Synthesis of new class dipeptide analogues with improved permeability and antithrombotic activity

Bioorganic & Medicinal Chemistry 14 (2006) 4761–4774

Synthesis of new class dipeptide analogues

with improved permeability and antithrombotic activity

Ming Zhao,^bLanrong Bi,^aWei Bi,^cChao Wang,^bZhe Yang,^b

Jingfang Ju^d,*and Shiqi Peng^a,*

^aCollege of Pharmaceutical Sciences, Capital University of Medical Sciences, Beijing, 100054, PR China

^bCollege of Pharmaceutical Sciences, Peking University, Beijing 100083, PR China

^cDepartment of Surgery, Second Aﬃliated Hospital of Hebei Medical University, Shijiazhuang 050000, PR China

^dUSA Mitchell Cancer Institute, 307 N. University Blvd, Mobile, AL 36688-0002, USA

Received 8 February 2006; revised 13 March 2006; accepted 15 March 2006

Available online 17 April 2006

Abstract—3-(S)-1,2,3,4-Tetrahydro-b-carboline-3-carboxylic acid isolated from A. Chinese G. Don was found to possess moderate

anti-aggregation activity, but with poor bioavailability. To improve its pharmacological property, we designed and synthesized a

series of novel dipeptide analogues by incorporating tetrahydro-b-carboline-3-carboxylic acid skeleton as an amino acid surrogate

( Trp). It turned out these dipeptide analogues exhibited good membrane permeability based on in vitro Caco-2 cell monolayers

*

permeability assay. As a result, the overall biological properties of these molecules were signiﬁcantly improved depending on the

nature of the amino acid residues introduced onto the 3-position of the tetrahydro-b-carboline moiety. It was very interesting to

notice that these dipeptide analogues (5b,c,h,i,n,o,p,q) displayed a remarkable dual antiaggregatory activity in both of ADP- and

PAF-induced platelet aggregation assay, and their aggregation response was signiﬁcantly higher than that of aspirin (p < 0.01).

In addition, these dipeptide analogues were observed for the dose-dependent antithrombotic eﬀect using in vivo rat arterial throm-

bosis model. The potency of antithrombotic activity of 5h,i,n,p was signiﬁcantly higher than that of aspirin (n = 12, p < 0.01) at

equal dose (5 lmol/kg).

1. Introduction

the limited membrane permeability of antithrombotic

drug candidates is thought to be a major barrier leading

to the low oral activity of such compounds.^5,6Various

strategies have been attempted to transiently alter the

unfavorable physicochemical properties of these com-

pounds to overcome their absorption problem.^7–11In

this regard, a promising strategy for overcoming this

problem is to target the intestinal peptide transport sys-

tem. With this in mind, the drug candidates were de-

signed in the form of di-/tripeptide analogues that can

be readily absorbed across the intestinal brush border

membrane via the peptide transport system. There are

number of reports demonstrating that some orally

administered peptide analogues could be eﬀectively ab-

sorbed through the intestinal peptide transport system.

These studies provided useful information on the struc-

tural requirements of substrates for the intestinal pep-

tide transport system. In general, the minimum

structural features appearing to be required by the intes-

tinal peptide transport system are: a free C-terminal car-

boxyl group or a group capable of hydrogen bond

formation, either an amino group or a weakly basic

Thromboembolic diseases are the leading cause of mor-

tality and morbidity worldwide.¹Despite the well-estab-

lished application of antithrombotic drugs, such as

anticoagulants, antiplatelet drugs, and thrombolytic

drugs, there is still an urgent need for more potent and

safer compounds for the prevention and treatment of

ischemic symptoms.^2–4Many antithrombotic drug can-

didates fail to exert their therapeutic potential due to

their poor bioavailability. For the oral antithrombotic

drug, the major challenge of reaching the action sites

is to cross the intestinal epithelial cells to get into the cir-

culation system. Most oral drugs currently on the mar-

ket rely on passive diﬀusion to cross cell membranes.

Although many factors aﬀect the oral activity of a drug,

Keywords: Dipeptide analogue; Antithrombotic activity; Membrane

permeability.

*

Corresponding authors. Tel.: +1 251 460 7393; fax: +1 251 460 6994

(J.J.); tel./fax: +86 10 8391 1528 (S.P.); e-mail addresses: jju@

usouthal.edu; sqpeng@mail.bjum.edu.cn

doi:10.1016/j.bmc.2006.03.026

4762

M. Zhao et al. / Bioorg. Med. Chem. 14 (2006) 4761–4774

group at the N-terminus, an overall charge of less than

two positive units, and preferably a C-terminal amino

acid in its ^L-conﬁguration.^12–22

of absorption of drug candidates after oral administra-

tion is crucial during the preclinical evaluation. The oral

bioavailability of drug candidates is inﬂuenced by many

factors, including dissolution, absorption, pre-systemic

and systemic metabolism, and elimination. However,

the intestinal mucosa is a signiﬁcant barrier to oral

delivery of drugs into the systemic circulation. Caco-2

cells possess many structural and functional similarities

to human enterocytes. Recently, Caco-2 cell permeabil-

ity test has been generally used as a screening tool for

assessing drug oral bioavailability during the early stage

of drug development.^34–38

In this study, we have synthesized and evaluated a series

of novel dipeptide analogues as a strategy to improve

pharmacological and pharmacokinetic properties.^23–32

The main feature of our design is to synthesize small

peptide analogues that are metabolically stable and rec-

ognized by the intestinal peptide transport system. In

this context, we designed and synthesized a series of

novel dipeptide analogues by incorporating a tetrahy-

dro-b-carboline-3-carboxylic acid skeleton as an amino

acid surrogate ( Trp). 3-(S)-1,2,3,4-Tetrahydro-b-carb-

Caco-2 cell monolayers were utilized as an in vitro mod-

el of the intestinal mucosa to assess the membrane

permeability for these dipeptide analogues. Drug perme-

ability is diﬃcult to measure, thus this assay provides a

convenient way to measure permeability based on the

apparent permeability coeﬃcient (P_app). P_app(A ! B)

is the permeability from the apical to the basolateral side

(intestine to blood), and P_app(B ! A) is the permeabil-

ity from the basolateral to the apical side (blood to intes-

tine). It was initiated by adding the test solution to the

apical or basolateral side of the monolayer. The various

dipeptide analogues across Caco-2 cell monolayers were

valuated (n = 3) in the apical to basolateral (A ! B) and

basolateral to apical directions (B ! A). The inﬂuence

of eﬄux carriers on the permeability of the diﬀerent

dipeptide analogues was also examined by comparing

the permeability ratio P_ratioof absorptive transport P_app

(A ! B) to the secretory one P_app(B ! A). Permeabili-

ty coeﬃcients for the synthetic compounds are summa-

rized in Table 1.

*

oline-3-carboxylic acid isolated from A. Chinese G.

Don was found to possess moderate anti-aggregation

activity, but with poor bioavailability.^31,33To improve

its biological property, we focused on improving the

absorption of 3-(S)-1,2,3,4-tetrahydro-b-carboline-3-

carboxylic acid by converting to a dipeptide analogue

for better peptide transport. Herein, we report the de-

sign, synthesis, and biological characterization of these

unique dipeptide analogues.

2. Results and discussion

2.1. Synthesis of dipeptide analogues based on 3-(S)-

1,2,3,4-tetrahydro-b-carboline-3-carboxylic acid scaﬀold

A series of dipeptide analogues were designed and syn-

thesized utilizing 3-(S)-1,2,3,4-tetrahydro-b-carboline-

3-carboxylic acid scaﬀold as ^L-tryptophane surrogate

( Trp). The synthetic route is outlined in Scheme 1.

*

Starting from the optically active ^L-tryptophane, com-

pound 1 was synthesized after the Pictect–Spengler reac-

tion, subsequently subject to protection, coupling, and

deprotection to generate the desired dipeptide analogues

5a–q with 88–98% yield. In general, these dipeptide ana-

logues fulﬁlled the minimal structural requirements for

the intestinal peptide transport system with a free

carboxyl group and one amide group.²²

According to the previous study, the compound with

permeability coeﬃcients P_app< 1 · 10^ꢀ6, 1–10 · 10^ꢀ6

,

and >10 · 10^ꢀ6cm/s was deﬁned as poorly, moderately,

and well absorbed, respectively.³⁶The parental com-

pound 1, 3-(S)-1,2,3,4-tetrahydro-b-carboline-3-carbox-

ylic acid, exhibited moderate permeation through the

Caco-2 cell monolayer (1: 8.95 · 10⁶cm/s). In contrast,

after converting the parental compound into the dipep-

tide analogue, the permeability was increased 2- to 3-

fold (except for 5f,m). For example, compound Cys-

2.2. In vitro membrane permeation study

(5h), Met-(5i), Arg-(5n), Lys-(5p) Trp exhibited the

*

considerably increased permeation through Caco-2 cells

compared to the parent compound 1 when applied to

Oral administration is the most commonly used drug-

dosing route. Therefore, the ability to predict the extent

O

OH

III

OH

I

II

N

NH₂

NH

N

H

N

H

Boc

N

H

2

1

O

R

O

R

O

R

NH

COOH

NH

Boc

COOH

IV

NH

COOCH₃

V

N

NH

N

H

N

H

Boc

H

5a-q

3a-q

4a-q

Scheme 1. Synthesis of the dipeptide analogues 5a–q based on 3-(S)-1,2,3,4-tetrahydro-b-carboline-3-carboxylic acid scaﬀold. Reagents and

conditions: (I) formaldehyde and sulfuric acid; (II) Boc₂O and triethylamine; (III) L-amino acid methyl ester and DCC; (IV) aqueous NaOH (2 mol/

L); (V) hydrogen chloride in ethyl acetate (4 mol/L). In a: R = CH₃; b: R = CH₂C₆H₅; c: R = CH(CH₃)₂; d: R = CH₂OH; e: R = CH(OH)CH₃; f:

R = CH₂C₆H₄-OH-p; g: R = tetrahydropyrrol-2-yl; h: R=CH₂SH; i: R = CH₂CH₂SCH₃; j: R = CH₂CH₂COOH; k: R = CH₂COOH; l: R = 1,3-

imidazol-5-methylene; m: R = indol-3-methylene; n: R = CH₂(CH₂)₂NHC(NH₂)@NH; o: R = H; p: R = CH₂(CH₂)₃NH₂; q: R = CH₂CH₂CONH₂.

M. Zhao et al. / Bioorg. Med. Chem. 14 (2006) 4761–4774

Table 1. Apparent permeability coeﬃcients of 1,5b–d,f,h,i,m–q

4763

for 5f,m). Compared to their parental compound 1,

the dipeptide analogues were clearly more permeable

to Caco-2 cell monolayers from the A ! B direction

than their counterpart 1. The results suggested that the

dipeptide analogues imparted greater membrane perme-

ation characteristics than the parental compound 1.

Since P_app(A ! B) of these compounds was substantial-

ly greater than P_app(B ! A), the asymmetric perme-

ation proﬁle implied that the involvement of apically

polarized eﬄux systems was likely to be excluded.

Compound

P_app· 10⁶(cm/s)

A ! B

B ! A

A ! B/B ! A

1

8.95

12.52

12.35

12.27

9.33

8.78

6.00

6.12

6.20

8.11

5.97

6.01

8.77

6.02

6.03

6.00

5.98

1.02

2.09

2.02

2.00

1.15

3.05

3.08

1.02

3.25

2.08

3.30

2.11

5b

5c

5d

5f

5h

5i

18.20

18.54

8.96

5m

5n

5o

5p

5q

19.54

12.55

19.82

12.64

Moreover, it was noted that the dipeptide analogue,

containing polar charged amino acid, that is, Lys- Trp

*

(5n), Arg- Typ (5p), was shown to have increased level

*

of membrane permeability compared to other dipeptide

analogues. We speculate that the dipeptide transport

system might be preferred to transport polar charged

residues better than other types of dipeptide analogues.

The standard deviations were generally less than 10% (n = 4); A ! B:

From apical side to basolateral side; B ! A: From basolateral side to

apical side.

Surprisingly, the dipeptide analogues Tyr- Trp (5f) and

*

the apical side of the monolayer, as shown by the 3-fold

increased permeability coeﬃcients P_app(A ! B) (5h:

18.20; 5i: 18.54; 5n: 19.54; 5p: 19.82 · 10⁶cm/s).

Trp- Trp (5m) exhibited a slightly improved permeabil-

*

ity (5f: 9.33 · 10⁶; 5m: 8.96 · 10⁶cm/s). This result might

imply that factors other than the polarity and charge,

for example, spatial arrangement of the molecule, might

also inﬂuence membrane permeability.

Recent studies indicated that polarized eﬄux system was

a barrier to the absorption of peptides and peptidomi-

metics.³⁹In our study, among the dipeptide analogues

tested, the absorptive A ! B transport was consistently

greater than that observed in the opposite direction

(Table 2), and the ratio of the apparent permeability

coeﬃcients (P_app) of these dipeptide analogues calculat-

ed from the A ! B and B ! A transport of the dipep-

tides analogues varied between 2.00 and 3.30 (except

In vitro membrane permeability experiment indicated

that this new class of dipeptide analogues was better ab-

sorbed in the intestine than its parental compound 1. In

this study, it is suggested that, the membrane permeabil-

ity of the dipeptide analogue was well correlated to the

chemical property of the amino acid residue (i.e., polar-

ity, charge, and spatial arrangement). These compounds

will be more likely to target the gastrointestinal trans-

porters involved in the absorption of amino acids and

small peptides thereby resulting in improved oral

bioavailability.

Table 2. Eﬀect of 5a–q on ADP-induced platelet aggregation

Compound

Am %ðX ꢁ SDÞ

10^ꢀ7mol/L

10^ꢀ6mol/L

10^ꢀ5mol/L

Although Caco-2 cells have been extensively used to

screen the permeation of drug candidates, further vali-

dation utilizing in vivo model will be needed due to

the diﬀerences between in vitro and in vivo models such

as the diﬀerent levels of esterases or the presence of

esterases (lipases) in the intestinal lumen.

NS

1

43.62 2.15

30.16 2.76^a

29.32 2.38^a

29.50 2.80^a

35.43 1.99^a

33.46 2.25^a

34.37 2.34^a

26.25 2.41^a

25.38 2.02^a

23.46 2.25^a,b,e

Aspirin

5a

5b

5c

30.28 2.45^a,b,d25.23 2.55^a,b,d18.64 2.21^a,b,d

28.68 2.66^a,b,d23.46 2.77^a,b,d15.92 2.90^a,b,d

31.56 2.51^a,b,e27.13 3.01^a,c,e20.81 2.61^a,b,d

5d

5e

35.24 2.38^a

34.97 3.11^a

34.89 3.00^a

30.78 3.20^a

30.11 3.40^a

31.01 3.23^a

25.99 3.02^a

25.87 3.15^a

25.59 2.85^a

2.3. Antiplatelet aggregation in vitro

5f

Antiplatelet aggregation assay was used to evaluate the

eﬀects of these analogues utilizing in vitro rabbit platelet

aggregation induced by adenosine-5-diphosphate (ADP)

or platelet activating factor (PAF). Incubation of dipep-

tide analogues with ADP or PAF in the presence of

platelets for 120 min clearly aﬀected the in vitro induced

platelet aggregation at a concentration of 100 nM. The

duration of incubation had no signiﬁcant inﬂuence on

the antiaggregatory capacity (data not shown).

5g

5h

5i

27.89 2.02^a,b,d21.88 2.30^a,b,d14.01 2.23^a,b,d

25.97 2.34^a,b,d20.86 2.25^a,b,d14.62 1.99^a,b,d

5j

33.41 2.25^a

32.88 2.57^a,c

27.82 2.36^a,c

24.01 2.58^a,c

5k

5l

26.69 2.62^a,b,e21.74 2.56^a,b,d

31.40 2.57^a,b,e25.45 2.74^a,b,d20.77 2.81^a,b,d

5m

5n

5o

5p

5q

35.01 3.03^a

30.42 3.29^a

25.66 3.32^a

25.22 2.26^a,b,d20.04 2.30^a,b,d13.84 1.87^a,b,d

28.50 2.52^a,b,d23.12 2.55^a,b,d15.55 2.73^a,b,d

25.01 2.12^a,b,d19.94 2.27^a,b,d13.62 1.83^a,b,d

30.05 2.38^a,b,d25.10 2.42^a,b,d18.26 2.25^a,b,d

The initial screening at 100 nM concentration revealed

that the most tested compounds were active for anti-ag-

gregation. However, the diﬀerences in chemical structure

of side chain had inﬂuence on ADP- or PAF-induced

aggregation. Compound 5h,i,n,p (Cys- Trp, Met- Trp,

n = 12, NS = vehicle.

^aCompare to NS, P < 0.01.

^bCompare to 1, P < 0.01.

^cCompare to 1, P < 0.05.

^dCompare to aspirin, P < 0. 01.

^eCompare to aspirin, P < 0.05.

*

Arg- Trp, Lys- Trp) was found to be the most potent

*

4764

M. Zhao et al. / Bioorg. Med. Chem. 14 (2006) 4761–4774

antiplatelet analogue based on the ADP- or PAF-in-

duced platelet assay (Tables 2 and 3).

ed with 10 lM of compound 5a,c,d, although antiaggre-

gatory eﬀect was signiﬁcant (compared to NS, 1, aspirin,

p < 0.01 for both), the platelet aggregation was inhibited

to a lesser extent compared with that of 5n,p (5a:

23.46 2.25; 5c: 15.92 2.90; 5d: 20.81 2.61; 5e:

25.99 3.02 vs 5n: 13.84 1.87; 5p: 13.62 1.83).

At the concentration of 100 nM or above, the dipeptide

analogues of Phe-(5b), Val-(5c), Cys-(5h), Met-(5i), Arg-

(5n), Gly-(5o), Lys-(5p), and Gln-(5q) Trp were ob-

*

served to have a dual eﬀect against ADP- and PAF-in-

duced platelet aggregation, and the antiaggregatory

potency was signiﬁcantly improved compared to com-

pound 1, and even considerably higher than that of aspi-

rin (compared to NS, 1, aspirin, p < 0.01, for both). For

example, when ADP activated platelets were treated

with 10^ꢀ6mol/L of 5b,c,h,i,n,o,p,q, the platelet aggrega-

tion rate (%) was considerably lowered from

43.62 2.15 (for NS, control) to 25.23 2.55 (5b),

23.46 2.77 (5c), 21.88 2.30 (5h), 20.86 2.55 (5i),

20.04 2.30 (5n), 23.12 2.55 (5o), 19.94 2.27 (5p),

and 25.10 2.42 (5q) versus 30.16 2.76 (1) and

29.32 2.38 (aspirin), respectively. Interestingly, at the

same concentration range, compound 5b,c,h,i,n,o,p,q

was detected with comparable antiaggregatory potency

in PAF-induced platelet aggregation assay.

Since Arg- and Lys-side chain signiﬁcantly promoted

biological activity, we assumed the presence of the posi-

tive charged residue in the dipeptide analogue might be

enhancing its antiaggregatory property. Compound 5j,k

displayed a good antiaggregatory eﬀect but weaker

potency than that of Arg-, and Lys- Trp with the substi-

*

tution of positive charged amino acid with negative

charged residue, that is, Glu- Trp (5j), Asp- Trp (5k)

*

in PAF- or ADP-induced platelet aggregation assay.

The results suggest that the basic positive charges in

the dipeptide analogues may be important for their

biological activity.

More interestingly, the introduction of sulfur-containing

side chain (Cys and Met) bestowed the dipeptide

analogue (5h,i) with the considerably enhanced antiag-

gregatory eﬀect. In ADP-induced platelet assay, at the

concentration of 10 lM of 5h,i, the platelet aggregation

rate (%) was signiﬁcantly decreased with 14.01 2.23,

14.62 1.99, respectively, compared to that of 1:

26.25 2.41 (p < 0.01). Similar observation was

observed in PAF-induced platelet aggregation assay.

To examine the impact of basic amino acid residue (argi-

nine, lysine) within the dipeptide analogues, the neutral

residue was introduced by replacing arginine and lysine

with neutral amino acid, that is, alanine, valine, and ser-

ine. It was noticed that, when the dipeptide analogues

containing basic residue, that is, Arg- Trp (5n), Lys-

*

that is, Ala- Trp (5a), Val- Trp (5c), Ser- Trp (5d),

*

Trp (5p), switched to the neutral dipeptide analogues,

Compared to Lys-, Arg-, Met-, Cys- Trp, the introduc-

*

the antiaggregatory potency was shown to be decreased.

*

tion of amino acid in aromatic rings, that is, Tyr- Trp

*

(5f), Trp- Trp (5m), resulted in a partial loss of antiag-

For example, when ADP activated platelet was incubat-

*

gregatory potency in both of PAF- and ADP-induced

platelet aggregation assay. In a similar manner, the

introduction of a rigid proline ring in the dipeptide ana-

logue generated a moderate anti-aggregation derivative

Table 3. Eﬀect of 5a–q on PAF-induced platelet aggregation

Compound

Am %ðX ꢁ SDÞ

Pro- Trp (5g). We deduced that these conformationally

*

10^ꢀ7mol/L

10^ꢀ6mol/L

10^ꢀ5mol/L

restricted dipeptide analogues (5f,m,g) will be most

likely to assume an inappropriate position in the recep-

tor–ligand recognition process thereby leading to a

decreased anti-aggregation eﬀect.

NS

46.01 2.34

32.45 2.28^a

31.87 2.44^a

32.02 2.67^a

36.98 2.43^a

36.06 2.38^a

36.78 2.44^a

29.01 2.50^a

27.00 2.52^a

27.28 2.33^a

Aspirin

5a

5b

5c

32.67 2.60^a,b,d27.45 2.62^a,b,d21.04 2.37^a,b,d

30.48 3.01^a,b,d25.57 2.69^a,b,d18.07 2.87^a,b,d

33.27 2.66^a,c,e29.48 2.95^a,c,e23.00 2.69^a,b,d

It seems that the presence of amino acid residue with

aromatic ring or rigid ring might hinder any interaction

between molecules and platelets. As a consequence, we

attempted to insert a smaller amino acid residue to lower

5d

5e

35.55 2.78^a

36.04 2.89^a

36.40 3.11^a

31.88 2.95^a

32.02 2.89^a

33.01 3.37^a

29.02 2.97^a

28.99 3.21^a

29.20 2.77^a

5f

5g

5h

5i

the steric hindrance, that is, Gly- Trp (5o). Interestingly,

*

30.30 2.23^a,b,d24.04 2.72^a,b,d16.56 2.45^a,b,d

28.04 2.77^a,b,d23.08 2.90^a,b,d17.17 2.55^a,b,d

a positive inﬂuence on antiaggregatory activity was

observed in these two series studied. For example, in

ADP-induced platelet assay, at the concentration of

10 lM, the platelet aggregation rate (%) was signiﬁcant-

ly decreased with 15.55 2.73, compared to that of 1:

26.25 2.41 (p < 0.01). A similar observation was seen

in PAF-induced platelet aggregation assay.

5j

36.06 2.64^a

34.22 2.29^a,c

29.38 2.88^a,c,e25.69 2.73^a,b

29.75 2.88^a,c24.04 2.66^a,b,e

5k

5l

33.70 2.88^a,c,e27.80 2.91^a,b,d23.38 2.60^a,b,d

5m

5n

5o

5p

5q

35.98 2.75^a

31.99 2.92^a

28.27 3.15^a

26.53 2.69^a,b,d21.88 2.78^a,b,d15.63 2.48^a,b,d

30.02 2.97^a,b,d25.22 2.54^a,b,d17.99 2.65^a,b,d

25.88 2.54^a,b,d21.38 2.62^a,b,d15.27 2.44^a,b,d

32.12 2.57^a,b,d27.07 2.59^a,b,d20.97 2.41^a,b,d

The in vitro assay indicated that the new class of dipep-

tide analogues was very likely to be a potent platelet

aggregation inhibitor based on in vitro induced platelet

aggregation. It was interesting to notice that, Val-, Phe-,

n = 12, NS = vehicle.

^aCompare to NS, P < 0.01.

^bCompare to 1, P < 0.01.

^cCompare to 1, P < 0.05.

^dCompare to aspirin, P < 0.01.

^eCompare to aspirin, P < 0.05.

Cys-, Met-, Arg-, Lys-, Gly-, Gln- Trp displayed a

*

remarkable dual antiplatelet activity in both of ADP-

and PAF-induced platelet aggregation assay. Surprisingly,

M. Zhao et al. / Bioorg. Med. Chem. 14 (2006) 4761–4774

4765

Table 5. Eﬀect of 5b–d,h,I,n–q at diﬀerent doses on the thrombus

weight

the introduction of Pro-, Trp-, Tyr-side chain was not

beneﬁcial to antiplatelet activity in the ADP- and

PAF-induced platelet aggregation assay. Thus, the rigid

side chain seemed to be detrimental to antiplatelet activ-

ity against ADP. In contrast, the presence of the polar

side chain rendered the dipeptide analogues, Lys-,

Compound

Dosage/wet thrombus (X ꢁ SD mg)

5.0 lmol/kg

2.5 lmol/kg

1.2 lmol/kg

5b

5c

5d

5h

5i

24.35 3.28^a

23.67 3.18^a

24.52 3.25^a

21.47 2.92^a

20.69 3.44^a

20.01 3.12^a

23.22 3.11^a

19.87 3.01^a

24.11 3.24^a

29.23 3.11^b

28.00 3.04^b

29.84 3.26^b

27.00 3.02^b

26.80 3.30^b

26.03 3.22^b

27.80 3.00^b

26.00 3.06^b

29.01 3.14^b

38.99 3.43

38.20 3.31

37.06 3.13

36.11 3.40

36.00 3.35

36.43 3.50

38.86 3.24

36.47 3.16

38.94 3.13

Arg-, Cys-, Met- Trp, to be the most potent compound

*

in this assay. Therefore, it seems reasonable to speculate

that, within this series, the nature of the amino acid res-

idues introduced in tetrahydrocarboline skeleton may be

crucial to determine the potential biological activity.

The aggregation response of these dipeptide analogues

was comparable to or better than that of aspirin even

when diﬀerent inducing agents (ADP or PAF) were used

(n = 12, p < 0.01). Longer incubation time has no impact

on platelet response, which may be due to a fast metab-

olization of the test compound (data not shown).

5n

5o

5p

5q

N = 12.

^aCompare to 2.5 lmol/kg group, P < 0.01.

^bCompare to 1.2 lmol/kg group, P < 0.01.

weight after the treatment with 5h,i,n,p was

20.69 3.44,

respectively

p < 0.01). The potency of antithrombotic activity of

5h,i,n,p was signiﬁcantly higher than that of aspirin

(aspirin: 28.95 3.32, n = 12, p < 0.01) at equal dose

(5 lmol/kg).

2.4. Evaluation of antithrombotic eﬀect in vivo

21.47 2.92,

19.87 3.01 mg,

20.01 3.12,

(NS: 38.67 3.26,

and

The antithrombotic eﬀect of dipeptide analogues was

evaluated using in vivo rat model. Initially, the dipeptide

analogues were administered at a dose of 1.2, 2.5, and

5 lmol/kg, respectively. From Tables 4 and 5, it was ob-

served that the antithrombotic activity of these dipeptide

analogues was following dose-dependent manners. At

the dose of 1.2 lmol/kg, only compound 5h,i,n,p exhib-

ited a weak antithrombotic eﬀect. While at a dose of

2.5 lmol/kg, the antithrombotic activity of all of dipep-

tide analogues tested was obvious and comparable to

that of aspirin (5 lmol/kg). Interestingly, at equivimolar

doses, some of the dipeptide analogues (5h,i,n,p) pre-

sented signiﬁcant antithrombotic eﬀect. The thrombus

3. Conclusion

The in vitro antiplatelet assay and in vitro antithrom-

botic assessment conﬁrmed that 3-(S)-1,2,3,4-tetrahy-

dro-b-carboline-3-carboxylic acid 1 was responsible for

the antithrombotic activity as an important scaﬀold.

After converting into its dipeptide analogue, the parent

compound 1 was imparted a signiﬁcantly enhanced anti-

thrombotic activity, and its anti-aggregation response

was also signiﬁcantly improved.

Table 4. Eﬀect of 5a–q on the thrombus weight

Compound

Wet thrombus

(X ꢁ SD mg)

38.67 3.26

Dry thrombus

(X ꢁ SD mg)

7.69 1.69

It was interesting to notice that the nature of the amino

acid residues introduced to the 3-position of tetrahydro-

b-carboline scaﬀold apparently had an impact on the

antiplatelet and antithrombotic activities of these ana-

logues. The most signiﬁcant antiplatelet and antithrom-

NS

1

29.69 3.22^a

5.88 1.26^a

Aspirin

5a

5b

5c

28.95 3.32^a

28.97 3.56^a

24.35 3.28^a,b,d

23.67 3.18^a,b,d

24.52 3.25^a,b,d

29.24 3.18^a

30.22 2.67^a

29.34 3.34^a

21.47 2.92^a,b,d

20.69 3.44^a,b,d

29.03 3.31^a

28.78 3.46^a

26.15 3.23^a,c,e

31.00 2.59^a

20.01 3.12^a,b,d

23.22 3.11^a,b,d

19.87 3.01^a,b,d

24.11 3.24^a,b,d

5.11 1.99^a

5.13 1.34^a

4.37 1.88^a,c,e

4.14 1.77^a,c,e

4.29 1.54^a,c,e

5.75 1.81^a

5.62 1.53^a

5.56 1.66^a

3.55 1.07^a,b,d

3.16 1.55^a,b,d

5.81 1.50^a

5.64 1.26^a

4.78 1.25^a,c

6.02 1.49^a

3.00 1.62^a,b,d

4.00 1.75^a,c,e

2.89 1.58^a,b,d

4.12 1.82^a,c,e

botic proﬁle of dipeptide analogues Lys- Trp, Arg- Trp

*

could be correlated with the inﬂuence of the polar

charged side chain. In addition, introduction of sulfur-

containing side chain (Cys and Met) bestowed the

parent compound 1 with the considerably improved

biological activity. Clearly, the polarity, charge,

molecular size, and the spatial arrangement of these

compounds appeared to be some of the key factors in

inﬂuencing their biological potencies.

5d

5e

5f

5g

5h

5i

5j

5k

5l

5m

5n

5o

5p

5q

Our results indicated by converting them into dipeptide

analogues, it rendered 3-(S)-1,2,3,4-tetrahydro-b-carbo-

line-3-carboxylic acid more easily to penetrate the cell

membrane, thereby increasing its penetration ability a

cross the cell membrane and leading to the enhanced

biological activity. The mechanism of the antithrombot-

ic activity of these compounds has not been elucidated,

and their abilities to prevent the aggregation stimulated

by diﬀerent aggregating agents led us to hypothesize that

they might intercept a common signaling pathway in the

Dosage, 5 lmol/kg; n = 12, NS = vehicle.

^aCompare to NS, P < 0.01.

^bCompare to 1, P < 0.01.

^cCompare to 1, P < 0.05.

^dCompare to aspirin, P < 0.01.

^eCompare to aspirin, P < 0.05.

4766

M. Zhao et al. / Bioorg. Med. Chem. 14 (2006) 4761–4774

platelet aggregation cascade. The notable anti-aggregat-

ing potency, regardless of the cause of activation, was

regarded as an attractive approach to develop novel

eﬀective antiplatelet drugs, for instance, for the GPIIb/

IIIa antagonists, phosphodiesterase inhibitors, and nat-

ural or synthetic platelet activating factor antagonists.

tion mixture, 5 mL of citrate solution (20%) was added

and the solution was extracted with ethyl acetate (3·

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

anhydrous MgSO₄. After removal of MgSO₄by ﬁltra-

tion, the ﬁltrate was dried by evaporation. The residue

obtained was crystallized in CHCl₃to yield 1.20 g

(76%) of the title compound. Mp 165–170 ꢁC; TOF/

MS: 317 [M+H]⁺339 [M+Na]⁺, 355 [M+K]; IR

(KBr): 3452, 3205, 3001, 2952, 2848, 1705, 1645, 1600,

In conclusion, these results suggested that the new class

of dipeptide analogues might be a promising antithrom-

botic agent. The antithrombotic eﬀect of these com-

pounds is most likely based on a direct, inhibitory

action on platelets.

1

1450, 1072, 901 cm^ꢀ1; H NMR (BHSC-500, DMSO-

d₆): d = 10.87 (s, 1H), 9.86 (s, 1H), 7.32 (t, J = 7.6 Hz,

1H), 7.21 (t, J = 7.9 Hz, 1H), 7.00 (d, J = 7.9 Hz, 1H),

6.84 (t, J = 7.6 Hz, 1H), 4.84 (t, J = 5.0 Hz, 1H), 4.20

(dd, J = 10.2 Hz, J = 4.8 Hz, 1H), 3.98 (dd,

J = 10.2 Hz, J = 3.2 Hz, 1H), 2.93 (d, J = 10.2 Hz, 2H),

1.46 (s, 9H). Anal. Calcd for C₁₇H₂₀N₂O₄: C, 64.54;

H, 6.37; N, 8.86. Found: C, 64.41; H, 6.25; N, 8.74.

4. Experimental

4.1. Chemistry

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 Qing-

dao silica gel H. The purities of the intermediates and

the products were conﬁrmed by TLC (Merck silica gel

plates of type 60 F₂₅₄, 0.25 mm layer thickness) and

HPLC (Waters, C₁₈column 4.6 · 150 mm). The amino

acid analysis was determined with Hitachi 835-50 instru-

ment. FAB-MS was determined by VG-ZAB-MS high

resolution GC/MS/DS and HP ES-5989x. Optical rota-

tions were measured with a Schmidt + Haensch Polar-

tromic D instrument.

4.4. General procedure for preparation of 3a–q

At 0 ꢁC, to a solution of 2.0 g (6.33 mmol) of 2 in 30 mL

of anhydrous THF, 1.2 g (8.9 mmol) of HOBt was add-

ed. After stirring for 10 min, 1.75 g (8.5 mmol) of DCC

was added. The suspension of 6.96 mmol of HClÆL-AA-

OMe in 3 mL of anhydrous THF was adjusted with

N-methyl morpholine to pH 8–9 and stirred at room

temperature for additional 20 min. This suspension

was then added to the solution of 2, and the reaction

mixture was continued stirring at 0 ꢁC for 2 h and then

at room temperature for 16 h. After evaporation, the

residue was dissolved in 30 mL of ethyl acetate. The

solution was washed extensively with 5% sodium bicar-

bonate, 5% citric acid, and saturated sodium chloride,

and the organic phase was separated and dried over

anhydrous sodium sulfate. After ﬁltration and evapora-

tion under reduced pressure, N-(N-Boc-3S-1,2,3,4-tetra-

hydro-b-carboline-3-carboxyl)-^L-amino acid methylester

(3a–q) was obtained.

4.2. 3S-1,2,3,4-Tetrahydro-b-carboline-3-carboxylic

acid (1)

To a mixture of 5.0 g (24.5 mmol) of ^L-tryptophane,

25 mL of H₂SO₄(1 mol/L), 80 mL of water, and 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 a concentrated ammonia solu-

tion. The mixture obtained was kept at 0 ꢁC for 12 h and

the formed precipitates were collected by ﬁltration.

After recrystallization, 3.97 g (75%) of the title com-

pound was obtained as a colorless powder. Mp 280–

282 ꢁC; EI/MS: 217 [M+H]⁺; IR (KBr): 3450, 3200,

4.4.1. N-(N-Boc-3S-1,2,3,4-tetrahydro-b-carboline-3-car-

boxyl)-^L-alanine methylester (3a). Yield 96%; mp 144–

146 ꢁC; ESI/MS 402 [M+H]⁺; IR (KBr): 3451, 3011,

2949, 2847, 1730, 1604, 1450, 1392, 1370, 1066,

1

897 cm^ꢀ1; H NMR (BHSC-500, DMSO-d₆): d = 9.89

(s, 1H), 7.98 (s, 1H), 7.32 (t, J = 7.5 Hz, 1H), 7.23 (t,

J = 7.8 Hz, 1H), 6.97 (d, J = 7.8 Hz, 1H), 6.81 (d,

J = 7.5 Hz, 1H), 4.88 (d, J = 5.2 Hz, 1H), 4.59 (m,

J = 5.5 Hz, 1H), 4.25 (dd, J = 10.0 Hz, J = 4.7 Hz,

1H), 4.17 (dd, J = 10.1 Hz, J = 3.5 Hz, 1H), 3.64 (s,

3H), 2.94 (d, J = 10.1 Hz, 2H), 1.55 (d, J = 5.2 Hz,

3H), 1.43 (s, 9H). Anal. Calcd for C₂₁H₂₇N₃O₅: C,

62.83; H, 6.78; N, 10.47. Found: C, 62.92; H, 6.74; N,

10.30.

3000, 2950, 2850, 1700, 1601, 1452, 1070, 900 cm^ꢀ1

;

¹H NMR (BHSC-500, DMSO-d₆): d = 10.99 (s, 1H),

9.89 (s, 1H), 7.30 (t, J = 7.5 Hz, 1H), 7.22 (t,

J = 8.0 Hz, 1H), 7.01 (d, J = 8.0 Hz, 1H), 6.81 (d,

J = 7.5 Hz, 1H), 4.01 (t, J = 4.8 Hz, 1H), 3.75 (dd,

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

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

1H). Anal. Calcd for C₁₂H₁₂N₂O₂: C, 66.65; H, 5.59;

N, 12.96. Found: C, 66.45; H, 5.72; N, 12.79.

4.4.2. N-(N-Boc-3S-1,2,3,4-tetrahydro-b-carboline-3-car-

boxyl)-^L-phenylalanine methylester (3b). Yield 98%; mp

150–152 ꢁC; ESI/MS 478 [M+H]⁺; IR (KBr): 3446,

3205, 3006, 2948, 2847, 1731, 1645, 1603, 1451, 1392,

4.3. N-Boc-3S-1,2,3,4-Tetrahydro-b-carboline-3-carbox-

ylic acid (2)

1

A suspension of 1.1 g (5.0 mmol) of 1 in 15 mL DMF

and 1.4 mL of triethylamine was vigorously stirred at

room temperature, 1.1 g (7.7 mmol) of Boc-N₃was add-

ed for 30 min. The reaction mixture was stirred at room

temperature for 24 h and at 40 ꢁC for 80 h. To the reac-

1370, 1069, 904 cm^ꢀ1; H NMR (BHSC-500, DMSO-

d₆): d = 9.92 (s, 1H), 7.97 (s, 1H), 7.31 (t, J = 7.5 Hz,

1H), 7.28 (t, J = 7.9 Hz, 2H), 7.19 (t, J = 7.6 Hz, 1H),

7.14 (d, J = 7.6 Hz, 2H), 7.02 (t, J = 7.6 Hz, 1H), 6.96

(d, J = 7.8 Hz, 1H), 6.80 (d, J = 7.6 Hz, 1H), 4.93 (d,

M. Zhao et al. / Bioorg. Med. Chem. 14 (2006) 4761–4774

4767

J = 5.4 Hz, 1H), 4.82 (t, J = 5.4 Hz, 1H), 4.27 (dd,

J = 10.2 Hz, J = 4.5 Hz, 1H), 4.18 (dd, J = 10.2 Hz,

J = 3.4 Hz, 1H), 3.62 (s, 3H), 3.17 (d, J = 5.4 Hz, 2H),

2.93 (d, J = 10.2 Hz, 2H), 1.48 (s, 9H). Anal. Calcd for

C₂₇H₃₁N₃O₅: C, 67.91; H, 6.54; N, 8.80. Found: C,

67.72; H, 6.62; N, 8.67.

65.71; H, 6.33; N, 8.51. Found: C, 65.67; H, 6.50; N,

8.67.

4.4.7. N-(N-Boc-3S-1,2,3,4-tetrahydro-b-carboline-3-car-

boxyl)-^L-proline methylester (3g). Yield 97%; mp 139–

141 ꢁC; ESI/MS 428 [M+H]⁺; IR (KBr): 3435, 3202,

3000, 2950, 2846, 1732, 1645, 1602, 1454, 1390, 1371,

4.4.3. N-(N-Boc-3S-1,2,3,4-tetrahydro-b-carboline-3-car-

boxyl)-^L-valine methylester (3c). Yield 95%; mp 138–

140 ꢁC; ESI/MS 430 [M+H]⁺. IR (KBr): 3443, 3202,

3001, 2951, 2845, 1729, 1648, 1602, 1450, 1392, 1370,

1063, 900 cm^ꢀ^{1 1}H NMR (BHSC-500, DMSO-d₆):

;

d = 10.01 (s, 1H), 7.35 (t, J = 7.4 Hz, 1H), 7.20 (t,

J = 7.7 Hz, 1H), 7.07 (d, J = 7.7 Hz, 1H), 6.91 (d,

J = 7.4 Hz, 1H), 4.88 (t, J = 5.4 Hz, 1H), 4.35 (t,

J = 5.6 Hz, 1H), 4.22 (d, J = 5.3 Hz, 2H), 3.59 (s, 3H),

3.47 (t, J = 5.6 Hz, 2H), 2.95 (d, J = 5.6 Hz, 2H), 2.29

(d, J = 5.6Hz, 2H), 1.97 (t, J = 4.9 Hz, 2H), 1.45 (s,

9H). Anal. Calcd for C₂₃H₂₉N₃O₅: C, 64.62; H, 6.84;

N, 9.83. Found: C, 64.57; H, 6.90; N, 9.67.

1067, 902 cm^ꢀ^{1 1}H NMR (BHSC-500, DMSO-d₆):

;

d = 10.04 (s, 1H), 7.96 (s, 1H), 7.29 (t, J = 7.4 Hz, 1H),

7.21 (t, J = 7.7 Hz, 1H), 7.00 (d, J = 7.7Hz, 1H), 6.89

(d, J = 7.4 Hz, 1H), 4.84 (t, J = 5.4 Hz, 1H), 4.42 (d,

J = 5.4 Hz, 1H), 4.22 (dd, J = 10.2 Hz, J = 4.5 Hz, 1H),

4.03 (dd, J = 10.2 Hz, J = 3.7 Hz, 1H), 3.62 (s, 3H),

3.10 (m, J = 5.4 Hz, 1H), 2.95 (d, J = 6.7 Hz, 2H), 1.47

(s, 9H), 1.05 (d, J = 5.4 Hz, 6H). Anal. Calcd for

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

64.43; H, 7.09; N, 9.67.

4.4.8. N-(N-Boc-3S-1,2,3,4-tetrahydro-b-carboline-3-car-

boxyl)-^L-cysteine methylester (3h). Yield 92%; mp 151–

153 ꢁC; ESI/MS 420 [M+H]⁺; IR (KBr): 3445, 3203,

3000, 2944, 2840, 1731, 1643, 1601, 1453, 1390, 1372,

1061, 898 cm^ꢀ^{1 1}H NMR (BHSC-500, DMSO-d₆):

;

4.4.4. N-(N-Boc-3S-1,2,3,4-tetrahydro-b-carboline-3-car-

boxyl)-^L-serine methylester (3d). Yield 92%; mp 139–

141 ꢁC; ESI/MS 418 [M+H]⁺. IR (KBr): 3442, 3200,

3001, 2952, 2845, 1730, 1644, 1606, 1455, 1392, 1370,

d = 9.93 (s, 1H), 7.97 (s, 1H), 7.32 (t, J = 7.5 Hz, 1H),

7.22 (t, J = 7.8 Hz, 1H), 7.00 (d, J = 7.8 Hz, 1H), 6.88

(d, J = 7.5 Hz, 1H), 4.93 (t, J = 5.3 Hz, 1H), 4.72 (t,

J = 5.5 Hz, 1H), 4.21 (d, J = 5.3 Hz, 2H), 3.68 (s, 3H),

3.16 (d, J = 5.5 Hz, 2H), 2.90 (d, J = 5.6 Hz, 2H), 1.45

(s, 9H), 1.62 (s, 1H). Anal. Calcd for C₂₁H₂₇N₃O₅S: C,

58.18; H, 6.28; N, 9.69. Found: C, 58.27; H, 6.33; N, 9.57.

1067, 900 cm^ꢀ^{1 1}H NMR (BHSC-500, DMSO-d₆):

;

d = 9.95 (s, 1H), 7.97 (s, 1H), 7.29 (t, J = 7.6 Hz, 1H),

7.22 (t, J = 7.9 Hz, 1H), 6.99 (d, J = 7.9 Hz, 1H), 6.83

(t, J = 7.6 Hz, 1H), 4.87 (d, J = 5.4 Hz, 1H), 4.52 (t,

J = 5.6 Hz, 1H), 4.19 (d, J = 5.2 Hz, 2H), 4.13 (d,

J = 5.6 Hz, 2H), 3.63 (s, 3H), 2.95 (d, J = 5.6 Hz, 1H),

2.92 (d, J = 5.6 Hz, 1H), 2.28 (s, 1H), 1.45 (s, 9H). Anal.

Calcd for C₂₁H₂₇N₃O₆: C, 60.42; H, 6.52; N, 10.07.

Found: C, 60.31; H, 6.36; N, 10.24.

4.4.9. N-(N-Boc-3S-1,2,3,4-tetrahydro-b-carboline-3-car-

boxyl)-^L-methionine methylester (3i). Yield 97%; mp

159–161 ꢁC; ESI/MS 462 [M+H]⁺;IR (KBr): 3441,

3203, 3004, 2953, 2847, 1732, 1641, 1603, 1454, 1390,

1

1372, 1061, 900 cm^ꢀ1; H NMR (BHSC-500, DMSO-

d₆): d = 10.04 (s, 1H), 7.97 (s, 1H), 7.32 (t, J = 7.5 Hz,

1H), 7.22 (t, J = 7.8 Hz, 1H), 6.99 (d, J = 7.8 Hz, 1H),

6.81 (d, J = 7.5 Hz, 1H), 4.86 (t, J = 5.3 Hz, 1H), 4.45

(t, J = 5.5 Hz, 1H), 4.28 (d, J = 5.1 Hz, 2H), 3.68 (s,

3H), 2.93 (d, J = 5.3 Hz, 2H), 2.42 (t, J = 5.4 Hz, 2H),

2.28 (d, J = 5.6 Hz, 2H), 2.10 (s, 3H), 1.44 (s, 9H). Anal.

Calcd for C₂₃H₃₁N₃O₅S: C, 59.85; H, 6.77; N, 9.10.

Found: C, 59.67; H, 6.59; N, 9.04.

4.4.5. N-(N-Boc-3S-1,2,3,4-tetrahydro-b-carboline-3-car-

boxyl)-^L-threonine methylester (3e). Yield 92%; mp 140–

142 ꢁC; ESI/MS 432 [M+H]⁺; IR (KBr): 3437, 3200,

3002, 2951, 2844, 1735, 1649, 1600, 1450, 1392, 1370,

1065, 901 cm^ꢀ^{1 1}H NMR (BHSC-500, DMSO-d₆):

;

d = 9.98 (s, 1H), 7.87 (s, 1H), 7.34 (t, J = 7.4 Hz, 1H),

7.25 (t, J = 7.6 Hz, 1H), 6.95 (d, J = 7.6 Hz, 1H), 6.72

(d, J = 7.4 Hz, 1H), 4.87 (t, J = 5.4 Hz, 1H), 4.67 (m,

J = 5.6 Hz, 1H), 4.48 (t, J = 5.6 Hz, 1H), 3.99 (m,

J = 5.2 Hz, 2H), 3.65 (s, 3H), 2.97 (d, J = 5.7 Hz, 2H),

2.19 (d, J = 3.7 Hz, 1H), 1.47 (s, 9H), 1.19 (d,

J = 5.6 Hz, 3H). Anal. Calcd for C₂₂H₂₉N₃O₆: C,

61.24; H, 6.77; N, 9.74. Found: C, 61.40; H, 6.91; N,

9.55.

4.4.10.

N-(N-Boc-3S-1,2,3,4-tetrahydro-b-carboline-3-

carboxyl)-^L-glutamic acid di-methylester (3j). Yield 93%;

mp 154–156 ꢁC; ESI/MS 474 [M+H]⁺; IR (KBr): 3441,

3203, 3000, 2944, 2831, 1731, 1645, 1604, 1455, 1390,

1

1372, 1067, 903 cm^ꢀ1; H NMR (BHSC-500, DMSO-

d₆): d = 9.89 (s, 1H), 8.04 (s, 1H), 7.39 (t, J = 7.6 Hz,

1H), 7.28 (t, J = 7.6 Hz, 1H), 7.01 (d, J = 7.7 Hz, 1H),

6.84 (d, J = 7.6 Hz, 1H), 4.90 (d, J = 5.4 Hz, 1H), 4.43

(t, J = 5.6 Hz, 1H), 4.22 (d, J = 5.5 Hz, 2H), 3.66 (s,

3H), 3.64 (s, 3H), 2.96 (d, J = 5.4 Hz, 2H), 2.28 (t,

J = 5.6 Hz, 2H), 2.24 (t, J = 5.7 Hz, 2H), 1.43 (s, 9H).

Anal. Calcd for C₂₄H₃₁N₃O₇: C, 60.88; H, 6.60; N, 8.87.

Found: C, 60.73; H, 6.49; N, 8.69.

4.4.6. N-(N-Boc-3S-1,2,3,4-tetrahydro-b-carboline-3-car-

boxyl)-^L-tyrosine methylester (3f). Yield 93%; mp 143–

145 ꢁC; ESI/MS 494 [M+H]⁺; IR (KBr): 3439, 3203,

3001, 2955, 2847, 1732, 1644, 1601, 1453, 1391, 1372,

1062, 903 cm^ꢀ^{1 1}H NMR (BHSC-500, DMSO-d₆):

;

d = 9.99 (s, 1H), 8.02 (s, 1H), 7.37 (t, J = 7.6 Hz, 1H),

7.22 (t, J = 7.7 Hz, 1H), 7.15 (d, J = 7.5 Hz, 2H), 7.02

(d, J = 7.5 Hz, 1H), 6.96 (d, J = 7.7 Hz, 1H), 6.91 (d,

J = 7.5 Hz, 2H), 4.98 (s, 1H), 4.93 (d, J = 5.4 Hz,1H),

4.80 (t, J = 5.6 Hz, 1H), 4.29 (m, J = 5.2 Hz, 2H), 3.64

(s, 3H), 3.15 (d, J = 5.2 Hz, 2H), 2.97 (d, J = 5.0 Hz,

2H), 1.49 (s, 9H). Anal. Calcd for C₂₇H₃₁N₃O₆: C,

4.4.11.

N-(N-Boc-3S-1,2,3,4-tetrahydro-b-carboline-3-

carboxyl)-^L-aspartic acid di-methylester (3k). Yield

90%; mp 158–160 ꢁC; ESI/MS 460 [M+H]⁺; IR (KBr):

3441, 3210, 3004, 2955, 2841, 1732, 1643, 1604, 1453,

1390, 1371, 1061, 903 cm^ꢀ^{1 1}H NMR (BHSC-500,

;

4768

M. Zhao et al. / Bioorg. Med. Chem. 14 (2006) 4761–4774

DMSO-d₆): d = 10.05 (s, 1H), 8.05 (s, 1H), 7.37 (t,

J = 7.4 Hz, 1H), 7.25 (t, J = 7.4 Hz, 1H), 7.00 (d,

J = 7.6 Hz, 1H), 6.95 (d, J = 7.4 Hz, 1H), 4.92 (d,

J = 5.5 Hz, 1H), 4.77 (t, J = 5.5 Hz, 1H), 4.24 (d,

J = 5.6 Hz, 2H), 3.62 (s, 3H), 3.58 (s, 3H), 2.91 (d,

J = 5.2 Hz, 2H), 2.85 (d, J = 5.4 Hz, 2H), 1.49 (s, 9H).

Anal. Calcd for C₂₃H₂₉N₃O₇: C, 60.12; H, 6.36; N,

9.14. Found: C, 60.03; H, 6.49; N, 8.99.

J = 10.2 Hz, J = 3.7 Hz, 1H), 3.66 (s, 3H), 2.95 (d,

20

D

J = 10.1 Hz, 2H), 1.45 (s, 9H). ½aꢂ ꢀ101ꢁ (c 0.36,

CHCl₃/CH₃OH, 1:1, v/v). Anal. Calcd for

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

62.15; H, 6.68; N, 10.68.

4.4.16. N-[(3S)-N-Boc-1,2,3,4-tetrahydro-b-carboline-3-

carboxyl]-^L-(Z)lysine methylester (3p). Yield 90%; mp

134–136 ꢁC; ESI/MS: 593 [M+H]⁺. IR (KBr): 3442,

3007, 2940, 2848, 1730, 1605, 1455, 1391, 1370, 1066,

4.4.12.

N-(N-Boc-3S-1,2,3,4-tetrahydro-b-carboline-3-

1

carboxyl)-^L-histidine methylester (3i). Yield 93%; mp

162–164 ꢁC; ESI/MS 454 [M+H]⁺; IR (KBr): 3442,

3206, 3004, 2949, 2839, 1730, 1643, 1601, 1454, 1391,

897 cm^ꢀ1; H NMR (BHSC-500, DMSO-d₆): d = 9.95

(s, 1H), 8.03 (s, 1H), 7.96 (s, 1H), 7.28 (t, J = 7.6 Hz,

1H), 7.22 (t, J = 7.2 Hz, 1H), 7.19 (t, J = 7.6 Hz, 1H),

7.17 (d, J = 7.2 Hz, 2H), 7.15 (t, J = 7.2 Hz, 2H), 6.96

(d, J = 7.6 Hz, 1H), 6.85 (d, J = 7.6 Hz, 1H), 5.36 (s,

2H), 4.90 (d, J = 5.5 Hz, 1H), 4.41 (t, J = 4.4 Hz, 1H),

4.20 (dd, J = 10.0 Hz, J = 4.5 Hz, 1H), 4.18 (dd,

J = 10.0 Hz, J = 3.7 Hz, 1H), 3.64 (s, 3H), 2.98 (t,

J = 4.4 Hz, 2H), 2.93 (d, J = 10.0 Hz, 2H), 1.91 (m,

1

1368, 1062, 902 cm^ꢀ1; H NMR (BHSC-500, DMSO-

d₆): d = 12.98 (s, 1H), 9.96 (s, 1H), 8.05 (s, 1H), 7.47

(s, 1H), 6.85 (s, 1H), 7.36 (t, J = 7.4 Hz, 1H), 7.20 (t,

J = 7.7 Hz, 1H), 7.16 (d, J = 7.7Hz, 1H), 6.98 (t,

J = 7.4 Hz, 1H), 4.93 (t, J = 5.3 Hz, 1H), 4.83 (t,

J = 5.4 Hz, 1H), 4.26 (d, J = 5.2 Hz, 2H), 3.64 (s, 3H),

3.19 (d, J = 5.4 Hz, 2H), 2.92 (d, J = 5.2 Hz, 2H), 1.49

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

6.25; N, 14.98. Found: C, 61.52; H, 6.38; N, 14.77.

J = 4.4 Hz, 2H), 1.55 (m, J = 4.4 Hz, 2H), 1.46 (s, 9H),

20

D

1.29 (m, J = 4.4 Hz, 2H). ½aꢂ ꢀ22ꢁ (c 0.39, CHCl₃/

CH₃OH, 1:1, v/v), Anal. Calcd for C₃₂H₄₀N₄O₇: C,

64.85; H, 6.80; N, 9.45. Found: C, 64.98; H, 6.69; N,

9.62.

4.4.13.

N-(N-Boc-3S-1,2,3,4-tetrahydro-b-carboline-3-

carboxyl)-^L-tryptophan methylester (3m). Yield 93%;

mp 161–163 ꢁC; ESI/MS 517 [M+H]⁺; IR (KBr): 3442,

3204, 3000, 2948, 2839, 1729, 1642, 1604, 1448, 1391,

4.4.17. N-[(3S)-N-Boc-1,2,3,4-tetrahydro-b-carboline-3-

carboxyl]-^L-glutamine methylester (3q). Yield 90%; mp

122–124 ꢁC; ESI/MS: 459 [M+H]⁺; IR (KBr): 3445,

3200, 3001, 2940, 2835, 1733, 1640, 1602, 1452, 1391,

1

1372, 1062, 900 cm^ꢀ1; H NMR (BHSC-500, DMSO-

d₆): d = 9.87 (s, 1H), 9.86 (s, 1H), 8.09 (s, 1H), 7.32 (t,

J = 7.5 Hz, 1H), 7.30 (t, J = 7.4 Hz, 1H), 7.12 (d,

J = 7.8 Hz, 1H), 7.11 (t, J = 7.8 Hz, 1H), 7.10 (d,

J = 7.6 Hz, 1H), 7.09 (t, J = 7.8 Hz, 1H), 7.04 (d,

J = 7.6 Hz, 1H), 6.98 (d, J = 7.5 Hz, 1H), 6.83 (s, 1H),

4.94 (d, J = 5.4 Hz, 1H), 4.76 (t, J = 5.3 Hz, 1H), 4.29

(d, J = 5.2 Hz, 2H), 3.64 (s, 3H), 3.19 (d, J = 5.4 Hz,

2H), 2.95 (d, J = 6.4 Hz, 2H), 1.49 (s, 9H). Anal. Calcd

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

67.55; H, 6.34; N, 10.72.

1

1370, 1065, 900 cm^ꢀ1; H NMR (DMSO-d₆): d = 9.91

(s, 1H), 8.00 (s, 1H), 7.29 (t, J = 7.4 Hz, 1H), 7.20 (t,

J = 7.4 Hz, 1H), 7.00 (d, J = 7.4 Hz, 1H), 6.80 (d,

J = 7.4 Hz, 1H), 6.05 (s, 2H), 4.92 (d, J = 5.5 Hz, 1H),

4.41 (t, J = 5.5 Hz, 1H), 4.24 (d, J = 5.6 Hz, 2H), 3.67

(s, 3H), 2.94 (d, J = 5.4 Hz, 2H), 2.18 (t, J = 5.5 Hz,

20

D

2H), 2.14 (t, J = 5.5 Hz, 2H), 1.46 (s, 9H). ½aꢂ ꢀ56ꢁ

(c 0.38, CHCl₃/CH₃OH, 1:1, v/v). Anal. Calcd for

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

60.73; H, 6.49; N, 8.69.

4.4.14.

N-(N-Boc-3S-1,2,3,4-tetrahydro-b-carboline-3-

carboxyl)-^L-arginine methylester (3n). Yield 88%; mp

168–170 ꢁC; ESI/MS 487 [M+H]⁺; IR (KBr): 3443,

3207, 3001, 2948, 2842, 1731, 1645, 1602, 1453, 1390,

4.5. General procedure for preparation of 4a–q

At 0 ꢁC to the solution of 1.0 g (2.5 mmol) of 3a–q in

4 mL of methanol and 2 mL of chloroform, 0.45 g

(11.34 mmol) of NaOH was added. The reaction

mixture was stirred at 0 ꢁC for 70 min. TLC analysis

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

pearance of 3a–q. After evaporation, the residue was

dissolved in 30 mL of water and extracted with ethyl

acetate (3· 20 mL). The organic phase was washed

successively with 5% sodium bicarbonate, 5% citric acid,

and saturated sodium chloride. The solution was then

dried over anhydrous sodium sulfate. Compounds

4a–q was obtained after ﬁltration and evaporation under

reduced pressure.

1

1372, 1061, 904 cm^ꢀ1; H NMR (BHSC-500, DMSO-

d₆): d = 10.22 (s, 1H), 8.45 (s, 2H), 8.27 (s, 1H), 8.22

(s, 1H), 8.01 (s, 1H), 7.29 (t, J = 7.6 Hz, 1H), 7.18 (t,

J = 7.7 Hz, 1H), 7.04 (d, J = 7.7 Hz, 1H), 6.96 (d,

J = 7.6 Hz, 1H), 4.90 (d, J = 5.3 Hz, 1H), 4.42 (t,

J = 4.2 Hz, 1H), 4.25 (d, J = 5.0 Hz, 2H), 3.65 (s, 3H),

2.94 (d, J = 4.1 Hz, 2H), 2.68 (t, J = 5.4 Hz, 2H), 1.92

(m, J = 5.5 Hz, 2H), 1.58 (m, J = 5.5 Hz, 2H), 1.57 (s,

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

N, 17.27. Found: C, 59.38; H, 7.19; N, 17.31.

4.4.15. N-[(3S)-N-Boc-1,2,3,4-tetrahydro-b-carboline-3-

carboxyl]-^L-glycine methylester (3o). Yield 97%; mp

133–135 ꢁC; ESI/MS 388 [M+H]⁺. IR (KBr): 3448,

3010, 2945, 2843, 1732, 1600, 1453, 1390, 1371, 1062,

4.5.1. N-(N-Boc-3S-1,2,3,4-tetrahydro-b-carboline-3-car-

boxyl)-^L-alanine (4a). Yield 79%; mp 169–171 ꢁC; ESI/

MS 388 [M+H]⁺. IR (KBr): 3439, 3234, 3215, 3000,

2952, 2847, 1732, 1645, 1602, 1453, 1390, 1373, 1061,

1

899 cm^ꢀ1; H NMR (BHSC-500, DMSO-d₆): d = 9.93

(s, 1H), 8.02 (s, 1H), 7.30 (t, J = 7.5 Hz, 1H), 7.20 (t,

J = 7.6 Hz, 1H), 6.95 (d, J = 7.6 Hz, 1H), 6.83 (d,

J = 7.6 Hz, 1H), 4.89 (d, J = 5.4 Hz, 1H), 4.22 (dd,

J = 10.2 Hz, J = 4.5 Hz, 1H), 4.18 (s, 2H), 4.19 (dd,

1

904 cm^ꢀ1; H NMR (BHSC-500, DMSO-d₆): d = 11.02

(s, 1H), 9.95 (s, 1H), 7.98 (s, 1H), 7.29 (t, J = 7.6 Hz,

1H), 7.17 (t, J = 7.9 Hz, 1H), 7.03 (d, J = 7.9 Hz, 1H),

M. Zhao et al. / Bioorg. Med. Chem. 14 (2006) 4761–4774

4769

20

D

6.94 (d, J = 7.6 Hz, 1H), 4.93 (d, J = 5.4 Hz, 1H), 4.66

(m, J = 5.4 Hz, 1H), 4.27 (d, J = 6.3 Hz, 2H), 2.97 (d,

J = 9.5 Hz, 2H), 1.48 (d, J = 5.4 Hz, 3H), 1.45 (s, 9H).

1.48 (s, 9H), 1.26 (d, J = 5.2 Hz, 3H). ½aꢂ ꢀ49ꢁ (c

0.39, CHCl₃/CH₃OH, 1:1, v/v). Anal. Calcd for

C₂₁H₂₇N₃O₆: C, 60.42; H, 6.52; N, 10.07. Found: C,

60.28; H, 6.39; N, 10.16.

20

½aꢂ ꢀ46ꢁ (c 0.39, CHCl₃/CH₃OH, 1:1, v/v). Anal. Calcd

D

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

62.18; H, 6.39; N, 10.71.

4.5.6. N-(N-Boc-3S-1,2,3,4-tetrahydro-b-carboline-3-car-

boxyl)-L-tyrosine (4f). Yield 67%; mp 147–149 ꢁC; ESI/

MS: 480 [M+H]⁺; IR (KBr): 3441, 3236, 3217, 3004,

2955, 2846, 1731, 1647, 1600, 1450, 1392, 1371, 1058,

4.5.2. N-[(3S)-N-Boc-1,2,3,4-tetrahydro-b-carboline-3-

carboxyl]-^L-phenylalanine (4b). Yield 94%; mp 129–

131 ꢁC; ESI/MS: 464 [M+H]⁺. IR (KBr): 3446, 3205,

3006, 2948, 2847, 1731, 1645, 1603, 1451, 1392, 1370,

899 cm^ꢀ1; H NMR (BHSC-500, DMSO-d₆): d = 10.97

1

(s, 1H), 9.60 (s, 1H), 8.15 (s, 1H), 7.32 (t, J = 7.2 Hz,

1H),7.14 (t, J = 7.4 Hz, 1H), 7.01 (d, J = 7.2 Hz, 1H),

6.96 (d, J = 7.2 Hz, 1H), 6.94 (d, J = 7.5 Hz, 2H), 6.88

(d, J = 7.2 Hz, 2H), 5.03 (s, 1H), 4.94 (d, J = 5.2 Hz,

1H), 4.84 (d, J = 5.3 Hz, 1H), 4.27 (d, J = 5.2 Hz, 2H),

1069, 904 cm^ꢀ^{1 1}H NMR (DMSO-d₆): d = 10.94 (s,

;

1H), 9.93 (s, 1H), 7.97 (s, 1H), 7.30 (t, J = 7.3 Hz,

1H), 7.26 (t, J = 7.4 Hz, 2H), 7.17 (t, J = 7.6 Hz, 1H),

7.15 (d, J = 7.4 Hz, 2H), 7.10 (t, J = 7.4 Hz, 1H), 7.02

(t, J = 7.4 Hz, 1H), 6.97 (d, J = 7.4 Hz, 1H), 4.93 (d,

J = 5.2 Hz, 1H), 4.78 (t, J = 5.2 Hz, 1H), 4.27 (d,

3.07 (m, J = 3.4 Hz, 2H), 2.92 (d, J = 4.5 Hz, 2H), 1.48

20

D

(s, 9H). ½aꢂ ꢀ61ꢁ (c 0.36, CHCl₃/CH₃OH, 1:1, v/v).

J = 5.2 Hz, 2H), 3.07 (d, J = 4.5 Hz, 2H), 2.98 (d,

Anal. Calcd for C₂₆H₂₉N₃O₆: C, 65.12; H, 6.10; N,

8.76. Found: C, 65.28; H, 6.17; N, 8.59.

20

D

J = 5.2 Hz, 2H), 1.49 (s, 9H). ½aꢂ ꢀ30ꢁ (c 0.36,

20

D

CHCl₃/CH₃OH, 1:1, v/v), ½aꢂ ꢀ66ꢁ (c 0.36, CHCl₃/

CH₃OH, 1:1, v/v), Anal. Calcd for C₂₆H₂₉N₃O₅: C,

67.37; H, 6.31; N, 9.07. Found: C, 67.22; H, 6.39; N,

10.21.

4.5.7. N-(N-Boc-3S-1,2,3,4-tetrahydro-b-carboline-3-car-

boxyl)-^L-proline (4g). Yield 83%; mp 136–138 ꢁC; ESI/

MS: 414 [M+H]⁺; IR (KBr): 3440, 3236, 3217, 3004,

2956, 2841, 1735, 1641, 1605, 1450, 1392, 1370, 1044,

4.5.3. N-(N-Boc-3S-1,2,3,4-tetrahydro-b-carboline-3-car-

boxyl)-^L-valine (4c). Yield 71%; mp 148–150 ꢁC; ESI/

MS: 416 [M+H]⁺. IR (KBr): 3441, 3236, 3212, 3002,

2951, 2845, 1731, 1643, 1600, 1450, 1392, 1374, 1060,

897 cm^ꢀ^{1 1}H NMR (DMSO-d₆): d = 10.95 (s, 1H),

;

9.70 (s, 1H), 7.32 (t, J = 7.2 Hz, 1H), 7.14 (t,

J = 7.5 Hz, 1H), 7.08 (d, J = 7.5 Hz, 1H), 6.96 (d,

J = 7.2 Hz, 1H), 4.92 (d, J = 5.1 Hz, 1H), 4.32 (t,

J = 5.3 Hz, 1H), 4.22 (m, J = 5.3 Hz, 2H), 3.47 (t,

J = 5.2 Hz, 2H), 2.95 (d, J = 5.2 Hz, 2H), 2.17 (dd,

900 cm^ꢀ^{1 1}H NMR (DMSO-d₆): d = 10.94 (s, 1H),

;

9.23 (s, 1H), 7.96 (s, 1H), 7.29 (t, J = 7.5 Hz, 1H), 7.18

(t, J = 7.6 Hz, 1H), 7.06 (d, J = 7.4 Hz, 1H), 6.97 (d,

J = 7.5 Hz, 1H), 4.95 (d, J = 5.2 Hz, 1H), 4.48 (d,

J = 5.2 Hz, 1H), 4.27 (d, J = 5.1 Hz, 2H), 2.92 (d,

J = 5.2 Hz, J = 3.4 Hz, 2H), 1.94 (t, J = 4.5 Hz, 2H),

20

D

1.47 (s, 9H). ½aꢂ ꢀ49ꢁ (c 0.35, CHCl₃/CH₃OH, 1:1,

v/v). Anal. Calcd for C₂₂H₂₇N₃O₅: C, 63.91; H, 6.58;

N, 10.16. Found: C, 64.14; H, 6.47; N, 10.32.

J = 5.5 Hz, 2H), 2.78 (m, J = 4.5 Hz, 1H), 1.49 (s, 9H),

20

D

1.25 (d, J = 5.6 Hz, 6H). ½aꢂ ꢀ72ꢁ (c 0.38, CHCl₃/

CH₃OH, 1:1, v/v). Anal. Calcd for C₂₂H₂₉N₃O₅: C,

63.60; H, 7.04; N, 10.11. Found: C, 63.42; H, 7.19; N,

10.21.

4.5.8. N-(N-Boc-3S-1,2,3,4-tetrahydro-b-carboline-3-car-

boxyl)-^L-cysteine (4h). Yield 78%; mp 150–152 ꢁC; ESI/

MS: 420 [M+H]⁺; IR (KBr): 3436, 3232, 3217, 3004,

2950, 2844, 1731, 1647, 1600, 1455, 1392, 1375, 1058,

4.5.4. N-(N-Boc-3S-1,2,3,4-tetrahydro-b-carboline-3-car-

boxyl)-^L-serine (4d). Yield 84%; mp 136–138 ꢁC; ESI/

MS: 390 [M+H]⁺; IR (KBr): 3437, 3236, 3213, 3005,

2950, 2846, 1730, 1641, 1600, 1451, 1392, 1370, 1058,

902 cm^ꢀ^{1 1}H NMR (DMSO-d₆): d = 10.89 (s, 1H),

;

9.95 (s, 1H), 7.99 (s, 1H), 7.27 (t, J = 7.3 Hz, 1H), 7.15

(t, J = 7.6 Hz, 1H), 7.05 (d, J = 7.6 Hz, 1H), 6.95 (t,

J = 7.2 Hz, 1H), 4.91 (t, J = 5.0 Hz, 1H), 4.75 (t,

J = 5.2 Hz, 1H), 4.24 (m, J = 5.0 Hz, 2H), 3.06 (d,

897 cm^ꢀ^{1 1}H NMR (DMSO-d₆): d = 10.92 (s, 1H),

;

9.93 (s, 1H), 7.97 (s, 1H), 7.30 (t, J = 7.3 Hz, 1H), 7.19

(t, J = 7.6 Hz, 1H), 7.06 (d, J = 7.6 Hz, 1H), 6.98 (d,

J = 7.3 Hz, 1H), 4.92 (d, J = 5.5 Hz, 1H), 4.46 (t,

J = 5.2 Hz, 1H), 4.27 (d, J = 5.0 Hz, 2H), 4.06 (d,

J = 5.0 Hz, 2H), 2.89 (d, J = 4.5 Hz, 2H), 1.55 (s, 1H),

20

D

1.52 (s, 9H). ½aꢂ ꢀ45ꢁ (c 0.38, CHCl₃/CH₃OH, 1:1,

v/v). Anal. Calcd for C₂₀H₂₅N₃O₅S: C, 57.26; H, 6.01;

N, 10.02. Found: C, 57.19; H, 6.13; N, 10.12.

J = 5.1 Hz, 2H), 2.93 (d, J = 5.4 Hz, 2H), 2.07 (s, 1H),

20

D

1.51 (s, 9H). ½aꢂ ꢀ70ꢁ (c 0.35, CHCl₃/CH₃OH, 1:1,

4.5.9. N-(N-Boc-3S-1,2,3,4-tetrahydro-b-carboline-3-car-

boxyl)-^L-methionine (4i). Yield 72%; mp 148–150 ꢁC;

ESI/MS: 448 [M+H]⁺; IR (KBr): 3436, 3232, 3213,

3007, 2953, 2849, 1735, 1648, 1600, 1450, 1393, 1375,

v/v). Anal. Calcd for C₂₀H₂₅N₃O₆: C, 59.54; H, 6.25;

N, 10.42. Found: C, 59.42; H, 6.19; N, 10.29.

4.5.5. N-(N-Boc-3S-1,2,3,4-tetrahydro-b-carboline-3-car-

boxyl)-^L-threonine (4e). Yield 78%; mp 143–145 ꢁC; ESI/

MS: 405 [M+H]⁺; IR (KBr): 3444, 3235, 3217, 3005,

2950, 2843, 1730, 1641, 1600, 1450, 1389, 1370, 1055,

1055, 897 cm^ꢀ^{1 1}H NMR (BHSC-500, DMSO-d₆):

;

d = 11.01 (s, 1H), 9.97 (s, 1H), 8.01 (s, 1H), 7.33 (t,

J = 7.0 Hz, 1H), 7.18 (t, J = 7.5 Hz, 1H), 7.05 (d,

J = 7.5 Hz, 1H), 6.95 (t, J = 7.0 Hz, 1H), 4.44 (t,

J = 5.5 Hz, 1H), 4.25 (m, J = 5.2 Hz, 1H), 4.23 (m,

J = 5.0 Hz, 2H), 2.89 (d, J = 4.4 Hz, 2H), 2.46 (t,

896 cm^ꢀ^{1 1}H NMR (DMSO-d₆): d = 10.97 (s, 1H),

;

8.92 (s, 1H), 7.95 (s, 1H), 7.29 (t, J = 7.2 Hz, 1H), 7.15

(t, J = 7.4 Hz, 1H), 7.05 (d, J = 7.4 Hz, 1H), 6.98 (d,

J = 7.2 Hz, 1H), 4.89 (d, J = 5.3 Hz, 1H), 4.46 (d,

J = 5.4 Hz, 1H), 4.37 (m, J = 5.1 Hz, 1H), 4.27 (d,

J = 4.8 Hz, 2H), 2.95 (d, J = 5.5 Hz, 2H), 2.16 (s, 1H),

J = 5.4 Hz, 2H), 2.16 (m, J = 5.6 Hz, 2H), 2.09 (s, 3H),

20

D

1.50 (s, 9H). ½aꢂ ꢀ38ꢁ (c 0.39, CHCl₃/CH₃OH, 1:1,

v/v). Anal. Calcd for C₂₂H₂₉N₃O₅S: C, 59.04; H, 6.53;

N, 9.39. Found: C, 59.20; H, 6.44; N, 9.22.

4770

M. Zhao et al. / Bioorg. Med. Chem. 14 (2006) 4761–4774

4.5.10. N-(N-Boc-3S-1,2,3,4-tetrahydro-b-carboline-3-car-

boxyl)-^L-glutamic acid (4j). Yield 76%; mp 160–162 ꢁC;

ESI/MS: 446 [M+H]⁺; IR (KBr): 3444, 3231, 3212, 3008,

2954, 2843, 1730, 1642, 1600, 1450, 1391, 1371, 1058,

d = 10.94 (s, 1H), 8.55 (s, 2H), 8.31 (s, 2H), 8.23 (s,

1H), 7.98 (s, 1H), 7.29 (t, J = 7.5 Hz, 1H), 7.11 (t,

J = 7.6 Hz, 1H), 7.02 (d, J = 7.6 Hz, 1H), 6.98 (d,

J = 7.5 Hz, 1H), 4.87 (t, J = 5.4 Hz, 1H), 4.41 (t,

J = 4.3 Hz, 1H), 4.25 (d, J = 4.8 Hz, 2H), 2.89 (d,

J = 4.9 Hz, 2H), 2.66 (t, J = 5.2 Hz, 2H), 1.81 (m,

1

900 cm^ꢀ1; H NMR (BHSC-500, DMSO-d₆): d = 11.02

(s, 2H), 9.87 (s, 1H), 8.11 (s, 1H), 7.28 (t, J = 7.2 Hz, 1H),

7.14 (t, J = 7.2 Hz, 1H), 7.06 (d, J = 7.4 Hz, 1H), 6.97 (t,

J = 7.2 Hz, 1H), 4.90 (t, J = 5.1 Hz, 1H), 4.42 (t,

J = 5.4 Hz, 1H), 4.21 (d, J = 5.1 Hz, 2H), 2.87 (d,

J = 5.1 Hz, 2H), 1.59 (m, J = 5.3 Hz, 2H), 1.56 (s, 9H).

20

D

½aꢂ ꢀ66ꢁ (c 0.37, CHCl₃/CH₃OH, 1:1, v/v). Anal. Calcd

for C₂₃H₃₂N₆O₅: C, 58.46; H, 6.83; N, 17.78. Found: C,

58.38; H, 6.96; N, 17.57.

J = 5.3 Hz, 2H), 2.22 (t, J = 5.2 Hz, 2H), 2.08 (t,

20

D

J = 5.2 Hz, 2H), 1.47 (s, 9H). ½aꢂ ꢀ51ꢁ (c 0.33, CHCl₃/

CH₃OH, 1:1, v/v). Anal. Calcd for C₂₂H₂₇N₃O₇: C,

59.32; H, 6.11; N, 9.43. Found: C, 59.24; H, 6.01; N, 9.29.

4.5.15. N-[(3S)-N-Boc-1,2,3,4-tetrahydro-b-carboline-3-

carboxyl]-^L-glycine (4o). Yield 95%; mp 148–150 ꢁC;

ESI/MS: 374 [M+H]⁺. IR (KBr): 3444, 3230, 3008,

2942, 2841, 1730, 1602, 1455, 1391, 1370, 1064,

4.5.11. N-(N-Boc-3S-1,2,3,4-tetrahydro-b-carboline-3-car-

boxyl)-^L-aspartic acid (4k). Yield 72%; mp 141–143 ꢁC;

ESI/MS: 432 [M+H]⁺; IR (KBr): 3442, 3236, 3217, 3005,

2950, 2843, 1728, 1642, 1600, 1450, 1392, 1370, 1058,

1

898 cm^ꢀ1; H NMR (BHSC-500, DMSO-d₆): d = 11.03

(s, 1H), 9.98 (s, 1H), 8.01 (s, 1H), 7.29 (t, J = 7.5 Hz,

1H), 7.18 (t, J = 7.6 Hz, 1H), 6.97 (d, J = 7.6 Hz, 1H),

6.85 (d, J = 7.6 Hz, 1H), 4.90 (d, J = 5.4 Hz, 1H), 4.24

(dd, J = 10.2 Hz, J = 4.5 Hz, 1H), 4.17 (s, 2H), 4.16

1

896 cm^ꢀ1; H NMR (DMSO-d₆): d = 10.99 (s, 2H), 9.94

(s, 1H), 7.98 (s, 1H), 7.26 (t, J = 7.1 Hz, 1H), 7.11 (t,

J = 7.1 Hz, 1H), 7.05 (d, J = 7.2 Hz, 1H), 6.94 (d,

J = 7.2 Hz, 1H), 4.87 (t, J = 5.2 Hz, 1H), 4.76 (t,

J = 5.3 Hz, 1H), 4.20 (m, J = 4.8 Hz, 2H), 2.89 (d,

(dd, J = 10.2 Hz, J = 3.7 Hz, 1H), 2.93 (d, J = 10.0 Hz,

20

D

2H), 1.46 (s, 9H). ½aꢂ (c 0.38, CHCl₃/CH₃OH, 1:1, v/

v). Anal. Calcd for C₁₉H₂₃N₃O₅: C, 61.11; H, 6.21; N,

11.25. Found: C, 61.30; H, 6.40; N, 11.09.

J = 5.2 Hz, 2H), 2.66 (d, J = 5.2 Hz, 2H), 1.47 (s, 9H).

20

D

½aꢂ ꢀ47ꢁ (c 0.35, CHCl₃/CH₃OH, 1:1, v/v). Anal. Calcd

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

58.55; H, 6.02; N, 9.91.

4.5.16. N-[(3S)-N-Boc-1,2,3,4-tetrahydro-b-carboline-3-

carboxyl]-^L-(Z)lysine (4p). Yield 92%; mp 155–157 ꢁC;

ESI/MS: 579 [M+H]⁺. IR (KBr): 3438, 3234, 3215,

3009, 2944, 2845, 1733, 1602, 1453, 1390, 1372, 1064,

4.5.12. N-(N-Boc-3S-1,2,3,4-tetrahydro-b-carboline-3-car-

boxyl)-^L-histidine (4l). Yield 76%; mp 156–158 ꢁC; ESI/

MS: 454 [M+H]⁺. IR (KBr): 3445, 3231, 3212, 3007,

2947, 2842, 1730, 1642, 1600, 1450, 1388, 1370, 1059,

899 cm^ꢀ1; ¹H NMR (DMSO-d₆): d = 11.85 (s, 1H), 11.62

(s, 1H), 9.94 (s, 1H), 7.89 (s, 1H), 7.45 (s, 1H), 7.29 (t,

J = 7.2 Hz, 1H), 7.15 (t, J = 7.5 Hz, 1H), 7.06 (d,

J = 7.5Hz, 1H), 6.98 (d, J = 7.2 Hz, 1H), 6.82 (s, 1H),

4.89 (t, J = 5.0 Hz,1H), 4.82 (t, J = 5.1 Hz, 1H), 4.20 (m,

899 cm^ꢀ^{1 1}H NMR (DMSO-d₆): d = 10.88 (s, 1H),

;

9.97 (s, 1H), 8.01 (s, 1H), 7.98 (s, 1H), 7.29 (t,

J = 7.5 Hz, 1H), 7.21 (t, J = 7.3 Hz, 1H), 7.18 (t,

J = 7.5 Hz, 1H), 7.17 (d, J = 7.3 Hz, 2H), 7.16 (t,

J = 7.3 Hz, 2H), 6.98 (d, J = 7.5 Hz, 1H), 6.88 (d,

J = 7.5 Hz, 1H), 5.33 (s, 2H), 4.92 (d, J = 5.6 Hz, 1H),

4.45 (t, J = 4.6 Hz, 1H), 4.23 (dd, J = 10.0 Hz,

J = 4.5 Hz, 1H), 4.19 (dd, J = 10.0 Hz, J = 3.7 Hz,

1H), 2.94 (t, J = 4.6 Hz, 2H), 2.95 (d, J = 10.0 Hz,

J = 5.1 Hz, 2H), 3.02 (d, J = 5.0 Hz, 2H), 2.92 (d,

20

D

J = 5.2 Hz, 2H), 1.50 (s, 9H). ½aꢂ ꢀ50ꢁ (c 0.36, CHCl₃/

2H), 1.84 (m, J = 4.6 Hz, 2H), 1.54 (m, J = 4.6 Hz,

20

D

CH₃OH, 1:1, v/v). Anal. Calcd for C₂₃H₂₇N₅O₅: C,

60.92;H,6.00;N,15.44. Found:C, 61.05;H,6.09;N,15.26.

2H), 1.46 (s, 9H), 1.29 (m, J = 4.6 Hz, 2H). ½aꢂ ꢀ34ꢁ

(c 0.36, CHCl₃/CH₃OH, 1:1, v/v). Anal. Calcd for

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

64.18; H, 6.44; N, 9.83.

4.5.13. N-(N-Boc-3S-1,2,3,4-tetrahydro-b-carboline-3-car-

boxyl)-^L-tryptophan (4m). Yield 70%; mp 158–160 ꢁC; ESI/

MS: 503 [M+H]⁺; IR (KBr): 3445, 3238, 3217, 3008, 2951,

4.5.17. N-[(3S)-N-Boc-1,2,3,4-tetrahydro-b-carboline-3-

carboxyl]-^L-glutamine (4q). Yield 90%; mp 129–131 ꢁC;

ESI/MS: 445 [M+H]⁺; IR (KBr): 3448, 3230, 3215,

3005, 2942, 2833, 1738, 1642, 1604, 1450, 1390, 1372,

2842, 1728, 1641, 1600, 1450, 1393, 1370, 1058, 897 cm^ꢀ1

;

¹H NMR (DMSO-d₆): d = 10.98 (s, 1H), 9.87 (s, 1H),

9.84 (s, 1H), 8.01 (s, 1H), 7.30 (t, J = 7.1 Hz, 1H), 7.16 (t,

J = 7.1 Hz, 1H), 7.14 (t, J = 7.4 Hz, 1H), 7.12 (t,

J = 7.2 Hz, 1H), 7.06 (d, J = 7.4 Hz, 1H), 7.05 (d,

J = 7.2 Hz, 1H), 6.99 (d, J = 7.1 Hz, 1H), 6.96 (d,

J = 7.1 Hz, 1H), 6.82 (s, 1H), 4.89 (t, J = 5.2 Hz, 1H),

4.84 (t, J = 5.1 Hz, 1H), 4.25 (m, J = 5.0 Hz, 2H), 2.91 (d,

1063, 901 cm^ꢀ^{1 1}H NMR (DMSO-d₆): d = 10.96 (s,

;

1H), 9.88 (s, 1H), 8.02 (s, 1H), 7.28 (t, J = 7.5 Hz,

1H), 7.21 (t, J = 7.5 Hz, 1H), 7.01 (d, J = 7.5 Hz, 1H),

6.82 (d, J = 7.5 Hz, 1H), 6.02 (s, 2H), 4.90 (d,

J = 5.6 Hz, 1H), 4.45 (t, J = 5.4 Hz, 1H), 4.25 (d,

J = 5.5 Hz, 2H), 2.92 (d, J = 5.5 Hz, 2H), 2.17 (t,

J = 5.0 Hz, 2H), 2.88 (d, J = 5.1 Hz, 2H), 1.53 (s, 9H).

20

D

½aꢂ ꢀ47ꢁ (c 0.38, CHCl₃/CH₃OH, 1:1, v/v). Anal. Calcd

J = 5.5 Hz, 2H), 2.07 (t, J = 5.5Hz, 2H), 1.42 (s, 9H).

20

D

for C₂₈H₃₀N₄O₅: C, 66.92; H, 6.02; N, 11.15. Found: C,

66.79; H, 5.94; N, 11.27.

½aꢂ ꢀ54ꢁ (c 0.39, CHCl₃/CH₃OH, 1:1, v/v). Anal. Calcd

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

59.63; H, 6.20; N, 12.79.

4.5.14.

N-(N-Boc-3S-1,2,3,4-tetrahydro-b-carboline-3-

carboxyl)-^L-arginine (4n). Yield 81%; mp 144–146 ꢁC;

ESI/MS: 473 [M+H]⁺; IR (KBr): 3441, 3230, 3212,

3007, 2950, 2844, 1730, 1640, 1600, 1450, 1392, 1375,

4.6. General procedure for preparation of 5a–o

At 0 ꢁC, solution of 0.7 g (1.85 mmol) of 4a–q in 10 mL

of hydrogen chloride/ethyl acetate (4 mol/L) was stirred

1057, 896 cm^ꢀ1

;

¹H NMR (BHSC-500, DMSO-d₆):

M. Zhao et al. / Bioorg. Med. Chem. 14 (2006) 4761–4774

4771

for 1 h and TLC analysis (chloroform/methanol, 5:1)

indicated complete disappearance of 4a–q. Upon evapo-

ration, the residue was dissolved in ethyl acetate (3·

10 mL) and evaporated under reduced pressure to re-

move hydrogen chloride. The residue was titrated with

20 mL of ethyl acetate and re-crystallized with buta-

nol/ether to provide 5a–q.

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

59.56; H, 5.81; N, 13.79.

4.6.5.N-(3S-1,2,3,4-Tetrahydro-b-carboline-3-carboxyl)-^L-

threonine (5e). Yield 91%; mp 180–182 ꢁC; ESI/MS: 318

[M+H]⁺; IR (KBr): 3444, 3235, 3217, 3005, 2950, 2843,

1730, 1641, 1600, 1450, 1055, 896 cm^ꢀ1

;

¹H

NMR(300 MHz, DMSO-d₆): d = 11.42 (s, 1H), 10.23 (s,

1H), 9.89 (s, 1H), 8.05 (s, 1H), 7.30 (t, J = 7.5 Hz, 1H),

7.18 (t, J = 8.1 Hz, 1H), 7.10 (d, J = 8.4 Hz, 1H), 7.00 (d,

J = 7.8 Hz, 1H), 4.52 (d, J = 5.3 Hz, 1H), 4.37 (m,

J = 5.4 Hz, 1H), 3.96 (d, J = 6.4 Hz, 1H), 3.86 (d,

4.6.1. N-(3S-1,2,3,4-Tetrahydro-b-carboline-3-carboxyl)-

L-alanine (5a). Yield 94%; mp 167–169 ꢁC; ESI/MS: 288

[M+H]⁺; IR (KBr): 3439, 3234, 3215, 3000, 2952, 2847,

1732, 1645, 1602, 1453, 1061, 904 cm^ꢀ^{1 1}H NMR

;

(300 MHz, DMSO-d₆): d = 11.86 (s, 1H), 10.00 (s, 1H),

9.96 (s,1H), 8.02 (s,1H), 7.31 (t, J = 6.6 Hz, 1H), 7.18

(t, J = 7.8 Hz, 1H), 6.87 (d, J = 7.9 Hz, 1H), 6.81 (d,

J = 7.6 Hz, 1H), 4.65 (m, J = 5.4 Hz, 1H), 3.95 (m,

J = 5.4 Hz, 2H), 2.80 (d, J = 5.4 Hz, 2H), 2.17 (s, 1H),

20

D

1.26 (d, J = 5.2 Hz, 3H). ½aꢂ ꢀ28ꢁ (c 0.36, CHCl₃/

CH₃OH, 1:1, v/v). Anal. Calcd for C₁₆H₁₉N₃O₄: C,

60.56;H,6.03;N,13.24.Found:C, 60.68;H,5.88;N,13.48.

J = 5.4 Hz, 1H), 3.91 (d, J = 5.3 Hz, 2H), 2.81 (d,

20

D

J = 5.6 Hz, 2H), 1.48 (d, J = 5.6 Hz, 3H). ½aꢂ ꢀ80ꢁ (c

4.6.6. N-(3S-1,2,3,4-Tetrahydro-b-carboline-3-carboxyl)-

L-tyrosine (5f). Yield 84%; mp 171–173 ꢁC; ESI/MS: 380

[M+H]⁺; IR (KBr): 3441, 3236, 3217, 3004, 2955, 2846,

0.39, CHCl₃/CH₃OH, 1:1, v/v). Anal. Calcd for

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

62.55; H, 5.79; N, 14.55.

1731, 1647, 1600, 1450, 1058, 899 cm^ꢀ1

;

¹H

NMR(300 MHz, DMSO-d₆): d = 11.44 (s, 1H), 10.19

(s, 1H), 9.96 (s, 1H), 8.05 (s, 1H), 7.29 (t, J = 7.5 Hz,

1H), 7.18 (t, J = 8.1 Hz, 1H), 7.01 (d, J = 8.1 Hz, 2H),

7.00 (d, J = 7.2 Hz, 2H), 6.97 (d, J = 7.8 Hz, 1H), 6.70

(d, J = 8.4 Hz, 2H), 5.02 (s, 1H), 4.85 (t, J = 5.3 Hz,

4.6.2. N-(3S-1,2,3,4-Tetrahydro-b-carboline-3-carboxyl)-

L-phenylalanine (5b). Yield 95%; mp 177–179 ꢁC; ESI/

MS: 364 [M+H]⁺; IR (KBr): 3435, 3231, 3213, 3003,

2950, 2844, 1730, 1642, 1600, 1450, 1060, 901 cm^ꢀ1

;

¹H NMR (300 MHz, DMSO-d₆): d = 11.33 (s, 1H),

10.05 (s, 1H), 9.93 (s, 1H), 8.01 (s, 1H), 7.33 (t,

J = 6.8 Hz, 1H), 7.30 (t, J = 6.4 Hz, 2H), 7.20 (t,

J = 7.6 Hz, 1H), 7.17 (d, J = 7.8 Hz, 2H), 7.10 (t,

J = 6.8 Hz, 1H), 7.02 (d, J = 7.6 Hz, 1H), 6.89 (d,

J = 7.4 Hz, 1H), 4.86 (t, J = 5.4 Hz, 1H), 4.00 (m,

1H), 3.97 (t, J = 6.2 Hz, 1H), 3.87 (d, J = 5.6 Hz, 2H),

20

D

3.06 (d, J = 5.6 Hz, 2H), 2.78 (d, J = 5.9 Hz, 2H). ½aꢂ

ꢀ88ꢁ (c 0.36, CHCl₃/CH₃OH, 1:1, v/v); Anal. Calcd

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

C, 66.48; H, 5.72; N, 11.17.

J = 5.4 Hz, 1H), 3.88 (d, J = 6.2 Hz, 2H), 3.07 (d,

4.6.7.N-(3S-1,2,3,4-Tetrahydro-b-carboline-3-carboxyl)-^L-

proline (5g). Yield 93%; mp 166–168 ꢁC; ESI/MS: 314

[M+H]⁺; IR (KBr): 3440, 3236, 3217, 3004, 2956, 2841,

20

D

J = 6.5 Hz, 2H), 2.81 (d, J = 6.2 Hz, 2H). ½aꢂ ꢀ30ꢁ (c

0.41, CHCl₃/CH₃OH, 1:1, v/v). Anal. Calcd for

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

69.56; H, 5.75; N, 11.34.

1735, 1641, 1605, 1450, 1044, 897 cm^ꢀ^{1 1}H NMR

;

(300 MHz, DMSO-d₆): d = 11.13 (s, 1H), 10.03 (s, 1H),

9.96 (s, 1H), 7.31 (t, J = 8.4 Hz, 1H), 7.18 (t, J = 7.5Hz,

1H), 7.02 (d, J = 7.8 Hz, 1H), 6.95 (d, J = 7.5 Hz, 1H),

4.32 (t, J = 5.1 Hz, 1H), 3.96 (t, J = 6.3 Hz, 1H), 3.86 (d,

J = 5.4 Hz, 2H), 3.47 (t, J = 6.3 Hz, 2H), 2.81 (t,

4.6.3. N-(3S-1,2,3,4-Tetrahydro-b-carboline-3-carboxyl)-

L-valine (5c). Yield 95%; mp 169–171 ꢁC; ESI/MS: 316

[M+H]⁺; IR (KBr): 3441, 3236, 3212, 3002, 2951,

2845, 1731, 1643, 1600, 1450, 1060, 900 cm^ꢀ1

;

¹H

J = 6.4 Hz, 2H), 2.26 (t, J = 6.5 Hz, 2H), 1.98 (m,

20

D

NMR (300 MHz, DMSO-d₆): d = 11.68 (s, 1H), 10.19

(s, 1H), 9.88 (s, 1H), 8.03 (s,1H), 7.32 (t, J = 7.5 Hz,

1H), 7.17 (t, J = 7.8 Hz, 1H), 7.05 (d, J = 7.4Hz, 1H),

6.98 (d, J = 7.4 Hz, 1H), 4.48 (t, J = 5.2 Hz, 1H), 3.94

(t, J = 5.2 Hz, 1H), 3.90 (d, J = 5.2 Hz, 2H), 2.82 (d,

J = 6.6 Hz, 2H). ½aꢂ ꢀ36ꢁ (c 0.36, CHCl₃/CH₃OH, 1:1,

v/v). Anal. Calcd for C₁₇H₁₉N₃O₃: C, 65.16; H, 6.11; N,

13.41. Found: C, 65.30; H, 6.20; N, 13.29.

4.6.8. N-(3S-1,2,3,4-Tetrahydro-b-carboline-3-carboxyl)-

L-cysteine (5h). Yield 98%; mp 200–201 ꢁC, ESI/MS: 320

[M+H]⁺; IR (KBr): 3436, 3232, 3217, 3004, 2950, 2844,

J = 5.4 Hz, 2H), 2.80 (m, J = 6.9 Hz, 1H), 1.06 (d,

20

D

J = 6.9 Hz, 6H). ½aꢂ ꢀ70ꢁ (c 0.38, CHCl₃/CH₃OH,

1:1, v/v). Anal. Calcd for C₁₇H₂₁N₃O₃: C, 64.74; H,

6.71; N, 13.32. Found: C, 64.89; H, 6.85; N, 13.44.

1731, 1647, 1600, 1455, 1058, 902 cm^ꢀ^{1 1}H NMR

;

(300 MHz, DMSO-d₆) d = 11.22 (s, 1H), 10.22 (s, 1H),

9.96 (s, 1H), 8.03 (s, 1H), 7.31 (t, J = 7.6 Hz, 1H), 7.17

(t, J = 7.8 Hz, 1H), 7.02 (d, J = 7.8 Hz, 1H), 6.95 (d,

J = 7.2 Hz, 1H), 4.79 (t, J = 5.6 Hz, 1H), 3.96 (t,

J = 5.8 Hz, 1H), 3.87 (d, J = 5.7 Hz, 2H), 3.07 (d,

4.6.4. N-(3S-1,2,3,4-Tetrahydro-b-carboline-3-carboxyl)-

L-serine (5d). Yield 89%; mp 162–164 ꢁC; ESI/MS: 304

[M+H]⁺; IR (KBr): 3437, 3236, 3213, 3005, 2950,

2846, 1730, 1641, 1600, 1451, 1058, 897 cm^ꢀ1

;

¹H

J = 6.7 Hz, 2H), 2.84 (d, J = 6.5 Hz, 2H), 1.62 (s, 1H).

20

D

NMR(300 MHz, DMSO-d₆): d = 11.50 (s, 1H), 10.19

(s, 1H), 9.96 (s, 1H), 8.10 (s, 1H), 7.31 (t, J = 7.5 Hz,

1H), 7.18 (t, J = 7.8 Hz, 1H), 7.09 (d, J = 7.8 Hz, 1H),

6.89 (d, J = 7.5 Hz, 1H), 4.59 (t, J = 5.1 Hz, 1H), 4.04

(d, J = 5.1 Hz, 2H), 3.96 (t, J = 5.1 Hz, 1H), 3.81 (d,

½aꢂ ꢀ86ꢁ (c 0.36, CHCl₃/CH₃OH, 1:1, v/v). Anal. Calcd

for C₁₅H₁₇N₃O₃S: C, 56.41; H, 5.37; N, 13.16. Found:

C, 56.28; H, 5.51; N, 13.03.

4.6.9. N-(3S-1,2,3,4-Tetrahydro-b-carboline-3-carboxyl)-

L-methionine (5i). Yield 83%; mp 186–189 ꢁC; ESI/MS:

348 [M+H]⁺; IR (KBr): 3436, 3232, 3213, 3007, 2953,

J = 5.9 Hz, 2H), 2.78 (d, J = 5.5 Hz, 2H), 2.30 (s, 1H).

20

D

½aꢂ ꢀ70ꢁ (c 0.36, CHCl₃/CH₃OH, 1:1, v/v). Anal. Calcd

4772

M. Zhao et al. / Bioorg. Med. Chem. 14 (2006) 4761–4774

2849, 1735, 1648, 1600, 1450, 1055, 897 cm^ꢀ1; ¹H NMR

(300 MHz, DMSO-d₆): d = 11.21 (s, 1H), 10.11 (s, 1H),

9.96 (s, 1H), 8.14 (s, 1H), 7.29 (t, J = 7.8 Hz, 1H), 7.16

(t, J = 7.8 Hz, 1H), 7.02 (d, J = 7.5 Hz, 1H), 6.93 (d,

J = 7.8 Hz, 1H), 4.48 (t, J = 5.5 Hz, 1H), 3.99 (t,

J = 5.4 Hz, 1H), 3.89 (d, J = 6.5 Hz, 2H), 2.79 (d,

7.31 (t, J = 7.2 Hz, 1H), 7.18 (t, J = 7.3 Hz, 1H), 7.14

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

J = 7.5 Hz, 1H), 7.10 (d, J = 7.5 Hz, 1H), 7.04 (d,

J = 7.5 Hz, 1H), 6.98 (d, J = 7.2 Hz, 1H), 6.85 (s, 1H),

4.87 (t, J = 5.5 Hz, 1H), 3.95 (t, J = 5.5 Hz, 1H), 3.86

(d, J = 5.4 Hz, 2H), 2.94 (d, J = 5.4 Hz, 2H), 2.79 (d,

20

D

J = 6.4 Hz, 2H), 2.51 (t, J = 5.7 Hz, 2H), 2.24 (m,

J = 5.5 Hz, 2H). ½aꢂ ꢀ64ꢁ (c 0.39, CHCl₃/CH₃OH,

20

D

J = 5.5 Hz, 2H), 2.09 (s, 3H). ½aꢂ ꢀ86ꢁ (c 0.39,

1:1, v/v). Anal. Calcd for C₂₃H₂₂N₄O₃: C, 68.64; H,

5.51; N, 13.92. Found: C, 68.55; H, 5.43; N, 13.79.

CHCl₃/CH₃OH, 1:1, v/v). Anal. Calcd for

C₁₇H₂₁N₃O₃S: C, 58.77; H, 6.09; N, 12.09. Found: C,

58.88; H, 5.89; N, 12.20.

4.6.14. N-(3S-1,2,3,4-Tetrahydro-b-carboline-3-carbox-

yl)-^L-arginine (5n). Yield 95%; mp 177–179 ꢁC; ESI/

MS: 373 [M+H]⁺; IR (KBr): 3441, 3230, 3212, 3007,

4.6.10. N-(3S-1,2,3,4-Tetrahydro-b-carboline-3-carbox-

yl)-^L-glutamic acid (5j). Yield 94%; mp 186–189 ꢁC;

ESI/MS: 346 [M+H]⁺; IR (KBr): 3444, 3231, 3212,

3008, 2954, 2843, 1730, 1642, 1600, 1450, 1058,

2950, 2844, 1730, 1640, 1600, 1450, 1057, 896 cm^ꢀ1

;

¹H NMR (300 MHz, DMSO-d₆): d = 11.50 (s, 1H),

10.48 (s, 1H), 10.39 (s, 2H), 10.02 (s, 1H), 9.88 (s, 1H),

8.05 (s, 1H), 7.31 (t, J = 7.5 Hz, 1H), 7.12 (t,

J = 7.6 Hz, 1H), 7.06 (d, J = 7.6 Hz, 1H), 6.95 (d,

J = 7.5 Hz, 1H), 6.89 (s, 1H), 4.48 (t, J = 5.4 Hz, 1H),

3.96 (t, J = 4.3 Hz, 1H), 3.85 (d, J = 4.8 Hz, 2H), 2.78

900cm^ꢀ^{1 1}H NMR (300 MHz, DMSO-d₆): d = 11.59

;

(s, 1H), 11.48 (s, 1H), 10.27 (s, 1H), 9.95 (s, 1H), 8.12

(s, 1H), 7.33 (t, J = 7.8 Hz, 1H), 7.18 (t, J = 7.8 Hz,

1H), 7.02 (d, J = 7.5 Hz, 1H), 6.95 (d, J = 7.5 Hz, 1H),

4.52 (t, J = 5.4 Hz, 1H), 3.98 (t, J = 5.5 Hz, 1H), 3.85

(d, J = 4.2 Hz, 2H), 2.66 (t, J = 5.2 Hz, 2H), 1.88 (m,

20

D

(d, J = 6.5 Hz, 2H), 2.81 (d, J = 5.1 Hz, 2H), 2.24 (t,

J = 4.1 Hz, 2H), 1.65 (m, J = 4.2 Hz, 2H). ½aꢂ ꢀ35ꢁ (c

20

D

J = 5.1 Hz, 2H), 2.11 (m, J = 5.5 Hz, 2H). ½aꢂ ꢀ66ꢁ (c

0.39, CHCl₃/CH₃OH, 1:1, v/v). Anal. Calcd for

C₁₈H₂₄N₆O₃: C, 58.05; H, 6.50; N, 22.57. Found: C,

58.19; H, 6.34; N, 22.39.

0.39, CHCl₃/CH₃OH, 1:1, v/v); Anal. Calcd for

C₁₇H₁₉N₃O₅: C, 59.12; H, 5.55; N, 12.17. Found: C,

59.24; H, 5.46; N, 12.02.

4.7. N-[(3S)-1,2,3,4-Tetrahydro-b-carboline-3-carboxyl]-

L-glycine (5o)

4.6.11. N-(3S-1,2,3,4-Tetrahydro-b-carboline-3-carbox-

yl)-^L-aspartic acid (5k). Yield 91%; mp 179–181 ꢁC;

ESI/MS: 332 [M+H]⁺; IR (KBr): 3442, 3236, 3217,

3005, 2950, 2843, 1728, 1642, 1600, 1450, 1058,

Yield 94%; mp 181–183 ꢁC; ESI/MS: 274 [M+H]⁺; IR

(KBr): 3434, 3230, 3213, 3004, 2950 2844, 1730, 1646,

896 cm^ꢀ1; H NMR (300 MHz, DMSO-d₆): d = 11.42

1601, 1455, 1062, 900 cm^ꢀ^{1 1}H NMR (BHSC-500,

;

1

(s, 1H), 11.40 (s, 1H), 10.32 (s, 1H), 9.99 (s, 1H), 8.05

(d, J = 5.5 Hz, 1H), 7.31 (d, J = 7.3 Hz, 1H), 7.16 (d,

J = 7.4 Hz, 1H), 7.11 (t, J = 7.4 Hz, 1H), 6.95 (t,

J = 7.3 Hz, 1H), 4.77 (t, J = 5.5 Hz, 1H), 3.96 (t,

DMSO-d₆): d = 11.05 (s, 1H), 9.94 (s, 1H), 8.00 (s,

1H), 7.27 (t, J = 7.6 Hz, 1H), 7.19 (t, J = 7.6 Hz, 1H),

6.95 (d, J = 7.6 Hz, 1H), 6.87 (d, J = 7.6 Hz, 1H), 4.16

(s, 2H), 4.08 (dd, J = 5.4 Hz, J = 4.5 Hz, 1H), 3.89 (d,

J = 5.4 Hz, 1H), 3.87 (d, J = 5.5 Hz, 2H), 2.81 (d,

J = 5.3 Hz, 2H), 2.83 (d, J = 5.4 Hz, 2H), 2.05 (s, 1H),

20

D

20

D

J = 5.2 Hz, 2H), 2.73 (d, J = 5.3 Hz, 2H). ½aꢂ ꢀ12ꢁ (c

½aꢂ ꢀ104ꢁ (c 0.38, CHCl₃/CH₃OH, 1:1, v/v). Anal.

0.39, CHCl₃/CH₃OH, 1:1, v/v). Anal. Calcd for

C₁₆H₁₇N₃O₅: C, 58.00; H, 5.17; N, 12.68. Found: C,

57.87; H, 5.09; N, 12.78.

Calcd for C₁₄H₁₅N₃O₃: C, 61.53; H, 5.53; N, 15.38.

Found: C, 61.38; H, 5.38; N, 15.51.

4.7.1. N-[(3S)]-1,2,3,4-Tetrahydro-b-carboline-3-carbox-

yl]-^L-(Z)lysine (5p). The solution of 1.5 g (2.50 mmol)

of 4p was mixed with 50 mg of Pd/C (5%) and 15 mL

of formic acid in methanol (4.4%), and agitated with

hydrogen at room temperature for 24 h. The reaction

mixture was ﬁlter and evaporated to give a colorless

powder. The powder was treated by use of the procedure

same as that used for 4a giving 0.9 g (90%) of the title

compound as a pale yellow powder. Mp 166–168 ꢁC;

ESI/MS: 479 [M+H]⁺; IR (KBr): 3440, 3236, 3217,

4.6.12. N-(3S-1,2,3,4-Tetrahydro-b-carboline-3-carbox-

yl)-^L-histidine (5l). Yield 94%; mp 184–186 ꢁC; ESI/

MS: 354 [M+H]⁺; IR (KBr): 3445, 3231, 3212, 3007,

2947, 2842, 1730, 1642, 1600, 1450, 1059, 899 cm^ꢀ1

;

¹H NMR (300 MHz, DMSO-d₆): d = 12.25 (s, 1H),

11.42 (s, 1H), 10.29 (s, 1H), 10.04 (s, 1H), 8.06 (s, 1H),

7.60 (s, 1H), 7.31 (d, J = 7.4 Hz, 1H), 7.17 (d,

J = 7.2 Hz, 1H), 7.03 (d, J = 7.2 Hz, 1H), 6.95 (t,

J = 7.4 Hz, 1H), 6.94 (s, 1H), 4.97 (t, J = 5.5 Hz, 1H),

3.97 (t, J = 5.5 Hz, 1H), 3.86 (d, J = 5.4 Hz, 2H), 3.05

3004, 2940, 2846, 1736, 1604, 1455, 1062, 898 cm^ꢀ1

;

20

D

(d, J = 5.3 Hz, 2H), 2.81 (d, J = 5.0 Hz, 2H). ½aꢂ ꢀ6ꢁ

¹H NMR (BHSC-500, DMSO-d₆): d = 10.92 (s, 1H),

9.95 (s, 1H), 8.03 (s, 1H), 8.00 (s, 1H), 7.27 (t,

J = 7.5 Hz, 1H), 7.20 (t, J = 7.3 Hz, 1H), 7.19 (t,

J = 7.5 Hz, 1H), 7.17 (d, J = 7.3 Hz, 2H), 7.14 (t,

J = 7.3 Hz, 2H), 6.96 (d, J = 7.5 Hz, 1H), 6.89 (d,

J = 7.5 Hz, 1H), 5.35 (s, 2H), 4.46 (t, J = 4.6 Hz, 1H),

3.99 (d, J = 5.6 Hz, 1H), 3.88 (dd, J = 10.0 Hz,

J = 4.5 Hz, 1H), 3.86 (dd, J = 10.0 Hz, J = 3.7 Hz,

1H), 2.96 (t, J = 4.6 Hz, 2H), 2.83 (d, J = 10.0 Hz,

(c 0.37, CHCl₃/CH₃OH, 1:1, v/v). Anal. Calcd for

C₁₈H₁₉N₅O₃: C, 61.18; H, 5.42; N, 19.82. Found: C,

61.23; H, 5.57; N, 19.69.

4.6.13. N-(3S-1,2,3,4-Tetrahydro-b-carboline-3-carbox-

yl)-^L-tryptophan (5m). Yield 86%; mp 170–172 ꢁC; ESI/

MS: 403 [M+H]⁺; IR (KBr): 3445, 3238, 3217, 3008,

2951, 2842, 1728, 1641, 1600, 1450, 1058, 897 cm^ꢀ1

;

¹H NMR(300 MHz, DMSO-d₆): d = 11.52 (s, 1H),

11.15 (s, 1H), 10.97 (s, 1H), 10.21 (s, 1H), 8.02 (s, 1H),

2H), 1.80 (m, J = 4.6 Hz, 2H), 2.10 (s, 1H), 1.56 (m,

20

D

J = 4.6 Hz, 2H), 1.29 (m, J = 4.6 Hz, 2H). ½aꢂ ꢀ39ꢁ (c

M. Zhao et al. / Bioorg. Med. Chem. 14 (2006) 4761–4774

4773

0.38, CHCl₃/CH₃OH, 1:1, v/v). Anal. Calcd for

C₂₆H₃₀N₄O₅: C, 65.26; H, 6.32; N, 11.71. Found: C,

65.41; H, 6.49; N, 11.53.

4.10. Apparent permeability coeﬃcient test

Caco-2 cells (from the American Type Culture Collec-

tion, Rockville, MD, USA) were cultivated on polycar-

bonate ﬁlters (transwell cell culture inserts, 12 mm in

diameter, 3.0 lM in mean pore size) as described else-

where.³⁶Caco-2 cells grown on ﬁlter supports for 21

days were used for all transport tests, and the integrity

of monolayers was routinely checked by measurements

of transepithelial electrical resistance (approximately

700 X cm²). All absorption tests were performed in

Hanks’ balanced salt solution (HBSS). Compound 1,

5b–d,f,h,i,m–q for evaluation was dissolved in HBSS to

prepare drug solutions at a ﬁnal concentration of

4 · 10^ꢀ3M. In apical to basolateral direction, transport

was initiated by adding drug solutions (total AP volume,

0.5 mL) to the apical compartment of inserts held in

transwells containing 1.5 mL of HBSS (basolateral com-

partment). In basolateral to apical direction, transport

was initiated by adding 1.5 mL of the solution of tested

compound to basolateral compartment and adding

0.5 mL of HBSS as receiving solution to apical side of

the monolayers. The monolayers were incubated in air

at 37 ꢁC and 95% relative humidity. At 30, 60, 90, and

120 min, samples were withdrawn from the receiving

side, and the concentrations of the samples were deter-

mined by HPLC analysis. The resistance of monolayers

was checked at the end of each experiment. Apparent

permeability coeﬃcients (P_app) were calculated accord-

ing to P_app= dQ/dt Æ 1/(A Æ C₀), wherein dQ/dt is the per-

meability rate, C₀is the initial concentration in the

donor chamber, and A is the surface area of monolayer

(1 cm²).

4.7.2. N-[(3S)-1,2,3,4-Tetrahydro-b-carboline-3-carbox-

yl]-^L-glutamine (5q). Using the procedure same as that

used for preparation of 5a, from 0.9 g (2.0 mmol) of

4q 0.6 g (91%) of the title compound was obtained as

a pale yellow powder. Mp 163–165 ꢁC; ESI/MS: 345

[M+H]⁺; IR (KBr): 3444, 3233, 3218, 3002, 2945,

2839, 1732, 1645, 1602, 1455, 1060, 900 cm^ꢀ^{1 1}H

;

NMR (BHSC-500, DMSO-d₆): d = 10.99 (s, 1H), 9.85

(s, 1H), 8.01 (s, 1H), 7.29 (t, J = 7.6 Hz, 1H), 7.20 (t,

J = 7.6 Hz, 1H), 7.00 (d, J = 7.6 Hz, 1H), 6.85 (d,

J = 7.6 Hz, 1H), 6.00 (s, 2H), 4.46 (t, J = 5.4 Hz, 1H),

3.98 (d, J = 5.8 Hz, 1H), 3.90 (d, J = 5.6 Hz, 1H), 3.82

(d, J = 5.5 Hz, 2H), 2.82 (d, J = 5.8 Hz, 2H), 2.19 (t,

J = 5.5 Hz, 2H), 2.07 (t, J = 5.5Hz, 2H), 2.02 (s, 1H).

20

D

½aꢂ ꢀ66ꢁ (c 0.38, CHCl₃/CH₃OH, 1:1, v/v). Anal. Calcd

for C₁₇H₂₀N₄O₄: C, 59.29; H, 5.85; N, 16.27. Found: C,

59.14; H, 5.68; N, 16.39.

4.8. In vitro anti-platelet aggregation activity assay^27,31

Platelet-rich plasma was prepared by centrifugation of

normal rabbit blood anticoagulation with sodium citrate

at a ﬁnal concentration of 3.8%. The platelet counts were

adjusted to 2 · 10⁵/lL by addition of autologous plasma.

Platelet aggregation tests were conducted in an aggregom-

eter using the standard turbidimetric technique. The

eﬀects of 5a–q on PAF (ﬁnal concentration 10^ꢀ5–10^ꢀ7

mol/L) or ADP (ﬁnal concentration 10^ꢀ5–10^ꢀ7mol/L)-

induced platelet aggregation were observed. The maximal

rate of platelet aggregation (Am%) was represented by the

peak height of aggregation curve.

Acknowledgments

4.9. In vivo antithrombotic activity in rat model^27,31

The project was supported by the National Natural Sci-

entiﬁc Foundation of China (S.P. 20372007, 20232020)

and Beijing Municipal Commission of Education

(200510025015).

The assessments described herein were performed based

on a protocol reviewed and approved by the Ethics

Committee of Peking University. The committee assures

taht the welfare of the animals was maintained in accor-

dance with the requirements of the animal welfare act

and according to the guide for care and use of laborato-

ry animals. The tested compound was dissolved in NS

just before use and kept in an ice bath. Male Wistar rats

weighing 250–300 g (purchased from Animal Center of

Peking University) were anesthetized with pentobarbital

sodium (80.0 mg/kg, ip), the right carotid artery and left

jugular vein were separated. A 6 cm thread with exact

weight was put into the middle of the polyethylene tube.

The polyethylene tube was full with heparin sodium

(50 IU/mL of 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, NS

or the solution of the tested compound in NS was inject-

ed and the polyethylene tube was inserted into the right

carotid artery. The blood was ﬂowed from the right

carotid artery to the left jugular vein via the polyethyl-

ene tube for 15 min. The thread was taken out and

weighed and the weight of the wet thrombus was record-

ed. The thread was kept in a desiccator for 2 weeks and

the weight of the dry thrombus was recorded.

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Article Doi

Synthesis of new class dipeptide analogues with improved permeability and antithrombotic activity

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