Discovery of a Potent and Selective TRPC5 Inhibitor, Efficacious in a Focal Segmental Glomerulosclerosis Model

Article abstract of DOI:10.1021/acsmedchemlett.9b00430

The nonselective Ca²⁺-permeable transient receptor potential (TRP) channels play important roles in diverse cellular processes, including actin remodeling and cell migration. TRP channel subfamily C, member 5 (TRPC5) helps regulate a tight balance of cytoskeletal dynamics in podocytes and is suggested to be involved in the pathogenesis of proteinuric kidney diseases, such as focal segmental glomerulosclerosis (FSGS). As such, protection of podocytes by inhibition of TRPC5 mediated Ca²⁺ signaling may provide a novel therapeutic approach for the treatment of proteinuric kidney diseases. Herein, we describe the identification of a novel TRPC5 inhibitor, GFB-8438, by systematic optimization of a high-throughput screening hit, pyridazinone 1. GFB-8438 protects mouse podocytes from injury induced by protamine sulfate (PS) in vitro. It is also efficacious in a hypertensive deoxycorticosterone acetate (DOCA)-salt rat model of FSGS, significantly reducing both total protein and albumin concentrations in urine.

Full text of DOI:10.1021/acsmedchemlett.9b00430

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Letter
Discovery of a Potent and Selective TRPC5 Inhibitor,
Efficacious in a Focal Segmental Glomerulosclerosis Model
Maolin Yu, Mark W. Ledeboer, Matthew Daniels, Goran Malojcic, Thomas
T Tibbitts, Marie Coeffet-Le Gal, Xin-Ru Pan-Zhou, Amy Westerling-Bui,
Maria Beconi, John F Reilly, Peter Mundel, and Jean-Christophe Harmange
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00430 • Publication Date (Web): 22 Oct 2019
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Discovery of a Potent and Selective TRPC5 Inhibitor, Efficacious in a
Focal Segmental Glomerulosclerosis Model
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Maolin Yu,*,† Mark W. Ledeboer,*,† Matthew Daniels,† Goran Malojcic,† Thomas T. Tibbitts,†
Marie Coeffet-Le Gal,# Xin-Ru Pan-Zhou,# Amy Westerling-Bui,# Maria Beconi,#,∇ John F. Reilly,#
Peter Mundel,# and Jean-Christophe Harmange†
†Drug Discovery, #Biology, Goldfinch Bio Inc., Cambridge, Massachusetts 02142, United States
KEYWORDS: TRPC5, focal segmental glomerulosclerosis, DOCA, podocyte, proteinuria, kidney disease
ABSTRACT: The non-selective Ca2+-permeable Transient Receptor Potential (TRP) channels play important roles in diverse
cellular processes, including actin remodeling and cell migration. TRP channel subfamily C, member 5 (TRPC5) helps
regulate a tight balance of cytoskeletal dynamics in podocytes and is suggested to be involved in the pathogenesis of
proteinuric kidney diseases, such as focal segmental glomerulosclerosis (FSGS). As such, protection of podocytes by
inhibition of TRPC5 mediated Ca2+ signaling may provide a novel therapeutic approach for the treatment of proteinuric
kidney diseases. Herein, we describe the identification of a novel TRPC5 inhibitor, GFB-8438, by systematic optimization
of a high-throughput screening hit, pyridazinone 1. GFB-8438 protects mouse podocytes from injury induced by protamine
sulfate (PS) in vitro. It is also efficacious in a hypertensive deoxycorticosterone acetate (DOCA)-salt rat model of FSGS,
significantly reducing both total protein and albumin concentrations in urine.
Progressive chronic kidney disease (CKD) is a global
health concern, affecting over 500 million people
worldwide and about 26 million in US alone.1,2 CKD is
regarded as an accelerator of cardiovascular disease (CVD),
which, along with renal failure are the primary cause of
morbidity and mortality in this underserved population.3
As one of the leading causes of glomerular disease, focal
segmental glomerulosclerosis (FSGS) in its most severe
form is associated with the nephrotic syndrome
characterized by symptomatic proteinuria (> 3.5 g protein
per day) in adults, scarring of the glomerulus, and loss of
terminally differentiated podocytes.4,5 Currently there are
few effective treatments for FSGS and many patients are
treated chronically with steroids, which have undesirable
side effects. Therefore, there is a significant unmet need for
novel therapeutic approaches that can prevent or slow
disease progression.
TRPC5 channel leads to further Rac1 activation in a feed-
forward loop, thereby driving cytoskeletal remodeling. The
ensuing cytoskeletal changes lead to effacement of
podocyte foot processes, podocyte loss and proteinuria.
Inhibition of TRPC5 by small molecules has been shown to
protect podocytes in vitro and suppresses proteinuria and
podocyte loss in vivo, in a transgenic rat model of FSGS. 10,11
TRPC5-knockout (KO) mice were also protected from
lipopolysaccharide
(LPS)-induced
albuminuria.10
Collectively, these results suggest that inhibition of TRPC5
may provide a novel, effective and safe approach for the
treatment of FSGS and related proteinuric kidney diseases.
Cl
N
N
NH
N
N
N
N
N
O
A hallmark of the pathogenesis of FSGS is podocyte
injury and loss. Podocyte foot process effacement, one of
the first morphologic alterations of the glomerular
filtration barrier, is caused by the disruption of the
podocyte actin cytoskeleton via activation of Rac1.6 TRPC5
has been identified as a key mediator of Rac1 activation in
podocytes.7,8 TRPC5 belongs to the mammalian
superfamily of transient receptor potential (TRP) Ca2+-
permeable non-selective cationic channels.9 Damage to
podocytes activates Rac1, leading to translocation of TRPC5
to the cell membrane. Activation of the membrane-located
Clemizole
ML204
AC1903
Cl
Cl
O
N
O
N
N
N
N
N
HO
HO
N
O
O
O
N
O
Cl
OCF3
HC-070
HC-608
Figure 1. Representative TRPC5 inhibitors from the
literature.
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1
2
3
4
Table 1. Summary of early dynamic SAR and core modification
5
R2
6
7
8
core
N
N
Br
R1
9
O
10
11
12
13
14
15
16
17
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19
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21
22
23
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TRPC5
Qpatch
IC50 (M)a
TRPC5
Qpatch
IC50 (M)a
Cmp
d
Cmpd
R2
Cores
R1
R2
Cores
R1
b
*
*
*
*
*
*
b
1
H
H
H
H
H
H
H
5.0
9
H
H
H
H
H
H
H
H
H
1.9
*
*
N
N
N
N
*
*
*
N
N
N
N
N
N
*
*
2
18
10
11
>30
>30
0.50
>30
27
*
*
*
*
*
*
*
*
*
*
*
N
N
N
N
N
N
N
N *
3
8.0
8.8
1.8
16
O
N
*
*
N
N
N
N
N
N
*
4
12
13
14
15
16
17
*
N
*
*
*
*
*
*
*
*
*
*
N
*
5
F
N
*
*
*
*
6-1c
6-2
7
*
N
N
*
N
*
N
N *
>30
27
>30
>30
2.8
N
*
*
* N
N *
Me
*
OH
*
N
N *
8
>30
a
Qpatch assay measuring the outward current at +80 mV in HEK 293 cells overexpressing human TRPC5 activated by 30 M
Rosiglitazone. Results are mean of at least two experiments. Experimental errors within 30% of value. This denotes the end
b
connecting to pyridazinone. cChloro analogue.
identified by Hydra/Boehringer-Ingelheim, are highly
N
TRPC5 Qpatch IC50
TRPC6 Qpatch IC50
MW
5.0 M
>30 M
363
potent.18 While these compounds have further helped
advance TRPC4 and TRPC5 biology, especially in the CNS,
this series of inhibitors suffers from poor physicochemical
properties such as low solubility (kinetic solubility of HC-
608 is 0.03 M at pH 7.4 in phosphate buffer) and very high
protein binding (>99.5% bound). In our search for novel
TRPC5 inhibitors with an improved overall profile, we
N
N
52 Å2
2.2
NH
TPSA
Br
cLogP
Ligand efficiency
1
O
0.34
Figure 2. HTS hit, 1
Several subtype-selective small molecule TRPC5
inhibitors have recently emerged in the literature.12,13,14 The
structures of representative compounds are summarized in
Figure 1. Chemical probes such as ML204,15 Clemizole,16
and AC190317 have been instrumental in understanding the
biological roles of TRPC5. In addition, this pioneering
work helped to underscore the therapeutic potential
associated with its inhibition. Although effective as
molecular probes, these molecules generally suffer from
low potency, off-target activities or unknown selectivity
and unfavorable pharmacokinetic properties. However,
xanthine analogues such as HC-608 and HC-070,
screened
a structurally diverse library of ~400,000
compounds against TRPC5, using a FLIPR assay, which
resulted in the identification of several low micromolar
hits. From the hit set, pyridazinone 1 (Figure 2) was
selected as an attractive starting point for further
exploration based on the encouraging potency, selectivity
over TRPC6, good ligand efficiency, low cLogP as well as
the potential for rapid SAR exploration. Another important
consideration was the ability of 1 to block the TRPC5
channel with similar potency, independent of the
activators used (Rosiglitazone A: IC50 5.0 M, Riluzole: IC50
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aromatic and aliphatic R2 groups were tolerated. However,
7.8 M, (−)-Englerin A: IC50 4.0 M, and Lanthanum: IC50
4.4 M).
Our initial chemistry efforts focused on the
improvement of the potency in blocking TRPC5 channel
Ca2+ current. A survey of benzylic groups indicated
dynamic SAR and suggested the possibility of improving
potency. As exemplified by compounds 1, 2 and 3 (Table 1),
1
2
3
4
5
6
7
incorporation of heteroaromatic groups tended to lead to
erosion of potency as represented by 6-1,6-2, 34 and 36. As
shown by 5, addition of a p-fluoro group boosted potency
2.5x relative to compound 1, while removal of the o-Me
group led to a 3x loss in potency. Racemic methyl
substitution on benzylic methylene was tolerated (4).
Turning our
8
9
Table 2. Summary of SAR in Br and benzyl region
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O
R2
N
N
N
NH
R1
O
TRPC5
Qpatch
IC50 (M)
TRPC5
Qpatch
IC50 (M)a
Compd R1
R2
Compd
R1
R2
*
*
12
18
19
20
-Br
0.5
28
29
30
31
-Cl
-Cl
-Cl
-Cl
14.5
*
*
*
*
*
*
-Me
-Et
28.0
11.0
2.4
0.38
0.49
0.65
*
-iPr
Cl
*
21
1.5
GFB-8438 -Cl
0.18
0.53
>30
11.2
0.65
8.2
*
F3C
*
*
*
*
*
*
O
22
23
24
25
>30
0.66
9.6
1.9
32
33
34
35
36
-Cl
-Cl
-Cl
-Cl
-Cl
F3CO
*
-Cl
MeO2S
*
-CF3
-CN
N
F3C
*
F
F3C
F3C
*
*
N
N
26
27
-OMe
>30
>30
*
*
* N
O
37
38
-Cl
-Cl
0.53
>30
*
O
aResults are mean of at least two experiments. Experimental errors within 30% of value. Activated (by 30 M Rosiglitazone)
TRPC5 overexpressed HEK 293 cells used for outward current measurement at +80 mV in Qpatch assay.
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attention to the pyridazinone ring, methylation and Having addressed several shortcomings related to the
original hit, the potency for blocking the Ca+2 current still
had room for further improvement (Table 2). Walking
methyl around the phenyl ring established that the ortho
mono-methyl substitution is most effective (23, 28, 29, 30).
Based on this result, we screened a range of ortho-
substituted analogues in order to identify a group to
further improve the potency and balance physicochemical
properties and microsomal stability. SAR suggested
lipophilic groups in this position were well tolerated with
CH3, Cl or CF3 (23, 31, 32, GFB-8438). While incorporating
nitrogen next to CF3 (34) improved the properties, it led to
dramatic loss in activity. Substitution in the para-position
with F to block the potential metabolic soft spot caused
slight erosion in potency (35). Polar motifs (33, 36, 38)
showed potency loss trend as well, suggesting this part of
the compound might bind in a lipophilic pocket. Indeed 4-
Me-cyclohexyl (37) was equally potent as 23, but
unfortunately with very unfavorable physiochemical
properties.
1
2
3
4
5
6
7
8
alkylation of the NH as in 7 and 8 respectively greatly
diminished the TRPC5 potency, suggesting that the NH
may be involved in a productive H-bond. Investigation of
modifications to the piperazine core proved informative.
Introduction of a chiral Me group provided enantiomers
with divergent activities as in 9 vs 10. However,
methylation adjacent to the basic piperazine N, as in 11, led
to ablation of all potency. As demonstrated with 13,
replacing the basic N with carbon resulted in complete loss
9
10
11
12
13
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8438
32
35
316
114
12
HT-solubility (pH = 7.4) (M)
rLM/hepatocyte
CLint 24 / 94
55 / 147
18 / 26
(mL/min/kg)
hLM/hepatocyte
CL 7.8 / 5.5 0.14 / 4.1 4.3 / 5.3
(mL/min/kg)
Pharmacokinetics
rat
31
rat
56
rat
27
CL (mL/min/kg)
T1/2 (h)
0.50
1.17
0.45
1.27
19 / -
1.05
1.81
Vss (L/Kg)
F (PO/SC) (%)
17 / 33
22 / -
Table 3. DMPK Profiling of GFB-8438, 32 and 35a
of potency. Homopiperazine (14) exhibited diminished
activity as well. Attempts to introduce bridged and spiro
features to the piperazine ring turned out to be
unproductive (15, 16), except analogue 17, which has
comparable activity to 1, but with much less favored
physiochemical properties. Interestingly, introduction of
the lactam carbonyl saw a 10x fold boost in potency (12).
Contrasting to the basic nature of the piperazine core, the
lactam displays very distinct electrostatic properties. The
switch from sp3 to sp2 also results in conformational and
trajectory change for piperazine and the adjacent benzyl
group (See Figure S1 for modelling study). These highly
specific SAR suggests that piperazine might sit in a well-
defined binding pocket and the potency gain observed
with the lactam is indicative of productive interactions
with the protein.
aSee Supporting Information for experimental details.
Several analogues identified in Table 2 have reasonable
potencies and potential to serve as tool compounds for in
vivo study. As such we determined the PK parameters for
GFB-8438, 32, and 35 (Table 3). All three compounds
demonstrated good metabolic stability when incubated
with both human liver microsomes and hepatocytes, but
with less stability in rat. The observed intrinsic microsomal
clearance correlated well with in vivo rat clearance, and
oral bioavailability in rats was modest and similar for each
of the three compounds. When dosed subcutaneously,
GFB-8438 exhibited significantly higher plasma exposure.
With the subcutaneous route overcoming the limited oral
bioavailability, GFB-8438 was deemed suitable for dosing
in our efficacy model.
Owing to the potential instability we turned our
attention to the bromide functionality in this series. We
speculated that it may play multiple roles: a) halogen
bonding with protein residue; b) fine tuning of the pKa of
the acidic proton in the warhead; c) steric and
conformational constraint of piperazinone scaffold. As
such, we replaced the Br with groups of varying size and
polarity. Replacing Br with a methyl (18) afforded
measurable potency. Increasing the alkyl size (18 to 21) led
to the increase in inhibitory activity with cyclopropyl as the
most optimal. However, larger groups such as THP were
not tolerated (22). Unsurprisingly, Cl analogue (23)
yielded similar potency to 12. Small electron-withdrawing
substituents were also tolerated albeit with 4- to 20-fold
reduction in potency (24, 25). Electron-donating
substitution (26, 27) proved to be ineffective, hinting at the
need to maintain pKa of the acidic NH proton. Through
this effort, we identified Cl as a suitable replacement for
Br, which is a common element in many approved
medicines.19
Table 4. in vitro profile of GFB-8438a
0.18
hTRPC5 Qpatch IC50 (M)
0.28
hTRPC5 manual patch clamp IC50 (M)
rTRPC5 Qpatch IC50 (M)
0.18
0.29
hTRPC4 Qpatch IC50 (M)
>30
hTRPC6 Qpatch IC50 (M)
>30
Nav1.5 manual patch clamp IC50 (M)
hERG manual patch IC50 (M)
Eurofins kinase panel (%Ctrl @10 M)
Eurofins receptor panel (%inh @10 M)
Equilibrium Solubility (pH=7.4) (M)
8.7
>50 (LOK 33%)
All < 50%
48
Human plasma protein binding (% 69
bound)
MDCK-MDR1 PA-B/ER (10-6 cm/s)
20 / 1.6
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aSee Supporting Information for experimental details.
when screened against the safety panels of kinases (59
kinases) and receptors (87 targets).
1
2
Having achieved a desired pharmacokinetic profile in
rat, we embarked on further characterizing GFB-8438. We
first confirmed the inhibition of human TRPC5 by manual
patch clamp. GFB-8438 inhibited the whole cell current
with an IC50 of 0.28 M comparable to the IC50 in Qpatch
assay (0.18 M) (Table 4). We then determined the potency
of GFB-8438 against the TRPC5 rat ortholog and found it
is equally potent in rodent TRPC5 Qpatch assay. Screening
for activity against a panel of TRP channels, we also found
that GFB-8438 was equipotent against TRPC4 and TRPC5,
and showed excellent selectivity against TRPC6, other TRP
family members (such as TRPC3/7, TRPA1, TRPV1/2/3/4/5
and TRPM2/3/4/8, data not shown), NaV 1.5, as well as
limited activity against the hERG channel. In addition,
GFB-8438 did not display significant off-target activity
3
4
5
6
7
8
9
Figure 3. in vitro efficacy study for GFB-8438 in PS-induced
mouse podocytes injury assay. (A) Control mouse podocytes
without PS and GFB-8438; (B) Mouse podocytes after
treatment with PS (300 M for 15 min); (C) Mouse podocytes
pretreated with GFB-8438 (1 M for 30 min), then insulted
with PS. The tiled images were acquired using a Zeiss LSM880
Airyscan super resolution confocal microscope. Green color:
Synaptopodin; Red color: Phalloidin; Blue color: Dapi.
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Figure 4. in vivo efficacy study for GFB-8438 in DOCA rat model (n = 8 in sham group, n = 15 in all other groups). Sprague
Dawley rats were unilaterally nephrectomized; after one-week recovery, rats were implanted with a DOCA pellet (45 mg)
and provided with high salts-containing tap water for 3 weeks of treatment. DOCA-salt rats received once daily
subcutaneous of GFB-8438 at 30 mg/kg or twice daily oral dosing of eplerenone at 50 mg/kg for 3 weeks. Hemodynamic
parameters and urinary albumin and protein were measured weekly. (A) total urinary protein excretion at week 0, 1, 2 and
3; (B) total urinary albumin excretion at week 0, 1, 2 and 3; (C) Mean arterial blood pressure measured at week 0, 1, 2 and 3.
No difference in creatinine clearance cross all groups. Data are presented as mean  SEM.
Based on the observed potency, selectivity, the overall
favorable physicochemical properties, and PK profile,
GFB-8438 was selected as an attractive tool compound for
further testing in in vitro and in vivo efficacy studies.
model of mineralocorticoid hypertension with renal
dysfunction, characterized by increased concentrations of
urinary protein and albumin excretion.22 Based on the
plasma exposure, we selected the highest tolerable
subcutaneous dose of 30 mg/kg QD for this study. As
shown in Figure 4, GFB-8438 demonstrated robust
benefits in this model. Statistically significant reduction in
urine protein concentrations were seen after three weeks
when compared to the DOCA control group. This trend
was even more robust when analyzing the urine albumin
concentrations, which were significantly lower at both
two- and three-week timepoints and similar to the
eplerenone control group. There was no significant
difference in body weight in all treated groups. Animals
receiving DOCA-GFB-8438 did not exhibit significant
differences in mean arterial blood pressure (BP), diastolic
and systolic BP compared to DOCA group, while
significant reduction of BP was observed at both week 2
and 3 in eplerenone treated group. The effect of GFB-8438
on proteinuria without affecting blood pressure contrasts
with mechanisms of action of angiotensin-converting
enzyme (ACE) inhibitor or an angiotensin II receptor
blocker (ARB) frequently used for the treatment of FSGS.
Thus, we sought to assess the ability of GFB-8438 to
elicit protective effect on podocytes following injury
induced by protamine sulfate (PS).10,20,21 Conditionally
immortalized mouse podocytes were incubated with PS, an
indirect activator of TRPC5. Extensive actin disruption and
loss of stress fibers was observed as demonstrated by the
loss of synaptopodin and a reorganization of the phalloidin
stained actin cytoskeleton (Figure 3, B). In contrast,
pretreatment of mouse podocyte with compound GFB-
8438, followed by incubation with PS, effectively blocked
synaptopodin loss and cytoskeletal remodeling (Figure 3,
C). This pharmacological effect is consistent with the prior
study using tool TRPC5 inhibitor ML204,10 further
validating the critical role of TRPC5 channel in
maintaining podocyte cytoskeletal integrity.
Next, GFB-8438 was evaluated in an in vivo
deoxycorticosterone acetate (DOCA)-rat model of FSGS.
The DOCA-salt hypertensive rat is a well-established
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DMPK profiling of GFB-8438, 32 and 35; Table S2. In vitro
These data are consistent with TRPC5 inhibitors such as
GFB-8438 having disease-modifying activity, addressing
the pathogenesis of FSGS by protecting the integrity of the
podocyte cytoskeleton.
1
2
3
4
5
6
7
8
activity profile of GFB-8438; PS-induced mouse podocytes
injury protection assay; Efficacy study in DOCA-salt rat
model; Figure S1; References (PDF)
The pyridazinone GFB-8438 was efficiently prepared in
a 3-step synthetic sequence described in Scheme 1 from
commercially available starting materials. Standard
alkylation of bromide with piperazinone under high
temperature, followed by Boc deprotection and SNAr
reaction with 4,5-dichloropyridazinone furnished the
synthesis of GFB-8438 in overall 17% yield after
recrystallization from acetic acid and water.
AUTHOR INFORMATION
Corresponding Author
*(M.L.) Tel: +1 (617) 337 4200. Email:
mledeboer@goldfinchbio.com
*(M.Y.) Tel: +1 (617) 337 4200. Email: myu@goldfinchbio.com
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Present Addresses
∇(M.B.) Disc Medicine, 400 Tech Square, 10th floor,
Cambridge, MA 02139, United States.
Scheme 1. Synthesis of Compound GFB-8438a
Author Contributions
CF3
O
CF3
O
The manuscript was written through contributions of all
authors. All authors have given approval to the final version
of the manuscript.
a
+
N
Br
Boc
HN
N
60%
N
Boc
1.2 eq.
1 eq.
1 eq.
O
Notes
Cl
Cl
CF3
N
NH
The authors declare the following competing financial
interest(s): M.Y., M.W.L., M.D., G.M., T.T.T., M.C-L.G., X-
R.P-Z., A.W-B., M.B., J.F.R., P.M., J-C.H are current or past
employee of Goldfinch Bio Inc.
N
CF3
O
O
N
1 eq.
b
c
N
N
74%
38%
NH
NH
Cl
O
1 eq.
ACKNOWLEDGMENT
aReagents and conditions: (a) NaH (2 equiv), DMF, 0 oC to
The authors would like to thank Liron Walsh, Adam Tebbe
and Mark Duggan for assistance in manuscript preparation;
Evotec for HTS screening; Pharmaron for chemical synthesis
and DMPK support; Plato Biopharma for DOCA study; Icagen
and SB Drug Discovery for assays support.
o
o
110 C; (b) TFA (7 equiv), DCM, 0 C to rt; (c) DIEA (1.5
equiv), DMA, rt to 100 oC
In summary, starting from HTS screening hit 1, a series
of systematic medicinal chemistry SAR explorations led to
the identification of GFB-8438, a novel, potent and sub-
type selective TRPC5 inhibitor with favorable overall in
vitro and in vivo properties. GFB-8438 was shown to
protect mouse podocytes from injury induced by
protamine sulfate in vitro. More importantly, GFB-8438
demonstrated robust efficacy in the hypertensive DOCA-
salt rat model without affecting blood pressure, further
validating the critical role of TRPC5 channel in the
pathogenesis of kidney disease.
ABBREVIATIONS
FSGS, focal segmental glomerulosclerosis; CKD, chronic
kidney disease; TRP, transient receptor potential; TRPC5,
TRP channel subfamily C, member 5; TRPA, TRP channel
subfamily A; TRPV, TRP channel subfamily V; TRPM, TRP
channel subfamily M; DOCA, deoxycorticosterone acetate; PS,
protamine sulfate; CVD, cardiovascular disease; KO,
knockout; LPS, lipopolysaccharide; ROS, reactive oxygen
species; HTS, high throughput screening; SAR, structure
activity relationship; CL, clearance; Vss, volume of
distribution; F, bioavailability; T1/2, half-life; MDCK-MDR1,
Madin-Darby Canine Kidney-multidrug resistance protein 1;
ER, efflux ratio; hERG, human ether-a-go-go-related gene;
HEK, human embryonic kidney; BP, blood pressure; SNAr,
nucleophilic aromatic substitution; SC, subcutaneous dose;
Progress toward the identification of TRPC5 inhibitors
suitable for testing in clinical trials will be reported in due
course.
PO,
oral
dose;
DMPK,
drug
metabolism
and
ASSOCIATED CONTENT
Supporting Information
pharmacokinetics; hLM, human liver microsomes; rLM, rat
liver microsomes; DMF, dimethylformamide; TFA,
trifluoroacetic acid; DCM, dichloromethane; DIEA,
diisopropylethylamine; DMA, dimethylacetamide.
The Supporting Information is available free of charge on the
ACS Publications website.
General chemistry methods; General Synthetic protocols;
Synthetic procedures for the preparation of selected
compounds; hTRPC5 fluorescence-based assay (FLIPR)
protocols; hTRPC5 automated patch clamp assay (Qpatch)
protocol; hTRPC5 manual patch clamp assay protocol;
automated patch clamp assay (Qpatch) protocol; hTRPC6
automated patch clamp assay (Qpatch) protocol; Table S1.
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Figure 3. in vitro efficacy study for GFB-8438 in PS-induced mouse podocytes injury assay
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Figure 4. in vivo efficacy study for GFB-8438 in DOCA rat model
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