Cholinesterase inhibitors: SAR and enzyme inhibitory activity of 3-[ω-(benzylmethylamino)alkoxy]xanthen-9-ones

Article abstract of DOI:10.1016/j.bmc.2006.09.026

In this work, we further investigated a previously introduced class of cholinesterase inhibitors. The removal of the carbamic function from the lead compound xanthostigmine led to a reversible cholinesterase inhibitors 3. Some new 3-[ω-(benzylmethylamino)alkoxy]xanthen-9-one analogs were designed, synthesized, and evaluated for their inhibitory activity against both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The length of the alkoxy chain of compound 3 was increased and different substituents were introduced. From the IC₅₀ values, it clearly appears that the carbamic residue is crucial to obtain highly potent AChE inhibitors. On the other hand, peculiarity of these compounds is the high selectivity toward BuChE with respect to AChE, being compound 12 the most selective one (6000-fold). The development of selective BuChE inhibitors may be of great interest to clarify the physiological role of this enzyme and to provide novel therapeutics for various diseases.

Full text of DOI:10.1016/j.bmc.2006.09.026

Bioorganic & Medicinal Chemistry 15 (2007) 575–585
Cholinesterase inhibitors: SAR and enzyme inhibitory activity
of 3-[x-(benzylmethylamino)alkoxy]xanthen-9-ones
Lorna Piazzi, Federica Belluti, Alessandra Bisi, Silvia Gobbi, Stefano Rizzo,
Manuela Bartolini, Vincenza Andrisano, Maurizio Recanatini and Angela Rampa*
Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy
Received 12 July 2006; revised 7 September 2006; accepted 13 September 2006
Available online 27 September 2006
Dedicated to prof. Piero Valenti on the occasion of his retirement
Abstract—In this work, we further investigated a previously introduced class of cholinesterase inhibitors. The removal of the car-
bamic function from the lead compound xanthostigmine led to a reversible cholinesterase inhibitors 3. Some new 3-[x-(benzylmeth-
ylamino)alkoxy]xanthen-9-one analogs were designed, synthesized, and evaluated for their inhibitory activity against both
acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The length of the alkoxy chain of compound 3 was increased
and different substituents were introduced. From the IC50 values, it clearly appears that the carbamic residue is crucial to obtain
highly potent AChE inhibitors. On the other hand, peculiarity of these compounds is the high selectivity toward BuChE with respect
to AChE, being compound 12 the most selective one (6000-fold). The development of selective BuChE inhibitors may be of great
interest to clarify the physiological role of this enzyme and to provide novel therapeutics for various diseases.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
of neurons and synapses in many areas of the CNS, espe-
cially in the regions involving higher order cognitive func-
tions, such as hippocampus and the association cortex.3
In addition, there is a global and dramatic reduction of
the neurotransmitters level, for example, noradrenaline,
dopamine, serotonin, glutamate, substance P, and acetyl-
choline (ACh). This depletion of neurotransmitters,
among which ACh is the most important one, is almost
certainly the cause for the broad clinical manifestations
of AD: memory impairment, hallucinations, paranoia,
restlessness and depression.4
Alzheimer’s disease (AD), the most common form of
dementia, is a chronic, neurodegenerative disorder,
which is characterized by loss of cognitive ability, severe
behavioral abnormalities and ultimately total degrada-
tion of intellectual and mental activities.
Although the origin of AD is still unknown, three path-
ological hallmarks have been identified, for example,
amyloid-b plaques, neurofibrillary tangles (NFTs) and
synaptic loss.1,2 The neuritic senile plaques consist of a
fibrillar amyloid core surrounded by dystrophic neurites
and reactive microglia.
These findings led to the development of the cholinergic
hypothesis that, at least in its earlier formulation, pre-
dicted a favourable effect for compounds able to sustain
the central cholinergic tone.5–7 The cholinergic hypothe-
sis provided the first rational approach to the treatment
of AD. To date only acetylcholinesterase (AChE) inhib-
itors, such as tacrine, donepezil, rivastigmine, and galan-
tamine and uncompetitive moderate affinity NMDA
receptor antagonist, memantine, are available for AD
treatment. On the other side in recent times, the role
of butyrylcholinesterase (BuChE) inhibition in AD has
received increasing attention, both from the medicinal
chemistry and the clinical points of view.8,9 Several lines
of evidence indicate that BuChE might be a co-regulator
The ante mortem diagnosis of AD is clinically problemat-
ic, and ambiguity arises from numerous other causes of
dementia that are pathologically unrelated to AD. At
autopsy, the AD brain is characterized by a number of
important pathological changes. There is a marked loss
Keywords: Alzheimer; Cholinesterase inhibitors; Xanthones; Xantho-
stigmine analogs.
*
Corresponding author. Fax: +39 051 2099734; e-mail: angela.
rampa@unibo.it
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.09.026

576
L. Piazzi et al. / Bioorg. Med. Chem. 15 (2007) 575–585
of the activity of the neurotransmitter acetylcholine.10
Remarkably, cortical levels of BuChE show a significant
increase in AD. In this respect, the research efforts are
focused on the development of selective inhibitors that
are necessary to clearly evaluate the role of the enzyme
and the therapeutic feasibility of its inhibition.
In previous papers we introduced modifications in the
linker chain, the carbamoyl substituent and the aryl
moiety of xanthostigmine in order to investigate the struc-
ture–activity relationships (SAR) of this series of com-
pounds.13,14
A three-dimensional model of the quaternary complex
between AChE and xanthostigmine showed that, by
properly extending the alkoxy chain of aminoalkoxyaryl
derivatives, it might be possible to reach the peripheral
anionic binding site in such a way that the heteroaryl
moiety would have the possibility to establish p-stacking
interactions with the indole ring of Trp286 located at the
entrance of the human AChE gorge. This aromatic res-
idue, together with other ones, is considered to consti-
tute an essential part of the AChE peripheral binding
site,15 and recent studies have shown that inhibitors
binding to this site are able to block the Ab aggregation
induced by AChE.16,17
Our research group has been involved for many years in
the development of AChE inhibitors as potential drugs
for AD. We designed and studied a class of N-methyl-
N-(3-carbamoyloxyphenyl)methylamino
derivatives
characterized by the presence of a heteroaryl moiety
linked to the tertiary amino nitrogen through an alkoxy
chain. Xanthostigmine (28a, Chart 1), the lead com-
pound, showed the highest activity (IC50 = 0.3 nM).11,12
O
CH
3
In this work, we intended to further investigate the inter-
actions of xanthostigmine analogues with the most
important residues of the AChE gorge by removing
the carbamic function from xanthostigmine. With this
modification, a reversible inhibitor was obtained (3,
Table 1). The length of the alkoxy chain of compound
3 was increased up to 12 methylene units (compounds
O
O(CH ) NCH
2 n 2
OCONHCH
3
n = 3
n = 7
28a (xanthostigmine)
28b
Chart 1.
Table 1. Physicochemical and analytical data of the compounds studied
O
R1
R3
R4
R6
R2
(CH ) NCH
R5
R
O
O
2 n
2
Compound
n
R
R1
R2
R3
R4
R5
R6
Mp (ꢁC)
Yield (%)
3
4
3
4
5
6
7
8
9
H
H
H
H
H
H
H
H
H
H
H
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Et
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
H
H
51–52a
60
50
50
50
50
50
40
60
80
50
50
50
90
90
70
70
40
30
30
25
30
90
40
40
70
H
H
67–69a
5
H
H
55–58a
6
H
H
158–160b
50–55a
7
H
H
8
H
H
86–89b
9
H
H
132–133a
108–109b
140–141b
100–102b
110–113b
97–98b
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
10
11
12
7
H
H
H
H
H
H
OCH3
OCH3
H
H
7
OCH3
H
H
3
OCH3
OCH3
H
189–191a
120–122b
101–103b
153–154b
131–133b
144–147b
88–90b
131–133b
161–164b
138–139b
oila
54–55b
124–126b
7
H
H
7
H
H
OCH3
H
7
H
H
H
Cl
OCH3
H
7
H
H
H
Cl
7
H
H
H
H
Cl
7
H
H
H
H
7
H
H
CH3
NO2
OCH3
OCH3
OCH3
OCH3
H
H
7
H
H
H
H
7
H
H
OCH3
H
H
7
H
H
H
7
H
H
OCH3
H
OCH3
H
7
H
H
a Free base (crystallizing from ligroin).
b Hydrochlorides (crystallizing from methanol–ether).

L. Piazzi et al. / Bioorg. Med. Chem. 15 (2007) 575–585
577
4–12) to identify the optimal distance between the xan-
2. Chemistry
thone moiety and the amino function. One or two meth-
oxy groups were introduced in positions 6 and 7 of the
xanthone nucleus (compounds 13 and 14), as these sub-
stituents could favour the interaction with the PAS, as
seen for both Donepezil18 and the inhibitor AP2238.19
The basicity of the amino function is crucial for the
inhibitory activity: the nitrogen has not to be protonated
to cross the BBB, but has to be protonated for the inter-
action with the central anionic site (AS). Therefore we
modified the basicity of the amino group introducing
some substituents on the phenyl ring (compounds 15–
26). Finally, the importance of the substituent on the
protonable amine was studied by replacing the methyl
group with an ethyl group (compound 27).
The synthesis of the studied compounds was accom-
plished as illustrated in Scheme 1. The 3-hydroxyxan-
then-9-one was treated with 1-bromo-x-chloroalkanes
or x-dibromoalkanes (according to commercial avail-
ability) in the presence of K2CO3 to afford x-haloalkoxy
derivatives 1a–j. The x-chloroalkyl derivatives 1a–d
were treated with NaI in refluxing methyl ethyl ketone
to give the corresponding x-iodoalkoxy intermediates
2a–d.
For the synthesis of 1k and 1l different routes were fol-
lowed. The 6-chloro-2,3-dimethoxyxanthen-9-one 30
was synthesized from 2-chloro-4,5-dimethoxybenzoic
O
O
O
O
O
R3
R4
R6
iii
i
R2
O
O(CH2)nNCH2
R5
O(CH2)nX
OH
1a-d: n=3-6; X=Cl
1e-j: n=7-12; X=Br
2a-d: n=3-6; X=I
ii
3-12: n=3-12; R2=CH3; R3=R4=R5=R6=H
15-27: n=3,7; see Table 1
COOH
H3CO
H3CO
COOH
Cl
H3CO
iv
v
+
HO
Cl
H3CO
O
O
Cl
29
O
O
O
H3CO
H3CO
H3CO
H3CO
H3CO
H3CO
vii
vi
O
Cl
O
OBn
OH
30
31
32
i
O
O
R1
H3CO
R1=H,OCH3
O
O
O
i
O(CH2)7Br
viii
1k-l
HO
OH
H3CO
O
OH
R1
33
iii
O
O
CH3
H3CO
O(CH2)7NCH2
13-14
Scheme 1. Reagents and conditions: (i) Br(CH2)nCl or Br(CH2)nBr, K2CO3 reflux, 24 h; (ii) NaI, methylethylketone, reflux 4 h; (iii) selected
N-benzylalkylamine, toluene, reflux 15 h; (iv) K2CO3, Cu, CuI, nitrobenzene, 170 ꢁC, 8 h; (v) P2O5, H3PO4, 110 ꢁC, 4 h; (vi) C6H5CH2OH, KOH,
N(C4H8)4Br; (vii) H2, Pd/C, rt; (viii) CH3I, C6H5CH2N(C2H5)3Cl.

578
L. Piazzi et al. / Bioorg. Med. Chem. 15 (2007) 575–585
acid through reaction with 3-chlorophenol in nitroben-
zene in the presence of K2CO3, Cu and CuI affording
the corresponding diphenyl ether 29 and subsequent
cyclization by means of polyphosphoric acid. The chlo-
roxanthone 30 was then treated with benzyl alcohol and
KOH in the presence of the transfer-phase catalyst tetra-
butylammonium bromide to afford the benzyloxy deriv-
ative 31, which was then debenzylated by H2 over Pd/C
at room temperature and pressure to give the hydroxy
derivative 32.
the method of Ellmann21 to determine the rate of hydro-
lysis of acetylthiocholine or butyrylthiocholine in the
presence of the inhibitor.
4. Results and discussion
The inhibitory activities against both erythrocyte AChE
and serum BuChE of 3-[x-(benzylmethylamino)alk-
oxy]xanthen-9-one derivatives together with that of the
reference compounds xanthostigmine (28a) and 28b
are reported in Table 2, expressed as IC50 values.
3,6-Dihydroxyxanthen-9-one20 was methylated by
means of methyl iodide and NaOH in the presence of
the transfer-phase catalyst benzyltriethylammonium
chloride to afford 3-hydroxy-6-methoxyxanthen-9-one
33. Compounds 32 and 33 were then alkylated as previ-
ously described to give 1k and 1l. The x-haloalkoxy
derivatives 1e–l and 2a–d were then condensed with
the selected N-benzylalkylamine to give the final com-
pounds 3–27.
From the IC50 values of compounds 3–12, it appears
that variations of the chain length (n in the general for-
mula) influenced both AChE and BuChE activities.
Remarkably, the behavior of the series is rather uniform
for AChE and BuChE inhibition up to a chain length of
seven methylene units, while it dramatically diverges
from eight carbon units forward. This clearly appears
from the plot of Figure 1, where the variations of inhib-
itory potency (expressed as pIC50) against both AChE
and BuChE are reported as a function of the number
of methylene units of compounds 3–12. As already dis-
cussed in previous works,12,13 in which 28a and its seven
methylene analogue 28b showed the highest inhibitory
potency against AChE, also in this series of non-car-
bamic analogues the best results were obtained for the
same cholinesterase with compounds 3 and 7, having
three and seven methylene units, respectively (3
IC50 = 2.68 lM and 7 IC50 = 2.82 lM). However, a pe-
culiar feature of compounds 8–12 is the high inhibitory
activity for BuChE, and in particular compound 8 (eight
The N-benzylalkylamines were prepared by condensa-
tion of the selected benzaldehydes and amines followed
by NaBH4 reduction.
The structure and the physicochemical data of com-
pounds 3–27 are reported in Table 1.
3. Enzyme inhibition
The inhibitory activity of the newly synthesized com-
pounds against both cholinesterases was studied using
Table 2. Inhibitory activity on isolated AChE and BuChE and IC50 ratio of the compounds studied
Compound
IC50(lM)
AchE
IC50 (lM)
BuChE
Ratio IC50
BuChE/AchE
Ratio IC50
AChE/BuChE
3
4
2.68 0.09
6.25 0.85
7.78 0.54
2.89 0.23
2.82 0.61
10.3 6.50
152
8.33 0.89
2.65 0.14
2.49 0.22
1.43 0.06
0.80 0.04
0.08 0.01
0.10 0.02
0.10 0.01
0.12 0.01
0.15 0.02
46.1 3.90
3.08 0.35
1.91 0.17
0.93 0.03
1.85 0.08
2.11 0.13
269 75
3.11
0.42
0.32
0.49
0.28
0.008
6.6
0.32
2.36
3.12
2.02
3.52
129
5
6
7
8
9
1520
3880
5217
6053
0.17
0.75
1.78
2.34
21.6
171
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28a
28b
388
626
2.6
1.92
1.65
5.9
908
7.84 0.51
2.32 0.13
3.40 0.51
2.18 0.16
40.0 3.80
360
1.33
0.56
0.43
0.05
0.006
0.62
0.10
0.12
0.05
0.03
0.014
0.01
0.006
0.10
160
433
594
1.6
9.3
63.8 3.1
127 48
1050
565
8.26
19.15
29.5
68.65
100
29.5 1.1
14.0 1.1
2.01 0.10
1.27 0.08
1.00 0.04
0.32 0.01
48 4 nM
16.5 1.4 nM
413
138
127
155
155
9.8
3.15 0.20
0.30 0.01 nM
0.32 0.09 nM
0.0062
0.019
51.6

L. Piazzi et al. / Bioorg. Med. Chem. 15 (2007) 575–585
579
Considering these results, we introduced electron with-
drawing and electron donating substituents in the ortho
position, to evaluate the importance of this modifica-
tion in stabilizing the formal positive charge on the
amine function, that is known to be crucial for the
interaction with the AS. As already shown in the liter-
ature for AChE activity,22 for this class of compounds
as well, the only accepted substituent on the benzylami-
no moiety seems to be the ortho methoxy group. This
hypothesis was confirmed by the introduction of one
or two methoxy groups in compound 16, which led
to less active compounds (24–26). Any substituent
which decreased activity toward AChE increased the
selectivity to BuChE. In an attempt to rationalize this
SAR, we considered that in this latter enzyme,
Lys286 and Val288 line the acyl pocket and that these
aminoacid residue side chains are not large, compared
to the phenylalanine side chains of the corresponding
residues of AChE. For this reason, it may be that
BuChE tolerated all the substituents we introduced in
the phenyl ring positions, with the exception of chlo-
rine atom. Accordingly to this hypothesis, also the
introduction of two or three methoxy groups led to
compounds showing a good BuChE inhibitory activity:
in particular 26 was 155 times more active on BuChE
then on AChE.
7
6
5
4
3
AChE
BuChE
2
4
6
8
n
10
12
14
Figure 1. Plot of pIC50 values for AChE (Ç) and BuChE (h) inhibition
against the number of methylene units of compounds 3–12.
methylene units) is a potent BuChE inhibitor having
IC50 = 80 nM. For these compounds, it appears that
the loss of the carbamic function and of the consequent
ability to covalently bind the BuChE active site is at
least partly compensated by a higher propensity to fill
the enzyme cavity and to stabilize its occupancy possibly
by means of hydrophobic interactions. In fact, the previ-
ously reported carbamic analogs of 8–12 showed IC50
values against BuChE in the range of 13.9–31.6 nM,13
which are only 3–6 times lower than those of the present
derivatives. On the contrary, the loss of activity against
AChE resulting from the elimination of the carbamic
function is of the order of thousands of times. Summa-
rizing, the effects of varying the chain length of the pres-
ent non-carbamic xanthostigmine derivatives, we point
out that the elongation of the chain proportionally in-
creased the selectivity of these compounds for BuChE,
which resulted more than 100-fold for 8 and 6000-fold
for 12, bearing a 12 methylene units chain.
Finally, the substitution of the methyl group on the ami-
no function (27) with an ethyl group maintained the
activity for AChE, while it increased the potency toward
BuChE with respect to compound 7.
5. Conclusions
A SAR study of non-carbamic derivatives of xantho-
stigmine (28a) was performed with the aim of explor-
ing the anti-cholinesterase potential of corresponding
reversible inhibitors. From the IC50 values shown in
Table 2, it clearly appears that the carbamic residue
is crucial to obtain highly potent AChE inhibitors.
However, a peculiar feature of compounds 8–12 of
this class is the high selectivity for BuChE shown
by derivatives in which the distance between the xan-
thone moiety and the benzylamino group is more
than seven methylene units. This could be explained
by the presence of a larger cavity in BuChE with re-
spect to the narrow gorge of AChE, that could allow
longer compounds to fold and better fill the hydro-
phobic cavity.
We further investigated the interaction of non-carbam-
ic xanthostigmine analogues with AChE and BuChE
by introducing one or two methoxy groups in posi-
tions 6 and 7 of the xanthone moiety. Both the meth-
oxy and the dimethoxy derivatives 13 and 14 inverted
the selectivity of the parent compound 7, allowing us
to speculate about a possible interaction of the meth-
oxy substituent with aminoacids that are missing in
BuChE.
To complete the SAR of this class of compounds, we
introduced different substituents on the benzyl ring, in
particular methoxy and chlorine groups in ortho, meta
and para position (15–21). Compounds 15–16, with
the methoxy group in the ortho position, were the
only compounds that retained the inhibitory potency
with respect to the unsubstituted parent compounds
3 and 7, respectively. Substituents in meta and in par-
ticular in para position drammatically reduced the
activity. The same trend was also shown with the
chlorine substitution, but in this case even the ortho
substitution was detrimental for activity, though less
than meta and para.
Unlike AChE, the physiological function of BuChE in
healthy and diseased humans is still unclear, and recent-
ly its involvement in developmental and neurobiological
processes has been suggested. In particular, BuChE is
thought to be involved in neurogenesis and regulation
of cell proliferation and differentiation, as well as in
changes in apoptotic events.23
Thus, the development of selective BuChE inhibitors, as
compounds 8–12 and 24–26, may be of great interest to
clarify the physiological role of this enzyme and to pro-
vide novel therapeutics for various diseases.

580
L. Piazzi et al. / Bioorg. Med. Chem. 15 (2007) 575–585
6. Experimental
6.1.7. 3-(8-Bromooctyloxy)xanthen-9-one (1f). Using the
previous procedure and starting from 2.12 g (0.01 mol)
of 3-hydroxyxanthen-9-one and 5.44 g (0.02 mol) of
1,8-dibromooctane, 2.8 g (70%) of 1f were obtained:
mp 138–141 ꢁC; H NMR d 1.45–1.55 (m, 8H), 1.80–
1.90 (m, 4H), 3.45 (t, 2H), 4.10 (t, 2H), 6.85–8.35 (m,
7H, Ar). Anal. (C21H23BrO3): C, H.
6.1. Chemistry
1
6.1.1. General methods. All melting points were deter-
mined in open glass capillaries using a Buchi apparatus
¨
and are uncorrected. 1H NMR spectra were recorded in
CDCl3 solution on a Varian Gemini 300 spectrometer
with Me4Si as the internal standard. Mass spectra were
recorded on a V.G. 7070 E spectrometer. Silica gel
Merck 230–400 mesh was used for purification with
flash chromatography. Wherever analyses are only indi-
cated with element symbols, analytical results obtained
for those elements were within 0.4% of the theoretical
values. Compounds’ names were obtained using AUTO-
NOM, PC software for nomenclature in organic chemis-
try, Beilstein-Institut and Springer.
6.1.8. 3-(9-Bromononyloxy)xanthen-9-one (1g). Using the
previous procedure and starting from 2.12 g (0.01 mol)
of 3-hydroxyxanthen-9-one and 5.72 g (0.02 mol) of
1,9-dibromononane, 3.3 g (80%) of 1 g were obtained:
1
mp 100–102 ꢁC; H NMR d 1.35–1.55 (m, 10H), 1.80–
1.90 (m, 4H), 3.45 (t, 2H), 4.10 (t, 2H), 6.85–8.35 (m,
7H, Ar). Anal. (C22H25BrO3): C, H.
6.1.9. 3-(10-Bromodecyloxy)xanthen-9-one (1h). Using
the previous procedure and starting from 2.12 g
(0.01 mol) of 3-hydroxyxanthen-9-one and 6.00 g
(0.02 mol) of 1,10-dibromodecane, 3.01 g (70%) of 1h
6.1.2. 3-(3-Chloropropoxy)xanthen-9-one (1a). A stirred
suspension of 2.12 g (0.01 mol) of 3-hydroxyxanthen-
9-one, 3.15 g (0.02 mol) of 1-bromo-3-chloropropane,
and 2.76 g (0.02 mol) of K2CO3 in dry acetone was
refluxed for 24 h. The reaction was monitored by
TLC. The hot reaction mixture was filtered and evap-
orated to dryness. The residue was crystallized from
EtOH to give 2.02 g (70%) of 1a: mp 122–125 ꢁC;
1H NMR d 2.30–2.40 (m, 2H), 3.90 (t, 2H), 4.42
(t, 2H), 6.98–8.40 (m, 7H, Ar). Anal. (C16H13ClO3):
C, H.
1
were obtained: mp 124–126 ꢁC; H NMR d 1.35–1.45
(m, 12H), 1.80–1.90 (m, 4H), 3.45 (t, 2H), 4.10 (t, 2H),
6.85–8.35 (m, 7H, Ar). Anal. (C23H27BrO3): C, H.
6.1.10.
3-(11-Bromoundecyloxy)xanthen-9-one
(1i).
Using the previous procedure and starting from 2.12 g
(0.01 mol) of 3-hydroxyxanthen-9-one and 6.28 g
(0.02 mol) of 1,11-dibromoundecane, 3.1 g (70%) of 1i
1
were obtained: mp 84–86 ꢁC; H NMR d 1.30–1.50 (m,
14H), 1.80–1.90 (m, 4H), 3.45 (t, 2H), 4.10 (t, 2H),
6.85–8.35 (m, 7H, Ar). Anal. (C24H29BrO3): C, H.
6.1.3. 3-(4-Chlorobutoxy)xanthen-9-one (1b). Using the
previous procedure and starting from 2.12 g (0.01 mol)
of 3-hydroxyxanthen-9-one and 3.43 g (0.02 mol) of 1-
bromo-4-chlorobutane, 2.1 g (70%) of 1b were obtained:
mp 123–125 ꢁC (EtOH); H NMR d 2.01–2.15 (m, 4H),
3.65 (t, 2H), 4.15 (t, 2H), 6.85–8.40 (m, 7H, Ar). Anal.
(C17H15ClO3): C, H.
6.1.11.
3-(12-Bromododecyloxy)xanthen-9-one
(1j).
Using the previous procedure and starting from 2.12 g
(0.01 mol) of 3-hydroxyxanthen-9-one and 6.56 g
(0.02 mol) of 1,12-dibromododecane, 3.2 g (70%) of 1j
1
1
were obtained: mp 80–81 ꢁC; H NMR d 1.25–1.50 (m,
16H), 1.80–1.90 (m, 4H), 3.45 (t, 2H), 4.10 (t, 2H),
6.85–8.35 (m, 7H, Ar). Anal. (C25H31BrO3): C, H.
6.1.4. 3-(5-Chloropentyloxy)xanthen-9-one (1c). Using
the previous procedure and starting from 2.12 g
(0.01 mol) of 3-hydroxyxanthen-9-one and 3.71 g
(0.02 mol) of 1-bromo-5-chloropentane, 2.2 g (70%) of
1c were obtained: mp 128–130 ꢁC (EtOH); H NMR d
1.65–1.75 (m, 2H), 1.85–1.95 (m, 4H), 3.60 (t, 2H),
4.10 (t, 2H), 6.85–8.40 (m, 7H, Ar). Anal. (C18H17ClO3):
C, H.
6.1.12. 3-(7-Bromoheptyloxy)-6-methoxyxanthen-9-one
(1k). Using the previous procedure and starting from
2.43 g (0.01 mol) of 3-hydroxy-6-methoxyxanthen-9-
one 33 and 5.16 g (0.02 mol) of 1,7-dibromoheptane,
1
1
3.1 g (80%) of 1k were obtained: mp 148–150 ꢁC; H
NMR d 1.45–1.55 (m, 6H), 1.80–1.90 (m, 4H), 3.45 (t,
2H), 3.95 (s, 3H), 4.10 (t, 2H), 6.85–8.25 (m, 6H, Ar).
Anal. (C21H23BrO4): C, H.
6.1.5. 3-(6-Chlorohexyloxy)xanthen-9-one (1d). Using
the previous procedure and starting from 2.12 g
(0.01 mol) of 3-hydroxyxanthen-9-one and 3.99 g
(0.02 mol) of 1-bromo-6-chloroexane, 2.64 g (80%) of
1d were obtained: mp 96–97 ꢁC (EtOH); H NMR d
1.45–1.55 (m, 4H), 1.80–1.90 (m, 4H), 3.55 (t, 2H),
4.05 (t, 2H), 6.85–8.35 (m, 7H, Ar). Anal. (C19H19ClO3):
C, H.
6.1.13. 3-(7-Bromoheptyloxy)-6,7-dimethoxyxanthen-9-
one (1l). Using the previous procedure and starting from
2.72 g (0.01 mol) of 3-hydroxy-6,7-dimethoxyxanthen-9-
one 32 and 5.16 g (0.02 mol) of 1,7-dibromoheptane,
3.5 g (80%) of 1l were obtained: mp 158–160 ꢁC; 1H
NMR d 1.45–1.55 (m, 6H), 1.80–1.90 (m, 4H), 3.45 (t,
2H), 3.90 (s, 3H), 3.95 (s, 3H), 4.10 (t, 2H), 6.85–8.30
(m, 5H, Ar). Anal. (C22H25BrO5): C, H.
1
6.1.6. 3-(7-Bromoheptyloxy)xanthen-9-one (1e). Using
the previous procedure and starting from 2.12 g
(0.01 mol) of 32 and 5.16 g (0.02 mol) of 1,7-dibromo-
heptane, 3.1 g (80%) of 1e were obtained: mp 90–
93 ꢁC; 1H NMR d 1.45–1.55 (m, 6H), 1.80–1.90 (m,
4H), 3.45 (t, 2H), 4.10 (t, 2H), 6.85-8.35 (m, 7H, Ar).
Anal. (C20H21BrO3): C, H.
6.1.14. 3-(3-Iodopropoxy)xanthen-9-one (2a). A mixture
of 2.88 g (0.01 mol) of 1a and 1.5 g (0.01 mol) of NaI
in 30 mL of methylethylketone was refluxed for 4 h.
After cooling, the separated solid was collected by filtra-
tion: 2.66 g (70%) of 2a were obtained: mp 119–121 ꢁC;

L. Piazzi et al. / Bioorg. Med. Chem. 15 (2007) 575–585
581
1H NMR d 2.30–2.40 (m, 2H), 3.45 (t, 2H), 4.30 (t, 2H),
6.90-8.40 (m, 7H, Ar). Anal. (C16H13IO3): C, H.
13.00), 134 (100), 91 (68.18). Anal. (C27H29NO3):
Calcd C, 78.04; H, 7.03; N, 3.37. Found: C, 77.99;
H, 7.01; N, 3.34.
6.1.15. 3-(4-Iodobutoxy)xanthen-9-one (2b). Using the
previous procedure and starting from 3.02 g (0.01 mol)
of 1b, 2.8 g (70%) of 2b were obtained: mp 113–
115 ꢁC; 1H NMR d 2.01–2.09 (m, 4H), 3.30 (t, 2H),
4.15 (t, 2H), 6.85–8.40 (m, 7H, Ar). Anal.
(C17H15IO3): C, H.
6.2.5. 3-[7-(Benzylmethylamino)-heptyloxy]xanthen-9-one
(7). d 1.32–1.61 (m, 8H, alkyl), 1.77–1.91 (m, 2H, al-
kyl), 2.24 (s, 3H, NCH3), 2.45 (t, 2H, CH2N), 3.50
(s, 2H, CH2Ar), 4.11 (t, 2H, OCH2), 6.80–8.33 (m,
12H, Ar). MS: m/z (rel.abundance): 429 (M+,
39.00), 134 (100), 91 (55.50). Anal. (C28H31NO3):
Calcd C, 78.29; H, 7.27; N, 3.26. Found: C, 78.31;
H, 7.04; N, 3.25.
6.1.16. 3-(5-Iodopentyloxy)xanthen-9-one (2c). Using the
previous procedure and starting from 3.16 g (0.01 mol)
of 1c, 2.9 g (70%) of 2c were obtained: mp 129–131 ꢁC;
1H NMR d 1.65–1.75 (m, 2H), 1.80–1.95 (m, 4H), 3.30
(t, 2H), 4.15 (t, 2H), 6.85–8.40 (m, 7H, Ar). Anal.
(C18H17IO3): C, H.
6.2.6. 3-[8-(Benzylmethylamino)octyloxy]xanthen-9-one
(8). d 1.18–1.50 (m, 8H, alkyl), 1.70–1.81 (m, 4H, alkyl),
2.50 (s, 3H, NCH3), 2.62 (t, 2H, CH2N), 4.10–4.30 (m,
4H, CH2Ar and OCH2), 6.98–8.22 (m, 12H, Ar). MS:
m/z (rel.abundance): 443 (M+, 6.13), 134 (64.70), 91
(100). Anal. (C29H33NO3): Calcd C, 78.52; H, 7.50; N,
3.16. Found: C, 78.49; H, 7.51; N, 3.14.
6.1.17. 3-(6-Iodohexyloxy)xanthen-9-one (2d). Using the
previous procedure and starting from 3.3 g (0.01 mol)
of 1d, 3.8 g (90%) of 2d were obtained: mp 75–78ꢁC;
1H NMR d 1.40–1.55 (m, 4H), 1.80–1.90 (m, 4H), 3.20
(t, 2H), 4.05 (t, 2H), 6.80–8.35 (m, 7H, Ar). Anal.
(C19H19IO3): C, H.
6.2.7. 3-[9-(Benzylmethylamino)nonyloxy]xanthen-9-one
(9). d 1.30–1.70 (m, 10H, alkyl), 1.79–1.94 (m, 4H,
alkyl), 2.70 (s, 3H, NCH3), 2.80–3.15 (m, 2H,
CH2N), 4.07–4.25 (m, 4H, CH2Ar and OCH2),
6.86–8.33 (m, 12H, Ar). MS: m/z (rel.abundance):
457 (M+, 30.30), 442 (14.2), 366 (100). Anal.
(C30H35NO3): Calcd C, 78.74; H, 7.71; N, 3.06.
Found: C, 78.69; H, 7.70; N, 3.04.
6.1.18. General method for the preparation of compounds
(3–27). A solution of the selected x-haloalkoxyxanthen-
9-one (5 mmol) and selected amine (10 mmol) in toluene
(100 mL) was refluxed for 30 h. After cooling, the tolu-
ene was evaporated and the residue was purified by flash
chromatography (toluene/acetone 3:2).
6.2.8. 3-[10-(Benzylmethylamino)decyloxy]xanthen-9-one
(10). d 1.22–1.58 (m, 14H, alkyl), 1.77–1.95 (m, 2H, al-
kyl), 2.20 (s, 3H, NCH3), 2.27 (t, 2H, CH2N), 3.50 (s,
2H, CH2Ar), 4.06 (t, 2H, OCH2), 6.94–8.27 (m, 12H,
Ar). MS: m/z (rel.abundance): 471 (M+, 31.50), 380
(100). Anal. (C31H37NO3): Calcd C, 78.95; H, 7.91; N,
2.97. Found: C, 78.99; H, 7.90; N, 2.94.
1
6.2. H NMR, mass spectrum data and elemental
analysis for compounds 3–27
6.2.1. 3-[3-(Benzylmethylamino)propoxy]xanthen-9-one
(3). d 2.00–2.10 (m, 2H, alkyl), 2.28 (s, 3H, NCH3),
2.61 (t, 2H, CH2N), 3.56 (s, 2H, CH2Ar), 4.16 (t, 2H,
OCH2), 6.89–8.48 (m, 12H, Ar). MS: m/z (rel.abun-
dance): 373 (M+, 9.55), 134 (100), 91 (91.92). Anal.
(C24H23NO3): Calcd C, 77.19; H, 6.21; N, 3.75. Found:
C, 77.08; H, 6.19; N, 3.71.
6.2.9. 3-[11-(Benzylmethylamino)undecyloxy]xanthen-9-
one (11). d 1.22–1.72 (m, 16H, alkyl), 1.80–1.97 (m,
2H, alkyl), 2.21 (s, 3H, NCH3), 2.35 (t, 2H, CH2N),
3.46 (s, 2H, CH2Ar), 4.06 (t, 2H, OCH2), 6.79–8.29
(m, 12H, Ar). MS: m/z (rel.abundance): 485 (M+,
28.40), 395 (100). Anal. (C32H39NO3): Calcd C, 79.14;
H, 8.09; N, 2.88. Found: C, 79.19; H, 8.10; N, 2.84.
6.2.2.
3-[4-(Benzylmethylamino)butoxy]xanthen-9-one
(4). d 1.60–1.92 (m, 4H, alkyl), 2.18 (s, 3H, NCH3),
2.44 (t, 2H, CH2N), 3.45 (s, 2H, CH2Ar), 3.98 (t, 2H,
OCH2), 6.73–8.29 (m, 12H, Ar). MS: m/z (rel.abun-
dance): 378 (M+, 14.10), 134 (100), 91 (55.52). Anal.
(C25H25NO3): Calcd C, 77.49; H, 6.50; N, 3.61. Found:
C, 77.41; H, 6.49; N, 3.69.
6.2.10. 3-[12-(Benzylmethylamino)dodecyloxy]xanthen-9-
one (12). d 1.30–1.61(m, 16H, alkyl), 1.81–1.99 (m, 2H,
alkyl), 2.20 (s, 3H, NCH3), 2.37 (t, 2H, CH2N), 3.48
(s, 2H, CH2Ar), 4.10 (t, 2H, OCH2), 6.74–8.37 (m,
12H, Ar). MS: m/z (rel.abundance): 499 (M+, 6.50),
409 (100), 407 (7.7). Anal. (C33H41NO3): Calcd C,
79.32; H, 8.27; N, 2.80. Found: C, 79.39; H, 8.30; N,
2.84.
6.2.3. 3-[5-(Benzylmethylamino)pentyloxy]xanthen-9-one
(5). d 1.73–1.90 (m, 4H, alkyl), 1.91–1.97 (m, 2H, alkyl),
2.12 (s, 3H, NCH3), 2.22 (t, 2H, CH2N), 3.50 (s, 2H,
CH2Ar), 4.10 (t, 2H, OCH2), 6.82–8.27 (m, 12H, Ar).
MS: m/z (rel.abundance): 401 (M+, 10.00), 134 (100),
91 (82.23). Anal. (C26H27NO3): Calcd C, 77.78; H,
6.78; N, 3.49. Found: C, 77.72; H, 6.74; N, 3.45.
6.2.11. 3-[7-(Benzylmethylamino)heptyloxy]-6-methoxy-
xanthen-9-one (13). d 1.3–1.6 (m, 8H, alkyl), 1.77–1.91
(m, 2H, alkyl), 2.20 (s, 3H, NCH3), 2.41 (t, 2H,
CH2N), 3.44 (s, 2H, CH2Ar), 3.95 (s, 3H, OCH3), 4.09
(t, 2H, OCH2), 6.82–8.25 (m, 11H, Ar). MS: m/z
(rel.abundance): 459 (M+, 37.00), 409 (17.0), 368 (100).
Anal. (C29H33NO4): Calcd C, 75.79; H, 7.24; N, 3.05.
Found: C, 75.72; H, 7.21; N, 3.04.
6.2.4. 3-[6-(Benzylmethylamino)hexyloxy]xanthen-9-one
(6). d 1.42–1.88 (m, 6H, alkyl), 1.78–1.92 (m, 2H, al-
kyl), 2.22 (s, 3H, NCH3), 2.43 (t, 2H, CH2N), 3.52
(s, 2H, CH2Ar), 4.12 (t, 2H, OCH2), 6.86–8.38 (m,
12H, Ar). MS: m/z (rel.abundance): 415 (M+,

582
L. Piazzi et al. / Bioorg. Med. Chem. 15 (2007) 575–585
6.2.12. 6-[7-(Benzylmethylamino)heptyloxy]-2,3-dimeth-
oxyxanthen-9-one (14). d 1.3–1.6 (m, 8H, alkyl), 1.77–
1.91 (m, 2H, alkyl), 2.20 (s, 3H, NCH3), 2.39 (t, 2H,
CH2N), 3.44 (s, 2H, CH2Ar), 3.95 (s, 3H, OCH3), 4.01
(s, 3H, OCH3), 4.09 (t, 2H, OCH2), 6.82–8.25 (m,
10H, Ar). MS: m/z (rel.abundance): 489 (M+, 100).
Anal. (C30H35NO5): Calcd C, 73.59; H, 7.21; N, 2.86.
Found: C, 73.58; H, 7.20; N, 2.84.
6.2.19. 3-{7-[(4-Chlorobenzyl)methylamino]-heptyloxy}-
xanthen-9-one (21). d 1.31–1.56 (m, 8H, alkyl), 1.78-
1.91 (m, 2H, alkyl), 2.19 (s, 3H, NCH3), 2.35 (t, 2H,
CH2N), 3.45 (s, 2H, CH2Ar), 4.09 (t, 2H, OCH2),
6.88–8.38 (m, 11H, Ar). MS: m/z (rel.abundance): 463
(M+, 25.00), 428 (100), 281 (29.20). Anal. (C28H30-
ClNO3): Calcd C, 72.48; H, 6.52; N, 3.02. Found: C,
72.47; H, 6.53; N, 3.04.
6.2.13. 3-{3-[(2-Methoxybenzyl)methylamino]propoxy}-
xanthen-9-one (15). d 2.00–2.10 (m, 2H, alkyl), 2.30 (s,
3H, NCH3), 2.59 (t, 2H, CH2N), 3.54 (s, 2H, CH2Ar),
3.89 (s, 3H, OCH3), 4.12 (t, 2H, OCH2), 6.75–8.33 (m,
11H, Ar). MS: m/z (rel.abundance): 403 (M+, 70.00),
164 (60.50), 121 (100). Anal. (C25H25-NO4): Calcd C,
74.42; H, 6.25; N, 3.47. Found: C, 74.49; H, 6.26; N,
3.44.
6.2.20. 3-{7-[Methyl-(2-Methylbenzyl)amino]-heptyloxy}-
xanthen-9-one (22). d 1.28–1.64 (m, 8H, alkyl), 1.78-1.92
(m, 2H, alkyl), 2.18 (s, 3H, NCH3), 2.30–2.43 (m, 5H,
OCH2 and ArCH3), 3.45 (s, 2H, CH2Ar), 4.07 (t, 2H,
OCH2), 6.82–8.36 (m, 11H, Ar). MS: m/z (rel.abun-
dance): 443 (M+, 7.55), 105 (100), 148 (54.64). Anal.
(C29H33NO3): Calcd C, 78.52; H, 7.50; N, 3.16. Found:
C, 78.50; H, 7.52; N, 3.14.
6.2.14. 3-{7-[(2-Methoxybenzyl)methylamino]heptyloxy}-
xanthen-9-one (16). d 1.32–1.61 (m, 8H, alkyl), 1.77–1.91
(m, 2H, alkyl), 2.20 (s, 3H, NCH3), 2.41 (t, 2H, CH2N),
3.44 (s, 2H, CH2Ar), 3.79 (s, 3H, OCH3), 4.09 (t, 2H,
OCH2), 6.75–8.38 (m, 11H, Ar). MS: m/z (rel.abun-
dance): 459 (M+, 9.00), 121 (100), 164 (45.50). Anal.
(C29H33NO4): Calcd C, 75.79; H, 7.24; N, 3.05. Found:
C, 75.79; H, 7.23; N, 3.04.
6.2.21.
3-{7-[Methyl-(2-nitrobenzyl)amino]heptyloxy}-
xanthen-9-one (23). d 1.25–1.58 (m, 8H, alkyl), 1.72–
1.90 (m, 2H, alkyl), 2.15 (s, 3H, NCH3), 2.39 (t, 2H,
CH2N), 3.38 (s, 2H, CH2Ar), 4.10 (t, 2H, OCH2),
6.87–8.37 (m, 11H, Ar). MS: m/z (rel.abundance): 474
(M+, 23.00), 427 (100), 457 (47.50). Anal. (C28H30-
N2O5): Calcd C, 70.87; H, 6.37; N, 5.90. Found: C,
70.89; H, 6.35; N, 5.94.
6.2.15. 3-{7-[(3-Methoxybenzyl)methylamino]heptyloxy}-
xanthen-9- one (17). d 1.30–1.60 (m, 8H, alkyl),
1.75–1.90 (m, 2H, alkyl), 2.18 (s, 3H, NCH3), 2.37
(t, 2H, CH2N), 3.45 (s, 2H, CH2Ar), 3.80 (s, 3H,
OCH3), 4.08 (t, 2H, OCH2), 6.73–8.36 (m, 11H,
Ar). MS: m/z (rel.abundance): 459 (M+, 11.22),
121 (100), 164 (60.79). Anal. (C29H33NO4): Calcd C,
75.79; H, 7.24; N, 3.05. Found: C, 75.75; H, 7.22;
N, 3.04.
6.2.22. 3-{7-[(2,3-Dimethoxybenzyl)methylamino]heptyl-
oxy}xanthen-9-one (24). d 1.80–2.02 (m, 6H, alkyl),
2.24–2.38 (m, 6H, alkyl), 3.12 (s, 3H, NCH3), 3.22–
3.30 (m, 2H, CH2N), 4.32–4.38 (m, 6H, OCH3), 4.63–
4.77 (m, 4H, OCH2 and CH2Ar), 7.43–8.75 (m, 10H,
Ar). MS: m/z (rel.abundance): 489 (M+, 8.31), 150
(100), 194 (80.21). Anal. (C30H35NO5): Calcd C, 73.59;
H, 7.21; N, 2.86. Found: C, 73.54; H, 7.23; N, 2.84.
6.2.23. 3-{7-[(2,5-Dimethoxybenzyl)methylamino]heptyl-
oxy}xanthen-9-one (25). d 1.31–1.57 (m, 8H, alkyl),
1.75-1.88 (m, 2H, alkyl), 2.21 (s, 3H, NCH3), 2.40 (t,
2H, CH2N), 3.46 (s, 2H, CH2Ar), 3.70 (s, 3H, OCH3),
3.80 (s, 3H, OCH3), 4.16 (t, 2H, OCH2), 6.68–8.35 (m,
10H, Ar). MS: m/z (rel.abundance): 489 (M+, 15.31),
150 (100), 194 (60.21). Anal. (C30H35NO5): Calcd C,
73.59; H, 7.21; N, 2.86. Found: C, 73.56; H, 7.22; N,
2.83.
6.2.16. 3-{7-[(4-Methoxybenzyl)methylamino]heptyloxy}-
xanthen-9-one (18). d 1.18–1.50 (m, 6H, alkyl), 1.62–
1.82 (m, 4H, alkyl), 2.60 (s, 3H, NCH3), 2.80–3.13
(m, 2H, CH2N), 3.80 (s, 3H, OCH3), 4.10–4.23 (m,
4H, OCH2 and CH2Ar), 6.96–8.22 (m, 11H, Ar).
MS: m/z (rel.abundance): 459 (M+, 8.00), 121 (100),
164 (19.78). Anal. (C29H33NO4): Calcd C, 75.79; H,
7.24; N, 3.05. Found: C, 75.77; H, 7.23; N, 3.03.
6.2.17. 3-{7-[(2-Chlorobenzyl)methylamino]-heptyloxy}-
xanthen-9-one (19). d 1.26-1.63 (m, 8H, alkyl), 1.73-
1.90 (m, 2H, alkyl), 2.21 (s, 3H, NCH3), 2.45 (t, 2H,
CH2N), 3.57 (s, 2H, CH2Ar), 4.04 (t, 2H, OCH2),
6.88-8.33 (m, 11H, Ar). MS: m/z (rel.abundance): 463
(M+, 100), 124 (100), 168 (75.90). Anal. (C28H30ClNO3):
Calcd C, 72.48; H, 6.52; N, 3.02. Found: C, 72.49; H,
6.53; N, 3.04.
6.2.24. 3-{7-[Methyl-(2,3,4-trimethoxybenzyl)amino]-hep-
tyloxy}xanthen-9-one (26). d 1.27–1.57 (m, 8H, alkyl),
1.68-1.84 (m, 2H, alkyl), 2.18 (s, 3H, NCH3), 2.37 (t,
2H, CH2N), 3.41 (s, 2H, CH2Ar), 3.83 (s, 3H, OCH3),
3.87 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 4.06 (t, 2H,
OCH2), 6.59–8.36 (m, 9H, Ar). MS: m/z (rel.abundance):
519 (M+, 5.85), 180 (100), 182 (13.74). Anal.
(C31H37NO6): Calcd C, 71.65; H, 7.18; N, 2.70. Found:
C, 71.64; H, 7.20; N, 2.71.
6.2.18. 3-{7-[(3-Chlorobenzyl)methylamino]-heptyloxy}-
xanthen-9-one (20). d 1.30–1.60 (m, 8H, alkyl), 1.78–
1.91 (m, 2H, alkyl), 2.18 (s, 3H, NCH3), 2.37 (t, 2H,
CH2N), 3.44 (s, 2H, CH2Ar), 4.07 (t, 2H, OCH2),
6.85–8.38 (m, 11H, Ar). MS: m/z (rel.abundance): 463
(M+, 7.85), 167 (100), 124 (75.96). Anal. (C28H30-
ClNO3): Calcd C, 72.48; H, 6.52; N, 3.02. Found: C,
72.46; H, 6.51; N, 3.01.
6.2.25. 3-{7-[Ethyl-(2-methoxybenzyl)amino]heptyloxy}-
xanthen-9-one (27). d 1.01–1.18 (m, 3H, CH2CH3),
1.32–1.61 (m, 8H, alkyl), 1.77–1.91 (m, 2H, alkyl), 2.20
(s, 3H, NCH3), 2.41 (t, 2H, CH2N), 2.61 (t, 2H,
CH2CH3), 3.44 (s, 2H, CH2Ar), 3.79 (s, 3H, OCH3),
4.09 (t, 2H, OCH2), 6.75–8.38 (m, 11H, Ar). MS: m/z
(rel.abundance): 473 (M+, 5.1), 445 (100). Anal.

L. Piazzi et al. / Bioorg. Med. Chem. 15 (2007) 575–585
583
(C30H35NO4): Calcd C, 76.08; H, 7.45; N, 2.96. Found:
C, 76.04; H, 7.43; N, 2.94.
6.3. The amine that were not commercially available were
synthesised using the following procedure
6.2.26. 2-(3-Chlorophenoxy)-4,5-dimethoxybenzoic acid
(29). A mixture of 7.4 g (0.034 mol) of 2-chloro-4,5-
dimethoxybenzoic acid, 4.4 g (0.034 mol) of 3-chloro-
phenol, 8 g of K2CO3, 0.5 g of Cu and 0.5 g of CuI in
80 mL of nitrobenzene was heated at 170–180 ꢁC for
8 h. The solvent was steam distilled and the residue
was filtered and acidified with HCl. The separated solid
was filtered, washed with water and suspended in NaH-
CO3 saturated solution. The suspension obtained was
filtered and acidified with HCl. The solid was collected
by filtration, washed with water and dried, obtaining
6.3.1.
Methylamine solution (30%, 10 mL) was rapidly added
to solution of 2-methoxybenzaldehyde (10 g,
N-Methyl-N-[(2-methoxyphenyl)methyl]imine.
a
0.07 mol) in ethanol (30 mL) at about 40 ꢁC. The mix-
ture was allowed to stand at 0–5 ꢁC for 2 h. The ethanol
was evaporated to dryness. The residue was dissolved in
CH2Cl2; the organic phase was washed with H2O and
evaporated to dryness to give the Schiff base (10.8 g,
1
100%) as an yellow oil; H NMR d 3.40–3.50 (m, 3H),
3.82 (s, 3H), 6.80–7.35 (m, 4H, Ar), 8.25 (s, 1H, CH).
1
4.22 g (41%) of 29: mp 170–173 ꢁC (toluene); H NMR
d 3.80 (s, 3H), 3.92 (s, 3H), 6.80–7.60 (m, 6H, Ar). Anal.
(C15H13ClO5): C, H.
6.3.2.
N-Methyl-N-[(3-methoxyphenyl)methyl]imine.
Using the previous procedure and starting from methyl-
amine solution and 3-methoxybenzaldehyde the Schiff
1
base (90%) was obtained as an oil; H NMR d 3.48–
3.52 (m, 3H), 3.84 (s, 3H), 6.92–7.35 (m, 4H, Ar), 8.23
(s, 1H, CH).
6.2.27. 3-Chloro-6,7-dimethoxyxanthen-9-one (30). 4.20 g
of 29 was added portionwise to 420 ml of 85% H3PO4
and 42 g of P2O5 and the reaction mixture was then
heated at 110 ꢁC under stirring for 4 h, cooled and
poured into ice. After filtration, the product was dried
and crystallized from toluene, affording 3.7 g (93%) of
6.3.3.
N-Methyl-N-[(4-methoxyphenyl)methyl]imine.
Using the previous procedure and starting from methyl-
amine solution and 4-methoxybenzaldehyde the Schiff
1
1
30: mp 238–240 ꢁC; H NMR d 3.90 (s, 3H), 3.95 (s,
3H), 6.80–8.20 (m, 5H, Ar). Anal. (C15H11ClO4): C, H.
base (90%) was obtained as an oil; H NMR d 3.41–
3.44 (m, 3H), 3.77 (s, 3H), 6.86–7.62 (m, 4H, Ar), 8.15
(s, 1H, CH).
6.2.28. 3-Benzyloxy-6,7-dimethoxyxanthen-9-one (31). A
solution of 3.62 g (0.01 mol) of 30 in 50 mL of benzyl
alcohol was added to a solution of KOH (5.60 g,
0.1 mol) in 20 mL of benzyl alcohol. 36,6 g of tetrabuty-
lammonium bromide were then added and the mixture
was heated to 75 ꢁC for 6h under stirring. The benzyl
alcohol was evaporated, the residue dissolved in HCl
50% and extracted with CH2Cl2. The organic phase
was washed with HCl 50%, then with water, dried and
evaporated. The residue was then purified by flash chro-
matography (toluene/acetone 3:2) to give 2.4 g (66%) of
6.3.4. N-Methyl-N-[(2-chlorophenyl)methyl]imine. Using
the previous procedure and starting from methylamine
solution and 2-chlorobenzaldehyde the Schiff base
(90%) was obtained as an oil; 1H NMR d 3.48–3.52
(m, 3H), 6.74–8.37 (m, 4H, Ar), 8.67 (s, 1H, CH).
6.3.5. N-Methyl-N-[(3-chlorophenyl)methyl]imine. Using
the previous procedure and starting from methylamine
solution and 3-chlorobenzaldehyde the Schiff base
(90%) was obtained as an oil; 1H NMR d 3.45–3.49
(m, 3H), 7.23–7.83 (m, 4H, Ar), 8.26 (s, 1H, CH).
1
31: mp 186–187 ꢁC; H NMR d 3.90 (s, 3H), 3.95 (s,
3H), 4.05 (s, 2H), 6.70-8.20 (m, 10H, Ar). Anal.
(C22H18O5): C, H.
6.3.6. N-Methyl-N-[(4-chlorophenyl)methyl]imine. Using
the previous procedure and starting from methylamine
solution and 4-chlorobenzaldehyde the Schiff base
(90%) was obtained as an oil; 1H NMR d 3.48–3.52
(m, 3H), 7.25–7.85 (m, 4H, Ar), 8.25 (s, 1H, CH).
6.2.29. 3-Hydroxy-6,7-dimethoxyxanthen-9-one (32). A
solution of 31 (2.4 g, 6.6 mmol) in THF (100 mL) was
hydrogenated at room temperature and pressure over
Pd/C. The solution was filtered from catalyst and evap-
orated to dryness to give 1.5 g (83%) of 32: mp > 300 ꢁC;
1H NMR d 3.90 (s, 3H), 3.95 (s, 3H), 6.80–8.20 (m, 5H,
Ar). Anal. (C15H12O5): C, H.
6.3.7. N-Methyl-N-[(2-methylphenyl)methyl]imine. Using
the previous procedure and starting from methylamine
solution and 2-methylbenzaldehyde the Schiff base
(90%) was obtained as an oil; 1H NMR d 2.48–2.51
(m, 3H), 3.51-3.55 (s, 3H), 7.15–7.85 (m, 4H, Ar), 8.59
(s, 1H, CH).
6.2.30. 3-Hydroxy-6-methoxyxanthen-9-one (33). 5 g
(0.022 mol) of 3,6-dihydroxyxanthen-9-one13 were sus-
pended in 250 mL of toluene, 5 g of N-benzyltriethylam-
monium chloride and 1.5 mL (0.022 mol) of methyl
iodide and then 125 mL of 50% NaOH were added.
The mixture was refluxed under stirring for 6 h and
the phases were separated: the toluene phase gave after
evaporation 1.4 g of 3,6-dimethoxyxanthen-9-one. The
aqueous phase was acidified with HCl and the solid
was filtered and dried. Crystallization from toluene
6.3.8. N-Methyl-N-[(2-nitrophenyl)methyl]imine. Using
the previous procedure and starting from methylamine
solution and 2-nitrobenzaldehyde the Schiff base (90%)
1
was obtained as an oil; H NMR d 3.58–3.62 (m, 3H),
7.52–8.05 (m, 4H, Ar), 8.69 (s, 1H, CH).
6.3.9. N-Methyl-N-[(2,3-dimethoxyphenyl)methyl]imine.
Using the previous procedure and starting from methyl-
amine solution and 2,3-dimethoxybenzaldehyde the
1
afforded 5.11 g (95%) of 33: mp 268–270 ꢁC; H NMR
d 3.9 (s, 3H), 6.80–8.10 (m, 6H, Ar). Anal.
(C14H10O4): C, H.
1
Schiff base (90%) was obtained as an oil; H NMR d

584
L. Piazzi et al. / Bioorg. Med. Chem. 15 (2007) 575–585
2.41–2.45 (m, 3H), 3.82 (s, 3H), 3.90 (s, 3H), 6.80–7.40
(m, 3H, Ar), 8.23 (s, 1H, CH).
6.3.18.
N-Methyl-N-[(4-chlorophenyl)methyl]amine.
Using the previous procedure and starting from the
Schiff base, the product (quantitative) was obtained as
an oil; H NMR d 2.45 (s, 3H), 3.74 (s, 2H), 7.20–7.45
(m, 4H, Ar). Anal. (C8H10ClN): C, H, N.
1
6.3.10. N-Methyl-N-[(2,5-dimethoxyphenyl)methyl]imine.
Using the previous procedure and starting from methyl-
amine solution and 2,5-dimethoxybenzaldehyde the
1
Schiff base (90%) was obtained as an oil; H NMR d
2.41–2.45 (m, 3H), 3.83 (s, 3H), 3.87 (s, 3H), 6.70–6.93
(m, 3H, Ar), 8.20 (s, 1H, CH).
6.3.19.
N-Methyl-N-[(2-methyphenyl)methyl]amine.
Using the previous procedure and starting from the
Schiff base, the product (quantitative) was obtained as
an oil; H NMR d 2.45 (s, 3H), 2.58 (s, 3H), 3.75 (s,
2H), 4.67 (broad, 1H, NH), 7.13–7.33 (m, 4H, Ar).
Anal. (C9H13N): C, H, N.
1
6.3.11. N-Methyl-N-[(2,3,4-trimethoxyphenyl)methyl]im-
ine. Using the previous procedure and starting from
methylamine solution and 2,3,4-trimethoxybenzalde-
1
hyde the Schiff base (90%) was obtained as an oil; H
NMR d 2.38–2.42 (m, 3H), 3.85 (s, 3H), 3.88 (s, 3H),
3.91 (s, 3H), 6.58–7.35 (m, 2H, Ar), 8.18 (s, 1H, CH).
6.3.20. N-Methyl-N-[(2-nitrophenyl)methyl]amine. Using
the previous procedure and starting from the Schiff base,
1
the product (quantitative) was obtained as an oil; H
NMR d 2.77 (s, 3H), 4.37 (s, 2H), 7.60–8.20 (m, 4H,
Ar). Anal. (C8H10N2O2): C, H, N.
6.3.12.
N-Ethyl-N-[(2-methoxyphenyl)methyl]imine.
Using the previous procedure and starting from ethyla-
mine solution (70%) and 2-methoxybenzaldehyde the
Schiff base (90%) was obtained as an oil; H NMR d
1.01–1.18 (m, 3H), 2.61 (q, 2H), 3.82 (s, 3H), 6.8–7.35
(m, 4H, Ar), 8.25 (s, 1H, CH).
6.3.21. N-Methyl-N-[(2,3-dimethoxyphenyl)methyl]amine.
Using the previous procedure and starting from the
Schiff base, the product (quantitative) was obtained as
an oil; H NMR d 2.42 (s, 3H), 3.78 (s, 2H), 3.80 (s,
3H), 3.88 (s, 3H), 6.80–7.10 (m, 3H, Ar). Anal.
(C10H15NO2): C, H, N.
1
1
6.3.13.
Sodium borohydride (7 g, 0.165 mol) was added por-
tionwise to solution of the Schiff base (5 g,
N-Methyl-N-[(2-methoxyphenyl)methyl]amine.
a
6.3.22. N-Methyl-N-[(2,5-dimethoxyphenyl)methyl]amine.
Using the previous procedure and starting from the
Schiff base, the product (quantitative) was obtained as
3.3 mmol) in ethanol (80 mL) at 0–5 ꢁC. The mixture
was stirred for 20 h at room temperature and
quenched with water. Ethanol was evaporated and
the remaining aqueous solution was extracted with
CH2Cl2. The organic phase was dried over Na2SO4,
and was evaporated to dryness to yield an oil (quan-
1
an oil; H NMR d 2.44 (s, 3H), 3.72 (s, 2H), 3.79 (s,
3H), 3.86 (s, 3H), 4.65 (broad, 1H, NH), 6.75–6.86 (m,
3H, Ar). Anal. (C10H15NO2): C, H, N.
1
titative); H NMR d 2.45 (s, 3H), 3.65 (s, 2H), 3.80 (s,
3H), 5.10 (broad, 1H, NH), 6.60–7.20 (m, 4H, Ar).
Anal. (C9H13NO): C, H, N.
6.3.23. N-Methyl-N-[(2,3,4-trimethoxyphenyl)methyl]-
amine. Using the previous procedure and starting from
the Schiff base, the product (quantitative) was obtained
1
as an oil; H NMR d 2.41 (s, 3H), 3.69 (s, 2H), 3.82 (s,
3H), 3.85 (s, 3H), 3.90 (s, 3H), 6.58–6.98 (m, 2H, Ar).
Anal. (C11H17NO3): C, H, N.
6.3.14.
N-Methyl-N-[(3-methoxyphenyl)methyl]amine.
Using the previous procedure and starting from the
Schiff base, the product (quantitative) was obtained as
1
an oil; H NMR d 2.45 (s, 3H), 3.73 (s, 2H), 3.80 (s,
3H), 4.63 (broad, 1H, NH), 6.85–7.25 (m, 4H, Ar).
Anal. (C9H13NO): C, H, N.
6.3.24.
N-Ethyl-N-[(2-methoxyphenyl)methyl]amine.
Using the previous procedure and starting from the
Schiff base, the product (quantitative) was obtained as
an oil; 1H NMR d 1.01–1.18 (m, 3H), 2.61 (q, 2H),
3.53 (m, 2H), 3.82 (s, 3H), 6.8-7.35 (m, 4H, Ar), 8.25
(broad, 1H, NH). Anal. (C10H15NO): C, H, N.
6.3.15.
N-Methyl-N-[(4-methoxyphenyl)methylamine.
Using the previous procedure and starting from the
Schiff base, the product (quantitative) was obtained:
1
mp 172–174 ꢁC; H NMR d 2.43 (s, 3H), 3.67 (s, 2H),
6.4. Inhibition of AChE and BuChE
3.79 (s, 3H), 5.28 (broad, 1H, NH), 6.80–7.22 (m, 4H,
Ar). Anal. (C9H13NO): C, H, N.
The method of Ellman20 was followed. Acetylthiocho-
line ATCh iodide solution (0.037 M) was prepared
in water. 5,50-dithio-bis(2-nitrobenzoic acid) (DTNB,
Ellman’s reagent) (0.01 M) was dissolved in pH 7.0
phosphate buffer, and 0.15% (w/v) NaHCO3 was added.
AChE solution was prepared dissolving 20 U in 5 mL of
0.2% aqueous gelatin by sonication at 35 ꢁC. A dilution
1:1 with water was performed before use, in order to ob-
tain the enzyme activity comprised between 0.130 and
0.100 AU/min. Stock solutions of the test compounds
(0.5–1 mM) were prepared in ethanol, as well as the phy-
sostigmine reference stock solution. The assay solutions
were prepared by diluting the stock solutions in water.
Five different concentrations of each compound were
6.3.16.
N-Methyl-N-[(2-chlorophenyl)methyl]amine.
Using the previous procedure and starting from
the Schiff base, the product (quantitative) was ob-
tained as an oil; 1H NMR d 2.44 (s, 3H), 3.81 (s,
2H), 7.18–7.38 (m, 4H, Ar). Anal. (C8H10ClN): C,
H, N.
6.3.17.
N-Methyl-N-[(3-chlorophenyl)methyl]amine.
Using the previous procedure and starting from the
Schiff base, the product (quantitative) was obtained as
1
an oil; H NMR d 2.44 (s, 3H), 3.75 (s, 2H), 7.18–7.25
(m, 4H, Ar). Anal. (C8H10ClN): C, H, N.

L. Piazzi et al. / Bioorg. Med. Chem. 15 (2007) 575–585
585
5. Bartus, R. T.; Dean, L. D., III; Beer, B.; Lippa, A. S.
Science 1982, 217, 408.
used in order to obtain inhibition of AChE activity com-
prised between 20 and 80%.
6. Poirier, J.; Iqbal, K.; Winblad, B.; Nishimura, T.; Takeda,
M.; Wisniewski, H. In Alzheimer’s Disease: Biology,
Diagnosis and Therapeutic; John Wiley: London, 1997; p
93.
7. Siddiqui, M. F.; Levey, A. I. Drugs Future 1999, 24, 417.
8. Greig, N. H.; Utsuki, T.; Yu, Q.; Zhu, X.; Holloway, H.
W.; Perry, T.; Lee, B.; Ingram, D. K.; Lahiri, D. K. Curr.
Med. Res. Opin. 2001, 17, 159.
The assay solution consisted of a 0.1 M phosphate
buffer pH 8.0, with the addition of 340 lM DTNB,
0.035 U/mL AChE derived from human erythrocytes
(Sigma Chemical), and 550 lM ATCh iodide. The final
assay volume was 1 mL. Test compounds were added to
the assay solution and preincubated with the enzyme for
20 min, the addition of substrate following.
9. Giacobini, E.; Spiegel, R.; Enz, A.; Veroff, A. E.; Cutler,
N. R. J. Neural. Transm. 2002, 109, 1053.
10. Darvesh, S.; Hopkins, D. A.; Geula, C. Nat. Rev.
Neurosci. 2003, 4, 131, and references therein.
11. Valenti, P.; Rampa, A.; Bisi, A.; Fabbri, G.; Andrisano,
V.; Cavrini, V. Med. Chem. Res. 1995, 5, 255.
12. Rampa, A.; Bisi, A.; Valenti, P.; Recanatini, M.; Cavalli,
A.; Andrisano, V.; Cavrini, V.; Fin, L.; Buriani, A.;
Giusti, P. J. Med. Chem. 1998, 41, 3976.
13. Rampa, A.; Piazzi, L.; Belluti, F.; Gobbi, S.; Bisi, A.;
Bartolini, M.; Andrisano, V.; Cavrini, V.; Cavalli, A.;
Recanatini, M.; Valenti, P. J. Med. Chem. 2001, 44, 3810.
14. Belluti, F.; Rampa, A.; Piazzi, L.; Bisi, A.; Gobbi, S.;
Bartolini, M.; Andrisano, V.; Cavalli, A.; Recanatini, M.;
Valenti, P. J. Med. Chem. 2005, 48, 4444.
Initial rate assays were performed at 37 ꢁC with a Jas-
co Uvidec-610 double beam Spectrophotometer: the
rate of absorbance increase at 412 nm was followed
for 5 min. Assays were performed with a blank con-
taining all components except AChE, in order to ac-
count for non-enzymatic reaction. The reaction rates
were compared and the percent inhibition due to the
presence of test compounds was calculated. Each con-
centration was analyzed in triplicate. The percent inhi-
bition of the enzyme activity due to the presence of
increasing test compound concentration was calculated
by the following expression: 100 ꢀ (vi/v0 · 100) where
vi is the rate calculated in the presence of inhibitor
and v0 is the enzyme activity. Inhibition curves were
obtained for each compound by plotting the % inhibi-
tion versus the logarithm of inhibitor concentration in
the assay solution. The linear regression parameters
were determined for each curve and the IC50
interpolated.
15. Harel, M.; Schalk, I.; Ehret-Sabatier, L.; Bouet, F.;
Goeldner, M.; Hirth, C.; Axelsen, P. H.; Silman, I.;
Sussman, J. L. Proc. Natl. Acad. Sci. U.S.A. 1993, 90,
9031.
´
16. Inestrosa, N. C.; Alvarez, A.; Perez, C. A.; Moreno, R. D.;
Vicente, M.; Linker, C.; Casanueva, O. I.; Soto, C.;
Garrido, J. Neuron 1996, 16, 881.
17. Recanatini, M.; Valenti, P. Curr. Pharm. Des. 2004, 10,
3157.
18. Sugimoto, H.; Yamanishi, Y.; Rimura, Y.; Kawakami, Y.
Curr. Med. Chem. 2000, 7, 303.
Inhibition of BuChE was measured as described above,
substituting 0.035 U/mL of BuChE from human serum
and 550 lM butyrylthiocholine (BTCh) for enzyme
and substrate, respectively.
19. Piazzi, L.; Rampa, A.; Bisi, A.; Gobbi, S.; Belluti, F.;
Cavalli, A.; Bartolini, M.; Andrisano, V.; Valenti, P.;
Recanatini, M. J. Med. Chem. 2003, 46, 2279.
20. Grover, P. K.; Shah, G. D.; Shah, R. C. J. Chem. Soc.
1955, 3982.
References and notes
21. Ellman, G. L.; Courtney, K. D.; Andres, V.; Featherstone,
R. M. Biochem. Pharmacol. 1961, 7, 88.
1. Kowall, N. W. Alzheimer Dis. Assoc. Disord. 1999, 13,
S11.
2. Fine, R. E. Alzheimer Dis. Assoc. Disord. 1999, 13, S82.
3. Salkoe, D. J. Trends Neurosci. 1993, 16, 403.
4. Parnetti, L.; Senin, U.; Mecocci, P. Drugs 1997, 53, 752.
22. Tumiatti, V.; Rosini, M.; Bartolini, M.; Cavalli, A.;
Marucci, G.; Andrisano, V.; Angeli, P.; Banzi, R.;
Minarini, A.; Recanatini, M.; Melchiorre, C. J. Med.
Chem. 2003, 46, 954.
23. Mack, A.; Robitzki, A. Prog. Neurobiol. 2000, 60, 607.