Towards the rational design of novel drugs based on solubility, partitioning/distribution, biomimetic permeability and biological activity exemplified by 1,2,4-thiadiazole derivatives

Article abstract of DOI:10.1039/c6md00545d

Novel 1,2,4-thiadiazole derivatives as potent neuroprotectors were synthesized and identified. Their ability to inhibit the glutamate stimulated Ca²⁺ uptake was investigated. The solubility of thiadiazoles was measured in a buffer solution (pH 7.4) at 298 K. The distribution coefficients in 1-octanol/buffer (pH 7.4) and 1-hexane/buffer (pH 7.4) immiscible phases as model systems imitating the gastrointestinal tract epithelium and the blood-brain barrier were determined. Permeation experiments the new Permeapad barrier using Franz diffusion cells were conducted and the apparent permeability coefficients were obtained. The influence of the compound structure on the physicochemical properties determining the bioavailability of drug-like substances was revealed. Solubility-permeability interplay has been assessed to evaluate potential bioavailability of the compounds studied.

Full text of DOI:10.1039/c6md00545d

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Terekhova, O. Silyukov, A. N. Proshin, A. Bauer-Brandl and G. L. Perlovich, Med. Chem. Commun., 2016,
DOI: 10.1039/C6MD00545D.
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ARTICLE
Towards the rational design of novel drugs based on solubility,
partitioning/distribution, biomimetic permeability and biological
activity exemplified by 1,2,4-thiadiazole derivatives†
T. V. Volkovaa, I. V. Terekhovaa, O. I. Silyukova, A. N. Proshinb, A. Bauer-Brandlc, G. L. Perlovicha,*
Novel 1,2,4-thiadiazole derivatives as potent neuroprotectors were synthesized and identified. Their ability to inhibit the
glutamate stimulated Ca2+ uptake was investigated. The solubility of thiadiazoles was measured in buffer solution (pH 7.4)
at 298 K. The distribution coefficients in 1-octanol/buffer (pH 7.4) and 1-hexane/buffer (pH 7.4) immiscible phases as
model systems imitating gastrointestinal tract epithelium and blood-brain barrier were determined. Permeation
experiments across new PermeapadTM barrier using Franz diffusion cells were conducted and the apparent permeability
coefficients were obtained. Influence of the compounds structure on the physicochemical properties determining the
bioavailability of drug-like substances was revealed. Solubility-permeability interplay has been assessed to evaluate
potential bioavailability of the compounds studied.
effective multitarget drugs [2]; searching for new targets
participating in the course of the disease. Moreover,
considering business purposes, repositioning the well-
1. Introduction
In the opinion of leading experts, Alzheimer’s disease is one
of the most frequent and progressive condition among
elderly people, comparable by occurrence with cardial and
cerebral infarctions [1]. As the etiology of Alzheimer’s
disease (Alzheimer type dementia) has not been found yet,
there is no rational standard therapy of the disease.
Currently, worldwide investigations in the sphere of
neurodegenerative drugs design are performing mainly in
the following directions: invention of neuroprotectors
efficacious in terms of pathogenesis of the disease and
capable to block a pathogenic mechanism, particularly,
known formulations to novel application is very attractive.
During the creation of new neuroprotective agents fast
screening of a large number of structures which exhibit
biological activity at the receptors comes forward.
Investigation of the drug delivery processes (transport
properties) is an important stage in the creation of
bioavailable drugs. Therefore, obtaining the screening
parameters characterizing solubility, partitioning, and
membrane permeability is an essential part of drug design.
The 1,2,4-thiadiazole derivatives are well known to have
the biologically active groups to specific targets of different
pathologies. Many of them reveal a wide spectrum of
biological activity including antioxidant and neuroprotective
ones [3-6]. Analysis of the influence of 1,2,4-thiadiazole
derivatives on the glutamate-induced calcium ions uptake
into synaptosomes of rat brain cortex is often used for
primary evaluation of the biological activity of newly
synthesized drug compounds [7]. The glutamate receptors
in the central nervous system are known to play an
important role both in regular neurophysiological processes
and pathogenesis of a series of neurodegenerative diseases
[8]. In this connection, the evaluation of a compound´s
a.Department of Physical Chemistry of Drugs, Krestov’s Institute of Solution
Chemistry, Russian Academy of Sciences, 153045 Akademicheskaya str. 1,
Ivanovo, Russia. E-mail: glp@isc-ras.ru
b.Institute of Physiologically Active Compounds, Russian Academy of Sciences,
142432, Chernogolovka, Russia.
c. Department of Physics, Chemistry and Pharmacy, University of Southern
Denmark, 5230 Odense M, Denmark
†The authors declare no compeꢀng interests.
Electronic Supplementary Information (ESI) available: Dependence of content of the
ionized forms of the molecules on the pH of buffer solution of compound 2 (Fig. 1SI);
Relationships of the permeability coefficients with the distribution coefficients (Fig.
2SI); Relationship of the permeability coefficient with physicochemical descriptor
HYBOT (Fig. 3SI). This material is available free of charge via the Internet at
http://pubs.acs.org.
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ability to affect the calcium channels mediated by the
glutamate receptors allows to assess their common
biological potential as possible neuroprotectors (in the case
of inhibiting) or activators of cognition functions (in the
case of activation). Members of a newly discovered class of
compounds, ampakines, boost the activity of glutamate and
thus make it easier to form memories. The cognition
enhancers work on the neural processes that underlie such
mental activities as attention, perception, learning,
memory, language, planning and decision-making, usually
by altering the balance of the chemical neurotransmitters
involved in these processes. At present, nootropics and
some neuronotrophic agents represent the available
approaches to symptomatic treatment of Alzheimer's
disease [9]. Attempts to improve cognitive function in
patients with brain disorders have become the focus of
intensive research efforts [10]. The task of developing
guidelines for creating effective modulators of brain
cognitive functions based on the derivatives of 1,2,4-
thiadiazole is relevant from the perspective of the creation
of new class of effective drugs for the treatment of
Alzheimer's disease.
occurring between the drug molecules and the rather tight
and lipophilic BBB environment. A parameter describing the
difference between the distribution coefficients in 1-
octanol/water and 1-hexane/water phases is often used to
account for specific interactions (donor-acceptor, hydrogen
bonding) to drug transport properties [16].
In the present study we propose the synthesis of new
1,2,4-thiadiazole derivatives with different substitution of
the phenyl ring and replacement of the phenyl ring by
cyclopropyl- and isoxazole-one. The aim focuses at
investigating the biological activity of the synthesized 1,2,4-
thiadiazoles and revealing correlations between the
structures, solubilities, lipophilicities and membrane
permeabilities of the respective substances. The present
work is a continuation of our previous attempts to study
the passive transport properties of 1,2,4-thiadiazoles [17-
23] and the relationship between the biological activity and
transport characteristics of drugs and drug-like compounds
[23,24].
2. Materials and methods
Lipophilicity is a property that has a major effect on in
vivo processes of absorption, distribution, metabolism,
excretion, and toxicity (ADMET) properties as well as
pharmacological activity [11]. The partition coefficient is
accepted by most scientists to be one of the most relevant
lipophilicity descriptors applied in pharmaceutical research
[12]. It can be measured by a rather simple experiment or
calculated. Lipophilicity has been correlated to many other
properties, such as solubility, permeability, metabolism,
toxicity, protein binding, and distribution. 1-Octanol is the
2.1. Synthesis
2.1.1. General Procedure for Preparing Novel 1,2,4-thiadiazoles
Synthetic approaches to novel 1,2,4-thiadiazoles were
carried out according to Scheme 1.
NH2
NH
b
preferred
organic solvent for phase-distribution
NH
R1NCS
R1
H3C
H3C
investigations or partition coefficient determination of
drugs. The 1-octanol/water partition coefficient is reported
in drug handbooks and is commonly used in various types
of analysis of drug properties [13]. The log D7.4
N
S
O+
N
O
a
log Doct /buf in the present study) of a compound stands
N
NH
NH
(
N
R1
R1
for its distribution coefficient at pH 7.4, and it is considered
to be a property of utmost importance because of its high
physiological relevance and its resemblance to real
biological partitions in the body [14]. As it has been
emphasized in [15], the implication of log D7.4 for drug
development leads to a general rule for optimal intestinal
absorption by passive diffusion permeability after oral
R2
R2
S
S
N
N
N
O
CH3
CH3
c
d
NH
NH
N
N
R1
R1
R2
NH
R2
S
S
N
NH
N
dosing. Drug substances should have a moderate log D7.4
.
CH3
CH3
Namely, a good balance of permeability and solubility exists
when log D7.4 = 0 – 3. At that, an optimum range for CNS
(central nervous system) narrows to log D7.4 = 1 – 3. Since
all drug compounds for the prevention of Alzheimer's
disease need to overcome the blood-brain barrier (BBB),
the analysis of drug interactions with these barriers is an
important objective in the design of effective drugs. As a
rule, the water/alkane systems (usually 1-hexane/water
distribution coefficients log Dhex / w ) are used for BBB
simulation. Selecting the alkane phase is dictated by the
fact that it is a good description of non-specific interactions
e
f
Scheme 1. Synthesis of the compounds studied.
5-N-Monoaminosubstituted
5-amino-3-(2-
aminopropyl)-[1,2,4]thiadiazoles (f) were synthesized in the
following matter:
A solution of 3-amino-5-methylisoxazole (a) (0.01 M) in
10 mL of acetonitrile was added drop wise to a solution of
isothiocyanate (0.01 M) in 10 mL of acetonitrile under
stirring. At that, the obtained isoxazole thiourea (b) in situ is
2 | J. Name., 2012, 00, 1-3
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transformed to [1,2,4]thiadiazole ring according to Boulton-
Katritskyi [25]. Upon completion of the dropping the
mixture was brought to boiling and left at room
temperature until the precipitation of 5-amino-3-(2-
oxopropyl)-[1,2,4]thiadiazole (c) occurs. The precipitate was
filtered off. If the crystallization was not taking place the
reaction mixture was evaporated and the resultant oil was
triturated in diethyl ether.
methanol, heated to 50 °C. Then sodium borohydride (0.01
M) was added portionwise under vigorous stirring. Under
the addition of sodium borohydride the reaction was
passed and the precipitate was dissolved. Upon the
completion of the reaction, methanol was evaporated, 50
mL of methylene chloride was added and washed with
water (2 x 50 mL), and then an organic layer was separated,
and dried over sodium sulfate. The drier was filtered off,
and the filtrate was evaporated to obtain 5-amino-3-(2-
hydroxypropyl)-[1,2,4]thiadiazole (f).
The
resulting
5-amino-3-(2-oxopropyl)-[1,2,4]
thiadiazole (c) (0.01 M) and primary amine (0.01 M) was
dissolved in 10 ml of methanol and left one day at room
temperature. The precipitate was filtered off. According to
NMR spectroscopy, the imine (d) forming during the
reaction was rearranged to the corresponding vinylamine 5-
amino-3-(2-amino-1-propenyl)-[1,2,4]thiadiazole (e) which
is introduced into the reduction reaction.
2.1.2. Chemical Procedures
5-N-Monoaminosubstituted
5-amino-3-(2-aminopropyl)-
[1,2,4]thiadiazoles were synthesized by standard and
accessible methods in the Institute of Physiologically Active
Compounds of the Russian Academy of Sciences. The
compounds synthesized are presented in Table 1.
The
resulting
5-amino-3-(2-amino-1-propenyl)-
[1,2,4]thiadiazole (e) (0.01 M) was suspended in 30 mL of
Table 1 Structures of studied compounds
Group I
Group II
CH3
CH3
N
N
R
N
N
R
N
S
N
S
OH
OH
R
R
CH3
CH3
S
N
N
CH3
N
CH3
S
N
N
N
H3C
H3C
H3C
CH3
H3C
CH3
№
1
R-
№
7
R-
№
R-
№
R-
H
H
H
H
13
18
N
Cl
N
Cl
N
S
N
H3C
H3C
Cl
Cl
Cl
2
3
4
8
14
19
H
N
H
N
H
N
H
S
Cl
N
H3C
H3C
Cl
MeO
F
H3C
Cl
9
15
16
20
21
H
H
H
N
H
N
N
N
F
F
MeO
F
10
H
H
N
H
H
N
N
Cl
N
Cl
N
O
H3C
H
N
5
6
11
12
17
22
H
H
N
H
N
N
N
O
Cl
Cl
H3C
H
H
N
N
H3C
Br
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N-{1-[5-(3,4-Dichlorophenylamino)-[1,2,4]thiadiazol-3-yl]-
propan-2-yl}-(2,2,6,6-tetramethylpiperidine-4-yl)amine (1).
(2H, d, J = 6.5 Hz, СН2CHМе), 3.03 (1H, m, C(4)H), 3.45 (1H,
m, СН2CHМе), 7.40 and 7.50 (4H, d and d, J = 8.4 Hz, Нarom).
[1-Methyl-2-(5-р-tolylamino-[1,2,4]thiadiazol-3-yl)-ethyl]-
White powder. Yield 67%. Calcd, %: С 54.29, H 6.61, N
15.83 C20H29Cl2N5S. Found, %: С 54.67, H 6.66, N 15.48.
NMR 1H [200 MHz] δ: 0.79 (2H, dt, J = 12.0, 21.2 Hz, С(3)НН,
С(5)НН), 1.08 and 1.12 (both s, 6 H, 2 CH3), 1.15 (3H, d, J =
6.6 Hz, СН2CHCH3), 1.84 (2H, dd, J = 3.3, 12.6 Hz, С(3)НН,
С(5)НН), 2.84 (1H, dd, J = 6.0, 14.2 Hz, СННCHМе), 2.92 (1H,
dd, J = 4.0, 14.2 Hz, СННCHМе), 3.00 (1H, m, C(4)H), 3.42
(1H, m, СН2CHМе), 7.17 (1H, dd, J = 2.2, 8.6 Hz, Нarom), 7.45
(1H, d, J = 8.6 Hz, Нarom), 7.51 (1H, d, J = 2.2 Hz, Нarom).
(2,2,6,6- tetramethylpiperidine-4-yl)-amine (6).
Light crystals. Yield 69%. Calcd, %: С 65.08, H 8.58, N
18.07. C21H33N5S. Found, %: С 65.01, H 8.52, N 18.09.
1
NMR H [200 MHz] δ: 0.71 (2H, m, С(3)НН, С(5)НН), 1.02,
1.05 (6H, s + s, 2x CH3), 1.07 (3H, d, J = 6.5 Hz, СН2CHCH3),
1.12, 1.14 (6H, s + s, 2xCH3), 1.75 (2H, dd, J = 3.1, 12.5 Hz,
С(3)НН, С(5)НН), 2.73 (2H, dd, J = 2.3, 6.5 Hz, СН2CHМе),
2.91 (1H, m, C(4)H), 3.32 (1H, m, СН2CHМе), 7.09 and 7.38
(4H, d + d, J = 8.6 Hz, Нarom), 10.56 (1Н, br.s, ArNH).
2-[5-(4-Methyl-3-chlorophenylamino-[1,2,4]thiadiazol-3-yl]-
1-methylethyl}-(2,2,6,6- tetramethylpiperidine-4-yl)-amine
2-[5-(3-Methylthiophenylamino-[1,2,4]thiadiazol-3-yl)-
(
2).
ethyl]-1-methylethyl}-(2,2,6,6- tetramethylpiperidine-4-yl)-
White powder. Yield 77%. Calcd, %: С 59.77, H 7.64, N
amine (7).
16.59 C21H32ClN5S. Found, %: 59.87, H 7.34, N 16.44.
1
NMR H [200 MHz] δ: 0.65 (1H, t, J = 12.0 Hz, СНH), 0.75
White powder. Yield 70%. Calcd, %: С 60.10, H 7.93, N
16.69 C21H33N5S2. Found, %: С 60.28, H 7.99, N 16.64.
NMR 1H [200 MHz] δ: 0.81 (2H, m, С(3)НН, С(5)НН), 1.13
and 1.20 (12H, s and s, 2xCH3), 1.21 (3H, d, J = 6.5 Hz,
СН2CHCH3), 1.86 (2H, d, J = 12.4 Hz, С(3)НН, С(5)НН), 2.54
(3Н, s, Ph-SСН3), 2.88 (2H, m, СН2CHМе), 3.01 (1H, m,
C(4)H), 3.47 (1H, m, СН2CHМе), 7.01 (2H, m, Нarom), 7.34
(2H, m, Нarom).
(1H, t, J = 12.0 Hz, СНH), 1.00 (6Н, s, (СН3)2), 1.06 (3Н, d, J =
6.3 Hz, ССН3), 1.10 (6Н, s, (СН3)2), 1.71 (2H, dd, J = 3.5, 12.3
Hz, 2СН), 2.31 (3Н, s, Ph-СН3), 2.71 (2H, dd, J = 6.3, 14.2 Hz,
СН2), 2.89 (1H, tt, J = 3.5, 11.6 Hz, NСН), 3.32 (1Н, m, NСН),
7.17 (1H, d, J = 8.4 Hz, Нarom), 7.29 (1H, dd, J = 2.2, 8.4 Hz,
Нarom), 7.71 (1H, d, J = 2.2 Hz, Нarom).
2-[5-(4-Fluoro-3-chlorophenylamino-[1,2,4]thiadiazol-3-yl]-
1-methylethyl}-(2,2,6,6- tetramethylpiperidine-4-yl)-amine
2-[5-(3-Methoxyphenylamino-[1,2,4]thiadiazol-3-yl]-1-
methylethyl}-(2,2,6,6- tetramethylpiperidine-4-yl)- amine
(
3).
(8).
White powder. Yield 67%. Calcd, %: С 56.39, H 6.86, N
White powder. Yield 70%. Calcd, %: С 62.50, H 8.24, N
16.44 C20H29ClFN5S. Found, %: С 56.26, H 6.81, N 16.55.
NMR 1H [200 MHz,] δ: 0.77 (2H, dt, J = 12.1, 21.0 Hz,
С(3)НН, С(5)НН), 1.06 and 1.14 (12H, c and с, 4xCH3), 1.15
(3H, d, J = 6.6 Hz, СН2CHCH3), 1.81 (2H, dd, J = 3.4, 12.6 Hz,
С(3)НН, С(5)НН), 2.83 (1H, dd, J = 6.0, 14.0 Hz, СННCHМе),
2.97 (1H, dd, J = 4.1, 14.0 Hz, СННCHМе), 3.00 (1H, m,
C(4)H), 3.40 (1H, m, СН2CHМе), 7.16 (2H, m, Нarom), 7.45
(1H, d, J = 8.0 Hz, Нarom).
17.35 C21H33N5OS. Found, %: С 62.66, H 8.14, N 17.34.
NMR 1H [200 MHz] δ: 0.81 (2H, m, С(3)НН, С(5)НН), 1.09
and 1.17 (12H, c and с, 4xCH3), 1.20 (3H, d, J = 6.2 Hz,
СН2CHCH3), 1.83 (2H, d, J = 12.2 Hz, С(3)НН, С(5)НН), 2.83
(2H, m, СН2CHМе), 2.97 (1H, m, C(4)H), 3.39 (1H, m,
СН2CHМе), 3.81 (3Н, с, Ph-ОСН3), 6.73 (2H, m, Нarom), 7.29
(2H, m, Нarom).
N-{1-[5-(4-Fluorophenylamino-[1,2,4]thiadiazol-3-yl]-
2-[5-(2-Methyl-5-chlorophenylamino-[1,2,4]thiadiazol-3-yl]-
1-methylethyl}-(2,2,6,6- tetramethylpiperidine-4-yl)-amine
propane-2-yl}-(2,2,6,6- tetramethylpiperidine-4-yl)-amine
(9).
(
4).
Light crystals. Yield 69%. Calcd, %: С 61.35, H 7.72, N
White powder. Yield 72%. Calcd, %: С 59.77, H 7.64, N
17.89 C20H30FN5S. Found, %: С 61.23, H 7.87, N 17.92.
1
NMR H [200 MHz] δ: 0.71 (2H, m, С(3)НН, С(5)НН), 1.02,
16.59 C21H32ClN5S. Found, %: C 59.80, H 7.30, N 16.49.
C21H32ClN5S. Found, %: 59.87, H 7.34, N 16.44.
1
NMR H [200 MHz] δ: 0.71 (1H, t, J = 12.0 Hz, СНH), 0.85
1.04 (12H, s, 4xCH3), 1.07 (3H, d, J = 6.6 Hz, СН2CHCH3), 1.72
(2H, dd, J = 3.0, 12.6 Hz, С(3)НН, С(5)НН), 2.72 (2H, dd, J =
2.3, 6.6 Hz, СН2CHМе), 2.91 (1H, m, C(4)H), 3.34 (1H, m,
СН2CHМе), 7.04 (2H, t, J = 8.6 Hz, Нarom), 7.58 (2H, dd, J =
4.7, 9.1 Hz, Нarom).
(1H, t, J = 12.0 Hz, СНH), 1.12 (6Н, s, (СН3)2), 1.14 (3Н, d, J =
6.2 Hz, ССН3), 1.19 (6Н, s, (СН3)2), 1.85 (2H, dd, J = 3.5, 12.3
Hz, 2СН), 2.32 (3Н, s, Ph-СН3), 2.831 (2H, m, СН2), 2.99 (1H,
tt, J = 3.5, 11.6 Hz, NСН), 3.41 (1Н, m, NСН), 7.17 (2H, m,
Нarom), 7.52 (1H, s, Нarom).
2-[5-(5-Methylisoxazol-3-ylamino)-[1,2,4]thiadiazol-3-yl]-1-
methylethyl}-(2,2,6,6- tetramethylpiperidine -4-yl)-amine
2-[5-(4-Chlorophenylamino-[1,2,4]thiadiazol-3-yl]-1-
(
10).
White powder. Yield 56%. Calcd, %: С 57.11, H 7.99, N
methylethyl}-(2,2,6,6- tetramethylpiperidine-4-yl)-amine (5).
White powder. Yield 70%. Calcd, %: С 58.88, H 7.41, N
17.16 C20H30ClN5S. Found, %: С 58.80, H 7.40, N 17.34.
NMR 1H [200 MHz] δ: 0.86 (2H, m, С(3)НН, С(5)НН), 1.14
and 1.20 (12H, s and s, 4x CH3), 1.27 (3H, d, J = 6.65 Hz,
СН2CHCH3), 1.87 (2H, d, J = 12.5 Hz, С(3)НН, С(5)НН), 2.84
22.20, C18H30N6OS. Found, %: С 56.91, H 8.10, N 22.44.
NMR 1H [200 MHz] δ: 0.74 (2H, m, С(3)НН, С(5)НН), 1.00
and 1.10 (12H, c and с, 4xCH3), 1.12 (3H, d, J = 6.2 Hz,
СН2CHCH3), 1.76 (2H, d, J = 12.2 Hz, С(3)НН, С(5)НН), 2.33
(1H, s, C=CCH3), 2.83 (2H, m, СН2CHМе), 3.00 (1H, m,
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C(4)H), 3.42 (1H, m, СН2CHМе), 4.12 (2Н, br.s, 2NH), 5.87
(1H, s, C=CH).
PermeapadTM barrier was prepared according to the
description of di Cagno and Bauer-Brandl [27]. In brief, a
thin layer of phosphatidylcholine (S-100) was applied on a
support sheet (PützGmbH, Taunusstein, Germany). The final
barrier was composed of support layer and a dried layer of
lipid. The final barrier appears mechanically flexible and
resistant, and can be cut to size by scissors or a punching
tool.
2-[5-Cyclopropylamino-[1,2,4]thiadiazol-3-yl]-1-
methylethyl}-(2,2,6,6-tetramethylpiperidine-4-yl)-amine
(
11).
White powder. Yield 63%. Calcd, %: С 60.49, H 9.26, N
20.75 C17H31N5S. Found, %: С 60.66, H 9.11, N 20.74.
NMR 1H [200 MHz] δ: 0.76 (6H, m, С(3)НН, С(5)НН, CH2CH2),
1.07 and 1.13 (12H, c and с, 4xCH3), 1.13 (3H, d, J = 6.35 Hz,
СН2CHCH3), 1.79 (2H, m, С(3)НН, С(5)НН), 2.61 (1H, m,
NСН), 2.75 (2H, m, СН2CHМе), 2.91 (1H, m, C(4)H), 3.33
(1H, m, СН2CHМе), 6.87 (1H, br.s, NН).
2.3. Solubility experiments
All the experiments were carried out by the shake flask
method [28] at 298 K (± 0.1 K). The essence of the above
mentioned method includes determination of the
compound concentration in the saturated solution. Glass
ampoules containing the test substance and the solvent
were placed into an air thermostat supplied by a stirring
device. To ensure the equilibration condition, the
dissolution profiles of the compounds were investigated.
Thermodynamic equilibrium was determined from
preliminary experiments including measuring the kinetic
dependences of the concentration of the saturated
solutions for the investigated substances. The time needed
for reaching the plateau of drug concentration against time
N-{1-[5-(4-Bromophenylamino-[1,2,4]thiadiazol-3-yl]-
propane-2-yl}-(2,2,6,6- tetramethylpiperidine-4-yl)-amine
(
12).
Yellow powder. Yield 72%. Calcd, %: С 53.09, H 6.68, N
15.48 C20H30BrN5S. Found, %: С 53.28, H 6.56, N 15.64.
NMR 1H [200 MHz] δ: 0.73 (2H, m, С(3)НН, С(5)НН), 1.02
and 1.05 (12H, c and с, 4xCH3), 1.08 (3H, d, J = 6.5 Hz,
СН2CHCH3), 1.72 (2H, dd, J = 3.1, 12.5 Hz, С(3)НН, С(5)НН),
2.75 (2H, dd, J = 2.3, 6.5 Hz, СН2CHМе), 2.91 (1H, m, C(4)H),
3.33 (1H, m, СН2CHМе), 7.40 and 7.52 (4H, d and d, J = 8.8
Hz, Нarom).
was considered
a suitable equilibration time. The
incubation time of 24 h was estimated to be high enough
for the equilibrium to be reached and all the solubility
2.2. Materials and Solvents
experiments lasted 24
h
before the solubility
1-Octanol (1-Octanol, CH3(CH2)7OH, MW 130.2, lot
11K3688) ARG was from Sigma Chemical Co (USA). 1-
Hexane (C6H14, MW 86.18, lot 07059903 C) ARG was from
SDS (Peypin, France).
measurements were performed. An aliquot of the saturated
solution was taken and centrifuged under temperature
control (Biofuge stratus, Germany) for 5 minutes at 298 K
temperature. The solid phase was removed through
Bidistilled water (with electrical conductivity 2.1 μS cm-
1) was used for preparation of buffer solutions.
isothermal filtration by the filter MILLEX®HA 0.45 ꢀm
(Ireland). The saturated solution was diluted with the
corresponding solvent to the required concentration. The
molar solubility of drugs was determined by means of
spectrophotometer Cary-50 (USA) in UV spectral region (λ =
200 – 400 nm) with an accuracy of 2–4 %. The experimental
results are reported as an average value of at least three
replicated experiments.
Phosphate buffer pH 7.4 (I=0.15 mol/L) for distribution
and solubility experiments was prepared by combining
potassium dihydrogen phosphate solution (KH2PO4) (9.1 g
in 1 L) and disodium hydrogen phosphate dodecahydrate
solution (Na2HPO4·12H2O) (23.6 g in 1 L) salts [26]. The pH
values were measured by using pH meter FG2-Kit (Mettler
Toledo, Switzerland) standardized with pH 1.68, 6.86 and
9.22 solutions.
2.4. Permeation studies
Phosphate buffer saline for permeation experiments
was prepared as described in [27] by mixing a 2.5%
(weight/volume) sodium dihydrogen phosphate dehydrate
solution with a 1.8% (weight/volume) disodium hydrogen
phosphate dodecahydrate solution in a ratio of 1 to 4 in
order to obtain a 74 mM phosphate solution.
2.4.1. Experiment design
Permeapad™ barrier adapted to Franz diffusion cell (SES
GmbH-Analytical Systems, Germany) was applied to the
determination of the permeability coefficients (
P
) as
app
recommended by di Cagno et al. [27]. In our experiment the
volume of the drug solution in the lower (donor) chamber
was 8 ml (approximately 5 mM concentration) and the
volume of the buffer solution in the upper (acceptor)
chamber was 1.5 ml. A surface area of the Permeapad™
barrier disposed between the lower and upper chambers
was 1 cm2. Aliquots of 0.5 mL were periodically withdrawn
from the upper chamber and after each withdrawal
The pH of the solution was measured by pH meter FG2-
Kit (Mettler Toledo, Switzerland) and adjusted to
physiologic conditions (pH 7.3–7.4) by the addition of
sodium hydroxide. The osmolality of the buffer solution was
measured by Semi-Micro Osmometer K-7400 (Herbert
Knauer GmbH, Berlin, Germany) and adjusted to 280–290
mOsm by the addition of NaCl.
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ARTICLE
Journal Name
replaced by 0.5 ml of fresh buffer. The amount of the
permeated substance was analyzed spectrophometrically
using Cary-50 (USA) spectrophotometer at the appropriate
wavelength. Permeability coefficients through the support
layer (without phospholipids) were determined similarly to
those measured through the Permeapad™ barrier. The
permeation experiments were performed at least in
The shake flask method was used to determine the
distribution coefficients ( Doct / buf ) in 1-octanol/buffer (pH
7.4) system [31-34]. The procedure was performed as
follows. Buffer (pH 7.4) and 1-octanol were mixed
vigorously for 24 h at 298 K (± 0.1 K), to promote solvent
saturation in both phases. The solvents were standing long
enough to allow the phases to separate, and the compound
was dissolved in the 1-octanol phase to obtain stock
solution. The buffer saturated 1-octanol phase with the
dissolved substance, and 1-octanol saturated buffer phase
were placed in a glass vials and mixed during 24 hours at
298 K. The substance concentrations were determined
spectrophotometrically using calibration curves. The
distribution coefficient was calculated by the following
equation:
triplicate for each compound and the final
determined from the average of the individual
calculated from each replicate. All experiments were
P
-value was
app
P
app
carried out at 298 K (± 0.1 K).
2.4.2. Data analysis
The calculation procedure includes the following consecutive
steps. Firstly, the cumulative amount of the permeated
substance (Q), expressed in number of moles was calculated for
each time interval and plotted against the time (t), expressed in
seconds, taking into account the surface area of the barrier. The
flux (J) - the slope of the linear regression was determined by
the following equation:
Doct / buf = Coct / buf /Cbuf / oct
where Coct /buf and Cbuf / oct are the molar
(4)
concentrations of solute in the mutually saturated phases
of 1-octanol and buffer. The accuracy of the distribution
coefficient value was verified by checking the mass balance
of the starting amount of compound i compared to the
total amount of the compound partitioned between the
two phases:
dQ
J
=
(1)
A×dt
mi = moct /buf + mbuf / oct
(5)
Secondly, the obtained flux was applied to calculate the
apparent permeability coefficient (Papp) using the following
equation:
where mi = Ci ⋅V i is the starting mass (in moles) of the
compound, moct / buf = Coct / buf ⋅V oct / buf is the mass of
the substance dissolved in the water-saturated 1-octanol
phase, and mbuf / oct = Cbuf / oct ⋅V buf / oct is the mass of
the substance dissolved in the 1-octanol saturated water
phase.
J
P
=
(2)
app
C
0
where C0 is the substance concentration in the donor
compartment.
2.6. Calcium-blocking property experiments
At last, based on the permeability coefficients measured
through the Permeapad™ barrier (Papp) and support layer (Psup
)
Synaptosomes of the rat brain cortex were isolated
according to a standard procedure from the brain of
newborn rats (9-10 days old). For accumulation of
radioactive lable, Р2-fraction of synaptosomes was
suspended in the incubation buffer А: 132 mМ NaCl, 5 mМ
KCl, 5 mМ HEPES, 10 mМ glucose, pH 7.4 (protein
concentration – 1.5-2 mg/mL.). Calcium concentration in
the final volume was 1.25 mM. Glutamate concentration of
200 µM was applied for stimulation of 45Са2+ uptake. After 5
min incubation at 335 K, the process was terminated by the
filtration on GF/B-filters (Whatman, UK), washed three
times with cold buffer B (145 mM HEPES, 10 mM Tris, 5.4
mM Trilon B, pH 7.4). All the experiments were performed
in triplicate. The samples were measured by means of fluid
scintillator beta-analyzer SL-4000 (TriCarb 2800, Perkin
Elmer).
the permeability coefficient through the lipid layer (Plip) was
determined by the following equation:
1
1
1
=
+
(3)
P
P
P
sup
app
lip
where the inverse values of the permeability coefficients
are the resistances (R) of the components (lipid and
support) of the Permeapad™ barrier.
Standard deviation calculations and student’s t-test were
evaluated, P≤0.05 was considered as significantly different.
Thomson Tau test was applied in order to identify possible
outlier values.
In preliminary experiments, all the compounds were
tested for the ability to inhibit glutamate stimulated Ca
2.5. Distribution coefficients determination
uptake at the concentration of 100 ꢀM. If the inhibition of
Glu-Ca-uptake was 50% and more, then further studies
6 | J. Name., 2012, 00, 1-3
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ARTICLE
oct /buf
hex / buf
were carried out to determine the concentration
dependence of inhibition. The amount of the uptake in
synaptosomes 45Са2+ was determined by the difference of
the label content with and without uptake stimulation and
expressed in % control (control - 100%). The corresponding
value of K43/ 21 was calculated according to the following
equation:
(
D
) and 1-hexane/buffer (
D
important characteristic’s commonly used as a measure to
) systems are
describe many biological, chemical and physical processes.
oct /buf
For example,
D
processes of intestinal tract membrane permeability,
is often used to estimate the
hex / buf
whereas D
Moreover, the parameter characterizing the difference
- the processes occurring in the BBB.
between the 1-hexane phase to 1-octanol one
log Doct / hex ) is often used to distinguish the contributions
K43/ 21 = [(Ca4 – Ca3)/(Ca2 ꢀ Ca1)]ꢁ100%
(6)
(
of non –specific and specific interactions (donor-acceptor,
hydrogen bonds) of the drug molecules with biological
environments. Based on this, we have tried to describe the
distribution of the selected class of compounds in the
assigned model systems of immiscible solvents. The
where Ca1 is the Ca2+ influx in the blank experiment
(without glutamate and test compounds); Ca2 is the Ca2+
influx in the presence of glutamate only (Glu-Ca-uptake);
Ca3 is the Ca2+ influx in the presence of test compound
(without glutamate); Ca4 is the Ca2+ influx in the presence of
both glutamate and test compound.
oct /buf
hex/ buf
experimental data for
D
presented in Table 2. It should be noted that for almost all
and
D
are
hex/ buf
the compounds (except 14 D
1. This indicates that these substances predominantly
)
-values are less than
2.7. Calculation procedure
dissolve in the aqueous phase. On the the contrary, in the
oct / buf
1-octanol/buffer system,
D
10), therefore, the substances prefer the 1-octanol phase.
-values exceed 1 (except
All the descriptors were calculated by program package
HYBOT-PLUS (version of 2003) in Windows [35].
oct /buf
We studied the trends between
D
and selected
Equilibrium constants for the compounds synthesized
were calculated by ACD/ChemSketch program [36]. Analysis
of the equilibrium of charged state of the molecules was
conducted by using program package presented in [37].
physicochemical descriptors. The main purpose of such
search was connected with an idea to develop an algorithm
for prediction of the experimental data on the basis of just
molecular structure knowledge. The HYBOT [35] descriptors
were selected as independent parameters, because they
describe the interaction of drug molecules with biological
2.8. DSC experiments
media. It should be noted that the access to obtain the
oct /buf
The fusion temperatures of the compounds under
investigation have been determined using a Perkin-Elmer
Pyris 1 DSC differential scanning calorimeter (Perkin-Elmer
Analytical Instruments, Norwalk, Connecticut, USA) with
Pyris software for Windows NT. DSC experiments were
conducted in an atmosphere of flowing (20 cm3·min-1) dry
helium gas of high purity 0.99996 (mass fraction) using
standard aluminum sample pans and a heating rate of 2
K·min-1. The accuracy of weight measurements was 0.005
mg. The DSC was calibrated with an indium sample from
Perkin-Elmer (P/N 0319-0033). The value determined for
the fusion enthalpy corresponded to 28.48 J·g-1 (the
reference value was 28.45 J·g-1). The fusion temperature
correlation equation was complemented by
D
-
values for the related compounds which were prepared in
the present work (group I) and those which have been
published by us previously (group II) [23] (Table 1).
However, the compounds of group I are in the ionized state
in buffer (pH 7.4), whereas drugs of group II - in the form of
neutral entities. The appropriate calculation of the
equilibrium forms of the compounds by using the approach
described in [37] and taking into account the calculated pKa
(pKa1 = 14.01 - 14.04; pKa2 = 1.29 - 4.67) [36] confirms the
neutral forms of the compounds of group II. For this group
of compounds the equality is realized: K0oct /buf
=
Doct / buf and K0hex /buf Dhex / buf
=
.
was 429.5
± 0.1 K (determined from ten measurements).
In order to recalculate the distribution coefficients, the
ionization state of their molecules was determined taking
into account that the presently investigated thiadiazoles
(group I) are bases and have several ionizable groups. The
macroscopic dissociation constants were calculated
according to [36].
3. Results and discussion
3.1. Distribution experiments
Based on the calculated pKa-values we determined the
content of a number of the forms with different degrees of
protonation in aqueous solutions depending on the рН of
the media by using [37] taking into account the following
ionization processes:
The processes of distribution of compounds in biological
media play an important role in drug delivery. These
processes
contribute
significantly
to
membrane
permeability determining the concentrations of drug
compounds in the membrane/lipophilic compartments
(biological medium phases). In addition, the distribution
coefficient of the compounds in 1-octanol/buffer
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Table 2 Experimental solubilitya values of the compounds studied in buffer ( S2298 X 2298 ), distribution coefficients 1-octanol/buffer (pH 7.4) ( Doct / buf ) and 1-hexane/buffer
,
(pH 7.4) ( Dhex / buf ), partitioning coefficients for neutral molecules in 1-octanol/buffer ( K0oct / buf ) and 1-hexane/buffer ( K0hex / buf ), biological coefficient describing ability to
inhibit Glu-Ca-uptake ( K43/ 21 ), melting points ( Tm ), molecular polarizability (α), acceptor molecular ability to create hydrogen bonds ( Σ(Ca ) ), acceptor molecular ability to
create hydrogen bonds normalized to molecular polarizability ( Σ(Ca )/
α
).
X2298
Doct / buf
K0oct / buf
Dhex / buf
K0hex / buf
K43/ 21
Tm
α
Σ(Ca ) Σ(Ca )/α
S2298
/ mol⋅l-1
/ mol fract
/ %
/ K
10-4 12.60 ± 0.02 8.28
×
104
0.011 ± 0.003
72.3
305 ± 18 347.2 ± 0.5
47.113
7.45
×
10-3
1
1.35
×
7.68
7.75
0.163
0.165
0.172
0.166
10-4 11.01 ± 0.03 7.31
10-4
10-5
×
×
×
×
104 0.0031 ± 0.0002
104 0.0064 ± 0.0005
20.6
42.0
214 ±12 319.3 ± 0.5 47.02
5.57
9.24
4.86
3.72
×
×
×
×
10-3
10-3
10-3
10-3
2
3
4
5
1.00
1.67
8.77
6.71
×
×
×
×
5.0 ± 0.4
6.9 ± 0.2
3.25
4.56
189 ± 14 333.5 ± 0.5 45.094 7.76
104
104
0.017 ± 0.004
0.011 ± 0.005
112.8
73.0
177 ± 9
172 ± 3
334.2 ± 0.5 47.02
7.83
7.69
10-5 1.83 ± 0.04 1.22
323.2 ± 0.5
45.185
0.17
8.37
1.20
×
×
10-3
10-2
6
7
1.51
2.17
×
×
10-4
10-4
6.6 ± 0.4
4.4 ± 0.2
4.44
2.92
×
×
104
104
0.013 ± 0.001
0.014 ± 0.002
89.3
92.9
128.0 ± 0.1 320.2 ± 0.5 45.092 7.73
0.171
117 ± 2
309.1 ± 0.5
47.792
45.366
43.166
8.16
8.33
7.66
0.171
0.184
0.177
2.13
1.82
1.41
×
×
×
10-2
10-2
10-2
8
9
3.87
3.30
2.55
×
×
×
10-4
10-4
10-4
3.5 ± 0.3
7.6 ± 0.3
2.34
5.06
2.41
×
×
×
104
104
103
0.013 ± 0.003
0.024 ± 0.003
0.041 ± 0.002
86.3
158.0
258.6
112 ± 25 314.7 ± 0.5
112 ± 12 317.6 ± 0.5
10
0.375 ±
0.005
108 ± 6
342.6 ± 0.5
41.113
38.328
45.883
8.48
7.32
0.206
0.191
3.10
3.49
×
×
10-1
10-3
11
12
6.19
6.29
×
×
10-3 1.21 ± 0.02 3.19
×
×
103
104
0.097 ± 0.004
0.013 ± 0.001
655.5
85.0
86 ± 14
82 ± 5
341.4 ± 0.5
318.5 ± 0.5
10-5
2.041 ±
0.004
1.36
7.69
0.168
0.178
0.181
13
ndb
10-4 d
nd
16 4.45
10-4 d
17 4.836
10-4 d 8.30
10-3 f
nd
ndb
610
±
30c
6.10
×
102
0.17
±
0.01c
0.17
1.68
0.16
0.56
0.46
0.31
0.48
0.58
0.32
0.18
51 ± 9c
87 ± 2c
75 ± 6c
nd
402.3 ± 0.2e
nd
29.307 5.21
29.214 5.28
27.288 5.29
29.214 5.35
27.379 5.22
27.286 5.26
29.986 5.68
14 4.83
15
×
8.70
×
10-6 d 1038
±
10c 1.038
5c
2.26
50c 1.049
×
103
102
1.68±0.07c
nd
226
±
±
±
×
0.16
0.56
0.46
0.31
0.48
0.58
0.32
0.18
±
±
±
±
±
±
±
±
0.04c
0.01c
0.05c
0.02c
0.02c
0.03c
0.02c
0.01c
0.194
0.183
×
8.00
×
×
10-6 d 1049
×
103
102
102
88 ± 16c 374.5 ± 0.2e
89 ± 13c 408.4 ± 0.2e
×
10-6 d
10-5 f
nd
380
445
5c
3.80
4.45
×
×
0.191
0.193
0.19
18
19
2.19
×
3.95
×
±
28c
85 ± 4c
390.1 ± 0.2f
94
±
13c
5c
9.4
2.05
3.16
×
10
86.7 ± 0.5c
nd
20 3.04
21 3.40
22
×
10-3 d
10-3 g
5.48
×
×
10-5 d
10-4 g
205
±
×
102
102
108 ± 11c 363.4 ± 0.2e
88 ± 16c 376.3 ± 0.2g
0.215
0.204
0.258
27.56
25.36
5.93
5.18
×
6.13
316 ± 3c
×
nd
nd
45
±
1c
4.5
×
10
109 ± 20c
nd
23.307 6.01
a relative standard uncertainty for solubility values ur (S2298 ) = 0.03
;
ur
(
X2298
)
=
0.03
b nd – not determined;
c Ref.(23);
d Ref.(20);
e Ref.(19);
f Ref.(17);
g Ref. (18);.
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HL+ = H++L
H2L2+ = HL++H+
H3L3+ = H2L++H+
(
pK1)
pK2)
pK3)
(7)
(8)
(9)
The K0oct / buf -values have been analyzed concerning 32
descriptors. The suitable correlation was obtained for the
(
independent variables which include polarizability (
acceptor molecular ability to create hydrogen bonds
Ca
).The results of the correlation analysis can be
presented by equation:
α) and
(
(
Σ
(
)
The pKa1 = 10.95 corresponds to the protonation process of
a nitrogen atom of NH-group of a pipyridine ring, pKa2
=
logK0oct / buf = (0.16
±
0.39) + (0.16
±
0.02)⋅α
Σ(Ca
ꢀ
)
7.31 -7.36 – to the protonation of a bridge NH-group and
pKa3 = 0.45 -3.83 - to the protonation process of a nitrogen
atom of a thiadiazole ring. K1
constants of the mentioned
ionized/unionized particle distributions (%) as a function of
рН for the compound (as an example) is presented in Fig.
,
K2 and K3 are equilibrium
ꢀ (0.37 0.14)
±
⋅
(13)
equations. The
R = 0.9631;
σ = 0.30; F = 121.6; n=22
2
With an increase in the polarizability of the molecule the
distribution coefficient grows. In other words, non-specific
interactions are shown in more extent in 1-octanol phase as
compared with aqueous one. While the molecular ability to
create hydrogen bonds increases, the distribution
coefficient in 1-octanol/buffer system decreases. The
acceptor ability of the molecule to hydrogen bond
formation should be revealed both in aqueous and 1-
octanol phases. However, as follows from the derived
equation (13), implementation of such bonds in aqueous
phase is favorable. Such phenomenon can be due to a
considerably smaller size of the water molecule in
comparison with 1-octanol one, resulting in better
conjugation between the solute and solvent molecules in
the solution and higher probability of hydrogen bond
formation. Appropriate analysis was performed for the
distribution processes (calculation of the distribution
coefficients for the uncharged species) of the selected
compounds in 1-hexane/buffer system. Like in the previous
1SI (Supporting Information). It is found that all the
compounds have similar dissociation constant values and,
respectively, the protonation degree. As follows from the
Fig. 1SI all the compounds are completely ionized at pH 7.4
and their molecules exist as approximately equal shares in
the form of mono- and di-cations.
For ionizable compounds the distribution coefficient
describing the overall lipophilicity of drugs is the ratio of the
concentrations of all molecular forms of compounds
existing in equilibrium in the aqueous phase and uncharged
particles in 1-octanol. For the investigated thiadiazoles the
equilibrium distribution can be represented by an equation
by which the apparent distribution coefficient ( Doct / buf
)
can be experimentally determined:
Doct / buf = [L]o/([L]w + [HL+]w + [HL2+]w + [HL3+]w) (10)
where [L]o – concentration of the neutral form of the
compound in 1-octanol, [L]w, [HL+]w, [HL2+]w, and [HL3+]w
–
case, log K0hex
molecular polarizability and acceptor molecular ability to
create hydrogen bonds ( Σ(Ca ):
-value correlates in a best way with
/
buf
the concentration of particles in an aqueous buffer
solution. In view of the above equilibrium constants the
equation can be represented as follows:
)
Doct / buf =[L]o/([L]w+[L][H+]/K1+[L][H+]2/K2+
/
buf
log K0hex
= ꢀ (5.2
±
0.6) + (0.037
(0.69 0.23)
σ = 0.47; F = 66.6; n=22
±
0.032)⋅α
ꢀ
+ [L][H+]3/K3
(11)
ꢀ
±
⋅
Σ
(Ca )
(14)
Further transformation leads to the equation, that relates
the intrinsic distribution coefficients to the experimentally
determined apparent distribution coefficients, hydrogen
ion concentration (H+) and the dissociation constants, K1
R = 0.9355;
A comparison of equations (13) and (14) shows that
K0hex /buf is approximately two times more sensitive to the
variation of
,
K2 and K3
:
Σ
(Ca ) -descriptor than K0oct /buf . Most probably,
this phenomenon can be explained by the specific
interactions of the dissolved molecule with the solvent both
in 1-octanol and buffer phases in 1-octanol/buffer system.
Therefore, the contribution of the descriptor is partly
equalized. As opposed to this, in 1-hexane/buffer system
the dissolved molecules interact specifically only with one
phase (buffer) which leads to a higher regression coefficient
value. Thus, correlation equations (13) and (14) allow
predicting the distribution coefficients for the studied class
of compounds both in 1-octanol/water and 1-hexane/water
systems. It is interesting to compare the distribution
coefficients for the molecules of group I compounds
(normalized to the neutral species) with those of group II
which differ from each other by only one fragment (tetra-
methylpiperidine-fragment (group I) instead of hydroxyl-
K0oct /buf
=
Doct / buf (1 + [H+]/K1 + [H+]2/K2 + [H+]3/K3) (12)
⋅
To make the conditions of the distribution processes
comparable, we recalculated the values of Doct /buf and
Dhex / buf for the neutral forms of the compounds of group I
by equation (12) (in Table 2, these coefficients are
designated as K0oct /buf and K0oct /buf ).
The data in Table 2 show that the calculated intrinsic
distribution coefficients (normalized to neutral species) of
the studied thiadiazoles significantly exceed (more than 4
times) the experimental apparent ones (belong to the
ionized forms of the molecules). The estimated ratios
indicate greater lipophilicity of the neutral particles
compared with the charged ones as expected.
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group (group II)) both in 1-octanol/water and 1-
hexane/water systems. The results of the comparative
analysis are given in Fig. 1. In 1-octanol/water system the
difference of the distribution coefficients between group I
and group II compounds is positive with an average
chlorine-atom in para-position of the phenyl-ring by a
methyl-group (compound ) increases the solubility twice,
and by methoxy-group (compound ) or fluorine-atom
(compound ) – about 6 times. Changing a methylphenyl-
6
8
9
substituent to methylisoxazole one slightly enhances the
solubility (about 1.5 times). Interestingly, the introduction
of the second chlorine atom in the meta- position of the
ꢁ
log K0oct / buf -value of 2.58
compounds 10 22). A similar tendency for 1-hexane/water
log K0hex / buf -value of 2.47
system with an average 0.23
(excluding the pair of compounds 14) is observed. The
derived values of
log K0 can be determined by: firstly,
± 0.20 (excluding the pair of
/
ꢁ
±
phenyl ring of compound
leads to double the solubility. At that, the solubility values
of the structure isomers 3-chloro-4-methyl- ( ) and 3-
chloro-6-methyl- ( ) are approximately equal. According to
the solubility increase the investigated thiadiazoles are
ranged in the following order: 12 (4-bromophenyl-) ≤ (4-
chlorophenyl-) < (3-chloro-6-methylphenyl-) ≤ (3-chloro-
4-methylphenyl-) (3,4-dichlorophenyl-)
5 (3,4-dichloro-derivative (1))
2/
ꢁ
2
conformational differences of the molecules of groups I and
II in water and 1-octanol and also water and 1-hexane
phases; secondly, variation of the energy of hydrogen bond
forming by a hydroxyl-group in the considered phases.
4
5
4
2
/
Analysis of Doct hex -values showed the existence of
≤
1
≤
6
(4-
(3-
(4-
strong specific interactions of the drug molecules with the
1-octanol (1.5 < log Doct / hex < 3.6). On this ground it may be
assumed that the redistribution of molecules from blood
plasma through the blood-brain barrier will be aggravated.
methylphenyl-)
≤
3
(3-chloro-4-fluorophenyl-) < 7
methylthiaphenyl-)
≤
10 (methylisoxazol-)
<
9
fluorophenyl -) ≤ 8 (4-methoxyphenyl-) < 11 (cyclopropyl-).
The solubility change regularities described above do not
have a forecasting power. Therefore, it was important to
obtain correlation equations between the solubility
characteristics and any kinds of descriptors. HYBOT
descriptors were used as physicochemical descriptors [25].
Crystal lattice thermodynamic characteristics are important
parameters in solid substance dissolution. To take this
factor into account, Yalkowsky et al. [38] introduced crystal
melting temperature as a descriptor into the correlation
equation (General Solubility Equation). Using this descriptor
to analyze the solubility thermodynamics is justified by the
fact that in many cases there is a correlation between the
molecular crystal sublimation Gibbs energies and melting
temperatures [39]. Besides, Yalkowsky et al. [38] described
water solubility of the compounds using 1-octanol/water
partitioning coefficients as descriptors (for imitation of
hydration term). The descriptor series suggested by
Yalkowsky was used to describe selected compounds
solubility. As a result, the correlation equation looks as
follows:
Fig.1. Comparative analysis of distribution coefficients for the molecules of
group I compounds (normalized to the neutral particles) with those of group
II differ from each other by only one fragment (tetra-methylpiperidine-
fragment (group I) instead of hydroxyl-group (group II) both in 1-
octanol/buffer and 1-hexane/buffer systems
log(S2298
ꢀ (0.19 ± 0.14)· log(Doct / buf
R = 0.8071;
)
= (0.4 ± 1.5) –
ꢀ (7.2 ± 4.7)·10ꢀ2·Tm
= 0.31; F = 12.4; n = 15
3.2. Solubility in buffer and melting
temperatures
)
(15)
The results of solubility experiments in buffer (pH 7.4) at
298 K and melting points of the compounds studied are
presented in Table 2.
σ
It is well known that the nature and position of the
substituent may influence essentially the solubility of the
compounds in structure analogues series. In this
connection, the comparative solubility analysis for the
synthesized thiadiazoles was performed. It was found that
the solubility among the investigated compounds with
tetramethylpiperidine fragment differs within two orders of
magnitude. Compound (11) with a cyclopropyl-substituent
Thus, the solubility of this class of compounds decreases
with increasing the lipophilicity (due to reducing the
hydration term) and the melting temperature growth
(crystal lattice energy strengthening).
We also tried to find a correlation equation using
HYBOT descriptors. Analysis of all the available descriptors
found that the best correlations were obtained with
polarizability (
α) and acceptor molecular ability to create
has the maximal solubility, and compounds (12) and (5)
hydrogen bonds ( Σ(Ca
)
):
with 4-bromophenyl- and 4-chlorophenyl- substituents,
respectively, – the minimal ones. The replacement of a
10 | J. Name., 2012, 00, 1-3
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298
sol
data in Table 3 confirmed these conclusions. This fact is a
clear indication of a significant retention effect induced by
the lipids present in the artificial barrier and that the lipid
layer is the dominant impediment to drug permeation.
From this it can be concluded that for all compounds
investigated the rate limiting step in permeability was
represented by the lipid layer of the artificial barrier. Due to
this fact it is proposed that for screening purposes, it is
ꢁ
G
(Buffer)/ kJ ⋅
mol−1 = (38.3 ± 2.6) +
+
(0.23 ± 0.13)·
α
+ (ꢀ3.4 ± 0.9)
⋅
Σ(Ca )
(16)
R = 0.8493;
σ
= 1.54; F = 18.11; n = 17
Thus, the solubility-values of this class of compounds in a
buffer solution can be estimated based on the knowledge
of the structural formula only.
reasonable to go without measuring the
P
- value.
sup
The values of the permeation coefficients vary
depending on the nature and number of substituents in the
3.3. Permeability Properties
molecule. The maximum permeability coefficient (
P
lip ) is
derivative
2). Replacement of the methyl- group in this
observed
for
3-chloro-4-methylphenyl-
Recently, much attention has been paid on the
development of better bioavailable drugs, which
significantly reduce both dosage and side effects. Water
solubility and permeability are considered to be the main
parameters determining the bioavailability of drugs.
Therefore, in our work we have tried to analyze these
specified parameters. PermeapadTM barrier developed and
described in [27] was chosen as a biomimetic artificial
barrier to study the permeability. Advantages of
PermeapadTM (compared to other artificial systems such as
PAMPA, PVPA) are: a unique design, usability, functional
stability in a wide range of pH values, compatibility with
biomimetic media, adaptability to Franz diffusion cell and
shelf life (more than 1 year). The apparent permeability
(compound
compound by a chlorine atom (dichlorophenyl-substituted
compound ( )) leads to a small reduction of permeability.
1
Thus, the permeability coefficient of 3-chloro-4-
methylphenyl- substituted compound is about 1.5 times
higher than that of its 3-chloro-6-methyl- isomer (4),
indicating the effect of not only the nature but also the
position of the substituent on the permeability of
thiadiazoles. Significant reduction in permeability
(minimum values among all compounds) occurs in
substances with isoxazole
(10) and cyclopropyl (11)
substituents. In the order of permeability decreasing
phenyl-substituted substances are ranged in the following
coefficients through the PermeapadTM barrier
support layer ( sup ) and lipid layer ( lip ) of the selected
( Papp ),
order: 3-chloro-4-methyl- (
chloro-6-methyl- ( ) ≥ 3,4-dichloro- (
methyloxy- ( ) > 4-fluoro- ( ) ≥ 3-methylthia- (
) > 4-bromo- (12).
Due to the fact that the distribution and permeability
2
) > 3- chloro-4-fluoro- (
) > 4-methyl- (
) > 4-chloro-
3
) > 3-
4
1
6) ≥ 3-
P
P
8
9
7
compounds, measured in a buffer solution (рН 7.4) at
T=298 K are summarized in Table 3.
(
5
Table 3 Apparent permeability coefficients measured through Permeapad™ barrier
processes for the selected compounds were studied in the
same buffer solution (pH 7.4) (i.e. ionization degree of the
particles and their ratio remained the same for the two
(
P
), support Layer (
P
) and extrapolated (
P
, thus the contribution of
app
sup
lip
the lipid layer to permeability resistance) of all compounds tested. Results are
reported as average and standard deviation of replicate experiments (n = 3–5).
/
buf
/ buf
designated processes), we tried Doct
and Dhex
as
,
Apparent permeability coefficients
Compound
descriptors for the permeability. In its turn, log P
app
PermeapadTM
barrier
Support layer
Lipid layer
log P
functions. It should be noted that there are no any
and log P were selected as the analyzed
lip
sup
106 / cm
⋅
s-1
P
×
106 /
P ×
lip
106 /
/
buf
app
sup
correlations between the selected functions and Dhex
P
×
cm⋅
s-1
cm⋅
s-1
(Fig. 2SI). In contrast, good correlations between log P
app
and log Doct / buf
,
as well as between log P
and
1
2
9.8 ± 0.8
15.7 ± 1.2
20.6 ± 0.1
25.4 ± 1.5
30.8 ± 3.0
21. 9 ± 1.4
26.3 ± 1.9
22.8 ± 1.4
16.5 ± 1.3
16.3 ± 1.1
31.7 ± 4.4
27.6 ± 1.3
25.8 ± 2.2
26.1 ± 4.0
39.5 ± 3.1
30.9 ± 7.3
26.9 ± 4.8
14.1 ± 0.2
22.8 ± 7.7
18.2 ± 1.9
21.7 ± 0.7
18.6 ± 2.0
3.7 ± 0.4
lip
13.5 ± 0.4
13.9 ± 2.3
14.4 ± 0.1
8.6 ± 0.9
12.2 ± 2.7
10.1 ± 0.5
9.4 ± 1.1
8.7 ± 1.1
3.4 ± 0.3
5.4 ± 0.4
6.5 ± 0.7
log Doct / buf (Fig. 2), exist, which can be described by the
following equations:
3
4
5
/
buf
log Papp = ꢀ (5.25
R = 0.8737;
log Plip = ꢀ (5.13 0.05) + (0.63
R = 0.9351;
It is easy to see that log P is in 1.7 times more sensitive
±
0.05) + (0.37
±
0.06)
10ꢀ2; n = 12
log(Doct / buf
⋅
log(Doct
)
(17)
6
7
σ
= 9.46
⋅
8
9
±
±
0.08)
⋅
)
(18)
10
11
12
6.8 ± 0.6
σ
= 0.11; n = 12
9.5 ± 0.6
lip
/
buf
to log(Doct
) changes in comparison with log Papp . Thus,
The results showed by [27] indicate that the
permeability coefficients through the sheer support layer
the supporting layer contributes to the sensitivity of this
coefficient, but, on the other hand, the correlation
parameters are not much deteriorated. Consequently,
(
P
sup ) were in all cases higher compared to the respective
measured for the entire Permeapad™ barrier. Our
P
app
log P can be used to analyze the lipid layer permeability.
app
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If we take into account that log P -value is burdened with
log P = ꢀ (1.47
±
0.80) ꢀ (18.9
±
4.5)
⋅
Σ(Ca )/
α
(20)
lip
lip
two experimental errors (sequential execution of two
experiments: permeability PermeapadTM barrier and
R = 0.7961;
σ
= 0.19; n = 12
Support layer), then
P
total time of the experiment is halved).
value is more accurate (and the
app
a
a
b
Fig.3. Dependencies log P vs Σ(Ca )/
α
(a) and log P vs
lip
app
Σ(Ca )/
α
(b)
Thus, the permeability coefficients of this class of
compounds may be predicted based solely on their
structural formulas. Due to the small number of data points
the paired correlation coefficients are not rather high.
However, the improvement should be expected if the
number of data points would increase. Just as in the case of
the distribution coefficients the correlation equations for
log P and log P retain the same trends on the selected
b
/
buf
Fig.2. Dependencies log P
vs log Doct
(a) and log P
vs
app
lip
/
buf
log Doct
(b)
app
lip
descriptor.
As the next step it was interesting to analyze the
relationships of HYBOT physicochemical descriptors with
the obtained permeability characteristics. Testing all the
descriptors revealed that only for log P and log P (as in
3.4. Integral Solubility - Permeability Properties
lip
app
/
buf
the previous cases for log Doct
) the correlation with
acceptor molecular ability to create hydrogen bonds
normalized to molecular polarizability ( Σ(Ca )/ ) was
The available data on the solubility of the test compounds
and the apparent permeability coefficients make it possible
to evaluate the potential bioavailability of the substances,
similarly to Sanphui et al. [40]. The approach consists of the
α
observed. Results of the analysis for log P and log Papp are
lip
estimation of diffusion fluxes through the lipid barrier ( Jlip
)
presented in Fig. 3, whereas the relationship between
and Permeapad (
J
app ) (area of 1 cm2 and nominal thickness
log P and Σ(Ca )/
correlation can be described by the following equations:
α
is shown in Fig. 3SI. The received
sup
of 1 cm) on the assumption that at one end of the barrier
the concentration is kept equal to thermodynamic solubility
value, while at the other end - the substance is effectively
removed and its concentration is practically equal to zero
(sink conditions). In other words, the value of the product
log P = ꢀ (2.95
±
0.48) ꢀ (11.9
±
2.7)
⋅
Σ(Ca )/
α
(19)
app
R = 0.8089;
σ
= 0.11; n = 12
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of thermodynamic solubility
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(
S2298 in mol
⋅
l-1), lipid
in cm
s-1) and Permeapad
s-1) is analyzed. The
permeability coefficient (
permeability coefficient (
P
⋅
lip
P
in cm⋅
app
analysis in logarithmic scale yields the following equations:
log Jlip
=
log(S2298
⋅
P )
=
log S2298
+
log P
(21a)
(21b)
lip
lip
log Japp
=
log(S2298
⋅
P
)
=
log S2298
+ log P
app
app
Fig. 4 depicts the analyzed values. It is easy to see that
according to the degree of decreasing the diffusion flux
value (a measure of absorption) the compounds can be
placed in the following order: for lipid layer - 11
> 8 > 9 > 3
>
2
>
7
>
≈
1 ≈ 6 > 4 > 5 ≈ 10 > 12; for Permeapad – 11 > 8 > 9
Fig.5. Dependences of log Jlip vs log S2298 and log Japp vs log S2298
> 3 > 7
6 > 2
≈
1
≈
4 > 10 > 5 > 12.
3.5. Calcium-blocking Properties
The substances synthesized in the present study belong to
essentially new group of compounds in a series of 1,2,4-
thiadiazole derivatives which, being bioisosteric analogues
of a model «ampakin» cyclothiazide, as proposed, can be
the positive modulators of AMPA-receptors. First of all the
ability of the synthesized substances to modulate
glutamate-dependent Ca-uptake in synaptosomes of rat
brain cortex was studied. The biological activity results
(
K
43 / 21 -values) are presented in Table 2.
The obtained results clearly indicate that the variation
of the structure of the substituent in the phenyl ring as well
as the replacement of the phenyl ring by the cyclopropyl-
and isoxazole-ones influence the ability of thiadiazole
derivatives to inhibit calcium ion uptake. The biological
a
activity ( K43 / 21 -values) of the investigated substances is
presented in Fig. 6 as a histogram to compare the
compounds of groups I and II with the same substituents.
300
Compounds 1-12
Compounds 13-22
250
200
150
100
50
b
Fig.4. Experimental solubility data ( S2298 ), lipid layer permeability coefficients
0
1/13 2/14 3/15 4/16 5/17 6/18 7/19 8/20 9/21 10/22 11
12
(
P ) and diffusion fluxes through lipid layer ( Jlip ) for the compounds studied
lip
(a); Experimental solubility data
(
S2298 ), Permeapad layer permeability
Compound
coefficients
(
P
)
and diffusion fluxes through Permeapad
(
Japp
) for the
app
compounds studied (b)
Fig.6. Dependence log(1/K43/21) vs. log Doct / buf
With this range of changing the
P P
and app -values it turns
lip
The comparative analysis of the activity of thiadiazole
derivatives newly synthesized and investigated by us before
indicated that, on the whole, the presence of
out that the solubility values have the main contribution to
the diffusion fluxes (Fig. 5).
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tetramethylpiperidine ring and additional NH-group instead
of hydroxyl group facilitates a considerable growth of
K43/21 -values (up to 6 times for the compounds with
often used descriptor during the estimation of the
quantitative structure-activity correlations.
In order to describe graphically the activity-lipophilicity
dependence we applied an approach introduced in [42] for
compounds of specific type of activity and close chemical
structure according to equation:
dichlorophenyl-substituent (compounds
1
and 13)).
However, it is interesting to note that if the replacement of
the phenyl ring by isoxazole-one takes place, the activities
of the respective structural analogues of groups I and II
practically coincide. The analysis of the structure-activity
relationship was conducted for thiadiazoles synthesized in
2
log(1/ Kba )
=
a0
+
a1
⋅
log Doct /buf
−
a2
⋅
(log Doct /buf
)
+
a3
⋅
σ
+
(22)
+a4 ⋅ Es
the present study (Group I – compounds 1-12). Compound
where Kba – any experimental value characterizing the
biological activity, Doct / buf – distribution coefficient of the
substance between 1-octanol and aqueous (buffer) phase,
σ and Es –parameters describe the electronic and steric
impacts of the substituents, respectively, ai – constants
derived from processing the experimental data by the least
square method.
12 with a bromine atom as a substituent in para-position of
the phenyl ring, revealing maximal inhibiting activity
K
(minimal 43/ 21 ), was chosen as a reference. As seen from
the histogram data, replacing a bromophenyl- substituent
by cyclopropyl- and methyl-isoxazole ones leads to a slight
consistent increase in
K
43/ 21 (compounds 11 and 10).
Further reduction of the inhibitory activity is observed for
compounds with 4-fluorophenyl-, 3-methoxyphenyl- and 3-
methylthiaphenyl- substituents, which have similar activity
Fig. 7 depicts the dependence of the biological activity
of the investigated thiadiazoles on the lipophilicity of the
molecules characterizing by the distribution coefficient in 1-
octanol/buffer system.
values (
the methyl group (compound
phenyl ring contributes to a further increase of
that, the inhibitory activity of the compound with
methylisoxazol- fragment 10 is higher than that of
methylphenyl- substituted one ( ).
9
(112%) =
8
(112%) ≤
7
(117% )). Introduction of
) in a para- position of the
43/ 21 . At
6
K
(
)
12
-1,9
-2,0
-2,1
-2,2
-2,3
-2,4
-2,5
11
6
10
The results presented in the histogram allowed us to
evaluate the total biological potential of the compounds,
not only as possible neuroprotectors, but also cognitive
8
9
7
6
4
5
stimulants (nootrops). Thus, for the five compounds (
1, 2, 3,
3
4, and 5) the values of 43/ 21 > 150 suggest them for
K
2
further testing as stimulants of cognitive functions using a
recognition test. It is interesting to note that these
compounds have chlorine atoms as substituents in the
phenyl ring, at the same time, di-substituted substances
with a chlorine atom in the meta-position reveal the
1
-0,6
-0,4
-0,2
0,0
0,2
0,4
0,6
0,8
1,0
1,2
logDoct/buf
K
maximum 43/ 21 -values and according to the reduction of
the activation function of glutamate-dependent uptake of
Fig.7. Dependence log(1/K43/21) vs. log Doct / buf
calcium ions the thiadiazoles can be arranged in the
following order:
methyl) > (3-chloro-4-fluoro-) >
(4-chloro-).
1
(3-chloro-4-chloro) >
2 (3-chloro-4-
The correlation equation, in which the biological activity
acts as a dependent variable and the distribution coefficient
in 1-octanol/buffer system acts as an independent one, is as
3
4
(3-chloro-6-methyl) > 5
follows (compound
5 is excluded from the correlation):
3.6. Activity correlations
log(1/K43/21) = ꢀ (1.91
±
0.05) + (0.04
±
0.10) log Doct / buf
ꢀ
During scientific prognostication of physiological activity in
QSAR [41] the influence of the following phenomena is
usually assessed: electronic effects (influencing the
ionization or polarity of the substance), steric
characteristics of the structure (playing an important role
for the evaluation of the binding strength of the
investigated compound with the biological target),
lipophilicity (ability to dissolve in fats, characterizing the
properties of the substance to penetrate through the cell
membranes). The distribution coefficient (and its logarithm)
in 1-octanol/buffer system is well known to be the most
ꢀ
(0.45±
0.14) (log Doct / buf
)
(23)
2
R = 0.7092;
σ = 0.08; F = 13.19; n = 11
It should be noted that the obtained correlation is not high;
this fact may be explained by the rather small number of
the experimental points. Enhancement of the correlation
can be expected at the extension of the number of the
compounds investigated. However, the obtained
regularities support the parabolic model to be an
appropriate manner for describing the dependence of the
specific activity of thiadiazoles synthesized in the present
14 | J. Name., 2012, 00, 1-3
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ARTICLE
5
A. M. MacLeod, R. Baker, S. B. Freedman, S. Patel, K. J.
Merchant, M. Roe, J. Saunders, J. Med. Chem., 1990, 33,
2052.
study on the lipophilicity expressed by the distribution
coefficient in 1-octanol/water system.
6
7
8
9
Y. Iizawa, K. Okonogi, R. Hayashi, T. Iwahi, T. Yamazaki, A.
Imada, Antimicrob. Agents Chemother., 1993, 37, 100.
L. N. Petrova, S. O. Bachurin, Bull. Expert. Biol. Med.,
2006, 142, 51.
J. N. Kew, J. A. Kemp, Psychopharmacology, 2005, 179 (1),
4.
L. Parnetti, U. Senin, P. Mecocci, Drugs, 1997, 53 (5), 752.
4. Conclusions
Novel
1,2,4-thiadiazole
derivatives
as
potent
neuroprotectors were synthesized and identified. Their
ability to inhibit the glutamate stimulated Ca2+ uptake was
investigated in vitro. The structure of thiadiazole molecules
was shown to influence the inhibition properties. Activity-
lipophilicity correlation was estimated based on the
parabolic model. The solubility of 1,2,4-thiadiazoles was
measured in buffer solution pH 7.4 at 298 K. The
distribution coefficients in 1-octanol/buffer (pH 7.4) and 1-
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Potential bioavailability of the substances was determined
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Novel 1,2,4-thiadiazole derivatives as potent neuroprotectors were synthesized and their
solubility, distribution coefficients, permeability and biological properties were studied.