Development of a Kilogram-Scale Synthesis of a Novel Anti-HCV Agent, CH4930808

Article abstract of DOI:10.1021/acs.oprd.7b00383

Herein, we report the kilogram-scale synthesis of CH4930808 (1), a novel anti-hepatitis C virus agent. While pursuing improved productivity using many through-process strategies, we conducted scrupulous impurity control. Finally, we successfully developed

Full text of DOI:10.1021/acs.oprd.7b00383

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Full Paper
Development of a Kilogram-scale Synthesis
of a Novel Anti-HCV Agent, CH4930808
Tsuyoshi Haneishi, Yoshiaki Kato, Hiroshi Fukuda, Tomoyuki Shimamura, Takemi Tanokura, Akira
Hiraide, Kaichiro Koyama, Masato Fudesaka, Kenji Maeda, Nobuyuki Nakata, Masahiro Nagase,
Takahiko Yabuzaki, Hiroaki Takao, Masaharu Kigawa, Hitoshi Shimizu, and Makoto Shimizu
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.7b00383 • Publication Date (Web): 16 Jan 2018
Downloaded from http://pubs.acs.org on January 16, 2018
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Development of a Kilogram-scale Synthesis of a
Novel Anti-HCV Agent, CH4930808
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Tsuyoshi Haneishi,†* Yoshiaki Kato,‡ Hiroshi Fukuda,† Tomoyuki Shimamura,‡ Takemi
Tanokura,‡ Akira Hiraide,‡ Kaichiro Koyama,‡ Masato Fudesaka,‡ Kenji Maeda,‡ Nobuyuki
Nakata,‡ Masahiro Nagase,‡ Takahiko Yabuzaki,‡ Hiroaki Takao,‡ Masaharu Kigawa,‡ Hitoshi,
Shimizu,‡ and Makoto Shimizu§
† Research Division, Chugai Pharmaceutical Co., Ltd., 1-135 Komakado, Gotemba, Shizuoka
412-8513, Japan
‡ Pharmaceutical Technology Division, Chugai Pharmaceutical Co., Ltd., 5-5-1 Ukima, Kita-ku,
Tokyo 115-8543, Japan
§ Department of Chemistry for Materials, Graduate School of Engineering, Mie University, Tsu,
Mie 514-8507, Japan
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ABSTRACT
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Herein, we report the kilogram-scale synthesis of CH4930808 (1), a novel anti-HCV agent.
While pursuing productivity using many through process, we conducted scrupulous impurity
control. Finally, we successfully developed a practical and scalable process for the synthesis of
(1·1.5 Na·2.5 H2O), by which we prepared 3.28 kg of the active pharmaceutical ingredient (API)
for clinical studies.
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Figure 1. Structure of CH4930808 (1)
KEYWORDS: Anti-HCV agent, telescoping, one-pot, impurity
INTRODUCTION
HCV is a worldwide infectious pathogen that causes chronic liver diseases, including hepatic
fibrosis, hepatic cirrhosis, and hepatocellular carcinoma (HCC).1 CH4930808 (1) showed potent
anti-HCV replicon activity and preferable pharmacokinetic profiles in rats.2 For clinical studies,
a multi-kilogram quantity of 1 was required. Because each intermediate had low crystallinity, we
used through-process strategies such as telescoping and one-pot procedure. By focusing on the
control of the critical side products in each step, which affected the quality of the API, we
successfully developed a robust manufacturing method of producing the API.
RESULTS AND DISCUSSIONS
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Initial Method for the API. In the initial route, a starting compound, L-tyrosine tert-butyl ester
2, was reacted with Boc2O in methanol to yield N-Boc-protected 3 (Step 1). O-2-butynylation
was performed using 1-bromo-2-butyne and potassium carbonate in DMF to afford 4 (Step 2),
and then N-Boc deprotection with 4N HCl in AcOEt followed by crystallization as an HCl salt
after liquid-liquid separation yielded the HCl salt of 5 (Step 3).
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A method capable of synthesizing a large amount of compound 6 as a (S)-1-phenethylamine
salt was developed previously.3 After de-salting 6 by liquid-liquid separation using aqueous
KHSO4 solution, amide-coupling with 5 using HATU/DIEPA in DMF gave 7 as a crude oil (Step
4). Then, both the O-TBS group and the ketal moiety were simultaneously deprotected with citric
acid at 60 ºC to afford 8 (Step 5), and a carboxylic acid salt 9 was obtained by 4-acetamido-
TEMPO oxidation4 of 8 and subsequent treatment with dicyclohexylamine (Step 6). Finally, de-
salting with liquid-liquid separation, followed by cleaving t-Bu ester using TFA/CH2Cl2, yielded
the target CH4930808 (1) as a free form (Step 7).5
In this route, isolation of compound 9·DCHA by crystallization after Step 6 was critical for
removing the impurities generated by the through process. However, since the crystallization was
an unusual oil-solid conversion in a poor solvent at 5 ºC, we could not perform the crystallization
on a large scale. Thus we decided to keep the API quality by avoiding the generation of the
critical contaminants in each step which were identified based on the detailed impurity analysis,
including isolation, characterization and generation cause analysis of the contaminants in
compound 1 obtained by the through process without isolation of compound 9·DCHA.
Scheme 1. Initial synthesis of CH4830808 (1)
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Steps1-3. By comparing the contaminants of 5 and the API, which were synthesized by initial
route without isolation of compound 9·DCHA, we identified the following four types of critical
contaminants in 5 and improved the process to minimize them.
Scheme 2. Generation process of impurities observed in 5
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In Step 1, Boc-protection was performed using a 1.00-equivalent of Boc2O in order to suppress
the formation of 10, from which Impurity-1 was formed. In addition, the reaction solvent used in
Step 2 was changed to DMF to enable one-pot synthesis of Steps 1 and 2.
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In the next Step 2, it was suggested that insufficient reaction conversion rate would lead to
contamination with Impurity-2 in 5. The conversion rate was improved by using fine-particle
K2CO3 and controlling the water content in the reaction system as described in Table 1. More
than 99.9% conversion was achieved at manufacturing (Entry 6).
Table 1. a Effect of water content for reaction conversion of Step 2
Entry Amount of additional water (v/w) KF (%) Conversion (%) at 30 min.
1
2
0
0.07
0.9
1.9
3.7
7.0
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99.8
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b 6
0.055
> 99.9
b
a shaking at rt, 600 mg scale. 25 ºC, 1 hr, at 6.00 kg scale manufacturing.
It was also found that residual 1-bromo-2-butyne gave rise to the formation of Impurity-3 on
the isolation of the compound 5 in the next step, resulting in the decreased yield of 5. This
problem was solved by adding diethylamine to quench the alkylating agent thoroughly after the
reaction was complete.6 In addition, contamination of a small amount of 1,3-dibromo-2-butene in
the 1-bromo-2-butyne that had been purchased in the early development stage caused Impurity-4
to form. This concern was addressed by setting the standard as high-quality at the time of
purchasing of the reagent.
In this way, a high-yield and high-purity synthesis of the intermediate 5 was successfully
developed.
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Steps 4-6. In the through process from Step 4 to Step 6, we identified two critical
contaminants affecting the quality of compound 1, which was synthesized by initial route
without isolation of compound 9·DCHA.
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One, which was found by correlating the amounts of 5, 12, and Impurity-5, was residual 5 in
compound 13, which we confirmed was transferred to Impurity-5, as depicted in Scheme 3.
Thus, after the condensation of 5 with 6 using less expensive WSC in AcOEt, the amount of
residual 5 was decreased to 0.23% by washing with aq. citric acid twice. As a result, Impurity-5
in the API was reduced to 0.07%. The other critical impurity was Impurity-6 generated in the
acidic condition (Step 5).7 Since the generation of Impurity-6 was inevitable under acidic
conditions, the route was modified to cleave the TBS group first under neutral conditions with
TBAF-AcOH and to deprotect the ketal separately after oxidation of alcohol to avoid the
formation of the lactone.
Scheme 3 Structure and plausible mechanism for the generation of Impurity-5
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Scheme 4 Generation of Impurity-6 at Step 5
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Step 7 and API Crystallization (Step 8). In Step 7, the reagent was changed from
TFA/CH2Cl2 to H2SO4/MeCN in order to reduce the burden on the environment and the
apparatus. As the API form of CH4930808 was determined to be 1.5 Na salt 2.5 hydrate, we
purified the final API by aqueous crystallization, adjusting the pH to 4.8.
Furthermore, in order to achieve the API at high quality and with a high recovery yield, we
established a subsequent purification process that involved 1) high-recovery crystallization, 2)
saponification, and 3) quality-raising crystallization. We performed the first aqueous
crystallization with a higher concentration of NaCl, and we also adjusted the pH to 4.8 to
increase the recovery yield. Then, before the next aqueous crystallization, we performed the
saponification process to hydrolyze the impurities8 so that they would be easier to remove.
Lastly, the second crystallization from aqueous MeCN afforded the API (99.62% purity).
Scheme 5. Improved process for preparing the API (1·1.5 Na ·2.5 H2O)
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SUMMARY
As described above, under our policy of using through process, we established a highly
efficient manufacturing method that can guarantee the quality of the API with high robustness.
Actually, we successfully manufactured 3.28 kg of API for clinical studies. The table below
summarizes the improvements made in the new route (Table 2).
Table 2. Improvement Summary
Initial Route Improved route
Total steps
Intermediate Isolation
Total organic solvent (v/w)
Kind of organic solvent
Filtration
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Concentration
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Vessel washing
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Total yield of 5·HCl (from 2, %)
82
99.0
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86
Purity of 5·HCl (HPLC %)
Total yield of 1 (from 6, %)
Purity of 1 (HPLC %)
99.8
a
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b
98.52
99.62
a Total yield of API (1·1.5Na·2.5 H2O), b purity of API.
EXPERIMENTAL SECTION
General. All reagents and solvents were purchased from commercial suppliers and used
without further purification. Optical rotations were measured on Horiba SEPA-500 automatic
polarimeter at the sodium D line (589 nm). 1H NMR spectra at 400 MHz and 13C NMR spectra at
100 MHz were recorded on Bruker Ascend 400, Chemical shifts for proton were reported in
parts per million (δ), downfield from tetramethylsilane as an internal standard and are referenced
13
to residual protium in the NMR solvent (CD3OD; δ 3.30 ppm). For C NMR, chemical shifts
were reported in the scale relative to NMR solvent (CD3OD; δ 49.0 ppm) as an internal
reference. NMR data are presented as follows: chemical shift (δ, ppm), integration, multiplicity
(s = singlet, d = doublet, dd = double-doublet, t = triplet, q = quartet, and m = multiplet) and
coupling constant. IR spectra were recorded by JASCO FT/IR-350. High resonance mass spectra
(HRMS) were recorded by Waters Xevo G2S TOF (ESI).
tert-butyl (tert-butoxycarbonyl)-L-tyrosinate (3)
To the solution of tert-butyl-L-tyrosinate (2, 6.00 kg, 25.3 mol) in DMF (19.81 kg) was added
Boc2O (5.52 kg, 25.3 mol) in DMF (2.81 kg). The mixture was stirred at 25 ºC for 1 h (>99%
conversion). The resulting mixture was used for the next step.
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tert-butyl
butoxycarbonyl)amino)propanoate (4)
(S)-3-(4-(but-2-yn-1-yloxy)phenyl)-2-((tert-
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To the solution of crude 3, H2O (0.33 kg) was added to adjust the water content (KF analysis,
1.0% weight), K2CO3 (14.00 kg, 101 mol) and 1-bromo-2-butyne (4.02 kg, 30.2 mol) were
successively added while maintaining the temperature below 40 ºC. The slurry was stirred at 25
ºC for 1 h. (>99% conversion) and stored at below 40 ºC overnight. Diethylamine (0.92 kg, 12.58
mol) was added and stirred at 25 ºC for 1 h. Water (72.0 kg) and AcOEt (37.8 kg) were added,
followed by liquid-liquid separation. The organic phase was washed with water (60.0 kg), 2% aq.
KHSO4 (60.0 kg), 5% aq. NaCl (60.0 kg), successively.
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The resulting organic phase was concentrated under reduced pressure. To the residue, AcOEt
(37.8 kg) was added and this solution was used for the next step.
tert-butyl (S)-2-amino-3-(4-(but-2-yn-1-yloxy)phenyl)propanoate hydrochloride (5·HCl)
To the solution of crude 4, 4N HCl in AcOEt (34.9 kg, 136.9 mol) was added and stirred at 25
ºC for 2 h (>97% conversion). To the reaction mixture, 10% aq. NaOH (90 L) was added below
25 ºC and the solution was stirred. The separated organic phase was washed with 10% aq. NaCl
(30.0 kg), and was cooled to below 0 ºC. Water (10.2 kg) and 4N HCl in AcOEt (5.8 kg) were
added successively and stirred at 0 ºC for 30 min. The solid was collected by filtration, and the
cake was washed with AcOEt (54.0 kg) and dried to afford 5·HCl (7.08 kg, 86% from compound
2, 99.8% purity by HPLC analysis, Impurity-1; 0.05 %, Imputity-4; 0.04 %). Colorless solid;
[α]D23 +17.4º (c 0.51, MeOH); 1H-NMR (400MHz, CD3OD) δ: 1.43 (s, 9H), 1.80 (t, 3H, J = 2.1
Hz), 3.12 (d, 2H, J = 7.0 Hz), 4.12 (t, 1H, J = 7.0 Hz), 4.66 (q, 2H, J = 2.1 Hz), 6.96 (d, 2H, J =
8.6 Hz), 7.21 (d, 2H, J = 8.6 Hz); 13C NMR (100MHz, CD3OD) δ: 3.1, 28.1(3 carbons), 36.9,
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55.7, 57.1, 75.1, 84.1, 85.1, 116.4(2 carbons), 127.8, 131.6(2 carbons), 158.9 169.2; HRMS m/z
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calcd for C17H23NO3 [M+H]+ 290.1756, found 290.1751.
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tert-butyl
(2S,3S,E)-3-(((S)-3-(4-(but-2-yn-1-yloxy)phenyl)-1-(tert-butoxy)-1-
oxopropan-2-yl)carbamoyl)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)-11-(2-heptyl-1,3-
dioxolan-2-yl)-2-hydroxyundec-4-enoate (7)
To the reactor charged with (S)-1-phenylethan-1-amine (S,E)-2-((S)-1-(tert-butoxy)-4-((tert-
butyldimethylsilyl)oxy)-2-hydroxy-1-oxobutan-2-yl)-10-(2-heptyl-1,3-dioxolan-2-yl)dec-3-
enoate (6, 6.00 kg, 96.1% purity, 99.39% ee, 93.4 w%, 7.47 mol), AcOEt (27.0 kg), 5% aq.
KHSO4 (30.0 kg) was added, then stirred at 20 ºC for 30 min. The separated organic phase was
washed with 5% aq. NaCl (60.0 kg). To the organic phase, DIPEA (4.70 kg, 36.4 mol), tert-butyl
(S)-2-amino-3-(4-(but-2-yn-1-yloxy)phenyl)propanoate hydrochloride (5·HCl, 2.71 kg, 98.9 w%,
8.23 mol), HOBt hydrate (1.50 kg, 9.79 mol) and WSC hydrochloride (1.80 kg, 9.39 mol) were
added and stirred at 20 ºC for 4 h (>95% conversion). The reaction mixture was washed with
10% aq. citric acid (60.0 kg), 10% aq. citric acid – 2% aq. NaCl (60.0 kg) and 5% aq. NaCl (60.0
kg) successively, and this solution was used for the next step.
tert-butyl (2S,3S,E)-3-(((S)-3-(4-(but-2-yn-1-yloxy)phenyl)-1-(tert-butoxy)-1-oxopropan-
2-yl)carbamoyl)-11-(2-heptyl-1,3-dioxolan-2-yl)-2-hydroxy-2-(2-hydroxyethyl)undec-4-
enoate (13)
To the solution of crude 7, AcOH (0.58 kg, 9.66 mol) and TBAF (1M in THF, d=0.903, 8.70
kg, 9.64 mol) were added and stirred at 20 ºC for 3 h (>95% conversion). 5% aq. NaHCO3 (60.0
kg) was added to the mixture, followed by liquid-liquid separation. The organic phase was
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concentrated under reduced pressure. To the residue, MeCN (23.5 kg) was added, and this
solution was used for the next step.
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(3S,4S,E)-4-(((S)-3-(4-(but-2-yn-1-yloxy)phenyl)-1-(tert-butoxy)-1-oxopropan-2-
yl)carbamoyl)-3-(tert-butoxycarbonyl)-12-(2-heptyl-1,3-dioxolan-2-yl)-3-hydroxydodec-5-
enoic acid (14)
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To the solution of crude 13, iodobenzene diacetate (7.16 kg, 22.2 mol) was added in 4 portions
at below 15 ºC, then water (3.00 kg) and 4-acetamido-TEMPO (0.34 kg, 1.59 mol) were added
and stirred at 15 ºC for 1 h (>95% conversion). 5% aq. Na2S2O3 (30.0 kg) and AcOEt (27.0 kg)
were added, followed by liquid-liquid separation. The separated organic phase was washed with
20% aq. NaCl (30.0 kg) and evaporated under reduced pressure. To the residue, MeCN (23.5 kg)
was added and evaporated under reduced pressure, then MeCN (23.5 kg) was added again, and
this solution was used for the next step.
(S)-2-((S,E)-1-(((S)-2-(4-(but-2-yn-1-yloxy)phenyl)-1-carboxyethyl)amino)-1,11-
dioxooctadec-3-en-2-yl)-2-hydroxysuccinic acid (1, CH4930808)
To the solution of crude 14, water (0.23 kg), sulfuric acid (2.80 kg, 28.6 mol) was added at
below 15 ºC and the solution was stirred at 25 ºC for 1 h. (>95% conversion). Water (30.0 kg)
and AcOEt (27.0 kg) were added, followed by liquid-liquid separation. The separated organic
phase was washed with 10% aq. NaCl twice (30.0 kg each time), and then concentrated under
reduced pressure. To the residue, MeCN (14.1 kg) was added and the solution was transferred to
a SUS container using MeCN (4.70 kg) and stored at 5 ºC, and this solution was used for the
final steps.
(S)-2-((S,E)-1-(((S)-2-(4-(but-2-yn-1-yloxy)phenyl)-1-carboxyethyl)amino)-1,11-
dioxooctadec-3-en-2-yl)-2-hydroxysuccinic acid (1·1.5Na·2.5H2O)
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The stored solution of crude 1 was charged to the reactor passed through the line filter, through
which water (12.0 kg) was added. After warming to 35 ºC, the solution was adjusted to pH 4.8
by adding 10% aq. NaOH (3.16 kg) and then 20% aq. NaCl (8.80 kg) was added. The solution
was re-adjusted to pH 4.8 by adding 10% aq. NaOH (2.10 kg). MeCN (0.45 kg) was added to
adjust the MeCN:water =1:1 (v/v). After the solution had been stirred at 5 ºC for 2 h, the solid
was filtered and washed with chilled MeCN:water = 4:1 (14.9 kg) to obtain the wet solid.
Step-2
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The wet solid and water (15.0 kg) were placed in the reactor, and 10% aq. NaOH (3.30 kg) and
MeCN (13.7 kg) were added (pH >13). After the mixture had been stirred at 35 ºC for 30 min.,
2N aq. HCl (3.98 kg) was added to adjust the pH to 4.8, then MeCN (2.96 kg) was added. The
resulting slurry was stirred at 35 ºC for 1 h, then filtered, and the cake was washed with
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MeCN:water = 4:1 (14.9 kg) and dried under 50% RH at 30 ºC to afford 1·1.5Na·2.5H2O;
Colorless solid. (3.28 kg, 55% from compound 6, 99.62 % purity by HPLC analysis, Impurity-5;
0.07 %) [α]D -18.2º (c 0.63, MeOH); 1H-NMR (400MHz, CD3OD) δ: 0.89 (t, 3H, J = 7.0 Hz)
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1.23-1.37 (m, 14H), 1.47-1.57 (m, 4H), 1.81 (t, 3H, J = 2.7 Hz), 1.94-2.00 (m, 2H), 2.40-2.45
(m, 4H), 2.53 (d, 1H, J = 16.1 Hz) 2.80, (d, 1H, J = 16.1 Hz), 2.96 (dd, 1H, J = 7.5, 14.0 Hz),
3.12 (dd, 1H J = 4.8, 14.0 Hz), 3.17-3.20 (m, 1H), 4.53 (dd, 1H, J =5.4, 7.5 Hz), 4.59 (q, 1H, J =
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2.7 Hz), 5.50-5.60 (m, 2H), 6.82 (d, 2H, J = 8.6 Hz), 7.15 (d, 2H, J = 8.6 Hz); C NMR
(100MHz, CD3OD) δ: 3.2, 14.4, 23.7, 24.9, 24.9, 30.0, 30.0, 30.1, 30.2, 30.3, 32.9, 33.6, 38.0,
43.5, 43.5, 43.7, 56.3, 57.1, 58.0, 75.4, 77.9, 83.9, 115.7(2 carbons), 125.4, 131.3, 131.5(2
carbons), 136.7, 158.1, 174.0, 175.2, 176.4, 177.9, 214.5; IR(KBr), 3392, 2926, 2850, 1705,
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1644, 1594, 1511, 1239, 1215 cm-1; HRMS m/z calcd for C35H49NO10[M+H]+ 644.3434, found :
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644.3414.
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ASSOCIATED CONTENT
Supporting Information
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1H-NMR and C-NMR spectra of all isolated compounds and isothermal adsorption curves
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and PXRD spectra of API. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel.: +81-550-87-9102; fax: +81-550-87-5326; e-mail: haneishitys@chugai-pharm.co.jp
ORCID
Tsuyoshi Haneishi: 0000-0002-8602-4331
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
We really thank Ms. Toshiko Ikeda, Ms. Nao Osaka, Ms. Yoko Kikai, Mr. Hiroshi Aoyama
and Mr. Masayuki Fujishima for IPC, Mr. Keiji Nii, Ms. Masako Arai and Ms. Keiko Yokoyama
for structural analyses, Dr. Noriyuki Takata for isothermal adsorption curves data of API, and
Dr. Takashi Emura and Dr. Tsutomu Sato for many scientific and constructive suggestions. We
also thank Mrs. Sally Matsuura at Editing Services at Chugai Pharmaceutical Co., Ltd for
assistance with English usage.
RERERENCES
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1. Sakamoto, H., Okamoto, K., Aoki, M., Kato, H., Katsume, A., Ohta, A., Tsukuda, T.,
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Aoki, Y., Arisawa, M., Kohara, M., Sudoh, M.; Nature Chemical Biology, 2005, 1, 333–337.
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2. (a) Aoki, M., Kato, H., Sudoh, M., Tsukuda, T., Masubuchi, M., Kawasaki, K.; PCT Int.
Appl. WO 2004071503. (b) Kawasaki, K., Masubuchi, M., Hayase, T., Komiyama, S.,
Watanabe, F., Fukuda, H., Murata, T., Matsubara, Y., Koyama, K., Shindoh, H., Sakamoto, H.,
Okamato, K., Ohta, A., Katsume, A., Aoki, M., Aoki, Y., Shimma, N., Sudoh, M., Tsukuda, T.
Bioorg. Med. Chem. Lett., 2013, 23, 336–339.
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3. (a) Kato, T., Kimura, N., Mizutani, A., Makino, T., Kawasaki, K., Fukuda, H., Komiyama,
S., Tsukuda, T.; PCT Int. Appl. WO 2006088071. (b) Kawasaki, K., Masubuchi, M., Fukuda H.,
Murata, T., Hayase, T., Komiyama, S., Watanabe, F., Makino, T., Mizutani, A., Katoh, T.,
Kimura, N., Takano, K., Sudoh, M., Aoki, M., Tsukuda, T., Shinma, N., at the 51st Symposium
on the Chemistry of Natural Products, Nagoya, Japan, October 7–9, 2009; P-29. Manufacturing
of compound 6 was outsourced to CRO.
4. (a) Mico, A. D., Margarita, R., Parlanti, L., Vescovi, A., Piancatelli, G., J. Org. Chem.,
1997, 62, 6974–6977. (b) Epp, J., B., Widlanski, T., S., J. Org. Chem., 1999, 64, 293–295. (c)
Schmidt, S., R., Catalysis of Organic Reactions : Twenty-first Conference, by CRC Press, 2006,
p119.
5. Alternative syntheses were reported. (CH4930808 as free form): (a) Takeuchi, S.,
Yasuda, S., Kumagai, N., Shibasaki, M., Angew. Chem Int. Ed., 2012, 51, 4218–4222. (b)
Murakata, M., Ikeda, T, Org. Biomol. Chem., 2017, 15, 6632–6639.
6. At the 6.00 kg scale manufacturing, 1-bromo-2-butyne was not detected by GC analysis of
organic phase in work-up.
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7. 5% of Impurity-6 was generated in citric acid, MeCN/H2O, 60 ºC 2 h.
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8. Although the structure of impurities has not been confirmed, these are assumed to be
esters, and they are decomposed by saponification process.
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9. Regarding the supply of the 2.5 hydrate of the API, it was indicated from the isothermal
adsorption curves (see Supporting Information) that the water in the crystal was lost when
relative humidity (RH) was 20% or less. Based on this data, the humidity conditioning was
performed.
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