A class of oral N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3- carbonyl]- N′-(amino-acid-acyl)hydrazine: Discovery, synthesis, in vitro anti-platelet aggregation/in vivo anti-thrombotic evaluation and 3D QSAR analysis

Article abstract of DOI:10.1016/j.ejmech.2011.04.037

The in vivo anti-thrombotic activities of amino acid modified tetrahydro-β-carbolines depended upon the proximity of the side chain of the amino acid residue to the carboline-cycle. Based on this proximity the computerized screening of various tetrahydro-

Full text of DOI:10.1016/j.ejmech.2011.04.037

European Journal of Medicinal Chemistry 46 (2011) 3237e3249
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
A class of oral N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-b-carboline-3-carbonyl]-
N0-(amino-acid-acyl)hydrazine: Discovery, synthesis, in vitro anti-platelet
aggregation/in vivo anti-thrombotic evaluation and 3D QSAR analysis
*
**
Kun Yao, Ming Zhao , Xiaoyi Zhang, Yuji Wang, Li Li, Meiqing Zheng, Shiqi Peng
College of Pharmaceutical Sciences, Capital Medical University, Beijing 100069, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
The in vivo anti-thrombotic activities of amino acid modified tetrahydro-
proximity of the side chain of the amino acid residue to the carboline-cycle. Based on this proximity the
computerized screening of various tetrahydro- -carboline derivatives was performed and N-[(1S,3S)-1-
methyl-1,2,3,4-tetrahydro-
-carboline-3-carbonyl]-N0-(amino-acid-acyl)hydrazines were explored having
b-carbolines depended upon the
Received 27 January 2011
Received in revised form
5 April 2011
Accepted 12 April 2011
Available online 29 April 2011
b
b
large proximity. The in vivo anti-thrombotic assays explored that at a dose of 10 nmol/kg eighteen novel N-
[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-
b
-carboline-3-carbonyl]-N0-(amino-acid-acyl)hydrazines were orally
efficacious.
Keywords:
Ó 2011 Elsevier Masson SAS. All rights reserved.
Tetrahydro-b-carboline
Anti-platelet aggregation
Anti-thrombotic activity
Conformation analysis
3D QSAR
1. Introduction
activation [7], mitogen activated protein kinase-activated protein
kinase 2 [8], cyclin-dependent kinases 4 [9,10], human mono-
amine oxidase [11], mitotic kinesin Eg5 [12], bacterial enoyl acyl
carrier protein reductase [13], acetyl-cholinesterases and butyr-
ylcholinesterases [14]; b) improving object recognition memory
[15]; c) stimulating insulin secretion [16]; d) interacting with DNA
[17,18]; e) blocking topoisomerases [19]; f) exhibiting trypanocidal
[6,20], antimalarial [21,22], cytotoxic [23e27], cardiovascular [28],
neuroprotective and neuron-differentiating [29], antiviral [30], as
well as antileishmanial actions [31]; g) binding to imidazoline
[32,33], mGluR1 [34], 5-HT2 serotonin receptors [35], and benzodi-
azepine receptors [36].
Cardiovascular events such as the deep vein thrombosis,
myocardial infarction, pulmonary embolism and stroke seriously
hazard the health of the human being, while intravascular throm-
bosis is known to be one of the most prominent causes of morbidity
and mortality [1]. The injury of blood vessel is implicated in the
arterial thrombosis [2]. Once vascular endothelium of the injured
blood vessel is damaged the platelets will adhere to the exposed
extracellular matrix [3]. Platelet adhesion primarily associates with
uncontrolled platelet activation and culminates in the intravascular
thrombosis [4]. The suppression of platelet activation can effectively
prevent the thrombosis [5], and the anti-platelet drugs have thera-
peutic utility [6]. Due to the current anti-platelet therapy usually
having limitedefficacy, anextraefforthasbeenmadetodiscovernew
Though a series of b-carbolines such as harmalol, harmaline,
norharmane, harmol, harmine and harmane have been known to
be able to prevent the platelet activation, and capable of inhibiting
collagen-induced platelet aggregation. The IC50 values of them to
leads, such as
more effectively inhibiting the platelet aggregation.
-Carbolines and tetrahydro- -carbolines are pharmacologically
b-carbolines and tetrahydro-b-carbolines, capable of
inhibit the platelet aggregation are higher than 130
mM [7]. While
b
b
(1S,3S)-1-methyl-1,2,3,4-tetrahydro- -carboline-3-carboxylic acids
b
importantindolealkaloids. Ithasbeenreported that theyhaveawide
spectrum of pharmacological actions such as a) inhibiting platelet
were reported to be able to effectively inhibit the platelet aggre-
gation induced by four aggregators including platelet-activating
factor (PAF), adenosine diphosphate (ADP), arachidonic acid (AA)
as well as thrombin (TH). The IC50 value of (1S,3S)-1-methyl-1,2,3,4-
* Corresponding author. Tel./fax: þ86 10 8280 2482.
** Corresponding author. Tel./fax: þ86 10 8391 1528.
E-mail addresses: mingzhao@mail.bjmu.edu.cn (M. Zhao), sqpeng@mail.bjmu.
edu.cn (S. Peng).
tetrahydro-
aggregation was lower than 1
could be a good lead compound [37]. In this context, we modified
b-carboline-3-carboxylic acid inhibited the platelet
mM, and its hydrazine derivative
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.04.037

3238
K. Yao et al. / European Journal of Medicinal Chemistry 46 (2011) 3237e3249
(1S,3S)-1-methyl-1,2,3,4-tetrahydro-
b
-carboline-3-carbonylhydra-
2.2. Synthesis of N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-
carboline-3-carbonyl]-N0-(amino-acid-acyl)hydrazines
b-
zine with amino acids preparing eighteen novel compounds,
evaluated their in vitro anti-platelet aggregation and in vivo anti-
thrombotic activities, and established their 3D QSAR equation.
The preparation of N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-b-
carboline-3-carbonyl]-N0-(amino-acid-acyl)hydrazines (5aer) was
carried out according to a five-step-route depicted in Scheme 1. The
2. Chemistry
racemic
b-carbolines were usually prepared via PicteteSpengler
condensation. To control the chirality in the PicteteSpengler
condensation for the enantiospecific synthesis of indole alkaloids
the effect of substitution on the stereospecificity was investigated
[39,40]. When L-tryptophan was used in the PicteteSpengler
condensation 1S,3S/1R,3S diasetereomers were formed in a ratio of
16:1 (79% yield of 1). After the esterification, 1S,3S diasetereomer of
the carboxylate methyl ester 2 was predominantly generated as
precipitates (89% yield). The increase of the stereoselectivity in favor
of the 1S,3S diasetereomer was probably due to the influence of the
substituents. In the presence of hydrazine hydrate the hydrazinol-
2.1. Discovery of N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-
carboline-3-carbo nyl]-N0-(amino-acid-acyl)hydrazines
b-
In the analysis of the stereoview derived from the QSAR module
of Cerius 2 it was found that the in vivo anti-thrombotic activities of
amino acid modified 3S-tetrahydro-b-carboline-3-carboxylic acid
and (3S,12aS)-hexahydropyrazino-[10,20:1,6]pyrido [3,4-b]indole-
1,4-dione depended on their stretching conformation [38]. In
3S-tetrahydro-b-carboline-3-carbonylamino acids (Fig. 1A) the
proximity of the side chain of the amino acid residue to the
carboline-cycle is larger than that in (3S,12aS)-hexahydropyrazino
[10,20:1,6]pyrido [3,4-b]indole-1,4-diones (Fig. 1B). On the in vivo
anti-thrombotic model the efficacy of the former is 10-fold lower
than that of the latter [37,38]. These observations suggest that for
ysis of 2 provided N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-b-carbo-
line-3-carbonyl]hydrazine (3, 72% yield). In DCC/HOBt/NMM
condition the coupling of 3 with Boc-amino acids gave N-[(1S,3S)-
1-methyl-1,2,3,4-tetrahydro-
noacid-acyl)hydrazine (4aer, 43e86% yield). On the removal of Boc
group of 4aer N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro- -carboline-
b
-carboline-3-carbonyl]-N0-(Boc-ami-
anti-thrombotic 3S-tetrahydro-b-carboline-3-carboxylic acid deri-
b
vatives the small proximity of the side chain of the amino acid
residue to the carboline-cycle benefits the in vivo activity. Following
up this clue the proximity of a series of amino acids modified car-
bolines were estimated, and the side chains of the amino acid
3-carbonyl]-N0-(amino-acid-acyl)hydrazine (5aer) were obtained
in 88e100% yields. To ensure the purity, 4aer were purified on
a chromatographic column, while 5aer were purified by ether-
promoted repeated solidification, and the HPLC purity was more
than 97%. These data demonstrated that the used procedures and
conditions were suitable for the preparation of 5aer with high
quality and acceptable yield.
residues in N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-b-carboline-3-
carbonyl]-N0-(amino-acid-acyl)hydrazines (Fig. 1C) were found
been far from the carboline-cycle, and this leaded to the discovery of
them as novel anti-thrombotic agents.
Fig. 1. QSAR module of Cerius 2 derived stereoview of 3S-tetrahydro-
pyrazino-[10,20:1,6]pyrido[3,4-b]indole-1,4-diones (B) and N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-
b
-carboline-3-carbonyl-amino acids (A), (3S, 12aS)-3-(aminoacid-side-chain)-2,3,6,7,12,12a-hexa-hydro-
b
-carboline-3-carbonyl]-N0-(amino-acid-acyl)hydrazines (C).

K. Yao et al. / European Journal of Medicinal Chemistry 46 (2011) 3237e3249
3239
CO H
CO H
2
CO CH
2
CO-NH-NH
2
2
3
i
ii
iii
NH
NH
NH
CH
2
NH
CH
N
N
H
1
N
H
2
N
H
3
H
CH
3
3
3
iv
CO-NH-NH-AA-Boc
NH
CH
CO-NH-NH-AA
v
NH
N
H
4a-r
N
H
5a-r
CH
3
3
Scheme 1. Synthetic route of N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-b
-caboline-3- carbonyl]-N0-(amino-acid-acyl)hydrazine. i) CH3CHO, H2SO4; ii) CH3OH, SOCl2; iii) NH2NH2$H2O;
iv) DCC, HOBt, NMM; v) 4 N HCl/EtOAc. In 4a and 5a, AA ¼ Gly; 4b and 5b, AA ¼ Ala; 4c and 5c, AA ¼ Ile; 4d and 5d, AA ¼ Leu; 4e and 5e, AA ¼ Val; 4f and 5f, AA ¼ Met; 4g and 5g,
AA ¼ Pro; 4h and 5h, AA ¼ Phe; 4i and 5i, AA ¼ Tyr; 4j and 5j, AA ¼ Thr; 4k and 5k, AA ¼ Ser; 4l and 5l, AA ¼ Trp; 4m, AA ¼ His(Boc); 5m, AA ¼ His; 4n AA ¼ Lys(Boc); 5n, AA ¼ Lys;
4o and 5o, AA ¼ Asp(OBzl); 4p and 5p, AA ¼ Glu(OBzl); 4q and 5q, AA ¼ Asn; 4r and 5r, AA ¼ Gln.
3. Pharmacology
On a model of extra-corporeal circulation of arterio-veinos
cannula the thrombus weights of the rats receiving 5aer were
measured, used to represent the in vivo anti-thrombotic activities,
and are listed in Table 2. As seen, the thrombus weights
(18.6e24.0 mg) of the rats receiving 10 nmol/kg of 5aer are
significantly lower than that (28.2 mg) of the rats receiving normal
saline (NS). This means that 5aer effectively inhibit the rats to form
thrombus. Besides, the thrombus weights (18.6e19.0 mg) of the
rats receiving 10 nmol/kg of 5f,j,k,l,n are significantly lower than
that (21.2 mg) of the rats receiving 100 nmol/kg of 3. This means
3.1. In vitro anti-platelet aggregation of 3 and 5aer
Considering the in vitro anti-platelet aggregation assay needs
a great quantity of blood, only a great quantity of pig blood is
commercially available in Beijing, and the in vitro anti-platelet
aggregation activity resulted from pig platelets matched the
in vivo anti-thrombotic activity resulted from rat [37,38], thus pig
platelets were used here. The in vitro activities of 3 and 5aer (at
a series of concentrations ranging from 1
evaluated by anti-platelet aggregation assay. PAF (final concentra-
tion 0.1 M), ADP (final concentration 10 M) and AA (final
concentration 350 M) were used as the aggregator. The in vitro
mM to 1.5 mM) were
Table 1
IC50 of 5aer inhibiting platelet aggregation.a
m
m
m
Compd.
IC50 (mM)
activities of 3 and 5aer inhibiting platelet aggregation were rep-
resented with IC50 values and are listed in Table 1.
The IC50 values indicate that 3 selectively inhibits AA-induced
in vitro platelet aggregation, and the inhibiting sequence is
AA > PAF > ADP. The IC50 values of 5aer inhibiting AA-induced
platelet aggregation range from 0.6 mM to 21.7 mM. The IC50 values
of 5a,b,f,g are equal to that of 3, while the IC50 values of 5cee,her are
significantly lower than that of 3. The IC50 values of 5aer inhibiting
ADP
PAF
AA
3
611.2 ꢀ 59.7
722.1 ꢀ 34.8
117.9 ꢀ 5.3f
51.1 ꢀ 4.6d
110.9 ꢀ 9.5
348.1 ꢀ 35.1n
162.4 ꢀ 11.8k
1.4 ꢀ 0.3b
21.5 ꢀ 1.0
20.7 ꢀ 1.5
20.0 ꢀ 1.3
2.4 ꢀ 0.2c
3.2 ꢀ 0.3d
3.7 ꢀ 0.3e
21.7 ꢀ 1.1
20.6 ꢀ 1.7
7.1 ꢀ 0.3i
13.0 ꢀ 0.7j
4.7 ꢀ 0.3f
19.9 ꢀ 1.0l
0.6 ꢀ 0.03b
4.8 ꢀ 0.3f
3.4 ꢀ 0.2d
5.2 ꢀ 0.3g
6.3 ꢀ 0.6h
19.7 ꢀ 0.7l
18.5 ꢀ 1.1k
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
31.6 ꢀ 1.7c
1.2 ꢀ 0.2b
156.0 ꢀ 10.7
417.1 ꢀ 18.0k
166.1 ꢀ 5.8h
306.9 ꢀ 26.8j
152.1 ꢀ 9.3g
531.0 ꢀ 41.2m
276.1 ꢀ 18.0i
21.7 ꢀ 2.2b
50.0 ꢀ 3.3g
52.9 ꢀ 5.0g
134.2 ꢀ 10.5j
h
PAF-induced platelet aggregation range from 1.2 mM to 506.8 mM.
108.9 ꢀ 13.0
12.7 ꢀ 1.2e
31.9 ꢀ 1.5f
119.1 ꢀ 9.0i
4.3 ꢀ 0.6c
The IC50 value of 5h is equal to that of 3, the IC50 values of 5cef,ien
are significantly lower than that of 3, while the IC50 values of
5a,b,g,oer are significantly higher than that of 3. The IC50 values of
5a-r inhibiting ADP-induced platelet aggregation range from
94.6 ꢀ 9.4e
7.7 ꢀ 0.6d
321.3 ꢀ 15.3j
117.0 ꢀ 8.6f
524.4 ꢀ 36.2l
609.1 ꢀ 35.5
598.4 ꢀ 41.1
106.1 ꢀ 5.1h
196.2 ꢀ 7.3l
362.3 ꢀ 22.6n
506.8 ꢀ 21.6
267.1 ꢀ 8.4m
21.7 mM to 722.1 mM. The IC50 values of 5q,r are equal to that of 3, the
IC50 values of 5bep are significantly lower than that of 3, while the
IC50 value of 5a is significantly higher than that of 3. Therefore
the IC50 values of 5f,h inhibiting the platelet aggregation induced
by three aggregators either are equal to or lower than that of 3,
while the IC50 values of 5cee,ien inhibiting the platelet aggregation
induced by three aggregators are significantly lower than that of 3.
For ADP: b) Compare to 3, 5aek,mer p < 0.01; c) Compare to 3, 5aec,eek,mer
p < 0.01; d) Compare to 3, 5a,b,eek,mer p < 0.01; e) Compare to 3, 5a,b,eek,ner
< 0.01; f) Compare to 3, 5a,eek,n,per p < 0.01; g) Compare to 3,
p
5a,e,f,h,ei,k,n,per p < 0.01 and to 5g p < 0.05; h) Compare to 3, 5a,f,h,k,n,per
p < 0.01; i) Compare to 3, 5a,f,n,per p < 0.01 and to 5h p < 0.05; j) Compare to 3,
5a,f,per p < 0.01; k) Compare to 3, 5a,per p < 0.01 and 5o p < 0.05; l) Compare to 3,
5a,q,r p < 0.01; m) Compare to 3, 5a,r p < 0.01 and to 5q p < 0.05.
3.2. In vivo anti-thrombotic activities of 5aer
For PAF: b) Compare to 3, 5a,b,eer p < 0.01; c) Compare to 3, 5a,b,eek,mer
p < 0.01; d) Compare to 3, 5a,b,eek,ner p < 0.01; e) Compare to 3, 5a,b,eeh,j,k,ner
p < 0.01; f) Compare to 3, 5a,b,eeh,k,ner p < 0.01; g) Compare to 3, 5a,b,g,h,k,ner
p < 0.01; h) Compare to 3, 5a,b, g,k,oer p < 0.01; i) Compare to 3, 5a,b,oer p < 0.01
and to 5g p < 0.05; j) Compare to 3, 5a,b,oer p < 0.01; k) Compare to 3, 5a,oer
p < 0.01; l) Compare to 3, 5a,per p < 0.01; m) Compare to 3, 5a,p,q p < 0.01; n)
Compare to 3, 5a,q p < 0.01.
To select the dose of 5aer some preliminary evaluations were
performed. The evaluations revealed that the effective dose
(0.1
line-3-carbonyl]hydrazine (3) was 50 fold lower than that (5
kg) of 3S-1,2,3,4-tetrahydro- -carboline-3-carboxylic acid, while
the effective dose of the latter was 10 fold higher than that
(0.5 mol/kg) of some of 3S-tetrahydro- -carboline-3-carbon-
ylaminoacids. This means that the introduction of -amino acid into
3S-1,2,3,4-tetrahydro- -carboline-3-carboxylic acid can result in
10-fold decrease in the effective dose. Therefore the effective dose of
5aer could be 500-fold lower than that (5 mol/kg) of 3S-1,2,3,4-
tetrahydro- -carboline-3-carboxylic acid and should be 10 nmol/kg.
m
mol/kg) of N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-
b
-carbo-
mmol/
b
For AA: b) Compare to 3, 5aek,mer p < 0.01; c) Compare to 3, 5a,b,dek,mer
p < 0.01; d) Compare to 3, 5a,b,eek,m,oer p < 0.01; e) Compare to 3, 5a,b,dek,mer
p < 0.01; f) Compare to 3, 5a,b,dei,k,m,n,qer p < 0.01 and to 5o p < 0.05; g)
Compare to 3, 5a, b,dei,k,m,n,per p < 0.01; h) Compare to 3, 5a,b,deg,i,k,m,n,q,r
p < 0.01 and to 5h p < 0.05; i) Compare to 3, 5a,b,deg,i,k,m,n,q,r p < 0.01; j)
Compare to 3, 5a,b,deg,k,m,n,q,r p < 0.01; k) Compare to 3, 5a,b,deg,m,n p < 0.01
and to 5k,q p < 0.05; l) Compare to 3 p < 0.05.
m
b
L
b
a
m
n ¼ 6, Am% of platelet aggregation induced by PAF, ADP and AA with of aspirin
b
(1.7 mM) were 33.22 ꢀ 2.02%, 31.14 ꢀ 2.28% and 34.50 ꢀ 2.49%, respectively.

3240
K. Yao et al. / European Journal of Medicinal Chemistry 46 (2011) 3237e3249
Table 3
that the anti-thrombotic efficacy of 5f,j,k,l,n is more than 10 fold
higher than that of 3. While the thrombus weights (19.8e21.4 mg)
of the rats receiving 10 nmol/kg of 5b,c,d,g,h,i,m,o,p are equal to
that of the rats receiving 100 nmol/kg of 3. This means that the anti-
thrombotic efficacy of 5b,c,d,g,h,i,m,o,p is 10-fold higher than that
Dose-dependent effect of 5j on the thrombus weight of the rats.a
Dose
10 nmol/kg
1 nmol/kg
0.1 nmol/kg
27.22 ꢀ 3.43d
5j
NS
18.67 ꢀ 1.51b
22.77 ꢀ 2.47c
28.21 ꢀ 1.11
a
of 3. Therefore the introduction of -amino acid into 3 substantially
L
Weight of wet thrombus is represented by X ꢀ SD mg, NS ¼ vehicle, n ¼ 12.
Compare to NS and 1 nmol/kg of 5j p < 0.01.
Compare to NS and 0.1 nmol/kg of 5j p < 0.01.
Compared to NS p > 0.05.
b
c
increases the in vivo anti-thrombotic efficacy.
On the other hand, it was also noticed that the in vivo activities
were not consistent with the in vitro activities. In vivo each member
of 5aer had to undergo individual absorption, distribution and
metabolism, while in vitro they did not. This perhaps should be
responsible for their inconsistent in vitro and in vivo activities.
d
tetrahydro-b-carboline-3-carbonyl]hydrazine that shared by 5aer.
This subset was used for the alignment. A rigid fit of atom pairings
was performed to superimpose each structure onto N-[(1S,3S)-1-
methyl-1,2,3,4-tetrahydro-b-carboline-3-carbonyl]hydrazine.
Stereoview of aligned 5aer is shown in Fig. 2. The alignment ster-
eoview explores that to superimpose onto N-[(1S,3S)-1-methyl-
1,2,3,4- tetrahydro-b-carboline-3-carbonyl]hydrazine the amino acid
chain of each structure has to take individual conformation, which
will affect on the anti-thrombotic activity.
3.3. Dose-dependent in vivo anti-thrombotic activity of 5j
Oral 5j was observed at three doses (10, 1 and 0.1 nmol/kg) to
produce a possible dose-dependent anti-thrombotic response in
rats. The thrombus weights are listed in Table 3. The data demon-
strate that the thrombus weight is progressively increased with the
decrease of the dose. Therefore, 5j had a dose-dependent anti-
thrombotic action.
4.2. MFA based Cerius 2 QSAR module of 5aeg,i,j,ler
4. 3D QSAR analysis of 5aer
Molecular field analysis (MFA) was performed for 5aeg,i,j,ler
using the QSAR module of Cerius 2 [42]. A five-step-procedure
consisted of generating conformers, energy minimization, match-
ing atoms and aligning molecules, setting preferences, and regres-
sion analysis was automatically practiced in MFA. Molecular
electrostatic and steric fields were created by use of proton, methyl
and hydroxyl anion as probes, respectively. These fields were
sampled at each point of a regularly spaced grid of 1 Å. An energy
cutoff of ꢀ30.0 kcal/mol was set for both electrostatic and steric
fields. The total grid points generated were 734. Though the spatial
and structural descriptors such as dipole moment, polarizability,
radius of gyration, number of rotatable bonds, molecular volume,
principal moment of inertia, AlogP98, number of hydrogen bond
donors and acceptors, and molar refractivity were also considered,
onlythe highestvariance holderprotonand methyldescriptors were
used. Regression analysis was carried out using the genetic partial
least squares (G/PLS) method consisting of 10,000 generations with
a population size of 100. The number of components was set to 5.
Cross-validation was performed with the leave-one-out procedure.
PLS analysiswas scaled, with all variables normalized toavariance of
1.0. The MFA model for the anti-thrombotic activities of 5aeg,i,j,ler
in terms of the most relevant descriptors including proton, methyl
and hydroxyl anion is expressed by Equation (1).
To understand the dependence of the in vivo anti-thrombotic
activities of 5aer upon their structures the 3D QSAR analysis was
performed. Training set (5aeg,i,j,ler)/test set (5h,k) selections
were done manually such that they populate the wide range of
anti-thrombotic activity in similar proportion. In the analysis, the
alignment, MFA based Cerius 2 QSAR module of 5aer and the
electrostatic and environments of them within the grid with 3D
points of the equation were involved.
4.1. Alignment of 5aer
Establishing the valid 3D-QSAR models a proper alignment
procedure of 5aer was practiced using the consensus model
align strategy in the align module within Cerius 2. Based on the
assumption that each structure of 5aer exhibits activity at the same
binding site of the receptor, they were aligned in a pharmacological
active orientation. To obtain a consistent alignment N-[(1S,3S)-
1-methyl-1,2,3,4-tetrahydro-
b-carboline-3-carbonyl]hydrazine was
selected as the template for superposing 5aer. The method used for
performing the alignment was the maximum common subgraph
(MCS) [41]. MCS looks at molecules as points and lines, and uses the
techniques out of graph theory to identify the patterns. Then MCS
finds the largest subset of atoms in N-[(1S,3S)-1-methyl-1,2,3,4-
Table 2
Effect of oral 5aer on the thrombus weight of the rats.a
Compd.
Thrombus weight
Compd.
Thrombus weight
NS
3
28.21 ꢀ 1.11
21.23 ꢀ 2.53b
23.74 ꢀ 2.33b
20.06 ꢀ 2.40d
20.62 ꢀ 2.13d
21.06 ꢀ 2.39d
24.01 ꢀ 1.97b
18.55 ꢀ 2.32c
20.39 ꢀ 2.81d
21.00 ꢀ 2.22d
20.24 ꢀ 2.39d
Aspirin
5j
5k
5l
5m
5n
5o
5p
5q
5r
18.24 ꢀ 1.23b
18.67 ꢀ 1.51c
19.04 ꢀ 2.51c
18.87 ꢀ 2.71c
19.75 ꢀ 2.85d
18.97 ꢀ 2.61c
19.65 ꢀ 2.40d
21.36 ꢀ 2.01d
20.42 ꢀ 1.81d
21.01 ꢀ 2.85d
5a
5b
5c
5d
5e
5f
5g
5h
5i
a
Weight of wet thrombus is represented by X ꢀ SD mg, NS ¼ vehicle, n ¼ 12;
mol/kg; Dose of 3: 0.1 mol/kg; Dose of 5aep: 10 nmol/kg.
Compare to NS p < 0.001.
Compare to NS p < 0.001 and to 3 p < 0.05.
Compare to NS p < 0.001 and to 3 p > 0.05.
Dose of aspirin: 160
m
m
b
c
d
Fig. 2. Alignment stereoview of 5aer used for molecular field generation.

K. Yao et al. / European Journal of Medicinal Chemistry 46 (2011) 3237e3249
3241
which means that at this position small groups or hydrophilic
group will decrease the thrombus weight. The terms of 0.030
(HOꢁ/320) and 0.068 (HOꢁ/407) have negative coefficients, which
means that at these positions hydrogen bond forming group will
decrease the thrombus weight.
As examples Fig. 4 gives four representatives 5f,h,k,p of which 5f
has hydrophobic group near CH3/298 and hydrogen bond forming
group near HOꢁ/320 and decrease the thrombus weight, 5h has
neither hydrophobic group near CH3/298 nor hydrogen bond form-
ing groups near HOꢁ/320 and HOꢁ/407 and increase the thrombus
weight, 5k has hydrophobic groups near CH3/298 and hydrogen
bond forming groups near HOꢁ/407 and decrease the thrombus
weight, while 5p has electron-releasing group near Hþ/399 and
hydrophobicgroupnearCH3/362thusincreasethethrombusweight.
4.3. Predicting the in vivo anti-thrombotic activity of 5h,k with
Equation (1)
The predict power of Equation (1) was demonstrated by
comparing the calculated and tested in vivo anti-thrombotic
activity of 5h,k (Table 4). The correlations of the predicting and
the testing values are also shown in Fig. 3. The results indicate that
Equation (1) rationally gives the thrombus weights of 5h,k treated
rats and the errors range from ꢁ0.83 to 0.85 mg. The calculated
weights are so approximate to the tested weights means
that Equation (1) is practical to accurately predict the thrombus
Fig. 3. Graph of tested vs. predicted anti-thrombotic activities of 5aer.
ꢀ
ꢁ
Thrombusweight ¼ 22:89ꢁ0:054 Hþ=394
ꢀ
ꢁ
ꢁ0:0054 Hþ=399 þ0:18ðCH3=298Þ
ꢀ
ꢁ
ꢁ0:032ðCH3=362Þꢁ0:030 HOꢁ=320
ꢁ0:068 HOꢁ=407
ꢀ
ꢁ
weights of N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-b-carboline-3-
(1)
carbonyl]-N0-(amino-acid-acyl)hydrazine treated rats.
In Equation (1) the data points (n), correlation coefficient (r) and
square correlation coefficient (r2) are 16, 0.984 and 0.968, respec-
tively. The parameters indicated that Equation (1) is able to predict
the in vivo activity for the analogs of 5aeg,i,j,ler. The tested
thrombus weights on rat thrombogenesis model and the calculated
thrombus weights based on Equation (1) were shown in Fig. 3.
Equation (1) contains 2 terms from proton descriptor, 2 terms
from methyl descriptor and 2 terms from hydroxyl anion descriptor.
The terms of 0.054 (Hþ/394) and 0.0054 (Hþ/399) have negative
coefficients, which means that at these positions electron-releasing
groups will increase the thrombus weight. The term of 0.18
(CH3/298) has positive coefficient, which means that at this posi-
tion small group or hydrophilic group will increase the thrombus
weight, while term of 0.032 (CH3/362) has negative coefficient,
5. Conclusions
The stretching conformation of amino acid modified 3S-tetra-
hydro- -carboline-3-carboxylic acids profoundly benefits the
b
in vivo anti-thrombotic activity and is characterized by the smaller
proximity of the side chain of the amino acid residue to the
carboline-cycle. The insertion of a hydrazine toward (1S,3S)-1-
methyl-1,2,3,4-tetrahydro-
crease the proximity of the side chain of the amino acid residue to
the carboline-cycle. Comparing to the known 3S-tetrahydro-
carboline-3-carbonylamino acids and (3S,12aS)-3-(aminoacid-side-
chanin)-2,3,6,7,12,12a-hexahydropyrazino[10,20:1,6]pyrido[3,4-b]
indole-1,4-diones
b-carboline-3-carbonylamino acids de-
b
-
these
novel
N-[(1S,3S)-1-methyl-1,2,3,4-
Fig. 4. Electrostatic and environments of 5f,h,k,p within the grid with 3D points of Equation (1).

3242
K. Yao et al. / European Journal of Medicinal Chemistry 46 (2011) 3237e3249
Table 4
with 2.0 g (8.7 mmol) of (1S,3S)-1-methyl-1,2,3,4-Tetrahydro-b-
Predicted and tested thrombus weight of 5h,k treated rats.
carboline-3-carboxylic acid (1). The reaction mixture was stirred at
room temperature for 12 h and TLC (CH2Cl2/CH3OH, 10:1) indicated
the complete disappearance of 1. The reaction mixture was
neutralized with aqueous sodium carbonate and then kept at 4 ꢃC
for 2 h. The formed precipitates were collected by filtration and
washed with water to give 1.9 g (89%) of the title compound as
yellowing powders. Mp 75e76 ꢃC; ESI-MS (m/z) 245 [M þ H]þ; IR
(KBr): 3400, 3204, 2961, 2903, 2814, 1745, 1622, 1594, 1505, 1453,
Compd.
Thrombus weight (mg)
Predicted
Tested
Error
Error%
5h
5k
20.17
19.89
21.00
19.04
ꢁ0.83
ꢁ3.95
0.85
4.46
tetrahydro-b
-carboline-3-carbonyl]-N0-(amino-acid-acyl)hydrazine
(5aer) have smaller proximity and 50-folde500-fold higher in vivo
anti-thrombotic activities.
1384, 1062, 895 cmꢁ1 1H NMR (500 MHz, CDCl3)
d
/ppm ¼ 10.06 (s,
1 H), 7.44 (t, J ¼ 6.4 Hz,1 H), 7.21 (t, J ¼ 8.6 Hz,1 H), 7.13 (d, J ¼ 7.5 Hz,
1 H), 7.02 (d, J ¼ 6.6 Hz, 1 H), 4.31 (q, J ¼ 6.6 Hz, 1 H), 3.79 (s, 3 H),
3.75 (m, J ¼ 6.9 Hz, 1 H), 3.10 (d, J ¼ 11.2 Hz, 1 H), 2.81 (t, J ¼ 11.2 Hz,
1 H), 2.27 (s, 1 H), 1.44 (d, J ¼ 6.0 Hz, 3 H).
The proximity of the side chain of the amino acid residue to the
carboline-cycle is also of critical importance for the in vivo anti-
thrombotic activity of 5aer themselves. Fig. 4 clearly shows this
importance, in 5f and 5k the proximity of the side chains to the
carboline-cycle is smaller, while in 5h and 5p the proximity of the
side chains to the carboline-cycle is larger, and consequently 5f and
5k have higher invivo anti-thromboticactivity, 5h and 5phave lower
in vivo anti-thrombotic activity. Therefore the proximity of the side
chain of the amino acid residue to the carboline-cycle can be used to
6.2.3. N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-b-carboline-3-carbonyl]-
hydrazine (3)
At room temperature with stirring to the solution of 1.0 g
(4.1 mmol) of (1S,3S)-1-methyl-1,2,3,4-tetrahydro- -carboline-3-
b
predict the in vivo anti-thrombotic activity of b-carbolines.
carboxylicacidmethylester(2)in5mlofchloroform4ml(80.0mmol,
85%) of hydrazine hydrate was added. The reaction mixture was
stirred at 60 ꢃC for 1 h and TLC (CH2Cl2/CH3OH, 10:1) indicated the
complete disappearance of 2. The reaction mixture was evaporated
under reduced pressure and the formed residue was dissolved in
50 ml of ethyl acetate. The solution was washed successively with 5%
sodium bicarbonate, 5% citric acid, and saturated sodium chloride,
and the organic phase was separated and dried over anhydrous
sodium sulfate. After filtration and evaporation under reduced pres-
sure, the title compound was provided as colorleþss powders, 720 mg
6. Experimental protocols
6.1. General
The protected amino acids with L-configuration were purchased
from Sigma Chemical Co. All coupling and deprotective reactions
were carried out under anhydrous conditions. Chromatography
was performed on Qingdao silica gel H. The purities of the inter-
mediates and the products were confirmed on thin layer chroma-
tography TLC (Merck silica gel plates of type 60 F254, 0.25 mm layer
thickness) and HPLC (Waters, C18 column 4.6 ꢂ 150 mm). 1H NMR
and 13C NMR spectra were recorded by Bruker Advance 300 and
500 spectrometers. FAB-MS was determined by VG-ZAB-MS high
resolution GC/MS/DS and HP ES-5989x. Optical rotations were
determined with a Schmidt þ Haensch Polartromic D instrument.
The statistical analysis of all the biological date was carried out by
use of ANOVA test with p < 0.05 as significant cutoff.
(72%). Mp176e177 ꢃC; ESI-MS (m/z):245[M þ H] ; ½a 2D0
¼ ꢁ103.13 (c
ꢄ
1.01, CH3OH); IR (cmꢁ1) 3326, 3277, 3019, 2978, 2913, 2843, 1650,
1605, 1687, 1552,1572, 1454, 1389, 1311, 1204, 1123, 996, 910, 751; 1H
NMR (300 MHz, DMSO-d6)
d
/ppm ¼ 10.75 (s,1 H), 9.05 (s,1 H), 7.37 (d,
J ¼ 7.5 Hz,1 H), 7.30 (m, 2 H), 7.03 (t, J ¼ 7.5 Hz,1 H), 6.95 (t, J ¼ 7.5 Hz,
1 H), 4.28 (s, 1 H), 4.05 (m, 1 H), 3.45 (dd, J ¼ 10.8 Hz, J ¼ 3.9 Hz, 1 H),
2.86 (dd, J ¼ 15.0 Hz, J ¼ 4.2 Hz,1 H), 2.60 (m,1 H), 2.07 (s,1 H),1.42 (d,
J ¼ 6.6 Hz, 3 H); 13C NMR (75 MHz, DMSO-d6)
/ppm ¼ 172.4, 138.3,
d
136.3, 127.4, 120.9, 118.8,111.4, 106.7, 65.3, 56.6, 48.7, 26.2, 20.5; Anal.
Calcd for C13H16N4O: C, 63.91; H, 6.60; N, 22.93. Found: C, 63.70; H,
6.45; N, 22.71.
6.2. Synthesis
6.2.1. (1S,3S)-1-methyl-1,2,3,4-tetrahydro-
acid (1)
b-carboline-3-carboxylic
6.2.4. N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-b-caboline-3-carbonyl]-
N0-(Boc-glycyl)-hydrazine (4a)
To the mixture of 2.0 g (9.9 mmol) of
L-tryptophan, 200 ml of
water, 0.2 ml of concentrated H2SO4 and 2 ml of ethyl aldehyde
(40%) were successively added. The reaction mixture was stirred at
room temperature for 8 h and TLC (CH2Cl2/CH3OH, 10:1) indicated
At 0 ꢃC to the solution of 394 mg (2.25 mmmol) of Boc-Gly,
304 mg (2.25 mmol) of HOBt, 507 mg (2.46 mmol) of DCC and
10 ml of anhydrous THF the solution of 500 mg (2.05 mmol) of N-
the complete disappearance of
L-tryptophan. The reaction mixture
[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-b-carboline-3-carbonyl]hydr-
was adjusted to pH 6e7 with concentrated aqueous solution of
ammonia and kept at 4 ꢃC for 2 h. The formed precipitates were
collected by filtration and washed with water to give 1.8 g (79%) of
the title compound as colorless powders. Mp 287e289 ꢃC; ESI/MS:
231 [M þ H]þ; IR (KBr): 3101-2405, 2962, 2905, 1703, 1624, 1595,
azine (3) and 10 ml of anhydrous THF was added. The reaction
solution was adjusted to pH 9.5 with 0.6 ml of N-methylmorpho-
line, stirred at 0 ꢃC for 1 h and at room temperature for 12 h and TLC
(chloroform/methanol, 15:1) indicated the complete disappearance
of 3. The reaction mixture was filtered to remove the resultant
precipitates. The filtrate was evaporated under reduced pressure to
remove the solvents. The residue was dissolved in 40 mL of ethyl
acetate and washed successively with 5% sodium bicarbonate, 5%
citric acid, and saturated sodium chloride, and the organic phase
was separated and dried over anhydrous sodium sulfate. After
filtration and evaporation under reduced pressure the residue was
purified on a chromatographic column (CH2Cl2/CH3OH, 20:1) to
provide 576 mg (70%) of the title compound as yellowing powders.
1506, 1453, 1376, 1072, 904 cmꢁ1 1H NMR (500 MHz, DMSO-d6)
d/
ppm ¼ 11.92 (s, 1 H), 10.97 (s, 1 H), 9.17 (s, 1 H), 7.45 (d, J ¼ 7.5 Hz,
1 H), 7.36 (t, J ¼ 8.0 Hz,1 H), 7.10 (t, J ¼ 8.0 Hz,1 H), 7.01 (t, J ¼ 7.5 Hz,
1 H), 4.22 (q, J ¼ 4.8 Hz, 1 H), 3.66 (dd, J ¼ 10.5 Hz, J ¼ 5.0 Hz, 1 H),
3.14 (dd, J ¼ 10.5 Hz, J ¼ 2.4 Hz, 1 H), 2.85 (ddd, J ¼ 10.5 Hz,
J ¼ 5.0 Hz, J ¼ 2.4 Hz, 1 H), 1.38 (d, J ¼ 5.0 Hz, 3 H).
6.2.2. (1S,3S)-1-methyl-1,2,3,4-tetrahydro-b-carboline-3-carboxylic
acid methyl ester (2)
Mp 146e47 ꢃC; ESI-MS (m/z): 402 [M þ H]þ; ½a 2D0
¼ ꢁ55.77 (c 1.1,
ꢄ
At 0 ꢃC and with stirring to 30 ml of methanol 1.2 ml of SOCl2
was added. This mixture was stirred for 40 min and then was mixed
CH3OH); IR (cmꢁ1) 3350, 3248, 2978, 2929, 1695, 1621, 1491, 1450,
1368, 1290, 1245, 1164, 1049, 861, 743; 1H NMR (300 MHz, DMSO-

K. Yao et al. / European Journal of Medicinal Chemistry 46 (2011) 3237e3249
3243
d6)
d
/ppm ¼ 10.77 (s, 1 H), 9.94 (s, 2 H), 7.37 (d, J ¼ 7.5 Hz, 1 H), 7.30
(d, J ¼ 7.5 Hz, 1 H), 7.03 (t, J ¼ 6.9 Hz, 1 H), 6.95 (t, J ¼ 7.5 Hz, 1 H),
6.86 (d, J ¼ 8.4 Hz, 1 H), 4.07 (m, 2 H), 3.57 (d, J ¼ 7.2 Hz, 1 H), 2.88
(dd, J ¼ 15.0 Hz, J ¼ 2.7 Hz, 1 H), 2.65 (m, 2 H), 1.71 (m, 1 H), 1.41
(d, J ¼ 7.8 Hz, 1 H), 7.01 (m, 2 H), 6.93 (t, J ¼ 6.9 Hz, 1 H), 4.08 (m,
1 H), 3.64 (d, J ¼ 6.0 Hz, 1 H), 3.57 (dd, J ¼ 10.8 Hz, J ¼ 3.9 Hz, 1 H),
2.88 (dd, J ¼ 15.0 Hz, J ¼ 3.0 Hz, 1 H), 2.63 (t, J ¼ 13.2 Hz, 1 H), 2.55
(m, 14 H), 0.95 (t, J ¼ 6.6 Hz, 6 H); 13C NMR (75 MHz, DMSO-d6)
d/
(m, 2 H),1.41 (m,12 H); 13C NMR (75 MHz, DMSO-d6)
d/ppm ¼ 171.7,
ppm ¼ 171.7, 170.7, 155.7, 138.3, 136.3, 130.7, 127.4, 120.9, 118.8,
117.8, 111.4, 106.5, 78.4, 60.2, 56.3, 48.7, 41.5, 28.6, 26.3, 23.4, 20.4,
14.5. Anal. Calcd for C24H35N5O4: C, 63.00; H, 7.71; N, 15.31.
Found: C, 62.81; H, 7.55; N, 15.54.
168.6, 156.2, 138.3, 136.3, 127.3, 121.0, 118.8, 117.8, 111.4, 106.5, 78.4,
60.2, 56.3, 48.7, 28.6, 26.3, 20.4. Anal. Calcd for C20H27N5O4: C,
59.84; H, 6.78; N, 17.44. Found: C, 59.65; H, 6.94; N, 17.66.
6.2.5. N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-
b-caboline-3-carbonyl]-
6.2.8. N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-b-caboline-3-carbonyl]-
N0-(Boc-valyl)-hydrazine (4e)
N0-(Boc-alanyl)-hydrazine (4b)
Using a procedure similar to that of preparing N-[(1S,3S)-1-methyl-
1,2,3,4-tetrahydro-
-caboline-3-carbonyl]-N0-(Boc-glycyl)-hydrazine
(4a) from 500 mg (2.05 mmol) of N-[(1S,3S)-1-methyl-1,2,3,4-
tetrahydro- -carboline-3-carbonyl]hydrazine (3) and 426 mg
(2.25 mmol) of Boc- -Ala 623 mg (73%) of the title compound were
Using a procedure similar to that of preparing N-[(1S,3S)-1-methyl-
1,2,3,4- tetrahydro-
-caboline-3-carbonyl]-N0-(Boc-glycyl)-hydrazine
(4a) from 500 mg (2.05 mmol) of N-[(1S,3S)-1-methyl-1,2,3,4-tetra-
hydro- -carboline-3-carbonyl]-hydrazine (3) and 489 mg (2.25 mmol)
of Boc-
b
b
b
b
L
L-Val 776 mg (86%) of the title compound were obtained as
obtainedas yellowingpowders. Mp128e130 ꢃC;ESI-MS(m/z):416
colorless powders. Mp 183e184 ꢃC; ESI-MS (m/z) 444 [M þ H]þ;
[M þ H]þ; ½a 2D0
ꢄ
¼ꢁ97.17 (c 1.3, CH3OH); IR (cmꢁ1) 3317, 2978, 2929,
½
a 2D0
ꢄ
¼ ꢁ83.77 (c 1.0, CH3OH); IR (cmꢁ1): 3432, 3329, 3211, 2970,
1687, 1503, 1454, 1368, 1315, 1249, 1164, 1044, 857, 743; 1H NMR
2929, 2847, 1679, 1613, 1527, 1478, 1364, 1315, 1294, 1241, 1160, 1021,
(300 MHz, DMSO-d6)
d
/ppm ¼ 10.76 (s, 1 H), 9.95 (s, 1 H), 7.89 (m,
751; 1H NMR (300 MHz, DMSO-d6)
d/ppm ¼ 10.76 (s,1 H), 9.97 (s,1 H),
2 H), 7.38 (d, J ¼7.5 Hz, 1 H), 7.30 (d, J ¼7.5 Hz,1 H), 7.37(t, J ¼6.9 Hz,
1 H), 6.95 (t, J ¼ 7.5 Hz, 1 H), 4.07 (m, 2 H), 3.57 (dd, J ¼ 10.8 Hz,
J¼4.2Hz,1H), 2.88(dd, J¼15.0 Hz, J¼2.7Hz,1H), 2.65(m,1H),1.41
(m, 12 H), 1.25 (d, J ¼ 6.9 Hz, 3 H). 13C NMR (75 MHz, DMSO-d6)
9.86 (s, 1 H), 7.37 (d, J ¼ 7.5 Hz, 1 H), 7.29 (d, J ¼ 7.5 Hz, 1 H), 7.05 (t,
J ¼ 6.9 Hz, 1 H), 6.95 (t, J ¼ 6.9 Hz, 1 H), 6.69 (d, J ¼ 9.0 Hz, 1 H), 4.09 (d,
J ¼ 5.7 Hz, 1 H), 3.88 (t, J ¼ 8.1 Hz, 1 H), 3.57 (d, J ¼ 10.5 Hz, 1 H), 2.88
(dd, J ¼ 2.7 Hz, J ¼ 15.0 Hz, 1 H), 2.65 (m, 1 H), 1.96 (m, 2 H), 1.41 (m,
12 H), 0.95 (d, J ¼ 6.9 Hz, 3 H), 0.90 (d, J ¼ 6.9 Hz, 3 H); 13C NMR
d
/ppm ¼ 171.7, 170.7, 155.7, 138.3, 136.3, 130.7, 127.4, 120.9, 118.8,
117.8, 111.4, 106.5, 78.4, 56.3, 48.7, 41.5, 28.6, 26.3, 23.4, 20.4, 14.5.
Anal. Calcd for C21H29N5O4: C, 60.71; H, 7.04; N, 16.86. Found: C,
60.92; H, 7.20; N, 16.63.
(75 MHz, DMSO-d6)
d/ppm ¼ 171.7, 170.5, 155.8, 138.3, 136.3, 130.7,
127.4, 120.9, 118.8, 117.8, 111.4, 106.5, 78.4, 58.7, 56.3, 48.7, 31.0, 28.6,
26.3, 20.4, 19.6, 18.8. Anal. Calcd for C23H33N5O4: C, 62.28; H, 7.50; N,
15.79. Found: C, 62.07; H, 7.34; N, 15.98.
6.2.6. N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-
b-caboline-3-carbonyl]-
N0-(Boc-isoleu-cyl)hydrazine (4c)
6.2.9. N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-b-caboline-3-carbonyl]-
N0-(Boc-methio-nyl)hydrazine (4f)
Using a procedure similar to that of preparing N-[(1S,3S)-1-methyl-
1,2,3,4-tetrahydro-
-caboline-3-carbonyl]-N0-(Boc-glycyl)-hydrazine
(4a) from 500 mg (2.05 mmol) of N-[(1S,3S)-1-methyl-1,2,3,4-
tetrahydro- -carboline-3-carbonyl]-hydrazine (3) and 521 mg
(2.25 mmol) of Boc- -Ile 558 mg (60%) of the title compound were
b
Using a procedure similar to that of preparing N-[(1S,3S)-1-methyl-
1,2,3,4-tetrahydro-
-caboline-3-carbonyl]-N0-(Boc-glycyl)-hydrazine
(4a) from 500 mg (2.05 mmol) of N-[(1S,3S)-1-methyl-1,2,3,4-tetra-
hydro- -carboline-3-carbonyl]-hydrazine (3) and 561 mg (2.25 mmol)
of Boc-
b
b
L
b
obtained as colorless powders. Mp 137e139 ꢃC; ESI-MS (m/z): 458
L-Met 591 mg (61%) of the title compound were obtained as
[M þ H]þ; ½a 2D0
ꢄ
¼ ꢁ87.30 (c 1.2, CH3OH); IR (cmꢁ1): 3333, 2970,
colorless powders. Mp 126e127 ꢃC; ESI-MS (m/z) 476 [M þ H]þ;
2933, 2876,1691,1625,1499,1458,1368,1315,1249,1164,1045,1012,
½
a 2D0
ꢄ
¼ ꢁ64.03 (c 1.4, CH3OH); IR (cmꢁ1): 3321, 2978, 2921, 2851, 1687,
743; 1H NMR (300 MHz, DMSO-d6)
d/ppm ¼ 10.79 (s, 1 H), 10.02 (s,
1499, 1454, 1368, 1311, 1245, 1164, 1049, 1017, 865, 743; 1H NMR
2 H), 7.37 (d, J ¼ 7.5 Hz,1 H), 7.30 (m, 2 H), 7.04 (t, J ¼ 6.9 Hz,1 H), 6.96
(t, J ¼ 6.9 Hz, 1 H), 6.76 (t, J ¼ 6.0 Hz, 1 H), 4.09 (d, 1 H), 3.94 (t,
J ¼ 8.4 Hz, 1 H), 3.56 (dd, J ¼ 10.5 Hz, J ¼ 3.6 Hz, 1 H), 2.88 (dd,
J ¼ 15.0 Hz, J ¼ 3.0 Hz,1 H), 2.63 (t, J ¼ 12.6 Hz,1 H),1.69 (m, 2 H),1.39
(m, 13 H), 0.92 (d, J ¼ 6.9 Hz, 3 H), 0.84 (t, J ¼ 7.5 Hz, 3 H). 13C NMR
(300 MHz, DMSO-d6)
d
/ppm ¼ 10.77 (s, 1 H), 9.99 (s, 2 H), 7.37 (d,
J ¼ 7.8 Hz, 1 H), 7.30 (d, J ¼ 7.8 Hz, 1 H), 7.03 (t, 2 H), 6.95 (t, J ¼ 7.5 Hz,
1 H), 4.04 (m, 2 H), 3.57 (d, J ¼ 7.2 Hz, 3 H), 2.90 (d, J ¼ 12.3 Hz, 1 H),
2.62 (d, 1 H), 2.55 (m, 3 H), 2.07 (s, 1 H),1.86 (m, 2 H), 1.41(m, 12 H); 13C
NMR (75 MHz, DMSO-d6)
d
/ppm ¼ 171.8, 171.1, 155.7, 138.3, 136.3,
(75 MHz, DMSO-d6)
d/ppm ¼ 171.6, 170.6, 155.7, 138.2, 136.2, 127.3,
127.3, 121.0, 118.8, 117.8, 111.4, 106.5, 78.6, 60.2, 56.3, 48.7, 32.4, 30.0,
28.6, 26.3, 20.4, 15.1. Anal. Calcd for C23H33N5O4S: C, 58.08; H, 6.99; N,
14.73. Found: C, 57.86; H, 6.82; N, 14.96.
121.0, 118.8, 117.8, 111.4, 106.5, 78.4, 57.6, 56.5, 48.6, 36.9, 28.6, 26.3,
24.9, 20.4, 15.6, 11.3; Anal. Calcd for C24H35N5O4: C, 63.00; H, 7.71; N,
15.31. Found: C, 62.81; H, 7.66; N, 15.54.
6.2.10. N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-
b-caboline-3-carbonyl]-
6.2.7. N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-
b-caboline-3-carbonyl]-
N0-(Boc-prolyl)-hydrazine (4g)
N0-(Boc-leucyl)-hydrazine (4d)
Using a procedure similar to that of preparing N-[(1S,3S)-1-methyl-
1,2,3,4-tetrahydro-
-caboline-3-carbonyl]-N0-(Boc-glycyl)-hydrazine
(4a) from 500 mg (2.05 mmol) of N-[(1S,3S)-1-methyl-1,2,3,4-tetra-
hydro- -carboline-3-carbonyl]-hydrazine (3) and 485 mg (2.25 mmol)
of Boc-
Using a procedure similar to that of preparing N-[(1S,3S)-1-methyl-
1,2,3,4- tetrahydro-
-caboline-3-carbonyl]-N0-(Boc-glycyl)-hydrazine
(4a) from 500 mg (2.05 mmol) of N-[(1S,3S)-1-methyl-1,2,3,4-
tetrahydro- -carboline-3-carbonyl]-hydrazine (3) and 521 mg
(2.25 mmol) of Boc- -Leu 784 mg (84%) of the title compound
b
b
b
b
L-Pro 655 mg (72%) of the title compound were obtained as
L
colorless powders. Mp 117e118 ꢃC; ESI-MS (m/z) 442 [M þ H]þ;
were obtained as colorless powders. Mp 173e173 ꢃC; ESI-MS
½
a 2D0
ꢄ
¼ ꢁ110.17 (c 1.3, CH3OH); IR (cmꢁ1): 3284, 2974, 2929, 2876,
(m/z) 458 [M þ H]þ; ½a D20
ꢄ
¼ ꢁ82.07 (c 1.1, CH3OH); IR (cmꢁ1):
1679, 1478, 1454, 1409, 1368, 1315, 1254, 1164, 1045, 739; 1H NMR
3329, 2958, 2933, 2872, 1695, 1662, 1499, 1454, 1388, 1367, 1318,
(300 MHz, DMSO-d6)
d
/ppm ¼ 8.92 (s, 1 H), 8.18 (s, 1 H), 7.44 (d,
1277, 1253, 1163, 1045, 857, 743. 1H NMR (300 MHz, DMSO-d6)
J ¼ 7.5 Hz, 1 H), 7.30 (d, J ¼ 7.5 Hz, 1 H), 7.17 (t, J ¼ 6.9 Hz, 1 H), 7.08 (t,
d
/ppm ¼ 10.76 (s, 1 H), 9.98 (s, 2 H), 7.43 (d, J ¼ 7.5 Hz, 1 H), 7.37
J ¼ 6.9 Hz, 1 H), 6.69 (d, J ¼ 9.0 Hz,1 H), 4.41 (s,1 H), 4.18 (m,1 H), 3.68

3244
K. Yao et al. / European Journal of Medicinal Chemistry 46 (2011) 3237e3249
(m, 1 H), 3.47 (m, 2 H), 3.20 (dd, J ¼ 15.6 Hz, J ¼ 3.0 Hz, 1 H), 2.75 (m,
J ¼ 7.5 Hz, 2 H), 6.39 (d, J ¼ 8.4 Hz, 1 H), 4.79 (s, 1 H), 4.08 (d, J ¼ 5.7 Hz,
1 H), 3.93(m, 2 H), 3.56 (d, J ¼ 7.2 Hz, 1 H), 2.89 (dd, J ¼ 11.7Hz,
J ¼ 3.0Hz,1 H), 2.63 (t, J ¼ 12.0 Hz,1 H),1.44 (m,12 H),1.14 (d, J ¼ 6.0 Hz,
2 H), 2.37 (m, 1 H), 1.89 (m, 3 H), 1.49 (m, 12 H); 13C NMR (75 MHz,
DMSO-d6)
d/ppm ¼ 171.1, 169.6, 136.7, 135.9, 127.2, 121.8, 119.6, 118.1,
110.9, 107.9, 80.9, 60.4, 56.9, 49.2, 47.1, 28.4, 25.2, 21.0, 20.1, 14.2; Anal.
Calcd for C23H31N5O4: C, 62.57; H, 7.08; N, 15.86. Found: C, 62.79; H,
7.23; N, 16.07.
3 H); 13C NMR (75 MHz, DMSO-d6)
d
/ppm ¼ 171.7, 169.6, 155.7, 138.3,
136.3, 127.3, 121.0, 118.8, 117.8, 111.4, 106.6, 78.7, 67.4, 65.4, 56.3, 48.7,
28.6, 26.3, 20.4. Anal. Calcd for C22H31N5O5: C, 59.31; H, 7.01; N, 15.72.
Found: C, 59.50; H, 7.17; N, 15.92.
6.2.11. N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-
b-caboline-3-carbonyl]-
N0-(Boc-phenyla-lanyl)hydrazine (4h)
6.2.14. N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-
b-caboline-3-carbonyl]-
N0-(Boc-seryl)-hydrazine (4k)
Using a procedure similar to that of preparing N-[(1S,3S)-1-methyl-
1,2,3,4-tetrahydro-
-caboline-3-carbonyl]-N0-(Boc-glycyl)-hydrazine
(4a) from 500 mg (2.05 mmol) of N-[(1S,3S)-1-methyl-1,2,3,4-tetra-
hydro- -carboline-3-carbonyl]hydrazine (3) and 598 mg (2.25 mmol)
of Boc-
b
Using a procedure similar to that of preparing N-[(1S,3S)-1-methyl-
1,2,3,4-tetrahydro-
-caboline-3-carbonyl]-N0-(Boc-glycyl)-hydrazine
(4a) from 500 mg (2.05 mmol) of N-[(1S,3S)-1-methyl-1,2,3,4-tetrahy-
dro- -carboline-3-carbonyl]hydrazine (3) and 462 mg (2.25 mmol) of
Boc-
b
b
L
-Phe 727 mg (72%) of the title compound were obtained as
b
colorless powder. Rf ¼ 0.41 (CH2Cl2: MeOH, 15 : 1), Mp 138e141 ꢃC;
L-Ser 500 mg (57%) of the title compound were obtained as
ESI-MS (m/z) 492 [M þ H]þ; ½a D20
ꢄ
¼ ꢁ56.70 (c 1.0, MeOH); IR (cmꢁ1
)
colorless powder. Mp 130e132 ꢃC; ESI-MS (m/z) 432 [M þ H]þ;
3333, 3231, 3060, 3027, 2974, 2925, 2859, 1711, 1683, 1625, 1527, 1495,
½
a 2D0
ꢄ
¼ ꢁ65.00 (c 1.2, CH3OH); IR (cmꢁ1): 3415, 3338, 2978, 2929, 2851,
1454,1368,1319, 1278, 1254, 1164,1025, 1025, 743; 1H NMR (300 MHz,
1703, 1679, 1568, 1503, 1454, 1364, 1315, 1249, 1164, 1057, 747; 1H NMR
DMSO-d6)
d
/ppm ¼ 10.79 (s, 1 H), 10.22 (s, 2 H), 7.35 (m, 6 H), 7.23 (d,
(300 MHz, DMSO-d6)
d
/ppm ¼ 10.77 (s, 1 H), 10.04 (s, 2 H), 7.37 (d,
J ¼ 6.9 Hz,1 H), 6.95 (m, 3 H), 4.30 (t, J ¼ 7.5 Hz,1 H), 4.10 (d, J ¼ 6.6 Hz,
1 H), 3.60 (dd, J ¼ 4.2 Hz, J ¼ 10.5 Hz, 1 H), 3.04 (dd, J ¼ 3.0 Hz,
J ¼ 13.5 Hz, 1 H), 2.88 (dd, J ¼ 3.0 Hz, J ¼ 15.0 Hz, 1 H), 2.80 (t,
J ¼ 12.3 Hz, 1 H), 2.66 (m, 2 H), 1.48 (d, J ¼ 6.6 Hz, 3 H),1.30 (s, 9 H); 13C
J ¼ 7.5 Hz, 1 H), 7.30 (d, J ¼ 8.1 Hz, 1 H), 7.05 (t, J ¼ 6.9 Hz, 1 H), 6.95 (t,
J ¼ 7.5 Hz, 2 H), 6.69 (d, J ¼ 8.1 Hz,1 H), 4.07 (m, 2 H), 3.58 (m, 3 H), 2.88
(dd, 1 H), 2.62 (m, 2 H), 1.41 (m, 12 H); 13C NMR (75 MHz, DMSO-d6)
d/
ppm ¼ 171.6, 169.5, 155.6, 138.3, 136.3, 127.3, 121.0, 118.8, 117.8, 111.4,
106.5, 78.7, 62.5, 60.2, 56.3, 48.7, 28.6, 26.3, 20.4. Anal. Calcd for
C21H29N5O5: C, 58.45; H, 6.77; N,16.23. Found: C, 58.64; H, 6.93; N,16.01.
NMR (75 MHz, DMSO-d6)
d/ppm ¼ 171.6, 171.0, 170.8, 155.7, 138.5,
138.3, 136.3, 129.7, 128.5, 127.3, 126.7, 121.0, 118.8, 117.9, 111.4, 110.5,
106.6, 78.4, 65.4, 56.4, 48.7, 28.6, 26.3, 20.4; Anal. Calcd for
C27H33N5O4: C, 65.97; H, 6.77; N, 14.25.
6.2.15. N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-
N0-(Boc-trpto-phanyl)hydrazine (4l)
b-caboline-3-carbonyl]-
6.2.12. N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-
b-caboline-3-carbonyl]-
N0-(Boc-tyrosyl)-hydrazine (4i)
Using a procedure similar to that of preparing N-[(1S,3S)-1-methyl-
1,2,3,4-tetrahydro-
-caboline-3-carbonyl]-N0-(Boc-glycyl)-hydrazine
(4a) from 500 mg (2.05 mmol) of N-[(1S,3S)-1-methyl-1,2,3,4-tetra-
hydro- -carboline-3-carbonyl]hydrazine (3) and 686 mg (2.25 mmol)
of Boc-
b
Using a procedure similar to that of preparing N-[(1S,3S)-1-
methyl-1,2,3,4-tetrahydro-
-caboline-3-carbonyl]-N0-(Boc-glycyl)-
hydrazine (4a) from 500 mg (2.05 mmol) of N-[(1S,3S)-1-methyl-
1,2,3,4-tetrahydro- -carboline-3-carbonyl]hydrazine (3) and
634 mg (2.25 mmol) of Boc- -Tyr 523 mg (50%) of the title
b
b
L
-Trp 795 mg (73%) of the title compound were obtained as
b
colorless powder. Mp 158e160 ꢃC; ESI-MS (m/z) 531 [M þ H]þ;
L
½
a 2D0
ꢄ
¼ ꢁ47.80 (c 1.2, CH3OH); IR (cmꢁ1) 3415, 3358, 3329, 3219, 3055,
compound were obtained as colorless powder. Mp 141e142 ꢃC; ESI-
2974, 2924, 2871, 1702, 1682, 1625, 1576, 1527, 1494, 1457, 1367, 1318,
MS (m/z) 508 [M þ H]þ; ½a D20
ꢄ
¼ ꢁ43.43 (c 1.1, CH3OH); IR (cmꢁ1
)
1273, 1249,1163, 743; 1H NMR (300 MHz, DMSO-d6)
d/ppm ¼ 10.83 (s,
3419, 3313, 3248, 2978, 2933, 2847, 1703, 1683, 1617, 1519, 1478,
1 H), 9.97 (s,1 H), 7.70 (d, J ¼ 7.8 Hz,1 H), 7.35 (m, 3 H), 7.24 (s,1 H), 7.05
(m, 4 H), 6.69 (d, J ¼ 7.8 Hz,1 H), 4.35 (m,1 H), 4.11 (d,1 H), 3.60 (d,1 H),
3.16 (dd, J ¼ 14.4 Hz, J ¼ 3.9 Hz, 1 H), 2.95 (m, 2 H), 2.67 (t, J ¼ 12.6 Hz,
1 H), 1.44 (d, J ¼ 6.6 Hz, 3 H), 1.33 (s, 12 H); 13C NMR (75 MHz, DMSO-
1454, 1364, 1315, 1249, 1164, 1053, 824, 743. 1H NMR (300 MHz,
DMSO-d6)
d
/ppm ¼ 10.79 (s, 1 H), 10.17 (s, 2 H), 9.17 (s, 1 H), 7.41 (d,
J ¼ 7.5 Hz, 1 H), 7.30 (d, J ¼ 7.8 Hz, 1 H), 7.15 (d, J ¼ 12.3 Hz, 2 H), 7.04
(d, J ¼ 7.5 Hz, 1 H), 6.96 (d, J ¼ 7.5 Hz, 1 H), 6.88 (d, J ¼ 8.7 Hz, 1 H),
6.67 (d, J ¼ 8.4 Hz, 2 H), 4.16 (m, 1 H), 4.10 (d, J ¼ 6.6 Hz, 1 H), 3.60
(dd, J ¼ 9.9 Hz, J ¼ 3.0 Hz, 1 H), 2.88 (m, 3 H), 2.66 (m, 2 H), 1.44 (d,
d6)
d
/ppm ¼ 171.6, 171.3, 170.8, 155.6, 138.3, 136.5, 136.3, 127.8, 127.3,
124.4,121.3,121.0,119.0,118.8,118.6,117.9,111.7,111.4,110.5,106.6, 78.4,
60.2, 56.4, 48.7, 28.6, 26.3, 21.2, 20.4. Anal. Calcd for C29H34N6O4: C,
65.64; H, 6.46; N, 15.84. Found: C, 65.43; H, 6.31; N, 15.62.
J ¼ 6.6 Hz, 3 H), 1.32 (s, 9 H); 13C NMR (75 MHz, DMSO-d6)
d/
ppm ¼ 171.5, 171.1, 156.2, 155.7, 154.8, 138.2, 136.3, 136.2, 130.6,
128.5, 127.3, 121.0, 118.8, 117.9, 115.3, 111.4, 106.6, 105.9, 78.4, 65.4,
56.4, 48.7, 37.3, 28.6, 26.2, 20.4. Anal. Calcd for C27H33N5O5: C,
63.89; H, 6.55; N, 13.80. Found: C, 63.68; H, 6.39; N, 13.57.
6.2.16. N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-
b-caboline-3-carbonyl]-
N0-(di-Boc-histi-dyl)-hydrazine (4m)
Using a procedure similar to that of preparing N-[(1S,3S)-1-methyl-
1,2,3,4-tetrahydro-
-caboline-3-carbonyl]-N0-(Boc-glycyl)-hydrazine
(4a) from 500 mg (2.05 mmol) of N-[(1S,3S)-1-methyl-1,2,3,4-tetra-
6.2.13. N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-
b-caboline-3-carbonyl]-
b
N0-(Boc-threo-nyl)hydrazine (4j)
hydro-b-carboline-3-carbonyl]hydrazine (3) and 801 mg (2.25 mmol)
Using a procedure similar to that of preparing N-[(1S,3S)-1-methyl-
1,2,3,4-tetrahydro-
-caboline-3-carbonyl]-N0-(Boc-glycyl)-hydrazine
(4a) from 500 mg (2.05 mmol) of N-[(1S,3S)-1-methyl-1,2,3,4-tetra-
hydro- -carboline-3-carbonyl]hydrazine (3) and 494 mg (2.25 mmol)
of Boc-
of Boc-
L
-His(Boc) 640 mg (54%) of the title compound were obtained
b
as colorless powder. Mp 154e155 ꢃC; ESI-MS (m/z) 582 [M þ H]þ;
½
a 2D0
ꢄ
¼ ꢁ36.57 (c 1.2, CH3OH); IR (cmꢁ1) 3346, 3190, 3060, 2978, 2929,
b
L
1756, 1695, 1609, 1491, 1458, 1392, 1294, 1254, 1155, 1049, 1012, 841,
-Thr 492 mg (54%) of the title compound were obtained as
743; 1H NMR (300 MHz, DMSO-d6)
d/ppm ¼ 10.79 (s, 1 H), 10.12 (s,
colorless powder. Mp 136e137 ꢃC; ESI-MS (m/z) 446 [M þ H]þ;
2 H), 8.12 (s,1 H), 7.37 (d, J ¼ 7.5 Hz,1 H), 7.30 (d, J ¼ 6.9 Hz,1 H), 7.03 (t,
J ¼ 7.5 Hz,1 H), 6.96 (t, J ¼ 6.9 Hz, 2 H), 4.35 (m,1 H), 4.08 (d, J ¼ 6.3 Hz,
1 H), 3.56 (d, J ¼ 6.9 Hz,1 H), 2.88 (m, 3 H), 2.63 (t, J ¼ 12.3 Hz,1 H),1.56
(m, 11 H), 1.44 (d, J ¼ 6.6 Hz, 3 H), 1.35 (s, 9 H); 13C NMR (75 MHz,
½
a 2D0
ꢄ
¼ ꢁ75.60 (c 1.1, CH3OH); IR (cmꢁ1): 3415, 3338, 2978, 2929, 2847,
1707, 1683, 1572, 1503, 1491, 1450, 1364, 1315, 1254, 1164, 743; 1H NMR
(300 MHz, DMSO-d6)
J ¼ 7.5 Hz, 1 H), 7.30 (d, J ¼ 7.5 Hz, 1 H), 7.03 (t, J ¼ 7.5 Hz, 1 H), 6.95 (t,
d
/ppm ¼ 10.78 (s, 1 H), 10.01 (s, 2 H), 7.37 (d,
DMSO-d6)
d/ppm ¼ 171.7, 170.8, 155.6, 147.2, 139.8, 138.2, 137.0, 136.3,

K. Yao et al. / European Journal of Medicinal Chemistry 46 (2011) 3237e3249
3245
127.3, 121.0, 118.8, 117.8, 114.9, 111.4, 106.5, 79.6, 78.6, 56.3, 52.8, 48.6,
31.2, 28.6, 27.8, 26.3, 20.4. Anal. Calcd for C29H39N7O6: C, 59.88; H, 6.76;
N, 16.86. Found: C, 59.67; H, 6.61; N, 17.08.
6.2.20. N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-
b-caboline-3-carbonyl]-
N0-(Boc-aspara-ginyl)hydrazine (4q)
Using a procedure similar to that of preparing N-[(1S,3S)-1-
methyl-1,2,3,4-tetrahydro-
-caboline-3-carbonyl]-N0-(Boc-glycyl)-
hydrazine (4a) from 500 mg (2.05 mmol) of N-[(1S,3S)-1-methyl-
1,2,3,4-tetrahydro- -carboline-3-carbonyl]hydrazine (3) and
523 mg (2.25 mmol) of Boc- -Asn 292 mg (31%) of the title
6.2.17. N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-
b
-caboline-3-carbonyl]-
b
N0-(di-Boc-lysyl)-hydrazine (4n)
b
Using a procedure similar to that of preparing N-[(1S,3S)-1-methyl-
1,2,3,4-tetrahydro-
-caboline-3-carbonyl]-N0-(Boc-glycyl)-hydrazine
(4a) from 500 mg (2.05 mmol) of N-[(1S,3S)-1-methyl-1,2,3,4-tetra-
L
b
compound were obtained as colorless powder. Mp 172e174 ꢃC; ESI-
MS(m/z) 459 [M þ H]þ; ½a D20
ꢄ
¼ ꢁ60.23 (c 1.3, CH3OH); IR (cmꢁ1
)
hydro-b-carboline-3-carbonyl]hydrazine (3) and 780 mg (2.25 mmol)
3333, 3182, 3056, 2978, 2933, 1695, 1679, 1613, 1499, 1454, 1392,
of Boc-
L
-Lys(Boc) 541 mg (46%) of the title compound were obtained as
1368, 1319, 1270, 1254, 1168, 1053, 1025, 857, 743; 1H NMR
colorless powder. Mp 129e130 ꢃC; ESI-MS (m/z) 573 [M þ H]þ;
(300 MHz, DMSO-d6)
d
/ppm ¼ 10.82 (s, 1 H), 9.50 (s, 2 H), 7.48 (d,
½
a 2D0
ꢄ
¼ ꢁ60.37 (c 1.2, CH3OH); IR (cmꢁ1) 3342, 2974, 2929, 2864, 1691,
J ¼ 6.3 Hz,1 H), 7.36 (d, J ¼ 7.5 Hz, 2 H), 7.03 (t, J ¼ 6.9 Hz,1 H), 6.95 (t,
J ¼ 6.6 Hz, 2 H), 4.36 (m, J ¼ 8.1 Hz, 1 H), 4.08 (d, J ¼ 6.3 Hz, 1 H), 4.00
(m, 1 H), 3.53 (dd, J ¼ 10.5 Hz, J ¼ 3.9 Hz,1 H), 3.39 (m, 1 H), 2.85 (dd,
J ¼ 14.7 Hz, J ¼ 2.4 Hz, 1 H), 2.62 (t, J ¼ 13.2 Hz, 1 H), 2.47 (m, 2 H),
1511, 1458, 1392, 1368, 1315, 1274, 1249, 1168, 1045, 1017, 861, 743; 1H
NMR (300 MHz, DMSO-d6)
d
/ppm ¼ 10.78 (s, 1 H), 10.07 (s, 2 H), 7.37
(d, J ¼ 7.5 Hz,1 H), 7.30 (d, J ¼ 7.8 Hz,1 H), 7.03 (t, J ¼ 6.9 Hz,1 H), 6.95 (t,
J ¼ 6.9 Hz,1 H), 6.86 (d, J ¼ 8.1 Hz,1 H), 6.74 (s,1 H), 4.09 (d, J ¼ 6.3 Hz,
1 H), 3.99 (m, 1 H), 3.57 (dd, J ¼ 10.5 Hz, J ¼ 3.9 Hz, 1 H), 2.88 (dd,
J ¼ 14.4 Hz, J ¼ 3.3 Hz, 3 H), 2.63 (m, 2 H), 1.51 (m, 27 H); 13C NMR
1.41 (m, 12 H); 13C NMR (75 MHz, DMSO-d6)
d/ppm ¼ 171.6, 171.1,
155.5, 138.2, 136.2, 127.3, 121.0, 118.8, 117.8, 111.4, 106.5, 78.6, 56.2,
50.4, 48.6, 37.9, 28.6, 28.4, 26.2, 20.4; Anal. Calcd for C22H30N6O5: C,
57.63; H, 6.59; N, 18.33. Found: C, 57.84; H, 6.75; N, 18.54.
(75 MHz, DMSO-d6)
d
/ppm ¼ 171.6, 156.0, 155.7, 138.2, 136.3, 127.3,
121.0, 118.8, 117.8, 111.4, 106.5, 78.6, 77.8, 56.5, 53.3, 49.0, 48.7, 32.2,
29.6, 28.7, 26.2, 23.2, 20.4. Anal. Calcd for C29H44N6O6: C, 60.82; H, 7.74;
N, 14.67. Found: C, 61.03; H, 7.90; N, 14.91.
6.2.21. N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-
b-caboline-3-carbonyl]-
N0-(Boc-glutami-nyl)hydrazine (4r)
6.2.18. N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-
b
-caboline-3-carbonyl]-
Using a procedure similar to that of preparing N-[(1S,3S)-1-methyl-
1,2,3,4-tetrahydro-
-caboline-3-carbonyl]-N0-(Boc-glycyl)-hydrazine
(4a) from 500 mg (2.05 mmol) of N-[(1S,3S)-1-methyl-1,2,3,4-tetra-
hydro- -carboline-3-carbonyl]hydrazine (3) and 555 mg (2.25 mmol)
of Boc-
N0-[Boc-(
b-OBzl-aspartyl)]hydrazine (4o)
b
Using a procedure similar to that of preparing N-[(1S,3S)-1-methyl-
1,2,3,4-tetrahydro-
-caboline-3-carbonyl]-N0-(Boc-glycyl)-hydrazine
(4a) from 500 mg (2.05 mmol) of N-[(1S,3S)-1-methyl-1,2,3,4-tetra-
b
b
L-Gln 420 mg (43%) of the title compound were obtained as
colorless powder. Mp 147e149 ꢃC; ESI-MS (m/z) 473 [M þ H]þ;
hydro-b-carboline-3-carbonyl]hydrazine (3) and 728 mg (2.25 mmol)
½
a 2D0
ꢄ
¼ ꢁ71.57 (c 1.2, CH3OH); IR (cmꢁ1) 3329, 2978, 2925, 2851, 1666,
of Boc-
L
-Asp(OBzl) 487 mg (43%) of the title compound were obtained
1642, 1507, 1454, 1392, 1368, 1315, 1249, 1160, 1049, 1025, 857, 743; 1H
as colorless powder. Mp 129e130 ꢃC; ESI-MS (m/z) 550 [M þ H]þ;
NMR (300 MHz, DMSO-d6)
d
/ppm ¼ 10.78 (s, 1 H), 10.00 (s, 2 H), 7.06
½
a 2D0
ꢄ
¼ ꢁ33.13 (c 1.1, CH3OH); IR (cmꢁ1) 3354, 3182, 3056, 2978, 2925,
(d, J ¼ 7.5 Hz, 1 H), 7.28 (m, 2 H), 7.03（t, J ¼ 6.9 Hz, 1 H), 6.93 (t,
J ¼ 6.6 Hz, 2 H), 6.77 (s,1 H), 4.10 (d, J ¼ 6.6 Hz,1 H), 4.00 (m,1 H), 3.57
(dd, J ¼ 10.8 Hz, J ¼ 3.9 Hz, 1 H), 2.88 (dd, J ¼ 15.0 Hz, J ¼ 3.0 Hz, 1 H),
2.79 (t, J ¼ 6.0 Hz, 1 H), 2.19 (m, 3 H), 1.85 (m, 2 H), 1.41 (m, 12 H); 13C
2847, 1711, 1683, 1613, 1519, 1495, 1454, 1392, 1368, 1343, 1274, 1254,
1164, 1049, 1025, 857, 739; 1H NMR (300 MHz, DMSO-d6)
d/
ppm ¼ 10.76 (s,1 H),10.01 (s, 2 H), 7.35 (m, 7 H), 7.16 (d, J ¼ 8.1 Hz,1 H),
7.03 (d, J ¼ 6.9 Hz, 1 H), 6.93 (d, J ¼ 6.9 Hz, 1 H), 5.16 (s, 2 H), 4.51 (m,
1 H), 4.08 (m, 1 H), 3.56 (dd, J ¼ 10.8 Hz, J ¼ 3.9 Hz, 1 H), 2.86 (m, 2 H),
NMR (75 MHz, DMSO-d6)
d
/ppm ¼ 174.3, 171.7, 171.1, 155.6, 138.2,
136.3, 127.3, 121.0, 118.8, 117.8, 111.4, 106.5, 78.6, 65.4, 56.3, 48.7, 31.9,
28.6, 28.4, 26.2, 20.4. Anal. Calcd for C23H32N6O5: C, 58.46; H, 6.83; N,
17.78. Found: C, 58.67; H, 6.99; N, 17.99.
2.67 (m, 3 H), 1.41 (m, 12 H); 13C NMR (75 MHz, DMSO-d6)
d/
ppm ¼ 173.9, 171.8, 170.3, 155.6, 138.3, 136.6, 128.9, 128.5, 128.2, 127.3,
127.0, 121.0, 118.9, 117.8, 111.4, 106.6, 106.3, 78.8, 66.1, 63.4, 56.3, 48.7,
37.0, 28.7, 26.3, 24.9, 20.4; Anal. Calcd for C29H35N5O6: C, 63.37; H,
6.42; N, 12.74. Found: C, 63.16; H, 6.27; N, 12.96.
6.2.22. N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-
b-caboline-3-carbonyl]-
N0-glycylhydrazine (5a)
6.2.19. N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-
b-caboline-3-carbonyl]-
At 0 ꢃC to the solution of 100 mg (0.249 mmol) of N-[(1S,3S)-1-
methyl-1,2,3,4-tetrahydro-
-caboline-3-carbonyl]-N0-(Boc-glycyl)-
N0-[Boc-(
g-OBzl-glutamoyl)]hydrazine (4p)
b
hydrazine (4a) in 6 ml hydrogen chloride/ethyl acetate (4 N) were
added dropwise. The reaction solution was stirred at 0 ꢃC for 2 h and
TLC (chloroform/methanol, 10:1) indicated the complete disap-
pearance of 4a. The reaction mixture was evaporated under reduced
pressure. The residue was dissolved in 5 ml of ethyl acetate and then
evaporated under reduced pressure. This procedure was repeated
for three times to remove the remained hydrogen chloride. The
residue was solidified in 20 ml of anhydrous ether to provide 70 mg
(93%) of the title compound as yellowing powders. Mp 229e231 ꢃC;
Using a procedure similar to that of preparing N-[(1S,3S)-1-methyl-
1,2,3,4-tetrahydro-
-caboline-3-carbonyl]-N0-(Boc-glycyl)-hydrazine
(4a) from 500 mg (2.05 mmol) of N-[(1S,3S)-1-methyl-1,2,3,4-tetra-
hydro- -carboline-3-carbonyl]hydrazine (3) and 760 mg (2.25 mmol)
of Boc-
b
b
L
-Glu(OBzl) 495 mg (50%) of the title compound were obtained
as colorless powder. Mp 110e111 ꢃC; ESI-MS (m/z) 564 [M þ H]þ;
½
a 2D0
ꢄ
¼ ꢁ51.87 (c 1.2, CH3OH); IR (cmꢁ1) 3329, 3031, 2978, 2929, 2851,
1715, 1642, 1499, 1454, 1392, 1368, 1315, 1249, 1164, 1053, 1025, 861,
743; H NMR (300 MHz, DMSO-d6)
d/ppm ¼ 10.77 (s, 1 H), 10.00 (s,
ESI-MS (m/z) 302 [M þ H]þ; ½a D20
ꢄ
¼ ꢁ43.43 (c 1.2, CH3OH); IR (cmꢁ1):
1
2 H), 7.37 (m, 7 H), 7.06 (m, 2 H), 6.93 (t,1 H), 5.16 (s, 2 H), 4.08 (m, 2 H),
3.57 (d, J ¼ 7.2 Hz, 1 H), 2.88 (dd, J ¼ 15.0 Hz, J ¼ 2.7 Hz, 1 H), 2.63 (t,
J ¼ 11.4 Hz, 1 H), 2.55 (m, 3 H), 1.90 (m, 2 H), 1.41(m, 12 H); 13C NMR
3187, 2978, 2463, 1723, 1687, 1572, 1478, 1388, 1318, 1274, 1213, 747;
1H NMR (300 MHz, DMSO-d6)
d
/ppm ¼ 11.50 (s, 1 H), 11.04 (s, 1 H),
9.71 (s, 1 H), 8.45 (m, 3 H), 7.46 (d, J ¼ 7.8 Hz, 1 H), 7.38 (d, J ¼ 7.8 Hz,
1 H), 7.13 (t, J ¼ 7.5 Hz,1 H), 7.06 (t, J ¼ 7.5 Hz,1 H), 4.80 (s,1 H), 4.40 (s,
1 H), 3.71 (s, 2 H), 3.40 (m, 1 H), 3.00 (t, J ¼ 12.9 Hz, 1 H), 1.73 (d,
(75 MHz, DMSO-d6)
d/ppm ¼ 172.7, 171.8, 170.8, 155.6, 138.3, 136.7,
136.3,128.9,128.4,127.3,121.0,118.8,117.8,111.4,106.5, 78.6, 65.9, 56.3,
52.6, 48.7, 30.5, 28.6, 27.8, 26.3, 21.2. Anal. Calcd for C30H37N5O6: C,
63.93; H, 6.62; N, 12.43. Found: C, 63.71; H, 6.47; N, 12.21.
J ¼ 6.6 Hz, 3 H); 13C NMR (75 MHz, DMSO-d6)
/ppm ¼ 167.5, 165.4,
d
136.9, 131.5, 126.1, 122.2, 119.6, 118.3, 120.0, 104.7, 65.3, 55.1, 50.0,

3246
K. Yao et al. / European Journal of Medicinal Chemistry 46 (2011) 3237e3249
24.0, 16.8; Anal. Calcd for C15H19N5O2: C, 59.79; H, 6.36; N, 23.24.
Found: C, 59.97; H, 6.51; N, 23.01.
6.2.26. N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-b-caboline-3-
carbonyl]-N0-valylhydra-zine (5e)
Using a procedure similar to that of preparing N-[(1S,3S)-1-
methyl-1,2,3,4-tetrahydro-
-caboline-3-carbonyl]-N0-glycylhydrazine
(5a) from 100 mg (0.226 mmol) of N-[(1S,3S)-1-methyl-1,2,3,4-tetra-
hydro-
-caboline-3-carbonyl]-N0-(Boc-valyl)-hydrazine (4e) 72 mg
6.2.23. N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-b-caboline-3-
b
carbonyl]-N0-alanylhydrazine (5b)
b
Using a procedure similar to that of preparing N-[(1S,3S)-1-methyl-
1,2,3,4-tetrahydro- (5a)
-caboline-3-carbonyl]-N0-glycylhydrazine
from 100 mg (0.241 mmol) of N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-
-caboline-3-carbonyl]-N0-(Boc-alanyl) hydrazine (4b) 70 mg (92%) of
(93%) of the title compound were obtained þas yellowing powders. Mp
b
209e211 ꢃC; ESI-MS (m/z) 344 [M þ H] ; ½a 2D0
¼ ꢁ23.57 (c 1.3,
ꢄ
CH3OH); IR (cmꢁ1): 3170, 2974, 2929, 2472, 1724, 1687, 1573, 1507,
b
1454, 1385, 1319, 1279, 1217, 747; 1H NMR (300 MHz, DMSO-d6)
d/
the title compound were obtained as yellowing powders. Mp
201e203 ꢃC; ESI-MS (m/z) 316 [M þ H]þ; ½a 2D0
¼ ꢁ34.87 (c 1.1,
ꢄ
ppm ¼ 11.51 (s, 1 H), 10.42 (d, J ¼ 8.1 Hz, 1 H), 9.67 (m, J ¼ 9.6 Hz, 1 H),
8.54 (s, 3 H), 7.44 (d, J ¼ 7.8 Hz, 1 H), 7.38 (d, J ¼ 8.1 Hz, 1 H), 7.15 (t,
J ¼ 7.5 Hz, 1 H), 7.03 (t, J ¼ 7.5 Hz, 1 H), 4.80 (s, 1 H), 4.40 (m, 1 H), 3.74
(s, 1 H), 3.45 (m, 1 H), 3.00 (t, J ¼ 13.2 Hz, 1 H), 2.18 (m, 1 H), 1.71 (d,
J ¼ 6.3 Hz, 3 H), 1.08 (d, J ¼ 6.9 Hz, 6 H); 13C NMR (75 MHz, DMSO-d6)
CH3OH). IR (cmꢁ1) 3175, 2982, 2925, 2472, 1724, 1687, 1569, 1503,
1450, 1389, 1319, 1270, 1209, 751; 1H NMR (300 MHz, DMSO-d6)
d/
ppm ¼ 11.50 (s, 1 H), 11.02 (s, 1 H), 9.67 (m, 1 H), 8.56 (m, 3 H), 7.44 (d,
J ¼ 7.8 Hz, 1 H), 7.38 (d, J ¼ 8.1 Hz, 1 H), 7.15 (t, J ¼ 7.5 Hz, 1 H), 7.03 (t,
J ¼ 7.5 Hz, 1 H), 4.80 (s, 1 H), 4.40 (m, 1 H), 4.00 (s, 1 H), 3.40 (m, 1 H),
3.00 (t, J ¼ 13.2 Hz,1 H),1.73 (d, J ¼ 6.6 Hz, 3 H),1.48 (d, J ¼ 6.9 Hz, 3 H);
d
/ppm ¼ 167.6, 167.5, 136.9, 131.5, 126.1, 122.2, 119.6, 118.3, 112.0, 104.8,
65.4, 55.0, 50.0, 30.4, 24.0, 18.6, 16.8; Anal. Calcd for C18H25N5O2: C,
62.95; H, 7.34; N, 20.39. Found: C, 62.74; H, 7.20; N, 20.61.
13C NMR (75 MHz, DMSO-d6)
122.2, 119.6, 118.3, 112.0, 104.8, 65.3, 55.0, 50.0, 24.0, 17.8, 16.8. Anal.
Calcd for C16H21N5O2: C, 60.94; H, 6.71; N, 22.21. Found: C, 61.15; H,
6.88; N, 22.43.
d
/ppm ¼ 169.0, 167.6, 136.9, 131.5, 126.1,
6.2.27. N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-b-caboline-3-
carbonyl]-N0-methionylhy-drazine (5f)
Using a procedure similar to that of preparing N-[(1S,3S)-1-
methyl-1,2,3,4-tetrahydro-
-caboline-3-carbonyl]-N0-glycylhydrazine
(5a) from 100 mg (0.211 mmol) of N-[(1S,3S)-1-methyl-1,2,3,4-tetra-
hydro-
-caboline-3-carbonyl]-N0-(Boc-methionyl)hydrazine (4f) 72
6.2.24. N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-b-caboline-3-
b
carbonyl]-N0-isoleucylhydrazine (5c)
b
Using a procedure similar to that of preparing N-[(1S,3S)-1-
methyl-1,2,3,4-tetrahydro-
-caboline-3-carbonyl]-N0-glycylhydrazine
(5a) from 100 mg (0.219 mmol) of N-[(1S,3S)-1-methyl-1,2,3,4-tetra-
hydro-
-caboline-3-carbonyl]-N0-(Boc-isoleucyl)hydrazine (4c) 72 mg
mg (91%) of the title compound were obtained as yellowing powders.
Mp 200e202 ꢃC; ESI-MS (m/z) 376 [M þ H]þ; ½a 2D0
ꢄ
¼ ꢁ11.73 (c 1.2,
b
CH3OH); IR (cmꢁ1): 3175, 2949, 2468, 1724, 1687, 1569, 1495, 1454,
1433, 1385, 1319, 1270, 1213, 747; 1H NMR (300 MHz, DMSO-d6)
d/
b
(92%) of the title compound were obtained as yellowing powders. Mp
ppm ¼ 11.48 (s, 1 H), 10.42 (d, J ¼ 7.5 Hz, 1 H), 9.65 (m, J ¼ 9.9 Hz, 1 H),
8.68 (s, 3 H), 7.44 (d, J ¼ 7.5 Hz, 1 H), 7.38 (d, J ¼ 8.1 Hz, 1 H), 7.13 (t,
J ¼ 7.5 Hz, 1 H), 7.03 (t, J ¼ 7.5 Hz, 1 H), 4.80 (s, 1 H), 4.40 (m, 1 H), 4.03
(m, 1 H), 3.41 (m, 1 H), 3.00 (t, J ¼ 13.2 Hz, 1 H), 2.68 (m, 2 H), 2.18 (m,
2 H), 2.18 (s, 3 H),1.72 (d, J ¼ 6.9 Hz, 3 H); 13C NMR (75 MHz, DMSO-d6)
204e206 ꢃC; ESI-MS (m/z): 358 [M þ H]þ; IR (cmꢁ1) 3170, 2970, 2472,
1724,1683, 1573, 1499, 1454, 1389, 1319, 1279, 1217, 747. ½a D20
¼ ꢁ22.10
ꢄ
(c 1.0, CH3OH); 1H NMR (300 MHz, DMSO-d6)
d
/ppm ¼ 11.48 (s, 1 H),
11.05 (s,1 H),10.90 (s,1 H),10.41 (d, J ¼ 8.1 Hz, 1 H), 9.65 (m, J ¼ 9.9 Hz,
1 H), 8.53 (s, 3 H), 7.44 (d, J ¼ 7.8 Hz,1 H), 7.38 (d, J ¼ 8.1 Hz,1 H), 7.13 (t,
J ¼ 7.5 Hz, 1 H), 7.03 (t, J ¼ 7.5 Hz, 1 H), 4.80 (s, 1 H), 4.40 (m, 1 H), 3.78
(s, 1 H), 3.40 (m, 1 H), 3.00 (t, J ¼ 13.2 Hz, 1 H), 1.93 (m, 1 H)，1.71 (d,
J ¼ 6.6 Hz, 3 H),1.66 (m, 2 H),1.33 (m, 2 H),1.01 (d, J ¼ 6.9 Hz,1 H), 0.89
d
/ppm ¼ 167.8, 167.6, 136.9, 131.5, 126.1, 122.2, 119.6, 118.3, 112.0, 104.8,
60.2, 55.0, 49.9, 31.6, 28.2, 24.0, 16.8, 14.6. Anal. Calcd for C18H25N5O2S:
C, 57.58; H, 6.71; N, 18.65. Found: C, 57.80; H, 6.88; N, 18.86.
(t, J ¼ 6.6 Hz, 1 H); 13C NMR (75 MHz, DMSO-d6)
/ppm ¼ 167.6, 166.9,
d
6.2.28. N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-b-caboline-3-
carbonyl]-N0-prolylhy-drazine (5g)
136.9, 131.5, 126.1, 122.2, 119.6, 118.3, 112.0, 104.8, 65.4, 55.0, 49.8, 36.8,
24.6, 24.0, 16.8, 14.6, 11.8; Anal. Calcd for C19H27N5O2: C, 63.84; H, 7.61;
N, 19.59. Found: C, 63.62; H, 7.45; N, 19.35.
Using a procedure similar to that of preparing N-[(1S,3S)-1-
methyl-1,2,3,4-tetrahydro-
-caboline-3-carbonyl]-N0-glycylhydrazine
(5a) from 100 mg (0.227 mmol) of N-[(1S,3S)-1-methyl-1,2,3,4-tetra-
hydro-
-caboline-3-carbonyl]-N0-(Boc-prolyl)-hydrazine (4g) 72 mg
b
6.2.25. N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-b-caboline-3-
carbonyl]-N0-leucylhydra-zine (5d)
b
(94%) of the title compound were obtained þas yellowing powders. Mp
Using a procedure similar to that of preparing N-[(1S,3S)-1-
methyl-1,2,3,4-tetrahydro-
-caboline-3-carbonyl]-N0-glycylhydrazine
(5a) from 100 mg (0.219 mmol) of N-[(1S,3S)-1-methyl-1,2,3,4-tetra-
hydro-
-caboline-3-carbonyl]-N0-(Boc-leucyl)-hydrazine (4d) 71 mg
234e236 ꢃC; ESI-MS (m/z) 342 [M þ H] ; ½a 2D0
¼ ꢁ60.07 (c 1.2,
ꢄ
b
CH3OH); IR (cmꢁ1): 3170, 2978, 2921, 2733, 2472, 1724, 1687, 1556,
1454, 1389, 1315, 1274, 1221, 747; 1H NMR (300 MHz, DMSO-d6)
d/
b
ppm ¼ 11.48 (s, 1 H), 11.02 (s, 1 H), 10.42 (m, 2 H), 9.68 (m, J ¼ 9.6 Hz,
1 H), 8.76 (s,1 H), 7.44 (d, J ¼ 7.8 Hz, 1 H), 7.38 (d, J ¼ 8.1 Hz,1 H), 7.15 (t,
J ¼ 7.5 Hz, 1 H), 7.03 (t, J ¼ 7.5 Hz, 1 H), 4.80 (s, 1 H), 4.37 (m, 2 H), 3.43
(m, 3 H), 3.00 (t, J ¼ 13.2 Hz, 1 H), 2.38 (m, 1 H), 1.92 (m, 2 H), 1.72 (d,
(91%) of the title compound were obtained þas yellowing powders. Mp
198e200 ꢃC; ESI-MS (m/z) 358 [M þ H] ; ½a 2D0
¼ ꢁ35.47 (c 1.2,
ꢄ
CH3OH); IR (cmꢁ1): 3174, 2962, 2471, 1723, 1683, 1576, 1499, 1450,
1388, 1319, 1274, 1213, 747; 1H NMR (300 MHz, DMSO-d6)
d/
J ¼ 6.9 Hz, 3 H); 13C NMR (75 MHz, DMSO-d6)
/ppm ¼ 167.9, 167.6,
d
ppm ¼ 11.48 (s, 1 H), 11.07 (s, 1 H), 11.02 (s, 1 H), 10.44 (d, J ¼ 5.4 Hz,
1 H), 9.65 (m, J ¼ 8.7 Hz, 1 H), 8.60 (s, 3 H), 7.44 (d, J ¼ 7.8 Hz,1 H), 7.36
(d, J ¼ 6.3 Hz,1 H), 7.19 (t, J ¼ 7.5 Hz,1 H), 7.03 (t, J ¼ 7.5 Hz,1 H), 4.80 (s,
1 H), 4.40 (m, 1 H), 3.88 (s, 1 H), 3.40 (m, 1 H), 3.00 (t, J ¼ 13.2 Hz, 1 H),
2.10 (m, 2 H), 1.68 (m, 6 H), 0.98 (d, J ¼ 6.6 Hz, 1 H), 0.92 (d, J ¼ 6.6 Hz,
136.9, 131.5, 126.0, 122.2, 119.7, 118.3, 112.0, 104.8, 60.2, 55.0, 49.9, 46.0,
30.3, 24.0, 21.6, 16.8; Anal. Calcd for C18H23N5O2: C, 63.32; H, 6.79; N,
20.51. Found: C, 63.11; H, 6.65; N, 20.74.
6.2.29. N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-b-caboline-3-
carbonyl]-N0-phenylalanylhydrazine (5h)
1 H); 13C NMR (75 MHz, DMSO-d6)
126.1, 122.2, 119.6, 118.3, 112.0, 104.8, 65.4, 55.0, 50.0, 23.9, 23.1, 22.8,
16.8; Anal. Calcd for C19H27N5O2: C, 63.84; H, 7.61; N, 19.59. Found: C,
63.62; H, 7.55; N, 19.83.
d
/ppm ¼ 168.1, 167.5, 136.9, 131.5,
Using a procedure similar to that of preparing N-[(1S,3S)-1-
methyl-1,2,3,4-tetrahydro-
-caboline-3-carbonyl]-N0-glycylhydrazine
b

K. Yao et al. / European Journal of Medicinal Chemistry 46 (2011) 3237e3249
3247
(5a) from 100 mg (0.204 mmol) of N-[(1S,3S)-1-methyl-1,2,3,4-tetra-
hydro-
-caboline-3-carbonyl]-N0-(Boc-phenyl-alanyl)hydrazine (4h)
CH3OH); IR (cmꢁ1) 3187, 2982, 2937, 2476, 1724, 1687, 1573, 1503,
1450, 1385, 1319, 1274, 1217, 751; 1H NMR (300 MHz, DMSO-d6)
b
d/
73 mg (92%) of the title compound were obtained as yellowing
ppm ¼ 11.48 (s, 1 H), 10.40 (s, 1 H), 9.68 (s, 1 H), 8.48 (s, 3 H), 7.44 (d,
J ¼ 7.5 Hz, 1 H), 7.38 (d, J ¼ 8.1 Hz, 1 H), 7.15 (t, J ¼ 7.5 Hz, 1 H), 7.05 (t,
J ¼ 7.5 Hz,1 H), 5.62 (s, 1 H), 4.81 (s, 1 H), 4.40 (s,1 H), 4.04 (s,1 H), 3.90
(dd, J ¼ 11.4 Hz, J ¼ 3.9 Hz, 1 H), 3.78 (m, 1 H), 3.43 (m, 1 H), 3.00 (t,
J ¼ 13.2 Hz,1 H),1.73 (d, J ¼ 6.9 Hz, 3 H); 13C NMR (75 MHz, DMSO-d6)
powders. Mp 185e187 ꢃC; ESI-MS (m/z): 392 [M þ H]þ; ½a 2D0
¼ ꢁ4.03
ꢄ
(c 1.3, CH3OH); IR (cmꢁ1) 3178, 2978, 2925, 2472, 1724, 1687, 1577,
1499, 1450, 1389, 1319, 1274, 1213, 747; 1H NMR (300 MHz, DMSO-d6)
d
/ppm ¼ 11.49 (s, 1 H), 10.45 (s, 1 H), 9.69 (s, 1 H), 8.56 (s, 3 H), 7.38 (m,
7 H), 7.13 (t, J ¼ 7.5 Hz, 1 H), 7.03 (t, J ¼ 7.5 Hz, 1 H), 4.80 (s, 1 H), 4.39
(m, 1 H), 4.21 (m, 1 H), 3.39 (m, 1 H), 3.25 (dd, J ¼ 13.8 Hz, J ¼ 6.0 Hz,
1 H), 3.14 (m, J ¼ 6.9 Hz, 1 H), 3.00 (t, J ¼ 13.2 Hz, 1 H), 1.72 (d,
d
/ppm ¼ 167.3,166.2, 136.9, 131.5, 126.1, 122.2, 119.6, 118.3, 112.0, 104.8,
65.3, 60.9, 55.0, 50.0, 24.0,16.8. Anal. Calcd for C16H21N5O3: C, 57.99; H,
6.39; N, 21.13. Found: C, 58.18; H, 6.55; N, 20.90.
J ¼ 6.9 Hz, 3 H); 13C NMR (75 MHz, DMSO-d6)
/ppm ¼ 167.4, 167.2,
d
136.9, 135.1, 131.5, 130.3, 128.9, 127.6, 126.1, 122.2, 119.6, 118.3, 112.0,
104.8, 65.3, 55.0, 49.9, 24.0, 16.8. Anal. Calcd for C22H25N5O2: C, 67.50;
H, 6.44; N, 17.89. Found: C, 67.68; H, 6.59; N, 18.11.
6.2.33. N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-b-caboline-3-
carbonyl]-N0-tryptophanyl-hydrazine (5l)
Using a procedure similar to that of preparing N-[(1S,3S)-1-
methyl-1,2,3,4-tetrahydro-
-caboline-3-carbonyl]-N0-glycylhydrazine
(5a) from 100 mg (0.189 mmol) of N-[(1S,3S)-1-methyl-1,2,3,4-tetra-
hydro-
-caboline-3-carbonyl]-N0-(Boc-trptophanyl)hydrazine (4l) 74
6.2.30. N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-b-caboline-3-
b
carbonyl]-N0-tyrosylhydra-zine (5i)
b
Using a procedure similar to that of preparing N-[(1S,3S)-1-
methyl-1,2,3,4-tetrahydro-
-caboline-3-carbonyl]-N0-glycylhydrazine
(5a) from 100 mg (0.197 mmol) of N-[(1S,3S)-1-methyl-1,2,3,4-tetra-
hydro-
-caboline-3-carbonyl]-N0-(Boc-tyrosyl)-hydrazine (4i) 73 mg
mg (91%) of the title compound were obtained as yellowing powders.
Mp 213e215 ꢃC; ESI-MS (m/z) 431 [M þ H]þ; ½a 2D0
ꢄ
¼ ꢁ9.07 (c 1.1,
b
CH3OH); IR (cmꢁ1): 3399, 3191, 2982, 2925, 2472, 1724, 1691, 1577,
1491, 1454, 1434, 1385, 1319, 1274, 1234, 747; 1H NMR (300 MHz,
b
(90%) of the title compound were obtained as yellowing powders. Mp
DMSO-d6)
d
/ppm ¼ 11.49 (s, 1 H), 11.12 (s, 1 H), 10.51 (s, 1 H), 9.70 (s,
216e218 ꢃC; ESI-MS (m/z) 408 [M þ H]þ; ½a 2D0
¼ 3.17 (c 1.2, CH3OH);
ꢄ
1 H), 8.45 (s, 3 H), 7.83 (d, J ¼ 7.5 Hz, 1 H), 7.46 (d, J ¼ 7.5 Hz, 1 H), 7.40
(d, J ¼ 7.8 Hz, 3 H), 7.08 (m, 4 H), 4.80 (s, 1 H), 4.39 (m, 1 H), 4.16 (m,
1 H), 3.39 (m, 2 H), 3.23 (dd, J ¼ 15.0 Hz, J ¼ 7.5 Hz, 1 H), 3.00 (t,
J ¼ 13.2 Hz,1 H),1.72 (d, J ¼ 6.6 Hz, 3 H); 13C NMR (75 MHz, DMSO-d6)
IR (cmꢁ1) 3175, 2983, 2929, 2472, 1720, 1695, 1614, 1516, 1450, 1385,
1319, 1270, 1230, 751; 1H NMR (300 MHz, DMSO-d6)
d/ppm ¼ 11.48 (s,
1 H), 11.04 (s, 1 H), 10.42 (d, J ¼ 7.2 Hz, 1 H), 8.45 (s, 3 H), 7.46 (d,
J ¼ 7.8 Hz, 1 H), 7.38 (d, J ¼ 7.8 Hz, 1 H), 7.20 (d, J ¼ 7.8 Hz, 2 H), 7.13 (t,
J ¼ 7.5 Hz, 1 H), 7.03 (t, J ¼ 7.5 Hz, 1 H), 6.75 (d, J ¼ 8.4 Hz, 3 H), 4.80 (s,
1 H), 4.39 (m, 1 H), 4.10 (s, 1 H), 3.49 (m, 1 H), 3.15 (dd, J ¼ 14.1 Hz,
J ¼ 5.7 Hz, 1 H), 2.99 (m, 2 H), 1.72 (d, J ¼ 6.6 Hz, 3 H); 13C NMR
d
/ppm ¼ 167.6, 167.2, 136.9, 136.7, 131.5, 127.7, 126.1, 125.6, 122.2, 121.5,
119.6, 119.1, 118.9, 112.0, 111.9, 107.1, 104.8, 60.2, 55.1, 50.0, 27.8, 24.1,
16.8. Anal. Calcd for C24H26N6O2: C, 66.96; H, 6.09; N, 19.52. Found: C,
66.75; H, 5.93; N, 19.75.
(75 MHz, DMSO-d6)
d
/ppm ¼ 167.4, 167.3, 157.1, 136.9, 131.5, 131.2,
126.1, 124.9, 122.2, 119.6, 118.3, 115.8, 112.0, 103.8, 65.3, 55.0, 49.9, 36.6,
24.0, 16.8. Anal. Calcd for C22H25N5O3: C, 64.85; H, 6.18; N, 17.19.
Found: C, 64.64; H, 6.03; N, 17.42.
6.2.34. N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-b-caboline-3-
carbonyl]-N0-histidylhydra-zine (5m)
Using a procedure similar to that of preparing N-[(1S,3S)-1-
methyl-1,2,3,4-tetrahydro-
-caboline-3-carbonyl]-N0-glycylhydrazine
(5a) from 100 mg (0.172 mmol) of N-[(1S,3S)-1-methyl-1,2,3,4-tetra-
hydro-
-caboline-3-carbonyl]-N0-(di-Boc-histidyl)hydrazine (4m) 61
6.2.31. N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-b-caboline-3-
b
carbonyl]-N0-threonylhydrazine (5j)
b
Using a procedure similar to that of preparing N-[(1S,3S)-1-
methyl-1,2,3,4-tetrahydro-
-caboline-3-carbonyl]-N0-glycylhydrazine
(5a) from 100 mg (0.225 mmol) of N-[(1S,3S)-1-methyl-1,2,3,4-tetra-
hydro-
-caboline-3-carbonyl]-N0-(Boc-threonyl)-hydrazine (4j) 71 mg
mg (94%) of the title compound were obtained as yellowing powders.
Mp 213e215 ꢃC; ESI-MS (m/z) 382 [M þ H]þ; ½a 2D0
ꢄ
¼ 25.27 (c 1.3,
b
CH3OH); IR (cmꢁ1) 3399, 3142, 2987, 2901, 2480, 1720, 1683, 1621,
1556, 1507, 1434, 1385, 1315, 1213, 751; 1H NMR (300 MHz, DMSO-d6)
b
(92%) of the title compound were obtaþined as yellowing powders. Mp
d
/ppm ¼ 14.72 (s, 1 H), 11.45 (s, 1 H), 10.34 (s, 1 H), 9.76 (s, 1 H), 9.11 (s,
221e223 ꢃC; ESI-MS (m/z) 346 [M þ H] ; ½a 2D0
¼ ꢁ40.10 (c 1.1, CH3OH);
ꢄ
1 H), 8.30 (s, 3 H), 7.62 (s, 1 H), 7.46 (d, J ¼ 5.8 Hz, 1 H), 7.38 (d,
J ¼ 8.1 Hz, 1 H), 7.13 (t, J ¼ 7.5 Hz, 1 H), 7.03 (t, J ¼ 7.5 Hz, 1 H), 4.80 (s,
1 H), 4.44 (m, 2 H), 3.39 (m, 3 H), 3.00 (t, J ¼ 13.2 Hz, 1 H), 1.72 (d,
IR (cmꢁ1) 3195, 2982, 2933, 2472, 1724, 1683, 1577, 1503, 1458, 1389,
1319, 1279, 1217, 751; 1H NMR (300 MHz, DMSO-d6)
d/ppm ¼ 11.50 (s,
J ¼ 6.6 Hz, 3 H); 13C NMR (75 MHz, DMSO-d6)
/ppm ¼ 167.8, 166.8,
d
1 H),10.45 (s,1 H), 9.66 (s,1 H), 8.45 (s, 3 H), 7.44 (d, J ¼ 7.2 Hz,1 H), 7.38
(d, J ¼ 7.8 Hz,1 H), 7.15 (t, J ¼ 7.5 Hz, 1 H), 7.03 (t, J ¼ 7.5 Hz,1 H), 5.70 (s,
1 H), 4.78 (s,1 H), 4.38 (s,1 H), 3.99 (m,1 H), 3.71 (d, J ¼ 7.2 Hz, 1 H), 3.41
(m, 1 H), 3.00 (t, J ¼ 13.2 Hz, 1 H), 1.72 (d, J ¼ 6.6 Hz, 3 H), 1.39 (d,
136.8, 134.4, 131.4, 126.7, 126.0, 122.3, 119.7, 118.4, 112.0, 104.8, 55.0,
50.5, 49.9, 26.7, 24.0, 16.8. Anal. Calcd for C19H23N7O2: C, 59.83; H,
6.08; N, 25.70. Found: C, 59.61; H, 5.91; N, 25.93.
J ¼ 6.0 Hz, 3 H); 13C NMR (75 MHz, DMSO-d6)
/ppm ¼ 167.6, 166.2,
d
136.9, 131.5, 126.1, 122.2, 119.6, 118.3, 112.0, 104.8, 66.5, 65.3, 55.0, 49.9,
24.0, 20.1, 16.8; Anal. Calcd for C17H23N5O3: C, 59.12; H, 6.71; N, 20.28.
Found: C, 58.93; H, 6.57; N, 20.52.
6.2.35. N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-b-caboline-3-
carbonyl]-N0-lysylhydra-zine (5n)
Using a procedure similar to that of preparing N-[(1S,3S)-1-
methyl-1,2,3,4-tetrahydro-
-caboline-3-carbonyl]-N0-glycylhydrazine
(5a) from 100 mg (0.175 mmol) of N-[(1S,3S)-1-methyl-1,2,3,4-tetra-
hydro-
-caboline-3-carbonyl]-N0-(di-Boc-lysyl)-hydrazine (4n) 60 mg
6.2.32. N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-
carbonyl]-N0-serylhydrazi-ne (5k)
b-caboline-3-
b
b
Using a procedure similar to that of preparing N-[(1S,3S)-1-
methyl-1,2,3,4-tetrahydro-
-caboline-3-carbonyl]-N0-glycylhydrazine
(5a) from 100 mg (0.232 mmol) of N-[(1S,3S)-1-methyl-1,2,3,4-tetra-
hydro-
-caboline-3-carbonyl]-N0-(Boc-seryl)-hydrazine (4k) 71 mg
(93%) of the title compound were obtained þas yellowing powders. Mp
b
216e218 ꢃC; ESI-MS (m/z) 373 [M þ H] ; ½a 2D0
¼ ꢁ20.47 (c 1.1,
ꢄ
CH3OH); IR (cmꢁ1) 3166, 2982, 2938, 2472, 1724, 1695, 1577, 1507,
b
1454, 1377, 1319, 1270, 751; 1H NMR (300 MHz, DMSO-d6)
d/
(92%) of the title compound were obtained þas yellowing powders. Mp
ppm ¼ 11.46 (s, 1 H), 10.39 (d, J ¼ 7.8 Hz, 1 H), 9.72 (m, J ¼ 9.0 Hz, 1 H),
226e228 ꢃC; ESI-MS (m/z) 332 [M þ H] ; ½a 2D0
¼ ꢁ46.33 (c 1.2,
ꢄ
8.61 (s, 3 H), 8.24 (s, 3 H), 7.44 (d, J ¼ 7.8 Hz, 1 H), 7.39 (d, J ¼ 8.1 Hz,

3248
K. Yao et al. / European Journal of Medicinal Chemistry 46 (2011) 3237e3249
1 H), 7.13 (t, J ¼ 7.5 Hz, 1 H), 7.03 (t, J ¼ 7.5 Hz, 1 H), 4.80 (s, 1 H), 4.39
(m, 1 H), 3.94 (m, 3 H), 3.00 (t, J ¼ 13.2 Hz, 1 H), 2.74 (s, 2 H), 1.72 (d,
ppm ¼ 170.8, 167.5, 167.3, 136.9, 131.5, 126.0, 122.2, 119.6, 112.0, 104.8,
65.3, 54.9, 49.9, 35.9, 24.0, 16.8. Anal. Calcd for C17H22N6O3: C, 56.97;
H, 6.19; N, 23.45. Found: C, 56.76; H, 6.04; N, 23.68.
J ¼ 6.6 Hz, 3 H), 1.58 (m, 4 H); 13C NMR (75 MHz, DMSO-d6)
d/
ppm ¼ 170.7, 167.9, 166.7, 136.9, 131.5, 126.0, 119.6, 112.0, 104.8, 60.2,
55.0, 49.9, 38.8, 30.8, 26.7, 24.0, 21.2, 16.8. Anal. Calcd for C19H28N6O2:
C, 61.27; H, 7.58; N, 22.56. Found: C, 61.50; H, 7.74; N, 22.78.
6.2.39. N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-b-caboline-3-
carbonyl]-N0-glutaminyl)-hydrazine (5r)
6.2.36. N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-
b-caboline-3-
Using a procedure similar to that of preparing N-[(1S,3S)-1-
methyl-1,2,3,4-tetrahydro-
-caboline-3-carbonyl]-N0-glycylhydrazine
(5a) from 100 mg (0.212 mmol) of N-[(1S,3S)-1-methyl-1,2,3,4-tet-
rahydro-
-caboline-3-carbonyl]-N0-(Boc-glutaminyl)hydrazine (4r)
72 mg (91%) of the title compound were obtained as yellowing
carbonyl]-N0-[(
b
-OBzl)-as-partyl]hydrazine (5o)
b
Using a procedure similar to that of preparing N-[(1S,3S)-1-
methyl-1,2,3,4-tetrahydro-
-caboline-3-carbonyl]-N0-glycylhydrazine
(5a) from 100 mg (0.182 mmol) of N-[(1S,3S)-1-methyl-1,2,3,4-tet-
rahydro- -OBzl-aspartyl)]hydrazine
-caboline-3-carbonyl]-N0-[Boc-(
(4o) 59 mg (90%) of the title compound were obtained as yellowing
b
b
powders. Mp 215e217 ꢃC; ESI-MS (m/z) 373 [M
þ
H]þ;
b
b
½
a 2D0
ꢄ
¼ ꢁ8.57 (c 1.3, CH3OH); IR (cmꢁ1) 3179, 2983, 2925, 2476,
1724, 1662, 1605, 1507, 1454, 1389, 1319, 1274, 1217, 751; 1H NMR
powders. Mp 254e256 ꢃC; ESI-MS (m/z) 360 [M
þ
H]þ;
(300 MHz, DMSO-d6)
d
/ppm ¼ 11.46 (s, 1 H), 10.44 (d, J ¼ 8.4 Hz,
½
a 2D0
ꢄ
¼ ꢁ25.80 (c 1.3, CH3OH); IR (cmꢁ1) 3182, 2972, 2927, 2475,
1 H), 9.68 (m, J ¼ 9.6 Hz, 1 H), 8.63 (s, 3 H), 7.52 (s, 1 H), 7.44 (d,
J ¼ 7.8 Hz, 1 H), 7.39 (d, J ¼ 8.1 Hz, 1 H), 7.13 (t, J ¼ 7.5 Hz, 1 H), 7.03
(t, J ¼ 7.5 Hz, 1 H), 6.95 (s, 1 H), 4.80 (s, 1 H), 4.39 (m, 1 H), 3.97 (s,
1 H), 3.39 (m, 1 H), 3.00 (t, J ¼ 13.2 Hz, 1 H), 2.37 (m, 2 H), 2.06 (m,
1724, 1687, 1507, 1454, 1388, 1319, 1274, 1210, 750; 1H NMR
(300 MHz, DMSO-d6)
d
/ppm ¼ 11.49 (s, 1 H), 11.08 (s, 1 H), 11.04
(s, 1 H), 10.04 (m, 1 H), 9.73 (m, 1 H), 8.68 (m, 2 H), 7.44 (d, J ¼ 7.5 Hz,
1 H), 7.39 (d, J ¼ 7.8 Hz, 1 H), 7.13 (t, J ¼ 7.5 Hz, 1 H), 7.03 (t, J ¼ 7.5 Hz,
1 H), 4.80 (d, J ¼ 6.3 Hz, 1 H), 4.39 (dd, J ¼ 14.4 Hz, J ¼ 4.2 Hz, 1 H),
4.21 (t, J ¼ 6.0 Hz, 1 H), 3.35 (m, 1 H), 3.00 (t, J ¼ 13.2 Hz, 3 H), 1.71 (d,
2 H), 1.72 (d, J ¼ 6.6 Hz, 3 H); 13C NMR (75 MHz, DMSO-d6)
d/
ppm ¼ 173.9, 167.7, 136.9, 131.5, 126.0, 122.2, 119.6, 118.3, 112.0,
104.8, 65.3, 55.0, 49.9, 30.7, 27.5, 24.0, 16.8. Anal. Calcd for
C18H24N6O3: C, 58.05; H, 6.50; N, 22.57. Found: C, 57.84; H, 6.36; N,
22.81.
J ¼ 6.6 Hz, 3 H); 13C NMR (75 MHz, DMSO-d6)
/ppm ¼ 170.9, 167.4,
d
136.9, 131.4, 126.0, 122.2, 119.7, 118.3, 112.0, 104.8, 65.3, 54.8, 49.8,
35.6, 23.9, 16.8. Anal. Calcd for C17H21N5O4: C, 56.82; H, 5.89; N,
19.49. Found: C, 56.61; H, 5.74; N, 19.71.
6.3. In vitro anti-platelet aggregation activity assay
6.2.37. N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-
b-caboline-3-
carbonyl]-N0-[(
g
-OBzl)glutamoyl]hydrazine (5p)
An H-10 cell counter was used to determine the platelet count
and a two-channel Chronolog aggregometer was used to evaluate
platelet aggregation. The pig blood (6 pigs, purchased from Animal
Center of Peking University) was centrifuged at 120g for 10 min and
the platelet rich plasma (PRP) was collected. The remaining blood
was centrifuged for an additional 10 min at 850g to prepare platelet
poor plasma (PPP). The final platelet count of the PRP was adjusted
to 2 ꢂ 108 platelets/mL with autologous PPP. To an optical aggreg-
ometry testing tuber, 0.5 mL of the adjusted plasma sample and
Using a procedure similar to that of preparing N-[(1S,3S)-1-
methyl-1,2,3,4-tetrahydro-
-caboline-3-carbonyl]-N0-glycylhydrazine
(5a) from 100 mg (0.178 mmol) of N-[(1S,3S)-1-methyl-1,2,3,4-tetra-
hydro- -OBzl-glutamoyl)]hydrazine
-caboline-3-carbonyl]-N0-[Boc-(
b
b
g
(4p) 58 mg (88%) of the title compound were obtained as yellowing
powders. Mp 211e213 ꢃC; ESI-MS (m/z) 374 [M þ H]þ; ½a 2D0
¼ ꢁ9.30
ꢄ
(c 1.3, CH3OH); IR (cmꢁ1) 3170, 2978, 2933, 2476, 1720, 1573, 1507,
1450, 1389, 1319, 1274, 1213, 751; 1H NMR (300 MHz, DMSO-d6)
d/
5
m
L of NS or 5
10) or 5 L of the solution of 5aer (in a series of final concentrations
of 100, 10, 1, 0.1, 0.01 and 0.001 M, prepared by diluting 10 mM of
stock solutions of 5aer in DMSO/NS (1/10) with NS) was added.
After adjustment of the baseline, 5 L of the solution of PAF (final
concentration 0.1 M, prepared by diluting 10 mM of stock solution
of PAF in DMSO/NS (1/10) with NS or 5 L of the solution of ADP
(final concentration 10 M, prepared by diluting 10 mM of stock
solution of ADP in DMSO/NS (1/10) with NS) or 5 L of the solution
of arachidonic acid in NS (AA, final concentration 350 M, prepared
mL of the solution (1.7 mM) of aspirin in DMSO/NS (1/
ppm ¼ 11.49 (s,1 H),11.08 (s,1 H),11.04 (s,1 H),10.04 (m,1 H), 9.73 (m,
1 H), 8.68 (m, 2 H), 7.44 (d, J ¼ 7.5 Hz, 1 H), 7.39 (d, J ¼ 8.1 Hz, 1 H), 7.13
(t, J ¼ 7.5 Hz, 1 H), 7.03 (t, J ¼ 7.5 Hz,1 H), 4.80 (s,1 H), 4.39 (s,1 H), 3.98
(s, 1 H), 3.45 (m, 1 H), 3.00 (t, J ¼ 13.2 Hz, 1 H), 2.60 (m, 2 H), 2.11 (m,
m
m
m
2 H), 1.71 (d, J ¼ 6.6 Hz, 3 H); 13C NMR (75 MHz, DMSO-d6)
d/
m
ppm ¼ 173.8, 167.8, 136.7, 131.4, 126.0, 122.2, 119.7, 118.3, 112.0, 104.8,
65.3, 54.9, 49.8, 29.2, 26.8, 23.9, 16.8. Anal. Calcd for C18H23N5O4: C,
57.90; H, 6.21; N, 18.76. Found: C, 58.09; H, 6.04; N, 18.52.
m
m
m
m
6.2.38. N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-
b
-caboline-3-
by diluting 100 mM of stock solution of AA in DMSO/NS (1/10) with
NS) was added and aggregation was measured at 37 ꢃC for 5 min.
The effects of 5aer (at a series of concentrations ranging from
carbonyl]-N0-asparaginyl-hydrazine (5q)
Using a procedure similar to that of preparing N-[(1S,3S)-1-
methyl-1,2,3,4-tetrahydro-
-caboline-3-carbonyl]-N0-glycylhydrazine
(5a) from 100 mg (0.218 mmol) of N-[(1S,3S)-1-methyl-1,2,3,4-tetra-
hydro- (4q)
-caboline-3-carbonyl]-N0-(Boc-asparaginyl)]hydrazine
100 mM to 1 nM) and aspirin (positive control, 1.7 mM) on PAF or
b
ADP or AA-induced platelet aggregation were observed. All these
anti-platelet aggregation tests in sixplicate tubers were carried out.
The maximum platelet aggregation (Am) of control group (NS) or
sample group (5aer) was represented by the peak height of
aggregation curve (equals to the maximum light transmission). The
inhibition rate was calculated according to the following formula:
Inhibition (%) ¼ [(Am of NS) ꢁ (Am of 5aer)]/(Am of NS) ꢂ 100%. Am%
of NS is the value of platelet aggregation induced by PAF, ADP and
AA without 5aer which are 52.30 ꢀ 1.78%, 50.16 ꢀ 3.65% and
49.62 ꢀ 2.90%, respectively. The concentration vs. inhibition rate
curve is plotted to determine the IC50 values with GWBASIC.EXE
program.
b
70 mg (90%) of the title compound were obtainþed as yellowing
powders. Mp 253e255 ꢃC; ESI-MS (m/z) 359 [M þ H] ; ½a 2D0
¼ ꢁ31.23
ꢄ
(c 1.2, CH3OH); IR (cmꢁ1) 3175, 2974, 2806, 2472, 1728, 1675, 1605,
1495, 1450, 1425, 1385, 1315, 1274, 1221, 747; 1H NMR (300 MHz,
DMSO-d6)
d
/ppm ¼ 11.50 (s, 1 H), 10.44 (s, 1 H), 9.68 (s, 1 H), 8.46 (s,
3 H), 7.87 (s, 1 H), 7.44 (d, J ¼ 7.8 Hz, 1 H), 7.39 (d, J ¼ 8.1 Hz, 1 H), 7.28
(s, 1 H), 7.13 (t, J ¼ 7.5 Hz,1 H), 7.03 (t, J ¼ 7.5 Hz,1 H), 4.80 (s,1 H), 4.37
(s, 1 H), 4.21 (s, 1 H), 3.39 (m, 1 H), 3.00 (t, J ¼ 13.2 Hz, 1 H), 2.78 (m,
2 H), 1.72 (d, J ¼ 6.6 Hz, 3 H); 13C NMR (75 MHz, DMSO-d6)
d/

K. Yao et al. / European Journal of Medicinal Chemistry 46 (2011) 3237e3249
3249
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6.4. In vivo anti-thrombotic assay
The assessments described here were performed based on
a protocol reviewed and approved by the ethics committee of
Capital Medical University. The committee assures the welfare of
the animals was maintained in accordance to the requirements
of the animal welfare act and according to the guide for care and
use of laboratory animals. Aspirin and 5aer were dissolved in NS
before administration and kept in an ice bath. Male Wista rats
weighing 250e300 g (purchased from Animal Center of Peking
University) were used. Thirty minutes after orally given aspirin
(positive control), NS (negative control) or 5aer, the rats were
anesthetized with pentobarbital sodium (80.0 mg/kg, i.p.) and the
right carotid artery and left jugular vein were separated. A weighed
6 cm thread was inserted into the middle of a polyethylene tube.
The polyethylene tube was filled with heparin sodium (50 IU/mL in
NS) and one end was inserted into the left jugular vein. From the
other end of the polyethylene tube, heparin sodium was injected as
anticoagulant, and this end was inserted into the right carotid
artery. Blood was allowed to flow from the right carotid artery to
the left jugular vein, through the polyethylene tube for 15 min. The
thread was removed to obtain the wet weight of the thrombus.
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This work was completed in the Beijing area major laboratory of
peptide and small molecular drugs, supported by PHR (IHLB, KZ
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Foundation of China (81072522, 30801426), and Special Project
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