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Cyclin-dependent kinases are serine/threonine kinases regulating the cell cycle. Similar to checkpoint kinases, they contain an N-terminal region with β sheets and a C-terminal region with α helices. The ATP-binding pocket is located in the linker region between the two lobes. CdKs complex

with cyclins for activation; the kinase domain represents the catalytic sub-unit in the complex, while the cyclin is the regulatory subsub-unit. At present, 9 CdKs and 11 cyclins have been identified. The different complexes fulfill different functions; CdKs 1, 2, 4 and 6 are cell cycle-related CdKs, while the others are involved in transcriptional functions.²³ CdK2 complexed with cyclin E regulates G1/S transition, but CdK2 acts during S-phase if com-plexed with cyclin A.²⁴ The various complexes are regulated by phospho-rylation and dephosphophospho-rylation. To active a CdK2/cyclin complex, Thr¹⁶⁰ is phosphorylated by H-CdK7/MAT1, and the dual specificity phosphatase Cdc25 dephosphorylates Thr¹⁴ and Thr¹⁵of CdK2.

CdK2 is an intensively studied cyclin-dependent kinase, but it is not suitable as a therapeutic target because it is not essential to cellular function.

If CdK2 is not expressed, cyclin E can target another CdK that can replace CdK2 in function.²⁵In addition, few CdK2 inhibitors are selective enough to be effective due to CdK2’s similarity to other kinases, e.g. Checkpoint Inhibitors (ChkIs).

A better target may be CdK4, which plays a role in mid-G1 phase regula-tion. In normal cells, a CdK4/cyclin D complex phosphorylates retinoblas-toma protein (Rb) causing the transcription factor E2F/DP to stay active.

Rb is a tumor suppressor that binds to E2F/DP and stops entry into S phase.²⁶

In cancer cells, CdKs or/and cyclins are often overexpressed or mutated.

In the CdK4 pathway, the Rb gene can be mutated or deleted. There are also endogenous inhibitors such as INK4 (inhibitor of CdK4) or CIP-KIP (CdK inhibitor protein-kinase inhibitor protein) which are frequently mutated, deleted or silenced in tumor cells.

As already mentioned, indolocarbazoles are potent kinase inhibitors that function by competitive blockage of ATP-binding sites. The interaction in the binding pocket via hydrogen bonds is similar to the one shown in Fig. 11.

To test the inhibitory effects of the indocarbazoles against D1-CdK4, phosphorylation of Rb was measured²⁷ Selective inhibitors were identi-fied by derivatization from indolocarbazoles, but without the sugar moiety found in UCN-01 or staurosporine. Both maleimide and carbonyl groups are important for enzyme interaction. Reduction to an alcohol or methy-lene group at one carbonyl decreased potency.

Natural Products from Terrestrial Microbial Organisms with Cytotoxic Cell Cycle Inhibitors 165

NH

N N

O O

R¹ R⁴

R² R³

(22)

Figure 16 Structure of bis-indolylmaleimides.

Arcyriaflavin A (10) was frequently used as parent compound. It is a naturally occurring substance that is potent against CdK4, inhibits cell cycle progression in G1 phase and is selective against E-CdK2.

To sum up, indolocarbazoles are better inhibitors than bis-indolyl-maleimides (22) (Fig. 16) and exert stronger antiproliferative activity.

Moreover, bis-indolylmaleimides are less selective against E-CDK2 and inhibit G2/M transition rather than G1 arrest. Consequently, it may be possible that bis-indolylmaleimides inhibit targets other than D1-CdK4.

Therefore, the Arcyriaflavin skeleton was used as a starting point to gener-ate better inhibitors. Alkylation at one indole nitrogen leads to increased inhibition of CdK4. Polar groups at the alkyl moiety give rise to even more potent inhibitors. The most potent alkylated indolocarbazoles can inhibit other kinases as well, so they are unselective. Arcyriaflavin A and com-pounds (23)–(25) (Fig. 17) were CdK4 inhibitors with IC50values between 80 and 180 nM.²⁶

Furthermore, the indole ring can be replaced by another aryl or het-eroaryl ring. The easiest alteration is to use naphthalene, which can be connected in three different ways (Fig. 18).

Only the first compound, in which a naphthyl-moiety is linked to maleimide, revealed good inhibitory effect and selectivity; the other two substances were less potent. Compounds with a phenyl ring, or tetrahy-dronaphthalene or heteroaryl rings, such as imidazole, pyridine or thio-phene, were less potent as well.²⁸ Inhibitory activities were also observed with up-angular naphthyl[2,1-a]carbazoles such as isoquinolinyl[6,5-a]pyrrolo[3,4-c]carbazole (IC50= 69 nM) and carbazoles with a nitrogen

N N NH O

R¹ R⁴

R³ R²

O

(10) R¹= H R²= H R³= H R⁴= H Arcyriaflavin A (23) R¹= H R²= CH₃ R³= H R⁴= H

(24) R¹= H R²= H R³= (CH₂)₃N(CH₃)₂ R⁴= H (25) R¹= OCH₃ R²= H R³= H R⁴= OCH₃

Figure 17 Structures of indolo-[2,3-a]pyrrolo[3,4-c]carbazoles.

NH

NH

O O NH

NH

O O NH

NH

O O

against D1-CdK4: IC₅₀ = 45nM IC₅₀ = 430nM IC₅₀ = >10µM

Figure 18 Three ways to replace indole by naphthalene.

in 4-position of the isoquinoline (26) (Fig. 19). Quinolinyl[a]pyrrolo [3,4-c]carbazoles and other isoquinolinylcarbazoles are weaker inhibitors with micromolar IC50values.²⁹

In another investigation, the indole ring was connected to the maleimide not by the pyrrole but by a benzene ring and substituted on both indole nitrogens and on the phenyl (Fig. 20).

Substituents larger than ethylene at N-5 decrease the compound’s ability to inhibit D1-CdK4. C-9 and C-10 substitutions also lower the potency except in the case of fluorine, but this derivate lacks an antiproliferative effect. The most tolerant position for substituents with minor effects on CdK4 inhibition is C-11. Hydroxyalkylation at C-12 results

Natural Products from Terrestrial Microbial Organisms with Cytotoxic Cell Cycle Inhibitors 167

Figure 19 Structures of isoquinoliny l[6,5-a]pyrrolo[3,4-c]carbazole.

N13

Figure 20 Structure of indolo-[6,7-a]pyrrolo[3,4-c]carbazoles.

in potent inhibitors, which are selective towards E-Cdk2 and PKA. How-ever, hydroxalkylation at N-13 leads to E-Cdk2 inhibitors. This demon-strates that the bond with C-12 substituents in the ATP-binding pocket is very specific yet does not interact with the same substituents if they are at N-13. In addition, if there is an alkyl moiety at C-1 and also a sub-stituent at N-13, the inhibition is also more selective for E-Cdk2 versus D1-Cdk4. All these mentioned compounds, however, lack aqueous solubil-ity and hence show little antiproliferative effect in cells. Therefore, more polar C-12 aminoalkylated compounds possessing more than 20-fold bet-ter selectivity against CdK2 were designed. These compounds are also more selective towards 36 other kinases, although they do not show specificity for two lymphocytic kinases Lyn and Blk. Aminoalkylated compounds such as (27) (Fig. 21) are antiproliferative and the aqueous solubility is further increased by converting them into their methanesulfate salts, which are quite stable in solution.²⁷

(27) NH

13 12 11

9 10

NH O

1 2

O N5

CH₃

NH

OH

Figure 21 Structure of 12-aminoalkylated indolo-[6,7-a]pyrrolo[3,4-c]carbazoles.