INCB24360 (Epacadostat), a Highly Potent and Selective Indoleamine-2,3-dioxygenase 1 (IDO1) Inhibitor for Immuno-oncology

Article abstract of DOI:10.1021/acsmedchemlett.6b00391

A data-centric medicinal chemistry approach led to the invention of a potent and selective IDO1 inhibitor 4f, INCB24360 (epacadostat). The molecular structure of INCB24360 contains several previously unknown or underutilized functional groups in drug subs

Full text of DOI:10.1021/acsmedchemlett.6b00391

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Letter
pubs.acs.org/acsmedchemlett
INCB24360 (Epacadostat), a Highly Potent and Selective
Indoleamine-2,3-dioxygenase 1 (IDO1) Inhibitor for Immuno-
oncology
Eddy W. Yue, Richard Sparks, Padmaja Polam, Dilip Modi, Brent Douty, Brian Wayland, Brian Glass,
Amy Takvorian, Joseph Glenn, Wenyu Zhu, Michael Bower, Xiangdong Liu, Lynn Leffet, Qian Wang,
Kevin J. Bowman, Michael J. Hansbury, Min Wei, Yanlong Li, Richard Wynn, Timothy C. Burn,
Holly K. Koblish, Jordan S. Fridman, Tom Emm, Peggy A. Scherle, Brian Metcalf,
and Andrew P. Combs*
Incyte Corporation, 1801 Augustine Cut-Off, Wilmington, Delaware 19803, United States
S
* Supporting Information
ABSTRACT: A data-centric medicinal chemistry approach
led to the invention of a potent and selective IDO1 inhibitor
4f, INCB24360 (epacadostat). The molecular structure of
INCB24360 contains several previously unknown or underu-
tilized functional groups in drug substances, including a
hydroxyamidine, furazan, bromide, and sulfamide. These
moieties taken together in a single structure afford a
compound that falls outside of “drug-like” space. Nevertheless,
the in vitro ADME data is consistent with the good cell
permeability and oral bioavailability observed in all species
(rat, dog, monkey) tested. The extensive intramolecular hydrogen bonding observed in the small molecule crystal structure of 4f
is believed to significantly contribute to the observed permeability and PK. Epacadostat in combination with anti-PD1 mAb
pembrolizumab is currently being studied in a phase 3 clinical trial in patients with unresectable or metastatic melanoma.
KEYWORDS: Epacadostat, INCB24360, IDO1, data-centric medicinal chemistry, immuno-oncology
he central tenet of cancer immunotherapy is that the
immune system can be induced to recognize and eliminate
T
malignant cells within the human body. The harnessing of our
innate immune system to treat a wide variety of cancers has been
proposed for decades. The recent unprecedented durable
responses observed in melanoma patients when treated with
anti-CTLA4 mAb (ipilimumab),1 and more recently anti-PD1
mAb (pembrolizumab and nivolumab)2 therapies have provided
the first clinical validation for this novel immune mediated
mechanism of action for cancer therapy (immuno-oncology or I-
O). Cancer therapy is currently undergoing a paradigm shift
toward identifying agents (mAb and small molecules) that afford
restoration and/or activation of the immune system, often in
combination strategies.3−6
Figure 1. IDO1 inhibitors.
null mice16 or IDO1 inhibitors in combination with anti-PD1
mAbs have shown synergy in efficacy models.17 These studies
provide strong support for the clinical testing of IDO1 inhibitors
in combination with anti-PD1 mAbs and anti-PDL1 mAbs to
improve the observed low response rates while maintaining their
remarkable durability.
IDO1, IDO2, and tryptophan 2,3-dioxygenase (TDO) are
members of the heme containing myoglobin family of enzymes
and oxygen transporter proteins that catalyze the initial and rate-
limiting enzymatic step in the kynurenine biochemical pathway
for the metabolism of the essential amino acid tryptophan to N-
Indoleamine-2,3-dioxygenase-1 (IDO1) was first shown by
Munn and Mellor to play an immunomodulatory role in fetal
protection from the maternal immune system.7 Multiple tumor
types, includingmelanoma, ovarian, andcolon, overexpress IDO1
and are believed to subvert this immunomodulatory mechanism
and promote tolerance local to the cancer.8,9 Since Munn and
Mellor’s seminal research, numerous articles have appeared
further validating IDO1 as a therapeutic target for cancer
immunotherapy, including studies with si-RNA,10 IDO null
mice,11,12 andsmallmoleculeinhibitors1-methyltryptophan1(1-
MT)13 and hydroxyamidine 2 (5l)14 (Figure 1).15 Notably, IDO1
Received: October 6, 2016
Accepted: February 24, 2017
© XXXX American Chemical Society
A
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Letter
formylkynurenine.18 IDO1 is expressed throughout the body
where it can be locally up-regulated in response to inflammation
and infection by cytokines, such as interferon gamma (IFN-γ).
IDO1 overexpression is associated with several diseases, but most
convincingly implicated in cancer.19 IDO1 induction in dendritic
cells may suppress T-cell responses and promote tolerance
through either direct effects on T-cells mediated by tryptophan
depletion or cytotoxic effects on T-cells from tryptophan
metabolites, such as quinolinic acid and kynurenic acid. The
role of IDO2 in immunomodulation is less well understood, as
there are functionally inactive polymorphisms occurring in
approximately 50% of the examined population with only recent
correlations to cancer.20−23 Conversely, TDO is predominantly
expressedintheliverwhereitisresponsible forthehomeostasis of
tryptophan levels throughout the body in response to dietary
intake. Though there is recent evidence that TDO is upregulated
in some tumor microenvironments,24 the vast majority of
research supports the predominant role of IDO1 in regulating
the immune response. A selective IDO1 inhibitor was therefore
desired to avoid potential untoward toxicities associated with
altering global tryptophan levels through TDO inhibition.
Table 1. Selected SAR of Furazan (3)
IDO IC50
(nM)
HeLa IC50
(nM)
P2
P1 + P2
a
a
cmpd
X
R
Cl
Cl
2
Cl
Br
Br
Br
Br
Br
NH2
75
19
>2.2
>2.2
>2.2
>2.2
1.4
>2.2
1.4
3a
3b
3c
3d
3e
NH2
50
10
NHMe
NMe2
NHEt
NHBn
100
>5000
180
230
14
>2.2
>2.2
>2.2
>2.2
>5000
35
280
2.1
a
Rat IntCl (L/h/kg). Rat hepatic blood flow = 3.3 L/h/kg.
presumably due to the electron deficiency of the furazan. A
general and robust alternate route to secondary amino-furazan
derivatives was developed via a Boulton−Katritzky rearrange-
ment of the amidooxime furazan (Scheme 1).30 Syntheses and
analytical data for all compounds are provided in the Supporting
Information.
We previously reported the discovery of a hydroxyamidine hit
with micromolar potency from a high throughput screen of our
internal collection (>300,000 compounds) that was rapidly
improved to a proof-of-concept (PoC) lead (2 or previously
reported as 5l).14 Compound 2 demonstrated nanomolar
potency in our IDO1 biochemical and cellular assays and
excellent selectivity over TDO, but poor oral bioavailability in
rodent pharmacokinetics (PK). The PoC study with 2
demonstrated single agent efficacy in a B16-GMCSF mouse
melanoma model, although it required subcutaneous dosing due
tothe compounds short in vivohalf-life. Incomparison to1-MT 1,
the first and only other confirmed competitive IDO1 inhibitor
(IDO1 Ki ≈ 34 μM) reported at the time of this research,25
hydroxyamidinelead2providedanexcellentstartingpointforour
medicinal chemistry program. We report herein the further
optimization of the PoC lead 2 to afford our clinical candidate
INCB24360, epacadostat 4f, a highly potent and selective IDO1
inhibitor with good oral bioavailability across multiple species.
An in-depth analysis of the ADME factors that may be limiting
the oral bioavailability of this novel chemotype was undertaken.
The metabolism studies revealed that the oxygen of the
hydroxyamidine was subject to phase two glucuronidation in
vivoandwas themajormetabolic pathwayforthehydroxyamidine
class of compounds.26−28 Two in vitro clearance counter screens
using liver S9 fractions were established to test our compounds’
susceptibility to glucuronidation (P2) alone and to P450 mediated
metabolism plus glucuronidation (P1 + P2). Metabolic profiling
of our previously reported meta-substituted phenyl derivatives of
3 did not provide significant improvements in P2 stability.14 The
meta-Br-phenyl 3a was identified as a slightly more potent IDO1
inhibitor in a HeLa cellular assay compared to the meta-Cl-phenyl
2 and was therefore incorporated into most subsequent
compounds within our structure−activity relationship (SAR)
studies (Table 1). Replacement of the furazan by a variety of
heterocycles was also investigated and revealed that the furazan
was essential for potent IDO1 inhibition.29 It was hypothesized
that substitution at the C3 position of the furazan might disrupt
binding to the proximal glucuronidase active site through a steric
and/or electronic clash and thus diminish the propensity of the
hydroxyamidine to be glucuronidated. Synthesis of secondary 3-
amino-furazan derivatives by direct alkylation or reductive
amination of the primary 3-amino substituent were low yielding,
a
Scheme 1. Synthesis of Substituted 3-Amino Furazans
a
Reagents: (a) KOH, 100 °C, 15 h, 19−99%; (b) NaNO2, NaCl, HCl,
H2O, 0 °C, 1.5 h, 7−99%; (c) R2NH2, Et3N, EtOH, 0 → 20 °C, 62−
99%.
InitialSARatthe3-aminopositionofthefurazandemonstrated
that a variety of secondary amino substituents, such as 3b and 3d,
were tolerated in the biochemical and cellular assays, but did not
improvetheinvitromeasuredP2clearance(Table1). Thetertiary
amino derivatives, such as 3c, were inactive. Extension of the
secondary amino side-chain to a variety of larger, more
hydrophobic substituents, such as benzyl derivative 3e,
maintained similar biochemical potency, and provided the initial
evidence that C3 substituents on the furazan are projected into a
solvent exposed region (see Figures S8 and S9) when bound to
IDO1. Unfortunately, no improvements in in vitro clearance were
observed and cellular activity was severely diminished, presum-
ably due to the very high protein binding (3e: fu < 0.5%) of these
lipophilic compounds. In an attempt to reduce the protein
binding and restore the potent cellular activity, a series of polar
side-chains at C3 of the amino-furazan were designed and
synthesized. The addition of polar capping groups to an amino-
ethyl C3 substituent provided a highly potent series of IDO1
inhibitors 4a−g (Table 2).
Thesimilarbiochemicalpotenciesofthesepolarinhibitors4d−
g compared to nonpolar analog 3e suggest that the improvements
B
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Table 2. Selected SAR of Amino-Furazan (4)
b
in vitro ADME
calculated properties and ligand efficiency metrics
IDO IC50
(nM)
HeLa IC50
(nM)
P2
P1 + P2
Caco2
FF
(%)
a
a
c
d
e
f
g
g
Cmpd
X
R
Cl
Cl
Pm
PSA cLogP HBD HBA MW
LE
LLE
4a
4b
4c
4d
4e
4f
Br
Br
Br
Br
Br
Br
Cl
OMe
OH
200
170
290
210
100
73
100
22
>2.2
>2.2
1.1
>2.2
>2.2
0.9
44
31
0.9
2.3
6.1
4.4
2.4
3.1
3.8
100
111
117
120
137
163
163
2.7
1.8
2.0
1.9
2.0
1.5
1.4
3
4
5
4
4
6
6
8
374
360
359
401
437
438
394
0.45
0.51
0.47
0.45
0.44
0.46
0.45
4.3
5.8
5.0
5.9
5.8
6.6
6.6
8
NH2
98
1.8
19
8
NHAc
17
0.9
2.0
9
NHSO2Me
NHSO2NH2
NHSO2NH2
16
0.7
1.5
24
10
11
11
7.4
11
0.4
0.7
4.0
4g
120
0.5
0.8
4.3
a
b
c
d
Rat IntCl (L/h/kg). Rat hepatic blood flow = 3.3 L/h/kg. ChemDraw Ultra 10. Caco2 values Pm × (10)−6 cm/s. Protein binding free fraction
e
f
g
(FF). PSA in Å2. Total number of nitrogen (N) and oxygens (O). Ligand efficiency (LE) and lipophilic ligand efficiency (LLE) calculated using
HeLa pIC50.
in protein binding free fractions were responsible for the dramatic
improvements (up to 40-fold) observed in cellular potencies. In
addition, the in vitro metabolic clearances (P1 and P2) were
significantly reduced, which strongly correlated with the
increasing polarity (lower cLogP, higher PSA) of compounds
4c−g.
potencies and near identical mouse PK for the two closely related
analogs (Figure 2). An immune-mediated mechanism-of-action
The sulfonamide 4e and sulfamide 4f (INCB24360, epacado-
stat) were both highly potent IDO1 inhibitors and demonstrated
moderate to low glucuronidation in vitro (0.4 and 0.2 L/h/kg,
respectively). Pharmacokinetics in rats were evaluated for both
compounds and demonstrated that the sulfamide 4f achieved
significantly higher exposure and a longer half-life than
sulfonamide 4e, in good agreement with the in vitro (P1 + P2)
clearance data.
A final round of SAR at the meta-position of the phenyl ring
identifiedthebromo4fandchloro4gderivativesascomparablein
IDO1 enzyme and cell potency, as well as PK in both rodents and
cynomologous monkeys (Table 3). A head-to-head efficacy study
with oral dosing(30 mg/kg) of 4f and 4gin aCT26 tumor growth
model in immunocompetent mice was designed to differentiate
the two leads. The bromo 4f analog proved to be superior in
efficacy (tumor growth control (TGC) = 56%) compared to the
chloro 4g analog (TGC = 13%) despite the similar HeLa cellular
Figure 2. INCB24360 (4f) suppresses CT26 tumor growth in
immunocompetent mice (4f) vs chloro (4g).
for 4f was supported by a parallel study in immune-compromised
mice (nu/nu) in which no difference in tumor growth inhibition
was observed between 4f treated and control animals.
Based on these findings, the bromo analog 4f was fully profiled
in vitro and in vivo (Table 4). In vitro characterization
demonstrated 4f is a highly potent IDO1 inhibitor in cells
(HeLa IC50 = 7.4 nM) and in an IFN-γ induced whole blood
(WB)assay(IC50 =125nM).31 Absoluteselectivity(>1,000-fold)
was observed over the related dioxygenases, TDO and IDO2. In
addition, 4f was clean in in vitro toxicology studies, including the
hERG patch clamp, PXR, Cyp inhibition, and CEREP panel of
over 50 receptors and enzymes (see Supporting Information).
PK profiling of 4f in rats, dogs, and cynomologous monkeys
demonstrated good exposures and oral bioavailabilities (Po F =
11%, 59%, 33%, respectively) in all species (Table 4). The PK is
consistent with the moderate permeability and clearance data
measured in in vitro (P1 + P2) assays (Table 4).
Table 3. Comparisons of Bromo (4f) vs Chloro (4g)
in vitro Potency
IDO1 IC50 (nM)
HeLa IC50 (nM)
Pharmacokinetics
rat PK AUC (μM·h)
1/2 (h)
Cyno PK AUC (μM·h)
1/2 (h)
4f
73
4g
120
11
7.4
1.3
2.2
9.3
2.7
3.3
2.7
15
t
Pharmacokinetic/pharmacodynamic (PK/PD) studies in mice
established a strong correlation between the coverage of the WB
IC50 at trough and reduction in kynurenine concentrations in the
plasma (Figure 3).32 Once daily oral dosing of 4f at 50 mg/kg
reducedkynureninelevelsinwild-typemicetobasallevelspresent
t
4.0
Efficacy
TGC @ day 24 −CT26 model (%)
AUC (μM·h)
56
13
15.0
15.1
C
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Table 4. PK Profile of INCB24360 (4f) Across Species
Cl
Vss
iv
AUC
Po F
(%)
Po
species
(L/h/kg)
(L/kg)
t
1/2 (h)
(μM·h)
t1/2 (h)
rat
1.1
0.5
0.8
2.0
0.7
1.8
1.4
3.1
3.3
1.3
29
11
59
33
2.2
4.9
2.7
dog
cyno
9.3
Figure 5. Model of INCB24360 (4f) bound to IDO1. The hydroxyl of
the requisite hydroxyamidine forms a coordinate covalent bond with the
ferrous iron of the heme, while the m-Br-phenyl group binds deep into
the active site (pocket A) of IDO1. The aminoethyl-sulfamide
substituent projects out of the active site toward solvent (pocket B).
IDO1 crystal structure used in the modeling was PDB entry 4PK5.
Figure 3. INCB24360 (4f) PK/PD in mice.
versus the measured LogD (@ pH 7.4) values of 2.3 and 1.5,
respectively. We subscribe to the principle that ligand efficiency
(LE), a measure of the free energy of binding per atom, and
lipophilic ligand efficiency (LLE = pIC50 − cLogP) provide a
more meaningful characterization of the quality of a clinical
candidate.35 The calculated LE and LLE based on the HeLa cell
assay, 0.46 and 6.6, respectively, for 4f are nearly ideal.
in IDO null mice (∼400 nM kynurenine). TDO metabolism is
believed to be responsible for the observed baseline levels of
kynurenine in these studies. Similar PK/PD correlations were
observed in dogs and cynomologous monkeys. Allometric scaling
using in vivo and in vitro ADME data to cover the WB IC50 at
troughresultedinapredictedoraldosingof4finhumansof50mg
twice-a-day.
Preclinical 28-day IND toxicology studies in mice and dogs
demonstrated 4f is well-tolerated, in accord with the high
selectivity observed in the in vitro profiling data. No adverse
findings were identified in any parameter (clinical observations
and pathology, histopathology, body weight, food consumption),
and there were no signs of autoimmunity. A maximum tolerated
dose (MTD) was not established at doses up to 2000 mg/kg/d in
mice and 500 mg/kg/d in dogs. Plasma levels of 4f achieved
multiples of the WB IC90 at trough during these safety studies.
The FDA granted a clinical safe starting dose of 960 mg based on
these IND safety study results.
Evaluation of the physiochemical and calculated properties of
4f confirmed the molecular structure to be outside of classical
“drug-like” space, including Lipinski’s rule-of-five33 (>5 HBD; 4f
= 6 and >10 HBD + HBA; 4f = 11) and Veber’s permeability
rules34 (PSA < 140 Å2; 4f PSA = 163 Å2) (Table 2). Contrary to
thepredictedlowpermeabilityofthesecompoundsbasedontheir
highpolarsurface area(PSA)andlargenumberofhydrogenbond
donors (HBD), these molecules maintained moderate perme-
ability in the Caco-2 assay. The good permeability and oral
bioavailability of 4f are attributed to the two intramolecular
hydrogen bonds observed in the crystal structure of 4f (Figure 4).
In summary, a potent and selective IDO1 inhibitor 4f
(INCB24360, epacadostat) was invented through a series of
SAR studies primarily focused on improving the PK by reducing
therateofglucuronidationwhilemaintainingcellpotency. Adata-
centric medicinal chemistry approach was taken, and therefore
calculated properties and/or perceived “drug-likeness” did not
dictate the design of new molecules. Ultimately, INCB24360 4f
was identified as a novel chemotype containing several previously
unidentified and/or underutilized substituents (furazan, hydrox-
yamidine, sulfamide, bromide) in known drug substances.
INCB24360 4f proved to be efficacious in rodent models of
melanoma and well-tolerated in preclinical IND toxicology
studies. Epacadostat (INCB24360) 4f in combination with anti-
PD1 mAb, pembrolizumab, is currently being tested in a pivotal
trial for the treatment of patients with unresectable or metastatic
melanoma.36,37
Figure4. Crystalstructure ofINCB24360(4f). Intramolecular hydrogen
bonds stabilize the cis-conformation.
One hydrogen bond exists between the aniline NH and the
oxygen of the hydroxyamidine and the second between the imino
nitrogen of the hydroxyamidine and the furazan C3 NH. This
networkofhydrogen bondsnotonly reducestheeffective polarity
of the compound and thus increases its permeability but also
stabilizes the typically high energy cis-conformation of the
amidine. This is critical since our SAR and modeling is consistent
with 4f existing in the cis-conformation for binding to IDO1
(Figure5). Furthersupporting evidencethat 4fissignificantlyless
polar (∼8-fold) than predicted from its calculated properties is
provided by the comparison of the calculated LogP (cLogP)
ASSOCIATED CONTENT
■
S
* Supporting Information
TheSupportingInformationisavailablefreeofchargeontheACS
Publications website at DOI: 10.1021/acsmedchemlett.6b00391.
D
DOI: 10.1021/acsmedchemlett.6b00391
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Experimental protocols and characterization data(PDF)
AUTHOR INFORMATION
Corresponding Author
*Tel: 302-498-6832. E-mail: acombs@incyte.com.
ORCID
■
Andrew P. Combs: 0000-0003-1418-5168
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Laurine Galya, Mei Li, James Doughty, and Karl Blom
for their analytical assistance. We thank Mary Becker-Pasha and
MarkRuparforproductionoftheenzymeanddevelopmentofthe
IDO and TDO enzyme assays, respectively. We thank Ravi Jalluri
for creating the graphics of the epacadostat/IDO1 model.
ABBREVIATIONS
■
IDO1, indoleamine-2,3-dioxygenase-1; TDO, tryptophan 2,3-
dioxygenase; I-O, immuno-oncology; WB, whole blood; PK/PD,
pharmacokinetics/pharmacodynamics; TGC, tumor growth
control; LE, ligand efficiency; LLE, lipophilic ligand efficiency;
HBD, hydrogen bond donor; HBA, hydrogen bond acceptor;
PSA, polar surface area; MW, molecular weight; IND, investiga-
tional new drug
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