A class of 3 S -2-aminoacyltetrahydro-β-carboline-3-carboxylic acids: Their facile synthesis, inhibition for platelet activation, and high in vivo anti-thrombotic potency

DOI: 10.1021/jm901816j

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Journal of Medicinal Chemistry p. 3106 - 3116 (2010)

Update date:2022-07-29

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Authors:

Liu, Jiawang

Jiang, Xueyun

Zhao, Ming

Zhang, Xiaoyi

Zheng, Meiqing

Peng, Li

Peng, Shiqi

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Article abstract of DOI:10.1021/jm901816j

3S-Tetrahydro-β-carboline-3-carboxylic acid (TCCA) effectively inhibits ADP-induced platelet activation. This paper used TCCA as a lead, modified its 2-position with amino acids, and provided 20 novel 3S-2-aminoacyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acids (5a-t). With the in vitro assay, it was demonstrated that this modification diminished the IC₅₀ values from 701 nM of TCCA to 10 nM of 5a-t. With the in vivo assay, it was demonstrated that this modification reduced the efficacious dose from 5.0 μmol/kg of TCCA to 0.1 μmol/kg of 5a-t. Comparing the Cerius² based conformation of them with that of their analogues, the 3-position modified TCCA, it was suggested that the comparatively unfolded conformation was one of the important factors of enhancing the in vivo antithrombotic potency.

Full text of DOI:10.1021/jm901816j

3106 J. Med. Chem. 2010, 53, 3106–3116

DOI: 10.1021/jm901816j

A Class of 3S-2-Aminoacyltetrahydro-β-carboline-3-carboxylic Acids: Their Facile Synthesis,

Inhibition for Platelet Activation, and High in Vivo Anti-Thrombotic Potency

Jiawang Liu, Xueyun Jiang,^†Ming Zhao,* Xiaoyi Zhang, Meiqing Zheng, Li Peng, and Shiqi Peng*

College of Pharmaceutical Sciences, Capital Medical University, Beijing 100069, PR China. ^†Co-first author.

Received November 23, 2009

3S-Tetrahydro-β-carboline-3-carboxylic acid (TCCA) effectively inhibits ADP-induced platelet activa-

tion. This paper used TCCA as a lead, modified its 2-position with amino acids, and provided 20 novel

3S-2-aminoacyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acids (5a-t). With the in vitro assay, it was

demonstrated that this modification diminished the IC₅₀values from 701 nM of TCCA to 10 nM of

5a-t. With the in vivo assay, it was demonstrated that this modification reduced the efficacious dose

from 5.0 μmol/kg of TCCA to 0.1 μmol/kg of 5a-t. Comparing the Cerius²based conformation of them

with that of their analogues, the 3-position modified TCCA, it was suggested that the comparatively

unfolded conformation was one of the important factors of enhancing the in vivo antithrombotic

potency.

Introduction

secretion,³¹binding to 5-HT₂serotonin receptors,³²inhibiting

bacterial enoyl acyl carrier protein reductase,³³and inhibiting

acetyl- and butyryl-cholinesterases³⁴as well as binding to

benzodiazepine receptor.³⁵

Intravascular thrombosis is implicated in the most frequent

cardiovascular events, such as deep vein thrombosis, pulmon-

ary embolism, myocardial infarction, and stroke, and it is one

of the most prominent causes of morbidity and mortality all

over the world.¹Once vascular endothelium of the injured

blood vessel is damaged, platelets adhere to the exposed

extracellular matrix.²Adhesion of platelets following

endothelial injury is the primary event usually associated with

uncontrolled platelet activation culminating into intravascu-

lar thrombosis.³The suppression of platelet function,

particularly through targeting such secondary regulatory

mechanisms, has proven effective in the prevention of inap-

propriate platelet activation that results in thrombosis.⁴Anti-

platelet therapy has an established role in the treatment of this

cardiovascular event.⁵Since the most commonly used anti-

platelet drugs lack clinical efficacy, in the past decades,

continuous efforts have been made to discover new com-

pounds capable of inhibiting platelet uncontrolled activation.

Natural and synthetic β-carbolines and tetrahydro-β-car-

bolines possess a variety of pharmacologic functions. So far, a

wide spectrum of pharmacological actions has been investi-

gated, such as trypanocidal action,^6,7inhibiting mitogen

activated protein kinase-activated protein kinase 2,⁸inhibit-

ing cyclin-dependent kinases 4,^9,10antimalarial action,^11,12

cytotoxic action,^13-17cardiovascular effects,¹⁸neuroprotec-

tive and neuron-differentiating actions,¹⁹inhibiting human

monoamine oxidase,²⁰improving object recognition mem-

ory,²¹antiviral action,²²binding to imidazoline receptors,^23,24

inhibiting mitotickinesinEg5,²⁵interacting towardDNA,^26,27

binding to MGluR1 receptor,²⁸antileishmanial action,²⁹

blocking the activity of topoisomerases,³⁰stimulating insulin

A series of β-carbolines such as harmalol, harmaline,

norharmane, harmol, harmine, and harmane were used as

the leads of the platelet activation inhibitors, but these

compounds were capable of inhibiting collagen-induced pla-

telet aggregation only, and the IC₅₀values were more than

130 μM.³⁶In a previous paper, we revealed that 3S-tetrahydro-

β-carboline-3-carboxylic acid(TCCA^a) mayeffectively inhibit

platelet-activating factor (PAF), adenosine diphosphate

(ADP), and arachidonic acid (AA) as well as thrombin

(TH) induced platelet aggregation, and the IC₅₀values were

less than 1 μM.³⁷In this study, we used amino acids modifying

the 2-position of TCCA, prepared 20 novel derivatives,

evaluated the in vitro antiplatelet aggregation and in vivo

antithrombotic activities, and demonstrated the effect of

Cerius²based conformation on the antithrombotic activity.

Results and Discussion

Facile Synthesis of 5a-t. A facile synthesis of 3S-2-ami-

noacyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acids

(5a-t) was performed by using a five-step route depicted in

Scheme 1. The esterification of TCAA from Pictet-Spengler

condensation of ^L-Trp and formaldehyde provided the

methylester (2, 92% yield). In the presence of DCC, HOBt,

and NMM, 2 was conjugated with 20 amino acids to give

^aAbbreviations: TCCA, 3S-tetrahydro-β-carboline-3-carboxylic acid;

ADP, adenosine diphosphate; AA, arachidonic acid; PAF, platelet-activat-

ing factor; TH, thrombin; NS, normal saline; Boc, tert-butyloxylcarbonyl;

HOBt, N-hydroxybenzotriazole; DCC, dicyclohexylcarbodiimide; NMM,

N-methyl morpholine; EtOAc, ethyl acetate; Et₃N, triethylamine;

PRP, platelet rich plasma; PPP, platelet poor plasma; TLC, thin layer

chromatography; MGluR1, metabotropic glutamate receptor-subtype 1;

5-TH 5-hydroxytryptamine.

*To whom correspondence should be addressed. For S.Q.P.: phone,

86-10-8391-1528; fax, 86-10-8391-1528; E-mail, sqpeng@mail.bjmu.

edu.cn. For M.Z.: phone, þ86 10 8280 2482; fax, þ86 10 8280 2482;

E-mail: mingzhao@mail.bjmu.edu.cn.

pubs.acs.org/jmc

Published on Web 03/23/2010

2010 American Chemical Society

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3107

Scheme 1. Five-Step Procedure for Preparing 3S-2-Ammonoa-

cyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic Acids^a

Table 1. IC₅₀of 5a-t Inhibiting Platelet Aggregation Induced by Four

Aggregators^a

inhibition of platelet aggregation IC₅₀(nM)

compd

ADP

PAF

701.01 ( 3.28 866.14 ( 2.39 752.28 ( 2.28 981.52 ( 3.55

6.71 ( 0.54

9.80 ( 0.59

7.80 ( 0.65

8.66 ( 0.63

7.81 ( 0.59

7.76 ( 0.62

9.40 ( 0.66

7.10 ( 0.64

6.50 ( 0.50

10.25 ( 0.70

6.62 ( 0.64

7.31 ( 0.62

10.40 ( 0.69

9.47 ( 0.72

8.56 ( 0.68

11.12 ( 0.72

7.70 ( 0.65

9.19 ( 0.69

8.70 ( 0.59

6.56 ( 0.67

24.60 ( 1.55

28.62 ( 1.60

27.26 ( 1.60

26.22 ( 1.52

24.55 ( 1.51

25.17 ( 1.55

25.70 ( 1.60

24.44 ( 1.49

23.52 ( 1.56

29.61 ( 1.64

23.98 ( 1.46

24.86 ( 1.62

27.94 ( 1.69

27.52 ( 1.51

26.50 ( 1.62

30.11 ( 1.72

28.20 ( 1.58

28.95 ( 1.64

26.77 ( 1.56

23.86 ( 1.57

13.60 ( 1.10

18.98 ( 1.55

17.70 ( 1.50

16.10 ( 1.45

14.77 ( 1.48

15.11 ( 1.51

16.66 ( 1.61

14.68 ( 1.47

12.45 ( 1.41

14.75 ( 1.50

13.14 ( 1.35

15.47 ( 1.60

16.82 ( 1.65

18.25 ( 1.59

16.80 ( 1.60

17.04 ( 1.64

17.18 ( 1.61

16.20 ( 1.53

16.95 ( 1.59

12.60 ( 1.45

27.41 ( 1.62

30.16 ( 1.80

32.52 ( 1.87

30.75 ( 1.90

27.80 ( 1.70

28.40 ( 1.68

30.09 ( 1.76

27.60 ( 1.59

25.31 ( 1.55

30.85 ( 1.83

26.65 ( 1.63

28.96 ( 1.70

29.60 ( 1.80

29.70 ( 1.78

31.95 ( 1.81

33.40 ( 1.85

30.10 ( 1.81

30.00 ( 1.78

34.10 ( 1.86

26.96 ( 1.73

^a(i) Formaldehyde and H₂SO₄; (ii) methanol and thionyl chloride, rt,

92% yield; (iii) Boc-AA-OH, DCC and NMM; (iv) aqueous NaOH (2N)

and hydrochloric acid; (v) hydrogen chloride in ethyl acetate (4 mol/L)

and aqueous NaOH. In 3a, 4a, 5a; R₁= CH₃; 3b, 4b, 5b R = CH₂C₆H₅; 3c,

4c,5c R=CH(CH₃)₂;3d,4d,5d R=CH₂OH; 3e,4e,5e R=CH(OH)CH₃;

3f, 4f, 5f R = CH₂C₆H₄-OH-p; 3g, 4g, 5g R = tetrahydropyrrol-2-yl; 3h, 4h,

5h R=CH₂SH; 3i, 4i , 5i R=CH₂CH₂SCH₃;3j R=CH₂CH₂CO₂CH₃;4j,

5j R=CH₂CH₂CO₂H; 3k R=CH₂CO₂CH₂C₆H₅;4k, 5k R=CH₂CO₂H;

3l, 4l, 5l R = 1,3-imidazol-5-methylene; 3m, 4m, 5m R = indol-3-yl-

methylene; 3n, 4n, 5n R = CH₂(CH₂)₂NHC(NH₂)dNH; 3o, 4o, 5o R =

H; 3p, 4p R = CH₂(CH₂)₃NHZ; 5p R = CH₂(CH₂)₃NH₂; 3q, 4q, 5q R =

CH₂CH₂CONH₂; 2r, 4r, 5r R = CH₂CONH₂; 3s, 4s, 5s R = CH₂CH-

(CH₃)₂; 3t, 4t, 5t R = CH(CH₃)CH₂CH₃.

^aIC₅₀is represented by mean ( SD nM, n = 6

Table 2. Thrombus Weights of Intravenous 5a-t Treated Rats^a

3S-N-(Boc-aminoacyl)-1,2,3,4-tetrahydro-β-carboline-3-car-

boxylic acid methylesters (3a-t, 86-95% yield). The saponi-

fication of 3a-t provided 3S-2-(Boc-aminoacyl)-1,2,3,4-tet-

rahydro-β-carboline-3-carboxylic acids (4a-t, 78-95%

yield). Removing Boc groups from 4a-t resulted in 5a-t

(81-95% yield). These data suggest that the reaction condi-

tions are mild and the yield of the individual reaction is

acceptable. Therefore the present synthetic route is suitable

for the introduction of amino acids into 2 position of TCCA.

It is worth indicating that the removal of the methylester

group occurs prior to the removal of the Boc group and the

saponification products of 3a-t adjusted to pH 7 are critical

for the formation of 5a-t. Once the removal of the Boc

group occurs prior to the removal of the methylester group,

the intramolecular cyclization spontaneously proceed to

form 7a-t instead of the Boc-removed products 6a-t.

Similarly, once the saponification products are adjusted to

pH less than 7, the intramolecular cyclization also sponta-

neously proceed to form 7a-t instead of the Boc-removed

products 6a-t.

5a-t Inhibiting ADP-, AA-, PAF-, and TH-Mediated

Platelet Activation. To evaluate the effect of 2-amino acid

modification of TCCA on the in vitro platelet aggregation

induced by different aggregators, the in vitro antiplatelet

aggregation assays of 5a-t were performed by following a

general procedure and using four aggregators, adenosine

diphosphate (ADP, final concentration 10 μM), arachidonic

acid (AA, final concentration 350 μM), platelet-activating

factor (PAF, final concentration 0.1 μM), and thrombin

(TH, final concentration 0.1 U/mL). The IC₅₀values were

listed in Table 1.

compd

thrombus weight

compd

thrombus weight

aspirin

TCCA

24.47 ( 3.21

15.21 ( 2.35

18.79 ( 2.01^b

14.61 ( 1.84^b

17.25 ( 2.06^b

18.56 ( 1.83^b

16.25 ( 2.28^b

15.72 ( 2.47^b

15.49 ( 2.66^b

17.24 ( 2.23^b

15.25 ( 2.85^b

13.00 ( 2.17^c

16.60 ( 2.47^b

14.40 ( 2.32^b

15.89 ( 2.84^b

18.43 ( 1.98^b

16.68 ( 2.69^b

19.29 ( 2.48^b

16.54 ( 2.46^b

17.30 ( 2.38^b

16.34 ( 2.51^b

25.26 ( 3.67^d

13.74 ( 2.25^c

^aThrombus weight is represented by mean ( SD mg, NS = vehicle,

n = 12; Aspirin dose = 167 μmol/kg; TCCA dose = 5 μmol/kg; 5a-t

dose = 100 nmol/kg. ^bCompared to NS and 5s p < 0.01. ^cCompared to

NS and 5b,c,g,m,o,q,s p < 0.01. ^dAt 1 μmol/kg of dose, the thrombus

weight is 10.55 ( 1.70 mg and compared to NS p < 0.01.

platelet activation induced by ADP than the other aggregators.

Interestingly, the IC₅₀value of TCCA against ADP-mediated

platelet activation is 701 nM and 63-108-fold higher than

those of 5a-t. Thus the 2-amino acid modification leads the

potency of TCCA inhibiting ADP-mediated platelet activation

to increase by 63-108-fold.

5a-t Possess High in Vivo Antithrombotic Potency. The in

vivo antithrombotic activities of 5a-t were assayed on an

extracorporeal circulation of arterioveinos cannula model of

rats. The individual stock solution of aspirin (positive con-

trol) and 5a-t in normal saline (NS) was administered

intravenously, the thrombus was weighed, and the data are

listed in Table 2. The thrombus weights of the rats receiving

100 nmol/kg of 5a-t range from 13.00 (5i) to 19.29 mg (5o)

and are significantly lower than that (24.47 mg) of the rats

receiving NS (p < 0.01). Though 100 nmol/kg of 5s pos-

sessed no antithrombotic action at the dose of 1 μmol/kg, the

thrombus weight dropped to 10.55 ( 1.70 mg (compared to

NS, p < 0.01). Table 2 further indicates that the efficacious

The IC₅₀values of 5a-t against ADP-, AA-, PAF-, and TH-

mediated platelet activation are 6.50-11.12, 23.52-30.11,

12.45-18.98, and 25.31-34.10 nM, respectively. The IC₅₀

values of 5a-t against ADP-mediated platelet activation are

2.7-3.6-, 1.7-1.9-, and 3.1-3.9- fold lower than those against

AA-, PAF-, and TH-mediated platelet activation, respectively.

Thus to a certain degree, 5a-t show higher inhibition on

3108 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8

Liu et al.

dose of TCCA is 5 μmol/kg. This dose is 5- and 50-fold higher

than that of 5s and 5a-r,t, respectively. Thus 2-amino acid

modification leads the potency of TCCA to increase by

5-50-fold.

Dose Dependence of in Vivo Antithrombotic Action of 5i and

5t. To clarify the effect of the dose on the in vivo antithrom-

botic activities, 100, 10, and 1 nmol/kg of the most potent 5i

and 5t were assayed. The data (Table 3) indicate that the

antithrombotic potency of 100 nmol/kg dose is significantly

higher than that of 10 nmol/kg dose, while the antithrombo-

tic potency of 10 nmol/kg dose is significantly higher than

that of 1 nmol/kg dose. These comparisons demonstrate that

5i and 5t dose-dependently inhibit the thrombosis of the

treated rats.

In an extracorporeal circulation of arterioveinos cannula

model, the rats were orally treated with NS (negative con-

trol), the individual stock solution of aspirin (positive con-

trol), and 5i or 5t in NS, and the data are listed in Table 4. The

evaluation explores that the thrombus weights of the rats

orally receiving 100 nmol/kg of 5i and 5t are significantly

lower than that of the rats receiving NS. The evaluation

further indicates that the thrombus weights of the intrave-

nously and orally treated rats gave no significant difference.

These results imply that 5i and 5t may be potent oral

antithrombotic agents due to the similarity between the

intravenous and oral potency of them.

Effect of Conformation on the in Vivo Antithrombotic

Activities of 5a-t. In a previous paper, we demonstrated

that at a dose of 5 μmol/kg, the 3-amino acid modified

TCCA, 3S-1,2,3,4-tetrahydro-β-carboline-3-carboxylamino

acids, may successfully inhibit the thrombosis of the treated

rats.³⁷Herein we explored the hypothesis that the effective

dose of 2-amino acid modified TCCA, 3S-2-aminoacyl-1,2,3,

4-tetrahydro-β-carboline-3-carboxylic acids, is 100 nmol/kg.

To understand this position isomerism-induced huge change of

the in vivo antithrombotic potency, the conformations of

2- and 3-amino acid modified TCCA were analyzed. Using

TCCA as the template of Cerius²based alignment, the distinct

stereoview for these two kinds of position isomers could be

described as shown in Figure 1. The stereoview-represented

conformation explores the idea that 3S-1,2,3,4-tetrahydro-β-

carboline-3-carboxylamino acids (A in Figure 1) have their

amino acid residues over the carboline ring, while 3S-2-ami-

noacyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acids (B in

Figure 1) have their amino acid residues aside the carboline

ring. This means a comparatively stretched conformation

is particularly important for the high in vivo antithrombotic

activity. This conformational requirement also explains the

difference between the in vivo antithrombotic activities

In Vivo Antithrombotic Activity of Oral 5i and 5t. To

explore the possibility of 5a-t as oral antithrombotic agents,

the oral antithrombotic effects of 5i and 5t were investigated.

Table 3. Effect of Dose of 5i,t on the Thrombus Weight of Intrave-

nously Treated Rats^a

thrombus weight at the following doses

compd

100 nmol/kg

10 nmol/kg

1 nmol/kg

13.00 ( 2.17^b

17.67 ( 2.65^c

22.59 ( 2.82

25.43 ( 3.34

13.74 ( 2.25^b

18.12 ( 2.77^c

^aThrombus weight is represented by mean ( SD mg, n = 12.

^bCompared to 10 nmol/kg p < 0.01. ^cCompared to 1 nmol/kg p < 0.01.

Table 4. Effect of Oral 5i,t on Thrombus Weight of the Treated Rats^a

compd

thrombus weight

compd

thrombus weight

25.02 ( 3.01

13.49 ( 2.07^b

aspirin

17.90 ( 2.50^b

13.88 ( 2.22^b

^aWeight of wet thrombus is represented by X ( SD mg, NS = vehicle,

n = 12; Dose of 5i,t = 100 nmol/kg; dose of aspirin = 167 μmol/kg.

^bCompared to NS, p < 0.01.

Figure 1. Stereoview of 3- and 2-amino acid modified TCCAs, (A) and (B); 5c (C); 5s (D).

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3109

of the structurally similar 5c (C in Figure 1) and 5s (D in

Figure 1).

1 H), 6.99 (t, J = 7.5 Hz, 1 H), 4.22 (d, J = 4.8 Hz, 2 H), 3.69 (dd,

J = 10.5 Hz, J = 5.0 Hz, 1 H), 3.56 (s, 3 H), 3.14 (dd, J = 10.5 Hz,

J = 2.4 Hz, 1 H), 2.83 (ddd, J = 10.5 Hz, J = 5.0 Hz, J = 2.4 Hz,

1 H), 2.66 (s, 1 H).

Conclusions

General Procedure for the Preparation of 3S-2-(Boc-aminoacyl)-

1,2,3,4-tetrahydro-β-carboline-3-carboxylic Acid Methylester

(3a-t). At 0 °C, to the solution of (2.2 mmol) Boc-^L-amino acids

in 30 mL of anhydrous THF, 300 mg (2.2 mmol) of HOBt were

added. After 10 min, 490 mg (2.4 mmol) of DCC were then added.

The suspension of 500 mg (2.17 mmol) of 3S-1,2,3,4-tetrahydro-β-

carboline-3-carboxylic acid methylester in 3 mL of anhydrous

THF was adjusted to pH 8-9 with N-methylmorpholine and

stirred at room temperature for another 20 min. This suspension

was then added to the solution of Boc-^L-amino acids, and the

reaction mixture was stirred at 0 °C for 2 h 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 saturated 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 title compound was obtained as

powder.

In conclusion, comparing with 3-amino acid modified

TCCA, the 2-position isomers, 3S-2-aminoacyl-1,2,3,4-tetra-

hydro-β-carboline-3-carboxylic acids, more effectively inhibit

ADP-, AA-, PAF-, and TH-induced in vitro platelet aggrega-

tion and highly inhibit the in vivo thrombosis of the intrave-

nously treated rats. The Cerius²based alignment resulted in a

comparatively unfolded conformation for the present 2-ami-

no acid modified TCCA. The fact that the efficacious dose of

the in vivo antithrombotic action of the 3-position isomers is

50-foldhigher thanthatofthe 2-positionisomerssuggests that

the comparatively unfolded conformation could be used as a

criterion in the design of the derivatives having high in vivo

antithrombotic potency. The fact that the oral and the

intravenous in vivo antithrombotic potencies of the most

potent 5i and 5t are close to each other suggests that 5a-t

could be the oral antithrombotic agents.

3S-2-(Boc-^L-alanyl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic

Acid Methylester (3a). Yield 93%; mp 148-150 °C. ESI/MS: 402

[M þ H]^þ. IR (KBr): 3340, 3006, 2953, 2840, 1748, 1642, 1605,

1450, 1391, 1072, 902 cm^-1. ¹H NMR (500 MHz, DMSO-d₆): δ

9.93 (s, 1 H), 8.03 (s, 1 H), 7.28 (t, J = 7.5 Hz, 1 H), 7.18 (t, J = 7.5

Hz, 1 H), 6.97 (t, J = 7.5 Hz, 1 H), 6.94 (t, J = 7.5 Hz, 1 H), 4.76 (t,

J = 5.8 Hz, 1 H), 4.66 (m, J = 5.3 Hz, 1 H), 3.84 (s, 2 H), 3.62 (dd,

J = 10.2 Hz, J = 5.2Hz, 1 H), 3.61 (s, 3 H), 3.20 (dd, J = 10.2 Hz,

J = 2.8 Hz, 1 H), 1.48 (d, J = 5.3 Hz, 3 H), 1.45 (s, 9 H). Anal.

Calcd for C₂₁H₂₇N₃O₅: C, 62.83; H, 6.78; N, 10.47. Found: C,

62.66; H, 6.61; N, 10.29.

3S-2-(Boc-^L-phenylalanyl)-1,2,3,4-tetrahydro-β-carboline-3-car-

boxylic Acid Methylester (3b). Yield 90%; mp 147-149 °C. ESI/

MS: 478 [M þ H]^þ. IR (KBr): 3338, 3010, 2943, 2840, 1752, 1640,

1602, 1458, 1390, 1070, 902 cm^-1. ¹H NMR (500 MHz, DMSO-

d₆): δ 9.97 (s, 1 H), 7.89 (s, 1 H), 7.27 (t, J = 7.4 Hz, 1 H), 7.20 (t,

J = 7.8 Hz, 2 H), 7.16 (t, J = 7.4 Hz, 1 H), 7.11 (d, J = 7.8 Hz, 2

H), 7.07 (t, J = 7.8 Hz, 1H), 6.99 (t, J = 7.4 Hz, 1 H), 6.95 (t, J =

7.4 Hz, 1 H), 5.01 (t, J = 5.6 Hz, 1 H), 4.79 (t, J = 5.6 Hz, 1 H),

3.88 (s, 2 H), 3.64 (dd, J = 10.0 Hz, J = 5.1 Hz, 1 H), 3.63 (s, 3 H),

3.26 (dd, J = 10.0 Hz, J = 2.9 Hz, 1 H), 3.06 (d, J = 5.6 Hz, 2 H),

1.46 (s, 9H).; Anal. Calcd for C₂₇H₃₁N₃O₅: C, 67.91; H, 6.54; N,

8.80. Found: C, 67.73; H, 6.70; N, 9.00.

3S-2-(Boc-^L-valinyl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic

Acid Methylester (3c). Yield 87%; mp 137-139 °C. ESI/MS: 430

[M þ H]^þ. IR (KBr): 3343, 3000, 2952, 2845, 1743, 1640, 1605,

1455, 1391, 1375, 1070, 900 cm^-1. ¹H NMR (500 MHz, DMSO-

d₆): δ 9.92 (s, 1 H), 8.00 (s, 1 H), 7.25 (t, J = 7.2 Hz, 1 H), 7.13 (t,

J= 7.2 Hz, 1 H), 6.95 (t, J= 7.2 Hz, 1 H), 6.91 (t, J=7.2Hz, 1H),

4.73 (t, J = 5.6 Hz, 1 H), 4.52 (d, J = 5.2 Hz, 1 H), 3.86 (s, 2 H),

3.62 (dd, J = 10.3 Hz, J = 5.1 Hz, 1 H), 3.64 (s, 3 H), 3.22 (dd, J =

10.3 Hz, J = 3.2 Hz, 1 H), 2.66 (m, J = 5.2 Hz, 1 H), 1.45 (s, 9 H),

1.03 (d, J = 5.4 Hz, 6 H). Anal. Calcd for C₂₃H₃₁N₃O₅: C, 64.32;

H, 7.27; N, 9.78. Found: C, 64.16; H, 7.20; N, 9.92.

3S-2-(Boc-^L-serinyl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic

Acid Methylester (3d). Yield 93%; mp 155-157 °C. ESI/MS: 418

[M þ H]^þ. IR (KBr): 3339, 3006, 2945, 2842, 1750, 1643, 1605,

1459, 1391, 1072, 900 cm^-1. ¹H NMR (500 MHz, DMSO-d₆): δ

10.01 (s, 1 H), 8.03 (s, 1 H), 7.29 (t, J= 7.6 Hz, 1 H), 7.19 (t, J=7.6

Hz, 1H), 7.02 (t, J = 7.6 Hz, 1 H), 6.90 (t, J = 7.6 Hz, 1 H), 4.78 (t,

J = 5.7 Hz, 1 H), 4.65 (t, J = 5.5 Hz, 1 H), 4.05 (d, J = 5.5 Hz, 1

H), 3.91 (s, 2 H), 3.62 (dd, J = 10.1 Hz, J = 5.0 Hz, 1 H), 3.60 (s, 3

H), 3.28 (dd, J = 10.1 Hz, J = 2.7 Hz, 1 H), 3.08 (d, J = 5.5 Hz, 2

H), 1.48 (s, 9 H). Anal. Calcd for C₂₁H₂₇N₃O₆: C, 60.42; H, 6.52;

N, 10.07. Found: C, 60.60; H, 6.71; N, 9.88.

Experimental Section

General. The protected amino acids with L-configuration

were purchased from Sigma Chemical Co. All coupling and

deprotective reactions were carried out under anhydrous con-

ditions. Chromatography was performed on Qingdao silica gel

H. The purity (>97%) of the intermediates and the final

products was confirmed on both TLC (Merck silica gel plates

of type 60 F₂₅₄, 0.25 mm layer thickness) and HPLC (Waters,

₁₈column 4.6 mm ꢀ 150 mm). ¹H NMR spectra were recorded

on Bruker Advance 500 spectrometers. FAB-MS was deter-

mined by VG-ZAB-MS high resolution GC/MS/DS and HP

ES-5989x. Optical rotations were determined with a Schmidtþ

Haensch Polartromic D instrument. The statistical analysis

of all the biological data was performed by ANOVA test with

p < 0.05 cutoff.

3S-1,2,3,4-Tetrahydro-β-carboline-3-carboxylic Acid (1). To a

mixture of 5.0 g (24.5 mmol) of ^L-tryptophan, 25 mL of H₂SO₄

(1 mol/L), and 80 mL of water, 8 mL of formaldehyde (36-38%)

were added. The reaction mixture was stirred at room temperature

for 2 h and adjusted to pH 6-7 with concentrated ammonia

liquor. The mixture obtained was kept at 0 °C for 12 h, and the

formed precipitates were collected by filtration. After recrystalli-

zation, 3.97 g (75%) of the title compound were obtained as a

colorless powder; mp 280-282 °C. ESI/MS: 217 [M þ H]^þ. IR

(KBr): 3450, 3200, 3000, 2950, 2850, 1700, 1601, 1452, 1070, 900

cm^-1. ¹H NMR (BHSC-500, DMSO-d₆): δ 10.99 (s, 1 H), 9.89 (s,

1 H), 7.30 (t, J = 7.5 Hz, 1 H), 7.22 (t, J = 8.0 Hz, 1 H), 7.01 (d,

J = 8.0 Hz, 1 H), 6.81 (d, J = 7.5 Hz, 1 H), 4.01 (t, J = 4.8 Hz, 1

H), 3.75 (dd, J = 10.5Hz, J = 5.0 Hz, 1 H), 3.64 (dd, J = 10.5 Hz,

J = 2.4 Hz, 1 H), 2.91 (d, J = 10.5 Hz, 2 H), 2.86 (s, 1 H).

3S-1,2,3,4-Tetrahydro-β-carboline-3-carboxylic Acid Methy-

lester (2). At 0 °C, 10 mL of thionyl chloride was added dropwise

to 50 mL of methanol, after which 5.0 g (23.1 mmol) of 3S-1,2,3,

4-tetrahydro-β-carboline-3-carboxylic acid was added. The reac-

tion mixture was stirred at room temperature for 15 h and then

TLC (ethyl acetate/petroleum, 5:12) was used to indicate the

completion of the reaction. The excess methanol and thionyl

chloride were removed by evaporation. The residue was dissolved

in 30 mL of ethyl acetate and washed successively with saturated

NaCO₃in water (3 ꢀ 30 mL) and saturated NaCl in water (3 ꢀ

30 mL). The separated ethyl acetate layer was dried with anhy-

drous MgSO₄, evaporated, and purified with flash chromatogra-

phy (CHCl₃/CH₃OH, 30:1) to provide 4.9 g (92%) of the title

compound as a colorless powder; mp 143-145 °C. ESI^þ/MS: 231

[M þ H]^þ. ¹H NMR (BHSC-500, DMSO-d₆): δ 9.79 (s, 1H), 7.28

(t, J = 7.5 Hz, 1 H), 7.18 (t, J = 7.5 Hz, 1 H), 7.01 (t, J = 7.5 Hz,

3S-2-(Boc-^L-threoninyl)-1,2,3,4-tetrahydro-β-carboline-3-car-

boxylic Acid Methylester (3e). Yield 90%; mp 128-130 °C. ESI/

MS: 432 [M þ H]^þ. IR (KBr): 3433, 3205, 3000, 2955, 2841,

3110 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8

Liu et al.

1730, 1643, 1605, 1452, 1390, 1372, 1060, 904 cm^-1. ¹H NMR

(500 MHz, DMSO-d₆): δ 10.02 (s, 1 H), 7.89 (s, 1 H), 7.30 (t, J =

7.3 Hz, 1 H), 7.22 (t, J = 7.3 Hz, 1 H), 6.90 (d, J = 7.4 Hz, 1 H),

6.70 (d, J = 7.3 Hz, 1 H), 4.82 (t, J = 5.3 Hz, 1 H), 4.63 (m, J =

5.3 Hz, 1 H), 4.44 (t, J = 5.3 Hz, 1 H), 4.03 (m, J = 5.2 Hz, 2 H),

3.62 (s, 3 H), 2.95 (d, J = 5.4 Hz, 2 H), 2.21 (d, J = 3.6 Hz, 1 H),

1.45 (s, 9 H), 1.21 (d, J = 5.6 Hz, 3 H). Anal. Calcd for

C₂₂H₂₉N₃O₆: C, 61.24; H, 6.77; N, 9.74. Found: C, 61.11; H,

6.65; N, 9.89.

DMSO-d₆): δ 9.95 (s, 1 H), 8.02 (s, 1 H), 7.29 (t, J = 7.6 Hz, 1 H),

7.22 (t, J = 7.2 Hz, 2 H), 7.20 (d, J = 7.2 Hz, 2 H), 7.18 (t, J = 7.5

Hz, 1 H), 7.16 (t, J = 7.2 Hz, 1H), 6.97 (t, J = 7.5 Hz, 1 H), 6.93

(t, J = 7.5 Hz, 1 H), 5.36 (s, 2 H), 5.14 (t, J = 5.5 Hz, 1H), 4.77 (t,

J = 5.7 Hz, 1 H), 3.62 (dd, J = 10.2 Hz, J = 5.1 Hz, 1 H), 3.64 (s,

3 H), 3.24 (dd, J = 10.2 Hz, J = 2.7 Hz, 1 H), 3.06 (d, J = 5.6 Hz,

2 H), 2.75 (d, J = 5.5 Hz, 2 H), 1.46 (s, 9 H). Anal. Calcd for

C₂₉H₃₃N₃O₇: C, 65.03; H, 6.21; N, 7.85. Found: C, 65.18; H, 6.06;

N, 7.99.

3S-2-(Boc-^L-tyrosinyl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic

Acid Methylester (3f). Yield 87%; mp 145-147 °C. ESI/MS: 494

[M þ H]^þ. IR (KBr): 3337, 3004, 2945, 2845, 1752, 1641, 1600,

1452, 1390, 1070, 903 cm^-1. ¹H NMR (500 MHz, DMSO-d₆): δ

10.02 (s, 1 H), 8.00 (s, 1 H), 7.27 (t, J = 7.5 Hz, 1 H), 7.19 (t, J =

7.5 Hz, 1 H), 6.96 (d, J = 7.6 Hz, 2 H), 6.89 (d, J = 7.5 Hz, 1 H),

6.88 (d, J = 7.5 Hz, 1 H), 6.68 (d, J = 7.6 Hz, 2 H), 5.02 (s, 1 H),

4.94 (t, J = 5.4 Hz, 1 H), 4.83 (t, J = 5.4 Hz, 1H), 3.93 (s, 2 H), 3.64

(dd, J = 10.0 Hz, J = 5.1 Hz, 1 H), 3.63 (s, 3 H), 3.31 (dd, J = 10.0

Hz, J = 2.7 Hz, 1 H), 3.07 (d, J = 5.4 Hz, 2 H), 1.46 (s, 9 H). Anal.

Calcd for C₂₇H₃₁N₃O₆: C, 65.71; H, 6.33; N, 8.51. Found: C, 65.54;

H, 6.46; N, 8.70.

3S-2-(Boc-^L-histidinyl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic

Acid Methylester (3l). Yield 92%; mp 140-142 °C. ESI/MS: 468

[M þ H]^þ. IR (KBr): 3445, 3203, 3005, 2944, 2837, 1731, 1645,

1602, 1451, 1393, 1370, 1060, 900 cm^-1. ¹H NMR (500 MHz,

DMSO-d₆): δ 12.95 (s, 1 H), 9.98 (s, 1 H), 8.02 (s, 1 H), 7.45 (s, 1

H), 7.32 (t, J = 7.2 Hz, 1 H), 7.18 (t, J = 7.5 Hz, 1 H), 7.18 (d,

J = 7.5 Hz, 1 H), 6.99 (t, J = 7.2 Hz, 1 H), 6.87 (s, 1 H), 4.94 (t,

J = 5.1 Hz, 1 H), 4.81 (t, J = 5.3 Hz, 1 H), 4.23 (d, J = 5.1 Hz,

2 H), 3.67 (s, 3 H), 3.17 (d, J = 5.2 Hz, 2 H), 2.95 (d, J = 5.1

Hz, 2 H), 1.47 (s, 9 H). Anal. Calcd for C₂₄H₂₉N₅O₅: C, 61.66;

H, 6.25; N, 14.98. Found: C, 61.79; H, 6.34; N, 14.82.

3S-2-(Boc-^L-tryptophanyl)-1,2,3,4-tetrahydro-β-carboline-3-

carboxylic Acid Methylester (3m). Yield 89%; mp 126-128 °C.

ESI/MS: 517 [M þ H]^þ. IR (KBr): 3335, 3009, 2942, 2842,

3S-2-(Boc-^L-prolinyl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic

Acid Methylester (3g). Yield 92%; mp 118-120 °C. ESI/MS: 428

[M þ H]^þ. IR (KBr): 3431, 3206, 3003, 2954, 2842, 1730, 1643,

1750, 1643, 1604, 1453, 1391, 1072, 900 cm^-1. H NMR (500

1605, 1456, 1394, 1370, 1065, 904 cm^-1. H NMR (500 MHz,

MHz, DMSO-d₆): δ 9.99 (s, 1 H), 9.96 (s, 1 H), 8.02 (s, 1 H),

7.29 (t, J = 7.6 Hz, 1 H), 7.21 (t, J = 7.4 Hz, 1 H), 7.19 (t, J =

7.4 Hz, 1 H), 7.18 (t, J = 7.4 Hz, 1 H), 7.17 (t, J = 7.4 Hz, 1 H),

7.16 (t, J = 7.6 Hz, 1 H), 7.01 (t, J = 7.6 Hz, 1H), 6.89 (t, J =

7.6 Hz, 1 H), 6.82 (s, 1 H), 4.90 (t, J = 5.5 Hz, 1 H), 4.85 (t, J =

5.4 Hz, 1 H), 3.92 (s, 2 H), 3.62 (dd, J = 10.1 Hz, J = 5.0 Hz, 1

H), 3.65 (s, 3 H), 3.32 (dd, J = 10.1 Hz, J = 2.9 Hz, 1 H),

2.92 (t, J = 5.5 Hz, 2 H), 1.48 (s, 9H). Anal. Calcd for

DMSO-d₆): δ 9.97 (s, 1 H), 7.32 (t, J = 7.2 Hz, 1 H), 7.22 (t, J = 7.4

Hz, 1H), 7.03 (d, J = 7.4 Hz, 1H), 6.93 (d, J = 7.2 Hz, 1 H), 4.85 (t,

J= 5.3 Hz, 1 H), 4.31 (t, J= 5.5 Hz, 1 H), 4.25 (d, J= 5.4 Hz, 2 H),

3.62 (s, 3 H), 3.44 (t, J= 5.5 Hz, 2 H), 2.97 (d, J= 5.5 Hz, 2 H), 2.25

(d, J = 5.4 Hz, 2 H), 1.95 (t, J = 5.1 Hz, 2 H), 1.47 (s, 9 H). Anal.

Calcd for C₂₃H₂₉N₃O₅: C, 64.62; H, 6.84; N, 9.83. Found: C, 64.75;

H, 6.92; N, 9.97.

3S-2-(Boc-^L-cysteinyl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic

Acid Methylester (3h). Yield 93%; mp 133-135 °C. ESI/MS: 434

[M þ H]^þ. IR (KBr): 3441, 3205, 3004, 2940, 2842, 1734, 1640,

1605, 1450, 1392, 1370, 1064, 900 cm^-1. ¹H NMR (500 MHz,

DMSO-d₆): δ 10.02 (s, 1 H), 7.99 (s, 1 H), 7.30 (t, J = 7.3 Hz, 1

H), 7.20 (t, J = 7.5 Hz, 1 H), 7.03 (d, J = 7.5 Hz, 1 H), 6.85 (d,

J = 7.3 Hz, 1 H), 4.90 (t, J = 5.1 Hz, 1H), 4.73 (t, J = 5.3 Hz,

1H), 4.23 (d, J = 5.4 Hz, 2 H), 3.63 (s, 3 H), 3.13 (d, J = 5.2 Hz,

2H), 2.99 (d, J = 5.3 Hz, 2 H), 1.47 (s, 9 H), 1.64 (s, 1 H). Anal.

Calcd for C₂₁H₂₇N₃O₅S: C, 58.18; H, 6.28; N, 9.69. Found: C,

58.25; H, 6.38; N, 9.86.

3S-2-(Boc-^L-methioninyl)-1,2,3,4-tetrahydro-β-carboline-3-

carboxylic Acid Methylester (3i). Yield 91%; mp 133-135 °C.

ESI/MS: 462 [M þ H]^þ. IR (KBr): 3443, 3205, 3007, 2950,

2844, 1730, 1645, 1602, 1455, 1392, 1375, 1064, 902 cm^-1. ¹H

NMR (500 MHz, DMSO-d₆): δ 10.01 (s, 1 H), 7.99 (s, 1 H),

7.30 (t, J = 7.3 Hz, 1 H), 7.20 (t, J = 7.5 Hz, 1H), 6.97 (d, J =

7.5 Hz, 1 H), 6.83 (d, J = 7.3 Hz, 1 H), 4.83 (t, J = 5.2 Hz, 1H),

4.47 (t, J = 5.3 Hz, 1 H), 4.27 (d, J = 5.3 Hz, 2 H), 3.66 (s, 3 H),

2.95 (d, J = 5.4 Hz, 2H), 2.45 (t, J = 5.3 Hz, 2 H), 2.25 (d, J =

5.3 Hz, 2 H), 2.11 (s, 3 H), 1.47 (s, 9 H). Anal. Calcd for

C₂₃H₃₁N₃O₅S: C, 59.85; H, 6.77; N, 9.10. Found: C, 59.69; H,

6.85; N, 9.26.

C₂₉H₃₂N₄O₅: C, 67.43; H, 6.24; N, 10.85. Found: C, 67.59;

H, 6.40; N, 10.72.

3S-2-(Boc-^L-argininyl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic

Acid Methylester (3n). Yield 88%; mp 137-139 °C. ESI/MS: 487

[M þ H]^þ. IR (KBr): 3445, 3209, 3004, 2945, 2840, 1733, 1642,

1600, 1451, 1392, 1370, 1064, 900 cm^-1. ¹H NMR (500 MHz,

DMSO-d₆): δ 10.20 (s, 1 H), 8.43 (s, 2 H), 8.25 (s, 1 H), 8.20 (s, 1

H), 8.03 (s, 1 H), 7.27 (t, J = 7.4 Hz, 1 H), 7.17 (t, J = 7.5 Hz, 1

H), 7.01 (d, J = 7.5 Hz, 1 H), 6.93 (d, J = 7.4 Hz, 1 H), 4.91 (d,

J = 5.1 Hz, 1 H), 4.40 (t, J = 4.3 Hz, 1 H), 4.27 (d, J = 5.1 Hz,

2 H), 3.67 (s, 3 H), 2.92 (d, J = 4.3 Hz, 2 H), 2.66 (t, J = 5.5 Hz,

2 H), 1.93 (m, J = 5.3 Hz, 2 H), 1.57 (m, J = 5.4 Hz, 2 H), 1.55

(s, 9 H). Anal. Calcd for C₂₄H₃₄N₆O₅: C, 59.24; H, 7.04; N,

17.27. Found: C, 59.10; H, 6.95; N, 17.40.

3S-2-(Boc-glycyl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic

Acid Methylester (3o). Yield 95%; mp 150-152 °C. ESI/MS: 388

[M þ H]^þ. IR (KBr): 3342, 3003, 2950, 2844, 1745, 1645, 1603,

1452, 1390, 1070, 900 cm^-1. ¹H NMR (500 MHz, DMSO-d₆): δ

9.91 (s, 1 H), 8.11 (s, 1 H), 7.29 (t, J = 7.6 Hz, 1 H), 7.16 (t, J =

7.6 Hz, 1H), 7.00 (t, J = 7.6 Hz, 1 H), 6.97 (t, J = 7.6 Hz, 1 H),

4.76 (t, J = 5.6 Hz, 2 H), 4.52 (d, J = 4.9 Hz, 2 H), 3.87 (s, 2 H),

3.65 (dd, J = 10.5 Hz, J = 5.0 Hz, 1 H), 3.58 (s, 3 H), 3.17 (dd,

J = 10.5 Hz, J = 2.4 Hz, 1 H), 1.44 (s, 9 H). Anal. Calcd for

C₂₀H₂₅N₃O₅: C, 62.00; H, 6.50; N, 10.85. Found: C, 62.18; H,

6.34; N, 10.67.

3S-2-[Boc-^L-lysinyl(Z)]-1,2,3,4-tetrahydro-β-carboline-3-carboxylic

Acid Methylester (3p). Yield 92%; mp 95-97 °C. ESI/MS: 593 [M þ

H]^þ. IR (KBr): 3342, 3003, 2940, 2845, 1752, 1641, 1602, 1456, 1390,

1070, 902 cm^-1. ¹H NMR (500 MHz, DMSO-d₆): δ 9.97 (s, 1 H),

8.05 (s, 1 H), 8.00 (s, 1 H), 7.27 (t, J = 7.5 Hz, 1 H), 7.22 (t, J = 7.2

Hz, 1H), 7.17 (t, J = 7.5 Hz, 1 H), 7.15 (d, J = 7.2 Hz, 2H), 7.13 (t,

J = 7.2 Hz, 2 H), 7.00 (t, J = 7.5 Hz, 1H), 6.88 (t, J = 7.5 Hz, 1 H),

5.36 (s, 2 H), 4.76 (t, J = 5.6 Hz, 1H), 4.55 (t, J = 5.6 Hz, 1H), 3.63

(dd, J = 10.2 Hz, J = 5.1 Hz, 1 H), 3.64 (s, 3 H), 3.30 (dd, J = 10.1

Hz, J = 2.7 Hz, 1 H), 3.08 (d, J = 5.5 Hz, 2 H), 2.95 (t, J= 5.4 Hz, 2

H), 1.75 (t, J = 5.5 Hz, 2 H), 1.58 (t, J = 5.3 Hz, 2 H), 1.48 (s, 9 H),

1.27 (m, J = 5.6 Hz, 2 H). Anal. Calcd for C₃₂H₄₀N₄O₇: C, 64.85; H,

6.80; N, 9.45. Found: C, 64.69; H, 6.71; N, 9.62.

3S-2-[Boc-^L-glutamyl(OMe)]-1,2,3,4-tetrahydro-β-carboline-3-

carboxylic Acid Methylester (3j). Yield 91%; mp 122-124 °C.

ESI/MS: 474 [M þ H]^þ. IR (KBr): 3443, 3205, 3004, 2940, 2834,

1735, 1642, 1601, 1452, 1395, 1370, 1063, 901 cm^-1. H NMR

(500 MHz, DMSO-d₆): δ 9.92 (s, 1 H), 8.01 (s, 1 H), 7.35 (t, J =

7.2 Hz, 1 H), 7.26 (t, J = 7.2 Hz, 1 H), 7.00 (d, J = 7.5 Hz, 1 H),

6.86 (d, J = 7.3Hz, 1 H), 4.87 (d, J = 5.3Hz, 1 H), 4.45 (t, J = 5.5

Hz, 1 H), 4.24 (d, J = 5.4 Hz, 2 H), 3.67 (s, 3 H), 3.65 (s, 3 H), 2.97

(d, J = 5.3 Hz, 2 H), 2.29 (t, J = 5.4 Hz, 2 H), 2.23 (t, J = 5.5 Hz,

2 H), 1.47 (s, 9 H). Anal. Calcdfor C₂₄H₃₁N₃O₇: C, 60.88;H, 6.60;

N, 8.87. Found: C, 60.75; H, 6.47; N, 9.05.

3S-2-[Boc-^L-Aspartyl(OBzl)]-1,2,3,4-tetrahydro-β-carboline-3-

carboxylic Acid Methylester (3k). Yield 92%; mp 144-146 °C.

ESI/MS: 536 [M þ H]^þ. IR (KBr): 3340, 3004, 2948, 2845, 1748,

1642, 1600, 1455, 1391, 1072, 904 cm^-1. H NMR (500 MHz,

Article

3S-2-(Boc-L-glutaminyl)-1,2,3,4-tetrahydro-β-carboline-3-

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3111

3S-2-(Boc-^L-phenylalanyl)-1,2,3,4-tetrahydro-β-carboline-3-car-

boxylic Acid (4b). Yield 89%; mp 125-127 °C. ESI/MS: 464 [M þ

H]^þ. IR (KBr): 3338, 3010, 2943, 2840, 1752, 1640, 1602, 1458,

1390, 1070, 902 cm^-1; [R]_D²⁰= -42 (c = 0.5, CH₃OH). ¹H NMR

(500 MHz, DMSO-d₆): δ 10.99 (s, 1 H), 9.98 (s, 1 H), 7.98 (s, 1 H),

7.28 (d, J = 7.5 Hz, 1 H), 7.18 (m, 6 H), 6.96 (t, J = 7.5 Hz, 1 H),

6.81 (t, J = 7.5 Hz, 1 H), 4.53 (dd, J = 10.5 Hz, J = 4.5 Hz, 1 H),

4.36 (m, 1 H), 4.27 (d, J = 13.0 Hz, 1 H), 3.79 (d, J = 13.0 Hz, 1

H), 2.95 (m, 3 H), 2.58 (t, 1 H), 1.41 (s, 9 H). Anal. Calcd for

C₂₆H₂₉N₃O₅: C, 67.37; H, 6.31; N, 9.07. Found: C, 67.18; H, 6.16;

N, 9.31.

carboxylic Acid Methylester (3q). Yield 86%; mp 145-147 °C.

ESI/MS: 459 [M þ H]^þ. IR (KBr): 3342, 3011, 2949, 2844, 1750,

1640, 1603, 1456, 1391, 1072, 905 cm^-1. H NMR (500 MHz,

DMSO-d₆): δ 10.00(s, 1 H), 8.02(s, 1 H), 7.29 (t, J = 7.6 Hz, 1 H),

7.17 (t, J = 7.6 Hz, 1 H), 6.86 (d, J = 7.6 Hz, 1 H), 6.83 (d, J = 7.6

Hz, 1 H), 6.12 (s, 2 H), 4.90 (t, J = 5.4 Hz, 1 H), 4.55 (t, J = 5.4

Hz, 1 H), 3.95 (s, 2 H), 3.62 (dd, J = 10.2 Hz, J = 5.0 Hz, 1 H),

3.60 (s, 3 H), 3.32 (dd, J = 10.1 Hz, J = 2.9 Hz, 1 H), 2.19 (t, J =

4.9 Hz, 2 H), 2.01 (m, J = 4.9 Hz, 2 H), 1.42 (s, 9 H). Anal. Calcd

for C₂₃H₃₀N₄O₆: C, 60.25; H, 6.59; N, 12.22. Found: C, 60.38; H,

6.77; N, 12.37.

3S-2-(Boc-^L-valinyl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic

Acid (4c). Yield 92%; mp 135-137 °C. ESI/MS: 416 [M þ H]^þ. IR

(KBr): 3343, 3000, 2952, 2845, 1743, 1640, 1605, 1455, 1391, 1375,

1070, 900 cm^-1; [R]_D²⁰= -94 (c = 0.5, CH₃OH). ¹H NMR (500

MHz, DMSO-d₆): δ 11.01 (s, 1 H), 9.99 (s, 1 H), 7.96 (s, 1 H), 7.30

(d, J = 7.5 Hz, 1 H), 7.18 (d, J = 7.5 Hz, 1 H), 6.98 (t, J = 7.5 Hz,

1 H), 6.79 (t, J = 7.5 Hz, 1 H), 4.55 (dd, J = 10.5 Hz, J = 4.5 Hz,

1H), 4.37(m, 1H), 4.19(d, J=13.0Hz, 1H), 3.78(d, J= 13.0 Hz,

1 H), 2.92 (m, 1 H), 2.72 (m, J = 4.5 Hz, 1 H), 2.54 (t, 1 H), 1.41 (s,

9 H), 1.15 (d, J = 5.6 Hz, 6 H). Anal. Calcd for C₂₂H₂₉N₃O₅: C,

63.60; H, 7.04; N, 10.11. Found: C, 63.79; H, 6.90; N, 9.80.

3S-2-(Boc-^L-serinyl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic

Acid (4d). Yield 89%; mp 123-125 °C. ESI/MS: 404 [M þ H]^þ. IR

(KBr): 3339, 3006, 2945, 2842, 1750, 1643, 1605, 1459, 1391, 1072,

900 cm^-1; [R]²_D⁰= -81 (c = 0.5, CH₃OH). ¹H NMR (500 MHz,

DMSO-d₆): δ 10.95 (s, 1 H), 9.95 (s, 1 H), 7.97 (s, 1 H), 7.29 (d, J =

7.5 Hz, 1 H), 7.17 (d, J = 7.5 Hz, 1 H), 7.02 (t, J = 7.5 Hz, 1 H),

6.88 (t, J = 7.5 Hz, 1 H), 4.52 (dd, J = 10.5 Hz, J = 4.5 Hz, 1 H),

4.35 (m, 1 H), 4.14 (d, J = 13.0 Hz, 1 H), 4.05 (m, 2 H), 3.82 (d, J =

13.0 Hz, 1 H), 2.93 (m, 1 H), 2.51 (t, 1 H), 1.44 (s, 9 H). Anal. Calcd

for C₂₀H₂₅N₃O₆: C, 59.54; H, 6.25; N, 10.42. Found: C, 59.72; H,

6.10; N, 10.65.

3S-2-(Boc-^L-threoninyl)-1,2,3,4-tetrahydro-β-carboline-3-car-

boxylic Acid (4e). Yield 87%; mp 148-150 °C. ESI/MS: 418

[M þ H]^þ. IR (KBr): 3433, 3205, 3000, 2955, 2841, 1730, 1643,

1605, 1452, 1390, 1372, 1060, 904 cm^-1; [R]_D²⁰= -60 (c = 0.5,

CH₃OH). ¹H NMR (500 MHz, DMSO-d₆): δ 10.96 (s, 1 H), 9.92

(s, 1 H), 7.95 (s, 1 H), 7.29 (d, J = 7.5 Hz, 1 H), 7.16 (d, J = 7.5

Hz, 1 H), 7.06 (t, J = 7.5 Hz, 1 H), 6.95 (t, J = 7.5 Hz, 1 H), 4.58

(dd, J = 10.5 Hz, J = 4.5 Hz, 1 H), 4.33 (m, 1 H), 4.20 (m, 3 H),

3.85 (d, J = 13.0 Hz, 1 H), 2.95 (m, 1 H), 2.52 (t, 1 H), 1.41 (s, 9

H), 1.25 (d, J = 5.2 Hz, 3 H). Anal. Calcd for C₂₁H₂₇N₃O₆: C,

60.42; H, 6.52; N, 10.07. Found: C, 60.64; H, 6.68; N, 9.83.

3S-2-(Boc-^L-tyrosinyl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic

Acid (4f). Yield 90%; mp 144-145 °C. ESI/MS: 480 [M þ H]^þ.

IR (KBr): 3337, 3004, 2945, 2845, 1752, 1641, 1600, 1452, 1390,

1070, 903 cm^-1; [R]_D²⁰= -66 (c = 0.5, CH₃OH).; ¹H NMR (500

MHz, DMSO-d₆): δ 10.97 (s, 1 H), 9.90 (s, 1 H), 8.05 (s, 1 H),

7.32 (d, J = 7.5 Hz, 1 H), 7.15 (d, J = 7.5 Hz, 1 H), 7.02 (t, J =

7.5 Hz, 1 H), 6.90 (t, J = 7.5 Hz, 1 H), 6.94 (d, J = 7.2 Hz, 2 H),

6.88 (d, J = 7.2 Hz, 2 H), 4.54 (dd, J = 10.5 Hz, J = 4.5 Hz,

1 H), 4.34 (m, 1 H), 4.07 (d, J = 13.0 Hz, 1 H), 3.82 (d, J = 13.0

Hz, 1 H), 3.05 (m, 3 H), 2.52 (t, 1 H), 1.42 (s, 9 H). Anal. Calcd

for C₂₆H₂₉N₃O₆: C, 65.12; H, 6.10; N, 8.76. Found: C, 65.33; H,

5.85; N, 9.00.

3S-2-(Boc-^L-asparaginyl)-1,2,3,4-tetrahydro-β-carboline-3-

carboxylic Acid Methylester (3r). Yield 91%; mp 137-139 °C.

ESI/MS: 445 [M þ H]^þ. IR (KBr): 3443, 3205, 3001, 2932, 2833,

1734, 1630, 1604, 1457, 1391, 1370, 1061, 900 cm^-1. H NMR

(500 MHz, DMSO-d₆): δ 9.97 (s, 1 H), 8.01 (s, 1 H), 7.22 (t, J =

7.3 Hz, 1 H), 7.14 (t, J = 7.1 Hz, 1 H), 7.01 (d, J = 7.3 Hz, 1 H),

6.85 (d, J = 7.3 Hz, 1 H), 6.03 (s, 2 H), 4.91 (d, J = 5.3 Hz, 1 H),

4.41 (t, J = 5.3 Hz, 1 H), 4.24 (d, J = 5.3 Hz, 2 H), 3.65 (s, 3 H),

2.92 (d, J = 5.1 Hz, 2 H), 2.55 (t, J = 5.3 Hz, 2 H), 1.49 (s, 9 H).

Anal. Calcd for C₂₂H₂₈N₄O₆: C, 59.45; H, 6.35; N, 12.60. Found:

C, 59.62; H, 6.44; N, 12.43.

3S-2-(Boc-^L-leucyl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic

Acid Methylester (3s). Yield 87%; mp 135-137 °C. ESI/MS: 444

[M þ H]^þ. IR (KBr): 3344, 3002, 2950, 2842, 1745, 1640, 1603,

1452, 1390, 1070, 900 cm^-1. ¹H NMR (500 MHz, DMSO-d₆): δ

9.87 (s, 1H), 8.00 (s, 1H), 7.27 (t, J = 7.4 Hz, 1 H), 7.15 (t, J = 7.4

Hz, 1 H), 6.99 (t, J= 7.4 Hz, 1 H), 6.96 (t, J= 7.4 Hz, 1 H), 4.75 (t,

J = 5.9 Hz, 1 H), 4.55 (d, J = 5.4 Hz, 1 H), 3.88 (s, 2 H), 3.60 (dd,

J = 10.0Hz, J = 5.0Hz, 1 H), 3.62 (s, 3 H), 3.22 (dd, J = 10.0Hz,

J = 2.9 Hz, 1 H), 1.88 (m, J = 5.4 Hz, 1 H), 1.78 (t, J = 5.0 Hz, 2

H), 1.46 (s, 9 H), 1.03 (d, J = 5.4 Hz, 6 H). Anal. Calcd for

C₂₄H₃₃N₃O₅: C, 64.99; H, 7.50; N, 9.47. Found: C, 65.16; H, 7.63;

N, 9.32.

3S-2-(Boc-^L-isoleucyl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic

Acid Methylester (3t). Yield 88%; mp 130-132 °C. ESI/MS: 444

[Mþ H]^þ. IR (KBr): 3344, 3002, 2950, 2842, 1745, 1640, 1603, 1452,

1390, 1070, 900 cm^-1. ¹H NMR (500 MHz, DMSO-d₆): δ 9.89 (s,

1 H), 8.03 (s, 1 H), 7.27 (t, J = 7.4Hz, 1H), 7.15 (t, J= 7.4 Hz, 1H),

6.99 (t, J = 7.4 Hz, 1 H), 6.96 (t, J = 7.4 Hz, 1 H), 4.75 (t, J =

5.9 Hz, 1 H), 4.55 (d, J = 5.4 Hz, 1 H), 3.88 (s, 2 H), 3.60 (dd, J =

10.0 Hz, J = 5.0 Hz, 1 H), 3.62 (s, 3 H), 3.22 (dd, J = 10.0 Hz, J =

2.9 Hz, 1 H), 2.48 (m, J = 5.4 Hz, 1 H), 1.33 (m, J = 5.0 Hz, 2 H),

1.46 (s, 9 H), 1.06 (d, J = 5.4 Hz, 3 H), 1.00 (t, J = 5.0 Hz, 3 H).

Anal. Calcd for C₂₄H₃₃N₃O₅: C, 64.99; H, 7.50; N, 9.47. Found: C,

65.16; H, 7.63; N, 9.32.

General Procedure for Preparation of 4a-t. At 0 °C, to the

solution of 0.50 g (1.25 mmol) of 3a-t in 4 mL of methanol and

2 mL of chloroform, 0.24 g (6.00 mmol) of NaOH was added. The

reaction mixture was stirred at 0 °C for 60 min. TLC analysis

(chloroform/methanol, 30:1) indicated a complete disappearance

of 3a-t. After evaporation, the residue was dissolved in 30 mL of

water, and the solution was adjusted to pH 7 with dilute hydro-

chloric acid and extracted with ethyl acetate (3 ꢀ 20 mL). The

organic phase was washed successively with 5% sodium bicarbo-

nate and saturated sodium chloride. The solution was then dried

over anhydrous sodium sulfate. Compounds 4a-t was obtained

after filtration and evaporation under reduced pressure.

3S-2-(Boc-^L-alanyl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic

Acid (4a). Yield81%; mp 175-177 °C. ESI/MS: 388 [M þ H]^þ. IR

(KBr): 3340, 3006, 2953, 2840, 1748, 1642, 1605, 1450, 1391, 1072,

902 cm^-1; [R]²_D⁰= -67 (c = 0.5, CH₃OH). ¹H NMR (500 MHz,

DMSO-d₆): δ 10.99 (s, 1 H), 10.01 (s, 1 H), 7.95 (s, 1 H), 7.27 (d,

J = 7.5 Hz, 1 H), 7.18 (d, J = 7.5 Hz, 1 H), 6.98 (t, J = 7.5 Hz,

1 H), 6.88 (t, J = 7.5 Hz, 1 H), 4.58 (dd, J = 10.5 Hz, J = 4.5 Hz,

1 H), 4.46 (m, 1 H), 4.25 (d, J = 13.0 Hz, 1 H), 3.83 (d, J = 13.0

Hz, 1 H), 2.97 (m, 1 H), 2.57 (t, 1 H), 1.47 (d, J = 5.4Hz, 3 H), 1.41

(s, 9 H). Anal. Calcd for C₂₀H₂₅N₃O₅: C, 62.00; H, 6.50; N, 10.85.

Found: C, 62.18; H, 6.64; N, 10.62.

3S-2-(Boc-^L-prolinyl)-1,2,3,4-tetrahydro-β-carboline-3-car-

boxylic Acid (4g). Yield 93%; mp 131-133 °C. ESI/MS: 414

[M þ H]^þ. IR (KBr): 3431, 3206, 3003, 2954, 2842, 1730, 1643,

1605, 1456, 1394, 1370, 1065, 904 cm^-1; [R]_D²⁰= -60 (c = 0.5,

CH₃OH). H NMR (500 MHz, DMSO-d₆): δ 10.95 (s, 1 H),

9.90 (s, 1 H), 7.35 (d, J = 7.5 Hz, 1 H), 7.16 (d, J = 7.5 Hz, 1

H), 7.06 (t, J = 7.5 Hz, 1 H), 6.86 (t, J = 7.5 Hz, 1 H), 4.52 (dd,

J = 10.5 Hz, J = 4.5 Hz, 1 H), 4.26 (m, 1 H), 4.12 (d, J = 13.0

Hz, 1 H), 3.98 (d, J = 13.0 Hz, 1 H), 3.37 (t, J = 5.2 Hz, 2 H),

2.96 (m, 1 H), 2.50 (t, 1 H), 1.84 (m, 2 H), 1.62 (m, 2 H), 1.41 (s,

9 H). Anal. Calcd for C₂₂H₂₇N₃O₅: C, 63.91; H, 6.58; N, 10.16.

Found: C, 63.70; H, 6.42; N, 9.93.

3S-2-(Boc-^L-cysteinyl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic

Acid (4h). Yield 78%; mp 142-144 °C. ESI/MS: 420 [M þ H]^þ. IR

3112 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8

Liu et al.

(KBr): 3441, 3205, 3004, 2940, 2842, 1734, 1640, 1605, 1450, 1392,

1370, 1064, 900 cm^-1; [R]_D²⁰= -51 (c = 0.5, CH₃OH). ¹H NMR

(500 MHz, DMSO-d₆): δ 10.97 (s, 1 H), 9.92 (s, 1 H), 7.85 (s, 1 H),

7.35 (d, J = 7.5 Hz, 1 H), 7.12 (d, J = 7.5 Hz, 1 H), 7.02 (t, J = 7.5

Hz, 1 H), 6.92 (t, J = 7.5 Hz, 1 H), 4.52 (dd, J = 10.5 Hz, J = 4.2

Hz, 1 H), 4.25 (m, 1 H), 4.14 (d, J = 13.0 Hz, 1 H), 3.88 (d, J =

13.0 Hz, 1 H), 3.02 (m, 3 H), 2.58 (t, 1 H), 1.42 (s, 9 H). Anal. Calcd

for C₂₀H₂₅N₃O₅S: C, 57.26; H, 6.01; N, 10.02. Found: C, 57.04; H,

6.16; N, 9.80.

7.31 (d, J = 7.5 Hz, 1 H), 7.14 (d, J = 7.5 Hz, 1 H), 6.97 (t, J = 7.5

Hz, 1H), 6.81 (t, J= 7.5 Hz, 1 H), 4.62 (dd, J= 10.5 Hz, J=4.5Hz,

1H),4.26(m,1H),4.11(d,J=13.0Hz,1H),3.86(d,J=13.0Hz,1

H),3.12(m,1H),2.58(m,3H),1.87(m,1H),1.71(m,1H),1.53(m,

2H),1.40(s,9H).Anal.CalcdforC₂₃H₃₂N₆O₅:C,58.46;H,6.83;N,

17.78. Found: C, 58.69; H, 6.97; N, 17.55.

3S-2-(Boc-glycyl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic

Acid (4o). Yield 95%; mp 142-144 °C. ESI/MS: 374 [M þ H]^þ.

IR (KBr): 3342, 3003, 2950, 2844, 1745, 1645, 1603, 1452, 1390,

1070, 900 cm^-1; [R]_D²⁰= -64 (c = 0.5, CH₃OH). ¹H NMR (500

MHz, DMSO-d₆): δ 10.99 (s, 1 H), 10.00 (s, 1 H), 7.95 (s, 1 H),

7.26 (d, J = 7.5 Hz, 1 H), 7.16 (d, J = 7.5 Hz, 1 H), 6.95 (t, J =

7.5 Hz, 1 H), 6.86 (t, J = 7.5 Hz, 1 H), 4.56 (dd, J = 10.5 Hz, J =

4.2 Hz, 1 H), 4.20 (d, J = 13.0 Hz, 1H), 4.12 (d, 2 H), 3.77 (d, J =

13.0 Hz, 1 H), 2.99 (m, 1 H), 2.54 (m, 1 H), 1.43 (s, 9 H). Anal.

Calcd for C₁₉H₂₃N₃O₅: C, 61.11; H, 6.21; N, 11.25. Found: C,

61.33; H, 6.38; N, 11.48.

3S-2-[Boc-^L-lysinyl(Z)]-1,2,3,4-tetrahydro-β-carboline-3-carboxylic

Acid (4p). Yield 92%; mp 151-153 °C. ESI/MS: 579 [M þ H]^þ. IR

(KBr): 3342, 3003, 2940, 2845, 1752, 1641, 1602, 1456, 1390, 1070,

902 cm^-1; [R]²_D⁰= -50 (c = 0.5, CH₃OH). ¹H NMR (500 MHz,

DMSO-d₆):δ10.96 (s, 1 H), 9.94 (s, 1 H), 8.04 (s, 1 H), 7.97 (s, 1 H),

7.32 (d, J = 7.5 Hz, 1 H), 7.18 (m, 6 H), 6.98 (t, J = 7.5 Hz, 1 H),

6.82 (t, J = 7.5 Hz, 1 H), 5.32 (s, 2 H), 4.62 (dd, J = 10.5 Hz, J =

4.5 Hz, 1 H), 4.42 (t, J = 4.5 Hz, 1 H), 4.14 (d, J = 13.0 Hz, 1 H),

3.88 (d, J = 13.0 Hz, 1 H), 2.95 (m, 3 H), 2.54 (m, 1 H), 1.82 (m, 2

H), 1.55 (m, 2 H), 1.43 (s, 9 H), 1.25 (m, 2 H). Anal. Calcd for

C₃₁H₃₈N₄O₇: C, 64.34; H, 6.62; N, 9.68. Found: C, 64.11; H, 6.45;

N, 9.94.

3S-2-(Boc-^L-glutaminyl)-1,2,3,4-tetrahydro-β-carboline-3-car-

boxylic Acid (4q). Yield 85%; mp 134-136 °C. ESI/MS: 445 [M

þ H]^þ. IR (KBr): 3342, 3011, 2949, 2844, 1750, 1640, 1603, 1456,

1391, 1072, 905 cm^-1; [R]_D²⁰= -67 (c = 0.5, CH₃OH). ¹H NMR

(500 MHz, DMSO-d₆): δ 11.00 (s, 1 H), 9.89 (s, 1 H), 7.85 (s,

1 H), 7.35 (d, J = 7.5 Hz, 1 H), 7.18 (d, J = 7.5 Hz, 1 H), 6.97 (t,

J = 7.5 Hz, 1 H), 6.83 (t, J = 7.5 Hz, 1 H), 6.34 (s, 2 H), 4.53 (dd,

J = 10.5 Hz, J = 4.5 Hz, 1 H), 4.26 (m, 1H), 4.08 (d, J = 13.0 Hz,

1 H), 3.84 (d, J = 13.0 Hz, 1 H), 2.96 (m, 1 H), 2.50 (m, 1 H), 2.18

(m, 2 H), 2.11 (m, 1 H), 1.98 (m, 1 H), 1.42 (s, 9 H). Anal. Calcd

for C₂₂H₂₈N₄O₆: C, 59.45; H, 6.35; N, 12.60. Found: C, 59.22; H,

6.20; N, 12.38.

3S-2-(Boc-^L-methioninyl)-1,2,3,4-tetrahydro-β-carboline-3-car-

boxylic Acid (4i). Yield 87%; mp 120-122 °C. ESI/MS: 448 [M

þ H]^þ. IR (KBr): 3443, 3205, 3007, 2950, 2844, 1730, 1645,

1602, 1455, 1392, 1375, 1064, 902 cm^-1; [R]_D²⁰= -35 (c = 0.5,

CH₃OH). H NMR (500 MHz, DMSO-d₆): δ 11.00 (s, 1 H),

9.90 (s, 1 H), 7.83 (s, 1 H), 7.36 (d, J = 7.5 Hz, 1 H), 7.14 (d, J =

7.5 Hz, 1 H), 6.98 (t, J = 7.5 Hz, 1 H), 6.82 (t, J = 7.5 Hz, 1 H),

4.52 (dd, J = 10.5 Hz, J = 4.5 Hz, 1 H), 4.20 (m, 1 H), 4.05 (d,

J = 13.0 Hz, 1 H), 3.89 (d, J = 13.0 Hz, 1 H), 2.98 (m, 1 H),

2.53 (t, 1 H), 2.35 (m, 2 H), 2.10 (m, 5 H), 1.41 (s, 9 H). Anal.

Calcd for C₂₂H₂₉N₃O₅S: C, 59.04; H, 6.53; N, 9.39. Found: C,

59.26; H, 6.69; N, 9.15.

3S-2-(Boc-^L-glutamyl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic

Acid (4j). Yield 86%; mp 168-170 °C. ESI/MS: 446 [M þ H]^þ. IR

(KBr): 3443, 3205, 3007, 2950, 2844, 1730, 1645, 1602, 1455, 1392,

1375, 1064, 902 cm^-1; [R]_D²⁰= -55 (c = 0.5, CH₃OH). ¹H NMR

(500 MHz, DMSO-d₆): δ 11.00 (s, 1 H), 9.89 (s, 2 H), 7.85 (s, 1 H),

7.36 (d, J = 7.5 Hz, 1 H), 7.15 (d, J = 7.5 Hz, 1 H), 6.97 (t, J = 7.5

Hz, 1 H), 6.82 (t, J = 7.5 Hz, 1 H), 4.55 (dd, J = 10.5 Hz, J = 4.5

Hz, 1 H), 4.28 (m, 1 H), 4.12 (d, J = 13.0 Hz, 1 H), 3.85 (d, J =

13.0 Hz, 1 H), 3.09 (m, 1 H), 2.52 (t, 1 H), 2.20 (t, 2 H), 2.15 (m,

1 H), 2.03 (m, 1 H), 1.42 (s, 9 H). Anal. Calcd for C₂₂H₂₇N₃O₇: C,

59.32; H, 6.11; N, 9.43. Found: C, 59.55; H, 6.27; N, 9.20.

3S-2-(Boc-^L-aspartyl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic

Acid (4k). Yield 91%; mp 133-135 °C. ESI/MS: 432 [M þ H]^þ. IR

(KBr): 3340, 3004, 2948, 2845, 1748, 1642, 1600, 1455, 1391, 1072,

904 cm^-1; [R]²_D⁰= -54 (c = 0.5, CH₃OH). ¹H NMR (500 MHz,

DMSO-d₆): δ 11.02 (s, 1 H), 9.90 (s, 2 H), 7.85 (s, 1 H), 7.35 (d, J =

7.5Hz, 1H), 7.15(d, J=7.5Hz, 1H), 6.99(t,J= 7.5 Hz, 1 H), 6.83

(t, J = 7.5 Hz, 1 H), 4.54 (dd, J = 10.5 Hz, J = 4.5 Hz, 1 H), 4.32

(m, 1 H), 4.16 (d, J = 13.0 Hz, 1 H), 3.88 (d, J = 13.0 Hz, 1 H), 3.06

(m, 1 H), 2.72(m, 1 H), 2.53 (m, 2 H), 1.42 (s, 9 H). Anal. Calcd for

C₂₁H₂₅N₃O₇: C, 58.46; H, 5.84; N, 9.74. Found: C, 58.55; H, 6.02;

N, 9.91. Found: C, 58.77; H, 6.19; N, 10.15.

3S-2-(Boc-^L-histidinyl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic

Acid (4l). Yield 89%; mp 160-162 °C. ESI/MS: 454 [M þ H]^þ. IR

(KBr): 3445, 3203, 3005, 2944, 2837, 1731, 1645, 1602, 1451, 1393,

1370, 1060, 900 cm^-1; [R]_D²⁰= -72 (c = 0.5, CH₃OH). ¹H NMR

(500 MHz, DMSO-d₆): δ 11.00 (s, 1 H), 9.95 (s, 1 H), 7.87 (s, 1 H),

7.45 (s, 1 H), 7.32 (d, J = 7.5 Hz, 1 H), 7.15 (d, J = 7.5 Hz, 1 H),

6.99(t, J =7.5Hz, 1 H), 6.88(s, 1H), 6.82 (t, J =7.5Hz, 1H), 4.50

(dd, J = 10.5, 4.5 Hz, 1 H), 4.35 (m, 1 H), 4.24 (d, J = 13.0 Hz,

1 H), 3.94 (d, J = 13.0 Hz, 1 H), 3.03 (m, 3 H), 2.52 (t, 1 H), 1.44 (s,

9 H). Anal. Calcd for C₂₃H₂₇N₅O₅: C, 60.92; H, 6.00; N, 15.44.

Found: C, 60.71; H, 6.15; N, 15.66.

3S-2-(Boc-^L-tryptophanyl)-1,2,3,4-tetrahydro-β-carboline-3-car-

boxylic Acid (4m). Yield 90%; mp 140-142 °C. ESI/MS: 503 [M þ

H].; IR (KBr): 3335, 3009, 2942, 2842, 1750, 1643, 1604, 1453,

1391, 1072, 900 cm^-1; [R]_D²⁰= -52 (c = 0.5, CH₃OH). ¹H NMR

(500 MHz, DMSO-d₆): δ 11.00 (s, 1 H), 10.03 (s, 1 H), 9.97 (s, 1 H),

7.90 (s, 1 H), 7.56 (d, J = 7.5 Hz, 1 H), 7.42 (d, J = 7.5 Hz, 1 H),

7.28 (d, J = 7.5 Hz, 1 H), 7.15 (d, J = 7.5 Hz, 1 H), 7.01 (s, 1 H),

6.88 (m, 4 H), 4.52 (dd, J = 10.5 Hz, J = 4.2 Hz, 1 H), 4.38 (m,

1H), 4.20(d, J= 13.0 Hz, 1 H), 3.89 (d, J= 13.0 Hz, 1 H), 3.01 (m,

3 H), 2.50 (t, 1 H), 1.42 (s, 9 H). Anal. Calcd for C₂₈H₃₀N₄O₅: C,

66.92; H, 6.02; N, 11.15. Found: C, 66.73; H, 6.18; N, 11.38.

3S-2-(Boc-^L-argininyl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic

Acid (4n). Yield 88%; mp 148-150 °C. ESI/MS: 473 [M þ H]^þ. IR

(KBr): 3445, 3209, 3004, 2945, 2840, 1733, 1642, 1600, 1451, 1392,

1370, 1064, 900 cm^-1; [R]_D²⁰= -84 (c = 0.5, CH₃OH). ¹H NMR

(500 MHz, DMSO-d₆): δ 10.97 (s, 1 H), 9.85 (s, 1 H), 7.85 (s, 1 H),

3S-2-(Boc-^L-asparaginyl)-1,2,3,4-tetrahydro-β-carboline-3-car-

boxylic Acid (4r). Yield 83%; mp 139-141 °C. ESI/MS: 431 [M þ

H]^þ. IR (KBr): 3443, 3205, 3001, 2932, 2833, 1734, 1630, 1604,

1457, 1391, 1370, 1061, 900 cm^-1; [R]_D²⁰= -58 (c = 0.5,

CH₃OH). H NMR (500 MHz, DMSO-d₆): δ 10.95 (s, 2 H),

9.87 (s, 1 H), 7.91 (s, 1 H), 7.32 (d, J = 7.5 Hz, 1 H), 7.15 (d, J =

7.5 Hz, 1 H), 6.98 (t, J = 7.5 Hz, 1 H), 6.82 (t, J = 7.5 Hz, 1 H),

6.20 (s, 2 H), 4.50 (dd, J = 10.5 Hz, J = 4.5 Hz, 1 H), 4.34 (m, 1

H), 4.15 (d, J = 13.0 Hz, 1 H), 3.89 (d, J = 13.0 Hz, 1H), 2.98 (m,

1 H), 2.77 (m, 1 H), 2.59 (m, 1 H), 2.52 (m, 1 H), 1.41 (s, 9 H).

Anal. Calcd for C₂₁H₂₆N₄O₆: C, 58.59; H, 6.09; N, 13.02. Found:

C, 58.36; H, 5.91; N, 13.27.

3S-2-(Boc-^L-leucyl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic

Acid (4s). Yield 94%; mp 138-140 °C. ESI/MS: 430 [M þ H]^þ. IR

(KBr): 3344, 3002, 2950, 2842, 1745, 1640, 1603, 1452, 1390, 1070,

900 cm^-1; [R]²_D⁰= -77 (c = 0.5, CH₃OH). ¹H NMR (500 MHz,

DMSO-d₆):δ 10.89 (s, 1 H), 9.95 (s, 1 H), 7.93 (s, 1 H), 7.29 (d, J =

7.5 Hz, 1 H), 7.16 (d, J = 7.5 Hz, 1 H), 6.96 (t, J = 7.5 Hz, 1 H),

6.78 (t, J = 7.5 Hz, 1 H), 4.58 (dd, J = 10.5 Hz, J = 4.5 Hz, 1H),

4.39 (m, 1 H), 4.22 (d, J = 13.0 Hz, 1 H), 3.81 (d, J = 13.0 Hz, 1

H), 2.92 (m, 1 H), 2.53 (m, 1 H), 1.85 (m, 1 H), 1.78 (m, 1 H), 1.66

(m, 1 H), 1.41 (s, 9 H), 1.08 (d, J = 5.6 Hz, 6H). Anal. Calcd for

C₂₃H₃₁N₃O₅: C, 64.32; H, 7.27; N, 9.78. Found: C, 64.10; H, 7.10;

N, 9.54.

3S-2-(Boc-^L-isoleucyl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic

Acid (4t).Yield 90%; mp134-136 °C. ESI/MS: 430 [M þ H]^þ. IR

(KBr):3344, 3002, 2950, 2842, 1745, 1640, 1603, 1452, 1390, 1070,

900 cm^-1; [R]²_D⁰= -67 (c = 0.5, CH₃OH). ¹H NMR (500 MHz,

DMSO-d₆): δ 10.92 (s, 1 H), 9.90 (s, 1 H), 7.93 (s, 1 H), 7.28 (d,

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3113

J = 7.5 Hz, 1 H), 7.14 (d, J = 7.5 Hz, 1 H), 6.96 (t, J = 7.5 Hz,

1 H), 6.78 (t, J = 7.5 Hz, 1 H), 4.57 (dd, J = 10.5 Hz, J = 4.5 Hz,

1 H), 4.32 (m, 1 H), 4.22 (d, J = 13.0 Hz, 1 H), 3.80 (d, J = 13.0

Hz, 1 H), 2.91 (m, 1 H), 2.53 (m, 2 H), 1.49 (m, 2 H), 1.41 (s, 9 H),

0.92 (d, J = 6.6 Hz, 3 H), 0.86 (t, 3 H). Anal. Calcd for

C₂₃H₃₁N₃O₅: C, 64.32; H, 7.27; N, 9.78. Found: C, 64.11; H,

7.13; N, 9.54.

General Procedure for Preparation of 5a-t. At 0 °C, solution

of 0.5 g (1.28 mmol) of 4a-t in 8 mL of hydrogen chloride/ethyl

acetate (4 mol/L) was stirred for 1 h and TLC analysis

(chloroform/methanol, 5:1) indicated complete disappearance

of 4a-t. Upon evaporation, the residue was dissolved in ethyl

acetate (3 ꢀ 10 mL) and evaporated under reduced pressure to

remove hydrogen chloride. At 0 °C, the residue was dissolved in

2 mL of water and titrated with the solution of sodium hydro-

xide (0.32 mmol/L, 4 mL). The formed precipitates were col-

lected by filtration and dried to provide 5a-t.

3S-2-^L-Threoninyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic

Acid (5e). Yield 90%; mp 156-158 °C. ESI/MS: 318 [M þ H]^þ.

IR (KBr): 3433, 3205, 3000, 2955, 2841, 1730, 1643, 1605, 1452,

1390, 1372, 1060, 904 cm^-1; [R]_D²⁰= -75 (c = 0.34, CH₃OH). ¹H

NMR (500 MHz, DMSO-d₆): δ 10.90 (s, 1 H), 7.59 (d, J = 7.5

Hz, 2 H), 7.28 (d, J = 7.5 Hz, 1 H), 7.16 (d, J = 7.5 Hz, 1 H), 7.06

(t, J = 7.5 Hz, 1 H), 6.94 (t, J = 7.5 Hz, 1 H), 4.57 (dd, J = 10.5

Hz, J = 4.5 Hz, 1 H), 4.45 (m, 1 H), 4.22 (m, 3 H), 3.84 (d, J =

13.0 Hz, 1 H), 2.95 (m, 1 H), 2.52 (m, 1 H), 1.21 (d, J = 5.0 Hz, 3

H). ¹³C NMR (125 MHz, DMSO-d₆): δ 171.2, 170.1, 136.6,

129.6, 126.5, 121.3, 119.5, 118.0, 111.4, 105.3, 64.8, 54.6, 50.2,

37.5, 23.6, 19.0. Anal. Calcd for C₁₆H₁₉N₃O₄: C, 60.56; H, 6.03;

N, 13.24. Found: C, 60.75; H, 6.17; N, 13.46.

3S-2-^L-Tyrosinyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic

Acid (5f). Yield 87%; mp 155-157 °C. ESI/MS: 380 [M þ H]^þ.

IR (KBr): 3337, 3004, 2945, 2845, 1752, 1641, 1600, 1452, 1390,

1070, 903 cm^-1; [R]_D²⁰= -84 (c = 0.32, CH₃OH). ¹H NMR (500

MHz, DMSO-d₆): δ 10.91 (s, 1 H), 7.85 (d, J = 7.5 Hz, 2 H), 7.34

(d, J = 7.5 Hz, 1 H), 7.17 (d, J = 7.5 Hz, 1 H), 7.03 (t, J = 7.5

Hz, 1 H), 6.90 (t, J = 7.5 Hz, 1 H), 6.93 (d, J = 7.2 Hz, 2 H), 6.85

(d, J = 7.2 Hz, 2H), 4.54 (dd, J = 10.5, J = 4.5 Hz, 1 H), 4.42

(m, 1 H), 4.10 (d, J = 13.0 Hz, 1H), 3.81 (d, J = 13.0 Hz, 1 H),

3.02 (m, 3 H), 2.53 (m, 1 H). ¹³C NMR (125 MHz, DMSO-d₆): δ

172.1, 170.8, 155.6, 136.6, 132.2, 129.3, 128.7, 127.8, 126.7,

126.0, 121.6, 119.1, 105.3, 54.9, 51.2, 47.1, 35.2, 23.6. Anal.

Calcd for C₂₁H₂₁N₃O₄: C, 66.48; H, 5.58; N, 11.08. Found: C,

66.71; H, 5.74; N, 10.89.

3S-2-^L-Prolinyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic

Acid (5g). Yield 92%; mp 158-160 °C. ESI/MS: 314 [M þ H]^þ.

IR (KBr): 3431, 3206, 3003, 2954, 2842, 1730, 1643, 1605, 1456,

1394, 1370, 1065, 904 cm^-1; [R]_D²⁰= -104 (c = 0.38, CH₃OH).

¹H NMR (500 MHz, DMSO-d₆): δ 10.98 (s, 1 H), 7.66 (d, J =

7.5 Hz, 2 H), 7.36 (d, J = 7.5 Hz, 1 H), 7.19 (d, J = 7.5 Hz, 1 H),

7.10 (t, J = 7.5 Hz, 1 H), 6.95 (t, J = 7.5 Hz, 1 H), 4.58 (dd, J =

10.5 Hz, J = 4.5 Hz, 1 H), 4.39 (m, 1 H), 4.14 (d, J = 13.0 Hz, 1

H), 3.99 (d, J = 13.0 Hz, 1 H), 3.36 (t, J = 5.2 Hz, 2 H), 2.95 (m,

1 H), 2.51 (m, 1 H), 1.87 (m, 2 H), 1.61 (m, 2 H). ¹³C NMR (125

MHz, DMSO-d₆): δ 172.2, 170.6, 136.8, 129.8, 126.7, 121.6,

119.1, 111.6, 105.3, 59.9, 45.2, 35.6, 32.6, 23.6, 19.6. Anal.

Calcd for C₁₇H₁₉N₃O₃: C, 65.16; H, 6.11; N, 13.41. Found: C,

65.35; H, 6.26; N, 13.62.

3S-2-^L-Cysteinyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic

Acid (5h). Yield 93%; mp 145-147 °C; ESI/MS: 320 [M þ H]^þ.

IR (KBr): 3441, 3205, 3004, 2940, 2842, 1734, 1640, 1605, 1450,

1392, 1370, 1064, 900 cm^-1; [R]_D²⁰= -56 (c = 0.35, CH₃OH).

IR (KBr): 3441, 3205, 3004, 2940, 2842, 1734, 1640, 1605, 1450,

1392, 1370, 1064, 900 cm^-1. ¹H NMR (500 MHz, DMSO-d₆): δ

10.90 (s, 1 H), 7.58 (d, J = 7.5 Hz, 2 H), 7.32 (d, J = 7.5 Hz, 1

H), 7.10 (d, J = 7.5 Hz, 1 H), 6.97 (t, J = 7.5 Hz, 1 H), 6.84 (t,

J = 7.5 Hz, 1 H), 4.52 (dd, J = 10.5 Hz, J = 4.2 Hz, 1 H), 4.38

(m, 1 H), 4.13 (d, J = 13.0 Hz, 1 H), 3.88 (d, J = 13.0 Hz, 1 H),

9.97 (m, 3 H), 2.53 (m, 1 H). ¹³C NMR (125 MHz, DMSO-d₆): δ

172.2, 170.6, 136.8, 129.8, 126.7, 121.6, 119.1, 111.6, 105.3, 58.9,

54.1, 35.6, 30.6, 20.6. Anal. Calcd for C₁₅H₁₇N₃O₃S: C, 56.41;

H, 5.37; N, 13.16. Found: C, 56.59; H, 5.52; N, 13.39.

3S-2-^L-Alanyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic

Acid (5a). Yield 93%; mp 175-177 °C. ESI/MS: 288 [M þ H]^þ;

[R]²_D⁰= -89 (c = 0.35, CH₃OH). IR (KBr): 3340, 3006, 2953,

2840, 1748, 1642, 1605, 1450, 1391, 1072, 902 cm^-1. ¹H NMR (500

MHz, DMSO-d₆): δ 11.02 (s, 1 H), 7.69 (d, J = 7.5 Hz, 2 H), 7.28

(d, J = 7.5 Hz, 1 H), 7.18 (d, J = 7.5 Hz, 1 H), 6.97 (t, J = 7.5 Hz,

1 H), 6.86 (t, J = 7.5 Hz, 1 H), 4.58 (dd, J = 10.5 Hz, J = 4.5 Hz,

1 H), 4.42 (m, 1 H), 4.27 (d, J= 13.0 Hz, 1 H), 3.84 (d, J= 13.0 Hz,

1 H), 2.98 (m, 1 H), 2.56 (m, 1 H), 1.47 (d, J = 5.4 Hz, 3 H). ¹³

NMR (125 MHz, DMSO-d₆): δ 172.2, 170.6, 136.8, 129.8, 126.7,

121.6, 119.1, 118.1, 111.6, 105.3, 54.9, 51.2, 47.1, 23.6. Anal. Calcd

for C₁₅H₁₇N₃O₃: C, 62.71; H, 5.96; N, 14.63. Found: C, 62.89; H,

6.10; N, 14.40.

3S-2-^L-Phenylalanyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic

Acid (5b). Yield 93%; mp 171-173 °C. ESI/MS: 364 [M þ H] ^þ

[R]²_D⁰= -96 (c = 0.36, CH₃OH). IR (KBr): 3338, 3010, 2943,

2840, 1752, 1640, 1602, 1458, 1390, 1070, 902 cm^-1. ¹H NMR (500

MHz, DMSO-d₆): δ 10.96 (s, 1 H), 7.85 (d, J = 7.5 Hz, 2 H),

7.27 (d, J = 7.5 Hz, 1 H), 7.18 (m, 6 H), 6.97 (t, J = 7.5 Hz, 1 H),

6.83 (t, J = 7.5 Hz, 1 H), 4.54 (dd, J = 10.5 Hz, J = 4.5 Hz, 1 H),

4.47 (m, 1 H), 4.26 (d, J= 13.0 Hz, 1 H), 3.79 (d, J=13.0Hz, 1H),

2.94 (m, 3 H), 2.52 (m, 1 H). ¹³C NMR (125 MHz, DMSO-d₆): δ

172.1, 170.8, 136.6, 129.8, 128.7, 127.8, 126.7, 126.0, 121.6, 119.1,

118.1, 111.6, 105.3, 54.9, 51.2, 47.1, 23.6. Anal. Calcd for

C₂₁H₂₁N₃O₃: C, 69.41; H, 5.82; N, 11.56. Found: C, 69.22; H,

5.67; N, 11.31.

3S-2-^L-Valinyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic

Acid (5c). Yield 87%; mp 165-167 °C. ESI/MS: 316 [M þ H]^þ.

[R]²_D⁰= -92 (c = 0.32, CH₃OH). IR (KBr): 3343, 3000, 2952,

2845, 1743, 1640, 1605, 1455, 1391, 1375, 1070, 900 cm^-1. H

NMR (500 MHz, DMSO-d₆): δ 10.98 (s, 1 H), 7.74 (d, J = 7.5

Hz, 2 H), 7.31 (d, J = 7.5 Hz, 1 H), 7.18 (d, J = 7.5 Hz, 1 H), 6.98

(t, J = 7.5 Hz, 1 H), 6.79 (t, J = 7.5 Hz, 1 H), 4.56 (dd, J = 10.5

Hz, J = 4.5 Hz, 1 H), 4.48 (m, 1 H), 4.20 (d, J = 13.0 Hz, 1 H),

3.79 (d, J = 13.0 Hz, 1 H), 2.92 (m, 1 H), 2.73 (m, J = 4.5 Hz, 1

H), 2.52 (m, 1 H), 1.13 (d, J = 5.5 Hz, 6 H); ¹³C NMR (125 MHz,

DMSO-d₆): δ 173.2, 171.6, 135.8, 129.6, 126.5, 121.6, 119.1,

118.1, 111.6, 105.3, 59.4, 57.8, 35.2, 34.1, 23.6, 17.6. Anal. Calcd

for C₁₇H₂₁N₃O₃: C, 64.74; H, 6.71; N, 13.32. Found: C, 64.51; H,

6.57; N, 13.10.

3S-2-^L-Serinyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic

Acid (5d). Yield 93%; mp 161-163 °C. ESI/MS: 304 [M þ H]^þ.

IR (KBr): 3339, 3006, 2945, 2842, 1750, 1643, 1605, 1459, 1391,

1072, 900 cm^-1; [R]_D²⁰= -68 (c = 0.35, CH₃OH). ¹H NMR (500

MHz, DMSO-d₆): δ 10.94 (s, 1 H), 7.57 (d, J = 7.5 Hz, 2 H), 7.28

(d, J = 7.5 Hz, 1 H), 7.17 (d, J = 7.5 Hz, 1 H), 7.02 (t, J = 7.5 Hz,

1 H), 6.86 (t, J = 7.5 Hz, 1 H), 4.52 (dd, J = 10.5 Hz, J = 4.5 Hz,

1 H), 4.39 (m 1 H), 4.13 (d, J = 13.0 Hz, 1 H), 4.01 (m, 2 H), 3.82

(d, J = 13.0 Hz, 1 H), 2.95 (m, 1 H), 2.52 (m, 1 H). ¹³C NMR (125

MHz, DMSO-d₆): δ 172.2, 170.6, 136.8, 129.8, 126.7, 121.8,

119.5, 118.0, 111.4, 105.3, 64.9, 54.9, 51.2, 37.1, 23.6. Anal. Calcd

for C₁₅H₁₇N₃O₄: C, 59.40; H, 5.65; N, 13.85. Found: C, 59.59; H,

5.70; N, 13.61.

3S-2-^L-Methioninyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic

Acid (5i). Yield 91%; mp 163-165 °C. ESI/MS: 348 [M þ H]^þ. IR

(KBr): 3443, 3205, 3007, 2950, 2844, 1730, 1645, 1602, 1455, 1392,

1375, 1064, 902 cm^-1; [R]_D²⁰= -88 (c = 0.30, CH₃OH). ¹H NMR

(500 MHz, DMSO-d₆): δ 10.91 (s, 1 H), 7.62 (d, J = 7.5 Hz, 2 H),

7.35 (d, J = 7.5 Hz, 1 H), 7.16 (d, J = 7.5 Hz, 1 H), 6.97 (t, J = 7.5

Hz, 1 H), 6.82 (t, J = 7.5 Hz, 1 H), 4.55 (dd, J = 10.5 Hz, J = 4.5

Hz, 1 H), 4.41 (m, 1 H), 4.02 (d, J = 13.0 Hz, 1 H), 3.86 (d, J =

13.0 Hz, 1 H), 2.98 (m, 1 H), 2.52 (m, 1 H), 2.33 (m, 2 H), 2.11 (m, 5

H). ¹³C NMR (125 MHz, DMSO-d₆): δ = 172.2, 170.6, 136.8,

129.8, 126.7, 121.6, 111.6, 105.3, 58.8, 51.1, 35.4, 34.2, 30.6, 20.6,

17.4. Anal. Calcd for C₁₇H₂₁N₃O₃S: C, 58.77; H, 6.09; N, 12.09.

Found: C, 58.96; H, 6.26; N, 11.88.

3S-2-^L-Glutamyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic

Acid (5j). Yield 91%; mp 172-174 °C. ESI/MS: 346 [M þ H]^þ.

3114 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8

Liu et al.

IR (KBr): 3443, 3205, 3007, 2950, 2844, 1730, 1645, 1602, 1455,

1392, 1375, 1064, 902 cm^-1; [R]_D²⁰= -84 (c = 0.32, CH₃OH). ¹H

NMR (500 MHz, DMSO-d₆): δ 10.89 (s, 1 H), 9.85 (s, 1 H), 7.64

(d, J = 7.5 Hz, 2 H), 7.37 (d, J = 7.5 Hz, 1 H), 7.18 (d, J = 7.5

Hz, 1 H), 6.97 (t, J = 7.5 Hz, 1 H), 6.83 (t, J = 7.5 Hz, 1 H), 4.58

(dd, J = 10.5 Hz, J = 4.5 Hz, 1 H), 4.46 (m, 1 H), 4.13 (d, J =

13.0 Hz, 1 H), 3.85 (d, J = 13.0 Hz, 1 H), 3.06 (m, 1 H), 2.51 (t,

1 H), 2.26 (t, 2 H), 2.10 (m, 2 H). ¹³C NMR (125 MHz, DMSO-d₆):

δ 172.2, 170.6, 136.8, 129.8, 126.7, 121.6, 119.1, 111.6, 105.3, 59.9,

51.2, 35.2, 27.1, 20.6. Anal. Calcd for C₁₇H₁₉N₃O₅: C, 59.12; H,

5.55; N, 12.17. Found: C, 58.93; H, 5.40; N, 12.41.

J = 7.5 Hz, 1 H), 6.82 (t, J = 7.5 Hz, 1 H), 4.56 (dd, J = 10.5 Hz,

J = 4.2 Hz, 1 H), 4.36 (d, 2 H), 4.19 (d, J = 13.0 Hz, 1 H), 3.78

(d, J = 13.0 Hz, 1H), 2.98 (m, 1 H), 2.53 (m, 1 H). ¹³C NMR (125

MHz, DMSO-d₆): δ 172.2, 170.6, 136.6, 129.8, 126.7, 121.6,

118.1, 111.6, 105.3, 54.9, 51.2, 47.1, 23.6. Anal. Calcd for

C₁₄H₁₅N₃O₃: C, 61.53; H, 5.53; N, 15.38. Found: C, 61.72; H,

5.69; N, 15.61.

3S-2-^L-Lysinyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic Acid

(5p). To the solution of 500 mg (0.86 mmol) of 4p in 15 mL of

methanol, 30 mg of Pd/C (5%) was added. The solution was

agitated with hydrogen at room temperature for 24 h. The reaction

mixture was filtered and evaporated to give a colorless powder. The

powder was treated by use of the procedure same as that used for

4a, giving 240 mg (81%) of the title compound; mp 159-161 °C.

ESI/MS: 345 [M þ H]^þ. IR (KBr): 3342, 3003, 2940, 2845, 1752,

1641, 1602, 1456, 1390, 1070, 902 cm^-1; [R]_D²⁰= -72 (c = 0.30,

3S-2-^L-Aspartyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic

Acid (5k). Yield 92%; mp 178-180 °C. ESI/MS: 332 [M þ H]^þ.

IR (KBr): 3340, 3004, 2948, 2845, 1748, 1642, 1600, 1455, 1391,

1072, 904 cm^-1; [R]_D²⁰= -98 (c = 0.34, CH₃OH). H NMR

(500 MHz, DMSO-d₆): δ 10.92 (s, 1 H), 9.88 (s, 1 H), 7.70 (d,

J = 7.5 Hz, 2 H), 7.36 (d, J = 7.5 Hz, 1 H), 7.15 (d, J = 7.5 Hz,

1 H), 6.99 (t, J = 7.5 Hz, 1 H), 6.84 (t, J = 7.5 Hz, 1 H), 4.57

(dd, J = 10.5 Hz, J = 4.5 Hz, 1 H), 4.42 (m, 1 H), 4.12 (d, J =

13.0 Hz, 1 H), 3.87 (d, J = 13.0 Hz, 1 H), 3.05 (m, 1 H), 2.78(m,

1 H), 2.56 (m, 2 H). ¹³C NMR (125 MHz, DMSO-d₆): δ 172.2,

170.6, 136.8, 128.8, 126.7, 122.6, 119.2, 111.6, 105.3, 59.9, 47.2,

43.2, 35.2, 20.6. Anal. Calcd for C₁₆H₁₇N₃O₅: C, 58.00; H, 5.17;

N, 12.68. Found: C, 58.20; H, 5.34; N, 12.89.

CH₃OH). H NMR (500 MHz, DMSO-d₆): δ 10.94 (s, 1 H),

7.94-7.86 (m, 4 H), 7.34 (d, J= 7.5 Hz, 1 H), 7.19 (d, J=7.5Hz, 1

H), 6.97 (t, J= 7.5 Hz, 1 H), 6.82 (t, J= 7.5 Hz, 1 H), 4.66 (dd, J=

10.5 Hz, J =4.5 Hz, 1 H), 4.48 (m, 1 H), 4.16 (d, J = 13.0 Hz, 1 H),

3.89 (d, J = 13.0 Hz, 1 H), 3.02 (m, 3 H), 2.52 (m, 1 H), 1.85 (m, 2

H), 1.60 (m, 2 H), 1.26 (m, 2 H). ¹³C NMR (125 MHz, DMSO-d₆):

δ 172.8, 171.6, 136.5, 129.8, 126.7, 121.6, 118.1, 111.6, 105.3, 54.9,

52.2, 43.1, 35.1, 32.2, 20.6. Anal. Calcd for C₁₈H₂₄N₄O₃: C, 62.77;

H, 7.02; N, 16.27. Found: C, 62.56; H, 7.20; N, 16.06.

3S-2-^L-Glutaminyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic

Acid (5q). Yield 86%; mp 177-179 °C. ESI/MS: 345 [M þ H]^þ.

IR (KBr): 3342, 3011, 2949, 2844, 1750, 1640, 1603, 1456, 1391,

1072, 905 cm^-1; [R]_D²⁰= -76 (c = 0.34, CH₃OH). ¹H NMR (500

MHz, DMSO-d₆): δ 10.90 (s, 1 H), 7.68 (d, J = 7.5 Hz, 2 H), 7.35

(d, J = 7.5 Hz, 1 H), 7.19 (d, J = 7.5 Hz, 1 H), 6.98 (t, J = 7.5 Hz,

1 H), 6.83 (t, J = 7.5 Hz, 1 H), 6.45 (s, 2 H), 4.55 (dd, J = 10.5 Hz,

J = 4.5 Hz, 1 H), 4.43 (m, 1 H), 4.09 (d, J = 13.0Hz, 1 H), 3.84 (d,

J = 13.0 Hz, 1 H), 2.95 (m, 1 H), 2.51 (m, 1 H), 2.25 (m, 2 H), 2.08

(m, 2 H). ¹³C NMR (125 MHz, DMSO-d₆): δ 172.2, 170.6, 136.6,

129.8, 126.7, 121.6, 118.1, 111.6, 105.3, 54.9, 51.2, 37.1, 32.6, 30.5,

20.6. Anal. Calcd for C₁₇H₂₀N₄O₄: C, 59.29; H, 5.85; N, 16.27.

Found: C, 59.10; H, 5.69; N, 16.51.

3S-2-^L-Asparaginyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic

Acid (5r). Yield 91%; mp 173-175 °C. ESI/MS: 331 [M þ H]^þ. IR

(KBr): 3443, 3205, 3001, 2932, 2833, 1734, 1630, 1604, 1457, 1391,

1370, 1061, 900 cm^-1; [R]_D²⁰= -88 (c = 0.32, CH₃OH). ¹H NMR

(500 MHz, DMSO-d₆): δ 10.88 (s, 1 H), 7.78 (d, J = 7.5 Hz, 2 H),

7.32 (d, J = 7.5 Hz, 1 H), 7.16 (d, J = 7.5 Hz, 1 H), 6.97 (t, J = 7.5

Hz, 1 H), 6.82 (t, J = 7.5 Hz, 1 H), 6.29 (s, 2 H), 4.51 (dd, J = 10.5

Hz, J = 4.5 Hz, 1 H), 4.39 (m, 1 H), 4.14 (d, J = 13.0 Hz, 1 H),

3.89 (d, J = 13.0 Hz, 1 H), 2.96 (m, 1 H), 2.78 (m, 1 H), 2.62 (m,

1 H), 2.51 (m, 1 H). ¹³C NMR (125 MHz, DMSO-d₆): δ 173.2,

171.6, 136.5, 129.6, 126.5, 121.4, 118.1, 111.8, 105.3, 59.1, 48.1,

40.1, 35.3, 23.6. Anal. Calcd for C₁₆H₁₈N₄O₄: C, 58.17; H, 5.49; N,

16.96. Found: C, 58.36; H, 5.65; N, 16.74.

3S-2-^L-Leucyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic Acid

(5s). Yield 87%; mp 161-163 °C. ESI/MS: 330 [M þ H]^þ. IR

(KBr): 3344, 3002, 2950, 2842, 1745, 1640, 1603, 1452, 1390, 1070,

900 cm^-1; [R]²_D⁰= -90 (c = 0.35, CH₃OH). ¹H NMR (500 MHz,

DMSO-d₆): δ 10.95 (s, 1 H), 7.78 (d, J = 7.5 Hz, 2 H), 7.31 (d, J =

7.5 Hz, 1 H), 7.15 (d, J = 7.5 Hz, 1 H), 6.96 (t, J = 7.5 Hz, 1 H),

6.79 (t, J = 7.5 Hz, 1 H), 4.58 (dd, J = 10.5 Hz, J = 4.5 Hz, 1 H),

4.44 (m, 1 H), 4.23 (d, J = 13.0 Hz, 1 H), 3.82 (d, J = 13.0 Hz, 1

H), 2.93 (m, 1 H), 2.52 (m, 1 H), 1.86 (m, 1 H), 1.78 (m, 1 H), 1.64

(m, 1 H), 1.01 (d, J = 5.6 Hz, 6 H). ¹³C NMR (125 MHz, DMSO-

d₆): δ 172.2, 170.6, 136.6, 129.8, 126.7, 121.6, 118.1, 111.6, 105.3,

59.9, 50.2, 45.1, 35.1, 23.6, 20.4. Anal. Calcd for C₁₈H₂₃N₃O₃: C,

65.63; H, 7.04; N, 12.76. Found: C, 65.82; H, 7.21; N, 12.54.

3S-2-^L-Isoleucyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic

Acid (5t). Yield 88%; mp 166-168 °C. ESI/MS: 330 [M þ H]^þ.

IR (KBr): 3344, 3002, 2950, 2842, 1745, 1640, 1603, 1452, 1390,

1070, 900 cm^-1; [R]_D²⁰= -85 (c = 0.33, CH₃OH). ¹H NMR (500

MHz, DMSO-d₆): δ 10.92 (s, 1 H), 7.72 (d, J = 7.5 Hz, 2 H), 7.29

(d, J = 7.5 Hz, 1 H), 7.13 (d, J = 7.5 Hz, 1H), 6.98 (t, J = 7.5 Hz,

3S-2-^L-Histidinyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic

Acid (5l). Yield 92%; mp 159-161 °C. ESI/MS: 354 [M þ H]^þ.

IR (KBr): 3445, 3203, 3005, 2944, 2837, 1731, 1645, 1602, 1451,

1393, 1370, 1060, 900 cm^-1; [R]_D²⁰= -72 (c = 0.35, CH₃OH). ¹H

NMR (500 MHz, DMSO-d₆): δ 10.96 (s, 1 H), 7.76 (d, J =

7.5 Hz, 2 H), 7.43 (s, 1 H), 7.32 (d, J = 7.5 Hz, 1 H), 7.14 (d, J =

7.5 Hz, 1 H), 6.96 (t, J = 7.5 Hz, 1 H), 6.87(s, 1 H), 6.80 (t, J = 7.5

Hz, 1 H), 4.51 (dd, J = 10.5 Hz, J = 4.5 Hz, 1 H), 4.39 (m, 1 H),

4.24 (d, J = 13.0 Hz, 1 H), 3.95 (d, J = 13.0 Hz, 1 H), 3.09 (m,

3 H), 2.52 (m, 1 H). ¹³C NMR (125 MHz, DMSO-d₆): δ 172.2,

136.7, 135.5, 133.1, 129.8, 126.5, 121.6, 119.6, 119.0, 111.6, 105.4,

59.9, 51.2, 35.2, 33.1, 20.6. Anal. Calcd for C₁₈H₁₉N₅O₃: C, 61.18;

H, 5.42; N, 19.82. Found: C, 61.37; H, 5.25; N, 19.60.

3S-2-^L-Tryptophanyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic

Acid (5m). Yield 89%; mp 171-173 °C. ESI/MS: 403 [M þ H]^þ. IR

(KBr): 3335, 3009, 2942, 2842, 1750, 1643, 1604, 1453, 1391, 1072,

900 cm^-1; [R]²_D⁰= -123 (c = 0.30, CH₃OH). ¹H NMR (500 MHz,

DMSO-d₆): δ 11.04 (s, 1 H), 10.98 (s, 1 H), 7.82 (d, J = 7.5 Hz, 2

H), 7.57 (d, J = 7.5 Hz, 1 H), 7.42 (d, J = 7.5 Hz, 1 H), 7.28 (d, J =

7.5 Hz, 1 H), 7.14 (d, J = 7.5 Hz, 1 H), 7.03 (s, 1 H), 6.87 (m, 4 H),

4.60 (dd, J = 10.5 Hz, J = 4.2 Hz, 1 H), 4.48 (m, 1 H), 4.21 (d, J =

13.0 Hz, 1 H), 3.89 (d, J = 13.0 Hz, 1 H), 3.06 (m, 3 H), 2.51 (m, 1

H). ¹³C NMR (125 MHz, DMSO-d₆): δ 172.2, 170.6, 136.8, 136.2,

129.8, 127.1, 126.7, 122.3, 121.6, 120.2, 119.1, 111.6, 105.3, 59.9,

53.2, 35.2, 34.1, 20.6. Anal. Calcd for C₂₃H₂₂N₄O₃: C, 68.64; H,

5.51; N, 13.92. Found: C, 68.83; H, 5.33; N, 14.16.

3S-2-^L-Argininyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic

Acid (5n). Yield 88%; mp 162-164 °C. ESI/MS: 373 [M þ H]^þ.

IR (KBr): 3445, 3209, 3004, 2945, 2840, 1733, 1642, 1600, 1451,

1392, 1370, 1064, 900 cm^-1; [R]_D²⁰= -76 (c = 0.32, CH₃OH).

¹H NMR (500 MHz, DMSO-d₆): δ 10.89 (s, 1 H), 7.82 (d, J =

7.5 Hz, 2 H), 7.64 (s, 1 H), 7.32 (d, J = 7.5 Hz, 1 H), 7.15 (d, J =

7.5 Hz, 1 H), 6.97 (t, J = 7.5 Hz, 1 H), 6.83 (t, J = 7.5 Hz, 1 H),

4.62 (dd, J = 10.5 Hz, J = 4.5 Hz, 1 H), 4.49 (m, 1 H), 4.12 (d,

J = 13.0 Hz, 1 H), 3.86 (d, J = 13.0 Hz, 1 H), 3.10 (m, 1 H), 2.62

(m, 3 H), 1.88 (m, 1 H), 1.75 (m, 1 H), 1.54 (m, 2 H). ¹³C NMR

(125 MHz, DMSO-d₆): δ 172.2, 170.6, 158.6, 136.8, 129.8,

126.7, 121.6, 119.1, 111.6, 105.3, 59.2, 51.2, 37.1, 35.2, 32.1,

23.6, 20.4. Anal. Calcd for C₁₈H₂₄N₆O₃: C, 58.05; H, 6.50; N,

22.57. Found: C, 57.83; H, 6.34; N, 22.35.

3S-2-Glycyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic Acid

(5o). Yield 95%; mp 174-176 °C. ESI/MS: 274 [M þ H]^þ. IR

(KBr): 3342, 3003, 2950, 2844, 1745, 1645, 1603, 1452, 1390,

1070, 900 cm^-1; [R]_D²⁰= -54 (c = 0.35, CH₃OH).; H NMR

(500 MHz, DMSO-d₆): δ 11.01 (s, 1 H), 7.83 (t, J = 7.5 Hz, 2 H),

7.29 (d, J = 7.5 Hz, 1 H), 7.15 (d, J = 7.5 Hz, 1 H), 6.95 (t,

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3115

1 H), 6.81 (t, J = 7.5 Hz, 1 H), 4.56 (dd, J = 10.5 Hz, J = 4.5 Hz,

1 H), 4.42 (m, 1 H), 4.20 (d, J = 13.0 Hz, 1 H), 3.80 (d, J = 13.0

Hz, 1 H), 2.92 (m, 1 H), 2.56 (m, 2 H), 1.52 (m, 2 H), 0.91(d, J =

6.6 Hz, 3 H), 0.89 (m, 3 H). ¹³C NMR (125 MHz, DMSO-d₆): δ

172.2, 170.6, 136.6, 129.8, 126.7, 121.6, 118.1, 111.6, 105.3, 59.9,

55.5, 40.1, 35.1, 23.6, 20.4, 14.2, 11.5. Anal. Calcd for

C₁₈H₂₃N₃O₃: C, 65.63; H, 7.04; N, 12.76. Found: C, 65.86; H,

7.20; N, 12.99.

carotid artery and left jugular vein of the rat were separated. A

weighed 6 cm thread was inserted into the middle of a poly-

ethylene tube. The polyethylene tube was filled with heparin

sodium (50 IU/mL in NS) and one end was inserted into the left

jugular vein while another end was inserted into the right carotid

artery. Blood flowed from the right carotid artery to the left

jugular vein through the polyethylene tube for 15 min. The

thread was taken out and the weight of the wet thrombus was

recorded.

In Vitro Antiplatelet Aggregation Activity Assay. An H-10 cell

counter was used to determine the platelet count and a two-

channel Chronolog aggregometer was used to evaluate platelet

aggregation. The pig blood (six pigs, purchased from Animal

Center of Peking University) was centrifuged at 1000 rpm for 10

min and the platelet rich plasma (PRP) was collected. The

remaining blood was centrifuged for an additional 10 min at

1500g to prepare platelet poor plasma (PPP). The final platelet

count of the PRP was adjusted to 2 ꢀ 10⁸platelets/mL with

autologous PPP. To an optical aggregometry testing tube, 0.5

mL of the adjusted plasma sample and 5 μL of NS or 5 μL of the

solution of 5a-t (in a series of final concentrations of 100, 10, 1,

0.1, 0.01, and 0.001 μM, prepared by diluting 10 mM of stock

solutions of 5a-t in DMSO/NS, 1/10(v/v), with NS) was added.

After adjustment of the baseline, 5 μL of the solution of PAF

(final concentration 0.1 μM, prepared with normal saline) or

5 μL of the solution of ADP (final concentration 10 μM, prepared

with normal saline) or 5 μL of the solution of arachidonic acid in

NS (AA, final concentration 350 μM, prepared with 1 M sodium

carbonate) or 50 μL of the solution of TH (final concentration

0.1 U/mL, prepared with normal saline) was added and aggregation

was measured at 37 °C for 5 min. The effects of 5a-t (at a series of

concentrations ranging from 100 μM to 1 nM) on PAF or ADP or

AA or TH-induced platelet aggregation were observed. All these

antiplatelet aggregation tests in sextuplicate tubes were carried out.

The maximum platelet aggregation (A_m) of control group (NS) or

sample group (5a-t) was represented by the peak height of

aggregation curve (equal to the maximum light transmission).

The inhibition rate was calculated according to the following

formula: inhibition (%) = [(A_mof NS) - (A_mof 5a-t)]/(A_mof

NS) ꢀ 100%. A_mof NS is the value of platelet aggregation induced

by PAF, ADP, AA, and TH without 5a-t and are 52.30 ( 1.78,

50.16 ( 3.65, 49.62 ( 2.90, and 61.20 ( 2.97, respectively. The

concentration vs inhibition rate curve is plotted to determine the

IC₅₀values with GWBASIC.EXE program.

In Vivo Antithrombotic Assay of Intravenously Injection of

5a-t in Rat Model. The assessments described here were per-

formed based on a protocol reviewed and approved by the ethics

committee of Capital Medical University. The committee as-

sures 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

5a-t were dissolved in NS before administration and kept in an

ice bath. Male Wister rats weighing 250-300 g (purchased from

Animal Center of Peking University) were used. The rats were

anesthetized with pentobarbital sodium (80.0 mg/kg, ip), and

the right carotid artery and left jugular vein were separated. A

weighed 6 cm thread was inserted into the middle of a poly-

ethylene tube. The polyethylene tube was filled with heparin

sodium (50 IU/mL in NS) and one end was inserted into the left

jugular vein. From the other end of the polyethylene tube

heparin sodium was injected as anticoagulant, then NS or

5a-t was injected, and this end was inserted into the right

carotid artery. Blood was allowed to flow from the right carotid

artery to the left jugular vein through the polyethylene tube for

15 min. The thread was removed to obtain the weight of the wet

thrombus.

Acknowledgment. This work was completed in the Beijing

Area Major Laboratory of Peptide and Small Molecular

Drugs, supported by PHR (IHLB, KZ 200810025010 and

200910025004), the National Natural Scientific Foundation

of China (20772082), and Special Project (2008ZX09401-002)

of China.

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ꢀ

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