The synthesis and evaluation of indolylureas as PKCα inhibitors

Article abstract of DOI:10.1016/j.bmc.2011.02.036

PKCα and PKA have crucial but opposing roles in the regulation of calcium handling within myocytes. Identification of compounds that inhibit PKCα, but not PKA, are potential therapeutic targets for the treatment of heart disease. The synthesis of indolylureas are described, and a compound displaying nanomolar inhibition towards PKCα with significant selectivity over PKA has been identified.

Full text of DOI:10.1016/j.bmc.2011.02.036

Bioorganic & Medicinal Chemistry 19 (2011) 2742–2750
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
The synthesis and evaluation of indolylureas as PKCa inhibitors
b
b
Jane F. Djung a, Richard. J. Mears b, , Christian A. G. N. Montalbetti , Thomas S. Coulter ,
Adam Golebiowski a, , Andrew N. Carr a, Oliver Barker b, Kenneth D. Greis a,à, Songtao Zhou a,§
Elizabeth Dolan a, Gregory F. Davis a
⇑
,
a Procter & Gamble Pharmaceuticals Inc., Health Care Research Center, 8700 Mason-Montgomery Road, Mason, OH 45040-9462, USA
b Evotec, 114 Milton Park, Abingdon, Oxfordshire OX14 4SA, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
PKC
Identification of compounds that inhibit PKC
ment of heart disease. The synthesis of indolylureas are described, and a compound displaying nanomolar
inhibition towards PKC with significant selectivity over PKA has been identified.
Ó 2011 Elsevier Ltd. All rights reserved.
a
and PKA have crucial but opposing roles in the regulation of calcium handling within myocytes.
Received 16 January 2011
Revised 17 February 2011
Accepted 21 February 2011
Available online 26 February 2011
a, but not PKA, are potential therapeutic targets for the treat-
a
Keywords:
Protein kinase C alpha
Kinase inhibitor
Heart disease
Indolylurea
1. Introduction
has revealed that PKC
in the regulation of calcium handling within myocytes.8a–d The
function of PKC in cardiomyocytes appears to be to reduce cal-
a and PKA play crucial, but opposing roles
Heart failure is one of the leading causes1 of morbidity and mor-
tality in developed nations. In the US, 5 million2 people suffer from
heart disease with over 700,000 new cases presenting every year.
Over 1 million hospitalizations/year result from this condition in
the US, with an estimated cost of $30–40 billion/annum.2b,c,3 The
mechanism of heart failure progression involves multiple causes,
including hypertension, atherosclerosis and a variety of risk factors
which lead to cardiac hypertrophy, impaired calcium handling by
cardiomyocytes and ultimately heart failure.4 While this disease
is well characterised5 and several therapies are available for treat-
ment, patients’ outcomes remain poor with a five-year mortality
rate of approximately 50%.
a
cium cycling capacity within the myocyte, leading to a reduction
in cell function (impaired contraction and relaxation), in part via
dephosphorylation of phospholamban8a and inhibition of the sar-
coplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) or SR Ca2+
pump. This decrease in SERCA activity prolongs the time for cyto-
solic calcium removal (decreased cardiac relaxation time) and may
lead to elevated cytosolic calcium concentrations (increased dia-
stolic tension). In turn, as less cytosolic calcium is sequestered by
SERCA, less calcium is available for subsequent contractions,
diminishing contractile force. In the chronic setting, there is evi-
dence that PKCa may be a key intracellular signalling kinase
A hallmark of heart failure is depressed cardiac function due to
impaired calcium handling within the cells of the heart or cardio-
myocytes.6 The goal of several experimental therapies is to im-
prove calcium handling and thus, restore the heart’s function
(inotropic/lusitropic therapy7) which would be beneficial in
patients with emergent or acute heart failure. Recent evidence
involved in activating signalling pathways which promote cardio-
myocyte hypertrophy.9 These data supported by reports10 demon-
strating that PKCa activity/expression is increased in the human
failing heart, provide evidence that inhibition of this kinase may
provide therapeutic benefit to heart failure patients.
A number of PKC inhibitors have been prepared11 several of
which have progressed to the clinical trial stage12a–d for the treat-
ment of various disorders. Among them, the bis-indolylmaleimides
A (Ruboxistaurin [LY333531], macular edema, diabetic kidney
disease, PKCb), B (Enzastaurin [LY317615], malignant glioma,
pancreatic, colorectal and non-small cell lung cancer, PKCb),
C (Midostaurin [PKC412], acute myeloid leukaemia, aggressive
⇑
Corresponding author. Tel.: +44 0 1235 861561; fax: +44 0 1235 838931.
E-mail address: richard.mears@evotec.com (Richard. J. Mears).
Present address: The Institute for Pharmaceutical Discovery, 23 Business Park
Drive, Brandford, CT 06405, USA.
à
Present address: Department of Cancer and Cell Biology, University of Cincinnati
College of Medicine, Cincinnati, OH 45237, USA.
§
systemic mastocytosis and mast cell leukaemia, PKCa, b, c) and
Present address: 1 Procter & Gamble Plz, Cincinnati, OH 45202-3393, USA.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.02.036

J. F. Djung et al. / Bioorg. Med. Chem. 19 (2011) 2742–2750
2743
D (AEB071,12e transplant rejection, psoriasis, PKC
a
, b, h) are note-
similarities to staurosporine. This paper describes our efforts to
identify PKC inhibitors based upon indolylurea template 1 in a
programme focused on the development of new therapies for heart
worthy (Fig. 1). The role of PKC isoforms in disease processes have
a
been the subject of a number of recent reviews.12f–j
Staurosporine is a broad spectrum kinase inhibitor, binding
tightly to the ATP pocket.13 We used the structure of staurosporine
as a starting point to design a template that mimics some of the
structural features of this molecule with the goal of achieving
failure.
2. Chemistry
selectivity for the ATP binding pocket of PKC
a over PKA. It was
The preparation of 3-indolylureas have been reported via Cur-
tius rearrangement of indolyl-3-carbonyl azides14 followed by
addition of amines to the isocyanate, and it was hoped that this
methodology would allow access to indolylureas of type 1. Prepa-
ration of the acyl azide precursor (Scheme 1) commenced with the
alkylation of methyl indole-3-carboxylate 2 with 3-(tert-butyldi-
methylsilyloxy)-1-bromopropane to surrender N-alkylated indole
3 in quantitative yield. A fluoride mediated deprotection of the
TBS group furnished alcohol 4, which was subsequently converted
to the amines 5 and 6 via the O-triflate intermediate. Hydrolysis of
the methyl ester group of 5 and 6 gave the corresponding amino
acids 7 and 8 after neutralisation and evaporation. These amino
acids were very hygroscopic and difficult to purify away from inor-
ganic impurities without significant loss of material. Although the
amino acids were isolated at neutral pH, observed 1H NMR spectra
were found to be very sensitive to the pH of isolation making it dif-
ficult to obtain consistent NMR spectra between different batches
of the same compound. Conversion of the amino acids 7 and 8 to
acyl azides 9 and 10 via the acid chloride derivatives was straight-
forward, and the acyl azide was found to be sufficiently stable and
envisaged that indolylureas of type 1 would have the potential to
form conformationally restricted structures via an intramolecular
H-bond ( Fig. 2), assuming a three dimensional structure with
Figure 1. Bis-indolylmaleimide derived PKC inhibitors progressed to clinical trial
stage.
Scheme 1. Preparation of acyl azides 9 and 10. Reagents and conditions: (a)
TBSO(CH2)3Br, K2CO3, DMF, 50 °C, 24 h, 100%; (b) TBAF, THF, rt, 93%; (c) Tf2O, 2,6-
lutidine, DCM, 0 °C then Me2NH or Et2NH, 87% (5), 59% (6); (d) NaOH, MeOH, H2O,
60 °C, 44% (7), 100% (8); (e) (COCl)2, DCM, rt, 4 h (f) NaN3 aq, THF, 0 °C, 61% (9), 50%
(10).
Figure 2. Representation of staurosporine docked into the homology model of
PKCa (showing key interactions) and design intention for indolylureas 1 (in
proposed intramolecular H-bonding conformation) to make the same amino acid
contacts.

2744
J. F. Djung et al. / Bioorg. Med. Chem. 19 (2011) 2742–2750
soluble in organic solvents to allow an aqueous workup to remove
any inorganic impurities carried through from the ester hydrolysis
step.
synthesis, and as a tool to explain observed activity trends. In the
absence of a publicly available crystal structure of human PKC
a,
it was necessary to construct a homology model. The 2.0 Å struc-
ture of the PKCh isoform19 was used as a template for this purpose
and sequence alignment was performed in MOE.20 The overall se-
quence homology was found to be 60%, but in the ATP binding
pocket this homology value increased to 80%. Model building and
refinement were also performed in MOE. Twenty-five intermediate
Preparation of biarylanilines 12, 13, 14 and 15 was undertaken
by a heteroaromatic cyclisation, or by Suzuki coupling to a pre-
formed aminoheterocyclic halide. Biarylaniline 12 was isolated in
good yield from a two-step synthesis commencing from acetophe-
none (Scheme 2) according to literature precedents.15a,b
Biarylanilines 13, 14 and 15 were prepared via Suzuki
coupling (Scheme 3) with phenylboronic acid from commercially
available 2-amino-6-chloropyridine and 2-amino-6-chloropyra-
zine. 4-Amino-2-chloropyrimidine was prepared according to
literature precedent.16 The Suzuki conditions employed17 fur-
nished the desired biarylanilines in good yield and circumvented
the requirement for amino group protection often necessary when
carrying out Suzuki couplings with substrates bearing basic NH
functionality.
Having isolated the precursor acyl azides 9 and 10, and biary-
lanilines 12–15, preparation of the indolylureas was attempted.
The Curtius rearrangement of both 9 and 10 was found to be facile
at 100 °C in anhydrous toluene (Scheme 4), and the isocyanate
formed proved sufficiently stable to allow reaction monitoring by
LC–MS (mobile phase: water/acetonitrile gradient). Complete
conversion to isocyanate was observed after 30–60 min at this
temperature, whereupon the appropriate biarylaniline was intro-
duced and heating continued for a further 2 h. The ureas 16–19
were then isolated chromatographically (silica-gel chromatogra-
phy or prep-LC).
models of PKCa were constructed using a Boltzman-weighted ran-
domised modelling procedure21a,b and each minimised to a gradi-
ent of 0.1 using the MOE implementation of the AMBER94
forcefield.22 The staurosporine ligand from the PKCh structure
was retained and fixed in the active site during this process in or-
der to maintain the shape of the cavity. Each intermediate was
scored with MOE’s residue packing function and the highest rank-
ing model selected. This was further optimised with the AMBER94
forcefield to remove unfavourable stereochemistry and atom
contacts.
The urea compounds 16–19 were prepared for docking in MOE.
Protonation states were assigned using in-house EVOwash soft-
ware and a low energy 3D conformer generated for each molecule
using the MOE implementation of the MMFF94x force field.23 All
molecules were docked into the hinge region24 of the PKC
a homol-
ogy model with GOLD v3.0.1,25 using the ChemScore26 scoring
function, with the active site defined as a 12 Å sphere centred in
the binding pocket. The 20 highest scoring poses for each molecule
were retained and clustered at an RMSD of 1.5 Å to remove redun-
dancy in the set of conformations for each ligand.
This docking study suggested a possible explanation for the ob-
served PKCa activity profile. Our initial assumption that the urea
3. Results and discussion
carbonyl would form an H-bond with Val420 (NH) and the urea
N3H would H-bond with Glu418 (C:O) in a manner analogous to
staurosporine proved invalid (Supplementary data, Figs. 7 and 8
show key domain contact representations of staurosporine and
The ureas 16–19 were screened for inhibition of PKCa and PKA
(Table 1). Evaluation of PKA inhibition in the primary screen re-
flects the importance of this kinase in enhancing calcium tran-
16 after docking into the PKCa homology model to illustrate the
sients within cardiomyocytes. Hence, selective inhibition of PKC
a
relative shift of 16 in the binding pocket). As illustrated for urea
16 (Figs. 4 and 5), the homology model suggests H-bonds exist be-
tween N3H and Val420 (C:O) and the urea carbonyl to Val420 (NH),
with the phenyldiazine unit orientated out of the ATP binding
pocket rather than occupying the hydrophobic pocket according
to our initial assumption. In this binding orientation an (ionic)
interaction between ammonium N5 and Asp467 carboxylate can
still occur (as it could if bound in a staurosporine like mode), but
additional interactions between diazine N1 and Tyr419 and a pos-
over PKA was a crucial hurdle to overcome for progression of com-
pounds suitable for further biological evaluation. The primary
screen was an isolated enzyme mass spectrometry (MS) assay18a
evaluating the ability of test compounds to inhibit the phosphory-
lation of a pseudo-substrate peptide by the respective kinases.
During the course of our investigations to discover PKCa inhib-
itors, molecular modelling was used to evaluate new templates for
sible
p-stacking effect between the phenyl ring and Tyr427 may
stabilise the 4-phenylpyrimidine group in this conformation whilst
maintaining an intramolecular (N2–N4H) H-bond to the urea. The
alternative 4-phenylpyrimidine conformation in which pyrimidine
N1 is H-bonded to urea N4H orientates the phenyl towards the low-
er part of the ATP pocket (i.e., towards Asp467) which contains sev-
eral carboxylate residues (Asp424 and Asp481). The electrostatic
repulsion between these residues and the
this conformation.
p-system may disfavour
Scheme 2. Preparation of biarylaniline 12 from acetophenone. Reagents and
conditions: (a) N,N-dimethylformamide dimethyl acetal, 115 °C, 20 h, 74%; (b)
(NH2)2CNH.HCl, NaOEt, EtOH, rfx, 20 h, 84%.
Ureas 17–19 can also adopt the same preferred conformation as
16, but at the expense of the intramolecular H-bonding between
urea N4H and the pyridine/diazine ring nitrogen N1. The alternative
conformation (as shown in Fig. 3) in which intramolecular H-bond-
ing to urea N4H can occur, brings the phenyl groups of 17–19 into
juxtaposition with carboxylate residues Asp424 and Asp467, result-
ing in repulsive interactions, and loss of H-bonding to Tyr419.
Hence in the optimum orientation shown in Figure 5, urea 16 is
stabilised by two H-bonds (intramolecular27 N2–N4H and intermo-
lecular N1–Tyr419), whereas ureas 17–19 are stabilised by a single
N1 intermolecular H-bond to Tyr 419.
Scheme 3. Preparation of biarylanilines 13, 14 and 15 by Suzuki coupling. Reagents
and conditions: (a) PhB(OH)2, K3PO4, Pd(OAc)2 cat., DtBPF cat., 1,4-dioxane, 100 °C,
82% (13), 36% (14), 55% (15).
Compounds 17–19 also show distortion of the planarity of the
biaryl group with respect to the indolylurea moiety, presumably

J. F. Djung et al. / Bioorg. Med. Chem. 19 (2011) 2742–2750
2745
Scheme 4. Preparation of indolylureas via Curtius rearrangement of acylazides 9 and 10. Reagents and conditions: (a) (i) 10, toluene, 100 °C, 60 min; (ii) 13, 14 or 15, 2 h,
100 °C, 11% (17), 6% (18), 3% (19); (b) (i) 9, toluene, 100 °C, 30 min; (ii) 12, 2 h, 100 °C, 33%.
Table 1
In vitro inhibition of PKC
a
and PKA by indolylureas 16–19
a
a
Compound
Biaryl substituent
PKC
a
IC50 (nM)
PKA IC50 (nM)
PKA/PKC
248
a
NH
NH
NH
NH
N
N
N
16
17
95
23550
N
2685
ntb
18
19
4810
nt
N
N
N
8070
3
nt
Staurosporinec
33
11
a
Data collected in triplicate from three successive days for a total of nine dose curves per test compound (n = 9). Estimated error of 10 nM associated with PKC
used for compounds 16–19.
a
assay
b
nt: not tested.
c
IC50 determination of staurosporin (PKCa
and PKA) using this MS assay, discussion of errors and comparison to literature values have been described.18a
Figure 3. Numbering of nitrogen atoms in ureas 16–19 for discussion purposes.
due to repulsive interactions between the aromatic C–H and N4–H
in the preferred binding conformation (Fig. 6). The effect of this
change can be observed by comparison of 16 (near planar orienta-
tion of phenylpyrimidine group with respect to the indolylurea
moiety imposed by the N4–H to N2 H-bond) and 17 (repulsive aryl
C–H N4–H steric clash, skewing the phenylpyridine group 20° out
Figure 4. Homology model of urea 16 in optimum binding mode with ATP pocket
of PKC (key interactions only shown).
a

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J. F. Djung et al. / Bioorg. Med. Chem. 19 (2011) 2742–2750
4. Conclusion
A number of indolylureas bearing heterobiaryl side chains on
the urea moiety were prepared during this study. One of these
compounds demonstrated nanomolar inhibition of PKC and an
acceptable selectivity profile against PKA. Using a homology model
of the PKC ATP binding site, a rationale was developed to explain
a
a
the activity trend observed with the series of compounds prepared.
5. Experimental
5.1. General methods
Thin-layer chromatography was performed on Merck Kieselgel
60 F254 silica gel plates and visualised by UV (254 nm). Flash chro-
matography was conducted using Silicycle silica gel (40–60 lM, pH
6.5–7.0). IR was recorded on a Nicolet 360 FTIR as a thin film or
with a golden-gate ATR device if solid. 1H and 13C NMR were re-
corded on Bruker Avance 400 or Avance 250 systems operating
at 400 or 250 MHz (1H), 100.6 or 62.9 Hz (13C). LC–MS were ana-
lysed on an Atlantis C18 reverse phase column using a water/ace-
tonitrile mobile phase (0.1% formic acid) linked to a Waters
MicromassÒ ZQ™ mass spectrometer. Accurate mass measure-
ments were recorded on an LC–MS system incorporating a Waters
1525 binary HPLC pump, a Waters 2488 UV detector and a Waters
LCT Premier mass spectrometer. Melting points were determined
on a Gallenkamp (model: MPD350) apparatus and are uncorrected.
Figure 5. Homology model of urea 16 in optimum binding mode with ATP pocket
of PKCa showing surface polarities (green: hydrophobic; blue: polar; purple:
charged residues).
of planarity) in their side-on docking views (Figs. 9–11, Supple-
mentary data). This skewing has little effect on the N1 to Tyr419
(OH) bond length (3.07 Å for 17 compared to 3.17 Å for 16) but
C4 of the pyridine ring of 17 is now placed in close proximity to
the top of the binding pocket with the potential for steric interac-
tions destabilising this skewed orientation relative to the planar
16.
5.2. PKCa and PKA inhibition assay
The decrease in IC50 of 16 versus 17–19 could also be ascribed to
the change in substitution of the N5 group (Me2N (16) to Et2N
(17–19)). Examination of the homology model of this pocket,
however, demonstrates that the region surrounding Asp467 is
sterically unencumbered (Figs. 9 and 10, Supplementary data,
show side-on views of docking 16 and 17 into the ATP pocket)
and should be able to accommodate a diethylamino group as easily
as dimethylamino. The possibility that the more hydrophobic
diethylamino group may seek out a less solvent exposed area of
the binding pocket, which could have an influence upon binding
affinity cannot be excluded, although substrate docking suggests
a similar spatial orientation of both dimethylamino and diethyl-
amino substituted chains. Hence the change in substitution at N5
is unlikely to cause the change in inhibition observed between
16 and 17–19, but cannot be entirely discounted as the origin of
the IC50 trends.
All enzyme inhibition assays were performed as described pre-
viously.18 Briefly, 10 point enzyme inhibitor dilution series were
prepared as 10 Â 10 arrays in the centre of 384 well plates. Com-
pound dilutions and the peptide substrate containing the neces-
sary cofactors (i.e., ATP, lipids, etc.) were prepared on separate
plates using a Beckman–Coulter Biomek 2000 workstation. The
reactions were initiated, allowed to proceed for 30–45 min,
stopped by removing 5 lL of the reaction mixture into 50 lL of
MALDI-MS matrix solution and transferred onto a MALDI-TOF MS
target plate, maintaining the original 10 Â 10 array. The specific as-
say conditions for each enzyme reaction were as follows: PKA—en-
zyme activity was measured using 2.7 units/well PKA (Upstate
Biotech #V5161) in 40 mM Tris–HCl, pH 7.5 containing 20 mM
MgCl2, 0.1 mg/mL BSA, 50
Standard incubations for PKA were 30 min at rt. PKC
activity was measured using 6 ng/well PKC (Promega #12,946)
in Promega activation buffer, plus 0.1 mg/mL BSA, 50 M ATP,
200 M EDTA, 400 M CaCl2, and 25 M neurogranin substrate.
Standard incubation conditions for PKC were 45 min at rt.
l
M ATP, and 50
lM kemptide substrate.
a—enzyme
We propose the activity of urea 16 can be assigned to the con-
formational rigidity imposed by the two H-bonding interactions of
the pyrimidine nitrogen atoms.
a
l
l
l
l
a
A cellular assay for PKC utilising MALDI-TOF was developed (see
Supplementary data).
5.3. Mass spectrometry readout
All spectra were collected on an Applied Biosystems (Foster
City, CA) 4700 Proteomics Analyzer, Matrix Assisted Laser Desorp-
tion Ionization-Time of Flight Mass Spectrometer (MALDI-TOF MS)
equipped with a Nd–YAG laser (355 nm, 3 ns pulse width, 200 Hz
repetition rate) in negative ion reflector mode as indicated. The
system was operated with the 4700 Explorer software, version
2.0 or 3.0. Automated acquisition parameters were adjusted to cap-
ture and average only those individual spectra within defined suc-
cess criteria. Specifically, signal intensities for the substrate
peptide were set to a minimum threshold of 3000 counts and a
maximum intensity of 65,000 counts. This ensured that neither
Figure 6. Compounds 16 and 17–19 in preferred binding conformation.

J. F. Djung et al. / Bioorg. Med. Chem. 19 (2011) 2742–2750
2747
null spectra nor saturated spectra were averaged into the final
readout. Depending on the substrate peptide used, between 1000
and 2000 laser shots were averaged from a minimum of 25 random
positions from each sample spot. Data were collected in triplicate
from three successive days (unless otherwise noted) to capture
the maximum variability related to preparation of the enzyme
reactions, transfer of samples to MALDI target plates, data collec-
tion, and data extraction. For data analysis, the isotope cluster
areas for the peptide substrate and product peaks were extracted
into a Microsoft Excel worksheet with the relative inhibition plot-
ted as % Maximal Activity as described previously.18a,b
CDCl3) d 8.10–8.13 (1H, m), 7.78 (1H, s), 7.31–7.33 (1H, m), 7.21–
7.25 (2H, m), 4.17 (2H, t, J = 6.9 Hz), 3.81 (3H, s), 2.17 (2H, t,
J = 6.9 Hz), 2.16 (6H, s), 1.94 (2H, quint., J = 6.9 Hz); 13C NMR
(100.6 MHz, CDCl3) d 165.5, 136.5, 134.6, 126.7, 122.6, 122.0,
121.8, 110.1, 106.8, 55.9, 50.9, 45.4, 44.4, 27.8; LC–MS: 99% UV
(215 nm), MS 261 (M+1)+ 100%; HRMS M+H+ calcd for
C15H21N2O2 261.1603, found 261.1597.
5.3.4. Methyl 1-(3-diethylaminopropyl)-1H-indole-3-carboxyl-
ate 6
Prepared on a 0.64 mmol scale according to procedure described
for dimethylamine analogue 5. The diethylamine 6 was isolated
(0.110 g, 0.38 mmol, 59%) by flash column chromatography as a
yellow oil: Rf 0.38 (10% Et3N in EtOAc); IR (film) 2969, 1696,
5.3.1. Methyl 1-[3-(tert-butyldimethylsilyloxy)propyl]-1H-
indole-3-carboxylate 3
A solution of methyl indole-3-carboxylate (1.87 g, 10.68 mmol),
1-bromo-3-tert-butyldimethylsilyloxypropane (5.0 mL, 21.5 mmol),
cesium carbonate (10.43 g, 32 mmol) and anhydrous DMF (90 mL)
was heated to 50 °C for 16 h under a nitrogen atmosphere. After
cooling, the reaction contents were poured into water (90 mL)
and extracted with EtOAc (2 Â 100 mL). The combined extracts
were dried over MgSO4, filtered and evaporated. The residue was
purified by flash column chromatography to yield the N-alkylated
indole 3 (3.74 g, 10.68 mmol, 100%) as a colourless oil: Rf 0.24 (9:1
heptane/EtOAc); IR (film) 1696, 1533, 1465, 1375, 1228, 1172,
1532, 1465, 1381, 1234, 1194, 1155, 1093, 1037 cmÀ1 1H NMR
;
(400 MHz, MeOH-d4) d 8.07 (1H, d, J = 7.7 Hz), 7.94 (1H, s), 7.45
(1H, d, J = 8.1 Hz), 7.18–7.26 (2H, m), 4.19 (2H, t, J = 7.0 Hz), 3.86
(3H, s), 2.49 (4H, q, J = 7.3 Hz), 2.40–2.44 (2H, m), 1.92–1.99 (2H,
m), 0.95 (6H, t, J = 7.3 Hz); 13C (100.6 MHz, MeOH-d4) d 167.3,
138.0, 136.2, 128.1, 123.9, 122.9, 122.4, 111.5, 107.5, 51.5, 50.5,
47.7, 45.7, 27.5, 11.3; LC–MS: 98% UV (215 nm), MS 289 (M+1)+
100%; HRMS M+H+ calcd for C17H25N2O2 289.1916, found 289.1903.
5.3.5. 1-(3-Dimethylaminopropyl)-1H-indole-3-carboxylic acid
7
1088 cmÀ1 1H NMR (400 MHz, CDCl3) d 8.10–8.14 (1H, m), 7.78
;
(1H, s), 7.33–7.36 (1H, m), 7.19–7.22 (2H, m), 4.22 (2H, t,
J = 6.8 Hz), 3.85 (3H, s), 3.49 (2H, t, J = 5.6 Hz), 1.96 (2H, quint.,
J = 6.2 Hz), 0.88 (9H, s), 0.00 (6H, s); 13C NMR (100.6 MHz, CDCl3)
d 165.5, 136.5, 134.7, 126.7, 122.4, 121.8, 121.7, 110.1, 106.8,
59.1, 50.9, 43.3, 32.5, 26.0, 18.4, À5.5; LC–MS: 100% UV
(215 nm), MS 348 (M+1)+ 100%; HRMS M+H+ calcd for C19H30NO3Si
348.1955, found 348.1990.
To a solution of methyl ester 5 (0.105 g, 0.40 mmol) in metha-
nol/water (10 mL of a 4:1 methanol/water mixture) was intro-
duced sodium hydroxide (0.081 g, 2.00 mmol). The solution was
heated to 60 °C for 36 h. After neutralisation of the reaction solu-
tion with 2 M aq HCl and evaporation to dryness, the residue
was diluted with methanol (2.0 mL) and the insoluble material
(NaCl) removed by filtration. The filtrate was evaporated to furnish
acid 7 as a tan amorphous solid (0.170 g, 0.69 mmol, 172%) con-
taining inorganic residues. Typically the material was carried
through to subsequent steps at this level of purity, but could be
purified by flash column chromatography (MeCN/MeOH gradient
neat MeCN to 1:1 MeCN/MeOH), evaporation, re-dilution in MeCN
and filtration. The filtrate was evaporated to surrender the acid 7
(0.043 g, 0.17 mmol, 44%) as a tan solid: Rf 0.27 (methanol); mp
140–180 °C (sample slowly darkens but does not melt up to
400 °C); IR (ATR) 3113, 2903, 1672, 1526, 1275, 1199, 1135,
5.3.2. Methyl 1-(3-hydroxypropyl)-1H-indole-3-carboxylate 4
To a solution of silylether 3 (3.693 g, 10.64 mmol) in anhydrous
THF (75 mL) at room temperature was introduced TBAF (10.64 mL
of a 1.0 M solution in THF, 10.64 mmol). After 30 min stirring, the
solution was evaporated to dryness. The residual oil was purified
by flash column chromatography (1:1–2:1 EtOAc/heptanes gradi-
ent) to furnish alcohol 4 (2.303 g, 9.88 mmol, 93%) as a colourless
waxy solid: Rf 0.30 (EtOAc); mp 58–59 °C; IR (ATR) 3493, 1674,
1527, 1459, 1375, 1223, 1161, 1099, 1065, 1026 cmÀ1
;
1H NMR
1018 cmÀ1 1H (400 MHz, MeOH-d4)
; d 8.25 (1H, dd, J = 7.0,
(400 MHz, CDCl3) d 8.09–8.12 (1H, m), 7.79 (1H, s), 7.32–7.36
(1H, m), 7.19–7.23 (1H, m), 4.25 (2H, t, J = 6.7 Hz), 3.83 (3H, s),
3.53 (2H, dt, J = 5.7, 4.7 Hz), 2.01 (2H, quint., J = 6.3 Hz), 1.72 (1H,
t, J = 4.5 Hz); 13C NMR (100.6 MHz, CDCl3) d 165.9, 136.5, 134.8,
126.6, 122.8, 121.9, 121.7, 110.1, 106.8, 58.7, 51.1, 43.3, 32.1; LC–
MS: 100% UV (215 nm), MS 234 (M+1)+ 100%; HRMS M+H+ calcd
for C13H16NO3 234.1130, found 234.1124.
1.1 Hz), 7.73 (1H, s), 7.38 (1H, d, J = 7.7 Hz), 7.17, (1H, dt, J = 6.9,
1.4 Hz), 7.12 (1H, dt, J = 7.4, 1.1 Hz), 4.19 (2H, t, J = 6.8 Hz), 2.31–
2.27 (2H, m), 2.19 (6H, s), 1.99 (2H, quint., J = 6.9 Hz); 13C
(100.6 MHz, MeOH-d4) d 168.9, 138.0, 136.2, 128.3, 124.1, 123.0,
122.6, 111.42, 108.8, 56.3, 44.7, 43.6, 26.5; LC–MS: 100% UV
(215 nm), MS 247 (M+1)+ 100%; HRMS M+H+ calcd for
C14H19N2O2 247.1446., found 247.1442.
5.3.3. Methyl 1-(3-dimethylaminopropyl)-1H-indole-3-carbox-
ylate 5
To a solution of alcohol 4 (0.245 g, 1.05 mmol) in anhydrous
DCM (7.0 ml) at 0 °C under an atmosphere of nitrogen was intro-
duced trifluoromethanesulfonic anhydride (0.26 mL, 1.6 mmol).
5.3.6. 1-(3-Diethylaminopropyl)-1H-indole-3-carboxylic acid 8
Prepared on a 42 mmol scale according to procedure described
for dimethylaminocarboxylic acid 7. The crude carboxylic acid 8
was isolated (22.27 g, 81 mmol, 192%) containing inorganic resi-
dues. A small sample was purified by flash column chromatogra-
phy (as carboxylic acid 7) for analysis purposes only, furnishing
acid 8 as a viscous orange/brown oil: Rf 0.34 (methanol), IR (film)
After 2–3 min,
a pre-cooled (0 °C) solution of 2,6-lutidine
(0.25 mL, 2.1 mmol) in anhydrous DCM (10.0 mL) was added via
canula over several minutes. The reaction solution was kept at
0 °C for 5 h. Dimethylamine (6.85 mL of a 2.0 M solution in THF,
13.7 mmol) was then introduced and the reaction mixture warmed
to room temperature for 16 h. The reaction solution was evapo-
rated to dryness and purified by flash column chromatography
(EtOAc containing a 1–10% gradient of triethylamine) to yield
amine 5 (0.237 g, 0.91 mmol, 87%) as a yellow oil: Rf 0.17 (10%
Et3N in EtOAc); IR (film) 2952, 1769, 1696, 1532, 1460, 1375,
3493 (br), 1550, 1431, 1262, 1166, 1110, 1037 cmÀ1 1H NMR,
;
MeOH-d4) d 8.21 (1H, dd, J = 7.1, 1.6 Hz), 7.76 (1H, s), 7.46 (1H,
dd, J = 7.0, 1.6 Hz), 7.16 (2H, app. d quint., J = 7.1, 1.4 Hz), 4.29
(2H, t, J = 6.8 Hz), 2.91 (4H, q, J = 7.3 Hz), 2.82–2.89 (2H, m),
2.11–2.23 (2H, m), 1.11 (6H, t, J = 7.3 Hz); 13C (62.9 MHz, MeOH-
d4) d 173.9, 137.9, 134.1, 129.1, 123.15, 123.10, 121.8, 114.5,
110.8, 49.3, 48.0, 44.7, 26.2, 9.6; LC–MS: 97% UV (215 nm), MS
275 (M+1)+ 100%; HRMS M+H+ calcd for C16H23N2O2 275.1759,
found 275.1749.
1268, 1234, 1189, 1150, 1087, 1031 cmÀ1 1H NMR (400 MHz,
;

2748
J. F. Djung et al. / Bioorg. Med. Chem. 19 (2011) 2742–2750
5.3.7. 1-(3-Dimethylaminopropyl)-1H-indole-3-carbonylazide 9
To a suspension of carboxylic acid 7 (0.115 g, 0.47 mmol) in
anhydrous DCM (30 mL) under an atmosphere of nitrogen was
introduced oxalyl chloride (0.122 mL, 1.40 mmol) and catalytic
DMF (ꢀ10 mg). The reaction mixture was stirred at room temper-
ature for 5 h, then evaporated to dryness. The residue was immedi-
ately re-dissolved in anhydrous THF (3.0 mL) and cooled to 0 °C. An
aqueous solution of sodium azide (0.151 g, 2.33 mmol in 0.5 mL of
water) was then introduced dropwise over several minutes. After
4 h at 0 °C, the reaction mixture was cautiously evaporated (bath
temp <21 °C) to remove THF and the aqueous residue transferred
to a separating funnel. Water (10 mL) was introduced and the pH
adjusted to 7.0 (if required), then the aqueous solution was ex-
tracted with EtOAc (2 Â 10 mL). The combined extracts were dried
over MgSO4, filtered and evaporated (bath <21 °C) to furnish the
crude acyl azide 9 (0.077 g, 0.28 mmol, 61%) as an oil. The purity
of this oil was found to be acceptable for use in subsequent steps
and no attempts were made to purify this substrate further [note:
this material was stored in a freezer at À25 °C]. IR (film) 2944,
br s); 13C NMR (100.6 MHz, CDCl3) 158.8, 156.6, 140.0, 138.5,
128.6, 126.9, 110.9, 107.8 [note: DEPT 135 experiment shows have
3 quaternary carbons, but only 5 methine carbons, assumed two
methines with coincident d’s]; LC–MS: 95% UV (215 nm), MS 171
(M+1)+ 100%; HRMS M+H+ calcd for C11H11N2 171.0922, found
171.0920.
5.3.9.2. 4-Amino-2-phenylpyrimidine 14.
Prepared on a
7.8 mmol scale. Purified product isolated as a colourless wax
(0.477 g, 2.78 mmol, 36%): mp 111–112 °C; Rf 0.38 (neat EtOAc);
IR (ATR): 3393, 3323, 3177, 1654, 1584, 1532, 1479, 1392,
1246 cmÀ1 1H NMR (250 MHz, MeOH-d4) d 8.18–8.22 (2H, m),
;
8.14 (1H, d, J = 6.0 Hz), 7.41–7.47 (3H, m), 6.44 (1H, d, J = 6.0 Hz);
13C NMR (100.6 MHz, MeOH-d4) d 162.3, 162.1, 152.4, 135.9,
127.8, 126.0, 125.7, 125.2, 100.8; LC–MS: 96% UV (215 nm), MS
172 (M+1)+ 100%; HRMS M+H+ calcd for C10H10N3 172.0874, found
172.0874.
5.3.9.3. 2-Amino-6-phenylpyrazine 15.
Prepared on
a
2769, 2133, 1666, 1520, 1462, 1380, 1188, 1141, 1059 cmÀ1
;
3.9 mmol scale. Purified product isolated as a tan, amorphous solid
(0.365 g, 2.1 mmol, 55%): mp 127.5–128.5 °C; Rf 0.32 (EtOAc); IR
(ATR): 3369, 3095, 3048, 1631, 1573, 1532, 1415, 1333, 1269,
1H NMR (400 MHz, CDCl3) d 8.24–8.28 (1H, m), 7.88 (1H, s),
7.40–7.43 (1H, m), 7.30–7.34 (2H, m), 4.26 (2H, t, J = 6.7 Hz), 2.24
(6H, s), 2.23 (2H, t, J = 6.6 Hz), 2.01 (2H, quint., J = 6.6 Hz); 13C NMR
(62.9 MHz, CDCl3) d 167.6, 136.7, 135.8, 126.3, 123.1, 122.4, 121.5,
110.1, 108.1, 55.4, 44.9, 44.3, 27.1; LC–MS: 100% UV (215 nm), MS
272 (M+1)+ 100%. HRMS M+H+ calcd for C14H18N5O 272.1511,
found 272.1501.
1211, 1071 cmÀ1 1H NMR (400 MHz, MeOH-d4) d 8.18 (1H, s),
;
7.92–7.95 (2H, m), 7.84 (1H, s), 7.39–7.47 (3H, m); 13C NMR
(100.6 MHz, MeOH-d4) d 157.1, 151.8, 138.2, 131.4, 130.5, 129.8,
129.6, 128.1; LC–MS: 100% UV (215 nm), MS 172 (M+1)+ 100%;
HRMS M+H+ calcd for C10H10N3 172.0874, found 172.0857.
5.3.8. 1-(3-Diethylaminopropyl)-1H-indole-3-carbonylazide 10
Prepared on a 5.46 mmol scale according to procedure outlined
for acyl azide 9. The crude acyl azide 10 (0.81 g, 2.73 mmol, 50%)
was isolated as a brown oil of acceptable purity for subsequent
reactions and stored in a freezer at À25 °C until used. IR (film)
5.3.10. 1-[1-(3-Dimethylaminopropyl)-1H-indol-3-yl]-3-(4-phenyl-
pyrimidin-2-yl)urea 16
A solution of acyl azide 9 (0.020 g, 0.074 mmol) in anhydrous
toluene (1.5 mL) was heated in a sealed tube at 100 °C for
30 min. After cooling, aniline 12 (0.025 g, 0.15 mmol) was added,
the tube re-capped and the solution heated to 100 °C for a further
2 h. The solution was evaporated, re-dissolved in DMSO and puri-
fied by mass directed prep-LC. The urea 16 was isolated (0.013 g,
0.024 mmol, 33%) as an amorphous pale yellow solid: mp 172 °C;
IR (ATR) 3142, 2938, 1683, 1586, 1544, 1404, 1322, 1293, 1230,
1187, 1007, 826, 767, 727 cmÀ1; Rf 0.17 (20% Et3N in EtOAc);
1H NMR (400 MHz, MeOH-d4) d 8.69 (1H, d, J = 5.5 Hz), 8.15 (2H,
d, J = 7.0 Hz), 7.62 (1H, s), 7.50–7.60 (5H, m), 7.45 (1H, d,
J = 8.0 Hz), 7.24 (1H, t, J = 7.7 Hz), 7.06 (1H, t, J = 7.5 Hz), 4.30 (2H,
t, J = 6.6 Hz), 3.05–3.16 (2H, m), 2.83 (6H, s), 2.26 (2H, quint.,
2135, 1668, 1527, 1465, 1375, 1262, 1189, 1026 cmÀ1 1H NMR
;
(CDCl3, 400 MHz) d 8.20–8.23 (1H, m), 7.89 (1H, s), 7.41–7.44
(1H, m), 7.30–7.34 (2H, m), 4.30 (2H, t, J = 6.9 Hz), 3.00 (4H, q,
J = 7.3 Hz), 2.92–2.95 (2H, m), 2.28 (2H, quint., J = 7.3 Hz), 1.20
(6H, t, J = 7.2 Hz); 13C NMR (62.9 MHz, CDCl3) d 166.9, 135.8,
134.9, 125.5, 122.5, 121.7, 120.7, 109.3, 107.5, 48.3, 45.7, 43.6,
25.2, 9.3; LC–MS: 92% UV (215 nm), MS 300 (M+1)+ 100%; HRMS
M+H+ calcd for C16H22N5O 300.1824, found 300.1832.
5.3.9. General procedure for the synthesis of compounds 13, 14
and 15
J = 6.9 Hz); 13C NMR (100.6 MHz, CDCl3)
d 165.3, 158.7 (br),
To a solution of the requisite chloro-amino-substituted hetero-
aromatic in anhydrous 1,4-dioxane (30 volumes wrt chloride) in a
sealed tube was introduced phenylboronic acid (1.5 equiv) and fi-
nely ground potassium phosphate (2.0 equiv). The solution was de-
gassed (N2 bubbling) for 5 min, Pd(OAc)2 (5 mol % wrt chloride)
and di-tert-butylphosphinoferrocene (5 mol % wrt chloride) intro-
duced and degassing continued for a further 5 min. The tube was
sealed under nitrogen and heated with rapid stirring at 100 °C
for 5 h. After cooling, the reaction mixture was filtered in vacuo
through a celite pad and the precipitated material washed with
1,4-dioxane. The combined filtrates were evaporated and purified
by flash column chromatography (neat hexane to 1:1 hexane/
EtOAc gradient containing 2.5% by volume Et3N) to furnish the
biarylanilines 13, 14 and 15.
158.1, 151.6, 136.3, 133.9, 131.4, 129.1, 127.5, 127.1, 121.9,
121.0, 118.4, 118.0, 117.2, 114.3, 110.8, 109.5, 56.5, 45.3, 44.1,
28.2; LC–MS: 100% UV (215 nm), MS 415 (M+1)+ 100%; HRMS
M+H+ calcd for C24H27N6O 415.2246, found 415.2233.
5.3.11. Generic procedures for synthesis of compounds 17, 18
and 19
A solution of acyl azide 10 (0.640 g, 2.14 mmol) in anhydrous
toluene (30 mL) was heated at 100 °C for 45 min. After cooling,
the solution was divided equally between three sealed tubes and
anilines 13, 14 and 15 (1.42 mmol, 2 equiv) added to the appropri-
ate sealed tube. The tubes were re-capped and heated to 100 °C for
a further 2 h. After cooling the toluene was evaporated and the res-
idue purified by flash column chromatography (1:1 hexane/EtOAc
to neat EtOAc gradient containing 2.5% by volume Et3N) to furnish
the ureas 17, 18 and 19.
5.3.9.1. 2-Amino-6-phenylpyridine 13.
Prepared on
a
7.8 mmol scale. Purified product isolated as a viscous orange oil
(1.092 g, 6.4 mmol, 82%): Rf 0.51 (neat EtOAc); IR (film): 3475,
3387, 3311, 3177, 1608, 1567, 1444, 1345, 1269, 1177,
5.3.11.1. 1-[1-(3-Diethylaminopropyl)-1H-indol-3-yl]-3-(6-phe-
nylpyridin-2-yl)-urea 17.
Purified product isolated as an or-
1018 cmÀ1
;
1H NMR (400 MHz, CDCl3) 7.94 (2H, d, J = 7.3 Hz),
ange wax (0.034 g, 0.077 mmol, 11%): Rf 0.23 (20% Et3N in
7.51 (1H, t, J = 7.9 Hz), 7.45 (2H, t, J = 7.5 Hz), 7.38 (1H, t,
J = 7.3 Hz), 7.10 (1H, d, J = 7.4 Hz), 6.46 (1H, d, J = 8.1 Hz), 4.7 (2H,
EtOAc); IR (ATR): 2956, 1643, 1579, 1433, 1386, 1158,
1071 cmÀ1 1H NMR (400 MHz, CDCl3 d 11.97 (1H, br s), 8.79 (1H,
;

J. F. Djung et al. / Bioorg. Med. Chem. 19 (2011) 2742–2750
2749
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br s), 7.96–7.99 (2H, m), 7.76 (1H, s), 7.72 (1H, t, J = 7.8 Hz), 7.49–
7.54 (4H, m), 7.35 (1H, d, J = 8.4 Hz), 7.21 (1H, t, J = 7.3 Hz), 6.93
(1H, t, J = 7.5 Hz), 6.90 (1H, br d, J = 7.7 Hz), 4.21 (2H, t,
J = 7.0 Hz), 2.60 (4H, q, J = 7.0 Hz), 2.55 (2H, t, J = 7.0 Hz), 2.09
(2H, quint., J = 6.9 Hz), 1.05 (6H, t, J = 7.1 Hz); 13C NMR
(62.9 MHz, MeOH-d4) d 156.6, 155.0, 154.6, 140.54, 140.48, 135.5,
130.3, 130.1, 128.3, 123.2, 122.7, 119.6, 119.3, 118.7, 115.7,
115.4, 111.7, 110.6, 50.8, 47.6, 45.1, 27.9, 11.2; LC–MS: 98% UV
(215 nm), MS 442 (M+1)+ 100%; HRMS M+H+ calcd for C27H32N5O
442.2607, found 442.2589.
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IR (film): 3212, 2961, 1678, 1549, 1468, 1427, 1380, 1211,
1065 cmÀ1 1H NMR (400 MHz, CDCl3) d 11.61 (1H, br s), 9.67
;
(1H, br s), 8.58 (1H, d, J = 5.9 Hz), 8.38 (2H, d, J = 6.6 Hz), 7.75
(1H, br s), 7.66 (1H, d, J = 8.0 Hz), 7.52–7.57 (3H, m), 7.40 (1H, d,
J = 8.0 Hz), 7.26 (1H, t, J = 7.5 Hz), 7.06 (1H, t, J = 7.5 Hz), 6.88 (1H,
br s), 4.24 (2H, t, J = 7.0 Hz), 2.60 (4H, q, J = 7.1 Hz), 2.55 (2H, t,
J = 7.1 Hz), 2.09 (2H, quint., J = 6.5 Hz), 1.05 (6H, t, J = 7.3 Hz);
13C NMR (62.9 MHz, MeOH-d4)
d 163.2, 158.6, 156.4, 151.8,
136.7, 133.6, 130.1, 127.7, 127.3, 121.3, 120.8, 117.91, 117.8,
116.3, 112.9, 108.73, 105.7, 48.8, 45.8, 43.1, 25.8, 9.1; LC–MS:
98% UV (215 nm), MS 443 (M+1)+ 100%; HRMS M+H+ calcd for
C26H31N6O 443.2559, found 443.2559.
5.3.11.3. 1-[1-(3-Diethylaminopropyl)-1H-indol-3-yl]-3-(6-phe-
nylpyrazin-2-yl)-urea 19. Purified product isolated as an yel-
low wax (0.008 g, 0.018 mmol, 3%): Rf 0.25 (20% Et3N in EtOAc); IR
(ATR): 3200, 2967, 1678, 1584, 1549, 1501, 1398, 1287, 1182,
1147, 1065 cmÀ1 1H NMR (400 MHz, CDCl3) d11.53 (1H, br s),
;
9.78 (1H, br s), 8.62 (1H, s), 8.44 (1H, br s), 8.00–8.04 (2H, m),
7.78 (1H, br s), 7.56–7.59 (3H, m), 7.55 (1H, d, J = 8.1 Hz), 7.39
(1H, d, J = 8.5 Hz), 7.24 (1H, t, J = 7.9 Hz), 7.02 (1H, br t,
J = 7.3 Hz), 4.23 (2H, t, J = 7.1 Hz), 2.57 (4H, q, J = 7.3 Hz), 2.52
(2H, t, J = 7.0 Hz), 2.06 (2H, quint., J = 7.0 Hz), 1.03 (6H, t,
J = 7.1 Hz); 13C NMR (62.9 MHz, CDCl3) d 153.4, 148.9, 136.3,
134.4, 134.21, 134.15, 133.9, 130.1, 129.2, 127.5, 122.0, 120.9,
118.5, 118.1, 117.4, 113.9, 109.64, 50.0, 46.7, 44.5, 27.8, 11.6; LC–
MS: 99% UV (215 nm), MS 443 (M+1)+ 100%; HRMS M+H+ calcd
for C26H31N6O 443.2559, found 443.2549.
Acknowledgements
The authors thank Mr. Marcus Halton for the accurate mass
measurements used in this study, Mr. Paul Mortensen for initial
studies towards development of the PKCa homology model and
Mr. Shane Pereira for assistance with graphic artwork.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.02.036.
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