N-[(3S)-Pyrrolidin-3-yl]benzamides as novel dual serotonin and noradrenaline reuptake inhibitors: Impact of small structural modifications on P-gp recognition and CNS penetration

Article abstract of DOI:10.1016/j.bmcl.2009.07.049

The structure-activity relationship and the synthesis of novel N-[(3S)-pyrrolidin-3-yl]benzamides as dual serotonin and noradrenaline monoamine reuptake inhibitors (SNRI) is described. Preferred compound 9 aka PF-184,298 is a potent SNRI with good selecti

Full text of DOI:10.1016/j.bmcl.2009.07.049

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5078–5081
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
N-[(3S)-Pyrrolidin-3-yl]benzamides as novel dual serotonin and noradrenaline
reuptake inhibitors: Impact of small structural modifications on P-gp
recognition and CNS penetration
c
a
a
c
b
Florian Wakenhut a, , Gill A. Allan , Paul V. Fish , M. Jonathan Fray , Anthony C. Harrison , Rachel McCoy ,
*
Stephen C. Phillips b, Alan Stobie a, Dominique Westbrook b, Simon L. Westbrook b, Gavin A. Whitlock a
a Department of Genitourinary Chemistry, Pfizer Global Research and Development, Sandwich Laboratories, Ramsgate Road, Sandwich, Kent CT13 9NJ, UK
b Department of Genitourinary Biology, Pfizer Global Research and Development, Sandwich Laboratories, Ramsgate Road, Sandwich, Kent CT13 9NJ, UK
c Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer Global Research and Development, Sandwich Laboratories, Ramsgate Road, Sandwich, Kent CT13 9NJ, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
The structure–activity relationship and the synthesis of novel N-[(3S)-pyrrolidin-3-yl]benzamides as dual
serotonin and noradrenaline monoamine reuptake inhibitors (SNRI) is described. Preferred compound 9
aka PF-184,298 is a potent SNRI with good selectivity over dopamine reuptake inhibition (DRI), good
in vitro metabolic stability, weak CYP inhibition and drug-like physicochemical properties consistent
with CNS target space. Evaluation in an in vivo preclinical model of stress urinary incontinence showed
9 significantly increased urethral tone at free plasma concentrations consistent with its in vitro primary
pharmacology.
Received 5 June 2009
Revised 3 July 2009
Accepted 7 July 2009
Available online 31 July 2009
Keywords:
N-[(3S)-Pyrrolidin-3-yl]benzamides
SNRI
Ó 2009 Elsevier Ltd. All rights reserved.
Serotonin reuptake
Noradrenaline reuptake
CYP2D6 inhibition
P-gp recognition
MDCK-mdr1
Urinary incontinence
Selective inhibition of serotonin (5-HT) and noradrenaline (NA)
reuptake constitutes an attractive dual pharmacology approach to
the treatment of a number of diseases. For example dual 5-HT/NA
reuptake inhibitor duloxetine 1 has shown clinical efficacy in the
treatment of depression,1,2 diabetic neuropathic pain3 and stress
urinary incontinence (SUI).4 For SUI, it has been reported that the
prevention of urinary leakage could be mediated by stimulation
of central 5-HT2 and a1 adrenergic receptors resulting in more
effective closure of the urethral sphincter.5 This finding reinforces
the importance of identifying inhibitors exhibiting properties com-
patible with CNS penetration.
(PUP) at free plasma concentrations consistent with its in vitro pri-
mary pharmacology and its progression was thus halted.
We postulated that the lack of efficacy was due to poor CNS
penetration as 3 was subsequently shown to have very low cere-
brospinal fluid (CSF) to free plasma ratio of ꢀ0.1 in rat and dog
at steady state.13 Our hypothesis was confirmed when the pharma-
cological profile of the compound was evaluated in vivo in micro-
dialysis experiments.14 Indeed, in these experiments, striatal 5-HT
levels were markedly increased only after systemic administration
of doses of compound 3 that were significantly higher than
As part of our research efforts to identify new potential drug
candidates, we have recently reported several new templates6–11
that have delivered potent and selective SNRIs, and notably a 3-
amino-pyrrolidine template that furnished compounds 28 and 310
(Fig. 1). Unfortunately, when compound 3 was assessed in an
in vivo preclinical efficacy model for SUI12 (Fig. 2), it did not pro-
duce statistically significant increase in Peak Urethral Pressure
H
N
H
N
O
NHMe
S
O
N
N
O
Me
Cl
Me
Cl
3
2
1 duloxetine
* Corresponding author. Tel.: +44 1304 640950; fax: +44 1304 651987.
E-mail address: florian.wakenhut@pfizer.com (F. Wakenhut).
Figure 1. Structures of duloxetine and disclosed SNRIs 2 and 3.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.049

F. Wakenhut et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5078–5081
5079
Boc
Boc
N
N
a
R1
S
S
N
H
H2N
4
5
Figure 2. Schematic of urethral tone model.12 A pressure transducer is introduced
in the urethra, up to the bladder neck then taken out slowly whilst recording the
pressure. The difference in PUP between control and drug expressed as a percentage
is the direct efficacy measure.
b
expected based on the data observed after intrastriatal perfusion of
compound 3. As 3 exhibited physicochemical properties consistent
with CNS target space15 (m. wt. 315; clog P 3.3; log D7.4 0.8; pKa
9.4; HBD 1; HBA 3; TPSA 32 Å2) we further hypothesised that the
poor CNS penetration was probably the result of recognition by
the P-glycoprotein transporter (P-gp). Indeed, a significant degree
of efflux was observed when 3 was assessed in the MDCK-mdr1
cell line (15/64 ER = 4.3).16
Boc
Boc
N
N
c
R1
O
R1
O
S
S
N
N
R
R
In order to reduce the level of P-gp recognition we elected to
disrupt the H-bonding capability of the molecule as it has been
demonstrated to be a key factor for recognition.17 As preliminary
structure activity relationship (SAR) investigations had already
established that the pyrrolidine secondary amine was key to po-
tency, we decided to focus our attention on the amide group. Our
main strategy was to isomerize the amide into the benzylic posi-
tion hoping that in doing so we would disrupt P-gp recognition.
This modification led to the discovery of a novel series of N-
[(3S)-pyrrolidin-3-yl]benzamide derivatives (Fig. 3). The SAR of
these amides 7–30 is discussed in this Letter. Furthermore the
ADME, safety and efficacy profile of compound 9 aka PF-184,298
is also reported.
Target test compounds were conveniently prepared from com-
mercially available homochiral N-BOC-3-amino pyrrolidine 4 by
the general methods described in Scheme 1. Secondary amines 5
were prepared either by (i) standard reductive amination under
hydride or hydrogenolysis conditions of amine 4 with the appro-
priate aldehydes or ketones, or (ii) coupling of amine 4 with the
appropriate activated acid followed by reduction of the corre-
sponding amide with borane–THF complex, or (iii) palladium cata-
lysed N-arylation of amine 4 with bromobenzene. Amines 5 were
subsequently converted into amides 6 by benzoylation with the
appropriate benzoyl chloride. Finally HCl or TFA deprotection of
the N-BOC amines provided required targets 7–30. It is worth not-
ing that this synthetic route was highly amenable to parallel syn-
thesis therefore facilitating SAR exploration.
7-30
6
Scheme 1. Reagents and conditions: (a) (i) aldehyde/ketone, MeOH–PhMe, rt, then
NaBH4, MeOH, rt or aldehyde/ketone, H2 (60 psi), 10% Pd-C, EtOH, rt; (ii) R0COCl,
NEt3, CH2Cl2, rt or R0CO2H, 1-propanephosphonic acid (T3P), NEt3, CH2Cl2, rt then
BH3ꢁTHF, THF, reflux; (iii) tris(dibenzylideneacetone)dipalladium(0) (5 mol %), 1,10-
binaphthalene-2,20-diylbis-(diphenyl)phosphine (10 mol %), PhBr, PhMe, 100 °C; (b)
ArCOCl, NEt3, CH2Cl2 or dioxane rt; (c) TFA, CH2Cl2, rt or 4 N HCl in dioxane, rt.
find that selectivity over DRI, metabolic stability and selectivity
over ion channel activity (K+, hERG) consistent with our project
objectives could be achieved as illustrated by compounds 9 and
13. Increasing polarity, for example by introduction of a branched
4-tetrahydropyranyl (compound 15), or replacing the alkyl substi-
tuent by a phenyl (compound 16) was also tolerated providing
additional potent and balanced SNRIs. From this analysis the isobu-
tyl group was selected as the R1 group and a broader set of R sub-
stituents with different substitution patterns was investigated
(compounds 17–29). This analysis demonstrated that (i) 2,3-disub-
stitution was generally the preferred substitution pattern for dual
activity as can be seen when comparing compound 9 with com-
pounds 17–20; (ii) replacing one or both of the chlorine atoms with
other substituents generally resulted in an erosion in NRI activity
as illustrated by compounds 21 to 27; (iii) the series could also de-
liver selective inhibitors of the serotonin transporter (SRI) as illus-
trated by compound 28 with a single 2-Cl, 4-F substituent or
selective inhibitors of the noradrenaline transporter (NRI), for
example compound 29 with a single 2-iPr substituent. Finally, it
is also worth noting that although the (R)-enantiomer possessed
SRI activity its NRI potency was inferior to the (S) (13 vs 30, SRI:NRI
1:13).
Initial SAR investigations focused on the variation of the R1
group with the benzamide substitution R maintained as 2,3-di-Cl
(Table 1). A series of analogues 7–14 were prepared with alkyls
of increasing size. Interestingly, with the exception of the ethyl
substituent, probably too small (compound 7), all groups at R1
were accommodated, affording several potent and balanced SNRIs
(ratios of activities SRI:NRI, 1:1 to 1:4). We were also pleased to
From these experiments, a small set of compounds emerged as
preferred, exhibiting potent SNRI activity in combination with
good selectivity over DRI and were selected for further profiling
in the MDCK-mdr1 cell line (Table 2).
H
We were very pleased to find that our strategy of disrupting P-gp
recognition had been successful, as compound 9 exhibited good
membrane permeability combined with a reduced efflux ratio com-
pared to 3 (ER = 2.7 vs 4.3). However not all benzamide derivatives
demonstrated reduced efflux ratios and small structural modifica-
tions did result in significant differences. This is well illustrated
when comparing 9 and 13. Indeed, despite having similar physico-
chemical properties these compounds exhibited different efflux ra-
tios (2.7 for 9 and 6.4 for 13). Finally, the worst compound in this
assay was 15 as it demonstrated poorer membrane permeability,
H
N
N
R1
N
O
N
O
R
compounds 7-30
Cl
Cl
Figure 3. Structure of new N-[(3S)-pyrrolidin-3-yl]benzamides 7–30.

5080
F. Wakenhut et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5078–5081
Table 1
In vitro inhibition of human monoamine reuptakea,b, metabolic stability, K+ hERG channel activity, clog P for compounds 7–30c
H
N
H
N
R1
N
N
O
O
R
Cl
30
7-29
Cl
Compound
R
R1
5-HT Ki (nM)
NA Ki (nM)
DA Ki (nM)
HLM Clint
(
ll/min/mg)
K+, hERG IC50 (nM)
clog P
Duloxetine
3
7
8
9
—
—
—
—
5
9
29
16
6
39
15
4
6
20
6
3
3
45
52
217
36
21
95
24
13
11
37
10
11
37
198
43
435
640
1090
630
544
3730
404
273
983
2600
945
501
2400
2430
3070
430
1330
2070
516
1610
2370
2560
NT
2540
3590
3090
4.3
3.4
2.6
3.2
3.6
2.9
3.7
4
<7.6
<13
<7
11
<8
26
NT
<7
34
13
<25
26
29
NT
36
23.5
NT
26
21
20
26
NT
20
24,700
>20,000
>18,600
20,000
>10,000
7140
16,800
10,300
7740
>40,000
7920
NT
19,800
>10,000
17,200
NT
2,3-Di-Cl
2,3-Di-Cl
2,3-Di-Cl
2,3-Di-Cl
2,3-Di-Cl
2,3-Di-Cl
2,3-Di-Cl
2,3-Di-Cl
2,3-Di-Cl
2,3-Di-Cl
2,4-Di-Cl
2,5-Di-Cl
3,5-Di-Cl
3,4-Di-Cl
3-Cl
2-Cl, 3-CF3
2-Me,3-Cl
2-Me,3-F
2-Cl, 3-F
2-F, 3-Cl
2-CF3, 3-F
2-Cl, 4-F
2-i-Propyl
2,3-Di-Cl
Ethyl
n-Propyl
i-Butyl
CH2CF3
CH2CH2c-Propyl
CH2t-Butyl
c-Pentyl
CH(Ethyl)2
4-Tetrahydropyranyl
Phenyl
i-Butyl
i-Butyl
i-Butyl
i-Butyl
i-Butyl
i-Butyl
i-Butyl
i-Butyl
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
3.6
4
1.8
3.7
3.7
3.7
3.7
3.7
3
3.7
3.5
2.9
3.1
3.1
3.4
3.1
3.6
3.6
54
6
4
50
19
11
3
13
15
10
25
7
135
103
29
69
67
139
26
263
65
4120
52,400
NT
i-Butyl
i-Butyl
i-Butyl
i-Butyl
i-Butyl
c-Pentyl
NT
>10,000
>10,000
NT
12,900
NT
1320
13
<7
NT
169
a
See Ref. 9 and 11 for definitions of terms and complete details of assay conditions.
Monoamine reuptake Ki are geometric means of a minimum of 2 experiments. Differences of <2-fold should not be considered significant.
NT denotes not tested.
b
c
Table 2
Table 3
Profile of compounds 3, 9, 13 and 15 in the MDCK-mdr1 cell line
Physicochemical properties, ADME profiles and K+ channel affinities of compound 9a
MDCK-mdr1 AB/BA Papp ꢂ 10ꢃ6 cm sꢃ1
MDCK-mdr1, ER
9
3
9
13
15
15/64 (n = 2)
16/43 (n = 3)
10/64 (n = 3)
1/20 (n = 1)
4.3
2.7
6.4
20
MW
315
3.6/0.6
1/3
10
32
clog P/log D7.4
HBD/HBA count
pKa
TPSA, Å2
HLM, Cli
h.heps, Cli
l
L/min/mg
L/min/millions cells
11
3
l
probably as a result of its lower lipophilicity and extra oxygen atom,
in combination with an increased efflux ratio ER = 20.
CYP2D6 inh. (dextromethorphan), IC50 (nM)
CYP1A2 inh. (tacrine), IC50 (nM)
>30,000
>30,000
>30,000
>30,000
>30,000
>30,000
>30,000
12/26
20,000
CYP2C9 inh. (diclofenac), IC50 (nM)
CYP2C19 inh. (S-methphenytoin), IC50 (nM)
CYP3A4 inh. (felodipine), IC50 (nM)
CYP3A4 inh. (midazolam), IC50 (nM)
CYP3A4 inh. (testosterone), IC50 (nM)
Compound 9 was selected for further evaluation based on the
combination of its superior profile in the MDCK-mdr1 cell line,
metabolic stability in human liver microsomes (HLM) predictive
of low in vivo clearance, and selectivity over ion channel activity
consistent with our project objectives as measured by binding to
the hERG channel. (Table 3).
Additional screening in in vitro ADME screens showed 9 to have
excellent metabolic stability in human hepatocytes. Compound 9
also exhibited weak inhibition of all major CYP450 enzymes (2D6,
CaCO-2, AB/BA @10
l
M, Papp ꢂ 10ꢃ6 cm sꢃ1
K+, hERG, Ki (nM)
a
See Ref. 9 for definitions of terms and assays.
3A4, 1A2, 2C9, 2C19, IC50s >30
ity in the CaCO-2 cell line, therefore predicting for good oral absorp-
tion. Finally, when compound was evaluated for broader
l
M) and good membrane permeabil-
To demonstrate the impact of the reduced P-gp recognition on
blood brain barrier (BBB) penetration, the CSF to unbound plasma
concentration ratio was measured in the rat following a steady
state infusion regimen. We were pleased to find that the observed
ratio of 0.45 was superior to the ratio of 0.1 observed for com-
pound 3. Now confident that 9 had significantly improved CNS
penetration compared to 3, its performance was evaluated in the
anaesthetized dog model described above (Fig. 4).12 From this
9
pharmacological activity in a panel of receptors, ion channels and
TM
enzymes (Cerep, BIOPRINT ), it was found to be highly selective
exhibiting binding affinity (>50% inhibition at 10
Na channel (site 2; IC50 = 3.6 M), the sigma receptor (IC50
5.1 M) and the kappa opioid receptor (IC50 = 5.8 M).
lM) only for the
l
=
l
l

F. Wakenhut et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5078–5081
5081
PUP (% change relative to control)
References and notes
80
70
60
*
1. (a) Jann, M. W.; Slade, J. H. Pharmacotherapy 2007, 11, 1571; (b) Montgomery, S.
Int. J. Psych. Clin. Pract. 2006, 10, 5; (c) Jackson, S. Curr. Med. Res. Opin. 2005, 21,
1669.
2. (a) Wernicke, J. F.; Iyengar, S.; Ferrer-Garcia, M. D. Curr. Drug Ther. 2007, 2, 161;
(b) Bauer, M.; Moeller, H.-J.; Schneider, E. Exp. Opin. Pharmacotherapy 2006, 7,
421.
3. Kajdasz, D. K.; Iyengar, S.; Desaiah, D. Clin. Ther. 2007, 29, 2536.
4. (a) Mariappan, P.; Alhasso, A.; Ballantyne, Z.; Grant, A.; N’Dow, J. Eur. Urol.
2007, 51, 67; (b) Steers, W. D.; Herschorn, S.; Kreder, K. J.; Moore, K.; Strohbehn,
K.; Yalcin, I.; Bump, R. C. BJU Int. 2007, 100, 337.
5. Thor, K. B.; Kirby, M.; Viktrup, L. Int. J. Clin. Pract. 2007, 61, 1349.
6. (a) Fray, M. J.; Bish, G.; Brown, A. D.; Fish, P. V.; Stobie, A.; Wakenhut, F.;
Whitlock, G. A. Bioorg. Med. Chem. Lett. 2006, 16, 4345; (b) Fray, M. J.; Bish, G.;
Fish, P. V.; Stobie, A.; Wakenhut, F.; Whitlock, G. A. Bioorg. Med. Chem. Lett.
2006, 16, 4349.
*
*
*
*
50
40
30
20
10
0
0 nM
0.5 nM
4 nM
7.5 nM
27 nM
103 nM 227 nM
Compound 9 free plasma concentration
Figure 4. The effects of compound 9 on peak urethral pressure expressed as
percentage change relative to control. Data is expressed as the mean SEM and
significant difference relative to control is denoted thus *p <0.05 (n = 4, n denotes
the number individual animals used to generate a cumulative dose response via iv
7. Fish, P. V.; Fray, M. J.; Stobie, A.; Wakenhut, F.; Whitlock, G. A. Bioorg. Med.
Chem. Lett. 2007, 17, 2022.
8. Whitlock, G. A.; Blagg, J.; Fish, P. V. Bioorg. Med. Chem. Lett. 2008, 18, 596.
9. Fish, P. V.; Deur, C.; Gan, X.; Greene, K.; Hoople, D.; Mackenny, M.; Para, K. S.;
Reeves, K.; Ryckmans, T.; Stiff, C.; Stobie, A.; Wakenhut, F.; Whitlock, G. A.
Bioorg. Med. Chem. Lett. 2008, 18, 2562.
infusion of compound to
a steady state free plasma concentration. Each bar
represents the mean response versus mean free plasma concentration for each
step). Dog functional potency for compound 9 was dSRI IC50 = 7.4 nM (platelet,
n = 18), dNRI IC50 = 28.6 nM (carotid artery, n = 15).19
10. Wakenhut, F.; Fish, P. V.; Fray, M. J.; Gurrell, I.; Mills, J. E.; Stobie, A.; Whitlock,
G. A. Bioorg. Med. Chem. Lett. 2008, 18, 4308.
11. SNRI activity of target compounds was established using scintillation proximity
assay (SPA) technology by assessing their ability to inhibit binding of selective
radioligands at the human 5-HT, NA and DA transporters. Cellular membrane
preparations generated from recombinant HEK293 cells expressing a single
monoamine transporter were utilised for these experiments. For details of the
assay conditions, see: Andrews, M. D.; Brown, A. D.; Fish, P. V.; Fray, M. J.;
Lansdell, M. I.; Ryckmans, T.; Stobie, A.; Wakenhut, F.; Gray, D. L. F. WO Patent
064351, 2006, pp 56–59. The BioByte software was used to assess clog P.
12. Adult, female, nulliparous beagle dogs were anaesthetised initially with
sodium pentobarbitone to induce surgical anaesthesia (30 mg/kg iv), and
study, it is evident that 9 significantly increases urethral tone in a
dose dependent manner. Furthermore, at an unbound plasma con-
centration of 4 nM, 9 increases PUP in the dog by 35%. To put this
result into context, this is superior to the 26% increase induced in
this model by 4.5 nM duloxetine, a concentration we estimate to
be close to human therapeutic free plasma concentrations based
on published human pharmacokinetic data18 and in-house human
plasma protein binding determinations (estimated free Cmax 5–
10 nM at 40 mg bid).
Finally, 9 was found to be clean in a package of in vitro and
in vivo genetic toxicology screens, produced no dose-limiting tox-
icity in acute and sub-chronic (14-day) oral rat and dog toxicology
screens and did not exhibit significant effect on spontaneous loco-
motor activity vs vehicle control in rats when administered orally
up to 15 mg/kg.
In summary, we have described the discovery of novel benzam-
ide derivatives as potent SNRIs. More specifically, compound 9 aka
PF-184,298 exhibited potent SNRI activity in combination with
good DRI selectivity, good metabolic stability, weak CYP inhibition,
low affinity for ion channels and no significant off-target pharma-
cology. Furthermore, isomerisation of the amide moiety to the ben-
zylic position resulted in a significant reduction in P-gp recognition
that translated into improved BBB penetration. When PF-184,298
was assessed in a dog in vivo preclinical model for stress urinary
incontinence, it elicited a dose dependent increase in PUP superior
to that observed with SNRI duloxetine. Finally, PF-184,298 did not
produce dose-limiting toxicity in preclinical toxicology screens and
was progressed to human clinical studies. The results of these
studies will be reported in future publications.
then transferred to
a-chloralose anaesthetic (70–100 mg/kg iv induction
followed by constant infusion to deliver 10–15 mg/kg/h iv for the duration of
the experiment), following completion of surgery. Animals were intubated, and
respiration maintained at a constant rate of 14 breaths per minute. Tidal
volume was adjusted to maintain expired air within normal physiological
limits. Throughout the experiment the animal was maintained at a core body
temperature of approximately 37 °C using a thermocouple heated blanket
(Harvard Apparatus Ltd., Kent, UK). Cannulation of superficial vessels was
carried out to allow administration of compounds, blood sampling, infusion of
anaesthetic, and monitoring of arterial pressure (Millar, 7F, Millar Instruments,
US). A midline incision was made in the abdomen to expose the bladder. The
ureters were cannulated to drain urine throughout the experiment. The dome
of the bladder was cannulated and the cannula fed through the bladder to the
external urethra. This catheter was used to introduce the urethral pressure
catheter (Millar SUPC-380C), into the urethra. The bladder was filled with
saline to achieve an intravesical pressure of approximately 8–10 mmHg, and
bladder pressure was measured by connecting the bladder catheter to
a
pressure transducer (Model DTX plus, Becton-Dickenson UK Ltd, Oxford, UK).
Following completion of surgery, animals were allowed to stabilise for at least
60 minutes before starting urethral pressure profilometry measurements.
Urethral pressure profilometry (UPP) was measured by withdrawing the
pressure transducer through the urethra at a constant rate. A full profile
measurement was obtained approximately every 6 min, and readings were
taken continuously throughout the experiment. Baseline measurements were
performed until 4 consistent measurements were identified, after which drug
administration commenced. From the profilometry data, peak urethral
pressure was measured (PUP, mmHg). Changes in PUP were compared with
mean baseline value and expressed as % change from baseline, recorded over
the 4 consecutive profiles obtained prior to test drug or vehicle administration.
All experiments involving animals were carried out in compliance with
national legislation, specifically the UK Animal (Scientific Procedures) Act
1986, and subject to local ethical review.
Acknowledgements
13. Measuring CSF levels is commonly used as a way to assess Blood Brain Barrier
penetration, see: (a) Lin, L. H. Curr. Drug. Metab. 2008, 9, 46; (b) Feng, M. R. Curr.
Drug. Metab. 2002, 3, 647.
14. Deecher, D. C.; Beyer, C. E.; Johnston, G.; Bray, J.; Shah, S.; Abou-Gharbia, M.;
Andree, T. H. J. Pharm. Exp. Ther. 2006, 318, 657.
15. Mahar Doan, K. M.; Humphreys, J. E.; Webster, L. O.; Wring, S. A.; Shampine, L.
J.; Serabjit-Singh, C. J.; Adkison, K. K.; Polli, J. W. J. Pharm. Exp. Ther. 2002, 303,
1029.
16. The MDCK-mdr1 cell line is commonly used to assess P-gp recognition, see:
Hammarlund-Udenaes, M.; Bredberg, U.; Friden, M. Curr. Top. Med. Chem. 2009,
9, 148.
We wish to thank Carol Bains, Gerwyn Bish, Timothy Buxton,
Edelweiss Evrard, Arnaud Lemaitre, Debbie Lovering, Edward Peg-
den, Bhairavi Patel, and Melanie Skerten for compound synthesis.
We are also grateful to Caroline Tolley and Doreen Davey for
screening data, Katherine Fenner and Ian Gurrell for generating
the MDCK-mdr1 data and to Kelly Conlon for providing the ure-
thral tone model materials and methods summary.
17. For a substrate model of the P-gp transporter, see: Seelig, A. Eur. J. Biochem.
1998, 251, 252.
18. (a) Chan, C.; Yeo, K. P.; Pan, A. X.; Lim, M.; Knadler, M. P.; Small, D. S. Br J. Clin.
Pharm. 2006, 63, 310; (b) kinner, M. H.; Kuan, H.; Pan, A.; Sathirakul, K.;
Knadler, M. P.; Gonzales, C. R.; Yeo, K. P.; Reddy, S.; Lim, M.; Ayan-Oshodi, M.;
Wise, S. D. Clin. Pharm. Ther. 2003, 73, 170.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.07.049.
19. For detailed experimental procedures see Supplementary data.