Synthesis of 2,6-disubstituted benzylamine derivatives as reversible selective inhibitors of copper amine oxidases

Article abstract of DOI:10.1016/j.bmc.2014.01.037

In order to obtain substrate-like inhibitors of copper amine oxidases (CAOs), a class of enzymes involved in important cellular processes as well as in crosslinking of elastin and collagen and removal of biogenic primary amines, we synthesized a set of benzylamine derivatives properly substituted at positions 2 and 6 and studied their biological activity towards some members of CAOs. With benzylamines 6, 7, 8 containing linear alkoxy groups we obtained reversible inhibitors of benzylamine oxidase (BAO), very active and selective toward diamine oxidase (DAO), lysyl oxidase (LO) and monoamine oxidase B (MAO B) characterized by a certain toxicity consequent to the crossing of the brain barrier. Poorly toxic, up to very active, reversible inhibitors of BAO, very selective toward DAO, LO and MAO B, were obtained with benzylamines 10, 11, 12 containing hydrophilic ω-hydroxyalkoxy groups. With benzylamines 13, 14, 15, containing linear alkyl groups endowed with steric, but not conjugative effects for the absence of properly positioned oxygen atoms, we synthesized moderately active inhibitors of BAO reversible and selective toward DAO, LO and MAO B. The cross examination of the entire biological data brought us to the conclusion that the bioactive synthesized compounds most likely exert their physiological role of reversible inhibitors in consequence of the formation of a plurality of hydrogen bonds or hydrophobic non-covalent interactions with proper sites in the protein. Accordingly, the reported inhibitors may be considered as a set of research tools for general biological studies and the formation of enzyme complexes useful for X-ray structure determinations aimed at the design of more sophisticated inhibitors to always better modulate the protein activity without important side effects.

Full text of DOI:10.1016/j.bmc.2014.01.037

Bioorganic & Medicinal Chemistry 22 (2014) 1558–1567
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of 2,6-disubstituted benzylamine derivatives as reversible
selective inhibitors of copper amine oxidases
b,
Francesco Lucchesini a, Marco Pocci a, Silvana Alfei a, Vincenzo Bertini a, , Franca Buffoni
⇑
a Dipartimento di Farmacia, Università di Genova, Via Brigata Salerno, 13, 16147 Genova, Italy
b Dipartimento di Farmacologia Preclinica e Clinica, Università di Firenze, Viale G. Pieraccini, 6, 50139 Firenze, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
In order to obtain substrate-like inhibitors of copper amine oxidases (CAOs), a class of enzymes involved
in important cellular processes as well as in crosslinking of elastin and collagen and removal of biogenic
primary amines, we synthesized a set of benzylamine derivatives properly substituted at positions 2 and
6 and studied their biological activity towards some members of CAOs.
With benzylamines 6, 7, 8 containing linear alkoxy groups we obtained reversible inhibitors of benzyl-
amine oxidase (BAO), very active and selective toward diamine oxidase (DAO), lysyl oxidase (LO) and
monoamine oxidase B (MAO B) characterized by a certain toxicity consequent to the crossing of the brain
barrier. Poorly toxic, up to very active, reversible inhibitors of BAO, very selective toward DAO, LO and
Received 14 September 2013
Revised 29 December 2013
Accepted 21 January 2014
Available online 30 January 2014
Keywords:
Copper amine oxidases
Reversible inhibitors
2,6-Disubstituted benzylamines
Metalation
MAO B, were obtained with benzylamines 10, 11, 12 containing hydrophilic
x-hydroxyalkoxy groups.
With benzylamines 13, 14, 15, containing linear alkyl groups endowed with steric, but not conjugative
effects for the absence of properly positioned oxygen atoms, we synthesized moderately active inhibitors
of BAO reversible and selective toward DAO, LO and MAO B.
Formylation
The cross examination of the entire biological data brought us to the conclusion that the bioactive syn-
thesized compounds most likely exert their physiological role of reversible inhibitors in consequence of
the formation of a plurality of hydrogen bonds or hydrophobic non-covalent interactions with proper
sites in the protein. Accordingly, the reported inhibitors may be considered as a set of research tools
for general biological studies and the formation of enzyme complexes useful for X-ray structure determi-
nations aimed at the design of more sophisticated inhibitors to always better modulate the protein activ-
ity without important side effects.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
having one copper ion Cu2+ and one quinone cofactor per subunit,
and variable percentages of a carbohydrate portion depending on
the enzyme source.
Copper amine oxidases1 (CAOs, EC 1.4.3.6) are ubiquitous
enzymes which control important cellular processes as well as
crosslinking of elastin and collagen, by catalyzing the oxidative
deamination of primary amines RCH2NH2 according to Eq. (1).
For the various typologies of CAOs, excluding lysyl oxidase (LO),
the cofactor is 2,4,5-trihydroxyphenylalanine quinone (TPQ) which
is connected to the protein through one covalent bond enabling
TPQ to assume two conformations, active and inactive, influencing
the enzyme reaction mechanism. For LO the cofactor is lysine tyro-
sylquinone (LTQ)8 stably connected to the protein through two
covalent bonds.
Different typologies of CAOs have ‘substrate channels’ of differ-
ent dimensions, from very narrow to very wide, in agreement with
the needs of their preferential substrates.
The X-ray structures of CAOs and of several complexes of CAOs
with inhibitors of different types, together with the bulk of results
from previous studies, allowed to conceive a reasonable pathway
for a ping-pong enzyme reaction mechanism corresponding to a
sequence of reductive and oxidative half reactions. In the reductive
half reaction the substrate amine reacts with TPQ to form a
RCH2NH2 þ O2 þ H2O ! RCH@O þ NH3 þ H2O2
ð1Þ
Pluridecennial studies and several X-ray structures of copper
amine oxidases from bacteria [Escherichia coli amine oxidase
(ECAO)2 and Arthrobacter globiformis amine oxidase (AGAO)3],
yeast [Hansenula polymorpha amine oxidase (HPAO)4 and Pichia
pastoris lysyl oxidase (PPLO)5], plants [Pisum sativum pea seedling
amine oxidase (PSAO)]6 and mammals [Homo sapiens diamine oxi-
dase (hDAO)]7 allowed to ascertain that all CAOs are homodimers
⇑
Corresponding author. Tel.: +39 010 3532685.
E-mail address: bertini@difar.unige.it (V. Bertini).
Professor Emeritus of the University of Florence, deceased on November 1, 2012.
http://dx.doi.org/10.1016/j.bmc.2014.01.037
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

F. Lucchesini et al. / Bioorg. Med. Chem. 22 (2014) 1558–1567
1559
quinoneimine (substrate Schiff base) in tautomeric equilibrium
O2
H2O
O
HO
with a quinoaldimine (product Schiff base) which hydrolyzes to
release aldehyde and to produce the reduced aminoresorcinol form
of TPQ (Scheme 1).
O
NH2
NH3
H2O2
OH
OH
In the oxidative half reaction the reduced cofactor in the pres-
ence of molecular oxygen is reoxidized to TPQ with releasing of
hydrogen peroxide and ammonia (Scheme 2).
Scheme 2. Oxidative half-reaction of TPQ cofactor.
Inhibitors of different structure succeed in trapping CAOs in
stable complexes through different mechanisms of action mainly
disclosed through X-ray structure determinations as cited in the
following examples.
The 2-hydrazinopyridine inhibits ECAO by covalently binding at
position 5 of the quinone ring of TPQ in a manner similar to a sub-
strate, but the produced hydrazone analogue to a substrate Schiff
base, having a nitrogen atom in place of a CH group, prevents
any proton abstraction, thus trapping the enzyme in a covalent
complex.9
With AGAO the inhibitor 4-(2-naphthyloxy)-2-butyn-1-amine
begins to react as a substrate producing aldehyde and transforming
the cofactor into the 5-amino form of the reduced TPQ, but such
amino group binds covalently to the triple bond present in the
aldehydic product giving a stable system which does not allow
enzyme regeneration.10
Among the two trans enantiomers (1S,2R)-(+)-trans-2-phenylcy-
clopropylamine [(+)-TCP] and (1R,2S)-(ꢀ)-trans-2-phenylcyclopro-
pylamine [(ꢀ)-TCP] only the first one inhibits ECAO. Such
enantiomer can place itself into the substrate channel and form
the Schiff base at position 5 of TPQ, but the hydrogen on the carbon
of the cyclopropyl ring adjacent to the nitrogen linked to TPQ,
pointing away from the proton abstraction site on the protein
chain, cannot be removed and the enzyme reaction is blocked.11
Nevertheless, extensive dialysis of the inhibited enzyme removed
TCP, restoring the enzyme activity and indicating that binding is
reversible.12
adducts with TPQ, structural analogous to the substrate Schiff base
of TPQ14 in accordance with the behaviour of 2-hydrazinopyridine
towards ECAO (see above). The comparative study of the structures
of such adducts with the three different hydrazine derivatives pro-
vided relevant structural insights into the substrate specificity of
AGAO.
All the above reports prove the current and prominent interest
for new inhibitors of CAOs as tools for disclosing the complex bio-
logical role of each of these enzymes often endowed with broad
substrate specificity adapted to a variety of physiological needs.
In view of pharmaceutical use of CAOs inhibitors, especially if irre-
versible, such peculiar substrate specificity entails severe risk of a
complete enzyme inactivation which insidiously can open the way
to noxious interferences.7,15–19
In this light, reversible inhibitors of CAOs forming rather stable
complexes with the protein on the basis of non-covalent interac-
tions give rise to relevant expectations for bioactive molecules able
to selectively modulate the protein activity, avoiding the possible
risks inherent in irreversible inhibitors.7,20
Some of us prepared and studied21 the first substrate-like, very
active, fully reversible inhibitors of different CAOs corresponding
to derivatives of 4-aminomethylpyridine with alkoxy (Series 1),
alkylthio (Series 2) and alkylamino groups (Series 3) at positions
3 and 5 of the ring, or with alkylamino groups at position 3 (Series
4).
The inhibitory activity of the prepared compounds was tested
on different CAOs such as diamine oxidase of porcine kidney
(DAO), benzylamine oxidase of porcine serum (BAO), lysyl oxidase
of porcine aorta (LO), pea seedling amine oxidase (PSAO), Hansen-
ula polymorpha amine oxidase (HPAO), and FAD monoamine oxi-
dases of rat liver (MAO A and MAO B).
Series 1 contained reversible, very active inhibitors of BAO,
selective with respect to DAO, LO, PSAO, HPAO, MAO A and MAO B.
Series 2 contained reversible inhibitors of BAO very selective
with respect to DAO and MAO, unexpectedly affording an interest-
ing new type of good substrate of DAO.
Series 3 allowed to suppress the selectivity especially between
BAO and DAO, containing reversible inhibitors very active on both
enzymes and active on MAO A, MAO B, PSAO and HPAO.
Series 4 contained reversible non-selective inhibitors very
active on DAO, BAO and PSAO, and active on HPAO, MAO A and
MAO B.
Kinetic experiments on the enzymatic reactions performed with
4-aminomethylpyridine and benzylamine as substrates of DAO and
BAO showed an interaction for the pyridine ring stronger than that
of benzene ring with both the enzymes.
Berenil [1,3-bis(40-amidinophenyl) triazene] and Pentamidine
[1,5-bis(4-amidinophenoxy) pentane] are excellent inhibitors of
hDAO through non-covalent binding.7 They occupy the narrow
asymmetrical cone-shaped cavity of the substrate channel adopt-
ing a conformation essentially straight for Berenil and horseshoe-
shaped for Pentamidine, blocking the access of substrates to TPQ.
Though not covalently bonded to the protein, these substances
form a very stable system based on hydrogen bonds and hydropho-
bic interactions.
Two racemic aryl 2,3-butadienamine analogues, such as
5-phenoxy-2,3-pentadienylamine (POPDA) and 6-phenyl-2,3-
hexadienylamine (PHDA), are inhibitors of AGAO.13 Initially they
react with AGAO as substrates giving the corresponding aldehyde
and the 5-amino form of the reduced TPQ, but the amino group
further reacts with the activated system of the allene aldehydes
attacking different carbon atoms either with POPDA or PHDA. In
both cases, trapping TPQ in covalent complexes causes the inacti-
vation of the enzyme.
Three hydrazine derivatives, such as benzylhydrazine (BHZ),
4-hydroxybenzylhydrazine (4-OH-BHZ) and phenylhydrazine
(PHZ), structural analogues of 2-phenylethylamine, tyramine and
benzylamine proved to be the first two good substrates of AGAO
and the last poor. The X-ray structures of complexes of these inhib-
itors with AGAO evidenced the formation of covalent hydrazone
It is interesting to note that the fully reversible inhibitor
3,5-diethoxy-4-aminomethylpyridine is well absorbed orally in
rats with a bioavailability of 51.5% undergoing a rather fast trans-
formation with a blood half-life of 2.0 h.22
H2O
O
O
HO
HO
NH2CH2R
H
O
N
H
CHR
NH2
N
H
CHR
RCH=O
OH
O
OH
O
Scheme 1. Reductive half-reaction of TPQ cofactor.

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F. Lucchesini et al. / Bioorg. Med. Chem. 22 (2014) 1558–1567
CH2NH2
OCH3
CH2NH2
OH
It is reasonable to assume that the heterocyclic nitrogen atom of
HCl
H3CO
HO
4-aminomethylpyridine allows the pyridine ring to place itself in
proximity of the enzymatic active site differently from how the
benzene ring of benzylamine does, affecting both the reactivity of
the two substrates and the activity of substrate-like inhibitors
derived from them.
1) HBr/H2O
2) HCl
5
4
Scheme 3. Synthesis of inhibitor 4.
Since the study of such inhibitors and the comparison of their
structures and behaviours appeared to us worthy of attention, in
this work we report syntheses and properties of a logical set of
2,6-disubstituted benzylamines, consisting of very selective, very
active, reversible inhibitors of BAO from porcine serum which
has benzylamine as preferred substrate, like other CAOs such as
plasma amine oxidase (PAO) and tissular semicarbazide sensitive
amine oxidase (SSAO).
CH=O
RO
OR
RO
OR
.
NH2OH HCl
1) BuLi/THF
2) DMF
pyridine/EtOH
18 R=Et
19 R=Pr
22 (R=Et, 74%)
23 (R=Pr, 80%)
24 (R=Bu, 84%)
25 (R=iPr, 72%)
20 R=Bu
21 R=iPr
The set of the biological data characterizes the prepared inhib-
itors as interesting bioactive materials for biological studies and
specifically for the preparation of a variety of stable crystalline
adducts with proper CAOs for X-ray structure determinations.
CH2NH2
OR
HCl
CH=NOH
OR
RO
RO
1) Ni Raney
2) HCl/Et2O
22a (R=Et, 90%)
23a (R=Pr, 89%)
24a (R=Bu, 74% )
25a (R=iPr, 87%)
6 (R=Et, 80%)
7 (R=Pr, 25%)
8 (R=Bu,16% )
2. Results and discussion
9 (R=iPr, 53%)
In search for substrate-mimicking inhibitors of those CAOs for
which benzylamine is a good substrate, we modified the benzyl-
amine structure by introducing substituents in the benzene ring
at positions ortho to the aminomethyl group, and recorded signifi-
cant inhibitory activities.23 On that basis, we successively planned
the present study concerning mono- and disubstituted benzylam-
ines having at positions 2 and 6, either hydrocarbon substituents
with expected steric effects, or alkoxy substituents capable of both
steric and coordinative effects due to the presence of oxygen
atoms.
Scheme 4. Synthesis of inhibitors 6–9.
O(CH2)nOTHP
1) NaH/DMF
OH
1) BuLi/Et2O
2) DMF
3) concd HCl
MeOH/H2O
2) Br(CH2)nOTHP
O(CH2)nOTHP
n=2,3,4
OH
26 (n=2, 60%)
27 (n=3, 72%)
28 (n=4, 80%)
Both adequate hydrocarbon chains and properly positioned
oxygen atoms resulted essential for a good inhibitory action and
a good control, through the balance of hydrophobic and hydro-
philic groups, of the toxic effect connected to the crossing of the
blood-brain barrier.
O(CH2)nOH
CH=O
O(CH2)nOH
CH2NH2
1) H2/NH3/MeOH
Ni Raney
HCl
2) HCl/THF
O(CH2)nOH
O(CH2)nOH
10 (n=2, 78%)
11 (n=3, 78%)
12 (n=4, 78%)
29 (n=2, 28%)
30 (n=3, 43%)
31 (n=4, 43%)
Chart 1 summarizes the structures of the benzylamine deriva-
tives discussed in this paper.
2.1. Chemistry
Scheme 5. Synthesis of inhibitors 10–12.
2,6-Dihydroxybenzylamine hydrochloride (4) was obtained
from the known 2,6-dimethoxybenzylamine (5)24 through
demethylation with hydrobromic acid at reflux followed by halide
exchange with concentrated hydrochloric acid (Scheme 3).
2,6-Dialkoxybenzylamine hydrochlorides 6–9 were synthesized
through metalation of the corresponding 1,3-dialkoxybenzenes
18–21 in THF, followed by formylation,25 conversion into oximes
22a–25a, reduction of oximes with Raney alloy and reaction with
gaseous HCl (Scheme 4).
through dialkylation of resorcinol with the appropriate 2-(x-bro-
moalkoxy)tetrahydropyran to obtain 26, 27 and 28, metalation
with BuLi in diethyl ether, formylation to afford benzaldehydes
29,27 30 and 31 in moderate yields, and reductive amination
followed by reaction with gaseous HCl in THF. The reductive ami-
nation of benzaldehydes 29–31 was necessary in this case since the
conversion to the corresponding oximes proved difficult.
In order to improve the metalation/formylation step, 26 was
made to react with BuLi and DMF in THF,25 but the formation of
products which did not contain any formyl group was recorded,
presumably originating from reactions involving the 2-(2-tetrahy-
dropyranyloxy)ethoxy substituent (Scheme 6).
26
AlH3 was explored as an alternative reducing agent of the
oxime function, but when employed in a test reduction of 22a it
caused partial elimination of the ethoxy group with formation of
2-ethoxybenzylamine and it was abandoned.
The synthesis of 2,6-bis(x-hydroxyalkoxy)benzylamines
hydrochlorides 10–12 was carried out according to Scheme 5,
Reasonably, vinylether 32 was obtained by b-elimination of tet-
rahydropyranyl anion promoted by BuLi,27 while diol 33 appeared
CH2NH2 HCl
CH2NH2 HCl
R
R
R
1 (R=OH), 2 (R=OEt), 3 (R=CH2OH)
4 (R=OH), 5 (R=OMe), 6 (R=OEt), 7 (R=OPr), 8 (R=OBu),
9 (R=OiPr),10 (R=OCH2CH2OH), 11 (R=OCH2CH2CH2OH),
12 (R=OCH2CH2CH2CH2OH), 13 (R=Et), 14 (R=Pr), 15
(R=Bu), 16 (R=CH2OCH3), 17 (R=CH2OCH2CH3)
Chart 1. Molecular structures of the prepared inhibitors.

F. Lucchesini et al. / Bioorg. Med. Chem. 22 (2014) 1558–1567
1561
HO
OH
O
O
O
O
O
O
1) BuLi
26 (22%)
+
+
THPO
OTHP
2) DMF
3) H3O+
O
O
O
O
O
O
26
33 (23%)
32 (25%)
H
O
O
H
O
O
B
A
Scheme 6. Formation of side-products 32 and 33 in metalation of 26.
to originate from reaction of metalated 26 and the carbonyl form B
of the eliminated tetrahydropyranyl anion A (Scheme 6)28 (see
Supplementary data for proposed reaction schemes, experimental
details and NMR assignments).
CH2NH2
R
HCl
CH=O
CH=NOH
R
R
R
R
R
1) AlH3/THF
2) HCl/Et2O
NH2OH HCl
pyridine
14 (R= C3H7, 75%)
15 (R= C4H9, 73%)
34 (R=C3H7)
35 (R=C4H9)
36 (R=C3H7, 79%)
37 (R=C4H9, 86%)
The synthesis of hydrochloride 13 was carried out without dif-
ficulty from 2,6-diethylbenzonitrile29 (Scheme 7).
For the synthesis of hydrochlorides 14 and 15 we resorted to
benzaldehydes 34 and 35 as key intermediates (Scheme 8). The
new benzaldehyde 35 was prepared in analogy to known 3430
through a three step sequence exploiting 2-(2,6-difluoro phenyl)-
4,5-dihydro-4,4-dimethyl-1,3-oxazole31 (Supplementary data).
Scheme 9 shows the synthesis of the hydrochlorides 16–17
starting from 2,6-bis(bromomethyl)benzonitrile.32
Scheme 8. Synthesis of inhibitors 14 and 15.
such as the isopropoxy of 9, probably sterically unsuitable for the
substrate channel of the enzyme active site.
For compounds 5–8 the toxicity increased, and the LD50
decreased with the growth of the hydrophobic character of the alk-
oxy groups, being accompanied by convulsive effects on the trea-
ted animals before death, in consequence of the brain barrier
crossing.
2.2. Biology
The introduction at positions 2 and 6 of benzylamine of
-hydroxyalkoxy groups having a good hydrophilic character
The activity of benzylamines 1–17 as substrate-mimicking
inhibitors of CAOs was assayed with different copper amine oxi-
dases such as BAO from porcine serum having benzylamine as a
preferred substrate, DAO from porcine kidney having putrescine,
cadaverine, histamine as preferred substrates, LO from porcine aor-
ta having lysine residues contained in elastine or collagen as pre-
ferred substrate, and also with the FAD monoamine oxidase MAO
B from rat liver which counts among its substrates 2-phenylethyl-
amine and benzylamine.
Table 1 summarizes the results of the biological assays.
Monosubstituted benzylamines 1–3, and benzylamine deriva-
tive 4 substantially did not show any inhibitory activity (Table 1,
Chart 1).
The inhibitory activities of benzylamines 5–17 (Table 1, Chart 1)
towards DAO, LO and the FAD enzyme MAO B were negligible,
showing IC50 values always higher than 10ꢀ3 mol/L with the excep-
tion of value 1.9 ꢁ 10ꢀ4 mol/L corresponding to a modest activity
of compound 5 towards MAO B.
On the contrary, several 2,6-disubstituted benzylamines were
active towards BAO at a concentration as low as 10ꢀ7 down to
6.6 ꢁ 10ꢀ8 mol/L corresponding to very active, very selective and
reversible inhibitors.
x
(10–12, Table 1, Chart 1), gave poorly toxic, very active, reversible
inhibitors of BAO with IC50 down to 6.6 ꢁ 10ꢀ8 mol/L. These com-
pounds endowed with peripheral OHs, were considered also
attractive for the possibility of anchoring the inhibitor to polymers
or resins through one of the hydroxyl groups with a good chance of
preserving its activity, in analogy with what we obtained with the
synthesis of the bioactive resins of 2-methoxy-6-[(4-vinyl)benzyl-
oxy]benzylamine hydrochloride.33
The 2,6-dialkylbenzylamines 13, 14 and 15 proved reversible
and selective inhibitors of BAO, but significantly less active than
the dialkoxy derivatives 6, 7 and 8 which can profit from conjuga-
tive effects of the oxygen atoms bonded to the benzene ring.
The 2,6-dialkoxymethylbenzylamines 16 and 17, bearing sub-
stituents with the same number of carbon and oxygen atoms as
6 and 7, but with the oxygens not directly connected to the ben-
zene ring, were negligibly active as inhibitors of BAO. This con-
firmed the importance of conjugative effects of properly
positioned oxygen atoms, which, if far from benzene ring, repre-
sented only slightly hydrophilic segments of the substituent
chains.
The introduction of alkoxy groups at positions 2 and 6 of ben-
zylamine produced selective inhibitors of BAO with an inhibitory
activity that was modest in the case of the small methoxy groups
(5), but in the case of longer aliphatic chains as ethoxy (6), propoxy
(7) and butoxy groups (8) suddenly increased (IC50 ꢂ 10ꢀ7 mol/L),
the values recorded representing the maximum inhibition limit
for such type of compounds.
3. Conclusions
With benzylamines 6, 7, 8, (Table 1) we synthesized reversible
inhibitors of BAO, very active and selective toward DAO, LO and
MAO B. Their significant toxicity, characterized by convulsive
effects on treated animals before death, increased with the hydro-
phobic character of the inhibitor, evidencing their ability to cross
the brain barrier.
It is remarkable that the inhibitory activity dropped (IC50
>10ꢀ3 mol/L), in the presence of bulkier branched alkoxy groups
With benzylamines 10, 11, 12, we synthesized reversible inhib-
itors of BAO, poorly toxic, up to very active and selective toward
DAO, LO and MAO B, and very attractive for the possibility of
anchoring inhibitor residues to polymers or resins through the
OH groups preserving the inhibitory activity.
CN
CH2NH2
HCl
CH2CH3
H3CH2C
CH2CH
H3CH2C
3 1) LiAlH4/Et2O
2) HCl/Et2O
13 (77%)
With benzylamines 13, 14, 15, endowed with steric, hydropho-
bic, but not conjugative effects, we synthesized moderately active
Scheme 7. Synthesis of inhibitor 13.

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F. Lucchesini et al. / Bioorg. Med. Chem. 22 (2014) 1558–1567
CN
CN
CH2NH2
HCl
CH2OR
ROH2C
CH2OR
BrH2C
CH2Br
ROH2C
1) LiAlH4/Et2O
2) HCl/Et2O
RONa
38 (R=CH3, 47%)
39 (R=C2H5, 37%)
16 (R=CH3, 94%)
17 (R=C2H5, 88%)
Scheme 9. Synthesis of inhibitors 16 and 17.
Table 1
IC50(mol/L) values of the inhibitory activity of benzylamines 1–17 towards different amine oxidases
Benzylamine
BAO⁄
DAO#
LO
MAO Bà
LD50 (mg/kg)
1 R1 = H, R2 = OH
>1.0 ꢁ 10ꢀ3 ws
>1.0 ꢁ 10ꢀ3 ws
>1.0 ꢁ 10ꢀ3 ws
>1.0 ꢁ 10ꢀ3 ws
>1.0 ꢁ 10ꢀ3
>1.0 ꢁ 10ꢀ3
>1.0 ꢁ 10ꢀ3
>1.0 ꢁ 10ꢀ3
>1.0 ꢁ 10ꢀ3
>1.0 ꢁ 10ꢀ3
>1.0 ꢁ 10ꢀ3
>1.0 ꢁ 10ꢀ3
>1.0 ꢁ 10ꢀ3
>1.0 ꢁ 10ꢀ3
>1.0 ꢁ 10ꢀ3 ws
>1.0 ꢁ 10ꢀ3 ws
>1.0 ꢁ 10ꢀ3 ws
>1.0 ꢁ 10ꢀ3
>1.0 ꢁ 10ꢀ3
>1.0 ꢁ 10ꢀ3 ws
>1.0 ꢁ 10ꢀ3
>1.0 ꢁ 10ꢀ3 ws
>1.0 ꢁ 10ꢀ3 ns
>1.0 ꢁ 10ꢀ3 ws
>1.0 ꢁ 10ꢀ3 ns
>1.0 ꢁ 10ꢀ3
—
—
—
—
2 R1 = H, R2 = OCH2CH3
3 R1 = H, R2 = CH2OH
4 R1 = R2 = OH
>1.0 ꢁ 10ꢀ3 ws
>1.0 ꢁ 10ꢀ3 ws
>1.0 ꢁ 10ꢀ3 ns
5 R1 = R2 = OCH3
1.2 0.2 ꢁ 10ꢀ4 r, m, ns
1.8 0.8 ꢁ 10ꢀ7 pr, m, ns
2.4 0.6 ꢁ 10ꢀ7 pr, m, ns
1.4 0.01 ꢁ 10ꢀ7 pr, nc, ns
>1.0 ꢁ 10ꢀ3ws
>1.0 ꢁ 10ꢀ3 ws
>1.0 ꢁ 10ꢀ3 ws
>1.0 ꢁ 10ꢀ3 ws
1.9 ꢁ 10ꢀ4 ns
>1.0 ꢁ 10ꢀ3 ns
120
74 20
37
25
—
8
6 R1 = R2 = OCH2CH3
7 R1 = R2 = O(CH2)2CH3
8 R1 = R2 = O(CH2)3CH3
9 R1 = R2 = OCH(CH3)2
10 R1 = R2 = O(CH2)2OH
11 R1 = R2 = O(CH2)3OH
12 R1 = R2 = O(CH2)4OH
13 R1 = R2 = CH2CH3
14 R1 = R2 = (CH2)2CH3
15 R1 = R2 = (CH2)3CH3
16 R1 = R2 = CH2OCH3
17 R1 = R2 = CH2OCH2CH3
>1.0 ꢁ 10ꢀ3
s
5
5
>1.0 ꢁ 10ꢀ3
s
>1.0 ꢁ 10ꢀ3 ns
>1.0 ꢁ 10ꢀ3 ws
>1.0 ꢁ 10ꢀ3 ns
>1.0 ꢁ 10ꢀ3 ws
>1.0 ꢁ 10ꢀ3 ns
>1.0 ꢁ 10ꢀ3 ws
>1.0 ꢁ 10ꢀ3 ws
>1.0 ꢁ 10ꢀ3 ws
>1.0 ꢁ 10ꢀ3 ns
2.6 0.2 ꢁ 10ꢀ6 pr, m, ns
5.5 1.5 ꢁ 10ꢀ7 pr, m, ns
6.6 0.6 ꢁ 10ꢀ8 pr, m, ws
4.3 0.4 ꢁ 10ꢀ4 r, m, ns
1.5 0.2 ꢁ 10ꢀ5 r, m, ns
1.4 0.3 ꢁ 10ꢀ5 r, m, ns
>1.0 ꢁ 10ꢀ3 r, m, ns
>1.0 ꢁ 10ꢀ3 ns
>1000
690 108
540 305
92 11
55 22
54 20
240 24
—
>1.0 ꢁ 10ꢀ3
s
>1.0 ꢁ 10ꢀ3
s
>1.0 ꢁ 10ꢀ3 ws
>1.0 ꢁ 10ꢀ3 ns
>1.0 ꢁ 10ꢀ3 ws
>1.0 ꢁ 10ꢀ3 ns
>1.0 ꢁ 10ꢀ3 ws
>1.0 ꢁ 10ꢀ3 ns
>1.0 ꢁ 10ꢀ3 ns
>1.0 ꢁ 10ꢀ3 ws
Used enzymes and best substrates:⁄porcine serum benzylamine oxidase, benzylamine; #porcine kidney diamine oxidase, putrescine; porcine aorta lysyl oxidase, protein-
bound lysine; àrat liver monoamine oxidase, 2-phenylethylamine. pr = partially reversible, restored enzyme activity between 50% and 80%; r = reversible, restored enzyme
activity over 80%; nc = non competitive; m = mixed; s = substrate; ws = weak substrate; ns = non substrate.
inhibitors of BAO reversible and selective toward DAO, LO and
MAO B.
4. Experimental section
This work and the previous one,21 both aimed at the discovery
of reversible substrate-like inhibitors of CAOs able to form stable
complexes with the protein through non-covalent interactions,
allowed us to find that CAOs having benzylamine as the preferred
substrate can be reversibly inhibited by two different classes of
compounds: one correlated to benzylamine (present work) and
the other correlated to 4-aminomethylpyridine (previous work)
having a pyridine ring capable of coordinative bonds that the ben-
zene ring cannot set up.
In spite of an expected significant diversity in forming non-
covalent interactions with the protein, the present work evidences
that benzylamine-based inhibitors as those pyridine-based give
rise to reversible selective inhibitors with IC50 in the order of
10ꢀ7 mol/L.
In all likelihood, our selective, very active, site-directed, revers-
ible inhibitors of BAO exert their bioactive interactions with the
protein by forming, in virtue of their structures and distribution
of chemical functions, a plurality of non-covalent bonds, whose
knowledge could turn useful for a future inhibitor design.
On such basis the main features of the prepared inhibitors qual-
ify as a set of research tools useful for general biological studies
and the formation of enzyme complexes with CAOs for X-ray struc-
ture determinations.
The hope of obtaining new knowledge on the substrate specific-
ity of the enzyme from a comparative study of X-ray structures of
complexes obtained from a CAO with different inhibitors, finds a
significant support in the work of Murakawa et al.14 where X-ray
crystal structures of AGAO complexed with three irreversible inhib-
itors are determined and compared. By such a way, accounting for
unknown naturally broad substrate specificities of the various CAOs
and deleterious side effects observed with irreversible inhibi-
tors,7,15–19 the expectation of attaining useful suggestions for the
design of a new generation of reversible inhibitors is founded.
4.1. Chemical compounds and methods
Melting points, determined on a Leica Thermogalen III hot stage
apparatus, and boiling points are uncorrected. FTIR spectra were
recorded as films or KBr pellets on a Perkin Elmer System 2000
spectrophotometer. 1H and 13C NMR spectra were acquired on a
Bruker Avance DPX 300 Spectrometer at 300 and 75.5 MHz, respec-
tively, using TMS as internal standard.
High-resolution mass spectra were obtained by using ESI-QTOF
Agilent 6540 spectrometer interfaced with HPLC Agilent 1290
system.
Flash-chromatographic separations were performed on Merck
Silica gel (0.040–0.063 mm). TLCs were obtained on Merck F254 sil-
ica gel aluminum sheets. PLCs were performed on Merck F254 60
silica gel plates (20 ꢁ 20 cm, 0.5 mm).
All commercial reagents and products were from Sigma–Aldrich
and were purified by standard procedures. Petroleum ether refers
to the 40–60 °C fraction.
2,6-Dimethoxybenzylamine24 and its hydrochloride (5) were
obtained from commercial 2,6-dimethoxybenzonitrile. AlH3,26
2,6-diethylbenzonitrile,29 2,6-dipropylbenzaldehyde,30 2-(2,6-
difluorophenyl)-4,5-dihydro-4,4-dimethyl-1,3-oxazole,31 2,6-bis
(bromomethyl)benzonitrile,32 2-(2-bromoethoxy)tetrahydropyran,34
2-(3-bromopropoxy)tetrahydropyran,35 and 2-(4-bromobutoxy)
tetrahydropyran36 were prepared according to known
procedures.
2-Hydroxybenzylamine37 and 2-ethoxybenzylamine38 were
converted into the corresponding hydrochlorides 1 and 2 by
dissolution in dry Et2O and saturation with dry gaseous HCl. 2-
Hydroxymethylbenzylamine hydrochloride (3) was obtained from
2-hydroxymethylbenzamide39 after reduction with lithium alumi-
num hydride40 and treatment with dry gaseous HCl as described
above.

F. Lucchesini et al. / Bioorg. Med. Chem. 22 (2014) 1558–1567
1563
The 1,3-dialkoxybenzenes 18–21 were prepared by making
react 1,3-benzenediol with NaH (as 60% by weight dispersion in min-
eral oil) in DMF followed by the appropriate alkyl halide, in analogy
to the synthesis of 1,3-bis(methoxymethoxy)benzene.41 1,3-Dieth-
oxybenzene (18): from bromoethane, 78%, bp 105–107 °C/4 torr
(Lit.:42 234–235 °C). 1,3-Dipropoxybenzene (19): from 1-bromo-
propane, 66%, bp 85–87 °C/0.3 torr (Lit.43: 127–128 °C/12 torr).
1,3-Bis(1-methylethoxy)benzene (20): from 2-bromopropane,
44%, bp 103–104 °C/4 torr (Lit.44: 237–238 °C). 1,3-Dibutoxyben-
zene (21): from 1-bromobutane, 73%, bp 106–109 °C/0.3 torr
(Lit.45: 290 °C).
4.1.2.4. 2,6-Bis(1-methylethoxy)benzaldehyde (25)25
.
Yield
72%. Flash chromatography using a mixture petroleum ether/
EtOAc 15/1 as eluent. Oil. IR (film, cmꢀ1): 2774, 1688 (CHO),
1252, 1113 (C–O), 780, 722 (ring). 1H NMR (CDCl3) d: 10.49
(s, 1H), 7.35 (t, 1H, J = 8.4 Hz), 6.53 (d, 2H, J = 8.4 Hz), 4.62
(sept, 2H, J = 6.1 Hz), 1.37 (d, 12H, J = 6.1 Hz). 13C NMR (CDCl3)
d: 189.77, 160.76; 135.03, 116.88, 106.32, 71.57, 22.04.
4.1.3. Oximes 22a–25a
A solution of 2,6-dialkoxybenzaldehyde (4 mmol) in absolute
ethanol (7 mL) was added with pyridine (0.7 mL), then with
hydroxylamine hydrochloride (1.11 g, 16 mmol) and heated for
1 h at reflux under magnetic stirring. The mixture was poured into
iced water (150 mL) to precipitate the oxime initially as oil which
rapidly turned to crystals. After 2 h the oxime was filtered, washed
with water, dried at reduced pressure and crystallized.
4.1.1. 2,6-Dihydroxybenzylamine hydrochloride (4)
2,6-Dimethoxybenzylamine24 (1.60 g, 9.6 mmol) was treated
with 48% aqueous HBr (10 mL) at reflux for 3 h. After removal of
the solvent at reduced pressure, the residue was in the order trea-
ted with concd HCl (10 mL) and brought to dryness three times up
to the disappearance of bromide ion to afford 4 (1.46 g, yield 87%).
4.1.3.1. 2,6-Diethoxybenzaldehyde oxime (22a).
Yield 90%.
Mp 160–162 °C (from benzene, white crystals). 1H NMR (CDCl3)
d: 10.96 (s, 1H), 8.18 (s, 1H, OH), 7.24 (t, 1H, J = 8.4 Hz), 6.65 (d,
2H, J = 8.4 Hz), 4.04 (q, 4H, J = 7.0 Hz), 1.31 (t, 6H, J = 7.0 Hz).
Mp 203–205 °C (ethanol/Et2O). IR (KBr, cmꢀ1
) 3211, 3045
(NH+OH), 1362 (C–O), 785, 687 (ring). 1H NMR (DMSO-d6) d:
10.03 (s, 2H), 7.96 (br s, 3H), 6.97 (t, 1H, J = 8.1 Hz), 6.43 (d, 2H,
J = 8.1 Hz), 3.89 (br q, 2H, J = 3.8 Hz). 13C NMR (DMSO-d6) d:
156.91, 129.62, 106.79, 106.13, 31.78. C7H10ClNO2 requires: C
47.88; H 5.74; N 7.98. Found: C 47.72; H 5.84; N 7.94.
C11H15NO3 requires: C 63.14; H 7.23; N 6.69. Found: C 63.44; H
7.40; N 6.73.
4.1.3.2. 2,6-Dipropoxybenzaldehyde oxime (23a).
Yield 89%.
Mp 111–113 °C (from benzene/hexane, white crystals). 1H NMR
(CDCl3) d: 10.25 (s, 1H), 8.55 (s, 1H, OH), 7.20 (t, 1H, J = 8.4 Hz),
6.54 (d, 2H, J = 8.4 Hz), 4.00 (t, 4H, J = 6.7 Hz), 1.90–1.78 (m, 4H),
1.02 (t, 6H, J = 7.4 Hz). C13H19NO3 requires: C 65.80; H 8.07; N
5.90. Found: C 66.13; H 8.30; N 6.05.
4.1.2. 2,6-Dialkoxybenzaldehydes 22–25
A solution of 1,3-dialkoxybenzene (5.2 mmol) in dry THF
(31 mL) was cooled to 0 °C and treated with a 1.66 M solution of
butyllithium in hexane (6.2 mmol). The mixture was left under
magnetic stirring at room temperature for 2 h, added with dry
DMF (1.0 mL, 12.9 mmol), left under stirring for additional 2 h
and finally hydrolyzed with 0.5 M HCl (50 mL). The two phases
were stirred for 30 min then separated. The aqueous phase was
extracted with Et2O (3 ꢁ 20 mL) and the extracts were combined
with the organic phase and dried (Na2SO4). After removal of the
solvent at reduced pressure the crude benzaldehydes were
obtained and purified as described below.
4.1.3.3. 2,6-Dibutoxybenzaldehyde oxime (24a).
Yield 74%.
Mp 90–91 °C (from benzene/hexane, white crystals). 1H NMR
(CDCl3) d: 9.97 (s, 1H), 8.53 (s, 1H, OH), 7.20 (t, 1H, J = 8.4 Hz),
6.55 (d, 2H, J = 8.4 Hz), 4.04 (t, 4H, J = 6.6 Hz), 1.85–1.76 (m, 4H),
1.59–1.42 (m, 4H), 0.96 (t, 6H, J = 7.4 Hz). C15H23NO3 requires: C
67.90; H 8.74; N 5.28. Found: C 68.17; H 8.94; N 5.16.
4.1.3.4. 2,6-Bis(1-methyethoxy)benzaldehyde
oxime
(25a).
Yield 87%. Mp 153–155 °C (from benzene/hexane,
4.1.2.1. 2,6-Diethoxybenzaldehyde (22).
Yield 74%. Crystalli-
white crystals). 1H NMR (CDCl3) d: 10.03 (s, 1H), 8.48 (s, 1H, OH),
7.18 (t, 1H, J = 8.4 Hz), 6.54 (d, 2H, J = 8.4 Hz), 4.57 (sept, 2H,
J = 6.1 Hz), 1.37 (d, 12H, J = 6.1 Hz). C13H19NO3 requires: C 65.80;
H 8.07; N 5.90. Found: C 66.16; H 8.40; N 5.88.
zation from hexane. Mp 56–58 °C. IR (KBr, cmꢀ1) 2792, 1682
(CHO), 1249, 1118 (C–O), 779, 721 (ring). 1H NMR (CDCl3) d:
10.54 (s, 1H), 7.39 (t, 1H, J = 8.5 Hz), 6.54 (d, 2H, J = 8.5 Hz), 4.11
(q, 4H, J = 7.0 Hz), 1.45 (t, 6H, J = 7.0 Hz). 13C NMR (CDCl3) d:
189.61, 161.52, 135.61, 114.73, 104.63, 64.54, 14.65. C11H14O3 re-
quires: C 68.02; H 7.27. Found: C 67.98; H 6.98.
4.1.4. 2,6-Dialkoxybenzylamine hydrochlorides 6–9
A solution of oxime (2.7 mmol) in ethanol (8.3 mL) was added
with a 2 M solution of NaOH (8.3 mL) and treated in small amounts
with Nickel Raney alloy (0.65 g) under magnetic stirring. At the end
of the hydrogen evolution, the mixture was stirred for 1 h and fil-
tered. The black solid was washed with ethanol (5 ꢁ 10 mL) and
the washings were combined with the filtrate and concentrated
at reduced pressure and 60 °C. The residue was taken with water
(3 mL), brought to pH <1 with concd HCl and concentrated at
reduced pressure and 60 °C to remove most of the volatiles. The
residue was dissolved in water (5 mL), brought to pH >12 with a
2 M solution of NaOH and extracted with Et2O (3 ꢁ 20 mL). After
drying over solid NaOH, the extracts were concentrated at reduced
pressure to afford the crude amine which was dissolved in dry Et2O
(15 mL), treated with a 1.2 M solution of gaseous HCl in Et2O until
pH strongly acid to precipitate the hydrochloride which was fil-
tered, dried over P2O5 and crystallized.
4.1.2.2. 2,6-Dipropoxybenzaldehyde (23).
Yield 80%. Flash
chromatography using a mixture petroleum ether/EtOAc 10/1 as
eluent. Oil. IR (film, cmꢀ1): 2772, 1688 (CHO), 1254, 1114 (C–O),
779, 732 (ring). 1H NMR (CDCl3) d: 10.56 (s, 1H), 7.38 (t, 1H,
J = 8.5 Hz), 6.53 (d, 2H, J = 8.5 Hz), 3.99 (t, 4H, J = 6.5 Hz), 1.91–
1.79 (m, 4H), 1.05 (t, 6H, J = 7.4 Hz). 13C NMR (CDCl3) d: 189.33,
161.69, 135.54, 114.89, 104.61, 70.43, 22.47, 10.53.
HRMS (ESI) m/z: [M+H]+ calcd for C13H19O3: 223.1334; found:
223.1327.
4.1.2.3. 2,6-Dibutoxybenzaldehyde (24)25
.
Yield 84%. Flash
chromatography using a mixture petroleum ether/EtOAc 15/1 as
eluent. Oil. IR (film, cmꢀ1): 2772, 1689 (CHO), 1251, 1105 (C–O),
778, 732 (ring). 1H NMR (CDCl3) d: 10.54 (s, 1H), 7.38 (t, 1H,
J = 8.4 Hz), 6.53 (d, 2H, J = 8.4 Hz), 4.03 (t, 4H, J = 6.5 Hz), 1.87–
1.76 (m, 4H), 1.58–1.44 (m, 4H), 0.97 (t, 6H, J = 7.4 Hz). 13C NMR
(CDCl3) d: 189.37, 161.70, 135.54, 114.82, 104.54, 68.63, 31.12,
19.23, 13.82.
4.1.4.1. 2,6-Diethoxybenzylamine hydrochloride (6).
Yield
80%. Mp 199–202 °C (from acetonitrile, white crystals). IR (KBr,
cmꢀ1): 2980, 2931 (NH+CH), 1258, 1134 (C–O), 775, 724 (ring).

1564
F. Lucchesini et al. / Bioorg. Med. Chem. 22 (2014) 1558–1567
1H NMR (DMSO-d6) d: 8.22 (br s, 3H), 7.30 (t, 1H, J = 8.4 Hz), 6.67
(d, 1H, J = 8.4 Hz), 4.07 (q, 4H, J = 7.0 Hz), 3.93 (s, 2H), 1.37 (t, 6H,
J = 7.0 Hz). 13C NMR (DMSO-d6) d: 158.12, 131.07, 109.88, 104.98,
64.34, 31.65, 15.03. C11H18ClNO2 requires: C 57.02; H 7.83; N
6.04; Cl 15.30. Found: C 57.15; H 7.82; N 6.08; Cl 15.35.
6.53–6.46 (m, 3H), 4.63–4.57 (m, 2H), 4.12–4.02 (m, 4H), 3.96–
3.80 (m, 4H), 3.61–3.45 (m, 4H), 2.07 (quintet, 4H, J = 6.5 Hz),
1.88–1.46 (m, 4H). 13C NMR (CDCl3) d: 160.25, 129.76, 106.78,
101.55, 98.94, 64.92, 64.03, 62.30, 30.70, 29.70, 25.47, 19.59.
C22H34O6 requires: C 66.98; H 8.69. Found: C 66.96; H 8.89.
4.1.4.2. 2,6-Dipropoxybenzylamine hydrochloride (7).
Yield
4.1.7. 4,40-(1,3-Phenylenedioxy)bisbutanol, tetrahydropyranyl
ether (28)
25%. Mp 145–147 °C (from acetonitrile, white crystals). IR (KBr,
cmꢀ1): 2963, 2937, 2906 (NH+CH), 1259, 1134 (C–O) 778, 731
(ring). 1H NMR (DMSO-d6) d: 8.18 (br s, 3H); 7.30 (t, 1H,
J = 8.4 Hz); 6.67 (d, 2H, J = 8.4 Hz); 3.97 (t, 4H, J = 6.5 Hz); 3.94 (s,
2H); 1.85–1.73 (m, 4H), 1.00 (t, 6H, J = 7.3 Hz). 13C NMR (DMSO-
d6) d: 157.68, 130.55, 109.41, 104.44, 69.56, 31.08, 21.85, 10.37.
C13H22ClNO2 requires: C 60.11; H 8.54; N 5.39; Cl 13.65. Found:
C 60.48; H 8.60; N 5.42; Cl 13.57.
Following a similar procedure, 28 was synthesized from 1,3-
benzenediol and 2-(4-bromobutoxy)tetrahydropyran.36 Clear oil.
Yield 80%. IR (KBr, cmꢀ1) 1255, 1130, 1030 (tetrahydropyranyl
ring). 1H NMR (CDCl3) d: 7.17–7.11 (m, 1H), 6.50–6.45 (m, 3H),
4.60 (t, 2H, J = 2.7 Hz), 3.97 (t, 4H, J = 6.3 Hz), 3.91–3.77 (m, 4H),
3.54–3.41 (m, 4H), 1.92–1.52 (m, 20H). 13C NMR (CDCl3) d:
160.27, 129.76, 106.63, 101.38, 98.83, 67.63, 67.11, 62.31, 30.73,
26.35, 26.21, 25.48, 19.62. C24H38O6 requires: C 68.22; H 9.06.
Found: C 68.49; H 9.34.
4.1.4.3. 2,6-Dibutoxybenzylamine hydrochloride (8).
Yield
16%. Mp 147–149 °C (from Et2O/hexane, white solid). IR (KBr,
cmꢀ1): 2960, 2873 (NH+CH), 1255, 1127 (C–O), 775, 722 (ring).
1H NMR (DMSO-d6) d: 8.14 (br s, 3H), 7.30 (t, 1H, J = 8.4 Hz), 6.68
(d, 2H, J = 8.4 Hz), 4.01 (t, 4H, J = 6.5 Hz), 3.93 (s, 2H), 1.80–1.71
(m, 4H), 1.52–1.39 (m, 4H), 0.94 (t, 6H, J = 7.4 Hz). 13C NMR
(DMSO-d6) d: 157.69, 130.54, 109.37, 104.42, 67.80, 31.12, 30.58,
18.64, 13.70. C15H26ClNO2 requires: C 62.59; H 9.10; N 4.87; Cl
12.32. Found: C 62.88; H 9.25; N 4.87; Cl 12.45.
4.1.8. 2,6-Bis(2-hydroxyethoxy)benzaldehyde (29)27
A
solution of bis(tetrahydropyranyl) ether 26 (4.04 g,
11.0 mmol) in dry Et2O (15 mL) was treated at 0 °C under nitrogen
and stirring with a 1.66 M solution of butyllithium in hexane
(7.6 mL, 12.6 mmol). The mixture was refluxed for 4 h, cooled to
0 °C, treated with dry DMF (1.25 mL, 16.2 mmol), left overnight un-
der stirring at room temperature and hydrolyzed with water
(20 mL). The aqueous phase was separated, extracted with Et2O
(2 ꢁ 20 mL) and combined with the organic phase. All the extracts
were washed twice with a 5% solution of NaOH and dried (Na2SO4).
The removal of the solvent at reduced pressure afforded a residue
which was purified by Flash Chromatography (FC) using a mixture
of petroleum ether/acetone 6/1 as eluent to yield the bistetrahy-
dropyranyl derivative of 29 as a viscous oil (2.44 g, yield 56%).
Spectral data coincident with those known.27
The oil was taken with MeOH (18 mL), diluted with water
(8 mL), treated with concd HCl (4 mL) and left at room temperature
for 3 h. The clear solution was neutralized with solid K2CO3 and
extracted with EtOAc (8 ꢁ 15 mL). The extracts were washed with
water, dried (Na2SO4) and concentrated at reduced pressure to af-
ford 29 as oil which was crystallized from EtOAc/Et2O at ꢀ18 °C
(0.702 g, total yield 28%). Mp 101–103 °C (from EtOAc, white leaf-
lets, Lit.27 101–103 °C from EtOAc). IR (KBr, cmꢀ1) 3431, 3331 (OH),
2964, 2814 (CH2), 1678 (CH@O), 1253, 1113 (C–O), 784, 726 (ring).
1H NMR (CDCl3) d: 10.48 (s, 1H), 7.41 (t, 1H, J = 8.5 Hz), 6.56 (d, 2H,
J = 8.5 Hz), 4.17–4.11 (m, 4H), 4.01–3.95 (m, 4H), 4.33–3.87 (very
broad, 2H). 13C NMR (CDCl3) d: 190.46, 161.63, 136.38, 114.95,
105.80, 71.08, 60.88. C11H14O5 requires: C 58.40; H 6.24. Found:
C 58.23; H 6.44.
4.1.4.4. 2,6-Bis(1-methylethoxy)benzylamine hydrochloride
(9).
Yield 53%. Mp 132–134 °C (from EtOAc, white needles). IR
(KBr, cmꢀ1): 2978, 2935, 2873 (NH+CH), 1260, 1126 (C–O), 781,
729 (ring). 1H NMR (DMSO-d6) d: 8.10 (br s, 3H), 7.29 (t, 1H,
J = 8.4 Hz), 6.66 (d, 2H, J = 8.4 Hz), 4.63 (sept, 2H, J = 6.0 Hz), 3.90 (s,
2H), 1.31 (d, J = 6.0 Hz, 12H). 13C NMR (DMSO-d6) d: 156.79, 130.31,
110.80, 105.28, 70.17, 31.38, 21.70. C13H22ClNO2 requires: C 60.11;
H 8.54; N 5.39; Cl 13.65. Found: C 60.23; H 8.61; N 5.36; Cl 13.40.
4.1.5. 2,20-(1,3-Phenylenedioxy)bisethanol, tetrahydropyranyl
ether (26)
NaH (3.18 g, 79.5 mmol as 60% by weight dispersion in mineral
oil) was washed under nitrogen and stirring with dry petroleum
ether (3 ꢁ 20 mL) and suspended in dry DMF (25 mL). The suspen-
sion was treated with a solution of 1,3-benzenediol (4.00 g,
36.3 mmol) in dry DMF (15 mL) in 15 min, left under stirring at
room temperature for 3 h and added with 2-(2-bromoethoxy)tet-
rahydropyran34 (15.03 g, 71.9 mmol). The mixture was left over-
night at room temperature, hydrolyzed with water (150 mL) and
extracted with Et2O (3 ꢁ 30 mL). The extracts were washed twice
with a 10% NaOH solution, dried (Na2SO4), concentrated at reduced
pressure and freed from the volatiles at 120 °C/0.1 torr. The residue
was filtered through a short basic alumina plug (15 g) using pen-
tane (150 mL) as eluent affording after removal of pentane at
reduced pressure 26 as a clear oil (7.95 g, 60%). For characterization
purposes a sample of 26 was chromatographed (PLC) using a mix-
ture of petroleum ether/ethyl acetate = 3/1 as eluent. IR (KBr,
cmꢀ1) 1184, 1158, 1141, 1126, 1079, 1036 (tetrahydropyranyl
ring). 1H NMR (CDCl3) d: 7.19–7.11 (m, 1H), 6.56–6.51 (m, 3H),
4.74–4.67 (m, 2H), 4.19–3.77 (m, 10H), 3.58–3.46 (m, 2H), 1.93–
1.47 (m, 12H). 13C NMR (CDCl3) d: 160.10, 129.77, 107.14,
101.98, 98.97, 67.42, 65.80, 62.18, 30.52, 25.43, 19.36. C20H30O6 re-
quires: C 65.55; H 8.25. Found: C 65.64; H 8.01.
4.1.9. 2,6-Bis(3-hydroxypropoxy)benzaldehyde (30)
Benzaldehyde 30 was prepared from 27 as described above
with the difference that the bistetrahydropyranyl derivative of 30
was not submitted to FC but directly deprotected through acid
hydrolysis to afford crude 30 which was purified by FC using a mix-
ture of petroleum ether/acetone 2/1 followed by petroleum ether/
acetone 1.5/1 as eluents. Yield 43%. Mp 73–75 °C (from EtOAc,
white crystals). IR (KBr, cmꢀ1) 3396, 3208 (OH), 2944, 2782
(CH2), 1678 (CH@O), 1253, 1114 (C–O), 776, 719 (ring). 1H NMR
(CDCl3) d: 10.46 (s, 1H), 7.43 (t, 1H, J = 8.5 Hz), 6.56 (d, 2H,
J = 8.5 Hz), 4.18 (t, 4H, J = 5.9 Hz), 3.86 (t, 4H, J = 5.5 Hz), 3.68 (br
s, 2H), 2.14–2.03 (m, 4H). 13C NMR (CDCl3) d: 189.61, 161.64,
136.55, 113.79, 104.45, 67.46, 60.45, 31.84. C13H18O5 requires: C
61.40; H 7.13. Found: C 61.26; H 7.27.
4.1.6. 3,30-(1,3-Phenylenedioxy)bispropanol, tetrahydropyranyl
ether (27)
Following a similar procedure, 27 was synthesized from 1,3-
benzenediol and 2-(3-bromopropoxy)tetrahydropyran.35 Clear oil.
Yield 72%. IR (KBr, cmꢀ1) 1183, 1155, 1140, 1124, 1077, 1035
(tetrahydropyranyl ring). 1H NMR (CDCl3) d: 7.18–7.11 (m, 1H),
4.1.10. 2,6-Bis(4-hydroxybutoxy)benzaldehyde (31)
Benzaldehyde 31 was prepared as described above after direct
deprotection through acid hydrolysis of the product of lithiation/

F. Lucchesini et al. / Bioorg. Med. Chem. 22 (2014) 1558–1567
1565
formylation of 28. Crude 31 was purified by FC using a mixture of
petroleum ether/acetone 2/3 followed by petroleum ether/acetone
1/4 as eluents. Yield 43%. Mp 79–80 °C (from EtOAc, white crys-
tals). IR (KBr, cmꢀ1) 3443 (OH), 2954, 2875 (CH2), 1675 (CH@O),
1252, 1106 (C–O), 787 (ring). 1H NMR (CDCl3) d: 10.48 (s, 1H),
7.40 (t, 1H, J = 8.5 Hz), 6.54 (d, 2H, J = 8.5 Hz), 4.07 (t, 4H,
J = 5.9 Hz), 3.71 (t, 4H, J = 6.2 Hz), 2.93 (br s, 2H), 1.99–1.91 (m,
4H), 1.80–1.71 (m, 4H). 13C NMR (CDCl3) d: 189.68, 161.71,
3034, 2969, 2935 (NH+CH) 804, 761 (ring). 1H NMR (DMSO-d6) d:
8.50 (br s, 3H), 7.32–7.25 (m, 1H), 7.12 (d, 2H, J = 7.6 Hz), 4.01 (s,
2H), 2.74 (q, 4H, J = 7.5 Hz), 1.17 (t, 6H, J = 7.5 Hz). 13C NMR
(DMSO-d6) d: 143.64, 129.39, 129.05, 126.12, 34.72, 25.18, 15.17.
C11H18ClN requires: C 66.15; H 9.08; N 7.01. Found: C 65.84; H
8.99; N 6.79.
4.1.13. 2,6-Dipropylbenzylamine hydrochloride (14)
136.22, 114.24, 104.53, 68.98, 61.78, 29.72, 25.31.
requires: C 63.81; H 7.85. Found: C 63.44; H 7.90.
C
15H22O5
A
solution of 2,6-dipropylbenzaldehyde (34)30 (2.50 g,
13.1 mol) in pyridine (20 mL) was treated with hydroxylamine
hydrochloride (2.74 g, 39.4 mol) for 1 h over a boiling water bath.
The mixture was hydrolyzed with water (40 mL), treated with con-
cd HCl up to pH = 1 and extracted with Et2O (5 ꢁ 50 mL). The
organic phase and extracts were combined, dried (Na2SO4) and
concentrated at reduced pressure to afford the 2,6-dipropylbenzal-
dehyde oxime (36) (2.13 g, yield 79%). Oil. IR (KBr, cmꢀ1) 3326
(OH), 1632 (w, C@N), 774, 749 (ring). 1H NMR (CDCl3) d: 8.40 (s,
1H), 7.85 (br s, 1H), 7.24–7.18 (m, 1H), 7.10–7.04 (m, 2H), 2.72–
2.63 (m, 4H), 1.65–1.52 (m, 4H), 0.94 (t, 6H, J = 7.3 Hz). 13C NMR
(CDCl3) d: 149.62, 142.17, 128.81, 127.46, 35.99, 24.40, 14.07
(one aromatic signal is hidden). HRMS (ESI) m/z: [M+H]+ calcd for
4.1.11. 2,6-Bis(x-hydroxyalkoxy)benzylamine hydrochlorides
10–12
A solution of benzaldehyde 29–31 (40 mmol) in MeOH (7 mL)
was treated in a 125 mL autoclave with a 6.6 N ammonia solution
in MeOH (19 mL) at 80 °C and 80 atm of hydrogen pressure for 7 h
in the presence of Ni Raney (1.60 g). After filtration, the catalyst
was washed with MeOH, the washings were combined with the fil-
trate and concentrated at reduced pressure. The crude benzyl-
amine derivatives were dissolved in dry THF (200 mL) and
treated with gaseous HCl up to complete precipitation of the
hydrochlorides which were filtered, washed with THF and dried
in vacuo.
C13H20NO: 206.1545; found: 206.1538.
Oxime 36 (2.13 g, 10.4 mmol) was dissolved in dry THF (24 mL)
and treated under nitrogen and stirring at reflux for 1 h with a THF
26
4.1.11.1. 2,6-Bis(2-hydroxyethoxy)benzylamine hydrochloride
solution of AlH3 (29 mL; 0.72 M). The mixture was hydrolyzed
(10).
Yield 78%. Mp 166–168 °C (from acetonitrile/ethanol,
with water (2 mL), 10% aqueous NaOH (2 mL), filtered and the filter
cake washed with Et2O (3 ꢁ 50 mL). Filtrate and washings were
combined and dried over KOH. After removal of the solvent at
reduced pressure the oily residue was dissolved in dry Et2O
(40 mL) and saturated with gaseous HCl to afford 14 which was fil-
tered, washed with Et2O and dried at reduced pressure (1.78 g,
yield 75%). Mp 220–222 °C (acetonitrile, white solid). IR (KBr,
cmꢀ1) 2961, 2933, 2872 (NH+CH), 789, 771 (ring). 1H NMR
(DMSO-d6) d: 8.43 (br s, 3H), 7.28–7.22 (m, 1H), 7.10 (d, 2H,
J = 7.6 Hz), 4.00 (s, 2H), 2.67 (t, 4H, J = 7.8 Hz), 1.62–1.48 (m, 4H),
0.96 (t, 6H, J = 7.3 Hz). 13C NMR (DMSO-d6) d: 142.24, 129.73,
128.68, 127.08, 34.86, 34.34, 23.98, 13.89. C13H22ClN requires: C
68.55; H 9.74; N 6.15; Cl 15.57. Found: C 68.30; H 10.11; N 5.96;
Cl 15.28.
white crystals). IR (KBr, cmꢀ1) 3293 (OH), 2962, 2939 (NH+CH),
1260, 1138 (C–O). 1H NMR (DMSO-d6) d: 8.15 (br s, 3H), 7.31
(t, 1H; J = 8.4 Hz), 6.70 (d, 2H, J = 8.4 Hz), 5.17 (br s, 2H), 4.08–
4.00 (m, 6H), 3.75 (br s, 4H). 13C NMR (DMSO-d6) d: 157.70,
130.52, 110.02, 105.08, 70.41, 59.32, 31.31. C11H18ClNO4 requires:
C 50.10; H 6.88; N 5.31. Found C 49.89; H 6.87; N 5.31.
4.1.11.2. 2,6-Bis(3-hydroxypropoxy)benzylamine hydrochloride
(11).
Yield 78%. Mp 156–158 °C (from ethanol/acetonitrile 1/
3, white crystals). IR (KBr, cmꢀ1) 3325 (OH), 2947, 2886 (NH+CH),
1259, 1126 (C–O), 773, 737 (ring). 1H NMR (DMSO-d6) d: 8.12 (br s,
3H), 7.31 (t, 1H, J = 8.4 Hz), 6.69 (d, 2H, J = 8.4 Hz), 4.75 (br t, 2H,
J = 4.7 Hz), 4.09 (t, 4H, J = 6.2 Hz), 3.95 (s, 2H), 3.64-3.56 (m, 4H),
1.91 (quintet, 4H, J = 6.1 Hz). 13C NMR (DMSO-d6) d: 157.62,
130.55, 109.32, 104.41, 65.52, 57.46, 31.75, 31.28. C13H22ClNO4
requires: C 53.51; H 7.60; N 4.80. Found: C 53.12; H 7.68; N 4.35.
4.1.14. 2,6-Dibutylbenzylamine hydrochloride (15)
A solution of 2,6-dibutylbenzaldehyde (35) (Supplementary
data) (2.00 g, 9.2 mol) in pyridine (20 mL) was treated with
hydroxylamine hydrochloride (1.90 g, 27.3 mol) for 1 h at 60 °C.
The mixture was hydrolyzed with water (40 mL), treated with con-
cd HCl up to pH = 1 and extracted with Et2O (5 ꢁ 50 mL). The
organic phase and extracts were combined, dried (Na2SO4) and
concentrated at reduced pressure to afford 2,6-dibutylbenzalde-
hyde oxime (37) (1.85 g, yield 86%). Oil. IR (KBr, cmꢀ1) 3326
(OH), 1635 (w, C@N), 762, 750 (ring). 1H NMR (CDCl3) d: 8.65 (br
s, 1H), 8.39 (s, 1H), 7.23–7.18 (m, 1H), 7.06 (d, 2H, J = 7.7 Hz),
2.73–2.64 (m, 4H), 1.59–1.47 (m, 4H), 1.41–1.27 (m, 4H), 0.91 (t,
6H, J = 7.3 Hz). 13C NMR (CDCl3) d: 149.39; 142.37; 128.88;
127.28; 33.56; 33.44; 22.63; 13.95 (one aromatic signal is hidden).
HRMS (ESI) m/z: [M+H]+ calcd for C15H24NO: 234.1858; found:
234.1850.
4.1.11.3. 2,6-Bis(4-hydroxybutoxy)benzylamine hydrochloride
(12).
Yield 78%. Mp 126–128 °C (from acetonitrile, white crys-
tals). IR (KBr, cmꢀ1) 3412 (OH), 2949, 2872 (NH+CH), 1257, 1126,
(C–O), 778, 726 (ring). 1H NMR (DMSO-d6) d: 8.00 (br s, 3H), 7.30
(t, 1H, J = 8.4 Hz), 6.68 (d, 2H, J = 8.4 Hz), 4.51 (br s, 2H), 4.02 (t,
2H, J = 6.5 Hz), 3.95 (br q, 2H, J = 4.4 Hz), 3.46 (t, 2H, J = 6.4 Hz),
1.85–1.76 (m, 2H), 1.63–1.54 (m, 2H). 13C NMR (DMSO-d6) d:
157.64, 130.60, 109.29, 104.47, 68.09, 60.31, 31.26, 28.80, 25.38.
C15H26ClNO4 requires: C 56.33; H 8.19; N 4.38. Found: C 56.24; H
8.43; N 4.16.
4.1.12. 2,6-Diethylbenzylamine hydrochloride (13)
A solution of 2,6-diethylbenzonitrile29 (1.59 g, 10.0 mmol) in
dry Et2O (50 mL) was added dropwise to a suspension of LiAlH4
(0.90 g, 23.7 mol) in dry Et2O (60 mL). The mixture was refluxed
under nitrogen and stirring for 6 h, hydrolyzed with 10% aqueous
NaOH (35 mL). The aqueous phase was separated and extracted
with fresh Et2O and the extracts combined with the organic phase
were dried over KOH. After removal of the solvent at reduced pres-
sure, the residue was dissolved in Et2O (50 mL) and saturated with
gaseous HCl to precipitate hydrochloride 13 which was dried at
reduced pressure and crystallized (1.54 g, yield 77%). Mp 240–
Oxime 37 (1.80 g, 7.7 mmol) was dissolved in dry THF (20 mL)
and treated under nitrogen and stirring at reflux for 1 h with a
26
THF solution of AlH3 (30 mL; 0.64 M). The mixture was hydro-
lyzed with water (4 mL), 10% aqueous NaOH (6 mL), filtered and
the filter cake washed with Et2O (3 ꢁ 50 mL). Filtrate and washings
were combined and dried over KOH. After removal of the solvent at
reduced pressure the oily residue was dissolved in dry Et2O
(40 mL) and saturated with gaseous HCl to afford 15 which was fil-
tered, washed with Et2O and dried at reduced pressure (1.44 g,
243 °C (from acetonitrile, fluffy, white needles). IR (KBr, cmꢀ1
)
yield 73%). Mp 146–147 °C (hexane, white solid). IR (KBr, cmꢀ1
)

1566
F. Lucchesini et al. / Bioorg. Med. Chem. 22 (2014) 1558–1567
2957, 2931, 2872 (NH+CH), 805, 762 (ring). 1H NMR (DMSO-d6) d:
8.41 (br s, 3H), 7.27–7.22 (m, 1H), 7.09 (d, 2H, J = 7.6 Hz), 3.99 (s,
2H); 2.72–2.64 (m, 4H), 1.56–1.32 (m, 8H), 0.93 (t, 6H, J = 7.2 Hz).
13C NMR (DMSO-d6) d: 142.46, 129.60, 128.75, 127.00, 34.91,
33.10, 32.08, 22.11, 13.77. C15H26ClN requires: C 70.42; H 10.24;
N 5.48; Cl 13.86. Found: C 70.18; H 10.48; N 5.17; Cl 14.20.
100 8.9 nmol mLꢀ1 hꢀ1) was obtained from Sigma. LO from por-
cine aorta was prepared according to Buffoni and Raimondi.47
Mitochondria used as source of MAO
B (specific activity
12.5 0.7 nmol mLꢀ1 minꢀ1
)
were obtained from rat liver by
homogeneization 1:10 in 0.01 M phosphate buffer pH 7.4 contain-
ing 0.25 M sucrose and sequential centrifugation at 1000g for
20 min and 10,000g for 20 min.
4.1.15. 2,6-Bis(alkoxymethyl)benzonitriles 38 and 39
Solid 2,6-bis(bromomethyl)benzonitrile32 (2.00 g, 6.9 mmol)
was added to a suspension of sodium methoxide or ethoxide
(17.5 mmol) in dry DMF (12 mL) cooled to 0 °C. The dark mixture
was stirred at 0 °C for 3 h, diluted with water (20 mL) and
extracted with Et2O (5 ꢁ 30 mL). The extracts were washed with
water (20 mL), dried (Na2SO4), removed from the solvent at
reduced pressure to afford crude nitriles 38 and 39 purified as de-
scribed below.
The enzymatic activity was systematically determined using
radioactive labeled substrates as reported by Ignesti et al.48 The
labeled
substrates
such
as
[7-14C]benzylamine,
2-
phenyl[1-14C]ethylamine, and [1,4-14C]putrescine were obtained
from Amersham Biosciences. Tritiated protein-bound lysine was
prepared according to Pinnel and Martin49 with successive modifi-
cation.50 The enzymatic activity of MAO-B was assayed using
2-phenyl[1-14C]ethylamine as described by Pino et al.51 In some
cases the enzymatic reaction was followed by measuring the pro-
duction of H2O2 with the method of 4-aminoantipyrine52 either
for the activity of BAO, DAO and LO, or the evaluation of the various
compounds as substrates of the same enzymes. The protein con-
tent was determined by the method of Lowry et al.53
4.1.15.1. 2,6-Bis(methoxymethyl)benzonitrile (38).
Yield
47%. Sublimation at 80 °C/0.01 torr. Mp 80–81 °C (from hexane,
white solid). IR (KBr, cmꢀ1) 2220 (CN), 1119 (C–O), 810, 742 (ring).
1H NMR (CDCl3) d: 7.62–7.47 (m, 3H); 4.66 (s, 4H); 3.48 (s, 6H). 13
C
The type of inhibition was evaluated by examining the effect of
the inhibitor on the enzymatic reaction velocity (V) at 5 different
substrate concentrations (S) and plotting the results with the clas-
sical double reciprocal method 1/V versus 1/S proposed by Linewe-
aver and Burk.54
NMR (CDCl3) d: 142.39; 132.70; 127.47; 115.49; 110.32; 72.26;
58.87. C11H13NO2 requires: C 69.09; H 6.85; N 7.32. Found: C
69.37; H 7.15; N 7.14.
4.1.15.2. 2,6-Bis(ethoxymethyl)benzonitrile (39).
Yield 37%.
The IC50 values for the examined inhibitors were established,
as means of 4 determinations, pre-incubating the enzyme for
30 min at 30 °C with the inhibitor at 6 different concentrations,
then adding the proper labeled substrate at a saturating concen-
tration, and performing the enzyme activity tests. IC50 were esti-
mated from plots of the percentage enzyme activity remaining
after inhibition versus negative logarithm of the inhibitor
concentration.
Oil. Flash chromatography using a mixture petroleum ether/
EtOAc = 10/2 as eluent. IR (KBr, cmꢀ1) 2219 (CN),1130, 1115 (C–
O), 792, 733(w) (ring). 1H NMR (CDCl3) d: 7.61–7.47 (m, 3H),
4.69 (s, 4H), 3.64 (q, 4H, J = 7.0 Hz), 1.28 (t, 6H, J = 7.0 Hz). 13C
NMR (CDCl3) d: 142.76, 132.67, 127.38, 115.56, 110.25, 70.32,
66.71, 15.12. C13H17NO2 requires: C 71.21; H 7.81; N 6.39. Found:
C 70.98; H 7.87; N 6.30.
Preliminary tests of reversibility were performed to show that
the inhibition produced by inhibitor at IC50 concentrations was
reverted by dialysis, then the reversibility was quantified as fol-
lows. From weighted mother solutions, two initial solutions were
prepared, a solution containing enzyme and inhibitor at a con-
centration of 4 ꢁ IC50 and a control solution where the inhibitor
was replaced by an equivalent volume of phosphate buffer pH
7.4. After 5 min at 37 °C the two initial solutions were employed
in preparing diluted solutions 1:10 and 1:100 and withdrawing
aliquots for activity tests, while the residual portions were dia-
lyzed against water at 4 °C for 24 h. The initial, 1:10 and 1:100
diluted, and dialyzed solutions, with or without inhibitor, were
submitted to radiochemical enzyme activity tests using
[7-14C]benzylamine as substrate at a saturating concentration,
stopping the enzymatic reaction with 3 N HCl, extracting with
ethyl acetate and measuring the produced radioactive ben-
zaldheyde. The inhibition reversibility was determined as per-
centage of activity with respect to a reaction performed under
the same conditions without inhibitor.
4.1.16. 2,6-Bis(alkoxymethyl)benzylamine hydrochlorides 16
and 17
A solution of nitrile 38 or 39 (2.6 mmol) in dry Et2O (50 mL) was
added in 15 min under N2 and stirring to a suspension of LiAlH4
(0.40 g, 10.5 mmol) in Et2O (25 mL). The suspension was refluxed
for 5 h, cooled to 0 °C and hydrolyzed with 20% aqueous NaOH
(1.5 mL). The solid was filtered and washed with Et2O
(5 ꢁ 10 mL). Filtrate and washings were combined and concen-
trated at reduced pressure to afford an oily residue which was dis-
solved in dry Et2O (30 mL) and treated under stirring with 1.2 M
gaseous HCl in Et2O (3 mL) to afford hydrochloride 16 or 17.
4.1.16.1. 2,6-Bis(methoxymethyl)benzylamine hydrochloride
(16).
Yield 94%. Mp 167–168 °C (from acetonitrile, white crys-
tals). IR (KBr, cmꢀ1) 3152, 2998, 2935 (NH+CH), 1102, 1080 (C–O),
797, 765 (ring). 1H NMR (DMSO-d6) d: 8.28 (br s, 3H), 7.39 (s, 3H),
4.62 (s, 4H), 4.08 (s, 2H), 3.34 (s, 6H). 13C NMR (DMSO-d6) 138.43,
131.62, 129.38, 128.69, 72.02, 57.68, 34.98. C11H18ClNO2 requires:
C 57.02; H 7.83; N 6.04. Found: C 56.80; H 8.11; N 5.88.
Tests of acute toxicity (LD50) were performed on Mus musculus,
Swiss white by intraperitoneal administration of five scaled doses
to 10 animals for each dose. The mortality data were elaborated
and the standard errors calculated according to Randhawa.55 The
toxic symptomatology began after 15–20 min and was character-
ized by tonic-clonic convulsions followed by respiratory block.
No apparent toxic symptoms were observed for 48 h on survived
animals.
4.1.16.2. 2,6-Bis(ethoxymethyl)benzylamine hydrochloride
(17).
Yield 88%. Mp 129–130 °C (from EtOAc, white needles).
IR (KBr, cmꢀ1) 3144, 2970, 2862 (NH+CH), 1089, 1072 (C–O), 800,
767 (ring). 1H NMR (DMSO-d6) d: 8.30 (br s, 3H), 7.42–7.34 (m,
3H), 4.66 (s, 4H), 4.11 (s, 2H), 3.56 (q, 4H, J = 7.0 Hz), 1.18 (t, 6H,
J = 7.0 Hz). 13C NMR (DMSO-d6) 138.68, 131.49, 129.20, 128.73,
70.00, 65.28, 35.00, 14.92. C13H22ClNO2 requires: C 60.11; H 8.54;
N 5.39. Found: C 60.17; H 8.43; N 5.08.
4.2. Biological materials and methods
Acknowledgment
Pure BAO from porcine serum was prepared by the method of
Buffoni and Blaschko.46 DAO from porcine kidney (specific activity
We thank Ms Jane Erkkila for language help and University of
Genoa for financial support (Progetti di Ricerca di Ateneo).

F. Lucchesini et al. / Bioorg. Med. Chem. 22 (2014) 1558–1567
1567
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.01.037.
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