Нові сполуки конденсованого 1,2,4-триазину: молекулярне моделювання, синтез та біотестування
Мета. Розширити спектр біологічно активних сполук 1,2,4-триазино[5,6-b][1,4]бензотіазинів (1,2,4-ТБТ) та виявити з-поміж них інгібітори синтезу РНК. Методи. Неемпірична квантово-хімічна оптимізація структури 3-оксо-1,2,4-ТБТ фрагментно-орієнтованим заміщенням, їхній молекулярний докінг у віртуальну...
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| Cite this: | Нові сполуки конденсованого 1,2,4-триазину: молекулярне моделювання, синтез та біотестування / Л.Г. Пальчиковська, І.В. Алексєєва, М.О. Платонов, О.М. Костенко, Л.С. Усенко, В.В. Негруцька, А.Д. Швед // Biopolymers and Cell. — 2009. — Т. 25, № 6. — С. 491-499. — Бібліогр.: 14 назв. — англ. |
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Пальчиковська, Л.Г. Алексєєва, І.В. Платонов, М.О. Костенко, О.М. Усенко, Л.С. Негруцька, В.В. Швед, А.Д. 2019-06-13T18:01:08Z 2019-06-13T18:01:08Z 2009 Нові сполуки конденсованого 1,2,4-триазину: молекулярне моделювання, синтез та біотестування / Л.Г. Пальчиковська, І.В. Алексєєва, М.О. Платонов, О.М. Костенко, Л.С. Усенко, В.В. Негруцька, А.Д. Швед // Biopolymers and Cell. — 2009. — Т. 25, № 6. — С. 491-499. — Бібліогр.: 14 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.0007FC https://nasplib.isofts.kiev.ua/handle/123456789/153309 547.836:573.3:577.15.04 Мета. Розширити спектр біологічно активних сполук 1,2,4-триазино[5,6-b][1,4]бензотіазинів (1,2,4-ТБТ) та виявити з-поміж них інгібітори синтезу РНК. Методи. Неемпірична квантово-хімічна оптимізація структури 3-оксо-1,2,4-ТБТ фрагментно-орієнтованим заміщенням, їхній молекулярний докінг у віртуальну мішень, раціональний хімічний синтез теоретично спрогнозованих речовин та їхнє тестування у модельній ферментативній системі транскрипції. Результати. Отримано низку похідних 1,2,4-ТБТ із замісниками у бензольному і триазиновому циклах базової молекули. Тестування сполук у системі транскрипції in vitro ДНК-залежною РНК-полімеразою бактеріофага Т7 визначило структурно- та концентраційно-залежне пригнічення ними синтезу РНК. Для всіх досліджуваних сполук експериментальні дані задовільно корелюють з результатами віртуального скринінгу. Найефективнішим інгібітором виявився 3-оксо-8-бутил-1,2,4- ТБТ, який у концентрації 6 мкг/мл забезпечує повне блокування транскрипції. Висновки. Аналіз даних тестування дозволяє припустити, що пригнічення синтезу РНК обумовлено зв’язуванням 3-оксо-8-бутил-1,2,4-ТБТ як з неасоційованою РНК-полімеразою, так і з РНК-полімеразою у складі транскрипційного комплексу. Aim. The addition of the new biologically active compounds to the series of the 1,2,4-triazino[5,6-b] [1,4]benzothiazine (1,2,4-TBT) derivatives and reveal among them the RNA synthesis inhibitors. Methods. The methods of structure optimization the 3-oxo-1,2,4-TBT by fragment-oriented substitution, the molecular doking of the new structures in a virtual target, the rational chemical synthesis of the theoretically prognoses compounds and their testing in the model transcription in vitro. Results. The series of 1,2,4-TBT derivatives with substituents in the benzene and triazine cycles of the base molecule were synthesized. Testing of the synthesized compounds in in vitro transcription system directed by T7 RNA polymerase revealed the structure- and concentration-dependent inhibition of the RNA synthesis by some of compounds. The experimental and virtual screening data for all investigated compounds have a good correlation. It was found that most effective derivative is the 3-oxo-8-butyl-1,2,4-TBT which completely inhibited transcription at the concentration of 6 mg/ml. Conclusions. Analysis of the testing data allows us to assume that the inhibition of the RNA synthesis is caused by binding of the 3-oxo-8-butyl-1,2,4-TBT both as to free RNA polymerase molecules, as to those consisting in transcriptional complex with DNA. Цель. Расширить спектр биологически активных соединений в ряду производных 1,2,4-триазино[5,6-b][1,4]бензотиазина (1, 2,4-TБT) и выявить среди них ингибиторы синтеза РНК. Методы. Неэмпирическая, квантово-химическая оптимизация структуры 3-оксо-1,2,4-ТБТ фрагментно-ориентированным замещением, молекулярный докинг новых структур в виртуальную мишень, рациональный химический синтез теоретически спрогнозированных веществ и их тестирование в модельной ферментативной системе транскрипции. Результаты. Получена серия производных 1,2,4-ТБТ с заместителями в бензольном и триазиновом циклах базовой молекулы. Тестирование веществ в системе транскрипции in vitro ДНК-зависимой РНК-полимеразой бактериофага Т7 определило структурно- и концентрационно-зависимое ингибирование синтеза РНК. Для всех исследованных соединений экспериментальные данные удотвлетворительно коррелируют с результатами виртуального скрининга. Наиболее эффективным ингибитором оказался 3-оксо-8-бутил-1,2,4-ТБТ, который в концентрации 6 мкг/мл обеспечивает полное блокирование транскрипции. Выводы. Анализ данных тестирования позволяет предположить, что угнетение синтеза РНК обусловлено связыванием 3-оксо-8-бутил-1,2,4-ТБТ как с неассоциированной РНК-полимеразой, так и с РНК-полимеразой в составе транскрипционного комплекса. uk Інститут молекулярної біології і генетики НАН України Вiopolymers and Cell Біоорганічна хімія Нові сполуки конденсованого 1,2,4-триазину: молекулярне моделювання, синтез та біотестування New 1,2,4-triazine bearing compounds: molecular modelling, synthesis and biotesting Новые соединения конденсированного 1,2,4-триазина: молекулярное моделирование, синтез и биотестирование Article published earlier |
| institution |
Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| collection |
DSpace DC |
| title |
Нові сполуки конденсованого 1,2,4-триазину: молекулярне моделювання, синтез та біотестування |
| spellingShingle |
Нові сполуки конденсованого 1,2,4-триазину: молекулярне моделювання, синтез та біотестування Пальчиковська, Л.Г. Алексєєва, І.В. Платонов, М.О. Костенко, О.М. Усенко, Л.С. Негруцька, В.В. Швед, А.Д. Біоорганічна хімія |
| title_short |
Нові сполуки конденсованого 1,2,4-триазину: молекулярне моделювання, синтез та біотестування |
| title_full |
Нові сполуки конденсованого 1,2,4-триазину: молекулярне моделювання, синтез та біотестування |
| title_fullStr |
Нові сполуки конденсованого 1,2,4-триазину: молекулярне моделювання, синтез та біотестування |
| title_full_unstemmed |
Нові сполуки конденсованого 1,2,4-триазину: молекулярне моделювання, синтез та біотестування |
| title_sort |
нові сполуки конденсованого 1,2,4-триазину: молекулярне моделювання, синтез та біотестування |
| author |
Пальчиковська, Л.Г. Алексєєва, І.В. Платонов, М.О. Костенко, О.М. Усенко, Л.С. Негруцька, В.В. Швед, А.Д. |
| author_facet |
Пальчиковська, Л.Г. Алексєєва, І.В. Платонов, М.О. Костенко, О.М. Усенко, Л.С. Негруцька, В.В. Швед, А.Д. |
| topic |
Біоорганічна хімія |
| topic_facet |
Біоорганічна хімія |
| publishDate |
2009 |
| language |
Ukrainian |
| container_title |
Вiopolymers and Cell |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| format |
Article |
| title_alt |
New 1,2,4-triazine bearing compounds: molecular modelling, synthesis and biotesting Новые соединения конденсированного 1,2,4-триазина: молекулярное моделирование, синтез и биотестирование |
| description |
Мета. Розширити спектр біологічно активних сполук 1,2,4-триазино[5,6-b][1,4]бензотіазинів (1,2,4-ТБТ) та виявити з-поміж них інгібітори синтезу РНК. Методи. Неемпірична квантово-хімічна оптимізація структури 3-оксо-1,2,4-ТБТ фрагментно-орієнтованим заміщенням, їхній молекулярний докінг у віртуальну мішень, раціональний хімічний синтез теоретично спрогнозованих речовин та їхнє тестування у модельній ферментативній системі транскрипції. Результати. Отримано низку похідних 1,2,4-ТБТ із замісниками у бензольному і триазиновому циклах базової молекули. Тестування сполук у системі транскрипції in vitro ДНК-залежною РНК-полімеразою бактеріофага Т7 визначило структурно- та концентраційно-залежне пригнічення ними синтезу РНК. Для всіх досліджуваних сполук експериментальні дані задовільно корелюють з результатами віртуального скринінгу. Найефективнішим інгібітором виявився 3-оксо-8-бутил-1,2,4- ТБТ, який у концентрації 6 мкг/мл забезпечує повне блокування транскрипції. Висновки. Аналіз даних тестування дозволяє припустити, що пригнічення синтезу РНК обумовлено зв’язуванням 3-оксо-8-бутил-1,2,4-ТБТ як з неасоційованою РНК-полімеразою, так і з РНК-полімеразою у складі транскрипційного комплексу.
Aim. The addition of the new biologically active compounds to the series of the 1,2,4-triazino[5,6-b] [1,4]benzothiazine (1,2,4-TBT) derivatives and reveal among them the RNA synthesis inhibitors. Methods. The methods of structure optimization the 3-oxo-1,2,4-TBT by fragment-oriented substitution, the molecular doking of the new structures in a virtual target, the rational chemical synthesis of the theoretically prognoses compounds and their testing in the model transcription in vitro. Results. The series of 1,2,4-TBT derivatives with substituents in the benzene and triazine cycles of the base molecule were synthesized. Testing of the synthesized compounds in in vitro transcription system directed by T7 RNA polymerase revealed the structure- and concentration-dependent inhibition of the RNA synthesis by some of compounds. The experimental and virtual screening data for all investigated compounds have a good correlation. It was found that most effective derivative is the 3-oxo-8-butyl-1,2,4-TBT which completely inhibited transcription at the concentration of 6 mg/ml. Conclusions. Analysis of the testing data allows us to assume that the inhibition of the RNA synthesis is caused by binding of the 3-oxo-8-butyl-1,2,4-TBT both as to free RNA polymerase molecules, as to those consisting in transcriptional complex with DNA.
Цель. Расширить спектр биологически активных соединений в ряду производных 1,2,4-триазино[5,6-b][1,4]бензотиазина (1, 2,4-TБT) и выявить среди них ингибиторы синтеза РНК. Методы. Неэмпирическая, квантово-химическая оптимизация структуры 3-оксо-1,2,4-ТБТ фрагментно-ориентированным замещением, молекулярный докинг новых структур в виртуальную мишень, рациональный химический синтез теоретически спрогнозированных веществ и их тестирование в модельной ферментативной системе транскрипции. Результаты. Получена серия производных 1,2,4-ТБТ с заместителями в бензольном и триазиновом циклах базовой молекулы. Тестирование веществ в системе транскрипции in vitro ДНК-зависимой РНК-полимеразой бактериофага Т7 определило структурно- и концентрационно-зависимое ингибирование синтеза РНК. Для всех исследованных соединений экспериментальные данные удотвлетворительно коррелируют с результатами виртуального скрининга. Наиболее эффективным ингибитором оказался 3-оксо-8-бутил-1,2,4-ТБТ, который в концентрации 6 мкг/мл обеспечивает полное блокирование транскрипции. Выводы. Анализ данных тестирования позволяет предположить, что угнетение синтеза РНК обусловлено связыванием 3-оксо-8-бутил-1,2,4-ТБТ как с неассоциированной РНК-полимеразой, так и с РНК-полимеразой в составе транскрипционного комплекса.
|
| issn |
0233-7657 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/153309 |
| citation_txt |
Нові сполуки конденсованого 1,2,4-триазину: молекулярне моделювання, синтез та біотестування / Л.Г. Пальчиковська, І.В. Алексєєва, М.О. Платонов, О.М. Костенко, Л.С. Усенко, В.В. Негруцька, А.Д. Швед // Biopolymers and Cell. — 2009. — Т. 25, № 6. — С. 491-499. — Бібліогр.: 14 назв. — англ. |
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New 1,2,4-triazine bearing compounds: molecular
modeling, synthesis and biotesting
L. G. Palchykovska, I. V. Alexeeva, M. O. Platonov, O. M Kostenko, L. S. Usenko,
V. V. Negrutska, A. D. Shved
Institute of molecular biology and genetics NAS of Ukraine
150, Zabolotnogo Str, Kyiv Ukraine, 03680
l.pàlñhykovska@imbg.org.ua
Aim. To enlarge a spectrum of biologically active compounds in the series of the 1,2,4-triazino[5,6-b]
[1,4]benzothiazine (1,2,4-TBT) derivatives and reveal among them efficient inhibitors of RNA synthesis
Methods. The methods of structure optimization of the 3-oxo-1,2,4-TBT by fragment-oriented substitution,
the molecular doking of new structures in a virtual target, the rational chemical synthesis of the
theoretically predicted compounds and their testing in the system of transcription in vitro. Results. The
series of 1,2,4-TBT derivatives with substituents in the benzene and triazine cycles of a base molecule were
synthesized. The testing of synthesized compounds in the in vitro transcription system directed by T7 RNA
polymerase revealed the structure- and concen- tration-dependent inhibition of the RNA synthesis by some
of these compounds. The experimental and virtual screening data for all investigated compounds have a
good correlation. It was found that most effective derivative is the 3-oxo-8-butyl-1,2,4-TBT which
completely inhibited transcription at the concentration of 6 mg/ml. Conclusions. The biotesting results
allow us to assume that the inhibition of RNA synthesis is caused by binding of the 3-oxo- 8-butyl-1,2,4-TBT
to both free RNA polymerase molecules and tho- se including in a transcriptional complex with DNA.
Keywords: 1,2,4-triazino[5,6-b][1,4]benzothiazines, design, virtual screening, synthesis, model
transcription system.
Introduction. Biological effect of polycyclic planar
molecules is commonly ascribed to their interaction
with DNA [1]. The compounds of such nature may
inhibit the function of enzyme complexes in the system
of nucleic acid biosynthesis involving DNA, such as
RNA- and DNA-polymerase, -topoisomerase,
-helicase complexes, etc. Preliminary studies on the
effect of a number of new tricyclic
1,2,4-triazine-bearing bases and their nucleosides on
reproduction of viruses from Íårpes viridae family,
virus of common herpes (VCH) and Epstein-Barr virus
(EBV) [3, 4] prompted us for further optimization of
the parent base to obtain more effective antiviral
substances.
491
ISSN 1993-6842. Biopolymers and cell. 2009. vol. 25. N 6. Translated from Ukrainian
Ó Institute of Molecular Biology and Genetics NAS of Ukraine, 2009
The aim of this work is to extend the spectrum of
effective biologically active compounds of tricyclic
system, 1,2,4-triazino[5,6-b][1,4]-benzothiazines
(1,2,4-TBT), bioisosters of the alloxazine and
isoalloxazine (that present natural components of
riboflavin coenzymes), and structural analogs of some
polycyclic antibiotics. Given the possibility of viral
polymerase inhibition it seemed reasonable to estimate
the effect of new derivatives on the RNA synthesis in
model system of transcription directed by
DNA-dependent Ò7 RNA-polymerase (RNAP) in vitro.
To achieve the desired aim it was necessary to
tackle such problems: to perform computer design of
starting compound, 3-oxo-1,2,4-TBT, through
modifying triazine and benzothiazine fragments of the
molecule and clear up the putative mechanism of
action for the designed new structures by their
molecular docking to virtual target of the T7 RNAP
catalytic site.
Materials and methods All calculations were
performed in program QXP /Flo+ [8]. Docking was
carried out using flexible ligand and fixed models of the
receptor to employ the algorithm of systematic docking
(SDOCK+), which demonstrated satisfactory ability to
reproduce ligand conformation with minimal RMSD
(medium square deviation) relative to crystallographic
results [9]. Maximal number of calculation steps was
determined on 300 and 10 optimal complexes
(proceeding from inherent scoring functions of QXP)
that are maintained for [the] analysis. The receptor
model was based on X-ray results for Ò7 RNAP–DNA
(PDB X-Ray code: 1SOV) transcription complex [10].
Chemical synthesis. For the synthesis of condensed
triazine derivatives 6-bromo-1,2,4-triazin-3,5(2Í,4Í)-
dione (1), its 2-b-D-triacetylribofuranoside (2) and
2-amino-4(5)-R-benzothiols (3) were used, synthesized
according to described earlier techniques [11, 12].
Other reagents and solvents were purchased from
UkrOrgSynthesis (Ukraine) and “Fluka” (Switzerland).
The obtained compounds were controlled through
thin layer chromatography (TLC) on the plates Silica
gel 60 F254 («Ìårñk», Germany) in the system of
chloroform/ethanol (9:1) or (98:2) solvents. For
preparative chromatography on silica gel a gradient of
ethanol concentrations in chloroform was used.
1Í-NMR spectra of synthesized compounds were
recorded on spectrometer «Mercury-400» («Varian»,
USA) in DMSO-d6, electronic absorption spectra – on
spectrometer Shimadzu UV-3100 (Japan). Melting
points (mp) were estimated on Boetius device.
General technique for production of
2,4-dihydro-3-oxo-7(8)-R-1,2,4-triazino[5,6-b][1,4]b
enzothiazines (4à–c). 5-mMol triazine bromoderivative
(1) and 5 mmîl respective 2-aminobenzothiol (3) were
boiled in 10 ml of dioxane-pyridine mixture (9:1) with
backflow condenser for 4 h. The first hour reaction
results in voluminous precipitate which is gradually
dissolved during its further occurrence. Following the
reaction mixture cooling, the solvents were removed in
vacuum, to crystallize the residue with
N,N-dimethylformamide (physico-chemical
characteristics of the novel compounds are presented in
the table).
Synthesis of 2-b-D-ribofuranosyl-3(4Í)-oxo-
7-trifluoromethyl-1,2,4-triazino[5,6-b][1,4]benzothia
zine (5a). M e t h o d I. Suspension of acylnucleoside 2
bromoderivative (452 mg, 1 mmol) in 5 ml [of]
ethanol:dimethylformamide (4:1) mixture was added
by 0.08 ml [of] pyridine (1 mmol) and
2-amino-4-trifluoromethyl-benzothiol (3a) (0.193 g, 1
mmol) and boiled for 8 h. After solvent evaporating,
acylnucleoside was isolated by chromatography on
column with Silica gel followed by subsequent
deblocking with 25% hydroxide ammonium solution
(10 ml) for 20 h at room temperature. Target
ribonucleoside was crystallized with ethanol. 112 mg
(27%) of analytically pure 5a were produced.
M e t h o d II. To a suspension of base 4a (290 mg,
1 mmol) and tetraacetylribose (350 mg, 1.1 mmol) in
10 ml of anhydrous acetonitrile was added 0,2 ml (1,6
mmol) of trimethylclorosilane (TMCS), 0,17 ml (0,8
mmol) [of] hexamethyldisilane (HMDS) and 0.15 ml
(1,6 mmol) [of] tin tetrachloride. The reaction mixture
was stirred for 7 h at room temperature and evaporated
to dryness in vacuum. The residue was dissolved in
chloroform, washed with water and solution sodium
acetate, dried over magnesium sulfate, filtered and
evaporated to a dark syrup. The acylnucleoside [was]
purified by flash chromatography on silica gel by
subsequent removal of acetylic groups with hydroxide
ammonium solution. The crystalline nucleoside 5a was
obtained: yield 138 mg (33%).
492
PALCHYKOVSKA L. G. ET AL.
Synthesis of 2-b-D-ribofuranosyl-3(4Í)-oxo-7-
chloro-1,2,4-triazino[5,6-b][1,4]benzothiazine (5b)
was performed similarly to 5a by the both methods.
Yield of the target ribonucleoside by the method I made
up 42.3 mg (11%), by the method II – 87.5 mg (23%) of
analytically pure 5b.
Transcription reaction ³n v³trî. The technique for
testing the synthesized substances on [a] model system
of transcription was presented in previous report [13].
There were used such reagents: linearized DNA of
pTZ19R plasmide as a template, four canonical
ribonucleoside triphosphates, RNA inhibitor
(RiboLock), transcription buffer (tris-HCl, pH 7.5,
MgCl2, spermidine, dithiothreitol) of the firm
“Fermentas” (Lithuania).
Test-agent pre-incubation with DNA-template or
RNA-polymerase was conducted in the presence of
other system components during 15 min at room
temperature. Following pre-incubation the system was
added with proper lacking biopolymer, and the reaction
proceeded for 40 min.
Results and discussion. Molecular modeling of
condensed triazines. Through computer studies it was
scheduled to solve such tasks: determine the topology
and tautomere status of the 3-oxo-1,2,4-TBT parent
base, accomplish the design by introducing the
substituents of various structure and nature in triazine
and benzothiazine fragments of molecule; elucidate
behavior of the basic molecule and its derivatives in the
model of Ò7 RNAÐ binding site; construct more active
derivatives of the basic structure consistent with
numerical trials.
Constructing the novel molecular structures
suggests the invariance of the functional group
position, donor-acceptor centers in triazine 1,2,4-ÒBÒ
fragment, capable to interact with both matrix bases
and conservative amino acid surrounding.
Benzothiazine fragment being linked to triazine
nucleus fills the extended cleft of polymerase catalytic
site concurrently promoting realization of the
numerous possibilities for stacking- and
cation-p-interaction. To our mind substitution of the
benzene nucleus may enhance the interaction of new
derivatives with the enzyme at the expense of increase
in their volume and hydrophobicity.
The presence of four nitrogen atoms and mobile
protons results in tautomeric conversions of the basic
compound and its derivatives.
493
NEW 1,2,4-TRIAZINE BEARING COMPOUNDS
Compound Yield, % M.p., ( °Ñ) 1Í-NMR ( DMSO-d6: d (ppm) UV, lmax, nm
4à 67 ³300
7,03 (m, 2H, Ph); 7,17 (d, 1Í, Ðh ); 11,27 (s, 1Í, NH);
12,09 (br. s, 1H, NH)
232; 246; [280]*; 369
4b 80 330
7,26 (s,1H, Ph); 7,34 (d, lH,Ph); 7,26 (s, 1H, Ph); 11,25 (s,
1H, NH); 12,15 (s, 1H, NH)
212; 243; [268]*; 379
4ñ 53 279-282
0,88 (t, 3Í, CH3); 1,25 (m, 2H, CH2 ); 1,49 (m, 2H, CH2);
2,46 (m, 2H, CH2); 6,94 (m, 2H, Ph); 7,00 (s, CH, Ph);
10,98 (br. s, 1H, NH); 11,80 (br. s, 1H, NH)
243; [268]*; 385
5à 27 (33)** 165-167
3,41 (m, 1H, Hb-5'); 3,50 (m, 1H, Ha-5'); 3,78 (d,1H, H-4',
J = 4,8); 3,98 (dd, 1H, H-3'); 4,15 (dd, 1H, H-2'); 4,39 (t,
1H, OH-5'); 4,79 (d, 1H, OH-3', J = 5,6); 5,00 (d, 1H,
OH-2', J = 4,8); 5,85 (d, 1H, H-1', J = 0,8); 7,26-7,30 (m,
3H, Ph); 11,59 (s, 1H, NH)
212; 249,5; [280]*; 379
5b 11 (23)** 199-202
3,40 (m, 1H, Hb-5'); 3,49 (m, 1H, Ha-5'); 3,77 (dd,1H, H-4',
J = 4,8); 3,97 (dd, 1H, H-3'); 4,15 (dd, 1H, H-2'); 4,39 (t,
1H, OH-5'); 4,78 (d, 1H, OH-3', J = 6,0); 4,99 (d, 1H,
OH-2', J = 5,2); 5,85 (d, 1H, H-1', J = 2,4); 6,99–7,15 (m,
3H, Ph); 11,46 (s, 1H, NH)
244; [266]*; 387
N o t e. UV spectra were recorded in ethanol; *concealed maximum; **compound yield by glycosilation method
Physico-chemical characteristics of the synthesized 1,2,4-triazino[5,6-b][1,4]benzothiazine derivatives(4à–ñ, 5à, 5b)
The first step of the computer studies was to
determine more possible and energetically more
advantageous tautomers of basic 3-oxo-1,2,4-TBT and
their topology. Above characteristics may reflect in
their behavior in the enzyme target and, hence, in the
mechanism of action of these compounds, in other
words, may affect their interaction with biopolymeres.
The variations in energy of such interaction for a
number of tautomers appeared to be insignificant while
amino acid surrounding tended to be cardinally
different, sometimes provoking emergence of so called
“false positive hits”. Optimization of the
3-oxo-1,2,4-TBT structure was accomplished by DFT.
The spatial structures of the most probable
tautomers of basic compound were constructed by
means of molecular design. Only one of three
tautomers structures was close to planar, which appears
to be energetically most favorable. According to
spectral results exactly this tautomer was realized upon
synthesis and used in docking to study the interaction
between the compounds investigated and Ò7 RNAP
active site. Fig.1 presents the structures of
3-oxo-1,2,4-TBT tautomers (4).
Further design of 3-oxo-1,2,4-TBT was aimed at
enhancing the interaction of novel derivatives with the
enzyme target and determining the putative mechanism
of their interaction. To fill the volume of the enzyme
complex pocket more densely the benzothiazine
fragment was modified by introducing the haloid,
haloidalkyl and alkyl groups in various positions.
While constructing the model of active site we
considered all amino acid residues within the radius of
0.1 nm around the catalytic pocket as those that may be
involved in ligand binding and those that ensure
polymerase functioning (Fig. 2). It is important to
define the enzyme dynamics during the catalytic event.
It is necessary in order to avoid the appearance of the
above mentioned “false positive hits”, i.e. the
compounds which should be active according to
calculations but do not show any activity in biotestng.
Through the docking in the chosen region of
transcription complex in the model of Ò7 RNAP site
there was carried out the virtual screening for modelled
structures. The analysis of the obtained data revealed
that ligands were situated for certain in the catalytic
pocket and kept in it at the expense of hydrogen bonds,
stacking- and cation-p-interaction with amino acid
residues and matrix (template) nucleotide bases. The
triazine fragment for most of considered structures is
localized within immediate proximity to the catalytic
Mg2+ ion and Tyr639 residue which takes part in
recognition of the regular ribonucleoside triphosphate
involved in the catalytic site. (Fig. 3, see pasting-in).
For derivatives with substitutes in the benzene ring
two various models of binding proved to be acceptable.
For the compounds with halogen-substitute or
trifluoromethyl group at the 7th position of benzene
ring, the triazine cycle is fixed by stacking interaction
with the matrix base (adenine) and hydrogen bonds
with residues Asp812 and Arg425 ( the distance
494
PALCHYKOVSKA L. G. ET AL.
N
N
N
S
NO
2
3
4 5
6
7
8
910
His784
Tyr639
Arg423
Arg425 Asp812
Cys540
Met635
Fig. 2. Schematic representation of inhibitors binding site with
marked key aminoacids residues
N
N
N
S
NO
H
H
2
3
4 5
6
7
8
910
N
N
N
S
NO
H
2
3
4 5
6
7
8
910
H
N
N
N
S
NO
H
H
2
3
4 5
6
7
8
910
0 24,98 2,62a b c
Fig. 1. The most possible tautomers(à–ñ) of 3-oxo-1,2,4-triazino[5,6-b][1,4]benzothiazine (4) and there relative Gibbs energy(Kcal/mol)
between respective atoms makes up 0.23 and 0.20 nm)
with substitutes establishing slight electrostatic bind
with Lys441 (Fig. 4, see pasting-in).
There is other situation for 3-Oxo-1,2,4-TBT: the
triazine fragment is kept in the active site owing to
hydrogen bonds with residues Arg422 and Tyr427 (the
distance between the atoms comprises 0.28 and 0.21
nm, respectively), while benzothiazine one – due to
hydrogen bonds with His784 residue (the distance
between the proper atoms is 0.10 nm). Alkyl strand
seems to be plunged into the bulky pocket, formed by
the Ñys540 and Met635 residues (Fig. 5, see pasting-in)
and fixed in it by the hydrophobic interactions. In such
case the compound bonds with the matrix base within
the complex are lacking, hence, its retention does occur
only due to the interaction with enzyme.
Analysis of behaviour of the above mentioned
bases’ derivatives that carry glycoside residue in the
triazine fragment in the model of catalytic site
demonstrated the lack of unambiguous superposition
upon identification of the most advantageous versions
of their arrangement within the target. It should be
emphasized that significant distinctions in the
respective positions of the molecule in a virtual target
may indicate a low energy of binding and as a result low
inhibitory capacity of this substance despite the
presence of five hydrogen bonds (Tyr639, His784,
Asp812 Lys441, Mg2+) and minor distances between
the atoms which form them (0.16; 0.22; 0.29; 0.26 ànd
0.25, respectively).
The results of virtual screening allow us to suggest
that the considered compounds may be potential
inhibitors, the extended alkyl substitute of
benzothiazine fragment being able to increase complex
stability and in this way enhance their inhibitory
activity. On the contrary, introduction of glycoside
residue into the triazine fragment may result in an
opposite effect.
Confirmation or rejection of this working
hypothesis may come from testing the derived
compounds’ activity in the in vitro system of
transcription catalysed by Ò7 RNAP.
In earlier studies we proposed rather simple scheme
for the synthesis of tricyclic triazine-bearing system
and its functionalization [3, 4]. Just this approach was
extended to the synthesis of a number of new
derivatives of basic 3-oxo-1,2,4-TBT (4).
Manufacturability and effectiveness are the positive
attributes of the above method - the reaction occurs in
unistage manner in the absence of specific conditions,
thus allowing production in preparative quantities.
Such approach was used to obtain new
triazine-bearing bases, substituted for chlorine atom or
trifluoromethyl group at 7 or n-butyl substituent at 8
position of the benzothiazine fragment (4à–ñ) and
respective ribonucleosides 5à, 5b with sugar moiety at
position 2 of the triazine fragment.
The efficiency of synthesis appeared to be over 50
%, reaching for 4à 67 %, 4b – 80%, 4c – 53 %, but
lower than that for non-substituted 3-oxo-1,2,4-TBT
yielded up to 90%. These data undoubtedly suggest the
essential effect of the chemical nature of the
substituents in the 2-aminobenzothiol molecule on the
process of triazine-bearing heterosystem formation. As
evidenced from Fig. 6, ribonucleosides 5à and 5b were
obtained by two techniques to define which of them is
more productive. In the case of triacetylriboside
5-bromo-6-azauracil (2) interaction with
2-aminobenzothiol 3a under above conditions (Fig. 6)
the yield of the target product was 27%, while upon
using reagent 3b it is essentially less – 11% (compound
5b). Considerable decrease in the efficiency of
synthesis of the ribofuranosides by this method may be
ascribed first of all to acylnucleoside susceptibility (2)
to the reaction conditions (temperature over 100°C),
and, like upon bases synthesis , to rather considerable
influence of Cl- and CF3-substitutes of corresponding
2-aminobenzothiols.
Direct glycosylation of the triazine-bearing bases
(4à, 4b) by the described procedure [3] somewhat
increased a yield of the final product for [the]
ÑF3-substituted [basis] (33%) and significantly raised
outcome for the Cl-substituted base (23%). Negative
contribution to the glycosylation process and obtainng
nucleoside of needed structure is determined by the
presence of at least two reaction centres (nitrogen
atoms in positions 2 and 4), i.e there is a possibility of
formation of two isomeric products. Analysis of the
reaction mixture showed that total yield of acylated
glycosides was about 60%, but the proportion of acetate
of the prevailing 2-N-nucleoside did not exceed 45%.
Free ribofuranosides 5à, 5b were derived by
495
NEW 1,2,4-TRIAZINE BEARING COMPOUNDS
deacetylation of corresponding acetates with
aqua-alcohol solution of ammonium hydroxide.
The comparison of tricyclic ribonucleosides
preparations, obtained in alternative ways, showed
their identity.
Investigation of the regulatory properties of the
obtained compounds in the model transcription complex
in vitro. The primary base (4) and its ribofuranoside
derivative (5) as compounds for comparison, the
synthesized bases (4à–ñ) and two ribonucleosides (5à,
5b) were tested in the enzyme system. The
concentration- and structure-dependent inhibition of total
RNA synthesis by the 1.2.4-ÒBÒ derivatives was
observed in the model system of Ò7 RNAP transcription
in vitro . Contrary to the initial base 4 , RNA synthesis
was reliably suppressed by three bases - with Ñ1-, CF3-
and n-butyl- substitutes in the benzothiazine fragment of
compounds 4à, 4b and 4ñ (Fig. 7). The n-butyl derivative
4c proved to be the most effective inhibitor of the
transcription process in vitro among the compounds under
study as was predicted by the docking results. Practically
complete absence of the transcript was found at 4c
concentration of 6 mg/ml.
Our hypothesis concerning glycoside derivatives
5à and 5b was also affirmed. Their testing in “living”
enzyme system did not reveal any inhibition of RNA
synthesis as compared with the corresponding bases,
i.e. these ribonucleosides (5à, 5b) appeared to be
“erroneously found hits” as was predicted by the
results of docking.
To establish to which of two biopolymers, DNA
and Ò7 RNAP, the 1.2.4-ÒBÒ derivatives possess
higher affinity the compounds were pre-incubated
with each of them. As is exemplified from butyl 4c
derivative, the pre-incubation with Ò7 RNAP
practically did not change the course of
concentration-dependent transcription reaction, as
compared with the standard conditions, but almost half
reduced the minimal inhibiting concentration of this
substance (Fig. 8). Instead, pre-incubation of 4c with
DNA considerably affects the course of
concentration-dependent RNA synthesis. The
substance distribution along the template seems to
reduce substantially the inhibitor working
concentration and as a result RNA transcript appears at
its higher concentrations (starting from 25 mg/ml).
496
PALCHYKOVSKA L. G. ET AL.
NH
N
H
N
O
O
Br
O AcAc O
O
Ac O
NH
N
N
O
O
Br
O HOH
O
OH
NH
N
N
O
S
N
R1
NH
N
N
H
S
NO
R
N H2
SH
R
1
2
3a -c
4 a- c
5 a, b
i
i
ii
ii
ii i
where R = R1 = H (4, 5); R = R1 = CF3 (a); R = R1 = Cl (b); R = n-Bu (c).
Rea gent s andreaction condit ions: ³) 1– EtOH–DMFA, Ðó, 120 °Ñ; 2 –NÍ
4
OH/EtOH, 20 °Ñ; i i)
dioxane–Ðó, 100 °Ñ; ³³ ³) 1 –acetoni trile, t etraacetylribose, TMCS, HMDS, SnCl
4
, 25 °Ñ; 2 –
NÍ4OH/EtOH, 20 °Ñ
5a, b
4a-c
3a-c
1
2
iii
i ii
iii
Fig. 6. Scheme of 7(8)R-3-oxo-
1,2,4-triazino[5,6-b][1,4]benzothiazine
synthesis
Probably, interaction of the compound 4c with Ò7
RNAP is critical in RNA synthesis suppression.
Conclusions.
1) A series of new
1,2,4-triazino[5,6-b][1,4]benzothiazine derivatives
was desihned by the methods of computational
biology.
2) The compounds of predicted structure with
lateral substituents in positions 2, 7 and 8 in
3-oxo-1,2,4-triazino[5,6-b][1,4]benzothiazine were
synthesized using technologically advantageous
methods, among them 2-N-ribonucleosides with CF3-
group or chlorine atom in the benzene ring; the identity
of the glycoside derivatives obtained by two
independent methods was verified.
3) Rather effective inhibitors of RNA synthesis
were identified by testing the synthesized substances in
Ò7 RNAÐ transcription system in vitro . Analysis of the
results allowed us to suggest that the compounds
studied are able to interact with both biopolymers, the
enzyme and DNA. According to the biological test,
3-oxo-8-n-butyl-triazinobenzothiazine appeared to be
497
NEW 1,2,4-TRIAZINE BEARING COMPOUNDS
0 0 0
9
94
0
20
40
60
80
100
Ê 1 2 3 4 5
à á
Ê 50 25 12,5 6 3
Êîíöåíòðàö³ÿ 4ñ, ìêã/ìë
%
Fig. 7. RNA synthesis inhibition in vitro (Ò7 RNAP transcription) by compound 4c under standard conditions. The total inhibition was
determined by absence of RNA transcripts in agarose gel: à – typical electroforegramm of RNA transcripts from three different experiments
(Ê – positive control; 1 – 50; 2 – 25; 3 – 12,5; 4 – 6; 5 – 3 mg/ml 4ñ); b – average results, presented as a percent of RNA transcript from
positive control (Ê) (Scion program)
0 3
12
47
73
0
20
40
60
80
100
0 0 0
8
56
0
20
40
60
80
100
à á
Ê 50 25 12,5 6 3
Êîíöåíòðàö³ÿ 4ñ, ìêã/ìë
Ê 50 25 12,5 6 3
Êîíöåíòðàö³ÿ 4ñ, ìêã/ìë
%%
Fig. 8. Influence of pre-incubation (15 min, at room temperature) of 4c with DNA-template (à) or Ò7 RNAP (b) on RNA synthesis
suppression in vitro (Ò7 RNAP transcription). Average results presented as a percent of RNA transcript from positive control (Ê) (Scion
program)
b
4c concentration, mg/ml
4c concentration, mg/ml4c concentration, mg/ml b
the most effective inhibitor; to our mind, a critical role
in this effect belongs to its interaction with both free
RNA-polymerase molecules and those inside the
transcription complex.
Ë. Ã. Ïàëü ÷è êî âñüêà, ². Â. Àëeêñººâà, Ì. Î. Ïëà òî íîâ, Î. Ì.
Êîñ òåí êî, Ë. Ñ. Óñåí êî, Â. Â. Íåã ðóöü êà, À. Ä. Øâåä
Íîâ³ ñïî ëó êè êîí äåí ñî âà íî ãî 1,2,4-òðè à çè íó: ìî ëå êó ëÿð íå ìî -
äå ëþ âàí íÿ, ñèí òåç òà á³îò åñ òó âàí íÿ
Ðå çþ ìå
Ìåòà. Ðîç øè ðè òè ñïåêòð á³îëîã³÷íî àê òèâ íèõ ñïî ëóê
1,2,4-òðè à çè íî[5,6-b][1,4]áåí çîò³àçèí³â (1,2,4-ÒÁÒ) òà âè ÿ âè -
òè ç-ïîì³æ íèõ ³íã³á³òîðè ñèí òå çó ÐÍÊ. Ìå òî äè. Íå åìï³ðè÷ -
íà êâàí òî âî-õ³ì³÷íà îïòèì³çàö³ÿ ñòðóê òó ðè 3-îêñî-1,2,4-ÒÁÒ
ôðàã ìåí òíî-îð³ºíòî âà íèì çàì³ùåí íÿì, ¿õí³é ìî ëå êó ëÿð íèé
äîê³íã ó â³ðòó àëü íó ì³øåíü, ðàö³îíàëü íèé õ³ì³÷íèé ñèí òåç òå î -
ðå òè÷ íî ñïðîã íî çî âà íèõ ðå ÷î âèí òà ¿õíº òåñ òó âàí íÿ ó ìî -
äåëüí³é ôåð ìåí òà òèâí³é ñèñ òåì³ òðàíñ êðèïö³¿. Ðå çóëü òà òè.
Îòðè ìà íî íèç êó ïîõ³äíèõ 1,2,4-ÒÁÒ ³ç çàì³ñíè êà ìè ó áåí çîëü íî -
ìó ³ òðè à çè íî âî ìó öèê ëàõ áà çî âî¿ ìî ëå êó ëè. Òåñ òó âàí íÿ ñïî ëóê
ó ñèñ òåì³ òðàíñ êðèïö³¿ in vitro ÄÍÊ-çà ëåæ íîþ ÐÍÊ-ïîë³ìå ðà -
çîþ áàê òåð³îôà ãà Ò7 âèç íà ÷è ëî ñòðóê òóð íî- òà êîí öåí -
òðàö³éíî-çà ëåæ íå ïðè ãí³÷åí íÿ íèìè ñèí òå çó ÐÍÊ. Äëÿ âñ³õ
äîñë³äæó âà íèõ ñïî ëóê åê ñïå ðè ìåí òàëüí³ äàí³ çà äîâ³ëüíî êî ðå -
ëþ þòü ç ðå çóëü òà òà ìè â³ðòó àëü íî ãî ñêðèí³íãó. Íà éå ôåê -
òèâí³øèì ³íã³á³òî ðîì âè ÿ âèâ ñÿ 3-îêñî-8-áó òèë-1,2,4- ÒÁÒ,
ÿêèé ó êîí öåí òðàö³¿ 6 ìêã/ìë çà áåç ïå ÷óº ïî âíå áëî êó âàí íÿ
òðàíñ êðèïö³¿. Âèñ íîâ êè. Àíàë³ç äà íèõ òåñ òó âàí íÿ äîç âî ëÿº
ïðè ïóñ òè òè, ùî ïðè ãí³÷åí íÿ ñèí òå çó ÐÍÊ îá óìîâ ëå íî çâ’ÿ çó -
âàí íÿì 3-îêñî-8-áó òèë-1,2,4-ÒÁÒ ÿê ç íå à ñîö³éî âà íîþ
ÐÍÊ-ïîë³ìå ðà çîþ, òàê ³ ç ÐÍÊ-ïîë³ìå ðà çîþ ó ñêëàä³ òðàíñ -
êðèïö³éíî ãî êîì ïëåê ñó.
Êëþ ÷îâ³ ñëî âà: 1,2,4-òðè à çè íî[5,6-b][1,4]-áåí çîò³àçè íè,
êîì ï’þ òåð íå ìî äå ëþ âàí íÿ, â³ðòó àëü íèé ñêðèí³íã, ñèí òåç, ìî -
äåëü íà ñèñ òå ìà òðàíñ êðèïö³¿.
Ë. È. Ïàëü ÷è êîâ ñêàÿ , È. Â. Àëeêñååâà, Ì. Î. Ïëà òî íîâ,
À. Í. Êîñ òåí êî, Ë. Ñ.Óñåí êî, Â. Â. Íåã ðóö êàÿ, À. Ä. Øâåä
Íî âûå ñî å äè íå íèÿ êîí äåí ñè ðî âàí íî ãî 1,2,4-òðè à çè íà:
ìî ëå êó ëÿð íîå ìî äå ëè ðî âà íèå, ñèí òåç è áè î òåñ òè ðî âà íèå
Ðå çþ ìå
Öåëü. Ðàñ øè ðèòü ñïåêòð áè î ëî ãè ÷åñ êè àê òèâ íûõ ñî å äè íå íèé â
ðÿäó ïðî èç âîä íûõ 1,2,4-òðè à çè íî[5,6-b][1,4]áåí çî òè à çè íà (1,
2,4-TÁT) è âû ÿ âèòü ñðå äè íèõ èí ãè áè òî ðû ñèí òå çà ÐÍÊ. Ìå -
òî äû. Íå ýì ïè ðè ÷åñ êàÿ, êâàí òî âî-õè ìè ÷åñ êàÿ îïòè ìè çà öèÿ
ñòðóê òó ðû 3-îêñî-1,2,4-ÒÁÒ ôðàã ìåí òíî-îðè åí òè ðî âàí íûì
çà ìå ùå íè åì, ìî ëå êó ëÿð íûé äî êèíã íî âûõ ñòðóê òóð â âèð òó -
àëü íóþ ìè øåíü, ðà öè î íàëü íûé õè ìè ÷åñ êèé ñèí òåç òå î ðå òè -
÷åñ êè ñïðîã íî çè ðî âàí íûõ âå ùåñòâ è èõ òåñ òè ðî âà íèå â
ìî äåëü íîé ôåð ìåí òà òèâ íîé ñèñ òå ìå òðàíñ êðèï öèè. Ðå çóëü -
òà òû. Ïî ëó ÷å íà ñå ðèÿ ïðî èç âîä íûõ 1,2,4-ÒÁÒ ñ çà ìåñ òè òå ëÿ -
ìè â áåí çîëü íîì è òðè à çè íî âîì öèê ëàõ áà çî âîé ìî ëå êó ëû.
Òåñ òè ðî âà íèå âå ùåñòâ â ñèñ òå ìå òðàíñ êðèï öèè in vitro ÄÍÊ-
çà âè ñè ìîé ÐÍÊ-ïî ëè ìå ðà çîé áàê òå ðèî ôà ãà Ò7 îïðå äå ëè ëî
ñòðóêòóð íî- è êîí öåí òðà öè îí íî-çà âè ñè ìîå èí ãè áè ðî âà íèå
ñèí òå çà ÐÍÊ. Äëÿ âñåõ èñ ñëå äî âàí íûõ ñî å äè íå íèé ýêñ ïå ðè ìåí -
òàëü íûå äàí íûå óäîò âëåò âî ðè òåëü íî êîð ðå ëè ðó þò ñ ðå çóëü -
òà òà ìè âèð òó àëü íî ãî ñêðè íèí ãà. Íà è áî ëåå ýô ôåê òèâ íûì
èí ãè áè òî ðîì îêà çàë ñÿ 3-îêñî-8-áó òèë-1,2,4-ÒÁÒ, êî òî ðûé â
êîí öåí òðà öèè 6 ìêã/ìë îá åñ ïå ÷è âà åò ïî ëíîå áëî êè ðî âà íèå
òðàíñ êðèï öèè. Âû âî äû. Àíàëèç äàí íûõ òåñ òè ðî âà íèÿ ïî çâî ëÿ -
åò ïðåä ïî ëî æèòü, ÷òî óãíå òå íèå ñèí òå çà ÐÍÊ îá óñëîâ ëå íî
ñâÿ çû âà íè åì 3-îêñî-8-áó òèë-1,2,4-ÒÁÒ êàê ñ íå àñ ñî öè è ðî âàí -
íîé ÐÍÊ-ïî ëè ìå ðà çîé, òàê è ñ ÐÍÊ-ïî ëè ìå ðà çîé â ñî ñòà âå
òðàíñ êðèï öè îí íî ãî êîì ïëåê ñà.
Êëþ ÷å âûå ñëî âà: 1,2,4-òðè à çè íî [5,6-b][1,4]áåí çî òè à çè íû,
êîì ïüþ òåð íîå ìîäåëèðîâà íèå, âèð òó àëü íûé ñêðè íèíã, cèí òåç,
ìî äåëü íàÿ ñèñ òå ìà òðàíñ êðèï öèè.
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UDC 547.836:573.3:577.15.04
Received 10.07.09
499
NEW 1,2,4-TRIAZINE BEARING COMPOUNDS
Figure to article by Palchykovska L. G. et al.
ISSN 1993-6842. Biopolymers and Cell. 2009. Vol. 25. N 6 (pasting-in)
Tyr 639
Arg 425
Mg2+
Asp 812
Fig. 3. Localization of 7-trifluoromethyl-3-oxo-
triazinobenzothiazin in the model of Ò7 RNAP active site (data of
molecular doking)
Met 635
His 784
Arg 423
Cys 540
Arg 425
Tyr 427
Fig. 4. Localization of 8-butyl-3-oxo-triazinobenzothiazin in the
model of Ò7 RNAP active site (method of molecular doking)
Tyr 427
Arg 425
Cys 540
Met 635
His 784
Arg 423
Tyr 639
Fig. 5. Superposition of the most probable positions of 8-butyl-3-oxo-1,2,4-triazinobenzothiazin (à) and
N2-ribonucleoside 7-trifluoromethyl-3-oxo-1,2,4-triazinobenzothiazin (b) according to doking results
b
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