Synthesis of novel Ral inhibitors: An in vitro and in vivo study

Article abstract of DOI:10.1016/j.bmcl.2016.10.021

Chemical synthesis was performed to produce a series of 6-amino-1,3-disubstituted-4-phenyl-1,4-dihydro pyrano[2,3-c]pyrazole-5-carbonitrile compounds (14–57) which were characterized by¹H NMR,¹³C NMR and LC/MS–MS. These compounds were assessed for their effect on the in vitro anchorage independent growth of human lung cancer cell line H2122 and IC₅₀values calculated. Two of the more potent compounds, BQU057 40 and BQU082 57 also displayed a dose dependent effect on RalA and RalB activity in H2122 spheroids using the common RalBP1 pull-down assay. Mouse PK and tissue distribution studies on 40 and 57 were performed and demonstrated that parent drug was present in tumor 3.0 h post ip (50 mg/Kg) dose.

Full text of DOI:10.1016/j.bmcl.2016.10.021

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of novel Ral inhibitors: An in vitro and in vivo study
Chao Yan a,b,y, Dan Theodorescu a,b,c, Bettina Miller d, Amit Kumar d, Vijay Kumar d, David Ross d,
Michael F. Wempe d,
⇑
a Department of Pharmacology, University of Colorado, Aurora, CO 80045, USA
b Department of Surgery, University of Colorado, Aurora, CO 80045, USA
c University of Colorado Comprehensive Cancer Center, Aurora, CO 80045, USA
d Department of Pharmaceutical Sciences, University of Colorado, Aurora, CO 80045, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Chemical synthesis was performed to produce a series of 6-amino-1,3-disubstituted-4-phenyl-1,4-dihy-
dro pyrano[2,3-c]pyrazole-5-carbonitrile compounds (14–57) which were characterized by 1H NMR, 13C
NMR and LC/MS–MS. These compounds were assessed for their effect on the in vitro anchorage indepen-
dent growth of human lung cancer cell line H2122 and IC50 values calculated. Two of the more potent
compounds, BQU057 40 and BQU082 57 also displayed a dose dependent effect on RalA and RalB activity
in H2122 spheroids using the common RalBP1 pull-down assay. Mouse PK and tissue distribution studies
on 40 and 57 were performed and demonstrated that parent drug was present in tumor 3.0 h post ip
(50 mg/Kg) dose.
Received 25 July 2016
Accepted 8 October 2016
Available online xxxx
Keywords:
Ral inhibitor
Chemical synthesis
Tissue distribution
Ó 2016 Elsevier Ltd. All rights reserved.
The Ras family of evolutionary related proteins is ubiquitously
expressed in cells and organs. The Ras genes (H-, K-, R-, N-)—a pro-
totypical Ras superfamily member belongs to the class of protein
known as ‘small GTPase’ and are involved in cellular signal trans-
duction.1 Ras is frequently mutated in human cancer2 and even
when not mutated, over-active (from upstream mediators) Ras sig-
naling can drive cancer growth.3 While efforts to develop targeted
therapy against Ras have not yet been successful, targeting Ras
effectors is a viable alternative approach. Examples include target-
ing the Raf-MEK-ERK mitogen activated protein kinase (MAPK)
cascade and phosphoinositide 3-kinase (PI3K)-AKT-mTOR path-
ways.4,5 However, until recently the third canonical effector path-
way, RalGEF-Ral has not been targeted.6 Ral (Ras-like) is an
important member of the Ras small GTPase superfamily and shares
a high structural similarity with the Ras protein. Like all GTPases,
Ral cycles between an inactive GDP-bound state and an active
GTP-bound state. Ral gets activated by guanine exchange factors
(RalGEFs) which are effectors of Ras. Once activated, Ral will bind
to its own specific downstream effectors such as RalBP17 and
Sec5/Exo84,8 which mediate the biological functions of Ral in cells.
The contributions of Ral signaling to tumorigenesis, progression
and metastasis in cancer have been well established by us9,10 and
others.11–15
We sought to identify potential small molecular weight Ral
inhibitors. An in silico search (ꢀ500,000 compounds) was per-
formed and was followed by in vitro ELISA screening (88 com-
pounds) to probe selective binding of active RalA-GTP to its
effector protein RALBP1.16 From these data, two compounds
(RBC8 and RBC10, Fig. 1) were identified as potential ‘lead’ tem-
plates and these two compounds were further studied via tumor
xenograft in vivo model (H2122 human lung cancer cell line).
We were not only interested in the ability to decrease in vivo
tumor growth, but also wanted to define tumor and tissue distribu-
tion for both compounds. The tissue distribution of RBC8 and
RBC10 (3.0 hr post ip dose; Table 1) in a mouse model illustrated
that drugs were detectable in all tissues tested (brain, heart,
kidney, liver and lung) with liver demonstrating the highest
O
O
Cl
O
O
N
N
HN
N
H
O
NH
2
RBC8
RBC10
⇑
Corresponding author. Tel.: +1 303 724 8982.
E-mail address: michael.wempe@ucdenver.edu (M.F. Wempe).
Chao Yan’s new contact information: State Key Laboratory of Pharmaceutical
CLogP: 5.52
CLogP: 5.43
y
Biotechnology, College of Life Sciences, Nanjing University, Nanjing, China.
Figure 1. RBC8 and RBC10.
http://dx.doi.org/10.1016/j.bmcl.2016.10.021
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Yan, C.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.10.021

2
C. Yan et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Table 1
Table 2
RBC8 and RBC10 tissue distribution data
1,3-Disubstituted-1H-pyrazol-5(4H)-one analogs
Tissue
RBC8
0.57 0.37
13.73 9.46
24.62 12.72
39.68 14.67
21.61 10.84
5.83 3.46
RBC10
R2
O
R2
Brain
0.21 0.09
3.54 0.60
5.73 1.80
11.47 1.02
3.18 0.98
4.17 1.45
3.95 0.30
Ethanol /
Δ
N
N
NH2
NH
O
O
Heart
Kidney
Liver
Lung
Tumor
Plasma
+
R1
R1
OEt
1
2
3 - 11
*
3.91 2.29
Compound #
R1
R2
*
Plasma expressed as
dose (50 mg/Kg).
lg/mL while tissues are
lg/g; data n = 3 SD; 3.0 h post ip
3
Ph
Me
4
5
6
Me
Me
Ph
Me
Ph
Ph
7
8
CH2-Ph
Me
Ph
N
O
3,4-Di-OMe-Ph
3,4-Di-OMe-Ph
4-OMe-Ph
4-OMe-Ph
O
9
10
11
Ph
Ph
Me
NH2
O
N
NH
RBC8
Mono-Oxidation
5.2 min
O-Demethylation
R3
O
N
O
- CH2
R7
R4
O
N
NH2
O
NC
O
CN
CN
R6
R5
O
+ O
+
NH2
O
NC
N
R2
N
NH
4.6 min
major
M1
CN
R6
R7
R5
M2
N
NH
4.7 min
minor
R6
R7
3 - 11
R5
R4
+ O
O
NH2
N
R4
O
N
- CH2
O
R3
R1
14 - 57
O
13
R3
N
NH2
O
12
2-(substituted)-
6-amino-1,3-disubstituted-4-phenyl-1,4-
dihydropyrano[2,3-c]pyrazole-5-carbonitriles
benzylidenemalononitrile
aromatic aldehydes
O
NH2
4.9 min
minor
N
NH
O
M3
O
Figure 3. Synthesis scheme for RBC8 analogs.
N
NH
5.0 min
major
M4
+ O
N
O
NH2
Table 2). This allows control of R1 and R2 substitutions in the final
compound.
O
N
5.2 min
minor
NH
The various 1,3-disubstituted-1H-pyrazol-5(4H)-one com-
pounds (3–11) were then utilized in the next reaction which used
various substituted aromatic aldehydes (12). The aromatic aldehy-
des (Fig. 3) reacted with malononitrile to give the corresponding
alkene; these alkenes then reacted in tandem with the 1,3-disub-
stituted-1H-pyrazol-5(4H)-one compound to produce the
corresponding 6-amino-1,3-disubstituted-4-phenyl-1,4-dihydro
pyrano[2,3-c]pyrazole-5-carbonitrile (14–57; Table 3). The general
synthesis has been previously described in the literature;17 the
chemical synthesis and chemical characterization for the prepared
analogs has been summarized in the Supplementary materials sec-
tion. The compounds were then tested in vitro to produce IC50
growth values.
M5
Figure 2. RBC8 proposed Phase I biotransformation.
concentrations. As the liver is a major site of drug metabolism, we
sought to probe mouse liver metabolism. Mouse (CD-1) liver
microsomes were used to probe CYP catalyzed biotransformationof
RBC8 (Fig. 2). LC/MS–MS methods were used to illustrate the for-
mation of three mono-oxidation products (M1, M3, and M5), and
two dealkylation (demethylation) products (M2 and M4); metabo-
lites numbers are listed in order of retention time. These in vitro
generated metabolites were also observed in vivo (Supplementary
materials section, S-Table 1). Overall M1 and M4 were the two
major in vivo metabolites. In an analogous fashion, RBC10 mouse
liver Phase I metabolism was probed. Two major metabolites were
produced in vitro (M6 and M7) and these two metabolites were
confirmed in the in vivo samples (Supplementary materials sec-
tion, S-Table 2). The distribution and metabolism data illustrate
that parent drug was distributed and that parent drug was present
in the tumors. Consequently, we decided to probe the RBC8 chem-
ical template. We sought to prepare a series of RBC8 analogs and
probe how the change in structure might alter in vitro biological
activity (i.e., Structure–Activity Relationship (SAR)).
The various synthesized analogs demonstrated IC50 values from
1.1 to >10 lM in H2122 cells and provided limited SAR informa-
tion. For example, if we compare compound 14 (R1 and
R2 = methyl) to compound 29, the data supports that size rather
than electronics on the aromatic ring are important factors; this
appears to also be present when one compares 32 to 37 and 38,
the bulkier 38 being more potent in the in vitro growth assay.
Comparing 18 to 35 illustrates that subtle chemical modifications
can give rise to significant changes; from this series, compounds
such as 35, 38, 40 and 57 were appealing. Consequently, we choose
two analogs for further study (BQU057 40 and BQU082 57, Fig. 4).
Compounds 40 and 57 were then used to determine the Ral
activity inhibition in cells. As presented in Figure 5, both com-
pounds displayed a dose dependent in vitro effect on RalA and RalB
activity.
The chemical synthesis to prepare RBC8 analogs begins with the
reaction of a hydrazine (1) with a 3-oxo-ester (2) to produce vari-
ous 1,3-disubstituted-1H-pyrazol-5(4H)-one compounds (3–11;
Please cite this article in press as: Yan, C.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.10.021

C. Yan et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
Table 3
RBC8 analogs prepared and tested in vitro
Compound #
R1
R2
R3
R4
R5
R6
R7
IC50 (lM)
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
Me
Me
Ph
Ph
Ph
Me
Me
Me
Ph
Me
Ph
Me
Ph
Ph
Me
Me
Me
Ph
Me
Me
Ph
Me
Ph
Me
Ph
H
H
H
H
H
H
H
F
F
F
F
F
F
F
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
F
H
H
H
H
H
H
F
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OMe
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
N:*
N:*
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OMe
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
9.1 0.3
>10
2.7 0.1
4.1 0.3
7.2 0.6
>10
>10
>10
6.6 0.5
>10
4.1 0.3
>10
6.0 0.5
7.6 0.3
>10
4.2 0.1
6.6 0.4
4.1 0.5
8.6 1.1
>10
3.1 0.7
1.1 0.2
>10
5.8 0.3
1.6 0.4
>10
2.2 0.3
4.1 0.9
2.9 0.4
5.5 1.0
5.6 0.5
5.2 0.3
4.0 0.5
5.4 0.6
8.6 0.5
8.4 0.9
4.1 0.8
5.2 0.6
8.8 0.4
>10
4-OMe-Ph
4-OMe-Ph
3,4-DiOMe-Ph
Me
Me
Ph
Ph
3,4-DiOMe-Ph
4-OMe-Ph
3,4-DiOMe-Ph
4-OMe-Ph
Me
Ph
Me
Me
Ph
H
F
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
CF3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Ph
Ph
p-OMe-Ph
p-OMe-Ph
Me
Me
Ph
Me
Ph
Me
Ph
Me
Ph
Me
Ph
Me
Ph
Me
Ph
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
CF3
O-CF3
O-CF3
CN
CN
CH(CH3)2
CH(CH3)2
O-CH2-
O-CH2-
N:*
H
H
CH2-O-
CH2-O-
H
N:*
H
H
H
H
Me
Ph
Me
Ph
Me
Ph
N:*
*
N:
Br
Br
H
2.4 0.4
1.7 0.1
1.7 0.1
0.3
H
Imidazole
Imidazole
H
n = 3–4 SD.
*
Attached to the aromatic ring.
N
N
CF3
N
N
N
N
N
N
O
NH2
O
NH2
BQU082 (57)
BQU057 (40)
CLogP: 3.52
CLogP: 4.17
Figure 4. Chemical structures of BQU057 and BQU082.
Figure 5. BQU057 and BQU082 Ral activity inhibition data.
Lastly, we also performed mouse PK experiments (ip dosing of
BQU057 and BQU082) to obtain plasma concentration versus time
profile data 5 h post-dose. These data illustrate that the two com-
pounds have very short plasma half-lives, T1/2 < 1.5 h.
In conclusion, we have prepared novel compounds, which dis-
play reasonable Ral inhibitory potential (i.e., approaching
1.0 lM). While this initial chemistry and biology effort has pro-
As summarized in Table 4, tissue distribution was probed at
3.0 h post ip dose. BQU057 and BQU082 were found to be well dis-
tributed in the mouse xenograft model and similar to RBC8 and
RBC10 were drug was detected in tumor.
duced interesting analogs, we continue to seek compounds which
are more potent and have improved ADME properties (i.e., lower
IC50, improved metabolic stability, longer half-life) through struc-
ture based design.
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C. Yan et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Table 4
References and notes
BQU057 and BQU082 mouse tissue distribution data
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Tissue
BQU057 (40)
BQU082 (57)
Brain
1.39 0.42
2.08 0.42
2.40 0.91
4.07 1.76
1.87 0.63
1.22 0.27
5.64 0.64
0.17 0.07
0.93 0.24
0.63 0.26
0.98 0.43
1.09 0.41
0.38 0.21
0.49 0.19
Heart
Kidney
Liver
Lung
Tumor
Plasma
*
*
Plasma expressed as
dose (50 mg/Kg).
l
g/mL while tissues are
l
g/g. Data n = 4 SD; 3.0 h post ip
11. Zipfel, P. A.; Brady, D. C.; Kashatus, D. F.; Ancrile, B. D.; Tyler, D. S.; Counter, C.
M. Oncogene 2010, 29, 4859.
12. Peschard, P.; McCarthy, A.; Leblanc-Dominguez, V.; Yeo, M.; Guichard, S.;
Stamp, G.; Marshall, C. J. Curr. Biol. 2012, 22, 2063.
Acknowledgments
The research use the services of the Medicinal Chemistry Core
Facility (MCCF) housed within the Department of Pharmaceutical
Sciences (DOPS); the MCCF receives funding via CCTSI, an institute
at the University of Colorado Denver supported in part by
NIH/NCATS Colorado CTSI Grant Number UL1TR001082. Work
was supported by grant from Colorado Center for Drug Discovery
(www.C2D2.org) (to DT) and CA091846 (to DT).
13. Lim, K. H.; O’Hayer, K.; Adams, S. J.; Kendall, S. D.; Campbell, P. M.; Der, C. J.;
Counter, C. M. Curr. Biol. 2006, 16, 2385.
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D. L.; Paschal, B. M.; Schwartz, M. A.; Jones, D. N.; Ross, D.; Meroueh, S. O.;
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.10.
021.
Please cite this article in press as: Yan, C.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.10.021