Probing the mid-gorge of cholinesterases with spacer-modified bivalent quinazolinimines leads to highly potent and selective butyrylcholinesterase inhibitors

Article abstract of DOI:10.1016/j.bmc.2010.12.034

The spacer structure of homobivalent quinazolinimes acting as potent acetyl-(AChE)- and butyrylcholinesterase (BChE) inhibitors was chemically modified introducing tertiary amine and acyl-amide moieties, and the activities at both ChEs were evaluated. Molecular docking was applied to explain the data and probe the capacity of the mid-gorge site of both ChEs. The novel spacer structures considerably alter the biological profile of bivalent quinazolinimines with regard to both inhibitory activity and selectivity. Mutual interaction of binding to the various sites of the enzymes was further investigated by applying also different spacer lengths and ring sizes of the alicycle of the tricyclic quinazolinimines. In order to achieve selectivity toward BChE and to improve inhibitory activities, the spacer structure was optimized and identified a highly potent and selective BChE inhibitor.

Full text of DOI:10.1016/j.bmc.2010.12.034

Bioorganic & Medicinal Chemistry 19 (2011) 1222–1235
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Probing the mid-gorge of cholinesterases with spacer-modified bivalent
quinazolinimines leads to highly potent and selective
butyrylcholinesterase inhibitors
Xinyu Chen a,b, Irina G. Tikhonova a, Michael Decker a,b,
⇑
a School of Pharmacy, Queen’s University Belfast, 97 Lisburn Road, Belfast BT9 7BL, UK
b Institut für Pharmazie, Universität Regensburg, Universitätstraße 31, Regensburg 93053, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
The spacer structure of homobivalent quinazolinimes acting as potent acetyl-(AChE)- and butyrylcholin-
esterase (BChE) inhibitors was chemically modified introducing tertiary amine and acyl-amide moieties,
and the activities at both ChEs were evaluated. Molecular docking was applied to explain the data and
probe the capacity of the mid-gorge site of both ChEs. The novel spacer structures considerably alter
the biological profile of bivalent quinazolinimines with regard to both inhibitory activity and selectivity.
Mutual interaction of binding to the various sites of the enzymes was further investigated by applying
also different spacer lengths and ring sizes of the alicycle of the tricyclic quinazolinimines. In order to
achieve selectivity toward BChE and to improve inhibitory activities, the spacer structure was optimized
and identified a highly potent and selective BChE inhibitor.
Received 23 September 2010
Revised 13 December 2010
Accepted 14 December 2010
Available online 21 December 2010
Keywords:
Alzheimer’s disease
Cholinesterase
Molecular docking
Bivalent inhibitors
Quinazolinimines
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
play a role both in AD pathology and cognition in normal brains.5
In rats, a BChE selective inhibitor (cymserine) elevated brain ACh
Two cholinesterases (ChEs ), acetylcholinesterase (AChE) and
butyrylcholinesterase (BChE), regulate cholinergic neurotransmis-
sion, the deterioration of which is responsible for the decline in
memory and cognition in patients suffering from Alzheimer’s disease
(AD).1 Although the decline of cholinergic neurotransmission is most
probably a downstream process of AD pathophysiology, until now
the only therapeutic intervention uses (apart from the N-methyl
D-aspartate (NMDA) antagonist memantine) AChE inhibitors for sta-
bilizing memory and cognition decline in AD patients.2 Approved
drugs are in general characterized by comparatively low inhibitory
activity and/or a pronounced side-effect profile (e.g., hepatotoxicity
of the otherwise potent inhibitor tacrine). In the last couple of years
BChE has gained special attention, since its amount in the cortex and
hippocampus on post mortem AD brains is up-regulated, whereas
the AChE amount is significantly down-regulated.3 An AChE nullizy-
gous rodent model seems to maintain full CNS functions and BChE
seems to be able to compensate for AChE in ACh hydrolysis which
is necessary for normal brain function.4 In general, BChE seems to
levels and augmented long term potentiation and learning.6 Interest-
ingly, cymserine application also lowered the amount of b-amyloid
peptide in the rats’ brain.6 Therefore, either BChE selective com-
pounds or inhibitors with mixed activities might be superior to
established AChE inhibitors for improvement of cognitive deficits
in more advanced stages of AD.
Several novel strategies have been applied in medicinal chemis-
try to target AD drug development, for example, well-known inhib-
itors (like tacrine; Chart 1) have been used in so-called hybrid
molecules that covalently connected two distinct drug mole-
cules.7–10 Additionally, a bivalency approach (formerly mainly ap-
plied to GPCR ligands, like opioid agonists and dopamine
antagonists 11,12) has been applied to AChE inhibitors (i.e., to con-
nect covalently two inhibitor molecules by a long hydrocarbon-
spacer).10,13 Most work on AChE inhibitors is based on tacrine and
its derivatives.10,14 Bivalent inhibitors were synthesized with im-
proved inhibitory activities and often increased selectivity toward
AChE.14 But not so much work has been done using the bivalency
approach to achieve high inhibitory activity at BChE.15,16 Recently,
we have reported strongly increased BChE inhibition for bivalent
compounds derived from tri- and tetracyclic tetahydro-6H-isochi-
nolino-[1,2-a]isoquinolines and -isoquinolinium salts.15 Interest-
ingly, BChE selectivity was also achieved with some homo- and
especially heterobivalent tacrine-based inhibitors by introducing
an alkylated or acetylated nitrogen atom into the spacer.17,18
⇑
Corresponding author. Tel.: +49 941 943 3289; fax: +49 941 943 4990.
E-mail address: michael.decker@chemie.uni-regensburg.de (M. Decker).
Abbreviations: AD, Alzheimer’s disease; (h)ACh(E), (human) acetylcholine(ester-
ase); BChE, butyrylcholinesterase; BOC, tert-butoxycarbonyl; ChE, cholinesterase;
DMAP, 4-dimethylaminopyridine; EDCI, 1-ethyl-3-(3-dimethylaminopropyl) carbo-
diimide; HOBt, hydroxybenzotriazole; MOE, molecular operating environment;
NMDA, N-methyl D-aspartate; SAR, structure–activity relationship.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.12.034

X. Chen et al. / Bioorg. Med. Chem. 19 (2011) 1222–1235
1223
Chart 1. Approved AD drug and ChE inhibitor tacrine; univalent quinazolinimines with the degree of BChE selectivity depending on alicycle size;19 bivalent quinazolinimines
a–d (see Table 1 for inhibition data).16
With regard to the work presented in this paper, we have pre-
viously described tricyclic quinazolinimines as a novel class of
ChE inhibitors, in which optimization of the alicycle size was used
to achieve higher BChE selectivity (Chart 1).19 Furthermore, biva-
lent quinazolinimines turned out to be highly potent ChE-inhibi-
tors (with an activity increase of 100-fold compared to the
univalent quinazolinimines) (cf. Table 1).16 Mixed AChE/BChE inhi-
bition was observed for heptamethylene (seven carbon atoms)
spacers, irrespective of the ring size of the alicycle (five- to eight-
membered rings) (compounds a and b in the Table 1).16 By using
an octamethylene (eight atoms) spacer an increase in BChE selec-
tivity could be observed with increasing alicycle ring size.
Maximum BChE selectivity (almost 190-fold) was achieved with
an eight-membered ring system, albeit a concomitant loss in BChE
inhibitory activity was observed with increasing alicycle size (com-
pounds c and d in the Table 1).16
Using the results obtained with chemically modified spacers in
heterobivalent tacrines and also results obtained with bivalent opi-
oid ligands, in which chemical modifications of the hydrocarbon
spacer influenced the binding profile,20 we decided to incorporate
a (tertiary) basic nitrogen atom or an acylated nitrogen atom,
respectively, into the spacer structure for both five- and eight-
membered ring homobivalent quinazolinimines by applying both
seven and eight atom spacers to link the inhibitor parts.18 Since
Table 1
Inhibition results of target compounds at AChE and BChE and resulting selectivities toward BChEa
Compound
Galanthamine
IC50 (AChEb), nM (pIC50 SEM)
IC50 (BChEc), nM (pIC50 SEM)
Selectivity IC50 (AChE)/IC50 (BChE)
640 (6.197 0.052)19,22
46.5 (7.333 0.037)
8400 (5.076 0.033)19,22
6.4 (8.195 0.038)
0.08
7.3
Tacrine
a
79 (7.104 0.030)16
541.0 (6.267 0.061)
330.7 (6.481 0.038)
88 (7.056 0.104)16
547.1 (6.262 0.066)
267.0 (6.573 0.031)
0.916
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
17
18
1
N
N
N
CH3
1.2
N
N
N
O
H3C
N
N
b
65 (7.185 0.096)16
27 (7.573 0.133)16
2.416
N
N
N
N
N
N
19
202.7 (6.693 0.121)
197.0 (6.705 0.089)
1
CH3
N
(continued on next page)

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X. Chen et al. / Bioorg. Med. Chem. 19 (2011) 1222–1235
Table 1 (continued)
Compound
IC50 (AChEb), nM (pIC50 SEM)
IC50 (BChEc), nM (pIC50 SEM)
Selectivity IC50 (AChE)/IC50 (BChE)
N
N
N
N
N
20
49.0 (7.310 0.041)
72.7 (7.139 0.034)
0.7
N
O
N
H3C
c
58 (7.236 0.054)16
560.7 (6.251 0.067)
20.9 (7.680 0.051)
4.8 (8.318 0.170)16
81.3 (7.090 0.045)
4.0 (8.403 0.087)
1216
6.9
5
N
N
N
N
N
N
N
N
N
N
N
N
21
N
N
N
N
N
N
CH3
N
N
N
22
25
N
N
N
N
N
O
H3C
N
18.0 (7.746 0.053)
5.9 (8.230 0.075)
3
O
CH3
N
N
N
N
N
N
N
N
N
27
6.7 (8.174 0.087)
10.6 (7.974 0.090)
0.6
N
N
N
N
N
O
N
N
N
N
N
H2C
CH3
d
14400 (4.842 0.059)16
605.8 (6.218 0.066)
177.4 (6.751 0.055)
289.0 (6.539 0.089)
76 (7.120 0.082)16
51.4 (7.289 0.033)
43.1 (7.365 0.028)
46.3 (7.335 0.125)
18916
11.8
4.2
N
N
N
N
N
N
N
N
N
N
N
N
N
N
23
24
26
CH3
O
H3C
N
6.2
O
CH3

X. Chen et al. / Bioorg. Med. Chem. 19 (2011) 1222–1235
1225
Table 1 (continued)
Compound
IC50 (AChEb), nM (pIC50 SEM)
IC50 (BChEc), nM (pIC50 SEM)
Selectivity IC50 (AChE)/IC50 (BChE)
N
N
N
N
N
N
N
N
N
28
153.2 (6.815 0.163)
37.7 (7.424 0.100)
4.1
N
O
N
N
H2C
HN
CH3
N
29
75.6 (7.122 0.060)
122.1 (6.913 0.149)
0.62
N
O
O
CH3
N
N
N
N
N
N
N
N
30
536.0 (7.271 0.044)
1056.0 (5.976 0.109)
621.0 (6.207 0.056)
25.5 (7.593 0.025)
12.9 (7.889 0.037)
8.1 (8.093 0.084)
21
N
O
N
N
H3C
H3C CH3
N
N
31
81.9
76.7
N
O
H3C
N
N
N
N
N
N
N
N
N
32
N
H3C
H3C
O
O
N
N
N
33
792.1 (6.101 0.024)
3.0 (8.525 0.019)
264
N
H3C
CH3
a
Values are means of at least three independent determinations, pIC50 = ꢀlog IC50
EC 3.1.1.7, type VI-S, from Electric Eel.
EC 3.1.1.8, from equine serum.
.
b
c
the bivalent quinazolinimines had previously been optimized in
terms of heterocycle structure,19,21 the size of the alicycle,19,22
and the spacer length,16 respectively, the spacer’s chemical struc-
ture remained to be optimized in this set of structure–activity rela-
tionships (SARs) using knowledge about the enzymes’ binding sites.
In this work we have applied molecular docking parallel to the
syntheses to understand the binding profiles of available inhibitors
that we synthesized and to predict novel structures with optimal
spacer interaction with both ChEs or with selectivity towards BChE.
To probe the mid-gorge binding sites of the ChEs involving interac-
tion with the spacer substructures, we synthesized a set of com-
pounds in which a central nitrogen atom in the spacer was
modified as a basic N-CH3 group or various N-acylated groups. To
probe successively the mid-gorge binding site (while using both
five- and eight-membered alicycle size and seven and eight atom
spacers) we incorporated N-acyl groups containing H-bond accep-
tors, donors, as well as hydrophobic moieties. The preference of
hydrophobic substitutions to increase inhibitory activity of the com-
pounds was further investigated in terms of their length and steric
expansion to yield optimal activity and selectivity toward BChE.
2. Chemistry
The target spacer-modified bivalent quinazolinimines (17–33)
were synthesized from quinazolinethiones (16a,b) and appropriate
a,x-diamines (5–15) (Scheme 1). The heterocyclic key inter-
mediate five- and eight-membered ring quinazolinethiones
(2,3-dihydropyrrolo[2,1-b]quinazoline-9(1H)-thione 16a and 8,9,
10,11-tetrahydro-6H-azocino[2,1-b]quinazoline-13(7H)-thione 16b)
were synthesized from the respective five-membered ring lactam
(pyrrolidin-2-one) and eight-membered ring lactam (azocan-2-
one) by reaction with Meerwein salt (triethyloxonium tetrafluoro-
borate) to yield the reactive iminium ethers, which reacted with
anthranilic acid to yield the respective tricyclic five-membered
(2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one) and eight-mem-
bered ring quinazolinones (8,9,10,11-tetrahydro-6H-azocino
[2,1-b]quinazolin-13(7H)-one).19,23 The oxygen-atom of the qui-
nazolinones could be replaced by a sulphur atom using Lawesson’s
reagent to yield compounds 16a,b.16,19
For selective alkylation (5 and 6) and acylation (7–15) of the
middle secondary amine-group of dipropylenetriamine (1) and

1226
X. Chen et al. / Bioorg. Med. Chem. 19 (2011) 1222–1235
O
O
CH3
CH3
CH3
CH3
N
N
N
O
CH3
2
N
N
N
H3C
H2N
OH
2
+ 2
(i)
N
H
NH2
(ii)
n = 1,2
1
: n = 1
2: n = 2
3: n = 1
BocHN
N
H
NHBoc
n = 1,2
4
: n = 2
O
O
+
R
(iii)
+ R
(iv)
(v)
OH
Cl
BocHN
N
NHBoc
n = 2
BocHN
N
CH3
NHBoc
n = 1,2
BocHN
N
O
NHBoc
n = 1,2
O
R
R
5: n = 1
7: n = 1, R = CH3
14: R = CH2NHCOCH3
6
: n = 2
8
9
15
: R = CH2CH2CH(CH3)2
:
:
n = 2, R = CH3
n = 2, R = CH2CH3
10: n = 2, R = C(=CH2)CH3
11
: n = 2, R = C(CH3)3
12: n = 2, R = (CH2)3CH3
13
: n = 2, R = CH2CH(CH3)2
S
S
N
S
N
N
N
(vi)
(vi)
(vi)
m =1,4
m =1,4
N
N
16a
16a
16b
: m = 1
: m = 1
16b: m = 4
16b: m = 4
m = 1,4
N
N
N
N
N
N
n = 1,2
N
R
m = 1,4
27: n = 2, m = 1, R = COC(=CH2)CH3
17: n = 1, m = 1, R = CH3
28
29
18
19
: n = 2, m = 4, R = COC(=CH2)CH3
: n = 2, m = 4, R = COCH2NHCOCH3
: n = 1, m = 1, R = COCH3
: n = 1, m = 4, R = CH3
30: n = 2, m = 4, R = COC(CH3)3
20: n = 1, m = 4, R = COCH3
31
: n = 2, m = 4, R = CO(CH2)3CH3
21
: n = 2, m = 1, R = CH3
32: n = 2, m = 4, R = COCH2CH(CH3)2
33
22: n = 2, m = 1, R = COCH3
23
24
: n = 2, m = 4, R = COCH2CH2CH(CH3)2
: n = 2, m = 4, R = CH3
: n = 2, m = 4, R = COCH3
25: n = 2, m = 1, R = COCH2CH3
26
: n = 2, m = 4, R = COCH2CH3
Scheme 1. Synthesis of spacer-modified bivalent quinazolinimines 17–33 with varying spacer lengths and modified chemical structures of the spacer. Reagents and
conditions: (i) KOH, toluene, 120 °C, 4 h; (ii) dipropylenetriamine (1) or spermidine (2), 60 °C, 4 h; (iii) CH3I, K2CO3, CH3CN, rt, 24 h; (iv) NEt3, cat. DMAP, CH2Cl2, 5 °C?rt, 3 h;
(v) EDCI, HOBt, CH2Cl2, rt, 12 h; (vi) (1) 4 M HCl in 1,4-dioxane, CH2Cl2, rt, 12 h; (2) HgBr2, Na2CO3, EtOH/toluene, 135 °C, 2 h.
spermidine (2), respectively, selective protection of both primary
amino-groups was necessary, which was achieved using N,N0-car-
bonyldiimidazole, tert-butanol and KOH in toluene to yield the
di-Boc-protected compounds with a free secondary amine function
(3 and 4; Scheme 1).24 Methylation of di-Boc-protected dipropy-
lenetriamine (5) and spermidine (6) was achieved by methyl iodide
and potassium carbonate in acetonitrile, whereas acylation was
achieved using the respective acid chlorides, triethylamine and
catalytic amounts of DMAP (Scheme 1). Alternatively, acylation
of 4 could be achieved using the free carbonic acid, EDCI and cata-
lytic amounts of HOBt (14 and 15, Scheme 1).
starting materials and the use of the spacer hydrochlorides: A 1/
1 mixture of toluene and ethanol was used as solvent, sodium car-
bonate and mercury(II) bromide as reagents (Scheme 1).
3. Results and discussion
We not only wanted to gain information on whether and how
the binding and inhibition profile of bivalent quinazolinimines is
influenced by the spacer structure, but also to investigate if the
spacer structure alone, or if the length of the spacer and the size
of the alicycle concomitantly modify the inhibition profile. There-
fore, five- and eight-membered ring systems, in which five-mem-
bered alicycles in tricyclic quinazolinimines exhibit the highest
activity at AChE in the set of alkylene bridged bivalent com-
pounds,16 were investigated and spacer lengths of seven and eight
atoms were synthesized and their inhibition profiles investigated
systematically in correlation to different spacer structures. Enzyme
inhibition was determined applying the colorimetric Ellman assay
For the formation of bivalent quinazolinimines (17–33) we used
N-methylated and N-acylated
a,x-diamines as starting materials
for the spacer structure in the form of their hydrochlorides, which
were obtained from the di-Boc-protected forms (5–15) using 4 M
HCl in 1,4-dioxane. The method for condensation with quinazolin-
ethiones had to be slightly modified compared to previously ap-
plied method16,19 due to altered solubility properties of the

X. Chen et al. / Bioorg. Med. Chem. 19 (2011) 1222–1235
1227
using AChE (EC 3.1.1.7, type VI-S, from electric Eel) and BChE (E.C.
3.1.1.8, from equine serum), with acetylthiocholine (ATC) and
butyrylthiocholine (BTC) respectively as substrates.19,21
ligand-binding cavity of AChE (Fig. 1B). The extra-volume in BChE
is gained due to non-conserved residues, which exhibit different
physicochemical and geometrical properties compared to the
properties of residues in AChE. Thus, aromatic residues N96,
Q147, V305, L314, A356 lining the binding cavity of BChE are
substituted by Y94, Y146, W304, F315, Y355 correspondently in
AChE. The large volume of the BChE binding cavity could explain
the higher binding tolerance to the structural modifications of
the compounds.
The detailed pictures of the superimposed docking poses of 22
and 24 are also shown in Figure 1 (C for BChE and D for AChE
respectively). As expected, the heterocyclic parts of bivalent qui-
nazolinimine inhibitors form characteristic aromatic interactions
in these enzymes. Thus, in BChE, one aromatic moiety is engaged
Altogether, seventeen novel bivalent inhibitors were synthe-
sized. In the set of seven atom spacer bridged compounds (17–
20), activities were generally lower than for the alkylene-bridged
parent compounds (a, b), with both N-CH3 (17 and 19) and N-
COCH3 (18 and 20) groups incorporated into the spacer (inhibitory
activities ranged from IC50 = 49 nM for 20 to 541 nM for 17 at
AChE). Especially the N-CH3 groups led to a three to sevenfold
(17) loss in activity at AChE and at BChE as well. As for the parent
compound, activity was almost identical at both ChEs. The intro-
duction of an acetyl group into the spacer of heterobivalent tac-
rines was described in the literature to improve BChE selectivity
suggesting that the mid-gorge interaction site might be a promis-
ing binding spot to shift selectivity towards BChE.17 For the N-
COCH3 compounds in the set of bivalent quinazolinimines, loss in
activity was observed, but it was far less pronounced than for the
N-CH3 compounds (with the only positive exception of a slightly
increased activity at AChE for the eight-membered ring bivalent
compound 20, IC50(AChE) = 49 nM and IC50(BChE) = 73 nM).
The binding data was different for quinazolinimines with eight
atoms spacers. And as for the seven atom bridged quinazolini-
mines, their binding data was also completely different from the
data of bivalent tacrines.17,18 For five-membered ring quinazolini-
mines, N-CH3 led to a dramatic activity decrease at both ChEs
(21, IC50(AChE) = 561 nM and IC50(BChE) = 81 nM).7 For the eight-
membered ring quinazolinimines there was a slight increase in
BChE activity, and surprisingly a 24-fold activity increase at AChE
also, which reduced the selectivity from 189-fold to 12-fold (23).
An N-COCH3 group led to activity increase (22 and 24) compared
to the alkylene parent compound: The high activity at BChE was
very slightly enhanced with both five- and eight-membered alicy-
cle quinazolinimines, activity at AChE was improved threefold for
five-membered rings (22, IC50(AChE) = 21 nM and IC50(B-
ChE) = 4 nM) and 81-fold for eight-membered rings (24, IC50(A-
ChE) = 177 nM and IC50(BChE) = 43 nM). Notably, inhibition data
at both AChE and BChE was therefore considerably different from
the data of the homobivalent compounds connected by a hydrocar-
bon spacer.
We then investigated computationally the binding of N-acety-
lated bivalent compounds 22 (five-membered ring alicycles) and
24 (eight-membered ring alicycles) with AChE and BChE to provide
structural insights and potential to functional modifications. For
that we constructed the homology models of the investigated spe-
cies, that is, equine BChE and electric eel AChE, using the crystal
structure of Torpedo californica AChE in the complex with a biva-
lent inhibitor (PDB code: 2CEK),25 thus we generated a bivalent
inhibitor-adapted conformation to simplify docking studies. We
built the 3D models of the enzyme species used for our biological
experiment for docking studies, as it has shown that the close ser-
ies of analogs binds to enzyme species differently,17,26,27 despite
the high sequence homology among species (more then 80%).28
Thus, we noted that V305, L313 and I426 of the binding site (7Å
from the ligand) in the equine species of BChE are substituted to
corresponding ALA, PRO and PHE in human BChE. In analogy S98,
F309, S310, G311 in the electric eel AChE located at the entrance
to the binding cavity correspond to LEU, GLN, GLU, SER in human
AChE. We hypothesize that these residues might be important
for species selectivity. The structural models of AChE and BChE in
cartoon representation coloured based on the secondary structure
and the ligand-binding site shown in the form of the Connolly sur-
face with docked 22 (i.e., acetylated eight atoms spacer compounds
with five-membered ring systems) in the space filling representa-
tion are shown in Figure 1A (BChE) and B (AChE). The ligand-bind-
ing cavity of BChE (Fig. 1A) is more spacious compared to the
in p-p stacking interactions with W110 of BChE, whilst the second
moiety locates around V305, D311 and F306 according to our dock-
ing studies.
In AChE, the bivalent inhibitor has two
p-p stacking interac-
tions between one quinazolinimine moiety and W108, and another
quinazolinimine moiety and W304 and Y94. The acyl group of the
linker forms hydrogen bond with Q147 in BChE and Y146 in AChE,
that provides a new favourable interaction with the enzyme. We
used the Site Map of Schrödinger software to predict the preferable
regions for hydrophobic and hydrophilic interactions, which are
also shown in Figure 1C and D. Accordingly, the regions with the
preference of hydrophobic groups are represented in yellow, while
the regions with the preference of acceptor and donor groups are
shown in red and blue, respectively. The Site Map shows that there
is possibility to increase the size of the functional group of the
spacer using hydrophobic substituents in order to increase potency
of inhibition especially toward BChE. This information was applied
in the next synthetic sequence.
In the case of N-acetyl substituent compounds, the remarkable
increase in AChE activity together with the slight increase at BChE
activity actually led to decreased selectivity. Given that there are
preferable regions around the mid-gorge of BChE we wanted to in-
crease this selectivity while maintaining high BChE activity by tak-
ing advantage of the mid-gorge region. For this reason the acyl
moiety of bulky eight-membered quinazolinimines was further
modified, whereas N-alkyl substituents were not investigated fur-
ther because of their poor biological profile in relation to potency
and BChE selectivity. A similar observation was later also made
for a different class of AChE hybrid inhibitors described by Pisani
et al.7 Apart from the available non-conserved residues in the
mid-gorge regions of the ChE binding site, which could be targeted
to design selective ligands, the available crystal structures of ChEs
as well as molecular dynamics studies of unoccupied and occupied
states of the enzymes have shown that there is a high degree of en-
zyme flexibility during the binding of an allosteric ligand that
causes substantial structural changes in the catalytic site.29–32
Importantly, the bioinformatics analysis of available crystal struc-
tures shows that the residues in the mid-gorge are likely in coop-
erative motions.18 Intuitively, the blockade of their mobility could
lead to modulation of enzyme inhibition. We therefore persisted in
modification of the mid-gorge spacer to modulate enzyme selectiv-
ity and improve potency.
We replaced the acetyl by a propionyl group (26 with eight-
membered alicycles, 25 with five-membered alicycles) and found
that the propionyl showed the same binding profile as acetyl at
both ChEs, with a slightly increased BChE selectivity for 26. Meth-
ylacryloyl substituents (27 and 28), which should be able to be
polarized and thus interact with polar groups of the mid-gorge
predicted by Site Map, led to an around twofold increase in AChE
activity compared to the propionyl compounds (both for five-
and eight-membered compounds) without significantly affecting
BChE activity. As a result, five-membered rings were abandoned

1228
X. Chen et al. / Bioorg. Med. Chem. 19 (2011) 1222–1235
Figure 1. The binding site of spacer-modified (N-acetylated) bivalent quinazolinimines 22 and 24 in butyrylcholinesterase (BChE) and acetylcholinesterase (AChE). (A and
B)—the ligand-binding cavity of BChE (A) and AChE (B) in the Connolly surface representation coloured based on the hydrophilic and hydrophobic properties (hydrophobic
regions are in green, receptor donor regions are in purple and receptor acceptor regions are in blue) with docked compound 22 having a eight atoms spacer and the N-COCH3
group in the space filling representation. The enzyme structure is shown in cartoons and coloured based on the secondary structure. The non-conserved residues are labelled
in yellow. (C and D)—the superimposed binding site of compounds 22 and 24 in BChE (C) and in AChE (D). The ligands are shown in stick-like representation with the carbon
atoms coloured in dark and light green. The residues of the binding sites are in stick-like representation with the carbon atoms coloured in grey. The preferable regions for
hydrophobic and hydrophilic interactions predicted by the Site Map (Schrödinger software) are shown in yellow (hydrophobic group preference), red (acceptor group
preference) and blue (donor group preference).
at this stage because they represented no promising contribution
to the improvement of the potency and selectivity focusing on
BChE. Yet the potential interaction of the substituent with polar
groups in the gorge led to the incorporation of an N-acetylglycine
moiety (29), which contains both H-bond-accepting and -donating
groups and might exhibit the interaction with other amino acids
within the mid-gorge of the enzymes. Surprisingly, despite its long
and sterically demanding spacer group, 29 shows IC50 values of
122 nM at BChE and 76 nM at AChE (Table 1). In contrast to previ-
ous results obtained with bis-tacrines, this sterically demanding
moiety is obviously able to interact well with AChE resulting in
low IC50 value at AChE. To explain this unexpected biological re-
sult, 29 was docked to the binding sites of both ChEs (Fig. 2A and
B). Our docking studies on the N-acetylglycine compound 29 (BChE
in Fig. 2A and AChE in B) show that the acetylglycine substituent
occupies a different position in the enzymes, thus it faces Q147
forming hydrogen bonds with its side chain and the backbone of
L314 in BChE (Fig. 2A), whilst it orientates in somewhat the oppo-
site direction forming a hydrogen bond with the backbones of F313
in AChE (Fig. 2B). The docking score of compound 29 is ꢀ17 kcal/
mol in AChE whereas ꢀ11.4 kcal/mol in BChE, indicating the pref-
erence in binding to AChE. This is in agreement with the predicted
hydrophilic binding sites by Site Map. Different location of the ace-
tylglycine group might account for the observed comparatively
high potency in both ChEs. Since our search was aiming to design
BChE-selective compounds, the comparatively low IC50 value at
BChE and the availability of preferable hydrophobic zones calcu-
lated by Site Map encouraged us to probe sterically even more
demanding moieties into the mid-gorge that lack the possibility
to interact with polar groups.
A sterically demanding 2,2-dimethylpropanoyl moiety (30)
showed improved activity at BChE (compared to methylacryloyl
and propionyl) with an IC50(BChE) of 26 nM. But despite the three-
fold increase in BChE activity compared to the alkylene-bridged
parent compound (d), the compound’s 21-fold BChE selectivity is
still lower. An interesting development was observed with even
longer chains: an unbranched pentanoyl moiety attached to the
spacer (31) showed 82-fold selectivity with an IC50(BChE) of
13 nM followed by a shorter 3-methylbutanoyl moiety (32) that
caused a further activity increase to an IC50(BChE) of 8 nM with
similar 77-fold selectivity. In the end, the combination of the long-
er chain with additional methyl-substitution to 4-methylpenta-
noyl-(33) led to the most potent and selective compound in the
whole series of compounds with an IC50(BChE) of 3 nM and 260-
fold selectivity over AChE (IC50 = 792 nM). Compound 33 therefore
surpasses the most selective compound in the alkylene-bridged
series of bivalent quinazolinimines (d) being 25-fold more active
at BChE with even improved selectivity. Even the most potent,
but unselective alkylene-bridged bivalent quinazolinimine with
five-membered ring alicycles (c) could be surpassed in BChE activ-
ity with 33.
In order to explain the molecular basis of high potency and
selectivity of 33 to BChE we have docked this compound to BChE
(Fig. 2C) and AChE binding sites as well (Fig. 2D). The most

X. Chen et al. / Bioorg. Med. Chem. 19 (2011) 1222–1235
1229
Figure 2. The preferable binding pose of compounds 29 and 33 in butyrylcholinesterase (BChE), (A and C) and acetylcholinesterase (AChE), (B and D) obtained by Induced Fit
docking (Schrödinger software). The ligands and the binding sites are in stick-like representation, compound 29 is in purple, compound 33 is in cyan and the residues of the
binding site are in grey. The prefarable regions for hydrophobic and hydrophilic interactions predicted by the Site Map (Schrödinger software) are shown in yellow
(hydrophobic group preference), red (acceptor group preference) and blue (donor group preference).
populated docking poses are shown in Figure 2C and D. Thus, in BChE
the functional group of the spacer locates in the hydrophobic bind-
ing pocket composed of L314, V316 and W259 which is shown in
yellow in Figure 2C. This preferable interaction leads to an increase
of compound affinity. Since L314 and V316 in BChE correspond to
F313 and F315 in AChE, the new hydrophobic interaction may be
responsible for selectivity of 33. On the contrary, there is no empty
pocket with preference to hydrophobic substituents in AChE be-
cause the corresponding F313 and F315 are involved into the intra-
molecular aromatic interaction network. However, there is the
hydrophobic pocket involving Y94, W304 and F315 mapped in yel-
low on Figure 2D. Indeed, our docking studies show that the pref-
erable pose for the aliphatic substituent of 33 is in the region of
these residues in AChE. However, this interaction in AChE partially
fills the space between W304 and F315 and thus prevents the for-
ing profile in terms of activity and especially selectivity. Intensive
investigation into the different structural parameters identified
compound 33 (showing an eight atom spacer, eight-membered
ring alicycles and a 4-methylpentanoyl moiety as the acyl part)
as a highly potent and selective inhibitor of BChE. Molecular dock-
ing of N-acetylglycine derivative (29) incorporating H-bond-
accepting and -donating properties was performed and yielded
low energy differences, which on the contrary suggested that the
hydrophobic interaction of the 4-methylpentanoyl moiety with
the mid-gorge site of the enzyme is responsible for high potency
of compound 33. Taken together, the different shape and physico-
chemical properties of the mid-gorge site of cholinesterases as well
as its flexibility provide the possibility to modify the spacer of biva-
lent ligands leading to novel binding profiles with high BChE po-
tency and selectivity.
mation of the
p-p electron sandwich with one quinazolinimine,
one of the key interactions of these bivalent inhibitors with ChEs.
The loss of this interaction likely leads to the significant loss of po-
tency of 33 in AChE. Accordingly, the quantitative docking score of
33 in the chosen poses is ꢀ9 kcal/mol in AChE and ꢀ10.5 kcal/mol
in BChE supporting high affinity of 33 to bind BChE. But in general,
it turned out to be hard to get a reliable prediction of experimental
inhibitory activity from the docking procedures performed due to
interactions of the spacer moieties with the enzymes’ structure
that impede reliable predictions.
5. Experimental section
5.1. Chemistry
Melting points are uncorrected and were measured in open cap-
illary tubes, using a Barnstead Electrothermal IA9100 melting point
apparatus. 1H and 13C NMR spectral data were obtained from a
Bruker Advance spectrometer (400 MHz and 125 MHz, respec-
tively). TLC was performed on silica gel on aluminium foils with
fluorescent indicator 254 nm (Fluka) or aluminium oxide on
TLC-PET foils with fluorescent indicator 254 nm (Fluka). For detec-
tion iodine vapour or UV light (254 nm), respectively, were used.
ESI-MS samples were analyzed using electrospray ionisation
ion-trap mass spectrometry in nanospray mode using a Thermo
Finnigan LCQ Deca. The CHN analyses were undertaken using
Perkin Elmer Elemental Analyser PE2400CHNS. For column
chromatography, silica gel 60, 230–400 mesh (Merck) or activated
neutral aluminium oxide, approximately 150 mesh, 58Å
(Sigma–Aldrich) were used.
4. Conclusion
Novel bivalent quinazolinimines were synthesized in which a
nitrogen atom was introduced into the alkylene hydrocarbon spac-
ers to yield tertiary amines or N-acyl amides. Apart from the spacer
structure the alicycle ring size of the heterocyclic quinazolinimines
and the spacer length were also modified to investigate mutual
interactions on compound activity and selectivity. Chemical mod-
ification of the spacer structure shows great influence on the bind-

1230
X. Chen et al. / Bioorg. Med. Chem. 19 (2011) 1222–1235
5.1.1. N-(t-Butoxycarbonyl)-N0-[3-(t-butoxycarbonylamino)-
propyl]-1,3-propanediamine (3)
a solution of propionyl chloride (0.67 mL, 4.4 mmol) in dichloro-
methane (10 mL) at 5 °C. After the addition, the solution was stir-
red at room temperature for 3 h. To the mixture 5% citric acid
aqueous solution (15 mL) was added and the aqueous layer was
separated. The organic layer was dried over MgSO4, filtered and
solvent evaporated. The crude product was purified by column
chromatography (dichloromethane/methanol, 20:1) to yield the
product (1.27 g, 79 %) as a viscous yellow liquid. 1H NMR (CDCl3,
400 MHz) d: 1.07 (t, 3H, J = 7.42 Hz, COCH2CH3), 1.35–1.70 (m,
24H, 2 ꢁ Boc, NCH2(CH2)2CH2NH, NCH2CH2CH2NH), 2.26 (q, 2H,
J = 7.46 Hz, NCOCH2), 2.96–3.34 (m, 8H, 2 ꢁ NCH2, 2 ꢁ BocNHCH2),
4.69 (br s, 1H, NHBoc), 5.41 (br s, 1H, NHBoc) ppm. 13C NMR (CDCl3,
400 MHz) d: 9.71 (COCH2CH3), 24.95, 25.97, 26.22, 27.58, 27.91,
28.38, 28.44, 37.17, 39.85, 42.32, 45.40, 78.79 (OC(CH3)3), 79.26
(OC(CH3)3), 156.05 (NHCOOtBu), 173.38 (NCOCH2CH3), 174.18
(NCOCH2CH3) ppm. ESI-MS m/z 402.3 [M+H]+.
Compound 3 was synthesized according to the method described
in Ref. 24. Colourless oil, yield 66 %. 1H NMR (CDCl3, 300 MHz) d: 1.37
(s, 18H, 2 ꢁ Boc), 1.56 (m, 4H, 2 ꢁ NHCH2CH2), 2.58 (t, 4H, J = 3.0 Hz,
2 ꢁ NHCH2), 3.14 (t, 4H, J = 6.0 Hz, 2 ꢁ BocNHCH2), 5.15 (br s, 1H,
NH) ppm. 13C NMR (CDCl3, 300 MHz) d: 27.43 (C(CH3)3), 28.79
(NHCH2CH2), 37.96 (BocNHCH2), 46.43 (NHCH2), 79.28 (NHC-
OOC(CH3)3), 155.12 (NHCOOtBu) ppm. Spectral data is in accordance
with literature data.24
5.1.2. N-(t-Butoxycarbonyl)-N0-[3-(t-butoxycarbonylamino)-
propyl]-1,4-butanediamine (4)
Compound 4 was synthesized according to the method described
in Ref. 28. Colourless oil, yield 61 %. 1H NMR (CDCl3, 300 MHz) d:
1.36–1.45 (m, 22H, 2 ꢁ Boc, NHCH2(CH2)2CH2NH), 1.57 (m, 2H,
NHCH2CH2CH2NH), 2.53 (t, 2H, J = 6.64 Hz, NHCH2(CH2)3NHBoc),
2.58(t, 2H, J = 6.61 Hz, NHCH2(CH2)2NHBoc),3.05(m, 2H,BocNHCH2),
3.12 (m, 2H, BocNHCH2), 4.99 (br s, 1H, NHBoc), 5.28 (br s, 1H, NHBoc)
ppm. 13C NMR (CDCl3, 300 MHz) d: 27.69, 28.16, 28.78, 30.21, 39.50,
40.76, 48.00, 49.76, 79.16 (OC(CH3)3), 156.45 (NHCOOtBu) ppm.
Spectral data is in accordance with literature data.28
5.1.7. N-Methacryloyl-N0-(t-butoxycarbonyl)-N-[3-(t-butoxycar-
bonylamino)propyl]-1,4-butanediamine (10)
This compound was prepared analogously to compound 9. Com-
pound 4 (1.5 g, 4.35 mmol), triethylamine (0.73 mL, 5.25 mmol) and
methacryloyl chloride (0.467 mL, 4.78 mmol) were used and the
crude product was purified by column chromatography (dichloro-
methane/methanol, 20:1 as eluent) to yield the product (1.6 g, 89 %)
as a viscous colourless liquid. 1H NMR (CDCl3, 400 MHz) d: 1.36–
1.52 (m, 22H, 2 ꢁ Boc, NCH2(CH2)2CH2NH), 1.64–1.65 (m, 2H, NCH2-
CH2CH2NH), 1.88 (s, 3H, COC(CH3)@CH2), 3.00–3.04 (m, 4H, 2 ꢁ
NCH2), 3.24–3.39 (m, 4H, 2 ꢁ BocNHCH2), 4.70 (br s, 1H, NH), 4.92
(s, 1H, @CH), 5.06 (s, 1H, @CH), 5.40 (br s, 1H, NH) ppm. 13C NMR
(CDCl3, 400 MHz) d: 20.65 (COC(CH3)@CH2), 26.14, 27.39, 27.79,
28.37, 28.39, 37.31, 39.95, 41.05, 48.11, 78.84 (OC(CH3)3), 79.14
(OC(CH3)3), 114.55 (@CH2), 141.00 (COC(CH3)@CH2), 156.01 (NHCO-
OtBu), 173.19 (COC(CH3)@CH2) ppm. ESI-MS m/z 414.4 [M+H]+.
5.1.3. N-(t-Butoxycarbonyl)-N0-[3-(t-butoxycarbonylamino)-
propyl]-N0-methyl-1,4-butanediamine (6)
Compound 6 was synthesized according to the method described
in Ref. 18. Viscous yellow liquid, yield 30 %. 1H NMR (CDCl3,
300 MHz) d: 1.37 (s, 18H, 2 ꢁ Boc), 1.44 (m, 4H, NHCH2(CH2)2-
CH2NH), 1.59 (m, 2H, NHCH2CH2CH2NH), 2.13 (s, 3H, NCH3), 2.28–
2.35 (m, 4H, 2 ꢁ CH2N(CH3)), 3.05–3.12 (m, 4H, 2 ꢁ CH2NHBoc),
4.87 (s, 1H, NH), 5.32 (s, 1H, NH) ppm. 13C NMR (CDCl3, 300 MHz)
d: 24.90, 27.22, 28.19, 28.88 (C(CH3)3), 40.22 (NHCH2), 40.80(NH-
CH2), 42.15 (NCH3), 56.66 (NCH2), 57.69 (NCH2), 79.30 (OC(CH3)3),
79.37 (OC(CH3)3), 156.48 (NHCOOtBu), 156.52 (NHCOOtBu) ppm.
Spectral data is in accordance with literature data.18
5.1.8. N-[4-(t-Butoxycarbonylamino)butyl]-N-[3-(t-butoxycar-
bonylamino)propyl]pivalamide (11)
5.1.4. N,N-Bis[3-(t-butoxycarbonylamino)propyl]acetamide (7)
Compound 7 was synthesized according to the method de-
scribed in Ref. 18. Viscous yellow liquid, yield 100 %. 1H NMR
(CDCl3, 300 MHz) d: 1.37 (d, 18H, 2 ꢁ Boc), 1.57–1.62 (m, 2H,
NHCH2CH2), 1.68–1.75 (m, 2H, NHCH2CH2), 2.02 (s, 3H, NCOCH3),
3.00 (q, 2H, J = 6.14 Hz, NCH2), 3.08 (q, 2H, J = 6.33 Hz, NCH2),
3.20 (t, 2H, J = 7.78 Hz, NHCH2), 3.32 (t, 2H, J = 6.46 Hz, NHCH2),
4.62 (br s, 1H, NH), 5.29 (br s, 1H, NH) ppm. 13C NMR (CDCl3,
300 MHz) d: 21.79 (COCH3), 28.36, 28.80, 28.88, 30.09, 37.66,
38.53, 42.81, 46.63, 79.32 (COOC(CH3)3), 80.00 (COOC(CH3)3),
156.42 (NHCOOtBu), 156.57 (NHCOOtBu), 171.22 (COCH3) ppm.
Spectral data is in accordance with literature data.18
This compound was prepared analogously to compound 9. Com-
pound 4 (0.5 g, 1.45 mmol), triethylamine (0.24 mL, 1.74 mmol) and
trimethylacetyl chloride (0.19 mL, 1.5 mmol) were used and the
crude product was purified by column chromatography (dichloro-
methane:methanol, 20:1 as eluent) to yield the product (0.5 g, 80
%) as a colourless oil.1H NMR (CDCl3, 400 MHz) d: 1.19 (s, 9H,
NHCOC(CH3)3), 1.37–1.66 (m, 24H, 2 ꢁ Boc, NCH2(CH2)2CH2NH,
NCH2CH2CH2NH), 3.00–3.07 (m, 4H, 2 ꢁ NCH2), 3.25–3.33 (m, 4H,
2 ꢁ BocNHCH2), 4.82 (br s, 1H, NH), 5.21 (br s, 1H, NH) ppm. 13C
NMR (CDCl3, 400 MHz) d: 26.44, 27.12, 27.38, 28.35, 28.37, 28.47,
38.04, 38.32, 39.00, 40.14, 40.24, 50.32, 79.27 (OC(CH3)3), 79.37
(OC(CH3)3), 156.27 (NHCOOtBu), 156.32 (NHCOOtBu), 177.64
(NCOC(CH3)3) ppm. ESI-MS m/z 430.2 [M+H]+.
5.1.5. N-Acetyl-N0-(t-butoxycarbonyl)-N-[3-(t-butoxycarbonyl-
amino)propyl]-1,4-butanediamine (8)
Compound 8 was synthesized according to the method described
in Ref. 18. Viscous yellow liquid, yield 80 %. 1H NMR (CDCl3,
300 MHz) d: 1.46–1.60 (m, 24H, 2 ꢁ Boc, NHCH2(CH2)2CH2NH,
NHCH2CH2CH2NH), 2.09 (s, 3H, NCOCH3), 3.08–3.41 (m, 8H,
2 ꢁ NCH2, 2 ꢁ NHCH2), 4.57 (br s, 1H, NH), 5.32 (br s, 1H, NH) ppm.
13C NMR (CDCl3, 300 MHz) d: 18.56 (COCH3), 25.65, 26.17, 27.22,
27.58, 27.91, 28.47, 28.53, 37.24, 39.76, 43.40, 45.52, 78.68
(OC(CH3)3), 79.15 (OC(CH3)3), 156.42 (NHCOOtBu), 172.54
(NCOCH3) ppm. ESI-MS m/z 388.3 [M+H]+. Spectral data is in
accordance with literature data.18
5.1.9. N-[4-(t-Butoxycarbonylamino)butyl]-N-[3-(t-butoxycar-
bonylamino)propyl] pentanamide (12)
This compound was prepared analogously to compound 9. Com-
pound 4 (0.5 g, 1.45 mmol), triethylamine (0.24 mL, 1.74 mmol) and
n-pentanoyl chloride (0.182 mL, 1.5 mmol) were used and the crude
product was purified by column chromatography (dichlorometh-
ane:methanol, 20:1 as eluent) to yield the product (0.51 g, 82 %) as
a colourless oil. 1H NMR (CDCl3, 400 MHz) d: 0.85 (t, J = 7.32 Hz,
CH3), 1.18–1.72 (m, 28H, 2 ꢁ Boc, NCH2(CH2)2CH2NH, NCH2CH2CH2-
NH, COCH2(CH2)2CH3), 2.22 (q, 2H, J = 7.60 Hz, COCH2), 2.95–3.34
(m, 8H, 2 ꢁ NCH2, 2 ꢁ BocNHCH2), 4.62 (br s, 1H, NH), 5.40 (br s,
1H, NH) ppm. 13C NMR (CDCl3, 400 MHz) d: 13.87 (CH3), 22.56
(CH2CH3), 27.61, 27.67, 28.38, 28.44, 32.80 (COCH2), 42.45, 45.40,
45.57, 47.47, 53.38, 78.87 (OC(CH3)3), 79.35 (OC(CH3)3), 156.04
(NHCOOtBu), 172.90 (NCOCH2), 173.60 (NCOCH2) ppm. ESI-MS m/z
430.2 [M+H]+.
5.1.6. N-Propionyl-N0-(t-butoxycarbonyl)-N-[3-(t-butoxycar-
bonylamino)propyl]-1,4-butanediamine (9)
To a solution of compound 4 (1.4 g, 4 mmol), triethylamine
(0.67 mL, 4.8 mmol) and a catalytic amount of 4-dimethylamino-
pyridine (DMAP) in dichloromethane (10 mL) was added dropwise

X. Chen et al. / Bioorg. Med. Chem. 19 (2011) 1222–1235
1231
5.1.10. 3-Methyl-N-[4-(t-butoxycarbonylamino)butyl]-N-[3-(t-
butoxycarbonylamino)propyl] butanamide (13)
The reaction mixture was concentrated and the crude product
hydrochloride was used in the next step without further purifica-
tion. Alternatively, N-Me-N-(3-propylamino)propanediamine is
also commercially available. A mixture of 2,3-dihydropyrrolo-
[2,1-b]quinazoline-9(1H)-thione 16a19 (202 mg, 1 mmol), N-Me-
N-(3-propylamino) propanediamine (0.08 mL, 0.5 mmol; alterna-
tively 0.5 mmol of the hydrochloride obtained as described above),
and sodium carbonate (318 mg, 3 mmol) was refluxed in ethanol
(10 mL)/toluene (10 mL) in an 135 °C oil bath, and finely grinded
mercury(II) bromide (0.72 g, 0.2 mmol) was added. The mixture
was heated and stirred vigorously for 2 h. The mixture was then fil-
tered and the filtrate concentrated. The crude product obtained
was purified by column chromatography (chloroform/methanol/
ammonia, 7:1:0.1). The pure compound was obtained as a yellow
grease (36 %). 1H NMR (CDCl3, 400 MHz) d: 1.83–1.88 (m, 4H,
CH3N(CH2CH2CH2N@)2), 2.02–2.07 (m, 4H, 2 ꢁ NCH2CH2), 2.28 (s,
3H, NCH3), 2.56 (t, 4H, J = 7.62 Hz, 2 ꢁ N(CH3)CH2), 2.93 (t, 4H,
J = 7.86 Hz, 2 ꢁ CH2C), 3.80 (t, 4H, J = 6.47 Hz, 2 ꢁ CH2N@), 3.88 (t,
4H, J = 7.08 Hz, 2 ꢁ NCH2), 7.06–7.12 (m, 2H, 2 ꢁ C(7)H),
7.39–7.41 (m, 4H, 2 ꢁ C(6, 8)H), 8.03 (d, 2H, J = 8.26 Hz,
2 ꢁ C(5)H) ppm. 13C NMR (CDCl3, 400 MHz) d: 18.71, 30.77,
32.83, 42.82, 48.23, 48.61, 55.57, 119.83 (arom), 123.81 (arom),
126.78 (arom), 128.05 (arom), 131.58 (arom), 145.33 (arom),
148.85 (arom), 160.17 (arom) ppm. ESI-MS m/z 482 [M+H]+. Elem.
Anal. Calcd for (C29H35N7ꢂ0.3CH2Cl2ꢂ0.6CH3OH): C, 68.23; H, 7.28;
N, 18.63. Found: C, 67.88; H, 7.45; N, 18.72.
This compound was prepared analogously to compound 9. Com-
pound 4 (0.5 g, 1.45 mmol), triethylamine (0.24 mL, 1.74 mmol) and
3-methylbutyryl chloride (0.182 mL, 1.5 mmol) were used and the
crude product was purified by column chromatography (dichloro-
methane:methanol, 20:1 as eluent) to yield the product (0.5 g, 80
%) as a colourless oil. 1H NMR (CDCl3, 400 MHz) d: 0.87–0.89 (m,
6H, CH(CH3)2), 1.31–1.71 (m, 25H, 2 ꢁ Boc, NCH2(CH2)2CH2NH,
NCH2CH2CH2NH, CH(CH3)2), 2.96–3.35 (m, 8H, 2 ꢁ NCH2, 2 ꢁ BocN-
HCH2), 4.67 (br s, 1H, NH), 5.43 (br s, 1H, NH) ppm. 13C NMR (CDCl3,
400 MHz) d: 22.42 (CH(CH3)2), 22.66 (CH(CH3)2), 25.00, 25.80, 26.21,
27.61, 28.38, 28.43, 29.99, 37.23, 39.94, 41.83, 41.88, 42.39, 45.38,
45.60, 47.46, 78.77 (OC(CH3)3), 79.25 (OC(CH3)3), 156.01 (NHCOOt-
Bu), 156.15 (NHCOOtBu), 172.11 (NCOCH2), 172.84 (NCOCH2) ppm.
ESI-MS m/z 430.2 [M+H]+.
5.1.11. 2-Acetamido-N-[4-(t-butoxycarbonylamino)butyl]-N-[3-
(t-butoxycarbonylamino) propyl]acetamide (14)
A mixture of 4 (0.69 g, 2 mmol), N-acetylglycine (0.234 g,
2 mmol), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI,
0.383 g, 2 mmol) and catalytic amount of 1-hydroxybenzotriazole
(HOBt) in anhydrous dichloromethane (10 mL) was stirred at room
temperature for 12 h. Then the reaction solution was quenched
with water, the separated organic phase was washed with 5 % citric
acid aqueous solution followed by 10 % ammonium chloride aque-
ous solution. Then the organic phase was dried over anhydrous
MgSO4 and concentrated under vacuum. The product was yielded
as colourless oil, which was used in the next step without further
purification. 1H NMR (CDCl3, 400 MHz) d: 1.37–1.72 (m, 24H,
2 ꢁ Boc, NCH2(CH2)2CH2NH, NCH2CH2CH2NH), 1.98 (s, 3H,
NHCOCH3), 3.00–3.37 (m, 8H, 2 ꢁ NCH2, 2 ꢁ NHCH2), 3.96–3.99
(m, 2H, COCH2NH), 4.64 (s, 1H, BocNH), 5.16 (s, 1H, BocNH), 6.57
(s, 1H, NHCOCH3) ppm. 13C NMR (CDCl3, 400 MHz) d: 23.01
(NHCOCH3), 24.71, 25.73, 27.56, 28.37, 28.40, 28.42, 28.44, 41.18,
41.23, 42.94, 44.39, 45.62, 46.46, 79.13 (OC(CH3)3), 79.34
(OC(CH3)3), 156.04 (NHCOOtBu), 167.71 (NHCOCH2NH), 168.20
(NHCOCH2NH), 170.09 (NHCOCH3) ppm. ESI-MS m/z 445.2 [M+H]+.
5.1.14. N,N-Bis(3-(2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-
ylideneamino)propyl) acetamide (18)
Compound 18 was synthesized analogously to compound 17
from compounds 7 (187 mg, 0.5 mmol) and 16a (202 mg, 1 mmol).
The crude product was purified by column chromatography
(chloroform:methanol:ammonia, 7:1:0.1). The pure compound
was obtained as
a
yellow grease (24 %). 1H NMR (CDCl3,
400 MHz) d: 1.90–2.12 (m, 11H, CH3CON(CH2CH2CH2N@)2,
2 ꢁ NCH2CH2), 2.95 (t, 4H, J = 8.0 Hz, 2 ꢁ CH2C), 3.44–3.54 (m,
4H, 2 ꢁ N(COCH3)CH2), 3.77–3.98 (m, 8H, 2 ꢁ CH2N@, 2 ꢁ NCH2),
7.12–7.20 (m, 2H, 2 ꢁ C(7)H), 7.44 (m, 4H, 2 ꢁ C(6, 8)H),
7.99–8.06 (m, 2H, 2 ꢁ C(5)H) ppm. 13C NMR (CDCl3, 400 MHz) d:
19.17, 22.06, 32.65, 33.18, 44.30, 47.39, 47.80, 48.72, 120.01
(arom), 124.48 (arom), 127.42 (arom), 128.26 (arom), 132.37
(arom), 146.50 (arom), 149.25 (arom), 160.55 (arom), 170.94
(COCH3) ppm. ESI-MS m/z 510.4 [M+H]+. Elem. Anal. Calcd for
(C30H35N7OꢂH2OꢂMeOH): C, 66.52; H, 7.38; N, 17.52. Found: C,
66.48; H, 7.71; N, 17.69.
5.1.12. 4-Methyl-N-[4-(t-butoxycarbonylamino)butyl]-N-[3-(t-
butoxycarbonylamino)propyl] pentanamide (15)
This compound was prepared analogously to compound 14.
Compound 4 (0.5 g, 1.45 mmol), EDCI (0.28 g, 1.45 mmol) and 4-meth-
ylpentanoic acid (0.17 g, 1.45 mmol) were used as starting materials
and reagents, respectively, and the crude product was used for the next
step without further purification. 1H NMR (CDCl3, 400 MHz) d: 0.83 (d,
6H, CH(CH3)2), 1.36–1.59 (m, 27H, 2 ꢁ Boc, NCH2(CH2)2CH2NH,
NCH2CH2CH2NH, CH2CH(CH3)2), 2.19–2.24 (m, 2H, NCOCH2),
2.96–3.33 (m, 8H, 2 ꢁ NCH2, 2 ꢁ BocNHCH2), 4.84 (br s, 1H, NH), 5.46
(br s, 1H, NH) ppm. 13C NMR (CDCl3, 400 MHz) d: 22.32 (CH(CH3)2),
27.54, 27.82, 27.91, 28.33, 28.38, 31.03, 34.32, 34.44, 39.87, 42.44,
45.41, 45.61, 47.50, 78.72 (OC(CH3)3), 79.11 (OC(CH3)3), 156.07
(NHCOOtBu), 156.16 (NHCOOtBu), 173.19 (NCOCH2), 173.80 (NCOCH2)
ppm. ESI-MS m/z 444.2 [M+H]+.
5.1.15. N1-Methyl-N3-(8,9,10,11-tetrahydro-6H-azocino[2,1-
b]quinazolin-13(7H)-ylidene)-N1-(3-(8,9,10,11-tetrahydro-6H-
azocino[2,1-b]quinazolin-13(7H)-ylideneamino)propyl)pro-
pane-1,3-diamine (19)
Compound 19 was synthesized analogously to compound 17
from 8,9,10,11-tetrahydro-6H-azocino[2,1-b]quinazoline-13(7H)-
thione 16b19 (244 mg, 1 mmol) and N-Me-N-(3-propylamino)pro-
panediamine (0.08 mL, 0.5 mmol; alternatively 0.5 mmol of its
hydrochloride salt as described above). Purification by column
chromatography (chloroform:methanol:ammonia, 9:1:0.1) yielded
a colourless oil (16 %). 1H NMR (CDCl3, 400 MHz) d: 1.33 (m, 4H,
alkylene-H), 1.50 (m, 4H, alkylene-H), 1.76 (m, 4H, alkylene-H),
1.83–1.85 (m, 8H, alkylene-H), 2.28 (s, 3H, NCH3), 2.56 (t, 4H,
J = 6.05 Hz, 2 ꢁ N(CH3)CH2), 2.79 (t, 4H, J = 7.41 Hz, 2 ꢁ CH2C),
3.79 (t, 4H, J = 5.94 Hz, 2 ꢁ CH2N@), 4.17 (m, 4H, 2 ꢁ NCH2),
7.05–7.08 (m, 2H, 2 ꢁ C(2)H), 7.35–7.40 (m, 4H, 2 ꢁ C(1, 3)H),
7.95 (d, 2H, J = 8.10 Hz, 2 ꢁ C(4)H) ppm. 13C NMR (CDCl3,
400 MHz) d: 24.84, 27.02, 28.88, 31.03, 31.11, 36.50, 43.13, 44.38,
49.49, 56.14, 120.06 (arom), 123.90 (arom), 126.85 (arom),
128.30 (arom), 131.80 (arom), 145.96 (arom), 147.78 (arom),
Preparation and spectral data of quinazolinethiones
2,3-dihydropyrrolo[2,1-b]quinazoline-9(1H)-thione (16a) and
8,9,10,11-tetrahydro-6H-azocino[2,1-b]quinazoline-13(7H)-thione
(16b) and their quinazolinone precursors have been described in
detail recently.19,21
5.1.13. N1-(2,3-Dihydropyrrolo[2,1-b]quinazolin-9(1H)-
ylidene)-N3-(3-(2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-
ylideneamino)propyl)-N3-methylpropane-1,3-diamine (17)
To a solution of 5 (173 mg, 0.5 mmol) in anhydrous dichloro-
methane (5 mL) was added 4 M HCl in 1,4-dioxane (5 mL) and
the resulting mixture was stirred at room temperature for 12 h.

1232
X. Chen et al. / Bioorg. Med. Chem. 19 (2011) 1222–1235
160.68 (arom) ppm. ESI-MS m/z 566 [M+H]+. Elem. Anal. Calcd for
(C35H47N7ꢂ0.8CHCl3): C, 65.02; H, 7.29; N, 14.83. Found: C, 64.77;
H, 7.31; N, 14.55.
[M+H]+. Elem. Anal. Calcd for (C31H37N7OꢂMeOH): C, 69.16; H,
7.44; N, 17.64. Found: C, 69.28; H, 7.14; N, 17.69.
5.1.19. N-(4-(2,3-Dihydropyrrolo[2,1-b]quinazolin-9(1H)-
ylideneamino)butyl)-N-(3-(2,3-dihydropyrrolo[2,1-
5.1.16. N,N-Bis(3-(8,9,10,11-tetrahydro-6H-azocino[2,1-b]qui-
nazolin-13(7H)-ylideneamino)propyl)acetamide (20)
b]quinazolin-9(1H)-ylideneamino)propyl) propionamide (25)
Compound 25 was synthesized analogously to compound 17
from compounds 9 (201 mg, 0.5 mmol) and 16a (202 mg, 1 mmol).
Purification by column chromatography (chloroform:metha-
nol:ammonia, 16:1:0.1) yielded a yellow grease (37 %). 1H NMR
(CDCl3, 400 MHz) d: 1.07 (t, 3H, J = 7.42 Hz, NCOCH2CH3),
1.61–1.74 (m, 4H, alkylene-H), 1.87–1.88 (m, 2H, alkylene-H),
1.96–2.09 (m, 4H, 2 ꢁ NCH2CH2), 2.24–2.38 (dq, J1 = 7.43 Hz,
J2 = 30.40 Hz, NCOCH2), 2.87–2.95 (m, 4H, 2 ꢁ CH2C), 3.25–3.50
(m, 4H, 2 ꢁ N(COCH2CH3)CH2), 3.74–3.77 (m, 4H, 2 ꢁ CH2N@),
3.84–3.89 (m, 4H, 2 ꢁ NCH2), 7.10–7.16 (m, 2H, 2 ꢁ C(7)H), 7.37–
7.44 (m, 4H, 2 ꢁ C(6, 8)H), 7.98–8.01 (m, 2H, 2 ꢁ C(5)H) ppm. 13C
NMR (CDCl3, 400 MHz) d: 9.72 (NCOCH2CH3), 9.80 (NCOCH2CH3),
18.66, 25.70, 26.36, 26.38, 26.91, 30.49, 30.74, 31.29, 32.39,
32.77, 44.13, 45.69, 45.81, 47.43, 48.10, 48.21, 50.05, 50.35,
119.64 (arom), 119.77 (arom), 123.84 (arom), 123.97 (arom),
126.77 (arom), 126.98 (arom), 127.83 (arom), 128.02
(arom), 131.60 (arom), 131.85 (arom), 145.95 (arom), 148.85
(arom), 160.08 (arom), 160.20 (arom), 173.53 (COCH3), 173.38
(COCH3) ppm. ESI-MS m/z 538.4 [M+H]+. Elem. Anal. Calcd for
(C36H49N7ꢂH2O): C, 72.32; H, 8.60; N, 16.40. Found: C, 72.38; H,
8.86; N, 16.57.
Compound 20 was synthesized analogously to compound 17
from compounds 7 (187 mg, 0.5 mmol) and 16a (202 mg, 1 mmol).
Purification by column chromatography (chloroform:metha-
nol:ammonia, 10:1:0.1) yielded a yellow grease (20 %). 1H NMR
(CDCl3, 400 MHz) d: 1.33–1.35 (m, 4H, alkylene-H), 1.50–1.51 (m,
4H, alkylene-H), 1.74–1.78 (m, 12H, alkylene-H), 2.06 (s, 3H,
NCOCH3), 2.80 (t, 4H, J = 6.24 Hz, 2 ꢁ CH2C), 3.42 (t, 2H, J = 7.72 Hz,
N(COCH3)CH2), 3.50 (t, 2H, J = 7.66 Hz, N(COCH3)CH2), 3.79 (t, 4H,
J = 4.90 Hz, 2 ꢁ CH2N@), 4.20 (m, 4H, 2 ꢁ NCH2), 7.10–7.14 (m, 2H,
2 ꢁ C(2)H), 7.37–7.45 (m, 4H, 2 ꢁ C(1, 3)H), 7.96 (t, 2H, J = 7.65 Hz,
2 ꢁ C(4)H) ppm. 13C NMR (CDCl3, 400 MHz) d: 22.06, 24.86, 24.89,
26.95, 27.04, 28.90, 31.04, 31.07, 31.93, 32.85, 36.59, 44.37, 44.47,
44.63, 47.74, 48.50, 48.16, 119.92 (arom), 120.03 (arom), 124.04
(arom), 124.12(arom), 126.93(arom), 127.16(arom), 128.18(arom),
128.32 (arom), 131.93 (arom), 132.12 (arom), 146.16 (arom), 146.79
(arom), 147.79(arom), 147.85(arom), 160.60 (arom), 160.69 (arom),
170.76 (COCH3) ppm. ESI-MS m/z 594.1 [M+H]+. Elem. Anal. Calcd for
(C36H47N7Oꢂ2HClꢂ5.3H2O): C, 56.73; H, 7.88; N, 12.86. Found: C,
57.03; H, 7.50; N, 12.48.
5.1.17. N1-(2,3-Dihydropyrrolo[2,1-b]quinazolin-9(1H)-
ylidene)-N4-(3-(2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-
ylideneamino)propyl)-N4-methylbutane-1,4-diamine (21)
Compound 21 was synthesized analogously to compound 17
from compounds 6 (180 mg, 0.5 mmol) and 16b (202 mg, 1 mmol).
Purification by column chromatography (chloroform:metha-
nol:ammonia, 9:1:0.1) yielded a yellow grease (20 %). 1H NMR
(CDCl3, 300 MHz) d: 1.67–1.75 (m, 4H, alkylene-H), 1.90–1.92 (m,
2H, alkylene-H), 2.02–2.06 (m, 4H, 2 ꢁ NCH2CH2), 2.39 (s, 3H,
NCH3), 2.60–2.72 (m, 4H, 2 ꢁ N(CH3)CH2), 2.93 (t, 4H, J = 7.99 Hz,
2 ꢁ CH2C), 3.77–3.83 (m, 4H, J = 6.24 Hz,2 ꢁ CH2N@), 3.86–3.90
(m, 4H, J = 7.03 Hz, 2 ꢁ NCH2), 7.12–7.16 (m, 2H, 2 ꢁ C(7)H), 7.40–
7.45 (m, 4H, 2 ꢁ C(6, 8)H), 8.01 (d, 2H, J = 8.19 Hz, 2 ꢁ C(5)H) ppm.
13C NMR (CDCl3, 300 MHz) d: 17.71, 29.87, 31.80, 40.77, 47.32,
47.37, 49.15, 54.01, 56.05, 118.65 (arom), 118.69 (arom), 122.91
(arom), 122.99(arom), 125.86(arom), 127.01(arom), 130.74(arom),
130.80 (arom), 144.60 (arom), 144.82 (arom), 147.82 (arom), 147.87
(arom), 159.16 (arom), 159.25 (arom) ppm. ESI-MS m/z 496.4
[M+H]+. Elem. Anal. Calcd for (C30H37N7ꢂ1.5CHCl3ꢂ2.5MeOH): C,
58.08; H, 7.17; N, 13.94. Found: C, 57.73; H, 6.85; N, 13.93.
5.1.20. N-(4-(2,3-Dihydropyrrolo[2,1-b]quinazolin-9(1H)-ylid-
eneamino)butyl)-N-(3-(2,3-dihydropyrrolo[2,1-b]quinazolin-
9(1H)-ylideneamino)propyl) methacrylamide (27)
Compound 27 was synthesized analogously to compound 17
from compounds 10 (207 mg, 0.5 mmol) and 16a (202 mg, 1 mmol).
Purification by column chromatography (chloroform:metha-
nol:ammonia, 20:1:0.1) yielded a yellow grease (29 %). 1H NMR
(CDCl3, 400 MHz) d: 1.59–2.09 (m, 13H, alkylene-H, 2 ꢁ NCH2CH2,
CH2@CCH3), 2.91 (t, J = 5.80 Hz, 2 ꢁ CH2C), 3.35–3.52 (m, 4H,
2 ꢁ CH2N(COC(CH3)@CH2)), 3.76–3.79 (m, 4H, 2 ꢁ CH2N@), 3.86–
3.88 (m, 4H, 2 ꢁ NCH2), 4.92–5.02 (dd, 2H, J1 = 10.64 Hz,
J2 = 29.50 Hz, COC@CH2), 7.12–7.15 (m, 2H, 2 ꢁ C(7)H), 7.41–7.43
(m, 4H, 2 ꢁ C(6, 8)H), 7.98–8.02 (m, 2H, 2 ꢁ C(5)H) ppm. 13C NMR
(CDCl3, 400 MHz) d: 14.02 (COC(CH3)@CH2), 18.70, 20.78, 22.96,
23.75, 25.29, 26.83, 28.91, 30.36, 30.47, 30.89, 32.41, 32.79, 38.73,
42.32, 44.19, 46.67, 47.68, 48.25, 48.71, 50.22, 114.17 (COC@CH2),
119.67 (arom), 123.86 (arom), 123.96 (arom), 125.27 (arom),
126.89 (arom), 127.93 (arom), 129.00 (arom), 131.71 (arom),
131.81 (arom), 141.60 (COC@CH2), 148.88 (arom), 148.91
(arom), 160.11 (arom), 160.19 (arom), 172.60 (COC@CH2) ppm.
ESI-MS m/z 550.5 [M+H]+. Elem. Anal. Calcd for (C38H51N7Oꢂ0.9-
CHCl3): C, 64.06; H, 7.17; N, 13.44. Found: C, 64.05; H, 6.87; N, 13.48.
5.1.18. N-(4-(2,3-Dihydropyrrolo[2,1-b]quinazolin-9(1H)-ylid -
eneamino)butyl)-N-(3-(2,3-dihydropyrrolo[2,1-b]quinazolin-
9(1H)-ylideneamino)propyl)acetamide (22)
Compound 22 was synthesized analogously to compound 17
from compounds 8 (194 mg, 0.5 mmol) and 16a (202 mg, 1 mmol).
Purification by column chromatography (chloroform:metha-
nol:ammonia, 9:1:0.1) yielded a yellow grease (31 %). 1H NMR
(CDCl3, 300 MHz) d: 1.64–1.76 (m, 4H, alkylene-H), 1.88–1.95 (m,
2H, alkylene-H), 2.03–2.08 (m, 7H, 2 ꢁ NCH2CH2, NCOCH3), 2.92–
2.96 (m, 4H, 2 ꢁ CH2C), 3.28–3.50 (m, 4H, 2 ꢁ N(COCH3)CH2),
3.77–3.81 (m, 4H, 2 ꢁ CH2N@), 3.88–3.98 (m, 4H, 2 ꢁ NCH2), 7.14–
7.18 (m, 2H, 2 ꢁ C(7)H), 7.44–7.47 (m, 4H, 2 ꢁ C(6, 8)H), 7.80–8.02
(m, 2H, 2 ꢁ C(5)H) ppm. 13C NMR (CDCl3, 400 MHz) d: 17.76, 20.66,
24.58, 25.82, 29.37, 31.73, 31.78, 42.90, 44.58, 44.90, 45.66, 46.36,
47.30, 48.17, 49.99, 119.67 (arom), 119.73 (arom), 123.85 (arom),
123.94 (arom), 126.87 (arom), 126.97 (arom), 128.09 (arom),
128.20 (arom), 131.75 (arom), 131.88 (arom), 145.63
(arom), 148.88 (arom), 148.91 (arom), 160.13 (arom), 160.19
(arom), 170.16 (COCH3), 170.29 (COCH3) ppm. ESI-MS m/z 524.7
5.1.21. (Z)-N1-Methyl-N4-(8,9,10,11-tetrahydro-6H-azocino[2,1-
b]quinazolin-13(7H)-ylidene)-N1-(3-((E)-8,9,10,11-tetrahydro-
6H-azocino[2,1-b]quinazolin-13(7H)-ylideneamino)propyl)-
butane-1,4-diamine (23)
Compound 23 was synthesized analogously to compound 17
from compounds 6 (180 mg, 0.5 mmol) and 16b (244 mg, 1 mmol).
Purification by column chromatography (dichloromethane/metha-
nol/triethylamine, 30:1:0.05) yielded a yellow grease (22 %). 1H
NMR (CDCl3, 400 MHz) d: 1.31–1.32 (m, 4H, alkylene-H), 1.49 (m,
4H, alkylene-H), 1.64–1.84 (m, 14H, alkylene-H), 2.28 (s, 3H,
NCH3), 2.48–2.60 (m, 4H, 2 ꢁ N(CH3)CH2), 2.76–2.80 (m, 4H,
2 ꢁ CH2C), 3.77–3.81 (m, 4H, 2 ꢁ CH2N@), 4.13–4.19 (m, 4H,
2 ꢁ NCH2), 7.07–7.11 (m, 2H, 2 ꢁ C(2)H), 7.35–7.42 (m, 4H,
2 ꢁ C(1, 3)H), 7.97 (t, 2H, J = 7.63 Hz, 2 ꢁ C(4)H) ppm. 13C NMR
(CDCl3, 400 MHz) d: 24.41, 24.43, 26.60, 26.62, 28.49, 28.53, 30.57,

X. Chen et al. / Bioorg. Med. Chem. 19 (2011) 1222–1235
1233
30.65, 31.40, 36.16, 42.76, 43.93, 44.04, 49.22, 50.92, 55.88, 58.00,
119.67 (arom), 119.71 (arom), 123.44 (arom), 123.51 (arom),
126.51 (arom), 126.54 (arom), 127.91 (arom), 131.35 (arom),
131.43 (arom), 145.43 (arom), 145.88 (arom), 147.48
(arom), 147.50 (arom), 160.128 (arom), 160.32 (arom) ppm. ESI-
MS m/z 580.5 [M+H]+. Elem. Anal. Calcd for (C32H39N7Oꢂ1.3CH2Cl2):
C, 61.71; H, 6.47; N, 15.13. Found: C, 61.99; H, 6.76; N, 14.89.
Purification of the crude product by column chromatography (chlo-
roform:methanol:ammonia, 30:1:0.1) yielded a yellow grease (27
%). 1H NMR (CDCl3, 400 MHz) d: 1.31–1.88 (m, 25H, 2 ꢁ NCH2(CH2)4,
@NCH2CH2, @NCH2CH2CH2, NCOC(CH3)@CH2), 2.76–2.79 (m, 4H,
2 ꢁ CH2C), 3.32–3.51 (m, 4H, 2 ꢁ CH2N(COC(CH3)@CH2)), 3.76–
3.77 (m, 4H, 2 ꢁ CH2N@), 4.13–4.20 (m, 4H, 2 ꢁ NCH2), 4.93–5.01
(dd, 2H, J1 = 5.08 Hz, J2 = 27.39 Hz, COC@CH2), 7.07–7.12 (m, 2H,
2 ꢁ C(2)H), 7.36–7.42 (m, 4H, 2 ꢁ C(1, 3)H), 7.92–7.96 (m, 2H,
2 ꢁ C(4)H) ppm. 13C NMR (CDCl3, 400 MHz) d: 14.67 (COC(CH3)@
CH2), 21.43, 20.78, 24.40, 24.43, 26.55, 26.58, 28.47, 30.61, 30.78,
36.11, 43.91, 48.39, 48.58, 50.71, 114.17 (COC@CH2), 119.55 (arom),
119.61 (arom), 123.47 (arom), 123.60 (arom), 126.13 (arom), 126.56
(arom), 127.81(arom), 128.18(arom), 128.77(arom), 128.99(arom),
131.42 (arom), 131.53 (arom), 141.56 (COC@CH2), 147.45 (arom),
147.47 (arom), 160.15 (arom), 160.24 (arom), 172.58 (COC@CH2)
5.1.22. N-(4-(8,9,10,11-Tetrahydro-6H-azocino[2,1-b]quinazo-
lin-13(7H)-ylideneamino)butyl)-N-(3-(8,9,10,11-tetrahydro--
6H-azocino[2,1-b]quinazolin-13(7H)-ylideneamino)propyl)-
acetamide (24)
Compound 24 was synthesized analogously to compound 17
from compounds 8 (194 mg, 0.5 mmol) and 16b (244 mg, 1 mmol).
Purification by column chromatography (chloroform:metha-
nol:ammonia, 25:1:0.1) yielded a yellow grease (20 %). 1H NMR
(CDCl3, 300 MHz) d: 1.33–1.37 (m, 4H, alkylene-H), 1.51 (m, 4H, alk-
ylene-H), 1.64–1.77 (m, 14H, alkylene-H), 2.06 (s, 3H, NCOCH3),
2.80–2.82 (m, 4H, 2 ꢁ CH2C), 3.25–3.49 (m, 4H, N(COCH3)CH2),
3.79–3.81 (m, 4H, 2 ꢁ CH2N@), 4.17–4.21 (m, 4H, 2 ꢁ NCH2), 7.12–
7.13 (m, 2H, 2 ꢁ C(2)H), 7.41–7.42 (m, 4H, 2 ꢁ C(1, 3)H), 7.95–7.97
(m, 2H, 2 ꢁ C(4)H) ppm. 13C NMR (CDCl3, 300 MHz) d: 22.01, 24.84,
26.15, 26.94, 27.00, 27.39, 28.89, 31.04, 32.84, 36.49, 44.41, 44.71,
46.06, 47.37, 48.51, 48.99, 49.72, 51.00, 51.21, 119.90 (arom),
124.00 (arom), 124.14 (arom), 127.00 (arom), 127.15 (arom),
128.17 (arom), 128.29 (arom), 131.98 (arom), 132.11
(arom), 142.79(arom), 147.65(arom), 147.80(arom), 160.57(arom),
160.80 (arom), 170.62 (COCH3), 170.67 (COCH3) ppm. ESI-MS m/z
607.6 [M+H]+. Elem. Anal. Calcd for (C39H52N8O2ꢂ1.4CH2Cl2): C,
61.79; H, 6.30; N, 14.66. Found: C, 61.90; H, 6.25; N, 14.63.
ppm. ESI-MS m/z 634.6 [M+H]+. Elem. Anal. Calcd for (C39H51
-
N7Oꢂ1.4CH2Cl2): C, 64.46; H, 7.20; N, 13.02. Found: C, 64.21; H,
7.36; N, 12.98.
5.1.25. 2-Acetamido-N-(4-(8,9,10,11-tetrahydro-6H-azocino-
[2,1-b]quinazolin-13(7H)-ylideneamino)butyl)-N-(3-(8,9,10,-
11-tetrahydro-6H-azocino[2,1-b]quinazolin-13(7H)-ylidene-
amino)propyl)acetamide (29)
Compound 29 was synthesized analogously to compound 17
from compounds 14 (223 mg, 0.5 mmol) and 16b (244 mg, 1 mmol).
Purification of the crude product by column chromatography (chlo-
roform:methanol:ammonia, 18:1:0.1) yielded a yellow grease (18
%). 1H NMR (CDCl3, 400 MHz) d: 1.32–1.86 (m, 22H, 2 ꢁ NCH2(CH2)4,
@NCH2CH2, @NCH2CH2CH2), 1.98 (s, 3H, NCOCH3), 2.77–2.83 (m, 4H,
2 ꢁ CH2C), 3.22–3.52 (m, 4H, 2 ꢁ CH2NCOCH2), 3.77–3.80 (m, 4H,
2 ꢁ CH2N@), 4.00–4.22 (m, 6H, NCOCH2NH, 2 ꢁ NCH2), 6.60–6.62
(m, 1H, NHCOCH3), 7.10–7.15 (m, 2H, 2 ꢁ C(2)H), 7.41–7.44 (m,
4H, 2 ꢁ C(1, 3)H), 7.93–7.96 (m, 2H, 2 ꢁ C(4)H) ppm. 13C NMR
(CDCl3, 400 MHz) d: 14.20 (NCOCH3), 23.08, 24.40, 26.54, 26.57,
28.46, 30.62, 30.64, 36.02, 41.46, 44.00, 44.12, 44.92, 46.01, 47.30,
47.91, 48.45, 50.60, 50.70, 119.41 (arom), 123.67 (arom), 123.72
(arom), 126.42(arom), 126.52(arom), 126.70(arom), 127.74(arom),
127.79 (arom), 127.83 (arom), 131.61 (arom), 131.72 (arom), 146.59
(arom), 147.25 (arom), 147.37 (arom), 160.31 (arom), 167.50
(NCOCH2NH), 167.66 (NCOCH2NH), 169.95 (NCOCH3), 170.01
(NCOCH3) ppm. ESI-MS m/z 665.7 [M+H]+. Elem. Anal. Calcd for
(C39H52N8O2ꢂ1.4CH2Cl2): C, 61.91; H, 7.05; N, 14.30. Found: C,
62.31; H, 7.12; N, 13.93.
5.1.23. N-(4-(8,9,10,11-Tetrahydro-6H-azocino[2,1-b]quinaz-
olin-13(7H)-ylideneamino)butyl)-N-(3-(8,9,10,11-tetrahydro-
6H-azocino[2,1-b]quinazolin-13(7H)-ylideneamino)propyl)-
propionamide (26)
Compound 26 was synthesized analogously to compound 17
from compounds 9 (201 mg, 0.5 mmol) and 16b (244 mg, 1 mmol).
Purification by column chromatography (chloroform:metha-
nol:ammonia, 25:1:0.1) yielded a yellow grease (26 %). 1H NMR
(CDCl3, 400 MHz) d: 1.05–1.09 (dt, 3H, J1 = 7.43 Hz, J2 = 2.74 Hz,
NCOCH2CH3), 1.32–1.35 (m, 4H, alkylene-H), 1.45–1.52 (m, 4H,
alkylene-H), 1.62–1.85 (m, 14H, alkylene-H), 2.24–2.34 (dq, 2H,
J1 = 19.23 Hz, J2 = 7.44 Hz, NCOCH2CH3), 2.76–2.82 (m, 4H,
2 ꢁ CH2C), 3.23–3.48 (m, 4H, 2 ꢁ N(COCH2CH3)CH2), 3.75–3.79 (m,
4H, 2 ꢁ CH2N@), 4.14–4.18 (m, 4H, 2 ꢁ NCH2), 7.07–7.13 (m, 2H,
2 ꢁ C(2)H), 7.35–7.43 (m, 4H, 2 ꢁ C(1, 3)H), 7.92–7.97 (m, 2H,
2 ꢁ C(4)H) ppm. 13C NMR (CDCl3, 400 MHz) d: 9.79 (NCOCH2CH3),
24.43, 24.46, 25.82, 26.36, 26.52, 26.57, 26.62, 27.08, 28.47, 28.50,
30.62, 30.78, 31.00, 31.51, 32.55, 36.13, 43.89, 43.96, 43.99, 44.48,
45.79, 45.79, 45.98, 48.13, 48.29, 48.67, 50.58, 50.87, 119.51 (arom),
119.57 (arom), 123.47 (arom), 123.50 (arom), 123.57 (arom), 123.67
(arom), 126.48(arom), 126.55(arom), 126.64(arom), 126.76(arom),
127.75 (arom), 127.77 (arom), 127.91 (arom), 131.37 (arom), 131.46
(arom), 131.51(arom), 131.65(arom), 145.74(arom), 145.87(arom),
147.47 (arom), 160.13 (arom), 160.21 (arom), 160.26 (arom), 160.30
(arom), 173.37 (COCH2CH3), 173.48 (COCH2CH3) ppm. ESI-MS m/z
622.5 [M+H]+. Elem. Anal. Calcd for (C37H49N7Oꢂ0.9CH2Cl2): C,
66.52; H, 7.48; N, 14.33. Found: C, 66.13; H, 7.53; N, 14.23.
5.1.26. N-(4-(8,9,10,11-Tetrahydro-6H-azocino[2,1-b]quinaz-
olin-13(7H)-ylideneamino)butyl)-N-(3-(8,9,10,11-tetrahydro-
6H-azocino[2,1-b]quinazolin-13(7H)-ylideneamino)propyl)-
pivalamide (30)
Compound 30 was synthesized analogously to compound 17
from compounds 11 (215 mg, 0.5 mmol) and 16b (244 mg, 1 mmol).
Purification by column chromatography (chloroform:metha-
nol:ammonia, 32:1:0.1) yielded a yellow grease (19 %). 1H NMR
(CDCl3, 400 MHz) d: 1.22 (s, 9H, COC(CH3)3), 1.32 (m, 4H, alkylene-
H), 1.49–1.91 (m, 18H, alkylene-H), 2.76–2.79 (m, 4H, 2 ꢁ CH2C),
3.35–3.49 (m, 4H, 2 ꢁ CH2NCOtBu), 3.77–3.79 (m, 4H, 2 ꢁ CH2N@),
4.12–4.25 (m, 4H, 2 ꢁ NCH2), 7.07 (m, 2H, 2 ꢁ C(2)H), 7.36–7.41
(m, 4H, 2 ꢁ C(1, 3)H), 7.93–7.98 (m, 2H, 2 ꢁ C(4)H) ppm. 13C NMR
(CDCl3, 400 MHz) d: 24.42, 26.53, 26.60, 28.46, 28.49, 28.64, 30.62,
31.01, 36.14, 36.19, 39.02, 43.78, 43.91, 48.90, 50.82, 119.63 (arom),
123.46 (arom), 123.57 (arom), 126.58 (arom), 126.69 (arom), l28.78
(arom), 131.41(arom), 131.51(arom), 145.61(arom), 145.97(arom),
147.50 (arom), 160.17 (arom), 160.26 (arom), 177.02 (NCOtBu)
5.1.24. N-(4-(8,9,10,11-Tetrahydro-6H-azocino[2,1-b]quinaz-
olin-13(7H)-ylideneamino)butyl)-N-(3-(8,9,10,11-tetrahydro-
6H-azocino[2,1-b]quinazolin-13(7H)-ylideneamino)propyl)-
methacrylamide (28)
ppm. ESI-MS m/z 650.7 [M+H]+. Elem. Anal. Calcd for (C40H55
-
Compound 28 was synthesized analogously to compound 17
from compounds 10 (207 mg, 0.5 mmol) and 16b (244 mg, 1 mmol).
N7OꢂMeOH): C, 72.21; H, 8.72; N, 14.38. Found: C, 72.19; H, 8.80;
N, 14.27.

1234
X. Chen et al. / Bioorg. Med. Chem. 19 (2011) 1222–1235
5.1.27. N-(4-(8,9,10,11-Tetrahydro-6H-azocino[2,1-b]quinaz-
olin-13(7H)-ylideneamino)butyl)-N-(3-(8,9,10,11-tetrahydro-
6H-azocino[2,1-b]quinazolin-13(7H)-ylideneamino)propyl)-
pentanamide (31)
2 ꢁ CH2C), 3.24–3.48 (m, 4H, 2 ꢁ CH2NCO), 3.75–3.80 (m, 4H,
2 ꢁ CH2N@), 4.13–4.28 (m, 4H, 2 ꢁ NCH2), 7.08–7.14 (m, 2H,
2 ꢁ C(2)H), 7.36–7.44 (m, 4H, 2 ꢁ C(1, 3)H), 7.93–7.97 (m, 2H,
2 ꢁ C(4)H) ppm. 13C NMR (CDCl3, 400 MHz) d: 22.42 (CHCH3),
22.48 (CHCH3), 24.42, 26.54, 26.58, 27.96, 28.00, 28.48, 30.61,
32.61, 34.48, 34.51, 36.01, 43.95, 44.03, 45.76, 46.11, 48.19,
48.41, 48.65, 50.62, 50.87, 119.51 (arom), 123.52 (arom), 123.61
(arom), 123.69 (arom), 126.51 (arom), 126.62 (arom), 126.73
(arom), 127.75 (arom), 127.91 (arom), 131.52 (arom), 131.67
(arom), 147.43 (arom), 160.15 (arom), 160.29 (arom), 173.03
(NCOCH2) ppm. ESI-MS m/z 664.7 [M+H]+. Elem. Anal. Calcd for
(C41H57N7Oꢂ1.1MeOHꢂ0.2CH2Cl2): C, 70.94; H, 8.70; N, 13.69.
Found: C, 70.56; H, 8.42; N, 13.41.
Compound 31 was synthesized analogously to compound 17
from compounds 12 (215 mg, 0.5 mmol) and 16b (244 mg, 1 mmol).
Purification by column chromatography (chloroform:methanol:
ammonia, 32:1:0.1) yielded a yellow grease (35 %). 1H NMR (CDCl3,
400 MHz) d: 0.84 (t, 3H, J = 7.32 Hz, NCO(CH2)3CH3), 1.18–1.85 (m,
26H, 2 ꢁ NCH2(CH2)4, @NCH2CH2, @NCH2CH2CH2, NCOCH2(CH2)2-
CH3), 2.22–2.30 (m, 2H, NCOCH2), 2.78–2.81 (m, 4H, 2 ꢁ CH2C),
3.24–3.48 (m, 4H, 2 ꢁ CH2NCO), 3.77–3.80 (m, 4H, 2 ꢁ CH2N@),
4.13–4.24 (m, 4H, 2 ꢁ NCH2), 7.09–7.14 (m, 2H, 2 ꢁ C(2)H), 7.39–
7.42 (m, 4H, 2 ꢁ C(1,3)H), 7.93–7.96 (m, 2H, 2 ꢁ C(4)H) ppm. 13C
NMR (CDCl3, 400 MHz) d: 13.92 (NCO(CH2)3CH3), 13.97, 22.63,
24.42, 25.79, 26.52, 26.56, 26.60, 27.70, 27.72, 28.46, 28.52, 30.59,
30.77, 30.96, 32.59, 32.87, 32.99, 36.07, 43.92, 43.96, 44.01, 44.36,
45.71, 46.07, 48.12, 48.37, 48.64, 50.58, 50.82, 119.49 (arom),
119.55 (arom), 119.62 (arom), 123.49 (arom), 123.58 (arom),
126.43 (arom), 126.50 (arom), 126.61 (arom), 126.73
(arom), 127.72(arom), 127.87(arom), 131.36(arom), 131.43(arom),
131.47 (arom), 131.61 (arom), 146.35 (arom), 147.37 (arom), 147.44
(arom), 147.46 (arom), 160.08 (arom), 160.16 (arom), 160.22
(arom), 160.26 (arom), 172.77 (NCO(CH2)3CH3), 172.80
(NCO(CH2)3CH3) ppm. ESI-MS m/z 650.7 [M+H]+. Elem. Anal. Calcd
for (C40H55N7OꢂCH2Cl2): C, 67.01; H, 7.82; N, 13.34. Found: C,
66.80; H, 7.60; N, 13.28.
5.2. Cholinesterase inhibition
AChE (E.C.3.1.1.7, Type VI-S, from Electric Eel) and BChE
(E.C.3.1.1.8, from equine serum) were purchased from Sigma-
Aldrich, UK. 5,50-Dithiobis(2-nitrobenzoic acid) (Ellman’s reagent,
DTNB), acetylthiocholine (ATC) and butyrylthiocholine (BTC) were
also purchased from Sigma–Aldrich, UK. UV measurements were
performed on Varian Cary 50 Scan UV–vis spectrometer and 4 mL
disposable cuvettes were used for the testing. As references for
ChE inhibition the established drugs galanthamine and tacrine
were used. Galanthamine hydrobromide was purchased from
JANSSEN-CILAG GmbH, Neuss, Germany. Tacrine hydrochloride
was purchased from Sigma–Aldrich, UK.
The assay was performed as described in the following proce-
5.1.28. 3-Methyl-N-(4-(8,9,10,11-tetrahydro-6H-azocino-
[2,1-b]quinazolin-13(7H)-ylideneamino)butyl)-N-(3-(8,9,-
10,11-tetrahydro-6H-azocino[2,1-b]quinazolin-13(7H)-
dure:19,21 A 3.32 ꢁ 10ꢀ3 M stock solution of the test compound
was prepared in 50% ethanol/buffer (pH 8), and 100 lL of this
solution were applied in 3.32 mL of final volume in the testing
cuvette, so that the highest concentration of the test compounds
applied in the assay was 10ꢀ4 M (final concentration). In order to
obtain an inhibition curve, at least five different concentrations
(normally in the range of 10ꢀ4–10ꢀ9 M) of the test compound were
measured at 25 °C at 412 nm, each concentration in triplicate.19,21
For buffer preparation, 1.36 g of potassium dihydrogen phosphate
(10 mmol) were dissolved in 100 mL of water and adjusted with
NaOH to pH 8.0 0.1. AChE solution was prepared to give
2.5 units/mL in 1.4 mL aliquots whereas for BChE the concentration
was 25 units/mL in 1.4 mL aliquots because of BChE’s lower activ-
ity. Furthermore, 0.01 M DTNB solution, 0.075 M ATC and BTC solu-
tions, respectively, were used. A cuvette containing 3.0 mL of
ylideneamino)propyl)butanamide (32)
Compound 32 was synthesized analogously to compound 17
from compounds 13 (215 mg, 0.5 mmol) and 16b (244 mg, 1 mmol).
Purification by column chromatography (chloroform:metha-
nol:ammonia, 30:1:0.1 as eluent) yielded a yellow grease (28 %).
1H NMR (CDCl3, 400 MHz) d: 0.87 (d, 6H, 2 ꢁ CH3), 1.32–1.88 (m,
23H, 2 ꢁ NCH2(CH2)4, @NCH2CH2, @NCH2CH2CH2, CH(CH3)2), 2.12–
2.15 (m, 2H, NCOCH2), 2.75–2.81 (m, 4H, 2 ꢁ CH2C), 3.24–3.48 (m,
4H, 2 ꢁ CH2NCO), 3.75–3.78 (m, 4H, 2 ꢁ CH2N@), 4.12–4.24 (m,
4H, 2 ꢁ NCH2), 7.06–7.13 (m, 2H, 2 ꢁ C(2)H), 7.35–7.42 (m, 4H,
2 ꢁ C(1, 3)H), 7.92–7.95 (m, 2H, 2 ꢁ C(4)H) ppm. 13C NMR (CDCl3,
400 MHz) d: 22.72 (CHCH3), 22.73 (CHCH3), 24.40, 25.82, 25.84,
25.87, 26.54, 26.57, 26.60, 28.45, 30.61, 30.80, 31.00, 32.63, 36.11,
41.89, 42.08, 43.95, 45.74, 46.14, 48.12, 48.44, 48.66, 50.62, 50.84,
119.50 (arom), 119.57 (arom), 123.50 (arom), 123.58 (arom),
123.66 (arom), 126.46 (arom), 126.52 (arom), 126.63 (arom),
126.74 (arom), 127.76 (arom), 127.90 (arom), 131.36
(arom), 131.44(arom), 131.49(arom), 131.64(arom), 145.70(arom),
145.80 (arom), 147.42 (arom), 147.45 (arom), 147.49 (arom), 160.10
(arom), 160.17 (arom), 160.24 (arom), 160.28 (arom), 172.11
(NCOCH2), 172.16 (NCOCH2) ppm. ESI-MS m/z 650.7 [M+H]+. Elem.
Anal. Calcd for (C40H55N7Oꢂ1.2CH2Cl2): C, 65.82; H, 7.70; N, 13.04.
Found: C, 65.67; H, 7.48; N, 13.13.
phosphate buffer, 100
and 100 L of the test compound solution was allowed to stand
for 3.0 min accurately, then 100 L of DTNB were added, then after
another 1.5 min the reaction was started by addition of 20 L of
lL of the respective enzyme (AChE/BChE)
l
l
l
the substrate solution (ATC or BTC according to corresponding
enzyme). The solution was mixed immediately, and exactly
2.5 min after the addition of substrate the absorption was mea-
sured using UV–vis spectrometer. For the reference value 100
lL
of water replaced the test compound solution. For determining
the blank value, additionally 100
solution.
lL of water replaced the enzyme
The inhibition curve was obtained by plotting percentage en-
zyme activity (100 % for the reference) versus logarithm of test
compound concentration using GraphPad Prism software (version
5.01).
5.1.29. 4-Methyl-N-(4-(8,9,10,11-tetrahydro-6H-azocino-
[2,1-b]quinazolin-13(7H)-ylideneamino)butyl)-N-(3-(8,9,-
10,11-tetrahydro-6H-azocino[2,1-b]quinazolin-13(7H)-
ylideneamino)propyl)pentanamide (33)
Compound 33 was synthesized analogously to compound 17
from compounds 15 (222 mg, 0.5 mmol) and 16b (244 mg,
1 mmol). Purification by column chromatography (chloro-
form:methanol:ammonia, 32:1:0.05) yielded a yellow grease (18
%). 1H NMR (CDCl3, 400 MHz) d: 0.82–0.83 (d, 6H, CH(CH3)2),
1.32–1.86 (m, 25H, 2 ꢁ NCH2(CH2)4, @NCH2CH2, @NCH2CH2CH2,
CH2CH(CH3)2), 2.22–2.30 (m, 2H, NCOCH2), 2.77–2.82 (m, 4H,
5.3. Molecular modelling
Homology modelling of the enzymes based on the crystal struc-
ture of Torpedo californica AChE (2CEK) was conducted using
Molecular Operating Environment (MOE) software33 with a default
protocol. The ligands were docked using the Induced Fit docking
protocol of the Schrödinger software34 with the Extra Precision

X. Chen et al. / Bioorg. Med. Chem. 19 (2011) 1222–1235
1235
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cedure using the LigPrep module35 of the Schrödinger software.
The best poses were selected based on the energy score and the
general position of bivalent ligands in the available crystal
structures (PDB: code 2CMF, 2CKM and 2CEK). The hydrophobic,
acceptor and donor regions of the binding site located at the radius
of 4 Å around the ligand were calculated using the Site Map.36 The
OPLS2005 force fields were used in all calculations. The pictures
were generated using MOE and Schrödinger software.
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Acknowledgements
We would like to thank Dr. John King for a generous donation to
18. Butini, S.; Campiani, G.; Borrielo, M.; Gemma, S.; Panico, A.; Persico, M.;
Catalanotti, B.; Ros, S.; Brindisi, M.; Agnusdei, M.; Fiorini, I.; Nacci, V.;
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establish
a medicinal chemistry laboratory at the School of
Pharmacy of Queen’s University Belfast and for providing a Ph.D.
studentship for X. Chen. We especially thank Professor Brian
Walker for his support during the course of this work.
Supplementary data
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Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.12.034. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
26. Carlier, P. R.; Chow, E. S.-H.; Han, Y.; Liu, J.; El Yazal, J.; Pang, Y.-P. J. Med. Chem.
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34. Schrödinger Suite 2008 Induced Fit Docking protocol; Glide version 5.0,
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New York, NY, 2005.
35. LigPrep, version 2.2, Schrödinger, LLC, New York, NY, 2009.
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