Developing structure-activity relationships from an HTS hit for inhibition of the Cks1-Skp2 protein-protein interaction

Article abstract of DOI:10.1016/j.bmcl.2015.09.067

Structure-activity relationships have been developed around 5-bromo-8-toluylsulfonamidoquinoline 1 a hit compound in an assay for the interaction of the E3 ligase Skp2 with Cks1, part of the SCF ligase complex. Disruption of this protein-protein interaction results in higher levels of CDK inhibitor p27, which can act as a tumor suppressor. The results of the SAR developed highlight the relationship between the sulfonamide and quinoline nitrogen, while also suggesting that an aryl substituent at the 5-position of the quinoline ring contributes to the potency in the interaction assay. Compounds showing potency in the interaction assay result in greater levels of p27 and have been shown to inhibit cell growth of two p27 sensitive tumor cell lines.

Full text of DOI:10.1016/j.bmcl.2015.09.067

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Developing structure–activity relationships from an HTS hit for
inhibition of the Cks1–Skp2 protein–protein interaction
Rajinder Singh, Arvinder Sran, David C. Carroll, Jianing Huang, Lyuben Tsvetkov, Xiulan Zhou,
⇑
Julie Sheung, John McLaughlin, Sarkiz D. Issakani, Donald G. Payan, Simon J. Shaw
Rigel Pharmaceuticals Inc., 1180 Veterans Boulevard, South San Francisco, CA 94080, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Structure–activity relationships have been developed around 5-bromo-8-toluylsulfonamidoquinoline 1 a
hit compound in an assay for the interaction of the E3 ligase Skp2 with Cks1, part of the SCF ligase com-
plex. Disruption of this protein–protein interaction results in higher levels of CDK inhibitor p27, which
can act as a tumor suppressor. The results of the SAR developed highlight the relationship between the
sulfonamide and quinoline nitrogen, while also suggesting that an aryl substituent at the 5-position of
the quinoline ring contributes to the potency in the interaction assay. Compounds showing potency in
the interaction assay result in greater levels of p27 and have been shown to inhibit cell growth of two
p27 sensitive tumor cell lines.
Received 31 August 2015
Revised 25 September 2015
Accepted 28 September 2015
Available online xxxx
Keywords:
E3 ubiquitin ligases
Protein–protein interactions
Structure–activity relationships
Anti-cancer
Ó 2015 Elsevier Ltd. All rights reserved.
p27
Ubiquitination of a protein provides a signal for its targeted
degradation and recycling via the ubiquitin–proteasome path-
way.1–3 The process of ubiquitination takes place in a series of
steps, beginning with the activation of ubiquitin through a ubiqui-
tin-activating enzyme E1 followed by transfer to a ubiquitin-conju-
gating enzyme E2. Finally the ubiquitin is linked to the lysine of the
target protein in the presence of an ubiquitin-protein ligase E3
(referred to as a ubiquitin ligase). Chains of four or more ubiquitin
domains activates the degradation process by the proteasome.
The E3 ubiquitin ligase acts as a substrate recognition module
for the ubiquitination system in which each E3 provides specificity
for only a small number of substrates. This specificity makes E3
ligases attractive targets for drug discovery (analogous to kinases),
for instance by preventing degradation of pro-apoptotic proteins in
cancer cells.4 Indeed the proteasome inhibitor Bortezomib (mar-
keted as VelcadeÒ) is indicated for the treatment of multiple mye-
loma and is thought to be preventing degradation of pro-apoptotic
proteins.5 There is now a second class of proteasome inhibitors
based on the natural product epoxomicin—the recently approved
Carfilzomib (marketed as KyprolisÒ) and Oprozomib, which is still
in clinical trials.
protein–protein interactions (PPI’s). PPI’s are an area that has not
been well explored in small-molecule drug-discovery since the
interaction surfaces are often large with flat or shallow grooves
at the interfaces.6 This is in contrast to the tight, well defined pock-
ets present in traditional enzymes or receptors. However, the dis-
covery of molecules targeting PPI’s has gained recognition and
provides potential for novel treatments directed at significant
human maladies.7,8 Indeed there have been several examples of
groups studying the disruption of E3 ligase binding. One of the ini-
tial investigations in this area came from the Roche group, working
on the disruption of binding between p53 and MDM2. MDM2
serves as the E3 ligase for p53 promoting degradation. The work
resulted in the identification of cis-imidazolines known as Nutlins,
which displace p53 from its complex with MDM2 in the
100–300 nM range.9 These efforts have spurred a number of
groups to develop structure–activity relationships (SAR) around
these and similar structures, resulting in compounds that inhibit
the p53–MDM2 interaction with single digit nanomolar potencies
and below.10–12 Further there have been several reports of small
molecules being used to target E3 ligases including the von Hip-
pel-Lindau ligase to disrupt the VHL–HIF-1
a
interaction13 as well
The development of small molecule E3 ligase inhibitors is
challenging due to the requirement of the molecules to disrupt
as a non-ligase PPI between HIF1
a
and HIF1b.14
The Skp1-Cullin 1-F-Box (SCF) family of E3 ligases are a well
characterized family held together through PPI’s. The complex con-
sists of the scaffold protein Cullin-1, which binds Roc1 (recruiting
the E2) and Skp1 (recruiting the F-Box protein). For this study we
⇑
Corresponding author. Tel.: +1 650 624 1100; fax: +1 650 624 1101.
E-mail address: sshaw@rigel.com (S.J. Shaw).
http://dx.doi.org/10.1016/j.bmcl.2015.09.067
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Singh, R.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.09.067

2
R. Singh et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Table 1
were interested in the E3 ligase complex responsible for p27, the
substrate recognition component Skp2 and an adaptor protein
Cks1 (Fig. 1). p27, a CDK inhibitor, is a negative regulator of cell
cycle progression. Low levels of p27 have been implicated in a
number of cancers,15 while elevated levels of Cks1 have been asso-
ciated with low levels of p27 and poor prognosis in cancer
patients.16,17 To our knowledge there have not been any reports
of compounds targeted to the Cks1–Skp2 PPI; however, there have
been several groups that have targeted the SCF ligases with the
goal of increasing levels of p27. Molecules have been identified dis-
rupting PPI’s between Skp1 and Skp218 and Skp2–Cks1–p27.19 Fur-
ther there have been several reports of molecules that interact
with alternative E3 ligases in the SCF system including Cdc4,20
Potency of compounds 1, 2, 8–18 in the interaction assay and their ability to increase
p27-GFP
Compounds
Cks1–Skp2 (
l
M)
GFP-p27
1
2
8
9
10
11
12
13
14
15
16
17
18
0.84
2.18
0.54
0.55
1.73
5.54
7.84
Inactive
Inactive
Inactive
3.57
1.15
1.74
+
+
+
+
+
+
+
À
À
À
+
Met3021 and bTRCP1.22 The inhibitors are in the 1–10
lM range
À
NA
and were identified through the screening of libraries with little
SAR maturation.
Our study began by developing a high through-put screen (HTS)
for inhibitors of the Cks1–Skp2 PPI. From the screen a number of
hit compounds were identified representing a range of structurally
diverse scaffolds. One intriguing hit was the sulfonamidoquinoline
1 (Fig. 1), a sub-micromolar inhibitor of the Cks1–Skp2 PPI. Sup-
port for inhibition of this interaction was observed in cells express-
ing a GFP tagged p27, where we were able to see an increase in p27
GFP both on Western blot and using an ELISA format, suggesting
that in a cellular system the compound indeed inhibits the ubiqui-
tination and degradation of p27 (Fig. 1 and Supplementary Mate-
rial). A similar effect was also observed with compound 2 (Table 1).
With a hit compound in hand our investigations began by prob-
ing the relationship between the quinoline nitrogen and the sul-
fonamide linkage using compounds available in our compound
library. The corresponding amide, naphthalene, N-methylsulfon-
amide, isoquinoline and reversed sulfonamide (3–7) were
investigated, all of which were inactive in the interaction assay
suggesting that the relationship between the quinoline and sulfon-
amide was important for potency (Fig. 1).
The bromo group on the quinoline of 1 was an ideal handle for
the introduction of a range of substituents through cross-coupling
chemistries. The Suzuki reaction yielded a series of biaryl com-
pounds (8–12) while Buchwald–Hartwig reaction allowed for the
introduction of cyclic amines (13–15) (Scheme 1).
Compounds 8–15 were screened in the interaction assay and
those compounds that showed promising potency were further
screened in the p27-GFP assay (Table 1). Both the phenyl 8 and
the furanyl 9 derivatives showed improved potency over the hit
compound 1 (in the 500 nM range). Both compounds also increase
the levels of p27 in the p27-GFP assay suggesting that the
molecules are indeed inhibiting the SCFSkp2 ligase complex in cells.
Figure 1. (A) Schematic representation of the p27-SCFSkp2 complex and the assay to investigate inhibitors of the Cks1–Skp2 interaction, (B) Western blot showing increase in
p27-GFP in presence of Velcade or 1, (C) p27-GFP assay in ELISA format showing the increase in p27-GFP with compound (blue line) relative to DMSO (red line), (D) hit
compound 1 and analogues to probe the relationship between the sulfonamide and the quinoline.
Please cite this article in press as: Singh, R.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.09.067

R. Singh et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
Scheme 1. Synthesis of 5-aryl, 5-amino- and 6-O-arylquinoline compounds.
The pyridyl compound 10 is also potent in the interaction assay,
while the thiophene 11 and benzothiophene 12 compounds are
less potent. Interestingly, despite the weaker potency of com-
pounds 11 and 12 in the interaction assay both raised levels of
the p27 in the p27-GFP assay as did the pyridyl compound 10
(see Supplementary Material). By contrast, the benzylpiperidinyl,
morpholino and N-methylpiperazinyl analogues 13–15 were inac-
tive in both the interaction and p27-GFP assays. Moving the aryl
group in 8 to both the 4- or 6-position of the quinoline ring
resulted in compounds that showed weak or no activity in the
interaction assay (see Supplementary Material for a list of com-
pounds). However, the 6-O-(phenyl)quinolyl ethers 16–18 synthe-
sized by Evans–Chan–Lam reaction between the corresponding
arylboronic acid and 6-hydroxyquinoline resulted in a compounds
that were able to inhibit the Cks1–Skp2 interaction (Scheme 1).23–25
Finally we examined the nature of the sulfonamido group.
Beginning from the 8-amino-5-bromoquinoline 19 a series of
sulfonamides 20a–20f were generated from the corresponding
sulfonyl chlorides. In each series a group of three Suzuki reactions
was carried out using the phenyl, 3- and 4-pyridylboronic acids to
generate a 6 Â 3 matrix of compounds (Scheme 2). The sulfonyl
group was varied to investigate both alkyl groups (methyl,
cyclopropyl), and aryl groups, with the aryl groups were chosen
to explore the effect of electron-withdrawing and electron-donat-
ing substituents on inhibition of the Cks1–Skp2 interaction.
The results showed that the aromatic group was required with
the both the methyl and cyclopropyl compounds 21a,b–23a,b
showing weak activity in the interaction assay and did not increase
p27-GFP. However, both the p-methoxysulfonamido 22c, 23c and
in particular the m-trifluorophenylsulfonamido compound 22d
showed excellent potency, especially 22c and 22d the analogues
with the 3-pyridyl group at the 5-position of the quinoline.26 Fur-
ther these compounds increased levels of p27-GFP. Indeed 22d was
the more potent compound in the interaction assay and one of the
most potent inhibitors of an SCF ligase interaction reported
(Table 2). The pyridylsulfonamido compounds 21e–23e showed
reasonable potency in the interaction assay, but mixed effects in
p27-GFP levels. The acetamidosulfonamido compounds 21f–23f
were poor inhibitors. Taken as a whole the results suggest that
the substituent on the sulfonamido group may extend into a
hydrophobic region in the protein.
We were interested to know if the compounds were able to
inhibit tumor cell growth. Thus, compounds that were able to inhi-
bit the interaction of Cks1–Skp2 were screened in both the lung
tumor cell line A549 and the fibrosarcoma HT1080 in a six-day cell
proliferation assay. The cell lines have been shown to be sensitive
to p27 (Table 3) both in the literature and in our own laboratories
(see Supplementary Material).27,28 The data was compared with
Table 2
Potency of the sulfonamide analogues in the Cks1–Skp2 interaction assay (
their ability to increase p27-GFP in parentheses
lM), and
21
2.20 (+)
12.10 (À)
8.02 (+)
6.70 (+)
2.20 (+)
7.60
22
23
a
b
c
d
e
f
15.13 (À)
7.16 (À)
0.58 (+)
0.17 (+)
1.67 (+/À)
14.49
Inactive (À)
10.94 (À)
2.45 (+/À)
Not synthesized
1.21 (À)
6.55
Scheme 2. Synthesis of compounds to probe the sulfonamido group.
Please cite this article in press as: Singh, R.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.09.067

4
R. Singh et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Table 3
Supplementary data
Potency of selected compounds against p27 sensitive cell lines in
proliferation assay and in a two-day toxicity assay
a six-day
A list of 4- and 6-substituted compounds screened, schemes for
the synthesis of 6 and 7, all experimental procedures including
descriptions of the assays, data for the p27-GFP ELISA assay, a time
course for proliferation of A549 cells in the presence of Skp2 siRNA
and compound characterization.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.09.
067. These data include MOL files and InChiKeys of the most
important compounds described in this article.
Interaction
Cks1–Skp2
6-Day proliferation
2-Day proliferation
IC50 M)
GI50
A549
2.48
48.5
2.36
26.0
(l
M)
(l
IC50
(l
M)
HT1080
H1299 A549
2
4
8
9
2.18
2.46
27.36
1.64
9.78
4.22
0.64
0.15
0.08
0.74
6.83
0.44
1.32
0.40
1.07
1.07
5.35
7.54
Inactive
0.54
0.55
1.73
5.54
7.84
3.57
1.15
1.74
0.58
2.45
0.17
1.67
1.21
Inactive
8.56
Inactive
7.22
Inactive
6.95
Inactive
7.13
10
11
12
16
17
18
22c
23c
22d
22e
23e
6.02
2.36
0.39
4.11
4.09
6.48
1.24
3.06
0.91
5.24
5.88
4.09
4.63
Inactive
2.46
Inactive
2.94
References and notes
11.34
Inactive
5.31
16.92
Inactive
7.94
1. Cardozo, T.; Pagano, M. Nat. Rev. Mol. Cell Biol. 2004, 5, 739.
2. Nalepa, G.; Rolfe, M.; Harper, J. W. Nat. Rev. Drug Discov. 2006, 5, 596.
3. Nakayama, K. I.; Nakayama, K. Nat. Rev. Cancer 2006, 6, 369.
4. Cohen, P.; Tcherpakov, M. Cell 2010, 143, 686.
5. Adams, J.; Behnke, M.; Chen, S.; Cruickshank, A. A.; Dick, L. R.; Grenier, L.;
Klunder, J. M.; Ma, Y.-T.; Plamondon, L.; Stein, R. L. Bioorg. Med. Chem. Lett.
1998, 8, 333.
3.90
5.91
1.05
1.49
Inactive
9.48
Inactive
11.83
6. Arkin, M. R.; Wells, J. A. Nat. Rev. Drug Discov. 2004, 3, 301.
7. Wells, J. A.; McClendon, C. L. Nature 2007, 450, 1001.
8. Dömling, A. Curr. Opin. Chem. Biol. 2008, 12, 281.
9. Vassilev, L. T.; Vu, B. T.; Graves, B.; Carvajal, D.; Podlaski, F.; Filipovic, Z.; Kong,
N.; Kammlott, U.; Lukacs, C.; Klein, C.; Fotouhi, N.; Liu, E. A. Science 2004, 303,
844.
our standard two-day cell proliferation assay used to screen for
toxicity in two cell lines—H1299 and the same A549 line.29
The results suggest that inhibition of the Cks1–Skp2 PPI results
in inhibition of cell proliferation in p27 sensitive cell lines, with
compounds exhibiting micromolar potencies. By contrast, com-
pound 4, which is inactive in the interaction assay is the weakest
compound screened in the p27 sensitive six-day assay (IC50
10. Ding, Q.; Zhang, Z.; Liu, J.-J.; Jiang, N.; Zhang, J.; Ross, T. M.; Chu, X.-J.;
Bartkovitz, D.; Podlaski, F.; Janson, C.; Tovar, C.; Filipovic, Z. M.; Higgins, B.;
Glenn, K.; Packman, K.; Vassilev, L. T.; Graves, B. J. Med. Chem. 2013, 56, 5979.
11. Gonzalez-Lopez de Turiso, F.; Sun, D.; Rew, Y.; Bartberger, M. D.; Beck, H. P.;
Canon, J.; Chen, A.; Chow, D.; Correll, T. L.; Huang, X.; Julian, L. D.; Kayser, F.; Lo,
M-C.; Long, A. M.; McMinn, D.; Oliner, J. D.; Osgood, T.; Powers, J. P.; Saiki, A. Y.;
Schneider, S.; Shaffer, P.; Xiao, S-H.; Yakowec, P.; Yan, X.; Ye, Q.; Yu, D.; Zhao,
X.; Zhou, J.; Medina, J. C.; Olson, S. H. J. Med. Chem. 2013, 56, 4053.
12. Miyazaki, M.; Naito, H.; Sugimoto, Y.; Kawato, H.; Okayama, T.; Shimizu, H.;
Miyazaki, M.; Kitagawa, M.; Seki, T.; Fukutake, S.; Aonuma, M.; Soga, T. Bioorg.
Med. Chem. Lett. 2013, 23, 728.
13. Buckley, D. L.; van Molle, I.; Gareiss, P. C.; Tae, H. S.; Michel, J.; Noblin, D. J.;
Jorgensen, W. L.; Ciulli, A.; Crews, C. M. J. Am. Chem. Soc. 2012, 134, 4465;
Galdeano, C.; Gadd, M. S.; Soares, P.; Scaffidi, S.; van Molle, I.; Birced, I.; Hewitt,
S.; Dias, D. M.; Ciulli, A. J. Med. Chem. 2014, 57, 8657.
14. Miranda, E.; Nordgren, I. K.; Male, A. L.; Lawrence, C. E.; Hoakwie, F.; Cuda, F.;
Court, W.; Fox, K. R.; Townsend, P. A.; Packham, G. K.; Eccles, S. A.; Tavassoli, A.
J. Am. Chem. Soc. 2013, 135, 10418.
15. Chu, I. M.; Hengst, L.; Slingerland, J. M. Nat. Rev. Cancer 2008, 8, 253.
16. Masuda, T-a; Inoue, H.; Nishida, K.; Sonoda, H.; Yoshikawa, Y.; Kakeji, Y.;
Utsunomiya, T.; Mori, M. Clin. Cancer Res. 2003, 9, 5693.
17. Shapira, M.; Ben-Izhak, O.; Bishara, B.; Futerman, B.; Minkov, I.; Krausz, M. M.;
Pagano, M.; Hershko, D. D. Cancer 2004, 100, 1615.
18. Chen, Q.; Xie, W.; Kuhn, D. J.; Voorhees, P. M.; Lopez-Girona, A.; Mendy, D.;
Corral, L. G.; Plantevin Krenitsky, V.; Xu, W.; Moutouh-de Parseval, L.; Webb, D.
R.; Mercurio, F.; Nakayama, K. I.; Nakayama, K.; Orlowski, R. Z. Blood 2008, 111,
4690.
19. Wu, L.; Grigoryan, A. V.; Li, Y.; Hao, B.; Pagano, M.; Cardozo, T. J. Chem. Biol.
2012, 19, 1515.
20. Orlicky, S.; Tang, X.; Neduva, V.; Elowe, N.; Brown, E. D.; Sicheri, F.; Tyers, M.
Nat. Biotechnol. 2010, 28, 733.
21. Aghajan, M.; Jonai, N.; Flick, K.; Fu, F.; Luo, M.; Cai, X.; Ouni, I.; Pierce, N.; Tang,
X.; Lomenick, B.; Damoiseaux, R.; Hao, R.; del Moral, P. M.; Verma, R.; Li, Y.; Li,
C.; Houk, K. N.; Jung, M. E.; Zheng, N.; Huang, L.; Deshaies, R. J.; Kaiser, P.;
Huang, J. Nat. Biotechnol. 2010, 28, 738.
22. Nakajima, H.; Fujiwara, H.; Furuichi, Y.; Tanaka, K.; Shimbara, N. Biochem.
Biophys. Res. Commun. 2008, 368, 1007.
23. Evans, D. A.; Katz, J. L.; West, T. R. Tetrahedron Lett. 1998, 39, 2937.
24. Lam, P. Y. S.; Clark, C. G.; Saubern, S.; Adams, J.; Winters, M. P.; Chan, D. M. T.;
Combs, A. Tetrahedron Lett. 1998, 39, 2941.
25. Chan, D. M. T.; Monaco, K. L.; Wang, R.-P.; Winters, M. P. Tetrahedron Lett. 1998,
39, 2933.
26. At the time of the study material to generate 23d was not available; however,
we believe it should be possible to generate the compound.
27. Ishii, T.; Fujishiro, M.; Masuda, M.; Goshima, Y.; Kitamura, H.; Teramoto, S.;
Matsuse, T. Eur. Respir. J. 2004, 23, 665.
>25 lM). The most potent compound in the interaction assay, the
trifluoromethylsulfonamidoquinoline 22d, shows sub-micromolar
potency against both cell lines, which compares favorably with
the compounds identified in previous studies targeting p27.18,19
In the two-day assay the compound shows the most activity of
those screened, however, 22d is approximately two-fold more
potent in the six-day assay. In general, the HT1080 cell line appears
to be more sensitive with several compounds (12, 16, 22c and 22d)
showing sub-500 nM potency. An outlier is the benzothio-
phenequinoline 12, a weak inhibitor in the PPI assay but the most
potent compound against both cell lines in the six-day assay and
inactive in the two-day assay. The cell data suggests the effect is
p27-related, which is lent further credence by the ability of the
compound to increase levels of p27-GFP in cells. It may require a
more diverse set of compounds to fully interpret the data for com-
pound 12.
In summary we have shown that the ability of compounds to
inhibit the Cks1–Skp2 PPI results in greater levels of p27 in cells,
which in turn translates to effects on tumor cell growth. The SAR
developed from the hit sulfonamidoquinoline 1, demonstrates a
key role for the sulfonamide NH and quinoline nitrogen for inhibi-
tion of the PPI. In addition, it appears that there are binding inter-
actions that can be obtained by placing aromatic groups on the
sulfonamide and at the 5-position of the quinoline ring. It is antic-
ipated that developing further SAR will result in low nanomolar
inhibitors of the Cks1–Skp2 PPI, analogous to the example of
MDM2-p53.
28. Hoshino, R.; Tanimura, S.; Watanabe, K.; Kataoka, T.; Kohno, M. J. Biol. Chem.
2001, 276, 2686.
Acknowledgments
29. McLaughlin, J.; Markovtsov, V.; Li, H.; Wong, S.; Gelman, M.; Zhu, Y.; Franci, C.;
Lang, D. W.; Pali, E.; Lasaga, J.; Low, C.; Zhao, F.; Chang, B.; Gururaja, T. L.; Xu,
W.; Baluom, M.; Sweeny, D.; Carroll, D.; Sran, A.; Thota, S.; Parmer, M.;
Romane, A.; Clemens, G.; Grossbard, E.; Qu, K.; Jenkins, Y.; Kinoshita, T.; Taylor,
V.; Holland, S. J.; Argade, A.; Singh, R.; Pine, P.; Payan, D. G.; Hitoshi, Y. J. Cancer
Res. Clin. Oncol. 2010, 136, 99.
We are grateful to Mark Irving, Van Ybarra and Duayne Tokush-
ige for purification of some samples and high-resolution mass
spectral determinations and Guoqiang (John) Dong for protein
preparations.
Please cite this article in press as: Singh, R.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.09.067