Rational Design and Optimization of a Novel Class of Macrocyclic Apoptosis Signal-Regulating Kinase 1 Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.9b01206

Structural analysis of a known apoptosis signal-regulating kinase 1 (ASK1) inhibitor bound to its kinase domain led to the design and synthesis of the novel macrocyclic inhibitor 8 (cell IC₅₀ = 1.2 μM). The profile of this compound was optimized for CNS penetration following two independent strategies: a rational design approach leading to 19 and a parallel synthesis approach leading to 26. Both analogs are potent ASK1 inhibitors in biochemical and cellular assays (19, cell IC₅₀ = 95 nM; 26, cell IC₅₀ = 123 nM) and have moderate to low efflux ratio (ER) in an MDR1-MDCK assay (19, ER = 5.2; 26, ER = 1.5). In vivo PK studies revealed that inhibitor 19 had moderate CNS penetration (K_puu = 0.17) and analog 26 had high CNS penetration (K_puu = 1.0).

Full text of DOI:10.1021/acs.jmedchem.9b01206

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Article
Rational Design and Optimization of a
Novel Class of Macrocyclic ASK1 Inhibitors
Martin Himmelbauer, Zhili Xin, John Howard Jones, Istvan Enyedy, Kristopher King, Douglas
J Marcotte, Paramasivam Murugan, Joseph Santoro, Thomas Hesson, Kerri Spilker,
Joshua L Johnson, Michael J. Luzzio, Rab Gilfillan, and Felix Gonzalez-Lopez de Turiso
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01206 • Publication Date (Web): 11 Nov 2019
Downloaded from pubs.acs.org on November 12, 2019
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Rational Design and Optimization of a Novel Class of
Macrocyclic ASK1 Inhibitors
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Martin K. Himmelbauer,a Zhili Xin,a Howard Jones,a Istvan Enyedy,a Kristopher King,b
Douglas J. Marcotte,c Paramasivam Murugan,d Joseph C. Santoro,d Thomas Hesson,d Kerri
Spilker,c Joshua L. Johnson,b Michael J. Luzzio,a Rab Gilfillan,a and Felix Gonzalez-Lopez de
Turiso.a,*
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aMedicinal Chemistry; bDrug Metabolism and Pharmacokinetics;cPhysical Biochemistry and Molecular Design;
dBioassays. Biotherapeutic and Medicinal Science. Biogen. 225 Binney Street, Cambridge, MA 02142. United
States.
KEYWORDS: Apoptosis signal-regulating kinase
neurodegenerative diseases.
1
(ASK1), rational design, parallel synthesis,
ABSTRACT: Structural analysis of a known ASK1 inhibitor bound to its kinase domain led to the design and synthesis
of the novel macrocyclic inhibitor 8 (cell IC50 = 1.2 M). The profile of this compound was optimized for CNS
penetration following two independent strategies: a rational design approach leading to 19 and a parallel synthesis
approach leading to 26. Both analogs are potent ASK1 inhibitors in biochemical and cellular assays (19, cell IC50 = 95
nM; 26, cell IC50 = 123 nM) and have moderate to low efflux ratio (ER) in an MDR1-MDCK assay (19, ER = 5.2; 26, ER
= 1.5). In vivo PK studies revealed that inhibitor 19 had moderate CNS penetration (Kpuu = 0.17) and analog 26 had
high CNS penetration (Kpuu = 1.0).
INTRODUCTION
Apoptosis signal-regulating kinase 1 (ASK1, MAP3K5) is a mitogen-activated protein kinase kinase kinase (MAP3K)
that plays a central role in the cellular stress response by modulating inflammation and apoptosis.1 Mechanistically,
activation of ASK1 leads to downstream phosphorylation of the mitogen-activated protein kinase kinases (MAP2Ks)
MKK3/6 and MKK4/7 which in turn phosphorylate the downstream mitogen-activated protein kinases (MAPKs) p38
and c-Jun N-terminal kinase (JNK) respectively.2 These two kinases are ultimately responsible for the inflammatory
and apoptotic stress response and modulation of ASK1 activity might be relevant in the treatment of neurological
disorders including amyotrophic lateral sclerosis (ALS),3 multiple sclerosis (MS),4 ischemic stroke,5 Parkinson’s
disease (PD)6 and Alzheimer’s disease (AD).7 To test the potential benefit of modulating this pathway in neurological
disease, several research groups have performed in vivo studies with small molecules designed to inhibit the kinase
activity of ASK1 in the CNS.8 Specifically, two ASK1 inhibitors: K811 (1) and K812 (2) (Figure 1) have been reported
to be efficacious in a SOD1 ALS model of disease.3 In this study, chronic treatment of SOD1G93A-transgenic mouse
with 1 (at 100 mg/kg) or 2 (at 30 mg/kg) starting at disease onset (week 28), led to increased survival and decreased
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motor-neuron death in the spinal cord of the treated animals.9 In a separate report, the CNS penetrant ASK1 inhibitor
MSC20232964A (3) was tested in an experimental autoimmune encephalomyelitis (EAE) model of MS demonstrating
that animals treated with 3 (at 30 mg/kg) had reduced inflammation in the spinal cord and the optic nerve thus
positioning ASK1 as a potential target for the treatment of MS.4 Despite this data, the hypothesis that modulation of
the ASK1 pathway might be beneficial for the treatment of neurological disease has not been tested in the clinic and
the main focus has been centered on peripheral human disease.10 Specifically, the ASK1 inhibitor Selonsertib (GS-
4997, 4), developed by Gilead Sciences, is being tested in Phase II clinical trials for the treatment of liver fibrosis.11
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O
CF3
N
O
N
N
N
O
F
H
O
NH
O
N
N
N
N
O
N
N
N
H
N
O
N
HN
N
X
1, X=N, K811
2, X=C, K812
3, MSC2032964A
4, Selonsertib (GS-4997)
IC50 = 5.9 nM
IC50 = 18 nM
IC50 = 9 nM
IC50 = 131 nM
Cell IC50 = 20 nM
Papp (ER)= 13 10-6 cm/s (68)
Cell IC50 = 869 nM
Papp (ER)= 0.6 10-6 cm/s (19)
Cell IC50 = 277 nM
Papp (ER)= 1.6 10-6 cm/s (0.5)
Cell IC50 = 13 M
Papp (ER)= 13 10-6 cm/s (2.3)
Cl/Clu (L/hr/kg) = 0.18/14.7
Figure 1. Profile of selected ASK1 inhibitors
RESULTS AND DISCUSSION
With the goal of identifying a novel series of ASK1 inhibitors with suitable profile for the treatment of human
neurological disease, we initiated a medicinal chemistry program by profiling a selected number of known
compounds from the literature (Figure 1). Among these, compound 4 had the best overall profile, with excellent
biochemical (IC50 = 5.9 nM)12 and cellular potency (IC50 = 20 nM).13 This analog also had favorable pharmacokinetic
(PK)14 profile (rat Cl/Clu = 0.18/14.7 L/hr/kg)15 but, in contrast to compounds 2 and 3, it was a substrate of P-
glycoprotein (P-gp) in an MDR1-MDCK assay as evidenced by its high efflux ratio (ER) making it unsuitable for CNS
indications.16,17 To enable a structure-based drug design approach, the X-ray cocrystal structure of compound 4
bound to the ASK1 kinase domain was elucidated.18 This X-ray confirmed the previously proposed binding mode19 in
which 4 interacts via the carbonyl group of the amide with the back-bone NH of Val757 in the hinge binder region of
ASK1. In addition, a hydrogen bond interaction between one of the N atoms of the triazole ring in 4 and the catalytic Lys
(709) was observed while the cyclopropyl-imidazole substituent appeared to be solvent exposed.20 An intra-molecular
hydrogen bond between the amide NH and the fluoro-substituent in its ortho-position also contributed to locking the
conformation of 4 into this favorable binding pose.
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Hinge
Binder
Val 757
6
7
8
9
Catalytic
Lysine
N
O
N
N
Lys 709
N
N
N
H
N
F
10
11
12
13
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Solvent
exposed
region
4 (GS-4997)
a)
b)
Figure 2. a) Cocrystal structure of 4, (GS-4997, green) with the kinase domain of ASK1 highlighting key interactions in the active
site (PDB code: 6OYT); b) Schematic representation of binding mode of GS-4997 to the kinase domain of ASK1. Solvent exposed
region in blue
At the outset the design strategy involved optimization of compound 4 for CNS penetration by deconstructing the
solvent exposed substituents and attempting to regain binding affinity via macrocyclization of the resulting
simplified pharmacophore (i.e. analog 8, Figure 3). Conceptually, this approach relied on the observation that the
phenyl group and triazole ring are in close spatial proximity and on the insight that macrocyclic structures typically
have reduced conformational freedom which can result in ligands with increased binding affinity.21
N
O
O
O
O
F
N
N
N
Cyclization
N
Deconstruct
N
N
N
N
N
N
N
N
N
H
H
H
N
N
N
solvent
exposed
substituents
R
n = 0-2
R = H, Me
n
R
Solvent
exposed
region
4 (GS-4997)
5, R = F
8, n=1, R = H
11, n=1, R=Me
6, R = OMe
Figure 3. Strategy for the design of novel ASK1 inhibitors
To test this hypothesis, initial truncation of the solvent exposed imidazole and methyl groups in 4 revealed an analog
(5) that retained some potency in the biochemical assay and had low ER (Table 1, IC50 = 76 nM, ER = 1.6). Subsequent
evaluation of functional groups with the ability to form an intramolecular hydrogen bond with the amide and link
the phenyl and triazole rings was performed. These studies showed that replacement of the fluoro substituent in 5
with a methoxy group led to an analog (6) with improved potency (Table 1, cell IC50 = 299 nM). This modification also
enabled implementation of our macrocyclization strategy and compounds with a range of differently sized tethers
were synthesized (Table 1, analogs 7-9). It was found that all the macrocyclic compounds synthesized were potent
ASK1 inhibitors in a biochemical assay (IC50 = 11-161 nM, Table 1, entries 3-5) and the 14-member inhibitor 8 had
the best balance of cellular potency (IC50 = 1.2 M) and ER (2.9). Comparison of analog 8 with its corresponding open
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form (10), revealed that the alkoxy-tether had contributed to an 8-fold improvement in biochemical potency (Table
1, compound 8, IC50 = 11 nM vs compound 10, IC50 = 92 nM).
6
7
8
9
Table 1. Identification of new ASK1 pharmacophore
O
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N
MDR1-
MDCK
Cell
IC50
RLM/HLM
(mL/min/kg
)
RPPB/
HPPB
(fu)
N
N
Biochem
N
H
Analog
N
MW/PSA/clogP
IC50 (nM)a
R1
R2
(nM)a,b
Papp/ERc
R1
F
R2
31.1/22.
3
5
6
iPr
iPr
76
-
8164
299
14.8/1.6
6.1/4.4
3.8/13.6
8.0/2.9
6.2/1.3
9.7/1.8
33/<5
36/34
325/73/1.8
337/82/2.1
321/82/1.5
335/82/2.1
349/82/2.6
309/82/1.3
OMe
10.8/2.7
7
161
11
20
92
6065
1211
1776
3735
201/55
130/71
141/56
82/17
54/-
O
8
8.2/3.2
1.6/0.14
24/8.3
O
9
O
10
OMe
Me
aResults represent the geometric mean of a minimum of two determinations. bInhibition of ASK1 autophosphorylation.
cMDR1-MDCK human P-gp transfected cell line. Papp – Apparent permeability Apical-to-Basolateral (10-6cm/s); ER = B-A/A-
B efflux ratio
To mimic the role of the isopropyl group present in analog 6, a methyl group  to the triazole was first introduced in
the macrocyclic analog 8 (Figure 3, analog 11). This compound was synthesized as the racemic form and when the
enantiomers were separated (Table 2, analogs 11 and 12) a 4-fold improvement in cellular potency was observed
with the more potent enantiomer (11, cell IC50 = 73 nM vs 6, cell IC50 = 299 nM). The absolute stereochemistry of
compound 11 was determined by an X-ray with human ASK1 (Figure 4). This cocrystal structure also revealed a
binding mode in which the same H-bond interactions of the carbonyl group of the amide to the backbone NH of
Val757 and the triazole N to the catalytic Lys observed in 4 were also present with 11 (Figure 4).
Figure 4. Overlay of the cocrystal structure of 11 (magenta) with 4, (GS-4997, green) in the kinase domain of ASK1. (PDB code:
6OYW)
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To systematically evaluate the impact of substituting other positions of the tether in analog 8, all the methylated
compounds were synthesized as the racemates and the enantiomers were separated (Table 2, analogs 13-15, only
the more potent enantiomers are shown). This optimization effort revealed that analog 13, in which the methyl group
had been incorporated at the  position of the triazole, also had improved cellular potency relative to 6 (Table 2, cell
IC50 = 232 nM).
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11
12
13
14
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16
17
18
19
20
21
22
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43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 2. Optimization of the tether
Cell
IC50
RPPB/
HPPB
(fu)
Biochem.
IC50 (nM)a
RLM/HLM
(mL/min/kg)
MDR1-MDCK
Papp/ERc
Analog
8
Structure
MW/PSA/clogP
335/82/2.1
(nM)a,b
O
N
N
N
H
N
N
11
-
1210
299
8.0/2.9
6.1/4.4
130/71
36/34
70/78
N
N
N
N
O
O
10.8/2.
7
N
H
6
337/82/2.1
349/82/2.6
O
O
N
N
H
N
11
4
73
6.4/3.7
109/79
4.5/2.4
N
N
O
O
N
N
N
N
H
N
N
N
12
13
350
14
5152
232
8.4/2.8
9.4/3.5
92/27
74/34
4.8/4.1
-
349/82/2.6
349/82/2.6
N
N
N
N
N
N
O
O
N
H
O
O
N
H
14d
15d
13
351
3.7/3.5
66/64
3.5/1.2
349/82/2.6
O
O
N
N
H
N
165
969
4.6/6.8
168/12
2.4/3.1
349/82/2.6
N
N
O
aResults represent the geometric mean of a minimum of two determinations. bInhibition of ASK1 autophosphorylation.
cMDR1-MDCK human P-gp transfected cell line. Papp – Apparent permeability Apical-to-Basolateral (10-6cm/s); ER = B-A/A-
B efflux ratio. dThe compounds were synthesized as racemates and the enantiomers were separated and tested. Only the
more potent enantiomer is shown. The absolute stereochemistry of compounds 13-15 was arbitrarily assigned.
Despite the significantly improved properties of analogs 11 and 13, these macrocyclic inhibitors suffered from poor
microsomal stability. Comprehensive metabolite profiling of the parent unsubstituted compound 8 revealed that
after one-hour incubation in rat or human liver microsomes the main metabolic soft spots were in the salicylate and
butylene linker (in rat liver microsomes) or in the amide bond (in human liver microsomes). To block the metabolic
soft spots in the salicylate, analogs 16 and 17, in which different positions of the phenyl group had been fluorinated,
were synthesized (Table 3). Notably, both compounds had improved microsomal stability and cellular potency
relative to analog 8. A follow up rat PK experiment showed that analog 17 had superior in vivo clearance relative to
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16 (and 8) and this substitution pattern was used in subsequent optimization efforts. Having improved the PK
properties of compound 8, introduction of the methyl group at both the  or  position of the triazole in 17 was
carried out. It was found that both analogs had good cellular potency (18, cell IC50 = 43 nM; 19, cell IC50 = 95 nM) and
good in vivo clearance (compound 18, Cl/Clu = 0.82/18 L/hr/kg; compound 19, Cl/Clu = 0.5/30 L/hr/kg) while
retaining a moderate ER (compound 18, ER = 4.9; compound 19, ER = 5.2).22
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 3. Improving microsomal stability by fluorination
MDR1-
MDCK
Biochem.
IC50
RPPB/
HPPB
(fu)
Cell
RLM/HLM
(mL/min/kg)
Cl/Clu
MW/PSA/
clogP
Analog
8
Structure
IC50 (nM)b
[L/hr/kg]d
(nM)a
Papp/ERc
O
335/82/2.
1
N
N
H
N
11
17
1210
658
8.0/2.9
5.7/3.1
130/71
75/21
8.2/3.2
3.6/2.8
2.9/35
N
N
O
O
O
O
353/82/2.
2
F
N
N
H
N
16
2.06/57
N
N
353/82/2.
2
N
N
H
N
17
18e
19e
8
7
8
867
43
8.0/2.6
4.5/4.9
3.6/5.2
66/23
70/20
90/16
3.3/1.1
4.4/3.7
2/0.3
1.06/32
0.82/18
0.5/30
N
N
F
F
O
O
367/82/2.
8
N
N
H
N
N
N
N
O
O
367/82/2.
8
N
N
H
N
95
N
F
O
aResults represent the geometric mean of a minimum of two determinations. bInhibition of ASK1 autophosphorylation.
cMDR1-MDCK human P-gp transfected cell line. Papp – Apparent permeability Apical-to-Basolateral (10-6cm/s); ER = B-A/A-
d
B efflux ratio. IV dosing using a DMA:EtOH:PG:water (1:1:3:5) formulation for compound 8 and DMSO:PG (1:1) for
e
analogs 16-19. The absolute stereochemistry was assigned by VCD experiments using the less potent enantiomer (see
supporting information).
Concurrent with this rational design effort, an alternative approach that relied on the parallel synthesis of a small
library of compounds starting with bromo intermediate 20 was pursued. The structure-activity relationship (SAR)
for some representative compounds from these studies is summarized in Table 4 and it showed that introduction of
a morpholine or a 2-oxa-6-azaspiro[3,3]heptane substituent onto the salicylate led to compounds with improved
potency and microsomal stability (Table 4). Unfortunately, despite the improved microsomal stability, analogs 21
and 22 had high in vivo clearance in rodents (analog 21, rat Cl = 4.1 L/hr/kg and analog 22, rat Cl = 3.0 L/hr/kg).
Compounds with a sulfone group intended to establish an interaction with Gln756 led to analogs that were potent in
a biochemical assay but had poor cellular potency (i.e. analog 23), likely due to diminished permeability.23 In contrast,
introduction of a substituted imidazole ring (Table 4, compound 24) led to a compound with good potency in
biochemical and cellular assays (IC50 = 4 nM and cell IC50 = 28 nM), which also had improved in vivo PK (Cl/Clu =
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0.65/74 L/hr/kg). This analog was found to be a P-gp substrate (24, ER = 69), thus limiting its ability to enter the
CNS and making it a good candidate for targeting therapeutic indications for which brain penetration is not desired.24
Interestingly, the pyrazole analog 25 was found to be one of the more potent analogs in the cellular assay (IC50 = 25
nM) and analog 26, on which a dimethylamino group attached to the pyrazole had been introduced, had the lowest
ER in this series (analog 26, ER = 1.5). Surprisingly, attempts to combine the best features of the optimized
macrocyclic structures (from Table 3) with the optimized substituents in Table 4 did not lead to compounds with
improved CNS-like properties (i.e., analog 27, Table 4).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 4. Parallel synthesis effort
RLM/
MDR1-
MDCK
Biochem.
IC50
Cell
IC50
RPPB/
HPPB
(fu)
HLM
(mL/min
/kg)
Cl/Clu
MW/PSA/
clogP
Analog
Structure
[L/hr/kg]d
(nM)a
(nM)b
Papp/ERc
O
Br
N
N
N
H
N
20
21
22
23
24
25
26
27 e
31
8
-
0.7/1.4
1.6/21
150/7.4
78/13
68/9
0.7/0.6
4.0/-
-/-
4.1/104
3.0/50
-/-
414/82/3.0
420/94/1.6
432/94/1.4
N
N
O
O
O
O
N
N
N
N
107
253
1437
28
N
H
O
O
6.0/15.
7
N
N
28
8
5.9/4.8
0.5/48.2
0.9/69
N
N
N
H
N
O
O
O
O
13.5/24
.0
413/126/1.
2
S
N
N
H
N
43/<5
47/11
53/11
114/31
52/42
N
N
O
N
O
O
415/100/2.
2
N
N
4
0.9/2.1
0.65/74
0.5/487
3.66/163
-/-
N
H
N
N
N
N
O
0.10/0.
17
415/100/2.
1
N
5
25
1.7/9.4
6.1/1.5
0.5/12.4
N
N
N
N
H
N
O
N
N
O
472/104/2.
7
N
N
6
148
2.2/6.6
N
H
N
N
N
N
N
O
O
N
N
504/103/3.
0
N
8
37
0.7/1.5
N
N
H
N
F
O
aResults represent the geometric mean of a minimum of two determinations. bInhibition of ASK1 autophosphorylation.
cMDR1-MDCK human P-gp transfected cell line. Papp – Apparent permeability Apical-to-Basolateral (10-6cm/s); ER = B-A/A-
B efflux ratio. dIV dosing using a solutol:PEG:EtOH:water (1:1:1:1) vehicle for 21 and DMA:EtOH:PEG:water (1:1:3:5) for 25
and 26. eThe absolute stereochemistry was arbitrarily assigned.
Chemistry
The synthesis of analogs 5, 6 and 10 was carried starting with the readily available picolinate 28 which after
hydrazide formation and condensation with N,N-dimethylformamide-dimethylacetal (DMF-DMA) afforded
intermediate 29. This compound was then heated under acidic conditions in the presence of either methylamine or
isopropylamine and hydrolyzed with aqueous NaOH to provide intermediates 30 and 31 respectively (Scheme 1).
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5
6
The ASK1 inhibitors 5 was accessed by reaction of aminopyridine 30 with 2-fluoro-benzoylchloride in the presence
of DMAP. Inhibitors 6 and 10 were synthesized by coupling of 2-methoxybenzoic acid with aminopyridine 30 and
31 and using propylphosphonic anhydride (T3P).
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 1. Synthesis of linear analogs
O
H
a
b
c
N
N
N
Me2N
N
N
N
NMe2
H2N
N
N
N
N
N
H
H2N
N
CO2Me
O
N
N
X
R
R
i
28
29
30,
31,
5,
R = Me
X = F, R = Pr
i
i
6
R = Pr
, X = OMe, R = Pr
10
, X = OMe, R = Me
Reagents and conditions: (a) N2H4•H2O, MeOH, reflux, 5 h then DMF-DMA, reflux 2 d, 64% over 2 steps; (b) compound 30:
MeNH2, CH3CN/AcOH 4/1, µW 110 °C, 1 h then NaOH, MeOH, reflux, 3 h, 68%; compound 31: isopropylamine, CH3CN/AcOH
4/1, reflux, 12 h then 1M aq. NaOH, 54%; (c) compound 5: 2-fluoro-benzoylchloride, DMAP, DCM, rt, 4 h, 63%; compounds 6
and 10: 2-methoxybenzoic acid, Et3N, T3P, 60 °C, 2 h, 35-40%.
The synthesis of macrocyclic compounds was carried out following two strategies: a macrolactamization approach
of an amino acid precursor (60-68, Scheme 2) or a ring closing metathesis (RCM) of a bis-olefin precursor (86-87,
Scheme 3). The synthesis of macrocyclic inhibitors following a macrolactamization approach, started with
installation of the linker via a Mitsunobu reaction between readily available building blocks 32 or 33 and protected
aminoalcohols 34-41 to provide intermediates 42-50. Deprotection of these intermediates using TFA, hydrazine or
CAN and TFA, gave the desired amines 51-59 in good yield. Reaction of primary free amine 51-59 with
dimethylhydrazonamide 29 by refluxing in acetic acid gave the desired condensation products as an inseparable
mixture with other partially hydrolyzed intermediates. This mixture was saponified with sodium hydroxide to
provide the desired amino acid 60-68 in acceptable overall yield. Amide coupling conditions with propylphosphonic
acid anhydride proved to be most reliable for the formation of the 13- to 15-membered macrocycles. In particular,
the desired 14-membered macrolactam (rac)-71 was obtained in 25% isolated yield without formation of any
undesired intra-molecular amide bond or dimerization. Chiral separation of intermediates 72, 74 and 76 followed
by Pd-mediated reduction of the precursors of the more potent enantiomers led to 13, 15 and 19. Reduction of bromo
intermediates 20, 69-71, 73 and 75 led to analogs 7-12, 14 and 18 after chiral separation of the racemic
intermediates.
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Scheme 2. Synthesis of macrocyclic analogs following a macrolactamization approach
4
5
6
7
8
9
O
O
O
O
Br
Br
OMe
n
Br
OMe
n
PG1
N
b,c or d
a
OMe
OH
NH2
X
PG1
N
X
O
PG2
Rx
Rx

X
PG2
HO

n
32, X=H
33, X=F
Rx
42, X=H, n=0, Rx=H, PG1=Boc, PG2=H
43, X=H, n=1, Rx=H, PG1=Boc, PG2=H
44, X=H, n=2, Rx=H, PG1=Boc, PG2=H
51, X=H, n=0, Rx=H
52, X=H, n=1, Rx=H
53, X=H, n=2, Rx=H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
34, n=0, Rx=H, PG1=Boc, PG2=H
35, n=1, Rx=H, PG1=Boc, PG2=H
36, n=2, Rx=H, PG1=Boc, PG2=H
54, X=H, n=1, R =Me
55, X=H, n=1, R =Me
56, X=H, n=1, R =Me
57, X=H, n=1, R =Me
58, X=F, n=1, R =Me
59, X=F, n=1, R =Me






45, X=H, n=1, R =Me, PG =Boc, PG =H
46, X =H, n=1, R =Me, PG =PG =Pht
47, X=H, n=1, R =Me, PG =Boc, PG =H
48, X=H, n=1, R =Me, PG =PG =Pht
49, X=F, n=1, R =Me, PG =Boc, PG =H
50, X=F, n=1, R =Me*, PG =Boc, PG =PMB






1
2
37, n=1, R =Me, PG =Boc, PG =H
38, n=1, R =Me, PG =PG =Pht
39, n=1, R =Me, PG =Boc, PG =PMB
40, n=1, R =Me, PG =Boc, PG =H




1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
41, n=1, R =Me, PG =Boc, PG =PMB

1
2
1
2
H
N
O
O
Me2N
N
N
N
NMe2
O
O
H2N
OH
N
O
Br
29
Br
N
N
N
g,h
N
N
N
H
e,f
N
N
X
n
X
Rx
n
Rx
60, X=H, n=0, Rx=H 63, X=H, n=1, R =Me
69, X=H, n=0, Rx=H 71, X=H, n=1, R =Me







61, X=H, n=1, Rx=H
62, X=H, n=2, Rx=H
20, X=H, n=1, Rx=H
70, X=H, n=2, Rx=H
64, X=H, n=1, R =Me
65, X=H, n=1, R =Me
66, X=H, n=1, R =Me
67, X=F, n=1, R =Me
72, X=H, n=1, R =Me

73, X=H, n=1, R =Me
74, X=H, n=1, R =Me
75, X=F, n=1, R =Me



68, X=F, n=1, R =Me
76, X=F, n=1, R =Me

O
O
N
11, X=H, n=1, R =Me (S)
12, X=H, n=1, R =Me (R)
13, X=H, n=1, R =Me
14, X=H, n=1, R =Me
15, X=H, n=1, R =Me
18, X=F, n=1, R =Me (S)
19, X=F, n=1, R =Me (R)







7, X=H, n=0, Rx=H
8, X=H, n=1, Rx=H
9, X=H, n=2, Rx=H
N
N
i,j

N
H
N

X
Rx
n
Reagents and conditions: (a) 34-41, PPh3, DIAD, THF, 0-25 °C, 12 h, 46-100%; (b) 42-45, 47 or 49, TFA, DCM, 40 °C, 5 h, 89%-
94%; (c) 46 or 48, NH2NH2, EtOH, reflux, 70-90%; (d) 50, 1. CAN; 2. TFA, 67%; (e) 29, AcOH, CH3CN, reflux, 24 h, 60%; (f)
NaOH, MeOH, THF, 70 °C, 10 h; (g) T3P, Et3N, EtOAc, 80 °C, 4 h, 23-45% over 2 steps; (h) 72, 74 and 76, chiral separation by
SFC; (i) Pd(dppf)Cl2, K2CO3, dioxane/water, 90 °C, 2 h; (j) chiral separation by SFC.
The synthesis of 16 and 17 started with the preparation of triazole 77 by condensation of 29 with allylamine in refluxing
acetic acid. Fragments 82 and 83 were synthesized by deprotonation of commercially available phenols 78 and 79 with NaH
followed by reaction with allyl bromide. The corresponding esters 80 and 81 were converted to the acyl chlorides 82 and 83
in a two-step procedure involving saponification and reaction of the acid intermediates with oxalyl chloride with catalytic
DMF. Amide bond formation between 77 and 82 (or 83) was carried out in DCM with DMAP to provide intermediates 84
and 85. Initial attempts to carry out the RCM with these precursors were unsuccessful and a Boc protecting group was
installed using Boc2O and DMAP to provide intermediates 86 and 87. The desired macrocyclic compounds 88 and 89 as a
mixture of E- and Z-isomers were obtained by refluxing 86 and 87 in toluene in the presence of Hoveyda-Grubbs 1st
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generation (HG 1st) catalyst. Hydrogenation of macrocyclic intermediates 88 and 89 with Pd/C under a H2 atmosphere
followed by deprotection of the Boc group with TFA furnished analogs 16 and 17.
6
7
8
Scheme 3. Synthesis of macrocyclic analogs following a ring closing metathesis approach
9
H
N
a
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N
H2N
N
N
Me2N
N
O
N
N
NMe2
O
O
N
O
N
N
N
e
f
29
77
N
F
H
N
O
O
O
5
b
c,d
OMe
OMe
Cl
84, X = 5-F
85, X = 4-F
F
F
F
OH
4
O
78, X = 5-F
79, X = 4-F
80, X = 5-F
81, X = 4-F
82, X = 5-F
83, X = 4-F
O
O
O
O
O
O
5
F
N
N
N
N
N
N
N
N
g
h
N
N
i
N
N
N
H
N
N
F
Boc
F
Boc
F
N
Boc
N
N
N
N
O
4
O
86, X = 5-F
87, X = 4-F
88, X = 5-F
89, X = 4-F
90, X = 5-F
91, X = 4-F
16, X = 5-F
17, X = 4-F
Reagents and conditions: (a) AcOH, allyl amine, reflux, 38%; (b) allyl bromide, NaH, DMF, 46-88%; (c) NaOH (aq), MeOH; (d)
(COCl)2, DCM, DMF (cat), 99-100% over 2 steps; (e) DMAP, DCM, 32-47%; (f) Boc2O, DMAP, DCM, 72-79%; (g) HG-1st
generation, toluene, reflux, 8-35%; (h) H2(g), Pd/C, 100%; (i) TFA, DCM, 29-32%.
The synthesis of intermediate 20 was scaled up early in the project for a parallel synthesis effort according to the
route described in Scheme 2. With large quantities of intermediate 20 in hand, analogs featuring a new carbon-
nitrogen bond such as 21 - 23 were generally accessed from primary or secondary amines via a Buchwald-Hartwig
coupling using a Pd2dba3 and Xphos catalyst system. The sulfone compound 23 was synthesized using a Cu-mediated
coupling with the corresponding sodium sulfinate salt. Suzuki-coupling between macrocycle 20 or 76b and the
corresponding boronates provided access to analogs 24 - 26 and 27 respectively.
Scheme 4. Derivatization of macrocyclic inhibitors
N
O
N
21, R=
22, R=
23, R=
24, R=
25, R=
26, R=
N
O
O
O
O
R
Br
N
O
O
N
a, b or c
N
N
N
N
N
N
N
N
H
H
N
N
N
O
N
N
20
S
Me2N
O
O
Me2N
N
N
O
O
Br
F
c
N
N
H
N
N
N
N
N
N
H
N
N
F
76b
27
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Reagents and conditions: (a) Analogs 21, 22: Amine, Pd2(dba)3, XPhos, NatBuO, 21-35%; (b) Analog 23: MeSO2Na, CuI, L-
proline sodium salt, 22%; (c) Analogs 24-27: Boronic ester, K2CO3, Pd(dtbpf)Cl2 (24) or Pd(dppf)Cl2 (25-27), 13-66%.
5
6
7
CNS penetration
8
9
To evaluate the CNS penetration of this series of inhibitors, several compounds with satisfactory profile were selected
for in vivo rat Kpuu studies. In these experiments the compounds were administered via an IV infusion25 in the carotid
artery for a period of four hours (1 mg/kg, 0.1 mg/mL) to reach steady state. After this time the plasma and brain
concentration levels were quantified, and the values were adjusted by the measured protein binding in plasma and
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
brain homogenate to calculate the Kpuu.
26 This experiment was conducted with analogs 18, 19, 24, 26 and 27 (Table
5) and, as expected, analog 24 and 27, which had a high ER in the MDR1-MDCK assay, were found to be peripherally
restricted. In contrast, analog 26, with an ER = 1.5 had a Kpuu of 1. The data for analogs 18 and 19 revealed that analog
19 had superior CNS penetration than 18 (Kpuu = 0.17 for analog 19 vs Kpuu = 0.09 for compound 18).
Table 5. In vivo Kpuu studies with selected compounds
Brain
Homogenate
(fu)
Biochem.
IC50 (nM)a
Cell
MDR1-MDCK
Papp/ERc
Analog
18
Structure
Cl/Clu
0.82/18
0.5/30
Kpuu
0.09
0.17
IC50 (nM)a,b
O
N
N
N
H
N
N
7
8
43
95
4.5/4.9
3.6/5.2
0.34
0.30
N
N
N
N
F
F
O
O
N
H
19
O
N
O
N
N
N
24
26
N
N
4
6.5
8
28
148
37
0.9/69
6.1/1.5
0.5/12
0.65/74
67/61
-
0.32
1.0
<0.1
1
N
H
N
O
N
O
N
N
N
H
N
N
N
O
N
N
N
O
27d
0.33
<0.1
N
N
N
N
H
N
F
O
aBiochemical assay as the geometric mean of at least two measurements. bInhibition of ASK1 autophosphorylation. cMDR1-
MDCK human P-gp transfected cell line. A-B = Apical-to-Basolateral; ER = B-A/A-B efflux ratio. dThe absolute
stereochemistry was arbitrarily assigned.
CONCLUSION
In summary, the X-ray cocrystal structure of compound 4 bound to the kinase domain of human ASK1 led to the
design and synthesis of the novel macrocyclic ASK1 inhibitor 8 following a deconstruction-cyclization approach
(Figure 5). The profile of analog 8 was optimized by alkylating the tether to improve potency and by fluorinating the
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7
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salicylate ring to improve microsomal stability. Combination of these two structural modifications led to the
identification of compounds 18 and 19 which had good cellular potency and PK as well as low to moderate CNS
penetration. Alternatively, the brain-permeability of macrocyclic compound 8 was modulated by the substituent
attached to the salicylate. This strategy led to the identification of compounds with good cellular potency and PK
properties that were peripherally restricted (such as analog 24) and compounds with good cellular potency and
excellent brain exposure (such as analog 26). These compounds might be useful tools to further understand the role
of ASK1 inhibition in neurodegenerative disease.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
O
N
N
N
N
N
N
N
N
H
H
N
N
F
O
F
O
Rational
Design
18
cell IC50 = 43 nM
19
cell IC50 = 95 nM
Cl/Clu = 0.5/30 L/hr/kg
Kpuu = 0.17
N
O
F
O
O
N
Cl/Clu = 0.82/18 L/hr/kg
Kpuu = 0.09
N
N
N
Deconstruction
Cyclization
N
N
N
N
N
H
H
N
N
4
8
Parallel
Synthesis
N
Me2N
N
N
O
O
O
N
N
N
N
N
N
N
N
H
N
H
N
N
O
24
26
cell IC50 = 123 nM
cell IC50 = 28 nM
Cl/Clu = 0.65/74 L/hr/kg
Kpuu < 0.1
Cl/Clu = 3.66/163 L/hr/kg
Kpuu = 1
Figure 5. Summary of identification and optimization of novel ASK1 macrocyclic inhibitors.
EXPERIMENTAL SECTION
All solvents and chemicals used were reagent grade. Anhydrous solvents were purchased from Sigma-Aldrich and
used as received. Analytical thin layer chromatography (TLC) and silica gel column chromatography were performed
on Merck silica gel 60 (230-400 mesh). Removal of solvents was conducted by using a rotary evaporator and residual
solvents were removed from non-volatile compounds using a vacuum manifold maintained at approximately 1 Torr.
NMR spectra were recorded on a Bruker Avance 400 MHz and 500 MHz NMR spectrometer. Chemical shifts () are
reported in parts per million (ppm) relative to residual undeuterated solvent as internal reference and coupling
constants (J) are reported in hertz (Hz). Splitting patterns are indicated as follows: s = singlet; d = doublet; t = triplet;
q = quartet; qn = quintet; dd = doublet of doublets; dt = doublet of triplets; tt = triplet of triplets; m = multiplet; br =
broad peak. All yields reported are isolated yields. All final compounds were purified to ≥95% purity as determined
by LC/MS analysis (using a lineal gradient of elution: 90% water in trifluoroacetic acid (containing 0.1% v/v)/10%
CH3CN in trifluoroacetic acid (containing 0.1% v/v) to 10% water in trifluoroacetic acid (containing 0.1% v/v) and
90% CH3CN in trifluoroacetic acid (0.1% v/v) for 2 minutes and then holding at 10% water in trifluoroacetic acid
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(0.1% v/v) and 90% CH3CN in trifluoroacetic acid (0.1% v/v) up to 3 minutes at a flowrate of 3 mL/min. (injection
volume 5µL and using a Waters Sunfire C18 3.5 M 4.6x20mm IS Column)). MS mode: MS:ESI+ scan range 100-1000
daltons PDA detection 210-400 nm. Final compounds were analyzed using UHPLC (Thermo Scientific Vanquish
(Thermo Fisher Scientific Waltham, MA) coupled with a Fusion Lumos Tribrid Orbitrap mass spectrometer (Thermo
Fisher Scientific). A Water’s (Milford, MA) Acquity BEH C18 (1.7um beads, 2.1x50mm) column was used for
separation. Mobile phase A was 10mM ammonium formate pH 4.0 and mobile phase B was acetonitrile. The flow
rate was 0.45 ml/min and the following gradient was used from 0-0.5 minutes 2% B and increased linearly to 50%
B at 7.3 minutes and 95% at 10.3 min and remained there for 0.7 minutes, dropped back to 2% B over 0.1 minutes
and remained there for 0.9 minutes. The mass spectrometer was operated in positive ion mode with accurate mass
measurements performed using the orbitrap set at 120,000K resolution (at m/z 200). A heated electrospray
ionization source was used with the following parameters: spray voltage 3500 V, sheath gas 35 (arbitrary units),
auxiliary gas 10 (arbitrary units), sweep gas 0 (arbitrary units), ion transfer tube temperature 350 °C, and vaporizer
temperature 300 °C.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2-Fluoro-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)benzamide (5). To a solution of amine 31 (41
mg, 0.2 mmol) and DMAP (29 mg, 2.4 mmol) in DCM (1 mL) was added 2-fluorobenzoyl chloride (25 µL, 0.2 mmol)
at room temperature. The resulting reaction mixture was stirred at room temperature for 4 h upon which a white
precipitate formed. The solids were filtered off and washed with CH3CN yielding the title compound (41 mg, 63%) as
an off-white solid. 1H NMR (400 MHz, DMSO- d6) δ ppm 10.89 (s, 1H), 8.86 (s, 1H), 8.22 (d, J = 8.3 Hz, 1H), 8.03 (t, J =
7.9 Hz, 1H), 7.90 (d, J = 7.3 Hz, 1H), 7.73 (dt, J = 7.5, 1.6 Hz, 1H), 7.67 - 7.57 (m, 1H), 7.43 - 7.32 (m, 2H), 5.69 (quin, J
= 6.7 Hz, 1H), 1.43 (d, J = 6.5 Hz, 6H). HRMS (m/z): [M + H]+ calcd. for C17H17FN5O 326.1412; found: 326.1416. HPLC
(CH3CN in 0.1% TFA): tR = 1.54 min (99% purity).
N-(6-(4-Isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-2-methoxybenzamide (6). To
a
solution of 2-
methoxybenzoic acid (17 mg, 0.1 mmol), compound 31 (21 mg, 1.0 mmol) and triethylamine (140 µL, 1.0 mmol) in
EtOAc (2 mL) was added propylphosphonic anhydride (190 µL, ≥50 wt. % in EtOAc, 0.3 mmol). The resulting reaction
mixture was heated at 60 °C for 2 h, cooled to room temperature and quenched with MeOH (2 mL). The volatiles
were removed, and the resulting residue was purified by column chromatography (12 g, SiO2, 0-100% EtOAc/EtOH
3/1 in heptane). The title compound (12 mg, 35%) was obtained as an off-white solid. 1H NMR (400 MHz, CD3OD) δ ppm
8.85 (s, 1H), 8.43 (dd, J = 8.4, 0.9 Hz, 1H), 8.09 (dd, J = 7.8, 1.8 Hz, 1H), 7.99 (dd, J = 8.4, 7.7 Hz, 1H), 7.85 - 7.81 (m,
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1H), 7.63 - 7.53 (m, 1H), 7.22 (dd, J = 8.5, 0.8 Hz, 1H), 7.13 (dt, J = 7.7, 1.0 Hz, 1H), 5.54 (quin, J = 6.8 Hz, 1H), 4.06 (s,
3H), 1.63 (d, J = 6.8 Hz, 6H). HRMS (m/z): [M + H]+ calcd. for C18H20N5O2 338.1612; found: 338.1616. HPLC (CH3CN in
0.1% TFA): tR = 1.63 min (100% purity).
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14H-6-Oxa-3-aza-2(2,6)-pyridina-1(3,4)-triazola-5(1,2)-benzenacyclononaphan-4-one (7). The reaction was
carried out following general procedure 5 and using compound 69 (12 mg 0.03 mmol), Pd(dppf)Cl2 (5 mg) yielding
the title compound (6 mg, 65%) as a colorless residue. 1H NMR (500 MHz, CDCl3) δ ppm 9.30 (br s, 1H), 8.27 (s, 1H),
8.01 - 7.91 (m, 2H), 7.88 (dd, J = 7.6, 1.5 Hz, 1H), 7.70 (d, J = 8.5 Hz, 1H), 7.51 (dt, J = 7.9, 1.8 Hz, 1H), 7.16 - 7.10 (m,
1H), 6.96 (d, J = 8.5 Hz, 1H), 4.35 - 4.16 (m, 2H), 2.55 (br s, 2H), 1.62 (br s, 2H). 13C NMR (100 MHz, CDCl3) δ ppm
164.4, 157.7, 151.4, 150.8, 145.4, 142. 9, 139.5, 133.9, 131.3, 121.1, 121.0, 119.6, 114.0, 111.9, 55.6, 49.1, 22.2. MS
(ESI): 322.1 [(M + H)]+. HPLC (CH3CN in 0.1% TFA): tR = 0.71 min (99% purity).
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14H-6-Oxa-3-aza-2(2,6)-pyridina-1(3,4)-triazola-5(1,2)-benzenacyclodecaphan-4-one (8). The reaction was
carried out according to general procedure 5 and using compound 20 (25 mg, 0.06 mmol) to give the title compound
(13 mg, 48%). 1H NMR (400 MHz, CDCl3) δ ppm 11.41 (s, 1H), 8.49 (s, 1H), 8.21 (dd, J = 7.8, 1.8 Hz, 1H), 8.05 (dd, J =
7.9, 0.9 Hz, 1H), 7.92 (d, J = 0.7 Hz, 1H), 7.90 - 7.85 (m, 1H), 7.51 - 7.42 (m, 1H), 7.11 (t, J = 7.6 Hz, 1H), 6.97 (d, J = 8.3
Hz, 1H), 4.34 - 4.20 (m, 4H), 2.74 - 2.60 (m, 2H), 2.08 – 1.98 (m, 2H). 13C NMR (100 MHz, CDCl3) δ ppm 163.0¸ 157.2,
151.8, 150.8, 145.8, 144.5, 140.3, 134.0, 132.4, 122.1, 121.3, 118.0, 114.0, 113.1, 68.2, 48.8, 27.1, 26.3. HRMS (m/z):
[M + H]+ calcd. for C18H18N5O2 336.1455; found: 336.1460. HPLC (CH3CN in 0.1% TFA): tR = 1.56 min (100% purity).
14H-6-Oxa-3-aza-2(2,6)-pyridina-1(3,4)-triazola-5(1,2)-benzenacycloundecaphan-4-one (9). To a suspension
of compound 70 (40 mg, 0.09 mmol) in THF (2 mL) was added n-BuLi in hexanes (0.37 mL of a 2.5 M solution, 0.93
mmol) at -78 °C. After 2 h at this temperature MeOH (1 mL) was added. The reaction mixture was then allowed to
warm to room temperature and concentrated under vacuum. The resulting residue was purified by column
chromatography (4 g SiO2, using a gradient of elution of MeOH and DCM, from 0 to 15%) to give the title compound
(8 mg, 25%) as an off-white solid. 1H NMR (400 MHz, CDCl3) δ ppm 10.76 (s, 1H), 8.60 (dd, J = 8.4, 0.9 Hz, 1H), 8.39
(d, J = 6.0 Hz, 1H), 8.32 (s, 1H), 8.16 (dd, J = 7.7, 0.9 Hz, 1H), 7.94 (s, 1H), 7.60 - 7.51 (m, 1H), 7.19 (td, J = 7.6, 0.9 Hz,
1H), 7.07 - 7.01 (m, 1H), 4.38 - 4.30 (m, 2H), 4.29 - 4.22 (m, 2H), 2.34 - 2.17 (m, 2H), 2.15 - 2.03 (m, 2H), 2.03 – 1.91
(m, 2H). MS (ESI): 350.2 [M + H]+. HPLC (CH3CN in 0.1% TFA): tR = 1.34 min (95% purity).
N-(6-(4-Methyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-2-methoxybenzamide (10). The reaction was carried out
according to the procedure for compound 6 and using compound 30 (192 mg, 1.1 mmol) and 2-methoxybenzoic acid
(201 mg, 1.3 mmol) to give the title compound (136 mg, 40%) as an off-white solid. 1H NMR (400 MHz, CDCl3) δ ppm
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10.44 (s, 1H), 8.47 (dd, J = 8.3, 0.8 Hz, 1H), 8.30 (dd, J = 7.9, 1.9 Hz, 1H), 8.19 (s, 1H), 8.06 (dd, J = 7.7, 0.9 Hz, 1H), 7.92
- 7.83 (m, 1H), 7.60 - 7.50 (m, 1H), 7.21 - 7.13 (m, 1H), 7.07 (d, J = 8.3 Hz, 1H), 4.14 (s, 3H), 4.08 (s, 3H). 13C NMR (100
MHz, CDCl3) δ ppm 163.5, 157.4, 151.5, 151.1, 146.3, 145.8, 139.5, 133.9, 132.6, 121.8, 121.2, 119.3, 115.0, 11.7, 56.2,
33.8. HRMS (m/z): [M + H]+ calcd. for C16H16N5O2 310.1299; found: 310.1304. HPLC (CH3CN in 0.1% TFA): tR = 1.45
min (100% purity).
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rac-10-Methyl-14H-6-oxa-3-aza-2(2,6)-pyridina-1(3,4)-triazola-5(1,2)-benzenacyclodecaphan-4-one (rac-
11). Step 1: The reaction was carried out according to general procedure 5 and using compound 71 (61 mg, 0.14
mmol) to give the title compound (20 mg, 40%) as a white solid. MS (ESI): 350.1 [M + H]+. Step 2: Chiral separation.
Compound rac-11 (11 mg) was purified by SFC (using a Chiralpak OD-H, 5m 250x300mm column and using 45%
MeOH (containing 0.1% Et2NH) in CO2 as the mobile phase at a flow rate of 100 mL/min (ABPR 120 BAR, MBPR 40
PSI)) to give in order of elution: Peak 1: (S)-10-methyl-14H-6-oxa-3-aza-2(2,6)-pyridina-1(3,4)-triazola-5(1,2)-
benzenacyclodecaphan-4-one (11, 4 mg, 36%, absolute stereochemistry was arbitrarily assigned) and Peak 2 (R)-
10-methyl-14H-6-oxa-3-aza-2(2,6)-pyridina-1(3,4)-triazola-5(1,2)-benzenacyclodecaphan-4-one (12, 4 mg, 36%,
absolute stereochemistry was arbitrarily assigned). 1H NMR (400 MHz, CDCl3) δ ppm 11.44 (s, 1H), 8.30 (s, 1H), 8.19
(dd, J = 7.8, 1.8 Hz, 1H), 7.96 - 7.92 (m, 1H), 7.91 - 7.87 (m, 1H), 7.84 (d, J = 7.8 Hz, 1H), 7.45 (ddd, J = 8.3, 7.4, 1.9 Hz,
1H), 7.12 - 7.06 (m, 1H), 6.94 (d, J = 7.8 Hz, 1H), 4.74 - 4.63 (m, 1H), 4.39 (dt, J = 9.2, 3.2 Hz, 1H), 4.14 - 4.07 (m, 1H),
3.40 - 3.34 (m, 1H), 2.24 - 2.10 (m, 1H), 1.88 - 1.68 (m, 2H), 1.57 (d, J = 7.0 Hz, 3HMS (ESI): 350.1 [M + H]+. ). HPLC/MS
(Chiralpak AD-H 5m, 4.6mmx250mm; 45% MeOH in 0.1% DEA in CO2, flow rate: 3mL/min), (11) tR = 4.20 min
(100% purity), (12) tR = 4.87 min (100% purity).
(R)-9-Methyl-14H-6-oxa-3-aza-2(2,6)-pyridina-1(3,4)-triazola-5(1,2)-benzenacyclodecaphan-4-one
(13).
The reaction was carried out according to general procedure 5 and using chiral 72a (12 mg, 0.03 mmol), Pd(dppf)Cl2
(5 mg) yielding the title compound (2 mg, 15%) as a colorless residue. 1H NMR (500 MHz, CDCl3) δ ppm 11.46 (br s,
1H), 8.30 (dd, J = 7.3, 1.8 Hz, 1H), 8.17 (s, 1H), 8.06 (dd, J = 9.5, 7.6 Hz, 2H), 7.95 - 7.89 (m, 1H), 7.54 (dt, J = 7.6, 1.8
Hz, 1H), 7.22 - 7.14 (m, 1H), 7.05 (d, J = 7.9 Hz, 1H), 4.62 (dd, J = 13.4, 3.1 Hz, 1H), 4.49 (td, J = 9.0, 3.4 Hz, 1H), 4.29 -
4.17 (m, 1H), 3.74 (dd, J = 13.4, 11.6 Hz, 1H), 3.59 - 3.43 (m, 1H), 2.20 - 2.12 (m, 1H), 1.86 - 1.75 (m, 1H), 1.12 (d, J =
6.1 Hz, 3H). . MS (ESI): 350.1 [M + H]+. HPLC (CH3CN in 0.1% TFA): tR = 0.34 min (99% purity).
rac-8-Methyl-14H-6-oxa-3-aza-2(2,6)-pyridina-1(3,4)-triazola-5(1,2)-benzenacyclodecaphan-4-one
(14).
Step 1: The reaction was carried out according to general procedure 5 and using compound 73 (63 mg, 0.15 mmol)
to give the title compound (30 mg, 44%). MS (ESI): 350.1 [M + H]+. Step 2: Chiral separation. Compound rac-14 (24
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mg) was purified by SFC (using a Chiralpak OD-H, 5m 250x30mm column and using 45% MeOH (containing 0.1%
Et2NH) in CO2 as the mobile phase at a flow rate of 100 mL/min (ABPR 120bar, MBPR 40 psi)) to give in order of
elution: (R)-8-methyl-14H-6-oxa-3-aza-2(2,6)-pyridina-1(3,4)-triazola-5(1,2)-benzenacyclodecaphan-4-one (14, 8
mg, 33%, absolute stereochemistry arbitrarily assigned) and (S)-8-methyl-14H-6-oxa-3-aza-2(2,6)-pyridina-1(3,4)-
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triazola-5(1,2)-benzenacyclodecaphan-4-one (8 mg, 33%, absolute stereochemistry arbitrarily assigned). H NMR
(400 MHz, CDCl3) δ ppm 11.41 (s, 1H), 8.25 (br d, J = 1.8 Hz, 1H), 8.24 (s, 1H), 8.02 (d, J = 8.0 Hz, 1H), 7.99 (d, J = 7.5
Hz, 1H), 7.90 (d, J = 8.0 Hz, 1H), 7.55 - 7.47 (m, 1H), 7.16 (t, J = 7.4 Hz, 1H), 7.02 (d, J = 8.3 Hz, 1H), 4.40 (dt, J = 12.9,
4.0 Hz, 1H), 4.30 - 4.15 (m, 2H), 3.92 (t, J = 9.7 Hz, 1H), 3.55 - 3.41 (m, 1H), 2.11 (br d, J = 7.0 Hz, 1H), 1.73 - 1.58 (m,
1H), 1.24 (d, J = 7.0 Hz, 3H). MS (ESI): 350.1 [M + H]+. HPLC (Chiralpak AD-H 5m, 4.6mmx250mm; 45% MeOH in
0.1% DEA in CO2, flow rate: 3mL/min): (14) tR = 2.40 (100% purity).
(S)-7-Methyl-14H-6-oxa-3-aza-2(2,6)-pyridina-1(3,4)-triazola-5(1,2)-benzenacyclodecaphan-4-one (15a). A
mixture of compound 74a (100 mg, 0.23 mmol) and Pd (100 g, 10% on C) in MeOH (20 mL) was stirred under H2 (15
psi) at 18 °C for 4 h. After this time, the mixture was filtered and concentrated to give the crude product which was
purified by SFC (using an OD 250mmx30mmx5m column and 40% EtOH (with 0.1% ammonium hydroxide) in CO2
as the mobile phase at a flow rate of 50 mL/min) to give the title compound (50 mg, 61%) as a yellow solid. 1H NMR
(400 MHz, DMSO-d6) δ ppm 11.31 (s, 1H), 8.70 (s, 1H), 8.06 (d, J = 6.8 Hz, 2H), 7.97 - 7.84 (m, 2H), 7.62 (s, 1H), 7.30
(d, J = 8.0 Hz, 1H), 7.15 (s, 1H), 5.08 (s, 1H), 4.27 (s, 2H), 3.01 (s, 1H), 1.95 (s, 3H), 1.35 (d, J = 6.0 Hz, 3H). HRMS (m/z):
[M + H]+ calcd. for C19H20N5O2 350.1612; found: 350.1618. HPLC (CH3CN in 0.1% TFA): tR = 3.35 min (98% purity).
(R)-7-Methyl-14H-6-oxa-3-aza-2(2,6)-pyridina-1(3,4)-triazola-5(1,2)-benzenacyclodecaphan-4-one (15b). A
mixture of compound 74b (100 mg, 0.23 mmol) and Pd (100 mg, 10% on C) in MeOH (10 mL) was stirred at under
H2 at 18 °C for 4 h. After this time the mixture was filtered, and the filtrate was concentrated to provide the title
compound (30 mg, 37%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 11.31 (s, 1H), 8.70 (s, 1H), 8.06 (d, J =
6.8 Hz, 2H), 7.97 - 7.84 (m, 2H), 7.62 (s, 1H), 7.30 (d, J = 8.0 Hz, 1H), 7.15 (s, 1H), 5.08 (s, 1H), 4.27 (s, 2H), 3.01 (s,
1H), 1.95 (s, 3H), 1.35 (d, J = 6.0 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ ppm 163.1, 156.5, 151.2, 150.5, 147.6, 145.7,
141.0, 134.8, 132.0, 121.4, 121.2, 117.6, 114.3, 113.1, 75.1, 48.4, 32.1, 23.7, 16.8. HRMS (m/z): [M + H]+ calcd. for
C19H20N5O2 350.1612; found: 350.1618. HPLC (CH3CN in 0.1% TFA): tR = 3.33 min (99% purity).
55-Fluoro-14H-6-oxa-3-aza-2(2,6)-pyridina-1(3,4)-triazola-5(1,2)-benzenacyclodecaphan-4-one (16). The
title compound was prepared according to the procedure 12 and using 90 (300 mg, 0.7 mmol). The product was
purified by HPLC (using a Phenomenex Synergi C18, 4 m 150x30mm column and using water (containing 0.05%
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HCl)/CH3CN, from 36% to 46% as the mobile phase at a flow rate of 25 mL/min) to give the title compound (60 mg,
29%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 11.33 (s, 1H), 8.84 (s, 1H), 8.16 - 8.03 (m, 1H), 7.94 - 7.86
(m, 2H), 7.73 (dd, J = 9.2, 3.5 Hz, 1H), 7.54 - 7.46 (m, 1H), 7.37 (dd, J = 9.2, 4.4 Hz, 1H), 4.39 - 4.25 (m, 4H), 2.46 - 2.41
(m, 2H), 2.03 – 1.93 (m, 2H). HRMS (m/z): [M + H]+ calcd. for C18H17FN5O2 354.1361; found: 354.1366. HPLC (CH3CN
in 0.1% TFA): tR = 1.67 min (100% purity).
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54-Fluoro-14H-6-oxa-3-aza-2(2,6)-pyridina-1(3,4)-triazola-5(1,2)-benzenacyclodecaphan-4-one (17). The
title compound was prepared according to general procedure 12 and using 91 (400 mg, 0.89 mmol) to give the title
compound (100 mg, 32%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 11.06 (s, 1H), 8.91 (s, 1H), 8.05 - 8.00
(m, 2H), 7.89 - 7.84 (m, 2H), 7.20 (m, , 1H), 6.98 (m, 1H), 4.32 - 4.24 (m, 4H), 2.43 - 2.41 (m, 2H), 1.93 - 1.92 (m, 2H).
HRMS (m/z): [M + H]+ calcd. for C18H17FN5O2 354.1361; found: 354.1367. HPLC (CH3CN in 0.1% TFA): tR = 1.64 min
(100% purity).
(S)-54-Fluoro-10-methyl-14H-6-oxa-3-aza-2(2,6)-pyridina-1(3,4)-triazola-5(1,2)-benzenacyclodecaphan-4-
one (18). Step 1: The reaction was carried out according to general procedure 5 and using compound 75 (500 mg,
1.3 mmol) to give (85 mg, 18%) of the racemic title compound. Step 2. Chiral separation. Compound rac-18 (85
mg) was purified by SFC (Chiralcel AD-H 5m, 30x250mm and using 50% ethanol (containing 0.1% Et2NH) in CO2 as
the mobile phase at a flow rate of 100 mL/min (ABPR 120 bar, MBPR 40 psi)) to give in order of elution: (S)-54-fluoro-
10-methyl-14H-6-oxa-3-aza-2(2,6)-pyridina-1(3,4)-triazola-5(1,2)-benzenacyclodecaphan-4-one (18) 1H NMR (400
MHz, DMSO-d6) δ ppm 11.13 (s, 1H), 8.86 (s, 1H), 8.07 (t, J = 7.8 Hz, 1H), 8.02 (t, J = 7.4 Hz, 1H), 7.88 - 7.80 (m, 2H),
7.19 (dd, J = 11.2, 2.4, Hz, 1H), 7.00 (dt, J = 8.4, 2.3 Hz, 1H), 4.59 (dt, J = 6.5, 2.4 Hz, 1H), 4.50 - 4.45 (m, 1H), 4.16 (t, J
= 10.0 Hz, 1H), 2.18 - 2.04 (m, 2H), 1.85 - 1.65 (m, 2H), 1.54 (d, J = 6.8 Hz, 3H). HRMS (m/z): [M + H]+ calcd. for
C19H19FN5O2 368.1517; found: 368.1524. HPLC (Chiralcel AD-H 5m, 4.6x250mm; 50% EtOH in 0.1% DEA in CO2,
flow rate: 3mL/min): tR = 2.48 min (100% purity). (30 mg, 35%) and (R)-54-fluoro-10-methyl-14H-6-oxa-3-aza-
2(2,6)-pyridina-1(3,4)-triazola-5(1,2)-benzenacyclodecaphan-4-one (18b) (32 mg, 38%, absolute stereochemistry
was assigned by VCD). 1H NMR (400 MHz, DMSO-d6) δ ppm 11.13 (s, 1H), 8.86 (s, 1H), 8.07 (t, J = 7.8 Hz, 1H), 8.02 (t,
J = 7.4 Hz, 1H), 7.88 - 7.80 (m, 2H), 7.19 (dd, J = 11.2, 2.4, Hz, 1H), 7.00 (dt, J = 8.4, 2.3 Hz, 1H), 4.59 (dt, J = 6.5, 2.4 Hz,
1H), 4.50 - 4.45 (m, 1H), 4.16 (t, J = 10.0 Hz, 1H), 2.18 - 2.04 (m, 2H), 1.85 - 1.65 (m, 2H), 1.54 (d, J = 6.8 Hz, 3H). MS
(ESI): 368.1 [M + H]+. HPLC (Chiralcel AD-H 5m, 4.6x250mm; 50% EtOH in 0.1% DEA in CO2, flow rate: 3mL/min):
tR = 2.48 min (100% purity).
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(R)-54-Fluoro-9-methyl-14H-6-oxa-3-aza-2(2,6)-pyridina-1(3,4)-triazola-5(1,2)-benzenacyclodecaphan-4-
one (19). The reaction was carried out according to general procedure 5 and using compound 76b to give the title
compound (23 mg, 27%). 1H NMR (500 MHz, DMSO-d6) δ ppm 11.13 (s, 1H), 8.87 (s, 1H), 8.07 (t, J = 7.8 Hz, 1H), 8.02
(t, J = 7.6 Hz, 1H), 7.85 (d, J = 7.9 Hz, 1H), 7.82 (d, J = 7.3 Hz, 1H), 7.19 (dd, J = 11.0, 2.4 Hz, 1H), 7.00 (dt, J = 8.4, 2.1,
1H), 4.60 (ddd, J = 10.4, 7.0, 3.4 Hz, 1H), 4.50 - 4.46 (m, 1H), 4.16 (br t, J = 9.8 Hz, 1H), 3.68 - 3.57 (m, 1H), 2.16 - 2.09
(m, 1H), 1.84 - 1.74 (m, 2H), 1.54 (d, J = 6.7 Hz, 3H). HRMS (m/z): [M + H]+ calcd. for C19H19FN5O2 368.1517; found:
368.1524. HPLC (Chiralpak AD-H 5m, 4.6mmx250mm; 40% MeOH in 0.1% DEA in CO2, flow rate: 3mL/min): tR =
3.14 min (100% purity).
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55-Bromo-14H-6-oxa-3-aza-2(2,6)-pyridina-1(3,4)-triazola-5(1,2)-benzenacyclodecaphan-4-one, (20). The
reaction was carried out according to general procedure 4 and using 61 (16.5 g, 95.4 mmol) to give the title
compound (9.0 g, 41%) as a white solid. 1H NMR (400 MHz, CDCl3) δ ppm 11.32 (s, 1H), 8.37 (s, 1H), 8.20 (s, 1H), 8.01
(d, J = 7.8 Hz, 2H), 7.94 - 7.86 (m, 1H), 7.59 (dd, J = 8.8, 2.5 Hz, 1H), 6.91 (d, J = 8.6 Hz, 1H), 4.33 - 4.21 (m, 4H), 2.67
(s, 2H), 2.12 - 2.01 (m, 2H). 13C NMR (100 MHz, CDCl3) δ ppm 160.5, 155.1, 150.7, 149.3, 145.1, 144.1, 139.2, 135.4,
134.0, 122.0, 117.1, 113.9, 113.7, 112.6, 67.7, 47.4, 26.0, 25.2. HRMS (m/z): [M + H (81Br)]+ calcd. for C18H17BrN5O2
414.0560; found: 414.0568. HPLC (CH3CN in 0.1% TFA): tR = 1.89 min (94% purity).
55-Morpholino-14H-6-oxa-3-aza-2(2,6)-pyridina-1(3,4)-triazola-5(1,2)-benzenacyclodecaphan-4-one (21).
The reaction was carried out according to general procedure 13 and using compound 20 (130 mg, 0.3 mmol) and
morpholine. Purification by column chromatography (4 g silica gel, 5-40% MeOH in DCM) gave the title compound
(46 mg, 35%) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ ppm 11.49 (s, 1H), 8.14 (s, 1H), 7.95 (d, J = 8.3 Hz, 1H), 7.93
- 7.89 (m, 1H), 7.84 - 7.78 (m, 1H), 7.74 (d, J = 3.0 Hz, 1H), 6.99 (dd, J = 8.9, 3.1 Hz, 1H), 6.90 (d, J = 8.8 Hz, 1H), 4.22 -
4.14 (m, 4H), 3.83 - 3.77 (m, 4H), 3.13 - 3.05 (m, 4H), 2.56 (br dd, J = 2.8, 1.5 Hz, 2H), 2.02 – 1.93 (m, 2H). 13C NMR
(100 MHz, CDCl3) δ ppm 162.8¸ 152.5, 151.7, 151.6, 150.6¸ 146.0¸ 145.6, 145.0, 140.1, 122.0, 119.3, 117.9, 115.2,
113.6, 69.0, 63.8, 50.3, 48.4, 27.2, 26.3. HRMS (m/z): [M + H]+ calcd. for C22H25N6O3 421.1983; found: 421.1989. HPLC
(CH3CN in 0.1% TFA): tR = 1.31 min (95% purity).
55-(2-Oxa-6-azaspiro[3.3]heptan-6-yl)-14H-6-oxa-3-aza-2(2,6)-pyridina-1(3,4)-triazola-5(1,2)-
benzenacyclodecaphan-4-one (22). The reaction was carried out according to general procedure 13 and using
compound 20 (150 mg, 0.4 mmol) and morpholine 2-oxa-6-azaspiro[3.3]heptane and heating at reflux for 8 h.
Purification by column chromatography (4 g silica gel, MeOH in DCM 1-15%) gave the title compound (33 mg,
21%) as a pale-yellow solid. 1H NMR (400 MHz, CDCl3) δ ppm 11.60 (s, 1H), 8.27 (s, 1H), 8.05 (d, J = 8.5 Hz, 1H), 8.00
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(d, J = 7.4 Hz, 1H), 7.96 - 7.87 (m, 1H), 7.36 (d, J = 3.0 Hz, 1H), 6.95 (d, J = 8.8 Hz, 1H), 6.62 (dd, J = 8.8, 3.3 Hz, 1H),
4.85 (s, 4H), 4.32 - 4.25 (m, 2H), 4.22 (t, J = 4.9 Hz, 2H), 4.05 (s, 4H), 2.66 (br s, 2H), 2.12 – 1.99 (m, 2H). HRMS (m/z):
[M + H]+ calcd. for C23H25N6O3 433.1983; found: 433.1989. HPLC (CH3CN in 0.1% TFA): tR = 1.30 min (100% purity).
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55-(Methylsulfonyl)-14H-6-oxa-3-aza-2(2,6)-pyridina-1(3,4)-triazola-5(1,2)-benzenacyclodecaphan-4-one
(23). A suspension of compound 20 (50 mg, 0.12 mmol), sodium methanesulfinate (15 mg, 0.15 mmol), copper iodide
(2 mg, 0.01 mmol) and L-proline sodium salt (3 mg, 0.02 mol) in NMP (1 mL) was degassed by purging with N2 for
15 min. The resulting mixture was heated at 100 °C for 8 h. After this time the reaction mixture was filtered and
purified by HPLC (using a Sunfire Prep C18 OBD, 5µm 30x50mm column and using water/CH3CN (containing 0.1%
TFA) from 90/10 to 30/70 as the mobile phase at a flow rate of 50 mL/min) to give the title compound (14 mg, 22%).
1H NMR (400 MHz, CD3CN) δ ppm 11.26 (br s, 1H), 8.64 (d, J = 2.8 Hz, 1H), 8.50 - 8.44 (m, 1H), 8.14 - 8.07 (m, 1H),
8.06 – 7.99 (m, 2H), 7.99 - 7.93 (m, 1H), 7.43 - 7.36 (m, 1H), 4.48 - 4.43 (m, 2H), 4.35 - 4.25 (m, 2H), 3.12 (s, 3H), 2.71
- 2.58 (m, 2H), 2.12 - 2.02 (m, 2H). HRMS (m/z): [M + H]+ calcd. for C19H20N5O4S 414.1231; found: 414.1239. HPLC
(CH3CN in 0.1% TFA): tR = 1.26 min (95% purity).
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55-(4-Methyl-1H-imidazol-1-yl)-14H-6-oxa-3-aza-2(2,6)-pyridina-1(3,4)-triazola-5(1,2)-
benzenacyclodecaphan-4-one (24). A reaction vial was charged with compound 20 (300 mg, 0.7 mmol), sodium
tert-butoxide (209 mg, 2.2 mmol) and 4-methyl-1H-imidazole (178 mg, 2.2 mmol). The reaction vial was purged with
N2, tert-butanol (3 mL) was added and the mixture was heated at 85 °C. Simultaneously, a vial containing a solution
of dtbpf (70 mg, 0.14 mmol) and Pd2dba3 (66 mg, 0.07 mmol) in toluene (1.5 mL) was heated at reflux for 15 minutes,
cooled to room temperature and then added to the first vial. The resulting mixture was heated at reflux for 4 h. After
this time the reaction was cooled to room temperature and MeOH (2 mL) was added and the reaction mixture was
filtered. The resulting product was washed with MeOH (2 x 4mL) and the combined organic extracts were evaporated
under vacuum and purified by HPLC (using an XTerra Prep RP18 OBD, 10 µm 50x250mm column and using water
(containing 0.1% TFA)/CH3CN from 90/10 to 10/90 as the mobile phase at a flow rate of 50 mL/min) to give the title
compound (48 mg, 16%) as a white solid. 1H NMR (400 MHz, CD3OD) δ ppm 9.34 (d, J = 1.8 Hz, 1H), 8.76 (s, 1H), 8.31
(d, J = 3.0 Hz, 1H), 8.05 – 7.96 (m, 2H), 7.85 (s, 2H), 7.79 (d, J = 1.5 Hz, 1H), 7.44 (d, J = 9.0 Hz, 1H), 4.43 (t, J = 4.9 Hz,
2H), 4.39 - 4.31 (m, 2H), 2.62 (br d, J = 6.3 Hz, 2H), 2.46 (d, J = 1.0 Hz, 3H), 2.09 (br s, 2H). HRMS (m/z): [M + H]+ calcd.
for C22H22N7O2 416.1829; found: 416.1836. HPLC (CH3CN in 0.1% TFA): tR = 1.09 min (100% purity).
55-(1-Methyl-1H-pyrazol-4-yl)-14H-6-oxa-3-aza-2(2,6)-pyridina-1(3,4)-triazola-5(1,2)-
benzenacyclodecaphan-4-one (25). The reaction was carried out according to general procedure 14 and using
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compound 20 (300 mg, 0.72 mmol) and 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (151
mg, 0.72 mmol). Purification by reverse phase HPLC (using a Phenomenex Synergi C18, 4m 150x30mm column and
using water (containing 0.05% HCl)/CH3CN from 22 to 42% as the mobile phase at a flow rate of 25 mL/min) gave
the title compound (40 mg, 13%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 11.27 (s, 1H), 8.85 (s, 1H),
8.15 (s, 1H), 8.09 - 8.06 (m, 2H), 7.91 - 7.89 (m, 1H), 7.85 - 7.84 (m, 2H), 7.77 - 7.74 (m, 1H), 7.28 - 7.26 (m, 1H), 4.32
- 4.29 (m, 4H), 3.82 (s, 3H), 2.48 - 2.46 (m, 2H), 1.94 - 1.93 (m, 2H). HRMS (m/z): [M + H]+ calcd. for C22H22N7O2
416.1829; found: 416.1837. HPLC (CH3CN in 0.1% TFA): tR = 1.45 min (100% purity).
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55-(1-(2-(Dimethylamino)ethyl)-1H-pyrazol-4-yl)-14H-6-oxa-3-aza-2(2,6)-pyridina-1(3,4)-triazola-5(1,2)-
benzenacyclodecaphan-4-one (26). The reaction was carried out according to general procedure 14 and using
compound 20 (700 mg, 1.7 mmol) and N,N-dimethyl-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-
1-yl)ethan-1-amine in dioxane/H2O (0.08 M, 10/1 v/v) and heating at 90 °C under a N2 atmosphere for 3 h.
Purification by column chromatography on silica gel using DCM/MeOH (from 100/1 to 1/1) as eluent gave the title
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compound (600 mg, 66%) as a white solid. H NMR (400 MHz, CD3OD) δ ppm 8.61 (s, 1H), 8.21 (d, J = 2.4 Hz, 1H),
8.00 (s, 1H), 7.98 - 7.94 (m, 2H), 7.85 (d, J = 6.0 Hz, 1H), 7.81 (s, 1H), 7.64 (dd, J = 8.8, 2.4 Hz, 1H), 7.13 (d, J = 8.8 Hz,
1H), 4.38 - 4.26 (m, 6H), 2.95 (t, J = 6.8 Hz, 2H), 2.62 - 2.47 (m, 2H), 2.40 (s, 6H), 2.08 - 2.05 (m, 2H). 13C NMR (100
MHz, CD3OD) δ ppm 162.0, 156.1, 151.3, 150.3, 144.3, 140.0, 136.8, 130.4, 127.5, 127.3, 125.4, 121.9, 120.5, 117.3,
114.2, 113.5, 113.2, 68.8, 56.8, 49.4, 45.7, 42.4, 26.6, 25.7. HRMS (m/z): [M + H]+ calcd. for C25H29N8O2 473.2408;
found: 473.2415. HPLC (CH3CN in 0.1% TFA): tR = 1.18 min (100% purity).
(R)-55-(1-(2-(Dimethylamino)ethyl)-1H-pyrazol-4-yl)-54-fluoro-9-methyl-14H-6-oxa-3-aza-2(2,6)-pyridina-
1(3,4)-triazola-5(1,2)-benzenacyclodecaphan-4-one (27). To a stirred solution of compound 76b (150 mg, 0.34
mmol) in EtOH (5 mL) and H2O (0.5 mL) was added N,N-dimethyl-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)-1H-pyrazol-1-yl)ethan-1-amine (91 mg, 0.34 mmol), PEPPSI (46 mg, 0.07 mmol) and K2CO3 (116 mg, 0.84 mmol)
and the reaction mixture was stirred at 90 °C for 2 h. After this time the reaction suspension was poured into water
(20 mL) and extracted with EtOAc (3 x 25 mL). The combined organic extracts were dried over Na2SO4, filtered and
concentrated. The crude product was purified by HPLC (using an Xtimate C18, 5m 150x25mm column and using
water (containing 10 mM NH4HCO3)/CH3CN, from 30% to 60% as the mobile phase at a flow rate of 25 mL/min) to
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give the title compound (31 mg, 18%) as a white solid. H NMR (400 MHz, DMSO-d6) δ ppm 11.11 (s, 1H), 8.67 (s,
1H), 8.26 - 8.21 (m, 2H), 8.07 - 8.05 (m, 1H), 7.91 - 7.87 (m, 3H), 7.32 - 7.28 (m, 1H), 4.54 - 4.42 (m, 2H), 4.29 - 4.22
(m, 3H), 3.92 - 3.85 (m, 1H), 3.26 - 3.22 (m, 1H), 2.70 - 2.66 (m, 2H), 2.18 (s, 6H), 2.08 – 2.01 (m, 1H), 1.70 - 1.64 (m,
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1H), 0.95 (d, J = 6.4 Hz, 3H). HRMS (m/z): [M + H]+ calcd. for C26H29FN8O2 505.2470; found: 505.2477.HPLC (CH3CN
in 0.1% TFA): tR = 1.28 min (100% purity). HPLC (CH3CN in 0.1% TFA): tR = 2.88 min (100% purity).
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(E)-N'-(6-(2-((E)-(Dimethylamino)methylene)hydrazine-1-carbonyl)pyridin-2-yl)-N,N-
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dimethylformimidamide (29). Step 1: A mixture of methyl 6-aminopyridine-2-carboxylate (6.0 g, 39.4 mmol) and
hydrazine hydrate (7.2 g, 78.9 mmol, 7.0 mL) was refluxed in MeOH (50 mL) for 5 h. The reaction mixture was
concentrated under vacuum and the resulting pale-yellow residue was recrystallized from MeOH (220 mL). Filtration
resulted in 4.4 g of a white solid. MS (ESI): 153.1 [(M + H)]+. Step 2: A mixture of the white solid (3.9 g, 25.3 mmol)
and N,N-dimethylformamide-dimethylacetal (DMF-DMA,16.75 mL, 126.5 mmol) was heated to reflux for 2 days. The
volatiles were removed, and the resulting residue was taken up in EtOAc (15 mL) and refluxed for 2 h. The mixture was
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gradually cooled to 0 °C before filtration to yield the title compound (5.8 g, 64% over 2 steps) as a pale-yellow solid. H
NMR (400 MHz, DMSO-d6) δ ppm 10.67 (s, 1H), 8.85 (s, 1H), 8.06 (s, 1H), 7.73 - 7.60 (m, 1H), 7.48 (dd, J = 7.3, 1.0 Hz,
1H), 6.91 (dd, J = 1.0, 8.0 Hz, 1H), 3.14 (s, 3H), 3.00 (s, 3H), 2.86 (s, 6H). MS (ESI): 263.2 [(M + H)]+.
6-(4-Methyl-4H-1,2,4-triazol-3-yl)pyridin-2-amine (30). To a solution of compound 29 (1.0 g, 3.8 mmol) in
CH3CN/AcOH (4/1, 1.25 mL) and methylamine (2M in THF, 11.4 mmol) was heated in a microwave (Biotage) at 110
°C for 1 h. The volatiles were removed under reduced pressure. The resulting residue was dissolved in MeOH (10
mL) and sodium hydroxide (610 mg, 15.2 mmol) was added and the resulting mixture was heated at reflux for 3 h.
The volatiles were removed, and the resulting residue was purified by column chromatography (24 g, SiO2, 0-
100% EtOAc/EtOH 3/1 in heptane). The title compound (450 mg, 68%) was obtained as a white solid. 1H NMR (400
MHz, CDCl3) δ ppm 9.66 (br s, 2H), 8.20 (s, 1H), 7.59 (d, J = 0.8 Hz, 1H), 7.58 (d, J = 6.3 Hz, 1H), 6.57 (dd, J = 7.2, 1.9
Hz, 1H), 4.05 (s, 3H). MS (ESI): 176.1 [(M + H)]+.
6-(4-Isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-amine (31). To a solution of compound 29 (200 g, 0.760 mol) in
CH3CN/AcOH (4/1 ratio, 1.25 L) isopropylamine (330 mL, 3.81 mol) was added dropwise at room temperature
followed by heating at reflux overnight. The volatiles were removed under reduced pressure and 1 M aq. NaOH was
added until pH 8 was reached. The precipitate was collected by filtration and the filtrate was repeatedly extracted
with EtOAc. The combined organic layers were dried over sodium sulfate and concentrated to a volume of 50 mL. A
precipitate was formed upon the addition of heptane yielding the title compound (84.2 g, 54%) as a white solid. 1H
NMR (400 MHz, CD3OD) δ ppm 9.28 (s, 1H), 7.77 (dd, J = 8.8, 7.3 Hz, 1H), 7.24 (dd, J = 7.3, 0.8 Hz, 1H), 6.90 (dd, J =
8.5, 0.8 Hz, 1H), 5.39 (quin, J = 6.7 Hz, 1H), 1.60 (s, 3H), 1.58 (s, 3H). MS (ESI): 204.1 [(M + H)]+.
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Methyl 5-bromo-2-(3-((tert-butoxycarbonyl)amino)propoxy)benzoate (42). The reaction was carried out
according to general procedure 1 and using compound 32 (2.5 g) and 34 to give the title compound (3.7 g, 89%) as
a colorless oil. 1H NMR (400 MHz, DMSO- d6) δ ppm 7.77 (d, J = 2.8 Hz, 1H), 7.69 (dd, J = 8.9, 2.6 Hz, 1H), 7.11 (d, J =
9.0 Hz, 1H), 6.85 (br t, J = 5.1 Hz, 1H), 4.07 - 4.01 (m, 2H), 3.82 - 3.79 (m, 3H), 3.14 - 3.07 (m, 2H), 1.82 (quin, J = 6.5
Hz, 2H), 1.36 (s, 9H). MS (ESI): 388.1 [(M + H) (79Br)]+.
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Methyl 5-bromo-2-(4-((tert-butoxycarbonyl)amino)butoxy)benzoate (43). The reaction was carried out
according to general procedure 1 and using compounds 32 (100 g) and 35. The product was purified by column
chromatography on silica gel, using a gradient of elution: petroleum ether/EtOAc (100/1 to 5/1) to give the title
compound (130 g, 75%) as a white solid. 1H NMR (400 MHz, CDCl3) δ ppm 7.91 (d, J = 2.5 Hz, 1H), 7.53 (dd, J = 8.9,
2.6 Hz, 1H), 6.84 (d, J = 9.0 Hz, 1H), 4.82 (br s, 1H), 4.04 (t, J = 6.0 Hz, 2H), 3.84 - 3.97 (m, 3H), 3.20 (q, J = 6.4 Hz, 2H),
1.83 - 1.91 (m, 2H), 1.63 - 1.75 (m, 2H), 1.40 - 1.49 (m, 9H). MS (ESI): 424.1 [(M + Na) (79Br)]+.
Methyl 5-bromo-2-((5-((tert-butoxycarbonyl)amino)pentyl)oxy)benzoate (44). The reaction was carried out
according to general procedure 1 and using 32 (2.5 g) and 36 (2.2 g) to give the crude title compound which was
used without further purification in the Boc-deprotection leading to compound 54 as a yellow oil. MS (ESI): 416.1
[(M + H) (79Br)]+.
rac-Methyl 5-bromo-2-((4-((tert-butoxycarbonyl)amino)pentyl)oxy)benzoate (45). The reaction was carried
out according to general procedure 1 and using 32 (5 g) and 37 to give the title compound (5.8 g, 64%) as a colorless
oil. 1H NMR (400 MHz, CDCl3) δ ppm 7.90 (d, J = 2.5 Hz, 1H), 7.53 (dd, J = 8.8, 2.5 Hz, 1H), 6.85 (d, J = 8.8 Hz, 1H), 4.62
- 4.43 (m, 1H), 4.03 (t, J = 6.0 Hz, 2H), 3.89 (s, 3H), 3.70 (br s, 1H), 1.94 - 1.80 (m, 2H), 1.71 - 1.61 (m, 1H), 1.43 (s, 9H),
1.33 - 1.25 (m, 1H), 1.17 (d, J = 6.5 Hz, 3H). MS (ESI): 438.0 [(M + Na) (79Br)]+.
rac-Methyl 5-bromo-2-(4-(1,3-dioxoisoindolin-2-yl)-3-methylbutoxy)benzoate (46). The reaction was carried
out according to general procedure 1 and using 32 (0.5 g) and 38. The crude product was purified by column
chromatography on silica gel, using a gradient of elution: petroleum ether/EtOAc (10/1 to 3/1) to give the title
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compound as a colorless oil (0.6 g, 63%). H NMR (400 MHz, CDCl3) δ ppm 7.84 (d, J = 2.5 Hz, 1H), 7.83 – 7.80 (m,
2H), 7.74 – 7.68 (m, 2H), 7.49 (dd, J = 9.0, 2.5 Hz, 1H), 6.82 (d, J = 9.0 Hz, 1H), 4.18 - 4.02 (m, 2H), 3.86 (s, 3H), 3.70 –
3.56 (m, 2H), 2.37 – 2.27 (m, 1H), 1.98 – 1.91 (m, 1H), 1.71 – 1.65 (m, 1H), 1.01 (d, J = 6.5 Hz, 3H). MS (ESI): 448.0 [(M
+ H) (81Br)]+.
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rac-Methyl 5-bromo-2-(4-((tert-butoxycarbonyl)amino)-2-methylbutoxy)benzoate (47). The reaction was
carried out according to general procedure 1 and using 32 (10 g) and 39 to give the title compound (8.8 g, 49%) as
a white solid. 1H NMR (400 MHz, CDCl3) δ ppm 8.08 – 7.80 (m, 1H), 7.64 - 7.41 (m, 1H), 6.96 - 6.71 (m, 1H), 4.93 - 4.79
(m, 1H), 3.89 (s, 3H), 3.88 - 3.79 (m, 2H), 3.30 - 3.13 (m, 2H), 2.17 - 2.10 (m, 1H), 1.80 - 1.65 (m, 1H), 1.59 - 1.50 (m,
1H), 1.43 (s, 9H), 1.08 (d, J = 6.8 Hz, 3H). MS (ESI): 438.0 [(M + Na) (79Br)]+.
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rac-Methyl 5-bromo-2-((5-(1,3-dioxoisoindolin-2-yl)pentan-2-yl)oxy)benzoate (48). The reaction was carried
out according to general procedure 1 and using 32 (110 mg, 0.5 mmol) and 40. The crude product was purified by
column chromatography on silica gel, using a gradient of elution: petroleum ether/EtOAc (10/1 to 3/1) to give the
title compound as a colorless oil (140 mg, 73%). 1H NMR (400 MHz, CDCl3) δ ppm 7.86-7.79 (m, 3H), 7.73-7.67 (m,
2H), 7.46 (dd, J = 8.8, 2.4 Hz, 1H), 6.83 (d, J = 8.8 Hz, 1H), 4.50-4.38 (m, 1H), 3.86 (s, 3H), 3.71 (t, J = 6.6Hz, 2H), 1.95-
1.75 (m, 3H), 1.74-1.64 (m, 1H), 1.30 (d, j = 6.4 Hz, 3H). MS (ESI): 467.8 [(M + Na) (79Br)]+.
rac-Methyl 5-bromo-2-((4-((tert-butoxycarbonyl)amino)pentyl)oxy)-4-fluorobenzoate (49). The reaction
was carried out according to general procedure 1 and using compounds 33 (30 g) and 37. The product (52 g) was
isolated as a brown gum and used without further purification in the next step. MS (ESI): 456.1 [(M + Na) (79Br)]+.
rac-Methyl
5-bromo-2-(4-((tert-butoxycarbonyl)(4-methoxybenzyl)amino)-3-methylbutoxy)-4-
fluorobenzoate (50). The reaction was carried out according to general procedure 1 and using compounds 33 (4 g)
and 40. The product was purified by columm chromatography on silica gel using petroleum ether/EtOAc (10/1 to
3/1) as eluent to give the title compound (5 g, 46%) as a colorless gum. 1H NMR (400 MHz, CDCl3) δ ppm 8.04 (d, J =
8.4 Hz, 1H), 7.14 - 7.13 (m, 2H), 6.85 - 6.80 (m, 2H), 6.71 - 6.70 (m, 1H), 4.45 - 4.37 (m, 2H), 3.99 - 3.98 (m, 2H), 3.85
(s, 3H), 3.78 (s, 3H), 3.16 - 3.08 (m, 2H), 2.12 - 2.11 (m, 1H), 1.88 - 1.87 (m, 1H), 1.62 - 1.61 (m, 1H), 1.46 (s, 9H), 0.94
(d, J = 6.8 Hz, 3H).
Methyl 2-(3-aminopropoxy)-5-bromobenzoate (51). The reaction was carried out according to general
procedure 2 and using compound 42 (1.6 g) to give the title compound (1.1 g, 93%). 1H NMR (400 MHz, DMSO- d6) δ
ppm 7.83 (d, J = 2.8 Hz, 1H), 7.80 (br s, 2H), 7.74 (dd, J = 9.0, 2.8 Hz, 1H), 7.15 (d, J = 9.0 Hz, 1H), 4.15 (t, J = 5.8 Hz,
2H), 3.81 (s, 3H), 3.03 (sxt, J = 6.3 Hz, 2H), 2.02 (quin, J = 6.4 Hz, 2H). MS (ESI): 288.0 [(M + H) (79Br)]+.
Methyl 2-(4-aminobutoxy)-5-bromobenzoate (52). The reaction was carried out according to general procedure
2 and using compound 43 (130 g) to give the title compound (90 g, 92%) as yellow solid. 1H NMR (400 MHz, DMSO-
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d6) δ ppm 7.77 (d, J = 2.8 Hz, 1H), 7.70 (dd, J = 8.9, 2.6 Hz, 1H), 7.14 (d, J = 8.8 Hz, 1H), 4.11 - 4.01 (m, 2H), 3.81 (s, 3H),
3.71 - 3.10 (m, 2H), 2.92 - 2.77 (m, 2H), 1.85 - 1.61 (m, 4H). MS (ESI): 302.0 [(M + H) (79Br)]+.
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Methyl 2-((5-aminopentyl)oxy)-5-bromobenzoate (53). The reaction was carried out according to general
procedure 2 and using compound 44 (16.6 g) to give the title compound (11.9 g, 94%) as colorless oil. 1H NMR (400
MHz, CDCl3) δ ppm 8.18 – 7.93 (m, 2H), 7.89 (d, J = 2.5 Hz, 1H), 7.55 (dd, J = 8.8, 2.5 Hz, 1H), 6.84 (d, J = 9.0 Hz, 1H),
4.03 (t, J = 5.9 Hz, 2H), 3.86 (s, 3H), 3.14 – 2.99 (m, 2H), 1.90 - 1.79 (m, 4H), 1.71 - 1.57 (m, 2H). MS (ESI): 316.1 [M +
H (79Br)]+.
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rac-Methyl 2-((4-aminopentyl)oxy)-5-bromobenzoate (54). The reaction was carried out according to general
procedure 2 and using compound 45 (16.6 g) to give the title compound (12.6 g, 99%) as a colorless oil. 1H NMR (400
MHz, CDCl3) δ ppm 7.94 (d, J = 2.8 Hz, 1H), 7.55 (dd, J = 8.9, 2.6 Hz, 1H), 6.83 (d, J = 8.8 Hz, 1H), 6.39 (br s, 2H), 4.08 –
3.99 (m, 2H), 3.84 (s, 3H), 3.45 - 3.33 (m, 1H), 2.03 – 1.77 (m, 4H), 1.33 (d, J = 6.5 Hz, 3H). MS (ESI): 316.1 [(M + H)
(79Br)]+.
rac-Methyl 2-(4-amino-3-methylbutoxy)-5-bromobenzoate (55). A mixture of compound 46 (400 mg, 0.9 mmol)
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and N2H4 H2O (45 mg, 0.9 mmol) in MeOH (8 mL) was stirred at 50 °Cfor 47 h. After this time the solvent was removed
in vacuum to give the crude product which was dissolved in H2O (10 mL) and extracted with DCM/MeOH (10/1, 3 x
20 mL). The separated organic phase was dried over Na2SO4, filtered and concentrated in vacuum to give the crude
title compound (200 mg, 70% crude) as colorless gum. MS (ESI): 318.0 [(M + H) (81Br)]+.
rac-Methyl 2-(4-amino-2-methylbutoxy)-5-bromobenzoate (56). The reaction was carried out according to
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general procedure 2 and using compound 47 (12.7 g) to give the title compound (9.1 g, 94%) as a colorless oil. H
NMR (400 MHz, CDCl3) δ ppm 7.96 (d, J = 2.5 Hz, 1H), 7.56 (dd, J = 8.9, 2.6 Hz, 1H), 6.85 (d, J = 9.0 Hz, 1H), 7.08 – 6.14
(m, 2H), 3.93 - 3.86 (m, 2H), 3.85 (s, 3H), 3.21 - 3.02 (m, 2H), 2.30 - 2.13 (m, 1H), 1.98 - 1.84 (m, 1H), 1.84 - 1.70 (m,
1H), 1.05 (d, J = 6.8 Hz, 3H). MS (ESI): 316.1 [(M + H) (79Br)]+.
rac-Methyl 2-((5-aminopentan-2-yl)oxy)-5-bromobenzoate (57). A mixture of compound 48 and N2H4.H2O (944
mg, 18.5 mmol) in MeOH (50 mL) was stirred at 50 °C for 17 h. After this time the volatiles were removed under
vacuum and the resulting residue was dissolved in H2O (20 mL). The pH was adjusted to 11 – 12 by addition of aq.
NaOH and the mixture was extrated with EtOAc (3 x 50 mL). The combined organic extracts were dried over Na2SO4,
filtered and concentrated to give the title compound (3.5 g, 90%) as a brown solid. MS (ESI): 315.9 [(M + H) (79Br)]+.
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rac-Methyl 2-((4-aminopentyl)oxy)-5-bromo-4-fluorobenzoate (58). The reaction was carried out according to
general procedure 2 and using compound 49 (51 g) to give the title compound (44 g, 89%) as a white solid. MS (ESI):
333.9 [(M + H) (79Br)]+.
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rac-Methyl 2-(4-amino-3-methylbutoxy)-5-bromo-4-fluorobenzoate (59).
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Step 1: A mixture of compound 50 (4.5 g, 8.1 mmol) and CAN (13.2 g, 25 mmol) in CH3CN/water (1/1, 100 mL) was
stirred at 10 °C for 30 min. After this time water (500 mL) was added and the mixture was extracted with EtOAc (3 x
200 mL). The combined organic extracts were dried over Na2SO4, filtered and concentrated to give the title compound
(5 g) as a colorless gum. MS (ESI): 434.0 [(M + H) (79Br)]+. Step 2: The reaction was carried out according to general
procedure 2 using the compound isolated in Step 1 (3.5 g) to give the title compound (1.8 g, 67% over 2 steps) as an
orange gum which was used without purification in the next step. MS (ESI): 334.0 [(M + H) (79Br)]+.
2-(3-(3-(6-Aminopyridin-2-yl)-4H-1,2,4-triazol-4-yl)propoxy)-5-bromobenzoic acid (60). Step 1. The reaction
was carried out according to general procedure 3, step 1 and using compound 51 (1.18 g) and compound 29 (0.54
g). The product was purified by column chromatography using a gradient of elution: DCM/MeOH (100/1 to 1/1) to
give the desired product as a mixture of partially hydrolyzed products which was used without further purification
in the next step (670 mg). The main product had: MS (ESI): 460.0 [(M + H) (79Br)]+. Step 2. The reaction was carried
out according to general procedure 3, step 2 and using the product from Step 1 (476 mg) to give the title compound
(275 mg, 30% over 2 steps) as a yellow oil. MS (ESI): 418.1 [(M + H) (79Br)]+.
2-(4-(3-(6-Aminopyridin-2-yl)-4H-1,2,4-triazol-4-yl)butoxy)-5-bromobenzoic acid (61). Step 1. The reaction
was carried out according to general procedure 3, step 1 and using compound 52 (17 g) and compound 29 (15.5 g).
The product was purified by column chromatography using a gradient of elution: DCM/MeOH (100/1 to 1/1) to give
the desired product as a mixture of partially hydrolyzed products which was used without further purification in the
next step (26 g). The main product had: MS (ESI): 474.1 [(M + H) (79Br)]+. Step 2. The reaction was carried out
according to general procedure 3, step 2 and using the product from Step 1 (26 g) to give the title compound (23 g,
97% over 2 steps) as a yellow oil. MS (ESI): 432.1 [(M + H) (79Br)]+.
2-((5-(3-(6-Aminopyridin-2-yl)-4H-1,2,4-triazol-4-yl)pentyl)oxy)-5-bromobenzoic acid (62). Step 1. The
reaction was carried out according to general procedure 3, step 1 and using compound 53 (4.8 g) and compound 29
(2.0 g)The product was purified by column chromatography using a gradient of elution: DCM/MeOH (100/1 to 1/1)
to give the desired product as a mixture of partially hydrolyzed products which was used without further purification
in the next step (3.0 g). The main product had: MS (ESI): 488.1 [(M + H) (79Br)]+. Step 2. The reaction was carried out
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according to general procedure 3, step 2 and using the product from Step 1 (3.0 g) to give the title compound (495
mg, 19% over 2 steps) as a yellow oil. MS (ESI): 446.1 [(M + H) (79Br)]+.
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rac-2-((4-(3-(6-Aminopyridin-2-yl)-4H-1,2,4-triazol-4-yl)pentyl)oxy)-5-bromobenzoic acid (63). Step 1. The
reaction was carried out according to general procedure 3, step 1 and using compound 54 (12.6 g) and compound
29 (5.2 g). The product was purified by column chromatography using a gradient of elution: DCM/MeOH (100/1 to
1/1) to give the desired product as a mixture of partially hydrolyzed products which was used without further
purification in the next step (2.4 g). The main product had: MS (ESI): 488.1 [(M + H) (79Br)]+. Step 2. The reaction
was carried out according to general procedure 3, step 2 and using the product from Step 1 (2.3 g) to give the title
compound (1.1 g, 94% over 2 steps) as a yellow oil. MS (ESI): 446.1 [(M + H) (79Br)]+.
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rac-2-(4-(3-(6-Aminopyridin-2-yl)-4H-1,2,4-triazol-4-yl)-3-methylbutoxy)-5-bromobenzoic acid (64). Step
1. The reaction was carried out according to general procedure 3, step 1 and using compound 55 (200 mg) and
compound 29 (138 mg). The product was purified by column chromatography using a gradient of elution:
DCM/MeOH (30/1 to 1/1) to give the desired product as a mixture of partially hydrolyzed products which was used
without further purification in the next step (50 mg). The main product had: MS (ESI): 490.0 [(M + H) (81Br)]+. Step
2. The reaction was carried out according to general procedure 3, step 2 and using the product from Step 1 (50 mg)
to give the title compound (60 mg, 19%) as a yellow oil. MS (ESI): 447.9 [(M + H) (81Br)]+.
rac-2-(4-(3-(6-Aminopyridin-2-yl)-4H-1,2,4-triazol-4-yl)-2-methylbutoxy)-5-bromobenzoic acid (65). Step
1. The reaction was carried out according to general procedure 3, step 1 and using compound 56 (9.1 g, 1.0 equiv.)
and compound 29 (3.8 g). The product was purified by column chromatography using a gradient of elution:
DCM/MeOH (30/1 to 1/1) to give the desired product as a mixture of partially hydrolyzed products which was used
without further purification in the next step (4.9 g). The main product had: MS (ESI): 488.0 [(M + H) (79Br)]+. Step 2.
The reaction was carried out according to general procedure 3, step 2 and using the product from Step 1 (4.9 g) to
give the title compound (4.4 g, 68%) as a yellow solid. MS (ESI): 446.1 [(M + H) (79Br)]+.
rac-2-((5-(3-(6-Aminopyridin-2-yl)-4H-1,2,4-triazol-4-yl)pentan-2-yl)oxy)-5-bromobenzoic acid (66). Step
1. The reaction was carried out according to general procedure 3, step 1 and using compound 57 (3.5 g) and
compound 29 (4.9 g). The product was purified by column chromatography using a gradient of elution: DCM/MeOH
(30/1 to 1/1) to give the desired product as a mixture of partially hydrolyzed products which was used without
further purification in the next step (2.0 g). The main product had: MS (ESI): 480.0 [(M + H) (81Br)]+. Step 2. The
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reaction was carried out according to general procedure 3, step 2 and using the product from Step 1 (2.0 g) to give
the title compound (1.83 g, 48%) as a yellow solid. MS (ESI): 446.1 [(M + H) (79Br)]+.
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rac-2-((4-(3-(6-Aminopyridin-2-yl)-4H-1,2,4-triazol-4-yl)pentyl)oxy)-5-bromo-4-fluorobenzoic acid (67).
Step 1. The reaction was carried out according to general procedure 3, step 1 and using compound 58 (15 g, 1.2
equiv.) and compound 29 (4.9 g). The product was purified by column chromatography using a gradient of elution:
DCM/MeOH (30/1 to 1/1) to give the desired product as a mixture of partially hydrolyzed products which was used
without further purification in the next step (16 g). The main product had: MS (ESI): 508.0 [(M + H) (81Br)]+. Step 2.
The reaction was carried out according to general procedure 3, step 2 and using the product from Step 1 (9.0 g) to
give the title compound (9.0 g, 100%) as a yellow solid. MS (ESI): 466.1 [(M + H) (81Br)]+.
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rac-2-(4-(3-(6-Aminopyridin-2-yl)-4H-1,2,4-triazol-4-yl)-3-methylbutoxy)-5-bromo-4-fluorobenzoic acid
(68). Step 1. The reaction was carried out according to general procedure 3, step 1 and using compound 59 (1.8 g)
and compound 29 (1.2 g). The product was purified by column chromatography using a gradient of elution:
DCM/MeOH (30/1 to 1/1) to give the desired product as a mixture of partially hydrolyzed products which was used
without further purification in the next step (800 mg). The main product had: MS (ESI): 508.0 [(M + H) (81Br)]+. Step
2. The reaction was carried out according to general procedure 3, step 2 and using the product from Step 1 (800 mg)
to give the title compound (700 mg, 34%) as a yellow solid. MS (ESI): 464.0 [(M + H) (79Br)]+.
55-Bromo-14H-6-oxa-3-aza-2(2,6)-pyridina-1(3,4)-triazola-5(1,2)-benzenacyclononaphan-4-one (69). The
reaction was carried out according to general procedure 4 and using 60 (471 mg, 1.1 mmol) to give the title
compound (140 mg, 31%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 10.42 (s, 1H), 8.73 (s, 1H), 7.95 (d, J
= 7.8 Hz, 1H), 7.85 (dd, J = 7.5, 0.8 Hz, 1H), 7.46 (s, 1H), 7.44 (d, J = 2.5 Hz, 1H), 7.32 (d, J = 7.8 Hz, 1H), 7.01 (d, J = 9.0
Hz, 1H), 4.78 (br s, 2H), 4.07 (br s, 2H), 2.19 (br s, 2H). MS (ESI): 400.0 [(M + H) (79Br)]+.
55-Bromo-14H-6-oxa-3-aza-2(2,6)-pyridina-1(3,4)-triazola-5(1,2)-benzenacycloundecaphan-4-one (70). The
reaction was carried out according to general procedure 4 and using 62 (231 mg, 0.52 mmol) to give the desired
product (100 mg, 45%). 1H NMR (400 MHz, CDCl3) δ ppm 10.62 (s, 1H), 8.53 (dd, J=8.4, 0.9 Hz, 1H), 8.49 (d, J = 2.8
Hz, 1H), 8.21 (s, 1H), 8.17 (dd, J = 7.7, 0.9 Hz, 1H), 7.93 (t, J = 8.0 Hz, 1H), 7.62 (dd, J = 8.7, 2.6 Hz, 1H), 6.92 (d, J = 8.8
Hz, 1H), 4.33 - 4.26 (m, 2H), 4.25 - 4.19 (m, 2H), 2.29 - 2.17 (m, 2H), 2.11 - 2.02 (m, 2H), 1.99 - 1.89 (m, 2H). MS (ESI):
428.1[(M + H) (79Br)]+.
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rac-55-Bromo-10-methyl-14H-6-oxa-3-aza-2(2,6)-pyridina-1(3,4)-triazola-5(1,2)-benzenacyclodecaphan-4-
one (71). The reaction was carried out according to general procedure 4 and using 63 (0.5 g, 1.1 mmol) to give the
desired product (178 mg, 39%) as a beige solid. 1H NMR (400 MHz, CD3OD) δ ppm 9.14 (s, 1H), 8.13 (s, 1H), 8.06 -
8.01 (m, 1H), 7.97 - 7.93 (m, 1H), 7.86 (d, J = 7.3 Hz, 1H), 7.65 (br d, J = 8.8 Hz, 1H), 7.12 (d, J = 9.0 Hz, 1H), 4.82 - 4.71
(m, 1H), 4.52 - 4.42 (m, 1H), 4.24 - 4.13 (m, 1H), 3.31 - 3.23 (m, 1H), 2.33 - 2.17 (m, 1H), 1.95 - 1.82 (m, 2H), 1.68 (d,
J = 6.8 Hz, 3H). MS (ESI): 428.1 [(M + H) (79Br)]+.
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rac-55-Bromo-9-methyl-14H-6-oxa-3-aza-2(2,6)-pyridina-1(3,4)-triazola-5(1,2)-benzenacyclodecaphan-4-
one (72). A mixture of compound 64 (40 mg, 0.09 mmol) in T3P (1 mL, ≥50 wt. % in EtOAc) and Et3N (1 mL) was
stirred at 50 °C for 2 h. After this time the solvent was removed in vacuum to give the crude product which was
dissolved in H2O (10 mL) and extracted with DCM (2 x 10 mL). The organic phase was dried over Na2SO4, filtered and
concentrated in vacuum. The crude product was purified by prep-TLC (DCM/MeOH = 10/1) to provide the title
compound (15 mg, 33%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 11.19 (s, 1H), 8.69 (s, 1H), 8.07 - 8.05
(m, 2H), 7.91 - 7.87 (m, 2H), 7.80 - 7.78 (m, 1H), 7.29 (d, J = 8.8 Hz, 1H), 4.52 - 4.42 (m, 2H), 4.28 - 4.23 (m, 1H), 3.92
- 3.86 (m, 1H), 3.22 - 3.21 (m, 1H), 2.08 - 2.02 (m, 1H), 1.71 - 1.65 (m, 1H), 0.96 (d, J = 6.8 Hz, 3H). MS (ESI): 429.8 [(M
+ H) (81Br)]+. Chiral separation: compound 72 (15 mg) was purified by SFC (using a Chiralpak AD-3 3m, 50x4.6mm
column and using 40% ethanol (containing 0.05% Et2NH) in CO2 as the mobile phase at a flow rate of 4 mL/min (at
a column temperature of 40 °C)) to give in order of elution. Peak 1 (absolute stereochemistry arbitrarily assigned):
(R)-55-bromo-9-methyl-14H-6-oxa-3-aza-2(2,6)-pyridina-1(3,4)-triazola-5(1,2)-benzenacyclodecaphan-4-one (72a,
5 mg, 33%) as a white solid. δ ppm 11.19 (s, 1H), 8.69 (s, 1H), 8.07 - 8.05 (m, 2H), 7.91 - 7.87 (m, 2H), 7.80 - 7.78 (m,
1H), 7.29 (d, J = 8.8 Hz, 1H), 4.52 - 4.42 (m, 2H), 4.28 - 4.23 (m, 1H), 3.92 - 3.86 (m, 1H), 3.22 - 3.21 (m, 1H), 2.08 -
2.02 (m, 1H), 1.71 - 1.65 (m, 1H), 0.96 (d, J = 6.8 Hz, 3H). MS (ESI): 429.8 [(M + H) (81Br)]+. Peak 2 (absolute
stereochemistry arbitrarily assigned): (S)-55-bromo-9-methyl-14H-6-oxa-3-aza-2(2,6)-pyridina-1(3,4)-triazola-
5(1,2)-benzenacyclodecaphan-4-one (72b, 5 mg, 33%) as a white solid. δ ppm 11.19 (s, 1H), 8.69 (s, 1H), 8.07 - 8.05
(m, 2H), 7.91 - 7.87 (m, 2H), 7.80 - 7.78 (m, 1H), 7.29 (d, J = 8.8 Hz, 1H), 4.52 - 4.42 (m, 2H), 4.28 - 4.23 (m, 1H), 3.92
- 3.86 (m, 1H), 3.22 - 3.21 (m, 1H), 2.08 - 2.02 (m, 1H), 1.71 - 1.65 (m, 1H), 0.96 (d, J = 6.8 Hz, 3H). MS (ESI): 430.0 [(M
+ H) (81Br)]+. Compound 72a was found to be more potent in the biochemical assay.
rac-55-Bromo-8-methyl-14H-6-oxa-3-aza-2(2,6)-pyridina-1(3,4)-triazola-5(1,2)-benzenacyclodecaphan-4-
one (73). The reaction was carried out according to general procedure 4 and using 65 (0.5 g, 1.1 mmol) to give the
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desired product (220 mg, 36%) as a beige solid. H NMR (400 MHz, CD3OD) δ ppm 9.20 (s, 1H) 8.21 (d, J = 2.8 Hz,
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1H), 8.13 - 8.01 (m, 2H), 7.95 (d, J = 7.3 Hz, 1H), 7.72 (dd, J = 8.8, 2.8 Hz, 1H), 7.23 (d, J = 9.0 Hz, 1H), 4.52 (s, 2H), 4.41
- 4.34 (m, 1H), 4.06 – 3.96 (m, 1H), 2.24 - 2.10 (m, 1H), 1.86 - 1.70 (m, 1H), 1.38 - 1.30 (m, 1H), 1.27 (d, J = 7.0 Hz, 3H).
MS (ESI): 428.1 [(M + H) (79Br)]+.
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rac-55-Bromo-7-methyl-14H-6-oxa-3-aza-2(2,6)-pyridina-1(3,4)-triazola-5(1,2)-benzenacyclodecaphan-4-
one (74). A mixture of 66 (1.83 g, 4.1 mmol) in T3P (15 mL, ≥50 wt. % in EtOAc) and Et3N (15 mL) was stirred at 70
°C for 3 h. After this time the volatiles were removed under reduced pressure to give the crude product. H2O (50 mL)
was then added and the resulting solid was collected by filtration and dried under vacuum to give the title compound
(1.0 g, 57%) as a brown solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 11.19 (s, 1H), 8.69 (s, 1H), 8.12 - 8.04 (m, 2H), 7.94
- 7.90 (m, 2H), 7.77 (dd, J = 8.8, 2.8 Hz, 1H), 7.29 (d, J = 8.8 Hz, 1H), 5.11 - 5.03 (m, 1H), 4.34 - 4.19 (m, 2H), 3.02 – 2.88
(m, 1H), 2.09 – 1.88 (m, 3H), 1.34 (d, J = 6.4 Hz, 3H). MS (ESI): 429.9 [(M + H) (81Br)]+. Chiral separation: Compound
74 (100 mg) was purified by SFC (using a Chiralpak AD-3 3m, 50x4.6mm column and using 40% ethanol (containing
0.05% Et2NH) in CO2 as the mobile phase at a flow rate of 4 mL/min (at a column temperature of 40 °C)) to give in
order of elution. Peak 1 (absolute stereochemistry arbitrarily assigned): (S)-55-bromo-7-methyl-14H-6-oxa-3-aza-
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1
2(2,6)-pyridina-1(3,4)-triazola-5(1,2)-benzenacyclodecaphan-4-one (74a, 30 mg, 30%) as a white solid. H NMR
(400 MHz, DMSO-d6) δ ppm 11.19 (s, 1H), 8.69 (s, 1H), 8.12 - 8.04 (m, 2H), 7.94 - 7.90 (m, 2H), 7.77 (dd, J = 8.8, 2.8
Hz, 1H), 7.29 (d, J = 8.8 Hz, 1H), 5.11 - 5.03 (m, 1H), 4.34 - 4.19 (m, 2H), 3.02 – 2.88 (m, 1H), 2.09 – 1.88 (m, 3H), 1.34
(d, J = 6.4 Hz, 3H). MS (ESI): 429.9 [(M + H) (81Br)]+. Peak 2 (absolute stereochemistry arbitrarily assigned): (R)-55-
bromo-7-methyl-14H-6-oxa-3-aza-2(2,6)-pyridina-1(3,4)-triazola-5(1,2)-benzenacyclodecaphan-4-one (74b, 40
mg, 40%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 11.19 (s, 1H), 8.69 (s, 1H), 8.02 - 8.15 (m, 2H), 7.92
(d, J = 7.8 Hz, 2H), 7.77 (dd, J = 9.0, 2.7 Hz, 1H), 7.29 (d, J = 8.8 Hz, 1H), 5.12 - 5.01 (m, 1H), 4.35 - 4.19 (m, 2H), 3.03 –
2.88 (m, 1H), 2.09 – 1.88 (m, 3H), 1.33 (d, J = 5.9 Hz, 3H). MS (ESI): 427.9 [(M + H) (79Br)]+.
rac-55-Bromo-54-fluoro-10-methyl-14H-6-oxa-3-aza-2(2,6)-pyridina-1(3,4)-triazola-5(1,2)-
benzenacyclodecaphan-4-one (75). The reaction was carried out according to general procedure 4 and using 67
(4.2 g, 9 mmol) to give the desired product (1.0 g, 25%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 11.01
(s, 1H), 8.85 (s, 1H), 8.11 - 8.04 (m, 2H), 7.82 - 7.78 (m, 2H), 7.41 (d, J = 11.2 Hz, 1H), 4.53 - 4.43 (m, 2H), 4.15 - 4.10
(m, 1H), 3.11 - 3.09 (m, 1H), 2.06 - 2.04 (m, 1H), 1.78 - 1.69 (m, 2H), 1.50 (d, J = 6.8 Hz, 3H). MS (ESI): 446.0 [(M + H)
(79Br)]+.
rac-55-Bromo-54-fluoro-9-methyl-14H-6-oxa-3-aza-2(2,6)-pyridina-1(3,4)-triazola-5(1,2)-
benzenacyclodecaphan-4-one (76). A mixture of 68 (800 mg, 1.7 mmol) in T3P (20 mL, ≥50 wt. % in EtOAc) and
29
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