Synthesis of tetrakis(multifluoro-4-pyridyl)porphin derivatives as acetylcholinesterase inhibitors

Article abstract of DOI:10.1016/S0960-894X(00)00254-7

New tetrakis(multifluoro-4-pyridyl)porphin derivatives (2-4) and water soluble porphyrin (5) were synthesized to investigate their interactions with acetylcholinesterase from electric eel. These compounds have been found to be the potent reversible inhibitors of the enzyme with K(i) values of μM range. In addition, porphyrin (5) showed broad spectrum of anticancer activities. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00254-7

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1435±1438
Synthesis of Tetrakis(multi¯uoro-4-pyridyl)porphin Derivatives
as Acetylcholinesterase Inhibitors
Sin Chul Moon,a Jung-Hyu Shin,a Bong Ho Jeong,b Hee Seok Kim,b
Byung Soo Yu,b Ji-Sook Lee,c Bang Sook Leec and Sung Keon Namgoongc,*
aDepartment of Chemistry, Seoul National University, Seoul 151-742, South Korea
bDepartment of Chemistry, Wonkwang University, Iksan, Chonbuk 570-749, South Korea
cDepartment of Chemistry, Seoul Women's University, Seoul 139-774, South Korea
Received 10 February 2000; accepted 22 April 2000
AbstractÐNew tetrakis(multi¯uoro-4-pyridyl)porphin derivatives (2±4) and water soluble porphyrin (5) were synthesized to inves-
tigate their interactions with acetylcholinesterase from electric eel. These compounds have been found to be the potent reversible
inhibitors of the enzyme with Ki values of mM range. In addition, porphyrin (5) showed broad spectrum of anticancer activities.
# 2000 Elsevier Science Ltd. All rights reserved.
The porphyrins play important roles in divergent ®eld
of research, including catalysis, solar energy conversion,
spectroscopy and the development of organic material.1
Furthermore, these compound have been well known to
show selective anity to tumor cells2,3 and applied for
photodynamic cancer therapy4 7 as a photosensitizer
and even for antiviral treatment.8 Another recent topic
relating with porphyrins is the studies on the treatment
for Alzheimer's Disease (AD) in connection with acetyl-
cholinesterase (AChE) inhibitors.9 AChE is a signi®cant
enzyme in the central and peripheral nervous systems
for the transmission of nerve impulses across nerve±
nerve and neuromuscular synapses.10 Among the vari-
ous approaches to cholinergic enhancement, inhibition
of the acetylcholine (ACh) degrading enzyme, AChE is
presently the most promising in terms of providing
candidate drugs for alleviating the symptoms of AD.
Porphyrin inhibitors of AChE are especially valuable in
the sense that they usually have low cytotoxicity and
possibility to reach to the central nervous system through
the blood brain barrier.11 Currently, several synthetic
meso-porphyrin derivatives (1a, 1b) which have
di¯uorophenyl substituents on the periphery of the por-
phin skeleton turned out to be potent AChE inhibitors.9
di€erent active sites in the enzyme; hydrogen bonding site,
negatively charged site and two di€erent hydrophobic
sites. These porphyrins might be ®t to this model. The
peripheral aryl groups in the porphyrins can bind to the
hydrophobic region 2. The partial positive charge on the
porphyrin skeleton developed by the electronegative
¯uorine atoms on the aromatic rings can interact with the
negatively charged site in the enzyme.9 In case of water
soluble porphyrin salts, the positive charge on the peri-
pheral pyridinium salt can also interact with this enzyme
active site.12 The precise sculpturing of the porphyrin
environments to be a promising inhibitor seems that the
peripheral tetra-aryl in the porphyrin should have at
least a ¯uorine substituent at either 2 or 6-position of
them.
We designed new porphyrin compounds (2±4) and water
soluble porphyrin (5) as potential AChE inhibitors. As
shown in Figure 1, porphyrins (2±5) have four multi-
¯uoropyridyl functionalities to ful®ll the above assump-
tion. Particularly porphyrin (5) could be the more useful
inhibitor in view of its solubility in aqueous solution. In
addition, it is interesting to examine how the number of
¯uorine substituents present on the aromatic rings
a€ects the interaction with AChE.
Recently, Sugimoto et al. proposed a model of AChE
active cites.12 According to this model, there are four
Herein we describe the synthesis and preliminary inhi-
bition data of porphyrins (2±5). Electric eel AChE was
used to determine inhibition constant, Ki on the enzyme
assay. All the synthetic compounds were isolated either
by ¯ash chromatography or recrystallization. Porphyrin
*Corresponding author. Tel.: +82-970-5660; e-mail: sknam@swu.ac.kr;
sknam@yahoo.com
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00254-7

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S. C. Moon et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1435±1438
(2) was successfully prepared in two steps from 2,3,5,6-
tetra¯uoropyridine-4-carbonitrile (6) in overall 3.2%
yield (Scheme 1). The highly volatile aldehyde (7) can be
obtained by the reduction of (6) with diisobutylalumi-
num hydride (DIBAH) at low temperature.13 The for-
mation of porphyrin is known to depend on a variety of
factors such as oxidant, catalyst, solvent and reaction
concentration. Macrocyclization to form porphyrin (2)
seems not to be a€ected by these factors.14 Typical reac-
tion between equimolar amount of the aldehyde (7) and
pyrrole in re¯uxing propionic acid a€orded reasonable
yield (7%) of the porphyrin (2). The synthesis of por-
phyrin (3) was achieved in six steps (overall 5% yield).
The hydride reduction of the aldehyde (7) and the
selective displacement of 6-¯uorine with hydrazine pro-
duced the 6-hydrazinopyridine-4-carbinol (9).15 The
sequential removal of hydrazine by copper sulfate and
Swern oxidation16 of the subsequent alcohol (10) gave the
aldehyde (11). The condensation reaction between equi-
molar amount of the aldehyde (11) and pyrrole in the
presence of BF3.etherate and the subsequent oxidation
with p-chloranil led to produce the corresponding por-
phyrin (3). The optimized condition typically gave 29%
of porphyrin (3). All the intermediates (8±11) in this
synthesis were also new compounds.
The porphyrins (2, 3) were not basic enough to form
ionic, water-soluble porphyrins. Porphyrin (4) was
Figure 1.
Scheme 1.

S. C. Moon et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1435±1438
1437
Scheme 2.
synthesized in two steps from 3-¯uoropyridine (12) in
overall 5% yield (Scheme 2). Lithiation of the pyridine
(12) and the sequential reaction with methyl formate
introduced the formyl group exclusively at C-4 position of
the pyridine.17 The typical reaction of the aldehyde (13)
with pyrrole in propionic acid generated 9% of porphyrin
(4). This method was usually best to produce porphyrin
(4). It is remarkable that suciently basic porphyrin (4)
was extracted with dichloromethane in concentrated
HCl solution. Methylation of porphyrin (4) with methyl
p-toluene sulfonate a€orded the excellent yield (90%) of
the water soluble porphyrin (5).18 Porphyrins (4, 5) are
also new compounds. The structures of porphyrins (2±
5) were con®rmed by spectroscopic analyses.
the penta¯uorophenylporphin (F20TPP) and ¯uoropyr-
idylporphins (2±5) are in mM range. All the inhibitors (2±
5) having ¯uorine atoms are reversible competitive
inhibitors since they have the increased Km values and
little e€ect on the Vmax values in these inhibition reac-
tions. In the case of the porphyrins containing hydroxyl
groups, they are either reversible noncompetitive inhi-
bitors or reversible mixed type inhibitors for AChE. The
¯uoropyridylporphins are more potent inhibitors than
penta¯uorophenylporphin. Neither 2-hydroxyphenyl nor
3-hydroxyphenyl group gives much di€erence in the
potency of the porphyrin inhibitors. The 2,5-dihydroxy-
phenyl group increased the inhibition potency of the
porphyrin by 10-fold compared to 3-hydroxyphenyl
group. All these results suggest that the compounds
should have 2 or 6-¯uoroaryl or 2-hydroxyaryl substitu-
tion at the periphery of the porphyrin to be the good
porphyrin-based AChE inhibitors. Similar porphyrin
compounds without ¯uorine or hydroxyl group are
found to be ine€ective in the AChE inhibition, which
suggests that the presence of these groups in the inhibi-
tors might be e€ective in the process of the enzyme
inhibition.
The inhibition constants, KI, of the porphyrin inhibitors
determined by Km=Vmax versus inhibitor concentration
replot are shown in Table 1. The inhibition constants of
Table 1. The inhibition constants of the porphyrin inhibitorsa
Inhibitors
Ki (m M)b
(Km=Ki)c
F20TPP9
(1a)9
15.2Æ4.30
0.005Æ0.001
0.012Æ0.002
7.29Æ1.24
2.85Æ0.42
5.08Æ0.8
3.06Æ0.4
50.9Æ2.5
9.7Æ0.6
9.0
26,000
12,000
19.2
(1b)9
(2)
Km=Ki Ratio roughly estimates the relative binding a-
nity of an inhibitor to the enzyme compared to the
substrate.9 Replacing carbon with nitrogen in phenyl
groups of porphyrin mostly increased the binding a-
nity by 2±5-fold except the porphyrins (1a, 1b). We have
also reported the porphyrins (1a, 1b) were much more
potent inhibitors of AChE than ordinary inhibitors con-
taining quaternary ammonium salt functionalities.9 How-
ever these compounds (1a, 1b) are rarely soluble in water.
Therefore it had diculties in controlling the concentration
of inhibitors when animal test was carried out. Water
soluble porphyrin (5) is more useful AchE inhibitors in
this sense.
(3)
(4)
(5)
49.1
27.6
45.8
2.7
14.3
7.6
4.7
55.6
2-FTPP20
3-FTPP20
2-(OH)TPP20
3-(OH)TPP20
2,5-(OH)2TPP20
18.3Æ0.7
29.6Æ1.2
2.5Æ0.1
aTimecourses for the AChE-catalyzed hydrolysis of ATCh were fol-
lowed by monitoring the formation of thioanion of nitrobenzoic acid
at 412 nm by Ellman's coupled enzyme assay. Reactions were run in
duplicate.
bKi is determined from the Km=Vmax versus inhibitor concentration replot.
cKm of acetylthiocholine (ATCh) for AChE is 0.14 mM. Porphyrins
(2±5) contain ¯uoropyridyl groups and the others have ¯uorophenyl
groups in their structures.
Moreover porphyrin (5) showed broad spectrum of
anticancer activities; based on our preliminary studies,19

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S. C. Moon et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1435±1438
porphyrin (5) had potent antiangiogenic activity in vivo
(more than 60% inhibition at 10 mg does per egg) on chor-
ioallantoic membrane (CAM) of growing chick embryos.
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It also exhibited good anticancer activity ex vivo on HL-
60 leukemia cell, B16 BL6 melanoma cell and in vitro
antimutational activities in Ames tests (data not shown
here). Broad anticancer activities of porphyrin (5) are
presumably considered to depend on the ¯uorine sub-
stituent and its proper position on the pyridine ring.
Considering all the results mentioned in this study, syn-
thetic manipulation of its derivatives could be another
issue in the ®eld of chemotherapeutics. The detailed bio-
logical tests of the water soluble porphyrin (5) and its
derivatization is recently in progress.
11. Yu, B. S.; Kim, S. M.; Kim, K. Y.; Lee, B. H. Unpub-
lished results.
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Acknowledgements
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This work was supported by a grant from the Seoul
Women's University, 1998 and from Wonkwang Uni-
versity, 1999.
References and Notes
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