New potent calcimimetics: II. Discovery of benzothiazole trisubstituted ureas

Article abstract of DOI:10.1016/j.bmcl.2013.01.077

Following the identification of trisubstituted ureas as a promising new chemical series of allosteric modulators of the calcium sensing receptor (CaSR), we further explored the SAR around the urea substitution, leading to the discovery of benzothiazole ur

Full text of DOI:10.1016/j.bmcl.2013.01.077

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2455–2459
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
New potent calcimimetics: II. Discovery of benzothiazole
trisubstituted ureas
a
a
a
b
c
Pierre Deprez a, , Taoues Temal , Hélène Jary , Marielle Auberval , Sarah Lively , Denis Guédin ,
⇑
Jean-Paul Vevert d
a Galapagos SASU, 102 Avenue Gaston Roussel, 93230 Romainville, France
b Amgen Inc, 1120 Veterans Boulevard, South San Francisco, CA 94080, USA
c Institut de Recherche Servier, 125 chemin de ronde, 78290 Croissy-sur-Seine, France
d 102 route de Noisy, 93230 Romainville, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 8 February 2013
Following the identification of trisubstituted ureas as a promising new chemical series of allosteric mod-
ulators of the calcium sensing receptor (CaSR), we further explored the SAR around the urea substitution,
leading to the discovery of benzothiazole urea compound 13. This compound is a potent calcimimetic
with an EC50 = 20 nM (luciferase assay). Evaluated in an in vivo model of chronic renal failure (short term
and long term in 5/6 nephrectomized rats), benzothiazole urea 13 significantly decreased PTH levels after
oral administration while keeping calcemia within the normal range.
Keywords:
Calcium sensing receptor
Benzothiazole urea
Positive allosteric modulators
Ó 2013 Elsevier Ltd. All rights reserved.
Secondary hyperparathyroidism (sHPT) is characterized by an
excessive production of parathyroid hormone (PTH) following re-
nal failure.1,2 PTH is secreted by parathyroid glands and PTH level
is controlled by the extracellular calcium concentration via the cal-
cium sensing receptor (CaSR), a family C GPCR.3,4 Treatment of
sHPT by positive allosteric modulators of the CaSR also called calc-
imimetics-proved to be a successful approach with the discovery of
the exploratory compound R-568,5,6 followed by cinacalcet (AMG-
073, SensiparÒ).7,8 Other calcimimetics have also been indentified,
carboxylic acid (Scheme 1). The intermediate acyl chloride derived
from gem-diphenyl ethyl carboxylic acid was generated with thio-
nyl chloride and was coupled to amino-ethylmorpholine to give
amide 2 which was collected by filtration and used in the next step
without additional purification. The reduction of the amide func-
tion of compound 2 was achieved using LiAlH4 in diethyl ether.
However, for kg scale synthesis, replacement of diethyl ether by
THF led to low yield and eventually, the use of the combination
of LiAlH4/AlCl3 in THF16 proved to be critical for improving the
yield to 72% in 3 h.
most of them containing the a
-methylbenzylamine group.9–11
Earlier, we described the identification of a new chemical series
of trisubstituted ureas represented by 1. Oxazole 1 is a potent
in vitro agonist of the CaSR, leading to the in vivo reduction of ser-
um PTH level in normal rats.12 The gem diphenyl-propyl group and
the N-ethylmorpholinyl substituent of the urea 1 appeared to be
important for in vitro potency, while some flexibility on the aro-
matic substitution on the phenyl ring was tolerated.
The secondary amine 3 was then purified by crystallization in
ethanol after acidic treatment (see Scheme 1).
Conversion of 3–13 was accomplished by stirring a mixture of
2-amino benzothiazole and carbonyl di-imidazole in dichloro-
methane overnight, followed by addition of 3. After 5 h of stirring,
the desired urea benzothiazole 13 was isolated in 84% yield. The
In our search towards new compounds with improved biologi-
cal profile on the CaSR, we describe here the identification of the
benzothiazole compound 13 (Fig. 1) including a SAR analysis and
pharmacological profile, indicating that benzothiazole 13 is a
potent calcimimetic in vitro and in vivo.
Urea compounds were prepared as described in previous
publications.12–14 We present here the optimized synthesis15 of
benzothiazole 13 from two commercially available building
blocks: amino-ethyl morpholine and the gem-diphenyl ethyl
O
N
O
N
H
N
H
N
N
N
N
O
S
O
O
13
1
N
CaSR EC50 = 20 nM
Potent in vivo activity
CaSR EC50 = 60 nM
⇑
Corresponding author. Tel.: +33 149424687; fax: +33 149424658.
E-mail address: pierre.deprez@glpg.com (P. Deprez).
Figure 1. Identification of lead compound benzothiazole urea 13.
Current address.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.01.077

2456
P. Deprez et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2455–2459
O
N
OH
O
H
H
N
H
N
N
N
c
N
N
a
b
N
O
O
O
S
O
+
O
2
3
13
N
H2N
Scheme 1. Reagents and conditions: (a) SOCl2, CH2Cl2, rt, 88%; (b) 1 equiv LiAlH4, 0.1 equiv AlCl3, THF, 0 °C to rt, 3 h, then EtOH/ HCl 72%; (c) 2-aminobenzothiazole, CDI,
CH2Cl2, rt, overnight, then addition of compound 3, CH2Cl2, rt, 84%.
Table 1
Table 1 (continued)
Calcimimetic activity of ureas 4–17a: replacement of phenyl by various heterocyclic
Compound
Ar
EC50 (nM)
20
rings
O
N
H
S
N
13
14
H
N
N
N
Ar
60
O
N
N
N
Compound
Ar
EC50 (nM)
1000
15
16
150
4
Ph
N
N
CH3
300
80
N
5
80
N
O
CH3
N
17
6
7
450
400
N
Cl
N
N
a
EC50 values in lM (n P1) have been determined by luciferase assay.
O
N
HCl salt was formed in EtOH and the compound was crystallized
with more than 98% purity. The overall yield was 61% without
any chromatography and this optimized process has been used
on a kilogram scale.
CH3
N
N
Urea compounds were tested in a dose response luciferase as-
say, with increasing calcium concentration.17 We observed a left-
ward shift of the hCaSR calcium responses by increasing
concentration of calcimimetics. The values indicated in this paper
correspond to an EC50 at 2 mM of calcium. In our assay conditions,
reference compound R-568 and urea compound 1 exhibited potent
activity with EC50 values of 80 nM and 60 nM, respectively.
Previously, we described the optimization of the trisubstituted
urea series by varying the meta substitution of the aniline ring with
a large number of functional groups.12
In this paper, we describe the replacement of the phenyl ring by
five- and six-membered ring heterocycles and fused aromatics.
The potent activity (EC50 <200 nM) of a few compounds in this
set confirmed the importance of the substitution of the urea for
modulating potency, and also the flexibility in this region. Pyridine
(5), thiazole (9) and isoxazole (10) substitutions of the urea proved
to be active, and a heterocycle bearing a nitrogen atom adjacent to
the –NH– of the urea was most efficient, as indicated by the
comparison of pyridine 5 and 4 (80 nM vs 1000 nM), or isoxazole
10 and 11 (200 nM vs 1700 nM). Based on this observation,
bicyclic aromatic rings with a nitrogen atom suitably positioned
8
9
200
300
S
N
S
N
10
11
200
CH3
N
O
O
CH3
1700
CH3
N
12
400
O
S
O

P. Deprez et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2455–2459
2457
were further explored (Table 1), leading to potent benzimidazole,
250
200
150
100
50
2.5
2.3
2.1
1.9
1.7
1.5
benzoxazole and benzothiazole compounds. Benzothiazole 13
proved to be the most potent compound identified (EC50 = 20 nM),
fourfold more potent than R-568.
Exploration of various substitutions around the benzothiazole
moiety was then undertaken (Table 2).
No further improvement was observed upon substitution of the
4-, 5- and 6-positions of benzothiazole. Most of the substitutions at
R1 and R3 resulted in analogs that were potent (EC50 below
100 nM).
Based on in vitro SAR studies, benzothiazole urea 13 was se-
lected for further characterization of its pharmacological and
safety profile.
Compound 13 was a potent candidate (EC50 = 20 nM) with four-
fold improvement over R-568. It was evaluated in in vivo models as
a dihydrochloride salt. This compound was stable over time in ser-
um, intestinal fluid, and over a pH range of 2–12. Its aqueous sol-
ubility was 0.3 mg/mL at pH 2.
A PK experiment in CRF rats (Chronic Renal Failure) with benzo-
thiazole 13 (iv dose, 0.3 mg/kg) indicated an AUC of 162 ng h/mL
associated with a Cmax of 546 ng/mL. The clearance was moderate
**
***
***
*
**
**
***
0
0
2
4
6
8
10 12 14 16 18 20 22 24
Time post-dosing (h)
Figure 2. In vivo serum PTH and Ca after a single oral dose of compound 13 (10 mg/
kg) in 5/6 nephrectomized male Sprague–Dawley rats (PTH (j), Ca (ꢀ)). n = 9–12, ⁄
p
<0.05, ⁄⁄p <0.01, ⁄⁄⁄p <0.0001 significantly different from ‘CRF vehicle’ group,
ANOVA, t-test.
(1.85 L/h/kg), and due to
a high volume of distribution
(Vd = 5.17 L/kg), the T1/2 was 6.73 h.
Further profiling of this compound was carried out to evaluate
the in vivo effect on serum PTH levels.
phosphorus. As indicated in Figure 2, compound 13 exhibited a
rapid and significant decrease in PTH (95% decrease at 2 h, ꢀ75%
at 6 h) and this effect was still significant after 24 h (ꢀ49%).
As described with other calcimimetics,19 this strong decrease in
PTH is usually associated with a significant decrease in serum cal-
cium. However, interestingly, only a slight decrease in serum cal-
cium was observed, with serum calcium being maintained above
2 mM. Therefore, no hypocalcemia was observed at the dose of
10 mg/kg despite the strong effect on PTH. In addition to its po-
tency, this was a striking feature of benzothiazole urea 13, indicat-
ing that a window between strong PTH decrease and hypocalcemia
was possible with this compound. Serum phosphorus levels in-
creased as expected (Fig. 3).
Benzothiazole urea 13 was evaluated18 in CRF rats (Fig. 2), a rel-
evant model for secondary hyperparathyroidism linked to renal
insufficiency. One week following a 5/6 nephrectomy (left kidney
and 2/3 of right kidney surgically removed), CRF rats were charac-
terized by high PTH level (177 pg/mL vs 38 pg/mL for intact rats) as
well as increased serum creatinin (63 vs 28
4 mM).
lM) and urea (10 vs
Benzothiazole 13 was administered orally at 10 mg/kg in CRF
rats and PTH was measured over 24 h along with calcium and
Table 2
The in vivo potency was confirmed in a long term (2-month
treatment) CRF model (Figs. 4 and 5). 20 days after surgery, 5/6
nephrectomized rats were provided with a high level of phospho-
rous in their drinking water (6 g/L NaHPO4) for a total of 114 days.20
Calcimimetic activity of urea 18–34a: variation of substitution on the benzothiazole
ring
O
N
8
H
3.5
1
R
N
N
N
7
*
2
S
O
R
3.1
2.7
2.3
1.9
1.5
6
5
4
3
2
1
3
H
R
**
**
Compound
R1
R2
R3
EC50 (nM)
***
***
**
13
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
H
H
H
H
H
H
H
H
H
H
20
20
30
600
30
40
90
60
80
30
Cl
OCH3
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
NHSO2CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
OCF3
Cl
CH2OH
NHSO2CH3
CH3
30
0
COOH
100
200
200
80
1000
200
60
COOCH2CH3
0
2
4
6
8
10 12 14 16 18 20 22 24
CONH(CH2)2-N(CH3)2
SO2CH3
OCH2CH3
Time post dosing (h)
F
Cl
Figure 3. In vivo serum phosphorus and CaxP after a single oral dose of compound
13 (10 mg/kg) in 5/6 nephrectomized male Sprague–Dawley rats (P (d), CaxP (N)).
n = 9–12, ⁄p <0.05, ⁄⁄p <0.01, ⁄⁄⁄p <0.0001 significantly different from ‘CRF vehicle’
group, ANOVA, t-test.
a
EC50 values in lM (n P1) have been determined by luciferase assay.

2458
P. Deprez et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2455–2459
out and none of the rats at either dose were affected by aortic
calcification.
In conclusion, benzothiazole compound 13 was identified as a
promising compound for preclinical studies. In addition to its phar-
macological properties, this compound proved to be selective over
##
180
160
140
120
100
80
a large panel of receptors and enzymes at 10
without any genotoxicity (Ames) and hERG inhibition at 10
l
M (Cerep screen),
M.
l
A 14-day toxicity study carried out did not show serious side effect
up to 1000 mg/kg.
However, despite the efficacy of benzothiazole 13 in CRF rats,
we observed a difference in the PK profile between CRF rats and
normal rats, with lower bioavailability in normal rats and higher
clearance (4.96 L h/kg vs 1.85 L h/kg), which made it difficult to
predict PK parameters in humans, thus preventing further develop-
ment of this compound in clinical trials.
60
-76%
**
40
-88%
**
Acknowledgments
20
0
The authors thank Séverine Hebbe from Galapagos and Paul
Harrington from Amgen for careful review of the manuscript.
Intact
Compound 13
CRF rats
10 30 mg/kg/d
0
References and notes
Figure 4. In vivo serum PTH after a two month-oral administration of compound 13
(10 and 30 mg/kg/day) in male 5/6 nephrectomized Sprague–Dawley rats, 24 h after
last administration. n = 6–10, ##p <0.01 significantly different intact rats, ⁄p <0.05,
⁄⁄p <0.01, significantly different from ‘CRF vehicle’ group, ANOVA, t-test.
1. Nemeth, E. F. In Principles of Bone Biology; Bilezikian, J. P., Raisz, L. G., Rodan, G.
A., Eds., 2nd ed.; Academic Press: San Diego, 2002; p 1339.
2. Wuthrich, R. P.; Martin, D.; Bilezikian, J. P. Eur. J. Clin. Invest. 2007, 37, 915.
3. Brown, E. M.; Gamba, G.; Riccardi, D.; Lombardi, M.; Butters, R.; Kifor, O.; Sun,
A.; Hediger, M. A.; Lytton, J.; Herbert, S. C. Nature 1993, 366, 575.
4. Rodriguez, M.; Nemeth, E. F.; Martin, D. Am. J. Physiol. 2005, 288, F253.
5. Nemeth, E. F. Van Wagenen, B. C.; Balandrin, M. F.; Delmar, E. G.; Moe, S. T. U.S.
Patent 6,031,003, 2002.
2.7
6. Nemeth, E. F. Endocrine 1999, 10, 97.
7. Sorbera, L. A.; Castaner, R. M.; Bayes, M. Drugs Future 2002, 27, 831.
8. Nemeth, E. F.; Heaton, W. H.; Miller, M.; Fox, J.; Balandrin, M. F.; Van Wagenen,
B. C.; Colloton, M.; Karbon, W.; Scherrer, J.; Shatzen, E.; Rishton, G.; Scully, S.;
Qi, M.; Harris, R.; Lacey, D.; Martin, D. J. Pharmacol. Exp. Ther. 2004, 308, 627.
9. Cohen, J. H.; Combs, D. W.; Rybczynski, P. J. U.S. Patent 6,172,091, 2001.
10. Kessler, A.; Faure, H.; Petrel, C.; Ruat, M.; Dodd, R. H. Bioorg. Med. Chem. Lett.
2001, 2000, 10.
11. Miyazaki, H.; Tsubakimoto, J.; Yasuda, K.; Takamuro, I.; Sakurai, O.; Yanagida,
T.; Hisada, Y. W.O. Patent 115,975, 2005.
12. Temal, T.; Jary, H.; Auberval, M.; Lively, S.; Guédin, D.; Vevert, J.-P.; Deprez, P.
Bioorg. Med. Chem. Lett. 2013, http://dx.doi.org/10.1016/j.bmcl.2013.01.078.
13. Deprez, P.; Patek, M. W.O. Patent 059,102, 2002.
2.5
*
2.3
2.1
14. Deprez, P.; Jary, H.; Temal, T. W.O. Patent 117,211, 2006.
1.9
1.7
15. Chemical procedure for synthesis of benzothiazole urea compound 13: 60.1 g
(0.37 mol, 1.5 equiv) of 1,10-carbonyl-diimidazole were dissolved in 660 mL of
CH2Cl2 in a 5-L reaction vessel. 37.1 g (0.25 mol) of 2-aminobenzothiazole
solution in 660 mL of CH2Cl2 was then added drop wise at a temperature of
between 20 and 24 °C. The mixture was left to stand with stirring at room
temperature for 15 h.
diphenyl-propyl)-(2-morpholin-4-yl-ethyl)-amine dihydrochloride
A
solution of 117.4 g (0.30 mol, 1.2 equiv) of (3,3-
and
3
59.8 g (0.60 mol, 2.4 equiv) of triethylamine in 660 mL of CH2Cl2 was added
over 15 min with continuous stirring of the reaction medium at room
temperature for 2 h. The reaction was monitored by TLC (SiO2, mobile phase
CH2Cl2/MeOH, 95/5, visualisation: UV).
1.5
Intact
Compound 13
CRF rats
10 30 mg/kg/d
0
600 mL of water and then 600 mL of saturated aqueous NaHCO3 solution were
added to the reaction medium. After separation of the organic phase, the
aqueous phase was extracted with 1 L of CH2Cl2. The organic fractions were
combined, washed with 1.5 L of a saturated aqueous NaCl solution, dried over
Na2SO4, filtered and concentrated to dryness under reduced pressure to give
143 g of an orange solid. This solid was dissolved in 1.4 L of acetonitrile at 70 °C
during 30 min. The resulting suspension was cooled to 0 °C and filtered. The
white solid isolated was washed with 500 mL of acetonitrile then dried under a
high vacuum obtained using a vane-type pump to give 103 g (84%) of 3-
benzothiazol-2-yl-1-(3,3-diphenyl-propyl)-1-(2-morpholin-4-yl-ethyl)-urea
13 in the form of a white crystalline solid.
Figure 5. In vivo serum Ca after a two month-oral administration of compound 13
(10 and 30 mg/kg/day) in male 5/6 nephrectomized Sprague–Dawley rats, 24 h after
last administration. n = 6–10, ⁄p <0.05 significantly different from ‘CRF Vehicle’
group, ANOVA, t-test.
After surgery, the rats were left without treatment for 65 days.
Then, compound 13 was administered orally at 10 and 30 mg/kg
once a day for 2-months. No mortality or side effects were observed
during the period of the treatment. Rats were then sacrificed and
PTH levels measured 24 h after the last administration. We ob-
served a decrease in PTH level of ꢀ76% (at 10 mg/kg/day) and of
ꢀ88% (at 30 mg/kg/day), confirming the potency and safety of the
compound in the two month-treatment (Fig. 4). As observed previ-
ously, calcium levels stayed within the normal range, with no statis-
tical difference at 10 mg/kg/day compared with the untreated CRF
rats (ꢀ4% ns), and decreased by 9% (p <0.05) at 30 mg/kg/day,
(Fig. 5). Histological analysis of aortic calcification was also carried
1H NMR (400 MHz, CDCl3) d 7.78 (d, 1H, aromatic H), 7.70 (d, 1H, aromatic H),
7.40 (t, 1H, aromatic H), 7.35–7.10 (m, 11H, aromatic H), 4.05 (m; 4H, 2ꢁCH2),
4.00 (t, 1H, CH), 3.37 (t, 4H, CH2), 2.65 (m, 6H, CH2), 2.40 (q, 2H, CH2).
16. Pettit, G. R.; Baumann, M. F.; Rangammal, K. N. J. Med. Pharm. Chem. 1962, 5,
800.
17. Luciferase in vitro assay: The human parathyroid cell Ca2+ receptor cDNA was
subcloned into the mammalian expression vector PECE (Ref. 1). The luciferase
reporter was subcloned into the mammalian expression vector pGL3basic
(Promega). Resistance to neomycin (pSV2-neo) and resistance to puromycin
(pSG5-puro) were used as selection markers. All these plasmids were
simultaneously transfected into CHO cells by calcium phosphate
precipitation. Transfected cells were grown in F12 medium containing 7.5%

P. Deprez et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2455–2459
2459
foetal bovine serum, 100 U/mL penicillin and 100
l
g/mL streptomycin (as 1%
The rats were fasted 24 h before necropsy and were sacrificed by
decapitation at various times (2, 6, 24 h) after the gavage with vehicle
Pen-Strep, BioWithaker), neomycin (750 g/mL) and puromycin (5
l
lg/mL).
Neomycin and puromycin resistant colonies were subcloned and assayed for
activation against a range of calcium concentration. Clone 8-5-5 was used to
assess the effects of compounds on [Ca2+]i. This stably transfected cell line is
termed ET8-5-5.
or calcimimetic. Blood samples were collected in Sarstedt Z tubes,
allowed to clot for 20 min and centrifuged (3000 rpm, Jouan CR422) at
4 °C. Serums were removed and stored at ꢀ20 °C until assayed. Serum
PTH was quantified according to the provider’s instructions using a rat
IRMA kit (Immutopics, 50-2000). Serum Ca, P, creatinin and urea were
measured using a blood chemistry analyzer (Cobas Mira) according to the
manufacturer’s protocols. Increased levels of urea and creatinin confirmed
the renal failure. Comparisons among the groups were made using one-
way analysis of variance (ANOVA) and unpaired t-test.
For measurements of [Ca2+]i, the cells were recovered from tissue culture flasks
by brief treatment with Trypsin–EDTA (Invitrogen; containing 0.53 mM
EDTAꢂ4Na in HBSS) and then seeded in Culture treated 96-well plates
(Greiner) at 50 K cells per well in the growth media (same as above, except
neomycin 400 lg/mL). Cells were grown in 37 °C TC incubator for 24 h. The
culture medium was then removed and replaced with F12 medium, 1% Pen-
Strep for an overnight foetal bovine serum starvation in 37 °C TC incubator.
Then the starvation medium was removed and replaced with a test buffer
(20 mM HEPES pH 7.4, 125 mM NaCl, 5 mM KCl, 1 mM MgCl2, 5.5 mM, glucose,
19. Fox, J.; Lowe, S. H.; Petty, B. A.; Nemeth, E. F. J. Pharmacol. Exp. Ther. 1999, 290, 473.
20. In vivo measurements:
Two-month treatment in long-term chronic renal failure:
Male Sprague–Dawley rats (250–300 g BW, CERJ, France) underwent surgical
5/6 nephrectomy or sham-operation according to a protocol approved by
Internal Ethical Committee for the care and use of animals.
2 g/L lysosyme and 0.3 mM CaCl2) supplemented with
a range of test
compound concentrations crossed against superadded range of CaCl2
a
concentrations. The cells were incubated with the test compounds for 5 h in
37 °C TC incubator. Then the test buffer was discarded, and cells were added
with the substrate for Luciferase from SteadyLite Kit (Perkin–Elmer). The
luminescence was recorded.
The dosings started 12 weeks post-nephrectomy and lasted 7 weeks. The rats
were orally dosed at 10 and 30 mg/kg/day with compound 13,
(dihydrochloride salt) formulated in methylcellulose 0.5%. Vehicle-treated
control rats received the same volume of vehicle (5 mL/kg).
18. In vivo measurements:
The rats were fasted 24 h before necropsy and were sacrificed by decapitation.
Blood samples were collected in Sarstedt Z tubes, allowed to clot for 20 min
and centrifuged (3000 rpm, Jouan CR422) at 4 °C. Serums were removed and
stored at ꢀ20 °C until assayed. Serum PTH was quantified according to the
provider’s instructions using a rat IRMA kit (Immutopics, 50-2000). Serum Ca,
P, creatinin and urea were measured using a blood chemistry analyzer (Cobas
Mira) according to the manufacturer’s protocols. Increased levels of urea and
creatinin confirmed the renal failure. Comparisons among the groups were
made using one-way analysis of variance (ANOVA) and unpaired t-test.
Single oral administration: Male Sprague–Dawley rats (250–300 g BW, CERJ,
France) underwent surgical 5/6 nephrectomy or sham-operation according
to a protocol approved by Internal Ethical Committee for the care and use
of animals. One week post-nephrectomy, rats received
a single oral
gavage (5 mL/kg) of compound or vehicle. Compound 13 (dihydrochloride
salt) was formulated in methylcellulose 0.5% and administered at 10 or
30 mg/kg. Vehicle-treated control rats were given the same volume of
vehicle.