Discovery of Orally Bioavailable Selective Inhibitors of the Sodium-Phosphate Cotransporter NaPi2a (SLC34A1)

Article abstract of DOI:10.1021/acsmedchemlett.8b00013

Sodium-phosphate cotransporter 2a, or NaPi2a (SLC34A1), is a solute-carrier (SLC) transporter located in the kidney proximal tubule that reabsorbs glomerular-filtered phosphate. Inhibition of NaPi2a may enhance urinary phosphate excretion and correct maladaptive mineral and hormonal derangements associated with increased cardiovascular risk in chronic kidney disease-mineral and bone disorder (CKD-MBD). To date, only nonselective NaPi inhibitors have been described. Herein, we detail the discovery of the first series of selective NaPi2a inhibitors, resulting from optimization of a high-throughput screening hit. The oral PK profile of inhibitor PF-06869206 (6f) in rodents allows for the exploration of the pharmacology of selective NaPi2a inhibition.

Full text of DOI:10.1021/acsmedchemlett.8b00013

Letter
Cite This: ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
Discovery of Orally Bioavailable Selective Inhibitors of the Sodium-
Phosphate Cotransporter NaPi2a (SLC34A1)
Kevin J. Filipski,*,† Matthew F. Sammons,† Samit K. Bhattacharya,† Jane Panteleev,‡,∥ Janice A. Brown,‡
Paula M. Loria,‡ Markus Boehm,† Aaron C. Smith,‡ Andre Shavnya,‡ Edward L. Conn,‡ Kun Song,†,⊥
Yan Weng,† Carie Facemire,†,# Harald Juppner,§ and Valerie Clerin†
̈
†Pfizer Worldwide Research & Development, 1 Portland Street, Cambridge, Massachusetts 02139, United States
‡Pfizer Worldwide Research & Development, 558 Eastern Point Road, Groton, Connecticut 06340, United States
§Endocrine Unit and Pediatric Nephrology Unit, Thier 10, Massachusetts General Hospital and Harvard Medical School, 50 Blossom
Street, Boston, Massachusetts 02114, United States
S
* Supporting Information
ABSTRACT: Sodium-phosphate cotransporter 2a, or NaPi2a (SLC34A1), is a solute-
carrier (SLC) transporter located in the kidney proximal tubule that reabsorbs glomerular-
filtered phosphate. Inhibition of NaPi2a may enhance urinary phosphate excretion and
correct maladaptive mineral and hormonal derangements associated with increased
cardiovascular risk in chronic kidney disease−mineral and bone disorder (CKD-MBD).
To date, only nonselective NaPi inhibitors have been described. Herein, we detail the
discovery of the first series of selective NaPi2a inhibitors, resulting from optimization of a
high-throughput screening hit. The oral PK profile of inhibitor PF-06869206 (6f) in
rodents allows for the exploration of the pharmacology of selective NaPi2a inhibition.
KEYWORDS: SLC34A1, NaPi2a, sodium-phosphate cotransporter, kidney, proximal tubule, SLC
hosphate is an essential mineral component of bone,
MBD.9−11 While oral Pi binders are used to manage sustained
Pmembranes, nucleic acids, nucleotides, and second hyperphosphatemia in end stage renal disease, their use has not
been recommended in earlier stages of the disease, and recent
studies revealed no major improvement in Pi homeostasis.8,12,13
Therefore, we pursued the development of a NaPi2a inhibitor
as a novel approach to promote renal Pi excretion, reduce Pi
and FGF23 overload, and decrease cardiovascular risk in
predialysis CKD-MBD patients.14−16
To date, only nonselective NaPi2 inhibitors such as 1 and 2
(Figure 1) or NaPi2b inhibitors have been disclosed.17−21
Selective inhibition of NaPi2a over NaPi2b will avoid the risk of
pulmonary alveolar microlithiasis, a lung calcification disease
associated with human NaPi2b mutations,22−24 or limited
efficacy in part caused by a compensatory upregulation of
kidney Pi reabsorption associated with NaPi2b inhibition.22,25
Furthermore, NaPi2a inhibitors are envisioned to be systemi-
cally available, unlike some intestinally restricted NaPi2b
agents, and could offer straightforward pharmacokinetic
monitoring.
messengers and is key to cellular processes involving kinases
and phosphatases.1,2 As such, a complex multiorgan regulatory
interplay exists to maintain inorganic phosphate (Pi) homeo-
stasis. Intestinal absorption of dietary Pi and urinary excretion
control the flux of Pi into and out of the body, respectively,
while bone is used as a reservoir in the regulation of Pi blood
levels.
The three members of the NaPi2 family of solute carrier
(SLC) sodium-phosphate cotransporters play a key role in
phosphate homeostasis. NaPi2a (SLC34A1) and NaPi2c
(SLC34A3) are located on the apical membrane of the kidney
proximal tubule and function to reabsorb glomerular-filtered
Pi.3 NaPi2b (SLC34A2) is present on the apical membrane of
the small intestine where it absorbs a portion of dietary Pi. The
bone-derived hormone fibroblast growth factor 23 (FGF23)
and parathyroid-derived parathyroid hormone (PTH) down-
regulate NaPi2a and NaPi2c to increase urinary Pi excretion,
while 1,25(OH)2-vitamin D3 increases intestinal absorption of
Pi by upregulating NaPi2b.4,5
Chronic kidney disease−mineral and bone disorder (CKD-
MBD) is characterized by reduced glomerular filtration rate
(GFR), altered calcium and Pi homeostasis, deranged levels of
their regulatory hormones PTH, FGF23, and 1,25(OH)2-
vitamin D3, and compromised bone health.6−8 Multiple lines of
evidence indicate that elevated serum Pi and FGF23 excess
directly and independently contribute to increased risk for
cardiovascular mortality and morbidity in patients with CKD-
In order to find chemical leads, a high-throughput screen
(HTS) was performed by measuring the uptake of 33P-
radiolabeled Pi into HEK293 cells stably expressing NaPi2a.26
The parental HEK293 cell line was shown to endogenously
express another sodium-phosphate cotransporter, PiT-1
(SLC20A1)28 (Figures S1 and S2) and, as such, showed a
Received: January 10, 2018
Accepted: April 4, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.8b00013
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
a
Scheme 1. Synthesis of Azaindole Analogues
Figure 1. Representative nonselective literature NaPi2 inhibitors.
measurable, albeit lower, basal uptake of Pi. A series of related
cell lines were also developed to probe the selectivity of
analogues against other sodium-phosphate cotransporters:
NaPi2b, NaPi2c, PiT-1, and PiT-2 (SLC20A2). Furthermore,
cell lines containing the rat and mouse isoforms of NaPi2a and
NaPi2c were developed. Compounds were also evaluated in a
competition binding assay using membranes from NaPi2a-
transfected cells (vide infra).
From the NaPi2a HTS of ∼225,000 compounds in the Pfizer
collection, 3a was found as a hit and was profiled further (Table
1). Compound 3a showed micromolar (IC50 = 6.5 μM) activity
against NaPi2a and poor ligand and lipophilic efficiencies29 (LE
= 0.26, LipE = 1.2). However, in contrast to reference
compounds 1 and 2, hit 3a showed some, albeit modest,
selectivity against the other NaPi2 and PiT transporters as well
as the parental cell line. Hit 3a is very lipophilic (logD = 4.3,
a
Reagents and conditions: (a) amine, i-Pr2NEt, CH3CN, 80 °C or
amine, i-Pr2NEt, CsF, CH3CN, 120 °C; (b) NCS, DMF, 20−30 °C.
shake-flask method) and not surprisingly has poor apparent
aqueous solubility measured at pH 6.5 (2 μM), very high
human and rat liver microsome (HLM, RLM) clearance (HLM
= 240 μL/min/mg; RLM > 560 μL/min/mg), and poor
permeability as measured in RRCK cells.27
The synthesis of derivatives of 3a was performed as follows
(Scheme 1). A literature method was used to synthesize
compounds 4a−b.30 Standard SNAr chemistry was used to
install a 7-amino group on the azaindole core, leading to
compounds 3a−c. Alternatively, chlorination of the azaindole at
the 3-position was effected with N-chlorosuccinimide to
Table 1. SAR around Azaindole Core and Literature Comparators
a
b
IC50’s and Ki’s are reported as the geometric mean of n ≥ 3 standard error of the mean, except where otherwise noted. Passive permeability
(Papp) from the apical to basolateral (AB) direction was measured in Ralph Russ Canine Kidney (RRCK) cells as previously described.27 Aqueous
c
solubility at pH 6.5.
B
DOI: 10.1021/acsmedchemlett.8b00013
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ACS Medicinal Chemistry Letters
Letter
Table 2. SAR around 7-Position of Core
a
b
c
d
IC50’s and Ki’s are reported as the geometric mean of n ≥ 3 standard error of the mean, except where otherwise noted. n = 1. n = 2. Passive
permeability (Papp) from the apical to basolateral (AB) direction was measured in Ralph Russ Canine Kidney (RRCK) cells as previously described.27
e
f
Aqueous solubility at pH 6.5. NT = not tested.
of the azaindole core was first modified. Replacement of the
piperidine 4-substituent with a spirocyclic lactam provided 3b.
This change resulted in increased LipE (3.6 vs 1.2) and
significantly improved selectivity for inhibition of NaPi2a over
the other sodium-phosphate cotransporters tested (all other
IC50’s > 25 μM, Figure S3). We also observed that the
maximum inhibitory effect of 3b in the NaPi2a-transfected cells
was ∼75% of total Pi uptake (Figure S4). Since the HEK293
cell line endogenously expresses PiT-1, and the nonselective 1
(25 μM) was used to define 100% inhibition, our interpretation
is that, after NaPi2a-transfection, the cells contain a ≤3:1
functional ratio of NaPi2a/PiT-1. This Emax attribute was
maintained with future potent analogues from this series.
The SAR of the 2- and 3-positions of the azaindole core was
examined next. Deletion of the 2-methyl group (3c) led to a
significant loss of potency and LipE (<2.8), suggesting a
beneficial lipophilic interaction in this location. The addition of
a 3-chloro substituent (6a) yielded a 100 nM inhibitor with
improved LipE (4.0), permeability, and excellent selectivity.
However, the RLM clearance and aqueous solubility of 6a were
still very poor. Subsequent work to improve the ADME
properties was done while maintaining the potent 3-chloro-2-
methylazaindole core (Table 2).
Table 3. Rodent Potency of 6f
a
IC50 (μM)
rat NaPi2a
rat NaPi2c
>25
mouse NaPi2a
0.54 0.099
mouse NaPi2c
>25
0.40 0.047
a
IC50’s are reported as the geometric mean of n ≥ 3 standard error
of the mean.
a
Scheme 2. Synthesis of Azaindole Analogue 6f
a
Reagents and conditions: (a) i-Pr2NEt, CH3CN, −10 to −5 °C, 87%;
(b) DBU, CH3CN, 90 °C, 70%; (c) POCl3, CH3CN, 60 °C, 90%; (d)
NCS, DMF, 30 °C, 76%; (e) i-Pr2NEt, CH3CN, 80 °C, 86%.
The N-methylated lactam (6b) showed similar potency to
6a; however, this did lead to a useful radioligand. The tritiated
(C[3H]3) version of 6b showed saturable binding to
membranes from the NaPi2a-transfected cells with a Kd =
320 nM (Figures S5 and S6). This differs from parental cell
provide intermediate 5, and analogous SNAr chemistry led to
compounds 6a−g.
In order to turn the initial hit 3a into a useful tool to probe
the pharmacology of selective NaPi2a inhibition, the 7-position
C
DOI: 10.1021/acsmedchemlett.8b00013
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ACS Medicinal Chemistry Letters
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a
Table 4. Rat and Mouse PK of 6f
species
route
dose (mg/kg)
Cl (mL/min/kg)
15
Vdss (L/kg)
t1/2 (h)
Cmax (ng/mL)
tmax (h)
AUC0−24h (ng·h/mL)
F (%)
b
Wistar−Han rat
Wistar−Han rat
Wistar−Han rat
C57BL6 mouse
C57BL6 mouse
C57BL6 mouse
IV
1
3.1
4.8
1100
4800
c
PO
PO
IV
5
720
0.75
1.0
87
c
50
6000
77,000
760
140
b
1
22
0.86
0.75
c
PO
PO
5
540
0.38
1.5
1000
27
c
50
11,000
92,000
240
a
All values are the arithmetic mean of data from n = 2 animals. Rat plasma protein binding fraction unbound (PPB FU) = 0.0243; mouse PPB FU =
b
c
0.0287. Vehicle: 10% DMSO/30% PEG-400/60% water (2 mL/kg). Vehicle: suspension in 0.5% methylcellulose (10 mL/kg).
membranes where saturable binding was not observed. This
radioligand was used to develop a competition binding assay
where the ability of test compounds to displace tritiated 6b was
measured. Ki’s shown in Tables 1 and 2 exhibit good agreement
between the functional and binding potencies. Although the
radioligand is competitive with nonselective 1, the structural
reasoning behind this difference remains unknown. Further-
more, the new inhibitors are functionally selective; it is
unknown if they bind to the other phosphate transporters
beyond the parental cells.
Through further SAR efforts, the presence of a hydrogen-
bond acceptor (HBA) near the 3- or 4-position of the
piperidine proved key to obtaining good potency. This can be
observed with 4-hydroxypiperidine with one of the highest
ligand efficiencies (LE = 0.41) observed for the entire series.
This compound still had poor solubility, so we pursued
multiple approaches to improve this property. By adding a
charged center as in the primary amine 6d (pKa = 8.9),
solubility was greatly improved (230 μM), unfortunately at the
cost of potency (2.5 μM). The amino amide 6e improved
potency, further confirming our HBA hypothesis, but since this
is a much weaker base (pKa = 5.7), the solubility at pH 6.5 was
greatly reduced. An alternative strategy of increasing flexibility
in an effort to break efficient crystal packing interactions led to
6f (PF-06869206). Morpholino-methanol 6f showed a balance
of attributes with 380 nM NaPi2a inhibition potency, excellent
subtype selectivity, and acceptable aqueous solubility (46 μM).
Furthermore, permeability is good (14 × 10−6 cm/s), and RLM
clearance is low (<14 μL/min/mg; HLM = 39 μL/min/mg).
The enantiomer, 6g, was also synthesized and has similar
potency.
Compound 6f was further profiled for potency in the rodent
NaPi2a and NaPi2c cell lines (Table 3). This analogue showed
comparable submicromolar activity for the human, rat, and
mouse NaPi2a isoforms and was selective over rodent NaPi2c.
To support chronic in vivo studies, a large-scale synthesis of
6f was developed (Scheme 2). Pyrrole 731 was reacted with
enol tosylate 832 to form 9. DBU was used to effect pyridine
ring cyclization to give azaindole 10. Conversion of 7-
hydroxyazindole 10 to the corresponding 7-chloroazaindole
4a was accomplished using phosphorus oxychloride. N-
Chlorosuccinimide chlorination of the 3-position yielded 5
and SNAr with (S)-morpholin-2-ylmethanol provided the
desired 6f.
Compound 6f was evaluated in rodent PK studies to
determine suitability for in vivo pharmacology exploration.
Results showed moderate clearance in both rat and mouse
(Table 4, Figure S7). Oral bioavailability at 5 mg/kg was good
in rat and moderate in mouse. At higher oral doses of 50 mg/
kg, supraproportional increases in exposure were observed in
both species, suggestive of saturation of clearance.
Figure 2. Inhibition of phosphate uptake in human proximal tubule
cells by (A) 1, (B) 6b, and (C) 6e compared with PFA. All three
experiments from one donor. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001,
****p ≤ 0.0001; one-way ANOVA with Dunnett’s comparison to 0
μM control.
D
DOI: 10.1021/acsmedchemlett.8b00013
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ACS Medicinal Chemistry Letters
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The ability of two selective NaPi2a inhibitors from this newly
identified series, 6b and 6e, to inhibit Pi uptake in human
proximal tubule cells was characterized in vitro. As determined
by qPCR, these cells retained expression of NaPi2a, NaPi2c,
PiT-1, and PiT-2 (Figure S8). Nonselective inhibitor
phosphonoformic acid (PFA)3,28 at 5 mM inhibited on average
84% of Pi uptake and was used as a positive control across
experiments (Figures 2 and S9). At a common test
concentration of 1 μM and using cells from three different
donors, nonselective inhibitor 1 and selective NaPi2a inhibitors
6b and 6e showed mean Pi inhibition values of 79, 41, and 32%,
respectively. All three inhibitors displayed concentration-
dependent inhibition of Pi uptake. These results confirm the
selectivity profile of the newly identified series in a highly
relevant in vitro functional assay and indicate that NaPi2a plays
a significant role in human proximal tubule Pi reabsorption.
In summary, PF-06869206 (6f) is the first orally bioavailable
selective NaPi2a inhibitor and, as such, represents a
pharmacological tool to probe the functional effects of selective
NaPi2a inhibition in vivo. Results of these efforts will be
communicated in a future disclosure.
ACKNOWLEDGMENTS
■
The authors wish to thank Professor Colin D. A. Brown, Git
Chung, Karen Atkinson, Jeffrey Chabot, Brian Chekal, Steven
B. Coffey, Kentaro Futatsugi, Daniel W. Kung, Chia-Chin
(Wilson) Lee, John Litchfield, Suman Luthra, Kim McClure,
Klaas Schildknegt, and WuXi AppTec. H.J. is supported by
NIH grants DK11794 and DK46718.
ABBREVIATIONS
■
Pi, inorganic phosphate; NaPi2a, sodium-phosphate transporter
2a; SLC34A1, solute carrier transporter 34A1; FGF23,
fibroblast growth factor-23; PTH, parathyroid hormone;
CKD-MBD, chronic kidney disease−mineral and bone
disorder; GFR, glomerular filtration rate; HTS, high throughput
screen; LipE, lipophilic efficiency; RLM, rat liver microsome;
RRCK, Ralph Russ Canine Kidney; ADME, absorption,
distribution, metabolism, and excretion; LE, ligand efficiency;
HBA, hydrogen bond acceptor
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Corresponding Author
*E-mail: kevin.filipski@pfizer.com.
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∥(J.P.) Amgen Inc., 360 Binney Street, Cambridge, Massachu-
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⊥(K.S.) IMED Oncology, AstraZeneca Pharmaceuticals,
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#(C.F.) EBSCO Health, 10 Estes Street, Ipswich, Massachusetts
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Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
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(1) All procedures on animals were in accordance with
regulations and established guidelines and were reviewed and
approved by Pfizer’s Institutional Animal Care and Use
Committee. (2) Research was conducted on human tissue
acquired from a third party that has been verified as compliant
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Compound 6f (PF-06869206) has been made commercially
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DOI: 10.1021/acsmedchemlett.8b00013
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