A Scalable Route for the Regio- and Enantioselective Preparation of a Tetrazole Prodrug: Application to the Multi-Gram-Scale Synthesis of a PCSK9 Inhibitor

Article abstract of DOI:10.1021/acs.oprd.7b00304

The synthesis of multigram quantities of small molecule PCSK9 inhibitor (R,S)-3 is described. The route features a safe, multikilogram method to prepare 5-(4-iodo-1-methyl-1H-pyrazol-5-yl)-2H-tetrazole (10). A three-component dynamic kinetic resolution between tetrazole 10, acetaldehyde, and isobutyric anhydride was catalyzed by a chiral DMAP catalyst to afford enantiomerically enriched hemiaminal ester (S)-12 on multikilogram scale. Magnesiation, transmetalation, and Negishi coupling provided access to Boc-intermediate (R,S)-13, which was deprotected to provide (R,S)-3 in multigram quantities.

Full text of DOI:10.1021/acs.oprd.7b00304

Subscriber access provided by READING UNIV
Full Paper
A Scalable Route for the Regio- and Enantioselective
Preparation of a Tetrazole Prodrug: Application to
the Multi-Gram Scale Synthesis of a PCSK9 Inhibitor
Anne Akin, Mark T Barrila, Thomas A Brandt, Anne-Marie Dechert-Schmitt, Pascal
Dube, David D Ford, Adam S. Kamlet, Chris Limberakis, Andrew Pearsall, David W.
Piotrowski, Brian Quinn, Sarah Rothstein, Jerry Salan, Liuqing Wei, and Jun Xiao
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.7b00304 • Publication Date (Web): 08 Nov 2017
Downloaded from http://pubs.acs.org on November 9, 2017
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 free 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 accessible to all
readers and 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.
Organic Process Research & Development 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 38
Organic Process Research & Development
1
2
3
4
A Scalable Route for the Regioꢀ and Enantioselective
Preparation of a Tetrazole Prodrug: Application to the
MultiꢀGram Scale Synthesis of a PCSK9 Inhibitor
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
‡
‡
‡
†
#
Anne Akin, Mark T. Barrila, Thomas Brandt, Anne-Marie R. Dechert-Schmitt, Pascal Dube, David
#
†
†
#
,†
D. Ford, Adam S. Kamlet, Chris Limberakis, Andrew Pearsall, David W. Piotrowski* , Brian
#
#
, #
†
†
Quinn, Sarah Rothstein, Jerry Salan,* Liuqing Wei, Jun Xiao
†
Medicine Design, Pfizer, Inc., Eastern Point Road, Groton, Connecticut, 06340, United States
‡
Chemical Research and Development, Pfizer, Inc., Eastern Point Road, Groton, Connecticut 06340,
United States
#
Nalas Engineering Services, Inc., 85 Westbrook Road, Centerbrook Connecticut 06409, United States
ACS Paragon Plus Environment

Organic Process Research & Development
Page 2 of 38
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
TOC/Abstract graphic
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
ACS Paragon Plus Environment

Page 3 of 38
Organic Process Research & Development
1
2
3
4
5
6
7
8
9
ABSTRACT: The synthesis of multiꢀgram quantities of a small molecule PCSK9 inhibitor (R,S)ꢀ3 is
described. The route features a safe, multiꢀkilogram method to prepare 5ꢀ(4ꢀiodoꢀ1ꢀmethylꢀ1Hꢀ
pyrazolꢀ5ꢀyl)ꢀ2Hꢀtetrazole (10). A threeꢀcomponent dynamic kinetic resolution (DKR) between
tetrazole 10, acetaldehyde and isobutyric anhydride was catalyzed by a chiral DMAP catalyst to
afford enantiomerically enriched hemiaminal ester (S)ꢀ12 on multiꢀkilogram scale. Magnesiation,
transmetallation and Negishi coupling provided access to Bocꢀintermediate (R,S)ꢀ13, which was
deprotected to provide (R,S)ꢀ3 in multiꢀgram quantities.
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: PCSK9, tetrazole, DKR, crossꢀcoupling
Introduction
High serum LDLꢀcholesterol (LDLꢀC) is a risk factor for atherosclerosis and a widely recognized
biomarker for increased risk of coronary heart disease (CHD). When pharmaceutical intervention is
®
®
required to lower LDLꢀC, statins (e.g. Lipitor , Crestor ) are often used as frontline treatment.
However, in some cases, these agents do not provide sufficient lowering of LDLꢀC. Inhibition of
proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a promising new target for
decreasing serum LDLꢀC levels and reducing risk of CHD. Of note, mAbs such as alirocumab and
1
evolocumab are approved injectable drugs. Previous disclosures from the Pfizer laboratories
2
described small molecule inhibitors of PCSK9, PFꢀ00932239 (1) and PFꢀ06446846 (2).
Subsequent optimization work led to the identification of tetrazole proꢀdrug (R,S)ꢀ3. The synthetic
challenges associated with the large scale synthesis of (R,S)ꢀ3 include the hazards of preparing a
tetrazole, regioꢀ and enantioselective formation of a tetrazole hemiaminal ester, and the subsequent
3
ACS Paragon Plus Environment

Organic Process Research & Development
Page 4 of 38
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
chemistry and handling of these baseꢀsensitive species. Herein, we disclose a safe and scalable route
directed toward multiꢀgram quantities of (R,S)ꢀ3 to support preclinical toxicology studies.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 1. Small molecule PCSK9 inhibitors
Medicinal Chemistry Routes to 3
Two versatile synthetic intermediates, tertiary amide 5 and iodopyrazole 8, were critical for
implementation of the early medicinal chemistry routes (Scheme 1). Amide 5 was synthesized in
two steps via Buchwald amination followed by acylation with 4ꢀbromobenzoyl chloride in the
presence of base. Conversion of 5 into pinacol boronate 6 then offered a range of opportunities to
append a distal heteroaryl group by Negishi (from 5) or SuzukiꢀMiyaura (from 6) crossꢀcoupling
methodologies (Scheme 1). Nitrile 8 was synthesized in three steps from the commercially available
4ꢀiodoꢀ3ꢀmethylpyrazoleꢀ5ꢀcarboxylic acid (7) by conversion to the acyl imidazole and
ammonolysis followed by dehydration. The Negishi coupling of 5 with 8 provided cyanopyrazole 9
in 80% yield, but the SuzukiꢀMiyaura coupling of 6 with heteroaryl halides (e.g. coupling of
pyrazole 8 to give 9) was generally favored for analog synthesis.
4
ACS Paragon Plus Environment

Page 5 of 38
Organic Process Research & Development
1
2
3
4
5
6
7
8
9
Scheme 1: Medicinal Chemistry Routes to Synthetic Intermediates
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
With due caution, installation of the tetrazole was investigated next. The conversion of nitriles
3
into tetrazoles with hydrazoic acid is well known. Small scale preparation (ca. 0.5 g) of tetrazole
1
0 could be effected by reaction of 8 with hydrazoic acid, made in situ from NaN and NH Cl, at
3 4
high temperature. However, the toxic and shockꢀsensitive nature of hydrazoic acid based reagents
led to exploration of alternative routes to tetrazoles. In brief, nitriles 8 or 9 could be converted into
tetrazoles 10 or 11 by reaction of hydrazine with a thioamide to provide the amidrazone, formed in
situ, and cyclized with nitrous acid. While this method avoided the use of hydrazoic acid, it
employed the toxic hydrazine and the unstable nitrous acid. Further work toward tetrazoles via this
route was abandoned. However, the availability of tetrazole 10 allowed for investigation of crossꢀ
5
ACS Paragon Plus Environment

Organic Process Research & Development
Page 6 of 38
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
coupling reactions of it or a protected version thereof (e.g. compound 12). The successful outcome
of the crossꢀcoupling with either tetrazole substrate 10 or 12 was not clear since the presence of an
acidic hydrogen on 10 could interfere with typical coupling conditions or the baseꢀlabile hemiaminal
ester 12 could suffer premature hydrolysis under typical SuzukiꢀMiyaura reaction conditions. Prior
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
to the recent report from Buchwald, crossꢀcoupling of aryl or heteroaryl halides containing acidic
hydrogens was viewed as difficult; a trace of desired coupled product 11 was obtained with these
reported conditions. After extensive investigation and highꢀthroughput screening, an optimized set
®
of conditions (aq. NaOH, Pd(OAc) , cataCXium A, dioxane 70ꢀ100 °C) allowed for facile coupling
2
of 10 with 6 on 1 g scale to provide tetrazole 11 in 92% isolated yield. Likewise, using racemic
5
hemiaminal substrate 12, highꢀthroughput screening led to identification of a set of mild, fluorideꢀ
based SuzukiꢀMiyaura coupling conditions that provided a high yield of coupled product 13. The
medicinal chemistry routes as described above were practical for analog generation when coupled
with chromatography on chiral support to separate either hemiaminal ester 12 or 13 (Scheme 2). The
late stage separation and NꢀBoc deprotection not only gave desired product (R,S)ꢀ3, but also
provided access to valuable diastereomeric standards (R,R)ꢀ13, (R,S)ꢀ13, and (R,R)ꢀ3. Furthermore,
6
the absolute stereochemistry of (R,R)ꢀ3 was determined from the Xꢀray crystal structure. The
stereochemistry of the desired diastereomer (R,S)ꢀ3, could thus be inferred from this structure.
6
ACS Paragon Plus Environment

Page 7 of 38
Organic Process Research & Development
1
2
3
4
5
6
7
8
9
Scheme 2: Medicinal Chemistry Routes to Tetrazole Intermediates and Product 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
As (R,S)ꢀ3 emerged as a potential candidate compound, our attention turned toward a safe,
scalable route. Our preferred approach for the construction of 3 was to perform a crossꢀcoupling
between hemiaminal ester 12 and benzamide 5 or 6. This option afforded the ability to
independently investigate the large scale preparation of tetrazole 10, a method to synthesize a single
enantiomer of 12 and optimized coupling conditions for 13. With these bond connections in mind,
an efficient, scalable route to 3 was undertaken.
7
ACS Paragon Plus Environment

Organic Process Research & Development
Page 8 of 38
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The original conditions used to prepare tetrazole 10 relied on the treatment of nitrile 8 with
hydrazoic acid generated in situ from NaN and NH Cl in DMF at temperatures above 100 °C.
3
4
Although convenient for small scale preparation, the generation of anhydrous hydrazoic acid posed
serious safety concerns. Hydrazoic acid is highly toxic, and is a sensitive energetic compound in
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7
concentrated solutions and vapors with a low detonation threshold. Additionally, it has a boiling
point of 37 °C, and it can potentially condense in cooler areas of the reactor, from mechanical parts
to ductwork. Condensed hydrazoic acid could then explode when the equipment is being
disassembled or cleaned. Moreover, the original conditions risk the formation of ammonium azide,
8
another powerful explosive, as a byꢀproduct. Furthermore, the target tetrazole 10 had the potential
to be an energetic compound itself, a concern that warranted further safety evaluation.
The medicinal chemistry route involved the treatment of nitrile 8 with an excess of NaN and
3
NH Cl in DMF at 120 °C. After reaching completion (ca. 20 h), the excess azide was neutralized
4
with nitrous acid (prepared in situ from the stepwise addition of aq NaNO and dilute H SO ). This
2
2
4
operation, while very effective in consuming the residual azide, was found to be exothermic with
concurrent generation of nitrogen gas. Moreover, even on small scale, the quench resulted in the
formation of a solid mass that impeded stirring. This significant coꢀprecipitation of inorganic salts,
potentially including ammonium azide, necessitated extensive washing of the solids obtained by
filtration before isolation of tetrazole 10 in ca. 85–90% yield.
Given the risk of explosion associated with producing hydrazoic acid at high levels during the
reaction, we reasoned that using water as a coꢀsolvent could facilitate the reaction by increasing the
solubility of the inorganic salts while desensitizing the hydrazoic acid contained in the gas phase
9
towards a potential explosion. This hypothesis was confirmed when >99.5% conversion was
8
ACS Paragon Plus Environment

Page 9 of 38
Organic Process Research & Development
1
2
3
4
5
6
7
8
9
obtained in 16 h at 90 °C using DMF/water (2:1). Further dilution of the reaction mixture with
additional water followed by the standard neutralization protocol mitigated risks in the isolation
procedure while producing 10 as a crystalline white solid. This procedure allowed for reproducible
preparation of high purity material in 88–93% yield. Tetrazole 10 was found to be insensitive to
impact using the Bruceton method on a BAM Fallhammer, showing no sign of decomposition in six
trials at 100 cm with a 10 kg hammer weight. Moreover, a similar evaluation of its sensitivity
towards friction using a BAM friction instrument again showed no event even at 360 N, thus
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
1
0
confirming the stability of tetrazole 10 towards impact and friction.
A recent report summarized the broad range of reaction conditions to synthesize tetrazoles from
1
1
nitriles. In light of the reports of the use of Lewis acids, in particular zinc salts, to catalyze
12
tetrazole formation with reduced hydrazoic acid levels, we evaluated these catalysts for the
conversion of nitrile 8 into tetrazole 10. The use of zinc oxide in THF/water led to modest
conversion (<40%) after prolonged reaction times at reflux. A broader screen of catalysts revealed
that ca. 70% conversion could be obtained using 10 mol% zinc bromide in THF/water (1:2).
Interestingly, the reaction pH was measured to be of 7.5, suggesting that the equilibrium between
1
3
azide and hydrazoic acid (pK = 4.6) was shifted significantly toward azide, offering hope that
a
levels of hydrazoic acid could be managed to safe levels. The slow reaction rate led us to screen a
variety of organic coꢀsolvents to identify an alternative to THF that would allow us access to higher
temperatures. We performed these reactions using 10 mL of water per gram of starting material, and
5
mL of the organic solvent per g of 10 (Table 1). Our study revealed that alternative organic coꢀ
solvents provided a significant increase in conversion compared with the original THF solvent
system. Interestingly, the reactions with ethanol and ethylene glycol both showed significant
9
ACS Paragon Plus Environment

Organic Process Research & Development
Page 10 of 38
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
accumulation of nitrile 8 on the walls of the test tube used to run the reaction, presumably due to
sublimation. This makes those solvents less practical. We were pleased to find that DMF and IPA
both led to high conversion at this small scale with traces of 8 accumulated on the walls for DMF
and none detectable with IPA. We thus selected IPA as the coꢀsolvent of choice for the system under
evaluation.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Table 1. Solvent Screen for the Formation of the Tetrazole 10 with ZnBr2.
Entry
Organic co-solvent
MeTHF
Conversion after 24 h
1
2
3
4
5
45%
86%
80%
89%
90%
DMF
EtOH
IPA
Ethylene glycol
a
Reaction conditions: 100 mg nitrile 8 in 1 mL water, 0.5 mL organic solvent, 2 eq NaN and 0.1 eq ZnBr at 80 °C.
3
2
The next step was to evaluate the impact of concentration and solvent ratio on the reaction
outcome. A series of reactions was conducted using 20 mL of solvent/g in order to stress the
reaction conversion under high dilution (Table 2). Although the solvent range evaluated was broad,
the study showed that a 1:1 ratio of water to IPA seemed to provide a suitable environment for the
smooth formation of 10. Additional control experiments showed that only trace amounts of 10 are
formed when the reaction is conducted in water or IPA alone. As full conversion was achieved after
48 h at 80 °C, these conditions routinely provided >80% yield of >98% potency of 10 after
treatment under the standard isolation procedure.
Table 2. Impact of Solvent Composition for the Formation of Tetrazole 10 catalyzed by ZnBr2
10
ACS Paragon Plus Environment

Page 11 of 38
Organic Process Research & Development
1
2
3
4
5
6
7
8
9
Entry
Ratio
Conversion
Accumulation of nitrile
Water-IPA after 24 h (48 h)
8 on walls?
1
2
3
1:3
1:1
3:1
77% (92%)
88% (97%)
61% (82%)
No
No
Yes
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
*
Reaction conditions: 100 mg nitrile 8 in 2 mL of solvent (water + IPA), 2 eq NaN and 0.1 eq ZnBr at 80 °C.
3 2
With an improved solvent composition determined, we turned our attention back to the effect of
the stoichiometry of NaN and ZnBr on conversion. We found that 2 equiv of NaN were enough to
3
2
3
obtain 98% conversion after 48 h at 80 °C. While lower quantities led to a more sluggish reaction,
increased catalyst loading showed no further advantage from a conversion and purity standpoint.
The subsequent evaluation of the impact of the ZnBr stoichiometry revealed that a 10 mol%
2
loading was necessary to achieve high conversion (98% after 48 h at 80 °C). Poor conversion was
obtained when using 5 mol% ZnBr . It is noteworthy that using 20 mol% of catalyst was found to
2
increase the rate of the reaction and provide 95% conversion in 16 h. As higher catalyst loadings
may lead to a greater quantity of hydrazoic acid produced via hydrolysis of ZnBr to form HBr, we
2
opted to maintain a low stoichiometry of ZnBr at the expense of reaction time. Thus, the optimal
2
conditions on small scale featured 2 equiv NaN , 10 mol% ZnBr in 5 volumes of water and 5
3
2
volumes of IPA at 80 °C to provide 98% conversion in 48 h while maintaining the reaction mixture
above pH 7.
Upon optimization of the stoichiometry, the selected process was evaluated to determine the levels
of hydrazoic acid present in the reactor headspace. This was achieved using gasꢀphase FTIR to
monitor gases leaving the reactor vent while a constant gas sweep was applied to the reactor. Offꢀ
line ion chromatography was also used to provide complementary data for this study by analyzing a
scrubber downstream of the IR cell. The original conditions (NaN , NH Cl, DMF, 120 °C) were also
3
4
11
ACS Paragon Plus Environment

Organic Process Research & Development
Page 12 of 38
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
evaluated for comparison. To test if the removal of hydrazoic acid in the gas sweep was negatively
impacting the reaction, we executed a control experiment by performing a small scale reaction using
the ZnBr conditions subjected to a constant air sweep. A 200 mg reaction was thus performed in an
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
EasyMax™ reaction tube with magnetic stirring equipped with an air sweep vented into two water
scrubbers in series to capture any hydrazoic acid that would escape. This safety measure was found
to only have minimal impact on the chemistry, with 86% conversion achieved after 24 h at 80°C and
92% after 40 h. Excess azide was destroyed by sequential addition of aq NaNO and H SO , and
2
2
4
tetrazole 10 was obtained in 80% yield after filtration and drying. Analyzing the water scrubber by
ion chromatography revealed the presence of less than 200 ppm (w/v) of hydrazoic acid in the first
10 mL scrubber (i.e. 2 mg hydrazoic acid trapped), supporting the assertion that only traces of
hydrazoic acid had formed during the reaction and its removal does not negatively impact the
chemistry. The absence of hydrazoic in the second water trap confirms that the hydrazoic acid
evolved was effectively trapped in the first water scrubber.
Control experiments with ZnBr in the absence of nitrile 8 were conducted to quantify the
2
maximum possible levels of hydrazoic acid under these conditions. A reaction was performed
following the material balance (Table 3). It is noteworthy that the reaction was performed using the
same reaction volume, solvent, and head space available as in the calibration experiments to ensure
the direct correlation of the data. The reagents were thus mixed at ambient temperature and heated to
80 °C while under monitoring by gasꢀphase FTIR. The FTIR profile of this reaction showed that no
detectable level of hydrazoic acid is present in the head space prior to heating the mixture (Figure 2).
Upon equilibration at 80 °C, the system reached a steady state where the absorption was constant
over time at approximately 400−500 ppm. Cooling the reaction mixture back to 20 °C led to the
12
ACS Paragon Plus Environment

Page 13 of 38
Organic Process Research & Development
1
2
3
4
5
6
7
8
9
disappearance of the hydrazoic acid peak by FTIR. The reactor was swept with N at a gas flow rate
2
of 0.79 mL/s, so at the average concentration of 410 ppm, this corresponds to a mass flow rate of 2.1
mg/h of hydrazoic acid. The area under the curve was calculated to be 189.4 AU s, corresponding to
a total of 3.1 mg of hydrazoic acid produced during the course of this experiment. At the end of the
experiment, a sample of the scrubber solution was analyzed by ion chromatography, revealing a 136
ppm concentration of azide (w/v as hydrazoic acid), representing 1.4 mg of hydrazoic acid dissolved
in the 10 mL scrubber. This number suggests that the scrubber was only partially adept at capturing
the hydrazoic acid formed, or that some hydrazoic acid was adsorbed in the tubing leading to the
scrubber.
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. Material Balance for the Evaluation of ZnBr Catalyst in the Absence of Nitrile
2
Reagent
NaN3
Quantity
1.73 g
mmol (ratio)
26.66 (20)
Water
IPA
15.0 mL
15.0 mL
0.30 g
ZnBr2
1.33 (1)
26.66
a
HN3
1.15 g
a
expected maximum amount of hydrazoic acid
13
ACS Paragon Plus Environment

Organic Process Research & Development
Page 14 of 38
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
00
Cooling to 20 °C
500
4
3
00
00
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
200
1
00
0
Heating to 80 °C
50
0
100
150
Time (min)
Figure 2. Headspace hydrazoic acid levels measured by gas phase FTIR under ZnBr conditions.
2
The advantages and disadvantages of the three different processes are compared to highlight the
differences between them (Table 4). The original conditions offered a slow release of hydrazoic acid
due to its heterogeneous nature but the isolation conditions were not suitable for scaleꢀup. Either of
the modified conditions, generation 1 or 2, was suitable for scale up. Generation 1 provided a higher
yield and a higher throughput. However, the homogeneity of the reaction led to the highest levels of
hydrazoic acid measured, which could be mitigated by an appropriate sweep of the reaction
headspace to a scrubber to ensure the neutralization of hydrazoic acid. Alternatively, the second
generation conditions offered the lowest levels of hydrazoic acid at the expense of reaction rate and
throughput. With either of these last two processes, tetrazole 10 was isolated in >99% potency
through the improved isolation protocol.
Table 4: Comparison of Methods to Convert Nitrile 8 into Tetrazole 10
Process
Yield
Throughput
HN in
Pros
Cons
3
14
ACS Paragon Plus Environment

Page 15 of 38
Organic Process Research & Development
1
2
3
4
5
headspace
a
Original
85%
90 g/L
> 3800 ppm
•
Heterogeneous;
slow release of
HN3
•
High HN levels
3
2
.4 equiv NaN3,
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
.0 equiv NH Cl,
4
•
Heterogeneous;
mass transfer issues
potential
DMF, 120 °C
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Generation 1
85ꢀ90% 80 g/L
8000 ppm
410 ppm
•Homogeneous
•High HN levels
3
2
2
equiv NaN3,
•
HN hazards
mitigated by
3
equiv NH Cl,
4
DMF/water, 90 °C
water scrubber
Generation 2
80%
< 40 g/L
•
Low HN3
•
•
Longer reaction time (48 h)
Potential for reaction to stall
2
0
equiv NaN3,
.1 equiv ZnBr2,
levels
(catalyst deactivation)
IPA/water, 75ꢀ80 °C
•
Lower yield
a
Forms a solid mass preventing stirring. Majority of precipitate washed away on filter through extensive washing.
Volume not included in calculation.
With a safe, scalable method to synthesize tetrazole 10, we turned our attention to improvement of
the final steps in the sequence. Our previously reported conditions for the regioꢀ and
5
enantioselective synthesis of tetrazole hemiaminal esters demonstrated that the more sterically
accessible nitrogen of the tetrazole (Nꢀ2) reacts with aldehydes at a faster rate to provide the
hemiaminal intermediate. In the presence of a (chiral) DMAP catalyst, the tetrazoleꢀ2ꢀhemiaminal
is preferentially acylated while the tetrazoleꢀ1ꢀheminal can be acylated (slower) or revert back to
aldehyde and tetrazole starting materials. The conditions were optimized for the scaled synthesis of
12 by examination of solvent, concentration, equivalents of acetaldehyde, and catalyst loading.
Reactions employed 2 equiv of acetaldehyde, 1.5 equiv of isobutyric anhydride, and 1.5 equiv of
Et N per equivalent of tetrazole 10. MTBE was used in place of diethyl ether as it is less prone to
3
form hazardous peroxides and favored for process scale reactions. Next, it was determined that in
order to drive the reaction to completion, 3 mol% of Connon’s catalyst 14 was required (overnight
reaction time). The use of 1 mol% of catalyst led to reaction stalling and approximately 15% of the
15
ACS Paragon Plus Environment

Organic Process Research & Development
Page 16 of 38
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
starting material tetrazole unconsumed. To optimize the enantioselectivity on large scale, the
°
reaction was executed at various temperatures. Holding the reaction temperature at 0 C led to
improved enantioselectivity compared to ambient temperature (92 vs 83% ee), though lowering the
°
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
temperature to ꢀ20 C provided a marginal improvement (93% ee). Finally, various dilution
conditions ranging from 20 mL/g to 75 mL/g were tested for effects on enantioselectivity (see Table
°
5
, results at 0 C). With higher dilution, tetrazole hemiaminal ester 12 was formed in higher
°
enantiomeric ratio. Ultimately, 50 mL/g and 0 C were chosen for scaleꢀup conditions due to tank
capacity and convenience. On a 2.5 kg scale, tetrazole 10 was converted into 12 in a regioselective
and enantioselective manner using catalyst 14 (189 g, 3 mol%). The prodrug fragment 12 was
obtained in quantitative yield and an enantiomeric ratio (97:3) before removal of the minor
enantiomer by chiral preparative chromatography.
Table 5. Small Scale Assessment of Dilution/Enantioselectivity Correlation for Tetrazole 12
Dilution
concentration %ee 12
(mL MTBE/g 10)
2
0
0.18 M
0.14 M
0.10 M
0.07 M
0.05 M
87
90
91
92
94
2
5
36
50
75
With a reliable route to 12 in hand, the merits of the previously examined crossꢀcoupling methods
were assessed. The Suzuki coupling was favored for routine analog synthesis primarily for reasons
of smallꢀscale reliability and convenience. However, in the interest of minimizing stepꢀcount and
1
4
avoiding the introduction of a potentially genotoxic boronic acid or ester, the appeal of the Negishi
16
ACS Paragon Plus Environment

Page 17 of 38
Organic Process Research & Development
1
2
3
4
5
6
7
8
9
coupling came to the fore for large scale reactions. In the event, magnesiation of tetrazole (S)ꢀ12 was
°
conducted by treatment with 1.3 equiv iPrMgCl at ꢀ45 C. After 10 min, transmetalation to the
organozinc was accomplished by addition of a 1.9 M solution of ZnCl in 2ꢀMeTHF (ꢀ45–23 °C, 1
2
°
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
h). Addition of bromide 5 and Pdꢀcatalyst effected a rapid Negishi coupling (45 C, 10 min, > 96%
conversion). In order to ensure high purity of the base labile (R,S)ꢀ3, minor impurities from the
Negishi coupling step were removed by chromatography to provide (R,S)ꢀ13. The intermediate 13
was deprotected with HCl to provide 241 g (89%) of (R,S)ꢀ3 as a partially crystalline solid suitable
for use in in vivo studies (Scheme 3).
Scheme 3. Hemiaminal Ester Synthesis and Negishi Coupling
Conclusions and Summary
Scalable methods to synthesize multiꢀkilogram quantities of 5ꢀ(4ꢀiodoꢀ1ꢀmethylꢀ1Hꢀpyrazolꢀ5ꢀyl)ꢀ
2Hꢀtetrazole (10) were evaluated. A thorough safety assessment was conducted to ensure proper
handling of hydrazoic acid/ammonium azide and tetrazole 10, which allowed for identification of a
few scalable methods. A threeꢀcomponent DKR between tetrazole 10, acetaldehyde and isobutyric
anhydride was catalyzed by a chiral DMAP catalyst to afford enantiomerically enriched hemiaminal
17
ACS Paragon Plus Environment

Organic Process Research & Development
Page 18 of 38
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ester (S)ꢀ12. However, chromatography on chiral support was required to purge the undesired
enantiomer from this oil. Further optimization of the acylation would be required to avoid
chromatography. The endꢀgame was enabled by a convergent Negishi coupling of hemiaminal ester
(S)ꢀ12 with 4ꢀbromobenzamide 5. Further optimization of the Negishi coupling would be required to
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
avoid the late stage chromatography. Uneventful NꢀBoc deprotection with HCl in Et O provided
2
>240 g of (R,S)ꢀ3. Replacement of the ethereal solution of HCl for the NꢀBoc deprotection would be
an improvement to the process.
Experimental Section
All chemicals, reagents, and solvents were purchased from commercial sources when available and
used without further purification. Small scale reactions were magnetically stirred, and monitored by
thin layer chromatography (TLC) using preꢀcoated Whatman 250 ꢀm silica gel plates and visualized
by fluorescence quenching under UV light. Airꢀ and moistureꢀsensitive reactions were carried out
as described. Silica gel chromatography was performed using an ISCO system using ISCO preꢀ
packaged columns. Concentration under reduced pressure (in vacuo) was performed by rotary
evaporation at appropriate temperature and pressure. Purified compounds were further dried under
high vacuum to remove residual solvent. Yields refer to purified compounds. NMR spectra were
1
recorded with a Bruker spectrometer using a 5 mm BBFO probe at 400 MHz and 101 MHz for H
13
1
and C acquisitions unless otherwise noted. Chemical shifts were referenced to the residual H
1
3
solvent signals (CDCl , δ 7.27) and solvent C signals (CDCl , δ 77.16). Signals are listed as
3
3
follows: chemical shift in ppm (multiplicity identified as s = singlet, d = doublet, t = triplet, q =
quartet, m = multiplet, br = broad; coupling constants in Hz; integration). ELS detection was
18
ACS Paragon Plus Environment

Page 19 of 38
Organic Process Research & Development
1
2
3
4
5
6
7
performed using a Polymer Labs (Varian) 2100 ELS detector. Purities are reported as ELSD area
percent. High resolution mass spectroscopy (HRMS) was performed on a LCꢀMS TOF using a C18
o
2
.5 ꢁm 3.0 X 5.0 mm column at 60 C; ammonium formate: water as mobile phase A and 50:50
MeOH:MeCN as mobile phase B. Infrared spectra (FTꢀIR) were collected as neat liquid or solids on
ZnSe surface.
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The level of hydrazoic acid in aqueous streams was measured using ion chromatography. It is
noteworthy that a water scrubber had to be used since aqueous hydroxide ions interfered with the
quantification of azide ions. A calibration curve was built using sodium azide solutions.
o
Ion Chromatography Method: Waters IonꢀPak anion HC (4.6 mm x 150 mm, Column T = 30 C));
Mobile Phase: 12/2/86 acetonitrile/butanol/borate–gluconate buffer; Flow rate: 2 mL/min; Injection:
50 ꢁL; Detection: Conductivity; Calibration: Sodium azide standard curve (~17ꢁg/mL – 170 ꢁg/mL
as hydrazoic acid).
Analytical UPLCꢀMS Method 1: Waters Acquity HSS T3 column (C 2.1 x 5 0 mm, 1.7 µ; Column
1
8
T = 60 °C); Gradient initial conditions: Aꢀ95%:Bꢀ5%; hold at initial from 0.0–0.1 min; linear Ramp
to Aꢀ5%:Bꢀ95% over 0.1–1.0 min; hold at Aꢀ5%:Bꢀ95% from 1.0–1.1 min; return to initial
conditions 1.1–1.5 min; mobile phase A: 0.1% formic acid in water (v/v); mobile phase B: 0.1%
formic acid in MeCN (v/v); flow rate: 1.25 mL/min.
Analytical UPLCꢀMS Method 2: Waters Acquity HSS T3 column (C18 2.1 x 5 0 mm, 1.7 µ,
Column T = 60 °C); Gradient initial conditions: Aꢀ95%:Bꢀ5%; hold at initial from 0.00–0.10 min;
19
ACS Paragon Plus Environment

Organic Process Research & Development
Page 20 of 38
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
linear Ramp to Aꢀ5%:Bꢀ95% over 0.10–2.60 min; hold at Aꢀ5%:Bꢀ95% from 2.60–2.95 min; return
to initial conditions 2.95–3.00 min; mobile phase A: 0.1% formic acid in water (v/v); mobile phase
B: 0.1% formic acid in MeCN (v/v); flow rate: 1.25 mL/min.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Chiral Preparative Chromatography Method 1: Lux Amyloseꢀ2 column (500 mm x 21.2 mm, 5 ꢁ,
ambient temperature); mobile phase: 95% CO /5% 1:1 MeOH/MeCN; isocratic conditions; flow
2
rate: 80.0 mL/min.
Chiral Preparative Chromatography Method 2: Lux Amyloseꢀ2 column (250 mm x 50.0 mm, 5 ꢁ,
ambient temperature); mobile phase: 65% CO /35% MeOH; isocratic conditions; flow rate: 250
2
mL/min.
Analytical UPLC Method: BEH C column (2.1 x 100 mm, 1.7 ꢁ); mobile phase A: 50 mM
8
NaClO
4
/0.1% H PO4; mobile phase B: MeCN; UV detection at 210 nm; flow rate 0.5 mL/min;
3
gradient: initial conditions: Aꢀ95%:Bꢀ5%; linear ramp from 0.25–6.25 min; hold from 6.25–6.75
min; return to initial conditions 6.85–9.50 min.
tertꢀbutyl (3R)ꢀ3ꢀ[(4ꢀbromobenzoyl)(3ꢀmethylpyridinꢀ2ꢀyl)amino]piperidineꢀ1ꢀcarboxylate (5)
20
ACS Paragon Plus Environment

Page 21 of 38
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
Step 1: A 500 L reactor was charged with 70.6 kg (10 volumes) of toluene, SPhos (320.0 g, 780
mmol, 0.02 equiv) and Pd(OAc) (87.0 g, 360 mmol, 0.01 equiv) and the air in the headspace was
2
exchanged with N (3x). After 2 h, 2ꢀbromoꢀ3ꢀmethylpyridine (8.0 kg, 46.8 mol, 1.25 equiv), tertꢀ
2
butyl (3R)ꢀ3ꢀaminopiperidineꢀ1ꢀcarboxylate (7.5 kg, 37.5 mol, 1.00 equiv) and tBuONa (5.5 kg,
°
5
7.8 mol, 1.50 equiv) were added under N atmosphere. The mixture was stirred at 80–90 C for 5 h,
2
°
then cooled to 45 C. The mixture was filtered through diatomaceous earth (3.9 kg). The pad was
rinsed with toluene (20 kg). The organic layer was washed with brine (2x 72 kg), concentrated to 10
volumes, treated with decolorizing carbon, filtered (2x diatomaceous earth), and concentrated to 3–4
°
volumes. The toluene was exchanged with heptane and the mixture was cooled to 0 C. The solids
were filtered, the cake was washed with heptane and dried to give tertꢀbutyl (3R)ꢀ3ꢀ[(3ꢀ
methylpyridinꢀ2ꢀyl)amino]piperidineꢀ1ꢀcarboxylate 6.25 kg, (49%) with 96% area purity, 98.9%
1
assay. H NMR (CDCl ) δ 8.00 (d, 1H), 7.20 (d, 1H), 6.51(dd, 1H), 4.36 (br s, 1H), 4.16 (br s, 1H),
3
3.63 (d, 1H), 3.52 (br s, 2H), 3.36ꢀ3.30 (m, 1H), 2.06 (s, 3H), 1.90 (br s, 1H), 1.73 (br s 2H), 1.59
13
(
br s, 1H), 1.38 (br s, 9H). C NMR (CDCl ) δ 156.0, 155.3, 145.5, 136.8, 116.5, 112.7, 79.5, 49.0,
3
+
4
6.3, 43.8, 29.9, 28.4, 22.5, 17.0. HRMS (m/z) [M+Na] calcd for C H N NaO , 314.1839; found,
16 25
3
2
314.1833.
21
ACS Paragon Plus Environment

Organic Process Research & Development
Page 22 of 38
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Step 2: A 500 L reactor was charged with toluene (78 kg, 14 volumes), DMF (250 g, 0.15 equiv)
and 4ꢀbromobenzoic acid (5.2 kg, 25.7 mol, 1.2 equiv). The conduit was rinsed with toluene (5 kg).
°
The mixture was cooled to 10–20 C, the reactor was charged with oxalyl chloride (4.2 kg, 33.1 mol,
°
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1.6 equiv) and the head tank was rinsed with toluene. The mixture was warmed to 35 C. After 4 h,
the mixture was concentrated to 5 volumes (residual oxalyl chloride < 0.05%). The tank was then
charged with toluene (48 kg), Et N (2.9 kg, 28.9 mol, 1.4 equiv) and tertꢀbutyl (3R)ꢀ3ꢀ[(3ꢀ
3
methylpyridinꢀ2ꢀyl)amino]piperidineꢀ1ꢀcarboxylate (6.2 kg, 21.3 mol, 1.0 equiv). The mixture was
°
°
stirred for 1 h at 10–20 C, then heated at 80–90 C. After about 10 h, the reaction was cooled to 10
°
C and filtered. The cake was washed with toluene (10 kg). The filtrates were washed 2x 10 % citric
acid solution (65 kg) and 1x 1N NaOH (65 kg). The aqueous NaOH layer was extracted 1x toluene
(54 kg). The combined organic layers were concentrated to 7 volumes, then solvent exchanged with
°
heptane and cooled to 0–10 C. After 5–10 h, the solids were isolated by centrifugation and dried for
°
1
6 h at 45 C. Assay showed > 0.5% impurity. The solids were dissolved in toluene (42.5 L), washed
°
with 10% NaOH (2 h, 40–45 C) and water (8.5 L). The organic layer was concentrated to 3–4
°
volumes. Heptane (4x 10 L) was added and the mixture concentrated at < 60 C. After cooling to
°
°
2
0–30 C, the solids were filtered, the cake was washed 1x heptane (4 L) and dried at 40–45 C to
afford tertꢀbutyl (3R)ꢀ3ꢀ[(4ꢀbromobenzoyl)(3ꢀmethylpyridinꢀ2ꢀyl)amino]piperidineꢀ1ꢀcarboxylate 5
1
as a solid (7.0 kg, 77 %) with 99.3% HPLC area purity, 101.1 % assay. H NMR (CDCl ) δ 8.41 (br
3
s, 1H), 7.34 (br s, 1H), 7.25 (d, 2H), 7.16ꢀ7.14 (m, 3H), 4.65 (br s, 1H), 4.48 (br d, 1H), 4.15ꢀ4.04
(br m, 2H), 3.39 (br s, 1H), 2.55 (br s, 1H), 2.37 (br s, 1H), 2.01ꢀ1.98 (br d, 3H), 1.74 (br s, 1H),
1
3
°
1.47ꢀ1.43 (br d, 10H). C NMR (CDCl , 50 C) δ 168.9, 154.8, 153.2, 146.8, 140.1, 135.6, 131.7,
3
22
ACS Paragon Plus Environment

Page 23 of 38
Organic Process Research & Development
1
2
+
130.8, 130.0, 124.4, 123.4, 79.6, 54.9, 48.0, 43.7, 28.4, 27.5, 25.1, 17.8. HRMS (m/z) [M+Na]
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
calcd for C H BrN NaO , 498.1189; found, 498.1190.
2
3
28
3
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
tertꢀbutyl
(3R)ꢀ3ꢀ{(3ꢀmethylpyridinꢀ2ꢀyl)[4ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀ
yl)benzoyl]amino}piperidineꢀ1ꢀcarboxylate (6)
A roundꢀbottom flask was charged with tertꢀbutyl (3R)ꢀ3ꢀ[(4ꢀbromobenzoyl)(3ꢀmethylpyridinꢀ2ꢀ
yl)amino]piperidineꢀ1ꢀcarboxylate 5 (150 g, 317 mmol), bis(pinacolato)diboron (97.8 g, 381 mmol),
potassium acetate (100 g, 1.01 mol, and 2ꢀMeTHF (750 mL). The reaction mixture was warmed to
°
.
7
5 C. Pd(dppf)Cl CH Cl (5.12 g, 6.21 mmol) was added and the reaction mixture was heated
2 2 2
under reflux for 19 h. The reaction mixture was cooled to rt and water was added. The reaction
mixture was passed through a pad of diatomaceous earth and the layers were separated. The organic
layer was concentrated in vacuo. The brown residue was purified by column chromatography on
silica gel, eluting with a gradient of 30–50% EtOAc in heptane. The productꢀcontaining fractions
were concentrated in vacuo. The residue was filtered through a pad of diatomaceous earth using
warm heptane and DCM to solubilize product. The reaction mixture was concentrated in vacuo until
product started to crystallize. The solids were granulated for 16 h at rt, collected via filtration and
23
ACS Paragon Plus Environment

Organic Process Research & Development
Page 24 of 38
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
dried in a vacuum oven to afford tertꢀbutyl (3R)ꢀ3ꢀ{(3ꢀmethylpyridinꢀ2ꢀyl)[4ꢀ(4,4,5,5ꢀtetramethylꢀ
1,3,2ꢀdioxaborolanꢀ2ꢀyl)benzoyl]amino}piperidineꢀ1ꢀcarboxylate 6 as a light pink solid (142 g,
1
8
6%). H NMR (CDCl ) δ 8.40 (m, 1H), 7.53ꢀ7.27 (m, 5H), 7.14ꢀ6.92 (m, 1H), 4.75ꢀ4.45 (m, 2H),
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4.20ꢀ3.90 (m, 1H), 3.63ꢀ3.21 (m, 1H), 2.84ꢀ2.10 (m, 3H), 2.06ꢀ1.88 (m, 3H), 1.81ꢀ1.56 (m, 2H),
13
o
1
.53ꢀ1.37 (m, 9H), 1.31 (s, 12H). C NMR (CDCl , 50 C) δ 169.9, 154.8, 153.2, 146.6, 139.9,
3
139.1, 133.8, 131.9, 130.6, 127.5, 123.2, 83.9, 79.6, 54.7, 48.0, 43.7, 28.4, 27.6, 25.1, 24.8, 17.8.
+
+
UPLC (UPLCꢀMS Method 1): t = 1.08 min. MS (ES+): 522.4 (M+H) . HRMS (m/z) [M+H] calcd
R
for C H BN O , 522.3139; found, 522.3144.
2
9
41
3
5
4ꢀiodoꢀ1ꢀmethylꢀ1Hꢀpyrazoleꢀ5ꢀcarbonitrile (8)
I
N
CN
N
Step 1: A roundꢀbottom flask was charged with 4ꢀiodoꢀ1ꢀmethylꢀ1Hꢀpyrazoleꢀ5ꢀcarboxylic acid
297 g, 1.18 mol), DCM (2.97 L), and 1,1’ꢀcarbonyldiimidazole (CDI) (207 g, 97% by mass, 1.24
mol). The reaction mixture was stirred at rt for 45 min. NH Cl (189 g, 3.53 mol) and Et N (498
(
4
3
mL, 3.53 mol) were added and the reaction mixture was stirred at rt overnight. The reaction mixture
was concentrated in vacuo and the residue was suspended in water (~3 L) and granulated at rt for 1
h. The solid was collected via filtration, washed with water, and dried in a vacuum oven to afford 4ꢀ
1
iodoꢀ1ꢀmethylꢀ1Hꢀpyrazoleꢀ5ꢀcarboxamide as a colorless solid (222 g, 75% yield). H NMR
(CDCl ) δ: 7.53 (s, 1H), 6.56 (br s, 1H), 6.01 (br s, 1H), 4.21 (s, 3H). UPLC (UPLCꢀMS Method 1):
3
+
t = 0.15 min. MS (ES+): 251.1 (M+H) .
R
24
ACS Paragon Plus Environment

Page 25 of 38
Organic Process Research & Development
1
2
3
4
5
6
7
8
9
Step 2: A roundꢀbottom flask was charged with 4ꢀiodoꢀ1ꢀmethylꢀ1Hꢀpyrazoleꢀ5ꢀcarboxamide (222
°
g, 886 mmol) and DCM (2.22 L) and the reaction mixture was cooled to 0 C. 2,6ꢀLutidine (310
mL, 2.66 mol) and TFAA (253 mL, 1.77 mol) were added. After reaction was complete, saturated aq
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
NaHCO (800 mL) was added and the layers separated. The aqueous layer was washed with DCM
3
(
800 mL). The organic layers were combined and washed with saturated aq NH Cl (800 mL), 1N
4
HCl (800 mL), and brine (800 mL). The organic layer was dried over MgSO , filtered, and
4
°
concentrated in vacuo. The residue was suspended in heptanes (~2 L) and granulated at 0–5 C for
3
0 min. The solid was collected via filtration and dried in a vacuum oven to afford 4ꢀiodoꢀ1ꢀmethylꢀ
1
Hꢀpyrazoleꢀ5ꢀcarbonitrile as a colorless solid (196 g, 95% yield). Steps 1 and 2 were repeated with
1
13
minor modifications to provide 7.2 kg of 8. H NMR (CDCl ) δ 7.60 (s, 1H), 4.09 (s, 3H). C NMR
3
(151 MHz, CDCl ) δ:144.8, 120.9, 110.0, 66.7, 39.4. UPLC (UPLCꢀMS Method 1): t = 0.70 min.
3
R
+
MS (ES+): 233.8 (M+H) .
tertꢀbutyl
(3R)ꢀ3ꢀ{[4ꢀ(5ꢀcyanoꢀ1ꢀmethylꢀ1Hꢀpyrazolꢀ4ꢀyl)benzoyl](3ꢀmethylpyridinꢀ2ꢀ
yl)amino}piperidineꢀ1ꢀcarboxylate (9)
4
ꢀBromobenzamide 6 (2.67 g, 5.12 mmol), nitrile 8 (1.19 g, 5.12 mmol), Pd (dba) (234 mg, 0.256
2 3
mmol), and XPhos (257 mg, 0.512 mmol) were dissolved in dioxane (27 mL) under nitrogen
25
ACS Paragon Plus Environment

Organic Process Research & Development
Page 26 of 38
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
atmosphere. A solution of Na CO (1.63 g, 15.4 mmol) in water (3 mL) was added. The mixture
2
3
º
was heated at 80 C for 6 h, then stirred at rt overnight. The reaction mixture was diluted with
EtOAc (150 mL) and washed with 50% brine solution (100 mL). The separated organic phase was
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
dried over MgSO , filtered and concentrated. The residue was purified by silica gel column
4
chromatography eluting with a gradient of 15–100% EtOAc/heptane to afford tertꢀbutyl (3R)ꢀ3ꢀ{[4ꢀ
(5ꢀcyanoꢀ1ꢀmethylꢀ1Hꢀpyrazolꢀ4ꢀyl)benzoyl](3ꢀmethylpyridinꢀ2ꢀyl)amino}piperidineꢀ1ꢀcarboxylate
1
as a colorless solid (1.87 g, 73% yield). H NMR (CDCl ) δ: 8.43 (m, 1H), 7.72 (s, 1H), 7.40–7.35
3
(m, 5H), 7.21–7.02 (m, 1H), 4.87–4.32 (m, 2H), 4.07 (s, 3H), 3.49 (d, 1H), 2.82–2.21 (m, 2H), 2.05
1
3
(
s, 3H), 1.80–1.48 (m, 4H), 1.47 (d, 9H). C NMR (CDCl ) δ: 169.1, 154.8, 153.2, 146.8, 140.0,
3
137.1, 136.7, 131.8, 130.7, 129.3, 128.7, 125.5, 123.3, 112.1, 111.1, 79.6, 55.0, 48.2, 43.7, 38.5,
+
2
8.4, 28.2, 27.5, 25.1, 17.8. UPLC (UPLCꢀMS Method 1): t = 1.01 min. MS (ES+): 501.4 (M+H) .
R
+
HRMS (m/z) [M+H] calcd for C H N O , 502.2638; found, 502.265.
28 33
6
3
5ꢀ(4ꢀiodoꢀ1ꢀmethylꢀ1Hꢀpyrazolꢀ5ꢀyl)ꢀ2Hꢀtetrazole (10)
Caution: This reaction generates hydrazoic acid and requires appropriate safety measures. Sodium
azide is highly toxic.
Original Method:
The nitrile 8 (462 mg, 1.98 mmol, 1 eq) was dissolved in DMF (2 mL). To this solution was added
NaN (387 mg, 4.66 mmol, 2.4 eq) and NH Cl (318 mg, 5.95 mmol, 3 eq). A blast shield was
3
4
placed in front of the reaction set up, then the reaction mixture was heated to 120 °C. After 3 h,
26
ACS Paragon Plus Environment

Page 27 of 38
Organic Process Research & Development
1
2
3
4
5
6
7
8
9
LCMS indicated reaction was complete. The mixture was diluted with water, cooled to 0 °C and
quenched using 20% aq NaNO followed by addition of 20% aq H SO until the pH of the mixture
2
2
4
was 3. A solid precipitated and was collected via suction filtration using a Buchner funnel with a
paper filter to afford 478 mg (88%) of tetrazole 10.
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
Generation 1:
A 50 L reactor was charged with DMF (16.9 kg) and water (5.8 kg) followed by nitrile 8 (4.5 kg,
19.3 mol). To the slurry was added NH Cl (2.1 kg, 38.6 mol) and NaN (2.5 kg, 38.6 mol). The inlet
4
3
and outlet were positioned at opposite sides of the glass lid and the reactor content was swept with
nitrogen to a 25% NaOH scrubber. The flow rate was not measured, but remained strong enough to
ensure bubbling through the scrubber to mitigate the risk of exposure to hydrazoic acid during the
°
course of the reaction. The slurry was then heated to 90 C for 16 h after which time HPLC assay
°
showed 99.6% conversion. The mixture was cooled to 5 C and water (11.6 kg) was added and
proper reactor venting was ensured. The reaction was quenched by the addition of a 40 wt% NaNO2
solution (3.4 kg, 19.3 mol of NaNO ). Next, a 32% HCl solution (7.2 kg) was added to the reactor to
2
a final pH of 0.5, inducing precipitation of tetrazole 10 in the process. (Note: aq HCl was preferred
over sulfuric acid for large scale work since it provided a mixture without precipitate because of the
formation of NaCl instead of the less soluble Na SO ). A Nutsche filter was pressurized with
2
4
nitrogen, then the mixture was filtered and the solids were washed with water (10.1 kg). The solids
were dried initially by flowing air through the Nutsche filter followed by air drying on trays to
1
afford tetrazole 10 (4.8 kg, 99.8 area% by HPLC, 89.1% yield). H NMR (MeOHꢀd ) δ 7.69 (s, 1H),
4
1
3
4.08 (s, 3H). C NMR (MeOHꢀd ) δ 151.8, 146.2, 132.8, 62.2, 39.9. UPLC (UPLCꢀMS Method 1):
4
+
t = 0.52 min. MS (ES+): 276.9 (M+H) .
R
27
ACS Paragon Plus Environment

Organic Process Research & Development
Page 28 of 38
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Generation 2:
A 500 mL reactor was charged with IPA (100 mL) and water (100 mL) followed by nitrile 8 (20 g,
6 mmol). To the slurry was added ZnBr (1.9 g, 8.6 mmol). The reactor content was swept with
8
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
nitrogen to a 20% NaOH scrubber to mitigate the risk of exposure to hydrazoic acid during the
°
course of the reaction. The slurry was then heated to 75 C. To the homogeneous solution was added
NaN (11.2 g, 172 mmol). After 47 h HPLC assay showed 95.7% conversion. The mixture was
3
°
cooled to 5 C. The reaction was quenched by the addition of a 20 wt% NaNO solution (44.42 g,
2
1
29 mmol of NaNO ). Next, a 20% HCl solution (58.4 g) was added to the reactor to a final pH of
2
ca. 1, inducing precipitation of tetrazole 10 in the process. The mixture was filtered on a Buchner
filter, the solids were washed with cold water, and dried in air to afford tetrazole 10 (20.06 g, 98
area% by HPLC, 84.6% yield).
tertꢀbutyl
(3R)ꢀ3ꢀ(4ꢀ(1ꢀmethylꢀ5ꢀ(2Hꢀtetrazolꢀ5ꢀyl)ꢀ1Hꢀpyrazolꢀ4ꢀyl)ꢀNꢀ(3ꢀmethylpyridinꢀ2ꢀ
yl)benzamido)piperidineꢀ1ꢀcarboxylate (11)
N
O
O
N
O
N
N N
NH
N
N N
A roundꢀbottom flask was charged with tertꢀbutyl (3R)ꢀ3ꢀ{(3ꢀmethylpyridinꢀ2ꢀyl)[4ꢀ(4,4,5,5ꢀ
tetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)benzoyl]amino}piperidineꢀ1ꢀcarboxylate 6 (1.53 g, 2.93 mmol)
28
ACS Paragon Plus Environment

Page 29 of 38
Organic Process Research & Development
1
2
3
4
5
6
7
8
9
and 5ꢀ(4ꢀiodoꢀ1ꢀmethylꢀ1Hꢀpyrazolꢀ5ꢀyl)ꢀ2Hꢀtetrazole 10 (674 mg, 2.44 mmol). To this was added
dioxane (12.2 mL) and aq NaOH (4.07 mL, 3M solution, 12.2 mmol). The reaction mixture was
°
heated to 70 C under nitrogen atmosphere for 30 min. To a vial was added the Pd(OAc) (34.3 mg,
2
®
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
0.153 mmol) and cataCXium A (123 mg, 0.342 mmol). The atmosphere was exchanged for
nitrogen, and toluene was added. The catalyst was stirred at rt for 15 min, until a bright yellow
slurry formed. At this point, the catalyst was added to the reaction mixture, and the temperature was
°
°
adjusted to 100 C. The reaction mixture was stirred for 3 h at 100 C. After complete consumption
of the starting material, the reaction was concentrated under reduced pressure, then water (10 mL)
was added. The reaction mixture was extracted with MTBE (3x 25 mL). The aq phase was cooled to
°
0 C, and 1M HCl was added dropwise to form a precipitate which was collected via suction
filtration and dried in vacuo to obtain tertꢀbutyl (3R)ꢀ3ꢀ(4ꢀ(1ꢀmethylꢀ5ꢀ(2Hꢀtetrazolꢀ5ꢀyl)ꢀ1Hꢀ
1
pyrazolꢀ4ꢀyl)ꢀNꢀ(3ꢀmethylpyridinꢀ2ꢀyl)benzamido)piperidineꢀ1ꢀcarboxylate 13 (1.2 g, 90%). H
NMR (MeOHꢀd ) δ: 8.42 (d, 1H), 7.80 (s, 1H), 7.72–7.51 (m, 1H), 7.30–7.05 (m, 5H), 4.62–4.44
4
(m, 2H), 4.18–3.98 (m, 2H), 3.95 (s, 3H), 2.75–2.62 (m, 1H), 2.18–2.04 (m, 2H), 1.79–1.70 (m, 2H),
1
3
o
1.53–1.42 (m, 4H), 1.31 (s, 9H). C NMR (101 MHz, CDCl , 55 C) δ: 172.2, 156.6, 154.0, 152.4,
3
147.9, 142.0, 139.0, 136.2, 135.4, 133.8, 129.8, 129.0, 127.9, 125.3, 124.7, 81.4, 56.5, 45.2, 38.4,
+
3
3.1, 28.8, 26.2, 18.1. UPLC (UPLCꢀMS Method 1): t = 0.82 min. MS (ES+): 544.4 (M+H) .
R
+
HRMS (m/z) [M+H] calcd for C H N O , 544.2779; found, 544.2771.
2
8
34
9
3
1ꢀ[5ꢀ(4ꢀiodoꢀ1ꢀmethylꢀ1Hꢀpyrazolꢀ5ꢀyl)ꢀ2Hꢀtetrazolꢀ2ꢀyl]ethyl 2ꢀmethylpropanoate (racꢀ12)
29
ACS Paragon Plus Environment