A class of novel N-(3S-1,2,3,4-tetrahydroisoquinoline-3-carbonyl)-l-amino acid derivatives: their synthesis, anti-thrombotic activity evaluation, and 3D QSAR analysis

Article abstract of DOI:10.1016/j.ejmech.2009.08.002

To find new anti-thrombotic agents, a natural amino acid was introduced into the 3-position of anti-platelet aggregation active 3S-tetrahydroisoquinoline-3-carboxylic acid (THIQA), and 20 novel dipeptide derivatives, 3S-tetrahydroisoquinoline-3-carboxyamino acids (6a-t), targeting the intestinal peptide transport system were provided. In vitro anti-platelet aggregation assay of 6a-t indicated that their potencies of inhibiting adenosine diphosphate (ADP), arachidonic acid (AA), platelet-activating factor (PAF), and thrombin (TH) induced platelet aggregations were higher than that of THIQA, and the in vivo anti-thrombotic assay of 6a-t indicated that their potencies of inhibiting thrombogenesis in rats were also higher than that of THIQA. According to MFA based Cerius2 QSAR module, using training/test set of 6a,b,d,g-p/6c,e,f,q and training/test set of 6a-p/6q-t, two equations (r, 0.984 and 0.996) correlating the structures with in vitro or in vivo activity of 6a-t were established.

Full text of DOI:10.1016/j.ejmech.2009.08.002

European Journal of Medicinal Chemistry 44 (2009) 4904–4919
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
A class of novel N-(3S-1,2,3,4-tetrahydroisoquinoline-3-carbonyl)- -amino
L
acid derivatives: their synthesis, anti-thrombotic activity evaluation,
and 3D QSAR analysis
,
a
b,
***
Shenling Cheng a, Xiaoyi Zhang a, Wei Wang b, Ming Zhao a , Meiqing Zheng , Heng Wei Chang
,
**
,
Jianghui Wu a, Shiqi Peng a
*
a College of Pharmaceutical Sciences, Capital Medical University, Beijing 100069, PR China
b CS Bio Company, 20 Kelly Court, Menlo Park, California 94025, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
To find new anti-thrombotic agents, a natural amino acid was introduced into the 3-position of anti-
platelet aggregation active 3S-tetrahydroisoquinoline-3-carboxylic acid (THIQA), and 20 novel dipeptide
derivatives, 3S-tetrahydroisoquinoline-3-carboxyamino acids (6a–t), targeting the intestinal peptide
transport system were provided. In vitro anti-platelet aggregation assay of 6a–t indicated that their
potencies of inhibiting adenosine diphosphate (ADP), arachidonic acid (AA), platelet-activating factor
(PAF), and thrombin (TH) induced platelet aggregations were higher than that of THIQA, and the in vivo
anti-thrombotic assay of 6a–t indicated that their potencies of inhibiting thrombogenesis in rats were
also higher than that of THIQA. According to MFA based Cerius2 QSAR module, using training/test set of
6a,b,d,g–p/6c,e,f,q and training/test set of 6a–p/6q–t, two equations (r, 0.984 and 0.996) correlating the
structures with in vitro or in vivo activity of 6a–t were established.
Received 2 May 2009
Received in revised form
2 July 2009
Accepted 3 August 2009
Available online 6 August 2009
Keywords:
Tetrahydroisoquinoline
Modification
Anti-thrombotic
3D QSAR
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
compounds are urgently needed, and a lot of efforts have been
devoted to the design of anti-thrombotic drug. However, many
Intravascular thrombosis is known as one of the most frequent
pathological events and a leading cause of morbidity and mortality
all over the world. In the onset and progression of acute coronary
syndromes, there are critical steps such as the rift, rupture and
pathogenesis of atheromatous plaque with the formation of either
partial or complete occlusive thrombus [1–3], while vascular
damage, stimulus of platelets, and initiation of the clotting cascade
are the essential factors of thrombosis. In the cases of unstable
Rougnon–Heberden disease, cardiac infarction, transient attack of
myocardial ischemia, apoplexy and atherosclerotic occlusion, the
so-called thromboembolic disorders of cardiovascular and cere-
brovascular, the subendothelium surfaces of the vessels are injured
and function as the adherent target of platelets [3–7]. Though the
anti-thrombotic therapy of anticoagulants, anti-platelet drugs and
thrombolytic drugs are well established, to improve the prevention
and treatment of ischemic symptoms, more potent and safer
candidates of anti-thrombotic drug fail to exert their therapeutic
potential mainly due to their poor bioavailability, which led us to
pay more attention to the development of GPIIb/IIIa antagonist of
small molecules with desirable pharmacodynamic and pharmaco-
kinetic properties [7–13]. Recently, intestinal peptide transport
system is successfully utilized to increase bioavailability [14–16]
and we reported our new design as drug candidates in the form of
dipeptide analogues that can be readily absorbed across the
intestinal brush border membrane via the peptide transport
system. For instance, 3S-1,2,3,4-tetrahydro-
b-carboline-3-carbox-
ylic acid (THCA) and 3S-1,2,3,4-tetrahydroisoqunoline-3-carbon-
ylamino acid (THIQA), the anti-platelet aggregation compounds,
were converted to their dipeptide analogues to enhance their anti-
thrombotic activity and permeability [8,9].
Besides a number of bioactivities [17–31], tetrahydroisoquino-
lines were identified as anti-platelet aggregation agents. The
investigations of the anti-platelet aggregation mechanism of
tetrahydroisoquinolines indicated that in addition to receptor and
* Corresponding author. Tel./fax: þ86 10 8391 1528.
** Tel./fax: þ86 10 8280 2482.
b-adrenergic/a2-adrenergic receptor system [32], thromboxane
A2/prostaglandin H2 receptor system was commonly involved, for
instance they may exert their inhibitory effects on AA-induced
platelet aggregation partly by inhibiting the production of TXA2
*** Tel.: þ650 322 1111; fax: þ650 322 2278.
E-mail addresses: mingzhao@mail.bjmu.edu.cn (M. Zhao), chang@csbio.com
(H.W. Chang), sqpeng@mail.bjmu.edu.cn (S. Peng).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.08.002

S. Cheng et al. / European Journal of Medicinal Chemistry 44 (2009) 4904–4919
4905
from AA and partly by directly blocking the TXA2 receptor [33–35].
Based on the results of these investigations some derivatives of
benzyltetrahydroisoquinoline alkaloid were reported having anti-
platelet aggregation activities [36–39].
yield of the individual reaction suggest that the present synthetic
route is suitable for preparing these novel 3S-1,2,3,4-tetrahy-
droisoquinoline-3-carbonylamino acids.
In our previous paper, THIQA was used as the anti-thrombotic
2.2. In vitro anti-platelet aggregation activities of 6a–t
pharmacophore and converted to 3S-N-(
rahydroisoquinolines for 3D QSAR analysis. The stereoview of indi-
vidual 3S-N-( -aminoacyl)-1,2,3,4-tetrahydroisoquinoline explored
L-aminoacyl)-1,2,3,4-tet-
To evaluate the effect of introducing amino acid into the 3-
position of THIQA, the in vitro anti-platelet aggregation activities of
6a–t were measured following the common procedure by using
four aggregators, namely platelet-activating factor (PAF, final
L
that due to proximity its side chain of 2-amino acid residue and the
3-carboxylic group may form hydrogen bonding leading to low anti-
thrombotic activity [9], while the corresponding 3S-1,2,3,4-tetra-
hydroisoquinoline-3-carbonyl- amino acid does not form this kind
of intramolecular hydrogen bonding and therefore it will have
higher anti-thrombotic activity (Fig. 1). In this context, the present
paper introduced the natural amino acids into the 3-position of
THIQA to provide 20 novel 3S-1,2,3,4-tetrahydroisoquinoline-3-
carbonylamino acids for anti-thrombotic evaluation and 3D QSAR
analysis.
concentration 0.1
mM), adenosine diphosphate (ADP, final concen-
tration 10 M), arachidonic acid (AA, final concentration 350
m
mM),
and thrombin (TH, final concentration 0.1 U/ml). The IC50 values are
listed in Table 1.
The data demonstrate that the IC50 values of THIQA against PAF,
ADP, AA and TH induced platelet aggregation are 0.55, 0.45, 0.96
and 0.88
droisoquinolines were investigated generally as the inhibitors of
ADP or AA and had 270–100 M and 3.3–140 M of IC50 values,
mM, respectively. As lead compounds tetrahy-
m
m
respectively [33,38]. Comparing with them THIQA belongs to
desirable lead and is more suitable as the inhibitor of ADP and AA
than that of PAF and TH. The IC50 values of 6a–t against ADP, AA,
PAF and TH range from 0.12 to 0.66, 0.10 to 0.49, 0.14 to 1.40 and
2. Results and discussion
2.1. Preparing 3S-1,2,3,4-tetrahydroisoquinoline-3-carbonylamino
acids
0.16 to 0.97 mM, respectively. Thus these dipeptides similarly
belong to desirable leads and are more suitable for inhibiting ADP
and AA than that of PAF and TH. It is known that the representa-
tives of tetrahydroisoquinolines, Higenamine, YS-49 and YS-51, are
able to inhibit both AA- and ADP-induced platelet aggregation
The preparation of 3S-1,2,3,4-tetrahydroisoquinoline-3-car-
bonylamino acids (6a–t) was carried out according to the five-step-
route depicted in Scheme 1. Using a common procedure L-Phe was
successively converted into THIQA (2, 84% yield) and Boc-THIQA (3,
80% yield) via Pictet–Spengler condensation and amidation,
respectively [40,41]. To determine if any racemization of the chiral
with IC50 values ranging from 3.3 to 270
mM, and other analogs
give 5.3–800 M of IC50 values in inhibiting U46619-induced
m
platelet aggregation [38]. In Table 1 the inhibitory potencies of 13
and 10 dipeptides are defined as 1.4–4.6 and 1.2–4.3-fold higher
than that of THIQA in inhibiting ADP- and AA-induced platelet
aggregation, respectively. Therefore the inhibition potencies of
both ADP- and AA-induced platelet aggregations derived benefit
from the modification. Besides, in AA- and ADP-induced platelet
aggregations Table 1 identifies 6a,b,t and 6f,m,s having the best
potency, respectively.
center in both
Spengler–Pictet reaction the enantiomeric purity of 2 was deter-
mined by analytical HPLC. After 20 l of the sample of 2 (10 M)
L-Phe and 2 had occurred during the course of
m
m
was loaded, the Daicel Chiralpak AS chiral column was eluted with
aqueous methanol (50%) for 40 min. The flow rate was 1 ml/min,
the sample was monitored with UV detector at 254.8 nm and the
peak area was recorded. The relative proportion of the peak area
suggested that 2 was a single enantiomer. In the presence of DCC,
HOBt and NMM, 3 was conjugated with 20 amino acid methylesters
to give 3S-N-Boc-1,2,3,4- tetrahydroisoquinoline-3-carbonylamino
acid methylesters (4a–t, 45–95% yield). The saponification of 4a–t
2.3. In vivo anti-thrombotic activities of 6a–t
provided
3S-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-carbonyl-
To evaluate the effect of the introduction of amino acid into
the 3-position of THIQA, the in vivo anti-thrombotic activity of
6a–t were measured by use of an extracorporeal circulation of
arterioveinous cannula model of rats. According to the general
amino acids (5a–t, 65–96% yield). Removing Boc groups from 5a–t
resulted in 3S-1,2,3,4-tetrahydroisoquinoline-3-carbonylamino
acids (6a–t, 48–100% yield). The mild condition and the moderate
Fig. 1. Stereoview of 3S-N-(L-argininyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid and N-(3S-1,2,3,4-tetrahydroisoquinoline-3-carbonyl)-L-arginine.

4906
S. Cheng et al. / European Journal of Medicinal Chemistry 44 (2009) 4904–4919
CO2H
CO2H
ii
CO2H
N-Boc
i
NH2
NH
1
2
3
iii
R
R
R
O
O
O
OMe
OH
v
OH
iv
NH
NH
NH
N-Boc
NH
6a-t
N-Boc
O
O
O
4a-t
5a-t
Scheme 1. (i) HCHO and HCl; (ii) (Boc)2O, 1 N NaOH; (iii) AA–OCH3, HOBt, DCC and NMM; (iv) NaOH aqueous (2 N); (v) Hydrogen chloride in ethyl acetate (6 N). In 4a–6a R ¼ CH3,
4b–6b R ¼ H, 4c–6c R ¼ CH(CH3)2, 4d–6d R ¼ CH2CH(CH3)2, 4e–6e R ¼ CH(CH3)CH2CH3, 4f–6f R ¼ indol-2-ylmethylene, 4g–6g R ¼ CH2OH, 4h–6h R ¼ 4-hydroxylbenzyl, 4i–6i
NHCHR ¼ cyclobutylamino, 4j–6j R ¼ CH2CONH2, 4k–6k R ¼ CH2CH2CONH2, 4l–6l R ¼ imidazol-4-ylmethylene, 4m R ¼ N-benzyloxycarbonylbutylamino-4-yl, 5m
& 6m
R ¼ butylamino-4-yl, 4n R ¼ methoxycarbonylmethylene, 5n & 6n R ¼ carboxylmethylene, 4o R ¼ methoxycarbonylethylene, 5o & 6o R ¼ carboxylethylene, 4p–6p R ¼ CH2SH, 4q–
6q R ¼ benzyl, 4r–6r R ¼ CH(OH)CH3, 4s–6s AA ¼ CH2(CH2)2NHC(NH2) ¼ NH, 4t–6t R ¼ CH2CH2SCH3.
procedure, the individual stock solution of Aspirin (positive
control) and 6a–t in NS was administered intravenously. The
thrombus weights were weighed, and the data are listed in
Table 2.
which has the biggest side chain, indol- 2-ylmethylene, 6q and 6s
exhibit the highest in vivo anti-thrombotic activity (Table 2). This
implies that the dipeptides, of which the side chains are proper,
exhibiting the best in vitro inhibitory potency in ADP-induced
platelet aggregation will also exhibit the highest in vivo anti-
thrombotic activity. In addition, the fact that the thrombus weights
of the rats receiving 167 mmol/kg of aspirin and 5 mmol/kg of 6a–t
are comparable indicates that the anti-thrombotic potency of 6a–t
is over 30-fold higher than that of aspirin (Table 2).
The thrombus weights of the rats receiving 5 mmol/kg of 6a–t
range from 18.73 to 23.58 mg, and are significantly lower than that
(28.54 mg) of rats receiving NS (p < 0.001), which demonstrate that
6a–t are in vivo anti-thrombotic active. On the other hand, the
thrombus weights of the rats receiving 5
range from 18.73 to 20.24 mg, and are significantly lower than that
(21.52 mg) of rats receiving 15 mol/kg of THIQA (p < 0.05–0.01).
The thrombus weights of the rats receiving 5 mol/kg of 6a,f,g,i,k–
n,r,t equal to that of rats receiving 15 mol/kg of THIQA. In other
mmol/kg of 6b,d,e,j,o,q,s
To clarify the benefit of 3-position modification the thrombus
weights of the rats receiving 5 mmol/kg of 3S-N-(L-argininyl)-1,2,3,4-
m
m
tetrahydroisoquinoline-3-carboxylic acids (7a–s), the 2-amino acid
modified THIQA, are incorporated into Table 2. The data indicate that
the anti-thrombotic activities of 6a–e,i,k–n are equal that of 7a–
e,i,k–n, the anti-thrombotic activities of 6h,j,o–q,s are significantly
higher than that of 7h,j,o–q,s, and only the anti-thrombotic activities
of 6f,g,t are significantly lower than that of 7f,g,t. Thus, 3-position
amino acid modification totally has one advantage over 2-position
amino acid modification.
m
words, modifying 3-position of THIQA with amino acids leads the
anti-thrombotic activity a 2-fold to more than 3-fold increase.
Therefore the modification is beneficial to the improvement of in
vivo anti-thrombotic activity of THIQA.
From Table 1 it is noticed that 6f,q,s are the best inhibitors for
ADP-induced platelet aggregation. Parallel with this, except 6f
Table 1
IC50 of 6a–t against four aggregators induced aggregation of pig platelets.a
R
O
OH
NH
6a-t
IC50 (µM)
NH
O
No/R
ADP
AA
PAF
TH
No
ADP
AA
PAF
TH
IQ
6a/CH3
6b/H
6c/CH(CH3)2
0.552 ꢀ 0.032 0.452 ꢀ 0.016 0.960 ꢀ 0.041 0.881 ꢀ 0.040 IQ
0.350 ꢀ 0.031 0.104 ꢀ 0.026 0.352 ꢀ 0.018 0.485 ꢀ 0.032 7a
0.496 ꢀ 0.028 0.108 ꢀ 0.031 0.791 ꢀ 0.041 0.306 ꢀ 0.018 7b
0.382 ꢀ 0.026 0.388 ꢀ 0.034 0.943 ꢀ 0.034 0.199 ꢀ 0.016 7c
0.478 ꢀ 0.029 0.290 ꢀ 0.025 1.611 ꢀ 0.036 0.454 ꢀ 0.031 7d
0.540 ꢀ 0.034 0.443 ꢀ 0.032 0.938 ꢀ 0.044 0.657 ꢀ 0.026 7e
0.120 ꢀ 0.019 0.374 ꢀ 0.033 0.668 ꢀ 0.033 0.234 ꢀ 0.028 7f
0.388 ꢀ 0.033 0.416 ꢀ 0.026 0.942 ꢀ 0.028 0.841 ꢀ 0.036 7g
0.481 ꢀ 0.030 0.390 ꢀ 0.030 1.152 ꢀ 0.042 0.970 ꢀ 0.034 7h
0.364 ꢀ 0.021 0.103 ꢀ 0.015 1.398 ꢀ 0.027 0.185 ꢀ 0.021 7i
0.470 ꢀ 0.029 0.166 ꢀ 0.018 0.153 ꢀ 0.016 0.551 ꢀ 0.029 7j
0.235 ꢀ 0.022 0.494 ꢀ 0.029 0.138 ꢀ 0.021 0.935 ꢀ 0.044 7k
0.661 ꢀ 0.034 0.491 ꢀ 0.034 0.724 ꢀ 0.032 0.370 ꢀ 0.019 7l
0.552 ꢀ 0.032 0.452 ꢀ 0.016 0.960 ꢀ 0.041 0.881 ꢀ 0.040
0.651 ꢀ 0.026 0.452 ꢀ 0.031 0.945 ꢀ 0.038 0.741 ꢀ 0.042
0.365 ꢀ 0.029 0.440 ꢀ 0.034 0.875 ꢀ 0.042 0.761 ꢀ 0.040
0.389 ꢀ 0.033 0.423 ꢀ 0.029 0.632 ꢀ 0.034 0.653 ꢀ 0.031
0.336 ꢀ 0.019 0.573 ꢀ 0.030 0.891 ꢀ 0.040 0.730 ꢀ 0.026
0.315 ꢀ 0.030 0.238 ꢀ 0.019 0.831 ꢀ 0.044 0.674 ꢀ 0.028
0.194 ꢀ 0.016 0.465 ꢀ 0.032 0.422 ꢀ 0.026 0.720 ꢀ 0.044
0.103 ꢀ 0.018 0.572 ꢀ 0.031 0.267 ꢀ 0.021 0.498 ꢀ 0.031
0.327 ꢀ 0.031 0.535 ꢀ 0.026 0.668 ꢀ 0.032 0.736 ꢀ 0.038
0.373 ꢀ 0.032 0.526 ꢀ 0.025 0.507 ꢀ 0.029 0.590 ꢀ 0.025
0.534 ꢀ 0.034 0.430 ꢀ 0.030 0.360 ꢀ 0.018 0.495 ꢀ 0.030
0.302 ꢀ 0.023 0.407 ꢀ 0.033 0.392 ꢀ 0.031 0.555 ꢀ 0.031
0.324 ꢀ 0.019 0.351 ꢀ 0.022 0.670 ꢀ 0.026 0.684 ꢀ 0.034
6d/CH2CH(CH3)2
6e/CH(CH3)C2H5
6f/indol-2-ylmethylene
6g/CH2OH
6h/p-HO–C6H4CH2
a6i/cyclobutylamino
6j/CH2CONH2
6k/CH2CH2CONH2
6l/imidazol-4-ylmethylene
6m/CH2(CH2)3NH2
6n/CH2CO2Me
6o/CH2CH2CO2Me
6p/CH2SH
0.194 ꢀ 0.015 0.277 ꢀ 0.031 1.042 ꢀ 0.034 0.733 ꢀ 0.031 7m 0.323 ꢀ 0.021 0.521 ꢀ 0.029 0.472 ꢀ 0.034 0.829 ꢀ 0.042
0.329 ꢀ 0.033 0.391 ꢀ 0.029 0.633 ꢀ 0.028 0.157 ꢀ 0.016 7n
0.482 ꢀ 0.026 0.356 ꢀ 0.028 0.176 ꢀ 0.017 0.545 ꢀ 0.041 7o
0.345 ꢀ 0.034 0.344 ꢀ 0.034 0.383 ꢀ 0.022 0.902 ꢀ 0.034
0.494 ꢀ 0.032 0.369 ꢀ 0.026 0.544 ꢀ 0.031 0.589 ꢀ 0.027
0.231 ꢀ 0.018 0.460 ꢀ 0.034 0.780 ꢀ 0.036 0.386 ꢀ 0.032
–
–
–
–
–
6q/C6H5CH2
6r/CH(OH)CH3
0.188 ꢀ 0.015 0.553 ꢀ 0.030 0.218 ꢀ 0.021 0.336 ꢀ 0.027 7q
0.164 ꢀ 0.022 0.418 ꢀ 0.028 0.588 ꢀ 0.032 0.763 ꢀ 0.031 7r
0.370 ꢀ 0.029 0.666 ꢀ 0.025 0.527 ꢀ 0.028 0.851 ꢀ 0.039
0.240 ꢀ 0.027 0.215 ꢀ 0.019 0.404 ꢀ 0.031 0.879 ꢀ 0.041
0.210 ꢀ 0.022 0.298 ꢀ 0.027 0.517 ꢀ 0.030 0.565 ꢀ 0.028
0.265 ꢀ 0.026 0.382 ꢀ 0.031 0.409 ꢀ 0.026 0.607 ꢀ 0.026
6s/CH2(CH2)2NHC(NH2)]NH 0.124 ꢀ 0.020 0.361 ꢀ 0.025 1.344 ꢀ 0.036 0.496 ꢀ 0.030 7s
6t/CH2CH2SCH3
0.229 ꢀ 0.018 0.191 ꢀ 0.015 0.376 ꢀ 0.029 0.312 ꢀ 0.026 7t
a
NHCHR ¼ cyclobutylamin, 7a–t ¼ 3S-N-(L-aminoacyl)-1,2,3,4- tetrahydroisoquinoline-3-carboxylic acids, n ¼ 6.

S. Cheng et al. / European Journal of Medicinal Chemistry 44 (2009) 4904–4919
4907
Table 2
circulation. While Caco-2 cells possess many structural and func-
tional similarities to human enterocytes. After converting THIQA
into the deceptive analogue 6s, the permeability can be increased
2.3 folds.
Effect of 6a–t on the weight of the wet thrombus of rats.a
Compd.
Thrombus weight
Compd.
Thrombus weight
NS
IQ
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
6s
6t
28.54 ꢀ 2.62
21.52 ꢀ 1.49b
21.40 ꢀ 1.39b
20.24 ꢀ 1.37c
19.20 ꢀ 1.57d
20.23 ꢀ 1.49c
20.03 ꢀ 1.29c
21.30 ꢀ 1.06b
23.58 ꢀ 1.93b
19.31 ꢀ 1.48d
20.25 ꢀ 1.50b
20.24 ꢀ 1.50c
22.08 ꢀ 1.54b
20.86 ꢀ 1.94b
21.28 ꢀ 1.17b
21.67 ꢀ 1.91b
19.69 ꢀ 1.25d
20.91 ꢀ 1.27b
19.11 ꢀ 1.58d
21.25 ꢀ 1.93b
18.73 ꢀ 1.56d
21.74 ꢀ 1.50b
Aspirin1
Aspirin2
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7q
7r
–
7s
27.60 ꢀ 1.89
13.22 ꢀ 1.67
20.30 ꢀ 1.33c
21.17 ꢀ 1.47b
20.12 ꢀ 1.59c
19.12 ꢀ 1.82d
21.14 ꢀ 1.24b
19.47 ꢀ 1.64d
18.00 ꢀ 1.59d
20.92 ꢀ 1.44b
21.09 ꢀ 1.68b
22.66 ꢀ 1.45b
21.30 ꢀ 1.69b
21.78 ꢀ 1.96b
21.31 ꢀ 1.75b
20.30 ꢀ 1.34c
20.87 ꢀ 1.45b
22.23 ꢀ 1.72b
20.73 ꢀ 1.53b
–
2.5. 3D QSAR analysis of amino acid modified THIQA
To elucidate the effects of 3-position amino acid modification on
the anti-thrombotic action the activities were converted into the
corresponding anti-platelet aggregation and thrombus weights.
The training/test set of 6a,b,d,g–p/6c,e,f,q and training/test set of
6a–p/6q–t selections were done manually such that they populate
the wide range of anti-platelet aggregation and anti-thrombotic
activities in similar proportions, respectively.
2.5.1. Alignment of amino acid modified THIQA
To establish the valid 3D QSAR models, a proper alignment
procedure of 6a,b,d,g–p or 6a–p was practiced using the target
model align strategy in the align module within Cerius2. Based on
the assumption that each structure of 6a,b,d,g–p or 6a–p exhibits
activity at the same binding site of the receptor, they were aligned
in a pharmacological active orientation. To obtain a consistent
alignment, the pharmacophore tetrahydroisoquinoline ring was
selected as the template for superposing 6a,b,d,g–p or 6a–p. The
method used for performing the alignment was the maximum
common subgraph (MCS) [42]. MCS looks at molecules as points
and lines, and uses the techniques out of graph theory to identify
the patterns. MCS then finds the largest subset of atoms in tetra-
hydroisoquinoline ring that shared by 6a,b,d,g–p or 6a–p, which
was used for the alignment. A rigid fit of atom pairings was
performed to superimpose each structure onto the target model
tetrahydroisoquinoline ring. Stereoview of aligned 6a,b,d,g–p or
6a–p is shown in Fig. 2 or Fig. 7. The alignment stereoview explores
that to superimpose onto tetrahydroisoquinoline ring, the intro-
duced amino acid residue on each structure has to take individual
conformation. This individual conformation of the amino acid
residue will affect the anti-thrombotic activity [9].
20.06 ꢀ 1.48c
7t
19.56 ꢀ 1.72d
a
Weight of wet thrombus is represented by X ꢀ SD mg, NS ¼ vehicle, n ¼ 12;
IQ ¼ 3S-isoquinoline- 3-carboxylic acid and intravenous dose ¼ 15
mmol/kg.
b
Compared to NS p < 0.001.
Compared to NS p < 0.001 and to IQ p < 0.05.
Compared to NS p < 0.001 and to IQ p < 0.01.
c
d
1
Aspirin intravenous dose ¼ 15
mmol/kg.
2
Aspirin intravenous dose ¼ 0
m
mol/kg; 6a–t ¼ 3S-1,2,3,4-tetrahydroisoquino-
line-3-carbonylamino
acids,
7a–t ¼ 3S-N-(L-aminoacyl)-1,2,3,4-tetrahy-
droisoquinoline-3-carboxylic acids and dose ¼ 5
mmol/kg.
2.4. In vitro membrane permeation test of 6s
To gain the proof that amino acid modification can enhance the
permeability of THIQA through intestinal peptide transport system
6s, the most interesting dipeptide in Table 2 was fed to Male Wistar
rats orally, THIQA was used as the reference compound, and their
oral activities were measured. Accordingly, the membrane perme-
abilities of THIQA and 6s were also measured on Caco-2 cell
monolayers. The data are summarized in Table 3. The data indicate
an orally and dose-dependently anti-thrombotic action for 6s. The
2.5.2. MFA based cerius2 QSAR module of amino acid modified
THIQA
Molecular field analysis (MFA) was performed for 6a,b,d,g–p or
6a–p using the QSAR module of Cerius2 [42]. A five-step-procedure
included generating conformers, energy minimization, matching
atoms and aligning molecules, setting preferences and regression
data also indicate that modifying THIQA with
a significant increase of the oral anti-thrombotic activity. The data
further indicate that modifying THIQA with
a significant increase of the permeability.
L-Arg results in
L-Arg results in
Though the oral bioavailability of a drug is influenced by a series
of factors, including dissolution, absorption, pre-systemic and
systemic metabolism, and elimination, the intestinal mucosa is
a significant barrier to oral delivery of drugs into the systemic
Table 3
Apparent permeability coefficients and oral activities of THIQA and 6s.
Compd. Dose, thrombus weight (X ꢀ SD mg)a Papp ꢁ 106 (cm/s)b
A / B B / A A / B/B / A
THIQA
6s
15
m
mol/kg, 23.88 ꢀ 2.34
9.00 8.71
17.12 7.34
1.03
2.33
5
1
m
m
mol/kg, 19.01 ꢀ 2.42c
mol/kg, 24.38 ꢀ 2.61d
0.2
m
mol/kg, 29.11 ꢀ 2.37e
a
n ¼ 12, Thrombus weight of the rats receiving 0.6 ml of NS (vehicle):
29.07 ꢀ 2.62 mg.
b
THIQA and 6s were dissolved in HBSS (final concentration: 4 mM), the standard
deviations were generally less than 9.2% (n ¼ 4); A / B: from apical side to baso-
lateral side; B / A: from basolateral side to apical side.
c
Compare to 15
Compare to 15
Compare to NS p > 0.05.
m
m
mol/kg of THIQA and 1
mol/kg of THIQA p > 0.05 and to 0.2
m
mol/kg of 6s, p < 0.01.
mol/kg of 6s, p < 0.01.
d
e
m
Fig. 2. Alignment stereoview of 6a,b,d,g–p used for molecular field generation.

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S. Cheng et al. / European Journal of Medicinal Chemistry 44 (2009) 4904–4919
analysis was automatically carried out in MFA. By use of proton,
methyl, and hydroxyl anion as probes, molecular electrostatic and
steric fields were created. These fields were sampled at each point
of a regularly spaced grid of 1 Å. A ꢀ 30.0 kcal/mol energy cutoff
was set for both electrostatic and steric fields. Totally 672 grid
points were generated. Among the considered spatial and struc-
tural descriptors such as dipole moment, polarizability, radius of
gyration, number of rotatable bonds, molecular volume, principal
moment of inertia, A log P98, number of hydrogen bond donors and
acceptors, and molar refractivity, only the highest variance holder
proton and methyl descriptors were used. Regression analysis was
performed by use of genetic partial least squares (G/PLS) method
consisted of 50,000 generations with 100 population size. The
component numbers were set to 5. Cross-validation was performed
with the leave-one-out procedure. PLS analysis was scaled, with all
variables normalized to a variance of 1.0.
2.5.3. 3D QSAR equation of amino acid modified THIQA against
ADP-induced platelet aggregation
Fig. 4. Graph of tested versus predicted inhibitions of 6a–t to ADP-induced platelet
Based on the module, the regions where variations in the steric
or electrostatic features of 6a,b,d,g–p lead to the increase or
decrease of their inhibition of ADP-induced platelet aggregation
were specified as Fig. 3 corresponding to the IC50 values.
The MFA model for the activities of 6a,b,d,g–p inhibiting ADP-
induced platelet aggregation in terms of the most relevant
descriptors including proton, methyl and hydroxyl anion is
expressed by equation (1).
aggregation.
equation (1) is able to predict the in vitro activity for the analogs of
6a–t. Accordingly the 3D QSAR equations for the inhibitions of
6a,b,d,g–p to PAF, AA and TH can be established to reflect the steric
and electrostatic effects.
Equation (1) contains 3 terms from proton descriptor, 8 terms
from methyl descriptor, and 2 terms from hydroxyl anion descriptor.
The terms of 3.78 (Hþ/188), 6.12 (Hþ/251) and 2.34 (Hþ/309) have
positive coefficients, which means that at these positions electron-
withdrawing groups will increase the activity. The terms of 3.78
(CH3/181), 2.17 (CH3/182) and 0.41 (CH3/261) have positive coeffi-
cients, which means that at these positions small groups will
increase the activity, while terms of 5.86 (CH3/179), 8.56 (CH3/259),
4.73 (CH3/262), 0.86 (CH3/348) and 8.5 (CH3/413) have negative
coefficients, which means that at these positions large group will
increase the activity. The term of 6.04 (HOꢂ/251) has positive
coefficient, which means that at this position electron-withdrawing
group will increase the activity, while the term of 8.9 (HOꢂ/190) has
negative coefficient, which means that at this position electron-
releasing group will increase the activity.
Activity [ 351:1 D 3:78ðHD=188Þ D 6:12ðHD=251Þ
D 2:34ðHD=309Þ L5:86ðCH3=179Þ D3:78ðCH3=181Þ
D2:17ðCH3=182Þ L8:56ðCH3=259Þ D0:41ðCH3=261Þ
L 4:73ðCH3=262Þ L 0:86ðCH3=348ÞL 8:5ðCH3=413Þ
L 8:9ðHOL=190ÞD 6:04ðHOL=251Þ
(1)
The correlation of the activities tested on the in vitro platelet
aggregation model and the activities calculated using equation (1)
is explained by Fig. 4. In equation (1), the data points (n), correla-
tion coefficient (r), square correlation coefficient (r2) were 16,
0.984, and 0.968, respectively. The tested activities on ADP-induced
platelet aggregation model and the calculated activities based on
equation (1) were shown in Fig. 4. The parameters indicated that
Figs. 5 and 6 give four representatives, 6e,l,r,s as examples.
Compounds 6r has electeon-releasing groups at Hþ/251 and OHꢂ/
190, moderate group at CH3/261 and CH3/262 as well as small group
at CH3/181 and CH3/182, and therefore it possesses higher anti-
platelet aggregation activity; 6s has electeon-releasing groups at Hþ/
188 and OHꢂ/190, small group at CH3/261 as well as big group at
CH3/262, CH3/348 and CH3/413, and therefore it possesses also
higher anti-platelet aggregation activity; 6e has electeon-releasing
group at OHꢂ/251, and small groups at CH3/262, CH3/259 and CH3/
179, and therefore it possesses lower anti-platelet aggregation
activity; 6l has electeo-releasing group at HOꢂ/251 as well as big
group at CH3/181, CH3/182 and CH3/261, and therefore it has lower
anti-platelet aggregation activity.
2.5.4. Predicting anti-platelet aggregation activities for 6c,e,f,q
The predicted power of equation (1) was demonstrated with the
comparison of the calculated and tested anti-platelet aggregation
activities of 6c,e,f,q (Table 4). The correlation of the predicted and
test values is also shown in Fig. 4. The results indicate that comparing
to the test value equation (1) may result in an error% ranging from 1.9
to 3.8% for 6c,e,f,q, which means that this equation is able to
accurately give an anti-platelet aggregation activity for unknown
3S-1,2,3,4-tetrahydroisoquinoline-3-carbonylamino acids.
Fig. 3. Steric and electrostatic features of 6a,b,d,g–p leading to the in vitro activity
changes.

S. Cheng et al. / European Journal of Medicinal Chemistry 44 (2009) 4904–4919
4909
Fig. 5. Steric and electrostatic environments of 6r,s with higher activities within the grid with 3D points of equation (1).
2.5.5. 3D QSAR equation of amino acid modified THIQA inhibiting
rat thrombogenesis
coefficient which means that at this position electron-releasing
group will increase the activity. The terms of 0.020 (CH3/197), 0.039
(CH3/322), 0.023 (CH3/386) and 0.0043 (CH3/463) have positive
coefficients, which means that at these positions small groups will
increase the activity, while terms of 0.062 (CH3/242) and 0.020
(CH3/327) have negative coefficients, which means that at these
positions big groups will increase the activity. The term of 0.027
(HOꢂ/310) and 0.0037 (HOꢂ/461) have positive coefficients, which
means that at these positions electron-withdrawing group will
increase the activity, while the term of 0.023 (HOꢂ/251), 0.030
(HOꢂ/253), 0.071 (HOꢂ/317) and 0.032 (HOꢂ/397) have negative
coefficient, which means that at this position electron-releasing
group will increase the activity.
Figs. 9 and 10 give four representatives, 6c,g,h,k as examples.
Compound 6c has electron-releasing groups at Hþ/325, OHꢂ/251,
OHꢂ/253 and OHꢂ/397, as well as electron-withdrawing groups
OHꢂ/310, and therefore it possesses higher in vivo anti-thrombotic
activity; 6h has electron-releasing groups at OHꢂ/253 and OHꢂ/317,
an electron-withdrawing group at Hþ/325, as well as has small
groups at CH3/197 and CH3/463, and therefore it possesses higher
in vivo anti-thrombotic; 6g has moderate groups at CH3/322 and
CH3/386, and an electron-releasing group at OHꢂ/461, and there-
fore it possesses lower in vivo anti-thrombotic activity; 6k has an
electron-withdrawing group at HOꢂ/397 and an electron-releasing
group at HOꢂ/461, as well as a small group at CH3/197 and CH3/463,
and therefore it has lower in vivo anti-thrombotic activity.
Based on the module, the regions where variations in the steric
or electrostatic features of 6a–p lead to the inhibition or
enhancement of thrombogenesis on rat model were specified as
Fig. 7 corresponding to the thrombus weights of 6a–p.
The MFA model for anti-thrombotic activities of 6a–p in terms of
the most relevant descriptors including proton, methyl and
hydroxyl anions is expressed as equation (2).
Activity [ 21:57 L0:023ðHD=251Þ D0:057ðHD=325Þ
D0:020ðCH3=197Þ L0:062ðCH3=242ÞD 0:039ðCH3=322Þ
L 0:020ðCH3=327Þ D 0:023ðCH3=386Þ D0:0043ðCH3=463Þ
L 0:023ðHOL=251ÞL 0:030ðHOL=253Þ D0:027ðHOL=310Þ
L 0:071ðHOL=317Þ L0:032ðHOL=397Þ
D 0:0037ðHOL=461Þ
(2)
The correlation of the activities tested on the in vivo thrombo-
genesis model and the activities calculated using equation (2) is
explained by Fig. 7. In equation (2), the data points (n), correlation
coefficient (r), and square correlation coefficient (r2) were 16, 0.996,
and 0.991, respectively. The tested activities on rat thrombogenesis
model and the calculated activities based on equation (2) were
shown in Fig. 8. The parameters indicated that equation (2) is able
to predict the in vivo activity for 6a–p.
Equation (2) contains 2 terms from proton descriptor, 6 terms
from methyl descriptor, and
6 terms from hydroxyl anion
descriptor. The terms of 0.057 (Hþ/325) has positive coefficient,
which means that at this position electron-withdrawing groups
will increase the activity, while term of 0.023 (Hþ/251) has negative
2.5.6. Predicting anti-thrombotic activity for 6q–t
To examine the predicting power of the equations (2) the in vivo
anti-thrombotic activities of 6q–t were calculated, listed in Table 5
Fig. 6. Steric and electrostatic environments of 6e,l with lower activities within the grid with 3D points of equation (1).

4910
S. Cheng et al. / European Journal of Medicinal Chemistry 44 (2009) 4904–4919
Table 4
Predicted and tested IC50 of 6c, e, f, q against ADP-induced aggregation.
Comp.
IC50 (nM)
Predict value
Test value
Error
Error%
6c
6e
6f
371.139
558.254
115.401
191.319
382.324
540.4
119.97
187.97
ꢂ11.19
17.854
ꢂ4.569
3.491
2.9
3.3
3.8
1.9
6q
and compared to the tested values. The correlation of the predicted
and tested in vivo anti-thrombotic activities of 6p–t is also shown
in Fig. 8. The results indicate that comparing to the test value
equation (2) may predict the anti-thrombotic activities with error%
ranging from 0.1 to 6.8% for 6p–t, which means that this equation is
able to accurately predict an anti-thrombotic activity for unknown
3S-1,2,3,4-tetrahydroisoquinoline-3-carbonylamino acid.
3. Conclusion
Fig. 8. Graph of tested versus predicted anti-thrombotic activities of 6a–p.
Despite great advances have been made in the prevention of
thrombotic disorders by various treatments, thromboembolic
diseases are still the leading cause of mortality and morbidity
worldwide, and there is still an urgent need for developing anti-
thrombotic drugs. In this paper, we modified anti-thrombotic active
3S-tetrahydroisoquinoline-3-carboxylic acid with natural amino
acids to give 20 novel dipeptide analogs of N-(3S-tetrahy-
droisoquinoline-3-carboxyl)amino acids with higher anti-throm-
botic activity than 3S-tetrahydroisoquinoline-3-carboxylic acid
itself. As anti-thrombotic agents, the new candidates were capable
of utilizing the intestinal peptide transport system as target for
good absorption, optimal aqueous solubility, and good drug-like-
ness so as to have enhanced anti-thrombotic activity. In a previous
paper, it has been demonstrated that the intestinal mucosa is
a significant barrier to oral delivery of drugs into the systemic
circulation, Caco-2 cells possess many structural and functional
similarities to human enterocytes, and the apparent permeability of
THCA is increased by amino acid modification [43]. By analyzing
their 3D QSAR, the present paper is not only able to help us getting
insight into the quantitative correlation of the structure and the in
vitro and in vivo activities of these 20 novel dipeptide analogs, but
also able to help us predicting the in vitro and in vivo activities of
unknown dipeptide analogs.
4. Materials and methods
4.1. General
All the reactions were carried out under nitrogen (1 bar). 1H
(300 and 500 MHz) and 13C (75 and 125 MHz) NMR spectra were
recorded on Bruker AMX-300 and AMX-500 spectrometers for
solution DMSO-d6, or CDCl3 with tetramethylsilane as internal
standard. IR spectra were recorded with a Perkin-Elmer 983
instrument. FAB/MS was determined on VG-ZAB-MS and TOF-MS
was recorded on MDS SCIEX QSTAR. Melting points were measured
on a XT5 hot stage microscope (Beijing key electro-optic factory).
All L-amino acids were purchased from China Biochemical Corp. TLC
was made with Qingdao silica gel GF254. Chromatography was
performed with Qingdao silica gel H60 or Sephadex-LH20. All
solvents were distilled and dried before use by reference to litera-
ture procedures. Optical rotations were determined with a Jasco
P-1020 Polarimeter at 20 ꢃC. The statistical analysis of all the
biological data was carried out by use of ANOVA test, p < 0.05 is
considered significant.
4.2. Chemical synthesis
4.2.1. (3S)-1,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid (2)
To the suspension of 5.0 g (0.03 mmol) of L-Phe–OH 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 ꢃC for 10 h, and TLC (CHCl3/CH3OH, 10:1) indicates
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 white powder. Mp 302–
¼ ꢂ68 (c ¼ 1.0, H2O); ESI-MS (m/e) 178[M þ H]þ; 1H
20
303 ꢃC; [
a
]
D
NMR (300 MHz, CDCl3)
d
/ppm ¼ 11.0 (s,1H), 7.25 (m, J ¼ 6.4 Hz, 2H),
7.02 (d, J ¼ 6.5 Hz, 1H), 6.98 (t, J ¼ 6.6 Hz, 1H), 3.80 (m, 3H), 3.03 (d,
J ¼ 7.5 Hz, 1H), 2.78(d, J ¼ 8.4 Hz, 1H), 2.0(s, 1H); 13C NMR (75 MHz,
CDCl3)
d
/ppm ¼ 174.9, 136.2, 134.2, 127.2, 126.0, 57.6, 47.4, 29.4.
4.2.2. 3S-2-Tert-butoxycarbonyl-1,2,3,4-tetrahydroisoquinoline-3-
carboxylic acid (3)
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-tetrahydroisoquinoline-
Fig. 7. Steric and electrostatic features of 6a–p leading to in vivo activity changes.

S. Cheng et al. / European Journal of Medicinal Chemistry 44 (2009) 4904–4919
4911
Fig. 9. Steric and electrostatic environments of 6c,h with higher in vivo anti-thrombotic activity within the grid with 3D points of equation (2).
3-carboxylic acid, the solution of 14.8 g (67.8 mmol) of Boc2O in
40 ml of THF was added at 0 ꢃC. 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
successively 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
under vacuum and the residue was triturated with petroleum ether
ether, 1:3) indicated the complete disappearance 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
successively with saturated aqueous solution of NaHCO3
(30 ml ꢁ 3), 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
under vacuum to give the title compound.
to give 12.5 g (80%) of the title compound as a colorless powder.
4.2.3.1. N-(3S-2-Tertbutoxycarbonyl-1,2,3,4-tetrahydroisoquinoline-
20
ESI-MS (m/e) 278 [M þ H]þ; [
a]
¼ ꢂ6.78 (c ¼ 1.0, methanol); 1H
3-carbonyl)-
L
-alanine methylester (4a). Yield: þ89%. Colorless
D
20
NMR (300 MHz, DMSO-d6)
d
/ppm ¼ 11.2 (s,1 H), 7.56 (d, J ¼ 7.5 Hz,1
powder, M.p. 84–85 ꢃC; ESI-MS (m/e) 363 [M þ H] ; [
a]
¼ ꢂ47.46
D
H), 7.44 (d, J ¼ 8.0 Hz, 1 H), 7.23 (t, J ¼ 8.2 Hz, 1 H), 7.22 (t, J ¼ 7.3 Hz,
1 H), 4.14 (m, J ¼ 5.2 Hz, 3 H), 3.13 (m, J ¼ 4.1 Hz, 2 H), 2.72(m,
(c ¼ 1.0, methanol); 1H NMR (300 MHz, DMSO-d6)
/ppm ¼ 8.72 (d,
d
J ¼ 8.4 Hz, 1 H), 7.55 (d, J ¼ 7.4 Hz, 1 H), 7.34 (d, J ¼ 8.1 Hz, 1 H), 7.22
(t, J ¼ 7.1 Hz, 1 H), 7.12(t, J ¼ 7.3 Hz, 1 H), 4.44 (t, J ¼ 5.4 Hz, 1 H), 4.42
(m, J ¼ 5.1 Hz, 6 H), 3.13 (m, J ¼ 4.4 Hz, 2 H), 2.75 (m, J ¼ 5.5 Hz, 2 H),
J ¼ 4.5 Hz, 2 H), 1.42 (s, 9 H); 13C NMR (75 MHz, DMSO-d6)
d/
ppm ¼ 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.
1.41 (s, 9 H), 1.38 (d, J ¼ 6.6 Hz, 3 H); 13C NMR (75 MHz, DMSO-d6)
d/
ppm ¼ 172.8, 168.9, 137.2, 132.4, 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. Anal. Calcd for C19H26N2O5: C,
62.97; H, 7.23; N, 7.73. Found C, 62.70; H, 7.41; N, 7.50.
4.2.3. General procedure for preparing 3S-2-Boc-1,2,3,4-
tetrahydro- isoquinoline-3-carbonylamino acid methylesters (4a–t)
At 0 ꢃC and with stirring, to the solution of 0.256 g
(0.924 mmol) of 3S-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. And then 0.228 g
(0.108 mmol) of DCC was added to form reaction mixture A. To
the suspension of 0.102 mmol of HCl$AA-OMe in 4 ml of anhy-
drous THF, 1 ml of N-methylmorpholine 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
4.2.3.2. N-(3S-2-Tertbutoxycarbonyl-1,2,3,4-tetrahydroisoquinoline-
3-carbonyl)-glycine methylester (4b). Yield: 85%. Colorless powder,
20
M.p. 125–126 ꢃC; ESI-MS (m/e) 349 [M þ H]þ;
[a
]
¼ ꢂ15.83
D
(c ¼ 1.0, methanol); 1H NMR (300 MHz, DMSO-d6)
/ppm ¼ 8.82 (d,
d
J ¼ 8.6 Hz, 1 H), 7.58 (d, J ¼ 7.9 Hz, 1 H), 7.54 (d, J ¼ 8.6 Hz, 1 H), 7.43
(t, J ¼ 7.4 Hz,1 H), 7.15 (t, J ¼ 8.5 Hz,1 H), 4.29 (t, J ¼ 6.3 Hz,1 H), 4.15
(m, J ¼ 4.3 Hz, 6 H), 3.25 (m, J ¼ 5.5 Hz, 2 H), 2.95 (m, J ¼ 4.6 Hz, 2 H),
1.52 (s, 9 H); 13C NMR (75 MHz, DMSO-d6)
d
/ppm ¼ 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,
Fig. 10. Steric and electrostatic environments of 6g,k with lower in vivo anti-thrombotic activity within the grid with 3D points of equation (2).

4912
S. Cheng et al. / European Journal of Medicinal Chemistry 44 (2009) 4904–4919
methanol); 1H NMR (300 MHz, DMSO-d6)
d
/ppm ¼ 8.75 (d,
Table 5
Predicted and tested thrombus weights 6q–t.
J ¼ 8.5 Hz,1 H), 7.56 (d, J ¼ 7.6 Hz,1 H), 7.35 (d, J ¼ 8.6 Hz,1 H), 7.32 (t,
J ¼ 7.7 Hz, 1 H), 7.12 (t, J ¼ 8.2 Hz, 1 H), 5.25 (s, 1 H), 4.46 (m,
J ¼ 5.6 Hz, 6 H), 4.36 (t, J ¼ 4.9 Hz, 1 H), 3.15 (m, J ¼ 4.9 Hz, 2 H), 2.69
Comp.
Thrombus weight (mg)
Predict weight
Test weight
Error
Error%
(m, J ¼ 4.1 Hz, 3 H), 1.56 (s, 9 H); 13C NMR (75 MHz, DMSO-d6)
d/
6q
6r
6s
6t
19.83
21.34
21.47
20.02
19.11
21.25
21.24
18.73
ꢂ0.72
0.09
0.23
1.29
3.7
0.4
0.1
6.8
ppm ¼ 172.8,171.9, 169.7, 137.8, 134.2, 128.9, 127.5, 127.3, 125.9, 81.3,
61.9, 61.2, 57.1, 54.9, 54.4, 51.8, 29.5, 27.9. Anal. Calcd for C19H26N2O6:
C, 60.30; H, 6.93; N, 7.40. Found C, 60.05; H, 6.80; N, 7.16.
51.9, 41.2, 31.0, 26.2. Anal. Calcd for C18H24N2O5: C, 62.05; H, 6.94; N,
8.04. Found C, 61.82; H, 6.79; N, 8.29.
4.2.3.8. N-(3S-2-Tertbutoxycarbonyl-1,2,3,4-tetrahydroisoquinoline-
3-carbonyl)-
L
-tyrosine methylester (4h). Yield: þ82%. Colorless
20
powder, M.p. 62–65 ꢃC; ESI-MS (m/e) 455 [M þ H] ; [
a]
¼ ꢂ23.26
D
4.2.3.3. N-(3S-2-Tertbutoxycarbonyl-1,2,3,4-tetrahydroisoquinoline-
(c ¼ 1.0, methanol); 1H NMR (300 MHz, DMSO-d6)
/ppm ¼ 7.73 (d,
d
3-carbonyl)-
L
-valine methylester (4c). Yield: 86%. Colorless powder,
J ¼ 8.6 Hz, 1 H), 7.56 (d, J ¼ 7.4 Hz, 1 H), 7.39 (d, J ¼ 8.3 Hz, 1 H), 7.23
(t, J ¼ 7.7 Hz,1 H), 7.13 (t, J ¼ 8.2 Hz, 5 H), 4.30 (t, J ¼ 4.4 Hz,1H), 6.70
(d, J ¼ 8.4 Hz, 2 H), 4.28 (m, J ¼ 4.2 Hz, 6 H), 3.13 (m, J ¼ 4.3 Hz, 2 H),
2.94 (m, J ¼ 4.8 Hz, 3 H), 1.43 (s, 9 H); 13C NMR (75 MHz, DMSO-d6)
20
M.p. 87–88 ꢃC; ESI-MS (m/e) 391 [M þ H]þ; [
a]
¼ ꢂ37.57 (c ¼ 1.0,
D
methanol); 1H NMR (300 MHz, DMSO-d6)
d
/ppm ¼ 8.78 (d,
J ¼ 8.4 Hz, 1 H), 7.66 (d, J ¼ 7.5 Hz, 1 H), 7.56 (d, J ¼ 8.0 Hz, 1 H), 7.31
(t, J ¼ 7.7 Hz, 1 H), 7.22 (t, J ¼ 8.2 Hz, 1 H), 4.45 (m, J ¼ 5.4 Hz, 6 H),
4.19 (t, J ¼ 5.6 Hz, 1 H), 3.42 (m, J ¼ 5.3 Hz, 2 H), 2.91 (m, J ¼ 4.4 Hz,
2H), 2.17 (dd, J ¼ 5.7 Hz, 2 H), 1.44 (s, 9 H), 0.97 (m, J ¼ 3.2 Hz, 6 H);
d
/ppm ¼ 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. Anal. Calcd for C25H30N2O6: C, 66.06; H, 6.65; N,
6.16. Found C, 66.25; H, 6.79; N, 6.40.
13C NMR (75 MHz, DMSO-d6)
d/ppm ¼ 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. Anal. Calcd for C21H30N2O5: C, 64.59; H, 7.74; N,
7.17. Found C, 64.36; H, 7.60; N, 6.91.
4.2.3.9. N-(3S-2-Tertbutoxycarbonyl-1,2,3,4-tetrahydroisoquinoline-
3-carbonyl)-L-proline methylester (4i). Yield: 95%. Colorless powder,
M.p. 103–104 ꢃC; ESI-MS (m/e) 388 [M þ H]þ;
[
a]
¼ ꢂ22.22
20
D
(c ¼ 1.0, methanol); 1H NMR (300 MHz, DMSO-d6)
/ppm ¼ 8.75 (d,
d
4.2.3.4. N-(3S-2-Tertbutoxycarbonyl-1,2,3,4-tetrahydroisoquinoline-
3-carbonyl)-
L
- leucine methylester (4d). Yielþd:91%. Colorless powder,
J ¼ 8.4 Hz, 1 H), 7.56 (d, J ¼ 7.5 Hz, 1 H), 7.35 (d, J ¼ 8.0 Hz, 1 H), 7.20
(t, J ¼ 7.7 Hz, 1 H), 7.12 (t, J ¼ 8.2 Hz, 1 H), 4.42 (m, J ¼ 5.8 Hz, 6 H),
4.28 (t, J ¼ 4.3 Hz, 1 H), 3.76 (d, J ¼ 6.3 Hz, 1 H), 3.68 (s, 1 H), 3.12 (m,
J ¼ 4.7 Hz, 2 H), 2.75 (m, J ¼ 4.5 Hz, 3 H), 1.97 (d, J ¼ 8.7 Hz, 4 H), 1.54
20
M.p. 81–83 ꢃC; ESI-MS (m/e) 405 [M þ H] ; [
a]
¼ ꢂ14.1 (c ¼ 1.0,
D
methanol); 1H NMR (300 MHz, DMSO-d6)
d/ppm ¼ 8.72 (d, J ¼ 8.4 Hz,
1 H), 7.55 (d, J ¼ 7.5 Hz, 1 H), 7.34 (d, J ¼ 8.1 Hz,1 H), 7.24 (t, J ¼ 7.3 Hz, 1
H), 7.14 (t, J ¼ 8.2 Hz, 1 H), 4.25 (t, J ¼ 5.4 Hz, 1 H), 4.14 (m, J ¼ 5.5 Hz, 6
H), 3.14 (m, J ¼ 4.3 Hz, 2 H), 2.72 (m, J ¼ 5.3 Hz, 2 H),1.47 (s, 9 H),1.44 (s,
(s, 9 H); 13C NMR (75 MHz, DMSO-d6)
d
/ppm ¼ 173.8, 172.1, 169.7,
137.1, 133.3, 127.9, 127.5, 127.2,125.9, 82.1, 65.6, 58.6, 56.8, 53.8, 52.1,
47.1, 29.9, 28.8, 22.6. Anal. Calcd for C21H28N2O5: C, 64.93; H, 7.27;
N, 7.21. Found C, 64.73; H, 7.11; N, 7.45.
9 H), 0.93 (m, J ¼ 4.1 Hz, 6 H); 13C NMR (75 MHz, DMSO-d6)
d/
ppm ¼ 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. Anal. Calcd for
C22H32N2O5: C, 65.32; H, 7.97; N, 6.93. Found C, 65.55; H, 7.81; N, 6.70.
4.2.3.10. N-(3S-2-Tertbutoxycarbonyl-1,2,3,4-tetrahydroisoquino-
line-3-carbonyl)-L-asparagine methylester (4j). Yield: 55%. Colorless
20
powder, M.p.101–102 ꢃC; ESI-MS (m/e) 407 [M þ H]þ; [
a]
¼ ꢂ6.50
4.2.3.5. N-(3S-2-Tertbutoxycarbonyl-1,2,3,4-tetrahydroisoquinoline-
D
3-carbonyl)-
L
-isoleucine methylester (4e). Yield:þ 89%. Colorless
(c ¼ 1.0, methanol); 1H NMR (300 MHz, DMSO-d6)
/ppm ¼ 8.72 (d,
d
20
powder, M.p. 73–74 ꢃC; ESI-MS (m/e) 405 [M þ H] ; [
a]
¼ ꢂ12.39
J ¼ 8.4 Hz, 1 H), 7.55 (d, J ¼ 7.6 Hz, 1 H), 7.34 (d, J ¼ 8.2 Hz, 1 H), 7.23
(t, J ¼ 7.7 Hz, 1 H), 7.12 (t, J ¼ 8.2 Hz, 1 H), 6.20 (s, 1 H), 4.41(m,
J ¼ 5.4 Hz, 6 H), 4.29 (t, J ¼ 4.3 Hz, 1 H), 3.12 (m, J ¼ 4.5 Hz, 2 H), 2.74
D
(c ¼ 1.0, methanol); 1H NMR (300 MHz, DMSO-d6)
/ppm ¼ 8.73 (d,
d
J ¼ 8.4 Hz, 1 H), 7.55 (d, J ¼ 7.3 Hz, 1 H), 7.34 (d, J ¼ 8.5 Hz, 1 H), 7.27
(t, J ¼ 7.3 Hz, 1 H), 7.13 (t, J ¼ 8.2 Hz, 1 H), 4.33 (m, J ¼ 4.7 Hz, 6 H),
4.29 (t, J ¼ 5.6 Hz, 1 H), 3.13 (m, J ¼ 4.1 Hz, 2 H), 2.74 (m, J ¼ 4.8 Hz, 3
H), 1.29 (dd, J ¼ 5.7 Hz, 2 H), 1.56 (s, 9 H), 0.94 (m, J ¼ 3.9 Hz, 6 H);
(m, J ¼ 4.5 Hz, 4 H), 1.42 (s, 9 H); 13C NMR (75 MHz, DMSO-d6)
d/
ppm ¼ 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. Anal. Calcd for C20H27N3O6: C, 59.25; H,
6.71; N, 10.36. Found C, 59.07; H, 6.56; N, 10.61.
13C NMR (75 MHz, DMSO-d6)
d/ppm ¼ 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. Anal. Calcd for C22H32N2O5: C, 65.32; H,
7.97; N, 6.93. Found C, 65.11; H, 7.82; N, 7.19.
4.2.3.11. N-(3S-2-Tertbutoxycarbonyl-1,2,3,4-tetrahydroisoquinoline-
3-carbonyl)-L-glutamine methylester (4k). Yield: 45%. Colorless
powder, M.p. 100–102 ꢃC; ESI-MS (m/e) 420 [M þ H]þ;
20
4.2.3.6. N-(3S-2-Tertbutoxycarbonyl-1,2,3,4-tetrahydroisoquinoline-
[a
]
¼ ꢂ28.78 (c ¼ 1.0, methanol); 1H NMR (300 MHz, DMSO-d6)
d/
D
3-carbonyl)-
L
-tryptophan methylester (4f). Yield: 87%. Colorless
ppm ¼ 8.72 (d, J ¼ 8.5 Hz, 1 H), 8.12 (s, 1 H), 7.55 (d, J ¼ 7.7 Hz, 1 H),
7.34 (d, J ¼ 8.2 Hz, 1 H), 7.24 (t, J ¼ 7.7 Hz, 1 H), 7.13 (t, J ¼ 8.2 Hz, 1
H), 4.33 (m, J ¼ 5.6 Hz, 6 H), 4.24 (t, J ¼ 4.3 Hz, 1 H), 3.13 (m,
J ¼ 4.8 Hz, 2 H), 2.73 (m, J ¼ 4.3 Hz, 6 H), 1.46 (s, 9 H); 13C NMR
20
powder, M.p. 71–72 ꢃC; ESI-MS (m/e) 478 [M þ H]þ; [
a
]
¼ ꢂ7.99
D
(c ¼ 1.0, methanol); 1H NMR (300 MHz, DMSO-d6)
/ppm ¼ 10.97 (s,
d
1 H), 9.06 (d, J ¼ 8.4 Hz, 1 H), 7.72 (m, J ¼ 7.5 Hz, 9 H), 4.38 (m,
J ¼ 5.3 Hz, 6 H), 4.30 (t, J ¼ 5.1 Hz, 1 H), 3.13 (m, J ¼ 4.5 Hz, 2 H), 2.69
(75 MHz, DMSO-d6)
d
/ppm ¼ 173.9, 171.9, 171.6, 168.9, 137.1, 133.5,
(m, J ¼ 4.0 Hz, 2 H), 1.65 (s, 9 H); 13C NMR (75 MHz, DMSO-d6)
d/
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. Anal. Calcd for C21H29N3O6: C, 60.13; H, 6.97; N,10.02. Found C,
60.30; H, 6.81; N, 10.25.
ppm ¼ 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. Anal. Calcd for C27H31N3O5: C, 67.91; H, 6.54; N, 8.80.
Found C, 67.68; H, 6.40; N, 8.57.
4.2.3.12. N-(3S-2-Tertbutoxycarbonyl-1,2,3,4-tetrahydroisoquino-
line-3-carbonyl)-
L
-histidine methylester (4l). Yield:þ65%. Colorless
20
powder, M.p. 116–117 ꢃC; ESI-MS (m/e) 429 [M þ H] ; [
a
]
¼ ꢂ6.67
4.2.3.7. N-(3S-2-Tertbutoxycarbonyl-1,2,3,4-tetrahydroisoquinoline-
D
3-carbonyl)-
L
-serin methylester (4g). Yield: 80%. Colorless powder,
(c ¼ 1.0, methanol); 1H NMR (300 MHz, DMSO-d6)
/ppm ¼ 9.12 (s,
d
20
M.p. 69–71 ꢃC; ESI-MS (m/e) 379 [M þ H]þ; [
a
]
¼ ꢂ7.45 (c ¼ 1.0,
J ¼ 8.0 Hz, 1 H), 8.72 (d, J ¼ 8.4 Hz, 1 H), 7.55 (d, J ¼ 7.5 Hz, 1 H), 7.35
D

S. Cheng et al. / European Journal of Medicinal Chemistry 44 (2009) 4904–4919
4913
(d, J ¼ 8.0 Hz, 1H), 7.21 (t, J ¼ 7.7 Hz,1 H), 7.13 (t, J ¼ 8.2 Hz,1 H), 4.29
(t, J ¼ 4.4 Hz, 1 H), 4.26 (m, J ¼ 4.3 Hz, 6H), 3.15 (m, J ¼ 5.1 Hz, 2 H),
3.14 (m, J ¼ 5.0 Hz, 2 H), 2.74 (m, J ¼ 4.5 Hz, 2 H), 1.43 (s, 9 H); 13C
27.5. Anal. Calcd for C25H30N2O5: C, 68.47; H, 6.90; N, 6.39. Found C,
68.71; H, 7.15; N, 6.63.
NMR (75 MHz, DMSO-d6)
d
/ppm ¼ 171.9, 171.6, 168.9, 137.1, 135.6,
4.2.3.18. N-(3S-2-Tertbutoxycarbonyl-1,2,3,4-tetrahydroisoquino-
133.8, 133.1, 128.9, 127.5, 127.2, 125.8, 118.7, 81.3, 68.2, 56.1, 53.4,
52.2, 51.3, 30.0, 28.9, 27.2. Anal. Calcd for C22H28N4O5: C, 61.67; H,
6.59; N, 13.08. Found C, 61.86; H, 6.72; N, 13.34.
line-3-carbonyl)-
L
-threonine methylester (4r). Yielþd: 89%. Colorless
20
powder, M.p. 80–81 ꢃC; ESI-MS (m/e) 393 [M þ H] ; [
a
]
¼ ꢂ42.43
D
(c ¼ 1.0, methanol); 1H NMR (300 MHz, DMSO-d6)
/ppm ¼ 8.73 (d,
d
J ¼ 8.4 Hz, 1 H), 7.56 (d, J ¼ 7.5 Hz, 1 H), 7.33 (d, J ¼ 8.1 Hz, 1 H), 7.22
(t, J ¼ 7.7 Hz,1 H), 7.14 (t, J ¼ 8.6 Hz,1 H), 4.28 (t, J ¼ 4.5 Hz,1 H), 4.29
(m, J ¼ 4.3 Hz, 6 H), 3.16 (m, J ¼ 4.2 Hz, 2 H), 2.74 (m, J ¼ 4.1 Hz, 3 H),
4.2.3.13. N-(3S-2-Tertbutoxycarbonyl-1,2,3,4-tetrahydroisoquino-
line-3-carbonyl)-L-lysine(Z) benzylester (4m). Yield: 95%. Syrupy.
20
ESI-MS (m/e) 630 [M þ H]þ; [
a
]
¼ ꢂ19.9 (c ¼ 1.0, CH3OH); 1H NMR
2.15 (s, 1 H), 1.52 (s, 9 H); 13C NMR (75 MHz, DMSO-d6)
d/
D
(300 MHz, CDCl3)
d
/ppm ¼ 8.72 (d, J ¼ 8.4 Hz, 1H), 6.99–7.60 (m,
ppm ¼ 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. Anal. Calcd for C20H28N2O6: C,
61.21; H, 7.19; N, 7.14. Found C, 61.00; H, 7.02; N, 7.41.
14H), 5.34 (d, J ¼ 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 ¼ 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.
Anal. Calcd for C36H43N3O7: C, 68.66; H, 6.88; N, 6.67. Found C,
68.47; H, 6.72; N, 6.90.
4.2.3.19. N-(3S-2-Tertbutoxycarbonyl-1,2,3,4-tetrahydroisoquino-
line-3-carbonyl)-L-arginine methylester (4s). Yield: 51%. Colorless
powder, M.p. 151–152 ꢃC; ESI-MS (m/e) 448.3[M þ H]þ;
20
[a
]
¼ ꢂ11.56 (c ¼ 1.0, methanol); 1H NMR (300 MHz, DMSO-d6)
d/
D
4.2.3.14. N-(3S-2-Tertbutoxycarbonyl-1,2,3,4-tetrahydroisoquino-
line-3-carbonyl)-aspartic acid dimethylester (4n). Yield: 75þ%.
ppm ¼ 8.99(s, 3 H), 8.72 (d, J ¼ 8.5 Hz, 1 H), 7.54 (d, J ¼ 7.3 Hz, 1 H),
7.35 (d, J ¼ 8.4 Hz, 1 H), 7.24 (t, J ¼ 7.7 Hz, 1H), 7.13 (t, J ¼ 8.3 Hz, 1 H),
4.24 (t, J ¼ 5.3 Hz,1 H), 4.12 (m, J ¼ 4.3 Hz, 6 H), 3.53 (m, J ¼ 4.6 Hz, 6
H), 3.15(m, J ¼ 4.6 Hz, 2 H), 2.73 (m, J ¼ 4.3 Hz, 4 H), 2.11 (m,
J ¼ 4.3 Hz, 1 H), 1.96 (m, J ¼ 3.9 Hz, 1 H), 1.45 (s, 9H); 13C NMR
Colorless powder, M.p. 106–107 ꢃC; ESI-MS (m/e) 407 [M þ H] ;
20
[
a]
¼ ꢂ3.23(c ¼ 1.0, methanol); 1H NMR (300 MHz, DMSO-d6)
d/
D
ppm ¼ 8.72 (d, J ¼ 8.4 Hz, 1 H), 7.55 (d, J ¼ 7.6 Hz, 1 H), 7.35 (d,
J ¼ 8.3 Hz, 1 H), 7.28 (t, J ¼ 7.7 Hz, 1 H), 7.13 (t, J ¼ 8.2 Hz, 1 H), 4.31
(m, J ¼ 4.3 Hz, 3 H), 4.24 (t, J ¼ 4.4 Hz, 1 H), 3.73 (m, J ¼ 4.6 Hz, 6 H),
3.12 (m, J ¼ 4.6 Hz, 2 H), 2.89 (m, J ¼ 4.3 Hz, 3 H), 2.76 (m, J ¼ 4.5 Hz,
(75 MHz, DMSO-d6)
d/ppm ¼ 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. Anal. Calcd for C22H33N5O5: C, 59.04; H, 7.43; N,
15.65. Found C, 59.23; H, 7.60; N, 15.81.
2 H), 1.42 (s, 9 H); 13C NMR (75 MHz, DMSO-d6)
d/ppm ¼ 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. Anal. Calcd for C21H28N2O7: C,
59.99; H, 6.71; N, 6.66. Found C, 59.80; H, 6.57; N, 6.91.
4.2.3.20. N-(3S-2-Tertbutoxycarbonyl-1,2,3,4-tetrahydroisoquino-
line-3-carbonyl)-L-methionine methylester (4t). Yield: 49%. Color-
less powder, M.p. 143–144 ꢃC; ESI-MS (m/e) 409 [M þ H]þ;
20
4.2.3.15. N-(3S-2-Tertbutoxycarbonyl-1,2,3,4-tetrahydroisoquino-
[a
]
¼ ꢂ12.90 (c ¼ 1.0, methanol); 1H NMR (300 MHz, DMSO-d6)
d/
D
line-3-carbonyl)-
L
- glutamic acid dimethylester (4o). Yield: 78þ%.
ppm ¼ 8.72 (d, J ¼ 8.7 Hz, 1 H), 7.55 (d, J ¼ 7.6 Hz, 1 H), 7.34 (d,
J ¼ 8.3 Hz,1 H), 7.23 (t, J ¼ 7.7 Hz,1 H), 7.12 (t, J ¼ 8.2 Hz,1 H), 4.32 (t,
J ¼ 4.5 Hz, 1H), 4.25 (m, J ¼ 4.9 Hz, 6 H), 3.12 (m, J ¼ 4.3 Hz, 2 H),
2.81 (m, J ¼ 4.9 Hz, 2 H), 2.35 (m, J ¼ 4.6 Hz, 2 H), 1.42 (s, 9 H), 1.27
Colorless powder, M.p. 107–109 ꢃC; ESI-MS (m/e) 420 [M þ H] ;
20
[
a]
¼ ꢂ4.05 (c ¼ 1.0, methanol); 1H NMR (300 MHz, DMSO-d6)
d/
D
ppm ¼ 8.72 (d, J ¼ 8.5 Hz, 1 H), 7.57 (d, J ¼ 7.6 Hz, 1 H), 7.33 (d,
J ¼ 8.7 Hz, 1 H), 7.28 (t, J ¼ 7.7 Hz, 1 H), 7.12 (t, J ¼ 8.2 Hz, 1 H), 4.45
(m, J ¼ 5.6 Hz, 3 H), 4.29 (t, J ¼ 5.5 Hz, 1 H), 3.53 (m, J ¼ 4.6 Hz, 6 H),
3.12 (m, J ¼ 4.5 Hz, 2 H), 2.72 (m, J ¼ 4.8 Hz, 2 H), 1.96 (m, J ¼ 5.2 Hz,
(m, J ¼ 4.2 Hz, 2 H); 13C NMR (75 MHz, DMSO-d6)
/ppm ¼ 171.8,
d
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. Anal. Calcd for C21H30N2O5S: C,
59.69; H, 7.16; N, 6.63. Found C, 59.88; H, 7.00; N, 6.88.
4 H), 1.42 (s, 9 H); 13C NMR (75 MHz, DMSO-d6)
d/ppm ¼ 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. Anal. Calcd for
C22H30N2O7: C, 60.82; H, 6.96; N, 6.45. Found C, 60.99; H, 7.13; N,
6.71.
4.2.4. General procedure preparing 3S-2-Boc-1,2,3,4-
tetrahydroisoquinoline-3-carbonylamino acids (5a–t)
At 0 ꢃC to the solution of 1.0 mmol of 3S-2-Boc-1,2,3,4-tetrahy-
droisoquinoline-3-carbonylamino acid methylester in 5 ml of
methanol, was added 7 ml of 2 N aqueous of NaOH to adjust pH 11.
The reaction mixture was stirred at 0 ꢃC for 3 h, and TLC (CCl3/
CH3OH, 5:1) indicated the complete disappearance of 3S-2-Boc-
1,2,3,4-tetrahydroisoquinoline-3-carbonylamino acid methylester.
The reaction mixture was adjusted to pH 7 with aqueous solution of
KHSO4. The solution was evaporated under vacuum to remove
methanol, adjusted 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 filtra-
tion, the filtrate was evaporated to provide the title compound.
4.2.3.16. N-(3S-2-Tertbutoxycarbonyl-1,2,3,4-tetrahydroisoquino-
line-3-carbonyl)-L- cysteine methylester (4p). Yield: 50%. Colorless
powder, M.p. 107–110 ꢃC; ESI-MS (m/e) 395 [M þ H]þ;
20
[
a]
¼ ꢂ25.12 (c ¼ 1.0, methanol); 1H NMR (300 MHz, CDCl3)
d/
D
ppm ¼ 8.00 (d, J ¼ 7.6 Hz, 1 H), 7.02 (m, J ¼ 8.2 Hz, 2 H), 6.95 (d,
J ¼ 8.8 Hz, 2 H), 4.90 (t, J ¼ 8.4 Hz, 1 H), 4.71 (m, 1 H), 4.20 (t,
J ¼ 8.3 Hz, 2 H), 3.65 (s, 3 H), 3.07 (m, J ¼ 5.4 Hz, 4 H), 1.5 (t,
J ¼ 6.4 Hz, 1 H), 1.41 (s, 9 H). Anal. Calcd for C19H26N2O5S: C, 57.85;
H, 6.64; N, 7.10. Found C, 57.66; H, 6.50; N, 6.89.
4.2.3.17. N-(3S-2-Tertbutoxycarbonyl-1,2,3,4-tetrahydroisoquino-
line-3-carbonyl)-
L
-phenylalanine methylester (4q). Yield: 87þ%.
Colorless powder, M.p. 91–93 ꢃC; ESI-MS (m/e) 439 [M þ H] ;
4.2.4.1. N-(3S-2-Tertbutoxycarbonyl-1,2,3,4-tetrahydroisoquinoline-
20
[
a]
¼ ꢂ7.61 (c ¼ 1.0, methanol); 1H NMR (300 MHz, DMSO-d6)
d/
3-carbonyl)-
123 ꢃC; ESI-MS (m/e) 348 [M-H]ꢂ; [
1H NMR (300 MHz, CDCl3)
1 H), 7.02 (m, J ¼ 8.2 Hz, 2 H), 6.95 (d, J ¼ 8.8 Hz, 2 H), 4.93 (d,
J ¼ 5.4 Hz, 1H), 4.63 (m, 1 H), 4.20(d, J ¼ 5.4 Hz, 2 H), 3.18 (d,
J ¼ 5.5 Hz, 1 H), 2.92 (d, J ¼ 8.5 Hz, 1 H), 1.45 (d, J ¼ 6.3 Hz, 3 H), 1.41
L-alanine (5a). Yield: 96%. Colorless powder, M.p. 122–
D
20
ppm ¼ 9.06(d, J ¼ 8.4 Hz, 1 H), 7.25(m, J ¼ 7.5 Hz, 9 H), 4.25(t,
J ¼ 5.6 Hz, 1 H), 4.15(m, J ¼ 5.2 Hz, 6 H), 3.02(m, J ¼ 4.2 Hz, 4 H),
2.93(m, J ¼ 5.6 Hz, 2 H), 1.45(s, 9 H), 1.41(m, J ¼ 4.1 Hz, 2 H); 13C
a
]
¼ ꢂ7.98 (c ¼ 1.0, methanol);
D
d
/ppm ¼ 11.00 (s, 1 H), 8.00 (d, J ¼ 7.6 Hz,
NMR (75 MHz, DMSO-d6)
d
/ppm ¼ 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,

4914
S. Cheng et al. / European Journal of Medicinal Chemistry 44 (2009) 4904–4919
(s, 9 H). Anal. Calcd for C18H24N2O5: C, 62.05; H, 6.94; N, 8.04. Found
C, 62.24; H, 6.80; N, 7.78.
H), 3.19 (d, J ¼ 5.3 Hz, 1 H), 3.05 (d, J ¼ 6.5 Hz, 1 H), 2.92 (m, 2 H),
1.41(s, 9 H). Anal. Calcd for C24H28N2O6: C, 65.44; H, 6.41; N, 6.36.
Found C, 65.23; H, 6.56; N, 6.60.
4.2.4.2. N-(3S-2-Tertbutoxycarbonyl-1,2,3,4-tetrahydroisoquinoline-
4.2.4.9. N-(3S-2-Tertbutoxycarbonyl-1,2,3,4-tetrahydroisoquinoline-
3-carbonyl)-glycine (5b). Yield:ꢂ99%. Colorless powder, M.p. 147–
20
149 ꢃC; ESI-MS (m/e) 333 [M-H] ; [
a]
D
¼ ꢂ0.13 (c ¼ 1.0, methanol);
3-carbonyl)-
170 ꢃC; ESI-MS (m/e) 373 [M-H]ꢂ; [
1H NMR (300 MHz, CDCl3)
L-proline (5i). Yield: 93%. Colorless powder, M.p. 169–
20
1H NMR (300 MHz, CDCl3)
d
/ppm ¼ 11.00 (s,1 H), 8.01 (d, J ¼ 7.6 Hz,1
a
]
¼ ꢂ6.06 (c ¼ 1.0, methanol);
D
d
/ppm ¼ 11.00 (s, 1 H), 7.02 (m,
H), 7.02 (m, J ¼ 8.2 Hz, 2 H), 6.95 (d, J ¼ 8.8 Hz, 2 H), 4.90 (d, J ¼ 5.4 Hz,
1 H), 4.21 (m, J ¼ 8.2 Hz, 2 H), 4.15(m, J ¼ 8.2 Hz, 2 H), 3.15 (d,
J ¼ 6.2 Hz, 1 H), 2.85 (d, J ¼ 6.2 Hz, 1 H), 1.41 (s, 9 H). Anal. Calcd for
C17H22N2O5: C, 61.07; H, 6.63; N, 8.38. Found C, 61.25; H, 6.50; N, 8.16.
J ¼ 8.2 Hz, 2 H), 6.95 (d, J ¼ 8.8 Hz, 2 H), 4.90 (t, J ¼ 7.4 Hz, 1 H), 4.31
(m,1 H), 4.20 (t, J ¼ 8.3 Hz, 2 H), 3.46 (m, 2 H), 3.01 (m, 2 H), 2.20 (m,
2 H), 1.97 (m, 2 H), 1.41 (s, 9 H). Anal. Calcd for C20H26N2O5: C, 64.15;
H, 7.00; N, 7.48. Found C, 64.34; H, 6.84; N, 7.70.
4.2.4.3. N-(3S-2-Tertbutoxycarbonyl-1,2,3,4-tetrahydroisoquinoline-
4.2.4.10. N-(3S-2-Tertbutoxycarbonyl-1,2,3,4-tetrahydroisoquino-
3-carbonyl)-
119 ꢃC; ESI-MS (m/e) 407 [M-H]ꢂ; [
1H NMR (300 MHz, CDCl3)
L-valine (5c). Yield: 93%. Colorless powder, M.p. 116–
20
line-3-carbonyl)-
M.p. 137–139 ꢃC; ESI-MS (m/e) 407 [M-H]ꢂ; [
methanol); 1H NMR (300 MHz, CDCl3)
L-asparagine (5j). Yield: 90%. Colorless powder,
a
]
¼ ꢂ6.53 (c ¼ 1.0, methanol);
D
20
a
]
¼ ꢂ9.42 (c ¼ 1.0,
D
d
/ppm ¼ 11.00 (s,1 H), 8.00 (d, J ¼ 6.7 Hz,1
d
/ppm ¼ 11.00 (s,1 H), 8.00 (d,
H), 7.01 (m, J ¼ 8.1 Hz, 2 H), 6.96 (d, J ¼ 8.8 Hz, 2 H), 4.90 (d, J ¼ 7.4 Hz,
1 H), 4.51 (d, J ¼ 5.3 Hz,1 H), 4.24 (m, 2 H), 3.05 (m, 2 H), 2.80 (m,1 H),
1.41 (s, 9 H), 1.21 (d, J ¼ 8.7 Hz, 6 H). Anal. Calcd for C20H28N2O5: C,
63.81; H, 7.50; N, 7.44. Found C, 63.60; H, 7.66; N, 7.68.
J ¼ 5.6 Hz, 1 H), 7.02 (m, J ¼ 8.2 Hz, 2 H), 6.95 (d, J ¼ 8.8 Hz, 2 H), 5.85
(s, 2 H), 4.85 (m,1 H), 4.71 (m, J ¼ 7.1 Hz,1 H), 4.21 (m, J ¼ 8.0 Hz, 2 H),
3.16 (m, 2 H), 2.68 (m, 2 H), 1.41 (s, 9 H). Anal. Calcd for C19H25N3O6:
C, 58.30; H, 6.44; N, 10.74. Found C, 58.11; H, 6.30; N, 10.52.
4.2.4.4. N-(3S-2-Tertbutoxycarbonyl-1,2,3,4-tetrahydroisoquinoline-
4.2.4.11. N-(3S-2-Tertbutoxycarbonyl-1,2,3,4-tetrahydroisoquinoline-
3-carbonyl)-
L
- leucine (5d). Yielꢂd: 90%. Colorless powder, M.p. 119–
3-carbonyl)-
L
-glutamine (5k). Yield: 98ꢂ%. Colorless powder, M.p.
20
120 ꢃC; ESI-MS (m/e) 389 [M-H] ; [
a]
¼ ꢂ8.91 (c ¼ 1.0, methanol);
D
20
154–157 ꢃC; ESI-MS (m/e) 421 [M-H] ; [
a
]
¼ ꢂ45.38 (c ¼ 1.0,
D
1H NMR (300 MHz, CDCl3)
d/ppm ¼ 11.00 (s, 1 H), 8.02 (d, J ¼ 8.6 Hz,
methanol); 1H NMR (300 MHz, CDCl3)
d/ppm ¼ 11.00 (s, 1 H), 8.00
1 H), 7.04 (m, J ¼ 8.2 Hz, 2 H), 6.97 (d, J ¼ 8.8 Hz, 2 H), 4.90 (d,
J ¼ 7.4 Hz, 1 H), 4.50 (m, 1 H), 4.22 (m, J ¼ 8.6 Hz, 2 H), 3.10 (m, 2 H),
1.78 (m, 3 H), 1.41 (s, 9 H), 1.01 (d, J ¼ 7.6 Hz, 6 H). Anal. Calcd for
C21H30N2O5: C, 64.59; H, 7.74; N, 7.17. Found C, 64.80; H, 7.89; N, 7.40.
(d, J ¼ 5.6 Hz, 1 H), 7.02 (m, J ¼ 8.2 Hz, 2 H), 6.95 (d, J ¼ 8.8 Hz, 2 H),
5.85 (s, 2 H), 4.85 (m, 1 H), 4.51 (m, J ¼ 7.1 Hz, 1 H), 4.22 (m,
J ¼ 8.0 Hz, 2 H), 3.06 (m, 2 H), 2.18 (m, J ¼ 7.2 Hz, 2 H), 2.06 (m, 2 H),
1.41 (s, 9 H). Anal. Calcd for C20H27N3O6: C, 59.25; H, 6.71; N, 10.36.
Found C, 59.44; H, 6.56; N, 10.13.
4.2.4.5. N-(3S-2-Tertbutoxycarbonyl-1,2,3,4-tetrahydroisoquinoline-
3-carbonyl)-
105–108 ꢃC; ESI-MS (m/e) 389 [M-H]ꢂ;
methanol); 1H NMR (300 MHz, CHCl3)
(d, J ¼ 5.6 Hz, 1 H), 7.01 (m, J ¼ 8.2 Hz, 2 H), 6.95 (d, J ¼ 8.8 Hz, 2 H),
4.91 (d, J ¼ 6.4 Hz, 1 H), 4.51 (m, 1 H), 4.21 (m, J ¼ 8.6 Hz, 2 H), 3.10
(m, 2 H), 2.58 (m, 1 H), 1.41 (s, 9 H),1.29 (m, 2 H), 1.06 (d, J ¼ 7.6 Hz, 3
H), 0.96 (d, J ¼ 8.5 Hz, 3 H). Anal. Calcd for C21H30N2O5: C, 64.59; H,
7.74; N, 7.17. Found C, 64.80; H, 7.89; N, 7.51.
L-isoleucine (5e). Yield: 94%. Colorless powder, M.p.
4.2.4.12. N-(3S-2-Tertbutoxycarbonyl-1,2,3,4-tetrahydroisoquino-
20
[
a
]
¼ ꢂ9.13 (c ¼ 1.0,
D
line-3-carbonyl)-
150–151 ꢃC; ESI-MS (m/e) 413 [M-H]ꢂ;
methanol); 1H NMR (300 MHz, CDCl3)
L-histidine (5l). Yield: 75%. Colorless powder, M.p.
d
/ppm ¼ 11.00 (s, 1 H), 8.00
20
[
a
]
¼ ꢂ12.8 (c ¼ 1.0,
D
d
/ppm ¼ 13.40 (d, J ¼ 5.6 Hz,
1 H), 11.01 (s, 1 H), 8.00 (s, 1 H), 7.44 (s, 1 H), 7.02 (m, J ¼ 8.2 Hz, 2 H),
6.95 (d, J ¼ 8.8 Hz, 2 H), 6.80 (d, J ¼ 7.6 Hz, 1 H), 4.92 (t, J ¼ 8.5 Hz, 1
H), 4.80 (t, J ¼ 6.4 Hz, 1 H), 4.21 (m, 2 H), 3.17 (m, 2 H), 2.92 (m, 2 H),
1.41 (s, 9 H). Anal. Calcd for C21H26N4O5: C, 60.86; H, 6.32; N, 13.52.
Found C, 60.65; H, 6.48; N, 13.72.
4.2.4.6. N-(3S-2-Tertbutoxycarbonyl-1,2,3,4-tetrahydroisoquinoline-
3-carbonyl)-
L
-tryptophan (5f). Yield: 90ꢂ%. Colorless powder, M.p.
4.2.4.13. N-(3S-2-Tertbutoxycarbonyl-1,2,3,4-tetrahydroisoquino-
20
115–116 ꢃC; ESI-MS (m/e) 462 [M-H] ; [
a
]
¼ ꢂ17.67 (c ¼ 1.0,
D
line-3-carbonyl)-
3S-2-Boc-1,2,3,4-tetrahydroisoquinoline-3-
L
-lysine (5m). The solution of 1.5 g (2.38 mmol) of
carbonyl- -lysine(Z)
methanol); 1H NMR (300 MHz, CDCl3)
d
/ppm ¼ 11.00 (s, 1 H), 10.10
L
(s, 1 H), 8.00 (d, J ¼ 5.6 Hz, 1 H), 7.18 (m, J ¼ 6.4 Hz, 4 H), 7.02 (m,
J ¼ 8.2 Hz, 2H), 6.97 (d, J ¼ 8.8 Hz, 2 H), 6.80 (d, J ¼ 6.4 Hz, 1H), 4.90
(d, J ¼ 7.1 Hz, 1 H), 4.81 (d, J ¼ 7.4 Hz, 1 H), 4.21 (m, 2 H), 3.10 (m, 4
H), 1.41(s, 9 H). Anal. Calcd for C26H29N3O5: C, 67.37; H, 6.31; N, 9.07.
Found C, 67.15; H, 6.14; N, 9.30.
benzylester (4p) in 5 ml anhydrous ethanol was mixed with 0.200 g
of Pt/C. To the suspension hydrogen was bubbled for 96 h and TLC
(CHCl3/CH3OH, 1:1) indicated complete disappearance of 4p. After
filtration the filtrate was evaporated to give 0.92 g (96%) of title
20
compound as a syrupy. ESI-MS (m/e) 404 [M-H]ꢂ; [
a
]
D
¼ ꢂ3.4
(c ¼ 1.0, CH3OH); 1H NMR (300 MHz, CDCl3)
/ppm ¼ 11.70 (s, 1H),
d
4.2.4.7. N-(3S-2-Tertbutoxycarbonyl-1,2,3,4-tetrahydroisoquinoline-
8.72 (d, J ¼ 8.4 Hz, 1H), 7.29 (m, 14H), 4.28 (t, 1H), 4.18 (m, 3H), 3.12
(m, 2H), 2.82 (m, 2H), 2.21 (m, 5H),1.36 (m,13H); 13C NMR (75 MHz,
3-carbonyl)-
L
-serin (5g). Yield:ꢂ93%. Colorless powder, M.p. 102–
20
104 ꢃC; ESI-MS (m/e) 363 [M-H] ; [
a]
D
¼ ꢂ3.30 (c ¼ 1.0, methanol);
CDCl3)
d
/ppm ¼ 172.9, 171.6, 169.6, 142.8, 137.6, 136.6, 134.7, 125.2–
1H NMR (300 MHz, CDCl3)
d
/ppm ¼ 11.00 (s,1 H), 8.00 (d, J ¼ 5.6 Hz,1
128.7, 82.7, 68.8, 67.8, 56.8, 54.8, 53.7, 52.8, 32.0, 30.2, 28.9, 27.9,
22.7. Anal. Calcd for C21H31N3O5: C, 62.20; H, 7.71; N,10.36. Found C,
62.00; H, 7.58; N, 13.59.
H), 7.02 (m, J ¼ 8.2 Hz, 2 H), 6.95 (d, J ¼ 8.8 Hz, 2 H), 4.83 (m, 1 H),
4.53 (m,1 H), 4.21 (m, 2 H), 3.96 (m, 2 H), 3.19 (d, J ¼ 8.2 Hz,1 H), 2.95
(d, J ¼ 6.4 Hz, 1 H), 1.41 (s, 9 H). Anal. Calcd for C18H24N2O6: C, 59.33;
H, 6.64; N, 7.69. Found C, 59.52; H, 6.79; N, 7.90.
4.2.4.14. N-(3S-2-Tertbutoxycarbonyl-1,2,3,4-tetrahydroisoquino-
line-3-carbonyl)-aspartic acid (5n). Yield:ꢂ65%. Colorless powder,
20
4.2.4.8. N-(3S-2-Tertbutoxycarbonyl-1,2,3,4-tetrahydroisoquinoline-
M.p. 131–133 ꢃC; ESI-MS (m/e) 391 [M-H] ; [
a]
¼ ꢂ7.93 (c ¼ 1.0,
D
3-carboxyl)-
L
-tyrosine (5h). Yield:ꢂ90%. Colorless powder, M.p. 99–
methanol); 1H NMR (300 MHz, CDCl3)
d
/ppm ¼ 11.00 (s, 2 H), 8.00
20
100 ꢃC; ESI-MS (m/e) 439 [M-H] ; [
a]
¼ ꢂ11.52 (c ¼ 1.0, meth-
(d, J ¼ 8.6 Hz, 1 H), 7.02 (m, J ¼ 8.2 Hz, 2 H), 6.95 (d, J ¼ 8.8 Hz, 2 H),
4.91 (m,1 H), 4.78 (m,1 H), 4.21 (m, 2 H), 3.09 (m, 2H), 2.68 (m, 2 H),
1.41 (s, 9 H). Anal. Calcd for C19H24N2O7: C, 58.16; H, 6.16; N, 7.14.
Found C, 58.35; H, 6.30; N, 7.39.
D
anol); 1H NMR (300 MHz, CDCl3)
J ¼ 8.6 Hz,1 H), 7.02 (m, J ¼ 8.2 Hz, 2 H), 6.98 (d, J ¼ 8.8 Hz, 2 H), 6.95
d/ppm ¼ 11.00 (s, 1H), 8.00 (d,
(d, J ¼ 8.8 Hz, 2 H), 6.72 (d, J ¼ 5.5 Hz, 2 H), 4.92 (m, 3 H), 4.21 (m, 2

S. Cheng et al. / European Journal of Medicinal Chemistry 44 (2009) 4904–4919
4915
4.2.4.15. N-(3S-2-Tertbutoxycarbonyl-1,2,3,4-tetrahydroisoquino-
and TLC (CCl3/CH3OH, 5:1) indicated the complete disappearance of
3S-2-Boc-1,2,3,4-tetrahydroisoquinoline-3-carbonyl- -amino acid.
line-3-carbonyl)-
L
- glutamic acid (5o). Yielꢂd: 89%. Colorless powder,
L
20
M.p. 125–128 ꢃC; ESI-MS (m/e) 405 [M-H] ; [
a
]
¼ ꢂ10.46 (c ¼ 1.0,
The reaction mixture was evaporated under vacuum to dry and the
residue was dissolved in 5 ml of ethyl acetate. The solution was
evaporated under vacuum to dry and the residue was re-dissolved
in 5 ml of ethyl acetate. This procedure was repeated for three times
to provide the title compound as colorless powder.
D
methanol); 1H NMR (300 MHz, CDCl3)
d/ppm ¼ 11.00 (s, 2 H), 8.00
(d, J ¼ 8.6 Hz, 1 H), 7.02 (m, J ¼ 8.2 Hz, 2 H), 6.95 (d, J ¼ 8.8 Hz, 2 H),
4.91 (m,1 H), 4.48 (m,1 H), 4.21 (m, 2 H), 3.09 (m, 2H), 2.24 (m, 2 H),
2.04 (m, 2 H), 1.41 (s, 9 H). Anal. Calcd for C20H26N2O7: C, 59.10; H,
6.45; N, 6.89. Found C, 59.30; H, 6.51; N, 7.12.
4.2.5.1. N-(3S-1,2,3,4-Tetrahydroisoquinoline-3-carbonyl)-L-alanine
4.2.4.16. N-(3S-2-Tertbutoxycarbonyl-1,2,3,4-tetrahydroisoquino-
(6a). Yield: 95%. Colorless powder, M.p. 121–123 ꢃC; ESI-MS (m/e)
20
line-3-carbonyl)-
L
- cysteine (5p). Yield:8ꢂ6%. Colorless powder, M.p.
248 [M þ H]þ; [
a]
¼ ꢂ164 (c ¼ 1.0, H2O); 1H NMR (300 MHz,
D
20
146–148 ꢃC; ESI-MS (m/e) 757 [M-H] ; [
a
]
¼ ꢂ25.12 (c ¼ 1.0,
DMSO-d6)
d
/ppm ¼ 12.9 (s, 1 H), 8.91 (m, J ¼ 5.9 Hz, 1 H), 8.72 (d,
D
methanol); 1H NMR (300 MHz, CDCl3)
d
/ppm ¼ 11.00 (s, 1 H), 8.00
J ¼ 8.4 Hz, 1 H), 7.56 (d, J ¼ 7.6 Hz, 1 H), 7.31 (d, J ¼ 8.1 Hz, 1 H), 7.23
(t, J ¼ 7.2 Hz,1 H), 7.13 (t, J ¼ 8.2 Hz,1 H), 4.44 (t, J ¼ 8.2 Hz,1 H), 4.35
(m, J ¼ 4.6 Hz, 3 H), 2.73 (m, J ¼ 4.3 Hz, 2 H), 1.38 (d, J ¼ 6.6 Hz, 3 H);
(d, J ¼ 7.6 Hz, 1 H), 7.02 (m, J ¼ 8.2 Hz, 2 H), 6.95 (d, J ¼ 8.8 Hz, 2 H),
4.90 (t, J ¼ 8.4 Hz,1 H), 4.81 (m,1 H), 4.20 (t, J ¼ 8.3 Hz, 2 H), 3.17 (m,
J ¼ 5.4 Hz, 2 H), 2.92 (m, J ¼ 5.4 Hz, 2 H), 1.5 (t, J ¼ 6.4 Hz, 1 H), 1.41
(s, 9 H). Anal. Calcd for C18H24N2O5S: C, 56.82; H, 6.36; N, 7.36.
Found C, 56.61; H, 6.22; N, 7.27.
13C NMR (75 MHz, DMSO-d6)
d
/ppm ¼ 174.8, 168.9, 137.2, 132.4,
129.0, 127.5, 127.1, 124.9, 69.5, 57.2, 53.1, 50.8, 25.6, 16.7. Anal. Calcd
for C13H16N2O3: C, 62.89; H, 6.50; N, 11.28. Found C, 63.11; H, 6.64;
N, 11.51.
4.2.4.17. N-(3S-2-Tertbutoxycarbonyl-1,2,3,4-tetrahydroisoquino-
line-3-carbonyl)-
M.p. 121–122 ꢃC; ESI-MS (m/e) 423 [M-H]ꢂ; [
methanol); 1H NMR (300 MHz, CDCl3)
J ¼ 7.6 Hz, 1 H), 7.11 (m, J ¼ 8.2 Hz, 2H), 7.02 (m, J ¼ 8.2 Hz, 2 H), 6.97
(d, J ¼ 8.9 Hz, 2 H), 6.81 (d, J ¼ 8.8 Hz, 3 H), 4.92 (d, J ¼ 5.5 Hz, 1 H),
4.81 (d, J ¼ 6.5 Hz, 1 H), 4.21 (m, J ¼ 8.6 Hz, 2 H), 3.17 (m, J ¼ 5.6 Hz, 2
H), 2.90 (m, J ¼ 5.6 Hz, 2 H), 1.41 (s, 9 H). Anal. Calcd for C24H28N2O5:
C, 67.91; H, 6.65; N, 6.60. Found C, 67.70; H, 6.51; N, 6.38.
L
-phenylalanine (5q). Yield: 96%. Colorless powder,
4.2.5.2. N-(3S-1,2,3,4-Tetrahydroisoquinoline-3-carbonyl)-glycine
20
a
]
¼ ꢂ9.73 (c ¼ 1.0,
(6b). Yield: 95%. Colorless powder, M.p.150–152 ꢃC;ESI-MS (m/e) 235
D
[M þ H]þ; [
a]
D
¼ ꢂ32.0 (c ¼ 1.0, H2O); 1H NMR (300 MHz, DMSO-d6)
20
d
/ppm ¼ 11.00 (s,1 H), 8.00 (d,
d
/ppm ¼ 12.9(s, 1 H), 8.91 (m, J ¼ 8.1 Hz, 1 H), 8.73 (d, J ¼ 8.4 Hz, 1 H),
7.65 (d, J ¼ 7.6 Hz, 1 H), 7.36 (d, J ¼ 8.0 Hz, 1 H), 7.25 (t, J ¼ 7.7 Hz, 1 H),
7.13 (t, J ¼ 8.3 Hz, 1 H), 4.45 (t, J ¼ 5.1 Hz, 1 H), 4.24 (m, J ¼ 5.3 Hz, 3 H),
2.74 (m, J ¼ 5.6 Hz, 2 H), 1.38 (d, J ¼ 4.6 Hz, 3 H); 13C NMR (75 MHz,
DMSO-d6)
d
/ppm ¼ 172.1, 169.1, 138.2, 132.3, 128.6, 127.7, 127.1, 124.7,
69.1, 57.2, 54.8, 42.2, 26.2. Anal. Calcd for C12H14N2O3: C, 61.53; H, 6.02;
N, 11.96. Found C, 61.34; H, 5.86; N, 11.73.
4.2.4.18. N-(3S-2-Tertbutoxycarbonyl-1,2,3,4-tetrahydroisoquino-
line-3-carbonyl)-
M.p. 116–117 ꢃC; ESI-MS (m/e) 377 [M-H]ꢂ; [
methanol); 1H NMR (300 MHz, CDCl3)
(d, J ¼ 6.6 Hz, 1 H), 7.02 (m, J ¼ 8.2 Hz, 2 H), 6.95 (d, J ¼ 8.8 Hz, 2 H),
4.80 (t, J ¼ 7.6 Hz, 1 H), 4.61 (d, 1 H), 4.35 (m, 1 H), 4.21 (m, 2 H), 3.29
(d, J ¼ 8.6 Hz, 1 H), 3.05 (d, J ¼ 5.5 Hz, 1 H), 2.0 (s, 1 H), 1.41 (s, 9 H),
1.21 (d, J ¼ 7.6 Hz, 3 H). Anal. Calcd for C19H26N2O6: C, 60.30; H,
6.93; N, 7.40. Found C, 60.49; H, 6.80; N, 7.62.
L-threonine (5r). Yield: 98%. Colorless powder,
20
a
]
¼ ꢂ59.69 (c ¼ 1.0,
4.2.5.3. N-(3S-1,2,3,4-Tetrahydroisoquinoline-3-carbonyl)-L-valine
D
d
/ppm ¼ 11.00 (s, 1 H), 8.00
(6c). Yield: 95%. Colorless powder, M.p. 203–204 ꢃC; ESI-MS (m/e)
277 [M þ H]þ; IR (cmꢂ1) 3286, 2963, 1669, 1576, 1457, 1399, 750;
20
[a
]
¼ ꢂ14.0 (c ¼ 1.0, H2O); 1H NMR (300 MHz, DMSO-d6)
d/
D
ppm ¼ 12.19 (s,1 H), 8.91 (m, J ¼ 8.7 Hz,1 H), 8.72 (d, J ¼ 8.4 Hz,1 H),
7.60 (d, J ¼ 7.5 Hz, 1 H), 7.43 (d, J ¼ 8.1 Hz, 1 H), 7.26 (t, J ¼ 7.7 Hz, 1
H), 7.13 (t, J ¼ 8.0 Hz, 1 H), 4.46 (t, J ¼ 5.6 Hz, 1 H), 4.13 (m, J ¼ 6.1 Hz,
3 H), 2.72 (m, J ¼ 4.5 Hz, 2 H), 2.18 (dd, J ¼ 5.7 Hz, 1 H), 0.95 (m,
4.2.4.19. N-(3S-2-Tertbutoxycarbonyl-1,2,3,4-tetrahydroisoquino-
J ¼ 3.5 Hz, 6 H); 13C NMR (75 MHz, DMSO-d6)
/ppm ¼ 171.8, 171.2,
d
line-3-carbonyl)-
L
-arginine (5s). Yield: 8ꢂ6%. Colorless powder, M.p.
137.2, 133.6, 128.9, 127.8, 127.5, 125.6, 68.9, 56.8, 56.0, 53.4, 30.1,
27.6,17.8,17.2. Anal. Calcd for C15H20N2O3: C, 65.20; H, 7.30; N,10.14.
Found C, 65.39; H, 7.44; N, 10.37.
20
182–184 ꢃC; ESI-MS (m/e) 432 [M-H] ; [
a
]
¼ ꢂ20.52 (c ¼ 1.0,
D
methanol); 1H NMR (300 MHz, CDCl3)
d
/ppm ¼ 11.00 (s, 1 H), 8.00
(d, J ¼ 5.6 Hz, 2 H), 7.02 (m, J ¼ 8.2 Hz, 2 H), 6.95 (d, J ¼ 8.8 Hz, 2 H),
4.94 (m, 1 H), 4.53 (m, 1 H), 4.21 (m, 2 H), 3.17 (m, 1 H), 2.94 (m,1 H),
2.65 (m, 2 H), 2.0 (d, J ¼ 6.2 Hz, 2 H), 1.79 (m, 2 H), 1.55 (m, 2 H), 1.41
(s, 9 H). Anal. Calcd for C21H31N5O5: C, 58.18; H, 7.21; N, 16.16. Found
C, 58.40; H, 7.36; N, 16.40.
4.2.5.4. N-(3S-1,2,3,4-Tetrahydroisoquinoline-3-carbonyl)-L-leucine
(6d). Yield: 90%. Colorless powder, M.p. 145–147 ꢃC; ESI-MS (m/e)
20
291 [M þ H]þ; [
a
]
¼ ꢂ16 (c ¼ 1.0, H2O); 1H NMR (300 MHz, DMSO-
D
d6)
d
/ppm ¼ 12.22 (s,1 H), 8.92 (m, J ¼ 8.7 Hz,1 H), 8.74 (d, J ¼ 8.3 Hz,
1 H), 7.56 (d, J ¼ 7.6 Hz,1 H), 7.34 (d, J ¼ 8.4 Hz,1 H), 7.23 (t, J ¼ 7.7 Hz,
1 H), 7.14 (t, J ¼ 8.3 Hz,1 H), 4.46 (t, J ¼ 5.3 Hz,1 H), 4.28 (t, J ¼ 5.7 Hz,
1 H), 4.27 (m, J ¼ 5.7 Hz, 3 H), 2.74 (m, J ¼ 4.7 Hz, 2 H), 1.80 (m,
J ¼ 4.2 Hz, 3 H), 0.93 (m, J ¼ 3.8 Hz, 6 H); 13C NMR (75 MHz, DMSO-
4.2.4.20. N-(3S-2-Tertbutoxycarbonyl-1,2,3,4-tetrahydroisoquino-
line-3-carbonyl)-
M.p. 144–145 ꢃC; ESI-MS (m/e) 407 [M-H]ꢂ; [
methanol); 1H NMR (300 MHz, CDCl3)
L-methionine (5t). Yield: 95%. Colorless powder,
20
a
]
¼ ꢂ9.83 (c ¼ 1.0,
D
d
/ppm ¼ 11.00 (s, 1 H), 8.00
d6)
d
/ppm ¼ 172.3, 170.2, 137.2, 134.9, 128.6, 127.8, 127.3, 125.9, 68.9,
(d, J ¼ 7.6 Hz, 1 H), 7.02 (m, J ¼ 8.2 Hz, 2 H), 6.95 (d, J ¼ 8.8 Hz, 2 H),
4.90 (t, J ¼ 8.4 Hz, 1 H), 4.51 (m, 1 H), 4.20 (t, J ¼ 8.3 Hz, 2 H), 3.02 (d,
J ¼ 5.4 Hz, 2 H), 2.44 (m, 2 H), 2.16 (t, J ¼ 6.4 Hz, 2 H), 2.09 (s, 3 H),
1.41 (s, 9 H). Anal. Calcd for C20H28N2O5S: C, 58.80; H, 6.91; N, 6.86.
Found C, 58.99; H, 6.77; N, 7.10.
55.9, 53.4, 49.5, 41.0, 27.6, 22.4, 21.9. Anal. Calcd for C16H22N2O3: C,
66.18; H, 7.64; N, 9.65. Found C, 66.37; H, 7.79; N, 9.86.
4.2.5.5. N-(3S-1,2,3,4-Tetrahydroisoquinoline-3-carbonyl)-L-isoleu-
cine (6e). Yield: 95%. Colorless powder, M.p. 126–129 ꢃC; ESI-MS
20
(m/z) 291 [M þ H]þ; [
a
]
¼ ꢂ10 (c ¼ 1.0, H2O); 1H NMR (300 MHz,
D
4.2.5. General procedure preparing 3S-1,2,3,4-
DMSO-d6)
d
/ppm ¼ 12.91 (s, 1H), 8.92 (m, J ¼ 8.7 Hz, 1 H), 8.73 (d,
tetrahydroisoquinoline-3-carbonyl-
To the solution of 0.86 mmol of 3S-2-Boc-1,2,3,4-tetrahy-
droisoquinoline-3-carbonyl- -amino acid in 5 ml of ethyl acetate,
L
-amino acids (6a–t)
J ¼ 8.3 Hz, 1 H), 7.58 (d, J ¼ 7.5 Hz, 1 H), 7.33 (d, J ¼ 8.1 Hz, 1 H), 7.24
(t, J ¼ 7.6 Hz,1 H), 7.13 (t, J ¼ 8.3 Hz,1 H), 4.53 (t, J ¼ 4.7 Hz,1 H), 4.33
(m, J ¼ 5.7 Hz, 3 H), 4.27 (t, J ¼ 4.8 Hz, 1 H), 2.74 (m, J ¼ 3.9 Hz, 3 H),
1.28 (dd, J ¼ 5.7 Hz, 2 H), 0.96 (m, J ¼ 3.2 Hz, 6 H); 13C NMR (75 MHz,
L
6 ml of 4 N hydrogen chloride/ethyl acetate solution was added at
0 ꢃC. The reaction mixture was stirred at room temperature for 4 h,
DMSO-d6)
d
/ppm ¼ 172.8, 170.8, 137.2, 133.4, 128.9, 127.9, 127.3,

4916
S. Cheng et al. / European Journal of Medicinal Chemistry 44 (2009) 4904–4919
125.6, 68.9, 57.0, 53.4, 53.2, 36.4, 27.2, 16.8, 12.3. Anal. Calcd for
C16H22N2O3: C, 66.18; H, 7.64; N, 9.65. Found C, 66.00; H, 7.51; N,
9.88.
4.2.5.11. N-(3S-1,2,3,4-Tetrahydroisoquinoline-3-carbonyl)-L-gluta-
mine (6k). Yield: 95%. Colorless powder, M.p. 87–89 ꢃC; ESI-MS
20
(m/z) 306.2[M þ H]þ; [
a
]
D
¼ ꢂ62 (c ¼ 1.0, H2O); 1H NMR (300 MHz,
DMSO-d6)
d
/ppm ¼ 11.91 (s, 1H), 9.07 (d, J ¼ 8.6 Hz, 1 H), 8.87 (m,
4.2.5.6. N-(3S-1,2,3,4-Tetrahydroisoquinoline-3-carbonyl)-
L
-trypto-
J ¼ 6.7 Hz, 1 H), 8.73 (d, J ¼ 8.1 Hz, 1 H), 8.1 (m, J ¼ 7.9 Hz, 1 H), 7.47
(d, J ¼ 7.6 Hz, 1 H), 7.29 (d, J ¼ 8.7 Hz, 1 H), 7.33 (t, J ¼ 6.7 Hz, 1 H),
7.24 (t, J ¼ 8.1 Hz, 1 H), 4.28 (t, J ¼ 4.9 Hz, 1 H), 4.26 (m, J ¼ 5.7 Hz, 3
phan (6f). Yield: 91%. Colorless powder, M.p. 146–147 ꢃC; ESI-MS
(m/e) 364 [M þ H]þ; IR (cmꢂ1) 3056, 1733, 1554, 1500, 1434, 743;
20
[
a]
¼ ꢂ152 (c ¼ 1.0, H2O); 1H NMR (300 MHz, DMSO-d6)
d
/
H), 2.83 (m, J ¼ 4.9 Hz, 4 H); 13C NMR (75 MHz, DMSO-d6)
d/
D
ppm ¼ 10.97 (s, 1 H), 10.91 (s, 1 H), 9.06 (d, J ¼ 8.3 Hz, 1 H), 8.86 (m,
J ¼ 8.1 Hz,1 H), 7.21 (m, J ¼ 7.1 Hz, 9 H), 4.55 (m, J ¼ 4.8 Hz, 3 H), 4.26
(t, J ¼ 5.9 Hz, 1 H), 2.81 (m, J ¼ 3.8 Hz, 2 H); 13C NMR (75 MHz,
ppm ¼ 173.8, 172.9, 171.6, 137.1, 133.5, 128.9, 127.5, 127.2, 125.8, 68.2,
56.2, 53.2, 51.3, 34.1, 27.3, 26.8. Anal. Calcd for C15H19N3O4: C, 59.01;
H, 6.27; N, 13.76. Found C, 58.83; H, 6.12; N, 13.99.
DMSO-d6)
d/ppm ¼ 172.9, 171.9, 168.9, 137.6, 136.8, 128.9, 128.4,
127.9, 127.3, 125.8, 122.9, 122.6, 122.1, 119.7, 112.8, 111.9, 69.9, 57.2,
54.3, 29.5, 26.6. Anal. Calcd for C21H23N3O4: C, 66.13; H, 6.08; N,
11.02. Found C, 65.92; H, 5.87; N, 11.26.
4.2.5.12. N-(3S-1,2,3,4-Tetrahydroisoquinoline-3-carbonyl)-L-histi-
dine (6l). Yield: 82%. Colorless powder, M.p. 126–127 ꢃC; ESI-MS
20
(m/z) 314 [M þ H]þ; [
a
]
¼ ꢂ34 (c ¼ 1.0, H2O); 1H NMR (300 MHz,
D
DMSO-d6)
d
/ppm ¼ 9.26 (s, 1 H), 9.05 (m, J ¼ 7.6 Hz, 1 H), 8.82 (m,
4.2.5.7. N-(3S-1,2,3,4-Tetrahydroisoquinoline-3-carbonyl)-
L
-serin
J ¼ 8.3 Hz, 1 H), 7.42 (d, J ¼ 7.3 Hz, 1 H), 7.35 (d, J ¼ 8.4 Hz, 1 H), 7.13
(t, J ¼ 7.1 Hz, 1 H), 7.07 (t, J ¼ 6.9 Hz, 1 H), 4.48 (t, J ¼ 5.5 Hz, 1 H),
4.36 (m, J ¼ 5.6 Hz, 3 H), 3.19 (m, J ¼ 4.7 Hz, 2 H), 2.74 (m, J ¼ 4.7 Hz,
(6g). Yield: 95%. Colorless powder, M.p. 101–104 ꢃC; ESI-MS (m/z)
265 [M þ H]þ; IR (cmꢂ1) 3321, 2982, 1683, 1556, 1500, 1434, 750;
20
[
a]
¼ ꢂ22.52 (c ¼ 1.0, H2O); 1H NMR (300 MHz, DMSO-d6)
d/
2 H); 13C NMR (75 MHz, DMSO-d6)
d/ppm ¼ 171.9,171.6,137.1,135.6,
D
ppm ¼ 11.91 (s, 1 H), 8.86 (m, J ¼ 8.3 Hz, 1 H), 8.83 (d, J ¼ 8.4 Hz, 1
H), 7.55 (d, J ¼ 7.6 Hz, 1 H), 7.21 (d, J ¼ 8.6 Hz, 1 H), 7.13 (t, J ¼ 8.3 Hz,
1 H), 7.03 (t, J ¼ 7.7 Hz, 1 H), 5.25 (s, 1 H), 4.33 (m, J ¼ 5.3 Hz, 3 H),
4.04 (t, J ¼ 5.3 Hz, 1 H), 2.74 (m, J ¼ 3.9 Hz, 2 H); 13C NMR (75 MHz,
133.8, 133.1, 128.9, 127.5, 127.2, 125.8, 118.7, 68.2, 56.1, 53.4, 52.2,
30.0, 27.2. Anal. Calcd for C16H18N4O3: C, 61.13; H, 5.77; N, 17.82.
Found C, 61.35; H, 5.91; N, 18.05.
DMSO-d6)
d/ppm ¼ 172.8, 171.6, 137.8, 134.2, 128.9, 127.5, 127.3,
4.2.5.13. N-(3S-1,2,3,4-Tetrahydroisoquinoline-3-carbonyl)-L-lysine
(5m). Yield: 86%. Colorless powder, M.p. 106–109 ꢃC; ESI-MS (m/z)
125.9, 61.9, 61.2, 57.1, 54.9, 54.4, 28.9. Anal. Calcd for C13H16N2O4: C,
59.08; H, 6.10; N, 10.60. Found C, 59.30; H, 6.25; N, 10.39.
306 [M þ H]þ; [
a
]
¼ ꢂ48 (c ¼ 1.0, H2O); 1H NMR (300 MHz,
20
D
DMSO-d6)
d
/ppm ¼ 9.07 (m, J ¼ 8.3 Hz, 2 H), 8.87 (m, J ¼ 8.3 Hz, 2
4.2.5.8. N-(3S-1,2,3,4-Tetrahydroisoquinoline-3-carbonyl)-
L
-tyrosine
H), 8.73 (d, J ¼ 8.6 Hz, 1 H), 7.71 (m, J ¼ 7.1 Hz, 4 H), 4.58 (m,
J ¼ 5.4 Hz, 3 H), 4.28 (t, J ¼ 5.1 Hz, 1 H), 2.99 (m, J ¼ 5.7 Hz, 2 H), 2.13
(m, J ¼ 5.4 Hz, 4 H), 1.29 (m, J ¼ 5.5 Hz, 4 H), 0.92 (s, 1 H); 13C NMR
(6h). Yield: 96%. Colorless powder, M.p. 120–124 ꢃC; ESI-MS (m/z)
20
341 [M þ H]þ; [
a
]
D
¼ ꢂ72 (c ¼ 1.0, H2O); 1H NMR (300 MHz,
DMSO-d6)
d
/ppm ¼ 11.91 (s, 1 H), 9.06 (d, J ¼ 8.3 Hz, 1 H), 8.97 (d,
(75 MHz, DMSO-d6)
d
/ppm ¼ 172.9, 171.6, 142.8, 137.6, 136.6, 134.7,
J ¼ 7.3 Hz, 1 H), 8.86 (m, J ¼ 8.2 Hz, 1 H), 7.46 (d, J ¼ 7.6 Hz, 1 H), 7.29
(d, J ¼ 8.3 Hz,1 H), 7.22 (t, J ¼ 6.8 Hz,1 H), 7.21 (t, J ¼ 8.1 Hz,1 H), 7.15
(m, J ¼ 7.2 Hz, 4 H), 6.70 (d, J ¼ 8.4 Hz, 2 H), 4.32 (m, J ¼ 5.7 Hz, 3 H),
4.23 (t, J ¼ 5.4 Hz, 1 H), 2.94 (m, J ¼ 4.5 Hz, 2 H); 13C NMR (75 MHz,
126.2, 68.8, 67.8, 56.8, 54.8, 53.7, 52.8, 32.0, 30.2, 27.9, 22.7. Anal.
Calcd for C16H23N3O3: C, 62.93; H, 7.59; N, 13.76. Found C, 62.70; H,
7.43; N, 13.99.
DMSO-d6)
d
/ppm ¼ 171.9, 171.6, 156.8, 133.1, 132.4, 129.9, 129.6,
4.2.5.14. N-(3S-1,2,3,4-Tetrahydroisoquinoline-3-carbonyl)-aspartic
acid (6n). Yield: 95%. Colorless powder, M.p. 109–110 ꢃC; ESI-MS
128.1, 127.8, 127.3, 125.1, 117.1, 115.7, 69.1, 57.2, 53.8, 53.1, 37.8, 37.2,
22.2. Anal. Calcd for C19H20N2O4: C, 67.05; H, 5.92; N, 8.23. Found C,
67.24; H, 5.77; N, 8.00.
(m/z) 293 [M þ H]þ; IR (cmꢂ1) 3451, 2937, 1725, 1455, 1358, 745;
20
[a
]
¼ ꢂ34 (c ¼ 1.0, H2O); 1H NMR (300 MHz, DMSO-d6)
d/
D
ppm ¼ 12.4 (s, 1 H), 11.7 (s, 1 H), 9.05 (m, J ¼ 8.6 Hz, 1 H), 8.91 (m,
J ¼ 10.1 Hz, 1 H), 7.58 (d, J ¼ 7.5 Hz, 1 H), 7.36 (d, J ¼ 8.4 Hz, 1 H), 7.25
(t, J ¼ 7.3 Hz,1 H), 7.13 (t, J ¼ 8.2 Hz,1 H), 4.44 (t, J ¼ 5.3 Hz,1 H), 4.36
(d, J ¼ 5.3 Hz, 3 H), 3.02 (d, J ¼ 4.6 Hz, 2 H), 2.62 (m, J ¼ 5.3 Hz, 3 H);
4.2.5.9. N-(3S-1,2,3,4-Tetrahydroisoquinoline-3-carbonyl)-L-proline
(6i). Yield: 98%. Colorless powder, M.p. 115–116 ꢃC; ESI-MS (m/z)
275 [M þ H]þ; IR (cmꢂ1) 3321, 2949, 1739, 1564, 1448, 1434, 750;
20
[
a]
¼ ꢂ14.06 (c ¼ 1.0, H2O); 1H NMR (300 MHz, DMSO-d6)
d/
13C NMR (75 MHz, DMSO-d6)
d/ppm ¼ 173.2, 172.8, 171.6, 137.8,
D
ppm ¼ 11.14 (s, 1 H), 8.87 (m, J ¼ 5.7 Hz, 1 H), 7.46 (d, J ¼ 7.6 Hz, 1 H),
7.28 (d, J ¼ 8.1 Hz, 1 H), 7.21 (t, J ¼ 8.1 Hz, 1 H), 7.02 (t, J ¼ 6.7 Hz, 1
H), 4.35 (m, J ¼ 4.6 Hz, 3 H), 4.25 (t, J ¼ 4.5 Hz, 1 H), 3.76 (d,
J ¼ 6.3 Hz, 1 H), 3.68 (s, 1 H), 2.83 (m, J ¼ 5.6 Hz, 2 H), 1.97 (d,
133.6, 127.6, 127.1, 126.9, 124.1, 69.6, 56.7, 53.8, 48.3, 37.9, 28.6. Anal.
Calcd for C14H16N2O5: C, 57.53; H, 5.52; N, 9.58. Found C, 57.74; H,
5.69; N, 9.81.
J ¼ 8.7 Hz, 4 H); 13C NMR (75 MHz, DMSO-d6)
d/ppm ¼ 173.8, 172.2,
4.2.5.15. N-(3S-1,2,3,4-Tetrahydroisoquinoline-3-carbonyl)-L-gluta-
137.1, 133.3, 127.9, 127.5, 127.2, 125.9, 66.6, 58.6, 56.8, 53.8, 52.1,
28.9, 22.7. Anal. Calcd for C15H18N2O3: C, 65.68; H, 6.61; N, 10.21.
Found C, 65.47; H, 6.47; N, 10.00.
mic acid (6o). Yield: 95%. Colorless powder, M.p. 103–106 ꢃC; ESI-
MS (m/z) 307 [M þ H]þ; IR (cmꢂ1) 3451, 2981,1734, 1404, 1358, 754;
20
[a
]
¼ ꢂ64 (c ¼ 1.0, H2O); 1H NMR (300 MHz, DMSO-d6)
d/
D
ppm ¼ 12.51 (s, 1 H), 11.17 (s, 1H), 8.73 (d, J ¼ 8.6 Hz, 1 H), 8.49 (m,
J ¼ 9.2 Hz, 1 H), 7.56 (d, J ¼ 7.5 Hz, 1 H), 7.35 (d, J ¼ 8.0 Hz, 1 H), 7.28
(t, J ¼ 7.7 Hz, 1 H), 7.15 (t, J ¼ 8.1 Hz, 1 H), 4.47 (t, J ¼ 5.7 Hz, 1H), 4.34
(m, J ¼ 5.7 Hz, 3 H), 2.76 (d, J ¼ 5.6 Hz, 2 H), 2.16 (m, J ¼ 4.6 Hz, 1 H),
4.2.5.10. N-(3S-1,2,3,4-Tetrahydroisoquinoline-3-carbonyl)-L-aspara-
gine (6j). Yield: 95%. Colorless powder, M.p. 100–101 ꢃC; ESI-MS
20
(m/z) 292 [M þ H]þ; [
a
]
D
¼ ꢂ22 (c ¼ 1.0, H2O); 1H NMR (300 MHz,
DMSO-d6)
d
/ppm ¼ 11.91 (s, 1 H), 9.06 (d, J ¼ 8.5 Hz, 1 H), 8.87 (m,
1.96 (m, J ¼ 3.6 Hz, 1 H); 13C NMR (75 MHz, DMSO-d6)
d/
J ¼ 8.3 Hz, 1 H), 8.73 (d, J ¼ 8.3 Hz, 1 H), 7.47 (d, J ¼ 7.6 Hz, 1 H), 7.36
(t, J ¼ 6.9 Hz, 1 H), 7.29 (d, J ¼ 8.4 Hz, 1 H), 7.27 (t, J ¼ 8.1 Hz, 1 H),
6.21 (s,1 H), 4.37 (m, J ¼ 5.9 Hz, 3 H), 4.27 (t, J ¼ 5.6 Hz,1 H), 2.85 (m,
ppm ¼ 173.1, 172.8, 171.6, 137.9, 127.6, 127.0, 124.1, 123.6, 69.5, 56.7,
53.8, 50.7, 27.8, 27.6, 26.7. Anal. Calcd for C15H18N2O5: C, 58.82; H,
5.92; N, 9.15. Found C, 58.63; H, 5.77; N, 8.92.
J ¼ 5.3 Hz, 4 H); 13C NMR (75 MHz, DMSO-d6)
/ppm ¼ 173.5, 171.8,
d
137.2, 134.7, 128.6, 127.5, 127.1, 125.9, 69.0, 53.3, 37.3, 17.9. Anal.
Calcd for C14H17N3O4: C, 57.72; H, 5.88; N, 14.42. Found C, 57.91; H,
5.72; N, 14.65.
4.2.5.16. N-(3S-1,2,3,4-Tetrahydroisoquinoline-3-carbonyl)-L-
cysteine (6p). Yield: 93%. Colorless powder, M.p. 195–198 ꢃC; ESI-
20
MS (m/z) 281 [M þ H]þ; [
a]
¼ ꢂ23 (c ¼ 1.0, H2O); 1H NMR
D

S. Cheng et al. / European Journal of Medicinal Chemistry 44 (2009) 4904–4919
4917
(300 MHz, DMSO)
d
/ppm ¼ 11.25 (s, 1 H), 9.86 (m, J ¼ 8.9 Hz, 1 H),
removed. The remaining blood was centrifuged for an additional
10 min at 1500g to prepare platelet poor plasma (PPP). The final
platelet count of the citrated plasma samples was adjusted to
2 ꢁ 108 platelets/ml with autologous PPP. To an optical aggregom-
8.92 (d, J ¼ 8.4 Hz, 1 H), 7.54 (d, J ¼ 7.5 Hz, 1H), 7.34 (d, J ¼ 8.2 Hz, 1
H), 7.24 (t, J ¼ 7.9 Hz,1 H), 7.13 (t, J ¼ 8.2 Hz,1 H), 4.34 (t, J ¼ 5.8 Hz,1
H), 4.24 (m, J ¼ 5.6 Hz, 3 H), 2.76 (m, J ¼ 4.6 Hz, 2 H), 2.01 (m,
J ¼ 4.5 Hz, 1 H), 1.98 (m, J ¼ 4.3 Hz, 1 H); 13C NMR (75 MHz, DMSO-
etry testing tuber, 0.5 ml of the adjusted plasma sample and 5 ml of
d6)
d
/ppm ¼ 171.6, 171.2, 137.1, 133.6, 128.6, 127.6, 127.1, 125.1, 68.7,
NS or 5
of 100, 10, 1, 0.1, 0.01 and 0.001
the baseline, 5 l of the solution of platelet-activating factor in NS
(PAF, final concentration 0.1 M) or 5 l of the solution of adenosine
diphosphate in NS (ADP, final concentration 10 M) or 5 l of the
solution of arachidonic acid in NS (AA, final concentration 350 M),
ml of the solution of 6a–t (in a series of final concentrations
56.8, 55.2, 53.2, 27.2, 26.7. Anal. Calcd for C13H16N2O3S: C, 55.70; H,
5.75; N, 9.99. Found C, 55.89; H, 5.91; N, 10.22.
m
M) was added. After adjustment of
m
m
m
4.2.5.17. N-(3S-1,2,3,4-Tetrahydroisoquinoline-3-carbonyl)-
L
-phenyl-
m
m
alanine (6q). Yield: 93%. Colorless powder, M.p. 106–108 ꢃC; ESI-
m
MS (m/e) 325 [M þ H]þ; IR (cmꢂ1) 3423, 2963, 1670, 1634, 1576,
or 50 ml of the solution of thrombin in NS (TH, final concentration
0.1 U/ml) was added and aggregation was measured at 37 ꢃC for
5 min. The effects of 6a–t (at a series of concentrations ranging
from 10 mM to 10 nM) on PAF or ADP or AA or TH induced platelet
aggregation were observed. All these anti-platelet aggregation tests
in sixplicate tubers were carried out. The maximal rate of platelet
aggregation (Am%) was represented by the peak height of aggre-
gation curve. The inhibition rate was calculated by % Inhib-
ition ¼ [(Am% of NS) ꢂ (Am% of 6a–t)]/(Am% of NS), where Am% of
NS ¼ 50.16 ꢀ 3.65%. The concentration vs. inhibition rate curve is
plotted to determine the IC50 values via GWBASIC.EXE program.
20
1457, 1400, 740; [
a
]
¼ ꢂ42 (c ¼ 1.0, H2O); 1H NMR (300 MHz,
D
DMSO-d6)
d
/ppm ¼ 12.68 (s, 1 H), 9.06 (d, J ¼ 8.4 Hz, 1 H), 7.21 (m,
J ¼ 8.3 Hz, 9 H), 4.35 (m, J ¼ 5.6 Hz, 3 H), 4.26 (t, J ¼ 5.2 Hz, 1 H), 2.81
(m, J ¼ 4.1 Hz, 2 H), 1.28 (m, J ¼ 3.3 Hz, 2 H); 13C NMR (75 MHz,
DMSO-d6)
d
/ppm ¼ 174.9, 171.8, 133.4, 128.2, 128.1, 127.9, 127.8,
127.5, 127.3, 126.2, 125.7, 69.5, 53.9, 53.5, 37.5, 27.5. Anal. Calcd for
C19H20N2O3: C, 70.35; H, 6.21; N, 8.64. Found C, 70.56; H, 6.37; N,
8.87.
4.2.5.18. N-(3S-1,2,3,4-Tetrahydroisoquinoline-3-carbonyl)-L-threo-
nine (6r). Yield: 68%. Colorless powder, M.p. 100–102 ꢃC; ESI-MS
20
(m/z) 279 [M þ H]þ; [
a
]
¼ ꢂ14 (c ¼ 1.0, H2O); 1H NMR (300 MHz,
4.3.2. In vivo anti-thrombotic assay of intravenously injection of
6a–t in rat model
D
DMSO-d6)
d
/ppm ¼ 11.91 (s, 1 H), 8.93 (d, J ¼ 8.5 Hz, 1 H), 8.87 (m,
J ¼ 8.2 Hz, 1 H), 7.47 (d, J ¼ 7.6 Hz, 1 H), 7.38 (d, J ¼ 8.4 Hz, 1 H), 7.31
(t, J ¼ 8.2 Hz, 1 H), 7.15 (t, J ¼ 7.7 Hz, 1 H), 4.35 (m, J ¼ 5.1 Hz, 3 H),
4.27 (t, J ¼ 5.3 Hz, 1 H), 2.84 (m, J ¼ 4.3 Hz, 2 H), 2.25 (s, 1 H); 13C
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 animals was maintained in accordance to the requirements
of the animal welfare act and according to the guide for care and
use of laboratory animals. Aspirin and 6a–t were dissolved in NS
before administration and kept in an ice bath. Male Wistar rats
weighing 250–300 g (purchased from Animal Center of Peking
University) were used. The rats were anesthetized with pentobar-
bital sodium (80.0 mg/kg, i.p.) and the right carotid artery and left
jugular vein were separated. A weighed 6 cm thread was inserted
into the middle of a polyethylene tube. The polyethylene tube was
filled with heparin sodium (50 IU/ml in NS) and one end was
inserted into the left jugular vein. From the other end of the poly-
ethylene tube heparin sodium was injected as anticoagulant, then
NS or 6a–t was injected, 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.
NMR (75 MHz, DMSO-d6)
d/ppm ¼ 172.8, 171.6, 137.2, 129.1, 127.9,
127.2, 125.6, 68.9, 68.2, 58.2, 53.7, 52.2, 27.8, 19.6. Anal. Calcd for
C14H18N2O4: C, 60.42; H, 6.52; N, 10.07. Found C, 60.23; H, 6.38; N,
10.30.
4.2.5.19. N-(3S-1,2,3,4-Tetrahydroisoquinoline-3-carbonyl)-L-argi-
nine (6s). Yield: 80%. Colorless powder, M.p. 201–204 ꢃC; ESI-MS
20
(m/z) 334 [M þ H]þ, [
a]
¼ ꢂ26 (c ¼ 1.0, H2O); 1H NMR (300 MHz,
D
DMSO-d6)
d
/ppm ¼ 11.72 (s, 1 H), 8.93 (s, 3 H), 8.91 (m, J ¼ 7.5 Hz, 1
H), 8.73 (d, J ¼ 8.6 Hz, 1H), 7.62 (d, J ¼ 7.5 Hz, 1 H), 7.53 (d, J ¼ 8.0 Hz,
1 H), 7.26 (t, J ¼ 7.7 Hz,1 H), 7.21 (t, J ¼ 8.2 Hz,1 H), 4.47 (t, J ¼ 5.7 Hz,
1 H), 4.15 (m, J ¼ 5.7 Hz, 3 H), 2.74 (m, J ¼ 5.7 Hz, 4 H), 1.96 (m,
J ¼ 5.7 Hz, 4 H); 13C NMR (75 MHz, DMSO-d6)
/ppm ¼ 171.9, 171.6,
d
137.2,133.6,128.9,127.9,127.2,125.6, 68.5, 57.2, 53.7, 52.7, 37.2, 26.7,
25.5. Anal. Calcd for C16H23N5O3: C, 57.64; H, 6.95; N, 21.01. Found C,
57.75; H, 7.10; N, 21.26.
4.2.5.20. N-(3S-1,2,3,4-Tetrahydroisoquinoline-3-carbonyl)-L-methi-
onine (6t). Yield: 91%. Colorless powder, M.p. 101–105 ꢃC; ESI-MS
4.3.3. In vivo anti-thrombotic assay of orally administration
of 6s in rat model
(m/z) 309 [M þ H]þ; IR (cmꢂ1) 3451, 2974, 1630, 1455, 1358, 739;
20
[
a]
¼ ꢂ48 (c ¼ 1.0, H2O); 1H NMR (300 MHz, DMSO-d6)
d/
Three doses (5, 1 and 0.2
mmol/kg) of 6s in NS or NS (0.6 ml)
D
ppm ¼ 11.93 (s,1 H), 9.06 (d, J ¼ 8.4 Hz,1 H), 8.86 (m, J ¼ 7.9 Hz,1 H),
7.46 (d, J ¼ 7.6 Hz, 1 H), 7.33 (t, J ¼ 6.6 Hz, 1 H), 7.29 (d, J ¼ 8.4 Hz, 1
H), 7.21 (t, J ¼ 8.1 Hz,1 H), 4.35 (m, J ¼ 4.6 Hz, 3 H), 4.27 (t, J ¼ 5.7 Hz,
1 H), 2.88 (m, J ¼ 5.3 Hz, 2 H), 2.51 (m, J ¼ 5.3 Hz, 5 H), 1.36 (m,
alone were fed to Male Wistar rats orally. Then the rats were
anesthetized with pentobarbital sodium (80.0 mg/kg, ip). 30 min
later the right carotid artery and left jugular vein of the rat were
separated. A weighed 6-cm thread was inserted into the middle of
a polyethylene tube. The polyethylene tube was filled with heparin
sodium (50 IU/ml in NS) and one end was inserted into the left
jugular vein while another end was inserted into the right carotid
artery. Blood flowed from the right carotid artery to the left jugular
vein through the polyethylene tube for 15 min. The thread was
taken out and the weight of the wet thrombus was recorded.
J ¼ 3.7 Hz, 2 H); 13C NMR (75 MHz, DMSO-d6)
/ppm ¼ 172.6, 171.8,
d
137.6, 128.9, 127.6, 127.3, 125.8, 69.1, 56.7, 53.2, 52.9, 31.2, 27.9, 18.8.
Anal. Calcd for C15H20N2O3S: C, 58.42; H, 6.54; N, 9.08. Found C,
58.63; H, 6.69; N, 9.31.
4.3. Bioassays
4.3.1. In vitro anti-platelet aggregation activity assay
4.3.4. Apparent permeability coefficient test of THIQA and 6s [43]
Caco-2 cells (from the American Type Culture Collection, Rock-
ville, MD, USA) were cultivated on polycarbonate filters (transwell
An H-10 cell counter was used to determine the platelet count
and a two-channel Chronolog aggregometer was used to evaluate
platelet aggregation. After collection, the pig blood was centrifuged
at 1000g for 10 min and the platelet rich plasma (PRP) was
cell culture inserts, 12 mm in diameter, 3.0
mM in mean pore size).
Caco-2 cells were grown on filter supports and the integrity of

4918
S. Cheng et al. / European Journal of Medicinal Chemistry 44 (2009) 4904–4919
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monolayers was routinely checked by measurements of trans-
epithelial electrical resistance (approximately 700
cm2). THIQA
U
and 6s for evaluation were dissolved in HBSS to prepare drug
solutions at a final concentration of 4 mM. In apical to basolateral
direction, transport was initiated by adding drug solutions (total AP
volume, 0.5 ml) to the apical compartment of inserts held in
transwells containing 1.5 ml of HBSS (basolateral compartment). In
basolateral to apical direction, transport was initiated by adding
1.5 ml of the solution of THIQA or 6s to basolateral compartment
and adding 0.5 ml of HBSS as receiving solution to apical side of the
monolayers. The monolayers were incubated in air at 37 ꢃC and 95%
relative humidity. At 30, 60, 90, and 120 min, samples were with-
drawn from the receiving side, and the concentrations of the
samples were determined by HPLC analysis. The resistance of
monolayers was checked at the end of each test. Apparent
permeability coefficients (Papp
) were calculated according to
Papp ¼ dQ/dt$1/(A$C0), wherein dQ/dt is the permeability rate, C0 is
the initial concentration in the donor chamber, and A is the surface
area of monolayer (1 cm2).
Acknowledgements
This work was supported by the Beijing area major laboratory of
peptide and small molecular drugs, PHR (IHLB, KZ200910025004),
Special Project (2008ZX09401-002) and the National Natural
Science Foundation of China (30672513 and 20772082).
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