Synthesis, biological activity and HPLC validation of 1,2,3,4- tetrahydroacridine derivatives as acetylcholinesterase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2011.04.038

The synthesis and biochemical evaluation of new hybrids of tacrine (THA) and 4-fluorobenzoic acid (4-FBA) possessing activity towards acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibition are presented. The compounds of interest were ob

Full text of DOI:10.1016/j.ejmech.2011.04.038

European Journal of Medicinal Chemistry 46 (2011) 3250e3257
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, biological activity and HPLC validation of 1,2,3,4-tetrahydroacridine
derivatives as acetylcholinesterase inhibitors
,
b
a
Pawe1 Szymanski a , Adam Karpinski , Elzbieta Mikiciuk-Olasik
*
ꢀ
a
ꢀ
Medical University, Department of Pharmaceutical Chemistry and Drug Analyses, ul. Muszynskiego 1, 90-151 Lodz, Poland
b Gerard Laboratories a Mylan Inc., Quality Control Department, Baldoye Industrial Estate, Unit 80, Dublin 13, Co. Dublin, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and biochemical evaluation of new hybrids of tacrine (THA) and 4-fluorobenzoic acid
(4-FBA) possessing activity towards acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE)
inhibition are presented. The compounds of interest were obtained from the reaction of activated 4-FBA
and diamino derivatives of 1,2,3,4-tetrahydroacridine. The compounds C6-2KW/HCl, C6-4KW/HCl and
C6-3KW/HCl have four-fold higher antiacetylcholinesterase activity than THA. All of the acquired
compounds present higher selectivity towards AChE than THA and lower selectivity towards BuChE. In
addition, a rapid, selective and stability-indicating HPLC method was developed and validated for the
determination of C6-2KW/HCl, C6-3KW/HCl and C6-4KW/HCl. THA and 4-FBA were found to be the main
Received 31 January 2011
Received in revised form
7 April 2011
Accepted 12 April 2011
Available online 22 April 2011
Keywords:
Tacrine
impurities. Chromatographic separation was achieved isocratically on
a Waters Symmetry C18
Alzheimer’s disease (AD)
Acetylcholinesterase
Butyrylcholinesterase
HPLC validation
4-Fluoroacetic acid
150 ꢀ 3.9 mm, 4
m
m column with a mobile phase of acetonitrile/buffer (17 mM sodium dodecyl sulphate
and 8.3 mM sodium dihydrogen phosphate, 50:50 v/v) (overall pH 4). A 1.5 ml/min flow rate and
a 247 nm wavelength were chosen for this method. C6-2KW/HCl, C6-3KW/HCl and C6-4KW/HCl were
subjected to acidic and basic hydrolysis, chemical oxidation, thermal exposition at 60 ꢁC and intense UV
light. The limits of detection (LOD) and quantification (LOQ) were less than 2
C6-2KW/HCl, C6-3KW/HCl and C6-4KW/HCl, 0.04 g/ml and 0.12 g/ml for THA, 0.42
1.41 g/ml for 4-FBA, respectively.
m
g/ml and 6
m
g/ml for
m
m
mg/ml and
m
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
One of the most popular therapeutic strategies in AD is the
control of cholinergic neurotransmission by the slow decline of
neuronal degeneration or increasing cholinergic transmission [6].
The reduction in the activity of cholinergic neurons have used
acetylcholinesterase inhibitors decreases the rate at which acetyl-
choline (ACh) is broken down, thereby increasing the concentration
of ACh in the brain. Cholinesterase inhibitors have been approved
as an efficacious treatment to reduce the symptoms of early-
medium stages of AD. Well known commercial ACh inhibitors
like tacrine, donepezil, rivastigmine and galantamine provide some
evidence for their applicability in the advanced stages [7e9].
Current research is going towards new types of compounds,
modelled by molecular modelling to estimate structure and inhi-
bition activity towards AChE/BuChE [10e12].
Alzheimer’s disease (AD) is a degenerative disease of the central
nervous system, characterised by a noticeable cognitive decline and
defined by a loss of memory and learning ability and a reduced
ability to perform the basic activities of daily living. The diagnosis of
AD is very difficult and still under development [1,2] or in clinical
studies [3,4]. The testing of intellectual functions to determine the
degree of cognitive status during medical examination is useful as
a supplementary method of diagnosing dementia. Incorporating
nuclear medicine into the diagnosis of dementia may not only lead
to improvements in the quality of medical examinations but could
also help to improve our understanding of the roots and develop-
ment of the disease. To investigate drug delivery and define the
mechanism of action, some atoms in a pharmaceutical ingredient
can be replaced by their radioisotopes (e.g., 18F) and then investi-
gated in the body [5].
Gonzales-Munoz and co-workers [13] presented a series of
N-Acylaminophenothiazines as a potential BuChE inhibitors. Simi-
lar activity was observed by Tasso [14] with quinolizidinyl deriva-
tives of bi- and tricyclic structures.
Gholivand [15] provide interesting studies about new phos-
phorus(V) hydrazides. Synthesised compounds have not only some
antibacterial activity but are able to slow down AChE and BuChE
(mainly) activity measured by Ellman’s method [16].
* Corresponding author. Tel./fax: þ48 42 677 92 50.
E-mail addresses: pawel.szymanski@umed.lodz.pl (P. Szymanski), adas_chem@
ꢀ
yahoo.pl (A. Karpinski).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.04.038

P. Szymanski et al. / European Journal of Medicinal Chemistry 46 (2011) 3250e3257
3251
NH2
(CH2)n
n= 2, 3, 4
O
HN
Cl
OH
a
b
NH2
N
N
1
2a-2c
Scheme 1. Synthesis of compounds 1, 2ae2c. Reagents: (a) cyclohexanone, POCl3, reflux; (b) 1,
u
-diamine, phenol, NaI, reflux.
2a, 3a n=2
2b, 3b n=3
2c,
n=4
O
O
3c
HN
(CH2)n
NH2
HN
(CH2)n
(CH2)n
F
HN
HN
N
F
HN
N
HCl
a
b
*
N
C6-2KW/HCl n=2
C6-3KW/HCl n=3
C6-4KW/HCl n=4
2a-2c
3a-3c
Scheme 2. Synthesis of final compounds. Reagents: (a) 4-fluorobenzoic acid, CDMT, methylmorpholine, THF; (b) HCl/ether.
However, there is intensive research towards a new type of drug
based on tacrine, which was registered by the American FDA as the
first drug in AD treatment. The therapeutic effects of tacrine for the
treatment of cognitive defects in Alzheimer’s disease (AD) were
reported in 1986 [17]. Newly designed drugs must eliminate tacrine
side effects like hepatotoxicity and cardiovascular system impair-
ment and also increase oral bioavailability.
The THA derivatives as AChE/BuChE inhibitors were often
developed. In 2004 Frideling [18] and co-workers synthesised
chiral THA derivatives from 3-methylcyclohexananone, menthone,
pulegone, carvone and dihydrocarvone. Pisoni [19] proposed
enentioselective synthesis of THA derivatives where inhibition
Germany) and a UV lamp (254 nm). The melting points were
measured on an electrothermal apparatus with open capillaries.
Anhydrous sodium sulphate (SigmaeAldrich GmbH, Steinheim,
Germany) was used to dry organic solutions during the work-up,
and the removal of solvents was carried out under vacuum with
a rotary evaporator. Column chromatography was performed using
silica-gel 60 (230e400 mesh, Merck KGaA, Damstad, Germany).
DTNB, both enzymes (C2629 and C4290) and acetylthiocholine
iodide were purchased from SigmaeAldrich GmbH, Steinheim,
Germany for biological-activity calculation.
Samples of C6-2KW/HCl (4-fluoro-N-[2-(1,2,3,4-tetrahydroa-
cridin-9-ylamino)ethyl]-benzamide hydrochloride), C6-3KW/HCl
(4-fluoro-N-[3-(1,2,3,4-tetrahydroacridin-9-ylamino)propyl]-benza-
mide hydrochloride), and C6-4KW/HCl (4-fluoro-N-[4-(1,2,3,4-tet-
rahydroacridin-9-ylamino)butyl]-benzamide hydrochloride) were
synthesised in-house (see Supplementary data, [23]). In addition
compounds were purified for validation purposes using micro scale
Dry Column Chromatography (DCC) [25].
activity has been checked (IC50 ¼ 0.06e7.22
mM).
The aim of this work was the synthesis of compounds tested by
molecular-modelling techniques, the calculation of biological
activity and the development and validation of a rapid reversed-
phase HPLC-UV method that will be useful for further early-stage
pharmaceutical testing.
Previously developed HPLC methods for tacrine, derivatives and
its metabolites have used fluorescence detection with a polar
stationary phases at a LOD of 0.25 ng/ml to 5.95 ng/ml [20e22].
HPLC-grade water and sodium dihydrogen phosphate
(SigmaeAldrich GmbH, Steinheim, Germany), HPLC-grade aceto-
nitrile (Merck KGaA, Damstad, Germany) and sodium dodecyl
sulphate (Romil LTD, Cambridge, UK) were used.
2. Experimental
2.2. Synthesis
2.1. Materials
The first step of the synthesis (Scheme 1) involves the reaction
between anthranilic acid and cyclohexanone in freshly prepared
1,2,3,4-Tetrahydroacridin-9-amine trihydrate (Tacrine, 99%) and
4-fluorobenzoic acid (>99%) were received from SigmaeAldrich
GmbH (Steinheim, Germany). The reactions were monitored by TLC
using 25 DC-Alufolien Kieselgel 60F254 (Merck KGaA, Damstad,
Table 2
IC50 values for activities on acetylcholinesterase and butyrylcholinesterase.
Compounds
AChE inhibition
IC50
BChE inhibition
IC50
Selectivity
for AChEa
Selectivity
for BChEb
Table 1
,
m
M
, mM
Statistical parameters value of Km and Vmax for AChE and BChE.
C6-2KW/HCl
C6-3KW/HCl
C6-4KW/HCl
THA
8.15 ꢀ 10ꢂ4
8.09 ꢀ 10ꢂ4
7.84 ꢀ 10ꢂ4
3.16 ꢀ 10ꢂ3
4.49 ꢀ 10ꢂ5
2.12 ꢀ 10ꢂ5
3.54 ꢀ 10ꢂ5
2.67 ꢀ 10ꢂ5
0.047
0.033
0.047
0.008
21.27
30.72
21.17
Parameters
AChE
BChE
Km
0.092683
m
M
0.100176
2.526429
0.9858
m
m
M
Vmax
2.29645
0.9954
0.098
m
M/min/ml
M/min/ml
118.63
r2
a
Selectivity for AChE is defined as IC50(BChE)/IC50(AChE).
Selectivity for BChE is defined as IC50(AChE)/IC50(BChE).
Standard error
0.021
b

3252
P. Szymanski et al. / European Journal of Medicinal Chemistry 46 (2011) 3250e3257
Fig. 1. Separation of tested compounds: Waters Symmetry C18, 250 mm ꢀ 4.6 mm,
m, 30 ꢁC, mobile phase: 62.5e37.5% (buffer*-ACN, v/v, *17 mM SDS and 8.3 mM
Fig. 3. A typical chromatogram of the separation of 4-FBA, tacrine and C6-2KW/HCl.
4
m
NaH2PO4) (overall pH 4).
2.3. Biological activity
POCl3. A similar reaction was reported by M.K. Hu and C.F. Lu [26].
The efficiency of this reaction was about 54%. The obtained
compound, 1, was then combined with two equivalents of the
Ellman’s spectrophotometrical method [16] was used with some
modifications to determine the activity of the newly synthesised
acetylcholinesterase inhibitors. Measurements were conducted at
37 ꢁC in phosphate-buffered solution (0.1 M, pH 8.0). The reaction
mixture in a final volume of 3 ml contained the AChE enzyme
(5 units/ml) and 5,50-dithiobisnitrobenzoic acid (DTNB, 0.05 ml,
0.5 M), which is a chromogen and an adequate inhibitor. Ace-
tylthiocholine iodide was used as a substrate. The inhibition curves
for every derivative were made with seven concentrations of ace-
tylthiocholine iodide. The activity of the acquired derivatives was
marked by measuring the absorbance at 412 nm. The measurement
was taken after 1 min of incubation of the reaction mixture. Each
measurement was made in triplicate. To acquire all of the AChE,
activity tests without inhibitors were always done.
The activity towards the inhibition of butyrylcholinesterase
(BuChE) was determined in the same way as that for AChE. In these
experiments, BuChE was used in the same concentration as AChE
(5 units/ml).
Non-linear and linear regressions were used to estimate the
drug concentration inducing 50% inhibition of the AChE or BuChE
activity.
appropriate 1,u-diamine. This reaction was accompanied by the
addition of sodium iodide (catalyst) in the presence of phenol at
180 ꢁC. The yields of this reaction were: 91% (2a), 88% (2b) and 72%
(3c). The generated compounds were subsequently used as
substrates for further synthesis (Scheme 2). The synthesis of
compounds 3ae3c have involved the activation of 4-fluorobenzoic
acid by stoichiometric amounts of 2-chloro-4,6-dimethyl-1,3,5-
triazine (CMDT) and N-methylmorpholine at low temperature [27].
We report that the results of this reaction stage are significantly
better when N-methylmorpholine is added dropwise into the
solution in an adequate solvent, such as dichloromethane, aceto-
nitrile, dioxane or tetrahydrofuran, at ꢂ5 ꢁC. The progress of this
reaction was verified by TLC. After approximately 2 h, a solution of
compounds 2ae2c in the proper solvent was added to the activated
4-fluorobenzoic acid. The final step of the synthesis was conducted
in the presence of hydrochloric acid in ether to produce the
hydrochloride form of the products (C6-2KW/HCl (3a), C6-3KW/
HCl (3b) and C6-4KW/HCl (3c)).
2.4. Apparatus
IR spectra were recorded in KBr using a Mattson Infinity Series
FT-IR spectrophotometer. 1H NMR spectra were recorded with
a Varian Mercury 300 MHz spectrometer using tetramethylsilane
as an internal standard. Mass spectra and elemental analyses were
Fig. 2. UV-absorption spectra of 4-FBA, THA and derivatives from 200 nm to 400 nm.
Concentration: C6-2KW/HCl, C6-3KW/HCl and C6-4KW/HCl at 5
ml and THA at 2.5 g/ml.
mg/ml, 4-FBA at 10 mg/
m
Fig. 4. A typical chromatogram of the separation of 4-FBA, tacrine and C6-3KW/HCl.

P. Szymanski et al. / European Journal of Medicinal Chemistry 46 (2011) 3250e3257
3253
Table 4
Method precision summary (intra and inter-assay).
Compound
RSD (%) Method precision
Intermediate precision (%)
4-FBA
THA
C6-2KW/HCl
C6-3KW/HCl
C6-4KW/HCl
0.90
0.48
0.56
0.41
0.62
0.91
0.51
0.65
0.72
0.39
2.7. Method validation
The analytical-method validation was carried out as per the ICH
Q2(R1) method-validation guidelines [24]. The following validation
parameters were addressed: selectivity, precision, linearity, accu-
racy, limit of detection, limit of quantification, robustness and the
stability of the tested compounds in diluent.
Fig. 5. A typical chromatogram of the separation of 4-FBA, tacrine and C6-4KW/HCl.
performed by the Centre of Molecular and Macromolecular Studies
in Lodz (Polish Academy of Sciences).
3. Results and discussion
The HPLC instrument consisted of a Waters 2695 Alliance
separation module with a Waters 2487 VWD (variable-wavelength
detector) and a Waters 2996 PDA detector (photodiode-array
detector) (Waters Corp., Milford, USA). However, an Agilent 1200
series with a VWD detector (Agilent Technologies, Waldbronn,
Germany) was used throughout the validation process. The soft-
ware used for data collection, integration and computation was
Millennium 32 (Waters) and Chromatography Data System (CDS)
Software (Agilent). A reversed phase Symmetry C18 column
3.1. Biochemical studies
The spectrophotometrical method pioneered by Ellman was
used to determine the inhibitory activity of the synthesised
compounds towards acetylcholinesterase (AChE) and butyr-
ylcholinesterase (BuChE) [16]. The inhibition type was justified by
the linear LineweavereBurk equation. The constant parameters Km
and Vmax were evaluated by the linear regression of the reaction
rate versus the substrate concentration. A non-linear regression
was used to estimate the apparent Ki constants. In Table 1, the
statistical parameters and values of Km and Vmax are presented for
both AChE and BuChE enzymes. Table 2 shows the acquired data of
the inhibitory activities of C6-2KW/HCl, C6-3KW/HCl and C6-4KW/
HCl towards AChE and BuChE. Newly synthesised constituents
C6-2KW/HCl, C6-3KW/HCl and C6-4KW/HCl, in comparison with
tacrine, possess 4-fold higher inhibitory activity towards AChE
(3.88, 3.91, 4.03 respectively). For all of the obtained compounds,
slightly higher IC50 values were recorded for BuChE than for tacrine.
Our structural modifications focused on the amino group of tacrine:
small aliphatic chain was inserted and coupling with 4-fluo-
robenzoic acid to assess the importance change in this part of
molecules. Compounds C6-2KW/HCl, C6-3KW/HCl and C6-4KW/
HCl showed a remarkable increase in activity, underlying the
importance of the longer alifatic chain. This shows that it was good
way to introduce this alteration.
(150 mm ꢀ 3.9 mm i.d.; particle size 4
mm) (Waters, USA) was used
for the separation.
2.5. Chromatographic conditions
The mobile phase consisted of acetonitrile and buffer (50:50,
v/v). The buffer consisted of 17 mM SDS and 8.3 mM sodium
dihydrogen phosphate. The overall pH was set to 4 ꢃ 0.2 with
phosphoric acid. Before injection into the system, mobile phase was
filtered through a 0.45 mm filter and degassed under vacuum. The
analysis was carried out under isocratic conditions using a flow rate
of 1.5 ml/min at ambient temperature with an injection volume of
10 ml. Chromatograms were recorded at 247 nm. A mixture of the
acetonitrile and water (50:50, v/v) was used as the diluent.
The PDA detector was used during the validation procedure.
2.6. Preparation of solutions
Furthermore, we estimated the selectivity of the inhibitory
effects of all received components and tacrine towards acetylcho-
linesterase (ratio of IC50 BuChE/AChE) and butyrylcholinesterase
(ratio of IC50 AChE/BuChE). All synthesised compounds display
higher selectivity towards AChE than tacrine (between 4.13 and
5.88-fold more selective). For the inhibition of BuChE, tacrine pre-
sented the highest selectivity than AChE. Generally it was shown
that after coupling tacrine with fluorobenzoic acid, we obtained
compounds more selective to BuChE. This is very important
because it is know that in Alzheimer Disease we can observe higher
A stock solution of 0.5 mg/ml was prepared by dissolving the
appropriate amount of the sample (C6-2KW/HCl, C6-3KW/HCl, or
C6-4KW/HCl) in the diluent. A working solution of 0.05 mg/ml was
prepared from the stock solution for the related-substance and
assay determination. Stock solutions of THA at 0.1 mg/ml and 4-FBA
at 1 mg/ml were also prepared in the diluent. Working impurity
solutions were prepared by 1:10 dilution in diluent for the related-
substance determination. Appropriate dilutions of stock solutions
were used for linearity studies and assay purposes.
Table 3
System precision summary.
Compound
RSD(%) Day 1
RSD(%) Day 2
RSD(%) Day 10
RSD(%) Separate HPLC
Theoretical plates (average)
Asymmetry (average)
4-FBA
THA
C6-2KW/HCl
C6-3KW/HCl
C6-4KW/HCl
0.08
0.27
0.05
0.11
0.13
0.21
0.22
0.06
0.09
0.10
0.14
0.15
0.09
0.10
0.15
0.11
0.31
0.09
0.01
0.23
1482
4217
5994
6030
5810
1.3
1.3
1.3
1.2
1.2

3254
P. Szymanski et al. / European Journal of Medicinal Chemistry 46 (2011) 3250e3257
Table 5
Linearity results.
Compound
Range
mg/ml
R2
Slope
CI of the slope
Intercept
LOD
mg/ml
LOQ mg/ml
C6-2KW/HCl
C6-3KW/HCl
C6-4KW/HCl
THA
5e200
5e200
5e200
0.05e10
0.5e100
0.99995
0.99999
0.99999
0.99999
0.99999
28380
17918
15296
44412
3730
277
76
11
174
6
ꢂ21414
ꢂ8429
ꢂ8026
153
1.96
0.84
0.81
0.04
0.42
5.94
2.55
2.45
0.12
1.41
4-FBA
568
Table 8
Table 6
Recovery of 4-FBA and tacrine in C6-3KW/HCl solution.
Summary for recovery of tacrine derivatives in diluent.
Impurity at 20%
Spiking
Impurity at 100%
Spiking
Impurity at 200%
Spiking
C6-2KW/HCl (%)
C6-3KW/HCl (%)
C6-4KW/HCl (%)
80%
100%
120%
101.46 ꢃ 0.46
100.39 ꢃ 0.43
100.82 ꢃ 0.92
99.41 ꢃ 0.95
99.40 ꢃ 0.61
99.02 ꢃ 0.93
99.15 ꢃ 0.57
99.03 ꢃ 0.39
98.71 ꢃ 1.04
Sample
4-FBA
THA
4-FBA
THA
4-FBA
THA
1
2
3
102.12
100.98
100.90
108.81
108.77
108.98
100.89
101.86
100.89
101.45
101.74
101.75
98.07
97.63
98.05
99.48
99.82
99.98
For each level samples were prepared in triplicate, and presented as average of
content.
Mean
SD
R.S.D. (%)
101.33
0.68
0.67
108.85
0.11
0.10
101.21
0.56
0.55
101.65
0.17
0.16
97.92
0.25
0.25
99.76
0.26
0.26
level of this enzyme in central nervous system. We can claim that
this tag of these compounds could be strong advantage as a new
potential AChEI’s.
diluent and scanned by a UV spectrophotometer in the range of
200e400 nm. The UV spectra of five substances are illustrated on
Fig. 2.
3.2. Validation and method development
The maximum absorbance for the three derivative substances
appears at 247 nm compared to the tacrine maximum at 240 nm.
However, the response of tacrine is twice as high as that of the
derivatives. The maximum absorbance of 4-FBA appears at 229 nm,
and it produces a four-fold smaller absorbance at 247 nm. However,
it was concluded that 247 nm is the most appropriate wavelength
for analysing the tested compounds with suitable sensitivity
despite the smaller absorbance for 4-FBA. The linearity shows that
During method development, a non-buffered mobile phase was
investigated in gradient mode, but both the separation and elution
time were unsatisfactory. Instead, a mobile phase consisting of
a SDS-buffer and acetonitrile switch C18 column was chosen. This
procedure improved the separation between base compounds via
octadecyl chains and the alkyl “bridge” of tacrine derivatives. The
working pH of the mobile phase was determined using the esti-
mated pKas from Marvin Software (ChemAxon) for all tested
compounds. The mobile phase was prepared at a pH in the range of
3e4.2 by the addition of sodium dihydrogen phosphate and
adjusted to 4 ꢃ 0.2 with phosphoric acid. The use of this mobile
phase led to a successful separation. Due to economical and prac-
tical considerations, a linear method was developed that is able to
separate impurities (synthesis substrates) from tacrine derivatives.
During the method-development process, a separation test of all
tacrine derivatives performed, including the optimisation of
the mobile-phase composition and temperature with a Waters
this method is linear in range from 0.5 to 100 mg/ml.
3.4. Selectivity
The HPLC chromatograms recorded for the mixture of C6-2KW/
HCl (C6-3KW/HCl or C6-4KW/HCl), 4-FBA and THA (Figs. 3e5)
clearly show that the tacrine derivatives are fully separated from
the related compounds. The resolution factor obtained for 4-FBA
and THA was 10.4, and the resolution factors for THA and the
derivatives were 15.1 for C6-2KW/HCl, 14.6 for C6-3KW/HCl and
12.8 for C6-4KW/HCl. In this study, a PDA detector was used to
confirm that the tacrine-derivative peaks are free from other
co-eluting substances. It was concluded that the developed method
is selective to impurities, mobile phase and diluent.
Symmetry column (250 mm ꢀ 4.6 mm, C18, 4
mm). However, due to
its lengthy run time, this method has little practical application
(Fig. 1).
3.3. Determination of a suitable wavelength
3.5. Precision and repeatability
Prior to the chromatographic-method development, the detec-
tion wavelength was determined by obtaining UV spectra of 4-FBA,
tacrine and tested compounds solutions. Samples were dissolved in
The RSD (%) for the system precision was lower than 1% for
all tests, which complies with the proposed acceptance criteria (NMT
Table 7
Table 9
Recovery of 4-FBA and tacrine in C6-2KW/HCl solution.
Recovery of 4-FBA and tacrine in C6-4KW/HCl solution.
Impurity at 20%
Spiking
Impurity at 100%
Spiking
Impurity at 200%
Spiking
Impurity at 20%
Spiking
Impurity at 100%
Spiking
Impurity at 200%
Spiking
Sample
4-FBA
THA
4-FBA
THA
4-FBA
THA
97.97
97.93
Sample
4-FBA
THA
4-FBA
THA
4-FBA
THA
1
2
3
98.51
97.65
97.83
94.52
91.67
91.62
99.88
99.80
101.44
96.79
96.72
96.50
100.42
100.53
100.74
1
2
3
104.30
103.00
104.82
103.79
101.85
103.23
103.24
102.78
102.48
102.70
102.19
101.50
98.93
99.50
99.61
102.24
103.34
103.33
100.06
Mean
SD
R.S.D. (%)
97.99
0.45
0.46
92.60
1.66
1.80
100.37
0.92
0.92
96.67
0.15
0.16
100.56
0.16
0.16
98.65
1.22
1.24
Mean
SD
R.S.D. (%)
104.04
0.94
0.90
102.96
1.00
0.97
102.83
0.38
0.37
102.13
0.60
0.59
99.34
0.37
0.37
102.97
0.63
0.61

P. Szymanski et al. / European Journal of Medicinal Chemistry 46 (2011) 3250e3257
3255
Table 10
Summary of forced degradation results for C6-2KW/HCl.
Stress conditions
Time
% Assay of active substance
Remarks
Acid hydrolysis (0.5 M HCl at RT)
Base hydrolysis (0.5 M NaOH at RT)
Oxidation (3% H2O2 at RT)
Thermal 60 ꢁC
48 h
48 h
48 h
8 h
101.1%
99.7%
100.3%
100.5%
97.8%
No degradation products formed (total unknown less than 0.1%)
No degradation products formed (total unknown less than 0.1%)
No degradation products formed (total unknown less than 0.1%)
No degradation products formed (total unknown less than 0.1%)
No degradation products formed
UV (254 nm)
8 h
Table 11
Summary of forced degradation results for C6-3KW/HCl.
Stress conditions
Time
% Assay of active substance
Remarks
Acid hydrolysis (0.5 M HCl at RT)
Base hydrolysis (0.5 M NaOH at RT)
Oxidation (3% H2O2 at RT)
48 h
48 h
48 h
8 h
99.4%
99.8%
100.3%
100.8%
100.5%
No degradation products formed (total unknown less than 0.1%)
No degradation products formed (total unknown less than 0.2%)
Some unknown degradation products were formed (total unknown about 0.3%)
No degradation products formed (total unknown less than 0.1%)
No degradation products formed (total unknown less than 0.1%)
Thermal 6 0 ꢁ
UV (254 nm)
C
8 h
Table 12
Summary of forced degradation results for C6-4KW/HCl.
Stress conditions
Time
% Assay of active substance
Remarks
Acid hydrolysis (0.5 M HCl at RT)
Base hydrolysis (0.5 M NaOH at RT)
Oxidation (3% H2O2 at RT)
Thermal 60 ꢁC
48 h
48 h
48 h
8 h
99.5%
98.1%
94.7%
99.1%
102.1%
Some unknown degradation products were formed (total unknown about 0.4%)
Some unknown degradation products were formed (total unknown about 0.3%)
Some unknown degradation products were formed (total unknown about 0.5%)
Some unknown degradation products were formed (total unknown about 0.3%)
No degradation products formed (total unknown less than 0.1%)
UV (254 nm)
8 h
2%). The method and intermediate have a relative standard deviation
(RSD) below 1%, which also complies with the proposed criteria.
coefficients greater than 0.999. Table 3 lists the linearity parame-
ters of the calibration curves for tacrine and tacrine derivatives.
Each point in the regression is the mean of three experiments.
The linear equations given in the linearity results were used to
calculate the limit of detection (LOD) and the limit of quantification
(LOQ). The LOD and LOQ are also given in this table and were found
to be lower than 2 mg/ml for LOD and less than 6 mg/ml for LOQ of
the tested tacrine derivatives.
3.6. Stability of solutions
The standard solutions of the active samples and the impurities
and a standard solution prepared from old standard stock were run
on Day 1, Day 2 and Day 10. The standard stock solution and the
working-standard solution were determined to be stable for up to
ten days. There was no response difference between Day 1 and Day
10, and no degradation products were detected.
3.8. Accuracy (% recovery)
The accuracy was determined by the recovery of the tacrine
derivatives prepared at 80%, 100% and 120% of the working stan-
dard in triplicate. The recovery was found to be within 98e102% for
all levels, which meets the proposed criteria. The summary results
are presented in Table 4.
3.7. Linearity, LOD and LOQ
The calibration curves for 4-FBA, THA, C6-2KW/HCl, C6-3KW/
HCl and C6-4KW/HCl were found to be linear with correlation
Fig. 6. Representative chromatography for C6-2KW/HCl at 0.5 mg/ml for the oxidation
Fig. 7. Representative chromatography for C6-3KW/HCl at 0.5 mg/ml for the base test
test with 3% H2O2. At 0.7 min, the blank peak is visible.
with NaOH.

3256
P. Szymanski et al. / European Journal of Medicinal Chemistry 46 (2011) 3250e3257
Fig. 8. Representative chromatography for C6-4KW/HCl at 0.5 mg/ml for the acid test with HCl.
To determine the accuracy of the related-substance method,
HCl, NaOH, H2O2, heat and UV lead to major degradation of tacrine
derivatives. Tacrine and 4-FBA impurities were not observed during
stress studies. Representative chromatograms of stress studies are
presented on Figs. 6e8.
Photodiode-array investigations of the active peak after stress
testing confirm the active peak to be pure, demonstrating the
stability-indicating nature of the method over the full spectrum of
stress testing. The total recovery of the active substance was
between 94.7% and 102.1%.
sample solutions were spiked at three different concentrations:
20%, 100% and 200% of the working-standard concentration. Trip-
licate samples were prepared and analysed. The results shown in
Tables 5e7, are expressed as percent recoveries of the particular
sample components. All of the results were within specification:
the recovery at the 20% level should be within 85e115% and that at
the 100% and 200% levels should be within 90e110% (Tables 8e12).
3.9. Robustness
The method robustness checked after deliberate alterations of
the mobile-phase composition, buffer composition, pH and flow
shows that slight changes in the operational parameters do not lead
to fundamental changes of the performance of the chromato-
graphic system within the specified limits. The tailing factor for
C6-2KW/HCl, C6-3KW/HCl, C6-4KW/HCl and Tacrine always
ranged from 1 to 1.4, and the components were well separated
under all of the changes performed. The percent recoveries of the
tested compounds were good under most conditions and did not
show a significant change when the critical parameters were
modified. Considering the results of the modifications of the
system-suitability parameters and the specificity of the method, it
is concluded that the method conditions are robust.
4. Conclusions
Treatment of AD, the most common form of dementia among
the elderly, still remains a challenge for scientists and doctors. In
the recent decades, there has been an accelerating general effort to
determine the risk factors and causes of AD development. Studies
have also been conducted to find better ways of treating this illness
and delaying its onset.
AChE inhibitors like tacrine, donepezil, galantamine and riva-
stigmine are established drugs in the treatment of AD. These
medicines are commonly approved for mild- to moderate-AD
treatment. However, the use of these drugs is frequently limited
because of their adverse effects. Thus, there is still a need to search
for new compounds with AChE inhibitory activity. A drug design
allowed us to synthesise a new series of AChEIs as analogues of
tacrine.
3.10. Degradation study
For the acid hydrolysis, 0.2 ml of 1 M HCl per 5 ml of test solution
was added and kept in ambient temperature for 48 h. After this
time, the relevant volume of 1 M NaOH was added to neutralise the
solution. The alkali hydrolysis used the same procedure with 1 M
NaOH used for the degradation procedure for 48 h and 1 M HCl
used for neutralisation. For the oxidation test, 0.2 ml of 5% H2O2 was
added for each 5 ml of test solution, kept for 48 h and injected into
the HPLC. For the UV-light degradation, a 0.5 mg/ml solution was
prepared and exposed for 8 h to intense UV light (254 nm). For the
thermal degradation, 0.5 mg/ml test solutions were kept for 8 h in
a 60 ꢁC water batch, cooled and injected into the HPLC system. In
each case, a blank solution was prepared as per the degradation
test.
All of the chromatograms of the degradation studies were
analysed against chromatograms of fresh solutions and relevant
blank solutions. All peaks from the fresh solutions were identified
with the difference checked and calculated. In all base and H2O2
solutions, the blank peak at 0.7 min was visible. For the presented
degradation studies, there is no evidence that conditions such as
The acquired compounds C6-2KW/HCl, C6-3KW/HCl and C6-
4KW/HCl are characterised by 4-fold higher activity towards
AChE inhibition than the reference tacrine compound. Further-
more, all obtained components demonstrate better selectivity for
AChE than does tacrine. The different structural modifications
carried out on tacrine especially on amino group were able to
improve the AChE inhibitory potency of the new derivatives. This
finding is very significant, especially in the context of AD diagnosis,
because this illness is associated with AChE deficits.
An isocratic RP-HPLC method has been developed and validated
for the purity evaluation and the determination of tacrine deriva-
tives. The developed method is selective, sensitive, precise and
stability indicating. The method is also capable of detecting THA
and 4-FBA at trace levels.
Synthesised in a simple and affordable way compounds shows
a
fairly good inhibitory activity. We have elaborated HPLC
method for this compounds too, and that is way they could be
considered as a new lead for further optimisation as a new
potential AChEI.

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