Tricyclic Indazoles - A Novel Class of Selective Estrogen Receptor Degrader Antagonists

Article abstract of DOI:10.1021/acs.jmedchem.8b01837

Herein, we report the identification and synthesis of a series of tricyclic indazoles as a novel class of selective estrogen receptor degrader antagonists. Replacement of a phenol, present in our previously reported tetrahydroisoquinoline scaffold, with an indazole group led to the removal of a reactive metabolite signal in an in vitro glutathione trapping assay. Further optimization, guided by X-ray crystal structures and NMR conformational work, varied the alkyl side chain and pendant aryl group and resulted in compounds with low turnover in human hepatocytes and enhanced chemical stability. Compound 9 was profiled as a representative of the series in terms of pharmacology and demonstrated the desired estrogen receptor α degrader-antagonist profile and demonstrated activity in a xenograft model of breast cancer.

Full text of DOI:10.1021/acs.jmedchem.8b01837

Subscriber access provided by United Arab Emirates University | Libraries Deanship
Article
Tricyclic indazoles – a novel class of selective
estrogen receptor degrader antagonists.
James S. Scott, Andrew Bailey, David Buttar, Rodrigo J. Carbajo, Jon Curwen, Paul Davey,
Robert D. M. Davies, Sebastien L. Degorce, Craig Donald, Eric Gangl, Ryan David Robert
Greenwood, Sam Groombridge, Tony Johnson, Scott Lamont, Mandy Lawson, Andrew
Lister, Christopher Morrow, Thomas A. Moss, Jennifer H Pink, and Radoslaw Polanski
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01837 • Publication Date (Web): 14 Jan 2019
Downloaded from http://pubs.acs.org on January 14, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the
course of their duties.

Page 1 of 53
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
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
Tricyclic indazoles – a novel class of selective estrogen
receptor degrader antagonists.
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
.*
.
.
.
.
.
James S. Scott, Andrew Bailey, David Buttar, Rodrigo J. Carbajo, Jon Curwen, Paul R. J. Davey,
.
.
.

.
Robert D. M. Davies, Sébastien L. Degorce, Craig Donald, Eric Gangl, Ryan Greenwood, Sam D.
.
.
.
.
.
Groombridge, Tony Johnson, Scott Lamont, Mandy Lawson, Andrew Lister, Christopher J.
.
.
.

Morrow, Thomas A. Moss, Jennifer H. Pink, Radoslaw Polanski.
.
Oncology, IMED Biotech Unit, AstraZeneca, Cambridge CB4 0WG, United Kingdom.

Oncology IMED Biotech Unit, AstraZeneca, R&D Boston, 35 Gatehouse Drive, Waltham,
Massachusetts 02451, United States.

Discovery Sciences, IMED Biotech Unit, AstraZeneca, Cambridge CB4 0WG, United Kingdom.
ABSTRACT
Herein we report the identification and synthesis of a series of tricyclic indazoles as a novel
class of selective estrogen receptor degrader antagonists. Replacement of a phenol, present in
our previously reported tetrahydroisoquinoline scaffold, with an indazole group led to removal of
a reactive metabolite signal in a in vitro glutathione trapping assay. Further optimization, guided
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 53
1
2
3
4
5
6
7
8
9
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
by X-ray crystal structures and NMR conformational work, varied the alkyl sidechain and
pendant aryl group and resulted in compounds with low turnover in human hepatocytes and
enhanced chemical stability. Compound 9 was profiled as a representative of the series in terms
of pharmacology and demonstrated the desired ER degrader-antagonist profile and
demonstrated activity in a xenograft model of breast cancer.
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
INTRODUCTION
Breast cancer is the second most common type of cancer and the most frequently occurring cancer
amongst women, with an estimated 1.67 million new cancer cases diagnosed in 2012, constituting 25% of
1
all cancers. In 2012 in the US it is estimated that 233,000 women were diagnosed with breast cancer and
4
4,000 women died from the disease; in the same year in the European Union an estimated 367,000
1
women were diagnosed with breast cancer and 91,000 died from the disease. Approximately 80% of
post-menopausal women with breast cancer have estrogen receptor  (ER)-positive disease.2
In patients with ER-positive breast cancer, endocrine therapies such as tamoxifen and aromatase
inhibitors (AI) have been the mainstay treatment. However, advanced breast cancer remains incurable,
with patients ultimately relapsing due to acquired or de novo resistance. Numerous resistance
mechanisms to anti-estrogens have been suggested. These include mutations in ERα that lead to ligand-
3
independent activation, activation of signaling pathways that can cause ER activation in the absence of
4
estradiol and changes in expression of ER-cofactors which result in ER modulators, such as
tamoxifen, becoming agonists.4 In all these cases, ER activity is still required. This continual
2
ACS Paragon Plus Environment

Page 3 of 53
1
Journal of Medicinal Chemistry
2
3
4
5
6
7
8
9
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
dependence of tumours on ERα highlights a currently unmet medical need to achieve a more complete
inhibition of the pathway.
Fulvestrant is a selective estrogen receptor degrader (SERD) which has demonstrated superiority over the
5
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
aromatase inhibitor, anastrozole. Introduced into clinical practice in 2002, fulvestrant is an ERα
antagonist with no known confounding agonist properties and has the unique ability to increase the rate of
turnover of the receptor which limits the amount available for potential further activation. This has led to
a focus on SERDs as a strategy for inhibition and removal of ERα. However, the use of fulvestrant is
limited in the clinic by a lack of oral bioavailability and is administered intramuscularly in doses of 500
5
mg once a month which could limit clinical efficacy. Oral SERDs could potentially achieve higher free
drug levels than has been possible with fulvestrant and this, in turn, might lead to a more complete
removal of ERα and a more durable response in patients.
Given the importance of ER as an oncology target, considerable effort has been devoted to identifying
an orally bioavailable SERD. A number of promising SERD compounds featuring a phenyl substituted
acrylic acid motif have been described recently. Initially reported on a triarylethylene scaffold GW76046
7
8
this group also appears in the development candidates GDC-810 from Seragon/Genentech, AZD9496
9
1
b from AstraZeneca and LSZ102 from Novartis. Other scaffolds containing the phenyl substituted
acrylic acid have been reported by AstraZeneca (coumarin10 and tetrahydroisoquinoline ), GSK
11
1
2
13
(
quinoline ) and Novartis (tetrahydroisoquinoline ) as well as the Trinity Biomedical Sciences Institute
1
4
15
16
(
benzoxepin ), the State University of New York at Stony Brook, (coumarin and triarylethylene ), the
1
7
University of Illinois at Chicago (benzothiophene ) and the University of Illinois at Urbana−Champaign
1
8
19
(
bicyclononane and adamantyl ). With the notable exceptions of 1b and GDC-810, the key compounds
described in each case relied on a phenol for potency. Recent work from the RCMI Cancer Research
Center has shown that a boronic acid can act as a surrogate for the phenol on a triarylethylene scaffold,
leading to GLL398.20
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 53
1
2
3
4
5
6
7
8
9
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
2
1
22
Other SERDs have entered clinical development such as RAD1901 from Radius and GDC-927 from
Seragon/Genentech which have basic groups showing that groups other than the acyclic acid are capable
of giving a SERD phenotype.
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
RESULTS AND DISCUSSION
We previously reported a series of tetrahydroisoquinoline (THIQ) phenols that showed an ER degrader-
1
1
antagonist profile. These compounds were observed to trap glutathione (GSH) in an in vitro microsome
23
based trapping assay, potentially indicative of a reactive metabolite risk. The GSH trapping was shown
to be attributable to the phenol functionality but the phenol was also demonstrated to be critical for
potency. We were thus drawn to investigate potential phenol replacements and targeted tricyclic indole
24
and indazole cores that had not been previously described in the chemical literature. We were intrigued
to explore whether these cores might be able to mimic the interaction with the salt bridge exploited by the
phenol by providing a suitably oriented hydrogen bond donor. In viewing the indole as a structural isomer
of the core present in 1b, we hypothesized that the alteration in connectivity of the ring system from a
[
6.5.6] to a [5.6.6] system (Figure 1.) might switch the interaction made by the respective indole NH’s
from a backbone carbonyl to the salt bridge.
Figure 1. Core designs based on hybridization of THIQ and tricyclic indole equity
O
OH
O
OH
Novel tricyclic cores
Ar
F
H
N
F
cyclise
R
N
[
6.5.6]
N
N
F
[5.6.6]
HO
HN
A
A=CH,N
THIQ 1a
AZD9496 1b
Synthesis of compounds
4
ACS Paragon Plus Environment

Page 5 of 53
1
Journal of Medicinal Chemistry
2
3
4
5
6
7
8
9
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
We therefore developed chemical routes towards these motifs as shown in Schemes (1-7). Our initial
attempts to synthesize the tricyclic indole core involved a Pictet-Spengler cyclization between indole
amine 2a and (E)-methyl 3-(4-formylphenyl)acrylate. Unfortunately, the major product from this reaction
was the unusual [5.6.7]-fused tricyclic amine 2b where the cyclization had occurred via the C3 position of
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
25
the indole ring (mode A) in preference to the desired reaction through the phenyl ring (mode B). This
was subsequently alkylated and deprotected to give 2 (Scheme 1). In order to access the targeted tricyclic
core, we resorted to a Bischler-Napieralski approach with the cyclization of amide 3a followed by
reduction of the imine intermediate to give a 33% yield of the desired cyclization product 3b (via mode
B). Subsequent alkylation to give 3c was followed by a Heck reaction to introduce the acrylate group 3d
and ester hydrolysis to give 3.
Scheme 1. Pictet-Spengler and Bischler-Napieralski approaches to tricyclic indolea
mode B
H
N
O
O
N
NH2
(a)
(b)
O
OR
HN
2a
R=Me, 2c
R=H, 2
N
H
N
H
(c)
mode A
2b
Br
O
OR
Br
mode B
O
(d)
(e)
(f)
HN
NH
N
N
Br
HN
HN
HN
HN
3a
3b
3c
mode A
R=Me, 3d
R=H, 3
(g)
a
o
Reagents and Conditions: (a) (E)-methyl 3-(4-formylphenyl)acrylate, AcOH, toluene, 80 C, 2 h, 31%;
(b) isobutyl trifluoromethanesulfonate, DIPEA, 1,4-dioxane, RT, 4 d, 72%; (c) NaOH, THF/MeOH, RT,
o
o
2
4 h, 86%; (d) POCl , 110 C, 3 h, then NaBH , MeOH, 0 C, 16 h 33%; (e) isobutyl
3 4
trifluoromethanesulfonate, DIPEA, 1,4-dioxane, RT, 24 h, 67%; (f) methyl acrylate, NMeCy , Pd-118,
2
o
TBAC, MeCN, 150 C, 1 h, 63%; (g) NaOH, THF/MeOH, RT, 24 h, 61%.
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 53
1
2
3
4
5
6
7
8
9
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
Indazole 4 was synthesized racemically from commercially available 4-bromo-1H-indazole. Heck
reaction with acrylamide gave the alkene 4a, which was hydrogenated with Pd/C to give
saturated amide 4b. Curtius rearrangement followed by Boc deprotection gave the amine 4d,
which was reacted with 4-bromobenzoic acid and HATU to afford the amide 4e. Bischler-
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
Napieralski cyclization, followed by reduction of the imine with NaBH gave the tricyclic indazole
4
4f, which was alkylated via reductive amination to give 4g. A Heck reaction on the bromide with
tert-butylacrylate gave ester 4h, and subsequent hydrolysis of the tert-butyl ester afforded the
racemic acid which was separated by chiral chromatography into enantiomerically pure 4
(Scheme 2).
Scheme 2. Bischler-Napieralski route to tricyclic indazolea
H N
2
O
H N
2
O
O
Br
H N
2
N
H
(a)
(b)
(c,d)
(e)
Br
N
N
N
N
N
N
H
N
H
N
H
N
H
N
H
4a
4b
4d
(f)
N
4e
R
N
N
(i,j)
(h)
N
N
N
O
HO
N
H
N
H
N
Br
H
O
O
4
4h
R=H, 4f
(g)
i
R= Bu, 4g
6
ACS Paragon Plus Environment

Page 7 of 53
Journal of Medicinal Chemistry
1
a
o
2
3
4
5
6
7
8
9
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
Reagents and Conditions: (a) acrylamide, NMeCy , Pd-118, TBAC, MeCN, 130 C, 2 h, 41%; (b) Pd/C,
2
t
o
H , EtOH, RT, 23 h, 94%; (c) Pb(OAc) , BuOH, 80 C, 45 min, 67%; (d) TFA, DCM, RT, 1 h, 93%; (e)
2
4
4
-bromobenzoic acid, HATU, DIPEA, DMF, RT, 1 h, 73%; (f) POCl , P O , reflux, 9 h, then NaBH ,
3 2 5 4
MeOH, THF, RT, 1 h, 77%; (g) isobutyrylaldehyde, Na(OAc) BH, AcOH, DCE, NMP, RT, 1 h, 93%; (h)
3
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
o
tert-butyl acrylate, NMeCy , Pd-118, TBAC, MeCN, 150 C, 45 min, 77%; (i) DCM, TFA, RT, 45 min;
2
(j) chiral separation, 38% over 2 steps.
Introduction of geminal-dimethyl substitution into the saturated ring could be achieved from the
THP-protected 4-bromo-indazole. Lithium-halogen exchange with n-BuLi followed by quenching
with dimethyloxirane and BF .Et O afford the tertiary alcohol 5a. Ritter reaction with
3
2
chloroacetonitrile, followed by cleavage of the amide of 5b with thiourea furnished the gem-
dimethyl amine 5c. The saturated ring of 5d was then constructed with a Pictet-Spengler
cyclization using 4-bromobenzaldehyde. In this case, the imine was pre-formed using pH 6
phosphate buffer, before heating in TFA to effect cyclization. THP protection of the indazole
gave 5e then alkylation with isobutyl triflate and DIPEA afforded 5f. Subsequent Heck reaction
installed the acrylic ester to give 5g, which was hydrolyzed under acidic conditions with
concomitant deprotection of the THP group. A final chiral separation of the enantiomers
afforded enantiopure 5 (Scheme 3).
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 53
1
2
3
4
5
6
7
8
9
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
Scheme 3. Pictet-Spengler route to gem-dimethyl substituted tricyclic indazolea
O
Cl
Br
H N
HO
N
H
2
HN
(e)
(a)
(b)
(c)
(d)
N
N
N
N
N
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
N
N
H
N
H
N
N
H
Br
THP
THP
5a
5b
5c
R=H, 5d
R=THP, 5e
(f)
N
N
N
(i,j)
(h)
N
N
N
N
THP
O
HO
N
N
H
Br
THP
O
O
5
5g
5f
a
o
Reagents and Conditions: (a) n-BuLi, 2,2-dimethyloxirane, BF .Et O, THF, −78 C, 1 h, 62%; (b) 2-
3
2
chloroacetonitrile, AcOH, H SO , RT, 48 h, 59%; (c) thiourea, EtOH, AcOH,  16 h, 83%; (d) potassium
2
4
o
o
phosphate buffer (0.1M, pH 6), MeCN, 50 C, 16 h, then TFA, 130 C, 5 h, 51%; (e) 3,4-dihydro-2H-
o
pyran, PTSA hydrate, DCM, 40 C, 16 h; (f) isobutyl trifluoromethanesulfonate, DIPEA, dioxane, RT, 24
o
h, 22% over 2 steps; (g) tert-butyl acrylate, NMeCy , Pd-118, TBAC, MeCN, 150 C, 1 h, 89%; (h) TFA,
2
DCM, RT, 2 h; (i) chiral chromatography, 35% over 2 steps.
Incorporation of a single methyl substituent on the saturated ring provided a significant synthetic
challenge. Traditional Bischler-Napieralski or Pictet-Spengler cyclizations where the amine was
2
6
unalkylated afforded the undesired cis-isomer as the major product. A trans relationship between methyl
and aryl substituents could be achieved in low yield via Pictet-Spengler cyclization of alkylated
tryptamine 6b which was synthesized by ring opening of a chiral cyclic sulfamidate to give 6a, followed
2
7
by reductive amination with isobutrylaldehyde. This established a chiral relay where the chirality of the
methyl group established the trans-relationship and controlled the absolute stereochemistry of the newly
formed chiral centre. However, the harsh conditions required meant that only robust alkyl sidechains such
as iBu were tolerated. Heck reaction on the Pictet-Spengler product 6c, followed by hydrolysis of the tert-
butyl ester of 6d afforded the desired acrylic acid 6 (Scheme 4).
8
ACS Paragon Plus Environment

Page 9 of 53
Journal of Medicinal Chemistry
1
2
3
4
Scheme 4. Pictet-Spengler route to 1,3-trans methyl substituted tricyclic indazolea
5
6
7
8
9
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
1
R
N
H
Br
N
N
O
O
O
(d)
S
(a)
(c)
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
NBoc
N
N
N
+
N
2 O
N
N
H
Br
N
H
R
N
H
H
1
O
R =H, 6a
R = Bu, 6b
2 t
6c
R = Bu, 6d
R =H, 6
(b)
1
i
(e)
2
a
o
o
Reagents and Conditions: (a) n-BuLi, THF, −78 C to 0 C, 1 h, 78% then 4N HCl/dioxane, RT, 1 h,
o
8
7%; (b) isobutyrylaldehyde, Na(OAc) BH, THF, 5 C, 30 min, 80%; (c) 4-bromobenzaldehyde, TFA,
3
o
o
toluene, 150 C, 40 h, 14%; (d) tert-butyl acrylate, NMeCy , Pd-118, TBAC, MeCN, 150 C, 45 min,
2
80%; (e) TFA, DCM, RT, 45 min, 19%.
To achieve variation of the N-alkyl sidechain and aryl ring, a more flexible approach was
developed that was tolerant to a range of functionality. This involved performing the Pictet-
Spengler cyclization on an aniline substrate and subsequently constructing the indazole ring.
o
Treatment of 2,6-dibromotoluene with n-BuLi in THF at −78 C, followed by addition of cyclic
sulfamidate gave the aliphatic amine 19 after Boc deprotection. N-Alkylation could be achieved
by heating this amine with the requisite alkyl triflate and DIPEA in 1,4-dioxane to give 20a-c.
Buchwald amination with benzophenone imine, followed by hydrolysis with aqueous acid
afforded the anilines 21a-c. The anilines were then subjected to Pictet-Spengler cyclization with
aldehydes already bearing the acrylic ester to furnish the cyclized products 22a-g as the desired
trans isomers. Interestingly, this cyclization proceeded more smoothly with a small amount of
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 53
1
2
3
4
5
6
7
8
9
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
water present in the AcOH; it was hypothesized that the water helps to hydrolyse the imine
formed from condensation of the aniline with the aldehyde, allowing cyclization to occur.
Indazole formation could then be achieved by either heating the aniline with isopentyl nitrite and
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
acetic anhydride under basic conditions (‘Method A’), or with NaNO in a mixture of propionic
2
acid and water under cooling (‘Method B’). Hydrolysis of the acrylic esters (and N-acetyl where
Method A was used) afforded the acrylic acids 7-12 and 15 (Scheme 5).
nd
Scheme 5. 2 generation route with late stage indazole formation.
1
1
2
R
2
R
R
R
Br
H N
2
X
N
Y
N
H
O
O
O
S
(a)
(b)
(d)
NBoc
+
O
Br
Br
NH2
X
O
19
X=Br, 20a-c
R3=Ac (Method A)
R =H (Method B)
22a-g
(c)
(e)
X=NH , 21a-c
3
2
1
1
2 R
2 R
1
2
7
8
9
R =F; R =Me; X,Y=H
R
R
1
2
R =H; R =Me; X,Y=F
1
2
R =F; R =Me; X,Y=F
X
N
X
N
(f)
1
2
10 R ,R =F; X,Y=F
11 R =F; R =Me; X=H; Y=OMe
2 R ,R =F; X=H; Y=OMe
5 R ,R =F; X=F; Y=OMe
1
2
1
2
N
N
1
1
HO
O
1
2
N
H
N
Y
Y
3
O
O
23a-g
R
a
o
o
Reagents and Conditions: (a) n-BuLi, THF, −78 C to 0 C, 1 h, then 4N HCl/dioxane, RT, 1 h, 61%; (b)
o
o
alkyl triflate, DIPEA, 1,4-dioxane, 90 C, 66-74% or isobutyrylaldehyde, Na(OAc) BH, THF, 0 C, 56%;
3
t
o
(
c) benzophenone imine, Pd dba , Rac-BINAP, NaO Bu, toluene, 90 C, then 1N aq. HCl, 71-91%; (d)
2 3
o
aldehyde, AcOH (1% H O), 60-100 C, 30-57%; (e) Method A: isopentyl nitrite, Ac O, KOAc, 18-crown-
2
2
o
o
6
, chloroform, 70 C, 48 h, or Method B: NaNO , propionic acid, water, −10 C, 1 h; (f) Method A and
2
o
Method B: aq. NaOH, THF, MeOH, RT-60 C, 1-16 h, 65-96%.
1
0
ACS Paragon Plus Environment

Page 11 of 53
Journal of Medicinal Chemistry
1
2
3
4
Further variation in the pendant phenyl ring was achieved via a closely related route. In this case, the
5
6
7
8
9
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
Pictet-Spengler cyclization was carried out on aldehydes containing a 4-bromo substituent.
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
Following construction of the indazole ring, this was used to introduce the acrylic acid using a
Heck reaction on methyl acrylate followed by hydrolysis of the ester (Scheme 6).
Scheme 6. Variation of the pendant phenyl ringa
F
F
F
F
F
Y
F
Y
F
F
N
O
N
O
N
O
N
H
X
Z
X
X
(e)
(a)
(b,c)
N
N
HO
N
N
H
Br
NH2
NH2
THP
O
Y
13a
4a
13 X=H; Y=F
14 X=F; Y=H
20c
Z=Br, 13c, 14c
(d)
1
Z=HC=CCO Me, 13d, 14d
2
a
o
Reagents and Conditions: (a) aldehyde, AcOH (1% H O), 90 C, 16 h, 50% for 13a, 26% for 14a; (b)
2
o
NaNO , propionic acid, H O, −20 C, 45-60 min, 70% for 13b, 92% for 14b; (c) 3,4-dihydro-2H-pyran,
2
2
PTSA hydrate, DCM, reflux, 3 h, 95% for 13c, 93% for 14c; (d) methyl acrylate, Pd-118, DIPEA, 1,4-
o
dioxane, 125 C, 2 h, 63% for 13d, 53% for 14d; (e) 4N HCl dioxane, RT, 15-30 min, then LiOH.H O,
2
THF/MeOH/ H O, RT, 16 h, 4% for 13, 22% for 14.
2
In order to access the quaternary-methyl analogues, the indazole was protected as the N-Boc derivative. In
the cases where the parent acrylic acid was used as starting material, this also formed the tert-butyl ester.
Treatment of the protected indazole with cerium(IV) ammonium nitrate in acetonitrile/water formed the
iminium ion, which could be reacted with methylmagnesium bromide to form the quaternary centre as a
single diastereoisomer, albeit in low yield. Hydrolysis of the N-Boc and ester groups afforded the acrylic
acids 16-18 (Scheme 7).
1
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 53
1
2
3
4
5
6
7
8
9
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
Scheme 7. Oxidation / Grignard approach to the quaternary substituted corea
1
1
1
2 R
2
R
2
R
R
R
R
X
N
Y
X
N
Y
X
N
Y
(a)
(b,c)
N
N
N
3
O
HO
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
3 O
N
H
N
N
H
R
R
Boc
O
O
O
C
1
2
3
16 R =F; R =Me; X,Y=F
17 R ,R =F; X,Y=F
R =Boc for 16a and 17a
1
2
3
R =Me for 18a
1
2
18 R =F; R =Me; X=H; Y=OMe
a
t
o
Reagents and Conditions: (a) R =H: Boc O, DMAP, BuOH, 80 C, 4 h, 97% for 16a, 36% for 17a;
3
2
R =Me: Boc O, DMAP, DCM, RT, 1 h, 100% for 18b; (b) cerium(IV) ammonium nitrate, MeCN, H O,
3
2
2
o
o
t
RT, 2 h, then MeMgBr, THF, −78 C to 0 C, 1 h, 12-41%; (c) R = Bu: 4N HCl in dioxane, RT, 2-3 h,
3
8
8% for 16, 94% for 17; R =Me: 4N HCl in dioxane, RT, 1 h, then 2N NaOH, THF, MeOH, RT, 2 h,
3
51% for 18.
Biological evaluation
Profiling of the isomeric indole 2 by-product showed only weak binding in the enzyme and no activity in
the ER degradation assay as expected. In contrast, the desired indole 3 showed good binding potency
and maintained the desired degrader-antagonist profile, although it was >10 fold less potent than the
THIQ 1a. Given the weaker activity of the racemate of 3, the individual enantiomers were not separated.
28
In contrast however, the most active enantiomer of indazole 4 was of similar potency and only slightly
1
1
more lipophilic (logD7.4 +0.4) relative to the THIQ phenol matched pair 1a (10a in previous paper ).
This also showed no evidence of trapping in the microsome based GSH assay (Table 1).
Table 1. Data for phenol, indazole and indole cores.
1
2
ACS Paragon Plus Environment

Page 13 of 53
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
O
OH
O
OH
O
OH
O
OH
N
HN
N
N
N
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
HO
HN
HN
1
a
2
3
4
N
ER
ER
LogD7.4c
Rat/Human Rat/Human
Cpd
GSH
DR
% Freed
hepatocyte
CLint
bind
pIC50a
trapping
pIC50b
(
µL/min/106
ratiof
cells)e
7
4
6
.6
8.4
1.3
1.0
1.7
1.9
1
2
3
4
a
3.7/6.3
- /11
38/38
<0.04
.4h
<5h
10/ -
20/8
-
.5
7.2
8.2
6.8/3.3
3.6/7.0
-
7.6g
10/51
<0.04
a
b
ER binding based on n  3 with SEM within 0.2 units, unless otherwise stated; ER degradation based on
c
n  3 with SEM within 0.2 units, unless otherwise stated; logD determined by shake flask method;
7
.4
dDetermined from DMSO stock solution by equilibrium dialysis in 10% plasma from Alderley Park Han
e
6
Wistar rats or 10% human plasma supplied by Quintiles; Rate of metabolism (µl/min/10 cells)
6
f
determined from DMSO stock solution in isolated hepatocytes diluted to 1x10 cells/mL; Ratio of GSH
g
h
trapping in human liver microsomes as a ratio to clozapine as detailed in reference 23; SEM 0.50; n=1.
An X-ray structure (PDBcode 6iar) of the indazole 4 complexed with an ER ligand binding domain
construct (Figure 2a) showed that the nitrogens of the tricyclic indazole interacted with a conserved water
and directly with Glu-353 with distances of 3.3 and 2.8 Å respectively. The indazole scaffold showed a
similar interaction network and occupied a similar binding pose in the ER binding site (Figure 2b) with
1
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 53
1
2
3
4
5
6
7
8
9
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
1
1
minimal perturbation of the ligand position with respect to the THIQ scaffold. Furthermore, overlay
with the structure of 1b (Figure 2c) showed close alignment of the [5.6.6] ring system of the tricyclic
8
indazole with the [6.5.6] core of the tricyclic indole. For the tricyclic indazole core the interaction
network differed with additional interactions with the indazole and the salt bridge (Figure 2a). The
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
hydrogen bond between the indole of 1b and Leu-346 was not replicated in the indazole scaffold structure
(
Figure 2c). The different ligand binding site interaction networks observed between the different series
resulted in slight perturbation of the key binding motifs (Figure 2d) of the series within the ER binding
domain.
a)
b)
c)
d)
1
4
ACS Paragon Plus Environment

Page 15 of 53
1
Journal of Medicinal Chemistry
2
3
4
5
6
7
8
9
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
Figure 2. (a) X-ray crystallography of indazole 4 bound to the ERα ligand binding domain construct
(pdbcode 6iar); (b) overlaid with THIQ 1a coloured orange (10a from ref 11; pdbcode 5fqr); (c) overlaid
with 1b coloured blue (from ref 8; pdbcode 5acc); (d) superimposition of ligands 1a and 1b with 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
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Given the high similarity of ligand binding between the three scaffolds, we applied some of the known
1
1
SAR from our previous work to increase potency. The 3,3-gem-dimethyl substituted core (5) was more
potent than the unsubstituted core (4) in agreement with previous SAR. The mono substituted 3-(R)-
methyl group (6) increased potency further providing our first sub-nanomolar degrader-antagonist (pIC50
2
9
9
.2). The core modification from 3-(R)-methyl to 3,3-gem-dimethyl (exemplified by 6 to 5) had been
shown previously to be beneficial for lowering human hepatocyte clearance however, in this series the
3
0
turnover was consistently higher. We then added fluorine atoms to the alkyl chain in the -position to
the nitrogen (7) and in the 2,6-positions of the aryl ring (8) as well as making the combination of both
changes (9). Indazole 9 has the same alkyl and aryl substitution pattern as 1b, allowing a direct
comparison of the scaffolds, and was a key compound for our evaluation of the indazole series.
Although the compounds were reasonably metabolically stable in rat hepatocytes, the high turnover in
human hepatocytes for compounds 5-7 was cause for concern. Whilst fluorination of the aryl ring (8, 9)
ameliorated this to some degree, further improvements were sought. Notably switching the alkyl chain
from the 2-fluoro-isobutyl to a 2,2-difluoro-isopropyl (10) substituent, lowered both lipophilicity and
human hepatocyte clearance whilst maintaining downregulation potency. Notably, none of these
indazoles showed any evidence of trapping in the GSH assay, in stark contrast to the previously reported
phenols (Table 2).31
Table 2. Data for methyl and fluoro substitutions of the core, alkyl chain and aryl ring.a
1
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 53
1
2
3
4
5
6
7
8
9
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
O
OH
R1
A =
2
2
R
R
B =
C =
1
F
F
R
N
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
HN
3
F
R
N
R1
R2
R3
ER
ER
LogD7.4
Rat/Human Rat/Human
Cpd
GSH
bind
pIC50
DR
% Free
hepatocyte
Clint
trapping
ratio
pIC50
(
µL/min/106
cells)
A
A
B
A
B
C
H
H
H
F
Me
H
7.7
8.0
8.8
8.9
9.5
9.4
8.8
9.2
9.5
9.1
9.6
9.4
2.0
-
5
6.6/3.6
6/93
<0.04
<0.04
-
6
3.4/2.2
7/30
H
2.9
3.0
2.6
2.3
7
1.5/0.87
0.52/0.48
0.83/0.58
1.3/1.1
8/78
H
8
19/22
26/17
18/8
<0.04
<0.04
<0.04
F
H
9
10
F
H
aLegend as Table 1.
Further optimization revealed that a 2-methoxy substituent was tolerated in the aryl ring
resulting in similar levels of potency and lipophilicity (compounds 11 & 12 are matched pairs of
9 & 10). This change further lowered turnover in human hepatocytes and the compounds were
clean when tested in the GSH trapping assay. Systematic exploration of the effect of fluorine
1
6
ACS Paragon Plus Environment

Page 17 of 53
Journal of Medicinal Chemistry
1
2
substitution on the aryl ring (13-15) showed that the 3-F (13) lost potency (20x relative to 5), the
5-F (14) was equipotent and the 6-F (15) was the most potent compound synthesized in this
series (Table 3).
3
4
5
6
7
8
9
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 3. Data for alkyl chain and aryl ring variation in the 2-methoxy aryl series.
O
OH
R1
2
R
5
6
3
B =
C =
F
F
O
1
R
N
F
HN
N
R1
R2
ER
ER
DR
LogD7.4
Rat/Human Rat/Human
Cpd
GSH
bind
pIC50
% Free
hepatocyte
Clint
trapping
ratio
pIC50
(µL/min/106
cells)
B
C
C
C
C
H
8.8
9.2
9.0
8.2
9.5
9.7
2.2
2.5
2.6
2.6
2.8
1
1
2
4.6/0.98
2.2/0.92
- /0.82
- /1.2
17/6
18/5
17/7
22/5
32/6
<0.04
.0b
H
9
1
<0.04
3-F
5-F
6-F
8.9
9.3
9.5
13
14
15
-
-
-
- /0.7
a
b
Legend as Table 1; SEM 0.50.
1
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 53
1
2
3
4
5
6
7
8
9
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
We then turned our attention to the 1-methyl substitution. We had previously investigated this
transformation (HMe) in the THIQ phenol series and found it added lipophilicity and lowered
potency (logD7.4 +0.9; pIC –0.4 for 1-H (10a) to 1-Me (11a) in ref 11). Despite this discouraging
50
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
result, we made the corresponding 1-methyl matched pairs in the indazoles and found with the 2,6-di-
fluoro aryl ring a more modest rise in lipophilicity and small drop in potency (16 & 9 logD7.4 +0.3;

pIC –0.3 and 17 & 10 logD +0.7; pIC –0.3). By contrast, with the 2-methoxy aryl, addition of
5
0
7.4
50
the 1-Me decreased lipophilicity and showed a bigger drop in potency (18 & 11 logD –0.3; pIC –
7
.4
50
0
.6) as shown in Table 4. To probe this surprising and divergent SAR, we considered the effect of the aryl
substituent in terms of conformation using a combination of computational and NMR techniques.
Table 4. Data for 1-methyl substituted indazoles.a
O
OH
R1
2
R
B =
C =
F
F
6
2
1
R
N
F
HN
N
R1
R2
ER
ER
DR
LogD7.4
Rat/Human Rat/Human
Cpd
GSH
bind
pIC50
% Free
hepatocyte
Clint
trapping
ratio
pIC50
µL/min/106
(
cells)
1
6
7
B
C
2,6-F,F
2,6-F,F
9.4
9.5
9.2
9.1
2.9
3.0
0.23/0.33
0.76/0.27
12/10
<0.04
-
1
20/6
1
8
ACS Paragon Plus Environment

Page 19 of 53
Journal of Medicinal Chemistry
1
2
3
4
1
8
B
2-OMe
8.4
8.6
1.9
- /7.7
10/4
-
aLegend as Table 1
5
6
7
8
9
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 investigate the influence of the ortho-substituent on the mobility of the aryl ring further we
carried out an NMR conformational analysis study on compounds 10, 12, 15 and 18 with the
1
19
results shown in Figure 3. Di-fluoro aryl compound 10 showed one single H and F resonance
respectively for the 2 hydrogen and 2 fluorine atoms in the aryl ring, indicating that both ortho-F
and meta-H were exposed to the same chemical environment in the NMR time-scale (i.e. the
1
19
aryl group is freely rotating). H and F n.O.e. based experiments established that the N-alkyl
chain adopted a preferred conformation with the hydrogens in the -position and the fluorine
atoms in the -position pointed away from the aryl ring, in close agreement with the X-ray
structure of 1b (pdbcode 5acc). Incorporation of an ortho-OMe group in the aryl ring (12) had a
very significant effect on the rotational flexibility of the ring as observed by NMR. In this case,
the aryl ring showed a restricted rotation, with the n.O.e.’s between the ortho-OMe/H and the
rest of the molecule consistent with the methoxy group pointing away from the core ring. The
restricted rotation of the aryl ring did not affect the conformation of the N-alkyl chain, which was
identical to that observed for compound 10. Further substitution of the ortho-H with a F (15) did
not alter the rigidity and orientation of the aryl ring or the N-alkyl chain compared with 12, with
1
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 53
1
2
3
4
5
6
7
8
9
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
the OMe group still favouring the alignment away from the ring. The incorporation of a methyl
group in the benzylic position (18) however, inverts the orientation of the aryl ring, with the OMe
group now positioned towards and on top of the core ring to avoid clashing with the Me group.
This switch in conformational locking occurs presumably due to steric reasons and we believe is
responsible for the divergent behavior observed in terms of both the potency and lipophilicity
with pairs 16 & 9 and 18 & 11. This is an example of the importance of considering molecular
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
conformation when interpreting data and highlights the utility of computational and NMR based methods
to provide an insight into the rigidity and flexibility of ligands in solution.
2
0
ACS Paragon Plus Environment

Page 21 of 53
Journal of Medicinal Chemistry
1
2
Figure 3. Experimentally derived NMR based conformations of 10, 12, 15 and 18 showing free
rotation of the 2,6-F,F aryl ring of 10 in contrast to the locked conformations of 12 & 15 (which
orient the 2-OMe substituent away from the core) and 18 (which contrastingly orients the 2-OMe
substituent towards the core).
3
4
5
6
7
8
9
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
Physicochemical & DMPK profiling
In terms of physicochemical properties, the indazole acids were found to have high permeability
with minimal efflux (based on Caco-2 and MDCK assays) and good solubility in aqueous media
with representative examples shown in Table 5. The plasma protein binding data was measured
in mouse and dog, with similar values being obtained to those in rat and human respectively.
Screening against a panel of five cytochrome P450 enzymes in a high throughput human liver
microsome assay showed no activity (IC values > 30 µM) against 3 isoforms (CYP1A2, CYP2C19,
5
0
CYP2D6) however modest inhibition of CYP2C9 was observed in both cases. Indazole 9 showed weak
activity (IC 13 µM) against CYP3A4 however this could be removed by subtle changes to the alkyl
5
0
chain (e.g. 10 IC > 30 µM). No discernable activity (IC > 30 µM) against the hERG ion channel was
5
0
50
observed for any of the examples tested from this series.
Table 5. Physicochemical & in vitro DMPK data for selected indazoles.
Caco2
Sol (µM)b
Mu/Rat/Dog % CYP 2C9 CYP 3A4 hERG
Cpd
MDCK Papp
2
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 53
1
2
3
4
5
6
7
8
9
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
(efflux
Free
(µM)c
(µM)c
(µM)d
P
app
ratio)a
(efflux
ratio)a
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
9
6.9 (1.4)
5.8 (2.6)
6.2 (2.2)
11 (0.6)
10 (1.3)
8.4 (1.7)
>1000
760
0.95/0.83/0.32 2.5
13
>30
-
>100
>100
-
10
2.1/1.3/0.62
1.2/2.2/0.87
6.6
-
12
810
aCompounds were incubated at 10 M in cultured Caco-2 or MDCK cells. Permeability was
measured in both the apical to basolateral (A to B) and basolateral to apical (B to A) directions.
b
Efflux ratio is expressed as (B to A) / (A to B); Solubility of compounds in aqueous phosphate
o
c
d
buffer at pH 7.4 after 24 h at 25 C; Inhibition of cytochrome P450 enzymes IC . Inhibition of
50
the hERG tail current was measured using a plate-based planar patch clamp system
(
IonWorks™).
We also investigated the chemical stability of these compounds, initially using an NMR based
1
approach. Compounds were dissolved in deuterated buffers (pH 4 and 7.4) and H NMR
spectra were recorded at several time points over a period of 8 d. Unchanged spectra after that
time indicate that the compound was stable under those conditions but modification of the NMR
spectra (e.g. reduction of parent signals, appearance of new resonances) was indicative that a
2
2
ACS Paragon Plus Environment

Page 23 of 53
Journal of Medicinal Chemistry
1
2
degradative process was taking place. Whilst 9 was completely stable at pH 7.4, some
instability was noted at pH 4 (58% of compound remaining after 8 d in buffer). By contrast,
compound 12 showed no degradation after 8 d at either pH 4 or 7.4. Subsequent profiling
showed that 12 showed no instability under forcing conditions (at pH 1, 4, or 10). This stability
was in notable contrast to that reported by Novartis on their phenolic THIQ scaffold where
3
4
5
6
7
8
9
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
13
racemization was observed and proposed to occur via a quinoline intermediate. We believe
that the combination of the less electron rich indazole together with the transannular methyl
group, which favours the thermodynamically more stable 1,3-trans arrangement, is responsible
for the enhanced stability of this scaffold relative to geminal-disubstituted phenolic THIQs.
The in vitro hepatocyte and in vivo pharmacokinetic (non-compartmental) properties of 9, 10
and 12 were investigated in rat and dog and their values are reported in Table 6. In vitro
6
6
clearance was moderate in both rat (18-26 L/min/10 cells) and dog (7-17 L/min/10 cells)
with 10 and 12, which contain the 2-OMe aryl ring, showing lower turnover than 9 which
contains the 2,6-F,F aryl. In vivo clearance in rats was low to moderate and ranged from 33-
46% and 7-15% of hepatic blood flow in rat and dog respectively. Scaling of in vitro clearance
with free fraction and blood/plasma ratio yielded an in vitro / in vivo correlation within 2-fold
2
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 53
1
2
3
4
5
6
7
8
9
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
(
data not shown). Volume of distribution was low to moderate in both rat and dog and is
consistent with the highly protein bound and acidic properties of these compounds.
Bioavailability was generally good (16-51%) for 9, 10 and 12 and is reflective of the high in vitro
permeability combined with low/moderate clearance. These properties were deemed suitable to
pursue in vivo pharmacodynamics studies.
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 6. Summary of Pharmacokinetic Parameters after IV and PO dosing in rat and dog.a
Species Clint
Dosea
CL
Vdss
t1/2
%F
PO AUC
Cpd
(
µL/min/106 IV/PO
(mL/min/kg) (L/kg)
(hr)
(M.hr)
cells)
26
(mg/kg)
0.5/1
9
Rat
23
1.6
1.2
3.7
1.3
6.1
4.8
6.8
31
16
51
45
36
45
0.83
0.57
0.21
3.2
10
Rat
18
18
17
7.1
9.6
0.5/1
0.5/1
0.5/1
0.5/1
0.5/1
26
1.6
1
9
1
1
2
Rat
32
1.0
Dog
Dog
Dog
5.9
5.4
2.8
0.58
0.63
0.36
0
2
3.0
6.6
aCompounds were dosed in rat and dog intravenously at 0.5 and orally at 1 mg/kg. The
formulation by both routes was 5% DMSO:95% hydroxylpropyl -cyclodextrin (30% w/v) at a
volume of 1 mL/kg and 4 mL/kg, IV and PO respectively.
2
4
ACS Paragon Plus Environment

Page 25 of 53
Journal of Medicinal Chemistry
1
2
Pharmacological profiling of compound 9
3
4
5
6
7
8
9
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
3
2
The pharmacology of compound 9 was profiled as previously described for 1b. As shown in
Figure 4, the curves from the ER downregulation assay (Figure 4a), tamoxifen competition
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
assay (Figure 4b) and ER antagonism assay (Figure 4c) were consistent with the desired
degrader-antagonist profile with no evidence of ER agonism as measured in the progesterone receptor
(PR) assay, a downstream marker for ER transcriptional activation (Figure 4a).
Figure 4. (a) ER (black) and PR (blue) protein expression in the MCF-7 cell line after treatment with
compound 9 showing a concentration dependent decrease in ER expression and no increase in PR
expression consistent with no ER agonism; (b) ER protein expression curve (black) and decrease in
potency in the presence of 0.25 μM tamoxifen (red) due to competition at the ER ligand binding site; (c)
Concentration-dependent decrease by 9 in estradiol-driven PR response as a measure of ER antagonism.
The ability of 9 to downregulate ER was also investigated by Western blot. MCF-7 cells were incubated
with either 9 or fulvestrant for 24 h at a range of concentrations and total protein was extracted and
immunoblotting performed to detect effects on ER protein expression. As shown in Figure 5, both 9 and
fulvestrant proved to be potent degraders of ER in MCF-7 cells.
2
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 53
1
2
3
4
5
6
7
8
9
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
Figure 5. Concentration-dependent degradation of ER by 9 and fulvestrant by Western blot analysis.
Compound 9 was also investigated in terms of its antiproliferative capability. As shown in Figure
6, a concentration-dependent decrease in estradiol-driven cell growth of the MCF-7 cell line was
observed with 9 (Figure 6a) giving a similar potency (pIC 8.2) to that observed with fulvestrant
50
(
pIC 8.3; Figure 6b).
5
0
Figure 6. Concentration-dependent decrease in estradiol-driven cell growth of the MCF-7 cell line with 9
(A) and fulvestrant (B).
2
6
ACS Paragon Plus Environment

Page 27 of 53
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
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
In vivo profiling was carried out to investigate the ability of 9 to effect reduction of PR levels
(
Figure 7) and tumour growth (Figure 8) in an MCF-7 xenograft model. MCF-7 xenografts, grown in
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
male SCID mice, were orally dosed once daily with either PEG/captisol (vehicle) or 9 at the doses shown.
After 3 d of dosing, tumours were collected 24 h after the last dose and 5 mg/kg was found to
reduce the normalized PR levels by 74% (p = 0.007) and at 50 mg/kg by 93% (p <0.001).
Figure 7. Dose-dependent decrease in PR levels, measured by Western blotting, following oral dosing of
9
to mice once daily in an MCF7 mouse tumour xenograft model. MCF-7 xenografts were dosed
for 3 d with doses shown and tumours collected at 24 h after the last dose.
Compound 9 was then evaluated for effect on tumour growth in the same MCF-7 xenograft model
(Figure 8). At doses of 20 mg/kg administered orally once daily, compound 9 was able to effect
significant tumour growth inhibition (80%) at 20 d in this model and was well tolerated.
2
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 53
1
2
3
4
5
6
7
8
9
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
Figure 8. Antitumour activity of 9 administered orally once daily at 20 mg/kg in MCF-7 xenografts.
CONCLUSIONS
In conclusion, we have demonstrated that the phenol of our previously reported
tetrahydroisoquinoline scaffold could be successfully replaced by an indazole group and that
this led to removal of a reactive metabolite signal in a glutathione in vitro trapping assay.
Optimization of the alkyl sidechain and pendant aryl group gave compounds that showed low
turnover in human hepatocytes and good chemical stability. Compound 9 was profiled as a
representative of the series in terms of pharmacology and demonstrated the desired ER
degrader-antagonist profile and demonstrated activity in a xenograft model.
EXPERIMENTAL SECTION
2
8
ACS Paragon Plus Environment

Page 29 of 53
1
Journal of Medicinal Chemistry
2
3
4
5
6
7
8
9
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
General Procedures: All solvents and chemicals used were reagent grade. Anhydrous solvents THF,
DCM, and DMF were purchased from Aldrich. Solutions were dried over anhydrous magnesium sulfate
or sodium sulfate, and solvent was removed by rotary evaporation under reduced pressure. Microwave
reactions were run in a Biotage ‘Initiator Robot 60’ (100-120/220-240 V, 50-60 Hz, 100 VA). Flash
column chromatography was carried out using prepacked silica cartridges (from 4 g up to 330 g) from
Grace, Redisep or Silicycle and eluted using an Isco Companion system. The purity of compounds
submitted for screening was >95% as determined by UV analysis of liquid chromatography-mass
spectroscopy (LCMS) chromatograms at 254 nM and substantiated using the TAC (Total Absorption
Chromatogram). Further support for the purity statement was provided using the MS TIC (Total Ion
Current) trace in ESI +ve and –ve ion modes, HRMS and NMR analysis. NMR spectra were recorded on
a Varian INOVA (600 MHz), Varian Gemini 2000 (300 MHz), Bruker Avance 700 (700 MHz), Bruker
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
Avance 500 (500 MHz) or Bruker Avance DPX400 (400 MHz) and were determined in CDCl , DMSO-
3
d , or MeOH-d . Chemical shifts are reported in ppm relative to TMS (0.00 ppm) or solvent peaks as the
6
4
internal reference. Splitting patterns are indicated as follows: s, singlet; d, doublet; t, triplet; m, multiplet;
br, broad peak. Analytical LCMS was carried out using a suitable system, such as a Waters 2790/95 LC
system with a 2996 PDA and a 2000 amu ZQ single quadrupole mass spectrometer, or a UPLC system
utilising a Waters Aquity Binary pump with sample manager, Aquity PDA and SQD Mass spectrometer.
Accurate mass and MSMS fragmentation data were obtained using a Thermo Scientific hybrid LTQ-FT
Mass Spectrometer with an Agilent 1100 Quaternary pump with PDA and Autosampler; 5 L of sample
dissolved in 50:50 acetonitrile:water 0.1% formic acid was injected onto a Thermo Scientific Hypersil
Gold 50 x 2.1 mm 5 m particle LC Column and eluted with a gradient of 5 to 100% B over 17 min with
3
min re-equilibration time at 5% B. The flow rate was 0.5 mL/min with A being 0.1% formic acid in
water and B 0.1% formic acid in acetonitrile. The MS and MSMS spectra were obtained in ESI +ve
mode in both the ion trap and Ion Cyclotron Resonance (ICR) cell using helium as the collision gas at a
normalised collision energy of 35 eV. The ICR cell was run at resolution settings of 25000 in MS mode
and 12500 in MSMS mode.
2
9
ACS Paragon Plus Environment