A new class of anti-thrombosis hexahydropyrazino-[1′,2′:1,6]pyrido-[3,4-b]-indole-1,4-dions: Design, synthesis, log K determination, and QSAR analysis

Article abstract of DOI:10.1016/j.bmc.2007.06.012

Based on the fact that the cyclization of N-[(3S)]-1,2,3,4-tetrahydro-β-carboline-3-carboxyl]-l-lysine in both of acetic acid aqueous (5%) and rat plasma gave the same product and the hypothesis that the cyclization product is antithrombotic active, we report the synthesis, in vitro anti-aggregation, and in vivo anti-thrombosis activity of 20 hexahydropyrazino[1′,2′:1,6]pyrido[3,4-b]indole-1,4-dions (5a-t) as potential anti-thrombosis agents in this study. Two intermediates (tetrahydro-β-carboline-3-carboxy-l-amino acid benzylesters, 2-aminoacyltetrahydro-β-carboline-3-carboxylic acid benzylesters) were prepared and used for the cyclization to form 5a-t. Coupling hydrochloric acid salt of tetrahydro-β-carboline-3-carboxylic acid esters and Boc-amino acids in the reported literature usually generates very low yield products accompanied by racemization. However, in our case, the free base of tetrahydro-β-carboline-3-carboxylic acid benzylester produced the desired products in high yields and without racemization. The anti-thrombosis results from both in vitro and in vivo studies revealed that 5a-t may be a new class of anti-thrombosis agents with potent effective concentration at 0.5 μmol/kg with oral administration. Moreover, a QSAR analysis was performed on these 20 compounds by using molecular descriptors generated by e-dragon server. Although the activities of these compounds are weakly correlated with the log P values, the current QSAR analysis revealed that the anti-thrombotic activity of these compounds can be explained by their steric and electrostatic effects.

Full text of DOI:10.1016/j.bmc.2007.06.012

Bioorganic & Medicinal Chemistry 15 (2007) 5672–5693
A new class of anti-thrombosis hexahydropyrazino-
[10,20:1,6]pyrido-[3,4-b]-indole-1,4-dions: Design, synthesis,
logK determination, and QSAR analysis
Jiawang Liu,a Guofeng Wu,a Guohui Cui,b Wei-Xuan Wang,c Ming Zhao,b,*
Chao Wang,a Ziding Zhangc,* and Shiqi Pengb,*
aCollege of Pharmaceutical Sciences, Peking University, Beijing 100083, PR China
bCollege of Pharmaceutical Sciences, Capital Medical University, Beijing 100069, PR China
cCollege of Biological Sciences, China Agricultural University, Beijing 100094, PR China
Received 26 April 2007; revised 4 June 2007; accepted 5 June 2007
Available online 9 June 2007
Abstract—Based on the fact that the cyclization of N-[(3S)]-1,2,3,4-tetrahydro-b-carboline-3-carboxyl]-L-lysine in both of acetic acid
aqueous (5%) and rat plasma gave the same product and the hypothesis that the cyclization product is antithrombotic active, we
report the synthesis, in vitro anti-aggregation, and in vivo anti-thrombosis activity of 20 hexahydropyrazino[10,20:1,6]pyrido[3,4-
b]indole-1,4-dions (5a–t) as potential anti-thrombosis agents in this study. Two intermediates (tetrahydro-b-carboline-3-carboxy-
L-amino acid benzylesters, 2-aminoacyltetrahydro-b-carboline-3-carboxylic acid benzylesters) were prepared and used for the
cyclization to form 5a–t. Coupling hydrochloric acid salt of tetrahydro-b-carboline-3-carboxylic acid esters and Boc-amino acids
in the reported literature usually generates very low yield products accompanied by racemization. However, in our case, the free
base of tetrahydro-b-carboline-3-carboxylic acid benzylester produced the desired products in high yields and without racemization.
The anti-thrombosis results from both in vitro and in vivo studies revealed that 5a–t may be a new class of anti-thrombosis agents
with potent effective concentration at 0.5 lmol/kg with oral administration. Moreover, a QSAR analysis was performed on these 20
compounds by using molecular descriptors generated by e-dragon server. Although the activities of these compounds are weakly
correlated with the logP values, the current QSAR analysis revealed that the anti-thrombotic activity of these compounds can
be explained by their steric and electrostatic effects.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
subendothelium surfaces of injured vessels with various
pathological conditions, including cardiovascular and
cerebrovascular thromboembotic disorders, such as
unstable angina, myocardial infarction, transient ische-
mic attack, stroke, and atherosclerosis.3–7
Intravascular thrombosis is one of the most frequent
pathological events and a major cause of morbidity
and mortality worldwide. Critical steps in the develop-
ment of acute coronary syndromes are the disruption,
rupture, erosion of atherosclerotic plaque with the for-
mation of either partial or complete occlusive throm-
bus.1–3 Factors contribute to thrombosis including
vascular damage, stimulation of platelets, and activation
of the coagulation cascade. Platelets adhere to exposed
Despite the well-established application of antithrom-
botic drugs, such as anticoagulants, antiplatelet drugs,
and thrombolytic drugs, there is still an urgent need
for more potent and safer compounds for the prevention
and treatment of ischemic symptoms. Many antithrom-
botic drug candidates fail to exert their therapeutic
potential due to their poor bioavailability. To improve
the pharmacological and the pharmacokinetic proper-
ties, we have developed a series of small-molecule
antithrombotic and thrombolytic agents.8–14 Recently,
a promising strategy for overcoming this poor bioavail-
ability problem was to target the intestinal peptide
transport system. In this context, we have synthesized
Keywords: Anti-thrombotic agent; Hexahydropyrazino[10,20:1,6]-pyri-
do[3,4-b]indole-1,4-dions; Intramolecular cyclization; Tetrahydro-b-c-
arboline-3-carboxylic acid.
*
Corresponding authors. Tel.: +86 10 6273 4376; fax: +86 10 6273
1332 (Z.Z.); tel./fax: +86 10 8280 2482 (M.Z.); tel./fax: +86 10 8391
1528 (S.P.); e-mail addresses: zidingzhang@cau.edu.cn; sqpeng@
mail.bjum.edu.cn
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.06.012

J. Liu et al. / Bioorg. Med. Chem. 15 (2007) 5672–5693
5673
and evaluated a series of novel dipeptide analogues as a
new strategy to improve pharmacological and pharma-
cokinetic properties. The main feature of our approach
is to design drug candidates in the form of dipeptide
analogues that can be readily absorbed across the
intestinal brush border membrane via the peptide
transport system. More specifically, 3-(S)-1,2,3,4-tetra-
hydro-b-carboline-3-carboxylic acid isolated from A.
Chinese G. Don was found to possess moderate anti-
aggregation activity, but with poor bioavailability. To
improve its biological property, we focused on improving
the absorption of 3-(S)-1,2,3,4-tetrahydro-b-carboline-
3-carboxylic acid by converting it into a dipeptide
analogue for improved peptide transport.9
intramolecular cyclization of N-[(3S)-1,2,3,4-tetrahy-
dro-b-carboline-3-carboxyl]-L-amino acid benzylesters
and (3S)-N-(L-aminoacyl)-1,2,3,4-tetrahydro-b-carbo-
line-3-carboxylic acid benzylesters, and their in vitro
and in vivo anti-thrombotic activities were evaluated.
Considering the significant correlation between logP
and retention time in HPLC (alternatively represented
as logK),15–17 moreover, the retention time of these new-
ly characterized anti-thrombotic agents was measured to
replace the time-consuming logP determination. Final-
ly, an optimal QSAR model was constructed by analyz-
ing the determined logK values and other molecular
descriptors generated from e-dragon server.
In the investigation we found that these dipeptide ana-
logues exhibited a strong tendency of intramolecular
cyclization. In order to confirm their cyclization and
the possible use N-[(3S)]-1,2,3,4-tetrahydro-b-carbo-
line-3-carboxyl]-L-lysine was selected as a representative
compound, incubated in acetic acid aqueous (5%) and in
rat plasma. Then 3.5 mg of N-[(3S)]-1,2,3,4-tetrahydro-
b-carboline-3-carboxyl]-L-lysine was incubated in 15 ml
of acetic acid aqueous (5%) at 37 ꢁC for 30 min and then
exposed to a gentle nitrogen-flow to evaporate to dry-
ness. The obtained residue was dissolved in 100 ll of
aqueous methanol (50%, v/v) to prepare sample solution
of HPLC-MS analysis. After 20 ll of the sample solu-
tion was loaded, the column was eluted with aqueous
methanol (50%, v/v) as the mobile phase. The flow rate
was 1 ml/min. The sample was monitored with UV
detector at 254.8 nm. The MS analysis was carried out
on HP ES-5989x instrument (cone, 30 V; source temper-
ature, 120 ꢁC; desolvation temperature, 350 ꢁC; desolva-
tion gas, 350 L/h; cone gas, 50 L/h) and a molecule ion
with 327 amu was found, which was deduced as 3-(4-
aminobutyl)-2,3,6,7,12,12a-hexahydropyrazino[10,20:1,6]
pyrido[3,4-b]indole-1,4-dione. Then its incubation pro-
cedure was further used with 15 ml of rat plasma instead
of acetic acid aqueous, a molecule ion with 327 amu was
again found and the same product was deduced. The
cyclization may be presented by Scheme 1. Based on
these observations the cyclized product was supposed
as antithrombotic active and was consequently
synthesized for bioassay. The data indicated that 3-(4-
aminobutyl)-2,3,6,7,12,12a-hexahydropyrazino[10,20:1,6]
pyrido [3,4-b]indole-1,4-dione was in vitro and in vivo
anti-thrombotic active.
2. Results and discussion
2.1. Coupling, cyclization, and stereochemistry
In general, 3-aminoacyl-2,3,6,7,12,12a-hexahydropy-
razino[10,20:1,6]pyrido[3,4-b]indole-1,4-diones can be
synthesized from the intramolecular cyclization of
N-[(3S)-1,2,3,4-tetrahydro-b-carboline-3-carboxyl]amino
acids (Scheme 2) or (3S)-N-(L-aminoacyl)-1,2,3,4-tet-
rahydro-b-carboline-3-carboxylic acids (Scheme 3).
For these two cyclization models, (3S)-1,2,3,4-tetra-
hydro-b-carboline-3-carboxylic acid is the key inter-
mediate. The results of the preparation reactions
and the stereochemistry were described as follows.
2.1.1. Cyclization of N-[(3S)-1,2,3,4-tetrahydro-b-carbo-
line-3-carboxyl]-L-amino acid benzylesters (3a–t). (3S)-
1,2,3,4-Tetrahydro-b-carboline-3-carboxylic acid 1 was
first transformed into N-Boc-tetrahydro-b-carboline-
carboxylic acid 2 via the acylation with Boc-N3 (76%),
which was then conjugated with amino acid benzylesters
to give 3a–t (85–96% yield). Upon removal of their
N-Boc group 4a-t were obtained. Interestingly, without
the additional coupling agents, in polar solvent the
intramolecular cyclization occurred forming a diketo-
piperazine ring to give 5a–l; 50j,k; 5m–o; 50p; 5q–t
(87–92%).
2.1.2. Coupling phosphoric salt of 3S-1,2,3,4-tetrahydro-
b-carboline-3-carboxylic acid benzylester (6). After
benzyl esterification of 1 in polyphosphoric acid/benzyl
alcohol system the phosphoric salt of 3S-1,2,3,4-tetrahy-
dro-b-carboline-3-carboxylic acid benzylester (6) was
obtained in 89% yield. A general coupling of phosphoric
salt 6 and Boc-amino acids provided N-(Boc-aminoacyl)
tetrahydro-b-carbolinecarboxylic acid benzylesters 8a–t
in low yield (7–60%).
Thus in this study 20 hexahydropyrazino[10,20:1,6]pyri-
do[3,4-b]-indole-1,4-diones were prepared via the
O
O
2.1.3. Coupling free base 3S-1,2,3,4-tetrahydro-b-carbo-
line-3-carboxylic acid benzylester (7). Treating phospho-
ric salt 6 with Et3N gave the free base 3S-1,2,
3,4-tetrahydro-b-carboline-3-carboxylic acid benzylester
(7), which was then coupled with Boc-amino acids. In con-
trast to phosphoric salt 6, the coupling yields of free base 7
were quite enormous (86–95%). With respect to the differ-
ence between the yields of coupling phosphoric salt 6 and
free base 7 it could be considered that under the general
coupling condition N-methyl morpholine is unable to
NH
NH
Rat plasma
NH
HO
N
N
(CH ) NH
2 4
(CH ) NH
2
N
2
2
4
H
H
O
O
Scheme 1. When N-[(3S)]-1,2,3,4-tetrahydro-b-carboline-3-carboxyl]-
L-lysine was incubated in 15 ml of rat plasma or acetic acid aqueous
(5%) at 37 ꢁC for 30 min, a cyclization product with 326 amu was
monitored by HPLC-MS analysis, which may be deduced as 3-(4-
aminobutyl)-2,3,6,7,12,12a-hexahydropyrazino[10,20:1,6]pyrido[3,4-b]-
indole-1,4-dione.

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J. Liu et al. / Bioorg. Med. Chem. 15 (2007) 5672–5693
R
O
NH
O
O
H
H
H
CO2Bzl
CO2Bzl
OH
ii
OH
iii
3a-t
NBoc
NH
HCl
NBoc
NH
N
N
N
1
2
H
H
H
R
iv
i
O
O
O
NH
O
H
H
H
OH
v
N
NH
HCl
NH2
N
R
N
N
H
H
H
4a-t
5a-i,l-o,q-t & 5'j,k,p
vi
O
H
NH
N
5j,k,p
N
R
H
O
Scheme 2. Preparing 3-aminoacyl-2,3,6,7,12,12a-hexahydropyrazino[10,20:1,6]pyrido[3,4-b]indole-1,4-dione via the intramolecular cyclization of
N-[(3S)-1,2,3,4-tetrahydro-b-carboline-3-carboxyl]-L-amino acid benzylester. (i) Formaldehyde and H2SO4; (ii) Triethylamine and Boc-N3; (iii)
H-AA-OBzl/DCC/NMM; (iv) Hydrogen chloride in ethyl acetate (4 mol/L); (v) Methanol, ethanol or acetone, and NMM; (vi) Pd/C, H2. In 3a,4a,
and 5a, R = CH3; 3b, 4b, and 5b, R = CH2C6H5; 3c,4c, and 5c, R = CH(CH3)2; 3d, 4d, and 5d, R = CH2OH; 3e, 4e, and 5e, R = CH(OH)CH3; 3f, 4f,
and 5f, R = CH2C6H4-OH-p; 3g, 4g, and 5g, R = tetrahydropyrrol-2-yl; 3h, 4h, and 5h, R = CH2SH; 3i, 4i, and 5i, R = CH2CH2SCH3; 3j, 4j, and 5j0,
R = CH2CH2COOCH2C6H5; 5j, R = CH2CH2COOH; 3k, 4k, and 50k, R = CH2COOCH2C6H5; 5k, R = CH2COOH; 3l, 4l and 5l, R = 1,3-imidazol-
5-methylene; 3m, 4m, and 5m, R = indol-3-methylene; 3n, 4n, and 5n, R = CH2(CH2)2NHC(NH2)@NH, 3o, 4o and 5o, R = H; 3p, 4p, and 50p,
R = CH2(CH2)3NHZ; 5p, R = CH2(CH2)3NH2; 3q, 4q, and 5q, R = CH2CH2CONH2; 3r, 4r, and 5r, R = CH2CONH2; 3s, 4s and 5s,
R = CH2CH(CH3)2; 3t, 4t, and 5t, R = CH(CH3)CH2CH3.
O
O
H
O
H
H
OBzl
H+
OBzl
OH
HCl
i
ii
NH
NH
NH
N
N
N
1
6
7
H
H
H
iii
O
O
H
N
H
OH
OBzl
v
iv
5a-i,l-o,q-t & 5'j,k,p
Boc-HN
Boc-HN
N
N
N
vi
R
O
R
O
H
H
8a-t
9a-t
v
5j,k,p
vi
5j,k,p
5a-i,l-o,q-t & 5'j,k,p
Scheme 3. Preparing 3-substituted-2,3,6,7,12,12a-hexahydropyrazino[10,20:1,6]pyrido[3,4-b]-indole-1,4-diones via the intramolecular amination of
N-aminoacylcarbolinecarboxylic benzylesters 8a–t, and the intramolecular acylation of N-aminoacylcarbolinecarboxylic acids 9a–t. (i) polyphos-
phoric acid, benzyl alcohol/80 ꢁC; (ii) Triethylamine; (iii) Boc-AA-OH, DCC, and NMM; (iv) Aqueous NaOH (2 N) and hydrochloric acid; (v)
Hydrogen chloride in ethyl acetate (4 mol/L); (vi) Pd/C, H2. In 5a, 8a, and 9a, R1 = CH3; 5b, 8b, and 9b, R = CH2C6H5; 5c, 8c, and 9c, R =
CH(CH3)2; 5d, 8d, and 9d, R = CH2OH; 5e, 8e, and 9e, R = CH(OH)CH3; 5f, 8f, and 9f, R = CH2C6H4-OH-p; 5g, 8g, and 9g, R = tetrahydropyrrol-
2-yl; 5h, 8h, and 9h, R = CH2SH; 5i, 8i and 9i, R = CH2CH2SCH3; 5j0 and 8j, R = CH2CH2CO2CH2C6H5; 5j and 9j, R = CH2CH2CO2H; 50k and 8k,
R = CH2CO2CH2C6H5; 5k and 9k, R = CH2CO2H; 5l, 8l, and 9l, R = 1,3-imidazol-5-methylene; 5m, 8m, and 9m, R = indol-3-yl-methylene; 5n, 8n,
and 9n, R = CH2(CH2)2NHC(NH2)@NH; 5o, 8o, and 9o, R = H; 50p, 8p, and 9p, R = CH2(CH3)3NHZ; 5q, 8q, and 9q, R = CH2CH2CONH2; 5r, 8r,
and 9r, R = CH2CONH2; 5s, 8s, and 9s, R = CH2CH(CH3)2; 5t, 8t, and 9t, R = CH(CH3)CH2CH3.
neutralize phosphoric acid and convert 6 into free base 7
to undergo the coupling.
90–94% yield. After removing Boc group 9a–t also
simultaneously underwent intramolecular cyclization
to form 5a–t in 86–91% yields (Scheme 3).
2.1.4. Cyclization of (3S)-N-(Boc-L-aminoacyl)-1,2,3,4-
tetrahydro-b-carboline-3-carboxylic acid benzylesters
(8a–t). Upon removal of Boc group an intramolecular
cyclization may simultaneously occur for 8a–t. This
two-step-one-pot reaction gave 3-substituted-2,3,6,7,12,
12a-hexahydropyrazino-[10,20:1,6]pyrido-[3,4-b]indole-1,
4-diones in good yield (87–92%).
2.1.6. Removing the benzyloxyl group of 50j,k and the
benzyloxycarbonyl of 50p by platinum catalyzed hydro-
genolysis. The platinum catalyzed hydrogenolysis of
5j0,k,p resulted in a smooth removal of both benzyloxyl
group and benzyloxycarbonyl group, and 50j,k,p were
converted into 5j,k,p in 95–95% yields (Scheme 3).
2.1.5. Cyclization of (3S)-N-(Boc-L-aminoacyl)-1,2,3,4-
tetrahydro-b-carboline-3-carboxylic acids (9a–t). In
aqueous NaOH, 8a–t may be converted into 9a–t in
2.1.7. Optical purity of 5a–t and 8a–t. In a solid
phase synthesis the coupling of hydrochloric salt of 3S-
1,2,3,4-tetrahydro-b-carboline-3-carboxylic acid benzyl-

J. Liu et al. / Bioorg. Med. Chem. 15 (2007) 5672–5693
5675
ester was reported inevitably leading to racemization
and giving a product consisted of four diastereoisomers.
Thus the reported bioassays in the literature were gener-
ally related to diastereomeric mixtures.18,19 To deter-
mine if any racemization of the preexisting stereogenic
center had occurred during the course of the coupling,
upon removal of the solvent and volatile side products,
the coupling products (5a–t and 8a–t) were then directly
analyzed by HPLC, which was carried out on Agilent
1100 series, the column was a reversed-phase C18 col-
umn (Agilent Zorbax Extend-C18, 4.6 · 15 mm, 5um).
After 20 ll of the sample (10 lM) was loaded, the col-
umn was eluted with aqueous methanol (50%) as the
mobile phase for 40 min. The flow rate was 1 ml/min,
5a–t and 8a–t in the sample were monitored with UV
detector at 254.8 nm and the peak area was recorded.
Based on the relative proportion of the peak areas it
was found that the diastereopurities of 5a–t and 8a–t
were higher than 98%.
range from 1 lmol/L to 0.01 lmol/L) were added. After
adjustment of the baseline, 5 ll of the solution of plate-
let-activating factor in NS (PAF, final concentration
10ꢀ7 mol/L), or 5 ll of the solution of adenosine diphos-
phate in NS (ADP, final concentration 10ꢀ5 mol/L) was
added and aggregation was recorded at 37 ꢁC for 5 min.
The effects of 5a–t (final concentration 1–0.01 lmol/L)
on PAF or ADP induced platelet aggregation were ob-
served. The maximal rate of platelet aggregation
(Am%) was represented by the peak height of aggrega-
tion curve. The inhibition rate was calculated by %Inhi-
bition = (Am% of NS, 50.16 3.65%) ꢀ (Am% of 5a–
t) ꢁ (Am% of NS, 50.16 3.65%) and the IC50 values
were determined. The range of the IC50 values of 5a–t
for ADP-induced aggregation of platelet in PRP plasma
of pig is 0.112 0.013 lmol/L to 2.045 0.095 lmol/L,
for PAF-induced aggregation of platelet in PRP plasma
of pig is 0.294 0.035 lmol/L to 1.134 0.102 lmol/L.
The results clearly indicate that the platelet aggregations
induced by the two aggregators are sensitive to 5a–t
(Fig. 1).
2.2. In vitro anti-platelet aggregation activities of 5a–t
The in vitro anti-platelet aggregation activities of 5a–t
were tested by use of a general procedure. After collec-
tion, the pig blood was centrifuged at 100 g for 10 min
and the platelet rich plasma (PRP) was removed. The
remaining blood was centrifuged for a further 10 min
at 1,500 g 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 a testing tube of the optical aggregometry 0.5 ml of
the adjusted plasma sample and 5 ll of NS or 5 ll of
the solution of 5a–t (in a series of concentrations of a
2.3. In vivo anti-thrombotic activities of intravenous 5a–t
The in vivo anti-thrombosis activities of 5a–t were tested
on Wistar rats using standard derivative circulation via
polyethylene tube. The anti-thrombosis potencies of
5a–t were represented by reduced weights of thrombus
and the results are listed in Table 1. Statistical analysis
of the data is carried out using one way ANOVA test
with p < 0.05 as significant cut-off. The in vivo activities
listed in Table 1 demonstrated that at 0.5 lmol/kg, 5a–t
were able to prevent rats from developing thrombus.
2.5
ADP
2
1.5
1
PAF
k
k
j
i
h
g
j
i
f
h
h
h
g
e
e
e
e
e
f
e
e
e
e
0.5
0
e
d
c
c
d
d
b
c
b
a
b
b
a
5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l 5m 5n 5o 5p 5q 5r 5s 5t
Compd.
Figure 1. In vitro anti-platelet aggregation activities of 5a–t. n = 6; for ADP induced aggregation, a = 5c. Compare to 5a,b,d–t p < 0.001; b = 5b,n.
Compare to 5a,d–m, o–t p < 0.001; c = 5h. Compare to 5a,d–g,i–m, o–t p < 0.001; d = 5d,r. Compare to 5a,e–g,i–m, o–q,s,t p < 0.001; e = 5f,i,j,o,s.
Compare to 5a,e,g,k–m, p,q,t p < 0.001; f = 5q. Compare to 5l p < 0.05, 5a,e,g,k,m,p,t p < 0.001; g = 5l. Compare to 5k,m,p,t p < 0.001; h = 5a,e,g.
Compare to 5m,p,t p < 0.001; i = 5k. Compare to 5p p < 0.001; j = 5t. Compare to 5m, p p < 0.01. for PAF induced aggregation, a = 5c. Compare to
5a,b,d–t p < 0.001; b = 5b,n. Compare to 5a,d–m, o–t p < 0.001; c = 5d,h. Compare to 5a,e–g,i–m, o–t p < 0.001; d = 5r. Compare to 5a,e–g,i–m,o–q,s,t
p < 0.001; e = 5f,i,j,o,s. Compare to 5e p < 0.05, to 5a,g,k–m, p,q,t p < 0.001; f = 5e. Compare to 5a,g,k–m,p,q,t p < 0.001; g = 5q. Compare to 5a,g,k–
m,p,t p < 0.001; h = 5l. Compare to 5k,m,p,t p < 0.001; i = 5a. Compare to 5g p < 0.05, to 5k,m,p,t p < 0.001; j = 5g. Compare to 5k,m,p,t p < 0.001;
k = 5k,t. Compare to 5m p < 0.05, to 5p p < 0.001.

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J. Liu et al. / Bioorg. Med. Chem. 15 (2007) 5672–5693
Table 1. Effect of intravenous 5a–t on the thrombus weight
ðX ꢂ SD mgÞ
2.4. Dose-dependent in vivo anti-thrombotic activities of
intravenous 5b,c,n
Compound
Wet thrombus
Dry thrombus
Compounds with significant activity (5b,c,n) were
administered using three dosages to conduct detailed
pharmacological activity profile study (Table 2). Statisti-
cal analysis was carried out using one way ANOVA test
with p < 0.05 as significant cut-off. The data indicated
that the in vivo anti-thrombotic activities of intravenous
5b,c,n were dose-dependent.
NS
Aspirin
5a
5b
5c
25.75 2.21
7.56 1.31a
12.05 1.08a
8.58 1.24b
8.19 1.76b
9.44 1.17c
11.49 1.27a
10.49 1.75d
11.28 1.07a
9.06 1.03c
10.41 0.93d
10.42 1.82d
12.26 1.83a
11.43 0.57a
12.42 1.65a
8.62 1.82b
10.68 1.55d
12.78 1.57a
11.10 1.29a
9.54 1.61c
10.40 1.88d
12.45 0.23a
5.43 0.62
2.40 0.5a
3.83 0.49a
2.72 0.44b
2.59 0.36b
2.98 0.46c
3.65 0.49
3.30 0.39d
3.58 0.42a
2.88 0.39c
3.28 0.54d
3.31 0.32
3.89 0.54a
3.63 0.27a
3.94 0.54a
2.74 0.33b
3.39 0.41d
4.06 0.31a
3.52 0.53a
3.03 0.51c
3.30 0.39d
3.95 0.31a
5d
5e
5f
5g
5h
5i
2.5. In vivo anti-thrombotic activities of oral 5k,o,p
To expand the administration route for all compounds
5k,o,p, the moderately bioactive compounds, with neu-
tral, acidic, and alkaline side chain, were administered
orally. Results from oral and intravenous are listed to-
gether in Table 3. The statistical analysis was carried
out using one way ANOVA test with p < 0.05 as signif-
icant cut-off. The data of Table 3 indicated that at the
dose of 0.5 lmol/kg the oral administration of 5k,o,p
effectively reduced rat thrombus and the potency was
equal to that of intravenous administration. These
observations indicate that this class of compounds are
promising as oral antithrombotic agents.
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
5t
n = 11; Aspirin: dosage (iv) = 1.8 · 105lmol/kg; 5a–t: dosage
(iv)=0.5 lmol/kg.
a Compare to NS, p < 0.001.
2.6. The retention time of 5a–t
b Compare to 5a,m,t,p and NS p < 0.001, to 5f,i,j,o,s p < 0.05.
c Compare to 5e,g,l,q p < 0.05, to NS p < 0.001.
d Compare to 5a,t,p p < 0.05, to NS p < 0.001.
The HPLC analysis was carried out to determine the
retention time (tR) of 5a–t, which were further repre-
sented as the logK values (Table 4). More details about
the measurement of retention time can be found in the
Experimental section. The logP value, defined as the
logarithm of the partition coefficient between n-octanol
and water, is an important parameter to reflect a
compound’s drugability. Meanwhile, it has been widely
used as an important molecular descriptor in QSAR
analysis such as the classical Hansch approach.20 Since
it has been well established that the logK values are
highly correlated with the corresponding logP values,
therefore the determination of logK was carried out in
this study to replace the time-consuming logP
determination.
The inhibiting potency was significantly greater
than that of NS and equal to Aspirin (at 1.8 ·
105lmol/kg). These data again explored that the substit-
uents at the 3-position of 2,3,6,7,12,12a-hexahydropy-
razino[10,20:1,6]pyrido[3,4-b]indole-1,4-dione
affected
the anti-thrombotic activity. Comparing to 5o (3-posi-
tion without substituent), compounds with 3-phenyl
(5b), isopropyl (5c), hydroxymethyl (5d), and mer-
captomethyl (5h) had higher anti-thrombosis activity.
Compounds with 3-carboxyl (5k), heterocyclic ring
(5m), and long aliphatic chain (5t) had lower anti-
thrombotic activity. Compounds with 3-hydroxylphenyl
(8f) and methylmercapto (5i) had equal anti-thrombotic
activity. It is possible that the anti-thrombosis activities
relate to the stabilities of the compounds in vivo, which
is also consistent with their chemical stabilities.
2.7. QSAR analysis of 5a–t
In the present study, the in vivo anti-thrombotic activi-
ties of intravenous 5a–t, represented as the weights of
Table 2. Effect of intravenous 5b,c,n at different dosages on the thrombus weight
Compound
Wet thrombus ðX ꢂ SD mgÞ
5 nmol/kg
Dry thrombus ðX ꢂ SD mgÞ
5 nmol/kg
0.5 lmol/kg
0.5 nmol/kg
0.5 lmol/kg
0.5 nmol/kg
NS
5b
5c
25.75 2.21
13.30 2.19c
14.79 1.33b
14.52 1.10c
5.43 0.62
2.08 0.33c
2.31 0.43b
2.27 0.27c
8.58 1.24a
8.19 2.76a
8.62 1.82a
15.30 2.20d
16.88 1.56d
15.71 0.88d
1.33 0.30a
1.20 0.26a
1.22 0.32a
2.39 0.40d
2.64 0.36d
2.45 0.39d
5n
n = 11.
a Compare to 5 nmol/kg and NS groups, p < 0.001.
b Compare to 0.5 nmol/kg group p < 0.01, to NS group p < 0.01.
c Compare to 0.5 nmol/kg group p < 0.05, to NS group, p < 0.001.
d Compare to NS group, p < 0.001.

J. Liu et al. / Bioorg. Med. Chem. 15 (2007) 5672–5693
Table 3. Effect of oral 5k,o,p on the thrombus weight
5677
Compound
Wet thrombus ðX ꢂ SD mgÞ
Wet thrombus ðX ꢂ SD mgÞ
Intravenous
Oral
Intravenous
Oral
NS
5k
5o
26.03 2.17
12.42 2.83a
11.57 2.00a
11.75 2.97a
25.75 2.21
12.26 1.83a
10.68 1.55a
12.78 1.57a
5.50 0.32
3.94 0.47a
3.67 0.39a
3.73 0.34a
5.43 0.62
3.89 0.54a
3.39 0.41a
4.06 0.31a
5p
n = 11, dosage = 0.5 lmol/kg.
a Compare to NS group, p < 0.001.
Table 4. logK values of compounds 5a–t
2.5
2.0
Compound
logK
y = -0.02x + 0.6182
R2 = 0.0025
5a
5b
5c
5d
5e
5f
0.47
1.05
1.5
0.97
1.0
ꢀ0.16
ꢀ0.14
0.01
0.5
5g
5h
5i
0.94
0.71
0.0
-0.5
-1.0
-1.5
0.97
0.21
5j
5k
5l
0.15
0.41
6.0
7.0
8.0
9.0
10.0 11.0
WWT
12.0
13.0
14.0
5m
5n
5o
5p
5q
5r
5s
5t
0.66
ꢀ0.58
0.19
Figure 2. The correlation between the WWT values and the measured
logK values for 5a–t.
ꢀ0.26
0.28
0.21
1.32
1.26
in the Experimental section. Grouped into Weighted
Holistic Invariant Molecular (WHIM) descriptors, Gm
encodes information about the overall symmetry of a
molecule weighted by atomic masses.22 The RDF060m
and RDF130m are classified into the RDF molecular
descriptors, which were obtained by radial distribution
wet thrombus (WWT), are regarded as the anti-throm-
botic activities in the QSAR analysis. The detailed pro-
cedures to measure the WWT value can be found in the
Experimental section. Since only very weak correlation
between the logK values and the activities (i.e., WWT)
was observed in 5a–t (cf. Fig. 2), the classical Hansch
method is not possible to obtain a suitable QSAR model
for 5a–t. Therefore, the QSAR analysis based on other
molecular descriptors was carried out. By selecting the
molecular descriptors generated from e-dragon webserver,21
the multiple linear regression method (MLR) was
employed to derive the QSAR equation of 5a–t. The
resubstitution and Leave-One-Out (LOO) tests were car-
ried out to validate the established equations. For 5a–t
Eq. 1 could give the highest predictive accuracy.
functions centered on different interatomic distances
23
˚
˚
(from 0.5 A to 15.5 A). The RDF molecular descrip-
tors can be interpreted as the probability distribution
of finding an atom in a spherical volume of certain ra-
dius. For RDF060m and RDF130m, the atomic mass
weights are used and the corresponding sphere radius
˚
is of 6.0 and 13.0 A, respectively. The JGI10 belongs
to the descriptors of charge indices, which evaluate the
global charge transferred between pairs of atoms inside
the molecule.24 A pair-wise correlation analysis between
the above four variables (Gm, RDF060m, RDF130m,
and JGI10) and activity (i.e., WWT) was carried out
and is summarized in Table 5. Generally, the selected
four molecular descriptors share medium correlation
with the in vivo anti-thrombotic activity, and the corre-
lation between Gm and WWT is the most significant
(the correlation coefficient is equal to 0.308). Addition-
ally, all the pair-wise correlation coefficients between
different molecular descriptors are in the range from
ꢀ0.308 to 0.325 (Table 5), implying that there is no sig-
nificant correlation existing between any two molecular
descriptors.
WWT ¼ 28:79ðꢂ5:52Þ ꢀ 124:97ðꢂ31:21Þ ꢃ Gm þ 0:53ðꢂ0:12Þ ꢃ RDF060m
ꢀ1:61ðꢂ0:34Þ ꢃ RDF130m þ 299:96ðꢂ60:86Þ ꢃ JGI10
N ¼ 20; R2 ¼ 0:74; S ¼ 0:68;ꢀe ¼ 0:50; RL2 OO ¼ 0:64; SLOO ¼ 0:83;ꢀeLOO ¼ 0:66;
F ¼ 11:27; p ¼ 0:0003
ð1Þ
The squared correlation coefficients in the resubstitution
test (R2) and LOO test ðRL2 OOÞ are 0.74 and 0.64, respec-
tively. Generally, R2 measures goodness-of-fit, whereas
R2LOO measures goodness-of-prediction. In addition, the
resubstitution analysis gave an absolute average error
ðꢀeÞ of 0.50, whereas the absolute average error for the
LOO test ðꢀeLOOÞ is 0.66. The detailed description of
the other statistical measurements in Eq. 1 is available
Considering the standard deviation in measuring the
WWT value for each compound (cf. Table 1), the

5678
J. Liu et al. / Bioorg. Med. Chem. 15 (2007) 5672–5693
Table 5. Correlation matrix of different parameters, including four
molecular descriptors and the in vivo anti-thrombotic activity (i.e.,
WWT)
tion occurring in the coupling of some tetrahydro-b-
carboline-3-carboxylic acid benzylester with Boc-amino
acids. The good in vitro anti-platelet and in vivo anti-
thrombotic activities of 5a–t confirmed that the intra-
molecular cyclization of the dipeptide analogues,
tetrahydro-b-carboline-3-carboxylamino acids and
2-aminoacyltetrahydro-b-carboline-3-carboxylid acids,
indeed resulted in antithrombotic active products. The
data from three doses of the most potential 5b,c,n sug-
gested that the in vivo anti-thrombolysis action may
be dose-dependent. The substantially equal potency of
intravenous and oral administration of 5k,o,p (the mod-
erately bioactive and with neutral, acidic, and alkaline
side chain compounds) implies that the cyclization
products of the mentioned dipeptides are promising as
oral anti-thrombotic agents.
WWT Gm
RDF060m RDF130m JGI10
WWT
Gm
1.000 ꢀ0.308
0.302
1.000 ꢀ0.173
ꢀ0.267
ꢀ0.202
0.325
0.265
ꢀ0.308
0.298
RDF060m
RDF130m
JGI10
1.00
1.000
ꢀ0.144
1.000
14
13
12
11
10
4. Experimental
9
General. The protected amino acids with L-configuration
were purchased from Sigma Chemical Co. All coupling
and deprotective reactions were carried out under anhy-
drous conditions. Chromatography was performed on
Qingdao silica gel H. The purities of the intermediates
and products were confirmed by TLC (Merck silica gel
plates of type 60 F254, 0.25 mm layer thickness) and
HPLC (Waters, C18 column 4.6 · 150 mm). The amino
acid analysis was determined with a Hitachi 835-50
instrument. FAB-MS was determined by VG-ZAB-MS
high resolution GC/MS/DS and HP ES-5989x. Optical
rotations were determined with a Schmidt + Haensch
Polartromic D instrument. Statistical analysis of all bio-
logical data was carried out using one way ANOVA test
with p < 0.05 as significant cut-off.
8
7
6
6
7
8
9
10
11
12
13
14
Measured WWT
Figure 3. Comparison of the predicted anti-thrombotic activities of
compounds 5a–t in the LOO test with the measured activities (i.e., the
weight of wet thrombus). The data points situated at the zone between
the two diagonal-dash lines correspond to the compounds, in which
the absolute values of the prediction errors are less than 1.0.
current predictive accuracy of 5a–t is relatively high
(Fig. 3). Therefore, Eq. 1 can be practically used to fore-
cast the anti-thrombotic activities of new derivatives. In
Eq. 1, three descriptors (Gm, RDF060m, RDF130m)
are significantly related to a molecule’s steric property,
while the remained descriptor JGI10 is more correlated
with the electrostatic character of a molecule. Therefore,
the overall anti-thrombotic activities of 5a–t can be rea-
sonably explained by their electrostatic and steric effects,
although they are weakly correlated with the logK values.
4.1. Preparation of 3S-1,2,3,4-Tetrahydro-b-carboline-3-
carboxylic acid and derivatives
4.1.1.
3S-1,2,3,4-Tetrahydro-b-carboline-3-carboxylic
acid (1). To a mixture of 5.0 g (24.5 mmol) of L-trypto-
phane, 25 ml of H2SO4 (1 mol/L), and 80 ml of water,
8 ml of formaldehyde (36–38%) was added. The reaction
mixture was stirred at room temperature for 2 h and ad-
justed to pH 6–7 with concentrated ammonia liquor.
The mixture was kept at 0 ꢁC for 12 h and the formed
precipitates were collected by filtration. After recrystal-
lization (ethanol/aqueous ammonia) 3.97 g (75%) of
the title compound was obtained as a colorless powder.
Mp 280–282 ꢁC; ESI/MS (m/z) 217 [M+H]+; IR (KBr):
3450, 3200, 3000, 2950, 2850, 1700, 1601, 1452, 1070,
3. Conclusion
In conclusion, the dipeptide analogues of tetrahydro-b-
carboline-3-carboxylamino acids and 2-aminoacyltetra-
hydro-b-carboline-3-carboxylic acids were easy to
cyclize to 3-aminoacyl-hexahydropyrazino[10,20:1,6]pyr-
ido-[3,4-b]indole-1,4-diones 5a–t via either 2-Boc-b-
carboline-3-carboxylamino acid benzylesters (3a–t) or
2-(Boc-aminoacyl)-tetrahydro-b-carboline-3-carboxylic
acid benzylesters (8a–t) in satisfactory yield. In the prep-
aration of 8a–t the comparisons of low yields/high
racemization and high yields/low racemization were ob-
served in the couplings of the phosphoric salts and the
free base of tetrahydro-b-carboline-3-carboxylic acid
benzylester with Boc-amino acids, respectively. This
result demonstrated that the 2-position binding acid
should be responsible for the low yields/high racemiza-
1
900 cmꢀ1; H NMR (BHSC-500, DMSO-d6): d = 10.99
(s, 1H),9.89 (s, 1H), 7.30 (t, J = 7.5 Hz, 1H), 7.22 (t,
J = 8.0 Hz, 1H), 7.01 (d, J = 8.0 Hz, 1H), 6.81 (d,
J = 7.5 Hz, 1H), 4.01 (t, J = 4.8 Hz, 1H), 3.75 (dd,
J = 10.5 Hz, J = 5.0 Hz, 1H), 3.64 (dd, J = 10.5 Hz,
J = 2.4 Hz, 1H), 2.91 (d, J = 10.5 Hz, 2H), 2.86 (s,
1H). Anal. Calcd for C12H12N2O2 C 66.65, H, 5.59, N
12.96. Found C 66.45, H 5.72, N 12.79.
4.1.2. N-Boc-3S-1,2,3,4-tetrahydro-b-carboline-3-carbox-
ylic acid (2). The suspension of 1.1 g (5.0 mmol) of com-
pound 1 in 15 ml of DMF and 1.4 ml of triethylamine

J. Liu et al. / Bioorg. Med. Chem. 15 (2007) 5672–5693
5679
was vigorously stirred at room temperature, to which
1.1 g (7.7 mmol) of Boc-N3 was added for 30 min. The
reaction mixture was stirred at room temperature for
24 h and at 40 ꢁC for 80 h. To the reaction mixture,
5 ml of citrate in water (20%) was added and the solu-
tion was extracted with ethyl acetate (30 ml · 3). The
separated ethyl acetate layer was dried with anhydrous
MgSO4. After removal of MgSO4 by filtration, the fil-
trate was evaporated to dryness. The residue obtained
was crystallized in CHCl3 to give 1.20 g (76%) of the title
compound. Mp 165–170 ꢁC; ESI/MS (m/z) 317 [M+H]+;
IR (KBr): 3452, 3205, 3001, 2952, 2848, 1705, 1645,
J = 8.1 Hz, 1H), 7.00 (t, J = 7.3 Hz, 1H), 6.95 (t,
J = 7.3Hz, 1H), 4.24 (d, J = 4.7 Hz, 1H), 3.67 (dd,
J = 10.1 Hz, J = 5.2 Hz, 1H), 3.55 (s, 2H), 3.15 (dd,
J = 10.1 Hz, J = 2.4 Hz, 1H), 2.84 (ddd, J = 10.1 Hz,
J = 5.2 Hz, J = 2.4 Hz, 1H), 2.81 (s, 1H).
4.2. General procedure for the preparation of N-(N-Boc-
3S-1,2,3,4-tetrahydro-b-carboline-3-carboxyl)-L-amino
acid benzylesters (3a–t)
At 0 ꢁC to the solution of 2.0 g (6.33 mmol) N-Boc-3S-
1,2,3,4-tetrahydro-b-carboline-3-carboxylic acid in
30 ml of anhydrous THF, 1.2 g (8.9 mmol) of HOBt
was added, after 10 min, 1.75 g (8.5 mmol) of DCC
was then added. The suspension of 6.96 mmol of
HCl Æ L-AA-OBzl in 3 ml of anhydrous THF was ad-
justed to pH 8–9 with N-methyl morpholine and stirred
at room temperature for another 20 min. This suspen-
sion then was added to the solution of N-Boc-3S-
1,2,3,4-tetrahydro-b-carboline-3-carboxylic acid and
the reaction mixture was stirred at 0 ꢁC for 2h and at
room temperature for 16 h. On evaporation the residue
was dissolved in 30 ml of ethyl acetate. The solution was
washed successively with 5% sodium bicarbonate, 5%
citric acid, and saturated sodium chloride and the or-
ganic phase was separated and dried over anhydrous so-
dium sulfate. After filtration and evaporation under
reduced pressure the title compound was obtained as
powder.
1600, 1450, 1072, 901 cmꢀ1 1H NMR (BHSC-500,
;
DMSO-d6): d = 10.87 (s, 1H), 9.86 (s, 1H), 7.32 (t,
J = 7.6 Hz, 1H), 7.21 (t, J = 7.9 Hz, 1H), 7.00 (d,
J = 7.9 Hz, 1H), 6.84 (t, J = 7.6 Hz, 1H), 4.84 (t,
J = 5.0 Hz, 1H), 4.20 (dd, J = 10.2 Hz, J = 4.8 Hz,
1H), 3.98 (dd, J = 10.2 Hz, J = 3.2 Hz, 1H), 2.93 (d,
J = 10.2 Hz, 2H), 1.46 (s, 9H). Anal. Calcd for
C17H20N2O4 C 64.54, H, 6.37, N 8.86. Found C 64.41,
H 6.25, N 8.74.
4.1.3. Phosphoric salt of 3S-1,2,3,4-tetrahydro-b-carbo-
line-3-carboxylic acid benzylester (6). At 70 ꢁC the mix-
ture of 60 g polyphosphoric acid and 600 ml of benzyl
alcohol was stirred for 20 min to form a clean solution.
The temperature of the solution was raised to 80 ꢁC, 3S-
1,2,3,4-tetrahydro-b-carboline-3-carboxylic acid (30 g)
was then added. The reaction mixture was stirred for an-
other 24h and TLC (CHCl3/MeOH, 10/1) indicated the
complete disappearance of 3S-1,2,3,4-tetrahydro-b-
carboline-3-carboxylic acid. The reaction mixture was
cooled to room temperature, diluted with 500 ml of
ether and 2100 ml of water, and stirred at room temper-
ature for 48h. The formed precipitates were collected by
filtration, washed with water and ether, dried over
anhydrous CaCl2 to provide 35 g (85%) of phosphoric
salt of 3S-1,2,3,4-tetrahydro-b-carboline-3-carboxylic
4.2.1. N-[(3S)-N-Boc-1,2,3,4-tetrahydro-b-carboline-3-
carboxyl]-L-alaninebenzylester (3a). Yield: 95%. Mp
110–112 ꢁC; ESI/MS (m/z) 478 [M+H]+. IR (KBr):
3447, 3342, 3001, 2945, 2842, 1761, 1733, 1602, 1455,
1391, 1373, 1062, 899 cmꢀ1 1H NMR (BHSC-500,
;
DMSO-d6): d = 9.93 (s, 1H), 8.04 (s, 1H), 7.30 (t,
J = 7.3 Hz, 1H), 7.24 (t, J = 7.3 Hz, 1H), 7.22 (t,
J = 7.2 Hz, 2H), 7.14 (d, J = 7.2 Hz, 2H), 7.11 (t,
J = 7.2 Hz, 1H), 6.95 (d, J = 7.3 Hz, 1H), 6.83 (d,
J = 7.3 Hz, 1H), 5.35 (s, 2H), 4.86 (d, J = 5.4 Hz, 1H),
4.56 (m, J = 5.2 Hz, 1H), 4.22 (dd, J = 10.1 Hz,
J = 4.5 Hz, 1H), 4.15 (dd, J = 10.0 Hz, J = 3.7 Hz,
acid benzylester as
a
colorless powder. Mp
192–194 ꢁC; ESI/MS (m/z) 307 [M+H]+; 1H NMR
(BHSC-500, DMSO-d6): d = 8.79 (s, 1H), 8.22 (s, 1H),
7.48 (d, J = 7.5 Hz, 1H), 7.40 (d, J = 7.2 Hz, 2H), 7.33
(t, J = 8.0 Hz, 1H), 7.20 (t, J = 7.6 Hz, 2H), 7.08 (t,
J = 8.0 Hz, 1H), 7.01 (t, J = 7.5Hz, 1H), 6.99 (t,
J = 7.5Hz, 1H), 4.22 (d, J = 4.8 Hz, 1H), 3.69 (dd,
J = 10.5 Hz, J = 5.0 Hz, 1H), 3.56 (s, 2H), 3.14
(dd, J = 10.5 Hz, J = 2.4 Hz, 1H), 2.83 (ddd,
J = 10.5 Hz, J = 5.0 Hz, J = 2.4 Hz, 1H).
1H), 2.92 (d, J = 10.0 Hz, 2H), 1.57 (d, J = 5.0 Hz,
20
D
3H), 1.46 (s, 9H). ½aꢄ ꢀ 100ꢅ (c 0.38, CHCl3/CH3OH,
1:1, v/v). Anal. Calcd for C27H31N3O5 C 67.91, H,
6.54, N 8.80. Found C 67.72, H 6.40, N 8.90.
4.2.2. N-[(3S)-N-Boc-1,2,3,4-tetrahydro-b-carboline-3-
carboxyl]-L-phenylalanine benzyl-ester (3b). Yield: 96%.
Mp 144–146 ꢁC; ESI/MS (m/z) 554 [M+H]+. IR (KBr):
3442, 3350, 3202, 3009, 2944, 2842, 1758, 1734, 1642,
4.1.4. 3S-1,2,3,4-Tetrahydro-b-carboline-3-carboxylic
acid benzylester (7). At 0 ꢁC to the stirring suspension
of 402 mg (1.0 mmol) of 6 in 30 ml of ethyl acetate,
303 mg (3.0 mmol) of triethylamine was added. The
mixture was stirred at room temperature until a clean
solution was formed. The solution was then washed with
aqueous NaHCO3 (5%, 50 ml · 6) and saturated aque-
ous NaCl (50 ml · 3), and dried over anhydrous
Na2SO4. After filtration the filtrate was evaporated to
provide 272 mg (89%) of 8 as a pale yellow powder.
1601, 1455, 1390, 1371, 1065, 902 cmꢀ1 1H NMR
;
(BHSC-500, DMSO-d6): d = 9.95 (s, 1H), 7.99 (s, 1H),
7.28 (t, J = 7.3 Hz, 1H), 7.25 (t, J = 7.6 Hz, 2H), 7.22
(t, J = 7.3 Hz, 2H),7.17 (t, J = 7.3 Hz, 1H), 7.15 (d,
J = 7.4 Hz, 2H), 7.13 (d, J = 7.3 Hz, 2H), 7.11 (t,
J = 7.3 Hz, 1H), 7.03 (t, J = 7.3 Hz, 1H), 6.98 (d,
J = 7.6 Hz, 1H), 6.82 (d, J = 7.2 Hz, 1H), 5.34 (s, 2H),
4.95 (d, J = 5.2 Hz, 1H), 4.84 (t, J = 5.2 Hz, 1H), 4.25
(dd, J = 10.0 Hz, J = 4.1 Hz, 1H), 4.15 (dd,
1
Mp 133–135 ꢁC; ESI/MS (m/z) 307 [M+H]+; H NMR
(BHSC-500, DMSO-d6): d = 8.74 (s, 1H) 7.45 (d,
J = 7.4 Hz, 1H), 7.37 (d, J = 7.2 Hz, 2H), 7.30 (t,
J = 8.1 Hz, 1H), 7.17 (t, J = 7.4 Hz, 2H), 7.03 (t,
J = 10.0 Hz, J = 3.5 Hz, 1H), 3.19 (d, J = 5.2 Hz, 2H),
20
D
0.38, CHCl3/CH3OH, 1:1, v/v). Anal. Calcd for
2.92 (d, J = 10.0 Hz, 2H), 1.49 (s, 9H). ½aꢄ ꢀ 52ꢅ (c

5680
J. Liu et al. / Bioorg. Med. Chem. 15 (2007) 5672–5693
C33H35N3O5 C 71.59, H 6.37, N 7.59. Found C 71.74, H
6.22, N 7.77.
J = 7.4 Hz, 2H), 5.40 (s, 2H), 4.99 (s, 1H), 4.91 (d,
J = 5.5 Hz, 1H), 4.82 (t, J = 5.7 Hz, 1H), 4.26 (m,
J = 5.3 Hz, 2H), 3.17 (d, J = 5.3 Hz, 2H), 2.95 (d,
J = 5.1 Hz, 2H), 1.47 (s, 9H). Anal. Calcd for
C33H35N3O6 C 69.58, H 6.19, N 7.38. Found C 69.41, H
6.10, N 7.53.
4.2.3. N-(N-Boc-3S-1,2,3,4-tetrahydro-b-carboline-3- car-
boxyl)-L-valine benzylester (3c). Yield: 94%. Mp
169–171 ꢁC; ESI/MS (m/z) 506 [M+H]+. IR (KBr):
3441, 3336, 3205, 3004, 2953, 2843, 1757, 1732, 1645,
1600, 1453, 1390, 1372, 1064, 900 cmꢀ1
;
1H NMR
4.2.7. N-(N-Boc-3S-1,2,3,4-tetrahydro-b-carboline-3-
carboxyl)-L-proline benzylester (3g). Yield: 93%. Mp
133–135 ꢁC; ESI/MS (m/z) 504 [M+H]+; IR (KBr):
3439, 3206, 3005, 2952, 2843, 1730, 1641, 1600, 1452,
(BHSC-500, DMSO-d6): d = 10.00 (s, 1H), 7.98 (s, 1H),
7.27 (t, J = 7.4 Hz, 1H), 7.23 (t, J = 7.0 Hz, 2H), 7.20 (t,
J = 7.4 Hz, 1H), 7.02 (d, J = 7.4 Hz, 1H), 7.13
(d, J = 7.0 Hz, 2H), 7.10 (t, J = 7.0 Hz, 1H), 6.87 (d,
J = 7.4 Hz, 1H), 5.34 (s, 2H), 4.86 (t, J = 5.2 Hz, 1H),
4.40 (d, J = 5.1 Hz, 1H), 4.24 (dd, J = 10.0 Hz,
J = 4.3 Hz, 1H), 4.05 (dd, J = 10.0 Hz, J = 3.5 Hz, 1H),
3.12 (m, J = 5.2 Hz, 1H), 2.93 (d, J = 6.4 Hz, 2H), 1.49
(s, 9H), 1.03 (d, J = 5.1 Hz, 6H). Anal. Calcd for
C29H35N3O5 C 68.89, H 6.98, N 8.31. Found C 68.74, H
7.08, N 8.48.
1395, 1373, 1060, 905 cmꢀ1 1H NMR (BHSC-500,
;
DMSO-d6): d = 10.04 (s, 1H), 7.33 (t, J = 7.3 Hz, 1H),
7.22 (t, J = 7.6 Hz, 1H), 7.20 (t, J = 7.2 Hz, 2H), 7.13
(d, J = 7.2 Hz, 2H), 7.09 (t, J = 7.2 Hz, 1H), 7.05 (d,
J = 7.6 Hz, 1H), 6.93 (d, J = 7.3 Hz, 1H), 5.30 (s, 2H),
4.86 (t, J = 5.3 Hz, 1H), 4.33 (t, J = 5.5 Hz, 1H), 4.24
(d, J = 5.2 Hz, 2H), 3.49 (t, J = 5.5 Hz, 2H), 2.93 (d,
J = 5.5 Hz, 2H), 2.27 (d, J = 5.5Hz, 2H), 1.96 (t,
J = 4.8 Hz, 2H), 1.45 (s, 9H). Anal. Calcd for
C29H33N3O5 C 69.17, H 6.60, N 8.34. Found C 69.02,
H 6.50, N 8.51.
4.2.4. N-[(3S)-N-Boc-1,2,3,4-tetrahydro-b-carboline-3-
carboxyl]-L-serine benzylester (3d). Yield: 93%. Mp
125–127 ꢁC; ESI/MS (m/z) 494 [M+H]+. IR (KBr):
3445, 3338, 3207, 3005, 2956, 2843, 1762, 1733, 1642,
4.2.8. N-(N-Boc-3S-1,2,3,4-tetrahydro-b-carboline-3-
carboxyl)-L-cysteine benzylester (3h). Yield: 90%. Mp
151–153 ꢁC; ESI/MS (m/z) 510 [M+H]+; IR (KBr):
3441, 3205, 3003, 2940, 2842, 1735, 1640, 1603, 1450,
1601, 1457, 1390, 1371, 1062, 903 cmꢀ1 1H NMR
;
(BHSC-500, DMSO-d6): d = 9.97 (s, 1H), 7.99 (s, 1H),
7.27 (t, J = 7.2 Hz, 1H), 7.23 (t, J = 7.3 Hz, 1H), 7.21
(t, J = 7.0 Hz, 2H), 7.13 (d, J = 7.0 Hz, 2H), 7.10 (t,
J = 7.0 Hz, 1H), 6.99 (d, J = 7.3 Hz, 1H), 6.85 (t,
J = 7.2 Hz, 1H), 5.33 (s, 2H), 4.89 (d, J = 5.2 Hz, 1H),
4.50 (t, J = 5.0 Hz, 1H), 4.17 (d, J = 5.0Hz, 2H), 4.15
1392, 1375, 1062, 899 cmꢀ1 1H NMR (BHSC-500,
;
DMSO-d6): d = 9.96 (s, 1H), 7.95 (s, 1H), 7.30
(t, J = 7.4 Hz, 1H), 7.24 (t, J = 7.6 Hz, 1H), 7.22 (t,
J = 7.5 Hz, 2H), 7.15 (d, J = 7.5 Hz, 2H), 7.11
(t, J = 7.5 Hz, 1H), 7.07 (d, J = 7.6 Hz, 1H), 6.86 (d,
J = 7.4 Hz, 1H), 5.32 (s, 2H), 4.95 (t, J = 5.4 Hz, 1H),
4.74 (t, J = 5.6 Hz, 1H), 4.23 (d, J = 5.4 Hz, 2H), 3.14
(d, J = 5.4 Hz, 2H), 3.02 (d, J = 5.5 Hz, 2H), 1.47 (s,
9H), 1.65 (s, 1H). Anal. Calcd for C27H31N3O5S C
63.63, H 6.13, N 8.25. Found C 63.45, H 6.02, N 8.10.
(d, J = 5.2 Hz, 2H), 2.94 (d, J = 5.3 Hz, 1H), 2.90 (d,
20
D
J = 5.3 Hz, 1H), 2.25 (s, 1H), 1.47 (s, 9H). ½aꢄ ꢀ 51ꢅ
(c 0.35, CHCl3/CH3OH, 1:1, v/v). Anal. Calcd for
C27H31N3O6 C 65.71, H 6.33, N 8.51. Found C 65.58,
H 6.39, N 8.70.
4.2.5. N-(N-Boc-3S-1,2,3,4-tetrahydro-b-carboline-3-
carboxyl)-L-threonine benzylester (3e). 90%. Mp 145–
147 ꢁC; ESI/MS (m/z) 508 [M+H]+; IR (KBr): 3440,
3334, 3201, 3002, 2951, 2845, 1760, 1731, 1640, 1600,
4.2.9. N-(N-Boc-3S-1,2,3,4-tetrahydro-b-carboline-3-
carboxyl)-L-methionine benzylester (3i). Yield: 91%.Mp
167–169 ꢁC; ESI/MS (m/z) 538 [M+H]+; IR (KBr):
3445, 3200, 3007, 2956, 2849, 1730, 1645, 1600, 1452,
1
1452, 1395, 1375, 1060, 900 cmꢀ1; H NMR (BHSC-
500, DMSO-d6): d = 9.95 (s, 1H), 7.84 (s, 1H), 7.30 (t,
J = 7.2 Hz, 1H), 7.24 (t, J = 7.2 Hz, 1H), 7.19 (t,
J = 7.0 Hz, 2H), 7.10 (d, J = 7.0 Hz, 2H), 7.07 (t,
J = 7.0 Hz, 1H), 6.97 (d, J = 7.2 Hz, 1H), 6.85 (t,
J = 7.2 Hz, 1H), 5.33 (s, 2H), 4.85 (t, J = 5.5 Hz, 1H),
4.63 (m, J = 5.5 Hz, 1H), 4.45 (t, J = 5.5 Hz, 1H), 3.95
(m, J = 5.3 Hz, 2H), 2.94 (d, J = 5.6Hz, 2H), 2.20 (s,
1H), 1.49 (s, 9H), 1.22 (d, J = 5.5 Hz, 3H). Anal. Calcd
for C28H33N3O6 C 66.26, H 6.55, N 8.28. Found C
66.11, H 6.38, N 8.44.
1395, 1370, 1064, 905 cmꢀ1 1H NMR (BHSC-500,
;
DMSO-d6): d = 10.07 (s, 1H), 7.94 (s, 1H), 7.33 (t,
J = 7.4 Hz, 1H), 7.23 (t, J = 7.6 Hz, 1H), 7.20
(t, J = 7.5 Hz, 2H), 7.17 (d, J = 7.5 Hz, 2H), 7.12 (t,
J = 7.5 Hz, 1H), 6.95 (d, J = 7.4 Hz, 1H), 6.83
(d, J = 7.4 Hz, 1H), 5.32 (s, 2H), 4.87 (t, J = 5.1 Hz,
1H), 4.46 (t, J = 5.4 Hz, 1H), 4.26 (d, J = 5.2 Hz, 2H),
2.95 (d, J = 5.1 Hz, 2H), 2.44 (t, J = 5.3 Hz, 2H), 2.25
(d, J = 5.4 Hz, 2H), 2.13 (s, 3H), 1.49 (s, 9H). Anal.
Calcd for C29H35N3O5S C 64.78, H 6.56, N 7.82. Found
C 64.62, H 6.45, N 7.99.
4.2.6. N-(N-Boc-3S-1,2,3,4-tetrahydro-b-carboline-3-
carboxyl)-L-tyrosine benzylester (3f). Yield: 90%. Mp
155–157 ꢁC; ESI/MS (m/z) 570 [M+H]+; IR (KBr):
3444, 3200, 3006, 2950, 2842, 1730, 1647, 1600, 1456,
4.2.10. N-(N-Boc-3S-1,2,3,4-tetrahydro-b-carboline-3-
carboxyl)-L-glutamic acid dibenzyl-ester (3j). Yield:
90%. Mp 144–146 ꢁC; ESI/MS (m/z) 626 [M+H]+; IR
(KBr): 3445, 3204, 3007, 2948, 2826, 1735, 1640, 1600,
1395, 1370, 1060, 900 cmꢀ1 1H NMR (BHSC-500,
;
1
DMSO-d6): d = 9.95 (s, 1H), 8.04 (s, 1H), 7.37
(t, J = 7.4 Hz, 1H), 7.23 (t, J = 7.6 Hz, 1H), 7.16 (d,
J = 7.4 Hz, 2H), 7.14 (t, J = 7.1 Hz, 2H), 7.12
(d, J = 7.1 Hz, 2H), 7.09 (t, J = 7.1 Hz, 1H), 7.05 (d,
J = 7.4 Hz, 1H), 6.93 (d, J = 7.6 Hz, 1H), 6.90 (d,
1452, 1391, 1375, 1062, 900 cmꢀ1; H NMR (BHSC-500,
DMSO-d6): d = 9.92 (s, 1H), 8.03 (s, 1H), 7.36 (t,
J = 7.5 Hz, 1H), 7.27 (t, J = 7.5 Hz, 1H), 7.24 (t,
J = 7.3 Hz, 2H), 7.22 (t, J = 7.4 Hz, 2H), 7.21
(d, J = 7.3 Hz, 2H), 7.19 (d, J = 7.4 Hz, 2H), 7.16 (t,

J. Liu et al. / Bioorg. Med. Chem. 15 (2007) 5672–5693
5681
J = 7.3 Hz, 1H), 7.14 (t, J = 7.4 Hz, 1H), 7.04(d, J = 7.5 Hz,
1H), 6.86 (d, J = 7.5 Hz, 1H), 5.36 (s, 2H), 5.33 (s, 2H),
4.93 (d, J = 5.3 Hz, 1H), 4.46 (t, J = 5.5 Hz, 1H), 4.25
(d, J = 5.4 Hz, 2H), 2.95 (d, J = 5.2 Hz, 2H), 2.25 (t,
J = 5.5 Hz, 2H), 2.22 (t, J = 5.6 Hz, 2H), 1.48 (s, 9H).
Anal. Calcd for C36H39N3O7 C 69.10, H 6.28, N 6.72.
Found C 69.27, H 6.18, N 6.89.
3446, 3203, 3005, 2944, 2846, 1725, 1642, 1603, 1450,
1395, 1370, 1064, 900 cmꢀ1 1H NMR (BHSC-500,
;
DMSO-d6): d = 10.20 (s, 1H), 8.42 (s, 2H), 8.25 (s,
1H), 8.20 (s, 1H), 8.00 (s, 1H), 7.27 (t, J = 7.4 Hz,
1H), 7.24 (t, J = 7.2 Hz, 2H), 7.17 (d, J = 7.2 Hz, 2H),
7.15 (t, J = 7.5 Hz, 1H), 7.13 (t, J = 7.2 Hz, 1H), 7.01
(d, J = 7.5 Hz, 1H), 6.93 (d, J = 7.4 Hz, 1H), 5.34 (s,
2H), 4.92 (d, J = 5.2 Hz, 1H), 4.40 (t, J = 4.4 Hz, 1H),
4.27 (d, J = 5.2 Hz, 2H), 2.90 (d, J = 4.4 Hz, 2H), 2.67
(t, J = 5.2 Hz, 2H), 1.95 (m, J = 5.3 Hz, 2H), 1.56 (m,
J = 5.3 Hz, 2H), 1.55 (s, 9H). Anal. Calcd for
C30H38N6O5 C 64.04, H 6.81, N 14.94. Found C
64.21, H 6.69, N 14.78.
4.2.11. N-(N-Boc-3S-1,2,3,4-tetrahydro-b-carboline-3-
carboxyl)-L-aspartic acid dibenzyl-ester (3k). Yield:
92%. Mp 132–134 ꢁC; ESI/MS (m/z) 612 [M+H]+; IR
(KBr): 3445, 3340, 3214, 3002, 2952, 2843, 1758, 1730,
1646, 1602, 1455, 1388, 1369, 1064, 901 cmꢀ1 1H
;
NMR (BHSC-500, DMSO-d6): d = 10.01 (s, 1H), 8.02
(s, 1H), 7.30 (t, J = 7.2 Hz, 1H), 7.24 (t, J = 7.2 Hz,
1H), 7.22 (t, J = 7.0 Hz, 2H), 7.20 (t, J = 7.0 Hz, 2H),
7.15 (d, J = 7.0 Hz, 2H), 7.13 (d, J = 7.0 Hz, 2H), 7.11
(t, J = 7.0 Hz, 1H), 7.10 (t, J = 7.0 Hz, 1H), 7.02 (d,
J = 7.2 Hz, 1H), 6.98 (d, J = 7.2 Hz, 1H), 5.36 (s, 2H),
5.34 (s, 2H), 4.94 (d, J = 5.2 Hz, 1H), 4.79 (t,
J = 5.2 Hz, 1H), 4.27 (d, J = 5.2 Hz, 2H), 2.94 (d,
J = 5.0 Hz, 2H), 2.87 (d, J = 5.2 Hz, 2H), 1.47 (s, 9H).
Anal. Calcd for C35H37N3O7 C 68.72, H 6.10, N 6.87.
Found C 68.54, H 6.19, N 7.00.
4.2.15. N-[(3S)-N-Boc-1,2,3,4-tetrahydro-b-carboline-
3-carboxyl]-L-glycine benzylester (3o). Yield: 95%. Mp
139–141 ꢁC; ESI/MS (m/z) 464 [M+H]+. IR (KBr):
3444, 3336, 3004, 2940, 2845, 1761, 1730, 1602, 1455,
1392, 1375, 1060, 903 cmꢀ1 1H NMR (BHSC-500,
;
DMSO-d6): d = 9.96 (s, 1H), 8.05 (s, 1H), 7.27 (t,
J = 7.4 Hz, 1H), 7.23 (t, J = 7.2 Hz, 2H), 7.18 (t,
J = 7.4 Hz, 1H), 7.15 (d, J = 7.2 Hz, 2H), 7.11 (t,
J = 7.2 Hz, 1H), 6.93 (d, J = 7.4 Hz, 1H), 6.85 (d,
J = 7.4 Hz, 1H), 5.36 (s, 2H), 4.86 (d, J = 5.2 Hz, 1H),
4.25 (dd, J = 10.0 Hz, J = 4.3 Hz, 1H), 4.17 (dd,
4.2.12. N-(N-Boc-3S-1,2,3,4-tetrahydro-b-carboline-3-
carboxyl)-L-histidine benzylester (3l). Yield: 91%. Mp
153–155 ꢁC; ESI/MS (m/z) 544 [M+H]+; IR (KBr):
3445, 3202, 3007, 2943, 2832, 1735, 1640, 1600, 1452,
J = 10.0 Hz, J = 3.4 Hz, 1H), 4.15 (s, 2H), 2.93 (d,
20
D
J = 10.0 Hz, 2H), 1.48 (s, 9H). ½aꢄ ꢀ 84ꢅ (c 0.36,
CHCl3/CH3OH, 1:1, v/v). Anal. Calcd for C26H29N3O5
C 67.37, H, 6.31, N 9.07. Found C 67.25, H 6.18, N 9.19.
1395, 1371, 1060, 905 cmꢀ1 1H NMR (BHSC-500,
;
DMSO-d6): d = 12.95 (s, 1H), 9.98 (s, 1H), 8.01 (s, 1H),
7.45 (s, 1H), 7.35 (t, J = 7.2 Hz, 1H), 7.22 (t, J = 7.3 Hz,
2H), 7.17 (t, J = 7.4 Hz, 1H), 7.15 (d, J = 7.3 Hz, 2H),
7.13 (d, J = 7.4 Hz, 1H), 7.11 (t, J = 7.3 Hz, 1H), 6.95 (t,
J = 7.2 Hz, 1H), 6.88 (s, 1H), 5.38 (s, 2H), 4.91
(t, J = 5.2 Hz, 1H), 4.80 (t, J = 5.1 Hz, 1H), 4.22 (d,
J = 5.1 Hz, 2H), 3.21 (d, J = 5.2 Hz, 2H), 2.90
(d, J = 5.1 Hz, 2H), 1.47 (s, 9H). Anal. Calcd for
C30H33N5O5 C 66.28, H 6.12, N 12.88. Found C 66.11,
H 6.00, N 13.07.
4.2.16. N-[(3S)-N-Boc-1,2,3,4-tetrahydro-b-carboline-
3-carboxyl]-L-lysine(Z) benzylester (3p). Yield: 92%.
Mp 123–125 ꢁC; ESI/MS (m/z) 669 [M+H]+. IR (KBr):
3445, 3339, 3002, 2942, 2845, 1761, 1735, 1602, 1459,
1392, 1371, 1063, 899 cmꢀ1 1H NMR (BHSC-500,
;
DMSO-d6): d = 9.98 (s, 1H), 8.02 (s, 1H), 7.99 (s, 1H),
7.27 (t, J = 7.3 Hz, 1H), 7.22 (t, J = 7.2Hz, 2H), 7.20
(t, J = 7.0Hz, 1H), 7.17 (t, J = 7.3 Hz, 1H), 7.14 (d,
J = 7.0 Hz, 2H), 7.13 (d, J = 7.2Hz, 2H), 7.12 (t,
J = 7.0 Hz, 2H), 7.10 (t, J = 7.2Hz, 1H), 6.98 (d,
J = 7.3 Hz, 1H), 6.88 (d, J = 7.3 Hz, 1H), 5.35 (s, 2H),
5.33 (s, 2H), 4.93 (d, J = 5.2 Hz, 1H), 4.45 (t,
J = 4.2 Hz, 1H), 4.25 (dd, J = 10.2 Hz, J = 4.3 Hz,
1H), 4.15 (dd, J = 10.2 Hz, J = 3.4 Hz, 1H), 2.96 (t,
J = 4.2 Hz, 2H), 2.94 (d, J = 10.2 Hz, 2H), 1.93 (m,
4.2.13. N-[(3S)-N-Boc-1,2,3,4-tetrahydro-b-carboline-
3-carboxyl]-L-tryptophan benzylester (3m). Yield: 92%.
Mp 136–138 ꢁC; ESI/MS (m/z) 593 [M+H]+. IR (KBr):
3444, 3337, 3208, 3005, 2945, 2835, 1758, 1736, 1645,
1602, 1449, 1390, 1370, 1067, 903 cmꢀ1
;
1H NMR
J = 4.2 Hz, 2H), 1.55 (m, J = 4.4 Hz, 2H), 1.48 (s, 9H),
20
D
CH3OH, 1:1, v/v). Anal. Calcd for C38H44N4O7
(BHSC-500, DMSO-d6): d = 9.96 (s, 1H), 9.89 (s, 1H),
8.03 (s, 1H), 7.29 (t, J = 7.3 Hz, 1H), 7.27 (t,
J = 7.2 Hz, 1H), 7.22 (t, J = 7.0 Hz, 2H), 7.14
(d, J = 7.0 Hz, 2H), 7.12 (d, J = 7.5 Hz, 1H), 7.11 (t,
J = 7.0 Hz, 1H), 7.10 (t, J = 7.5 Hz, 1H), 7.09 (d,
J = 7.3 Hz, 1H), 7.07 (t, J = 7.5 Hz, 1H), 7.02
(d, J = 7.3 Hz, 1H), 6.97 (d, J = 7.2 Hz, 1H), 6.85 (s,
1H), 5.36 (s, 2H), 4.96 (d, J = 5.2 Hz, 1H), 4.73
(t, J = 5.1 Hz, 1H), 4.27 (d, J = 5.0 Hz, 2H), 3.17 (d,
1.27 (m, J = 4.2 Hz, 2H). ½aꢄ ꢀ 41ꢅ (c 0.39, CHCl3/
C
68.24, H, 6.63, N 8.38. Found C 68.08, H 6.77, N 8.53.
4.2.17. N-[(3S)-N-Boc-1,2,3,4-tetrahydro-b-carboline-
3-carboxyl]-L-glutamine benzylester (3q). Yield: 87%.
Mp 130–132 ꢁC; ESI/MS (m/z) 535 [M+H]+. IR (KBr):
3441, 3207, 3004, 2942, 2830, 1736, 1642, 1600, 1455,
1390, 1375, 1062, 903 cmꢀ1 1H NMR (BHSC-500,
;
J = 5.2 Hz, 2H), 2.97 (d, J = 6.2 Hz, 2H), 1.47 (s, 9H).
DMSO-d6): d = 9.95 (s, 1H), 8.04 (s, 1H), 7.27 (t,
J = 7.3 Hz, 1H), 7.24 (t, J = 7.2 Hz, 2H), 7.18 (t,
J = 7.3 Hz, 1H), 7.16 (d, J = 7.2 Hz, 2H), 7.13 (t,
J = 7.2 Hz, 1H), 7.04 (d, J = 7.3 Hz, 1H), 6.85 (d,
J = 7.3 Hz, 1H), 6.08 (s, 2H), 5.35 (s, 2H), 4.94 (d,
J = 5.4 Hz, 1H), 4.43 (t, J = 5.4 Hz, 1H), 4.27 (d,
J = 5.5 Hz, 2H), 2.90 (d, J = 5.2 Hz, 2H), 2.15 (t,
J = 5.4 Hz, 2H), 2.16 (t, J = 5.4Hz, 2H), 1.49 (s, 9H).
20
D
½aꢄ ꢀ 77ꢅ (c 0.36, CHCl3/CH3OH, 1:1, v/v). Anal.
Calcd for C35H36N4O5 C 70.93, H 6.12, N 9.45. Found
C 70.80, H 6.01, N 9.60.
4.2.14. N-(N-Boc-3S-1,2,3,4-tetrahydro-b-carboline-3-
carboxyl)-L-arginine benzylester (3n). Yield: 85%. Mp
152–154 ꢁC; ESI/MS (m/z) 563 [M+H]+; IR (KBr):

5682
20
J. Liu et al. / Bioorg. Med. Chem. 15 (2007) 5672–5693
½aꢄ ꢀ 50ꢅ (c 0.38, CHCl3/CH3OH, 1:1, v/v). Anal.
for 20 min. Then the reaction mixture was stirred at
room temperature for 50 min and TLC(ethyl acetate/
petroleum, 5:10) indicated the completion of removing
Boc. The reaction mixture was evaporated under vac-
uum. The residue was diluted in 10 ml of ethyl acetate
and then evaporated to dryness, which was repeated
for three times to thoroughly remove the free hydrogen
chloride. The residue was dissolved in 10 ml of methanol
and treated with 1.0 ml of triethylamine. The reaction
mixture was stirred at room temperature for 20h and
TLC(ethyl acetate/petroleum, 5:10) indicated the com-
pletion of reaction. The reaction mixture was evapo-
rated under vacuum and the residue was purified by
column of silica gel (ethyl acetate/petroleum, 5:12) to
give the title compound as colorless powder.
D
Calcd for C29H34N4O6 C 65.15, H 6.41, N 10.48. Found
C 65.32, H 6.52, N 10.31.
4.2.18. N-[(3S)-N-Boc-1,2,3,4-tetrahydro-b-carboline-
3-carboxyl]-L-asparagine benzylester (3r). Yield: 90%.
Mp 142–144 ꢁC; ESI/MS (m/z) 521 [M+H]+. IR (KBr):
3443, 3207, 3004, 2932, 2833, 1735, 1630, 1604, 1451,
1392, 1375, 1064, 900 cmꢀ1 1H NMR (BHSC-500,
;
DMSO-d6): d = 9.96 (s, 1H), 8.05 (s, 1H), 7.28 (t,
J = 7.3 Hz, 1H), 7.23 (t, J = 7.4 Hz, 2H), 7.19 (t,
J = 7.3 Hz, 1H), 7.17 (d, J = 7.4 Hz, 2H), 7.15 (t,
J = 7.4 Hz, 1H), 7.06 (d, J = 7.3 Hz, 1H), 6.87 (d,
J = 7.3 Hz, 1H), 6.05 (s, 2H), 5.33 (s, 2H), 4.97 (d,
J = 5.3 Hz, 1H), 4.45 (t, J = 5.3 Hz, 1H), 4.24 (d,
J = 5.3 Hz, 2H), 2.92 (d, J = 5.1 Hz, 2H), 2.17 (t,
20
D
J = 5.1 Hz, 2H), 1.47 (s, 9H). ½aꢄ ꢀ 42ꢅ (c 0.30,
4.3.1. (3S,12aS)-3-Methyl-2,3,6,7,12,12a-hexahydropy-
razino[10,20:1,6]pyrido[3,4-b]-indole-1,4-dion (5a). Yield;
91%. Mp 233–235 ꢁC, ESI-MS (m/z) 270 [M+H]+,
CHCl3/CH3OH, 1:1, v/v). Anal. Calcd for C28H32N4O6
C 64.60, H 6.20, N 10.76. Found C 64.78, H 6.29, N 10.57.
1
IR(KBr): 3337, 2926, 1678, 1455, 1326, 744 cmꢀ1; H
4.2.19. N-[(3S)-N-Boc-1,2,3,4-tetrahydro-b-carboline-3-
carboxyl]-L-leucine benzylester (3s). Yield: 92%. Mp
140–142 ꢁC; ESI/MS (m/z) 520 [M+H]+. IR (KBr):
3440, 3207, 3005, 2951, 2842, 1735, 1644, 1603, 1452,
NMR (DMSO-d6, 300 MHz): d = 10.97 (s,1H), 8.46 (d,
J = 2.1Hz, 1H), 7.46 (d, J = 7.5 Hz, 1H), 7.35 (t,
J = 7.5 Hz, 1H), 7.07 (t, J = 7.5 Hz, 1H), 6.96 (t,
J = 7.5 Hz, 1H), 5.33 (d, J = 16.8 Hz, lH), 4.73 (m,
J = 6.9 Hz, 1H), 4.28 (dd, J = 4.2 Hz, J = 11.7 Hz,
1H), 4.20 (d, J = 16.5 Hz, 1H), 3.26 (dd, J = 4.2 Hz,
J = 15.0 Hz, 1H), 2.80 (t, J = 4.8 Hz, 1H), 1.35 (d,
J = 6.9 Hz, 3H). 13C NMR (DMSO-d6, 300 MHz)
d = 167.21, 166.82, 136.02, 134.55, 131.00, 126.26,
1393, 1370, 1060, 904 cmꢀ1 1H NMR (BHSC-500,
;
DMSO-d6): d = 9.95 (s, 1H), 8.06 (s, 1H), 7.27 (t,
J = 7.3 Hz, 1H), 7.24 (t, J = 7.1 Hz, 2H), 7.21 (t,
J = 7.3 Hz, 1H), 7.15 (d, J = 7.1 Hz, 2H), 7.11 (t,
J = 7.1 Hz, 1H), 7.06 (d, J = 7.3 Hz, 1H), 6.82 (d,
J = 7.3 Hz, 1H), 5.33 (s, 2H), 4.93 (t, J = 5.2 Hz, 1H),
4.41 (t, J = 5.2 Hz, 1H), 4.26 (dd, J = 10.0 Hz,
J = 4.5 Hz, 1H), 4.09 (dd, J = 10.0 Hz, J = 3.8 Hz,
1H), 2.91 (d, J = 6.2 Hz, 2H), 2.83 (d, J = 5.1 Hz, 2H),
121.36, 120.12, 119.15, 111.09, 59.80, 52.98, 42.87,
20
D
24.75, 17.31. ½aꢄ ꢀ 150ꢅ (c 1.0, CH3OH); Anal. Calcd
for C15H15N3O2 C 66.90, H 5.61, N 15.60. Found C
66.70, H 5.41, N 15.81.
1.53 (s, 9H), 1.34 (m, J = 5.1 Hz, 1H), 1.07 (d,
20
D
J = 5.3 Hz, 6H). ½aꢄ ꢀ 33ꢅ (c 0.31, CHCl3/CH3OH,
4.3.2. (3S,12aS)-3-Benzyl-2,3,6,7,12,12a-hexahydropy-
razino[10,20:1,6]pyrido[3,4-b]-indole-1,4-dione (5b). Yield:
90%. Mp 242–245 ꢁC; EI-MS (m/z) 346 [M+H]+;
1:1, v/v). Anal. Calcd for C30H37N3O5 C 69.34, H
7.18, N 8.09. Found C 69.25, H 7.08, N 8.26.
1
IR(KBr): 3328, 2936, 1669, 1455, 1328, 744 cmꢀ1; H
4.2.20. N-[(3S)-N-Boc-1,2,3,4-tetrahydro-b-carboline-3-
carboxyl]-L-isoleucine benzylester (3t). Yield: 90%. Mp
134–136 ꢁC; ESI/MS (m/z) 520 [M+H]+. IR (KBr):
3445, 3339, 3210, 3010, 2955, 2840, 1758, 1732, 1645,
NMR (DMSO-d6, 300MHz) d = 10.81 (s,1H), 8.46 (d,
J = 1.8 Hz, 1H), 7.42 (d, J = 7.5 Hz, 1H), 7.31 (t,
J = 7.5 Hz, 1H), 7.22 (t, J = 7.2 Hz, 2H), 7.14 (d,
J = 7.2 Hz, 2H), 7.06 (t, J = 7.2 Hz, 1H), 7.02 (t,
J = 7.5 Hz, 1H), 6.93 (t, J = 7.5 Hz, 1H), 5.32 (d,
J = 16.5 Hz, 1H), 4.37 (s, 2H), 4.07 (d, J = 16.8 Hz,
1H), 3.98 (dd, J = 11.7 Hz, J = 4.5 Hz, 1H), 3.17 (dd,
J = 13.2 Hz, J = 3.3 Hz, 1H), 2.88 (dd, J = 13.5 Hz,
1600, 1452, 1390, 1371, 1064, 903 cmꢀ1 1H NMR
;
(BHSC-500, DMSO-d6): d = 9.96 (s, 1H), 8.00 (s, 1H),
7.27 (t, J = 7.1 Hz, 1H), 7.22 (t, J = 7.0 Hz, 2H), 7.20
(t, J = 7.1 Hz, 1H), 7.00 (d, J = 7.1 Hz, 1H), 7.13 (d,
J = 7.0 Hz, 2H), 7.10 (t, J = 7.0 Hz, 1H), 6.88 (d,
J = 7.1 Hz, 1H), 5.35 (s, 2H), 4.95 (t, J = 5.0 Hz, 1H),
4.42 (t, J = 5.0 Hz, 1H), 4.25 (dd, J = 10.2 Hz,
J = 4.0 Hz, 1H), 4.05 (dd, J = 10.2 Hz, J = 3.5 Hz,
1H), 2.94 (d, J = 6.0 Hz, 2H), 2.91 (m, J = 5.1 Hz,
J = 5.1 Hz, 1H), 2.64 (dd, J = 14.7 Hz, J = 3.6 Hz,
20
D
1H); ½aꢄ ꢀ 53ꢅ (c 1.0, CH3OH); Anal. Calcd for
C21H19N3O2 C 73.03, H 5.54, N 12.17. Found C
73.30, H 5.76, N 12.01.
1H), 1.47 (s, 9H), 1.30 (m, J = 5.2 Hz, 2H), 1.05 (d,
4.3.3. (3S,12aS)-3-(Isopropyl)-2,3,6,7,12,12a-hexahydro-
pyrazino[10,20:1,6]-pyrido[3,4-b]-indole-1,4-dione
20
D
J = 5.2 Hz, 3H), 0.95 (t, J = 5.2 Hz, 3H). ½aꢄ ꢀ 36ꢅ (c
(5c).
Yield: 92%. Mp 216–218 ꢁC, ESI-MS (m/z) 298
0.37, CHCl3/CH3OH, 1:1, v/v). Anal. Calcd for
C30H37N3O5 C 69.34, H 7.18, N 8.09. Found C 69.50,
H 7.29, N 8.24.
[M+H]+; IR(KBr): 3331, 2939, 1662, 1457, 1384, 1365,
1
746 cmꢀ1; H NMR (DMSO-d6, 300 MHz): d = 10.82
(s, 1H), 8.60 (d, J = 2.7 Hz, 1H), 7.36 (d, J = 7.1 Hz,
1H), 7.25 (t, J = 7.1 Hz, 1H), 7.02 (t, J = 7.1 Hz, 1H),
6.93 (t, J = 7.1 Hz, 1H), 5.22 (d, J = 15.2 Hz, 1H), 4.21
(dd, J = 11.3 Hz, J = 4.2 Hz, 1H), 4.19 (d, J = 17.0 Hz,
1H), 3.27 (dd, J = 14.6 Hz, J = 3.7 Hz, 1H), 2.99 (m,
J = 13.3 Hz, 1H), 2.89 (t, J = 13.3 Hz, 1H), 2.74 (m,
J = 6.2 Hz, 1H), 1.07 (d, J = 6.2 Hz, 6H). 13C NMR
4.3. General procedure for the preparation of hexahydropy-
razino[10,20:1,6]pyrido-[3,4-b]indole-1,4-diones 5a–i,l–o, q–t
and 50j,kp from 3a–t
At 0 ꢁC 2.0 mmol of 3a–t was dissolved in 10 ml of
hydrogen chloride/ethyl acetate (4 mol/L) and stirred

J. Liu et al. / Bioorg. Med. Chem. 15 (2007) 5672–5693
5683
20
D
(DMSO-d6, 300 MHz) d = 169.05, 168.48, 135.09,
133.71, 130.93, 122.59, 121.47, 118.99, 113.44, 111.27,
J = 5.4 Hz, 2H), 1.95 (t, J = 5.4 Hz, 2H). ½aꢄ ꢀ 89:1ꢅ
(c 1.0, CH3OH). Anal. Calcd for C17H17N3O2
C 69.14, H 5.80, N 14.23. Found C 69.01, H 5.70, N
14.14.
20
D
66.99, 63.10, 41.72, 28.75, 24.38, 16.82. ½aꢄ ꢀ 84ꢅ (c
1.0, CH3OH). Anal. Calcd for C17H19N3O2 C 68.67, H
6.44, N 14.13. Found C 68.50, H 6.27, N 14.00.
4.3.8. (3S,12aS)-3-Mercaptomethyl-2,3,6,7,12,12a-hexa-
hydropyrazino[10,20:1,6]pyrido-[3,4-b]indole-1,4-dione (5h).
Yield: 91%. Mp 237–239 ꢁC; ESI/MS (m/z) 302 [M+H]+;
4.3.4. (3S,12aS)-3-Hydroxymethyl-2,3,6,7,12,12a-hexa-
hydropyrazino[10,20:1,6]pyrido-[3,4-b]indole-1,4-dione(5d).
Yield: 90%. Mp 264–267 ꢁC, ESI-MS (m/z) 286 [M+H]+,
IR (KBr): 3343, 2936, 1647, 1641, 1459, 1333, 741cmꢀ1
;
IR(KBr): 3344, 2926, 1683, 1642, 1463, 1334, 744cmꢀ1
;
1H NMR (BHSC-500, DMSO-d6): d = 10.03 (s, 1H),
8.00 (s, 1H), 7.28 (t, J = 7.0 Hz, 1H), 7.15 (d,
J = 7.0 Hz, 1H), 7.08 (d, J = 7.0 Hz, 1H), 6.89 (d,
J = 7.0 Hz, 1H), 4.93 (t, J = 5.4 Hz, 1H), 4.79 (t,
J = 5.6 Hz, 1H), 4.29 (d, J = 5.2 Hz, 2H), 2.92 (d,
1H NMR (DMSO-d6, 300MHz) d = 10.92 (s,1H), 8.24
(d, J = 2.4 Hz, 1H), 7.43 (d, J = 7.5 Hz, 1H), 7.32 (t,
J = 7.5 Hz, 1H), 7.04 (t, J = 7.5 Hz, 1H), 6.93 (t,
J = 7.5 Hz, 1H), 5.42 (d, J = 16.5 Hz, 1H), 5.23 (t,
J = 4.8 Hz, 1H), 4.25 (dd, J = 11.4 Hz, J = 4.2 Hz,
1H), 4.15 (d, J = 16.5 Hz, 1H), 4.05 (d, J = 4.8 Hz,
1H), 3.94 (s, 1H), 3.17 (dd, J = 15.0 Hz, J = 3.6 Hz,
1H), 2.98 (t, J = 13.5 Hz, 2H); 13C NMR (DMSO-d6,
300 MHz) d = 166.88, 163.82, 135.86, 129.77, 126.33,
J = 5.4 Hz, 2H), 3.07 (d, J = 5.6 Hz, 2H), 1.65 (s, 1H).
20
D
½aꢄ ꢀ 48:7ꢅ (c 1.00, CH3OH); Anal. Calcd for
C15H15N3O2S C 59.78, H 5.02, N 13.94. Found C
59.92, H 5.13, N 13.76.
120.86, 118.56, 117.46, 110.96, 105.82, 62.66, 57.37,
4.3.9. (3S,12aS)-3-Methylmercaptoethyl-2,3,6,7,12,12a-
hexahydropyrazino[10,20:1,6]-pyrido[3,4-b]indole-1,4-dione
(5i). Yield: 90%. Mp 216–217 ꢁC; ESI/MS (m/z) 330
[M+H]+; IR (KBr): 3341, 2934, 1648, 1643, 1458,
20
D
55.82, 27.00. ½aꢄ ꢀ 141ꢅ (c 1.0, CH3OH). Anal. Calcd
for C15H15N3O3 C 63.15, H 5.30, N 14.73. Found C
63.48, H 5.52, N 14.54.
1332, 742 cmꢀ1 1H NMR (BHSC-500, DMSO-d6):
;
4.3.5. (3S,12aS)-3-(10-Hydroxyeth-10-yl)-2,3,6,7,12,12a-
hexahydropyrazino[10,20:1,6]-pyrido[3,4-b]indole-1,4-dio-
ne(5e). Yield: 89%. Mp 242–244 ꢁC; ESI/MS (m/z) 300
[M+H]+; IR (KBr): 3340, 2929, 1686, 1642, 1465,
d = 10.00 (s, 1H), 7.99 (s, 1H), 7.27 (t, J = 7.0 Hz, 1H),
7.19 (t, J = 7.0 Hz, 1H), 7.08 (t, J = 7.0 Hz, 1H), 6.87
(d, J = 7.0 Hz, 1H), 4.87 (t, J = 5.4 Hz, 1H), 4.49 (t,
J = 5.2 Hz, 1H), 4.29 (d, J = 5.3 Hz, 2H), 2.94 (d,
1333, 744 cmꢀ1
;
1H NMR (BHSC-500, DMSO-d6):
J = 5.4 Hz, 2H), 2.43 (t, J = 5.3 Hz, 2H), 2.17 (d,
20
D
d = 10.00 (s, 1H), 7.98 (s, 1H), 7.29 (t, J = 7.1 Hz, 1H),
7.25 (t, J = 7.1 Hz, 1H), 7.09 (t, J = 7.1 Hz, 1H), 6.97
(d, J = 7.1 Hz, 1H), 4.81 (t, J = 5.3 Hz, 1H), 4.63 (m,
J = 5.2 Hz, 1H), 4.45 (t, J = 5.4 Hz, 2H), 4.22 (m,
J = 5.3 Hz, 2H), 2.10 (s, 3H). ½aꢄ ꢀ 65:3ꢅ (c 1.0,
CH3OH). Anal. Calcd for C17H19N3O2S C 61.98, H
5.81, N 12.76. Found C 62.14, H 5.89, N 12.90.
J = 5.6 Hz, 1H), 2.90 (d, J = 5.4 Hz, 2H), 2.15 (s, 1H),
4.3.10. (3S,12aS)-3-Benzyloxycarbonylethyl-2,3,6,7,12,12a-
hexahydropyrazino[10,20:1,6]-pyrido[3,4-b]indole-1,4-dione
(50j). Yield: 92%. Mp 236–238 ꢁC; ESI/MS (m/z) 417
[M+H]+; IR (KBr): 3342, 3000, 2940, 1675, 1602, 1586,
20
D
1.22 (d, J = 5.6 Hz, 3H). ½aꢄ ꢀ 108:5ꢅ (c 1.0, CH3OH);
Anal. Calcd for C16H17N3O3 C 64.20, H 5.72, N 14.04.
Found C 64.32, H 5.80, N 14.22.
1
1457, 1333, 750 cmꢀ1; H NMR (BHSC-500, DMSO-
4.3.6. (3S,12aS)-3-(p-Hydroxyphenylmethyl)-2,3,6,7,12,12a-
hexahydropyrazino[10,20:1,6]-pyrido[3,4-b]indole-1,4-dione
(5f). Yield: 88%. Mp 249–251 ꢁC; ESI/MS (m/z) 362
[M+H]+; IR (KBr): 3342, 2936, 1682, 1643, 1464, 1333,
d6): d = 9.99 (s, 1H), 8.00 (s, 1H), 7.30 (t, J = 7.1 Hz,
1H), 7.22 (t, J = 7.1 Hz, 1H), 7.18 (t, J = 7.0 Hz, 2H),
7.13 (d, J = 7.0 Hz, 2H), 7.08 (t, J = 7.0 Hz, 1H), 7.06 (t,
J = 7.0 Hz, 1H), 7.01 (d, J = 7.2 Hz, 1H), 6.85 (d,
J = 7.2 Hz, 1H), 5.33 (s, 2H), 4.92 (d, J = 5.0 Hz, 1H),
4.43 (t, J = 5.2 Hz, 1H), 4.21 (d, J = 5.1 Hz, 2H), 2.94
1
743 cmꢀ1; H NMR (BHSC-500, DMSO-d6): d = 10.02
(s, 1H), 8.01 (s, 1H), 7.31 (t, J = 7.0 Hz, 1H), 7.23 (t,
J = 7.2 Hz, 1H), 7.16 (d, J = 7.0 Hz, 2H), 7.13 (d,
J = 7.0 Hz, 2H), 7.01 (t, J = 7.2 Hz, 1H), 6.89 (d,
J = 7.0 Hz, 1H), 4.97 (s, 1H), 4.85 (d, J = 5.2 Hz, 1H),
4.78 (t, J = 5.4 Hz, 1H), 4.21 (m, J = 5.2 Hz, 2H), 3.13
(d, J = 5.2 Hz, 2H), 2.25 (t, J = 5.5 Hz, 2H), 2.22
20
D
(t, J = 5.5 Hz, 2H). ½aꢄ ꢀ 109:4ꢅ (c 1.0, CH3OH); Anal.
Calcd for C24H23N3O4 C 69.05, H 5.55, N 10.07. Found C
68.90, H 5.62, N 10.21.
(d, J = 5.2 Hz, 2H), 2.90 (d, J = 5.4 Hz, 2H).
20
D
½aꢄ ꢀ 122:3ꢅ (c 1.0, CH3OH); Anal. Calcd for
4.3.11. (3S,12aS)-3-Benzyloxycarbonylmethyl-2,3,6,7,12,12a-
hexahydro pyrazino[10,20:1,6]-pyrido[3,4-b]indole-1,4-dione
(50k). Yield: 90%. Mp 255–257 ꢁC, ESI-MS (m/z) 404
[M+H]+; (KBr): 3346, 2923, 1678, 1600, 1589, 1507,
C21H19N3O3 C 69.79, H 5.30, N 11.63. Found C 69.61,
H 5.41, N 11.47.
1
4.3.7. (5aS,14aS)-1,2,3,5,5a,6,11,12,14,14a-Decahydro-
pyrrolo[100,200: 4050]pyrazino[10,20:1,6]-pyrido[3,4-b]indole-
1,4-dione(5g). Yield: 89%. Mp 233–235 ꢁC; ESI/MS
(m/z) 296 [M+H]+; IR (KBr): 3344, 2934, 1684, 1643,
1460, 1335, 747 cmꢀ1; H NMR (DMSO-d6, 300 MHz)
d = 9.98 (s,1H), 8.20 (s,1H), 7.30 (d, J = 7.4 Hz, 1H),
7.23 (t, J = 7.4 Hz, 1H), 7.20 (t, J = 7.2 Hz, 2H), 7.13 (t,
J = 7.2 Hz, 2H), 7.10 (t, J = 7.2 Hz, 1H), 7.03 (t,
J = 7.4 Hz, 1H), 6.92 (t, J = 7.3 Hz, 1H), 5.35 (d,
J = 16.6 Hz, 1H), 5.33 (s, 2H), 4.22 (dd, J = 11.0 Hz,
J = 3.6 Hz, 1H), 4.17 (d, J = 16.5 Hz, 1H), 4.12 (d,
J = 14.3 Hz, 1H), 3.16 (dd, J = 14.0 Hz, J = 3.6 Hz, 1H),
1460, 1332, 743 cmꢀ1; H NMR (BHSC-500, DMSO-
1
d6): d = 10.03 (s, 1H), 7.21 (t, J = 7.2 Hz, 1H), 7.15
(t, J = 7.2 Hz, 1H), 7.05 (t, J = 7.2 Hz, 1H), 6.95 (d,
J = 7.2 Hz, 1H), 4.86 (t, J = 5.5 Hz, 1H), 4.33
(t, J = 5.4 Hz, 1H), 4.24 (d, J = 5.5 Hz, 2H), 3.45
(t, J = 5.4 Hz, 2H), 2.93 (d, J = 5.3 Hz, 2H), 2.25 (d,
2.96 (t, J = 13.0 Hz, 1H), 2.81 (d, J = 10.0 Hz, 2H).
20
D
½aꢄ ꢀ 70ꢅ (c 1.0, CH3OH); Anal. Calcd for

5684
J. Liu et al. / Bioorg. Med. Chem. 15 (2007) 5672–5693
C23H21N3O4 C 68.47, H 5.25, N 10.42. Found C 68.35, H
5.09, N 10.27.
J = 13.5 Hz, 1H), 2.88 (t, J = 13.5 Hz, 1H). 13C NMR
(DMSO-d6, 300 MHz) d = 168.87, 167.66, 136.11,
134.02, 130.34, 124.63, 121.55, 120.21, 119.44, 111.22,
20
4.3.12. (3S,12aS)-3-(10,30-Imidazol-50-methylene)-2,3,6,7,12,12a-
hexahydropyrazino-[10,20:1,6]pyrido[3,4-b]indole-1,4-dione
(5l). Yield: 91%. Mp 207–209 ꢁC; ESI/MS (m/z) 336
[M+H]+; IR (KBr): 3344, 2925, 1677, 1601, 1587, 1505,
59.75, 52.76, 42.80, 24.77. ½aꢄ ꢀ 135 (c 1.0, CH3OH).
D
Anal. Calcd for C14H13N3O2 C 65.87, H 5.13, N 16.46.
Found C 65.65, H 5.01, N 16.67.
1
1458, 1333, 751 cmꢀ1; H NMR (BHSC-500, DMSO-
4.3.16. (3S,12aS)-3-(40-Benzyloxycarbonylaminobutyl)-
2,3,6,7,12,12a-hexahydro pyrazino-[10,20:1,6]pyrido[3,4-
b]indole-1,4-dione (50p). Yield: 90% Mp 212–214 ꢁC;
ESI/MS (m/z) 461 [M+H]+. IR (KBr): 3445, 3308,
d6): d = 12.99 (s, 1H), 10.03 (s, 1H), 7.98 (s, 1H), 7.45 (s,
1H), 7.33 (t, J = 7.0 Hz, 1H), 7.25 (t, J = 7.0 Hz, 1H),
7.09 (t, J = 7.0 Hz, 1H), 6.99 (t, J = 7.0 Hz, 1H), 6.87 (s,
1H), 4.92 (t, J = 5.2 Hz, 1H), 4.83 (t, J = 5.5 Hz, 1H),
1
2985, 1601, 1450, 1064, 900 cmꢀ1; H NMR (BHSC-
4.27 (d, J = 5.5 Hz, 2H), 3.20 (d, J = 5.5 Hz, 2H), 2.92
500, DMSO-d6): d = 10.03 (s, 1H), 8.00 (s, 1H), 7.99
(s, 1H), 7.27 (t, J = 7.2 Hz, 1H), 7.21 (t, J = 7.1 Hz,
2H), 7.19 (t, J = 7.1 Hz, 1H), 7.17 (t, J = 7.2 Hz, 1H),
7.16 (d, J = 7.1 Hz, 2H), 7.07 (t, J = 7.2 Hz, 1H), 6.89
(d, J = 7.2 Hz, 1H), 5.32 (s, 2H), 4.93 (d, J = 5.2 Hz,
1H), 4.47 (t, J = 4.5 Hz, 1H), 4.29 (dd, J = 10.0 Hz,
J = 4.2 Hz, 1H), 4.19 (dd, J = 10.0 Hz, J = 3.8 Hz,
1H), 2.98 (t, J = 4.7 Hz, 2H), 2.95 (d, J = 5.2 Hz, 2H),
20
D
(d, J = 5.1 Hz, 2H). ½aꢄ ꢀ 88:3ꢅ (c 1.0, CH3OH); Anal.
Calcd for C18H17N5O2 C 64.47, H 5.11, N 20.88. Found
C 64.62, H 5.20, N 21.03.
4.3.13. (3S,12aS)-3-Indolymethyl-2,3,6,7,12,12a-hexa-
hydropyrazino[10,20:1,6] pyrido[3,4-b]-indole-1,4-dione (5m).
Yield: 87%. Mp 176–180 ꢁC; ESI-MS (m/z) 385 [M+H]+;
1
IR(KBr): 3329, 2938, 1683, 1465, 1338, 746 cmꢀ1; H
1.91 (m, J = 4.7 Hz, 2H), 1.55 (m, J = 4.7 Hz, 2H),
20
D
NMR (DMSO-d6, 300 MHz): d = 10.76 (s, 1H), 10.74
(s, 1H), 8.43 (d,1H, J = 1.8 Hz, 1H), 7.50 (d,
J = 7.5 Hz, 1H), 7.28 (t, J = 7.5 Hz, 1H), 7.26 (t, J =
7.2 Hz, 1H), 7.24 (t, J = 7.2 Hz, 1H), 7.22 (d,
J = 7.2 Hz, 1H), 7.20 (t, J = 7.2 Hz, 1H), 7.08 (t,
J = 7.5 Hz, 1H), 6.95 (t,1H, J = 7.5 Hz, 1H), 6.82 (s,
1H), 5.27 (d, J = 16.2 Hz, 1H), 4.31 (d, J = 2.4 Hz,
1H), 4.05 (d, J = 16.5 Hz, 1H), 3.95 (dd, J = 12.0 Hz,
J = 4.5 Hz, 1H), 3.28 (dd, J = 14.1 Hz, J = 3.6, Hz,
1H), 3.07 (dd, J = 14.1 Hz, J = 4.2 Hz, 1H), 2.93 (d,
J = 3.5 Hz, 2H); 13C NMR (DMSO-d6, 300MHz)
d = 165.65, 164.64, 135.80, 135.71, 129.11, 127.62,
126.19, 124.00, 120.58, 118.45, 118.36, 118.10, 117.25,
1.29 (m, J = 4.7 Hz, 2H). ½aꢄ ꢀ 44ꢅ (c 1.0, CH3OH).
Anal. Calcd for C26H28N4O4 C 67.81, H 6.13, N
12.17. Found C 67.96, H 6.21, N 12.33.
4.3.17. (3S,12aS)-3-(Propionamide-20-yl)-2,3,6,7,12,12a-
hexahydropyrazino[10,20:1,6]pyrido-[3,4-b]indole-1,4-dione
(5q). Yield: 92%. Mp 251–253 ꢁC; ESI/MS (m/z) 327
[M+H]+. IR (KBr): 3344, 2939, 1684, 1465, 1332, 746
1
cmꢀ1; H NMR (BHSC-500, DMSO-d6): d = 10.01 (s,
1H), 7.98 (s, 1H), 7.27 (t, J = 7.2 Hz, 1H), 7.19 (t,
J = 7.2 Hz, 1H), 7.03 (d, J = 7.2 Hz, 1H), 6.85 (d,
J = 7.2 Hz, 1H), 6.11 (s, 2H), 4.93 (d, J = 5.5 Hz, 1H),
4.44 (t, J = 5.4 Hz, 1H), 4.27 (d, J = 5.5 Hz, 2H), 2.92
110.81, 108.02, 105.42, 79.06, 55.77, 55.36, 30.17,25.6;
(d, J = 5.2 Hz, 2H), 2.17 (t, J = 5.4 Hz, 2H), 2.11 (t,
20
D
20
D
½aꢄ ꢀ 182ꢅ (c 0.34, CHCl3/CH3OH, 1:1, v/v). Anal.
J = 5.4 Hz, 2H). ½aꢄ ꢀ 62ꢅ (c 1.0, CH3OH); Anal.
Calcd for C23H20N4O2 C 71.86, H 5.24, N 14.57. Found
C 72.04, H 5.56, N 14.33.
Calcd for C17H18N4O3 C 62.57, H 5.56, N 17.17. Found
C 62.71, H 5.64, N 17.35.
4.3.14. (3S,12aS)-3-(30-Guanidinopropyl)-2,3,6,7,12,12a-
hexahydropyrazino[10,20:1,6]pyrido-[3,4-b]indole-1,4-dione
(5n). Yield: 90%. Mp 240–242 ꢁC; ESI/MS (m/z) 355
[M+H]+; IR (KBr): 3339, 2941, 1680, 1456,
4.3.18. (3S,12aS)-3-(Acetylamine-10-yl)-2,3,6,7,12,12a-
hexahydropyrazino[10,20:1,6]pyrido-[3,4-b]indole-1,4-dione
(5r). Yield: 91%. Mp 247–249 ꢁC; ESI/MS (m/z) 313
[M+H]+. IR (KBr): 3341, 2942, 1681, 1463, 1336, 741
1
1342, 743 cmꢀ1
;
1H NMR (BHSC-500, DMSO-d6):
cmꢀ1; H NMR (BHSC-500, DMSO-d6): d = 10.03 (s,
d = 10.03 (s, 1H), 8.44 (s, 2H), 8.20 (s, 1H), 8.16 (s,
1H), 8.01 (s, 1H), 7.23 (t, J = 7.0 Hz, 1H), 7.15
(t, J = 7.0 Hz, 1H), 7.02 (d, J = 7.0 Hz, 1H), 6.95
(d, J = 7.0 Hz, 1H), 4.92 (d, J = 5.2 Hz, 1H), 4.35
(t, J = 4.6 Hz, 1H), 4.23 (d, J = 5.2 Hz, 2H), 2.91
1H), 8.00 (s, 1H), 7.27 (t, J = 7.2 Hz, 1H), 7.19 (t,
J = 7.2 Hz, 1H), 7.05 (d, J = 7.2 Hz, 1H), 6.87 (d,
J = 7.2 Hz, 1H), 6.06 (s, 2H), 4.93 (d, J = 5.3 Hz, 1H),
4.45 (t, J = 5.2 Hz, 1H), 4.23 (d, J = 5.3 Hz, 2H), 2.92
(d, J = 5.4 Hz, 2H), 2.65 (t, J = 5.3 Hz, 2H).
20
D
(d, J = 4.7 Hz, 2H), 2.66 (t, J = 5.2 Hz, 2H), 1.95 (m,
½aꢄ ꢀ 45:2ꢅ (c 1.0, CH3OH). Anal. Calcd for
20
J = 5.2 Hz, 2H), 1.56 (m, J = 5.2 Hz, 2H). ½aꢄ ꢀ 90:6ꢅ
C16H16N4O3 C 61.53, H 5.16, N 17.94. Found C
61.38, H 5.08, N 17.76.
D
(c 1.0, CH3OH); Anal. Calcd for C18H22N6O2 C 61.00,
H 6.26, N 23.71. Found C 61.18, H 6.33, N 23.53.
4.3.19. (3S,12aS)-3-(20-Methylpropyl)-2,3,6,7,12,12a-
hexahydropyrazino[10,20:1,6]pyrido-[3,4-b]indole-1,4-dione
(5s). Yield: 89%. Mp 231–233 ꢁC; ESI/MS (m/z) 312
[M+H]+. IR (KBr): 3343, 2943, 1682, 1465, 1335, 743
4.3.15. (12aS)-2,3,6,7,12,12a-Hexahydropyrazino[10,20:1,6]pyr-
ido[3,4-b]indole-1,4-dione (5o). Yield: 92%. Mp 247–
249 ꢁC; ESI-MS (m/z) 256 [M+H]+; IR (KBr) : 3307,
2986, 1646, 1455, 1328, 748 cmꢀ1; 1H NMR (DMSO-d6,
300 MHz): d = 10.94 (s,1 H), 8.26 (s, 1H), 7.43 (d,
J = 7.5 Hz, 1H), 7.33 (t, J = 7.5 Hz, 1H), 7.08 (t,
J = 7.5 Hz, 1H), 6.96 (t, J = 7.5 Hz, 1H), 5.36 (d,
J = 16.5 Hz, 1H), 4.22 (m, J = 13.0 Hz, 2H), 4.05 (d,
J = 17.7 Hz, 1H), 3.86 (d, J = 17.7 Hz, 1H), 3.20 (m,
1
cmꢀ1; H NMR (BHSC-500, DMSO-d6): d = 10.02 (s,
1H), 7.98 (s, 1H), 7.27 (t, J = 7.2 Hz, 1H), 7.19 (t,
J = 7.2 Hz, 1H), 7.03 (d, J = 7.2 Hz, 1H), 6.85 (d,
J = 7.2 Hz, 1H), 4.93 (t, J = 5.3 Hz, 1H), 4.45 (t,
J = 5.2 Hz, 1H), 4.26 (dd, J = 10.0 Hz, J = 4.4 Hz,
1H), 4.15 (dd, J = 10.0 Hz, J = 3.9 Hz, 1H), 2.92 (d,

J. Liu et al. / Bioorg. Med. Chem. 15 (2007) 5672–5693
5685
J = 6.2 Hz, 2H), 2.65 (t, J = 5.3 Hz, 2H), 1.83 (m,
4.4.2. (3S)-N-(Boc-L-phenylalanyl)-1,2,3,4-tetrahydro-b-
carboline-3-carboxylic acid benzylester (8b). Yield: 95%.
Mp 129–131 ꢁC; ESI/MS (m/z) 554 [M+H]+; IR (KBr):
3345, 3014, 2942, 2833, 1751, 1647, 1603, 1452, 1389,
20
J = 5.3 Hz, 1H), 1.07 (d, J = 5.3 Hz, 6H). ½aꢄ ꢀ 52ꢅ
D
(c 1.0, CH3OH). Anal. Calcd for C18H21N3O2 C 69.43,
H 6.80, N 13.49. Found C 69.60, H 6.71, N 13.66.
1062, 901 cmꢀ1 1H NMR (BHSC-500, DMSO-d6):
;
4.3.20. (3S,12aS)-3-(10-Methylpropyl)-2,3,6,7,12,12a-hexa-
hydropyrazino[10,20:1,6]pyrido-[3,4-b]indole-1,4-dione(5t).
Yield: 87%. Mp 219–220 ꢁC, ESI-MS (m/z) 312 [M+H]+;
d = 10.01 (s, 1H), 7.89 (s, 1H), 7.27 (t, J = 7.3 Hz, 1H),
7.20 (t, J = 7.5 Hz, 2H), 7.18 (t, J = 7.1 Hz, 2H), 7.17
(t, J = 7.3 Hz, 1H), 7.13 (d, J = 7.6 Hz, 2H), 7.10 (d,
J = 7.1 Hz, 2H), 7.07 (t, J = 7.1 Hz, 1H), 7.05 (t,
J = 7.6 Hz, 1H), 6.95 (t, J = 7.2 Hz, 1H), 6.90 (t,
J = 7.3 Hz, 1H), 5.33 (s, 2H), 5.01 (t, J = 5.3 Hz, 1H),
4.79 (t, J = 5.4 Hz, 1H), 3.91 (s, 2H), 3.65 (dd,
J = 10.1 Hz, J = 5.2 Hz, 1H), 3.23 (dd, J = 10.1 Hz,
IR(KBr): 3328, 2936, 1669, 1455, 1388, 1369, 744 cmꢀ1
;
1H NMR (DMSO-d6, 300 MHz): d = 11.12 (s,1H), 8.64
(d, J = 2.1 Hz, 1H), 7.40 (d, J = 7.3 Hz, 1H), 7.30 (t,
J = 7.3 Hz, 1H), 7.00 (t, J = 7.3 Hz, 1H), 6.95 (t,
J = 7.3 Hz, 1H), 5.30 (d, J = 16.0 Hz, 1H), 4.25 (dd,
J = 11.0 Hz, J = 4.0 Hz, 1H), 4.17 (d, J = 17.3 Hz, 1H),
3.22 (dd, J = 15.0 Hz, J = 3.3 Hz, 1H), 2.96 (m,
J = 13.0 Hz, 1H), 2.83 (t, J = 13.0 Hz, 1H), 2.61 (m,
J = 6.0 Hz, 1H), 1.35 (m, J = 6.0 Hz, 2H), 1.07 (d,
J = 6.0 Hz, 3H), 0.97 (t, J = 6.0 Hz, 6H). 13C NMR
(DMSO-d6, 300 MHz) d = 167.00, 166.48, 136.13,
130.37, 126.51, 121.44, 118.96, 117.40, 111.20, 105.81,
J = 2.9 Hz, 1H), 3.09 (d, J = 5.3 Hz, 2H), 1.47 (s, 9H).
20
D
½aꢄ ꢀ 80ꢅ (c 0.35, CHCl3/CH3OH, 1:1, v/v). Anal.
Calcd for C33H35N3O5 C 71.59, H 6.37, N 7.59. Found
C 71.74, H 6.46, N 7.73.
4.4.3. (3S)-N-(Boc-L-valinyl)-1,2,3,4-tetrahydro-b-carbo-
line-3-carboxylic acid benzylester (8c). Yield: 89%. Mp
127–129 ꢁC; ESI/MS (m/z) 506 [M+H]+; IR (KBr):
3343, 3002, 2941, 2844, 1743, 1642, 1600, 1453, 1392,
56.30, 53.11, 46.12, 26.75, 34.34, 23.30, 14.82, 11.77.
20
D
½aꢄ ꢀ 73:7ꢅ (c 1.0, CH3OH); Anal. Calcd for
1
C18H21N3O2 C 9.43, H 6.80, N 13.49. Found C 69.22,
H 6.61, N 13.68.
1375, 1070, 901 cmꢀ1; H NMR (BHSC-500, DMSO-
d6) d = 10.02 (s,1H), 8.03 (s,1H), 7.25 (t, J = 7.2 Hz,
1H), 7.20 (t, J = 7.0 Hz, 2H), 7.13 (t, J = 7.2 Hz, 1H),
7.12 (d, J = 7.0 Hz, 2H), 7.08 (t, J = 7.0 Hz, 1H), 6.96
(t, J = 7.2 Hz, 1H), 6.91 (t, J = 7.2 Hz, 1H), 5.33 (s,
2H), 4.72 (t, J = 5.7 Hz, 1H), 4.53 (d, J = 5.1 Hz, 1H),
3.87 (s, 2H), 3.62 (dd, J = 10.1 Hz, J = 5.3 Hz, 1H),
3.23 (dd, J = 10.1 Hz, J = 3.3 Hz, 1H), 2.65 (m, J = 5.1
4.4. General procedure for the preparation of (3S)-N-
(Boc-aminoacyl)-1,2,3,4-tetrahydro-b-carboline-3-car-
boxylic acid benzylester (8a–t)
At 0 ꢁC to the stirring solution of 2.50 mmol of Boc-AA-
OH, 350 mg(2.59 mmol) of HOBt, and 600 mg
(2.90 mmol) of DCC in 15 ml of anhydrous dichloro-
methane, 664 mg (2.17 mmol) of 3S-1,2,3,4-tetrahydro-
b-carboline-3-carboxylic acid benzylester (7) was added.
After stirring at 0 ꢁC for 30 min the reaction mixture
was stirred at room temperature for 30 min and TLC
(ethyl acetate/petroleum, 5:12) indicated the completion
of the reaction. The formed precipitates of DCU were
filtered and the filtrate was evaporated. The residue
was dissolved in 30 ml of ethyl acetate and the solution
was washed successively with saturated aqueous NaH-
CO3 (30 ml · 3), saturated aqueous NaCl (30 ml · 3),
and aqueous KHSO4 (5%, 30 ml · 3). The separated
ethyl acetate layer was dried with anhydrous MgSO4, fil-
tered, evaporated, and purified with flash chromatogra-
phy (CHCl3/CH3OH, 30:1) to provide the title
compound as powder.
Hz, 1H), 1.47 (s, 9H), 1.07 (d, J = 5.3 Hz, 6H).
20
D
½aꢄ ꢀ 58ꢅ (c 0.39, CHCl3/CH3OH, 1:1, v/v). Anal.
Calcd for C29H35N3O5 C 68.89, H 6.98, N 8.31. Found
C 68.73, H 6.87, N 8.47.
4.4.4. (3S)-N-(Boc-L-serinyl)-1,2,3,4-tetrahydro-b-carbo-
line-3-carboxylic acid benzylester (8d). Yield: 90%. Mp
127–129 ꢁC; ESI/MS (m/z) 494 [M+H]+; IR (KBr):
3345, 3013, 2940, 2844, 1750, 1642, 1600, 1457, 1392,
1073, 902 cmꢀ1 1H NMR (BHSC-500, DMSO-d6):
;
d = 10.01 (s, 1H), 8.03 (s, 1H), 7.25 (t, J = 7.2 Hz, 1H),
7.21 (t, J = 7.0 Hz, 2H), 7.15 (t, J = 7.2 Hz, 1H), 7.11
(d, J = 7.0 Hz, 2H), 7.08 (t, J = 7.0 Hz, 1H), 7.01 (t,
J = 7.2 Hz, 1H), 6.90 (t, J = 7.1 Hz, 1H), 5.35 (s, 2H),
4.73 (t, J = 5.6 Hz, 1H), 4.60 (t, J = 5.1 Hz, 1H), 4.05
(d, J = 5.3 Hz, 1H), 3.90 (s, 2H), 3.62 (dd, J = 10.1 Hz,
J = 5.3 Hz, 1H), 3.23 (dd, J = 10.1 Hz, J = 2.9 Hz,
20
D
1H), 3.10 (d, J = 5.1 Hz, 2H), 1.45 (s, 9H). ½aꢄ ꢀ 51ꢅ
4.4.1. (3S)-N-(Boc-L-alanyl)-1,2,3,4-tetrahydro-b-carbo-
line-3-carboxylic acid benzylester (8a). Yield: 94%. Mp
130–132 ꢁC; ESI/MS (m/z) 478 [M+H]+; IR (KBr):
3347, 3005, 2951, 2843, 1745, 1642, 1604, 1457, 1391,
(c 0.35, CHCl3/CH3OH, 1:1, v/v). Anal. Calcd for
C27H31N3O6 C 65.71, H 6.33, N 8.51. Found C 65.50,
H 6.25, N 8.68.
1
1371, 1071, 902 cmꢀ1; H NMR (BHSC-500, DMSO-
4.4.5. (3S)-N-(Boc-L-threoninyl)-1,2,3,4-tetrahydro-b-
carboline-3-carboxylic acid benzylester (8e). Yield: 91%.
Mp 131–133 ꢁC; ESI/MS (m/z) 508 [M+H]+; IR (KBr):
3443, 3336, 3208, 3003, 2950, 2841, 1762, 1735, 1643,
d6): d = 9.97 (s,1H), 8.03 (s,1H), 7.24 (t, J = 7.2 Hz,
1H), 7.20 (t, J = 7.1 Hz, 2H), 7.13 (t, J = 7.2 Hz, 1H),
7.10 (d, J = 7.1 Hz, 2H), 7.07 (t, J = 7.1 Hz, 1H), 6.97
(t, J = 7.2 Hz, 1H), 6.93 (t, J = 7.2 Hz, 1H), 5.33 (s,
2H), 4.75 (m, J = 5.5 Hz, 1H), 4.66 (m, J = 5.4 Hz,
1H), 3.83 (s, 2H), 3.64 (dd, J = 10.1 Hz, J = 5.2 Hz,
1602, 1455, 1393, 1373, 1062, 904 cmꢀ1 1H NMR
;
(BHSC-500, DMSO-d6): d = 10.02 (s, 1H), 7.87 (s,
1H), 7.27 (t, J = 7.3 Hz, 1H), 7.23 (t, J = 7.3 Hz, 1H),
7.17 (t, J = 7.1 Hz, 2H), 7.13 (d, J = 7.1 Hz, 2H), 7.05
(t, J = 7.1 Hz, 1H), 6.99 (d, J = 7.3 Hz, 1H), 6.87 (t,
J = 7.3 Hz, 1H), 5.30 (s, 2H), 4.83 (t, J = 5.4 Hz, 1H),
4.61 (m, J = 5.4 Hz, 1H), 4.43 (t, J = 5.3 Hz, 1H), 3.97
1H), 3.25 (dd, J = 10.1 Hz, J = 2.9 Hz, 1H), 1.49 (s,
20
D
9H), 1.45 (d, J = 5.1 Hz, 3H). ½aꢄ ꢀ 1248ꢅ (c 0.35,
CHCl3/CH3OH, 1:1, v/v). Anal. Calcd for C27H31N3O5
C 67.91, H 6.54, N 8.80. Found C 67.76, H 6.65, N 8.97.

5686
J. Liu et al. / Bioorg. Med. Chem. 15 (2007) 5672–5693
(m, J = 5.5 Hz, 2H), 2.92 (d, J = 5.3 Hz, 2H), 2.17 (s,
1H), 1.46 (s, 9H), 1.20 (d, J = 5.3 Hz, 3H). Anal. Calcd
for C28H33N3O6 C 66.26, H 6.55, N 8.28. Found C
66.12, H 6.43, N 8.42.
4.44 (t, J = 5.3 Hz, 1H), 4.23 (d, J = 5.1 Hz, 2H), 2.97
(d, J = 5.2 Hz, 2H), 2.41 (t, J = 5.2 Hz, 2H), 2.21 (d,
J = 5.3 Hz, 2H), 2.11 (s, 3H), 1.47 (s, 9H). Anal. Calcd
for C29H35N3O5S C 64.78, H 6.56, N 7.82. Found C
64.60, H 6.47, N 7.97.
4.4.6. (3S)-N-(Boc-L-tyrosinyl)-1,2,3,4-tetrahydro-b-
carboline-3-carboxylic acid benzylester (8f). Yield: 89%.
Mp 133–135 ꢁC; ESI/MS (m/z) 570 [M+H]+; IR (KBr):
3442, 3206, 3003, 2954, 2840, 1734, 1642, 1603, 1452,
4.4.10. (3S)-N-[Boc-L-glutamyl(OBzl)]-1,2,3,4-tetrahy-
dro-b-carboline-3-carboxylic acid benzylester (8j). Yield:
91%. Mp 125–127 ꢁC; ESI/MS (m/z) 626 [M+H]+; IR
(KBr): 3442, 3206, 3003, 2945, 2823, 1736, 1641, 1604,
1391, 1373, 1062, 903 cmꢀ1 1H NMR (BHSC-500,
;
1
DMSO-d6): d = 10.01 (s, 1H), 8.03 (s, 1H), 7.33 (t,
J = 7.2 Hz, 1H), 7.21 (t, J = 7.4 Hz, 1H), 7.14 (d,
J = 7.2 Hz, 2H), 7.11 (t, J = 7.2 Hz, 2H), 7.07 (d,
J = 7.2 Hz, 2H), 7.03 (t, J = 7.2 Hz, 1H), 7.01 (d,
J = 7.2 Hz, 1H), 6.90 (d, J = 7.4 Hz, 1H), 6.88 (d,
J = 7.2 Hz, 2H), 5.41 (s, 2H), 4.95 (s, 1H), 4.88 (d,
J = 5.3 Hz, 1H), 4.80 (t, J = 5.5 Hz, 1H), 4.23 (m,
J = 5.4 Hz, 2H), 3.15 (d, J = 5.4 Hz, 2H), 2.92 (d,
J = 5.2 Hz, 2H), 1.48 (s, 9H). Anal. Calcd for
C33H35N3O6 C 69.58, H 6.19, N 7.38. Found C 69.43,
H 6.12, N 7.55.
1450, 1390, 1373, 1060, 896 cmꢀ1; H NMR (BHSC-
500, DMSO-d6): d = 9.98 (s, 1H), 8.01 (s, 1H), 7.34 (t,
J = 7.3 Hz, 1H), 7.24 (t, J = 7.3 Hz, 1H), 7.21 (t,
J = 7.1 Hz, 2H), 7.18 (t, J = 7.2 Hz, 2H), 7.17 (d,
J = 7.1 Hz, 2H), 7.15 (d, J = 7.2 Hz, 2H), 7.14 (t,
J = 7.1 Hz, 1H), 7.12 (t, J = 7.2 Hz, 1H), 7.00 (d,
J = 7.3 Hz, 1H), 6.87 (d, J = 7.3 Hz, 1H), 5.36 (s, 2H),
5.34 (s, 2H), 4.91 (d, J = 5.2 Hz, 1H), 4.44 (t, J = 5.4
Hz, 1H), 4.23 (d, J = 5.3 Hz, 2H), 2.97 (d, J = 5.1 Hz,
2H), 2.23 (t, J = 5.4 Hz, 2H), 2.20 (t, J = 5.4 Hz, 2H),
1.47 (s, 9H). Anal. Calcd for C36H39N3O7 C 69.10, H
6.28, N 6.72. Found C 68.97, H 6.21, N 6.88.
4.4.7. (3S)-N-(Boc-L-prolinyl)-1,2,3,4-tetrahydro-b-carbo-
line-3-carboxylic acid benzylester (8g). Yield: 91%. Mp
113–115 ꢁC; ESI/MS (m/z) 504 [M+H]+; IR (KBr):
3441, 3209, 3001, 2954, 2840, 1733, 1644, 1603, 1450,
4.4.11. (3S)-N-[Boc-L-aspartyl(OBzl)]-1,2,3,4-tetrahy-
dro-b-carboline-3-carboxylic acid benzylester (8k). Yield:
90%. Mp 139–141 ꢁC; ESI/MS (m/z) 612 [M+H]+; IR
(KBr): 3441, 3342, 3210, 3000, 2954, 2841, 1754, 1732,
1392, 1370, 1062, 903 cmꢀ1 1H NMR (BHSC-500,
;
DMSO-d6): d = 10.01 (s, 1H), 7.31 (t, J = 7.2 Hz, 1H),
7.19 (t, J = 7.4 Hz, 1H), 7.15 (t, J = 7.0 Hz, 2H), 7.10
(d, J = 7.0 Hz, 2H), 7.07 (t, J = 7.0 Hz, 1H), 7.03 (d,
J = 7.4 Hz, 1H), 6.91 (d, J = 7.2 Hz, 1H), 5.33 (s, 2H),
4.84 (t, J = 5.4 Hz, 1H), 4.30 (t, J = 5.6 Hz, 1H), 4.21
(d, J = 5.3 Hz, 2H), 3.46 (t, J = 5.6 Hz, 2H), 2.91 (d,
J = 5.4 Hz, 2H), 2.29 (d, J = 5.4 Hz, 2H), 1.93 (t,
J = 4.7 Hz, 2H), 1.47 (s, 9H). Anal. Calcd for
C29H33N3O5 C 69.17, H 6.60, N 8.34. Found C 69.32,
H 6.68, N 8.49.
1643, 1600, 1452, 1389, 1368, 1061, 900 cmꢀ1 1H
;
NMR (BHSC-500, DMSO-d6): d = 9.97 (s, 1H), 8.00
(s, 1H), 7.28 (t, J = 7.1 Hz, 1H), 7.22 (t, J = 7.1 Hz,
1H), 7.19 (t, J = 6.9 Hz, 2H), 7.17 (t, J = 6.9 Hz, 2H),
7.13 (d, J = 6.9 Hz, 2H), 7.11 (d, J = 6.9 Hz, 2H), 7.09
(t, J = 6.9 Hz, 1H), 7.06 (t, J = 6.9 Hz, 1H), 7.00 (d,
J = 7.1 Hz, 1H), 6.96 (d, J = 7.1 Hz, 1H), 5.34 (s, 2H),
5.31 (s, 2H), 4.92 (d, J = 5.1 Hz, 1H), 4.76 (t, J = 5.1
Hz, 1H), 4.25 (d, J = 5.1 Hz, 2H), 2.92 (d, J = 5.1 Hz,
2H), 2.85 (d, J = 5.1 Hz, 2H), 1.45 (s, 9H). Anal. Calcd
for C35H37N3O7 C 68.72, H 6.10, N 6.87. Found C
68.86, H 6.18, N 7.02.
4.4.8. (3S)-N-(Boc-L-cysteinyl)-1,2,3,4-tetrahydro-b-
carboline-3-carboxylic acid benzyl-ester (8h). Yield:
90%. Mp 131–133 ꢁC; ESI/MS (m/z) 510 [M+H]+; IR
(KBr): 3445, 3201, 3000, 2942, 2845, 1732, 1643, 1601,
4.4.12. (3S)-N-(Boc-L-histidinyl)-1,2,3,4-tetrahydro-b-
carboline-3-carboxylic acid benzyl-ester (8l). Yield:91%.
Mp 137–139 ꢁC; ESI/MS (m/z) 544 [M+H]+; IR (KBr):
3442, 3201, 3004, 2941, 2830, 1732, 1643, 1602, 1455,
1
1452, 1391, 1373, 1060, 901 cmꢀ1; H NMR (BHSC-
500, DMSO-d6): d = 10.00 (s, 1H), 8.01 (s, 1H), 7.27
(t, J = 7.2 Hz, 1H), 7.21 (t, J = 7.4 Hz, 1H), 7.19 (t,
J = 7.2 Hz, 2H), 7.13 (d, J = 7.2 Hz, 2H), 7.08 (t,
J = 7.2 Hz, 1H), 7.06 (d, J = 7.4 Hz, 1H), 6.87 (d,
J = 7.2 Hz, 1H), 5.33 (s, 2H), 4.96 (t, J = 5.3 Hz, 1H),
4.75 (t, J = 5.5 Hz, 1H), 4.25 (d, J = 5.3 Hz, 2H), 3.12
(d, J = 5.3 Hz, 2H), 3.05 (d, J = 5.4 Hz, 2H), 1.48 (s,
9H), 1.63 (s, 1H). Anal. Calcd for C27H31N3O5S C
63.63, H 6.13, N 8.25. Found C 63.79, H 6.20, N 8.11.
1393, 1374, 1062, 901 cmꢀ1 1H NMR (BHSC-500,
;
DMSO-d6): d = 12.97 (s, 1H), 10.01 (s, 1H), 8.03 (s,
1H), 7.47 (s, 1H), 7.33 (t, J = 7.1 Hz, 1H), 7.20 (t,
J = 7.1 Hz, 2H), 7.15 (t, J = 7.2 Hz, 1H), 7.13 (d,
J = 7.1 Hz, 2H), 7.11 (d, J = 7.2Hz, 1H), 7.05 (t,
J = 7.1 Hz, 1H), 6.97 (t, J = 7.1 Hz, 1H), 6.86 (s, 1H),
5.35 (s, 2H), 4.93 (t, J = 5.1 Hz, 1H), 4.81 (t, J = 5.3
Hz, 1H), 4.24 (d, J = 5.2 Hz, 2H), 3.23 (d, J = 5.3 Hz,
2H), 2.93 (d, J = 5.2 Hz, 2H), 1.48 (s, 9H). Anal. Calcd
for C30H33N5O5 C 66.28, H 6.12, N 12.88. Found C
66.13, H 6.04, N 13.04.
4.4.9. (3S)-N-(Boc-L-methioninyl)-1,2,3,4-tetrahydro-
b-carboline-3-carboxylic acid benzylester (8i). Yield:
90%. Mp 144–146 ꢁC; ESI/MS (m/z) 538 [M+H]+; IR
(KBr): 3443, 3205, 3002, 2954, 2845, 1733, 1642, 1603,
4.4.13. (3S)-N-(Boc-L-tryptophanyl)-1,2,3,4-tetrahydro-
b-carboline-3-carboxylic acid benzylester (8m). Yield:
90%. Mp 144–146 ꢁC; ESI/MS (m/z) 593 [M+H]+. IR
(KBr): 3441, 3332, 3211, 3002, 2941, 2830, 1755, 1732,
1
1455, 1392, 1373, 1061, 902 cmꢀ1; H NMR (BHSC-
500, DMSO-d6): d = 10.02 (s, 1H), 7.97 (s, 1H), 7.30
(t, J = 7.2 Hz, 1H), 7.21 (t, J = 7.4 Hz, 1H), 7.17 (t,
J = 7.4 Hz, 2H), 7.14 (d, J = 7.4 Hz, 2H), 7.10 (t,
J = 7.4 Hz, 1H), 6.93 (d, J = 7.2 Hz, 1H), 6.85 (d,
J = 7.2 Hz, 1H), 5.34 (s, 2H), 4.85 (t, J = 5.2 Hz, 1H),
1641, 1600, 1443, 1392, 1373, 1062, 900 cmꢀ1 1H
;
NMR (BHSC-500, DMSO-d6): d = 9.99 (s, 1H), 9.97
(s, 1H), 8.01 (s, 1H), 7.27 (t, J = 7.1 Hz, 1H), 7.25 (t,

J. Liu et al. / Bioorg. Med. Chem. 15 (2007) 5672–5693
5687
J = 7.0 Hz, 1H), 7.21 (t, J = 6.9 Hz, 2H), 7.15 (d,
J = 6.9 Hz, 2H), 7.11 (d, J = 7.3 Hz, 1H), 7.07 (t,
J = 6.9 Hz, 1H), 7.05 (t, J = 7.3 Hz, 1H), 7.03 (d,
J = 7.1 Hz, 1H), 7.00 (t, J = 7.3 Hz, 1H), 6.99 (d,
J = 7.1 Hz, 1H), 6.96 (d, J = 7.0 Hz, 1H), 6.83 (s, 1H),
5.32 (s, 2H), 4.93 (d, J = 5.1 Hz, 1H), 4.75 (t,
J = 5.2 Hz, 1H), 4.23 (d, J = 5.1 Hz, 2H), 3.15 (d,
Calcd for C38H44N4O7 C 68.24, H, 6.63, N 8.38. Found
C 68.37, H 6.71, N 8.22.
4.4.17. (3S)-N-(Boc-L-glutaminyl)-1,2,3,4-tetrahydro-
b-carboline-3-carboxylic acid benzyl-ester (8q). Yield:
88%. Mp 137–139 ꢁC; ESI/MS (m/z) 535 [M+H]+. IR
(KBr): 3443, 3211, 3007, 2940, 2835, 1732, 1644, 1605,
1
J = 5.1 Hz, 2H), 2.95 (d, J = 6.0 Hz, 2H), 1.48 (s, 9H).
1453, 1393, 1372, 1060, 900 cmꢀ1; H NMR (BHSC-
20
D
½aꢄ ¼ ꢀ70ꢅ (c 0.34, CHCl3/CH3OH, 1:1, v/v). Anal.
500, DMSO-d6): d = 9.99 (s, 1H), 8.01 (s, 1H), 7.25 (t,
J = 7.1 Hz, 1H), 7.21 (t, J = 7.0 Hz, 2H), 7.16 (t,
J = 7.1 Hz, 1H), 7.13 (d, J = 7.0 Hz, 2H), 7.10 (t,
J = 7.0 Hz, 1H), 7.01 (d, J = 7.1 Hz, 1H), 6.87 (d,
J = 7.1 Hz, 1H), 6.09 (s, 2H), 5.37 (s, 2H), 4.92 (d,
J = 5.3 Hz, 1H), 4.41 (t, J = 5.3 Hz, 1H), 4.25 (d,
J = 5.4 Hz, 2H), 2.93 (d, J = 5.1 Hz, 2H), 2.17 (t,
Calcd for C35H36N4O5 C 70.93, H 6.12, N 9.45. Found
C 71.08, H 6.20, N 9.62.
4.4.14. (3S)-N-(Boc-L-argininyl)-1,2,3,4-tetrahydro-b-
carboline-3-carboxylic acid benzyl-ester (8n). Yield:
87%. Mp 133–135 ꢁC; ESI/MS (m/z) 563 [M+H]+; IR
(KBr): 3443, 3205, 3001, 2946, 2842, 1727, 1645, 1600,
J = 5.2 Hz, 2H), 2.14 (t, J = 5.2 Hz, 2H), 1.47 (s, 9H).
20
D
1
1453, 1393, 1372, 1061, 903 cmꢀ1; H NMR (BHSC-
½aꢄ ¼ ꢀ47ꢅ (c 0.37, CHCl3/CH3OH, 1:1, v/v). Anal.
500, DMSO-d6): d = 10.01 (s, 1H), 8.40 (s, 2H), 8.23
(s, 1H), 8.15 (s, 1H), 8.03 (s, 1H), 7.25 (t, J = 7.2 Hz,
1H), 7.22 (t, J = 7.0 Hz, 2H), 7.15 (d, J = 7.0 Hz, 2H),
7.13 (t, J = 7.2 Hz, 1H), 7.11 (t, J = 7.0 Hz, 1H), 7.00
(d, J = 7.2 Hz, 1H), 6.93 (d, J = 7.2 Hz, 1H), 5.36 (s,
2H), 4.90 (d, J = 5.1 Hz, 1H), 4.37 (t, J = 4.5 Hz, 1H),
4.25 (d, J = 5.1 Hz, 2H), 2.93 (d, J = 4.6 Hz, 2H), 2.68
(t, J = 5.1 Hz, 2H), 1.93 (m, J = 5.1 Hz, 2H), 1.54 (m,
J = 5.1 Hz, 2H), 1.53 (s, 9H). Anal. Calcd for
C30H38N6O5 C 64.04, H 6.81, N 14.94. Found C
64.19, H 6.90, N 14.79.
Calcd for C29H34N4O6 C 65.15, H 6.41, N 10.48. Found
C 65.01, H 6.30, N 10.65.
4.4.18. (3S)-N-(Boc-L-asparginyl)-1,2,3,4-tetrahydro-b-
carboline-3-carboxylic acid benzyl-ester (8r). Yield:
89%. Mp 136–138 ꢁC; ESI/MS (m/z) 521 [M+H]+. IR
(KBr): 3441, 3203, 3001, 2935, 2837, 1732, 1634, 1601,
1
1455, 1393, 1376, 1062, 901 cmꢀ1; H NMR (BHSC-
500, DMSO-d6): d = 9.99 (s, 1H), 8.02 (s, 1H), 7.26 (t,
J = 7.1 Hz, 1H), 7.21 (t, J = 7.2 Hz, 2H), 7.17 (t,
J = 7.1 Hz, 1H), 7.13 (d, J = 7.2 Hz, 2H), 7.10 (t,
J = 7.2 Hz, 1H), 7.02 (d, J = 7.1 Hz, 1H), 6.89 (d,
J = 7.1 Hz, 1H), 6.03 (s, 2H), 5.35 (s, 2H), 4.95 (d,
J = 5.1 Hz, 1H), 4.43 (t, J = 5.1 Hz, 1H), 4.20 (d,
4.4.15. (3S)-N-(Boc-L-glycinyl)-1,2,3,4-tetrahydro-b-
carboline-3-carboxylic acid benzyl-ester (8o). Yield:
93%. Mp 133–135 ꢁC; ESI/MS (m/z) 464 [M+H]+. IR
(KBr): 3441, 3332, 3001, 2944, 2843, 1760, 1735, 1601,
J = 5.1 Hz, 2H), 2.93 (d, J = 5.2 Hz, 2H), 2.15 (t,
20
D
J = 5.2 Hz, 2H), 1.46 (s, 9H). ½aꢄ ¼ ꢀ37ꢅ (c 0.32,
1
1459, 1390, 1376, 1062, 900 cmꢀ1; H NMR (BHSC-
CHCl3/CH3OH, 1:1, v/v). Anal. Calcd for C28H32N4O6
C 64.60, H 6.20, N 10.76. Found C 64.75, H 6.27, N
10.89.
500, DMSO-d6): d = 9.99 (s, 1H), 8.02 (s, 1H), 7.25 (t,
J = 7.2 Hz, 1H), 7.20 (t, J = 7.0 Hz, 2H), 7.19 (t,
J = 7.2 Hz, 1H), 7.13 (d, J = 7.0 Hz, 2H), 7.09 (t,
J = 7.0 Hz, 1H), 6.95 (d, J = 7.2 Hz, 1H), 6.87 (d,
J = 7.2 Hz, 1H), 5.35 (s, 2H), 4.84 (d, J = 5.1 Hz, 1H),
4.23 (dd, J = 10.1 Hz, J = 4.1 Hz, 1H), 4.15 (dd,
4.4.19. (3S)-N-(Boc-L-leucinyl)-1,2,3,4-tetrahydro-b-
carboline-3-carboxylic acid benzyl-ester (8s). Yield:
91%. Mp 153–155 ꢁC; ESI/MS (m/z) 520 [M+H]+. IR
(KBr): 3443, 3205, 3002, 2954, 2846, 1733, 1648, 1601,
J = 10.1 Hz, J = 3.5 Hz, 1H), 4.11 (s, 2H), 2.95 (d,
20
D
1
J = 10.1 Hz, 2H), 1.47 (s, 9H). ½aꢄ ꢀ 80ꢅ (c 0.37,
1455, 1391, 1372, 1063, 900 cmꢀ1; H NMR (BHSC-
CHCl3/CH3OH, 1:1, v/v). Anal. Calcd for C26H29N3O5
C 67.37, H, 6.31, N 9.07. Found C 67.49, H 6.39, N 9.23.
500, DMSO-d6): d = 9.99 (s, 1H), 8.02 (s, 1H), 7.25 (t,
J = 7.1 Hz, 1H), 7.21 (t, J = 7.0 Hz, 2H), 7.19 (t,
J = 7.1 Hz, 1H), 7.13 (d, J = 7.0 Hz, 2H), 7.09 (t,
J = 7.0 Hz, 1H), 7.02 (d, J = 7.1 Hz, 1H), 6.80 (d,
J = 7.1 Hz, 1H), 5.35 (s, 2H), 4.95 (t, J = 5.1 Hz, 1H),
4.43 (t, J = 5.0 Hz, 1H), 4.24 (dd, J = 10.1 Hz,
J = 4.3 Hz, 1H), 4.11 (dd, J = 10.1 Hz, J = 3.7 Hz,
1H), 2.93 (d, J = 6.1 Hz, 2H), 2.80 (d, J = 5.2 Hz, 2H),
4.4.16. (3S)-N-[Boc-L-lysinyl(Bzl)]-1,2,3,4-tetrahydro-b-
carboline-3-carboxylic acid benzyl-ester (8p). Yield:
90%. Mp 129–131 ꢁC; ESI/MS (m/z) 669 [M+H]+. IR
(KBr): 3441, 3334, 3005, 2946, 2847, 1763, 1730,
1600, 1454, 1390, 1374, 1062, 902 cmꢀ1 1H NMR
;
(BHSC-500, DMSO-d6): d = 10.01 (s, 1H), 8.01 (s,
1H), 7.97 (s, 1H), 7.25 (t, J = 7.1 Hz, 1H), 7.20 (t,
J = 7.0 Hz, 2H), 7.17 (t, J = 6.9 Hz, 1H), 7.15 (t,
J = 7.1 Hz, 1H), 7.12 (d, J = 6.9 Hz, 2H), 7.10 (d,
J = 7.0 Hz, 2H), 7.08 (t, J = 6.9 Hz, 2H), 7.05 (t,
J = 7.0 Hz, 1H), 6.95 (d, J = 7.1 Hz, 1H), 6.87 (d,
J = 7.1 Hz, 1H), 5.33 (s, 2H), 5.30 (s, 2H), 4.91 (d,
J = 5.1 Hz, 1H), 4.43 (t, J = 4.3 Hz, 1H), 4.27 (dd,
J = 10.1 Hz, J = 4.1 Hz, 1H), 4.17 (dd, J = 10.1 Hz,
J = 3.6 Hz, 1H), 2.97 (t, J = 4.3 Hz, 2H), 2.93 (d,
J = 10.1 Hz, 2H), 1.95 (m, J = 4.1 Hz, 2H), 1.53 (m,
1.51 (s, 9H), 1.33 (m, J = 5.2 Hz, 1H), 1.09 (d, J = 5.1
20
D
Hz, 6H). ½aꢄ ¼ ꢀ39ꢅ (c 0.33, CHCl3/CH3OH, 1:1, v/
v). Anal. Calcd for C30H37N3O5 C 69.34, H 7.18, N
8.09. Found C 69.48, H 7.29, N 7.94.
4.4.20. (3S)-N-(Boc-L-isoleucinyl)-1,2,3,4-tetrahydro-
b-carboline-3-carboxylic acid benzyl-ester (8t). Yield:
89%. Mp 143–145 ꢁC; ESI/MS (m/z) 520 [M+H]+. IR
(KBr): 3441, 3335, 3216, 3012, 2951, 2844, 1755, 1730,
1641, 1603, 1455, 1392, 1374, 1060, 900 cmꢀ1 1H
;
NMR (BHSC-500, DMSO-d6): d = 10.01 (s, 1H), 8.03
(s, 1H), 7.25 (t, J = 7.0 Hz, 1H), 7.20 (t, J = 6.9 Hz,
2H), 7.17 (t, J = 7.0 Hz, 1H), 7.11 (d, J = 6.9 Hz, 2H),
J = 4.5 Hz, 2H), 1.47 (s, 9H), 1.25 (m, J = 4.3 Hz, 2H).
20
D
½aꢄ ¼ ꢀ36ꢅ (c 0.37, CHCl3/CH3OH, 1:1, v/v). Anal.

5688
J. Liu et al. / Bioorg. Med. Chem. 15 (2007) 5672–5693
7.07 (t, J = 6.9 Hz, 1H), 7.00 (d, J = 7.0 Hz, 1H), 6.86 (d,
J = 7.0 Hz, 1H), 5.33 (s, 2H), 4.93 (t, J = 5.2 Hz, 1H),
4.40 (t, J = 5.1 Hz, 1H), 4.23 (dd, J = 10.1 Hz,
J = 4.2 Hz, 1H), 4.03 (dd, J = 10.1 Hz, J = 3.3 Hz,
1H), 2.95 (d, J = 6.1 Hz, 2H), 2.90 (m, J = 5.2 Hz,
ESI/MS (m/z) 416 [M+H]+; IR (KBr): 3446, 3345,
3007, 2950, 2843, 1729, 1645, 1600, 1452, 1393, 1370,
1072, 904 cmꢀ1 1H NMR (BHSC-500, DMSO-d6):
;
d = 11.37 (s, 1H), 9.94 (s,1H), 8.03 (s,1H), 7.27 (t,
J = 7.4 Hz, 1H), 7.15 (t, J = 7.4 Hz, 1H), 6.97 (t,
J = 7.4 Hz, 1H), 6.94 (t, J = 7.4 Hz, 1H), 4.75 (t,
J = 5.2 Hz, 1H), 4.55 (d, J = 5.4 Hz, 1H), 3.89 (s, 2H),
3.65 (dd, J = 10.1 Hz, J = 5.3 Hz, 1H), 3.25 (dd,
1H), 1.45 (s, 9H), 1.32 (m, J = 5.1 Hz, 2H), 1.03 (d,
20
D
J = 5.1 Hz, 3H), 0.94 (t, J = 5.3 Hz, 3H). ½aꢄ ꢀ 37ꢅ (c
0.35, CHCl3/CH3OH, 1:1, v/v). Anal. Calcd for
C30H37N3O5 C 69.34, H 7.18, N 8.09. Found C 69.21,
H 7.10, N 8.26.
J = 10.1 Hz, J = 3.0 Hz, 1H), 2.68 (m, J = 5.4 Hz, 1H),
20
D
1.49 (s, 9H), 1.05 (d, J = 5.2 Hz, 6H). ½aꢄ ꢀ 58ꢅ (c
0.35, CHCl3/CH3OH, 1:1, v/v). Anal. Calcd for
C22H29N3O5 C 63.60, H 7.04, N 10.11. Found C
63.76, H 7.16, N 9.99.
4.5. General procedure for the preparation of 9a–t from
8a–t
At 0 ꢁC to the solution of 2.5 mmol of 8a–t in 6 ml of
methanol and 3 ml of chloroform, 450 mg (11.34 mmol)
of NaOH was added. The reaction mixture was stirred
at 0 ꢁC for 50 min and TLC (chloroform/methanol,
30:1) indicated the completion of the reaction. The reac-
tion mixture was adjusted to pH 2 with hydrochloric
acid (2 mol/L). On evaporation the residue was dis-
solved in 30 ml of ethyl acetate. The organic phase
was washed with saturated aqueous sodium chloride
and then dried over anhydrous sodium sulfate. After fil-
tration, evaporation under reduced pressure, and purifi-
cation with flash chromatography (CHCl3/CH3OH,
30:1) the title compounds were obtained as colorless
powder.
4.5.4. (3S)-N-(Boc-L-serinyl)-1,2,3,4-tetrahydro-b-carbo-
line-3-carboxylic acid (9d). 90%. Mp 170–172 ꢁC; ESI/
MS (m/z) 404 [M+H]+; IR (KBr): 3441, 3342, 3010,
2942, 2847, 1732, 1647, 1600, 1454, 1393, 1070,
1
905 cmꢀ1; H NMR (BHSC-500, DMSO-d6): d = 11.21
(s, 1H), 10.00 (s, 1H), 8.00 (s, 1H), 7.27 (t, J = 7.3 Hz,
1H), 7.16 (t, J = 7.2 Hz, 1H), 7.00 (t, J = 7.2 Hz, 1H),
6.93 (t, J = 7.2 Hz, 1H), 4.75 (t, J = 5.3 Hz, 1H), 4.66
(t, J = 5.3 Hz, 1H), 4.07 (d, J = 5.3 Hz, 2H), 3.64 (dd,
J = 10.3 Hz, J = 5.2 Hz, 1H), 3.26 (dd, J = 10.3 Hz,
J = 2.9 Hz, 1H), 3.11 (d, J = 5.3 Hz, 2H), 2.20 (s, 1H),
20
D
1.49 (s, 9H). ½aꢄ ꢀ 54ꢅ (c 0.39, CHCl3/CH3OH, 1:1,
v/v). Anal. Calcd for C20H25N3O6 C 59.54, H 6.25, N
10.42. Found C 59.37, H 6.12, N 10.61.
4.5.1. (3S)-N-(Boc-L-alanyl)-1,2,3,4-tetrahydro-b-carbo-
line-3-carboxylic acid (9a). Yield: 91%.Mp 160–162 ꢁC;
ESI/MS (m/z) 388 [M+H]+; IR (KBr): 3451, 3343,
3010, 2956, 2847, 1734, 1643, 1600, 1455, 1390, 1070,
4.5.5. (3S)-N-(Boc-L-threoninyl)-1,2,3,4-tetrahydro-b-
carboline-3-carboxylic acid (9e). Yield: 89%. Mp 155–
157 ꢁC; ESI/MS (m/z) 418 [M+H]+; IR (KBr): 3440,
3345, 3008, 2952, 2845, 1733, 1646, 1602, 1456, 1393,
1
1
906 cmꢀ1; H NMR (BHSC-500, DMSO-d6): d = 11.30
1370, 1063, 901 cmꢀ1; H NMR (BHSC-500, DMSO-
(s, 1H), 9.96 (s,1H), 8.06 (s,1H), 7.27 (t, J = 7.3Hz,
1H), 7.16 (t, J = 7.3 Hz, 1H), 6.94 (t, J = 7.3 Hz, 1H),
6.90 (t, J = 7.3 Hz, 1H), 4.73 (t, J = 5.6 Hz, 1H), 4.65
(m, J = 5.3 Hz, 1H), 3.86 (s, 2H), 3.60 (dd,
J = 10.0 Hz, J = 5.0 Hz, 1H), 3.22 (dd, J = 10.0 Hz,
d6): d= 11.12 (s, 1H), 9.99 (s, 1H), 8.00 (s, 1H), 7.27 (t,
J = 7.1 Hz, 1H), 7.20 (t, J = 7.1 Hz, 1H), 6.92 (d,
J = 7.2 Hz, 1H), 6.73 (d, J = 7.1 Hz, 1H), 4.77 (t,
J = 5.1 Hz, 1H), 4.60 (m, J = 5.1 Hz, 1H), 4.42 (t,
J = 5.3 Hz, 1H), 4.00 (m, J = 5.2 Hz, 2H), 2.93 (d,
J = 5.2 Hz, 2H), 2.23 (d, J = 3.7 Hz, 1H), 1.47 (s, 9H),
1.24 (d, J = 5.2 Hz, 3H). Anal. Calcd for C21H27N3O6
C 60.42, H 6.52, N 10.07. Found C 60.59, H 6.62, N
9.92.
J = 2.9 Hz,1H), 1.46 (d, J = 5.6 Hz, 3H), 1.48 (s, 9H).
20
D
½aꢄ ꢀ 128ꢅ (c 0.38, CHCl3/CH3OH, 1:1, v/v). Anal.
Calcd for C20H25N3O5 C 62.00, H 6.50, N 10.85. Found
C 62.19, H 6.68, N 10.67.
4.5.2. (3S)-N-(Boc-L-phenylalanyl)-1,2,3,4-tetrahydro-b-
carboline-3-carboxylic acid (9b). Yield: 90%. Mp
167–169 ꢁC; ESI/MS (m/z) 464 [M+H]+; IR (KBr):
3448, 3339, 3004, 2947, 2845, 1732, 1645, 1605, 1453,
4.5.6. (3S)-N-(Boc-L-tyrosinyl)-1,2,3,4-tetrahydro-b-
carboline-3-carboxylic acid (9f). Yield: 88%. Mp 161–
163 ꢁC; ESI/MS (m/z) 480 [M+H]+; IR (KBr): 3442,
3335, 3001, 2943, 2846, 1750, 1643, 1602, 1450, 1393,
1
1392, 1067, 905 cmꢀ1
;
1H NMR (BHSC-500,
1371, 1072, 900 cmꢀ1; H NMR (BHSC-500, DMSO-
DMSO-d6): d= 11.29 (s, 1H), 9.98 (s, 1H), 7.99 (s,
1H), 7.28 (t, J = 7.3Hz, 1H), 7.22 (t, J = 7.5Hz, 2H),
7.14 (t, J = 7.2 Hz, 1H), 7.13 (d, J = 7.5Hz, 2H), 7.09
(t, J = 7.5Hz, 1H), 6.97 (t, J = 7.2 Hz, 1H), 6.93 (t,
J = 7.2 Hz, 1H), 4.98 (t, J = 5.2 Hz, 1H), 4.77
(t, J = 5.3 Hz, 1H), 3.96 (s, 2H), 3.62 (dd, J = 10.3 Hz,
d6): d = 11.24 (s, 1H), 10.00 (s, 1H), 8.02 (s, 1H), 7.23
(t, J = 7.2 Hz, 1H), 7.15 (t, J = 7.2 Hz, 1H), 6.93 (d,
J = 7.3 Hz, 2H), 6.85 (d, J = 7.2 Hz, 1H), 6.82 (d,
J = 7.2 Hz, 1H), 6.66 (d, J = 7.3 Hz, 2H), 5.00 (s, 1H),
4.90 (t, J = 5.3 Hz, 1H), 4.81 (t, J = 5.3 Hz, 1H), 3.90
(s, 2H), 3.61 (dd, J = 10.1 Hz, J = 5.3 Hz, 1H), 3.34
J = 5.0 Hz, 1H), 3.23 (dd, J = 10.3 Hz, J = 2.6 Hz,
(dd, J = 10.1 Hz, J = 2.9 Hz, 1H), 3.05 (d, J = 5.2 Hz,
20
D
20
D
1H), 3.04 (d, J = 5.3 Hz, 2H), 1.46 (s, 9H). ½aꢄ ꢀ 75ꢅ
2H), 1.49 (s, 9H). ½aꢄ ꢀ 37ꢅ (c 0.34, CHCl3/CH3OH,
(c 0.37, CHCl3/CH3OH, 1:1, v/v). Anal. Calcd for
C26H29N3O5 C 67.37, H 6.31, N 9.07. Found C 67.23,
H 6.20, N 9.25.
1:1, v/v). Anal. Calcd for C26H29N3O6 C 65.12, H
6.10, N 8.76. Found C 65.00, H 6.01, N 8.93.
4.5.7. (3S)-N-(Boc-prolinyl)-1,2,3,4-tetrahydro-b-carbo-
line-3-carboxylic acid (9g). Yield: 89%. Mp 140–142 ꢁC;
ESI/MS (m/z) 414 [M+H]+; IR (KBr): 3445, 3430, 3203,
4.5.3. (3S)-N-(Boc-L-valinyl)-1,2,3,4-tetrahydro-b-carbo-
line-3-carboxylic acid (9c). Yield: 92%.Mp 157–159 ꢁC;

J. Liu et al. / Bioorg. Med. Chem. 15 (2007) 5672–5693
5689
3007, 2952, 2840, 1733, 1641, 1602, 1453, 1391, 1372,
J = 10.0 Hz, J = 2.9 Hz, 1H), 3.02 (d, J = 5.3 Hz, 2H),
20
D
1
1062, 901 cmꢀ1; H NMR (BHSC-500, DMSO-d6): d=
2.73 (d, J = 5.2 Hz, 2H), 1.49 (s, 9H). ½aꢄ ꢀ 69ꢅ (c
11.04 (s, 1H), 9.99 (s, 1H), 7.30 (t, J = 7.0 Hz, 1H),
7.20 (t, J = 7.2 Hz, 1H), 7.01 (d, J = 7.2 Hz, 1H), 6.90
(d, J = 7.0 Hz, 1H), 4.83 (t, J = 5.1 Hz, 1H), 4.33 (t,
J = 5.4 Hz, 1H), 4.22 (d, J = 5.2 Hz, 2H), 3.41 (t,
J = 5.4 Hz, 2H), 2.95 (d, J = 5.4 Hz, 2H), 2.27 (d,
J = 5.3 Hz, 2H), 1.93 (t, J = 5.2 Hz, 2H), 1.49 (s, 9H).
Anal. Calcd for C22H27N3O5 C 63.91, H 6.58, N
10.16. Found C 63.98, H, 6.61, N 10.20.
0.39, CHCl3/CH3OH, 1:1, v/v). Anal. Calcd for
C21H25N3O7 C 58.46, H 5.84, N 9.74. Found C 58.31,
H 5.77, N 9.92.
4.5.12. (3S)-N-(Boc-L-histidinyl)-1,2,3,4-tetrahydro-b-
carboline-3-carboxylic acid (9l). Yield: 91%. Mp 162–
164 ꢁC; ESI/MS (m/z) 454 [M+H]+; IR (KBr): 3447,
3441, 3211, 3002, 2942, 2834, 1733, 1640, 1600, 1455,
1390, 1372, 1063, 904 cmꢀ1 1H NMR (BHSC-500,
;
4.5.8. (3S)-N-(Boc-cysteinyl)-1,2,3,4-tetrahydro-b-carbo-
line-3-carboxylic acid (9h). Yield: 91%. Mp 151–153 ꢁC;
ESI/MS (m/z) 420 [M+H]+; IR (KBr): 3442, 3433, 3201,
3002, 2943, 2845, 1730, 1643, 1602, 1453, 1391, 1372,
DMSO-d6): d = 12.97 (s, 1H), 11.02 (s, 1H), 10.01 (s,
1H), 8.00 (s, 1H), 7.43 (s, 1H), 7.28 (t, J = 7.0 Hz,
1H), 7.15 (t, J = 7.2 Hz, 1H), 7.12 (d, J = 7.2 Hz, 1H),
6.97 (t, J = 7.0 Hz, 1H), 6.85 (s, 1H), 4.92 (t, J = 5.0
Hz, 1H), 4.83 (t, J = 5.1 Hz, 1H), 4.21 (d, J = 5.0 Hz,
2H), 3.13 (d, J = 5.0 Hz, 2H), 2.94 (d, J = 5.0 Hz, 2H),
1.49 (s, 9H). Anal. Calcd for C24H29N5O5 C 60.92, H
6.00, N 15.44. Found C 61.10, H 6.08, N 15.59.
1061, 903 cmꢀ1 1H NMR (BHSC-500, DMSO-d6):
;
d = 10.96 (s, 1H), 10.00 (s, 1H), 8.01 (s, 1H), 7.27 (t,
J = 7.1 Hz, 1H), 7.18 (t, J = 7.3 Hz, 1H), 7.00 (d,
J = 7.3 Hz, 1H), 6.87 (d, J = 7.1 Hz, 1H), 4.87 (t,
J = 5.3 Hz, 1H), 4.71 (t, J = 5.1 Hz, 1H), 4.20 (d,
J = 5.3 Hz, 2H), 3.15 (d, J = 5.1 Hz, 2H), 2.97 (d,
J = 5.4 Hz, 2H), 1.66 (s, 1H), 1.49 (s, 9H). Anal. Calcd
for C20H25N3O5S C 57.26, H 6.01, N 10.02. Found C
57.12, H 6.10, N 10.19.
4.5.13. (3S)-N-(Boc-L-tryptophanyl)-1,2,3,4-tetrahydro-
b-carboline-3-carboxylic acid (9m). Yield: 87%. Mp 141–
143 ꢁC; ESI/MS (m/z) 503 [M+H]+; IR (KBr): 3443,
3339, 3010, 2945, 2846, 1730, 1642, 1600, 1455, 1393,
1070, 906 cmꢀ1 1H NMR (BHSC-500, DMSO-d6):
;
4.5.9. (3S)-N-(Boc-methioninyl)-1,2,3,4-tetrahydro-b-
carboline-3-carboxylic acid (9i). Yield: 90%. Mp 154–
156 ꢁC; ESI/MS (m/z) 448 [M+H]+; IR (KBr): 3445,
3440, 3205, 3002, 2953, 2841, 1732, 1646, 1600, 1450,
d = 11.32 (s, 1H), 9.95 (s, 1H), 9.93 (s, 1H), 8.05
(s, 1H), 7.27 (t, J = 7.3 Hz, 1H), 7.22 (t, J = 7.2 Hz,
1H), 7.20 (t, J = 7.2 Hz, 1H), 7.19 (t, J = 7.2 Hz, 1H),
7.18 (t, J = 7.2 Hz, 1H), 7.13 (t, J = 7.3 Hz, 1H), 7.03
(t, J = 7.3 Hz, 1H), 6.92 (t, J = 7.3 Hz, 1H), 6.84 (s,
1H), 4.93 (t, J = 5.2 Hz, 1H), 4.88 (t, J = 5.1 Hz, 1H),
3.96 (s, 2H), 3.60 (dd, J = 10.3 Hz, J = 5.2 Hz, 1H),
1393, 1372, 1062, 901 cmꢀ1 1H NMR (BHSC-500,
;
DMSO-d6): d = 11.01 (s, 1H), 9.98 (s, 1H), 7.96 (s,
1H), 7.28 (t, J = 7.1 Hz, 1H), 7.17 (t, J = 7.3 Hz, 1H),
6.99 (d, J = 7.3 Hz, 1H), 6.85 (d, J = 7.1 Hz, 1H), 4.81
(t, J = 5.0 Hz, 1H), 4.45 (t, J = 5.1 Hz, 1H), 4.25 (d,
J = 5.1 Hz, 2H), 2.93 (d, J = 5.2 Hz, 2H), 2.43 (t,
J = 5.1 Hz, 2H), 2.22 (d, J = 5.1 Hz, 2H), 2.13 (s, 3H),
1.49 (s, 9H). Anal. Calcd for C22H29N3O5S C 59.04, H
6.53, N 9.39. Found C 59.21, H 6.62, N 9.56.
3.35 (dd, J = 10.3 Hz, J = 2.6 Hz, 1H), 2.95 (t,
20
D
J = 5.3 Hz, 2H), 1.46 (s, 9H). ½aꢄ ꢀ 55ꢅ (c 0.38,
CHCl3/CH3OH, 1:1, v/v). Anal. Calcd for C28H30N4O5
C 66.92, H 6.02, N 11.15. Found C 67.09, H 6.17, N
11.01.
4.5.14. (3S)-N-(Boc-L-argininyl)-1,2,3,4-tetrahydro-b-
carboline-3-carboxylic acid (9n). Yield: 90%.Mp 148–
150 ꢁC; ESI/MS (m/z) 473 [M+H]+; IR (KBr): 3443,
3441, 3211, 3006, 2942, 2841, 1735, 1644, 1603, 1450,
4.5.10. (3S)-N-(Boc-glutamyl)-1,2,3,4-tetrahydro-b-carbo-
line-3-carboxylic acid (9j). Yield: 92%. Mp 149–151 ꢁC;
ESI/MS (m/z) 446 [M+H]+; IR (KBr): 3445, 3440,
3211, 3001, 2943, 2830, 1732, 1640, 1603, 1450, 1392,
1391, 1374, 1061, 903 cmꢀ1 1H NMR (BHSC-500,
;
1
1371, 1060, 899 cmꢀ1; H NMR (BHSC-500, DMSO-
DMSO-d6): d = 11.03 (s, 1H),10.04 (s, 1H), 8.41 (s,
2H), 8.22 (s, 1H), 8.20 (s, 1H), 8.01 (s, 1H), 7.25 (t,
J = 7.2 Hz, 1H), 7.16 (t, J = 7.3 Hz, 1H), 7.00 (d,
J = 7.3 Hz, 1H), 6.95 (d, J = 7.2 Hz, 1H), 4.93 (d,
J = 5.2 Hz, 1H), 4.42 (t, J = 4.5 Hz, 1H), 4.29 (d,
J = 5.2 Hz, 2H), 2.93 (d, J = 4.5 Hz, 2H), 2.64 (t,
J = 5.4 Hz, 2H), 1.91 (m, J = 5.4 Hz, 2H), 1.55 (m,
J = 5.4 Hz, 2H), 1.50 (s, 9H). Anal. Calcd for
C23H32N6O5 C 58.46, H 6.83, N 17.78. Found C
58.61, H 6.92, N 17.61.
d6): d = 11.03 (s, 1H), 11.00 (s, 1H), 9.96 (s, 1H), 8.00
(s, 1H), 7.32 (t, J = 7.0 Hz, 1H), 7.23 (t, J = 7.0 Hz,
1H), 7.02 (d, J = 7.2 Hz, 1H), 6.89 (d, J = 7.0 Hz, 1H),
4.85 (d, J = 5.2 Hz, 1H), 4.47 (t, J = 5.3 Hz, 1H), 4.25
(d, J = 5.2 Hz, 2H), 2.95 (d, J = 5.2 Hz, 2H), 2.25 (t,
J = 5.3 Hz, 2H), 2.21 (t, J = 5.5 Hz, 2H), 1.48 (s, 9H).
Anal. Calcd for C22H27N3O7 C 59.32, H 6.11, N 9.43.
Found C 59.49, H 6.19, N 9.26.
4.5.11. (3S)-N-(Boc-L-aspartyl)-1,2,3,4-tetrahydro-b-
carboline-3-carboxylic acid (9k). Yield: 90%. Mp 159–
161 ꢁC; ESI/MS (m/z) 432 [M+H]+; IR (KBr): 3446,
3441, 3343, 3002, 2943, 2842, 1730, 1648, 1602, 1452,
4.5.15. (3S)-N-(Boc-glycyl)-1,2,3,4-tetrahydro-b-carbo-
line-3-carboxylic acid (9o). Yield: 92%. Mp 171–173 ꢁC;
ESI/MS (m/z) 374 [M+H]+; IR (KBr): 3446, 3340, 3233,
3008, 2942, 2841, 1730, 1644, 1605, 1456, 1392, 1066,
1
1390, 1075, 901 cmꢀ1; H NMR (BHSC-500, DMSO-
1
d6): d = 11.39 (s, 1H), 11.06 (s, 1H), 9.97 (s, 1H), 8.05
(s, 1H), 7.27 (t, J = 7.4 Hz, 1H), 7.19 (t, J = 7.3 Hz,
1H), 7.17 (t, J = 7.4 Hz, 1H), 6.99 (t, J = 7.3 Hz, 1H),
5.16 (t, J = 5.3 Hz, 1H), 4.75 (t, J = 5.4 Hz, 1H), 3.60
(dd, J = 10.0 Hz, J = 5.3 Hz, 1H), 3.27 (dd,
990 cmꢀ1; H NMR (BHSC-500, DMSO-d6): d= 11.22
(s, 1H), 9.95 (s, 1H), 8.07 (s,1H), 7.27 (t, J = 7.3 Hz,
1H), 7.17 (t, J = 7.3 Hz, 1H), 6.97 (t, J = 7.3 Hz, 1H),
6.93 (t, J = 7.3 Hz, 1H), 4.79 (t, J = 5.3 Hz, 1H), 4.53
(d, J = 4.7 Hz, 2H), 3.88 (s, 2H), 3.67 (dd, J = 10.1 Hz,

5690
J. Liu et al. / Bioorg. Med. Chem. 15 (2007) 5672–5693
J = 5.2 Hz, 1H), 3.13 (dd, J = 10.1 Hz, J = 2.6
J = 7.4 Hz, 1H), 6.93 (t, J = 7.2 Hz, 1H), 4.73 (t,
J = 5.7 Hz, 1H), 4.57 (d, J = 5.4 Hz, 1H), 3.86 (s, 2H),
3.62 (dd, J = 10.0 Hz, J = 5.0 Hz, 1H), 3.24 (dd,
J = 10.1 Hz, J = 2.7 Hz, 1H), 1.89 (m, J = 5.3 Hz, 1H),
20
D
Hz,1H),1.47 (s, 9H). ½aꢄ ꢀ 123ꢅ (c 0.34, CHCl3/
CH3OH, 1:1, v/v). Anal. Calcd for C19H23N3O5
C 61.11, H 6.21, N 11.25. Found C 61.28, H 6.39, N
11.07.
1.76 (t, J = 5.2 Hz, 2H), 1.49 (s, 9H), 1.05 (d,
20
D
J = 5.4 Hz, 6H). ½aꢄ ꢀ 46ꢅ (c 0.35, CHCl3/CH3OH,
4.5.16. (3S)-N-[Boc-L-lysinyl(Bzl)]-1,2,3,4-tetrahydro-b-
carboline-3-carboxylic acid (9p). YiEld: 90%. ESI/MS
(m/z) 579 [M+H]+; IR (KBr): 3448, 3340, 3001, 2943,
2841, 1750, 1643, 1600, 1452, 1391, 1372, 1071, 900
1:1, v/v). Anal. Calcd for C23H31N3O5 C 64.32, H
7.27, N 9.78. Found C 64.50, H 7.37, N 9.61.
4.5.20. (3S)-N-(Boc-L-isoleucyl)-1,2,3,4-tetrahydro-b-
carboline-3-carboxylic acid (9t). Yield: 87%. Mp
144–146 ꢁC; ESI/MS (m/z) 430 [M+H]+; IR (KBr):
3447, 3346, 3004, 2952, 2846, 1742, 1643, 1600, 1450,
1
cmꢀ1; H NMR (BHSC-500, DMSO-d6): d = 11.18 (s,
1H), 10.02 (s, 1H), 8.03 (s, 1H), 8.01 (s, 1H), 7.25 (t,
J = 7.2 Hz, 1H), 7.19 (t, J = 7.0 Hz, 1H), 7.13 (t,
J = 7.2 Hz, 1H), 7.11 (d, J = 7.0 Hz, 2H), 7.07 (t,
J = 7.0 Hz, 2H), 7.01 (t, J = 7.2 Hz, 1H), 6.84 (t,
J = 7.2 Hz, 1H), 5.33 (s, 2H), 4.72 (t, J = 5.5 Hz, 1H),
4.51 (t, J = 5.5 Hz, 1H), 3.65 (dd, J = 10.1 Hz,
J = 5.0 Hz, 1H), 3.32 (dd, J = 10.0 Hz, J = 2.9 Hz,
1H), 3.09 (d, J = 5.4 Hz, 2H), 2.93 (t, J = 5.2 Hz, 2H),
1393, 1372, 1071, 903 cmꢀ1 1H NMR (BHSC-500,
;
DMSO-d6): d = 10.97 (s,1H), 9.96 (s,1H), 8.01 (s,1H),
7.25 (t, J = 7.2 Hz, 1H), 7.13 (t, J = 7.2 Hz, 1H), 6.97
(t, J = 7.2 Hz, 1H), 6.93 (t, J = 7.2 Hz, 1H), 4.73 (t,
J = 5.7 Hz, 1H), 4.52 (d, J = 5.3 Hz, 1H), 3.85 (s, 2H),
3.63 (dd, J = 10.1 Hz, J = 5.2 Hz, 1H), 3.25 (dd,
J = 10.1 Hz, J = 2.7 Hz, 1H), 2.44 (m, J = 5.2 Hz, 1H),
1.77 (t, J = 5.4 Hz, 2H), 1.56 (t, J = 5.2 Hz, 2H),
20
D
1.47 (s, 9H), 1.29 (m, J = 5.5 Hz, 2H). ½aꢄ ꢀ 24ꢅ (c
1.35 (m, J = 5.1 Hz, 2H), 1.47 (s, 9H), 1.08 (d,
20
D
0.35, CHCl3/CH3OH, 1:1, v/v). Anal. Calcd for
C31H38N4O7 C 64.34, H 6.62, N 9.68. Found C 64.21,
H 6.51, N 9.83.
J = 5.2 Hz, 3H), 1.04 (t, J = 5.2 Hz, 3H). ½aꢄ ꢀ 47ꢅ (c
0.36, CHCl3/CH3OH, 1:1, v/v). Anal. Calcd for
C23H31N3O5 C 64.32, H 7.27, N 9.78. Found C 64.48,
H 7.36, N 9.63.
4.5.17. (3S)-N-(Boc-L-glutaminyl)-1,2,3,4-tetrahydro-b-
carboline-3-carboxylic acid (9q). 92%. Mp 161–163 ꢁC;
ESI/MS (m/z) 445 [M+H]+; IR (KBr): 3344, 3438,
3010, 2944, 2841, 1752, 1643, 1601, 1452, 1393, 1369,
4.6. General procedure for the preparation of hexahyd-
ropyrazino[10,20:1,6]pyrido[3,4-b]-indole-1,4-diones 5a–t
from 8a–t
1070, 901 cmꢀ1 1H NMR (BHSC-500, DMSO-d6):
;
d = 11.03 (s, 1H), 9.98 (s, 1H), 8.00 (s, 1H), 7.27 (t,
J = 7.3 Hz, 1H), 7.15 (t, J = 7.3 Hz, 1H), 6.82 (d,
J = 7.3 Hz, 1H), 6.80 (d, J = 7.3 Hz, 1H), 6.10 (s, 2H),
4.93 (t, J = 5.2 Hz, 1H), 4.57 (t, J = 5.2 Hz, 1H), 3.91
(s, 2H), 3.60 (dd, J = 10.2 Hz, J = 5.0 Hz, 1H), 3.30
(dd, J = 10.0 Hz, J = 2.7 Hz, 1H), 2.17 (t, J = 4.8 Hz,
At 0 ꢁC 2.0 mmol of 8a–t was dissolved in 10 ml of
hydrogen chloride/ethyl acetate (4 mol/L) and stirred
for 10 min. Then the reaction mixture was stirred at
room temperature for 20–25 min and TLC(ethyl ace-
tate/petroleum, 5:12) indicated the completion of the
reaction. The reaction mixture was evaporated under
vacuum. The residue was diluted in 10 ml of ethyl ace-
tate and then evaporated to dryness, which was repeated
for three times to give the title compound as colorless
powder in 84–90% yields.
2H), 2.00 (m, J = 4.9 Hz, 2H), 1.45 (s, 9H).
20
D
½aꢄ ¼ ꢀ32ꢅ (c 0.36, CHCl3/CH3OH, 1:1, v/v). Anal.
Calcd for C22H28N4O6 C 59.45, H 6.35, N 12.60. Found
C 59.61, H 6.26, N 12.77.
4.5.18. (3S)-N-(Boc-L-asparaginyl)-1,2,3,4-tetrahydro-b-
carboline-3-carboxylic acid (9r). Yield: 91%. Mp 166–
168 ꢁC; ESI/MS (m/z) 431 [M+H]+. IR (KBr): 3446,
3339, 3207, 3003, 2930, 2838, 1730, 1633, 1600, 1452,
4.7. General procedure for the preparation of hexahyd-
ropyrazino[10,20:1,6]pyrido[3,4-b]-indole-1,4-diones 5a–t
from 9a–t
1390, 1373, 1064, 903 cmꢀ1
;
1H NMR (BHSC-500,
At 0 ꢁC 2.0 mmol of 9a–t was dissolved in 10 ml of
hydrogen chloride/ethyl acetate (4 mol/L) and stirred
for 10 min. Then the reaction mixture was stirred at
room temperature for 100–140 min and TLC(ethyl ace-
tate/petroleum, 5:12) indicated the completion of the
reaction. The reaction mixture was evaporated under
vacuum. The residue was diluted in 10 ml of ethyl ace-
tate and then evaporated to dryness, which was repeated
for three times to give the title compound as colorless
powder in 86–91% yields.
DMSO-d6): d = 11.03 (s, 1H), 9.99 (s, 1H), 8.03 (s,
1H), 7.20 (t, J = 7.1 Hz, 1H), 7.11 (t, J = 7.0 Hz, 1H),
7.00 (d, J = 7.1 Hz, 1H), 6.87 (d, J = 7.1 Hz, 1H), 6.00
(s, 2H), 4.93 (d, J = 5.2 Hz, 1H), 4.43 (t, J = 5.2 Hz,
1H), 4.26 (d, J = 5.1 Hz, 2H), 2.90 (d, J = 5.1 Hz, 2H),
20
D
2.57 (t, J = 5.1 Hz, 2H), 1.45 (s, 9H). ½aꢄ ꢀ 31ꢅ (c
0.32, CHCl3/CH3OH, 1:1, v/v). Anal. Calcd for
C21H26N4O6 C 58.59, H 6.09, N 13.02. Found C
59.77, H 6.18, N 13.20.
4.5.19. (3S)-N-(Boc-L-leucyl)-1,2,3,4-tetrahydro-b-carbo-
line-3-carboxylic acid (9s). Yield: 89%. Mp 147–149 ꢁC;
ESI/MS (m/z) 430 [M+H]+; IR (KBr): 3445, 3347,
3001, 2953, 2845, 1741, 1642, 1601, 1455, 1393, 1371,
4.7.1. (3S,12aS)-3-Carboxyethyl-2,3,6,7,12,12a-hexahyd-
ropyrazino[10,20:1,6]-pyrido[3,4-b]-indole-1,4-dione (5j). A
colorless powder of 834 mg (2.0 mmol) of 5j0 was mixed
with 50 mg of Pd/C (5%) and 25 ml of formic acid in
methanol (4.4%), and agitated with hydrogen at room
temperature for 24h. The reaction mixture was filtered
and evaporated to give 634 mg (97%) of the title
1072, 903 cmꢀ1 1H NMR (BHSC-500, DMSO-d6):
;
d = 10.97 (s, 1H), 9.87 (s, 1H), 8.03 (s, 1H), 7.25 (t,
J = 7.2 Hz, 1H), 7.13 (t, J = 7.2 Hz, 1H), 6.97 (t,

J. Liu et al. / Bioorg. Med. Chem. 15 (2007) 5672–5693
5691
compound as colorless powder. Mp 255–257 ꢁC; ESI/
MS (m/z) 328 [M+H]+; IR (KBr): 3445, 3332, 2940,
peak of 5a–t, the appearance time of acetone peak (t0,
1.591 min) was used as a control. As an alternative rep-
resentation of tR, the parameter logK was defined based
on the equation logK = log[(tR ꢀ t0)/t0].
1
1684, 1338, 746 cmꢀ1; H NMR (BHSC-500, DMSO-
d6): d = 11.02 (s, 1H), 10.01 (s, 1H), 8.02 (s, 1H), 7.29
(t, J = 7.0 Hz, 1H), 7.21 (t, J = 7.0 Hz, 1H), 7.03 (d,
J = 7.0 Hz, 1H), 6.83 (d, J = 7.0 Hz, 1H), 4.90 (d,
J = 5.2 Hz, 1H), 4.41 (t, J = 5.3 Hz, 1H), 4.25 (d,
J = 5.2 Hz, 2H), 2.93 (d, J = 5.3 Hz, 2H), 2.27 (t,
4.9. In vitro anti-platelet aggregation activity assay
Platelet-rich plasma was prepared by centrifugation of
pig blood anticoagulation with sodium citrate at a final
concentration of 0.38%. The platelet counts were ad-
justed to 2 · 108/ml by addition of autologous plasma.
Platelet aggregation tests were conducted in an aggre-
gometer using the standard turbidimetric technique.
The effects of 5a–t (in a series of concentrations of a
range from 1 lmol/L to 0.01 lmol/L) on PAF (final con-
centration 10ꢀ7 mol/L) or ADP (final concentration
10ꢀ5 mol/L) induced platelet aggregation were observed.
The maximal rate of platelet aggregation (Am %) was
represented by the peak height of aggregation curve.
J = 5.3 Hz,
2H),
2.02
(q,
J = 5.3 Hz,
2H).
20
D
½aꢄ ꢀ 100:1ꢅ (c 1.0, CH3OH); Anal. Calcd for
C17H17N3O4 C 62.38, H 5.23, N 12.84. Found C
62.55, H 5.32, N 12.71.
4.7.2. (3S,12aS)-3-Carboxymethyl-2,3,6,7,12,12a-hexahyd-
ropyrazino[10,20:1,6]pyrido-[3,4-b]indole-1,4-dione
(5k).
Using the same procedure for 5j from 806 mg (2.0 mmol)
of 50k 601 mg (96%) of the title compound was obtained
as colorless powder. Mp 271–273 ꢁC, ESI-MS (m/z) 314
[M+H]+; (KBr): 3442, 3336, 2943, 1688, 1337, 743 cmꢀ1
;
1H NMR (DMSO-d6, 300 MHz) d = 11.00 (s, 1H), 9.99
(s,1H), 8.01 (s,1H), 7.28 (d, J = 7.2 Hz, 1H), 7.20
(t, J = 7.2 Hz, 1H), 7.05 (t, J = 7.2 Hz, 1H), 6.90
(t, J = 7.2 Hz, 1H), 5.05 (t, J = 6.6 Hz, 1H), 4.85
(t, J = 5.3 Hz, 1H), 4.24 (d, J = 5.6 Hz, 2H),
4.10. In vivo anti-thrombotic activity in rat model
The assessments of in vivo anti-thrombotic activity were
performed based on a protocol reviewed and approved
by the Ethics Committee of Peking University. The com-
mittee assures the welfare of the animals which were
maintained in accordance to the requirements of the ani-
mal welfare act and according to the guide for care and
use of laboratory animals.
2.96 (t, J = 5.6 Hz, 2H), 2.85 (d, J = 5.3 Hz, 2H).
20
D
C 61.34, H 4.83, N 13.41. Found C 61.27, H 4.77, N 13.59.
½aꢄ ꢀ 59ꢅ (c 1.0, CH3OH); Anal. Calcd for C16H15N3O4
4.7.3. (3S,12aS)-3-Aminobutyl-2,3,6,7,12,12a-hexahydro-
pyrazino[10,20:1,6]pyrido[3,4-b] indole-1,4-dione (5p). Using
the same procedure for 5j from 922 mg (2.0 mmol) of 50p
619 mg (95%) of the title compound was obtained as col-
orless powder. Mp 216–218 ꢁC, EI-MS (m/z) 327 [M+H]+;
4.11. Intravenous administration
Aspirin, 5a–t were dissolved in NS just before use and
kept in an ice bath. Male Wistar rats weighing 250–
300 g (purchased from Animal Center of Peking Univer-
sity) 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. When the rats were intravenously adminis-
tered from the other end of the polyethylene tube hepa-
rin sodium was injected as anticoagulant, then NS,
Aspirin, 5a–t were injected, and this end was inserted
into the right carotid artery. Blood flowed from the right
carotid artery to the left jugular vein through the poly-
ethylene tube for 15 min. The thread was removed.
The weight of the wet thrombus thread was recorded.
The thread was kept in a desiccator for 2 weeks and
the weight of the dry thrombus was recorded.
IR(KBr): 3324, 2936, 1683, 1463, 1336, 745 cmꢀ1 1H
;
NMR (DMSO-d6, 300 MHz) d = 9.92 (s,1H), 8.21
(d, J = 1.8 Hz, 1H), 7.41 (d, J = 7.5 Hz, 1H), 7.28
(t, J = 7.5 Hz, 1H), 7.08 (t, J = 7.5 Hz, 1H), 6.95 (t,1H,
J = 7.5 Hz, 1H), 5.36 (d. J = 16.5 Hz, 1H), 4.60
(t, J = 6.8 Hz, 1H), 4.30 (dd, J = 11.7 Hz, J = 4.5 Hz,
1H), 4.13 (dd, J = 11.7 Hz, J = 4.5 Hz, 1H), 3.26 (dd,
J = 13.2 Hz, J = 3.3 Hz, 1H), 2.79 (dd, J = 13.5 Hz,
J = 5.1 Hz, 1H), 2.70 (t, J = 4.8 Hz, 2H), 2.01 (s, 2H),
1.80 (m, J = 4.8 Hz, 2H), 1.60 (m, J = 4.8 Hz, 2H), 1.32
20
D
(m, J = 4.8 Hz, 2H). ½aꢄ ꢀ 48ꢅ (c 1.0, CH3OH); Anal.
Calcd for C18H22N4O2 C 66.24, H 6.79, N 17.17. Found
C 66.01, H 6.58, N 17.41.
4.8. Determination of the retention time of 5a–t
Hexahydropyrazino[10,20:1,6]pyrido-[3,4-b]indole-1,4-
dions 5a–t were dissolved in aqueous methanol (50%) to
prepare sample solution of 10 lM. The HPLC analysis
was carried out on Agilent 1100 series, the column was
a reversed-phase C18 column (Agilent Zorbax Extend-
C18, 4.6 · 15 mm, 5 lm). After 20 ll of the sample (10
lM) was loaded, the column was eluted with 50% solu-
tion of MeOH as the mobile phase for 40 min. The flow
rate was 1 ml/min. The peak of 5a–t in the sample was
monitored with UV detector at 254.8 nm and the reten-
tion time (tR) corresponding to its peak was recorded.
With the same HPLC conditions the retention time of
acetone peak was recorded as t0. In order to offset the
influence of the solvent on the appearance time of the
4.12. Oral administration
The solution of Aspirin, 5a–t in NS was fed to Male
Wistar rats (weighing 250–300 g, purchased from Ani-
mal Center of Peking University) 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 so-
dium (50 IU/ml in NS) and one end was inserted into

5692
J. Liu et al. / Bioorg. Med. Chem. 15 (2007) 5672–5693
the left jugular vein while another end was inserted into
the right carotid artery. Blood flowed from the right car-
otid artery to the left jugular vein through the polyeth-
ylene tube for 15 min. The thread was taken out and
the weight of the wet thrombus was recorded. The
thread was kept in a desiccator for 2 weeks and the
weight of the dry thrombus was recorded.
prepare sample solution of HPLC-MS analysis on HP
ES-5989x instrument.
In the HPLC-MS analysis the column was a reversed-
phase C18 column (Agilent Zorbax Extend-C18,
4.6 · 15 mm, 5 lm). After 20 ll of the sample solution
was loaded, the column was eluted with 50% solution
of MeOH as the mobile phase for 40 min. The flow rate
was 1 ml/min. The formed metabolite in the sample was
monitored with UV detector at 254.8 nm. The MS ana-
lysis was carried out using ESI-model (cone, 30 V;
source temperature, 120 ꢁC; desolvation HPLC-MS
analysis desolvation gas, 350 L/h; cone gas, 50 L/h).
4.13. Dosage
For intravenous anti-thrombotic assay, 5a–t were given
to the rats at a dose of 0.5 lmol/kg. For dose-dependent
experiments, 5b,c,n were given to the rats intravenously
at three doses 0.5 lmol/kg, 5 nmol/kg, and 0.5 nmol/kg,
respectively. For oral anti-thrombotic assay, 5k,o,p were
given to the rats at a dose of 0.5 lmol/kg.
4.16. QSAR analysis
To perform a QSAR analysis, first the SMILES formats of
compounds 5a–t were generated using JME server (http://
www.molinspiration.com/cgi-bin/properties). Moreover,
theobtainedSMILESfilesweresubmittedtoe-dragonserver
(http://www.vcclab.org/lab/edragon/) to calculate molec-
ular descriptors. Finally, the 1664 different molecular
parameters for each compound were obtained.
4.14. Deducing the cyclization product of N-[(3S)]-1,2,3,4-
tetrahydro-b-carboline-3-carboxyl]-L-lysine in rat plasma
Male Wistar rats weighing 250 10 g (purchased from
Animal Center of Peking University) were anesthetized
with pentobarbital sodium (80.0 mg/kg, ip), and the left
jugular vein was separated. A 6-cm polyethylene tube
was inserted into the left jugular vein and the whole
blood was drawn into a heparin sodium containing cen-
trifugal tube. The whole blood was centrifuged at 0 ꢁC
and 3500 g for 5 min to isolate the plasma. To the
plasma (15 ml) a solution of 3.5 mg of N-[(3S)]-1,2,3,4-
tetrahydro-b-carboline-3-carboxyl]-L-lysine in 100 ll of
normal saline was added. At 37 ꢁC the mixture was incu-
bated and shaken in a shaker. Thirty minutes later the
incubated mixture was centrifuged at 0 ꢁC and 3500 g
for 5 min, and the supernatant was exposed to a gentle
nitrogen-flow to evaporate. The obtained residue was
dissolved in 100 ll of aqueous methanol (50%, v/v) to
prepare sample solution of HPLC-MS analysis on HP
ES-5989x instrument.
The MLR method was employed to derive the QSAR
equation of 5a–t. Concerning the anti-thrombotic effect
of a molecule, the weight of wet thrombus (i.e., WWT)
was considered in the current QSAR analysis. As the
number of compounds is only 20, the optimum number
of molecular descriptors used in the QSAR analysis is
around 4. To obtain the best regression equation, a
two-step descriptor selection procedure was employed.
The first step consisted in the elimination of the descrip-
tors that are not able to provide useful statistical infor-
mation. For instance, the absolute value of the
correlation coefficient between the selected descriptor
(Xi) and the activity (WWT) under investigation should
be larger than 0.2. At the second step, the descriptor
subset was optimized via a genetic algorithm. Finally,
the optimal QSAR equation in describing the anti-
thrombotic effects of 5a–t was determined.
In the HPLC-MS analysis the column was a reversed-
phase C18 column (Agilent Zorbax Extend-C18,
4.6 · 15 mm, 5 lm). After 20 ll of the sample solution
was loaded, the column was eluted with 50% solution
of MeOH as the mobile phase for 40 min. The flow
rate was 1 ml/min. The formed metabolite in the sam-
ple was monitored with UV detector at 254.8 nm. The
MS analysis was carried out using ESI-model (cone,
30 V; source temperature, 120 ꢁC; desolvation HPLC-
MS analysis desolvation gas, 350 L/h; cone gas,
50 L/h).
To systematically assess a QSAR model, a reliable vali-
dation is required. Usually, a QSAR model is evaluated
by the predictive results for a training dataset (resubsti-
tution test) and a testing dataset (cross-validation),
respectively. Of the various cross-validation tests, the
LOO test is thought to be a reliable one. By the LOO
test, the WWT value of each compound in the dataset
is predicted by the rules derived using all other
compounds except the one that is being predicted. Both
tests of resubstitution and LOO were used to evaluate
the QSAR model in the current work. In the resubstitu-
tion test, three statistical parameters (R2, S, and ꢀeÞ were
used to evaluate the performance. The squared correla-
tion coefficient R2 is defined as follows:
4.15. Deducing the cyclization product of N-[(3S)]-
1,2,3,4-tetrahydro-b-carboline-3-carboxyl]-L-lysine
in 5% acetic acid aqueous
To water (15 ml) a solution of 3.5 mg of N-[(3S)]-1,2,3,4-
tetrahydro-b-carboline-3-carboxyl]-L-lysine in 100 ll of
normal saline was added. At 37 ꢁC the mixture was incu-
bated and shaken in a shaker. Thirty minutes latter the
incubated mixture was centrifuged at 0 ꢁC and 3500 g
for 5 min, and the supernatant was exposed to a gentle
nitrogen-flow to evaporate. The obtained residue was
dissolved in 100 ll of aqueous methanol (50%, v/v) to
P
N
2
ðyi ꢀ ^yiÞ
R2 ¼ 1 ꢀ
ð2Þ
i¼1
P
N
i¼1
2
ðyi ꢀ ꢀyÞ
where yi, ^yi, and ꢀy are predicted, actual, and mean val-
ues of the activity, respectively. The Nis the number of

J. Liu et al. / Bioorg. Med. Chem. 15 (2007) 5672–5693
5693
7. Willerson, J. T.; Golino, P.; Eidt, J.; Campbell, W. B.;
Buja, L. M. Circulation 1989, 80, 198.
8. Wu, Y.; Bi, L.; Bi, W.; Li, Z.; Zhao, M.; Wang, C.; Ju, J.;
Peng, S. Bioorg. Med. Chem. 2006, 14, 5711.
9. Zhao, M.; Bi, L.; Bi, W.; Wang, C.; Yang, Z.; Ju, J.; Peng,
S. Bioorg. Med. Chem. 2006, 14, 4761.
10. Zhao, M.; Liu, J.; Wang, C.; Wang, L.; Liu, H.; Peng, S.
J. Med. Chem. 2005, 48, 4285.
11. Zhao, M.; Wang, C.; Liu, J.; Zhou, K.; Peng, S. Bioorg.
Med. Chem. 2004, 12, 2275.
compounds in the resubstitution test. The root mean
square of errors S was calculated with the following
equation:
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
P
To illustrate the predictive accuracy more explicitly, the
N
i¼1
2
ðyi ꢀ ^yiÞ
S ¼
ð3Þ
N
ꢀe (absolute average error) is defined as follows:
P
In the LOO test, the above three statistical parameters,
N
i¼1
12. Zhao, M.; Wang, C.; Wu, Y.; Zhou, K.; Peng, S. Prep.
Biochem. Biotechnol. 2004, 34, 57.
13. Zhao, M.; Wang, C.; Yang, J.; Liu, J.; Xu, Y.; Wu, Y.;
Peng, S. Bioorg. Med. Chem. 2003, 11, 4913.
14. Liu, J.; Zhao, M.; Wang, C.; Peng, S. Bioorg. Med. Chem.
Lett. 2003, 13, 4065.
jyi ꢀ ^yij
ꢀe ¼
ð4Þ
N
that is R2LOO; SLOO, and ꢀeLOO, were also calculated.
15. Griffin, S.; Grant Wyllie, S.; Markham, J. J. Chromatogr.
A 1999, 864, 221.
16. Kugel, C.; Heintzelmann, B.; Wagner, J. J. Chromatogr. A
1994, 667, 29.
Acknowledgments
This work was supported by the Beijing area major
laboratory of peptide and small molecular drugs, the
National Natural Scientific Foundation of China
(20372007, 20232020) and Beijing Municipal Commis-
sion of Education (KZ 200510025015). Z.Z. was sup-
ported by Program for New Century Excellent Talents
in University (NCET-06-0116).
17. Reichelova, V.; Albertioni, F.; Liliemark, J. J. Chroma-
togr. A 1994, 667, 37.
18. Van Loevezijn, A.; Allen, J. D.; Schinkel, A. H.; Koomen,
G. J. Bioorg. Med. Chem. Lett. 2001, 11, 29.
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