Novel hybrid compounds based on amino derivatives of 1,2,4-thiadiazole and docosahexaenoic acid

Full text of DOI:10.1134/S0012500812110031

ISSN 0012ꢀ5008, Doklady Chemistry, 2012, Vol. 447, Part 1, pp. 238–240. © Pleiades Publishing, Ltd., 2012.
Original Russian Text © I.V. Serkov, A.N. Proshin, L.N. Petrova, N.M. Gretskaya, V.V. Bezuglov, S.O. Bachurin, 2012, published in Doklady Akademii Nauk, 2012, Vol. 447, No. 1,
pp. 49–52.
CHEMISTRY
Novel Hybrid Compounds Based on Amino Derivatives
of 1,2,4ꢀThiadiazole and Docosahexaenoic Acid
I. V. Serkova, A. N. Proshina, L. N. Petrovaa, N. M. Gretskayab,
V. V. Bezuglovb, and Corresponding Member of the RAS S. O. Bachurina
Received April 18, 2012
DOI: 10.1134/S0012500812110031
In this paper, we describe the synthesis of hybrid
compounds of new series based on amino derivatives
of 1,2,4ꢀthiadiazole containing the DHA residue as a
supplementary pharmacophore. It was shown in preꢀ
liminary experiments that DHA can block
uptake in synaptosomes of rat cerebral cortex at conꢀ
centrations of 0.01–0.1 mM.
Docosahexaenoic acid (DHA, C22 : 6 n – 3) is the
most widespread polyunsaturated fatty acid of brain. It
is a component of phospholipids of neuronal memꢀ
branes and is essential for the growth and development
of brain and for the implementation of cognitive funcꢀ
tions. DHA participates in the processes of signal
transmission, synaptogenesis, and neurite growth,
regulates membrane fluidity, and is a precursor of varꢀ
ious oxylipins, including resolvins and neuroprotecꢀ
tins [1]. Recent studies showed an important role of
DHA in neuron protection in neurodegenerative disꢀ
eases, in particular, Alzheimer disease [2].
45Ca2+
We used tetramethylpiperidine derivatives of 1,2,4ꢀ
thiadiazole (4а, 4b) as amines. These compounds were
obtained by the reaction of 3ꢀaminoisoxazole with aryl
isothiocyanates followed by the Boulton–Katrizky
rearrangement of resultant thioureas into 5ꢀaminoꢀ3ꢀ
The design of hybrid compounds combining fragꢀ
ments of DHA and another pharmacologically active
compound is a promising approach in medicinal
chemistry. For example, anticancer properties of
Paclitaxel and other pharmaceuticals were considerꢀ
ably enhanced after their conjugation with DHA [3].
The introduction of DHA residue in the molecules of
potential neuroprotectors to enhance their neuroproꢀ
tecting activity may be especially efficient. The hyperꢀ
activation of glutamate receptors of central nervous
system and the massive influx of calcium into neurons
of the cell is known to be one of the main reasons of
nerve cell death and development of neurodegeneraꢀ
tive diseases, while the blockers of calcium flows are
considered as promising neuroprotector agents [4, 5].
We have shown recently that 1,2,4ꢀthiadiazole derivaꢀ
tives containing NOꢀproducing fragments are efficient
blockers for the glutamateꢀmediated uptake of calꢀ
cium ions [6]. The nitro group in these hybrid comꢀ
pounds behaves as a supplementary functional eleꢀ
ment that produces nitric oxide.
(2'ꢀoxopropyl)ꢀ1,2,4ꢀthiadiazoles (
oles were involved into condensation with 2,2,6,6ꢀ
tetramethylaminopiperidine followed by the reducꢀ
tion of enamines ( ) with sodium borohydride in
methanol to give amino derivatives of 1,2,4ꢀthiadiazꢀ
ole (4а 4b) (Scheme 1) [8], which were used for the
introduction of DHA residue. DHA amides of these
derivatives were prepared by the activation of the carꢀ
boxy group of the acid using chloroformate method
(A) or by N,N 'ꢀdisuccinimidyl carbonate (B) [9].
1) [7]. Thiadiazꢀ
1
3
,
According to method A, DHA (5) was first reacted
with isobutyl chloroformate to give a mixed anhydride
that was combined without isolation with the amine to
give target amides (6а, 6b). In method B, the carboxy
group was activated by N,N'ꢀdisuccinimidyl carbonꢀ
ate. The mixed anhydride also without isolation was
reacted with the amine.
The neuroprotective activity of the obtained comꢀ
pounds was assessed from the direct effect on the
glutamate receptors of the Р2 fraction of synaptosomes
of rat cerebral cortex by the glutamateꢀstimulated
45Са2+ uptake in synaptosomes [10].
a Institute of Physiologically Active Substances,
Russian Academy of Sciences, Severnyi pr. 1,
Chernogolovka, Moscow oblast, 142432 Russia
b Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry,
Russian Academy of Sciences, ul. MiklukhoꢀMaklaya 16/10,
Moscow, 117997 Russia
Our studies show that the effect of DHA amides 6a
and 6b on glutamate receptors fundamentally differs
from that of initial amines 4a and 4b. Amines 4a and
4b stimulate the glutamateꢀinduced calcium uptake,
whereas DHA amides 6a and 6b (like DHA itself)
238

NOVEL HYBRID COMPOUNDS BASED ON AMINO DERIVATIVES
239
R
R
NH2
Cl
Cl
H
N
4
N
O
3
N
S
a
5
2'
1'
+
N
N
N
H
N
NH
H
S
2
N
1
H
1a,
1b
2
3a,
3b
R
Cl
O
H
b
N
N
5
or d
OH
N
H
c
N
NH
S
4a, 4b
4
5
7
8
10 11
13 14
16 17
19 20
22
R
Cl
3
6
9
12
15
18
21
2
O
1
2'
23
N
N
24
1'
N
H
25
N
NH
6a, 6b
S
R = Cl(a), Me(b).
Scheme 1.
a
, MeOH, r. t., 2–5 h;
, MeCN, N,N'ꢀdisuccinimidyl carbonate, 0–4°C, 15 min, then Et N, 4a, 4b, r. t., 1.5 h.
3
b, MeOH, NaBH , 40–50°C, 2 h; c, MeCN, Et N, isobutyl chloroformate, 0–4°C, 20 min,
4
3
then 4a 4b, r. t., 1 h;
,
d
inhibit this uptake with IC50 60–70 µM (table). Thus, (Chemapol) and silica gel Kieselgel 60 (Merck), and
the introduction of the DHA fragment fundamentally
alters the neurotropic properties of initial molecules
(1,2,4ꢀthiadiazole derivatives), which can be used for
the tailored design of original neuroprotective agents.
separation was monitored by TLC. Solutions were
concentrated on a rotary evaporator in a vacuum of a
waterꢀjet pump at a bath temperature no higher
than 30°C
.
EXPERIMENTAL
Synthesis of Amides of Docosahexaenoic Acid
1H NMR spectra were recorded on a Bruker
CXPꢀ200 spectrometer. The chemical shifts were
Method A. Triethylamine (0.35 mmol) was added
to a solution of 0.3 mmol of docosahexaenoic acid (
5)
given on the
δ scale relative to Me4Si. Melting points
in 5 mL of acetonitrile in argon atmosphere, the mixꢀ
were determined on a Boetius hot stage apparatus and ture was stirred for 10–15 min at 0–4°С, 0.35 mmol of
were uncorrected. Thin layer chromatography was isobutyl chloroformate was added and stirred for
carried out with Silufol UV 254 plates (Kavalier) and 20 min at 0–4°С. A solution of 0.4 mmol of amine in
Kieselgel 60 F254 (Merck), visualization with a 5% 0.5 mL of methanol was added to the resultant mixed
solution of phosphomolybdic acid in alcohol. Column anhydride. The reaction mixture was stirred for 1 h at
chromatography was carried out using silica gel L ambient temperature and concentrated, and the resiꢀ
Table 1. Effect of thiadiazoles on glutamateꢀstimulated 45Са2+ uptake in synaptosomes of rat cerebral cortex
Parameter
, % *
IC50, µM **
4a
4b
6a
6b
DHA
K
304.6 17.7
–
213.7 12.4
–
29.7 6.3
58.9
30.3 4.8
67.6
10.4 2.1
10.1
45 2+
*
K is the percentage of Са uptake in synaptosomes of rat cerebral cortex toward control (control is 100%).
** IC was determined for active compounds.
50
DOKLADY CHEMISTRY Vol. 447
Part 1
2012

240
SERKOV et al.
Determination of the 45Са2+ uptake in synaptoꢀ
somes. Synaptosomes of rat cerebral cortex were isoꢀ
lated by Hayosh standard procedure from the cerebral
cortex of newborn rats (9–10 days old). To accumulate
due was diluted with water and extracted with ethyl
acetate ( 10 mL). The organic extracts were comꢀ
bined, washed with water, saturated sodium chloride
3
×
solution, and dried with anhydrous Na2SO4. The dryꢀ
ing agent was filtered off, and the filtrate was concenꢀ radiolabel, the Р2 fraction of synaptosomes was susꢀ
trated. The residue was purified by column chromaꢀ
tography on silica gel in a chloroform–isopropanol
gradient system. Fractions containing the product
(monitoring by TLC) were combined, and the solvent
was evaporated in a vacuum.
pended in incubation buffer A: 132 mM NaCl,
5 mM KCl, 5mM HEPES, 10 mM glucose, pH 7.4
(protein concentration 1.5–2 mg/mL). The calcium
concentration in final volume was 1.25 mM
(1.4
μ
Ci/mL). To stimulate the 45Са2+ uptake in synꢀ
M glutamate was used. After 5ꢀmin
aptosomes, 200
μ
Method B.
(0.45 mmol) was added to a solution of 0.3 mmol of
docosahexaenoic acid ( ) in 2 mL of acetonitrile in an
argon atmosphere. The mixture was stirred for 10–
15 min at 0–4 ; then, 0.9 L of triethylamine and a
N,N 'ꢀDisuccinimidyl carbonate
incubation with glutamate at 37°C, the uptake was
terminated by filtration through fiberglass filters
GF/B (Whatman), washed three times with cold
buffer solution B: 145 mM HEPES, 10 mM Tris,
5.4 mM Trilon B, pH 7.4. All assays were performed in
triplicate in two independent experiments. The samples
5
°
С
μ
solution of 0.4 mmol of amine in 0.5 mL of methanol
were added, and themixture was stirred for another 1.5 h.
The reaction mixture was treated as described above.
were analyzed using a TriCarb 2800 liquid scintillation
β
analyzer (PerkinElmer). The 45Са2+ uptake in synaptoꢀ
somes was determined from the difference in label conꢀ
tent between the glutamateꢀstimulated uptake and that
without stimulation and was expressed in percent of conꢀ
trol (control was taken as 100%). The statistical treatꢀ
Docosahexaenoic acid amide of {2'ꢀ[5ꢀ(3,4ꢀdichloꢀ
rophenylamino)ꢀ1,2,4ꢀthiadiazolꢀ3ꢀyl]ꢀ1'ꢀmethylethyl}ꢀ
(2",2",6",6"ꢀtetramethylpiperidinꢀ4"ꢀyl)amine (6a).
Method B. The reaction of 98 mg of docosahexaenoic
acid gave 67.4 mg of amide 6a, yield 30%, thick viscous
ment of the results was carried out using Student’s
tꢀtest.
colorless oil,
1H NMR (CDCl3);
1.02 and 1.09 (s + s, 6H, 2CH3), 1.24 and 1.25 (s + s,
6H, 2CH3), 1.39 (d, 3H, 6.6 Hz, NCHCH3), 1.41
(dt, 2H, = 12.0, 21.2 Hz, Hꢀ23,
2H, 3.3, 12.6 Hz, H ꢀ23, H ꢀ25), 2.08 (m, 2H, Hꢀ21),
Rf 0.72 (chloroform–methanol, 10 : 1).
δ, ppm: 0.97 (m, 3H, Hꢀ22),
ACKNOWLEDGMENTS
We are grateful to V.A. Karnaukhova (Institute of
Bioorganic Chemistry, RAS) for her help with the synꢀ
J
J
Н
НHꢀ25), 1.86 (dd, thesis of the target compounds.
J
Н
Н
This work was supported in part by the Presidium of
the Russian Academy of Sciences (program no. 7
“Development of Methods for the Preparation of Chemꢀ
ical Compounds and Design of New Materials”) and the
Ministry of Education and Science of the Russian Federꢀ
ation (State Contract no. 14.740.11.0810).
2.47 (m, 4H, Hꢀ2, Hꢀ3), 2.82 (m, 10H, Hꢀ6, Hꢀ9, Hꢀ12,
Hꢀ15, Hꢀ18), 3.25 (m, 2H, СН2CHМе), 3.74 (m, 1H,
Hꢀ24), 4.48 (m, 1H, NCHМе), 5.34 (m, 12H, Hꢀ4,
Hꢀ5, Hꢀ7, Hꢀ8, Hꢀ10, Hꢀ11, Hꢀ13, Hꢀ14, Hꢀ16, Hꢀ17,
Hꢀ19, Hꢀ20), 7.17 (dd, 1H,
J
2.2, 8.6 Hz, Harom), 7.45 (d,
1H, 8.6 Hz, Harom), 7.51 (d, 1H,
J
J
2.2 Hz, Harom).
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Docosahexaenoic acid amide of {2'ꢀ[5ꢀ(3ꢀchloroꢀ4ꢀ
methylphenylamino)ꢀ1,2,4ꢀthiadiazolꢀ3ꢀyl]ꢀ1'ꢀmethylꢀ
ethyl}ꢀ(2",2",6",6"ꢀtetramethylpiperidinꢀ4ꢀyl)amine
(6b). Method A. Reaction of 91 mg of docosaꢀ
hexaenoic acid gave 69 mg of amide 6b, yield 30%,
thick viscous colorless oil, Rf 0.32 (chloroform–methꢀ
anol, 20 : 1).
1H NMR (CDCl3,
1.02 and 1.09 (s + s, 6H, 2CH3), 1.23 and 1.24 (s + s,
6H, 2CH3), 1.41 (d, 3H, 6.6 Hz, NCHCH3), 1.43
(dt, 2H, = 12.0, 21.2 Hz, Hꢀ23, Hꢀ25), 1.84 (dd,
2H, 3.3, 12.6 Hz, H ꢀ23, H ꢀ25), 2.08 (m, 2H, Hꢀ
δ, ppm): 0.99 (m, 3H, Hꢀ22),
J
J
Н
Н
J
Н
Н
21), 2.34 (s, 3H, ArCH3), 2.46 (m, 4H, Hꢀ2, Hꢀ3),
2.80 (m, 10H, Hꢀ6, Hꢀ9, Hꢀ12, Hꢀ15, Hꢀ18), 3.23
(m, 2H, СН2CHМе), 3.75 (m, 1H, Hꢀ24), 4.49 (m,
1H, NCHMe), 5.36 (m, 12H, Hꢀ4, Hꢀ5, Hꢀ7, Hꢀ8,
Hꢀ10, Hꢀ11, Hꢀ13, Hꢀ14, Hꢀ16, Hꢀ17, Hꢀ19, Hꢀ20),
7.35 (m, 3H, Harom).
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DOKLADY CHEMISTRY Vol. 447
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2012