Naphthol derivatives as TRPV1 inhibitors for the treatment of urinary incontinence

Article abstract of DOI:10.1016/j.bmcl.2011.04.013

We have identified naphthol derivatives as inhibitors of the vanilloid receptor TRPV1 by high throughput screening. The initial lead showed high clearance in rats and has been optimized by enhancing the acidity of the phenol group. Compound 6b has reduced

Full text of DOI:10.1016/j.bmcl.2011.04.013

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3354–3357
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Naphthol derivatives as TRPV1 inhibitors for the treatment
of urinary incontinence
Klaus Urbahns a, , Takeshi Yura a, Muneto Mogi a, Masaomi Tajimi a, Hiroshi Fujishima a, Tsutomu Masuda a,
⇑
Nagahiro Yoshida a, Toshiya Moriwaki a, Timothy B. Lowinger a, Heinrich Meier b, Fiona Chan c,
David Madge c, Jang B. Gupta a
a Research Center Kyoto, Bayer Yakuhin, Ltd, Kizu, Soraku, Kyoto 619-0216, Japan
b Bayer Healthcare, Aprather Weg, 42096 Wuppertal, Germany
c Xention Limited, Iconix Park, London Road, Pampisford, Cambridge, CB22 3EG, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
We have identified naphthol derivatives as inhibitors of the vanilloid receptor TRPV1 by high throughput
screening. The initial lead showed high clearance in rats and has been optimized by enhancing the acidity
of the phenol group. Compound 6b has reduced clearance, improved potency and is active in rat cystom-
etry models of urinary incontinence after intravenous administration.
Received 21 February 2011
Revised 29 March 2011
Accepted 3 April 2011
Available online 9 April 2011
Ó 2011 Elsevier Ltd. All rights reserved.
TRPV1 (Transient receptor potential vanilloid 1, vanilloid recep-
tor 1 or VR1) is an ion channel that gates after a variety of stimuli
such as heat, acid or chemical agents like capsaicin (1a) or resinif-
eratoxin (1b). It has been suggested that inhibiting TRPV1 could
O
H
O
O
HO
O
O
HN
O
O
MeO
HO
HO
MeO
1a: Capsaicin
1b: Resiniferatoxin
O
S
Cl
N
H
N
HN
N
HO
HO
H
HO
3a: Lead from High Throughput Screening
2: Capsazepine
Figure 1. Structural comparison of HTS hit 3a with the agonists 1a/b and the known antagonist 2.
⇑
Corresponding author at present address: Merck KGaA, Frankfurter Strasse 250, 64293 Darmstadt, Germany. Tel.: +49 6151 72 6283; fax: +49 6151 72 3129.
E-mail address: Klaus.Urbahns@merck.de (K. Urbahns).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.04.013

K. Urbahns et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3354–3357
3355
Table 1
B
a)
HN
Y
Structure–activity relationship of naphthol-based TRPV-1 antagonists Variation of the
anilino substitution R
I
3a-m, r-z
X
O
A
O
a’)
R
3n-q
HN
N
H
HO
b)
c)
e)
f)
a)
IIb
IV
4a
I:
A=H, B=H, X=H, Y=H
IIa: A=Me, B=H, X=H, Y=H
IIb: A=H, B=Me, X=H, Y=H
IIIa: A=H, B=H, X=Cl, Y=H
IIIb: A=H, B=H, X=Cl, Y=Cl
IIIc: A=H, B=H, X=Cl, Y=Br
IIId: A=H, B=H, X=Br, Y=Br
d)
Compd
R
r-IC50 [nM]
h-IC50 [nM]
c Log P
4b
5c
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
H
53
64
29
87
4.1
4.1
4.9
4.9
4.0
4.5
4.5
4.0
4.0
3.6
3.9
3.5
4.7
4.6
2.5
2.8
3.6
4.1
4.2
4.8
4.8
5.2
5.3
4.9
5.2
5.5
2-CF3
3-CF3
4-CF3
2-Cl
3-Cl
4-Cl
3-Me
4.5
4.9
38
3.2
11
3.5
5.4
52
6.1
12
11
14
8.0
7.5
28
4c
4d
IIa
19
21
3-F
g)
h)
a)
a)
3-OMe
3-NO2
3-COMe
3-Br
8.6
14
O
39
O
8.0
1.6
520
670
270
7.1
>1000
30
5.7
1.7
>1000
>1000
520
3.9
>1000
24
TIPSO
3-COOEt
3-CH2OH
3-CH(CH3)OH
3-COOH
3-SMe
2,6-Cl2
2,5-Cl2
2,4-Cl2
3,4-Cl2
3,5-Cl2
2-CF3, 4-Cl
5-CF3, 2-Cl
3-CF3, 4-Cl
IIIa
i)
6a
a)
IIIb
IIIc
6b
6c
IV
3s
3t
j)
a)
3u
3v
3w
3x
3y
3z
16
12
4.9
12
31
13
O
4.5
12
O
19
12
k)
a)
l)
IIId
V
6d
5a
1.9
2.2
IC50 values are medians of three dose–response curves.
V
Figure 2. Synthesis of TRPV1 inhibitors. Reactions and conditions: (a) ArNCO,
dioxane, reflux, 3 h, 20–95%. (a’) 1,1’-carbonyldi-(1,2,4 triazole) (1 equiv) THF, rt
1 h, then ArNH2, 50 °C, 15 h, (40–80%). (b) Pyridine (2 equiv), (CF3CO)2O (1.5 equiv),
THF, rt, 1.5 h 30%; (2) MeI (1.2 equiv), K2CO3 (5 equiv), Bu4NI, acetone, rt, 2.5 h
(63%). (3): NaBH4 (0.95 equiv) EtOH, rt, 87%. (c) (1) NaNO2 (1.1 equiv), KI
(1.1 equiv), THF/3 N HCl (1:2), 1 h, 0 °C, 26%; (2). Bu3SnCHCH2 (1.2 equiv), Pd(PPh3)4
(0.2 equiv), toluene, 90 °C, 16 h, 100%; (3) Imidazole (1.5 equiv), (iPr)3SiCl
(1.1 equiv), DMF, 50 °C, 16 h, 63%; (4) 9-BBN (1.1 equiv), THF, rt, 5 h, then aq NaOH
(3 N), aq H2O2 (30%), rt 6 h, 56%. (5) HIO4 (1 equiv), CrO3 (1 equiv) CH3CN/H2O (3:1),
0 °C, 30 min, 24%. (d) (1) DMAP (0.2 equiv), 1-(3-diethylaminopropyl)3-ethylcar-
bodiimide (1.2 equiv), CH2Cl2, rt, 16 h, 89%; (2) Bu4NF (3 equiv), THF, rt, 30 min,
65%. (e) (EtS)2NCN (2 equiv), (nBu)2O, reflux, 3 h, then 4Cl-3CF3ArNH2 (3 equiv)
reflux, 12 h, 30%. (f) 3-F-C6H4CH2COOH, DMAP (0.2 equiv), 1-(3-diethylamino-
propyl)3-ethylcarbodiimide (1.2 equiv), CH2Cl2, rt, 16 h, 62%. (g) (1) PhCHO
(1.1 equiv), Na2SO4 (5 equiv), THF, reflux, 12 h, 98%. (2) MeI (2 equiv), NaOH
(2 equiv), acetone, rt, 2 h, then 2 N HCl/THF (2:1), rt 2 h, 93%. (h) NCS (1 equiv), THF,
rt, 16 h, 69%. (i) NCS (2.2 equiv), THF, rt 16 h, 70%. (j) NBS (1 equiv), THF, rt, 16 h,
41%. k) NBS (2 equiv), THF, rt, 16 h, 21%. (l) (1) NH2OHHCl (3 equiv), K2CO3
(4 equiv), MeOH, reflux, 16 h, quant. (2) Pd/C (10%), AcOH (cat.), H2 (1 atm), MeOH,
rt, 16 h, quant. (3) BBr3 (1.3 equiv), CH2Cl2, 0 °C, 98%. (4) 4-Cl-3-CF3-C6H3NHCOPh,
DMSO, 90 °C, 16 h, 60%.
Table 2
Structure–activity relationship of naphthol-based TRPV-1 antagonists Variation of the
urea portion R
B
R
A
C
HO
Compd
A
B
C
R
h-IC50 [nM]
4a
4b
4c
4d
NMe
CH2
NH
O
O
NCN
O
NH
NH
NH
CH2
3-CF3, 4-Cl
3-CF3, 4-Cl
3-CF3, 4-Cl
3-F
44
34
340
>1000
NH
IC50 values are medians of three dose–response curves.
Using a Ca-flux primary assay system, we performed a high
throughput screen and identified 3a as a lead with selectivity
against the P2X1 ion channel.3 This compound is a nanomolar
inhibitor of both rat and human TRPV1. Whilst its phenol moiety
is structurally related to the agonists 1a and 1b, its bicyclic struc-
ture is reminiscent to the known antagonist Capsazepine (2) and
offers ample opportunity for structural modification (Fig. 1). Inter-
estingly, other groups have identified related HTS-hits, but fol-
lowed optimization strategies different to ours, indicating the
versatility of the lead.4
provide potential treatments for pain, cough or urinary inconti-
nence. First TRPV1 antagonists have now reached clinical trials
with promising results for nociception, however, most compounds
exhibit mechanism-based hyperthermia as a side effect. Strategies
aimed at widening the therapeutic window include co-administra-
tion of anti-pyretics, shortening the compounds’ half life or
addressing alternate indications, such as urinary incontinence
(UI).1
UI is characterized by involuntary leakage of urine and can have
profound and distressing impact on the quality of life for affected
individuals. The current treatment of UI involves muscarinic
antagonists and is associated with mechanism-based side effects
such as dry mouth symptoms.2
The majority of compounds were prepared from naphthol I by
alkylation or halogenation to furnish intermediates IIa–b and
IIIa–d. Ureas were synthesized from these intermediates or I by
coupling directly with the corresponding aryl isocyanate.

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K. Urbahns et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3354–3357
Table 3
Structure–activity relationship of naphthol-based TRPV-1 antagonists Variation of the
naphthalene system
Cl
CF3
F
HN
HN
HN
O
HN
O
HO
HO
5b
5a
Cl
CF3
Cl
HN
CF3
HN
HN
O
HN
O
HO
X
O
Y
6a-d
h-IC50 [nM]
5c
Compd
X
Y
r-IC50 [nM]
AUC [ng h ml-1
]
5a
5b
5c
6a
6b
6c
6d
—
—
—
Cl
Cl
Cl
Br
—
—
—
H
Cl
Br
Br
nd
>1000
19
4.5
2.1
4.9
16
260
>1000
32
3.4
5.9
nd
nd
114
3
84
6.8
7.5
477
596
The area under the curve (AUC) is obtained after oral administration to rats and
relates to a 1 mg kgÀ1 dose.
Figure 3. Cystometry evaluation in the cyclophosphamide (CYP)-induced cystitis
model. Compound 6b dose-dependently inhibits voiding frequency and bladder
capacity after intravenous administration to female rats.
Table 4
or isoelectronic replacement (4b) resulted in drop of activity by an
order of magnitude.
Cystometry evaluation in the capsaicin-induced overactive bladder (OAB) model
Compd
Capsaicin-induced OAB
The carbonyl group (B) appeared to be even more critically in-
volved in binding as the corresponding cyano-guanidine 4c was
100-fold less active. Comparison of 4d with 3i highlighted the
importance of the distal urea NH (C) for molecular recognition.
We also undertook first variations of the naphthalene system
(Table 3). Compared to 3z, the tetrahydro-naphthalene 5a showed
a 100-fold drop in activity. Similarly, eliminating the ring alto-
gether reduced activity markedly (5b vs 3i).
3 mg kgÀ1
10 mg kgÀ1
6b
6c
6d
n.d.
88%
11%
55%
n.d.
65%
Compounds 6b–d were given intravenously to rats. Inhibition of micturition fre-
quency is expressed in percent as compared to control.7
Whilst 3z was the most potent compound of the series, we
didn’t consider it for in vivo investigations. This naphthol was
highly cleared in rats (23 l hÀ1 kgÀ1), probably as a result of phase
II metabolism. Simple alkylation of the hydroxyl group reduced
clearance 10-fold (5c: 2.0 l hÀ1 kgÀ1) and furnished detectable
AUC’s after oral administration. Unfortunately, 5c’s TRPV1 activity
was also reduced indicating the importance of the hydroxyl group
for the pharmacophore.
We were speculating that enhancing the naphthol’s acidity by
introducing electron withdrawing substituents would reduce
clearance and enhance oral exposure, whilst leaving the pharma-
cologically important hydroxyl group intact. Whereas potency
was maintained, 6a–d showed a good correlation of naphthol
acidity with oral AUC and 6b–d reduced capsaicin-induced
micturition frequency in female rats (Table 4).8 Due to its low
clearance (0.16 l hÀ1 kgÀ1), 6b was selected for further character-
isation in the cyclophosphamide-induced cystitis model and
favourably altered voiding frequency as well as bladder capacity
at intravenous doses above 0.05 mg kgÀ1 (Fig. 3).9 Whilst useful
Compounds 3n–q display sensitive functional groups and therefore
we used the CDT coupling method5,6 (Fig. 2). Compound 5b is com-
mercially available.7
We explored the substitution pattern of 3a’s aniline fragment
with compounds 3b–g and the 3-position appeared to contribute
most strongly to TRPV1 activity (Table 1): 3c and 3f offered by
an order of magnitude better potency than 3a. Therefore, we inves-
tigated the 3-position with further substituents (3h–r). Not sur-
prisingly, TRPV1 activity correlated well with c log P for all
derivatives 3a–z, indicating the lipophilic nature of the binding
site. The 2,6-disubstituted 3s was inactive, probably indicating
the importance of an in-plane urea/aniline conformation for activ-
ity. Comparing the di-substituted derivatives 3s–z, we concluded
that the 4-Cl, 3-CF3 substitution pattern in 3z was most potent, al-
beit most lipophilic. Our compounds showed no notable differ-
ences between rat and human TRPV1.
We also explored the lead’s urea moiety (Table 2). 3c’s proximal
urea NH (A) seemed to be involved in a H-bond as methylation (4a)

K. Urbahns et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3354–3357
3357
measured at 250 msec intervals and IC50 values were determined after agonist
stimulation (200 nM ,b methylene ATP). All compounds showed activity
>1000 nM. 2’,3’-O-(2,4,6-trinitrophenyl)adenosine-5’-triphosphate was used as
a reference.
as
a pharmacological tool for intravenous studies, 6b’s oral
a
bioavailability was still low (3%). This was probably due to 6b’s
limited solubility (0.03 mg lÀ1), a feature not uncommon for
urea-based leads.
Starting from an HTS lead, we have identified single-digit nano-
molar TRPV1 antagonists. In preparing more acidic compounds, we
have reduced rat clearance by two orders of magnitude. Compound
6b showed intravenous activity in two rat cystometry models,
highlighting the potential of TRPV1 antagonists for the treatment
of UI. Further modifications of this lead towards orally active
compounds will be reported shortly.
4. The initial hit compound identified from HTS was compound 3r (singleton), a
commercially available material, which was apparently present in HTS
compound collections of several competitors. Therefore, it is not surprising
that related leads have been described by other groups and optimised in an
independent, different way following our initial patent applications: Gomtsyan,
A.; Rayburt, E.K.; Schmidt, R.G.; Zheng, G.Z., Perner, R.J.; Didomenico, S.; Koenig,
J.R.; Turner, S.; Jinkerson, T.; Drizin, I.; Hannick, S.M.; Macri, B.S., McDonald,
H.A.; Honore, P.; Wismer, C.T., Marsh, K.C.; Wetter, J.; Stewart, K.D.; Oie, T.;
Jarvis, M.F.; Surowy, C.S.; Faltynek, C.R.; Lee, C.H.; J. Med. Chem, 2005, 48, 744.
This group immediately abandons the phenolic hydroxy moiety whilst our
strategy includes maintaining this structural feature. McDonnell, M.E.; Zhank,
S.P.; Nasser; N. Dubin, A.E.; Dax, S.L.; Bioorg. Med. Chem. Lett., 2004, 14, 531. In
contrast to the work presented here, this group describes mainly benzylic
analogs of our lead.
References and notes
5. Synthetic details: Yura, T.; Mogi, M.; Ikegami, Y.; Masuda, T.; Kokubo, T.;
Urbahns, K.; Lowinger, T. B.; Yoshida, N.; Freitag, J.; Meier, H.; Wittka-Nopper,
R.; Marumo, M.; Shiroo, M.; Tajimi, M.; Takeshita, K.; Moriwaki, T.; Tsukimi, Y.
(Bayer Yakuhin) WO 03014064 2003, Chem. Abstr. 2003, 139, 79169. Yura, T.;
Mogi, M.; Ikegami, Y.; Masuda, T.; Kokubo, T.; Urbahns, K.; Yoshida, O.;
Marushige, M.; Shiroo, M.; Tajimi, M.; Takeshita, K.; Moriwaki, T.; Tsukimi, Y.
(Bayer Yakuhin) WO 03055848, 2003, Chem.Abstr. 2003, 139, 79170.
Analytical data for 6a: mp 260 decomp.; MS(ESI): 415; 6b: mp 255, decomp.;
MS(ESI): 449; 6c: mp 209, decomp.; 6d: mp 242–243, MS(ESI): 536.
6. Beyerman, H. C.; van den Brink, W. Rec. Trav. Chim. Pays Bas 1961, 80, 1372.
7. [CAS Nr. 1183505-20-5].
8. Acidities were calculated using ACD/PhysChem Suite Vers.12 (Advanced
Chemistry Development, Toronto, On, Canada, 2010). pKa’s: 3z: 9.66; 6a:9.28;
6b: 8.83; 6c: 8.81; 6d: 8.79.
9. Cystometry models were performed according to Lecci, A.; Giuliani, S.;
Santicioli, P.; Maggi, C.A. Eur. J. Pharmacol., 1994, 259 129.
1. Szallasi, A.; Cortright, D. N.; Blum, C. A.; Eis, S. R. Nat. Rev. Drug Discovery 2007,
6, 357; (b) Kym, P. R.; Kort, M. E.; Hutchins, C. W. Biochem.Pharmacol. 2009, 78,
211; (c) Gunthorpe, M. J.; Chizh, B. A. Drug Discovery Today 2009, 14, 56; (d)
Gavva, N. Open Drug Discovery J. 2009, 1, 1; (e) Alawi, K.; Keeble, J. Pharmacol.
Ther. 2010, 152, 181; (f) Stec, M.M.; Bo, Y.; Chakrabarti, P.P.; Liao, L.; Ncube,
M.; The use of the TRPV1 receptor has been patent-protected: Curtis, R.A.J.
(Millenium Pharmaceuticals) WO 00/029577, 2000, Chem. Abstr. 2000, 133,
351661.; (g) Use of TRPV1 antagonists for UI: Shiroo, M.; Yura, T.; Yamamoto,
N.; Tajimi, M.; Tsukimi, Y. (Bayer Yakuhin), WO 04028440 2004, Chem. Abstr.
2004, 140, 297515.; Capsazepine’s activity in UI: (h) Bevan, S.; Hothi, S.; James,
I. F.; Rang, H. P.; Shah, K.; Walpole, C. S. J.; Teats, J. C. Br. J. Pharmacol. 1992,
107, 544; (i) Walpole, C. S.; Bevan, S.; Boverman, G.; Boelsterli, J. J.;
Breckenridge, R.; Davies, J. W.; Hughes, G. A.; James, I.; Oberer, L. J. Med.
Chem. 1994, 37, 1942; Strategies to overcome hyperthermia: (j) Palin, R.;
Abernethy, L.; Ansar, N.; Cameron, K.; Clarkson, T.; Dempster, M.; Dunn, D.;
Easson, A.-M.; Edwards, E.; Maclean, J.; Everett, K.; Feilden, H.; Ho, K.-K.;
Kultgen, S.; Littlewood, P.; McArthur, D.; McGregor, D.; McLuskey, H.; Neagu,
I.; Neale, S.; Nisbet, L. A.; Ohlmeyer, M.; Pham, Q.; Ratcliffe, P.; Rong, Y.;
Roughton, A.; Sammons, M.; Swanson, R.; Tracey, H.; Walker, G. Bioorg. Med.
Chem. Lett. 2011, 21, 892.
Cyclophosphamide-induced cystitis: cystitis was induced by cyclophosphamide
administration (150 mg/kg, ip, saline) to female Sprague Dawley rats (180–
250 g). After 48 h
a bladder catheter was implanted and saline at room
temperature infused into the bladder for 20 min (3.6 ml/min, micturition
cycle). Intravesical pressure was recorded (Viggo-Spectramed PTe Ltd, DT-
2. Shaban, A.; Drake, M.; Hashim, H. Auton. Neurosci. 2010, 152, 4; Sorbera, L. A.;
Bozzo, J.; Rosa, E.; Dulsat, C. Drugs of the Future 2008, 33, 455; Tiwar, A.;
Naruhanahalli, K. S. Exp. Opin. Invest. Drugs 2006, 15, 1017; (d) Chancellor, M. B.;
de Groat, W. C. J. Urology 1999, 162, 3.
3. Assay descriptions: h/r-VR1 c-DNA was transfected into a CHOluc9aeq cell line
(pcDNA3) containing aequorin and CRE-luciferase reporter genes. After loading
XXAD) and after
3 micturition cycles, 6b was administered intravenously
(ethanol, Tween 80, saline 1:1:8). The cystometry parameters were determined
from the micturition interval and the volume of infused saline and evaluated
using unpaired Student’s t-test. Probability levels of less than 5% were accepted
as statistically significant. Data shown are mean SEM.
Capsaicin-induced micturition: following the protocol above, instead of
cyclophosphamide administration 48 h prior to the experiment, Capsaicin
(30 lM in saline) was infused at room temperature 2 min after compound
administration (6b, 6c, 6d, iv in ethanol, Tween 80, saline 1:1:8). Micturition
frequency is expressed as percent of control.
with Fluo-3AM (2 lM, Molecular Probes) and stimulation with capsaicin
(10 nM), fluorescence changes were recorded for 1 min (kex = 488 nm/
kem = 540 nm, Hamamatsu photonics) and IC50values determined.
A h-P2X1 transfected CHOluc9aeq cell line was used as a counterscreen using
Fluo-3AM (1 lM). Fluorescence intensity (kex = 410 nm, kem = 510 nm) was