2-Substituted (S)-2-(3,3-dimethyl-1-oxo-10,10a-dihydroimidazo[1,5-b] isoquinolin-2(1H,3H,5H)-yl)acetic acids: Conformational prediction, synthesis, anti-thrombotic and vasodilative evaluation

Article abstract of DOI:10.1016/j.bmc.2010.12.005

(S)-1,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid (TIC) can inhibit thrombosis by inhibiting platelet aggregation. The investigation of amino acids modified TIC reveals that a stretching conformation is critical for high anti-thrombotic activity. The conformational modeling shows that introducing a ring into amino acid modified TIC results in a desirable stretching conformation. According to this hypothesis, we synthesized seventeen novel 2-substituted (S)-2-(3,3-dimethyl-1-oxo-10,10a-dihydroimidazo[1,5-b]isoquinolin- 2(1H,3H,5H)-yl)acetic acids (5a-q). In the in vitro anti-platelet aggregation assay, for ADP-induced platelet aggregation the IC₅₀ values of 5a-q are 1.8-3.4-folds lower than that of TIC. In the in vivo anti-thrombotic assay, the effective dose of 5a-q was 167-folds lower than that of TIC. The vessel strip assay showed that 5a-q had mild vasorelaxation activity.

Full text of DOI:10.1016/j.bmc.2010.12.005

Bioorganic & Medicinal Chemistry 19 (2011) 871–882
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
2-Substituted (S)-2-(3,3-dimethyl-1-oxo-10,10a-dihydroimidazo[1,5-b]
isoquinolin-2(1H,3H,5H)-yl)acetic acids: Conformational prediction, synthesis,
anti-thrombotic and vasodilative evaluation
⇑
⇑
Hong Wang, Le Peng, Ming Zhao , Jiawang Liu, Xiaoyi Zhang, Yuji Wang, Jianhui Wu, Li Li, Shiqi Peng
College of Pharmaceutical Sciences, Capital Medical University, Beijing 100069, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
(S)-1,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid (TIC) can inhibit thrombosis by inhibiting platelet
aggregation. The investigation of amino acids modified TIC reveals that a stretching conformation is crit-
ical for high anti-thrombotic activity. The conformational modeling shows that introducing a ring into
amino acid modified TIC results in a desirable stretching conformation. According to this hypothesis,
we synthesized seventeen novel 2-substituted (S)-2-(3,3-dimethyl-1-oxo-10,10a-dihydroimidazo[1,5-
b]isoquinolin-2(1H,3H,5H)-yl)acetic acids (5a–q). In the in vitro anti-platelet aggregation assay, for
ADP-induced platelet aggregation the IC50 values of 5a–q are 1.8–3.4-folds lower than that of TIC. In
the in vivo anti-thrombotic assay, the effective dose of 5a–q was 167-folds lower than that of TIC. The
vessel strip assay showed that 5a–q had mild vasorelaxation activity.
Received 29 September 2010
Revised 1 December 2010
Accepted 2 December 2010
Available online 5 December 2010
Keywords:
Tetrahydroisoquinoline
Anti-thrombotic activity
Vasorelaxation
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
In our previous work, (S)-1,2,3,4-tetrahydroisoquinoline-3-car-
boxylic acid (TIC) was used as a lead and C- and/or N-terminal
In the most frequent cardiovascular events such as deep vein
thrombosis, myocardial infarction, pulmonary embolism, and
stroke, the intravascular thrombosis is one of the most prominent
causes of morbidity and mortality.1 The injury of blood vessel has
been correlated with the arterial thrombosis.2 Upon the damage
of the vascular endothelium of the injured blood vessel, the platelets
adhere to the exposed extracellular matrix.3 Platelet adhesion is the
primary event that usually associates with the uncontrolled platelet
activation and culminates in the intravascular thrombosis.4 The
suppression of the platelet adhesion and activation, particularly
through targeting such secondary regulatory mechanism is effec-
tive in the prevention of the thrombosis.5 In the management of
the cardiovascular event, anti-platelet therapy has an established
role.6 Therefore, a continuous effort has been made to discover
new leads capable of inhibiting platelet activation and aggregation.
Tetrahydroisoquinoline is one of the reported platelet-aggregation
inhibitors. Besides anti-platelet aggregation action, tetrahydro-
isoquinolines have a number of additional bioactivities.7–12 The
anti-platelet aggregation mechanism of tetrahydroisoquinolines
amino acid modifications were performed.21–23 In this continuing
work on the modification of TIC with a substituted-dihydroimidaz-
ole functional group, seventeen novel 2-substituted (S)-2-(3,3-di-
methyl-1-oxo-10,10a-dihydroimidazo[1,5-b]isoquinolin-2(1H,3H,
5H)-yl)acetic acids (5a–q) were synthesized, and evaluated in an
in vitro anti-platelet aggregation assay, and an in vivo anti-
thrombotic model.
2. Results and discussion
2.1. Stretching conformation of 5a–q
The SAR analysis revealed that the in vivo anti-thrombotic activ-
ity of amino acids modified TIC derivatives depended on the stretch-
ing level of their conformation.21 As seen in the stereoview derived
from the QSAR module of Cerius,2 the in vivo anti-thrombotic
activities of N-(
1,2,3,4-tetrahydroisoquinoline-3-carboxylamino acids (B) and
N-( -amino-acyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxyl amino
L-aminoacyl)-1,2,3,4-tetrahydroisoquinolines (A),
includes the b-adrenergic/a
2-adrenergic receptor system,13 and
L
the thromboxane A2/prostaglandin H2 receptor system.14–16 Based
on these receptor systems the design of tetrahydroisoquinolines
attracts many interests.17–20
acids (C) are enhanced with the increase of the stretching level of the
conformation (Fig. 1). This means that to get derivatives with
stretching conformation is a key step to TIC modification. In the
stretching conformation screening, it was found that (S)-2-(3,3-di-
methyl-1-oxo-10,10a-dihydroimidazo[1,5-b]isoquinolin-2(1H,3H,
5H)-yl)acetic acids (5a–q) derived from the cyclization of 3S-N-
⇑
Corresponding authors. Tel./fax: +86 10 8280 2482 (M.Z.); tel./fax: +86 10 8391
1528 (S.P.).
(L-aminoacyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylamino acids
E-mail addresses: mingzhao@mail.bjmu.edu.cn (M. Zhao), sqpeng@mail.bjmu.
(B) had desirable stretching conformation (Fig. 1, D). Consequently
edu.cn (S. Peng).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.12.005

872
H. Wang et al. / Bioorg. Med. Chem. 19 (2011) 871–882
Increase of the stretching level of the conformation
O
O
R
O
R
OH
O
N
H
CO2H
N
CO2H
H
N
R
N
R
O
NH
NH2
NH2
Increase of the in vivo anti-thrombotic efficacy
O
R
N
CO2H
N
Figure 1. The correlation of in vivo anti-thrombotic activity with the stretching level of the conformation and the desirable stretching conformation of 2-substituted (S)-2-
(3,3-dimethyl-1-oxo-10,10a-dihydroimidazo[1,5-b]isoquinolin-2(1H,3H,5H)-yl)-acetic acids (5a–q).
the stretching conformation of 5a–q was correlated with the
2.3. In vitro anti-platelet aggregation of 5a–q
desirable anti-thrombotic activity.
In the in vitro assays, the inhibition of 5a–q (final concentra-
2.2. Synthesis of 5a–q
tions ranging from 10
factor (PAF, final concentration 0.1
(ADP, final concentration 10 M), arachidonic acid (AA, final
concentration 350 M), and thrombin (TH, final concentration
l
M
to 10 nM) to platelet-activating
l
M), adenosine diphosphate
The preparation of 5a–q was achieved by following the route of
Scheme 1. Via Pictet–Spengler condensation L-Phe-OH was con-
verted into (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid
(TIC, 1, 84% yield). After Boc-protection the Boc-TIC (2) was cou-
l
l
0.1 U/mL) induced platelet aggregation was tested. The anti-
aggregation activity was represented by IC50 values listed in
Table 1. The IC50 values demonstrate that the activities of
5a–q are significantly higher than that of TIC (1). This means
that introducing tricyclic unit into TIC could generally increase
the in vitro anti-platelet aggregation activity. Besides, the IC50
values reflect an inhibition order of ADP > AA > PAF > TH. The
differences of the IC50 values suggest that introducing tricyclic
unit into TIC benefits the inhibition of ADP induced platelet
aggregation in particular. Therefore, ADP induced platelet
pled with L-amino acid methylester to form seventeen Boc-TIC-L-
aminoacyl-OCH3s (3a–q) in 45–95% yield. The reaction of 3a–q
with acetone provided dihydroimidazo[1,5-b]isoquinolinyl carbox-
ylic acid methylesters (4a–q) in 37–66% yield. After removing
methylester group, 4a–q were converted into dihydroimidazo
[1,5-b]isoquinolinyl carboxylic acids (5a–q) in 85%–94% yield.
Therefore, seventeen novel compounds 5a–q were smoothly pre-
pared via this route.
O
O
O
ii
i
OH
OH
OH
NH2
N
NH
Boc
O
2
1
R
iii
H2N
CO2Me
O
O
R
R
R
v
N
CO2Me
iv
N
CO2H
N
CO2Me
N
N
H
N
Boc
5a-q
3a-q
4a-q
Scheme 1. Synthetic route of 2-substituted (S)-2-(3,3-dimethyl-1-oxo-10,10a-dihydroimidazo-[1,5-b] isoquinolin-2(1H,3H,5H)-yl)-acetic acids. Reagents: (i) HCHO and HCl;
(ii) (Boc)2O and NaHCO3; (iii) L-AA-OCH3, DCC and NMM; (iv) 4 N HCl/EtOAc, methanol, acetone and triethylamine; (v) NaOH in methanol (2 N). In 3a–5a, R = CH2CH(CH3)2;
3b–5b, R = CH(CH3)2; 3c–5c, R = CH(CH3)CH2CH3; 3d–5d, R = CH3; 3e–5e, R = H; 3f–5f, R = CH2C6H5; 3g–5g, R = CH2C6H4–OH-p; 3h–5h, R = indole-5-yl–CH2; 3i & 4i,
R = CH2CO2CH3; 5i, R = CH2CO2H; 3j & 4j, R = CH2CH2CO2CH3; 5j, R = CH2CH2CO2H; 3k & 4k, R = CH2CH2CH2CH2NHCOOCH2C6H5; 5k, R = CH2CH2CH2CH2NH2; 3l–5l,
R = CH(OH)CH3; 3m–5m, R = CH2CONH2; 3n–5n, R = CH2CH2CONH2; 3o & 4o, R = CH2CH2CH2NHC(NH)NHNO2; 5o, R = CH2CH2CH2NHC(NH)NH2; 3p–5p, R = CH2CH2SCH3;
3q–5q, R = CH2SH.

H. Wang et al. / Bioorg. Med. Chem. 19 (2011) 871–882
873
Table 1
IC50 of 5a–q against four aggregators induced aggregation of pig plateletsa
2.5. Dose-dependent in vivo anti-thrombotic activity of 5a
Intravenously injection of 5a was observed at the doses of 100,
10 and 1 nmol/kg to produce a possible dose-dependent anti-
thrombotic response in the rats. The thrombus weights are listed
in Table 3, which demonstrate that the thrombus weight is pro-
gressively increased with dose decrease. Therefore, 5a exhibited
dose-dependent anti-thrombotic action.
Compd
IC50 (nM)
ADP
AA
PAF
TH
1
541.7 36.6
171.0 25.4
175.2 30.3
158.5 27.5
199.5 32.6
172.9 29.0
163.1 28.1
272.9 31.7
198.0 32.2
293.9 33.1
248.3 30.5
190.7 32.1
207.4 31.6
283.3 32.8
182.0 31.2
158.8 27.1
264.2 31.0
179.7 30.8
481.4 34.0
287.3 28.2
292.4 32.3
271.1 31.0
324.3 32.8
289.8 29.7
277.0 30.5
419.7 33.4
322.4 32.4
447.1 34.5
387.8 36.0
312.9 32.7
334.6 32.9
433.3 33.7
301.6 30.9
271.1 29.6
408.5 32.7
298.6 29.1
943.8 63.4
514.9 36.0
524.7 36.8
485.8 34.0
576.0 40.0
519.4 36.4
496.5 34.8
752.4 52.7
577.8 40.1
801.3 56.1
695.0 48.7
560.8 39.3
599.7 42.0
776.6 54.4
540.6 37.8
485.8 34.0
499.1 35.0
535.2 37.5
980.3 65.3
598.9 41.9
610.3 40.1
565.0 39.6
677.3 47.7
604.1 42.3
577.5 40.4
875.1 61.3
672.1 47.1
932.0 65.2
808.4 56.6
652.3 45.7
697.6 48.8
903.2 63.2
628.7 44.0
566.7 40.1
851.5 59.6
622.5 43.6
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
2.6. Vasorelaxation activities of 4a–q and 5a–q
Vasoconstriction may disturb the vascular tone and blood flow,
and subsequently influence the supply of oxygen and nutrients for
the organs. In the discovery of the novel vasodilators, (11aS)-1,2,
3,5,11,11a-hexahydro-3,3-dimethyl-1-oxo-6H-imidazo-[30,40:1,2]
pyridin[3,4-b]indol-2-substituted acetates (Fig. 2, E) were re-
ported.24 As is shown in Figure 2, the removing of a pyrrole ring
from E derives 4a–q. Based on this structural correlation, the
vasorelaxation assays of 4a–q were performed and the data are
listed in Table 4. The data indicate that 3 ꢀ 10ꢁ4 M of 4a–q exhibit
moderate vasorelaxation, and the effective concentration is 6-folds
higher than that of E (5 ꢀ 10ꢁ5 M). This great increase of the effec-
tive concentration emphasizes that the pyrrole ring has essential
contribution to the vasorelaxation of E.
a
IC50 was represented by X SD nM, n = 6.
aggregation is more sensitive to 5a–q, and 5c,o are the most
potential compounds.
To clarify the contribution of the methylester group to the
vasorelaxation of 4a–q the vasorelaxation assays of 5a–q were
2.4. In vivo anti-thrombotic activity of 5a–q
Table 3
Effect of different doses of 5a on the thrombus weight of the ratsa
On an extra-corporeal circulation of arterio-veinos cannula
model the in vivo assays were performed. In these assays
10 nmol/kg of 5a–q were intravenously administered and the
thrombus weights of the treated rats are listed in Table 2. The
thrombus weights of 5a–q treated rats (ranged from 12.99 to
21.41 mg) are significantly lower than that of normal saline
(NS) treated rats (24.09 mg, p <0.01), which indicates that 5a–q
effectively inhibit the rats to form thrombus. On the other hand,
except the thrombus weights (20.10–21.41 mg) of 10 nmol/kg of
Dose
100 nmol/kg
10.02 2.07b
10 nmol/kg
1 nmol/kg
Thrombus weight
NS
12.99 2.05c
22.09 3.33
20.67 2.16d
a
b
c
Weight of wet thrombus is represented by X SD mg, NS = vehicle, n = 12.
Compared to 1 nmol/kg of 5a p <0.01, to 10 nmol/kg group p <0.05.
Compared to NS and 1 nmol/kg groups p <0.01.
d
Compared to NS group p >0.05.
5g,i,m treated rats, which equal that of 15 lmol/kg of 1 treated
rats (22.01 mg), the thrombus weights of 10 nmol/kg of 5a–f,
h,j–l,n–q treated rats are significantly lower than that of
O
O
R
R
15
l
mol/kg of 1 treated rats. These comparisons mean that the
N
CO2Me
N
CO2Me
N
N
present modification benefits the in vivo anti-thrombotic efficacy.
Table 2 further indicates that most compounds with lower in vivo
anti-thrombotic activity have polar side chain, while most com-
pounds with higher in vivo anti-thrombotic activity have apolar
side chain.
N
H
4a-q
E
Figure 2. Structural correlation of (11aS)-1,2,3,5,11,11a-hexahydro-3,3-dimethyl-
1-oxo-6H-imidazo[30,40:1,2]pyridin[3,4-b]indol-2-substituted acetates (E) with
4a–q.
Table 2
Effect of 5a–q on the thrombus weight of the ratsa
Compd
Dose
Thrombus weight
Compd
Dose
167
Thrombus weight
NS
1
24.09 2.33
22.01 2.14e
Aspirin
5i
5j
5k
5l
5m
5n
5o
5p
5q
l
mol/kg
13.22 1.67c
21.41 2.12d
18.57 2.21b
14.98 1.94b
16.04 2.01b
20.75 2.49d
14.39 2.29b
13.01 2.44b
19.56 2.10c
14.36 2.14b
15
lmol/kg
10 nmol/kg
10 nmol/kg
10 nmol/kg
10 nmol/kg
10 nmol/kg
10 nmol/kg
10 nmol/kg
10 nmol/kg
10 nmol/kg
b
5a
5b
5c
5d
5e
5f
10 nmol/kg
10 nmol/kg
10 nmol/kg
10 nmol/kg
10 nmol/kg
10 nmol/kg
10 nmol/kg
10 nmol/kg
13.79 2.02
14.01 2.10b
12.99 2.05b
15.45 1.89b
13.89 2.00b
13.27 2.27b
20.10 2.36d
15.43 1.89b
5g
5h
a
b
c
Weight of wet thrombus is represented by X SD mg, NS = vehicle, n = 12.
Compared to NS and 1, p <0.01.
Compared to NS p <0.01, to 1 p <0.05.
Compared to NS p <0.01, to 1 p >0.05.
Compared to NS p <0.05.
d
e

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H. Wang et al. / Bioorg. Med. Chem. 19 (2011) 871–882
Table 4
Effect of 3 ꢀ 10ꢁ4 M of 4a–q and 5a–q on NE induced vasoconstrictiona
Compd
Vasorelaxation
Compd
Vasorelaxation
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
NS
74.68 6.72
54.99 4.70
99.00 7.00
34.10 2.04
75.35 3.52
32.18 3.34
15.56 2.78
28.08 2.44
64.09 5.12
42.48 4.72
68.21 4.62
35.21 2.22
47.89 4.61
38.61 3.73
6.78 0.40
43.06 9.36
96.30 3.84
0
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
30.14 2.75
16.00 1.91
34.29 2.23
8.41 1.25
21.11 2.20
26.61 2.71
1.59 0.40
11.86 1.38
2.89 0.62
4.33 0.65
12.01 1.41
9.98 1.07
3.89 0.39
12.24 1.72
32.58 1.92
5.58 0.49
16.57 1.81
Figure 3. The comparison of the stretching level of 5o,q with high activity to that of
5g,i with low activity.
a
Vasorelaxation is represented by X SD%, n = 6.
300 and 500 spectrometers. FAB-MS was determined by VG-ZAB-
MS high resolution GC/MS/DS and HP ES-5989x. Optical rotations
were determined with a Schmidt+Haensch Polartromic D instru-
ment. The statistical analysis of all the biological date was carried
out by use of ANOVA test with p <0.05 as significant cut-off.
performed and the data are listed in Table 4. The data indicate
that at the dose of 3 ꢀ 10ꢁ4 M 5a–q exhibit a weak vasorelaxa-
tion. Except 5f, the vasorelaxation of 5a–q is significantly lower
than that of 4a–q. This great decrease of the vasorelaxation im-
plies that the methylester group has important contribution to
the vasorelaxation of 4a–q. However, the data further indicate
that the vasorelaxation order of 5a–q is substantially similar to
that of their in vivo anti-thrombotic activities. This similarity
suggests that the vasorelaxation may enhance the anti-throm-
botic activity.
4.1.2. (S)-1,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid (1)
To the suspension of 5.0 g (0.03 mmol) of L-Phe in 50 mL of
chloroform and 27 ml of formaldehyde, 45 mL of concentrated
hydrochloric acid was added drop-wise. The reaction mixture
was stirred at 80–90 °C for 10 h, and TLC (CHCl3/CH3OH, 10:1) indi-
cates the complete disappearance of L-Phe. The reaction mixture
was cooled to room temperature and the formed precipitates were
collected by filtration. The collected solids were successively
washed with water (30 mL ꢀ 3) and acetone (30 mL ꢀ 3) to give
4.5 g (84%) of the title compound as a colorless powder. Mp 302–
3. Conclusion
The introduction of an imidazole ring into the amino acid
modified TIC is optionally correlated with conformation rigidity
design. Based on conformational rigidity, seventeen novel dihy-
droimidazo[1,5-b]isoquinolinyl carboxylic acids (5a–q) do have
desirable stretching conformation and consequently do have high
in vitro anti-platelet aggregation and in vivo anti-thrombotic
activities. The pharmacological benefit of the stretching confor-
mation is not only reflected by the in vivo anti-thrombotic
activity of the cyclic derivatives (5a–q) over that of their parents
(C-terminal modified TICs, B), but also reflected by the in vivo
anti-thrombotic activities of 5c,a,o,f,e,q over that of 5l,j,p,g,m,i.
The stretching level of the conformation of 5c,a,o,f,e,q is higher
than that of 5l,j,p,g,m,i, which was visualized in Figure 3. Besides,
a week vasorelaxation of 5a–q contributes to their in vivo anti-
thrombotic activity. This provides an additional approach for
the design of the derivatives of 5a–q.
303 °C; ½a 2D0
ꢂ
= ꢁ68 (c 1.0, H2O); ESI-MS (m/e) 178 [M+H]+; 1H
NMR (300 MHz, CDCl3) d/ppm = 11.0 (s, 1H), 7.25 (m, J = 6.4 Hz,
2H), 7.02 (d, J = 6.5 Hz, 1H), 6.98 (t, J = 6.6 Hz, 1H), 3.80 (m, 3H),
3.03 (d, J = 7.5 Hz, 1H), 2.78 (d, J = 8.4 Hz, 1H), 2.0 (s, 1H); 13C
NMR (75 MHz, CDCl3) d/ppm = 174.9, 136.2, 134.2, 127.2, 126.0,
57.6, 47.4, 29.4.
4.1.3. (S)-2-tert-Butoxycarbonyl-1,2,3,4-tetrahydroisoquin-
oline-3-carboxylic acid (2)
To the suspension of 2.49 g (62.2 mmol) of NaOH, 62.2 mL of
water, and 10.0 g (56.5 mmol) of 3S-1,2,3,4-tetrahydroisoquino-
line-3-carboxylic acid, the solution of 14.8 g (67.8 mmol) of Boc2O
in 40 mL of THF was added at 0 °C. The suspension was stirred at
room temperature for 48 h to form a clean solution, and TLC (ethyl
acetate/petroleum ether, 1:3) indicated complete disappearance of
3S-1,2,3,4-tetra-hydroisoquinoline-3-carboxylic acid. The reaction
mixture was evaporated under vacuum, and the residue was dis-
solved in 100 ml of ethyl acetate. The solution was washed succes-
sively with 5% aqueous solution of KHSO4 (30 mL ꢀ 3) and
saturated aqueous solution of NaCl (30 mL ꢀ 3), and dried with
anhydrous Na2SO4. After filtration, the filtrate was evaporated un-
der vacuum and the residue was triturated with petroleum ether to
give 12.5 g (80%) of the title compound as a colorless powder. ESI-
4. Experimental section
4.1. Synthesis
4.1.1. General
The protected amino acids with L-configuration were purchased
MS (m/e) 278 [M+H]+; ½a D20
ꢂ
= ꢁ6.78 (c 1.0, methanol); 1H NMR
from Sigma Chemical Co. All coupling and deprotective reactions
were carried out under anhydrous conditions. Chromatography
was performed on Qingdao silica gel H. The purities of the
intermediates and the products were confirmed on thin layer chro-
matography TLC (Merck silica gel plates of type 60 F254, 0.25 mm
layer thickness) and HPLC (Waters, C18 column 4.6 ꢀ 150 mm).
1H NMR and 13C NMR spectra were recorded by Bruker Advance
(300 MHz, DMSO-d6) d/ppm = 11.2 (s, 1H), 7.56 (d, J = 7.5 Hz, 1H),
7.44 (d, J = 8.0 Hz, 1H), 7.23 (t, J = 8.2 Hz, 1H), 7.22 (t, J = 7.3 Hz,
1H), 4.14 (m, J = 5.2 Hz, 3H), 3.13 (m, J = 4.1 Hz, 2H), 2.72 (m,
J = 4.5 Hz, 2H), 1.42 (s, 9H); 13C NMR (75 MHz, DMSO-d6)
d/ppm = 176.8, 169.8, 137.5, 132.8, 129.0, 127.8, 126.9, 125.2,
82.9, 60.7, 56.9, 51.3, 28.2, 25.5.

H. Wang et al. / Bioorg. Med. Chem. 19 (2011) 871–882
875
4.1.4. General procedure for preparing (S)-2-Boc-1,2,3,4-tetrahy-
droisoquinoline-3-carbonylamino acid methyl esters (3a–q)
At 0 °C and with stirring, to the solution of 0.256 g (0.924 mmol)
of (S)-2-Boc-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, 2 mL
of anhydrous THF 0.151 g (0.109 mmol) of HOBt was added, and
stirred for 10 min. Then, 0.228 g (0.108 mmol) of DCC was added
to form reaction mixture A. To the suspension of 0.102 mmol of
HClꢃAA-OMe in 4 ml of anhydrous THF, 1 mL of N-methylmorpho-
line was added and stirred at room temperature for 35 min to form
reaction mixture B (pH 9). The reaction mixture A and B were
combined, stirred at room temperature for 12 h, and TLC (ethyl
acetate/petroleum ether, 1:3) indicated the complete disappear-
ance of 3S-2-Boc-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid.
The formed precipitate of DCU was removed by filtration, and the
filtrate was evaporated under vacuum. The residue was dissolved
in 50 ml of ethyl acetate, the formed solution was washed succes-
sively with saturated aqueous solution of NaHCO3 (30 mL ꢀ 3), 5%
aqueous solution of KHSO4 (30 mL ꢀ 3) and saturated aqueous solu-
tion of NaCl (30 mL ꢀ 3), and dried with anhydrous Na2SO4. After fil-
tration, the filtrate was evaporated under vacuum to give 3a–q.
129.0, 127.5, 127.1, 124.9, 83.2, 69.5, 57.2, 52.0, 53.1, 49.8, 29.2,
25.6, 16.6.
4.1.4.5. N-((S)-2-tert-Butoxycarbonyl-1,2,3,4-tetrahydroisoquin-
oline-3-carbonyl)-glycine methylester (3e). Yield: 85%, colorless
powder, mp 125–126 °C; ESI-MS (m/e) 349 [M+H]+; ½a D20
= ꢁ15.83
ꢂ
(c 1.0, methanol); 1H NMR (300 MHz, DMSO-d6) d/ppm = 8.82 (d,
J = 8.6 Hz, 1H), 7.58 (d, J = 7.9 Hz, 1H), 7.54 (d, J = 8.6 Hz, 1H),
7.43 (t, J = 7.4 Hz, 1H), 7.15 (t, J = 8.5 Hz, 1H), 4.29 (t, J = 6.3 Hz,
1H), 4.15 (m, J = 4.3 Hz, 6H), 3.25 (m, J = 5.5 Hz, 2H), 2.95 (m,
J = 4.6 Hz, 2H), 1.52 (s, 9H); 13C NMR (75 MHz, DMSO-d6)
d/ppm = 171.1, 169.1, 168.9, 138.2, 132.3, 128.6, 128.1, 127.6,
124.6, 82.0, 68.1,57.5, 54.8, 51.9, 41.2, 31.0, 26.2.
4.1.4.6. N-((S)-2-tert-Butoxycarbonyl-1,2,3,4-tetrahydroisoquin-
oline-3-carbonyl)-L-phenylalanine methylester (3f). Yield: 87%,
colorless powder, mp 91–93 °C; ESI-MS (m/e) 439 [M+H]+;
½
a 2D0
ꢂ
= ꢁ7.61 (c 1.0, methanol); 1H NMR (300 MHz, DMSO-d6)
d/ppm = 9.06 (d, J = 8.4 Hz, 1H), 7.25 (m, J = 7.5 Hz, 9H), 4.25 (t,
J = 5.6 Hz, 1H), 4.15 (m, J = 5.2 Hz, 6H), 3.02 (m, J = 4.2 Hz, 4H),
2.93 (m, J = 5.6 Hz, 2H), 1.45 (s, 9H), 1.41 (m, J = 4.1 Hz, 2H); 13C
NMR (75 MHz, DMSO-d6) d/ppm = 169.6, 133.3, 128.2, 128.1,
127.9, 127.5, 127.3, 126.2, 125.7, 80.2, 69.5, 53.9, 53.5, 52.0, 37.5,
29.3, 28.5, 27.5.
4.1.4.1. N-((S)-2-tert-Butoxycarbonyl-1,2,3,4-tetrahydroisoquin-
oline-3-carbonyl)-L-leucine methylester (3a). Yield: 91%, color-
less powder, mp 81–83 °C; ESI-MS (m/e) 405 [M+H]+;
½
a 2D0
ꢂ
= ꢁ14.1 (c 1.0, methanol); 1H NMR (300 MHz, DMSO-d6)
d/ppm = 8.72 (d, J = 8.4 Hz, 1H), 7.55 (d, J = 7.5 Hz, 1H), 7.34 (d,
J = 8.1 Hz, 1H), 7.24 (t, J = 7.3 Hz, 1H), 7.14 (t, J = 8.2 Hz, 1H), 4.25
(t, J = 5.4 Hz, 1H), 4.14 (m, J = 5.5 Hz, 6H), 3.14 (m, J = 4.3 Hz, 2H),
2.72 (m, J = 5.3 Hz, 2H), 1.47 (s, 9H), 1.44 (s, 9H), 0.93 (m,
J = 4.1 Hz, 6H); 13C NMR (75 MHz, DMSO-d6) d/ppm = 171.6,
170.2, 168.2, 137.2, 133.9, 128.6, 127.8, 127.3, 125.9, 80.2, 68.9,
55.9, 53.4, 51.8, 49.5, 41.0, 29.5, 27.9, 22.9, 22.2.
4.1.4.7. N-((S)-2-tert-Butoxycarbonyl-1,2,3,4-tetrahydroisoquin-
oline-3-carbonyl)-L-tyrosine methylester (3g). Yield: 82%, color-
less powder, mp 62–65 °C; ESI-MS (m/e) 455 [M+H]+;
½
a 2D0
ꢂ
= ꢁ23.26 (c 1.0, methanol); 1H NMR (300 MHz, DMSO-d6)
d/ppm = 7.73 (d, J = 8.6 Hz, 1H), 7.56 (d, J = 7.4 Hz, 1H), 7.39 (d,
J = 8.3 Hz, 1H), 7.23 (t, J = 7.7 Hz, 1H), 7.13 (t, J = 8.2 Hz, 5H), 4.30
(t, J = 4.4 Hz, 1H), 6.70 (d, J = 8.4 Hz, 2H), 4.28 (m, J = 4.2 Hz, 6H),
3.13 (m, J = 4.3 Hz, 2H), 2.94 (m, J = 4.8 Hz, 3H), 1.43 (s, 9H); 13C
NMR (75 MHz, DMSO-d6) d/ppm = 171.9, 171.6, 167.1, 156.8,
133.1, 132.4, 129.9, 129.6, 128.1, 127.8, 125.1, 127.3, 116.1,
115.7, 83.2, 69.1, 57.2, 53.8, 53.1, 52.0, 37.8, 37.2, 29.7, 27.2.
4.1.4.2. N-((S)-2-tert-Butoxycarbonyl-1,2,3,4-tetrahydroisoquin-
oline-3-carbonyl)-L-valine methylester (3b). Yield: 86%, color-
less powder, mp 87–88 °C; ESI-MS (m/e) 391 [M+H]+;
½
a 2D0
ꢂ
= ꢁ37.57 (c 1.0, methanol); 1H NMR (300 MHz, DMSO-d6)
d/ppm = 8.78 (d, J = 8.4 Hz, 1H), 7.66 (d, J = 7.5 Hz, 1H), 7.56 (d,
J = 8.0 Hz, 1H), 7.31 (t, J = 7.7 Hz, 1H), 7.22 (t, J = 8.2 Hz, 1H), 4.45
(m, J = 5.4 Hz, 6H), 4.19 (t, J = 5.6 Hz, 1H), 3.42 (m, J = 5.3 Hz, 2H),
2.91 (m, J = 4.4 Hz, 2H), 2.17 (dd, J = 5.7 Hz, 2H), 1.44 (s, 9H), 0.97
(m, J = 3.2 Hz, 6H); 13C NMR (75 MHz, DMSO-d6) d/ppm = 171.8,
171.2, 169.6, 137.2, 133.6, 128.9, 127.8, 127.5, 125.6, 82.0, 68.9,
56.8, 56.0, 53.4, 52.0, 30.1, 29.3, 27.6, 17.6, 17.3.
4.1.4.8. N-((S)-2-tert-Butoxycarbonyl-1,2,3,4-tetrahydroisoquin-
oline-3-carbonyl)-L-tryptophan methylester (3h). Yield: 87%,
colorless powder, mp 71–72 °C; ESI-MS(m/e) 478 [M+H]+;
½
a 2D0
ꢂ
= ꢁ7.99 (c 1.0, methanol); 1H NMR (300 MHz, DMSO-d6)
d/ppm = 10.97 (s, 1H), 9.06 (d, J = 8.4 Hz, 1H), 7.72 (m, J = 7.5 Hz,
9H), 4.38 (m, J = 5.3 Hz, 6H), 4.30 (t, J = 5.1 Hz, 1H), 3.13 (m,
J = 4.5 Hz, 2H), 2.69 (m, J = 4.0 Hz, 2H), 1.65 (s, 9H); 13C NMR
(75 MHz, DMSO-d6) d/ppm = 172.9, 171.9, 168.9, 137.6, 136.8,
128.4, 127.9, 127.3, 125.8, 122.9, 122.6, 122.1, 119.8, 112.0,
110.9, 81.2, 68.9, 57.2, 54.3, 51.8, 30.9, 29.4, 27.6.
4.1.4.3. N-((S)-2-tert-Butoxycarbonyl-1,2,3,4-tetrahydroisoquin-
oline-3-carbonyl)-L-isoleucine methylester (3c). Yield: 89%, col-
orless powder, mp 73–74 °C; ESI-MS (m/e) 405 [M+H]+;
½
a 2D0
ꢂ
= ꢁ12.39 (c 1.0, methanol); 1H NMR (300 MHz, DMSO-d6)
4.1.4.9. N-((S)-2-tert-Butoxycarbonyl-1,2,3,4-tetrahydroisoquin-
oline-3-carbonyl)-aspartic acid dimethylester (3i). Yield: 75%,
colorless powder, mp 106–107 °C; ESI-MS (m/e) 407 [M+H]+;
d/ppm = 8.73 (d, J = 8.4 Hz, 1H), 7.55 (d, J = 7.3 Hz, 1H), 7.34 (d,
J = 8.5 Hz, 1H), 7.27 (t, J = 7.3 Hz, 1H), 7.13 (t, J = 8.2 Hz, 1H), 4.33
(m, J = 4.7 Hz, 6H), 4.29 (t, J = 5.6 Hz, 1H), 3.13 (m, J = 4.1 Hz, 2H),
2.74 (m, J = 4.8 Hz, 3H), 1.29 (dd, J = 5.7 Hz, 2H), 1.56 (s, 9H), 0.94
(m, J = 3.9 Hz, 6H); 13C NMR (75 MHz, DMSO-d6) d/ppm = 172.8,
171.8, 169.9, 137.2, 133.4, 128.9, 127.9, 127.3, 125.6, 81.2, 68.9,
57.0, 53.4, 53.2, 51.8, 36.4, 30.0, 27.2, 26.2, 15.8, 11.5.
½
a 2D0
ꢂ
= ꢁ3.23 (c 1.0, methanol); 1H NMR (300 MHz, DMSO-d6)
d/ppm = 8.72 (d, J = 8.4 Hz, 1H), 7.55 (d, J = 7.6 Hz, 1H), 7.35 (d,
J = 8.3 Hz, 1H), 7.28 (t, J = 7.7 Hz, 1H), 7.13 (t, J = 8.2 Hz, 1H), 4.31
(m, J = 4.3 Hz, 3H), 4.24 (t, J = 4.4 Hz, 1H), 3.73 (m, J = 4.6 Hz, 6H),
3.12 (m, J = 4.6 Hz, 2H), 2.89 (m, J = 4.3 Hz, 3H), 2.76 (m,
J = 4.5 Hz, 2H), 1.42 (s, 9H); 13C NMR (75 MHz, DMSO-d6) d/
ppm = 173.1, 172.8, 171.6, 169.6, 137.9, 133.6, 127.6, 127.0,
126.9, 124.1, 81.8, 69.5, 56.7, 53.8, 51.7, 48.2, 37.9, 29.1, 27.6.
4.1.4.4. N-((S)-2-tert-Butoxycarbonyl-1,2,3,4-tetrahydroisoquin-
oline-3-carbonyl)-L-alanine methylester (3d). Yield: 89%, color-
less powder, mp 84–85 °C; ESI-MS (m/e) 363 [M+H]+;
½
a 2D0
ꢂ
= ꢁ47.46 (c 1.0, methanol); 1H NMR (300 MHz, DMSO-d6)
4.1.4.10.
quinoline-3-carbonyl)-
N-((S)-2-tert-Butoxycarbonyl-1,2,3,4-tetrahydroiso-
-glutamic acid dimethylester (3j).
d/ppm = 8.72 (d, J = 8.4 Hz, 1H), 7.55 (d, J = 7.4 Hz, 1H), 7.34 (d,
J = 8.1 Hz, 1H), 7.22 (t, J = 7.1 Hz, 1H), 7.12 (t, J = 7.3 Hz, 1H), 4.44
(t, J = 5.4 Hz, 1H), 4.42 (m, J = 5.1 Hz, 6H), 3.13 (m, J = 4.4 Hz, 2H),
2.75 (m, J = 5.5 Hz, 2H), 1.41 (s, 9H), 1.38 (d, J = 6.6 Hz, 3H); 13C
NMR (75 MHz, DMSO-d6) d/ppm = 172.8, 168.9, 137.2, 132.4,
L
Yield: 78%, colorless powder, mp 107–109 °C; ESI-MS (m/e) 420
[M+H]+;
½
a 2D0
ꢂ
= ꢁ4.05 (c 1.0, methanol); 1H NMR (300 MHz,
DMSO-d6) d/ppm = 8.72 (d, J = 8.5 Hz, 1H), 7.57 (d, J = 7.6 Hz, 1H),
7.33 (d, J = 8.7 Hz, 1H), 7.28 (t, J = 7.7 Hz, 1H), 7.12 (t, J = 8.2 Hz,

876
H. Wang et al. / Bioorg. Med. Chem. 19 (2011) 871–882
1H), 4.45 (m, J = 5.6 Hz, 3H), 4.29 (t, J = 5.5 Hz, 1H), 3.53 (m,
J = 4.6 Hz, 6H), 3.12 (m, J = 4.5 Hz, 2H), 2.72 (m, J = 4.8 Hz, 2H),
1.96 (m, J = 5.2 Hz, 4H), 1.42 (s, 9H); 13C NMR (75 MHz, DMSO-
d6) d/ppm = 173.2, 172.8, 171.6, 169.6, 137.9, 133.6, 127.6, 127.0,
126.9, 124.1, 81.8, 69.5, 56.7, 53.8, 51.6, 50.7, 29.1, 27.8, 27.6, 26.6.
½
a 2D0
ꢂ
= ꢁ12.90 (c 1.0, methanol); 1H NMR (300 MHz, DMSO-d6)
d/ppm = 8.72 (d, J = 8.7 Hz, 1H), 7.55 (d, J = 7.6 Hz, 1H), 7.34 (d,
J = 8.3 Hz, 1H), 7.23 (t, J = 7.7 Hz, 1H), 7.12 (t, J = 8.2 Hz, 1H), 4.32
(t, J = 4.5 Hz, 1H), 4.25 (m, J = 4.9 Hz, 6H), 3.12 (m, J = 4.3 Hz, 2H),
2.81 (m, J = 4.9 Hz, 2H), 2.35 (m, J = 4.6 Hz, 2H), 1.42 (s, 9H), 1.27
(m, J = 4.2 Hz, 2H); 13C NMR (75 MHz, DMSO-d6) d/ppm = 171.8,
171.6, 169.2, 137.6, 128.9, 127.6, 127.2, 125.8, 81.3, 69.1, 56.7,
53.2, 52.9, 52.3, 31.2, 29.1, 27.9, 17.8.
4.1.4.11. N-((S)-2-tert-Butoxycarbonyl-1,2,3,4-tetrahydroisoquin-
oline-3-carbonyl)-L-lysine(Z) benzylester (3k). Yield: 95%. Syr-
upy. ESI-MS (m/e) 630 [M+H]+; ½a D20
ꢂ
= ꢁ19.9 (c 1.0, CH3OH); 1H
NMR (300 MHz, CDCl3) d/ppm = 8.72 (d, J = 8.4 Hz, 1H), 6.99–7.60
(m, 14H), 5.34 (d, J = 6.3 Hz, 2H), 4.28 (t, 1H), 4.18 (m, 9H), 3.12
(m, 2H), 2.82 (m, 2H), 2.21 (m, 5H), 1.36 (m, 13H); 13C NMR
(75 MHz, CDCl3) d/ppm = 171.9, 171.6, 169.6, 142.8, 137.6, 136.5,
133.7, 125.2–128.9, 82.7, 68.8, 67.8, 56.8, 54.8, 53.7, 52.8, 49.5,
32.0, 30.2, 28.7, 27.9, 21.7.
4.1.4.17.
N-((S)-2-tert-Butoxycarbonyl-1,2,3,4-tetrahydroiso-
-cysteine methylester (3q). Yield: 50%,
quinoline-3-carbonyl)-
L
colorless powder, mp 107–110 °C; ESI-MS (m/e) 395 [M+H]+;
½
a 2D0
ꢂ
= ꢁ25.12 (c 1.0, methanol); 1H NMR (300 MHz, CDCl3)
d/ppm = 8.00 (d, J = 7.6 Hz, 1H), 7.02 (m, J = 8.2 Hz, 2H), 6.95 (d,
J = 8.8 Hz, 2H), 4.90 (t, J = 8.4 Hz, 1H), 4.71 (m, 1H), 4.20
(t, J = 8.3 Hz, 2H), 3.65 (s, 3H), 3.07 (m, J = 5.4 Hz, 4H), 1.5 (t,
J = 6.4 Hz, 1H), 1.41 (s, 9H); 13C NMR (75 MHz, DMSO-d6)
d/ppm = 174.8, 172.6, 159.2, 139.6, 137.1, 128.1, 127.4, 126.2,
125.6, 70.1, 66.1, 63.2, 52.1, 46.3, 31.8, 29.0, 26.8.
4.1.4.12. N-((S)-2-tert-Butoxycarbonyl-1,2,3,4-tetrahydroisoquin-
oline-3-carbonyl)-L-threonine methylester (3l). Yield: 89%, col-
orless powder, mp 80–81 °C; ESI-MS (m/e) 393 [M+H]+;
½
a 2D0
ꢂ
= ꢁ42.43 (c 1.0, methanol); 1H NMR (300 MHz, DMSO-d6)
d/ppm = 8.73 (d, J = 8.4 Hz, 1H), 7.56 (d, J = 7.5 Hz, 1H), 7.33 (d,
J = 8.1 Hz, 1H), 7.22 (t, J = 7.7 Hz, 1H), 7.14 (t, J = 8.6 Hz, 1H), 4.28
(t, J = 4.5 Hz, 1H), 4.29 (m, J = 4.3 Hz, 6H), 3.16 (m, J = 4.2 Hz, 2H),
2.74 (m, J = 4.1 Hz, 3H), 2.15 (s, 1H), 1.52 (s, 9H); 13C NMR
(75 MHz, DMSO-d6) d/ppm = 172.8, 171.6, 167.9, 137.1, 129.1,
127.9, 127.2, 125.6, 83.1, 68.2, 67.9, 58.2, 53.7, 51.2, 28.2, 27.8, 18.6.
4.1.5. General procedure for preparing 2-substituted (S)-2-(3,3-
dimethyl-1-oxo-10,10a-dihydroimidazo[1,5-b]isoquinolin-
2(1H,3H,5H)-yl)-acetic acid methylesters (4a–q)
At 0 °C to the solution of 4.51 mmol of substituted (S)-2-Boc-
1,2,3,4-tetrahydro-isoquinoline-3-carbonylamino acid methylest-
ers (3) in 8 mL of ethyl acetate 16 mL of hydrogen chloride/ethyl
acetate (4 N) were added drop-wise. The reaction solution was stir-
red at 0 °C for 90 min and evaporated under reduced pressure. The
residue was dissolved in 60 mL of methanol and 20 mL of acetone.
With triethylamine, the reaction solution was adjusted to pH 9, at
room temperature and in dark stirred for 240 h, and thin layer
chromatography (TLC) (CHCl3/MeOH, 10:1) indicated the complete
disappearance of 3. On evaporation, the residue was dissolved in
200 mL of ethyl acetate. The solution was washed successively
with 5% sodium bicarbonate, 5% citric acid, and saturated sodium
chloride and the organic phase was separated and dried over anhy-
drous sodium sulfate. After filtration and evaporation under re-
duced pressure, the residue was dissolved in 5 mL of methanol to
crystallize 4a–q.
4.1.4.13.
N-((S)-2-tert-Butoxycarbonyl-1,2,3,4-tetrahydroiso-
-asparagine methylester (3m). Yield:
quinoline-3-carbonyl)-
L
55%, colorless powder, mp 101–102 °C; ESI-MS (m/e) 407 [M+H]+;
½
a 2D0
ꢂ
= ꢁ6.50 (c 1.0, methanol); 1H NMR (300 MHz, DMSO-d6)
d/ppm = 8.72 (d, J = 8.4 Hz, 1H), 7.55 (d, J = 7.6 Hz, 1H), 7.34 (d,
J = 8.2 Hz, 1H), 7.23 (t, J = 7.7 Hz, 1H), 7.12 (t, J = 8.2 Hz, 1H), 6.20
(s, 1H), 4.41 (m, J = 5.4 Hz, 6H), 4.29 (t, J = 4.3 Hz, 1H), 3.12 (m,
J = 4.5 Hz, 2H), 2.74 (m, J = 4.5 Hz, 4H), 1.42 (s, 9H); 13C NMR
(75 MHz, DMSO-d6) d/ppm = 174.5, 171.8, 168.9, 137.2, 133.7,
128.6, 127.5, 127.3, 125.9, 82.9, 69.0, 53.3, 52.0, 37.2, 17.8.
4.1.4.14. N-((S)-2-tert-Butoxycarbonyl-1,2,3,4-tetrahydroisoquin-
oline-3-carbonyl)-L-glutamine methylester (3n). Yield: 45%, col-
orless powder, mp 100–102 °C; ESI-MS (m/e) 420 [M+H]+;
4.1.5.1. (S)-2-((S)-3,3-Dimethyl-1-oxo-10,10a-dihydroimidazo-
[1,5-b]isoquinolin-2(1H,3H,5H)-yl)-4-methylpentanoic acid me-
thylester (4a). Yield: 46%, colorless powder, mp 186–188 °C.
½
a 2D0
ꢂ
= ꢁ28.78 (c 1.0, methanol); 1H NMR (300 MHz, DMSO-d6)
d/ppm = 8.72 (d, J = 8.5 Hz, 1H), 8.12 (s, 1H), 7.55 (d, J = 7.7 Hz,
1H), 7.34 (d, J = 8.2 Hz, 1H), 7.24 (t, J = 7.7 Hz, 1H), 7.13 (t,
J = 8.2 Hz, 1H), 4.33 (m, J = 5.6 Hz, 6H), 4.24 (t, J = 4.3 Hz, 1H), 3.13
(m, J = 4.8 Hz, 2H), 2.73 (m, J = 4.3 Hz, 6H), 1.46 (s, 9H); 13C NMR
(75 MHz, DMSO-d6) d/ppm = 173.9, 171.9, 171.6, 168.9, 137.1,
133.5, 127.5, 127.2, 125.8, 82.3, 68.2, 56.1, 53.4, 51.3, 51.2, 34.2,
28.9, 27.2, 26.8.
½
a 2D0
ꢂ
= ꢁ58.73 (c 0.25, CHCl3); ESI/MS: 345 [M+H]+; IR (KBr disk):
3456, 2957, 1744, 1697, 1457, 1425, 1367, 1262, 1245, 1191,
750 cmꢁ1 1H NMR (500 MHz, DMSO) dH = 7.16 (t, J = 7.6 Hz, 1H),
.
7.12 (d, J = 7.6 Hz, 1H), 7.10 (d, J = 7.6 Hz, 1H), 7.09 (t, J = 7.6 Hz,
1H), 4.05 (t, J = 10.0 Hz, 1H), 3.95 (t, J = 15.0 Hz, 1H), 3.74 (s, 2H),
3.63 (s, 3H), 2.90 (dd, J = 5.0 Hz, J = 15.0 Hz, 1H), 2.65 (dd,
J = 10.0 Hz, J = 15.0 Hz, 1H), 1.90 (m, 2H), 1.65 (m, 1H), 1.41 (s,
3H), 1.26 (s, 3H), 0.94 (d, J = 5.0 Hz, 3H), 0.92 (d, J = 5.0 Hz, 3H);
13C NMR (125 MHz, DMSO) dC = 19.33, 22.62, 22.82, 25.09, 31.00,
46.69, 52.48, 52.57, 52.90, 56.38, 78.79, 126.26, 126.60, 127.38,
129.91, 133.78, 135.16, 171.19, 171.66. Elemental Anal. Calcd for
4.1.4.15. N-((S)-2-tert-Butoxycarbonyl-1,2,3,4-tetrahydroisoquin-
oline-3-carbonyl)-L-arginine methylester (3o). Yield: 51%, color-
less powder, mp 151–152 °C; ESI-MS (m/e) 448 [M+H]+;
½
a 2D0
ꢂ
= ꢁ11.56 (c 1.0, methanol); 1H NMR (300 MHz, DMSO-d6)
d/ppm = 8.99 (s, 3H), 8.72 (d, J = 8.5 Hz, 1H), 7.54 (d, J = 7.3 Hz,
1H), 7.35 (d, J = 8.4 Hz, 1H), 7.24 (t, J = 7.7 Hz, 1H), 7.13 (t,
J = 8.3 Hz, 1H), 4.24 (t, J = 5.3 Hz, 1H), 4.12 (m, J = 4.3 Hz, 6H), 3.53
(m, J = 4.6 Hz, 6H), 3.15 (m, J = 4.6 Hz, 2H), 2.73 (m, J = 4.3 Hz, 4H),
2.11 (m, J = 4.3 Hz, 1H), 1.96 (m, J = 3.9 Hz, 1H), 1.45 (s, 9H); 13C
NMR (75 MHz, DMSO-d6) d/ppm = 171.9, 171.6, 169.6, 149.7,
137.2, 133.6, 128.9, 127.9, 127.2, 125.6, 83.2, 68.5, 57.2, 53.7, 52.7,
51.0, 37.2, 29.1, 28.7, 26.7, 24.5.
C20H28N2O3: C, 69.74; H, 8.19; N, 8.13. Found: C, 69.96; H, 8.01;
N, 8.36.
4.1.5.2. (S)-2-((S)-3,3-Dimethyl-1-oxo-10,10a-dihydroimidazo-
[1,5-b]isoquinolin-2(1H,3H,5H)-yl)-3-methylbutanoic acid me-
thylester (4b). Yield: 66%, colorless powder, mp 181–183 °C.
½
a 2D0
ꢂ
= ꢁ23.68 (c 0.25, CHCl3); ESI/MS: 331 [M+H]+; IR (KBr disk):
3468, 2965, 1722, 1668, 1445, 1415, 1358, 1268, 1191, 749 cmꢁ1
.
1H NMR (500 MHz, DMSO) dH = 7.15 (t, J = 7.5 Hz, 1H), 7.11 (d,
J = 7.5 Hz, 1H), 7.08 (d, J = 7.5 Hz, 1H), 7.06 (t, J = 7.5 Hz, 1H), 3.95
(d, J = 15.0 Hz, 1H), 3.92 (d, J = 5.0 Hz, 1H), 3.71 (d, J = 15.0 Hz,
1H), 3.64 (s, 3H), 3.30 (dd, J = 4.0 Hz, J = 10.0 Hz, 1H), 2.85 (dd,
4.1.4.16. N-((S)-2-tert-Butoxycarbonyl-1,2,3,4-tetrahydroisoquin-
oline-3-carbonyl)-
L-methionine methylester (3p). Yield: 49%,
colorless powder, mp 143–144 °C; ESI-MS (m/e) 409 [M+H]+;

H. Wang et al. / Bioorg. Med. Chem. 19 (2011) 871–882
877
J = 4.0 Hz, J = 15.0 Hz, 1H), 2.61 (dd, J = 10.0 Hz, J = 15.0 Hz, 1H),
1.85 (m, 1H), 1.39 (s, 3H), 1.24 (s, 3H), 0.98 (t, J = 6.0 Hz, 6H); 13C
NMR (125 MHz, DMSO) dC = 21.12, 21.22, 22.49, 23.40, 28.07,
52.64, 56.45, 56.76, 65.38, 81.74, 127.37, 127.50, 128.57, 128.69,
132.94, 133.34, 171.18, 171.77. Elemental Anal. Calcd for
3.14 (dd, J = 4.0 Hz, J = 11.0 Hz, 1H), 2.94 (dd, J = 4.0 Hz, J = 16.0 Hz,
1H), 2.66 (dd, J = 11.0 Hz, J = 15.5 Hz, 1H), 1.26 (s, 3H), 0.93 (s, 3H);
13C NMR (125 MHz, DMSO) d /ppm = 17.75, 24.73, 30.86, 34.15,
46.67, 52.78, 56.46, 78.63, 126.24, 126.60, 127.09, 127.13, 128.75,
129.88, 130.24, 133.64, 135.03, 138.69, 170.87, 171.58. Elemental
Anal. Calcd for C23H26N2O3: C, 72.99; H, 6.92; N, 7.40. Found: C,
72.80; H, 6.78; N, 7.21.
C19H26N2O3: C, 69.06; H, 7.93; N, 8.48. Found: C, 69.27; H, 8.10;
N, 8.25.
4.1.5.3. (2S)-2-((S)-3,3-Dimethyl-1-oxo-10,10a-dihydroimidazo-
[1,5-b]isoquinolin-2(1H,3H,5H)-yl)-3-methylpentanoic acid me-
thylester (4c). Yield: 61%, colorless powder, mp 192–194 °C.
4.1.5.7.
(S)-2-((S)-3,3-Dimethyl-1-oxo-10,10a-dihydroimidazo
[1,5-b]isoquinolin-2(1H,3H,5H)-yl)-3-(4-hydroxyphenyl)propa-
noic acid methylester (4g). Yield: 53%, colorless powder, mp
½
a 2D0
ꢂ
= ꢁ58.73 (c 0.25, CHCl3); ESI/MS: 345 [M+H]+; IR (KBr disk):
204–207 °C. ½a 2D0
ꢂ
= ꢁ134.50 (c 0.25, CHCl3); ESI/MS: 395 [M+H]+;
3448, 2968, 1728, 1683, 1448, 1423, 1384, 1261, 1215, 1201,
IR (KBr disk): 3326, 2932, 2774, 1743, 1698, 1627, 1540, 1423,
743 cmꢁ1 1H NMR (500 MHz, DMSO) dH = 7.19 (t, J = 7.4 Hz, 1H),
.
1372, 1260, 1107, 747 cmꢁ1 1H NMR (500 MHz, DMSO) dH = 7.22
.
7.16 (d, J = 7.4 Hz, 1H), 7.13 (d, J = 7.4 Hz, 1H), 7.11 (t, J = 7.4 Hz,
1H), 4.01 (d, J = 15.0 Hz, 1H), 3.96 (d, J = 6.5 Hz, 1H), 3.71 (d,
J = 15.0 Hz, 1H), 3.62 (s, 3H), 3.30 (dd, J = 5.0 Hz, J = 10.0 Hz, 1H),
2.92 (dd, J = 5.0 Hz, J = 15.0 Hz, 1H), 2.64 (dd, J = 10.0 Hz,
J = 15.0 Hz, 1H), 2.22 (m, 2H), 1.69 (m, 1H), 1.39 (s, 3H), 1.22 (s,
3H), 0.96 (t, J = 6.5 Hz, 6H); 13C NMR (125 MHz, DMSO)
dC = 11.89, 16.02, 25.04, 26.53, 30.89, 31.08, 37.32, 46.29, 51.5,
55.60, 56.76, 78.53, 126.24, 126.38, 127.18, 129.20, 134.34,
135.35, 171.97, 173.40. Elemental Anal. Calcd for C20H28N2O3: C,
69.74; H, 8.19; N, 8.13. Found: C, 69.92; H, 8.00; N, 8.34.
(d, J = 7.8 Hz, 2H), 7.19 (d, J = 7.8 Hz, 2H), 7.13 (t, J = 7.6 Hz, 1H),
7.10 (d, J = 7.6 Hz, 1H), 7.07 (d, J = 7.6 Hz, 1H), 7.05 (t, J = 7.6 Hz,
1H), 5.22 (s, 1H), 4.13 (dd, J = 6.5 Hz, J = 11.0 Hz, 1H), 3.89
(d, J = 15.5 Hz, 1H), 3.67 (s, 3H), 3.60 (t, J = 11.0 Hz, 1H), 3.42
(d, J = 16.0 Hz, 1H), 3.28 (dd, J = 6.5 Hz, J = 16.0 Hz, 1H), 3.23 (dd,
J = 7.0 Hz, J = 7.5 Hz, 1H), 2.87 (dd, J = 7.0 Hz, J = 16.0 Hz, 1H), 2.64
(dd, J = 8.0 Hz, J = 15.5 Hz, 1H), 1.25 (s, 6H); 13C NMR (125 MHz,
DMSO) dC = 24.12, 24.87, 30.85, 33.67, 46.78, 52.9, 56.55, 57.53,
78.78, 115.26, 115.51, 126.56, 127.22, 129.44, 129.86, 130.11,
131.07, 131.54, 133.85, 135.22, 156.26, 171.223, 172.14. Elemental
Anal. Calcd for C23H26N2O4: C, 70.03; H, 6.64; N, 7.10. Found: C,
69.81; H, 6.50; N, 7.33.
4.1.5.4. (S)-2-((S)-3,3-Dimethyl-1-oxo-10,10a-dihydroimidazo-
[1,5-b]isoquinolin-2(1H,3H,5H)-yl)propanoic acid methylester
(4d). Yield: 51%, colorless powder, mp 201–203 °C.
½
a 2D0
=
ꢂ
ꢁ38.96 (c 0.25, CHCl3); ESI/MS: 303 [M+H]+; IR (KBr disk): 3396,
4.1.5.8. (S)-2-((S)-3,3-Dimethyl-1-oxo-10,10a-dihydroimidazo-
[1,5-b]isoquinolin-2(1H,3H,5H )-yl)-3-(1H-indol-3-yl)propanoic
acid methylester (4h). Yield: 51%, colorless powder, mp 174–
2988, 2973, 1747, 1700, 1626, 1436, 1328, 1243, 1121, 748 cmꢁ1
.
1H NMR (500 MHz, DMSO) dH = 7.17 (t, J = 7.2 Hz, 1H), 7.14 (d,
J = 7.2 Hz, 1H), 7.12 (d, J = 7.2 Hz, 1H), 7.10 (t, J = 7.2 Hz, 1H), 4.27
(q, J = 7.0 Hz, 1H), 3.98 (d, J = 14.0 Hz, 1H), 3.74 (d, J = 15.0 Hz,
1H), 3.62 (s, 3H), 3.22 (dd, J = 3.5 Hz, J = 10.5 Hz, 1H), 2.95 (d,
J = 15.5 Hz, 1H), 2.68 (dd, J = 12.0 Hz, J = 14.5 Hz, 1H), 1.43 (d,
J = 6.0 Hz, 3H), 1.41 (s, 3H), 1.277 (s, 3H); 13C NMR (125 MHz,
DMSO) dC = 16.85, 17.68, 25.43, 30.95, 33.82, 46.77, 49.02, 52.51,
56.76, 78.64, 126.27, 126.78, 127.22, 129.93, 133.76, 135.17,
170.77, 171.65. Elemental Anal. Calcd for C17H22N2O3: C, 67.53;
H, 7.33; N, 9.26. Found: C, 67.31; H, 7.18; N, 9.58.
176 °C. ½a 2D0
ꢂ
= ꢁ127.80 (c 0.25, CHCl3); ESI/MS: 418 [M+H]+; IR
(KBr disk): 3396, 2931, 2769, 1747, 1698, 1516, 1457, 1368,
1339, 1259, 1101, 743 cmꢁ1
.
1H NMR (500 MHz, DMSO)
dH = 10.86 (s, 1H), 7.57 (d, J = 5.0 Hz, 1H), 7.53 (d, J = 5.0 Hz, 1H),
7.34 (d, J = 5.0 Hz, 1H), 7.30 (d, J = 5.0 Hz, 1H), 7.16 (t, J = 7.5 Hz,
1H), 7.13 (d, J = 7.5 Hz, 1H), 7.10 (d, J = 7.5 Hz, 1H), 7.07 (t,
J = 7.5 Hz, 1H), 6.92 (s, 1H), 4.28 (dd, J = 4.5 Hz, J = 10.0 Hz, 1H),
3.74 (d, J = 12.5 Hz, 1H), 3.70 (s, 3H), 3.47 (dd, J = 10.0 Hz,
J = 16.5 Hz, 2H), 3.46 (d, J = 14.0 Hz, 1H), 3.34 (m, 1H), 3.08 (dd,
J = 4.0 Hz, J = 11.5 Hz, 1H), 2.96 (dd, J = 3.5 Hz, J = 15.5 Hz, 1H),
2.67 (dd, J = 11.0 Hz, J = 15.0 Hz, 1H), 1.22 (s, 3H), 0.81 (s, 3H);
13C NMR (125 MHz, DMSO) dC = 17.63, 24.18, 24.85, 30.94, 46.68,
52.67, 56.09, 56.62, 78.65, 111.01, 111.93, 118.44, 118.93, 121.27,
124.82, 126.23, 126.59,126.74, 127.12, 128.08, 129.88, 133.68,
135.03, 136.47, 171.06, 171.29. Elemental Anal. Calcd for
4.1.5.5. (S)-2-(3,3-Dimethyl-1-oxo-10,10a-dihydroimidazo[1,5-
b]isoquinolin-2(1H,3H,5H)-yl)acetic acid methylester (4e). Yield:
52%, colorless powder, mp 188–190 °C. ½a D20
= ꢁ54.20 (c 0.25,
ꢂ
CHCl3); ESI/MS: 289 [M+H]+; IR (KBr disk): 3358, 2953, 2709,
1726, 1697, 1612, 1417, 1323, 1296, 1218, 976, 751 cmꢁ1 1H
.
C25H27N3O3: C, 71.92; H, 6.52; N, 10.06. Found: C, 71.73; H, 6.36;
NMR (500 MHz,DMSO) dH = 7.17 (t, J = 7.3 Hz, 1H), 7.15 (d,
J = 7.3 Hz, 1H), 7.12 (d, J = 7.3 Hz, 1H), 7.09 (t, J = 7.3 Hz, 1H), 4.09
(s, 2H), 3.98 (d, J = 14.0 Hz, 1H), 3.76 (s, 2H), 3.672 (s, 3H), 2.99
(dd, J = 3.5 Hz, J = 15.5 Hz, 1H), 2.70 (dd, J = 12.0 Hz, J = 15.0 Hz,
1H), 1.36 (s, 3H), 1.24 (s, 3H); 13C NMR (125 MHz, DMSO)
dC = 18.07, 24.93, 31.02, 33.82, 46.64, 52.36, 56.65, 78.08, 126.31,
126.61, 127.23, 129.93, 133.75, 135.20, 169.80, 171.51. Elemental
Anal. Calcd for C16H20N2O3: C, 66.65; H, 6.99; N, 9.72. Found: C,
66.46; H, 6.85; N, 7.20.
N, 10.74.
4.1.5.9.
(S)-2-((S)-3,3-Dimethyl-1-oxo-10,10a-dihydroimidazo
[1,5-b]isoquinolin-2(1H,3H,5H)-yl)succinic acid dimethylester
(4i). Yield: 60%, colorless powder, mp 134–136 °C.
½
a 2D0
=
ꢂ
ꢁ131.12 (c 0.25, CHCl3); ESI/MS: 361 [M+H]+; IR (KBr disk):
3402, 2984, 2778, 1740, 1694, 1558, 1436, 1373, 1257, 1232,
1169, 756 cmꢁ1
.
1H NMR (500 MHz, DMSO) dH = 7.16 (t,
J = 7.8 Hz, 1H), 7.14 (d, J = 7.8 Hz, 1H), 7.10 (d, J = 7.8 Hz, 1H),
7.07 (t, J = 7.8 Hz, 1H), 4.43 (t, J = 5.0 Hz, 1H), 3.97 (d, J = 15.0 Hz,
1H), 3.74 (d, J = 15.0 Hz, 1H), 3.65 (s, 3H), 3.64 (s, 3H), 3.33 (dd,
J = 5.0 Hz, J = 15.0 Hz, 1H), 3.27 (dd, J = 10.0 Hz, J = 15.0 Hz, 1H),
2.93 (dd, J = 5.0 Hz, J = 15.0 Hz, 1H), 2.80 (dd, J = 5.0 Hz,
J = 15.0 Hz, 1H), 2.64 (dd, J = 10.0 Hz, J = 15.0 Hz, 1H), 1.45 (s, 3H),
1.24 (s, 3H); 13C NMR (125 MHz, DMSO) dC = 19.12, 24.93, 30.79,
34.81, 46.64, 50.51, 52.18, 53.06, 56.24, 78.72, 126.31, 126.63,
127.19, 129.91, 133.62, 135.07, 170.26, 171.38, 171.45. Elemental
Anal. Calcd for C19H24N2O5: C, 63.32; H, 6.71; N, 7.77. Found: C,
63.53; H, 6.87; N, 7.56.
4.1.5.6. (S)-2-((S)-3,3-Dimethyl-1-oxo-10,10a-dihydroimidazo-
[1,5-b]isoquinolin-2(1H,3H,5H)-yl)-3-phenylpropanoic acid me-
thylester (4f). Yield: 51%, colorless powder, mp 191–193 °C.
½
a 2D0
ꢂ
= ꢁ96.18 (c 0.25, CHCl3); ESI/MS: 379 [M+H]+; IR (KBr disk):
3423, 2923, 2774, 1745, 1698, 1457, 1434, 1417, 1372, 1245, 1220,
1177, 745, 703 cmꢁ1 1H NMR (500 MHz, DMSO) dH = 7.23 (t,
.
J = 7.5 Hz, 2H), 7.18 (d, J = 7.5 Hz, 2H), 7.10 (t, J = 7.5 Hz, 1H), 7.13
(t, J = 7.4 Hz, 1H), 7.11 (d, J = 7.4 Hz, 1H), 7.09 (d, J = 7.4 Hz, 1H),
7.06 (t, J = 7.4 Hz, 1H), 4.36 (dd, J = 4.5 Hz, 1H), 3.82 (d, J = 14.0 Hz,
1H), 3.68 (s, 3H), 3.53 (t, J = 13.5 Hz, 1H), 3.32 (d, J = 4.5 Hz, 2H),

878
H. Wang et al. / Bioorg. Med. Chem. 19 (2011) 871–882
4.1.5.10. (S)-2-((S)-3,3-Dimethyl-1-oxo-10,10a-dihydroimidazo
[1,5-b]isoquinolin-2(1H,3H,5H)-yl)pentanedioic acid dimethy-
lester (4j). Yield: 56%, colorless powder, mp 181–182 °C.
J = 15.5 Hz, 1H), 1.45 (s, 3H), 1.25 (s, 3H); 13C NMR (125 MHz,
DMSO) dC = 24.92, 25.04, 31.13, 34.18, 49.74, 51.96, 52.45, 58.86,
78.67, 126.54, 127.03, 127.23, 127.31, 134.00, 135.69, 172.40,
172.81, 173.86. Elemental Anal. Calcd for C18H23N3O4: C, 62.59;
H, 6.71; N, 12.17. Found: C, 64.82; H, 7.12; N, 8.61.
½
a 2D0
ꢂ
= ꢁ140.20 (c 0.25, CHCl3); ESI/MS: 375 [M+H]+; IR (KBr disk):
3423, 2988, 2769, 1745, 1684, 1423, 1357, 1268, 1173, 748 cmꢁ1
.
1H NMR (500 MHz, DMSO) dH = 7.24 (t, J = 7.8 Hz, 1H), 7.22 (d,
J = 7.8 Hz, 1H), 7.18 (d, J = 7.8 Hz, 1H), 7.12 (t, J = 7.8 Hz, 1H), 4.25
(m, 1H), 3.95 (d, J = 15.0 Hz, 1H), 3.72 (d, J = 15.0 Hz, 1H), 3.64 (s,
3H), 3.62 (s, 3H), 3.35 (dd, J = 5.0 Hz, J = 15.0 Hz, 1H), 3.29 (dd,
J = 10.0 Hz, J = 15.0 Hz, 1H), 2.95 (dd, J = 5.0 Hz, J = 15.0 Hz, 1H),
2.83 (dd, J = 5.0 Hz, J = 15.0 Hz, 1H), 2.68 (dd, J = 10.0 Hz,
J = 15.0 Hz, 1H), 1.38 (s, 3H), 2.34 (m, 1H), 2.21 (m, 1H), 1.29 (s,
3H); 13C NMR (125 MHz, DMSO) dC = 24.22, 24.56, 24.84, 28.62,
29.33, 39.44, 55.25, 55.56, 59.81, 78.54, 125.98, 126.43, 126.67,
127.48, 131.33, 131.99, 168.54, 170.91, 171.48, 172.10. Elemental
Anal. Calcd for C20H26N2O5: C, 64.15; H, 7.00; N, 7.48. Found: C,
64.33; H, 6.85; N, 7.70.
4.1.5.14.
(S)-5-Amino-2-((S)-3,3-dimethyl-1-oxo-10,10a-dihy-
droimidazo[1,5-b]-isoquinolin-2(1H,3H,5H)-yl)-5-oxopentanoic
acid methylester (4n). Yield: 42%, colorless powder, mp 159–
161 °C. ½a 2D0
ꢂ
= ꢁ140.20 (c 0.25, CHCl3); ESI/MS: 360 [M+H]+; IR
(KBr disk): 3356, 2955, 2748, 1740, 1684, 1436, 1364, 1236, 1174,
1123, 748 cmꢁ1. 1H NMR (500 MHz, DMSO) dH = 7.18 (d, J = 7.8 Hz,
1H), 7.16 (d, J = 7.8 Hz, 1H), 7.14 (t, J = 7.8 Hz, 1H), 7.12 (t,
J = 7.8 Hz, 1H), 6.03 (s, 2H), 4.16 (t, J = 7.0 Hz, 1H), 3.96 (d,
J = 14.5 Hz, 1H), 3.64 (s, 3H), 3.62 (s, 2H), 3.37 (dd, J = 4.0 Hz,
J = 11.0 Hz, 1H), 2.92 (dd, J = 3.5 Hz, J = 15.5 Hz, 1H), 2.65 (dd,
J = 11.0 Hz, J = 15.5 Hz, 1H), 2.48 (m, J = 7.0 Hz, 1H), 2.38 (m,
J = 7.0 Hz, 2H), 1.379 (s, 3H), 1.291 (s, 3H); 13C NMR (125 MHz,
DMSO) dC = 24.07, 24.83, 25.81, 30.89, 33.80, 47.92, 51.20, 56.53,
56.77, 78.58, 124.67, 126.19, 126.56, 127.12, 133.73, 135.08,
163.79, 171.01, 171.56, 172.86. Elemental Anal. Calcd for
4.1.5.11. (S)-6-(Benzyloxycarbonyl)-2-((S)-3,3-dimethyl-1-oxo-
10,10a-dihydro-imidazo[1,5-b]isoquinolin-2(1H,3H,5H)-yl)hex-
anoic acid methylester (4k). Yield: 42%, colorless powder, mp
136–138 °C. ½a 2D0
ꢂ
= ꢁ114.32 (c 0.25, CHCl3); ESI/MS: 494 [M+H]+; IR
C19H25N3O4: C, 63.49; H, 7.01; N, 11.69. Found: C, 63.28; H, 7.17;
N, 11.91.
(KBr disk): 3623, 3426, 2958, 2773, 1744, 1697, 1459, 1367, 1262,
1205, 1096, 750 cmꢁ1 1H NMR (500 MHz, DMSO) dH = 8.31 (t,
.
J = 9.0 Hz, 1H), 7.22 (t, J = 9.0 Hz, 1H), 7.20 (t, J = 9.0 Hz, 2H), 7.19 (t,
J = 7.8 Hz, 2H), 7.18 (d, J = 7.8 Hz, 1H), 7.17 (d, J = 7.8 Hz, 1H), 7.10 (t,
J = 7.8 Hz, 1H), 7.07 (t, J = 7.8 Hz, 1H), 5.10 (s, 2H), 4.12 (t, J = 6.0 Hz,
1H), 3.96 (d, J = 14.0 Hz, 1H), 3.74 (d, J = 14.5 Hz, 1H), 3.64 (s, 3H),
3.29 (dd, J = 10.0 Hz, J = 15.0 Hz, 1H), 3.21 (m, J = 6.0 Hz, 2H), 2.95
(dd, J = 5.0 Hz, J = 15.0 Hz, 1H), 2.68 (dd, J = 10.0 Hz, J = 15.0 Hz, 1H),
1.85 (m, J = 6.0 Hz, 2H), 1.65 (m, J = 6.0 Hz, 2H), 1.40 (s, 3H), 1.38 (s,
3H), 1.21 (m, J = 6.0 Hz, 2H); 13C NMR (125 MHz, DMSO) dC = 21.40,
27.23, 27.15, 28.45, 29.12, 29.72, 41.95, 51.93, 53.56, 55.01, 59.21,
69.56, 126.25, 126.60, 127.22, 127.38, 128.54, 129.91, 133.38,
133.80, 135.19, 135.28, 136.52, 170.15, 172.04, 172.53. Elemental
Anal. Calcd for C28H35N3O5: C, 68.13; H, 7.15; N, 8.51. Found: C,
64.33; H, 6.85; N, 7.70.
4.1.5.15. (S)-2-((S)-3,3-Dimethyl-1-oxo-10,10a-dihydroimidazo
[1,5-b]isoquinolin-2(1H,3H,5H)-yl)-6-(3-nitroguanidino)hexa-
noic acid methylester (4o). Yield: 43%, colorless powder, mp
186–188 °C. ½a 2D0
ꢂ
= ꢁ147.13 (c 0.25, CHCl3); ESI/MS: 433 [M+H]+;
IR (KBr disk): 3356, 3264, 2933, 2772, 1742, 1674, 1516, 1456,
1434, 1360, 1260, 1233, 1190, 744 cmꢁ1 1H NMR (500 MHz,
.
DMSO) dH = 9.12 (s, 1H), 7.82 (s, 1H), 7.17 (d, J = 7.8 Hz, 1H), 7.14
(d, J = 7.8 Hz, 1H), 7.12 (t, J = 7.8 Hz, 1H), 7.10 (t, J = 7.8 Hz, 1H),
4.24 (dd, J = 4.0 Hz, J = 10.5 Hz, 1H), 3.830 (d, J = 14.0 Hz, 1H),
3.682 (s, 3H), 3.55 (d, J = 14.0 Hz, 1H), 3.46 (dd, J = 10.0 Hz,
J = 15.0 Hz, 1H), 3.20 (dd, J = 5.0 Hz, J = 14.0 Hz, 1H), 3.14 (dd,
J = 5.0 Hz, J = 10.0 Hz, 1H), 2.94 (dd, J = 4.0 Hz, J = 15.0 Hz, 1H),
2.68 (t, J = 4.0 Hz, 2H), 1.98 (d, J = 4.0 Hz, 2H), 1.58 (m, J = 4.0 Hz,
2H), 1.29 (s, 3H), 0.94 (s, 3H); 13C NMR (125 MHz, DMSO)
dC = 17.92, 24.21, 30.91, 33.83, 46.72, 52.59, 56.56, 56.91, 78.70,
115.52, 126.22, 126.59, 127.14, 127.23, 128.52, 128.69, 131.14,
133.70, 135.09, 135.24, 156.47, 170.96, 171.42. Elemental Anal.
Calcd for C20H28N6O5: C, 55.54; H, 6.53; N, 19.43. Found: C,
55.73; H, 6.68; N, 19.22.
4.1.5.12. (2S)-2-((S)-3,3-Dimethyl-1-oxo-10,10a-dihydroimidazo-
[1,5-b]isoquinolin-2(1H,3H,5H)-yl)-3-hydroxybutanoic acid me-
thylester (4l). Yield: 42%, colorless powder, mp 201–203 °C.
½
a 2D0
ꢂ
= ꢁ59.20 (c 0.25, CHCl3); ESI/MS: 333 [M+H]+; IR (KBr disk):
3408, 2925, 2850, 1743, 1694, 1671, 1626, 1575, 1401, 1359,
1244, 1123, 747 cmꢁ1 1H NMR (500 MHz, DMSO) dH = 7.22 (d,
.
J = 7.8 Hz, 1H), 7.20 (d, J = 7.8 Hz, 1H), 7.18 (t, J = 7.8 Hz, 1H), 7.15
(t, J = 7.8 Hz, 1H), 4.28 (d, J = 7.0 Hz, 1H), 3.97 (d, J = 15.0 Hz, 1H),
3.73 (d, J = 15.0 Hz, 1H), 3.63 (s, 3H), 3.41 (m, 1H), 3.21 (dd,
J = 4.0 Hz, J = 10.5 Hz, 1H), 2.97 (dd, J = 5.0 Hz, J = 15.5 Hz, 1H), 2.68
(dd, J = 11.0 Hz, J = 15.0 Hz, 1H), 1.39 (s, 3H), 1.277 (s, 3H), 1.21 (d,
J = 7.0 Hz, 3H), 1.92 (s, 1H); 13C NMR (125 MHz, DMSO) dC = 19.74,
24.84, 25.87, 33.86, 44.66, 47.86, 51.87, 55.66, 77.55, 126.67,
127.08, 127.96, 129.24, 132.91, 133.61, 174.11, 175.89. Elemental
Anal. Calcd for C18H24N2O4: C, 65.04; H, 7.28; N, 8.43. Found: C,
64.82; H, 7.12; N, 8.61.
4.1.5.16. (S)-2-((S)-3,3-Dimethyl-1-oxo-10,10a-dihydroimidazo
[1,5-b]isoquinolin-2(1H,3H,5H)-yl)-4-(methylthio)butanoic acid
methylester (4p). Yield: 37%, colorless powder, mp 174–176 °C.
½
a 2D0
ꢂ
= ꢁ89.13 (c 0.25, CHCl3); ESI/MS: 363 [M+H]+; IR (KBr disk):
3422, 2997, 2787, 1733, 1705, 1456, 1418, 1350, 1268, 1240,
1148, 752 cmꢁ1
.
1H NMR (500 MHz, DMSO) dH = 7.16 (d,
J = 7.8 Hz, 1H), 7.13 (d, J = 7.8 Hz, 1H), 7.10 (t, J = 7.8 Hz, 1H), 7.08
(t, J = 7.8 Hz, 1H), 4.21 (t, J = 6.5 Hz, 1H), 3.96 (d, J = 14.5 Hz, 1H),
3.74 (d, J = 14.5 Hz, 1H), 3.65 (s, 3H), 3.36 (dd, J = 4.0 Hz,
J = 11.0 Hz, 1H), 2.93 (dd, J = 3.5 Hz, J = 15.5 Hz, 1H), 2.66 (dd,
J = 11.5 Hz, J = 15.5 Hz, 1H), 2.54 (d, J = 6.5 Hz, 2H), 2.32 (t,
J = 6.5 Hz, 2H), 2.12 (s, 3H), 1.39 (s, 3H), 1.30 (s, 3H); 13C NMR
(125 MHz, DMSO) dC = 14.95, 19.40, 24.83, 29.14, 30.93, 46.78,
52.71, 52.90, 56.31, 78.67, 126.28, 126.62, 127.18, 129.92, 133.76,
135.15, 171.18, 171.31. Elemental Anal. Calcd for C19H26N2O3S: C,
62.96; H, 7.23; N, 7.73. Found: C, 62.77; H, 7.38; N, 7.50.
4.1.5.13.
(S)-4-Amino-2-((S)-3,3-dimethyl-1-oxo-10,10a-dihy-
droimidazo[1,5-b]-isoquinolin-2(1H,3H,5H)-yl)-4-oxobutanoic
acid methylester (4m). Yield: 42%, colorless powder, mp 183–
185 °C. ½a 2D0
ꢂ
= ꢁ109.20 (c 0.25, CHCl3); ESI/MS: 346 [M+H]+; IR
(KBr disk): 3398, 2945, 2763, 1740, 1694, 1436, 1356, 1257,
1168, 751 cmꢁ1
.
1H NMR (500 MHz, DMSO) dH = 7.17 (d,
J = 7.8 Hz, 1H), 7.14 (d, J = 7.8 Hz, 1H), 7.12 (t, J = 7.8 Hz, 1H), 7.10
(t, J = 7.8 Hz, 1H), 6.01 (s, 2H), 4.43 (t, J = 7.0 Hz, 1H), 3.97 (d,
J = 14.5 Hz, 1H), 3.65 (s, 3H), 3.34 (d, J = 7.0 Hz, 2H), 3.30 (dd,
J = 8.0 Hz, J = 11.0 Hz, 1H), 2.93 (dd, J = 3.5 Hz, J = 15.5 Hz, 1H),
2.81 (dd, J = 5.5 Hz, J = 15.5 Hz, 1H), 2.65 (dd, J = 11.5 Hz,
4.1.5.17. (R)-2-((S)-3,3-Dimethyl-1-oxo-10,10a-dihydroimidazo-
[1,5-b]isoquinolin-2(1H,3H,5H)-yl)-3-mercaptopropanoic acid
methylester (4q). Yield: 40%, colorless powder, mp 146–148 °C.
½
a 2D0
ꢂ
= ꢁ89.13 (c 0.25, CHCl3); ESI/MS: 335 [M+H]+; IR (KBr disk):
3356, 2987, 2787, 1740, 1673, 1418, 1350, 1289, 1240, 1176,

H. Wang et al. / Bioorg. Med. Chem. 19 (2011) 871–882
879
1101, 748 cmꢁ1. 1H NMR (500 MHz, DMSO) dH = 7.16 (d, J = 7.7 Hz,
1H), 7.14 (d, J = 7.7 Hz, 1H), 7.11 (t, J = 7.7 Hz, 1H), 7.09 (t,
J = 7.7 Hz, 1H), 4.36 (t, J = 7.0 Hz, 1H), 3.98 (d, J = 14.0 Hz, 1H),
3.76 (d, J = 13.5 Hz, 1H), 3.68 (s, 3H), 3.56 (dd, J = 6.5 Hz,
J = 13.5 Hz, 1H), 3.33 (d, J = 7.0 Hz, 2H), 2.92 (dd, J = 3.5 Hz,
J = 15.5 Hz, 1H), 2.65 (dd, J = 12.5 Hz, J = 15.5 Hz, 1H), 2.54 (s, 1H),
1.44 (s, 3H), 1.31 (s, 3H); 13C NMR (125 MHz, DMSO) dC = 14.95,
19.40, 24.83, 29.14, 30.93, 46.78, 51.18, 52.71, 52.90, 56.31,
78.67, 126.28, 126.62, 127.18, 129.92, 133.76, 135.15, 171.18,
171.31. Elemental Anal. Calcd for C17H22N2O3S: C, 61.05; H, 6.63;
N, 8.38. Found: C, 60.84; H, 6.47; N, 8.61.
1682, 1456, 1428, 1386, 1259, 1217, 1191, 746 cmꢁ1
.
1H NMR
(300 MHz, DMSO) dH = 11.07 (s, 1H), 7.21 (d, J = 7.8 Hz, 1H), 7.18
(d, J = 7.8 Hz, 1H), 7.15 (t, J = 7.8 Hz, 1H), 7.11 (t, J = 7.8 Hz, 1H),
3.94 (t, J = 6.5 Hz, 1H), 3.98 (d, J = 7.2 Hz, 1H), 3.72 (d, J = 14.2 Hz,
1H), 3.31 (dd, J = 3.7 Hz, J = 11.1 Hz, 1H), 2.92 (dd, J = 3.7 Hz,
J = 16.1 Hz, 1H), 2.64 (dd, J = 11.4 Hz, J = 16.3 Hz, 1H), 2.22 (m,
J = 6.5 Hz, 1H), 1.69 (m, J = 6.5 Hz, 2H), 1.39 (s, 3H), 1.21 (s, 3H),
0.96 (t, J = 6.5 Hz, 6H); 13C NMR (75 MHz, DMSO) dC = 11.89,
16.02, 25.04, 26.53, 30.89, 31.08, 37.32, 46.29, 55.60, 56.76,
78.53, 126.24, 126.38, 127.18, 129.20, 134.34, 135.35, 171.97,
173.40. Elemental Anal. Calcd for C19H26N2O3: C, 69.06; H, 7.93;
N, 8.48. Found: C, 69.27; H, 7.77; N, 8.70.
4.1.6. General procedure for preparing 2-substituted (S)-2-(3,3-
dimethyl-1-oxo-10,10a-dihydroimidazo[1,5-b]isoquinolin-
2(1H,3H,5H)-yl)-acetic acids (5a–q)
4.1.6.4.
(S)-2-((S)-3,3-Dimethyl-1-oxo-10,10a-dihydroimidazo
[1,5-b]isoquinolin-2(1H,3H,5H)-yl)propanoic acid (5d). Yield:
At 0 °C to the solution of 1.45 mmol of 2-substituted (S)-2-(3,3-
dimethyl-1-oxo-10,10a-dihydroimidazo[1,5-b]isoquinolin-2(1H,-
3H,5H)-yl)-acetic acid methylesters (4) in 5 mL of methanol, 7 mL
of 2 N NaOH was added to adjust pH 11. The reaction mixture
was stirred at 0 °C for 3 h and TLC (CCl3/CH3OH, 5:1) indicated
the complete disappearance of 4. The reaction mixture was ad-
justed to pH 7 with aqueous solution of KHSO4. The solution was
evaporated under vacuum to remove methanol, adjusted to pH 2
with aqueous solution of KHSO4, and extracted with ethyl acetate
(30 mL ꢀ 3). The combined ethyl acetate was successively washed
with saturated aqueous solution of NaCl (20 mL ꢀ 2) and dried
with anhydrous Na2SO4. After filtration, the filtrate was evaporated
to provide 5a–q.
92%, colorless powder, mp 197–199 °C. ½a D20
= ꢁ45.06 (c 0.25,
ꢂ
CH3OH); ESI/MS: 289 [M+H]+; IR (KBr disk): 2978, 1708, 1450,
1424, 1378, 1364, 1258, 1220, 739 cmꢁ1 1H NMR (300 MHz,
.
DMSO) dH = 11.13 (s, 1H), 7.15 (d, J = 7.8 Hz, 1H), 7.13 (d,
J = 7.8 Hz, 1H), 7.10 (t, J = 7.8 Hz, 1H), 7.08 (t, J = 7.8 Hz, 1H), 4.13
(q, J = 6.9 Hz, 1H), 3.95 (d, J = 14.4 Hz, 1H), 3.62 (d, J = 14.4 Hz,
1H), 3.29 (dd, J = 3.3 Hz, J = 11.1 Hz, 1H), 2.89 (dd, J = 3.3 Hz,
J = 15.6 Hz, 1H), 2.59 (dd, J = 11.4 Hz, J = 15.6 Hz, 1H), 1.43 (d,
J = 6.9 Hz, 3H), 1.40 (s, 3H), 1.26 (s, 3H); 13C NMR (75 MHz, DMSO)
dC = 15.68, 18.97, 24.94, 30.90, 46.69, 49.00, 56.68, 78.50, 126.25,
126.58, 127.20, 129.94, 133.84, 135.25, 170.24, 172.61. Elemental
Anal. Calcd for C16H20N2O3: C, 66.65; H, 6.99; N, 9.72. Found: C,
66.44; H, 6.83; N, 9.94.
4.1.6.1.
(S)-2-((S)-3,3-Dimethyl-1-oxo-10,10a-dihydroimidazo
4.1.6.5. (S)-2-(3,3-Dimethyl-1-oxo-10,10a-dihydroimidazo[1,5-
b]isoquinolin-2(1H,3H,5H)-yl)acetic acid (5e). Yield: 86%, color-
[1,5-b]isoquinolin-2(1H,3H,5H)-yl)-4-methylpentanoic
(5a). Yield: 88%, colorless powder, mp 188–190 °C.
acid
½
a 2D0
ꢂ
=
less powder, mp 186–188 °C. ½a D20
= ꢁ127.63 (c 0.25, CH3OH); ESI/
ꢂ
ꢁ41.23 (c 0.25, CH3OH); ESI/MS: 331 [M+H]+; IR (KBr disk):
MS: 275 [M+H]+; IR (KBr disk): 2958, 1713, 1450, 1423, 1372, 1366,
1207, 744 cmꢁ1. 1H NMR (300 MHz, DMSO) dH = 11.11 (s, 1H), 7.21
(d, J = 7.8 Hz, 1H), 7.19 (d, J = 7.8 Hz, 1H), 7.15 (t, J = 7.8 Hz, 1H),
7.12 (t, J = 7.8 Hz, 1H), 3.97 (d, J = 14.2 Hz, 1H), 3.93 (s, 2H), 3.74
(d, J = 14.1 Hz, 1H), 3.296 (dd, J = 3.6 Hz, J = 10.8 Hz, 1H), 2.978
(dd, J = 3.9 Hz, J = 15.6 Hz, 1H), 2.68 (dd, J = 11.7 Hz, J = 15.0 Hz,
1H), 1.35 (s, 3H), 1.22 (s, 3H); 13C NMR (75 MHz, DMSO)
dC = 25.05, 26.38, 31.06, 46.66, 56.74, 63.34, 77.99, 126.27,
126.87, 128.49, 129.92, 133.92, 135.26, 170.72, 171.28. Elemental
Anal. Calcd for C15H18N2O3: C, 65.68; H, 6.61; N, 10.21. Found: C,
65.87; H, 6.78; N, 10.00.
2963, 1731, 1663, 1464, 1424, 1365, 1261, 1228, 1191, 745 cmꢁ1
.
1H NMR (300 MHz, DMSO) dH = 11.11 (s, 1H), 7.17 (d, J = 7.8 Hz,
1H), 7.15 (d, J = 7.8 Hz, 1H), 7.12 (t, J = 7.8 Hz, 1H), 7.08 (t,
J = 7.8 Hz, 1H), 3.95 (d, J = 14.1 Hz, 1H), 3.88 (t, J = 6.9 Hz, 1H),
3.72 (d, J = 14.4 Hz, 1H), 3.31 (dd, J = 3.6 Hz, J = 10.8 Hz, 1H), 2.89
(dd, J = 3.6 Hz, J = 16.2 Hz, 1H), 2.65 (dd, J = 11.4 Hz, J = 15.6 Hz,
1H), 1.96 (t, J = 6.9 Hz, 2H), 1.69 (m, 1H), 1.40 (s, 3H), 1.19 (s,
3H), 0.92 (t, J = 6.0 Hz, 6H); 13C NMR (75 MHz, DMSO) dC = 19.21,
21.92, 22.70, 23.25, 24.72, 30.97, 46.41, 55.72, 56.49, 78.76,
126.23, 126.34, 126.58, 129.24, 134.29, 135.22, 170.95, 172.78. Ele-
mental Anal. Calcd for C19H26N2O3: C, 69.06; H, 7.93; N, 8.48.
Found: C, 69.25; H, 7.79; N, 8.69.
4.1.6.6. (S)-2-((S)-3,3-Dimethyl-1-oxo-10,10a-dihydroimidazo-
[1,5-b]isoquinolin-2(1H,3H,5H)-yl)-3-phenylpropanoic acid (5f).
4.1.6.2. (S)-2-((S)-3,3-Dimethyl-1-oxo-10,10a-dihydroimidazo-
Yield: 94%, colorless powder, mp 190–192 °C. ½a D20
= ꢁ114.20 (c 0.25,
ꢂ
[1,5-b]isoquinolin-2(1H,3H,5H)-yl)-3-methylbutanoic
acid
CH3OH); ESI/MS: 365 [M+H]+; IR (KBr disk): 2978, 2956, 2774, 1744,
1698, 1457, 1433, 1417, 1372, 1245, 1219, 1170, 745, 703 cmꢁ1. 1H
NMR (500 MHz, DMSO) dH = 11.15 (s, 1H), 7.25 (t, J = 7.5 Hz, 2H),
7.22 (d, J = 7.5 Hz, 2H), 7.20 (t, J = 7.5 Hz, 1H), 7.18 (t, J = 7.4 Hz, 1H),
7.16 (d, J = 7.4 Hz, 1H), 7.13 (d, J = 7.4 Hz, 1H), 7.10 (t, J = 7.4 Hz, 1H),
4.18 (dd, J = 5.0 Hz, J = 10.0 Hz, 1H), 3.85 (d, J = 15.0 Hz, 1H), 3.59 (t,
J = 10.0 Hz, 1H), 3.51 (d, J = 15.0 Hz, 1H), 3.28 (dd, J = 5.0 Hz,
J = 15.0 Hz, 1H), 3.18 (dd, J = 5.0 Hz, J = 10.0 Hz, 1H), 2.88 (dd,
J = 5.0 Hz, J = 15.0 Hz, 1H), 2.64 (dd, J = 10.0 Hz, J = 15.0 Hz, 1H), 1.23
(s, 3H), 0.82 (s, 3H); 13C NMR (75 MHz, DMSO) dC = 24.67, 30.85,
34.15, 46.71, 56.68, 56.96, 78.57, 126.20, 126.58, 126.91, 127.13,
128.49, 128.68, 128.86, 129.89, 130.19, 130.60, 133.71, 135.09,
139.33, 171.30, 171.81. Elemental Anal. Calcd for C22H24N2O3: C,
72.50; H, 6.64; N, 7.69. Found: C, 72.69; H, 6.80; N, 7.47.
(5b). Yield: 92%, colorless powder, mp 174–176 °C. ½a D20
ꢂ
= ꢁ8.89
(c 0.25, CH3OH); ESI/MS: 317 [M+H]+; IR (KBr disk): 2968, 1728,
1669, 1443, 1417, 1359, 1263, 1226, 1170, 743 cmꢁ1 1H NMR
.
(300 MHz, DMSO) dH = 11.09 (s, 1H), 7.20 (d, J = 7.8 Hz, 1H), 7.19
(d, J = 7.8 Hz, 1H), 7.14 (t, J = 7.8 Hz, 1H), 7.10 (t, J = 7.8 Hz, 1H),
3.94 (d, J = 14.1 Hz, 1H), 3.99 (d, J = 6.9 Hz, 1H), 3.69 (d,
J = 14.2 Hz, 1H), 3.30 (dd, J = 3.6 Hz, J = 11.2 Hz, 1H), 2.89 (dd,
J = 3.7 Hz, J = 16.2 Hz, 1H), 2.65 (dd, J = 11.3 Hz, J = 16.1 Hz, 1H),
1.84 (m, 1H), 1.39 (s, 3H), 1.24 (s, 3H), 0.98 (t, J = 6.0 Hz, 6H); 13C
NMR (75 MHz, DMSO) dC = 21.12, 21.22, 22.49, 23.40, 28.07,
56.45, 56.76, 65.38, 81.74, 127.37, 127.50, 128.57, 128.69, 132.94,
133.34, 171.18, 171.77. Elemental Anal. Calcd for C18H24N2O3: C,
68.33; H, 7.65; N, 8.85. Found: C, 68.52; H, 7.81; N, 8.63.
4.1.6.3. (2S)-2-((S)-3,3-Dimethyl-1-oxo-10,10a-dihydroimidazo-
[1,5-b]isoquinolin-2(1H,3H,5H)-yl)-3-methylpentanoic acid (5c).
4.1.6.7.
(S)-2-((S)-3,3-Dimethyl-1-oxo-10,10a-dihydroimidazo
[1,5-b]isoquinolin-2(1H,3H,5H)-yl)-3-(4-hydroxyphenyl)propa-
Yield: 93%, colorless powder, mp 186–188 °C. ½a D20
= ꢁ22.47 (c
ꢂ
noic acid (5g). Yield: 92%, colorless powder, mp 196–198 °C.
0.25, CH3OH); ESI/MS: 331 [M+H]+; IR (KBr disk): 2965, 1735,
½
a 2D0
ꢂ
= ꢁ27.07 (c 0.25, CH3OH); ESI/MS: 381 [M+H]+; IR (KBr disk):

880
H. Wang et al. / Bioorg. Med. Chem. 19 (2011) 871–882
3236, 2929, 2774, 1716, 1683, 1626, 1576, 1516, 1449, 1372, 1260,
1245, 745 cmꢁ1. 1H NMR (300 MHz, DMSO) dH = 11.07 (s, 1H), 7.12
(d, J = 7.8 Hz, 2H), 7.10 (d, J = 7.8 Hz, 2H), 7.08 (t, J = 7.6 Hz, 1H),
7.07 (d, J = 7.6 Hz, 1H), 7.05 (d, J = 7.6 Hz, 1H), 7.03 (t, J = 7.6 Hz,
1H), 5.22 (s, 1H), 4.08 (dd, J = 6.6 Hz, J = 10.8 Hz, 1H), 3.84 (d,
J = 15.6 Hz, 1H), 3.58 (t, J = 11.1 Hz, 1H), 3.41 (d, J = 15.9 Hz, 1H),
3.27 (dd, J = 6.6 Hz, J = 15.6 Hz, 1H), 3.21 (dd, J = 6.9 Hz, J = 7.5 Hz,
1H), 2.88 (dd, J = 6.9 Hz, J = 15.9 Hz, 1H), 2.64 (dd, J = 7.8 Hz,
J = 15.3 Hz, 1H), 1.25 (s, 6H); 13C NMR (75 MHz, DMSO)
dC = 24.69, 24.92, 30.87, 33.77, 46.74, 56.40, 57.40, 78.64, 115.19,
115.39, 126.55, 127.14, 129.39, 129.88, 130.00, 131.03, 131.49,
133.76, 135.14, 156.21, 171.14, 171.99. Elemental Anal. Calcd for
4.1.6.11. (S)-6-Amino-2-((S)-3,3-dimethyl-1-oxo-10,10a-dihydro-
imidazo[1,5-b]-isoquinolin-2(1H,3H,5H)-yl)hexanoic acid (5k).
Yield: 87%, colorless powder, mp 141–143 °C. ½a D20
= ꢁ = ꢁ107.07
ꢂ
(c 0.25, CH3OH); ESI/MS: 346 [M+H]+; IR (KBr disk): 3648, 3326,
2930, 2774, 1735, 1627, 1596, 1558, 1423, 1205, 750, 574 cmꢁ1
.
1H NMR (500 MHz, DMSO) dH = 11.84 (s, 1H), 8.31 (t, J = 9.0 Hz,
2H), 7.16 (d, J = 7.8 Hz, 1H), 7.14 (d, J = 7.8 Hz, 1H), 7.12 (t,
J = 7.8 Hz, 1H), 7.09 (t, J = 7.8 Hz, 1H), 4.12 (t, J = 6.0 Hz, 1H), 3.96
(d, J = 14.0 Hz, 1H), 3.74 (d, J = 14.5 Hz, 1H), 3.29 (dd, J = 10.0 Hz,
J = 15.0 Hz, 1H), 3.22 (m, J = 6.0 Hz, 2H), 2.95 (dd, J = 5.0 Hz,
J = 15.0 Hz, 1H), 2.68 (dd, J = 10.0 Hz, J = 15.0 Hz, 1H), 1.86 (m,
J = 6.0 Hz, 2H), 1.65 (m, J = 6.0 Hz, 2H), 1.391 (s, 3H), 1.378 (s,
3H), 1.221 (m, J = 6.0 Hz, 2H); 13C NMR (125 MHz, DMSO)
dC = 21.28, 27.24, 27.18, 28.58, 29.25, 29.80, 41.97, 53.44, 55.11,
59.27, 69.58, 126.34, 127.38, 128.59, 133.45, 133.80, 136.52,
170.22, 172.57. Elemental Anal. Calcd for C19H27N3O3: C, 66.06;
H, 7.88; N, 12.16. Found: C, 65.87; H, 7.73; N, 12.37.
C22H24N2O4: C, 69.46; H, 6.36; N, 7.36. Found: C, 69.27; H, 6.20;
N, 7.55.
4.1.6.8.
(S)-2-((S)-3,3-Dimethyl-1-oxo-10,10a-dihydroimidazo
[1,5-b]isoquinolin-2(1H,3H,5H)-yl)-3-(1H-indol-3-yl)propanoic
acid (5h). Yield: 86%, colorless powder, mp 175–177 °C.
½
a 2D0
ꢂ
= ꢁ112.12 (c 0.25, CH3OH); ESI/MS: 404 [M+H]+; IR (KBr disk):
4.1.6.12.
(2S)-2-((S)-3,3-Dimethyl-1-oxo-10,10a-dihydroimi-
3256, 2931, 2769, 1716, 1686, 1516, 1456, 1367, 1339, 1259, 1242,
dazo[1,5-b]isoquinolin-2(1H,3H,5H)-yl)-3-hydroxybutanoic acid
743 cmꢁ1
.
1H NMR (500 MHz, DMSO) dH = 11.90 (s, 1H), 10.91 (s,
(5l). Yield: 85%, colorless powder, mp 196–198 °C. ½a D20
= ꢁ29.20
ꢂ
1H), 7.56 (d, J = 5.0 Hz, 1H), 7.53 (d, J = 5.0 Hz, 1H), 7.34 (d,
J = 5.0 Hz, 1H), 7.30 (d, J = 5.0 Hz, 1H), 7.16 (t, J = 5.0 Hz, 1H), 7.14
(d, J = 7.5 Hz, 1H), 7.11 (d, J = 7.5 Hz, 1H), 6.98 (t, J = 7.5 Hz, 1H),
4.12 (dd, J = 5.0 Hz, J = 10.0 Hz, 1H), 3.95 (dd, J = 6.6 Hz,
J = 10.8 Hz, 1H), 3.79 (d, J = 10.0 Hz, 1H), 3.71 (m, 1H), 3.41 (d,
J = 10.0 Hz, 1H), 3.25 (m, 1H), 3.05 (dd, J = 3.0 Hz, J = 10.0 Hz, 1H),
2.94 (dd, J = 3.0 Hz, J = 10.0 Hz, 1H), 2.67 (dd, J = 10.0 Hz,
J = 15.0 Hz, 1H), 1.21 (s, 6H); 13C NMR (125 MHz, DMSO)
dC = 24.08, 24.79, 30.91, 33.81, 47.95, 56.45, 56.76, 65.38, 78.60,
111.47, 111.91, 118.43, 121.26, 124.68, 126.20, 126.57, 127.13,
128.10, 129.89, 133.73, 135.09, 136.47, 171.04, 172.07. Elemental
Anal. Calcd for C24H25N3O3: C, 71.44; H, 6.25; N, 10.41. Found: C,
71.26; H, 6.10; N, 10.62.
(c 0.25, CH3OH); ESI/MS: 319 [M+H]+; IR (KBr disk): 3328, 2928,
2850, 1671, 1626, 1575, 1401, 1244, 732, 599 cmꢁ1 1H NMR
.
(500 MHz, DMSO) dH = 11.37 (s, 1H), 7.21 (d, J = 7.8 Hz, 1H), 7.19
(d, J = 7.8 Hz, 1H), 7.17 (t, J = 7.8 Hz, 1H), 7.13 (t, J = 7.8 Hz, 1H),
4.28 (d, J = 7.0 Hz, 1H), 3.96 (d, J = 15.0 Hz, 1H), 3.73 (d,
J = 15.0 Hz, 1H), 3.42 (m, 1H), 3.22 (dd, J = 4.0 Hz, J = 10.5 Hz, 1H),
2.96 (dd, J = 5.0 Hz, J = 15.5 Hz, 1H), 2.69 (dd, J = 11.0 Hz,
J = 15.0 Hz, 1H), 1.92 (s, 1H), 1.41 (s, 3H), 1.28 (s, 3H), 1.23 (d,
J = 7.0 Hz, 3H); 13C NMR (125 MHz, DMSO) dC = 16.78, 17.66,
25.48, 30.87, 33.85, 46.79, 49.11, 56.78, 78.62, 126.37, 126.68,
127.32, 129.98, 133.82, 135.26, 170.82, 171.57. Elemental Anal.
Calcd for C17H22N2O4: C, 64.13; H, 6.97; N, 8.80. Found: C, 64.32;
H, 6.81; N, 8.58.
4.1.6.9.
(S)-2-((S)-3,3-Dimethyl-1-oxo-10,10a-dihydroimidazo
4.1.6.13.
(S)-4-Amino-2-((S)-3,3-dimethyl-1-oxo-10,10a-dihy-
[1,5-b]isoquinolin-2(1H,3H,5H)-yl)succinic acid (5i). Yield:
droimidazo[1,5-b]-isoquinolin-2(1H,3H,5H)-yl)-4-oxobutanoic
85%, colorless powder, mp 126–128 °C. ½a D20
= ꢁ120.36 (c 0.25,
ꢂ
acid (5m). Yield: 88%, colorless powder, mp 192–194 °C.
CH3OH); ESI/MS: 333 [M+H]+; IR (KBr disk): 2984, 1733, 1683,
½
a 2D0
ꢂ
= ꢁ169.10 (c 0.25, CH3OH); ESI/MS: 332 [M+H]+; IR (KBr disk):
1558, 1404, 1206, 751 cmꢁ1
.
1H NMR (500 MHz, DMSO) dH = 7.17
2996, 1688, 1609, 1558, 1456, 1141, 750, 622 cmꢁ1 1H NMR
.
(m, 4H), 4.42 (t, J = 5.0 Hz, 1H),3.96 (d, J = 15.0 Hz, 1H), 3.74 (d,
J = 15.0 Hz, 1H), 3.32 (dd, J = 5.0 Hz, J = 15.0 Hz, 1H), 3.26 (dd,
J = 10.0 Hz, J = 15.0 Hz, 1H), 2.94 (dd, J = 5.0 Hz, J = 15.0 Hz, 1H),
2.81 (dd, J = 5.0 Hz, J = 15.0 Hz, 1H), 2.63 (dd, J = 10.0 Hz,
J = 15.0 Hz, 1H), 1.44 (s, 3H), 1.25 (s,3H); 13C NMR (125 MHz,
DMSO) dC = 19.25, 24.88, 30.57, 34.75, 46.45, 53.45, 56.75, 78.85,
126.28, 126.74, 127.21, 129.88, 133.48, 135.02, 170.22, 171.33,
171.47. Elemental Anal. Calcd for C17H20N2O5: C, 61.44; H, 6.07;
N, 8.43. Found: C, 61.66; H, 6.22; N, 8.65.
(500 MHz, DMSO) dH = 11.25 (s, 1H), 7.17 (d, J = 7.5 Hz, 1H), 7.15
(d, J = 7.5 Hz, 1H), 7.13 (t, J = 7.5 Hz, 1H), 7.12 (t, J = 7.5 Hz, 1H),
6.03 (s, 2H), 4.43 (t, J = 7.0 Hz, 1H), 3.97 (d, J = 14.5 Hz, 1H), 3.74
(d, J = 14.0 Hz, 1H), 3.35 (m, J = 7.0 Hz, 1H), 3.28 (dd, J = 8.0 Hz,
J = 11.0 Hz, 1H), 2.94 (dd, J = 3.5 Hz, J = 15.5 Hz, 1H), 2.81 (dd,
J = 5.5 Hz, J = 15.5 Hz, 1H), 2.65 (dd, J = 11.5 Hz, J = 15.5 Hz, 1H),
1.46 (s, 3H), 1.27 (s, 3H); 13C NMR (125 MHz, DMSO) dC = 24.97,
25.14, 31.21, 34.22, 49.84, 52.57, 58.98, 78.75, 126.59, 127.13,
127.33, 127.41, 134.11, 135.74, 172.54, 172.86, 173.76. Elemental
Anal. Calcd for C17H21N3O4: C, 61.62; H, 6.39; N, 12.68. Found: C,
61.41; H, 6.22; N, 12.44.
4.1.6.10. (S)-2-((S)-3,3-Dimethyl-1-oxo-10,10a-dihydroimidazo
[1,5-b]isoquinolin-2(1H,3H,5H)-yl)pentanedioic acid (5j). Yield:
87%, colorless powder, mp 172–175 °C. ½a D20
ꢂ
= ꢁ74.20 (c 0.25,
4.1.6.14. (S)-5-Amino-2-((S)-3,3-dimethyl-1-oxo-10,10a-dihy-
droimidazo[1,5-b]-isoquinolin-2(1H,3H,5H)-yl)-5-oxopentanoic
acid (5n). Yield: 89%, colorless powder, mp 156–158 °C.
CH3OH); ESI/MS: 347 [M+H]+; IR (KBr disk): 2998, 1684, 1635,
1576, 1457, 750 cmꢁ1 1H NMR (500 MHz, DMSO) dH = 11.58 (s,
.
1H), 11.43 (s, 1H), 7.22 (t, J = 7.8 Hz, 1H), 7.20 (d, J = 7.8 Hz, 1H),
7.19 (d, J = 7.8 Hz, 1H), 7.15 (t, J = 7.8 Hz, 1H), 4.25 (m, 1H), 3.94
(d, J = 15.0 Hz, 1H), 3.72 (d, J = 15.0 Hz, 1H), 3.36 (dd, J = 5.0 Hz,
J = 15.0 Hz, 1H), 3.28 (dd, J = 10.0 Hz, J = 15.0 Hz, 1H), 2.94 (dd,
J = 5.0 Hz, J = 15.0 Hz, 1H), 2.83 (dd, J = 5.0 Hz, J = 15.0 Hz, 1H),
2.67 (dd, J = 10.0 Hz, J = 15.0 Hz, 1H), 2.33 (m, 1H), 2.22 (m, 1H),
1.37 (s, 3H), 1.28 (s, 3H); 13C NMR (125 MHz, DMSO) dC = 24.21,
24.44, 24.78, 28.58, 29.36, 39.47, 55.52, 59.80, 78.50, 125.97,
126.42, 126.61, 127.55, 131.38, 131.87, 168.63, 170.93, 171.59,
172.33. Elemental Anal. Calcd for C18H22N2O5: C, 62.42; H, 6.40;
N, 8.09. Found: C, 62.60; H, 6.56; N, 8.33.
½
a 2D0
ꢂ
= ꢁ24.25 (c 0.25, CH3OH); ESI/MS: 346 [M+H]+; IR (KBr disk):
2984, 1733, 1684, 1635, 1576, 1418, 1206, 746 cmꢁ1 1H NMR
.
(500 MHz, DMSO) dH = 11.30 (s, 1H), 7.17 (d, J = 7.8 Hz, 1H), 7.15
(d, J = 7.8 Hz, 1H), 7.13 (t, J = 7.8 Hz, 1H), 7.10 (t, J = 7.8 Hz, 1H),
6.03 (s, 2H), 4.17 (t, J = 7.0 Hz, 1H), 3.96 (d, J = 14.5 Hz, 1H), 3.74
(d, J = 14.0 Hz, 1H), 3.64 (s, 2H), 3.37 (dd, J = 4.0 Hz, J = 11.0 Hz,
1H), 2.92 (dd, J = 3.5 Hz, J = 15.5 Hz, 1H), 2.65 (dd, J = 11.0 Hz,
J = 15.5 Hz, 1H), 2.48 (m, J = 7.0 Hz, 1H), 2.38 (m, J = 7.0 Hz, 1H),
1.38 (s, 3H), 1.30 (s, 3H); 13C NMR (125 MHz, DMSO) d (ppm) =
24.14, 24.93, 25.71, 30.79, 33.82, 47.82, 56.43, 56.87, 78.48,
124.77, 126.29, 126.66, 127.22, 133.83, 135.18, 163.69, 171.14,

H. Wang et al. / Bioorg. Med. Chem. 19 (2011) 871–882
881
171.47, 172.88. Elemental Anal. Calcd for C18H23N3O4: C, 62.59; H,
6.71; N, 12.17. Found: C, 62.40; H, 6.55; N, 12.40.
University) were used. Immediately after decapitation, rat aortic
strips were stripped and put in a perfusion bath with 15 mL
warmed (37 °C), oxygenated (95%O2/5%CO2) Kreb’s solution (pH
7.4). The aortic strip was connected to a tension transducer, and
the relaxation contraction curve of muscles was registered. Admin-
istration of 59 lM NE induced hypertonic contraction of the vessel
strip. As the contraction reaches its maximum, NE was washed out,
and the vessel strip was stabilized for 30 min. After renewal of the
4.1.6.15. (S)-2-((S)-3,3-Dimethyl-1-oxo-10,10a-dihydroimidazo-
[1,5-b]isoquinolin-2(1H,3H,5H)-yl)-5-guanidinopentanoic acid
(5o). Yield: 88%, colorless powder, mp 181–183 °C.
½
a 2D0
ꢂ
=
ꢁ34.98 (c 0.25, CH3OH); ESI/MS: 374 [M+H]+; IR (KBr disk):
3358, 2933, 1683, 1614, 1516, 1449, 1373, 1338, 1246, 746 cmꢁ1
.
1H NMR (500 MHz, DMSO) dH = 11.51 (s, 1H), 9.13 (s, 2H), 7.80 (s,
1H), 7.17 (d, J = 7.8 Hz, 1H), 7.15 (d, J = 7.8 Hz, 1H), 7.13 (t,
J = 7.8 Hz, 1H), 7.11 (t, J = 7.8 Hz, 1H), 4.24 (dd, J = 4.0 Hz,
J = 10.5 Hz, 1H), 3.81 (d, J = 14.0 Hz, 1H), 3.53 (d, J = 14.5 Hz, 1H),
3.44 (dd, J = 10.0 Hz, J = 15.0 Hz, 1H), 3.21 (dd, J = 5.0 Hz,
J = 14.0 Hz, 1H), 3.15 (dd, J = 5.0 Hz, J = 10.0 Hz, 1H), 2.95 (dd,
J = 4.0 Hz, J = 15.0 Hz, 1H), 2.68 (dd, J = 11.0 Hz, J = 15.0 Hz, 2H),
1.99 (d, J = 4.0 Hz, 2H), 1.56 (m, J = 4.0 Hz, 2H), 1.27 (s, 3H), 0.82
(s, 3H); 13C NMR (125 MHz, DMSO) dC = 17.94, 24.25, 30.94,
33.85, 46.77, 56.58, 56.99, 78.77, 126.23, 127.12, 128.45, 131.22,
133.79, 135.11, 156.45, 170.98, 171.41. Elemental Anal. Calcd for
solution, 59
value of the aortic strip reached the peak, 15
control) or 15 L solution of 5a–q in ethanol was administrated to
l
M NE was added. When the hypertonic contraction
l
L ethanol (negative
l
observe their vasodilation.
4.2.2. In vitro anti-platelet aggregation activity assay
An H-10 cell counter was used to determine the platelet count
and a two-channel Chronolog aggregometer was used to evaluate
platelet aggregation. The pig blood (six pigs, purchased from Ani-
mal Center of Peking University) was centrifuged at 1000 rpm for
10 min and the platelet rich plasma (PRP) was collected. The
remaining blood was centrifuged for an additional 10 min at
1500 rpm to prepare platelet poor plasma (PPP). The final platelet
count of the PRP was adjusted to 2 ꢀ 108 platelets/mL with autol-
ogous PPP. To an optical aggregometry testing tuber, 0.5 mL of the
C19H27N5O3: C, 61.11; H, 7.29; N, 18.75. Found: C, 61.32; H, 7.45;
N, 18.53.
4.1.6.16. (S)-2-((S)-3,3-Dimethyl-1-oxo-10,10a-dihydroimidazo
[1,5-b]isoquinolin-2(1H,3H,5H)-yl)-4-(methylthio)butanoic acid
adjusted plasma sample and 5
5a–q (in a series of final concentrations of 100, 10, 1, 0.1, 0.01
and 0.001 M, prepared by diluting 10 mM of stock solutions of
5a–q in DMSO/NS, 1/10, with NS) was added. After adjustment of
the baseline, 5 L of the solution of PAF (final concentration
0.1 M, prepared by diluting 10 mM of stock solution of PAF in
DMSO/NS, 1:10, with NS) or 5 L of the solution of ADP (final con-
centration 10 M, prepared by diluting 10 mM of stock solution of
ADP in DMSO/NS, 1:10, with NS) or 5 L of the solution of arachi-
donic acid in NS (AA, final concentration 350 M, prepared by
diluting 10 mM of stock solution of AA in DMSO/NS, 1:10, with
NS), or 50 L of the solution of TH (final concentration 0.1 U/mL,
lL of NS or 5 lL of the solution of
(5p). Yield: 90%, colorless powder, mp 182–184 °C. ½a D20
= ꢁ61.74
ꢂ
(c 0.25, CH3OH); ESI/MS: 349 [M++H]+; IR (KBr disk): 2998, 2916,
l
2580, 1723, 1688, 1654, 1437, 1368, 1268, 1245, 1191, 746 cmꢁ1
.
1H NMR (500 MHz, DMSO) dH = 11.61 (s, 1H), 7.17 (d, J = 7.8 Hz,
1H), 7.15 (d, J = 7.8 Hz, 1H), 7.12 (t, J = 7.8 Hz, 1H), 7.10 (t,
J = 7.8 Hz, 1H), 4.21 (t, J = 6.5 Hz, 1H), 3.97 (d, J = 14.0 Hz, 1H),
3.74 (d, J = 14.5 Hz, 1H), 3.36 (dd, J = 4.0 Hz, J = 11.0 Hz, 1H), 2.92
(dd, J = 3.5 Hz, J = 15.5 Hz, 1H), 2.66 (dd, J = 11.0 Hz, J = 15.5 Hz,
1H), 2.54 (d, J = 6.5 Hz, 2H), 2.30 (t, J = 6.5 Hz, 2H), 2.10 (s, 3H),
1.38 (s, 3H), 1.31 (s, 3H); 13C NMR (125 MHz, DMSO) dC = 14.91,
19.47, 24.74, 29.22, 30.87, 46.82, 52.89, 56.35, 78.69, 126.34,
126.572, 127.24, 129.98, 133.82, 135.24, 171.21, 171.35. Elemental
Anal. Calcd for C18H24N2O3S: C, 62.04; H, 6.94; N, 8.04. Found: C,
61.85; H, 6.78; N, 8.27.
l
l
l
l
l
l
l
prepared by diluting 100 U/mL of stock solution of TH in DMSO/
NS, 1:10, with NS) was added and aggregation was measured at
37 °C for 5 min. The effects of 5a–q (at a series of concentrations
ranging from 100 lM to 1 nM) on PAF or ADP or AA or TH-induced
4.1.6.17. (R)-2-((S)-3,3-Dimethyl-1-oxo-10,10a-dihydroimidazo
[1,5-b]isoquinolin-2(1H,3H,5H)-yl)-3-mercaptopropanoic acid
platelet aggregation were observed. All these anti-platelet aggrega-
tion tests in sixplicate tubers were carried out. The maximum
platelet aggregation (Am) of control group (NS) or sample group (
5a–q) was represented by the peak height of aggregation curve
(equals to the maximum light transmission). The inhibition rate
was calculated according to the following formula: Inhibition
(%) = [(Am of NS) ꢁ (Am of 5a–q)]/(Am of NS) ꢀ 100%. Am% of NS is
the value of platelet aggregation induced by PAF, ADP, AA and TH
without 5a–q and are 52.30 1.78%, 50.16 3.65%, 49.62 2.90%
and 61.20 2.97%, respectively. The concentration versus inhibi-
tion rate curve is plotted to determine the IC50 values with GWBA-
(5q). Yield: 90%, colorless powder, mp 152–154 °C.
½
a 2D0
=
ꢂ
ꢁ21.35 (c 0.25, CH3OH); ESI/MS: 321 [M+H]+; IR (KBr disk):
2986, 2503, 1713, 1676, 1417, 1289, 1211, 1101, 746 cmꢁ1 1H
.
NMR (500 MHz, DMSO) dH = 11.28 (s, 1H), 7.17 (d, J = 7.7 Hz, 1H),
7.15 (d, J = 7.7 Hz, 1H), 7.13 (t, J = 7.7 Hz, 1H), 7.11 (t, J = 7.7 Hz,
1H), 4.39 (t, J = 7.0 Hz, 1H), 3.97 (d, J = 14.0 Hz, 1H), 3.77 (d,
J = 13.5 Hz, 1H), 3.55 (dd, J = 6.5 Hz, J = 13.5 Hz, 1H), 3.34 (d,
J = 7.0 Hz, 2H), 2.93 (dd, J = 4.0 Hz, J = 15.5 Hz, 1H), 2.66 (dd,
J = 12.5 Hz, J = 15.5 Hz, 1H), 2.54 (s, 1H), 1.44 (s, 3H), 1.31 (s, 3H);
13C NMR (125 MHz, DMSO) dC = 14.85, 19.43, 24.86, 29.24, 30.83,
46.85, 52.68, 52.82, 56.41, 78.72, 126.38, 126.52, 127.28, 129.82,
133.81, 135.24, 171.26, 171.41. Elemental Anal. Calcd for
SIC.EXE program.
4.2.3. In vivo anti-thrombotic assay of intravenously injection
of 5a–q in rat model
C16H20N2O3S: C, 59.98; H, 6.29; N, 8.74. Found: C, 59.77; H, 6.14;
N, 8.51.
The assessments described here were performed based on a
protocol reviewed and approved by the ethics committee of Capital
Medical University. The committee assures the welfare of the ani-
mals was maintained in accordance to the requirements of the ani-
mal welfare act and according to the guide for care and use of
laboratory animals. Aspirin and 5a–q were dissolved in NS before
administration and kept in an ice bath. Male Wister rats weighing
250–300 g (purchased from Animal Center of Peking University)
were used. The rats were anesthetized with pentobarbital sodium
(80.0 mg/kg, ip) and the right carotid artery and left jugular vein
were separated. A weighed 6 cm thread was inserted into the mid-
dle of a polyethylene tube. The polyethylene tube was filled with
4.2. Bioassays
4.2.1. Vasodilation assay
A constant temperature trough (CS501, Chongqing YinHe exper-
imental apparatus Ltd of China) was used to insure the buffer
warmed; a tension transducer (Hang JZ101, Beidian Xinghang
machine and equipment Ltd) and a two-channel physiological
recorder (LMS-2B, Chengdu apparatus manufacturer) were used
to evaluate the vasodilative effect. The Male Wistar rats weighing
250–300 g (purchased from Animal Center of Capital Medical

882
H. Wang et al. / Bioorg. Med. Chem. 19 (2011) 871–882
7. Lane, J. W.; Estevez, A.; Mortara, K.; Callan, O.; Spencer, J. R.; Williams, R. M.
Bioorg. Med. Chem. Lett. 2006, 16, 3180.
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left jugular vein. From the other end of the polyethylene tube, hep-
arin sodium was injected as anticoagulant, then NS or 5a–q was in-
jected, and this end was inserted into the right carotid artery.
Blood was allowed to flow from the right carotid artery to the left
jugular vein, through the polyethylene tube for 15 min. The thread
was removed to obtain the weight of the wet thrombus.
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Acknowledgments
This work was completed in the Beijing area major laboratory of
peptide and small molecular drugs, supported by PHR (IHLB, KZ
200810025010, KZ 200910025004, KM200710025010), the Na-
tional Natural Scientific Foundation of China (20772082), and Spe-
cial Project (2008ZX09401-002) of China.
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Supplementary data
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Supplementary data associated with this article can be found, in
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