Synthesis, biological evaluation and molecular modelling of sulfonohydrazides as selective PI3K p110α inhibitors

Article abstract of DOI:10.1016/j.bmc.2007.08.062

A series of 2-methyl-5-nitrobenzenesulfonohydrazides were prepared and evaluated as inhibitors of PI3K. An isoquinoline derivative shows good selectivity for the p110α isoform over p110β and p110δ, and also demonstrates good in vitro activity in a cell pr

Full text of DOI:10.1016/j.bmc.2007.08.062

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 15 (2007) 7677–7687
Synthesis, biological evaluation and molecular modelling
of sulfonohydrazides as selective PI3K p110a inhibitors
Jackie D. Kendall,a,* Gordon W. Rewcastle,a,c Raphael Frederick,a Claire Mawson,a
William A. Denny,a,c Elaine S. Marshall,a Bruce C. Baguley,a Claire Chaussade,b
Shaun P. Jacksond and Peter R. Shepherdb,c
aAuckland Cancer Society Research Centre, School of Medical and Health Sciences, The University of Auckland,
Private Bag 92019, Auckland 1020, New Zealand
bDepartment of Molecular Medicine and Pathology, School of Medical and Health Sciences, The University of Auckland,
Private Bag 92019, Auckland 1020, New Zealand
cMaurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Private Bag 92019, Auckland 1020, New Zealand
dAustralian Centre for Blood Diseases, Monash University, Melbourne, Australia
Received 26 July 2007; revised 28 August 2007; accepted 29 August 2007
Available online 4 September 2007
Abstract—A series of 2-methyl-5-nitrobenzenesulfonohydrazides were prepared and evaluated as inhibitors of PI3K. An isoquino-
line derivative shows good selectivity for the p110a isoform over p110b and p110d, and also demonstrates good in vitro activity in a
cell proliferation assay. Molecular modelling provides a rationalisation for the observed SAR.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
sitide dependent protein kinase 1 (PDK1). Phosphory-
lated PKB is subsequently involved in a range of
processes which inhibit apoptosis and promotes cell
growth and cell motility.6,7 The lipid phosphatase PTEN
dephosphorylates PIP3 and acts as a tumour suppressor.
It is deleted or inactivated in a range of tumours, leading
toincreasedlevelsofPIP3andincreasedtumoursurvival.8
Phosphoinositide-3-kinases (PI3Ks) are a group of lipid
kinases which phosphorylate the 3-hydroxyl group of
phosphoinositides. They are split into three classes
(Class I, II and III) and play an important role in cellu-
lar signaling.1 Class I is further split into Class Ia and Ib
based on their mechanism of activation. The Class Ia
PI3Ks are heterodimeric, consisting of a catalytic sub-
unit (p110a, p110b or p110d) in complex with a regula-
tory subunit.2 There is a great deal of interest in PI3Ks
as cancer targets, particularly for the p110a isoform
which is mutated and/or over-expressed in more than
30% of tumours.3,4 Many of these cancer-specific muta-
tions lead to a gain in function which results in oncoge-
The exact roles of the individual isoforms are beginning
to emerge,9,10 and hence more potent and selective com-
pounds of this type would not only be useful as potential
drugs, but also as biological tools to help establish the
roles of individual PI3K isoforms in cellular systems.
Two inhibitors, wortmannin and LY-294002, have been
studied extensively; however, neither shows selectivity
within the Class I PI3K isoforms (Fig. 1). Wortmannin,
a fungal metabolite, has a low nanomolar IC50 against
PI3K and binds covalently in the ATP-binding pocket.
It is unstable in solution and has limited activity in
tumour xenograft models.11 LY-294002 is chemically
stable but inhibits only at micromolar concentrations.
nicity.4
A recent review notes that 22 different
chemotypes of PI3K inhibitors have been reported, with
several compounds approaching clinical trial.5
Phosphoinositide 3,4,5-triphosphate (PIP3), the product
of PI3K phosphorylation, recruits protein kinase B
(PKB) which in turn is phosphorylated by 3-phosphoino-
There is considerable interest in obtaining selective
inhibitors of the p110a isoform as novel cancer thera-
peutics; however, few selective inhibitors have been
Keywords: PI3K; p110a; Sulfonohydrazide.
Corresponding author. Tel.: +64 9 3737599; e-mail: j.kendall@
auckland.ac.nz
*
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.08.062

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J. D. Kendall et al. / Bioorg. Med. Chem. 15 (2007) 7677–7687
The syntheses of bromoisoquinolines 15a–c are depicted
in Scheme 2. Bromoacetophenones 8a and 8b were con-
verted to phenylpropionitriles 9a and 9b with TosMIC
under basic conditions.17 Borane reduction of the nitr-
iles followed by formylation afforded protected amines
11a and 11b in excellent yield. Cyclisation to isoquino-
lines 13a–c was achieved in two steps by heating with
P2O5 in polyphosphoric acid, followed by MnO2 oxida-
tion to install the remaining double bond. Oxidation of
the methyl group of 13a–c to the aldehyde proved to be
problematic. Direct oxidation with SeO2 was unsuccess-
ful and returned only unreacted starting material. It was
eventually achieved by radical bromination followed by
immediate substitution with NaOAc in one pot, then ba-
sic hydrolysis to afford alcohols 14a–c. The intermediate
bromides were unstable to isolation. Finally MnO2 oxi-
dation provided the required aldehydes 15a–c.
Figure 1. Structures of PI3K inhibitors.
Sulfonohydrazides 16a–p were prepared from the corre-
sponding aldehydes by one of three methods as depicted
in Scheme 3 and Table 1. Reaction of the aldehyde with
methylhydrazine followed by sulfonylation (Method A)
was used for the majority of the aldehydes. However in
our hands we found this method to be somewhat
capricious and irreproducible, which led us to investigate
alternative routes. More reliable methods were
condensation of the aldehyde with 2-methyl-5-nitro-
benzenesulfonohydrazide followed by methylation under
phase transfer conditions with iodomethane (Method B)
or with diazomethane under neutral conditions (Method
C). In all cases only one stereoisomer was observed.
reported in the literature. Imidazo[1,2-a]pyridines have
recently been demonstrated to show excellent p110a
selectivity.12–14 In particular, imidazo[1,2-a]pyridine 1
is reported to be a sub-nanomolar p110a inhibitor with
>100-fold selectivity over p110b and p110c. In addition
1 showed activity in a human cancer xenograft model.14
Thieno[3,2-d]pyrimidine 2 is also a very potent p110a
inhibitor (IC50 2.0 nM) with 8-fold selectivity over
p110b.15,16
The work we report here investigates the structure–activ-
ity relationship (SAR) around 1 exploring major changes
in the two-ring chromophore. We wanted to determine
whether the position of the two nitrogens in the ring sys-
tem was crucial and whether the ring size was important.
We then used that information to validate the molecular
modelling results to determine whether the model we
had was consistent with the observed data.
This also led to the development of a higher yielding
synthesis of sulfonohydrazide 1 (Scheme 3). The previ-
ous synthesis gave a 8% yield of 1 from aldehyde 17.14
We have improved this yield to 40% by reaction of 17
using Method C.
2.2. Biological activity
2. Results and discussion
The biological activity of the compounds was evaluated
using an isolated enzyme inhibition assay. All com-
pounds were assayed against the three Class I a PI3K
isoforms, and the results are summarised in Table 1.
2.1. Preparation of sulfonohydrazides
The sulfonohydrazides were prepared from the corre-
sponding heteroaryl aldehydes. Aldehyde 5 was pre-
pared by hydrogenation of the 6-membered ring of 3
followed by reoxidation of the benzylic alcohol with
MnO2 (Scheme 1). Benzo[c]isoxazole 7 was prepared
by reduction of carboxylic acid 6 via the benzylic
alcohol.
Removal of the bromo substituent of 1 (16a) gave a 100-
fold decrease in potency for p110a compared to 1, but
still retained selectivity over p110b and p110d. This
was deemed sufficient activity to allow use of unsubsti-
tuted (more easily accessible) chromophores for the ini-
tial comparative studies. Partial reduction of the
imidazo[1,2-a]pyridine ring (16b) and removal of the
pyridyl ring (16c) led to a complete loss of p110a activity
(IC50 > 10 lM against p110a), as did several heterocy-
clic replacements that still retained the 6,5-ring system
(16d–f, 16h, 16i). Benzo[c]isoxazole 16g possessed weak
activity against p110a (IC50 5.0 lM). Quinoline 16k
was only 2.7-fold less active than 16a, however moving
the position of the sulfonohydrazide group (16l) again
led to a complete loss of activity for p110a. The isomeric
isoquinoline 16m was the best unsubstituted hetereocy-
clic replacement tested, with a similar PI3K inhibition
profile to 16a.
Scheme 1. Reagents and conditions: (a) H2, Pd/C, MeOH; (b) MnO2,
CH2Cl2; (c) i—CDI, THF; ii—NaBH4; iii—MnO2.

J. D. Kendall et al. / Bioorg. Med. Chem. 15 (2007) 7677–7687
7679
t
Scheme 2. Reagents and conditions: (a) TosMIC, KOtBu, BuOH, DME; (b) i—BH3ÆSMe2, THF; ii—6 M HCl; (c) ethyl formate; (d) P2O5, PPA,
160 ꢁC; (e) MnO2, dioxane; (f) i—NBS, AIBN, benzene; ii—BHT; iii—NaOAc, DMF; iv—NaOH, MeOH; (g) MnO2, CH2Cl2.
mutation in p110a (as found in NZOV9) does not signif-
icantly affect the results. The compounds which are most
potent against p110a are also active in both cell lines (1,
16a, 16g, 16k, 16m, 16n). This would also suggest that
this series of compounds has good cell penetration.
Furthermore, 16a and 16m both inhibit cell proliferation
at <1 lM. Five compounds (16d, 16e, 16l, 16o and 16p)
all have activity in at least one of the two cell lines at
<10 lM, but do not possess activity against the PI3K
isoforms at the same concentration. This may be due
to effects on kinase targets other than PI3K.
We have developed a novel series of sulfonohydrazides.
Isoquinoline 16m shows good selectivity for p110a over
p110b and p110d, and in addition inhibits cell prolifera-
tion in two human cancer cell lines.
Scheme 3. Reagents and conditions: (a) i—methylhydrazine, EtOH;
ii—2-methyl-5-nitrobenzenesulfonyl chloride, NEt3, CH2Cl2; (b) i—2-
methyl-5-nitrobenzenesulfonohydrazide, MeOH; ii—MeI, PhMe3 N+Iꢁ,
15% aq NaOH, CH2Cl2; (c) i—2-methyl-5-nitrobenzenesulfonohydra-
zide, MeOH; ii—CH2N2, Et2O, THF.
2.3. Molecular modelling and discussion
We then investigated three bromo substituted isoquino-
lines analogues of 16m (16n–p) to establish whether a
similar dramatic increase in p110a activity to that seen
between 1 and 16a could be seen. The three regioisomers
were made because it was not immediately obvious from
a simple overlay of the structures which position on the
isoquinoline ring would be optimal. The 6-bromo iso-
mer 16n was the most active against p110a of these com-
pounds, but was 3-fold less active than 16m.
In order to rationalise the affinity and selectivity ob-
served for the lead structure 1, we studied its binding
mode within PI3K by means of molecular modelling.
The structure of p110a has not yet been experimentally
solved. However, the X-ray crystal structure of the
p110c isoform, alone or in complex with different li-
gands such as wortmannin and LY-294002, is available
in the Protein Data Bank (www.pdb.org),10,18,19 and the
p110a and p110c isoforms share good sequence identity
(ꢀ35%). The alignment of their sequences (Fig. 2a)
The compounds were also evaluated in a cellular assay
measuring inhibition of proliferation using two early
passage human cancer cell lines in a thymidine depletion
assay (Table 1). The NZB5 cell line was derived from a
brain tumour (medulloblastoma), and has the wild-type
gene for p110a. The NZOV9 cell line was derived from
an ovarian tumour (poorly differentiated endometrioid
adenocarcinoma), and contains a mutant p110a enzyme
with a single amino acid substitution in the kinase
domain (Y1021C).
clearly shows that residues lying in the active site region
˚
(within 15 A around the ATP-binding site, denoted by
*
in Fig. 2a) are mostly identical (coloured in cyan) or
similar (coloured in yellow). Only a few amino acids
are different (coloured in magenta). In addition to being
a highly potent inhibitor of p110a, 1 also exhibits good
inhibitory potency against p110c (IC50 76 nM).10 These
observations prompted us to investigate the binding of 1
within the p110c active site with a view to explain its
activity and selectivity profile.
There is overall good agreement between the two differ-
ent cell lines; there is at most a 2-fold difference between
NZB5 and NZOV9 for the same compound. Hence the
Compound 1 was docked within the p110c binding site
(PDB code: 2CHX) by means of the automated GOLD
program.20 In order to take into account protein

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J. D. Kendall et al. / Bioorg. Med. Chem. 15 (2007) 7677–7687
Table 1. Synthetic methods, and inhibition of PI3K isoforms and cell proliferation for compounds 1 and 16a–p
Methoda
PI3K IC50 (lM)
p110b
Cell line IC50 (lM)
NZB5 NZOV9
b
c
Compound
R
p110a
p110d
1
C
A
0.0078
0.34
0.91
0.069
0.35
0.066
0.68
N
N
N
16a
0.86
>10
>10
>10
>10
>10
5.0
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
5.4
N
N
N
16b
16c
16d
16e
16f
16g
16h
16i
A
A
A
A
A
C
A
A
A
A
B
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
3.6
10
10
>20
2.1
3.3
15
>20
1.2
Me
N
N
N
n.d.d
>20
3.3
N
Me
N
N
O
3.9
12
O
S
>10
>10
>10
2.3
8.5
>20
>20
1.5
3.0
0.89
5.1
6.6
5.7
>20
>20
0.8
16j
N
16k
16l
N
N
>10
0.80
2.2
6.0
16m
16n
16o
16p
B
0.50
4.2
N
N
C
C
C
9.0
Br
Br
>10
>10
>10
>10
11
Br
N
12
a Synthetic method from Scheme 3.
b Values are means of two experiments, variation between experiments is no more than 20%.
c Values are means of one to four experiments; variation between experiments is no more than 32%.
d n.d., not determined.
flexibility, the conformation with the highest score
(GoldScore) was further refined using the MINIMIZE
module as implemented in SYBYL version 7.3 (Tripos
¨
interactions which stabilise 1 within the cleft are de-
picted in Figure 2b. The imidazo[1,2-a]pyridine moiety
is deeply inserted in the ATP-binding site, interacting
through a T-shape interaction with the phenol of Tyr-
867. The heterocycle is further stabilised via a hydrogen
bond contact between N-1 of 1 and the backbone NH of
force field and Gasteiger–Huckel charges).21 The key

J. D. Kendall et al. / Bioorg. Med. Chem. 15 (2007) 7677–7687
7681
a
c
b
d
HIS (p110α)
e
HIS (p110α)
˚
Figure 2. (a) Sequence alignment between p110a and p110c isoforms. , Amino acid residue within 15 A of the ATP-binding site; cyan, conserved
*
residue; yellow, similar residue; magenta, different residue; (b) docking of 1 within the active site of p110c (carbon atom in cyan), in
magenta = histidine residue in p110a; (c) representation of lipophilic properties mapped onto the Connolly surface around p110c and 1 (picture made
using Molcad);21 (d) docking of 16n (carbon atom in cyan) and 16o (carbon atom in yellow) within the active site of p110c, in magenta = histidine
residue in p110a; (e) superimposition of 1 (carbon atom in green), 16n (carbon atom in cyan) and 16o (carbon atom in yellow) within the active site of
p110c. (Pictures made using Pymol.)22
Val-882. This interaction appears to be crucial as it is
observed in all p110c-inhibitor complexes de-
scribed.10,18,19 This is consistent with compounds 16d,
16e, 16h–j which do not possess a nitrogen at this
position available for hydrogen bonding being essen-
tially inactive (IC50s >10 lM for all Class Ia PI3Ks).
Compound 1 is also stabilised through hydrogen bond
interactions between the nitro group on the arylsulfonyl
moiety and the backbone NH of Asp-884 and Ala-885.
numbering) is not and could play a crucial role in selectiv-
ity. The Thr-886 in p110c corresponds to a histidine in
p110a. To evaluate the potential implication of this differ-
ence on the binding of 1, we virtually mutated Thr-886
into a histidine in the p110c-compound 1 complex (the
His residue is represented in magenta in Fig. 2b). A new
hydrogen bonding interaction between the histidine NH
and one of the oxygen atoms of the sulfonyl group of 1
could then be formed, which would further stabilise the
ligand in the p110a cavity. This additional interaction
could clearly account for the high selectivity of 1 against
p110a.
Aiming to identify key amino acids that could explain the
selectivity of 1 for p110a, we also analysed the non-con-
served residues between both isoforms (Fig. 2a in magen-
ta). Although most of these are situated far from the hinge
region, the residue in position 886 (according to the p110c
As expected, the bromine atom also plays a critical role
in the binding. The active site cavity is particularly

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J. D. Kendall et al. / Bioorg. Med. Chem. 15 (2007) 7677–7687
lipophilic (Fig. 2c), and the hydrophobic bromine atom
fits neatly in a lipophilic pocket.
sured in DMSO-d6. Low resolution mass spectra were
recorded on a Thermo Finnigan MSQ single quadrupole
mass spectrometer. Silica gel chromatography was per-
formed using 200–320 mesh silica gel obtained from
APS Finechem Ltd. THF was distilled from the sodium
benzophenone ketyl immediately before use. BHT is 2,6-
di-tert-butyl-4-methylphenol; TosMIC is p-toluenesulfo-
nylmethyl isocyanide. Yields have not been optimised.
We have also studied the binding of the bromoisoquino-
lines 16n and 16o with a view to rationalise the weak
p110a activity observed in that series compared to 1.
The docking of 16n and 16o in the p110c active site
revealed a slightly different binding conformation. Simi-
larly to 1, the critical hydrogen bond interaction between
the backbone NH of Val-882 and the N atom of the iso-
quinoline ring system is formed; however, the arylsulfonyl
moieties of 16n and16o are less deeply inserted in the ac-
tive site cavity (Fig. 2d). Importantly, in that orientation,
the hydrogen bond interaction between the histidine NH
(in p110a) and one oxygen atom of the sulfonyl group
could not be formed. Moreover, the favourable interac-
tions between the nitro group on the arylsulfonyl moiety
and the backbone NH of Asp-884 and Ala-885 are also
lost. Finally, the superimposition of 16n and 16o with 1
(Fig. 2e) clearly shows that in the isoquinoline series the
bromo substituent cannot adopt the same orientation as
observed with the imidazo[1,2-a]pyridine ring system.
All together, these differences probably account for the
weaker activity in this series compared to 1.
4.1.1. (5,6,7,8-Tetrahydroimidazo[1,2-a]pyridin-3-yl)meth-
anol (4). A solution of imidazo[1,2-a]pyridine-3-carbalde-
hyde23 3 (93 mg, 0.64 mmol) in MeOH (20 mL) was
hydrogenated at 40 psi in the presence of 10% Pd/C
(20 mg) at room temperature for 18 h. The reaction mix-
ture was filtered through Celite, washed with MeOH and
the solvent removed from the filtrate in vacuo to leave 4
1
as a white solid (91 mg, 94%). H NMR d (400 MHz,
CDCl3) 6.89 (s, 1H), 4.59 (s, 2H), 3.95–4.00 (m, 2H),
2.84–2.89 (m, 2H), 1.86–2.05 (m, 4H). LC–MS (APCI+)
153 (MH+, 100%).
4.1.2. 5,6,7,8-Tetrahydroimidazo[1,2-a]pyridine-3-carbal-
dehyde (5). MnO2 (520 mg, 6.0 mmol) was added to a
solution of 4 (91 mg, 0.60 mmol) in CH2Cl2 (10 mL)
and stirred at room temperature for 18 h. The reaction
mixture was filtered through Celite, washed with
CH2Cl2 and the solvent removed from the filtrate in va-
cuo to leave 5 as a white solid (85 mg, 94%). 1H NMR d
(400 MHz, CDCl3) 9.66 (s, 1H), 7.71 (s, 1H), 4.29–4.35
(m, 2H), 2.92–2.97 (m, 2H), 1.88–2.04 (m, 4H). LC–
MS (APCI+) 151 (MH+, 100%).
From this model it becomes evident that none of the iso-
quinolines (16n, 16o, 16p) can place a bromo substituent
in the same position as does the imidazo[1,2-a]pyridine,
thus accounting for the lack of p110a activity for the
bromoisoquinolines.
3. Conclusion
4.1.3. Benzo[c]isoxazole-3-carbaldehyde (7). A mixture of
benzo[c]isoxazole-3-carboxylic acid24 6 (3.31 g, 20 mmol)
and 1,10-carbonyldiimidazole (4.86 g, 30 mmol) in dry
THF (200 mL) was stirred at room temperature for
12 h, when a clear solution was obtained. The solution
was added to a stirred solution of sodium borohydride
(3.4 g, 90 mmol) in water (100 mL) at room temperature.
After 15 min the mixture was treated dropwise with concd
HCl until hydrogen evolution ceased. The THF was re-
moved in vacuo and the residue extracted with EtOAc,
washed with dilute ammonia solution and dried
(Na2SO4). MnO2 (5 g) was added and the mixture was
heated under reflux for 3 h. After filtering, the solvent
was removed and the residue was chromatographed on
silica, eluting with CH2Cl2, to give 1.03 g (44%) of 7. 1H
NMR d (400 MHz, CDCl3) 10.38 (s, 1H), 8.02 (br d, J
8.8 Hz, 1H), 7.78 (br d, J 9.1 Hz, 1H), 7.45 (ddd, J 9.1,
6.5, 0.9 Hz, 1H), 7.33 (ddd, J 8.8, 6.5, 0.6 Hz, 1H).
A range of heterocyclic analogues of 1 have been prepared
and were evaluatedagainsttheClass IaPI3Kisoforms. Iso-
quinoline 16m exhibited selectivity for p110a over p110b
and p110d. The compounds were also assayed for inhibi-
tion of cell proliferation in two different human cancer cell
lines, and furthermore compounds with good p110a
potency also showed good activity in the cellular assay.
Modelling of 1 in the active site of p110c helps to rational-
ise the observed SAR. In particular, a suitably positioned
nitrogen on the heterocycle available for hydrogen bond-
ing is required, and the bromine of 1 fits neatly into a lipo-
philic pocket to provide significant stabilisation of the
complex. Mutation of Thr-886 in p110c to His, as it would
appear in p110a, provides a rationalisation for the ob-
served selectivity of this series of compounds where the
sulfonyl group is now involved in a hydrogen bond with
the histidine. Further work in this area is ongoing and will
be reported in future publications.
4.1.4. 2-(3-Bromophenyl)propanenitrile (9a). A solution
t
of KOtBu (2.26 g, 20.1 mmol) in dry BuOH (10 mL)
was added to a solution of 30-bromoacetophenone 8a
(2.00 g, 10.0 mmol) and TosMIC (2.35 g, 12.0 mmol)
in dry 1,2-dimethoxyethane (30 mL) under an atmo-
sphere of N2, while cooled in an ice bath, at a rate to en-
sure the reaction temperature remained <10 ꢁC. After
1 h the ice bath was removed, the reaction mixture stir-
red at room temperature for a further 1 h, then diluted
with water and extracted twice with hexanes. The organ-
ic extracts were combined, dried (Na2SO4) and the sol-
vent removed in vacuo. The crude product was
4. Experimental
4.1. Chemistry
NMR spectra were recorded on a Bruker Avance 400
spectrometer; chemical shifts are reported in d using
SiMe4 as the internal standard when measured in
CDCl3, and the residual DMSO as standard when mea-

J. D. Kendall et al. / Bioorg. Med. Chem. 15 (2007) 7677–7687
7683
distilled on a Kugelrohr apparatus (oven temperature
130 ꢁC, oil pump) to afford 9a as a colourless oil
acid (13.4 g) were heated to 160 ꢁC, then P2O5 (3.14 g,
11.1 mmol) was added and the resulting mixture heated
at 160 ꢁC for 18 h. After cooling to room temperature,
ice was added and the mixture stirred with a glass rod until
all of the ice was melted. The resulting solution was made
basic with 6 M aqueous NaOH then extracted three times
with CH2Cl2. The combined extracts were dried (Na2SO4)
and the solvent removed in vacuo. Column chromatogra-
phy, eluting with hexanes/EtOAc 9:1 to 4:1 to 3:1 gave
first 12c as a yellow oil (468 mg, 38%). 1H NMR d
(400 MHz, CDCl3) 8.70 (m, 1H), 7.48 (d, J 7.8 Hz, 1H),
7.14–7.25 (m, 2H), 3.79 (ddd, J 16.1, 6.1, 2.3 Hz, 1H),
3.60 (ddd, J 16.1, 8.1, 2.2 Hz, 1H), 2.85 (m, 1H), 1.23 (d,
J 7.0 Hz, 3H). LC–MS (APCI+) 224 (MH+ with 79Br,
100%), 226 (MH+ with 81Br, 100%). Second to elute was
1
(1.24 g, 59%). H NMR d (400 MHz, CDCl3) 7.51 (m,
1H), 7.47 (m, 1H), 7.24–7.33 (m, 2H), 3.87 (q, J
7.3 Hz, 1H), 1.65 (d, J 7.3 Hz, 3H).
4.1.5. 2-(4-Bromophenyl)propanenitrile (9b). Compound
9b was prepared from 40-bromoacetophenone 8b by the
same procedure as that of 9a. Compound 9b was obtained
1
as a colourless oil (2.30 g, 73%). H NMR d (400 MHz,
CDCl3) 7.52 (d, J 8.4 Hz, 2H), 7.24 (d, J 8.4 Hz, 2H),
3.86 (q, J 7.3 Hz, 1H), 1.63 (d, J 7.3 Hz, 3H).
4.1.6. 2-(3-Bromophenyl)propan-1-amine (10a). BH3ÆSMe2
(1.77 mL, 17.7 mmol) was added to a solution of 9a
(1.24 g, 5.90 mmol) in dry THF (15 mL) at room tempera-
tureunder anatmosphereof N2, and thenrefluxed for 18 h.
The reaction was quenched by the dropwise addition of
6 M aqueous HCl (4 mL). After refluxing for a further
2 h, the solution was made basic with 6 M aqueous NaOH
then extracted three times with CH2Cl2. The combined
extracts were dried (Na2SO4) and the solvent removed in
vacuo. Column chromatography, eluting with CH2Cl2/
MeOH/concd aqueous NH3 99:1:0.1 to 98:2:0.2 to
1
12a, obtained as an off-white solid (618 mg, 50%). H
NMR d (400 MHz, CDCl3) 8.30 (m, 1H), 7.45 (dd, J
8.0, 1.8 Hz, 1H), 7.39 (d, J 1.8 Hz, 1H), 7.30 (d, J
8.0 Hz, 1H), 3.82 (ddd, J 16.2, 6.2, 2.2 Hz, 1H), 3.52
(ddd, 16.2, 9.0, 2.3 Hz, 1H), 2.88 (m, 1H), 1.25 (d, J
7.0 Hz, 3H). LC–MS (APCI+) 224 (MH+ with 79Br,
90%), 226 (MH+ with 81Br, 100%).
4.1.11. 7-Bromo-4-methyl-3,4-dihydroisoquinoline (12b).
Compound 12b was prepared from 11b by the same pro-
cedure as that of 12a. Compound 12b was obtained as a
brown oil (914 mg, 99%). 1H NMR d (400 MHz, CDCl3)
8.28 (dd, J 2.2, 2.2 Hz, 1H), 7.52 (dd, J 8.1, 2.0 Hz, 1H),
7.42 (d, J 2.0 Hz, 1H), 7.12 (d, J 8.1 Hz, 1H), 3.83 (ddd,
J 16.3, 6.2, 2.2 Hz, 1H), 3.56 (ddd, 16.3, 8.7, 2.2 Hz,
1H), 2.85 (m, 1H), 1.23 (d, J 7.0 Hz, 3H). LC–MS
(APCI+) 224 (MH+ with 79Br, 90%), 226 (MH+ with
81Br, 100%).
1
97:3:0.3 gave 10a as a colourless oil (1.18 g, 94%). H
NMR d (400 MHz, CDCl3) 7.32–7.37 (m, 2H), 7.10–7.20
(m, 2H), 2.63–2.88 (m, 3H), 1.24 (d, J 6.9 Hz, 3H). LC–
MS (APCI+) 214 (MH+ with 79Br, 85%), 216 (MH+ with
81Br, 100%).
4.1.7.2-(4-Bromophenyl)propan-1-amine(10b).Compound
10b was prepared from 9b by the same procedure as that of
10a. Compound 10b was obtained as a colourless oil
1
(1.99 g, 86%). H NMR d (400 MHz, CDCl3) 7.43 (d, J
8.4 Hz, 2H), 7.09 (d, J 8.4 Hz, 2H), 2.67–2.88 (m, 3H),
1.23 (d, J 6.9 Hz, 3H). LC–MS (APCI+) 214 (MH+ with
79Br, 90%), 216 (MH+ with 81Br, 100%).
4.1.12. 6-Bromo-4-methylisoquinoline (13a). Compound
12a (618 mg, 2.76 mmol) and MnO2 (3.60 g, 41.4 mmol)
were refluxed in dioxane (50 mL) for 18 h, then filtered
through a plug of Celite and washed with CH2Cl2.
The solvent was removed from the filtrate in vacuo. Col-
umn chromatography, eluting with hexanes/EtOAc 9:1
4.1.8. N-(2-(3-Bromophenyl)propyl)formamide (11a). Com-
pound 10a (1.18 g, 5.51 mmol) was refluxed in ethyl
formate (2.3 mL, 28 mmol) for 40 h. After cooling to room
temperature, the solvent was removed in vacuo to afford
11a as a colourless oil (1.33 g, 100%). 1H NMR d
(400 MHz, CDCl3) 8.12 (s, 0.85H, major rotamer), 7.93
(d, J 11.9 Hz, 0.15H, minor rotamer), 7.31–7.40 (m, 2H),
7.08–7.23 (m, 2H), 5.20–5.50 (m, 1H), 3.62–3.72 (m, 1H),
3.22–3.38 (m, 1H), 2.81–3.01 (m, 1H), 1.25–1.31 (m, 3H).
LC–MS (APCI+) 242 (MH+ with 79Br, 100%), 244 (MH+
with 81Br, 90%).
1
to 85:15 gave 13a as a yellow oil (212 mg, 35%). H
NMR d (400 MHz, CDCl3) 9.09 (s, 1H), 8.40 (s, 1H),
8.12 (d, J 1.8 Hz, 1H), 7.84 (d, J 8.7 Hz, 1H), 7.70 (dd,
J 8.7, 1.8 Hz, 1H), 2.59 (s, 3H). LC–MS (APCI+) 222
(MH+ with 79Br, 100%), 224 (MH+ with 81Br, 95%).
4.1.13. 7-Bromo-4-methylisoquinoline (13b). Compound
13b was prepared from 12b by the same procedure as
that of 13a. Compound 13b was obtained as a yellow so-
1
lid (342 mg, 38%). H NMR d (400 MHz, CDCl3) 9.04
4.1.9. N-(2-(4-Bromophenyl)propyl)formamide (11b). Com-
pound 11b was prepared from 10b by the same procedure
as that of 11a. Compound 11b was obtained as a colourless
oil (2.25 g, 100%). 1H NMR d (400 MHz, CDCl3) 8.10 (s,
0.85H, major rotamer), 7.90 (d, J 11.9 Hz, 0.15H, minor
rotamer), 7.42–7.47 (m, 2H), 7.01–7.13 (m, 2H), 5.25–
5.40 (m, 1H), 3.59–3.74 (m, 1H), 3.21–3.40 (m, 1H),
2.81–2.99 (m, 1H), 1.24–1.29 (m, 3H). LC–MS (APCI+)
242 (MH+ with 79Br, 100%), 244 (MH+ with 81Br, 90%).
(s, 1H), 8.40 (s, 1H), 8.13 (d, J 1.9 Hz, 1H), 7.84 (d, J
8.9 Hz, 1H), 7.80 (dd, J 8.9, 1.9 Hz, 1H), 2.62 (s, 3H).
LC–MS (APCI+) 222 (MH+ with 79Br, 90%), 224
(MH+ with 81Br, 100%).
4.1.14. 8-Bromo-4-methylisoquinoline (13c). Compound
13c was prepared from 12c by the same procedure as
that of 13a. Compound 13c was obtained as a white so-
1
lid (198 mg, 43%). H NMR d (400 MHz, CDCl3) 9.51
(s, 1H), 8.46 (s, 1H), 7.93 (d, J 8.4 Hz, 1H), 7.85 (d, J
7.5 Hz, 1H), 7.56 (dd, J 8.4, 7.5 Hz, 1H), 2.64 (s, 3H).
LC–MS (APCI+) 222 (MH+ with 79Br, 100%), 224
(MH+ with 81Br, 95%).
4.1.10. 6-Bromo-4-methyl-3,4-dihydroisoquinoline (12a)
and 8-bromo-4-methyl-3,4-dihydroisoquinoline (12c). A
mixture of 11a (1.33 g, 55.0 mmol) and polyphosphoric

7684
J. D. Kendall et al. / Bioorg. Med. Chem. 15 (2007) 7677–7687
4.1.15. (6-Bromoisoquinolin-4-yl)methanol (14a). A solu-
tion of 13a (212 mg, 0.95 mmol), NBS (187 mg,
1.05 mmol) and AIBN (16 mg, 0.097 mmol) in benzene
(25 mL) was refluxed under an atmosphere of N2 for
1 h. After cooling to room temperature, 2,6-di-tert-bu-
tyl-4-methylphenol (105 mg, 0.48 mmol) was added
and the solution stirred further 30 min. The reaction
was then diluted with DMF (25 mL), then NaOAc
(392 mg, 4.78 mmol) was added and the solution heated
at 60 ꢁC for 1 h. The solvent was removed in vacuo, the
residue taken up in MeOH (40 mL) and 1 M aqueous
NaOH (20 mL) and refluxed for 1 h. The MeOH was re-
moved in vacuo, the aqueous residue neutralised with
1 M aqueous HCl, extracted twice with CH2Cl2, the
combined extracts were dried (Na2SO4) and the solvent
removed in vacuo. Column chromatography, eluting
with hexanes/EtOAc 2:1 to 1:1 to EtOAc gave 14a as a
yellow solid (76 mg, 33%). 1H NMR d (400 MHz,
CDCl3) 9.19 (s, 1H), 8.54 (s, 1H), 8.36 (s, 1H), 7.88 (d,
J 8.7 Hz, 1H), 7.74 (d, J 8.7 Hz, 1H), 5.09 (s, 2H).
LC–MS (APCI+) 238 (MH+ with 79Br, 95%), 240
(MH+ with 81Br, 100%).
4.1.20. 8-Bromoisoquinoline-4-carbaldehyde (15c). Com-
pound 15c was prepared from 14c by the same proce-
dure as that of 15a. Compound 15c was obtained as
an off-white solid (43 mg, 60%). 1H NMR
d
(400 MHz, CDCl3) 10.41 (s, 1H), 9.87 (s, 1H), 9.22 (d,
J 8.6 Hz, 1H), 9.02 (s, 1H), 7.98 (d, J 7.6 Hz, 1H),
7.74 (dd, J 8.6, 7.6 Hz, 1H). LC–MS (APCI+) 236
(MH+ with 79Br, 75%), 238 (MH+ with 81Br, 75%),
268 (MH+ with 79Br + MeOH, 100%), 270 (MH+ with
81Br + MeOH, 85%).
4.1.21. N0-((6-Bromoimidazo[1,2-a]pyridin-3-yl)methylene)-
N,2-dimethyl-5-nitrobenzenesulfonohydrazide (1) Method C.
A mixture of 6-bromoimidazo[1,2-a]pyridine-3-carbal-
dehyde14 17 (2.25 g, 10 mmol) and 2-methyl-5-nitro-
benzenesulfonohydrazide25 (3.0 g, 13 mmol) in MeOH
(200 mL) was stirred at room temperature for 4 h
to give a precipitate that was collected, washed
with MeOH and dried to give N0-((6-bromoimi-
dazo[1,2-a]pyridin-3-yl)methylene)-2-methyl-5- nitro-
benzenesulfonohydrazide (2.11 g, 48%). 1H NMR d
(400 MHz, DMSO-d6) 12.16 (br, exchangeable with
D2O, 1H), 9.00 (dd, J 2.0, 0.7 Hz, 1H), 8.76 (d, J
2.5 Hz, 1H), 8.41 (dd, J 8.4, 2.5 Hz, 1H), 8.30 (s, 1H),
8.04 (s, 1H), 7.75 (d, J 8.5 Hz, 1H), 7.69 (dd, J 9.5,
0.7 Hz, 1H), 7.55 (dd, J 9.5, 2.0 Hz, 1H), 2.77 (s, 3H).
A solution of the crude hydrazone (1.30 g, 3 mmol) in
THF (150 mL) was treated with excess CH2N2 in ether
until the yellow colour persisted. The solvent was
removed in vacuo and the residue recrystallized from
ethanol to give 1 as a yellow solid (1.12 g, 83%).
4.1.16. (7-Bromoisoquinolin-4-yl)methanol (14b). Com-
pound 14b was prepared from 13b by the same proce-
dure as that of 14a. Compound 14b was obtained as a
yellow solid (89 mg, 32%). 1H NMR d (400 MHz,
CDCl3) 9.15 (s, 1H), 8.55 (s, 1H), 8.17 (d, J 2.0 Hz,
1H), 8.07 (d, J 9.0 Hz, 1H), 7.84 (dd, J 9.0, 2.0 Hz,
1H), 5.12 (s, 2H). LC–MS (APCI+) 238 (MH+ with
79Br, 100%), 240 (MH+ with 81Br, 95%).
1
Mp 221–223 ꢁC. H NMR d (400 MHz, CDCl3) 9.03
4.1.17. (8-Bromoisoquinolin-4-yl)methanol (14c). Com-
pound 14c was prepared from 13c by the same procedure
as that of 14a. Compound 14a was obtained as an off-
white solid (52 mg, 42%). 1H NMR d (400 MHz, CDCl3)
9.63 (s, 1H), 8.62 (s, 1H), 8.30 (d, J 8.3 Hz, 1H), 7.89 (d, J
7.5 Hz, 1H), 7.60 (dd, J 8.3, 7.5 Hz, 1H), 5.14 (d, J
5.8 Hz, 2H), 1.79 (t, J 5.8 Hz, 1H). LC–MS (APCI+)
238 (MH+ with 79Br, 95%), 240 (MH+ with 81Br, 100%).
(d, J 2.4 Hz, 1H), 9.01 (dd, J 2.0, 0.8 Hz, 1H), 8.35
(dd, J 8.4, 2.4 Hz, 1H), 7.95 (s, 1H), 7.86 (s, 1H),
7.55 (d, J 8.9 Hz, 1H), 7.53 (d, J 7.7 Hz, 1H), 7.38
(dd, J 9.5 Hz, 1H), 3.51 (s, 3H), 2.73 (s, 3H). LC–MS
(APCI+) 452 (MH+ with 79Br, 95%), 454 (MH+ with
81Br, 100%). Anal. Calcd for C16H14BrN5O4S: C,
42.49; H, 3.12; N, 15.48. Found: C, 42.65; H, 3.15;
N, 15.52.
4.1.18. 6-Bromoisoquinoline-4-carbaldehyde (15a). Com-
pound 14a (74 mg, 0.31 mmol) and MnO2 (541 mg,
6.22 mmol) were stirred in CH2Cl2 (20 mL) for 3 days,
then filtered through a plug of Celite and the solvent
removed from the filtrate in vacuo to afford 15a as a
yellow solid (36 mg, 49%). 1H NMR d (400 MHz,
CDCl3) 10.35 (s, 1H), 9.45 (d, J 1.8 Hz, 1H), 9.40
(s, 1H), 8.96 (s, 1H), 7.94 (d, J 8.7 Hz, 1H), 7.84
(dd, J 8.7, 1.8 Hz, 1H). LC–MS (APCI+) 236 (MH+
with 79Br, 70%), 238 (MH+ with 81Br, 85%), 268
(MH+ with 79Br + MeOH, 100%), 270 (MH+ with
81Br + MeOH, 90%).
4.1.22. N0-(Imidazo[1,2-a]pyridin-3-ylmethylene)-N,2-
dimethyl-5-nitrobenzenesulfonohydrazide (16a) Method
A. Methylhydrazine (19 mg, 0.41 mmol) was added to
a solution of imidazo[1,2-a]pyridine-3-carbaldehyde23
(40 mg, 0.27 mmol) in EtOH (2 mL). After 6 h at room
temperature, the solvent was removed in vacuo. The
residue was taken up in CH2Cl2 (5 mL), then NEt3
(0.07 mL, 0.50 mmol) and 2-methyl-5-nitrobenzene-
sulfonyl chloride (129 mg, 0.55 mmol) were added. After
2 h, water was added. The layers were separated, the
aqueous layer was extracted with CH2Cl2, the
combined organic layers dried (Na2SO4) and the solvent
removed in vacuo. Column chromatography, eluting
with hexanes/EtOAc 1:1 to EtOAc gave 16a as a yel-
4.1.19. 7-Bromoisoquinoline-4-carbaldehyde (15b). Com-
pound 15b was prepared from 14b by the same procedure
as that of 15a. Compound 15b was obtained as a yellow
solid (54 mg, 61%). 1H NMR d (400 MHz, CDCl3)
10.37 (s, 1H), 9.36 (s, 1H), 9.11 (d, J 9.1 Hz, 1H), 8.96
(s, 1H), 8.24 (d, J 2.0 Hz, 1H), 7.98 (dd, J 9.1, 2.0 Hz,
1H). LC–MS (APCI+) 236 (MH+ with 79Br, 50%), 238
(MH+ with 81Br, 45%), 268 (MH+ with 79Br + MeOH,
90%), 270 (MH+ with 81Br + MeOH, 100%).
1
low solid (26 mg, 25%). H NMR d (400 MHz, CDCl3)
9.06 (d, J 2.4 Hz, 1H), 8.98 (m, 1H), 8.31 (dd, J 8.4,
2.4 Hz, 1H), 7.97 (s, 1H), 7.87 (s, 1H), 7.68 (m, 1H),
7.52 (d, J 8.4 Hz, 1H), 7.36 (m, 1H), 6.93 (m, 1H),
3.49 (s, 3H), 2.74 (s, 3H). LC–MS (APCI+) 374
(MH+, 100%). Anal. Calcd for C16H15N5O4SÆ
0.15EtOAc: C, 51.57; H, 4.22; N, 18.11. Found: C,
51.62; H, 4.23; N, 18.12.

J. D. Kendall et al. / Bioorg. Med. Chem. 15 (2007) 7677–7687
7685
4.1.23. N,2-Dimethyl-5-nitro-N0-((5,6,7,8-tetrahydroim-
idazo[1, 2-a]pyridin-3-yl)methylene)benzenesulfonohyd-
razide (16b). Compound 16b was prepared from 5 by
Method A. Compound 16b was obtained as a yellow so-
lid (44 mg, 42%). 1H NMR d (400 MHz, CDCl3) 8.91 (d,
J 2.4 Hz, 1H), 8.30 (dd, J 8.4, 2.4 Hz, 1H), 7.60 (s, 1H),
7.51 (d, J 8.4 Hz, 1H), 7.19 (s, 1H), 3.86–3.93 (m, 2H),
3.38 (s, 3H), 2.83–2.87 (m, 2H), 2.72 (s, 3H), 1.77–1.91
(m, 4H). LC–MS (APCI+) 378 (MH+, 100%). Anal.
Calcd for C16H19N5O4SÆ0.25EtOAc: C, 51.12; H, 5.30;
N, 17.53. Found: C, 51.26; H, 5.25; N, 17.78.
obtained as a yellow solid (300 mg, 41%). Mp 168–
170 ꢁC. 1H NMR d (400 MHz, CDCl3) 9.04 (d, J
2.4 Hz, 1H), 8.32 (dd, J 8.4, 2.4 Hz, 1H), 7.92 (s, 1H),
7.61 (br d, J 8.8 Hz, 1H), 7.56 (br d, J 9.1 Hz, 1H), 7.51
(br d, J 8.4 Hz, 1H), 7.32 (ddd, J 9.1, 6.5, 0.8 Hz, 1H),
7.05 (dd, J 8.7, 6.5 Hz, 1H), 3.55 (s, 3H), 2.76 (s, 3H).
LC–MS (APCI+) 375 (MH+, 100%). Anal. Calcd for
C16H14N4O5S: C, 51.33; H, 3.77; N, 14.97; Found: C,
51.45; H, 3.77; N, 14.93.
4.1.29. N0-(Benzofuran-3-ylmethylene)-N,2-dimethyl-5-
nitrobenzenesulfonohydrazide (16h). Compound 16h was
prepared from benzofuran-3-carbaldehyde28 by Method
A. Compound 16h was obtained as a yellow solid
4.1.24. N,2-Dimethyl-N0-((1-methyl-1H-imidazol-5-yl)meth-
ylene)-5-nitrobenzenesulfonohydrazide (16c). Compound
16c was prepared from 1-methyl-1H-imidazole-5-carbal-
dehyde26 by Method A. Compound 16c was obtained as
ayellowsolid(43 mg, 16%). 1HNMRd(400 MHz,CDCl3)
8.89 (d, J 2.3 Hz, 1H), 8.30 (dd, J 8.4, 2.3 Hz, 1H), 7.63 (s,
1H), 7.51 (d, J 8.4 Hz, 1H), 7.41 (s, 1H), 7.26 (s, 1H), 3.64
(s, 3H), 3.42 (s, 3H), 2.72 (s, 3H). LC–MS (APCI+) 338
(MH+, 100%). Anal. Calcd for C13H15N5O4S: C, 46.28;
H, 4.48; N, 20.76. Found: C, 46.16; H, 4.55; N, 20.46.
1
(292 mg, 76%). H NMR d (400 MHz, CDCl3) 8.53 (d,
J 2.4 Hz, 1H), 8.28 (dd, J 8.4, 2.4 Hz, 1H), 7.56 (s, 1H),
7.36–7.44 (m, 3H), 7.16–7.32 (m, 3H), 3.86 (s, 3H), 2.56
(s, 3H). LC–MS (APCI+) 374 (MH+, 100%). Anal. Calcd
for C17H15N3O5S: C, 54.68; H, 4.05; N, 11.25. Found: C,
54.86; H, 4.03; N, 11.16.
4.1.30. N0-(Benzo[b]thiophen-3-ylmethylene)-N,2-dimethyl-
5-nitrobenzenesulfonohydrazide (16i). Compound 16i was
prepared from benzo[b]thiophene-3-carbaldehyde by
Method A. Compound 16i was obtained as an orange solid
(126 mg, 53%). 1H NMR d (400 MHz, CDCl3) 9.08 (d, J
2.4 Hz, 1H), 8.29 (dd, J 8.4, 2.4 Hz, 1H), 8.22 (d, J
8.0 Hz, 1H), 7.91 (s, 1H), 7.80 (d, J 8.0 Hz, 1H), 7.61 (s,
1H), 7.47 (d, J 8.4 Hz, 1H), 7.27–7.39 (m, 2H), 3.48 (s,
3H), 2.74 (s, 3H). LC–MS (APCI+) 390 (MH+, 100%).
Anal. Calcd for C17H15N3O4S2: C, 52.43; H, 3.88; N,
10.79. Found: C, 52.68; H, 3.90; N, 10.82.
4.1.25. N0-(Imidazo[1,5-a]pyridin-3-ylmethylene)-N,2-di-
methyl-5-nitrobenzenesulfonohydrazide (16d). Compound
16d was prepared from imidazo[1,5-a]pyridine-3-carbal-
dehyde27 by Method A. Compound 16d was obtained
1
as an orange solid (35 mg, 25%). H NMR d (400 MHz,
CDCl3) 9.07 (d, J 2.4 Hz, 1H), 8.85 (m, 1H), 8.30 (dd, J
8.4, 2.4 Hz, 1H), 8.02 (s, 1H), 7.49–7.58 (m, 3H), 6.93
(ddd, J 9.1, 6.6, 0.9 Hz, 1H), 6.74 (m, 1H), 3.51 (s, 3H),
2.73 (s, 3H). LC–MS (APCI+) 374 (MH+, 100%). Anal.
Calcd for C16H15N5O4S: C, 51.47; H, 4.05; N, 18.76.
Found: C, 51.70; H, 4.18; N, 18.65.
4.1.31. N,2-Dimethyl-N0-(naphthalen-1-ylmethylene)-5-nitro-
benzenesulfonohydrazide (16j). Compound 16j was
prepared from 1-naphthaldehyde by Method A. Com-
pound 16j was obtained as a yellow solid (272 mg, 74%).
1H NMR d (400 MHz, DMSO-d6) 8.75 (d, J 2.5 Hz,
1H), 8.55 (d, J 8.5 Hz, 1H), 8.39–8.44 (m, 2H), 7.92–
7.97 (m, 2H), 7.73–7.78 (m, 2H), 7.51–7.56 (m, 2H), 7.41
(m, 1H), 3.54 (s, 3H), 2.72 (s, 3H). LC–MS (APCI+) 384
(MH+, 100%). Anal. Calcd for C19H17N3O4S: C, 59.52;
H, 4.47; N, 10.96. Found: C, 59.69; H, 4.66; N, 11.06.
4.1.26. N0-(Imidazo[1,5-a]pyridin-1-ylmethylene)-N,2-di-
methyl-5-nitrobenzenesulfonohydrazide (16e). Compound
16e was prepared from imidazo[1,5-a]pyridine-1-carbal-
dehyde27 by Method A. Compound 16e was obtained as
1
an orange solid (127 mg, 34%). H NMR d (400 MHz,
CDCl3) 9.04 (d, J 2.4 Hz, 1H), 8.27 (dd, J 8.4, 2.4 Hz,
1H), 8.03 (s, 1H), 7.99 (s, 1H), 7.93 (m, 1H), 7.80 (m,
1H), 7.49 (d, J 8.4 Hz, 1H), 6.91 (ddd, J 9.2, 6.5,
0.9 Hz, 1H), 6.70 (m, 1H), 3.44 (s, 3H), 2.75 (s, 3H).
LC–MS (APCI+) 374 (MH+, 100%). Anal. Calcd for
C16H15N5O4S: C, 51.47; H, 4.05; N, 18.76. Found: C,
51.78; H, 4.12; N, 18.91.
4.1.32. N,2-Dimethyl-5-nitro-N0-(quinolin-4-ylmethylene)-
benzenesulfonohydrazide (16k). Compound 16k was pre-
pared from quinoline-4-carbaldehyde by Method A.
Compound 16k was obtained as an orange solid (87 mg,
1
4.1.27. N,2-Dimethyl-N0-((1-methyl-1H-indol-3-yl)methy-
lene)-5-nitrobenzenesulfonohydrazide (16f). Compound
16f was prepared from 1-methyl-1H-indole-3-carbalde-
hyde by Method A. Compound 16f was obtained as an or-
ange solid (113 mg, 36%). 1H NMR d (400 MHz, CDCl3)
9.01 (d, J 2.4 Hz, 1H), 8.28 (dd, J 8.4, 2.4 Hz, 1H), 8.08 (s,
1H), 7.86 (d, J 8.0 Hz, 1H), 7.46 (d, J 8.4 Hz, 1H), 7.25–
7.33 (m, 3H), 7.13 (m, 1H), 3.79 (s, 3H), 3.34 (s, 3H),
2.74 (s, 3H). LC–MS (APCI+) 387 (MH+, 100%). Anal.
Calcd for C18H18N4O4S: C, 55.95; H, 4.70; N, 14.50.
Found: C, 56.15; H, 4.74; N, 14.76.
24%). H NMR d (400 MHz, CDCl3) 9.01 (d, J 2.4 Hz,
1H), 8.91 (d, J 4.5 Hz, 1H), 8.37 (dd, J 8.5, 0.8 Hz, 1H),
8.31 (dd, J 8.4, 2.4 Hz, 1H), 8.13 (d, J 8.5, 0.7 Hz, 1H),
8.08 (s, 1H), 7.72 (m, 1H), 7.47–7.56 (m, 3H), 3.59 (s,
3H), 2.77 (s, 3H). LC–MS (APCI+) 385 (MH+, 100%).
Anal. Calcd for C18H16N4O4S: C, 56.24; H, 4.20; N,
14.57. Found: C, 56.50; H, 4.20; N, 14.57.
4.1.33. N,2-Dimethyl-5-nitro-N0-(quinolin-3-ylmethylene)-
benzenesulfonohydrazide (16l) Method B. A mixture of
quinoline-3-carbaldehyde (157 mg, 1 mmol) and of 2-
methyl-5-nitrobenzenesulfonohydrazide25 (230 mg, 1 mmol)
inMeOH(5 mL)wasstirredatroomtemperaturefor2 hto
give a precipitate of 0.32 g (86% yield) of 2-methyl-5-nitro-
N0-(quinolin-3-ylmethylene)benzenesulfonohydrazide.
4.1.28. N0-(Benzo[c]isoxazol-3-ylmethylene)-N,2-dimeth-
yl-5-nitrobenzenesulfonohydrazide (16g). Compound 16g
was prepared from 7 by Method C. Compound 16g was

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J. D. Kendall et al. / Bioorg. Med. Chem. 15 (2007) 7677–7687
The crude hydrazide was combined with a mixture of
iodomethane (0.25 g), phenyltrimethylammonium iodide
(25 mg), 15% NaOH (3 mL) and CH2Cl2 (5 mL), and the
two-phase solution was stirred overnight at room temper-
ature. The organic layer was separated, dried (Na2SO4)
and eluted through a column of alumina to remove polar
impurities. The solvent was removed and the residue was
recrystallized from MeOH to give 16l as white crystals
8.4 Hz, 1H), 3.58 (s, 3H), 2.75 (s, 3H). LC–MS (APCI+)
463 (MH+ with 79Br, 90%), 465 (MH+ with 81Br, 100%).
Anal. Calcd for C18H15BrN4O4S: C, 46.66; H, 3.26; N,
12.09. Found: C, 46.74; H, 3.42; N, 11.96.
4.2. Enzyme assays
The Class Ia PI3K assays were performed using a basic
thin-layer chromatography technique, as described previ-
ously.9 The p110d/p85 PI3K was from Upstate and the
p110a/p85 and p110b/p85 isoforms were prepared as de-
scribed previously.9 Reactions were made containing
0.1 lg recombinant enzyme, 10 lg L-a-phosphatidylinosi-
tol, inhibitor (DMSO only or DMSO + inhibitor to a final
concentration of 1%), 2· Lipid Kinase Buffer (40 mM
Tris–HCl, pH 7.4, 200 mM NaCl, 1 mM EDTA) and acti-
vated upon the addition of an ATP mix (5 mM MgCl2,
100 lM ATP, 0.1 lL [c33-P]ATP). Reactions were
incubated at room temperature for 1 h following which
the reactions were stopped by the addition of 1 M HCl.
The lipids were then extracted using a two step procedure.
First, 200 lL of chloroform/methanol (1:1) was added, the
biphasic reactions mixed and centrifuged briefly and the
inorganic phase was removed and discarded. Following
this 80 lL of methanol/hydrochloric acid (1:1) was added
and the same procedure followed. Next, 70 lL of the or-
ganic phase was transferred to a clean 1.6 mL tube and
the reactions were dried using a speed vac, with no heating,
for 30 min. The reactions were spotted onto TLC plates
(Merck Ltd) and developed for 1 h in 1-propanol/2 M ace-
tic acid (13:7). The TLC plates were then dried at room
temperature and quantified using a phosphorimager
(StormImager, Amersham). Nine inhibitor concentrations
were used to determine the IC50. Each experiment was per-
formed twice and the average IC50 value used.
1
(148 mg, 45%). Mp 130–132 ꢁC. H NMR d (400 MHz,
CDCl3) 9.08 (d, J 2.1 Hz, 1H), 8.93 (d, J 2.4 Hz, 1H),
8.30 (dd, J 8.4, 2.4 Hz, 1H), 8.24 (d, J 2.0 Hz, 1H), 8.08
(br d, J 8.5 Hz, 1H), 7.83 (dd, J 8.1, 1.1 Hz, 1H), 7.76–
7.72 (m, 2H), 7.57 (td, J 7.5, 1.1 Hz, 1H), 7.50 (d, J
8.4 Hz, 1H), 3.50 (s, 3H), 2.85 (s, 3H). LC–MS (APCI+)
385 (MH+, 100%). Anal. Calcd for C18H16N4O4S: C,
56.24;H,4.20;N,14.57;Found:C, 56.03;H,4.28;N,14.33.
4.1.34. N0-(Isoquinolin-4-ylmethylene)-N,2-dimethyl-5-nitro-
benzenesulfonohydrazide (16m). Compound 16m was pre-
pared from isoquinoline-4-carbaldehyde29 by Method B.
Compound 16m was obtained as a white solid (32%). Mp
(MeOH) 160–162 ꢁC. 1H NMR d (400 MHz, CDCl3) 9.17
(s, 1H), 9.07 (d, J 2.4 Hz, 1H), 8.65 (d, J 8.3 Hz, 1H), 8.55
(s, 1H), 8.30 (dd, J 8.4, 2.4 Hz, 1H), 8.00–7.96 (m, 2H),
7.71–7.62 (m, 2H), 7.49 (d, J 8.4 Hz, 1H), 3.58 (s, 3H),
2.76 (s, 3H). LC–MS (APCI+) 385 (MH+, 100%). Anal.
Calcd for C18H16N4O4S: C, 56.24; H, 4.20; N, 14.57; Found:
C, 56.31; H, 4.30; N, 14.85.
4.1.35. N0-((6-Bromoisoquinolin-4-yl)methylene)-N,2-
dimethyl-5-nitrobenzenesulfonohydrazide (16n). Com-
pound 16n was prepared from 15a by Method C.
Compound 16n was obtained as a cream-coloured solid
(8 mg, 11%). 1H NMR d (400 MHz, CDCl3) 9.14 (s,
1H), 9.01 (d, J 2.4 Hz, 1H), 8.93 (d, J 1.8 Hz, 1H), 8.58
(s, 1H), 8.31 (dd, J 8.4, 2.4 Hz, 1H), 7.96 (s, 1H), 7.83
(d, J 8.7 Hz, 1H), 7.72 (dd, J 8.7, 1.8 Hz, 1H), 7.50 (d, J
8.4 Hz, 1H), 3.59 (s, 3H), 2.76 (s, 3H). LC–MS (APCI+)
463 (MH+ with 79Br, 85%), 465 (MH+ with 81Br, 100%).
Anal. Calcd for C18H15BrN4O4SÆ1/3 EtOAc: C, 47.13;
H, 3.61; N, 11.38. Found C, 47.32; H, 3.56; N, 11.50.
4.3. Cellular assay
The early passage cell lines used in this study were
developed in this laboratory and cultured as previously
described.30 Cell lines were grown in a-modified mini-
mal essential growth medium supplemented with insu-
lin, transferrin, selenite and 5% foetal bovine serum.
Individual wells of 96-well tissue culture plates con-
tained 1000 cells in a volume of 150 lL. Drugs were
added at 10-fold concentration steps to a maximum
of 20 lM and plates were incubated under an atmo-
sphere of 5% O2, 5% CO2 and 90% N2 for five days,
with [3H]thymidine (0.04 lCi per well) being added
over the last 6 h. Cells were harvested and the incorpo-
rated radioactivity was measured. Duplicate samples
were analysed for each drug dose with multiple control
samples and data were fitted to a least-squares regres-
sion of the form y = y0 + aeꢁbx, where y is the incorpo-
rated radioactivity, x is the drug concentration and y0,
a and b are variables. The IC50 value was defined as the
drug concentration reducing [3H]thymidine incorpora-
tion by 50%.
4.1.36. N0-((7-Bromoisoquinolin-4-yl)methylene)-N,2-di-
methyl-5-nitrobenzenesulfonohydrazide (16o). Compound
16o was prepared from 15b by Method C. Compound 16o
was obtained as a cream-coloured solid (39 mg, 37%). 1H
NMR d (400 MHz, CDCl3) 9.10 (s, 1H), 9.01 (d, J 2.4 Hz,
1H), 8.64 (d, J 9.1 Hz, 1H), 8.56 (s, 1H), 8.31 (dd, J 8.4,
2.4 Hz, 1H), 8.14 (d, J 2.0 Hz, 1H), 7.94 (s, 1H), 7.78
(dd, J 9.1, 2.0 Hz, 1H), 7.50 (d, J 8.4 Hz, 1H), 3.57 (s,
3H), 2.76 (s, 3H). LC–MS (APCI+) 463 (MH+ with
79Br, 90%), 465 (MH+ with 81Br, 100%). Anal. Calcd for
C18H15BrN4O4S: C, 46.66; H, 3.26; N, 12.09. Found: C,
46.83; H, 3.44; N, 11.99.
4.1.37. N0-((8-Bromoisoquinolin-4-yl)methylene)-N,2-di-
methyl-5-nitrobenzenesulfonohydrazide (16p). Compound
16p was prepared from 15c by Method C. Compound
16p was obtained as a cream-coloured solid (25 mg,
1
30%). H NMR d (400 MHz, CDCl3) 9.61 (s, 1H), 9.04
4.4. Molecular modelling
(d, J 2.4 Hz, 1H), 8.70 (d, J 8.6 Hz, 1H), 8.63 (s, 1H),
8.30 (dd, J 8.4, 2.4 Hz, 1H), 7.98 (s, 1H), 7.88 (d, J
7.5 Hz, 1H), 7.54 (dd, J 8.6, 7.5 Hz, 1H), 7.49 (d, J
Molecular modelling studies were carried out using SYB-
YL software, version 7.3, running on a Linux worksta-

J. D. Kendall et al. / Bioorg. Med. Chem. 15 (2007) 7677–7687
7687
tion.21 Compound 1 was built using the Sketch module
and optimised using the Tripos force field. Docking sim-
ulations were performed on human p110c (PDB code
2CHX) with the automated GOLD program.20 20 confor-
mations were generated. In order to take into account
protein flexibility, the conformation with the best score
(GoldScore) was further refined using the MINIMIZE
module. The minimization process used the Powell meth-
od31 with the Tripos force field (dielectric constant 1r) to
12. Hayakawa, M.; Kaizawa, H.; Kawaguchi, K.; Matsuda,
K.; Ishikawa, N.; Koizumi, T.; Yamano, M.; Okada, M.;
Ohta, M. US Patent 6,403,588, 2001.
13. Hayakawa, M.; Kaizawa, H.; Kawaguchi, K.; Ishikawa,
N.; Koizumi, T.; Ohishi, T.; Yamano, M.; Okada, M.;
Ohta, M.; Tsukamoto, S.; Raynaud, F. I.; Waterfield, M.
D.; Parker, P.; Workman, P. Bioorg. Med. Chem. 2007, 15,
403.
14. Hayakawa, M.; Kawaguchi, K.; Kaizawa, H.; Koizumi,
T.; Ohishi, T.; Yamano, M.; Okada, M.; Ohta, M.;
Tsukamoto, S.; Raynaud, F. I.; Parker, P.; Workman, P.;
Waterfield, M. D. Bioorg. Med. Chem. 2007, 15, 5837.
15. Hayakawa, M.; Kaizawa, H.; Moritomo, H.; Kawaguchi,
K.; Koizumi, T.; Yamano, M.; Matsuda, K.; Okada, M.;
Ohta, M. US Patent 6,608,053, 2003.
16. Hayakawa, M.; Kaizawa, H.; Hiroyuki, M.; Koizumi, T.;
Ohishi, T.; Okada, M.; Ohta, M.; Tsukamoto, S.; Parker,
P.; Workman, P.; Waterfield, M. Bioorg. Med. Chem.
2006, 14, 6847.
reach a final convergence of 0.01 kcal molꢁ1
.
Mutation of Thr-886 into histidine was done using the
BIOPOLYMER module. The side chain conformation
was set according to the Lovell rotamer library32 and re-
fined using the MINIMIZE module to a final conver-
gence of 0.01 kcal molꢁ1
.
17. Oldenziel, O. H.; Van Leusen, D.; Van Leusen, A. M. J.
Org. Chem. 1977, 42, 3114.
Acknowledgment
18. Walker, E. H.; Pacold, M. E.; Perisic, O.; Stephens, L.;
Hawkins, P. T.; Wymann, M. P.; Williams, R. L. Mol. Cell
2000, 6, 909.
This work was funded in part by the Health Research
Council of New Zealand.
19. Camps, M.; Ruckle, T.; Hong, J.; Ardissone, V.; Rintelen,
¨
F.; Shaw, J.; Ferrandi, C.; Chabert, C.; Gillieron, C.;
Franc¸on, B.; Martin, T.; Gretener, D.; Perrin, D.; Leroy,
D.; Vitte, P.-A.; Hirsch, E.; Wymann, M. P.; Cirillo, R.;
Schwarz, M. K.; Rommel, C. Nat. Med. 2005, 11, 936.
20. Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor,
R. J. Mol. Biol. 1997, 267, 727.
References and notes
1. Stephens, L.; Williams, R.; Hawkins, P. Curr. Opin.
Pharmacol. 2005, 5, 357.
2. 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.
3. Samuels, Y.; Wang, Z.; Bardelli, A.; Silliman, N.; Ptak, J.;
Szabo, S.; Yan, H.; Gazdar, A.; Powell, S. M.; Riggins, G.
J.; Willson, J. K. V.; Markowitz, S.; Kinzler, K. W.;
Vogelstein, B.; Velculescu, V. E. Science 2004, 304, 554.
4. Vogt, P. K.; Kang, S.; Elsliger, M.-A.; Gymnopoulos, M.
Trends Biochem. Sci. 2007, 32, 342.
21. SYBYL 7.3, Tripos Inc., 1699 South Hanley Rd., St.
Louis, Missouri, 63144, USA.
22. DeLano, W. L. The PyMOL Molecular Graphics System.
DeLano Scientific: San Carlos, CA, USA, 2002.
23. Gueiffier, A.; Lhassani, M.; Elhakmaoui, A.; Snoeck, R.;
Andrei, G.; Chavignon, O.; Teulade, J.-C.; Kerbal, A.;
Essassi, E. M.; Debouzy, J.-C.; Witvrouw, M.; Blache, Y.;
Balzarini, J.; De Clercq, E.; Chapat, J.-P. J. Med. Chem.
1996, 39, 2856.
5. Ruckle, T.; Schwarz, M. K.; Rommel, C. Nat. Rev. Drug
¨
Disc. 2006, 5, 903.
24. Eckroth, D. R.; Cochran, T. G. J. Chem. Soc. (C) 1970,
2660.
6. Kim, D.; Cheng, G. Z.; Lindsley, C. W.; Yang, H.; Cheng,
J. Q. Curr. Opin. Invest. Drugs 2005, 6, 1250.
25. Fel’dman, I. Kh.; Mikhailova, V. N. Zh. Obshch. Khim.
1963, 33, 38.
7. Hennessy, B. T.; Smith, D. L.; Ram, P. T.; Lu, Y.; Mills,
G. B. Nat. Rev. Drug Discov. 2005, 4, 988.
26. Collman, J. P.; Zhong, M.; Costanzo, S.; Zhang, C. J.
Org. Chem. 2001, 66, 8252.
8. Maehama, T.; Dixon, J. E. Trends Cell Biol. 1999, 9, 125.
9. Chaussade, C.; Rewcastle, G. W.; Kendall, J. D.; Denny,
W. A.; Cho, K.; Grønning, L. M.; Chong, M. L.;
Anagnostou, S. H.; Jackson, S. P.; Daniele, N.; Shepherd,
P. R. Biochem. J. 2007, 404, 449.
10. Knight, Z. A.; Gonzalez, B.; Feldman, M. E.; Zunder, E.
R.; Goldenberg, D. D.; Williams, O.; Loewith, R.; Stokoe,
D.; Balla, A.; Toth, B.; Balla, T.; Weiss, W. A.; Williams,
R. L.; Shokat, K. M. Cell 2006, 125, 733.
11. Norman, B. H.; Shih, C.; Toth, J. E.; Ray, J. E.; Dodge, J.
A.; Johnson, D. W.; Rutherford, P. G.; Schultz, R. M.;
Worzalla, J. F.; Vlahos, C. J. J. Med. Chem. 1996, 39,
1106.
27. Fuentes, O.; Paudler, W. W. J. Heterocycl. Chem. 1975,
12, 379.
28. Zaidlewicz, M.; Chechlowska, A.; Prewysz-Kwinto, A.;
Wojtczak, A. Heterocycles 2001, 55, 569.
29. Lutz, C.; Graf, C.-D.; Knochel, P. Tetrahedron 1998, 54,
10317.
30. Marshall, E. S.; Baguley, B. C.; Matthews, J. H. L.; Jose,
C. C.; Furneaux, C. E.; Shaw, J. H. F.; Kirker, J. A.;
Morton, R. P.; White, J. B.; Rice, M. L.; Isaacs, R. J.;
Coutts, R.; Whittaker, J. R. Oncol. Res. 2004, 14, 297.
31. Powell, M. J. D. Math. Program 1977, 12, 241.
32. Lovell, S. C.; Word, J. M.; Richardson, J. S.; Richardson,
D. C. Proteins: Struct., Funct., Genet. 2000, 40, 389.