Original Article
Zergiebel Stephanie, Seeling Andreas. In vitro Studies
In Vitro Studies on Physiological and Chemical Stability of New
LE404-Derivatives with Extended Half-Life
Authors
Stephanie Zergiebel, Andreas Seeling
Affiliation
Institute of Pharmacy, Friedrich-Schiller University Jena,
Jena, Germany
Key words
azecines, antipsychotic drugs, prodrugs, octanol/
water-partition coefficients, esterase cleavage
received 04.01.2018
accepted 31.01.2018
Bibliography
DOI  https://doi.org/10.1055/s-0044-102096
Published online: 2018
Drug Res
© Georg Thieme Verlag KG Stuttgart · New York
ISSN 2194-9379
Correspondence
PD Dr. Andreas Seeling
Institute of Pharmacy
Friedrich-Schiller University Jena
Philosophenweg 14
07743 Jena
Introduction
Dibenzoazecines represent a class of high-affinity dopamine and
serotonin receptor antagonists [1, 2].
The neuroleptic potency of the substance LE404 was proved by
in vivo tests [3]. It has been shown that the in vivo tested azecines
in their efficacy are comparable to haloperidol and risperidone, but
the therapeutic index in most cases is larger. However, a very rapid
decrease in the effect of LE404 was published [3] (maximum effi-
ciency after 30 min). To solve this problem, derivatives of the
hexahydrodibenzo[d;g]acezine have been synthesized in further
work. Some of the substances have already been tested for effec-
tiveness and duration of action in vivo [4]. A significant inhibition
of the conditioned avoidance response (CAR) could be demonstrat-
ed up to 90 min after substance application by the use of an ester
prodrug. Based on this finding, further ester prodrugs as well as
structurally different LE404 derivatives were synthesized [5]. There
exist no pharmacokinetic data for the new substances. In order to
distinguish potential drugs from non-administrable or inadequate-
Zergiebel S, Seeling A. In Vitro Stability of LE404-Derivatives …  Drug Res
Thieme
on …  Drug Res 2018; 00: 00–00
Germany
Tel.:  + 49/641/9 49814, Fax:  + 49/3641/9 49802
b8sean@rz.uni-jena.de
Abs tr ac t
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Dibenzoazecines are a class of potential neuroleptics with high
affinity to dopamine and serotonin receptors. The efficacy
and high therapeutic range has already been demonstrated
in vivo with the lead structure 7-methyl-5,6,7,8,9,14-
hexahydrodibenzo[d,g]azecin-3-ol (LE404) and selected de-
rivatives. There is a variety of new synthesized structurally dif-
ferent dibenzoazecine derivatives with the aim to improve
pharmacokinetic parameters, all of which contain the lead
structure LE404. For a multitude of these substances is still a
lack of information, inclusive of stability, physicochemical pa-
rameters, pharmacokinetics and metabolism. Therefore, the
present study investigated the stability properties of 17 new
azecine derivatives, including esterase cleavage, stability in
simulated gastrointestinal fluid, stability at different pH-values
and determination of octanol/water-partition coefficients.
These findings, in correlation to the properties and efficacy of
the already in vivo tested substances, will be useful for safety
and efficacy in further in vivo tests.
ly stable candidates, further stability tests and pharmacokinetic
studies have to be conducted in vitro before further in vivo tests
will be carried out. The current work reports the investigation of
new synthesized prodrugs with respect to their octanol/water-par-
tition coefficient (log PO/W), pH dependent stability and esterase
stability. This is expected to reveal the speed and extent of ester
cleavage. In correlation with the results of the substances already
tested in animal experiments, a statement can be made as to
whether a half-life extension can possibly be achieved by certain
structural variations. The estimation of the stability of the sub-
stances can be used to predict a possible route of administration
of a new drug candidate, for example, whether the compound al-
lows an oral administration.
The data obtained here will help to increase safety and efficacy
for following in vivo tests.
To enable these stability tests, a selective and sensitive analyti-
cal method for any compound was developed and validated using
HPLC-UV system.
 Original Article
Materials and Methods
General
All solvents and reagents were purchased from Merck (Germany),
Sigma-Aldrich Chemie Ltd. (Germany), VWR International Ltd. (Ger-
many) or Fisher Scientific Ltd. (Germany) and correspond to the
purity “p.a.”.
For preparation of mobile phases HPLC gradient grade acetoni-
trile (HiPerSolv CHROMANORM™, VWR International, Belgium) and
deionized water (TKA GenPure water system, Thermo Electron LED
Ltd., Germany) were used. The tested azecine derivatives, synthe-
sized in the Institute of Pharmacy Jena [5], are shown in ▶Table 1,
with the HPLC methods required for their analysis and correspond-
ing retention times.
The used phosphate buffer pH 7.4 contains 26.5 g Na2HPO4 · 12
H2O and 2.3 g KH2PO4 dissolved ad 1000 ml with deionized water.
The pH was adjusted to 7.4 with 10 % (m/v) NaOH or 10 % H3PO4
(m/v).
Instrument and chromatographic conditions
The stability tests of the azecine derivatives were followed by re-
versed phase HPLC and the formed 1 was quantified using
β-naphthol as internal standard. HPLC analyses were performed
with 5 different methods (▶ Table 2) using a LC-10 AS pump, an
auto injector SIL-10 A with an UV detector SPD-10A in combination
with a communication bus module SCL-10AVP. Integration and cal-
culation were done with Class LC10™ software (all parts Shimadzu
Europe). Mobile phase A contains 4 mmol/L potassium dihydrogen
phosphate-buffer pH 2.5 and mobile phase B acetonitril. A mem-
brane filter of 0.2 μm porosity was used to filter the mobile phase
(RC-Membranfilter, Sartorius, Germany) then the mobile phase was
degassed by sonification. Separations were achieved using a C18
reversed-phase column (phenomenex® Gemini 5 u C18 110 Å,
250 × 4.60 mm) and a precolumn (phenomenex® Security Guard
Cartridge Gemini C18, 4 × 3 mm, Phenomenex Inc., 63741 Aschaf-
fenburg, Germany). Chromatography was conducted at ambient
temperature at a flow rate of 1 ml · min − 1 and the detection wave
length was 220 nm. The volume of the sample solution injected
was 50 μl. Thiourea was used to determine the dead time. The aze-
cine derivatives peak at incubation time 0 min respectively after
­direct extraction was set to 100 %. Peak areas of each after differ-
ent incubation times were normalized to this. Normalized peak
areas of each measurement in triplicate were averaged.
Analytical validation
The limit of detection (LOD) and limit of quantification (LOQ) were
determined based on the standard deviation of the response and
the slope according to the ICH Guideline Q2 (R1) [6]. The LOD and
LOQ of 1 were determined by injecting a series of diluted solutions
of 1 (6 concentrations between 5 · 10 − 4 mol · l − 1 and 10 − 6 mol · l − 1,
3 times per concentration) to get a calibration curve. Standard de-
viation, slope and coefficient of determination (R2) of the calibra-
tion curve were calculated to ascertain linearity of the method. The
resolution values were determined according to the Europaean
Pharmacopoeia 9.0 [7].
Thieme
pH dependent stability
For the investigation of acid-dependent hydrolysis, an aqueous HCl
solution (pH 0.5) was prepared containing one of the azecine de-
rivatives (2-18) each and β-naphthol as internal standard at con-
centrations 10 − 4 mol · l − 1 (azecine derivative), 5 · 10 − 5 mol · l − 1
(β-naphthol), and 20 % (v/v) acetonitrile.
The final injectable solution samples for the base-dependent
hydrolysis consist of 10  − 4 mol · l  − 1 azecine derivative (2-18),
5 · 10 − 5 mol · l − 1 β-naphthol as internal standard and 20 % (v/v) ace-
tonitrile in an aqueous NaOH solution pH 11.
Hourly, 50 μl of one of the mixtures (pH 0.5, pH 1.2, pH 6.8, pH
7.4, pH 11 respectively) are injected into the HPLC system. This sta-
bility test is carried out over a period of 6 h at room temperature.
For selected substances (7, 8) the stability at pH 7.4 was inves-
tigated. For this purpose, the substances were dissolved in the same
concentration as in the other pH-dependent stability test. The sol-
vent used was a phosphate buffer pH 7.4 with 20 % (v/v) acetoni-
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trile.
Stability in simulated gastrointestinal fluids
Preparation of simulated gastric and intestinal fluid was performed
according to the European Pharmacopoeia 9.0 (5.17.1) [7] without
enzymes.
Simulated gastric fluid was prepared by dissolving 0.5 g sodium
chloride in deionized water. After acidified with 20 ml hydrochloric
acid was supplemented with deionized water to 250 ml. The result-
ing pH was 1.2. Simulated intestinal fluid was prepared by dissolv-
ing 6.8 g potassium dihydrogen phosphate in 250 ml deionized
water, 77 ml 0.2 M-sodium hydroxide solution and 500 ml deion-
ized water were added. After pH adjustment to 6.8, the mixture
was diluted with deionized water to 1000 ml.
Samples for the study of stability in simulated gastrointestinal
fluids contained 10 − 4 mol · l − 1 azecine derivative (2-18), 5 · 10 − 5
mol · l − 1 β-naphthol as internal standard and 20 % (v/v) acetonitrile.
Over a period of 6 h, 50 µl of the test solution were injected into the
HPLC system every hour.
In vitro esterase stability
The esterase solution must be freshly prepared. A solution of 12.5
U/ml in phosphate buffer pH 7.4 was prepared from the lyophilized
porcine liver esterase. The lyophilized porcine liver esterase (spec-
ification: E 3019-3,5kU/LOT#028K7005V; Sigma-Aldrich, 91625
Schnelldorf) had an activity of 17 U/mg. The derivatives (2-18) were
pre-dissolved in acetonitrile and added as an aliquot to the phos-
phate buffer pH 7.4 (heated to 40  °C in a water bath), so that a final
concentration of 1 % acetonitrile is not exceeded. The appropriate
amount of esterase stock solution was added to get a final esterase
activity of 0.2 U per sample cup. Over the entire time of the exper-
iment, the samples were shaken gently at 40  °C. Samples were
taken as triplicate after 1; 2; 3; 4; 5; 10; 15; 20; 30; 60 and 120 min.
At each time 150 µl were transferred to Eppendorff-reaction-tubes,
which contain 130 μl acetonitrile (for denaturing the enzymes) and
20 μl β-naphthol standard solution in acetonitrile to get a final con-
centration of 10  − 4 mol · l  − 1 azecine derivative (2-18) and
5 · 10 − 5 mol · l − 1 β-naphthol. 50 μl of these samples were injected
into the HPLC system.
Zergiebel S, Seeling A. In Vitro Stability of LE404-Derivatives …  Drug Res
▶Table 1 The lead compound 1, novel derivatives (2–18) and their deter- ▶Table 1 The lead compound 1, novel derivatives (2–18) and their deter-
Continued.
mined retention times (tR) with the indicated HPLC method (a-e) from mined retention times (tR) with the indicated HPLC method (a-e) from

O HO O HO
R R
N N N N

1-17 18 1-17 18

R =  HPLC- tR (1) tR R =  HPLC- tR (1) tR

method (derivative) method (derivative)

H 1 - - - 14 e 5.42 8.73
N
aliphatic esters O
2 a 3.4 4.22 15 e 5.42 10.94
N
O
O
3 a 3.4 8.73
carbonate

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O 16 b 3.19 4.48
O
4 a 3.4 13.46
O

O propargyl derivatives
17 d 3.09 4.26
cyclic aliphatic esters
5 b 3.19 9.33 18 e 5.42 6.48

6 c 3.3 15.63 For the test of transesterification, samples were taken as tripli-
cate additionally at 25; 40 and 50 min. At the times 21; 26, 31, 41,
O 51 and 61 min, respectively, 10 − 5 mol of 1 were added.
amino acid esters
Chromatographic determination of octanol/water
NH2 7 e 5.42 3.61
partition coefficients (log PO/W)
O For the determination of octanol/water-partition coefficients a
HPLC gradient method was used with the mobile phase A: 1 % (v/v)
NH2 8 e 5.42 4.33
acetic acid in acetonitrile and the mobile phase B: 1 % (v/v) acetic
O
acid in deionized water. It was used a flow rate of 1 ml · min − 1 and
a detection wavelength of 220 nm. From 0 to 10 min, a linear gra-
aromatic esters
dient was achieved, starting with 10 % A, up to 95 % A. From 10 to
9 b 3.19 7.46
15 min an isocratic part followed with 95 % A. A further 10 min, the
system was rinsed on the starting conditions of 10 % A. Separations
O were achieved using a C18 reversed-phase column (Nucleodur C18
10 b 3.19 10.65 Isis 3 µm, 125 × 4 mm, Macherey-Nagel, Germany) and a precolumn
(EC 4/3 Universal RP, SN E15101206 LOT 4014, Machery-Nagel,
O Germany). As standard substances 1, 2, 3, 4 [5] and 7-methyl-
O 11 b 3.19 9.61 5,6,7,8,9,15-hexahydro-benzo[f][1, 3]dioxolo[4, 5]k]benzazecine
(RMD3) [8] were used. The substances were injected 3 times and
the average value of the obtained retention times went into the
O
calculation. The octanol/water partition coefficients of the refer-
12 b 3.19 9.28 ence substances were taken from literature [4].
S
O O
Results
carbamates
13 e 5.42 7.85
O HPLC method development
N
Separation of the prodrugs, metabolites and the internal standard
O
requires only for some substances (7, 8, 13, 14, 15, 18) a gradient
elution. In most cases, isocratic elution is sufficient. The aqueous
phase of the eluent contains a phosphate buffer pH 2.5. Also under

Zergiebel S, Seeling A. In Vitro Stability of LE404-Derivatives …  Drug Res

 Original Article Thieme

▶Table 2 Chromatographic conditions of the used HPLC methods (a-e). derivatives show a much stronger alkalic hydrolysis than an acidic
hydrolysis. Only the substances 2, 3, 5 and 8 were hydrolyzed under
Time
Method A [ % (v/v)] B [ % (v/v)] Comments acidic conditions (▶ Fig. 1). The lead compound 1 is formed as the
[min]
degradation product.
a 0-20 65 35 isocratically
b 0-20 60 40 isocratically
The larger and more sterically demanding the aliphatic residue,
the slower the hydrolysis takes place. The pivalic acid ester 4 and
c 0-20 62.5 37.5 isocratically
the cyclohexanecarboxylic acid ester 6 were stable at pH 0.5. The
d 0-20 57.5 42.5 isocratically
aromatic esters and the carbonate were not cleaved by acid. The
e 0-12 75.0 to 60.0 25.0 to 40.0 linear
para-substituted benzyl esters (10, 11) showed the highest base
gradient
resistances with a percent decrease of 6 % (10) and 11 % (11), re-
12-19 60.0 to 20.0 40.0 to 80.0 linear
gradient spectively, within the group of the hydrolysis-sensitive substances.
19-20 20.0 to 75.0 80.0 to 25.0 linear
The carbonate 16 is not cleaved by acid, but rapid hydrolysis of 67 %
gradient occurs within 6 h under basic conditions. The amino acid esters
20-30 75 25 linear show a surprisingly good acid resistance, but they are completely
gradient cleaved under basic conditions within 1 h.

Stability in simulated gastrointestinal fluids

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▶Fig. 1 Percentage remaining amount of substance within 6 h
under acidic respectively under basic conditions as well as standard
deviation.
basic conditions, at pH 10, the azecine derivatives are very easy to
determine [4]. However, the acidic buffer was preferred and vali-
dated in this study, because the test substances have a higher long-
term stability in this medium.
Analytical validation
The methods gave resolutions between 3.1 and 28.4 among the
azecine derivatives, degradation products and the internal stand-
ard.
The calibration curve for 1 was found to be linear in the range of
5 · 10 − 4 mol · l − 1 and 10 − 6 mol · l − 1 for all methods. The values of
coefficient of determination and the experimental LOD and LOQ
values were similar for all methods. The calculated LOD was
4.75 · 10 − 7 mol · l − 1 and the calculated LOQ was 1.44 · 10 − 6 mol · l − 1
with a coefficient of determination of 0.9978.
pH dependent stability
The carbamates (13, 14, 15), the propinyl derivatives (17, 18) and
the sulfonic acid ester 12 showed no pH-dependent instability over
the test period of 6 h. For all other derivatives, the percent decrease
within 6 h under basic conditions is illustrated in ▶Fig. 1. All ester
In these tests, first no enzymes were added, because all drug can-
didates are compounds which are not attacked by digestive en-
zymes due to their structure. Thus, instability in the gastrointesti-
nal tract would only be due to the prevailing pH conditions. Excep-
tions are the amino acid esters (7, 8). The pH-dependent
decomposition and the enzyme-dependent decomposition should
be tested separately. But because both substances were complete-
ly decomposed after 2 h in the simulated intestinal fluid, no further
results could be expected from the enzymatic tests. Because rapid
decomposition occurs at pH 6.8, the stability of the amino acid es-
ters was also tested under physiological pH conditions (pH 7.4).
Here as well, the substances completely hydrolyzed within 2 h.
However, in the simulated gastric fluid, the valine ester (7) and the
leucine ester (8) were stable for 6 h. This suggests that the stability
of 7 and 8 increases with decreasing pH conditions. A further sta-
bility test at pH 5 was carried out to pursue this. Here the concen-
tration of 7 decreases by 75 % and of 8 by 91 % in 6 h. This proves
that when the pH decreases, the stability of the starting compound
increases. Moreover, ▶ Table 3 shows that the valine ester has a
slightly higher stability than the leucine ester over all pH ranges.
All other tested substances are stable in simulated gastrointes-
tinal fluids.
In vitro esterase stability
The extent and speed of ester hydrolysis of the test compounds
was investigated by porcine liver esterase over a time period of
120 min. For the most part, the test substances are only moderate-
ly soluble in water and thus inaccessible to enzymatic tests. In other
studies, DMSO was used as a solubilizer [4]. In this case, however,
a sufficient solubility could not be achieved with all substances.
Therefore, the azecine derivatives were pre-dissolved in acetoni-
trile and added to the buffer solution. The final concentration of
acetonitrile has never exceeded 1 %, in order not to impair the en-
zyme activity. The ester cleavage for the substances 2-6 and 16 fol-
lowed a first order kinetic (▶Fig. 2). The proportion of starting ma-
terial, split per unit of time, was constant. So half-life of the ester
cleavage can be calculated from the linear equation (▶ Table 4).
The half-lives of the esters 2-6 are between 16 min and 32 min. The
larger and more sterically demanding the acid component of the
ester, the slower the cleavage takes place. The cyclic aliphatic es-

Zergiebel S, Seeling A. In Vitro Stability of LE404-Derivatives …  Drug Res

▶Table 3 Percentage remaining amount of the amino acid derivatives (7, 8) within 1 h, 2 h and 6 h under different pH conditions as well as standard deviation.

Remaining amounts [ %] of 7 and 8 under the corresponding pH

Substance Test period
pH 0.5 pH 1.2 pH 5 pH 6.8 pH 7.4 pH 11
7 100 ± 0.3 100 ± 1.5 85.3 ± 3.3 1.5 ± 0.05 1.7 ± 0.07 0
1h
8 96.8 ± 1.4 100 ± 2.0 82.9 ± 1.2 1.5 ± 0.08 2.0 ± 0.01 0
7 100 ± 0.7 100 ± 1.5 67.8 ± 0.8 0 0 0
2h
8 90.1 ± 2.3 100 ± 0.2 61.5 ± 0.4 0 0 0
7 100 ± 1.5 100 ± 0.7 25.2 ± 1.3 0 0 0
6h
8 87.1 ± 1.2 100 ± 1.6 9.4 ± 1.2 0 0 0

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▶Fig. 2 Semi logarithmic presentation of remaining amount of
various azecine derivatives by esterase cleavage which follow first ▶Fig. 3 Semi logarithmic presentation of the decrease of aromatic
order kinetics and the associated coefficient of determination (R2) of esters derivatives (9, 10, 11) by esterase cleavage.
the calibration curve.

▶Table 4 Half-life (t½) of the esterase cleavage and the associated
c­ oefficient of determination (R2) of the calibration curve.
Substance t½ [min]
aliphatic esters
2 21
3 23
4 32
cyclic aliphatic esters
5 16
6 18
carbonate
16 131
aromatic esters
9 22
10 41
11 16
ters (5, 6) showed a faster cleavage than the short chain fatty acid
esters (2-4). The carbonate 16 was degraded very slowly with a
half-life of 131 min.
The aromatic esters (9, 10, 11) showed a first order kinetic up
to 20 min after esterase addition. Thereafter, the rate of ester cleav-
age decreased. From 60 min, no significant decrease in the amount
of ester occurred (▶ Fig. 3). In the case of hydrophobic molecules,
the tendency of the transesterification by means of hCE-1 increas-
es [9]. Starting from a certain amount of the corresponding alco-
hol (which is released by hydrolysis of the ester), it is possible for
this alcohol to re-attack the benzoate-hCE-1 intermediate and thus
restore the starting substance (i. e. the ester). To prove that this is
R2
caused by transesterification, the test was carried out again. At the
0.9918
times 21; 26, 31, 41, 51 and 61 min, respectively, 10 − 5 mol of the
formed phenol 1 were added. In the first 20 min, the first order ki-
0.9838
netics were obtained again. After the first addition of the phenol
0.9829
1, the amount of starting substance (9, 10, 11 respectively) in-
creased again strongly. At the next additions of 1, a further contin-
0.9576
uous, but not so pronounced, increase of the ester was observed
0.9954
(▶ Fig. 4). It can be assumed that there is a permanent concentra-
tion balance in the body (continuous blood flow). Therefore, the
0.9702 concentration of the resulting phenol 1 near the esterase will never
be so great that reesterification takes place. Thus, it is possible to
0.9703 calculate approximate half-lives for the aromatic esters from the
0.9808 first 20 min of the test (▶Table 4).
0.9744
Octanol/water partition coefficients
For the analysis, a special polymeric cross-linked surface modified
column was selected, which is distinguished by its high steric se-
lectivity [10]. Thus it was possible to determine exact log P O/W
­values from structurally very similar substances within a maximum
analysis time of 15 min. There is a linear relationship between
­retention time and lipophilicity of a substance and therefore its log
PO/W value. By the approach partition coefficients for other sub-
stances can be calculated from the linear equation. This approxi-
mation is even more precisely if the reference substances are struc-
turally similar to the analytes [11]. Therefore, azecine derivatives

Zergiebel S, Seeling A. In Vitro Stability of LE404-Derivatives …  Drug Res

 Original Article Thieme

▶Table 5 Retention times (tR), and log PO/W values of the reference sub-
stances [4] and determined log PO/W values of the azecines.

tR [min] log P o/w

references substances
1 6.35 3.33
RMD3 6.96 3.9
2 7.19 4.02
3 7.6 4.32
4 7.87 4.47
test substances
8 4.17 0.95
7 5.53 2.6
18 6.36 3.37
▶Fig. 4 Semi logarithmic presentation of the decrease of aromatic 13 6.82 3.74
esters derivatives (9, 10, 11) by esterase cleavage up to 20 min. 16 6.88 3.79
Addition of 1 after 21, 26, 31, 41, 51 and 61 min (each 10 − 5 mol) to
14 6.89 3.8

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verify transesterification.
15 7.12 3.97
17 7.13 3.98
were used for the preparation of the calibration curve. The log PO/W 11 7.51 4.25
values of the reference substances 1, 2, 3, 4 and RMD3 were deter- 5 7.53 4.27
mined in previous work [4]. Octanol/water-partition coefficients 9 7.59 4.31
from the reference and the test substances are summarized in 10 7.84 4.48
▶ Table 5. All the tested azecine derivatives have a partition coef-
6 7.87 4.5
ficient below 5 and it is therefore assumed that these substances
12 8.01 4.59
exhibit a good absorption or permeation [12].

Discussion
The stability and metabolism studies carried out here, provide in-
dispensable indications of the stability in physiological media, du-
ration of action and so a possible prediction of an administration
route of the new azecine derivatives. Other influencing factors on
bioavailability are lipophilicity and membrane permeability. For the
better classification of the substances, octanol/water partition co-
efficients were also determined. In general, oral application is the
preferred administration route of drugs, because of low risk of mi-
crobial contamination, low invasiveness and cost-effectiveness
[13]. In case of neuropsychiatric disorders, e. g., schizophrenia, a
good patient compliance is particularly important. This is improved
by long-term application forms. Thus, it was a main aim of the me-
tabolism test, to select the active drug candidates, which have the
highest stability in vitro. The in vitro tests allow conclusions about
the real behavior of the substance in the body.
In the present study the stability of the substances in media sim-
ulating the route of orally administrated drugs, like the gastroin-
testinal tract, was tested. All derivatives were stable in simulated
gastric and intestinal fluid, with the exception of the amino acid es-
ters (7, 8). Thus, almost all substances, considered by their stabil-
ity, are suitable for an oral application.
Only 4 of the 17 derivatives show slightly instability within 6 h
under strong acidic conditions (pH 0.5). The extend of the base-
dependent hydrolysis depends on the steric demand of the ester
residue. The highest stabilities thereby show the aromatic com-
pounds (9, 10, 11).
The stability of the amino acid esters decreases with increasing
pH. Even in the simulated intestinal fluid at pH 6.8, the cleavage of
98 % of the ester takes place within 1 h. The actual aim, namely to
use amino acid transporters in the intestinal wall for an active trans-
port will possibly fail due to the insufficient stability. With Valaci-
clovir, good results have been achieved through this transport
mechanism [14]. However, this is an aliphatic ester. In contrast, the
phenol esters 7 and 8 have much lower stability.
Because esterases are ubiquitous in the human body, the imple-
mentation of potential drugs by these enzymes is of great interest.
The slower the esterase cleavage, the longer the substance stays
in the body and the longer is its duration of action. In case of the
isobutyric ester 3, the duration of the free phenol 1 could be in-
creased from 30 min to 90 min [4]. The carbamates and the propi-
nyl ether showed no esterase-induced cleavage. In this case, fur-
ther it would have to be tested, whether the effective phenol is lib-
erated from the prodrug via other mechanisms. One possibility is
the metabolism via butyrylcholinesterase, whose hydrolytic activ-
ity has been demonstrated against carbamates (e. g., for bamb-
uterol) [15]. The release of LE404 from the ether derivative 17 can
be catalyzed by CYP450 metabolism, as in the case of O-deethyla-
tion of phenacetin via CYP1A2 [16]. The carbonate 16 has by far
the longest half-life in vitro (131 min). All other derivatives are in
the range from 16 to 40 min. If isobutyric ester 3, which has already
been tested in vivo, is used as the basis for comparison, it could be
assumed that substances with similar half-lives in vitro also have
similar duration of action in vivo. Therefore, the esters 4 and 10 as
well as the carbonate 16 are to be emphasized, because they have
a significantly higher half-life than the other derivatives which lie
in the range of the isobutyric ester 3 or lower. A transesterification
was observed in the aromatic esters (9, 10, 11), which could be de-

Zergiebel S, Seeling A. In Vitro Stability of LE404-Derivatives …  Drug Res

tected by addition of the free phenol. In vivo, this phenomenon
plays a subordinate role because the constant blood flow will never
cause an overconcentration of 1 on the enzyme. Thus, half-lives
were also calculated for esters 9, 10 and 11.
The log PO/W values of all substances could be determined by
means of a calibration curve over the linear relationship between
the retention time and the lipophilicity. In any case, with the excep-
tion of the amino acid derivatives (7, 8), the derivatives are more
lipophilic than the phenolic compound 1. This is in line with expec-
tations, as with other ester prodrugs this tendency has already been
demonstrated [17]. The tested azecine derivatives have octanol/
water partition coefficients between 0.95 and 4.59. Therefore, it is
assumed that these substances exhibit a good absorption or per-
meation [12]. In principle, a log PO/W in the range of 3-5 is to be
classified as tendencial low soluble in water, which could lead to dif-
ficulties with an oral application. On the other hand, lipophilic sub-
stances are easier to overcome the blood-brain barrier, which is a
great advantage in psychoactive substances.
The analytical validation of the methods was carried out accord-
ing to ICH Guideline Q2 (R1) [6]. Different mobile phases were test-
ed in distinct proportions of organic solvent (acetonitrile) and phos-
phate buffer, at different pH values of aqueous phases (2.5, 7.4 and
10.0). The adequacy of mobile phase was decided on the basis of
selectivity and sensitivity and the separation between degradation
products formed during stress studies. There were 5 different
HPLC-UV methods by means of which all 17 derivatives and their
degradation products could be reproducibly quantified.
The tests presented herein provide first indications as to which
of the substances have a longer duration of action in vivo with con-
sistent activity and furthermore sufficient stability for an applica-
tion. In order to confirm the assumptions, it is necessary in further
research to carry out in vivo studies. Firstly, the esters 4 and 10 and
the carbonate 16 would be offered. The esters 4 and 10 have al-
most identical octanol/water partition coefficients (log P O/W
(4) = 4.47, log PO/W (10) = 4.48) in the range of substance 3 (log PO/W
(3) = 4.37) which has already been examined in vivo and showed a
longer half-life than LE404 [4]. 3 was administered orally as better
soluble maleate salt. The similarity of the octanol/water partition
coefficients suggests that in future in vivo tests, 4 and 10 should
be previously converted into the more soluble salt form in order to
compare the pharmacokinetic parameters of the substances. In
contrast, the carbonate 16 with a log PO/W (16) = 3.79 is more hy-
drophilic in its comparison and allows probably direct application
as free base. However, it would also be advantageous to perform
comparative in vivo tests as maleate salt comparing the effects of
the applied free base with those of the maleate salt of 16. In view
of the high esterase stability of 16, a significant increase in the half-
life compared to LE404 and 3 is to be expected. Also 4 and 10 show
a potential for half-life extension according to the esterase assays.
How strongly the insertion of a propargyl group (17, 18) prolongs
the half-life is to be verified by future CYP450-catalyzed metabo-
lism tests and confirmed with in vivo tests. The behavior of other
azecine derivatives in vivo is also of great interest. Thereby conclu-
sions can be drawn about the predictability which is made via es-
terase cleavage.
Zergiebel S, Seeling A. In Vitro Stability of LE404-Derivatives …  Drug Res
Conflict of Interest
The authors declare no competing financial interest.
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