Search for inhibitors of bacterial and human protein kinases among derivatives of diazepines[1,4] annelated with maleimide and indole cycles

Article abstract of DOI:10.1021/jm800758s

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 dialkylaminomethyl 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.

Full text of DOI:10.1021/jm800758s

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 Scha¨chtele,§ 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
from the conformation of planar maleimidoindolocarbazoles
formed from 3,4-bis(indol-3-yl)maleimides.13 This suggests that
the spectrum of protein kinases inhibited by annelated diazepines
might vary from that of annelated maleimidoindolocarbazoles.
In this study we developed the methods of preparation of
diazepine derivatives 2 and 3, aiming at identification of serine/
threonine and tyrosine protein kinase inhibitors within this
chemical class. The compounds with the dialkylaminomethyl
group in position 3 of the indole moiety of 2 or 3, as well as
the compounds containing a substituent at the nitrogen atom of
the maleimide cycle, were synthesized. We tested our novel
compounds for the ability to inhibit human protein kinases in
vitro and protein kinase signaling in Streptomyces. Our results
provide evidence that individual derivatives of diazepines[1,4]
annelated with maleimide and indole cycles are differentially
potent against human and bacterial protein kinases. Thus, some
hit compounds might be perspective anti-infective agents, while
the others are promising as candidate drugs for somatic diseases.
Protein kinases are the key regulators of cellular signaling,
thereby representing attractive targets for therapeutic intervention
in a variety of diseases. These enzymes represent the largest
family of signaling proteins in eukaryotic cells implicated in a
plethora of aspects of cell regulation.1,2 Protein kinases interact
with diverse substrates ranging from the enzymes, including
protein kinases, to transcription factors, receptors, and chromatin
components. Furthermore, the bacterial eukaryotic-type serine/
threonine protein kinases regulate biofilm formation and are
therefore pivotal for bacterial survival.3-5 Also, protein kinases
play a key role in control of virulence and growth of Strepto-
cocci and Mycobacteria6,7 and are involved in resistance of
Streptomyces to aminoglycoside antibiotics.8-10 Thus, targeting
protein kinases emerges as an important component of thera-
peutic regimens in a variety of diseases including somatic
disorders and infections.
Potent inhibitors of protein kinases, predominantly the serine/
threonine type kinases, have been found among the derivatives
of bis-3,4(indol-3-yl)maleimides and their congeners.1,2 In these
compounds the maleimide moiety interacts with the kinase ATP
binding pocket via two hydrogen bonds.11 We have developed
the method of preparation of 3,4-bis(indol-1-yl)maleimides (1)
that are isomeric to 3,4-bis(indol-3-yl)maleimides. 3,4-Bis(indol-
1-yl)maleimides under the action of protic acids were trans-
formed into derivatives of dihydrodiazepines[1,4], 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). Dehydrogenation of 2 leads to 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 1).12 The conformations of annelated
Chemistry
Introduction of Substituents at Position 3 of the Indole
Moiety. Aminomethylation (Mannich reaction) of compounds
2a or 2b under the action of dialkylamines and paraformalde-
hyde in acetic acid led to 4a, 4b, or 5b in approximately 50%
yields. Aminomethylation of diazepine 3b using morpholine
produced bis-morpholinomethyl derivative 6 (50% yield) (Scheme
2).
Introduction of Substituents at the Nitrogen Atom of
the Maleimide Cycle. The interaction of diazepine[1,4] 2a with
1,3-dibromopropane in boiling dioxane in the presence of K2CO3
yielded compound 7, which was separated by chromatography
in 50% yield. The interaction of 7 with HNEt2 or N-methylpip-
erazine in DMF led to 9a or 9b in 70% yields. Condensation
of 7 with thiourea or N-substituted thioureas in boiling
ethanol-dioxane yielded compounds 10a-f (Scheme 3). 3-Bro-
mopropyl derivative 8 was obtained from diazepine 3a used
for preparation of 11a,b that contain the isothiourea moiety
* To whom correspondence should be addressed. Phone: 7 499-245-
3753. Fax: 7499-245-0295. E-mail: mnp@space.ru.
†
Vavilov Institute of General Genetics.
Research Centre for Biotechnology of Antibiotics BIOAN.
Gause Institute of New Antibiotics.
Tumor Biology Center Freiburg.
‡
#
§
|
Bach Institute of Biochemistry.
Blokhin Cancer Center.
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-ꢀ, 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
are presented in Table 1. It is worth noting that propyl
carbamimidothioate derivatives of diazepine[1,4] 2 (10a,c,d,f)
were more specific toward cyclin dependent kinases (CDKs)
than the isothiourea-containing derivatives of 3 (11a,b). The
latter compounds were less selective toward the kinases in the
panel. Compounds 10 and 11 represent the maleimide deriva-
tives substituted at the maleimide nitrogen atom. It has been
reported that substitution of maleimide nitrogen of indolylma-
leimide derivatives leads to the loss of the activity against protein
kinases.14 To investigate the binding mode of N-substituted bis-
indolylmaleimides in the active site of protein kinases, molecular
docking was applied.
was modeled. Results of the docking studies showed that the
preferential mode of binding of 10a and 11a to CDK2 was
characterized by single hydrogen bond formed by maleimide
carbonyl oxygen and main chain amide of hinge residue Leu83.
However, this hydrogen bond was not observed for all CDK2
models. When it was present (7 cases out of 11), this bond was
relatively long (∼2.5 Å), suggesting that binding of N-
substituted bis-indolylmaleimides was not as specific compared
to other kinase inhibitors that form at least two hydrogen bonds
with the hinge region.11 Nevertheless, although docking to
particular CDK2 models revealed some alternative binding
modes of 10a and 11a, the described binding via single
conservative hydrogen bond prevailed. Moreover, this mode of
interaction was characterized by higher binding energy. The
Molecular Docking of 10a and 11a to CDK2. Binding of
10a and 11a with CDK2, a well-studied kinase representative,

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-
phorylation. Our derivatives can sensitize Streptomyces to
kanamycin (see below), whereas the effect of sensitization was
not observed in a E. coli aphVIII+ strain,10 the bacteria that
possess no eukaryotic-type serine/threonine protein kinases.16
proposed mode of binding of 10a and 11a with CDK2 is
depicted in Figure 1.
New Bacterial Cell-Based Test System for Screening of
Protein Kinase Inhibitors. Principle of the Test System. It
has been demonstrated8 that the resistance to aminoglycoside
antibiotics (e.g., kanamycin) of a Streptomyces liVidans strain
with exogenously expressed aminoglycoside phosphotransferase
type VIII (aphVIII), the enzyme that inactivates kanamycin, is
mediated by a serine/threonine kinase(s) that up-regulates
aphVIII. This protein contains Ser146, the site of phosphorylation
for serine/threonine protein kinases of Streptomyces coelicolor
or Streptomyces liVidans.8,9 Phosphorylation of aphVIII at this
site results in an increased resistance of Streptomyces liVidans
Validation of the Bacterial Test System. We examined the
ability of partially purified protein kinases of Streptomyces
coelicolor to phosphorylate aphVIII. A radiometric protein
kinase assay was used for determination of the kinase activity
of actinobacterial protein kinases in vitro. Protein kinases were
incubated in standard conditions in the presence of [γ-32P]ATP
and aphVIII with or without known modulators and inhibitors
of protein kinase activity and incorporation of γ-phosphoryl
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
4b
5b
9a
13
12
10a
10c
10b
10d
11a
10f
11b
10e
Bis-I
Bis-V
25
100
200
20
400
100
2000
40
100
100
2700
40
1000
1500
300
3
13.0 ( 0.5
14.0 ( 0.5
13.0 ( 0.5
12.5 ( 0.5
12.5 ( 0.5
11.5 ( 0.5
11.5 ( 0.5
10.5 ( 0.5
10.5 ( 0.5
10.0 ( 0.5
10.0 ( 0.5
9.0 ( 0.5
9.0 ( 0.5
9.0 ( 0.5
10 ( 0.5
Figure 2. Growth inhibition of Streptomyces liVidans aphVIII+ strain:
(1) kanamycin, 5 nM/disk; (2) Bis-I, 300 nM/disk; (3) Bis-I +
kanamycin, halo diameter ) 10 mm; (4) 4b, 100 nM/disk; (5) 4b +
kanamycin, halo diameter ) 14 mm.
7.5 ( 0.5
a Minimal toxic concentration was 2-fold higher than the concentration
used in the experiments. b Kanamycin alone did not inhibit growth of the
test culture (halo diameter ) 0).
in Supporting Information) indicated that at least three aphVIII-
phosphorylating activities, Ca2+-independent, Ca2+/calmodulin-
dependent, and Ca2+/phospholipid-dependent, were detectable
in the Streptomyces coelicolor cell lysates. The selective
inhibitor of Ca2+/calmodulin-dependent protein kinases, KN-
62,a blocked aphVIII phosphorylation, whereas its inactive
Chart 1. Bis(indol-3-yl)maleimides Bis-IV, Bis-V, and Bis-I
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,
Bis-V, 4a, 4b, and 9a to influence aphVIII induced kanamycin
phosphorylation. As shown in Table 3-SI (Supporting Informa-
tion), none of these compounds had a discernible effect on
kanamycin phosphorylation, indicating that bis-indolylmaleim-
ides and our novel compounds do not target kanamycin
phosphorylation by aphVIII in vitro. The present data validated
our bacterial test system as an appropriate tool for phenotypic
screening of protein kinase inhibitors.
Use of the Bacterial Test System for Screening of
Protein Kinase Inhibitors. We next used our bacterial test
system for screening novel compounds for their ability to inhibit
a Abbreviations: Bis-I: [1-(3-dimethylaminopropyl)-1H-indol-3-yl]-4-(1H-
indol-3-yl)pyrrole-2,5-dione; Bis-V: 1-methyl-3,4-bis(1H-indol-3-yl)pyrrole-
2,5-dione; KN-62, [(1-[N,O-bis(5-isoquinolinesulphonyl)-N-methyl-L-
tyrosyl]-4-phenylpiperazine); KN-92, 2-[N-(4′-methoxybenzenesulfonyl]amino-
N-(4′-chlorophenyl)-2-propenyl-N-methylbenzylamine phosphate; EGTA,
ethylene glycol bis(ꢀ-aminoethyl)-N,N,N′,N′-tetraacetic acid.

DeriVatiVes of Diazepines as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7735
saturated aqueous NaCl (50 mL), dried, and evaporated. The residue
after solvent evaporation was chromatographed (CHCl3-MeOH,
10:1) to give 4a as a violet amorphous compound (540 mg, 1.3
mmol, 55%): δH (DMSO-d6) 1.00-1.04 (6H, m), 2.54-2.56 (4H,
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 )
7.51), 7.23 (1H, d, J ) 7.33), 7.29 (1H, d, J ) 7.69), 7.74 (1H, d,
J ) 7.68), 8.39 (1H, s); ESI-HRMS calculated for (C25H24N4O2 +
H+) 413.1972, found 413.1976; purity 98.7% (HPLC).
6-[(Diethylamino)methyl]-2-methyl-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)-dione (4b).
Compound 4b was obtained from 2b (800 mg, 2.3 mmol) as
described for 4a. The residue after solvent evaporation was
chromatographed (hexane-EtOAc-Et3N, 3:1:0.1) to give 4b as a
violet solid (640 mg, 1.5 mmol, 65%); mp 86-88 °C (i-PrOH); δH
(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,
m), 7.07 (1H, t, J ) 8.59), 7.19-7.22 (3H, m), 7.77-7.79 (1H,
m), 8.43 (1H, s); ESI-HRMS calculated for (C26H26N4O2 + H+)
427.2129, found 427.2123; purity 99.1% (HPLC).
2-Methyl-6-(1-pyrrolidinylmethyl)-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)-dione (5b).
Compound 5b was obtained from 2b (800 mg, 2.3 mmol) as
described for 4a. The residue after solvent evaporaition was
chromatographed (hexane-EtOAc-Et3N, 3:1:0.1) to give 5b as a
violet solid (640 mg, 1.5 mmol, 65%): mp 69-71 °C (i-PrOH); δH
(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
(1H, t, J ) 6.77), 7.21-7.22 (2H, m), 7.71-7.73 (2H, m) 8.45
(1H, s); ESI-HRMS calculated for (C26H24N4O2 + H+) 425.1972,
found 425.1978; purity 98.4% (HPLC).
3-(1,3-Dioxo-1,3,9b,10-tetrahydro-2H-indolo[1′,7′:4,5,6]pyrro-
lo[3′,4′:2,3][1,4] diazepino[1,7-a]indol-2-yl)propyl Imidothiocar-
bamate (10a). The stirred solution or suspension of diazepine 7
(500 mg, 1.1 mmol) in EtOH (20 mL) was boiled for 7 h with
thiourea (250 mg, 3.3 mmol), cooled, and concentrated in vacuo,
diluted with EtOAc (50 mL), washed with saturated aqueous NaCl
(50 mL), dried, and evaporated. The residue after solvent evapo-
raition was chromatographed (CHCl3-MeOH, 10:1) to give 10a
as a violet amorphous compound (240 mg, 0.55 mmol, 50%): δH
(DMSO-d6) 1.88-1.92 (2H, m), 2.89-2.93 (2H, m), 3.58-3.62
(2H, m), 3.72-3.79 (2H, m), 5.27-5.31 (1H, m), 6.82 (1H, t, J )
7.32), 6.85 (1H, d, J ) 3.48), 6.89 (1H, d, J ) 7.87), 7.04 (1H, t,
J ) 7.29), 7.20 (1H, t, J ) 7.65), 7.26 (1H, d, J ) 7.32), 7.32 (1H,
d, J ) 7.65), 7.65 (1H, d, J ) 7.83), 8.45 (1H, d, J ) 3.62), 9.62
(3H, s); ESI-HRMS calculated for (C24H21N5O2S + H+) 444.1489,
found 444.1482; purity 95.2% (HPLC).
protein kinase signaling by sensitizing Streptomyces liVidans
aphVIII+ strain to kanamycin. Figure 2 shows that kanamycin
alone had virtually no growth inhibitory effect, nor did we
observe bacterial lysis by 4b or Bis-I alone. In constrast, 4b in
combination with kanamycin produced a zone of bacterial lysis
14 mm in diameter. A smaller size of lytic halo was observed
around the paper disk imbibed with both kanamycin and the
reference protein kinase inhibitor Bis-I (Figure 2). This repre-
sentative experiment demonstrated that our bacterial test system
can be used to study the kinase inhibitory activity of low
molecular weight compounds. Each compound was tested at a
subtoxic dose, i.e., at the concentration ∼50% of minimal
growth inhibitory dose. Therefore the concentrations of indi-
vidual compounds (Table 2) were equitoxic (see also Supporting
Information, Table 4sSI, for detailed dose response). As shown
in Table 2, the compounds can be divided into two groups
depending on their potency. The first group included 4a,b, 5b,
9a, and 13, with the growth inhibition zone (halo) 12.5-14 mm
in diameter. It is interesting to note also that the compounds
4b and 5b, bearing the methyl group at the maleimide nitrogen,
were as active as unsubstituted derivative 4a. Other compounds
including Bis-I demonstrated a lower activity (growth inhibition
zones smaller than 11.5 mm). Importantly, the compounds 4a,b,
5b, 9a, and 13, which were the most potent in the bacterial test
system, were less active against human protein kinases (Table
1). These latter compounds, being the most active modulators
of serine/threonine protein kinase/aphVIII mediated bacterial
response to kanamycin, are of special interest because they might
be candidates for future nontoxic inhibitors of pathogenicity and
virulence in clinical bacterial strains.6,7 In contrast, the isothio-
urea containing compounds 10a-f, 11a, and 11b that inhibited
human protein kinases (Table 1) were less potent against
bacterial protein kinase signaling in our test system (Table 2).
Table 3-SI (Supporting Information) shows the concentrations
of novel compounds used in combination with 5 µg/disk of
kanamycin. From this table it is clear that our compounds at
the concentrations presented in Table 2 did not inhibit growth
of Streptomyces liVidans aphVIII+, thereby ruling out a
possibility that novel compounds alone (i.e., in the absence of
kanamycin) could influence the bacterial viability. Finally, none
of the tested novel compounds influenced proliferation or
viability of human epithelial (colon, breast) and lymphoid cells
at 50 µM for 72 h of continuous cell exposure (not shown).
3-(1,3-Dioxo-1,3,9b,10-tetrahydro-2H-indolo[1′,7′:4,5,6]pyrro-
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
mg, 1.1mmol) as described for 10a. The residue was chromato-
graphed (CHCl3 s MeOH, 10:1), to give 10b as a violet amorphous
compound (240 mg, 0.55 mmol, 50%); δH (DMSO-d6) 1.93-1.97
(2H, m), 2.91 (3H, s), 3.28-3.32 (2H, m), 3.62-3.65 (2H, m),
3.75-3.91 (2H, m), 5.33-5.36 (1H, m), 6.82 (1H, t, J ) 7.43),
6.87 (1H, d, J ) 3.63), 6.90 (1H, d, J ) 8.06), 7.03 (1H, t, J )
7.28), 7.21 (1H, t, J ) 7.68), 7.27 (1H, d, J ) 7.23), 7.35 (1H, d,
J ) 7.69), 7.67 (1H, d, J ) 7.83), 8.44 (1H, d, J ) 3.48), 9.40
(2H, s); ESI-HRMS calculated for (C25H23N5O2S+ H+) 458.1645,
found 458.1649; purity 96.5% (HPLC).
3-(1,3-Dioxo-1,3-dihydro-2H-indolo[1′,7′:4,5,6]pyrrolo[3′,4′:
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
described for 10a. The residue was chromatographed (CHCl3-
MeOH, 10:1) to give 11a as a red amorphous compound (240 mg,
0.55 mmol, 50%): δH (DMSO-d6) 1.90-1.93 (2H, m), 3.19-3.22
(2H, m), 3.55-3.61 (2H, m), 6.78 (1H, d, J ) 3.66), 7.08 (1H, t,
J ) 7.14), 7.11-7.18 (3H, m), 7.45-7.49 (3H, m), 7.70 (1H, d, J
) 7.73), 8.11 (1H, d, J ) 3.62), 9.09 (3H, s). ESI-HRMS calculated
for (C24H19N5O2S + H+) 442.1332, found 442.1337; purity 98.9%
(HPLC).
Concluding Remarks
In this study we report the synthesis of novel derivatives of
diazepines[1,4] annelated with maleimide and indole cycles as
tentative inhibitors of bacterial and human protein kinases. We
developed a new Streptomyces-based test system for testing the
kinase inhibitory potency of novel compounds. Together with
the conventional in vitro kinase assay using recombinant kinases
and inhibitors, the bacterial test system allowed us to specify
the compounds preferentially potent for microbial or human
protein kinases. Such initial screening may be useful as an
informative and inexpensive tool for selection of active com-
pounds among large scale chemical libraries.
Experimental Section
Chemistry. 6-[(Diethylamino)methyl]-9b,10-dihydro-1H-indo-
lo[1′,7′:4,5,6]pyrrolo[3′,4′:2,3][1,4]diazepino[1,7-a]indole-1,3(2H)-
dione (4a). To a stirred solution or suspension of diazepine 2a (800
mg, 2.4 mmol) in AcOH (50 mL) were added paraformaldehide
(500 mg) and HNEt2 (0.73 g, 10 mmol). The mixture was stirred
at 50 °C for 20 h, concentrated in vacuo, diluted with EtOAc (100
mL), washed with saturated aqueous NaHCO3 to neutral pH,

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′:
2,3][1,4]diazepino[1,7-a]indol-2-yl)propyl N-Methylimidothiocar-
bamate (11b). Compound 11b was obtained from 8 (500 mg,
1.1mmol) as described for 10a. The residue was chromatographed
(CHCl3-MeOH, 10:1) to give 11b as a red amorphous compound
(240 mg, 0.55 mmol, 50%): δH (DMSO-d6) 1.89-1.91 (2H, m),
2.89 (3H, s), 3.17-3.20 (2H, m), 3.54-3.61 (2H, m), 6.77 (1H, d,
J ) 3.68), 7.06 (1H, t, J ) 7.13), 7.10-7.17 (3H, m), 7.43-7.48
(3H, m), 7.69 (1H, d, J ) 7.70), 8.10 (1H, d, J ) 3.58), 9.07 (2H,
s); ESI-HRMS calculated for (C25H21N5O2S+ H+) 456.1489, found
456.1483; purity 99.0% (HPLC).
Molecular Modeling. Binding of 10a and 11a with CDK2, a
representative protein kinase, was modeled in the following way.
Three dimensional structures of 10a and 11a were modeled at the
Hartree-Fock level of theory (semiempiric AM1 method) using
the Gaussian 98 program package.17 The full atomic models of
CDK2 structure were prepared from the available Protein Data Bank
(PDB) structures using Lead Finder software.18 In total, 11 models
were prepared starting from PDB files 1gz8, 1h00, 1ke9, 1oit, 1r78,
2aoc, 2b52, 2b55, 2r3h, 2r3r, 2vtt to imitate the ATP binding site
flexibility. The set of different CDK2 structures was selected from
PDB using the criteria of good structure resolution (<2 Å) and
chemicaldiversityofboundATP-competitiveinhibitors.Protein-ligand
docking was performed with Lead Finder software.
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Acknowledgment. We thank Drs. G. Chilov and F. Novikov
(MolTech, Ltd., Moscow, Russia) for help in molecular modeling
and Dr. E.Bykov (Gause Institute of New Antibiotics, Moscow)
for 3D-structures of compounds 10a and 11a. This work was
funded by Russian Foundation of Basic Research Grant 06-03-
32233 and EU grant “Protein KinasessNovel Drug Targets of
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Supporting Information Available: General experimental
information and details of the synthesis of compounds 6-8, 9a,b,
10c-f, 12, and 13; MS, HRMS, HPLC data of the novel
compounds; details of the in vitro kinase assays; data on bacterial
sensitization to kanamycin depending on the concentration of protein
kinase inhibitors. This material is available free of charge via the
Internet at http://pubs.acs.org.
JM800758S