Synthesis of 4-[(diethylamino)methyl]-phenol derivatives as novel cholinesterase inhibitors with selectivity towards butyrylcholinesterase

Article abstract of DOI:10.1016/j.bmcl.2010.04.059

A series of novel cholinesterase inhibitors, being composed of 4-[(diethylamino)methyl]-phenoxy and secondary amine which were linked with a different length alkyl chain, were designed and synthesized from the starting material p-hydroxybenzaldehyde. Thes

Full text of DOI:10.1016/j.bmcl.2010.04.059

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3254–3258
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of 4-[(diethylamino)methyl]-phenol derivatives as novel
cholinesterase inhibitors with selectivity towards butyrylcholinesterase
*
*
Liang Yu, Rihui Cao, Wei Yi, Qin Yan, Zhiyong Chen, Lin Ma, Wenlie Peng , Huacan Song
School of Chemistry and Chemical Engineering, Sun Yat-sen University, 135 Xin Gang West Road, Guangzhou 510275, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel cholinesterase inhibitors, being composed of 4-[(diethylamino)methyl]-phenoxy and
secondary amine which were linked with a different length alkyl chain, were designed and synthesized
from the starting material p-hydroxybenzaldehyde. These compounds were evaluated as acetylcholines-
terase and butyrylcholinesterase (AChE/BChE) inhibitors. Compounds 25–31 having a secondary amine
moiety connected to the phenyl ring via eight CH2 units spacer were found to be the most potent inhib-
itors with IC50 value lower than 220 nM and 48 nM against AChE and BChE, respectively. Interestingly,
these inhibitors showed a surprising selectively toward BChE, and compounds 26, 27, and 30 displayed
12.5, 18.6, and 18.8-fold higher affinity to BChE. The inhibition kinetics analyzed by Linewear–Burk plots
revealed that such compounds were mix-type inhibitors.
Received 18 January 2010
Revised 29 March 2010
Accepted 15 April 2010
Available online 18 April 2010
Keywords:
Alzheimer’s disease
p-Hydroxybenzaldehyde
Cholinesterase
Inhibitors
Ó 2010 Elsevier Ltd. All rights reserved.
Kinetics analysis
Alzheimer’s disease (AD), the most common form of dementia
accounting for about 50–60% of the overall cases of dementia
among persons over 65 years of age,1 is a neurodegenerative alter-
ation characterized by a low acetylcholine (ACh) in hippocampus
and cortex.2 ACh is a neurotransmitter that is hydrolyzed by ace-
tylcholinesterase (AChE, E.C. 3.1.1.7) and butyrylcholinesterase
(BChE, E.C. 3.1.1.8).3,4 Recently, the genesis of amyloid protein pla-
ques was associated with some alterations of both ChEs (AChE and
BChE), given that by using ChE inhibitors (ChEI) such plaques de-
creased considerably in patients with AD.5,6 Beside this, AChE
activity decreases progressively in certain brain regions from mild
to severe stages of AD to reach 10–15% of normal values, while
BChE activity is unchanged or even increased by 20%, therefore, a
large pool of BChE is available in glia neurons and neuritic plaques.
It may not be an advantage for a ChEI to be selective for AChE; on
the contrary a good balance between AChE and BChE even with
selectivity towards BChE may result in a higher efficacy.7
The crystallographic structure of AChE from Torpedo california
(TcAChE) has been established recently, enabling a close look at
its three-dimensional structure and a better understanding of its
mechanism of action.8,9 Within the structure, it exists (1) a cata-
lytic triad active center (Ser 200, His440, Glu327) in the bottom
of a deep and narrow gorge; (2) 14 aromatic residues lined a sub-
stantial portion of the surface of the gorge; (3) a peripheral anionic
binding site (PAS) of the enzyme at the gorge mouth.
On the basis of the crystallographic structure, recently, a series of
p-hydroxybenzaldehyde derivatives10,11 and 4-[(diethylamino)-
methyl]phenoxy compounds12 were designed and evaluated as
potent AChE and BChE inhibitors. Further study indicated that these
compounds exhibited the potent activity mainly due to its strong
interaction with the active site gorge. In addition, lots of investiga-
tions also showed that the introduction of amine cation moiety
remarkably improved the AChE inhibitory by the electrostatic inter-
action with the PAS of enzyme.13–15
Taking advantage of above information, we focused on the devel-
opment of more active compounds which are able to interact with
the active site gorge and bind the PAS of enzyme (i.e., a dual-site
inhibitor). In the present investigation, a series of novel ChE inhibi-
tors was designed by linking a 4-[(diethylamino)methyl]phenoxy
(DEAMP) group and secondary amine moiety with a different length
alkyl chain using p-hydroxybenzaldehyde as starting material. In
addition, the size of secondary amine moiety and the length of the
alkyl chain were explored systematically.
The synthetic routes of compounds 3–38 were outlined in Scheme
1. The p-hydroxybenzaldehyde was treated with
x-dibromoalkanes
in the presence of K2CO3 to afford
x-bromoalkoxy derivatives
1a–f.16,17 Compounds 1a–f reacted with diethyl amine in 1,2-
dichloroethane (DCE), the resulting product was reduced with NaB-
H(OAc)3 to obtain the DEAMP
x
-bromoalkyl derivatives 2a–f.18,19
Compounds 2a–f reacted with the selected secondary amines to give
the final compounds 3–38.20 The final compounds were treated with
3 N HCl/Et2OH to obtain their hydrochloride salts. All compounds
synthesized were characterized by chemical and spectral methods.
To determine the potential interest of compounds 3–38 for the
treatment of AD, their anticholinesterase activities were assayed
* Corresponding authors. Tel.: +86 20 84110918; fax: +86 20 84112245 (H.S.).
E-mail addresses: pengwl@mail.sysu.edu.cn (W. Peng), yjhxhc@mail.sysu.edu.cn
(H. Song).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.04.059

L. Yu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3254–3258
3255
OHC
N
OHC
iii
N
ii
i
O(CH2)nR
O(CH2)nBr
1a-f: n=2,4,5,6,8,10
O(CH2)nBr
OH
2a-f: n=2,4,5,6,8,10
3-38
Scheme 1. Synthesis of compounds 3–38. Reagents and conditions: (i) Br(CH2)nBr, K2CO3 reflux, 24 h; (ii) diethyl amine, NaBH(OAc)3, DCE, rt 1.5–4 h; (iii) the corresponding
secondary amine, KI, anhydrous ethanol, reflux 5–15 h.
according to Ellmann et al.21 against freshly prepared AChE from
Electrophorus electricus and horse BChE from plasma using galan-
thamine-HBr as reference compounds.22 Table 1 summaries the
data comparing AChE and BChE inhibition as well as the selectivity
for BChE inhibitory activities from IC50 values for the new 4-
[(diethylamino)methyl]phenol derivatives.
As shown in Table 1, all the compounds were active for both
AChE and BChE inhibition. The variations of the alkyl chain length
(n in the general formula) significantly affected ChE inhibitory po-
tency and the selectivity. Remarkably, the behavior of these com-
pounds is rather similar for AChE and BChE inhibition up to a
chain length of six methylene units, while it dramatically diverges
Table 1
Chemical structure and inhibitory activity against isolated AChEa and BChEb of compounds 3–38, respectively, and resulting selectivities expressed as the ratio of IC50 values
N
O(CH2)nR
Compd
n
R
Yieldd (%)
ChE inhibition IC50
(lM)
Selectivity (AChE/BchE)
AChEc
13.68
BChEc
3
4
5
6
7
8
2
2
4
4
4
4
91
85
82
92
88
86
10.10
1.35
0.56
2.44
9.40
0.57
3.87
N
N
N
11.45
3.42
20.27
1.40
0.71
9.73
2.74
N
N
6.74
N
N
N
5.58
10.63
9
4
4
83
82
13.68
10.26
11.69
2.52
1.17
4.01
N
N
10
N
N
N
N
N
11
12
13
14
15
5
5
5
5
5
80
92
86
83
85
0.50
4.31
2.48
1.76
8.37
1.54
0.67
9.35
3.08
4.42
0.32
6.44
0.27
0.57
1.89
N
N
16
17
5
5
N
N
83
84
5.59
2.46
1.78
1.90
3.13
1.29
(continued on next page)

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L. Yu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3254–3258
Table 1 (continued)
Compd
n
R
Yieldd (%)
ChE inhibition IC50
(lM)
Selectivity (AChE/BchE)
AChEc
BChEc
N
N
N
N
N
18
19
20
21
22
6
6
6
6
6
83
96
88
85
89
0.44
1.28
0.34
0.65
0.48
0.74
0.58
0.89
1.82
1.62
0.96
1.37
3.76
2.20
1.66
N
N
23
24
6
6
83
84
2.16
0.67
1.34
0.23
1.61
2.92
N
N
N
N
N
N
N
25
26
27
28
29
8
8
8
8
8
82
94
89
84
90
0.084
0.092
0.21
0.015
0.0073
0.011
0.014
0.048
5.42
12.50
18.60
5.38
N
N
0.077
0.14
2.99
30
31
8
8
N
N
87
85
0.17
0.14
0.0091
0.017
18.82
8.45
N
N
N
N
N
32
33
34
35
36
10
10
10
10
10
88
95
91
89
90
0.30
0.60
0.54
0.58
0.31
0.068
0.078
0.033
0.19
4.41
7.66
16.48
3.10
N
N
0.027
11.29
37
10
10
N
89
86
0.34
0.026
13.07
38
0.26
0.67
0.015
1.52
16.54
0.044
N
Galanthamine-HBre
a
AChE, E.C. 3.1.1.7, from electric eel.
BChE, E.C. 3.1.1.8, from horse serum.
IC50 values are means of three different experiments.
The yields of the final step.
IC50 values of AChE reported in the literature: 0.3–0.8 lM.
b
c
d
e
from eight to ten carbon units. Compounds 25–31 having an sec-
ondary amine moiety connected to the phenyl ring via eight CH2
unit spacer were found to be the most potent inhibitors with IC50
[(diethylamino)methyl]-phenoxy function and secondary amine
moiety is eight CH2 units.
We further investigated the inhibitory effect and the selectivity
of various secondary amine moieties on AChE and BChE. As shown
in Table 1, compounds 25, 26, and 28, bearing the cyclic amine
value lower than 0.21
lM and 0.048 lM against AChE and BChE,
respectively, suggesting that the optimal distance between 4-

L. Yu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3254–3258
3257
could be characterized by a linear mixed type of enzyme inhibi-
tion.23 For BChE the results show the same type of inhibition.
Therefore, we concluded that compound 26 caused a mixed type
of inhibition, that is, compound 26 as a dual-site inhibitor could
interact with both active site gorge and PAS of enzyme at the same
time.
In conclusion, a series of novel cholinesterase inhibitors, being
composed of 4-[(diethylamino)methyl]-phenoxy and secondary
aminewhichwerelinkedwithadifferentlengthalkylchain, werere-
ported. These compoundsexhibited the expected inhibitory potency
against AChE but were additionally found to be very potent inhibi-
tors of BChE. Structure–activity relationships analysis indicated that
the optimal distance between 4-[(diethylamino)methyl]-phenoxy
function and secondary amine moiety is eight CH2 units. The inhibi-
tion kinetics analyzed by Linewear–Burk plots revealed that such
compounds were mix-type inhibitors. All these results suggested
that such compounds might be utilized for the development of
new candidates for treatment of Alzheimer’s disease.
Figure 1. Lineweaver–Burk plots resulting from substrate–velocity curves of AChE
activity with different substrate concentrations (100–400 M) in the absence and
presence of compound 26 with concentration of 50 and 100 nM.
l
Acknowledgment
This work was supported by the Natural Science Foundation of
Guangdong Province, China (2004B30101007) and the Science and
Technology Project of Guangdong Province, China (2009B060700048).
References and notes
1. McDowell, I. Aging (Milano) 2001, 13, 143.
2. Terry, A. V.; Buccafusco, J. J. J. Pharmacol. Exp. Ther. 2003, 306, 821.
3. Silman, I.; Sussman, J. L. Curr. Opin. Pharmacol. 2005, 5, 293.
4. Nachon, F.; Nicolet, Y.; Masson, P. Ann. Pharm. Fr. 2005, 63, 194.
5. Greig, N. H.; Sambamurti, K.; Yu, Q. S.; Brossi, A.; Bruinsma, G. B.; Lahiri, D. K.
Curr. Alzheimer Res. 2005, 2, 1307.
6. Lahiri, D. K.; Farlow, M. R.; Hintz, N.; Utsuki, T.; Greig, N. H. Acta Neurol. Scand.
Suppl. 2000, 176, 60–67.
7. Giacobini, E. In Cognitive Enhancing Drugs; Buccafusco, J. J., Ed.; Birkhäuser:
Basel, Boston, Berlin, 2004; pp 11–36.
8. Sussman, J. L.; Harel, M.; Frolow, F.; Oefner, C.; Goldman, A.; Toker, L.; Silman, I.
Science 1991, 253, 872.
9. 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.
10. Wen, H.; Lin, C. L.; Que, L.; Peng, W. L.; Wang, Z. H.; Song, H. C. Eur. J. Med. Chem.
2008, 43, 166.
Figure 2. Lineweaver–Burk plots resulting from substrate–velocity curves of BChE
activity with different substrate concentrations (100–500 lM) in the absence and
presence of compound 26 with concentration of 10 and 20 nM.
11. Wen, H.; Zhou, Y. Y.; Lin, C. L.; Song, H. C. Bioorg. Med. Chem. Lett. 2007, 17,
2123.
12. Sheng, R.; Lin, X.; Li, J. Y.; Hu, Y. Z. Bioorg. Med. Chem. Lett. 2005, 15, 3834.
13. Tang, H.; Wei, Y. B.; Zhang, C.; Ning, F. X.; Qiao, W.; Huang, S. L.; Ma, L.; Huang,
Z. S.; Gu, L. Q. Eur. J. Med. Chem. 2009, 44, 2523.
14. Tang, H.; Ning, F. X.; Wei, Y. B.; Huang, S. L.; Huang, Z. S.; Albert Sun-Chi Chan,
A. S. C.; Gu, L. Q. Bioorg. Med. Chem. Lett. 2007, 17, 3765.
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Bioorg. Med. Chem. Lett. 2006, 16, 2170.
16. Piazzi, L.; Belluti, F.; Bisi, A.; Gobbi, S.; Rampa, A. Bioorg. Med. Chem. 2007, 15,
575.
17. General procedure for the preparation of compounds 1a–f. A stirred suspension of
groups, showed the highest activities against AChE with IC50 value
of 0.084, 0.092, and 0.077 lM, respectively. Compounds 26 and 30,
containing piperidine and dipropylamine moiety, respectively, pre-
sented the best BChE inhibitory potencies with an IC50 value of
0.0073 and 0.0091 lM. It was noted that these inhibitors showed
a surprising selectively toward BChE, and compounds 26, 27, and
30 displayed 12.50, 18.60, and 18.82-fold higher affinity to BChE.
Compound 26 was selected for kinetic measurements because it
showed the highest inhibitory activity against AChE and BChE. The
mechanism of inhibition was analyzed by recording substrate–
velocity curves in the absence and the presence of compound 26
at different concentrations. Substrate concentration was varied
20 mmol of p-hydroxybenzaldehyde, 40 mmol of
x-dibromoalkanes, and
40 mmol of K2CO3 in dry acetone was refluxed for 24 h. The reaction was
monitored by TLC. The hot reaction mixture was filtered and evaporated to
dryness. The residue was purified by chromatography using CH2Cl2 as eluent to
afford 1a–f in 60–75% yields. Compound 1e: yield 70%, colorless oil. 1H NMR
(CDCl3, 300 MHz) d, ppm: 9.86 (s, 1H, CHO), 7.82 (d, J = 8.8 Hz, 2H, ArH), 6.99
(d, J = 8.7 Hz, 2H, ArH), 4.04 (t, J = 6.5 Hz, 2H, H1), 3.20 (t, J = 7.0 Hz, 2H, H8),
1.88–1.78 (m, 4H, H7, H2), 1.42–1.30 (m, 8H, H3, H4, H5, H6).
from 100 to 400 lM. For AChE, 50 nM and 100 nM concentrations,
respectively, of compound 26 were applied. For BChE, 10 nM and
20 nM concentrations, respectively, of compound 26 were used.
Figure 1 showed the Lineweaver–Burk plots, which are reciprocal
rates versus reciprocal substrate concentrations for the different
inhibitor concentrations resulting from the substrate–velocity
curves for AChE. The results showed that the plots of 1/V versus
1/[S] gave a family of straight lines with different slopes but they
intersected on another in the third quadrant. Similar results were
obtained for BChE (Fig. 2). The inhibitory behavior of compound
26, as deduced from Figure 1, is strictly similar to that of some re-
ported compounds which could bind simultaneously at the cata-
lytic site and at the peripheral anionic site (PAS) of AChE and
18. Abdel-Magid, A.; Carson, K.; Harris, B. J. Org. Chem. 1996, 61, 3849.
19. General procedure for the preparation of compounds 2a–f. Compounds 1a–f
(10 mmol) and diethyl amine (10 mmol) were mixed in 1,2-dichloroethane
(35 mL) and then treated with sodium triacetoxyborohydride (3.0 g, 14 mmol).
The mixture was stirred at rt for 1.5–4 h. The reaction mixture was quenched
by adding aqueous saturated NaHCO3, and the product was extracted with
EtOAc. The EtOAc extract was dried (MgSO4), and the solvent was evaporated
to give 2a–f in 95–98% yields. Compound 2e: yield 96%, yellow oil. 1H NMR
(CDCl3, 300 MHz) d, ppm: 7.32 (d, J = 8.8 Hz, 2H, ArH), 6.85 (d, J = 8.8 Hz, 2H,
ArH), 3.93 (t, J = 6.6 Hz, 2H, H1), 3.72 (s, 2H, CH2Ph), 3.18 (t, J = 6.6 Hz, 2H, H8),
2.48 (q, J = 7.2 Hz, 4H, NCH2CH3), 1.86–1.72 (m, 4H, H7, H2), 1.42–1.30 (m, 8H,
H3, H4, H5, H6), 1.01 (t, 6H, J = 7.1 Hz, NCH2CH3).
20. General procedure for the preparation of compounds 3–38.
A mixture of
compounds 2a–f (2 mmol), KI (3 mmol) and secondary amine (10–20 mmol)
in anhydrous ethanol (50 mL) was refluxed for 5–15 h. After completion of the

3258
L. Yu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3254–3258
reaction as indicated by TLC, the solution was cooled and filtered, and then
22. Assay of the AChE and BChE inhibitory activity. The assay was performed as
described in the following procedure. Five different concentration of each
compound were measured at 412 nm for 1 min, each concentration in
triplicate. For buffer preparation, 0.1 M dipotassium hydrogen phosphate was
adjusted to pH 8.0 with 1 M potassium dihydrogen phosphate. Enzyme
solutions were prepared to give 2.5 units/mL in 1.5 mL aliquots. Furthermore,
0.01 M DTNB solution, 0.075 M ATC, and BTC solutions, respectively, were
concentrated under reduced pressure. The residue was dissolved in CH2Cl2, and
then washed with saturated NaHCO3 and brine, dried with anhydrous Na2SO4,
and solvent removed in vacuum. The crude product was purified by
chromatography using CH2Cl2/MeOH/aqueous NH3 (50:1:0.5) as eluent to
afford 3–38 in 80–96% yields. Compound 26: yield 94%, yellow oil. 1H NMR
(CDCl3, 300 MHz) d, ppm: 7.17 (d, J = 8.8 Hz, 2H, ArH), 6.80 (d, J = 8.8 Hz, 2H,
ArH), 3.90 (t, J = 6.6 Hz, 2H, H1), 3.47 (s, 2H, CH2Ph), 2.48 (q, J = 6.8 Hz, 4H,
NCH2CH3), 2.34 (m, 4H, Hpip-2,6), 2.24 (t, J = 6.4 Hz, 2H, H8), 1.76–1.69 (m, 2H,
H2), 1.60–1.52 (m, 4H, Hpip-3,5), 1.46–1.42 (m, 6H, H7, H6, H3), 1.37–1.30 (m,
6H, Hpip-4, H5, H4), 1.01 (t, 6H, J = 6.8 Hz, NCH2CH3). 13C NMR(CDCl3, 75 MHz)
used. A cuvette containing 880
enzyme, 50 L of DTNB, and 20
to pre-incubate for 15 min at 37 °C, and the reaction was started by addition of
40 L of the substrate solution (ATC/BTC). The reaction was monitored by
measuring the absorbance at 412 nm. For the reference value, 20 L of DMSO
replaced the test compound solution. For determining the blank value,
additionally 10 L of buffer replaced the enzyme solution.
l
L of phosphate buffer, 10
lL of the respective
l
lL of the test compound solution was allowed
l
d: 158.12(Cphenyl-1), 133.99 (Cphenyl-4), 130.31 (Cphenyl-3,5), 114.26 (Cphenyl
-
l
2,6), 68.19 (C1), 59.95 (CH2Ph), 56.99 (C8), 54.93 (Cpip-2,6), 46.70 (2NCH2CH3),
29.65 (C2), 29.43 (C3), 29.24 (C5), 28.05 (C7), 27.22 (C6), 26.69 (C4), 26.27
(Cpip-3,5), 24.82 (Cpip-4), 11.95 (2 NCH2CH3).
l
23. Pang, Y. P.; Quiram, P.; Jelacic, T.; Hong, F.; Brimijoin, S. J. Biol. Chem. 1996, 271,
21. Ellman, G. L.; Courtney, K. D.; Andres, V.; Featherstone, R. M. Biochem.
Pharmacol. 1961, 7, 88.
23646.