Discovery of CX-5461, the first direct and selective inhibitor of RNA polymerase I, for cancer therapeutics

Article abstract of DOI:10.1021/ml300110s

Accelerated proliferation of solid tumor and hematologic cancer cells is linked to accelerated transcription of rDNA by the RNA polymerase I (Pol I) enzyme to produce elevated levels of rRNA (rRNA). Indeed, upregulation of Pol I, frequently caused by mutational alterations among tumor suppressors and oncogenes, is required for maintenance of the cancer phenotype and forms the basis for seeking selective inhibitors of Pol I as anticancer therapeutics. 2-(4-Methyl-[1,4]diazepan-1-yl)-5-oxo-5H-7-thia-1,11b-diaza-benzo[c] fluorene-6-carboxylic acid (5-methyl-pyrazin-2-ylmethyl)-amide (CX-5461, 7c) has been identified as the first potent, selective, and orally bioavailable inhibitor of RNA Pol I transcription with in vivo activity in tumor growth efficacy models. The preclinical data support the development of CX-5461 as an anticancer drug with potential for activity in several types of cancer.

Full text of DOI:10.1021/ml300110s

Letter
pubs.acs.org/acsmedchemlett
Discovery of CX-5461, the First Direct and Selective Inhibitor of RNA
Polymerase I, for Cancer Therapeutics
Mustapha Haddach,* Michael K. Schwaebe, Jerome Michaux, Johnny Nagasawa, Sean E. O'Brien,
Jeffrey P. Whitten, Fabrice Pierre, Pauline Kerdoncuff, Levan Darjania, Ryan Stansfield, Denis Drygin,
Kenna Anderes, Chris Proffitt, Josh Bliesath, Adam Siddiqui-Jain, May Omori, Nanni Huser,
William G. Rice, and David M. Ryckman
Cylene Pharmaceuticals, 5820 Nancy Ridge Drive, Suite 200, San Diego, California 92121, United States
S
* Supporting Information
ABSTRACT: Accelerated proliferation of solid tumor and hematologic cancer cells is linked to
accelerated transcription of rDNA by the RNA polymerase I (Pol I) enzyme to produce elevated
levels of rRNA (rRNA). Indeed, upregulation of Pol I, frequently caused by mutational alterations
among tumor suppressors and oncogenes, is required for maintenance of the cancer phenotype and
forms the basis for seeking selective inhibitors of Pol I as anticancer therapeutics. 2-(4-Methyl-
[1,4]diazepan-1-yl)-5-oxo-5H-7-thia-1,11b-diaza-benzo[c]fluorene-6-carboxylic acid (5-methyl-pyra-
zin-2-ylmethyl)-amide (CX-5461, 7c) has been identified as the first potent, selective, and orally
bioavailable inhibitor of RNA Pol I transcription with in vivo activity in tumor growth efficacy models. The preclinical data
support the development of CX-5461 as an anticancer drug with potential for activity in several types of cancer.
KEYWORDS: CX-5461, rRNA, RNA polymerase I, p53
he establishment and maintenance of malignancy depends
not only on mutated proto-oncogenes but also on an
suppressor p53. On one hand, activation of p53 is known to
repress Pol I transcription through disruption of preinitiation
complex formation;11,12 on the other hand, Pol I transcription
negatively regulates p53 activation through sequestration of
ribosomal proteins in the nucleolus. The inhibition of Pol I
transcription was shown to trigger a phenomenon called
“nucleolar stress” that leads to the translocation of ribosomal
proteins from nucleolus to nucleoplasm, where some of them,
for example, rpL5 and rpL11, will bind to MDM2, triggering its
dissociation from and thus activation of p53.13 Thus, by
sustaining high levels of Pol I transcription, cancer cells
maintain nucleolar integrity and hence promote the suppres-
sion of p53.
T
array of equally essential nonmutated genes that have become
deregulated.1,2 Products of these nonmutated genes control
processes that enable cancer cells to meet the increased
demand in growth and proliferation as well as to deal with the
burden of stress imposed by malignant transformation. Such
gene products are required to maintain oncogenic signaling and
inhibition of nononcogenic signaling, and interference with
these proteins can disrupt essential processes upon which
cancer cells have become dependent. Surprisingly, such
deregulated proteins represent an underexploited group of
prime targets for the treatment of cancer.
RNA polymerase I (Pol I) is a prime example of a
nononcogene target that can be leveraged for cancer
therapeutics. Pol I catalyzes for the synthesis of rRNA
(rRNA), which forms the backbone of the ribosome.3−5 The
rate of Pol I transcription controls cellular growth and
proliferation,6 and under normal conditions, it is tightly
regulated. However, during tumorigenesis, the relationship
between extracellular signaling and Pol I transcription is
deregulated, and cancer cells gain the capacity for excessive
production of rRNA and ribosomes necessary to produce
proteins for unbridled cell proliferation.6−9 Indeed, upregula-
tion of rRNA is sufficient by itself to maintain the oncogenic
phenotype, as siRNA directed at a subunit of Pol I leads to
partial inhibition of rRNA synthesis and induction of cell death
in cancer cells.10 In addition to the regulation of proliferation,
Pol I transcription is also involved in the extensive bidirectional
crosstalk with various oncogenes and tumor suppressors.7 One
example of such interaction is a relationship of Pol I with tumor
Multiple approved cancer therapeutics have been reported to
act through inhibition of rRNA synthesis, but none are known
to directly target the Pol I multiprotein enzyme complex.7,14
Hence, the rationale and medical need to justify the search for
agents directed at the Pol I complex as a nononcogene target
for cancer therapeutics still exist. Therefore, agents that
selectively disrupt Pol I transcription are conceptually attractive
as anticancer therapeutics.
The goal of the current project was to create a set of
molecules that directly, selectively, and potently inhibit Pol I,
that are orally bioavailable, and that exert in vivo antitumor
activity, from which a clinical candidate could be selected. For
this purpose, we developed and deployed a cell-based qRT-
PCR assay that measures the differential effects of new chemical
Received: May 4, 2012
Accepted: May 8, 2012
Published: May 8, 2012
© 2012 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
entity (NCE) on Pol I- and Pol II-driven transcription, as
previously described.15 The in vitro antiproliferative activity of
analogues was evaluated using colorectal adenocarcinoma
HCT-116 cells and then expanded to additional cell lines. To
rapidly screen compounds for oral absorption, a cassette
format16,17 was used to dose ICR mice at 25 mg/kg for each
compound, and blood was collected at 0.25, 0.5, 1, 2, 4, 6, and 8
h. These efforts led to the rapid discovery of 2-(4-methyl-
[1,4]diazepan-1-yl)-5-oxo-5H-7-thia-1,11b-diaza-benzo[c]-
fluorene-6-carboxylic acid (5-methyl-pyrazin-2-ylmethyl)-amide
(CX-5461, 7c), a highly potent, selective, and specific inhibitor
of rRNA synthesis that suppresses Pol I transcription at the
initiation stage and exhibits antiproliferative activity in vitro and
antitumor activity in xenograft models.
systematic study of structure−activity relationships (SARs) was
executed for three regions (A−C) of compound 2. The general
synthetic route used to prepare these compounds is illustrated
in Scheme 1. The synthesis of analogues around compound 2
a
Scheme 1. Synthesis of Analogues
The first set of compounds derived from our chemical
library, represented by entry 1 (Figure 1 and Table 1),
a
Conditions: (a) MgCl2, Et3N. (b) Amines, NMP, heat. (c) AlCl3,
amines, CH2Cl2.
began with the reaction between substituted 2,6-dichloronico-
tinic acid chloride 3 and ethyl 2-(5-chlorobenzothiazol-2-
yl)acetate 4 in the presence of magnesium chloride to give the
desired quinolone.19 Conversion of intermediate 5 to the
corresponding substituted amino intermediate 6, followed by
formation of an amide group under aluminum chloride-
catalyzed reaction conditions, provided quinolone 7. This
versatile sequence allowed for the rapid investigation of SARs in
all three regions. The key SAR results for select analogues
within this series are summarized in Table 1.
Our initial efforts focused on exploring the effect of electron-
withdrawing groups on the core structure (region B). Of note,
we found that introduction of fluorine at the R1 position (2a)
led to a decrease in RNA Pol I selectivity with similar oral
absorption to compound 1a. Furthermore, replacement of
hydrogen by fluorine at R3 (4a) resulted in a dramatic loss as
Figure 1. Structures of 1 and 2. Screening lead 2.
demonstrated reasonable inhibition of Pol I transcription and
modest selectivity vis-a-vis a lack of inhibition of Pol II
̀
transcription but displayed poor PK characteristics and a lack of
oral bioavailability (Table 1). Using scaffold hopping,18 we
designed several alterations of the core framework of
compound 1 (data not shown) that permit a similar geometric
arrangement of the side chains (regions A and C, Figure 1).
Among the scaffolds generated from this exercise, the
benzothiazole-based scaffold 2 (Figure 1) emerged as a
potential lead for a core structure.
Compound 2 showed a significant increase in oral absorption
while retaining a good in vitro biological profile (Table 1). A
a
Table 1. Selected Analogues SAR
IC50 (μM)
entry
n
X
R1
R2
R3
HCT-116
rRNA
cMYC
RNA Pol I
selectivity cMYC/rRNA
mouse PO AUC
1
0.02
0.12
0.05
0.08
0.59
0.19
0.66
0.19
0.15
>10
0.75
0.29
0.07
0.16
1.25
0.02
0.50
0.15
0.22
>10
5
15.2
17.5
9.05
8.17
3
ND
ND
0.91
0.63
4.41
ND
ND
1.54
0.88
>10
0.64
0.44
6.6
52.4
250
57
NQ
2
2586
2398
2261
6010
159
1a
2a
3b
4a
5c
6c
7c
8c
9c
10c
1
1
1
1
1
1
2
2
2
2
O
H
F
A
A
B
H
H
H
F
O
O
F
6.5
O
H
H
H
H
H
H
H
A
C
C
C
C
C
C
150
4
O
H
H
H
H
H
H
2.2
32869
90598
93373
ND
NMe
NMe
O
4.57
30.93
>10
13.37
15.28
31
141
Net
0.17
0.29
0.13
0.46
103
33
ND
N-Cyclohexyl
ND
a
AUC(0−24h), ng h/mL; ND, mot determined; and NQ, mot quantifiable.
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ACS Medicinal Chemistry Letters
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compared to compound 2a in oral absorption, although the
cellular activity profile was retained.
process also known to activate p53,21 but rather to Pol I
inhibition. As Figure 2 in the Supporting Information shows, 7c
We next examined modifications to region C. The change of
the position of the basic nitrogen from C3 (compound 2) to
C2 (compound 1a) on the side chain of region C led to an
increase in antiproliferative activity as determined by inhibition
of HCT-116 cancer cells and an increase of potency in the
inhibition of rRNA synthesis.
This compound also demonstrated higher selectivity for
RNA Pol I (rRNA synthesis) versus Pol II (c-myc mRNA
synthesis). However, oral absorption was similar for both
compounds (2 and 1a). The addition of rigidity to region C
(3b) led to a dramatic decrease in cellular and Pol I enzyme
activities (IC50 of 0.59 and 4.41 μM, respectively). The
selectivity between RNA Pol I and Pol II was also substantially
lower (6.5-fold). However, compound 3b demonstrated an oral
absorption 2-fold greater than the oral absorption of compound
2a. A breakthrough in increasing AUC was realized when the
basic methylpyrrolidine in compound 1a was replaced with
methylpyrazine (5c). Although the cellular activity of
compound 5c was moderate (IC50 = 0.66 μM), the inhibition
of Pol I and Pol II enzymes was completely lost. To regain
potency for Pol I enzyme inhibition, we examined the SAR of
the morpholino moiety (region A) of compound 5c. A detailed
survey of substitutions in region A led to the identification of a
few preferred substituents at region A, including 1-methyl-
piperazine (6c) and 1-methyl-1,4-diazepane (7c). Compound
6c increased oral exposure in mice 3-fold (6c vs 5c) with good
overall cellular and enzyme activity profiles. Compound 7c also
exhibited higher oral exposure and better selectivity for Pol I as
compared to compounds 5c and 6c. Compound 7c presented
similar activity to compound 6c in inhibiting cell growth (IC50
= 0.15 μM). The removal of basic center in region A (8c) led to
complete loss of biological activity. Increasing the size of N-
substituents on piperazine ring (9c and 10c) did not alter the
overall cellular or enzymatic activities.
Figure 2. Tumor growth efficacy of 7c in MV 4;11 xenograft as
measured by tumor volume. Nude mice were administered 6.25, 12, or
25 mg/kg dose ip once a day on a 5-on, 2-off schedule over a period of
21 days. Also represented is the 21 days animal body weight. Error
bars denote the standard error of the mean (SEM).
had no noticeable effect on DNA integrity relative to the
positive control actinomycin, a known DNA-damaging agent, at
similar concentrations (100 nM). At 10 μM CX-5461, the
COMET formation was more evident, but this concentration of
7c is approximately 100-fold the antiproliferative EC50, and the
effect is likely to be secondary to the induction of cell death and
the associated DNA fragmentation.
In addition, 7c was shown to be neither a strong DNA
intercalator nor minor groove binding drug at a concentration
as high as 50 μM (Figure 3 and the Supporting Information).
Taken together, these mechanistic assays demonstrated that the
activation of p53 is due to Pol I inhibition.
On the basis of target selectivity, in vitro antiproliferative
activity, and preliminary in vivo PK properties, compound 7c
was selected for further in vitro and in vivo testing. Treatment
of biphenotypic B myelomonocytic leukemia cell line MV 4;11
and large cell immunoblastic lymphoma cell line SR with
compound 7c resulted in the suppression of Pol I transcription
in both cell lines with EC50 values of 95 and 135 nM,
respectively. Furthermore, compound 7c demonstrated approx-
imately 200-fold selectivity against Pol I relative to Pol II, while
exerting no inhibition of DNA replication or protein translation
at concentrations as high as 10 μM. Compound 7c also
demonstrated robust cell growth inhibition in both cell lines
with IC50 values of 11 and 13 nM, respectively. Compound 7c
caused a significant activation of p53 as well as its transcrip-
tional target p21 in MV 4;11 and SR cells at 1 μM
concentration (Table 2).
Figure 3. PD biomarker study. Nude mice bearing MV 4;11 xenografts
were administered single 6.25, 12, or 25 mg/kg ip dose of 7c. Eight
hours later, animals were sacrificed, and intratumoral levels of p21
were determined by Western hybridization. Error bars denote the
standard error of the mean (SEM).
COMET assay20 ruled out the possibility that the activation
of p53 observed in both cell lines is due to DNA damage, a
a
Table 2. Effect of 7c on Activation of p53 and p21
The PK profile of 7c was evaluated in four preclinical species
(mouse, rat, dog, and monkey). Favorable half-lives (T1/2) were
observed across species with this compound. Clearance was low
to moderate, and the volume of distribution (Vd) was larger
than total body water in dog and monkey, indicating extensive
extravascular distribution. The measured oral bioavailability
cell line
p53 protein levels
p21 protein levels
MV 4;11
SR
6.7
4.1
5.4
3.2
a
Activation of p53 and p21. Cells were treated for 6 h with 7c. The
induction of p53 and p21 was measured by Western hybridization.
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ACS Medicinal Chemistry Letters
Letter
ranged from 24% in mice to 45% in Cynomolgus monkey
(Table 3).
AUTHOR INFORMATION
Corresponding Author
*Tel: 858-204-9319. E-mail: mhaddach@san.rr.com.
Notes
■
a
Table 3. Preclinical PK Profile of 7c
The authors declare no competing financial interest.
iv dosing
oral dosing
species
Cls
Vd
T1/2
AUC
% F
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0.03
0.06
1.6
0.24
0.08
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6.3
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92057
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24
29
38
45
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ASSOCIATED CONTENT
* Supporting Information
Biological assays and experimental procedures. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
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