Synthesis and acetylcholinesterase inhibition of derivatives of huperzine B

Article abstract of DOI:10.1016/j.bmcl.2004.11.060

By targeting dual active sites of AChE, a number of new derivatives of HupB have been synthesized and tested as acetylcholinesterase inhibitors. The most potent compound, bis-HupB 5b is 72-fold more potent in AChE inhibition and 79-fold more selective for AChE versus BChE than HupB.

Full text of DOI:10.1016/j.bmcl.2004.11.060

Bioorganic & Medicinal Chemistry Letters 15 (2005) 523–526
Synthesis and acetylcholinesterase inhibition of derivatives of
huperzine B
Song Feng, Yu Xia, Dongmei Han, Chunyan Zheng, Xuchang He, Xican Tang and
Donglu Bai*
State Key Laboratory for New Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China
Received 12 October 2004; revised 19 November 2004; accepted 22 November 2004
Available online 16 December 2004
Abstract—By targeting dual active sites of AChE, a number of new derivatives of HupB have been synthesized and tested as acetyl-
cholinesterase inhibitors. The most potent compound, bis-HupB 5b is 72-fold more potent in AChE inhibition and 79-fold more
selective for AChE versus BChE than HupB.
ꢀ 2004 Elsevier Ltd. All rights reserved.
To date, AChE inhibitors are the major drugs approved
for the symptomatic treatment of AlzheimerÕs disease.
It has also been demonstrated that AChE could play
a key role during the early stage in the development of
the senile plaques by accelerating b-amyloid peptide
deposition.1
ited, only a few simple N-methylated and hydrogenated
derivatives of HupB were prepared and tested for their
inhibitory activity of AChE.6
Based on the hypothesis with respect to two binding
sites in the active-site gorge of AChE and the good
example of bis-tacrine (3)7 as well as the crystallographic
structure of the complex of AChE with bivalent ligands
related to HupA,8 a series of new derivatives of HupB
with two pharmacophoric moieties, which may interact
with both central and peripheral binding sites in the ac-
tive-site gorge of AChE, respectively, were synthesized
and tested for their inhibitory activity of AChE. For
the preparation of bis-HupB derivatives 5, a few meth-
ods were tested including the reaction of HupB with
a,x-dihaloalkane in the presence of silver carbonate or
with a,x-diacylalkane dihalides in the presence of triethyl-
amine, but no desired products were obtained. Further-
more, reductive amination of a,x-alkylenedial with
HupB, which successfully delivered six dimers of
HupA,9 also failed in furnishing bis-HupB.
Natural (ꢀ)-huperzine B (HupB, 1), a Lycopodium alka-
loid from Huperzia serrata, is demonstrated as a potent
and reversible inhibitor of AChE.2,3 In experiments per-
formed on unanaesthetized rabbits,4 HupB exhibited a
higher therapeutic index in comparison with (ꢀ)-huper-
zine A (HupA, 2), which is more potent than HupB in
AChE inhibition. (ꢀ)-HupA is the major Lycopodium
alkaloid and has now been approved in China as a drug
for the treatment of AD.5
However, the studies on chemical modification and
structure–activity relationships of HupB are very lim-
H
N
H
N
O
O
In this paper, we would like to describe the synthesis and
antiAChE activity of new derivatives of HupB.
N
NH ( CH2
)
7
NH
N
H
N
NH2
Because of the steric hindrance of the amino group, the
direct dimerization of HupB via a tether linked to the
amino group is very difficult. HupB was thus first acyl-
ated by chloroacetyl chloride, obtaining chloroacetyl
HupB 4 with a good yield of 96%. When 2 equiv of
chloroacylated HupB 4 and 1 equiv of piperazine or
1
2
3
Keywords: AChE inhibitors; Huperzine B; Derivatives; Bis-HupB.
*
Corresponding author. Tel.: +86 21 5080 5896; fax: +86 21 5080
7088; e-mail: dlbai@mail.shcnc.ac.cn
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.11.060

524
S. Feng et al. / Bioorg. Med. Chem. Lett. 15 (2005) 523–526
homopiperazine reacted in acetonitrile at 70 ꢁC in the
presence of potassium carbonate and potassium iodide,
the HupB dimers 5a and 5b were afforded, which were
further reduced by LiAlH4 to produce 6a and 6b,
respectively (Scheme 1). The overall yields of 6a and
6b are 45–50%.
The antiAChE activity of the HupB derivatives were
measured in vitro according to the modified Ellman
method.11 The results in Table 1 reveal that the homo-
dimeric bis-HupB 5 and 6 show much higher potency
and selectivity, however, the hetero-dimers 7, 8, 9 and
10 exhibit equal or lower activity in AChE inhibition
than their parent HupB. The homo-dimer 5b is 72-fold
more potent in inhibiting AChE and 79-fold more selec-
tive for AChE versus butyrocholinesterase (BChE) than
HupB. The length of the side chain in hetero-dimers 9
and 10 is crucial to the inhibitory activity of AChE.
Compounds 9d and 10d (n = 3) exhibit 7–8-fold increase
in AChE inhibition than 9b and 10b (n = 1). The inhib-
itory activities of the amides 5, 7 and 9 are nearly the
same as the corresponding amines 6, 8 and 10. The pre-
Considering that an aromatic ring in a bivalent anti-
AChE ligand is the important feature for the binding
of the ligand to the peripheral site of AChE,10 we also
designed a series of HupB hetero-dimers 7, 8, 9 and
10, in which HupB moiety was combined with an aro-
matic moiety via a nitrogen-containing tether. The syn-
thetic approach to these hetero-dimers is quite similar to
the homo-dimers 5 and 6 (Scheme 2).
H
N
H
N
H
N
H
N
O
O
O
O
b
a
N
N
N
N
CCH2N NCH2C
n
H
CCH2Cl
O
O
O
1
5
4
H
N
H
N
O
O
5a,6a: n=2
5b,6b: n=3
c
N
N
CH2CH2N
NCH2CH2
n
6
Scheme 1. Synthesis of bis-HupB. Reagents and conditions: (a) chloroacetyl chloride, Et3N, CH2Cl2, 0 ꢁC, 1 h, 96%; (b) piperazine or
homopiperazine, K2CO3, KI, CH3CN, 70 ꢁC, 8 h, 70–80%; (c) LiAlH4, THF, reflux 2 h, 60–70%.
H
N
H
N
a
O
O
b
N
N
Ar
HN NCH2
CH2CH2N NCH2 Ar
Ar
CCH2N NCH2
O
H
N
O
8
7
N
CCH2Cl
O
H
N
H
N
O
O
b
a
4
N
N
CH2CH2N (CH2
R
)
n
HN (CH2
R
)
n
CCH2N (CH2
)
n
R
O
10
9
7a, 8a : Ar =
7b, 8b : Ar =
9a, 10a : R =
, n=1
OCH3
CH3
9b, 10b : R =
9c, 10c : R =
9d, 10d: R =
, n=1
, n=2
, n=3
NO2
N
7c : Ar =
CH3
CH3
7d, 8d : Ar =
Scheme 2. Synthesis of HupB hetero-dimer derivatives. Reagents and conditions: (a) K2CO3, KI, CHCl3, reflux, 24 h, 40–90%; (b) LiAlH4, THF,
reflux 2 h, 60–80%.

S. Feng et al. / Bioorg. Med. Chem. Lett. 15 (2005) 523–526
525
Table 1. Physical properties and AChE inhibition of derivatives of (ꢀ)-HupB
25
Compd13
½aꢁD (c = 0.1, CHCl3)
Mp (ꢁC)
AChE IC50 (lM)a
BChE IC50 (lM)b
Selectivity for AChEc
HupB
5a
6a
270–271
>230 dec
>230 dec
>230 dec
202–204
98–100
8.202
1.173
0.407
0.114
0.218
33.30
31.60
5.747
10.297
5.58
157
>184
192
171
125
—
19
>157
472
107.3
52.4
94.8
5b
6b
1500
573
ꢀ3.5
65.4
7a
8a
ꢀ6.4
81.2
115–117
145–147
112–114
>140 dec
132–134
114–116
138–140
116–118
112–114
84–86
—
—
7b
8b
ꢀ8.4
77.1
—
—
7c
7d
88.4
4.6
38.10
26.70
>52
—
—
8d
9a
127.5
ꢀ26.8
104.8
ꢀ8.7
100.9
ꢀ13.9
121.7
ꢀ9.4
—
—
10a
9b
>54
50.70
66.45
23.50
20.70
6.19
—
—
10b
9c
68–70
75–77
—
—
10c
9d
103–105
69–70
—
—
10d
9.531
a Assay performed by the modified Ellman method12 using rat cortex homogenate. Values are means of three different experiments.
b Assay performed using rat serum.
c Selectivity for AChE is defined as IC50 (BChE)/IC50 (AChE).
5. Jiang, H. L.; Luo, X. M.; Bai, D. L. Curr. Med. Chem.
2003, 10, 2231–2252.
6. Tang, X. C.; Xu, H.; Feng, J.; Zhou, T. X.; Liu, J. S. Acta
Pharmacol. Sinica 1994, 15, 107–110.
7. Pang, Y. P.; Quiram, P.; Jelacic, T.; Hong, F.; Brimijoin,
S. J. Biol. Chem. 1996, 271, 23646–23649.
8. Wong, D. M.; Greenblatt, H. M.; Dvir, H.; Carlier, P. R.;
Han, Y. F.; Pang, Y. P.; Silman, I.; Sussman, J. L. J. Am.
Chem. Soc. 2003, 125, 363–373.
liminary docking (DOCK 4.0) studies of 5b based on the
structure of the complex of TcAChE with HupB,12 re-
vealed that one HupB moiety bound to central catalytic
site and another one bound to the opening of the active-
site gorge, with the homopiperazinyl group interacting
with the middle part of the gorge. The further molecular
modeling studies are now in progress.
In summary, we have succeeded in the synthesis of 19
new HupB derivatives, in which bis-HupB 5a, 5b, 6a
and 6b, are much more potent and selective in AChE
inhibition. The obviously increased AChE inhibition
and selectivity of bis-HupB are presumed to be related
with interaction with both central and peripheral active
sites of AChE. Further studies on bis-HupB derivatives
are in progress in our laboratory.
9. Jin, G. Y.; Luo, X. M.; He, X. C.; Jiang, H. L.; Zhang, H.
Y.; Bai, D. L. Arzneim. Forsch. 2003, 53, 753–757.
10. Sugimoto, H.; Yamanishi, Y.; Limura, Y.; Kawakami, Y.
Curr. Med. Chem. 2000, 7, 303–339.
11. (a) Ellman, G. L.; Courtney, K. D.; Valentino Andre, J.
R.; Featherstone, R. M. Biochem. Pharmacol. 1961, 7, 88–
95; (b) Cheng, D. H.; Tang, X. C. Pharmacol. Biochem.
Behav. 1998, 60, 377–386.
12. Dvir, H.; Jiang, H. L.; Wong, D. M.; Harel, M.; Chetrit,
M.; He, X. C.; Jin, G. Y.; Yu, G. L.; Tang, X. C.; Silman,
I.; Bai, D. L.; Sussman, J. L. Biochemistry 2002, 41,
10810–10818.
Acknowledgements
13. All new compounds showed satisfactory spectroscopic
data.
Selected analytical data:
This work was partially supported by the National Nat-
ural Science Foundation of China, and Shanghai SK
Foundation for Research and Development (project
2004010-h).
5b: IR (KBr): 3427, 2928, 1655, 1556, 1406, 1186, 1107,
1
833, 752 cmꢀ1; H NMR (400 MHz, CDCl3): 12.90 (br s,
2H), 75.6 (d, 2H, J = 9.47 Hz), 6.41 (d, 2H, J = 9.5 Hz),
5.42 (d, 2H, J = 4.3 Hz), 3.91 (d, 2H, J = 11.4 Hz), 3.42
(m, 4H, J = 14.7 Hz, 17.9 Hz), 3.28 (d, 2H, J = 14.1 Hz),
2.79–2.92 (m, 8H), 2.39–2.58 (m, 8H), 1.91 (m, 4H), 1.57–
1.66 (m, 10H), 1.21–1.39 (m, 6H); 13C NMR (100 MHz,
CDCl3): 173.4 · 2, 165.3 · 2, 142.8 · 2, 142.2 · 2, 133.4 ·
2, 123.8 · 2, 118.2 · 2, 117.3 · 2, 64.6 · 2, 61.4 · 2, 55.3 ·
2, 54.1 · 2, 45.5 · 2, 44.7 · 2, 40.7 · 2, 34.5 · 2, 29.6 · 1,
28.9 · 1, 27.9 · 1, 26.2 · 2, 25.6 · 2, 23.0 · 2; ESIMS
(m/z): 693.5 ((M+H)+, 100), 409.8 (11), 283.4.
6a: IR (KBr): 3419, 2925, 1658, 1604, 1552, 1457, 1299,
1112, 833, 638 cmꢀ1; 1H NMR (300 MHz, CDCl3) d 11.95
(br s, 2H), 7.63 (d, 2H, J = 9.3 Hz), 6.42 (d, 2H, J =
9.6 Hz), 5.47 (d, 2H, J = 4.1 Hz), 2.84 (dd, 2H, J = 17.6 Hz,
4.9 Hz), 2.46–2.72 (m, 8H), 2.38–2.52 (m, 8H), 2.28–2.36
References and notes
1. Barril, X.; Orozco, M.; Luque, F. J. Mini Rev. Med. Chem.
2001, 1, 255–266.
2. Liu, J. S.; Zhu, Y. Z.; Yu, C. M.; Zhou, Y. Z.; Han, Y. Y.;
Wu, F. W.; Qi, B. F. Can. J. Chem. 1986, 64, 837–839.
3. (a) Xu, H.; Tang, X. C. Acta Pharmacol. Sinica 1987, 8,
18–22; (b) Zhu, X. D.; Tang, X. C. Acta Pharmacol. Sinica
1988, 9, 492–497; (c) Liu, J.; Zhang, H. Y.; Wang, L. M.;
Tang, X. C. Acta Pharmacol. Sinica 1999, 20, 141–145.
4. Yan, X. F.; Lu, W. H.; Lou, W. J.; Tang, X. C. Acta
Pharm. Sinica 1987, 8, 117–123.

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S. Feng et al. / Bioorg. Med. Chem. Lett. 15 (2005) 523–526
(m, 8H), 2.05 (d, 2H, J = 16.5 Hz), 1.81 (m, 2H), 1.58 (s,
(1H, br s), 7.63 (1H, d, J = 9.6 Hz), 7.24–7.32 (5H, m),
6.40 (1H, d, J = 9.5 Hz), 5.43 (1H, d, J = 5.2 Hz), 3.52
(2H, s), 3.25–3.31 (1H, m), 2.85 (1H, dd, J = 16.8, 5.5 Hz),
2.26–2.67 (15H, m), 1.98 (1H, dd, J = 16.5 Hz), 1.74 (1H,
m), 1.59 (1H, m), 1.54 (3H, s), 1.48 (1H, m), 1.38 (1H, m),
1.14–1.27 (1H, m); EIMS (m/z): 458 (M+, 8), 285 (15), 269
(100), 84 (47).
6H), 1.48–1.72 (m, 4H), 1.42 (m, 2H), 1.28 (m, 2H); 13C
NMR (100 MHz, CDCl3) 163.8 · 2, 142.5 · 2, 141.2 · 2,
131.5 · 2, 124.8 · 2, 120.4 · 2, 116.7 · 2, 59.9 · 2, 57.0 · 2,
52.6 · 4, 47.2 · 2, 46.8 · 2, 43.5 · 2, 36.9 · 2, 33.6 · 2,
28.7 · 2, 25.1 · 2, 23.5 · 2, 22.1 · 2; ESIMS (m/z): 651.4
((M+H)+, 100), 395.2 (25), 283.2, 240.2, 226.2.
6b: IR: 3423, 2928, 2794, 1657, 1605, 1552, 1458, 1113,
752 cmꢀ1; H NMR (400 MHz, CDCl3): d 12.63 (2H, br,
10d: IR (KBr): 3431, 2933, 1660, 1612, 1462, 1404, 1186,
1107, 833, 700, 534 cmꢀ1 1H NMR (400 MHz, CDCl3)
;
1
s), 7.62 (2H, d, J = 9.5), 6.39 (2H, d, J = 9.3), 5.45 (2H, d,
J = 5.1), 3.28 (2H, m), 2.84 (4H, d.d, J = 5.4, 17.4), 2.62
(2H, J = 12.4), 2.26–2.47 (16H, m), 1.99 (2H, d, J = 16.35),
1.65–1.79 (12H, m), 1.49–1.58 (8H, m), 1.39 (2H, d,
J = 12.1), 1.17–1.25 (4H, m); ESIMS (m/z): 667.5
((M+H)+, 100), 689.6 ((M+Na)+, 3), 411.4 (5), 354.3 (2).
8a: IR (KBr): 3419, 2931, 2808, 1657, 1552, 1456, 1300,
1112, 833, 698 cmꢀ1; 1H NMR (400 MHz, CDCl3) d 12.64
d 13.20 (1H, br s), 7.56 (1H, d, J = 9.3 Hz), 7.15–7.28 (5H,
m), 6.40 (1H, d, J = 9.3 Hz), 5.40 (1H, d, J = 5.5 Hz), 3.98
(1H, m), 3.46 (1H, d, J = 14.3 Hz), 3.38 (1H, d,
J = 18.1 Hz), 3.01 (1H, d, J = 14.3 Hz), 2.89 (1H, dd,
J = 18.1, 5.5 Hz), 2.56–2.72 (2H, m), 2.34–2.52 (6H, m),
2.29 (3H, s), 1.73–2.00 (4H, m), 1.67 (2H, m), 1.57 (3H, s),
1.22–1.35 (1H, m); EIMS (m/z): 445 (M+, 2), 255 (20), 162
(100), 91 (15).