Heteroarylamide smoothened inhibitors: Discovery of N-[2,4-dimethyl-5-(1-methylimidazol-4-yl)phenyl]-4-(2-pyridylmethoxy)benzamide (AZD8542) and N-[5-(1H-imidazol-2-yl)-2,4-dimethyl-phenyl]-4-(2- pyridylmethoxy)benzamide (AZD7254)

Article abstract of DOI:10.1016/j.bmc.2019.115227

Aberrant hedgehog (Hh) pathway signaling is implicated in multiple cancer types and targeting the Smoothened (SMO) receptor, a key protein of the Hh pathway, has proven effective in treating metastasized basal cell carcinoma. Our lead optimization effort focused on a series of heteroarylamides. We observed that a methyl substitution ortho to the heteroaryl groups on an aniline core significantly improved the potency of this series of compounds. These findings predated the availability of SMO crystal structure in 2013. Here we retrospectively applied quantum mechanics calculations to demonstrate the o-Me substitution favors the bioactive conformation by inducing a dihedral twist between the heteroaryl rings and the core aniline. The o-Me also makes favorable hydrophobic interactions with key residue side chains in the binding pocket. From this effort, two compounds (AZD8542 and AZD7254) showed excellent pharmacokinetics across multiple preclinical species and demonstrated in vivo activity in abrogating the Hh paracrine pathway as well as anti- tumor effects.

Full text of DOI:10.1016/j.bmc.2019.115227

Journal Pre-proofs
Heteroarylamide Smoothened Inhibitors: Discovery of N-[2,4-dimethyl-5-(1-
methylimidazol-4-yl)phenyl]-4-(2-pyridylmethoxy)benzamide (AZD8542)
and N-[5-(1H-imidazol-2-yl)-2,4-dimethyl-phenyl]-4-(2- pyridylme-
thoxy)benzamide (AZD7254)
Bin Yang, Alexander W. Hird, Michael S. Bodnarchuk, Xiaolan Zheng, Les
Dakin, Qibin Su, Kevin Daly, Robert Godin, Maureen M. Hattersley, Patrick
Brassil, Sean Redmond, Daniel John Russell, James W. Janetka
PII:
S0968-0896(19)30668-6
https://doi.org/10.1016/j.bmc.2019.115227
BMC 115227
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
23 April 2019
9 November 2019
14 November 2019
Please cite this article as: B. Yang, A.W. Hird, M.S. Bodnarchuk, X. Zheng, L. Dakin, Q. Su, K. Daly, R. Godin,
M.M. Hattersley, P. Brassil, S. Redmond, D. John Russell, J.W. Janetka, Heteroarylamide Smoothened
Inhibitors: Discovery of N-[2,4-dimethyl-5-(1-methylimidazol-4-yl)phenyl]-4-(2-pyridylmethoxy)benzamide
(AZD8542) and N-[5-(1H-imidazol-2-yl)-2,4-dimethyl-phenyl]-4-(2- pyridylmethoxy)benzamide (AZD7254),
Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.115227
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Heteroarylamide Smoothened Inhibitors: Discovery of N-[2,4-dimethyl-5-(1-
methylimidazol-4-yl)phenyl]-4-(2-pyridylmethoxy)benzamide (AZD8542) and N-[5-
(1H-imidazol-2-yl)-2,4-dimethyl-phenyl]-4-(2- pyridylmethoxy)benzamide
(AZD7254)
Bin Yang,1* Alexander W. Hird,1 Michael S. Bodnarchuk,2 Xiaolan Zheng,1 Les Dakin,1 Qibin Su,1 Kevin
Daly,1 Robert Godin,3 Maureen M. Hattersley,3 Patrick Brassil,4 Sean Redmond,5 Daniel John Russell,1 James

W. Janetka1,
1Medicinal Chemistry,Oncology, IMED Biotech Unit, AstraZeneca, 35 Gatehouse Drive,
Waltham MA 02451,USA
2Medicinal Chemistry, Oncology, IMED Biotech Unit, AstraZeneca, Building 310, Cambridge Science
Park, Milton Road, Cambridge UK
3Bioscience, Oncology, IMED Biotech Unit, AstraZeneca, 35 Gatehouse Drive, Waltham MA 02451,
USA
4Drug Metabolism and Pharmacokinetics, Oncology, IMED Biotech Unit, AstraZeneca, 35 Gatehouse
Drive, Waltham MA 02451, USA
5Oncology Safety, Drug Safety and Metabolism, IMED Biotech Unit, AstraZeneca, 35 Gatehouse
Drive, Waltham MA 02451, USA
ABSTRACT
Aberrant hedgehog (Hh) pathway signaling is implicated in multiple cancer types and targeting the
Smoothened (SMO) receptor, a key protein of the Hh pathway, has proven effective in treating
metastasized basal cell carcinoma. Our lead optimization effort focused on a series of
heteroarylamides. We observed that a methyl substitution ortho to the heteroaryl groups on an aniline
core significantly improved the potency of this series of compounds. These findings predated the
availability of SMO crystal structure in 2013. Here we retrospectively applied quantum mechanics
calculations todemonstrate the o-Me substitution favors the bioactive conformation by inducing a

dihedral twist between the heteroaryl rings and the core aniline. The o-Me also makes favorable
hydrophobic interactions with key residue side chains in the binding pocket. From this effort, two
compounds (AZD8542 and AZD7254) showed excellent pharmacokinetics across multiple preclinical
species and demonstrated in vivo activity in abrogating the Hh paracrine pathway as well as anti-
tumor effects.
INTRODUCTION
The Hedgehog (Hh) pathway is a critical pathway during embryogenesis where it mediates cell
proliferation, differentiation, and patterning of the embryo. This pathway has been conserved through
evolution and has been identified throughout the animal kingdom including sea urchins, worms, flies
1
and mammals. The human genome contains three Hh genes, Sonic (SHH), Indian (IHH) and Desert
(DHH). Hh proteins are secreted from the cell and bind a transmembrane receptor Ptch1. The
function of Ptch1 is to inhibit a 7-pass membrane protein smoothened (SMO), a frizzled family G
protein–coupled receptor (GPCR) that transmits the Hh signal across the membrane to the
2
3
cytoplasm. Multiple SMO inhibitors have been identified, including vismodegib (1) , sonidegib
4
5-6
7
(2), glasdegib (3),
and taladegib (4) (Figure 1) and have been evaluated in clinical trials.
8-9
Compounds 1 and 2 have been approved for the treatment of Basal Cell Carcinoma.
Recently,
renewed attention to Hh pathway inhibitors has been inspired after the X-ray structures of SMO
10,11,12
became available.
These recent breakthroughs in structural biology helped elucidate a
mechanism where a large extracellular region and two allosterically linked ligand-binding sites in
SMO regulate its activity. Cholesterol likely functions as an endogenous SMO ligand that binds to
one of the two allosteric sites and stabilizes SMO to a resting or apo conformation poised to respond
to Hh signals. SMO inhibitors, such as vismodegib, bind to the second allosteric site to occlude the
12
cholesterol-binding site, inhibiting Hh signaling activity. A more recent report suggests that known
SMO inhibitors (e.g., cyclopamine and vismodegib) bind in and block a transmembrane hydrophobic

channel that could be used by the protein to shuttle cholesterol from the inner membrane to the
13
extracellular Cysteine-Rich-Domain (CRD) in order to activate SMO. We previously disclosed a
14
series heteroarylamides as potent SMO inhibitors with good preclinical pharmacokinetic profiles.
Further optimization on this heteroarylamide series revealed some intriguing structure-activity
relationships (SAR). Herein we report our work on these potent SMO inhibitors that was done before
the availability of X-ray crystal structure of SMO. With the available ligand-bound crystal structures
of SMO in the public domain, we also attempted to understand the observed SAR of these
heteroarylamides in a retrospective sense.
O
Cl
O
SO2
CF3O
N
N
N
H
N
O
N
H
Cl
1 (vismodegib)
2 (sonidegib)
F3C
F
O
CN
H
N
N
H
N
N
N
N
N
H
O
H
N
H
N
N
N
3 (glasdedib)
4 (taladegib)
O
N
N
H
N
N
H
O
5
Figure 1. Structures of SMOinhibitors
RESULTSANDDISCUSSION
GLI modulation and efficacy in mouse xenograft models.
We previously reported compound 5 as a potent SMO inhibitor with excellent rodent PK and good
physical properties.14 Compound 5 was further taken into in vivo studies and it demonstrated a dose

dependent pharmacologic effect (mouse GLI suppression) in both Colo2015 and NCI-H460 xenograft
models (Figures S1 and S2). Although we observed reduced GLI expression in mouse stroma cells
within the xenograft, no significant modulation of human GLI was observed in the tumour cells
(Figure S2 and Table S2). Others have shown that activation of the Hh pathway by SHH does not
affect endogenous GLI1 mRNA levels in tumor epithelial cells (Bxpc3 and CFPAC-1), and suggested
that Hh signaling acts in a paracrine fashion from tumor cells to stromal cells because SMO inhibition
in the mouse stroma of xenograft tumor models is required for growth inhibition.15 While this
explains the lack of GLI modulation in human tumor cells by 5 in our HT29 xenograft study, at the
time we also wanted to rule out potential inter-species potency differences between mouse and human
SMO as the cause. Our primary assay for potency optimization utilized a mouse cell
line(C3H10T1/2) and aligning human and mouse SMO sequences revealed 92% similarity which
raised our concern regarding inter-species binding affinity difference. Seeking to understand the risk
that we might have inadvertently optimized 5 for mouse SMO inhibition over its human isoform, we
developed a competition binding assay (Bodipy-labeled cyclopamine) to test the affinity difference of
5 to mouse and human SMO, using 1 as the control. Interestingly, we observed similar binding
potency for 1 in Hela cells transfected with either mouse or human SMO, whereas 5 showed a 7-fold
decrease in affinity from mouse to human (Figure 2). Furthermore, when 1 and 5 were tested in a
luciferase assay using Homo sapiens palatal mesenchyme cells (HEPM), we also observed notable
IC value differences between these two compounds (0.4 nM vs. 2.4 nM for 1 and 5, respectively.
50
These two showed less difference in potency in our primary cellular assay using the mouse cell line
C3H10T1/2 (4.7 nM for 1 vs. 12 nM for 5, Table 1). This unexpected potency drop-off for 5 led us to
C3H10T1/2 (4.7 nM for 1 vs. 12 nM for 5, Table 1). This unexpected potency drop-off for 5 led us to
deprioritize further preclinical studies on 5 and initiated additional exploration work for more potent
SMO inhibitors. We continued to use the C3H10T1/2 differentiation assay as our primary assay and
used the low throughput human SMO binding and HEPM assays to check key compounds to prevent
unexpected species-dependent potency difference.

A:
B:
C:
Binding EC of 1 in human and mouse SMO transfected Hela cells; B: Binding EC
50 50
Figure 2. A:
of 5 in human and mouse SMO transfected Hela cells; C: 5 and 1 induced
luciferase inhibition in
HPEM cells.
Synthesis of ortho substituted heteroarylamides.
The general synthetic route to a series of ortho substituted heteroarylamides is illustrated in Scheme 1.

The phenol in benzoyl ester 6 undergoes direct alkylation by reaction with chloromethylpyridine 7 in
the presence of potassium carbonate. Hydrolysis of the corresponding ester product 8 under basic
conditions results in carboxylic acid 9. Amide coupling between the acid 9 and the trisubstituted
anilines 10 using HATU as the coupling reagent and DIPEA as the base affords the bromoamide
compounds 11. The bromide can be converted to pinacol boronates 12 under Suzuki reaction
conditions, and here we used palladium tetrakis as the catalyst and cesium carbonate as the base in
anhydrous 1,4-dioxane. The resulting arylboronates 12 were further coupled with heteroaryl halide,
again using Suzuki coupling conditions particularly with 1′-bis(di-tert-butylphosphino)ferrocene]-
(Pd(dt-bpf)Cl ) as the catalyst, to generate a series of heteroaryl
substituted
dichloropalladium(II)
2
benzamide compounds (13).

O
K2CO3
O H
NaOH
N
Cl
+
O
+
N
H
EtOH
95%
O
DMA
96%
–
O
Cl
O
6
7
8
HATU
DIPEA
O
N
N H2
H
N
O
Br
R
N
Br
+
DMA
HO
O
R
O
10a R = Cl
10b R = Me
10c R = F
11a R = Cl
11b R = Me
11c R = F
9
O
O
B
B
O
O
O
Pd(PPh3)4
Cs2CO3
HetAryl-X
Suzuki
N
O
B
H
N
O
Dioxane
O
R
12a R = Cl
12b R = Me
12c R = F
O
N
H
N
HetAr
O
R
13
Scheme 1. Synthetic route to heteroaryl substituted benzamides (heteroarylamides).
4-Cl on aniline core and o-Me on imidazoles for torsional twist.
Suspecting that the chloro group ortho to the pyridyl in 1 increased the torsional twist between the
aniline phenyl and the pyridyl ring, we envisioned that tuning the dihedral angle between the
heteroaryl group and the aniline phenyl in our heteroarylamide series could also benefit the potency,
with the assumption that the heteroarylamides bind to the same pocket and with a similar pose in SMO
as 1. We then employed Quantum-Mechanics (QM) to perform a torsion scan of 1 (Supporting

Information, Figure S3), which indeed revealed two minima at 140° and 240° (Figure 3A).
We therefore sought to design and investigate heteroarylamides which showed similar torsional
preferences as 1, anticipating that one of these two dihedral twists may be representative of the
bioactive conformation. Although we lacked structural understanding of how the twisted
conformation might increase the potency of compounds 1 or analogues of 5 at the time, we reasoned
the twisted heteroaryl rings might allow for favourable π- πinteractions with the protein, allow the
nitrogen electron lone pairs of the heteroaryls to form polar interactions with the protein, or might
simply offer improved receptor-ligand complementarity in the ground state conformation of the
ligand.
We first synthesized three imidazole benzamides with the requisite 4-Cl on the aniline ring, and
compared their potency in a cell based shh stimulated C3H10T1/2 differentiation assay to the 4-H
analogues that were previous disclosed.16 We were gratified to observe consistent improvements in
potency upon introduction of the 4-Cl group, notably in the case of 1,2-dimethylimidazole
compounds(17 and 18), a ~10-fold improvement was achieved. While the gain in potency was
encouraging, we were intrigued by the likely contributing factors to the observed potency difference
between these 4-Cl and 4-H matched pairs. The introduction of the 4-Cl group resulted in notably
higher measured LogD vs. their 4-H matched pairs, demonstrated by two 1-methylimidazole
compounds (15 vs. 16) and two1,2-dimethyl imidazole compounds (17 vs. 18), suggesting that the
potency improvement was likely due to increased lipophilicity rather than a conformational change.
The increased LogD with 4-Cl substitution was also accompanied by decreased solubility (Table 1,
14 and 18 aqueous solubility < 1 µM).
Without observing significant potency improvement independent of the lipophilicity, we next
performed a QM based torsion scan between the aniline and imidazole rings of the compounds in

Table 1. Interestingly, a planar torsion was predicted to be adopted by the 4-Cl imidazole
compound(14), like the 4-H imidazole 5 (Figure 3B). We suspected an intramolecular hydrogen-bond
locked the Cl and the H-bond donor NH from the imidazole group.17 Similar behaviour was seen in
the other matched pairs 15 vs. 16 and 17 vs. 18, and in both matched pairs an internal hydrogen-bond
likely formed between the CH of the imidazole and the Cl atom. The free energy difference between
the favoured torsional angle and the perceived 140o/240o bioactive torsion was not seen to change
significantly for Cl-containing species compared to their H-containing matched pairs, supporting the
hypothesis that the increase in potency was predominantly driven by LogD. We characterised the free
energy difference as ΔE
calculated as the average energy required to adopt either the 140º/240º
bio,
bioactive dihedral relative to the lowest energy torsion.
A:
B:
HN
Cl
N
N
NH
Cl
0/360o
180o
Figure 3. A: Depiction of the minima dihedral angles between the pyridyl and o-Cl-phenyl from QM
based torsion scan; B: Illustration of 0º and 180º dihedral angles between the imidazole and phenyl rings
when two aromatic rings adopt co-planar conformation.

Table 1. 4-Cl substituted heterobiaryl amides increased potency but at the expense of LogD.
O
N
H
N
Ar
R
O
shh EC50
(nM)a
Solubility
(µM)b
ΔEbio
(kcal/mol)
c
140o
190
350
180
0
Compound
Ar
R
LogD
1
4.7
13
2.8
3.0
>3.0
2.5
3.1
2.7
3.3
6
N
5
H
Cl
H
25
<1
80
20
46
<1
2.5
2.6
2.6
2.5
2.6
2.3
N
H
14
15
16
17
18
7.5
89
N
Cl
H
23
N
27
180
10
N
Cl
2.9
N
a C3H10T1/2 differentiation stimulated by sonic Hh protein (shh) was previous described.16 EC50 values
are the geometric mean of at least two tests.
b Solids from evaporated DMSO solutions were used to measure solubility.
c Calculated torsional angles between the heteroaryl ring and the phenyl ring using the Becke3LYP
functional18 and the 6-311+G* basis set.
Given that the 4-Cl substitution did not appear to favour a notable torsional twist between the two
aromatic rings, together with the unwanted LogD increase and deterioration in solubility, we revisited
the impact on the torsion angle by the o-Me groups on the 5-membered imidazole rings that we
previously synthesized (Table 2). Matched pair analysis showed that o-Me substituted imidazoles all
resulted in a reduced ΔE
compared to o-H, indicating a lower barrier to twisting the biaryl away
bio
from coplanarity. However, it is noteworthy the o-Me substitution on the imidazole nitrogen (19 and

21) reduced the potency relative to their NH matched pairs, 5 and 20, respectively. In contrast, the o-
Me substitution on a carbon atom of the imidazole, while retaining the NH group, showed similar
potency (22 vs. 23). The subtle SAR led us to suspect that the NH group, likely as a H-bond donor,
might make a favorable polar interaction in the binding pocket of SMO. As a result, although the
reduced energy barrier (ΔE ) to adopt the perceived bioactive twisted conformation by o-Me
bio
substitutions might favor the SMO-ligand interaction, the elimination of a possible polar interaction
between the imidazole NH and SMO protein compromised the benefit to potency from o-Me
substitution.
Compound 24 is a matched pair to both 14 (4-Cl substitution on the aniline core) and 19 (N-Me
substitution on the imidazole group). The N-Me substitution neutralized the favourable torsion twist
induced by the 4-Cl substitution, resulting in 24 but with no potency gain from 14. The slight
improvement in potency for 24 over 19 was consistent with a reduced ΔE
and the increase of LogD
bio
(+ 0.4). Compound 24 also showed a reduction in solubility relative to 19, retaining the trend we
observed in compounds shown in Table 1. We consistently observed that the methyl substituents on
these imidazoles generally maintain or slightly reduce LogD, presumably through their electron
donating effect to increase the pK of the imidazole nitrogen.
a
However, a more notable effect of these
methyl groups on physical properties is that they sometimes strongly increased the solubility of this
series of compounds, in marked contrast to the 4-Cl substitution on the middle aniline ring.
Table 2: o-Me substitution on imidazole improves physical properties
O
N
H
N
Ar
R
O
ΔEbio
(kcal/mol)
shh EC50
(nM)a
Solubility
(µM)
Compound
Ar
R
LogD


N
19
1.1
H
18
2.8
192
210
N
H
N
1.7
0.5
1.6
0.6
0.2
20
21
22
H
H
H
H
Cl
6.7
30
39
21
6.8
3.0
2.8
2.8
2.9
3.2
15
371
90
160
140
160
160
300
N
N
N
H
N
N
N
H
N
23
24
123
1
N
N
a EC50 values are the geometric mean of at least two tests.
4-Me on aniline core to introduce torsional twist on heteroarylamides.
Having observed the potency gain with 4-Cl substitution on the middle aniline group and the
beneficial physical property improvement with methyl substitution on the imidazole groups we then
synthesized 4-Me analogues on the middle aniline, hypothesizing the 4-Me could improve the potency
while retaining the promising physical properties. Intrigued by the subtle potency changes when 4-Cl
was installed, we also looked for additional ortho substitution groups, such as 4-F. Here we observed
remarkable improvement in potency in the shh induced cell differentiation assay, particularly for 25
(EC50 1.4 nM) where the 4-Me substitution showed a near 10-fold increase over 5 (EC50 13 nM).
Compound 25 has both a lower ΔEbio and a more orthogonal ground state conformation (210°) relative
to 5, indicating that it can adopt the presumed bioactive conformation more readily (Table 3). The
substitution of Cl and Me on phenyl groups is normally considered to be bioisosteric due to their

similar size,19 yet here we observed remarkable potency differences between these two substituents.
The QM torsional analysis revealed that Cl-containing analogues can form an intramolecular H-bond
to either the ortho NH or CH on the linking heterocycle, whilst the Me-containing compounds cannot.
This results in a reduced energy barrier to adopt the perceived binding mode for the Me-containing
compounds which is consistent with their boost in potency relative to Cl-analogues.
The potency gain in 26 (EC50 2.6 nM) is even more noteworthy at 34-fold compared to its 4-H
analogue 15 (EC 89 nM).
Both 25 and 26 are also more potent than their 4-Cl analogues (14 and
50
), consistent with a reduction in ΔE .
16
Furthermore, they also showed a smaller LogD increase
bio
compared to the chloride analogues. Similar to the 4-Cl compounds, both 25 and 26 still showed a
trend for decreased solubility. We also tested 25 and 26 in rat and human hepatocyte stability assays,
and while both compounds showed moderate Cl values, there was an overall increase relative to
int
their
4-H matched pairs. The 4-Me substitution in 27 also showed 10-fold improvement in potency
4-H compound 21, but it came with some decreased metabolic stability (rat hepatocytes Cl
int
over the
32 µl/min/10-6 cells and human hepatocytes Cl 12 µl/min/10-6 cells) and hERG inhibition liability
int
also increased (IC50 12 µM).
The 4-F compound 28 showed no improvement of potency over its 4-H analogue 5, consistent with no
improvement ofΔE
.
bio A clear disadvantage of 4-F substitution as shown in 28 is its notably reduced
solubility. Anticipating the 2-Me substitution (R ) could induce a similar dihedral twist as 4-Me on
2
the aniline ring, we also synthesized 29. However, compound 29 showed no potency gain over 5,
potentially owing to conformational effects on the adjacent amide moiety.
Table 3. 4-Me and 4-F substituted heteroaryl amides.

O
R2
N
H
N
Ar
O
R1
Heps Clint
(ul/min/10-6)
ΔEbio
(kcal/
mol)
shh
hERG
IC50
(µM)
Solubility
(µM)c
a
Ar
R1
R2
EC50
LogD
(nM)b
Rat
Human
N
25
26
27
28
29
Me
Me
Me
F
H
H
1.0
2.6
2.9
17
3.1
3.0
2.8
3.0
2.8
2
8
18
9
6
>28
21
210
170
220
0
1.5
0.8
0.1
4.5
1.9
N
H
N
19
32
N
N
H
NDd
1
12
12
N
N
N
H
14
NDd
NDd
>100
>100
N
H
H
Me
13
69
NDd
150
N
H
a Calculated torsional angles between the heteroaryl ring and the phenyl ring using the Becke3LYP
functional and the 6-311+G* basis set.
b EC50 values are the geometric mean of at least two tests.
c Solids from evaporated DMSO solutions were used to measure solubility.
d Not determined.
Encouraged by the significant potency gain from the 4-Me substitution, we next synthesized a small
set of 4-Me heteroaryl amides, structurally diversifying from the imidazoles into other heteroaryls,
including pyrazole, thiazole, pyridyl, and pyridazine (Table 4). The potency difference among these
compounds is relatively insignificant, but it could also be that the measured potency was reaching the
limit of the test concentration range of our cellular assay. For most of these compounds, one concern
was the increased lipophilicity in comparison with the imidazole amides, as some of them failed to
give numerical readouts in LogD measurement, possibly due to their poor solubility and high
lipophilicity (e.g. the benzimidazole analogue 30 and 3-pyridyl analogue 35). The thiazole compound

31 showed excellent potency, but its HLM (human liver microsome) and RLM (rat liver microsome)
Cl were too high to beacceptable.
int
Two pyrazole analogues, 32 and 33, exhibited similar potency
and improved solubility, with 32 showing better metabolic stability in both rat and human liver
microsomes. 2-Pyridyl compound 34 and 3-pyridyl compound 35 showed subtle differences in
potency (0.8 vs. 2.3 nM) that may imply the 2-pyridyl nitrogen is making polar interaction with the
SMO protein while 3-pyridyl N is not as optimally positioned to make the same interaction. However,
neither compound was chosen for further studies due to their higher metabolic Cl s.
int Surprisingly,
the two aminopyridine analogues 36 and 37 showed little difference in potency despite the different
orientation of their amino groups. Although the amino group presumably affords additional
hydrophilicity, both 36 and 37 failed to show meaningful improvement in solubility. The
aminopyridazine compound 38 was of interest as it brought together excellent potency and metabolic
stability in both human and rat liver microsomes, although solubility continued to be a concern.
Table 4. Non-imidazole heterobiaryl amides with 4-Me substitution
O
N
H
N
HetAr
O
Compo
und
Name
shh EC50
Solubility
(µM)
RLM CLint
(µl/min/mg)
HLM CLint
(µl/min/mg)
HetAr
LogD
>3.5
>3.0
(nM)a
N
30
31
3.1
<1
<1
21
30
N
S
0.9
1.1
147
513
N
H
N
N
32
>3.0
33
55
20

N
N
33
3.1
>3.0
35
271
122
N
34
35
36
0.8
2.3
1
>3.0
>3.0
3.2
<1
9
296
78
209
62
9
N
H2N
<1
32
N
N
N
37
38
1.7
>3.0
2.6
2
6
155
8
88
<4
H2N
H2N
<1.5
N
Binding mode of heterobiaryl amides to SMO
As transmembrane proteins, GPCRs were notoriously difficult to crystallize and as a result the binding
modes of GPCR inhibitors, including SMO inhibitors, largely remained mysterious for decades.
However, recent advancements in protein crystallography have begun to unveil the complex signal
transduction mechanism of the Hh pathway, where SMO plays a key regulatory role.12-13 The co-
crystallization of cyclopamine and vismodegib with SMO offered insight into the transmembrane
binding pocket of SMO.10-11 It is interesting to note this binding pocket is close to the extracellular
domain CRD and is likely accessible to SMO ligands without the need for them to first enter the cell.
With the recently available X-ray structure of SMO, we retrospectively docked our heteroarylamides
into the SMO transmembrane pocket that vismogedib occupies (PDB 5L7I),12 and illustrated in
Figure 4 is the predicted binding mode of 25. As anticipated, the imidazole and the aniline rings are
predicted to adopt a significant twisted conformation of 228º, close to the dihedral angle adopted by
vismodegib in SMO (240º). This twisted conformation between the two aromatic rings in 25 allows

some key ligand-protein interactions: 1) the imidazole group forms edge-to-plane π-π interactions, one
with the indole group of Trp281 and another with His470, explaining the improved potency when the
dihedral twist is induced by 4-Me substitution; and 2) the hydrogen bond between the imidazole NH
and the phenol group of Tyr394. Interestingly, in addition to forcing the bioactive dihedral twist, 4-
Me also makes favourable hydrophobic contacts with some lipophilic residues in the binding pocket,
notably the isobutyl and the methyl sulfide sidechains of Leu522 and Met230. The core amide group
of this series of compounds also makes two key interactions, forming hydrogen bonds with the
guanidine group of Arg400 and the acid group of Asp384.
Figure 4. Predicted binding mode of 25 in SMO (25 and SMO ribbon are in green and key residue side
chains are in grey). Key polar interactions between the ligand and the protein residues were shown as
dotted lines. Key interacting residue were labelled.
In vivo studies with SMO inhibitors
For pharmacological evaluation, we chose Colo205 mouse xenografts as the in vivo model for
pharmacodynamics (PD) screening to measure the expression of murine Hh pathway genes and the
decrease in murine GLI (mGLI) after treatment with SMO inhibitors. Female nude mice were

implanted with 5MColo205 cells subcutaneously. Once tumors reached >200 mm3 in size, animals
were treated with a single oral dose of the treatment compound. At 1 and 8 h, mice were sacrificed,
and tumors were analyzed for mGLI levels by RT-PCR and blood plasma samples were assessed for
drug concentrations. Maximal inhibition of mGLI achieved in this model was observed at 6 and 8
hours post dose (Supporting Information, Figure S1). Summarized in Table 5 are the PK/PD results
for some of the SMO inhibitors 25, 26, 32, 38, and a few of their structurally matched pairs, as well as
the key tool compounds 1 and 5. For each compound, mGLI inhibition was measured after 8 hours of
the single dose (40 mg/kg) and normalized vs. the vehicle control group; Mouse plasma PK was
measured at two timepoints, 1 hour and 8 hours. Shown in Table 5 is also the human and mouse
plasma protein binding (PBB) free fraction to allow estimation of free concentration of compound in
mouse.
Compound 1 demonstrated excellent oral exposure in mouse and significantly decreased mGLI
mRNA expression (82%). Four compounds featuring imidazole groups (5, 25, 26, and 29) showed
different oral exposure levels as shown by plasma concentrations, with compound 5 and its 4-F
analogue 28 achieving significantly higher plasma concentration than the 4-Me analogue 25, but
nevertheless 25 showed the highest mGLI inhibition (88%). The decreased potency coupled with
poor plasma exposure of 29 resulted in no observable mGLI inhibition. Consistent with the potency
gain in the shh C3H10T1/2 differentiation assay for 4-Me analogues, two matched pairs, 15 vs. 26 and
22 vs. 27, showed notable difference in mGLI inhibition despite the similar plasma concentrations
between each matched pair. This further underlined the significance of 4-Me substitution in
enhancing the potency of this series of heteroaryl amide compounds. The pyrazole compound 32,
however, showed relatively poor exposure in mouse and as a result less inhibition of mGLI (75%).
The pyridazine compound 38, despite its higher concentration than 25, produced slightly less mGLI
inhibition. The human PPB free fractions of these compounds are generally similar, except the
noticeably lower free fraction for 1.

Table 5. Mouse GLI mRNA inhibition in a Colo205 xenograft PD model
O
R2
N
H
N
Ar
O
R1
Plasma
Concentration inhibition
(8 h) ((µM)
%
Plasma
Concentration
(1 h) (µM)
Compound
Name
Hu / mouse
PPB (% free)
R1
R2
mGLI (8
h)
1
<1.0a / 2.6
6.0 / 4.0
7.1 / 3.3
11 / -
10.3
18.8
7.5
82%
79%
88%
73%
0%
5
H
Me
F
25
28
29
15
26
21
27
32
38
H
22.3
58
1.9
20
H
Me
H
9 / -
3.3
63
0.14
16
H
9.5 / -
39%
87%
12%
81%
75%
80%
Me
H
5.5 / 1.2
19 / -
94
16
1.8
2.9
5.5
33
0.08
0.28
0.23
2.7
H
Me
Me
Me
9.5 / -
H
H
2.1 / -
6.4 / 2.9
a Compound 1 was reported to have species-dependent PPB due to its strong binding to human α-1-acid
glycoprotein (AAG).20
Based on their attractive in vivo activity, we prioritized compounds 25 and 26 for more extensive
preclinical evaluation. Both 25 and 26 were further tested in an HT29-MEF (murine embryonic
fibroblast) co-implant xenograft model for anti-tumour efficacy. The HT29 model was set up with a
suboptimal number of tumor cells (0.3 M) which were co-implanted with 1.5M MEF (GSC-6002,
Globalstem, Rockville, MD) in nude mice to model the Hh/SMO paracrine signaling pathway.
Treatment started when the average tumor volume reached approximately 85 mm3. Compounds 25
and 26 were dosed orally at 40 mg/kg twice daily for 10 days. As a result, both 25 and 26 exhibited
42% and 43% tumor growth inhibition respectively at p < 0.01 (Figure 4).

Figure 4. 25 and 26 in HT-29+MEF xenograft model showing tumor growth inhibition as single agents.
Human SMO potency and ADME properties of 25 and 26
Because the Colo205 xenograft model measured the inhibition of mGLI mRNA expression, and we
were conscious of the potential difference between mouse and human activity, we tested the respective
binding affinity of 25 and 26 to mouse and human SMO (Table 6). Both compounds showed
negligible difference in binding between these two species and demonstrated similar potency to 1 in
human cell lines (HEPM) in inhibiting the Hh pathway, in line with their excellent binding affinity to
human SMO. The potencies of 1 in these assays are also included in Table 6, illustrating that 26
possesses similar potency across these assays to 1, but 25 shows better potency across these cell-based
assays. The solubility of both 25 and 26 are low, but they were both able to be dosed as high as 100
mg/kg in rodents and still achieve dose proportional oral exposure (data not shown), indicating that
their low aqueous solubility did not limit their oral absorption, likely due to their excellent
permeability. We investigated their efflux potential and permeability using a human cell line
engineered to express the P-gp efflux pump (MDR1-LLC PK1 cells), and the efflux ratios are 5.3 and
2 for 25 and 26, respectively. The low efflux ratios indicate 25 and 26 as P-gp substrates, but the
efflux ratios are relatively low, and the permeability of both compounds as assessed by A>B drug
migration is deemed to be high (Papp A>B 7.5×10-6 cm/s and 16.5×10-6 cm/s for 25 and 26,

respectively).
Table 6. Summary of preclinical data of 1, 25, and 26
1
25
26
Mouse shh IC50 (nM)
Binding EC50 (nM) human/mouse
HPEM IC50 (nM)
4.7
1
2.6
2.0 /3.0
0.5 / 20
1.5 / 3.1
0.4
0.08
88%
0.2
mGLI inhibition (40 mg/kg)
LogD
82%
87%
2.8
6
3.1
2
3
8
Aqueous solubility pH7.4 (µM)
PPB (% free)
2.6 / < 1.0 / - / <1.0
- / 16 / - / < 1
3.3 / 6.0 / 11 / 6.7
6.6 / 18 / 1.4 / 9
31 / 24 / 4.0 / 18
1.2 / 1.0 / 7.2 / 5.5
<1 / 19 / 10 / 6
mouse/rat/dog/human
Heps Clint (µl/min/1E6)
mouse/rat/dog/human
Clearance (ml/min/kg)
mouse/rat/dog/Guinea pig
Vdss (L/kg)
5.6 / 2 / - / -
6.1 / 1.7 / 4.7 / 1.6
0.5 / 0.3 / - / -
1.0 / 1.9 / - / -
33 / 49 / - / -
1.6 / 1.2 / 0.9 / 1.8
0.7 / 1.1 / 3.3 / 1.5
75 / 100 / 100
0.4 / 0.2 / 0.5 / 0.5
0.8 / 2.4 / 1.4 / 4.2
70 / 90 / 32
mouse/rat/dog/guinea pig
t1/2 (h) mouse/rat/dog/guinea pig
% Oral bioavailability
mouse/rat/dog
CYP 3A4, 2D6, 2C9, 2C19, 1A2
IC50 (µM)
all > 20
>33
all > 20
25
2C9: 4.5
23
hERG IC50 (µM)
MDR1-LLC PK1 (A > B) Papp
(10-6 cm/s) / Efflux ratio
- / -
7.5 / 5.3
16.5 / 2
Compounds 25 and 26 have slightly different PK profiles in rodents. 25 shows moderate plasma
clearance in both mouse and rat, while 26 more resembles the rodent PK profile of 1, with remarkably
low plasma clearance. However, even though 25 showed higher clearance in both mouse and rat, its
plasma half-life (t ) is similar to 26 and 1, due to its higher volume of distribution.
1/2
Given their
similar physical properties and close structural resemblance, the difference in volume distribution
between 25 and 26 in mouse and rat can be explained by their different free fraction in PPB in these
two species, as 25 with higher free fraction in plasma produces a proportionally larger volume.
Likewise, the smaller difference in dog PPB between these two compounds also correlates to their
similar volumes in dog. Judging by their comparable human protein binding free fraction, it is

reasonable to expect the human volume of these two compounds will be similar, and considering their
small differences in human hepatocyte Cl , we can expect their human plasma half-lives are likely to
int
be in a similar range. In our hands both 25 and 26 demonstrated excellent oral bioavailability across
mice, rats, and dogs. The bioavailabilities of 1 in mice and rats were limited by its absorption
because1 demonstrated very low clearance in both species.8 We also evaluated the drug-drug
interaction potential of these compounds, and none of these three compounds showed cytochrome
P450 inhibition liability. Lastly, both 25 and 26 were evaluated in a panel of cardiac ion channel
assays; both compounds were shown to be inactive in Na+ or K+ ion channel assays but showed
moderate hERG inhibition. Given their excellent cellular potency and predicted low efficacious
concentrations in the clinic, the hERG activity did not prevent us from further preclinical safety
studies. Both 25 and 26 were subsequently nominated for further pre-clinical studies as candidate
drugs (AZD7254 and AZD8452,16 respectively).
Summary
In summary, our previous lead SMO inhibitor 5 showed an unexpected inter-species difference in its
binding affinity towards SMO between mouse and human. In the absence of protein structural
information, we methodically explored the SAR of this series of heteroarylamides and eventually
identified a subseries of SMO inhibitors with improved potency and in vivo activity in mGLI
expression inhibition. The 4-Me on the central aniline ring being ortho to the heteroaryl is critical for
the improved potency, and the potency gained from the methyl group was explained both by
introducing a twisted conformation between the two aromatic rings and its interaction with
hydrophobic residues in the SMO transmembrane binding site. These dual effects of the 4-Me group
in this subseries serves an example of the often sought-after “magic methyl” effect.21 Optimal
heteroaryl groups were also found to make polar interactions with the binding pocket residues via
hydrogen bonding interactions. Among these subseries of potent SMO inhibitors, compounds 25 and
26 were selected for their excellent in vivo activity in a Colo205 mouse xenograft model and both

compounds demonstrated good oral PK in multiple preclinical species and were predicted to have
acceptable human PK to allow once daily dosing. On the basis of the above overall assessment, both
25 and 26 advanced into preclinical safety studies. Compound 26 (AZD8452) was discontinued due
to potential cardiovascular risk found in GLP safety studies. Compound 25 (AZD7254) was late
discontinued due to shifted business priorities.
EXPERIMENTAL
General information
All solvents used were commercially available in anhydrous grade. Reagents were utilized without
further purification unless otherwise stated. Evaporation of solvent was carried out using a rotary
evaporator under reduced pressure at a bath temperature of up to 60 ºC. Temperatures are given in
degrees Celsius (ºC), and operations were carried out at room or ambient temperature, that is at a
temperature in the range of 18-30 ºC. In general, the course of reactions was followed by thin layer
chromatography or mass spectroscopy and reaction times are given for illustration only; where a
synthesis is described as being analogous to that described in a previous example, the amounts used
are the millimolar ratio equivalents to those used in the previous example. NMR data is in the form of
delta values for major diagnostic protons, given in parts per million (ppm) relative to
tetramethylsilane(TMS) as an internal standard, determined at 300 MHz or 400 MHz using deuterated
solvent. Analytical mass spectra were run with an electron energy of 70 eV in the chemical ionization
(CI) mode using a direct exposure probe; where indicated ionization was effected by electron impact
(EI), electrospray (ESP), or atmospheric pressure chemical ionization (APCI); values for m/z are
given; generally, only ions which indicate the parent mass are reported. Unless otherwise indicated, all
final compounds were purified to ≥95% purity, as assessed by analytical HPLC using an Agilent 1100
equipped with Waters columns (Atlantis T3, 2.1 × 50 mm, 3 μm or Atlantis C18, 2.1 × 50 mm, 5 μm)
eluted for >10 min with a gradient mixture of water and acetonitrile with either formic acid or
ammonium acetate added as a modifier, monitored at wavelengths of 220, 254, and 280 nm. All

experimental activities involving animals were carried out in accordance with AstraZeneca animal
welfare protocols, which are consistent with The American Chemical Society Publications rules and
ethical guidelines.
5-bromo-4-chloro-2-methylaniline (10a)
In a 200-mL round-bottomed flask was placed 1-bromo-2-chloro-4-methyl-5-nitrobenzene (4 g, 16 mmol)
and FeCl3 in silica gel (5%, 11.2 g) in MeOH (50 mL). The reaction mixture was heated to 70 C for 15
min and then hydrazine monohydrate (8.8 mL, 192 mmol) was slowly added and the reaction mixture was
refluxed overnight. After cooled to RT, the mixture was filtered, and concentrated in vacuo to afford the
1
title compound (3.4 g) in 95% yield. H NMR (DMSO-d6) δ 1.98 (s, 3 H), 6.90 (s, 1 H), 7.10 (s, 1 H).
N-(5-bromo-4-chloro-2-methylphenyl)-4-(pyridin-2-ylmethoxy)benzamide (11a). In a 100-mL
round-bottomed flask was dissolved 4-(pyridin-2-ylmethoxy)benzoic acid (800 mg, 4 mmol) in SOCl2 (6
mL). The solution was stirred for 1 h at RT. The solution was concentrated in vacuo to give 4-(pyridin-
2-ylmethoxy)benzoyl chloride. The crude product was dissolved in DCM (10 mL) followed by the
addition of 5-bromo-4-chloro-2-methylaniline (500 mg, 2.27 mmol), pyridine (5 mL), and TEA (10 mL).
The reaction mixture was heated to 50 C and stirred for 2h. The mixture was concentrated in vacuo and
the crude product was purified by ISCO MPLC (20-33% EtOAc/petroleum ether) to afford the title
1
compound (270 mg) in 28% yield. H NMR (DMSO-d6) δ 2.05 (s, 3 H), 2.25 (s, 2 H), 7.13 (m, 2 H), 7.34
(m, 1 H), 7.54 (m, 2 H), 7.77 (s, 1 H), 7.84 (m, 1 H), 7.92 (m, 2 H), 8.58 (m, 1 H), 9.81 (s, 1 H).
N-(4-chloro-2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-4-(pyridin-2-
ylmethoxy)benzamide (12a). In a 10 mL vial was added 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-
dioxaborolane) (0.441 g, 1.74 mmol), N-(5-bromo-4-chloro-2-methylphenyl)-4-(pyridin-2-
ylmethoxy)benzamide (0.5 g, 1.16 mmol), and KOAc (0.341 g, 3.47 mmol) in dioxane (80 mL) to give a

colorless suspension. Nitrogen was bubbled in for 20 min before Pd(PPh3)4 (0.134 g, 0.12 mmol) was
added. The reaction was stirred at 115 ºC in a microwave for 5h. After concentration under reduced
pressure, the crude product was purified by ISCO MPLC (0-5% MeOH in DCM) to give the title
1
compound. H NMR (DMSO-d6) δ 1.07 (s, 6 H), 1.16 (s, 6 H), 2.22 (s, 3 H), 5.28 (s, 2 H), 7.16 (d, 2 H),
7.36 (s, 1 H), 7.54 (d, 1 H), 7.59 (s, 1 H), 7.63 (m, 1 H), 7.85 (m, 1 H), 7.96 (m, 2 H), 8.60 (d, 1 H), 9.82
(s, 1 H).
N-[4-chloro-5-(1H-imidazol-2-yl)-2-methylphenyl]-4-(pyridin-2-ylmethoxy)benzamide (14). In a 10
mL vial was added N-(4-chloro-2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-4-
(pyridin-2-ylmethoxy)benzamide (0.15 g, 0.31 mmol), 2-bromo-1H-imidazole (0.070 g, 0.5 mmol), and
KOAc (0.077 g, 0.78 mmol) in dioxane (3.0 mL) to give a brown suspension. The reaction mixture was
diluted with water (1.0 mL). Nitrogen was bubbled in for 20 min before Pd(PPh3)4 (0.036 g, 0.03 mmol)
was added. The reaction was heated using a microwave at 130 °C for 2 h. After concentration in vacuo,
the residue was diluted with MeOH (0.5 mL) and DMSO (0.5 mL). The solution was filtered and purified
by Gilson HPLC (5-80% MeCN/0.1% TFA in water) to give the title compound as the product (0.012 g,
9% yield). 1H NMR (DMSO-d6) 2.37 (s, 3 H), 5.34 (s, 2 H), 7.20 (d, 2 H), 7.43 - 7.51 (m, 1 H), 7.63 (d, 1
H), 7.73 (s, 1 H), 7.86 - 7.91 (m, 3 H), 7.93 - 8.04 (m, 3 H), 8.66 (d, 1 H), 10.04 (s, 1 H), 14.85 (br s, 2
H); MS (M+H+) = 419.
Compounds 16, 18, and 20 were synthesized using the similar reaction as described in the synthesis of
compound 14 from intermediate 12a.
N-[4-chloro-2-methyl-5-(1-methyl-1H-imidazol-4-yl)phenyl]-4-(pyridin-2-ylmethoxy)benzamide
(16). 1H NMR (DMSO-d6) 2.31 (s, 3 H), 3.92 (s, 3 H), 5.34 (s, 2 H), 7.19 (d, 2 H), 7.44 -7.52 (m, 1 H),
7.60 - 7.68 (m, 2 H), 7.78 (s, 1 H), 7.94 - 8.04 (m, 3 H), 8.12 (s, 1 H), 8.66 (d, 1 H), 9.19 (s, 1 H), 9.98 (s,

1 H); MS (M+H+) = 433.
N-[4-chloro-5-(1,2-dimethyl-1H-imidazol-4-yl)-2-methylphenyl]-4-(pyridin-2-ylmethoxy)benzamide
1
(18). H NMR (DMSO-d6) 2.30 (s, 3 H), 2.63 (s, 3 H), 3.80 (s, 3 H), 5.34 (s, 2 H), 7.19 (d, 2 H), 7.47 (m,
1 H), 7.63 (m, 2 H), 7.77 (s, 1 H), 7.97 (m, 3 H), 8.07 (s, 1 H), 8.65 (d, 1 H), 9.98 (s, 1 H); MS (M+H+) =
447.
N-[4-chloro-2-methyl-5-(1-methyl-1H-imidazol-2-yl)phenyl]-4-(pyridin-2-ylmethoxy)benzamide
1
(20). H NMR (DMSO-d6) δ 2.38 (s, 3 H), 3.72 (s, 3 H), 5.33 (s, 2 H), 7.19 (d, 2 H), 7.46 (m, 1 H), 7.62
(d, 1 H), 7.76 (s, 1 H), 7.95 (m, 6 H), 8.65 (d, 1 H), 10.04 (s, 1 H). MS (M+H+) = 433.
N-(5-bromo-2,4-dimethyl-phenyl)-4-(2-pyridylmethoxy)benzamide (11b). In a round-bottomed flask
was placed 5-bromo-2,4-dimethylaniline (5 g, 25 mmol), 4-(pyridin-2-ylmethoxy)benzoic acid (6.3 g,
26.5 mmol), and DIPEA (8.9 mL, 50 mmol) in DMF (50 mL). The mixture was cooled to 0 °C with a
water-ice bath before HATU (11.5 g, 30 mmol) was added. The mixture was warmed to RT and stirred
overnight. To the reaction solution was added water (200 mL). The precipitate was collected by filtration
1
to afford the title compound (4 g, 41% yield). H NMR (DMSO-d6) δ 2.14 (s, 3 H), 2.27 (s, 3 H), 5.26 (s,
2 H), 7.13 (d, 2 H), 7.23 (s, 1 H), 7.34 (m, 1 H), 7.52 (t, 1 H), 7.56 (s, 1 H), 7.82 (m, 1 H), 7.92 (d, 2 H),
8.57 (m, 1 H).
N-(2,4-dimethyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-4-(pyridin-2-
ylmethoxy)benzamide (12b). In a round-bottomed flask was added N-(5-bromo-2,4-dimethylphenyl)-4-
(pyridin-2-ylmethoxy)benzamide (4g, 9.73mmol), bis(pinacolato)diboron (2.96 g,11.6mmol), and KOAc
(2.86 g, 29.2 mmol) in dioxane (50 mL) to give a suspension. To the mixture was added PdCl2(dppf)
(400 mg). The reaction was stirred at 80 C under a nitrogen atmosphere overnight. The reaction mixture
was concentrated in vacuo and water (80 mL) was added. The mixture was extracted with EtOAc (2 X 30

mL) and the combined organic layers were dried (Na2SO4), then concentrated in vacuo to afford the crude
product which was purified by ISCO MPLC (1% MeOH/DCM) to give the title compound (2.3 g, 52%
1
yield). H NMR (DMSO-d6) δ 1.26 (s, 12 H), 2.15 (s, 3 H), 2.41 (s, 3 H), 5.25 (s, 2 H), 7.06 (s, 1 H), 7.12
(m, 2 H), 7.35 (m, 1 H), 7.51 (m, 2 H), 7.81 (m, 1 H), 7.94 (m, 2 H), 8.58 (m, 1 H), 9.71 (s, 1 H).
N-[5-(1H-imidazol-2-yl)-2,4-dimethylphenyl]-4-(pyridin-2-ylmethoxy)benzamide (25). In a
10 mL vial was added N-(2,4-dimethyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-
4-(pyridin-2-ylmethoxy)benzamide (0.25 g, 0.55 mmol), 2-bromo-1H-imidazole (0.12 g, 0.82
mmol), and Cs2CO3 (0.44 g, 1.36 mmol) in dioxane (5 mL) to give a brown suspension. The
reaction mixture was diluted with water (2 mL). Nitrogen was bubbled in for 20 min before
Pd(PPh3)4 (0.063 g, 0.05 mmol) was added. The reaction was heated at 110 ºC for 4h under
microwave conditions. The reaction mixture was concentrated under reduced pressure. The
residue was purified by Gilson HPLC (MeCN/0.1% TFA in water). To the purified product was
added HCl in Et2O (0.5 mL, 1 mmol). The mixture was concentrated in vacuo to give the HCl
1
salt of the title compound (10 mg, 4.2%). H NMR (DMSO-d6)  2.31 (s, 3 H), 2.36 (s, 3 H),
5.32 (s, 2 H), 7.18 (d, 2 H), 7.39 (s, 1 H), 7.45 (br s, 1 H), 7.61 (s,2 H), 7.84 (s, 2 H), 7.96 (m, 3
H), 8.63 (d, 1 H), 9.90 (s, 1 H), 14.54 ( br s, 1 H); MS (M+H+) = 399.
Compounds below were synthesize following the general synthetic route as shown above for 25.
N-[2,4-dimethyl-5-(1-methyl-1H-imidazol-4-yl)phenyl]-4-(pyridin-2-ylmethoxy)benzamide (26). 1H
NMR (DMSO-d6) 2.26 (s, 3 H), 2.37 (s, 3 H), 3.92 (s, 3 H), 5.37 (s, 2 H), 7.18 (d, 2 H), 7.30 (s, 1 H), 7.53
(m, 2 H), 7.69 (d, 1 H), 7.91 (s, 1 H), 8.02 (m, 3 H), 8.69 (d, 1 H), 9.21 (s, 1 H), 9.87 (s, 1 H); MS
(M+H+) = 413.

N-[5-(1,2-dimethyl-1H-imidazol-5-yl)-2,4-dimethylphenyl]-4-(pyridin-2-ylmethoxy)benzamide (27).
1H NMR (DMSO-d6) 2.16 (s, 3 H), 2.28 (s, 3 H), 2.62 (m, 3 H), 3.46 (s, 3 H), 5.33 (s, 2 H), 7.17 (d, 2 H),
7.33 (d, 2 H), 7.47 (m, 1 H), 7.63 (m, 2 H), 7.97 (m, 3 H), 8.65 (d, 1 H), 9.83 (s, 1 H), 14.38 ( br s, 1 H);
MS (M+H+) = 427.
1
N-[5-(1H-benzimidazol-2-yl)-2,4-dimethylphenyl]-4-(pyridin-2-ylmethoxy)benzamide (30). H NMR
(DMSO-d6) 2.35 (s, 3 H), 2.55 (s, 3 H), 5.37 (s, 2 H), 7.20 (d, 2 H), 7.46 (s, 1 H), 7.52 (m, 1 H), 7.60 (m, 2
H), 7.68 (d, 1 H), 7.83 (s, 1 H), 7.87 (m, 2 H), 8.01 (m, 3 H), 8.68 (d, 1 H), 9.99 (s, 1 H); MS (M+H+) =
449.
N-[2,4-dimethyl-5-(4-methyl-1,3-thiazol-2-yl)phenyl]-4-(pyridin-2-ylmethoxy)benzamide
(31). 1H NMR (DMSO-d6) 9.81 (br. s., 1 H), 8.68 (d, 1 H), 8.03 - 8.28 (m, 1 H), 7.94 (d, 2 H), 7.65
- 7.80 (m, 2 H), 7.50 - 7.65 (m, 1 H), 7.31 (s, 1 H), 7.19 (s, 1 H), 7.12 (d, 2 H), 5.36 (s, 2 H), 2.46
(br. s., 3 H), 2.37 (s, 3 H), 2.17 (s, 3 H); MS (M+H+) = 430.
1
N-[2,4-dimethyl-5-(3-methyl-1H-pyrazol-4-yl)phenyl]-4-(pyridin-2-ylmethoxy)benzamide (32). H
NMR (DMSO-d6) 2.11 (s, 3 H), 2.16 (d, 6 H), 5.42 (s, 2 H), 7.02 - 7.18 (m, 5 H), 7.63 - 7.75 (m, 1 H),
7.83 (d, 1 H), 7.88 - 7.99 (m, 3 H), 8.22 (t,1 H), 8.73 (d, 1 H), 9.72 (s, 1 H); MS (M+H+) = 413.
N-[2,4-dimethyl-5-(1,3,5-trimethyl-1H-pyrazol-4-yl)phenyl]-4-(pyridin-2-ylmethoxy)benzamide
1
(33). H NMR (DMSO-d6) 1.94 - 2.11 (m, 9 H), 2.22 (s, 3 H), 3.78 (d, 3 H), 5.46 (d, 2 H), 7.03 (s, 1 H),
7.18 (d, 3 H), 7.72 (br. s., 1 H), 7.86 (br. s., 1 H), 7.99 (d, 2 H), 8.26 (br. s., 1 H), 8.79 (d, 1 H), 9.76 (br. s.,
1 H); MS (M+H+) = 441.
N-(2,4-dimethyl-5-pyridin-2-ylphenyl)-4-(pyridin-2-ylmethoxy)benzamide (34) 2.24 (d, 6 H), 5.34 (s,
2 H), 7.12 (d, 2 H), 7.26 (s, 1 H), 7.44 (s, 1 H), 7.54 (br. s., 1 H), 7.69 (d, 1 H), 7.88 - 8.01 (m, 3 H), 8.06

(s, 1 H), 8.40 (s, 1 H), 8.66 (d, 1 H), 8.80 (d, 1 H), 9.86 (s, 1 H); MS (M+H+) = 410.
N-(2,4-dimethyl-5-pyridin-3-ylphenyl)-4-(pyridin-2-ylmethoxy)benzamide
(35). 1H NMR (DMSO-d6) 2.20 (d, 6 H), 5.36 (s, 2 H), 7.12 (m, 2 H), 7.23 (s, 1 H), 7.31 (s, 1 H), 7.48 -
7.64 (m, 1 H), 7.72 (d, 1 H), 7.94 (m, 2 H),8.01 (m, 1 H),8.05 - 8.16 (m, 1 H),8.48 (d, 1 H),8.67 (d, 1 H),8.77
- 8.94 (m, 2 H),9.83 (s, 1 H); MS (M+H+) = 410.
N-[5-(5-aminopyridin-2-yl)-2,4-dimethylphenyl]-4-(pyridin-2-ylmethoxy)benzamide (36). 1H NMR
(DMSO-d6) 2.21 (s, 6 H), 5.34 (s, 2 H), 7.12 (d, 2 H), 7.24 (s, 1 H), 7.34 (s, 1 H), 7.54 (d, 1 H), 7.60 -
7.75 (m, 3 H), 7.87 - 8.01 (m, 3 H), 8.06 (t, 1 H), 8.66 (d, 1 H), 9.84 (s, 1 H); MS (M+H+) = 425.
1
N-[5-(6-aminopyridin-2-yl)-2,4-dimethylphenyl]-4-(pyridin-2-ylmethoxy)benzamide (37). H NMR
(DMSO-d6) 2.23 (d, 6 H), 5.40 (s, 2 H),6.78 (d, 1 H),6.95 (d,1 H),7.13 (d, 2 H),7.18 - 7.29 (m, 1 H),7.36 (s, 1
H),7.52 - 7.70 (m, 1 H),7.79 (d, 1 H),7.85 - 8.00 (m, 3 H),8.00 - 8.34 (m, 3 H),8.71 (d, 1 H),9.86 (s, 1 H),14.06 (br.
s., 1 H); MS (M+H+) = 425.
N-(5-(6-aminopyridazin-3-yl)-2,4-dimethylphenyl)-4-(pyridin-2-ylmethoxy)benzamide (38).
1H
NMR (DMSO-d6) 2.23 (s, 3 H), 2.28 (s, 3 H), 5.28 (s, 2 H), 6.41 (s, 2 H), 6.83 (d, 1 H), 7.16 (m, 3 H), 7.31
(s, 1H), 7.39 (m, 2 H), 7.54 (d, 1 H), 7.85 (m, 1 H), 7.96 (d, 2 H), 8.60 (m, 1 H), 9.76 (s, 1 H); MS (M+H+)
= 426.
QM calculations: All QM calculations were performed using Jaguar,22 embedded within Schrodinger’s
Maestro package. Torsion profiles were generated using the relaxed coordinate scan protocol, using
intervals of 10o between 0o and 360o. Calculations were performed at the DFT level theory, using the B3-
LYP functional18 and a 6-31G** basis set.