[page 92↓]

5  Activities of calcineurin inhibitors

5.1 Measurement of calcineurin inhibitory activity

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.

5.2 Calcineurin inhibitory activity of target molecules

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].


[page 94 - 95↓]

Table 5.1 Derivatives of pyrazolo[1,5-a]pyrimidine

Structure

Name

Activity

Structure

Name

Activity

131

Yl-133

77 %

activity

at 20 μM

113f

Yl-215

28 %

activity

at 20 μM

113g

Yl-298

77 %

activity

at 20 μM

113k

Yl-293

81 %

activity

at 20 μM

113i

Yl-354

91 %

activity

at 20 μM

   

128

Yl-193

IC50 =18.5 μm

129

Yl-217

51 %

activity

at 20 μm

127

Yl-308

not active

   

109

Yl-245

67%

activity

at 20 μM

114

Yl-286

78 %

activity

at 20 μM

106h

Yl-208

not active

106a

Yl-42

not active

107a

Yl-43

not active

   

113d

Yl-209

IC50 = 8.5 μM

113e

Yl-261

53 %

activity

at 20 μM

113b

Yl-197

IC50 = 11.2 μM

116b

Yl-266

not active

113a

Yl-199

IC50 = 13.2 μM

116a

Yl-328

84 %

activity

at 20 μM

113c

Yl-190

IC50 = 12.0 μM

   

117

Yl-282

not active

118

Yl-352

94 %

activity

at 20 μM

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.


[page 97↓]

Table 5.2 Derivatives of other bicyclic heterocycle

Structure

Name

Activity

Structure

Name

Activity

189

Yl-185

84%

activity

at 20 μM

Yl-202

190

not active

192

Yl-201

not active

   

186

Yl-368

95 %

activity

at 20 μM

187

Yl-376

87 %

activity

at 20 μM

194b

Yl-238

90 %

activity

at 20 μM

195

Yl-350

53 %

activity

at 20 μM

198

Yl-247

not active

200

Yl-354

not active

199

Yl-258

70 %

activity

at 20 μM

202

Yl-260

74 %

activity

at 20 μM

194a

Yl-377

78 %

activity

at 20 μM

227

Yl-252

72 %

activity

at 20 μM

225

Yl-237

81 %

activity

at 20 μM

226

Yl-240

not active

220

Yl-242

not active

221

Yl-331

57 %

active

at 20 μM

223

Yl-279

94 %

activity

at 20 μM

224

Yl-324

67 %

activity

at 20 μM

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.


[page 100↓]

Table 5.4 Derivatives of other monocyclic heterocycle

Structure

name

activity

Structure

name

activity

335

Yl-219

66 %

activity

at 20 μM

334

Yl-204

60 %

activity

at 20 μM

313a

Yl-249

91 %

activity

at 20 μM

313

Yl-269

89 %

activity

at 20 μM

311

Yl-235

78 %

activity

at 20 μM

312

Yl-367

88 %

activity

at 20 μM

314

Yl-263

68 %

activity

at 20 μM

   

339

Yl-373

87 %

activity

at 20 μM

340

Yl-377

71 %

activity

at 20 μM

322

Yl-321

not active

323

Yl-332

72 %

activity

at 20 μM

298

Yl-336

no active

299

Yl-341

not active

283

Yl-317

68 %

activity

at 20 μM

285

Yl-327

68 %

activity

at 20 μM

286

Yl-335

81 %

activity

at 20 μM

287

Yl-345

62 %

activity

at 20 μM

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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