Synthesis and biological evaluation of (-)-Linarinic acid derivatives as neuroprotective agents against OGD-induced cell damage

Article abstract of DOI:10.1002/ardp.201100424

A series of novel (-)-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1- carboxylic acid derivatives were designed and synthesized. All of the prepared compounds were screened for their neuroprotective effects using an in vitro oxygen glucose deprivation (OGD

Full text of DOI:10.1002/ardp.201100424

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
1
Full Paper
Synthesis and Biological Evaluation of (ꢀ)-Linarinic Acid
Derivatives as Neuroprotective Agents Against OGD-Induced
Cell Damage
Yu Tian1, Chao Ma1, Linyin Feng2, Lei Zhang2, Feiyue Hao1, Li Pan1, and Maosheng Cheng1
1 Key Laboratory of Structure-Based Drugs Design & Discovery of Ministry of Education, School of
Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang, China
2 Shanghai Institute of Materia Medica Chinese Academy of Sciences, Shanghai, China
A series of novel (ꢀ)-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline-1-carboxylic acid derivatives were
designed and synthesized. All of the prepared compounds were screened for their neuroprotective
effects using an in vitro oxygen glucose deprivation (OGD) model of ischemic stroke. Some of the target
compounds exhibited moderate to excellent protective potency. In particular, compounds 9d, 9e, 9g, and
9h showed significant protective effects in the SH-SY5Y cell line at all three concentrations tested.
Keywords: Cerebral ischemia / (ꢀ)-Linarinic acid derivatives / Neuroprotective effect / Oxygen glucose deprivation /
SH-SY5Y cells
Received: November 25, 2011; Revised: January 6, 2012; Accepted: January 13, 2012
DOI 10.1002/ardp.201100424
Introduction
deprivation (OGD) model of ischemic stroke in SH-SY5Y cells.
This result attracted our interest and prompted us to syn-
thesize (ꢀ)-linarinic acid analogs in an attempt to develop
this novel active scaffold as a neuroprotective agent [13].
In a continuing effort to find new potent analogs of
(ꢀ)-linarinic acid and to verify the importance of the carbox-
ylic acid moiety, compounds 4b–c, 5a–f, 6a, and 9a–h (Fig. 1)
were designed and synthesized. First, halogen substituents
were incorporated into the (ꢀ)-linarinic acid scaffold at the
C-7 position. Methyl ester and isopropyl ester analogs were
also introduced to investigate the influence of size and other
properties related to ester substitution. Finally, various
amides derivatives were prepared to study the effect of an
amide moiety on (ꢀ)-linarinic acid.
Cerebral ischemia, also known as brain ischemia, is one of
the primary causes of sudden death in the world. In the past
few decades, there has been a considerable increase in the use
of complementary treatments, such as herbal remedies.
Many traditional plants have been claimed to be useful for
the control of problems due to cerebral ischemia and associ-
ated pathologies [1–9].
Linaria vulgaris Mill., distributed in the northeastern and
Inner Mongolia regions of China, is known as a traditional
Chinese medicine with various pharmacological properties
[10]. (ꢀ)-Linarinic acid, namely (ꢀ)-1,2,3,9-tetrahydropyr-
rolo[2,1-b]quinazoline-1-carboxylic acid, is a novel tricyclic
quinazoline alkaloid that was isolated from L. vulgaris
Mill [11]. The total asymmetric synthesis and the assignment Results and discussion
of absolute configuration for this natural product were first
reported in 2006 [12]. In our recent study, a property of
(ꢀ)-linarinic acid was found that could protect against
ischemia-induced cell injury in an in vitro oxygen glucose
Chemistry
The synthetic routes to the target compounds 4a–c, 5a–f, 6a,
and 9a–h are illustrated in Scheme 1, Scheme 2, and
Scheme 3. The synthetic strategy began with the reductive
amination of 2-nitrobenzaldehydes 1a–c [14] using commer-
cially available L-glutamic acid followed by an intramolecular
cyclization and dehydration in refluxing ethanol to produce
the desired series of significant intermediates 3a–c [15, 16].
Next, compounds 3a–c were treated with an excess of 80%
hydrazine hydrate, ferric chloride, and activated carbon in
Correspondence: Maosheng Cheng, Key Laboratory of Structure-Based
Drugs Design & Discovery of Ministry of Education, School of
Pharmaceutical Engineering, Shenyang Pharmaceutical University, 103
Wenhua Road, Shenhe District, Shenyang 110016, P. R. China.
E-mail: mscheng@syphu.edu.cn
Fax: þ86 24 23995043
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2
Y. Tian et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
Figure 1. (S)-Linarinic acid and (S)-linarinic acid derivatives.
refluxing ethanol for 72 h, yielding three of the target com-
pounds 4a–c [12, 17–19]. Compounds 4a–c were then reacted
with thionyl chloride and alcohols to afford compounds 5a–f
via the esterification. In addition, treatment of compound 5a
with an aqueous solution of ammonia at 608C afforded the
corresponding amide derivative 6a [20]. However, the above
reaction conditions were not suitable for the synthesis of
other substituted amide derivatives. Thus, the target com-
pounds 9a–h were successfully prepared via the amidation of
intermediate 3a with different amines followed by reduction
and dehydration reactions. The structures of all target com-
pounds were identified by 1H NMR, 13C NMR, IR, and HRMS.
Biological evaluation
For the biological activities reported herein, we used an
in vitro OGD model of ischemia reperfusion on SH-SY5Y
Scheme 1. Synthesis of compounds 4a–c and 5a–f. Reaction conditions: (a) L-glutamic acid, NaOH, NaBH4, and HCl/H2O;
(b) CH3CH2OH, reflux, 68.6% (for two steps); (c) 80% NH2NH2 H2O, FeCl3, active carbon, CH3CH2OH, reflux, 54.4%; (d) SOCl2,
CH3OH or (CH3)2CHOH, 65.8%.
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
(ꢀ)-Linarinic Acid Derivatives
3
The results showed that OGD alone reduced cell viability
to 59%, compared to the control, whereas, treatment of
SH-SY5Y cells with (ꢀ)-linarinic acid (10 mM) prior to OGD
significantly increased cell viability (87%, compared to
control). It was also demonstrated that pharmacologically,
most of the (ꢀ)-linarinic acid derivatives exhibited good pro-
tective effects on the damaged SH-SY5Y cells, and some of
the derivatives were equally effective or more effective than
(ꢀ)-linarinic acid at various concentrations. As is shown in
Fig. 2, compounds 9d–h were found to be more potent
(cell viability at 82, 87, 83, 90, and 89%, respectively) than
(ꢀ)-linarinic acid at a concentration of 100 nM (cell viability
at 80%). At a concentration of 1 mM, compounds 9d, 9e, 9g,
and 9h exhibited a considerable protective effect and signifi-
cantly increased SH-SY5Y cell viability (at 90, 86, 87, and 87%,
respectively), in response to OGD-induced cellular damage,
Scheme 2. Synthesis of compound 6a. Reaction conditions:
(e) NH4OH, 608C, 53.6%.
human neuroblastoma cells, differentiated into neuron-like
cells, to evaluate whether (ꢀ)-linarinic acid and its derivatives
protect the cells against ischemia [21–25]. OGD of SH-SY5Y
cells is a well-established in vitro model that is widely used for
ischemic studies. Cell viability was evaluated with the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay. The neuroprotective effects of (ꢀ)-linarinic acid and its
derivatives on cell viability are shown in Fig. 2.
Scheme 3. Synthesis of compounds 9a–h. Reaction conditions: (f) Amines, ClCOOCH2CH3, TEA, 62.5%; (g) 80% NH2NH2 H2O, FeCl3,
active carbon, CH3CH2OH, reflux, 87.2%; (h) POCl3, 46.1%.
Figure 2. Neuroprotective effects of (S)-linarinic acid and its derivatives on cell viability. SH-SY5Y cells were pretreated with 0.1–10 mM
concentrations of test compounds and then treated with OGD for 10 h. Experimental parameters were assayed at 12 h after DMEM/F12
solution reoxygenation. Cell viability is indicated by the MTT values of each experimental group expressed as percentages of the value for the
sham-OGD group. Values are reported as means W SD. Significant difference from OGD treated group: ꢁp <0.05, ꢁꢁp <0.01, and
ꢁꢁꢁp <0.001. The results were obtained from four independent experiments performed in triplicate.
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

4
Y. Tian et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
showing greater activity than (ꢀ)-linarinic acid at the same
concentration (cell viability at 83%). At a concentration of
10 mM, only derivative 9e (cell viability at 91%) was more
effective than (ꢀ)-linarinic acid.
trations. Thus, the presence of a free carboxylic acid is not
essential for the protective effects. A further study of the
detailed biological mechanisms for these compounds is
currently in progress, which will pave the way to exploit
them as preventive or therapeutic strategies for cerebral
ischemia damage.
These preliminary structure–activity relationships (SARs)
suggested that, in general, amide derivatives of (ꢀ)-linarinic
acid showed better neuroprotective effects than both ester
analogs and (ꢀ)-linarinic acid itself (e.g., compounds 4a, 5a, Experimental
and 9e). The addition of a halogen substituent at the C-7
position of (ꢀ)-linarinic acid had little effect on the activity,
although for a given scaffold, the introduction of a bromine
substituent on C-7 resulted in slightly increased cell viability
compared to a chlorine substituent (e.g., compounds 4b and
4c). Furthermore, ester derivatives of (ꢀ)-linarinic acid showed
decreased cell viability compared to the corresponding car-
boxylic acid derivatives (e.g., compounds 4a and 5a), and
compounds with a smaller methyl ester were more effective
than those with an isopropyl ester (e.g., compounds 5a and 5d).
Biological data indicated that the amide derivatives exhib-
ited a moderate to significant influence on protective
activity, and the nature of the substituents on the amide
nitrogen affected the cell viability. Compounds 9f, 9g, and 9h,
which had piperidinyl, morpholinyl, and N-methylpipera-
zinyl amide substituents, respectively, possessed significant
advantages over compounds 6a, 9a, 9b, and 9c. This differ-
ence could be due to a more optimal hydrophilicity exhibited
by 9f–h when compared to 6a and 9a–c. The above results
indicate that the introduction of a heterocyclic amine could
increase the cell viability. Interestingly, comparison of the
activity of 9a and 9c showed that 9c, which has a cyclopropyl
substituent on the amide nitrogen, was more active than its
N-propyl isomer. Additionally, there seems to be a positive
correlation between the number of carbon atoms in the amide
nitrogen substituent (e.g., compounds 6a, 9a, and 9b) and the
neuroprotective effect of the compounds, although 9b began to
exhibit some toxicity at a concentration of 10 mM. In addition,
compounds 9d and 9e were more active than compound 9a,
indicating that the introduction of benzyl or 3,4-methylene-
dioxyphenylethyl substituents on the amide nitrogen atom
could enhance protective potency. The activities of all the
tested compounds are shown in Table 1.
Chemistry
All commercially available reagents and solvents were used with-
out further purification. Melting points were determined in a
Bu¨chi Melting Point B-540 apparatus and were uncorrected.
Infrared spectra were recorded in a Bruker IFS-55 spectropho-
tometer. Optical rotations were measured on a Perkin Elmer
241MC spectrometer. 1H NMR and 13C NMR spectra were
recorded in a Bruker ARX 300 MHz. Chemical shift values were
reported in d (ppm), and coupling constants were reported in
Hertz. High-resolution mass spectrometry (HRMS) data were
obtained using a Bruker microTOF-Q time of flight mass spec-
trometer or a Bruker APEXIII 7.0 Tesla FT-MS. Recycling prepara-
tive HPLC was conducted with a reverse phase column (JAIGEL
ODS-AP-L SP-120-15), using JAI LC-9103. Column chromatography
was performed on silica gel H, and analytical TLC data was
obtained using silica gel HF254
.
General procedure for the preparation of compounds 4a–c
An ethanol solution (400 mL) of 2-nitrobenzaldehyde 1a, 5-
chloro-2-nitrobenzaldehyde 1b, or 5-bromo-2-nitrobenzaldehyde
1c (300 mmol) was added dropwise at room temperature to an
aqueous solution of L-glutamic acid (315 mmol) and sodium
hydroxide (2 M, 300 mL). The mixture was stirred at room
temperature for 1 h and cooled to 08C. Sodium borohydride
(450.0 mmol) was added in small portions at 0–58C, and the
mixture was then warmed up to room temperature and stirred
for 3 h. The resulting solution was evaporated under vacuum till
its volume was reduced to 300 mL, and then extracted with
diethyl ether (70 mL ꢂ 3). The aqueous layer was acidified to
pH 3 with concentrated hydrochloric acid. The resulting suspen-
sion of 2a–c was filtered and then heated in refluxing ethanol
(800 mL) for 5 h. This solution was then filtered and concen-
trated in vacuum to give an ivory-white dusty solid 3a–c, which
was used in the next reaction without further purification. To a
solution of the above crude intermediate 3a–c in ethanol
(600 mL) was added activated carbon (6.00 g) and a catalytic
amount of ferric chloride (0.05 g). The suspension was then
heated to 508C and hydrazine hydrate (1200 mmol) was added
dropwise. This mixture was heated at reflux for approximately
72 h. At this point, the mixture was filtered and the solvent was
removed by vacuum evaporation. The residue was purified by
flash chromatography on silica gel using methanol–ethyl acetate
(5:4 v/v) to give compounds 4a–c.
Conclusion
In summary, (ꢀ)-linarinic acid and 17 (ꢀ)-linarinic acid
derivatives were synthesized as single isomers and screened
at three different concentrations for their neuroprotective
effects in an in vitro OGD model of ischemic stroke using
SH-SY5Y cells. Biological results revealed that most synthe-
sized compounds displayed neuroprotective effects against
ischemia, and among these compounds, 9d, 9e, 9g, and 9h
exhibited remarkable protective activities at various concen-
(S)-(ꢀ)-1,2,3,9-Tetrahydropyrrolo[2,1-b]quinazoline-1-
carboxylic acid 4a
White solid; yield 37.3%; mp: 229.4–232.58C; ½aꢃ2D5: ꢀ222.2
(c 0.25, MeOH); 1H NMR (300 MHz, CD3OD) d: 2.22–2.33
(m, 1H), 2.53–2.66 (m, 1H), 2.93–3.32 (m, 2H), 4.32
(dd, 1H, J ¼ 4.1, 9.3 Hz), 4.76 (d, 1H, J ¼ 15.4 Hz), 4.98
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
(ꢀ)-Linarinic Acid Derivatives
5
Table 1. In vitro cell viability of compounds 4a–4c, 5a–5f, 6a, and 9a–h against OGD-induced cell damage at three concentrations
Compound
Structure
R3
Cell viability (%)
R1
R2
R4
100 nM
1 mM
10 mM
Control
OGD
4a
4b
4c
5a
5b
5c
5d
100
59
83
78
82
73
70
78
66
67
71
61
H
Cl
Br
H
Cl
Br
H
Cl
Br
H
H
H
H
CH3
CH3
CH3
CH(CH3)2
CH(CH3)2
CH(CH3)2
80
75
77
73
72
75
65
61
66
61
87
76
80
77
73
71
63
68
69
70
5e
5f
6a
NH2
9a
H
64
68
77
9b
9c
H
H
65
72
67
78
15
84
9d
9e
H
H
82
87
90
86
79
91
9f
H
H
H
83
90
89
81
87
87
76
86
83
9g
9h
(d, 1H, J ¼ 15.4 Hz), 7.00–7.02 (m, 1H), 7.19–7.27 (m, 2H),
7.29–7.35 (m, 1H); 13C NMR (75 MHz, CD3OD) d: 25.6, 30.2,
46.5, 70.2, 117.7, 118.4, 128.1, 128.3, 130.3, 132.2, 165.0,
174.6; IR (KBr): 3419.7, 2921.7, 1667.3, 1613.4, 1584.2,
1499.5, 1385.3, 1291.4, 764.2 cmꢀ1; ESI-HRMS: calcd for
C12H13N2O2, 217.0972; found, 217.0969. [MþH]þ.
J ¼ 15.8 Hz), 7.00 (d, 1H, J ¼ 8.5 Hz), 7.27 (d, 1H, J ¼ 1.9 Hz),
7.33 (dd, 1H, J ¼ 1.9, 8.5 Hz); 13C NMR (75 MHz, CD3OD) d:
25.6, 30.2, 46.3, 70.1, 119.3, 120.5, 128.3, 130.3, 131.2, 133.1,
165.2, 174.7; IR (KBr): 3440.3, 2924.1, 1667.8, 1620.7, 1578.5,
1492.6, 1388.8, 1300.8, 1189.9, 667.6 cmꢀ1; ESI-HRMS: calcd
for C12H12ClN2O2, 251.0582; found, 251.0581. [MþH]þ.
(S)-(ꢀ)-7-Chloro-1,2,3,9-tetrahydropyrrolo[2,1-b]-
(S)-(ꢀ)-7-Bromo-1,2,3,9-tetrahydropyrrolo[2,1-b]-
quinazoline-1-carboxylic acid 4b
quinazoline-1-carboxylic acid 4c
White solid; yield 34.9%; mp: 265.4–268.18C; ½aꢃ2D5: ꢀ251.2
(c 0.25, MeOH); 1H NMR (300 MHz, CD3OD) d: 2.23–2.33
(m, 1H), 2.53–2.66 (m, 1H), 2.93–3.14 (m, 2H), 4.32 (dd,
1H, J ¼ 4.1, 9.3 Hz), 4.75 (d, 1H, J ¼ 15.7 Hz), 4.96 (d, 1H,
White solid; yield 30.8%; mp: 282.5–284.98C; ½aꢃ2D5: –224.0 (c 0.25,
MeOH); 1H NMR (300 MHz, CD3OD) d: 2.22–2.33 (m, 1H),
2.53–2.66 (m, 1H), 2.92–3.13 (m, 2H), 4.32 (dd, 1H, J ¼ 3.9,
9.3 Hz), 4.75 (d, 1H, J ¼ 15.7 Hz), 4.97 (d, 1H, J ¼ 15.8 Hz),
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

6
Y. Tian et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
6.94 (d, 1H, J ¼ 8.5 Hz), 7.43 (d, 1H, J ¼ 1.7 Hz), 7.48 (dd, 1H,
J ¼ 1.7, 8.5 Hz); 13C NMR (75 MHz, CD3OD) d: 25.6, 30.3,
46.1, 70.3, 119.4, 120.5, 120.8, 131.2, 131.7, 133.3, 165.2,
174.7; IR (KBr): 3424.7, 2926.1, 1657.7, 1577.2, 1497.1,
1421.1, 1373.5, 1296.3, 819.0 cmꢀ1; ESI-HRMS: calcd for
C12H12BrN2O2, 295.0077; found, 295.0079. [MþH]þ.
(S)-(ꢀ)-Isopropyl 1,2,3,9-tetrahydropyrrolo[2,1-b]-
quinazoline-1-carboxylate 5d
White solid; yield 61.5%; mp: 112.5–113.38C; ½aꢃ2D5: ꢀ301.2 (c
0.25, MeOH); 1H NMR (300 MHz, DMSO-d6) d: 1.21–1.25
(m, 6H), 1.86–1.98 (m, 1H), 2.24–2.36 (m, 1H), 2.43–2.61
(m, 2H), 4.14 (dd, 1H, J ¼ 4.5, 8.7 Hz), 4.44–4.54 (m, 2H),
4.93–5.05 (m, 2H), 6.83 (m, 1H), 6.93–6.94 (m, 2H), 7.05–
7.12 (m, 1H); 13C NMR (75 MHz, DMSO-d6) d: 21.5, 21.6,
23.5, 29.2, 45.0, 63.0, 68.7, 119.8, 123.5, 123.8, 126.1, 128.1,
142.9, 162.2, 170.7; IR (KBr): 3460.7, 2982.7, 1739.7, 1626.6,
1595.6, 1574.4, 1456.6, 1436.4, 1370.4, 1196.0, 1105.6,
722.9 cmꢀ1; ESI-HRMS: calcd for C15H19N2O2, 259.1441;
found, 259.1445. [MþH]þ.
General procedure for the preparation of compounds 5a–f
Thionyl chloride (30 mL) was added dropwise to a stirred
solution of 4a–c (38.0 mmol) in anhydrous methanol or isopro-
panol (300 mL) at 0–58C. The mixture was stirred at this tempera-
ture for 6 h and then concentrated under vacuum. The residue
was alkalified to pH 8 with a 5% aqueous solution of potassium
carbonate and then dissolved in ethyl acetate (300 mL). After
being washed with water and brine, the organic layers were
dried over Na2SO4 and concentrated under reduced pressure.
The residue was purified by flash chromatography on silica gel
using ethyl acetate to give compounds 5a–f.
(S)-(ꢀ)-Isopropyl 7-chloro-1,2,3,9-tetrahydropyrrolo[2,1-b]-
quinazoline-1-carboxylate 5e
White solid; yield 47.6%; mp: 112.6–113.88C; ½aꢃ2D5: ꢀ270.3
(c 0.25, MeOH); 1H NMR (300 MHz, DMSO-d6) d: 1.22–1.26
(m, 6H), 1.88–1.99 (m, 1H), 2.25–2.37 (m, 1H), 2.43–2.61 (m,
2H), 4.16 (dd, 1H, J ¼ 4.4, 8.7 Hz), 4.45–4.55 (m, 2H), 4.94–
5.06 (m, 2H), 6.84 (d, 1H, J ¼ 8.4 Hz), 7.05 (d, 1H, J ¼ 2.0 Hz),
7.12 (dd, 1H, J ¼ 2.4, 8.4 Hz); 13C NMR (75 MHz, DMSO-d6)
d: 21.4, 21.5, 23.4, 29.2, 44.6, 62.8, 68.7, 121.9, 125.0,
125.9, 127.0, 127.8, 142.1, 162.6, 170.5; IR (KBr): 3426.2,
2980.0, 2933.3, 1741.4, 1627.8, 1591.7, 1489.2, 1471.3,
1413.3, 1192.5, 1104.6, 828.1 cmꢀ1; ESI-HRMS: calcd for
C15H18ClN2O2, 293.1051; found, 293.1048. [MþH]þ.
(S)-(ꢀ)-Methyl 1,2,3,9-tetrahydropyrrolo[2,1-b]-
quinazoline-1-carboxylate 5a
White solid; yield 65.8%; mp: 87.4–88.18C; ½aꢃ2D5: ꢀ121.2 (c 0.25,
MeOH); 1H NMR (300 MHz, DMSO-d6) d: 1.92–2.02 (m, 1H),
2.24–2.37 (m, 1H), 2.473–2.61 (m, 2H), 3.72 (s, 3H), 4.19
(dd, 1H, J ¼ 4.4, 8.7 Hz), 4.45 (d, 1H, J ¼ 13.6 Hz), 4.54
(d, 1H, J ¼ 13.6 Hz), 6.88–6.97 (m, 3H), 7.08–7.13 (m, 1H);
13C NMR (75 MHz, DMSO-d6) d: 23.4, 29.2, 45.1, 52.2, 62.8,
119.7, 123.4, 123.8, 126.1, 128.0, 142.6, 162.0, 171.6; IR (KBr):
3407.1, 2953.4, 2845.2, 1741.9, 1629.9, 1595.8, 1489.3, 1429.5,
1205.4, 1179.7, 1042.1, 766.8 cmꢀ1; ESI-HRMS: calcd for
C13H15N2O2, 231.1128; found, 231.1127. [MþH]þ.
(S)-(ꢀ)-Isopropyl 7-bromo-1,2,3,9-tetrahydropyrrolo[2,1-b]-
quinazoline-1-carboxylate 5f
White solid; yield 53.2%; mp: 121.2–122.58C; ½aꢃ2D5: ꢀ225.6
(c 0.25, MeOH); 1H NMR (300 MHz, DMSO-d6) d: 1.22–1.26
(m, 6H), 1.89–1.99 (m, 1H), 2.25–2.38 (m, 1H), 2.45–2.63 (m,
2H), 4.19 (dd, 1H, J ¼ 4.5, 8.8 Hz), 4.46–4.56 (m, 2H), 4.93–
5.06 (m, 2H), 6.78 (d, 1H, J ¼ 8.4 Hz), 7.20 (d, 1H, J ¼ 2.0 Hz),
7.26 (dd, 1H, J ¼ 2.2, 8.4 Hz); 13C NMR (75 MHz, DMSO-d6)
d: 21.4, 21.5, 23.4, 29.2, 44.5, 63.0, 68.8, 115.3, 122.2,
125.0, 128.8, 130.8, 141.8, 162.9, 170.4; IR (KBr): 3419.3,
2978.0, 2932.0. 1738.1, 1623.8, 1589.1, 1566.8, 1490.0,
1408.6, 1193.5, 1106.2, 826.9 cmꢀ1; ESI-HRMS: calcd for
C15H18BrN2O2, 337.0546; found, 337.0543. [MþH]þ.
(S)-(ꢀ)-Methyl 7-chloro-1,2,3,9-tetrahydropyrrolo[2,1-b]-
quinazoline-1-carboxylate 5b
White solid; yield 56.9%; mp: 92.9–94.58C; ½aꢃ2D5: ꢀ128.6 (c 0.25,
MeOH); 1H NMR (300 MHz, DMSO-d6) d: 1.91–2.03 (m, 1H),
2.25–2.38 (m, 1H), 2.44–2.62 (m, 2H), 3.72 (s, 3H), 4.23
(dd, 1H, J ¼ 4.5, 8.7 Hz), 4.46 (d, 1H, J ¼ 14.1 Hz), 4.53
(d, 1H, J ¼ 14.1 Hz), 6.84 (d, 1H, J ¼ 8.4 Hz), 7.04 (d, 1H,
J ¼ 2.1 Hz), 7.12 (dd, 1H, J ¼ 2.3, 8.3 Hz); 13C NMR
(75 MHz, DMSO-d6) d: 23.4, 29.2, 44.7, 52.3, 62.7, 121.9,
124.9, 125.8, 127.1, 127.8, 142.0, 162.6, 171.5; IR (KBr):
3448.1, 2953.7, 2844.4, 1734.4, 1628.1, 1591.8, 1483.6,
1411.8, 1222.2, 1182.0, 833.0 cmꢀ1; ESI-HRMS: calcd for
C13H14ClN2O2, 265.0738; found, 265.0737. [MþH]þ.
(S)-(ꢀ)-1,2,3,9-Tetrahydropyrrolo[2,1-b]quinazoline-1-
carboxamide 6a
A suspension of 5a (5.17 g, 20.0 mmol) in an aqueous solution
of ammonia (20 mL) was heated to 608C for 2 h then cooled to
room temperature. The precipitate was filtered off, washed with
(S)-(ꢀ)-Methyl 7-bromo-1,2,3,9-tetrahydropyrrolo[2,1-b]-
quinazoline-1-carboxylate 5c
water, dried, and crystallized from ethanol to furnish compound
White solid; yield 40.6%; mp: 95.6–97.98C; ½aꢃ2D5: ꢀ120.8 (c 0.25,
MeOH); 1H NMR (300 MHz, DMSO-d6) d: 1.92–2.02 (m, 1H),
2.25–2.38 (m, 1H), 2.43–2.60 (m, 2H), 3.72 (s, 3H), 4.23
(dd, 1H, J ¼ 4.5, 8.8 Hz), 4.46 (d, 1H, J ¼ 14.1 Hz), 4.52
(d, 1H, J ¼ 14.1 Hz), 6.77 (d, 1H, J ¼ 8.4 Hz), 7.16 (d, 1H,
J ¼ 2.1 Hz), 7.25 (dd, 1H, J ¼ 2.3, 8.4 Hz); 13C NMR
(75 MHz, DMSO-d6) d: 23.3, 29.2, 44.5, 52.3, 62.7, 119.1,
122.4, 125.4, 128.7, 130.7, 142.5, 162.7, 171.5; IR (KBr):
3388.1, 2951.4, 1730.2, 1620.3, 1588.7, 1488.9, 1406.2,
1222.7, 1197.9, 1180.5, 816.2 cmꢀ1; ESI-HRMS: calcd for
C13H14N2O2, 309.0233; found, 309.0231. [MþH]þ.
25
6a (2.31 g, 53.6%) as a white solid. mp: 261.0–261.48C; ½aꢃD
:
ꢀ192.2 (c 0.25, MeOH); 1H NMR (300 MHz, DMSO-d6) d:
1.78–1.88 (m, 1H), 2.15–2.28 (m, 1H), 2.40–2.60 (m, 2H),
3.93 (dd, 1H, J ¼ 5.0, 8.3 Hz), 4.42 (s, 2H), 6.81–6.83 (m,
1H), 6.90–6.94 (m, 2H), 7.05–7.10 (m, 1H), 7.25 (s, 1H),
7.69 (s, 1H); 13C NMR (75 MHz, DMSO-d6) d: 24.0, 29.6,
44.9, 63.8, 119.8, 123.3, 123.4, 126.1, 127.9, 143.2, 162.7,
172.2; IR (KBr): 3357.5, 3193.5, 2922.5, 2852.1, 1680.5,
1621.2, 1596.5, 1573.4, 1490.2, 1291.2, 763.3 cmꢀ1; ESI-
HRMS: calcd for C12H14N3O, 216.1131; found, 216.1134.
[MþH]þ.
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
(ꢀ)-Linarinic Acid Derivatives
7
4.32–4.42 (m, 2H), 6.81–6.83 (m, 1H), 6.88–6.94 (m, 2H), 7.05–
7.10 (m, 1H), 8.34 (d, 1H, J ¼ 3.9 Hz); 13C NMR (75 MHz,
DMSO-d6) d: 5.7, 5.8, 22.4, 23.8, 29.6, 44.8, 63.9, 119.8, 123.4,
123.5, 126.1, 127.9, 143.3, 162.6, 171.3; IR (KBr): 3430.7,
2886.3, 1679.2, 1625.9, 1596.0, 1572.4, 1491.3, 1264.3,
; ESI-HRMS: calcd for C15H18N3O, 256.1444;
found, 256.1440. [MþH]þ.
General procedure for the preparation of compounds 9a–h
To a solution of 3a (38.0 mmol) and triethylamine (57.0 mmol) in
anhydrous THF (15 mL) was added ethyl chloroformate
(38.0 mmol) at 08C, and the mixture was stirred for 0.5 h at this
temperature. The amine corresponding to the desired product
(38.0 mmol) was added, and the mixture was stirred at room
temperature for 4 h. The suspension was filtered and the filtrate
was concentrated to give compounds 7a–h, which were used
without further purification. To a solution of 7a–h in ethanol
(200 mL) were added activated carbon (1.00 g) and a catalytic
amount of ferric chloride (0.005 g). Then, hydrazine hydrate
(228 mmol) was added dropwise and the suspension was refluxed
for 6 h. The resulting mixture was filtered, and the solvent was
concentrated to dryness to afford intermediates 8a–h. The crude
intermediates 8a–h were dissolved in phosphorus oxychloride
(5 mL), refluxed for 1 h, and then concentrated under vacuum.
The residue was alkalified to pH 8 with an aqueous solution of
ammonia (25%) and then dissolved in CH2Cl2 (200 mL). After
being washed with water and brine, the organic layers were
dried over Na2SO4 and concentrated under reduced pressure.
The residue was purified by flash chromatography on silica gel
using methanol–dichloromethane (1:10 v/v) to give compounds
9a–h.
767.9 cmꢀ1
(S)-(ꢀ)-N-Benzyl-1,2,3,9-tetrahydropyrrolo[2,1-b]-
quinazoline-1-carboxamide 9d
White solid; yield 36.6%; mp: 198.9–201.88C; ½aꢃ2D5: ꢀ216.0 (c 0.5,
CHCl3); 1H NMR (300 MHz, DMSO-d6) d: 1.81–1.92 (m, 1H),
2.18–2.31 (m, 1H), 2.42–2.63 (m, 2H), 4.03 (dd, 1H, J ¼ 5.0,
8.2 Hz), 4.33 (d, 2H, J ¼ 5.8 Hz), 4.40 (d, 1H, J ¼ 13.7 Hz),
4.45 (d, 1H, J ¼ 13.4 Hz), 6.82–6.84 (m, 1H), 6.91–6.92
(m, 1H), 7.05–7.11 (m, 1H), 7.26–7.37 (m, 5H), 8.80 (t, 1H,
J ¼ 5.7 Hz); 13C NMR (75 MHz, DMSO-d6) d: 24.0, 29.6, 42.2,
44.9, 64.1, 119.8, 123.4, 126.1, 126.9, 127.2, 127.9, 128.4,
139.2, 143.3, 162.5, 170.3; IR (KBr): 3425.7, 3277.3, 2923.4,
1655.1, 1626.6, 1594.5, 1574.5, 1260.4, 767.1 cmꢀ1; ESI-
HRMS: calcd for C19H20N3O, 306.1601; found, 306.1599.
[MþH]þ.
(S)-(ꢀ)-N-(3,4-Methylenedioxyphenylethyl)-1,2,3,9-
(S)-(ꢀ)-N-Propyl-1,2,3,9-tetrahydropyrrolo[2,1-
tetrahydropyrrolo[2,1-b]quinazoline-1-carboxamide 9e
White solid; yield 39.6%; mp: 179.3–180.78C; ½aꢃ2D5: ꢀ220.0 (c 0.5,
CHCl3); 1H NMR (300 MHz, DMSO-d6) d: 1.69–1.80 (m, 1H),
2.08–2.21 (m, 1H), 2.38–2.58 (m, 2H), 2.65–2.69 (m, 2H), 3.20–
3.32 (m, 2H), 3.88 (dd, 1H, J ¼ 4.9, 8.1 Hz), 4.20 (d, 1H,
J ¼ 13.5 Hz), 4.31 (d, 1H, J ¼ 13.5 Hz), 5.95 (d, 2H,
J ¼ 6.1 Hz), 6.64–6.67 (m, 1H), 6.80–6.84 (m, 3H), 6.86–6.94
(m, 2H), 7.05–7.10 (m, 1H), 8.26 (t, 1H, J ¼ 5.3 Hz); 13C NMR
(75 MHz, DMSO-d6) d: 25.2, 30.4, 36.0, 41.7, 46.2, 66.5, 102.2,
109.1, 110.2, 120.6, 123.0, 124.0, 125.5, 127.2, 129.3, 134.0,
142.8, 147.6, 149.2, 165.0, 172.8; IR (KBr): 3422.1, 3269.7,
b]quinazoline-1-carboxamide 9a
White solid; yield 25.1%; mp: 165.6–167.78C; ½aꢃ2D5: ꢀ180.0 (c 0.5,
CHCl3); 1H NMR (300 MHz, DMSO-d6) d: 0.85–0.87 (m, 3H),
1.43–1.46 (m, 2H), 1.79–1.85 (m, 1H), 2.17–2.25 (m, 1H), 2.42–
2.48 (m, 1H), 2.53–2.57 (m, 1H), 3.03–3.11 (m, 2H), 3.89–4.01
(m, 1H), 4.36 (d, 1H, J ¼ 6.7 Hz), 4.42 (d, 1H, J ¼ 6.6 Hz),
6.82–6.84 (m, 1H), 6.89–6.95 (m, 2H), 7.04–7.13 (m, 1H),
8.27 (s, 1H); 13C NMR (75 MHz, DMSO-d6) d: 11.4, 22.3,
24.0, 29.6, 44.9, 64.1, 119.8, 123.4, 126.1, 127.9, 143.4,
162.5, 170.0; IR (KBr): 3280.9, 2963.1, 2932.8, 2874.4,
1651.6, 1626.8, 1595.5, 1573.1, 1491.3, 1453.8, 1425.2,
1260.3, 1202.6, 1153.3, 760.5 cmꢀ1; ESI-HRMS: calcd for
C15H20N3O, 258.1601; found, 258.1600. [MþH]þ.
2922.4, 1659.2, 1627.0, 1502.4, 1487.7, 1244.7, 763.2 cmꢀ1
;
ESI-HRMS: calcd for C21H22N3O3, 364.1656; found,
364.1658. [MþH]þ.
(S)-(ꢀ)-N-Nonyl-1,2,3,9-tetrahydropyrrolo[2,1-b]-
(S)-(ꢀ)-1-(1,2,3,9-Tetrahydropyrrolo[2,1-b]quinazolin-1-yl)-
quinazoline-1-carboxamide 9b
formylpiperidine 9f
White solid; yield 23.7%; mp: 113.5–114.68C; ½aꢃ2D5: ꢀ182.6 (c 0.5,
CHCl3); 1H NMR (300 MHz, DMSO-d6) d: 0.83–0.87 (m, 3H),
1.24–1.28 (m, 12H), 1.40–1.42 (m, 2H), 1.75–1.86 (m, 1H),
2.13–2.25 (m, 1H), 2.40–2.61 (m, 2H), 3.06–3.12 (m, 2H),
3.97 (dd, 1H, J ¼ 5.0, 8.1 Hz), 4.41 (d, 1H, J ¼ 13.5 Hz), 4.34
(d, 1H, J ¼ 13.5 Hz), 6.81–6.84 (m, 1H), 6.90–6.91 (m, 2H),
7.05–7.11 (m, 1H), 8.23 (t, 1H, J ¼ 5.3 Hz); 13C NMR (75 MHz,
DMSO-d6) d: 14.0, 22.1, 24.0, 26.3, 28.7, 29.0, 29.6, 31.3, 38.5,
44.9, 64.2, 119.7, 123.4, 126.1, 127.9, 143.2, 162.6, 170.0; IR
(KBr): 3422.1, 3279.2, 2923.2, 1652.7, 1628.2, 1261.5,
White solid; yield 31.2%; mp: 263.4–264.98C; ½aꢃ2D5: ꢀ176.6 (c 0.5,
CHCl3); 1H NMR (300 MHz, DMSO-d6) d: 1.48–1.55 (m, 3H),
1.56–1.61 (m, 3H), 1.98–2.08 (m, 1H), 2.56–2.66 (m, 1H), 3.01–
3.06 (m, 2H), 3.42–3.48 (m, 4H), 4.66 (d, 1H, J ¼ 15.5 Hz), 4.74
(d, 1H, J ¼ 15.5 Hz), 5.13 (dd, 1H, J ¼ 3.7, 9.4 Hz), 7.18–7.26
(m, 3H), 7.31–7.37 (m, 1H); 13C NMR (75 MHz, DMSO-d6) d:
23.6, 23.8, 25.8, 28.8, 42.6, 45.0, 45.6, 64.0, 116.8, 116.9, 126.7,
127.2, 129.1, 131.0, 163.9, 165.6; IR (KBr): 3294.8, 2928.2,
;
ESI-HRMS: calcd for C17H22N3O, 284.1757; found, 284.1761.
[MþH]þ.
1672.6, 1636.9, 1582.7, 1500.9, 1462.3, 1246.8, 774.7 cmꢀ1
760.8 cmꢀ1
; ESI-HRMS: calcd for C21H32N3O, 342.2540;
found, 342.2542. [MþH]þ.
(S)-(ꢀ)-1-(1,2,3,9-Tetrahydropyrrolo[2,1-b]quinazolin-1-yl)-
(S)-(ꢀ)-N-Cyclopropyl-1,2,3,9-tetrahydropyrrolo[2,1-b]-
formylmorpholine 9g
White solid; yield 29.7%; mp: 190.2–190.98C; ½aꢃ2D5: ꢀ176.0 (c 0.5,
CHCl3); 1H NMR (300 MHz, DMSO-d6) d: 2.08–2.10 (m, 1H),
2.54–2.61 (m, 1H), 3.01–3.03 (m, 2H), 3.50–3.54 (m, 4H), 3.60–
3.64 (m, 4H), 4.67–4.74 (m, 2H), 5.10 (dd, 1H, J ¼ 1.6, 4.7 Hz),
7.13–7.14 (m, 1H), 7.21–7.25 (m, 2H), 7.33–7.35 (m, 1H);
quinazoline-1-carboxamide 9c
White solid; yield 38.4%; mp: 276.1–277.28C; ½aꢃ2D5: ꢀ280.4 (c 0.5,
CHCl3); 1H NMR (300 MHz, DMSO-d6) d: 0.42–0.49 (m, 2H),
0.59–0.68 (m, 2H), 1.75–1.86 (m, 1H), 2.10–2.22 (m, 1H), 2.39–
2.60 (m, 2H), 2.64–2.73 (m, 1H), 3.87 (dd, 1H, J ¼ 5.0, 8.2 Hz),
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

8
Y. Tian et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
13C NMR (75 MHz, DMSO-d6) d: 23.4, 28.8, 42.1, 45.1,
63.7, 65.9, 117.1, 126.6, 127.1, 129.1, 131.4, 164.0, 166.3; IR
(KBr): 3293.8, 2922.8, 1670.5, 1642.1, 1583.5, 1108.9,
772.8 cmꢀ1; ESI-HRMS: calcd for C16H20N3O2, 286.1550;
found, 286.1551. [MþH]þ.
[2] S. S. Kang, J. Y. Lee, Y. K. Choi, G. S. Kim, B. H. Han, Bioorg. Med.
Chem. Lett. 2004, 14, 2261–2264.
[3] R. W. Jiang, K. M. Lau, P. M. Hon, T. C. W. Mak, K. S. Woo, K. P.
Fung, Curr. Med. Chem. 2005, 12, 237–246.
[4] C. Y. Cheng, S. Y. Su, N. Y. Tang, T. Y. Ho, S. Y. Chiang, C. L.
Hsieh, Brain Res. 2008, 1209, 136–150.
0
(S)-(ꢀ)-N-Methyl-N -[1-(1,2,3,9-tetrahydropyrrolo[2,1-
[5] G. Zhang, M. L. Guo, R. P. Li, Y. Li, H. M. Zhang, Z. W. Su, Chem.
Nat. Compd. 2009, 45, 398–401.
b]quinazolin-1-yl)formyl]piperazine 9h
White solid; yield 19.7%; mp: 236.5–237.38C; ½aꢃ2D5: ꢀ142.0 (c 0.5,
CHCl3); 1H NMR (300 MHz, DMSO-d6) d: 2.16–2.26 (m, 1H),
2.35 (s, 3H), 2.52–2.77 (m, 5H), 3.02–3.08 (m, 2H), 3.54–3.77
(m, 2H), 4.70 (s, 2H), 5.08 (dd, 1H, J ¼ 3.8, 9.5 Hz), 7.02–7.05
(m, 1H), 7.14–7.17 (m, 1H), 7.24–7.29 (m, 1H), 7.33–7.37 (m,
1H); 13C NMR (75 MHz, DMSO-d6) d: 23.6, 28.9, 41.3, 44.3,
45.1, 45.2, 53.8, 54.2, 63.5, 117.4, 117.9, 126.2, 127.0, 128.9,
132.9, 163.8, 166.4; IR (KBr): 3421.7, 2922.0, 1669.9, 1639.3,
1579.5, 1495.7, 1455.4, 1252.8, 778.8 cmꢀ1; ESI-HRMS: calcd
for C17H23N4O, 299.1866; found, 299.1863. [MþH]þ.
[6] S. O. Cho, J. Y. Ban, J. Y. Kim, H. S. Ju, I. S. Lee, K. S. Song,
K. Bae, Y. H. Seong, Arch. Pharm. Res. 2009, 32, 923–932.
[7] L. Li, B. Zhang, Y. Q. Tao, Y. Wang, H. Wei, J. Zhao, R. X.
Huang, Z. Pei, Brain Res. 2009, 1290, 91–101.
[8] Z. H. Yang, K. Sun, Z. H. Yan, W. H. Suo, G. H. Fu, Y. Lu, Chem.
Biol. Interact. 2010, 183, 165–171.
[9] W. Wang, X. X. Cha, J. Reiner, Y. Gao, H. L. Qiao, J. X. Shen,
J. B. Chang, Eur. J. Med. Chem. 2010, 45, 1941–1946.
[10] Jiangsu College of New Medicine Science. Thesaurus of the
Traditional Chinese Medicines, Shanghai People’s Press,
Shanghai 1977, p. 1526.
Oxygen glucose deprivation protocol and cell
viability assay
[11] H. M. Hua, M. S. Cheng, X. Li, Y. H. Pei, Chem. Pharm. Bull. 2002,
50, 1393–1394.
The neuroprotective effects of these (ꢀ)-linarinic acid derivatives
were evaluated by an in vitro model (OGD) of ischemia reperfu-
sion using SH-SY5Y cells (human neuroblastoma cells). SH-SY5Y
cells were seeded in 96-well plates at a density of 1.0 ꢂ 105 cells/
well. The cells were washed once and maintained in glucose-free
DMEM solution. This medium had been bubbled with 95% N2/5%
CO2 at 3 L/min for 5 min and pre-warmed to 378C. Hypoxia
was induced by placing cells at 378C in a humidified, sealed
chamber that had been flushed with 95% N2/5% CO2 for 10 min.
The (ꢀ)-linarinic acid derivatives were also included in the OGD
solution during the OGD period. At the end of the OGD period
(10 h), cultures were returned to the conditioned medium for
another 12 h. Cell viability was assessed by the metabolism of
MTT. After 8 h of OGD treatment, an MTT solution (0.5 mg/mL)
was added to each well, and cells were maintained in the growth
medium for 4 h. After solubilization with DMSO (100 mL), the
absorbance of the formazan dye was measured using a micro-
plate reader (NovoStar, BMG) at 490 nm. In each experiment,
cultures exposed to OGD were always compared with normoxic
controls, which were supplied with DMEM/F12 and maintained
in standard incubation conditions. All assays were done in
triplicate.
[12] M. S. Cheng, Q. Li, B. Lin, Y. Sha, J. H. Ren, Y. He, Q. H. Wang,
H. M. Hua, R. Kenneth, Tetrahedron: Asymmetry 2006, 17, 179–
183.
[13] M. S. Cheng, C. Ma, Y. Tian, J. H. Liu, Y. Sha, Q. H. Wang,
Faming Zhuanli Shenqing Gongkai Shuomingshu. CN 101580505
A. 2009.
[14] V. Gundula, G. Hans, Chem. Ber. 1989, 122, 1199–1201.
[15] T. Helgaker, K. Ruud, K. L. Bak, P. Jørgensen, J. Olsen, Faraday
Discuss. 1994, 99, 165–180.
ˇ
[16] F. Szemes, S. Marchalın, N. Bar, B. Decroix, J. Heterocycl. Chem.
´
1998, 35, 1371–1375.
[17] T. Hirashima, O. Manabe, Chem. Lett. 1975, 259–260.
[18] J. W. Larsen, M. Freund, K. Y. Kim, M. Sidovar, J. L. Stuart,
Carbon 2000, 38, 655–661.
[19] B. H. Han, D. H. Shin, S. Y. Cho, Tetrahedron Lett. 1985, 26,
6233–6234.
[20] P. Franchetti, S. Marchetti, L. Cappellacci, H. N. Jayaram,
J. A. Yalowitz, B. M. Goldstein, J. L. Barascut, D. Dukhan,
J. L. Imbach, M. Grifantini, J. Med. Chem. 2000, 43, 1264–
1270.
[21] D. H. Lee, Y. J. Lee, K. H. Kwon, J Clin. Biochem. Nutr. 2010, 47,
121–129.
This work was supported by a grant from the National Natural Science
Foundation of China (No. 30973613).
´
´
[22] F. Domoki, B. Kis, T. Gaspar, J. A. Snipes, J. S. Parks, F. Bari, D.
W. Busija, Am. J. Physiol. Cell Physiol. 2009, 296, C97–C105.
The authors have declared no conflict of interest.
[23] G. Singh, M. A. Siddiqui, V. K. Khanna, M. P. Kashyap, S.
Yadav, Toxicol. Mech. Methods 2009, 19, 154–160.
[24] Z. Y. Zuo, Y. Wang, Y. M. Huang, Eur. J. Pharmacol. 2006, 542,
84–91.
References
[1] X. Y. Liu, R. Zhang, W. F. Xu, C. W. Li, Q. Q. Zhao, X. P. Wang,
Bioorg. Med. Chem. Lett. 2003, 13, 2123–2126.
[25] G. Miglio, F. Varsaldi, E. Francioli, A. Battaglia, P. L.
Canonico, G. Lombardi, Eur. J. Pharmacol. 2004, 489, 157–165.
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com