Adventures in Scaffold Morphing: Discovery of Fused Ring Heterocyclic Checkpoint Kinase 1 (CHK1) Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.7b01490

Checkpoint kinase 1 (CHK1) inhibitors are potential cancer therapeutics that can be utilized for enhancing the efficacy of DNA damaging agents. Multiple small molecule CHK1 inhibitors from different chemical scaffolds have been developed and evaluated in clinical trials in combination with chemotherapeutics and radiation treatment. Scaffold morphing of thiophene carboxamide ureas (TCUs), such as AZD7762 (1) and a related series of triazoloquinolines (TZQs), led to the identification of fused-ring bicyclic CHK1 inhibitors, 7-carboxamide thienopyridines (7-CTPs), and 7-carboxamide indoles. X-ray crystal structures reveal a key intramolecular noncovalent sulfur-oxygen interaction in aligning the hinge-binding carboxamide group to the thienopyridine core in a coplanar fashion. An intramolecular hydrogen bond to an indole NH was also effective in locking the carboxamide in the preferred bound conformation to CHK1. Optimization on the 7-CTP series resulted in the identification of lead compound 44, which displayed respectable drug-like properties and good in vitro and in vivo potency.

Full text of DOI:10.1021/acs.jmedchem.7b01490

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Article
Adventures in Scaffold Morphing: Discovery of Fused
Ring Heterocyclic Checkpoint Kinase 1 (CHK1) Inhibitors
Bin Yang, Melissa Vasbinder, Alexander W. Hird, Qibin Su, Haixia Wang, Yan Yu, Dorin
Toader, Paul D Lyne, Jon A. Read, Jason Breed, Stephanos Ioannidis, Chun Deng,
Michael Grondine, Nancy DeGrace, David Whitston, Patrick Brassil, and James W Janetka
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 04 Jan 2018
Downloaded from http://pubs.acs.org on January 4, 2018
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Adventures in Scaffold Morphing: Discovery of Fused Ring
Heterocyclic Checkpoint Kinase 1 (CHK1) Inhibitors
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Bin Yang *, Melissa M. Vasbinder, Alexander W. Hird , Qibin Su , Haixia Wang , Yan Yu, Dorin
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Toader , Paul D. Lyne , Jon A. Read , Jason Breed , Stephanos Ioannidis, Chun Deng , Michael
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1
¶

Grondine , Nancy DeGrace, David Whitston , Patrick Brassil and James W. Janetka
1Oncology Chemistry, IMED Biotech Unit, AstraZeneca, 35 Gatehouse Drive, Waltham MA
0
2451, USA
2Discovery Sciences, IMED Biotech Unit, AstraZeneca, Building 310, Cambridge Science
Park, Milton Road, Cambridge UK
ABSTRACT
Checkpoint kinase-1 (CHK1) inhibitors are potential cancer therapeutics which can be
utilized for enhancing the efficacy of DNA damaging agents. Multiple small molecule
CHK1 inhibitors from different chemical scaffolds have been developed and evaluated
in clinical trials in combination with chemotherapeutics and radiation treatment.
Scaffold morphing of thiophene carboxamide ureas (TCUs), such as AZD7762 (1) and
a related series of triazoloquinolines (TZQs), led to the identification of fused-ring
bicyclic CHK1 inhibitors, 7-carboxamide thienopyridines (7-CTPs) and 7-carboxamide
indoles. X-ray crystal structures reveal a key intramolecular non-covalent sulfur-
oxygen interaction in aligning the hinge-binding carboxamide group to the
thienopyridine core in a coplanar fashion. An intramolecular hydrogen bond to an
indole NH, was also effective in locking the carboxamide in the preferred bound
conformation to CHK1. Optimization on the 7-CTP series resulted in the identification
of lead compound 44, which displayed respectable drug-like properties and good in
vitro and in vivo potency.
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INTRODUCTION
Both normal and tumor cells employ strictly regulated DNA damage repair (DDR)
pathways to survive DNA damage induced by ultraviolet rays, chemical, or replication
errors, or chemotherapeutic and radiation treatments for cancer patients. It is well
established that disruption of DNA repair pathways is a promising therapeutic strategy
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that can be used to sensitize the effects of DNA-damaging treatments. An attractive
approach to modulating DNA repair activity and thus potentially improving the
therapeutic index of DNA-damaging therapy is to interfere with cell cycle checkpoint
signaling. Checkpoint kinase-1 (CHK1, CHEK1) and checkpoint kinase-2 (CHK2,
CHEK2) are Ser/Thr protein kinases that mediate the cellular response to DNA-
damage. In response to DNA-damage, CHK1 is activated by phosphorylation of
residues Ser-317 and/or Ser-345 by ATR kinase, and tumor cells subsequently
undergo S or G2/M phase cell-cycle arrest, primarily driven by CDK inhibition, allowing
cells to repair damaged DNA.2 CHK2 is activated by phosphorylation on Thr-68
mediated by ATM kinase in response to DNA damage and causes cell cycle arrest at
the G1 phase. A CHK1 inhibitor would allow a cell with damaged DNA to abrogate the
cell cycle arrest and progress through the cell-cycle, ultimately leading to mitotic
catastrophe and/or apoptosis.3,4 CHK1 inhibitors have attracted substantial interest
from both academia and industry as potential cancer therapeutics, and a number of
diverse CHK1 inhibitors have been disclosed and evaluated in human clinical trials.5-
1
2
We previously reported two series of structurally distinct CHK1 inhibitors, thiophene
carboxamide ureas (TCUs) and triazoloquinolones (TZQs) (Figure 1). Our past efforts
identified the clinical candidate 1 (AZD7762), a small molecule agent that could be
dosed intravenously in combination with standard chemo- or radiotherapies and
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selectively sensitize p53-negative tumors over normal cells.13-15 Herein, we report on
our expanded medicinal chemistry exploration of CHK1 inhibitors and discovery of two
new series of thienopyridine and indole carboxamide bicyclic CHK1 inhibitors, derived
from X-ray structure-based modification or ‘scaffold morphing’ of the TCU and TZQ
chemical series. Notably we first disclosed this work in an earlier patent publication16
which was subsequently followed by a report of similar compounds from others.18
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(AZD7762)
Ring fusion to
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Disconnect
Ring fusion to
mimic H-bond
mimic H-bond
triazolone ring
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Figure 1. TCU and triazoloquinolone CHK1 inhibitors and their fused ring analogs using a scaffold
morphing strategy.
RESULTS AND DISCUSSION
We previously reported X-ray crystallography data on our CHK1 inhibitors in which we
elucidated that the urea carbonyl group in the TCU and the triazolone carbonyl in the
TZQ compounds form the key hinge-binding interaction with the backbone NH of Cys-
14
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7 in the ATP-binding pocket of CHK1 (PDB code 2YDK and 2X8E ). Furthermore,
the urea NH forms an H-bond to the backbone carbonyl of Glu-85, in addition to three
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polar interactions between the protonated piperidine nitrogen and the CHK1 residues,
Asp-148, Glu-134, and Asn-135, at the edge of the ribose binding pocket (Figure 2).14
We made a key observation that compound 1 and its matched pair isomeric thiophene,
3
both adopt a bound conformation having an intramolecular H-bond between the
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internal urea NH to the C-2 (1) or C-3 carbonyl (3) of the amide group (blue dotted line
in Figure 1). Thus, we envisioned that a 5,6-bicyclic ring system, such as the
thienopyridine core in 4 and 5 (Figure 1), would mimic this intramolecular hydrogen
bond existing in the TCU inhibitors. Separately, disconnecting the hydrazide bond of
the 5-membered triazolone ring in the TZQ scaffold also generates a similar bicyclic
core that the thienopyridine group resembles. Guided by the above structural insight
from two distinct chemotypes, we undertook a ‘scaffold morphing’ strategy to explore
the potential for a variety of 5,6-bicyclic scaffolds as novel CHK1 inhibitors.
Scaffold morphing of TCU to 4-CTPs and 7-CTPs.
Due to its closest similarity to the clinical candidate TCU 1, we first pursued the
synthesis and evaluation of thienopyridine 4 and the des-fluoro analog 12. Shown in
Scheme 1, the synthesis of 4 starts with a Knoevenagel type condensation of glyoxylic
acid with 2-(3-thienyl)acetonitrile to afford the unsaturated carboxylate 7.
Subsequently the acid was transformed to the acyl chloride followed by reaction with
sodium azide to give acyl azide 8 in overall 82% yield. Curtius rearrangement of the
acyl azide followed by electrophilic cyclization onto the thiophene ring at high
temperatures yielded the fused ring thienopyridone 9. The 2-position of the thiophene
ring was selectively brominated with N-bromosuccinimide followed by treatment with
phosphorus oxychloride to give chloropyridine intermediate 10, which was further
displaced by 1-N-Boc-protected (S)-3-aminopiperidine in the presence of potassium
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carbonate in NMP. The resultant bromo-thienopyridine 11 was coupled to m-fluoro-
phenylboronic acid using a palladium-catalyzed Suzuki reaction. The desired
thienopyridine carboxamide 4 was generated by partial hydrolysis of the nitrile to the
carboxamide with simultaneous Boc deprotection using polyphosphoric acid. We
labeled this scaffold 4-CTP since the carboxamide group is substituted on C-4 of the
thienopyridine core. Compound 12 was generated in a similar fashion to 4 from 11
using phenylboronic acid.
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Scheme 1. Synthesis of 4-CTP 4.a
d
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4: R = F
12: R = H
aReagents and conditions: (a) glyoxylic acid, K
DCM, rt, 63% yield; (c) NaN , dioxane/water, rt, 1.5 h, 82% yield; (d) diphenyl ether, 230 C, 0.5 h, 44%
yield; (e) NBS, AcOH/DMF, 80C, 1 h, 96% yield; (f) POCl , 110 C, overnight, 82% yield; (g) (S)-3-
amino-Boc-piperidine, K CO , NMP, 80 C, quantitative yield; (h) Pd(Ph P) , m-F-PhB(OH) , Cs CO
dioxane/water, 80 C, 54% yield; (i) PPA, 110 C, 12 h.
CO , MeOH, reflux, 3h, 90% yield; (b) oxalyl chloride,
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Figure 2. X-ray crystal structure overlay of compounds 1 and 3 bound to human CHK1 kinase domain.
Carbon atoms and hydrogen bonds of 1 bound to CHK1 are colored green and carbon atoms of 3 are
colored yellow (PDB codes 2YDJ and 2YDK respectively. Selected residues and inhibitors are depicted
as sticks. Picture was generated in PYMOL).
Surprisingly, both compounds 4 and 12 showed very weak potency (500-1000 fold
drop vs. 1) in the CHK1 biochemical assay (Table 1). We suspected that the steric
repulsion between the carbonyl group of the C-4 carboxamide and C-3 hydrogen
forced the carboxamide out of the plane of the thienopyridine bicycle, rendering the
binding conformation of compounds 4 and 12 to CHK1 protein a high energy state. In
Figure 2, the urea groups in both compounds 1 and 3 share a coplanar conformation
with the thiophene ring, which likely contributes to their potent activity in CHK1
inhibition. A search of similar analogs in our internal crystal structure database
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revealed that in similar ligands with primary carboxamide groups substituted at C-4 of
the 5,6-bicyclic heteroaryl core, the dihedral angle was measured to be 25-30 degrees,
significantly larger than we measured in both TCUs (15.1 for 1 and 15 for 3). Despite
their weaker potency, we demonstrated that 4-CTPs 4 and 12 had improved properties
relative to TCU 1, including weaker hERG inhibition (Table 1).
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Table 1. CHK1 potency and hERG IC50s of 1 and its 4-CTP analogs.
CHK1
hERG
Compound
IC50 (µM)*a IC50 (µM)
1
4
0.005
6.8
15
>31.6
>31.6
12
2.6
*IC50 represents activity of the compound in a CHK1 biochemical assay.
aIC50 values are the geometric mean of at least two measurements.
We surmised that an alternative thienopyridine analog would be a direct mimic to
compound 3, essentially flipping the sulfur in 4-CTP to the carboxamide side of the
thienopyridine core to give compound 5. In this 7-carboxamide-thienopyridine (7-CTP)
scaffold, the close proximity of the carboxamide carbonyl group and the sulfur atom in
the thiophene ring would make the non-covalent oxygen-sulfur interaction possible,
and we hypothesized that it would enable the 7-CTP compounds to adopt a
conformation that better mimics the coplanar feature between the carboxamide group
and thiophene ring in 1 and 3. Therefore, we synthesized 7-CTP analogs using a
similar route to the syntheses of compounds 4 and 12, starting from commercially
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available 2-(2-thienyl)acetonitrile. CHK1 potency, aqueous solubility, and hERG
inhibition of these compounds are shown in Table 2. To our delight, the 7-CTP
compounds indeed showed excellent activity in CHK1 inhibition. Inhibitors 5 and 13
are matched pairs of 12 and 4, yet their IC50s are vastly improved and reach the same
levels of potency as the original TCU compounds 1 and 3. Based on TZQ analog 2,
two analogs containing thiophene substituents incorporated at the 2-position, were
also synthesized, also showing similar potency to 5. Encouraged by these promising
results, the 7-CTP inhibitors were tested in our previously described HT29 cell
abrogation assay.13 Compounds 5, 13, 14, and 15 indeed proved to be extremely
potent in abrogating the HT29 cell-cycle arrest caused by camptothecin-induced DNA
damage. Further profiling of these compounds revealed they have good aqueous
solubility and moderate to low activity in hERG inhibition (Table 2). However, when
tested for their in vivo pharmacokinetic properties, these compounds were found to
have very high systemic clearance in the rat (exceeding rat liver blood flow),
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warranting a further optimization effort to increase their half-life and improve their in
vivo exposure.
Table 2. Potency and properties of close analogs of 4 and 12 with 7-CTP core.
1
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CHK1
Abrogation
Solubility hERG IC50 Rat clearance
R
IC50 (µM) EC50 (µM)ab (µM)c
(µM)
CL (ml/min/kg)
3
5
1
1
1
0.007
0.007
0.019
0.026
0.024
0.012
0.010
>2000
>1000
334
>31.6
19.7
14.2
>30
28
49
Ph
>72
>72
>72
>72
3
4
5
m-F-Ph 0.012
2-thienyl 0.015
3-thienyl 0.006
>1000
980
aIC50 and EC50 values are the geometric mean of at least two measurements.
bEC50 represents cellular activity in cells pretreated with a DNA-damaging agent, camptothecin,
b
prior to compound treatment. Solubility is measured using solids from evaporated DMSO solution.
In our previous communications on TZQ CHK1 inhibitors, pyrazoles which occupy the
solvent channel of CHK1, in place of the thiophene group in compound 2 were
tolerated.15 Using this as precedent, we grafted those more hydrophilic heterocycles
to the new 7-CTP scaffold, installing them on C-2 of the 7-CTP core (Table 3), to
reduce the lipophilicity of 7-CTPs as a strategy to address metabolic stability issues
and to improve margin over hERG inhibition. Nevertheless, while we found CHK1
enzyme potency was retained with these heteroaryl substituents, pyrazoles, 16 and
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17, and imidazole, 19, caused a drop in cell potency, likely due to reduced cell
permeability, as lower cLogP (16 and 19 compared with 18) or an additional hydrogen
bond donor (16 compared with 17) is associated with larger biochemical to cellular
potency drop-off. Benzyl pyrazole 18 maintained cellular potency, but had high
lipophilicity (cLogP = 4.0) due to the hydrophobic benzyl substitution. We also
evaluated analogs with smaller C-2 substituents such as methyl group at C-2 (20) but
found significantly reduced CHK1 potency, suggesting an aromatic ring or larger
groups at C-2 is essential.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 3. Heteroaryl or alkyl substitutions at C-2 of 7-CTP reduced CHK1 cellular potency.
CHK1
Abrogation
R
CLogP
2.0
IC50 (µM)a EC50 (µM)a
1
6
0.020
0.006
0.014
2.3
1
1
1
7
8
9
0.15
0.06
2.0
4.0
0.024
0.15
10
1.7
2.3
20
Me
ND*
1
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aIC50 and EC50 values are the geometric mean of at least two measurements
*
Not determined.
Modification of thienopyridines to indoles
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Intramolecular sulfur-oxygen interactions are known to be isosteric to hydrogen-
bonding interactions.17 Recognizing the carbonyl oxygen and thiophene sulfur
interaction in 7-CTPs was critical for the potency of the already explored bicyclic
scaffolds, we were intrigued by the potential of an indole core that can offer an
intramolecular hydrogen bond between the carbonyl of the carboxamide group and
the indole NH (Figure 3). We surmised that a hydrogen bond can lock the 7-
carboxamide of the indole compound 21 in the desired conformation for its optimal
binding to CHK1.
S-O interaction
H-bond
5
21
Figure 3. Isosteric replacement of S-O interaction using the hydrogen bond of the indole NH.
Illustrated in Scheme 2 is the synthetic route to the initially designed indole analog 21,
based on 7-CTP 5. The nitro anthranilate derivative 22 was converted to methyl ester
23 using thionyl chloride in methanol under reflux conditions. Ester 23 then underwent
a novel one-pot multi-step transformation using potassium tert-butoxide in DMSO
which involved generation of an enamine by reaction with acetophenone and
nucleophilic aromatic substitution ortho to the activating nitro group, followed by air
oxidation to produce the indole carboxylic acid 24. The indole intermediate 24 was
1
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1
1
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
converted to amide 25 upon treatment of isobutyl chloroformate and ammonia.
Hydrogenation of the nitro group of 25 yielded the aniline compound 26, which was
then converted to the anticipated racemic version of the product 21 by reductive
amination with Boc protected 3-oxopiperidine in good yield. Chiral separation of the
racemic mixture of 21 by chrial preparative HPLC afforded the desired S-enantiomer
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
21 and its R-enantiomer (R)-21.
Scheme 2. Synthesis of indole analog 21.b
a
b
c
22
23
24
d
e, f, g
25
26
21
bReagents and conditions: (a) SOCl
DMSO, -15 C, 1 h, 90%; (c) isobutyl chloroformate, NH
Pd/C, rt, overnight, MeOH, 80% yield; (e) 3-oxo-Boc-piperidine, Na
, MeOH, reflux, 2 days, 63% yield; (b) acetophenone, KOt-Bu,
, DCM, -15 C to rt, 80% yield; (d) H , 10%
SO , NaBH(OAc) , rt, AcOH, 30%
2
3
2
2
4
3
yield; (f) 4N HCl in dioxane, MeOH, 2 h, 78% yield; (g) chiral preparative HPLC for enantiomeric
separation.
In addition to 21, three additional analogs bearing m-fluorophenyl, p-fluorophenyl, and
p-chlorophenyl groups at C-2 of the indole core were also synthesized (27, 28, and
29, Table 4). As we expected, these indole analogs proved to be equal or slightly less
1
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potent relative to their matched pairs of 5 and 13, both in the CHK1 inhibition assay
and in the cell-cycle abrogation assay. The aqueous solubility of compound 21 is still
respectable at 280 µM but lower than the matched 7-CTP analog 5. An unanticipated
concern for these indole compounds, however, is their hERG inhibition liability, as
shown in Table 4. All four analogs (21 and 27-29) are more potent hERG inhibitors
compared with their 7-CTP matched pairs. Compound 21, as a representative from
this sub-series of CHK1 inhibitors, was taken into rat in vivo PK studies, and similar to
its 7-CTP analog, (S)-1 showed high clearance (CL: 160 mL/min/mg), high volume of
distribution (Vss: 46 L/kg), and a short half-life (t1/2: 2.6 h).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
To further test the importance of the non-covalent intramolecular interaction perceived
to exist both in 7-CTPs and in the indole analogs (21 and 27-29), we synthesized a
furopyridine compound 30, aiming to deprive the hydrogen bond interaction between
the NH and the carboxamide carbonyl group of indole 21. As we expected, switching
indole NH in 21 to O in 30 caused a 10-fold drop in potency, confirming the critical role
of the intramolecular electrical interaction for these CHK1 inhibitors, existing in 7-CTPs
as an oxygen-sulfur interaction and in the indoles as a hydrogen bond. Consistent
with our observations first reported in 200616 and now a complete account herein, a
series of CHK1 inhibitors published by Zhao et. al. also contains additional 5,6-bicyclic
aromatic cores, such as thiazolopyridine and thiazolopyridazine, which embed the
sulfur atom next to the carboxamide group.18 Presumably, the oxygen-sulfur
interaction also strongly contributed to the potency in a similar fashion to that seen in
these 7-CTP examples.
Next, we obtained X-ray crystal structures for both compounds 13 and 21 bound to
1
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
CHK1. Undeniably, these two molecules superimposed on each other and TCUs 1
and 3 closely, both maintaining a near coplanar conformation between the
carboxamide group and the bicyclic heteroaryl core, and made analogous polar and
non-polar interactions with the CHK1 protein (Figure 4). Same as in both TCUs (1
and 3, Figure 1), the carboxamides of 13 and 21 make hydrogen-bonding interactions
with the hinge residues Glu-85 and Cys-87. Their protonated piperidine nitrogens
make three polar interactions in the ribose binding pocket, including to Asp-148, Glu-
134, and Asn-135. In both structures, the C-4 NH interacts with the terminal
carboxylate of Glu-91 through a water molecule. The indole or thienopyridine bicyclic
core is sandwiched between the hydrophobic side chains of Val-23 and Leu-15 of the
P-loop and Leu-137 of the catalytic loop (not shown in Figure 4). The indole NH of 21
makes no direct polar interactions with CHK1, unlikely to directly contribute to the
binding affinity of 21 to CHK1 other than the intramolecular hydrogen-bonding to the
carboxamide group. Here, we experimentally demonstrated that the indole NH was
an effective isosteric replacement to the sulfur atom in 7-CTPs and good potency was
achieved with the indole core. However, due to the concern on the hERG activity of
these indole compounds, we shifted our focus back to the thienopyridines.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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Figure 4. X-ray crystal structure overlay of compounds 13 and 21 bound to human CHK1 kinase
domain. Carbon atoms and hydrogen bonds of 13 bound to CHK1 are colored yellow and carbon atoms
of 21 are colored green (PDB codes are 6FC8 for 13 and 6FCK for 21). 13 is modelled as a dual
conformation. Selected residues and inhibitors are depicted as sticks. Picture was generated in
PYMOL).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 4. CHK1 potency, solubility, and hERG inhibition of indole and furopyridine analogs.
hERG
CHK1
Abrogation Solubility
Ar
X
Y
IC50
IC50 (µM)a EC50 (µM)a
(µM)
(
µM)
11.6
7.7
2
2
2
2
3
1
Ph
NH
NH
NH
NH
O
CH
CH
CH
CH
N
0.031
0.009
0.023
0.015
0.33
0.053
0.030
0.040
0.070
0.39
280
NDb
7
m-F-Ph
p-F-Ph
p-Cl-Ph
Ph
8
380
6.7
9
320
4.1
0c
>1000
NDb
aIC50 and EC50 values are the geometric mean of at least two measurements
bNot determined.
cFor the synthesis of the furopyridine analog 30, see Supporting Information.
Optimization of the amino-piperidine group
Our previous work on TCUs explored the SAR of the solvent channel aryl groups
(
equivalent to the C-2 aryl on 7-CTPs) extensively, and simple phenyl or halogen
1
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1
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
substituted phenyl groups were identified to offer a better overall profile.13 As
previously noted, we and others also published extensive C-2 aryl or heteroaryl SAR
exploration on the thienopyridine core as CHK1 inhibitors.17 As summarized in
Table 2 and Table 4, neither the variation on the C-2 aryl groups of the 7-CTP core
nor switching the core from CTP to indole effectively lowered the in vivo rat
clearance. Shown in Table 5 is the measured intrinsic clearance when compounds
(5, 14, 15, and 21) were incubated with human or rat liver microsomes. Consistent
with the observed high in vivo rat clearance, these compounds showed moderate to
high CLint in rat microsomal studies, although their human microsomal CLints were
more moderate. Our earlier SAR studies on the TCUs identified 3-(S)-
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0
1
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
aminopiperidine as a more potent group,13,14 and we suspected the high rat
clearance for both the thienopyridine and indole compounds at least was partially
due to the metabolic liability of the amino-piperidine group. Herein, we set out to
further study the SAR around the aminopiperidine group in 7-CTPs to address
metabolic stability and hERG issues associated with this series of compounds.
While it was not clear to us if the C-4 NH played a role in contributing to the metabolic
instability of the 7-CTPs, we were also intrigued to know how significant its water
molecule-bridged polar interaction with Glu-91 was to the potency. This interaction
can be disrupted by small alkyls substitutions on the C-4 nitrogen. A list of compounds
that were evaluated from this effort are shown in Table 6. The syntheses of these
analogs followed a route very similar to that of compound 5. Analogs 31 and 32 with
the C-4 oxygen and sulfur replacements for nitrogen, respectively, were synthesized
when 3-(S)-hydroxy or 3-sulfanyl Boc piperidine was used in step g, Scheme 1, but
using 7-CTP as the intermediate (Supporting Information). Analogs with alkyl
1
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substitutions on the C-4 nitrogen were obtained when N-substituted-Boc-piperidines
were used.
When tested for CHK1 activity, both 31 and 32 showed about a 10-fold drop in
potency compared with 5 in the CHK1 biochemical assay, and their cellular potency
decrease was similar or more significant. Small alkyl substituents at the C-4 nitrogen
of 7-CTP (compounds 33 and 34) were slightly better in maintaining biochemical
activity (3-5 fold drop), but cellular potency also dropped significantly. More polar
substitutions, such as compound 35, that contain a terminal hydroxy group on the alkyl
group, brought a similar drop in biochemical activity, but their toll on cellular activity
was more profound. Large hydrophobic substitution (36) were detrimental to potency.
Interestingly, the N-methyl compound (33) showed improved stability in both human
and rat microsomal assays, but C-4 O-linker analog 31 showed significantly reduced
microsomal stability (Table 5). Unfortunately, these compounds, except the
hydroxyethyl analog 35, also showed increased hERG liability, and their increased
lipophilicity (cLogP 3.87 for 5 and up to 4.60 for 34) likely contributed to their
promiscuous hERG inhibition.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 5. In vitro metabolic stability of 7-CTPs and indole 21
Hu mics CLint Rat mics CLint
Core
(
µL/min/mg)
(µL/min/mg)
5
1
1
2
3
7-CTP
7-CTP
7-CTP
Indole
7-CTP
9.2
9.1
29
32
36
4
5
1
1
72
21
120
>120
35
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1
1
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
32
7-CTP
7-CTP
<5
10
11
37
3
3
Table 6. CHK1 potency of analogs after replacement of the thienopyridine C-4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
nitrogen
hERG
CHK1
Abrogation
X
IC50
(µM)
3.8
IC50 (µM)a EC50 (µM)a
3
3
3
3
1
O
0.083
0.056
0.025
0.035
0.079
0.66
0.34
0.21
0.84
0.11
3.70
NDc
2b
3
S
4.9
NCH
NCH
3
2
2.7
4
CH
OH
CH Ph
3
2.4
35
36
N(CH
NCH
2
)
2
>31.6
2.1
2
2
aIC50 and EC50 values are the geometric mean of at least two measurements
bracemic version only
cNot determined
Scheme 3. Synthesis of 3-amino-2-methyl-piperidines.a
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a
b
37
38
39
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
c
racemic
racemic
3:2
40
41
42
aReagents and conditions: (a) MeLi, THF, 0 C to rt, 5 h, 14% yield; (b) 10% Pd/C, H
. MeOH, rt, 3 days,
2
100% yield; (c) CbzCl, DIPEA, DCM, rt, 1 h, 49% yield; (d) silica gel column separation, EtOH/hexane,
9% yield trans isomer and 35% yield for cis isomer; (e) 4N HCl in dioxane, MeOH, rt, 100% yield.
4
In order to address the metabolic stability concern of the 3-amino-piperidine group, we
next focused on the structural modifications close to the piperidine nitrogen, as it is
known that a basic nitrogen can be susceptible to oxidation by FMO (Flavin-containing
monooxygenase) enzymes in the hepatocytes.19 Reducing nitrogen pKa is a known
approach in mitigating FMO induced metabolic stability, and we were also aware that
the basic piperidine likely strongly contributed to the high volume of distribution of
these compounds in rat PK studies.20 However, as illustrated in the binding modes of
both TCUs (1 and 3) and compounds 13 and 21, the basic nitrogen was important for
potency in this scaffold, so we suspected significantly modulating the pKa of the
piperidine group would not be a viable strategy. We instead focused on introducing
different substitutions on the piperidine ring, anticipating that these substitutions could
afford more metabolically stable compounds while maintaining CHK1 potency. Shown
in Table 7 are a series of compounds that we constructed in this study.
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The key for the syntheses of the piperidine-modified compounds in Table 7 was to
obtain the piperidine intermediates with a variety of substitutions and stereoisomers.
Illustrated in Scheme 3 is the route to the Cbz protected 3-amino-2-methyl-piperidine
intermediate for compounds 44 and 45. Protected L-ornithine 37 reacted directly with
methyllithium to yield ketone 38 which underwent cyclization under hydrogenation
conditions to form a mixture of four piperidine diastereomers 39, due to racemization
at the alpha stereocenter. The Cbz group was reinstalled to the ring nitrogen to
facilitate the separation of trans and cis diastereomers, and Boc deprotection provided
the Cbz protected 3-amino-piperidine intermediates, setting the stage for synthesis of
the final inhibitors (c.f. steps g-I in Scheme 1). The trans racemic mixture 41 led to
the desired pure enantiomer 44 after chiral HPLC separation, while the cis racemic
mixture 42 led to 45.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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6
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Simple methyl substitution on the ring nitrogen in piperidine (43) caused a 20-fold
potency drop from 5, confirming the importance of the multi-point polar interaction
between the piperidine (Figure 4) and the CHK1 ribose binding pocket residues.
Interestingly, one of the two diastereomers synthesized from 3-amino-2-methyl-
piperidine intermediates, the (2R,3S)-diastereomer (44), showed superior potency
over the other diastereomer 45, offering similar potency to 5. We later obtained the
X-ray crystal structure for 44 (Figure 5). The binding mode of 44 is nearly identical to
3 and maintained all of the polar or hydrophobic interactions. As expected with 44,
the 2-methyl group trans to the 3-amino group on the piperidine ring adopted the
equatorial conformation, directing it toward Leu-137. The presumed axial 2-methyl in
45 is less preferred. A trans 4-hydroxyl substituted piperidine analog (46) retained
biochemical activity, but cellular potency dropped significantly. A more extended trans
2
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2-hydroxylethyl group on the piperidine ring was not well tolerated for potency (47).
The all cis tri-substituted piperidine analog 48 showed comparably good enzyme and
cellular potency. The cis-racemic mixture of 3-amino-6-methyl-piperidine analogs
were also synthesized (49) and it remained reasonably potent. Interestingly, the
diastereomeric pair 44 and 45 showed significantly different hERG activity, and to our
delight, the more potent 44 demonstrated improved hERG profile with IC50s greater
than 31.6 µM, the maximum tested concentration in the assay. In addition, when
tested in human and rat liver microsomal stability assays, compound 44 showed a
stability improvement over 5 (< 5 µL/min/mg in human microsomes and <10 µL/min/mg
in rat microsomes).
1
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2
3
4
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6
7
8
9
0
Figure 5. X-ray crystal structure overlay of compounds 44 and 3 bound to human CHK1 kinase domain.
Carbon atoms and hydrogen bonds of 44 bound to CHK1 are colored yellow and carbon atoms of 3 are
colored green (PDB codes are 6FCF for 44). Selected residues and inhibitors are depicted as sticks.
Pictures were generated in PYMOL.
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2
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3
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3
4
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4
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4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 7. CHK1 potency and hERG inhibition of substituted 3-aminopiperidine analogs.
Het =
0
1
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7
8
9
0
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43

46
48
49
CHK1
Abrogation
EC50 (µM)
hERG (µM)
IC50 (µM)a
a
4
4
4
4
4
4
4
3
0.14
0.006
0.88
0.008
0.050
0.41
NDb
>31.6
5.9
4
5
0.069
6*
7*
8*
9*
0.010
>31.6
>31.6
8.4
0.083
3.3
0.012
0.010
0.030
0.021
2.45
*racemic mixture
aIC50 and EC50 values are the geometric mean of at least two measurements
bND: not determined
For the detailed syntheses of 43, 46, 47, 48, and 49, see Supporting Information.
7-CTPs shows enhanced DNA-damaging effects in vitro and in vivo
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To evaluate the potentiating effect of our new lead 7-CTP CHK1 inhibitors to DNA-
damaging agents, such as gemcitabine, we employed a potentiation assay that was
described in detail previously.21 Briefly, the concentration-response of tumor cells to
proliferation inhibition by DNA-damaging agents was measured with or without the
presence of CHK1 inhibitors, which was dosed in a predefined constant concentration.
The ratio of the GI50 from the DNA-damaging agent alone over the GI50 from the
combination of DNA-damaging reagent and a CHK1 inhibitor was designated as the
potentiation factor (PF). Compounds 5, 13, and 44, along with previously reported 3,
were evaluated in this assay, each against three cell lines (SW620, MDA-MB-231, and
H460-DNp53). Their potentiating effects on gemcitabine-induced cell proliferation
inhibition are shown in Table 8. These four compounds all demonstrated synergistic
effects when dosed in combination with gemcitabine. The potentiation factor (PF) was
found to be more significant in the more gemcitabine-resistant cell line (H460-DNp53,
an engineered cell line that was developed by retroviral infection of a dominant-
negative p53 construct),21 and less so in the more gemcitabine-sensitive cell line
1
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5
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5
5
5
5
5
6
0
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8
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9
0
(
MDA-MB-231). Compound 3 exhibited higher PF among these four compounds when
tested in MDA-MB-231, but in the slightly less sensitive cell line SW620, all four
compounds tested showed a similar level of PF. However, compound 44 stood out
with a superior enhancing effect (PF = 124) when tested in H460-DNp53 cells,
lowering the gemcitabine GI50 from 2.1 nM to 0.017 nM. The enhancing effect
between 5 and 13 was marginal in both MDA-MB-231 and SW620 cells, but 13
exhibited a stronger effect in H460-DNp53 cells.
Table 8. Proliferation inhibition potentiation effect of CHK1 inhibitors when used in
combination with gemcitabine.a
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single agent single agent
combination
GI50 (nM)b
GI50 (nM)
GI25 (nM)
PFc
MDA-MB-231
0
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9
0
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0
3
596
536
641
306
300
270
320
150
0.027
0.052
0.068
0.052
9
5
4
5
5
13
44
gemcitabine 0.251
SW620
3
839
970
841
725
420
490
420
360
0.077
0.067
0.091
0.074
11
12
11
11
5
13
4
4
gemcitabine 0.84
H460-DNp53
3
5
1
4
1231
1798
2904
2453
620
0.031
0.075
0.055
0.017
67
900
28
3
4
1450
1230
38
124
gemcitabine 2.085
aGI50s and GI25 values are the mean value of two duplicated runs.
bCHK1 inhibitors concentration used in the combination with gemcitabine was its GI25 as a single agent.
cPotentiation factor
ꢀ
ꢁ50ꢂꢃꢄꢅꢆꢇꢈꢉꢊꢋꢈꢌꢅꢂꢊꢍꢎꢌꢅꢏ
ꢁ50ꢂꢃꢄꢅꢆꢇꢈꢉꢊꢋꢈꢌꢅꢂꢈꢌꢂꢇꢎꢆꢋꢈꢌꢊꢉꢈꢎꢌꢏ
ꢐ ꢑꢒ
ꢀ
2
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Compounds 5, 13, and 44 were dosed intravenously to mouse, rat, and dog to evaluate
their pharmacokinetic (PK) properties (Table 9). All three compounds were measured
to have high plasma clearance in rat, however, due to their high volume of distribution,
likely resulting from the presence of the basic piperidine group, these compounds still
showed reasonable half-lives. Their clearance was found to be more moderate in dog.
Both in terms of CL and t1/2, compound 5 was found to be slightly inferior in comparison
to 13 and 44. Compound 44 showed a more consistent profile across mouse and rat
with lower CL, longer t1/2, and lower Vss, and in dog its t1/2 is notably longer, likely
associated with its larger Vss in dog.
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We tested these three compounds for their in vivo effects in a mouse Hollow Fiber
(HF) model.14,21 In this model, HCT116 cells were treated with the DNA-damaging
agent topotecan to induce cell cycle arrest in the G2 phase. The hollow fibers were
filled with cell cycle arrest cells and subsequently were implanted subcutaneously in
mice before compounds were intravenously dosed. Implanted fibers were retrieved
after 30 h to determine the cell cycle profile of the enclosed cells. When dosed with
CHK1 compounds, the G2 arrest was overcome causing a reduction in the number of
cells in G2 and a concomitant increase in the number of cells re-entering the G1 phase.
The “odds value” was then calculated as the ratio of the G2/G1 populations for
topotecan alone and the CHK1 inhibitor combination. Based on the odds value, we
calculated an odds ratio (OR) that allowed quantitative assessment of the degree of
checkpoint abrogation. The greater the OR, the higher the level of checkpoint
abrogation. Shown in Figure 6 are the odds ratios for 5, 13, and 44. All three
compounds showed G2 arrest abrogation effect in the HCT116 HF assay, with 13
2
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6
showing the lowest OR (2.0) among these three compounds. Compounds 5 and 44
demonstrated similar PD effects in this model at the same dose (10 mg/kg), with OR
at 3.2 and 3.7, respectively.
0
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Table 9. In vivo PK of 5, 13, and 44
5
13
44
CL (mL/min/kg)
Mouse / rat / dog
45 / 295 / 11 28 / 196 / 10
1.5 / 3.1 / 5.4 8.5 / 4.4 / 5.1
29 / 105/ 10
t ½ (h)
mouse / rat / dog
3.4 / 5.1/ 9.4
4.6 / 24 / 7.1
Vss (L/kg)* mouse / 4.0 / 75 / 3.7
rat / dog
4.6 / 54.3 / 4.4
*Steady-state volume of distribution after intravenous dose.
5.0
4
.0
.0
3.7
3
.2
3
2.0
2.0
1
.0
1
.0
.0
0
Topotecan
5
13
44
The error bars represent the standard error of the mean odds ratio of multiple runs of the
study.
ꢀ2%
ꢓꢔꢔꢂꢃꢉꢎꢗꢎꢉꢅꢇꢊꢌꢂꢊꢍꢎꢌꢅꢏ
ꢓꢔꢔꢂꢃꢉꢖꢅꢊꢉꢆꢅꢌꢉꢏ
ꢓꢔꢔ ꢐꢂ
ꢂꢂꢂꢂꢂꢂꢂꢂꢓꢔꢔꢕꢂꢖꢊꢉꢈꢎ ꢐꢂ
ꢂꢂꢂꢂ
ꢀ1%
Figure 6. Odds ratio of 5, 13, and 44 in Hollow Fiber mouse PD assay (iv administration, using HCT116
cells)
CONCLUSION
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Using X-ray structure-based drug design, we employed a scaffold morphing strategy
to successfully design several new series of potent CHK1 inhibitors featuring 5,6-
bicyclic cores that mimic the productive conformation of TCU compounds bound to
CHK1. We discovered a key intramolecular oxygen-sulfur interaction was critical for
the potency of carboxamide thienopyridines (CTPs), as this interaction allows the
carboxamide group to orient in a coplanar conformation with the bicyclic aromatic core.
Replacement of the O-S interaction by an intramolecular hydrogen bond was proven
to be effective in maintaining the critical coplanar conformation and hence resulted in
similar CHK1 potency. Additional structural optimization of this class of thienopyridine
compounds has led to the identification of advanced lead compound 44, which
contains a trans 3-amino-2-methylpiperidine group to occupy the ribose pocket of
CHK1. Compound 44 encompasses a combination of excellent potency, reduced
hERG liability, and improved in vitro metabolic stability. When used in combination
with gemcitabine, it strongly enhanced the proliferation inhibition effect of gemcitabine
in multiple tumor cell lines. The in vivo plasma clearance of 44 in rodents was found
to be high, but in dog both the clearance and half-life were reasonable. When tested
for its in vivo PD activity in combination with topotecan, compound 44 demonstrated
the mode of action in abrogating HCT116 G2 cell cycle arrest induced by topotecan,
which presumably would lead to eventual apoptosis of HCT116 cells. In a subsequent
preclinical rat telemetry study, compound 44 was found to induce reversible
hypotension and reflex tachycardia when dosed to male Han Wistar rats at 12.5 mg/kg.
Given that cardiac dose-limiting toxicities occurred in patients of 1 and other CHK1
inhibitors in phase I clinical trials,22,23 the decision was not to progress compound 44
or other CHK1 inhibitors from this class into additional preclinical and clinical studies.
1
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Currently there are multiple CHK1 inhibitors (prexasertib, ARRY-575, and CCT-
245737) (Citeline/Trialtrove, as of October, 2016) in clinical trials and it remains a hope
that one or more of these inhibitors will eventually be approved, bringing important
new treatment options to cancer patients.
0
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0
EXPERIMENTAL SECTION
General information
All solvents used were commercially available in anhydrous grade. Reagents were
utilized without further purification unless otherwise stated. Evaporation of solvent
was carried out using a rotary evaporator under reduced pressure at a bath
temperature of up to 60 ºC. Temperatures are given in degrees Celsius (ºC), and
operations were carried out at room or ambient temperature, that is, at a temperature
in the range of 18-30 ºC. In general, the course of reactions was followed by thin
layer chromatography or mass spectroscopy and reaction times are given for
illustration only; where a synthesis is described as being analogous to that described
in a previous example, the amounts used are the millimolar ratio equivalents to those
used in the previous example. NMR data is in the form of delta values for major
diagnostic protons, given in parts per million (ppm) relative to tetramethylsilane
(TMS) as an internal standard, determined at 300 MHz or 400 MHz using deuterated
solvent. Analytical mass spectra were run with an electron energy of 70 eV in the
chemical ionization (CI) mode using a direct exposure probe; where indicated
ionization was effected by electron impact (EI), electrospray (ESP), or atmospheric
pressure chemical ionization (APCI); values for m/z are given; generally, only ions
which indicate the parent mass are reported. All final compounds were purified to
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≥95% purity, as assessed by analytical HPLC using an Agilent 1100 equipped with
Waters columns (Atlantis T3, 2.1 × 50 mm, 3 μm or Atlantis dC18, 2.1 × 50 mm, 5
μm) eluted for >10 min with a gradient mixture of water and acetonitrile with either
formic acid or ammonium acetate added as a modifier, monitored at wavelengths of
1
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0
220, 254, and 280 nm.
All experimental activities involving animals were carried out in accordance with
AstraZeneca animal welfare protocols, which are consistent with The American
Chemical Society Publications rules and ethical guidelines.
2
-(3-fluorophenyl)-7-[(3S)-piperidin-3-ylamino]thieno[2,3-c]pyridine-4-
carboxamide (4).
To a flask containing tert-butyl (3S)-3-{[4-cyano-2-(3-fluorophenyl)thieno[2,3-c]pyridin-
7-yl]amino}piperidine-1-carboxylate was added approximately 2.00 mL of PPA. The
o
reaction mixture was stirred at 110 C for 12 h. The crude reaction mixture was then
diluted with 10.0 mL of water and brought to a basic pH with 6N NaOH. The mixture
was then extracted with ethyl acetate (4 x 100 mL) followed by CH
2
Cl
2
/MeOH (1/1, 4
x 100 mL), dried over MgSO , and concentrated under reduced pressure to yield the
4
crude product which was purified by silica gel chromatography (100% CH
2
Cl to 20%
2
MeOH/CH
2
Cl
2
/3% NH
4
OH) to afford the title compound. 1H NMR (300 MHz; d
6
-
DMSO;  ppm) 8.01 (s, 1H), 7.91 (s, 1H), 7.51 (s, 1H), 7.38 (m, 1H), 7.36 (m, 2H),
7
4
.34 (br s, 1H), 6.80 (m, 1H), 4.21 (m, 1H), 3.15 (m, 2H), 2.87 (m, 2H), 1.92-1.46 (m,
H); LCMS (ES, M+H) = 371.
2
-phenyl-4-[(3S)-piperidin-3-ylamino]thieno[3,2-c]pyridine-7-carboxamide (5).
A solution of tert-butyl (3S)-3-{[7-cyano-2-(phenyl)thieno[3,2-c]pyridin-4-
2
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