Synthesis of substituted 4-(1H-indol-6-yl)-1H-indazoles as potential PDK1 inhibitors

Article abstract of DOI:10.1016/j.tet.2013.11.054

The development of a preparative route to a series of novel 4-(1H-indol-6-yl)-1H-indazole compounds as potential PDK1 inhibitors is described. The synthetic strategy centres on the late-stage Suzuki cross-coupling of N-unprotected indazole and indole frag

Full text of DOI:10.1016/j.tet.2013.11.054

Accepted Manuscript
Synthesis of substituted 4-(1H-indol-6-yl)-1H-indazoles as potential PDK1 inhibitors
Martin Brzozowski, Nathan J. O’Brien, David J.D. Wilson, Belinda M. Abbott
PII:
S0040-4020(13)01756-0
10.1016/j.tet.2013.11.054
TET 25031
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 12 August 2013
Revised Date: 1 November 2013
Accepted Date: 18 November 2013
Please cite this article as: Brzozowski M, O’Brien NJ, Wilson DJD, Abbott BM, Synthesis of substituted
4-(1H-indol-6-yl)-1H-indazoles as potential PDK1 inhibitors, Tetrahedron (2013), doi: 10.1016/
j.tet.2013.11.054.
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ACCEPTED MANUSCRIPT
Tetrahedron
journal homepage: www.elsevier.com
Synthesis of substituted 4-(1H-indol-6-yl)-1H-indazoles as potential PDK1 inhibitors
Martin Brzozowski, Nathan J. O’Brien, David J. D. Wilson and Belinda M. Abbott*
Department of Chemistry, La Trobe Institute for Molecular Science, La Trobe University, Victoria 3086, Australia
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
The development of a preparative route to a series of novel 4-(1H-indol-6-yl)-1H-indazole
compounds as potential PDK1 inhibitors is described. The synthetic strategy centres on the late
stage Suzuki cross-coupling of N-unprotected indazole and indole fragments. The use of a
monoligated palladium catalyst system was found to be highly beneficial in the cross-coupling
reaction. The indazole and indole fragments were constructed by diazotisation/cyclisation and
SNAr/reductive cyclisation sequences, respectively.
Available online
Keywords:
2009 Elsevier Ltd. All rights reserved.
3-Phosphoinositide-dependent kinase 1 (PDK1)
4-(1H-indol-6-yl)-1H-indazole
Inhibitor
Suzuki cross-coupling
1. Introduction
core possessed the correct geometry to fulfill both of these needs
(Figure 1). The heterocyclic ring nitrogen atoms form a hydrogen
3-Phosphoinositide-dependent kinase 1 (PDK1) is an integral
member of the PI3K/Akt signalling pathway. Its activity is
modulated by phosphatidylinositol 3-kinase (PI3K) through the
regulation of phosphatidylinositol 3,4,5-triphosphate (PtdInsP3).
The PI3K/Akt signalling pathway is responsible for the
regulation of cellular processes such as growth, metabolism,
proliferation and survival.1 PDK1 is known to activate at least 23
different members of this pathway including Akt,2,3 PKC
isoforms,4 p70 S6K,5 p90 SGK6 and RSK.7
bond donor/acceptor/donor motif that provides key interactions
with the backbone residues, Ser89 and Ala91. Functionalisation
of the indole ring at the 2- and 4-positions was anticipated to
provide access to the solvent exposed entrance regions (E0 and
E1) and the ribose pocket (R), respectively. Appending
functionality at the 6-position of the indazole ring would allow
the catalytic residues in the phosphate pocket (P) and also the
hydrophobic pocket (BP-I) to be targeted.
Many human cancers exhibit mutations that result in high
levels of the secondary messenger, PtdInsP3, leading to
constitutive activation of members of the PI3K/Akt pathway.8,9
Inappropriate activation of this pathway promotes tumour
progression and angiogenesis while inhibiting apoptosis.10
Experimental evidence has shown that inhibitors of this pathway
are able to sensitise cells to apoptosis and reduce tumour
formation.11 Given its regulatory role in the PI3K/Akt pathway,
PDK1 provides an attractive target for the development of
oncology therapeutics.*
We aimed to develop novel scaffolds that bound to the PDK1
hinge region and allowed for straightforward incorporation of
additional functionality to target various pockets within the ATP
binding site. Using
a molecular modelling-guided design
approach, we identified that a 4-(1H-indol-6-yl)-1H-indazole
———
Figure 1. Schematic illustrating how the 4-(1H-indol-6-yl)-1H-
indazole scaffold interacts with the hinge region of PDK1 and
displays potential to access various pockets within the binding site.
Corresponding author. Tel.: +61 3 9479 2520; fax: +61 3 9479
1266; e-mail: b.abbott@latrobe.edu.au

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2
Tetrahedron
2. Results and discussion
The synthetic strategy was centred on synthesising the
indazole and indole fragments separately and then forming the
biaryl linkage by a late-stage Suzuki cross-coupling reaction
(Scheme 1). The indole intermediate 5 was proposed to be
accessible by a SNAr/reductive cyclisation sequence of the
activated aryl fluoride 7. Nitration and decarboxylative
halogenation of the commercially available starting material 8
was anticipated as a means of providing the alkylation precursor.
Preparation of the requisite indazole fragments largely
followed that of similar 4,6-disubstituted indazoles reported in
the literature14,15 (Scheme 2). Ortho-toluidine 12 was synthesised
from 4-methyl-3-nitrobenzoic acid 9 according to literature
methods.16,17 Diazotisation and subsequent intramolecular
cyclisation of 12 then furnished the indazole 13. A hypervalent
iodine mediated Hofmann rearrangement was used to complete
the conversion of the acid 14 to the corresponding homologated
amine 17. Treatment of 17 with isocyanic acid, produced in situ,
gave the primary urea indazole fragment 18.
Figure 2. PDK1 inhibitors developed by Berlex Biosciences.
The side chain functionalities of the designed compounds
were based on fragments of known inhibitors that had been
shown to interact with the targeted pockets. Compounds 1 and 2
(Figure 2) are potent PDK1 inhibitors developed by Berlex
Biosciences,12,13 with X-ray crystal structures available of both
compounds in complex with PDK1 (PDB codes: 1Z5M and
2PE2). Both of these structures were utilised in the design of the
first 4-(1H-indol-6-yl)-1H-indazole target structure, 3a (Table 1).
4-Fluoro-3,5-dinitrobenzoic acid (19) was prepared in good
yield by the method of Nielsen18 (Scheme 3). A photoassisted
Hunsdiecker reaction was then employed to effect the
decarboxylative halogenation of acid 19. The method of Meyers
and Fleming19 provided the desired aryl bromide 7 in a moderate
yield of 59% (Table 2, entry 1). Efforts to substitute carbon
tetrachloride with less hazardous solvents led to unacceptable
reductions in yield or decomposition (Table 2, entries 2-4).
Compound 1 contains a pyrrolidine urea moiety that binds to
the ribose pocket of PDK1 and this side chain was incorporated
at the indole 4-position of 3a (R2 side chain). Compound 2
displays a primary urea group that interacts with catalytic
residues in the phosphate pocket and a N-(2-(piperidin-1-
yl)ethyl)amide group that binds in the E0 region. These two side
chain groups were integrated into 3a at the indazole 6-position
(R1) and the indole 2-position (R3).
We identified a small set of target structures (3a-c, Table 1) to
be synthesised and subject to biological evaluation. Herein, we
report the development of a synthetic route to this novel class of
heterocyclic compounds. The resulting synthesis is modular in
nature, with derivatisation of late stage intermediates giving
simple access to all target structures.
Table 1. 4-(1H-Indol-6-yl)-1H-indazoles designed as potential
PDK1 inhibitors.
Compound
R1
R2
R3
3a
3b
3c
3d
3e
Scheme 1. Retrosynthetic approach to the novel 4-(1H-indol-6-yl)-
1H-indazole series.

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Scheme 2. Reagents and conditions: (a) 1,3-dibromo-5,5-dimethylhydantion, H2SO4, 98%; (b) cat. H2SO4, 4 Å MS, MeOH, reflux, 90%; (c)
SnCl2 (5 eq.), EtOH, reflux, 30 min; (d) 15% aq. NaOH, CH2Cl2, 80%; (e) NaNO2, 90% aq. AcOH, 72%; (f) 15% aq. NaOH, EtOH, 83%; (g)
SOCl2, reflux; (h) NH4OH, THF, 0 °C, 87%; (i) PhI(OAc)2, KOH, MeOH, 0 °C, 51%; (j) KOH (10 eq.), 80% aq. MeOH, reflux, 2 d, 72%; (k)
NaOCN (2 eq.), 80% aq. AcOH, 0 °C, 3 h, 74%.
takes place at the solvent interface, where the intermediate acyl
hypohalite is prone to attack by water as it is formed.20 A two-
step procedure, involving Dean-Stark trapping of water prior to
halogen addition was found to be highly advantageous (Table 2,
entry 10). Further investigation revealed the reaction to be
concentration dependent, with poor results obtained at
concentrations higher than 0.2 M (Table 2, entries 9 and 11).
Scheme 3. Reagents and conditions: (a) fuming HNO3, fuming
H2SO4, 100 °C, 3 h, 68%; (b) See Table 2.
Treatment of the activated aryl fluoride 7 with the enolate of
1-tert-butyl
6-methyl
3-oxohexanedioate21,22
gave the
nucleophilic aromatic substitution product 20 (Scheme 4), which
existed exclusively in the (Z)-enol form due to a strong
intramolecular hydrogen bond. The enol 20 was then smoothly
decarboxylated in excellent yield to provide the ketone 6.
Catalytic hydrogenation with platinum(IV) oxide gave rapid
reductive cyclisation of ketone 6 to the N-hydroxyindole 21 in a
62% isolated yield. Careful reaction monitoring was required to
avoid hydrodehalogenation. The aminoindole 22 was obtained by
further reduction of 21 with 10 equivalents of zinc powder in a
1:3 mixture of acetic acid and methanol at room temperature.
Attempts
to
replace
mercury(II)
oxide
with
(diacetoxyiodo)benzene, and bromine with iodine, were all
unsuccessful with only traces of product observed at best (Table
2, entries 5-7). Upon reverting to the originally successful
conditions, the product yield was found to diminish significantly
when the reaction was scaled up. Sensitivity to water formed
during the reaction was identified as a plausible cause of this
difficulty. For substrates with poor solubility, the decarboxylation
Table 2. Optimisation of decarboxylative halogenation reaction conditions.
Entry
Conditionsa
Solventb Scale (g) Concentration (M) Yield %
1
Red HgO, Br2, 150 W bulb, reflux, 3 h
CCl4
CHCl3
CH2Cl2
PhCl
CCl4
0.2
0.2
0.2
0.2
0.1
0.1
0.1
2.0
8.0
2.0
10.0
15.0
0.08
0.08
0.08
0.10
0.04
0.04
0.04
0.15
1.2
59
38
2
Red HgO, Br2, 150 W bulb, reflux, 3 h
3
Red HgO, Br2, 150 W bulb, reflux, 3 h
16
4
Red HgO, Br2, 150 W bulb, reflux, 4 h
0 (dec.)
tracec
0
5
PhI(OAc)2, I2, 150 W bulb, reflux, 3 h
6
PhI(OAc)2, Br2, 150 W bulb, reflux, 3 h
CCl4
7
Red HgO, I2, 150 W bulb, reflux, 4 h
CCl4
0c
8
Red HgO, Br2, 150 W bulb, reflux, 4 h
CCl4
52
9
Red HgO, Br2, 150 W bulb, reflux, 5 h
CCl4
34
10
11
12
CCl4
0.2
72
1. red HgO, reverse phase Dean-Stark trap, reflux, 1.5 h; 2. Br2, 150 W bulb, reflux, 3 h
1. red HgO, reverse phase Dean-Stark trap, reflux, 7 h; 2. Br2, 150 W bulb, reflux, 5 d
1. red HgO, reverse phase Dean-Stark trap, reflux, 2 d; 2. Br2, 150 W bulb, reflux, 27 h
CCl4
1.5
25
CCl4
0.2
56
aAll reactions conducted under argon using oven-dried glassware. 1.5 eq. each of oxidising agent and halogen used in all cases.
bAll solvents were distilled prior to use and stored over 4 Å molecular sieves.
cAryl iodide product.

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4
Tetrahedron
Scheme 4. Reagents and conditions: (a) NaH, 1-tert-butyl 6-methyl 3-oxohexanedioate, THF, 0 °C → rt, 62%; (b) 10% HCl in MeOH, reflux,
93%; (c) H2 (1 atm.), PtO2 (2.5% w/w), MeOH, 15-20 min, 62%; (d) Zn (10 eq.), 1:3 AcOH/MeOH, 24 h, 78%; (e) Triphosgene, C5H5N,
CH2Cl2, 0 °C, 2.5 h, then pyrrolidine, 68%; (f) Ac2O, Et3N, CH2Cl2, 79%; (g) 15% aq. NaOH, MeOH, 82-88%; (h) EDAC·HCl, HOBt·H2O,
CH2Cl2, 0.5 h, then 2-(piperidin-1-yl)ethanamine or 2-methylpropan-1-amine, 57-81%.
Table 3. Synthesis of 4-(1H-indol-6-yl)-1H-indazoles by sequential
Miyaura borolation and Suzuki cross-coupling of indazole bromides.
Elaboration of the key indole intermediate 22 at the 2- and
4-positions provided facile access to all of the required indole
cross-coupling fragments. N,N-disubstituted urea 23a was
prepared in a 68% yield by reacting the amine 22 with
triphosgene and then pyrrolidine. A slow, inverse addition of the
amine to a triphosgene solution was required to avoid the
formation of a symmetrical dimer as the major product.
Acetylation of 22 under standard conditions yielded the
N-acetylindole 23b. Ester hydrolysis with aqueous sodium
hydroxide afforded the free acids 24a and 24b in high yields.
Diimide mediated coupling of the acids with the appropriate
amine then furnished the carboxamide fragments 5a-c.
Entry Substrate Conditionsa Bromide Product Yield %
The 4-(1H-indol-6-yl)-1H-indazoles 3a-e were synthesised by
Miyaura borolation of indazole bromides 18 and 13, followed by
Suzuki cross-coupling of the corresponding indazole boronate
esters 4a and 4b (Table 3). Miyaura borolation conditions23 gave
full conversion of the primary urea 18 to the boronate ester 4a,
but this compound could not be successfully purified by flash
chromatography or crystallisation due to poor solubility.
Attempts to employ impure 4a in the cross-coupling step were
unsuccessful and thus a one-pot procedure was developed to
avoid isolation of the boronate intermediate (Table 3, entry 1).
This one-pot procedure provided the desired biaryl 3a, albeit in a
poor yield of 24% over two steps, with multiple side products
observed by TLC. Fragments 4b and 5a gave no cross-coupled
product when treated with PdCl2(dppf) and caesium carbonate in
a refluxing 4:1 mixture of 1,4-dioxane and water (Table 3, entry
2). MS analysis indicated that significant hydrolysis of the
methyl ester 4b was observed under these reaction conditions.
Caesium carbonate is known to facilitate ester cleavage24,25 and it
is plausible that the carboxylate formed by the competing
hydrolysis reaction led to inhibition of the already sluggish
cross-coupling reaction by catalyst deactivation.
1
2
3
4
5
6
18
13
13
13
13
13
A
5a
5a
3a
3b
3b
3c
3d
3e
24
B, C
B, D
B, D
B, D
B, D
77, 0
5a
77, 56b
77, 70b
77, 67b
77, 44b
5c
5b
23a
aConditions: (A) PdCl2(dppf) (5 mol %), bis(pinacolato)diboron (1.4 eq.),
KOAc, MeOH, reflux, 16 h, then indole bromide (1.3 eq.), PdCl2(dppf) (5
mol %), Cs2CO3, 4:1 1,4-dioxane/water, reflux, 4 h; (B) PdCl2(dppf) (3 mol
%), bis(pinacolato)diboron (1.4 eq.), KOAc, MeOH, reflux, 16 h; (C)
PdCl2(dppf) (5 mol %), indole bromide (1.3 eq.), Cs2CO3, 4:1
1,4dioxane/water, reflux,
2
h. (D) Pd2(dba)3 (5 mol %),
2-dicyclohexylphosphino-2’,4’,6’-triisopropylbiphenyl (10 mol %), indole
bromide (0.6 eq.), Na2CO3, 4:1 1,4-dioxane/water, reflux, 2-4 h.
bYield based on bromide coupling partner.

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For the synthesis of biaryls 3b-e, more active catalyst systems
with a Phenomenex® Jupiter C18 300 Å column (250 mm × 10.0
mm, 10 µm) at a flow rate of 5 mL/min, monitored at 220 nm.
were sought to improve the efficiency of the cross-coupling
reaction and the milder base, sodium carbonate, was employed to
avoid the competing ester hydrolysis. A dialkylbiarylphosphine
palladium catalyst system developed by Buchwald and
co-workers26-28 gave smooth cross-coupling of boronate ester 4b
with the indole bromides 5a-c and 23a (Table 3, entries 3-6).
This catalyst system provided greatly enhanced coupling
efficiency, with only a single product observed by TLC in all
cases. This increased efficiency is attributed to the formation of a
highly active monoligated palladium complex,29 which rapidly
undergoes oxidative insertion with traditionally sluggish
substrates such as the N-unprotected heteroaryl halides studied in
this work.
4.2. Synthesis
4.2.1. Methyl 4-bromo-1H-indazole-6-carboxylate (13)
To a stirred solution of amine 12 (4.50 g, 18.4 mmol) in acetic
acid (80 mL), was added a solution of sodium nitrite (1.39 g, 20.3
mmol) in water (8 mL). The mixture was stirred overnight and
then concentrated under reduced pressure. The residue was
diluted with saturated sodium hydrogen carbonate (100 mL),
extracted with ethyl acetate (3 × 30 mL) and the combined
organic extracts were subject to standard workup. The crude
product was recrystallised from toluene and the mother liquors
were purified by flash chromatography (15% EtOAc/hexanes) to
afford the indazole 13 (3.38 g, 72%) as a pale orange solid; Rf
(15% EtOAc/hexanes) 0.25; mp 173-174 ºC; δH (DMSO-d6):
13.83 (1H, br s), 8.16 (2H, s), 7.78 (1H, d, J 1.0 Hz), 3.90 (3H,
s); δC (DMSO-d6): 165.3, 139.7, 133.4, 128.4, 125.9, 122.6,
113.2, 111.9, 52.5; m/z (ESI): 254.9 (M[79Br]H+), 256.9
(M[81Br]H+); HRMS (ESI): M[79Br]H+, found 254.9765.
C9H8BrN2O2 requires 254.9769.
3. Conclusion
In summary, we have developed an expedient route for the
preparation of novel 4-(1H-indol-6-yl)-1H-indazoles as potential
inhibitors of PDK1. A SNAr/reductive cyclization sequence gave
access to a variety of 6-bromo-2,4-disubstituted-1H-indoles,
which were then cross-coupled with 1H-indazol-4-yl-boronate
esters. The use of a highly active monoligated palladium catalyst
system was found to greatly improve the efficiency of the
late-stage Suzuki cross-coupling. Preliminary biological
screening of these compounds is currently underway and will be
reported shortly.
4.2.2. 4-Bromo-1H-indazole-6-carboxylic acid (14)
A mixture of ester 13 (3.38 g, 13.3 mmol) and 15% aqueous
sodium hydroxide (15 mL) in ethanol (30 mL) was stirred at
room temperature for 4 h and then concentrated under reduced
pressure. The residue was diluted with water, acidified to pH 4
with 1 M hydrochloric acid, and the resulting orange precipitate
was collected by filtration and dried in vacuo to give the acid 14
(2.64 g, 83%) as a pale orange solid; Rf (10% MeOH/CH2Cl2)
0.10; mp 294 - 296 ºC (dec); δH (DMSO-d6): 13.78 (1H, br s),
13.3 (1H, br s). 8.15 (2H, s), 7.80 (1H, d, J 0.5 Hz); δC
(DMSO-d6): 166.4, 139.9, 133.4, 129.9, 125.7, 123.0, 113.0,
111.8; m/z (ESI): 240.9 (M[79Br]H+), 242.9 (M[81Br]H+); HRMS
(ESI): M[79Br]H+, found 240.9607. C8H6BrN2O2 requires
240.9613.
4. Experimental section
4.1. General methods
Melting points were measured on a Reichert Thermopan
microscope hot stage apparatus and are uncorrected. All
glassware used in moisture sensitive reactions was oven dried
and then cooled under argon prior to use. The standard workup
procedure for an organic extract comprised washing with brine,
drying over anhydrous magnesium sulfate and filtration, followed
by concentration under reduced pressure to afford the crude
product. Analytical Thin Layer Chromatography (TLC) was
performed on Merck kieselgel 60 F254 plates aluminium backed
plates and visualised using a 254 nM UV lamp or by staining
with ninhydrin stain consisting of ninhydrin (0.2 g), acetic acid
(0.5 mL), and water (4.5 mL) in n-butanol (100 mL). Flash
chromatography was performed on silica gel (Davisil® LC60Å
40-63 micron) according to the method of Still et al.30 Flash
chromatography eluent systems containing dichloromethane
saturated with ammonia were freshly prepared as follows:
Dichloromethane (400 mL) and ammonium hydroxide (30%, 40
mL) were shaken in a separating funnel, and the cloudy
dichloromethane layer was separated. Upon addition of the
required volume of methanol, the solution became homogenous
and was used immediately. NMR spectra were recorded on a
4.2.3. 4-Bromo-1H-indazole-6-carboxamide (15)
A stirred suspension of acid 14 (1.46 g, 6.0 mmol) and thionyl
chloride (10 mL) was heated to reflux. After 1 h, the mixture
became homogeneous and the solution was concentrated under
reduced pressure. Dry toluene (30 mL) was added and the
mixture was evaporated to dryness to remove trace thionyl
chloride. The residue was suspended in dry tetrahydrofuran (50
mL), cooled to 0 ºC, and 30% ammonium hydroxide (20 mL)
was added dropwise. After being stirred overnight, the mixture
was diluted with water (100 mL), and the resulting precipitate
was collected by filtration and dried in vacuo to afford the
carboxamide 15 (1.26 g, 87%) as a yellow solid; Rf (5%
MeOH/CH2Cl2) 0.25; mp 262-263 ºC (dec); δH (DMSO-d6):
13.78 (1H, br s), 8.20 (1H, br s), 8.11 (1H, s), 8.10 (1H, s), 7.83
(1H, d, J 1.0 Hz), 7.55 (1H, br s); δC (DMSO-d6): 166.8, 140.0,
133.5, 133.3, 125.0, 122.2, 112.9, 109.7; m/z (ESI): 240.0
(M[79Br]H+), 242.0 (M[81Br]H+); HRMS (ESI): M[79Br]H+, found
239.9766. C8H7BrN3O requires 239.9772.
1
Bruker AV-500 at 500.19 MHz for H nuclei and at 125.78 MHz
for 13C nuclei. Chemical shifts are reported as δ values in parts
per million (ppm). In reporting spectral data the following
abbreviations have been used: s, singlet; br s, broad singlet; d,
doublet; t, triplet; q, quartet; m, multiplet. Coupling constants (J)
are reported to the nearest 0.5 Hz. Low-resolution electrospray
ionisation (ESI) mass spectra were recorded on a Bruker
Daltronics Esquire 6000 Ion Trap mass spectrometer in methanol
(0.1% formic acid) at 40 eV cone voltage. High-resolution
electrospray ionisation mass spectra were recorded on an Agilent
6224 TOF LC/MS mass spectrometer coupled to an Agilent 1290
Infinity and were reference mass corrected via a dual-spray
electrospray ionisation source. Preparative reverse phase high
performance liquid chromatography (HPLC) was performed on a
Beckman system (125 Solvent Module and 166 Detector) fitted
4.2.4. Methyl (4-bromo-1H-indazol-6-yl)carbamate (16)
Carboxamide 15 (750 mg, 3.12 mmol) was added to a stirred
solution of potassium hydroxide (438 mg, 7.80 mmol) in
methanol (50 mL). Once the reaction mixture was mostly
homogenous, it was cooled to 0 ºC and (diacetoxyiodo)benzene
(1.01 g, 3.12 mmol) was added in a single portion. After 3 h, the
reaction mixture was concentrated under reduced pressure,
diluted with saturated ammonium chloride (100 mL) and the
resulting precipitate collected by filtration. The precipitate was

ACCEPTED MANUSCRIPT
6
Tetrahedron
suspended in boiling methanol (30 mL) and filtered whilst hot
4.2.8. (Z)-1-tert-Butyl 6-methyl 2-(4-bromo-2,6-dinitrophenyl)-3-
to remove insoluble impurities. The filtrate was evaporated under
reduced pressure to give the crude product, which was purified
by flash chromatography (3% MeOH/CH2Cl2) to give the
carbamate 16 (429 mg, 51%) as a fawn coloured crystalline solid;
Rf (5% MeOH/CH2Cl2) 0.41; mp 198-204 ºC (dec); δH
(DMSO-d6): 13.21 (1H, br s), 9.93 (1H, br s), 7.90 (1H, s), 7.83
(1H, s), 7.38 (1H, d, J 1.5 Hz), 3.70 (3H, s); δC (DMSO-d6):
154.0, 141.0, 138.5, 133.0, 119.7, 115.8, 112.9, 96.8, 51.9; m/z
(ESI): 270.0 (M[79Br]H+), 271.9 (M[81Br]H+); HRMS (ESI):
M[79Br]H+, found 269.9872. C9H9BrN3O2 requires 269.9878.
hydroxyhex-2-enedioate (20)
Sodium hydride (60% dispersion in oil, 5.09 g, 0.127 mol)
was washed with dry hexanes then suspended in dry
tetrahydrofuran (100 mL) and cooled to 0 ºC. A solution of
1-tert-butyl 6-methyl 3-oxohexanedioate19,20 (15.4 g, 66.9 mmol)
in dry tetrahydrofuran (50 mL) was added dropwise over 10 min
(H2 gas evolved). After 15 min, a solution of bromide 7 (16.9 g,
63.7 mmol) in dry tetrahydrofuran (100 mL) was added and the
mixture was allowed to stir for a further 24 h at room
temperature. The reaction mixture was then concentrated under
reduced pressure, diluted with dichloromethane (400 mL) and the
organic fraction washed with 2 M hydrochloric acid (160 mL).
The organic fraction was subject to standard workup to give the
crude product as a dark orange solid. This was recrystallised
(MeOH) and the mother liquors were further purified by flash
chromatography (CH2Cl2) and recrystallisation (MeOH) to yield
the enol 20 (18.7 g, 62%) as pale yellow plates; Rf (15%
EtOAc/hexanes) 0.44; mp 137-139 ºC; δH (CDCl3): 13.26 (1H, br
s), 8.19 (2H, s), 3.61 (3H, s), 2.56 (2H, t, J 7.5 Hz), 2.28 (2H, t, J
7.5 Hz), 1.34 (9H, s); δC (CDCl3): 174.2, 172.6, 169.5, 151.5,
130.4, 123.4, 122.5, 95.0, 83.6, 52.0, 29.6, 28.3, 27.9; m/z (ESI):
497.0 (M[79Br]Na+), 499.0 (M[81Br]Na+); HRMS (ESI):
M[81Br]Na+, found 499.0144. C17H19BrN2O9 requires 499.0172.
4.2.5. 4-Bromo-1H-indazol-6-amine (17)
A stirred solution of carbamate 16 (338 mg, 1.60 mmol) and
potassium hydroxide (896 mg, 16.0 mmol) in a 4:1 mixture of
methanol/water (50 mL) was heated to reflux. After 2 d, the
reaction mixture was concentrated under reduced pressure,
diluted with saturated ammonium chloride (100 mL) and
extracted with ethyl acetate (6 × 20 mL). The combined organic
fractions were treated according to standard workup to give the
crude product, which was purified by flash chromatography (50%
EtOAc/hexanes) to afford the amine 17 (244 mg, 72%) as a dark
orange crystalline solid; Rf (50% EtOAc/hexanes) 0.23; mp
146-147 ºC (dec); δH (DMSO-d6): 12.57 (1H, br s), 7.66 (1H, s),
6.73 (1H, d, J 1.5 Hz), 6.48 (1H, t, J 1.0 Hz), 5.45 (2H, br s); δC
(DMSO-d6): 148.8, 142.5, 132.8, 116.4, 114.5, 113.0, 89.5; m/z
(ESI): 211.9 (M[79Br]H+), 213.9 (M[81Br]H+); HRMS (ESI):
M[79Br]H+, found 211.9816. C7H7BrN3 requires 211.9823.
4.2.9. Methyl 5-(4-bromo-2,6-dinitrophenyl)-4-oxopentanoate (6)
A stirred solution of enol 20 (18.7 g, 39.3 mmol) and 32%
hydrochloric acid (30 mL) in methanol (300 mL) was heated to
reflux overnight. The mixture was cooled to room temperature
and the solid that crystallised from the reaction mixture was
collected by filtration. The filtrate was concentrated under
reduced pressure, diluted with dichloromethane (200 mL) and
then washed with saturated sodium hydrogen carbonate (100
mL). The organic fraction was subject to standard workup to give
the crude product, which was recrystallised (MeOH) and
combined with the previously isolated material to give the ketone
6 (13.7 g, 93%) as off-white plates; Rf (30% EtOAc/hexanes)
0.60; mp 100-102 ºC; δH (CDCl3): 8.28 (2H, s), 4.31 (2H, s), 3.67
(3H, s), 2.92 (2H, t, J 6.6 Hz), 2.64 (2H, t, J 6.6 Hz); δC (CDCl3):
201.6, 172.6, 151.4, 131.7, 123.6, 121.8, 52.1, 41.5, 37.1, 27.8;
m/z (ESI): 396.9 (M[79Br]Na+), 298.9 (M[81Br]Na+); HRMS
(ESI): M[79Br]Na+, found 396.9643. C12H11BrN2O7Na requires
396.9647.
4.2.6. 1-(4-Bromo-1H-indazol-6-yl)urea (18)
To a stirred solution of amine 17 (500 mg, 2.40 mmol) in a 5:1
mixture of acetic acid/water (12 mL) at 0 ºC, was added sodium
cyanate (307 mg, 4.70 mmol). After 1 h, the reaction was diluted
with water (50 mL) and the precipitate collected by filtration. The
filtrate was concentrated to a small volume (5 mL) and cooled on
ice to yield more of the precipitate. The combined solids were
reslurried in warm n-butanol, filtered and dried in vacuo to give
the urea 18 (446 mg, 74%) as an off-white powder; Rf (5%
MeOH/CH2Cl2) 0.11; mp >300 ºC; δH (DMSO-d6): 13.04 (1H, br
s), 8.82 (1H, br s), 7.85 (1H, s), 7.81 (1H, s), 7.26 (1H, s), 5.95
(2H, br s); δH (DMSO-d6): 155.9, 141.3, 139.9, 132.8, 119.1,
115.9, 112.6, 96.1; m/z (ESI): 254.9 (M[79Br]H+), 256.9
(M[81Br]H+); HRMS (ESI): M[79Br]H+, found 254.9874.
C8H9BrN4O requires 254.9881.
4.2.10. Methyl 3-(6-bromo-1-hydroxy-4-nitro-1H-indol-2-
yl)propanoate (21)
4.2.7. 5-Bromo-2-fluoro-1,3-dinitrobenzene (7)
Acid 19 (15.0 g, 65.3 mmol) and red mercury(II) oxide (21.2
g, 98.0 mmol) were suspended in dry carbon tetrachloride (300
mL). The flask was equipped with a reverse phase Dean-Stark
trap, and the heterogeneous mixture was heated to reflux while
being stirred vigorously. After 2 d, approximately 1 mL of water
was observed in the Dean-Stark trap. A solution of bromine (5.0
mL, 98.0 mmol) in carbon tetrachloride (1 M) was added
dropwise while the reaction flask was irradiated with a 150W
bulb. After 27 h, the reaction was cooled to room temperature
and saturated sodium hydrogen carbonate (100 mL) was added.
The two-phase mixture was filtered through Celite®, and the
filtrate was extracted with carbon tetrachloride (3 × 50 mL). The
combined organic fractions were treated according to standard
workup to provide the bromide 7 (9.75 g, 56%) as off-white
coloured plates; Rf (10% EtOAc/hexanes) 0.32; mp 73-75 ºC; δH
(CDCl3): 8.45 (2H,d, J 5.5 Hz, 2H); δC (CDCl3): 147.7 (J 283
Hz), 139.53, 133.7 (J 2.5 Hz), 116.6 (J 6.3 Hz); Note: compound
7 did not ionize under ESI MS conditions.
A suspension of ketone 6 (500 mg, 1.50 mmol) and
platinum(IV) oxide (2.5% w/w, 14.0 mg) in methanol (20 mL)
was stirred under an atmosphere of hydrogen (balloon). Once the
mixture became homogeneous (10-15 min), stirring was
continued for
a further 5 min until TLC indicated full
consumption of the starting material. The reaction mixture was
then filtered through Celite® and concentrated under reduced
pressure to give
chromatography
a
black solid. Purification by flash
EtOAc/hexanes) afforded the
(30%
N-hydroxyindole 21 (270 mg, 62%) as a bright yellow crystalline
solid; Rf (40% EtOAc/hexanes) 0.49 (self stains bright yellow,
dark orange with ninhydrin); mp 163-165 ºC (dec); δH (CDCl3):
9.73 (1H, br s), 8.21 (1H, d, J 1.5 Hz), 7.96 (1H, d, J 1.0 Hz),
6.89 (1H, s), 3.68 (3H, s), 3.16 (2H, t, J 6.0 Hz), 2.89 (2H, t, J 6.0
Hz); δC (CDCl3): 176.4, 141.9, 139.6, 135.7, 120.6, 118.3, 116.5,
112.6, 96.1, 53.0, 35.0, 19.4; m/z (ESI): 343.0 (M[79Br]H+), 345.0
(M[81Br]H+); HRMS (ESI): M[81Br]H+, found 344.9901.
C12H12BrN2O5 requires 344.9930.

ACCEPTED MANUSCRIPT
4.2.11. Methyl 3-(4-amino-6-bromo-1H-indol-2-yl)propanoate
(22)
(3 × 20 mL). The combined organic fractions were treated
according to standard workup to give the crude product.
Purification by flash chromatography (5% MeOH/CH2Cl2)
afforded the acid 24a (649 mg, 88%) as a white solid; mp
200-202 ºC; Rf (5% MeOH/CH2Cl2) 0.21; δH (DMSO-d6): 12.22
(1H, br s), 11.02 (1H, s), 7.73 (1H, s), 7.45 (1H, d, J 1.0 Hz),
7.12 (1H, s), 6.33 (1H, s), 3.41-3.43 (4H, m), 2.90 (2H, t, J 8.0
Hz), 2.64 (2H, t, J 8.0 Hz), 1.85-1.88 (4H, m); δC (DMSO-d6):
173.5, 153.8, 138.2, 137.1, 132.5, 120.2, 113.2, 112.6, 108.0,
97.0, 45.7, 33.1, 25.1, 23.1; m/z (ESI): 380.1 (M[79Br]H+), 382.1
(M[81Br]H+); HRMS (ESI): M[79Br]H+, found 380.0606.
C16H19BrN3O3 requires 380.0610.
To a stirred solution of indole 21 (6.60 g, 19.2 mmol) in 3:1
methanol/acetic acid (300 mL), was added zinc dust (12.6 g,
0.192 mol), portionwise over 30 min. After being stirred
overnight, the reaction mixture was concentrated under reduced
pressure, diluted with ethyl acetate (100 mL) and filtered. The
organic layer was washed with saturated sodium hydrogen
carbonate (3 × 30 mL) and treated according to standard workup
to give a brown solid. Purification by flash chromatography
(0-5% EtOAc/CH2Cl2) afforded the aminoindole 22 (4.44 g,
78%) as fluffy white crystals; Rf (40% EtOAc/hexanes) 0.49
(dark purple with ninhydrin); mp 137-138 ºC; δH (CDCl3): 8.47
(1H, br s), 6.94 (1H, s), 6.49 (1H, d, J 1.5 Hz), 6.10 (1H, d, J 1.0
Hz), 3.88 (2H, br s), 3.72 (3H, s), 3.03 (2H, t, J 6.5 Hz), 2.71
(2H, t, J 6.5 Hz); δC (CDCl3): 174.5, 139.8, 137.5, 136.9, 116.5,
115.6, 107.5, 105.0, 96.5, 52.2, 34.0, 23.1; m/z (ESI): 297.0
(M[79Br]H+), 299.0 (M[81Br]H+); HRMS (ESI): M[79Br]H+, found
297.0231. C12H14BrN2O2 requires 297.0239.
4.2.15. 3-(4-Acetamido-6-bromo-1H-indol-2-yl)propanoic acid
(24b)
A mixture of methyl ester 23b (1.59 g, 4.69 mmol) and 15%
aqueous sodium hydroxide (20 mL) in methanol (50 mL) was
stirred at room temperature overnight. The mixture was acidified
with 2 M hydrochloric acid and the resulting precipitate was
collected by filtration and dried in vacuo to give the acid 24b
(920 mg, 82%) as an off-white solid; Rf (7.5% MeOH/CH2Cl2)
0.22; mp 220-224 ºC (dec); δH (DMSO-d6): 12.29 (1H, br s),
11.13 (1H, br s), 9.63 (1H, s), 7.84 (1H, s), 7.18 (1H, s), 6.52
(1H, s), 2.93 (2H, t, J 7.5 Hz), 2.65 (2H, t, J 7.5 Hz), 2.13 (3H,
s); δC (DMSO-d6): 173.5, 168.8, 138.7, 137.2, 131.2, 119.1,
112.6, 112.5, 109.0, 96.6, 33.0, 23.9, 23.1; m/z (ESI): 325.0
(M[79Br]H+), 327.0 (M[81Br]H+); HRMS (ESI): M[79Br]H+, found
325.0184. C13H13BrN2O3 requires 325.0188.
4.2.12. Methyl 3-(6-bromo-4-(pyrrolidine-1-carboxamido)-1H-
indol-2-yl)propanoate (23a)
A solution of amine 22 (1.17 g, 3.90 mmol) and pyridine (700
µL, 8.70 mmol) in dry dichloromethane (150 mL) was added
dropwise over 2.5 hours to a solution of triphosgene (444 mg,
1.50 mmol) in dry dichloromethane (20 mL) at 0 ºC. Once the
addition was complete, a solution of pyrrolidine (0.34 mL, 4.10
mmol) and pyridine (0.7 mL, 8.70 mmol) in dry dichloromethane
(5 mL) was added in a single portion. The reaction mixture was
stirred for a further 0.5 h at 0 ºC, then washed sequentially with
10% potassium bisulfate (30 mL) and 5% sodium hydrogen
carbonate (30 mL). The organic fraction was treated according to
standard workup to give the crude product, which was purified by
flash chromatography (1-2.5% MeOH/CH2Cl2) to give the urea
23a (1.06 g, 68%) as a white solid; Rf (2.5% MeOH/CH2Cl2)
0.35; mp 166-170 ºC; δH (CDCl3): 8.67 (1H, br s), 7.79 (1H, s),
7.15 (1H, s), 6.28 (1H, br s), 6.08 (1H, s), 3.72 (3H, s), 3.50-3.53
(4H, m), 3.02 (2H, t, J 6.5 Hz), 2.70 (2H, t, J 6.5 Hz), 1.98-2.01
(4H, m); δC (CDCl3): 174.3, 153.9, 138.2, 137.2, 131.4, 119.5,
115.1, 113.8, 109.2, 96.0, 52.1, 46.0, 33.8, 25.8, 23.2; m/z (ESI):
394.1 (M[79Br]H+), 396.0 (M[81Br]H+); HRMS (ESI): M[81Br]H+,
found 396.0742. C17H21BrN3O3 requires 396.0766.
4.2.16. Preparation of 3-(1H-indol-2-yl)propanamides by EDAC
mediated amide coupling
4.2.16.1. General procedure A. A mixture of the acid (1 eq.),
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
(1.8 eq.), and 1-hydroxybenzotriazole monohydrate (1.8 eq.) in
dry dichloromethane was stirred at room temperature for 0.5 h.
The amine (1.1 eq.) was added and the mixture was allowed to
stir until TLC indicated full consumption of the starting material.
The reaction mixture was washed with water, and treated
according to standard workup to afford the crude product.
4.2.16.2. N-(6-Bromo-2-(3-oxo-3-((2-(piperidin-1-
yl)ethyl)amino)propyl)-1H-indol-4-yl)pyrrolidine-1-carboxamide
(5a)
4.2.13. Methyl 3-(4-acetamido-6-bromo-1H-indol-2-
yl)propanoate (23b)
Acid 24a (100 mg, 0.26 mmol) was coupled with 2-(piperidin-
1-yl)ethanamine according to General procedure A. Purification
by flash chromatography (0-0.5% MeOH/CH2Cl2 sat. with NH3)
followed by recrystallisation (CH2Cl2) gave the carboxamide 5a
(73 mg, 57%) as white crystals; Rf (2% MeOH/CH2Cl2 sat. with
NH3) 0.20; mp 104-112 ºC; δH (DMSO-d6): 10.97 (1H, br s), 7.76
(1H, t, J 5.5 Hz), 7.73 (1H, s), 7.43 (1H, d, J 1.5 Hz), 7.11 (1H, d,
J 1.0 Hz), 6.28 (1H, s), 3.40-3.43 (4H, m), 3.13-3.17 (2H, m
(collapses to t, J 7.0 Hz upon treatment with D2O)), 2.90 (2H, t, J
7.5 Hz), 2.47 (2H, t, J 7.5 Hz), 2.25-2.29 (6H, m), 1.85-1.88 (4H,
m), 1.43-1.48 (4H, m), 1.34-1.36 (2H, m); δC (DMSO-d6): 170.9,
153.8, 138.6, 137.1, 132.4, 120.3, 113.5, 112.5, 108.0, 97.0, 57.8,
54.1, 45.7, 36.3, 34.8, 25.5, 25.1, 24.0, 23.7; m/z (ESI): 490.2
(M[79Br]H+), 492.2 (M[81Br]H+); HRMS (ESI): M[81Br]H+, found
492.1791. C23H33BrN5O2 requires 492.1818.
To a stirred solution of amine 22 (1.77 g, 5.96 mmol) in dry
dichloromethane (150 mL), was added triethylamine (870 µL,
6.25 mmol) and acetic anhydride (560 µL, 6.25 mmol). After 4.5
h, the reaction mixture was concentrated under reduced pressure.
The crude product was recrystallised from methanol to give the
acetamide 23b (1.60 g, 79%) as a white solid; Rf (2.5%
MeOH/CH2Cl2) 0.27; mp 209-211 ºC; δH (DMSO-d6): 11.13 (1H,
br s), 9.62 (1H, s), 7.84 (1H, s), 7.18 (1H, s), 6.51 (1H, s), 3.61
(3H, s), 2.97 (2H, t, J 7.5 Hz), 2.75 (2H, t, J 7.5 Hz), 2.13 (3H,
s); δC (DMSO-d6): 172.4, 168.7, 138.2, 137.1, 131.2, 119.0,
112.6, 112.4, 109.0, 96.7, 51.5, 32.7, 23.9, 23.0; m/z (ESI): 339.0
(M[79Br]H+), 341.0 (M[81Br]H+); HRMS (ESI): M[79Br]H+, found
339.0337. C14H16BrN2O3 requires 339.0344.
4.2.16.3. 3-(4-Acetamido-6-bromo-1H-indol-2-yl)-N-(2-
(piperidin-1-yl)ethyl)propanamide (5b)
4.2.14. 3-(6-Bromo-4-(pyrrolidine-1-carboxamido)-1H-indol-2-
yl)propanoic acid (24a)
Acid 24b (500 mg, 1.54 mmol) was coupled with 2-(piperidin-
1-yl)ethanamine according to General procedure A. Purification
by flash chromatography (0.5-2% MeOH/CH2Cl2 sat. with NH3)
gave the carboxamide 5b (435 mg, 65%) as an beige-coloured
A mixture of methyl ester 23a (768 mg, 1.95 mmol) and 15%
aqueous sodium hydroxide (10 mL) in methanol (10 mL) was
stirred at room temperature overnight. The mixture was acidified
with 2 M hydrochloric acid and then extracted with ethyl acetate

ACCEPTED MANUSCRIPT
8
Tetrahedron
4.2.18. Methyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
1H-indazole-6-carboxylate (4b)
solid; Rf (2.5% MeOH/CH2Cl2 sat. with NH3) 0.20; mp 84-92
ºC; δH (DMSO-d6): 11.07 (1H, br s), 9.63 (1H, br s), 7.81 (1H, s),
7.75 (1H, t, J 5.5 Hz), 7.17 (1H, s), 6.47 (1H, s), 3.12-3.16 (2H,
m (collapses to t, J 7.0 Hz upon treatment with D2O)), 2.91 (2H,
t, J 7.5 Hz), 2.47 (2H, t, J 7.5 Hz), 2.24-2.28 (6H, m), 2.13 (3H,
s), 1.42-1.47 (4H, m), 1.33-1.36 (2H, m); δC (DMSO-d6, 325 K):
170.7, 168.5, 139.0, 137.0, 131.0, 119.3, 112.6, 112.3, 109.0,
96.6, 57.6, 54.1, 36.3, 34.8, 25.4, 23.9, 23.6, 23.5; m/z (ESI):
435.1 (M[79Br]H+), 437.1 (M[81Br]H+); HRMS (ESI): M[81Br]H+,
found 437.1396. C20H28BrN4O2 requires 437.1371.
A
mixture of bromide 13 (850 mg, 3.33 mmol),
(1,1’-bis(diphenylphosphino)ferrocene)palladium(II) dichloride
(73.0 mg, 3 mol %), bis(pinacolato)diboron (1.18 g, 4.62 mmol)
and potassium acetate (981 mg, 9.99 mmol) in methanol (30 mL)
was degassed under high vacuum, purged with argon, and then
heated to reflux for 16 h. The reaction mixture was concentrated
under reduced pressure, diluted with dichloromethane (30 mL)
and washed with water (50 mL). The organic fraction was treated
according to standard workup to give the crude product.
Purification by flash chromatography (20% EtOAc/hexanes)
gave an inseparable 9:1 mixture of the dioxaborolane 4b (776
mg, 77%) and methyl 1H-indazole-6-carboxylate (50 mg, 9%) as
a dark orange crystalline solid; Rf (25% EtOAc/hexanes) 0.30;
mp 146-147 ºC (dec); δH (DMSO-d6): 13.51 (1H, br s), 8.28 (2H,
s), 8.07 (1H, s), 3.91 (3H, s), 1.36 (12H, s); m/z (ESI): 303.1
(MH+); This mixture was used in subsequent reactions without
further purification.
4.2.16.4. N-(6-Bromo-2-(3-(isobutylamino)-3-oxopropyl)-1H-
indol-4-yl)pyrrolidine-1-carboxamide (5c)
Acid 24a (584 mg, 1.54 mmol) was coupled with
2-methylpropan-1-amine according to General procedure A.
Purification by flash chromatography (1-5% MeOH/CH2Cl2)
gave the carboxamide 5c (540 mg, 81%) as an off-white solid; Rf
(2% MeOH/CH2Cl2) 0.25; mp 104-109 ºC; δH (DMSO-d6): 10.98
(1H, br s), 7.86 (1H, t, J 5.5 Hz), 7.72 (1H, s), 7.43 (1H, d, J 1.5
Hz), 7.11 (1H, d, J 0.5 Hz), 6.28 (1H, s), 3.40-3.43 (4H, m), 2.87-
2.92 (4H, m), 2.48 (2H, m (obscured by DMSO peak)), 1.85-1.88
(4H, m), 1.62-1.70 (1H, m), 0.81 (6H, d, J 7.0 Hz); δC
(DMSO-d6): 171.0, 153.8, 138.7, 137.1, 132.4, 120.4, 113.2,
112.5, 108.1, 96.9, 46.1, 45.7, 34.7, 28.1, 25.1, 23.7, 20.1; m/z
(ESI): 435.1 (M[79Br]H+), 437.1 (M[81Br]H+); HRMS (ESI):
M[81Br]H+, found 437.1371. C20H28BrN4O2 requires 437.1396.
4.2.19. Monoligated palladium catalyzed Suzuki cross-coupling
4.2.19.1. General procedure B. Aryl bromide (1 eq.),
2-dicyclohexylphosphino-2’,4’,6’-triisopropylbiphenyl (10 mol
%), tris(dibenzylideneacetone)dipalladium(0) (5 mol %), sodium
carbonate (2.0 eq.) and dioxaborolane 4b (1.5 eq.) were
suspended in a 4:1 mixture of 1,4-dioxane/water (5 mL). The
mixture was degassed under high vacuum, purged with argon,
and then heated to reflux for 2-4 h. Upon completion, the mixture
was cooled to room temperature, filtered and then concentrated
under reduced pressure to give the crude product.
4.2.17. N-(2-(3-Oxo-3-((2-(piperidin-1-yl)ethyl)amino)propyl)-6-
(6-ureido-1H-indazol-4-yl)-1H-indol-4-yl)pyrrolidine-1-
carboxamide (3a)
A mixture of bromide 18 (100 mg, 0.39 mmol), (1,1’-
bis(diphenylphosphino)ferrocene)palladium(II) dichloride (14.0
mg, 5 mol %), bis(pinacolato)diboron (139 mg, 0.55 mmol) and
potassium acetate (115 mg, 1.18 mmol) in methanol (5 mL) was
degassed under high vacuum, purged with argon, and then heated
to reflux for 16 h. A solution of caesium carbonate (345 mg, 1.06
mmol) in water (0.5 mL) was added and the mixture was stirred
at reflux for a further 3 h then concentrated under reduced
4.2.19.2. Methyl 4-(2-(3-oxo-3-((2-(piperidin-1-
yl)ethyl)amino)propyl)-4-(pyrrolidine-1-carboxamido)-1H-indol-
6-yl)-1H-indazole-6-carboxylate (3b)
Bromide 5a (100 mg, 0.20 mmol) was treated according to
General procedure B. Purification by flash chromatography
(2-5% MeOH/CH2Cl2 sat. with NH3) yielded the title compound
3b (67 mg, 56%) as a white solid; Rf (5% MeOH/CH2Cl2 sat.
with NH3): 0.40; mp 136-148 ºC (dec); δH (DMSO-d6): 13.55
(1H, br s), 10.98 (1H, d, J 1.5 Hz), 8.41 (1H, s), 8.09 (1H, s),
7.83 (1H, s), 7.80 (1H, t, J 5.5 Hz), 7.78 (1H, d, J 1.0 Hz), 7.70
(1H, d, J 1.0 Hz), 7.35 (1H, s), 6.33 (1H, s), 3.93 (3H, s),
3.45-3.48 (4H, m), 3.15-3.19 (2H, m (collapses to t, J 7.0 Hz
upon treatment with D2O)), 2.97 (2H, t, J 7.5 Hz), 2.53 (2H, m
(obscured by DMSO peak)), 2.27-2.30 (6H, m) 1.88-1.91 (4H,
m), 1.44-1.48 (4H, m), 1.34-1.36 (2H, m); δC (DMSO-d6): 171.2,
166.8, 154.4, 140.3, 139.4, 137.2, 136.2, 133.7, 131.5, 130.7,
127.7, 123.9, 122.0, 118.4, 112.1, 110.2, 105.5, 97.2, 57.9, 54.1,
52.4, 45.8, 36.4, 35.0, 25.5, 25.2, 24.1, 23.9; m/z (ESI): 586.3
(MH+); HRMS (ESI): MH+, found 586.3142. C32H40N7O4
requires 586.3142.
pressure.
Bromide
5a
(100
mg,
0.52
mmol),
(1,1’-bis(diphenylphosphino)ferrocene)palladium(II) dichloride
(14.0 mg, 5 mol %) and a 4:1 mixture of 1,4-dioxane/water (5
mL) were added to the residue. The mixture was degassed under
high vacuum, purged with argon, and then heated to reflux for 4
h. The reaction mixture was cooled to room temperature, filtered
and then concentrated under reduced pressure to give the crude
product. Flash chromatography (5-10% MeOH/CH2Cl2 sat. with
NH3) gave an off-white solid (29.0 mg, 24%), which was further
purified by preparative reverse phase HPLC (5-65% MeCN/10
mM aq. NH4CO3) to give the title compound 3a as a fluffy, white
solid (5.6 mg, 5%); Rf (10% MeOH/CH2Cl2 sat. with NH3): 0.17;
mp > 300 ºC; δH (DMSO-d6): 12.80 (1H, br s), 10.98 (1H, br s),
8.82 (1H, s), 8.13 (1H, s), 7.87 (1H, s), 7.82 (1H, t, J 5.5 Hz),
7.78 (1H, s), 7.66 (1H, d, J 1.0 Hz), 7.27 (1H, s), 6.98 (1H, d, J
1.5 Hz), 6.31 (1H, s), 5.89 (2H, br s), 3.36-3.47 (4H, m),
3.15-3.19 (2H, m (collapses to t, J 7.0 Hz upon treatment with
D2O)), 2.95 (2H, t, J 7.5 Hz), 2.53 (2H, m (obscured by DMSO
peak)), 2.32-2.26 (6H, m) 1.88-1.90 (4H, m), 1.43-1.48 (4H, m),
1.33-1.36 (2H, m); δC (DMSO-d6): 171.1, 156.2, 154.3, 141.6,
139.1, 139.0, 137.1, 135.6, 133.1, 131.5, 131.3, 121.7, 116.7,
112.0, 111.7, 105.1, 96.9, 95.0, 57.8, 54.1, 45.7, 36.3, 35.0, 25.5,
25.1, 24.0, 23.9; m/z (ESI): 586.3 (MH+); HRMS (ESI): MH+,
found 586.3252. C31H40N9O3 requires 586.3254.
4.2.19.3. Methyl 4-(2-(3-(isobutylamino)-3-oxopropyl)-4-
(pyrrolidine-1-carboxamido)-1H-indol-6-yl)-1H-indazole-6-
carboxylate (3c)
Bromide 5c (100 mg, 0.23 mmol) was treated according to
General procedure B. Purification by flash chromatography
(1-3% MeOH/CH2Cl2 sat. with NH3) yielded the title compound
3c (86 mg, 70%) as an off-white solid; Rf (2% MeOH/CH2Cl2
sat. with NH3): 0.21; mp 149-162 ºC (dec); δH (DMSO-d6): 13.56
(1H, br s), 10.99 (1H, br s), 8.41 (1H, s), 8.09 (1H, s), 7.90 (1H,
t, J 5.5 Hz), 7.82 (1H, s), 7.76 (1H, d, J 1.0 Hz), 7.69 (1H, d, J
1.5 Hz), 7.35 (1H, s), 6.34 (1H, s), 3.93 (3H, s), 3.44-3.47 (4H,
m), 2.98 (2H, t, J 7.5 Hz), 2.90 (2H, t, J 6.5 Hz), 2.54 (2H, t, J
7.5 Hz), 1.88-1.91 (4H, m), 1.64-1.72 (1H, m), 0.83 (6H, d, J 6.5

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Hz); δC (DMSO-d6): 171.1, 166.7, 154.3, 140.2, 139.4, 137.1,
8.
9.
Osaka, M.; Oshimura, M.; Ito, H. Apoptosis 2004, 9, 667-676.
136.1, 133.5, 131.5, 130.6, 127.5, 123.8, 122.0, 118.3, 111.9,
110.0, 105.4, 97.0, 52.3, 46.1, 45.7, 34.9, 28.1, 25.1, 23.9, 20.1;
m/z (ESI): 531.3 (MH+); HRMS (ESI): MH+, found 531.2718.
C29H35N6O4 requires 531.2720.
Vanhaesebroeck, B; Leevers, S. J.; Ahmadi, K.; Timms, J.; Katso,
R.; Driscoll, P. C.; Woscholski, R.; Parker, P. J.; Waterfield, M. D.
Ann. Rev. Biochem. 2001, 70, 535-602.
10. Raimondi, C.; Falasca, M. Curr. Med. Chem. 2011, 18, 2763-2769.
11. Falasca, M. Curr. Pharm. Des. 2010, 16, 1410-1416.
12. Feldman, R. I.; Wu, J. M.; Polokoff, M. A.; Kochanny, M. J.;
Dinter, H.; Zhu, D.; Biroc, S. L.; Alicke, B.; Bryant, J.; Yuan, S.;
Buckman, B. O.; Lentz, D.; Ferrer, M.; Whitlow, M.; Adler, M.;
Finster, S.; Chang, Z.; Arnaiz, D. O. J. Biol. Chem. 2005, 280,
19867-19874.
4.2.19.4. Methyl 4-(4-acetamido-2-(3-oxo-3-((2-(piperidin-1-
yl)ethyl)amino)propyl)-1H-indol-6-yl)-1HI-indazole-6-
carboxylate (3d)
Bromide 5b (100 mg, 0.23 mmol) was treated according to
General procedure B. Purification by flash chromatography
(2.5-5% MeOH/CH2Cl2 sat. with NH3) yielded the title
compound 3d (82 mg, 67%) as an off-white solid; Rf (2.5%
MeOH/CH2Cl2 sat. with NH3): 0.11; mp 147-154 ºC; δH (DMSO-
d6): 13.56 (1H, br s), 11.07 (1H, s), 9.71 (1H, s), 8.36 (1H, s),
8.10 (1H, s), 8.07 (1H, s), 7.77-7.79 (2H, m), 7.40 (1H, s), 6.50
(1H, s), 3.93 (3H, s), 3.15-3.19 (2H, m), 2.98 (2H, t, J 7.5 Hz),
2.53 (2H, m (obscured by DMSO peak)), 2.26-2.29 (6H, m), 2.16
(3H, s), 1.43-1.47 (4H, m), 1.32-1.35 (2H, m); δC (DMSO-d6):
171.0, 168.6, 166.7, 140.2, 139.7, 137.2, 136.0, 133.4, 130.7,
130.3, 127.6, 123.8, 120.7, 118.5, 111.0, 110.2, 106.3, 96.7,
57.85, 54.1, 52.4, 36.3, 35.0, 25.5, 24.0, 23.9 (2 overlapping
signals); m/z (ESI): 531.3 (MH+); HRMS (ESI): MH+, found
531.2722. C29H35N6O4 requires 531.2720.
13. Islam, I.; Brown, G.; Bryant, J.; Hrvatin, P.; Kochanny, M. J.;
Phillips, G. B.; Yuan, S.; Adler, M.; Whitlow, M.; Lentz, D.;
Polokoff, M. A.; Wu, J.; Shen, J.; Walters, J.; Ho, E.;
Subramanyam, B.; Zhu, D.; Feldman, R. I.; Arnaiz, D. O. Bioorg.
Med. Chem. Lett. 2007, 17, 3819-3825.
14. Kang, X.; Fine, R. M.; Banksy, B.; Long, W.; Ma, C.; Li, H.; Wang,
Y.; Hu, Y.; Wang, Y. Compounds and Compositions as Protein
Kinase Inhibitors. WO 2011038579, April 7, 2011.
15. Dillon, M. P.; Krauss, N. E. Indole, Indazole, and Benzimidazole
Arylamides as P2X3 and P2X2/3 Antagonists. US 20100324070.
Dec 23, 2010.
16. Leed, A. R.; Boettger, S. D.; Ganem, B. J. Org. Chem. 1980, 45,
1098-1106.
17. Gin, M. S.; Yokozawa, T.; Prince, R. B.; Moore, J. S. J. Am. Chem.
Soc. 1999, 121, 2643-2644.
4.2.19.5. Methyl 4-(2-(3-methoxy-3-oxopropyl)-4-
(pyrrolidine-1-carboxamido)-1H-indol-6-yl)-1H-indazole-6-
carboxylate (3e)
18. Neilsen, A. T.; Chefin. A. P.; Christian, S. L. J. Org. Chem. 1984,
49, 4575-4580.
19. Meyers, A. I.; Fleming, M. P. J. Org. Chem. 1979, 44, 3405-3406.
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21. Cason, J. Org. Synth. 1945, 25, 19.
22. Bunce, R. A.; Nammalwar, B. J. Heterocyclic Chem. 2009, 46,
172-177.
Bromide 23a (100 mg, 0.23 mmol) was treated according to
General procedure B. Purification by flash chromatography
(2.5% MeOH/CH2Cl2) yielded the title compound 3e (36 mg,
44%) as a white solid; Rf (2.5% MeOH/CH2Cl2): 0.20; mp
142-155 ºC; δH (DMSO-d6): 13.52 (1H, br s), 11.02 (1H, d, J 1.5
Hz), 8.41 (1H, s), 8.10 (1H, s), 7.80 (1H, s), 7.79 (1H, d, J 1.0
Hz), 7.73 (1H, d, J 1.0 Hz), 7.36 (1H. s), 6.39 (1H, s), 3.93 (3H,
s), 3.64 (3H, s), 3.46-3.49 (4H, m), 3.04 (2H, t, J 7.5 Hz), 2.79
(2H, t, J 7.5 Hz), 1.88-1.91 (4H, m); δC (DMSO-d6): 172.4,
166.6, 154.2, 140.1, 138.3, 137.2, 136.0, 133.5, 131.5, 130.8,
127.5, 123.8, 121.7, 118.3, 111.9, 110.0, 105.3, 97.1, 52.2, 51.4,
45.7, 32.9, 25.0, 23.1; m/z (ESI): 490.2 (MH+); HRMS (ESI):
MH+, found 490.2093. C26H28N5O5 requires 490.2091.
23. Ishiyama, T.; Murata, M.; Miyaura, N. J. Org. Chem. 1995, 60,
7508-7510.
24. Zaugg, H. E. J. Org. Chem. 1976, 41, 3419-3421.
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Am. Chem. Soc. 2005, 127, 4685-4696.
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3358-3366.
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2923-2925.
Acknowledgements
The authors would like to thank Dr. Sebastian Marcuccio of
Advanced Molecular Technologies Pt. Ltd. (AMT) for assistance
with cross-coupling chemistry and Dr. Les Deady (LIMS) for
helpful synthesis discussions. We thank Dr. Jason Dang (MIPS)
for HRMS analysis. MB is grateful to La Trobe University for
financial support through the provision of a La Trobe University
Postgraduate Scholarship and the LIMS Writing-Up Award.
References and notes
1.
2.
3.
4.
5.
Mora, A.; Kommander, D.; van Aalten, D. M. F.; Alessi, D. R.
Semin. Cell Dev. Biol. 2004, 15, 161-170.
Brazil, D. P.; Hemmings, B. A. Trends Biochem. Sci. 2001, 26,
657-664.
Scheid, M. P.; Woodgett, J. R. Nat. Rev. Mol. Cell Biol. 2001, 2,
760-768.
Le Good, J. A.; Ziegler, W. H.; Parakh, D. B.; Alessi, D. R.; Cohen,
P.; Parker, P. J. Science 1998, 281, 2042-2045.
Pullen, N.; Dennis, P. B.; Andjekovic, M.; Dufner, A.; Kozma, S.
C.; Hemmings, B. A.; Thomas, G. Science 1998, 279, 707-710.
Lang, F.; Cohen, P. Sci. STKE 2001, 108, RE17.
Frodin, M.; Gammeltoft, S. Mol. Cell. Endrocrinol. 1999, 151,
65-77.
6.
7.

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10
Tetrahedron
Graphical Abstract
Synthesis of substituted 4-(1H-indol-6-yl)-
1H-indazoles as potential PDK1 inhibitors
Leave this area blank for abstract info.
Martin Brzozowski, Nathan J. O’Brien and Belinda M. Abbott
Department of Chemistry, La Trobe Institute for Molecular Science, La Trobe University, Victoria 3086,
Australia

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Supplementary Information
Synthesis of substituted 4-(1H-indol-6-yl)-1H-indazoles as potential
PDK1 inhibitors
Martin Brzozowski, Nathan J. O’Brien, David J. D. Wilson and Belinda M. Abbott*
Department of Chemistry, La Trobe Institute for Molecular Science, La Trobe University, Victoria 3086, Australia
Computational Methodology
Molecular modelling protocol
A series of ligands were designed by hybridisation/fragmentation of known PDK1 inhibitors to provide novel
structures. These structures were then evaluated in silico by performing molecular dynamics (MD) simulations
followed by free binding energy calculations using the AMBER software package1. It should be noted that
generation of the protein/ligand complexes is intended to generate an appropriate starting geometry for MD
simulation rather than a fully optimised, low-energy structure. An optimised structure will be achieved following
minimisation and equilibration steps in the MD protocol.
Ligand setup and parameterisation
Initial ligand structures were generated using the Maestro module of Schrödinger2 and exported as pdb files.
Hydrogen atoms and connectivity information were added using the LEaP module of AMBER and double bonds
were added manually. Ligand atomic partial charges were obtained by performing quantum chemical calculations
using Gaussian 033. Single point energy calculations were carried out at the B3LYP level of theory using a ccp-
VTZ basis set. Solvent effects were included by a polarisable continuum model (PCM)4,5 self-consistent reaction
field (SCRF) method with an ether-type solvent (diethyl ether ε = 4.24). The resulting electrostatic potentials
(ESP) generated from the quantum calculations were then transformed into atomic charges in the antechamber
module of AMBER using the restrained ESP (RESP) methodology6. Force field parameters were automatically
generated in antechamber and these were imported into the General AMBER Force Field (GAFF)7.
Preparation of complexes
The structure of PDK1 in complex with ATP (PDB code: 2BIY)8 was selected for use in our modelling as it was
the most complete structure available at the time and was of high resolution. The crystallographic data was edited
to remove water, glycerol and sulfate ions. The ATP molecule was also removed to give a PDK1 structure with a
vacant ATP binding site. Missing protein side chains and hydrogen atoms added manually in LEaP using the ff03
force field9 to provide an ‘empty’ PDK1 template. Maestro was used to manually dock the ligand molecules into
the enzyme active site of the ‘empty’ PDK1 structure using a superimposition method. The PDK1/ligand
complexes were charge neutralised by the addition of counterions and solvated by a TIP3P solvent box with a
minimum solute wall distance of 10 Å.
MD simulations
Five successive minimization steps were performed: (i) 5000-step minimisation [1000 step steepest-descent (SD)
then 4000-step conjugate gradient (CG)] with protein/ligand system restrained to relax solvent and counterion
molecules, (ii) three rounds of 1000-step minimisation [100 with SD and 900 with CG] with decreasing restraints

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of 15, 10, 5 kcal/mol applied to the protein C-α backbone atoms and (iii) 1000-step minimisation [100 with SD
and 900 with CG] with all restraints removed. The system was then heated to 300 K using Langevin dynamics10 in
a slow, stepwise fashion (30 K every 2500 steps). Equilibration was then performed in the NPT ensemble for
50,000 steps (a total of 100 ps with a 2 fs timeframe) with all restraints removed. The SHAKE algorithm11 was
used to constrain bond lengths for bonds involving hydrogen with a cutoff of 12 Å for non-bonded interactions.
Pressure was regulated by way of isotropic position scaling and temperature was held constant at 300 K with
regulation by Langevin dynamics. A production phase of 5 ns was completed in five 1ns blocks, using the same
conditions as the equilibration phase. Post-production analysis was performed to ensure the system structure had
remained realistic and that the equilibrium structure had not changed drastically. This was done by viewing the
output trajectory file in VMD12 to ensure the protein remained folded and also by plotting the physical parameters
to ensure that they remained stable.
Free binding energy calculations
After the product dynamics, the resulting MD trajectory was used to perform free energy calculations. This was
done using the single trajectory approach, with all of the snapshots coming from the one trajectory rather than
separate trajectories for the complex, receptor and the ligand. Snapshots were generated from the MD trajectories
at 10 ps intervals to produce a series of uncorrelated structures for each of the complex, receptor and ligand. The
free binding energies were then calculated using the MM_PBSA script in AMBER.
References
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11. Ryckaert, J.-P.; Ciccotti, G.; Berendsen, H. J. C. J. Comput. Phys. 1977, 23, 327-341.
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