Synthesis and evaluation of trypanocidal activity of derivatives of naturally occurring 2,5-diphenyloxazoles

Bioorg. Med. Chem. 42 (2021) 116253

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Bioorganic & Medicinal Chemistry

journal homepage: www.elsevier.com/locate/bmc

Synthesis and evaluation of trypanocidal activity of derivatives of naturally

occurring 2,5-diphenyloxazoles

,

Koichi Narita^{a *}, Keisuke Suganuma^b, Toshihiro Murata^a, Ryutaro Kondo^a, Hiroka Satoh^a,

Kazuhiro Watanabe^a, Kenroh Sasaki^a, Noboru Inoue^b, Yuichi Yoshimura^a

^aFaculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japan

^bNational Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada, Obihiro, Hokkaido 080-8555, Japan

A R T I C L E I N F O

A B S T R A C T

Keywords:

African trypanosomiasis is a zoonotic protozoan disease affecting the nervous system. Various natural products

reportedly exhibit trypanocidal activity. Naturally occurring 2,5-diphenyloxazoles present in Oxytropis lanata,

and their derivatives, were synthesized. The trypanocidal activities of the synthesized compounds were evaluated

against Trypanosoma brucei brucei, T. b. gambiense, T. b. rhodesiense, T. congolense, and T. evansi. Natural product 1

exhibited trypanocidal activity against all the species/subspecies of trypanosomes, exhibiting half-maximal

2,5-Diphenyloxazole

Trypanocidal activity

Trypanosoma congolense

Trypanosomiasis

Natural product

inhibitory concentrations (IC₅₀) of 1.1–13.5 μM. Modiﬁcation of the oxazole core improved the trypanocidal

activity. The 1,3,4-oxadiazole (7) and 2,4-diphenyloxazole (9) analogs exhibited potency superior to that of 1.

However, these compounds exhibited cytotoxicity in Madin-Darby bovine kidney cells. The O-methylated analog

of 1 (12) was non-cytotoxic and exhibited selective trypanocidal activity against T. congolense (IC₅₀= 0.78 µM).

Structure-activity relationship studies of the 2,5-diphenyloxazole analogs revealed aspects of the molecular

structure critical for maintaining selective trypanocidal activity against T. congolense.

1. Introduction

of parasitism, when the protozoa are present in the blood, non-speciﬁc

symptoms such as anemia and intermittent fever are observed. As the

African trypanosomiasis is a zoonotic protozoan disease caused by

infection of a host mammal by trypanosomes. African trypanosomiasis

can be classiﬁed based on the causative species/subspecies of trypano-

some and the host mammals.¹Human African trypanosomiasis (HAT),

also known as sleeping sickness, is a human disease caused by Trypa-

nosoma brucei rhodesiense and T. b. gambiense.²Animal African trypa-

nosomosis (AAT), also known as Nagana, is caused by T. b. brucei,

T. congolense, and T. vivaxi affecting domestic and wild animals.³AAT

caused by T. congolense causes the most severe symptoms in infected

animals. Surra is a livestock disease caused by T. evansi.⁴Dourine is a

venereal disease in equine caused by T. equiperdum.⁵While HAT and

AAT are prevalent only in Sub-Saharan Africa, cases of surra and dourine

are seen around the world (surra: North-East Africa, Asia, Latin America;

dourine: Africa, Asia, the Middle East, South-East Europe, and South

America).

disease progresses, trypanosomes invade and proliferate within the

central nervous system, which causes neurological symptoms including

ataxia, paralysis, coma, and eventually death.²Since trypanosomes

frequently and randomly change the cell surface antigens, it is very

difﬁcult to develop a vaccine for the disease, and so far, none have been

developed.⁶Treatment with trypanocidal drugs remains the main

strategy for disease control. Pentamidine, melarsoprol, eﬂornithine,

suramin, and a combination of nifurtimox and eﬂornithine are available

to treat HAT.⁷In addition, diminazene, quinapyramine, and suramin are

used to treat AAT and surra.³However, most existing drugs were

developed over 50 years ago, and multidrug-resistant strains have

emerged as a result of protozoal mutations partly caused by improper

drug administration and abuse.⁸Fexinidazole, recently approved for use

in the Democratic Republic of Congo, based on positive reviews from the

European Medicines Agency, is the ﬁrst oral drug to treat HAT caused by

T. b. gambiense.⁹However, fexinidazole and nifurtimox act through

similar mechanisms and cross-resistance between them has been re-

ported.¹⁰Additionally, there has not been any signiﬁcant progress in the

At an early stage of infection, trypanosomes proliferate in the host’s

bloodstream before crossing the blood-brain barrier and parasitizing the

central nervous system and cerebrospinal ﬂuid. During the early stages

* Corresponding author.

E-mail address: k-narita@tohoku-mpu.ac.jp (K. Narita).

https://doi.org/10.1016/j.bmc.2021.116253

Received 23 March 2021; Received in revised form 25 May 2021; Accepted 28 May 2021

Available online 12 June 2021

K. Narita et al.

Bioorganic & Medicinal Chemistry 42 (2021) 116253

development of new drugs for AAT. Therefore, there is a need to design

and develop new, structurally diverse anti-trypanosomal drugs with

different mechanisms of action and improved safety.

2. Results and discussion

2.1. Chemistry

Tremendous efforts have been devoted to discovering naturally

occurring compounds with trypanocidal activity and various active

skeletons, including ﬂavonoids, xanthones, quinones, alkaloids, sesqui-

terpenoids, macrocycles, and cyclic peptides, have been isolated.¹¹In

2016, Banzragchgarav et al. reported the isolation and structure eluci-

dation of eleven 2,5-diphenyloxazoles from the roots of Oxytropis lanata,

which is found in Mongolia.¹²Some of these compounds exhibited try-

panocidal activity against T. congolense, with a half-maximal inhibitory

To date, several methods for the synthesis of 2,5-diaryloxazoles have

been developed.^26–34In 2013, Zhang et al. reported a highly practical

method for synthesizing 2,5-diaryloxazoles from bromoketones and

benzylamines using an I₂/K₂CO₃system.³⁵This method is noteworthy

because the reaction can be performed under metal-free conditions, the

reagents used are readily available and inexpensive, and a wide range of

functional groups are tolerated. These features make the method suit-

able for the synthesis of 1–4. The synthesis of compounds 1–4 is outlined

in Scheme 1. The 2,5-diphenyloxazoles 12¹⁹–15 were synthesized using

combinations of commercially available bromoacetophenones 10a, 10b

and benzylamines 11 a–c under Zhang’s condition. The demethylation

reaction using BBr₃in CH₂Cl₂afforded 1–4; however, these compounds

could not be separated from the borane residues at this step. Therefore,

1–4 were trapped as acetates 16–19, and pure 1–4 were obtained via

recrystallization after hydrolysis of the acetyl group using 3 M HCl. The

mass spectrometry (MS) and ¹H and ¹³C nuclear magnetic resonance

spectroscopy (NMR) data of 1–4 was identical to those of compounds

1–4 isolated from natural sources.¹²

concentration (IC₅₀) of 1.0–14.8 μM (1–5, Figure 1). The IC₅₀values of

these compounds were in the order: 1 > 5 > 3 ≈ 2 > 4. These results

indicate that both the number of hydroxy groups and their position in

the phenyl ring are important for trypanocidal activity. Although several

naturally occurring 2,5-diaryloxazoles have been isolated,^13–18some of

which are biologically active, 1–5 were the ﬁrst naturally occurring

trypanocidal 2,5-diphenyloxazoles to be reported. The synthesis and

evaluation of the anti-inﬂammatory activity of 1 have been reported in

the literature.¹⁹2,5-Bis[4-amidinophenyl]oxazole (A) is a synthetic try-

panocidal agent,²⁰and the trypanocidal activities of 2,5-diaryfurans,²¹

2,4-disubstituted arylthiazoles,^22,23and 3,5-diarylisoxazoles^24,25have

been investigated. However, the trypanocidal activities of 2,5-dipheny-

loxazoles possessing hydroxy groups on the phenyl ring have not been

investigated. Therefore, 1–5 are attractive synthetic scaffolds for the

design of new trypanocidal agents.

As shown in Scheme 2, heterocyclic analogs 6–9 were synthesized using

Lawesson’sreagentoracid-mediatedcyclizationreaction. Theaminoketone

20 ³⁶was condensed with the carboxylic acid 21, using 1-ethyl-3-(3-dime-

thylaminopropyl)carbodiimide hydrochloride (EDC⋅HCl) to afford the

amide 22. 2,5-Oxathiazole 6 was obtained by thionation, followed by

cyclization of 22 using Lawesson’s reagent,^37,38and demethylation. 1,3,4-

Oxadiazole 7 and 1,3,4-thiadiazole 8 were prepared from the common in-

termediate 25, which was synthesized by the condensation of the hydrazide

24³⁹with21.Cyclizationof25usingPOCl₃afforded26,whilecyclizationof

25 using Lawesson’s reagent afforded 27.⁴⁰Both were converted to the

corresponding phenols, 7 and 8, using BBr₃. Meanwhile, 2,4-diphenyloxa-

zole 9 was synthesized via alkylation of 21 with 10a, followed by cycliza-

tion using BF₃⋅OEt₂in the presence of acetamide,⁴¹and demethylation.

The initial evaluation of the trypanocidal activity of the synthesized

analogs revealed the selective inhibitory activity of 12 against

T. congolense (see below, Table 1). Therefore, we synthesized analogs

30–49 to investigate the effects of the number and position of methoxy

groups. The bromoacetophenones 10a ꢀ l and benzylamines 11a, b, d-i

were subjected to Zhang’s conditions to afford analogs 30–49 (Scheme

3). Compounds 30, 31, 33, 34 have been previously reported in the

literature.^19,42

We designed analogs of compound 1, the most potent of the ﬁve

compounds, by modifying the oxazole core (thiazole 6, 1,3,4-oxadiazole

7, and 1,3,4-thiadiazole 8) and changing the position of the hydrox-

yphenyl substituents (2,4-diphenyloxazole 9). In this study, we describe

the synthesis of various 2,5-diphenyloxazole analogs, including the

naturally occurring compounds 1–4, and the evaluation of their bio-

logical against various trypanosome species/subspecies, to move toward

the discovery of new trypanocidal drug leads.

Additionally, the methoxy group at the 2^′′position of 12 was

modiﬁed to assess the steric and electronic effects of substitutions at this

position. The benzyl group of 49 was removed by hydrogenolysis to

afford 50, which was then alkylated using alkyl halides, to afford

compounds 51–53, possessing alkoxy groups of different sizes at the 2^′′

position (Scheme 4). Compounds with nitrogen-containing groups

substituted at the 2^′′position 56–59 were also synthesized. The reaction

of 2-bromo-2^′-nitroacetophenone with 11a under the same conditions

produced a complex mixture and was found to be unsuitable for the

synthesis of 56. Therefore, the oxazole 56 was synthesized by acid-

mediated cyclization of the amide 55, which was obtained from the

43

condensation of 54

with 21. Reduction of the nitro group in 56

afforded the aniline analog 57, which was then converted to dimethy-

laniline 58 and acetamide 59.

2.2. Biological evaluation

The trypanocidal activity of the synthesized compounds was evalu-

ated against T. b. brucei, T. b. gambiense, T. b. rhodesiense, T. congolense,

and T. evansi. Additionally, preliminary safety was evaluated by

assessing the cytotoxic activity against normal Madin-Darby bovine

kidney (MDBK) cells. The selectivity index (SI) against each trypano-

some species was calculated.^44,45These results are summarized in

Fig. 1. Structures of naturally occurring 2,5-diphenyloxoazoles 1–5 with try-

panocidal activity, compound 1-analogs 6–9, and synthetic trypanocidal agent

2,5-bis[4-amidinophenyl]oxazole (A).

2

K. Narita et al.

Bioorganic & Medicinal Chemistry 42 (2021) 116253

Scheme 1. Synthesis of naturally occurring 2,5-diphenyloxazoles 1–4. Reagents and conditions: a) I₂, K₂CO₃, N,N-dimethylformamide, 60 ^◦C, 18 h, 60–74%. b) 1)

BBr₃, CH₂Cl₂, 0 ^◦C, 24 h. 2) acetic anhydride, 4-dimethylaminopyridine, pyridine, r.t., 5 h, 35–90% (2 steps). c) 3 M HCl, tetrahydrofuran, r.t., 3 d, 62–83%. OMe =

methoxy, Ac = acetyl.

Scheme 2. Synthesis of analogs 6–9.

Reagents and conditions: a) 21,

EDC⋅HCl, HOBt, triethylamine, CH₂Cl₂,

r.t., 12 h, 65% (22) or 83% (25). b)

Lawesson’s reagent, tetrahydrofuran,

reﬂux, 3 h, 85%. c) BBr₃, CH₂Cl₂, 0 ^◦C,

20 h, 79% (6) or 65% (7,8) or 92% (9).

d) POCl₃, CH₃CN, reﬂux, 24 h, 85% (26).

e) Lawesson’s reagent, toluene, reﬂux, 2

d, 54% (27). f) 21, DBU, dime-

thylformamide, 60 ^◦C,

3 h, 82%. g)

acetamide, BF₃⋅OEt₂, xylene, reﬂux, 2 d,

60%. EDC⋅HCl = 1-ethyl-3-(3-dimethy-

laminopropyl)carbodiimide hydrochlo-

ride, HOBt

= 1-hydroxybenzotriazole,

DBU = 1,8-Diazabicyclo[5.4.0]undec-7-

ene. Me = methyl.

Table 1.

Among the synthesized natural products, compound 1 exhibited the

(4.7 µM), T. b. brucei (9.1 µM), and T. b. gambiense (13.5 µM). Com-

pounds 2–4 also exhibited the lowest IC₅₀values against T. congolense.

These ﬁndings suggest that hydroxy-substituted 2,5-diphenyloxazoles

can effectively inhibit the growth of T. congolense.

most potent inhibitory activity in all the species/subspecies of try-

panosomes against which its activity was evaluated. Similar to the try-

panocidal activity against T. congolense,¹²these results suggest that the

number and positions of the hydroxy groups in 2,5-diphenyloxazole

signiﬁcantly affect the trypanocidal activity of other trypanosome spe-

cies as well, and the substitutional pattern of 1 is most favorable.

Compound 1 showed the highest potency (lowest IC₅₀value) against

T. congolense (1.1 µM), followed by T. b. rhodesiense (2.8 µM), T. evansi

Compounds 6–8, compound 1 analogs with a modiﬁed oxazole core,

exhibited activity comparable or superior to that of 1. The 1,3,4-oxadia-

zole (7) exhibited better potency than the thiazole derivative (6) and the

1,3,4-thiadiazole derivative (8) against all species of trypanosomes and

retained preferential inhibitory activity against T. congolense (IC₅₀

=

0.85 µM). The positional isomer 9 exhibited inhibitory activity against

3

Table 1

Trypanocidal activities of 2,5-diphenyloxazoles 1–4 and their analogs 6–9, 12–19, 23, 26, 27, 29.

Trypanosomes IC₅₀µM^a± SD (SI^c)

Normal cell IC₅₀µM^b

Compound

X

Y

R¹

R²

T. b. brucei

T. b. gambiense

T. b. rhodesiense

T. congolense

T. evansi

MDBK

1

O

S

CH

N

2^′, 3^′-OH

2^′, 5^′-OH

–––

2^′′-OH

9.1 ± 2.0

25.9 ± 4.1

20.6 ± 2.5

54.7 ± 22.1

7.7 ± 2.2

1.9 ± 0.6

3.9 ± 1.0

1.3 ± 0.6

> 80.3

(0.77)

(3.0)

(0.36)

(0.41)

(2.6)

(3.1)

(3.5)

(7.5)

ND

13.5 ± 0.7

80.9 ± 6.6

17.8 ± 1.1

78.3 ± 3.6

8.2 ± 1.7

1.9 ± 0.5

4.6 ± 0.4

1.6 ± 1.0

> 80.3

(0.52)

(0.96)

(0.42)

(0.29)

(2.4)

(3.1)

(3.0)

(6.1)

ND

2.8 ± 0.7

84.2 ± 0.2

18.1 ± 0.1

72.1 ± 10.4

7.8 ± 2.3

1.9 ± 0.6

4.4 ± 1.3

1.5 ± 0.8

> 80.3

(2.5)

(0.92)

(0.42)

(0.31)

(2.6)

(3.1)

(6.4)

ND

1.1 ± 0.1

9.5 ± 0.7

4.7 ± 0.5

8.6 ± 6.6

2.3 ± 0.1

0.67 ± 0.10

0.85 ± 0.19

0.85 ± 0.44

0.78 ± 0.27

> 80.3

(6.3)

(8.2)

(1.6)

(2.6)

(8.8)

(16)

4.7 ± 1.2

22.2 ± 5.3

12.2 ± 5.8

44.4 ± 26.9

4.0 ± 0.6

0.81 ± 0.25

2.1 ± 0.7

0.42 ± 0.07

> 80.3

(1.5)

(3.5)

(0.61)

(0.51)

(5.0)

(7.2)

(6.3)

(23)

7.0 ± 0.8

77.4 ± 10.0

7.5 ± 2.9

22.7 ± 9.0

20.0 ± 0.3

5.9 ± 0.6

13.6 ± 0.1

9.7 ± 0.6

> 321

2

2^′′-OH

3

2^′′, 5^′′-OH

2^′′-OH

4

2^′, 5^′-OH

2^′, 3^′-OH

6

7

O

S

2^′′-OH

8

N

2^′′-OH

9

See above

(11)

12

O

S

CH

N

2^′, 3^′-OMe

2^′, 5^′-OMe

–––

2^′′-OMe

(>414)

ND

13

2^′′-OMe

> 80.3

ND

> 80.3

ND

> 80.3

ND

> 80.3

ND

> 321

14

2^′′, 5^′′-OMe

2^′′-OAc

> 88.9

ND

> 88.9

ND

> 88.9

ND

15.4 ± 2.9

2.9 ± 0.0

0.80 ± 0.13

14.4 ± 2.7

3.9 ± 0.0

16.8 ± 4.4

5.7 ± 1.1

2.7 ± 0.5

3.8 ± 0.9

9.4 ± 1.0

7.2 ± 0.9

1.1 ± 0.2

(>23)

(>100)

(6.6)

(4.4)

(2.4)

(1.8)

(20)

> 88.9

ND

> 355

15

2^′, 5^′-OMe

2^′, 3^′-OAc

2^′, 5^′-OAc

–––

> 73.2

ND

> 73.2

ND

> 73.2

ND

> 73.2

ND

> 293

16

4.6 ± 1.9

33.6 ± 19.4

16.6 ± 0.1

35.2 ± 12.1

32.0 ± 2.5

13.3 ± 4.1

29.8 ± 4.3

38.1 ± 0.0

0.066 ± 0.005

4.7 ± 2.0

(1.2)

(1.9)

(0.58)

(0.85)

(3.6)

(2.4)

(>10)

(>8.4)

––

6.5 ± 1.1

55.3 ± 1.9

41.9 ± 26.1

49.8 ± 2.7

31.4 ± 0.6

13.3 ± 4.1

22.1 ± 0.6

38.7 ± 0.4

0.064 ± 0.002

4.6 ± 2.4

(0.82)

(1.2)

(0.23)

(0.60)

(3.6)

(2.4)

(>14)

(>8.3)

––

2.5 ± 1.2

56.0 ± 2.7

64.4 ± 4.9

47.4 ± 2.5

29.6 ± 5.7

13.4 ± 4.3

27.2 ± 7.7

31.6 ± 7.5

0.076 ± 0.011

4.4 ± 1.6

(2.1)

(1.1)

(0.15)

(0.63)

(3.9)

(2.4)

(>11)

(>10)

––

5.6 ± 2.8

34.0 ± 16.3

18.9 ± 1.8

44.7 ± 2.1

30.9 ± 4.1

7.5 ± 2.9

28.1 ± 5.7

37.9 ± 1.6

0.38 ± 0.06

2.6 ± 1.4

(0.96)

(1.9)

(0.51)

(0.67)

(3.7)

(4.3)

(>11)

(>8.5)

––

5.3 ± 0.8

64.2 ± 22.2

9.6 ± 6.5

30.0 ± 1.8

114.5 ± 9.3

32.3 ± 0.4

> 305

17

2^′′-OAc

18

2^′′, 5^′′-OAc

2^′′-OMe

19

2^′, 5^′-OAc

2^′, 3^′-OMe

23

26

O

S

2^′′-OMe

(12)

27

N

2^′′-OMe

(>80)

(>34)

––

29

See above

> 321

Suramine^d

––

Nifrutimox^d

––

a

Concentration that induces 50% inhibition of trypanosomatids. ^bConcentration that induces 50% inhibition of normal Madin-Darby bovine kidney (MDBK) cells. ^cSI: Selectivity index: IC₅₀in MDBK/IC₅₀in try-

panosomatids. ^dPositive control. ND: not determined.

K. Narita et al.

Bioorganic & Medicinal Chemistry 42 (2021) 116253

Scheme 3. Syntheses of compounds 30–49. Reagents and conditions: a) I₂, K₂CO₃, dimethylformamide, 60 ^◦C, 18 h. OMe = methoxy.

Scheme 4. Synthesis of the 2^′′-modiﬁed

analogs 49–59. Reagents and conditions:

a) H₂(balloon), Pd/C, tetrahydrofuran, r.

t., 24 h, 87%. b) ethyl iodide, K₂CO₃, DMF,

60 ^◦C, 2 h, 94% (51). c) n-propyl iodide,

K₂CO₃, DMF, 60 ^◦C, 2 h, 96% (52). d)

MOMCl, NaH, DMF, r.t., 1 h, 90%. (53). e)

21, EDC⋅HCl, HOBt, Et₃N, CH₂Cl₂, r.t., 12

h, 62%. f) POCl₃, reﬂux, 3 h, 71%. g) H₂

(balloon), Pd/C, methanol, rt, 1 h, 77%. h)

methyl iodide, K₂CO₃, 60 ^◦C, 24 h, 70%. i)

AcCl, Et₃N, CH₂Cl₂, 0 ^◦C, 3 h, 87%. Me =

methyl, Ac = acetyl, MOM = methox-

ymethyl, DMF

=

dimethylformamide,

1-

Et₃N trimethylamine, EDC⋅HCl

=

ethyl-3-(3-dimethylaminopropyl)carbodii-

mide hydrochloride,

hydroxybenzotriazole.

HOBt

=

1-

5

Table 2

Trypanocidal activities of 2,5-diphenyloxazoles 30–53, 56–59.

Trypanosomes IC₅₀µM^a± SD (SI^c)

Normal cell IC₅₀µM^b

Compound

R¹

R²

T. b. brucei

T. b. gambiense

T. b. rhodesiense

T. congolense

T. evansi

MDBK

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

56

57

58

59

2^′-OMe

2^′′-OMe

> 88.9

ND

> 88.9

ND

> 88.9

ND

2.8 ± 0.6

6.7 ± 0.6

21.4 ± 5.4

> 88.9

(>128)

(24)

> 88.9

ND

> 355

3^′-OMe

2^′′-OMe

> 88.9

(<1.8)

(>5.7)

(ND)

(0.47)

(0.79)

(1.9)

(1.3)

(0.30)

ND

> 88.9

(<1.8)

(>5.3)

ND

> 88.9

(<1.8)

(>5.3)

ND

> 88.9

(<1.8)

(>6.6)

ND

161.1 ± 0.2

> 355

3^′-OMe

3^′′-OMe

62.2 ± 5.7

> 88.9

66.9 ± 15.6

> 88.9

66.5 ± 1.0

> 88.9

(>17)

ND

53.9 ± 6.7

> 88.9

4^′-OMe

4^′′-OMe

> 355

2^′, 3^′-OMe

2^′, 4^′-OMe

2^′, 6^′-OMe

2^′, 3^′-OMe

2^′-OMe

–––

86.6 ± 1.9

75.3 ± 1.2

19.7 ± 3.5

76.0 ± 0.4

76.4 ± 0.9

> 80.3

84.7 ± 0.4

35.8 ± 0.1

24.6 ± 0.3

73.9 ± 0.9

74.5 ± 0.4

22.2 ± 1.0

69.2 ± 0.9

31.2 ± 0.9

> 73.2

(0.48)

(1.7)

(1.5)

(1.3)

(0.31)

(>14)

(1.3)

(>9.4)

ND

84.1 ± 0.0

59.2 ± 19.7

18.4 ± 1.0

76.1 ± 0.4

77.0 ± 0.9

> 80.3

(0.48)

(1.0)

(2.0)

(1.3)

(0.30)

ND

4.0 ± 0.0

15.3 ± 1.9

5.3 ± 0.0

10.3 ± 0.3

17.3 ± 1.2

4.3 ± 0.9

26.4 ± 0.3

1.5 ± 0.4

1.6 ± 0.1

7.7 ± 1.8

23.6 ± 6.9

51.3 ± 9.5

16.4 ± 2.1

45.8 ± 1.0

3.0 ± 0.5

7.7 ± 2.3

> 84.1

(10)

59.3 ± 20.6

65.1 ± 9.5

21.7 ± 2.2

68.2 ± 12.4

64.5 ± 4.2

> 80.3

(0.68)

(0.91)

(1.7)

(1.4)

(0.35)

ND

40.6 ± 11.2

59.5 ± 14.6

37.6 ± 5.1

96.3 ± 20.7

22.8 ± 0.5

> 321

2^′′-OMe

(3.9)

(7.1)

(9.3)

(1.3)

(>75)

(3.4)

(>190)

(>188)

(12)

2^′′-OMe

3^′′-OMe

4^′′-OMe

2^′′, 5^′′-OMe

2^′′, 3^′′-OMe

2^′′, 4^′′-OMe

2^′′, 5^′′-OMe

2^′′, 6^′′-OMe

2^′, 3^′, 4^′′-OMe

2^′′, 3^′′-OMe

2^′′, 3^′′, 4^′′-OMe

2-naphthyl

2^′′-Me

2^′, 3^′-OMe

2^′, 3^′, 4^′-OMe

2^′, 3^′-OMe

70.0 ± 0.3

35.4 ± 0.2

> 73.2

(1.3)

(>8.3)

ND

70.2 ± 0.1

28.7 ± 7.0

> 73.2

(1.3)

(>10)

ND

65.5 ± 2.8

28.2 ± 4.5

> 73.2

(1.4)

(>10)

ND

90.8 ± 3.9

> 293

31.6 ± 1.0

64.9 ± 0.1

64.1 ± 0.2

37.6 ± 15.0

71.5 ± 2.4

39.1 ± 0.1

15.5 ± 0.3

> 84.1

(2.9)

(1.5)

(>4.2)

(1.1)

(0.60)

(1.5)

(6.3)

ND

25.8 ± 4.7

63.4 ± 0.6

63.1 ± 0.1

40.5 ± 13.4

51.7 ± 4.4

40.0 ± 0.6

16.2 ± 0.4

> 84.1

(3.5)

(1.6)

(>4.3)

(0.98)

(0.83)

(1.4)

(6.0)

ND

32.2 ± 0.1

65.0 ± 0.2

64.5 ± 0.1

31.4 ± 0.8

71.7 ± 2.4

40.0 ± 0.5

15.8 ± 0.8

> 84.1

(2.8)

(1.5)

(>4.2)

(1.3)

(0.60)

(1.4)

(6.2)

ND

26.6 ± 5.6

59.8 ± 3.1

19.9 ± 8.7

60.4 ± 0.5

71.2 ± 1.7

36.0 ± 0.7

16.7 ± 1.3

> 84.1

(3.4)

(1.7)

(>14)

(0.66)

(0.60)

(1.6)

(5.8)

ND

91.6 ± 17.6

99.1 ± 13.7

> 269

(4.2)

(>5.3)

(2.4)

(0.93)

(19)

39.7 ± 5.1

42.7 ± 0.2

56.9 ± 13.3

97.6 ± 48.8

> 336

2^′′-Obenzyl

2^′′-OH

(13)

ND

2^′′-OEt

> 76.8

ND

> 76.8

ND

> 76.8

ND

4.4 ± 0.8

7.6 ± 2.3

3.9 ± 1.0

8.4 ± 1.6

9.1 ± 2.0

4.6 ± 0.9

38.2 ± 6.9

(>69)

(>39)

(21)

> 76.8

ND

> 307

2^′′-OnPr

20.3 ± 1.0

21.5 ± 2.4

49.6 ± 2.4

59.8 ± 18.9

20.4 ± 0.9

> 73.9

(>15)

(3.8)

(>6.2)

(0.80)

(>15)

ND

28.2 ± 7.4

19.6 ± 1.1

63.4 ± 11.0

46.2 ± 2.3

35.2 ± 0.9

> 73.9

(>10)

(4.2)

(>4.8)

(1.0)

(>8.8)

ND

27.1 ± 8.8

26.2 ± 7.9

56.4 ± 18.1

62.1 ± 20.5

23.3 ± 4.6

> 73.9

(>11)

(3.1)

(>5.4)

(0.77)

(>13)

ND

28.4 ± 6.9

25.8 ± 0.3

49.0 ± 9.8

105.3 ± 60.7

24.7 ± 0.7

> 73.9

(>10)

(3.2)

(>6.3)

(0.45)

(>12)

ND

> 295

2^′′-OMOM

2^′′-NO₂

82.2 ± 30.3

> 306

(>36)

(5.2)

(>66)

(>7.8)

2^′′-NH₂

47.6 ± 21.4

> 308

2^′′-NMe₂

2^′′-NHAc

> 296

a

b

c

Concentration that induces 50% inhibition of trypanosomatids. Concentration that induces 50% inhibition of normal Madin-Darby bovine kidney (MDBK) cells. SI: Selectivity index: IC₅₀in MDBK / IC₅₀in

trypanosomatids. ND: not determined.

K. Narita et al.

Bioorganic & Medicinal Chemistry 42 (2021) 116253

T. congolense, equal to that of 7. It was twice as active against T. evansi

(IC₅₀= 0.42 µM) as against T. congolense. This indicates that the posi-

tions of the phenyl rings in the oxazole scaffold affect the spectrum of

trypanocidal activity, and 2,4-diphenyloxazole skeleton may be suitable

for developing T. evansi inhibitors. However, the potencies of 1–9

showed a positive correlation with their cytotoxicity against MDBK cells,

and the compounds exhibited low SI values.

inactive. This result was unexpected since both 1 (2^′,3^′–OH, 2^′′–OH) and

12 (2^′,3^′-OMe, 2^′′-OMe) exhibited potent inhibitory activities. While the

reason for this is unclear, one possible explanation is that 1 and 12 may

engage with different targets, despite their structural similarity; the 2^′′-

demethylation of 12 may have led to the loss of trypanocidal activity

through both modes of action. Additionally, the steric size of the alkoxy

group at the 2^′′position in 12 also had a signiﬁcant impact on the ac-

tivity. The steric sizes were estimated to follow the order methoxy <

ethoxy < methoxy-methoxy (OMOM) ≈ O-n-propyl < O-benzyl, while

the trypanocidal activity against T. congolense was in the order 12 (2^′′-

OMe, IC₅₀= 0.78 µM) > 51 (2^′′-ethoxy, IC₅₀= 4.4 µM) > 53 (2^′′-OMOM,

IC₅₀= 5.1 µM) > 52 (2^′′-O-n-propyl, IC₅₀= 7.6 µM) ≈ 49 (2^′′-O-benzyl,

IC₅₀= 7.7 µM). Therefore, the substitution of a small alkoxy group is

preferable, suggesting that the 2^′′-methoxy group may act as a hydrogen

bond acceptor and occupy a small hydrophobic pocket, explaining the

restricted molecular size. Regarding the inﬂuence of the nitrogen-

containing functional groups, the 2^′′-nitro- (56) and 2^′′-amino-

substituted (57) analogs exhibited comparable anti-T. congolense activ-

ities, however, the cytotoxicity of 57 was found to be 6-fold higher than

that of 56. Compound 58, the dimethylated form of 57, exhibited a 2-

fold higher trypanocidal activity and a 12-fold higher SI than 57. On

the other hand, the cytotoxicity of 57 was reduced by acetamidation, but

the 2^′′-acetamide analog 59 also exhibited poor trypanocidal activity. As

with the alkoxy group, it appears that small hydrogen bond acceptors

are preferable at this position, and the electron density of the B ring does

not affect the trypanocidal activity.

Fortunately, this drawback could be overcome by methylating the

phenolic hydroxy groups. Compounds 12–15, the methoxy-substituted

synthetic precursors of 1–4, exhibited very low cytotoxicity against

MDBK cells. However, their trypanocidal activities against most strains

were also dramatically reduced, except for the anti-T. congolense activ-

ities of 12 and 15. The IC₅₀values of 12 and 15 against T. congolense

were 0.78 µM and 2.9 µM, respectively, which were 1.4- and 3-fold

lower than those of the corresponding OH derivatives, respectively (1:

IC₅₀= 1.1 µM, 4: IC₅₀= 8.6 µM). The SI values of 12 and 15 against

T. congolense were also very high (12: SI > 414, 15: SI > 100). Notably,

the IC₅₀and SI values of 12 were comparable to the hit anti-

trypanosomiasis activity criteria deﬁned by the World Health Organi-

zation: Tropical Disease Research.⁴⁶While the acetate derivatives 16–19

exhibited inhibitory proﬁles similar to those of the corresponding OH

derivatives, there were no improvements in their SI values. They might

have been converted to the corresponding phenols in culture conditions.

On the other hand, the O-methylated hetero aromatics 23, 26, 27, and

29 also exhibited reduced cytotoxicity but no improvements in the

trypanocidal activities against T. congolense, as seen with 12 and 15.

These results suggest that methylation of the phenolic hydroxy group in

2,5-diphenyloxazoles and related compounds reduces the cytotoxicity

against MDBK cells, and 2,5-diphenyloxazoles possessing methoxy

groups are preferable structure structures that selectively inhibit

T. congolense with low cellular toxicity.

As described above, 12 and its analogs selectively inhibited

T. congolense among the ﬁve species/subspecies evaluated, with low

cellular toxicity. The mode of action of these compounds, why they are

selective for T. congolense, and how T. congolense recognizes them, re-

mains unclear but transporters or target enzymes speciﬁc to

T. congolense may be responsible for this species-speciﬁc activity.

The unique inhibitory proﬁle of 12 prompted us to evaluate the

trypanocidal activities of 30–53 and 56–59; the results are summarized

in Table 2. Generally, the properties of all the methoxy-substituted 2,5-

diphenyloxazoles tested were similar to those of 12; they exhibited low

cytotoxicity and were selective toward T. congolense. While none of them

were superior to 12, this work revealed several aspects of the structur-

e–activity relationships (SAR) of the scaffold.

3. Conclusion

A total of 47 analogs of 2,5-diphenyloxazole (41 new compounds and

six known compounds) were synthesized, and the trypanocidal activities

of the synthesized compounds were evaluated against T. b. brucei, T. b.

gambiense, T. rhodesiense, T. congolense, and T. evansi. Although the

naturally occurring compound 1 exhibited high activity against all of

these species/subspecies, its high cytotoxicity presented a challenge.

The trypanocidal activities were improved by modifying the oxazole

core into 1,3,4-oxadiazole and 2,4-diphenyloxazole. The cytotoxicity

was reduced by replacing the hydroxy group with a methoxy group.

Compound 12 exhibited selective trypanocidal activity against

T. congolense with high potency and low cytotoxicity (IC₅₀= 0.78 µM, SI

= 414). The SAR of 12 was further evaluated, but none of the analogs

exhibited improved activity. However, the SAR studies of the 2,5-diphe-

nyloxazole scaffold revealed the following about maintaining selective

trypanocidal activity against T. congolense: i) the number and positions

of methoxy groups in phenyl rings are important, and 2^′,3^′-OMe and 2^′′-

OMe substituents are favorable for high potency and low cytotoxicity on

the mammalian cell line used; and ii) the molecular size of the substit-

uent at the 2^′′position affects its trypanocidal activity, and the small

methoxy group is most favorable. These ﬁndings may be useful in the

design and development of new trypanocidal drugs to treat AAT and the

design of biological tools to evaluate the species speciﬁcity of the mol-

ecules toward T. congolense. Further structural optimization of 2,5-

diphenyloxazole to enhance its trypanocidal activity and investigation

of its underlying mode of action are currently underway and will be

reported in due course.

Compounds 30–34 are dimethoxy-substituted analogs. Compound

30, which lacks the 3^′-methoxy group present in 12, exhibited reduced

potency compared to 12 but maintained the T. congolense-selective ac-

tivity and high SI value (IC₅₀= 2.8 µM, SI = 128). On the other hand,

compound 31, which lacks the 2^′-methoxy group of 12, exhibited a

higher IC₅₀(6.7 µM) and lower SI value (SI = 24). Compounds 32 (3^′-

OMe, 3^′′-OMe) and 33 (4^′-OMe, 4^′′-OMe) exhibited much higher IC₅₀

values (32: 21.4 µM, 33: 88.9 µM). Meanwhile, compound 34 (2^′-OMe,

3^′-OMe) exhibited comparable trypanocidal activity to 31 but was less

selective (IC₅₀= 4.0 µM, SI = 10). These results suggest that the

methoxy groups on the A and B rings neighboring the oxazole core (2^′

and 2^′positions) are essential for potent trypanocidal activity and high

selectivity. The introduction of additional methoxy groups on the phenyl

rings severely impacted the activity against T. congolense. Compounds

35–46, possessing various substitutional patterns of three to six methoxy

groups exhibited lower inhibitory activities than 12, although 39 (2^′-

OMe, 2^′′,5^′′-OMe), 41 (2^′,3^′-OMe, 2^′′,4^′′-OMe), and 42 (2^′,3^′-OMe, 2^′′,5^′′-

OMe) exhibited acceptable potencies and SI values. Therefore, the 4^′′

and/or 5^′′positions on the B ring appear to tolerate the introduction of

additional functional groups or carbon side chains.

The replacement of the 2^′′-OMe group on the B ring in 12, with a

methyl group, reduced its trypanocidal activity against T. congolense

(48: IC₅₀= 3.0 µM), resulting in an inhibitory proﬁle similar to that of

34 (2^′′-H). The replacement of the B ring with a 2-naphthyl group

signiﬁcantly reduced the trypanocidal activity of the scaffold (47: IC₅₀

= 45.8 µM). Therefore, the phenyl ring with an oxygen atom at the 2^′′

position, as seen in 12, was considered essential for its high potency and

selectivity. However, the 2^′′–OH derivative (50) was found to be

7

K. Narita et al.

Bioorganic & Medicinal Chemistry 42 (2021) 116253

4. Experimental

Puriﬁcation by recryatallization (hexane/CH₂Cl₂3:1) gave 14 (810 mg,

74%) as pale yellow needles. M.p. 133–134 ^◦C. ¹H NMR (400 MHz,

CDCl₃): δ = 3.86 (3H, s), 3.93 (3H, s), 6.84 (1H, dd, J = 8.8, 3.2 Hz), 6.91

(1H, d, J = 8.8 Hz), 7.42–7.51 (4H, m), 7.67 (1H, s), 8.10–8.13 ppm (2H,

m). ¹³C NMR (100 MHz, CDCl₃): δ = 55.87, 55.89, 111.5, 111.9, 113.7,

117.9, 126.3 (2C), 127.5, 128.0, 128.7 (2C), 130.2, 147.6, 150.2, 153.7,

160.0 ppm. FT-IR (KBr): 2955, 2840, 1586, 1571, 1506, 1449, 1343,

1290, 1277, 1240, 1205, 1188, 1133, 1042, 1017, 965, 866, 836, 800,

793, 774, 737, 708 cm^{ꢀ 1}. HRMS (EI): calcd for C₁₇H₁₅NO₃281.1052;

found 281.1051. Anal. calcd for C₁₈H₁₇NO₄: C 72.58, H 5.37, N 4.98;

found C 72.55, H 5.43, N 5.02.

4.1. Chemistry

All reactions involving air- and moisture-sensitive reagents were

carried out using oven-dried glassware and standard syringe–septum

cap techniques. Routine reaction monitoring was carried out using glass-

supported Merck silica gel 60 F₂₅₄TLC plates. Flash column chroma-

tography was performed on Kanto Chemical Silica Gel 60 N (spherical,

neutral 40–50 nm) with the solvents indicated. Commercially available

anhydrous solvent, tetrahydrofuran (THF), dichloromethane (CH₂Cl₂)

were used. Other all solvents and reagents were used as supplied. ¹H and

¹³C NMR spectroscopy was carried out on a JEOL AL-400 (400 MHz)

spectrometer. Chemical shifts are expressed in ppm using Me₄Si (δ = 0)

as an internal standard or residual solvent peak [¹H NMR, CDCl₃(7.26),

acetone‑d₆(2.05), DMSO‑d₆(2.50), CD₃OD (3.31): ¹³C NMR, CDCl₃

(77.2), acetone‑d₆(29.8, 206.3), DMSO‑d₆(39.5), CD₃OD (49.0)]. IR

spectral measurements were carried out on a JASCO FT/IR-4100 spec-

trophotometer. HRMS spectra were measured on a JEOL JMS-700 high-

resolution mass spectrometer.

4.1.4. 2-(2^′,5^′-Dimethoxyphenyl)-5-(2^′′,5^′′-dimethoxyphenyl)oxazole (15)

Compound 15 was synthesized from 2-bromo-2^′,5^′-methox-

yacetophenone (10b) (1.00 g, 3.9 mmol), 2,5-dimethoxybenzylamine

(11b) (0.65 mL, 4.3 mmol) in a manner similar to that described for

the synthesis of 12. Puriﬁcation by recrystallization (hexane/CH₂Cl₂

1:1) gave 15 (840 mg, 64%) as pale yellow granules. M.p. 127–129 ^◦C.

¹H NMR (400 MHz, CDCl₃): δ = 3.85 (3H, s), 3.86 (3H, s), 3.94 (3H, s),

3.97 (3H, s), 6.84 (1H, dd, J = 8.8, 2.9 Hz), 6.92 (1H, d, J = 8.8 Hz),

6.99–7.02 (2H, m), 7.44 (1H, d, J = 2.9 Hz), 7.60–7.61 (1H, m), 7.71

ppm (1H, s). ¹³C NMR (100 MHz, CDCl₃): δ = 55.8, 55.9, 56.0, 56.9,

111.5, 112.0, 113.8, 113.9, 114.4, 117.2, 117.4, 118.0, 127.8, 147.2,

150.2, 152.0, 153.5, 153.7, 158.3 ppm. FT-IR (KBr): 3408, 3005, 2962,

2835, 1609, 1569, 1509, 1459, 1318, 1278, 1219, 1182, 1118, 1051,

1019, 974, 852, 807, 743 cm^{ꢀ 1}. HRMS (EI): calcd for C₁₉H₁₉NO₅

341.1263; found 341.1272. Anal. calcd for C₁₈H₁₇NO₄: C 66.85, H 5.61,

N 4.10; found C 66.73, H 5.66, N 4.11.

4.1.1. 2-(2^′,3^′-Dimethoxyphenyl)-5-(2^′′-methoxyphenyl)oxazole (12)

Iodine (4.87 g, 19 mmol) was added to a stirred solution of 2-bromo-

2^′-methoxyacetophenone (10a) (2.00 g, 8.7 mmol), 2,3-dimethoxyben-

zylamine (11a) (1.6 mL, 11 mmol) and K₂CO₃(4.83 g, 35 mmol) in DMF

(87 mL). The mixture was stirred at 80 ^◦C for 18 h. After cooling to room

temperature, the reaction was quenched with 10% aqueous Na₂S₂O₃

(30 mL), and the resulting mixture was extracted with Et₂O (3 × 60 mL).

The combined extracts were washed with brine (2 × 60 mL), then dried

with Na₂SO₄. The mixture was concentrated in vacuo, and the residue

was puriﬁed by recrystallization (hexane/ CHCl₃3:1) to give 12 (1.65 g,

60%) as off-white powders. Spectroscopic data for 12 were consistent

with those previously reported for this compound.¹⁹M.p. 153–154 ^◦C.

¹H NMR (400 MHz, CDCl₃): δ = 3.92 (3H, s), 3.99 (3H, s), 4.01 (3H, s),

6.99–7.03 (2H, m), 7.07 (1H, t, J = 7.6 Hz), 7.16 (1H, t, J = 8.0 Hz),

7.28–7.33 (1H, m), 7.65 (1H, dd, J = 8.0, 1.5 Hz), 7.69 (1H, s), 7.90 ppm

(1H, dd, J = 8.0, 1.5 Hz). ¹³C NMR (100 MHz, CDCl₃): δ = 55.4, 56.1,

61.2, 110.8, 114.1, 117.4, 120.9, 121.4, 122.2, 124.2, 125.9, 127.5,

128.9, 147.7, 147.8, 153.8, 155.7, 158.3 ppm. FT-IR (KBr): 2940, 1566,

1529, 1493, 1474, 1421, 1344, 1303, 1261, 1185, 1165, 1128, 1082,

1043, 1021, 1002, 942, 837, 790, 750, 726 cm^{ꢀ 1}. HRMS (EI): calcd for

4.1.5. 2-(2^′,3^′-Diacetoxyphenyl)-5-(2^′′-acetoxyphenyl)oxazole (16)

Boron tribromide (1 M in CH₂Cl₂; 16.1 mL, 16 mmol) was added to a

stirred solution of 12 (1.00 g, 3.2 mmol) in CH₂Cl₂(16 mL) at 0 ^◦C under

argon. After 24 h, the reaction was quenched with saturated aqueous

NaHCO₃(30 mL), and the resulting mixture was extracted with EtOAc

(3 × 60 mL). The combined extracts were washed with brine (2 × 60

mL), then dried with Na₂SO₄. Concentration of the solvent in vacuo

afforded 2-(2^′,3^′-Dihydroxyphenyl)-5-(2^′′-hydroxyphenyl)oxazole (1)

(865 mg), which was used for the next reaction without further

puriﬁcation.

Acetic anhydride (1.5 mL, 16 mmol) was added was added to a

stirred solution of crude product 1 (862 mg) in pyridine (32 mL) con-

taining 4-dimethylaminopyridine (DMAP) (39.2 mg, 0.32 mmol) at

room temperature under argon. After 5 h, the reaction mixture was

diluted with Et₂O (200 mL). The organic layer was washed successively

with 3 M HCl (2 × 60 mL), saturated aqueous NaHCO₃(2 × 60 mL) and

brine (2 × 60 mL), then dried with Na₂SO₄. The mixture was concen-

trated in vacuo, and the residue was puriﬁed by column chromatog-

raphy (hexane/EtOAc 2:1) to give 16 (611 mg, 48% for 2 steps) as a pale

pink solid. Recrystallization from hexane/CHCl₃(3:1) gave an analytical

sample of 16 as pale pink needles. M.p. 130–131 ^◦C. ¹H NMR (400 MHz,

CDCl₃): δ = 2.32 (3H, s), 2.39 (3H, s), 2.41 (3H, s), 7.21 (1H, dd, J = 7.8,

1.5 Hz), 7.30–7.40 (4H, m), 7.46 (1H, s), 7.79 (1H, dd, J = 7.6, 1.7 Hz),

8.00 ppm (1H, dd, J = 7.8, 1.5 Hz). ¹³C NMR (100 MHz, CDCl₃): δ =

20.6, 20.8, 21.2, 120.7, 122.0, 123.4, 125.3, 126.2, 126.3, 126.6, 126.8,

126.9, 129.4, 140.2, 143.8, 146.7, 147.2, 157.3, 168.1, 168.5, 168.6

ppm. FT-IR (KBr): 3009, 2927, 1763, 1569, 1530, 1490, 1468, 1444,

1374, 1250, 1212, 1185, 1156, 1137, 1117, 1055, 1014, 949, 907, 858,

827, 796, 754, 727 cm^{ꢀ 1}. HRMS (EI): calcd for C₂₁H₁₇NO₇395.1005;

found 395.1007. Anal. calcd for C₂₁H₁₇NO₇: C 63.80, H 4.33, N 3.54;

found C 63.69, H 4.37, N 3.57.

C

₁₈H₁₇NO₄311.1158; found 311.1153. Anal. calcd for C₁₈H₁₇NO₄: C

69.44, H 5.50, N 4.50; found C 69.24, H 5.55, N 4.52.

4.1.2. 2-(2^′,5^′-Dimethoxyphenyl)-5-(2^′′-methoxyphenyl)oxazole (13)

Compound 13 was synthesized from 2-bromo-2^′-methox-

yacetophenone (10a) (1.00 g, 4.4 mmol), 2,5-dimethoxybenzylamine

(11b) (0.78 mL, 5.2 mmol) in a manner similar to that described for

the synthesis of 12. Puriﬁcation by column chromatography (hexane/

EtOAc 3:1) gave 13 (1.01 g, 74%) as a pale yellow solid. Recrystalliza-

tion from hexane/CH₂Cl₂(2:1) gave an analytical sample of 13 as pale

yellow needles. M.p. 85–87 ^◦C. ¹H NMR (400 MHz, CDCl₃): δ = 3.85

(3H, s), 3.96 (3H, s), 3.97 (3H, s), 6.97–6.99 (3H, m), 7.06 (1H, t, J = 8.0

Hz), 7.29 (1H, td, J = 8.0, 1.5 Hz), 7.60–7.61 (1H, m), 7.69 (1H, s), 7.88

ppm (1H, dd, J = 8.0, 1.5 Hz). ¹³C NMR (100 MHz, CDCl₃): δ = 55.4,

55.8, 56.8, 110.8, 113.9, 114.4, 117.2, 117.27, 117.32, 120.8, 125.9,

127.5, 128.8, 147.3, 152.0, 153.4, 155.7, 158.2 ppm. FT-IR (KBr): 3398,

3005, 2934, 2834, 1568, 1536, 1509, 1459, 1309, 1281, 1258, 1209,

1182, 1161, 1129, 1117, 1054, 1020, 872, 831, 802, 744 cm^{ꢀ 1}. HRMS

(EI): calcd for C₁₈H₁₇NO₄311.1158; found 311.1145. Anal. calcd for

C

₁₈H₁₇NO₄: C 69.44, H 5.50, N 4.50; found C 69.37, H 5.54, N 4.49.

4.1.6. 2-(2^′,5^′-Diacetoxyphenyl)-5-(2^′′-acetoxyphenyl)oxazole (17)

Compound 17 was synthesized from 13 (200 mg, 0.64 mmol) in a

manner similar to that described for the synthesis of 16. Puriﬁcation by

column chromatography (hexane/EtOAc 2:1) gave 17 (154 mg, 76% for

2 steps) as a white solid. Recrystallization from hexane/CH₂Cl₂(3:1)

4.1.3. 2-Phenyl-5-(2^′′,5^′′-dimethoxyphenyl)oxazole (14)

Compound 14 was synthesized from 2-bromo-2^′,5^′-methox-

yacetophenone (10b) (1.00 g, 3.9 mmol), benzylamine (11c) (0.51 mL,

4.6 mmol) in a manner similar to that described for the synthesis of 12.

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Bioorganic & Medicinal Chemistry 42 (2021) 116253

gave an analytical sample of 17 as colorless powders. M.p. 144–145 ^◦C.

¹H NMR (400 MHz, CDCl₃): δ = 2.33 (3H, s), 2.40 (3H, s), 2.41 (3H, s),

7.17–7.26 (3H, m), 7.31–7.40 (2H, m), 7.46 (1H, s), 7.81 (1H, dd, J =

7.3, 2.0 Hz), 7.85 ppm (1H, d, J = 2.9 Hz). ¹³C NMR (100 MHz, CDCl₃):

δ = 21.0, 21.2, 21.3, 120.6, 121.2, 121.9, 123.4, 124.5, 125.0, 126.4,

126.8, 126.9, 129.4, 145.6, 146.7, 147.2, 148.3, 157.0, 168.6, 169.0,

169.7 ppm. FT-IR (KBr): 3422, 2925, 2851, 1763, 1655, 1560, 1543,

1498, 1372, 1220, 1175, 1125, 1051, 1015, 950, 928, 912, 890, 819,

756, 741 cm^{ꢀ 1}. HRMS (EI): calcd for C₂₁H₁₇NO₇395.1005; found

395.1011. Anal. calcd for C₂₁H₁₇NO₇: C 63.80, H 4.33, N 3.54; found C

63.60, H 4.30, N 3.56.

(EI): calcd for C₁₅H₁₁NO₄269.0688; found 269.0689. Anal. calcd for

C

₁₅H₁₁NO₄⋅H₂O: C 62.72, H 4.62, N 4.88; found C 62.60, H 4.62, N 4.88.

4.1.10. 2-(2^′,5^′-Dihydroxyphenyl)-5-(2^′′-hydroxyphenyl)oxazole (2)

Compound 2 was synthesized from 17 (51.7 mg, 0.13 mmol) in a

manner similar to that described for the synthesis of 1. Puriﬁcation by

recrystallization (hexane/EtOAc 4:1) gave 2 (25.2 mg, 72%) as colorless

powders. Spectroscopic data for 2 were consistent with those previously

reported for this compound.¹²M.p. 269–271 ^◦C. ¹H NMR (400 MHz,

DMSO‑d₆): δ = 6.85–6.90 (2H, m), 6.97 (1H, t, J = 7.6 Hz), 7.01 (1H, d,

J = 8.3 Hz), 7.24 (1H, t, J = 7.8 Hz), 7.35 (1H, d, J = 2.4 Hz), 7.72 (1H,

s), 7.78 (1H, d, J = 7.8 Hz), 9.22 (1H, s), 10.46 (1H, s), 10.57 ppm (1H,

s). ¹³C NMR (100 MHz, DMSO‑d₆): δ = 110.5, 110.9, 114.2, 115.9,

117.6, 119.5, 120.2, 124.5, 125.4, 129.7, 147.0, 149.4, 150.1, 154.1,

158.9 ppm. FT-IR (KBr): 3376, 1655, 1638, 1604, 1572, 1560, 1543,

1508, 1491, 1459, 1448, 1338, 1201, 1159, 1121, 945, 889, 869, 812,

786, 762 cm^{ꢀ 1}. HRMS (EI): calcd for C₁₅H₁₁NO₄269.0688; found

269.0700. Anal. calcd for C₁₅H₁₁NO₄: C 66.91, H 4.12, N 5.20; found C

66.72, H 4.12, N 5.18.

4.1.7. 2-Phenyl-5-(2^′′,5^′′-diacetoxyphenyl)oxazole (18)

Compound 18 was synthesized from 14 (200 mg, 0.71 mmol) in a

manner similar to that described for the synthesis of 16. Puriﬁcation by

column chromatography (hexane/EtOAc 3:1) gave 18 (154 mg, 76% for

2 steps) as a pale yellow solid. Recrystallization from hexane/CH₂Cl₂

(5:1) gave an analytical sample of 18 as pale yellow needles. M.p.

126–127 ^◦C. ¹H NMR (400 MHz, CDCl₃): δ = 2.34 (3H, s), 2.41 (3H, s),

7.10 (1H, dd, J = 8.8, 2.4 Hz), 7.23 (1H, d, J = 8.8 Hz), 7.46–7.48 (3H,

m), 7.49 (1H, s), 7.60 (1H, d, J = 2.4 Hz), 8.08–8.10 ppm (2H, m). ¹³

C

4.1.11. 2-Phenyl-5-(2^′′,5^′′-dihydroxyphenyl)oxazole (3)

NMR (100 MHz, CDCl₃): δ = 21.0, 21.3, 119.4, 121.95, 121.98, 124.3,

126.4 (2C), 126.9, 127.3, 128.8 (2C), 130.7, 143.8, 146.2, 148.4, 161.2,

168.5, 169.1 ppm. FT-IR (KBr): 2922, 1762, 1618, 1542, 1498, 1448,

1376, 1342, 1201, 1170, 1142, 1054, 1016, 923, 887, 839, 778, 712

cm^{ꢀ 1}. HRMS (EI): calcd for C₁₉H₁₅NO₅337.0950; found 337.0958.

Anal. calcd for C₁₉H₁₅NO₅: C 67.65, H 4.48, N 4.15; found C 67.55, H

4.50, N 4.16.

Compound 3 was synthesized from 18 (217 mg, 0.64 mmol) in a

manner similar to that described for the synthesis of 1. Puriﬁcation by

recrystallization (acetone) gave 3 (120 mg, 83%) as orange needles.

Spectroscopic data for 3 were consisten₁t with those previously reported

for this compound.¹²M.p. 292–294 ^◦C. H NMR (400 MHz, DMSO‑d₆): δ

= 6.63 (1H, dd, J = 8.8, 2.9 Hz), 6.80 (1H, d, J = 8.8 Hz), 7.20 (1H, d, J

= 2.9 Hz), 7.50–7.58 (3H, m), 7.61 (1H, s), 8.04–8.06 (2H, m), 8.98 (1H,

s), 9.76 ppm (1H, s). ¹³C NMR (100 MHz, DMSO‑d₆): δ = 111.0, 114.5,

116.3, 116.7, 125.7 (2C), 126.7, 127.0, 129.2 (2C), 130.4, 146.8, 148.3,

150.0, 158.7 ppm. FT-IR (KBr): 3245, 3033, 2745, 1520, 1491, 1480,

1446, 1396, 1357, 1282, 1241, 1198, 1135, 1122, 923, 866, 818, 774,

708 cm^{ꢀ 1}. HRMS (EI): calcd for C₁₅H₁₁NO₃253.0739; found 253.0745.

Anal. calcd for C₁₅H₁₁NO₃: C 71.14, H 4.38, N 5.63; found C 70.85, H

4.49, N 5.49.

4.1.8. 2-(2^′,5^′-Diacetoxyphenyl)-5-(2^′′,5^′′-diacetoxyphenyl)oxazole (19)

Compound 19 was synthesized from 15 (628 mg, 1.8 mmol) in a

manner similar to that described for the synthesis of 16 (In the deme-

thylation step, 15 equivalent of Boron Tribromide (27.6 mL, 28 mmol)

was used. In the acetylation step, 6 equivalent of acetic anhydride (1.0

mL, 111 mmol) was used). Puriﬁcation by column chromatography

(hexane/CHCl₃2:1) gave 17 (154 mg, 76% for 2 steps) as a white solid.

Recrystallization from hexane/ CHCl₃(3:1) gave an analytical sample of

19 as colorless granules. M.p. 193–195 ^◦C. ¹H NMR (400 MHz, CDCl₃): δ

= 2.33 (3H, s), 2.34 (3H, s), 2.40 (3H, s), 2.41 (3H, s), 7.11 (1H, dd, J =

9.0, 2.9 Hz), 7.19 (1H, d, J = 9.0 Hz), 7.23–7.26 (3H, m), 7.49 (1H, s),

7.52 (1H, d, J = 2.9 Hz), 7.84 ppm (1H, d, J = 2.9 Hz). ¹³C NMR (100

MHz, CDCl₃): δ = 21.0 (2C), 21.1, 21.2, 119.6, 121.0, 121.5, 122.0,

122.4, 124.3, 124.7, 125.0, 127.50, 144.0, 145.6, 146.3, 148.30,

148.32, 157.2, 168.4, 169.0, 169.1, 169.7 ppm. FT-IR (KBr): 3504,

3083, 3019, 2940, 1766, 1572, 1500, 1429, 1369, 1208, 1172, 1052,

1015, 924, 891, 837, 783, 748 cm^{ꢀ 1}. HRMS (EI): calcd for C₂₃H₁₉NO₉

453.1060; found 453.1059. Anal. calcd for C₂₃H₁₉NO₉: C 60.93, H 4.22,

N 3.09; found C 60.93, H 4.24, N 3.12.

4.1.12. 2-(2^′,5^′-Dihydroxyphenyl)-5-(2^′′,5^′′-dihydroxyphenyl)oxazole (4)

Compound 4 was synthesized from 19 (59.1 mg, 0.13 mmol) in a

manner similar to that described for the synthesis of 1. Puriﬁcation by

recrystallization (hexane/EtOAc 3:1) gave 4 (23.1 mg, 62%) as pale pink

powders. Spectroscopic data for 4 were consistent with those previously

reported for this compound.¹²M.p. 276–278 ^◦C. ¹H NMR (400 MHz,

DMSO- d₆): δ = 6.67 (1H, dd, J = 8.8, 2.4 Hz), 6.82 (1H, d, J = 8.8 Hz),

6.84–6.90 (2H, m), 7.17 (1H, d, J = 2.4 Hz), 7.30 (1H, d, J = 2.4 Hz),

7.68 (1H, s), 9.02 (1H, s), 9.24 (1H, s), 9.85 (1H, s), 10.44 ppm (1H, s).

¹³C NMR (100 MHz, DMSO‑d₆): δ = 110.5, 110.8, 110.9, 114.2, 116.7,

116.8, 117.7, 120.2, 124.5, 146.9, 147.1, 149.4, 150.05, 150.10, 158.7

ppm. FT-IR (KBr): 3335, 1637, 1577, 1543, 1509, 1448, 1407, 1363,

1235, 1209, 1119, 1041, 972, 875, 827, 787, 754 cm^{ꢀ 1}. HRMS (EI):

calcd for C₁₅H₁₁NO₅285.0637; found 285.0650. Anal. calcd for

4.1.9. 2-(2^′,3^′-Dihydroxyphenyl)-5-(2^′′-hydroxyphenyl)oxazole (1)

3 M aqueous HCl (10 mL) was added to a stirred solution of 16 (488

mg, 1.2 mmol) in THF/MeOH (2:1, 30 mL) at room temperature. After 3

days, the reaction mixture was diluted with water (30 mL), and the

resulting mixture was extracted with EtOAc (3 × 60 mL). The combined

extracts were washed with brine (2 × 60 mL), then dried with Na₂SO₄.

The mixture was concentrated in vacuo, and the residue was puriﬁed by

recrystallization (hexane/EtOAc 3:1) to give 1 (206 mg, 62%) as

colorless powders. Spectroscopic data for 1 were consistent with those

previously reported for this compound.^12,19M.p. 196–197 ^◦C. ¹H NMR

(400 MHz, acetone‑d₆): δ = 6.90 (1H, t, J = 8.0 Hz), 6.99–7.04 (2H, m),

7.07 (1H, dd, J = 7.8, 1.0 Hz), 7.22–7.26 (1H, m), 7.52 (1H, dd, J = 8.0,

1.7 Hz), 7.78 (1H, s), 7.89 ppm (1H, dd, J = 7.8, 1.5 Hz). ¹³C NMR (100

MHz, acetone- d₆): δ = 111.8, 115.5, 116.7, 117.1, 118.5, 120.5, 120.8,

125.3, 126.5, 130.3, 146.3, 146.7, 148.1, 154.7, 160.6 ppm. FT-IR

(KBr): 3588, 3567, 3531, 3483, 3365, 3253, 1499, 1484, 1456, 1427,

1388, 1375, 1281, 1243, 1225, 1152, 1116, 778, 751, 726 cm^{ꢀ 1}. HRMS

C₁₅H₁₁NO₅: C 63.16, H 3.89, N 4.91; found C 63.02, H 3.85, N 4.85.

4.1.13. 2,3-Dimethoxy-N-(2-(2-methoxyphenyl)-2-oxoethyl)benzamide

(22)

Et₃N (3.9 mL, 28 mmol) was added dropwise to a stirred solution of

aminoketone 20 (1.88 g, 9.3 mmol) and carboxcylic acid 21 (1.70 g, 9.3

mmol) in CH₂Cl₂(45 mL) containing EDC⋅HCl (2.32 g, 12 mmol) and

HOBt (1.64 g, 12 mmol) at room temperature under argon. After 12 h,

the reaction was quenched with 3 M aqueous HCl (30 mL), and the

resulting mixture was extracted with EtOAc (3 × 100 mL). The com-

bined extracts were washed successively with saturated aqueous

NaHCO₃(2 × 100 mL) and brine (2 × 100 mL), then dried with Na₂SO₄.

The mixture was concentrated in vacuo, and the residue was puriﬁed by

column chromatography (hexane/EtOAc 1:2) to give 22 (1.98 g, 65%)

as a white solid. Recrystallization from hexane/CH₂Cl₂(3:1) gave an

analytical sample of 22 as colorless powders. M.p. 93–94 ^◦C. ¹H NMR

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K. Narita et al.

Bioorganic & Medicinal Chemistry 42 (2021) 116253

(400 MHz, CDCl₃): δ = 3.92 (3H, s), 3.99 (3H, s), 4.06 (3H, s), 4.96 (2H,

d, J = 4.4 Hz), 7.01–7.08 (3H, m), 7.15 (1H, t, J = 8.0 Hz), 7.52–7.56

(1H, m), 7.75 (1H, dd, J = 8.0, 1.7 Hz), 7.98 (1H, dd, J = 7.6, 1.7 Hz),

9.19 ppm (1H, br s). ¹³C NMR (100 MHz, CDCl₃): δ = 51.7, 55.6, 56.1,

61.5, 111.7, 115.6, 120.8, 122.8, 124.1, 124.7, 126.3, 131.0, 134.8,

148.1, 152.7, 159.7, 165.0, 195.1 ppm. FT-IR (KBr): 3369, 2939, 2837,

1650, 1597, 1577, 1518, 1464, 1437, 1356, 1285, 1264, 1207, 1163,

1059, 1020, 993, 808, 755, 641 cm^{ꢀ 1}. HRMS (EI): calcd for C₁₈H₁₉O₅

329.1263; found 329.1264. Anal. calcd for C₁₈H₁₉O₅: C 65.64, H 5.82, N

4.25; found C 65.61, H 5.83, N 4.25.

J = 2.9 Hz), 10.38 ppm (1H, d, J = 2.9 Hz). ¹³C NMR (100 MHz,

DMSO‑d₆): δ = 55.96, 55.98, 61.3, 112.1, 115.4, 120.6, 120.8, 121.0,

124.2, 127.7, 130.6, 133.0, 146.7, 152.6, 157,1, 163.4, 163.8 ppm. FT-

IR (KBr): 3855, 3842, 3736, 3691, 3650, 3353 2956 1617, 1571, 1448,

1268, 1184, 1109, 1060, 1015, 978, 962, 877, 817, 760 cm^{ꢀ 1}. HRMS

(EI): calcd for C₁₇H₁₈N₂O₅330.1216; found 330.1219. Anal. calcd for

C₁₇H₁₈N₂O₅: C 61.81, H 5.49, N 8.48; found C 61.84, H 5.47, N 8.48.

4.1.17. 2-(2^′,5^′-Dimethoxyphenyl)-5-(2^′′-methoxyphenyl)-1,3,4-

oxadiazole (26)

A solution of 25 (1.12 g, 3.4 mmol) in MeCN (14 mL) containing

phosphoryl chloride (1.4 mL, 15 mmol) was heated at reﬂux for 24 h.

After cooling to room temperature, the reaction mixture was diluted

with CH₂Cl₂(100 mL). The organic layer was washed successively with

saturated aqueous NaHCO₃(2 × 30 mL) and brine (2 × 30 mL), then

dried with MgSO₄. The mixture was concentrated in vacuo, and the

residue was puriﬁed by recrystallization (hexane/CHCl₃2:1) to give 26

(805 mg, 85%) as pale red powders. M.p. 117–118 ^◦C. ¹H NMR (400

MHz, CDCl₃): δ = 3.93 (3H, s), 3.99 (3H, s), 4.01 (3H, s), 7.07–7.12 (3H,

m), 7.19 (1H, t, J = 7.8 Hz), 7.49–7.54 (1H, m), 7.64 (1H, dd, J = 7.8,

1.0 Hz), 8.04 ppm (1H, dd, J = 7.8, 1.0 Hz). ¹³C NMR (100 MHz, CDCl₃):

δ = 55.9, 56.0, 61.4, 111.9, 113.2, 115.3, 119.0, 120.7, 121.6, 124.5,

130.5, 132.9, 148.0, 153.7, 157.9, 162.9, 163.6 ppm. FT-IR (KBr): 2940,

2843, 1604, 1580, 1534, 1497, 1456, 1366, 1283, 1265, 1234, 1170,

1132, 1086, 1042, 1019, 998, 844, 789, 775, 750, 722 cm^{ꢀ 1}. HRMS (EI):

calcd for C₁₇H₁₆N₂O₄312.1110; found 312.1102. Anal. calcd for

4.1.14. 2-(2^′,5^′-Dimethoxyphenyl)-5-(2^′′-methoxyphenyl)thiazole (23)

A solution of 22 (100 mg, 0.30 mmol) in THF (6 mL) containing

Lawesson’s reagent (243 mg, 0.60 mmol) was heated at reﬂux for 3 h.

After cooling to room temperature, concentration of the solvent in vacuo

afforded a residue, which was puriﬁed by column chromatography

(hexane/EtOAc 1:2) to give 23 (83.4 mg, 85%) as a pale yellow solid.

Recrystallization from hexane/CH₂Cl₂(1:1) gave an analytical sample of

23 as pale yellow needles. M.p. 83–84 ^◦C. ¹H NMR (400 MHz, CDCl₃): δ

= 3.92 (3H, s), 3.95 (3H, s), 3.99 (3H, s), 6.96 (1H, dd, J = 7.8, 1.5 Hz),

7.00–7.05 (2H, m), 7.16 (1H, t, J = 7.8 Hz), 7.31 (1H, td, J = 7.8, 1.5

Hz), 7.68 (1H, dd, J = 7.8, 1.5 Hz), 7.96 (1H, dd, J = 7.8, 1.5 Hz), 8.27

ppm (1H, s). ¹³C NMR (100 MHz, CDCl₃): δ = 55.5, 55.9, 60.1, 111.5,

113.0, 119.4, 120.9, 121.0, 124.2, 127.6, 128.7, 129.0, 135.5, 140.1,

146.5, 153.0, 155.7, 161.5 ppm. FT-IR (KBr): 2937, 2835, 1596, 1578,

1514, 1487, 1470, 1432, 1317, 1265, 1249, 1181, 1116, 1094, 1078,

1053, 1010, 994, 844, 788, 747 cm^{ꢀ 1}. HRMS (EI): calcd for C₁₈H₁₇NO₃S

327.0929; found 327.0932. Anal. calcd for C₁₈H₁₇NO₃S: C 66.03, H

5.23, N 4.28; found C 65.91, H 5.23, N 4.27.

C₁₇H₁₆N₂O₄: C 65.38, H 5.16, N 8.97; found C 65.10, H 5.24, N 8.93.

4.1.18. 2-(2^′,5^′-Dihydroxyphenyl)-5-(2^′′-hydroxyphenyl)-1,3,4-

oxadiazole (7)

4.1.15. 2-(2^′,5^′-Dihydroxyphenyl)-5-(2^′′-hydroxyphenyl)thiazole (6)

Boron tribromide (1 M in CH₂Cl₂; 1.6 mL, 1.6 mmol) was added to a

stirred solution of 22 (100 mg, 0.31 mmol) in CH₂Cl₂(3 mL) at 0 ^◦C

under argon. After 20 h, the reaction was quenched with saturated

aqueous NaHCO₃(3 mL), and the resulting mixture was extracted with

EtOAc (3 × 6 mL). The combined extracts were washed with brine (2 ×

6 mL), then dried with Na₂SO₄. The mixture was concentrated in vacuo,

and the residue was puriﬁed by column chromatography (hexane/

EtOAc 1:2) to give 6 (68.6 mg, 79%) as a yellow solid. Recrystallization

from hexane/CH₂Cl₂(3:1) gave an analytical sample of 6 as yellow

powders. M.p. 180–181 ^◦C. ¹H NMR (400 MHz, acetone‑d₆): δ = 6.78

(1H, t, J = 8.0 Hz), 6.88 (1H, dd, J = 7.8, 1.5 Hz), 6.91–6.95 (1H, m),

7.04 (1H, d, J = 8.3 Hz), 7.19 (1H, td, J = 3.9, 1.5 Hz), 7.21 (1H, td, J =

3.9, 1.5 Hz), 7.71 (1H, dd, J = 7.8, 1.5 Hz), 8.34 ppm (1H, s). ¹³C NMR

(100 MHz, acetone‑d₆): δ = 117.2, 117.5, 117.7, 118.2, 118.7, 120.4,

121.2, 129.0, 130.5, 134.3, 139.0, 146.1, 147.2, 154.4, 168.9 ppm. FT-

IR (KBr): 3855, 3751, 3736, 3713, 3691, 3677, 3650, 3630, 3407, 2922,

1603, 1489, 1470, 1352, 1269, 1195, 1162, 1101, 1060, 814, 774, 748,

721 cm^{ꢀ 1}. HRMS (EI): calcd for C₁₅H₁₁NO₃S 285.0460; found 285.0461.

Anal. calcd for C₁₅H₁₁NO₃S: C 63.14, H 3.89, N 4.91; found C 63.14, H

3.89, N 4.91.

Boron tribromide (1 M in CH₂Cl₂; 3.2 mL, 3.2 mmol) was added to a

stirred solution of 26 (200 mg, 0.64 mmol) in CH₂Cl₂(6 mL) at 0 ^◦C

under argon. After 20 h, the reaction was quenched with saturated

aqueous NaHCO₃(10 mL), and the resulting mixture was extracted with

EtOAc (3 × 20 mL). The combined extracts were washed with brine (2 ×

20 mL), then dried with Na₂SO₄. The mixture was concentrated in

vacuo, and the residue was puriﬁed by recrystallization (hexane/EtOAc

1:2) to give 7 (112 mg, 65%) as off-white powders. M.p. 201–202 ^◦C. ¹H

NMR (400 MHz, DMSO‑d₆): δ = 6.87 (1H, td, J = 8.0, 1.0 Hz), 7.02–7.06

(2H, m), 7.10 (1H, d, J = 8.0 Hz), 7.32 (1H, d, J = 8.0 Hz), 7.47 (1H, td,

J = 8.0, 1.0 Hz), 7.87 (1H, d, J = 8.0 Hz), 9.91 ppm (3H, br s). ¹³C NMR

(100 MHz, DMSO‑d₆): δ = 109.4, 109.6, 117.1, 118.2, 118.8, 119.8,

119.9, 128.8, 133.5, 145.4, 146.3, 156.4, 162.6, 163.1 ppm. FT-IR

(KBr): 3650, 3498, 3185, 1624, 1586, 1542, 1487, 1407, 1231, 1147,

1066, 1043, 1028, 991, 967, 882, 823, 793, 751, 735, 701 cm^{ꢀ 1}. HRMS

(EI): calcd for C₁₄H₁₀N₂O₄270.0641; found 270.0634. Anal. calcd for

C₁₄H₁₀N₂O₄: C 62.22, H 3.73, N 10.37; found C 61.96, H 3.77, N 10.28.

4.1.19. 2-(2^′,5^′-Dimethoxyphenyl)-5-(2^′′-methoxyphenyl)-1,3,4-

thiadiazole (27)

A solution of 25 (400 mg, 1.2 mmol) in toluene (12 mL) containing

Lawesson’s reagent (785 mg, 1.9 mmol) was heated at reﬂux for 2 days.

After cooling to room temperature, concentration of the solvent in vacuo

afforded a residue, which was puriﬁed by column chromatography

(hexane/EtOAc 2:1) to give 27 (215 mg, 54%) as a white solid.

Recrystallization from hexane/CHCl₃(2:1) gave an analytical sample of

27 as colorless needles. M.p. 150–151 ^◦C. ¹H NMR (400 MHz, CDCl₃): δ

= 3.94 (3H, s), 3.98 (3H, s), 4.04 (3H, s), 7.03 (1H, dd, J = 8.0, 1.2 Hz),

7.07 (1H, d, J = 8.0 Hz), 7.14 (1H, t, J = 8.0 Hz), 7.20 (1H, t, J = 8.0 Hz),

7.45–7.49 (1H, m), 8.10 (1H, dd, J = 8.0, 1.2 Hz), 8.56 ppm (1H, dd, J =

7.8, 2.0 Hz). ¹³C NMR (100 MHz, CDCl₃): δ = 55.7, 56.0, 60.4, 111.3,

114.0, 119.7, 120.0, 121.3, 124.5, 124.9, 128.7, 131.7, 146.4, 152.9,

156.0, 163.0, 163.7 ppm. FT-IR (KBr): 3073, 3005, 2945, 2836, 1921,

1597, 1581, 1471, 1409, 1267, 1160, 1101, 1053, 1013, 975, 885, 844,

786, 761 cm^{ꢀ 1}. HRMS (EI): calcd for C₁₇H₁₆N₂O₃S 328.0882; found

328.0888. Anal. calcd for C₁₇H₁₆N₂O₃S: C 62.18, H 4.91, N 8.53; found

4.1.16. 2,3-Dimethoxy-N^′-(2-methoxybenzoyl)benzohydrazide (25)

Et₃N (3.8 mL, 27 mmol) was added dropwise to a stirred solution of

hydrazide 24 (1.50 g, 9.0 mmol) and carboxcylic acid 21 (1.64 g, 9.0

mmol) in CH₂Cl₂(45 mL) containing EDC⋅HCl (2.25 g, 12 mmol) and

HOBt (1.59 g, 12 mmol) at room temperature under argon. After 12 h,

the reaction was quenched with 3 M aqueous HCl (30 mL), and the

resulting mixture was extracted with EtOAc (3 × 100 mL). The com-

bined extracts were washed successively with saturated aqueous

NaHCO₃(2 × 100 mL) and brine (2 × 100 mL), then dried with Na₂SO₄.

The mixture was concentrated in vacuo, and the residue was puriﬁed by

recrystallization (hexane/EtOAc 1:1) to give 25 (2.47 g, 83%) as

colorless needles. M.p. 149 ^◦C. ¹H NMR (400 MHz, DMSO‑d₆): δ = 3.85

(3H, s), 3.85 (3H, s), 3.92 (3H, s), 7.08 (1H, t, J = 7.6 Hz), 7.15–7.23

(4H, m), 7.51–7.55 (1H, m), 7.81 (1H, dd, J = 7.6, 1.7 Hz), 10.22 (1H, d,

10

K. Narita et al.

Bioorganic & Medicinal Chemistry 42 (2021) 116253

C 62.18, H 4.92, N 8.50.

725 cm^{ꢀ 1}. HRMS (EI): calcd for C₁₈H₁₇NO₄311.1158; found 311.1149.

Anal. calcd for C₁₈H₁₇NO₄: C 69.44, H 5.50, N 4.50; found C 69.31, H

5.60, N 4.50.

4.1.20. 2-(2^′,5^′-Dihydroxyphenyl)-5-(2^′′-hydroxyphenyl)-1,3,4-

thiadiazole (8)

Boron Tribromide (1 M in CH₂Cl₂; 1.5 mL, 1.5 mmol) was added to a

stirred solution of 27 (100 mg, 0.30 mmol) in CH₂Cl₂(3 mL) at 0 ^◦C

under argon. After 20 h, the reaction was quenched with saturated

aqueous NaHCO₃(5 mL), and the resulting mixture was extracted with

EtOAc (3 × 10 mL). The combined extracts were washed with brine (2 ×

10 mL), then dried with Na₂SO₄. The mixture was concentrated in

vacuo, and the residue was puriﬁed by column chromatography (hex-

ane/EtOAc 2:1) to give 8 (56.9 mg, 65%) as a yellow solid. Recrystal-

lization from hexane/CHCl₃(5:1) gave an analytical sample of 8 as

yellow powders. M.p. 237–238 ^◦C. ¹H NMR (400 MHz, DMSO‑d₆): δ =

6.81 (1H, t, J = 7.8 Hz), 6.94 (1H, d, J = 7.8 Hz), 7.00 (1H, t, J = 7.8 Hz),

7.08 (1H, d, J = 7.8 Hz), 7.36–7.41 (1H, m), 7.67 (1H, d, J = 7.8 Hz),

8.24 (1H, d, J = 7.8 Hz), 9.87 (1H, br s), 10.28 (1H, br s), 11.13 ppm

(1H, br s). ¹³C NMR (100 MHz, DMSO‑d₆): δ = 109.4, 109.6, 117.1,

118.2, 118.8, 119.8, 119.9, 128.8, 133.5, 145.4, 146.3, 156.4, 162.6,

163.1 ppm. FT-IR (KBr): 3397, 1617, 1593, 1576, 1559, 1503, 1478,

1456, 1362, 1259, 1160, 1132, 1091, 1066, 973, 949, 878, 820, 788,

756, 739, 727 cm^{ꢀ 1}. HRMS (EI): calcd for C₁₄H₁₀N₂O₃S 286.0412; found

286.0411. Anal. calcd for C₁₄H₁₀N₂O₃S: C 58.73, H 3.52, N 9.78; found

C 58.61, H 3.54, N 9.75.

4.1.23. 2-(2^′,3^′-Dihydroxyphenyl)-4-(2^′′-hydroxyphenyl)oxazole (9)

Boron tribromide (1 M in CH₂Cl₂; 22 mL, 22 mmol) was added to a

stirred solution of 29 (1.35 g, 4.3 mmol) in CH₂Cl₂(22 mL) at 0 ^◦C under

argon. After 20 h, the reaction was quenched with saturated aqueous

NaHCO₃(40 mL), and the resulting mixture was extracted with EtOAc

(3 × 50 mL). The combined extracts were washed with brine (2 × 50

mL), then dried with Na₂SO₄. The mixture was concentrated in vacuo,

and the residue was puriﬁed by column chromatography (hexane/

EtOAc 2:1) to give 9 (1.07 g, 92%) as a white solid. Recrystallization

from hexane/EtOAc (3:1) gave an analytical sample of 9 as yellow

powders. M.p. 203–204 ^◦C. ¹H NMR (400 MHz, CD₃OD): δ = 6.84 (1H, t,

J = 8.0 Hz), 6.92–6.98 (3H, m), 7.18 (1H, td, J = 7.8, 1.5 Hz), 7.39 (1H,

dd, J = 7.8, 1.5 Hz), 7.94 (1H, dd, J = 7.8, 1.5 Hz), 8.37 ppm (1H, s). ¹³

C

NMR (100 MHz, CD₃OD): δ = 112.4, 116.4, 117.6, 118.3, 119.0, 120.6,

120.7, 128.1, 130.0, 137.2, 137.4, 146.9, 147.1, 156.3, 161.8 ppm. FT-

IR (KBr): 3855, 3752, 3736, 3712, 3691, 3677, 3650, 3630, 3418, 1618,

1580, 1471, 1436, 1277, 1226, 1159, 1117, 1068, 814, 720 cm^{ꢀ 1}. HRMS

(EI): calcd for C₁₅H₁₁NO₄269.0688; found 269.0688. Anal. calcd for

C₁₅H₁₁NO₄: C 66.91, H 4.12, N 5.20; found C 66.87, H 4.27, N 5.12.

4.1.24. 2-(2^′-Methoxyphenyl)-5-(2^′′-methoxyphenyl)oxazole (30)

4.1.21. 2-(2-Methoxyphenyl)-2-oxoethyl 2,3-dimethoxybenzoate (28)

DBU (1.6 mL, 11 mmol) was added dropwise to a stirred solution of

2-bromo-2^′-methoxyacetophenone (10a) (2.00 g, 8.7 mmol) and car-

boxylic acid 21 (1.65 g, 9.1 mmol) in DMF (44 mL) at room temperature

under argon, and the resulting mixture was stirred at 60 ^◦C for 3 h. The

reaction was quenched with water (20 mL), and the resulting mixture

was extracted with Et₂O (3 × 20 mL). The combined extracts were

washed with brine (2 × 20 mL), then dried with Na₂SO₄. The mixture

was concentrated in vacuo, and the residue was puriﬁed by column

chromatography (hexane/EtOAc 1:1) to give 28 (2.36 g, 82%) as a white

solid. Recrystallization from hexane/CH₂Cl₂(5:1) gave an analytical

sample of 28 as colorless powders. M.p. 107–108 ^◦C. ¹H NMR (400 MHz,

CDCl₃): δ = 3.88 (3H, s), 3.93 (3H, s), 3.96 (3H, s), 5.47 (2H, s), 7.00

(1H, d, J = 8.3 Hz), 7.03–7.13 (3H, m), 7.50–7.55 (2H, m), 7.96 ppm

(1H, dd, J = 7.8, 2.0 Hz). ¹³C NMR (100 MHz, CDCl₃): δ = 55.5, 56.0,

61.6, 70.4, 111.5, 116.0, 121.0, 122.7, 123.8, 124.4, 125.7, 131.0,

134.8, 149.3, 153.4, 159.5, 165.6, 192.9 ppm. FT-IR (KBr): 3369, 2939,

2837, 1650, 1597, 1577, 1518, 1464, 1437, 1356, 1285, 1264, 1207,

1163, 1059, 1020, 993, 808, 755, 641 cm^{ꢀ 1}. HRMS (EI): calcd for

Compound 30 was synthesized from 2-bromo-2^′-methox-

yacetophenone (10a) (1.00 g, 4.4 mmol), 2-methoxybenzylamine (11d)

(0.67 mL, 5.2 mmol) in a manner similar to that described for the syn-

thesis of 12. Puriﬁcation by column chromatography (hexane/EtOAc

2:1) gave 30 (920 mg, 75%) as a white solid. Recrystallization from

hexane/CH₂Cl₂(4:1) gave an analytical sample of 30 as white powders.

Spectroscopic data for 30 were consistent with those previously reported

for this compound.¹⁹M.p. 117–119 ^◦C. ¹H NMR (400 MHz, CDCl₃): δ =

3.99 (3H, s), 4.01 (3H, s), 7.00 (1H, d, J = 8.3 Hz), 7.05–7.09 (3H, m),

7.30 (1H, td, J = 7.8, 1.5 Hz), 7.43 (1H, td, J = 7.8, 1.5 Hz), 7.69 (1H, s),

7.89 (1H, dd, J = 7.8, 1.5 Hz), 8.06 ppm (1H, dd, J = 7.8, 1.5 Hz). ¹³

C

NMR (100 MHz, CDCl₃): δ = 55.5, 56.1, 110.9, 112.0, 116.7, 117.5,

120.6, 120.8, 125.9, 127.6, 128.8, 130.1, 131.4, 147.2, 155.7, 157.6,

158.4 ppm. FT-IR (KBr): 3398, 2967, 2838, 1603, 1563, 1496, 1451,

1429, 1282, 1260, 1189, 1159, 1110, 1059, 1022, 953, 828, 759, 740,

703 cm^{ꢀ 1}. HRMS (EI): calcd for C₁₇H₁₅NO₃281.1052; found 281.1059.

Anal. calcd for C₁₇H₁₅NO₃: C 72.59, H 5.37, N 4.98; found C 72.45, H

5.43, N 4.98.

C

₁₈H₁₈O₆330.1103; found 330.1104. Anal. calcd for C₁₈H₁₈O₆: C 65.45,

4.1.25. 2-(3^′-Methoxyphenyl)-5-(2^′′-methoxyphenyl)oxazole (31)

Compound 31 was synthesized from 2-bromo-2^′-methox-

yacetophenone (10a) (1.00 g, 4.4 mmol), 3-methoxybenzylamine (11e)

(0.67 mL, 5.2 mmol) in a manner similar to that described for the syn-

thesis of 12. Puriﬁcation by column chromatography (hexane/EtOAc

3:1) gave 31 (924 mg, 75%) as a pale yellow solid. Recrystallization

from hexane/CH₂Cl₂(2:1) gave an analytical sample of 31 as pale yel-

low needles. Spectroscopic data for 31 were consistent with those pre-

viously reported for this compound.¹⁹M.p. 128–129 ^◦C. ¹H NMR (400

MHz, CDCl₃): δ = 3.90 (3H, s), 3.98 (3H, s), 6.99–7.01 (2H, m), 7.08

(1H, t, J = 7.8 Hz), 7.31 (1H, t, J = 7.8 Hz), 7.39 (1H, t, J = 7.8 Hz),

7.65–7.66 (2H, m), 7.72 (1H, d, J = 7.8 Hz), 7.89 ppm (1H, d, J = 7.8

Hz). ¹³C NMR (100 MHz, CDCl₃): δ = 55.4 (2C), 110.86, 110.91, 116.6,

117.2, 118.8, 120.8, 125.8, 127.6, 128.8, 129.0, 129.9, 147.8, 155.7,

159.85, 159.89 ppm. FT-IR (KBr): 2961, 2838, 1616, 1568, 1540, 1492,

1454, 1347, 1285, 1250, 1214, 1188, 1123, 1043, 1017, 940, 869, 836,

780, 749, 724 cm^{ꢀ 1}. HRMS (EI): calcd for C₁₇H₁₅NO₃281.1052; found

281.1052. Anal. calcd for C₁₇H₁₅NO₃: C 72.59, H 5.37, N 4.98; found C

72.58, H 5.48, N 4.95.

H 5.49; found C 65.45, H 5.45.

4.1.22. 2-(2^′,3^′-Dimethoxyphenyl)-4-(2^′′-methoxyphenyl)oxazole (29)

BF₃⋅OEt₂(1.1 mL, 8.6 mmol) was added to a stirred solution of 28

(2.36 g, 7.1 mmol) in xylene (36 mL) containing acetamide (2.11 g, 36

mmol) at room temperature under argon, and the resulting mixture was

heated at reﬂux for 2 days. After cooling to room temperature, the re-

action was quenched with water (20 mL), and the resulting mixture was

extracted with EtOAc (3 × 20 mL). The combined extracts were washed

with brine (2 × 20 mL), then dried with Na₂SO₄. The mixture was

concentrated in vacuo, and the residue was puriﬁed by column chro-

matography (hexane/EtOAc 4:1) to give 29 (1.35 g, 60%) as a white

solid. Recrystallization from hexane/CH₂Cl₂(5:1) gave an analytical

sample of 29 as colorless powders. M.p. 107–108 ^◦C. ¹H NMR (400 MHz,

CDCl₃): δ = 3.92 (3H, s), 3.98 (6H, s), 6.98 (1H, d, J = 8.0 Hz), 7.02 (1H,

dd, J = 8.0, 1.5 Hz), 7.09 (1H, td, J = 8.0, 1.0 Hz), 7.15 (1H, t, J = 8.0

Hz), 7.28–7.32 (1H, m), 7.64 (1H, dd, J = 8.0, 1.5 Hz), 8.27–8.29 ppm

(2H, m). ¹³C NMR (100 MHz, CDCl₃): δ = 55.3, 56.1, 61.4, 110.4, 114.2,

120.2, 120.8, 121.8, 122.4, 124.2, 128.0, 128.4, 136.9, 137.4, 147.9,

153.7, 156.4, 158.7 ppm. FT-IR (KBr): 3000, 2941, 1565, 1492, 1311,

1264, 1244, 1167, 1128, 1099, 1064, 1034, 997, 939, 837, 793, 754,

4.1.26. 2-(3^′-Methoxyphenyl)-5-(3^′′-methoxyphenyl)oxazole (32)

Compound

32

was

synthesized

from

2-bromo-3^′-

11

K. Narita et al.

Bioorganic & Medicinal Chemistry 42 (2021) 116253

methoxyacetophenone (10c) (1.00 g, 4.4 mmol), 3-methoxybenzyl-

amine (11f) (0.68 mL, 5.2 mmol) in a manner similar to that

described for the synthesis of 12. Puriﬁcation by column chromatog-

raphy (benzene/EtOAc 6:1) gave 32 (1.14 g, 93%) as a pale yellow solid.

Recrystallization from hexane/CH₂Cl₂(8:1) gave an analytical sample of

32 as pale yellow columnar crystals. M.p. 78–79 ^◦C. ¹H NMR (400 MHz,

CDCl₃): δ = 3.87 (3H, s), 3.89 (3H, s), 6.89 (1H, ddd, J = 8.0, 2.4, 1.2

Hz), 7.01 (1H, ddd, J = 8.0, 2.4, 1.2 Hz), 7.24 (1H, dd, J = 2.4, 1.2 Hz),

7.30 (1H, dt, J = 8.0, 1.2 Hz), 7.35 (1H, t, J = 8.0 Hz), 7.39 (1H, t, J =

8.0 Hz), 7.43 (1H, s), 7.63 (1H, dd, J = 2.4, 1.2 Hz), 7.70 ppm (1H, dt, J

= 7.8, 1.2 Hz). ¹³C NMR (100 MHz, CDCl₃): δ = 55.3, 55.4, 109.7, 110.9,

114.0, 116.7, 116.8, 118.7, 123.7, 128.6, 129.2, 129.9, 130.0, 151.1,

159.9, 160.0, 161.0 ppm. FT-IR (KBr): 3000, 2955, 2836, 1608, 1587,

1542, 1489, 1346, 1314, 1286, 1227, 1164, 1134, 1088, 1045, 994, 972,

865, 851, 775, 726 cm^{ꢀ 1}. HRMS (EI): calcd for C₁₇H₁₅NO₃281.1052;

found 281.1061. Anal. calcd for C₁₇H₁₅NO₃: C 72.59, H 5.37, N 4.98;

found C 72.57, H 5.48, N 5.04.

¹³C NMR (100 MHz, CDCl₃): δ = 55.3, 55.4, 56.0, 99.0, 105.0, 109.9,

110.7, 117.5, 120.7, 125.7, 127.3, 128.5, 131.0, 146.5, 155.5, 158.5,

158.9, 162.4 ppm. FT-IR (KBr): 2942, 2838, 1614, 1494, 1458, 1402,

1345, 1301, 1249, 1215, 1174, 1142, 1114, 1056, 1033, 929, 841, 820,

801, 758, 741 cm^{ꢀ 1}. HRMS (EI): calcd for C₁₈H₁₇NO₄311.1158; found

311.1165. Anal. calcd for C₁₈H₁₇NO₄: C 69.44, H 5.50, N 4.50; found C

69.17H 5.69, N 4.57.

4.1.30. 2-(2^′,6^′-Dimethoxyphenyl)-5-(2^′′-methoxyphenyl)oxazole (36)

Compound 36 was synthesized from 2-bromo-2^′-methox-

yacetophenone (10a) (1.00 g, 4.4 mmol), 2,6-dimethoxybenzylamine

47

(11 h)

(876 mg, 5.2 mmol) in a manner similar to that described

for the synthesis of 12. Puriﬁcation by column chromatography (hex-

ane/EtOAc 2:1) gave 36 (137 mg, 10%) as a white solid. Recrystalliza-

tion from hexane/Et₂O (2:1) gave an analytical sample of 36 as colorless

plates. M.p. 158–159 ^◦C. ¹H NMR (400 MHz, CDCl₃): δ = 3.78 (6H, s),

3.97 (3H, s), 6.62 (2H, d, J = 8.3 Hz), 6.97 (1H, d, J = 7.6 Hz), 7.01 (1H,

t, J = 7.6 Hz), 7.25–7.29 (1H, m), 7.37 (1H, t, J = 8.3 Hz), 7.71 (1H, s),

7.81 ppm (1H, dd, J = 7.6, 1.7 Hz). ¹³C NMR (100 MHz, CDCl₃): δ =

55.4, 56.0 (2C), 103.9, 107.1 (2C), 110.7, 117.6, 120.6, 126.1, 126.9,

128.6, 131.9, 148.0, 155.4, 155.5, 159.9 (2C) ppm. FT-IR (KBr): 2959,

2838, 1613, 1586, 1492, 1472, 1432, 1250, 1182, 1110, 1054, 1024,

956, 836, 791, 765, 734 cm^{ꢀ 1}. HRMS (EI): calcd for C₁₈H₁₇NO₄

311.1158; found 311.1161. Anal. calcd for C₁₈H₁₇NO₄: C 69.44, H 5.50,

N 4.50; found C 69.40H 5.61, N 4.57.

4.1.27. 2-(4^′-Methoxyphenyl)-5-(4^′′-methoxyphenyl)oxazole (33)

Compound 33 was synthesized from 2-bromo-4^′-methox-

yacetophenone (10d) (1.00 g, 4.4 mmol), 4-methoxybenzylamine (11f)

(0.68 mL, 5.2 mmol) in a manner similar to that described for the syn-

thesis of 12. Puriﬁcation by recrystallization (benzene/CH₂Cl₂4:1) gave

33 (787 mg, 64%) as yellow plates. Spectroscopic data for 33 were

consistent with those previously reported for this compound.⁴²M.p.

136–138 ^◦C. ¹H NMR (400 MHz, CDCl₃): δ = 3.85 (3H, s), 3.87 (3H, s),

6.95–7.00 (4H, m), 7.28 (1H, s), 7.61–7.65 (2H, m), 8.01–8.04 ppm (2H,

m). ¹³C NMR (100 MHz, CDCl₃): δ = 55.33, 55.35, 114.2 (2C), 114.3

(2C), 120.4, 121.0, 121.7, 125.6 (2C), 127.8 (2C), 150.7, 159.6, 160.6,

161.2 ppm. FT-IR (KBr): 2938, 2839, 1615, 1589, 1560, 1492, 1442,

1419, 1339, 1306, 1290, 1250, 1173, 1105, 1056, 1022, 950, 831, 740,

703 cm^{ꢀ 1}. HRMS (EI): calcd for C₁₇H₁₅NO₃281.1052; found 281.1051.

Anal. calcd for C₁₇H₁₅NO₃: C 72.59, H 5.37, N 4.98; found C 72.38, H

5.47, N 5.07.

4.1.31. 2-(2^′,3^′-Dimethoxyphenyl)-5-(3^′′-methoxyphenyl)oxazole (37)

Compound 37 was synthesized from 2-bromo-3^′-methox-

yacetophenone (10c) (1.00 g, 4.4 mmol), 2,3-dimethoxybenzylamine

(11a) (0.79 mL, 5.2 mmol) in a manner similar to that described for

the synthesis of 12. Puriﬁcation by column chromatography (hexane/

EtOAc 2:1) gave 37 (1.26 g, 93%) as a white solid. Recrystallization

from hexane/CH₂Cl₂(4:1) gave an analytical sample of 37 as colorless

needles. M.p. 84–85 ^◦C. ¹H NMR (400 MHz, CDCl₃): δ = 3.86 (3H, s),

3.92 (3H, s), 4.01 (3H, s), 6.87–6.90 (1H, m), 7.02 (1H, d, J = 8.0 Hz),

7.16 (1H, t, J = 8.0 Hz), 7.26–7.27 (1H, m), 7.30–7.37 (2H, m), 7.48

(1H, s), 7.61–7.64 ppm (1H, m). ¹³C NMR (100 MHz, CDCl₃): δ = 55.3,

56.0, 61.2, 109.5, 114.0, 114.2, 116.7, 121.3, 122.0, 123.5, 124.3,

129.3, 130.0, 147.7, 151.0, 153.8, 159.3, 159.9 ppm. FT-IR (KBr): 2961,

2830, 1601, 1525, 1480, 1424, 1349, 1298, 1268, 1232, 1188, 1133,

1083, 1051, 1005, 972, 847, 782, 742, 724 cm^{ꢀ 1}. HRMS (EI): calcd for

4.1.28. 2-(2^′,3^′-Dimethoxyphenyl)-5-phenyloxazole (34)

Compound 34 was synthesized from 2-bromo-acetophenone (10e)

(500 mg, 2.5 mmol), 2,3-dimethoxybenzylamine (11a) (0.45 mL, 3.0

mmol) in a manner similar to that described for the synthesis of 12.

Puriﬁcation by column chromatography (hexane/EtOAc 3:1) gave 34

(633 mg, 99%) as a yellow solid. Recrystallization from hexane/Et₂O

(10:1) gave an analytical sample of 34 as yellow columnar crystals.

Spectroscopic data for 34 were consistent with those previously reported

for this compound.¹⁹M.p. 61–62 ^◦C. ¹H NMR (400 MHz, CDCl₃): δ =

3.90 (3H, s), 4.01 (3H, s), 7.01 (1H, dd, J = 8.0, 1.5 Hz), 7.15 (1H, td, J

= 8.0, 5.4 Hz), 7.30–734 (1H, m), 7.42 (2H, t, J = 8.0 Hz), 7.49 (1H, s),

7.63 (1H, dd, J = 8.0, 1.5 Hz), 7.72 ppm (2H, d, J = 8.0 Hz). ¹³C NMR

(100 MHz, CDCl₃): δ = 56.0, 61.1, 114.2, 121.2, 121.9, 123.2, 124.1,

124.2 (2C), 128.0, 128.3, 128.8 (2C), 147.6, 151.1, 153.7, 159.3 ppm.

FT-IR (KBr): 3000, 2936, 2836, 1583, 1530, 1480, 1424, 1347, 1313,

1264, 1235, 1173, 1131, 1086, 1045, 1005, 965, 942, 844, 790, 763,

748, 728 cm^{ꢀ 1}. HRMS (EI): calcd for C₁₇H₁₅NO₃281.1052; found

281.1051. Anal. calcd for C₁₇H₁₅NO₃: C 72.59, H 5.37, N 4.98; found C

72.69, H 5.46, N 5.06.

C

₁₈H₁₇NO₄311.1158; found 311.1156. Anal. calcd for C₁₈H₁₇NO₄: C

69.44, H 5.50, N 4.50; found C 69.28H 5.59, N 4.50.

4.1.32. 2-(2^′,3^′-Dimethoxyphenyl)-5-(4^′′-methoxyphenyl)oxazole (38)

Compound 38 was synthesized from 2-bromo-4^′-methox-

yacetophenone (10d) (1.00 g, 4.4 mmol), 2,3-dimethoxybenzylamine

(11a) (0.79 mL, 5.2 mmol) in a manner similar to that described for

the synthesis of 12. Puriﬁcation by column chromatography (hexane/

EtOAc 3:1) gave 38 (1.22 g, 90%) as white solid. Recrystallization from

hexane/Et₂O (5:1) gave an analytical sample of 38 as colorless needles.

M.p. 61–63 ^◦C. ¹H NMR (400 MHz, CDCl₃): δ = 3.86 (3H, s), 3.93 (3H, s),

4.00 (3H, s), 6.96–6.999 (2H, m), 7.02 (1H, dd, J = 7.8, 1.5 Hz), 7.16

(1H, t, J = 8.0 Hz), 7.37 (1H, s), 7.62 (1H, dd, J = 7.8, 1.5 Hz),

7.64–7.68 ppm (2H, m). ¹³C NMR (100 MHz, CDCl₃): δ = 55.2, 55.9,

61.1, 113.9, 114.3 (2C), 120.9, 121.1, 121.7, 122.0, 124.2, 125.6 (2C),

147.5, 151.1, 153.7, 158.6, 159.6 ppm. FT-IR (KBr): 2935, 2836, 1614,

1570, 1532, 1500, 1480, 1465, 1421, 1318, 1302, 1257, 1177, 1129,

1082, 1048, 1027, 1004, 943, 833, 789, 744, 728 cm^{ꢀ 1}. HRMS (EI):

calcd for C₁₈H₁₇NO₄311.1158; found 311.1151. Anal. calcd for

4.1.29. 2-(2^′,4^′-Dimethoxyphenyl)-5-(2^′′-methoxyphenyl)oxazole (35)

Compound 35 was synthesized from 2-bromo-2^′-methox-

yacetophenone (10a) (1.00 g, 4.4 mmol), 2,4-dimethoxybenzylamine

(11 g) (0.79 mL, 5.2 mmol) in a manner similar to that described for

the synthesis of 12. Puriﬁcation by column chromatography (hexane/

EtOAc 1:1) gave 35 (859 mg, 63%) as a pale yellow solid. Recrystalli-

zation from hexane/CH₂Cl₂(10:1) gave an analytical sample of 35 as

pale yellow columnar crystals. M.p. 72–73 ^◦C. ¹H NMR (400 MHz,

CDCl₃): δ = 3.88 (3H, s), 3.98 (3H, s), 3.99 (3H, s), 6.58–6.62 (2H, m),

6.99 (1H, d, J = 8.0 Hz), 7.06 (1H, t, J = 8.0 Hz), 7.26–7.31 (1H, m),

7.65 (1H, s), 7.87 (1H, dd, J = 8.0, 1.5 Hz), 8.00 ppm (1H, d, J = 8.0 Hz).

C₁₈H₁₇NO₄: C 69.44, H 5.50, N 4.50; found C 69.36H 5.65, N 4.51.

4.1.33. 2-(2^′-Methoxyphenyl)-5-(2^′′,5^′′-dimethoxyphenyl)oxazole (39)

Compound 39 was synthesized from 2-bromo-2^′,5^′-dimethox-

yacetophenone (10b) (1.00 g, 3.9 mmol), 2-methoxybenzylamine (11d)

(0.59 mL, 4.6 mmol) in a manner similar to that described for the

12

K. Narita et al.

Bioorganic & Medicinal Chemistry 42 (2021) 116253

synthesis of 12. Puriﬁcation by column chromatography (hexane/EtOAc

2:1) gave 39 (809 mg, 67%) as a yellow solid. Recrystallization from

hexane/CH₂Cl₂(10:1) gave an analytical sample of 39 as yellow gran-

ules. M.p. 102–103 ^◦C. ¹H NMR (400 MHz, CDCl₃): δ = 3.85 (3H, s), 3.93

(3H, s), 4.00 (3H, s), 6.84 (1H, dd, J = 9.0, 2.9 Hz), 6.91 (1H, d, J = 9.0

Hz), 7.04–7.09 (2H, m), 7.41–7.45 (2H, m), 7.71 (1H, s), 8.05 ppm (1H,

dd, J = 7.6, 1.7 Hz). ¹³C NMR (100 MHz, CDCl₃): δ = 55.8, 55.9, 56.1,

111.4, 112.0 (2C), 113.4, 116.6, 118.1, 120.6, 127.9, 130.0, 131.5,

147.0, 150.2, 153.7, 157.6, 158.4 ppm. FT-IR (KBr): 3005, 2947, 2834,

1569, 1507, 1456, 1316, 1288, 1258, 1178, 1109, 1053, 1011, 971, 867,

830, 798, 762, 745, 703 cm^{ꢀ 1}. HRMS (EI): calcd for C₁₈H₁₇NO₄

311.1158; found 311.1153. Anal. calcd for C₁₈H₁₇NO₄: C 69.44, H 5.50,

N 4.50; found C 69.29H 5.57, N 4.59.

117.9, 121.4, 122.1, 124.3, 127.7, 147.66, 147.68, 150.2, 153.7, 153.8,

158.4 ppm. FT-IR (KBr): 2943, 2836, 1567, 1533, 1507, 1473, 1339,

1265, 1245, 1206, 1178, 1152, 1129, 1082, 1048, 1024, 1007, 971, 856,

837, 791, 750 cm^{ꢀ 1}. HRMS (EI): calcd for C₁₉H₁₉NO₅341.1263; found

341.1263. Anal. calcd for C₁₉H₁₉NO₅: C 66.85, H 5.61, N 4.10; found C

66.71, H 5.72, N 4.14.

4.1.37. 2-(2^′,3^′-Dimethoxyphenyl)-5-(2^′′,6^′′-dimethoxyphenyl)oxazole

(43)

Compound 43 was synthesized from 2-bromo-2^′,6^′-dimethox-

yacetophenone (10 h) ⁴⁹(1.00 g, 3.9 mmol), 2,3-dimethoxybenzylamine

(11a) (0.70 mL, 4.6 mmol) in a manner similar to that described for the

synthesis of 12. Puriﬁcation by column chromatography (hexane/EtOAc

2:1) gave 43 (240 mg, 18%) as a pale yellow solid. Recrystallization

from hexane/CH₂Cl₂(10:1) gave an analytical sample of 43 as pale

yellow needles. M.p. 137–138 ^◦C. ¹H NMR (400 MHz, CDCl₃): δ = 3.87

(6H, s), 3.91 (3H, s), 3.97 (3H, s), 6.65 (2H, d, J = 8.8 Hz), 6.99 (1H, dd,

J = 8.0, 1.5 Hz), 7.13 (1H, t, J = 8.0 Hz), 7.30 (1H, t, J = 8.8 Hz), 7.48

(1H, s), 7.64 ppm (1H, dd, J = 8.0, 1.5 Hz). ¹³C NMR (100 MHz, CDCl₃):

δ = 56.0 (2C), 56.1, 61.2, 104.2 (2C), 106.7, 113.8, 121.5, 122.7, 124.1,

129.2, 130.1, 144.7, 147.7, 153.7, 158.2 (2C), 158.8 ppm. FT-IR (KBr):

3005, 2936, 2838, 1587, 1542, 1480, 1425, 1343, 1265, 1234, 1188,

1110, 1079, 1048, 992, 969, 944, 838, 782, 754, 729 cm^{ꢀ 1}. HRMS (EI):

calcd for C₁₉H₁₉NO₅341.1263; found 341.1263. Anal. calcd for

C₁₉H₁₉NO₅: C 66.85, H 5.61, N 4.10; found C 66.81, H 5.81, N 4.15.

4.1.34. 2-(2^′,3^′-Dimethoxyphenyl)-5-(2^′′,3^′′-dimethoxyphenyl)oxazole

(40)

Compound 40 was synthesized from 2-bromo-2^′,3^′-dimethox-

yacetophenone (10f) ⁴⁸(1.00 g, 3.9 mmol), 2,3-dimethoxybenzylamine

(11a) (0.70 mL, 4.6 mmol) in a manner similar to that described for the

synthesis of 12. Puriﬁcation by column chromatography (hexane/EtOAc

2:1) gave 40 (822 mg, 62%) as a white solid. Recrystallization from

hexane/Et₂O (10:1) gave an analytical sample of 40 as colorless needles.

M.p. 55–56 ^◦C. ¹H NMR (400 MHz, CDCl₃): δ = 3.91 (3H, s), 3.92 (6H, s),

4.01 (3H, s), 6.90 (1H, dd, J = 8.0, 1.5 Hz), 7.02 (1H, dd, J = 8.0, 1.5

Hz), 7.14 (1H, t, J = 8.0 Hz), 7.16 (1H, t, J = 8.0 Hz), 7.47 (1H, dd, J =

8.0, 1.5 Hz), 7.65 (1H, dd, J = 8.0, 1.5 Hz), 7.77 ppm (1H, s). ¹³C NMR

(100 MHz, CDCl₃): δ = 55.9, 56.0, 59.7, 61.2, 112.1, 114.1, 117.7,

121.3, 122.0, 122.5, 124.2, 124.5, 127.5, 145.6, 147.6, 147.7, 153.1,

153.8, 158.6 ppm. FT-IR (KBr): 2945, 2831, 1586, 1528, 1488, 1433,

1343, 1315, 1263, 1193, 1131, 1082, 1051, 999, 974, 837, 782, 741,

725 cm^{ꢀ 1}. HRMS (EI): calcd for C₁₉H₁₉NO₅341.1263; found 341.1253.

Anal. calcd for C₁₉H₁₉NO₅: C 66.85, H 5.61, N 4.10; found C 66.96, H

5.69, N 4.17.

4.1.38. 2-(2^′,3^′-Dimethoxyphenyl)-5-(2^′′,3^′′,4^′′-trimethoxyphenyl)oxazole

(44)

Compound 44 was synthesized from 2-bromo-2^′,3^′,4^′-trimethox-

yacetophenone (10i) ⁵⁰(1.00 g, 3.5 mmol), 2,3-dimethoxybenzylamine

(11a) (0.62 mL, 4.2 mmol) in a manner similar to that described for the

synthesis of 12. Puriﬁcation by column chromatography (hexane/EtOAc

2:1) gave 44 (836 mg, 65%) as a yellow solid. Recrystallization from

hexane/CH₂Cl₂(10:1) gave an analytical sample of 44 as yellow gran-

ules. M.p. 102–104 ^◦C. ¹H NMR (400 MHz, CDCl₃): δ = 3.91 (3H, s), 3.92

(3H, s), 3.93 (3H, s), 3.98 (3H, s), 4.00 (3H, s), 6.78 (1H, d, J = 8.8 Hz),

7.01 (1H, dd, J = 8.0, 1.5 Hz), 7.16 (1H, t, J = 8.0 Hz), 7.55 (1H, d, J =

8.8 Hz), 7.62–7.65 ppm (2H, m). ¹³C NMR (100 MHz, CDCl₃): δ = 56.00,

56.02, 60.2, 60.9, 61.2, 107.8, 114.0, 115.7, 120.5, 121.3, 122.1, 124.2,

125.6, 142.6, 147.59, 147.62, 150.5, 153.8 (2C), 158.1 ppm. FT-IR

(KBr): 2971, 2841, 1605, 1561, 1534, 1487, 1337, 1287, 1268, 1229,

1209, 1130, 1087, 1045, 1011, 916, 849, 809, 788, 746 cm^{ꢀ 1}. HRMS

(EI): calcd for C₂₀H₂₁NO₆371.1369; found 371.1365. Anal. calcd for

C₂₀H₂₁NO₆: C 64.68, H 5.70, N 3.77; found C 64.55, H 5.79, N 3.84.

4.1.35. 2-(2^′,3^′-Dimethoxyphenyl)-5-(2^′′,4^′′-dimethoxyphenyl)oxazole

(41)

Compound 41 was synthesized from 2-bromo-2^′,4^′-dimethox-

yacetophenone (10 g) (1.00 g, 3.9 mmol), 2,3-dimethoxybenzylamine

(11a) (0.70 mL, 4.6 mmol) in a manner similar to that described for

the synthesis of 12. Puriﬁcation by column chromatography (hexane/

EtOAc 2:1) gave 41 (586 mg, 44%) as a pale yellow solid. Recrystalli-

zation from hexane/CH₂Cl₂(10:1) gave an analytical sample of 41 as

pale yellow needles. M.p. 122–124 ^◦C. ¹H NMR (400 MHz, CDCl₃): δ =

3.83 (3H, s), 3.90 (3H, s), 3.93 (3H, s), 3.99 (3H, s), 6.53 (1H, d, J = 2.4

Hz), 6.59 (1H, dd, J = 8.3, 2.4 Hz), 6.98 (1H, dd, J = 8.0, 1.5 Hz), 7.13

(1H, t, J = 8.0 Hz), 7.56 (1H, s), 7.63 (1H, dd, J = 8.0, 1.5 Hz), 7.79 ppm

(1H, d, J = 8.3 Hz). ¹³C NMR (100 MHz, CDCl₃): δ = 55.3, 55.4, 55.9,

61.2, 98.5, 104.9, 110.6, 113.8, 121.2, 122.2, 124.1, 125.4, 126.6,

147.5, 147.8, 153.7, 156.9, 157.6, 160.6 ppm. FT-IR (KBr): 2941, 2837,

1612, 1569, 1531, 1499, 1466, 1321, 1268, 1232, 1207, 1127, 1083,

1046, 1026, 948, 822, 788, 744, 721 cm^{ꢀ 1}. HRMS (EI): calcd for

C₁₉H₁₉NO₅341.1263; found 341.1256. Anal. calcd for C₁₉H₁₉NO₅: C

66.85, H 5.61, N 4.10; found C 66.98, H 5.71, N 4.08.

4.1.39. 2-(2^′,3^′,4^′-Trimethoxyphenyl)-5-(2^′′,3^′′-dimethoxyphenyl)oxazole

(45)

Compound 45 was synthesized from 2-bromo-2^′,3^′-dimethox-

yacetophenone (10f) (1.00 g, 3.9 mmol), 2,3,4-trimethoxybenzylamine

(11i) ⁵¹(0.84 mL, 4.6 mmol) in a manner similar to that described for

the synthesis of 12. Puriﬁcation by recrystallization (hexane/EtOAc 3:1)

gave 45 (535 mg, 37%) as yellow needles. M.p. 145–146 ^◦C. ¹H NMR

(400 MHz, CDCl₃): δ = 3.92 (6H, s), 3.93 (3H, s), 3.94 (3H, s), 4.04 (3H,

s), 6.80 (1H, d, J = 8.8 Hz), 6.90 (1H, dd, J = 8.0, 1.2 Hz), 7.15 (1H, t, J

= 8.0 Hz), 7.45 (1H, dd, J = 8.0, 1.2 Hz), 7.73 (1H, s), 7.78 ppm (1H, d,

J = 8.8 Hz). ¹³C NMR (100 MHz, CDCl₃): δ = 55.9, 56.1, 59.7, 61.0, 61.6,

107.7, 111.9, 115.1, 117.7, 122.7, 124.5, 124.6, 127.4, 143.1, 145.5,

147.0, 152.6, 153.1, 155.5, 158.6 ppm. FT-IR (KBr): 2938, 2834, 1597,

1486, 1459, 1410, 1341, 1268, 1248, 1211, 1131, 1092, 1025, 997, 972,

915, 854, 814, 784, 732, 701 cm^{ꢀ 1}. HRMS (EI): calcd for C₂₀H₂₁NO₆

371.1369; found 371.1365. Anal. calcd for C₂₀H₂₁NO₆: C 64.68, H 5.70,

N 3.77; found C 64.62, H 5.77, N 3.84.

4.1.36. 2-(2^′,3^′-Dimethoxyphenyl)-5-(2^′′,5^′′-dimethoxyphenyl)oxazole

(42)

Compound 42 was synthesized from 2-bromo-2^′,5^′-dimethox-

yacetophenone (10b) (1.00 g, 3.9 mmol), 2,3-dimethoxybenzylamine

(11a) (0.78 mL, 5.2 mmol) in a manner similar to that described for

the synthesis of 12. Puriﬁcation by recrystallization (hexane/CH₂Cl₂

4:1) gave 42 (987 mg, 75%) as pale yellow granules. M.p. 138–139 ^◦C.

¹H NMR (400 MHz, CDCl₃): δ = 3.84 (3H, s), 3.93 (3H, s), 3.94 (3H, s),

4.01 (3H, s), 6.85 (1H, dd, J = 9.0, 3.2 Hz), 6.92 (1H, d, J = 9.0 Hz), 7.02

(1H, dd, J = 8.0, 1.5 Hz), 7.16 (1H, t, J = 8.0 Hz), 7.46 (1H, d, J = 3.2

Hz), 7.66 (1H, dd, J = 8.0, 1.5 Hz), 7.71 ppm (1H, s). ¹³C NMR (100

MHz, CDCl₃): δ = 55.8, 55.9, 56.1, 61.2, 111.0, 112.1, 114.1, 114.2,

4.1.40. 2-(2^′,3^′,4^′-Trimethoxyphenyl)-5-(2^′′,3^′′,4^′′-trimethoxyphenyl)

oxazole (46)

Compound

46

was

synthesized

from

2-bromo-2^′,3^′,4^′-

13

K. Narita et al.

Bioorganic & Medicinal Chemistry 42 (2021) 116253

trimethoxyacetophenone (10i) (625 mg, 2.2 mmol), 2,3,4-trimethoxy-

benzylamine (11i) (512 mg, 2.6 mmol) in a manner similar to that

described for the synthesis of 12. Puriﬁcation by column chromatog-

raphy (benzene/EtOAc 5:1) gave 46 (344 mg, 40%) as a pale yellow

solid. Recrystallization from hexane/CH₂Cl₂(10:1) gave an analytical

sample of 46 as pale yellow crystals. M.p. 84–85 ^◦C. ¹H NMR (400 MHz,

CDCl₃): δ = 3.92 (3H, s), 3.93 (3H, s), 3.93 (3H, s), 3.94 (3H, s), 3.97

(3H, s), 4.03 (3H, s), 6.77 (1H, d, J = 6.3 Hz), 6.80 (1H, d, J = 6.3 Hz),

7.52 (1H, d, J = 8.8 Hz), 7.58 (1H, s), 7.76 ppm (1H, d, J = 8.3 Hz). ¹³C

NMR (100 MHz, CDCl₃): δ = 56.1 (2C), 60.2, 60.9, 61.1, 61.5, 107.7,

107.8, 115.2, 115.9, 120.4, 124.5, 125.5, 142.7, 143.1, 147.1, 150.5,

152.5, 153.7, 155.4, 158.1 ppm. FT-IR (KBr): 2987, 2938, 1599, 1489,

1468, 1431, 1415, 1292, 1245, 1210, 1191, 1173, 1130, 1090, 1015,

915, 849, 818, 728, 703 cm^{ꢀ 1}. HRMS (EI): calcd for C₂₁H₂₃NO₇

401.1475; found 401.1473. Anal. calcd for C₂₁H₂₃NO₇: C 62.84, H 5.78,

N 3.49; found C 62.80, H 5.65, N 3.43.

114.1, 117.6, 121.1, 121.3, 122.1, 124.2, 125.6, 127.8, 127.9 (2C),

128.3, 128.8 (2C), 128.9, 136.2, 147.68, 147.72, 153.8, 154.8, 158.2

ppm. FT-IR (KBr): 3004, 2934, 2878, 1567, 1530, 1493, 1468, 1420,

1388, 1345, 1259, 1133, 1087, 1046, 1003, 833, 787, 741, 701 cm^{ꢀ 1}

.

HRMS (EI): calcd for C₂₄H₂₁NO₄387.1471; found 387.1473. Anal. calcd

for C₂₄H₂₁NO₄: C 74.40, H 5.46, N 3.62; found C 74.52, H 5.63, N 3.64.

4.1.44. 2-(2^′,3^′-Dimethoxyphenyl)-5-(2^′′-hydroxyphenyl)oxazole (50)

10% Pd/C (235 mg) was added to a solution of 49 (911 mg, 2.4

mmol) in THF (24 mL) at room temperature. The mixture was stirred for

24 h under H₂(1 atm) at room temperature. The reaction mixture was

diluted with EtOAc (30 mL), and the catalyst was removed by ﬁltration

through a small pad of Celite. The ﬁltrate was concentrated in vacuo to

give a residue, which was puriﬁed by column chromatography (CHCl₃/

MeOH 40:1) to give 50 (608 mg, 87%) as a white solid. Recrystallization

from hexane/EtOAc (2:1) gave an analytical sample of 50 as colorless

needles. M.p. 186–187 ^◦C. ¹H NMR (400 MHz, acetone‑d₆): δ = 3.92

(3H, s), 4.00 (3H, s), 6.99–7.03 (1H, m), 7.06 (1H, d, J = 7.8 Hz),

7.16–7.24 (3H, m), 7.62 (1H, dd, J = 6.8, 2.9 Hz), 7.72 (1H, s), 7.87 (1H,

dd, J = 7.8, 1.5 Hz), 9.43 ppm (1H, br s). ¹³C NMR (100 MHz, ace-

tone‑d₆): δ = 56.3, 61.3, 115.2, 116.5, 116.7, 120.9, 122.0, 123.0, 125.1,

126.4, 127.7, 129.9, 148.5, 149.2, 154.6, 155.0, 159.1 ppm. FT-IR

(KBr): 3482, 3067, 1572, 1525, 1474, 1450, 1421, 1351, 1263, 1228,

1133, 1086, 1056, 1001, 945, 847, 786, 743, 719 cm^{ꢀ 1}. HRMS (EI):

calcd for C₁₇H₁₅NO₄297.1001; found 297.0995. Anal. calcd for

4.1.41. 2-(2^′,3^′-Dimethoxyphenyl)-5-(naphthalen-2-yl)oxazole (47)

Compound 47 was synthesized from 2-Bromo-2^′-acetonaphthone

(10j) (1.00 g, 4.0 mmol), 2,3-dimethoxybenzylamine (11a) (0.73 mL,

4.8 mmol) in a manner similar to that described for the synthesis of 12.

Puriﬁcation by column chromatography (hexane/EtOAc 3:1) gave 47

(1.26 g, 95%) as a pale yellow solid. Recrystallization from hexane/

CH₂Cl₂(5:1) gave an analytical sample of 47 as pale yellow needles. M.

p. 105–107 ^◦C. ¹H NMR (400 MHz, CDCl₃): δ = 3.93 (3H, s), 4.06 (3H, s),

7.04 (1H, dd, J = 8.0, 1.2 Hz), 7.19 (1H, t, J = 8.0 Hz), 7.54–7.47 (2H,

m), 7.61 (1H, s), 7.68 (1H, dd, J = 8.0, 1.2 Hz), 7.79 (1H, dd, J = 8.0, 1.2

Hz), 7.82–7.85 (1H, m), 7.88–7.90 (2H, m), 8.20 ppm (1H, s). ¹³C NMR

(100 MHz, CDCl₃): δ = 56.0, 61.3, 114.3, 121.3, 122.0, 122.1, 122.9,

123.8, 124.3, 125.4, 126.4, 126.7, 127.8, 128.2, 128.7, 133.0, 133.4,

147.8, 151.3, 153.8, 159.5 ppm. FT-IR (KBr): 2940, 1528, 1477, 1422,

1330, 1264, 1229, 1190, 1130, 1084, 1041, 1001, 972, 932, 892, 862,

820, 790, 751, 726 cm^{ꢀ 1}. HRMS (EI): calcd for C₂₁H₁₇NO₃331.1208;

found 331.1216. Anal. calcd for C₂₁H₁₇NO₃: C 76.12, H 5.17, N 4.23;

found C 75.92, H 5.22, N 4.24.

C₁₇H₁₅NO₄⋅H₂O: C 64.75, H 5.43, N 4.44; found C 64.77, H 5.44, N 4.45.

4.1.45. 2-(2^′,3^′-Dimethoxyphenyl)-5-(2^′′-ethoxyphenyl)oxazole (51)

EtI (82.0 L, 1.0 mmol) was added to a stirred solution of 50 (100

μ

mg, 0.34 mmol) in DMF (3.4 mL) containing K₂CO₃(139 mg, 1.0 mmol)

at room temperature, and the mixture was stirred at 60 ^◦C for 2 h. After

this time, the mixture was cooled to room temperature, and H₂O (5 mL)

was added. The resulting mixture was extracted with Et₂O (3 × 5 mL),

and the combined extracts were washed with brine (2 × 5 mL), then

dried with Na₂SO₄. The mixture was concentrated in vacuo, and the

residue was puriﬁed by column chromatography (hexane/EtOAc 2:1) to

give 51 (102 mg, 94%) as white solid. Recrystallization from hexane/

CH₂Cl₂(5:1) gave an analytical sample of 51 as colorless plates. M.p.

108–109 ^◦C. ¹H NMR (400 MHz, CDCl₃): δ = 1.56 (3H, t, J = 7.0 Hz),

3.92 (3H, s), 4.01 (3H, s), 4.19 (2H, q, J = 7.0 Hz), 6.97 (1H, d, J = 8.0

Hz), 7.00–7.07 (2H, m), 7.16 (1H, t, J = 8.0 Hz), 7.25–7.30 (1H, m), 7.65

(1H, dd, J = 8.0, 2.0 Hz), 7.77 (1H, s), 7.90 ppm (1H, dd, J = 8.0, 2.0

Hz). ¹³C NMR (100 MHz, CDCl₃): δ = 14.9, 56.0, 61.2, 63.9, 111.5,

114.0, 117.3, 120.7, 121.4, 122.2, 124.2, 125.8, 127.5, 128.9, 147.7,

147.9, 153.8, 155.1, 158.2 ppm. FT-IR (KBr): 2940, 2835, 1561, 1528,

1475, 1420, 1342, 1303, 1261, 1244, 1166, 1129, 1081, 1045, 1000,

943, 839, 793, 747, 725 cm^{ꢀ 1}. HRMS (EI): calcd for C₁₉H₁₉NO₄

325.1314; found 325.1311. Anal. calcd for C₁₉H₁₉NO₄: C 70.14, H 5.89,

N 4.31; found C 70.32, H 6.01, N 4.35.

4.1.42. 2-(2^′,3^′-Dimethoxyphenyl)-5-(2^′′-methylphenyl)oxazole (48)

Compound 48 was synthesized from 2-bromo-2^′-methyl-

acetophenone (10 k) (1.00 g, 4.7 mmol), 2,3-dimethoxybenzylamine

(11a) (0.85 mL, 5.6 mmol) in a manner similar to that described for

the synthesis of 12. Puriﬁcation by column chromatography (hexane/

EtOAc 2:1) gave 48 (1.11 g, 80%) as a yellow solid. Recrystallization

from hexane/CH₂Cl₂(10:1) gave an analytical sample of 48 as yellow

needles. M.p. 97–98 ^◦C. ¹H NMR (400 MHz, CDCl₃): δ = 2.55 (3H, s),

3.93 (3H, s), 3.99 (3H, s), 7.03 (1H, dd, J = 8.3, 1.5 Hz), 7.16 (1H, t, J =

8.3 Hz), 7.25–7.33 (3H, m), 7.40 (1H, s), 7.64 (1H, dd, J = 8.3, 1.5 Hz),

7.78–7.81 ppm (1H, m). ¹³C NMR (100 MHz, CDCl₃): δ = 21.9, 56.1,

61.3, 114.2, 121.4, 122.0, 124.3, 126.1, 126.2, 126.8, 127.4, 128.3,

131.2, 134.9, 147.8, 150.7, 153.8, 159.0 ppm. FT-IR (KBr): 2941, 2842,

1561, 1527, 1458, 1421, 1380, 1347, 1309, 1263, 1233, 1154, 1132,

1086, 1049, 1005, 970, 940, 837, 791, 762, 748, 728 cm^{ꢀ 1}. HRMS (EI):

calcd for C₁₈H₁₇NO₃295.1208; found 295.1207. Anal. calcd for

4.1.46. 2-(2^′,3^′-Dimethoxyphenyl)-5-(2^′′-n-propoxyphenyl)oxazole (52)

nPrI (98.0 μL, 1.0 mmol) was added to a stirred solution of 50 (100

C

₁₈H₁₇NO₃: C 73.20, H 5.80, N 4.74; found C 73.34, H 5.95, N 4.72.

mg, 0.34 mmol) in DMF (3.4 mL) containing K₂CO₃(139 mg, 1.0 mmol)

at room temperature, and the mixture was stirred at 60 ^◦C for 2 h. After

this time, the mixture was cooled to room temperature, and H₂O (5 mL)

was added. The resulting mixture was extracted with Et₂O (3 × 5 mL),

and the combined extracts were washed with brine (2 × 5 mL), then

dried with Na₂SO₄. The mixture was concentrated in vacuo, and the

residue was puriﬁed by column chromatography (hexane/EtOAc 2:1) to

give 52 (110 mg, 96%) as a white solid. Recrystallization from hexane/

CH₂Cl₂(5:1) gave an analytical sample of 52 as colorless needles. M.p.

82–83 ^◦C. ¹H NMR (400 MHz, CDCl₃): δ = 1.13 (3H, t, J = 7.6 Hz),

1.91–2.00 (2H, m), 3.92 (3H, s), 4.01 (3H, s), 4.09 (2H, q, J = 6.3 Hz),

6.97 (1H, d, J = 7.8 Hz), 7.00–7.06 (2H, m), 7.16 (1H, t, J = 7.8 Hz),

7.25–7.30 (1H, m), 7.66 (1H, dd, J = 7.8, 1.5 Hz), 7.77 (1H, s), 7.90 ppm

(1H, dd, J = 7.8, 1.5 Hz). ¹³C NMR (100 MHz, CDCl₃): δ = 10.9, 22.6,

4.1.43. 2-(2^′,3^′-Dimethoxyphenyl)-5-(2^′′-benzyloxyphenyl)oxazole (49)

Compound 49 was synthesized from 2-bromo-2^′-benzylox-

yacetophenone (10 l) ⁵²(2.00 g, 7.7 mmol), 2,3-dimethoxybenzylamine

(11a) (1.2 mL, 9.3 mmol) in a manner similar to that described for the

synthesis of 12. Puriﬁcation by column chromatography (hexane/EtOAc

4:1) gave 49 (1.36 g, 45%) as a white solid. Recrystallization from

hexane/CH₂Cl₂(5:1) gave an analytical sample of 49 as colorless

granules. M.p. 131–132 ^◦C. ¹H NMR (400 MHz, CDCl₃): δ = 3.91 (3H, s),

3.99 (3H, s), 5.21 (2H, s), 7.01 (1H, dd, J = 8.0, 1.2 Hz), 7.06–7.10 (2H,

m), 7.14 (1H, t, J = 8.0 Hz), 7.29 (1H, td, J = 8.0, 1.5 Hz), 7.35–7.43

(3H, m), 7.49 (2H, d, J = 7.3 Hz), 7.62–7.64 (2H, m), 7.93 ppm (1H, dd,

J = 8.0, 1.5 Hz). ¹³C NMR (100 MHz, CDCl₃): δ = 56.1, 61.2, 70.6, 111.9,

14

K. Narita et al.

Bioorganic & Medicinal Chemistry 42 (2021) 116253

56.0, 61.2, 70.0, 111.5, 114.0, 117.3, 120.6, 121.3, 122.1, 124.2, 125.8,

127.5, 128.9, 147.7, 147.9, 153.8, 155.2, 158.2 ppm. FT-IR (KBr): 2972,

2941, 2878, 1562, 1527, 1493, 1476, 1421, 1391, 1345, 1249, 1167,

1130, 1084, 1043, 1005, 841, 789, 748, 724 cm^{ꢀ 1}. HRMS (EI): calcd for

C₂₀H₂₁NO₄339.1471; found 339.1471. Anal. calcd for C₂₀H₂₁NO₄: C

70.78, H 6.24, N 4.13; found C 71.01, H 6.40, N 4.16.

yellow plates. M.p. 112–113 ^◦C. ¹H NMR (400 MHz, CDCl₃): δ = 3.91

(3H, s), 3.96 (3H, s), 7.04 (1H, d, J = 8.0 Hz), 7.15 (1H, t, J = 8.0 Hz),

7.50 (1H, t, J = 8.0 Hz), 7.56 (1H, s), 7.58 (1H, dd, J = 8.0, 1.2 Hz), 7.65

(1H, t, J = 7.3 Hz), 7.80 (1H, d, J = 7.3 Hz), 7.84 ppm (1H, d, J = 8.0

Hz). ¹³C NMR (100 MHz, CDCl₃): δ = 56.0, 61.2, 114.8, 121.1, 121.4,

121.8, 124.3, 124.4, 127.5, 129.2, 129.3, 132.5, 146.0, 147.2, 147.9,

153.7, 160.8 ppm. FT-IR (KBr): 3168, 3094, 2936, 1607, 1531, 1477,

1458, 1424, 1359, 1308, 1268, 1234, 1192, 1160, 1133, 1087, 1044,

1003, 967, 847, 791, 776, 744, 726 cm^{ꢀ 1}. HRMS (EI): calcd for

4.1.47. 2-(2^′,3^′-Dimethoxyphenyl)-5-(2^′′-methoxymethoxyphenyl)oxazole

(53)

NaH (55.7 mg, 1.4 mmol) was added portionwise to a stirred solution

C₁₇H₁₄N₂O₅326.0903; found 326.0906. Anal. calcd for C₁₇H₁₄N₂O₅: C

of 50 (276 mg, 0.93 mmol) in DMF (9 mL) at 0 ^◦C under argon, and the

62.57, H 4.32, N 8.59; found C 62.76, H 4.48, N 8.60.

mixture was stirred at 0 ^◦C for 30 min. After this time, MOMCl (105

μL,

1.4 mmol) was added and the mixture was stirred at room temperature

for a further 1 h. The reaction was then quenched with saturated

aqueous NH₄Cl (5 mL), and the resulting mixture was extracted with

Et₂O (3 × 10 mL). The combined extracts were washed with brine (2 ×

10 mL), then dried with Na₂SO₄. The mixture was concentrated in

vacuo, and the residue was puriﬁed by column chromatography (hex-

ane/EtOAc, 2:1) to give 53 (286 mg, 90%) as a white solid. Recrystal-

lization from hexane/CH₂Cl₂(10:1) gave an analytical sample of 53 as

colorless needles. M.p. 82–83 ^◦C. ¹H NMR (400 MHz, CDCl₃): δ = 3.52

(3H, s), 3.91 (3H, s), 4.02 (3H, s), 5.36 (2H, s), 7.01 (1H, dd, J = 8.0, 1.2

Hz), 7.09–7.17 (2H, m), 7.18–7.22 (1H, m), 7.24–7.30 (1H, m), 7.65

(1H, dd, J = 8.0, 1.2 Hz), 7.73 (1H, s), 7.91 ppm (1H, dd, J = 8.0, 1.2

Hz). ¹³C NMR (100 MHz, CDCl₃): δ = 56.0, 56.3, 61.1, 94.1, 114.1 (2C),

117.9, 121.3, 121.9, 122.0, 124.2, 125.9, 127.4, 128.8, 147.60, 147.64,

153.1, 153.7, 158.4 ppm. FT-IR (KBr): 2940, 2903, 1561, 1527, 1491,

1349, 1263, 1229, 1196, 1158, 1126, 1083, 1045, 1003, 920, 838, 788,

754, 723 cm^{ꢀ 1}. HRMS (EI): calcd for C₁₉H₁₉NO₅341.1263; found

341.1265. Anal. calcd for C₁₉H₁₉NO₅: C 66.85, H 5.61, N 4.10; found C

66.82, H 5.70, N 4.14.

4.1.50. 2-(2^′,3^′-Dimethoxyphenyl)-5-(2^′′-aminophenyl)oxazole (57)

10% Pd/C (42.6 mg) was added to a solution of 56 (139 mg, 0.43

mmol) in MeOH (8 mL) at room temperature. The mixture was stirred

for 1 h under H₂(1 atm) at room temperature. The reaction mixture was

diluted with EtOAc (10 mL), and the catalyst was removed by ﬁltration

through a small pad of Celite. The ﬁltrate was concentrated in vacuo to

give a residue, which was puriﬁed by column chromatography (hexane/

EtOAc 2:1) to give 57 (96.6 mg, 77%) as yellow solid. Recrystallization

from hexane/CH₂Cl₂(10:1) gave an analytical sample of 57 as yellow

granules. M.p. 130–131 ^◦C. ¹H NMR (400 MHz, CDCl₃): δ = 3.92 (3H, s),

3.94 (3H, s), 4.56 (2H, br s), 6.77 (1H, d, J = 7.3 Hz), 6.81 (1H, d, J =

7.3 Hz), 7.01 (1H, dd, J = 8.0, 1.5 Hz), 7.13–7.18 (2H, m), 7.42 (1H, s),

7.53 (1H, dd, J = 8.0, 1.5 Hz), 7.66 ppm (1H, dd, J = 8.0, 1.5 Hz). ¹³

C

NMR (100 MHz, CDCl₃): δ = 56.0, 61.5, 112.7, 114.0, 116.9, 118.2,

121.1, 121.7, 123.6, 124.4, 127.1, 129.7, 143.5, 147.5, 150.8, 153.6,

158.4 ppm. FT-IR (KBr): 3471, 3380, 2934, 1607, 1559, 1530, 1475,

1422, 1345, 1320, 1266, 1127, 1084, 1046, 993, 941, 812, 789, 763,

743, 721 cm^{ꢀ 1}. HRMS (EI): calcd for C₁₇H₁₆N₂O₃296.1161; found

296.1152. Anal. calcd for C₁₇H₁₆N₂O₃: C 68.91, H 5.44, N 9.45; found C

68.77, H 5.55, N 9.42.

4.1.48. 2,3-Dimethoxy-N-(2-(2-nitrophenyl)-2-oxoethyl)benzamide (55)

Et₃N (0.45 mL, 3.3 mmol) was added dropwise to a stirred solution of

aminoketone 54 (236 mg, 1.1 mmol) and carboxcylic acid 21 (198 mg,

1.1 mmol) in CH₂Cl₂(11 mL) containing EDC⋅HCl (272 mg, 1.4 mmol)

and HOBt (191 mg, 1.4 mmol) at room temperature under argon. After

12 h, the reaction was quenched with 3 M aqueous HCl (10 mL), and the

resulting mixture was extracted with EtOAc (3 × 20 mL). The combined

extracts were washed successively with saturated aqueous NaHCO₃(2 ×

20 mL) and brine (2 × 20 mL), then dried with Na₂SO₄. The mixture was

concentrated in vacuo, and the residue was puriﬁed by column chro-

matography (hexane/EtOAc, 2:1) to give 55 (231 mg, 62%) as a white

solid. Recrystallization from hexane/CH₂Cl₂(3:1) gave an analytical

sample of 55 as colorless needles. M.p. 129–130 ^◦C. ¹H NMR (400 MHz,

CDCl₃): δ = 3.90 (3H, s), 3.98 (3H, s), 4.71 (2H, d, J = 5.0 Hz), 7.07 (1H,

dd, J = 8.0, 1.0 Hz), 7.14 (1H, t, J = 8.0 Hz), 7.59–7.67 (3H, m), 7.77

(1H, td, J = 8.0, 1.0 Hz), 8.15 (1H, d, J = 8.0 Hz), 8.89 ppm (1H, br t, J

= 5.0 Hz). ¹³C NMR (100 MHz, CDCl₃): δ = 49.5, 56.0, 61.4, 115.8,

122.6, 124.2, 124.3, 125.5, 127.8, 131.0, 134.4, 135.8, 145.7, 147.9,

152.6, 165.4, 198.4 ppm. FT-IR (KBr): 3364, 2978, 2945, 2843, 1709,

1646, 1577, 1514, 1458, 1426, 1372, 1267, 1227, 1078, 1052, 995, 849,

811, 768, 736 cm^{ꢀ 1}. HRMS (EI): calcd for C₁₇H₁₆N₂O₆344.1008; found

344.1010. Anal. calcd for C₁₇H₁₆N₂O₆: C 59.30, H 4.68, N 8.14; found C

59.32, H 4.63, N 8.14.

4.1.51. 2-(2^′,3^′-Dimethoxyphenyl)-5-(2^′′-N,N-dimethylaminophenyl)

oxazole (58)

MeI (0.13 mL, 2.0 mmol) was added to a stirred solution of 57 (100

mg, 0.34 mmol) in DMF (3.4 mL) containing K₂CO₃(280 mg, 2.0 mmol)

at room temperature, and the mixture was stirred at 60 ^◦C for 24 h. After

this time, the mixture was cooled to room temperature, and H₂O (5 mL)

was added. The resulting mixture was extracted with Et₂O (3 × 5 mL),

and the combined extracts were washed with brine (2 × 5 mL), then

dried with Na₂SO₄. The mixture was concentrated in vacuo, and the

residue was puriﬁed by column chromatography (hexane/EtOAc 2:1) to

give 58 (76.0 mg, 70%) as a white solid. Recrystallization from hexane/

CH₂Cl₂(10:1) gave an analytical sample of 58 as colorless granules. M.p.

103–104 ^◦C. ¹H NMR (400 MHz, CDCl₃): δ = 2.75 (6H, s), 3.92 (3H, s),

4.02 (3H, s), 7.02 (1H, dd, J = 8.0, 1.0 Hz), 7.11–7.15 (1H, m), 7.16 (1H,

t, J = 8.0 Hz), 7.20 (1H, dd, J = 8.0, 1.0 Hz), 7.29 (1H, td, J = 8.0, 2.0

Hz), 7.66 (1H, dd, J = 8.0, 2.0 Hz), 7.81 (1H, s), 7.87 ppm (1H, dd, J =

8.0, 2.0 Hz). ¹³C NMR (100 MHz, CDCl₃): δ = 44.2 (2C), 56.1, 61.2,

114.1, 119.6, 121.4, 122.2, 122.8, 123.1, 124.3, 126.87, 126.91, 128.8,

147.7, 149.3, 151.1, 153.8, 158.3 ppm. FT-IR (KBr): 2941, 2831, 2784,

1561, 1527, 1457, 1342, 1303, 1260, 1230, 1183, 1127, 1083, 1044,

1002, 946, 841, 792, 744, 725 cm^{ꢀ 1}. HRMS (EI): calcd for C₁₉H₂₀N₂O₃

324.1474; found 324.1470. Anal. calcd for C₁₉H₂₀N₂O₃: C 70.35, H

6.21, N 8.64; found C 70.31, H 6.36, N 8.63.

4.1.49. 2-(2^′,3^′-Dimethoxyphenyl)-5-(2^′′-nitrophenyl)oxazole (56)

A solution of 55 (1.12 g, 3.4 mmol) in phosphoryl chloride (10 mL)

was heated at reﬂux for 3 h. After cooling to room temperature, con-

centration of the solvent in vacuo afforded a residue, which was diluted

with CH₂Cl₂(200 mL). The organic layer was washed successively with

water (2 × 50 mL), saturated aqueous NaHCO₃(2 × 50 mL) and brine (2

× 50 mL), then dried with MgSO₄. The mixture was concentrated in

vacuo, and the residue was puriﬁed by column chromatography (hex-

ane/EtOAc, 1:2) to give 56 (490 mg, 71%) as a yellow solid. Recrys-

tallization from hexane/EtOAc (3:1) gave an analytical sample of 56 as

4.1.52. 2-(2^′,3^′-Dimethoxyphenyl)-5-(2^′′-acetoamidophenyl)oxazole (59)

Acetyl chloride (0.13 mL, 1.9 mmol) was added dropwise to a stirred

solution of 57 (288 mg, 0.97 mmol) in CH₂Cl₂(10 mL) containing Et₃N

(0.54 mL, 3.9 mmol) at 0 ^◦C under argon. After 3 h, the reaction was

quenched with saturated aqueous NaHCO₃(5 mL), and the resulting

mixture was extracted with CHCl₃(3 × 10 mL). The combined extracts

were washed with brine (2 × 10 mL), then dried with Na₂SO₄. The

mixture was concentrated in vacuo, and the residue was puriﬁed by

column chromatography (hexane/EtOAc, 3:1) to give 59 (285 mg, 87%)

15

K. Narita et al.

Bioorganic & Medicinal Chemistry 42 (2021) 116253

5

6

Gizaw Y, Megersa M, Fayera T. Dourine: a neglected disease of equids. Trop Anim

Health Prod. 2017;49:887–897. https://doi.org/10.1007/s11250-017-1280-1.

La Greca F, Magez S. Vaccination against trypanosomiasis: can it be done or is the

trypanosome truly the ultimate immune destroyer and escape artist? Hum Vaccin.

2011;7:1225–1233. https://doi.org/10.4161/hv.7.11.18203.

as a white solid. Recrystallization from hexane/CHCl₃(3:1) gave an

analytical sample of 59 as colorless needles. M.p. 179–180 ^◦C. ¹H NMR

(400 MHz, CDCl₃): δ = 2.28 (3H, s), 3.93 (3H, s), 3.95 (3H, s), 7.05 (1H,

d, J = 8.0 Hz), 7.16–7.20 (2H, m), 7.39 (1H, t, J = 8.0 Hz), 7.44 (1H, s),

7.61 (1H, d, J = 8.0 Hz), 7.68 (1H, d, J = 8.0 Hz), 8.35 (1H, d, J = 8.0

Hz), 8.60 ppm (1H, br s). ¹³C NMR (100 MHz, CDCl₃): δ = 24.6, 56.0,

61.2, 114.6, 117.8, 121.1, 123.1, 124.4, 124.6, 125.2, 127.2, 129.7,

134.5, 147.6, 149.6, 153.7 (2C), 159.5, 168.9 ppm. FT-IR (KBr): 3264,

2939, 1656, 1584, 1568, 1524, 1476, 1351, 1302, 1262, 1150, 1134,

1088, 1045, 1006, 969, 844, 788, 768, 743, 722 cm^{ꢀ 1}. HRMS (EI): calcd

for C₁₉H₁₈N₂O₄338.1267; found 338.1269. Anal. calcd for C₁₉H₁₈N₂O₄:

C 67.45, H 5.36, N 8.28; found C 67.41, H 5.36, N 8.23.

7

Steverding D. The development of drugs for treatment of sleeping sickness: a

historical review. Parasites Vectors. 2010;3:1–9. https://doi.org/10.1186/1756-3305-

3-15.

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Capela R, Moreira R, Lopes F. An overview of drug resistance in protozoal diseases.

Int J Mol Sci. 2019;20:5748. https://doi.org/10.3390/ijms20225748.

Pelfrene E, Harvey Allchurch M, Ntamabyaliro N, et al. The European Medicines

Agency’s scientiﬁc opinion on oral fexinidazole for human African trypanosomiasis.

PLoS Negl Trop Dis. 2019;13, e0007381. https://doi.org/10.1371/journal.

pntd.0007381.

10 Sokolova AY, Wyllie S, Patterson S, Oza SL, Read KD, Fairlamb AH. Cross-resistance

to nitro drugs and implications for treatment of human African trypanosomiasis.

Antimicrob Agents Chemother. 2010;54:2893–2900. https://doi.org/10.1128/

AAC.00332.

4.2. Biological evaluation

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jnatprod.8b00159.

Trypanocidal activity of synthesized compounds against 5 trypano-

some species and cytotoxicity of synthesized compounds against MDBK

cell were evaluated in vitro using 96-well cell culture plate format as

previously established with slight modiﬁcation.^44,45Brieﬂy, for trypa-

nocidal activity evaluation, each well contain 100 µL of trypanosomes as

5 × 10³cells/mL (T. b. brucei, T. b. gambiense and T. b. rhodesiense) or 1

× 10⁴cells/mL (T. evansi) or 2 × 10⁵cells/mL (T. congolense) were

cultivated with compounds from 25 µg/mL to 1.6 ng/mL by 5 times

serial dilution. After incubation for 72 h in 37 ^◦C (T. b. brucei, T. b.

gambiense, T. b. rhodesiense and T. evansi) or 33 ^◦C (T. congolense), 25 µL

of CellTiter-Glo^TMLuminescent Cell Viability Assay reagent (Promega

Japan, Tokyo, Japan) was added to evaluate intracellular ATP concen-

tration according to the instruction. For cytotoxicity evaluation, each

well contain 1 × 10⁴cells/mL of MDBK cell were cultivated with com-

pounds from 100 µg/mL to 6.4 ng/mL by 5 times serial dilution. After

incubation for 72 h in 37 ^◦C, OD450 nm was measured at 0 and 4 h after

adding 10 µL of Cell Counting Kit-8 (Dojindo laboratories, Kumamoto,

Japan). The trypanocidal activity and cytotoxicity evaluations were

replicated three times. The IC₅₀s were calculated using GraphPad Prism

version 8 (GraphPad Software, San Diego, CA, USA).

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Declaration of Competing Interest

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The authors declare that they have no known competing ﬁnancial

interests or personal relationships that could have appeared to inﬂuence

the work reported in this paper.

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Acknowledgments

This study was ﬁnancially supported by a Cooperative Research

Grant (2019-joint-5, 2020-joint-13) of the National Research Center for

Protozoan Diseases, Obihiro University of Agriculture and Veterinary

Medicine, and the Japan Society for the Promotion of Science (JSPS)

KAKENHI (Grant Number JP20K15960 to K.N.).

24 Trefzger OS, das Neves AR, Barbosa NV, Carvalho DB, Pereira IC, Perdomo RT, et al..

Design, synthesis and antitrypanosomatid activities of 3, 5-diaryl-isoxazole

analogues based on neolignans veraguensin, grandisin and machilin G. Chem Biol

Drug Des 2019;93:313-324. https://doi.org/10.1111/cbdd.13417.

25 da Rosa R, de Moraes MH, Zimmermann LA, Schenkel EP, Steindel M, Bernardes LSC.

Design and synthesis of a new series of 3,5-disubstituted isoxazoles active against

Trypanosoma cruzi and Leishmania amazonensis. Eur J Med Chem. 2017;128:25–35.

https://doi.org/10.1016/j.ejmech.2017.01.029.

Appendix A. Supplementary material

26 Li J, Zhu SR, Xu Y, et al. Synthesis of 2,5-diaryloxazoles through rhodium-catalyzed

annulation of triazoles and aldehydes. RSC Adv. 2020;10:24795–24799. https://doi.

org/10.1039/D0RA03966G.

Supplementary data to this article can be found online at https://doi.

org/10.1016/j.bmc.2021.116253.

27 Yu X, Chen K, Wang Q, Zhang W, Zhu J. Synthesis of 2, 5-disubstituted oxazoles via

cobalt (iii)-catalyzed cross-coupling of N-pivaloyloxyamides and alkynes. Chem

Commun. 2018;54:1197–1200. https://doi.org/10.1039/C7CC08611C.

28 Chatterjee T, Cho JY, Cho EJ. Synthesis of substituted oxazoles by visible-light

photocatalysis. J Org Chem. 2016;81:6995–7000. https://doi.org/10.1021/acs.

joc.6b00989.

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Synthesis and evaluation of trypanocidal activity of derivatives of naturally occurring 2,5-diphenyloxazoles

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