Practical and Scalable Manufacturing Process for Plasma Kallikrein Inhibitor ASP5069

Article

Practical and Scalable Manufacturing Process for Plasma Kallikrein

Inhibitor ASP5069

Shun Hirasawa* and Yoshinori Kohmura

Cite This: https://dx.doi.org/10.1021/acs.oprd.0c00291

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ABSTRACT: The plasma kallikrein inhibitor ASP5069 is a promising drug candidate for the treatment of edema and hematoma

and for the prevention of bleeding during surgery. Here, we report the development of a practical and scalable process for

manufacturing ASP5069 that features a convergent synthetic approach, suppression of impurity formation, eﬀective puriﬁcation of

the amine compound by extraction, improved reproducibility of the reductive amination reaction, and a drying process for the

dihydrate form. This process was successfully used to prepare >100 g of ASP5069.

KEYWORDS: ASP5069, plasma kallikrein inhibitor, Mannich reaction, reductive amination, dihydrate form

INTRODUCTION

deprotection of Boc and tert-butyl groups by triﬂuoroacetic

acid (TFA) gave 1·TFA.

■

In the kallikrein−kinin system, the serine protease plasma

kallikrein cleaves high-molecular-weight kininogen to release

the active peptide bradykinin. Bradykinin is a vasodilator that

contributes to the activation of inﬂammation and production

of pain and may mediate hereditary angioedema (HAE).

Additionally, plasma kallikrein is implicated in the expansion of

After salt screening of 1 for the development form,

monohydrochloride dihydrate (1·HCl·2H O) was selected.

Because multi-kilograms of 1·HCl·2H O are required for

preclinical and clinical studies, the development of a scalable

and practical preparation process was needed. To determine

the scale-up synthetic route, we evaluated two alternative

approaches to obtain 6 (Scheme 2). Route A involved

reductive amination of 3 and 5-chloroanthranilic acid, followed

by amidation with the aniline 4, to give the intermediate 6.

However, the reductive amination conditions did not give the

compound 7. Route B was possibly a one-step shorter

synthetic method for obtaining the compound 5 because the

chronic subdural hematoma and hyperglycemia-induced

cerebral hematoma, and ﬁbrinolysis. The plasma kallikrein

inhibitor ASP5069 (1·HCl·2H O)a carboxylic acid deriva-

tive having three aromatic rings, amidine, and piperazine

(

Figure 1)is therefore a promising drug candidate for the

treatment of edema and hematoma and for the prevention of

bleeding during surgery.

7d,8

coupling of anthranilic acid or isatoic anhydride

with

various amines is widely known. In our case, however, the

target 5 was not obtained under a variety of conditions. Thus,

the failure of the alternative approaches and the convergent

and simple nature of the discovery approach suggested that the

latter may be suitable for scale-up. However, several issues

within the discovery procedure would make it diﬃcult to

manufacture multi-kilograms of 1·HCl·2H O, including the use

of column chromatography puriﬁcation, poor reproducibility,

and safety and environmental concerns. The challenges we

faced in solving these problems are described in detail below.

Figure 1. Structure of ASP5069 (1·HCl·2H O).

RESULTS AND DISCUSSION

■

Mannich Reaction and Alkylation. In the Mannich

reaction of 3-ethoxysalicylaldehyde (2) with methylpiperazine

and formaldehyde in ethanol solvent under discovery

Scheme 1 shows the discovery route for 1·TFA, which

comprises a convergent synthetic approach featuring reductive

amination of aldehyde 3 and aniline 5. Mannich reaction of 2

with methylpiperazine and formaldehyde, followed by

alkylation with tert-butyl bromoacetate, yielded aldehyde 3.

Aniline 5 was synthesized from 4 using the following three-step

sequence: Boc protection, amidation with 5-chloro-2-nitor-

obenzoic acid, and hydrogenation of the nitro group.

Reductive amination of 3 and 5 was performed using sodium

triacetoxyborohydride in acetic acid to give 6. Finally,

Received: June 22, 2020

XXXX American Chemical Society

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Article

Scheme 1. Discovery Route for 1·TFA (ref 6)

Scheme 2. Alternative Approaches to Obtain 6

conditions, 3.4 HPLC area percent (A %) of an unknown

impurity was observed as a major impurity (Table 1, entry 1).

Given that impurities derived from the unknown impurity

remained in the product of the subsequent step, minimizing

the formation of the unknown impurity was required.

solvent 2-propanol was used, the corresponding impurity was

reduced to a very low level (0.7 A %) (entry 2). Because the

purity of the resulting Mannich product 9 was suﬃciently high

without crystallization isolation, the reaction was telescoped to

the next alkylation reaction.

We predicted that the nucleophilicity of the ethanol solvent

The yield of the target 3 was very low under discovery

may generate impurities. When the more sterically hindered

conditions because of the relatively high levels of an impurity

(

approximately 30 A %) formed from the alkylation of 9 with

Table 1. Solvent Study of Mannich Reaction

tert-butyl bromoacetate, and column chromatography puriﬁ-

cation was required to isolate 3. We predicted that the

impurity was a quaternary ammonium compound obtained

from overalkylation of the piperazine nitrogen. To avoid the

overalkylation, ﬁrst, we attempted to inhibit the N-nucleophil-

icity by mixing the Lewis acid ZnBr with 9 to form a metal

complex with the piperazine moiety. Subsequent addition of

tert-butyl bromoacetate yielded 3 with a reduced level of the

overalkylated impurity (8 A %). While impurity formation was

suppressed, there was room for improvement. Second, we

attempted to use the harder electrophile tert-butyl chloroace-

HPLC A %

major impurity (HPLC retention

entry

solvent

start 2 target 9

time)

tate without a Lewis acid. As a result, the quaternary

ammonium did not form and the two-step yield improved from

ethanol

2-propanol

0.6

0.1

92.7

97.0

3.4 (9.5 min)

0.7 (14.9 min)

3% in the discovery method to 79%, as expected. However,

HPLC method A.

small amounts of impurities derived from the Mannich

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Scheme 3. Alkylation of 9 with tert-Butyl Chloroacetate

Scheme 4. Synthesis of Aniline 5 from Starting Material 4

Discovery conditions: (a) 4 M aq NaOH, Boc O, 1,4-dioxane, 0 °C, 7 h, 89%; (b) 5-chloro-2-nitrobenzoic acid, (COCl) , DMF, DCE, rt, 3 h;

concentration; then 8, pyridine, MeCN, rt, 18 h, 76%; (c) H , Raney nickel, 1,4-dioxane, rt, 2 d, 96%. Scale-up conditions: (a) 5 M aq NaOH,

Boc O, THF, 5 °C, 3 h, 97%; (b) 5-chloro-2-nitrobenzoic acid, SOCl , toluene, 100 °C, 6 h; concentration to 2 vol, 60 °C; then 8, i-Pr NEt, THF,

−

10 °C, 2 h, 87%; (c) H , Raney nickel, DMF, H O, 30 °C, 16 h, 87%.

Scheme 5. Reductive Amination and Boc Deprotection to Converge to Amidine 11

reaction contaminated the product, as shown in Scheme 3.

These neutral impurities could be removed to the organic layer

by extraction of the target 3 with an acidic aqueous solution.

Subsequently, basiﬁcation of the aqueous extract and

extraction of 3 with an organic solvent (isopropyl acetate (i-

PrOAc)) gave pure 3 in high quality (98.7 A %). This Mannich

reaction−alkylation sequence was successfully performed on a

replacing oxalyl chloride with thionyl chloride and DCE with

toluene, which gave amide 10 in 87% yield. A previous report

suggested that isolated 2-nitrobenzoyl chloride decomposes

violently in the presence of heat, suggesting the need to use a

diluted solution. In the case of our 5-chloro-2-nitorobenzoyl

chloride intermediate, exothermic decomposition was observed

at 170 °C using diﬀerential scanning calorimetry. We

concluded that 5-chloro-2-nitorobenzoyl chloride could be

safely concentrated in toluene solution by performing the

concentration process at a minimum of 100 °C below the

exothermic temperature and setting the concentration end

point to 2 vol. Finally, in the reduction of the nitro moiety

using hydrogen and Raney nickel, 1,4-dioxane was replaced

with N,N-dimethylformamide (DMF), and aniline 5 was

obtained in 87% yield.

00 g scale.

Preparation of 5. Aniline 5 was prepared from amidine 4

Scheme 4). The discovery method for 5 required improve-

(

ments to safety and environment-related processes. In the Boc

protection of 4, use of 1,4-dioxane needed to be eliminated for

scale-up because of its suspected carcinogenicity. Tetrahy-

drofuran (THF) was found to be a good alternative and Boc-

amidine 8 was obtained in 97% yield. In the discovery method,

oxalyl chloride was used to activate 5-chloro-2-nitorobenzoic

acid and the subsequent amidation was performed in 1,2-

dichloroethane (DCE). Because oxalyl chloride induces the

formation of carbon monoxide, it needed to be avoided. DCE

is inappropriate for scale-up because of potential mutagenic

Reductive Amination. In reductive amination of aniline 5

and aldehyde 3, the discovery method had poor reproducibility

because of the uncontrolled formation of impurities, Boc-

deprotected compound 11 and the possible cyclic aminal 14/

15. Column chromatography puriﬁcation was required to

and environmental impact. This problem was solved by

remove the impurities.

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Scheme 6. Formation of 14/15

Scheme 7. Potential Reaction Mechanism of Reductive Amination

Water caused Boc deprotection, and impurity 11 gradually

increased under the reaction conditions. Because water is a

byproduct of imine formation, it is impossible to inhibit the

water formation and Boc deprotection. Hence, we attempted

to converge to Boc-deprotected compound 11 without

isolating 6. After the reductive amination reaction, addition

of water to the mixture and stirring at 60 °C led to successful

conversion of 6 to 11 (Scheme 5).

In the discovery procedure, the initial mixing of aniline 5

and aldehyde 3 caused the formation of 14/15 by intra-

molecular cyclization of the imine intermediate 12/13, and

subsequent addition of a reductant did not lead to the

conversion of 14/15 to 11/6 (Scheme 6). We predicted that

addition of aldehyde 3 to the mixture of aniline 5 and

reductant would induce immediate reduction of the corre-

sponding imine intermediate 12/13 to yield the target 11/6

with suppression of formation of 14/15. Thus, we attempted

this method to change the order of the reagents added to the

reaction.

As a result, when 3 equiv of sodium triacetoxyborohydride

was added to a slurry of aniline 5 in acetic acid, the slurry

turned into a clear solution with the generation of hydrogen

gas. Thereafter, addition of the aldehyde 3 and subsequent Boc

deprotection yielded the target 11 as the main product (75.7 A

%), while successfully suppressing the production of 14 (0.4 A

%). When the reaction involved reducing the amount of

reductant from 3 equiv to 1.5 equiv, the target 11 and 14 were

obtained at 29.3 A % and 52.9 A %, respectively. Based on the

above results, we suggest the following potential reaction

mechanism in Scheme 7: (1) at the mixing of aniline 16/5 and

sodium triacetoxyborohydride, the boron complex 17/18 was

formed with the generation of hydrogen, which has a much

higher solubility than aniline 16/5, leading the reaction

mixture to gradually change from a slurry to a clear solution;

(2) aldehyde 3 was added, and boron-complexed imine 19/20

was generated from aldehyde 3 and boron-coordinated aniline

17/18; (3) because the imine was activated by boron, it was

immediately reduced by another sodium triacetoxyborohydride

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>6.0 led to contamination with an undesired polymorph or

Article

Scheme 8. tert-Butyl Ester Deprotection of 11

to give 21/22; and (4) the water byproduct of imine formation

induced hydrolysis to give the target 11/6. The theoretical

need for 2 equiv of sodium triacetoxyborohydride suggested by

the potential mechanism indicates that the above result of

obtaining 14 at 52.9 A % in the presence of 1.5 equiv of

sodium triacetoxyborohydride is reasonable.

aqueous acetone at pH < 5.0 (Figure 2). Adjusting the pH to

Meanwhile, the boron complex 17/18, which was observed

as 16/5 on HPLC, was unstable under the reaction conditions

and gradually decomposed. Because the melting point of acetic

acid is 16.7 °C, the reaction temperature cannot be lowered.

However, the use of acetic acid/isopropyl acetate as the solvent

enables the temperature to be lowered to <0 °C, and the

decomposition of 17/18 can be suppressed. Given that

isopropyl acetate was the extraction solvent for the puriﬁcation

of 3, the isopropyl acetate solution of 3 can be utilized without

solvent switching.

After Boc deprotection for conversion to amidine 11,

crystallization was performed in 2-butanol/tert-butyl methyl

ether (MTBE). This highly reproducible method solved the

problems associated with the discovery method and gave 11 in

Figure 2. Solubility of ASP5069 in aqueous acetone. Conditions:

ASP5069 (100 mg), acetone (10 vol), water (40 vol), NaCl (2 eq), 1

M aqueous HCl for pH adjustment, rt, >5 h.

pseudopolymorph, as observed by X-ray powder diﬀraction

8% yield at a 100 g scale.

Cleavage of the tert-Butyl Ester and Properties of 1·

HCl. Although tert-butyl ester deprotection of 11 under

(

XRPD) (Figure S2). Given that the pKa values were 11.2, 7.7,

.7, and 0.9 for base and 2.8 for acid, as calculated using ACD/

Percepta, compound 1 may exist mostly as a monohydro-

chloride in an approximately pH 5.5 aqueous solution. Based

on these ﬁndings, the target pH was set to 5.5.

hydrochloric acid/alcohol solvent conditions yielded 1·3HCl,

many impuritiessuch as amide, in which an amidine moiety

was hydrolyzed, and ester, in which tert-butyl ester was

substituted with an alcohol solventwere observed. In

addition, because 1·3HCl was obtained as an amorphous

solid, puriﬁcation of 1 was diﬃcult without using column

chromatography. We attempted to use an excess amount of

hydrochloric acid without an alcohol solvent to suppress the

solvent-mediated substitution reaction in the deprotection

reaction. As expected, the side reaction was inhibited, and the

subsequent addition of acetone as an antisolvent led to the

formation of a crystalline rather than an amorphous solid.

Elemental analysis of the obtained crystal indicated that it was

After polish ﬁltration of the aqueous 1·4HCl solution, pH

was adjusted with aqueous sodium hydroxide, and acetone was

added as an antisolvent for crystallization of 1·HCl·2H O.

Interestingly, the pH decreased with the precipitation of

crystals, leading to a corresponding increase in the loss of 1 to

the mother liquor. This phenomenon suggested that the

compound 1 may be in equilibrium with dihydrochloride and

monohydrochloride in pH 5.5 aqueous acetone, as shown in

Scheme 9. Precipitation of 1·HCl would move the reaction

Scheme 9. Possible Equilibrium of Compound 1 in pH 5.5

Aqueous Acetone

·4HCl·6H O, not 1·3HCl (Scheme 8). Following the

observation that the weight of 1·4HCl changed after drying,

we investigated the adsorption and desorption of hydrated

water at diﬀerent levels of relative humidity (RH). We

observed a unique hygroscopic proﬁle in which 1 ·4HCl

adsorbed and desorbed up to 13 equiv of water (Figure S1).

Given that the amount of hydrate water in this compound

could easily be altered depending on the humidity of its

toward the formation of more of 1·HCl and hydrogen chloride,

causing the pH to decrease. pH adjustment was repeated every

few hours until the pH stabilized at the target value, which was

achieved after several adjustments, and the monohydrochloride

form was obtained with decreased loss (4.3%) to the mother

liquor.

environment, 1·4HCl·xH O (x = 0−13) was immediately used

for the next reaction after drying.

Formation of ASP5069 (1·HCl·2H O) and Drying

Process for the Dihydrate form. Because 1 contains a

carboxylic acid moiety and several basic nitrogen atoms, the

Meanwhile, there was concern about losing the hydrated

water under drying conditions. Figures 3 and 4 show the

hygroscopic proﬁles at 25 °C and 50 °C. At 25 °C, according

solubility of ASP5069 (1·HCl·2H O) was largely dependent

on pH. The solubility of ASP5069 increased signiﬁcantly in

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Figure 3. Hygroscopic properties of 1 ·HCl at 25 °C.

Article

good yield. The ﬁnal steps were cleavage of the tert-butyl ester

and hydrochloride salt formation. tert-Butyl ester deprotection

was performed in the presence of excess amounts of

hydrochloric acid to yield 1·4HCl as a crystal, which had

unique hygroscopic properties. Wet ASP5069 was isolated at

pH 5.5 under pH-controlled crystallization in an aqueous acid

solution. Finally, drying the dihydrate form under controlled

vacuum pressure gave ASP5069. This process was successfully

used to yield >100 g of ASP5069 with a purity of 98.5 A %.

EXPERIMENTAL SECTION

■

General Methods. Unless otherwise noted, all reactions

were performed under a nitrogen atmosphere. All reagents and

solvents purchased from suppliers were used as received unless

otherwise noted. NMR spectra were recorded on a Bruker

ADVANCE III HD500. Chemical shifts (δ) are reported in

ppm in reference to the residual solvent signal (δ 2.50 for H

NMR in DMSO-d and δ 39.52 for C{ H} NMR in DMSO-

d₆). High-resolution mass spectra were obtained on a Thermo

Scientiﬁc EXACTIVE Plus. IR spectra were recorded on a

SHIMADZU IRAﬃnity-1S.

HPLC Methods. Method A: YMC-Pack Pro C18 (5 μm),

50 × 4.6 mm I.D.; eluent: 0.1 M aqueous NaClO (pH 2.0)/

MeCN (6/4); run time: 25 min; ﬂow rate: 1.0 mL/min;

temperature: 40 °C; detection: UV at 220 nm. Retention time:

Figure 4. Hygroscopic properties of 1·HCl at 50 °C.

6.5 min for 2, 2.2 min for 9, 6.2 min for 3, 1.9 min for 4, 3.5

to the adsorption curve (blue), approximately 2 equiv of water

were adsorbed at 10% RH. Thereafter, with increasing RH, the

water content gradually increased to 2.3 equiv. The target

range of the water content of 1·HCl was set to 2.1 ± 0.2H O,

which is the water content at normal humidity (approximately

min for 8, 2.1 min for 5-chloro-2-nitorobenzoic acid, and 10.1

min for N-butyl-5-chloro-2-nitrobenzamide.

Method B: YMC-Pack Pro C18 (5 μm), 150 × 4.6 mm I.D.;

eluent A: 0.1 M aqueous NaClO (pH 2.0); eluent B: MeCN;

ﬂow rate: 1.5 mL/min; temperature: 40 °C; detection: UV at

20 nm. Gradient: 0 → 15 min, A/B = 75/25; 15 → 40 min,

0% RH) at 25 °C. At 50 °C, the drying temperature,

according to the desorption curve (pink), loss of the hydrated

water would occur below 6% RH (0.7 kPa of water vapor

pressure) (see Figure 4). Previous studies have reported drying

A/B = 75/25 → 20/80; 40 → 50 min, A/B = 20/80. Retention

time: 19.6 min for 3, 4.7 min for 8, 22.0 min for 10, 23.4 min

for 5, 6.2 min for 16, 29.0 min for 6, 26.6 min for 11, and 14.7

min for 1.

methods for preventing anhydride contamination. In our

case, the hygroscopic proﬁle at 50 °C suggested that the target

Method C: YMC-Pack ODS-A (5 μm), 150 × 4.6 mm I.D.;

.1 ± 0.2 hydrate could be obtained by drying the wet 1·HCl

eluent: 0.03 M aqueous KH PO /THF (6/4); run time: 25

product at 50 °C under a vacuum at 0.7−10.5 kPa. In the >100

g scale experiment, drying was performed under a vacuum at

min; ﬂow rate: 1.0 mL/min; temperature: 40 °C; detection:

UV at 220 nm. Retention time: 12.3 min for 10 and 13.7 min

for 5.

−9 kPa until loss on drying (LOD) was ≤1.0%, to give the

.1 hydrate form of ASP5069 (KF titration: 5.6%) with 98.5 A

tert-Butyl {2-Ethoxy-6-formyl-4-[(4-methylpiperazin-1-yl)-

methyl]phenoxy}acetate (3). To a slurry of 3-ethoxysalicy-

laldehyde (2) (100 g, 602 mmol) in 2-propanol (800 mL) at

15−25 °C, 1-methylpiperazine (86.8 mL, 1.3 equiv) and

formaldehyde (37% in water, 67.2 mL, 1.5 equiv) at 15−25 °C

were added. After stirring for 10 min at 15−25 °C, the reaction

mixture was warmed to 80 °C and stirred for 67 h at 80 °C

until HPLC indicated that aldehyde 2 had been consumed (0.3

A % by HPLC method A). The reaction mixture was cooled to

60 °C and concentrated under a vacuum to 200 mL at 60 °C.

The brown solution was diluted with toluene (800 mL) and

concentrated under a vacuum at 60 °C to 200 mL. This

process was repeated three times to give 9 in a toluene

solution.

purity and ≤0.5 A % individual impurities.

CONCLUSIONS

■

This study describes the development of a practical, scalable,

and reproducible process for manufacturing ASP5069. The

process comprised a convergent synthetic approach, eliminated

the need for column chromatography, and solved safety and

environmental concerns associated with the discovery method.

In the Mannich reaction and alkylation sequence, the use of a

more sterically hindered solvent suppressed the impurity

formation, and N-overalkylation was markedly inhibited by

using a harder electrophile to give 3 in good yield. We also

developed an eﬀective puriﬁcation method for amine 3 by

using an acidic aqueous solution for extraction. For the

preparation of 5, we replaced the solvent with an environ-

mentally friendly alternative and developed the safe process for

isolating 5-chloro-2-nitrobenzoyl chloride. For the reductive

amination reaction, establishing an appropriate order for the

addition of reagents and appropriate reaction temperature

suppressed the production of 14/15 and improved process

reproducibility, and subsequent Boc deprotection yielded 11 in

The toluene solution of 9 was diluted with DMF (500 mL)

at rt. The resulting deep brown solution was cooled to −10 to

0 °C. K CO (91.3 g, 1.1 equiv) was added at −10 to 0 °C.

The resulting slurry was aged for 40 min at −10 to 0 °C. tert-

Butyl chloroacetate (94.7 mL, 1.1 equiv) was added at −10 to

0 °C. The resulting slurry was warmed gradually to 40 °C over

8 h. Another portion of K CO (74.9 g, 0.9 equiv) was added

at 40 °C. The resulting slurry was stirred for 1 h at 40 °C.

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Another portion of tert-butyl chloroacetate (8.61 mL, 0.1

equiv) was added, and the resulting slurry was stirred for 1.5 h

at 40 °C until HPLC indicated that 9 had been consumed (0.7

A % by HPLC method A). The reaction mixture was

added SOCl (72.5 mL, 2.0 equiv) at rt. The resulting slurry

was warmed to 100 °C and stirred for 6 h at 100 °C until

HPLC indicated that 5-chloro-2-nitorobenzoic acid had been

consumed (0.3 A % by HPLC method A: the reaction was

monitored by mixing the reaction aliquot with n-butylamine in

a HPLC vial). The reaction mixture was cooled to 60 °C and

concentrated under a vacuum to 233 mL at 60 °C. Toluene

(936 mL) was added and concentrated under a vacuum to 233

mL at 60 °C. This process was repeated to give 5-chloro-2-

nitrobenzoyl chloride in a toluene solution (98.1 A % by

HPLC method A: an aliquot of the solution was mixed with n-

butylamine in a HPLC vial, and checked using HPLC).

Boc amidine 8 (117 g, 497 mmol) was added to a mixed

transferred to H O (150 mL) with the seed 3 (100 mg) in

another ﬂask over 30 min at 10−20 °C. The resulting slurry

was aged for 1 h at 40 °C, cooled to rt, aged for 108 h at 20−

5 °C, and then ﬁltered. The wet cake was washed with DMF/

H O (1/4) (500 mL) and H O (1000 mL) and dried under a

vacuum at 50 °C for 21 h to give crude 3 (187 g, 476 mmol,

9%; 97.8 A % by HPLC method A; 88.3 A % by HPLC

method B) as a light brown solid.

Puriﬁcation of Aldehyde 3. To a solution of crude 3 (136 g,

47 mmol) in i-PrOAc/H O (680 mL/1000 mL) at rt was

solution of THF (468 mL) and i-Pr NEt (130 mL, 1.5 equiv)

added 6 M aqueous HCl (57.8 mL, 1.0 equiv). After vigorous

stirring at rt, the biphasic mixture was separated. The organic

layer was extracted with H O (136 mL) at rt. The combined

at rt. The resulting slurry was cooled to −15 to −10 °C. The

toluene solution of 5-chloro-2-nitrobenzoyl chloride (233 mL,

1.2 equiv) was added dropwise at −15 to −5 °C. The residual

acid chloride in the ﬂask was rinsed with THF (58.5 mL). The

resulting orange solution was stirred for 2 h at −15 to −5 °C

until HPLC indicated that amidine 8 had been consumed (0.2

A % by HPLC method B). The reaction mixture was quenched

with 5% aqueous Na CO (585 mL) at below 10 °C. The

aqueous layer was washed with i-PrOAc (136 mL), diluted

with i-PrOAc (1020 mL) and then basiﬁed with 5 M aqueous

NaOH (76.3 mL, 1.1 equiv) at rt. After vigorous stirring, the

biphasic mixture was separated. The organic layer was washed

with 10% brine (272 mL) and 20% brine (136 mL). The

organic layer was concentrated under a vacuum to 272 mL,

and i-PrOAc (680 mL) was added. This process was repeated.

The precipitated inorganic salts were removed by ﬁltration to

give aldehyde 3 in i-PrOAc solution (0.304 M, 1088 mL, 331

mmol, 95%, 98.7 A % by HPLC method B). The authentic

biphasic mixture was stirred vigorously for 20 min and then

separated at 10−30 °C. The organic layer was washed with

10% brine (293 mL × 2) and 20% brine (147 mL) and then

added dropwise to toluene (2630 mL) with the seed 10 (58.5

mg, 0.05 wt %) in another ﬂask at 48−52 °C over 0.5 h. The

residual organic layer in the ﬂask was rinsed with THF (58.5

mL), and the rinse solution was added to the slurry. The

resulting yellow slurry was aged for 1 h at 48−52 °C, cooled

gradually to 15−25 °C, aged for 68 h at 15−25 °C, and then

ﬁltered. The wet cake was washed with toluene (1170 mL × 2)

and dried under a vacuum at rt for 1 h and then at 50 °C for 14

sample was obtained by crystallization from i-PrOAc. H NMR

(

DMSO-d , 500 MHz): δ 1.37 (t, 3H, J = 7.0 Hz), 1.38 (s,

H), 2.14 (s, 3H), 2.16−2.53 (m, 8H), 3.42 (s, 2H), 4.10 (q,

H, J = 7.0 Hz), 4.76 (s, 2H), 7.19 (d, 1H, J = 1.9 Hz), 7.25

(

d, 1H, J = 1.9 Hz), 10.50 (s, 1H). C{ H} NMR (DMSO-d ,

25 MHz): δ 14.5, 27.6, 45.7, 52.4, 54.7, 61.2, 64.4, 69.4, 81.5,

17.7, 119.7, 128.6, 134.6, 148.9, 150.7, 168.3, 190.5. HRMS−

h to give 10 (180 g, 430 mmol, 87%, 94.0 A % by HPLC

ESI (m/z): [M + H] calcd for C H O N , 393.2384; found,

method B) as a colorless solid. H NMR (DMSO-d , 500

−

93.2383. IR (ATR, cm ): 2936, 2787, 1748, 1144, 812, 725.

MHz): δ 1.45 (s, 9H), 7.74 (d, 2H, J = 8.7 Hz), 7.86 (dd, 1H, J

= 8.8, 2.3 Hz), 7.99 (d, 2H, J = 8.7 Hz), 7.99 (d, 1H, J = 2.3

Hz), 8.20 (d, 1H, J = 8.7 Hz), 9.00 (br s, 2H), 10.96 (br s,

Mp: 88−90 °C. Assay (QNMR): 100 wt %.

tert-Butyl [(4-Aminophenyl) (imino)methyl]carbamate

(8). To a slurry of 4-aminobenzamidine dihydrochloride (4)

1H). C{ H} NMR (DMSO-d , 125 MHz): δ 28.0, 77.6,

(

51.1 g, 246 mmol) in THF (128 mL) was added 5 M aqueous

118.9, 126.4, 128.6, 129.2, 129.7, 130.9, 134.0, 138.9, 141.8,

NaOH (221 mL, 4.5 equiv) at 0−10 °C. The resulting biphasic

144.9, 162.9, 163.7, 165.4. HRMS−ESI (m/z): [M + H] calcd

solution was stirred for 30 min at 0−10 °C. To the mixture was

for C H O N Cl, 419.1117; found, 419.1118. IR (ATR,

−

added Boc O (57.5 mL, 1.0 equiv) at 0−10 °C. The resulting

cm ): 1618, 1607, 1533, 1497, 1277, 1148, 853, 839. Mp: 187

°C dec. Assay (QNMR): 90 wt %.

slurry was stirred for 3 h at 0−10 °C until HPLC indicated that

amidine 4 had been consumed (0.3 A % by HPLC method A).

tert-Butyl {[4-(2-Amino-5-chlorobenzamido)phenyl]-

(imino)methyl}carbamate (5). Nitro-amide 10 (60.0 g, 143

The slurry was added dropwise to H O (1022 mL) in another

ﬂask at 0−10 °C over 1 h. The reaction ﬂask was rinsed with

mmol) was dissolved in a mixed solvent of H O (43.8 mL) and

DMF (300 mL) at rt in a 1000 mL autoclave. To the solution

H O (153 mL), and the rinse solution was added to the

crystallization ﬂask. The resulting slurry was aged for 3 h at 0−

was added pre-rinsed Raney nickel [washed with H O (300

0 °C and ﬁltered. The wet cake was washed with H O (511

mL × 3)] (60.0 g, 100 wt %) in DMF (136 mL) at rt. The

resulting mixture was stirred for 16 h at 30 °C under a H₂

atmosphere (44 psi) until HPLC indicated that amide 10 had

been consumed (ND by HPLC method C). Then, the reaction

mixture was cooled to rt and ﬁltered. Filtration was performed

mL) and dried under a vacuum at 50 °C for 22 h to give 8

(

56.3 g, 239 mmol, 97%; 98.5 A % by HPLC method A) as a

white solid. H NMR (DMSO-d , 500 MHz): δ 1.43 (s, 9H),

.77 (s, 2H), 6.55 (d, 2H, J = 8.8 Hz), 7.72 (d, 2H, J = 8.8

Hz), 8.03−9.69 (m, 2H). C{ H} NMR (DMSO-d , 125

MHz): δ 28.1, 77.0, 112.6, 120.4, 129.2, 152.5, 164.0, 166.2.

using a pressure ﬁlter under a N atmosphere to avoid exposing

ﬂammable Raney Ni to air. The ﬁltered solid and vessels were

HRMS−ESI (m/z): [M + H] calcd for C H O N ,

rinsed with DMF/H O (10/1) (240 mL). The combined light

−

236.1394; found, 236.1392. IR (ATR, cm ): 3146, 1609,

1267, 1146, 1125, 862, 797. Mp:195 °C dec. Assay (QNMR):

99 wt %.

brown ﬁltrate was warmed to 50 °C. Water (240 mL) was

added, and then the mixture was seeded with 5 (30.0 mg, 0.05

wt %). The resulting slurry was aged for 1 h at 50 °C. Another

tert-Butyl {[4-(5-Chloro-2-nitrobenzamido)phenyl]-

portion of H O (360 mL) was added dropwise over 0.5 h. The

(

imino)methyl}carbamate (10). To a slurry of 5-chloro-2-

resulting slurry was aged for 1 h at 50 °C, cooled to rt, aged for

20 h at rt, and then ﬁltered. The wet cake was washed with

nitorobenzoic acid (120 g, 1.2 equiv) in toluene (816 mL) was

Org. Process Res. Dev. XXXX, XXX, XXX−XXX

Organic Process Research & Development

Article

DMF/H O (1/2) (300 mL) and then H O (600 mL) and

MHz): δ 1.34 (t, 3H, J = 7.0 Hz), 1.39 (s, 9H), 1.86 (s,

dried under a vacuum at 50 °C for 139 h to give 5 (48.5 g, 125

CH COOH, 3.9H), 2.10 (s, 3H), 2.13−2.45 (m, 8H), 3.31 (s,

mmol, 87%, 94.4 A % by HPLC method B) as a gray-brown

2H), 4.02 (q, 2H, J = 7.0 Hz), 4.50 (d, 2H, J = 5.4 Hz), 4.61 (s,

2H), 6.74 (d, 1H, J = 9.2 Hz), 6.79 (d, 1H, J = 1.5 Hz), 6.83

(d, 1H, J = 1.6 Hz), 7.29 (dd, 1H, J = 9.0, 2.5 Hz), 7.82 (d, 1H,

J = 2.5 Hz), 7.86 (d, 2H, J = 9.0 Hz), 7.84−7.89 (m, 1H), 7.95

(d, 2H, J = 9.0 Hz), 8.41−10.43 (m, 3H), 10.60 (br s, 1H).

solid. H NMR (DMSO-d , 500 MHz): δ1.45 (s, 9H), 6.51 (s,

H), 6.80 (d, 1H, J = 8.9 Hz), 7.24 (dd, 1H, J = 8.9, 2.5 Hz),

.71 (d, 1H, J = 2.6 Hz), 7.82 (d, 2H, J = 8.9 Hz), 7.97 (d, 2H,

J = 8.9 Hz), 8.49−9.55 (m, 2H), 10.30 (s, 1H). C{ H} NMR

(

DMSO-d , 125 MHz): δ 28.0, 77.6, 115.5, 117.8, 118.2,

C{ H} NMR (DMSO-d , 125 MHz): δ 14.7, 22.1

19.6, 128.0, 128.2, 129.1, 132.1, 142.3, 148.8, 163.7, 165.6,

(

CH COOH), 27.7, 41.5, 45.5, 52.2, 54.5, 61.6, 63.9, 69.5,

66.8. HRMS−ESI (m/z): [M + H] calcd for C H O N Cl,

81.1, 113.0, 113.7, 115.9, 117.9, 120.0, 122.5, 128.5, 128.8,

−

89.1375; found, 389.1377. IR (ATR, cm ): 3420, 3312,

976, 1609, 1508, 1489, 1308, 1292, 1236, 1140, 845, 831.

31.2, 132.6, 133.8, 143.7, 143.9, 148.2, 150.2, 165.0, 167.3,

68.4, 173.2 (CH COOH). HRMS−ESI (m/z): [M + H]

Mp: 195 °C dec. Assay (QNMR): 95 wt %.

calcd for C H O N Cl, 665.3213; found, 665.3218. IR (ATR,

cm ): 2970, 1508, 1497, 1489, 1207, 1146, 851, 745, 648.

tert-Butyl {2-({2-[(4-Carbamimidoylphenyl)carbamoyl]-4-

chloroanilino}methyl)-6-ethoxy-4-[(4-methylpiperazin-1-yl)-

methyl]phenoxy}acetate (11). To a slurry of aniline 5 (100 g,

−1

Mp: 140 °C dec. Anal. Calcd for C H ClN O ·

.3C H O .0.7HCl.1.7H O: C, 56.5; H, 6.9; N, 10.5; Cl, 7.5.

2 4 2 2

57 mmol) in i-PrOAc (700 mL) was added NaBH(OAc) in

Found: C, 56.8; H, 6.8; N, 10.1; Cl, 7.7. KF titration: 3.8%.

Assay (QNMR): 95 wt %.

AcOH [prepared by adding NaBH (29.2 g, 3.0 equiv) to

AcOH (800 mL) at 16−30 °C. Caution: It is important to add

{

2-({2-[(4-Carbamimidoylphenyl)carbamoyl]-4-

NaBH slowly while controlling the reaction temperature and

chloroanilino}methyl)-6-ethoxy-4-[(4-methylpiperazin-1-yl)-

methyl]phenoxy}acetic Acid Monohydrochloride Dihydrate

hydrogen production] at −10 to 0 °C over 45 min. Caution: It

is equally important to add NaBH(OAc)₃slowly while

controlling the reaction temperature and hydrogen production.

(

ASP5069, 1·HCl·2H O). Compound 11 (170 g, 213 mmol)

was suspended in 6 M aqueous HCl (850 mL, 24 equiv) at rt.

The suspension was stirred for 2 h at 40 °C until HPLC

indicated that 11 had been consumed (1.0 A % by HPLC

method B). Acetone (4.25 L) was added at 40 °C over 1 h.

The resulting slurry was stirred for 3 h at 40 °C, cooled to 20

Residual NaBH(OAc) in the ﬂask was rinsed with AcOH

(

200 mL) and added to the reaction ﬂask. The resulting thin,

light brown slurry was stirred for 70 min at −10 to 0 °C. Then,

aldehyde 3 in i-PrOAc (931 mL, 0.304 M, 1.1 equiv) was

added over 80 min at −10 to 0 °C. The resulting yellow slurry

was stirred for 4 h at −10 to 0 °C. Another portion of aldehyde

C (20 °C/h), stirred for 2 h, and then ﬁltered. The wet cake

was washed with acetone (1.7 L) and dried under a vacuum at

0 °C for 10 h to give 1·4HCl·xH O (x = 0−13, 155 g, 97.4 A

in i-PrOAc (84.5 mL, 0.304 M, 0.1 equiv) was added over 5

min at −10 to 0 °C. The resulting brown solution was stirred

for 2.5 h at −10 to 0 °C until HPLC indicated that aniline 5

and Boc-deprotected aniline 16 had been consumed (ND and

by HPLC method B). Compound 1·4HCl·xH O (x = 0−13,

45 g) was dissolved in water (668 mL) at rt. The solution was

ﬁltered through a 0.5 μm ﬁlter, and residual 1·4HCl·xH O (x =

.4 A %, respectively, by HPLC method B). Water (4.63 mL,

.0 equiv) was added at −10 to 0 °C. The resulting mixture

−13) in the ﬂask and on the ﬁlter was rinsed with water (158

mL). The ﬁltrate was stirred at rt, and acetone (826 mL) was

was warmed to 60 °C and stirred for 20 h at 60 °C until HPLC

indicated that the intermediate 6 had been consumed (0.2 A %

by HPLC method B). The mixture was cooled to 0 °C and

stirred for 9.5 h at 0 °C. To the mixture were added 7.5 M

aqueous NaOH (600 mL) and 2-butanol (400 mL) at 0−10

added at rt. The pH of the mixture was adjusted from 1.0 to

.6 by adding 5 M aqueous NaOH (108 mL). The solution

was warmed to 50 °C and stirred for 1 h at 50 °C. Acetone

2878 mL) was added to the slurry over 1 h. The slurry was

(

stirred for 1 h at 50 °C, cooled to 20 °C (10 °C/h), and stirred

at 20 °C for 11 h. The pH of the mixture was adjusted to

approximately 5.5 by adding 5 M aqueous NaOH and 6 M

aqueous HCl, and then the slurry was stirred for 2−14 h. This

pH adjustment was repeated until the pH stabilized at

approximately 5.5. The slurry was ﬁltered. The wet cake was

C. The resulting mixture was warmed to 30 °C and stirred for

h at 30 °C and then transferred to a separation funnel via a

glass ﬁlter to remove insoluble precipitates. The reaction ﬂask

was rinsed with i-PrOAc/2-butanol/H O (3/2/1) (90 mL),

and the rinsed solution was transferred to the funnel via the

ﬁlter. The biphasic mixture was separated. The organic layer

washed with acetone/H O (607 mL/121 mL) and acetone

was washed with H O (250 mL × 2) and 20% brine (200 mL).

(

1.46 L) and dried at 50 °C under a vacuum at 7−9 kPa for 3

The solution was concentrated under a vacuum at 50 °C to

days until LOD was ≤1.0% to give ASP5069 (117 g, 172

00 mL, and 2-butanol (500 mL) was added. This process was

mmol, 86%; 98.5 A % by HPLC method B) as a white solid.

repeated three times. Precipitated inorganic salts were removed

by ﬁltration, and the ﬁltrate was concentrated to 500 mL. The

solution was added dropwise to MTBE (2300 mL) in another

ﬂask at 50 °C over 1 h. The residual solution in the ﬂask was

rinsed with 2-butanol (280 mL), and the rinsed solution was

added to the crystallization ﬂask at 50 °C. The resulting yellow

slurry was stirred for 1 h at 50 °C, cooled to 20 °C, stirred for 4

h at 20 °C, and then ﬁltered. The wet cake was washed with

MTBE (2000 mL) and dried under a vacuum at 40 °C for 13 h

to give 11 (181 g, 226 mmol, 88%; 92.8 A % by HPLC method

B). KF, NMR, and elemental analysis indicated that 11

contained 1.3 equiv of AcOH, 0.7 equiv of HCl, and 1.7 equiv

H NMR (DMSO-d , 500 MHz): δ 1.34 (t, 3H, J = 6.9 Hz),

.30 (s, 3H), 2.32−2.72 (m, 8H), 3.36 (s, 2H), 4.02 (q, 2H, J

6.9 Hz), 4.53 (s, 2H), 4.59 (s, 2H), 6.76 (s, 1H), 6.76 (d,

1H, J = 9.1 Hz), 6.84 (s, 1H), 7.28 (dd, 1H, J = 9.0, 2.4 Hz),

7.81 (d, 1H, J = 2.4 Hz), 7.86 (d, 2H, J = 8.9 Hz), 7.84−7.88

(m, 1H), 7.95 (d, 2H, J = 8.8 Hz), 9.18 (br s, 2H), 9.96 (br s,

2H), 10.63 (s, 1H). C{ H} NMR (DMSO-d , 125 MHz): δ

14.7, 41.5, 44.2, 50.9, 53.6, 61.2, 63.8, 69.9, 113.0, 113.9, 115.9,

117.8, 119.9, 120.0, 122.5, 128.5, 128.8, 131.7, 132.58, 132.61,

143.9, 144.3, 148.3, 150.5, 165.0, 167.3, 172.4. HRMS−ESI

(m/z): [M + H] calcd for C H O N Cl, 609.2587; found,

−

of water. Yield and assay are calculated as 11·1.3AcOH·

609.2589. IR (ATR, cm ): 1489, 1204, 851, 716, 638. Mp:

.7HCl·1.7H O. Yellow solid. H NMR (DMSO-d , 500

209 °C dec. KF titration: 5.6%. Assay (QNMR): 99 wt %.

Org. Process Res. Dev. XXXX, XXX, XXX−XXX

Organic Process Research & Development

Xiong, L.; Song, H.; Li, Y.; Li, Z. Synthesis and insecticidal activities

Article

Kondou, A. Plasma kallikrein inhibitor. WO 2011118672 A1, March

4, 2011.

7) (a) Zhou, Y.; Feng, Q.; Di, F.; Liu, Q.; Wang, D.; Chen, Y.;

of 2,3-dihydroquinazolin-4(1 H )-one derivatives targeting calcium

channel. Bioorg. Med. Chem. 2013, 21, 4968−4975. (b) Mizutani,

T.; Nagase, T.; Ito, S.; Miyamoto, Y.; Tanaka, T.; Takenaga, N.;

Tokita, S.; Sato, N. Development of novel 2-[4-(aminoalkoxy)-

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ASSOCIATED CONTENT

sı Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.oprd.0c00291.

■

(

H and C{ H} NMR spectra of compounds 3, 8, 10, 5,

1, and ASP5069 (1·HCl·2H O) (PDF )

AUTHOR INFORMATION

Corresponding Author

Shun Hirasawa − Pharmaceutical Science & Technology Labs.,

Pharmaceutical Technology, Astellas Pharma Inc., Tsukuba-

shi, Ibaraki 305-8585, Japan; orcid.org/0000-0002-

■

008, 18, 6041−6045. (c) Kim, D.; Wang, L.; Hale, J. J.; Lynch, C. L.;

Budhu, R. J.; MacCoss, M.; Mills, S. G.; Malkowitz, L.; Gould, S. L.;

DeMartino, J. A.; Springer, M. S.; Hazuda, D.; Miller, M.; Kessler, J.;

Hrin, R. C.; Carver, G.; Carella, A.; Henry, K.; Lineberger, J.; Schleif,

W. A.; Emini, E. A. Potent 1,3,4-trisubstituted pyrrolidine CCR5

receptor antagonists: effects of fused heterocycles on antiviral activity

and pharmacokinetic properties. Bioorg. Med. Chem. Lett. 2005, 15,

659-3424 ; Email: shun.hirasawa@astellas.com

Author

2129−2134. (d) Wright, W. B., Jr.; Tomcufcik, A. S.; Chan, P. S.;

Yoshinori Kohmura − Pharmaceutical Science & Technology

Labs., Pharmaceutical Technology, Astellas Pharma Inc.,

Tsukuba-shi, Ibaraki 305-8585, Japan

Marsico, J. W.; Press, J. B. Thromboxane synthetase inhibitors and

antihypertensive agents. 4. N-[(1H-imidazol-1-yl)alkyl] derivatives of

quinazoline-2,4(1H,3H)-diones, quinazolin-4(3H)-ones, and 1,2,3-

benzotriazin-4(3H)-ones. J. Med. Chem. 1987, 30, 2277−2283.

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.oprd.0c00291

(e) Tani, J.; Yamada, Y.; Oine, T.; Ochiai, T.; Ishida, R.; Inoue, I.

Studies on biologically active halogenated compounds. 1. Synthesis

and central nervous system depressant activity of 2-(fluoromethyl)-3-

aryl-4(3 H )-quinazolinone derivatives. J. Med. Chem. 1979 , 22 , 95 −99.

(8) (a) Clark, R. H.; Wagner, E. C. Isatoic Anhydride. I. Reactions

with Primary and Secondary Amines and with Some Amides1. J. Org.

Chem. 1944, 09, 55−67. (b) Coppola, G. M. The Chemistry of Isatoic

Anhydride. Synthesis 1980 , 1980 , 505 −536.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We thank M. Fujimoto and Y. Hashimoto for analytical

support and T. Tsunoda, S. Ieda, T. Takahashi, and M. Okada

for project management advice.

(

9) Pearson, R. G. Hard and Soft Acids and Bases. J. Am. Chem. Soc.

963, 85, 3533−3539.

10) Anderson, N. G. Practical Process Research & Development, 2nd

ed.; Academic Press: Oxford, U.K.. 2012; p 129.

(11) Bretherick’s Handbook of Reactive Chemical Hazards, 5th ed.;

Urben, P., Ed.; Butterworth-Heinemann: Oxford, 1995; Vol. 1, p 856.

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16) The target quality of the active pharmaceutical ingredient is

≥97 A %.