Potential of aryl-urea-benzofuranylthiazoles hybrids as multitasking agents in Alzheimer's disease

ACCEPTED MANUSCRIPT

Potential of Aryl-urea-Benzofuranylthiazoles hybrids as multitasking agents in

Alzheimer’s disease

a

b

c

d

Belma Zengin Kurt, Isil Gazioglu, Livia Basile,* Fatih Sonmez,* Tiziana Ginex, Mustafa

e

b

Kucukislamoglu and Salvatore Guccione

a

Bezmialem Vakif University, Faculty of Pharmacy, Department of Analytical and Medicinal

Chemistry, 34093, Istanbul, TURKEY

b

Department of Drug Sciences, University of Catania, Viale A. Doria 6 Ed. 2, Città

Universitaria, I- 95125, Catania, ITALY

c

Sakarya University, Pamukova Vocational High School, 54900, Sakarya, TURKEY

d

Molecular Modelling Laboratory, Department of Food Science, University of Parma, Parco

Area delle Scienze 17/A, Parma 43124, ITALY

e

Sakarya University, Faculty of Arts and Science, Department of Chemistry, 54055, Sakarya,

TURKEY

*

Corresponding authors: University of Catania, Department of Drug Sciences, Viale A. Doria 6

Ed. 2, Città Universitaria, I- 95125, Catania, ITALY. Tel. +39 095 738-4020; fax +39 095 738-

208; email address: basilelivia@gmail.com (L. Basile); Sakarya University, Pamukova

Vocational High School, 54900, Sakarya, TURKEY. Tel.: +90-264-2953378; fax: +90-264-

953679; e-mail address: fsonmez@sakarya.edu.tr (F.Sonmez).

4

2

Keywords:

Aryl-urea-Benzofuranylthiazoles,

Inhibitors,

Acetylcholinesterase,

Butyrylcholinesterase, Antioxidant, Docking.

Abstract

New benzofuranylthiazole derivatives containing the aryl-urea moiety were synthesized and

evaluated in vitro as dual acetylcholinesterase (AChE)-butyrylcholinesterase (BuChE) inhibitors.

In addition, the cupric reducing antioxidant capacities (CUPRAC) and ABTS cation radical

scavenging abilities of the synthesized compounds were assayed. The result showed that all the

synthesized compounds exhibited inhibitory activity on both AChE and BuChE with 1-(4-(5-

bromobenzofuran-2-yl)thiazol-2-yl)-3-(2-fluorophenyl)urea (e25, IC value of 3.85 µM) and 1-

5

0

1

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(

4-iodophenyl)-3-(4-(5-nitrobenzofuran-2-yl)thiazol-2-yl)urea (e38, IC value of 2.03 µM) as the

5

0

strongest inhibitors against AChE and BuChE, respectively. Compound e38 was 8.5-fold more

potent than galanthamine. The selectivity index of e25 and e38 was 2.40 and 0.37 against AChE

and BuChE, respectively. Compound e2, e4 and e11 (IC = 0.2, 0.5 and 1.13 µM, respectively)

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0

showed a better ABTS cation radical scavenging ability than the standard quercetin (IC50= 1.18

µM). Best poses of compounds e38 on BuChE and e25 on AChE indicate that the thiazole ring

and the amidic moiety are important sites of interaction with both ChEs. In addition, the

benzofuran ring and phenyl ring are anchored to the side chains of both enzymes by π-π(pi-pi)

interactions.

1

. Introduction

Alzheimer’s disease (AD) is the most common neurodegenerative disease with symptoms of

memory loss, cognition defect and behavioural impairment [1-3]. The classical hypothesis of AD,

named “cholinergic hypothesis”, suggests that acetylcholinesterase inhibitors (AChEI) could

increase ACh levels in AD patients through the inhibition of AChE and, therefore, relieve some

symptoms experienced by AD patients [4,5]. AD is probably associated with multifaceted

etiologies and pathogenic phenomena. In any case, oxidative stress can be considered the

causative unifying factor [6].

Cholinergic system is the earliest and most profoundly affected neurotransmitter system in AD,

with substantial loss of the forebrain, cortex, and hippocampus. Ach and the above mentioned

brain regions are critical in the acquisition, processing, and storage of memories and have

supported the use of cholinomimetics in the treatment of AD [7]. It is well known that two forms

of cholinesterases coexist ubiquitously throughout the body, i.e., acetylcholinesterase (AChE; EC

3

.1.1.7) and butyrylcholinesterase (BuChE; EC 3.1.1.8). Among its functions, AChE regulates

the impulse transmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter

ACh. BuChE, also known as pseudocholinesterase, is primarily localized in plasma, liver, and

muscle tissues.

The pharmacological role of BuChE was not yet completely understood but it is supposed that it

may have a compensatory role in the modulation of the hydrolysis of ACh in brains causing

degenerative changes. Consequently, BuChE may be a target for increasing the cholinergic tone

in AD patients [8,9].

Based on these findings, many efforts have been made in the search for potent AChE inhibitors,

and a large number of naturally occurring and synthetic AChE inhibitors such as galantamine,

2

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huperzine A, physostigmine, ambenonium and tacrine have already been reported (Fig. 1) [10-

1

3].

Crystallographic structure of AChE from Torpedo californica [14] shows three main

binding sites, namely (a) the catalytic triad at the bottom of active site including Ser200, His440

and Glu327; (b) the catalytic anionic site (CAS) at the vicinity of the catalytic triad consisting of

Trp84, Tyr130, Gly199, His441 and His444; (c) peripheral anionic site (PAS) at the gorge rim

comprising Tyr70, Asp72, Tyr121, Trp279 and Tyr334 [15,16]. Inhibition of AChE can be

accomplished by three different ways depending on the nature of the interaction of the inhibitor

with the enzyme binding site [17]. Irreversible inhibitors, such as organophosphates, form a

strong covalent bond with the serine residue in the catalytic triad. Pseudo-irreversible inhibitors,

as carbamates, lead to formation of a carbamylated serine into the catalytic triad that is slowly

hydrolysed to regenerate the active enzyme. Reversible inhibitors give a transient non-covalent

binding through electrostatic interactions with the active and/or peripheral sites. The reversible

inhibitors may be classified as (a) active-site inhibitors directed toward the catalytic anionic sub-

site at the bottom of the gorge, (b) peripheral anionic site inhibitors which bind at the entrance to

the gorge, or (c) elongated gorge-spanning inhibitors which bridge the two sites [17].

Recently, some works on AChE inhibitors with a benzofuran moiety have been reported [18,19].

Amide or imide-based AChE inhibitors have also been reported [20-21] with both these

functionalities acting as hydrogen bond donors towards oxygen or nitrogen lone pairs of Tyr72,

Tyr124, Tyr203 and Tyr337 of the enzyme. Amidic or imidic fragments interact with the

catalytic triad Ser203-Glu334-His447 of the active binding site. Hydrogen bonds with Tyr70 and

His447 were also reported for compounds having urea, carbamate or sulphonamide moieties as

spacers [22-25].

Dibenzofuran and tricyclic tacrine-ferrulic acid derivatives as multifunctional anti-Alzheimer

agents were also previously reported by Fang and co-workers [26,27]. Tricyclic and heterocyclic

rings derivatives, such as tacrine, quinolizidinyl, piperidine and indolinone derivatives give

strong parallel π-π (pi-pi) stacking with residues at CAS [28-33]. An additional π-π (pi-pi)

stacking interaction was also observed between the furan ring and Phe330 [18,19]. Moreover, it

has been reported that thiazolo-triazin derivatives form a hydrogen bond with Tyr124 and π-π

interaction with Trp286 [34]. Docking into the BuChE gorge, which is larger than that of AChE,

showed that heterocyclic rings give (i) a cation-π interaction with Trp82 and Trp430 and also (i)

hydrophobic and/or π-π (pi-pi) stacking interactions with Phe118 and Trp231 [3,24].

3

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Overall, heterocyclic and aromatic rings can have strong parallel π-π stacking with residues at the

CAS of the enzyme whereas the urea moiety might contribute to inhibitor activity by additional

interactions.

On the basis of the above reported evidences a series of 38 novel urea substituted benzofuran

derivatives (e1-e38), including the thiazole ring as an additional spacer, was designed and

synthesized (Fig. 2). AChE/BuChE inhibition and antioxidant properties were evaluated.

Structure-activity relationships are described and rationalized by docking studies.

2

. Results and discussion

2

.1. Chemistry

The synthetic procedures are depicted in Scheme 1. 2-Acetyl benzofuran derivatives b1-b3 were

prepared as previously reported [35]. 1-(1-benzofuran-2-yl)-2-bromoethanone derivatives (c1-c3)

were prepared by brominating of 2-acetyl benzofuran using molecular bromine in chloroform.

The reaction of c1-c3 with thiourea in ethanol gave 4-(1-benzofuran-2-yl)-1,3-thiazol-2-amines

(

d1-d3). These compounds were reacted with arylisocyanates in THF to get the final products

e1-e38) at high yields.

(

1

13

All the new compounds were characterized through H NMR, C NMR, IR, MS and elemental

-1

analysis. Infrared spectra for e1-e38 show absorptions between 3500 and 3000 cm related to N-

-1

H stretching, absorptions at 1650-1700 cm from the urea carbonyl moiety stretching and

-1

absorptions at 1550 cm for the thiazole C=N moiety stretching. Furthermore, absorptions among

-1

3

111 cm and 2950 cm indicated C-H stretching for the thiazole and furan rings, respectively.

1

In case of H NMR spectra, the resonance for the hydrogen attached to the amide nitrogen was

between 8.20 and 11.50 ppm. Signals for aromatic protons were observed between 6.50 and 8.52

ppm and those for the proton of thiazole and furan ring were detected around 7.10 and 7.50 ppm

1

3

as a singlet. Regarding C NMR spectra, carbon atoms of urea carbonyl, benzofuran ring (C

2

and

C ) and thiazole ring (C ) were observed between 150.7 and 161.4 ppm.

8

3

2

.2. Biological activities

.2.1. Inhibitory activities on AChE and BuChE

The Ellman’s method with galanthamine as the reference compound [36] was applied to measure

AChE and BuChE inhibitory activity for all synthetized compounds (e1-e38). The IC values for

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0

AChE and BuChE inhibition are summarized in Table 1.

The results show that the synthesized compounds exhibit low to moderate AChE inhibition. IC₅₀

4

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values are between 3.85 and 78.85 µM for AChE and between 2.03 and 154.08 µM for BuChE.

Among the synthesized compounds, e25 (IC50 = 3.85 µM) shows the highest inhibitory activity

on AChE. The value was less than that of galantamine (IC = 2.41 µM) but close to that of

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rivastigmine (IC = 3.01 µM).

5

0

e38 exhibited the strongest inhibition against BuChE with an IC50 value of 2.03 µM, which was

.5-fold and 2.3 more than that of galanthamine (IC = 17.38 µM) and donepezil (IC = 4.66

8

5

0

50

µM), respectively. Furthermore, eight of the synthesized compounds (e8, e11, e20, e22, e24, e25,

e33 and e38) showed higher inhibition against BuChE than galanthamine.

Moving the fluorine atom in the phenyl ring from the orto-position (e25, IC = 3.85 µM for

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0

AChE, IC50 9.25 µM for BuChE) to the meta-position (e26, IC50 = 29.38 for AChE, IC50 = 40.16

for BuChE) or the para-position (e27, IC = 35.62 for AChE, IC = 64.32 for BuChE) led to a

5

0

50

major decline of the inhibitory activity for both ChEs (Table 1). An opposite trend was observed

for compounds e9 (IC = 30.92 µM for AChE, IC = 41.66 µM for BuChE), e10 (IC = 48.99

5

0

50

µM for AChE, IC = 17.98 µM for BuChE) and e11 (IC = 55.03 µM for AChE, IC = 7.45

5

0

50

µM for BuChE) were the above mentioned replacement led to a decrease of the inhibitory

activities against AChE (Table 1), but to an increase in the BuChE inhibition (Table 1). It can be

speculated on a more volume allowed in the active site of the latter enzyme to accommodate the

alternate meta and para substitutions (vide infra Docking Results). Hydrophobic interactions also

combined with the H-bond acceptor character of the fluorine atom might play a role.

Moving the chlorine atom in the phenyl ring from the meta-position (e12, IC50 = 9.67 µM for

AChE, IC₅₀= 21.52 µM for BuChE; e28, IC₅₀= 7.93 µM for AChE, IC₅₀= 26.32 µM for

BuChE) to the para-position (e13, IC50 = 42.55 µM for AChE, IC50 = 38.58 µM for BuChE; e29,

IC = 22.23 µM for AChE, IC = 36.93 µM for BuChE) led to a decrease of the inhibitory

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50

activity for both ChEs (Table 1).

Moving the nitro group in the phenyl ring from the orto-position (e6, IC50 = 41.91 µM for AChE,

IC = 79.93 µM for BuChE) to the meta-position (e7, IC = 41.19 µM for AChE, IC = 26.81

5

0

50

µM for BuChE) and above all the para-position (e8, IC50 = 5.23 µM for AChE, IC50 = 3.44 µM

for BuChE) led to a major enhancement of the inhibitory activity for both ChEs. This effect was

not observed for compounds e22 (IC = 8.82 µM for AChE, IC = 5.12 µM for BuChE), e23

5

0

50

(

IC50 = 17.45 µM for AChE, IC50 = 78.97 µM for BuChE) and e24 (IC50 = 4.78 µM for AChE,

IC = 3.12 µM for BuChE), with a R was bromine atom as R substituent. The bromine atom

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0

1

might lead to some unfavourable positioning of the nitro group which doesn’ t allow for good

interactions.

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Compounds with a methoxy group in the meta- position of the phenyl ring (e2, IC = 48.90 µM

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0

for AChE, IC50 = 59.56 µM for BuChE) exhibited higher inhibitory activity than that with the

same substituent in the ortho or para positions (e1, IC = 60.97 µM for AChE, IC = 81.28 µM

5

0

50

for BuChE; e3, IC = 52.47 µM for AChE, IC = 74.26 µM for BuChE) for both ChEs (Table

5

0

50

1

). It is supposed that methoxy substituent can better act as H-bond acceptor binding more tightly

to both enzymes finally enhancing the inhibitory effects.

Alternatively this favourable effect might be explained as a consequence of a different electron

density which is lower in the meta- position on respect to that ortho- and para.

Overall, the inhibitory activity on AChE seems to be strongly dependent on the size and

polarizability of the halogen substituent at the para-position of the phenyl ring (for size and

polarizability, I > Br > Cl > F; for AChE inhibitory activity, e16 (R =4-I, IC = 21.35 µM) > e15

2

50

(R

2

=4-Br, IC50 = 32.82 µM) > e13 (R

2 2

=4-Cl, IC50 = 42.55 µM) > e11 (R =4-F, IC50 = 55.03

µM); e31 (R =4-I, IC = 6.42 µM) > e30 (R =4-Br, IC = 11.94 µM) > e29 (R =4-Cl, IC =

2

50

2

50

2

50

2

2.23 µM) > e27 (R =4-F, IC = 35.62 µM); e38 (R =4-I, IC = 5.53 µM) > e37 (R =4-Br, IC

2

50

2

50

2

50

=

2 2

28.19 µM) > e36 (R =4-Cl, IC50 = 30.56 µM) > e35 (R =4-F, IC50 = 41.13 µM). This

relationship was not observed for BuChE.

Hydrophobic contacts and π-π stacking interactions between the phenyl, thiazole and

benzofuran rings of the synthesized compounds and CAS or PAS of AChE might take place. A

hydrogen bond might form between urea moiety and Tyr70 or His447 in the active site.

2

.2.2. Docking studies

AChE and BuChE share the 65% of amino acid sequence homology and overall have a similar

structure [37]. Six of the fourteen aromatic amino acid residues that constitute the active site

gorge of AChE are replaced by aliphatic amino acid residues in BuChE. Thereof the volume of

the BuChE active site gorge is larger (~ 200 Å3) than that of AChE. The replacement of aromatic

with aliphatic amino acids is also critical for the selectivity against different inhibitor of the two

enzymes [38]. Docking studies performed on both ChEs reveal that all the title compounds have

multiple binding modes. To reach a plausible positioning of the compounds into the active site of

both ChEs, the allowed search space was restricted to the region between CAS and PAS sites of

both AChE and BuChE. All the compounds gave a suitable occupation of the active binding site

of the two enzymes, assuming different geometries stabilized by interactions with the side chains

of CAS and PAS (Fig. 3). Overall, the thiazole ring has a critical role for its capacity to form H

bond interaction with Tyr121 of AChE. Docking scores for compounds e25 (IC = 3.85 ꢀM

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against AChE) and e38 (IC = 2.03 ꢀM against BuChE) are in agreement with the experimental

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in vitro data. In this regard, the former exhibits higher binding affinity for AChE whereas the

latter gave a better scoring result for BuChE binding. 2D LigPlot diagrams [39] showing the

interactions of the best poses for compounds e25 and e38 respectively into the active site of

AChE site and BuChE as visualized by Python Molecular viewer [40], are shown in Fig. 4 and

Fig. 5. Thiazole ring of e25 gives H bond interaction through its nitrogen that acts as acceptor

towards the hydroxyl group of Tyr121 (hydrogen bond interaction energy, EH: -8.10) of AChE.

Another H bond can be considered for nitrogen of the amidic moiety that acts as acceptor towards

the hydroxyl group of Ser122 (EH: -7.95). Furthermore, favourable steric interactions can be

established with Asp72, Gly117, Gly118, Gly123, Gly335, Phe330, Ser124, Trp279, Tyr116,

Tyr121 and Tyr130 of AChE. The phenyl moiety of e25 points toward the PAS pocket and gives

T-stacking with the phenolic side chain of Tyr84 (π-π interaction). The benzofuran group stacks

between the benzyl moiety of Phe331 (Steric Interaction Energy, ES: -15.56) and the phenolic

one of Tyr334 (ES: -27.82), stabilizing the accommodation of compound e25 at the entrance of

the active binding site. Another T-stacking interaction can be found between the thiazole ring of

e25 and the benzyl side chain of Phe330 (ES: -9.44) [41].

Binding of e38 into the BuChE gorge is characterized by the formation of the following three H

bond interactions: (1) the nitrogen of thiazole ring as H-bond acceptor for Thr120, (2) the

nitrogen of the nitro group as H-bond acceptor for Tyr128 hydroxyl group and (3) the oxygen of

nitro group as H-bond acceptor for nitrogen of Gly115. This result can be due to the fact that

BuChE binding site is bigger than the AChE one, therefore e38 gains a better positioning into the

BuChE binding site, finally leading to the formation of a more stable complex. Orto-Fluorine

atom lies outside the gorge of BuChE. The two nitrogen atoms of the amidic moiety form a salt

bridge with the side chain of Asp70. A strong π-π(pi-pi) interaction (ES: -35.21) is observed

between the benzofuran ring and the Trp82 indolyl moiety. In addition, favourable steric

interactions occur with the side chain of Asn68, Asn83, Gln71, Glu197, Gly116, Gly439, Ile69,

Tyr114, Trp112 and His438.

2

.3. Antioxidant activity assay

.3.1. ABTS cation radical scavenging assay

The ABTS method is based on the ability of hydrogen or electron-donating antioxidants to

decolourize the performed radical monocation of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic

acid) generated due to oxidation of ABTS with potassium persulfate [42]. Five of the synthesized

7

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compounds exhibited good radical scavenging ability (Table 1). Particularly compounds e2, e4

and e11 (IC50= 0.2, 0.5 and 1.13 µM, respectively) lacking of substituents on the benzofuran ring

showed significantly better activity than quercetin (IC = 1.18 µM). Compounds e18-e37,

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0

containing NO - and Br- substituents at 5-position of the benzofuran ring, showed significantly

2

less ABTS cation radical scavenging.

2

.3.2. CUPRAC assay

CUPRAC assays have a distinct advantage over other electron-transfer based assays (e.g., Folin,

FRAP, ABTS, DPPH). This advantage is its realistic pH close to that physiological, favourable

redox potential, accessibility and stability of reagents and applicability to lipophilic antioxidants

as well as hydrophilic ones [43]. The cupric reducing antioxidant capacities of the synthesized

compounds (e1-e38) were determined according to a reported method [41] using quercetin as the

reference compound. Among the synthesized compounds, e17 (A0.50 = 92.68 µM) showed the

highest cupric reducing antioxidant activity. The synthesized compounds have lower CUPRAC

activity than quercetin (A₀_.₅₀= 1.45 µM) (Table 1).

+

.

The antioxidant results coming from the ABTS and CUPRAC assays are, not comparable

because of the differences in the methods. In fact, the interaction between an antioxidant and

+

.

ABTS is an indication of the ability of the hydroxyl or amine functional group to react with or

+

.

scavenge free radicals. In addition, the trapping of ABTS by hydroxyl or amine-type

+

.

antioxidants can be regarded as a hydrogen atom transfer from O–H or N-H to ABTS [44]. The

CUPRAC method is based on reduction of Cu(II) to Cu(I) by reductants (antioxidants) present in

a sample [45]. Compounds e2 (A0.50 = 104.46 µM), e4 (A0.50 = 192.91 µM), e7 (A0.50 >200 µM)

and e11 (A₀_.₅₀= 110.31 µM)have a high electron or hydrogen atom transfer ability, but they don’t

have reducing ability of Cu(II) to Cu(I).

3

. Conclusion

A series of 38 novel urea substituted benzofuranylthiazoles derivatives (e1-e38) was synthesized

and their inhibitory activities on AChE and BuChE and antioxidant activities were evaluated. All

the synthesized compounds inhibited AChE and BuChE. Among them, e25 (IC₅₀= 3.85 ꢀM

against AChE) was found to be the most active as AChE inhibitor whereas e38 (IC50 = 2.03 ꢀM

against BuChE) exhibited the strongest inhibition against BuChE with IC value of 2.03 µM,

50

which was 8.5-fold more potent than that of galanthamine. Additionally, most of the synthesized

compounds (e2, IC = 48.90 µM for AChE, IC = 59.56 µM for BuChE; e4, IC =10.40 µM for

5

0

50

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AChE, IC₅₀= 56.32 µM for BuChE; e7, IC₅₀= 41.19 µM for AChE, IC₅₀= 26.81 µM for

BuChE; e9, IC50 = 30.92 µM for AChE, IC50 = 41.66 µM for BuChE; e11, IC50 = 55.03 µM for

AChE, IC = 7.45 µM for BuChE) exhibited good ABTS cation radical scavenging ability with

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0

e2 (IC = 48.90 µM for AChE, IC = 59.56 µM for BuChE), e4 (IC = 10.40 µM for AChE,

5

0

50

IC50 = 56.32 µM for BuChE) and e11 (IC50 = 55.03 µM for AChE, IC50 = 7.45 µM for BuChE)

showing significantly better activity than quercetin. The SAR revealed that the inhibitory activity

of the synthesized compounds could also be affected by the type and position of the halogen

substituent on the phenyl ring.

Overall benzofuranylthiazole shows an interesting potential as a new chemotype to develop

multifunctional agents in AD by properly modulating the substitution pattern.

4

. Experimental section

4

.1. Chemistry. General information

Melting points were taken on a Barnstead Electrothermal 9200. IR spectra were registered on a

1

13

Shimadzu Prestige-21 (200 VCE) spectrometer. H and C NMR spectra (Supplementary

Materials) were registered on a Varian Infinity Plus spectrometer at 300 and at 75 Hz,

1

13

respectively. H and C chemical shifts are referenced to the internal deuterated solvent. Mass

spectra were obtained using MICROMASS Quattro LC-MS-MS spectrometer. The elemental

analyses were carried out with a Leco CHNS-932 instrument. Spectrophotometric analyses were

performed by a BioTek Power Wave XS (BioTek, USA). The electric eel acetylcholinesterase

(

AChE, Type-VI-S, EC 3.1.1.7, 425.84 U/mg, Sigma) and horse serum butyrylcholinesterase

BuChE, EC 3.1.1.8, 11.4 U/mg, Sigma) were purchased from Sigma (Steinheim, Germany). The

(

other chemicals and solvents were purchased from Fluka Chemie, Merck, Alfa Easer and Sigma-

Aldrich.

4

2

.1.1. General procedures of synthesis and spectral data

.1.1.1. Synthesis of 2-acetyl benzofuran derivatives (b1-3)

-Acetyl benzofuran derivatives were prepared in accordance with previously reported methods

[35]. Salicylaldehydes (100 mmol) and KOH (100 mmol) were stirred in methanol (250 ml) for

3

2

0 minutes then chloroacetone (120 mmol) was dropped at 0–10°C. The mixture was reﬂuxed for

h (or 48 hours for b3). Methanol was removed using a rotary evaporator, and the mixture was

extracted with CH Cl (3x 50 mL). The organic layer was dried over anhydrous Na SO and

2

4

removed using a rotary evaporator. The product was recrystallized from ethanol.

9

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1

-(Benzofuran-2-yl)ethanone (b1): 78% yield, mp. 76-77 °C (lit. [35] mp. 75°C); H NMR

(

DMSO-d , 300 MHz) δ/ppm: 2.65 (3H, s), 7.31–7.34 (1H, m), 7.45–7.52 (2H, m), 7.60 (1H, d),

6

1

3

7

1

.73 (1H, d); C NMR (DMSO-d , 75 MHz) δ/ppm: 27.8, 111.8, 112.4, 124.6, 125.3, 127.4,

6

29.6, 151.8, 155.4, 187.2.

-(5-Bromo-benzofuran-2-yl)ethanone (b2): 85% yield, mp. 110-111 °C (lit. [35] mp. 109–

1

11°C); H NMR (DMSO-d , 300 MHz) δ/ppm: 2.72 (3H, s), 7.49 (1H, s), 7.52 (1H, d), 7.57

6

1

3

(

6

1H, d), 7.85 (1H, s); C NMR (DMSO-d , 75 MHz) δ/ppm: 27.9, 111.7, 113.3, 116.9, 125.8,

1

29.3, 132.2, 153.9, 155.6, 188.8.

-(5-Nitrobenzofuran-2-yl)ethanone (b3): 65% yield, mp. 174-176 °C (lit. [35] mp. 175–177°C);

1

6

H NMR (DMSO-d , 300 MHz) δ/ppm: 2.66 (3H, s), 7.62 (1H, s), 7.69 (1H, d), 8.38 (1H, d),

1

3

8

1

4

2

.66 (1H, s); C NMR (DMSO-d , 75 MHz) δ/ppm: 27.6, 111.8, 112.1, 118.8, 124.4, 126.7,

6

42.9, 154.8, 156.8, 187.5.

.1.1.2. Synthesis of 2-bromoacetylbenzofuran derivatives (c1-3)

-Bromoacetyl benzofuran derivatives were prepared in accordance with previously reported

methods [46]. A solution of bromine (1 mol) in chloroform was added to a solution of 2-acetyl

benzofuran (1 mol) in chloroform. The mixture was stirred at 50 °C for 15 min. The precipitate

was filtered and washed with ether. The product was recrystallized from acetic acid.

1

3

8

1

2

-(1-Benzofuran-2-yl)-2-bromoethanone (c1): 80% yield, mp. 90–92 °C; H NMR (DMSO-d ,

6

00 MHz) δ/ppm: 4.85 (2H, s), 7.40–7.45 (1H, t), 7.55–7.61 (1H, m), 7.88 (1H, t), 8.05 (1H, s),

13

.12 (1H, s); C NMR (DMSO-d

6

, 75 MHz) δ/ppm: 49.7, 111.7, 117.1, 120.9, 122.5, 123.9,

35.5, 151.4, 154.8, 159.8, 185.8.

1

-Bromo-1-(5-bromo-1-benzofuran-2-yl) ethanone (c2): 76% yield, mp. 140–142 °C; H NMR

(DMSO-d , 300 MHz) δ/ppm: 4.87 (2H, s), 7.52–7.62 (1H, m), 7.87 (1H, t), 8.07 (1H, s), 8.20

6

1

3

(

1H, s); C NMR (DMSO-d , 75 MHz) δ/ppm: 48.9, 112.5, 116.9, 117.2, 125.4, 130.1, 137.1,

6

1

53.3, 161.2, 185.0.

1

2

-Bromo-1-(5-nitro-1-benzofuran-2-yl) ethanone (c3): 72% yield, mp. 211–213 °C; H NMR

1

3

(

DMSO-d

6

, 300 MHz) δ/ppm: 4.59 (2H, s), 7.68–7.82 (2H, m), 8.40 (1H, d), 8.71 (1H, s); C

NMR (DMSO-d , 75 MHz) δ/ppm: 50.1, 114.7, 117.2, 118.3, 121.4, 132.5, 147.1, 155.1, 169.0,

6

1

4

83.8.

.1.1.3. Synthesis of 4-(benzofuran-2-yl)thiazol-2-amine derivatives (d1-3)

-(benzofuran-2-yl)thiazol-2-amine derivatives were prepared in accordance with previously

reported methods [47]. Thiourea (5 mmol) was added to a solution of 2-bromoacetylbenzofuran

derivatives (5 mmol) in hot ethanol (20 mL). The mixture was refluxed for 1 hour and cooled.

1

0

ACCEPTED MANUSCRIPT

The mixture was neutralized with aqueous ammonia. The precipitate was filtered and washed

with ethanol. The product was recrystallized from ethanol.

1

4

-(benzofuran-2-yl)thiazol-2-amine (d1): 85% yield; mp. 214–266 °C; H NMR (DMSO-d , 300

6

1

3

MHz) δ/ppm: 6.96 (1H, s), 7.06 (1H, s), 7.22-7.32 (4H, m), 7.57 (1H, d), 7.63 (1H, d); C NMR

(

DMSO-d

6

, 75 MHz) δ/ppm: 102.1, 105.3, 110.7, 118.2, 121.3, 122.5, 126.1, 140.3, 151.3, 154.1,

1

64.3.

1

4

-(5-bromobenzofuran-2-yl)thiazol-2-amine (d2): 85% yield; mp. 234–236 °C; H NMR

(DMSO-d6, 300 MHz) δ/ppm: 6.95 (1H, s), 7.10 (1H, s), 7.21 (2H, s), 7.42 (1H, d), 7.54 (1H, d),

1

3

7

1

4

d

.84 (1H, d); C NMR (DMSO-d , 75 MHz) δ/ppm: 102.2, 104.8, 112.1, 118.5, 122.4, 128.2,

6

29.5, 140.5, 151.1, 154.5, 164.7.

1

-(5-nitrobenzofuran-2-yl)thiazol-2-amine (d3): 85% yield; mp. 244–256 °C; H NMR (DMSO-

6

2

, 300 MHz) δ/ppm: 7.19 (2H, d), 7.36 (2H, s, NH ), 7.81 (1H, d), 8.17-8.21 (1H, m), 8.61 (1H,

1

3

d); C NMR (DMSO-d , 75 MHz) δ/ppm: 103.3, 106.8, 112.4, 118.2, 120.8, 130.8, 140.9, 144.6,

6

1

55.7, 157.6, 169.8.

4

.1.1.4. Synthesis of urea substituted benzofuranylthiazole derivatives (e1-e38)

Isocyanate derivatives (1 mmol) were added to a solution of thiazole derivatives (1 mmol) and

triethyl amine (1 mL) in dry THF. The mixture was refluxed for 12 h. Tetrahydrofuran was

removed using a rotary evaporator, and the mixture was washed with chloroform. The product

was recrystallizated from ethanol. e1-e38 were obtained in yields of 77-99%.

1

-(4-(benzofuran-2-yl)thiazol-2-yl)-3-(2-methoxyphenyl)urea (e1): Yellowish powder, 64% yield,

-1

1

mp. 263-265 °C; IR: 3260, 3190, 3113, 2957, 1694, 1527, 1292, 1255 cm ; H NMR (DMSO-

, 300 MHz) δ/ppm: 3.41 (3H, s, -OCH ), 6.35-6.51 (4H, m, H5,20,21,22), 6.73 (1H, d, J=8.8 Hz,

H ), 6.78 (1H, s, H ), 6.97 (1H, s, H ), 7.08 (1H, d, J=8.2 Hz, H ), 7.43 (1H, d, J=7.6 Hz, H ),

d

6

3

6

3

14

7

23

1

3

7

.68 (1H, t, J=2,1 Hz, H ), 8.21 (1H, s, NH), 10.68 (1H, s, NH); C NMR (DMSO-d , 75 MHz)

4 6

3

δ/ppm: 56.3(-OCH ), 102.7(C-3), 104.8(C-14), 111.4(C-7), 111.6(C-20), 119.8 (C-4), 121.1(C-

2

1

2), 121.9(C-23), 122.5(C-5), 123.8(C-18), 125.1(C-6), 129.2(C-21), 129.4(C-9), 141.9(C-14),

+

48.8(C-19), 152.9(C-2), 153.4(C-16), 154.6(C-8), 163.0(C-12); LC-MS (m/z): 366.17 [MH ].

Anal. Calcd. for C H N O S: C, 62.45; H, 4.14; N, 11.50. Found: C, 62.73; H, 4.09; N, 11.33.

1

9

15

3

1

-(4-(benzofuran-2-yl)thiazol-2-yl)-3-(3-methoxyphenyl)urea (e2): Cream powder, 80% yield,

-1

1

mp. 250-252 °C; IR: 3260, 3213, 3111, 2941, 1693, 1525, 1292, 1257 cm ; H NMR (DMSO-

, 300 MHz) δ/ppm: 3.77 (3H, s, -OCH ), 6.66 (1H, d, J=8.2 Hz, H21), 7.01 (1H, d, J=8.0 Hz,

d

6

3

1

ACCEPTED MANUSCRIPT

H ), 7.16 (1H, s, H ), 7.20–7.37 (4H, m, H

), 7.58 (1H, s, H ), 7.62 (1H, d, J=8.0 Hz, H ),

2

3

5,6,14,22

19

7

1

3

7

.69 (1H, d, J=7.3 Hz, H

4

), 8.96 (1H, s, NH), 10.87 (1H, s, NH); C NMR (DMSO-d

6

, 75 MHz)

δ/ppm: 55.7(-OCH ), 103.1(C-3), 105.1(C-14), 109.0(C-19), 110.2(C-7), 111.7(C-23), 118.6(C-

3

2

1

1), 122.0(C-4), 123.9(C-5), 125.3(C-6), 129.1(C-9), 130.4(C-22), 140.2(C-18), 140.9(C-10),

+

52.1(C-2), 152.4(C-16), 154.6(C-7), 154.8(C-20), 160.4(C-12); LC-MS (m/z): 366.17 [MH ].

Anal. Calcd. for C H N O S: C, 62.45; H, 4.14; N, 11.50; found: C, 62.82; H, 4.08; N, 11.27.

1

9

15

3

1

-(4-(benzofuran-2-yl)thiazol-2-yl)-3-(4-methoxyphenyl)urea (e3): White powder, 68% yield,

-1

1

mp. 236-238 °C; IR: 3259, 3188, 3116, 2997, 1674, 1566, 1307, 1252 cm ; H NMR (DMSO-

d

7

6

, 300 MHz) δ/ppm: 3.71 (3H, s, -OCH

3

), 6.90 (2H, d, J=9.1 Hz, H20,22), 7.11 (1H, s, H

3

), 7.24-

.33 (2H, m, H ), 7.38 (2H, d, J=8.8 Hz, H

), 7.52 (1H, s, H ), 7.59 (1H, d, J=7.6 Hz, H ),

5

,6

19,23

14

7

1

3

.65 (1H, d, J=7.3 Hz, H ), 8.73 (1H, s, NH), 10.80 (1H, s, NH); C NMR (DMSO-d , 75 MHz)

4

6

δ/ppm: 55.8(-OCH ), 103.1(C-3), 110.1(C-14), 111.7(C-7), 114.7(C-20,22), 121.3(C-19,23),

3

1

22.0(C-4), 123.9(C-5), 125.3(C-6), 129.1(C-9), 131.9(C-18), 140.8(C-10), 152.3(C-2), 152.5(C-

+

6), 154.8(C-8), 155.8(C-21), 160.7(C-12); LC-MS (m/z): 366.12 [MH ]. Anal. Calcd. for

C H N O S: C, 62.45; H, 4.14; N, 11.50; found: C, 62.58; H, 4.17; N, 11.43.

19

15

3

1

-(4-(benzofuran-2-yl)thiazol-2-yl)-3-(p-tolyl)urea (e4): Cream powder, 75% yield, mp. 269-271

-1

1

°

C; IR: 3323, 3271, 3121, 2916, 1687, 1556, 1288, 1242 cm ; H NMR (DMSO-d , 300 MHz)

6

δ/ppm2.24 (3H, s, 4-CH

3

), 7.11 (2H, d, J=8.2 Hz, H20,22) 7.12 (1H, s, H ) 7.24 –7 34 (2H, m,

H ), 7.36 (2H, d, J=8.2 Hz, H

), 7.53 (1H, s, H ) 7.59 (1H, d, J= 7.9 Hz, H ), 7.67 (1H, d,

5

,6

19,23

14

7

1

3

J=8.2 Hz, H ), 8.82 (1H, s, NH), 10.81 (1H, s, NH); C NMR (DMSO-d , 75 MHz) δ/ppm:

4

6

2

1

3

1.0(-CH ), 103.1(C-3), 110.1(C-14), 111.7(C-7), 119.5(C-4), 122.0(C-19,23), 123.9(C-5),

25.3(C-6), 129.1(C-20,22), 130.0(C-9), 132.5(C-18), 136.4(C-21), 140.8(C-10), 152.1(C-2),

+

52.4(C-16), 154.8(C-8), 160.6(C-12);LC-MS (m/z): 350.11 [MH ]. Anal. Calcd. for

C H N O S: C, 65.31; H, 4.33; N, 12.03; found: C, 65.44; H, 4.29; N, 12.08.

19

15

3

2

1

-(4-(benzofuran-2-yl)thiazol-2-yl)-3-phenylurea (e5): White powder, 78% yield, mp. 283-285

-1

1

°

C; IR: 3209, 3132, 3034, 2949, 1681, 1512, 1269, 1255 cm ; H NMR (DMSO-d , 300 MHz)

6

3

δ/ppm: 7.04 (1H, t, J=7.3 Hz, H21), 7.12(1H, s, H ), 7.24-7.34 (4H, m, H5,6,20,22), 7.50 (2H, d,

J=11.5 Hz, H₁₉_,₂₃), 7.54 (1H, s, H ), 7.59 (1H, d, J=8.2 Hz, H ), 7.65 (1H, d, J= 7.3 Hz, H ), 8.93

14

7

4

1

3

(

1H, s, NH), 10.86 (1H, s, NH); C NMR (DMSO-d , 75 MHz) δ/ppm: 103.1(C-3), 110.2(C-14),

6

1

11.7(C-7), 119.4(C-4), 122.0(C-19,23), 123.6(C-5), 123.9(C-6), 125.3(C-21), 129.1(C-20,22),

29.6(C-9), 139.0(C-18), 140.8(C-10), 152.2(C-2), 152.4(C-16), 154.8(C-8), 160.6(C-12); LC-

1

2

ACCEPTED MANUSCRIPT

+

MS (m/z): 335.89 [MH ]. Anal. Calcd. for C H N O S: C, 64.46; H, 3.91; N, 12.53; found: C,

1

8

13

3

2

6

4.57; H, 3.88; N, 12.50.

1

2

3

-(4-(benzofuran-2-yl)thiazol-2-yl)-3-(2-nitrophenyl)urea (e6): Orange powder, 70% yield, mp.

-1

1

53-255 °C; IR: 3323, 3286, 3126, 2953, 1697, 1492, 1332, 1266 cm ; H NMR (DMSO-d

6

,

00 MHz) δ/ppm: 7.12 (1H, s, H ), 7.24-7.33 (3H, m, H₅_,₆_,₇), 7.57-7.60 (2H, m, H₁₃_,₂₁), 7.67 (1H,

3

dd, J =1.2 Hz; J =7.0 Hz, H ), 7.73 (1H, td, J =1.4 Hz; J =7.1 Hz, H ), 8.12 (1H, dd, J =1.4 Hz;

1

2

4

1

2

22

1

J =8.5 Hz, H ), 8.32 (1H, dd, J =1.2 Hz; J =8.5 Hz, H ), 9.92 (1H, s, NH), 12.18 (1H, s, NH);

2

20

1

2

23

1

3

C NMR (DMSO-d , 75 MHz) δ/ppm: 103.2(C-3), 110.5(C-14), 111.7(C-7), 119.8(C-23),

6

1

22.1(C-4), 123.6(C-5), 124.0(C-6), 125.4(C-20), 126.2(C-21), 129.1(C-9), 134.3(C-18),

35.8(C-22), 138.7(C-19), 141.1(C-10), 152.1(C-2), 152.3(C-16), 154.8(C-8), 160.4(C-12); LC-

+

MS (m/z): 380.85 [MH ]. Anal. Calcd. for C H N O S: C, 56.84; H, 3.18; N, 14.73; found: C,

1

8

12

4

5

6.92; H, 3.14; N, 14.77.

1

-(4-(benzofuran-2-yl)thiazol-2-yl)-3-(3-nitrophenyl)urea (e7): Yellowish powder, 65% yield,

-1

1

mp. 257-259 °C; IR: 3325, 3210, 3151, 2980, 1691, 1527, 1344, 1294, 1246 cm ; H NMR

(

DMSO-d , 300 MHz) δ/ppm: 7.16 (1H, s, H ), 7.25-7.37 (2H, m, H ), 7.59-7.65 (3H, m,

6

3

5,6

4 1 2

H7,14,22), 7.69 (1H, d, J=8.2 Hz, H ), 7.82 (1H, d, J=8.2 Hz, H23), 7.91 (1H, dd, J =1.5 Hz; J =8.2

1

3

Hz, H ), 8.58 (1H, s, H ), 9.46 (1H, s, NH), 11.20 (1H, s, NH); C NMR (DMSO-d , 75 MHz)

2

1

19

6

δ/ppm: 103.2(C-3), 110.5(C-14), 111.7(C-7), 113.4(C-19), 118.0(C-21), 122.1(C-4), 124.0(C-5),

1

25.4(C-9), 125.7(C-6), 129.1(C-23), 130.9(C-22), 140.4(C-18), 140.9(C-10), 148.7(C-20),

+

52.3(C-2), 152.4(C-16), 154.8(C-8), 160.3(C-12); LC-MS (m/z): 381.22 [MH ]. Anal. Calcd. for

C

18

H

12

N

4

O

4

S: C, 56.84; H, 3.18; N, 14.73; found: C, 56.90; H, 3.15; N, 14.70.

1

-(4-(benzofuran-2-yl)thiazol-2-yl)-3-(4-nitrophenyl)urea (e8): Yellowish powder, 85% yield,

-1

1

mp. 304-306 °C; IR: 3466, 3213, 3126, 2976, 1693, 1560, 1512, 1330, 1298, 1253 cm ;

H

NMR (DMSO-d , 300 MHz) δ/ppm: 7.16 (1H, s, H ), 7.25-7.36 (2H, m, H ), 7.60-7.63 (2H, m,

6

3

5,6

4

H7,14), 7.68 (1H, d, J=7.6 Hz, H ), 7.75 (2H, d, J=9.1 Hz, H19,23), 8.23 (2H, d, J=9.1 Hz, H20,22),

1

3

9

1

.63 (1H, s, NH), 11.16 (1H, s, NH);

C NMR (DMSO-d , 75 MHz) δ/ppm: 103.2(C-3),

6

10.7(C-14), 111.7(C-7), 118.9(C-19,23), 122.1(C-4), 124.0(C-5), 125.4(C-20,22), 125.8(C-6),

29.1(C-9), 141.2(C-10), 142.5(C-21), 145.6(C-18), 152.0(C-2), 152.3(C-16), 154.8(C-8),

+

60.1(C-12); LC-MS (m/z): 380.89 [MH ]. Anal. Calcd. for C H N O S: C, 56.84; H, 3.18; N,

1

8

12

4

4.73; found: C, 56.89; H, 3.13; N, 14.76.

1

3

ACCEPTED MANUSCRIPT

1

2

3

-(4-(benzofuran-2-yl)thiazol-2-yl)-3-(2-fluorophenyl)urea (e9): Cream powder, 85% yield, mp.

-1

1

73-275 °C; IR: 3203, 3113, 3087, 2954, 1701, 1519, 1280, 1257 cm ; H NMR (DMSO-d

6

,

00 MHz) δ/ppm: 7.10-7.23 (3H, m, H₃_,₂₁_,₂₂), 7.25-7.37 (3H, m, H₅_,₆_,₂₀), 7.59 (1H, s, H ), 7.64

14

(1H, d, J=8.2 Hz, H ), 7.69 (1H, d, J=7.9 Hz, H ), 8.15 (1H, td, J =1.8 Hz; J =8.2 Hz, H ), 8.99

7 4 1 2 23

1

3

(

1H, s, NH), 11.15 (1H, s, NH); C NMR (DMSO-d

6

, 75 MHz) δ/ppm: 103.2(C-3), 110.3(C-14),

1

6

11.7(C-7), 115.7(C-20), 116.0(C-18), 121.5(C-4), 122.1(C-23), 124.0(C-5), 124.4(C-6),

25.4(C-9), 126.9(C-22), 127.1(C-21), 129.1(C-10), 140.9(C-2), 151.9(C-16), 152.3(C-8),

+

54.8(C-19), 160.4(C-12); LC-MS (m/z): 354.20 [MH ]. Anal. Calcd. for C H FN O S: C,

1

8

12

3

2

1.18; H, 3.42; N, 11.89; found: C, 61.23; H, 3.39; N, 11.87.

1

-(4-(benzofuran-2-yl)thiazol-2-yl)-3-(3-fluorophenyl)urea (e10): Cream powder, 80% yield, mp.

-1

1

2

93-295 °C; IR: 3258, 3205, 3116, 2962, 1689, 1573, 1516, 1294, 1255 cm ; H NMR (DMSO-

d , 300 MHz) δ/ppm: 6.86 (1H, td, J =1.8 Hz; J =8.2 Hz, H ), 7.12 (1H, s, H ), 7.17-7.38 (4H,

6

1

2

21

3

m, H₅_,₆_,₁₄_,₂₃), 7.48 (1H, dt, J =2.0 Hz; J =11.4 Hz, H ), 7.56 (1H, s, H ), 7.59 (1H, d, J=8.5 Hz,

1

2

22

19

1

3

H

7

), 7.67 (1H, dd, J

1

=1.2 Hz; J

2

4

=8.5 Hz, H ), 9.14 (1H, s, NH), 10.96 (1H, s, NH); C NMR

(

DMSO-d , 75 MHz) δ/ppm: 103.2(C-3), 106.0(C-14), 106.3(C-7), 109.8(C-4), 110.4(C-19),

6

1

3

11.7(C-21), 115.2(C-23), 122.1(C-5), 124.0(C-6), 125.4(C-9), 129.1(C-22), 131.2(C-18),

31.3(C-10), 140.8(C-2), 152.1(C-16), 152.4(C-8), 154.8(C-20), 160.4(C-12); LC-MS (m/z):

+

54.16 [MH ]. Anal. Calcd. for C H FN O S: C, 61.18; H, 3.42; N, 11.89; found: C, 61.13; H,

18

12

3

2

.45; N, 11.93.

1

2

3

7

-(4-(benzofuran-2-yl)thiazol-2-yl)-3-(4-fluorophenyl)urea (e11): White powder, 80% yield, mp.

-1

1

64-266 °C; IR: 3266, 3224, 3132, 2958, 1685, 1520, 1269, 1255 cm ; H NMR (DMSO-d

6

,

00 MHz) δ/ppm: 7.12 (1H, s, H ), 7.17 (2H, d, J=8.8 Hz, H ), 7.22-7.33 (2H, m, H ), 7.41-

3

20,22

5,6

7 1

.52 (2H, m, H19,23), 7.54 (1H, s, H14), 7.59 (1H, d, J=8.2 Hz, H ), 7.65 (1H, dd, J =0.9 Hz;

1

3

J =7.5 Hz, H ), 8.95 (1H, s, NH), 10.90 (1H, s, NH); C NMR (DMSO-d , 75 MHz) δ/ppm:

2

4

6

1

03.1(C-3), 110.2(C-14), 111.7(C-7), 116.0(C-22), 116.3(C-20), 120.6(C-23), 121.4(C-19),

21.5(C-4), 122.1(C-5), 123.9(C-6), 125.3(C-9), 129.1(C-18), 135.3(C-10), 140.8(C-2), 152.3(C-

+

6), 152.4(C-8), 154.8(C-21), 160.6(C-12); LC-MS (m/z): 352.79 [M ]. Anal. Calcd. for

C

18

H12FN

3

O

2

S: C, 61.18; H, 3.42; N, 11.89; found: C, 61.21; H, 3.46; N, 11.91.

1

2

3

-(4-(benzofuran-2-yl)thiazol-2-yl)-3-(3-chlorophenyl)urea (e12): White solid, 90% yield, mp.

-1

1

44-246 °C; IR: 3441, 3192, 3110, 2989, 1693, 1573, 1286, 1259 cm ; H NMR (DMSO-d

6

,

00 MHz) δ/ppm: 7.06-7.10 (1H, m, H ), 7.12 (1H, s, H ), 7.24-7.33 (4H, m, H₅_,₆_,₂₂_,₂₃), 7.56 (1H,

2

1

3

1

4

ACCEPTED MANUSCRIPT

s, H ), 7.59 (1H, d, J=7.6 Hz, H ), 7.65 (1H, dd, J =1.2 Hz; J =7.6 Hz, H ), 7.70 (1H, s, H ),

1

4

7

1

2

4

19

1

3

9

1

.11 (1H, s, NH), 10.99 (1H, s, NH);

6

C NMR (DMSO-d , 75 MHz) δ/ppm: 103.2(C-3),

10.4(C-14), 111.7(C-7), 117.9(C-23), 118.8(C-4), 122.1(C-5), 123.2(C-19), 124.0(C-6),

25.4(C-21), 129.1(C-9), 131.2(C-22), 133.9(C-20), 140.6(C-18), 140.9(C-10), 152.2(C-2),

+

52.4(C-16), 154.8(C-8), 160.4(C-12); LC-MS (m/z): 370.09 [MH ]. Anal. Calcd. for

C H ClN O S: C, 58.46; H, 3.27; N, 11.36; found: C, 58.50; H, 3.24; N, 11.39.

18

12

3

2

1

2

d

7

-(4-(benzofuran-2-yl)thiazol-2-yl)-3-(4-chlorophenyl)urea (e13): White powder, 80% yield, mp.

-1

1

94-296 °C; IR: 3228, 3112, 3047, 2953, 1689, 1510, 1497, 1269, 1253 cm ; H NMR (DMSO-

, 300 MHz) δ/ppm: 7.15 (1H, s, H ), 7.25-7.37 (2H, m, H5,6), 7.40 (2H, d, J=9.0 Hz, H20,22),

.55 (2H, d, J=8.8 Hz, H₁₉_,₂₃), 7.59 (1H, s, H ), 7.62 (1H, dd, J =1.2 Hz; J =8.2 Hz, H ), 7.69

6

3

14

1

2

7

1

3

(

1H, dd, J =1.2 Hz; J =7.3 Hz, H ), 9.09 (1H, s, NH), 10.97 (1H, s, NH); C NMR (DMSO-d ,

1

2

4

6

7

1

5 MHz) δ/ppm: 103.1(C-3), 110.3(C-14), 111.7(C-7), 121.0(C-19,23), 122.1(C-4), 123.9(C-5),

25.4(C-6), 127.1(C-20,22), 129.1(C-9), 129.4(C-21), 138.1(C-18), 140.8(C-10), 152.1(C-2),

+

52.4(C-16), 154.8(C-8), 160.4(C-12); LC-MS (m/z): 370.08 [MH ]. Anal. Calcd. for

C H ClN O S: C, 58.46; H, 3.27; N, 11.36; found: C, 58.52; H, 3.22; N, 11.34.

18

12

3

2

1

-(4-(benzofuran-2-yl)thiazol-2-yl)-3-(3,4-dichlorophenyl)urea (e14): Cream powder, 85% yield,

-1

1

mp. 285-287 °C; IR: 3242, 3116, 3053, 2949, 1701, 1517, 1284, 1253 cm ; H NMR (DMSO-

, 300 MHz) δ/ppm: 7.15 (1H, s, H ), 7.24-7.36 (2H, m, H5,6), 7.42 (1H, dd, J =2.4 Hz; J =8.8

Hz, H ), 7.57 (1H, s, H ), 7.60-7.63 (2H, m, H ), 7.69 (1H, dd, J =0.9 Hz; J =6.7 Hz, H ),

d

6

3

1

2

3

14

7,22

1

2

4

13

7

.90 (1H, d, J=2.3 Hz H ), 9.23 (1H, s, NH), 11.14 (1H, s, NH); C NMR (DMSO-d , 75 MHz)

19 6

δ/ppm: 103.2(C-3), 110.5(C-14), 111.7(C-7), 119.7(C-4), 120.6(C-22), 122.1(C-23), 124.0(C-5),

1

25.0(C-6), 125.4(C-9), 129.1(C-19), 131.4(C-21), 131.8(C-20), 139.3(C-18), 140.9(C-10),

+

52.1(C-2), 152.4(C-16), 154.8(C-8), 160.3(C-12); LC-MS (m/z): 404.10 [MH ]. Anal. Calcd. for

C H Cl N O S: C, 53.48; H, 2.74; N, 10.39; found: C, 53.53; H, 2.78; N, 10.37.

18

11

2

3

2

1

2

d

7

-(4-(benzofuran-2-yl)thiazol-2-yl)-3-(4-bromophenyl)urea (e15): White powder, 80% yield, mp.

-1

1

91-293 °C; IR: 3230, 3128, 3061, 2951, 1685, 1512, 1490, 1273, 1253 cm ; H NMR (DMSO-

, 300 MHz) δ/ppm: 7.12 (1H, s, H ), 7.22-7.33 (2H, m, H5,6), 7.45 (2H, d, J=9.7 Hz, H20,22),

.50 (2H, d, J=9.7 Hz, H₁₉_,₂₃), 7.55 (1H, s, H ), 7.59 (1H, d, J=7.9 Hz, H ), 7.65 (1H, d, J=7.6

6

3

14

7

1

3

Hz, H ), 9.06 (1H, s, NH), 10.94 (1H, s, NH); C NMR (DMSO-d , 75 MHz) δ/ppm: 103.3(C-

4

6

3

1

), 110.5(C-14), 111.9(C-7), 115.3(C-21), 118.8(C-19), 118.9(C-23), 121.6(C-4), 122.3(C-20),

24.2(C-22), 125.6(C-6), 129.3(C-9), 132.5(C-5), 138.7(C-18), 141.1(C-10), 152.3(C-2),

1

5

ACCEPTED MANUSCRIPT

+

1

52.6(C-16), 155.0(C-8), 160.6(C-12); LC-MS (m/z): 414.13 [MH ]. Anal. Calcd. for

S: C, 52.19; H, 2.92; N, 10.14; found: C, 52.15; H, 2.95; N, 10.16.

18 3 2

C H12BrN O

1

-(4-(benzofuran-2-yl)thiazol-2-yl)-3-(4-iodophenyl)urea (e16): White powder, 68% yield, mp.

-1

1

2

54-256 °C; IR: 3228, 3113, 3055, 2955, 1687, 1512, 1495, 1292, 1255 cm ; H NMR (DMSO-

), 7.57 (1H, s, H ), 7.61 (2H,

d , 300 MHz) δ/ppm: 7.14 (1H, s, H ), 7.24-7.37 (4H, m, H

6

3

5,6,20,22

14

dd, J =2.6 Hz; J =8.2 Hz, H ), 7.68 (2H, dd, J =2.4 Hz; J =8.5 Hz, H₁₉_,₂₃), 9.04 (1H, s, NH),

1

2

4,7

1

2

1

3

1

4

2

0.92 (1H, s, NH); C NMR (DMSO-d , 75 MHz) δ/ppm: 86.8(C-21), 103.1(C-3), 110.3(C-14),

6

11.7(C-7), 121.6(C-4), 122.0(C-19,23), 123.9(C-5), 125.3(C-6), 129.1(C-9), 138.1(C-20,22),

39.0(C-18), 140.9(C-10), 152.1(C-2), 152.4(C-16), 154.8(C-8), 160.4(C-12); LC-MS (m/z):

+

62.10 [MH ]. Anal. Calcd. for C H IN O S: C, 46.87; H, 2.62; N, 9.11; found: C, 46.84; H,

1

8

12

3

2

.65; N, 9.13.

1

-(4-(5-bromobenzofuran-2-yl)thiazol-2-yl)-3-(2-methoxyphenyl)urea (e17): Greenish powder,

-1 1

7

0% yield, mp. 271-273 °C; IR: 3242, 3120, 3060, 2935, 1697, 1523, 1282, 1257 cm ; H NMR

(

DMSO-d , 300 MHz) δ/ppm: 3.87 (3H, s, -OCH ), 6.89-7.06 (3H, m, H₂₀_,₂₁_,₂₂), 7.1 (1H, s, H₃),

6

3

7

.44 (1H, dd, J =2.1 Hz; J =8.8 Hz, H ), 7.57 (2H, t, J=4.1 Hz, H₆_,₁₄), 7.86 (1H, d, J=2.1 Hz,

1

2

7

1

3

H

4 6

23), 8.10 (1H, d, J=9.0 Hz, H ), 8.75 (1H, s, NH), 11.37 (1H, s, NH); C NMR (DMSO-d , 75

MHz) δ/ppm: 56.5(-OCH ), 102.6(C-3), 111.0(C-14), 111.6(C-20), 113.7(C-7), 116.2(C-5),

3

1

4

3

19.2(C-23), 121.3(C-22), 123.7(C-4), 124.4(C-18), 127.8(C-21), 128.1(C-6), 131.5(C-9),

40.3(C-10), 148.6(C-19), 152.0(C-2), 153.6(C-16), 153.7(C-8), 160.7(C-12); LC-MS (m/z):

+

44.26 [MH ]. Anal. Calcd. for C H BrN O S: C, 51.36; H, 3.18; N, 9.46; found: C, 51.33; H,

1

9

14

3

.14; N, 9.49.

1

-(4-(5-bromobenzofuran-2-yl)thiazol-2-yl)-3-(3-methoxyphenyl)urea (e18): Yellow powder,

-1

1

7

8% yield, mp. 264-266 °C; IR: 3266, 3217, 3107, 2953, 1691, 1525, 1290, 1255 cm ; H NMR

(

DMSO-d , 300 MHz) δ/ppm: 3.75 (3H, s, -OCH ), 6.62 (1H, dd, J =2.3 Hz; J =8.2 Hz, H ),

6

3

1

2

21

6

3 7

.97 (1H, d, J=7.9 Hz, H23), 7.10 (1H, s, H ), 7.16 (1H, t, J=2.3 Hz, H ), 7.21 (1H, t, J=8.2 Hz,

H ), 7.44 (1H, dd, J =2.0 Hz; J =8.8 Hz, H ), 7.57 (1H, s, H ), 7.60 (1H, s, H ), 7.86 (1H, d,

2

1

2

6

14

19

1

3

J=2.0 Hz, H

4

), 8.94 (1H, s, NH), 10.84 (1H, s, NH); C NMR (DMSO-d

6

, 75 MHz) δ/ppm:

5

1

5.7(-OCH ), 102.7(C-3), 105.2(C-14), 109.1(C-9), 111.2(C-7), 111.7(C-23), 113.7(C-5),

3

16.2(C-21), 118.6(C-4), 124.4(C-22), 127.9(C-6), 130.5(C-9), 131.4(C-18), 140.1(C-10),

+

40.3(C-2), 152.1(C-16), 153.6(C-8), 153.8(C-20), 160.3(C-12); LC-MS (m/z): 444.08 [MH ].

Anal. Calcd. for C H BrN O S: C, 51.36; H, 3.18; N, 9.46; found: C, 51.38; H, 3.20; N, 9.43.

1

9

14

3

1

6

ACCEPTED MANUSCRIPT

1

-(4-(5-bromobenzofuran-2-yl)thiazol-2-yl)-3-(4-methoxyphenyl)urea (e19): Cream powder, 75%

-1

1

yield, mp. 260-262 °C; IR: 3269, 3211, 3115, 2937, 1685, 1525, 1274, 1255 cm ; H NMR

DMSO-d , 300 MHz) δ/ppm: 3.72 (3H, s, -OCH ), 6.83 (1H, d, J=7.6 Hz, H ), 6.89 (2H, d,

(

6

3

19

J=7.9 Hz, H₂₀_,₂₂), 7.10 (1H, s, H ), 7.31 (1H, d, J=7.9 Hz, H ), 7.38 (1H, d, J=7.9 Hz, H ), 7.43

3

23

7

(

1H, d, J=8.8 Hz, H

6

4

), 7.57 (1H, s, H14), 7.86 (1H, s, H ), 8.75 (1H, s, NH), 10.79 (1H, s, NH);

1

3

C NMR (DMSO-d , 75 MHz) δ/ppm: 55.8(-OCH ), 102.6(C-3), 111.1(C-14), 113.7(C-7),

6

3

1

4

3

14.7(C-20,22), 116.2(C-5), 121.4(C-19,23), 124.4(C-4), 127.8(C-6), 131.5(C-9), 131.9(C-18),

33.7(C-10), 140.3(C-2), 152.3(C-16), 153.6(C-8), 155.9(C-21), 160.8(C-12); LC-MS (m/z):

+

44.16 [MH ]. Anal. Calcd. for C H BrN O S: C, 51.36; H, 3.18; N, 9.46; found: C, 51.39; H,

1

9

14

3

.15; N, 9.49.

1

-(4-(5-bromobenzofuran-2-yl)thiazol-2-yl)-3-(p-tolyl)urea (e20): Cream powder, 80% yield, mp.

-1

1

2

77-279 °C; IR: 3259, 3199, 3113, 2914, 1685, 1520, 1319, 1295, 1257 cm ; H NMR (DMSO-

d , 300 MHz) δ/ppm: 2.24 (3H, s, 4-CH ), 7.10 (1H, s, H ), 7.12 (2H, d, J=8.5 Hz, H₂₀_,₂₂), 7.35

6

3

(

2H, d, J=8.5 Hz, H19,23), 7.44 (1H, dd, J

1

=2.0 Hz; J

2

7

=8.8 Hz, H ), 7.57 (1H, s, H14), 7.59 (1H, d,

13

J=8.8 Hz, H ), 7.86 (1H, d, J=2.0 Hz, H ), 8.83 (1H, s, NH), 10.81 (1H, s, NH); C NMR

6

4

(

DMSO-d , 75 MHz) δ/ppm: 21.0(-CH ), 102.6(C-3), 111.2(C-5), 113.7(C19,23), 116.2(C-14),

6

3

1

19.5(C-7), 124.4(C-4), 127.8(C-20,22), 130.0(C-9), 131.5(C-6), 132.6(C-18), 136.4(C-21),

+

40.3(C-10), 152.1(C-2), 153.6(C-16), 153.7(C-8), 160.7(C-12); LC-MS (m/z): 428.69 [MH ].

Anal. Calcd. for C18

H12BrN

3

O

2

S: C, 53.28; H, 3.29; N, 9.81; found: C, 53.25; H, 3.27; N, 9.85.

1

-(4-(5-bromobenzofuran-2-yl)thiazol-2-yl)-3-phenylurea (e21): Cream powder, 72% yield, mp.

-1 1

3

29-331 °C; IR: 3232, 3200, 3111, 2943, 1695, 1519, 1498, 1274, 1252 cm ; H NMR (DMSO-

d , 300 MHz) δ/ppm: 7.04 (1H, t, J=7.3 Hz, H ), 7.10 (1H, s, H ), 7.25-7.38 (2H, m, H₂₀_,₂₂),

6

21

3

7

.41-7.53 (3H, m, H7,19,23), 7.56 (1H, s, H14), 7.59 (1H, s, H

6

4

), 7.86 (1H, d, J=2.0 Hz, H ), 8.92

1

3

(

1H, s, NH), 10.83 (1H, s, NH); C NMR (DMSO-d , 75 MHz) δ/ppm: 102.6(C-3), 111.2(C-14),

6

1

13.7(C-7), 116.2(C-5), 119.4(C-19,23), 123.6(C-4), 124.4(C-21), 127.8(C-20,22), 129.6(C-6),

31.5(C-9), 139.0(C-18), 140.3(C-10), 152.1(C-2), 153.6(C-16), 153.7(C-8), 160.7(C-12); LC-

+

MS (m/z): 414.77[MH ]. Anal. Calcd. for C H BrN O S: C, 52.19; H, 2.92; N, 10.14; found: C,

1

8

12

3

2

5

2.28; H, 2.87; N, 10.11.

1

-(4-(5-bromobenzofuran-2-yl)thiazol-2-yl)-3-(2-nitrophenyl)urea (e22): Greenish powder, 82 %

-1

1

yield, mp. 337-339 °C; IR: 3254, 3206, 3115, 2960, 1695, 1525, 1348, 1280, 1259 cm ;

H

NMR (DMSO-d , 300 MHz) δ/ppm: 7.11 (1H, s, H ), 7.28 (1H, t, J=7.3 Hz, H ), 7.44 (1H, dd,

6

3

21

1

7

ACCEPTED MANUSCRIPT

J =2.0 Hz; J =8.8 Hz, H ), 7.57 (1H, d, J=8.8 Hz, H ), 7.64 (1H, s, H ), 7.74 (1H, t, J=7.3 Hz,

1

2

7

6

14

4

H22), 7.87 (1H, d, J=1.8 Hz, H ), 8.12 (1H, d, J=8.2 Hz, H20), 8.30 (1H, d, J=8.2 Hz, H23), 9.91

1

3

(

1H, s, NH), 12.18 (1H, s, NH); C NMR (DMSO-d , 75 MHz) δ/ppm: 102.6(C-3), 111.2(C-14),

6

1

13.7(C-7), 116.2(C-5), 119.4 (C-6), 123.6(C-18), 124.4(C-4), 127.8(C-21), 129.6(C-20,22),

31.5(C-9), 139.0(C-19,23), 140.3(C-10), 152.1(C-2), 153.6(C-16), 153.7(C-8), 160.7(C-12);

+

LC-MS (m/z): 459.02 [MH ]. Anal. Calcd. for C H BrN O S: C, 47.07; H, 2.41; N, 12.20;

18

11

4

found: C, 47.18; H, 2.45; N, 12.22.

1

-(4-(5-bromobenzofuran-2-yl)thiazol-2-yl)-3-(3-nitrophenyl)urea (e23): Cream powder, 80%

-1

yield, mp. 292-294 °C; IR: 3331, 3280, 3095, 2940, 1699, 1552, 1520, 1325, 1284, 1247 cm ;

1

H NMR (DMSO-d , 300 MHz) δ/ppm: 7.10 (1H, s, H ), 7.42 (1H, dd, J =1.8 Hz; J =8.5 Hz,

6

3

1

2

H ), 7.57 (2H, d, J=8.5 Hz, H ), 7.62 (1H, s, H ), 7.73-7.89 (3H, m, H₄_,₂₂_,₂₃), 8.53 (1H, s, H₁₉),

7

6,21

14

1

3

9

1

.42 (1H, s, NH), 11.11 (1H, s, NH);

C NMR (DMSO-d , 75 MHz) δ/ppm: 102.7(C-3),

6

11.5(C-14), 113.4(C-7), 113.7(C-19), 116.2(C-5), 118.0(C-21), 124.4(C-4), 125.3(C-23),

25.6(C-22), 127.8(C-6), 130.8(C-9), 131.4(C-18), 140.4(C-10), 148.7(C-20), 152.2(C-2),

+

53.0(C-16), 153.6(C-8), 160.6(C-12); LC-MS (m/z): 459.07 [MH ]. Anal. Calcd. for

C H BrN O S: C, 47.07; H, 2.41; N, 12.20; found: C, 47.14; H, 2.43; N, 12.25.

18

11

4

1

-(4-(5-bromobenzofuran-2-yl)thiazol-2-yl)-3-(4-nitrophenyl)urea (e24): Orange powder, 85%

-1

1

yield, mp. 337-339 °C; IR: 3367, 3338, 3122, 2962, 1703, 1517, 1506, 1295, 1255 cm ;

H

NMR (DMSO-d , 300 MHz) δ/ppm: 7.11 (1H, s, H ), 7.42 (1H, dd, J =2.0 Hz; J =8.5 Hz, H ),

6

3

1

2

7

.55 (1H, d, J=8.8 Hz, H ), 7.63 (1H, s, H ), 7.70 (2H, d, J=9.0 Hz, H₁₉_,₂₃), 7.84 (1H, d, J=2.0

6 14

1

3

4

Hz, H ), 8.18 (2H, d, J=9.0 Hz, H20,22), 9.60 (1H, s, NH), 11.08 (1H, s, NH); C NMR (DMSO-

d₆, 75 MHz) δ/ppm: 102.7(C-3), 111.5(C-14), 113.5(C-7), 113.7(C-5), 116.2(C-19,23), 118.0(C-

4

1

), 124.5(C-20,22), 125.7(C-6), 127.9(C-9), 130.9(C-10), 131.5(C-21), 140.4(C-2), 148.7(C-18),

+

52.3(C-16), 153.6(C-8), 160.4(C-12); LC-MS (m/z): 459.59 [MH ]. Anal. Calcd. for

C H BrN O S: C, 47.07; H, 2.41; N, 12.20; found: C, 47.16; H, 2.40; N, 12.23.

18

11

4

1

-(4-(5-bromobenzofuran-2-yl)thiazol-2-yl)-3-(2-fluorophenyl)urea (e25): White powder, 87%

-1

1

yield, mp. 346-348 °C; IR: 3270, 3209, 3113, 2947, 1689, 1521, 1284, 1257 cm ; H NMR

DMSO-d , 300 MHz) δ/ppm: 7.07-7.17 (3H, m, H₃_,₂₁_,₂₂), 7.27 (1H, td, J =1.5 Hz; J =8.2 Hz,

(

6

1

2

H ), 7.43 (1H, dd, J =2.0 Hz; J =8.5 Hz, H ), 7.58 (1H, d, J=8.8 Hz, H ), 7.61 (1H, s, H ), 7.86

2

0

1

2

7

6

14

(

1H, d, J=2.0 Hz, H

4

1 2

), 8.11 (1H, td, J =1.8 Hz; J =8.2 Hz, H23), 8.94 (1H, s, NH), 11.09 (1H, s,

1

3

NH); C NMR (DMSO-d , 75 MHz) δ/ppm: 102.8(C-3), 111.7(C-14), 113.7(C-7), 116.3(C-20),

6

1

8

ACCEPTED MANUSCRIPT

1

18.6(C-5), 118.9(C-18), 124.5(C-23), 125.8(C-4), 127.9(C-22), 131.5(C-21), 140.5(C-6),

42.2(C-9), 142.5(C-10), 145.6(C-2), 146.3(C-16), 152.3(C-8), 153.7(C-19), 160.3(C-12); LC-

+

MS (m/z): 431.83 [MH ]. Anal. Calcd. for C H BrFN O S: C, 50.01; H, 2.56; N, 9.72; found:

1

8

11

3

2

C, 50.12; H, 2.50; N, 9.70.

1

-(4-(5-bromobenzofuran-2-yl)thiazol-2-yl)-3-(3-fluorophenyl)urea (e26): Cream powder, 65%

-1

1

yield, mp. 272-274 °C; IR: 3271, 3207, 3113, 2953, 1701, 1519, 1294, 1257 cm ; H NMR

(

DMSO-d , 300 MHz) δ/ppm: 6.86 (1H, td, J =2.0 Hz; J =8.5 Hz, H ), 7.10 (1H, s, H ), 7.18

6 1 2 21 3

(

1H, d, J=8.2 Hz, H ), 7.34 (1H, q, J=7.6 Hz, H ), 7.41-7.50 (2H, m, H₇_,₁₉), 7.58 (1H, d, J=8.5

23 22

1

3

6 4

Hz, H ), 7.60 (1H, s, H14), 7.86 (1H, d, J=1.8 Hz, H ), 9.14 (1H, s, NH), 10.95 (1H, s, NH); C

NMR (DMSO-d , 75 MHz) δ/ppm: 102.7(C-3), 106.0(C-14), 106.3(C-7), 109.9(C-19), 110.1(C-

6

2

1

4

2

1), 111.3(C-5), 113.7(C-23), 115.2(C-4), 116.2(C-22), 124.4(C-6), 127.8(C-9), 131.1(C-18),

31.4(C-10), 140.3(C-2), 140.9(C-16), 152.1(C-8), 153.6(C-20), 160.5(C-12); LC-MS (m/z):

+

32.19 [MH ]. Anal. Calcd. for C H BrFN O S: C, 50.01; H, 2.56; N, 9.72; found: C, 50.09; H,

1

8

11

3

2

.53; N, 9.89.

1

-(4-(5-bromobenzofuran-2-yl)thiazol-2-yl)-3-(4-fluorophenyl)urea (e27): White powder, 62%

-1

1

yield, mp. 258-260 °C; IR: 3278, 3109, 3080, 2951, 1697, 1527, 1276, 1259, 1207 cm ;

H

NMR (DMSO-d , 300 MHz) δ/ppm: 7.08-7.21 (3H, m, H₃_,₂₀_,₂₂), 7.41-7.55 (3H, m, H₇_,₁₉_,₂₃), 7.61

6

6 4

(1H, s, H14), 7.89 (1H, d, J=2.0 Hz, H ), 8.75 (1H, s, H ), 9.06 (1H, s, NH), 10.97 (1H, s, NH);

1

3

C NMR (DMSO-d , 75 MHz) δ/ppm: 102.6(C-3), 111.2(C-14), 113.7(C-7), 116.0(C-5),

6

1

16.3(C-19,23), 121.4(C-4), 121.5(C-20,22), 124.5(C-9), 127.9(C-6), 131.5(C-21), 135.4(C-18),

+

40.3(C-10), 152.3(C-2), 153.7(C-16), 157.1(C-8), 160.7(C-12); LC-MS (m/z): 431.75 [MH ].

Anal. Calcd. for C H BrFN O S: C, 50.01; H, 2.56; N, 9.72; found: C, 50.10; H, 2.51; N, 9.70.

1

8

11

3

2

1

-(4-(5-bromobenzofuran-2-yl)thiazol-2-yl)-3-(3-chlorophenyl)urea (e28): Orange powder, 72%

-1

1

yield, mp. 290-292 °C; IR: 3261, 3113, 3084, 2951, 1693, 1519, 1274, 1257 cm ; H NMR

DMSO-d , 300 MHz) δ/ppm: 7.10 (1H, s, H ), 7.26-7.33 (3H, m, H21,22,23), 7.43 (1H, dd, J =2.0

(

6

3

1

Hz; J =8.8 Hz, H ), 7.57 (1H, d, J=8.8 Hz, H ), 7.61 (1H, s, H ), 7.70 (1H, s, H ), 7.86 (1H, d,

2

7

6

14

4

1

3

J=1.8 Hz, H19), 9.11 (1H, s, NH), 10.99 (1H, s, NH); C NMR (DMSO-d

6

, 75 MHz) δ/ppm:

1

02.7(C-3), 111.3(C-14), 111.4(C-7), 113.7(C-5), 116.2(C-23), 117.9(C-19), 118.6(C-4),

18.8(C-21), 123.2(C-22), 124.4(C-6), 127.8(C-9), 131.2(C-20), 131.4(C-18), 133.9(C-10),

+

40.6(C-2), 152.2(C-16), 153.6(C-8), 160.5(C-12); LC-MS (m/z): 447.74 [MH ]. Anal. Calcd. for

C H BrClN O S: C, 48.18; H, 2.47; N, 9.36; found: C, 48.12; H, 2.45; N, 9.33.

18

11

3

2

1

9

ACCEPTED MANUSCRIPT

1

-(4-(5-bromobenzofuran-2-yl)thiazol-2-yl)-3-(4-chlorophenyl)urea (e29): White powder, 58%

-1

1

yield, mp. 306-308 °C; IR: 3269, 3196, 3128, 2997, 1693, 1610, 1525, 1282, 1242 cm ;

H

NMR (DMSO-d , 300 MHz) δ/ppm: 7.10 (1H, s, H ), 7.36 (2H, d, J=8.8 Hz, H₂₀_,₂₂), 7.44 (1H, td,

6

3

J =1.8 Hz; J =8.5 Hz, H ), 7.51 (2H, d, J=8.8 Hz, H

), 7.56 (1H, d, J=8.5 Hz, H ), 7.60 (1H, s,

6

1

2

7

19,23

1

3

H

14), 7.85 (1H, d, J=1.8 Hz, H

4 6

), 9.07 (1H, s, NH), 10.92 (1H, s, NH); C NMR (DMSO-d , 75

MHz) δ/ppm: 102.7(C-3), 111.3(C-14), 113.7(C-7), 116.2(C-5), 121.0(C-19,23), 124.4(C-4),

1

27.2(C-20,22), 127.8(C-6), 128.4(C-9), 131.5(C-21), 138.0(C-18), 140.3(C-10), 152.1(C-2),

+

53.6(C-16), 153.7(C-8), 160.6(C-12); LC-MS (m/z): 447.77 [MH ]. Anal. Calcd. for

C H BrClN O S: C, 48.18; H, 2.47; N, 9.36; found: C, 48.10; H, 2.44; N, 9.39.

18

11

3

2

1

-(4-(5-bromobenzofuran-2-yl)thiazol-2-yl)-3-(4-bromophenyl)urea (e30): Yellowish powder,

7

6% yield, mp. 315-317 °C; IR: 3242, 3190, 3107, 2947, 1693, 1521, 1497, 1311, 1276, 1257

-1

1

cm ; H NMR (DMSO-d , 300 MHz) δ/ppm: 7.10 (1H, s, H ), 7.42 (1H, d, J=2.0 Hz, H ), 7.46

6

3

7

(

2H, d, J=8.8 Hz, H₂₀_,₂₂), 7.50 (2H, d, J=8.8 Hz, H₁₉_,₂₃), 7.58 (1H, d, J=8.8 Hz, H ), 7.61 (1H, s,

6

1

3

H

4 6

14), 7.86 (1H, d, J=2.0 Hz, H ), 9.08 (1H, s, NH), 10.95 (1H, s, NH); C NMR (DMSO-d , 75

MHz) δ/ppm: 102.7(C-3), 107.5(C-21), 111.3(C-14), 113.7(C-7), 115.1(C-5), 116.2(C-19,23),

1

21.4(C-4), 124.5(C-6), 127.9(C-20,22), 131.5(C-9), 132.3(C-18), 138.5(C-10), 140.3(C-2),

+

52.1(C-16), 153.6(C-8), 160.6(C-12); LC-MS (m/z): 491.48 [MH ]. Anal. Calcd. for

C H Br N O S: C, 43.84; H, 2.25; N, 8.52; found: C, 43.89; H, 2.20; N, 8.50.

18

11

2

3

2

1

-(4-(5-bromobenzofuran-2-yl)thiazol-2-yl)-3-(4-iodophenyl)urea (e31): White powder, 67%

-1

1

yield, mp. 311-313 °C; IR: 3252, 3192, 3118, 2967, 1695, 1525, 1350, 1278, 1252 cm ;

NMR (DMSO-d , 300 MHz) δ/ppm: 7.13 (1H, s, H ), 7.30 (2H, d, J=8.8 Hz, H20,22), 7.46 (1H,

), 7.64 (1H, s, H ), 7.67 (1H, d,

H

6

3

dd, J =2.0 Hz; J =8.8 Hz, H ), 7.60 (2H, d, J=8.8 Hz, H

1

2

7

19,23

14

13

6 4

J=8.8 Hz, H ), 7.89 (1H, d, J=2.0 Hz, H ), 9.05 (1H, s, NH), 10.92 (1H, s, NH); C NMR

(

DMSO-d , 75 MHz) δ/ppm: 86.9(C-21), 102.7(C-3), 111.3(C-14), 113.7(C-7), 116.2(C-5),

6

1

21.6(C-19,23), 124.4(C-4), 127.8(C-6), 131.4(C-9), 138.1(C-20,22), 138.9(C-18), 140.3(C-10),

+

52.0(C-2), 153.6(C-16), 153.7(C-8), 160.5(C-12); LC-MS (m/z): 539.68 [MH ]. Anal. Calcd. for

C H BrIN O S: C, 40.02; H, 2.05; N, 7.78; found: C, 40.10; H, 2.01; N, 7.82.

18

11

3

2

1

-(4-methoxyphenyl)-3-(4-(5-nitrobenzofuran-2-yl)thiazol-2-yl)urea (e32): Brown powder, 60%

-1

yield, mp. 266-268 °C; IR: 3310, 3225, 3116, 2990, 1695, 1555, 1527, 1340, 1292, 1247 cm ;

1

H NMR (DMSO-d

6

3

, 300 MHz) δ/ppm: 3.70 (3H, s, -OCH ), 6.88 (2H, d, J=9.0 Hz, H20,22), 7.29

(

1H, s, H ), 7.36 (2H, d, J=9.0 Hz,

), 7.61 (1H, s, H ), 7.80 (1H, d, J=9.1 Hz, H ), 8.16 (1H,

3

19,23

14

7

2

0

ACCEPTED MANUSCRIPT

1

3

d, J=9.1 Hz, H ), 8.57 (1H, d, J=2.4 Hz, H ), 9.31 (1H, s, NH), 10.98 (1H, s, NH); C NMR

6

4

(

DMSO-d

6

, 75 MHz) δ/ppm: 55.8(-OCH

3

), 103.6(C-3), 111.9(C-14), 112.5(C-7), 114.7(C-20,22),

1

18.3(C-4), 120.3(C-19,23), 121.1(C-6), 129.9(C-9), 132.0(C-18), 133.6(C-10), 139.7(C-5),

+

44.5(C-2), 152.3(C-16), 155.3(C-21), 157.5(C-8), 160.9(C-12); LC-MS (m/z): 411.17 [MH ].

Anal. Calcd. for C19

H

14

N

4

O

5

S: C, 55.60; H, 3.44; N, 13.65; found C, 55.68; H, 3.47; N, 13.62.

1

-(4-(5-nitrobenzofuran-2-yl)thiazol-2-yl)-3-phenylurea (e33): Brown powder, 65% yield, mp.

-1

1

2

64-266 °C; IR: 3325, 3260, 3121, 2952, 1701, 1497, 1330, 1310, 1276 cm ; H NMR (DMSO-

d , 300 MHz) δ/ppm: 7.01 (1H, d, J=8.8 Hz, H ), 7.30-7.39 (2H, m, H₂₀_,₂₁), 7.41-7.48 (3H, m,

6

3

H

19,22,23), 7.64 (1H, s, H14), 7.82 (1H, d, J=8.8 Hz, H

7

6

), 8.18 (1H, d, J=8.8 Hz, H ), 8.57 (1H, s,

1

3

H ), 9.97 (1H, s, NH), 10.99 (1H, s, NH); C NMR (DMSO-d , 75 MHz) δ/ppm: 102.8(C-3),

4

6

1

11.6(C-14), 113.8(C-7), 116.3(C-4), 118.2(C-6), 124.5(C-19), 125.7(C-23), 127.9(C-9),

29.0(C-21), 131.0(C-20), 131.5(C-22), 137.2(C-18), 140.4(C-10), 145.5(C-5), 148.8(C-2),

+

53.6(C-16), 153.7(C-8), 161.9(C-12); LC-MS (m/z): 381.15 [MH ]. Anal. Calcd. for

C

18

H

12

N

4

O

4

S: C, 56.84; H, 3.18; N, 14.73; found C, 56.80; H, 3.14; N, 14.78.

1

-(4-(5-nitrobenzofuran-2-yl)thiazol-2-yl)-3-(4-nitrophenyl)urea (e34): Brown powder, 58%

-1

yield, mp. 286-288 °C; IR: 3418, 3223, 3128, 2966, 1697, 1512, 1337, 1320, 1298, 1270 cm ;

1

H NMR (DMSO-d , 300 MHz) δ/ppm: 7.33 (1H, s, H ), 7.71 (2H, d, J=8.5 Hz, H₁₉_,₂₃), 7.81 (1H,

6

3

7 4

d, J=8.5 Hz, H ), 7.93 (1H, s, H14), 8.16-8.22 (3H, m, H6,20,22), 8.60 (1H, d, J=2.4 Hz, H ), 9.96

1

3

(

1H, s, NH), 11.25 (1H, s, NH); C NMR (DMSO-d , 75 MHz) δ/ppm: 102.8(C-3), 111.7(C-

6

1

4), 113.8(C-7), 116.3(C-4), 118.6(C-19,23), 124.5(C-6), 125.8(C-20,22), 127.9(C-9), 131.5(C-

0), 142.2(C-21), 142.5(C-18), 145.7(C-5), 152.0(C-2), 152.5(C-16), 153.7(C-8), 160.3(C-12);

+

LC-MS (m/z): 426.10 [MH ]. Anal. Calcd. for C H N O S: C, 50.82; H, 2.61; N, 16.46; found

18

11

5

6

C, 50.89; H, 2.63; N, 16.42.

1

-(4-fluorophenyl)-3-(4-(5-nitrobenzofuran-2-yl)thiazol-2-yl)urea (e35): Yellowish powder, 54%

-1

1

yield, mp. 213-215 °C; IR: 3410, 3270, 3113, 2976, 1697, 1517, 1338, 1278, 1257 cm ;

H

NMR (DMSO-d , 300 MHz) δ/ppm: 7.14 (1H, t, J=8.2 Hz, H ), 7.26-7.39 (2H, m H₃_,₂₀), 7.46-

6

22

7

8

1

.51 (2H, m, H19,23), 7.65 (1H, s, H14), 7.82 (1H, d, J=8.8 Hz, H

7

), 8.18 (1H, d, J=8.8 Hz, H

6

),

1

3

.58 (1H, s, H ), 10.32 (1H, s, NH), 11.25 (1H, s, NH); C NMR (DMSO-d , 75 MHz) δ/ppm:

4

6

02.6(C-3), 111.5(C-14), 113.2(C-7), 116.1(C-20,22), 117.3(C-4), 118.4(C-19,23), 121.2(C-6),

22.8(C-9), 128.7(C-18), 139.1(C-10), 143.1(C-5), 144.2(C-2), 151.2(C-16), 152.4(C-8),

2

1

ACCEPTED MANUSCRIPT

+

1

5

55.6(C-21), 160.8(C-12); LC-MS (m/z): 399.11 [MH ]. Anal. Calcd. for C H FN O S: C,

1

8

11

4

4.27; H, 2.78; N, 14.06; found C, 54.21; H, 2.75; N, 14.09.

1

-(4-chlorophenyl)-3-(4-(5-nitrobenzofuran-2-yl)thiazol-2-yl)urea (e36): Yellowish powder, 68%

-1

1

yield, mp. 294-296 °C; IR: 3396, 3219, 3128, 2953, 1695, 1525, 1340, 1292, 1251 cm ;

H

NMR (DMSO-d , 300 MHz) δ/ppm7.31 (1H, s, H ), 7.35 (2H, d, J=8.8 Hz, H₂₀_,₂₂), 7.50 (2H, d,

6

3

J=8.8 Hz, H₁₉_,₂₃), 7.66 (1H, s, H ), 7.82 (1H, d, J=9.1 Hz, H ), 8.17 (1H, d, J=9.1 Hz, H ), 8.59

14

7

6

1

3

(

1H, s, H ), 9.83 (1H, s, NH), 11.15 (1H, s, NH);

C NMR (DMSO-d , 75 MHz) δ/ppm:

6

4

1

5

03.7(C-3), 112.1(C-14), 112.5(C-7), 118.3(C-4), 120.3(C-6), 120.9(C-19,23), 127.0(C-9),

29.4(C-20,22), 129.9(C-21), 138.2(C-18), 139.8(C-10), 144.5(C-5), 152.2(C-2), 155.3(C-16),

+

57.7(C-8), 160.6(C-12); LC-MS (m/z): 415.08 [MH ]. Anal. Calcd. for C H ClN O S: C,

1

8

11

4

2.12; H, 2.67; N, 13.51; found C, 52.19; H, 2.64; N, 13.54.

1

-(4-bromophenyl)-3-(4-(5-nitrobenzofuran-2-yl)thiazol-2-yl)urea (e37): Dark brown powder,

5

4% yield, mp. 234-236 °C; IR: 3466, 3230, 3121, 2982, 1701, 1550, 1515, 1342, 1292, 1253

-1

1

cm ; H NMR (DMSO-d , 300 MHz) δ/ppm: 7.32 (1H, s, H ), 7.44 (2H, d, J=7.0 Hz, H₂₀_,₂₂),

6

3

7

.50 (2H, d, J=7.0 Hz, H₁₉_,₂₃), 7.77 (1H, s, H ), 7.94 (1H, d, J=9.0 Hz, H ), 8.14 (1H, d, J=9.0

14 7

1

3

6 4 6

Hz, H ), 8.55 (1H, s, H ), 9.56 (1H, s, NH), 10.28 (1H, s, NH); C NMR (DMSO-d , 75 MHz)

δ/ppm: 92.6(C-21), 103.8(C-3), 112.2(C-14), 112.5(C-7), 114.8(C-4), 118.3(C-6), 120.5(C-

1

4

9,23), 121.0(C-9), 129.9(C-20,22), 132.3(C-18), 139.8(C-10), 144.6(C-5), 152.3(C-2), 155.3(C-

+

6), 157.7(C-8), 160.5(C-12); LC-MS (m/z): 459.04 [MH ]. Anal. Calcd. for C H BrN O S: C,

1

8

11

4

7.07; H, 2.41; N, 12.20; found C, 47.13; H, 2.45; N, 12.22.

1

-(4-iodophenyl)-3-(4-(5-nitrobenzofuran-2-yl)thiazol-2-yl)urea (e38): Brown powder 60%

-1

1

yield, mp. 239-241 °C; IR: 3418, 3211, 3121, 2967, 1695, 1512, 1340, 1278, 1255 cm ;

H

NMR (DMSO-d , 300 MHz) δ/ppm: 7.17 (1H, d, J=1.4 Hz, H ), 7.27-7.32 (2H, m, H₂₀_,₂₂), 7.28

6

3

(

2H, dd, J

1

=1.2 Hz; J

2

7

=8.8 Hz, H19,23), 7.57 (1H, d, J=1.2 Hz, H14), 7.79 (1H, d, J=8.2 Hz, H ),

8

.18 (1H, dt, J =1.2 Hz; J =8.0 Hz, H ), 8.51 (1H, s, NH), 8.59 (1H, d, J=1.2 Hz, H ), 8.83 (1H,

1 2 6 4

1

3

s, NH); C NMR (DMSO-d , 75 MHz) δ/ppm: 85.0(C-21), 103.3(C-3), 106.8(C-14), 112.4(C-

6

7

1

), 118.2(C-4), 120.8(C-6), 121.2(C-23), 122.4(C-19), 130.1(C-9), 137.5(C-22), 138.0(C-20),

40.9(C-18), 141.4(C-10), 144.6(C-5), 155.2(C-2), 155.8(C-16), 157.7(C-8), 160.6(C-12); LC-

+

MS (m/z): 507.04 [MH ]. Anal. Calcd. for C18

4 4

H11IN O S: C, 42.70; H, 2.19; N, 11.07; found C,

4

2.78; H, 2.15; N, 11.01.

4

.2. Anticholinesterase activity assays

2

ACCEPTED MANUSCRIPT

Acetyl- (AChE) and butyryl-cholinesterase (BuChE) inhibitory activities of the synthesized

compounds were determined as previously reported [36]. The IC50 of each substance was

determined by constructing an absorbance and/or inhibition (%) curve at five different

concentrations (12.5, 25, 50, 100 and 200 ꢀM). IC values were calculated for a given antagonist

5

0

by determining the concentration needed to inhibit half of the maximum biological response of

the agonist. The substrates of the reaction were acetylthiocholine iodide and butyrylthiocholine

iodide. 5,5'-dithio-bis(2-nitrobenzoic) acid (DTNB) was used to measure anticholinesterase

activity. Stock solutions of the compounds and galanthamine in n-propanol were prepared at a

concentration of 4000 µg/mL. Aliquots of 150 µL of 100 mM phosphate buffer (pH 8.0), 10 ꢀL

-4

of sample solution and 20 ꢀL AChE (2.476x10 U/ꢀL) (or 3.1813x10 U/ꢀL BChE) solution

were mixed and incubated for 15 min at 25ºC. 10 ꢀL of DTNB solution was prepared by adding

2

.0 mL of pH 7.0 and 4.0 mL of pH 8.0 phosphate buffers to a mixture of 1.0 mL of 16 mg/mL

DTNB and 7.5 mg/mL NaHCO in pH 7.0 phosphate buffers. The reaction was initiated by the

3

addition of 10 ꢀL (7.1 mM) acetylthiocholine iodide (or 0.79 mM butyrylthiocholine iodide). In

this method, the activity was measured by following the yellow colour produced as a result of the

thio anion produced by reacting the enzymatic hydrolysis of the substrate with DTNB. The

samples were prepared at an initial concentration of 4000 ꢀM then were diluted with propyl

alcohol which was also, used as a control solvent. The hydrolysis of the substrates was monitored

using a BioTek Power Wave XS at 412 nm.

4

.3. Antioxidant activity assays

In the CUPRAC assay, absorbance values were used to calculate the A0.50, whereas inhibition (%)

values were used in the in ABTS assay to calculate the IC .

5

0

·

+

ABTS scavenging activities of the synthesized compounds were determined as previously

·

+

reported [42]. The solution of ABTS radical was generated by dissolving 19.2 mg of 2,2'-azino-

bis(3-ethylbenzothiazoline-6-sulphonic acid) (7 mM ABTS) and 3.3 mg K S O in distilled water

2

3

(5 mL). This solution was kept in dark for 24 hours at room temperature, and absorbance of the

solution was fixed to ~0.70 at 734 nm by dilution. Solutions of the samples were prepared in n-

propanol at a concentration of 1000 µg/mL. Absorbance was measured at room temperature at

·

+

7

34 nm, after 6 minutes from ABTS addition. Decrease in the absorption was used to calculate

the activities. The results were expressed as IC . Propyl alcohol was used as a control solvent.

5

0

Cupric reducing antioxidant capacities of the synthesized compounds were determined in

accordance with the literature method [43]. Solutions of the compounds and standards were

2

3

ACCEPTED MANUSCRIPT

prepared in n-propanol at a concentration of 1000 µg/mL. Different volumes (1000 mg/L and

5

6

4.5 mL) of the sample were added to a solution prepared by adding 61.0 µL of 10 mM CuCl

2

,

1.0 µL 7.5 mM neocuproine and 61.0 µL of 1.0 mM NH CH COO buffer (pH 7), respectively.

4

3

Absorbance was measured at room temperature at 450 nm, after an hour. The results were

calculated as A0.50. Propyl alcohol was used as a controls.

4

.4. Docking

3

D structures of the ligands were built and energy-minimized using Sybyl v8.1 (Tripos, Inc., St.

Louis, MO) on an Intel (Xeon 4 core, HP Z820) using Linux 6 operating system. The crystal

structures of torpedo califonica AchE protein (PDB code: 1ACJ) and human BuchE (pdb code:

1

P0M) were retrieved from the Protein Data Bank. Docking and scoring per se were performed

using MolDock Optimizer and MolDock Grid, respectively, as implemented within Molegro

Virtual Docker (version 6). The hardware used was Intel Core i7, with 16 GB ram memory using

Linux/Ubuntu 10.04 operating system [48]. Only side-chain torsional angles were allowed to

relax during energy minimization. The protein backbone was kept rigid and a new receptor

conformation was generated for each pose after docking with flexible side chains. 30 runs for

each molecule were carried out with a population size of 50, maximum iteration of 2000, scaling

factor of 0.50, crossover rate of 0.90 and a variation-based termination scheme.

Acknowledgments

This work was supported by the Sakarya Research Fund of the Sakarya University. Project

Number: 2014-50-02-003.

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1] O. di Pietro, E. Viayna, E.V. Garcia, M. Bartolini, R. Ramon, J.J. Jimenez, M.V. Clos, B.

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Tetrahydrobenzo[h][1,6]naphthyridines as a new family of potent peripheral-to-midgorge-

site inhibitors of acetylcholinesterase: Synthesis, pharmacological evaluation and

mechanistic studies, Eur. J. Med. Chem. 73 (2014) 141-152.

[

2] P.M. Ruiz, L. Rubio, E.G. Palomero, I. Dorronsoro, M.M. Millan, R. Valenzuela, P. Usan, C.

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Martinez, Design, synthesis, and biological evaluation of dual binding site

acetylcholinesterase inhibitors: New disease-modifying agents for alzheimer’s disease, J.

Med. Chem. 48 (2005) 7223-7233.

2

4

ACCEPTED MANUSCRIPT

[

3] M. Ignasik, M. Bajda, N. Guzior, M. Prinz, Ul. Holzgrabe, B. Malawska, Design, synthesis

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Potential of aryl-urea-benzofuranylthiazoles hybrids as multitasking agents in Alzheimer's disease

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