Tacrine based human cholinesterase inhibitors: Synthesis of peptidic-tethered derivatives and their effect on potency and selectivity

Article abstract of DOI:10.1016/j.bmcl.2008.08.076

Tacrine based reversible inhibitors of cholinesterases (ChEIs) containing peptidic tethers were synthesized to interact with specific regions at the gorge level, and their potency was determined with human (h) acetylcholinesterase and butyrylcholinesteras

Full text of DOI:10.1016/j.bmcl.2008.08.076

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5213–5216
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Tacrine based human cholinesterase inhibitors: Synthesis of peptidic-tethered
derivatives and their effect on potency and selectivity
a,b
Stefania Butini a,b, Egeria Guarino a,b, Giuseppe Campiani a,b, , Margherita Brindisi
,
*
Salvatore Sanna Coccone a,b, Isabella Fiorini a,b, Ettore Novellino a,c, Tatyana Belinskaya d,
Ashima Saxena d, Sandra Gemma a,b
a European Research Centre for Drug Discovery and Development (NatSynDrugs), Via Aldo Moro 2, 53100 Siena, Italy
b Dip. Farmaco Chimico Tecnologico, via Aldo Moro 2, Università di Siena, 53100 Siena, Italy
c Dip. di Chimica Farmaceutica e Tossicologica, Università di Napoli Federico II, via D. Montesano 49, 80131 Napoli, Italy
d Division of Biochemistry, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Tacrine based reversible inhibitors of cholinesterases (ChEIs) containing peptidic tethers were synthe-
sized to interact with specific regions at the gorge level, and their potency was determined with human
(h) acetylcholinesterase and butyrylcholinesterase. Analogues 3i,j and 3l,m were identified as promising
hits and may pave the way for the development of a new series of tacrine based enzyme selective hChEIs.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 17 July 2008
Revised 20 August 2008
Accepted 21 August 2008
Available online 26 August 2008
Keywords:
Cholinesterases
Alzeheimer’s disease
Inhibitors
Tacrine
Alzheimer’s disease (AD) is the most common form of neurode-
generative dementia among older people, it is associated with a loss
of basal forebrain cholinergic neurons (particularly in neocortex,
hippocampus, and amygdala), with a consequent lack of acetylcho-
line (ACh) around brain cells showing degenerative changes.
Cholinesterases (ChEs) (acetylcholinesterase (AChE) and butyrylch-
olinesterase(BuChE))areAChhydrolyzingenzymesandtheirinhibi-
tion represents the currently employed approach for the treatment
of AD. AChE inhibitors (AChEIs) used to treat AD patients at present
are donepezil, rivastigmine and galantamine, while tacrine (1, Fig. 1)
was the first AChE inhibitor introduced in therapy.
While AChE has a well established ‘classical’ esterase activity, the
physiologic role of BuChE is still controversial. However, BuChE may
have a compensatory role in the hydrolysis of ACh in brains with
degenerative changes, thus making it an additional target for
increasing the cholinergic tone in AD patients affected by severe
symptoms.1–5 This raised the hypothesis that inhibitory action on
both enzymes could lead to an improved therapeutic benefit.4–6
AChE and BuChE are multifunctional enzymes characterized by
their classical esterase activity, and by non-classical functions that
H
N
NH2
S
( )8
N
N
N
1, Tacrine
2
X
NHR
H
N
R1
( )n
L
HN
N
O
3a-m
Figure 1. Reference and title compounds.
are unrelated to their hydrolytic function (e.g., modulation of glial
activation, participation in haematopoiesis, neuritic outgrowth, tau
phosphorylation, adhesion protein-like activity, and promotion of
amyloid-b aggregation).7 All these actions involve the peripheral
anionic site (PAS) or other AChE surface sites8 and are related to
specific protein conformations, sensitive to the concentration of
metal ions.9–13 Consequently, ChEIs may possibly have a variety
* Corresponding author. Address: Dipartimento Farmaco Chimico Tecnologico,
Universitá di Siena, via Aldo Moro 2, 53100 Siena Italy. Tel.: +39 0577 234172; fax:
+39 0577 234333.
E-mail address: campiani@unisi.it (G. Campiani).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.08.076

5214
S. Butini et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5213–5216
Table 1
of effects in the CNS, modulating classical and non-classical func-
tions of the enzymes. The array of protein conformations present
in different structural/functional forms of these enzymes suggests
the existence of a high degree of flexibility in ChEs structure, and
coherently, multiple AChE motions have been demonstrated.14
Very recently we uncovered specific hot spots at the gorge level
of both enzymes, and extremely potent tacrine-based heterobiva-
lent ligands were identified through binding to multiple interac-
tion sites in human (h) AChE and BuChE.15
Dissociation constants for the inhibition of hAChE and hBuChE by tacrine-related
compounds 3a–m and reference compounds
Compound Structurea
Kib (nM) ( SEM)
hAChE
hBChE
O
NH2
H
N
3a
1908 (63)
708 (44)
HN
THA
O
The relationship between fluctuation in AChE structure and
function has been the subject of intense research7,16 and confirms
a mutual allosteric modulation between the catalytic site (CAS)
and the PAS through the mid-gorge amino acid residues.
Me
O
NH
H
3b
6320 (610)
11190 (424)
4130 (202)
2214 (414)
3330 (235)
7970 (1520)
1198 (53)
252 (90)
580 (50)
251 (14)
1764 (31)
443 (27)
N
HN
A promising strategy for interfering with biological processes is
through the control of intra- and inter-protein interactions by
means of small molecules.17 We recently proposed an integrated
approach to the rational design of small molecule enzyme modula-
tors based on the control of protein–protein interactions between
ChE substructures (i.e., hot spots) to design novel and extremely
potent hChEIs.16 An in depth study of the amino acid composition
of the gorge of AChE drove the rational modification of the tether of
the bis-tacrine derivative 2 (Fig. 1) demonstrating that the tether is
crucial for potency and selectivity.16
THA
O
O
NH2
H
N
3c
HN
THA
O
O
HN
H
N
3d
HN
THA
O
We herein report the synthesis of a series of tacrine based
hChEIs (3a–m, Fig. 1 and Table 1) possessing a peptidic tether
NH
NH
O
NH2
NH
H
N
and one or two tacrine moieties.18 The natural or unnatural
L-ami-
3e
HN
no acid chosen to ‘span’ the gorge were hydrophobic in nature
when coupled to a tacrine monomer (3a–j), or chosen on the basis
of flexibility and adaptability to the gorge, when linked to two ta-
THA
O
Me
O
crine units as in 3k–m, (L-glutamate for 3k–l and L-proline for 3m).
H
3f
N
While these compounds were being investigated, similar ap-
HN
proaches were published by other authors.19,20
THA
O
For the synthesis of compounds 3a–h (Scheme 1), tacrine (1)
was reacted with the appropriate acid chloride to afford deriva-
tives 4a,b. Treatment of 4a with sodium azide followed by hydrog-
enolysis furnished the 1-aminoacetamido compound 5, the key
intermediate for the synthesis of compounds 3a–f. Compound 5
was coupled with protected amino acids and the resulting interme-
diates, after cleavage of the protecting group, afforded the desired
final compounds. Zinc chloride and sodium borohydride reduction
of the cyano group of 4b, in the presence of Boc anhydride, pro-
vided the 3-aminoprotected propionamide 6. Deprotection of 6,
Boc
NH
NH
O
HN
H
N
3g
( )2
HN
THA
O
O
NH2
H
N
3h
3i
34030 (8160) 173 (55)
( )2
HN
THA
O
and coupling with L-BocTrpOH afforded 3g from which 3h was ob-
tained after exposure to hydrochloric acid.
Boc
NH
NH
HN
H
N
130 (20)
1.87 (0.13)
1.33 (0.10)
12.63 (0.74)
25.15 (2.45)
Reaction of 9-chlorotetrahydroacridine 7 (Scheme 2) with the
appropriate diamines, afforded 8a,b. Compound 8a21 was synthe-
sized by applying a new, highly yielding (77%), microwave alkyl-
ation protocol, while 8b was prepared as previously described.22
Compounds 3i,k were obtained by coupling suitable amino acids
to 8a. Deprotection of 3i,k gave the final compounds 3j,l. By a mod-
ification of the literature protocol,23 9-chlorotetrahydroacridine 7
was reacted with ethanolamine and the corresponding alcohol
was efficiently brominated to 9. In order to synthesize 3m, com-
HN
THA
O
NH2
H
N
3j
280 (30)
HN
THA
O
NHBoc
NH
H
N
H
N
O
( )2
3k
3l
15.40 (1.43)
6.21 (0.15)
pound 10, obtained by coupling of
ated with the bromo-derivative 9.
L-BocPro-OH with 8b, was alkyl-
THA
THA
H
N
O
O
( )2
THA
THA
The inhibitory activity of the new tacrine-related compounds
was evaluated using purified recombinant hAChE and hBuChE.24,16
Results are reported in Table 1. The mono-tacrine derivatives 3a–h
and 3i,j show in general a preference for hBuChE. Compounds 3a–
h, lacking a basic tacrine nitrogen, one of the key determinant for
potency, proved to be weak inhibitors of hAChE. In particular com-
NH2
H
N
H
N
O
( )2
H
N
NH
( )2
pounds 3a–f, where a glycine unit was used to space aromatic L-
amino acids from the tacrine moiety, exhibited a micromolar affin-
ity for hAChE, and submicromolar for hBuChE. Compounds 3a,b

S. Butini et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5213–5216
5215
(N68, Q119, A277, L286, V288, and A328) in hBuChE).15a The intro-
duction of an extra fused phenyl ring (3c) dramatically affected
hAChE inhibition while hBuChE inhibitory activity was increased
by almost three times (3a vs 3c). For the conformationally con-
strained derivative 3d (3d vs 3b) the same trend of activity was ob-
served, being 3d two times more potent at hBuChE than 3b. In the
Table 1 (continued)
Compound
Structurea
Kib (nM) ( SEM)
hAChE
hBChE
H
N
N
( )2
THA
THA
3m
6.75 (0.54)
1.77 (0.14)
( )
5 N
L-tryptophan series tethered by glycine (3e,f), the activity of the
N
H
H
O
analogue 3e was similar to that of 3a on hAChE, but it was about
three times more potent on hBuChE (3e vs 3a). While on this latter
enzyme 3e showed a potency similar to that of the naphtyl deriv-
ative 3c, it proved to be five times more potent against hAChE. In
this case, N-methylation of the tryptophan primary amino group
moderately affected hAChE activity but significantly reduced
hBuChE inhibitory potency. The homologation of 3e, using beta ala-
nine in place of glycine at the tether level, provided a subset of ana-
logues (3g,h) which were in general much more selective for
hBuChE. In particular 3h, bearing the unprotected protonatable
function, provided the best hAChE/hBuChE ratio for the subset
(3h, hAChE/hBuChE ratio = 197). In a similar extent, tert-butyl car-
bamolylation of 3h (3g), increased affinity for hAChE and reduced
potency on hBuChE. Considering the larger void of hBuChE this
data is apparently discordant with the amino acid composition
and with the physico-chemical properties of the two enzymes that
are due to structural differences in the active site gorge of the two
enzymes, leading to a lower electrostatic gradient and a larger void
in hBuChE.25
1
2
—
—
36 (1)
27.90 (2.42)
7 (2)
1.65 (0.1)
a
THA, 1,2,3,4-tetrahydroacridin-9-yl.
Ki (nM) is the mean of at least three determinations.
b
O
NH2
d, e/f
HN
O
3a-f
b, c
X
HN
N
N
5
a
1
O
HN
NHBoc
f, d
4a, X = Cl
g
4b, X = CN
3g
Substantial improvement of hChE inhibitory activity was ob-
tained with analogues 3i and 3j endowed with (i) a more flexible
tether, and (ii) a protonatable function at the tacrine level, key
determinants for potency improvement. Indeed 3i and 3j showed
hAChE inhibitory potency in the high nanomolar range and dis-
played excellent hBuChE inhibitory properties (3j hAChE/hBuChE
ratio = 210). Indeed 3j was the more potent and selective hBuChE
inhibitor of the series.
N
f
6
3h
Scheme 1. Reagents and conditions: (a) ClCOCH2X, TEA, CH2Cl2, rt; (b) (from 4a)
NaN3, ethanol/water, reflux; (c) H2, Pd/C 10%, MeOH, rt; (d) Boc or Cbz protected
amino acid, EDCI, HOBt, dry CH2Cl2 or dry DMF, rt (3c); (e) H2, Pd/C 10%, MeOH, rt
(3a–c); (f) CH3COCl, MeOH, rt (3d–f); (g) (from 4b) ZnCl2Á6H2O, NaBH4, (Boc)2O,
MeOH, rt.
L
-
We therefore synthesized bis-tacrine analogues in which two
flexible ethylamino moieties were coupled with
L-glutamate
(3k,l), reaching a tether length of 11 atoms (improving also tether
flexibility) and introducing a protonatable function at the tether le-
vel to optimally bind the three interaction points at the gorge level
(CAS, mid-gorge and PAS).15 While the protected analogue 3k,
which lacks the protonatable glutamate function, showed a similar
nanomolar potency at both enzymes, the deprotected analogue 3l
proved to be more potent at hAChE with respect to hBuChE, in
agreement with the electrostatic gradient of the gorge of the two
enzymes. High potency for hChEs was also found for 3m in which
( )
n
Cl
HN
N
NH2
3i
d
d
3j
3l
c
a/b
N
e
8a, n = 2
3k
7
8b, n = 5
h, i
O
f, g
the
x-glutamate ethylamido moiety of 3l was replaced by an L-pro-
H
N
2HCl
linepentylamide (3m).
HN
In summary, we described herein the synthesis and biological
evaluation of tacrine based hChEIs containing specific peptidic
tethers the modification of which determines a substantial modu-
lation of hChEs inhibitory activity. The low potency of glycine con-
taining compounds and beta alanine derivatives (3a–h) was highly
improved enhancing tether length and flexibility. This work al-
lowed the identification of compounds 3i and 3j as the more potent
and selective hBuChE inhibitors of the series, demonstrating the
key role played by the protonatable tacrine moiety. Furthermore
the three interaction point bis-tacrine derivatives 3k–m proved
to be potent hChEIs as well.
Br
NH
HN
5
l
+
3m
N
10
N
9
Scheme 2. Reagents and conditions: (a) for 8a ethylendiamine, MW, 150W,
12 min; (b) for 8b 1,5-pentandiamine, TEA, 1-pentanol, 160 °C; (c) (from 8a) ( )-
N-Boc-tryptophan, EDCI, HOBt, TEA, dry DMF, rt; (d) CH3COCl, MeOH, rt; (e) (from
8a, 2 equiv), ( )-N-Boc-glutamate EDCI, HOBt, TEA, dry CH2Cl2, rt; (f) ethanolamine,
120 °C; (g) CBr4, PPh3, CH2Cl2, rt; (h) from 8b, ( )-N-Boc-proline EDCI, HOBt, TEA, dry
L
L
L
CH2Cl2, rt; (i) CH3COCl, MeOH, rt; (l) TEA, CH3CN, rt.
Acknowledgments
differ for the presence of a methyl group on the pendant basic
nitrogen which reduced hAChE potency of three times (3a vs 3b)
while hBuChE was less sensitive to the steric hindrance provided
by the methylamino group, according to its gorge larger void (6
of the 14 aromatic residues (Y72, Y124, W286, F295, F297 and
Y337) of the hAChE gorge are replaced by aliphatic residues
The authors thank the European Research Centre for Drug Dis-
covery and Development and MIUR Prin for financial support.
The opinions or assertions contained herein are the private views
of the authors, and are not to be construed as official, or as reflect-

5216
S. Butini et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5213–5216
Novellino, E.; Holloway, H. W.; Greig, N. H.; Belinskaya, T.; Fedorko, J. M.;
Saxena, A. J. Med. Chem. 2005, 48, 1919.
16. Butini, S.; Campiani, G.; Borriello, M.; Gemma, S.; Panico, A.; Persico, M.;
Catalanotti, B.; Ros, S.; Brindisi, M.; Agnusdei, M.; Fiorini, I.; Nacci, V.;
Novellino, E.; Belinskaya, T.; Saxena, A.; Fattorusso, C. J. Med. Chem. 2008, 51,
3154 (and references cited therein).
ing true views of the Department of the Army or the Department of
Defense.
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over
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