Development of a Robust Scale-Up Synthetic Route for BPR1K871: A Clinical Candidate for the Treatment of Acute Myeloid Leukemia and Solid Tumors

Julakanti Satyanarayana Reddy, Chih-Ming Chen, Mohane Selvaraj Coumar, Hsu-Yi Sun, Na Sun,

Article

Development of a Robust Scale-Up Synthetic Route for BPR1K871: A

Clinical Candidate for the Treatment of Acute Myeloid Leukemia

and Solid Tumors

and Hsing-Pang Hsieh*

Cite This: Org. Process Res. Dev. 2021, 25, 817 −830

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sı Supporting Information

ABSTRACT: Herein, a robust and scalable procedure for the synthesis of multikinase inhibitor BPR1K871 (1, a quinazoline

compound bearing a substituted thiazoline side chain), which is a clinical candidate for the treatment of acute myeloid leukemia and

solid tumors, is reported. The previously reported medicinal chemistry synthetic route A with seven steps had encountered several

issues during scale-up syntheses such as low yields (7.7% overall yield), the formation of inseparable impurities, particularly in the

chlorination step, use of hazardous reagents (NaH/DMF), and laborious column chromatography steps for the puriﬁcation of the

products. A step-by-step approach to overcome the above issues was planned and implemented through two similar routes (B1 and

B2) on a gram scale and ﬁnally through route B3 on a kilogram scale to synthesize 1. The ﬁnal optimized synthetic route B3 does not

require column chromatography puriﬁcation steps. It is one step shorter than the original route A and avoided hazardous reagents for

the alkylation reaction in step 2. Furthermore, the highlights of the new route B3 include liquid−liquid continuous extraction of

compound 13 in step 2, the use of POCl instead of SOCl to minimize the formation of impurities in the chlorination step 3, and

telescoped synthesis of key Boc-protected amino intermediate 15 from 13, in high purity. Using the scale-up route B3, the ﬁnal

product 1 (3.09 kg, yield of 16.5% over six steps with an HPLC purity of 97.8%) was obtained in a single batch for preclinical testing

and facilitated clinical testing of 1, which is underway.

KEYWORDS: acute myeloid leukemia (AML), BPR1K871, scale-up synthesis, quinazoline, clinical candidate, telescoped synthesis

. INTRODUCTION

In recent years, several research groups focused on the

development of small molecules targeting the Aurora kinases

and found a common approach for the discovery of new

molecularly targeted cancer therapeutics for the treatment of

Acute myeloid leukemia (AML) is a life-threatening hemato-

logical malignancy in children and adolescents. More than a

quarter of a million people get diagnosed annually worldwide

with AML. Despite signiﬁcant progress over the last three

decades in the treatment of AML, more than half of young

adult patients and about 90% of older adults still die due to its

solid tumors, hematological malignancies, and AML.

Barasertib (AZD1152), a quinazoline developed by AstraZe-

neca, CCT241736, an imidazopyridine established by UK

Cancer Therapeutics Unit, indolinone derivative from Chern

severity. Therefore, a more eﬀective and less toxic new

generation of AML therapeutics is required urgently.

et al. and pyrrolopyrimidine, and furanopyrimidine

derivatives developed from our research group are a few

agents that are reported for the treatment of AML and have

both Aurora and FLT3 kinase inhibition.

In this respect, targeting altered signaling molecules in

cancer is a time-tested strategy, resulting in the development of

several anti-cancer drugs. Kinase inhibitors are a class of

targeted anti-cancer drugs that block the overexpressed and/or

mutant kinase functions. Kinases act as a molecular switch,

The FMS-like tyrosine kinase-3 (FLT3) is a class-III

tyrosine kinase receptor family and plays a key role in the

survival, proliferation, and diﬀerentiation of hematopoietic

turning “on” or “oﬀ” the signaling in several cellular pathways.

Of the 518 kinases reported in the human genome, US FDA

cells. Meanwhile, Aurora kinases A, B, and C are the

members of the serine/threonine kinase family, which are

(

U.S.A. Food and Drug Administration) approved 62

essential in the cell mitosis process. BPR1K871 (1, Figure 1)

is a quinazoline derivative that was identiﬁed as a potent dual

inhibitors that target around 20 kinases. Additionally, many

kinase inhibitors are registered in clinical trials and are at

diﬀerent drug development phases. Analysis of the reported

Received: November 26, 2020

Published: February 18, 2021

kinase inhibitors has shown that the quinazoline core is a

privileged scaﬀold for inhibiting adenosine triphosphate

(

ATP)-dependent kinases. Moreover, among the 62 clinically

approved kinase inhibitors, 7 contained the quinazoline

scaﬀold.

2021 American Chemical Society

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acid (2) with formamidine acetate to aﬀord intermediate 3.

Subsequently, the S Ar reaction of 3 with 1,3-propanediol (4)

in the presence of NaH base gave 5. The quinazolinone 5 was

treated with thionyl chloride at reﬂux to furnish the dichloride

Subsequently, compound 6 underwent an S Ar reaction

with tert-butyl (5-(2-aminoethyl)thiazol-2-yl)carbamate (7)

synthesized from our lab in 17.0% yield over six steps), in the

(

presence of triethylamine base to furnish compound 8. In turn,

this was reacted with triﬂuoroacetic acid at 26 °C, followed by

the base treatment provided the free amine 9 in excellent yield

after column puriﬁcation. For the formation of the urea

functional group, amine intermediate 9 was reacted with 3-

chlorophenyl isocyanate (10) to yield 11 in a 63% yield. The

last stage of the synthesis installed the dimethylamino group

Figure 1. Structure of multikinase inhibitor BPR1K871 (1).

inhibitor of Aurora and FLT3 kinases (AURKA IC = 22 nM,

AURKB IC₅₀= 13 nM, FLT3 IC₅₀= 19 nM); it has a single-

digit nanomolar level cellular activities in AML cell lines

through the S 2 reaction of chloro compound 11 with

(

MOLM-13 and MV4-11, EC₅₀∼5 nM). The kinase proﬁle

dimethylamine in the presence of KI to aﬀord 1 with a 36%

of 1 using the KINOMEScan technology revealed that 77

therapeutically important kinases out of 395 nonmutant

kinases were inhibited (65% at 1000 nM). In vitro and in

vivo studies have proven 1 as a potent multikinase inhibitor

and a US FDA approved investigational new drug (IND) for

Phase I testing.

yield.

Synthetic route A (Scheme 1) is already reported and was

developed to produce the milligram scale of compound 1

during the in vitro and in vivo assessment of kinase inhibitory/

anti-cancer activity. Synthetic route A consisted of seven steps

from commercially available 2-amino-4-ﬂuorobenzoic acid (2)

and resulted in an overall yield of 7.7% on a milligram scale.

However, scale-up to gram-scale synthesis resulted in a

decrease in yield.

As candidate 1 is advanced to clinical development, large-

scale manufacturing of this compound in the multikilogram

scale requires a practical and robust synthetic method. Such a

multikilogram synthesis of active pharmaceutical ingredients

Medicinal chemistry synthetic routes aim to explore the

structure−activity relationship (SAR) in a series of compounds

under investigation. For such SAR exploration, there is a

requirement for synthesizing diverse analogues. Hence, it was

advantageous to introduce the chemical diversity as later as

possible in the synthetic route to obtain the desired analogues.

Hence, during the medicinal chemistry optimization of 1, two

important chemical diversity points, the urea functional group

and dimethylamino groups, were introduced at the 6th and 7th

(

API) is critical for the program progression. Herein, we

describe the synthetic process development activities through

routes A, B1, B2, and B3 resulting in a practical and scale-up

procedure that can operate on a 3 kg scale for the non-GMP

production of 1.

.1. Medicinal Chemistry Synthesis of 1. The initial

medicinal chemistry synthetic strategy for compound 1 is

shown in Scheme 1. The synthesis of compound 1

commenced with the condensation of 2-amino 4-ﬂuorobenzoic

Scheme 1. Synthetic Route A for 1

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Scheme 2. Retrosynthetic Analysis of 1

step of the seven-step synthetic route A, respectively. This

route was advantageous for optimization in the medicinal

chemistry program but was not suitable for the multigram

scale-up synthesis of 1 that is essential for preclinical

investigations.

Several drawbacks were identiﬁed during the scale-up

synthesis of 1 using route A including, (i) variable yields

route A, from compound 13 by a chlorination step followed by

the S Ar reaction of the resulting compound 14 with thiazole

intermediate 7. While the intermediate 13 could be assembled

via S Ar reaction between 7-ﬂuoroquinazolin-4(3H)-one (3)

and 3-(dimethylamino)propan-1-ol (12). Compound 3 could

be made from commercially available 2-amino-4-ﬂuorobenzoic

acid (2), as in route A.

during the chlorination and S Ar step to form the intermediate

and the ﬁnal dimethyl amination step. Moreover, a low yield

. RESULTS AND DISCUSSION

in the ﬁnal step leads to the increased use of the key thiazole

intermediate 7, which was synthesized in six steps. Hence the

overall cost of synthesis also increased, (ii) the use of unsafe

reagent NaH/DMF in step 2 for the formation of 5, and (iii)

the requirement for several column chromatography puriﬁca-

tions steps. Moreover, several unknown side products were

formed in the ﬁnal dimethylamino substitution reaction during

the scale-up of 1, resulting in a decreased yield and

necessitated an increased eﬀort during the puriﬁcation with

column chromatography.

The above disadvantages motivated us to seek an alternative,

safe, and eﬃcient route to provide multikilograms of 1 in a

high yield with easy to purify steps. As the ﬁnal installation of

the dimethylamino group in 1 was a major source of the

problem, installing this functionality at an early stage was

envisaged. For this purpose, a retro-synthetic analysis of 1 was

carried out for planning an alternative synthetic route.

2.1. Milligram-Scale Synthesis of 1 Using Route B1.

Initially, the intermediate 14 was planned to be synthesized

from the dichloride compound 6 (Scheme 1). However, the 4-

chloro group on the pyrimidine ring is more reactive than the

chloro group on the alkyl chain. Hence, the dimethylamino

group could not be installed before the thiazole side chain on

the dichloride intermediate 6. Further, attempts were made to

synthesize the monochloride intermediate or install another

leaving group triﬂate (OTf) on the alkyl chain terminal but

could not get satisfying results. Hence, as shown in Scheme 3

(synthetic route B1), the 3-(dimethylamino)propan-1-ol (12)

group was directly installed using an S Ar-type reaction on the

quinazolinone ring in the presence of the base KOH. This

avoided using sodium hydride to install the side-chain group at

the 7-position of the quinazoline ring. Moreover, this provided

a good yield of the intermediate 13 and avoided using a

laborious column chromatography procedure for the puriﬁca-

tion. Additionally, the dimethylamino intermediate 13 could

be synthesized in one-pot two-step synthesis from commer-

cially available 2-amino-4-ﬂuorobenzoic acid (2) with an 83%

.2. Retrosynthetic Analysis of 1. The retro-synthetic

analysis of target molecule 1 is shown in Scheme 2.

Accordingly, the method of assembling the target molecule 1

would be via a urea linkage between the subunit 16 and 3-

chlorophenyl isocyanate (10). Meanwhile, the amine 16 could

be obtained by the removal of Boc from protected amine 15.

Compound 15 could be constructed using a similar strategy to

16,19

yield.

Subsequently, the intermediate 13 underwent chlorination

by treatment with 20.0 equiv of thionyl chloride to provide the

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Scheme 3. Synthetic Route B1 for 1

-chloro quinazoline intermediate 14. As this compound was

the inﬂuence of starting material purity. The second and the

most important problem is the chlorination reaction, which

needed 20−30 equiv of thionyl chloride to complete the

monochloro intermediate 14 formation. During the scale-up

synthesis, more impurities were formed after quenching the

reaction, perhaps due to ineﬃcient heat transfer while

unstable, it was directly reacted with the thiazole intermediate

, which was followed by acidic extraction for the deprotection

of the Boc group to furnish the ﬁnal amino intermediate 16 in

1% yield over three steps. Finally, the amine 16 was reacted

with 3-chlorophenyl isocyanate (10) to give 1 in a moderate

yield of 45%. Overall, route B1 was much more straightforward

than route A with six-step synthesis from the commercially

available starting material, with an overall yield of 11.6%. This

synthetic route’s major advantage is the easy puriﬁcation steps,

requiring only extraction and ﬁltration without column

chromatography. However, for kilogram-scale manufacturing

to supply 1 for preclinical and clinical studies, several changes

are still required to ensure a robust synthetic procedure.

quenching with saturated NaHCO solution. This dramatically

aﬀected both the yield and subsequent puriﬁcation process. To

overcome this issue, an alternative chlorination reagent was

sought. Phosphoryl trichloride was found to be a suitable

alternative reagent for pyrimidinone chlorination, as it was

also widely used, similar to that of thionyl chloride. The use of

this regent decreased the amounts (equivalent) of reagent

required to complete chlorination. It decreased the number of

impurities formed, thus simpliﬁed the puriﬁcation process. The

chloro intermediate 14, without puriﬁcation, was reacted with

the key thiazole intermediate 7, and the resultant product 15

isolated in 52% yield over two steps in multigram scales. Next,

the removal of the Boc protection was carried out with

triﬂuoroacetic acid. The amine 16 was isolated as triﬂuoro-

acetic acid salt by recrystallization from a mixture of methanol

diethyl ether. As the triﬂuoroacetate salt increased the

solubility of 16 in DCM, the ﬁnal urea formation reaction

proceeded in DCM assisted by the base triethylamine resulting

in an improved yield of 73% for the formation of ﬁnal product

1. Overall, the synthetic route B2 (Scheme 4) incorporating

.2. Gram-Scale Synthesis of 1 Using Route B2.

Although route B1 proved to be more eﬃcient for synthesizing

, this methodology could still not be applied for a multigram

scale-up synthesis due to two main problems. The ﬁrst one is

the variable yields in the one-pot synthesis of dimethylamino

pyrimidinone intermediate 13, which was aﬀected by the

unknown impurity present in the starting material 2-amino-4-

ﬂuorobenzoic acid (2) due to degradation. This problem could

be overcome by recrystallization in EtOH before reaction,

which caused lower yield and a concomitant increase in the

cost. However, the original two-step procedure for the

synthesis of 13, instead of the one-pot synthesis, overcame

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Scheme 4. Synthetic Route B2 for 1

the modiﬁed conditions for reactions with diﬀerent reagents/

solvents has resulted in an improved overall yield of 14.7% for

the synthesis of 1 on multigram scales.

Table 1. Optimization of Quinazolinone 3 Synthesis

.3. Kilogram-Scale Synthesis of 1 Using Route B3. As

a brief ﬁnding, the modiﬁed synthetic route B2 (Scheme 4) is

more advantages than synthetic route B1 (Scheme 3) due to

the replacement of SOCl with POCl for the chlorination

step, which required lower equivalents of reagents and at the

same time suppressed the number of impurities formed. The

isolation of 16 as a TFA salt facilitated the ﬁnal urea bond

formation reaction in DCM, as 16 TFA salt was highly soluble

in DCM. Due to these, the overall yield for 1 using the route

B2 was 14.7%, which is a signiﬁcant 2-fold improvement

compared to 7.7% for 1 using the original route A. Further,

route B2 has improved safety due to the avoidance of

entry

solvent

time (h)

temp (°C)

3 yield (%)

neat

DMSO

EtOH

120

reﬂux

Reaction performed with benzoic acid 2 (1.0 equiv) and

formamidine acetate (2.0 equiv). Isolated yields of product 3 and

purity determined by HPLC analysis (C18 HPLC column, 0.1% TFA

in ACN/0.1% TFA in H O).

dangerous NaH/DMF used in route A, which may have

potential safety issues during the scale-up. Since the route B2

was advantageous in several ways, we intended to use the same

for the large-scale (kilogram level) synthesis of 1. However, it

was intended to optimize further the reaction conditions and

reagents used in each step to improve the yield. This led to the

preparation of large quantities of compound 1 to enable

preclinical testing.

unknown impurities. The formation of impurities precluded

increasing the reaction time to drive it to completion.

However, the reaction of 2 with 2.0 equiv of formamidine

acetate in EtOH under reﬂux condition was found to be

excellent with an enhanced (> 90%) yield of product 3. In

this reaction system, the resulting product 3 was not soluble in

EtOH at room temperature, facilitating the easy isolation of a

pure product by simple ﬁltration.

.4. Optimization of Quinazolinone Intermediate 3

Synthesis. The ﬁrst step in the process development

campaign is similar to the medicinal chemistry synthesis of

the quinazolinone 3 from the starting material 2 by

condensation with formamidine acetate. The following three

conditions were studied to optimize this reaction for a 10.0 g

scale of starting material 2 (Table 1). Condensation of 2 with

formamidine acetate in solvent-free condition (neat) or the

presence of DMSO solvent gave a low yield due to incomplete

conversion of the starting material and formation of several

To execute the optimized reaction conditions on a bulk scale

(10.0 kg), the reaction was performed in ethanol under reﬂux

for 48 h. The completion of the reaction was monitored by

HPLC analysis, which indicated the presence of 4.3%

unreacted starting material (R = 10.15 min). The unreacted

starting material 2 could be removed successfully in the

workup process. Once the reaction is completed, the batch

temperature was gradually decreased to about 10−15 °C and

stirred for 4 h. The precipitated product was centrifuged to get

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the cake, rinsed with cold EtOH, and dried under vacuum at

demonstrated on a kilogram scale (9.22 kg), the desired

compound 13 provided a moderate yield (66.9%) with a 98.5%

HPLC purity. The lower yield during kilogram-scale synthesis

might be due to incomplete continuous extraction even after

reﬂux for 3 days. Also, 2.7% product 13 was found in 42 kg of

the ﬁltrate using a mixture of EtOH/EtOAc for washing.

Therefore, some process development works are still needed to

improve the yield and potential manufacture.

5 °C for 24 h to aﬀord 3 (8.70 kg, 89.4% yield) with an

HPLC purity of >99%.

.5. Formation of Intermediate 13. Next, for the

synthesis of 13, we set out to optimize the reaction condition

for kilogram-scale synthesis (Table 2). S Ar reaction of 3-

Table 2. Synthesis of Intermediate 13

2.6. Telescoped Synthesis of Boc-Protected Amino-

Thiazole Intermediate 15. As a next step, we set out to

optimize the telescoped reaction sequence involving the

chlorination of 13, followed by the S Ar reaction to produce

the intermediate 15 in kilogram scales. The chlorination

reaction of 13 with POCl /DCE was not suitable for

manufacturing on a kilogram scale due to the DCE solvent’s

high toxicity. Hence, we investigated the chlorination reactions

with two diﬀerent solvents and reaction conditions for the

eﬃcient production of 14 (Table 3). Completion of the

reaction was monitored using HPLC, both by monitoring the

disappearance of the reactant 13 and the formation of the

product 14. The purity of the product was inﬂuenced both by

the reaction solvent and reaction’s batch size. The chlorination

reaction of 13 in toluene aﬀords 14 with a large number of

impurities. In contrast, the reaction in acetonitrile was

promising with a higher purity of the product during a

multigram-scale synthesis of 14 (entry 5 in Table 2, HPLC

entry batch size (3, g)

solvent

temp (°C) yield (%) purity (%)

10.0

20.0

DMSO

neat

140

125

120

98.5

98.1

98.4

99.1

560.0

neat

Reaction performed with quinazolinone 3 (1.0 equiv), amino alcohol

2 (4.5 equiv), KOH (4.0 equiv), and reaction time 16 h. Isolated

yields of product 13 and purity determined by HPLC analysis (C18

HPLC column, 0.1% TFA in ACN/0.1% TFA in H O).

purity: 96.7%). The conversion rate of the reaction was

inﬂuenced by the increased batch size, temperature, and time.

(

dimethylamino) propan-1-ol (12) and compound 3 in a

The crude product 14 was directly used in the next step (S Ar

DMSO solvent using KOH as the base at 125 °C smoothly

produced 13 in 83% yield on a small scale (10.0 g). Even

though the current protocol had avoided the dangerous NaH

reaction) after workup, as it was not stable on isolation.

Moreover, it was found that the presence of 7 helps the

intermediate 14 to maintain stability. Therefore, compound 7

was added during the reaction workup process for 14, and then

the reaction mixture was concentrated. The solvent was

exchanged with dichloromethane to perform the second step of

the telescoped synthesis.

originally used in the medicinal chemistry synthesis of 1,₂

KOH/DMSO is still hazardous for large-scale manufacturing.

Hence, other conditions were tested to replace the DMSO

solvent. Thus, the reaction could be progressed in neat 3-

(

dimethylamino)propan-1-ol (12) using KOH as the base, the

reaction was quenched with water, and then the product 13

For the large-scale synthesis quenching and workup of the

reaction mixture to get the crude product 14 with high purity

required careful handling, as the chlorinated product 14 at pH

isolated by ethyl acetate extraction in good yields (88%

isolated yield) in a 10.0 g scale reaction.

This reaction proceeds smoothly, but the isolation of the

desired product 13 on a large scale via the traditional batch

extraction method is ineﬃcient due to the product’s poor

solubility. This would particularly be a major problem in

kilogram-scale synthesis, as the amount of solvent required for

extraction would be enormous. Therefore, a special apparatus

≤ 3 or ≥ 10 hydrolyzed to the starting material 13.

Therefore, upon completion of the chlorination reaction, the

batch temperature was decreased to 26 °C; this resulted in the

solidiﬁcation of the reaction mixture, which was diﬃcult to

handle. Moreover, when the reaction was diluted with DCM at

the batch temperature of 26 °C, the brown sticky precipitate

was suspended in the reaction mixture. It was diﬃcult to

transfer into another reactor for workup. As the reaction’s

quenching process was exothermic in nature, the crude

reaction temperature was decreased to −5 °C and neutralized

(Figure 2) was used for the continuous extraction of the

aqueous phase for a longer time (3 days) using ethyl acetate or

CH Cl under diﬀerent pH levels using a test batch of 20.0 g

reaction. While continuous extraction with EtOAc under pH

9−10 (pH adjusted by adding 6 N HCl) gave compound 13

along with one major impurity 17 (LCMS: 163.0 [M + 1] and

85.0 [M + Na]), possibly from the ether bond cleavage of 13,

whereas continuous extraction with CH Cl under the pH

∼

with a 10% K

resulting clear solution was further neutralized with a 10%

CO aqueous solution by maintaining the same temperature

2 3

CO aqueous solution to raise the pH to 3. The

until pH 9. For the workup process, the quenched solution was

transferred to another reactor, warmed to ∼15 °C, agitated for

20 min, and settled to split layers and extracted with DCM to

isolate the product. Unfortunately, during this workup process,

it was found that over 50% of 14 hydrolyzed back to the

starting material 13.

∼

9−10 gave 13 along with another impurity 18 (LCMS: 296.1

M + 1]; quaternary ammonium salt, Figure 2) possibly from

the addition of 13 and CH Cl .

[

In contrast, continuous extraction with EtOAc without

adjustment of pH by adding 6 N HCl to the reaction mixture

(

pH >10) successfully provides the desired product 13 with

The hydrolysis of 14 increased in the reaction with side

product HCl autocatalyzing the reaction. Therefore, in the next

attempt, we successfully applied the quenching and workup

relatively good yield and purity (after slurry puriﬁcation

disposal). Based on the optimized extraction conditions with

EtOAc, the isolated yield of 13 for a medium-scale reaction of

process by adopting the method developed by Pesti et al.

Cold aqueous K HPO would be an advantageous quench

60.0 g was 79%. However, when this procedure was

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Figure 2. Continuous liquid−liquid extraction. (A) Liquid−liquid continuous extractor for solvents less dense than water (for example. EtOAc/

H O). The ﬂow of the organic (EtOAc) gas phase is shown using black-color arrows, and the movement of the liquid phase is shown using blue-

color arrows. The organic liquid phase extracts the product from the aqueous layer present in ﬂask 4 and gets collected in a separate ﬂask 8 and (B)

structure of impurities (17 and 18) formed during the extraction process.

Table 3. Chlorination of 13 with POCl under Diﬀerent

Conditions

in the next attempt, the batch temperature was carefully

decreased to 30−35 °C (this prevented solidiﬁcation of the

reaction mixture), and then the reaction mixture was diluted

with CH Cl . Next, the reaction mixture was quenched by the

slow addition of 12.5% K HPO aqueous solution to reach a

pH of 4 to 5 by maintaining the reaction temperature at −5 to

5 °C. The reaction mixture was further neutralized with a

0% K CO aqueous solution to reach a pH of 9 to 10 by

_H_P_L_C₍_%₎b

maintaining the reaction mixture at low temperatures.

Subsequently, the organic layer was separated, and the aqueous

batch size

temp

time

(h)

layer was extracted with CH Cl and concentrated under

entry

(13, g)

(°C)

solvent

toluene

impurities

vacuum to a minimum amount of solvent; this was centrifuged

to get crude 14. The unstable intermediate 14 is eﬃciently

accessible under careful workup conditions with a purity of

0.70

5.00

20.00

108.00

164.00

2.0

1.5

8.0

77.4

93.9

94.1

90.2

96.7

19.2

2.7

3.0

5.4

2.1

CH CN

96.7% in kilogram scales. The optimized chlorination process

CH CN

was scaled up eﬀectively for 14, with a batch size of 1.45 kg of

the starting material.

We tested diﬀerent types of bases and solvent conditions for

CH CN

Reaction performed with quinazolinone 13 (1.0 equiv), POCl (2.0

equiv). Purity determined by HPLC analysis (C18 HPLC column,

.1% TFA in ACN/0.1% TFA in H O).

the next telescoped S Ar displacement reaction of 14 with 7

(

commercially available) (Table 4). Use of Et N, or K CO in

3 2 3

EtOH, i-PrOH, DMF, DMAC, or CH CN solvents, resulted in

solution; as in the case of an accidental overcharge, it would

not result in an overly caustic quench mixture. Moreover, using

a base with more strength to neutralize acid, i.e., K CO , that

a high level of impurities (7.4−16.5%) with a mass of 688.4 (M

+ 1) at 11.7 min (HPLC analysis) (Table 4). The impurity

with M + 1 of 688.4 is likely to be a pseudo-dimeric impurity

required less water to dissolve per base equivalent, was not

advantageous in the initial neutralization of POCl . Therefore,

formed by S Ar reaction between the dimethylamino nitrogen

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Table 4. Optimization of S Ar Reaction of 14 under Diﬀerent Reaction Conditions

HPLC (%)

entry

14 (purity)

base^b

solvent

temp (°C)

time (h)

impurities^c

0.60 g (95.4%)

5.00 g (86.3%)

3.30 g (91.3%)

3.34 g (92.0%)

0.50 g (93.2%)

1.20 g (89.8%)

517.30 g (91.0%)

Et N

i-PrOH

EtOH

DMF

DMAC

i-PrOH

EtOH

2.5

2.0

2.5

3.0

2.0

7.0

83.0

83.3

72.4

75.1

79.1

68.3

96.4

95.0

8.9

8.0

9.7

12.2

11.3

16.5

3.5

Et N

K CO

DIPEA

CH CN

9.0

3.3

Reaction performed with chloroimidate 14 (1.0 equiv), amine 7 (1.0 equiv). Base (1.0 equiv). Purity determined by HPLC analysis (C18

HPLC column, 0.1% TFA in ACN/0.1% TFA in H O). Moreover, the di ﬀ erent bases generated the same types of impurities and are provided in %.

Figure 3. Structure of impurities of 19, 20, and 21.

Scheme 5. Synthesis of Amino-thiazole 16

of product 15 and starting material 14, followed by

demethylation. The possible structure of impurity (19) is

shown in Figure 3. The formation of such pseudo-dimeric

limit. In contrast, the use of a mixture of EtOAc/MeOH

(10:1.5) solvent to wash the product removed the impurity

successfully (due to diﬀerential solubility of impurity and the

product 15), and only a minimum level (0.8−0.9%) was

detected in the ﬁnal product 15.

compounds is reported earlier. However, the use of

diisopropylethylamine (DIPEA) in acetonitrile gave promising

results for this reaction. Therefore, we attempted bulk-scale

synthesis using 14 (entry 8, 517.3 g, purity: 91.0% HPLC)

under the same condition, which produced 15 (95.0% purity),

2.7. Synthesis of Intermediate 16. We used the same

reaction method in route B2 to synthesize penultimate

compound 16 (Scheme 5) for kilogram-scale synthesis. To

obtain high purity on a bulk scale, compound 15 was reacted

with TFA in dichloromethane at around 40−45 °C to remove

with a low level of impurity (3.3%). This indicates that the

formation of impurity 19 is suppressed to a minimum level.

Even though the above reaction condition minimized the

amount of impurity 19 formed, there was a need to remove the

the Boc protective group to get 16 as TFA salt. During the

process development, it was found that the impurity 19 (688.4

[M + 1]) from the previous step remained, which gave a

corresponding de-Boc impurity 20 (Figure 3) at 5.8 min

(588.3 [M + 1], HPLC analysis) after the reaction. Further, it

was found that the purity of 16 depends only on the quality of

∼

3% level of impurity present in the ﬁnal product. For this, the

crude compound 15 was washed with several solvents such as

EtOAc, MeOH, EtOH, i-PrOH, CH CN, CH Cl , and

acetone, which could not remove the impurity to a satisfactory

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Table 5. Solvent Screening for Urea Bond Formation to Get 1

entry

16 (purity)

solvent

time (h)

yield (%)^b

purity (%)^c

10.0 g (95.0%)

10.0 g (98.1%)

16.6 g (98.1%)

130.0 g (98.2%)

CH Cl₂

65.2

66.0

69.3

92.9

89.4

96.5

96.9

97.7

CH Cl /MeOH

CH Cl /CH CN

Reaction performed with amine 16 (1.0 equiv), isocyanate 9 (1.5 equiv), and Et N (3.0 equiv). Isolated yields of product 1. Purity determined

by HPLC analysis (C18 HPLC column, 0.1% TFA in ACN/0.1% TFA in H O).

Scheme 6. Kilogram-Scale Synthesis of 1 (Route B3)

starting material 15 and does not change with minor variations

2.8. Synthesis of BPR1K871 (1). The ﬁnal compound 1

in the reaction conditions. Hence, the pure product’s isolation

from the reaction mixture required a robust recrystallization

procedure, for which MTBE (methyl tert-butyl ether) and

MeOH solvent mixture was used.

was synthesized using the same coupling reaction used in route

B2, where the reaction between intermediate 16 and 3-

chlorophenyl isocyanate (10) was carried out in DCM using

12,24

Et N as a base (Scheme 4).

At ﬁrst, the reaction on a large

Consequently, using the above reaction and workup method,

intermediate 16 was obtained in an excellent yield (99%) and

HPLC purity (98.2%) on a single large-scale batch of 5.5 kg

without the need for column puriﬁcation. As it was found that

scale using DCM as a solvent alone resulted in the incomplete

reaction due to the formation of a sticky suspension. Hence,

unreacted starting material 16 was still present even after

prolonged reaction time. Hence, we sought an alternative

solvent system to carry out the reaction (Table 5). The use of

the CH Cl /MeOH solvent system to conduct the reaction

16 absorbed moisture easily over a short time (4.6% to 9.0%

over 24 h, as determined by Karl Fischer titration), it was

stored under anhydrous condition (desiccant) until consumed

in the next step.

showed that the coupling reaction did not occur eﬃciently.

Moreover, the use of an additional quantity of 10 to drive the

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Article

reaction to completion resulted in the formation of an impurity

suppliers and used without further puriﬁcation. The process

campaign reactions are performed in a glass or glass-lined

jacketed reactor ﬂushed with dry nitrogen. The compounds

1 (679.0 [M + 1]) at 14.2 min in HPLC (Figure 3) due to

diurea formation. Finally, the reaction in CH Cl /CH CN

(

1:1) solvent mixture went on to completion with a good yield

were characterized by H-NMR and C-NMR spectra using

of the ﬁnal product 1, which was isolated by recrystallization

Varian Mercury 400 MHz spectrometers. H-NMR chemical

from a mixture of CH CN and MeOH (1:1). The CH Cl /

shifts are reported as δ values in part per million (ppm) and

relative to the deuterated solvents’ residual resonance. In-

process control analysis coducting by HPLC for intermediates

using method 1 and the ﬁnal product was conducted using

HPLC method 1 and 2. HPLC method 1: Agilent 1100 DAD

system, Agilent Zorbax Eclipse Plus C18 column, 150 × 4.6

mm, 3.5 μM, 1.0 mL/min, at 30 °C, DAD detector, UV

detection at 220, 240, 254, 260 nm, injection volume 5 μL and

CH CN (1:1) solvent system avoided sticky gel formation

during the reaction process. Moreover, it was found that the

starting material’s moisture content needs to be kept at a

minimum so that 16 could be consumed completely during the

reaction to get the ﬁnal product in high purity. This procedure

was used on a kilogram scale to get 3.09 kg of 1 (71.8% yield)

with 97.8% HPLC purity.

.9. Final Scale-Up Synthetic Route. The optimized

Mobile phase A (0.1% TFA in H O), and mobile phase B

process manufacturing was run on a multikilogram scale in a

kilo lab facility using a six-step reaction sequence (Scheme 6).

All the steps provided the product as solid and were

centrifugation either from the reaction mixture directly or

recrystallized from solvents to get the product in high purity. In

the ﬁrst step, the quinazolinone 3 was formed from the

commercially available material 2. Once the reaction was

completed, simple centrifugation provided the product in high

purity. While in the second step, the isolation of 13 from the

reaction mixture required continuous liquid−liquid extraction

due to poor solubility of 13 in EtOAc. Product 14 from step 3

was telescoped directly to step 4 due to instability of the chloro

product 14. A well-controlled quenching process with a 12.5%

K HSO aqueous solution and saturated K CO aqueous

(0.1%TFA in ACN). The gradient elution program at 0 min

was mobile phase A 95 and 5% mobile phase B, at 14 min was

mobile phase A 40 and 60% mobile phase B, 18−22 min was

mobile phase A 10 and 90% mobile phase B with an

acquisition time of 22 min. Method 2: Agilent 1200 DAD

system, Agilent Zorbax SB-CN column, 150 × 4.6 mm, 3.5 μm,

1.0 mL/min, at 30 °C, DAD detector, UV detection at 254,

nm, injection volume 5 μL and mobile phase A (10 mM

KH PO in water (adjusted pH = 2.5 by H PO )), and mobile

phase B (ACN/THF = 95/5). The gradient elution program at

0 min was mobile phase A 95 and 5% mobile phase B and at 22

min 10% mobile phase A and 90% mobile phase B with an

acquisition time of 22 min. High-resolution mass spectra

(HRMS) were recorded using a VARIAN 901-MS.

solution ensured the preparation of 14 in high purity.

Recrystallization from MTBE and MeOH solvents provided

4.2. Synthetic Route A. The experimental details and

compound characterization (NMR and HRMS) for the

synthesis of BPR1K871, according to route A, were previously

6 in excellent purity (98.2%) in step 5, ready for the ﬁnal

reaction. Finally, urea formation in a mixture of the CH Cl /

reported.

CH CN (1:1) solvent system produced 3.09 kg of ﬁnal

4.3. Synthetic Route B1. 4.3.1. 7-(3-(Dimethylamino)-

propoxy)quinazolin-4(3H)-one (13). To a stirred solution of

product 1 in a single batch.

-amino-4-ﬂuorobenzoic acid (2) (1.00 g, 6.45 mmol) in

. CONCLUSIONS

DMSO (2 mL) was added formamidine acetate (1.07 g, 10.31

mmol) at room temperature, and the resulting mixture was

heated to 120 °C for 4 h. Further, 3-dimethylamino-1-

propanol (7.98 g, 77.35 mmol) was added to the reaction

mixture and stirred at the same temperature for 1 h. To the

resulting mixture at 120 °C, KOH (4.34 g, 77.35 mmol) was

added as one portion, and stirring was continued at 125 °C for

In summary, we established a robust and scalable process for

the synthesis of multitargeted kinase inhibitor BPR1K871 (1)

as a clinical candidate. During the scale-up synthesis, we

overcame several inherent diﬃculties associated with the initial

medicinal chemistry synthetic method (route A). Particularly,

the terminal dimethyl amino functional groups in the earlier

step in the synthesis enabled easy puriﬁcation of the products.

The isolation of less soluble intermediate 13 was successfully

carried out by continuous liquid−liquid extraction. During the

scale-up process, the formation of various impurities 17−21

was minimized using optimized reaction conditions and careful

processing steps, which helped to avoid column chromatog-

raphy puriﬁcation steps. The scale-up synthetic method (route

B3) was one step shorter than the initial route A (seven steps;

6 h. The mixture was cooled to room temperature and diluted

with H O (200 mL), and the solution pH value adjusted to 10

with an 18% HCl solution before being extracted with CH Cl

(

6 × 200 mL). The combined organic phases were washed

with brine, dried over MgSO , and concentrated under

reduced pressure to aﬀord compound 13 (1.32 g, 83%) as a

yellow solid.

4.3.2. 3-((4-Chloroquinazolin-7-yl)oxy)-N,N-dimethylpro-

pan-1-amine (14). To a stirred solution of 13 (0.50 g, 2.02

mmol) and DMF (0.5 mL) in toluene (5 mL) was heated to

.7% yield) and provided 1 with an improved yield of 16.5%.

Product 1 was prepared in a single batch of 3.09 kg (97.8%

HPLC purity) for preclinical testing. Improved yield,

avoidance of several column chromatography puriﬁcation

steps, the use of safe reagents, and the minimum amounts of

reagents/solvents resulted in the synthesis of 1 cost-eﬀectively.

The developed methodology could be employed for the

synthesis of 1 to support the clinical development.

0 °C and then added SOCl (2.93 mL, 40.44 mmol). The

resulting solution was stirred at the same temperature for 2 h.

The reaction was cooled to room temperature, removed the

excess reagent under reduced pressure, and diluted with

CH Cl (40 mL) and sat. NaHCO aqueous solution (40 mL).

The organic phase was separated, and the aqueous layer was

extracted with CH Cl (2 × 50 mL). The combined organic

phases were washed with brine, dried over MgSO , and

. EXPERIMENTAL SECTION

.1. General Procedure. All the starting materials,

reagents, and solvents were obtained from commercial

concentrated under reduced pressure to aﬀord crude product

14 (0.52 g) as a brown solid. This compound was used

immediately for the next step without further puriﬁcation.

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.3.3. 5-(2-((7-(3-(Dimethylamino)propoxy)quinazolin-4-

Article

followed by being stirred for 15 min. The mixture was ﬁltered,

and the collected ﬁltrate concentrated under reduced pressure

to aﬀord the product 14 (8.24 g, 85%) as a red liquid.

4.4.4. tert-Butyl(5-(2-((7-(3-(dimethylamino)propoxy)-

quinazolin-4-yl)amino)ethyl)thiazol-2-yl) Carbamate (15).

To a solution of the above compound 14 (8.24 g, 31.00

yl)amino)ethyl)thiazol-2-amine (16). To a stirred solution

of the above compound 14 and tert-butyl (5-(2-aminoethyl)-

thiazol-2-yl)carbamate (7) (0.49 g, 2.02 mmol) in EtOH (10

mL) added triethylamine (0.61 g, 6.07 mmol) at room

temperature. The resulting mixture was heated to 85 °C for 16

h. The reaction was cooled to room temperature, the solvent

evaporated under reduced pressure, and the residue was

diluted with CH Cl (100 mL) and H O (100 mL). The

mmol) and Et N (4.32 mL, 31.00 mmol) at room temperature

were added i-PrOH (16 mL) and tert-butyl (5-(2-aminoethyl)-

thiazol-2-yl)carbamate (7) (9.05 g, 37.20 mmol). The resulting

organic layer was separated, and then 3 N HCl (100 mL) was

added and extracted. The organic layer was discarded. The

aqueous layer containing the compound was separated and

further diluted with a mixture of H O (100 mL) and CH Cl

solution was heated to reﬂux under a N atmosphere for 30

min. Next, the solvent was removed under reduced pressure

and diluted with CH Cl (50 mL) and H O (50 mL). The

organic phase was separated, and the aqueous phase was

(

100 mL), and the pH value adjusted to 12 with 2 N NaOH.

The organic phase was separated. The aqueous phase extract

extracted with CH Cl (2 × 100 mL); the combined organic

phases were washed with sat. NaHCO (50 mL) and brine (50

with CH Cl (2 × 100 mL); the combined organic phases were

mL), dried over MgSO , and concentrated under reduced

washed with brine, dried over MgSO , and concentrated under

pressure. The crude product was dissolved in a mixture of

EtOH (8 mL) and diethyl ether (80 mL) by heating to boiling,

ﬁltered, and then cooled to room temperature. This solution

was stirred overnight to precipitate the product, which was

collected by ﬁltration to aﬀord 15 (8.95 g, 52%) as a white

solid.

reduced pressure to aﬀord 16 (0.23 g, 31.1%, over three steps).

.3.4. 1-(3-Chlorophenyl)-3-(5-(2-((7-(3-(dimethylamino)-

propoxy)quinazolin-4-yl)amino)ethyl)thiazol-2-yl)urea (1).

A solution of compound 16 (0.23 g, 0.62 mmol) and 3-

chlorophenyl isocyanate (1.91 g, 12.46 mmol) in MeOH (0.7

mL) and CH Cl (7 mL) was stirred at room temperature for

4.4.5. 5-(2-((7-(3-(Dimethylamino)propoxy)quinazolin-4-

yl)amino)ethyl)thiazol-2-amine (16). To a solution of 15

(6.30 g, 13.33 mmol) in CH Cl (102 mL) at room

6 h. Once the reaction is over, the solvent was removed under

reduced pressure; acetone (20 mL) was added to the residue

and stirred at room temperature for 16 h. The resulting solid

was collected to aﬀord compound 1 (0.15 g, 45%) as a white

solid.

temperature was added TFA (21.42 mL, 0.27 mol). The

resulting mixture was stirred at the same temperature for 16 h.

The solvent was removed under reduced pressure, added

MeOH (6 mL) and diethyl ether (24 mL), and stirred for 2 h.

The precipitate formed was ﬁltered to get 16 (9.30 g, ∼99%)

as a white solid.

.4. Synthetic Route B2. 4.4.1. 7-Fluoroquinazolin-

(3H)-one (3). A stirred solution of 2-amino-4-ﬂuorobenzoic

acid (2) (20.00 g, 0.13 mol) in EtOH (200 mL) was heated to

reﬂux for 1 h. The resulting mixture at room temperature was

ﬁltered, and formamidine acetate (13.32 g, 0.13 mol) was

added to the collected ﬁltrate and heated to reﬂux for 3 days.

The reaction mixture was cooled to room temperature, and the

formed solid product was ﬁltered and washed with EtOH (30

mL) to aﬀord compound 3 (15.00 g, 71%) as a brown solid.

4.4.6. 1-(3-Chlorophenyl)-3-(5-(2-((7-(3-(dimethylamino)-

propoxy)quinazolin-4-yl)amino)ethyl)thiazol-2-yl)urea (1).

To a solution of compound 16 (9.5 g, 13.29 mmol) in dry

CH Cl (190 mL) under an N₂atmosphere at room

temperature was added Et N (3.70 mL, 26.58 mmol). The

resulting solution was stirred for 30 min and then added slowly

1-chloro-3-isocyanatobenzene (10) (3.24 mL, 26.59 mmol) for

about 80 min at room temperature and stirred for an additional

30 min. The precipitated product from the reaction mixture

was collected by ﬁltration. The crude product was dissolved in

.4.2. 7-(3-(Dimethylamino)propoxy)quinazolin-4(3H)-

one (13). To a stirred solution of compound 3 (15.00 g,

.09 mol), 3-(dimethylamino)propan-1-ol (42.42 g, 0.41

mmol) in DMSO (30 mL) at room temperature, was added

KOH (20.02 g, 0.37 mol). The resulting solution was stirred at

EtOAc (40 mL) and added Et N (3.7 mL) dropwise at 0 °C;

40 °C for 16 h. Then, the reaction was cooled down to room

then H O (40 mL)was added to the solution at that same

temperature and diluted with H O (150 mL) and EtOAc (100

temperature and stirred for 2 h. The product precipitated out

and was ﬁltered to obtain 1 (5.12 g, 73%) as a white solid with

HPLC purity of 99.1%.

mL). The solution pH was adjusted to 10 with a 6 N HCl and

then ﬁltered to remove insoluble particles. The resulting ﬁltrate

was continuously extracted with EtOAc (200 mL) for about 16

h using a continuous liquid−liquid extractor (shown in Figure

4.5. Synthetic Route B3. 4.5.1. 7-Fluoroquinazolin-

4(3H)-one (3). A 200.0 L glass-lined jacketed reactor was

charged with ethanol (70.0 kg) and 2-amino-4-ﬂuorobenzoic

acid (2) (9.70 Kg, 62.52 mol, 1.0 equiv). The resulting mixture

was stirred at room temperature and then added formamidine

acetate (13.13 kg, 125.04 mol, 2.0 equiv) in one portion at the

same temperature. The reaction mixture was warmed to reﬂux

and stirred for 2 days. When the HPLC analysis indicated <4%

of the starting material 2 remained, the batch temperature was

gradually decreased to 10−15 °C and stirred for 4 h at that

temperature. While maintaining the internal temperature of

10−15 °C, the compound was precipitated, the mixture was

centrifuged, and the cake was rinsed with ethanol (8.0 Kg).

The wet cake was dried in an oven under vacuum at 55 °C for

24 h to aﬀord compound 3 (9.17 kg, 89.4%) as an oﬀ-white

). The collected organic phase was cooled to room

temperature, concentrated to about 50 mL, and stirred at

room temperature for 4 h to precipitate the compound. The

precipitate was ﬁltered and dried to aﬀord compound 13

(

12.53 g, 55%) as a white solid.

.4.3. 3-((4-Chloroquinazolin-7-yl)oxy)-N,N-dimethylpro-

pan-1-amine (14). To a stirred solution of 13 (9.00 g, 36.39

mmol) in 1,2-dichloroethane (18 mL) was added POCl (9.18

mL, 98.26 mmol) under a nitrogen atmosphere at room

temperature. The resulting solution was heated to the reﬂux

temperature of the solvent for 4 h. The reaction was quenched

with ice water (200 mL), and then the pH was adjusted to 9

with sat. Na CO solution. The reaction mixture was extracted

with CH Cl (2 × 300 mL), and to the combined organic

solid with an HPLC purity of 99.8%. H-NMR (400 MHz,

phases were added acid clay (9.0 g) and MgSO (9.0 g),

DMSO-d ) δ 12.35 (brs, 1H), 8.16 (dd, J = 8.8, 6.4 Hz, 1H),

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.13 (s, 1H), 7.45 (dd, J = 10.4, 2.8 Hz, 1H), 7.39 (ddd, J =

sampled for HPLC purity and found to be 96.7%. Due to the

instability of chloro compound 14, the above ﬁltrate was

directly used for the next step.

.4, 8.8, 2.4 Hz, 1H). ¹³C NMR (100 MHz, DMSO-d ), δ

66.8−164.3 (d, J = 249.3 Hz), 160.0, 150.9 (d, J = 13.0 Hz),

46.8, 128.9 (d, J = 10.7 Hz), 119.6, 115.2 (d, J = 23.7 Hz),

12.3 (d, J = 21.4 Hz). HRMS (ESI) calcd for C H FN NaO

4.5.4. tert-Butyl(5-(2-((7-(3-(dimethylamino)propoxy)-

quinazolin-4-yl)amino)ethyl)thiazol-2-yl) Carbamate (15).

The amine 7 (1.45 kg, 5.97 mol, 0.9 equiv) was directly

charged into the ﬁltrate 14, which was obtained in the earlier

step. The reaction mixture was then concentrated under

vacuum at 20 °C to about 2.5 L volume and was charged with

[M + Na]: 187.0283; found 187.0283.

.5.2. 7-(3-(Dimethylamino)propoxy)quinazolin-4(3H)-

one (13). A 200.0 L glass-lined jacketed reactor was charged

with 3-(dimethylamino)propan-1-ol (12) (31.62 kg, 306.59

mol, 5.5 equiv) and powdered KOH (12.51 kg, 222.98 mol, 4.0

equiv). The resulting mixture was warmed to 120 °C and

stirred for 1 h. Then, quinazolinone 3 (9.15 kg, 55.75 mol, 1.0

equiv) was added to the reactor at that temperature. The

reaction mixture was stirred at the same temperature for 8 h;

HPLC analysis indicated only 0.3% of 3 remained (Rt = 6.9

min). The reaction mixture was cooled down to 15 °C, and

CH CN (8.2 kg) and then concentrated to about 4.1 L

volume. The mixture was transferred to a 50.0 L reactor, and

CH CN (6.6 kg) and DIPEA (0.856 kg, 6.63 mol, 1.0 equiv)

were charged into the reactor. The mixture was heated to 55

°C and held for 2 h; then the batch temperature raised to 65

°C over 30 min and held for 2 h with stirring. An additional

amount of amine 7 (0.161 kg, 0.663 mol, 0.1) was charged into

the reactor at that temperature. The reaction temperature was

raised to 75 °C and stirred for 4 h. The reaction mixture was

sampled by HPLC, which detected 3.5% unreacted starting

material 14. Then, MeOH (0.62 L) was added into the reactor

while the temperature was maintained at about 65 °C and held

for 1 h. The mixture was cooled to 20 °C over 2 h. The

mixture was stirred for about 5 h and then centrifuged to get

H O (100.0 L) was added to the reactor dropwise over a 1 h

period while maintaining the internal temperature at 20−25

°C. The resulting mixture was continuously extracted with

EtOAc (650.0 kg) for 3 days using a liquid−liquid continuous

extractor, as shown in Figure 2. Finally, the aqueous phase was

extracted twice with a mixture of EtOAc (150.0 kg × 2) and

EtOH (10.0 kg × 2); the combined organic phase was

concentrated under vacuum at 50 °C until the volume was

about 55.0 L. The mixture was treated with EtOH (5.0 kg) and

heated to 45 °C for 1 h. The solution temperature was

decreased to 15 °C and held for about 2 h to aﬀord the

product’s precipitation. The mixture was centrifuged, collected

the solid, and the cake rinsed with a mixture of EtOAc (4.9 kg)

and EtOH (0.46 kg), which gave 11.20 kg wet cake. The wet

cake was dried in an oven under vacuum at 45 °C for 18 h to

give the desired product 13 (9.22 kg, 66.9%) as a white solid,

the crude cake and washed with a mixture of CH CN (7.0 kg)

and MeOH (0.40 kg) to get 4.56 kg 15 as a wet-cake with

89.5% HPLC purity. A solution of EtOAc (6.32 kg) and

MeOH (1.26 kg) and the wet cake 4.56 kg in a 50 L reactor

was heated to 85 °C for 12 h. Then, the reaction temperature

cooled to 20 °C over 2.5 h and held for 3 h. The mixture was

centrifuged, and the cake was rinsed with EtOAc (4.0 kg) to

aﬀord a 1.30 kg product. The wet cake was dried in an oven

under vacuum at 50 °C for 10 h to get the product 15 (1.22 kg,

with an HPLC purity of 98.5%. H-NMR (400 MHz, DMSO-

d₆) δ 12.07 (brs, 1H), 8.04 (s, 1H), 8.00 (d, J = 9.6 Hz, 1H),

38.9% yield over two steps) as a light brown solid with 96.8%

HPLC purity. H-NMR (400 MHz, DMSO-d ) δ 11.16 (brs,

.08 (t, J = 7.6 Hz, 2H), 4.13 (t, J = 6.4 Hz, 2H), 2.37 (t, J =

.8 Hz, 2H), 2.15 (s, 6H), 1.91−1.84 (m, 2H). C-NMR (100

2H), 8.41 (s, 1H), 8.24 (t, J = 5.2 Hz, 1H), 8.10 (d, J = 9.2 Hz,

1H), 7.11 (dd, J = 9.2, 2.4 Hz, 2H), 7.07 (dd, J = 8.4, 2.4 Hz,

2H), 4.12 (t, J = 6.4 Hz, 2H), 3.70 (q, J = 12.8, 6.8 Hz, 2H),

3.06 (t, J = 6.8 Hz, 2H), 2.38 (t, J = 7.2 Hz, 2H), 2.15 (s, 6H),

MHz, DMSO-d ), δ 163.2, 160.2, 150.9, 145.9, 127.4, 116.3,

15.9, 108.8, 66.3, 55.5, 45.1, 26.6. HRMS (ESI) calcd for

C H N O [M + H]: 248.1399; found 248.1395.

1.92−1.85 (m, 2H), 1.44 (s, 9H). C-NMR (100 MHz,

.5.3. 3-((4-Chloroquinazolin-7-yl)oxy)-N,N-dimethylpro-

DMSO-d ) δ 161.7, 158.9, 158.3, 155.5, 152.7, 151.3, 134.9,

pan-1-amine (14). A 50.0 L glass-lined jacketed reactor was

128.3, 124.2, 116.9, 109.1, 107.4, 80.8, 66.1, 55.5, 45.1, 41.7,

27.8, 26.6, 25.7. HRMS (ESI) calcd for C H N NaO S [M +

charged with CH CN (7.8 kg) and 13 (1.64 kg, 6.63 mol, 1.0

23 32

equiv) and stirred at room temperature. Further, POCl (2.03

kg, 13.26 mol, 2.0 equiv) was added into the reaction mixture

over 10 min while maintaining the batch temperature below 30

Na]: 495.2154; found 495.2679.

4.5.5. 5-(2-((7-(3-(Dimethylamino)propoxy)quinazolin-4-

yl)amino)ethyl)thiazol-2-amine (16). A 100.0 L jacketed

reactor was ﬂushed with nitrogen and charged with Boc-amine

15 (4.00 kg, 8.46 mol, 1.0 equiv) and CH Cl (42.2 kg).

C. The temperature was increased to ∼80 °C over 45 min

(

the reaction mixture cleared at 56 °C) and held for 8 h. The

completion of the reaction was established by HPLC analysis,

which indicated that the unreacted starting material was

around 1%. The reaction was cooled to ∼35 °C over 1 h,

charged with CH Cl (46.0 kg), and then transferred into a

Triﬂuoroacetic acid (15.20 kg, 132.88 mol, 15.7 equiv) was

dropwise added into the reactor over a period of 1 h, while the

reaction temperature was maintained <30 °C. The resultant

mixture was heated to 45 °C and stirred at that temperature for

about 6 h. When the HPLC analysis indicated that <1% of 15

remained, then, the reaction was concentrated to about 10.0 L

volume. Subsequently, MeOH (3.2 kg) and MTBE (12.0 kg)

were charged into the reactor and stirred at room temperature

for about 6 h. The mixture was ﬁltered, and the solid obtained

was washed with MTBE (12.0 kg). The wet cake was dried in a

vacuum oven at 50 °C for about 2 days to yield 5.85 kg

dropping tank. The mixture in the dropping tank was

transferred into a 12.5% K HPO aqueous quench solution

(

97.4 kg) in a 200.0 L reactor over a 20 min period while

maintaining the temperature −5 to +5 °C to reach the target

pH 4−5. Then, 50% K CO aqueous solution (14.8 kg) was

charged into the reactor over 20 min at 5−15 °C until pH 9−

0. The mixture was stirred for 20 min at about 15 °C and

settled to split layers. The organic layer was separated, and the

aqueous layer was washed with CH Cl (46.0 kg) again. The

(∼99%) of 16 as a white solid with 98.2% HPLC purity. H-

NMR (400 MHz, DMSO-d ) δ 9.98 (brs, 1H), 9.83 (brs, 1H),

combined organic phase was washed with 5% brine (33.0 kg)

and dried over Na SO (6.6 kg) for 2 h. The mixture was

8.86 (s, 1H), 8.69 (s, 1H), 8.38 (d, J = 9.6 Hz, 1H), 7.40 (dd, J

= 9.2, 2.4 Hz, 1H), 7.25 (d, J = 2.4 Hz, 1H), 7.04 (s, 1H), 4.24

(t, J = 6.0 Hz, 2H), 3.86 (q, J = 12.4, 6.4 Hz, 2H), 3.25 (brs,

ﬁltered and rinsed with CH Cl (13.0 kg), the ﬁltrate was

Org. Process Res. Dev. 2021, 25, 817−830

Organic Process Research & Development

Complete contact information is available at:

Article

H), 3.03 (t, J = 6.4 Hz, 2H), 2.83 (s, 6H), 2.22−2.15 (m,

Chih-Ming Chen − Institute of Biotechnology and

Pharmaceutical Research, National Health Research

Institutes, Zhunan 35053, Taiwan, ROC

Mohane Selvaraj Coumar − Centre for Bioinformatics, School

of Life Sciences, Pondicherry University, Puducherry 605014,

India; orcid.org/0000-0002-0505-568X

Hsu-Yi Sun − Institute of Biotechnology and Pharmaceutical

Research, National Health Research Institutes, Zhunan

35053, Taiwan, ROC

H). C-NMR (100 MHz, DMSO-d ), δ 169.6, 163.6, 160.1,

58.9 (q, J = 64.9, 32.8 Hz, CO, triﬂuoroacetic acid), 151.4,

40.2, 125.7 (d, J = 98.4 Hz), 121.4 (CF , triﬂuoroacetic acid),

18.4 (d, J = 31.3 Hz), 115.3, 106.9, 101.2, 65.9, 53.9, 42.2,

1.8, 25.5, 23.6. HRMS (ESI) calcd for C H N OS [M + H]:

73.1810; found 373.1807.

.5.6. 1-(3-Chlorophenyl)-3-(5-(2-((7-(3-(dimethylamino)-

propoxy)quinazolin-4-yl)amino)ethyl)thiazol-2-yl)urea (1).

A 200.0 L jacketed reactor ﬂushed with nitrogen was charged

with 16 (5.57 kg, 8.05 mol, 1.0 equiv), CH Cl (61.0 kg), and

Na Sun − China Gateway Pharmaceutical Development Co,

Shanghai 201417, China

dry CH CN (36.8 kg) while stirring at 32 °C. Then, Et N

https://pubs.acs.org/10.1021/acs.oprd.0c00515

(

2.80 kg, 27.60 mol, 3.43 equiv) was added at that temperature

over 15 min and stirred for 10 min. Next, 3-chlorophenyl

isocyanate (2.06 kg, 13.44 mol, 1.67 equiv) was added at that

temperature over 5 min, and the mixture was stirred for about

Author Contributions

C.-M.C. and M.S.C. contributed equally to this work.

h while the temperature was maintained at about 35 °C;

Notes

HPLC analysis determined that <0.07% of the starting material

The authors declare no competing ﬁnancial interest.

6 remained unreacted. The reaction was cooled to 25 °C,

held for 1 h, and then centrifuged to obtain the product as a

ACKNOWLEDGMENTS

■

cake, which was recrystallized with a CH CN (46.0 kg) and

We are grateful to Dr. Hui-Yi Shiao, Dr. Donald C. T. Hou,

and Ms. Yi-Chen Chiu for their support during the

implementation of this campaign. Financially support from

the National Health Research Institute and Ministry of Science

and Technology, Taiwan (MOST 109-2113-M-400-003 for

H.-P. H.) and the Center of Applied Nanomedicine, National

Cheng Kung University from The Featured Areas Research

Center Program within the framework of the Higher Education

Sprout Project by the Ministry of Education (MOE) in Taiwan

is gratefully acknowledged. J.S.R. is supported by a

postdoctoral fellowship from the Value-Added MedChem

Innovation Center, Taiwan.

MeOH (36.8 kg) solvent mixture. The wet cake was dried in

an oven under vacuum at 50 °C for over 12 h to aﬀord the ﬁnal

product 1 (3.04 kg, 71.8%) as a white solid with purity of

7.8% (HPLC methods 1 and 2) and 97.2% assay purity with a

single maximum impurity of ∼0.6−0.7%. H-NMR (400 MHz,

DMSO-d ) δ 10.64 (brs, 1H), 9.20 (brs, 1H), 8.41 (s, 1H),

.25 (t, J = 5.6 Hz, 1H), 8.11 (d, J = 9.2 Hz, 1H), 7.70 (s, 1H),

.33−7.28 (m, 2H), 7.11 (dd, J = 9.2, 3.2 Hz, 2H), 7.05 (m,

H), 4.12 (t, J = 6.4 Hz, 2H), 3.72 (q, J = 12.8, 6.8 Hz, 2H),

.07 (t, J = 7.2 Hz, 2H), 2.38 (t, J = 7.2 Hz, 2H), 2.15 (s, 6H),

.92−1.85 (m, 2H). C-NMR (100 MHz, DMSO-d ) δ 161.7,

59.2, 158.9, 155.6, 152.5, 151.3, 140.5, 133.2, 132.7, 130.4,

27.3, 124.2, 122.0, 117.8, 116.9, 109.1, 107.4, 66.0, 55.5, 45.1,

1.6, 26.6, 25.8. HRMS (ESI) calcd for C H ClN NaO S [M

ABBREVIATIONS

■

AML, acute myeloid leukemia; FDA, food and drug

administration; FLT3, FMS-like receptor kinase tyrosine

inhibitor; AURKA, Aurora Kinase A; AURKB, Aurora kinase

B; MOLM-13, human leukemia cell lines; MV4-11, human

leukemia cell lines; IND, investigational new drug; API, active

pharmaceutical ingredient; GMP, good manufacturing prac-

Na]: 548.1611; found 548.1598.

ASSOCIATED CONTENT

■

sı Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.oprd.0c00515.

tice; S Ar, nucleophilic aromatic substitution; ATP, adenosine

triphosphate; DCM, dichloromethane; ACN, acetonitrile;

DMF, dimethylformamide; DMSO, dimethyl sulfoxide;

HPLC, high-performance liquid chromatography; LCMS,

liquid chromatography-mass spectrometry; DCE, 1,2-dichloro-

ethane; DMAC, dimethylacetamide; DIPEA, diisopropylethyl-

amine; TFA, triﬂuoroacetic acid; MTBE, methyl tert-butyl

ether; HRMS, high-resolution mass spectrometry; ESI, electro-

spray ionization

¹H and ¹³C NMR spectra and HPLC characterization

data for all new compounds (PDF)

AUTHOR INFORMATION

■

Corresponding Author

Hsing-Pang Hsieh − Institute of Biotechnology and

Pharmaceutical Research, National Health Research

Institutes, Zhunan 35053, Taiwan, ROC; Department of

Chemistry, National Tsing Hua University, Hsinchu 30013,

Taiwan, ROC; Biomedical Translation Research Centre,

Academia Sinica, Taipei 11520, Taiwan, ROC;

orcid.org/0000-0002-1332-005X ; Phone: +886-37-246-

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