| [page 92↓] |
The enzyme inhibitory effect of a compound is usually characterized by the IC50 value, the smaller the IC50 value, the higher is the inhibitory activity. For example the known good calcineurin inhibitors, cyclosporin A, microcystin IR and FK506, exhibit IC50 values of 0.5 μM, 0.2 μM and 0.2 μM, respectively.
Inhibiting activities of the new compounds synthesized by us are determined up to a value of 20 μM. If the inhibiting activity of a compound is lower (i.e. the inhibitor is weak), the remaining activity of calcineurin is recorded at a concentration of the inhibitor of 20 μM, e.g. “60 % activity at 20 μ M” means at 20 μM concentration of inhibitor, 60 % of calcineurin protein is still active. Again, the smaller the value, the higher is the activity. If the compound does not show inhibition at a concentration of 20 μM, it is termed “not active”.
In pharmacology and biochemistry, in order to determine the efficacy of a drug or inhibitor, the following terms are commonly used.
EC50: Clinical efficacy of a drug (Concentration required) to produce 50% of the maximum effect (may be inhibitory or stimulatory effect). This term is used usually with pharmaceuticals.
ED50: Medium effective dose (as opposed to concentration) at which 50 % of individuals exhibit the specified quantitative effect.
IC50: Concentration required producing 50 % inhibition.
Ki: Inhibitor concentration at which 50% inhibition is observed. It is calculated using Michaelis-Menten kinetics.
Parallel to the synthesis of new calcineurin inhibiting compounds presented in this thesis, other synthetic efforts in our group [16b] concentrated on pyrazolo[1,5-a]pyrimidine 341, with a side chain at the position 7. (Figure 5.1)
| [page 93↓] |
Figure 5.1 | ||
The best inhibiting activities of these pyrazolo[1,5-a]pyrimidines were achieved with compounds 4, 342 and 343. (Figure 5.2)
Figure 5.2 | ||
Calcineurin inhibitory activities of pyrazolo[1,5-a]pyrimidine compounds, synthsized by us, are listed inTable 5.1.
Our synthetic strategy allowed to synthesize pyrazolo[1,5-a]pyrimidines, where the ω-functionalized saturated side chain is attached to other positions than in 341, i.e. position 5 or position 3. This positional change is tolerated to a certain extent (see Table 5.1 compounds 128, 129, 116a, 116b), if an aminoalkylamino chain is found in position 5 (see compound 128). On the other hand, 3-aminoalkyl substituted pyrazolo[1,5-a]pyrimidines show almost no activity (see Table 5.1 compounds 116a and 116b). However, the aminopropynyl precursors 113a, 113 b, 113c and 113d exhibit an unexpectedly high activity. This indicates that the side chain of the general structure 8 can also be unsaturated. Comparison of compounds 113d and 113e reveal a massive effect of the aryl substitutent on the inhibition strength. If the terminal group in the side chain is not basic like in the phthalimide compound 118 or in the nitrile (compound 107a), the activity gets lost as found in other investigation performed in our group [16b].
|
Table 5.1 Derivatives of pyrazolo[1,5-a]pyrimidine
Table 5.2 shows the results of calcineurin inhibition tests of compounds fitting into the general structure 8, where the central cores are other N-containing bicyclic heterocycles. [page 96↓]Unexpectedly, none of the examples reached activities of pyrazolo[1,5-a]pyrimidines. This fact demonstrates that the central heterocycle of the structural model 8 plays a crucial role.
|
Table 5.2 Derivatives of other bicyclic heterocycle
Parallel work in our group has led to pyrimidines 344, which exhibit high calcineurin inhibiting activities and remarkably low toxicity. (Figure 5.3)
Figure 5.3 | ||
| [page 98↓] |
Calcineurin inhibitory activities of pyrimidine compounds, synthesized by us, are listed inTable 5.3.
Table 5.3 Derivatives of pyrimidine
Structure |
Name |
Activity |
Structure |
Name |
Activity |
|
261a Yl-211 |
IC50 =20 μM |
|
262 Yl-210 |
75 % activity at 20 μM |
|
261b Yl-314 |
61 % active at 20 μM |
|
263 Yl-212 |
76 % activity at 20 μM |
|
259a Yl-181 |
75 % activity at 20 μM |
|
260 Yl-383 |
46 % activity at 20 μM |
|
259b Yl-315 |
not active |
|
259c Yl-322 |
not active |
|
256 Yl-323 |
53 % activity at 20 μM |
|
257 Yl-330 |
50% activity at 20 μM |
|
258 Yl-346 |
53 % activity at 20 μM |
|
|
|
Our synthetic methodology allowed to synthesize 4-dimethylaminopropyl pyrimidine 257, which represents an example of structure 344 with X = CH2, and to provide access to isomers 260, 261a, 261b, 262 and 263, where the saturated side chains were attached to position 2. It turned out that all these analogous or isomers showed a comparable activity. For improvement variation of the aryl groups would be advisable.
| [page 99↓] |
Calcineurin inhibitory activities of other momocyclic N-heterocycles substituted by ω-functionalized side chains and two aryl groups fitting into the general structure 8, are shown in Table 5.4. Unfortunately all these compounds showed much lower activities than the corresponding pyrimidines 344. Nevertheless, some surprising results are mentioned here: the dimethylaminopropynyl oxazole 311 showed a higher activity than the structure analogue 312. 2-Amino-3,5-diphenylpyridine 283 and its oxygen-analogue 285 show a modest inhibiting activities, although they do not fit into the general structure 8.
|
Table 5.4 Derivatives of other monocyclic heterocycle
Considering the calcineurin inhibitory effects of compounds obtained in our group so far, the following structure-activity relations can be summarized:
(1) Effect of core heterocycle
Pyrazolo[1,5-a]pyrimidine, pyrazolo[1,5-a]triazine and pyrimidine are preferable as core heterocycles in the general structure 8 of potential calcineurin inhibitors.
Some corresponding active calcinerine inhibitors are shown below. (Figure 5.4)
Figure 5.4 | ||
| [page 101↓] |
(2) The position of side chain
The optimal position of attachment of the side chain depends on the type of heterocycles. In the pyrazolo[1,5-a]pyrimidine series, position 7 of the pyrazolo[1,5-a]pyrimidine core is most effective for calcineurin inhibition, compared with connection sites 5 and 3. In addition, the CH2NH(CH2)3NMe2 side chain is less effective than NH(CH2)3NMe2.(Figure 5.5)
Figure 5.5 | ||
In the pyrimidine series, many target molecules with side chains at position 4, and position 2 were synthesized and tested. It was found that the calcineurin inhibitory activities of these two kinds of compounds are similar. (Figure 5.6)
Figure 5.6 | ||
| [page 102↓] |
(3) The effect of substituted groups
Based on some preliminary conclusions of the effect of substituents at the side chain on the activity of calcineurin inhibitors of the general structure 8, the following trends can be mentioned:
(a) Heterocycles with Me2N(CH2)2S- side chain are more active than the compounds with Me2N(CH2)2O- and Me2N(CH2)3NH- side chain. (Figure 5.7)
Figure 5.7 | ||
(b) Heterocycles with a Me2NCH2C≡C- side chain are more active than compounds with a saturated Me2N(CH2)3- side chain. (Figure 5.8) This trend is similar to a report by Cheng [222] on the activities of nicotinic receptors [Me2N(CH2)2O- > Me2N(CH2)2- > Me2NCH2C≡C- > Me2N(CH2)3-].
Figure 5.8 | ||
| [page 103↓] |
(c) In general, heterocycles with one 4-chlorophenyl or one 3,4-dichlorophenyl are more active than the compounds with phenyl. (Figure 5.9)
Figure 5.9 | ||
On the other hand, our results demonstrated that it is impossible to deduce stringent roles for structure-activity relations from the data obtained so far. It would be extremely helpful if additional information (e.g. X-ray crystal analytical or NMR data), could be achieved about the interaction of an inhibitor with calcineurin. This would allow a prediction of optimized structure by the establishment of quantitative structure-activity-relation.
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