6, 121.5, 123.1,
127.9, 146.9, 147.1, 148.2, 149.7, 166.7, 168.8. [α]D (c = 1.0, MeOH): −79. IR (NaCl, cm−1): 813, 1180,
1262, 1633, 1694, 2934, 2978, 3445 (broad).
3.2. Biological Experiments
All experimental procedures were performed in accordance with the guidelines of the Brazilian
College for Animal Experimentation and were approved by the Ethics Committee at the University of
Campinas. Six-week old male Swiss albinus mice were obtained from the University of Campinas
Animal Breeding Center and maintained in individual cages at 21 ± 2 °C, with a 12/12 h dark-light cycle
with food and water ad libitum. Mice were fed on a high fat diet containing 31% (w/w) of fat from lard.
After 6 h of fasting (starting 0800 h), mice were divided into three groups treated by an intraperitoneal
(ip) injection with a single dose of either tarchonanthuslactone (3.0 mg/kg), dihydrocaffeic acid (3.0 mg/kg)
or caffeic acid (3.0 mg/kg). Then, blood glucose levels were measured after 30, 60 and 90 min.
Long-term and cumulative effects of tarchonanthuslactone were evaluated in Swiss mice fed on high
fat diet for eight weeks and treated three times a week, for four weeks with 3.0 mg/kg per dose ip of
tarchonanthuslactone. Two days after the last dose of the compound, the mice were submitted to a

Molecules 2015, 20
5047
6-hour fasting and blood glucose levels were determined and expressed as percentage of saline treated
group. Furthermore, lean non-diabetic Swiss mice fed on a commercial chow diet with approximately
4% (w/w) of fat were treated by an intraperitoneal injection with a single dose of tarchonanthuslactone
(3.0 mg/kg) and the control group received saline. Blood glucose levels were measured after 30, 60 and
90 min and the area under this curve was calculated and expressed as percentage of saline treated group.
The number of animals in each group was at least six. Single blood samples were obtained from the
tip of the tail and glucose levels were immediately measured using a glucometer from Abbott
(Opptimum, Abbott Diabetes Care Inc., Alameda, CA, USA).
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/20/03/5038/s1.
Acknowledgments
We thank the São Paulo State Research Foundation (FAPESP, proc. 13/07607-8) and the Brazilian
National Research Council (CNPq) for financial support. The laboratories “Laboratory of Cell
Signaling” and “Organic Synthesis Laboratory” belong to the Obesity and Comorbidities Research
Center. GFPS, LFTN, CMA and LFRN thank FAPESP for fellowships (11/50514-5, 12/09254-2,
10/08673-6, 2009/53606-8). The authors also thank to Institute of Chemistry/UNICAMP for financial
and academic support.
Author Contributions
Performed the biological experiments and the analysis of the results: G.F.P.S. and L.F.R.N. Designed
the synthesis and performed the chemical experiments: L.F.T.N. and C.M.A. Conceived the
experiments: L.A.V. and R.A.P. Wrote the paper: L.A.V., R.A.P. and G.F.P.S.
Conflicts of Interest
The authors declare no conflict of interest.
References
1. Akash, M.S.H.; Rehman, K.; Chen, S. Role of inflammatory mechanisms in pathogenesis of type 2
diabetes mellitus. J. Cell. Biochem. 2013, 114, 525–531.
2. Donath, M.Y. Targeting inflammation in the treatment of type 2 diabetes: time to start. Nat. Rev.
Drug Discov. 2014, 13, 465–476.
3. Goldfine, A.B.; Fonseca, V.; Jablonski, K.A.; Pyle, L.; Staten, M.A.; Shoelson, S.E. The effects of
salsalate on glycemic control in patients with type 2 diabetes: a randomized trial. Ann. Intern. Med.
2010, 152, 346–357.
4. Akash, M.S.H.; Rehman, K.; Chen, S. An overview of valuable scientific models for diabetes
mellitus. Curr. Diabetes Rev. 2013, 9, 286–293.

Molecules 2015, 20
5048
5. Oboh, G.; Agunloye, O.M.; Adefegha, S.A.; Akinyemi, A.J.; Ademiluyi, A.O. Caffeic and
chlorogenic acids inhibit key enzymes linked to type 2 diabetes (in vitro): A comparative study.
J. Basic Clin. Physiol. Pharmacol. 2014, 26, 165–170.
6. Akash, M.S.H.; Rehman, K.; Chen, S. Effects of coffee on type 2 diabetes mellitus. Nutrition 2014,
30, 755–763.
7. Akash, M.S.H.; Rehman, K.; Chen, S. Spice plant Allium cepa: Dietary supplement for treatment
of type 2 diabetes mellitus. Nutrition 2014, 30, 1128–1137.
8. Bohlmann F.; Suwita, A. Ein neues bisabolen-derivat und ein neues dihydro kaffesaure-derivat aus
tarchonanthus tribolus. Phytochemistry 1979, 18, 677–678.
9. Van Huyssteen, M.; Milne, P.J.; Campbell, E.E.; van de Venter, M. Antidiabetic and cytotoxicity
screening of five medicinal plants used by traditional African health practitioners in the Nelson
Mandela Metropole, South Africa. Afr. J. Tradit. Complement. Altern. Med. 2011, 8, 150–158.
10. Kalesse, M.; Christmann, M.; Bhatt, U.; Quitschalle, M.; Claus, E.; Saeed, A.; Burzlaff, A.;
Kasper, C.; Haustedt, L.O.; Hofer, E.; et al. The chemistry and biology of ratjadone. ChemBioChem
2001, 2, 709–714.
11. Kalesse, M.; Christmann, M. The chemistry and biology of the leptomycin family. Synthesis 2002,
8, 981–1003.
12. Bialy, L.; Waldmann, H. Synthesis and biological evaluation of cytostatin analogues.
Chem. Commun. 2003, 1872–1873.
13. Buck, S.B.; Hardouin, C.; Ichikawa, S.; Soenen, D.R.; Gauss, C.M.; Hwang, I.; Swingle, M.R.;
Bonness, K.M.; Honkanen, R.E.; Boger, D.L. Fundamental role of the fostriecin unsaturated lactone
and implications for selective protein phosphatase inhibition. J. Am. Chem. Soc. 2003, 125,
15694–15695.
14. Enders, D.; Steinbusch, D. An efficient asymmetric synthesis of tarchonanthuslactone. Eur. J.
Org. Chem. 2003, 22, 4450–4454.
15. Baktharama, S.; Selvakumar, S.; Singh, V.K. Asymmetric synthesis of all the stereoisomers of
tarchonanthuslactone. Tetrahed. Lett. 2005, 46, 7527–7529.
16. George, S.; Sudalai, A. Enantioselective synthesis of tarchonanthuslactone using proline-catalyzed
asymmetrica-aminooxylation. Tetrahedron Asymmetry 2007, 18, 975–981.
17. Yadav, J.S.; Kumar, N.N.; Reddy, M.S.; Prasad, A.R. Stereoselective synthesis of
tarchonanthuslactone via the Prins cyclisation. Tetrahedron 2007, 63, 2689–2694.
18. Lee, H.; Sampath, V.; Yoon, Y. A facile total synthesis of all stereoisomers of tarchonanthuslactone
and euscapholide from chiral epichlorohydrin. Synlett 2009, 2, 249–252.
19. Hsu, F.; Chen, Y.; Cheng, J. Caffeic acid as active principle from the fruit of Xanthium strumarium
to lower plasma glucose in diabetic rats. Planta Med. 2000, 66, 228–230.
20. Yadav, J.S.; Reddy, P.M.K.; Gupta, M.K.; Chary, J. Stereoselective total synthesis of
tarchonanthuslactone and formal synthesis of (–)-colletol. Synthesis 2007, 23, 3639–3646.
21. Scott, M.S.; Luckhurst, C.A.; Dixon, D.J. A total synthesis of tarchonanthuslactone exploiting
N-pyrrole carbinols as efficient stereocontrolling elements. Org. Lett. 2005, 7, 5813–5816.
22. Garaas, S.D.; Hunter, T.J.; O’Doherty, G.A. An enantioselective synthesis of tarchonanthuslactone.
J. Org. Chem. 2002, 67, 2682–2685.

Molecules 2015, 20
5049
23. Rossmeisl, M.; Rim, J.S.; Koza, R.A.; Kozak, L.P. Variation in type 2 diabetes-related traits in
mouse strains susceptible to diet-induced obesity. Diabetes 2003, 52, 1958–1966.
24. De Souza, C.T.; Araújo, E.P.; Stoppiglia, L.F.; Pauli, J.R.; Ropelle, E.; Rocco, S.A.; Marin, R.M.;
Franchini, K.G.; Carvalheira, J.B.; Saad, M.J.; et al. Inhibition of UCP2 expression reverses
diet-induced diabetes mellitus by effects on both insulin secretion and action. FASEB J. 2007, 21,
1153–1163.
25. Zarbin, P.H.G.; de Oliveira, A.R.M.; Delay, C.E. Diastereoselective route to (2R,5S)- and (2S,5S)-
2-methyl-1,6-dioxaspiro[4.5]decane, a pheromone component of the wasp Paravespula vulgaris.
Tetrahedron Lett. 2003, 44, 6849–6851.
26. Moore, C.G.; Murphy, P.J.; Williams, H.L.; McGown, A.T.; Smith, N.K. Synthetic studies towards
ptilomycalin A: Total synthesis of crambescidin 359. Tetrahedron 2007, 63, 11771–11780.
27. Wattanasereekul, S.; Maier, M.E. Synthesis of the 8-hydroxy acid of jasplakinolide. Adv. Synth. Catal.
2004, 346, 855–861.
28. Kumar, P.; Gupta, P.; Naidu, S.V. A simple and efficient approach to 1,3-polyols: Application to
the synthesis of cryptocarya diacetate. Chem. Eur. J. 2006, 12, 1397–1402.
29. Gupta, P.; Naidu, S.V.; Kumar, P. Enantioselective synthesis of tarchonanthuslactone via iterative
hydrolytic kinetic resolution. Tetrahedron Lett. 2005, 46, 6571–6573.
Sample Availability: Samples of the compounds 2 and 3 are available from the authors.
© 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/4.0/).