Evolution of the Synthesis of AMPK Activators for the Treatment of Diabetic Nephropathy: From Three Preclinical Candidates to the Investigational New Drug PF-06409577

Article abstract of DOI:10.1021/acs.oprd.8b00059

Indole acids 1, 2, and 3 are potent 5′-adenosine monophosphate-activated protein kinase (AMPK) activators for the potential treatment of diabetic nephropathy. Compounds 1-3 were scaled to supply material for preclinical studies, and indole 3 was selected for advancement to first-in-human clinical trials and scaled to kilogram quantities. The progression of the synthesis strategy for these AMPK activators is described, as routes were selected for efficient structure-activity relationship generation and then improved for larger scales. The developed sequences employed practical isolations of intermediates and APIs, reproducible cross-coupling, hydrolysis, and other transformations, and enhanced safety and purity profiles and led to the production of 40-50 g of 1 and 2 and 2.4 kg of 3. Multiple polymorphs of 3 were observed, and conditions for the reproducible formation of crystalline material suitable for clinical development were identified.

Full text of DOI:10.1021/acs.oprd.8b00059

Subscriber access provided by READING UNIV
Full Paper
The Evolution of the Synthesis of AMPK Activators for the
Treatment of Diabetic Nephropathy: from Three Preclinical
Candidates to the Investigational New Drug PF–06409577
Aaron C. Smith, Daniel W. Kung, Andre Shavnya, Thomas A Brandt, Philip D Dent, Nathan
Edward Genung, Shawn Cabral, Jane Panteleev, Michael Herr, Ka Ning Yip, Gary E Aspnes,
Edward L. Conn, Matthew S. Dowling, David J. Edmonds, Ian D Edmonds, Dilinie Fernando,
Paul Matthew Herrinton, Nandell F Keene, Sophie Lavergne, Qifang Li, Jana Polivkova,
Colin R. Rose, Benjamin A. Thuma, Michael G Vetelino, Guoqiang Wang, John D Weaver,
Daniel W Widlicka, Kristin E Price Wiglesworth, Jun Xiao, Todd Zahn, and Yingxin Zhang
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.8b00059 • Publication Date (Web): 10 May 2018
Downloaded from http://pubs.acs.org on May 10, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 51
Organic Process Research & Development
1
2
3
4
5
6
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
ACS Paragon Plus Environment

Organic Process Research & Development
Page 2 of 51
1
2
3
4
5
6
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
The Evolution of the Synthesis of AMPK Activators
for the Treatment of Diabetic Nephropathy: from
Three Preclinical Candidates to the Investigational
New Drug PF-06409577
Aaron C. Smith,*,† Daniel W. Kung,*,† Andre Shavnya,† Thomas A. Brandt,† Philip D. Dent,†
Nathan E. Genung,† Shawn Cabral,† Jane Panteleev,† Michael Herr,† Ka Ning Yip,# Gary E.
Aspnes,# Edward L. Conn,† Matthew S. Dowling,† David J. Edmonds,# Ian D. Edmonds, § Dilinie
P. Fernando,† Paul M. Herrinton,○ Nandell F. Keene,† Sophie Y. Lavergne,† Qifang Li,† Jana
Polivkova,† Colin R. Rose,† Benjamin A. Thuma,† Michael G. Vetelino,† Guoqiang Wang,† John
D. Weaver, III,† Daniel W. Widlicka,† Kristin E. Price Wiglesworth,† Jun Xiao,† Todd Zahn,○
Yingxin Zhang†
†Pfizer Worldwide Research & Development; Eastern Point Road; Groton, Connecticut 06340;
United States.
1
ACS Paragon Plus Environment

Page 3 of 51
Organic Process Research & Development
1
2
3
4
5
#Pfizer Worldwide Research & Development; 610 Main Street; Cambridge, Massachusetts
02139; United States.
6
7
8
9
§Bridge Organics; 311 W. Washington St.; Vicksburg, Michigan 49097; United States
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
○BoroPharm, Inc.; 39555 Orchard Hill Place, Suite 600; Novi, Michigan 48375; United States
Table of Contents Graphic
2
ACS Paragon Plus Environment

Organic Process Research & Development
Page 4 of 51
1
2
3
4
ABSTRACT
5
6
7
8
9
Indole acids 1, 2, and 3 are potent 5’-adenosine-monophosphate-activated protein kinase
(AMPK) activators for the potential treatment of diabetic nephropathy. Compounds 1–3 were
scaled to supply material for pre-clinical studies, and indole 3 was selected for advancement to
first-in-human clinical trials and was scaled to kilogram quantities. The progression of the
synthesis strategy for these AMPK activators is described, as routes were selected for efficient
SAR generation and then were improved for larger scales. The developed sequences employed
practical isolations of intermediates and APIs, reproducible cross-coupling, hydrolysis, and other
transformations, and enhanced safety and purity profiles leading to production of 40–50 g of 1
and 2, and 2.4 kg of 3. Multiple polymorphs of 3 were observed and conditions for the
reproducible formation of crystalline material suitable for clinical development were identified.
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
Keywords: Suzuki, Negishi, Borylation, Acylation, AMPK, Pinnick
3
ACS Paragon Plus Environment

Page 5 of 51
Organic Process Research & Development
1
2
3
4
5
6
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
Figure 1. Potent Activators of the AMPK α1β1γ1 Isoform
INTRODUCTION
5’-Adenosine monophosphate-activated protein kinase (AMPK) is a heterotrimeric serine-
threonine kinase (consisting of α-, β-, and γ-subunits), which plays a key role in energy
homeostasis. Indole acids 1, 2, and 3 (Figure 1) were recently identified as potent activators of
β1-containing isoforms of AMPK and were evaluated in safety and efficacy studies.1,2,3 In
addition, compound 3 was advanced to first-in-human clinical trials as a potential treatment for
diabetic nephropathy. The development of improved synthetic routes for producing multi-gram
quantities of 1 and 2, and kilogram quantities of 3, is described herein.
Scheme 1. General Synthetic Strategies Employed to Study SAR of Lead Series
4
ACS Paragon Plus Environment

Organic Process Research & Development
Page 6 of 51
1
2
3
4
5
6
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
During the discovery phase of the AMPK program, a lead series consisting of a 5-aryl-indole-
3-carboxylate was found to activate β1-containing isoforms.1 Several different synthetic
strategies (Scheme 1) were identified to quickly explore SAR of compounds in the lead series.
Initial syntheses generally relied upon installation of the 5-aryl moiety through a cross-coupling
reaction to a 5-bromo-indole, followed by introduction of the 3-carboxylic acid via formylation
and oxidation of the intermediate aldehyde [Eq. (1)]. The order of the reaction steps was also
reversed by introduction of the aldehyde first followed by cross-coupling with an aryl boronate.
Subsequent routes enabled late-stage diversification of the aryl group through installation of an
ester rather than an aldehyde to simplify chemistry downstream of the coupling [Eq. (2)]. A third
variation of this sequence was also employed where the reactivity of the Suzuki coupling
partners (halide, boronate) was inverted by employing an indole boronate [Eq. (3)], thereby
expanding the available reagent set to include aryl and heteroaryl halide coupling partners (rather
than the less readily available heteroaryl boronic acids or boronates). These general
disconnections and synthetic strategies served as the basis to develop improved syntheses of
5
ACS Paragon Plus Environment

Page 7 of 51
Organic Process Research & Development
1
2
3
4
5
6
7
8
indoles 1, 2, and 3 on larger scales while also managing reaction concentrations, impurity
profiles, reagent costs, and isolation and crystallization of solid intermediates and final products.
RESULTS AND DISCUSSION
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
Discovery Route to Indole 1
Scheme 2. Synthesis of THP-Aryl-Boronate 7
The discovery synthesis of 1 relied upon Suzuki coupling between boronate ester 7 and indole
aldehyde 12. The initial synthesis of boronate 7 started with a Prins cyclization between p-
bromobenzaldehyde 4 and 3-buten-1-ol to form bromotetrahydropyran 5 (Scheme 2).4
Elimination to afford the olefin and subsequent hydrogenation provided aryl bromide 6 which
was then converted to boronate ester 7. Although successful, the isolation of 7 was complicated
by the presence of excess potassium acetate and chromatography was required to achieve >95%
purity.
Scheme 3. Discovery Synthesis of 1
6
ACS Paragon Plus Environment

Organic Process Research & Development
Page 8 of 51
1
2
3
4
5
6
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
The difluoroindole was initially synthesized from isatin 8 (Scheme 3). A regioselective
bromination followed by reduction with borane5 afforded the indole in low yield (31% over two
steps). Difficulty in controlling the reduction (both over- and under-reduced byproducts were
identified) and complications in chromatographic separations contributed to the low yield.
Higher yields and simplified purification of 5-bromo-4,6-difluoroindole 9 were achieved in an
alternative three-step sequence consisting of reduction of commercially available 4,6-
difluoroindole 10 to the indoline, regioselective bromination and chromatography-free isolation
of bromoindoline 11 as its HCl salt, and oxidation of the indoline to indole with DDQ (44% over
three steps). Formylation under Vilsmeier-Haack conditions led to 3-aldehyde 12. Suzuki
coupling between 12 and boronate ester 7 followed by Pinnick oxidation provided the racemic
carboxylic acid 1. Although the coupling was successful, low solubilities of the coupling product
led to high dilutions (75 volumes), incomplete conversion of starting material, and difficulties in
isolation as HPLC purification was employed on analogue scale. SFC purification on chiral
phase was then used to isolate the eutomer, whose absolute configuration was determined by
single-crystal X-ray analysis.6
7
ACS Paragon Plus Environment

Page 9 of 51
Organic Process Research & Development
1
2
3
4
5
6
7
8
9
Although this route was successful for milligram scale, several problems needed to be
addressed to increase practicality for larger scale synthesis: the complex isolation of boronate
ester 7 and indole 9, the low solubility of aldehyde intermediates that led to inconsistent results
and complex isolations at the Suzuki coupling and Pinnick oxidation steps, and the use of SFC
on chiral phase to isolate the final product.
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
Improved Synthesis of 1
Scheme 4. Scalable Synthesis of Boronic Acid 16
To eliminate the need for chromatographic separation of enantiomers, an enantioselective
approach to the tetrahydropyran ring formation was envisioned. Starting from commercially
available ketone 13, several asymmetric reduction methods gave encouraging initial hits,
including Noyori hydrogenation (RuCl(p-cymene)[(S,S)-Ts-DPEN], 93.5:6.5 er of 15)7 and
ketoreductase-catalyzed biotransformation (Codex® KRED-P1 B12, 89% conversion, 98.0:2.0 er
of 14).8 However, CBS reduction9 (Scheme 4) was selected for further optimization due to the
balance of high selectivity, yield, and control over the process. On a 50-g scale, alcohol 14 was
obtained as a solid product after aqueous workup (95% yield, 98.4:1.6 er), with temperature
control (–20 °C) being a critical variable to ensure high enantioselectivity. Subsequent
8
ACS Paragon Plus Environment

Organic Process Research & Development
Page 10 of 51
1
2
3
4
5
6
7
8
9
cyclization generated tetrahydropyran 15 without the need for chromatography. Synthesis of the
corresponding boronic acid 16 (rather than the boronate ester) was proposed to facilitate isolation
without chromatography. After utilizing tetrahydroxydiboron10 in a palladium-catalyzed cross-
coupling, the resultant boronic acid was extracted into aqueous basic media, allowing many of
the organic impurities to be purged with an MTBE wash. Neutralization and crystallization then
produced boronic acid 16 with an isolated yield of 72% (26 g) over the first three steps of the
synthesis. Detectable amounts of boroxine were observed by 1H NMR, but this by-product was
not detrimental to downstream chemistry.
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 5. Improved Synthesis of 5-bromo-4,6-difluoro-1H-indole 9
An alternative ring-building strategy was employed to circumvent the challenging redox steps in
the synthesis of the difluoroindole coupling partner. Iodination of the symmetrical 4-bromo-3,5-
difluoroaniline 17 afforded 18 in high yield by precipitation after the addition of aqueous sodium
thiosulfate solution to the reaction mixture (Scheme 5). Sonogashira coupling with TMS-
acetylene (charged in two portions to ensure complete conversion of the iodide) proceeded
smoothly with 1.6 mol% each of copper and palladium catalysts. Solid 19 was isolated after
filtration through Celite and silica, followed by trituration with hexane. Initially, the cyclization
9
ACS Paragon Plus Environment

Page 11 of 51
Organic Process Research & Development
1
2
3
4
5
6
7
8
9
to the indole was effected by the stepwise cleavage of the silyl group followed by rhodium-
catalyzed cycloisomerization.11 Subsequently, the cyclization conditions were simplified by
employing potassium t-butoxide in NMP to effect both desilylation and cyclization.12 When this
conversion was carried out by addition of 19 in one portion to potassium t-butoxide in NMP, the
crude indole 9 was obtained in ~85% purity, but this addition was accompanied by a rapid
exotherm, from 33 to 87 °C on 100-g scale. Attempts to moderate this exotherm by slow addition
of compound 19 resulted in greater formation of impurities (<50% purity of crude product with
addition at 20–25 °C over 110 min, or at 58–65 °C over 30 min). With addition in one portion
the exotherm was complete within one min, such that the conditions were close to adiabatic and
thus were expected to be relatively insensitive to scale. Indeed, on conversion of 1.44 kg of
compound 19, the exotherm (33 to 89 °C) was very close to that observed on the 100-g run.
Although this procedure enabled formation of >0.75 kg of pure indole 9, additional studies are
needed to develop a more controlled process before further scale-up and the rhodium-catalyzed
method could be a potential alternative. For project expediency, this material was isolated by
silica gel chromatography followed by trituration; full optimization of conditions to directly
precipitate the product was not completed, although the purified compound 9 was crystalline
(m.p. 91–92 °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
Scheme 6. Acylations of Difluoroindole 9
10
ACS Paragon Plus Environment

Organic Process Research & Development
Page 12 of 51
1
2
3
4
5
6
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
To mitigate the challenges associated with the low solubility of aldehyde 12, two
complementary strategies were examined. The first approach replaced the aldehyde with an
alternative precursor to the carboxylate group, such as a ketone or ester. For many indoles, the
two-step esterification at C3 via acylation with trichloroacetyl chloride and subsequent
methanolysis13 was readily achieved; however, for indole 20 the yield was <20% (Scheme 6) due
to poor conversion, which we attributed to the electron-withdrawing effect of the two fluoro
substituents. Use of trifluoroacetic anhydride as a more reactive acylating agent provided the
trifluoromethyl ketone 21 in 60% yield. Unfortunately, the trifluoromethyl ketone proved to be
less reactive to methanolysis/hydrolysis than similar trichloromethyl ketones reported in the
literature. Methanolysis proceeded in low yield (31%), although hydrolysis at high temperature
(140 °C) with sodium hydroxide produced the carboxylic acid in 87% yield. Use of the
trifluoromethyl ketone 21 or the acid 23 in the subsequent Suzuki coupling led to low reaction
conversion and significant decarboxylation, respectively, and thus this approach was
discontinued.
Scheme 7. Endgame for Improved Synthesis of 1
11
ACS Paragon Plus Environment

Page 13 of 51
Organic Process Research & Development
1
2
3
4
5
6
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
The alternative strategy for addressing the complications caused by the low solubility of
aldehyde 12 consisted of re-ordering the synthetic steps in such a way that Suzuki coupling
preceded introduction of the aldehyde group (Scheme 7). The Suzuki coupling between boronic
acid 16 and bromoindole 9 proceeded under standard conditions and recrystallization of 24 from
ethyl acetate–heptane after aqueous workup was employed to provide 55% yield, but a consistent
purity profile. One-pot formylation by reaction with the Vilsmeier salt and subsequent hydrolysis
with aqueous sodium hydroxide afforded aldehyde 25, which crystallized directly from the
reaction mixture in 71% yield. The solubility of intermediates used in this sequence was
appropriate to facilitate material handling and to increase yields on this scale, and all
intermediates were isolated by precipitation/crystallization. To increase safety in the Pinnick
oxidation, DMSO14 was employed as a hypochlorite scavenger that was more easily controlled
than the volatile 2-methyl-2-butene. Additionally, the solubility of the reaction components was
improved by switching to 2-Me-THF/DMSO from acetonitrile (Scheme 3) which led to full
conversion of the starting material to greatly facilitate isolation of the final product. After
aqueous workup, the product acid 1 was isolated in 80% yield after precipitation from ethyl
acetate/2-Me-THF. In a separate step, slurrying in ethanol:water (95:5) led to isolation of
12
ACS Paragon Plus Environment

Organic Process Research & Development
Page 14 of 51
1
2
3
4
crystalline 1 (47 g, 96% yield, >99:1 er) in 99.5% purity suitable for exploratory toxicology
5
6
studies.
7
8
Discovery Synthesis of 2
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 8. Early Synthesis of Pyridyl Bromide 28 and Boronate 29
The synthesis of the pyridylindole 2 was initially expected to utilize a similar cross-coupling
disconnection as phenylindole 1. The pyridyl coupling partner was synthesized from
dichloropyridine 26 by two successive SNAr displacements with sodium methoxide and
dimethylamine to afford bromopyridine 28 (Scheme 8). The regioselectivity of the methoxide
addition was ~5:1 in favor of the desired 27, leading to low isolated yields of 28 over two steps,
primarily due to the difficulty in chromatographic separation of the closely eluting regioisomers.
The bromide was then converted to pinacol boronate 29. However, protodeboronation observed
even under mild conditions made isolation problematic and caused concerns about this
intermediate’s stability. Attempts to synthesize boronic acid 30 were also unsuccessful.
Scheme 9. Synthesis of Indole Boronate
13
ACS Paragon Plus Environment

Page 15 of 51
Organic Process Research & Development
1
2
3
4
5
6
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
Given the complexity of isolating the pyridyl boronate, work began on the synthesis of a 5-
borylindole and bromopyridine 28 to invert the roles in the Suzuki coupling. This route
modification also supported the discovery project needs for incorporation of varied (hetero)aryl
substituents at C5 of the indole by employing the widely available (hetero)aryl halide reagents.
Formylation of commercially-available 5-bromo-6-chloroindole 31 under Vilsmeier conditions
provided aldehyde 32 (Scheme 9). The corresponding borylated indole 33, however, could not be
successfully isolated, with the product of debromination/deboronation obtained as the major
product. However, revisiting the acylation approaches previously examined during the synthesis
of difluoroindole 1 showed that the relatively greater nucleophilicity of the chloroindole core
allowed the successful use of an alternative route. Acylation of indole 31 with trichloroacetyl
chloride provided trichloromethyl ketone 34 in good yield as compared to the less electron-rich
difluoroindole 9 (62% vs. 14%, respectively; see also Scheme 6). The trichloromethyl ketone
was treated with sodium methoxide in methanol to produce methyl ester 35. Borylation of this
indole was successful and a good yield (77%) of 36 was obtained; however, chromatography was
necessary to achieve >95% purity. Attempts to synthesize boronic acid 37 afforded no desired
14
ACS Paragon Plus Environment

Organic Process Research & Development
Page 16 of 51
1
2
3
4
5
6
product due to rapid protodeboronation or dehalogenation; further efforts to access the boronate
or to telescope the coupling reaction were not pursued.
7
8
Scheme 10. Small Scale Synthesis of 2
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
For the initial small-scale synthesis of 2 (Scheme 10), Suzuki coupling between bromopyridine
28 and indoleboronate 36 afforded the ester 38 as a solid product (49% after chromatography).
Saponification afforded the desired indole acid, albeit in low isolated yield due to
decarboxylation observed under these conditions. While this route successfully delivered
milligram quantities of 2 for in vitro characterization, critical disadvantages limited its
application on a larger scale. The main problems to be addressed included poor regioselectivity
in the preparation and isolation of bromopyridine 28, isolation and/or stability of 5-borylindole
36, and decarboxylation of 2, which in combination resulted in low yields.
Improved Synthesis of 2 to Support Larger Scale Deliveries
Scheme 11. Improved Synthesis of Bromide 28
15
ACS Paragon Plus Environment

Page 17 of 51
Organic Process Research & Development
1
2
3
4
5
6
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
To address the concerns with the synthesis of bromopyridine 28 on larger scale, the route was
redesigned (Scheme 11). Starting from the less expensive symmetrical 2,6-difluoropyridine 39,
SNAr reaction with dimethylamine hydrochloride in the presence of K2CO3 provided pyridine 40,
which was used directly in the next step. Bromination with N-bromosuccinimide in acetonitrile
was regioselective (>95%) and high-yielding, affording crystalline 41 in 83% yield over two
steps after aqueous workup and trituration. The second SNAr reaction with potassium methoxide
in methanol at reflux cleanly displaced the fluoride to provide 1.4 kg of the desired 28 as a
crystalline solid in 99% yield after aqueous workup.
Since the initial Suzuki coupling was promising, alternative synthetic conditions were
examined to improve formation of boronate ester 36. Unfortunately, screening and optimization
of conditions to improve the synthesis of boronate ester 36 or boronic acid 37 did not lead to the
identification of satisfactory conditions, as poor reactivity and debromination/deboronation were
recurrent problems.
Table 1. Protected Substrates for Negishi Coupling
16
ACS Paragon Plus Environment

Organic Process Research & Development
Page 18 of 51
1
2
3
4
5
6
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
Negishi cross-coupling was considered an attractive alternative because of the stability of the
halide substrates and the ability to form the organometallic intermediate in situ. The organozinc
was formed from bromopyridine 28 by metal-halogen exchange with n-butyllithium followed by
treatment with ZnCl2, and then subjected to Pd coupling with indole bromide 35 (Table 1).
Although it appeared that the organozinc reagent had successfully formed (observed
debromination of the pyridine), no desired product was obtained, which led to the hypothesis that
the acidic NH of the indole interfered with the cross-coupling. N-Protective groups were then
employed to test this hypothesis (Table 1). The N-acetyl protected 42a led to formation of N-
deacylated, uncoupled indole 35 when treated under the coupling conditions. The less labile N-
pivaloyl protected 42b provided the desired cross-coupling as a mixture of N-pivaloyl protected
43b and deprotected 38. After complete deprotection of the mixture in a separate step (1 M
NaOH:MeOH, 1:1, 30 min, rt), 39% of 38 was obtained. Finally, use of the more robust N-tosyl
group led to the most effective coupling without undesirable N-deprotection to afford N-tosyl
indole 43c. After aqueous workup, the cross-coupling product was purified by crystallization
17
ACS Paragon Plus Environment

Page 19 of 51
Organic Process Research & Development
1
2
3
4
(MeOH:EtOAc, 20:1) followed by trituration with MeOH (91% yield) to enhance purity to
5
6
97.2%.
7
8
Scheme 12. Improved Synthesis of 2
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
Removal of both the N-tosyl and the ester protecting groups in one pot was attempted under
various aqueous NaOH conditions, but incomplete deprotection of the sulfonamide or N-
methylation of the indole (with MeOH as cosolvent) was observed. Instead, a controlled two-step
process was successful, employing tetrabutylammonium fluoride (TBAF) in THF at reflux to
cleave the N-tosyl group to provide the pyridylindole ester 38 (Scheme 12) in 89% yield after
workup and trituration. Subsequent hydrolysis of the ester afforded 38 g of the carboxylic acid
which was isolated after workup and recrystallization from heptane. It is important to note that
decarboxylation was observed at higher temperatures (3–4% decarboxylation at 60 °C, 20–24 h;
>15% at 70 °C, 15 h) or with long reaction times (4% at 50 °C, 110 h); however, decarboxylation
by-product formation was reduced (2%) by maintaining the temperature between 50–55 °C and
employing a moderate reaction time (36–48 h).
Discovery Synthesis of 3
Scheme 13. Discovery Route to 3
18
ACS Paragon Plus Environment

Organic Process Research & Development
Page 20 of 51
1
2
3
4
5
O
O
O
O
B
B
n-BuLi
OH
O
Pd(dppf)Cl2, KOAc
dioxane, 110 °C
OH
Br
6
O
B
7
8
9
THF, < -75 °C
89%
>95%
chromatography
necessary
Br
Br
O
44
45
46
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
OH
OH
potential safety risk
46
1. , Pd(dppf)Cl2
O
O
(aq.) 2M K2CO3, EtOH
PhCH3, 95 °C, 70%
OH
NaClO2, NaH2PO4
2-methyl-2-butene
31
2. POCl3, DMF, 0%
benzylic 3o alcohol
CH3CN, t-BuOH,
water, (0.02 M)
57%
N
N
Cl
Cl
H
H
47
3
incompatible with Vilsmeier conditions
46
, Pd(dppf)Cl2
(aq.) 2M K2CO3, EtOH
PhCH3,110 °C, 64%
32
chromatography
necessary
The first synthesis of 3 began with the general strategy utilized for 1 and consisted of Suzuki,
formylation, and oxidation steps. Boronate 46 was synthesized starting from 1,4-
dibromobenzene 44 through metal-halogen exchange and quenching with cyclobutanone to
provide mono-bromide 45, followed by its conversion to the neopentylglycol boronate 46 in
good yields (Scheme 13). Suzuki coupling between boronate 46 and indole 31 installed the 5-
aryl group successfully. However, formylation in the presence of the tertiary benzylic alcohol
proved problematic and decomposition was observed, presumably due to facile formation of the
tertiary benzylic cation. This problem was not mitigated by protecting the hydroxyl group as
various ethers. The steps were reordered and aldehyde 32 was employed directly in the Suzuki
coupling to provide the desired indole 47 in 64% yield. Pinnick oxidation then provided the
desired carboxylic acid analog 3.
Although this route was successful for delivering milligram quantities of 3, its use was
precluded for accessing larger amounts of material due to issues similar to the other routes, as the
isolation of boronate 46 was not feasible without chromatography and the Suzuki coupling and
Pinnick oxidation were complicated by the low solubility of aldehydes 32 and 47.
19
ACS Paragon Plus Environment

Page 21 of 51
Organic Process Research & Development
1
2
3
4
Improved Synthesis of 3 for Hundred-Gram Scale
5
6
Scheme 14. Improved Synthesis of 3
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
In a strategy similar to the one used for 1, the boronic acid was targeted as the practical
coupling intermediate (Scheme 14) based on the hypothesis that it could be isolated in pure form
without chromatography unlike the boronate ester 46. The borylation of bromide 45 with
tetrahydroxydiboron proceeded smoothly on a hundred-gram scale, allowing for isolation of
boronic acid 48 through extraction into a basic aqueous phase followed by acidification and
crystallization in 65% yield.
The strategy of introducing the aldehyde after Suzuki coupling that was employed for aldehyde
25 (see Scheme 7) was impractical for aldehyde 47, due to the sensitivity of the tertiary benzylic
alcohol to the acidic formylation conditions. Therefore, an alternative sequence of bond
constructions was employed. The Suzuki reaction between boronic acid 48 and bromoindole
ester 35 proceeded smoothly to provide the desired product in 64% yield. Isolation and
purification was achieved through recrystallization from ethyl acetate:heptane (1:1.25) and
trituration from methanol and toluene to provide ester 49. To complete the synthesis, sodium
hydroxide and methanol were used to effect saponification of the methyl ester, although this
hydrolysis reaction was somewhat challenging. At lower temperature (50 °C, 15 h) incomplete
conversion (62%) of the electron rich indole-ester was observed, while at 70 °C or above,
significant decarboxylation of the resultant carboxylic acid occurred. Ultimately, for simplicity
20
ACS Paragon Plus Environment

Organic Process Research & Development
Page 22 of 51
1
2
3
4
5
6
7
8
9
of purification, we opted to heat this reaction at 70 °C for 18 h to favor complete conversion at
the expense of 17% observed decarboxylation. Indole 3 was isolated by first extracting into basic
aqueous media, then washing with MTBE to remove the non-acidic impurities. Neutralization
and crystallization from ethanol/water provided the desired product in 74% for the two steps.
This method was used to synthesize 79 g of 3 in 99.6% purity, but further process refinements
were desirable to provide material suitable for clinical studies.
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
Improved Synthesis of 3 for Kilogram Scale
Scheme 15. Synthesis of 3 on Kilogram Scale
Although the boronic acid 48 offered improved isolation as compared to the boronate ester, the
high cost and limited availability of the catalyst (Pd-XPhos G2 precatalyst), as well as concerns
about impurities derived from mutagenic tetrahydroxydiboron,15 made alternative solutions
desirable. The synthesis of 48 was modified to a sequential formation of pinacolboronate ester 50
followed by cleavage to the boronic acid (Scheme 15). Borylation of bromide 45 with
bis(pinacolato)diboron was achieved with the readily available catalyst Pd(dppf)Cl2. After
minimal purification of the boronate ester (simple silica gel plug filtration and crystallization),
treatment with diethanolamine released the pinacol to form the diethanolamine-boronate adduct16
21
ACS Paragon Plus Environment

Page 23 of 51
Organic Process Research & Development
1
2
3
4
5
6
7
8
which was isolated by precipitation from isopropanol-hexane. Hydrolysis with hydrochloric acid
and crystallization from the reaction mixture afforded the boronic acid 48 in 55% yield over
three steps on >5 kg scale.
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
To render the synthesis of indole ester 35 more reproducible, a two-step rather than the
telescoped sequence was employed (compare to Scheme 9). The intermediate trichloromethyl
ketone was isolated in 62% yield via crystallization, and subsequent methanolysis afforded the
ester in 86% yield. For the Suzuki coupling of boronic acid 48 and bromide 35, process
optimization focused on the efficiency of the reaction conversion, the Suzuki product isolation,
and removal of residual palladium and remaining boronic acid 48 (identified as a weak
mutagen).17 A small reaction screen studying the catalyst, solvent, base, and reaction time
identified many suitable catalysts; Pd(PPh3)2Cl2 was chosen due to its low cost. The reactants
were also significantly more soluble in 2-methyltetrahydrofuran (2-MeTHF), enabling increased
reaction concentration. The screen revealed that strong bases such as sodium hydroxide led to
increased impurity formation, while potassium carbonate provided complete conversion with no
evidence of degradation. In all cases, an excess of boronic acid 48 was required to ensure
complete consumption of bromoindole 35. These improved coupling conditions enabled a
decreased reaction time (5 h) on kilogram scale. After aqueous workup, palladium was visually
observed both before and after filtration through diatomaceous earth; therefore an additional
process was employed to reduce Pd content. After treatment with a silica-supported thiol to
scavenge residual Pd, the Suzuki product was isolated in 77% yield by crystallization from 2-
MeTHF and acetonitrile. Boronic acid and Pd residues were quantitated at acceptable levels (<30
and 45 ppm, respectively). Conditions for ester saponification were modified as follows: 5
equivalents of aqueous NaOH in 1:1 methanol:THF at reflux for 13–14 h. Use of this mixed
22
ACS Paragon Plus Environment

Organic Process Research & Development
Page 24 of 51
1
2
3
4
5
6
7
8
9
solvent system afforded a balance of minimizing the decarboxylation byproduct (4%) and
unreacted ester (2%) impurities. Under the strongly basic conditions, residual Pd from the prior
step precipitated as Pd black. An organic wash (MTBE) removed the decarboxylation byproduct
and the starting ester. Acidification of the aqueous layer in the presence of ethanol induced
crystallization, resulting in an 83% yield (2.4 kg) of compound 3.
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
Figure 2. Gravimetric Solubility of Crystalline Forms 1 and 2 of Compound 3
30
Ttrans = 30°C
25
20
Form 2
15
Form 1
10
5
0
20
30
40
50
60
Temperature (°C)
Three crystalline forms of 3 were identified. Forms 1 and 2 were enantiotropically related
anhydrous crystalline forms, and Form 3 was a transient hydrate that dehydrated to afford Form
2. Initial syntheses of 3 consistently provided Form 2; however, later syntheses provided Form 1
or mixtures of Forms 1 and 2 from the same binary solvent system (ethanol–water). Further
investigation revealed that the later re-crystallizations were performed at 45–50 °C, which
predominantly led to Form 1, while earlier experiments were performed at room temperature,
leading to Form 2. The gravimetric solubility values shown in Figure 2 were consistent with
these observations and ultimately a transition temperature of approximately 30 °C was
interpolated. For further development, Form 2 was considered more desirable, as uncontrolled
form conversion was unlikely to occur during storage under ambient conditions. In practical
23
ACS Paragon Plus Environment

Page 25 of 51
Organic Process Research & Development
1
2
3
4
5
6
7
8
9
terms, on the largest batch (2.4 kg), Form 1 was converted to a mixture of Forms 2 and 3 by
granulation in 50% ethanol–water at 25 °C; subsequent drying under positive nitrogen pressure
at 50 °C led to dehydration of Form 3 and complete conversion to Form 2 with monitoring by X-
ray powder diffraction (XRD) pattern analysis. Rehydration of Form 2 to Form 3 under ambient
conditions was not detected.
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
CONCLUSIONS
The evolution of the synthesis of three potent AMPK activators has been described. The
challenges associated with the early-stage syntheses including unstable intermediates, difficult
isolations, and poor reaction profiles were identified and overcome to deliver material for key in
vivo studies. Methods including heteroarene substitutions, borylation, Suzuki and Negishi cross-
couplings, and ester hydrolysis were developed with improved reaction reproducibility, enhanced
purity and safety profiles, and practical isolation of intermediates and APIs. These improvements
ultimately resulted in the successful completion of synthesis campaigns to produce 40–50 g of 1
and 2, and 2.4 kilograms of clinical candidate 318 for key efficacy and safety studies.
Additionally, the challenges in investigating, developing, and controlling the solid form for 3
were overcome to reproducibly prepare a stable crystalline form with properties suitable for
further development.
EXPERIMENTAL SECTION
All chemicals, reagents, and solvents were purchased from commercial sources and were used
without further purification. Reactions were carried out at room temperature (~23 °C) unless
otherwise indicated. Chemical shifts in NMR analyses were referenced to the residual 1H solvent
signals (CDCl3, δ 7.27; DMSO–d6, δ 2.50; CD3OD, δ 3.31) and solvent 13C signals (CDCl3, δ
77.0; DMSO–d6, δ 39.51; CD3OD, δ 49.15). Signals are listed as follows: chemical shift in ppm;
24
ACS Paragon Plus Environment

Organic Process Research & Development
Page 26 of 51
1
2
3
4
5
6
7
8
9
multiplicity identified as s = singlet, br = broad, d = doublet, t = triplet, q = quartet, m =
multiplet; coupling constants in Hz. Purity is reported by relative UV area from HPLC analysis.
Water content was determined by Karl Fischer titration. Pd content determined by inductively
coupled plasma mass spectrometry (ICP-MS).
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
(S)-1-(4-bromophenyl)-5-chloropentan-1-ol (14): A 1 L 3-neck oven-dried round-bottom flask
equipped with a digital thermometer and an addition funnel was charged with (R)-(+)-2-methyl-
CBS-oxazaborolidine (5.25 g, 17.8 mmol), anhydrous 2-MeTHF (60 mL), and borane in THF
(1.0 M, 185 mL, 185 mmol) under a N2 atmosphere. The clear colorless solution was cooled to
−20 °C using a cryocooler in an acetone bath. 13 (50.0 g, 180 mmol) in anhydrous 2-MeTHF
(150 mL, rinsed with 2 x 20 mL) was added dropwise over about 3 h while keeping the internal
temperature below −15 °C during addition. The solution was stirred at −20 °C for 1 h. The
reaction was monitored by TLC (25% EtOAc/heptane, KMnO4 stain) and GCMS until full
conversion of the substrate was observed. MeOH (120 mL) was added dropwise from an
addition funnel at a rate targeting an internal temperature of −20 °C. Some effervescence was
observed and the temperature rose to −13 °C. The solution was stirred at −10 °C for 1 h until gas
evolution ceased. 1 M HCl (150 mL) was added dropwise via addition funnel while stirring at
−10 °C. A colorless suspension formed, and the mixture was stirred at 0 °C for 1 h, then stirred
at room temperature for 15 h. The cloudy solution was poured into a separatory funnel
containing 100 mL of brine. EtOAc (~300 mL) was added, and the layers were separated. The
aqueous layer was extracted with EtOAc (2 x 400 mL). The organic phases were combined,
washed with brine (300 mL), dried over sodium sulfate, filtered, and concentrated to dryness.
The resultant oil was dissolved in ~300 mL petroleum ether and stirred at ambient temperature
for 20 min. During this time a colorless precipitate formed. The solution was cooled to 0 °C, the
25
ACS Paragon Plus Environment

Page 27 of 51
Organic Process Research & Development
1
2
3
4
5
6
7
8
slurry was filtered, and the solids were washed with ~100 mL petroleum ether. The mother
liquor was concentrated to dryness to give 14 (53.8 g, quant.), as a colorless oil which was
carried forward to the next step without further purification.
9
An analytical sample was purified via flash column chromatography for characterization. 1H
NMR (400 MHz, CDCl3) δ 7.48 (d, J=8.2 Hz, 2H), 7.22 (d, J=8.2 Hz, 2H), 4.65 (dd, J=7.4, 5.9
Hz, 1H), 3.52 (t, J=6.6 Hz, 2H), 1.93 (br. s, 1H), 1.85–1.36 (m, 6H). 13C NMR (101 MHz,
CDCl3) δ 143.5, 131.6, 127.6, 121.3, 73.7, 44.8, 38.2, 32.3, 23.0. GCMS calc. for C11H13BrO
(m/z): 240. Enantiomeric ratio (er): 98.4:1.6 as determined by chiral SFC analysis: (Chiral Tech
AD-H, 250mm x 4.6 mm, 5% MeOH/CO2 to 60% MeOH/CO2, 3.0 mL/min, 210 nm,
backpressure 120 Bar); tR = 6.035 min (major), tR = 7.266 min (minor).
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
(S)-2-(4-bromopenyl)tetrahydro-2H-pyran (15): 14 (180 mmol) was transferred to a round
bottom flask as a solution in THF (180 mL), and the mixture was cooled to −5 °C (brine/ice
bath). Potassium t-butoxide solution (250 mL, 250 mmol, 1.0 M in THF) was added slowly to
the reaction flask at a rate to maintain internal temperature below 5 °C. The reaction was
warmed to room temperature and stirred for 15 h. The reaction mixture was cooled in an ice bath
and 1 M HCl (150 mL) was added slowly at a rate to keep the temperature below 8 °C. The
reaction mixture was extracted with EtOAc (2 x 300 mL) and washed with brine (150 mL). The
combined organic phases were dried over sodium sulfate, filtered, concentrated, and dried under
high vacuum to yield 15 (44.2 g, quant.) as an orange-brown oil which solidified upon standing
The material was taken forward to the next reaction without further purification.
An analytical sample was purified via flash column chromatography for characterization:
GCMS calc. for C11H13BrO (m/z): 240, 242; found: 240, 242. 1H NMR (400 MHz, CDCl3) δ
7.46 (d, J=8.2 Hz, 2H), 7.23 (d, J=8.6 Hz, 2H), 4.29 (dd, J=11.1, 2.2 Hz, 1H), 4.17–4.10 (m,
26
ACS Paragon Plus Environment

Organic Process Research & Development
Page 28 of 51
1
2
3
4
5
6
7
8
9
J=11.2, 2.0, 2.0 Hz, 1H), 3.61 (td, J=11.5, 2.7 Hz, 1H), 1.98–1.91 (m, 1H), 1.85–1.77 (m, 1H),
1.73–1.48 (m, 4H). 13C NMR (101 MHz, CDCl3) δ 142.4, 131.3, 127.5, 120.9, 79.3, 68.9, 34.0,
25.8, 23.9. er: 98.11:1.89 as determined by chiral SFC analysis: (Lux Amylose-2, 250mm x 4.6
mm, 5% MeOH/CO2 to 60% MeOH/CO2, 3.0 mL/min, 210 nm, backpressure 120 Bar); tR =
3.829 min (major), tR = 4.100 min (minor).
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
(S)-(4-(tetrahydro-2H-pyran-2-yl)phenyl)boronic acid (16): A three-necked round bottom
flask was equipped with an overhead stirrer, reflux condenser, and a nitrogen line. Sodium t-
butoxide (251 mg, 2.61 mmol), potassium acetate (41.2 g, 420 mmol, oven dried), chloro(2-
dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2′-amino-1,1′-
biphenyl)]palladium(II) (Pd-XPhos G2, 734 mg, 0.933 mmol), XPhos (1250 mg, 2.61 mmol),
and tetrahydroxydiboron (33.5 g, 373 mmol) were added to the flask, and the flask was purged
with N2 for 10 min. EtOH (273 mL) was added to 15 (180 mmol) in a separate flask and the
solution was purged with N2 for 10 min. The solution was added to the three-necked reaction
flask, washing with more EtOH (100 mL). The yellow suspension was stirred at reflux
temperature for 16 h. The reaction mixture turned to a brown suspension. TLC indicated product
formation with a small amount of substrate remaining. The reaction was cooled to ambient
temperature, MTBE (500 mL) was added and the mixture was filtered through a plug of Celite,
washing with an additional 200 mL MTBE. The dark brown solution was concentrated to give a
brown foam. MTBE (500 mL) and water (100 mL) were added and the mixture was transferred
to a separatory funnel. The layers were separated and the aqueous was extracted with MTBE
(100 mL). The organic phases were combined and extracted with 1 M aqueous NaOH (3 x 100
mL, 1 x 50mL). The basic aqueous phases were combined and washed with MTBE (100 mL).
The basic aqueous phases were transferred to a round bottom flask fitted with a magnetic stir bar
27
ACS Paragon Plus Environment

Page 29 of 51
Organic Process Research & Development
1
2
3
4
5
6
7
8
9
and cooled in an ice bath. Conc. HCl was added to adjust pH to ~6. During this time oil
separated from the solution and the flask was agitated by sonication to induce crystallization,
then stirred vigorously for 16 h. Fluffy solids formed and were filtered off to provide 16 as a
colorless solid (26.83 g, 72%). m.p. 186–192 °C; 1H NMR (400 MHz, DMSO-d6) δ 7.95 (br. s,
2H), 7.73 (d, J=8.2 Hz, 2H), 7.27 (d, J=8.2 Hz, 2H), 4.29 (dd, J=11.1, 1.8 Hz, 1H), 4.06–3.96
(m, 1H), 3.57–3.47 (m, 1H), 1.90–1.73 (m, 2H), 1.71–1.49 (m, 3H), 1.48–1.34 (m, 1H), (some
boroxine formation observed by NMR, ratio of boroxine:boronic acid observed to change with
NMR solvent). 13C NMR (101 MHz, DMSO-d6) δ 145.3, 133.9, 124.6, 78.9, 67.9, 33.9, 25.5,
23.5, (carbon bonded to B is not observed).
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
4-bromo-3,5-difluoro-2-iodoaniline (18): To 4-bromo-3,5-difluoroaniline (1.39 kg, 6.67 mol)
in acetic acid (7.5 L) at 20 °C was added N-iodosuccinimide (1.53 kg, 6.80 mol). The mixture
was stirred at 20 °C for 4 h (initial exotherm to 22 °C was observed) then quenched by the
addition of 10% sodium thiosulfate (750 mL) followed by the addition of water (10.5 L) over 1 h
(mild initial exotherm to 23 °C was observed). The resulting slurry was stirred at 20 °C for 1 h
then the solid was filtered off and washed with water (4 x 1.5 L), air-dried, and then dried under
vacuum at 45 °C to afford 18 (2.15 kg, 6.42 mol, 96%) as a white solid. 1H NMR (500 MHz,
DMSO-d6) δ 6.58 (d, J=11.2 Hz, 1H) 6.03 (br. s, 2H). LCMS: calc. for C6H3BrF2IN (M+H):
333.9; found: 334.0.
4-bromo-3,5-difluoro-2-((trimethylsilyl)ethynyl)aniline (19): To an inert 20 L reactor was
charged 18 (2.19 kg, 6.56 mol), Pd(PPh3)4 (125 g, 108 mmol), CuI (20 g, 105 mmol) and toluene
(9 L). The mixture was deoxygenated using 3 x vacuum / nitrogen cycles and triethylamine (1.25
L, 8.97 mol) was added. The mixture was deoxygenated using 1 x vacuum / nitrogen cycles and
TMS-acetylene (1.15 L, 8.30 mol) was added. The mixture was heated to 41 °C for 18 h (TLC
28
ACS Paragon Plus Environment