Synthesis of peptidyl phosphonates containing 5-(4′-carboxyphenyl)- 10,15,20-tritolylporphyrin

Article abstract of DOI:10.1002/hc.20407

The synthesis and characterization of three new porphyrin-peptidyl- phosphonate derivatives, containing a diphenyl 3-pyridylmethylphosphonate moiety, is described. These compounds could serve as potential photosensitizers for the PDT method of tumor thera

Full text of DOI:10.1002/hc.20407

Heteroatom Chemistry
Volume 19, Number 1, 2008
Synthesis of Peptidyl Phosphonates
ꢀ
Containing 5-(4 -Carboxyphenyl)-10,15,20-
Tritolylporphyrin
Jan Habdas1 and Bogdan Boduszek2
1Institute of Chemistry, University of Silesia, 40-006 Katowice, Poland
2Department of Organic Chemistry, Faculty of Chemistry, Wroclaw University of Technology,
50-370 Wroclaw, Poland
Received 26 February 2007; revised 18 June 2007
or dipeptidyl moiety. It was an improvement, in com-
ABSTRACT: The synthesis and characteriza-
parison with the widely applied “Photofrin II” [4]
tion of three new porphyrin-peptidyl-phosphonate
for the PDT therapy, which is a mixture of various
derivatives, containing a diphenyl 3-pyridylmethyl-
hematoporphyrins.
phosphonate moiety, is described. These compounds
As it is known, aminophosphonic acids are
could serve as potential photosensitizers for the PDT
phosphorus analogs of regular amino acids with
method of tumor therapy and displayed activity as in-
great biological activity [5,6]. Hence, we are inter-
ꢁ
C
hibitors of aminopeptidase N. 2008 Wiley Periodicals,
ested in the porphyrin-aminophosphonate deriva-
Inc. Heteroatom Chem 19:107–111, 2008; Published online
tives, which might turn out to be compounds
in Wiley InterScience (www.interscience.wiley.com). DOI
with definite biological activity, such as enzyme
10.1002/hc.20407
inhibitors.
The aim of this work is the synthesis of new
porphyrin-peptidyl-phosphonates, the substances
with potential biological activity.
INTRODUCTION
Our earlier investigations concerning the prepara-
tion of some photosensitizers, used for the PDT
method for the diagnosis and therapy of tumors,
have shown that certain derivatives of porphyrins
containing an amide bond in the molecule demon-
strated the properties of the photosensitizers [1–3].
These porphyrin–peptide conjugates possessed a
strictly defined porphyrin structure in which the por-
phyrin ring was covalently linked with an amino acid
RESULTS AND DISCUSSION
We have synthesized three new tolylpor-
phyrin derivatives containing pyridine peptidyl-
phosphonate fragments in a side chain of the
porphyrin ring. The compounds were obtained
in the reaction of 5-(4ꢀ-carboxyphenyl)-10,15,20-
tritolylporphyrin (1) with pyridine derivatives of
the diphenyl esters of selected phosphonodipep-
tides. The choice of the diphenyl phosphono-esters
for synthesis was made because such esters of
aminophosphonic acids showed great biological
activity [5].
Correspondence to: Bogdan Boduszek; e-mail: bogdan.boduszek
@pwr.wroc.pl
Contract grant sponsor: Institute of Chemistry, University of
Silesia, Katowice, Poland.
Contract grant sponsor: Faculty of Chemistry, Wroclaw
The pyridine-3-yl Cbz-phosphonodipeptides
2a–c as single diastereomers, were synthesized
University of Technology, Wroclaw, Poland.
c
ꢀ
2008 Wiley Periodicals, Inc.
107

108 Habdas and Boduszek
SCHEME 1 Structures of the Cbz-phosphono-peptides.
earlier from protected L-amino acids [7] and were
transformed into the corresponding hydrobromides
2aꢀ–cꢀ with nonsubstituted amino groups, suitable
for this work.
Because of the existence of phosphono-dipeptide
moieties in the final products, we have decided to
perform preliminary inhibitory tests by using the se-
lected enzyme, aminopeptidase N (APN/CD13). As it
is known, the aminopeptidase N [11] is a cell surface
peptidase, which occurs in a wide variety of tissues
and cells. This enzyme is usually overfilled in tumor
cells and plays a critical role in angiogenesis, which
is, in turn, a complex cascade process playing a cen-
tral role in tumor growth.
The results of the inhibitory activity of the
diphenyl porphyrin-peptidyl-phosphonates 3a–c to-
ward APN/CD13 are given in Table 1. The method
used for the inhibitory tests was previously described
in the literature [12].
The products 3a–c showed a moderate inhibi-
tion of aminopeptidase N. Among them, the best
inhibitor of APN/CD 13 was the 3b [TTP-Val-
3PyP(OPh)] (IC50 = 19.3 µM). Surprisingly, the start-
ing material, that is, 5-(4ꢀ-carboxyphenyl)-10,15,20-
tritolylporphyrin (1), with IC50 equal to 18.5 µM, was
the best inhibitor.
The preliminary tests showed that the pep-
tidyl derivatives of tolylporphyrin and the carboxy-
tolylporphyrin itself were significant inhibitors of
aminopeptidase N and may be promising biologi-
cally active agents for further investigation in this
field, which are already in progress.
Coupling the phosphonates 2aꢀ–cꢀ with the tolyl-
porphyrin 1 enabled the combination of the biolog-
ical properties of the porphyrins with the known in-
hibitory activity of the peptidyl diphenyl phospho-
nates, which are well recognized as inhibitors of the
serine proteases [6,8]. Also, an introduction of the
pyridine derivatives of phosphonopeptides to such
structures should increase the solubility in aqueous
systems.
The formulas of the 2a–c (Cbz-phosphono-
peptides) used for the preparation of the correspond-
ing hydrobromides 2aꢀ–cꢀ are shown in Scheme 1.
The side peptidyl chains in the synthesized
compounds were composed with chosen amino
acids and pyridine-3-yl-methyl diphenyl phospho-
nate ester: namely, alanine-[(1-amino)-1-(pyridine-
3-yl)-methylphosphonate diphenyl ester] (in the
case of 3a), valine-[(1-amino)-1-(pyridine-3-yl)-
methylphosphonate diphenyl ester] (in the case
of 3b), and proline-[(1-amino)-1-(pyridine-3-yl)-
methylphosphonate diphenyl ester] (in the case of
3c).
The method applied for synthesis of similar
peptidyl-phosphonate derivatives of the porphyrin
is already elaborated by us in 2005 [9].
The considered products, that is, the tritolyl-
porphyrin-pyridine-phospho-dipeptides 3a–c were
obtained from the tritolylporphyrin 1 [10], by the
use of the DCC coupling method. A course of the
reaction is shown in Scheme 2.
In the near future, we are planning to obtain the
free phosphonic acid derivatives of the porphyrin,
without the diphenyl ester functions, which, as it
is expected, should show higher inhibitory activity
toward aminopeptidase N.
The isolation of the 3a–c was done in a classical
way, which was applied in the preparation of short
peptides, and the products were additionally purified
by means of column chromatography.
EXPERIMENTAL
General
The obtained products were porphyrin-like sub-
stances, unstable at elevated temperatures.
NMR spectra were recorded on a Bruker Avance
TM DRX 300 MHz in CDCl3using 300 MHz for H
1
Heteroatom Chemistry DOI 10.1002/hc

Synthesis of Peptidyl Phosphonates Containing 5-(4ꢀ-Carboxyphenyl)-10,15,20-Tritolylporphyrin 109
SCHEME 2 Synthesis of the tolylporphyrin pyridine peptidyl diphenyl phosphonates 3a–c.
TABLE 1 Inhibition of Aminopeptidase N [CD 13]a
Compound No.
Compound Abbreviation
Molar Weight
IC50 (µM)b,c
3a
3b
3c
1
TTP-Ala-3PyP(OPh)
TTP-Val-3PyP(OPh)
TTP-Pro-3PyP(OPh)
TTP-COOH
1094
1122
1120
701
87.3
19.3
32.4
18.5
aConcentration of enzyme: 5 µg/mL.
bConcentration of substrate, L-leucylo-(4-nitroanilide): 250 µM/mL.
cBuffer: KH2PO4-Na2HPO4 with pH 7.2 was used.
Heteroatom Chemistry DOI 10.1002/hc

110 Habdas and Boduszek
NMR and 121.51 MHz for 31P NMR spectra in the
Department of Organic Chemistry at the Wroclaw
University of Technology. IR and UV spectra were
measured on a Perkin Elmer 1600 FTIR spectropho-
tometer in the Department of Organic Chemistry at
the Wroclaw University of Technology and in the
Institute of Chemistry at the University of Silesia,
Katowice. MS analyses were performed by electro-
spray ionization with a Finnigan TSQ 700 instru-
ment on mode ESI + Q1MS at the Department of
Chemistry at the University of Wroclaw.
eluate with the second purple band containing the
tolylporphyrin 1. After the evaporation of the sol-
vent, a dark-violet product (1) was obtained.
1: Yield: 400 mg (5%). The spectroscopic data
(UV, IR) of the 1 was in agreement with the literature
[10].
1H NMR (CDCl3), δ, ppm: 8.86 (d, 2H, J= 4.8 Hz,
arom.), 8.76 (d, 2H, J = 4.8 Hz, arom.), 8.28 (d, 2H,
J = 8.19 Hz, arom.), 8.10 (d, 8H, J = 7.8 Hz, Phs),
7.96 (d, 2H, J = 8.19 Hz, arom.), 7.56 (d, 8H, J = 7.8
Hz, Phs), 2.71 (s, 6H, CH3), 2.21 (s, 3H, CH3), −2.75
(bs, 2H, NH-pyrrole). MS: ESI + Q1MS, 701 (M + 1).
All reagents were purchased from the Sigma
Aldrich Co.
Diphenyl pyridine-3-methyl-(Cbz-amino) phos-
phonate alanine derivative (2a), diphenyl pyridine-
3-methyl-(Cbz-amino)phosphonate valine deriva-
tive (2b), and diphenyl pyridine-3-methyl-(Cbz-
amino)phosphonate proline derivative (2c) were
obtained as described in [7]. For stability and long
storage, the amine groups in the 2a–c were protected
by the Cbz groups, which could be easily removed by
means of the 30% HBr solution in anhydrous acetic
acid, in a one-pot procedure.
Preparation of Hydrobromides 2aꢀ –cꢀ
The diphenyl peptidyl-pyridine phosphonate 2a–c
[7] (0.5 mmol) was mixed with 30% HBr solution
in acetic acid (1.0 mL). The mixture was kept for
1–2 h, protected against moisture and then treated
with anhydrous diethyl ether (20 mL) with stirring.
The supernatant ethereal layer was removed by de-
cantation, and a fresh portion of diethyl ether was
added. The procedure was repeated three times, and
the product was filtered to give a whitish solid, be-
ing the hydrobromide of the 2aꢀ, 2bꢀ or2cꢀ, which
was used directly in the next step.
5-(4-Carboxyphenyl)-10,15,20-tritolylporphyrin
(1) was generally obtained by the Adler’s method
[10], with some modifications. In our case, 5-
(4-carboxyphenyl)-10,15,20-tritolylporphyrin
(1)
was prepared by the condensation of 4-
methylbenzaldehyde and 4-carboxybenzaldehyde
with pyrrole in boiling propionic acid. The applied
molar ratio of the reactants (3:1:4) was optimized
and gave the highest yield of the 1 in the conditions
used.
General Procedure for Preparation of the
Tolylporphyrin-pyridine-peptidyl-phosphonates
3a–c
1,3-Dicyclohexylcarbodiimide (21 mg, 0.1 mmol)
(DCC, a coupling agent) was added with stirring
to the solution of 5-(4-carboxyphenyl)-10,15,20-p-
tritolylporphyrin (1, 70 mg, 0.1 mmol) in dry methy-
lene chloride (10 mL). Then, a solution of the hydro-
bromide of the corresponding aminophosphonate
2aꢀ–cꢀ (0.1 mmol) with triethylamine (11 mg) and
4-(dimethylamino)pyridine (12 mg, DMAP, a cata-
lyst) in methylene chloride (10 mL) was added and
the mixture was stirred for 2 h at 0◦C and left for 24 h
at room temperature. After this, the reaction mixture
was evaporated to dryness, treated with ethyl acetate
(50 mL), stirred, and filtered. The filtrate was subse-
quently washed with 0.1 M aqueous solution of citric
acid (20 mL), 1.0 M aq. NaHCO3, (2 × 20 mL), and
water (3 × 20 mL), dried (anh. Na2SO4), filtered, and
evaporated to give the crude product as a dark-violet
solid. The product was purified by column chro-
matography (silica gel: 60–230 mesh, eluant: CHCl3–
MeOH, v/v 20:1), collecting the band containing the
tritolylporphyrin-peptidyl-phosphonate 3a–c. After
the evaporation of the eluate, the particular prod-
uct (3a–c) was obtained as a dark-violet solid.
Procedure for Preparation of
5-(4-Carboxyphenyl)-10,15,20-tris-
p-tolylporphyrin (1)
In a 1-L round-bottom flask, equipped with an effi-
cient mechanical stirrer was placed consecutively:
propionic acid (600 mL), 4-tolylaldehyde (3.6 g,
30 mmol), and 4-carboxybenzaldehyde (1.5 g, 10
mmol). The mixture was heated to 140◦C and pyrrole
(6.7 g, 40 mmol) was added dropwise for 30 min with
stirring. Heating was continued for the next 30 min
and then the mixture cooled. The mixture was left
for 24 h and then a separated product was filtered
off, washed several times with water and a mixture
of water–methanol (v/v: 4:1). Washing was contin-
ued until the filtrate become colorless and odorless.
The product was dried to give a dark violet solid
(m= 2.1 g, 30%, which was a mixture of various por-
phyrins). The desired product was separated by col-
umn chromatography (silica gel 60–230 mesh, elu-
ant: chloroform–methanol; v/v 9:1), collecting the
Heteroatom Chemistry DOI 10.1002/hc

Synthesis of Peptidyl Phosphonates Containing 5-(4ꢀ-Carboxyphenyl)-10,15,20-Tritolylporphyrin 111
3a: Yield, 55 mg (50%), 1H NMR(CDCl3), δ
(ppm): 8.88–6.90 (m, 38H, aromatic protons), 6.42
(d, 1H, CH P, J = 7.67 Hz), 3.70–3.50 (m, 3H, NH,
CH protons), 3.06 (s, 3H, CH3 protons), 2.71 (s, 6H,
CH3 protons), 1.25 (m, CH3), −2.78 (bs, 2H, NH-
pyrrole). 31P NMR (CDCl3), δ (ppm): 12.84 (s). IR
(KBr) (cm−1): 3326; 2963; 2927; 2850; 1627 (C O);
1573; 1262 (P O); 891; 801; 692; 640. MS: ESI +
Q1MS, 1094 (M).
could serve as a new class of inhibitors of some
proteases.
ACKNOWLEDGMENTS
We thank Ms. Renata Grzywa and Dr. Marcin
Sien´czyk from the Wroclaw University of Technol-
ogy for performing the inhibitory tests.
3b: Yield: 60 mg (54%), 1H NMR (CDCl3), δ
(ppm): 8.88–6.50 (m, 40H, aromatic, NH and CH P
protons), 4.4–3.8 (m, 5H, NH, CH, CH3 protons),
3.03 (s, 3H, CH3), 2.71 (s, 6H, CH3), 1.1 (m., 3H,
CH3), −2.77 (bs, 2H, NH-pyrrole). 31P NMR (CDCl3),
δ (ppm): 13.29 (s). IR (KBr) (cm−1): 3326; 2928; 2850;
1626 (C O); 1575; 1311; 1244 (P O); 1227; 1088;
892; 801; 641. MS: ESI + Q1MS, 1121 (M – 1).
3c: Yield: 59 mg (53%), 1H NMR (CDCl3), δ
(ppm): 8.89–7.17 (m, 38H, aromatic protons), 7.04
(d, 1H, CH P, J = 7.72 Hz), 4.2–3.7 (m, 9H, NH, CH2,
CH protons), 2.94 (s 3H, CH3), 2.71 (s, 6H, CH3), –
2.77 (bs, 2H, NH-pyrrole). 31P NMR (CDCl3), δ (ppm):
14.29 (s). IR (KBr) (cm−1): 3323; 2927; 2850; 1646;
1626 (C O); 1473; 1222 (P O); 1069; 966; 891; 800;
693; 641. MS: ESI + Q1MS, 1120 (M).
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CONCLUSIONS
The synthesis of the three new p-tritolylporphyrin-
peptidyl-pyridine-3-yl phosphonates is described.
These compounds were obtained in a satisfactory
yield from the carboxy-p-tritolylporphyrin 1 and
phosphonodipeptides 2aꢀ–cꢀ with protected phos-
phonic acid functions by a typical peptide-synthesis
method. The obtained products showed a mod-
erate inhibitory activity toward aminopeptidase
N, an enzyme responsible for tumor cell growth.
However, these compounds and their derivatives
Heteroatom Chemistry DOI 10.1002/hc