1,1-Difluoroethyl-substituted triazolothienopyrimidines as inhibitors of a human urea transport protein (UT-B): New analogs and binding model

Article abstract of DOI:10.1016/j.bmcl.2013.03.089

The kidney urea transport protein UT-B is an attractive target for the development of small-molecule inhibitors with a novel diuretic ('urearetic') action. Previously, two compounds in the triazolothienopyrimidine scaffold (1a and 1c) were reported as UT-B inhibitors. Compound 1c incorporates a 1,1-difluoroethyl group, which affords improved microsomal stability when compared to the corresponding ethyl-substituted compound 1a. Here, a small focused library (4a-4f) was developed around lead inhibitor 1c to investigate the requirement of an amidine-linked thiophene in the inhibitor scaffold. Two compounds (4a and 4b) with nanomolar inhibitory potency (IC₅₀ ≈ 40 nM) were synthesized. Computational docking of lead structure 1c and 4a-4f into a homology model of the UT-B cytoplasmic surface suggested binding with the core heterocycle buried deep into the hydrophobic pore region of the protein.

Full text of DOI:10.1016/j.bmcl.2013.03.089

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3338–3341
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
1,1-Difluoroethyl-substituted triazolothienopyrimidines as
inhibitors of a human urea transport protein (UT-B):
New analogs and binding model
Y. Liu a, C. Esteva-Font b, C. Yao b, P. W. Phuan b, A. S. Verkman b, M. O. Anderson a,
⇑
a Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, CA 94132-4236, United States
b Departments of Medicine and Physiology, University of California, San Francisco, CA 94143-0521, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The kidney urea transport protein UT-B is an attractive target for the development of small-molecule
inhibitors with a novel diuretic (‘urearetic’) action. Previously, two compounds in the triazolothienopyr-
imidine scaffold (1a and 1c) were reported as UT-B inhibitors. Compound 1c incorporates a 1,1-difluoro-
ethyl group, which affords improved microsomal stability when compared to the corresponding
ethyl-substituted compound 1a. Here, a small focused library (4a–4f) was developed around lead inhib-
itor 1c to investigate the requirement of an amidine-linked thiophene in the inhibitor scaffold. Two com-
pounds (4a and 4b) with nanomolar inhibitory potency (IC50 ꢀ 40 nM) were synthesized. Computational
docking of lead structure 1c and 4a–4f into a homology model of the UT-B cytoplasmic surface suggested
binding with the core heterocycle buried deep into the hydrophobic pore region of the protein.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 8 March 2013
Revised 21 March 2013
Accepted 22 March 2013
Available online 1 April 2013
Keywords:
Urea transport protein
Triazolothienopyrimidine
Microsomal stability
Molecular docking
Kidney urea transporters (UTs) are required for the formation of
a concentrated urine.1,2 Epithelial cells in some kidney tubules ex-
press UT-A proteins, which are encoded by the SLc14A2 gene, and
endothelial cells in some microvessels express UT-B, which is en-
coded by the SLc14A1 gene.3–5 Knockout mice lacking various
UTs manifest impaired urinary concentrating ability, suggesting
the application of UT inhibitors as ‘urearetics’ with a novel diuretic
mechanism of action.6–9
Early high-throughput screening using a human erythrocyte
lysis assay identified phenylsulfoxyoxozole inhibitors of human
UT-B with IC50 ꢀ 100 nM.10 Limitations of the original compounds
included poor metabolic stability and poor activity against rodent
UT-B. Further screening using a mouse erythrocyte lysis assay
yielded triazolothienopyrimidine UT-B inhibitors (Fig. 1), with
the most potent compound, 1a, having IC50 ꢀ 11 nM for inhibition
of human UT-B and 25 nM for inhibition of mouse UT-B.11 Admin-
istration of 1a to mice reduced urine osmolality and increased
urine output. Compound 1a and related analogs inhibit UT-B by a
competitive mechanism.
R1 position as well as variations at the amidine linkage) were eval-
uated, with no improved leads identified.12 One interesting analog
(1b) was synthesized containing a trifluoromethyl group at posi-
tion R1, which resisted microsomal degradation, but suffered sig-
nificant loss of potency against UT-B. Reasoning that a two
carbon atom substituent was required for potency, but that ben-
zylic hydrogens are prone to radical abstraction, we synthesized
the 1,10-difluoroethyl inhibitor (1c), and found that the inhibitor
resisted microsomal degradation with little effect on UT-B inhibi-
tion activity (IC50 ꢀ 14 nM) of the original lead 1a.12 We chose to
incorporate the amidine-linked thiophene-2-methyl group into
1c, which has generally been associated with more potent and sta-
ble inhibitors in this class. Herein, we prepared a small library of
analogs of 1c, in which alternative amidine linked groups were
evaluated for UT-B inhibitory potency and hepatic microsomal sta-
bility. Indeed, while the stability of 1c was enhanced greatly by the
incorporation of the 1,10-difluoroethyl substituent, a slow micro-
somal oxidation reaction could still be observed by LCMS, which
we speculate might be modulated by variation of the amidine
substituent.
Though 1a was effective in mice when administered in high
concentration, it underwent rapid oxidation by hepatic enzymes,
mostly at the benzylic position of the ethyl substituent. A collec-
tion of 273 commercially available analogs of 1a (exploring the
The synthetic approach to generate this library of inhibitors,
with differing amidine substituents tethered from the core hetero-
cycle is summarized in Scheme 1. As was described previously, 4-
bromo-(1,1-difluoroethyl)-benzene (2) was transformed to the
corresponding triazolothienopyrimindine (3) by [2+3] cycloaddi-
tion with an azidothiophene ester.12,11 Subsequently, lactam 3
⇑
Corresponding author.
E-mail address: marc@sfsu.edu (M.O. Anderson).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.03.089

Y. Liu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3338–3341
3339
Table 1
UT-B inhibition activitya and microsomal stabilityb of synthesized compounds
Figure 1. Previously characterized trizaolothienopyrimidine inhibitors of UT-B.
Compd
R1
R2
IC50 (nM)
11
% Remainingb
1a
<5
96
1c
4a
14
38
22
4b
4c
4d
43
47
14
79
353
>800
Scheme 1. Synthesis of amidine analogs of triazolothienopyrimidine lead structure
1c. Reagents and conditions: (a) described previously;12 (b) 1° amines, PyBOP,
CH3CN, DBU, microwave irradiation, 100 °C, 30 min.
4e
4f
>800
>800
75
24
was coupled with various primary amines, using PyBOP and DBU
under microwave irradiation, to generate a library of amidine
derivatives (4a–4f). While amidine formation using PyBOP and
DBU has been described previously,13,14 we observed that micro-
wave irradiation was necessary to effect quantitative conversion
in this class of molecules. Typically the compounds could be iso-
lated with simple aqueous workup, and purified by trituration with
cold methanol. All of the compounds were characterized by 1H
NMR and HRMS, and had a characteristic triplet (3J = ꢁ18 Hz) due
to coupling between the terminal methyl and neighboring difluo-
romethylene groups. Yields and spectroscopic characterization of
these compounds are provided in Supplementary data.
The library was designed to explore the effect of the amidine
substituent on UT-B inhibition potency and on microsomal stabil-
ity (Table 1). Compounds 1a and 1c were the previously character-
ized inhibitors.11,12 The new inhibitors synthesized in this library
had amidine substituents designed to explore: methyl substitution
on the thiophene (4a), an isomeric thiophene connection point
(4b), replacement of the thiophene heterocycle for furan (4c) or
benzyl (4d), and the use of small hydrophobic alkyl ring systems
such as cyclopropylmethyl (4e) or cyclobutylmethyl (4f).
UT-B inhibition potency was determined by an erythrocyte lysis
assay, as previously described10 in which lysis is measured in acet-
amide-loaded erythrocytes following dilution into isosmolar PBS
(Supplementary data). Erythrocytes express the UT-B protein,
which facilitates the transmembrane transport of urea, acetamide
and other small polar solutes. UT-B inhibition increases hypotonic
lysis, as measured by near-infrared light absorbance, because
water influx is not opposed by acetamide efflux. Microsomal stabil-
ity was determined by incubation of compounds in the presence of
a
Determined by mouse erythrocyte lysis assay (n = 3). IC50 determined by fit to a
single-site saturation model.
Percentage of parent compound remaining after 30 min incubation with rat
liver microsomes.
b
rat liver microsomes and NADPH, followed by organic extraction
and LCMS analysis, as described12 (Supplementary data).
In general, modification of the amidine substituent reduced
inhibition potency and microsomal stability. Two of the more po-
tent new inhibitors evaluated were 4a (containing 4-methylthi-
ophene) and 4b (thiophene connected at the 3 position). Both of
these inhibitors had modest potency, but suffered in terms of
microsomal stability when compared to 1c. An analog containing
furan (4c) instead of thiophene (1c) showed poor potency and sta-
bility, which was surprising due to the similarity between the thi-
ophene and furan heterocycles. This is consistent with another
observation, in which the furan analog of 1a was was found to have
greatly reduced potency.12 Replacement of the amidine-linked thi-
ophene with non-heterocyclic rings such as benzyl (4d), cyclopro-
pylmethyl (4e), and cyclobutylmethyl (4f) also greatly reduced
potency. In the case of 4d and 4e, these modifications mildly im-
proved microsomal stability compared to 1a.
Molecular modeling and docking was done to investigate the
potential inhibitor binding site. While a high-resolution structural
model of UT-B has not been reported, we constructed a homology
model for UT-B using the SWISS MODEL utility, using the full hu-
man UT-B1 protein sequence, and have previously used this model
to postulate a mode of binding of 1a.11 Compounds 1c and 4a–4f

3340
Y. Liu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3338–3341
the inhibitor has a good complementary fit to the surface of UT-
B, and aside from the one potential hydrogen bonding interaction,
most likely is bound due to hydrophobic interactions with non-po-
lar amino acid residues. Inhibitor 4b is an exception compared to
the other compounds, in that the lowest energy conformation pre-
sented the thiophene deep into the pocket.
(b) The docking software used has six independent scoring
functions that evaluate protein–ligand interactions using different
algorithms. Of the scoring functions available, five reliably distin-
guished the relatively active inhibitors (1a, 1c, 4a and 4b) from
the relatively inactive inhibitors (4c–4f): Shapegauss, PLP, Chem-
gauss3, OEChemScore, and Screenscore. The sixth scoring function
(ZapBind), which weights heavily toward electrostatic interactions,
was not useful at discriminating active from inactive inhibitors in
this series, which is expected given the limited electrostatic inter-
actions in our postulated binding mode.
(c) Deciphering the structural determinants of the active and
inactive compounds is challenging. The thiophenemethyl amidine
compounds (1a and 1c) appear to have the most complementary
overlap, giving rise to the most potent compounds. Alkylation of
the thiophene (4a) or simple use of an isomerically connected thi-
ophene (4b) decreases potency, albeit not catastrophically. Most
puzzling is the severe drop in potency in the furan-based inhibitor
(4c), for which docking identified a similar mode of binding as the
more potent thiophene-based inhibitor 1c. Nevertheless, with the
FRED scoring functions named above (excluding ZapBind) all cor-
rectly ranked 1c as a better scoring ligand than 4c. The remaining
less active inhibitors (4d–4f) were scored poorly by FRED, and
apparently the amidine linked substituent had less complementary
fit compared to the more active compounds.
In summary, a focused library of six analogs of UT-B inhibitor 1c
was prepared to characterize the structural requirements for po-
tency and microsomal stability. Two additional compounds with
modest potency (4a and 4b) were discovered, but none were able
to match 1c. Computational docking into a homology model of UT-
B gave a tentative mode of binding for 1c and 4a–4f. While caution
should be used in over-interpreting docking results from homology
models, we were gratified to see that docking with multiple inde-
pendent scoring functions effectively distinguished the active from
inactive analogs, and that with exception of one compound (4b),
the docked conformations were consistent.
c
Acknowledgements
This work was supported by grant DK35124 from the National
Institutes of Health. The authors acknowledge OpenEye Scientific
(Santa Fe, NM) for its Academic Site License program.
Figure 2. Model of the binding of 1c with the cytoplasmic channel region of human
UT-B based on a homology model: (a) zoomed-in and (b) zoomed-out depictions;
(c) potential interactions between 1c and UT-B cytoplasmic surface residues.
Supplementary data
Supplementary data (these data include synthetic procedures
and spectroscopic characterization of compounds 4a–4f, and an
additional figure related to the computational modeling experi-
ments.) associated with this article can be found, in the online ver-
sion, at http://dx.doi.org/10.1016/j.bmcl.2013.03.089.
were docked into the same homology model using FRED v2.2.5
(OpenEye Scientific).15,16 Figure 2 shows a tentative model for
binding of inhibitor 1c, zoomed in and out, with the key interac-
tions shown. We offer several observations from the modeling:
(a) The computed mode of binding of inhibitor 1c is consistent
with our previously reported model of 1a. A similar mode of bind-
ing was found for 4a and 4c–4f, with the orientation of the core
heterocycle, aryl sulfone, and amidine substituents being consis-
tent (Supplementary Fig. S1). In the postulated binding mode, the
core heterocycle is buried deep into a hydrophobic pocket sur-
rounded by Leu121, Leu365, Phe301, Phe71, and other amino acids.
There is a potential hydrogen bonding interaction between the
Asn165 sidechain and an exposed triazole nitrogen. The aryl sulfone
is surrounded by Ser169, Ser365, and Asp172, while the thiophene
group resides near the Thr368 methyl group and Lys366. Overall,
References and notes
1. Bankir, L.; Yang, B. Kidney Int. 2012, 81, 1179.
2. Sands, J. M.; Layton, H. E. Semin. Nephrol. 2009, 29, 178.
3. Sands, J. M. Curr. Opin. Nephrol. Hypertens. 2004, 13, 525.
4. Smith, C. P. Exp. Physiol. 2009, 94, 180.
5. Stewart, G. Br. J. Pharmacol. 2011, 164, 1780.
6. Yang, B.; Bankir, L.; Gillespie, A.; Epstein, C. J.; Verkman, A. S. J. Biol. Chem. 2002,
277, 10633.
7. Fenton, R. A.; Chou, C. L.; Stewart, G. S.; Smith, C. P.; Knepper, M. A. Proc. Natl.
Acad. Sci. U.S.A. 2004, 101, 7469.

Y. Liu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3338–3341
3341
8. Fenton, R. A. Curr. Opin. Nephrol. Hypertens. 2008, 17, 513.
9. Uchida, S.; Sohara, E.; Rai, T.; Ikawa, M.; Okabe, M.; Sasaki, S. Mol. Cell. Biol.
2005, 25, 7357.
13. Wan, Z. K.; Wacharasindhu, S.; Binnun, E.; Mansour, T. Org. Lett. 2006, 8, 2425.
14. Wan, Z.-K.; Wacharasindhu, S.; Levins, C. G.; Lin, M.; Tabei, K.; Mansour, T. S. J.
Org. Chem. 2007, 72, 10194.
10. Levin, M. H.; de la Fuente, R.; Verkman, A. S. FASEB J. 2007, 21, 551.
11. Yao, C.; Anderson, M. O.; Zhang, J.; Yang, B.; Phuan, P. W.; Verkman, A. S. J. Am.
Soc. Nephrol. 2012, 23, 1210.
12. Anderson, M. O.; Zhang, J.; Liu, Y.; Yao, C.; Phuan, P. W.; Verkman, A. S. J. Med.
Chem. 2012, 55, 5942.
15. McGann, M. J. Chem. Inf. Model. 2011, 51, 578.
16. McGaughey, G. B.; Sheridan, R. P.; Bayly, C. I.; Culberson, J. C.; Kreatsoulas, C.;
Lindsley, S.; Maiorov, V.; Truchon, J. F.; Cornell, W. D. J. Chem. Inf. Model. 2007,
47, 1504.