The Novel 4-Phenyl-2-Phenoxyacetamide Thiazoles modulates the tumor hypoxia leading to the crackdown of neoangiogenesis and evoking the cell death

Article abstract of DOI:10.1016/j.ejmech.2017.10.082

Tumor microenvironment is a complex multistep event which involves several hallmarks that transform the normal cell into cancerous cell. Designing the novel antagonistic molecule to reverse the tumor microenvironment with specific target is essential in modern biological studies. The novel 4-phenyl-2-phenoxyacetamide thiazole analogues 8_a-ab were synthesized in multistep process, then screened and assessed for cytotoxic and anti-proliferative effects in vitro against multiple cancer cells of different origin such as MCF-7, A549, EAC and DLA cells which revealed that compound 8f with fluoro and methyl substitute has potential cytotoxic efficacy with an average IC₅₀ value of ? 13 μM. The mechanism of cytotoxicity assessed for anti-tumor studies both in ascites and solid tumor models in-vivo inferred the regressed tumor activity. This is due to changes in the cause of tumor microenvironment with crackdown of neovascularization and evoking apoptosis process as assessed by CAM, corneal vascularization and apoptotic hallmarks in 8f treated cells. The molecular gene studies inferred involvement of HIF-1upregulation and stabilization of p53 which are interlinked in signaling as conferred by immunoblot analysis.

Full text of DOI:10.1016/j.ejmech.2017.10.082

Accepted Manuscript
The Novel 4-Phenyl-2-Phenoxyacetamide Thiazoles modulates the tumor hypoxia
leading to the crackdown of neoangiogenesis and evoking the cell death
Yasser Hussein Eissa Mohammed, Vikas H. Malojirao, Prabhu Thirusangu,
Mohammed Al-Ghorbani, B.T. Prabhakar, Shukath Ara Khanum
PII:
S0223-5234(17)30884-X
10.1016/j.ejmech.2017.10.082
EJMECH 9874
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 July 2017
Revised Date: 20 October 2017
Accepted Date: 30 October 2017
Please cite this article as: Y.H.E. Mohammed, V.H. Malojirao, P. Thirusangu, M. Al-Ghorbani, B.T.
Prabhakar, S.A. Khanum, The Novel 4-Phenyl-2-Phenoxyacetamide Thiazoles modulates the tumor
hypoxia leading to the crackdown of neoangiogenesis and evoking the cell death, European Journal of
Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.10.082.
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ACCEPTED MANUSCRIPT
Graphical abstract

ACCEPTED MANUSCRIPT
The novel 4-phenyl-2-phenoxyacetamide thiazoles modulates the tumor
hypoxia leading to the crackdown of neoangiogenesis and evoking the cell
death.
Yasser Hussein Eissa Mohammed1,3, Vikas H.Malojirao2, Prabhu Thirusangu2,
Mohammed Al-Ghorbani1, Prabhakar B.T2, Shukath Ara Khanum1*.
1Department of Chemistry, Yuvaraja’s College, University of Mysore, Mysore- 570005
Karnataka, India.
2Molecular Onco-medicine Laboratory, Postgraduate Department of Studies and Research in
Biotechnology, Sahyadri Science College (A), Kuvempu University, Shivamogga- 577203,
Karnataka, India.
3Department of Biochemistry, Faculty of Applied Science College, University of Hajjah, Yemen.
*Correspondence Author:
Dr. Shaukath Ara Khanum
Associate Professor and Head
Department of Chemistry,
Yuvaraja’s College,
University of Mysore, Mysuru.
Phone: +91 - 99018 88755
Fax: +821- 2419239
Email: shaukathara@yahoo.co.in
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ABSTRACT
Tumor microenvironment is a complex multistep event which involves several hallmarks
that transform the normal cell into cancerous cell. Designing the novel antagonistic molecule to
reverse the tumor microenvironment with specific target is essential in modern biological
studies. The novel 4-phenyl-2-phenoxyacetamide thiazole analogues 8a-ab were synthesized in
multistep process, then screened and assessed for cytotoxic and anti-proliferative effects in vitro
against multiple cancer cells of different origin such as MCF-7, A549, EAC and DLA cells
which revealed that compound 8f with fluoro and methyl substitute has potential cytotoxic
efficacy with an average IC50 value of ̴ 13 µM. The mechanism of cytotoxicity assessed for anti-
tumor studies both in ascites and solid tumor models in-vivo inferred the regressed tumor
activity. This is due to changes in the cause of tumor microenvironment with crackdown of
neovascularization and evoking apoptosis process as assessed by CAM, corneal vascularization
and apoptotic hallmarks in 8f treated cells. The molecular gene studies inferred involvement of
HIF-1upregulation and stabilization of p53 which are interlinked in signaling as conferred by
immunoblot analysis.
Key words: Phenoxy acetic acid; Thiazole; Hypoxia; Tumor Microenvironment; HIF-1a; p53.
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1. INTRODUCTION
Cancer is the multistep disease condition where divergent tissue mass or a cell expand in
an uncoordinated manner with that of bordering normal tissues or a cell and endure to be same
even after the termination of the cause that provoked it [1]. Diverse line of demonstration reveals
that neoplastic cells intrinsically mutate the normal cells into cancerous one. Communication of
neoplastic cells with adjacent cell population by discharging and drawing constant impulse
resulting in unending biological vista termed as tumor microenvironment which render provision
for maturation of malignant cells [2]. A key environmental provocation linked with tumor
metastasis and scarce clinical diagnosis is cancerous cell oxygen deficiency, designated as
hypoxia [3]. This condition is known to prompt the genes winding in the regulation of cell
proliferation, extracellular matrix (ECM) production, cell adhesion, and other hallmarks of tumor
genesis including apoptosis. The mechanism behind these effects is often accomplished through
induction of the hypoxia-inducible factor (HIF) family of transcription factors which is regulated
by tumor suppressor gene p53 [4]. Thus, tumor microenvironment is accepted as the top cat in
the advancement of neoplastic cells and vulnerable target for therapy [5]. Conventional cancer
treatment can show earlier positive result, but down the line, diseases may relapse by developing
resistance to multiple drugs. This intimates for the search of lost interconnection between tumor
development mechanism, hypoxia and current drug development approach. Tumor
microenvironment may be a decisive factor for connecting lacking link.
The attention of medicinal chemists is attracted by thiazole ring due to not only their synthetic
feasibility but also to their incorporation into the diversity of therapeutic active agents. Thiazole
derivatives exemplify an extensive variety of biological potencies including anti-cancer activity
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[6-8]. Thiazole, and its derivatives have been given special attention due to their widely potential
applications as medicinal drug. For instance, tiazofurin, dasatinib, and bleomycin are known
anti-neoplastic drugs. Several phenoxy acid analogues such as methotrexate, nizatidine and
alunbirg are known anti-neoplastic drugs [9-11]. Newly, most of the compounds, isolated from
the natural products, containing thiazole moiety, exhibit considerable cytotoxicities and anti-
tumor potentials [12-15]. Moreover, phenoxy acids and their analogues are related with a
diversity of biological activities not only anti-inflammatory, analgesic and anti-oxidant but also
anti-cancer activity [16, 17].
Earlier, our lab has reported the BP-1T (benzophenone-thiazole) molecule as potent
anti-angiogenic pharamacophore which regulates p53&HIF-1α pathway [18, 19]. These findings
have made it clear that conjugation of two moieties will lead to possible anti-cancerous
molecule. With this background, we have synthesized active phenoxy acetic acid at the position
4 of potent 2- amino phenyl thiazole moiety using different aryl acids having active groups like;
fluoro, difluoro, chloro, nitro, bromo etc, with phenyl thiazole backbone. It has been hoped that
the combination of these active groups in the new molecular design would lead to better anti-
tumor agents. In this regard, we report the synthesis of newly designed 2-phenoxy-N-(4-
phenylthiazol-2-yl)acetamide analogues (8a-ab) and evaluated these compounds for the anti-
cancer activity.
2. RESULT AND DISCUSSION
2.1. Chemistry
Synthesis of the title compounds 8a-ab was accomplished by a synthetic procedure as
shown in Scheme 1. All the synthesized compounds were established by IR, NMR and mass
spectral data. Substituted phenols 1a-j on esterification with ethyl chloroacetate (2) using dry
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acetone as a solvent gave substituted ethyl phenoxy acetates 3a-j, which were confirmed by the
disappearance of OH stretching and appearance of carbonyl stretching band for the ester group in
the IR absorption spectra. The proton NMR observations revealed that broad singlet for OH
proton disappeared and a triplet and quartet signals for CH3 and CH2 protons respectively
appeared. The compounds 3a-j on refluxing with aqueous sodium hydroxide in ethanol gave
phenoxy acetic acids 4a-j, which was clearly evident with the appearance of carbonyl group
stretching band of a carboxylic acid in the IR spectra. In proton NMR, the appearance of COOH
proton and disappearance of triplet and quartet peaks for CH3 and CH2 protons, respectively has
confirmed the formation of the product. Further, 2-amino-4-phenyl thiazoles (7a-c) were obtained
by the reaction of substituted acetophenones and thiourea in the presence of iodine as a catalyst
and ethanol as a solvent to give 7a-c, which was established by the disappearance of carbonyl
stretching of acetophenone in the IR spectra. Besides, an increase in one aromatic proton of
thiazole ring and the disappearance of one NH2 proton of thiourea in the NMR spectra also
confirms the product. Finally, substituted phenoxy acetic acids (4a-j), on treatment with 2-amino-
4- phenyl thiazoles (7a-c) in the presence of lutidine and TBTU (O-(benzotriazol-1-yl)-
N,N,No,No-tetramethyl uronium tetrafluoroborate) as a coupling reagent, delivered the expected
final products 8a-ab in a good yield (80-85%). This was supported by the disappearance of NH2
and COOH stretching of the compounds 7a-c and 4a-j, respectively in the IR spectra. Also by the
disappearance of COOH proton and appearance of CONH proton in the NMR spectra.
2.2. Biology
2.2.1. Compound 8f is an effective cytotoxic molecule
Literature survey reveals that the anti-tumor properties of amino phenyl thiazole and
phenoxy acetic acid derivatives are well known [16, 17]. In the current investigation, cytotoxic
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potential of the amino phenyl thiazole analogues conjugated with phenoxy acetic acid derivatives
was carried out by cell based screening against multiple cancer cell lines, such as MCF-7, A549,
EAC and DLA cells in-vitro. Out of twenty eight compounds, compound 8f showed significant
cytotoxicity effect against these cell lines with an average IC50 value of ~13 µM as listed in
table-1 which was inferred by MTT and Tryphan blue assays. In spite of modest anti-
proliferative and cytotoxic effect of other compounds of the series as discussed in SAR, two
separate cytotoxic assays strongly recommend the compound 8f as an effective compound which
was selected for further studies. In addition to that we tested the potentiality of the compound 8f
against one of the drug resistance cell lines MDA-MB-468 cells which a triple negative breast
cancer cell line. The cell lines are resistant to chemotherapy since the lack of biomarkers, which
is major factor of failure in establishing a therapeutic strategy [20]. To test whether the
synthesized analogues have any effect on these cell lines, all 28 analogues were screened against
MDA-MB-468 cell lines, out of these compound 8f was showed moderate effect against these
cancer cells with IC50 value of ~10 µM whereas 5-fluorouracil exhibited no significant
cytotoxicity as compared to the compound 8f which is a promisable drug ability which could be
further investigated for multidrug resistance [20].
2.2.2. Structure activity Relationship (SAR)
Heterocyclic compounds containing thiazole scaffold as a core unit is known to be
pharmacologically active molecules against various pathological conditions including cancer
[1-9]. Phenoxy acetic acids have a potential pharmacological activity [16]. Also 2- amino phenyl
thiazole derivatives are known to be active anti-cancer agents against many cancer cells [18].
The current study involves the multistep synthesis of 2- amino phenyl thiazole analogues hybrids
with phenoxy acetic acid derivatives. Structurally, the title compounds are having a basic
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backbone of 2- amino phenyl thiazole with phenoxy acetic acid link [Figure 1]. The IC50 values
for compounds as depicted in Table-1 which suggest that the compound 8f with two fluoro
groups at ortho and para position in ring A and methyl group at the para position in ring C
showed IC50 value ̴ 13 µM on MCF-7, A549, EAC and DLA cells and ̴ 10 µM on MDA-MB-468
a drug resistant cell line as verified by in-vitro studies. In addition, the compound 8i with fluoro
group at para position in ring A and methyl group at the para position in ring C exhibited
moderate IC50 ~19 µM. on MCF-7, A549, EAC and DLA cells and ̴ 24 µM on MDA-MB-468
cells , Other compounds, 8s, 8v have a structure similar to 8f but lacking one methyl group and
showed increased IC50 ~26 µM & ~35 µM respectively on MCF-7 ,A549, EAC and DLA cells
and ̴ 32 µM & 41 µM on MDA-MB-468 cells . 5-fluorouracil is used as a positive control which
showed good anti-neoplastic effect against MCF-7, A549, EAC and DLA cells but did not
showed any significant effect against MDA-MB-468 cells. Over all in the title compounds 8a-ab
increasing the number of methyl groups and the number of fluoro groups at the para & ortho
position played a significant role in the biological activity of the compounds. Thus, fluoro groups
at ortho and para position in ring A of pheoxy acetic acid and a methyl group at the para position
in ring C of thiazole are important for the biological activity. The other molecules which contain
substituents like chloro and bromo does not show any significant cytotoxicity [Table 1].
Compound 8f was selected as a lead compound, based on its significant structure activity
relationships and increased in IC50 values, for further evaluation of its angiogenesis activity.
2.2.3. Compound 8f exhibits anti-tumor potentiality
Ehrlich ascites carcinoma (EAC) and solid tumor have become most widely used
experimental animal model for assessing the in vivo tumor development and also for
angiogenesis studies. The mouse mammary carcinoma cells are known to secrete the ascites fluid
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in the exponentially, which is direct nutritional and the growth factors source for tumor
development. Therefore, shuttering secretion of ascites by molecules may contribute to tumor
reduction [19, 21]. In the current investigation, EAC tumor models were administered with
compound 8f at 25 mg/kg and 50 mg/kg body weight (bw) on every alternative day for three
doses. The experimental results indicate that compound 8f was decreased the tumor volume by
74.35% and 80.2% of inhibition in a dose dependent manner [Figure 2a and 2e] which resulted
in diminished cell density with six fold reduction in a concentration dependent manner [Figure
2b]. The treatment of compound 8f markedly counteracted the secretion of ascites with three to
four fold decrease in dose dependent manner [Figure 2c] which reflected in extended
survivability with 23 and 25 plus days compared to untreated animals [Figure 2d]. The
administration of compound 8f had not shown any symptoms of adverse effect on the normal
lymphoid organs which was evident from the morphology of the liver and spleen of treated and
untreated groups [Figure 2f and 2g]. For further confirmation, EAC solid tumor models were
developed and administered with compound 8f at 25 mg and bw intraperitoneally. The results
inferred that compound 8f has reduced the tumor growth as observed by animal morphology
[Figure 3a and 3b] dissected tumor mass up to 10 g compared to control [Figure 3d].The
graphical representation of the tumor volume in a dose dependent manner is illustrated in [Figure
3c]. Compound 8f prolonged lifespan of the animal for 110 days compared to that of untreated
animals [Figure 3e].
2.2.4. Compound 8f regresses the neoangiogenesis and actuates the tumor cell apoptosis in-vivo.
Angiogenesis is a central event of tumor development and aggressiveness. It plays a vital
role in supplying oxygen, growth factors in tumor cells [22] and confrontation of tumor cells to
apoptosis which is one of the significant strategies of cancerous cells for their survivability [23].
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Counter attacking physiological conditions is essential to tackle the tumor progression and
development. Inhibition of tumor growth by compound 8f prompted to investigate the
anti-angiogenic efficacy on neovascularization by reliable models such as alkali injured rat
cornea and rVEGF165 induced CAM assay and tumor induced peritoneal angiogenesis assay. The
results demonstrate that the compound 8f restrains the sprouting angiogenesis in cornea to about
60% [Figure 4a and 4b] and with the CAM model to about 80% [Figure 4c and 4d] compared to
control. The administration of compound 8f regressed EAC induced angiogenesis in peritoneum
with 30 and 10 % respectively in a concentration dependent manner [Figure 4e and 4f]. Further,
compound 8f altered another hallmark i.e. apoptotic parameters which was assessed by giemsa
stain and DNA fragmentation assay. The results demonstrate that the compound 8f induced the
apoptosis in EAC cells by forming membrane blabbing, cell shrinkage and irregular shape which
resulted in DNA degradation, a chief event in cell death [Figure 5a and 5c].
2.2.5. Compound 8f effectively blocks HIF-1α by p53/MDM2-mediated proteasomal degradation
During tumor condition, hypoxia microenvironment persists and up regulates the HIF-1-α
a transcription factor which promotes the angiogenic event and evades apoptosis of the cells.
This process is regulated by down regulation of p53 expression in the tumor cells [21, 23]. The
interaction between HIF-1α, p53 and MDM2 has a crucial part in the regulation of HIF-1α. In
reaction to hypoxia which will be present in tumor micro environment, HIF-1α is vastly
expressed and interacts with p53 thereby, HIF-1α protects the p53 from MDM2-mediated
ubiquitination [24] but HIF-1α is negatively regulated by p53 and provides the HIF-1α to MDM2
for ubiquitination process, leading to HIF-1α proteosomal degradation resulting in the tumor
inhibition [25-27]. In the current investigation the decrease in HIF-1α expression and increase in
the p53 expression after compound 8f treatment reveals the possible involvement of alteration in
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p53/MDM2 mediated stabilization of HIF-1α. HIF -1-α is the key candidate mainly involved in
the crackdown of the extracellular matrix (ECM) and sprouting of newer blood vessels in the
tumor as well as in the resistance of apoptosis. A decrease in HIF-1 α will contribute the
overexpression of p53, a prime transcription factor for induction of programmed cell death.
Blocking HIF 1-α and activating p53 expression by molecule in the neoplastic condition may
effectively modulate the tumor angiogenesis and apoptosis. Biochemical modulation of tumor
inhibition by compound 8f was assessed by immunoblots. The experimental evidences postulate
that compound 8f effectively decreased the expression of HIF-1α and upregulated the p53 and
MDM-2 which is evident from immunoblots [Figure 5b]. VEGF being the predominant activator
of angiogenesis mediated tumor establishment, drastically reduced tumor growth after compound
8f treatment was observed as assessed by ELISA. The compound 8f directly targets HIF-1α
stabilization and there by downstream angiogenic gene expression eventually leading to
decreased tumor growth
[Figure 5d].
3. Conclusion
In our current research, the 2-amino phenyl thiazole analogues (8a-ab) were synthesized
then screened for cytotoxic effects in vitro against multiple cancer cells of different origin, such
as MCF-7, A549, EAC and DLA cells , which revealed that compound 8f has potential cytotoxic
efficacy with an average IC50 value of
̴
against a drug resistant MDA-MB-468 cell with the IC50 value of
̴
postulated the angiogenic inhibitory modulation in various rVEGF165 induced model as assessed
by CAM and corneal vascularization. The potent lead compound 8f was further assessed for anti-
tumor studies, both in ascites and solid tumor models in vivo, which revealed the regressed tumor
activity. The compound 8f acts against the tumor cells by repressing HIF-1α by p53/MDM-2
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mediated degradation .The molecular gene studies inferred involvement of HIF-1α upregulation
and stabilization of p53, which are interlinked in signaling as conferred by immunoblot analysis.
4. Materials and methods
4.1. Experimental section
Unless stated otherwise, all reagents and solvents required for the synthesis of the title
compounds 8a-ab were procured from Sigma Aldrich Chemical Co. The purity of the compounds
was checked by TLC which was performed on aluminium-backed silica plates and the spots were
detected by exposure to UV-lamp at λ= 254 nm. Melting points and boiling point were measured
on a Chemiline, Microcontroller Based Melting Point/ Boiling Point-Cl725 Apparatus with a
digital thermometer. IR spectra were recorded on the Agilent Technologies Cary 630 FT-IR
spectrometer, 1H and 13C NMR spectra were recorded on VNMRS-400 Agilent-NMR
spectrophotometer. Chemical shifts are given in parts per million downfield from
tetramethylsilane. The mass spectra were obtained with a VG70-70H spectrophotometer and the
elemental analysis (C, H, and N) was performed on Elementar Vario EL III elemental analyzer.
The results of elemental analyses were within ±0.4%.
Sodium bicarbonate and protease inhibitor cocktail and another reagents and solvents
which were required for the biological assays were obtained from Sigma Aldrich, USA,
Anti- HIF 1 -α obtained from Santa Cruz, USA, Anti -p53 and anti-β- Actin were obtained from
BD bioscience, USA, DMEM medium, whereas anti-biotic- anti-mycotic solution, fetal bovine
serum (FBS) were obtained from Invitrogen, USA.
4.1.1. Chemistry
4.1.1.1. General synthetic procedure for phenoxy acetic ethyl ester derivatives (3a-j).
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A mixture of substituted phenols (1a-j, 0.05 mol) and ethyl chloroacetate (2, 0.075 mol) in dry
acetone (40 ml) with anhydrous potassium carbonate (0.075 mol) was refluxed for 10-12 h. The
reaction mixture was cooled and solvent removed by distillation. The residual mass was
triturated with cold water to remove potassium carbonate, and extracted with ether (3 × 30 ml).
The ether layer was washed with 10% sodium hydroxide solution (3 × 30 ml) followed by water
(3 × 30 ml) and then dried over anhydrous sodium sulfate and evaporated to afford compounds
(3a-j). Finally, these compounds were purified by recrystallization and distillation method.
4.1.1.1.1. Ethyl 2-phenoxyacetate (3a)
1
Yield 80%; B.P. 100oC; FT-IR (KBr, νmaxcm-1): 1735-1759 (ester, C=O), 1150(ester, C-O); H
NMR (400 MHz, CDCl3)δ(ppm): 1.32 (t, 3H, CH3 of ester), 4.24 (q, 2H, CH2 of ester), 5.01 (s,
2H, OCH2), 6.77–7.34 (m, 5H, Ar-H); LC–MS m/z 181 [M+1]+. Anal. Calcd. for C10H12O3
(180): C, 66.65; H, 6.71. Found: C, 66.62; H, 6.68 %.
4.1.1.1.2. Ethyl 2-(2-bromophenoxy)acetate (3b)
o
Yield 91%; B.P. 279 C; FT-IR (KBr, νmaxcm-1): 1730-1755 (ester, C=O), 1155 (ester, C-O); 1H
NMR (CDCl3) δ(ppm): 1.20 (t, 3H, CH3of ester), 4.16 (q, 2H, CH2 of ester), 4.76 (s, 2H, OCH2),
6.87-7.23 (m, 4H, Ar-H); LC–MS m/z 259 [M+] and 261 [M+2]. Anal. Calcd. for C10H11BrO3
(259): C, 46.36; H, 4.28. Found: C, 46.15; H, 3.97 %.
4.1.1.1.3. Ethyl 2-(4-chloro-3-methylphenoxy)acetate (3c)
o
1
Yield73%; B.P. 114 C; FT-IR (KBr, νmaxcm-1): 1720-1745 (ester, C=O), 1120 (ester, C-O); H
NMR (400 MHz, CDCl3) δ(ppm): 1.29 (t, 3H, CH3 of ester), 2.38 (s, 3H, CH3), 4.34 (q, 2H, CH2
of ester), 5.01 (s, 2H, OCH2), 6.77–7.34 (m, 3H, Ar-H); LC–MS m/z 229 [M+] and 231 [M+2].
Anal. Calcd. for C11H13ClO3 (229): C, 57.78; H, 5.73. Found: C, 57.63; H, 5.65 %.
4.1.1.1.4. Ethyl 2-(2-methylphenoxy)acetate (3d)
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o
1
Yield 88%; B.P. 259 C; FT-IR (KBr, νmaxcm-1): 1730-1740 (ester, C=O), 1115 (ester, C-O); H
NMR (400 MHz, CDCl3) δ(ppm): 1.43 (t, 3H, CH3 of ester), 2.41 (s, 3H, CH3), 4.24 (q, 2H, CH2
of ester), 5.08 (s, 2H, OCH2), 6.97–7.34 (m, 4H, Ar-H); LC–MS m/z 195 [M+1]+. Anal. Calcd.
for C11H14O3 (194): C, 68.02; H, 7.27. Found: C, 67.88; H, 7.21 %.
4.1.1.1.5. Ethyl 2-(2-isopropylphenoxy)acetate (3e)
o
1
Yield 85%; B.P. 300 C; FT-IR (KBr, νmaxcm-1): 1730-1745 (ester, C=O), 1123 (ester, C-O); H
NMR (400 MHz, CDCl3) δ(ppm): 1.17 (d, 6H, J=7 Hz, 2CH3), 1.36 (t, 3H, CH3 of ester), 3.18
(m, 1H, CH), 4.74 (q, 2H, CH2 of ester), 4.91 (s, 2H, OCH2), 6.17–7.24 (m, 4H, Ar-H); LC–MS
m/z 223 [M+1]+. Anal. Calcd. for C13H18O3 (222): C, 70.24; H, 8.16. Found: C, 70.15; H, 8.11
%.
4.1.1.1.6. Ethyl 2-(2,4-difluorophenoxy)acetate (3f)
o
1
Yield 75%; B.P. 263 C; FT-IR (KBr, νmaxcm-1): 1728-1738 (ester, C=O), 1115 (ester, C-O); H
NMR (400 MHz, CDCl3) δ(ppm): 1.44 (t, 3H, CH3 of ester), 4.32 (q, 2H, CH2 of ester), 4.41 (s,
2H, OCH2), 6.77–7.44 (m, 3H, Ar-H); LC–MS m/z 217 [M+1]+. Anal. Calcd. for C10H10F2O3
(216): C, 55.56; H, 4.66. Found: C, 55.48; H, 4.49 %.
4.1.1.1.7. Ethyl 2-(4-fluorophenoxy)acetate (3g)
o
1
Yield 71%; B.P. 145 C; FT-IR (KBr, νmaxcm-1): 1729-1743 (ester, C=O), 1126 (ester, C-O); H
NMR (400 MHz, CDCl3) δ(ppm): 1.29 (t, 3H, CH3 of ester), 4.64 (q, 2H, CH2 of ester), 5.11 (s,
2H, OCH2), 7.07–7.12 (d, 2H,J=7 Hz, Ar-H),7.13-7.14 (d, 2H, J=8 Hz, Ar-H); LC–MS m/z 198
[M+]. Anal. Calcd. for C10H11FO3 (197): C, 60.60; H, 5.59. Found: C, 60.58; H, 5.57 %.
4.1.1.1.8. Ethyl 2-(2,4-diisopropylphenoxy)acetate (3h)
Yield75%; Pasty mass; FT-IR (KBr, νmaxcm-1): 1735-1750 (ester, C=O), 1120-1270 (ester, C-O);
1H NMR (400 MHz, CDCl3) δ(ppm): 1.17 (d, 6H, J=8 Hz, 2CH3),1.25 (d, 6H, J=8 Hz,
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2CH3),1.29 (t, 3H, CH3 of ester), 2.38 (m, 1H, CH),3.59 (m, 1H, CH), 4.84 (q, 2H, CH2 of ester),
4.81 (s, 2H, OCH2), 6.87–7.84 (m, 3H, Ar-H); LC–MS m/z 265 [M+1]+. Anal. Calcd. for
C16H24O3 (264): C, 72.69; H, 9.15. Found: C, 72.53; H, 9.03 %.
4.1.1.1.9. Ethyl 2-(4-Methylphenoxy)acetate (3i)
o
1
Yield 88%; B.P. 141 C; FT-IR (KBr, νmaxcm-1): 1731-1760 (ester, C=O), 1127 (ester, C-O); H
NMR (400 MHz, CDCl3) δ(ppm): 1.46 (t, 3H, CH3 of ester), 2.38 (s, 3H, CH3), 4.55 (q, 2H, CH2
of ester), 5.32 (s, 2H, CH2), 6.97–7.14 (d, 4H, J=8 Hz, Ar-H), 7.21–7.44 (d, 4H, J=8 Hz, Ar-H);
LC–MS m/z 195 [M+1]+. Anal. Calcd. for C11H14O3 (194): C, 68.02; H, 7.27. Found: C, 67.89;
H, 7.13 %.
4.1.1.1.10. Ethyl 2-(4-chlorophenoxy)acetate (3j)
o
1
Yield 70%; B.P. 178 C; FT-IR (KBr, νmaxcm-1): 1733-1751 (ester, C=O), 1131 (ester, C-O); H
NMR (400 MHz, CDCl3) δ(ppm): 1.53 (t, 3H, CH3 of ester), 4.54 (q, 2H, CH2 of ester), 4.38 (s,
2H, OCH2), 7.04-7.21 (d, 2H, J=8 Hz, Ar-H), 7.33-7.41 (d, 2H, J=8 Hz, Ar-H); LC–MS m/z 215
[M+] and 217 [M+2]. Anal. Calcd. for C10H11ClO3 (215): C, 55.96; H, 5.17. Found: C, 55.87; H,
5.13 %.
4.1.1.2. General synthetic procedure for phenoxyacetic acid analogues (4a-j).
Compounds (3a-j ,0.02 mol) were dissolved in ethanol (15 mL), sodium hydroxide (0.035 mol) in
water (5 mL) was added, andthe mixture was refluxed for 5–9 h. The reaction mixture was
cooled and acidified with 2 N hydrochloric acid. The precipitate was filtered, washed with water,
and finally recrystallized from methanol to afford desired compounds (4a-j). Compound (4a) is
takenas a representative example to explain physical and characterization data.
4.1.1.2.1. 2-Phenoxyacetic acid (4a)
14

ACCEPTED MANUSCRIPT
Yield 81%; M.P. 98-101ºC; FT-IR (KBr, νmaxcm-1):1714-1725 (C=O), 1246 (Ar–O–C), 3377-
3388 (OH); 1H NMR (400 MHz, CDCl3) δ(ppm): 4.66 (s, 2H, OCH2), 6.79-7.35 (m, 5H, Ar-H),
13.11 (s, 1H, COOH); LC–MS m/z 153 [M+1]+. Anal. Calcd. for C8H8O3 (152): C, 63.15; H,
5.30. Found: C, 63.12; H, 5.27 %.
4.1.1.2.2.2-Bromo phenoxy acetic acid (4b)
Yield 91%; M.P. 217-219oC; FT-IR (KBr, νmaxcm-1):1715-1730 (C=O), 1250 (Ar–O–C), 3375-
3399 (OH); 1H NMR (400 MHz, CDCl3) δ(ppm): 4.71 (s, 2H, OCH2), 6.89-7.45 (m, 4H, Ar-H),
14.09 (s, 1H, COOH); LC–MS m/z 231 [M+], 233 [M+2]. Anal. Calcd. for C8H7BrO (231): C,
41.59; H, 3.05. Found: C, 41.52; H, 3.15 %.
4.1.1.2.3.2-(4-Chloro-3-methylphenoxy)acetic acid (4c)
Yield 77%; M.P. 115-118 oC; FT-IR (KBr, νmaxcm-1): 1710-1720 (C=O), 1245 (Ar–O–C), 3366-
1
3396 (OH); H NMR (400 MHz, CDCl3) δ(ppm): 2.46 (s, 3H, CH3), 4.56 (s, 2H, OCH2), 6.61-
7.54 (m, 3H, Ar-H), 13.14 (s, 1H, COOH); LC–MS m/z 201 [M+], 203 [M+2]. Anal. Calcd. for
C9H9ClO3 (201): C, 53.88; H, 4.52. Found: C, 53.73; H, 4.33 %.
4.1.1.2.4. 2-(2-Methylphenoxy)acetic acid (4d)
Yield 81%; M.P. 142-144 ºC; FT-IR (KBr, νmaxcm-1): 1730-1745 (C=O), 1253 (Ar–O–C), 3375-
1
3380 (OH); H NMR (400 MHz, CDCl3) δ(ppm): 2.39 (s, 3H, CH3), 4.66 (s, 2H, OCH2), 6.88-
8.35 (m, 4H, Ar-H), 13.13 (s, 1H, COOH); LC–MS m/z 167 [M+1]+. Anal. Calcd. for C9H10O3
(166): C, 65.05; H, 6.07. Found: C, 64.91; H, 6.10 %.
4.1.1.2.5. 2-(2-Isopropylphenoxy)acetic acid (4e)
Yield 69%; M.P. 132-134 ºC; FT-IR (KBr, νmaxcm-1): 1715-1735 (C=O), 1249 (Ar–O–C), 3355-
1
3473 (OH); H NMR (400 MHz, CDCl3) δ(ppm): 1.17 (d, 6H, J=8 Hz, CH3), 3.06 (m, H, CH),
15

ACCEPTED MANUSCRIPT
4.89 (s, 2H, OCH2), 6.92-8.01 (m, 4H, Ar-H), 14.02 (s, 1H, COOH); LC–MS m/z 195 [M+1]+.
Anal. Calcd. for C11H14O3 (194): C, 68.02; H, 7.27. Found: C, 67.89 H, 7.11 %.
4.1.1.2.6. 2-(2,4-Difluorophenoxy)acetic acid (4f)
Yield 79%; M.P. 124-127 ºC; FT-IR (KBr, νmaxcm-1): 1724-1745 (C=O), 1253 (Ar–O–C), 3375-
1
3391 (OH); H NMR (400 MHz, CDCl3) δ(ppm): 4.77 (s, 2H, OCH2), 6.92-7.38 (m, 3H, Ar-H),
13.20 (s, 1H, COOH); LC–MS m/z 188 [M+]. Anal. Calcd. for C8H6F2O3 (188): C, 51.08; H,
3.21. Found: C, 51.18; H, 3.16 %.
4.1.1.2.7. 2-(4-Fluorophenoxy)acetic acid (4g)
Yield73%; M.P. 103-106 ºC; FT-IR (KBr, νmaxcm-1): 1725-1755 (C=O), 1250 (Ar–O–C), 3345-
3380 (OH); 1H NMR (400 MHz, CDCl3) δ(ppm): 4.59 (s, 2H, OCH2), 7.09-7.25 (d, 2H, J=8 Hz,
Ar-H),7.31-7.42 (d, 2H, J=8 Hz, Ar-H) 13.93 (s, 1H, COOH); LC–MS m/z 170 [M+]. Anal.
Calcd. for C8H7FO3 (170): C, 56.48; H, 4.15. Found: C, 56.41; H, 4.02 %.
4.1.1.2.8.2-(2,4-Diisopropylphenoxy)acetic acid (4h)
Yield 78%; M.P. 82-85ºC; FT-IR (KBr, νmaxcm-1): 1725-1748 (C=O), 1258 (Ar–O–C), 3348-
1
3399 (OH); H NMR (400 MHz, CDCl3) δ(ppm): 1.15 (d, 6H, J=8Hz, 2CH3),1.25 (d, 6H,
J=8Hz, 2CH3), 3.09 (m, H, CH),3.89 (m, H, CH), 4.69 (s, 2H, OCH2), 5.99-7.45 (m, 3H, Ar-
H), 13.26 (s, 1H, OH); LC–MS m/z 237 [M+1]+. Anal. Calcd. for C14H20O3 (236): C, 71.16; H,
8.53. Found: C, 71.06; H, 8.34 %.
4.1.1.2.9. (4-Methylphenoxy)acetic acid(4i)
Yield 81%; M.P. 142-144 ºC; FT-IR (KBr, νmaxcm-1): 1730-1745 (C=O), 1253 (Ar–O–C), 3375-
1
3380 (OH); H NMR (400 MHz, CDCl3) δ(ppm): 2.39 (s, 3H, CH3), 4.66 (s, 2H, OCH2), 6.88-
7.15 (d, 2H, J=8 Hz, Ar-H), 7.23-7.35 (d, 2H, J=8 Hz, Ar-H), 13.13 (s, 1H, COOH); LC–MS
16

ACCEPTED MANUSCRIPT
m/z 167 [M+1]+. Anal. Calcd. for C9H10O3 (166): C, 65.05; H, 6.07. Found: C, 64.84; H, 6.01
%.
4.1.1.2.10.4-Chlorophenoxyacetic acid (4j)
Yield 88%; M.P. 155-158ºC; FT-IR (KBr, νmaxcm-1): 1701-1725 (C=O), 1243 (Ar–O–C), 3375-
3376 (OH); 1H NMR (400 MHz, CDCl3) δ(ppm): 4.58 (s, 2H, OCH2), 7.09-7.15 (d, 2H, J=8 Hz,
Ar-H), 7.23-7.45 (d, 2H, J=8 Hz, Ar-H), 14.02 (s, 1H, COOH); LC–MS m/z 187 [M+], 189
[M+2]. Anal. Calcd. for C8H7ClO3 (187): C, 51.50; H, 3.78. Found: C, 51.42; H, 3.65 %.
4.1.1.3. General procedure for the synthesis of 2-amino-4-phenylthiazoles (7a-c)
2-Amino-4-phenyl thiazoles (7a-c) were synthesized by using substituted acetophenones (5a-c,
0.0025 mol) and thiourea (6, 0.003 mol). The reaction mixture was refluxed for 14 h in the
presence of iodine (0.0062 mol) and ethanol as a solvent. The product 7a-c were basified with
sodium hydroxide solution to get the white solid. The product was recrystallized from ethanol to
get needle like crystals.
4.1.1.3.1. 2-Amino-4-(4-chlorophenyl)thiazole (7a).
o
Yield 82%; M.P. 170-172 C; FT-IR (KBr, νmaxcm-1): 1696 (C=N), 3374-3376 (N-H);1H NMR
(400 MHz, DMSO)δ(ppm): 6.91-7.12 (s, 1H, thiazole ring), 7.14-7.22 (d, 2H, J=8. Hz, Ar-H),
7.25-7.32 (d, 2H, J=8 Hz, Ar-H), 7.45 (bs, 2H, NH2). LC–MS m/z 210 (M+), 212 (M+2). Anal.
Calcd. for C9H7ClN2S (210): C, 51.31; H, 3.35 N, 13.30. Found: C, 51.21; H, 3.17; N, 13.26%.
4.1.1.3.2. 2-Amino-4-(4-methylphenyl)thiazole (7b).
o
1
Yield 88%; M.P. 150-152 C; FT-IR (KBr, νmaxcm-1): 1692 (C=N), 3373-3375 (N-H); H NMR
(400 MHz, DMSO) δ(ppm): 2.31 (s, 3H, CH3), 7.01-7.22 (s, 1H, thiazole ring), 7.25-7.32 (d, 2H,
J=8.50 Hz, Ar-H), 7.35-7.42 (d, 2H, J=8 Hz, Ar-H), 7.50 (bs, 2H, NH2). LC–MS m/z 191 (M+).
17

ACCEPTED MANUSCRIPT
Anal. Calcd. for C10H10N2S (191): C, 63.13; H, 5.30; N, 14.72. Found: C, 63.15; H, 5.23; N,
14.70%.
4.1.1.3.3. 2-Amino-4-(4-bromophenyl)thiazole (7c).
1
Yield 78%; M.P. 184-186 oC; FT-IR (KBr, νmaxcm-1): 1698 (C=N), 3371-3373 (N-H); H NMR
(400 MHz, DMSO) δ(ppm): 6.96-7.22 (s, 1H, thiazole ring), 7.28-7.36 (d, 2H, J=8 Hz, Ar-H),
7.37-7.40 (d, 2H, J=8.50 Hz, Ar-H), 7.48 (bs, 2H, NH2). LC–MS m/z 255 (M+), 257(M+2).
Anal. Calcd. for C9H7BrN2S (255): C, 42.37; H, 2.77; N, 10.98. Found: C, 42.21; H, 2.52; N,
10.95%.
4.1.1.4. General synthetic procedure for 4-Phenyl-2-Phenoxyacetamide Thiazoles analogues
(8a-ab).
o
phenoxyacetic acids (4a-j, 2 mmol) in dry DCM (20 ml) was stirred at 25–30 C, and then
lutidine (3mmol) was added, followed by the addition of substituted amino-4-phenyl-1,3-
thiazoles (2 mmol). The reaction mixture was stirred at the same temperature for 30 min, then
o
cooled to 0–5 C and TBTU (2 mmol) was added over a period of 30 min maintaining the
o
temperature below 5 C. The reaction mass was stirred overnight and monitored by TLC using
chloroform: methanol (9:1) as the mobile phase. The purity of the compounds was determined
using high performance liquid chromatography (HPLC) by reversed phase agilent zorbax SB-
C18 column method, with methanol (50%): acetonitrile (30%): water (20%) as mobile phase.
The solvent was evaporated at reduced pressure, quenched by the addition of crushed ice and the
obtained solid was filtered, dried and recrystallized from ethanol to afford compounds 8a-ab in
good yield [supplementary files].
4.1.1.4.1. 2-Phenoxy-N-(4-(p-tolyl) thiazol-2-yl) acetamide (8a)
18

ACCEPTED MANUSCRIPT
o
Yield 83%; Purity ˃88% HPLC; M.P. 154-156 C; FT-IR (KBr, νmaxcm-1): 1679 (C=O), 1700
1
(C=N), 3173-3176(N-H); H NMR (400 MHz, DMSO) δ(ppm): 2.35 (s, 3H, CH3), 4.84 (s, 2H,
CH2), 6.92-7.77(m, 10H, Ar-H), 12.49 (bs, 1H, NH); 13C NMR (100 MHz, DMSO): δ 21.22,
66.61, 105.94, 114.12, 121.09, 125.73, 129.72, 130.01, 131.99, 150.60, 159.01, 164,57, 169.53;
LC–MS m/z (M+) 324. Anal. Calcd. For C18H16N2O2S (324): C, 66.65; H, 4.97; N, 8.64. Found
C, 66.69; H, 5.07; N, 8.58%.
4.1.1.4.2.2-(2-Bromophenoxy)-N-(4-(p-tolyl) thiazol-2-yl) acetamide (8b).
o
Yield 70%;Purity ˃85% HPLC; M.P. 187-189 C; FT-IR (KBr, νmaxcm-1): 1675 (C=O), 1698
(C=N), 3174-3180 (N-H);1H NMR (400 MHz, DMSO) δ(ppm): 2.47 (s, 3H, CH3), 4.86 (s, 2H,
CH2), 6.92-7.77(m, 9H, Ar-H), 12.46 (bs, 1H, NH); 13C NMR (100 MHz, DMSO): δ 21.22,
66.62, 106.04, 115.12, 118.40, 125.09, 129.11, 130.01 131.61, 132.69, 150.60, 158.01, 164.57,
169.13; LC–MS m/z 403 (M+) 405 (M+2). Anal. Calcd. For C18H15BrN2O2S (403): C, 53.61; H,
3.75; N, 6.95. Found: C, 53.47; H, 3.66; N, 6.88%.
4.1.1.4.3. 2-(4-Chloro-3-methylphenoxy)-N-(4-(p-tolyl) thiazol-2-yl) acetamide (8c).
o
Yield 90%;Purity ˃89% HPLC; M.P. 193-195 C; FT-IR (KBr, νmaxcm-1): 1665 (C=O), 1690
(C=N), 3155-3162 (N-H);1H NMR (400 MHz, DMSO) δ(ppm): 2.35 (s, 6H, 2CH3), 4.86 (s, 2H,
CH2), 6.92-7.77(m, 8H, Ar-H), 12.46 (bs, 1H, NH); 13C NMR (100 MHz, DMSO): δ 20.01,
21.22, 66.61, 106.04, 113.70, 125.73, 129.32, 130.01, 131.99, 133.99, 150.60, 159.01, 164,57,
169.13; LC–MS m/z 373 (M+) 375 (M+2). Anal. Calcd. For C19H17ClN2O2S (373): C, 61.20; H,
4.60; N, 7.51. Found: C, 61.12; H, 4.64; N, 7.39%.
4.1.1.4.4. N-(4-(P-tolyl) thiazol-2-yl)-2-(2-methylphenoxy) acetamide (8d)
o
Yield 88%; Purity ˃84% HPLC; M.P. 156-158 C; FT-IR (KBr, νmaxcm-1): 1670(C=O), 1687
1
(C=N), 3178-3183 (N-H); H NMR (400 MHz, DMSO) δ(ppm): 2.15 (s, 3H, CH3), 2.34 (s, 3H,
19

ACCEPTED MANUSCRIPT
CH3), 4.63 (s, 2H, CH2), 6.88-7.96(m, 9H, Ar-H), 12.46 (bs, 1H, NH); 13C NMR (100 MHz,
DMSO): δ15.41, 15.64, 21.32, 66.61, 105.01, 112.12, 120.40, 125.09, 126.21, 126.96, 129.21,
130.01, 131.99, 150.60, 159.01, 164.57, 169.13; LC–MS m/z (M+) 338. Anal. Calcd. For
C19H18N2O2S (338): C, 67.43; H, 5.36; N, 8.28.Found: C, 67.39; H, 5.32; N, 8.25%.
4.1.1.4.5.2-(2-Isopropylphenoxy)-N-(4-(p-tolyl) thiazol-2-yl) acetamide (8e)
o
Yield 83%; Purity ˃90% HPLC; M.P.130-132 C; FT-IR (KBr, νmaxcm-1): 1679 (C=O), 1699
(C=N), 3179-3188(N-H); 1H NMR (400 MHz, DMSO) δ(ppm): 1.20 (d, 6H, J=8Hz,2CH3), 2.34
(s, 3H, CH3),3.57 (m, 1H, CH), 4.83 (s, 2H, CH2), 6.86 -7.96(m, 9H, Ar-H), 12.48 (bs, 1H, NH);
13C NMR (400 MHz, DMSO): σ 21.33, 23.23, 27.33, 66.82, 105.01, 112.12, 121.01, 125.23,
126.70, 127.12,129.22, 130.01, 131.99, 136.09, 150.60, 155.01, 164.53, 169.13; LC–MS m/z
(M+) 366. Anal. Calcd. For C21H22N2O2S (366): C, 68.83; H, 6.05; N, 7.64.Found: C, 68.75; H,
5.89; N, 7.60%.
4.1.1.4.6. 2-(2, 4-Difluorophenoxy)-N-(4-(p-tolyl) thiazol-2-yl) acetamide (8f)
o
Yield 87%;Purity ˃96% HPLC; M.P. 136-138 C; FT-IR (KBr, νmaxcm-1): 1680 (C=O), 1701
(C=N), 3182-3188 (N-H);1H NMR (400 MHz, DMSO) δ(ppm): 2.34 (s, 3H, CH3), 4.65 (s, 2H,
CH2), 6.67-7.96 (m, 8H, Ar-H), 12.49 (bs, 1H, NH); 13C NMR (100 MHz, DMSO): δ 21.22,
66.64, 105.03, 106.13, 112.07, 117.09, 125.73, 129.53, 130.02, 131.99, 145.23, 150.60, 153.01,
153.78, 164.13, 169.23; LC–MS m/z (M+1) 361. Anal. Calcd. For C18H14F2N2O2S (360): C,
59.99; H, 3.92; N, 7.77. Found C, 59.76; H, 3.89; N, 7.58%.
4.1.1.4.7. N-(4-(4-Bromophenyl) thiazol-2-yl)-2-(4-chloro-2-methylphenoxy) acetamide (8g).
o
Yield 84%;Purity ˃88% HPLC; M.P. 198-200 C; FT-IR (KBr, νmaxcm-1): 1676 (C=O), 1690
1
(C=N), 3170- 3175(N-H); H NMR (400 MHz, DMSO) δ(ppm): 2.15 (s, 3H, CH3), 4.66 (s, 2H,
CH2), 6.82-7.78(m, 8H, Ar-H), 12.46 (bs, 1H, NH); 13C NMR (100 MHz, DMSO): δ 14.94,
20

ACCEPTED MANUSCRIPT
66.86, 105.44, 111.84, 123.40, 125.09, 126.73, 127.12, 128.03, 129.82, 132.12, 150.60, 156.72,
164.21, 169.26; LC–MS m/z 438 (M+) 440 (M+2) 442 (M+4). Anal. Calcd. For
C18H14BrClN2O2S (438): C, 49.39; H, 3.22; N, 6.40. Found: C, 49.32; H, 3.11; N, 6.28%.
4.1.1.4.8. N-(4-(4-Bromophenyl) thiazol-2-yl)-2-(4-fluorophenoxy) acetamide (8h)
o
Yield 87%;Purity ˃98% HPLC; M.P. 225-227 C; FT-IR (KBr, νmaxcm-1): 1671 (C=O), 1697
(C=N), 3179-3183 (N-H);1H NMR (400 MHz, DMSO) δ(ppm): 4.86 (s, 2H, CH2), 7.09 -7.98(m,
9H, Ar-H), 12.48 (bs, 1H, NH); 13C NMR (100 MHz, DMSO): δ 66.61, 105. 42, 115.92, 116.43,
123.13, 128.34, 132.04, 133.11, 150.60, 153.74, 155.24, 164.23, 169.26; LC–MS m/z 407 (M+)
409 (M+2). Anal. Calcd. For C17H12BrFN2O2S (407): C, 50.14; H, 2.97; N, 6.88. Found: C,
50.03; H, 2.90; N, 6.76%.
4.1.1.4.9. 2-(4-Fluorophenoxy)-N-(4-(p-tolyl) thiazol-2-yl) acetamide (8i)
o
Yield 81%;Purity ˃92% HPLC; M.P.186-188 C; FT-IR (KBr, νmaxcm-1): 1678 (C=O), 1693
1
(C=N), 3181-3184 (N-H); H NMR (400 MHz, DMSO) δ(ppm): 2.34 (s, 3H, CH3), 4.65 (s, 2H,
CH2), 7.26-7.96 (m, 9H, Ar-H), 12.49 (bs, 1H, NH); 13C NMR (100 MHz, DMSO): δ 21.33,
66.63, 105.02, 115.92, 116.07, 125.71, 129.51, 130.22, 131.99, 150.60, 153.01, 155.22, 164.13,
169.32; LC–MS m/z (M+) 342. Anal. Calcd. For C18H15FN2O2S (342): C, 63.14; H, 4.42; N,
8.18. Found C, 63.10; H, 4.32; N, 8.06%.
4.1.1.4.10. 2-(4-Bromophenoxy)-N-(4-(4-bromophenyl) thiazol-2-yl) acetamide (8j)
o
Yield 87%;Purity ˃86% HPLC; M.P. 220-222 C; FT-IR (KBr, νmaxcm-1): 1669 (C=O), 1695
1
(C=N), 3170-3177 (N-H); H NMR (400 MHz, DMSO) δ(ppm): 4.86 (s, 2H, CH2), 6.99 -
7.96(m, 9H, Ar-H), 12.48 (bs, 1H, NH); 13C NMR (100 MHz, DMSO): δ 66.63, 105.42, 115.93,
118.42, 123.13, 128.33, 132.01, 132.76, 133.12, 150.60, 156.72, 164.22, 169.26; LC–MS m/z
21

ACCEPTED MANUSCRIPT
468 (M+) 470 (M+2) 472 (M+4). Anal. Calcd. For C17H12Br2N2O2S (468): C, 43.61; H, 2.58; N,
5.98. Found: C, 43.53; H, 2.46; N, 5.85%.
4.1.1.4.11. N-(4-(4-Bromophenyl) thiazol-2-yl)-2-phenoxyacetamide (8k)
o
Yield 89%;Purity ˃80% HPLC; M.P. 190-192 C; FT-IR (KBr, νmaxcm-1): 1677 (C=O), 1686
(C=N), 3182-3187 (N-H);1H NMR (400 MHz, DMSO) δ(ppm): 4.86 (s, 2H, CH2), 6.99 -7.96(m,
10H, Ar-H), 12.48 (bs, 1H, NH); 13C NMR (100 MHz, DMSO): δ 66.62, 105. 43, 114.93,
121.45, 123.15, 128.35, 130.15, 132.09, 133.65, 150.60, 157.75, 164.25, 169.26; LC–MS m/z
389 (M+) 391 (M+2). Anal. Calcd. For C17H12Br2N2O2S (389): C, 43.61; H, 2.58; N, 5.98.
Found: C, 43.53; H, 2.46; N, 5.75%.
4.1.1.4.12. N-(4-(4-Bromophenyl) thiazol-2-yl)-2-(o-tolyloxy) acetamide (8l)
o
Yield 88%;Purity ˃83% HPLC; M.P. 184-186 C; FT-IR (KBr, νmaxcm-1): 1675 (C=O), 1698
(C=N), 3176-3182 (N-H); 1H NMR (400 MHz, DMSO) δ(ppm): 2.15 (s, 3H, CH3), 4.63 (s, 2H,
CH2), 6.82-7.96(m, 9H, Ar-H), 12.26 (bs, 1H, NH); 13C NMR (100 MHz, DMSO): δ 16.05,
67.12, 106.03, 112.93, 121.63, 123.72, 126.03, 126.91, 128.33, 131.62, 132.02, 133.11, 151.60,
158.72, 164.22, 169.26; LC–MS m/z 403 (M+) 405 (M+2). Anal. Calcd. For C18H15BrN2O2S
(403): C, 53.61; H, 3.75; N, 6.95. Found: C, 55.41; H, 3.57; N, 6.89%.
4.1.1.4.13. N-(4-(4-Bromophenyl) thiazol-2-yl)-2-(2-isopropylphenoxy) acetamide (8m)
o
Yield 83%;Purity ˃93% HPLC; M.P. 131-133 C; FT-IR (KBr, νmaxcm-1): 1672 (C=O), 1692
1
(C=N), 3172-31779 (N-H); H NMR (400 MHz, DMSO) δ(ppm): 1.14 (d, 6H, J=8Hz, CH3),
3.05 (m, 1H, CH), 4.63 (s, 2H, CH2), 6.90 -7.96(m, 9H, Ar-H), 12.48 (bs, 1H, NH); 13C NMR
(100 MHz, DMSO): δ 23.62, 27.32, 67.13, 106.01, 113.12, 121.00, 123.73, 126.61,127.12,
128.22, 132.03 , 132.72, 136.61, 150.60, 156.01, 164.5, 169.13; LC–MS m/z 431 (M+) 433
22

ACCEPTED MANUSCRIPT
(M+2). Anal. Calcd. For C20H19BrN2O2S (431): C, 55.69; H, 4.44; N, 6.49. Found: C, 55.47; H,
4.40; N, 6.27%.
4.1.1.4.14. N-(4-(4-Bromophenyl) thiazol-2-yl)-2-(2,4-difluorophenoxy) acetamide (8n)
o
Yield 89%; Purity ˃94% HPLC; M.P. 176-178 C; FT-IR (KBr, νmaxcm-1): 1665 (C=O), 1690
1
(C=N), 3155-3165 (N-H); H NMR (400 MHz, DMSO) δ(ppm): 4.63 (s, 2H, CH2), 6.68 -
7.96(m, 8H, Ar-H), 12.34 (bs, 1H, NH); 13C NMR (100 MHz, DMSO): δ 66.73, 104.99, 106.01,
112.42, 117.62, 123.32, 128.42, 132.02, 132.44, 145.12, 150.22, 153.11, 153.92,164.21, 169.26;
LC–MS m/z 425 (M+) 427 (M+2). Anal. Calcd. For C17H11BrF2N2O2S (425): C, 48.02; H, 2.61;
N, 6.59. Found: C, 48.07; H, 2.56; N, 6.62%.
4.1.1.4.15. 2-(4-Bromophenoxy)-N-(4-(4-chlorophenyl) thiazol-2-yl) acetamide (8o)
o
Yield 83%;Purity ˃92% HPLC; M.P. 216-218 C; FT-IR (KBr, νmaxcm-1): 1675 (C=O), 1695
(C=N), 3175-3180 (N-H);1H NMR (400 MHz, DMSO) δ(ppm): 4.89 (s, 2H, CH2), 6.98 -7.96(m,
9H, Ar-H), 12.48 (bs, 1H, NH); 13C NMR (100 MHz, DMSO): δ 66.71, 105. 92, 115.32, 118.42,
128.71, 129.33, 131.12, 132.62, 134.34, 149.99, 156.92, 163.99, 170.01; LC–MS m/z 424 (M+)
426 (M+2) 428 (M+4). Anal. Calcd. For C17H12BrClN2O2S (424): C, 48.19; H, 2.85; N, 6.61.
Found: C, 48.11; H, 2.77; N, 6.74%.
4.1.1.4.16. N-(4-(4-Chlorophenyl) thiazol-2-yl)-2-phenoxyacetamide (8p)
o
Yield 88%; Purity ˃89% HPLC; M.P. 197-200 C; FT-IR (KBr, νmaxcm-1): 1665 (C=O), 1685
1
(C=N), 3170-3174 (N-H); H NMR (400 MHz, DMSO) δ(ppm): 4.85 (s, 2H, CH2), 6.90-7.96
(m, 10H, Ar-H), 12.46 (bs, 1H, NH); 13C NMR (100 MHz, DMSO): δ 66.61, 105.04, 114.32,
121.02, 128.91, 129.22, 130.01, 131.11, 135.01, 151.00, 159.11, 165, 07, 168.97; LC–MS m/z
345 (M+) 347 (M+2). Anal. Calcd. For C17H13ClN2O2S (345): C, 59.22; H, 3.80; N, 8.12. Found:
C, 59.16; H, 3.87; N, 8.16%.
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4.1.1.4.17. N-(4-(4-Chlorophenyl) thiazol-2-yl)-2-(2-methylphenoxy) acetamide (8q)
o
Yield 84%;Purity ˃81% HPLC; M.P. 191-193 C; FT-IR (KBr, νmaxcm-1): 1676 (C=O), 1692
1
(C=N), 3180-3189 (N-H); H NMR (400 MHz, DMSO) δ(ppm): 2.15 (s, 3H, CH3), 4.63 (s, 2H,
CH2), 6.85-7.96 (m, 9H, Ar-H), 12.56 (bs, 1H, NH); 13C NMR (100 MHz, DMSO): δ 16.11,
67.01, 105.01, 112.32, 121.31, 126.55, 127.01, 128.92, 129.33, 131.01, 132.12, 134.43, 149.99,
159.33, 164, 57, 169.13; LC–MS m/z 359 (M+) 361 (M+2). Anal. Calcd. For C18H15ClN2O2S
(359): C, 60.25; H, 4.21; N, 7.81. Found: C, 60.20; H, 4.23; N, 7.72%.
4.1.1.4.18. N-(4-(4-Chlorophenyl) thiazol-2-yl)-2-(2-isopropylphenoxy) acetamide (8r)
o
Yield 80%;Purity ˃89% HPLC; M.P. 137-139 C; FT-IR (KBr, νmaxcm-1): 1677 (C=O),
1695(C=N), 3171-3176 (N-H); 1H NMR (400 MHz, DMSO) δ(ppm): 1.18 (d, 6H, J=8Hz, CH3),
3.05 (m, 1H, CH), 4.63 (s, 2H, CH2), 6.95 -8.21(m, 9H, Ar-H), 12.67 (bs, 1H, NH); 13C NMR
(100 MHz, DMSO): δ 24.03, 28.13, 66.75, 106.03, 113.12, 121.09, 126.64, 127.71, 128.97,
130.01, 131.45, 135,13, 137.24, 151.21, 156.17, 164.52, 169.13; LC–MS m/z 387 (M+) 389
(M+2). Anal. Calcd. For C20H19ClN2O2S (387): C, 62.09; H, 4.95; N, 7.24. Found: C, 62.13; H,
4.79; N, 7.18%.
4.1.1.4.19. N-(4-(4-Chlorophenyl) thiazol-2-yl)-2-(2,4-difluorophenoxy) acetamide (8s)
Yield 85%; Purity ˃86% HPLC; M.P. 119-121oC; FT-IR (KBr, νmaxcm-1): 1678 (C=O), 1697
1
(C=N), 3178-3183 (N-H); H NMR (400 MHz, DMSO) δ(ppm): 4.63 (s, 2H, CH2), 6.68-7.96
(m, 8H, Ar-H), 12.46 (bs, 1H, NH); 13C NMR (100 MHz, DMSO): δ 66.81, 104.99, 106.31,
113.11, 117.85, 129.52, 130.32, 131.61, 134.81, 146.01, 151.01, 153.52, 154.11, 164.57, 169.13;
LC–MS m/z 381 (M+) 383 (M+2). Anal. Calcd. For C17H11ClF2N2O2S (381): C, 53.62; H, 2.91;
N, 7.36. Found: C, 53.45; H, 2.84; N, 7.39%.
4.1.1.4.20. 2-(4-Chloro-2-methylphenoxy)-N-(4-(4-chlorophenyl) thiazol-2-yl) acetamide (8t)
24

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o
Yield 88%; Purity ˃87% HPLC; M.P. 232-235 C; FT-IR (KBr, νmaxcm-1): 1679 (C=O), 1699
(C=N), 3179-3189 (N-H);1H NMR (400 MHz, DMSO) δ(ppm): 2.15 (s, 3H, CH3), 4.63 (s, 2H,
CH2), 6.85-7.96 (m, 9H, Ar-H), 12.33 (bs, 1H, NH); 13C NMR (100 MHz, DMSO): δ 15.11,
67.31, 105.84, 113.14, 125.62, 126.41, 127.61, 128.90, 129.33, 130.16, 131.41, 134.93, 150.31,
157.32, 164.57, 169.13; LC–MS m/z 393 (M+) 395 (M+2) 397 (M+4). Anal. Calcd. For
C18H14Cl2N2O2S (393): C, 54.97; H, 3.59; N, 7.12. Found: C, 54.81; H, 3.42; N, 7.17%.
4.1.1.4.21. N-(4-(4-Chlorophenyl) thiazol-2-yl)-2-(4-fluorophenoxy) acetamide (8u)
o
Yield 86%;Purity ˃95% HPLC; M.P. 236-238 C; FT-IR (KBr, νmaxcm-1): 1678 (C=O), 1695
(C=N), 3178-3187 (N-H);1H NMR (400 MHz, DMSO) δ(ppm): 4.63 (s, 2H, CH2), 7.09 - 8.04
(m, 9H, Ar-H), 12.46 (bs, 1H, NH); 13C NMR (100 MHz, DMSO): δ 66.81, 105.08, 115.81,
116.61, 128.41, 130.03, 131.32, 134.41, 150.31, 154.22, 156.22, 166,37, 170.03; LC–MS m/z
363 (M+) 365 (M+2). Anal. Calcd. For C17H12ClFN2O2S (363): C, 56.28; H, 3.33; N, 7.72.
Found: C, 56.20; H, 3.29; N, 7.56%.
4.1.1.4.22. N-(4-(4-Chlorophenyl) thiazol-2-yl)-2-(2,4-diisopropylphenoxy) acetamide (8v)
o
Yield 80%; Purity ˃98% HPLC; M.P. 133-135 C; FT-IR (KBr, νmaxcm-1): 1676 (C=O), 1695
1
(C=N), 3179-3189 (N-H); H NMR (400 MHz, DMSO) δ(ppm): 1.14-1.20 (d, 6H, J=8 Hz,
2CH3),(d, 6H, J=8 Hz 2CH3), 2.87 (m, 1H, CH), 3.05 (m, 1H, CH), 4.63 (s, 2H, CH2), 6.76 -
7.96(m, 8H, Ar-H), 12.59 (bs, 1H, NH); 13C NMR (100 MHz, DMSO): δ 23.92, 24.87, 28.33,
34.32, 67.52, 106.01, 112.34, 125.33, 127.13,129.23, 130.13, 131.34, 135.33, 138.42, 141.42,
151.60, 153.67, 165.52, 169.93; LC–MS m/z 429 (M+) 431 (M+2). Anal. Calcd. For
C23H25ClN2O2S (429): C, 64.40; H, 5.87; N, 6.53. Found: C, 64.26; H, 5.89; N, 6.61%.
4.1.1.4.23. N-(4-(p-Tolyl) thiazol-2-yl)-2-(4-methylphenoxy) acetamide (8w)
25

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o
Yield 84%; Purity ˃92% HPLC; M.P. 173 -175 C; FT-IR (KBr, νmaxcm-1): 1680 (C=O), 1685
(C=N), 3166-3172 (N-H); 1H NMR (400 MHz, DMSO) δ(ppm): 2.29 (s, 6H, 2CH3), 4.89 (s, 2H,
CH2), 6.89-7.96 (m, 9H, Ar-H), 12.49 (bs, 1H, NH); 13C NMR (100 MHz, DMSO): δ 20.22,
21.45, 66.64, 105.06, 114.33, 125.65, 129.66, 130.01, 130.72, 131.03, 131.82, 150.60, 155.25,
164.13, 169.23; LC–MS m/z 338 (M+). Anal. Calcd. For C19H18N2O2S (338): C, 67.43; H, 5.36;
N, 8.28. Found C, 67.47; H, 5.22; N, 8.15%.
4.1.1.4.24. N-(4-(4-Bromophenyl) thiazol-2-yl)-2-(4-methylphenoxy) acetamide (8x)
o
Yield 89%;Purity ˃84% HPLC; M.P. 195-197 C; FT-IR (KBr, νmaxcm-1): 1683 (C=O), 1686
(C=N), 3170- 3173 (N-H);1H NMR (400 MHz, DMSO) δ(ppm): 2.25 (s, 3H, CH3), 4.89 (s, 2H,
CH2), 6.89-7.96 (m, 9H, Ar-H), 12.46 (bs, 1H, NH); 13C NMR (100 MHz, DMSO): δ 21.44,
66.62, 105.04, 114.41, 123.13, 128.03, 130.03, 131.11, 132.11, 132.58, 150.60, 155.13, 164.23,
169.46; LC–MS m/z 403 (M+) 405 (M+2). Anal. Calcd. For C18H15BrN2O2S (403): C, 53.61; H,
3.75; N, 6.95. Found: C, 55.54; H, 3.70; N, 6.82%.
4.1.1.4.25. N-(4-(4-Chlorophenyl) thiazol-2-yl)-2-(4-methylphenoxy) acetamide (8y)
o
Yield 81%; Purity ˃83% HPLC; M.P. 216 – 219 C; FT-IR (KBr, νmaxcm-1): 1670 (C=O), 1690
1
(C=N), 3170-3175 (N-H); H NMR (400 MHz, DMSO) δ(ppm): 2.28 (s, 3H, CH3), 4.89 (s, 2H,
CH2), 6.80 -7.86(m, 9H, Ar-H), 12.38 (bs, 1H, NH); 13C NMR (100 MHz, DMSO): δ 21.42,
66.92, 105.11, 114.41, 128.81,129.22, 130.12 , 130.72, 131.03, 134.54, 150.60, 155.94, 164.52,
169.13; LC–MS m/z 359 (M+) 361 (M+2). Anal. Calcd. For C18H15ClN2O2S (359): C, 60.25; H,
4.21; N, 7.81. Found: C, 60.13; H, 4.27; N, 7.79%.
4.1.1.4.26. 2-(4-Chlorophenoxy)-N-(4-(p-tolyl) thiazol-2-yl) acetamide (8z)
o
Yield 86%;Purity ˃85% HPLC; M.P. 173 – 175 C; FT-IR (KBr, νmaxcm-1): 1671 (C=O), 1690
(C=N), 3171-3177 (N-H);1H NMR (400 MHz, DMSO) δ(ppm): 2.34 (s, 3H, CH3), 4.89 (s, 2H,
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CH2), 7.03 -7.96(m, 9H, Ar-H), 12.69 (bs, 1H, NH); 13C NMR (100 MHz, DMSO): σ 22.02,
66.91, 105.32, 117.33, 125.83,126.84, 129.42, 130.12, 131.22, 132.74, 150.60, 156.72, 164.52,
169.53; LC–MS m/z 359 (M+) 361 (M+2). Anal. Calcd. For C18H15ClN2O2S (359): C, 60.25; H,
4.21; N, 7.81. Found: C, 60.02; H, 4.28; N, 7.88%.
4.1.1.4.27. N-(4-(4-Bromophenyl)thiazol-2-yl)-2-(4-chlorophenoxy)acetamide (8aa)
o
Yield 84%; Purity ˃92% HPLC; M.P. 205 – 207 C; FT-IR (KBr, νmaxcm-1): 1675 (C=O), 1680
1
(C=N), 3176-3182 (N-H); H NMR (400 MHz, DMSO) δ(ppm): 4.89 (s, 2H, CH2), 7.03-7.96
(m, 9H, Ar-H), 12.46 (bs, 1H, NH); 13C NMR (100 MHz, DMSO): δ 66.59, 105.41, 117.43,
123.23, 126.71, 128.43, 131.09, 132.09, 133.03, 150.60, 156.46, 164.34, 169.36; LC–MS m/z
424 (M+) 426 (M+2) 428 (M+4). Anal. Calcd. For C17H12BrClN2O2S (424): C, 48.19; H, 2.85;
N, 6.61. Found: C, 48.02; H, 2.88; N, 6.48%.
4.1.1.4.28. 2-(4-Chlorophenoxy)-N-(4-(4-chlorophenyl) thiazol-2-yl) acetamide (8ab)
o
Yield 80%; Purity ˃85% HPLC; M.P. 206 – 208 C; FT-IR (KBr, νmaxcm-1): 1673 (C=O), 1705
1
(C=N), 3180-3186 (N-H); H NMR (400 MHz, DMSO) δ(ppm): 4.89 (s, 2H, CH2), 7.03 -
7.96(m, 9H, Ar-H), 12.49 (bs, 1H, NH); 13C NMR (100 MHz, DMSO): δ 66.92, 107.12, 117.92,
126.83, 128.94, 129.57, 130.84, 131.20, 134.64, 152.10, 156.44, 164.54, 169.13; LC–MS m/z
379 (M+) 381 (M+2) 383 (M+4). Anal. Calcd. For C17H12Cl2N2O2S (379): C, 53.84; H, 3.19; N,
7.39. Found: C, 53.80; H, 3.13; N, 7.30%.
4.1.2. Biology
4.1.2.1. Animal cell culture and in vitro compound treatment
MCF-7, A549, EAC, DLA and MDA-MB-468 cells were used for the present study and
the cells were cultured in-vitro as described earlier [28]. All the above cells were treated with
increasing concentrations of 2-phenoxy-N-(4-phenylthiazol-2-yl)acetamide analogues (0, 10, 20,
27

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50, and 100 µM in DMSO) for 48 hours and further used for future experiments. Using
appropriate vehicle and positive control, every experiment was repeated for three times
independently.
4.1.2.2. Trypan blue dye exclusion assay
The effect of 4-phenyl-2-phenoxyacetamide thiazole derivatives 8a-ab on the cell toxicity
against MCF-7, A549, EAC, DLA and MDA-MB-468 cells were assessed by trypan blue dye
exclusion assay [29]. DLA cells treated with and without the compounds were pulled out and
resuspended in 0.4% trypan blue, and the viable cells were counted using a hemocytometer. The
inhibitory concentration (IC50) value was estimated after 48 h of treatment.
4.1.2.3. MTT assay
The effect of 4-phenyl-2-phenoxyacetamide thiazole derivatives 8a-ab on cell survivality
and differentiation of MCF-7, A549, EAC, DLA and MDA-MB-468 cells were assessed by MTT
assay, as explained earlier [28]. Referred cells that were treated with or without compounds were
cultured for 48 h. MTT reagent (5 mg/mL) was incorporated, and the change in color because of
differentiating cells was determined.
4.1.2.4. Animal ethics and determination of LD50 value
Swiss female albino mice and Whistler rats weighing between 28–30g and 100-120g
respectively were used throughout the study. The animals were grouped and housed in
polyacrylic cages with not more than ten animals per cage with adequate food and water supply.
All procedures described were reviewed and approved by the National College of Pharmacy
Ethical Committee, Shivamogga, India, in accordance with the CPCSEA guidelines for
laboratory animal facility (NCP/IAEC/CL/101/05/ 2012-13). LD50 of the compounds 8f was
analyzed and treatment doses were fixed accordingly.
28