MK-8353: Discovery of an Orally Bioavailable Dual Mechanism ERK Inhibitor for Oncology

Article abstract of DOI:10.1021/acsmedchemlett.8b00220

The emergence and evolution of new immunological cancer therapies has sparked a rapidly growing interest in discovering novel pathways to treat cancer. Toward this aim, a novel series of pyrrolidine derivatives (compound 5) were identified as potent inhibitors of ERK1/2 with excellent kinase selectivity and dual mechanism of action but suffered from poor pharmacokinetics (PK). The challenge of PK was overcome by the discovery of a novel 3(S)-thiomethyl pyrrolidine analog 7. Lead optimization through focused structure-activity relationship led to the discovery of a clinical candidate MK-8353 suitable for twice daily oral dosing as a potential new cancer therapeutic.

Full text of DOI:10.1021/acsmedchemlett.8b00220

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Letter
MK-8353: Discovery of an Orally Bioavailable
Dual Mechanism ERK Inhibitor for Oncology
Sobhana Babu Boga, Yongqi Deng, Liang Zhu, Yang Nan, Alan Cooper, Gerald W Shipps, Ronald Doll,
Neng-Yang Shih, Hugh Zhu, Robert Sun, Tong Wang, Sunil Paliwal, Hon-Chung Tsui, Xiaolei Gao, Xin
Yao, Jagdish Desai, James Wang, Abdul Basit Alhassan, Joseph Kelly, Mehul Patel, Kiran K Muppalla,
Subramanyam Gudipati, Li-Kang Zhang, Alexei V. Buevich, David Hesk, Donna Carr, Priya Dayananth,
Stuart Black, Hong Mei, Kathleen Cox, Bradley Sherborne, Alan Hruza, Li Xiao, Weihong Jin, Brian Long,
Gongjie Liu, Stacey A Taylor, Paul Kirschmeier, William Windsor, Robert Bishop, and Ahmed Samatar
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00220 • Publication Date (Web): 14 Jun 2018
Downloaded from http://pubs.acs.org on June 14, 2018
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Windsor, William; Merck,
Bishop, Robert; Merck and Co Inc
Samatar, Ahmed; Merck and Co Inc
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MKꢀ8353: Discovery of an Orally Bioavailable Dual Mechanism ERK Inhibitor
for Oncology
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Sobhana Babu Boga,*‡ Yongqi Deng,† Liang Zhu,† Yang Nan,† Alan B. Cooper,‡ Gerald W. Shipps Jr.,† Ronald Doll,‡,
Neng-Yang Shih,‡, Hugh Zhu,‡ Robert Sun,‡ Tong Wang,† Sunil Paliwal,‡ Hon-Chung Tsui,‡ Xiaolei Gao,‡ Xin Yao,‡ Jag-
dish Desai,‡ James Wang,‡ Abdul Basit Alhassan,‡ Joseph Kelly,‡ Mehul Patel,† Kiran Muppalla,† Subrahmanyam Gudipati,‡
Li-Kang Zhang,‡ Alexei Buevich,‡ David Hesk,‡ Donna Carr,‡ Priya Dayananth,‡ Stuart Black,‡ Hong Mei,‡ Kathleen Cox,‡
‡
‡
‡
Bradley Sherborne,‡ Alan W. Hruza,‡ Li Xiao,‡ Weihong Jin,‡ Brian Long, Gongjie Liu, Stacey A. Taylor, Paul
Kirschmeier,‡ William T. Windsor,‡ Robert Bishop,‡ Ahmed A. Samatar‡
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†Merck & Co., Inc., 33 Avenue Louis Pasteur, Boston, MA 02115, USA
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‡Merck & Co., Inc., 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
KEYWORDS: MK-8353, MAPK pathway, ERK inhibitor, kinase selectivity, lead optimization, oncology
ABSTRACT: The emergence and evolution of new immunological cancer therapies has sparked a rapidly growing interest in
discovering novel pathways to treat cancer. Toward this aim, a novel series of pyrrolidine derivatives (compound 5) were idenꢀ
tified as potent inhibitors of ERK1/2 with excellent kinase selectivity and dual mechanism of action but suffered from poor
pharmacokinetics (PK). The challenge of PK was overcome by the discovery of a novel 3(S)ꢀthiomethyl pyrrolidine analog 7.
Lead optimization through focused SAR led to the discovery of a clinical candidate MKꢀ8353 suitable for twice daily oral dosꢀ
ing as a potential new cancer therapeutic.
The MAPK pathway plays a central role in regulating
mammalian cell growth, differentiation, survival and migration by
relaying extracellular signals. Activation of the MAPK pathway ocꢀ
curs via a cascade of protein phosphorylation events. One of the
critical components in this pathway is the small GTPase RAS which
recruits and activates the serine/threonine kinase RAF. Activated
RAF phosphorylates and activates MEK1/2 which in turn phosphorꢀ
ylates and activates ERK1/2. Once activated, ERK1/2 promotes the
transcription of genes that are involved in cell cycle regulation,
differentiation and survival1. Aberrant activation of the MAPK pathꢀ
way has been demonstrated to be an important feature common to
several human tumor types and several components of the pathꢀ
way have become attractive drug targets. Gain of function mutaꢀ
tions of the RAS oncogene are found in several human cancers
including pancreatic (90%), colorectal (50%) and lung (30%). Gain
of function BꢀRAF mutations, which lead to constitutive activation of
the MEKꢀERK pathway, are found in melanoma(~ 70%); colorecꢀ
tal(15%) and thyroid tumors(50%).2 In addition, nonꢀsmall cell lung
cancer and pancreatic tumors have been shown to have high inciꢀ
dence of RAS mutation. Tumors with mutations in BRAF or RAS
are usually nonꢀoverlapping in occurrence. The transforming activiꢀ
ty of the RAS and BRAF mutations suggests that inhibition of the
downstream targets such as ERK would be a good strategy for the
development of therapeutic agents. Inhibition of ERK should effecꢀ
tively inhibit all signal transduction downstream from MEK since
ERK is the only known substrate for MEK. To date, several signal
transduction inhibitors e.g., Vemurafenib(BRAF), Dabrafꢀ
enib(BRAF), and Trametinib(MEK)) targeting this pathway have
been approved and various RASꢀERK inhibitors are being studied
at various phases of development.3 Reactivation of the ERK1/2
phosphorylation and signaling has been frequently associated with
secondary resistance to these upstream inhibitors.4 Recently sevꢀ
eral highly optimized ERK inhibitors such as the pyrrole 1 by Verꢀ
tex/Biomed Valley Discoveries,5 the pyridone 2 by Genentech,6
fused pyrroloꢀdiazepanone 3 by Novartis7 and irreversible acrylate
inhibitor 4 by Astrazeneca8 have been reported (Figure 1) highꢀ
lighting the importance of exploring targeted inhibition of more
downstream components of MAPK signaling.
Figure 1. Representative structures of ERK inhibitors 1ꢀ5
Compound 5 suffered from poor pharmacokinetics (PK) which preꢀ
cluded further development due to high clearance and low permeaꢀ
bility leading to poor absorption and bioavailability in rat, as seen in
the plasma drug concentration measured by area under the conꢀ
centrationꢀtime curve (AUC) (rat AUC @ 10 mpk = 0 ꢁM.h; F % = 0;
Figure 2). The conventional approaches of lowering the clearance
by reducing hydrophobicity and lowering logD did not improve oral
exposure as lowering logD further deteriorated the permeability.
Although compound 5 provided an excellent starting point for our
medicinal chemistry program, our goal was to develop a potent,
selective and orally bioavailable ERK1/2 inhibitor for oncology. Cold
metabolite identification studies revealed pyrrolidine amide hydrolyꢀ
sis between centre core and Left Hand Side (LHS) due to proteolytꢀ
ic enzymes. We suspected that this hydrolysis was responsible for
poor PK. Systematic introduction of steric hindrance groups at the
3ꢀposition of the pyrrolidine led to the discovery of 3(S)ꢀthiomethyl
pyrrolidine analog 7(Figure 2). This group vastly improved the PK
(rat/dog AUC PK @10 mpk = 26/17 ꢁM.h; rat/dog F % = 70/75, see
reference12 for detailed SAR analysis) but with the loss of 2ꢀ3 fold
cell potency when compared to PoC compound 5. Herein we report
further optimization of the lead 7 to which led to the discovery of a
clinical candidate MKꢀ8353, a highly potent and selective ERK1/2
inhibitor with good oral bioavailability across multiple preꢀclinical
species. MKꢀ8353 displayed antiꢀtumor efficacy in several BRAFꢀ
mutant models.
We previously reported the discovery of a pyrrolidine
based ERK inhibitor 5 (SCHꢀ772984)9 which was derived from an
initial high throughput screening hit 6 utilizing an automated ligand
identification system (ALIS)10 and validation11 (Figure 2). Comꢀ
pound 5 showed excellent kinase selectivity against a panel of
kinases while displaying nanomolar ERK1/2 potency in both bioꢀ
chemical and cellular assays. Interestingly, compound 5 displayed
dual mechanism of action effecting both the inhibition of MEK
phosphorylation of ERK1/2 and inhibition of its intrinsic kinase funcꢀ
tion with nanomolar potency. This pyrrolidine compound 5 served
as a ProofꢀofꢀConcept (PoC) lead, that demonstrated single agent
efficacy in several xenograft mouse models with either KRAS or
BRAF mutant cell lines.
Although thiomethyl compound 7 improved PK properties from the
PoC lead compound 5, it required further optimization to improve
potency, reduce hERG and improve the overall profile in selecting a
single clinical candidate for progression. Crystal structure of comꢀ
pound 5 and other related molecules in this series revealed that the
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pyrimidine group in the RHS forms a key πꢀπ interaction with Tyrꢀ
Attempted replacement of the phenyl group between the piperidineꢀ
ene ring and the pyrimidine ring in 7 with a 5ꢀmembered thiazole
ring resulted in two regioꢀisomers 12 and 13 with much decreased
ERK1/2 potency. This limited SAR illustrated that the piperidineꢀene
phenyl pyrimidine construction of the RHS of the molecule 7 was
optimal in maintaining the ERK potency with improved PK.
62 of the ERK protein.12,13 The nature of the linker connecting the
pyrimidine to the centre core is critical for optimal binding as it oriꢀ
ents the pyrimidine to pick up the πꢀπ interaction with Tyrꢀ62. We
began SAR scrutinizing the piperidineꢀene (double bond oxidation–
a potential liability) on the RHS of the molecule 7 while holding the
pꢀfluorophenyl indazole on the LHS constant and the results are
summarized in Table 1. Replacing piperidineꢀene in 7 with piperaꢀ
zine to obtain compound 8 maintained ERK1/2 potency but showed
a decreased rat AUC by 7ꢀfold. Substitution of fluorine on the oꢀ
position of phenyl ring to obtain compound 9 resulted in 16 fold loss
in ERK2 potency, potentially changing the trajectory angle of pyrimꢀ
idine to a less optimal position. Reducing the double bond of piperiꢀ
dineꢀene 7 to a tetrahydropyridine group to obtain compound 10
and substitution of fluorine on the oꢀposition of phenyl ring to obtain
compound 11 both resulted in 2ꢀ3 fold loss in ERK2 potency.
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Concurrently, we rapidly investigated 3(S)ꢀthiomethoxy pyrrolidine
compound 7 that provided the balanced enzymatic ERK potency
and PK properties while we continued medicinal chemistry efforts to
further improve physicochemical properties such as cell potency
(HTꢀ29 and coloꢀ205) and other offꢀtargets viz., hERG by combinꢀ
ing various LHS and RHS modifications and the results are summaꢀ
rized in Table 2. Change of the pꢀflouro phenyl group attached to
the indazole on the LHS of compound 7 to a 4,5ꢀmethylenedioxy
phenyl compound 14 gave an excellent 4ꢀfold boost in rat AUC
(106 ꢁM at 10 mpk) and 1.5 fold improvement in cellular potency
but suffered from strong CYP 3A4 inhibition (preꢀincubation) with an
in vitro IC50 = 0.3 ꢁM. In general, this series of ERK compounds are
not potent inhibitors of human CYPs 1A2, 2C9, 2C19 or 2D6 but
are time dependent inhibitors of CYP3A4, and thus potentially raise
the concerns for drugꢀdrug interactions when coꢀadministered with
drugs that are primarily metabolized by CYP 3A4. The 5ꢀmethoxy
pyridyl, compound 15 showed a similar profile but with only marginꢀ
al improvement in hERG activity and rat AUC. Various heterocyclic
rings were explored on the RHS and LHS. For example, 2ꢀ
ethylaminoꢀ1,3,4ꢀoxadiazole compound 16 (with pꢀflouro phenyl
indazole in LHS) and 2ꢀethylaminoꢀ1,3ꢀthiazole compound 17 (with
2ꢀmethylpyridine indazole in LHS) showed only a modest rat AUC
4.9 ꢁM and 4.8 ꢁM at 10 mpk respectively. However, a more elecꢀ
tron deficient 1ꢀmethylꢀ1,2,4ꢀtriazole compound 18 improved both
ERK1/2 enzymatic and cellular (HTꢀ29) potency by 3 to 4ꢀfold and
at the same time restored the rat oral AUC exposure. To remove
the potential metabolic liability of the methoxy group in 5ꢀmethoxy
pyridyl compound 18, introduction of a 5ꢀisopropoxy pyridyl group
to obtain compound 19 led to a 10ꢀfold loss of ERK1/2 potency
potentially due to steric hindrance and nonꢀalignment of favorable
Lys112 interaction of ERK. Enabled by structure based drug design
(SBDD), an effort to reꢀorient 5ꢀisopropoxy O atom which forms a
key Hꢀbond with Lys112 of ERK when changed to 4ꢀisopropoxy
pyridyl indazole on the LHS (see Xꢀray, Figureꢀ4) and in combinaꢀ
tion with the potent, optimal 1ꢀmethylꢀ1,2,4ꢀtriazole RHS resulted in
the discovery of compound 20. Compound 20 displayed overall
improved ERK1/2 enzymatic, cell potency, hERG selectivity and
good rat oral AUC. Further replacement of 1ꢀmethylꢀ1,2,4ꢀtriazole
with pyrimidine, compound 21 led to a loss of ERK1/2 and cell
potency(2 fold) with no advantage in hERG selectivity. At this point,
compound 20 was selected as a preꢀclinical candidate (MKꢀ8353)
for further development and evaluated in vivo studies based on its
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Figure 2 Progression of discovery of ERK inhibitor MKꢀ8353
Compound
Enzymatic ERK1/2 IC50 (nM)
Cell Proliferation HTꢀ29/Coloꢀ205 IC50 (nM)
CYP 3A4 (co/pre) (mM)
hERG Rb (%I) @1.5/5 mg/ml
Hu & Dog hep. CL (mL/min/M)
106Caco II (Papp/cm.Sꢀ1)
Rat AUC @ 10 mpk (ꢁM.hr.)
Clp (mL/min/kg)
5
8/1
59/16
7.8/5.7
12/3
ND
4
7
41/7
118/45
>30/2
8/21
18/7
60
26
8.4
2.5
2.5
in vitro potency (ERK1/2 IC50
= 20/7 nM), cellular activity
(HT29/Coloꢀ205 IC50 = 51/23 nM) and in conjunction with its favorꢀ
able offꢀtarget hERG activity and pharmacokinetic profile. Interestꢀ
ingly, the sulfone methyl pyrrolidine analog of compound 20 gave
compound 22 and the opposite enantiomer 3(R)ꢀthiomethoxy of
compound 20 gave compound 23. Both compounds were shown to
be weaker in ERK1/2 potency, illustrating the importance of stereoꢀ
specific structural requirement for this class of inhibitors for optimal
potency.12
0
122
1.5
1.6
0
t½ (h)
Mean residence time (hr.)
Bioavailability F %
Table 1. Targeted modifications on the right hand side portion of
compound 7
70
Dog AUC @ 10 mpk (nM.hr.)
Clp (mL/min/kg)
ND
ND
ND
ND
ND
17
7.6
2.0
5.1
t½ (h)
Mean residence time (hr.)
F %
75
ND, Not Determined.
Compound
#
aERK1/2
IC50 (nM)
HT29
IC50 (nM)
Coloꢀ205
IC50 (nM)
hERG Rb %I @
1.5/5 ꢁg/ml
R
Rat AUC (ꢁM.hr)a
25.9
7
41/7
118
45
8/21
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8
9
25/5.6
663/114
43/23
87
47
ND
76
3/47
ND
3.6
ND
ND
ND
1
2
3
4
5
6
7
ND
245
209
10
11
22/12
11/32
135/25
83
8
9
12
207/36
543
ND
>111
ND
ꢀ5/10
6/19
ND
ND
10
11
12
13
14
15
16
17
18
19
20
21
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13
952/291
a Rat AUC (ꢁM.hr) @ 10 mpk
The excellent selectivity of MKꢀ8353 for ERK1/2 can be attributed
to a large compound induced conformational change in the polyꢀ
glycine loop of ERK (see crystal structure, Figureꢀ4). A2058 melaꢀ
noma cells were used to characterize the inꢀcell target engagement
profile of MKꢀ8353. A typical western analysis profile of MKꢀ8353
(see supporting info.) caused a dose proportional decrease in
pERK1/2 and pRSK levels with complete suppression of pERK1/2
at 30 nM in A2058 cells, illustrating the dual mechanism of action.
MKꢀ8353 was not a potent inhibitor of human CYPs 1A2, 2C9,
2C19 or 2D6 but inhibits CYP 3A4 (preꢀincubation) in vitro and
showed inhibition of CYP 3A4 and 2C8 (IC50 = 1.7 & 3.5 ꢁM), which
can cause drugꢀdrug interactions when co administered with drugs
that are primarily metabolized by CYP 2C8 or 3A4. MKꢀ8353 is a
weak inhibitor of hERG current, producing 16% inhibition at 0.6 ꢁM.
There were no test articleꢀrelated changes in PR, QRS, and
QT/QTc intervals in telemetered guineaꢀpigs exposed at 30 & 100
mg/kg with exposure multiples of 2ꢀ3 fold (based on total Cmax).
Based on the metabolite characterization in rat bile, urine and
plasma, the primary metabolic pathways appeared to be oxidative
dealkylation of the parent compound leading to a loss of the isoproꢀ
pyl group as well as the amide cleavage of the parent with subseꢀ
quent oxidative and conjugation reactions. No glutathione conjuꢀ
gate metabolites were found.18
The full profile of the clinical compound MKꢀ8353 is deꢀ
picted in Figure 3. MKꢀ8353 is a potent and selective inhibitor of
both active and inactive ERK1 and ERK2 kinases (IC50=20 and 7
nM, respectively). The overall kinase selectivity profile of MKꢀ8353
was characterized against
a panel of 233 mammalian serꢀ
ine/threonine and tyrosine kinases. Only three kinases showed
greater than 50% inhibition at the 1 ꢁM test concentration and none
of the 231 offꢀtarget kinases were inhibited at 100 nM.
Table 2. Combination of LHS and RHS modifications of the 3(S)ꢀ
thiomethoxy pyrrolidine core.
Compound
#
aERK1/2
IC50 (nM)
HT29
IC50 (nM)
Coloꢀ205
IC50 (nM)
hERG Rb %I @
1.5/5 ꢁg/ml
Rat AUC
(ꢁM.hr)a
R1
R2
7
41/7
118
77
45
8/21
25.9
14
45/13
29
19/16
106
2/15
15
16
17
19/5
17/5
34/6
60
49
34
22
30
30.8
4.9
7/19b
ꢀ3/6
131
ꢀ7/ꢀ3
4.8
18
19
7.9/1.7
74/27
20/7
80
99
51
10
41
23
0.8/7.2b
3/10a
16.9
ND
20
ꢀ3.5/6
34.5
(MKꢀ8353)
ꢀ1.7/ꢀ0.2b
21
93/13
228/29
ND/729
79
243
ND
57
86
ND
ND
ND
25/37
4/5b
2214
2315
>1000
6/10b
ND Not Determined; a Rat AUC (ꢁM.hr) @ 10 mpk; b hERG (IW) %I@ 1/10 ꢁM.
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Figure 4. Xꢀray crystal structure of MKꢀ8353 (PDB:6DCG) in the
active site of ERK2 with hydrogen bond interactions to the key
residues highlighted in dashed lines.
1
2
3
4
5
6
7
8
Figure 3. Detailed profile of MKꢀ8353
S
O
O
N
NH
N
HN
N
N
N
O
N
MKꢀ8353
(clinical candidate)
N
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Compound
Molecular weight
Melting point (°C)
MKꢀ8353
691.8
133
ERK 1 & 2 IC (nM)
20 & 7
50
Table 3. Mean Pharmacokinetic Parameters of MKꢀ8353 following
Cell proliferation IC (nM)
intravenous administrationb
50
371, 51, 23
185
A2058, HTꢀ29, Coloꢀ205
Caspase (nM)
CYP Co/Preꢀincubation IC (ꢁM)
AUC
(0ꢀ∞)
(µM.hr)
Dose
(mg/kg)
Halfꢀ
life
(hr)
MRT
(hr)
Clearance
(mL/min/k
g)
Vd,ss
(L/kg)
Species
n
50
Mouse
SD Rat
Dog
10
3
3
30
3
3
19.4
85
20
1.8
1.6
2.8
1.3
1.5
2.3
4.0
1.9
38
1.0
3.9
3.3
0.1
0.9
2.0
>5/1.7; >5/3.5; >5/>5
3A4; 2C8; 2C9
hERG voltage clamp
Caco II AP>BL/ BL>AP(nM/sec)
Plasma protein binding:
rat/dog/monkey/mice/human
16% I @ 600 nM
134 / 400
Monkey
1
3
1.3
17.9
b aqueous solution of 20% hydroxypropylꢀβꢀcyclodextrin (HPβCD)
>99% bound
Table 4. Mean Pharmacokinetic Parameters of MKꢀ8353 following
The in vivo pharmacokinetics and metabolism of MKꢀ
8353 were evaluated in male CD1 mice, Sprague Dawley (SD) rats,
guinea pigs, beagle dogs, and cynomologus monkeys. With the
exception of monkeys, MKꢀ8353 showed moderate clearance after
IV administration in all species, with a halfꢀlife range of 1.3ꢀ2.8 hr
and a mean residence time range of 1.5ꢀ4.0 hr (Table 3 and 4).
Acceptable oral bioavailability was seen in mice, rats and dogs (23ꢀ
80%) but low oral bioavailability in monkeys (2%). The permeability
observed in Cacoꢀ2 cells was high (135 nm/sec), suggesting that
intestinal absorption and permeability in humans should also be
high. The steadyꢀstate volume of distribution in mice, dogs and
monkeys was in the range of 0.9ꢀ3.3 L/kg, while in rats it was 0.1
L/kg.
oral administrationc
AUC
(0ꢀ∞)
(µM.hr)
Bioavailꢀ
ability
(%)
Cmax
(µM)
Dose
(mg/kg)
Tmax
(hr)
Species
n
Mouse
SD rat
Dog
30
10
30
10
27
3
3
4.6
24
6.5
1.0
1.7
4.0
1.4
15.3
143
45
80
50
23
2
Monkey
3
0.06
0.2
c 0.4% hydroxypropyl methylcellulose (0.4% MC)
In addition to inhibiting the kinase activity of ERK, MKꢀ
8353 prevents the phosphorylation of ERK by MEK.18 By contrast, a
published Vertex ERK inhibitor,4 which maintains the original apoꢀ
ERK conformation of Tyr34, inhibits the enzymatic activity of ERK,
but not its phosphorylation by MEK. Apparently, phosphorylation of
ERK requires not only the full length ERK protein, but also a preꢀ
cise conformation of ERK. Successful ERK phosphorylation by
MEK appears to critically depend on retaining the native conforꢀ
mation within Tyr34 and the Glyꢀrich loop. Efficacy of antiꢀtumor
activity of MKꢀ8353 was established in the Coloꢀ205 human colon
xenograft model. Female nude mice bearing Coloꢀ205 human colon
tumor xenografts were randomized for treatment with MKꢀ8353 at
the doses shown Figure 5. After 28 days of treatment, MKꢀ8353 at
all the doses tested significantly inhibited Coloꢀ205 tumor growth
compared to vehicle control (P < 0.05 or P < 0.0001). At the 20 and
40 mg/kg, p.o., bid dose MKꢀ8353 caused 37% and 88% TGI reꢀ
spectively. At the 60 mg/kg, p.o., bid doses MKꢀ8353 caused mean
tumor regressions of 40%. Projection of the human dose regimen
was carried out based on an effective average plasma exposure of
31 uM.hr, corresponding to a 72% tumor growth inhibition in the
coloꢀ205 mouse model. The projected human dose regimen is bid
with a dose range of 400 mg (based on dogs) to 1800 mg (based
on monkeys).18
The crystal structure of ERK with the ATP competitive inꢀ
hibitor MKꢀ8353 has been determined at 1.45 Å resolutions. An
overall view of this structure is shown below (Figure 4). The indaꢀ
zole ring of the inhibitor inserts into the ATP binding cavity and
mimics the hydrogen bonding of adenine to the hinge region of
ERK and Hꢀbonds to the backbone Asp104 CO and Met106 NH.
Another strong interaction is the Hꢀbond from the catalytic Lys52 to
the pyrrolidine nitrogen which requires the deprotonated form of the
amino nitrogen; a pKa near neutral pH is essential. Tyr34 folds
under the Glyꢀrich loop (conformational shift) and stacks with the
pyrrolidine ring. This rearrangement opens a cavity that is occupied
by the pendant methylꢀtriazole where it stacks with Tyr62. 3(S)ꢀ
thiomethoxy group that allows to retain strong binding to ERK point
towards the binding region defined by residues Asn152 and
Cys164. The numerous interactions which MKꢀ8353 makes with
ERK contribute to its potency and selectivity versus other kinases.
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Scheme 1 Synthesis of MKꢀ8353
Figure 5. Antiꢀtumor activity of MKꢀ8353 on the Coloꢀ205 human
colon tumor xenograft model
1
2
3
4
O
O
N
Br
B
Br
a
b
N
NH
O
O
O
O
I
O
O
B B
O
24
5
26
25
6
7
8
9
Ph3C
Ph3C
N
N
N
N
c
d
NH2
NO2
NO2
Br
10
11
12
13
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
58
59
60
N
N
O
O
29
Ph3C N
28
N
27
LHS
NH
NH
H
N
CN
f
g
e
O
N
H
HCl
Br
Br
Br
Commercial
31
32
The discovery synthesis of ERK inhibitor MKꢀ8353 relied on a conꢀ
vergent approach utilizing three fragments viz., LHS substituted isoꢀ
propoxy indazole (LHS 29), RHS piperidineꢀene phenyl triazole
(RHS 37), 3(S)ꢀthiomethyl pyrrolidine centre core 41 and final couꢀ
pling as depicted in Scheme 1. Chemoꢀselective alkylation of 5ꢀ
bromopyridinꢀ2(1H)ꢀone 24 with isopropyl iodide using K2CO3 folꢀ
lowed by borylation gave 2ꢀisopropoxyꢀ5ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀ
dioxaborolanꢀ2ꢀyl)pyridine 26. Suzuki reaction of the commercially
available 3ꢀbromoꢀ5ꢀnitroꢀ1ꢀtritylꢀ1Hꢀindazole 2717 with 26 gave the
nitro indazole 28 in good yield after chromatography. Reduction of
28 gave LHS 29 as an oil in good yield without chromatography,
with an overall yield 20%. RHS 37 was prepared by reaction of the
commercially available bromoꢀ4ꢀcyanobenzene 30 with ethanol
under acidic conditions to give 31 followed by methyl hydrazine to
form the hydrazinoimidate 32 in modest yield. After reaction with
formic acid the bromophenylꢀNꢀmethyl triazole intermediate 33 was
obtained. The tetrahydropyridine ring was introduced by a Suzuki
reaction of the commercially available Boc protected tetrahydroꢀ
pyridineꢀboronate 35 to obtain the tricyclic ring system 36. RHS
fragment, chloroacetamide 37, was obtained in excellent yield by
reaction of the deprotected 36 with chloroacetyl chloride. The cenꢀ
tre core, 3(S)ꢀthiomethyl pyrrolidine 41 was obtained in good yield
in 3 steps starting from commercially available pyrrolidine intermeꢀ
diate 38. Deprotonation with LDA at _78 oC and quenching with
dimethyl disulfide gave the centre core 3ꢀthiomethyl Pyrrolidine 39
in moderate yield.16 Subsequent Boc deprotection gave 40 and
chiral resolution by crystallization with Lꢀtartaric acid and filtration
from methanol gave the pure enantiomer 3(S)ꢀthiomethyl pyrroliꢀ
dine 41 in good yield with >99% purity. Boc protection followed by
hydrolysis of the methyl ester gave 43 in good yield. The final couꢀ
pling of the intermediates is proceeded by coupling LHS 29 with
centre core derivative 43 using HATU to give 44 in good yield. After
deprotection of both trityl and Boc groups with TFA gave 44 in 73%
after purification. Final coupling between 45 and RHS 37 using
Hünig's base at 50 °C resulted MKꢀ8353 in good yield after chroꢀ
matography. The convergent discovery synthesis of MKꢀ8353 is 19
steps with the longest linear synthesis of 9 steps proceeding in an
unoptimized 3 % overall yield (Scheme1). Optimization of this
synthesis to provide kg quantities will be discussed in future publiꢀ
cations.
30
N
h
N
N
Boc N
N
N
N
O
Boc N
B
O
Br
35
33
34
O
N
N
j
N
N
i
HN
N
N
N
Cl
HCl
37
RHS
36
k
l
MeO2C
MeS
MeS
N
N
NH
Boc
Boc
MeO2C
MeO2C
40
38
39
S
S
o
S
n
m
N
N
O
HO
Boc
Boc
NH
O
O
O
LꢀTartaric acid
O
43
42
41
Centre Core
Ph3C
O
O
S
S
N
HN
N
N
N
N
H
H
N
NH
Boc
q
p
r
MKꢀ8353
N
N
O
44
O
45
i
Reagents and conditions: (a) PrI, K2CO3 DMF, 59%; (b) B2Pin2,
Pd(dppf)2Cl2, KOAc, DMSO; (c) 27, Pd(Ph3P)4, Na2CO3 TolꢀEtOHꢀ
H2O; Pd/C, 100 °C, 80%; (d) Pd/C, H2 iPrOHꢀToluene, 100%; e)
HCl, EtOH, 56%; (f) MeNHNH2, pyridine, 85%; (g) Formic acid,
84%; (h) 34, Pd(dppf)2Cl2, K2CO3, DME/water, 73%; (i) HCl, Dioxꢀ
ane, 100%; (j) Chloro acetyl chloride, Et3N, CH2Cl2, 67%; (k) LDA,
(SMe)2, THF, ꢀ78°C, 67%; (l) TFA, CH2Cl2, 90%; (m) Lꢀtartaric acid,
MeOH, 78%; (n) (Boc)2O, Et3N, DMF, 100%; (o) aq. LiOH (2.0M),
THF/MeOH, 92%; (p) LHS 29, HATU, iPr2EtN, DMF, 60%; (q) TFA,
CH2Cl2, 73%; (r) RHS 37, iPr2EtN, DMF, 50 °C, 70%.
In conclusion, structureꢀbased drug design afforded a novel conꢀ
formationally restricted potent pyrrolidine series of ERK inhibitors.
3(S)ꢀthiomethyl substitution of the pyrrolidine core retarded amide
metabolism and ameliorated the challenge of PK in this chemotype.
A systematic SAR exploration of LHS and RHS led to the discovery
of orally bioavailable ERK inhibitor MKꢀ8353. MKꢀ8353 displayed
excellent potency, high kinase selectivity and dual mechanism of
action inhibition. MKꢀ8353 also demonstrated significant in vivo
efficacy, demonstrated by tumor growth inhibition & regressions in
BRAF/KRAS tumor models. The detailed pharmacological and
clinical evaluation of MKꢀ8353 as a potential treatment of cancer
showed that it was well tolerated up to 400 mg twice daily and exꢀ
hibited antitumor activity in patients with BRAFV600ꢀmutant melaꢀ
noma.19
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ACS Medicinal Chemistry Letters
ASSOCIATED CONTENT\
Jones, C. D.; Jones, C. R.; Renshaw, J.; Roberts, K.; Snow,
L.; Tonge, M.; Yeung, K. StructureꢀGuided Design of Highly
Selective and Potent Covalent Inhibitors of ERK1/2. J. Med.
Chem. 2015, 58, 4790..
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Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website.
Kinase Selectivity, PK Profiles, Xꢀray Dataꢀcollection, Experimental
procedures, NMR, MS data
9. Morris, E. J.; Jha, S.; Restaino, C. R.; Dayananth, P.; Zhu, H.;
Cooper, A.; Carr, D.; Deng, Y.; Jin, W.; Black, S.; Long, B.;
Liu, J.; Dinunzio, E.; Windsor, W.; Zhang, R.; Zhao, S.; Angaꢀ
gaw, M. H.; Pinheiro, E. M.; Desai, J.; Xiao, L.; Shipps, G.;
Hruza, A.; Wang, J.; Kelly, J.; Paliwal, S.; Gao, X.; Babu, B.
S.; Zhu, L.; Daublain, P.; Zhang, L.; Lutterbach, B. A.; Pelleꢀ
tier, M. R.; Philippar, U.; Siliphaivanh, P.; Witter, D.;
Kirschmeier, P.; Bishop, W. R.; Hicklin, D.; Gilliland, D. G.;
Jayaraman, L.; Zawel, L.; Fawell, S.; Samatar, A. A. Discovery
of a novel ERK inhibitor with activity in models of acquired reꢀ
sistance to BRAF and MEK inhibitors. Cancer Discovery,
2013, 3, 742ꢀ750.
10. Deng, Y.; Shipps Jr, G. W.; Cooper, A. B., English, J. M.; Anꢀ
nis, D. A.; Carr, D.; Nan, Y.; Wang, T.; Zhu, H.Y.; Chuang, Cꢀ
C.; Dayananth, P.; Hruza, A. W.; Xiao, Li.; Jin, W.; Kirschmeiꢀ
er, P.; Windsor, W. T.; Samatar, A.A. Discovery of Novel, Dual
Mechanism ERK Inhibitors by Affinity Selection Screening of
an Inactive Kinase. J. Med. Chem. 2014, 57, 8817ꢀ8826
11. Zhu, H. Y.; Desai, J.; Deng, Y.; Cooper, A. B.; Wang, J.;
Shipps, J.; Samatar, A.; Carr, D.; Windsor, W. Discovery of
hydroxyaniline amides as selective Extracellular Regulated
Kinase (Erk) inhibitors. Bioorg. Med. Chem. Lett. 2015, 25,
1627ꢀ1629
12. Boga, S. B.; Alhassan, AꢀB.; Cooper, A. B.; Doll, R.; Shih, Nꢀ
Y.; Shipps, G. Jr.; Deng, Y.; Zhu, H.; Nan, Y.; Sun, R.; Zhu, L.;
Desai, J.; Patel, M.; Muppalla, K.; Gao, X.; Wang, J.; Yao, X.;
Kelly, J.; Gudipati, S.; Paliwal, S.; Tsui, HꢀC.; Wang, T.; Sherꢀ
borne, B.; Xiao, L.; Hruza, A.; Buevich, A.; Zhang, LꢀK.; Hesk,
D.; Samatar, A. A.; Carr, D.; Long, B.; Black, S.; Dayananth,
P.; Windsor, W.; Kirschmeier, P.; Bishop, R. Discovery of
3(S)ꢀthiomethyl pyrrolidine ERK inhibitors for oncology.
Bioorg. Med. Chem. Lett. 2018, 28, 2029ꢀ2034
13. Chaikuad, A.; Tacconi, E. M.; Zimmer, J.; Liang, Y.; Gray, N.
S.; Tarsounas, M.; Knapp, S. A unique inhibitor binding site in
ERK1/2 is associated with slow binding kinetics. Nat Chem
Biol. 2014, 10, 853ꢀ860
AUTHOR INFORMATION
Corresponding Author
* Tel.: +1 9087405351
Email address: sobhana.babu.boga@merck.com (S. B. Boga)
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Author Contributions
The manuscript was written through contributions of all authors. All
authors have given approval to the final version of the manuscript.
ACKNOWLEDGMENT
We thank Drs. John Piwinski, Cathy Strader, Cecil Pickett, Malcolm
MacCoss at Merck & Co., Inc., Kenilworth, NJ USA for their support
and encouragement. Drs. William Greenlee and Robert Aslanian for
support of the program.
ABBREVIATIONS
ERK, extracellular signalꢀregulated kinases, MAPK, mitogenꢀ
activated protein kinases; SBDD, structure based drug design;
SAR, structureꢀactivity relationship; PK, pharmacokinetics; AUC,
area under curve; TGI, tumor growth inhibiition; PoC, Proof of Conꢀ
cept
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(methylthio)pyrrolidineꢀ1,3ꢀdicarboxylate 42 with mCPBA afꢀ
forded the sulfone 1ꢀ(tertꢀbutyl) 3ꢀmethyl (S)ꢀ3ꢀ
of
1ꢀ(tertꢀbutyl)
3ꢀmethyl
(S)ꢀ3ꢀ
(methylsulfonyl)pyrrolidineꢀ1,3ꢀdicarboxylate centre core for
the synthesis of compound 22
15. Crystallization
of
methyl
3ꢀ(methylthio)pyrrolidineꢀ3ꢀ
carboxylate 40 with Dꢀtartaric acid gave the 3(R)ꢀthiomethyl
pyrrolidine centre core for the synthesis of compound 23
16. Boga, S. B.; Alhassan, A. B.; Cooper, A. B.; Shih, NꢀY.; Doll,
R. J. [3+2] Cycloadditionꢀmediated synthesis of 3ꢀ
methylsulfanylꢀpyrrolidineꢀ3ꢀcarboxylic acid methyl ester Tet-
rahedron Lett., 2009, 505315ꢀ5316
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no, B. H.; Robert, L.; Ribas, A. Development of MKꢀ8353, an
orally administered ERK1/2 inhibitor, in patients with advanced
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(S)ꢀ1ꢀ(1ꢀ(4ꢀChloroꢀ3ꢀfluorophenyl)ꢀ2ꢀ
hydroxyethyl)ꢀ4ꢀ(2ꢀ((1ꢀmethylꢀ1Hꢀpyrazolꢀ5ꢀ
yl)amino)pyrimidinꢀ4ꢀyl)pyridinꢀ2(1H)ꢀone (GDCꢀ0994), an Exꢀ
tracellular SignalꢀRegulated Kinase 1/2 (ERK1/2) Inhibitor in
Early Clinical Development. J. Med. Chem., 2016, 59, 5650ꢀ
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Eckersley, K.; Fairley, G.; Feron, L.; Flemington, V.; Graham,
C. R.; Greenwood, R.; Hopcroft, P.; Howard, T. D.; James, M.;
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MK-8353: Discovery of an Orally Bioavailable Dual Mechanism ERK Inhibitor
for Oncology
Sobhana Babu Boga,*‡ Yongqi Deng,† Liang Zhu,† Yang Nan,† Alan B. Cooper,‡ Gerald W. Shipps Jr.,† Ronald
Doll,‡, Neng-Yang Shih,‡, Hugh Zhu,‡ Robert Sun,‡ Tong Wang,† Sunil Paliwal,‡ Hon-Chung Tsui,‡ Xiaolei Gao,‡
Xin Yao,‡ Jagdish Desai,‡ James Wang,‡ Abdul Basit Alhassan,‡ Joseph Kelly,‡ Mehul Patel,† Kiran Muppalla,†
Subrahmanyam Gudipati,‡ Li-Kang Zhang,‡ Alexei Buevich,‡ David Hesk,‡ Donna Carr,‡ Priya Dayananth,‡ Stuart
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Black,‡ Hong Mei,‡ Kathleen Cox,‡ Bradley Sherborne,‡ Alan W. Hruza,‡ Li Xiao,‡ Weihong Jin,‡ Brian Long,
‡
Gongjie Liu, ‡ Stacey A. Taylor, ‡ Paul Kirschmeier,‡ William T. Windsor,‡ Robert Bishop,‡ Ahmed A. Samatar‡
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