Synthesis and evaluation of diverse analogs of amygdalin as potential peptidomimetics of peptide T

Article abstract of DOI:10.1016/j.bmcl.2004.12.071

Peptide T (ASTTTNYT) is a promising molecule to prevent the neuropsychometric symptoms of patients suffering AIDS and for the treatment of psoriasis. In order to fully prove its therapeutic benefits, efforts were put forward to design peptidomimetics of the peptide. In this direction, in a recent computational study the natural product amygdalin was identified as a prospective peptidomimetic of the peptide and later proved to exhibit a similar chemotactic profile to the peptide. However, the cyanide moiety of amygdalin provides to the molecule a toxic profile. The present study reports the synthesis of a set of amygdalin analogs lacking the cyanide group with improved chemotactic profiles.

Full text of DOI:10.1016/j.bmcl.2004.12.071

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1493–1496
Synthesis and evaluation of diverse analogs of amygdalin
as potential peptidomimetics of peptide T
Eyleen Araya,a Alex Rodriguez,b Jaime Rubio,b Alessandro Spada,a
Jesus Joglar,a Amadeu Llebaria,a Carmen Lagunas,d Andres G. Fernandez,d
Susanna Spisanie and Juan J. Perezc,*
a
´
RUBAM, Dept. de Quımica Organica Biologica, (IIQAB-CSIC), Jordi Girona Salgado, 18-26, E-08034 Barcelona, Spain
bDepartament de Quimica fisica, Universitat de Barcelona, Mari i Franques, 1, E-08028 Barcelona, Spain
cDepartament d’Enginyeria Quimica, Technical University of Catalonia, Av. Diagonal, 647, E-08028 Barcelona, Spain
dDepartamento de Investigacion preclinica, Laboratorios Salvat SA, Gall, 30, E-08950 Esplugues de Llobregat, Spain
eDepartment of Biochemistry and Molecular Biology, Universita di Ferrara, I-44100 Ferrara, Italy
Received 24 August 2004; revised 22 December 2004; accepted 23 December 2004
Available online 22 January 2005
Abstract—Peptide T (ASTTTNYT) is a promising molecule to prevent the neuropsychometric symptoms of patients suffering AIDS
and for the treatment of psoriasis. In order to fully prove its therapeutic benefits, efforts were put forward to design peptidomimetics
of the peptide. In this direction, in a recent computational study the natural product amygdalin was identified as a prospective pep-
tidomimetic of the peptide and later proved to exhibit a similar chemotactic profile to the peptide. However, the cyanide moiety of
amygdalin provides to the molecule a toxic profile. The present study reports the synthesis of a set of amygdalin analogs lacking the
cyanide group with improved chemotactic profiles.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
diverse peptide T analogs,7 using a computational ap-
proach, a model of the bioactive conformation of the
peptide was recently proposed.8 This study led to define
Peptide T (ASTTTNYT) is a fragment of the gp120 en-
velop protein of the human immunodeficiency virus
(HIV), shown to inhibit binding of gp120 to the CD4
receptors expressed in T4 helper/inducer lymphocytes,1
as well as to trigger human monocyte chemotaxis
through the CD4/T4 antigen.2 Different studies suggest
that peptide T is a promising bioactive molecule for pre-
venting the neuropsychometric symptoms of patients
with AIDS,3 and for the treatment of psoriasis.4 In this
direction, the beneficial effects of peptide T on keratino-
cytes have recently been demonstrated.5,6 However, due
to the poor pharmacological profile of peptides, it is
sometimes difficult to prove fully their therapeutic bene-
fits. To this end, efforts were recently put forward to find
peptidomimetics of peptide T. From previous structure–
activity studies based on the chemotactic properties of
a
pharmacophore incorporating all the chemical
moieties, as well as their relative positions necessary to
exhibit good monocyte chemotactic activity.9 This
pharmacophore was used for in silico screening of differ-
ent databases of compounds, leading to the identifica-
tion of amygdalin as a candidate peptidomimetic of
peptide T. A subsequent monocyte chemotaxis study
proved the compound to exhibit a chemotactic profile
similar to that of peptide T.10
Amygdalin is a natural product that exhibits a toxic pro-
file known to be due to the release of cyanide ions in
vivo, produced in the hydrolysis of the glycoside bond
by the action of glucosidases, releasing in a first step glu-
cose and mandelonitrile, that is, later transformed into
benzaldehyde and cyanhydric acid.11 The goal of the
present work was to design amygdalin analogs with
the cyanide group replaced by other functionalities
aimed at reducing its toxic profile. Accordingly, different
structural modifications were studied, including the
Keywords: Amygdalin derivatives; Psoriasis; Peptidomimetics.
*
Corresponding author. Tel.: +34 93 4016111; fax: +34 93 4016210;
e-mail: juan.jesus.perez@upc.edu
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.12.071

1494
E. Araya et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1493–1496
removal or substitution of this moiety. Compounds
reported in the present work have the structural formula
(1):
The protection of the hydroxyl groups of amygdalin was
performed by acetylation,12 followed by hydrolysis of the
glycoside bond to produce heptaacetylgentiobiose (A).
This compound was the precursor to produce an interme-
diate that was used to glycosylate the selected alcohols.
OH
O
HO
Of the derivatives of heptaacetylgentiobiose (A) tested in
the glycosylation reaction (Scheme 1), the halides (C)
(where X = Br, F) used in the Koenigs–Knorr method,
showed poorer results than the trichloracetimidate (B)
in yield, generality, and selectivity with the different
alcohols tested. From the trichloroacetimido intermedi-
ate (B) the diverse derivatives can be synthesized by
alcohol glycosylation according to Scheme 2.
O
HO
OH
HO
HO
O
O
OH
R
(1)
where R are benzyl or arylalkyl alcohol derivatives,
substituted or not by diverse groups.
Compounds were synthesized as glycosylation products
of diverse alcohols with hydroxyl protected gentiobiose
(the corresponding disaccharide of amygdalin), using
amygdalin itself as starting product. For this purpose,
it was required to produce a glycosyl donor intermediate
as starting product. Starting from the protected gentio-
biose (A), two different intermediates were considered (B
and C). The synthetic route used is shown in Scheme 1.
2. Results and discussion
Analogs synthesized in the present work as well as re-
sults of the monocyte chemotaxis assays are listed in Ta-
ble 1. Some of the analogs, like compounds 9, 10, and 11
(the only three compounds with a substituted aromatic
ring) exhibit an improved monocyte chemotactic activity
in regard to amygdalin at sub-nanomolar concentra-
OH
O
OAc
O
Ac2O
Pyridine,12h
HO
O
AcO
AcO
O
H
HO
O
H
HO
O
O
HO
AcO
AcO
O
HO
OH
AcO
0
º
to rt.
OAc
CN
CN
OAc
O
OAc
AcO
AcO
O
Pd(OH)2 cat
O
AcO
AcO
O
O
Cl3CCN, NaH (cat.)
CH2Cl2, 15' r.t.
AcO
AcO
AcO
O
AcO
Acetone / Toluene
3h, r.t.
AcO
AcO
OH
AcO
O
OAc
NH
CCl3
B
A
OAc
O
AcO
AcO
O
AcO
AcO
O
X
AcO
C
AcO
Scheme 1.
OAc
O
AcO
O
OAc
AcO
O
AcO
ROH, BF3.Et2O
AcO
AcO
O
AcO
AcO
O
AcO
O
O
AcO
CH2Cl2
-50º 15', r.t 3h .
O
R
AcO
AcO
NH
CCl3
OAc
B
OH
O
cat NaOMe/ MeOH
HO
HO
O
Reflux
10'
O
OH
O
HO
R
HO
OH
1—13
Scheme 2.

E. Araya et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1493–1496
Table 1. Peak values of the chemotactic index together with the
1495
1.20
1.00
0.80
0.60
0.40
0.20
0.00
peptide T
corresponding concentrations (ꢀlog[A]) for the different analogs
synthesized
[DAla1]pepT NH2
Molecule Substituent
Chemotactic index p[A] Rmsd
˚
(A)
analog 10
analog 9
1
0.76
0.84
8
9
0.72
0.70
HO
analog11
amygdalin
2a
3b
4
HO
-15
-10
-5
0
0.77
0.71
0.75
0.70
0.79
0.84
0.87
0.94
0.84
0.94
8
9
0.72
0.27
0.74
0.69
0.74
0.32
0.74
0.72
0.81
0.64
0.17
HO
HO
concentration/M
Figure 1. Plot of the chemotactic index of different compounds versus
T (h);
their concentration. Compounds displayed are: peptide
[DAla1]peptideTNH2 (Æ); Amygdalin (j); analog 9 (d); analog 10
(m); and analog 11 (n).
COOH
5b
6
HO
HO
activity at micromolar concentrations, although it may
the case that only one of the enantiomers is active.
Although this may explain in part the difference of activ-
ity observed between compounds 2 and 5, it should also
be argued that the higher chemotactic activity exhibited
by compound 2 is due to the constrained conformation
adopted by the aromatic ring due to its interaction with
the bulky neighboring tert-butyl group. Similarly, com-
pound 8 shows a loss of chemotactic activity in regard to
compound 4, suggesting that the pharmacophore re-
quires only a proton accepting moiety in this position.
6
9
HO
HO
7b
6
8
7
CONH2
In order to get some insights into the structure–activity
relationships among this set of compounds, for each of
the analogs, the conformation that best fitted the phar-
macophore requirements was computed. This was car-
ried out by finding the geometry with the lowest root
mean square deviation between the corresponding
chemical moieties of the analogs and the pharmaco-
phore groups, by varying all rotatable angles without
energy minimization. Last column of Table 1 lists the
values of the root mean square deviation (rmsd) between
the most fitted conformation of each analog and the
four point pharmacophore. Analysis of the results sug-
gests that compounds can be classified into two catego-
OCH3
9
10
10
10
8
HO
HO
Br
10
11
12
H3CO
HO
OCH3
˚
ries, those that exhibit rmsd values around 0.7 A and
˚
those with values of 0.3 A and lower. Analogs in the lat-
HO
ter category fulfill one of the proton accepting centers
with the carboxyl (4), carboxamide (8), and cyanide
(13) moieties, respectively, whereas those compounds
of the former category use the ether oxygen as proton
accepting center. As can be seen, good fitting of the
compounds to the pharmacophore is not enough to ex-
plain the chemotactic indexes observed. Accordingly,
steric hindrance, the energy burden to access the most
fitted conformation or electronic effects on the aromatic
ring, are features that are not included in the pharmaco-
phore in its present status and may account for the
activity of the compounds.
13
14
15
Amygdalin
Peptide T
[Dala1]pepTNH2
0.63
1.09
0.89
10
11
11
a Assayed as 3:2 mixture of epimers at the benzylic carbon.
b Assayed as 1:1 mixture of epimers at the benzylic carbon.
tions. This can be seen in more detail in Figure 1, where
the chemotactic index of these compounds, measured as
a function of the concentration is compared to that of
peptide T, [DAla1]peptide T, and amygdalin. Com-
pounds 2, 4, and 6 exhibit lower activity than amygda-
lin, being their chemotactic activity one order of
magnitude lower. Similarly, compounds 1, 3, and 12
exhibit even poorer activities, two orders of magnitude
lower than amygdalin. Compounds 5 and 7 both as-
sayed as a 1:1 mixture of epimers show chemotactic
In summary, there were identified several analogs of
amygdalin that can be considered peptidomimetics of
peptide T, based on the similar chemotactic profile

1496
E. Araya et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1493–1496
exhibited. Moreover, since these analogs lack the cya-
nide group they are expected not to be toxic. However,
further studies need to be carried out in order to identify
differences in the pharmacological profile in regard to
peptide T. Work in this direction is presently being
undertaken.
been supported by the Spanish Ministry of Science and
Technology through grant PTR1995-0404-OP-02-02.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.
2004.12.071. Typical procedure for glycosylation of
alcohols and characteristics of the analogs described in
the present work is available as supplementary data.
3. Experimental section
Monocyte chemotaxis measurements were carried out
following the procedure described in Ref. 9.
References and notes
4. Synthesis
1. Pert, C. B.; Hill, J. M.; Ruff, M. R.; Bernan, R. M.;
Robey, W. G.; Arthur, L. O.; Ruscetti, F. W.; Farrar, W.
L. Octapeptides deduced from the neuropeptide receptor-
like pattern of antigen T4 in brain potently inhibit human
immunodeficiency virus receptor binding and T-cell infec-
tivity. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 9254–9258.
2. Ruff, M. R.; Martin, B. M.; Ginns, E. I.; Farrar, W. L.;
Pert, C. B. CD4 receptor binding peptides that block HIV
infectivity cause human monocyte chemotaxis. Relation-
ship to vasoactive intestinal polypeptide. FEBS Lett. 1987,
211, 17–22.
3. Wetterberg, L.; Alexius, B.; Saaf, J.; Sonnenberg, A.;
Britton, S.; Pert, C. B. Peptide T in treatment of AIDS.
Lancet 1987, 1, 159.
4. Farber, E. M.; Cohen, E. N.; Trozak, D. J.; Wilkinson, D.
I. Peptide T improves psoriasis when infused into lesions
in nanogram amounts. J. Am. Acad. Dermatol. 1991, 25,
658–664.
5. Raychaudhuri, S. K.; Raychaudhuri, S. P.; Farber, E. M.
Anti-chemotactic activities of peptide-T: a possible mech-
anism of actions for its therapeutic effects on psoriasis. Int.
J. Immunopharmacol. 1998, 20, 661–667.
6. Tufano, M. A.; Greco, R.; Paoletti, I.; Donnarumma, G.;
Canozo, N.; Baroni, A. Immunomodulatory effects of
peptide T on human keratinocyte cells. Br. J. Dermatol.
2002, 147, 663–669.
7. Marastoni, M.; Salvadori, S.; Baloni, G.; Scaranari, V.;
Spisani, S.; Reali, E.; Traniello, S.; Tomatis, R. Structure–
activity relationships of cyclic and linear peptide T
analogues. Int. J. Pept. Protein Res. 1993, 41, 441–454.
8. Centeno, N. B.; Perez, J. J. A proposed bioactive
conformation of peptide T. J. Compd.-Aided Mol. Design.
1998, 12, 7–14.
9. Llorens, O.; Centeno, N. B.; Filizola, M.; Spisani, S.;
Marastoni, M.; Calafell, M.; Herranz, C.; Perez, J. J. A
proposed bioactive form of peptide T and the design of
peptidomimetics. Lett. Pept. Sci. 1998, 5, 179–182.
10. Llorens, O.; Filizola, M.; Spisani, S.; Marastoni, M.;
Herranz, C.; Perez, J. J. Amygdalin binds to the CD4
receptor as suggested from molecular modeling studies.
Bioorg. Med. Chem. Lett. 1998, 8, 781–786.
4.1. 2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl (1!6)-
2,3,4-tri-O-acetyl-b-D-glucopyranoside, hepta-O-acetyl-
D-gentiobiose (A)
Following an adaptation of a literature procedure
describing the synthesis of the benzoyl derivative,13
a
suspension of heptaacetylgentiobiose14 (9.0 g, 12 mmol)
and 20% Pd(OH)2/C (3.6 g) in a toluene–acetone mix-
ture (3:2 v:v, 500 mL) is hydrogenated at 25 °C and
1.0 bar until total consumption of the staring material
(TLC). The reaction mixture is filtered and evaporated.
The residue obtained is dissolved in EtOAc (200 mL)
and washed with 1 N HCl and saturated NaCl. Drying
and evaporation produce a yellow solid that is re-crys-
tallized from ethanol to give 6.80 g (86%) of product
as white crystals.
1H NMR (300 MHz, CDCl3) d (ppm): 5.50 (1H, dd,
J = 9.6, J = 10.2), 5.19 (1H, t, J = 9.6), 5.07 (1H, t,
J = 9.6), 4.94 (1H, dd, J = 8.1, J = 9.6), 4.88–4.81 (2H,
m), 4.55 (1H, d, J = 8.0), 4.26–4.12 (2H, m), 3.85–3.80
(1H, m), 3.72–3.65 (1H, m), 3.63–3.56 (2H). 13C NMR
(75 MHz, CDCl3) d (ppm): 170.27, 170.17, 170.09,
169.70, 169.39, 169.36, 169.21, 101.05, 89.95, 72.55,
71.96, 71.18, 71.00, 69.85, 69.13, 68.76, 68.07, 67.96,
62.51, 61.60, 20.5–20.3 (7C).
4.2. O-(2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl
(1!6)-2,3,4-tri-O-acetyl-b-D-glucopyranosyl)trichloro-
acetimidate (B)
A catalytic amount of 60% dispersion of NaH in mineral
oil (approx 0.025 mmol) is added to a solution of hepta-
O-acetylgentiobiose (500 mg, 0.8 mmol) and trichloro-
acetonitrile (0.5 mL, 5.0 mmol) in dichloromethane
(20 mL) and the mixture is stirred at 25 °C under nitro-
gen until the total reaction (TLC) of the protected disac-
charide (usually 15–30 min). The mixture is evaporated
to dryness and the residue employed in the next step
without further purification.
11. Holzbecher, M.; Moss, M.; Ellenberger, H. The cyanide
content of Laetrile preparations, apricot, peach and apple
seeds. Clin. Toxicol. 1984, 22, 341–347.
12. Ziegler, T.; Seidl, U. Preparation of some amygdalin-
derived gentiobiosyl donors and acceptors for oligosacch-
acaride synthesis. J Carbohydr. Chem. 1991, 10, 813–831.
13. Bliard, C.; Massiot, G.; Nazabadioko, S. Amygdalin as
building block in oligosaccharide synthesis. Tetrahedron
Lett. 1993, 34, 5083–5084.
Acknowledgements
14. The
R enantiomer of this product was previously
J.J.P. in indebted to the Fundacion Inquifarma for finan-
cial support through a research award. This research has
described in: Hirata, T.; Shimoda, K., et al. Bull. Chem.
Jpn. 2001, 74, 539–542.