Discovery, synthesis and structure-activity analysis of symmetrical 2,7-disubstituted fluorenones as urea transporter inhibitors

Article abstract of DOI:10.1039/c5md00198f

Kidney urea transporters are targets for development of small-molecule inhibitors with action as salt-sparing diuretics. A cell-based, functional high-throughput screen identified 2,7-bisacetamido fluorenone 3 as a novel inhibitor of urea transporters UT-A1 and UT-B. Here, we synthesized twenty-two 2,7-disubstituted fluorenone analogs by acylation. Structure-activity relationship analysis revealed: (a) the carbonyl moiety at C9 is required for UT inhibition; (b) steric limitation on C2, 7-substituents; and (c) the importance of a crescent-shape structure. The most potent fluorenones inhibited UT-A1 and UT-B urea transport with IC₅₀ ～ 1 μM. Analysis of in vitro metabolic stability in hepatic microsomes indicated metabolism of 2,7-disubstituted fluorenones by reductase and subsequent elimination. Computational docking to a homology model of UT-A1 suggested UT inhibitor binding to the UT cytoplasmic domain at a site that does not overlap with the putative urea binding site.

Full text of DOI:10.1039/c5md00198f

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Discovery, synthesis and structure–activity analysis
of symmetrical 2,7-disubstituted fluorenones as
urea transporter inhibitors†
Cite this: DOI: 10.1039/c5md00198f
Sujin Lee,a Cristina Esteva-Font,a Puay-Wah Phuan,a Marc O. Andersonb and
a
*
A. S. Verkman
Kidney urea transporters are targets for development of small-molecule inhibitors with action as salt-
sparing diuretics. A cell-based, functional high-throughput screen identified 2,7-bisacetamido fluorenone 3
as a novel inhibitor of urea transporters UT-A1 and UT-B. Here, we synthesized twenty-two 2,7-disubsti-
tuted fluorenone analogs by acylation. Structure–activity relationship analysis revealed: (a) the carbonyl
moiety at C9 is required for UT inhibition; (b) steric limitation on C2, 7-substituents; and (c) the importance
of a crescent-shape structure. The most potent fluorenones inhibited UT-A1 and UT-B urea transport with
IC50 ~ 1 μM. Analysis of in vitro metabolic stability in hepatic microsomes indicated metabolism of 2,7-
disubstituted fluorenones by reductase and subsequent elimination. Computational docking to a homology
model of UT-A1 suggested UT inhibitor binding to the UT cytoplasmic domain at a site that does not over-
lap with the putative urea binding site.
Received 7th May 2015,
Accepted 29th May 2015
DOI: 10.1039/c5md00198f
www.rsc.org/medchemcomm
The kidney expresses urea transporter (UT) proteins, which
facilitate the passive transport of urea across cell plasma
membranes in a subset of kidney tubules and microvessels.1
SLc14A1 and SLc14A2 genes encode UT-A and UT-B urea
transporters, respectively.2 Studies in mice lacking UTs3–7 and
in rodents treated with UT inhibitors8–10 indicate that UT-A1,
the UT-A isoform expressed at the apical membrane of epithe-
lial cells in inner medullary collecting duct, is the principal
target for diuretic development. Absence or inhibition of UTs
impairs urinary concentrating function, producing a diuretic
response. UT inhibitors are thus development candidates as
first-in-class salt-sparing diuretics for therapy of various
edema states and hyponatremias, such as those associated
with congestive heart failure and cirrhosis.11–13
Our lab previously developed high-throughput functional
assays of UT-A14 and UT-B15 urea transporters. Several classes
of small-molecule inhibitors of the target UT-A1 were identi-
fied.10,14 In proof-of-concept studies, two classes of inhibitors
with low micromolar IC50 produced a diuretic response in
rats;10 however, their inhibition potency and metabolic stabil-
ity were not optimal for further development. Additional
screening reported here identified symmetrical, disubstituted
fluorenones as novel UT inhibitors. Because of the drug-like
properties of tricyclic fluorenones and the absence of a com-
mercial source to obtain analogs for structure–activity rela-
tionship analysis, here we synthesized 22 symmetrical, disub-
stituted fluorenones, measured their UT inhibition activity
and selectivity, analyzed their inhibition and metabolism
mechanisms, and used homology modeling and computa-
tional docking to propose binding sites on UT-A and UT-B.
A UT-A1 inhibition screen of 50 000 compounds identified
2,7-bisacetamidofluorenone 3 as a UT-A1 inhibitor with IC50
~ 1 μM that produced complete inhibition at higher concen-
trations (Fig. 1A). Fluorenone 3 also inhibited UT-B with sim-
ilar potency. The fluorenone scaffold has not been previously
reported for the inhibition of urea transporters, though there
are prior reports of biological activities of this compound
class. Tiloron is an orally bioavailable antiviral agent16,17 and
an immunomodulator.18 The antitumor activity of fluorenone
derivatives has been shown to result from inhibition of
telomerase and DNA topoisomerase I.19–21 Most reported
fluorenone analogs focused on 2,7-bis-ester or ether moieties,
unlike the bis-acetamidofluorenone 3 identified from the UT-
A1 screen. The 2,7-bis-acetamido fluorenone structure has
drug-like properties, including favorable molecular weight
(294 Da), topological surface area (75.2 Å2) and c log P IJ2.12),
which fall within the Lipinski22 and Veber23 criteria for orally
bioavailable drugs.
a Departments of Medicine and Physiology, University of California, San
Francisco, CA, 94143-0521, USA. E-mail: Alan.Verkman@ucsf.edu
b Department of Chemistry and Biochemistry, San Francisco State University, San
Francisco, CA, 94132-4136, USA
Based on the potency and physicochemical properties of
3, a series of 2,7-disubstituted fluorenone analogs were ratio-
nally designed to identify more potent urea transport
† Electronic supplementary information (ESI) available: See DOI: 10.1039/
c5md00198f
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Fig. 1 Discovery of 2,7-disubstituted fluorenone 3 as UT-A1 inhibitor. A. Structure of 3 and concentration-inhibition data for inhibition of rat UT-
A1 and UT-B urea transporters Fitted parameters: IC50 1μM and 1.5μM, Hill coefficient 0.9 and 1.1, for UT-A1 and UT-B respectively. B. Strategy for
analog synthesis for SAR analysis.
inhibitors and to establish structure–activity relationships.
Structurally, fluorenone 3 is a symmetrical, rigid crescent-
shaped molecule with a carbonyl group at the 9-position and
bisacteamido groups at the 2 and 7 positions.
16. Further substitution reaction of 11 using methoxyethanol
with sodium hydride afforded 13, and with sodium hydroxide
and sodium azide gave the corresponding amide analogs 12
and 14, respectively.
As diagrammed in Fig. 1B, analogs were designed to
include: i) different functional groups on the 2,7-diamino
position; ii) different non-carbonyl functional groups at the
9-position; and iii) flexible and ring strain-released scaffolds.
In a preliminary study, testing of ~70 commercially available
fluorenone analogs did not identify active analogs.
To investigate the importance of the carbonyl at the
9-position for urea transport inhibition, we synthesized
oxime 17, hydroxyl 18 and sulfone 20 (Scheme 3). Condensa-
tion of 2,7-bisacetamido fluoren-9-one 3 with hydroxylamine
Scheme 1 shows the synthetic approaches for the prepara-
tion of 2,7-bisIJalkylamido)fluorenones 3–10. Reduction of
commercially available 2,7-dinitro-fluoren-9-one
1 using
sodium sulfide nonahydrate and sodium hydroxide afforded
the key intermediate 2,7-diamino-fluoren-9-one 2.19 The re-
synthesis of 3 was accomplished by acetylation of 2 using
acetic anhydride. Additional acyl analogs of 3 were similarly
prepared by reaction of 2 with the respective acyl reagents
under basic condition to yield propionyl, isobutyryl and
butyryl analogs 4, 5 and 6 respectively. We next prepared the
bio-isostere analogs of 3, trifluoroacetamide 7, carbamate
8
and methanesulfonamide 9. Treatment of
2
with
trifluoroacetic acid and heating to reflux in a sealed tube
afforded trifluoroacetamide 7.24 The carbamate analog 8 was
synthesized by reaction of 2 with potassium isocyanate.25 Sul-
fonamide analog 9 was prepared by mesylation in pyridine.
The γ-sultam 10 was synthesized using chloropropansulfonyl
chloride and subsequent cyclization with potassium
carbonate.
As shown in Scheme 2, analogs with extended chains on
the amide bond were synthesized. Bromoacetamido 11 and
benzyloxyacetamido 15 were synthesized from 2 using corre-
sponding acyl halides in refluxing xylene. Using maleic anhy-
dride in refluxing chloroform gave the maleamic acid analog
Scheme
1 Synthesis of 2,7-bisalkylamidofluorenone and 2,7-
bissufoneamidofluorence analogs. Reaction conditions and reagents:
(a) Na2S·9H2O, NaOH, EtOH, 78%; (b) Et3N, THF, Ac2O for 3, propionly
chloride for 4; isobutyryl chloride for 5; butyryl chloride for 6; (c)
CF3CO2H in a sealed tube for 7; (d) KNCO, AcOH/H2O for 8; (e) MsCI,
pyridine, THF for 9; (f) 3-chloropropansulfonyl chloride, pyridine,
CH2CI2; then, K2CO3, CH3CN, reflux, for 10.
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Scheme
2 Synthesis of extended 2,7-bisacetylamidofluorenone
analogs. Reaction condition and reagents: (a) bromoacetyl bromide,
xylene, reflux, 40%; (b) benzyloxyacetyl chloride, xylene, reflux, 26% for
15; (c) maleic anhydride, CHCI3, 26% for 16; (d) NaOH, EtOH, 86% for
12; (e) methoxyethanol, NaH, CH3CN, 18% for 13; (f) NaN3, DMF, 24%
for 14.
hydrochloride in pyridine afforded oxime analog 17. Ketone
reduction of 3 with sodium borohydride in methanol gave
hydroxyl analog 18. Acylation of commercially available 2,7-
diaminodiphenylenesulfone 19 afforded the bisacetamido-
diphenylsulfone 20. To prepare flexible ring acyclic analogs,
commercially available 3,3′-diaminobenzophenone 21 was
acylated and sulfonated to give bisacetamide 22,
Scheme 3 Synthesis of carbonyl-modified fluorenone analogs. Reac-
tion conditions and reagents: (a) NH2OH·HCI, DMSO/H2O, 22%; (b)
NaBH4, CH3CN/MeOH IJ10/1), 28%; (c) Et3N, THF, Ac2O, 65%; (d) Et3N,
THF, Ac2O, 62% for 22; benzyloxyacetyl chloride, xylene, reflux, 46%
for 23; (e) MsCI, pyridine, THF, 23%.
benzyloxyacetamide
23
and
bismethanesulfonamido-
benzophenone 24.
Table 1 summarizes IC50 values for inhibition of UT-A1
and UT-B urea transport by the 2,7-disubstituted fluorenones
1–24. Most analogs showed similar IC50 for inhibition of UT-
A1 and UT-B urea transport. Non-substituted fluorenones (1,
2 and 21) were inactive. Elongation of the carbon chain on
the amide reduced inhibition, comparing acetamide 3 to
propionyl 4, isobutyryl 5, maleamic acid 16 and butyryl 6.
Linear ether linkage analogs with elongated side chain also
reduced inhibition. Hydroxyacetamide 12 had low activity,
and benzyloxyacetamides 15 and 13 were inactive. Replacing
suggesting that a hydrogen bond acceptor is necessary on the
carbonyl. Interestingly, sulfone 20 showed UT-B selectivity
with IC50 of 2 μM for UT-B compared to 15 μM for UT-A1.
Fig. 2A shows concentration-inhibition data for UT-A1
and UT-B inhibition by 3 and analogs 7, 9 and 22. Fig. 2B
shows original concentration-inhibition curves for 3 (left
panel). The in vitro characterization of 3 revealed complete
reversibility for inhibition of UT-A1 (second panel), rapid
inhibition kinetics with complete inhibition by 40 seconds
(third panel), and a non-competitive inhibition mechanism
in which apparent IC50 was independent of urea concentra-
tion (right panel).
acetamide with methanesulfonylamide
9 and carbamate
8 slightly reduced activity. Addition to the acetamide of one
more atom – hydroxyl 12, methyl 4 or bromide 11 – reduced
activity. These findings implicate a narrow size limitation for
substituents. Interestingly, trifluoroacetamide 7, which has
the same carbon length as 3, was much less active, indicating
that steric and electronic effects also influence inhibition.
The influence of the carbonyl moiety was determined by
comparing UT-A1 inhibition by 3, 17, 18 and 20. These ana-
logs contain acetamide on the C2 and C7 positions, but activ-
ity was reduced greatly by replacing the carbonyl with oxime
17, sulfone 20 or hydroxyl 18 moieties (IC50 12 to >50 μM),
To
investigate
the
potential
utility
of
disubstituted fluorenones for UT inhibition in animal studies,
in vitro microsomal stability measurements were made using
an established assay in which compounds were incubated
with rat liver microsomes in the presence of NADPH.26 For 3,
60% remained after 15 min (Fig. 3A). Oxidation catalyzed by
the cytochrome P450 monooxygenase, a well-known meta-
bolic reaction on aromatic compounds, was not detected
from LC/MS analysis. Instead, a peak at [M + H − 16] was
detected that increased over time (Fig. 3B). Generation of a
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Table 1 Inhibition of UT-A1 and UT-B urea transport by 2,7-disubstituted
fluorenone analogs
UT-A1 UT-B
IC50
IC50
Structure
Functional group
(μM) (μM)
1
>50
>50
R
2 H
3 COCH3
4 COCH2CH3
>50
1
15
>50
1.5
5
5 COCHIJCH3)
18
50
2
6 COIJCH2)2CH3
7 COCF3
8 CONH2
>50
10
2
1.5
>50
30
>50
7.5
1.2
2.5
>50
10
9 SO2CH3
10 SO2IJCH2)3
11 COCH2Br
12 COCH2OH
20
25
13 COCH2OIJCH2)2OMe >50
50
14 COCH2N3
15 COCH2OBn
16 COIJCH)2CO2H
X
20
>50
30
30
>50
>50
Fig.
3
In vitro matabolic stability of disubstituted fluorenones.
17 CNHOH
18 CHOH
20 SO2
R
21 H
22 COCH3
23 COCH2OBn
24 SO2CH3
12
>50
15
25
20
2
Compounds incubated with hepatic micromes in the presence of
NADPH. A. LC/MS showing disapperance of 3 and appearance of
metabolite over 60 min. B. Kinetics of disappearance of 3, 17, 20, and
22 over 60 min.
>50
18
>50
>50
>50
20
>50
30
dinitrofluorenone by rat liver enzymes utilizing NADH/
NADPH has been reported, although the specific carbonyl
reduction enzyme was not established.27 Following reduction
of carbonyl on fluorenone, the amide proton is readily
removed by base-catalyzed elimination to give 2-imino-
fluorene 25. To support this proposed mechanism, fluorenol
[M + H − 16] metabolite suggests loss of oxygen, suggesting
that bisacetamidofluorenone metabolism involves the mecha-
nism proposed in Fig. 4. Carbonyl reduction of 2,7-
Fig. 2 Urea transport inhibition by 2,7-disubstituted fluorenone. A. Concentration-inhibition data for UT-A1 and UT-B for 3, 11, 14 and 19. B. Pri-
mary data from UT-A inhibition assay showing reversibility, inhibition kinetics and urea competition for 3. Phloretin concentration was 0.35 mM.
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shape, which is lost when substituted with biphenylacetone.
Increasing flexiblility of the acetophenone moiety reduced UT
inhibition activity.
Computational docking simulations were done to propose
binding modes for the most potent UT-A1 inhibitor 3, and
the most potent UT-B inhibitor 8. Structures for rat UT-A1
and UT-B were generated by homology modeling as
described,10 based on the high resolution X-ray crystal struc-
ture of bovine UT-B solved at 2.5 Å.29 Our homology models
of these two proteins contain sites that appear homologous
to narrow constriction (Sm) and low energy urea binding
sites (So and Si) observed in the bovine UT-B template
structure.
Fig. 4 Proposed mechanism of hepatic metabolism of 3.
18 was subjected to basic condition (1M NaOH). The elimi-
nated product 25 could be isolated after 1 hour in ~60%
yield, and matched [M + H − 16].28 Also, to confirm this pro-
posed mechanism, metabolic stability was studied for oxime
17, bisacetaminobiphenylacetone 22 and bisacetamino-
sulfone 20, which do not contain a carbonyl moiety and thus
cannot undergo carbonyl reduction/base-catalyzed elimina-
tion sequence. We found that 17, 20 and 22 were relatively
stable compared to fluorenone 3 (>80% remaining at 15
min), and [M + H − 16] was not detected by LC/MS (data not
shown).
Interestingly, acetophenone analog 22 had less UT inhibi-
tion activity than 3 and sulfonamide 24 was inactive com-
pared to corresponding fluorenone analog 9, which showed
strong inhibition. This result suggests that the 3D structure
of the fluorenone core is important to UT inhibition.
Fig. 5A shows docked conformations of inhibitor 3 bound
to UT-A1 and UT-B. The lowest energy conformation of this
molecule docked into both proteins in similar orientations.
The planar inhibitor scaffold docked into the outer part of
the UT-A1 and UT-B pore region, not binding deeply into the
channel. In the docked conformation of 3 with UT-A1 the
amido NH2 group projects into a pocket surrounded by
Glu572, Val603, Asn604, Phe832 and Tyr900. The bis-urea inhibi-
tor 8, which shows a greater potency for UT-B, is shown for
comparison (Fig. 5B), and docks in a similar manner as 3.
Other active analogs of 3 also docked similarly. The position
of the inhibitor scaffolds do not overlap the putative cytoplas-
mic urea binding site (Si) near Gln599, which is homologous
to Gln63 in the selenourea-bound template structure.28 Taken
together with urea competition experiments for 3, the pro-
posed binding site in Fig. 5 is consistent with a non-
competitive inhibition mechanism.
Fluorenones with 2,7-disubstituents have
a crescent-like
Fig. 5 Computational docking of 3 and 8 to homology models of rat UT-A and UT-B. Zoomed-in and zoomed-out representations of (A) inhibitor
3 bound to rat UT-A and UT-B cytoplasmic domains; and (B) the most potent UT-B inhibitor 8 bound to the rat UT-B cytoplasmic domain.
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Conclusions
using different concentrations of urea (80 to 1,600 mM) in
the UT-A1 inhibition assay.
In conclusion, we identified by high-throughput screening
symmetrical 2,7-disubstituted fluorenones as novel UT inhibi-
tors, and established structure–activity relationships by syn-
thesis and characterization of 22 analogs. Functional studies
indicated reversible inhibition of UT-A1 urea transport by the
2,7-disubstituted fluorenone analogs by a non-competitive
inhibition mechanism. Docking computations suggested
inhibitor binding at the UT outer pore regions at a site dis-
tinct from the putative urea binding site. Finally, analysis of
inhibitor metabolism indicated carbonyl reduction by reduc-
tase and subsequent base-catalyzed elimination.
Acknowledgements
Supported by grants DK101373, DK35124, DK72517, EB00415
and EY13574 from the National Institutes of Health. The
authors acknowledge OpenEye Scientific (Santa Fe, NM, USA)
for its Academic Site License program.
Notes and references
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Experimentals
Synthesis
All chemical synthetic procedures and characterizations are
described in supplementary information.
Cell culture
Triply transfected MDCK cells expressing rat UT-A1, yellow
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–
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Hz), 5.57 (brs, 1H), 2.55 (s, 3H), 2.05 (s, 3H); LRMS (ESI) m/z
279 (M + H)+.
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