J. Med. Chem.

2008, 51, 7731–7736 7731

Search for Inhibitors of Bacterial and Human Protein Kinases among Derivatives of
Diazepines[1,4] Annelated with Maleimide and Indole Cycles

Valery N. Danilenko,†,‡ Alexander Y. Simonov,# Sergey A. Lakatosh,# Michael H. G. Kubbutat,§ Frank Totzke,§
Christoph Schächtele,§ Sergey M. Elizarov,| Olga B. Bekker,†,‡ Svetlana S. Printsevskaya,# Yuryi N. Luzikov,#
Marina I. Reznikova,# Alexander A. Shtil,⊥ and Maria N. Preobrazhenskaya*,#
Gause Institute of New Antibiotics, 11 B. PirogoVskaya Street, Moscow 119021, Russia, VaViloV Institute of General Genetics, 3 Gubkin Street,
Moscow 119991, Russia, Research Centre for Biotechnology of Antibiotics BIOAN, 27 Solzhenitsyn Street, Moscow 109004, Russia, Bach
Institute of Biochemistry, 33 Leninsky Prospekt, Moscow 119071, Russia, ProQinase GmbH, Tumor Biology Center Freiburg, Breisacher
Strasse 117, 79106 Freiburg, Germany, and Blokhin Cancer Center, 24 Kashirskoe shosse, Moscow 115478, Russia

ReceiVed June 20, 2008

Aminomethylation of 9b,10-dihydro-1H-indolo[1,7:4,5,6]pyrrolo[3,4:2,3][1,4]diazepino-[1,7-a]indole-1,3(2H)-
diones or 1H-indolo[1,7:4,5,6]pyrrolo[3,4:2,3][1,4]diazepino[1,7-a]indole-1,3(2H)-diones resulted in dialky-
laminomethyl derivatives. Alkylation of the nitrogen atom of maleimide moiety of polyannelated diazepines
with 1,3-dibromopropane and subsequent reaction with thiourea or its N-alkyl derivatives gave isothiourea-
carrying compounds. The compounds containing isothiourea moiety were active against individual human
serine/threonine and tyrosine kinases at low micromolar concentrations. Dialkylaminomethyl derivatives of
diazepines sensitized Streptomyces liVidans with overexpressed aminoglycoside phosphotransferase type
VIII (aphVIII) to kanamycin by inhibiting serine/threonine kinase(s) mediated aphVIII phosphorylation.

Introduction diazepines 2 and 3 are shell-like and therefore differ substantially

Protein kinases are the key regulators of cellular signaling, from the conformation of planar maleimidoindolocarbazoles
thereby representing attractive targets for therapeutic intervention formed from 3,4-bis(indol-3-yl)maleimides.13 This suggests that
in a variety of diseases. These enzymes represent the largest the spectrum of protein kinases inhibited by annelated diazepines
family of signaling proteins in eukaryotic cells implicated in a might vary from that of annelated maleimidoindolocarbazoles.
plethora of aspects of cell regulation.1,2 Protein kinases interact In this study we developed the methods of preparation of
with diverse substrates ranging from the enzymes, including diazepine derivatives 2 and 3, aiming at identification of serine/
protein kinases, to transcription factors, receptors, and chromatin threonine and tyrosine protein kinase inhibitors within this
components. Furthermore, the bacterial eukaryotic-type serine/ chemical class. The compounds with the dialkylaminomethyl
threonine protein kinases regulate biofilm formation and are group in position 3 of the indole moiety of 2 or 3, as well as
therefore pivotal for bacterial survival.3-5 Also, protein kinases the compounds containing a substituent at the nitrogen atom of
play a key role in control of virulence and growth of Strepto- the maleimide cycle, were synthesized. We tested our novel
cocci and Mycobacteria6,7 and are involved in resistance of compounds for the ability to inhibit human protein kinases in
Streptomyces to aminoglycoside antibiotics.8-10 Thus, targeting vitro and protein kinase signaling in Streptomyces. Our results
protein kinases emerges as an important component of thera- provide evidence that individual derivatives of diazepines[1,4]
peutic regimens in a variety of diseases including somatic annelated with maleimide and indole cycles are differentially
disorders and infections. potent against human and bacterial protein kinases. Thus, some
Potent inhibitors of protein kinases, predominantly the serine/ hit compounds might be perspective anti-infective agents, while
threonine type kinases, have been found among the derivatives the others are promising as candidate drugs for somatic diseases.
of bis-3,4(indol-3-yl)maleimides and their congeners.1,2 In these
Chemistry
compounds the maleimide moiety interacts with the kinase ATP
binding pocket via two hydrogen bonds.11 We have developed Introduction of Substituents at Position 3 of the Indole
the method of preparation of 3,4-bis(indol-1-yl)maleimides (1) Moiety. Aminomethylation (Mannich reaction) of compounds
that are isomeric to 3,4-bis(indol-3-yl)maleimides. 3,4-Bis(indol- 2a or 2b under the action of dialkylamines and paraformalde-
1-yl)maleimides under the action of protic acids were trans- hyde in acetic acid led to 4a, 4b, or 5b in approximately 50%
formed into derivatives of dihydrodiazepines[1,4], 9b,10-dihydro- yields. Aminomethylation of diazepine 3b using morpholine
1H-indolo[1,7:4,5,6]pyrrolo[3,4:2,3][1,4]diazepino[1,7-a]indole- produced bis-morpholinomethyl derivative 6 (50% yield) (Scheme
1,3(2H)-diones (2). Dehydrogenation of 2 leads to 1H-indolo[1,7: 2).
4,5,6]pyrrolo[3,4:2,3][1,4]diazepino[1,7-a]indole-1,3(2H)- Introduction of Substituents at the Nitrogen Atom of
diones (3) (Scheme 1).12 The conformations of annelated the Maleimide Cycle. The interaction of diazepine[1,4] 2a with
1,3-dibromopropane in boiling dioxane in the presence of K2CO3
* To whom correspondence should be addressed. Phone: 7 499-245- yielded compound 7, which was separated by chromatography
3753. Fax: 7499-245-0295. E-mail: mnp@space.ru. in 50% yield. The interaction of 7 with HNEt2 or N-methylpip-
†
Vavilov Institute of General Genetics. erazine in DMF led to 9a or 9b in 70% yields. Condensation
‡
Research Centre for Biotechnology of Antibiotics BIOAN. of 7 with thiourea or N-substituted thioureas in boiling
#
Gause Institute of New Antibiotics.
§
Tumor Biology Center Freiburg. ethanol-dioxane yielded compounds 10a-f (Scheme 3). 3-Bro-
|
Bach Institute of Biochemistry. mopropyl derivative 8 was obtained from diazepine 3a used
⊥
Blokhin Cancer Center. for preparation of 11a,b that contain the isothiourea moiety
10.1021/jm800758s CCC: $40.75  2008 American Chemical Society
Published on Web 12/03/2008
7732 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 Danilenko et al.

Scheme 1. Bis(3,4-indol-1-yl)maleimides (1) and Their Transformation into Annelated Maleimidoindolodiazepines

9b,10-dihydro-1H-indolo[1,7:4,5,6]pyrrolo[3,4:2,3][1,4]diazepino[1,7-a]indole-1,3(2H)-diones (2) and
1H-indolo[1,7:4,5,6]pyrrolo[3,4:2,3]-[1,4]diazepino[1,7-a]indole-1,3(2H)-diones (3)

Scheme 2. Aminomethylation of Compounds 2 and 3 Scheme 3. Synthesis of N-Substituted Polyannelated

Maleimidoindolodiazepines[1,4]

(Scheme 3). Interaction of 3a with 1-(N,N-dimethylamino)-3-

chloropropane in boiling dioxane in the presence of K2CO3 gave
13 in 70% yield. Similarly, compound 12 was obtained from
compound 2a (Scheme 4). Properties of the new compounds
are available in Supporting Information.

Biological Evaluation
Inhibitory Activities of Novel Compounds against
Human Protein Kinases. The compounds were tested against
a panel of 25 human protein kinases (AKT1, ARK5, Aurora-
A, Aurora-B, B-RAF-V600E, CDK2/CycA, CDK4/CycD1,
CK2-R1, COT, INS-R, MET, PDGFR-β, PLK1, SAK, SRC,
TIE2, AXL, EGF-R, EPHB4, ERBB2, FAK, IGF1-R, VEGF-
R2, VEGF-R3, and PKC-R). The compounds containing the
dialkylaminomethyl substituent at the position 3 of indole ring(s)
(4-6) or dialkylaminopropyl substituent at the position 1 of
maleimide ring (9, 12, and 13) were inactive against tested
enzymes at micromolar concentrations. In constrast, the com-
pounds containing a propyl carbamimidothioate substituent at
the maleimide nitrogen (10 and 11) attenuated the activity of
individual kinases. The inhibitory activities of these compounds
was modeled. Results of the docking studies showed that the
are presented in Table 1. It is worth noting that propyl
preferential mode of binding of 10a and 11a to CDK2 was
carbamimidothioate derivatives of diazepine[1,4] 2 (10a,c,d,f)
characterized by single hydrogen bond formed by maleimide
were more specific toward cyclin dependent kinases (CDKs)
than the isothiourea-containing derivatives of 3 (11a,b). The carbonyl oxygen and main chain amide of hinge residue Leu83.
latter compounds were less selective toward the kinases in the However, this hydrogen bond was not observed for all CDK2
panel. Compounds 10 and 11 represent the maleimide deriva- models. When it was present (7 cases out of 11), this bond was
tives substituted at the maleimide nitrogen atom. It has been relatively long (∼2.5 Å), suggesting that binding of N-
reported that substitution of maleimide nitrogen of indolylma- substituted bis-indolylmaleimides was not as specific compared
leimide derivatives leads to the loss of the activity against protein to other kinase inhibitors that form at least two hydrogen bonds
kinases.14 To investigate the binding mode of N-substituted bis- with the hinge region.11 Nevertheless, although docking to
indolylmaleimides in the active site of protein kinases, molecular particular CDK2 models revealed some alternative binding
docking was applied. modes of 10a and 11a, the described binding via single
Molecular Docking of 10a and 11a to CDK2. Binding of conservative hydrogen bond prevailed. Moreover, this mode of
10a and 11a with CDK2, a well-studied kinase representative, interaction was characterized by higher binding energy. The
DeriVatiVes of Diazepines as Inhibitors Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7733

Scheme 4. Synthesis of Compounds 12 and 13 aphVIII+ to kanamycin. Thirty-four serine/threonine protein

kinases have been identified in Streptomyces coelicolor A(3)2
strain closely related to Streptomyces liVidans 66.15 At least one
Ca2+-dependent serine/threonine protein kinase in Streptomyces
coelicolor, pk25 (as well as its homologue in Streptomyces
liVidans) can phosphorylate aphVIII.8 Thus, sensitization of
bacteria to kanamycin, the phenomenon dependent on aphVIII
phosphorylation, can be used for testing the ability of low
molecular weight compounds to attenuate the activity of protein
kinase(s) in a Streptomyces-based test system. If the protein
kinase(s) is inhibited, the aphVIII is not phosphorylated and
the zone of bacterial lysis is increased compared with control
(no inhibitor). Together, these data strongly suggest that the
inhibitors of serine/threonine protein kinases can sensitize
Streptomyces liVidans aphVIII+ to kanamycin via attenuation
of serine/threonine protein kinase(s) mediated aphVIII phos-
proposed mode of binding of 10a and 11a with CDK2 is phorylation. Our derivatives can sensitize Streptomyces to
depicted in Figure 1. kanamycin (see below), whereas the effect of sensitization was
New Bacterial Cell-Based Test System for Screening of not observed in a E. coli aphVIII+ strain,10 the bacteria that
Protein Kinase Inhibitors. Principle of the Test System. It possess no eukaryotic-type serine/threonine protein kinases.16
has been demonstrated8 that the resistance to aminoglycoside Validation of the Bacterial Test System. We examined the
antibiotics (e.g., kanamycin) of a Streptomyces liVidans strain ability of partially purified protein kinases of Streptomyces
with exogenously expressed aminoglycoside phosphotransferase coelicolor to phosphorylate aphVIII. A radiometric protein
type VIII (aphVIII), the enzyme that inactivates kanamycin, is kinase assay was used for determination of the kinase activity
mediated by a serine/threonine kinase(s) that up-regulates of actinobacterial protein kinases in vitro. Protein kinases were
aphVIII. This protein contains Ser146, the site of phosphorylation incubated in standard conditions in the presence of [γ-32P]ATP
for serine/threonine protein kinases of Streptomyces coelicolor and aphVIII with or without known modulators and inhibitors
or Streptomyces liVidans.8,9 Phosphorylation of aphVIII at this of protein kinase activity and incorporation of γ-phosphoryl
site results in an increased resistance of Streptomyces liVidans residue into aphVIII was determined. The results (Table 1-SI
Table 1. Inhibition of Human Protein Kinases by Isothiourea Containing Compoundsa

a
(/) IC50, the concentration of the inhibitor that down-regulated the kinase activity by 50% (mean of three independent measurements). The kinase
activity in the absence of the inhibitor was considered as 100%.
7734 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 Danilenko et al.

Figure 1. Binding of 10a (a) and 11a (b) to the ATP-binding pocket of CDK2 predicted by molecular docking. The model was prepared from PDB
structure 1r78.

Table 2. Inhibition of Growth of Streptomyces liVidans aphVIII+ Strain

by Polyannelated Diazepine Derivatives
subtoxic
concentration, halo diameter (mm),
compd nM/diska kanamycin 5 µg/diskb + inhibitor
4a 25 13.0 ( 0.5
4b 100 14.0 ( 0.5
5b 200 13.0 ( 0.5
9a 20 12.5 ( 0.5
13 400 12.5 ( 0.5
12 100 11.5 ( 0.5
10a 2000 11.5 ( 0.5
10c 40 10.5 ( 0.5
10b 100 10.5 ( 0.5
10d 100 10.0 ( 0.5
11a 2700 10.0 ( 0.5
10f 40 9.0 ( 0.5
11b 1000 9.0 ( 0.5
Figure 2. Growth inhibition of Streptomyces liVidans aphVIII+ strain: 10e 1500 9.0 ( 0.5
(1) kanamycin, 5 nM/disk; (2) Bis-I, 300 nM/disk; (3) Bis-I + Bis-I 300 10 ( 0.5
kanamycin, halo diameter ) 10 mm; (4) 4b, 100 nM/disk; (5) 4b + Bis-V 3 7.5 ( 0.5
kanamycin, halo diameter ) 14 mm. a
Minimal toxic concentration was 2-fold higher than the concentration
in Supporting Information) indicated that at least three aphVIII- used in the experiments. b Kanamycin alone did not inhibit growth of the
test culture (halo diameter ) 0).
phosphorylating activities, Ca2+-independent, Ca2+/calmodulin-
dependent, and Ca2+/phospholipid-dependent, were detectable Chart 1. Bis(indol-3-yl)maleimides Bis-IV, Bis-V, and Bis-I
in the Streptomyces coelicolor cell lysates. The selective
inhibitor of Ca2+/calmodulin-dependent protein kinases, KN-
62,a blocked aphVIII phosphorylation, whereas its inactive
analogue, KN-92, had no effect. Bis-I, the inhibitor of Ca2+/
phospholipid-dependent serine/threonine protein kinases, at-
tenuated Ca2+/phospholipid-dependent aphVIII phosphorylation
by approximately 50%, whereas Bis-V, a compound with little
kinase inhibitory potency, evoked only marginal effect on
aphVIII phosphorylation. Importantly, compound 4b decreased
aphVIII phosphorylation down to the basal level (Table 3-SI in
Supporting Information). Next, we tested the ability of Bis-I, tion), none of these compounds had a discernible effect on
Bis-V, 4a, 4b, and 9a to influence aphVIII induced kanamycin kanamycin phosphorylation, indicating that bis-indolylmaleim-
phosphorylation. As shown in Table 3-SI (Supporting Informa- ides and our novel compounds do not target kanamycin
phosphorylation by aphVIII in vitro. The present data validated
a
Abbreviations: Bis-I: [1-(3-dimethylaminopropyl)-1H-indol-3-yl]-4-(1H- our bacterial test system as an appropriate tool for phenotypic
indol-3-yl)pyrrole-2,5-dione; Bis-V: 1-methyl-3,4-bis(1H-indol-3-yl)pyrrole- screening of protein kinase inhibitors.
2,5-dione; KN-62, [(1-[N,O-bis(5-isoquinolinesulphonyl)-N-methyl-L- Use of the Bacterial Test System for Screening of
tyrosyl]-4-phenylpiperazine); KN-92, 2-[N-(4′-methoxybenzenesulfonyl]amino-
N-(4′-chlorophenyl)-2-propenyl-N-methylbenzylamine phosphate; EGTA, Protein Kinase Inhibitors. We next used our bacterial test
ethylene glycol bis(β-aminoethyl)-N,N,N′,N′-tetraacetic acid. system for screening novel compounds for their ability to inhibit
DeriVatiVes of Diazepines as Inhibitors Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7735

protein kinase signaling by sensitizing Streptomyces liVidans saturated aqueous NaCl (50 mL), dried, and evaporated. The residue
aphVIII+ strain to kanamycin. Figure 2 shows that kanamycin after solvent evaporation was chromatographed (CHCl3-MeOH,
alone had virtually no growth inhibitory effect, nor did we 10:1) to give 4a as a violet amorphous compound (540 mg, 1.3
observe bacterial lysis by 4b or Bis-I alone. In constrast, 4b in mmol, 55%): δH (DMSO-d6) 1.00-1.04 (6H, m), 2.54-2.56 (4H,
combination with kanamycin produced a zone of bacterial lysis m), 3.67-3.83 (4H, m), 5.21-5.25 (1H, m), 6.79 (1H, t, J ) 7.51),
6.84 (1H, d, J ) 7.87), 7.01 (1H, t, J ) 8.87), 7.18 (1H, t, J )
14 mm in diameter. A smaller size of lytic halo was observed 7.51), 7.23 (1H, d, J ) 7.33), 7.29 (1H, d, J ) 7.69), 7.74 (1H, d,
around the paper disk imbibed with both kanamycin and the J ) 7.68), 8.39 (1H, s); ESI-HRMS calculated for (C25H24N4O2 +
reference protein kinase inhibitor Bis-I (Figure 2). This repre- H+) 413.1972, found 413.1976; purity 98.7% (HPLC).
sentative experiment demonstrated that our bacterial test system 6-[(Diethylamino)methyl]-2-methyl-9b,10-dihydro-1H-indolo[1′,7′:
can be used to study the kinase inhibitory activity of low 4,5,6]pyrrolo[3′,4′:2,3][1,4]diazepino[1,7-a]indole-1,3(2H)-dione (4b).
molecular weight compounds. Each compound was tested at a Compound 4b was obtained from 2b (800 mg, 2.3 mmol) as
subtoxic dose, i.e., at the concentration ∼50% of minimal described for 4a. The residue after solvent evaporation was
growth inhibitory dose. Therefore the concentrations of indi- chromatographed (hexane-EtOAc-Et3N, 3:1:0.1) to give 4b as a
vidual compounds (Table 2) were equitoxic (see also Supporting violet solid (640 mg, 1.5 mmol, 65%); mp 86-88 °C (i-PrOH); δH
Information, Table 4sSI, for detailed dose response). As shown (CDCl3) 1.05-1.09 (6H, m), 2.52-2.58 (4H, m), 3.10 (3H, s), 3.69
(2H, s), 3.79-3.81 (2H, m), 5.10-5.13 (1H, m), 6.81-6.85 (2H,
in Table 2, the compounds can be divided into two groups
m), 7.07 (1H, t, J ) 8.59), 7.19-7.22 (3H, m), 7.77-7.79 (1H,
depending on their potency. The first group included 4a,b, 5b, m), 8.43 (1H, s); ESI-HRMS calculated for (C26H26N4O2 + H+)
9a, and 13, with the growth inhibition zone (halo) 12.5-14 mm 427.2129, found 427.2123; purity 99.1% (HPLC).
in diameter. It is interesting to note also that the compounds 2-Methyl-6-(1-pyrrolidinylmethyl)-9b,10-dihydro-1H-indolo[1′,7′:
4b and 5b, bearing the methyl group at the maleimide nitrogen, 4,5,6]pyrrolo[3′,4′:2,3][1,4]diazepino[1,7-a]indole-1,3(2H)-dione (5b).
were as active as unsubstituted derivative 4a. Other compounds Compound 5b was obtained from 2b (800 mg, 2.3 mmol) as
including Bis-I demonstrated a lower activity (growth inhibition described for 4a. The residue after solvent evaporaition was
zones smaller than 11.5 mm). Importantly, the compounds 4a,b, chromatographed (hexane-EtOAc-Et3N, 3:1:0.1) to give 5b as a
5b, 9a, and 13, which were the most potent in the bacterial test violet solid (640 mg, 1.5 mmol, 65%): mp 69-71 °C (i-PrOH); δH
system, were less active against human protein kinases (Table (CDCl3) 1.74-1.77 (4H, m), 2.54-2.57 (4H, m), 3.09 (3H, s),
3.77-3.82 (2H, m), 5.07-5.10 (2H, m), 6.81-6.84 (2H, m), 7.07
1). These latter compounds, being the most active modulators
(1H, t, J ) 6.77), 7.21-7.22 (2H, m), 7.71-7.73 (2H, m) 8.45
of serine/threonine protein kinase/aphVIII mediated bacterial (1H, s); ESI-HRMS calculated for (C26H24N4O2 + H+) 425.1972,
response to kanamycin, are of special interest because they might found 425.1978; purity 98.4% (HPLC).
be candidates for future nontoxic inhibitors of pathogenicity and 3-(1,3-Dioxo-1,3,9b,10-tetrahydro-2H-indolo[1′,7′:4,5,6]pyrro-
virulence in clinical bacterial strains.6,7 In contrast, the isothio- lo[3′,4′:2,3][1,4] diazepino[1,7-a]indol-2-yl)propyl Imidothiocar-
urea containing compounds 10a-f, 11a, and 11b that inhibited bamate (10a). The stirred solution or suspension of diazepine 7
human protein kinases (Table 1) were less potent against (500 mg, 1.1 mmol) in EtOH (20 mL) was boiled for 7 h with
bacterial protein kinase signaling in our test system (Table 2). thiourea (250 mg, 3.3 mmol), cooled, and concentrated in vacuo,
Table 3-SI (Supporting Information) shows the concentrations diluted with EtOAc (50 mL), washed with saturated aqueous NaCl
of novel compounds used in combination with 5 µg/disk of (50 mL), dried, and evaporated. The residue after solvent evapo-
kanamycin. From this table it is clear that our compounds at raition was chromatographed (CHCl3-MeOH, 10:1) to give 10a
as a violet amorphous compound (240 mg, 0.55 mmol, 50%): δH
the concentrations presented in Table 2 did not inhibit growth
(DMSO-d6) 1.88-1.92 (2H, m), 2.89-2.93 (2H, m), 3.58-3.62
of Streptomyces liVidans aphVIII+, thereby ruling out a (2H, m), 3.72-3.79 (2H, m), 5.27-5.31 (1H, m), 6.82 (1H, t, J )
possibility that novel compounds alone (i.e., in the absence of 7.32), 6.85 (1H, d, J ) 3.48), 6.89 (1H, d, J ) 7.87), 7.04 (1H, t,
kanamycin) could influence the bacterial viability. Finally, none J ) 7.29), 7.20 (1H, t, J ) 7.65), 7.26 (1H, d, J ) 7.32), 7.32 (1H,
of the tested novel compounds influenced proliferation or d, J ) 7.65), 7.65 (1H, d, J ) 7.83), 8.45 (1H, d, J ) 3.62), 9.62
viability of human epithelial (colon, breast) and lymphoid cells (3H, s); ESI-HRMS calculated for (C24H21N5O2S + H+) 444.1489,
at 50 µM for 72 h of continuous cell exposure (not shown). found 444.1482; purity 95.2% (HPLC).
3-(1,3-Dioxo-1,3,9b,10-tetrahydro-2H-indolo[1′,7′:4,5,6]pyrro-
Concluding Remarks lo[3′,4′:2,3][1,4] diazepino[1,7-a]indol-2-yl)propyl N-Methylimi-
dothiocarbamate (10b). Compound 10b was obtained from 7 (500
In this study we report the synthesis of novel derivatives of mg, 1.1mmol) as described for 10a. The residue was chromato-
diazepines[1,4] annelated with maleimide and indole cycles as graphed (CHCl3 s MeOH, 10:1), to give 10b as a violet amorphous
tentative inhibitors of bacterial and human protein kinases. We compound (240 mg, 0.55 mmol, 50%); δH (DMSO-d6) 1.93-1.97
developed a new Streptomyces-based test system for testing the (2H, m), 2.91 (3H, s), 3.28-3.32 (2H, m), 3.62-3.65 (2H, m),
kinase inhibitory potency of novel compounds. Together with 3.75-3.91 (2H, m), 5.33-5.36 (1H, m), 6.82 (1H, t, J ) 7.43),
the conventional in vitro kinase assay using recombinant kinases 6.87 (1H, d, J ) 3.63), 6.90 (1H, d, J ) 8.06), 7.03 (1H, t, J )
and inhibitors, the bacterial test system allowed us to specify 7.28), 7.21 (1H, t, J ) 7.68), 7.27 (1H, d, J ) 7.23), 7.35 (1H, d,
the compounds preferentially potent for microbial or human J ) 7.69), 7.67 (1H, d, J ) 7.83), 8.44 (1H, d, J ) 3.48), 9.40
protein kinases. Such initial screening may be useful as an (2H, s); ESI-HRMS calculated for (C25H23N5O2S+ H+) 458.1645,
found 458.1649; purity 96.5% (HPLC).
informative and inexpensive tool for selection of active com-
3-(1,3-Dioxo-1,3-dihydro-2H-indolo[1′,7′:4,5,6]pyrrolo[3′,4′:
pounds among large scale chemical libraries. 2,3][1,4]diazepino[1,7-a]indol-2-yl)propyl Imidothiocarbamate (11a).
Compound 11a was obtained from 8 (500 mg, 1.1mmol) as
Experimental Section described for 10a. The residue was chromatographed (CHCl3-
Chemistry. 6-[(Diethylamino)methyl]-9b,10-dihydro-1H-indo- MeOH, 10:1) to give 11a as a red amorphous compound (240 mg,
lo[1′,7′:4,5,6]pyrrolo[3′,4′:2,3][1,4]diazepino[1,7-a]indole-1,3(2H)- 0.55 mmol, 50%): δH (DMSO-d6) 1.90-1.93 (2H, m), 3.19-3.22
dione (4a). To a stirred solution or suspension of diazepine 2a (800 (2H, m), 3.55-3.61 (2H, m), 6.78 (1H, d, J ) 3.66), 7.08 (1H, t,
mg, 2.4 mmol) in AcOH (50 mL) were added paraformaldehide J ) 7.14), 7.11-7.18 (3H, m), 7.45-7.49 (3H, m), 7.70 (1H, d, J
(500 mg) and HNEt2 (0.73 g, 10 mmol). The mixture was stirred ) 7.73), 8.11 (1H, d, J ) 3.62), 9.09 (3H, s). ESI-HRMS calculated
at 50 °C for 20 h, concentrated in vacuo, diluted with EtOAc (100 for (C24H19N5O2S + H+) 442.1332, found 442.1337; purity 98.9%
mL), washed with saturated aqueous NaHCO3 to neutral pH, (HPLC).
7736 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 Danilenko et al.

3-(1,3-Dioxo-1,3-dihydro-2H-indolo[1′,7′:4,5,6]pyrrolo[3′,4′: References
2,3][1,4]diazepino[1,7-a]indol-2-yl)propyl N-Methylimidothiocar- (1) Goekjian, P.; Jirousek, M. Protein kinase C inhibitors as novel
bamate (11b). Compound 11b was obtained from 8 (500 mg, anticancer drugs. Expert Opin. InVest. Drugs 2001, 10, 2117–2140.
1.1mmol) as described for 10a. The residue was chromatographed (2) Goekjian, P.; Jirousek, M. Protein kinase C in the treatment of disease:
signal transduction pathways, inhibitors and agents in development.
(CHCl3-MeOH, 10:1) to give 11b as a red amorphous compound Curr. Med. Chem. 1999, 6, 877–903.
(240 mg, 0.55 mmol, 50%): δH (DMSO-d6) 1.89-1.91 (2H, m), (3) Hussain, H.; Branny, P.; Allan, E. A eukaryotic-type serine/threonine
2.89 (3H, s), 3.17-3.20 (2H, m), 3.54-3.61 (2H, m), 6.77 (1H, d, protein kinase is required for biofilm formation, genetic competence,
J ) 3.68), 7.06 (1H, t, J ) 7.13), 7.10-7.17 (3H, m), 7.43-7.48 and acid resistance in Streptococcus mutans. J. Bacteriol. 2006, 188,
1628–1632.
(3H, m), 7.69 (1H, d, J ) 7.70), 8.10 (1H, d, J ) 3.58), 9.07 (2H, (4) Gopalaswamy, R.; Narayanan, S.; Jacobs, W.; Av-Gay, Y. Mycobac-
s); ESI-HRMS calculated for (C25H21N5O2S+ H+) 456.1489, found terium smegmatis biofilm formation and sliding motility are affected
456.1483; purity 99.0% (HPLC). by the serine/threonine protein kinase PknF. FEMS Microbiol. Lett.
Molecular Modeling. Binding of 10a and 11a with CDK2, a 2008, 278, 121–127.
representative protein kinase, was modeled in the following way. (5) Papavinasasundaram, K.; Chan, B.; Chung, J.; Colston, M.; Davis,
E.; Av-Gay, Y. Deletion of the Mycobacterium tuberculosis pknH gene
Three dimensional structures of 10a and 11a were modeled at the confers a higher bacillary load during the chronic phase of infection
Hartree-Fock level of theory (semiempiric AM1 method) using in BALB/c mice. J. Bacteriol. 2005, 187, 5751–5760.
the Gaussian 98 program package.17 The full atomic models of (6) Echenique, J.; Kadioglu, A.; Romao, S.; Andrew, P. W.; Trombe, M. C.
CDK2 structure were prepared from the available Protein Data Bank Protein serine/threonine kinase StkP positively controls virulence and
competence in Streptococcus pneumoniae. Infect. Immun. 2004, 72,
(PDB) structures using Lead Finder software.18 In total, 11 models 2434–2437.
were prepared starting from PDB files 1gz8, 1h00, 1ke9, 1oit, 1r78, (7) Scherr, N.; Honnappa, S.; Kunz, G.; Mueller, P.; Jayachandran, R.;
2aoc, 2b52, 2b55, 2r3h, 2r3r, 2vtt to imitate the ATP binding site Winkler, F.; Pieters, J.; Steinmetz, M. Structural basis for the specific
flexibility. The set of different CDK2 structures was selected from inhibition of protein kinase G, a virulence factor of Mycobacterium
tuberculosis. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 12151–12156.
PDB using the criteria of good structure resolution (<2 Å) and (8) Elizarov, S.; Sergienko, S.; Sizova, I.; Danilenko, V. Dependence of
chemicaldiversityofboundATP-competitiveinhibitors.Protein-ligand aminoglycoside 3′-phosphotransferase VIII activity on protein serine/
docking was performed with Lead Finder software. threonine kinases in Streptomyces rimosus. Mol. Biol. (Russian). 2005,
Biological Methods. The protocol of in vitro kinase assay is 39, 255–263.
given in Supporting Information. A strain of Streptomyces liVidans (9) Bekker, O.; Alekseeva, M.; Elizarov, S.; Lubimova, I.; Danilenko, V.
Ca2+-dependent modulation of resistance to antibiotics of Streptomyces
harboring pSU23 plasmid carrying the aphVIII gene (Streptomyces liVidans 66 and Streptomyces coelicolor A3(2). J. Microbiol.(Rus),
liVidans aphVIII+ strain) was used as a test culture to analyze the 2008, 77 (5), 559–567.
inhibitors of bacterial serine-treonine protein kinases. The gene (10) Sizova, L. A.; Hegemann, P.; Furmann, M.; Danilenko, V. N.
product, aminoglycoside phosphotransferase aphVIII, phosphory- Streptomyces rimosus aminoglycoside 3′-phosphotransferase VIII:
comparisons with aminoglycoside 3′-phosphotransferases of aminogly-
lates and inactivates kanamycin, thereby rendering bacteria resistant cosidesproducing strains and with eukaryotic protein kinases. Mol.
to this antibiotic. This activity of aphVIII is dependent on Biol. (Russ.). 2002, 36, 27–36.
phosphorylation by a serine/threonine protein kinase(s). The kinase (11) Komander, D.; Kular, G. S.; Schuettelkopf, A.,W.; Deak, M.; Prakash,
inhibitory activity of new compounds was investigated by the paper K. R. C.; Bain, J.; Elliott, M.; Garrido-Franco, M.; Kozikowski, A. P.;
Alessi, D. R.; van Aalten, D. M. F. Interactions of LY333531 and
disk method. Paper disks (7 mm in diameter) containing kanamycin other bisindolyl maleimide inhibitors with PDK1. Structure 2004, 12,
(5 µg/disk) and various amounts of tested compounds were applied 215–226.
on the plates with logarithmically growing Streptomyces liVidans (12) Lakatosh, S; Luzhikov, Yu.; Preobrazhenskaya, N. Org. Biomol. Chem.
aphVIII+ and incubated at 28 °C for 20 h. The halo diameters 2003, 1, 826–833.
(13) Bykov, E.; Lakatosh, S.; Preobrazhenskaya, M. Transformations of
formed after exposure of bacteria with the combination of kana- 3,4-bisindolylmaleimides with differently bonded indole and maleimide
mycin and potential inhibitors were compared with the respective moieties under the action of protic acids: a quantum chemical study.
sizes for kanamycin alone or the combinations of kanamycin and Russ. Chem. Bull, Int. Ed. 2006, 55, 781–787.
known inhibitor of serine/threonine protein kinases, Bis-I.19 As a (14) Pindur, U.; Kim, Y.-S.; Meharbani, F. Advances in indolo[2,3-a]-
negative control, Bis-V was used.20 The structures of Bis-I and carbazole chemistry: design and synthesis of protein kinase C and
topoisomerase I inhibitors. Curr. Med. Chem. 1999, 6, 29–70.
Bis-V are shown in Chart 1. To rule out the cytotoxicity of novel (15) Petrickova, K.; Petricek., M. Eukaryotic-type protein kinases in
compound as a factor that might increase the diameter of the zone Streptomyces coelicolor: variations on a common theme. Microbiology
of lysis, we used subtoxic concentrations of tested compounds. The 2003, 149, 1609–1621.
subtoxic concentrations were approximately 2 times lower than the (16) http://eecoli.kaist.ac.kr/search?term)kinase&category)pname.
(17) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb,
minimal toxic concentrations, i.e., minimal doses that inhibited M. A.; Cheeseman, J. R.; Zakrzewski, V. G.; Montgomery, J. A., Jr.;
growth of test bacteria. At subtoxic concentrations no inhibition of Stratmann, R. E.; Burant, J. C.; Dapprich, S.; Millam, J. M.; Daniels,
bacterial growth was observed. The detailed methods are given in A. D.; Kudin, K. N.; Strain, M. C.; Farkas, O.; Tomasi, J.; Barone, V.;
Supporting Information. Cossi, M.; Cammi, R.; Mennucci, B.; Pomelli, C.; Adamo, C.; Clifford,
S.; Ochterski, J.; Petersson, G. A.; Ayala, P. Y.; Cui, Q.; Morokuma, K.;
Salvador, P.; Dannenberg, J. J.; Malick, D. K.; Rabuck, A. D.; Ragha-
Acknowledgment. We thank Drs. G. Chilov and F. Novikov vachari, K.; Foresman, J. B.; Cioslowski, J.; Ortiz, J. V.; Baboul, A. G.;
(MolTech, Ltd., Moscow, Russia) for help in molecular modeling Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.;
and Dr. E.Bykov (Gause Institute of New Antibiotics, Moscow) Gomperts, R.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.;
Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; Johnson,
for 3D-structures of compounds 10a and 11a. This work was B.; Chen, W.; Wong, M. W.; Andres, J. L.; Gonzalez, C.; Head-Gordon,
funded by Russian Foundation of Basic Research Grant 06-03- M.; Replogle, E. S.; Pople, J. A. Gaussian 98, revision A.1x; Gaussian,
32233 and EU grant “Protein KinasessNovel Drug Targets of Inc.: Pittsburgh, PA, 2001; http://www.nersc.gov/nusers/resources/software/
apps/chemistry/gaussian/g98/g94.htm.
Postgenomic Era”, Contract No. LSHB-CT-2004-503467. (18) www.biomoltech.com.
(19) Martiny-Baron, M.; Kazanietz, M.; Mischak, H.; Blumberg, P.; Kochs,
Supporting Information Available: General experimental G.; Hug, H.; Marme, D.; Schachtele, C. Selective inhibition of protein
information and details of the synthesis of compounds 6-8, 9a,b, kinase C isozymes by the indolocarbazole Go 6976. J. Biol. Chem.
10c-f, 12, and 13; MS, HRMS, HPLC data of the novel 1993, 268, 9194–9197.
(20) Toullec, D.; Pianetti, P.; Coste, H.; Bellevergue, P.; Grand-Perret, T.;
compounds; details of the in vitro kinase assays; data on bacterial Ajakane, M.; Baudet, V.; Boissin, P.; Boursier, E.; Loriolle, F. The
sensitization to kanamycin depending on the concentration of protein bisindolylmaleimide GF 109203X is a potent and selective inhibitor
kinase inhibitors. This material is available free of charge via the of protein kinase C. J. Biol. Chem. 1991, 266, 15771–15781.
Internet at http://pubs.acs.org. JM800758S