ELSEVIER Chemico-Biological Interactions 105 (1997) 199-228

A comparative QSAR analysis of

acetylcholinesterase inhibitors currently studied for
the treatment of Alzheimer’s disease

M. Recanatini ‘s*, A. Cavalli ‘, C. Hansch b

a Depurtment ofPharmaceutical Sciences, University of Bologna, Via Belmeloro 6,
I-40126 Bologna, Italy
b Department of Chemistry, Pomona College, Claremont, CA 91711, USA

Received 28 January 1997; accepted 12 June 1997

Abstract

Considering the relevance of acetylcholinesterase inhibitors as potential agents for the

treatment of the Alzheimer’s disease, we have undertaken a comparative QSAR analysis
aimed at individuating the physico-chemical properties governing the inhibitory activity of
such compounds. The QSAR equations for 13 series of derivatives have been calculated and
discussed. The series studied are all those we found in the literature suitable for a QSAR
analysis and represent the three main classes of acetylcholinesterase inhibitors currently
investigated, namely, physostigmine analogues, 1,2,3,4_tetrahydroacridines and benzy-
lamines. The equations we obtained show that, within each class, the main physico-chemical
properties affecting the inhibitory activity are almost the same for all the series and can be
individuated by the use of proper parameters. The conclusions of this study can be
summarized as follows: (a) hydrophobicity plays a critical role in both the physostigmine-
and the benzylamine-derived classes; (b) electronic effects are important for the interactions
carried out by the variable portion of benzylamine derivatives; and (c) steric factors are also
significant, but, as in other cases, the collinearity between steric and hydrophobic parameters
does not allow one to draw any final conclusion. 0 1997 Elsevier Science Ireland Ltd.

Keywords: Alzheimer’s disease; Acetylcholinesterase inhibitors; QSAR; Structure-activity

relationships; Enzyme inhibition; Computer-assisted drug design

* Corresponding author. E-mail: mreca@alma.unibo.it

0009-2797/97/$17.00 Q 1997 Elsevier Science Ireland Ltd. All rights reserved.

PI1 SOOO9-2797(97)00047- 1
200 M. Recanatini et al. jChemico-Biological Interactions 105 (1997) 199-228

1. Introduction

Acetylcholinesterase (AChE) inhibitors form one of the most actively investigated

classes of compounds as potential agents for the treatment of Alzheimer’s disease
(AD). AD is a chronic neurodegenerative disorder characterized by loss of memory,
behavioral abnormalities, physical debility and, ultimately, death. It is among the
leading causes of death in the industrialized countries and, so far, no definitive
treatment or cure for this devastating disease has been found. However, in the last
few years, a formidable research effort is being expended to develop drugs able of
successfully treating AD patients.
Pathological changes in the AD brain are known and they are mainly represented
by a dramatic loss of neurons in many areas of the central nervous system and by
a great reduction of the levels of central neurotransmitters. The most important
therapeutic approaches currently followed [l] may be related to these pathological
markers and they are dealing with the amyloid plaque formation and toxicity [2],
and with the decrease of neurotransmitters levels [l]. Within the latter, the so-called
cholinergic hypothesis [3,4] defines a strategy aimed at enhancing the cholinergic
transmission through several mechanisms. Inhibition of AChE, the enzyme respon-
sible for the acetylcholine metabolic breakdown, is one of these mechanisms,
actually the one that allowed the development of the only drug presently approved
for the treatment of AD, i.e. tacrine [5].
Inhibitors of: AChE have been known for a long time [6], and structure-activity
studies, particularly in the field of insecticides, date back to 1956 [7].
When a class of compounds or a biological system are intensely investigated with
the aim of developing new drugs, the need arises of taking advantage in the most
effective manner of the knowledge already accumulated in that field. A way of
doing this is provided by the QSAR methodology [8], which aims at developing
simple mathematical models correlating changes in biological activity with varia-
tions in the structure of molecules. These variations are accounted for by parame-
ters (experimentally determined or calculated) related to physico-chemical
properties of the compounds. If QSAR equations can be calculated for many series
of derivatives acting on the same biological system, and if the equations contain the
same (or similar) parameters, a direct comparison can be performed on the
structure-activity relationships of all the series. In addition, all the QSAR models
taken together can contribute to hypothesize an overall picture of the system
investigated, by revealing common properties governing the ligand-target interac-
tion. Examples of this procedure can be found in a recent compilation [9].
The QSAR of AChE inhibitors were reviewed in recent times [9- 121, but, in these
works, most attention was devoted to the insecticidal properties of these com-
pounds and the classes of organophosphorous derivatives and carbamates were
mainly studied. Insect and vertebrate AChE, though, bear differences that probably
affect the structure-activity relationships of compounds inhibiting the two enzymes
[lo]. Such differences might lead to the design of safer insecticides, but, on the other
hand, question the use of the QSAR derived so far for, say, carbamates, for
assisting the design of new AChE inhibitors as agents for the treatment of AD.
 M. Recanatini et al. /Chemico-Biological Interactions 105 (1997) 199-228 201

Table 1
Data used in the calculation of Eq. (1) and Eq. (2) (dataset 1)[17]

Number Compound Log l/IC,, observed Log l/IC,, calculated” A Log P’ L

1 n=2 6.155 5.329 0.826 -2.584 9.400

2 n=3 5.420 5.029 0.392 - 3.003 10.210
3 n=4 4.678 5.608 -0.930 -2.194 11.460
4 ?I=5 5.119 5.729 -0.610 -2.025 12.260
5 n=6 6.032 6.106 - 0.074 - 1.496 13.510
6 n=7 6.420 6.419 -0.059 -0.967 14.320
7 n=8 7.097 6.838 0.259 -0.438 15.560
8 n=9 1.222 7.154 0.067 0.091 16.370
9 n= 10 7.398 7.366 0.032 0.620 17.620
10 n=ll 7.398 7.407 - 0.009 1.149 18.860
11 n= 12 7.155 7.296 -0.141 1.678 19.670
12 Hep.phys. 6.538 6.508 0.030 3.602 ~
13 Phys. 7.523 7.307 0.216 0.428 ~

a Values calculated by means of Eq. (2).

Consequently, we thought it was worthwhile to undertake a comparative QSAR

analysis of AChE inhibitors studied on vertebrate enzymes and designed with the
aim of finding new compounds useful against AD. Our study was focused on the
physico-chemical properties affecting the ligand-enzyme interaction and pointed to
the rationalization of the mode of action of the presently most interesting deriva-
tives. The results presented in this paper show that, through the calculation and the
examination of QSAR equations for several series of inhibitors, it is possible to
individuate probable interaction modes and the properties governing them.

Table 2
Data used in the calculation of Eq. (3) (dataset 2)[18]

Number R Logl/IC,, observed Log 1/IC,, calculated” A Log P’

1 ‘=3 7.215 7.230 -0.015 0.428

2 H 7.252 7.237 0.015 0.422
3 CH,CH = CH, 7.161 7.237 - 0.076 0.422
4 CHGH, 6.004 5.845 0.159 1.636
5 CH2CH,CsH, 5.987 5.663 0.324 1.795
6 CONHCH3 5.032 5.438 - 0.407 1.991b

a Values calculated by means of Eq. (3).

b Log P value for the unionized compound.
Table 3
Data used in the calculation of Eq. (4) (dataset 3)[19]

Number X R, Log I/lC,, observed Log 1IIC,, calculated” A k(6) n(4)

2’-CH, CH, 7.987 7.905 0.083 0.000 0.000

H CH, 7.620 7.905 -01285 0.000 0.000
4’-CH, CH, 6.856 7.354 - 0.497 0.000 0.560
2’,4’-(CH,), CH, 7.866 7.354 0.513 0.000 0.560
2’,4’,6’-(CH,), CH, 5.889 5.803 0.086 - 1.240 0.560
2’-C,H, CH, 8.013 7.905 0.109 0.000 0.000
2’,6’-(C,H&,4’-CH, CH.3 5.826 6.266 -0.441 - 1.310 0.560
8 2’-i-C,H, CH, 7.810 7.905 -0.095 0.000 0.000
9 4’-i-C,H, CH, 6.120 6.399 - 0.279 0.000 1.530
10b 2’-Cl CK 6.305 7.905 - 1.600 0.000 0.000
11 2’,6’-(Cl), CJ% 7.177 6.691 0.485 -0.970 0.000
12 2’-CH, H 7.770 7.905 -0.135 0.000 0.000
13 2’,4’-(CH,), H 7.762 7.354 0.408 0.000 0.560
14 H H 7.860 7.905 -0.044 0.000 0.000
15 4’-i-C,H, H 6.492 6.399 0.092 0.000 I .530

d Values calculated by means of Eq. (4).

’ Data point omitted in the calculation of Eq. (4).
Table 4
Data used in the calculation of Eq. (5) (dataset 4)[20]

k
Number R R, R2 Log l/IC,, observed Log 1/I& calculated” A B](R) I-Me 2
2
n-W-h CH, CH, 6.943 7.060 -0.117 1.520 1.ooo
2 n-W-h -3 CH3 7.114 7.060 0.053 1.520 1.ooo
3 n-W-4 5 CA CH3 7.237 7.060 0.176 1.520 1.ooo
4 n-C&,, CA CH3 7.347 7.060 0.286 1.520 1.000
5 n-C,H, C,H, CH, 7.046 7.060 -0.015 1.520 1.ooo
6 t-C,H, W, CH, 4.904 4.903 0.001 2.600 1.ooo
7 n-C,H, ‘3, CHJ 6.812 7.060 - 0.248 1.520 1.ooo
8 CH, ‘3, ‘=J 7.420 7.060 0.360 1.520 1.ooo
9 C,H, CA CK 6.754 6.681 0.074 1.710 1.ooo
10 CH(CH&,H, C,H, CH, 5.876 5.902 - 0.026 2.100 1.ooo
11 CH&H, W-b CH3 7.161 7.060 0.101 1.520 1.oOO
12 n-C,H,, n-cd-b CH3 7.032 7.060 - 0.029 1.520 1.000
13 n-Cd,3 n -CJH, ‘=3 7.208 7.060 0.147 1.520 1.ooo
14 n-W,5 CHGH, CH3 6.296 7.060 -0.764 1.520 1.000
15 n-V,5 C,H, H 6.419 6.256 0.163 1.520 0.000
16 n-cd,, C,H, H 6.482 6.256 0.426 1.520 0.000
17 n-C,H, C,H, H 5.968 6.256 -0.287 1.520 0.000
18 n-C,H, GH, H 5.659 6.256 - 0.596 1.520 0.000
19 CH, C,HS H 6.597 4.256 0.341 1.520 0.000
20 n-C,H,, n-C,H, H 6.201 6.256 - 0.054 1.520 0.000
21 n-GH,, fi-C,H, H 6.263 6.256 0.007 1.520 0.000

a Values calculated by means of Eq. (5).

204 M. Recanatini et al. /Chemico-Biological Interactions 105 (1997) 199-228

Table 5
Data used in the calculation of Eq. (6) (dataset 5)[21]

Number R Log l/K, observed Log l/K, calculated” A L

H 7.301 7.312 -0.011 2.060

2 CH, 7.097 7.074 0.023 2.870
3 C,Hs 6.699 6.144 -0.045 4.110
4 n-C,H, 6.523 6.651 -0.128 4.920
5 n-C,H, 6.699 6.847 -0.148 6.170
6 n-C,H, I 7.155 7.055 0.099 6.970
I CH,CH=CH, 6.854 6.658 0.196 5.110
8 c-C,H, , 6.886 6.847 0.039 6.170
9 CW& 6.824 6.664 0.160 4.620
10 CH,‘=K,Hs 7.398 7.424 - 0.027 8.330
11 CH&H,NH, 6.495 6.652 -0.157 4.830

AValues calculated by means of Eq. (6)

2. Methods

AChE inhibition data were taken from the literature and are reported in Tables
1- 13. For each set, the consistency of the data was checked and, in few cases, only
congeneric subsets were taken into consideration.
All the parameters were automatically retrieved and loaded into the tables by the
program C-QSAR [13], that was used also for the calculation of the regression
analyses. The statistical significance was checked by calculating the F-test and all
the reported equations resulted significant at the 99% level, unless otherwise stated.
Moreover, for each equation, the q2 (cross validated r2) calculated by the program
(20% points dropped each run, 100 iterations) is reported.
Partition coefficients (log P values) were calculated with the CLOGP method
[14], as implemented in the C-QSAR program.
With regard to the log P values for the ionized compounds (log P’ values of
Tables 1 and 2) they were calculated manually, by modifying the CLOGP
procedure in a way to take into account the presence of charged N atoms. For all
the compounds of datasets 1 and 2, the protonation of the eseroline N-l atom was
considered by using the value of - 3.86 for the NH + fragment [15] and adding the
proper geometric and electronic bond factors [14]. The proximity effects [14]
between the two nuclear nitrogen’s were also recalculated. For the morpholinoalkyl
derivatives (dataset l), also the variable length of the alkyl chain had to be taken
into account, which was accomplished by adding the mentioned geometric and
electronic bond factors related to the protonated morpholinic nitrogen (fragment
value - 3.86). Again, proximity effects were calculated, regarding the interactions
Table 6
Data used in the calculation of Eq. (7) (dataset 6)[22]

Number R R* Log l/ICSO observed Log 1/I& calculated” A L I-Cl

H H 5.398 5.988 -0.590 2.060 0.000

2 H 6-Cl 7.932 7.496 0.435 2.060 1.000
3 H 6-OCH, 5.699 5.988 - 0.289 2.060 0.000
4 H 6-CF, 5.752 5.988 -0.236 2.060 0.000
5 H 6-F 6.535 5.988 0.547 2.060 0.000
6 CH, H 5.754 5.537 0.217 2.870 0.000
7 n-C,H, H 4.876 4.672 0.204 4.920 0.000
8 CH,CH,N(CH,), H 5.115 5.018 0.096 6.760 0.000
9 CH,CH,C,H, H 5.470 5.512 - 0.042 8.330 0.000
10 CH,C,H, H 4.492 4.724 -0.231 4.620 0.000
11 CH,(2’-C&H,) H 4.987 4.724 0.264 4.620 0.000
12 CH,(3’-ClC,H,) H 4.816 4.724 0.153 4.620 o.cloo
13 CH2(4’-ClC,H,) H 4.565 4.670 -0.104 5.290 0.000
14 CH,(2’-F&H,) H 4.384 4.724 - 0.340 4.620 0.000
15 CH,(3’-F&H,) H 4.883 4.724 0.159 4.620 0.000
16 CH,(4’-FC,H,) H 4.777 4.665 0.112 5.210 0.000
17 CH,(2’-CH30C,H,) H 4.444 4.724 - 0.280 4.620 0.000
18 CH,(3 -CH,OC,H,) H 4.676 4.724 - 0.048 4.620 0.000
19 CH,(4’-CH,OC,H,) H 4.963 4.952 0.011 6.540 0.000
20 CH,(2’-CH,C,H,) H 5.230 4.724 0.506 4.620 0.000
21 CH,(3’-CH&,H,) H 4.611 4.724 -0.113 4.620 0.000
22 CH,(4’-CH,C,H,) H 4.678 4.484 - 0.006 5.430 0.000
23 CHQ-CF,C,H,) H 4.876 4.724 0.153 4.620 0.000
24 CH,(3’-CF,C,H,) H 4.848 4.724 0.124 4.620 0.000
25 CH,(4’-CF,C,H,) H 4.742 4.765 - 0.023 5.860 0.000
26 CH,C& 6-Cl 5.886 6.232 -0.346 4.620 1.000
27 CH,(4’-FC,H,) 6-Cl 6.085 6.174 - 0.089 5.210 I .ooo
28 CH,C,H, 6-F 5.064 4.724 0.340 4.620 0.000
29 CH,(2’-CF,C,H,) 6-F 4.697 4.724 - 0.027 4.620 0.000
30 CH,C,H, GCF, 4.166 4.124 -0.577 4.620 0.000

a Values calculated by means of Eq. (7).

206 M. Recanatini et al. / Chemico-Biological Interactions 105 (1997) 199-228

Table I
Data used in the calculation of Eq. (9) (dataset 7)[23]

Number X Log I/G, Log l/K,, calculated” A F Log P

observed

I H 5.412 5.670 -0.198 0.000 4.572

2 NO, 6.820 6.729 0.091 0.650 4.477
3 CH, 5.757 5.565 0.192 0.010 5.071
4 Cl 5.876 6.148 ~ 0.272 0.420 5.361
5b OH 5.611 6.227 -0.616 0.330 4.446
6 OCH, 6.154 6.079 0.075 0.290 4.792
7 OSO,CH, 6.421 6.451 -0.030 0.400 3.982
8 NH, 5.971 5.972 - 0.002 0.080 3.855
9 NHCOCH, 6.484 6.289 0.195 0.310 4.060
10 NHSO,CH, 6.331 6.319 0.011 0.280 3.738
11 NHSO,C,H,CH, 5.660 5.738 - 0.079 0.240 5.866
12 CO&H s 5.97 1 5.845 0.125 0.310 5.885
13 COOCH, 6.245 6.186 0.059 0.340 4.679
14 COOC,H, 6.168 6.058 0.110 0.340 5.208
15 CONHCH, 6.420 6.429 - 0.009 0.350 3.745
16 CON(C,H,), 6.009 6.277 - 0.269 0.350 4.370

a Values calculated by means of Eq. (9).

b Data point omitted in the calculation of Eq. (9).

of the polar morpholinic fragments between them and, eventually, of the proto-
nated nitrogen with the carbamate fragment. An example of the calculation of log
P’ is reported in Table 14.

3. Results

The screening of the literature allowed us to find only a limited number of series
of AChE inhibitors suitable for the QSAR analysis. This was rather surprising,
given the great emphasis cast on the cholinergic approach to the treatment of AD.
However, we found enough data concerning the three main classes of currently
investigated AChE inhibitors: physostigmine analogues, 1,2,3,4_tetrahydroacridine
derivatives and benzylamines.
Physostigmine is a natural product that has long been known as an irreversible
AChE inhibitor [16]. Modifications on its structure regard principally the car-
bamoylic group and, to a lesser extent, the eseroline nucleus. Even if many series of
derivatives were synthesized, only few could be analyzed with the QSAR procedure.
Compounds in dataset 1 [17] have the general formula
n -- -
O~N-(CH+$COO
Table 8
Data used in the calculation of Eq. (IO) (dataset 8)[24]

Number X R Log l/K,, observed Log I iIC,, calculated” A Log P

1 H COW, 5.638 5.572 0.067 4.089 0.000

2 3-NO, H 5.533 5.314 0.219 4.556 0.650
3 3-NO, COCH; 6.280 6.202 0.077 3.832 0.650
4 4-N& H 4.991 5.021 - 0.029 3.586 0.080
5 4-NHCOCH, H 5.246 5.170 0.076 3.832 0.310
6 4-U H 4.810 4.BYY -0.090 5.526 0.420
7 4x1 COCH, 5.670 5.788 -0.119 4.802 0.420
8 4-CH, H 4.801 4.583 0.218 5.312 0.010
9 4-CN H 5.305 5.257 0.048 4.246 0.510
10 4-CN COCH; 6.343 6.146 0.197 3.522 0.510
I1 4-OH H 4.900 5.120 - 0.220 4.146 U.330
12 4-OCH, H 4.788 4.957 -0.169 4.732 0.290
13 3-OCH, H 5.031 4.957 0.074 4.732 0.290
14 3-UCH, CUCH ? 5.241 5.846 -0.605 4.008 0.2’10
I5 4-SCH? H 4.678 4.765 - 0.087 5.372 0.230
16 4-SUCH? H 5.125 5.489 -0.364 3.222 0.520
17 4-SOzCH3 H 5.502 5.508 ~ 0.007 3.172 0.530
18 4-W&H, COCH, 6.785 6.397 0.388 2.448 0.530
19 3-N0,.4-Cl H 5.726 5.574 0.152 5.069 I .070
20 3-N0,,4-C’l COCH, 6.389 6.462 ~ 0.073 4.345 1.070
21 3,4,5-(OC’H,), H 5.389 5.645 ~ 0.256 3.926 0.870
22 H H 4.930 4.683 0.247 4.813 0.000
23 4-NO, H 5.520 5.314 0.206 4.556 0.650
24 4-NO, COCH, 6.270 6.202 0.068 3.832 0.650
2s 3.4.(OCH,), H 5.270 5.290 - 0.020 4.381 0.580

d Values cahlated by means of Eq. (10).

208 M. Recanatini et al. /Chemico-Biological Interactions 105 (1997) 199-228

Table 9
Data used in the calculation of Eq. (11) (dataset 9)[25]

Number X Log l/G, Log l/G, A Log P F

observed calculated”

H 6.921 7.249 -0.329 3.259 0.000

4-CH, 7.114 7.083 0.030 3.758 0.010
4-OCH, 7.585 7.440 0.145 3.460 0.290
4-Cl 7.523 7.306 0.217 4.174 0.420
3-NO, 7.824 7.713 0.051 3.434 0.650
4-C,H, 6.638 6.694 -0.056 5.147 0.120
3,4-(Cl), 7.260 7.455 -0.195 4.827 0.840
3,4(-CH=CHCH=CH-) 7.000 6.864 0.136 4.433 0.030

a Values calculated by means of Eq. (11).

where IZ varies from 2 to 12; physostigmine and eptylphysostigmine are also in the
series. In this dataset, the alkyl substituent on the carbamic function of physostigmine
was linearly increased and a second ionizable function was introduced at the end of
it. In the view of the authors [17], these compounds should be able to bind to two
anionic centers of the enzyme active site with both the protonated N-l atom of the
eseroline ring and the protonated morpholinic nitrogen. In this respect, the length
of the alkyl chain should be crucial. Following this hypothesis, we calculated Eq. (1)
which relates in a parabolic way the inhibitory activity to the length of the substituents
attached to the carbamic nitrogen; the parameter used is the Verloop’s L:
IC,, Human Erythrocyte Acetylcholinesterase

Log l/K,, = 2.367(0.346)L - 0.066(0.01 1)L2 - 13.924(2.638)

IZ= 9 r2 = 0.995(q2 = 0.995) s = 0.086 F,,, = 567.82 L,,, = 18.016
(omitted: YI= 2; n = 3) (1)

This equation indicates that, when the length of the morpholinoalkyl chain is optimal
(L about 18 A), the interaction is good, while for longer or shorter chains, one of
the two contacts is weakened. For calculating Eq. (1) the two reference compounds
physostigmine and eptylphysostigmine were not taken into consideration, because
they do not bear the ionizable morpholine moiety. In addition, two data points,
corresponding to both the n = 2 and the n = 3 analogues, were omitted, as they did
not fit the parabolic relationship. Another major concern with Eq. (1) is that, for the
set of nine compounds considered, the L parameter is perfectly collinear with log P
(r2,ogpIL= 0.984), thus rendering ambiguous any interpretation of the QSAR. How-
ever, assuming that, at the site of action, compounds of dataset 1 are completely
ionized at both aminic functions, the calculated log P’ values were used and a bilinear
relationship was obtained, accounting for all the 13 derivatives tested:
Table 10
Data used in the calculation of Eq. (12) and Eq. (13) (dataset 10)[26]

Number X Log l/I&, observed Log 1/I&, calculated” A I-P I-O F Log P

1 H 6.252 4.354 -0.102 0.000 0.000 0.000 4.075

2 2-CH, 6.000 5.731 0.263 0.000 1.OOo 0.010 4.174
3 3-CH, 6.328 6.361 - 0.033 0.000 0.000 0.010 4.574
4 4-CH, 6.745 4.825 - 0.080 0.000 0.000 0.010 4.574
5 2-NO, 6.056 6.201 -0.145 0.000 1.000 0.650 3.350
6 3-NO, 6.638 6.825 -0.187 0.000 0.000 0.650 4.250
7 4-NO, 7.260 7.289 - 0.029 1.000 0.000 0.650 4.250
8 4-OCH, 7.056 7.028 0.028 1.oOO 0.000 0.290 4.276
9 4-CHO 6.921 7.057 -0.136 1.000 0.000 0.330 3.986
10 4-Cl 6.745 7.122 -0.378 1.000 0.000 0.420 4.990
11 4-F 7.071 7.144 - 0.073 1.000 0.000 0.450 4.420
12 4-COCH, 7.292 1.057 0.235 1.000 0.000 0.330 3.968
13 4-SO,CH,C,HS 7.538 1.209 0.328 1.000 0.000 0.540 4.650
14 2-(-N=) 6.097 6.215 -0.118 0.000 1.000 0.670 3.522
15 3-(-N=) 7.161 6.840 0.322 0.000 0.000 0.670 3.522
16 4-(-N=) 7.409 7.304 0.105 1.000 0.000 0.670 3.522

a Values calculated by means of Eq. (12).

210 M. Recanatini et al. / Chemico-Biological Interactions 105 (1997) 199-228

Table 11
Data used in the calculation of Eq. (14) (dataset 11)[27]

Number X Log 1PC,,, Log 1/I%, A MR(4) MR(3)

observed calculated”

H 7.523 7.959 - 0.436 0.103 0.103

2 4-NO2 7.903 8.107 -0.204 0.736 0.103
3 4-NH, 8.056 8.062 -0.006 0.542 0.103
4 4-NHCOCH, 8.553 8.284 0.269 1.493 0.103
5 4-NHCOC,H, 8.921 8.745 0.176 3.464 0.103
6 4-OCH, 8.097 8.119 ~ 0.022 0.787 0.103
7 4CONHCH,C,H, 8.658 8.853 -0.196 3.929 0.103
8 4COC,H, 8.620 8.644 ~ 0.024 3.033 0.103
9 3-NO, 8.046 7.695 0.350 0.103 0.736
10 3-NH, 7.959 7.776 0.182 0.103 0.542
II 3-NHCOC,H, 6.469 6.558 - 0.090 0.103 3.464

il Values calculated by means of Eq. (14)

IC,, Human Erythrocyte Acetylcholinesterase

Log l/ICSo = 0.717(0.284) log P’ - 1.158(0.689) log(pP’+ 1) + 7.181(0.468)

n = 13 r* = 0.792(q2 = 0.772) s = 0.497 F3,Y = 11.44 log p = - 0.792
log Pbnt = 1.003 (2)

Table 12
Data used in the calculation of Eq. (15) (dataset 12)[28]

Number X Log I /ICso observed Log 1/IC,,, calcula- A rrb

ted”

I H 7.260 7.987 -0.727 0.000

2 5-CH, 8.108 8.204 ~ 0.096 -0.170
3 5,6-(CH,)Z 8.237 8.293 - 0.056 -0.240
4 5-OCH, 8.143 8.331 -0.188 ~ 0.270
5 6-OCH, 8.081 7.834 0.247 0.120
6 7-OCH, 8.149 7.987 0.162 0.000
7 6-NHCOCH, 8.553 7.719 0.833 0.210
8 6-NHCOC,H, 8.027 7.962 0.065 0.020
9 6-NHSO,C,H, 7.854 7.783 0.071 0.160
IO h-N-morpholinyl 9.097 8.624 0.473 -0.500
11 6-NH, 7.699 8.191 - 0.492 -0.160
12 6-OH 7.585 7.834 - 0.249 0.120
13 6-Br 7.301 7.490 -0.189 0.390
14 6-CN 6.996 7.274 ~ 0.278 0.560
15 6-CONH, 8.056 7.630 0.425 0.280

a Values calculated by means of Eq. (15).

b Substituents in positions 5, 6 and 7 on the benzisoxazole ring were considered in positions para, meta
and who, respectively, to the 0 atom, and the corresponding crnaril, o,,,,~~ and cortho values were used.
 M. Recanatini et al. /Chemico-Biological Interactions 105 (1997) 199-228 211

Table 13
Data used in the calculation of Eq. (16) (dataset 13)[29]

Number X Log l/IC,, observed Log l/K.& calculated” A F

1 H 6.602 6.593 0.009 0.000

2 4-OCH, 6.824 6.453 0.370 0.290
3 4-COCH, 6.398 6.434 - 0.036 0.330
4 3-NO, 6.155 6.281 -0.126 0.650
5 3-CF, 6.618 6.410 0.268 0.380
6 2-CH, 6.398 6.588 -0.190 0.010
7 2-F 6.071 6.377 -0.306 0.450
8 2,3,4,5,6-(F), 5.523 5.512 0.011 2.250

a Values calculated by means of Eq. (16).

The high s value of Eq. (2) is mostly due to the deviation of two data points (n = 2
and II = 4). Interestingly, for the whole dataset, the collinearity between L and log
P'is absent (T&,,~ = 0.049, which might confirm the actual role of hydrophobicity
in the interaction of the series of compounds 1 with AChE.
The compounds of dataset 2 [18] are analogues of physostigmine where only the
N-substituent in position 1 is varied, while the N-methylcarbamic moiety is
conserved.

Table 14
Calculation of log P’ for compound 2 of Table 1

Class Log P contribution description Value

Fragment # 1 Tertiary amine -0.93

Fragment # 2 Protonated amine -3.86
Fragment # 3 NH-Carbamate - 1.46
Fragmenr # 4 Protonated amine -3.86
Fragment # 5 Ether -1.82
Isolating carbon 14 Aliphatic isolating carbons 2.73
Isolating carbon 6 Aromatic isolating carbons 0.78
Exfragment branch 1 Chain and 2 cluster branches -0.39
Exfragment hydrogen 31 Hydrogens on isolating carbons 7.037
Exfragment bonds 4 Chain and 12 alicyclic -1.56
A Geometric factor: 5( - 0.08) - 0.40
Hecrronic factor: 2( - 0.X)+ (- 0.28)+( - 0.14)+( - 0.07) - 1.61
Benzylbond simple 1 Benzyl bond to simple aromatics -0.15
Proximity Y-C-Y -0.32 (-0.93 -3.86) 1.534
Proximity Y-C-C-Y -0.15 (-1.82 -3.86) 0.852
Electronic sigrho 1 Potential interaction: 1.00 used 0.104

Result - 3.003

Manual modifications to the computerized CLOGP calculation are reported in italics.

212 M. Recanatini et al. /Chemico-Biological Interactions 105 (1997) 199-228

w-pO*nJ~,

&Is lk
We expected that, for this series too, the main factor governing the anti-
cholinesterase activity might well be the hydrophobic character of the compounds.
Again, we used log P’ values calculated for the ionized molecules, except for the
derivative bearing the R = CONHCH, substituent: in this case, in fact, the amidic
group suppresses the ionization of the N-l atom. The following equation was
obtained:
IC,, Electric Eel Acetylcholinesterase
Log l/K,, = - 1.146(0.445) log P’ + 7.721(0.586)
(3)
n = 6 Y*= 0.927(q2 = 0.927) s = 0.275 F,,, = 51.07
The inverse relationship with log P’ expressed by Eq. (3) is rather surprising, but,
as will be evident in the following, not unusual in series of AChE inhibitors.
The dataset 3 [19] refers to the general formula

and the following equation describes the variation of inhibitory activity:

IC,, Human Erythrocyte Acetylcholinesterase
Log l/K,, = 1.251(0.427&(6) - 0.984(0.392)~(4) + 7.905(0.285)
n = 14 r2 = 0.848(q2 = 0.846) s = 0.348 F,,,, = 30.67
(omitted: X = 2’-Cl, R, = Me) (4)

In this equation all the parameters refer to substituents on the phenyl ring. One
point (I-Me,2’-Cl) strongly deviates from the correlation, being calculated 1.6 log
units more potent than is observed. The variation of the R, substituent is not
important, as we see that the four compounds with R, equal to H are rather well
accounted for by the equation. The E,(6) term indicates an unfavorable steric effect
for X-substituents in the 6 position of the carbamic phenyl ring, pointing out the
detrimental effect of two ortho groups simultaneously present on the same ring. The
negative n(4) term seems to indicate that less lipophilic para-X-substituents are
preferred. However, also in this case the collinearity between parameters strongly
affects the equation: lipophilic (z) and steric (Es) parameters for both 4’ and 6’
positions are collinear and Eq. (4) was chosen on the basis of considerations of bio-
and physico-chemical meaning.
 M. Recanatini et al. /Chemico-Biological Interactions 105 (1997) 199-228 213

The dataset 4 [20] contains %carba-analogues of physostigmine carrying different

substituents on both the remaining N atoms and varying also the methyl group in
position 3a of the eseroline ring (RJ.

R-fl-COOdN,

I
Rl
A significant equation could be obtained from this set, using one of the Verloop’s
steric parameters, Bl, and an indicator variable, I-Me, assuming the value of 1
when R, is Me (presence of the methyl group in position 3a of the eseroline ring):
IC,, Human Erythrocyte Acetylcholinesterase

Log l/IC,, = - 1.998(0.569)Bl(R) + 0.805(0.309)1-Me + 9.292(0.899)

(5)
IZ= 21 r2 = 0.792( q* = 0.781) s = 0.308 F,,,, = 34.37

The parameter Bl takes into account the minimum width of the substituent, and it
has to be noted that, in the present series, only three derivatives have a Bl value
higher than that of all the other compounds. This feature, plus the presence of the
methyl group in position 1, accounts for almost 80% of the variance of activity. It
is rather strange, but analogous to the previous case of dataset 3, that the different
substituents in N-l (R,) do not seem to influence the QSAR for this series. On the
other hand, the strong steric requisite expressed by the negative Bl(R) term might
recall the analogous situation depicted by Eq. (4) ( re ferring to dataset 3) containing
a highly positive Es term (Taft’s Es values are negative).
A recently approved drug for the treatment of the Alzheimer disease is the
tetrahydroacridine compound tacrine [5]. In the literature, we found only two
papers reporting extensive modifications of the parent compound.
In dataset 5 [21], we took into consideration tacrine derivatives carrying sub-
stituents on the amino group in position 9:
R

For this set, one equation correlating the variations of the inhibition constant Kr
(the dissociation constant for the enzyme-inhibitor complex) with structural
parameters is a bilinear one involving the length parameter L:
KI Electric Eel Acetylcholinesterase

Log l/K, = - 0.296(0.136)L + 0.569(0.220) log@ x 10L + 1) + 7.921(0.539)

n = 11 r* = 0.844(q2 = 0.842) s = 0.142 F,,, = 12.59 L,,, = 4.903 log /? = - 4.867

(6)
214 M. Recanatini et al. / Chemico-Biological Interactions 105 (1997) 199-228

This is an unusual bilinear equation showing a descending side at lower L values;

Lopt actually corresponds to a minimum of activity, after which the log l/K1 values
rise. In this case, compounds with extreme values of the parameter display the best
activity.
In the similar dataset 6 [22], substituents are introduced also in the position 6 of
the tetrahydroacridine ring, in addition to those in position 9.

The correlation equation for this larger dataset is quite similar to that of dataset 5,
as it contains a bilinear term in L and is characterized by a minimum instead of a
maximum of activity:
IC,, Rat Striatum Acetylcholinesterase

Log l/I& = - 0.560(0.132)L + 0.877(0.263) log@ x 10L + 1) +

1.509(0.386)1-Cl+ 7.141(0.528)

IZ= 30 r* = 0.861 (q2 = 0.857) s = 0.305 F4 25 = 38.83 Lopt = 5.116 log a = - 4.869(7)

In Eq. (7), the indicator variable Z-Cl takes the value of 1 when a 6-Cl substituent
is present on the molecule and accounts for the remarkably higher activity of such
analogues. Besides this feature, the rather narrow variation of activity within this
series is acceptably explained by the bilinear variation of L; as in Eq. (6), the minimum
value of activity is calculated at an L value of about 5 A.
For both datasets 5 and 6, the collinearity problem has to be considered, even if
for the series 6 it is less serious than for the 5. Log P and L values are intercorrelated
for the dataset 5 (r2 = 0.826) and this casts some doubt on Eq. (6). On the other hand,
in the larger set 6, the collinearity between log P and L is not so high (r2 = 0.384)
which can give some more confidence in Eq. (7). Moreover, the alternative equations
in log P are statistically worse and the above QSARs can reasonably be preferred.
A third class of AChE inhibitors is that of benzylamines. In these compounds,
generally, an arylcarboxamidoalkyl chain of varying length is linked either to a
benzyl,alkyl-disubstituted N atom, or to the C-4 of an N-benzyl-substituted pipe-
ridine. Many of these series have been recently published and some were studied from
the QSAR point of view.
In two related papers [23,24], Ishihara et al. studied several sets of benzylamine
derivatives and obtained some quantitative relationships, which we recalculated by
using their electronic parameters together with correct n values. In the dataset 7 [23],
isoindoldione compounds substituted in position 5 of the indole moiety are
considered:
 M. Recanatini et al. /Chemico-Biological Interactions IO5 (1997) 199-228 215

For a series of 16 derivatives of the above general formula the following equation
was calculated by Ishihara et al.:
IC,, Rat Brain Acetylcholinesterase

Log l,/ZC,, = -0.279(0.150)x + 0.926(0.418)(a, + 0,,)/2 + 5.873(0.141)

II = 16 r2 = 0.712 s = 0.209 (8)

In this equation, the electronic term introduced by the authors takes into account
the fact that the substituents are in position para to one of the carbonyls and at the
same time meta to the other. By using the field parameter F and the calculated log
P values instead, Eq. (9) was calculated, after omitting one data point:
IC,, Rat Brain Acetylcholinesterase

Log l/IC,, = - 0.243(0.131) log P+ 1.594(0.570)F+ 6.782(0.642)

y1= 15 r2 = 0.821(q2 = 0.813) s = 0.161 F,,,, = 24.49 (omitted: X = OH) (9)

Eq. (8) and Eq. (9) express the same QSAR, even if the terms of Eq. (9) are of more
general meaning and allow a comparison with other similar relationships.
In a subsequent paper [24], the same authors studied some analogues (dataset 8)
in which the isoindoldione (phthalimide) ring of the previous series was replaced
with a 3-arylpropenamide moiety:
0
CH=CH-8 C2Hs
‘N-(CH2)&CH2
R’

In this set, X substituents are located in either para or meta positions or, in a few
cases, in both, while the R substituent can be only H or CH,CO. The equation
calculated for this set is the following:
IC,, Rat Brain Acetylcholinesterase

Log l/I& = - 0.217(0.222) log P + 0.884(0.340)F+ 0.731(0.235)1+

5.730(0.670)

n = 25 r2 = 0.862(q2 = 0.857) s = 0.231 F3 21 = 43.57 (10)

In Eq. (lo), the indicator variable I takes the value of 1 when the acyl group is
present on the amidic nitrogen, thus pointing out a net gain of inhibitory activity
associated with this feature.
Eq. (9) and Eq. (10) are rather similar and suggest similar behavior of the two
216 M. Recanatini et al. / Chemico-Biological Interactions 105 (1997) 199-228

sets of compounds at the inhibition site of AChE. In both series 7 and 8,

electron-attracting groups on the aryl moiety exert a favorable effect. The low
negative coefficient with the hydrophobic term in both equations suggests that less
lipophilic substituents are slightly more favorable.
A series of compounds showing some structural similarity with the 7 and 8
derivatives is the dataset 9, which is made by benzoylthioureas bearing an N-
alkyl,N-benzylaminic moiety comparable to those of 7 and 8. This series was
recently published by Vidaluc et al. [25] and it may be represented by the following
general formula:

a i ‘i
,C&CH2-0-CH2CH2-N-CH2
H3

X
The X substituents are located in 4 position of the phenyl ring, except for two
3,4-disubstituted and one 3-substituted derivatives. The correlation equation calcu-
lated for this series of compounds is the following:
IC,, Rat Brain Acetylcholinesterase

Log i/K,, = - 0.351(0.309) log P + 0.900(0.677)F+ 8.394(1.253)

(11)
n = 8 r2 = 0.778(q2 = 0.767) s = 0.219 F,,, 2 8.76

Eq. (11) is significant at the 95% level and it contains the same hydrophobic and
electronic terms as Eq. (9) and Eq. (10). It is also interesting to note that the
coefficients associated to these terms are similar, even if in Eq. (9) the coefficient of
the electronic term F is somewhat higher. The quality of Eq. (11) is lower, but it
may still be accepted considering its strict similarity to the other two.
As will be discussed in the next section, the presence of the same parameters with
similar coefficients in Eqs. (9)-(11) will let us hypothesize that the members of the
sets represented by the general formulas 7, 8 and 9 bind to AChE at the same site.
Several series of benzylpiperidine derivatives have recently appeared in the
literature and this class of compounds is presently regarded with much interest as
a promising source of potent inhibitors of AChE.
Sugimoto et al. [26] synthesized and tested on mouse brain homogenates a rather
big series of benzylpiperidines bearing an acylaminoethyl chain in position 4 of the
piperidine. We studied a homogeneous subset of benzoylaminoethyl derivatives
(dataset 10) of general formula

, $,CH2CH2~NS,~
/

d;y
X
The X-substituted phenyl ring bears mostly para, but also meta and ortho sub-
stituents; the series also comprises the 2-(-N=), 3-(-N=) and 4-(-N=), i.e. the
 M. Recanatini et al. / Chernico-Biological Interactions 105 (1997) 199-228 217

2-picolyl-, nicotinyl- and isonicotinyl-derivatives. The QSAR we developed for this

series is represented by Eq. (12):
I& Mouse Brain Acetylcholinesterase
Log l/I& = 0.725(0.479)F+ 0.464(0.296)1-P - 0.624(0.377)1-O +
6.354(0.291)
n = 16 r2 = 0.845(q2 = 0.845) s = 0.224 F3.,2 = 21.82 (12)

In this equation the indicator variables I-P and I-O take the value of 1 if a para or
an ortho substituent, respectively, is present on the benzoylic ring, otherwise they
are equal to 0. From Eq. (12) it appears that structural characteristic of the
molecules are most important, in the sense that substituents in position para to the
amidic linkage exert a favorable effect on the inhibitory activity, while ortho
substituents are detrimental. In this equation, the F electronic term with almost the
same positive coefficient as in Eqs. (9))( 1 I) is present, but the log P term is lacking.
Eq. (13), containing the hydrophobic term, is not as good as Eq. (12) indicating
that the electronic effect, in this case overrides the hydrophobic one:
I& Mouse Brain Acetylcholinesterase
Log l/U& = - 0.355(0.314) log P + 0.597(0.337)1-P - 0.677(0.436)1-O +
8.036(1.301)
(13)
12= 16 r2 = 0.804(q2 = 0.803) s = 0.252 F3.,2 = 16.41
It has to be noted that almost no collinearity exists between log P and F (r&p!I;
= 0.227).
In a subsequent paper, Sugimoto et al. [27] studied a series of benzylpiperidines
bearing the phthalimide moiety linked to an ethyl spacer located in position 4 of the
piperidine ring (dataset 11).

This series resembles that of dataset 7 and most of the substituents are in a position
corresponding to that of the benzylamino derivatives 7; three substituents are in
position 3 of the phthalimidic nucleus and may be considered as ortho-substituents
with respect to one of the carbonyls. The best QSAR equation we obtained is the
following:
IC,, Mouse Brain Acetylcholinesterase
Log l/K,,, = 0.234(0.138)MR(4) - 0.417(0.202)MR(3) + 7.978(0.299)
(14)
n = 11 r2 = 0.881(q2 = 0.881) s = 0.260 F,,, = 29.47
Eq. (14), containing only molar refractivity terms, looks different from those
previously examined for benzylpiperidines (or benzylamines), although structure 11
218 M. Recanarini et al. / Chemico-Biological 1ntrrartion.v 105 (1997) 199-228

is very similar also to structure 10. Remarkably, a low collinearity exists between the
A4R and n parameters for both the 3 and 4 positions of 11 (r&,l~MRc4j= 0.383;
I.=
rr(3j.MR(3,= 0.102). A striking feature of this QSAR is the absence of any electronic
term.
In a recent paper, Villalobos et al. [28] reported the structure-activity relationships
of a series of 4-(3-benzisoxazolylethyl)-N-benzylpiperidines (dataset 12) and hypothe-
sized a binding mode of these inhibitors to the active site of AChE. These authors
also studied the docking of the inhibitors to the enzyme by means of molecular
dynamics simulations.

The substituents, mostly in 6 position of the benzisoxazole moiety, are well varied,
but, from the examination of the corresponding substituent constants for the
hydrophobic, electronic and steric effects, only the following very poor equation could
be calculated:
ICsO Human Erythrocyte Acetylcholinesterase
Log l/K,, = - 1.274(0.861@ + 7.987(0.231)
(15)
n = 15 r= = 0.44O(q’ = 0.398) s = 0.410 F,,,, = 10.22
Eq. (15) cannot be defined a QSAR at all, but it was reported to show a loose trend
in the SAR, which, however, is in agreement with the conclusions reached by other
authors via a different approach using molecular modeling techniques (see Section
4).
Finally, we considered a set of arylthioureas (dataset 13) described in a paper by
Vidaluc et al. [29].

X substituents are present in ortho, metu and paru positions and one penta-F-substi-
tuted derivative is also in the series. A not very bright equation was calculated for
this series too, but it might be of some interest to report it:
IC,, Rat Brain Acetylcholinesterase
Log l,‘IC,, = - 0.480(0.312)Ff 6.593(0.271)
(16)
n = 8 Y’ = 0.703(q2 = 0.656) s = 0.244 F,,, = 14.19
Eq. (16) contains a negative electronic term, pointing out that the previous
relationship 15 could not be merely an artifact. Also in the case of derivatives 13,
electron donation towards a polar moiety seems to favor the binding to the enzyme.
 M. Recanatini et al. / Chemico-Biological Interactions 105 (1997) 199-228 219

4. Discussion

One of the aims of a comparative QSAR analysis is that of bringing to light

common features of ligand-receptor interactions displayed by different series of
compounds. This can be done by detecting equal corresponding parameters (with
similar coefficients) in the equations describing the QSAR of each series. In order
to attain this goal, a great deal of work should be done a priori, by building
suitable sets of congeneric compounds and testing them homogeneously on the
biological target. Unfortunately, this is almost never done. What is usually done
instead, is an a posteriori analysis in which a number of series of compounds acting
on the same enzyme (or receptor) is studied, for obtaining significant QSAR
equations. The equations are then examined and, from their similarities or diffe-
rences, deductions are made about the ligand-target interaction. Problems often
come from the poor choice of congeners constituting series of analogues not
designed for QSAR studies. Collinearity between some physico-chemical properties
of the ligands can occur, thus weakening even the descriptive capability of the
equations. However, despite the risk of overemphasizing questionable relationships,
the comparative work performed on the basis of quantitative relationships via the
analysis of physico-chemical parameters is definitely worth doing. The results might
reveal not trivial, previously masked features of the interaction and, more impor-
tant, they can guide the selection of new compounds to be synthesized and tested.
In the case of the inhibitor-AChE system, the need for information about the
liganddenzyme interactions is particularly urgent for the reasons stated above
(Section 1). From screening of the recent literature, though, relatively few series
suitable for QSAR analysis were found and, among these, only some gave statisti-
cally significant equations. Eqs. (l))( 16), refer to three classes of compounds,
namely physostigmine analogues, 1,2,3,4-tetrahydro-9-aminoacridines and benzy-
lamines. The structures of these parent compounds are different, but they all show
a common characteristic, that is an ionizable N atom. Besides this common
anchoring function, whose binding counterpart is more or less precisely identified,
molecules belonging to the three classes stabilize their interaction with the enzyme
through a number of other bonds, which they eventually establish, depending on
the structure.
It is known from X-ray crystallographic studies that the AChE active site appears
as a deep gorge which penetrates into the enzyme widening to its base [30]. The
catalytic triad made by Serzoo, His44” and G~u’*~ (the numbering of aminoacids is
from the Torpedo Californica enzyme) lies at the bottom. A striking feature of the
gorge is that it is lined by 14 aromatic residues which make approximately 40% of
its surface and allow the AChE active site to be qualified as hydrophobic.
Crystallographic studies carried out by Sussman and his group [31] recently
revealed the localization of various inhibitors cocrystallized with the enzyme,
pointing out the role of the indole moiety of TrpX4 as the binding counterpart of
such ligands as edrophonium and tacrine. However, despite the atomic resolution
level at which the above features are detailed, the problem of depicting or even
predicting inhibitorAChE interactions is far from being solved. The flexibility of
220 M. Recunatini et al. 1 Chemico-Biological Inteructiom 105 (1997) 199-228

the ligands in conjunction with the rather large volume available to them inside the
gorge allow incredibly high numbers of binding modes. In recent docking studies,
Pang and Kozikowski [32,33] showed the possibility of identifying few likely
binding sites out of the billions of impossible ones.
The examination of the datasets taken into consideration and of the correspon-
ding equations, actually confirms a varied spectrum of structures and different
physico-chemical properties involved in the binding of inhibitors to AChE. As
expected, from the QSARs, it did not come out an unitary and common mode of
interaction of the three classes of inhibitors with the enzyme. However, taking a
closer look at the equations, common features within each class could be revealed
and at least partially rationalized.
Datasets 1, 2, 3 and 4 represent but a small sample of the great number of
modifications performed on the structure of the natural product physostigmine [16].
This alkaloid from Calabar beans is known to carbamoylate the catalytic Ser200 of
AChE with a longer regeneration time than that displayed by the normally acylated
enzyme. The modifications of the original structure of physostigmine embodied in
datasets l-4 regard mostly the carbamic nitrogen and the ionizable function at N-l
and, unfortunately, they are not ideal for QSAR analysis. Yet, significant equations
could be calculated.
Dataset 1 provides an example of the ambiguity that originates when examining
from a QSAR point of view series of data not designed with that aim. Eq. (1) could
be satisfying, considering the good statistics and considering also that it fits the
hypothesis stated by the authors [ 171. The optimum length of ca. 18 A referring to
the whole morpholinoalkyl substituent is consistent with the approximated distance
of 12- 14 A estimated between the two anionic sites of the gorge [17]. This ‘local’
QSAR can be useful for rationalizing the behavior of the series, nevertheless,
whenever a new QSAR is calculated, one has to refer it to the frame of knowledge
constituted by the other equations already existing for the same system. Recently,
it was shown that, for a number of representative series of AChE inhibitors as
different as phosphorothioates, piperidines, trimethylammonium salts and carba-
mates, hydrophobicity played a significant role, as revealed by the presence in the
equations of either log P or n terms [lo]. In this context, one may say that Eq. (2)
is more general and consistent with other results in the same field, and it should be
preferred.
Eq. (3) accounting for the inhibitory activity of dataset 2 can now be considered.
In this dataset, structural variations occur at the ionizable N-l atom of the eseroline
nucleus. At a first glance, one might hypothesize that variations of inhibitory
activity should parallel the extent of ionization. However, pK, values of these
compounds are not known, while the estimated partition coefficient of the ionized
species (log P’) allowed us to calculate a significant equation (Eq. (3)). The negative
coefficient of the log P’ term indicates that less hydrophobic compounds are
favored, and this might be related with the dynamic process that leads the ligands
to the active site inside the gorge. It might be that more hydrophobic compounds
(even if protonated) become trapped at some peripheral binding site, instead of
reaching the catalytic site.
 M. Recanatini et al. / Chemico-Biological Interactions 105 (1997) 199-228 221

In both datasets 1 and 2, it is the hydrophobic character of the whole molecules

that plays a critical role in allowing the electrostatic contacts to be established.
Dataset 3 contains phenserine derivatives and its QSAR is represented by Eq. (4).
In this equation, the E,(6) term indicates an unfavorable effect of the second
ortho-substituent. A global hydrophobicity parameter did not enter the equation,
but a negative rc term appears for the para position on the phenyl ring. This QSAR
might be interpreted as indicating the critical role of the phenyl ring: two ortho
groups on it can hinder some positive interaction at the edge of the gorge, either by
preventing the entry of the molecule, or by distorting the ring out of the plane of
conjugation with the carbamic moiety. Para-X-substituents probably do not con-
tact the enzyme surface, but are exposed to the solvent.
Steric effects seem to govern also the QSAR for dataset 4. The most important
parameter in Eq. (5) is, in fact, the Verloop’s Bl term that appears with a large
negative coefficient. From the structure of the parent compound of this dataset, we
see that one structural characteristic of physostigmine, i.e. the methylated N-8
atom, can be missing, while, from Eq. (5), it results that the methyl group in
position 3a gives an important positive contribution to the activity (high coefficient
of the indicator variable Z-Me). No significant hydrophobic effects appear, even if
they might be masked by the steric ones.
From the QSARs represented by Eqs. (2)-(5), one may conclude that, as far as
binding to the enzyme active site is concerned, there is not much more to be gained
from modifications on the physostigmine lead compound. The relationships exa-
mined show that the electrostatic anchoring interaction is modulated by hydropho-
bic and steric effects, which are not easy to untangle, due to the characteristics of
the active site gorge. In fact, this seems to be rather narrow at the top, then getting
wider and convoluted, thus allowing many different ways for ligands to bind. The
morpholinoalkyl derivatives appear as the most interesting ones and some effort
aimed at clarifying their QSAR could be worthwhile.
Datasets 5 and 6 are structurally strictly correlated and contain analogues of the
recently marketed anticholinesterase drug tacrine [5]. It was rather surprising to
realize that, despite the emphasis cast on tacrine and its development, only these
two series of derivatives have been published, one having tacrine itself as the parent
compound and the other the 1-hydroxy-analogue, presently in Phase III clinical
trial under the name of velnacrine [34]. In both datasets, substituents are present on
the 9-amino function; in dataset 6, few substituents are also introduced in position
6.
Eq. (6) and Eq. (7) are rather unusual and difficult to understand. The first
problem is collinearity: in Eq. (6), we are not quite sure that the L parameter is
modeling purely steric effects, because it varies linearly with log P. In Eq. (7), the
risk is lower, but cannot be excluded. However, the similarity of the two equations
(calculated from data originated independently in different laboratories) and their
acceptable statistics allow one to make some consideration.
The QSARs represented by Eq. (6) and Eq. (7) consist of inverse bilinear models
in L; the highly positive contribution of a Cl atom in position 6 of the tetrahy-
droacridine ring is also pointed out for dataset 6 by the Z-Cl indicator variable.
222 M. Recanatini rt al. / Chenzico-BioloRicul Interactions 10.5 (1997) 199-228

Inverse bilinear models are rather unusual in QSAR, however, at least one case is
reported in the literature [35] and ten datasets featuring this kind of model (either
in log P, 71, CT+or MR) can be found in the Pomona College database [13].
In the present case, the models indicate that the inhibitory activity decreases as
the length of the 9-N-substituent increases, up to a value of approximately 5 A (see
the Lopt values of Eq. (6) and Eq. (7)), then it slowly increases. Considering the
current knowledge about the binding mode of tacrine, two hypotheses may be
advanced, in order to explain the common behavior of the two series of compounds.
A first hypothesis can be based on the published X-ray crystallographic structure of
the complex between tacrine and AChE [31]. Sussman et al. showed that 1,2,3,4-te-
trahydro-9-aminoacridine binds to the catalytic site of the enzyme by stacking against
the indole ring of TrpX4 and forming a hydrogen bond between its protonated ring
nitrogen and the carbonyl of His440. Assuming that the compounds of datasets 5 and
6 bind to the same site, inspection of the enzymatic space available for substituents
stemming outwards from the 9-amino group reveals that this space is rather open.
In fact, in that direction, only residues 69-72 of the protein interpose between the
tetrahydroacridine binding site and the extra-enzymatic space. Given this situation,
the inverse bilinear model might indicate that analogues bearing shorter substituents
bind easily to the tacrine site; as the 9-substituent length increases, the short portion
of protein chain gives some disturbance; then, longer groups might overcome the
barrier and protrude beyond it. Strictly speaking, the L parameter says nothing about
the nature of the binding interaction, but, given the nature of the substituents in
datasets 5 and 6, it might be hydrophobic and/or n-stacking (most analogues of
dataset 6 are N-benzyl-substituted).
This hypothesis has some drawbacks. First, analogues of 6 carrying much longer
substituents (not present in the dataset) are not as active as expected. Second, in the
case of 9-N-substituted derivatives, the position of the 9-aminoacridine nucleus might
not be the same as that of tacrine. Actually, another hypothesis able to explain the
inverse bilinear models of Eq. (6) and Eq. 7 arises just from the latter observation.
There is experimental evidence that tacrine is able to bind also at a peripheral site
[36]. Moreover, in his theoretical work, Pang [32] calculated the position of tacrine
at this peripheral site and showed that the binding occurs through stacking of the
aromatic ring of the inhibitor against the indole ring of Trp2’” and interaction of the
protonated nitrogen with Trp27” and Phe2’“. One might hypothesize that some of the
analogues of datasets 5 and 6 bind at the catalytic site, while others bind at the
peripheral site. In this way, the two branches of the bilinear models might represent
different relationships for subsets of compounds acting at different sites: analogues
bearing shorter substituents bind at one site, those bearing longer groups bind at
another. Again, though, we cannot say exactly which kind of interactions take place.
As a final comment to Eq. (6) and Eq. (7) we have to note the relevant positive
contribution of the chlorine at position 6 in the series 6, pointed out by the high
positive coefficient of the Z-Cl term in Eq. (7). Too few substituents are varied in that
position for allowing the calculation of a QSAR. The effect could be hydrophobic
(interaction with a small pocket), as well as electronic (action on the rc-electron cloud
of the aromatic ring).
 M. Recanatini et al. / Chemico-Biological Interactions 105 (1997) 199-228 223

In summary, Eq. (6) and Eq. (7) expressing the QSARs for two series of tacrine
analogues seem to suggest more questions than answers. Nonetheless, they might
constitute a first step towards the physico-chemical explanation of the mode of action
of these important inhibitors of AChE. The next step is to study carefully designed
analogues able to fill the gaps in the parameter space and provide a sound basis on
which more informative QSAR can be built.
The last class of AChE inhibitors examined can be broadly defined as that of
benzylamines. As previously noted, both N-benzyl,N-alkylamines and N-benzylpipe-
ridines are currently being studied in search of effective drugs against Alzheimer’s
disease. The Japanese compound E-2020 [37] is the most important representative
of the class.
A lot of medicinal chemistry work has been done and published on these
compounds and, among the many papers recently appearing in the literature, some
deal with the QSAR and molecular modeling of series of analogues of the class
[28,32,33,38-401. An X-ray picture of some benzylamine derivatives bound inside the
active site gorge of the enzyme is still missing, but the few theoretical studies on these
compounds allowed investigators to work out reasonable hypotheses on how they
bind to AChE. The discussion of the QSAR of benzylamines will necessarily take
into consideration these contributions.
Turning to the equations, one can see that the structure-activity models for
datasets 7-9, expressed by Eqs. (9)-( 1 l), are in good agreement. All these QSARs
contain the same parameters with quite similar coefficients. In addition, Eq. (12) for
dataset 10 contains the same electronic parameter as the previous ones, while lacking
the log P term. Actually, a significant equation in log P (Eq. (13)) can be obtained
for this dataset, but the two parameters seem to be alternatives to one another. It
might be that, increasing the parameter’s range, both effects show up in the same
equation. Indicator variables appear in Eq. (10) and Eq. (12) showing the effects of
structural characteristics not accountable for by physico-chemical parameters.
The overall picture for datasets 7- 10 is consistent in a satisfactory way and allows
one to reasonably admit that all these congeners act in the same manner against the
enzyme. The most important effect seems to be the electronic one, exerted by
substituents on the aroyl moiety of the molecules: electron-attracting groups are
favored. As regards hydrophobicity, a decrease of log P throughout the series leads
to increasing inhibitory potency. Structural factors critical for the activity are the
presence of the acetyl group on the amidic nitrogen of 8 and the presence of
para-substituents on the benzoyl ring of 10; both exert positive effects. Ortho-sub-
stituents on the same benzoyl ring decrease the activity.
The negative hydrophobic effect is the most striking feature of these QSARs.
However, we found similar negative dependence on the hydrophobic character in
dataset 2 (Eq. (3)) and dataset 3 (Eq. (4)). In order to explain this result, one should
consider the following issues. Collinearity between steric and hydrophobic effects
might play a role: as compounds become more lipophilic, they also become bulkier
and the interaction with (or the entrance into) the gorge may be difficult. Secondly,
as mentioned before, the active site gorge is a deep hydrophobic cavity, at the bottom
of which the catalytic site is hosted. It might be that hydrophobic contacts of the
224 M. Recanatini rt crl. / Chmzico-Biol~~gical Interactions 105 (1997) 199-228

inhibitor molecules with the walls of the cavity prevent or slow down the access of
the cationic moiety to its binding site. If this occurs, and the ‘ionic’ bond does not
establish, the overall interaction might be weaker and the resulting inhibitory
activity lower. Finally, two molecular modeling studies [28,33] regarding ben-
zylpiperidines interacting with AChE agree on the conclusion that probably sub-
stituents on the aroyl part of these molecules do not contact the enzyme surface,
but remain outside the gorge exposed to the solvent. This is remarkable, because,
in all the benzylamine datasets considered, structural variations occur just on that
aroyl (or aryl) moiety. As a consequence, given that changes in the log P values are
mostly due to different aroyl (or aryl) substituents, if they are all or in part solvent
exposed, their desolvation is not required and a lower log P is more favorable.
Considering now the electronic effects revealed by Eqs. (9))( 12), they indicate the
need to decrease the electron density on the aromatic ring on which substituents are
placed, in order to obtain a better inhibitory activity. The effect is relevant,
observing the high coefficients associated with the F terms. A possible explanation
of this aspect of the QSARs of benzylamines is that electron-attracting substituents
render the aromatic rings more suitable for charge-transfer interactions with some
electron-rich counterpart on the enzyme. A likely candidate for such a role might
be the indole ring of Trp17’, which is located at the entrance of the gorge [30]. This
residue was shown by Sussman et al. [31] to be able to change its position upon
binding of ligands and, moreover, theoretical calculations demonstrated that it
might be involved in the formation of the binding complex between the enzyme and
E-2020, probably via n-stacking interactions with the indanone moiety of the
inhibitor [33].
A high percentage of the variance of the inhibitory activity of series 10 is
accounted for by the indicator variables Z-P and Z-O: they suggest a positive
contribution for pcrva-substituents and a negative one for o&o-substituents. At
present, there is no explanation for the positive para effect, and, moreover, more
ortlzo-substituents would be needed in the series to confirm the ortho effect.
On the other hand, the QSAR expressed by Eq. (I 2) can be compared with the
3D-QSAR coming from a CoMFA study [38] on a series of inhibitors that includes
those of dataset 10. There is agreement between the results of the two analyses,
inasmuch as the CoMFA steric contour plots reveal that placing substituents on the
benzoyl moiety enhances the inhibitory activity, while occupying the ortho position
has a negative effect. No comparison can be made with the electrostatic results of
the CoMFA analysis, because the training set contains molecules bearing an
electronically different aryl ring.
Eq. (14) calculated for dataset 11 appears completely different from those
examined thus far for series of benzylamines. Only two MR (molecular refractivity)
terms are present in the equation, but with different sign: positive for the 4 position,
negative for the 3 position. It is commonly assumed in QSAR studies that, when
MR appears with positive sign, it indicates favorable polar interactions, while, when
the sign with it is negative, unfavorable bulk effects are indicated [15]. In line with
this assumption, we might infer that substituents in position 4 of the phthalimide
ring of 11 experience favorable polar contacts. Actually, this might be the case,
 M. Recanatini et al. / Chemico-Biological Interactions 10.5 (1997) 199-228 225

considering that they probably are all or in part exposed to the extra-enzymatic
space and that they are mostly hydrophilic. Substituents in position 3 might disturb
some positive interaction of protein residues with the carbonyl of the imide group
or make some bad steric contact themselves. It can be noted that the situation
might not be much different from that depicted by Eq. (12) for dataset 10. No
electrostatic or hydrophobic term appears in Eq. (14) which might be the conse-
quence of a small range of variation of these properties.
Eq. (15) and Eq. (16) are two poor correlations, which, as we stated above,
should not be considered QSAR at all. However, there is a trend in the variation
of activity in datasets 12 and 13, that is revealed by these equations and that cannot
be denied. It is remarkable that the inhibitory activity increases as the electron-
donating power of substituents increases: this is the opposite of the previously
discussed results regarding datasets 7-10. There are two reasons for reporting these
apparently anomalous and certainly weak relationships: first, two datasets show the
same behavior; second, the theoretical study of Villalobos et al. [28] on the
compounds of dataset 12 has proposed a binding mode for these derivatives that is
in agreement with the vague relationship revealed by Eq. (15). In their simulations,
these authors observed a hydrogen bond between the benzisoxazole oxygen and the
backbone N-H of Phe288. In Eq. (15) the negative coefficient of the D term
suggests that electron-donating groups on the benzisoxazole ring increase the
inhibitory potency of the compounds. This might be reasonably explained with the
need of increasing the electron density on the oxygen atom, in view of its
involvement as a hydrogen bond acceptor in the binding interaction with the
enzyme. Of course, Eq. (15) alone would be of no value. The agreement with
another relationship and with another independent study can make it worthy of
some attention.
As a final observation about the QSARs of the benzylamine class, it might be
pointed out that electrostatic interactions seem to play a key role in the binding
mode of these compounds. However, the presence of the log P term in the best
established relationships Eqs. (9)-(11) reminds us of the hydrophobic nature of the
active site gorge of AChE and casts some attention on the need of carefully design
the lead compounds, in order to obtain the most productive interaction with the
enzyme. It is quite remarkable that the variation of the activity term (log l/l&) in
all these series is very narrow. Given that structural changes occur on the aryl part
of the molecules, one might conclude that this moiety is not so crucial in the
binding process. What seems to be inferred from QSARs Eqs. (9)-(16) is that the
body of the molecule is critical in its entirety, while aryl substituents can just fine
tune the global inhibitor-enzyme interaction.

5. Conclusions

The examination of the quantitative models for the structure-activity relation-

ships of several series of compounds belonging to the three main classes of AChE
inhibitors allowed us to individuate the physico-chemical properties involved in the
226 M. Recanatini et al. j’Chemico-Biological 1ntrrac.tion.c 105 (1997) 199-228

inhibitory activity. These properties affect differently the single classes, as seems
reasonable, considering that the AChE active site was shown to be deep and wide
enough to permit multiple binding modes. The conclusions of this study can be
summarized as follows:
(a) hydrophobicity plays a critical role in both the physostigmine- and the
benzylamine-derived classes; hydrophobic interactions might favor the binding of
inhibitors to the cavity, but, in some cases, they can also be an obstacle to reaching
the deep anchoring site;
(b) electronic effects are important for the interactions carried out by the variable
portion of benzylamine derivatives;
(c) steric factors are also significant, but, as in other cases, collinearity with
lipophilicity does not allow one to draw a clear picture; this is particularly evident
for the acridine series and for some series of physostigmine analogues.
As stated in Section 1, the comparative QSAR analysis presented here is an
attempt at organizing in a consistent way the knowledge available about com-
pounds able to inhibit AChE. This is an effort of rationalization of known data
that might provide a starting point for the design of new and more potent AChE
inhibitors. The QSAR methodology can greatly help this effort, being firmly
founded on physico-chemical and statistical bases. In rational drug design, these are
requirements that must be met even more strictly in fields like that of the agents for
the treatment of AD, where reliable data and careful use of the resources are
indispensable.

Acknowledgements

Investigation supported by University of Bologna (funds for selected research

topics).

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