Dual-acting agents that possess free radical scavenging and antithrombotic activities: Design, synthesis, and evaluation of phenolic tetrahydro-β-carboline RGD peptide conjugates

Article abstract of DOI:10.1016/j.bmcl.2006.06.024

A new approach to construct a single dual-acting agent is described. Compounds 6a-c are potent free radical scavengers as demonstrated by the EC₅₀ values in PC12 cell survival assay in term of NO, H₂O₂, and ^{{radical dot}}OH scavenging activity. The Ach-induced vaso-relaxation assay further confirms the potent NO scavenging activity of compounds 6a-c. In addition, 6a-c are efficacious in a rat arterial thrombosis, and are active in ADP- or PAF-induced in vitro platelet aggregation assay, suggesting that compounds 6a-c also possess anti-thrombotic activities. Since both free radical and thrombogenesis are important risk factors in myocardial ischemic/reperfusion injuries, these dual-acting agents having both free radical scavenging and antithrombolic activities may potentially be beneficial toward their treatment.

Full text of DOI:10.1016/j.bmcl.2006.06.024

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4523–4527
Dual-acting agents that possess free radical scavenging and
antithrombotic activities: Design, synthesis, and evaluation
of phenolic tetrahydro-b-carboline RGD peptide conjugates
Wei Bi,a,* Lanrong Bi,b Jianhui Cai,a Sanguang Liu,a Shiqi Peng,b
Nicholas O. Fischer,c Jeffrey B.-H. Tokc,* and Guohua Wanga,*
aSecond Affiliated Hospital of HeBei Medical University, Shijiazhuang 050000, PR China
bCollege of Pharmaceutical Sciences, Capital University of Medical Sciences, Beijing, 100054, PR China
cChemistry and Materials Science Directorate, Lawrence Livermore National Laboratory,
7000 East Avenue, Livermore, CA 94550, USA
Received 29 April 2006; revised 6 June 2006; accepted 8 June 2006
Available online 23 June 2006
Abstract—A new approach to construct a single dual-acting agent is described. Compounds 6a–c are potent free radical scavengers
as demonstrated by the EC50 values in PC12 cell survival assay in term of NO, H2O2, and ÅOH scavenging activity. The Ach-induced
vaso-relaxation assay further confirms the potent NO scavenging activity of compounds 6a–c. In addition, 6a–c are efficacious in a
rat arterial thrombosis, and are active in ADP- or PAF-induced in vitro platelet aggregation assay, suggesting that compounds 6a–c
also possess anti-thrombotic activities. Since both free radical and thrombogenesis are important risk factors in myocardial ische-
mic/reperfusion injuries, these dual-acting agents having both free radical scavenging and antithrombolic activities may potentially
be beneficial toward their treatment.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Cardiovascular disease is a major cause of disability
and mortality in the developed countries of the world.
Prolonged reduction in coronary blood flow due to
atherosclerotic plaques or vasospasm can result in severe
damage to the myocardium, thus leading to cellular
injury and eventually cellular death due to apoptosis
and/or necrosis. Re-instatement of blood flow, that is,
reperfusion, is not without significant negative conse-
quences and cellular damage. When oxygenated blood
re-enters previously hypoxic cardiac tissues, it initiates
a cascade of events that, paradoxically, results in further
cellular and tissue damages. This tissue destruction is
referred to as reperfusion injury. Reperfusion injury is
hypothesized to be associated with the production of
toxic free radical species after the re-supply of blood
(or oxygen) to previously ischemic (or hypoxic)
tissues.1–3 Therefore, enhancing the organ antioxidant
capacity may be viewed as a promising therapeutic
strategy to attenuate I/R injury. It has been reported that
a variety of free radical scavengers and antioxidants are
capable of ameliorating ischemia-reperfusion injury.4–10
Tetrahydro-b-carbolines are naturally occurring sub-
stances found in food, alcoholic and nonalcoholic
drinks, and fruit-derived products. It has been reported
tetrahydro-b-carbolines are potent antioxidants and
may thus be useful for the prevention of diseases associ-
ated with oxidative damage.11–15 Furthermore, it is well
recognized that phenolic compounds in foods possess
several interesting biological and chemical properties,
such as antioxidant activity and the ability to scavenge
reactive oxygen species.16,17 The phenolic compounds
may hence prevent various diseases associated with oxi-
dative stress, for example, cancers, cardiovascular dis-
eases, and inflammation. In addition, phenolic
tetrahydro-b-carbolines have been identified as effective
free radical scavenger and antioxidants.13
Since acute myocardial ischemia is usually due to a
thromboembolic event in the coronary arteries, causal
treatment and secondary prevention always include
one or more forms of anti-thrombotic therapies, such
Keywords: Anti-thrombotic; Free radical; Dual-action.
*
Corresponding authors. Tel.: +1 925 423 1549; fax: +1 925 422 3570
(J.B.-H.T); tel.: +86 10 0311 8722 2282 (W.B.); tel.: +86 10 0311 8722
2282 (G.W.); e-mail: tok2@llnl.gov
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.06.024

4524
W. Bi et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4523–4527
as anti-platelet agents, heparins, or fibrinolytic drugs.
RGD (Arg-Gly-Asp) containing peptides have been
investigated to develop therapeutic agents for treating
thrombosis.18–20 The mechanism of action of RGD pep-
tides is inhibiting cell adhesion to extracellular matrix
protein via binding of RGD peptide to the integrin
receptors on the cell surface.21 Several anti-thrombotic
agents have been derived from RGD peptides.18,22 For
example, Aggrastatꢁ, an RGD peptidomimetic, has
been used clinically for treating patients with thrombo-
sis.23 In our previous studies, RGD-S, RGD-V, and
RGD-F were used as the building block in the modifica-
tion of the oligopeptides with anti-thrombotic and/or
thrombolytic activity.24–26 Considering the free radical
scavenging properties possessed by phenolic tetrahy-
dro-b-carboline and the anti-thrombic activity demon-
strated by RGD peptides, we sought to link a phenolic
tetrahydro-b-carboline moiety to the thrombolytic
RGD peptides in hope that the resulting phenolic tetra-
hydro-b-carboline RGD peptide conjugates would
exhibit both NO scavenging activity and thrombolytic
activity.
For the synthesis of the phenolic tetrahydro-b-carboline
RGD peptide conjugates, L-tryptophan was first reacted
with phenolic aldehyde (Scheme 1A). Specifically,
syringaldehyde in acidic-aqueous conditions affords 1
in 46% yield.39 This reaction occurs through a Pictet–
Spengler intramolecular cyclization of the Schiff base
to afford the 1,3-disubstituted tetrahydro-b-carbolines.
1,3-Disubstituted tetrahydro-b-carbolines that arise
from L-tryptophan and syringaldehyde provided two
diastereoisomers (1S,3S-1 and 1R,3S-1), which were
then separated via RP-HPLC. Diastereoisomer assign-
ments were based on acquired positive NOE spectra of
H-1 and H-3 signals in the cis-isomer. H-1 signals corre-
sponding to the cis-isomer appeared at higher field than
in the trans-isomer, whereas C-1 and C-3 NMR signals
corresponding to the trans-isomer appeared at higher
field than those of the cis-isomer. This observation is
A
B
C
Scheme 1. Synthetic route for compounds 6a–c. Reagents: (i) H2SO4; (ii) Boc-N3; (iii) 4 N HCl/ethyl acetate; (iv) HF; (v) 2, DCC; (vi) HF.

W. Bi et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4523–4527
Table 2. Inhibition of Ach-induced vaso-relaxation
4525
in agreement with the expected pattern for 1,3-disubsti-
tuted tetrahydro-b-carbolines. Subsequent treatment of
1 with Boc-N3 gave the N-Boc protected 2 in 50% yield.
Compound
Inhibition percentage ðX ꢂ SD%Þ
10ꢁ6 mol/L
10ꢁ7 mol/L
10ꢁ8 mol/L
NS
1
1.63 1.45
57.22.4a,b
60.73.5a,b
71.21.9a,b
73.31.8a,b
The protected tetrapeptide intermediates were prepared
via the solution method according to the synthetic route
shown in Scheme 1B.40 The stepwise synthesis (C ! N
in 83–96% yield) was performed starting with benzyl es-
ter of L-Ser (Bzl), L-Val, and L-Phe as the C-terminal
residue, respectively. The tetrapeptide intermediates
were then deprotected with HF to afford 4a–c (4a,
RGD-S, 63% yield; 4b, RGD-V, 85% yield; 4c, RGD-
F, 83% yield). If the Boc groups of the protected tetra-
peptide intermediates were removed, the corresponding
N-terminal tetrapeptides 3a–c were obtained in theoret-
ical yield.
79.93.1a,b
91.92.9a,b
92.21.4a,b
90.90.7a,b
10.91.6
12.82.8
13.32.0
25.42.1a
6a
6b
6c
a In comparison with normal saline, NS, p < 0.001, n = 6.
b In comparison with 10ꢁ8 mol/L of the same compound, p < 0.001.
a concentration of 10ꢁ6 mol/L, compound 1 showed
good inhibition ability with 79.9%, whereas compounds
6a–c displayed higher inhibition activities at ꢀ91–92%,
respectively. We hypothesized that the NO scavenging
activity of compounds 6a–c led to attenuation of the
NO concentration in vitro, which then led to the inhibi-
tion of Ach-induced vaso-relaxation.34–36
As shown in Scheme 1C, deprotected tetrapeptides 3a–c
were acylated with 2 to give 5a–c in 50–56% yields. After
removal of the protecting groups, the final desired prod-
ucts 6a–c were obtained in ꢀ60% yields.
In vitro anti-platelet aggregation activities of com-
pounds 1, 4a–c, and 6a–c were again assayed using
our previously reported method.26,37,38 Briefly, platelet-
rich plasma was prepared by centrifugation of normal
rabbit blood anti-coagulation with sodium citrate at a fi-
nal concentration of 3.8%. The platelet counts were
adjusted to 2 · 105/lL through addition of autologous
plasma. Platelet aggregation studies were conducted in
an aggregometer using the standard turbidimetric tech-
nique. The agonists used were platelet activating factor
(PAF, final concentration 10ꢁ7 mol/L) and adenosine
diphosphate (ADP, final concentration 10ꢁ5 mol/L).
The effects of compounds 1, 4a–c, and 6a–c on PAF-
or ADP-induced platelet aggregation were then evaluat-
ed. The maximal rate of platelet aggregation (Am%) was
represented by the peak height of aggregation curve.
The obtained data are listed in Tables 3 and 4, respec-
tively. It was observed that compounds 6a–c displayed
a remarkable dual-anti-platelet aggregation activity in
both of ADP- and PAF-induced platelet aggregation as-
say. For the ADP-induced platelet assay, with 6a–c at a
concentration of 10ꢁ6 mol/L, the platelet aggregation
rates were collectively decreased to ꢀ26–28%. (NS:
56.40 5.23; 1: 46.65 4.60, n = 8, p < 0.001). Similar
observation was observed in PAF-induced platelet
The free radicalÅ scavenging activities of 6a–c against
NO, H2O2, and OH were evaluated in PC12 cells and
compared with that of 1 (which has previously been
identified as effective free radical scavenger and antioxi-
dant13) using a published method.27–29 The results are
expressed as EC50 (lM) values. As shown in Table 1,
the EC50 values of 6a–c were found to be similar to that
of 1, suggesting that they were effective scavengers of
Å
NO, H2O2, and OH as 1. The scavenging capacity
(EC50) among all of the tested compounds ranged from
ꢀ87 to 92 lM for NO, from ꢀ30 to 67 lM for H2O2,
Å
and ꢀ86 to 95 lM for OH.
Next, the NO scavenging abilities of 6a–c were further
evaluated in the acetylcholine (Ach)-evoked, endotheli-
um-mediated relaxation assay (normal saline, NS, was
used as a control).29,30 The endothelium controls the
tone of the underlying vascular smooth muscle through
the production of vasodilator mediators.31–33 In this
experimental model, Ach acts on the endothelium to re-
lease nitric oxide (NO), a potent vasodilating mediator.
The decreased relaxation response in rat arotic strip
could be attributed to a reduction in NO (synthesized
by the endothelium). Therefore, the present study was
undertaken to assess the NO scavenging capability of
these compounds. The results are expressed as a
percentage inhibition of acetylcholine (Ach)-induced
vaso-relaxation by test compounds and are summarized
in Table 2. It was observed that Ach-induced relaxation
was reversed significantly by compounds 1 and 6a–c. At
Table 3. Effects of 1, 4a–c, and 6a–c on ADP-induced platelet
aggregation
Compound
Am% ðX ꢂ SDÞ
10ꢁ7 mol/L
10ꢁ6 mol/L
10ꢁ5 mol/L
NS
1
56.40 5.23
54.88 2.43
52.50 2.43
51.78 3.20b
34.99 2.57a
49.37 3.22b
50.20 3.13b
38.00 3.11a,c
46.65 4.60a
39.62 3.00a
29.61 2.63a
22.71 2.64a
28.31 2.58a,c
29.60 3.03a,c
29.43 3.14a,c
27.33 2.02a
16.60 2.61a
11.65 1.84a
7.87 1.76a
11.33 1.71a,c
14.38 1.50a,c
13.11 2.51a,c
4a
4b
4c
6a
6b
6c
Table 1. The free radical scavenging activities in PC12 cell survival
assay
Compound
EC50 (lM) values ðX ꢂ SDÞ
EC50/NO
EC50/H2O2
EC50/OH
1
89.67 2.45
87.61 3.22
90.32 2.12
92.76 3.56
43.66 3.21
30.63 2.41
33.49 1.89
67.92 2.13
86.77 2.61
95.43 2.34
92.32 2.01
90.43 1.95
N = 8; NS, vehicle.
6a
6b
6c
a Compared to NS, p < 0.001.
b Compared to NS, p < 0. 01.
c Compared to 1, p < 0.001.

4526
W. Bi et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4523–4527
Table 4. Effects of 1, 4a–c, and 6a–c on PAF-induced platelet
aggregation
were active in ADP- or PAF-induced in vitro platelet
aggregation assay, which suggests that compounds 6a–
c also possess anti-thrombotic activity. Taken together,
a novel class of potential dual-action agents which pos-
sess both anti-thrombotic and free radical scavenging
capabilities is described. Such composite dual-acting
agent should constitute a promising approach in the
development for new protective therapy against ische-
mia-reperfusion related injuries.
Compound
Am% ðX ꢂ SDÞ
10ꢁ7 mol/L
10ꢁ6 mol/L
10ꢁ5 mol/L
NS
1
58.70 4.11
51.42 4.01
55.80 3.21
56.69 3.10
55.07 3.27
40.39 2.74a
55.62 3.20
54.06 3.22b
36.34 3.89a
4a
4b
4c
6a
6b
6c
36.47 3.00a,d 22.03 2.31a
34.84 2.51a,d 20.01 2.12a
24.02 2.20a,d
7.73 2.20a
34.59 2.19a,d 28.61 2.14a,c
35.80 2.70a,d 25.88 2.21a,c
36.59 3.11a,c 25.33 2.22a,d 21.52 2.19a, c
Acknowledgments
N = 8; NS, vehicle.
a Compared to NS, P < 0.001.
b Compared to NS, p < 0.01.
c Compared to 1, p < 0.001.
The authors are grateful to the Second Affiliated Hospi-
tal of HeBei Medical University for support of this re-
search. N.O.F. wishes to acknowledge LLNL’s CMS
directorate for a postdoctoral fellowship. J.B.-H.T. is
grateful to both T. Tarasow and C. Dolan for many
helpful discussions. Work performed in LLNL is carried
out under the auspices of the U.S. Department of Ener-
gy by the University of California under contract W-
7405-Eng-48.
aggregation assay. Thus, the in vitro assay indicated that
the new class of phenolic tetrahydro-b-carboline RGD
peptides conjugates was very likely to be potent platelet
aggregation inhibitors.
The anti-thrombotic effects of compounds 6a–c were
evaluated again using our previously reported in vivo
rat model method,26,37,38 and were expressed as the
reduction of thrombus mass. The obtained results are
summarized in Table 5. The thrombus weight after the
treatment with 6a–c was 5.00 1.04, 4.85 1.20, and
References and notes
1. Cannon, R. O. Nat. Clin. Pract. Cardiovasc. Med. 2005, 2,
88.
2. Neuzil, J.; Rayner, B. S.; Lowe, H. C.; Witting, P. K.
Redox Rep. 2005, 10, 187.
2.68 0.45 mg,
respectively.
(NS:
9.05 1.01,
p < 0.01). Thus, the anti-thrombotic activities of 6a–c
were in fact higher than that of RGD-S, RGD-V, and
RGD-F at a dosage of 5 lmol/kg.
3. Saeed, S. A.; Waqar, M. A.; Zubairi, A. J.; Bhurgri, H.;
Khan, A.; Gowani, S. A.; Waqar, S. N.; Choudary, M. I.;
Jalil, S.; Zaidi, A. H.; Ara, I. J. Coll. Physician Surg. Pak.
2005, 15, 507.
4. Parlakpinar, H.; Sahna, E.; Acet, A.; Mizrak, B.; Polat, A.
Toxicology 2005, 209, 1.
5. Parlakpinar, H.; Ozer, M. K.; Acet, A. Mol. Cell.
Biochem. 2005, 277, 137.
6. Esrefoglu, M.; Gul, M.; Parlakpinar, H.; Acet, A. Fundam.
Clin. Pharmacol. 2005, 19, 365.
7. Jin, Z. Q.; Zhou, H. Z.; Cecchini, G.; Gray, M. O.;
Karliner, J. S. Am. J. Physiol. Heart Circ. Physiol. 2005,
288, 2986.
In summary, compounds 6a–c were observed to demon-
strate good free radical scavenging activity as that
of parental compound 1 as demonstrated by the EC50
values in PC12 cell survival assay. The Ach-induced
vaso-relaxation test further confirmed the potent NO
scavenging activity of compounds 6a–c. In addition,
6a–c were efficacious in a rat arterial thrombosis, and
8. Sahna, E.; Acet, A.; Ozer, M. K.; Olmez, E. J. Pineal. Res.
2002, 33, 234.
Table 5. Effects of the compounds 1, 4a–c, and 6a–c on the thrombus
weight ðX ꢂ SDÞ
9. Lee, Y. M.; Chen, H. R.; Sheu, J. R.; Wang, J. J.; Yen, M.
H. J. Pineal. Res. 2002, 33, 72.
10. Sahna, E.; Olmez, E.; Acete, A. J. Pineal. Res. 2002, 32,
194.
Compound
Dosage
Wet thrombus
(mg)
Dry thrombus
(mg)
11. Herraiz, T.; Galisteo, J. J. Agric. Food Chem. 2003, 51,
7156.
12. Ichikawa, M.; Ryu, K.; Yoshida, J.; Ide, N.; Yoshida, S.;
Sasaoka, T.; Sumi, S. Biofactors 2002, 16, 57.
13. Herraiz, T.; Galisteo, J.; Chamorro, C. J. Agric. Food
Chem. 2003, 51, 2168.
14. Herraiz, T.; Galisteo, J. Free Radic. Res. 2002, 36, 923.
15. Pless, G.; Frederiksen, T. J.; Garcia, J. J.; Reiter, R. J. J.
Pineal Res. 1999, 26, 236.
NS
1
3 ml/kg
45.89 3.60
43.76 3.18
42.90 3.18
35.60 3.20b
26.68 3.15a
28.01 4.00a,c
29.05 2.94b,e
22.09 3.12a,f
9.05 1.01
7.27 0.79a
7.62 1.10
6.43 1.06b
4.05 0.98a
5.00 1.04b,d
4.85 1.20b,e
2.68 0.45a,g
5 lmol/kg
5 lmol/kg
5 lmol/kg
5 lmol/kg
5 lmol/kg
5 lmol/kg
5 lmol/kg
4a
4b
4c
6a
6b
6c
16. Hsu, L. Y.; Lin, C. F.; Hsu, W. C.; Hsu, W. L.; Chang, T.
C. Biol. Pharm. Bull. 2005, 28, 1211.
17. Yilmaz, Y.; Toledo, R. T. Trends Food Sci. Technol. 2004,
15, 422.
18. Nicholson, N. S.; Abood, N. A.; Panzer-Knodle, S. G.;
Frederick, L. G.; Page, J. D.; Salyers, A. K.; Suleymanov,
O. D.; Szalony, J. A.; Taite, B. B.; Anders, R. J. Med. Res.
Rev. 2001, 21, 211.
N = 8.
a In comparison with normal saline (NS), P < 0.001.
b In comparison with NS, P < 0.01.
c In comparison with 4a, p < 0.01.
d In comparison with 4a, p < 0.05.
e In comparison with 4b, p < 0.05.
f In comparison with 4c, p < 0.001.
g In comparison with 4c, p < 0.01.

W. Bi et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4523–4527
4527
19. Scarborough, R. M.; Gretler, D. D. J. Med. Chem. 2000,
43, 3453.
Nakamura, Y.; Kinoshitaa, M.; Horie, M. Life Sci. 2003,
73, 3083.
20. Zablocki, J. A.; Rico, J. G.; Garland, R. B.; Rogers, T. E.;
Williams, K.; Schretzman, L. A.; Rao, S. A.; Bovy, P. R.;
Tjoeng, F. S.; Lindmark, R. J., et al. J. Med. Chem. 1995,
38, 2378.
37. Zhao, M.; Bi, L.; Bi, W.; Wang, C.; Yang, Z.; Ju, J.; Peng,
S. Bioorg. Med. Chem. 2006, 14, 4761.
38. Liu, J.; Zhao, M.; Wang, C.; Peng, S. Bioorg. Med. Chem.
Lett. 2003, 13, 4065.
21. Ruoslahti, E. Integrins as Receptors for Extracellular
Matrix. In Cell Biology of Extracellular Matrix; Hay, E.
D., Ed.; Plenum Press: New York, NY, 1991.
22. Cheng, S.; Craig, W. S.; Mullen, D.; Tschopp, J. F.; Dixon,
D.; Pierschbacher, M. D. J. Med. Chem. 1994, 37, 1.
23. Batchelor, W. B.; Tolleson, T. R.; Huang, Y.; Larsen, R.
L.; Mantell, R. M.; Dillard, P.; Davidian, M.; Zhang, D.;
Cantor, W. J.; Sketch, M. H., Jr.; Ohman, E. M.; Zidar, J.
P.; Gretler, D.; DiBattiste, P. M.; Tcheng, J. E.; Califf, R.
M.; Harrington, R. A. Circulation 2002, 106, 1470.
24. Zhao, M.; Peng, S. J. Prakt. Chem. 1999, 341, 668.
25. Zhao, M.; Wang, C.; Yang, J.; Peng, S. Prep. Biochem.
Biotechnol. 2000, 30, 247.
26. Lin, N.; Zhao, M.; Wang, C.; Peng, S. Bioorg. Med. Chem.
Lett. 2002, 12, 585.
27. Desole, M. S.; Sciola, L.; Sircana, S.; Godani, C.; Migheli,
R.; Delogu, M. R.; Piras, G.; De, N. G.; Miele, E.
Neurosci. Lett. 1998, 247, 1.
28. Mosmann, T. J. Immunol. Methods 1983, 65, 55.
29. Wu, Y.; Bi, L.; Bi, W.; Zhao, M.; Li, Z.; Ju, J.; Peng, S.
Bioorg. Med. Chem., in press.
30. Cowart, M.; Kowaluk, E. A.; Daanen, J. F.; Kohlhaas, K.
L.; Alexander, K. M.; Wagenaar, F. L.; Kerwin, J. F. J.
Med. Chem. 1998, 41, 2636.
31. Komori, K.; Vanhoutte, P. M. Blood Vessels 1990, 27,
238–245.
32. Moncada, S.; Palmer, R. M. J.; Higgs, E. A. Pharmacol.
Rev. 1991, 43, 109.
33. Calles-Escandon, J.; Cippola, M. Endocr. Rev. 2001, 22, 36.
34. Chalupsky, K.; Lobysheva, I.; Nepveu, F.; Gadea, I.;
Beranova, P.; Entlicher, G.; Stoclet, J.; Muller, B.
Biochem. Pharmacol. 2004, 67, 1203.
35. Beranova, P.; chalupsky, K.; Kleschyov, A.; Schott, C.;
Boucher, J.; Mansuy, D.; Munzel, T.; Muller, B.; Stoclet,
J. Eur. J. Pharmacol. 2005, 516, 260.
39. Synthesis of (1S,3S)- and (1R,3S)-1-(40-hydroxy-30,50-
dimethoxyphenyl)-1,2,3,4-tetrahydro-b-carboline-3-carbox-
ylic acid (cis-1 and trans-1): L-tryptophan (306 mg,
1.5 mmol) dissolved in 0.05 N H2SO4 (13 mL) was
stirred with the syringaldehyde (1.65 mmol) (Aldrich).
The reaction mixture was allowed to react at 70 ꢂC for
6 days, and a precipitate of 1 was obtained (1.1:1)
(254 mg, 46%). cis-1: 1H NMR (CD3OD + TFA) 3.77
(m, 2H, H-4), 3.97–4.02 [2s, 6H, (OCH3)2], 4.80 (dd,
1H, H-3, J3,4a = 12.1 Hz, J3,4b = 5.3 Hz), 6.01 (s, 1H,
H-1), 6.85, 6.98 (2s, 2H, Ph), 7.2–7.65 (m, 4H, indol);
13C NMR (CD3OD + TFA) 23.73 (C-4), 56.91 (OCH3),
57.69 (C-3), 60.58 (C-1), 108.30 (C-4a), 108.47 (Ph),
112.56 (C-8), 119.25 (C-5), 120.84 (C-6), 124.04 (C-7),
124.45 (Ph), 127.16 (C-4b), 129.43 (C-9a), 138.78
(C-8a), 149.73, 158.84, 160.24 (Ph), 171.06 (COOH);
RP-HPLC-ESI-MS (tR = 15.95 min) (M+H)+ 369,
(M+Hꢁ73) 296. trans-1: 1H NMR (CD3OD + TFA)
4.72 (m, 1H, H-3), 6.22 (s, 1H, H-1); 13C NMR
(CD3OD + TFA) 53.60 (C-3), 57.37 (C-1); other signals
as cis-1: RP-HPLC-ESI-MS (tR = 16.8 min) (M+H)+
369, (M+Hꢁ73) 296.
40. Boc-Arg(Tos)-Gly-Asp(OcHex)-Ser(Bzl)-OBzl, mp 73–
20
75 ꢂC, ½aꢃD ꢁ34 (c 0.4, CHCl3), FAB-MS (m/e): 950
20
[M+H]+; BocArg-(Tos)-Gly-Asp(OcHex)-Val-OBzl, mp
79–81 ꢂC, ½aꢃD ꢁ24 (c 0.4, CHCl3), FAB-MS (m/e): 872
20
[M+H]+; Boc-Arg(Tos)-Gly-Asp(OcHex)-Phe-OBzl, mp
165–170 ꢂC, ½aꢃD ꢁ25 (c 0.4, CHCl3), FAB-MS (m/e):
20
920 [M+H]+; RGD-S, mp 110–111 ꢂC, ½aꢃD ꢁ20 (c 0.2,
20
6 N HCl), FAB-MS (m/e): 434 [M+H]+; RGD-V, mp 120–
121 ꢂC, ½aꢃD ꢁ20 (c 0.3, 6 N HCl), FAB-MS (m/e): 446
20
[M+H]+; RGD-F, mp 125–126 ꢂC, ½aꢃD ꢁ40 (c 0.2, 6 N
20
HCl), FAB-MS (m/e): 494 [M+H]+; compound 6a: ½aꢃD 38
(c 1, H2O), FAB-MS (m/e): 784 [M+H]+; compound 6b:
20
½aꢃD 18 (c 1, H2O), FAB-MS (m/e): 796 [M+H]+; com-
20
pound 6c: ½aꢃD 22 (c 1, H2O), FAB-MS (m/e): 844
36. Nakae, I.; Matsumoto, T.; Omura, T.; Takashima, H.;
Ohira, N.; Tarutani, Y.; Yasuda, Y.; Matsuo, S.; Koh, T.;
[M+H]+.