Novel 1,2,4-thiadiazole derivatives as potent neuroprotectors: Approach to creation of bioavailable drugs

Article abstract of DOI:10.1021/mp300011r

Novel 1,2,4-thiadiazole derivatives as potent neuroprotectors were synthesized and identified. Their ability to inhibit the glutamate stimulated Ca uptake was measured. Permeation experiments on the phospholipid membranes were conducted, and the apparent

Full text of DOI:10.1021/mp300011r

Article
pubs.acs.org/molecularpharmaceutics
Novel 1,2,4-Thiadiazole Derivatives as Potent Neuroprotectors:
Approach to Creation of Bioavailable Drugs
German L. Perlovich,*,†,‡ Alexey N. Proshin,‡ Tatyana V. Volkova,† Ludmila N. Petrova,‡
and Sergey O. Bachurin‡
†Institute of Solution Chemistry, Russian Academy of Sciences, 153045 Ivanovo, Russia
‡Institute of Physiologically Active Compounds, Russian Academy of Sciences, 142432 Chernogolovka, Russia
ABSTRACT: Novel 1,2,4-thiadiazole derivatives as potent
neuroprotectors were synthesized and identified. Their ability
to inhibit the glutamate stimulated Ca uptake was measured.
Permeation experiments on the phospholipid membranes were
conducted, and the apparent permeability coefficients were ob-
tained. The partition coefficients in n-octanol/buffer (pH 7.4)
and n-hexane/buffer (pH 7.4) immiscible phases (as model
systems for characterizing gastrointestinal tract membranes
and BBB) were determined. A classification of the studied
compounds from the standpoint of “permeability−activity”
properties was proposed.
KEYWORDS: 1,2,4-thiadiazole derivatives, neuroprotectors, permeability, phospholipid membrane, partitioning, specific activity
The interaction with target receptors is a key property in the
designing of drug molecules. However, the drug delivery pro-
cesses also play an important role in the creation of bioavailable
drugs. Therefore, obtaining the screening parameters character-
izing solubility, partitioning, and membrane permeability is an
essential part of drug design. In the present study, a variety of in
vitro and in vivo methods were used to perform the primary
screening and the selection of potential neuroprotectors in a
wide spectrum of GluR-ligands. Particularly, the properties of
novel 1,2,4-thiadiazoles (see Table 1 in the Experimental
Section) were analyzed to inhibit the specific glutamate-
stimulated calcium ions uptake (Glu-Ca-uptake) in rat brain
synaptosomes. The noted compounds correspond to molecules
which include a thiadiazole pharmacophoric fragment. This
class of substances demonstrates very interesting pharmaco-
logical10 and neuroprotector11 properties. Glutamate neuro-
mediator plays an important role in the working of the nervous
system. However, excess of the neuromediator leads to
superactivation of the glutamate receptors and can generate
programmed cell death (apoptosis).12 The inhibition ability of
45Ca2+ glutamate-dependent uptake in rat brain synaptosomes
gives an opportunity to estimate the influence of the com-
pounds on pathological processes at various neurodegenerative
diseases.13,14
1. INTRODUCTION
The N-methyl-D-aspartate (NMDA) receptor belongs to a
family of the ionotropic glutamate receptors and performs
important functions in the central nervous system. It is mainly
involved in the neuronal signaling processes, memory
consolidation, and synaptic plasticity.1,2 The neurotoxicity
which is induced by NMDA hyperactivation leads to a number
of pathological conditions, ranging from acute neurodegener-
ative disorders, such as stroke and trauma, to chronic forms
of neurodegenerative diseases, i.e., Huntington’s disease,
Parkinson’s disease, Alzheimer’s disease (AD), as well as lateral
amyotrophic sclerosis.3−6
It is generally recognized that the specific inhibition of
calcium ion influx via hyperactivated glutamate(Glu)-receptor-
channel complex (GluR), particularly NMDA-subtype receptor,
provides sufficient protection against a diverse group of neuro-
logical stresses related to the Glu excitotoxicity, such as brain
ischemia, AD, etc.7 In the case of AD, it is considered that
specific inhibitors of Glu-stimulated calcium uptake in nervous
cells provide sufficient neuronal protection against the neuro-
toxic effect associated with β-amyloid peptide (Aβ) as the
major pathogen in AD, which is particularly realized by the
potentiating of endogenous glutamate excitotoxic effects.8 On
the other hand, it is assumed that glutamate receptor agonists
(especially α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic
acid (AMPA)-subtype receptor agonists) exhibit strong
cognition-enhancing effects due to the activation of gluta-
matergic neurotransmission.9 Such dualism in properties of
glutamatergic compounds explains a strong interest in these
compounds as promising neuroprotectors and cognition-
enhancers.
Received: January 8, 2012
Revised: February 14, 2012
Accepted: February 20, 2012
Published: February 21, 2012
© 2012 American Chemical Society
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Table 1. Structures of Studied Compounds
It should be noted that, for the considered class of the
compounds, it keeps the Lipinski’s rule of five. This fact
confirms, once more, that selected 1,2,4-thiadiazoles could be
perspective candidates as druglike substances. This study is
aimed at developing a novel approach for the prediction of
bioavailable drug molecules with maximal specific activity.
Scheme 1
2. EXPERIMENTAL SECTION
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.
The compounds synthesized are presented in Table 1. 1-[5-
Amino-1,2,4-thiadiazol-3-yl]propan-2-ols (IV) were prepared
by a reduction of appropriated 1-[5-amino-1,2,4-thiadiazol-3-yl]-
propan-2-ones (III). The synthetic method for ketones (III) is
based on Boulton−Katrizky’s rearrangement15 of isoxazolic thio-
urea (II) (obtained by a standard reaction between isothio-
cyanates and 3-amino-5-methyl-izoxazole (way A) in dipolar
aprotic solvents (DMSO, acetonitrile) to 1,2,4-thiadiazoles). At
room temperature the reaction in DMSO passes completely in
5−6 h.16 It should be mentioned that this method of synthesis
makes it possible to produce only N-monosubstituted derivatives
of 1,2,4-thiadiazole, namely [5-N-monosubstituted-amino-1,2,4-
thiadiazol-3-yl]propan-2-ones (III, R2 = H). Therefore, 3-
isothiocyanato-5-methylisoxazole (I)17 was utilized to obtain
N,N-disubstituted derivatives of 1,2,4-thiadiazole synthesized in
the first instance. The received isothiacyanate was introduced in
the reaction with secondary amines (way B) (inaccessible for this
rearrangement before), and such an approach gave an oppor-
tunity for synthesizing the original N,N-disubstituted derivatives
of 1,2,4-thiadiazoles (III) with the yield of 60−95%. Next, the
keto-derivatives of thiadiazole (III) were reduced by sodium
borohydride in methanol to afford the desirable 1-[5-amino-
1,2,4-thiadiazol-3-yl]propan-2-ols (IV).18
2.1.2. Chemical Procedures. 1-[5-amino-1,2,4-thiadiazol-3-
yl]propan-2-ols were synthesized by standard and accessible
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methods at the Institute of Physiologically Active Compounds
of the Russian Academy of Sciences.
(R,S)-1-[5-(4-Chlorophenylamino)-1,2,4-thiadiazol-3-yl]-
propan-2-ol (5). Yield 83%; mp 132−134 °C. H NMR [200
MHz, CDCl3] δ: 1.33 (3H, d, J = 6.2, CH3), 2.86 (1H, dd, J =
3.0, 15.6 Hz, CH), 3.04 (1H, dd, J = 8.6, 15.6 Hz, CH), 4.30
(1H, br s, OH), 4.31 (1H, m, CH), 7.24 (2H, d, J = 8.9 Hz,
ArH), 7.43 (2H, d, J = 8.5 Hz, ArH), 8.42 (1H, br s, NH). Anal.
(C11H12ClN3OS) C,H,N.
1
3-Isothiocyanato-5-methylizoxazole (I). 3-Amino-5-meth-
ylizoxazole (49.05 g, 0.5 M) in CH2Cl2 (300 mL) was added
dropwise to the stirred mixture of thiophosgene (42.2 mL,
0.55 M) and NaHCO3 (92.4 g, 1.1 M) in H2O (200 mL). The
reaction mixture was cooled in an ice bath and kept under
stirring for 1 h. After that the dichloromethane layer was
separated and washed out with a saturated aqueous solution of
NaCl (2 × 50 mL). The solution was dried (Na2SO4), and the
solvent was evaporated. The obtained oil was distilled in
vacuum. The yield amounted to 36.8 g (52.6%), bp 39−41 °C
(1 mmHg). 1H NMR [200 MHz, CDCl3] δ: 2.50 (c, 3H, CH3),
6.00 (c, 1H, CH). 13C NMR [200 MHz, CDCl3] δ: 172.9 (C5),
153.1 (C3), 145.4 (C7), 100.1 (C4), 13.2 (C6).
2.1.3. General Procedure for the Preparation of (R,S)-1-[5-
Amino-1,2,4-thiadiazol-3-yl]propan-2-ols (IV). Step 1. 3-
Isothiocyanato-5-methylizoxazole (0.01 M) in CH3CN (10 mL)
was added slowly to the stirred solution of amine (0.01 M) in
CH3CN (10 mL). After the addition was over, the mixture was
brought to reflux. Then the solution was cooled to room
temperature. Generally, one day was required for precipitate
formation. The grown crystals were filtered.
Step 2. 1-[5-Amino-1,2,4-thiadiazol-3-yl]propan-2-one (0.01 M)
obtained at step 1 was dissolved in 20 mL of methanol. NaBH4
(0.015 M) was added portionwise to the solution, and the
mixture was stirred for 30 min, at the same time hydrogen
gassing was stopped. The solvent was removed until the
resultant residue was dry, and then the residue was taken up in
dichloromethane/water. The organic layer was separated, dried
over Na2SO4, and filtered. The solvent was evaporated. The
crude powder was recrystallized from isopropanol to derive the
final product. The overall yield was 60−90%.
(R,S)-1-[(5-Phenylamino)-1,2,4-thiadiazol-3-yl]propan-2-
ol (1). The overall yield for the two steps was 62%; mp 101−
103 °C. 1H NMR [200 MHz, CDCl3] δ: 1.32 (3H, d, J = 6.3 Hz,
CH3), 2.85 (1H, dd, J = 3.0, 15.5 Hz, CH), 3.02 (1H, dd, J =
8.8, 15.5 Hz, CH), 4.10 (1H, br s, OH), 4.32 (m, 1H, CH),
7.15−7.52 (5H, m, ArH), 9.16 (1H, br s, NH). Anal.
(C11H13N3OS) C,H,N.
(R,S)-1-{5-[4-(1-Hydroxyethyl)phenylamino]-1,2,4-thiadiazol-
1
3-yl}propan-2-ol (6). Yield 64%; mp 144−146 °C. H NMR
[200 MHz, DMSO-d6] δ: 1.14 (3H, d, J = 6.0 Hz, CH3), 1.33
(3H, d, J = 6.5 Hz, CH3), 2.66 (1H, dd, J = 6.1, 14.2 Hz, CH),
2.81 (1H, dd, J = 7.0, 14.2 Hz, CH), 4.13 (1H, m, CH), 4.42
(1H, d, J = 4.4 Hz, OH), 4.66 (1H, m, CH), 4.84 (1H, d,
J = 4.0 Hz, OH), 7.27 (2H, d, J = 8.4 Hz, ArH), 7.38 (2H, d, J =
8.4 Hz, ArH), 10.66 (1H, s, NH). Anal. (C13H17N3O2S) C,H,N.
(R,S)-1-[5-(4-Trifluoromethylphenylamino)-1,2,4-thiadiazol-
1
3-yl]propan-2-ol (7). Yield 85%; mp 143−145 °C. HNMR
[200 MHz, CDCl3] δ: 1.35 (3H, d, J = 6.3, CH3), 2.91 (1H, dd,
J = 8.6, 15.5 Hz, CH), 3.08 (1H, dd, J = 3.0, 15.5 Hz, CH), 3.80
(1H, br s, OH), 4.36 (1H, m, CH), 7.39 (2H, d, J = 8.6 Hz,
ArH), 7.70 (2H, d, J = 8.6 Hz, ArH), 9.30 (1H, br s, NH). Anal.
(C12H12F3N3O2S) C,H,N.
(R,S)-1-[5-(3-Chloro-4-methylphenylamino)-1,2,4-thiadiazol-
1
3-yl]propan-2-ol (8). Yield 76%; mp 104−106 °C. H NMR
[200 MHz, CDCl3] δ: 1.36 (3H, d, J = 6.2, CH3), 2.43 (3H, s,
CH3), 2.88 (1H, dd, J = 8.6, 15.5 Hz, CH), 3.05 (1H, dd, J =
3.0, 15.5 Hz, CH), 4.35 (1H, m, CH), 4.51 (1H, br s, OH),
7.14 (1H, dd, J = 2.4, 8.1 Hz, ArH), 7.34 (2H, d, J = 8.5 Hz,
ArH), 9.24 (1H, br s, NH). Anal. (C12H14ClN3OS) C,H,N.
(R,S)-1-[5-(3,4-Dichlorophenylamino)-1,2,4-thiadiazol-3-
1
yl]propan-2-ol (9). Yield 77%; mp 143−145 °C. H NMR
[200 MHz, CDCl3] δ: 1.35 (3H, d, J = 6.1, CH3), 2.88 (1H, dd,
J = 8.6, 15.6 Hz, CH), 3.06 (1H, dd, J = 2.8, 15.6 Hz, CH), 4.35
(1H, m, CH), 4.45 (1H, s, OH), 7.18 (1H, dd, J = 2.7, 8.6 Hz,
ArH), 7.48 (2H, m, ArH), 9.17 (1H, br s, NH). Anal.
(C11H11Cl2N3OS) C,H,N.
(R,S)-1-[5-(4-Fluorophenylamino)-1,2,4-thiadiazol-3-yl]-
1
propan-2-ol (10). Yield 82%; mp 103−105 °C. H NMR
[200 MHz, DMSO-d6] δ: 1.18 (3H, d, J = 6.3 Hz, CH3), 2.72
(1H, dd, J = 4.0, 14.2 Hz, CH), 2.85 (1H, dd, J = 7.0, 14.2 Hz,
CH), 4.17 (1H, m, CH), 4.35 (1H, d, J = 4.4 Hz, OH), 7.06
(2H, t, J = 8.3 Hz, ArH), 7.55 (2H, ddd, J = 2.38, 4.6, 9.1 Hz,
ArH), 10.69 (1H, s, NH). Anal. (C11H12FN3OS) C,H,N.
(R,S)-1-[(5-(2-Chloro-5-trifluoromethylphenylamino)-
1,2,4-thiadiazol-3-yl]propan-2-ol (11). Yield 66%; mp 116−
(R,S)-1-[5-(4-Methylphenylamino)-1,2,4-thiadiazol-3-yl]-
1
propan-2-ol (2). Yield 73%; mp 116−118 °C. H NMR [200
MHz, DMSO-d6] δ: 1.19 (3H, d, J = 5.5 Hz, CH3), 2.33 (3H, s,
CH3), 2.73 (1H, dd, J = 6.0, 13.7 Hz, CH), 2.84 (1H, dd, J =
7.0, 13.7 Hz, CH), 4.17 (1H, m, CH), 4.41 (1H, d, J = 4.5 Hz,
OH), 7.12 (2H, d, J = 8.3 Hz, ArH), 7.34 (2H, d, J = 8.3 Hz,
ArH), 10.64 (1H, s, NH). Anal. (C12H15N3OS) C,H,N.
(R,S)-1-[5-(4-Ethoxyphenylamino)-1,2,4-thiadiazol-3-yl]-
propan-2-ol (3). Yield 72%; mp 111−113 °C. 1H NMR
[200 MHz, CDCl3] δ: 1.30 (3H, d, J = 5.5, CH3), 1.48 (3H, t,
J = 7.0, CH3), 2.86 (2H, m, CH2), 4.07 (2H, q, J = 7.0, CH2),
4.27 (1H, m, CH), 4.30 (1H, br s, OH), 6.97 (2H, d, J = 8.6 Hz,
ArH), 7.23 (2H, d, J = 8.6 Hz, ArH), 8.68 (1H, br s, NH). Anal.
(C13H17N3O2S) C,H,N.
1
118 °C. H NMR [200 MHz, CDCl3] δ: 1.36 (3H, d, J = 6.2,
CH3), 2.94 (1H, dd, J = 8.4, 15.6 Hz, CH), 3.09 (1H, dd, J =
3.5, 15.6 Hz, CH), 3.61 (1H, br s, OH), 4.36 (1H, m, CH),
7.37 (1H, dd, J = 1.2, 8.3 Hz, ArH), 7.61 (1H, d, J = 8.0, ArH),
8.26 (1H, s, ArH), 8.31 (1H, br s, NH). Anal. (C12H11ClF3N3OS)
C,H,N.
(R,S)-1-[5-(3-Methylphenylamino)-1,2,4-thiadiazol-3-yl]-
propan-2-ol (12). Yield 72%; mp 88−89 °C. 1H NMR [200 MHz,
DMSO-d6] δ: 1.18 (3H, d, J = 6.2 Hz, CH3), 2.37 (3H, s, CH3),
2.72 (1H, dd, J = 6.2, 14.2 Hz, CH), 2.84 (1H, dd, J = 7.0,
14.2 Hz, CH), 4.17 (1H, m, CH), 4.46 (1H, d, J = 4.4 Hz,
OH), 7.12 (2H, d, J = 6.8 Hz, ArH), 7.14−7.30 (3H, m, ArH),
10.71 (1H, s, NH). Anal. (C12H15N3OS) C,H,N.
(R,S)-1-[5-(4-Methoxyphenylamino)-1,2,4-thiadiazol-3-yl]-
propan-2-ol (13). Yield 81%; mp 90−91 °C. 1H NMR [200 MHz,
DMSO-d6] δ: 1.18 (3H, d, J = 6.3 Hz, CH3), 2.69 (1H, dd, J =
7.0, 14.2 Hz, CH), 2.82 (1H, dd, J = 7.0, 14.2 Hz, CH), 3.78
(3H, s, CH3), 4.15 (1H, m, CH), 4.42 (1H, d, J = 4.5 Hz, OH),
(R,S)-1-[5-(3-Chloro-4-fluorophenylamino)-1,2,4-thiadia-
1
zol-3-yl]-propan-2-ol (4). Yield 68%; mp 124−126 °C. H
NMR [200 MHz, DMSO-d6] δ: 1.18 (3H, d, J = 6.3 Hz, CH3),
2.73 (1H, dd, J = 6.3, 14.4 Hz, CH), 2.87 (1H, dd, J = 6.7, 14.4
Hz, CH), 4.17 (1H, m, CH), 4.29 (1H, d, J = 4.7 Hz, OH),
7.20 (1H, t, J = 8.8 Hz, ArH), 7.45 (1H, ddd, J = 2.8, 4.2,
9.1 Hz, ArH), 7.82 (1H, dd, J = 2.8, 6.5 Hz, ArH), 8.98 (1H, br
s, NH). Anal. (C11H11ClFN3OS) C,H,N.
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6.87 (2H, d, J = 9.0 Hz, ArH), 7.40 (2H, d, J = 9.0 Hz, ArH),
10.54 (1H, s, NH). Anal. (C12H15N3O2S) C,H,N.
4.41 (1H, d, J = 4.2 Hz, OH), 8.65 (1H, s, NH). Anal.
(C8H13N3OS) C,H,N.
(R,S)-1-[5-(4-Pyridin-2-ylpiperazin-1-yl)-1,2,4-thiadiazol-3-
(R,S)-1-[5-(2-Chlorophenylamino)-1,2,4-thiadiazol-3-yl]-
propan-2-ol (14). Yield 77%; mp 86−88 °C. 1H NMR [200 MHz,
CDCl3] δ: 1.36 (3H, d, J = 6.1, CH3), 2.91 (1H, dd, J = 8.8,
15.6 Hz, CH), 3.08 (1H, dd, J = 3.1, 15.6 Hz, CH), 4.30 (1H,
br s, OH), 4.34 (1H, m, CH), 7.15 (1H, t, J = 7.5 Hz, ArH),
7.35−7.55 (2H, m, ArH), 7.67 (1H, d, J = 8.1 Hz, ArH), 8.25
(1H, br s, NH). Anal. (C11H12ClN3OS) C,H,N.
1
yl]propan-2-ol (23). Yield 63%; mp 106−107 °C. H NMR
[200 MHz, DMSO-d6] δ: 1.14 (3H, d, J = 6.0 Hz, CH3), 2.63
(1H, dd, J = 6.3, 14.4 Hz, CH), 2.74 (1H, dd, J = 7.0, 14.4 Hz,
CH), 3.60 (4H, m, N(CH2)2), 3.68 (4H, m, N(CH2)2), 4.08
(1H, m, CH), 4.31 (1H, d, J = 4.4 Hz, OH), 6.61 (1H, dd, J =
4.9, 7.9 Hz, PyH), 6.80 (1H, d, PyH), 7.50 (1H, td, J = 7.9,
2.0 Hz, PyH), 6.61 (1H, dd, J = 2.1, 4.9 Hz, PyH). Anal.
(C16H19F3N4OS) C,H,N.
(R,S)-1-[5-(2-Fluorophenylamino)-1,2,4-thiadiazol-3-yl]-
1
propan-2-ol (15). Yield 73%; mp 79−81 °C. H NMR [200
(R,S)-1-{5-[4-(3-Trifluoromethylphenyl)piperazin-1-yl]-
1,2,4-thiadiazol-3-yl}propan-2-ol (24). Yield 66%; mp 95−97
°C. 1H NMR [200 MHz, DMSO-d6] δ: 1.16 (3H, d, J = 6.1 Hz,
CH3), 2.65 (1H, dd, J = 6.0, 14.3 Hz, CH), 2.77 (1H, dd, J =
6.8, 14.3 Hz, CH), 3.42 (4H, m, N(CH2)2), 3.68 (4H, m,
N(CH2)2), 4.12 (1H, m, CH), 4.32 (1H, d, J = 4.4 Hz, OH),
7.02−7.26 (4H, m, ArH). Anal. (C16H19F3N4OS) C,H,N.
(R,S)-1-[3-(2-Hydroxypropyl)-[1,2,4]thiadiazol-5-yl]-
piperidine-4-carboxylic Acid Amide (25). Yield 60%; mp 134−
MHz, CDCl3] δ: 1.34 (3H, d, J = 6.1, CH3), 2.86 (1H, dd, J =
8.6, 15.7 Hz, CH), 3.03 (1H, dd, J = 2.8, 15.7 Hz, CH), 4.30
(1H, br s, OH), 4.33 (1H, m, CH), 7.20−7.48 (4H, m, ArH),
8.28 (1H, br s, NH). Anal. (C11H12FN3OS) C,H,N.
(R,S)-1-[5-(3-Methylthiophenylamino)-1,2,4-thiadiazol-3-
1
yl]-propan-2-ol (16). Yield 69%; mp 101−102 °C. H NMR
[200 MHz, CDCl3] δ: 1.32 (3H, d, J = 6.2, CH3), 2.53 (3H, s,
CH3), 2.83 (1H, dd, J = 8.6, 15.5 Hz, CH), 3.00 (1H, dd, J =
2.9, 15.5 Hz, CH), 4.28 (1H, br s, OH), 4.33 (1H, m, CH),
6.98−7.42 (4H, m, ArH), 8.35 (1H, br s, NH). Anal.
(C12H15N3OS2) C,H,N.
1
136 °C. H NMR [200 MHz, DMSO-d6] δ: 1.12 (3H, d, J =
6.1 Hz, CH3), 1.15−1.93 (4H, m, CH2CHCH2), 2.40 (1H, m,
CH2CHCH2), 2.58 (1H, dd, J = 6.0, 14.4 Hz, CH), 2.70 (1H,
dd, J = 7.2, 14.4 Hz, CH), 3.11−3.30 (2H, m, N(CHH)2),
3.75−3.92 (2H, m, N(CHH)2), 4.07 (1H, m, CH), 4.31 (1H, d,
J = 4.4 Hz, OH), 6.60 (1H, br s, NH), 7.17 (1H, br s, NH).
Anal. (C11H18N4O2S) C,H,N.
(R,S)-1-[5-(3-Methyl-4-chlorophenylamino)-1,2,4-thiadiazol-
1
3-yl]propan-2-ol (17). Yield 80%; mp 134−136 °C. H NMR
[200 MHz, DMSO-d6] δ: 1.18 (3H, d, J = 6.0 Hz, CH3), 2.34
(3H, s, CH3), 2.73 (1H, dd, J = 6.3, 14.2 Hz, CH), 2.86 (1H,
dd, J = 6.7, 14.2 Hz, CH), 4.16 (1H, m, CH), 4.44 (1H, d,
J = 4.7 Hz, OH), 7.21 (1H, d, J = 8.4 Hz, ArH), 7.31 (1H, dd,
J = 2.3, 8.3 Hz, ArH), 7.63 (1H, d, J = 2.1 Hz, ArH), 10.80
(1H, br s, NH). Anal. (C12H14ClN3OS) C,H,N.
(R,S)-1-[5-(5-Methylisoxazol-3-ylamino)-[1,2,4]thiadiazol-
1
3-yl]propan-2-ol (26). Yield 62%; mp 189−191 °C. H NMR
[200 MHz, DMSO-d6] δ: 1.15 (3H, d, J = 6.0 Hz, CH3), 2.41
(3H, s, CH3), 2.73 (1H, dd, J = 6.0, 14.1 Hz, CH), 2.85 (1H,
dd, J = 7.0, 14.1 Hz, CH), 4.12 (1H, m, CH), 5.70 (2H, br s,
OH, NH), 6.01 (1H, s, CH). Anal. (C9H12N4O2S) C,H,N.
(R,S)-1-{5-[4-(4-Nitrophenyl)piperazin-1-yl]-1,2,4-thiadiazol-
(R,S)-1-[5-(2-Methyl-3-chlorophenylamino)-1,2,4-thiadia-
1
zol-3-yl]propan-2-ol (18). Yield 71%; mp 124−125 °C. H
NMR [200 MHz, DMSO-d6] δ: 1.17 (3H, d, J = 6.0 Hz, CH3),
2.37 (3H, s, CH3), 2.68 (1H, dd, J = 6.0, 14.1 Hz, CH), 2.80
(1H, dd, J = 6.8, 14.1 Hz, CH), 4.12 (1H, m, CH), 4.36 (1H, d,
J = 4.5 Hz, OH), 7.17 (2H, m, ArH), 7.77 (1H, m, ArH), 10.08
(1H, br s, NH). Anal. (C12H14ClN3OS) C,H,N.
1
3-yl}propan-2-ol (27). Yield 74%; mp 91−93 °C. H NMR
[200 MHz, CDCl3] δ: 1.31 (3H, d, J = 6.3 Hz, CH3), 2.79 (1H,
dd, J = 8.9, 15.6 Hz, CH), 2.98 (1H, dd, J = 2.8, 15.6 Hz, CH),
3.58−3.81 (8H, m, N(CH2CH2)2N), 4.13 (1H, s, OH), 4.24
(1H, m, CH), 6.92 (2H, d, J = 9.5 Hz, ArH), 8.21 (2H, d, J =
9.5 Hz, ArH). Anal. (C15H19N5O3S) C,H,N.
(R,S)-1-[5-(3-Methoxyphenylamino)-1,2,4-thiadiazol-3-yl]-
1
propan-2-ol (19). Yield 69%; mp 123−124 °C. H NMR
[200 MHz, CDCl3] δ: 1.20 (3H, d, J = 6.3, CH3), 2.75 (1H, dd,
J = 8.6, 15.6 Hz, CH), 2.94 (1H, dd, J = 3.1, 15.6 Hz, CH), 3.60
(1H, br s, OH), 3.74 (3H, s, CH3), 4.19 (1H, m, CH), 6.55−
6.77 (3H, m, ArH), 7.15−7.25 (3H, m, ArH), 8.96 (1H, br s,
NH). Anal. (C12H15N3O2S) C,H,N.
(R,S)-1-[5-(6-Methylpyridin-2-ylamino)-[1,2,4]thiadiazol-3-
1
yl]propan-2-ol (28). Yield 65%; mp 146−148 °C. H NMR
[200 MHz, DMSO-d6] δ: 1.16 (3H, d, J = 6.0 Hz, CH3), 2.74
(1H, dd, J = 5.8, 14.2 Hz, CH), 2.85 (1H, dd, J = 6.8, 14.2 Hz,
CH), 4.14 (1H, m, CH), 4.45 (1H, d, J = 4.2 Hz, OH), 6.78
(1H, d, J = 7.4 Hz, PyH), 6.93 (1H, d, J = 8.4 Hz, PyH), 7.57
(1H, t, J = 7.5 Hz, PyH), 11.84 (1H, s, NH). Anal.
(C11H14N4OS) C,H,N.
(R,S)-1-[5-(3-Chlorophenylamino)-1,2,4-thiadiazol-3-yl]-
1
propan-2-ol (20). Yield 73%; mp 115−117 °C. H NMR
[200 MHz, CDCl3] δ: 1.34 (3H, d, J = 6.2, CH3), 2.87 (1H, dd,
J = 6.2, 15.5 Hz, CH), 3.05 (1H, dd, J = 3.0, 15.5 Hz, CH), 4.20
(1H, br s, OH), 4.31 (1H, m, CH), 7.12−7.44 (4H, m, ArH),
8.59 (1H, br s, NH). Anal. (C11H12ClN3OS) C,H,N.
(R,S)-1-{5-[2-(5-Methoxy-1H-indol-3-yl)ethylamino]-
[1,2,4]thiadiazol-3-yl}propan-2-ol (29). Yield 66%; mp 116−
1
118 °C. H NMR [200 MHz, DMSO-d6] δ: 1.17 (3H, d, J =
(R,S)-1-(5-Dibenzylamino-1,2,4-thiadiazol-3-yl)propan-2-
6.1 Hz, CH3), 2.64 (1H, dd, J = 5.8, 14.5 Hz, CH), 2.73 (1H,
dd, J = 7.0, 14.5 Hz, CH), 3.03 (2H, t, J = 6.9 Hz, CH2), 3.58
(2H, q, J = 6.7 Hz, CH2), 3.82 (3H, s, CH3), 4.11 (1H, m, CH),
4.42 (1H, br s, OH), 6.69 (1H, dd, J = 2.3, 8.6 Hz, ArH), 6.97
(1H, d, J = 2.1 Hz, ArH), 7.07 (1H, d, J = 2.1 Hz, ArH), 7.22
(1H, d, J = 2.1 Hz, ArH), 8.31 (1H, t, NH), 10.50 (1H, s, NH).
Anal. (C16H20N4O2S) C,H,N.
1
ol (21). Yield 77%; mp 65−66 °C. H NMR [200 MHz,
DMSO-d6] δ: 1.18 (3H, d, J = 6.1 Hz, CH3), 2.68 (1H, dd, J =
6.7, 14.1 Hz, CH), 2.80 (1H, dd, J = 6.8, 14.1 Hz, CH), 4.14
(1H, m, CH), 4.39 (1H, d, J = 4.4 Hz, OH), 4.65 (4H, s,
N(CH2)2), 7.22−7.40 (10H, m, ArH). Anal. (C19H21N3OS)
C,H,N.
(R,S)-1-(5-Cyclopropylamino-1,2,4-thiadiazol-3-yl)propan-
(R,S)-N-[3-(2-Hydroxypropyl)-[1,2,4]thiadiazol-5-yl]-
1
1
2-ol (22). Yield 67%; mp 121−123 °C. H NMR [200 MHz,
benzamide (30). Yield 69%; mp 73−75 °C. H NMR [200
DMSO-d6] δ: 0.59−0.84 (4H, m, (CH2)2), 1.13 (3H, d, J = 6.6 Hz,
CH3), 2.52 (1H, m, CH), 2.62 (1H, dd, J = 6.0, 14.2 Hz, CH),
2.71 (1H, dd, J = 6.7, 14.2 Hz, CH), 4.03 (1H, m, CH),
MHz, D2O] δ: 1.23 (3H, d, J = 6.3 Hz, CH3), 2.89−2.97 (2H,
m, CH2), 4.35 (1H, m, CH), 7.45−7.60 (3H, m, ArH), 8.05−
8.15 (2H, m, ArH). Anal. (C12H13N3O2S) C,H,N.
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(R,S)-1-[5-(2-Methyl-5-chlorophenylamino)-[1,2,4]-
thiadiazol-3-yl]propan-2-ol (31). Yield 81%; mp 102−103 °C.
1H NMR [200 MHz, DMSO-d6] δ: 1.26 (3H, d, J = 6.2 Hz,
CH3), 2.30 (s, 3H, CH3), 2.79 (1H, dd, J = 8.6, 15.5 Hz, CH),
2.95 (1H, dd, J = 2.2, 15.5 Hz, CH), 4.14 (s, 1H, OH), 4.25 (m,
1H, CH), 7.13 (1H, dd, J = 1.8, 8.1 Hz, ArH), 7.22 (1H, d, J =
8.1 Hz, ArH), 7.44 (1H, d, J = 1.8 Hz, ArH), 8.60 (s, 1H, NH).
Anal. (C12H14ClN3OS) C,H,N.
2.2. Materials and Solvents. 1-Octanol (n-octanol,
CH3(CH2)7OH, MW 130.2, lot 11K3688) ARG was from
Sigma Chemical Co. (USA). n-Hexane (C6H14, MW 86.18, lot
07059903 C) ARG was from SDS (Peypin, France).
Phosphate buffer solution (pH 7.4) was prepared by mixing
the solutions of appropriate phosphoric acid sodium and pot-
assium salts. All the chemicals were AR grade. The pH values
were measured by the use of a Toledo MP 220 pH meter
(Mettler, USA) standardized with pH 1.68 and 9.22 solutions.
Egg phosphatidyl choline, Lipoid E-80 was obtained from
Lipoid (Germany). Culture inserts (Transwell-Clear, d = 6.5 mm)
and plates were purchased from Corning Inc. Corning, USA,
filters in existence were removed, and mixed cellulose ester
filters (0.65 μm pore size) from Millipore, Billerica, USA were
fused on. Phospholipid vesicle-based barriers were prepared
according to the procedure described earlier.19
2.3. Permeation Studies. Stock solutions of the
compounds under study (2.5 × 10−5 to 2.5 × 10−3 mol·L−1)
were prepared by dissolving the drug in phosphate buffer
solution pH 7.4. Containing KH2PO4, Na2HPO4·12H2O, NaCl,
NaN3, and water, the solution was adjusted to pH 7.4 with
HCl/NaOH if necessary. Concentrations of different drug solu-
tions were chosen on the assumption of analytical consid-
erations as well as for obtaining the sink conditions during per-
meation studies. The concentrations of different drug solutions
had to be high enough for the obtainment of a definite amount
of a drug in an acceptor chamber during permeation studies to
be quantified by means of UV-absorbance and still be below the
solubility limit.
The method of the liposome preparation involves film
hydration/filter extrusion. Egg phosphatidyl choline (1.8 g)
was dissolved in a mixture of chloroform and methanol (2:1)
(6 mL) in a round-bottom flask. The organic solvent was re-
moved under vacuum at 55 °C. The deposited lipid film was
exposed to vacuum of 55 hPa at room temperature for an
additional period of 3 h to remove traces of solvent, before
hydration with phosphate buffer, containing 10% (v/v) ethanol
to obtain a 6% (w/v) liposomal dispersion. The liposome dis-
persion was then filter-extruded through 0.8 and 0.4 μm poly-
carbonate membrane filters (Millipore) at room temperature.
The extrusion was done by hand with a syringe through the
filters in a 25 mm Swinnex Filter Holder from Millipore. To
obtain liposomes of two different sizes, one portion was extrud-
ed five times through the 0.8 μm polycarbonate membrane
filter while another portion was extruded five additional
passages through filters with 0.4 μm pore size.
Permeation studies were performed after loading the inserts
(the facility for bearing the 0.65 μm pore size mixed cellulose
ester filter) with drug solution (100 μL) and placing them into
separate acceptor compartments containing phosphate buffer
solution (600 μL) (pH 7.4). The permeation experiment was
carried out at room temperature without agitation, and inserts
were moved to wells containing equal quantities of fresh buffer
solution. Taking into account the low solubility of the com-
pounds under investigation, the operational range of 12−24 h
was optimal. In conclusion of the permeation experiment,
samples (200 μL) from each acceptor compartment were trans-
ferred into 96-well UV 96-well black plates (Costar) and drug
concentrations were measured spectrophotometrically (Spec-
tramax 190; Molecular devices, Molecular Devices Corporation,
California, USA) at the most appropriate wavelength for each
drug. A blank well was filled with pure phosphate buffer pH 7.4.
The electrical resistance of the lipid barriers was measured
(Millicell-ERS, Millipore, USA) immediately after completion
of permeation studies. To find out the resistance of the lipid
barrier itself, a value of 119 Ω resulting from the filter char-
acteristics was subtracted from the observed resistance
meaning. Such a difference was then multiplied by the surface
area (0.33 cm2) to be normalized for the dimensions of the insert.
The experiments were performed at least in triplicate with three
inserts in each parallel for every compound. The mean values
and standard deviations are reported.
The standard time dependences of the cumulative amount of
permeated drug consisted of 6−9 points, and every point
represented the mean value of three parallels. The r2-meanings
were always higher than 0.99. Repeatability corresponded to
3.9−20.1%, and an intermediate precision of 2.7−29.4% was
observed for different drugs using such a novel method.
The linear part of the slope of the curve of the standard time
dependence of the cumulative amount represented the steady-
state flux rate. If a lag time was observed before the steady-state
conditions were attained, and the saturation conditions took
place at the end of the experiments, only the middle points of
the cumulative plot were used for the calculation procedure,
based on the steady-state flux. The obtained flux rates were
used to calculate the apparent permeability coefficient (Papp
,
cm·s−1) by means of the following equation:
P
= J/(A·(C − C ))
d a
app
(1)
where J is the observed flux rate at steady-state (nmol·s−1), A is
the surface area of the insert (cm2), and Cd and Ca are the
concentrations of the solutions in the donor and acceptor
chambers (nmol/mL), respectively. The experiments were
performed under the sink conditions; that is, the drug
concentration in the acceptor chamber did not exceed 10% of
the drug concentration in the donor chamber at any time.20
2.4. Determination of Partition Coefficients. The
isothermal saturation method was used to determine the
partition coefficients in n-octanol/buffer (pH 7.4), Doct/buf, and
n-hexane/buffer (pH 7.4), Dhex/buf, systems. All the experiments
were carried out at 25 °C. The substance concentrations were
measured spectrophotometrically using calibration curves. The
partition coefficient was calculated using the following
equation:
D = C /C
(2)
ow wo
where Cow and Cwo are the molar concentrations of solute in the
mutually saturated phases of octanol and water. The accuracy of
the D 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:
m = m + m
wo
(3)
i
ow
where mi = CiVi is the starting mass (in moles) of the
compound, mow = CowVow is the mass of the substance dissolved
in the water−saturated octanol phase, and mwo = CwoVwo is the
mass of the substance dissolved in the octanol−saturated water
phase.
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2.5. Calcium-Blocking Property Experiments. Inter-
action between the compounds and the glutamate-dependent
calcium uptake system was studied on newborn (8−11 days
old) rat brain synaptosomal P2-fraction isolated according to
the following method: synaptosomes were suspended in
incubation buffer A (132 mM NaCl, 5 mM KCl, 5 mM
HEPES) pH 7.4 stored at 0 °C during the experimental stage.
The aliquots of synaptosomes (50 μL) were transposed to
buffer A containing test compound and 45Ca-samples. Calcium
ion uptake was stimulated by introducing the glutamate solu-
tion (200 μM). After 5 min incubation at 37 °C, the process
was terminated by the filtration on GF/B-filters. The sample
was washed three times with cold buffer B (145 mM KCl,
10 mM Tris, 5 mM Trilon B) followed by radioactivity measuring
with scintillator counter SL-4000 Intertechnic.
In preliminary experiments, all the compounds were tested
for the ability to inhibit glutamate stimulated Ca uptake at the
concentration of 100 μM. If the inhibition of Glu-Ca-uptake
was 50% and more, then further studies were carried out to
determine the concentration dependence of inhibition and the
corresponding value of K43/21 was measured according to the
following equation:
Table 2. Biological Coefficient Describing Ability to Inhibit
Glu-Ca-uptake (K43/21), Partition Coefficients n-Octanol/
Buffer (pH 7.4) (Doct/buf) and n-Hexane/Buffer (pH 7.4)
(Dhex/buf), Apparent Permeability Coefficient (Papp) for 1,2,4-
Thiadiazole Derivatives
N
K43/21 (%)
Doct/buf
Dhex/buf
107Papp (cm/s)
1
84
85
6
4
149 36
445 28
478 16
0.14 0.04
0.31 0.02
0.12 0.03
0.16 0.04
0.46 0.05
0.14 0.01
0.81 0.05
1.68 0.07
0.17 0.01
0.32 0.02
6.3 0.4
3.36 0.08
3.87 0.19
2.48 0.15
7.47 0.41
8.33 0.23
2.60 0.04
23.7 0.4
7.6 0.9
2
3
110 10
75
89 13
108
66 13
4
6
226
380
45
5
5
5
6
8
1
7
560
7
8
87
51
2
9
1038 10
610 30
a
9
npm
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
88 16
52
316
568
457
205
400
3
2
2
5
5
10.7 0.6
94.7 3.6
5.59 0.06
2.48 0.02
7.60 0.16
6.60 0.19
1.77 0.07
4.08 0.05
1.30 0.03
2.52 0.19
2.30 0.01
18.4 0.2
6.48 0.77
5.22 0.05
1.08 0.04
3.85 0.17
12.5 0.2
14.7 0.9
3.49 0.06
3.21 0.12
8.73 0.02
2.44 0.03
4
101 14
108 11
112 21
79 13
0.36 0.03
0.58 0.03
0.40 0.01
0.115 0.008
0.48 0.02
0.80 0.01
1.061 0.012
0.140 0.014
0.104 0.012
9.0 0.5
180 10
94 13
86.7 0.5
K
= 100[(Ca − Ca )/(Ca − Ca )]
4 3 2 1
47
30
93
47
7
4
8
6
1205 10
(4)
43/21
250
9
where Ca1 is the 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); and Ca4 is the Ca2+ influx in the presence of
both glutamate and test compound.
2.6. Calculation Procedure. All the descriptors were
calculated with the program package HYBOT-PLUS (version
of 2003) in Windows.21 Structural similarity was estimated
using Tanimoto similarity indices (Tc) obtained by means of
the program MOLDIVS (MOLecular DIVersity & Similar-
ity).22
295 26
1381 49
98 10
338
19.2 0.1
50
6
87.0 0.6
0.227 0.008
0.42 0.03
16.2 0.1
111
86
2
3
8
145 22
97 14
1.30 0.06
0.046 0.003
0.18 0.01
1.8 0.1
109 20
45
1
105
123
6
4
3160 20
229
90
6
0.13 0.01
0.31 0.02
0.076 0.004
0.56 0.01
121 24
82 13
1
163
6
88
4
1049 50
T = N(A&B)/[N(A) + N(B) − N(A&B)]
(5)
c
a
npm − not possible to measure.
where N(A) is the number of fragments in molecule A, N(B) is
the number of fragments in molecule B, and N(A&B) is the
number of common fragments in molecules A and B.
In this program, molecular fragments are defined as atom
centered concentric environments. The fragment consists of a
central atom and neighboring atoms connected within a
predefined sphere size (the number of bonds between the
central and edge atoms). For each atom in a fragment, the
information on the atom and bond type, charge, valency, cycle
type, and size is coded into fixed-length variables, which are
subsequently used to define a pseudorandom hash value for this
fragment. The program permits estimation of the similarity of
each molecule in the database with all the other molecules by
sorting them according to the value of similarity with the initial
molecule.
Several approaches were used to find out the regularity of
biological activity (K43/21) behavior. First, we tried to disclose
the correlations between log(K43/21) and the physicochemical
descriptors HYBOT.21 Unfortunately, the regularities were not
observed. Second, we tried to reveal the correlations of bio-
logical activity against the distribution parameters: log(Doct/buf),
log(Dhex/buf), and log(Doct/buf) − log(Dhex/buf). Our efforts were
not successful again. Consequently, we assumed that the
interaction of the selected compounds with receptors is not
limited by drug delivery processes (absorption, distribution, and
others) and is connected directly with the geometry, topology,
and nature of the substitutes in the drug molecules. Such ob-
servation suggests an idea to analyze the connection of bio-
logical activity with the outlined parameters.
To check the correlation models, all the experimental values
were divided in a random way into a training set including 21
compounds and a test set including the other 10 substances (1,
2, 4, 7, 10, 11, 14, 15, 17, 19).
In order to carry out a comparative analysis of 1,2,4-
thiadiazole phenyl derivatives' biological activity beside the type
of the substituent, a separate table (Table 3) was constructed.
The diagonal elements of the matrix correspond to mono-
phenyl substituted fragments.
The structure activity relationship of the receptor shows the
maximal activity observed for disubstituted phenyl (notably
-CH3 and Cl-) isomers: 2-CH3-3-Cl- (18) (30%) and 3-CH3-4-
Cl- (17) (47%). On the other hand, the activities of the
3. RESULTS AND DISCUSSION
3.1. Calcium-Blocking Properties. The results of the
ability to inhibit Glu-Ca-uptake (K43/21) for the compounds are
shown in Table 2.
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introducing the CF3-group at the 5-position: this gives 2-Cl-5-
CF3- (11) with K43/21 = 52%. Moreover, the membrane per-
meability of the molecule increases by 12 times: 94.7 × 10−7
cm·s−1 for 11 relative to 7.6 × 10−7 cm·s−1 for 14. Thus, we
observed the effect of synchronic improvement of both biological
activity and membrane permeability. The comparison of Cl- and
F-phenyl isomers showed that the 2-F- (15) isomer is more active
(79%) than 2-Cl- (14) (112%). Whereas para-isomers have
approximately the same activities: 4-F- (10) (88%) and 4-Cl- (5)
(89%). Introducing an additional F-atom at the para-position of
the most active 3-Cl- (20) isomer (it gives 3-Cl-4-F- (4)) leads to
the decrease in the activity from 47 to 75%, respectively.
3.2. Membrane Permeability Properties. The apparent
permeability coefficients (Papp) of 31 novel 1,2,4-thiadiazoles
were measured using the phospholipid membrane approach
(Table 2).
Table 3. Biological Coefficient Describing the Ability to
Inhibit Glu-Ca-Uptake (K43/21, %) for Various Phenyl
Substituted 1,2,4-Thiadiazoles
As in the previous section, we used HYBOT21 descriptors
and the conception of the nearest neighbors (with analysis of
Tanimoto similarity indices (Tc))22 to find a correlation be-
tween Papp values and independent variables (the physicochem-
ical descriptors). The analysis of the descriptors demonstrated
that the log(Papp) function can be described by the descriptor
indicating the sum of positive charges on the atoms of the
molecule under study (∑Q(+)). The next step was to divide all
the experimental data of the training set (21 substances) into
clusters including the compounds with Tanimoto similarity
indices (Tc) from 0.7 up to 1 for each element of the test set. It
should be mentioned that the number of elements in clusters of
the compounds selected as a test set differed essentially (from 3
up to 8). The experimental data, entering the cluster, were
smoothed by a linear function, because the noted function
describes a relationship between the Papp value and the ∑Q(+)-
descriptor in the best way. After that, the unknown Papp value
(corresponding to the test set) was estimated by means of the
function. Comparative analysis of the predicted and the
experimental Papp values presented in Figure 1 can be described
by the correlation equation:
a
number 1\number 2: number 1 corresponds to the activity of i-
CH3-monosubstituted phenyl and number 2 corresponds to the activity of
i-Cl-monosubstituted phenyl. n: not measured.
compounds with the same substitutes located at the different
positions (3-Cl-4-CH3- (8) and 3-Cl-5-CH3- (31)) are
approximately of the same values (88%) (if they are compared
to each other), but they are more than twice lower in
comparison with the most active substance (18). Such an
observation indicates strictly the fact that the receptors studied
are sensitive to the topology of the selected molecules. It is
interesting to note that monophenyl substituted 4-CH3- (2)
and 4-Cl- (5) have approximately the same activity (89%),
which is comparable with the activities of the disubstituted (8
and 31) isomers. Thus, the activity of 4-Cl- (5) increases
essentially when an additional methyl group is introduced into
phenyl ring at the meta-position (3-CH3-4-Cl- (17)), and this
value becomes comparable with the activity of the mono-
Cl-substituted 3-Cl- (20) isomer. The above-mentioned approach
of molecule structural modification, when the biological activity
of the modified molecule is compared with mono-substituted
ones, is very useful, as it allows improvement of other
characteristics (for example, ADME or membrane perme-
ability) without degradation. As an example, we would like to
present the results of Table 2 concerning the apparent
coefficients of membrane permeability: for compound 17 this
value is twice higher (4.08 × 10−7 cm·s−1) in comparison with
that of 20 (2.3 × 10−7 cm·s−1). Introducing the methyl group at
the ortho-position into 3-Cl-substituted phenyl improves the
biological activity of the molecule (17) (30%), whereas
introducing such a substitute into positions 4 (8) and 5 (31)
leads to the degradation of the test property (88%).
Introducing an additional Cl-atom into the most biologically
active mono-Cl-isomer (3-Cl- (20)) at the para-position (it
gives 3-Cl-4-Cl- (9)) almost does not change the activity and
shows the same effect as the insertion of the methyl group at
position 3. Placing the CF3-group at the para-position of the
phenyl ring leads to the maximal increasing of the biological
activity (66%) in comparison with the other studied substitutes
at this position: CH3- (85%); OC2H5- (110); Cl- (89);
OHCHCH3- (108); F- (88); and OCH3- (108). In its turn, the
biological activity of 2-Cl- (14) increases almost twice after
Figure 1. Comparative analysis of the experimental (log(Papp
versus the predicted (log(Papp)cal) values for the test set.
) )
exp
log(P
)
= (0.04 0.82) + (0.99 0.13) log(P
)
app exp
app cal
(6)
R = 0.934; σ = 0.18; n = 10
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So, knowing only the structural formula of a substance and the
number of the nearest physicochemical neighbors in the
clusters gives an opportunity to predict the Papp value. It should
be noted that the training set of Papp values includes only 21
compounds (approximately 67% from the total number of
studied compounds) and this fact restricts the application capa-
bility of the approach, because some clusters have just several
structurally similar neighbors. The above-mentioned fact
reduces the statistical accuracy of the prognoses. Nevertheless,
the available database/set can predict a new Papp value safely (as
it follows from the test set). It should be noted that if experi-
mental data volume growth persists, the proposed calculation
algorithms will work more effectively. In this study we tried to
develop a conception of obtaining bioavailable drug molecules
on the basis of the available experimental materials. The experi-
ments generating permeability characteristics are going on, and
we hope that in the near future calculation procedure precision
will be improved essentially.
It is evident that a low correlation coefficient is observed.
Therefore, to improve the model, we used the conception of
the nearest neighbors which was also applied in the previous
section. For each substance, belonging to the test set, we
formed clusters based on the training set with Tanimoto
similarity indices from 0.7 up to 1. The Doct/buf value of the test
set was estimated from the linear dependence received from a
smoothing procedure of the experimental points within the
cluster. The results of the calculation are presented in Figure 3
and can be described by the correlation equation:
3.3. Partitioning Properties. 3.3.1. n-Octanol/Buffer
(pH 7.4) Partitioning. As the developed class of the com-
pounds is intended for oral introduction, it was interesting to
study partition processes taking place at the main types of
membranes, which the compounds overcome in order to reach
the targets at the cerebral cortex. Immiscible solvent pairs n-
octanol/buffer (pH 7.4) (describing gastrointestinal tract
membranes) and n-hexane/buffer (pH 7.4) (characterizing BBB
membranes) which are usually used for application purposes
were selected as the model systems. We wanted to analyze
some correlations/trends between partitioning coefficients, on
one hand, and apparent membrane permeability coefficients, on
the other hand. For this purpose, the partition coefficients
(Doct/buf) for 31 novel 1,2,4-thiadiazoles were obtained in the n-
octanol/buffer (pH 7.4) system (Table 2). As in the previous
sections, we used HYBOT descriptors in order to find out the
relationships between physicochemical characteristics and parti-
Figure 3. Comparative analysis of the experimental log(Doct/buf
)
exp
versus the predicted log(Doct/buf
)
values for the test set.
cal
oct/buf
log(D
)
= (−0.1 0.5) + (1.0 0.2)
oct/buf
exp
× log(D
)
cal
(8)
tion coefficients. The data analysis showed that the log(Doct/buf
)
function can be described by the descriptor indicating the sum
of H-bond acceptor and donor factors normalized to the
polarizability (Σ(Cad/α)) of the molecule (Figure 2).
R = 0.880; σ = 0.14; n = 10
The comparison of eqs 7 and 8 shows that the latter model
predicts Doct/buf values three times better (if the standard
deviation is taken into account) than the former one. Thus, the
partition coefficients of this class of the substances can be
predicted even if we know their structural formula only.
Approximately 90% of the studied 1,2,4-thiadiazoles have
log(Doct/buf) values within the range from 1.5 to 3.5. This fact
confirms that most of the compounds are lipophilic, and as a
consequence, they will prefer lipophilic drug delivery pathways
to overcome gastrointestinal tract membranes.
3.3.2. n-Hexane/Buffer (pH 7.4) Partitioning. Partition
processes in the n-hexane/buffer (pH 7.4) system (imitating
BBB) were studied in the same way as those in the system
described above. For this purpose, the partition coefficients for
31 novel 1,2,4-thiadiazoles were obtained (Dhex/buf) and the
correlations between these values and HYBOT physicochem-
ical descriptors were analyzed. The result of the analysis
showed that the experimental values can be described in the
best way by the descriptor ∑(Cad/α) (Figure 4).
Figure 2. Dependence of log(Doct/buf) versus ∑(Cad/α).
oct/buf
hex/buf
log(D
) = (2.2 0.4) − (8.1 1.2) (C /α)
ad
∑
(9)
log(D
) = (5.1 0.5) − (8.6 1.5) (C /α)
ad
∑
(7)
R = 0.722; σ = 0.45; n = 31
R = 0.771; σ = 0.37; n = 31
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enhance as well. The predictive power of the model for another
class of compounds will be determined by the number of struc-
turally relative neighbors at the cluster describing the unknown
substance.
3.3.3. Influence of Molecular Topology and Nature of
Substituents on Partitioning. As it was said before, the parti-
tion coefficients Doct/buf and Dhex/buf carry important information
about the interactions of drugs with biological barriers
(gastrointestinal tract and blood brain barrier in particular).
Therefore, it was interesting to analyze the influence of the
nature and position of different substitutes in the 1,2,4-thiadiazole
phenyl ring on the partition coefficients. Moreover, if we take
into account a direct connection of drug delivery to NMDA-
subtype and AMPA-subtype receptors with distribution pro-
cesses in gastrointestinal tract and blood brain barrier blood
currents, it becomes very timely to compare the values Doct/buf
and Dhex/buf and to study the possibilities of changing these
values by structural modification of the molecule.
Figure 4. Dependence of log(Dhex/buf) versus ∑(Cad/α).
Figure 6 shows distribution coefficient values of 1,2,4-
thiadiazole phenyl derivatives in logarithmical coordinates.
To improve the predictive power of the model, we used
the conception of the nearest neighbors explained before.
Figure 6. Experimental data in the coordinates log(Doct/buf) versus
log(Doct/buf) (numbering corresponds to Table 1).
Figure 5. Comparative analysis of the experimental log(Dhex/buf
)
exp
versus the predicted log(Dhex/buf
)
values for the test set.
cal
The red dotted lines, crossing the experimental point that
corresponds to unsubstituted phenyl, divide the space into four
sectors. Every sector describes the influence of substitutes on
distribution coefficient values as compared to the case of the
unsubstituted molecule. For example, the top right sector
corresponds to increasing both Doct/buf and Dhex/buf values, etc.
This fact means that, for most of the compounds, introduction
of substitute to the phenyl ring leads to simultaneously
increasing the distribution coefficients under study. The
exceptions represent several compounds: 3-SCH3 (16), for
which the decrease of the Doct/buf value and the increase of the
Dhex/buf value are observed, whereas for 2-F- (15), 3-Cl- (20), 4-
OC2H5 (3), and (28) the opposite results are revealed: the
Doct/buf value grows and Dhex/buf falls. It is important that for 4-
OHCH2CH3 (6) and 3-OCH3 (19) the Dhex/buf value does not
grow in comparison with unsubstituted compound, but the
Doct/buf value falls for one (6) and grows for one (19).
The results of the calculations are presented in Figure 5 and can
be described by the correlation equation:
hex/buf
log(D
)
= (0.20 0.15) + (1.43 0.28)
hex/buf
exp
log(D
)
cal
(10)
R = 0.872; σ = 0.17; n = 10
The model applying the nn-method predicts Dhex/buf values
much better in comparison with eq 9. Thus, the partition
coefficients of the test substances can be predicted knowing the
structural formula only. Approximately 85% of the studied
1,2,4-thiadiazoles have log(Dhex/buf) values below 1. This fact
confirms that most of the compounds will prefer aqueous drug
delivery pathways to overcome the BBB.
Several points should be mentioned about the applicability of
the model based on the conception of nearest neighbors. As
was shown before, the model using the training set from 21
compounds works well. While a number of substances at the
training set increase, the predictive power of the model will
When studying the position of the substitute in the phenyl
ring (isomerism), the following conclusions can be made. The
distribution coefficients, Doct/buf and Dhex/buf, are approximately
equal for 2- (14) and 4-Cl (5) isomers. For the meta-isomer
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(20), in its turn, a significant increase of the Doct/buf value and a
sharp rise for the Dhex/buf one are observed. The experimental
point corresponding to 3,4,-Cl- (9) lies along the straight line
connecting the values of the 4-Cl and 3-Cl isomers in the space
studied (dotted black line). In other words, the properties of
these isomers are additive. The significant difference of the
meta-isomer distribution coefficients can be caused by strong
conformation changes of the molecule during transfer from one
phase to another in comparison with the other isomers. At the
same time, it may be such a peculiarity of the meta-isomer that
determines partially the abnormally high biological activity
(K43/21) of phenyl derivate compounds with the Cl-atom in the
meta-position: 9, 18, and 4.
3.4. Permeability−Activity Relationship. In order to
analyze the relationship between permeability characteristics
(Papp) and specific biological activity (K43/21), the experimental
data for the compounds under study were presented in the
coordinates log(K43/21) versus log(Papp) (Figure 7). All the
The analysis of the available di-isomers Cl- and CH3-
(2-CH3-3-Cl- (18), 3-CH3-4-Cl- (17), 3-Cl-4-CH3- (8), and 3-
Cl-6-CH3- (31)) has the following results. For compounds 8,
17, and 31, the Doct/buf values do not differ a lot and are close to
the 3-Cl-isomer value. While for compound 18 the distribution
coefficient is 1 order of magnitude lower. The Dhex/buf values for
8 and 31 have three time differences, while for 17 and 18 the
difference is insignificant. The experimental values for methyl
isomers 3-CH3- (12) and 4-CH3- (2) do not differ much from
each other and lie along the trend line, connecting Cl- isomers.
Moreover, compound 2-CH3-3-Cl- (18) also lies along this line
(Figure 6, black dotted line), which proves the additivity of the
properties under consideration for some chlorine- and methyl-
isomers.
Such compounds as fluorine isomers (2-F- (15) and 4-
F- (10)), 4-CF3- (7), as well as disubstituted phenyl with fluorine
atoms (3-F-4-Cl- (4) and 2-Cl-5-CF3- (11)) lie in the
coordinates log(Doct/buf) versus log(Doct/buf) on the trend line
(blue dotted line in Figure 6). And the trend lines for chlorine-
and fluorine-derivatives differ a lot: they have different
inclination angles. Distribution coefficient values rise signifi-
cantly during the transfer from the 2-F- to 4-F-isomer (for both
immiscible phases). Replacing the substitute of fluorine at the
para-position with CF3- also leads to a significant increase in
both the Doct/buf and Dhex/buf values. Two trend lines
demonstrate that it is possible to change both the lipophilic
and hydrophilic properties of the considered class compounds
intentionally.
Comparing the properties of 4-CH3- (2) and 4-OCH3- (13)
shows that the introduction of an additional hydrophilic oxygen
atom into a substitute results in a decrease of the Doct/buf value
and an increase of the Dhex/buf value. Adding, in its turn, a
methylene group into the methoxy-substitute of 4-OCH3-
compound (13) (which gives compound 4-OC2H5- (3)) leads
to an increase of the Doct/buf value and a significant decrease of
the Dhex/buf value. It is interesting that placing the methoxy-
substitute in the meta-position leads to a considerable decrease
of the Dhex/buf value and a small rise of the Doct/buf value as
compared to the substitution in the para-position. Introducing
an oxygen atom into 3-CH3- (12) (which gives 3-OCH3- (19))
reduces its lipophilicity slightly and decreases the Dhex/buf value
considerably, whereas a similar introduction of an S-atom into
3-CH3- (12) (which gives 3-SCH3- (16)) leads to an
insignificant change of the Dhex/buf value but to a considerable
reduction of the Doct/buf value. And finally, a small modification
which involves the replacement of substitute 4-OC2H5- (3)
with 4-OHCH2CH3- (6) leads to a dramatic decrease of
lipophility (by an order) while the Dhex/buf value remains the
same.
Figure 7. Experimental data in coordinates log(Papp) versus log(K43/21
(numbering corresponds to Table 1).
)
values can be conditionally divided into four groups: (I) with
high parameters of permeability (for the considered com-
pounds) and high values of activity (on the tested receptors)
(1 × 10−6 < Papp (cm·s−1); 0 < K43/21 < 100%); (II) with high
permeability and low activity (1 × 10−6 < Papp; 100 < K43/21);
(III) with low permeability and high activity (Papp < 1 × 10−6;
0 < K43/21 < 100); (IV) with low permeability and low activity
(Papp < 1 × 10−6; 100 < K43/21). The compounds belonging to
classes I and III are promising in terms of their further testing.
Moreover, the substances of class I are more preferable to those
of class III. In their turn, 1,2,4-thiadiazoles of classes II and IV
are not suitable for further work and should be structurally
modified toward the improvement of the necessary “perme-
ability−activity” properties to replace them with the most
favorable group. The most promising “permeability−activity”
properties correspond to CF3-phenyl derivatives of 1,2,4-
thiadiazole (7, 11), which can be recommended as the
substances for the further biological and preclinical evalua-
tions/tests.
Comparison of 1,2,4-thiadiazole Cl-phenyl isomers (5, para;
20, meta; 14, ortho) leads to the following conclusions. The
permeability characteristics of 5 and 14 are approximately the
same; however, the activity values are different: substance 5
belongs to group III, whereas 14 belongs to group IV. Meta-
isomer 20 is much more active than the other two ones and for
the studied compounds has the minimal K43/2 value (together
with 17). Nevertheless, the permeability value of the compound
is 4 times lower than that of the para- and ortho-isomers. It is
difficult to make a decision unambiguously concerning the
recommendation for the further testing of substance 20. But if
we take into account the maximal biological effect, the
compound can be offered for further evaluation.
F-Phenyl isomers of 1,2,4-thiadiazole (10, para; 15, ortho)
are situated approximately in the frontier region of groups I and
III. This fact confirms insignificant property differences
between the compounds. Nevertheless, the para-isomer is
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more permeable, whereas the ortho-isomer has better affinity to
the biological receptor.
For methylphenyl isomers of 1,2,4-thiadiazole (2, para; 12,
meta), the substitution of the meta-isomer with the para-isomer
leads to an improvement of biological receptors inhibition,
whereas membrane permeability decreases. Compound 12
belongs to class IV, whereas 2 belongs to class III.
For methoxyphenyl isomers of 1,2,4-thiadiazole (13, para;
19, meta), a similar replacement as the one for methyl isomers
leads to the opposite effect. The inhibition of the biological
receptors becomes worse, whereas the membrane permeability
remains approximately the same.
It was interesting to analyze the influence of substitute nature
at the para-position of the phenyl ring (blue points) on the
“permeability−activity” properties. The results of the analysis
are shown in Figure 7 (red line), and the experimental values
can be described by the following equation:
maximum activity is produced by disubstituted phenyls with
substitutes 2-CH3-3-Cl- (18) (30%) and 3-CH3-4-Cl- (17)
(47%). On the other hand, the activity of the compounds with
the same substitutes 3-Cl-4-CH3- (8) and 3-Cl-5-CH3- (31) is
approximately equal (88%) and twice lower in comparison with
that of the most active compound (18).
We conducted correlation analysis of log(Doct/buf) and
log(Dhex/buf) with HYBOT physicochemical descriptors by
dividing the space into clusters. We received the correlation
models that allow us to predict the values in question. Besides,
we studied in detail the reciprocal influence of substitutes in the
1,2,4-thiadiazole phenyl ring on the values of the distribution
coefficients under study.
The experimental Papp values were divided into clusters
including the compounds with Tanimoto similarity indices (Tc)
lying in the interval from 0.7 up to 1. Within each cluster, log
Papp values can be described by the descriptor indicating the
sum of positive charges on the atoms of the studied molecule
(∑Q(+)). On the basis of both the received correlation
equation and the method of the nearest neighbors, we
proposed an algorithm for the prediction of Papp values when
only the molecular structural formula is known.
log(P ) = (2.4 1.6) − (4.4 0.8) log(K
)
43/21
app
(11)
R = 0.9103; σ = 0.18; n = 6
We proposed a classification of compounds in the space of
the “permeability−activity” properties.
Thus, it can be assumed that in the future membrane
permeability measurements of 1,2,4-thiadiazole para-phenyl
derivatives will make it possible to predict K43/21 values.
Let us consider the combined impact of two substitutes at
the 1,2,4-thiadiazole phenyl ring on the “permeability−activity”
properties in comparison with that of mono- derivatives. If we
take compound 17 (para-Cl and meta-methyl) and compare it
to 5 (para-Cl) and 12 (meta-methyl), the membrane
permeability of 17 decreases slightly, whereas the K43/21 value
decreases anomalously. In the case of comparing substances 4
(para-F and meta-Cl), 10 (para-F), and 20 (meta-Cl), an
absolutely opposite effect is observed. The anomalously low
AUTHOR INFORMATION
■
Corresponding Author
*Institute of Solution Chemistry, Russian Academy of
Sciences, 153045 Ivanovo, Russia. Telephone: +7 4932 533784.
Fax: +7 4932 336237. E-mail address: glp@isc-ras.ru.
Notes
The authors declare no competing financial interest.
K
43/21 value for 20 (47%) and a high one for 10 (88%) gives the
ACKNOWLEDGMENTS
■
average value for 4 (75%). The analogous regularity (but with
the opposite effect) is observed for the membrane permeability
data. So, we disclosed the contrary effect of the F- and Cl-
substitutes influence on the “permeability−activity” properties.
Thus, the proposed classification of compounds in the
space of “permeability−activity” properties allows us to select
the most promising substances for their further biological
evaluation/testing.
The present research was funded as a part of the basic research
program established by the Presidium of the Russian Academy
of Sciences “Fundamental sciences for medicine” and the
Russian Foundation for Basic Research (No. 09-03-00057).
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