2,3-Diamino acid modifying 3S-tetrahydroisoquinoline-3-carboxylic acids: Leading to a class of novel agents with highly unfolded conformation, selective in vitro anti-platelet aggregation and potent in vivo anti-thrombotic activity

Article abstract of DOI:10.1016/j.bmc.2010.01.009

In the preparation of anti-thrombotic agents the 2- and 3-positions of 3S-tetra-hydroisoquinoline-3-carboxylic acid (THIQA) were simultaneously modified with amino acids to form 20 novel N-(3S-N-aminoacyl-1,2,3,4-tetrahydroisoquinoline-3-carbonyl)amino acids (8a-t). On an in vitro platelet aggregation model 8a-t selectively inhibit ADP-induced platelet aggregation and their IC₅₀ values are leas than 3.5 nM. On an extracorporeal circulation of arterioveinos cannula model of rats both orally and intraveously effective doses of 8a-t are less than 30 nmol/kg. Cerius² based stereoview of explores 8a-t having highly unfolded conformation. 3D QSAR analysis gives the importance of the unfolded conformation to high in vitro anti-platelet aggregation and in vivo anti-thrombotic potency rational understanding.

Full text of DOI:10.1016/j.bmc.2010.01.009

Bioorganic & Medicinal Chemistry 18 (2010) 1536–1554
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
2,3-Diamino acid modifying 3S-tetrahydroisoquinoline-3-carboxylic acids:
Leading to a class of novel agents with highly unfolded conformation, selective
in vitro anti-platelet aggregation and potent in vivo anti-thrombotic activity
a
b,
Xiaoyi Zhang a, Wei Wang b, Shenling Cheng a, Ming Zhao a, , Meiqing Zheng , Heng Wei Chang
,
*
*
Jianhui Wu a, Shiqi Peng a,
*
a College of Pharmaceutical Sciences, Capital Medical University, Beijing 100069, PR China
b C S Bio Company, 20 Kelly Court, Menlo Park, CA 94025, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
In the preparation of anti-thrombotic agents the 2- and 3-positions of 3S-tetra-hydroisoquinoline-3-car-
boxylic acid (THIQA) were simultaneously modified with amino acids to form 20 novel N-(3S-N-amino-
acyl-1,2,3,4-tetrahydroisoquinoline-3-carbonyl)amino acids (8a–t). On an in vitro platelet aggregation
model 8a–t selectively inhibit ADP-induced platelet aggregation and their IC50 values are leas than
3.5 nM. On an extracorporeal circulation of arterioveinos cannula model of rats both orally and intrav-
eously effective doses of 8a–t are less than 30 nmol/kg. Cerius2 based stereoview of explores 8a–t having
highly unfolded conformation. 3D QSAR analysis gives the importance of the unfolded conformation to
high in vitro anti-platelet aggregation and in vivo anti-thrombotic potency rational understanding.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 2 November 2009
Revised 1 January 2010
Accepted 5 January 2010
Available online 13 January 2010
Keywords:
Tetrahydroisoquinoline
Modification
Anti-thrombotic
Conformation
3D QSAR
1. Introduction
the other hand, intestinal peptide transport system is particularly
utilized to increase bioavailability.15–17 Following this approach
Intravascular thrombosis is one of the most frequent patholog-
ical events as well as a leading cause of morbidity and mortality all
over the world. In the onset and progression of acute coronary syn-
dromes, the rift, rupture and pythogenesis of atheromatous plaque
with the formation of either partial or complete occlusive throm-
bus are critical steps, while vascular damage, stimulus of platelets,
and initiation of the clotting cascade are essential factors.1–3 In the
cases of thromboembotic disorders of cardiovascular and cerebro-
vascular the subendothelium surfaces of the vessels are injured
and function as the adherent target of platelets.3–7 Though antico-
agulants, anti-platelet drugs and thrombolytic drugs are clinically
used for anti-thrombotic therapy, to improve the treatment of
ischemic symptoms, more potent and safer agents are urgently
needed, and a lot of efforts have been devoted to anti-thrombotic
drug design. However, due to undesirable bioavailability the anti-
thrombotic candidates usually fail to exert therapeutic potential,
which leads to the development of GPIIb/IIIa antagonists having
good pharmacodynamic and pharmacokinetic property.7–14 On
3S-1,2,3,4-tetrahydro-isoqunoline-3-carbonylamino acid (THIQA),
an anti-platelet aggregation compound, was modified to its dipep-
tide analogues to improve their permeability and consequently to
enhance their anti-thrombotic activity.9,10
In addition to a number of bioactivities,18–32 tetrahydroisoquin-
olines were also reported as anti-platelet aggregation agents. In the
mechanism investigations of anti-platelet aggregation of tetrahy-
droisoquinolines not only b-adrenergic/a2-adrenergic receptor sys-
tem,33 but also thromboxane A2/prostaglandin H2 receptor system
was involved. Tetrahydroisoquinolines may exert their inhibitory
effects on arachidonic acid (AA) induced platelet aggregation partly
by inhibiting the production of TXA2 from AA and partly by directly
blocking the TXA2 receptor.34–36 Based on these investigations
some derivatives of benzyltetrahydroisoquinoline alkaloid were
reported having anti-platelet aggregation activities.37–40
In the previous paper, we used THIQA as the anti-thrombotic
lead, introducing amino acid into its 2- or 3-position, and prepared
two series of pseudopeptides 3S-N-(L-amino-acyl)-1,2,3,4-tetrahy-
droisoquinoline-3-carboxylic acids (9a–t),9 as well as 3S-1,2,3,4-tet-
rahydroisoquinoline-3-carbonylamino acids (10a–t).10 In our pre-
investigation it was found that a folded (Fig. 1a), unfolded (Fig. 1b)
and highly unfolded conformation (Fig. 1c) of 9s, 10s and N-(3S-N-
* Corresponding authors. Tel.: +86 10 8280 2482; fax: +86 10 8280 2482 (M.Z.);
tel.: +1 650 322 1111; fax: +1 650 322 2278 (H.W.C.); tel.: +86 10 8391 1528; fax:
+86 10 8391 1528 (S.P.).
E-mail addresses: mingzhao@mail.bjmu.edu.cn (M. Zhao), chang@csbio.com
(H.W. Chang), shiqipeng@163.com (S. Peng).
L
-argininyl-1,2,3,4-tetrahydroisoquinoline-3-carbonyl)-
L-arginine
matching a low, moderate and high in vivo anti-thrombotic activity,
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.01.009

X. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 1536–1554
1537
(4a–t, 45–95% yield). The saponification of 4a–t provided 3S-N-
(Boc-aminoacyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acids
(5a–t, 51–96% yield). In the presence of DCC, HOBt and NMM, 5a–t
were conjugated with 20 amino acid benzylesters to give N-(3S-N-
Boc-aminoacyl-1,2,3,4-tetrahydroisoquinoline-3-carbonyl)amino
acid banzylesters (6a–t, 46–96% yield). Catalytic hydrogenation of
6a–t provided N-(3S-N-Boc-aminoacyl-1,2,3,4-tetrahydroisoquino-
line-3-carbonyl)amino acids (7a–t, 83–97% yield). Removing Boc
groups from 7a–t resulted in N-(3S-N-aminoacyl-1,2,3,4-tetrahy-
dro-isoquinoline-3-carbonyl)amino acids (8a–t, 85–98% yield).
The mild condition and the acceptable yield of the individual reac-
tion suggest that the present synthetic route is suitable for preparing
these novel N-(3S-N-aminoacyl-1,2,3,4-tetrahydroisoquinoline-3-
carbonyl)amino acids.
2.2. 2,3-Diamino acid modification of THIQA enhancing the
in vitro activity
Figure 1. Cerius2 based stereoview of 3S-N-(
oline-3-carboxylic acid (a) and N-(3S-1,2,3,4-tetrahydroisoquinoline-3-carbonyl)-
arginine (b) and N-(3S-N-L-argininyl-1,2,3,4-tetrahydroisoquinoline-3-carbonyl)-
L-argininyl)-1,2,3,4-tetrahydroisoquin-
L
-
-
To evaluate the effect of 2,3-diamino acid modification of THIQA
on the in vitro activity, the in vitro anti-platelet aggregation assays
of 8a–t were performed by following the common procedure and
using four aggregators, namely platelet-activating factor (PAF, final
L
arginine (c).
respectively. This implies that 2,3-diamino-acid-substituted THI-
QAs should have highly unfolded conformation and exhibit high
in vivo anti-thrombotic potency.
concentration 0.1
l
M), adenosine diphosphate (ADP, final concen-
tration 10 M), arachidonic acid (AA, final concentration 350
l
l
M),
and thrombin (TH, final concentration 0.1 U/ml). The IC50 values
against the aggregation induced by them are listed in Table 1.
In the literature a number of tetrahydroisoquinolines were re-
ported as selective inhibitors of ADP and AA and the IC50 values
against platelet aggregation induced by them range from 100 to
In this context, the present paper prepared 20 novel N-(3S-N-
L-
aminoacyl-1,2,3,4-tetrahydroisoquinoline-3-carbonyl)- -amino
L
acids (8a–t), evaluated their anti-thrombotic activities, examined
their conformation, and analyzed their 3D QSAR.
270
that the IC50 values of THIQA against platelet aggregation induced
by ADP and AA are 0.55 and 0.45 M, respectively. The IC50 values
lM and 3.3 to 140 l
M, respectively.40,41 Our assay indicates
2. Results and discussion
l
2.1. A seven-step-procedure of preparing 8a–t
of THIQA are 182–491 and 7–311-fold lower than that of the men-
tioned tetrahydroisoquinolines. Therefore as the lead of anti-plate-
let aggregators THIQA obviously possesses advantage.
In Table 1 the IC50 values of 8a–t against ADP, AA, PAF and TH
induced platelet aggregation are less than 3.73, 10.04, 6.32 and
11.35 nM, respectively. The IC50 values of 8a–t against four aggre-
gator induced platelet aggregation are totally less than 11.35 nM
identifies 8a–t as excellent anti-platelet aggregators. Among the
mentioned IC50, the values of 8a–t against ADP-induced platelet
aggregation are the smallest ones (<3.73 nM). The mean IC50 values
The preparation of N-(3S-N-aminoacyl-1,2,3,4-tetrahydroiso-
quinoline-3-carbonyl)amino acids (8a–t) was carried out according
to the seven-step-route depicted in Scheme 1. After esterification
the formed L-Phe-OMe was treated with formaldehyde in Pictet–
Spengler condensation condition to provide methylester of THIQA
(3, 84% yield).41,42 In the presence of DCC, HOBt and NMM, 3 was
conjugated with 20 amino acids to give 3S-N-(Boc-aminoacyl)-
1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid methyl-esters
CO2CH3
CO2CH3
CO2H
i
ii
NH2
NH2
NH
2
1
3
iii
R
O
CO2H
NH-Boc
CO2CH3
NH-Boc
CO2 Bzl
NH
v
iv
NH-Boc
N
N
N
R
R
R
6a-t
5a-t
4a-t
O
O
O
vi
R
O
R
O
CO2
H
NH
CO2
H
NH
NH2
vii
NH-Boc
N
N
R
R
7a-t
8a-t
O
O
Scheme 1. Synthetic route of N-(3S-2-aminoacyl-1,2,3,4-tetrahydroisoquinoline-3-carbonyl)-
L-amino acids. Reagents: (i) SOCl2 and CH3OH; (ii) HCHO and HCl; (iii) Boc-AA,
HOBt, DCC and NMM; (iv) 2 N NaOH; (v) AA-OBzl, HOBt, DCC and NMM; (vi) Pd/C; (vii) hydrogen chloride in ethyl acetate (6 N). In (4–8)a R = CH3, (4–8)b R = H, (4–8)c
R = CH(CH3)2, (4–8)d R = CH2CH(CH3)2, (4–8)e R = CH(CH3)CH2CH3, (4–8)f R = indol-2-ylmethylene, (4–8)g R = CH2OH, (4–8)h R = 4-hydroxylbenzyl, (4–8)i NHCHR = cyc-
lobutylamino, (4–8)j R = CH2CONH2, (4–8)k R = CH2CH2CONH2, (4–8)l R = imidazol-4-ylmethylene, (4–7)m R = N-tert-butoxy, 8m R = aminobutyl, (4–6)n R = benzyloxycar-
bonylmethylene, 7n and 8n R = carboxylmethylene, (4–6)o R = benzyloxycarbonylethylene, 7o and 8o R = carboxylethylene, (4–6)p R = CH2S–But, 7p and 6p R = CH2SH, (4–
8)q R = benzyl, (4–6)r R = CH(OCH2C6H5)CH3, 7r and 8r R = CH(OH)CH3, (4–6)s AA = CH2(CH2)2NHC(NHNO2)@NH, 7s and 8s AA = CH2(CH2)2NHC(NH2)@NH, (4–8)t
R = CH2CH2SCH3.

1538
X. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 1536–1554
Table 1
IC50 of 8a–t against four aggregators induced aggregation of pig plateletsa
10a–t against ADP induced aggregation range from 2.17 to 3.73,
103 to 534 and 120 to 661 nM, respectively. Comparing to 2- and
3-monoaminoacid modification 2,3-diamino acid modification of
THIQA results in a 47–143 and 55–177-fold increase for the
in vitro inhibition to ADP-induced platelet aggregation.
Compd
ADP
AA
960 2.32
PAF
TH
881 2.52
IQ
8a
8b
8c
8d
8e
8f
8g
8h
8i
552 3.25
2.36 0.44
2.44 0.21
2.57 0.35
2.57 0.25
2.89 0.17
2.42 0.33
2.17 0.47
2.58 0.22
3.13 0.63
3.07 0.89
2.55 0.31
2.84 0.42
3.73 0.18
3.48 0.51
2.19 0.58
3.26 0.19
2.23 0.31
2.89 0.43
3.15 0.64
3.42 0.25
452 2.21
5.60 0.86
5.15 0.64
5.87 0.62
5.03 0.38
5.64 0.39
4.88 0.98
5.36 0.65
4.52 0.18
5.55 0.11
5.39 0.23
5.72 0.91
5.57 0.30
5.67 0.34
4.37 0.62
4.19 0.25
6.32 0.57
4.92 0.68
4.14 0.72
6.07 0.50
4.91 0.57
8.73 0.51
7.95 0.59
9.08 0.27
8.17 0.55
8.92 0.24
9.31 0.29
7.99 0.16
8.83 0.31
8.54 0.34
8.28 0.64
8.37 0.48
9.38 0.56
9.64 0.27
9.55 0.42
9.86 0.54
8.18 0.43
7.84 0.46
10.04 0.68
9.17 0.41
8.19 0.57
9.48 0.28
10.64 0.82
9.25 0.74
10.02 0.87
11.35 0.41
9.18 0.50
10.24 0.92
9.84 0.40
10.01 0.26
8.43 0.15
9.13 0.22
10.04 0.82
8.98 0.13
11.12 0.95
10.27 0.53
9.64 0.20
8.87 0.43
10.83 0.57
9.87 0.28
9.99 0.38
2.3. 2,3-Diamino acid modification enhancing the in vivo
activity
To get insight into the effect of 2,3-diamino acid modification of
THIQA on in vio activity 8a–t were assayed on an extracorporeal
circulation of arterioveinos cannula model of rats. The individual
stock solution of aspirin (positive control) and 8a–t in NS was
administered intravenously, the thrombus weights were weighe-
ded and the data are listed in Table 3. The thrombus weights of
the rats receiving 30 nmol/kg of 8a–t range from 10.61 (8s) to
25.20 mg (8b), which are significantly lower than that (28.54 mg)
of the rats receiving NS (p <0.01). Thus, 8a–t are highly anti-throm-
botic agents and 8s the most potency agent.
8j
8k
8l
8m
8n
8o
8p
8q
8r
8s
8t
To clarify the benefit of 2,3-diaminoacid modification of THIQA
in Table 3 the in vivo anti-thrombotic activities of 8a–t are com-
pared with those of 2-(9a–t) and 3-monoaminoacid modification
as well as THIQA itself (10a–t). The thrombus weights of the rats
a
IC50 is represented by mean SD nM.
intravenously receiving 30 nmol/kg of 8a–t, 5
lmol/kg of 9a–t
of 8a–t against four aggregators induced aggregation of pig plate-
lets are 2.55 nM, 8.80 nM, 5.244 nM and 9.859 nM, respectively.
The potency of 8a–t inhibiting ADP induced aggregation is 2.0–
3.9-fold higher than the other three aggregators.
and 10a–t, as well as 15 mol/kg of THIQA are 10.61–25.20,
l
18.00–22.66, 18.73–23.58 and 21.52 mg, respectively. Thus even
in the case that the dose of 8a–t is 167 and 500-fold lower than
that of (9,10)a–t and THIQA, respectively, 8a–t still possess the
comparable in vivo anti-thrombotic potency to that of (9,10)a–t
and THIQA. Therefore comparing to 2- and 3-modifications 2,3-dia-
minoacid modification of THIQA results in a greatly increase of the
in vivo anti-thrombotic activity.
In respect of ADP-induced platelet aggregation the IC50 values of
THIQA and 8a–t are 552 nM and 2.17–3.73 nM, respectively, 8a–t
are 159–254-fold lower than THIQA. According to the literature
some representatives of tetrahydroisoquinolines such as Higen-
amine, YS-49 and YS-51, effectively inhibit ADP-induced platelet
aggregations and the IC50 values range from 3.3 to 800 l
M,40,41,43
2.4. Highly unfolded conformation of 2,3-diamino-acid
modified THIQAs
which are 1520–214477-fold higher than 8a–t. Therefore 2,3-dia-
mino acid modification of THIQA greatly enhances the in vitro
anti-platelet aggregation potency.
To understand the structural basis of 2-/3-monoaminoacid and
2,3-diamino acid modifications of THIQA resulted in different
in vitro anti-platelet aggregation as well as in vivo anti-thrombotic
activities the Cerius2 based stereoview of 9a–t (Fig. 2a), 10a–t
(Fig. 2b) and 8a–t (Fig. 2c) was compared. Figure 2 indicates that
the conformational extensibility is 8a–t > 10a–t > 9a–t. As men-
tioned above, the in vitro anti-platelet aggregation and the
in vivo anti-thrombotic activities are also 8a–t > 10a–t > 9a–t.
The consistence of these orders reflects the correlation of confor-
mational extensibility with the in vitro anti-platelet aggregation
and/or the in vivo anti-thrombotic activities, as well as explores
the importance of highly conformational extensibility of 2,3-dia-
mino-acid modifications of THIQA to enhancing the in vitro anti-
platelet aggregation and/or the in vivo anti-thrombotic activities.
To clarify the benefit of 2,3-diamino acid modification of THIQA
in Table 2 the IC50 values of 8a–t against ADP-induced platelet
aggregation are compared with those of 2-(9a–t) and 3-monoami-
noacid modified THIQA (10a–t). The IC50 values of 8a–t, 9a–t and
Table 2
IC50 of (8–10)a–t against ADP-induced platelet aggregationa
Compd
IC50
Compd
IC50
Compd
IC50
8a
8b
8c
8d
8e
8f
8g
8h
8i
2.36 0.44a
2.44 0.21a
2.57 0.35a
2.57 0.25a
2.89 0.17a
2.42 0.33a
2.17 0.47a
2.58 0.22a
3.13 0.63a
3.07 0.89a
2.55 0.31a
2.84 0.42a
3.73 0.18a
3.48 0.51a
2.19 0.58a
3.26 0.19a
2.23 0.31a
2.89 0.43a
3.15 0.64a
3.42 0.25a
9a
9b
9c
9d
9e
9f
9g
9h
9i
651 26
365 29
389 33
336 19
315 30
194 16
103 18
327 31
373 32
534 34
302 23
324 19
323 21
345 34
494 32
—
10a
10b
10c
10d
10e
10f
10g
10h
10i
350 31
496 28
382 26
478 29
540 34
120 19
388 33
481 30
364 21
470 29
235 22
661 34
194 15
329 33
482 26
231 18
188 15
164 22
124 20
229 18
2.5. Dose dependence of in vivo anti-thrombotic activities of
intravenous 8a,r,s
8j
8k
8l
9j
9k
9l
10j
10k
10l
The effect of dose on the in vivo anti-thrombotic activity of the
mice of intravenously receiving 8a, 8r and 8s, the compounds pos-
sessing the highest activities at 30 nmol/kg of dose, was observed.
The doses of 8a,r are 30.00, 3.00 and 0.30 nmol/kg, of 8s are 30.00,
3.00 and 0.03 nmol/kg. The thrombus weights of the treated rats
are listed in Table 4. The data demonstrate that 8a,r,s dose-depen-
dently inhibit the thrombosis of the treated rats.
To clarify the benefit of 2,3-diamino acid modification of THIQA
the dose dependent in vivo anti-thrombotic activities of 8a,r,s are
compared with those of 2- (9g) and 3- (10s) mono-aminoacid mod-
ification of THIQA in Table 4. The thrombus weight of the rats
8m
8n
8o
8p
8q
8r
8s
8t
9m
9n
9o
9p
9q
9r
9s
9t
10m
10n
10o
10p
10q
10r
10s
10t
370 29
240 27
210 22
265 26
a
IC50 is represented by mean SD nM, 9a–t = 3S-N-(L-aminoacyl)-1,2,3,4-tetra-
hydroisoquino line-3-carboxylic acids, 10a–t = 3S-1,2,3,4-tetrahydroisoquinoline-
3-carbonylamino acids.

X. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 1536–1554
1539
Table 3
Effect of 8a–t on the thrombus weight of intravenously treated ratsa
Compd
Thrombus weight
Compd
Thrombus weight
Compd
Thrombus weight
NS
8a
8b
8c
8d
8e
8f
8g
8h
8i
28.54 2.62
17.12 1.31d
25.20 1.13e
19.42 1.55e
19.53 1.64e
24.30 1.88e
20.11 1.74e
20.97 1.89e
18.79 2.03e
19.27 1.98e
17.95 1.72e
18.13 1.48e
20.91 1.98e
18.65 1.31e
20.49 1.46e
18.95 2.69e
19.35 2.23e
22.51 0.91e
14.32 1.29c
10.61 1.85b
19.36 2.37e
IQ
9a
9b
9c
9d
9e
9f
9g
9h
9i
21.52 1.49e
20.30 1.33e
21.17 1.47e
20.12 1.59e
19.12 1.82e
21.14 1.24e
19.47 1.64e
18.00 1.59e
20.92 1.44e
21.09 1.68e
22.66 1.45e
21.30 1.69e
21.78 1.96e
21.31 1.75e
20.30 1.34e
20.87 1.45e
—
Aspirin
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
10m
10n
10o
10p
10q
10r
13.22 1.67e
21.40 1.39e
20.24 1.37e
19.20 1.57e
20.23 1.49e
20.03 1.29e
21.30 1.06e
23.58 1.93e
19.31 1.48e
20.25 1.50e
20.24 1.50e
22.08 1.54e
20.86 1.94e
21.28 1.17e
21.67 1.91e
19.69 1.25e
20.91 1.27e
19.11 1.58e
21.25 1.93e
18.73 1.56e
21.74 1.50e
8j
8k
8l
9j
9k
9l
8m
8n
8o
8p
8q
8r
8s
8t
9m
9n
9o
9p
9q
9r
9s
9t
22.23 1.72e
20.73 1.53e
20.06 1.48e
19.56 1.72e
10s
10t
a
Weight of wet thrombus is represented by mean SD mg, NS = vehicle, n = 12; IQ = 3S-isoquinoline-3-carboxylic acid and intravenous dose = 15
intravenous dose = 165 mol/kg; 8a–t dose = 30 nmol/kg; 9a–t = 3S-N-( -aminoacyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acids and dose = 5 mol/kg; 10a–t = 3S-
mol/kg.
lmol/kg; Aspirin
l
L
l
1,2,3,4-tetrahydroisoquinoline-3-carbonylamino acids and dose = 5
l
b
Compared to NS and 8a–r,t p <0.01.
Compared to NS and 8a–q,t p <0.01.
Compared to NS and 8b–g,l,n,q p <0.01, to 8h,i,p,t p <0.05.
Compared to NS p <0.01.
c
d
e
Figure 2. Cerius2 based stereoview of 3S-N-(
acids (b) and N-(3S-N- -aminoacyl-1,2,3,4-tetrahydroisoquinoline-3-carbonyl)-
L
-aminoacyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acids (a), N-(3S-1,2,3,4-tetrahydroisoquinoline-3-carbonyl)-
-amino acids (c).
L-amino
L
L
receiving intravenous 3 nmol/kg of 8a equals those of 50 nmol/kg
of 9g and 1 mol/kg of 10s, the thrombus weight of the rats receiv-
ing intravenous 3 nmol/kg of 8r is significantly lower than those of
the rats receiving intravenous 50 nmol/kg of 9g and 1 mol/kg of
two (8r,s) are significantly lower than thir intravenous activities.
These results imply that 8a–t could be orally effective anti-throm-
botic agents, and their oral potencies are either at the same level as
that of intravenous potencies or below the level of intravenous
potencies.
l
l
10s, while the thrombus weight of the rats receiving intravenous
3 nmol/kg of 8s is significantly lower than those of the rats receiv-
ing intravenous 5
l
mol/kg of 9g and 10s. These comparisons dem-
2.7. Computational chemistry
onstrate that the in vivo anti-thrombotic activity of 2,3-
diaminoacid modified THIQA is at least 17–333-fold higher than
those of 2- and 3-monoaminoacid modified THIQA.
To quantitatively analyse the relationship of the conformation
and anti-thrombotic activity of 8a–t on the valid 3D-QSAR models
a proper alignment procedure was performed by using the target
model align strategy in the align module within Cerius2. With an
assumption that each structure of 8a–t binds the same site of the
receptor and exhibits activity, they were aligned in a pharmacolog-
ical active orientation. To obtain a consistent alignment the tetrahy-
droisoquinoline ring was used as the template for superposing 8a–t.
The maximum common subgraph (MCS) was used to perform this
alignment.44 The alignment of 8a–t were shown as Figure 3, which
explored that to superimpose onto tetrahydroisoquinoline ring the
side chains of amino acid residue in each structure had to take its
individual conformation and finally affected their in vitro anti-plate-
let aggregation and in vivo anti-thrombotic activities.
2.6. In vivo anti-thrombotic activity of oral 8a,j,k,m,r,s
To explore the possibility of oral administration 8a,j,k,m,r,s
were selected as the representatives of 8a–t, their oral activities
were evaluated on an extracorporeal circulation of arterioveinos
cannula model of rats and the data are listed in Table 5. The eval-
uation explores that the thrombus weights of the rats receiving or-
ally administering of 30 nmol/kg of 8a,j,k,m,r,s are significantly
lower than that of the rats receiving NS. The evaluation also ex-
plores that 4 (8a,j,k,m) of these 6 compounds (8a,j,k,m,r,s) possess
identical intravenous and oral activity, and the oral activities of

1540
X. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 1536–1554
Table 4
that they populate the wide range of activities in similar propor-
tions. Based on the QSAR module of Cerius2 the MFA was per-
formed by use of the training set. Three probes were chosen to
describe 16 molecules. Methyl group was used to account for steric
contacts, while proton and hydroxyl ion probes were used to eval-
uate electrostatic potential fields on each ligand structure. The
MFA model of 8a–c,f–i,k–q,s,t inhibiting ADP-induced-platelet
aggregation in terms of the descriptors proton, methyl and hydro-
xyl ion was expressed by Eq. 1. The data points (n), correlation
coefficient (r) and square correlation coefficient (r2) of Eq. 1 were
16, 0.996 and 0.992, respectively. The tested and calculated
anti-platelet aggregation induced by ADP is graphically shown in
Figure 4:
Effect of dose of 8a,r,s on the thrombus weight of intravenously treated ratsa
Compd
Dose
—
Thrombus weight (mg)
NS
Aspirin
28.80 1.40a
13.22 1.67
27.60 1.89
17.12 1.31b
23.35 1.81c
29.77 2.52c
14.32 1.29b
20.95 1.88c
28.63 0.70
10.61 1.85b
16.29 2.41d
19.71 1.85
18.00 1.59
22.54 3.40
28.58 1.56
18.73 1.56
24.38 2.61
29.11 2.37
165
50
l
mol/kg
lmol/kg
8a
8r
30 nmol/kg
3 nmol/kg
0.3 nmol/kg
30 nmol/kg
3 nmol/kg
0.3 nmol/kg
30 nmol/kg
3 nmol/kg
0.03 nmol/kg
5 lmol/kg
50 nmol/kg
5 nmol/kg
8s
9g
10s
Activity ¼ 2:755 þ 0:021ðHþ=725Þ þ 0:0080ðHþ=448Þ
ꢀ 0:064ðHþ=876Þ ꢀ 0:0059ðHþ=773Þ
5
1
l
l
mol/kg
mol/kg
ꢀ 0:0018ðHþ=855Þ þ 0:0029ðCH3=785Þ
ꢀ 0:010ðCH3=633Þ þ 0:011ðHOꢀ=285Þ
þ 0:011ðHOꢀ=396Þ þ 0:0041ðHOꢀ=577Þ
þ 0:0053ðHOꢀ=767Þ þ 0:0019ðHOꢀ=865Þ
0.2 lmol/kg
a
Weight of wet thrombus is represented by mean SD mg, NS = vehicle, n = 12,
3 nmol/kg of 8a compared with 50 nmol/kg of 9g and 1
3 nmol/kg of 8r compared with 50 nmol/kg of 9g and 1
l
l
mol/kg of 10s p >0.05,
mol/kg of 10s p <0.05,
3 nmol/kg of 8s compared with 5 lmol/kg of 9g and 10s p <0.05.
ꢀ 0:0090ðHOꢀ=624Þ
ð1Þ
b
Compared with NS, 3 nmol/kg and 0.3 nmol/kg of 8a,r,s p <0.01.
Compared with NS and 0.3 nmol/kg of 8a,r p <0.01.
Compared with NS and 0.03 nmol/kg of 8s p <0.01.
c
d
Eq. 1 contains 5 terms from proton descriptor, 2 terms from
methyl descriptor, and 5 terms from hydroxyl anion descriptor.
The terms of 0.021(H+/725) and 0.0080(H+/448) have positive coef-
ficients, which means that at these positions electron-releasing
groups will increase the activity, while the terms of 0.064(H+/876),
0.0059(H+/773) and 0.0018(H+/855) have negative coefficients,
which means that at these positions electron-withdrawing groups
will increase the activity. The term of 0.0029(CH3/785) has positive
coefficient, which means that at this position small group will in-
crease the activity, while term of 0.010(CH3/633) has negative coef-
ficients, which means that at these positions large groups will
increase the activity. The terms of 0.011(HOꢀ/285), 0.011(HOꢀ/
396), 0.0041(HOꢀ/577), 0.0053(HOꢀ/767) and 0.0019(HOꢀ/865)
have positive coefficients, which means that at these positions elec-
tron-withdrawing groups will increase the activity, while term of
0.0090(HOꢀ/624) has negative coefficients, which means that at
these positions electron-releasing groups will increase the activity.
As examples, Figures 5 and 6 give the electrostatic and environ-
ments of 8a,o,p,t within the grid with 3D points of Eq. 1. Figure 5
indicates that besides the isoquinoline ring has same electrostatic
Table 5
Effect of oral 8a,j,k,m,r,s on thrombus weight of the treated ratsa
Compd
Thrombus weight
Compd
Thrombus weight
NS
8a
8j
28.80 1.40
18.89 2.49b
16.88 1.71b
18.23 1.60b
Aspirin
8m
8r
14.99 1.71b
19.72 1.88b
17.53 1.71b
15.15 2.23b
8k
8s
a
Weight of wet thrombus is represented by X SD mg, NS = vehicle, n=12; Dose
of 8a,j,k,m,r,s = 30 nmol/kg; Dose of Aspirin = 165
lmol/kg.
b
Compared with NS p < 0.01.
Figure 3. Alignment of 8a–t used for molecular field generation.
2.7.1. 3D QSAR analysis of 8a-t inhibiting ADP-induced platelet
aggregation
After energy-minimization using the MMFF94 (Merck Molecu-
lar Force Field), the Molecular Field Analysis (MFA) was performed
for 8a–t using the QSAR module of Cerius2.45 Training set (8a–c,f–
i,k–q,s,t)/test set (3d,e,j,r) selections were done manually such
Figure 4. Graph of tested versus predicted activities of 8a–t inhibiting ADP-induced
platelet aggregation.

X. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 1536–1554
1541
Figure 5. Electrostatic and steric environments of 8a (a) and 8o (b) with higher in vitro activity within the grid with 3D points of Eq. 1.
and environments, 8a has a small group near CH3/785 region, elec-
tron-withdrawing group near H+/448, as well as hydrogen bond
forming groups near OHꢀ/767,865 region and thus totally results
in great increase of in vitro anti-platelet aggregation activity; 8o
has small group near CH3/633 region, electron-withdrawing group
near H+/448 and hydrogen bond forming group near OHꢀ/
396,577,624 region and thus totally results in increase of in vitro
anti-platelet aggregation activity. Figure 6 indicates that besides
isoquinoline ring has same electrostatic and environments, 8p
has electron-withdrawing group near H+/855, small group near
CH3/785 region and hydrogen bond forming group near OHꢀ/
577,865 region, and thus totally results in no increase of in vitro
anti-platelet aggregation activity, while 8t has small groups near
CH3/633,785 region, hydrogen bond forming group near OHꢀ/577
region and thus totally results in no increase of in vitro anti-plate-
let aggregation activity.
droxyl ion was expressed by Eq. 2. The data points (n), correlation
coefficient (r) and square correlation coefficient (r2) of Eq. 2 were
16, 0.995 and 0.990, respectively. The tested and calculated anti-
thrombotic activities (thrombus weights) are graphically shown
in Figure 7:
Activity ¼ 20:978 þ 0:21ðHþ=646Þ þ 0:24ðHþ=856Þ
ꢀ 0:27ðHþ=536Þ ꢀ 0:31ðHþ=634Þ
ꢀ 0:068ðHþ=475Þ ꢀ 0:029ðHþ=766Þ
ꢀ 0:015ðHþ=863Þ þ 0:011ðCH3=874Þ
ꢀ 0:093ðCH3=449Þ ꢀ 0:030ðCH3=733Þ
ꢀ 0:027ðCH3=623Þ ꢀ 0:10ðHOꢀ=773Þ
ꢀ 0:0061ðHOꢀ=376Þ
ð2Þ
The predict power of Eq. 1 was examined by comparing the cal-
culated and measured anti-platelet aggregation activities of 8d,e,j,r
in Table 6. The predicting and measuring activities of them are also
graphically shown in Figure 4. The results indicate that Eq. 1 ratio-
nally gives anti-platelet aggregation activities of 8d,e,j,r with er-
rors ranging from ꢀ0.03 to ꢀ0.19. Most of the calculated
activities close the measured activities means that Eq. 1 is able
to accurately predict the inhibition of the derivatives of 2,3-diami-
no acid modified THIQA to ADP-induced-platelet aggregation.
Eq. 2 contains 7 terms from proton descriptor, 4 terms from
methyl descriptor, and 2 terms from hydroxyl anion descriptor.
The terms of 0.21(H+/646) and 0.24(H+/856) have positive coeffi-
cients, which means that at these positions electron-releasing
groups will increase the activity, while the terms of 0.27(H+/
536), 0.31(H+/634), 0.068(H+/475), 0.029(H+/766) and 0.015(H+/
863) have negative coefficients, which means that at these posi-
tions electron-withdrawing groups will increase the activity.
The term of 0.011(CH3/874) has positive coefficient, which means
that at this position small group will increase the activity, while
terms of 0.093(CH3/449), 0.030(CH3/733) and 0.027(CH3/623)
have negative coefficients, which means that at these positions
large groups will increase the activity. The terms of 0.10(HOꢀ/
773) and 0.0061(HOꢀ/376) have negative coefficients, which
means that at these positions electron-releasing groups will in-
crease the activity.
2.7.2. 3D QSAR analysis of 8a-t inhibiting thrombosis of treated
rats
Using the same protocol as that of item 2.4.1 and the training
set (8a–d,f,h–i,k–o,q–t)/test (8e,g,j,p) were selected. The MFA
model of thrombus weights of the rats receiving intravenous 8a–
d,f,h–i,k–o,q–t in terms of the descriptors proton, methyl and hy-
Figure 6. Electrostatic and steric environments of 8p (a) and 8t (b) with lower in vitro activity within the grid with 3D points of Eq. 1.

1542
X. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 1536–1554
Table 6
The predict power of Eq. 2 was examined by comparing the cal-
culated and measured anti-thrombotic activities of 8e,g,j,p in Table
7. The correlations of predicting and measuring values are also
graphically shown in Figure 6. The results indicate that Eq. 2 ratio-
nally gives anti-thrombotic activities of 8e,g,j,p with errors ranging
from ꢀ0.28 to 1.11 mg. The calculated activity closes the measured
activity means that Eq. 2 is able to accurately predict the in vivo
anti-thrombotic activity of the derivatives of 2,3-diamino acid
modified THIQA.
Predicted and tested IC50 of 8d,e,j,r against ADP-induced platelet aggregation
Compd
IC50 (nM)
Test value
Predict value
Error
Error%
8d
8e
8j
2.39
2.78
2.97
2.86
2.57
2.89
3.07
2.89
ꢀ0.19
ꢀ0.11
ꢀ0.10
ꢀ0.03
7.2
3.9
3.4
1.0
8r
3. Conclusion
In conclusion, an unfold conformation is crucial for enhancing
the activity of amino acid modified THIQA. Comparing with 2- (
9a–t) or 3-mono amino acid modified THIQAs (10a–t), 2,3-diamino
acid modified THIQAs (8a–t) possess highly unfolded conforma-
tion. This leads 8a–t having selective in vitro inhibition to ADP-in-
duced platelet aggregation, and potential in vivo inhibition to
thrombosis of the orally and intravenously treated rats. The IC50
values of 2- or 3-monoaminoacid modified THIQAs against ADP-in-
duced platelet aggregation are 47–143 and 55–177-fold higher
than that of 2,3-diamino acid modified THIQAs, respectively. To
reach the same in vivo anti-thrombotic potency the dose of 2- or
3-monoaminoacid modified THIQAs has to be 167-fold higher than
that of 2,3-diamino-acid modified THIQAs. The effect of the highly
unfolded conformation on the in vitro anti-platelet aggregation
and the in vivo anti-thrombotic activities could be explained with
3D QSAR.
Figure 7. Graph of tested versus predicted activities of 8a–t inhibiting thrombosis.
4. Experimental section
4.1. General method
As examples, Figures 8 and 9 give the electrostatic and environ-
ments of 8a,l,q,r within the grid with 3D points of Eq. 2. Figure 8
indicates that besides the isoquinoline ring has same electrostatic
and environments, 8a has a small group near CH3/733 region, elec-
tron-withdrawing group near H+/766, electron-releasing group
near H+/646, as well as electron-releasing group near OHꢀ/773 re-
gion and thus totally results in increase of in vitro anti-platelet
aggregation activity; 8r has large group near CH3/449 and CH3/
733 regions, electron-releasing groups near H+/646 and OHꢀ/376
regions and thus totally results in increase of in vivo anti-throm-
botic activity. Figure 9 indicates that besides isoquinoline ring
has same electrostatic and environments, 8l has electron-releasing
group near H+/863 H+/863, large group near CH3/874 and CH3/975
region, small group near CH3/623 region and thus totally results in
no increase of in vivo anti-thrombotic activity, while 8r has large
group near CH3/449 and CH3/623 regions, electron-releasing
groups near H+/863 region, electron-withdrawing group near H+/
766 region and thus totally results in no increase of in vivo anti-
thrombotic activity.
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 according to literature
procedures. Optical rotations were determined with a Jasco P-1020
Polarimeter at 20 °C. The statistical analysis of all the biological
data was carried out by use of ANOVA test, p<0.05 is considered
significant.
Figure 8. Electrostatic and steric environments of 8r (a) and 8a (b) with higher in vitro activity within the grid with 3D points of Eq. 2.

X. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 1536–1554
1543
Figure 9. Electrostatic and steric environments of 8q (a) and 8l (b) with lower in vitro activity within the grid with 3D points of Eq. 2.
Table 7
and stirred for 30 min to form mixture B. At 0 °C the mixture A
and B were mixed and then 0.26 g (0.12 mmol) of DCC was added.
The reaction mixture was stirred at 0 °C for 2 h, at room tempera-
ture for 12 h and TLC (ethyl acetate/petroleum ether, 1:2) indicated
the complete disappearance of (3S)-1,2,3,4-tetrahydroisoquino-
line-3-carboxylic acid methylester. The formed precipitates of
DCU were 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 aque-
ous 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 evap-
orated under vacuum to give 4a–t.
Predicted and tested thrombus weight of 8e,g,j,p treated rats
Compd
Thrombus weight (mg)
Predict value
Test value
Error
Error%
8e
8g
8j
24.02
22.08
18.58
20.19
24.30
20.97
17.95
19.35
ꢀ0.28
1.11
0.63
0.84
ꢀ1.2
5.1
3.4
8p
4.2
4.2. Preparing compounds
4.2.1. 3S-1,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid
methylester (3)
4.3.1. 3S-N-(Boc-L-alaninyl)-1,2,3,4-tetrahydroisoquinoline-3-
At 0 °C to 20 ml of methanol 5.2 ml of thionyl chloride was added
dropwise, which took 10 min. To this mixture 5.37 g (0.03 mmol) of
Phe-OCH3 was added. The reaction mixture was stirred at room tem-
perature for 100 h, and TLC (CCl3/CH3OH = 10:1) indicated the com-
plete disappearance of Phe-OCH3. The reaction mixture was
evaporated under vacuum. The residue was dissolved in 20 ml of
methanol and evaporated under vacuum. This procedure was re-
peated for three times. The residue was dissolved in 50 ml of chloro-
form and 27 ml of formaldehyde. To this solution 45 ml of
concentrated hydrochloric acid was added dropwise. The reaction
mixture was stirred at 80–90 °C for 10 h, and TLC (CHCl3/CH3OH,
10:1) indicates the complete disappearance of Phe-OCH3. 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.
This procedure was repeated for three times. The residue was crys-
tallized in the mixture of methanol/ether to provide 5.41 g (94%) of
carboxylic acid methylester (4a)
Using the general procedure of preparing 4a–t from 1.93 g
(10.21 mmol) Boc-Ala and 1.50 g (7.85 mmol) of (3S)-1,2,3,4-tetra-
hydroisoquinoline-3-carboxylic acid methylester 2.57 g (90%) of
the title compound was obtained as colorless powders. ESI-MS (m/
e) 363 [M+H]+; IR (cmꢀ1
) 2968, 1734, 1648, 1497, 1457,
1395,1380, 1365, 1198, 1111, 750, 600; ½a D20
¼ ꢀ28:5 (c = 1.2,
ꢂ
CH3OH); 1H NMR (300 MHz, CDCl3) d/ppm = 8.00 (d, J = 6.6 Hz,
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),4.83 (d, J = 7.5 Hz, 1H), 4.68 (m, 1H), 4.55 (d,
J = 5.6 Hz, 1H), 4.49 (d, J = 8.6 Hz, 1H), 3.63 (s, 3H), 3.30 (d,
J = 7.5 Hz, 1H), 3.10 (d, J = 8.6 Hz, 1H), 1.46 (d, J = 5.4 Hz, 3H), 1.41
(s, 9H); 13C NMR (75 MHz, CDCl3) d/ppm = 171.6, 172.9, 155.1,
132.1, 131.7, 127.2, 80.8, 55.8, 52.2, 46.9, 44.6, 28.4, 27.0, 19.0.
4.3.2. 3S-N-(Boc-glycinyl)-1,2,3,4-tetrahydroisoquinoline-3-
carboxylic acid methylester (4b)
Using the general procedure of preparing 4a–t from 1.19 g
(6.81 mmol) Boc-Gly and 1.0 g (5.24 mmol) of (3S)-1,2,3,4-tetrahy-
droisoquinoline-3-carboxylic acid methylester 1.73 g (95%) of the
title compound was obtained as colorless powders. ESI-MS (m/e)
349 [M+H]+; IR (cmꢀ1) 2969, 1735, 1648, 1496, 1457, 1395, 1365,
the title compound as a colorless powder. Mp 262–264 °C, ½a D20
¼
ꢂ
ꢀ61:0 (c = 1.2, CH3OH), ESI-MS (m/e) 192 [M+H]+. 1H NMR
(300 MHz, CDCl3) d/ppm = 7.0 (m, 4H), 3.80 (m, 3H), 3.67 (s, 3H),
3.13 (d, J = 8.6 Hz, 1H), 2.78 (d, J = 7.4 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.
1198, 1111, 750, 599; ½a D20
ꢂ
¼ ꢀ11:7 (c = 1.1, CH3OH); 1H NMR
(300 MHz, CDCl3) d/ppm = 8.00 (d, J = 5.6 Hz, 1H), 7.22 (m,
J = 6.4 Hz, 2H), 7.05 (d, J = 6.6 Hz, 1H), 6.95 (t, J = 6.5 Hz, 1H), 4.80
(d, J = 6.4 Hz, 1H), 4.51(m, 2H), 3.85 (d, J = 7.5 Hz, 2H), 3.63 (s,
3H), 3.25 (m, 2H), 1.41 (s, 9H); 13C NMR (75 MHz, CDCl3) d/
ppm = 171.6, 168.6, 155.8, 131.9, 130.9,128.4,127.2, 126.6, 80.8,
55.8, 52.0, 44.6, 43.0, 28.4, 27.0.
4.3. General procedure of preparing 3S-N-(Boc-aminoacyl)-
1,2,3,4-tetrahydroisoquino-line-3-carboxylic acid methylesters
(4a–t)
At 0 °C and with stirring to the solution of 1.0 mmol of Boc-AA
in 10 ml of anhydrous THF 0.14 g (1.0 mmol) of HOBt was added to
form reaction mixture A. The solution of 0.23 g (0.91 mmol) of 3S-
1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid methylester in
5 ml of anhydrous THF was adjusted pH 9 with triethylamine
4.3.3. 3S-N-(Boc-L-valinyl)-1,2,3,4-tetrahydroisoquinoline-3-
carboxylic acid methylester (4c)
Using the general procedure of preparing 4a–t from 1.48 g
(6.81 mmol) Boc-Val and 1.0 g (5.24 mmol) of (3S)-1,2,3,4-tetrahy-

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X. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 1536–1554
droisoquinoline-3-carboxylic acid methylester 1.28 g (63%) of the
title compound was obtained as colorless syrupy. ESI-MS (m/e)
391 [M+H]+; IR (cmꢀ1) 2970, 1734, 1648, 1498, 1457, 1395, 1385,
1395, 1365, 1198, 1111, 750, 599; ½a D20
¼ ꢀ7:61 (c = 1.0, CH3OH);
ꢂ
1H NMR (300 MHz, CDCl3) d/ppm = 8.00 (d, J = 5.6 Hz, 1H), 7.02
(m, J = 8.2 Hz, 2H), 6.95 (d, J = 8.8 Hz, 2H), 4.83 (m, J = 8.2 Hz, 1H),
4.63 (s, 2H), 4.51 (m, 2H), 3.86 (m, 2H), 3.63 (s, 3H), 3.29 (d,
J = 6.5 Hz, 1H), 3.05 (d, J = 8.6 Hz, 1H), 1.41 (s, 9H); 13C NMR
(75 MHz, CDCl3) d/ppm = 171.6, 170.1, 155.8, 137.2, 135.9, 134.2,
128.4, 127.2, 125.6, 79.5, 74.3, 70.2, 55.8, 52.0, 50.7, 44.6, 28.4,
27.0.
1375, 1365, 1198, 1111, 750, 599; ½a D20
¼ ꢀ15:8 (c = 1.2, CH3OH);
ꢂ
1H NMR (300 MHz, CDCl3) d/ppm = 8.00 (d, J = 6.6 Hz, 1H), 7.22
(m, J = 6.4 Hz, 2H), 7.07 (d, J = 6.5 Hz, 1H), 6.96 (t, J = 6.6 Hz, 1H),
4.80 (d, J = 5.4 Hz, 1H), 4.61 (d, J = 6.4 Hz, 1H), 4.51 (m, 2H), 4.24
(m, 1H), 3.63 (s, 3H), 3.25 (m, 2H), 2.0 (s, 1H), 1.41 (s, 9H), 1.21
(d, J = 7.8 Hz, 3H); 13C NMR (75 MHz, CDCl) d/ppm = 171.6, 168.6,
155.8, 135.9, 133.9, 128.4, 127.2, 126.6, 80.8, 67.8, 58.4, 55.8,
52.0, 44.6, 28.4, 27.0,18.9.
4.3.8. 3S-N-(Boc-L-tyrocinyl)-1,2,3,4-tetrahydroisoquinoline-3-
carboxylic acid methyl-ester (4h)
Using the general procedure of preparing 4a–t from 2.53 g
(6.81 mmol) Boc-Tyr and 1.00 g (5.24 mmol) of (3S)-1,2,3,4-tetra-
hydroisoquinoline-3-carboxylic acid methylester 2.25 g (79%) of
the title compound was obtained as colorless syrupy. ESI-MS (m/
e) 545 [M+H]+; IR (cmꢀ1) 3411, 2969, 1734, 1648, 1497, 1457,
4.3.4. 3S-N-(Boc-L-leucinyl)-1,2,3,4-tetrahydroisoquinoline-3-
carboxylic acid methylester (4d)
Using the general procedure of preparing 4a–t from 2.36 g
(10.21 mmol) Boc-Leu and 1.5 g (7.85 mmol) of (3S)-1,2,3,4-tetra-
hydroisoquinoline-3-carboxylic acid methylester 1.44 g (45%) of
the title compound was obtained as colorless syrupy. ESI-MS (m/
e) 405 [M+H]+; IR (cmꢀ1) 2970, 1734, 1648, 1497, 1457, 1395,
1395, 1380, 1365, 1198, 1111, 750, 599; ½a D20
¼ ꢀ6:86 (c = 1.0,
ꢂ
CH3OH); 1H NMR (300 MHz, CDCl3) d/ppm = 8.00 (d, J = 7.6 Hz,
1H), 7.02 (m, J = 8.2 Hz, 2H), 6.98 (d, J = 8.8 Hz, 2H), 6.95 (d,
J = 8.8 Hz, 2H), 6.72 (d, J = 5.5 Hz, 2H), 5.20 (s, 1H), 4.92 (m, 1H),
4.80 (t, J = 6.4 Hz, 1H), 4.51 (d, J = 7.6 Hz, 1H), 4.41 (d, J = 5.4 Hz,
1H), 3.63 (d, J = 7.5 Hz, 3H), 3.29 (d, J = 6.8 Hz, 1H), 3.05 (d,
J = 8.3 Hz, 1H), 2.92 (m, 2H), 1.41 (s, 9H); 13C NMR (75 MHz, CDCl3)
1385, 1375, 1365, 1198, 1111, 750, 599; ½a D20
¼ ꢀ16:3 (c = 1.0,
ꢂ
CH3OH); 1H NMR (300 MHz, CDCl3) d/ppm = 8.00 (d, J = 6.6 Hz,
1H), 7.02 (m, J = 8.2 Hz, 2H), 6.95 (d, J = 8.8 Hz, 2H), 4.80 (d,
J = 8.4 Hz, 1H), 4.51 (m, 3H), 3.63(s, 3H), 3.10 (m, 2H), 1.78 (m,
3H), 1.41 (s, 9H), 1.01(d, J = 7.4 Hz, 6H); 13C NMR (75 MHz, CDCl3)
d/ppm = 171.6, 170.1, 157.9, 155.8, 141.2, 136.5, 134.9, 131.2,
d
/ppm = 171.6, 170.1, 155.8, 135.9, 134.2, 128.4, 127.2, 126.6, 79.5,
129.0, 128.4, 127.2, 126.6, 114.2, 79.5, 70.9, 55.8, 52.9, 52.0, 44.6,
37.8, 28.4, 27.0.
55.8, 52.0, 50.7, 44.6, 41.6, 28.4, 27.0, 22.3.
4.3.5. 3S-N-(Boc-L-isoleucinyl)-1,2,3,4-tetrahydroisoquinoline-
4.3.9. 3S-N-(Boc-L-prolinyl)-1,2,3,4-tetrahydroisoquinoline-3-
3-carboxylic acid methyl-ester (4e)
carboxylic acid methylester (4i)
Using the general procedure of preparing 4a–t from 2.36 g
(10.21 mmol) Boc-Ile and 1.5 g (7.85 mmol) of (3S)-1,2,3,4-tetrahy-
droisoquinoline-3-carboxylic acid methylester 1.62 g (51%) of the
title compound was obtained as colorless syrupy. ESI-MS (m/e)
405 [M+H]+; IR (cmꢀ1) 2969, 1734, 1648, 1497, 1457, 1395,1380,
Using the general procedure of preparing 4a–t from 1.46 g
(6.81 mmol) of Boc-Pro and 1.00 g (5.24 mmol) of (3S)-1,2,3,4-tet-
rahydroisoquinoline-3-carboxylic acid methylester 1.73 g (85%) of
the title compound was obtained as colorless powders. ESI-MS (m/
e) 389 [M+H]+; IR (cmꢀ1) 3410, 2969, 1734, 1648, 1497, 1457,
1365, 1198, 1111, 750, 599; ½a D20
ꢂ
¼ ꢀ13:4 (c = 1.0, CH3OH); 1H
1395, 1365, 1360, 1198, 1111, 750, 599; ½a D20
¼ ꢀ54:6 (c = 1.0,
ꢂ
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), 4.80 (d, J = 5.4 Hz, 1H),
4.51 (m, 3H), 3.63 (s, 3H), 3.10 (m, 2H), 2.5 (m, 1H), 1.41 (s, 9H),
1.29 (m, 2H), 1.06 (d, J = 6.5 Hz, 3H), 0.96 (d, J = 8.1 Hz, 3H); 13C
NMR (75 MHz, CDCl3) d/ppm = 171.6, 170.1, 155.8, 135.9,
134.2,128.4, 127.2, 126.6, 79.5, 55.8, 52.0, 44.6, 28.4, 27.0, 24.3,
14.6, 10.9.
CH3OH); 1H NMR (300 MHz, CDCl3) d/ppm = 7.02 (m, J = 8.2 Hz,
2H), 6.95 (d, J = 8.8 Hz, 2H), 4.80 (t, J = 8.4 Hz, 1H), 4.51 (m, 2H),
4.29 (t, J = 7.6 Hz, 1H), 3.63 (s, 3H), 3.20 (m, 4H), 1.71 (m, 2H),
1.60 (m, 2H), 1.41 (s, 9H); 13C NMR (75 MHz, CDCl3) d/
ppm = 171.6, 170.1, 155.8, 135.9, 134.2, 128.4, 127.2, 126.6, 79.5,
55.8, 52.0, 47.1, 44.6, 29.7, 28.4, 27.0, 22.1.
4.3.10. 3S-N-(Boc-L-asparaginyl)-1,2,3,4-tetrahydroisoquinoline-
4.3.6. 3S-N-(Boc-L-tryptophanyl)-1,2,3,4-tetrahydroisoquinoline-
3-carboxylic acid methyl-ester (4j)
3-carboxylic acid methylester (4f)
Using the general procedure of preparing 4a–t from 4.74 g
(20.42 mmol) of Boc-Asn and 3.00 g (15.71 mmol) of (3S)-1,2,3,4-
tetrahydroisoquinoline-3-carboxylic acid methylester 5.13 g
(84%) of the title compound was obtained as colorless powders.
ESI-MS (m/e) 406 [M+H]+; IR (cmꢀ1) 3411, 2969, 1734, 1648,
1497, 1457, 1395, 1380, 1365, 1198, 1111, 750, 599;
Using the general procedure of preparing 4a–t from 1.03 g
(3.40 mmol) Boc-Trp and 0.50 g (2.62 mmol) of (3S)-1,2,3,4-tetra-
hydroisoquinoline-3-carboxylic acid methylester 0.64 g (52%) of
the title compound was obtained as yellowing powders. ESI-MS
(m/e) 478 [M+H]+; IR (cmꢀ1) 2968, 1734, 1648, 1497, 1457,
1395,1380, 1365, 1198, 1111, 750, 599; ½a D20
ꢂ
¼ ꢀ6:38 (c = 1.0,
½
a 2D0
ꢂ
¼ ꢀ10:4 (c = 1.0, CH3OH); 1H NMR (300 MHz, CDCl3) d/
CH3OH);1H NMR (300 MHz, CDCl3) d/ppm = 10.10 (s, 1H), 8.00 (d,
J = 7.6 Hz, 1H), 7.18 (m, J = 6.4 Hz, 4H), 7.02 (m, J = 8.2 Hz, 2H),
6.95 (d, J = 8.8 Hz, 2H), 6.80 (d, J = 6.4 Hz, 1H), 4.90 (d, J = 8.0 Hz,
1H), 4.81 (d, J = 5.4 Hz,1H), 4.51 (m, 2H), 3.63 (s, 3H), 3.10 (m,
4H), 1.41 (s, 9H); 13C NMR (75 MHz, CDCl3) d/ppm = 171.6, 170.1,
155.8, 135.9, 134.2, 128.4, 127.2, 126.6, 122.9, 119.0, 110.9, 79.5,
55.8, 52.0, 31.5, 44.6, 28.4, 27.0.
ppm = 8.00 (d, J = 5.6 Hz, 1H), 7.02 (m, J = 8.2 Hz, 2H), 6.95 (d,
J = 8.8 Hz, 2H), 6.0 (s, 2H), 4.78 (m, 2H), 4.51 (d, J = 6.4 Hz, 1H),
4.41 (d, J = 5.4 Hz, 1H), 3.63 (s, 3H), 3.29 (d, J = 7.2 Hz, 1H), 3.05
(d, J = 8.6 Hz, 1H), 2.68 (m, 2H), 1.41 (s, 9H); 13C NMR (75 MHz,
CDCl3) d/ppm = 174.5, 171.6, 170.1, 155.8, 135.9, 134.2, 128.4,
127.2, 126.6, 79.5, 55.8, 52.0, 51.6, 44.6, 37.9, 28.4, 27.0.
4.3.11. 3S-N-(Boc-L-glutaminyl)-1,2,3,4-tetrahydroisoquinoline-
4.3.7. 3S-N-(Boc-L-serinyl)-1,2,3,4-tetrahydroisoquinoline-3-
3-carboxylic acid methyl-esters (4k)
carboxylic acid methylester (4g)
Using the general procedure of preparing 4a–t from 2.51 g
(10.21 mmol) of Boc-Gln and 1.50 g (7.85 mmol) of (3S)-1,2,3,4-
tetrahydroisoquinoline-3-carboxylic acid methylester 3.02 g
(92%) of the title compound was obtained as colorless powders.
ESI-MS (m/e) 420 [M+H]+; IR (cmꢀ1) 3411, 2969, 1734, 1648,
Using the general procedure of preparing 4a–t from 1.15 g
(5.65 mmol) Boc-Ser and 0.83 g (4.35 mmol) of (3S)-1,2,3,4-tetra-
hydroisoquinoline-3-carboxylic acid methylester 0.99 g (49%) of
the title compound was obtained as colorless syrupy. ESI-MS (m/
e) 469 [M+H]+; IR (cmꢀ1) 3411, 2969, 1734, 1648, 1497, 1457,
1497, 1457, 1395,1380, 1365, 1198, 1111, 750, 599; ½a D20
¼ ꢀ14:7
ꢂ

X. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 1536–1554
1545
(c = 1.0, CH3OH); 1H NMR (300 MHz, CDCl3) d/ppm = 8.00 (d,
J = 5.6 Hz, 1H), 7.02 (m, J = 8.2 Hz, 2H), 6.95 (d, J = 8.8 Hz, 2H),
4.78 (t, J = 6.4 Hz, 1H), 4.48 (m, 3H), 3.63 (s, 3H), 3.29 (d,
J = 7.2 Hz, 1H), 3.05 (d, J = 6.6 Hz, 1H), 2.18 (t, J = 8.5 Hz, 2H), 2.07
(m, 2H), 1.41 (s, 9H); 13C NMR (75 MHz, CDCl3) d/ppm = 174.5,
171.6, 170.1, 155.8, 135.9, 134.2, 128.4, 127.2, 126.6, 79.5, 55.8,
52.0, 51.6, 44.6, 32.6, 28.4, 27.6, 27.0.
(c = 1.0, CH3OH);1H NMR (300 MHz, CDCl3) d/ppm = 8.00 (d,
J = 6.6 Hz, 1H), 7.19 (s, 5H), 7.02 (m, J = 8.2 Hz, 2H), 6.95 (d,
J = 8.8 Hz, 2H), 5.34 (s, 2H), 4.81 (t, J = 8.4 Hz, 1H), 4.53 (m, 1H),
4.51 (d, J = 5.4 Hz, 1H), 4.41 (d, J = 8.5 Hz, 1H), 3.63 (s, 3H), 3.29
(d, J = 7.6 Hz, 1H), 3.05 (d, J = 6.5 Hz, 1H), 2.21 (m, 4H), 1.41 (s,
9H); 13C NMR (75 MHz, CDCl3) d/ppm = 174.5, 171.6, 170.1,
155.8, 141.2, 135.9, 134.2, 129.4, 127.2, 126.6, 79.5, 68.5, 55.8,
52.0, 51.6, 44.6, 37.9, 28.4, 27.0.
4.3.12. 3S-N-(Boc-L-histidinyl)-1,2,3,4-tetrahydroisoquinoline-
3-carboxylic acid methyl-esters (4l)
4.3.16. 3S-N-[Boc-L-(S-p-methylbenzylcysteinyl)]-1,2,3,4-
Using the general procedure of preparing 4a–t from 3.46 g
(13.61 mmol) of Boc-His and 2.00 g (10.47 mmol) of (3S)-1,2,3,4-
tetrahydroisoquinoline-3-carboxylic acid methylester 3.23 g
(72%) of the title compound was obtained as colorless powders.
ESI-MS (m/e) 429 [M+H]+; IR (cmꢀ1) 3411, 2969, 1734, 1648,
tetrahydroisoquinoline-3-carboxylic acid methylester (4p)
Using the general procedure of preparing 4a–t from 3.32 g
(10.21 mmol) of Boc-Cys(S-CH2C6H4-Me-p) and 1.50 g (7.85 mmol)
of (3S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid methyles-
ter 3.40 g (87%) of the title compound was obtained as colorless
powders. ESI-MS (m/e) 499 [M+H]+. 1H NMR (300 MHz, CDCl3) d/
ppm = 8.00 (d, J = 6.6 Hz, 1H), 7.11 (s, 4H), 6.98 (d, J = 8.8 Hz, 2H),
6.91 (d, J = 8.8 Hz, 2H), 4.81 (t, J = 8.4 Hz, 1H), 4.53 (m, 1H), 4.51
(d, J = 5.4 Hz, 1H), 4.41 (d, J = 8.5 Hz, 1H), 3.72 (d, 2H), 3.63 (s,
3H), 3.29 (d, J = 7.6 Hz, 1H), 3.05 (d, J = 6.5 Hz, 1H), 2.82 (d,
J = 9.0 Hz, 2H), 2.36 (s, 3H), 1.41 (s, 9H); 13C NMR (75 MHz, CDCl3)
1497, 1457, 1395,1380, 1365, 1198, 1111, 750, 599; ½a D20
¼ ꢀ11:2
ꢂ
(c = 1.0, CH3OH);1H NMR (300 MHz, CDCl3) d/ppm = 13.40 (d,
J = 7.4 Hz, 1H), 8.00 (s, 1H), 7.44 (s, 1H), 7.02 (m, J = 8.2 Hz, 2H),
6.95 (d, J = 8.8 Hz, 2H), 6.80 (d, J = 6.3 Hz, 1H), 4.92 (t, J = 7.3 Hz,
1H), 4.80 (t, J = 5.4 Hz, 1H), 4.51 (d, J = 6.2 Hz, 1H), 4.41 (d,
J = 8.6 Hz, 1H), 3.63 (s, 3H), 3.17 (m, 1H), 2.92 (m, 1H), 1.41 (s,
9H); 13C NMR (75 MHz, CDCl3) d/ppm = 171.6, 170.1, 155.8,
135.9, 134.2, 133.5, 128.4, 127.2, 126.6, 119.6, 79.5, 55.8, 52.0,
51.6, 44.6, 30.5, 28.4, 27.0.
d/ppm = 171.6, 170.1, 155.8, 135.9, 134.2, 129.4, 127.2, 126.6,
125.9, 79.5, 55.8, 52.0, 51.8, 44.6, 37.9, 34.1, 28.4, 27.0, 24.3.
4.3.17. 3S-N-(Boc-L-phenylalaninyl)-1,2,3,4-tetrahydroisoquino-
line-3-carboxylic acid methylester (4q)
a
x
4.3.13. 3S-N-(N ,N -diBoc-
L-lysinyl)-1,2,3,4-tetrahydroisoquino-
line-3-carboxylic acid methylesters (4m)
Using the general procedure of preparing 4a–t from 2.71 g
(10.21 mmol) of Boc-Phe and 1.50 g (7.85 mmol) of (3S)-1,2,3,4-
tetrahydroisoquinoline-3-carboxylic acid methylester 2.58 g
(75%) of the title compound was obtained as colorless powders.
ESI-MS (m/e) 439 [M+H]+; IR (cmꢀ1) 2968, 1734, 1648, 1600,
1497, 1457, 1395, 1380, 1365, 1198, 1111, 750, 700, 599;
Using the general procedure of preparing 4a–t from 2.34 g
(6.81 mmol) of Boc2-Lys and 1.00 g (5.24 mmol) of (3S)-1,2,3,4-tet-
rahydroisoquinoline-3-carboxylic acid methylester 2.53 g (93%) of
the title compound was obtained as colorless powders. ESI-MS (m/
e) 520 [M+H]+; IR (cmꢀ1) 3411, 2969, 1734, 1648, 1497, 1457,
1395,1380, 1365, 1198, 1111, 750, 599; ½a D20
ꢂ
¼ ꢀ9:0 (c = 1.0,
½
a 2D0
ꢂ
¼ ꢀ35:8 (c = 1.0, CH3OH); 1H NMR (300 MHz, CDCl3) d/
CH3OH); 1H NMR (300 MHz, CDCl3) d/ppm = 8.00 (d, J = 7.6 Hz,
2H), 7.02 (m, J = 8.2 Hz, 2H), 6.95 (d, J = 8.8 Hz, 2H), 4.78 (t,
J = 8.4 Hz, 1H), 4.52 (m, 2H), 4.41 (d, J = 6.4 Hz, 1H), 3.63 (s, 3H),
3.29 (d, J = 5.6Hz, 1H), 3.05 (d, J = 8.5 Hz, 1H), 2.96 (m, 2H), 1.79
(m, 2H), 1.55 (m, 2H), 1.41 (s, 18H), 1.29 (m, 2H); 13C NMR
(75 MHz, CDCl3) d/ppm = 171.6, 170.1, 155.8, 135.9, 134.2, 128.4,
127.2, 126.6, 79.5, 55.8, 52.0, 51.6, 44.6, 41.9, 31.9, 28.4, 27.0, 20.7.
ppm = 8.00 (d, J = 6.6 Hz, 1H), 7.22 (m, J = 7.4 Hz, 2H), 7.15 (d,
J = 7.2 Hz, 2H), 7.10 (d, J = 7.8 Hz, 1H), 7.02 (m, J = 8.2 Hz, 2H),
6.95 (d, J = 8.8 Hz, 2H), 4.92 (d, J = 5.2 Hz, 1H), 4.80 (d, J = 8.4 Hz,
1H), 4.51 (d, J = 7.1 Hz, 1H), 4.41 (d, J = 7.3 Hz, 1H), 3.63 (s, 3H),
3.10 (m, 4H), 1.41 (s, 9H); 13C NMR (75 MHz, CDCl3) d/
ppm = 171.6, 170.1, 155.8, 139.5, 135.9, 134.2, 128.4, 127.2,
126.6, 79.5, 55.8, 52.0, 44.6, 37.8, 28.4, 27.0.
4.3.14. 3S-N-(Boc-L-aspartyl-b-benzylester)-1,2,3,4-tetrahydroiso-
4.3.18. 3S-N-(Boc-L-threoninyl)-1,2,3,4-tetrahydroisoquinoline-
quinoline-3-carboxylic acid methylester (4n)
3-carboxylic acid methyl-ester (4r)
Using the general procedure of preparing 4a–t from 1.16 g
(3.59 mmol) of Boc-Asp(OBzl) and 0.53 g (2.85 mmol) of (3S)-
Using the general procedure of preparing 4a–t from 2.23 g
(10.21 mmol) of Boc-Thr and 1.0 g (5.24 mmol) of (3S)-1,2,3,4-tet-
rahydroisoquinoline-3-carboxylic acid methylester 1.52 g (74%) of
the title compound was obtained as colorless syrupy. ESI-MS (m/e)
393 [M+H]+; IR (cmꢀ1) 3411, 2969, 1734, 1648, 1497, 1457, 1395,
1,2,3,4-tetrahydroisoquinoline-3-carboxylic
acid
methylester
0.89 g (63%) of the title compound was obtained as colorless pow-
ders. ESI-MS (m/e) 497 [M+H]+; IR (cmꢀ1) 3411, 2969, 1734, 1648,
1497, 1457, 1395, 1380, 1365, 1198, 1111, 750, 599; ½a D20
ꢂ
¼ ꢀ5:70
1380, 1365, 1198, 1111, 750; ½a D20
ꢂ
¼ ꢀ20:1 (c = 1.0, CH3OH); 1H
(c = 1.0, CH3OH); 1H NMR (300 MHz, CDCl3) d/ppm = 8.00 (d,
J = 6.6 Hz, 1H), 7.19 (s, 5H), 7.02 (m, J = 8.2 Hz, 2H), 6.95 (d,
J = 8.8 Hz, 2H), 5.34 (s, 2H), 5.17 (t, J = 7.3 Hz, 1H), 4.81 (t,
J = 8.4 Hz, 1H), 4.51 (d, J = 7.3 Hz, 1H), 4.41 (d, J = 5.6 Hz, 1H),
3.63 (s, 3H), 3.29 (d, J = 6.6 Hz, 1H), 3.05 (d, J = 8.6 Hz, 1H), 2.88
(m, 1H), 2.63 (m, 1H), 1.48 (s, 9H); 13C NMR (75 MHz, CDCl3) d/
ppm = 174.5, 171.6, 170.1, 155.8, 141.2, 135.9, 134.2, 129.4,
127.2, 126.6, 79.5, 68.5, 55.8, 52.0, 51.6, 49.7, 44.6, 37.9, 28.4, 27.0.
NMR (300 MHz, CDCl3) d/ppm = 8.00 (d, J = 6.6 Hz, 1H), 7.02 (m,
J = 8.2 Hz, 2H), 6.95 (d, J = 8.8 Hz, 2H), 4.80 (t, J = 7.1 Hz, 1H), 4.61
(d, J = 8.2 Hz, 1H), 4.46 (m, 2H), 4.24 (m, 1H), 3.63 (s, 3H), 3.29
(d, J = 5.4 Hz, 1H), 3.05 (d, J = 6.4 Hz, 1H), 2.0 (s, 1H), 1.48 (s, 9H),
1.21 (d, J = 7.1 Hz, 3H); 13C NMR (75 MHz, CDCl3)/ppm = 171.6,
170.1, 155.8, 135.9, 134.2, 128.4, 127.2, 126.6, 79.5, 67.8, 58.8,
55.0, 51.7, 44.6, 28.4, 27.0, 10.9.
4.3.19. 3S-N-(Boc-L
-NOG2 -argininyl)-1,2,3,4-tetrahydroisoquino-
4.3.15. 3S-N-(Boc-
L
-glutamoyl-
c
-benzylester)-1,2,3,4-
line-3-carboxylic acid methylesters (4s)
tetrahydroisoquinoline-3-carboxylic acid methylester (4o)
Using the general procedure of preparing 4a–t from 2.37 g
(7.04 mmol) of Boc-Glu(OBzl) and 1.19 g (6.23 mmol) of (3S)-
Using the general procedure of preparing 4a–t from 3.25 g
(10.21 mmol) of Boc-Arg(NO2) and 1.0 g (5.24 mmol) of (3S)-
1,2,3,4-tetrahydroisoquinoline-3-carboxylic
acid
methylester
1,2,3,4-tetrahydroisoquinoline-3-carboxylic
acid
methylester
1.24 g (48%) of the title compound was obtained as colorless syr-
1.59 g (50%) of the title compound was obtained as colorless pow-
upy. ESI-MS (m/e) 494 [M+H]+; IR (cmꢀ1) 3411, 2969, 1734, 1648,
ders. ESI-MS (m/e) 511 [M+H]+; IR (cmꢀ1) 3411, 2969, 1734, 1648,
1497, 1457, 1395,1380, 1365, 1198, 1111, 750, 599; ½a D20
¼ ꢀ2:55
ꢂ
1497, 1457, 1395,1380, 1365, 1198, 1111, 750, 599; ½a D20
ꢂ
¼ ꢀ57:6
(c = 1.0, CH3OH); 1H NMR (300 MHz, CDCl3) d/ppm = 8.00 (d,

1546
X. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 1536–1554
J = 7.6 Hz, 2H), 7.02 (m, J = 8.2 Hz, 2H), 6.95 (d, J = 8.8 Hz, 2H), 4.84
(m, 1H), 4.53 (m, 1H), 4.51 (d, J = 7.4 Hz, 1H), 4.41 (d, J = 8.6 Hz,
1H), 3.63 (s, 3H), 3.27 (m, 1H), 3.04 (m, 1H), 2.65 (m, 2H), 2.0 (d,
J = 8.5 Hz, 2H), 1.79 (m, 2H), 1.55 (m, 2H), 1.41 (s, 9H); 13CNMR
(75 MHz, CDCl3) d/ppm = 171.8, 170.1, 158.2, 155.8, 135.9, 134.2,
129.4, 127.2, 126.6, 79.5, 71.7, 55.8, 53.8, 51.2, 44.6, 37.1, 28.4,
27.0, 24.3.
4.4.5. 3S-N-(Boc-L-isoleucinyl)-1,2,3,4-tetrahydroisoquinoline-
3-carboxylic acid (5e)
Using the general procedure of preparing 5a–t from 0.60 g
(1.49 mmol) of 4e 0.52 g (89%) of the title compound was obtained
as colorless powders. ESI-MS (m/e) 391 [M+H]+.
4.4.6. 3S-N-(Boc-L-tryptophanyl)-1,2,3,4-tetrahydroisoquinoline-
3-carboxylic acid (5f)
Using the general procedure of preparing 5a–t from 0.61 g
(1.28 mmol) of 4f 0.56 g (95%) of the title compound was obtained
as colorless syrupy. ESI-MS (m/e) 464 [M+H]+.
4.3.20. 3S-N-(Boc-L-methioninyl)-1,2,3,4-tetrahydroisoquino-
line-3-carboxylic acid methyl-ester (4t)
Using the general procedure of preparing 4a–t from 2.54 g
(10.21 mmol) of Boc-Met and 1.0 g (5.24 mmol) of (3S)-1,2,3,4-tet-
rahydroisoquinoline-3-carboxylic acid methylester 1.77 g (80%) of
the title compound was obtained as colorless syrupy. ESI-MS (m/e)
423 [M+H]+; IR (cmꢀ1) 3411, 2969, 1734, 1648, 1497, 1457, 1395,
4.4.7. 3S-N-(Boc-L-serinyl)-1,2,3,4-tetrahydroisoquinoline-3-
carboxylic acid (5g)
Using the general procedure of preparing 5a–t from 0.97 g
(2.09 mmol) of 4g 0.88 g (94%) of the title compound was obtained
as colorless powders. ESI-MS (m/e) 450 [M+H]+.
1380, 1365, 1198, 1111, 750, 599; ½a D20
¼ ꢀ17:7 (c = 1.0, CH3OH);
ꢂ
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), 4.80 (t, J = 6.4 Hz, 1H),
4.51 (m, 3H), 3.63 (s, 3H), 3.29 (d, J = 5.3 Hz, 1H), 3.05 (d,
J = 7.6 Hz, 1H), 2.44 (m, 2H), 2.16 (t, J = 6.6 Hz, 2H), 2.09 (s, 3H),
1.41 (s, 9H); 13CNMR (75 MHz, CDCl3) d/ppm = 171.6, 170.1,
155.8, 135.9, 134.2, 128.4, 127.2, 126.6, 79.5, 55.8, 52.0, 51.6,
44.6, 31.9, 29.3, 28.4, 27.0, 17.4.
4.4.8. 3S-N-(Boc-L-tyrocinyl)-1,2,3,4-tetrahydroisoquinoline-3-
carboxylic acid (5h)
Using the general procedure of preparing 5a–t from 1.48 g
(3.73 mmol) of 4h 1.33 g (80%) of the title compound was obtained
as colorless syrupy. ESI-MS (m/e) 446 [M+H]+.
4.4.9. 3S-N-(Boc-L-prolinyl)-1,2,3,4-tetrahydroisoquinoline-3-
carboxylic acid (5i)
Using the general procedure of preparing 5a–t from 0.10 g
(0.26 mmol) of 4i 0.05 g (51%) of the title compound was obtained
as colorless powders. ESI-MS (m/e) 375 [M+H]+.
4.4. General procedure of preparing 3S-N-(Boc-aminoacyl)-
1,2,3,4-tetrahydroisoquino-line-3-carboxylic acids (5a–t)
At 0 °C to the solution of 1.0 mmol of 3S-N-(Boc-aminoacyl)-
1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid methylester in
5 ml of methanol 7 ml of aqueous of NaOH (2 N) 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 (3S)-N-
(Boc-aminoacyl)-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid
methylester. The reaction mixture was adjusted to pH 7 with aque-
ous 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 filtration the filtrate was evaporated to provide 5a–t.
4.4.10. 3S-N-(Boc-L-asparaginyl)-1,2,3,4-tetrahydroisoquinoline-
3-carboxylic acid (5j)
Using the general procedure of preparing 5a–t from 0.19 g
(0.47 mmol) of 4j 0.17 g (90%) of the title compound was obtained
as colorless powders. ESI-MS (m/e) 392 [M+H]+.
4.4.11. 3S-N-(Boc-L-glutaminyl)-1,2,3,4-tetrahydroisoquinoline-
3-carboxylic acid (5k)
Using the general procedure of preparing 5a–t from 1.33 g
(3.17 mmol) of 4k 1.26 g (98%) of the title compound was obtained
as colorless powders. ESI-MS (m/e) 406 [M+H]+.
4.4.1. 3S-N-(Boc-L-alaninyl)-1,2,3,4-tetrahydroisoquinoline-3-
carboxylic acid (5a)
4.4.12. (3S)-N-(Boc-
3-carboxylic acid (5l)
L-histidinyl)-1,2,3,4-tetrahydroisoquinoline-
Using the general procedure of preparing 5a–t from 1.50 g
(4.14 mmol) of 4a 1.29 g (89%) of the title compound was obtained
as colorless powders. ESI-MS (m/e) 349 [M+H]+.
Using the general procedure of preparing 5a–t from 0.83 g
(1.93 mmol) of 4l 0.74 g (92%) of the title compound was obtained
as colorless powders. ESI-MS (m/e) 415 [M+H]+.
a
x
4.4.13. 3S-N-(N ,N -diBoc-
L-lysinyl)-1,2,3,4-tetrahydroisoquino-
4.4.2. 3S-N-(Boc-glycinyl)-1,2,3,4-tetrahydroisoquinoline-3-
carboxylic acid (5b)
line-3-carboxylic acid (5m)
Using the general procedure of preparing 5a–t from 2.39 g
(4.60 mmol) of 4m 2.11 g (91%) of the title compound was ob-
tained as colorless powders. ESI-MS (m/e) 506 [M+H]+.
Using the general procedure of preparing 5a–t from 162 mg
(0.46 mmol) of 4b 137 mg (89%) of the title compound was ob-
tained as colorless powders. ESI-MS (m/e) 335 [M+H]+.
4.4.14. 3S-N-(Boc-L-aspartyl)-1,2,3,4-tetrahydroisoquinoline-3-
carboxylic acid (5n)
Using the general procedure of preparing 5a–t from 1.74 g
(3.50 mmol) of 4n 1.32 g (96%) of the title compound was obtained
as colorless powders. ESI-MS (m/e) 393 [M+H]+.
4.4.3. 3S-N-(Boc-L-valinyl)-1,2,3,4-tetrahydroisoquinoline-3-
carboxylic acid (5c)
Using the general procedure of preparing 5a–t from 0.445 g
(1.14 mmol) of 4c 0.357 g (83%) of the title compound was ob-
tained as colorless powders. ESI-MS (m/e) 377 [M+H]+.
4.4.15. 3S-N-(Boc-L-glutamoyl)-1,2,3,4-tetrahydroisoquinoline-
4.4.4. 3S-N-(Boc-L-leucinyl)-1,2,3,4-tetrahydroisoquinoline-3-
3-carboxylic acid (5o)
carboxylic acid (5d)
Using the general procedure of preparing 5a–t from 0.79 g
(1.54 mmol) of 4o 0.52 g (83%) of the title compound was obtained
as colorless powders. ESI-MS (m/e) 407 [M+H]+.
Using the general procedure of preparing 5a–t from 0.24 g
(0.59 mmol) of 4d 0.22 g (96%) of the title compound was obtained
as colorless syrupy. ESI-MS (m/e) 391 [M+H]+.

X. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 1536–1554
1547
4.4.16. 3S-N-[Boc-
hydroisoquinoline-3-carboxylic acid (5p)
L
-(S-p-methylbenzylcycteinyl)]-1,2,3,4-tetra-
29.7, 28.4, 28.3, 19.2, 18.3. Anal. Calcd for C28H35N3O6: C, 65.99;
H, 6.92; N, 8.25. Found: C, 65.76; H, 6.78; N, 8.00.
Using the general procedure of preparing 5a–t from 1.00 g
(2.01 mmol) of 4p 0.91 g (93%) of the title compound was obtained
as colorless powders. ESI-MS (m/e) 485 [M+H]+.
4.5.2. N-(3S-N-Boc-glycinyl-1,2,3,4-tetrahydroisoquinoline-3-
carbonyl)glycine benzyl-ester (6b)
Using the general procedure of preparing 6a–t from 108 mg
(0.39 mmol) of 5b and 75 mg (0.46 mmol) of Gly-oBzl 116 mg
(62%) of the title compound was obtained as colorless powders.
4.4.17. 3S-N-(Boc-L-phenylalaninyl)-1,2,3,4-tetrahydroisoquino-
line-3-carboxylic acid (5q)
Using the general procedure of preparing 5a–t from 0.33 g
(0.76 mmol) of 4q 0.31 g (96%) of the title compound was obtained
as colorless powders. ESI-MS (m/e) 425 [M+H]+.
Mp 97–98 °C, ESI-MS (m/e) 482 [M+H]+. ½a D20
¼ ꢀ20:0 (c = 1.2,
ꢂ
CH3OH); 1H NMR (300 MHz, DMSO-d6) d/ppm = 8.40 (d,
J = 4.5 Hz, 2H), 7.20 (m, 5H), 7.00 (m, 4H), 5.54 (s, 2H), 4.66 (m,
3H), 4.20 (d, J = 4.5 Hz, 2H), 3.82 (d, J = 4.5 Hz, 2H), 3.18 (dd,
J = 6.0 Hz, 1H), 2.91 (dd, J = 6.0 Hz, 1H), 1.78 (s, 9H). 13C NMR
(75 MHz, DMSO-d6): d/ppm = 172.5, 170.6, 164.7, 156.2, 135.0,
133.6, 133.2, 128.7, 128.4, 127.6, 127.3, 126.0, 80.2, 67.6, 55.6,
46.3, 40.6, 31.5, 28.4, 24.3. Anal. Calcd for C26H31N3O6: C, 64.85;
H, 6.49; N, 8.73. Found: C, 64.62; H, 6.31; N, 8.49.
4.4.18. 3S-N-(Boc-L-threoninyl)-1,2,3,4-tetrahydroisoquinoline-
3-carboxylic acid (5r)
Using the general procedure of preparing 5a–t from 0.78 g
(2.00 mmol) of 4r 0.70 g (93%) of the title compound was obtained
as colorless powders. ESI-MS (m/e) 379 [M+H]+.
4.4.19. 3S-N-(Boc-L
-NOG2 -argininyl)-1,2,3,4-tetrahydroisoquino-
line-3-carboxylic acid (5s)
Using the general procedure of preparing 5a–t from 1.15 g
(2.33 mmol) of 4s 0.95 g (85%) of the title compound was obtained
as colorless powders. ESI-MS (m/e) 480 [M+H]+.
4.5.3. N-(3S-N-Boc-
L-valinyl-1,2,3,4-tetrahydroisoquinoline-3-
carbonyl)- -valine benzylester (6c)
L
Using the general procedure of preparing 6a–t from 1.38 g
(2.86 mmol) of 5c and 0.77 g (3.72 mmol) of Val-oBzl 1.22 g
(74%) of the title compound was obtained as colorless powders.
Mp 85–87 °C, ESI-MS (m/e) 566 [M+H]+. ½a D20
¼ ꢀ49:3 (c = 1.1,
ꢂ
4.4.20. 3S-N-(Boc-L-methioninyl)-1,2,3,4-tetrahydroisoquinoline-
3-carboxylic acid (5t)
Using the general procedure of preparing 5a–t from 1.15 g
(2.72 mmol) of 4t 1.03 g (93%) of the title compound was obtained
as colorless powders. ESI-MS (m/e) 409 [M+H]+.
CH3OH); 1H NMR (300 MHz, DMSO-d6): d/ppm = 8.14 (d,
J = 9.8 Hz, 2H), 7.17 (m, J = 9.0 Hz, 9H), 5.15 (s, 2H), 4.91 (t,
J = 8.2 Hz, 1H), 4.46 (m, 4H), 3.18 (dd, J = 6.0 Hz, 1H), 2.91 (dd,
J = 6.0 Hz, 1H), 3.09 (m, 1H), 2.68 (m, 1H), 1.39 (s, 9H), 0.89 (d,
13
J = 3.6 Hz, 12H).
C NMR (75 MHz, DMSO-d6): d/ppm = 171.9,
171.6, 169.1, 155.6, 140.1, 136.9, 134.5, 129.9, 127.8, 127.5,
127.0, 125.9, 80.1, 68.5, 62.0, 57.8, 55.0, 43.8, 31.2, 30.0, 28.2,
27.5, 17.4, 17.0. Anal. Calcd for C32H43N3O6: C, 67.94; H, 7.66; N,
7.43. Found: C, 67.70; H, 7.51; N, 7.66.
4.5. General procedure of preparing N-(3S-N-Boc-aminoacyl-
1,2,3,4-tetrahydroisoquinoline-3-carbonyl)amino acid
benzylesters (6a–t)
At 0 °C and with stirring to the solution of 1.0 mmol of 3S-N-
(Boc-aminoacyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid
(5a–t) in 30 ml of anhydrous THF 0.14 g (1.0 mmol) of HOBt was
added to form reaction mixture A. The solution of 0.23 g
(0.91 mmol) of AA-OBzl in 5 ml of anhydrous THF was adjusted
pH 9 with triethylamine and stirred for 30 min to form mixture
B. At 0 °C the mixture A and B were mixed and then 0.26 g
(0.12 mmol) of DCC was added. The reaction mixture was stirred
at 0 °C for 2 h, at room temperature for12 h and TLC (ethyl ace-
tate/petroleum ether, 1:2) indicated the complete disappearance
of 5a–t. The formed precipitates of DCU were 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 satu-
rated aqueous solution of NaCl (30 ml ꢁ 3) and dried with anhy-
drous Na2SO4. After filtration the filtrate was evaporated under
vacuum to give 6a–t.
4.5.4. N-(3S-N-Boc-
L-leucinyl-1,2,3,4-tetrahydroisoquinoline-3-
carbonyl)- -leucine benzylester (6d)
L
Using the general procedure of preparing 6a–t from 1.10 g
(2.86 mmol) of 5d and 0.82 g (3.72 mmol) of Leu-OBzl 1.54 g
(92%) of the title compound was obtained as colorless powders.
Mp 50–52 °C, ESI-MS (m/e) 594 [M+H]+. ½a D20
¼ ꢀ189:7 (c = 1.2,
ꢂ
CH3OH); 1H NMR (300 MHz, DMSO-d6): d/ppm = 8.14 (d,
J = 9.8 Hz, 2H), 7.17 (m, J = 9.0 Hz, 9H), 5.35 (s, 2H), 4.91 (t,
J = 8.2 Hz, 1H), 4.46 (m, 4H), 3.18 (dd, J = 6.0 Hz, 1H), 2.91 (dd,
J = 6.0 Hz, 1H), 1.86 (m, 2H), 1.83 (m, 2H), 1.70 (m, 2H), 1.39 (s,
9H), 0.89 (d, J = 3.6 Hz, 12H).
ppm = 171.9, 171.6, 169.1, 155.6, 140.1, 136.9, 134.5, 129.9,
127.8, 127.5, 127.0, 125.9, 79.1, 68.0, 62.1, 55.0, 43.8, 41.2, 40.0,
28.2, 27.5, 22.9, 22.0. Anal. Calcd for C34H47N3O6: C, 68.78; H,
7.98; N, 7.08. Found: C, 69.01; H, 8.15; N, 7.30.
13
C NMR (75 MHz, DMSO-d6): d/
4.5.5. N-(3S-N-Boc-L-isoleucinyl-1,2,3,4-tetrahydroisoquinoline-
3-carbonyl)- -isoleucine benzylester (6e)
L
Using the general procedure of preparing 6a–t from 1.42 g
(3.65 mmol) of 5e and 1.05 g (4.74 mmol) of Ile-OBzl 1.20 g (56%)
of the title compound was obtained as colorless powders. Mp
4.5.1. N-(3S-N-Boc-
L-alaninyl-1,2,3,4-tetrahydroisoquinoline-3-
carbonyl)- -alanine benzylester (6a)
L
Using the general procedure of preparing 6a–t from 0.505 g
(1.45 mmol) of 5a and 0.303 g (1.87 mmol) of Ala-oBzl 0.40 g
(54%) of the title compound was obtained as colorless powders.
63–67 °C, ESI-MS (m/e) 594 [M+H]+. ½a D20
¼ ꢀ75:2 (c = 1.2, CH3OH);
ꢂ
1H NMR (300 MHz, DMSO-d6): d/ppm = 8.14 (d, J = 9.8 Hz, 2H), 7.17
(m, J = 9.0 Hz, 9H), 5.35 (s, 2H), 4.91 (t, J = 8.2 Hz, 1H), 4.46 (m, 4H),
3.18 (dd, J = 6.0 Hz, 1H), 2.91 (dd, J = 6.0 Hz, 1H), 2.51 (m, 2H), 1.39
(s, 9H), 1.28 (m, 4H), 1.08 (d, 6H), 1.01 (t, J = 8.4 Hz, 6H). 13 C NMR
(75 MHz, DMSO-d6): d/ppm = 171.8, 171.4, 169.0, 155.5, 140.2,
136.7, 134.6, 129.8, 127.7, 127.2, 126.8, 125.8, 79.4, 68.1, 62.5,
55.0, 53.1, 43.8, 37.2, 36.0, 28.2, 27.5, 24.9, 14.0, 10.2. Anal. Calcd
for C34H47N3O6: C, 68.78; H, 7.98; N, 7.08. Found: C, 68.57; H,
7.81; N, 7.32.
Mp 100–103 °C, ESI-MS (m/e) 510 [M+H]+. ½a D20
¼ ꢀ25:5 (c = 1.1,
ꢂ
CH3OH); 1H NMR (300 MHz, DMSO-d6) d/ppm = 8.26 (d,
J = 8.7 Hz, 2H), 7.20 (m, 5H), 7.00 (m, 4H), 5.53 (s, 2H), 4.97 (t,
J = 5.7 Hz, 1H), 4.73 (d, J = 6.3 Hz, 2H), 4.61 (m, 2H), 3.32 (d,
J = 5.5 Hz, 1H), 3.12 (d, J = 5.5 Hz, 1H), 1.99 (d, J = 7.5 Hz, 6H),
1.45 (s, 9H). 13C NMR (75 MHz, DMSO-d6) d/ppm = 173.2, 172.8,
171.3, 145.1, 133.8, 133.1, 128.3, 127.9, 127.7, 127.6, 126.9,
126.2, 125.9, 81.4, 81.3, 56.4, 52.4, 47.8, 47.6, 45.2, 32.1, 30.7,

1548
X. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 1536–1554
4.5.6. N-(3S-N-Boc-
line-3-carbonyl)-L-tryptophan benzylester (6f)
L
-tryptophanyl-1,2,3,4-tetrahydroisoquino-
28.9, 24.5, 23.8, 21.8, 21.1. Anal. Calcd for C32H39N3O6: C, 68.43;
H, 7.00; N, 7.48. Found: C, 68.21; H, 6.82; N, 7.23.
Using the general procedure of preparing 6a–t from 0.61 g
(1.33 mmol) of 5f and 0.51 g (1.72 mmol) of Trp-OBzl 0.84 g
(85%) of the title compound was obtained as colorless powders.
4.5.10. N-(3S-N-Boc-
L-asparaginyl-1,2,3,4-tetrahydroisoquino-
line-3-carbonyl)- -asparagine benzylester (6j)
L
Mp 154–157 °C, ESI-MS (m/e) 740 [M+H]+. ½a D20
ꢂ
¼ ꢀ177:4 (c = 1.2,
Using the general procedure of preparing 6a–t from 102 mg
(0.26 mmol) of 5j and 73 mg (0.33 mmol) of Asn-OBzl 102 mg
(66%) of the title compound was obtained as colorless powders.
CH3OH); 1H NMR (300 MHz, DMSO-d6): d/ppm = 10.92 (d,
J = 15.1 Hz, 2H), 8.52 (d, J = 7.5 Hz, 1H), 8.29 (d, J = 7.0 Hz, 1H),
7.29 (m, 13H), 7.19 (d, J = 7.8 Hz, 1H), 7.11 (d, J = 7.8 Hz, 1H),
7.06 (t, J = 7.5 Hz, 1H), 7.02 (t, J =7.4 Hz, 1H), 6.86 (s, 2H), 5.33 (s,
2H), 5.03 (t, J = 6.1 Hz, 1H), 5.00 (m, J = 8.6 Hz, 2H), 4.56 (s, 2H),
3.18 (dd, J = 6.0 Hz, 1H), 3.03 (m, 2H), 2.95 (m, 2H), 2.91 (dd,
J = 6.0 Hz, 1H), 1.40 (s, 9H). 13C NMR (75 MHz, DMSO-d6): d/
ppm = 172.7, 171.7, 155.1, 140.0, 136.8, 136.6, 136.4, 134.1,
129.6, 127.8, 127.6, 126.9, 125.8, 122.4, 120.4, 119.6, 111.9,
109.7, 79.0, 70.0, 68.4, 62.7, 54.3, 53.7, 44.4, 32.2, 30.1, 28.6,
27.5. Anal. Calcd for C44H45N5O6: C, 71.43; H, 6.13; N, 9.47. Found:
C, 71.62; H, 6.30; N, 9.71.
Mp 105–111 °C, ESI-MS (m/e) 596 [M+H]+. ½a D20
¼ ꢀ120:5 (c = 1.1,
ꢂ
CH3OH); 1H NMR (300 MHz, DMSO-d6): d/ppm = 8.19 (d, J = 8 Hz,
2H), 7.21 (m, 5H), 7.19 (d, J = 7.8 Hz, 1H), 7.11 (d, J = 7.8 Hz, 1H),
7.06 (t, J = 7.5 Hz, 1H), 7.02 (t, J = 7.4 Hz, 1H), 5.98 (s, 4H), 5.37 (s,
2H), 4.91 (t, J = 8.2 Hz, 1H), 4.46 (m, 4H), 3.18 (dd, J = 6.0 Hz, 1H),
2.91 (dd, J = 6.0 Hz, 1H), 2.83 (m, 4H), 1.37 (s, 9H). 13C NMR
(75 MHz, DMSO-d6): d/ppm = 174.8, 171.3, 170.4, 169.6, 156.3,
140.9, 136.7, 134.3, 129.1, 127.9, 127.8, 127.7, 127.0, 125.7, 80.1,
68.1, 63.9, 50.2, 48.1, 43.4, 40.2, 39.7, 28.5, 26.8. Anal. Calcd for
C30H37N5O8: C, 60.49; H, 6.26; N, 11.76. Found: C, 60.70; H, 6.42;
N, 11.51.
4.5.7. N-(3S-N-Boc-L-serinyl-1,2,3,4-tetrahydroisoquinoline-3-
carbonyl)- -serine benzylester (6g)
L
4.5.11. N-(3S-N-Boc-L-glutaminyl-1,2,3,4-tetrahydroisoquino-
Using the general procedure of preparing 6a–t from 99 mg
(0.22 mmol) of 5g and 57 mg (0.29 mmol) of Ser-OBzl 99 mg
(85%) of the title compound was obtained as colorless powders.
line-3-carbonyl)- -glutamine benzylester (6k)
L
Using the general procedure of preparing 6a–t from 709 mg
(1.75 mmol) of 5k and 236 mg (2.25 mmol) of Gln-OBzl 502 mg
(46%) of the title compound was obtained as colorless powders.
Mp 112–114 °C, ESI-MS (m/e) 542 [M+H]+. ½a D20
¼ ꢀ30:8 (c = 1.2,
ꢂ
CH3OH); 1H NMR (300 MHz, DMSO-d6): d/ppm = 8.30 (d,
J = 2.5 Hz, 2H), 7.24 (m, 5H), 7.19 (d, J = 7.8 Hz, 1H), 7.11 (d,
J = 7.8 Hz, 1H), 7.06 (t, J = 7.5 Hz, 1H), 7.02 (t, J =7.4 Hz, 1H), 5.30
(s, 2H), 4.76 (m, 5H), 4.13 (d, J = 8.8 Hz, 2H), 4.03 (d, J = 8.8 Hz,
2H), 3.18 (dd, J = 6.0 Hz, 1H), 2.91 (dd, J = 6.0 Hz, 1H), 1.40 (s,
9H). 13C NMR (75 MHz, DMSO-d6): d/ppm = 172.0, 171.8, 170.6,
159.5, 138.2, 136.8, 134.2, 128.6, 127.5, 127.4, 127.0, 126.2, 79.0,
66.3, 61.2, 58.2, 54.2, 43.1, 28.2, 27.2. Anal. Calcd for C28H35N3O8:
C, 62.09; H, 6.51; N, 7.76. Found: C, 62.29; H, 6.69; N, 8.00.
Mp 65–68 °C, ESI-MS (m/e) 624 [M+H]+. ½a D20
¼ ꢀ4:9 (c = 1.0,
ꢂ
CH3OH); 1H NMR (300 MHz, DMSO-d6): d/ppm = 8.19 (d,
J = 8.0 Hz, 2H), 7.21 (m, 5H), 7.19 (d, J = 7.8 Hz, 1H), 7.11 (d,
J = 7.8 Hz, 1H), 7.06 (t, J = 7.5 Hz, 1H), 7.02 (t, J = 7.4 Hz, 1H), 5.96
(s, 4H), 5.37 (s, 2H), 4.91 (t, J = 8.2 Hz, 1H), 4.46 (m, 4H), 3.18
(dd, J = 6.0 Hz, 1H), 2.91 (dd, J = 6.0 Hz, 1H), 2.23 (t, J = 8.2 Hz,
4H), 2.08 (m, 4H), 1.37 (s, 9H). 13C NMR (75 MHz, DMSO-d6): d/
ppm = 173.8, 171.3, 170.4, 169.6, 156.3, 140.9, 136.7, 134.3,
129.1, 127.9, 127.8, 127.7, 127.0, 125.7, 80.1, 68.1, 63.9, 50.2,
48.1, 43.4, 30.4, 28.5, 27.1, 26.8. Anal. Calcd for C32H41N5O8: C,
61.62; H, 6.63; N, 11.23. Found: C, 61.40; H, 6.47; N, 11.00.
4.5.8. N-(3S-N-Boc-L-tyrocinyl-1,2,3,4-tetrahydroisoquinoline-
3-carbonyl)- -tyrosine benzylester (6h)
L
Using the general procedure of preparing 6a–t from 0.61 g
(1.36 mmol) of 5h and 0.48 g (1.77 mmol) of Tyr-OBzl 0.86 g
(91%) of the title compound was obtained as colorless powders.
4.5.12. N-(3S-N-Boc-
L-histidinyl-1,2,3,4-tetrahydroisoquinoline-
3-carbonyl)- -histidine benzylester (6l)
L
Using the general procedure of preparing 6a–t from 1.94 g
(4.69 mmol) of 5l and 1.49 g (6.09 mmol) of His-OBzl 2.91 g
(85%) of the title compound was obtained as colorless powders.
Mp 141–143 °C, ESI-MS (m/e) 694 [M+H]+. ½a D20
¼ ꢀ20:9 (c = 1.2,
ꢂ
CH3OH); 1H NMR (300 MHz, DMSO-d6):d/ppm = 8.14 (d,
J = 7.8 Hz, 2H), 7.21 (m, 5H), 7.19 (d, J = 7.8 Hz, 1H), 7.11 (d,
J = 7.8 Hz, 1H), 7.06 (t, J = 7.5 Hz, 1H), 7.02 (t, J = 7.4 Hz, 1H), 6.91
(d, J = 8.8 Hz, 4H), 6.80 (d, J = 8.1 Hz, 4H), 5.30 (s, 2H), 4.91 (t,
J = 8.2 Hz, 1H), 4.46 (m, 4H), 3.06 (m, 6H), 1.44 (s, 9H). 13C NMR
(75 MHz, DMSO-d6): d/ppm = 172.0, 171.8, 170.6, 155.5, 138.2,
136.8, 134.2, 132.1, 128.6, 127.5, 127.4, 127.0, 126.2, 115.8, 79.0,
66.3, 61.2, 53.2, 52.1, 43.1, 37.8, 37.0, 28.2, 27.2. Anal. Calcd for
C40H43N3O8: C, 69.25; H, 6.25; N, 6.06. Found: C, 69.02; H, 6.09;
N, 6.29.
Mp 204–207 °C, ESI-MS (m/e) 642 [M+H]+. ½a D20
¼ ꢀ3:0 (c = 1.2,
ꢂ
CH3OH); 1H NMR (300 MHz, DMSO-d6): d/ppm = 13.5 (s, 2H),
8.19 (d, J = 8.0 Hz, 2H), 7.50 (s, 2H), 7.21 (m, 5H), 7.19 (d,
J = 7.8 Hz, 1H), 7.11 (d, J = 7.8 Hz, 1H), 7.06 (t, J = 7.5 Hz, 1H), 7.02
(t, J = 7.4 Hz, 1H), 6.85 (s, 2H), 5.37 (s, 2H), 4.91 (t, J = 8.2 Hz, 1H),
4.46 (m, 4H), 3.05 (m, 6H), 1.37 (s, 9H). 13C NMR (75 MHz,
DMSO-d6): d/ppm = 171.8, 170.2, 156.7, 141.3, 136.7,135.6, 134.3,
129.1, 127.9, 127.8, 127.7, 127.0, 125.7, 120.1, 80.1, 68.1, 63.9,
53.2, 51.1, 43.4, 30.4, 29.8, 28.5, 27.1. Anal. Calcd for C34H39N7O6:
C, 63.64; H, 6.13; N, 15.28. Found: C, 63.65; H, 6.31; N, 15.52.
4.5.9. N-(3S-N-Boc-
L-prolinyl-1,2,3,4-tetrahydroisoquinoline-3-
a
x
carbonyl)- -proline benzylester (6i)
L
4.5.13. N-(3S-N-N ,N -diBoc-
L-lysinyl-1,2,3,4-tetrahydroisoquin-
Using the general procedure of preparing 6a–t from 0.50 g
(1.34 mmol) of 5i and 0.35 g (1.70 mmol) of Pro-OBzl 0.50 g
(67%) of the title compound was obtained as colorless powders.
oline-3-carbonyl)- -lysine benzylester (6m)
L
Using the general procedure of preparing 6a–t from 793 mg
(1.57 mmol) of 5m and 685 mg (2.04 mmol) of Lys(Boc)-OBzl
1163 mg (90%) of the title compound was obtained as colorless
Mp 75–78 °C, ESI-MS (m/e) 562 [M+H]+. ½a D20
¼ ꢀ100:2 (c = 1.2,
ꢂ
CH3OH); 1H NMR (300 MHz, DMSO-d6): d/ppm = 7.21 (m, 5H),
7.19 (d, J = 7.8 Hz, 1H), 7.11 (d, J = 7.8 Hz, 1H), 7.06 (t, J = 7.5 Hz,
1H), 7.02 (t, J =7.4 Hz, 1H), 5.35 (s, 2H), 4.91 (t, J = 8.2 Hz, 1H),
4.46 (m, 4H), 3.46 (m, 4H), 3.18 (dd, J = 6.0 Hz, 1H), 2.91 (dd,
J = 6.0 Hz, 1H), 1.97 (m, 4H), 1.59 (m, 4H), 1.45 (s, 9H). 13C NMR
(75 MHz, DMSO-d6): d/ppm = 173.1, 171.8, 171.0, 155.6, 136.7,
135.7, 135.1, 128.8, 128.1, 127.8, 127.7, 127.5, 127.3, 127.1,
125.5, 80.0, 71.9, 61.0, 60.7, 60.2, 46.8, 44.7, 43.8, 32.4, 29.1, 29.0,
powders. Mp 45–46 °C, ESI-MS (m/e) 824 [M+H]+. ½a D20
¼ ꢀ27:2
ꢂ
(c = 1.3, CH3OH); 1H NMR (300 MHz, DMSO-d6): d/ppm = 8.19 (d,
J = 8.0 Hz, 4H), 7.21 (m, 5H), 7.19 (d, J = 7.8 Hz, 1H), 7.11 (d,
J = 7.8 Hz, 1H), 7.06 (t, J = 7.5 Hz, 1H), 7.02 (t, J =7.4 Hz, 1H), 5.37
(s, 2H), 4.91 (t, J = 8.2 Hz, 1H), 4.46 (m, 4H), 3.18 (dd, J = 6.0 Hz,
1H), 2.91 (dd, J = 6.0 Hz, 1H), 2.81 (t, J = 7.8 Hz, 4H), 1.77 (m, 8H),
1.37 (s, 27H), 1.20 (m, 4H). 13C NMR (75 MHz, DMSO-d6): d/
ppm = 171.8, 171.2, 170.2, 156.7, 141.3, 136.7, 134.3, 129.1,

X. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 1536–1554
1549
127.9, 127.8, 127.7, 127.0, 125.7, 80.1, 68.1, 63.9, 53.2, 43.4, 41.1,
31.7, 31.0, 29.8, 28.5, 27.1, 20.6. Anal. Calcd for C44H65N5O10: C,
64.13; H, 7.95; N, 8.50. Found: C, 64.32; H, 8.10; N, 8.74.
4H), 2.91 (dd, J = 6.0 Hz, 1H), 1.37 (s, 9H). 13C NMR (75 MHz,
DMSO-d6): d/ppm = 171.8, 171.3, 170.4, 156.3, 140.9, 136.7,
134.3, 129.1, 128.7, 127.9, 127.8, 127.7, 127.0, 126.0, 125.7, 80.1,
68.1, 63.9, 50.2, 48.1, 43.4, 37.8, 30.4, 27.1, 26.8. Anal. Calcd for
C40H43N3O6: C, 72.60; H, 6.55; N, 6.35. Found: C, 72.59; H, 6.40;
N, 6.11.
4.5.14. N-(3S-N-Boc-L-aspartyl-1,2,3,4-tetrahydroisoquinoline-
3-carbonyl)- -aspartic acid dibenzylester (6n)
L
Using the general procedure of preparing 6a–t from 2.08 g
(5.30 mmol) of 5n and 2.18 g (6.89 mmol) of Asp(OBzl)-OBzl
3.32 g (91%) of the title compound was obtained as colorless pow-
4.5.18. N-(3S-N-Boc-
L-threoninyl-1,2,3,4-tetrahydroisoquinoline-
3-carbonyl)- -threonine benzylester (6r)
L
ders. Mp 68–70 °C, ESI-MS (m/e) 778 [M+H]+. ½a D20
¼ ꢀ21:3 (c = 1.3,
ꢂ
Using the general procedure of preparing 6a–t from 1.21 g
(3.21 mmol) of 5r and 0.80 g (4.17 mmol) of Thr-OBzl 1.76 g
(96%) of the title compound was obtained as colorless powders.
CH3OH); 1H NMR (300 MHz, DMSO-d6): d/ppm = 8.19 (d, J = 8 Hz,
2H), 7.21 (m, 15H), 7.19 (d, J = 7.8 Hz, 1H), 7.11 (d, J = 7.8 Hz, 1H),
7.06 (t, J = 7.5 Hz, 1H), 7.02 (t, J =7.4 Hz, 1H), 5.37 (s, 6H), 4.91 (t,
J = 8.2 Hz, 1H), 4.46 (m, 4H), 3.18 (dd, J = 6.0 Hz, 1H), 2.91 (dd,
J = 6.0 Hz, 1H), 2.83 (m, 4H), 1.37 (s, 9H). 13C NMR (75 MHz,
DMSO-d6): d/ppm = 177.8, 171.3, 170.4, 169.6, 156.3, 140.9,
136.7, 134.3, 129.1, 127.9, 127.8, 127.7, 127.0, 125.7, 80.1, 68.1,
63.9, 50.2, 48.1, 43.4, 40.2, 39.7, 28.5, 26.8. Anal. Calcd for
C44H47N3O10: C, 67.94; H, 6.09; N, 5.40. Found: C, 67.72; H, 5.91;
N, 5.63.
Mp 136–138 °C, ESI-MS (m/e) 570 [M+H]+. ½a D20
¼ ꢀ5:5 (c = 1.0,
ꢂ
CH3OH); 1H NMR (300 MHz, DMSO-d6): d/ppm = 8.30 (d,
J = 2.5 Hz, 2H), 7.24 (m, 5H), 7.19 (d, J = 7.8 Hz, 1H), 7.11 (d,
J = 7.8 Hz, 1H), 7.06 (t, J = 7.5 Hz, 1H), 7.02 (t, J = 7.4 Hz, 1H), 5.30
(s, 2H), 4.76 (m, 5H), 4.30 (m, 2H), 3.18 (dd, J = 6.0 Hz, 1H), 2.91
(dd, J = 6.0 Hz, 1H), 2.11 (s, 2H), 1.40 (s, 9H), 1.21 (d, 6H). 13C
NMR (75 MHz, DMSO-d6): d/ppm = 172.0, 171.8, 170.6, 159.5,
138.2, 136.8, 134.2, 128.6, 127.5, 127.4, 127.0, 126.2, 79.0, 66.3,
61.2, 58.2, 54.2, 43.1, 28.2, 27.2, 18.9. Anal. Calcd for C30H39N3O8:
C, 63.25; H, 6.90; N, 7.38. Found: C, 63.44; H, 7.07; N, 7.14.
4.5.15. N-(3S-N-Boc-L-glutamoyl-1,2,3,4-tetrahydroisoquinoline-
3-carbonyl)- -glutamic acid dibenzylester (6o)
L
Using the general procedure of preparing 6a–t from 0.47 g
(1.15 mmol) of 5o and 0.49 g (1.49 mmol) of Glu(OBzl)-OBzl
0.76 g (92%) of the title compound was obtained as colorless pow-
4.5.19. N-(3S-N-(Boc-
line-3-carbonyl)-
-NOG2 -arginine benzylester (6s)
L
-NOG2 -argininyl-1,2,3,4-tetrahydroisoquino-
L
Using the general procedure of preparing 6a–t from 0.48 g
(1.00 mmol) of 5s and 0.29 g (1.30 mmol) of Arg(NO2)-OBzl
0.69 g (89%) of the title compound was obtained as colorless pow-
ders. Mp 66–67 °C, ESI-MS (m/e) 806 [M+H]+. ½a D20
¼ ꢀ30:8 (c = 1.3,
ꢂ
CH3OH); 1H NMR (300 MHz, DMSO-d6): d/ppm = 8.19 (d, J = 8.0 Hz,
2H), 7.21 (m, 15H), 7.19 (d, J = 7.8 Hz, 1H), 7.11 (d, J = 7.8 Hz, 1H),
7.06 (t, J = 7.5 Hz, 1H), 7.02 (t, J = 7.4 Hz, 1H), 5.37 (s, 6H), 4.91 (t,
J = 8.2 Hz, 1H), 4.46 (m, 4H), 3.18 (dd, J = 6.0 Hz, 1H), 2.91 (dd,
J = 6.0 Hz, 1H), 2.23 (t, J = 8.2 Hz, 4H), 2.08 (m, 4H), 1.37 (s, 9H).
13C NMR (75 MHz, DMSO-d6): d/ppm = 177.8, 171.3, 170.4, 169.6,
156.3, 140.9, 136.7, 134.3, 129.1, 127.9, 127.8, 127.7, 127.0,
125.7, 80.1, 68.1, 63.9, 50.2, 48.1, 43.4, 30.4, 28.5, 27.1, 26.8. Anal.
Calcd for C46H51N3O10: C, 68.55; H, 6.38; N, 5.21. Found: C, 68.33;
H, 6.21; N, 5.00.
ders. Mp 109–110 °C, ESI-MS (m/e) 772 [M+H]+. ½a D20
¼ ꢀ19:6
ꢂ
(c = 1.2, CH3OH); 1H NMR (300 MHz, DMSO-d6): d/ppm = 8.19 (d,
J = 8.0 Hz, 2H), 7.21 (m, 5H), 7.19 (d, J = 7.8 Hz, 1H), 7.11 (d,
J = 7.8 Hz, 1H), 7.06 (t, J = 7.5 Hz, 1H), 7.02 (t, J = 7.4 Hz, 1H), 5.37
(s, 2H), 4.91 (t, J = 8.2 Hz, 1H), 4.46 (m, 4H), 3.18 (dd, J = 6.0 Hz,
1H), 2.91 (dd, J = 6.0 Hz, 1H), 2.61 (t, J = 7.8 Hz, 4H), 2.02 (s, 4H),
1.77 (m, 4H), 1.54 (m, 4H), 1.37 (s, 9H). 13C NMR (75 MHz,
DMSO-d6): d/ppm = 171.8, 171.2, 170.2, 158.1, 156.7, 141.3,
136.7, 134.3, 129.1, 127.9, 127.8, 127.7, 127.0, 125.7, 80.1, 68.1,
63.9, 53.2, 43.4, 41.1, 37.2, 31.7, 31.0, 29.8, 28.5, 27.1, 24.6. Anal.
Calcd for C34H49N11O10: C, 60.07; H, 7.27; N, 18.54. Found: C,
60.28; H, 7.45; N, 18.79.
4.5.16. [3S-N-Boc-L-(S-p-methylbenzylcysteinyl)-1,2,3,4-tetrahy-
droisoquinoline-3-carbonyl]-L-S-p-methylbenzylcysteine benzy-
lester (6p)
Using the general procedure of preparing 6a–t from 0.60 g
(1.24 mmol) of 5p and 0.51 g (1.62 mmol) of Cys(S-CH2C6H4-Me-
p)-OBzl 0.75 g (83%) of the title compound was obtained as
colorless powders. Mp 70–74 °C, ESI-MS (m/e) 782 [M+H]+.
4.5.20. N-(3S-N-Boc-
L-methioninyl-1,2,3,4-tetrahydroisoquino-
line-3-carbonyl)- -methionine benzylester (6t)
L
Using the general procedure of preparing 6a–t from 0.96 g
(2.36 mmol) of 5t and 0.62 g (2.60 mmol) of Met-OBzl 0.71 g
(48%) of the title compound was obtained as colorless powders.
½
a 2D0
ꢂ
¼ ꢀ52:2 (c = 1.2, CH3OH); 1H NMR (300 MHZ, DMSO-d6):d/
ppm = 8.19 (d, J = 8.0 Hz, 2H), 7.21 (m, 5H), 7.19 (d, J = 7.8 Hz,
1H), 7.11 (d, J = 7.8 Hz, 1H), 7.06 (t, J = 7.5 Hz, 1H), 7.02 (t, J
=7.4 Hz, 1H), 6.94 (d, J = 8.6 Hz, 4H), 6.85 (d, J = 8.6 Hz, 4H), 5.37
(s, 2H), 4.91 (t, J = 8.2 Hz, 1H), 4.46 (m, 4H), 3.18 (dd, J = 6.0 Hz,
1H), 2.91 (dd, J = 6.0 Hz, 1H), 2.41 (t, J = 7.8 Hz, 4H), 2.22 (m, 4H),
2.10 (s, 6H), 1.37 (s, 9H). 13C NMR (75 MHz, DMSO-d6): d/
ppm = 171.8, 171.2, 170.2, 158.1, 156.7, 141.3, 136.7, 134.3,
129.1, 127.9, 127.8, 127.7, 127.0, 125.7, 80.1, 68.1, 63.9, 53.2,
43.4, 31.7, 31.0, 29.8, 28.5, 27.1, 17.6. Anal. Calcd for C44H51N3O6S2:
C, 67.58; H, 6.57; N, 5.37. Found: C, 67.80; H, 6.73; N, 5.60.
Mp 77–80 °C, ESI-MS (m/e) 630 [M+H]+. ½a D20
¼ ꢀ30:9 (c = 1.2,
ꢂ
CH3OH); 1H NMR (300 MHz, DMSO-d6): d/ppm = 8.19 (d,
J = 8.0 Hz, 2H), 7.21 (m, 5H), 7.19 (d, J = 7.8 Hz, 1H), 7.11 (d,
J = 7.8 Hz, 1H), 7.06 (t, J = 7.5 Hz, 1H), 7.02 (t, J = 7.4 Hz, 1H), 5.37
(s, 2H), 4.91 (t, J = 8.2 Hz, 1H), 4.46 (m, 4H), 3.18 (dd, J = 6.0 Hz,
1H), 2.91 (dd, J = 6.0 Hz, 1H), 2.41 (t, J = 7.8 Hz, 4H), 2.22 (m, 4H),
2.10 (s, 6H), 1.37 (s, 9H). 13C NMR (75 MHz, DMSO-d6): d/
ppm = 171.8, 171.2, 170.2, 158.1, 156.7, 141.3, 136.7, 134.3,
129.1, 127.9, 127.8, 127.7, 127.0, 125.7, 80.1, 68.1, 63.9, 53.2,
43.4, 31.7, 31.0, 29.8, 28.5, 27.1, 17.6. Anal. Calcd for C32H43N3O6S2:
C, 61.02; H, 6.88; N, 6.67. Found: C, 61.23; H, 7.07; N, 6.91.
4.5.17. N-(3S-N-Boc-L-phenylalaninyl-1,2,3,4-tetrahydroisoquin-
oline-3-carbonyl)- -phenylalanine benzylester (6q)
L
4.6. General procedure of preparing N-(3S-N-Boc-aminoacyl-
1,2,3,4-tetrahydroisoquinoline-3-carbonyl)amino acids (7a–t)
Using the general procedure of preparing 6a–t from 0.23 g
(0.55 mmol) of 5q and 0.17 g (0.71 mmol) of Phe-OBzl 0.34 g
(94%) of the title compound was obtained as colorless powders.
Mp 78–81 °C, ESI-MS (m/e) 662 [M+H]+. ½a D20
¼ ꢀ14:4 (c = 1.1,
ꢂ
To the suspension of 0.50 mmol of 6a–t, 15 ml of methanol,
4 ml of water, 20 mg of Pd/C (10%) hydrogen gas was bubbled for
20 h and TLC indicates complete disappear of 6a–t. After filtration
the filtrate was evaporated under vacuum and the residue was
CH3OH); 1H NMR (300 MHz, DMSO-d6):d/ppm = 8.19 (d,
J = 8.0 Hz, 2H), 7.21 (m, 13H), 7.10 (m, 6H), 5.37 (s, 2H), 4.91 (t,
J = 8.2 Hz, 1H), 4.46 (m, 4H), 3.18 (dd, J = 6.0 Hz, 1H), 3.14 (m,

1550
X. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 1536–1554
triturated with ether repeatedly to provide the title compound as
colorless powder.
4.6.10. N-(3S-N-Boc-
L-asparaginyl-1,2,3,4-tetrahydroisoquinoline-
3-carbonyl)- -asparagine (7j)
L
Using the general procedure of preparing 7a–t from 0.13 g
(0.21 mmol) of 6j 0.10 g (91%) of the title compound was obtained
as colorless powders. Mp 300–303 °C, ESI-MS (m/e) 504 [MꢀH]ꢀ.
4.6.1. N-(3S-N-Boc-
L-alaninyl-1,2,3,4-tetrahydroisoquinoline-3-
carbonyl)- -alanine (7a)
L
Using the general procedure of preparing 7a–t from 0.37 g
(0.73 mmol) of 6a 0.29 g (95%) of the title compound was obtained
as colorless powders. Mp 155–157 °C, ESI-MS (m/e) 418 [MꢀH]ꢀ.
½
a 2D0
ꢂ
¼ ꢀ15:7 (c = 1.1, CH3OH).
4.6.11. N-(3S-N-Boc-L-glutaminyl-1,2,3,4-tetrahydroisoquinoline-
½
a 2D0
ꢂ
¼ ꢀ2:1 (c = 1.2, CH3OH).
3-carbonyl)- -glutamine (7k)
L
Using the general procedure of preparing 7a–t from 0.50 g
(0.80 mmol) of 6k 0.40 g (93%) of the title compound was obtained
as colorless powders. Mp 191–195 °C, ESI-MS (m/e) 532 [MꢀH]ꢀ.
4.6.2. N-(3S-N-Boc-glycinyl-1,2,3,4-tetrahydroisoquinoline-3-
carbonyl)glycine (7b)
Using the general procedure of preparing 7a–t from 0.11 g
(0.23 mmol) of 6b 0.086 g (89%) of the title compound was ob-
tained as colorless powders. Mp 142–144 °C, ESI-MS (m/e) 390
½
a 2D0
ꢂ
¼ ꢀ11:3 (c = 1.2, CH3OH).
[MꢀH]ꢀ. ½a 2D0
ꢂ
¼ ꢀ10:2 (c = 1.1, CH3OH).
4.6.12. N-(3S-N-Boc-L-histidinyl-1,2,3,4-tetrahydroisoquinoline-
3-carbonyl)- -histidine (7l)
L
Using the general procedure of preparing 7a–t from 0.61 g
(0.83 mmol) of 6l 0.41 g (89%) of the title compound was obtained
as colorless powders. Mp 279–280 °C, ESI-MS (m/e) 550 [MꢀH]ꢀ.
4.6.3. N-(3S-N-Boc-
L
-valinyl-1,2,3,4-tetrahydroisoquinoline-3-
carbonyl)- -valine (7c)
L
Using the general procedure of preparing 7a–t from 0.66 g
(1.14 mmol) of 7c 0.45 g (83%) of the title compound was obtained
as colorless powders. Mp 100–103 °C, ESI-MS (m/e) 474 [MꢀH]ꢀ.
½
a 2D0
ꢂ
¼ ꢀ5:4 (c = 1.0, CH3OH).
a
x
½
a 2D0
ꢂ
¼ ꢀ16:2 (c = 1.3, CH3OH).
4.6.13. N-(3S-N-N ,N -diBoc-
L-lysinyl-1,2,3,4-tetrahydroisoquino-
line-3-carbonyl)- -lysine (7m)
L
Using the general procedure of preparing 7a–t from 0.84 g
(1.02 mmol) of 6m 0.70 g (93%) of the title compound was ob-
tained as colorless powders. Mp 152–154 °C, ESI-MS (m/e) 732
4.6.4. N-(3S-N-Boc-
L
-leucinyl-1,2,3,4-tetrahydroisoquinoline-3-
carbonyl)- -leucine (7d)
L
Using the general procedure of preparing 7a–t from 0.50 g
(0.84 mmol) of 6d 0.40 g (94%) of the title compound was obtained
as colorless powders. Mp 143–147 °C, ESI-MS (m/e) 502 [MꢀH]ꢀ.
[MꢀH]ꢀ. ½a 2D0
¼ ꢀ20:7 (c = 1.3, CH3OH).
ꢂ
½
a 2D0
ꢂ
¼ ꢀ59:2 (c = 1.1, CH3OH).
4.6.14. N-(3S-N-Boc-L-aspartyl-1,2,3,4-tetrahydroisoquinoline-
3-carbonyl)- -aspartic acid (7n)
L
4.6.5. N-(3S-N-Boc-
L
-leucinyl-1,2,3,4-tetrahydroisoquinoline-3-
Using the general procedure of preparing 7a–t from 0.26 g
(0.38 mmol) of 6n 0.70 g (96%) of the title compound was obtained
as colorless powders. Mp 167–168 °C, ESI-MS (m/e) 506 [MꢀH]ꢀ.
carbonyl)- -isoleucine (7e)
L
Using the general procedure of preparing 7a–t from 0.50 g
(0.84 mmol) of 6e 0.41 g (96%) of the title compound was obtained
as colorless powders. Mp 77–78 °C, ESI-MS (m/e) 502 [MꢀH]ꢀ.
½
a 2D0
ꢂ
¼ ꢀ12:1 (c = 1.2, CH3OH).
½
a 2D0
ꢂ
¼ ꢀ59:8 (c = 1.2, CH3OH).
4.6.15. N-(3S-N-Boc-L-glutamoyl-1,2,3,4-tetrahydroisoquinoline-
3-carbonyl)- -glutamic acid (7o)
L
4.6.6. N-(3S-N-Boc-L-tryptophanyl-1,2,3,4-tetrahydroisoquino-
Using the general procedure of preparing 7a–t from 0.26 g
(0.37 mmol) of 6o 0.18 g (90%) of the title compound was obtained
as colorless powders. Mp 150–152 °C, ESI-MS (m/e) 534 [MꢀH]ꢀ.
line-3-carbonyl)- -trypto-phan (7f)
L
Using the general procedure of preparing 7a–t from 0.72 g
(0.97 mmol) of 6f 0.59 g (93%) of the title compound was obtained
as colorless powders. Mp 213–215 °C, ESI-MS (m/e) 648 [MꢀH]ꢀ.
½
a 2D0
ꢂ
¼ ꢀ56:2 (c = 1.1, CH3OH).
½
a 2D0
ꢂ
¼ ꢀ21:2 (c = 1.1, CH3OH).
4.6.16. N-(3S-Boc-L-cysteinyl-1,2,3,4-tetrahydroisoquinoline-3-
carbonyl)- -cysteine (7p)
L
4.6.7. N-(3S-N-Boc-L-serinyl-1,2,3,4-tetrahydroisoquinoline-3-
Using the general procedure of preparing 7a–t from 0.39 g
(0.50 mmol) of 6p 0.22 g (91%) of the title compound was obtained
as colorless powders. Mp 70–74 °C, ESI-MS (m/e) 482 [MꢀH]ꢀ.
carbonyl)- -serine (7g)
L
Using the general procedure of preparing 7a–t from 0.52 g
(0.97 mmol) of 6g 0.41 g (93%) of the title compound was obtained
as colorless powders. Mp 213–215 °C, ESI-MS (m/e) 450 [MꢀH]ꢀ.
½
a 2D0
ꢂ
¼ ꢀ52:2 (c = 1.2, CH3OH).
½
a 2D0
ꢂ
¼ ꢀ21:2 (c = 1.1, CH3OH).
4.6.17. N-(3S-N-Boc-
L-phenylalaninyl-1,2,3,4-tetrahydroisoquin-
4.6.8. N-(3S-N-Boc-
L
-tyrocinyl-1,2,3,4-tetrahydroisoquinoline-
oline-3-carbonyl)- -phenylalanine (7q)
L
3-carbonyl)- -tyrosine (7h)
L
Using the general procedure of preparing 7a–t from 0.60 g
(0.91 mmol) of 6q 0.49 g (92%) of the title compound was obtained
as colorless powders. Mp 113–114 °C, ESI-MS (m/e) 570 [MꢀH]ꢀ.
Using the general procedure of preparing 7a–t from 0.70 g
(1.01 mmol) of 6h 0.59 g (97%) of the title compound was obtained
as colorless powders. Mp 251–252 °C, ESI-MS (m/e) 602 [MꢀH]ꢀ.
½
a 2D0
ꢂ
¼ ꢀ19:8 (c = 1.1, CH3OH).
½
a 2D0
ꢂ
¼ ꢀ6:2 (c = 1.1, CH3OH).
4.6.18. N-(3S-N-Boc-
L-threoninyl-1,2,3,4-tetrahydroisoquinoline-
4.6.9. N-(3S-N-Boc-
L
-prolinyl-1,2,3,4-tetrahydroisoquinoline-3-
3-carbonyl)- -threonine (7r)
L
carbonyl)- -proline (7i)
L
Using the general procedure of preparing 7a–t from 0.47 g
(0.83 mmol) of 6r 0.39 g (98%) of the title compound was obtained
as colorless powders. Mp 98–99 °C, ESI-MS (m/e) 478 [MꢀH]ꢀ.
Using the general procedure of preparing 7a–t from 0.56 g
(0.71 mmol) of 6i 0.31 g (94%) of the title compound was obtained
as colorless powders. Mp 172–175 °C, ESI-MS (m/e) 470 [MꢀH]ꢀ.
a 2D0
ꢂ
¼ ꢀ2:5 (c = 1.1, CH3OH).
½
a 2D0
ꢂ
¼ ꢀ71:3 (c = 1.1, CH3OH).
½

X. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 1536–1554
1551
4.6.19. N-(3S-N-Boc-
L
-argininyl-1,2,3,4-tetrahydroisoquinoline-
for C20H29N3O4: C, 63.98; H, 7.79; N, 11.19. Found: C, 64.17; H,
7.99; N, 11.00.
3-carbonyl)- -arginine (7s)
L
Using the general procedure of preparing 7a–t from 0.30 g
(0.39 mmol) of 6s 0.22 g (95%) of the title compound was obtained
as colorless powders. Mp 215–216 °C, ESI-MS (m/e) 588 [MꢀH]ꢀ.
4.7.4. N-(3S-N-
L-Leucinyl-1,2,3,4-tetrahydroisoquinoline-3-
carbonyl)- -leucine (8d)
L
½
a 2D0
ꢂ
¼ ꢀ15:4 (c = 1.1, CH3OH).
Using the general procedure of preparing 7a–t from 0.20 g
(0.40 mmol) of 6d 0.15 g (94%) of the title compound was obtained
as colorless powders. Mp 381–383 °C, ESI-MS (m/e) 404 [M+H]+.
4.6.20. N-(3S-N-Boc-L-methioninyl-1,2,3,4-tetrahydroisoquinoline-
3-carbonyl)- -methionine (7t)
L
½
a 2D0
ꢂ
¼ ꢀ18:9 (c = 1.1, H2O). 1H NMR (300 MHz, DMSO-d6): d/
Using the general procedure of preparing 7a–t from 0.40 g
(0.64 mmol) of 6t 0.33 g (94%) of the title compound was obtained
as colorless powders. Mp 201–204 °C, ESI-MS (m/e) 538 [MꢀH]ꢀ.
ppm = 12.09 (s, 1H), 8.26 (d, J = 8.7 Hz, 1H), 7.00 (m, 4H), 4.97 (t,
J = 5.7 Hz, 1H), 4.46 (m, 3H), 3.56 (d, J = 8.6 Hz, 1H), 3.18 (dd,
J = 6.0 Hz, 1H), 2.91 (dd, J = 6.0 Hz, 1H), 1.99 (s, 2H), 1.83 (m, 2H),
1.74 (t, J = 8.0 Hz, 4H), 0.98 (d, J = 7.8 Hz, 12H). 13C NMR (75 MHz,
DMSO-d6) d/ppm = 174.2, 172.3, 169.5, 136.8, 133.1, 128.3, 127.7,
126.9, 125.9, 62.4, 53.1, 48.8, 44.0, 43.6, 40.6, 28.4, 22.8. Anal. Calcd
for C22H33N3O4: C, 65.48; H, 8.24; N, 10.41. Found: C, 65.26; H,
8.07; N, 10.66.
½
a 2D0
ꢂ
¼ ꢀ28:4 (c = 1.1, CH3OH).
4.7. General procedure of preparing N-(3S-N-aminoacyl-1,2,3,4-
tetrahydroisoquinoline-3-carbonyl)amino acids (8a–t)
To the solution of 1 mmol of 7a–t in 5 ml of ethyl acetate, 10 ml
of 4 N hydrogen chloride/ethyl acetate solution was added at 0 °C.
The reaction mixture was stirred at room temperature for 4 h, and
TLC (CCl3/CH3OH, 5:1) indicated the complete disappearance of
7a–t. 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-dis-
solved in 5 ml of ethyl acetate. This procedure was repeated for
three times to provide 8a–t.
4.7.5. N-(3S-N-
L-isoleucine-1,2,3,4-tetrahydroisoquinoline-3-
carbonyl)- -isoleucine (8e)
L
Using the general procedure of preparing 8a–t from 0.20 g
(0.40 mmol) of 7e 0.14 g (87%) of the title compound was obtained
as colorless powders. Mp 390–392 °C, ESI-MS (m/e) 404 [M+H]+.
½
a 2D0
ꢂ
¼ ꢀ68:8 (c = 1.1, H2O). 1H NMR (300 MHz, DMSO-d6): d/
ppm = 12.06 (s, 1H), 8.26 (d, J = 8.7 Hz, 1H), 7.00 (m, 4H), 4.97 (t,
J = 5.7 Hz, 1H), 4.46 (m, 3H), 3.56 (d, J = 8.6 Hz, 1H), 3.18 (dd,
J = 6.0 Hz, 1H), 2.91 (dd, J = 6.0 Hz, 1H), 2.61 (m, 1H), 2.12 (m,
1H), 1.99 (s, 2H), 1.30 (m, 4H), 1.02 (m, 12H). 13C NMR (75 MHz,
DMSO-d6) d/ppm = 174.2, 172.3, 169.5, 136.8, 133.1, 128.3, 127.7,
126.9, 125.9, 62.4, 58.8, 53.1, 44.0, 40.6, 36.6, 28.4, 24.6, 14.4,
10.2. Anal. Calcd for C22H33N3O4: C, 65.48; H, 8.24; N, 10.41. Found:
C, 65.27; H, 8.08; N, 10.64.
4.7.1. N-(3S-N-
L-Alaninyl-1,2,3,4-tetrahydroisoquinoline-3-
carbonyl)- -alanine (8a)
L
Using the general procedure of preparing 8a–t from 0.21 g
(0.50 mmol) of 7a 0.15 g (95%) of the title compound was obtained
as colorless powders. Mp 387–388 °C, ESI-MS (m/e) 320 [M+H]+.
½
a 2D0
ꢂ
¼ ꢀ61:1 (c = 1.1, H2O). 1H NMR (300 MHz, DMSO-d6): d/
ppm = 10.50 (s, 1H), 8.26 (d, J = 8.7 Hz, 1H), 7.00 (m, 4H), 4.97 (t,
J = 5.7 Hz, 1H), 4.53 (m, 3H), 3.61 (m, 1H), 3.18 (dd, J = 6.0 Hz, 1H),
2.91 (dd, J = 6.0 Hz, 1H), 1.99 (s, 2H), 1.44 (d, J = 8.8 Hz, 3H), 1.28
(d, J = 8.8 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) d/ppm = 174.2,
171.3, 170.1, 136.8, 133.1, 128.3, 127.7, 127.6, 126.9, 125.9, 52.4,
47.8, 45.2, 28.4, 19.2, 18.3. Anal. Calcd for C16H21N3O4: C, 60.17; H,
6.63; N, 13.16. Found: C, 60.36; H, 6.78; N, 13.41.
4.7.6. N-(3S-N-L-Tryptophanyl-1,2,3,4-tetrahydroisoquinoline-
3-carbonyl)- -tryptophan (8f)
L
Using the general procedure of preparing 8a–t from 0.25 g
(0.39 mmol) of 7f 0.20 g (93%) of the title compound was obtained as
colorless powders. Mp 392–394 °C, ESI-MS (m/e) 550 [M+H]+.
½
a 2D0
ꢂ
¼ ꢀ45:4 (c = 1.2, H2O). 1H NMR (300 MHz, DMSO-d6): d/
ppm = 10.92 (s, 1H), 10.62 (s, 2H), 8.52 (d, J = 7.5 Hz, 1H), 7.29 (m,
8H), 7.00 (m, 4H), 6.86 (s, 2H), 5.03 (t, J = 6.1 Hz, 1H), 5.00 (m,
J = 8.6 Hz, 2H), 4.56 (s, 2H), 3.18 (dd, J = 6.0 Hz, 1H), 3.03 (m, 2H),
2.95 (m, 2H), 2.91 (dd, J = 6.0 Hz, 1H), 1.96 (s, 2H). 13C NMR (75 MHz,
DMSO-d6): d/ppm = 174.7, 171.7, 170.2, 136.8, 136.6, 136.4, 134.1,
129.6, 127.8, 126.9, 125.8, 122.4, 120.4, 119.6, 111.9, 109.7, 62.7,
54.3, 56.7, 52.7, 44.4, 34.1, 30.6, 27.5. Anal. Calcd for C32H31N5O4: C,
69.93; H, 5.69; N, 12.74. Found: C, 69.70; H, 5.51; N, 12.98.
4.7.2. N-(3S-N-Glycinyl-1,2,3,4-tetrahydroisoquinoline-3-
carbonyl)glycine (8b)
Using the general procedure of preparing 8a–t from 70 mg
(0.18 mmol) of 7b 49 mg (89%) of the title compound was obtained
as colorless powders. Mp 380–381 °C, ESI-MS (m/e) 292 [M+H]+.
½
a 2D0
ꢂ
¼ ꢀ12:9 (c = 1.1, H2O). 1H NMR (300 MHz, DMSO-d6): d/
ppm = 11.09 (s, 1H), 8.26 (d, J = 8.7 Hz, 1H), 7.00 (m, 4H), 4.97 (t,
J = 5.7 Hz, 1H), 4.46 (m, 2H), 4.16 (s, 2H), 3.55 (s, 2H), 3.18 (dd,
J = 6.0 Hz, 1H), 2.91 (dd, J = 6.0 Hz, 1H), 1.99 (s, 2H). 13C NMR
(75 MHz, DMSO-d6) d/ppm = 173.2, 172.3, 165.1, 136.8, 133.1, 128.3,
127.7, 126.9, 125.9, 62.4, 44.8, 42.2, 28.4. Anal. Calcd for C14H17N3O4:
C, 57.72; H, 5.88; N, 14.42. Found: C, 57.50; H, 5.71; N, 14.65.
4.7.7. N-(3S-N-L-Serinyl-1,2,3,4-tetrahydroisoquinoline-3-
carbonyl)- -serine (8g)
L
Using the general procedure of preparing 8a–t from 0.40 g
(0.89 mmol) of 7g 0.30 g (93%) of the title compound was obtained
as colorless powders. Mp 373–375 °C, ESI-MS (m/e) 352 [M+H]+.
½
a 2D0
ꢂ
¼ ꢀ5:8 (c = 1.1, H2O). 1H NMR (300 MHz, DMSO-d6): d/
4.7.3. N-(3S-N-
L-Valinyl-1,2,3,4-tetrahydroisoquinoline-3-
ppm = 12.80 (s, 1H), 8.30 (d, J = 2.5 Hz, 1H), 7.00 (m, 4H), 4.76
(m, 5H), 4.13 (d, J = 8.8 Hz, 2H), 4.03 (d, J = 8.8 Hz, 2H), 3.18 (dd,
J = 6.0 Hz, 1H), 2.91 (dd, J = 6.0 Hz, 1H), 1.98 (s, 2H). 13C NMR
(75 MHz, DMSO-d6): d/ppm = 174.0, 171.8, 170.6, 136.8, 134.2,
128.6, 127.5, 127.4, 127.0, 66.3, 61.2, 60.9, 58.2, 57.1, 54.2, 43.1,
27.2. Anal. Calcd for C16H21N3O6: C, 54.69; H, 6.02; N, 11.96. Found:
C, 54.90; H, 6.21; N, 12.19.
carbonyl)- -valine (8c)
L
Using the general procedure of preparing 8a–t from 0.20 g
(0.42 mmol) of 7c 0.16 g (98%) of the title compound was obtained
as colorless powders. Mp 112–114 °C, ESI-MS (m/e) 376 [M+H]+.
½
a 2D0
ꢂ
¼ ꢀ32:1 (c = 1.1, H2O). 1H NMR (300 MHz, DMSO-d6): d/
ppm = 12.09 (s, 1H), 8.26 (d, J = 8.7 Hz, 1H), 7.00 (m, 4H), 4.97 (t,
J = 5.7 Hz, 1H), 4.46 (m, 3H), 3.56 (d, J = 8.6 Hz, 1H), 3.18 (dd,
J = 6.0 Hz, 1H), 2.91 (dd, J = 6.0 Hz, 1H), 2.86 (m, 1H), 2.30 (m,
1H), 1.99 (s, 2H), 0.98 (d, J = 7.8 Hz, 12H). 13C NMR (75 MHz,
DMSO-d6) d/ppm = 174.2, 172.3, 169.5, 136.8, 133.1, 128.3, 127.7,
126.9, 125.9, 62.4, 58.6, 57.1, 44.8, 34.1, 30.2, 28.4, 17.5. Anal. Calcd
4.7.8. N-(3S-N-L-Tyrocinyl-1,2,3,4-tetrahydroisoquinoline-3-
carbonyl)- -tyrosine (8h)
L
Using the general procedure of preparing 8a–t from 0.60 g
(1.00 mmol) of 7h 0.48 g (93%) of the title compound was obtained

1552
X. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 1536–1554
as colorless powders. Mp 375–376 °C, ESI-MS (m/e) 504 [M+H]+.
4.7.13. N-(3S-N-
L-Lysinyl-1,2,3,4-tetrahydroisoquinoline-3-
½
a 2D0
ꢂ
¼ 32:7 (c = 1.1, H2O). 1H NMR (300 MHz, DMSO-d6): d/
carbonyl)- -lysine (8m)
L
ppm = 11.19 (s, 1H), 8.14 (d, J = 7.8 Hz, 1H), 7.02 (m, 4H), 6.91 (d,
J = 8.8 Hz, 4H), 6.80 (d, J = 8.1 Hz, 4H), 4.91 (t, J = 8.2 Hz, 1H), 4.46
(m, 4H), 3.06 (m, 6H), 1.95 (s, 2H). 13C NMR (75 MHz, DMSO-d6):
d/ppm = 174.0, 171.8, 170.6, 155.5, 138.2, 136.8, 134.2, 132.1,
128.6, 127.5, 127.4, 127.0, 126.2, 115.8, 61.2, 55.2, 52.1, 43.1,
40.8, 37.0, 27.2. Anal. Calcd for C28H29N3O6: C, 66.79; H, 5.80; N,
8.34. Found: C, 66.58; H, 5.64; N, 8.57.
Using the general procedure of preparing 8a–t from 0.55 g
(0.75 mmol) of 7m 0.24 g (90%) of the title compound was ob-
tained as colorless powders. Mp 299–303 °C, ESI-MS (m/e) 434
[M+H]+. ½a 2D0
ꢂ
¼ ꢀ5:5 (c = 1.3, H2O). 1H NMR (300 MHz, DMSO-d6):
d/ppm = 11.02 (s, 1H), 8.19 (d, J = 8.0 Hz, 1H), 7.03 (m, 4H), 4.91
(t, J = 8.2 Hz, 1H), 4.46 (m, 4H), 3.18 (dd, J = 6.0 Hz, 1H), 2.91 (dd,
J = 6.0 Hz, 1H), 2.81 (t, J = 7.8 Hz, 4H), 1.77 (m, 8H), 1.97 (s, 6H),
1.20 (m, 4H). 13C NMR (75 MHz, DMSO-d6): d/ppm = 174.8, 171.2,
170.2, 136.7, 134.3, 129.1, 127.9, 127.8, 127.7, 125.7, 63.9, 55.2,
51.4, 43.1, 41.7, 34.0, 32.1, 29.8, 27.1, 20.6. Anal. Calcd for
C22H35N5O4: C, 60.95; H, 8.14; N, 16.15. Found: C, 60.76; H, 8.29;
N, 16.36.
4.7.9. N-(3S-N-
L-Prolinyl-1,2,3,4-tetrahydroisoquinoline-3-
carbonyl)- -proline (8i)
L
Using the general procedure of preparing 8a–t from 0.20 g
(0.42 mmol) of 7i 0.48 g (93%) of the title compound was obtained
as colorless powders. Mp 372–376 °C, ESI-MS (m/e) 372 [M+H]+.
a 2D0
¼ ꢀ60:2 (c = 1.2, H2O). 1H NMR (300 MHz, DMSO-d6): d/
4.7.14. N-(3S-N-L-Aspartyl-1,2,3,4-tetrahydroisoquinoline-3-
½ ꢂ
carbonyl)- -aspartic acid (8n)
L
ppm = 12.08 (s, 1H), 7.01 (m, 4H), 4.91 (t, J = 8.2 Hz, 1H), 4.46 (m,
4H), 3.46 (m, 4H), 3.18 (dd, J = 6.0 Hz, 1H), 2.91 (dd, J = 6.0 Hz,
1H), 2.05 (s, 1H), 1.97 (m, 4H), 1.59 (m, 4H). 13C NMR (75 MHz,
DMSO-d6): d/ppm = 174.1, 173.8, 170.0, 136.7, 134.7, 128.8,
128.1, 127.8, 127.7, 127.5, 125.5, 61.0, 60.7, 58.2, 46.8, 44.7, 43.8,
32.4, 29.1, 28.9, 27.6, 24.5, 22.8. Anal. Calcd for C20H25N3O4: C,
64.67; H, 6.78; N, 11.31. Found: C, 64.44; H, 6.61; N, 11.56.
Using the general procedure of preparing 8a–t from 0.20 g
(0.39 mmol) of 7n 0.15 g (94%) of the title compound was obtained
as colorless powders. Mp 376–377 °C, ESI-MS (m/e) 408 [M+H]+.
½
a 2D0
ꢂ
¼ ꢀ10:3 (c = 1.1, H2O). 1H NMR (300 MHz, DMSO-d6): d/
ppm = 12.01 (s, 1H), 11.20 (s, 2H), 8.19 (d, J = 8 Hz, 1H), 7.02 (m,
4H), 4.91 (t, J = 8.2 Hz, 1H), 4.46 (m, 4H), 3.18 (dd, J = 6.0 Hz, 1H),
2.91 (dd, J = 6.0 Hz, 1H), 2.83 (m, 4H), 1.98 (s, 2H). 13C NMR
(75 MHz, DMSO-d6): d/ppm = 177.8, 174.3, 170.4, 169.6, 136.7,
134.3, 129.1, 127.9, 127.7, 125.7, 63.9, 50.2, 48.1, 47.2, 43.4, 39.7,
26.8. Anal. Calcd for C18H21N3O8: C, 53.07; H, 5.20; N, 10.31. Found:
C, 52.84; H, 5.03; N, 10.55.
4.7.10. N-(3S-N-
L-Asparaginyl-1,2,3,4-tetrahydroisoquinoline-
3-carbonyl)- -asparagine (8j)
L
Using the general procedure of preparing 8a–t from 0.20 g
(0.40 mmol) of 7j 0.15 g (95%) of the title compound was obtained
as colorless powders. Mp 376–377 °C, ESI-MS (m/e) 406 [M+H]+.
4.7.15. N-(3S-N-L-Glutamoyl-1,2,3,4-tetrahydroisoquinoline-3-
½
a 2D0
ꢂ
¼ ꢀ14:9 (c = 1.2, H2O). 1H NMR (300 MHz, DMSO-d6): d/
carbonyl)- -glutamic acid (8o)
L
ppm = 11.28 (s, 1H), 8.19 (d, J = 8 Hz, 1H), 7.02 (m, 4H), 5.98 (s,
4H), 4.91 (t, J = 8.2 Hz, 1H), 4.46 (m, 4H), 3.18 (dd, J = 6.0 Hz, 1H),
2.91 (dd, J = 6.0 Hz, 1H), 2.83 (m, 4H), 1.97 (s, 2H). 13C NMR
(75 MHz, DMSO-d6): d/ppm = 174.8, 173.3, 170.4, 169.6, 136.7,
134.3, 129.1, 127.9, 127.8, 127.7, 125.7, 59.9, 56.2, 48.1, 47.4,
43.2, 39.7, 28.5. Anal. Calcd for C18H23N5O6: C, 53.33; H, 5.72; N,
17.27. Found: C, 53.11; H, 5.54; N, 17.52.
Using the general procedure of preparing 8a–t from 0.15 g
(0.28 mmol) of 7o 0.11 g (92%) of the title compound was obtained
as colorless powders. Mp 291–293 °C, ESI-MS (m/e) 436 [M+H]+.
½
a 2D0
ꢂ
¼ ꢀ40:7 (c = 1.1, H2O). 1H NMR (300 MHz, DMSO-d6): d/
ppm = 11.13 (s, 3H), 8.19 (d, J = 8.0 Hz, 1H), 7.03 (m, 4H), 4.91 (t,
J = 8.2 Hz, 1H), 4.46 (m, 4H), 3.18 (dd, J = 6.0 Hz, 1H), 2.91 (dd,
J = 6.0 Hz, 1H), 2.23 (t, J = 8.2 Hz, 4H), 2.08 (m, 4H), 1.99 (s, 2H).
13C NMR (75 MHz, DMSO-d6): d/ppm = 177.8, 174.3, 171.3, 169.6,
136.7, 134.3, 129.1, 127.9, 127.0, 125.7, 63.9, 54.2, 51.1, 43.4,
30.4, 28.5, 27.1, 26.8. Anal. Calcd for C20H25N3O8: C, 55.17; H,
5.79; N, 9.65. Found: C, 55.38; H, 5.95; N, 9.89.
4.7.11. N-(3S-N-
L-Glutaminyl-1,2,3,4-tetrahydroisoquinoline-3-
carbonyl)- -glutamine (8k)
L
Using the general procedure of preparing 8a–t from 0.30 g
(0.56 mmol) of 7k 0.23 g (96%) of the title compound was obtained
as colorless powders. Mp 375–376 °C, ESI-MS (m/e) 434 [M+H]+.
4.7.16. N-(3S-L-Cysteinyl-1,2,3,4-tetrahydroisoquinoline-3-
½
a 2D0
ꢂ
¼ ꢀ9:7 (c = 1.1, H2O). 1H NMR (300 MHz, DMSO-d6): d/
carbonyl)- -cysteine (8p)
L
ppm = 8.19 (d, J = 8.0 Hz, 1H), 7.02 (m, 4H), 5.96 (s, 4H), 4.91 (t,
J = 8.2 Hz, 1H), 4.46 (m, 4H), 3.18 (dd, J = 6.0 Hz, 1H), 2.91 (dd,
J = 6.0 Hz, 1H), 2.23 (t, J = 8.2 Hz, 4H), 2.08 (m, 4H), 1.97 (s, 2H).
13C NMR (75 MHz, DMSO-d6): d/ppm = 174.8, 173.3, 170.4, 169.6,
136.7, 134.3, 127.9, 127.8, 127.7, 127.0, 125.7, 63.9, 54.2, 50.2,
48.1, 43.4, 32.6, 30.4, 27.1, 26.8. Anal. Calcd for C20H27N5O6: C,
55.42; H, 6.28; N, 16.16. Found: C, 55.61; H, 6.45; N, 16.41.
Using the general procedure of preparing 8a–t from 68 mg
(0.14 mmol) of 7p 49 mg (92%) of the title compound was obtained
as colorless powders. Mp 291–293 °C, ESI-MS (m/e) 384 [M+H]+.
½
a 2D0
ꢂ
¼ ꢀ18:1 (c = 1.1, H2O). 1H NMR (300 MHz, DMSO-d6): d/
ppm = 11.02 (s, 1H), 8.19 (d, J = 8.0 Hz, 1H), 7.05 (m, 4H), 4.91 (t,
J = 8.2 Hz, 1H), 4.46 (m, 4H), 3.18 (dd, J = 6.0 Hz, 1H), 2.91 (dd,
J = 6.0 Hz, 1H), 2.41 (t, J = 7.8 Hz, 4H), 1.96 (s, 2H). 13C NMR
(75 MHz, DMSO-d6): d/ppm = 174.8, 171.2, 170.2, 136.7, 134.3,
129.1, 127.9, 127.8, 127.0, 125.7, 63.9, 57.8, 53.2, 43.4, 31.0, 28.5,
27.1. Anal. Calcd for C16H21N3O4S2: C, 50.11; H, 5.52; N, 10.96.
Found: C, 50.33; H, 5.69; N, 10.71.
4.7.12. N-(3S-N-
L-Histidinyl-1,2,3,4-tetrahydroisoquinoline-3-
carbonyl)- -histidine (8l)
L
Using the general procedure of preparing 8a–t from 0.35 g
(0.64 mmol) of 7l 0.24 g (85%) of the title compound was obtained
as colorless powders. Mp 395–397 °C, ESI-MS (m/e) 452 [M+H]+.
4.7.17. N-(3S-N-L-Phenylalaninyl-1,2,3,4-tetrahydroisoquinoline-
½
a 2D0
ꢂ
¼ ꢀ6:6 (c = 1.2, H2O). 1H NMR (300 MHz, DMSO-d6): d/
3-carbonyl)- -phenylala-nine (8q)
L
ppm = 13.50 (s, 2H), 11.05 (s, 1H), 8.19 (d, J = 8.0 Hz, 1H), 7.50 (s,
2H), 7.03 (m, 4H), 6.85 (s, 2H), 4.91 (t, J = 8.2 Hz, 1H), 4.46 (m,
4H), 3.05 (m, 6H), 1.97 (s, 2H). 13C NMR (75 MHz, DMSO-d6): d/
ppm = 174.9, 171.8, 170.2, 136.7, 135.6, 134.3, 129.1, 127.9,
127.7, 125.7, 120.1, 63.9, 55.2, 51.1, 43.4, 33.4, 29.8, 27.1. Anal.
Calcd for C22H25N7O4: C, 58.53; H, 5.58; N, 21.72. Found: C,
58.72; H, 5.74; N, 21.95.
Using the general procedure of preparing 8a–t from 0.41 g
(0.70 mmol) of 7q 0.31 g (95%) of the title compound was obtained
as colorless powders. Mp 291–293 °C, ESI-MS (m/e) 472 [M+H]+.
½
a 2D0
ꢂ
¼ ꢀ34:5 (c = 1.2, H2O). 1H NMR (300 MHz, DMSO-d6): d/
ppm = 12.07 (s, 1H), 8.19 (d, J = 8.0 Hz, 1H), 7.10 (m, 14H), 4.91
(t, J = 8.2 Hz, 1H), 4.46 (m, 4H), 3.18 (dd, J = 6.0 Hz, 1H), 3.14 (m,
4H), 2.91 (dd, J = 6.0 Hz, 1H), 2.01 (s, 2H). 13C NMR (75 MHz,

X. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 1536–1554
1553
DMSO-d6): d/ppm = 174.8, 171.3, 170.4, 140.9, 136.7, 134.3, 129.1,
128.7, 127.9, 127.8, 127.0, 126.0, 125.7, 63.9, 55.1, 51.2, 48.1, 43.4,
40.4, 37.1, 26.8. Anal. Calcd for C28H29N3O4: C, 71.32; H, 6.20; N,
8.91. Found: C, 71.51; H, 6.38; N, 8.70.
centration 0.1 U/ml) was added and aggregation was measured at
37 °C for 5 min. The effects of 6a–t (at a series of concentrations
ranging from 10 lM to 10 nM) on PAF or ADP or AA or TH induced
platelet aggregation were observed. All these anti-platelet aggrega-
tion tests in sixplicate tubers were carried out. The maximal rate of
platelet aggregation (Am%) was represented by the peak height of
aggregation curve. The inhibition rate was calculated by % Inhibi-
tion = [(Am% of NS) ꢀ (Am% of 8a–t)] ꢃ (Am% of NS), where Am% of
NS = 50.16 3.65%. The concentration versus inhibition rate curve
is plotted to determine the IC50 values via GWBASIC.EXE program.
4.7.18. N-(3S-N-
L-Threoninyl-1,2,3,4-tetrahydroisoquinoline-3-
carbonyl)- -threonine (8r)
L
Using the general procedure of preparing 8a–t from 0.40 g
(0.84 mmol) of 7r 0.31 g (96%) of the title compound was obtained
as colorless powders. Mp 329–330 °C, ESI-MS (m/e) 380 [M+H]+.
½
a 2D0
ꢂ
¼ ꢀ12:9 (c = 1.2, H2O). 1H NMR (300 MHz, DMSO-d6): d/
ppm = 12.05 (s, 1H), 8.30 (d, J = 2.5 Hz, 1H), 7.04 (m, 4H), 4.76
(m, 5H), 4.30 (m, 2H), 3.18 (dd, J = 6.0 Hz, 1H), 2.91 (dd,
J = 6.0 Hz, 1H), 2.11 (s, 2H), 1.98 (s, 2H), 1.21 (d, 6H). 13C NMR
(75 MHz, DMSO-d6): d/ppm = 175.0, 171.8, 170.6, 136.8, 134.2,
128.6, 127.5, 127.0, 126.2, 70.0, 66.3, 63.2, 62.8, 61.2, 43.1, 27.2,
18.9. Anal. Calcd for C18H25N3O6: C, 56.98; H, 6.64; N, 11.08. Found:
C, 56.77; H, 6.50; N, 10.84.
4.9. In vivo anti-thrombotic assay of intravenously injection of
8a–t in rat model
The assessments described here were performed based on a
protocol reviewed and approved by the ethics committee of Cap-
ital 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 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
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 polyethylene
tube heparin sodium was injected as anticoagulant, then NS or
8a–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.
4.7.19. N-(3S-N-
L-Argininyl-1,2,3,4-tetrahydroisoquinoline-3-
carbonyl)- -arginine (8s)
L
Using the general procedure of preparing 8a–t from 0.25 g
(0.43 mmol) of 7s 0.19 g (90%) of the title compound was obtained
as colorless powders. Mp 391–393 °C, ESI-MS (m/e) 490 [M+H]+.
½
a 2D0
ꢂ
¼ ꢀ4:5 (c = 1.2, H2O). 1H NMR (300 MHz, DMSO-d6): d/
ppm = 12.09 (s, 1H), 8.19 (d, J = 8.0 Hz, 1H), 7.02 (m, 4H), 4.91 (t,
J = 8.2 Hz, 1H), 4.46 (m, 4H), 3.18 (dd, J = 6.0 Hz, 1H), 2.91 (dd,
J = 6.0 Hz, 1H), 2.61 (t, J = 7.8 Hz, 4H), 2.02 (s, 4H), 1.77 (m, 4H),
1.54 (m, 4H), 1.98 (s, 2H). 13C NMR (75 MHz, DMSO-d6): d/
ppm = 174.8, 171.2, 170.2, 158.1, 136.7, 134.3, 129.1, 127.9,
127.8, 127.0, 125.7, 63.9, 54.2, 51.8, 43.4, 37.2, 31.7, 28.2, 27.1,
24.6. Anal. Calcd for C22H35N9O4: C, 53.97; H, 7.21; N, 25.75. Found:
C, 53.75; H, 7.03; N, 25.51.
4.7.20. N-(3S-N-
L-Methioninyl-1,2,3,4-tetrahydroisoquinoline-
3-carbonyl)- -methionine (8t)
L
4.10. In vivo anti-thrombotic assay of orally administration of
6s in rat model
Using the general procedure of preparing 8a–t from 0.40 g
(0.74 mmol) of 7s 0.30 g (91%) of the title compound was obtained
as colorless powders. Mp 391–393 °C, ESI-MS (m/e) 440 [M+H]+.
Three doses (5, 1 and 0.2
lmol/kg) of 6s in NS or NS (0.6 ml)
½
a 2D0
ꢂ
¼ ꢀ34:6 (c = 1.2, H2O). 1H NMR (300 MHz, DMSO-d6): d/
alone were fed to Male Wistar rats orally. Then the rats were
anesthetized with pentobarbital sodium (80.0 mg/kg, ip). Thirty
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.
ppm = 11.13 (s, 1H), 8.19 (d, J = 8.0 Hz, 1H), 7.02 (m, 4H), 4.91 (t,
J = 8.2 Hz, 1H), 4.46 (m, 4H), 3.18 (dd, J = 6.0 Hz, 1H), 2.91 (dd,
J = 6.0 Hz, 1H), 2.41 (t, J = 7.8 Hz, 4H), 2.22 (m, 4H), 2.10 (s, 6H),
1.99 (s, 2H). 13C NMR (75 MHz, DMSO-d6): d/ppm = 174.8, 171.2,
170.2, 136.7, 134.3, 129.1, 127.9, 127.8, 127.0, 125.7, 63.9, 53.2,
50.3, 43.4, 34.7, 31.0, 29.8, 27.1, 17.6. Anal. Calcd for C20H29N3O4S2:
C, 54.64; H, 6.65; N, 9.56. Found: C, 54.83; H, 6.82; N, 9.79.
4.8. 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. After collection, the pig blood was centrifuged
at 1000g for 10 min and the platelet rich plasma (PRP) was re-
moved. 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-
Acknowledgments
This work was finished in the Beijing Area Major Laboratory of
Peptide and Small Molecular Drugs, supported by PHR (IHLB,
KZ200910025010), Special Project (2008ZX09401–002) and the
National Natural Science Foundation of China (30672513 and
20772082).
etry testing tuber, 0.5 ml of the adjusted plasma sample and 5
NS or 5 l of the solution of 8a–t (in a series of final concentrations
of 100, 10, 1, 0.1, 0.01 and 0.001 M) was added. After adjustment
of 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 aden-
osine diphosphate in NS (ADP, final concentration 10 M) or 5 l of
the solution of arachidonic acid in NS (AA, final concentration
350 M), or 50 l of the solution of thrombin in NS (TH, final con-
ll of
l
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