Alkylamino derivatives of 4-aminomethylpyridine as inhibitors of copper-containing amine oxidases

Article abstract of DOI:10.1021/jm0408316

The first substratelike, reversible inhibitors of different copper amine oxidases (CAOs) with IC₅₀ (M) as low as 2.0 × 10^-8 corresponding to derivatives of 4-aminomethylpyridine with alkoxy (1a-d), alkylthio (2a,b), and alkylamino (3

Full text of DOI:10.1021/jm0408316

664
J. Med. Chem. 2005, 48, 664-670
Articles
Alkylamino Derivatives of 4-Aminomethylpyridine as Inhibitors of
Copper-Containing Amine Oxidases
Vincenzo Bertini,*,† Franca Buffoni,‡ Giovanni Ignesti,‡ Nevio Picci,§ Sonia Trombino,§ Francesca Iemma,§
Silvana Alfei,† Marco Pocci,† Francesco Lucchesini,† and Angela De Munno|
Dipartimento di Chimica e Tecnologie Farmaceutiche e Alimentari, Universita` di Genova, Via Brigata Salerno,
16147 Genova, Italy, Dipartimento di Farmacologia Preclinica e Clinica, Universita` di Firenze, Viale G. Pieraccini, 6,
50139 Firenze, Italy, Dipartimento di Scienze Farmaceutiche, Universita` della Calabria, 87030 Arcavacata di Rende, Italy,
and Dipartimento di Chimica e Chimica Industriale, Universita` di Pisa, Via Risorgimento, 35, 56126 Pisa, Italy
Received April 26, 2004
The first substratelike, reversible inhibitors of different copper amine oxidases (CAOs) with
IC50 (M) as low as 2.0 × 10-8 corresponding to derivatives of 4-aminomethylpyridine with alkoxy
(1a-d), alkylthio (2a,b), and alkylamino (3a-e, 4a-j) groups in the positions 3 and 5 have
been prepared and studied. The inhibitors 1a-d are active on benzylamine oxidase and
semicarbazide-sensitive amine oxidase and are very selective with respect to diamine oxidase,
lysyl oxidase, and monoamine oxidases. The inhibitors 2a,b are selective for benzylamine
oxidase whereas 2a is also a new type of good substrate of diamine oxidase. The inhibitors
3a-e and 4a-j are substratelike, reversible, nonselective inhibitors of various CAOs including
pea seedling amine oxidase and Hansenula polymorpha amine oxidase, whose enzymatic sites
are known from X-ray structure determinations. The inhibitors 3b,c and 4b,c are excellent
substratelike tools for studies correlating CAOs that afford crystals suitable for X-ray structure
determinations with CAOs from mammals.
Introduction
oxidation of aminoquinol to TPQ with consumption of
oxygen and release of hydrogen peroxide and ammonia.
The mechanism implies the initial formation of a Schiff
base between the substrate amino group and the
quinone carbonyl group in position 5 of the cofactor,
followed by the abstraction of a proton from the CH2
next to the NH2 group, catalyzed by the carboxylate of
Asp383 in ECAO or Asp300 in PSAO.7 In substantial
agreement with the proposed mechanism, in the struc-
ture of the complex between the nonsubstratelike,
nonselective, 2-hydrazinopyridine inhibitor and ECAO,5
the inhibitor exploits its NHNH2 system for the forma-
tion of an imine double bond between the NH2 and the
C-5 carbonyl group of TPQ and of a hydrogen bond
between the NH and the protein carboxylate Asp383.
CAO inhibitors containing the CH2NH2 function have
not received proper attention for many years.8 Our
contribution in this field has been the synthesis of
substratelike, reversible CAO inhibitors, very active on
porcine serum benzylamine oxidase (BAO) and tissular
semicarbazide sensitive amine oxidase (SSAO) and
highly selective with respect to porcine kidney diamine
oxidase (DAO) and porcine aorta lysyl oxidase (LO) as
well as mitochondrial monoamine oxidase (MAO) A and
B. They are a series of 2,6-dialkoxybenzylamines9 and
3,5-diethoxy-4-aminomethylpyridine,10 the pyridine de-
rivative being more selective and less toxic.
Copper-containing amine oxidases (CAOs) are widely
distributed in prokaryotes and eukaryotes where they
catalyze the oxidative deamination of primary amines
containing the CH2NH2 group to the corresponding
aldehyde with formation of ammonia and hydrogen
peroxide. CAOs constitute a family of enzymes, often
present in the same organism, very effective on different
substrates, but poorly selective as their widespread
moderate activity shows. The progress on the knowledge
of such enzymes regarding important points such as
physiological role, nature of the cofactor, mechanism of
the enzymatic reaction, and role of the copper has
received increased attention in the last years with the
X-ray structure determination of crystalline CAOs from
various sources such as Escherichia coli (ECAO),1 pea
seedling (PSAO),2 Arthrobacter globiformis (AGAO),3
Hansenula polymorpha (HPAO),4 and of a covalent
complex ECAO-inhibitor5 useful for confirming some
aspects of the proposed ping-pong mechanism of the
enzymatic reaction.6 Such a mechanism is divided into
a first reductive half-reaction involving reduction of the
cofactor 2,4,5-trihydroxyphenylalanine quinone (TPQ)
to aminoquinol and oxidation of the amine to the
aldehyde and a second oxidative half-reaction involving
* Author to whom correspondence should be addressed. Phone: +39
010 3532670. Fax: +39 010 3532684. E-mail: bertini@dictfa.unige.it.
† Universita` di Genova.
With this work, we explore the effect on the inhibitory
activity toward different CAOs of previously prepared
alkoxy, alkylthio, and alkylamino disubstituted deriva-
tives of the 4-aminomethylpyridine dihydrochloride (12)
‡ Universita` di Firenze.
§ Universita` della Calabria.
|| Universita` di Pisa.
10.1021/jm0408316 CCC: $30.25 © 2005 American Chemical Society
Published on Web 01/11/2005

Derivatives of 4-Aminomethylpyridine
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3 665
Chart 1
Scheme 1
(1-3, Chart 1) and of totally new alkylamino monosub-
stituted derivatives of 12 (4). Two benzylamine deriva-
tives (5) have been prepared for comparison purposes
aimed to ascertain the importance of the pyridine ring.
MAO enzymes have been tested for recording their
possible sensitivity to the same products.
free bases 10a-j owing to their low stability were
identified by GC-MS and immediately transformed into
the corresponding dihydrochlorides 4a-j, which were
crystallized, some in the form of hydrates.
It is to be noted that both monoaminated (4) and
bisaminated (3) derivatives afford dihydrochlorides such
as the progenitor 12, probably by protonating the
aminomethyl group and ring nitrogen, whose positive
charge reduces the basicity of the amino or alkylamino
groups in the positions 3 and 5 of the aromatic ring.14
Scheme 2 represents the synthesis of benzylamines
11a,b and their transformation into the corresponding
dihydrochlorides 5a,b.
Chemistry
The new 3-amino- and 3-alkylamino-4-aminometh-
ylpyridine derivatives in the form of stable dihydrochlo-
rides 4a-j (Chart 1) were synthesized starting from
3-chloro-4-pyridinecarbonitrile (6)11 or 3,5-dichloro-4-
pyridinecarbonitrile (7)12 through the initial nucleophilic
substitution of a chlorine atom with ammonia or amines,
in analogy to a previous report.13 The catalytic reduction
of the cyano group, which followed the substitution, also
performed the hydrogenolysis of the residual C-Cl bond
in the intermediates 9 (Scheme 1). The compounds 4a-c
were prepared from both 6 and 7 to compare the two
procedures. Because the reagent 6, obtained according
to a known method,11 needed laborious crystallizations
to remove the 2-chloro isomer, 7 has to be considered
as a more satisfactory starting material.
Results and Discussion
Before the inhibitory activity of all of the products
containing the skeleton of 12 on different CAOs such
as DAO from porcine kidney, BAO from porcine serum,
PSAO, HPAO, and also rat liver MAO A and B was
assayed, 12 was tested as a substrate of the same
enzymes (Table 1). The deamination of 12 was modest
with DAO and MAO A and B, barely detectable with
PSAO, and remarkable with BAO and HPAO.
The use of ethanol in place of glacial acetic acid in
the catalytic hydrogenation of the pyridinecarbonitriles
8a-c and 9a-j afforded incomplete transformations of
the reagents even with prolonged reaction times. The
Table 2 compares the inhibitory activity of 1-5 for
the various enzymes and for DAO, BAO, PSAO, and

666 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3
Bertini et al.
Scheme 2
positions 3 and 5 of 12, we have found the first
substratelike, fully reversible, very active inhibitors of
BAO, selective with respect to DAO, LO, and also MAO
A and B. They are weak substrates of DAO, never above
15% of the rate of putrescine.
The compound 1a shows a low toxicity (Table 3),
which further decreases going to 1b through 1c in
accordance with the increasing hydrophilicity of the
molecule and the increasing difficulty in crossing the
brain barrier. Compound 1a is 100% reversible and
suitable for in vitro and in vivo experiments also under
oral administration.10
The compounds 1b,c, chosen to increase the hydro-
philicity of the inhibitor, can be suitable also for the
preparation of interesting glycoconjugates or polymeric
systems, properly exploiting one of the two OH groups.
In this respect, the benzyl-substituted 1d represents a
model for a polymerizable molecule having a styryl in
place of the phenyl group.
Table 1. Substrate Activity of 1 mM 12 as Percentage of the
Activity of the Best Substrate (1 mM) for Various Amine
Oxidasesa
DAO
a
BAO
b
MAO A
c
MAO B
d
PSAO
a
HPAO
d
8%
87%
3%
4%
<0.1%
45%
The inhibitor 1a tested toward PSAO and HPAO
confirmed its high selectivity for BAO.
a Enzymes: DAO ) diamine oxidase of porcine kidney; BAO )
benzylamine oxidase of porcine serum; MAO A and B ) monoam-
ine oxidases of rat liver; PSAO ) pea seedling amine oxidase;
HPAO ) Hansenula polymorpha amine oxidase. Substrates: a )
putrescine; b ) benzylamine; c ) 5-hydroxytryptamine; d )
â-phenylethylamine.
The replacement of the alkoxy with linear or branched
alkylthio groups (compounds 2a,b) still gives reversible
inhibitors of BAO, very selective with respect to DAO
and also MAO, but unexpectedly with 2a, we have found
a totally new type of good substrate of DAO (45% of the
putrescine), much better than the progenitor 12 (8%),
thus indicating an active role of the sulfur atoms. The
tert-butylthio groups of 2b still favor the inhibition of
BAO, but their bulkiness makes the sulfur atoms unable
to influence the reactivity of 2b as substrate of DAO
through the formation of coordinate bonds.
HPAO assigns the letter s to the compounds that behave
as substrates showing an oxidation rate higher than the
5% of that of the best substrate (putrescine for DAO and
PSAO, benzylamine for BAO, and â-phenylethylamine
for HPAO) or the letter n if their oxidation rate is lower
than the 5% or is under the limit of the experimental
detectability.
The replacement of the alkylthio with alkylamino
groups (compounds 3a-e) has the very important
consequence of causing the loss of selectivity in the
With 1a-d corresponding to the introduction of
alkoxy groups of different length and structure in the
Table 2. IC50(M) Values of the Inhibitors of 1-5 Series for Different Amine Oxidasesa,b
DAO
a
BAO
b
LO
c
MAO A
d
MAO B
e
PSAO
a
HPAO
e
compd
1a
1b
1c
1d
2a
2b
3a
3b
3c
3d
3e
4a
4b
4c
4d
4e
4f
>1.0 × 10-3
>1.0 × 10-3
>1.0 × 10-3
>1.0 × 10-3
1.0 × 10-3
>1.0 × 10-3
1.2 × 10-7
6.1 × 10-8
2.5 × 10-6
1.0 × 10-6
1.0 × 10-5
3.0 × 10-7
1.6 × 10-7
5.0 × 10-7
1.6 × 10-5
5.0 × 10-6
1.9 × 10-5
1.6 × 10-5
1.8 × 10-5
5.0 × 10-5
>1.0 × 10-4
>1.0 × 10-4
>1.0 × 10-4
s
1.7 × 10-7
3.2 × 10-6
1.5 × 10-7
5.0 × 10-6
1.3 × 10-7
1.6 × 10-6
1.7 × 10-6
4.4 × 10-7
2.0 × 10-8
>1.0 × 10-7
3.5 × 10-4
1.6 × 10-5
8.0 × 10-6
2.0 × 10-6
1.0 × 10-4
>1.0 × 10-4
n
n
n
n
n
n
n
s
>1.0 × 10-3
1.0 × 10-4
1.0 × 10-4
>1.0 × 10-3
n
n
n
n
>1.6 × 10-3
>1.0 × 10-3
>1.0 × 10-3
>1.0 × 10-3
1.0 × 10-3
3.3 × 10-4
n
>1.0 × 10-3
n
n
s
>1.6 × 10-3
n
s
6.3 × 10-4
>1.0 × 10-3
>1.0 × 10-3
>1.7 × 10-4
2.5 × 10-4
n
n
n
n
n
n
s
1.7 × 10-4
4.4 × 10-5
6.3 × 10-5
1.7 × 10-5
5.5 × 10-5
s
n
1.0 × 10-4
3.5 × 10-4
n
n
n
n
n
n
s
3.0 × 10-4
1.0 × 10-5
>2.0 × 10-3
4.2 × 10-4
3.0 × 10-4
2.2 × 10-3
>1.0 × 10-4
>1.0 × 10-4
>1.0 × 10-4
1.5 × 10-4
9.5 × 10-5
>1.0 × 10-4
2.0 × 10-5
>2.0 × 10-3
>1.7 × 10-4
>1.7 × 10-4
2.0 × 10-3
n
n
s
7.1 × 10-7
9.3 × 10-6
n
n
5.3 × 10-5
8.7 × 10-5
>1.0 × 10-3
>1.0 × 10-3
7.0 × 10-4
>1.0 × 10-3
>3.0 × 10-4
5.4 × 10-4
>1.0 × 10-3
7.5 × 10-4
1.5 × 10-3
n
n
s
s
s
n
n
n
n
n
n
n
n
n
9.0 × 10-5
s
1.6 × 10-5 nc
5.6 × 10-6 nc
7.9 × 10-5 nc
5.6 × 10-6 nc
1.0 × 10-4 nc
>1.0 × 10-4 sc
>1.0 × 10-4 sc
>1.0 × 10-4
>1.0 × 10-4
>1.0 × 10-4
7.0 × 10-5
n
s
4 g
4h
4i
1.0 × 10-3
8.9 × 10-4
s
s
n
n
s
4j
5a
5b
>1.0 × 10-4
>1.0 × 10-4
>1.0 × 10-4
>1.0 × 10-4
n
n
n
n
>1.0 × 10-4
a The values of IC50 (M) are the means obtained from five concentrations of each inhibitor studied in duplicate at a saturating
concentration (1 mM) of the substrate. The variability of the measures averaged 7% for DAO, BAO, LO, and HPAO, 6% for MAO A and
B, and 3% for PSAO. b Enzymes: DAO ) diamine oxidase of porcine kidney; BAO ) benzylamine oxidase of porcine serum; LO ) lysyl
oxidase of porcine aorta; MAO A and B ) monoamine oxidases of rat liver; PSAO ) pea seedling amine oxidase; HPAO ) Hansenula
polymorpha amine oxidase. Best substrates: a ) putrescine; b ) benzylamine; c ) protein-bound lysine; d ) 5-hydroxytryptamine; e )
â-phenylethylamine. s ) the compound is substrate with an oxidation rate above 5% of that of the best substrate; n ) the compound
shows an oxidation rate under 5% of that of the best substrate or it is a nonsubstrate in the limit of the experimental detectability. c Data
obtained with porcine serum from ICN.

Derivatives of 4-Aminomethylpyridine
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3 667
Table 3. Intraperitoneal LD50 of 1a-c in Mus musculus
Table 4. Kinetic Parameters of the Reactions of DAO and
BAO with 12 and Benzylamine Determined by the
Michaelis-Menten Equation
compound
LD50 mg/kg
1a
1b
1c
342
>600
>500
reagents
DAO + 12
Vmax nmol mL-1 h-1
Km µmol
21 ( 0.9
22 ( 0.3
1247 ( 169
1653 ( 39
4 ( 0.1
557 ( 39
16 ( 0.7
124 ( 17
DAO + benzylamine
BAOa+ 12
reversible inhibition of different CAOs. Compounds
3a-e are similarly active on both BAO and DAO. The
enzymes MAO A and B are always moderately inhibited.
The highest inhibitory activity is attained for DAO with
3b and for BAO with 3c. The fact that 3a, containing
an unsubstituted NH2 in the position 3, is a little less
active than 3b, more active than 3c for DAO, and less
active than 3b and 3c for BAO provides evidence that
the inhibition is essentially determined by the coordi-
native effect of the nitrogen atoms in the positions 3
and 5, even though the activity increase for BAO going
from 3b to 3c shows that for this enzyme a certain
hindrance of the alkyl chain can bring a significant
contribution.
BAOa+ benzylamine
a Porcine serum from ICN.
4b,c and test them with the main enzymes considered
in this work. The replacement of the pyridine with the
benzene ring caused a generalized collapse of the
inhibitory activity and stimulated a careful examination
of the kinetics of the enzymatic oxidations with DAO
and BAO of the substrates 12 and benzylamine, char-
acterized by a significant substrate-inhibition effect. The
kinetic parameters, determined by the Michaelis-
Menten equation, are reported in Table 4. The observed
Km values concerning the reactions performed with 12
always are smaller than those with benzylamine, indi-
cating a greater affinity of 12 for either DAO or BAO,
thus assigning to the pyridine ring an interaction with
both the enzymes stronger than that of the benzene
ring.
The inhibitors 3b,c, in accordance with their nonse-
lective character for DAO and BAO, tested toward
PSAO and HPAO showed a good inhibitory activity for
the first and modest for the second enzyme, with 3b
being more active than 3c.
Inhibitors active as weak substrates were investigated
for the effect of the preincubation time on the inhibitory
activity, and they showed a decrease of activity with the
preincubation time, in agreement with what was found
in a careful study of the mechanism of the inhibition
accomplished using 1a and a crystalline preparation of
the porcine serum benzylamine oxidase.10 Such study
indicated that kinetically 1a appears to be a mixed-
competitive site-directed inhibitor, which increases the
Km and decreases the Vm, but it can appear as a pure
noncompetitive inhibitor if it is preincubated at a
concentration lower than that of the enzyme. The
inhibitor 1a forms with the quinone in the active site
of BAO a substrate Schiff base, which probably proceeds
to a product Schiff base endowed with good stability that
sluggishly hydrolyzes to produce the aldehyde derived
from 1a and the aminoquinol which regenerates the
oxidized enzyme by oxidation with molecular oxygen.
In fact when the molar ratio 1a/BAO is higher than 1
in the presence of a saturating concentration of benzyl-
amine, the enzyme is totally inhibited excluding the
possibility of transimination, but gradually the inhibi-
tion reverts, and benzylamine is oxidized at a rate that
is proportional to the freed enzyme.
The introduction of only one alkylamino group in the
position 3 of 12 (4a-j) still produces nonselective,
reversible inhibitors for CAOs, whereas MAOs are
poorly or moderately inhibited. The highest inhibitory
activity is obtained for DAO with 4b and for BAO with
4c containing a methylamino or ethylamino group,
respectively, in analogy with the observations reported
for 3b and 3c.
By examination of the inhibitory activity for DAO and
BAO of 4a-j where the amino group at position 3 is
unsubstituted (4a), or substituted with linear, branched,
or cyclic alkyl moieties (4b-j), it appears evident that
the bioactivity strongly depends on the coordinative
effect of the nitrogen atom at the position 3. The alkyl
moiety, which transforms the amino group from primary
(4a) into secondary (4b-i) or tertiary (4j), can enhance
the bioactivity only if it is a methyl for DAO and BAO
(4b) or possesses an adequate shape for BAO (4c,f,g,i).
A comparison between 3b and 4b for DAO and 3c and
4c for BAO shows that two alkylamino groups (3b,c)
are more effective than one (4b,c), probably because the
double substitution provides the inhibitor with two
equivalent faces for the interaction with the enzymatic
site. The positive effect of the double substitution is
greater for BAO, for which 4c is also a modest substrate.
The nonselective inhibitors for DAO and BAO 4b,c,
tested toward PSAO and HPAO, proved to be better
than 3b,c, attaining a very good activity for PSAO and
good for HPAO, 4b being more active than 4c for both
the enzymes.
To ascertain if inhibitors of the series 4 with large
alkylamino groups in the position 3 could show some
selective activity for different CAOs, 4d,j were tested
toward PSAO and HPAO obtaining only modest values
of inhibitory activity.
Because very good selective inhibitors of BAO having
the structure of 2,6-dialkoxybenzylamines are known,9
it seemed right to prepare the monoalkylamino benzyl-
amines 5a,b analogous to the nonselective inhibitors
Conclusions
With 1a-d, we have found the first substratelike,
very active, nontoxic, fully reversible, useful for oral
administration inhibitors selective for BAO.
With 2a,b, we have found a new type of substratelike,
reversible inhibitors selective for BAO and also a totally
new type of good substrate for DAO.
With 3b,c and 4b,c, we have found the first nonselec-
tive inhibitors of CAOs, very active on DAO, BAO, and
also PSAO and HPAO that, contrarily to hydrazine
derivatives, are substratelike and fully reversible. The
inhibition activity of 3b,c and 4b,c decreases going from
DAO to HPAO through PSAO, and for each enzyme it
decreases going from methylamino to ethylamino sub-
stituents, i.e., from 3b to 3c and from 4b to 4c, in

668 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3
Bertini et al.
matic activities of DAO, BAO, and PSAO, as well as the
evaluations of the various products as substrate of all of the
considered enzymes, were obtained by measuring the produc-
tion of H2O2 with the method of 4-aminoantipyrine.19 The
protein content was assayed by the method of Lowry et al.20
The inhibition reversibility was ordinarily ascertained by
incubating the enzyme with the inhibitor and determining the
specific activity of the mixture after progressive dilutions in
the presence of constant substrate concentration. The revers-
ibility of the BAO inhibitor 1a was measured from the
recovered enzyme activity after exhaustive dialysis against
0.07 M phosphate buffer pH 7.4. The acute toxicity (LD50) was
studied in Mus musculus, Swiss white. Tests were carried out
by intraperitoneal administration of five scaled doses to 10
animals for each dose. The toxic symptomatology began after
15-20 min and was characterized by tonic-clonic convulsions
followed by respiratory block. The survived animals, observed
for 48 h, did not show apparent symptoms.
accordance with an active site of DAO having similari-
ties mostly with that of PSAO, within the enzymes
whose structures are known. As a result, PSAO is
expected to be a reasonable model for DAO useful for
structural studies in the presence of substratelike
inhibitors. In agreement with the observed peculiar
sensitivity of BAO to steric effects, the inhibition of such
an enzyme increases from 3b to 3c and from 4b to 4c,
with an opposite trend with respect to PSAO and HPAO,
therefore these enzymes are not fully satisfactory
models for BAO. Nevertheless, HPAO resembles BAO
in preferring primary monoamine substrates, as con-
firmed by the noticeable activity of both HPAO and BAO
toward 12 (Table 1).
The inhibition mechanism of the prepared inhibitors
deduced from kinetic data foresees the formation of a
stable Schiff base that sluggishly hydrolyzes producing
the aldehyde derived from the inhibitor and allowing
the regeneration of the enzyme.
Melting points were determined on Electrothermal ap-
1
paratus and are uncorrected. H NMR spectra were obtained
on Bruker WM-300 or AcP-300 spectrometers. Chemical shifts
are reported on the δ scale and are referred to tetramethyl-
silane. Mass spectra were recorded on Hewlett-Packard GC-
MSD 5972 instrument. IR spectra were recorded on 1000 PC
FT-IR Paragon spectrometer. Microanalyses for C, H, N, and
Cl were performed at the Microanalysis Service of the Depart-
ment of Chemistry, University of Calabria and were within
(0.4% of the theoretical values. 4-Aminomethylpyridine,
reagents, and solvents were purchased from Aldrich. Com-
pounds 1a-d,12 2a,b,21 3a-e,13 6,11 7,12 9a-d,13 11a,22 and
N-ethylisatoic anhydride23 were prepared according to known
procedures. 4-Aminomethylpyridine dihydrochloride (12) was
obtained from the corresponding commercial amine as de-
scribed for 10a-j.
The nonselective inhibitors of the series 3 and 4, for
which a plurality of factors such as the coordinative
effects of the nitrogen atoms of the amino or alkylamino
groups ortho to the aminomethyl function, the signifi-
cant interaction of the pyridine ring with the enzyme,
and also the steric effects in the case of BAO contribute
in stabilizing the enzyme-inhibitor complex, appear to
be suitable for comparative studies of CAOs from
different sources. Interesting insights regarding the
enzymatic and inhibition reactions and useful structural
indications for devising new selective inhibitors of
various CAOs in mammals are expected to be obtained
from new X-ray structure determinations of CAO com-
plexes with the best substratelike inhibitors 3b,c and
4b,c.
General Procedure for the Reaction of 3-Chloro-4-
pyridinecarbonitrile (6) and 3,5-Dichloro-4-pyridinecar-
bonitrile (7) with Amines or Ammonia. Compound 6 or 7
(2.0 mmol) and excess of the proper amine or ammonia used
as solvent were introduced at -50 °C under nitrogen into a
50-100 mL pressure resistant glass vial with a Rotaflo
stopcock and allowed to react in thermostatic bath at 80 °C
(room temperature for the synthesis of 9e). The reaction times
were determined by checking with GC-MS the increase of the
product. The mixture, after evaporation at reduced pressure
of all the volatiles, treatment with water, and extraction with
chloroform, was dried over anhydrous Na2SO4. After removal
of the solvent the crude products 8a-c and 9a-j were purified
by column chromatography on Merck neutral alumina grade
I using a mixture hexane/chloroform 1:1 as eluent and
characterized. Compounds 8a24 and 9a-d13 showed results
identical to the known ones. Products (including 8a prepared
according to Scheme 1), starting nitriles, reaction times, yields,
melting points, IR, 1H NMR, and GC-MS data are listed below.
Experimental Section
Pure BAO from porcine serum was prepared by the method
of Buffoni and Blaschko15 and used in all the applications
unless otherwise stated. Porcine serum with BAO specific
activity 1492 ( 90 nmol mL-1 h-1 was obtained from ICN
(Irvine, CA). DAO from porcine kidney, with specific activity
100 ( 8.9 nmol mL-1 h-1, was obtained from Sigma. LO from
porcine aorta was prepared according to Buffoni and Rai-
mondi.16 Tritiated protein-bound lysine was prepared accord-
ing to Melet et al.17 Homogeneate of pea seedling was prepared
from 8 days old seedling, homogeneized 1:8 in 0.1 M phosphate
buffer pH 7.4, centrifuged at 2000g for 20 min, filtered on
paper, and further centrifuged at 3000g for 30 min (PSAO
specific activity 100.1 ( 8.7 nmol mL-1 min-1). Hansenula
polymorpha wild-type NCYC-495 was incubated for 24 h in a
medium containing 2% glucose, 1 µM CuSO4, 1.7% yeast
nitrogen base without ammonium sulfate and amino acids (Y
1251, Sigma), and 0.25% methylamine hydrochloride, then it
was centrifuged four times at 1500g for 5 min. The precipitate,
stored at -80 °C, was suspended in 10 mL of 0.1 M phosphate
buffer pH 7.4, homogeneized, and centrifuged at 2500g for 30
min (HPAO specific activity 54.3 ( 0.2 nmol mL-1 h-1).
Mitochondria were obtained from rat liver by homogeneization
1:10 in 0.01 M phosphate buffer pH 7.4 containing 0.25 M
sucrose and sequential centrifugation at 1000g for 20 min and
10 000g for 20 min (specific activity MAO A 43.4 ( 2.5, MAO
B 12.5 ( 0.7 nmol mL-1 min-1).
3-Amino-4-pyridinecarbonitrile (8a). 6; 54 days; 14%;
137-138 °C (lit.24 137-138 °C). IR (KBr): 3398, 2225, 1561,
808, 593 cm-1 1H NMR (CDCl3) δ: 4.52 (br s, 2H); 7.24 (d,
.
1H, J ) 5.5 Hz); 8.04 (d, 1H, J ) 5.5 Hz); 8.28 (s, 1H). GC-MS
(EI) m/z: 119 (M+, 100%).
3-Methylamino-4-pyridinecarbonitrile (8b). 6; 24 h;
46%; 143-145 °C. IR (KBr): 3279, 2219, 1571, 805, 593 cm-1
.
1H NMR (CDCl3) δ: 3.02 (d, 3H, J ) 5.5 Hz); 4.58 (br s, 1H);
7.21 (d, 1H, J ) 5.0 Hz); 8.00 (d, 1H, J ) 5.0 Hz); 8.20 (s, 1H).
GC-MS (EI) m/z: 133 (M+, 100%). Anal. (C7H7N3): C, H, N.
3-Ethylamino-4-pyridinecarbonitrile (8c). 6; 24 h; 54%;
84-86 °C. IR (KBr): 3213, 2219, 1569, 809, 593 cm-1. 1H NMR
(CDCl3) δ: 1.35 (t, 3H, J ) 7.2 Hz); 3.36 (dq, 2H, J1 ) 7.2 Hz,
J2 ) 5.3 Hz); 4.44 (br s, 1H); 7.22 (d, 1H, J ) 5.3 Hz); 7.99 (d,
1H, J ) 5.3 Hz); 8.21 (s, 1H). GC-MS (EI) m/z: 147 (M+, 33%);
132 (100%). Anal. (C8H9N3): C, H, N.
3-Cyclopropylamino-5-chloro-4-pyridinecarbonitrile
(9e). 7; 53 h; 67%; 109-110 °C. IR (KBr): 3343, 2229, 1559,
862, 585 cm-1. 1H NMR (CDCl3) δ: 0.68 (m, 2H); 0.94 (m, 2H);
The 14C-substrates such as benzylamine, â-phenylethyl-
amine, putrescine, and 5-hydroxytryptamine were obtained
from Amersham Biosciences. All of the other reagents for
biological assays were analytical reagent grade from either
BDH, Merck, or Sigma.
The enzymatic activities of MAO and HPAO were assayed
using 14C-substrates as described by Pino et al.18 The enzy-

Derivatives of 4-Aminomethylpyridine
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3 669
2.61 (m, 1H); 5.09 (br s, 1H); 8.04 (s, 1H); 8.48 (s, 1H). GC-MS
(EI) m/z: 193 (M+, 68%); 192 (100%). Anal. (C9H8N3Cl): C, H,
N, Cl.
0.69 (m, 2H); 0.94 (m, 2H); 2.59 (m, 1H); 4.27 (s, 2H); 7.83 (m,
1H); 8.19 (m, 1H); 8.46 (s, 1H). GC-MS (EI) m/z: 163 (M+,
36%), 145 (100%). Anal.(C9H18N3Cl2O1.5): C, H, N, Cl.
3-Cyclopentylamino-5-chloro-4-pyridinecarbonitrile
(9f). 7; 36 h; 48%; 119-120 °C. IR (KBr): 3239, 2223, 1559,
846, 589 cm-1. 1H NMR (CDCl3) δ: 1.56 (m, 2H); 1.75 (m, 4H);
2.12 (m, 2H); 3.95 (m, 1H); 4.60 (br s, 1H); 7.96 (s, 1H); 8.10
(s, 1H). GC-MS (EI) m/z: 221 (M+, 40%); 192 (100%). Anal.
(C11H12N3Cl): C, H, N, Cl.
3-Cyclopentylamino-4-aminomethylpyridine Dihydro-
chloride Hemihydrate (4f). 9f; 79%; 258 °C (dec). IR (KBr):
3543, 3312, 1545, 804, 578 cm-1 1H NMR (CD3OD) δ: 1.69
.
(m, 4H); 1.84 (m, 2H); 2.15 (m, 2H); 3.94 (m, 1H); 4.33 (s, 2H);
7.80 (d, 1H, J ) 5.9 Hz); 8.12 (d, 1H, J ) 5.9 Hz); 8.19 (s, 1H).
GC-MS (EI) m/z: 191 (M+, 33%), 122 (100%). Anal. (C11H20N3-
Cl2O0.5): C, H, N, Cl.
3-Cyclohexylamino-5-chloro-4-pyridinecarbonitrile
(9g). 7; 24 h; 59%; 124-125 °C. IR (KBr): 3341, 2231, 1560,
844, 587 cm-1. 1H NMR (CDCl3) δ: 1.34 (m, 5H); 1.69 (m, 1H);
1.82 (m, 2H); 2.07 (m, 2H); 3.47 (m, 1H); 4.53 (br d, 1H, J )
7.4 Hz); 7.93 (s, 1H); 8.08 (s, 1H). GC-MS (EI) m/z: 235 (M+,
33%); 192 (100%). Anal. (C12H14N3Cl): C, H, N, Cl.
3-Cycloheptylamino-5-chloro-4-pyridinecarbonitrile
(9h). 7; 24 h; 74%; 83-84 °C. IR (KBr): 3355, 2227, 1561, 847,
3-Cyclohexylamino-4-aminomethylpyridine Dihydro-
chloride Monohydrate (4g). 9g; 76%; 246 °C (dec). IR
(KBr): 3437, 3326, 1545, 807, 578 cm-1. 1H NMR (CD3OD) δ:
1.46 (m, 5H); 1.73 (m, 1H); 1.85 (m, 2H); 2.08 (m, 2H); 3.48
(m, 1H); 4.31 (s, 2H); 7.78 (d, 1H, J ) 6.1 Hz); 8.08 (d, 1H, J
) 6.1 Hz); 8.22 (s, 1H). GC-MS (EI) m/z: 205 (M+, 15%), 122
(100%). Anal. (C12H23N3Cl2O): C, H, N, Cl.
586 cm-1 1H NMR (CDCl3) δ: 1.64 (m, 11H); 2.04 (m, 1H);
.
3.64 (m, 1H); 4.57 (br d, 1H, J ) 7.4 Hz); 7.94 (s, 1H); 8.01 (s,
1H). GC-MS (EI) m/z: 249 (M+, 23%); 192 (100%). Anal.
(C13H16N3Cl): C, H, N, Cl.
3-Cycloheptylamino-4-aminomethylpyridine Dihydro-
chloride Monohydrate (4h). 9h; 47%; 249 °C (dec). IR
1
(KBr): 3425, 1550, 799, 547 cm-1. H NMR (CD3OD) δ: 1.69
(m, 11H); 2.03 (m, 1H); 3.31 (m, 1H); 4.28 (s, 2H); 7.78 (d, 1H,
J ) 5.5 Hz); 8.08 (s, 1H); 8.09 (d, 1H, J ) 5.5 Hz). GC-MS (EI)
m/z: 219 (M+, 35%), 122 (100%). Anal. (C13H25N3Cl2O): C, H,
N, Cl.
3-Cyclohexylmethylamino-5-chloro-4-pyridinecarbo-
nitrile (9i). 7; 24 h; 49%; 149-150 °C. IR (KBr): 3346, 2231,
1
1566, 847, 585 cm-1. H NMR (CDCl3) δ: 1.01 (m, 2H); 1.23
(m, 3H); 1.73 (m, 6H); 3.14 (m, 2H); 4.75 (br s, 1H); 7.96 (s,
1H); 8.07 (s, 1H). GC-MS (EI) m/z: 249 (M+, 19%); 166 (100%).
Anal. (C13H16N3Cl): C, H, N, Cl.
3-Cyclohexylmethylamino-4-aminomethylpyridine Di-
hydrochloride Monohydrate (4i). 9i; 48%; 242 °C (dec). IR
(KBr): 3382, 3308, 1551, 809, 553 cm-1. 1H NMR (CD3OD) δ:
1.49 (m, 11H); 3.15 (d, 2H, J ) 7.8 Hz); 4.28 (s, 2H); 7.76 (d,
1H, J ) 5.5 Hz); 8.08 (d, 1H, J ) 5.5 Hz); 8.14 (s, 1H). GC-MS
(EI) m/z: 219 (M+, 19%), 119 (100%). Anal. (C13H25N3Cl2O):
C, H, N, Cl.
3-(1-Piperidinyl)-5-chloro-4-pyridinecarbonitrile (9j).
7; 1 h; 91%; 55-56 °C. IR (KBr): 2230, 1554, 863, 583 cm-1
.
1H NMR (CDCl3) δ: 1.66 (m, 2H); 1.79 (m, 4H); 3.34 (m, 4H);
8.20 (s, 1H); 8.27 (s, 1H). GC-MS (EI) m/z: 221 (M+, 66%);
220 (100%). Anal. (C11H12N3Cl): C, H, N, Cl.
3-(1-Piperidinyl)-4-aminomethylpyridine Dihydro-
chloride Hemihydrate (4j). 9j; 59%; 245 °C (dec). IR (KBr):
3449, 1520, 820, 559 cm-1. 1H NMR (CD3OD) δ: 1.70 (m, 2H);
1.82 (m, 4H); 3.07 (m, 4H); 4.50 (s, 2H); 8.09 (d, 1H, J ) 6.1
Hz); 8.63 (d, 1H, J ) 6.1 Hz); 8.65 (s, 1H). GC-MS (EI) m/z:
191 (M+, 24%), 173 (100%). Anal. (C11H20N3Cl2O0.5): C, H, N,
Cl.
General Procedure for the Reduction of Nitriles 8a-c
and 9a-j to Amines 10a-j and Their Transformation
into Dihydrochlorides 4a-j. A 0.3 M solution of 8a-c or
9a-j in glacial acetic acid was treated with H2 at 1.5 atm in
the presence of 10% Pd on activated charcoal (3.5 g per mmol
of nitrile) for 1-16 h. The slurry was then filtered, and the
filtrate was concentrated at reduced pressure, treated with 1
N aqueous NaOH, extracted with chloroform, and dried over
anhydrous Na2SO4. After removal of the solvent at reduced
pressure, the crude amines 10a-j were dissolved in anhydrous
THF and saturated with gaseous HCl to afford the dihydro-
chlorides 4a-j, which were crystallized from 95% ethanol and
characterized. Products, starting nitriles, yields, melting
points, IR, 1H NMR data, and GC-MS of the free bases are
listed below.
2-Methylaminobenzylamine Dihydrochloride (5a). Ac-
cording to Coyne and Cusic,22 the commercial N-methylisatoic
anhydride was transformed into 2-methylaminobenzamide.
(Yield 80%; mp 169-171 °C (95% ethanol). IR (KBr): 3401,
1
3357, 3172, 1660, 1617, 747 cm-1. H NMR (CDCl3) δ: 2.84
(d, 3H, J ) 5.1 Hz); 6.53 (m, 1H); 6.67 (dd, 1H, J1 ) 7.6 Hz, J2
) 0.9 Hz); 6.97-7.53 (very br, NH2); 7.30 (m, 1H); 7.63 (dd,
1H, J1 ) 7.9 Hz, J2 ) 1.5 Hz); 8.09 (br s, NH). GC-MS (EI)
m/z: 150 (M+, 100%), 133 (49%).) This was subsequently
transformed into 2-methylaminobenzylamine (11a). (Oil; yield
3-Amino-4-aminomethylpyridine Dihydrochloride (4a).
8a; 72%; 279 °C (dec). IR (KBr): 3472, 3289, 1531, 795, 604
cm-1. H NMR (CD3OD) δ: 4.28 (s, 2H); 7.78 (d, 1H, J ) 5.8
91%. IR (film): 3367, 3330, 1607, 1587, 1519, 1470, 749 cm-1
.
1
Hz); 8.08 (d, 1H, J ) 5.8 Hz); 8.20 (s, 1H). GC-MS (EI) m/z:
GC-MS (EI) m/z: 136 (M+, 100%), 118 (91%).) The compound
11a (4.0 mmol) was converted into its dihydrochloride by
dissolution in dry ethyl ether (50 mL) and saturation with
gaseous HCl. The crude product was crystallized to give white
crystals of 5a. (Yield 89%; mp 205 °C (95% ethanol) (lit.25 208-
210 °C). IR (KBr): 3008-2589 (broad), 1591, 1500, 1475, 1457
cm-1. 1H NMR (CD3OD) δ: 3.13 (s, 3H); 4.39 (s, 2H); 4.95 (br
123 (M+, 100%). Anal. (C6H11N3Cl2): C, H, N, Cl.
3-Methylamino-4-aminomethylpyridine Dihydrochlo-
ride (4b). 8b; 60%; 256 °C (dec). IR (KBr): 3286, 1551, 801,
592 cm-1. 1H NMR (CD3OD) δ: 2.97 (s, 3H); 4.26 (s, 2H); 7.76
(d, 1H, J ) 5.5 Hz); 8.13 (d, 1H, J ) 5.5 Hz); 8.18 (s, 1H).
GC-MS (EI) m/z: 137 (M+, 43%), 119 (100%). Anal. (C7H13N3-
Cl2): C, H, N, Cl.
+
s, NH2 + NH3+); 7.52-7.74 (m, 4H). Anal. (C8H14N2Cl2): C,
H, N, Cl.)
3-Ethylamino-4-aminomethylpyridine Dihydrochlo-
ride (4c). 8c; 65%; 217 °C (dec). IR (KBr): 3362, 3294, 1569,
2-Ethylaminobenzylamine Dihydrochloride (5b). The
N-ethylisatoic anhydride, prepared from isatoic anhydride
according to Hardtmann,23 was transformed as described above
into 2-ethylaminobenzamide. (Yield 20%; mp 127-128 °C (95%
ethanol). IR (KBr): 3404, 3185, 1642, 1619, 748 cm-1. 1H NMR
(CDCl3) δ: 1.29 (t, 3H, J ) 7.2 Hz); 3.20 (q, 2H, J ) 7.2 Hz);
5.78 (br s, NH2); 6.56 (m, 1H); 6.69 (dd, 1H, J ) 7.9 Hz); 7.34
(m, 2H); 7.70 (br s, NH). GC-MS (EI) m/z: 164 (M+, 60%), 132
(100%).) This was subsequently transformed into 2-ethylami-
nobenzylamine (11b). (Oil; yield 98%. IR (film): 3374, 3301,
1606, 1588, 1519, 1471, 749 cm-1. GC-MS (EI) m/z: 150 (M+,
54%), 118 (100%).) And this was finally transformed into white
crystals of 5b. (Yield 66%; mp 185-188 °C (propanol/95%
ethanol 1:1). IR (KBr): 3038-2645 (br), 1592, 1519, 1505, 1473
1
816, 550 cm-1. H NMR (CD3OD) δ: 1.35 (t, 3H, J ) 7.3 Hz);
3.33 (q, 2H, J ) 7.3 Hz); 4.27 (s, 2H); 7.71 (d, 1H, J ) 5.3 Hz);
8.11 (d, 1H, J ) 5.3 Hz); 8.15 (s, 1H). GC-MS (EI) m/z: 151
(M+, 29%), 119 (100%). Anal. (C8H15N3Cl2): C, H, N, Cl.
3-[(1-Methylethyl)amino]-4-aminomethylpyridine Di-
hydrochloride (4d). 9d; 70%; 197 °C (dec). IR (KBr): 3279,
1562, 806, 524 cm-1 1H NMR (CD3OD) δ: 1.29 (d, 6H, J )
.
6.4 Hz); 3.79 (sept, 1H, J ) 6.4 Hz); 4.11 (s, 2H); 7.22 (d, 1H,
J ) 5.0 Hz); 7.88 (d, 1H, J ) 5.0 Hz); 8.03 (s, 1H). GC-MS (EI)
m/z: 165 (M+, 38%), 133 (100%). Anal.(C9H17N3Cl2): C, H, N,
Cl.
3-Cyclopropylamino-4-aminomethylpyridine Dihydro-
chloride Sesquihydrate (4e). 9e; 47%; 254 °C (dec). IR
(KBr): 3412, 3288, 1554, 812, 578 cm-1. 1H NMR (CD3OD) δ:
1
cm-1. H NMR (CD3OD) δ: 1.46 (t, 3H, J ) 7.3 Hz); 3.53 (q,

670 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3
Bertini et al.
+
2H, J ) 7.3 Hz); 4.45 (s, 2H); 4.99 (br s, NH2 + NH3+); 7.64
(10) Buffoni, F.; Bertini, V.; Dini, G. The Mechanism of Inhibition of
Benzylamine Oxidase by 3,5-Diethoxy-4-aminomethylpyridine
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(m, 3H); 7.78 (m, 1H). Anal. (C9H16N2Cl2): C, H, N, Cl.)
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Acknowledgment. This work was financially sup-
ported by MIUR (Programmi di Ricerca di Rilevante
Interesse Nazionale 2002) and university funds.
(12) Bertini, V.; Lucchesini, F.; Pocci, M.; De Munno, A. 3,5-Dichloro-
4-Pyridinecarbonitrile as a Key Reagent in a Synthesis of Copper
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Supporting Information Available: Elemental analyses.
This material is available free of charge via the Internet at
http://pubs.acs.org.
(14) Beltrame, P.; Cadoni, E.; Floris, C.; Gelli, G.; Lai, A. 15N and
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