Synthesis and vasodilator effects of rutaecarpine analogues which might be involved transient receptor potential vanilloid subfamily, member 1 (TRPV1)

Article abstract of DOI:10.1016/j.bmc.2009.02.015

Rutaecarpine is the major alkaloid component of Wu-Chu-Yu, a well known Chinese herbal drug. It has been reported that rutaecarpine causes the vasodilator, hypotensive effects by stimulation of CGRP synthesis and release via activation of TRPV1. In present study, 23 rutaecarpine analogues were designed and synthesized. Then, the vasodilator effects of theses compounds were screened by rat aortic ring experiment. The result showed that the 14-N atom of rutaecarpine might be the key site for the activity. The 5-carbonyl might make lower contribution to the effect. And simple substitute in indole-ring or quinazoline-ring would not enhance the vasodilator effect unless in proper position with proper group. One of these compounds, 10-methylrutaecarpine, exhibited similar effect with rutaecarpine. Further functional experiments showed its vasodilator and hypotensive effect were related to the stimulation of CGRP release via activation of TRPV1. The vasodilator effects of these compounds were evaluated and the structure-activity relationship was elucidated for the first time. The results suggested a new direction of valuable TRPV1 agonist as anti-hypertensive drugs.

Full text of DOI:10.1016/j.bmc.2009.02.015

Bioorganic & Medicinal Chemistry 17 (2009) 2351–2359
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and vasodilator effects of rutaecarpine analogues which might be
involved transient receptor potential vanilloid subfamily, member 1 (TRPV1)
b
a
b
a
a
Zhuo Chen a, Gaoyun Hu a, , Dai Li , Jun Chen , Yuanjian Li , Huayong Zhou , Ye Xie
*
a Chemistry Section, Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Central South University, No. 172, Tong Zi Po Road, Changsha 410013, Hunan, China
b Pharmacology Section, Department of Pharmacology, School of Pharmaceutical Sciences, Central South University, Changsha, Hunan, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Rutaecarpine is the major alkaloid component of Wu-Chu-Yu, a well known Chinese herbal drug. It has
been reported that rutaecarpine causes the vasodilator, hypotensive effects by stimulation of CGRP syn-
thesis and release via activation of TRPV1. In present study, 23 rutaecarpine analogues were designed and
synthesized. Then, the vasodilator effects of theses compounds were screened by rat aortic ring experi-
ment. The result showed that the 14-N atom of rutaecarpine might be the key site for the activity. The 5-
carbonyl might make lower contribution to the effect. And simple substitute in indole-ring or quinazo-
line-ring would not enhance the vasodilator effect unless in proper position with proper group. One of
these compounds, 10-methylrutaecarpine, exhibited similar effect with rutaecarpine. Further functional
experiments showed its vasodilator and hypotensive effect were related to the stimulation of CGRP
release via activation of TRPV1. The vasodilator effects of these compounds were evaluated and the struc-
ture–activity relationship was elucidated for the first time. The results suggested a new direction of valu-
able TRPV1 agonist as anti-hypertensive drugs.
Received 30 December 2008
Revised 6 February 2009
Accepted 7 February 2009
Available online 14 February 2009
Keywords:
Rutaecarpine
Transient receptor potential vanilloid
subfamily, member 1
Calcitonin gene-related peptide
Vasodilator
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
According to molecular mechanisms recently described to play a
critical role in pain transmission is the TRPV1 receptor, which pro-
Evodiamine (1) and rutaecarpine (2) are the two major quinazo-
linocarbolin alkaloidal components in Evodiae Fructus, also known
as ‘Wu-Chu-Yu’, which has been prescribed for the treatment of
hypertension in traditional Chinese medicine. Both of them can re-
lax vascular smooth muscle,1 and markedly decrease the blood
pressure.2
vides an opportunity for development of selective agonists or antag-
onists as agents to treat pathological pain.7 But only few
cardiovascular researches focus on the receptor’s agonists or antag-
onists were reported.
TRPV1 is a nonselective cation channel.8 Upon TRPV1 activation,
both sodium and calcium ions enter the cell through the channel
pore, resulting in cell membrane depolarization. TRPV1 activation
also leads to release of substance P (SP) and calcitonin gene related
peptide (CGRP), which enhance peripheral sensitization of tissue.6
CGRP is a very potent vasodilator, approximately 100–1000 times
more potent than other vasodilators such as adenosine, SP or acetyl-
choline,anditpossessespositivechronotropicandinotropiceffects.9
SeverallinesofevidencesuggestthatCGRPplaysanimportantrolein
regulating vascular resistance and regional organ blood flow, all the
more, in the initiation, progression and maintenance of hyperten-
sion.10 Researchers have been pursuing agents that can stimulate
the CGRP synthesis and release via activation of TRPV1.
8
7
O
O
9
6
10
11
5
N
N
N
13a
4
2
13b
N
H
13
N
N
3
12
H
14
H3C
1
2
1
Previous investigations have demonstrated that the rutaecar-
pine produces the vasodilator,3 positive inotropic and chrono-
tropic4 and myocardium-protective effects5 via stimulation of
CGRP synthesis and release by activation of Transient receptor po-
tential vanilloid subfamily, member 1 (TRPV1). Furthermore, the
hypotensive effect of rutaecarpine is mediated by activation of
TRPV1, a new anti-hypertensive target.1
Since modifications of rutaecarpine structure have been investi-
gated for a long time, it has been concluded that rutaecarpine der-
ivates have variety of biological activities such as cytotoxicity,11 or
inhibitory effects on COX-II.12 But few studies concern the TRPV-1-
related vasodilator effects of rutaecarpine derivates. The potential
structure–activity relationship remains unclear.
TRPV1, also known as vanilloid receptor subtype 1(VR1), is now
recognized as a molecular integrator of inflammatory mediators.6
In order to investigate the key region of rutaecarpine which
keeps its vasodilator effect and improves its drug-like property,
* Corresponding author. Tel./fax: +86 731 2650371.
E-mail address: hugaoyun@yahoo.com.cn (G. Hu).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.02.015

2352
Z. Chen et al. / Bioorg. Med. Chem. 17 (2009) 2351–2359
several rutaecarpine analogues were designed and synthesized in
present study. Then, the vasodilator effect of theses compounds
were screened by rat aortic ring experiments. On the other side,
in order to explore whether the mechanism of the vasodilator
and hypotensive effects of rutaecarpine derivates is related to
TRPV1, further aortic ring experiments and anti-hypertensive ef-
fect on rat model were conducted by using 10-methyl-rutaecar-
pine which exerts similar as rutaecarpine. Meanwhile the
similarity of structures of rutaecarpine with that of the traditional
agonists of TRPV1 is very limited, suggesting that this novel class of
enhancers could be a new type of pharmacological activity for
TRPV1 function with vasodilator effect.
(8a–n), the target compounds were obtained by Fischer condensa-
tion in high temperature in PPA.13,14
In order to find out whether the 14-N contributes to the activity,
we designed and synthesized the 14-O and 14-C analogues of ruta-
ecarpine. The 14-O derivates (13a–d) could be obtained by reac-
tions of salicyl chloride and substituted dihydrocarbolines (see
Scheme 2).15 Meanwhile, the 14-C derivates (14a, 14b) could be
synthesized from tryptamine or its substitute refluxed with ninhy-
drin (see Scheme 3).16
Furthermore, rutaecarpine was reduced by LiAlH4 (see Scheme
4). Two products were obtained (15a, 15b). Finally, we synthesized
18 and 20 to discuss the situation of the connection between in-
dole and quinazoline (see Schemes 5 and 6).17
2. Chemistry
3. Result and discussion
We synthesized 23 rutaecarpine analogues systematically,
which included several different skeletons. The synthesis of rutae-
carpine and its simple substitutes (9a–m) are shown in a general
sense in Scheme 1. Most of these compounds have been synthe-
sized before in several ways by many others. In this case, anthra-
nilic acid or its substituted derivates were used, they were
converted to the activated intermediate (4 or 5) by SOCl2, followed
reaction with piperdin-2-one to obtain the derivates of 8,9-dihydo-
6H-pyrido[2,1-b]quinazolin-11(7H)-one (6a–e). After the reaction
with substituted diazobenzene (7a–g) to get hydrazone analogues
Vasodilator responses to the target compounds were evaluated
by the rat aortic ring experiments. The effects caused by the com-
pounds in diverse concentration were measured and the EC50 were
calculated, respectively. The results were shown in Table 1.
Compounds 9a–f are substituted on the indole-ring with group
of alkyl, alkoxyl, acyl or halide. The vasodilator effect of these com-
pounds had no much difference with rutaecarpine, of which com-
pound 9a (10-methylrutaecarpine) was the best candidate.
Unfortunately, in the cases of compounds 9g–m, which are halides
of rutaecarpine on the quinazoline-ring, showed lower activity of
O
O
COOH
CO
ii
N
i
O
S
R2
R2
R2
R2
N
NH2
NH
N
H
4a-e
O
6a-e
3a-e
5a-e
O
O
N
iii
iv
N
R2
R2
N
N
Cl
N
N
N
HN
R2
N
R1
R1
H
2, 9a-m
8a-n
7a-g
Scheme 1. Synthesis of rutaecarpine and its substitutes. Reagents and conditions: (i) SOCl2, benzene, N2, reflux, 2 h; (ii) 2-piperidinone, benzene, N2, rt; (iii) 50% HOAc, ꢀ5 °C,
3 h; (iv) PPA, 160 °C, 2 h.
O
R1
O
R1
NH2
N
N
H
R1
R1
R3
v
vi
vii
N
N
H
N
H
12a-d
R3 O
N
H
N
H
R3
13a-d
11a-d
10a-b
Scheme 2. Synthesis of 14O-rutaecarpine and its derivates. Reagents and conditions: (v) R3 = H:HCO2Et, reflux, 5 h; R3 = Me:Ac2O, Et3 N, DCM, rt; (vi) R3 = H:POCl3, DCM, rt;
R3 = Me:P2O5, xylene, reflux; (vii) salicyl chloride, benzene rt.
R1
O
R1
NH2
N
NH
R1
O
viii
N
N
H
HO
N
H
HO
10a-b
14a-b
Scheme 3. Synthesis of 14C-rutaecarpine and its derivates. Reagents and conditions: (viii) ninhydrin, EtOH/H2O/H2SO4, 80 °C, 12 h.

Z. Chen et al. / Bioorg. Med. Chem. 17 (2009) 2351–2359
2353
O
N
N
N
N
iv
+
N
N
N
H
HN
N
H
H
15b
15a
1
Scheme 4. Reduction of rutaecarpin. Reagents: (iv) LiAlH4, THF.
O
O
N
O
N
O
xi
xii
N
H
x
N
H
O
O
HN
N
F3 C
F3C
N
H
O
N
CF3
18
17
16
Scheme 5. Synthesis of 13b-trifluororutaecarpine. Reagents and conditions: (x) (CF3CO)2O, pyridine, 25 °C, 15 min, 115 °C, 5 min; (xi) tryptamine, 115 °C, 30 min; (xii) HCl/
HOAc, reflux.
bonyl might be less important to the activity. Associating with
other TRPV1 ligands, quinazoline-ring might be the pharmacore
of rutaecarpine.
CO2Me
O
CO2Me
O
O
HN
H2N
N
xiii
xiv
O
N
H
N
H
N
For the studies on the involvement of TRPV1 and endogenous
CGRP in vasodilator responses to the compounds, using the com-
pound 9a as a representative, the rat aortic rings were exposed
to capsaicin, a TRPV1 agonist as the desensitizer, or capsazepin, a
competitive TRPV1 antagonists, or CGRP-(8-37) a selective CGRP
receptor antagonist, for 10 min, respectively. Then, the vasodilator
effect to compound 9a was tested. The results were shown in
Figure 1. The results suggest that the vasodilator effect of the com-
pound 9a is similar to that of rutaecarpine, it might involve the
endogenous CGRP via activation of TRPV1.
In addition, we tested the depressor effect of the compound 9a
and the possible mechanism in the phenol-induced hypertensive
rats. Acute administration of the compound 9a (30, 100 or 300 g/
kg, i.v.) caused a depressor effect in a dose-dependent manner
(Fig. 2), which was blocked by capsaicin (used to deplete the CGRP
from sensory nerves) as shown by an 85% decrease in mean arterial
pressure (MAP) (Fig. 3). These results suggest that the hypotensive
effect of compound 9a, just as rutaecarpine, might be mediated by
stimulation of CGRP release via activation of TRPV1 in the phenol-
induced hypertensive rats.
In conclusion, some compounds synthesized acted similarly as
rutaecarpine, while some had negative responses. The functional
experiments and anti-hypertensive effects on rat model showed
that the vasodilator and hypotensive activity of rutaecarpine ana-
logues might be related to the stimulation of CGRP release via acti-
vation of VR1. But the binding site of the compounds with TRPV1’s
structure is still unknown and the mechanism responsible for the
depressor and vasodilator effects of rutaecarpine and its derivates
are not clearly elucidated. The molecular pharmacological studies
of these compounds are proceeding in order to find the direct evi-
dence of activating TRPV1 and their possible binding sites. The pre-
liminary structure–activity relationships of rutaecarpine analogues
described in present this study might be more descriptive than
mechanistic. But this suggest that rutaecarpine can be a potential
anti-hypertensive leading compound with novel mechanism.
N
H
O
19
20
16
Scheme 6. Synthesis of methyl 3-(1H-indol-3-yl)-2-(4-oxoquinazolin-3(4H)-
yl)propanoate. Reagents and conditions: xiii) tryptophan methyl ester hydrochlo-
ride, MeCN, Et3N, 1 h; (xiv) HC(OEt)3, reflux, 5 h.
vasodilatation. It may be due to their higher lipophilicity which re-
duces their solubility in water.
In order to test our hypothesis that the 14-N contributes to the
activity, compounds of 14-O or 14-C instead of 14-N in rutaecar-
pine skeleton were prepared. The changes sharply decreased the
activity of vasodilatation, especially in the 14-C analogues. It may
be the result that the compounds 14a and 14b are both replaced
the 14-N by enol structure which is rather more different to a
nitrogen atom than an oxygen atom. All these results suggest that
the 14-N is the key structure to keep the vasodilator effect and it is
irreplaceable.
Compounds 15a and 15b are two 5-carbonyl-reduced products
of rutaecarpine. Both of their 5-carbonyls were converted to meth-
ylenes. Furthermore, according to compound 15b, the double bond
between 13b and 14-N was also reduced. It is interesting result that
they kept the vasodilator effect of rutaecarpine to some extent. It
may lead to the conclusion that the absence of 5-carbonyl is accept-
able. While in the other hand, the difference between the two com-
pounds, whether 14-N is saturated or not, have no much effect on
the activity result. These results further confirmed the key role of
14-N in keeping the vasodilator effect of rutaecarpine.
Compounds 18 and 20 were synthesized in order to investigate
the significance of the region around 13a and 13b sites of rutaecar-
pine. If a group such as trifluoro-methyl was introduced to the 13b
site of rutaecarpine (18), the dihedral angle between indole-ring
and qunazoline-ring would be changed which may be similar with
evodiamine (2). The result showed that this change decreased the
activity somewhat. Because the trifluoromethyl is not a very repre-
sentative group, the introductions of other more general groups to
13b site are proceeding. Interestingly, the result of compound 20
was significant. Because of the absence of the bond between
13a–13b sites, the flat conformation of rutaecarpine was broken
and the vasodilator activity was abolished.
4. Experimental
4.1. Chemical synthesis
In summary, simple substitution cannot improve the vasodila-
tor activity of rutaecarpine significantly. The active center of ruta-
ecarpine analogues should be the irreplaceable 14-N and its
neighbour sites. The connection between 13a and 13b sites and
flat-like conformation are vital to the vasodilator effect. The 5-car-
4.1.1. Materials and instrumentation
All materials were obtained and used as received from commer-
cial sources unless specially noted. The purity of all compounds
were examined by high performance liquid chromatography. 1H
NMR and 13C NMR spectra were obtained at 300 MHz or

2354
Z. Chen et al. / Bioorg. Med. Chem. 17 (2009) 2351–2359
Table 1
The vasodilator effects of rutaecarpine and its derivates
EC50 (mol Lꢀ1
)
Emax REM (%)
a
Compound
R1
R2
R3
O
N
R2
N
HN
R1
2, 9a-m
2
H
H
H
H
H
H
H
H
2-Cl
3-Cl
4-Cl
2-Cl
3-Cl
4-Cl
—
—
—
—
—
—
—
—
—
—
—
—
—
1.33 ꢁ 10ꢀ6
1.24 ꢁ 10ꢀ6
2.99 ꢁ 10ꢀ6
3.25 ꢁ 10ꢀ6
5.51 ꢁ 10ꢀ5
2.44 ꢁ 10ꢀ6
2.31 ꢁ 10ꢀ6
2.11 ꢁ 10ꢀ5
3.23 ꢁ 10ꢀ5
1.68 ꢁ 10ꢀ5
2.25 ꢁ 10ꢀ5
2.11 ꢁ 10ꢀ6
1.97 ꢁ 10ꢀ5
79.3 3.5
9a
9b
9c
9d
9e
9f
9g
9h
9i
10-CH3
12-CH3
10-COCH3
10-OCH3
12-Cl
9,10,12-TriCl
H
H
85.1 3.2*
47.0 5.5**
68.2 2.1*
42.6 3.1**
50.1 1.1**
51.0 1.3**
36.6 1.5**
40.6 4.6**
39.6 2.6**
33.9 1.7**
55.2 3.2**
37.1 2.1**
H
9j
9k
9l
10-OCH3
10-OCH3
10-OCH3
O
R1
9m
10-OCH3
3-Br
9.40 ꢁ 10ꢀ6
40.3 2.8**
N
N
H
R3
O
13a-d
13a
13b
H
H
—
—
H
CH3
2.20 ꢁ 10ꢀ4
1.59 ꢁ 10ꢀ3
39.8 4.2**
29.9 5.3**
13c
13d
OCH3
OCH3
—
—
H
6.36 ꢁ 10ꢀ2
13.0 2.2**
CH3
9.34 ꢁ 10ꢀ4
31.6 6.7**
O
R1
N
N
H
HO
14a-b
14a
14b
H
—
—
—
—
1.54 ꢁ 10ꢀ2
5.70 ꢁ 10ꢀ3
14.1 3.1**
13.2 2.0**
OCH3
N
N
N
N
H
N
H
HN
15b
15a
15a
15b
—
—
—
—
—
—
2.51 ꢁ 10ꢀ5
2.01 ꢁ 10ꢀ5
68.9 2.4*
63.6 1.9*
CO2Me
O
N
O
N
N
H
N
H
HN
N
F3C
20
18
18
20
—
—
—
—
—
—
7.86 ꢁ 10ꢀ6
5.46 ꢁ 10ꢀ3
42.5 4.3*
15.5 2.6**
Emax REM, n = 6–8, *P < 0.5 versus rutaecarpine, **P < 0.01 versus rutaecarpine.
a
400 MHz using Me4Si as an internal standard. Mass spectras were
recorded on Qstar LC/MS instrument. Chromatography was per-
formed with commercial silica gel (300–400 mesh).
was evaporated under 25 °C. The residue was dissolved in dry
benzene (300 ml), followed adding piperdin-2-one (9.9 g,
0.1 mol). The mixture was stirred at rt for 14 h under N2.
The mixture was concentrated in vacuum and then diluted
with CHCl3. The solution was basified by 10% Na2CO3, then
washed by H2O, brine and dried (Na2SO4). The derivates of
8,9-dihydo-6H-pyrido [2,1-b]quinazolin-11(7H)-one (6a–e) was
4.1.2. General procedure for preparation of rutaecarpine
substitutes (2, 9a–m)
To a solution of anthranilic acid (or its substituted derivates,
3a–e 0.1 mol) in dry benzene (300 ml) was added SOCl2 (60 g,
0.5 mol) dropwise under N2. After refluxed for 2 h, the mixture
obtained
by
column
chromatography
(EtOAc/petroleum
ether = 1:4) .

Z. Chen et al. / Bioorg. Med. Chem. 17 (2009) 2351–2359
2355
100
80
60
40
20
0
70
MAP
compound 9a
60
50
40
30
20
10
+ capsazepine(10-5 M)
+ capsaicin(10-5 M)
+ CGRP
(10-6 M)
8-37
**
7
6.5
6
5.5
5
0
Compound 9a (300 µg/kg)
+ Capsaicin
Compound 9a (-log M)
Figure 3. Effect of pre-treating with capsaicin on depressor effect of compound 9a
(values represent mean REM, n = 5–9, **P < 0.01 vs compound 9a). MAP: mean
arterial pressure.
Figure 1. Concentration–response curve of vasodilator effect of compound 9a to rat
aortic rings. Capsaicin was exposed for 20 min, and then washed with fresh Krebs’
solution. CGRP-(8-37) or capsazepine was exposed for 10 min before vasodilator
response to compound 9a (value represent mean REM, n = 8, **P < 0.01 vs
compound 9a).
4.1.2.1. Rutaecarpine (2). Starting with anthranilic acid and
using aniline. C18H13N3O. Mp: 255–256 °C; MS (m/z): 287 (M+);
1H NMR (400 MHz, CDCl3): d 3.23 (t, J = 6.8 Hz, 2H), 4.6 (t,
J = 6.8 Hz, 2H), 7.17 (t, J = 7.2 Hz, 1H), 7.31 (t, J = 7.2 Hz, 1H), 7.37
(d, J = 4.2 Hz, 1H), 7.42 (m, 1H), 7.62 (d, J = 4.0 Hz, 1H), 7.65 (d,
J = 7.2 Hz, 1H), 7.70 (m, 1H), 8.31 (dd, J = 0.8, 8.0 Hz, 1H), 9.56
(1H, NH); 13C NMR (100 MHz, CDCl3): d 19.7, 41.3, 112.2, 118.6,
120.1, 120.7, 121.1, 125.6, 125.7, 126.3, 126.5, 127.0, 127.3,
134.4, 138.4, 145.1, 147.3, 161.5.
80
Systolic Pressure
Diastolic Pressure
Mean Arterial Pressure
**
60
**
40
20
0
**
4.1.2.2. 10-Methylrutaecarpine (9a). Starting with anthranilic
acid and using p-toluidine. C19H15N3O. Mp: 295–297 °C; MS (m/
z): 301 (M+); 1H NMR (300 MHz, DMSO-d6): d 2.39 (s, 3H), 3.14
(t, J = 6.9 Hz, 2H), 4.44 (t, J = 6.9 Hz, 2H), 7.10 (dd, J = 1.5, 8.7 Hz,
1H), 7.39 (d, J = 4.8 Hz, 1H), 7.41 (d, J = 2.1 Hz, 1H), 7.46 (t,
J = 6.3 Hz, 1H), 7.67 (d, J = 7.4 Hz, 1H), 7.80 (t, J = 7.8 Hz, 1H), 8.16
(dd, J = 1.2,7.8 Hz, 1H), 11.0 (1H, NH); 13C NMR (75 MHz, DMSO-
d6): d 19.2, 21.3, 41.1, 112.5, 117.6, 119.4, 120.9, 125.4, 126.1,
126.7, 126.8, 126.9, 127.2, 128.8, 134.6, 137.4, 145.6, 147.7, 160.9.
Figure 2. Effect of compound 9a on blood pressure (value represent mean REM,
n = 5–9, *P < 0.05, **P < 0.01 vs vehicle).
4.1.2.3. 12-Methylrutaecarpine (9b). Starting with anthranilic
acid and using o-toluidine. C19H15N3O. Mp: 291–293 °C; MS (m/
z): 301 (M+); 1H NMR (300 MHz, DMSO-d6): d 2.38 (s, 3H), 3.11
(t, J = 6.9 Hz, 2H), 4.40 (t, J = 6.9 Hz, 2H), 7.08 (dd, J = 1.5, 8.7 Hz,
1H), 7.36 (m, 1H), 7.40 (d, J = 8.7 Hz, 1H), 7.44 (m, 1H), 7.67 (d,
J = 7.4 Hz, 1H), 7.82 (t, J = 7.8 Hz, 1H), 8.15 (dd, J = 1.2, 7.8 Hz,
1H), 11.61 (1H, NH); 13C NMR (75 MHz, DMSO-d6): d 19.3, 21.4,
41.2, 112.6, 117.7, 119.5, 121.1, 125.5, 126.2, 126.8, 126.9, 127.3,
128.8, 134.6, 137.4, 145.6, 145.7, 147.8, 161.0.
To a solution of aniline (or its substitutes, 0.01 mol) in 20% HCl
was added NaNO2 aqueous solution (6.9 g in 5 ml) dropwise in
0 °C. The diazobenene chloride (7a–g) was obtained by continued
stirring for further 30 min in this temperature, whereafter the pH
of this reaction solution was adjust to pH 4 by adding sodium ace-
tate. The mixture was diluted with glacial acid (5 ml) and a solu-
tion of 6a–e in 50% acetic acid (10 ml) was added dropwise. The
reaction mixture was stirred for 3 h at 5 °C. The mixture is then al-
lowed to stand overnight in a refrigerator. The precipitated crystals
were collected and washed with water, The obtained 6-aromatic
hydrazono-11-oxo-tetrahydro-11H-pyrido[2,1-b]quinazolines’
derivates (8a–n) could be used without further purification or
recrystallized from n-propanol if necessary.
Compounds 8a–n (0.01 mol) were heated in 20 g of polyphos-
phoric acid at 160 °C for 60 min under N2. After cooling, the mix-
ture was diluted with water and the pH was adjusted to 9 by
ammonium hydroxide solution. The precipitated solid was filtered,
then recrystallized from DMF. The products (2 or 9a–m) were ob-
tained by column chromatography (EtOAc/petroleum ether = 1:5)
and recrystallized from EtOAc. The purification of products were
determinated by HPLC.
4.1.2.4. 12-Acetylrutaecarpine (9c). Starting with anthranilic
acid and using p-acteyl-aniline. C20H15N3O. Mp: 267–268 °C; 1H
NMR (400 MHz, DMSO): d 2.74 (s, 3H), 3.23 (t, J = 6.8 Hz, 2H),
4.58 (t, J = 6.8 Hz, 2H), 7.23 (m, 1H), 7.43 (t, J = 3.6 Hz, 1H), 7.74
(m, 2H), 7.87 (m, 2H), 8.29 (d, J = 8.0 Hz, 1H), 11.00 (1H, NH); 13C
NMR (100 MHz, DMSO): d 19.5, 26.6, 41.0, 117.5, 119.7, 121.1,
121.2, 126.2, 126.4, 126.9, 127.0, 127.1, 128.0, 129.0, 134.4,
136.7, 144.2, 147.5, 161.6, 199.6.
4.1.2.5. 10-Methoxylrutaecarpine (9d). Starting with anthra-
nilic acid and using p-methoxyl aniline. C19H15N3O2. Mp: 252–
254 °C; MS (m/z): 317 (M+); 1H NMR (400 MHz, DMSO-d6): d 3.16
(t, J = 6.8 Hz, 2H), 3.80 (s, 3H), 4.45 (t, J = 6.8 Hz, 2H), 6.92 (dd,

2356
Z. Chen et al. / Bioorg. Med. Chem. 17 (2009) 2351–2359
J = 2.0, 8.8 Hz,1H), 7.12 (d, J = 2.0 Hz, 1H), 7.38 (d, J = 8.8 Hz, 1H),
7.47 (t, J = 7.2 Hz, 1H), 7.67 (d, J = 7.6 Hz, 1H), 7.81 (t, J = 7.6 Hz,
1H), 8.16 (d, J = 7.2 Hz, 1H), 11.74 (s, 1H, NH); 13C NMR
C19H14N3O2Cl. Mp: 283–284 °C; MS (m/z): 351 (M+); 1H NMR
(300 MHz, DMSO-d6): d 3.15 (t, J = 6.9 Hz, 2H), 3.79 (s, 3H), 4.43
(t, J = 6.9 Hz, 2H), 6.93 (dd, J = 2.4, 9.0 Hz, 1H), 7.09 (d, J = 2.4 Hz,
1H), 7.39 (d, J = 9.0 Hz, 1H), 7.64 (d, J = 8.7 Hz, 1H), 7.77 (dd,
J = 2.4, 8.4 Hz, 1H), 8.05 (d, J = 2.4 Hz, 1H); 13C NMR (75 MHz,
DMSO-d6): d 19.3, 41.4, 55.8, 101.1, 113.7, 116.5, 118.1, 122.1,
125.4, 125.8, 127.4, 128.9, 130.2, 134.4, 134.7, 145.9, 146.6,
154.3, 160.0.
(100 MHz, DMSO-d6):
d 19.06, 40.90, 55.37, 100.49, 113.45,
115.99, 117.47, 120.66, 125.11, 125.91, 126.43, 126.63, 127.44,
133.99, 134.45, 145.37, 147.46, 153.82, 160.66.
4.1.2.6. 12-Chlororutaecarpine (9e). Starting with anthranilic
acid and using p-chloro-aniline. C18H12N3OCl. Mp: 215–216 °C;
MS (m/z): 321 (M+); 1H NMR (300 MHz, DMSO-d6): d 3.18 (t,
J = 6.9 Hz, 2H), 4.46 (t, J = 6.9 Hz, 2H), 7.11 (t, J = 7.8 Hz, 1H), 7.34
(d, J = 7.5 Hz, 1H), 7.49 (m, 1H), 7.63 (d, J = 8.1 Hz, 1H), 7.75 (d,
J = 8.4 Hz, 1H), 7.81 (m, 1H), 8.17 (dd, J = 1.2, 7.8 Hz, 1H), 11.83
(1H, NH); 13C NMR (75 MHz, DMSO-d6): d 19.3, 41.02, 117.2,
119.3, 119.4, 121.2, 124.8, 126.6, 126.9, 127.1, 127.3, 128.8,
134.7, 135.8, 134.0, 145.1, 147.6, 160.9.
4.1.2.13. 4-Chloro-10-methoxylrutaecarpine (9l). Starting
with 2-amino-6-chlorobenzoicacid and using 4-methoxylaniline.
C19H14N3O2Cl. Mp: 300–301 °C; MS (m/z): 351 (M+); 1H NMR
(300 MHz, DMSO-d6): d 3.15 (t, J = 6.9 Hz, 2H), 3.80 (s, 3H), 4.38
(t, J = 6.9 Hz, 2H), 6.94 (dd, J = 2.4, 8.7 Hz, 1H), 7.11 (d, J = 2.1 Hz,
1H), 7.40 (dd, J = 0.6, 9 Hz, 1H), 7.44 (dd, J = 1.2, 4.8 Hz, 1H), 7.59
(dd, J = 1.2, 8.1 Hz, 1H), 7.69 (t, J = 8.1, 1H); 13C NMR (75 MHz,
DMSO-d6): d 19.3, 41.2, 55.9, 101.3, 113.7, 116.5, 117.9, 118.3,
125.4, 126.3, 127.2, 128.5, 133.2, 134.3, 134.5, 146.1, 150.3,
154.3, 159.0.
4.1.2.7. 9,10,12-Trichlororutaecarpine (9f). Starting with
anthranilic acid and using 2,4,5-tri-chloroaniline. C18H10N3OCl3.
Mp: 260.9–263.7 °C; MS (m/z): 391 (M+); 1H NMR (300 MHz,
DMSO-d6): d 3.44 (t, J = 6.9 Hz, 2H), 4.45 (t, J = 6.9 Hz, 2H), 7.48
(m, 1H), 7.54 (s, 1H), 7.74 (d, J = 7.5 Hz, 1H), 7.80 (m, 1H), 8.15
(d, J = 7.2 Hz, 1H); 13C NMR (75 MHz, DMSO-d6): d 20.5, 40.8,
117.0, 118.7, 121.3, 123.0, 123.3, 124.7, 125.0, 126.8, 126.9,
127.2, 130.9, 134.7, 135.1, 144.3, 147.3, 160.6.
4.1.2.14. 2-Bromo-10-methoxylrutaecarpine (9m). Starting
with 2-amino-4-bromobenzoicacid and using 4-methoxylaniline.
C19H14N3O2Br. Mp: 288–289 °C; MS (m/z): 395 (Mꢀ1), 397
(M++1); 1H NMR (300 MHz, DMSO-d6): d 3.14 (t, J = 6.9 Hz, 2H),
3.79 (s, 3H), 4.43 (t, J = 6.9 Hz, 2H), 6.93 (dd, J = 2.4, 8.1 Hz, 1H),
7.09 (d, J = 2.1 Hz, 1H), 7.38 (d, J = 9.0 Hz, 1H), 7.57 (d, J = 8.7 Hz,
1H), 7.90 (dd, J = 2.4, 8.7 Hz, 1H), 8.19 (d, J = 2.4 Hz, 1H); 13C
NMR (100 MHz, DMSO-d6): d 19.3, 41.5, 55.8, 101.9, 113.8, 116.6,
118.2, 118.3, 122.5, 125.4, 127.3, 128.9, 129.1, 134.4, 137.4,
146.1, 146.9, 154.3, 159.9.
4.1.2.8. 2-Chlororutaecarpine (9g). Starting with 2-amino-4-
chlorobenzoicacid and using aniline. C18H12N3OCl. Mp: 296–
298 °C; MS (m/z): 321 (M+); 1H NMR (300 MHz, DMSO-d6): d 3.19
(t, J = 6.9 Hz, 2H), 4.44 (t, J = 6.9 Hz, 2H), 7.10 (m, 1H), 7.28 (m,
1H), 7.47 (dd, J = 2.1, 9.0 Hz, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.63 (m.
2H), 8.14 (d, J = 8.4, 1H); 13C NMR (75 MHz, DMSO-d6): d 19.2,
41.2, 112.9, 118.9, 119.8, 120.2, 120.3, 125.2, 125.3, 125.7, 126.3,
126.9, 129.0, 139.1, 139.2, 146.8, 148.9, 160.4.
4.1.3. General procedure for preparation of 14-O rutaecarpines
(Method 1) (13a and 13c)
A
solution of tryptamine (or 5-methoxyl- tryptamine,
0.125 mol) in ethyl formate (150 ml) was refluxed for 6 h. After
evaporating the excess solvent in vacuum, the residue was dis-
solved in CHCl3, followed washed with saturated NaHCO3 solution,
2% HCl, dried by Na2SO4. The N-formyltryptamine derivates (11a or
11c) can be used in the next step by evaporating the excess solvent
without further purification.
To a solution of 11a (or 11c, 0.12 mol) in dry CH2Cl2 (60 ml) was
added POCl3 (10 ml, 0.12 mol) dropwise in ice-bath. The mixture
was stirred for 2 h. After evaporating the excess solvent, the resi-
due was dissolved in water, washed with ether. The pH of aqueous
layer was adjusted by ammonium hydroxide to pH 10. Then the
solution was extracted with CH2Cl2, and washed with saline, dried
by Na2SO4. The crude 4,9-dihydro-carboline derivates (12a or 12c)
were obtained by evaporating solvent in vacuum followed recrys-
tallized from ethyl acetate.
To a solution of 12a (or 12c, 0.04 mol) and salicyl chloride (pre-
pared from salicyl acid in sulphoxide chloride, 0.04 mol) in dry
benzene (200 ml) was stirred in rt for 2 h. The mixture was evap-
orated under reduced pressure, which followed residue was di-
luted by CH2Cl2. After washed with 10% NaOH, water, saline, the
solution was dried by Na2SO4. The products (13a or 13c) were ob-
tained by column chromate- graphy (CHCl3) followed recrystal-
lized from ethyl acetate.
4.1.2.9. 3-Chlororutaecarpine (9h). Starting with 2-amino-5-
chlorobenzoicacid and using aniline. C18H12N3OCl. Mp: 320–
322 °C; MS (m/z): 321 (M+); 1H NMR (300 MHz, DMSO-d6): d 3.21
(t, J = 6.9 Hz, 2H), 4.45 (t, J = 6.9 Hz, 2H), 7.10 (m, 1H), 7.28 (m,
1H), 7.49 (m, 1H), 7.64 (m, 1H), 7.69 (s, 1H), 7.81 (dd,
J = 1.2,9 Hz, 1H), 8.07 (d, J = 2.7 Hz, 1H); 13C NMR (75 MHz,
DMSO-d6): d 19.4, 41.6, 113.1, 118.8, 120.4, 120.6, 122.4, 125.3,
125.5, 126.1, 127.2, 129.2, 130.6, 135.1, 139.2, 146.2, 146.7, 160.3.
4.1.2.10. 4-Chlororutaecarpine (9i). Starting with 2-amino-6-
chlorobenzoicacid and using aniline. C18H12N3OCl. Mp: 313–
314 °C; MS (m/z): 321 (M+); 1H NMR (300 MHz, DMSO-d6): d 3.18
(t, J = 6.9 Hz, 2H), 4.39 (t, J = 6.9 Hz, 2H), 7.10 (m, 1H), 7.28 (m, 1H),
7.46 (dd, J = 1.2, 7.2 Hz, 1H), 7.50 (m, 1H), 7.61 (dd, J = 1.2, 8.4 Hz,
1H), 7.65 (m, 1H), 7.71 (t, J = 8.1 Hz, 1H); 13C NMR (75 MHz,
DMSO-d6): d 19.3, 41.3, 113.0, 118.0,118.9, 120.2, 120.4, 125.2,
155.3, 126.5, 126.9, 128.7, 133.3, 134.5, 139.1, 146.2, 150.3, 159.0.
4.1.2.11. 2-Chloro-10-methoxylrutaecarpine (9j). Starting with
2-amino-4-chlorobenzoicacid
and
using
4-methoxylaniline.
C19H14N3O2Cl. Mp: 333–334 °C; MS (m/z): 351 (M+); 1H NMR
(300 MHz, DMSO-d6): d 3.16 (t, J = 6.9 Hz, 2H), 3.80 (s, 3H), 4.43 (t,
J = 6.9 Hz, 2H), 6.94 (dd, J = 2.4, 8.7 Hz, 1H), 7.10 (d, J = 2.1 Hz, 1H),
7.40 (dd, J = 0.6, 8.7 Hz, 1H), 7.45 (dd, J = 2.1, 8.1 Hz, 1H), 7.60 (d,
J = 0.6, 2.1 Hz, 1H), 8.13 (dd, J = 0.6, 8.7, 1H); 13C NMR (75 MHz,
DMSO-d6): d 19.3, 41.3, 55.8, 101.1, 113.8, 116.6, 118.4, 119.8,
125.3, 125.6, 126.2, 127.3, 128.9, 134.5, 139.2, 146.9, 148.9, 154.3,
160.4.
4.1.3.1. 6,7,8,13b-Tetrahydro-5-oxo-5H-b-carbolin[1,2-b] qui-
nazoline-8(6H)-one (13a). C18H14N2O2. Mp: 225–227 °C; MS
(m/z): 291.5 (M++1; 1H NMR (400 MHz, CDCl3): d 3.02 (m, 2H),
3.19 (m, 1H), 4.94 (dd, J = 4.4, 2.8 Hz, 1H), 6.47 (s, 1H), 7.03 (d,
J = 8 Hz, 1H), 7.15 (d, J = 7.2, 1H), 7.19 (d, J = 7.2, 1H), 7.29 (t,
J = 8 Hz, 1H), 7.43 (d, J = 8 Hz, 1H), 7.49 (t, J = 8 Hz, 1H), 7.61 (d,
J = 8 Hz, 1H), 8.06 (d, J = 8 Hz, 1H), 8.34 (s, 1H); 13C NMR
(100 MHz, CDCl3): d 20.2, 39.1, 81.2, 111.6, 113.7, 116.3, 118.6,
4.1.2.12. 3-Chloro-10-methoxylrutaecarpine (9k). Starting
with 2-amino-5-chlorobenzoicacid and using 4-methoxylaniline.

Z. Chen et al. / Bioorg. Med. Chem. 17 (2009) 2351–2359
2357
119.4, 120.3, 123., 123.7, 125.9, 127.0, 128.8, 134.2, 137.2, 156.7,
163.0.
1 M H2SO4 (10 ml) was added a aqueous solution of ninhydrin
(1.78 g, 0.010 mol) in 50% ethanol dropwise under N2. The mixture
was heated to 80 °C with stirring under N2 for 18 h. The mixture
was filtered and washed with ethanol. The product (14a or 14b)
was obtained by column chromatography (CHCl3) followed recrys-
tallized from methanol.
4.1.3.2. 6,7,8,13b-Tetrahydro-10-methoxyl-5-oxo-5H-b-carbo-
lin[1,2-b] quinazoline-8(6H)-one (13c). C19H16N2O3. Mp: 221–
223 °C; MS (m/z): 321.4 (M++1); 1H NMR (400 MHz, CDCl3): d
2.96 (m, 2H), 3.10 (m, 1H), 3.88 (s, 3H), 4.93 (dd, J = 5.2, 13.2 Hz,
1H), 6.45 (s, 1H), 6.95 (dd, J = 2.4, 8.8 Hz, 1H), 7.02 (s, 1H), 7.03
(d, J = 8 Hz, 1H), 7.17 (t, J = 7.2 Hz, 1H), 7.32 (d, J = 8.8 Hz, 1H),
7.49 (m, 1H), 8.06 (d, J = 7.2 Hz, 1H), 8.21 (s,1H); 13C NMR
(100 MHz, CDCl3): d 20.2, 39.1, 55.9, 81.2, 100.9, 112.4, 113.5,
114.1, 116.3, 118.6, 123.0, 126.3, 127.8, 128.8, 132.3, 134.2,
154.6, 156.7, 163.0.
4.1.5.1. 14-Hydroxy-(3,14,15,16,17,18,19,20)-octade-hydro-21-
yohimbanone (14a). C19H14N2O2. Mp: 238–240 °C; MS (m/z):
302 (M+); 1H NMR (400 MHz, DMSO-d6): d 3.05 (t, J = 6.4 Hz, 2H),
4.41 (t, J = 6.4 Hz, 2H), 7.04 (t, J = 7.6 Hz, 1H), 7.15 (t, J = 7.6 Hz,
1H), 7.53 (m, 2H), 7.62 (d, J = 8.4 Hz, 1H), 7.79 (t, J = 7.2 Hz, 1H),
7.98 (d, J = 8.0 Hz, 1H), 8.27 (d, J = 7.6 Hz, 1H), 9.25 (s, 1H), 11.09
(s, 1H); 13C NMR(100 MHz, DMSO-d6): d 19.8, 41.07, 110.9, 112.7,
118.5, 119.4, 120.1, 121.8, 122.7, 124.8, 125.3, 126.9, 127.6,
128.0, 130.5, 132.4, 133.5, 137.6, 159.6.
4.1.4. General procedure for preparation of 14-O rutaecarpines
(Method 2) (13b and 13d)
To
a solution of tryptamine (or 5-methoxyl-tryptamine,
0.050 mol) in CH2Cl2 (135 ml) was added a solution of acetic anhy-
dride (9 ml) and triethylamine (16 ml) in CH2Cl2 (30 ml) dropwise.
The mixture was stirred at rt for 1 h. The mixture was then washed
with saturated NaHCO3 aqueous solution, saline and dried by
Na2SO4. After evaporated the solvent, the crude N-acetic-trypt-
amine derivates (11b or 11d) were obtained and it can be recrys-
tallized in EtOAc–petroleum ether if necessary.
To a solution of 11b (or 11d, 0.040 mol) in xylene (150 ml) was
added P2O5 (24 g, 0.169 mol) at rt with stirring. The mixture was
heated to reflux for another 1 h. After cooling, the mixture was di-
luted with water, washed with ether. The pH of aqueous layer was
adjusted by 25% NaOH solution to pH 10. The solution was ex-
tracted with CH2Cl2. The organic layer was washed with saline,
dried by Na2SO4. The crude 1-methyl-4,9-dihydro-carboline deri-
vates (12b or 12d) were obtained by evaporating solvent in vac-
uum followed recrystallized from ethyl acetate.
A solution of 12b (or 12d, 0.05 mol) and salicyl chloride (7.8 g,
0.05 mol) in dry CH2Cl2 (200 ml) was stirred at rt for 2 h. The mix-
ture was evaporated under reduced pressure, and the residue was
diluted by CH2Cl2. After washed with 10% NaOH, water, saline, the
solution was dried by Na2SO4. The products (13b or 13d) were ob-
tained by column chromatography (CHCl3) followed recrystallized
from ethyl acetate.
4.1.5.2. 10-Methoxyl-14-hydroxy-(3,14,15,16,17,18,19,20)-octa-
de-hydro-21-yohimbanone (14b). C20H16N2O3. Mp: 237–
239 °C; MS (m/z): 332 (M+); 1H NMR (400 MHz, DMSO-d6): d 3.05
(t, J = 6.4 Hz, 2H), 3.78 (s, 3H), 4.41 (t, J = 6.4 Hz, 2H), 6.81 (dd,
J = 2.0, 6.4 Hz, 1H), 7.04 (s, 1H), 7.51 (m, 2H), 7.78 (t, J = 7.2 Hz,
1H), 7.97 (d, J = 8.0 Hz, 1H), 8.27 (d, J = 7.6 Hz, 1H), 9.21 (s, 1H),
10.95 (s, 1H); 13C NMR (100 MHz, DMSO-d6): d 19.9, 41.1, 55.6,
99.7, 110.7, 113.3, 113.5, 120.2, 121.7, 124.7, 125. 3, 126.8, 128.0,
130.3, 132.3, 132.8, 133.6, 153.8, 159.6.
4.1.6. Preparation of rutaecarpene (15a) and rutaecarpane
(15b)
To a solution of rutaecarpine (prepared followed the general
procedure described above, 2.87 g, 0.01 mol) in dry tetrahydrofu-
ran (100 ml) was added lithium aluminium hydride (1.52 g,
0.04 mol) in portion. The mixture was stirred at rt for about 6 h.
The mixture was quenched with 0.1 N HCl. After evaporating the
great mass of solvent in vacuum, the residue was extracted with
CH2Cl2, washed with saline and dried by Na2SO4. The two products
were obtained by column chromatography (EtOAc/petroleum
ether = 1:4) followed recrystallized from ethyl acetate.
4.1.6.1. Rutaecarpene (15a). C18H15N3. Mp: 165–168 °C; MS
(m/z): 272 (M+–1); 1H NMR (400 MHz, CDCl3):
d 3.15 (t,
4.1.4.1. 6,7,8,13b-Tetrahydro-13b-methyl-5-oxo-5H-b-carbolin-
[1,2-b] quinazoline-8(6H)-one (13b). C19H16N2O2. Mp:161–
163 °C; MS (m/z): 305.4 (M++1); 1H NMR (400 MHz, CDCl3): d
1.90 (s, 3H), 2.91 (m, 1H), 2.99 (m, 1H), 3.19 (m, 1H), 5.18 (m,
1H), 6.97 (d, J = 7.6 Hz, 1H), 7.12 (m, 1H), 7.17 (m, 1H), 7.27 (m,
1H), 7.43 (d, J = 8 Hz, 1H), 7.46 (m, 1H), 7.57 (d, J = 8 Hz, 1H),8.04
(dd, J = 1.6,6.4 Hz, 1H), 8.23 (1H); 13C NMR (100 MHz, CDCl3): d
20.6, 23.5, 38.1, 87.8, 111.5, 111,5, 116.8, 117.6, 119.3, 120.2,
122.5, 123.4, 126.1, 128.5, 131.9, 134.3, 136.8, 154.5, 160.6.
J = 6.8 Hz, 2H), 4.57 (t, J = 6.8 Hz, 2H), 4.59 (s, 2H), 6.98 (d,
J = 6.8 Hz, 1H), 7.04 (t, J = 7.6 Hz, 1H), 7.11 (t, J = 7.6 Hz, 1H), 7.18
(t, J = 7.6 Hz, 1H), 7.23 (m, 2H), 7.34 (d, J = 8.4, 1H), 7.54 (d,
J = 8.4 Hz, 1H); 13C NMR (100 MHz, CDCl3): d 20.2, 49.9, 50.5,
112.3, 116.4, 119.5, 120.0, 123.1, 124.8 125.4, 125.5, 128.7, 137.8,
149.4.
4.1.6.2. Rutaecarpane (15b). C18H17N3. Mp: 238–240 °C; MS
(m/z): 274 (M+–1); 1H NMR (400 MHz, CDCl3): d 2.81 (m, 2H),
2.92 (m, 1H), 3.22 (m, 2H), 3.93 (m, 2H), 4.14 (m, 1H), 4.68 (s,
1H), 6.78 (d, J = 3.6 Hz, 1H), 6.89 (t, J = 7.2, 1H), 7.02 (d, J = 7.2 Hz,
1H), 7.10 (t, J = 7.2 Hz, 1H), 7.17 (t, J = 8.0 Hz, 1H), 7.23 (d,
J = 8 Hz, 2H), 7.49 (d, J = 8 Hz, 1H), 8.59 (s, 1H); 13C NMR
(100 MHz, CDCl3): d 21.45, 48.8, 55.5, 68.2, 109.8, 110.2, 111.2,
118.7, 119.2, 119.6, 121.0, 122.0, 123.6, 126.7, 127.3, 127.4,
131.6, 136.3, 141.4.
4.1.4.2. 6,7,8,13b-Tetrahydro-10-methoxyl-13b-methyl-5-oxo-
5H-b-carbolin[1,2-b] quinazoline-8(6H)-one (13d). C20H18
N2O3. Mp: 223–225 °C; MS (m/z): 335.4 (M++1); 1H NMR
(400 MHz, CDCl3): d 1.89 (s, 3H), 2.86 (m, 1H), 2.97 (m, 1H), 3.17
(m, 1H), 3.87 (s, 3H), 5.18 (dd, J = 4.4,8.8 Hz, 1H), 6.92 (m, 2H),
6.99 (m, 1H), 7.12 (t, J = 7.6 Hz, 1H), 7.31 (d, J = 8.8 Hz, 1H), 7.46
(m, 1H), 8.04 (dd, J = 1.2,6.8 Hz, 1H), 8.16 (1H); 13C NMR
(100 MHz, CDCl3): d 20.6, 23.5, 38.1, 55.9, 87.9, 101.0, 111.2,
112.3, 113.7, 116.8, 117.6, 122.4, 126.5, 128.5, 131.8, 132.6,
134.3, 154.5, 154.5, 160.6.
4.1.7. Preparation of 13b-trifluoromethyl-13b,14-
dihydrorutaecarpine (18)
To a solution of isatoic anhydride (4.0 g, 0.025 mol) in dry pyr-
idine (80 ml) was added trifluoroacetic anhydride (3.7 g,
0.025 mol) during 15 min with well-stirred at rt. Then the mixture
was refluxed for 15 min, whereupon tryptamine (4.0 g, 0.025 mol)
was added and refluxed for continued 2 h. After cooling, the mix-
ture was poured into water, and collect the solid formed. The inter-
4.1.5. General procedure for preparation of 14-C rutaecarpine
(14a and 14b)
To
a solution of tryptamine (or 5-methoxyl-tryptamine,
0.010 mol) in a mixture of ethanol (25 ml), water (25 ml) and

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Z. Chen et al. / Bioorg. Med. Chem. 17 (2009) 2351–2359
mediate (17), 3-[2-(3-indolyl)-ethyl]-2-(trifluoromethy1)-4(3H)-
quinazolinone was recrystallized from methanol without further
treatment.
aorta were rapidly isolated cleaned of fat and connective tissues,
and then cut into rings of 4 mm length. The rings were sus-
pended horizontally between two stainless steel wires and
mounted in a 5-ml organ chamber filled with warmed (37 °C)
and oxygenated (95% O2 and 5% CO2) Krebs’ solution, which
had the following composition (mM): NaCl, 119.0; NaHCO3,
25.0; KCl, 4.7; KH2PO4, 1.2; MgSO4, 1.2; CaCl2, 2.5; and glucose,
11.0. Each of the rings’ end was connected to a force transducer.
The aortic rings were stretched with 2 g resting force, equili-
brated for 60 min, and then precontracted with KCl (60 mM).
After a maximal response to KCl was observed, the rings were
washed with Krebs’ solution and equilibrated for another
Compound 17 was refluxed in a mixture of acetic acid (30 ml)
and HCl (5 ml) for 2 h. The mixture was diluted with water
(50 ml) and collected the solid formed. The product was obtained
by recrystallized from ethyl acetate. C19H14F3N3O. Mp: 300–
301 °C; MS (m/z): 357 (M+); 1H NMR (300 MHz, DMSO-d6): d 2.80
(m, 1H), 2.96 (m, 1H), 3.30 (m, 1H), 5.15 (dd, J = 6.8, 18 Hz, 1H),
6.87 (m, 2H), 7.10 (m, 1H), 7.24 (m, 1H), 7.39 (m, 1H), 7.54 (m,
1H), 7.59 (m, 1H), 7.79 (dd, J = 2, 10.8 Hz, 1H), 10.84 (1H, NH);
13C NMR (100 MHz, DMSO-d6):
d 20.1, 37.5, 112.5(split),
114.8(split), 115.3, 119.3(split), 119.9, 123.5, 123.9, 125.2,
125.3(split), 125.4(split), 127.9, 128.1, 131.9, 134.2, 137.2(split),
144.2(split), 161.8.
30 min. The rings were contracted with phenylephrine (2 lM).
After the contraction had stabilized, a cumulative concentra-
tion–response curve to rutaecarpine and other candidates
(10ꢀ7–10ꢀ5 M) was observed.
4.1.8. Preparation of methyl 3-[2-(3-Indolyl)ethyl]-2-(4-
quinazolinone-3(4H)-yl)-propanoate (20)
For the study on the involvement of TRPV1 and endogenous
CGRP in vasodilator effect of compound 9a, the preparations were
divided into four groups. After the contraction caused by phenyl-
A mixture of tryptophan methyl ester hydrochloride (2.54 g,
0.01 mol), isatoic anhydride (1.63 g, 0.01 mol) and triethylamine
(1.4 ml, 0.01 mol) was refluxed in acetonitrile (20 ml) for 2 h. The
crystals formed were collected after cooling. The product (19) N-
(20-aminobenzoyl)tryptophan methyl ester could be used to the
next step without further purification.
ephrine (2
lM) had stabilized, three groups were exposed to cap-
saicin (10ꢀ5 M), a TRPV1 agonist as the desensitizer, for 20 min;
capsazepin (10ꢀ5 M),
a competitive TRPV1 antagonists, for
10 min; CGRP-(8-37) (10ꢀ6 M), a selective CGRP receptor antago-
nist, for 10 min, respectively. Then, the cumulative vasodilator re-
sponse to compound 9a was recorded.
A
mixture of N-(20-aminobenzoyl)tryptophan methyl ester
(1.69 g, 0.005 mmol) and triethyl orthoformate (20 ml) was re-
fluxed for 10 h. After evaporated excess reagent, the product was
obtained by column chromatography (CH2Cl2). C20H17N3O3. Mp:
139–140 °C; MS (m/z): 348 (M++1); 1H NMR (400 MHz, CDCl3): d
3.63 (m, 1H), 3.76 (m, 1H), 3.80 (s, 3H), 5.37 (dd, J = 4.8,9.6 Hz,
1H), 6.83 (d, J = 2.4 Hz, 1H), 7.07 (m, 1H), 7.15 (m, 1H), 7.29 (d,
J = 8.4 Hz, 1H), 7.49 (m, 1H), 7.53 (d, J = 7.6 Hz, 1H), 7.62 (m, 2H),
7.73 (m, 1H), 8.18 (1H, NH), 8.28 (dd, J = 1.2, 8.4 Hz, 1H); 13C
NMR (100 MHz, CDCl3): d 26.0, 52.9, 59.1, 109.7, 111.5, 118.0,
119.9, 121.7, 122.5, 123.1, 126.6, 126.8, 127.3, 134.5, 136.2,
145.6, 147.4, 160.6, 169.5.
4.2.3. Hypotensive effect of compound 9a
Fifteen days after phenol treatment, rats were divided randomly
into six groups and subjected to the following experimental proto-
cols: (1) hypertension group; (2) vehicle group, rats were intrave-
nously treated with 0.1 ml vehicle (a mixture of dimethyl sulfoxide
and ethanol, 2:8, v/v); (3) compound 9a (30
pound 9a (100 g/kg) group; (5) compound 9a (300
rats of these three groups received a intravenous injection of com-
pound 9a at the dose of 30, 100 or 300 g/kg, respectively; (6) cap-
saicin plus compound 9a (300 g/kg) group, which was given
l
g/kg) group; (4) com-
l
lg/kg) group,
l
l
capsaicin (50 mg/kg, s.c.) 4 days before compound 9a administra-
4.2. Biological methods
tion under anesthesia with sodium pentobarbital.
After anesthesia with sodium pentobarbital, the right jugular
vein and carotid artery of rat were cannulated for administration
of drugs or for monitoring of blood pressure with a pressure trans-
ducer coupled to computerized recorder (BL-NewCentury 410,
Chengdu, China).
4.2.1. Animals and reagents
All animals received humane care in compliance with Guide for
the Care and Use of Laboratory Animals published by the National
Institutes of Health. For the development of phenol-induced hyper-
tensive rats, male Sprague-Dawley rats weighing 250–300 g were
used in the present study. After anesthesia with sodium pentobar-
bital (60 mg/kg, i.p.), the left kidney was exposed and injected with
4.3. Statistical analysis
50
l
l of 10% phenol in the parenchyma of the lower pole of the kid-
Data are expressed as means SEM (n = 8–12). All values were
analyzed using ANOVA and multiple comparison test (the Stu-
dent–Newman–Keuls t-test), using SPSS 10.0 (SPSS Inc., Chicago,
Illinois, USA). The acceptable value of significance was P < 0.05.
ney. Control rats received 50
ll of 0.9% NaCl in the lower pole of
the kidney as a normaotensive control for the hypertensive rats.
The rats were kept in cages in a room on a 12-h light: 12-h dark
cycle and with free access to tap water and standard rat chow.
Capsaicin, capsazepine, CGRP-(8-37) and dimethyl sulfoxide
were purchased from Sigma chemical company (St. Louis, USA). In
the study of vasodilator effects, the candidate compounds, capsaicin,
and capsazepine were initially dissolved in dimethyl sulfoxide, and
further diluted in Kreb’s solution to the proper final concentration.
The final concentration of dimethyl sulfoxide did not exceed 0.1%,
which had no effect on vascular tension. CGRP-(8-37) was dissolved
in distilled water. In the case of the hypotensive effect of the com-
pound 9a, compound 9a wad dissolved in a mixture of dimethyl sulf-
oxide and ethanol (2:8, v/v), and capsaicin was dissolved in a vehicle
containing 10% Tween 80, 10% ethanol and 80% saline.
Acknowledgement
This research was supported by the Grant (No. 30572251) from
the National Natural Science Foundation, China.
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