Design, synthesis and biological evaluation of α-substituted isonipecotic acid benzothiazole analogues as potent bacterial type II topoisomerase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.10.058

The discovery and optimisation of a new class of benzothiazole small molecules that inhibit bacterial DNA gyrase and topoisomerase IV are described. Antibacterial properties have been demonstrated by activity against DNA gyrase ATPase and potent activity against Staphylococcus aureus, Enterococcus faecalis, Streptococcus pyogenes and Haemophilus influenzae. Further refinements to the scaffold designed to enhance drug-likeness included analogues bearing an α-substituent to the carboxylic acid group, resulting in excellent solubility and favourable pharmacokinetic properties.

Full text of DOI:10.1016/j.bmcl.2013.10.058

Bioorganic & Medicinal Chemistry Letters 23 (2013) 6598–6603
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and biological evaluation of
a-substituted
isonipecotic acid benzothiazole analogues as potent bacterial
type II topoisomerase inhibitors
d
a
a
c
Lorraine C. Axford a, , Piyush K. Agarwal , Kelly H. Anderson , Laura N. Andrau , John Atherall ,
Stephanie Barker b, James M. Bennett b, Michael Blair a, Ian Collins b, Lloyd G. Czaplewski b, David T. Davies b,
Carlie T. Gannon a, Dushyant Kumar d, Paul Lancett b, Alastair Logan b, Christopher J. Lunniss a,
Dale R. Mitchell c, Daniel A. Offermann a, James T. Palmer a, Nicholas Palmer c, Gary R. W. Pitt a,
Stéphanie Pommier b, Daniel Price b, B. Narasinga Rao d, Rashmi Saxena d, Tarun Shukla d, Amit K. Singh d,
Mahipal Singh d, Anil Srivastava d, Christopher Steele c, Neil R. Stokes b, Helena B. Thomaides-Brears b,
Edward M. Tyndall a, David Watson c, David J. Haydon b
⇑
a Biota Holdings Ltd, 10/585 Blackburn Road, Notting Hill, Victoria 3168, Australia
b Biota Europe Ltd, Begbroke Science Park, Begbroke, Oxfordshire OX5 1PF, United Kingdom
c BioFocus, Chesterford Research Park, Saffron Walden, Essex CB10 1XL, United Kingdom
d Jubilant Chemsys Ltd, B-34, Sector-58, Noida 201301, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The discovery and optimisation of a new class of benzothiazole small molecules that inhibit bacterial
DNA gyrase and topoisomerase IV are described. Antibacterial properties have been demonstrated by
activity against DNA gyrase ATPase and potent activity against Staphylococcus aureus, Enterococcus faecal-
is, Streptococcus pyogenes and Haemophilus influenzae. Further refinements to the scaffold designed to
Received 25 June 2013
Revised 26 October 2013
Accepted 28 October 2013
Available online 4 November 2013
enhance drug-likeness included analogues bearing an
a-substituent to the carboxylic acid group, result-
ing in excellent solubility and favourable pharmacokinetic properties.
Keywords:
Ó 2013 Elsevier Ltd. All rights reserved.
DNA gyrase
Topoisomerase
Antibacterial
Drug resistance in pathogenic bacteria is increasing throughout
the world, leading to higher mortality and increased healthcare
costs.1 There is a huge need to develop new classes of therapeutic
agents to address this problem.
GyrB and ParE subunits are functionally similar,3 having an ATPase
domain in the N-terminal domain and a C-terminal domain that
interacts with the other subunit (GyrA and ParC respectively) and
the DNA. The conservation between the DNA gyrase and topoiso-
merase IV active sites suggests that inhibitors of the sites should
simultaneously target both type II topoisomerases.3 Such ‘dual-tar-
geting’ inhibitors are attractive because they have the potential to
reduce the development of target-based resistance. Ideally, an
antibiotic based on the inhibition of bacterial type II topoisomeras-
es would be selective for the bacterial enzymes and relatively inac-
tive against the eukaryotic type II topoisomerases.
Type II topoisomerases catalyse the interconversion of DNA
topoisomers by transporting one DNA segment through another.2
Bacteria encode two type II topoisomerase enzymes, DNA gyrase
and DNA topoisomerase IV.3 DNA gyrase controls DNA supercoiling
and relieves topological stress. Topoisomerase IV decatenates
daughter chromosomes following replication and can also relax
supercoiled DNA. Bacterial type II topoisomerases form a hetero-
tetrameric complex composed of two subunits. DNA gyrase forms
an A2B2 complex comprised of GyrA and GyrB, whereas topoiso-
merase IV forms a C2E2 complex comprised of ParC and ParE.3
The bacterial type II topoisomerases are highly conserved enzymes
allowing the design of broad-spectrum inhibitors. Furthermore, the
The authors have previously described a class of benzothiaz-
oles4–7 which inhibits bacterial DNA gyrase activity. In this com-
munication,
a
class of related compounds that include
a
carbocyclic or heterocyclic ring comprising of an
a-substituted car-
boxylate, is described. Early hits such as 1 (Fig. 1)4 showed antibac-
terial activity but had limited solubility. As solubility is important
in clinical administration of antibacterial drugs targeting both
intravenous (IV) and oral delivery, solubilising moieties were
⇑
Corresponding author. Tel.: +61 3 9915 3700; fax: +61 3 9915 3701.
E-mail address: l.offermann@biota.com.au (L.C. Axford).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.10.058

L. C. Axford et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6598–6603
6599
O
O
HO
N
N
N
N
HO
O
O
O
N
N
H
N
H
N
H
N
S
N
S
N
S
5
2
N
H
N
H
N
H
7
N
N
N
1
2
3
IC50a (µg/mL)
0.014
0.12
6.25
0.001
0.12
50
0.010
0.015
>800
E. fs MIC (µg/mL)
Solubility (µg/mL)
Figure 1. Progression of early hits to isonipecotic acid containing analogues. aDNA gyrase ATPase (S. aureus).
explored that would not interfere with the promising antimicrobial
profile. It was discovered that the addition of a carboxylic acid moi-
ety appended to the C-5 aryl group, 2, indeed improved the solubil-
(CF3) or methanesulfonyl chloride (SO2Me). Some analogues were
prepared via a modified synthetic route or from alternative starting
materials as described elsewhere.9
ity8 at physiological pH from 6.25
lg/mL to 50
lg/mL, whilst
Compounds were tested in a DNA gyrase ATPase activity
assay.10 The antimicrobial activity of the compounds was mea-
sured by susceptibility testing using the broth microdilution
method11 and MICs were determined against Staphylococcus aureus
(ATCC 29213), E. faecalis (ATCC 29212), Streptococcus pyogenes
(ATCC 51339) and Haemophilus influenzae (ATCC 49247).
Table 1 indicates the structure activity relationship (SAR) with-
in the series. All analogues tested displayed potent activity in the
DNA gyrase ATPase assay. Compounds 3 and 18 have IC50 values
maintaining antibacterial activity against the target pathogens
(minimal inhibitory concentration (MIC) data for Enterococcus fae-
calis shown as an example).
By linking the carboxylate to the C-5 aryl group by a cyclic
amine, for example the isonipecotic acid appendage, 3, both the
antibacterial activity and the solubility increased. It was discov-
ered that the addition of a substituent
a to the carboxylic acid im-
proved the PK profile of the series without loss of potency or
solubility. Compounds of this class possessed enhanced solubilities
at physiologically acceptable pH, enabling IV administration, along
with excellent oral bioavailability, lower clearance, and higher
for S. aureus topoisomerase IV (0.012 and 0.008 lg/mL respec-
tively)10 comparable to their S. aureus DNA gyrase ATPase IC50 val-
ues. Compounds 3 and 18 also showed specificity for bacterial
topoisomerases, with no inhibition of human topoisomerase II
being observed.7 A dual targeting intracellular inhibitor of GyrB
and ParE would be expected to show a low frequency of resistance
(FoR).13 The FoR to compound 3 and 18 in S. aureus (ATCC 29213)
was determined.7 At concentrations equivalent to four- and eight-
fold the MICs no spontaneous mutants were isolated, giving mea-
sured FoRs of 610À10. Taken together this suggests the compounds
are potent and specific inhibitors of both DNA gyrase and topoiso-
merase IV. Small alkyl groups gave the best antibacterial profile as
well as excellent solubility and stability in mouse microsomes
(intrinsic clearance, CLint). For example, methyl 18 and ethyl 19
groups were shown to have the most favourable activity across
the panel. Aryl containing groups such as phenyl 20 lost activity
against S. pyogenes (S. py) and H. influenzae (H. in) and demon-
strated poor solubility. Addition of a fluorine-containing group
such as CF3 21 led to a marked decrease in activity, particularly
against S. aureus (S. au). Other electron withdrawing groups such
as SO2Me 22 led to a further decrease in activity across the species
tested. Electron rich groups such as OH 23 and NH2 24 generally
showed poor activity, particularly against S. aureus, E. faecalis (E.
exposure compared to compounds without an
a substituent. The
discovery and preparation of this class of compounds are now
described.
During the exploration of C-5, a 2-pyridyl group was main-
tained at the C-7 position of the benzothiazole core. Boronic acid
intermediate 4 was prepared as an advanced precursor, according
to Scheme 1.
Synthesis commenced with bromination of 2-amino-5-nitro-
phenol 5 in acetonitrile followed by deamination with sodium ni-
trite and sulfuric acid in ethanol. Benzylation of phenol 6 and
reduction of the nitro group afforded 7. Addition of benzoyl isothi-
ocyanate to 7 furnished N-benzoylthiourea 8, which was readily
cleaved to the thiourea 9 with sodium hydroxide. Bromine-medi-
ated cyclisation to the benzothiazole core followed, with subse-
quent conversion to the ethyl urea 10. Stille coupling installed
the C-7 pyridyl group. Treatment of 11 with methane sulfonic acid
yielded alcohol 12, which upon conversion to triflate 13 and a
Miyaura borylation reaction yielded the key intermediate 4 for fur-
ther derivatisation.
Initially, the introduction of an
a substituent to the carboxylic
acid was investigated. As shown in Scheme 2, commercially avail-
able ethyl N-Boc-piperidine-4-carboxylate 14 was treated with
lithium diisopropylamide at À78 °C, quenched with an alkyl halide,
then warmed to room temperature to furnish 15. Removal of the
Boc group with hydrogen chloride in 1,4-dioxane gave crude 16
which was then used in an aromatic nucleophilic substitution reac-
tion with 5-bromo-2-chloropyrimidine to yield bromide 17. Suzuki
cross-coupling with boronic acid 4, followed by saponification,
yielded the target products.
fs) and H. influenzae. The nature of the a-substituent coupled with
the carboxylate was key to fine tuning the ideal profile of the ser-
ies. With this information in mind, subsequent analogues were
prepared with an
SAR.
a-methyl substituent in order to obtain further
Next, the effect of the heterocycle directly attached to the C-5
position of the core was investigated. A set of analogues was
prepared9 using the
a-methyl isonipecotic acid group as the opti-
mal group from the earlier exploration. Selected compounds are
described in Table 2. The 3,5-pyrimidyl group 18 (data in Table 1)
gave the best overall profile, followed by the 3-pyridyl 25. The
3,5-pyrimidyl and 3-pyridyl groups showed an improvement in
Alternative
a-substituents were prepared by quenching the
lithiated species (step a) with alternative electrophiles that is, S-
(trifluoromethyl)dibenzothiophenium trifluoromethanesulfonate

6600
L. C. Axford et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6598–6603
HO
NO
HO
NO
HO
NO
BnO
NO
2
2
2
2
a
c
d
b
H2N
H2N
Br
Br
Br
5
6
H
N
H
N
H
N
BnO
NH2
BnO
Ph
BnO
NH2
BnO
N
S
NH2
f
e
g
S
O
S
Br
Br
Br
Br
7
8
9
O
O
O
BnO
BnO
HO
N
H
N
H
N
H
N
S
N
S
N
S
N
H
N
H
N
H
j
h
i
Br
N
N
10
11
12
OH
B
O
O
TfO
N
N
N
N
S
HO
H
H
N
H
N
H
k
l
S
N
N
13
4
Scheme 1. Reagents and conditions: (a) bromine, MeCN, 30 °C, 1 h; 85%; (b) sulfuric acid (conc.), EtOH, À10 to 50 °C then NaNO2, reflux, 3 h; 84%; (c) benzyl bromide, K2CO3,
acetone, rt, 15 min then reflux, 3 h; 51%; (d) SnCl2Á2H2O, THF, reflux, 3 h; 97%; (e) benzoyl isothiocyanate, acetone, rt, 45 min; 85%; (f) NaOH, H2O, 70 °C, overnight; 78%; (g)
bromine, MeCN, 0 °C, 30 min then rt, 1 h; 59%; (h) ethyl isocyanate, 1,4-dioxane, 80 °C, overnight; 75%; (i) 2-tributylstannyl pyridine, Pd(PPh3)4, DMF, 100 °C, 16 h; 70%; (j)
methane sulfonic acid, dichloromethane, rt, 3 h; 97%; (k) N-phenylbis(trifluoromethane sulfonamide), DIPEA, DMF, rt, 3 h; 68%; (l) bis(neopentylglycolato) diboron,
Pd(dppf)Cl2ÁDCM, KOAc, DMSO, 80 °C, 1.5 h; 88%.
O
O
O
O
O
O
O
a
b
c
N
O
N
O
NH
HCl
O
O
14
15
16
O
O
HO
d, e
N
N
N
N
O
N
N
H
N
S
N
Br
N
H
17
N
18
Scheme 2. Reagents and conditions: (a) LDA, THF, À78 °C, 45 min, then methyl iodide, À78 °C to rt, 6 h; 65%; (b) 4 M HCl in 1,4-dioxane, rt, 30 min; (c) 5-bromo-2-
chloropyrimidine, DIPEA, EtOH, 70 °C, 1 h; 75% (over two steps); (d) 4, Pd(PPh3)4, Na2CO3 (2 M, aq), EtOH, 80 °C; 2 h; 22%; (e) KOtBu, DMSO, rt, 2 h; 65%.
activity against S. aureus and H. influenzae compared to other ana-
logues. Switching to a pyrazine analogue 26 resulted in a fourfold
drop in activity against S. aureus and H. influenzae relative to 3,5-
pyrimidyl. Regiochemical substitution also had an effect on antimi-
crobial activity. Meta substitution 27 was less favourable than para
(relative to the benzothiazole core) particularly illustrated by com-
paring MICs against S. aureus. Switching to a five-membered ring
such as 1,2,4-thiadiazole 28 led to poor activity against all patho-
gens as well as a minor drop-off in enzyme activity. In all cases,
data suggest that solubility and microsomal stability were rela-
tively unchanged by these modifications. 3,5-Pyrimidine was se-
lected as the optimal heterocycle for the preparation of further
analogues.
Modifications of the isonipecotic acid ring were then explored
(Table 3). Five-, six- and seven-membered rings were synthesised.9
Again, all analogues showed good enzyme data. Pyrrolidine com-
pound 29 showed good activity against S. pyogenes and H. influen-
zae, however the alternative substitution pattern 30 displayed
more modest activity against all pathogens tested. The piperidine
analogue 18 (data in Table 1) showed improved activity, with the

L. C. Axford et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6598–6603
6601
Table 1
Effect of various
a
-substituents on antibacterial activity
O
HO
R
N
N
O
N
N
H
N
S
N
H
N
Solubility8
(lg/mL)
CLint (mL/min/kg)
a
12
#
R
IC50
(lg/mL)
MIC (
lg/mL)
E. fs
S. au
S. py
H. in
3
18
19
20
21
22
23
24
H
Me
Et
0.010
0.008
0.012
0.12
0.12
0.06
0.5
2
>16
8
>16
0.03
0.06
0.06
0.5
0.25
4
0.015
0.015
0.015
0.06
0.25
4
2
2
4
>16
16
>16
8
>800
>800
>800
12.5
>800
>800
100
<5.8
<5.8
<5.8
<5.8
N.D.
N.D.
N.D.
N.D.
Ph
0.013b
0.016
CF3
SO2Me
OH
NH2
0.015b
0.003b
0.025b
0.12
4
0.5
>16
>16
25
N.D. Not determined.
a
DNA gyrase ATPase (S. aureus).
DNA gyrase ATPase (Escherichia coli).
b
Table 2
Effect of the heterocycle on antibacterial activity
O
HO
N
O
Het
N
H
N
S
N
H
N
Solubility8
(
lg/mL)
CLint (mL/min/kg)
a
12
#
Het
IC50
(l
g/mL)
MIC (
S. py
l
g/mL)
S. au
E. fs
H. in
N
N
25
26
27
0.012
0.02
0.12
0.5
1
0.12
0.12
0.06
0.03
2
8
4
>800
>800
>800
<5.8
<5.8
N.D.
0.03
0.06
N
N
0.015
N
28
0.056
1
1
N.D.
>16
N.D.
N.D.
N
S
N.D. Not determined.
a
DNA gyrase ATPase (S. aureus).
alternate regiochemisty 31 being disfavoured as well as exhibiting
poor solubility. The azepane analogue 32 showed comparable
potency, solubility and microsomal stability to 18, apart from a de-
crease in H. influenzae activity. All analogues were prepared race-
Suitable compounds were selected and their PK profile deter-
mined in mouse and/or rat.14 The maximum concentration follow-
ing oral administration (Cmax), area under the curve (AUCIV),
bioavailability (F) and clearance following IV administration (CL)
were calculated from the measured plasma concentrations. Se-
lected results are shown in Table 4.
mically apart from 18 which offered
a fortuitous synthetic
advantage due to its achiral nature.

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L. C. Axford et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6598–6603
Table 3
Effect of the acid attachment ring
R
N
O
N
N
H
N
S
N
H
N
Solubility8
(lg/mL)
CLint (mL/min/kg)
a
12
#
R
IC50
(lg/mL)
MIC (
lg/mL)
S. au
S. py
E. fs
H. in
N
HO
HO
29
30
0.005
0.04
0.5
0.06
0.5
0.25
1
>800
>800
N.D.
N.D.
O
O
2
N.D.
0.5
4
8
N
N
N
31
32
0.007b
0.006
0.5
0.5
6.25
N.D.
<5.8
HO
HO
O
O
0.12
0.12
N.D.
8
>800
N.D. Not determined.
a
DNA gyrase ATPase (S. aureus).
DNA gyrase ATPase (E. coli).
b
References and notes
Table 4
Calculated PK parameters following administration at 3 mg/kg
1. Magiorakos, A.-P.; Srinivasan, A.; Carey, R. B.; Carmeli, Y.; Falagas, M. E.; Giske,
C. G.; Harbarth, S.; Hindler, J. F.; Kahlmeter, G.; Olsson-Liljequist, B.; Paterson,
D. L.; Rice, L. B.; Stelling, J.; Struelens, M. J.; Vatopoulos, A.; Weber, J. T.; Monnet,
D. L. Clin. Microbiol. Infect. 2012, 18, 268.
2. Levine, C.; Hiasa, H.; Marians, K. J. Biochim. Biophys. Acta, Gene Struct. Expr.
1998, 1400, 29.
3. East, S. P.; zaplewski, L. G.; Haydon, D. J. In Designing Multi-Target Drugs;
Morphy, J. R., Harris, C. J., Eds.; RSC Publishing, 2012; pp 335–352. Chapter 20.
4. Haydon, D.J.; Czaplewski, L.G.; Palmer, N.J.; Mitchell, D.R.; Atherall, J.F.; Steele,
C.R.; Ladduwahetty, T.; PCT Int. Appl. WO2007/148093.
#
Species
Cmax
(lg/mL)
AUCIV
(lg hr/mL)
F (%)
CL (mL/min/kg)
3
Mouse
Rat
Mouse
Rat
Mouse
Rat
0.07
1.8
1.7
0.6
6.1
3.5
0.9
17.4
7.6
6.6
14.3
27.7
8
48
33
31
87
80
55
18
25
25
19
19
2.8
6.5
7.6
3.5
1.5
5. Haydon, D.J.; Czaplewski, L.G.; PCT Int. Appl. WO2009/074810.
6. Haydon, D.J.; Czaplewski, L.G.; PCT Int. Appl. WO2009/074812.
7. Stokes, N. R.; Thomaides-Brears, H. B.; Barker, S.; Bennett, J. M.; Berry, J.;
Collins, I.; Czaplewski, L. G.; Gamble, V.; Lancett, P.; Logan, A.; Lunniss, C.;
Peasley, H.; Pommier, S.; Price, D.; Smee, C.; Haydon, D. J. Antimicrob. Agents
Chemother. 2013, in press. http://dx.doi.org/10.1128/AAC.00719-13
8. The solubility of the compounds was measured using a turbidimetric method.
A series of doubling dilutions of compounds were prepared in neat dimethyl
The results clearly demonstrate that the addition of a substitu-
ent to the carboxylic acid significantly improved the PK profile of
this class of compound when compared to compound 3. Pleasingly,
the maximum oral concentration, AUC and bioavailability all
showed much greater values and decreased clearance rates were
a
sulfoxide (DMSO). 5 lL samples were diluted 20-fold into 95 lL volumes of
observed. For example, the addition of an ethyl group at the
a po-
100 mM phosphate buffered saline (PBS, pH 7.4) in microtitre assay plates and
allowed to reach equilibrium at room temperature for 24 h. The absorbance
within each well of the plate was read spectrophotometrically at a wavelength
of 620 nm. A precipitate forms when the maximum aqueous solubility level is
reached. The value quoted is the highest concentration where the compound
was in solution that is, where no measurable precipitate was visible.
9. For experimental details on these and similar compounds see: Palmer, J.T.;
Lunniss, C.J.; Offermann, D.A.; Axford, L.C.; Blair, M.; Mitchell, D.; Palmer, N.;
Steele, C.; Atherall, J.; Watson, D.; Haydon, D.; Czaplewski, L.; Davies, D.;
Collins, I.; Tyndall, E.M.; Andrau, L.; Pitt, G.R. W.; PCT Int. Appl. WO2012/
045124.
sition, compared to a hydrogen substituent (compounds 3 vs 19 in
mouse), gave an 87-fold improvement in maximum concentration,
a 16-fold improvement in AUCIV and a 10-fold increase in bioavail-
ability. Clearance was reduced by more than 15-fold. This trend
was shown to be consistent over all compounds tested. Further-
more, the rat plasma protein binding remains the same for both
compounds 3 and 18 (99.3% in each case)7 giving weight to
improvements in the pharmacology by the addition of an
a substi-
10. ATPase assay: Enzymes were purchased from Inspiralis Ltd (Norwich, United
Kingdom). For the S. aureus DNA gyrase ATPase assay, the final assay
composition was 10 nM DNA gyrase enzyme (A2B2 complex), 0.08 mg/mL
DNA, 40 mM HEPES pH 7.6, 10 mM magnesium acetate, 0.5 M potassium
glutamate, 0.01 mg/mL BSA and 2 mM DTT, 1 mM ATP and 5% DMSO solution
containing the compounds. For the E. coli DNA gyrase ATPase assay, the final
assay composition was 10 nM DNA gyrase enzyme (A2B2 complex), 0.08 mg/
mL DNA, 35 mM Tris pH 7.5, 24 mM KCl, 2 mM MgCl2, 6.5% glycerol, 0.1 mg/mL
BSA and 2 mM DTT, 1 mM ATP and 5% DMSO solution containing the
compounds. For the S. aureus topoisomerase IV ATPase assay, the final assay
composition was 25 nM topoisomerase IV enzyme (C2E2 complex), 0.01 mg/mL
DNA, 50 mM Tris–HCl pH 7.5, 5 mM magnesium chloride, 0.35 M potassium
tuent, rather than merely variation in protein binding. Other com-
pounds have similar values.
In summary, the design, synthesis and antibacterial activity of a
new class of compound have been reported. The compounds dis-
play outstanding antibacterial activity with inhibition of both
DNA gyrase and topoisomerase IV, promising PK profiles, good
microsomal stability and excellent solubility. Further investigation
of these compounds as potential antibacterial agents is underway
and will be reported elsewhere in due course.

L. C. Axford et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6598–6603
6603
glutamate, 0.05 mg/mL BSA and 5 mM DTT, 1 mM ATP and 1% DMSO solution
containing the compounds. The reactions, in a volume of 25 L, were started by
Reactions were stopped at 0, 10, 30 and 60 min by the addition of DMSO
(100 L). Samples were extracted using acetonitrile and centrifugation, and the
l
l
the addition of the ATP and allowed to incubate at 30 °C for 60 min. Reactions
were stopped by adding 0.2 mL malachite green solution (0.034% malachite
green, 10 mM ammonium molybdate, 1 M HCl, 3.4% ethanol, 0.01% tween 20).
Colour was allowed to develop for 5 min and the absorbance at 600 nm was
supernatants were analysed by LCMS. T1/2 values were determined from the
slope of the peak areas over time, which was then used to calculate the
intrinsic clearance.
13. O’Neil, A. J.; Chopra, I. Expert Opin. Investig. Drugs 2004, 13, 1045.
14. The compound concentration in plasma was measured by LC/MS/MS following
a single IV or oral administration of the compounds at a dose of 3 mg/kg. IV
dose formation was administered as a single bolus dose through the tail vein,
whilst oral dose formulation was administered by an oral gavage needle. In
both cases, the dose volume was 5 mL/kg. Blood was collected from rats using a
jugular vein catheter and from anesthetised mice through a capillary guided
into the retro-orbital plexus. The collected blood was centrifuged to obtain
plasma and the compounds extracted into methanol prior to determining the
compound concentration by LC/MS/MS.
measured
spectrophotometrically.
The
half-maximum
inhibitory
concentration (IC50) values were determined from the absorbance readings
using no compound and no enzyme controls.
11. Clinical and Laboratory Standards Institute, Approved Standard—Seventh
Edition; 2006, Vol. 26, No. 2, Document M7–A7.
12. Mouse microsome stability assay: Compounds were tested at 10 lM in duplicate
with a final concentration of 0.1% v/v DMSO. Mouse liver microsomes (Life
Technologies) were added to a final protein concentration of 1 mg/mL and
reactions were started by the addition of NADPH to a concentration of 1 mM.