Карцев В.Г.Избранные методы с-за и модифик. гетероциклов т.1 , 2003
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Table 1. Continued
07 |
H |
H |
4-MeSO2 |
F |
F |
0.012 |
1.921 |
1.92 |
1.79 |
1.77 |
08 |
Me |
H |
4-MeSO2 |
H |
H |
0.012 |
1.921 |
1.92 |
1.79 |
1.77 |
09 |
H |
Me |
4-MeSO2 |
H |
H |
3.0 |
1.910 |
–0.48 |
–0.47 |
–0.47 |
10 |
Me |
Me |
4-MeSO2 |
H |
H |
5.0 |
–0.699 |
–0.7 |
–0.92 |
–1.17 |
11 |
H |
Me |
H |
4-MeSO2 |
H |
1.0 |
0.000 |
0 |
0.28 |
0.31 |
12 |
H |
CH2CO- 4-MeSO2 |
H |
H |
0.9 |
0.046 |
0.05 |
–0.47 |
–0.58 |
|
|
|
2Et |
|
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13 |
Cl |
H |
4-MeSO2 |
Cl |
H |
0.016 |
1.796 |
1.8 |
1.79 |
1.79 |
14 |
Cl |
Cl |
4-MeSO2 |
Cl |
H |
3.6 |
–0.556 |
–0.56 |
–0.47 |
–0.45 |
Table 2. Observed and calculated activities of the test set
Molecule |
|
Observed activity |
Calculated activity |
Residual |
O |
S |
3.6 |
3.6 |
0 |
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O |
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Br |
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S |
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F |
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SC-558 |
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O |
Cl |
1.585 |
1.79 |
–0.205 |
|
|
|
N
MeO
CO2H
Indomethacin
Br |
2.698 |
3.3 |
–0.602 |
F3C N N
O
OS NH2
DUP-697
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Table 3. 3D-QSAR model describing correlation and statistical reliability for COX-2 inhibition activity
Model |
RMSA |
RMSP |
R2 |
Chance |
Size |
Match |
Variable |
No. of |
no. |
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compounds |
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|
01 |
0.37 |
0.40 |
0.91 |
0.00 |
3 |
0.59 |
2 |
14 |
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Molecular modeling and 3D QSAR studies were performed on a silicon graphics Indy R 4000 work station employing molecular simulations incorporations (MSI) software (Insight II [28], Discover [29], and Apex-3D [30]). 3D molecular structures of all the compounds were built in the builder module of Insight II software. These 3D structures were later optimized for their geometry using CVFF force field [31] and the energy minimization was performed using the steepest descent, conjugate gradient, Newton– Raphsons algorithms in sequence followed by Quasi-Newton–Raphson (va09a) implemented in the discover module by using 0.001 kcal/mol energy gradient convergence and maximum number of iteration set to 1000. Energy minimized structures were stored in MDL format. In order to check the validity of the above energy minimized techniques vis-a-vis other low energy conformations near global minimum, one of the most active compound (no. 7) was subjected to molecular dynamics (MD) simulations using CVFF force field [31]. In this procedure, the optimized conformations of compound no. 7 were randomized by setting random velocities and carrying out MD simulations at 0.1 ps at T = 1000 K. The obtained average conformations of compound no. 7 by this calculation was used as starting point for another 5 ps of MD simulations at T = 1000 K. The purpose of high temperature MD was to explore the conformational space extensively. An annealing procedure was subsequently applied to each average conformations obtained in high temperature simulations. The annealing was carried out, as slow cooling down of the structure from 1000 to 300 K. The last step of an annealing procedure was energy minimization. The total energy of these 20 conformations obtained in 75–150 ps simulation time ranged between 185.42 to 185.72 kcal that was near to the conformational energy (185.4056 kcal) obtained from the standard energy minimization procedure described above. Hence, the same energy minimized conformations were used in the 3D-QSAR model development.
The energy minimized structures were subjected to different computational chemistry programs including MOPAC 6.0 ver. (MNDO Hamiltonian) [32] for the calculations of different physicochemical and quantum-chemical parameters: atomic charge, π-popula- tion, electron donor (DON_01) and acceptor (ACC_01) index, HOMO, LUMO, hydrophobicity, and molar refractivity based on atomic contributions [33]. These parameters were then used by APEX-3D program for automated identification of pharmacophore and 3D QSAR model building [34]. The 5,6-diarylimidazo[2,1-b]thiazoles with definite COX-2 inhibitory activity (–log IC50) were subdivided into the following classes: (i) very active (> 1.7), (ii) active (< 1.7 and ≥ –0.60), (iii) less active/inactive (> –0.60). The 3D QSAR equations were derived by defining COX-2 inhibitory activity (–log IC50) as a dependent variable and biophoric center properties (π-population, charge, HOMO, LUMO, ACC_01, Don_01, hydrophobicity, refractivity), global properties (total hydrophobicity and total refractivity), secondary sites [(H-acceptor (presence), H-donor (presence), heteroatom (presence), hydrophobic (hydrophobicity), steric (refractivity) and ring
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(presence)] as independent variables with the occupancy set at 5, site radius at 0.80, sensitivity at 0.80, and randomization value at 100. Quality of each model was estimated from the r (coefficient of correlation), RMSA (calculated root mean square error based on all compounds with degree of freedom of correction), RMSP (root mean square error based on 'leave-one-out' with no degree of freedom correction), chance statistics, and match parameter as described in our earlier paper [35].
Results and discussion
In view of several 3D QSAR models generated for the training set, only one model (Fig. 2) containing all the compounds was selected based on the statistical criteria: correlation coefficient (r2 > 0.9), 100% reliability (chance = 0.00), good superimposition (match value > 0.50). This model (Table 3) was found to describe most accurately the distribution of the biophores for the COX-2 inhibition activity.
|
R3 |
SS1 |
|
|
B |
R2 |
C |
R |
SS2 |
|
|
R1 |
N |
R4 |
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||
S |
N |
|
A |
|
|
Fig. 2. Pictorial representation of pharmacophoric sites and secondary sites (SS) in the model.
There are biophoric sites: first site (A) being sulfur at position one, second site (B) being sulfur or any of the two oxygen atoms present at R3 or R4, and third site (C) being ring center attached at fifth or sixth positions. Two of the three biophoric sites (A and B), in the model are for hydrogen bonding, while an electron rich site (C) is probably involved in electrostatic interactions.
The spatial disposition of the biophoric sites in above model for selective COX-2 inhibition not only depends on physicochemical properties of biophoric centers corresponding to site A (π-population: 0.171 ± 0.0078, DON_01: 7.292 ± 0.452), site B (π-population: 0.302 + 0.011, DON_01: 6.265 + 0.050), and site C (cycle size: 6 ± 0.00, π-population: 6 ± 0.00) but also on their spatial arrangements, the mean interatomic distances of the three biophoric sites A, B, C, are as follows: A–B (10.005 ± 0.277), A–C (8.160 + 0.372), B–C (6.355 + 0.025).
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a |
b |
c |
Fig. 3. Mapping of the test set molecules to the biophoric sites (solid spheres) and secondary sites (grey circles): (a) Indomethacin, (b) DUP-697, (c) SC-558 (conformation derived from its x-ray crystal structure with COX-2 [36]).
Tyr 385
PheArg 5138 |
C
A
B
His 90 |
Tyr 355 |
Arg 120
Fig. 4. Stereo-view of the interactions of SC-558 with the cyclooxygenase-2 active site [36] vis-a-vis biophoric sites A, B, C.
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In order to explain the variations in COX-2 inhibitory activity data and to understand the drug-enzyme interactions, the 3D QSAR equation (Eq. 1) was also derived using the biophore as a template for superimposition. COX-2 inhibition was related to two secondary site parameters (variable): presence of mean electron donor reactivity (DON_01) at site SS1 in the vicinity of sulfur or any of the two oxygen atoms present at R3/R4 (10.005 ± 0.277, 8.160 ± 0.372 from the biophoric sites A, B, C respectively and steric bulk in terms of atomic refractivity increments (steric Refractivity) at site SS2 in the vicinity of the nitrogen at the fourth/seventh position of the thiazole ring (3.795 ± 0.009, 8.973 ± 0.294, 2.639 ± 0.0497 from the biophoric sites A, B, C, respecttively). This equation showed a good correlation coefficient values (r = 0.952) of high (> 99.9 %) statistical significance (F1,12 α:0.001 = 22.2; F1,12 = 52.790).
–log IC50 = 1.001 ± 0.236 DON_01 at site SS1 – 0.539 ± 0.055 Refractivity at site SS2 –5.711,
n = 14, r = 0.952, F1,12 = 52.790 |
(1) |
This 3D QSAR model also has good superimposition (match value = 0.59) with good predictive power as evidenced by low chance value (0.01) with almost similar RMSA and RMSP values. The model well explained the variation in the observed activity in most of the cases (Table 1).
The 3D QSAR equation indicates that electron donor reactivity at site SS1 positively contribute to the COX-2 inhibitory activity while the steric bulk at site SS2 is not favorable for the activity. A comparison of the observed versus calculated/predicted values indicate that the most active compounds, no. 1 and no. 13, showed calculated and predicted activity very near to the reported biological activity. The other compounds also showed good agreement between observed and calculated/predicted activity, except for three compounds nos. 3, 5, and 12) where the observed activity was little lower/higher than the calculated activity. In order to further validate this model, the activities of three standard compounds (test set) were predicted by this model (Fig. 3) where a good agreement was observed between the predicted and reported activity (Table 2). In view of the prediction of SC-558 by this model whose X-ray crystal structure with COX-2 is known, the mapping of SC-558 of this conformation to this model led to the comparison of the biophoric sites with the binding site analysis of SC-558 with COX-2 proposed by Kurumbail et al. [37] (Fig. 4). It suggests that biophoric site C of this model corresponds to the hydrophobic site cavity formed by Tyr 385 and other amino acids, such as Phe381, Leu384, Trp387, Phe513 and Ser530, where the bromophenyl ring of SC-558 has been shown to bind. The trifluoromethyl group of SC-558 is involved in a hydrogen-bonding interaction with Arg120, which corresponds to biophoric site B, and has been suggested to be essential for the activation of the COX-2 enzyme.
Finally, the third and the most predominant interactive site influencing the selectivity of SC-558 has been suggested to result from the phenyl sulphonamide moiety which binds in a pocket that is more restricted in COX-1 and is unoccupied in complexes of COX-2. The phenyl ring is surrounded by hydrophobic residues Leu352, Try 355, Phe518, Val 523, and backbone of Ser353, and sulfonamide group extends into region near the surface of COX-2 that is relatively polar where one of the oxygen atoms forms hydrogen bond to Arg513 while the other oxygen is linked by a hydrogen bond to His90
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and the amide nitrogen forms a hydrogen bond to the carbonyl oxygen of Phe518. This important site corresponds to the biophoric site A in the model (Figure 5).
These studies suggests that, in 5,6-diarylimidazo[2.1-b]thiazoles, the sulfur/oxygen of R3/R4 group, sulfur of the thiazole and phenyl ring at 5/6 position of thiazole of the training set molecules aligns with the fluorine of CF3, sulfonamide group and bromophenyl of SC-558, respectively. Among the two secondary sites SS1 and SS2, the SS1 at biophoric site B is involved in hydrogen bonding interaction while the other site SS2, in vicinity of N4/N7 of thiazole, disfavours steric bulk probably to maintain interactions in the polar region around bioiphoric site A.
Fig. 5. Superimposition of all the molecules of the training set mapped to the biophoric sites A, B, C represented by some interacting active site residues of COX-2.
Conclusions
The 3D QSAR model describing the COX-2 inhibition by 5,6-diarylimidazo[2,1-b]thi- azoles has led to the identification of essential structural features (which are consistent with the active site of the enzyme) in terms of the physicochemical properties (π-popu- lation, DON_01 and 6π-electron cloud) and their spatial dispositions., where the hydrogen bonding groups at site A and B and a 6π-elctron cloud at site C are essential and crucial for the activity in the present set of compounds.
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According to the 3D QSAR equation, the COX-2 inhibitory activity is enhanced by the hydrogen bonding interactions influenced by the electron donor reactivity of group R3/R4 and decreased by steric hindrance at N4/N7 of thiazole. As the model also has good predictions for a test set, it may be useful in designing new active and selective COX-2 inhibitors.
References
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Генеральный спонсор и организатор – InterBioScreen Ltd. |
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Взаимодействие 2-трифторметилхромонов
салкилмеркаптоацетатами – новая редокс-реакция
сширокими синтетическими возможностями
Сосновских В.Я., Усачев Б.И.
Уральский государственный университет 620083, Екатеринбург, пр. Ленина, 51
Известно [1, 2], что взаимодействие этил- 1а и метил- 1b меркаптоацетатов с α,β-не- предельными кетонами протекает как нуклеофильное присоединение НS-группы к активированной двойной связи с последующей циклизацией по карбонилу в соответствующие тиофеновые производные. Аналогичные реакции с эфирами 3-мето- кси-4,4,4-трифторкротоновой [3], α-фторалкилуксусных [4] и фторалкилпропиоловых [5] кислот дают алкил 3-гидрокси-5-фторалкилтиофен-2-карбоксилаты, а с β-хлор-α,β-енонами [6] и α-фторалкилкетонами [4] – алкил 5-фторалкилтиофен-2- карбоксилаты. о-Гидроксихалконы [7, 8] также реагируют с эфирами 1, однако в этом случае, благодаря присутствию в ароматическом кольце о-НО-группы, реакция сопровождается циклизацией в 2-арил-1,2-дигидро-4Н-тиено[2,3-c]хромен-4- оны (2, дигидротиенокумарины).
Ar |
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O |
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OH |
OR' |
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O H |
O |
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S |
R |
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O O |
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OR' |
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CF3 |
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O O |
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Когда двойная связь инкорпорирована в цикл, как в циклогекс-2-еноне [9], направление взаимодействия меняется и первоначальный аддукт присоединения по Михаэлю подвергается циклизации в дикетон 3, существующий в енольной форме. В то же время реакция 3,3-диалкил-6-трифторметил-2,3-дигидро-4-пиронов с эфирами 1 протекает с участием обоих электрофильных центров по типу мета- бриджинга без раскрытия пиронового кольца, давая производные 2-окса-7-тиаби-
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цикло[3,2,1]октана 4 [10]. В связи с этим, а также с учетом уникальных биологических свойств, проявляемых многими фторсодержащими гетероциклическими соединениям [11], мы изучили взаимодействие 2-трифторметилхромонов с эфиром 1a.
Принимая во внимание данные работ [7, 8, 10], можно было ожидать, что эта реакция будет протекать либо без раскрытия пиронового кольца с образованием мостиковой системы 5, либо с его раскрытием, сопровождающимся атакой по кетогруппе и циклизацией в тиено[2,3-c]кумарины 6. Однако взаимодействие 2-три- фторметилхромонов 7a–k с эфиром 1а при молярном соотношении 1 : 3 при 80°C в присутствии Et3N в качестве основания неожиданно привело к дигидротиенокумаринам 8a–k с выходами 66–93% (схема 1). Вторым продуктом был диэтил- 3,4-дитиаадипат, что указывает на окислительно-восстановительный характер этой трансформации. Интересно, что ожидаемые соединения 5 и/или 6 не были обнаружены даже в следовых количествах.
Схема 1
CF3
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3HSCH2CO2Et |
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−EtOH, −H2O, |
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CF3 −(SCH |
CO Et) |
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R = R' = R" = R"' = H (a); R = R" = R"' = H, R' = Me (b);
R = R" = R"' = H, R' = MeO (c); R = R' = R"' = H, R" = MeO (d);
R = R" = R"' = H, R' = NH2 (e); R = R" = R"' = H, R' = Cl (f);
R = R" = R"' = H, R' = Br (g); R = R" = H, R' = R"' = Br (h);
R = R" = Me, R' = R"' = H (i); R = R' = H, R"+R"' = бензо (j);
R+R' = бензо, R" = R"' = H (k)
Природа и положение заместителей в бензольном кольце не оказывают существенного влияния на ход реакции, однако она оказалась типичной только для 2-трифторметилхромонов и не протекала при замене CF3-группы на CF2H, (CF2)2H, CCl3 и Ме-группы. Строение кумаринов 8 хорошо согласуется с данными элементного анализа, ЯМР 1H, 19F, 13C, ИК и масс-спектров, а структура 8b подтверждена с помощью РСА [12]. В спектрах ЯМР 1Н помимо сигналов ароматических протонов присутствуют сигналы алифатических протонов CH2 и CH, образующих АВХ-систему (JAВ = 17.7–19.2, JAX = 10.3–12.3, JВХ = 2.6–4.5 Гц) при δ 3.6–4.4 и 4.3–5.2 м.д., соответственно.
Для прояснения механизма этой необычной реакции было решено расширить ряд исходных хромонов в расчете на то, что серьезные изменения в их структуре (более существенные, чем простой перебор заместителей в бензольном кольце) позволят остановить реакцию на одной из промежуточных стадий. С этой целью мы изучили взаимодействие 7-полифторалкилноркеллинов 9, синтезированных при конденсации келлинона с эфирами полифторалкановых кислот [13], с алкилмеркаптоацетатами 1a, b и нашли, что эти соединения, являясь хромонами, реаги-
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