Design, synthesis and biological evaluation of new 1,3-thiazole derivatives as potential anti-inflammatory agents

Article abstract of DOI:10.1016/j.molstruc.2021.130526

Thiazoles are widely recognized as nuclei of great value for obtaining molecules with various biological activities, including analgesic, anti-inflammatory, anti-HIV, antidiabetic, antitumor and antimicrobial. A library of 26 thiazole derivatives as fragments were designed, synthesized and evaluated for their anti-inflammatory activities. Some screened compounds showed promising results and were found to be potent in the series by inhibiting LPS-induced TNFα and IL-8 release with IC₅₀ values in μM range without cytotoxic activity.

Full text of DOI:10.1016/j.molstruc.2021.130526

Journal of Molecular Structure 1239 (2021) 130526
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Design, synthesis and biological evaluation of new 1,3-thiazole
derivatives as potential anti-inflammatory agents
Marina Modric´ a,∗, Marin Božicˇevic´ b, Ivan Faraho c, Martina Bosnarc, Irena Škoric´ b,∗
a Chemistry, Fidelta Ltd., Prilaz baruna Filipovi´ca 29, HR-10000 Zagreb, Croatia
b Department of Organic Chemistry, Faculty of Chemical Engineering and Technology, University of Zagreb, Maruli´cev trg 19, HR-10000 Zagreb, Croatia
c Pharmacology in vitro, Fidelta Ltd., Prilaz baruna Filipovi´ca 29, HR-10000 Zagreb, Croatia
a r t i c l e i n f o
a b s t r a c t
Article history:
Thiazoles are widely recognized as nuclei of great value for obtaining molecules with various biological
activities, including analgesic, anti-inflammatory, anti-HIV, antidiabetic, antitumor and antimicrobial. A
library of 26 thiazole derivatives as fragments were designed, synthesized and evaluated for their anti-
inflammatory activities. Some screened compounds showed promising results and were found to be po-
tent in the series by inhibiting LPS-induced TNFα and IL-8 release with IC50 values in μM range without
cytotoxic activity.
Received 1 April 2021
Revised 13 April 2021
Accepted 15 April 2021
Available online 21 April 2021
Keywords:
Thiazole
© 2021 Elsevier B.V. All rights reserved.
Small chemical fragments
Library design
Anti-inflammatory activity
1. Introduction
the drug-like molecules. Their in vitro anti-inflammatory activities
have been screened in two assays. Compounds were screened in
Small-ring heterocycles including nitrogen and sulfur have been
under investigation for a long time on account of their synthetic
diversity and therapeutic relevance. Among the wide range of het-
erocycles explored to privileged candidates in drug discovery, thi-
azoles have been identified to play a necessary role in medicinal
chemistry.
in vitro cell viability assays to evaluate potential cytotoxicity. Some
screened compounds were found to be potent in the series by in-
hibiting LPS-induced TNFα and IL-8 release with IC50 values in μM
range without cytotoxic activity. Prompted by obtained results a
new series of thiazole derivatives are in plan to be designed in
drug like molecules with anti-inflammatory activity.
1,3-Thiazole nuclei is already present in a variety of pharmaco-
logically active substances such as antibiotics, antimycotics, anti-
histamines, vitamin B1 (thiamine) supplements and xanthine ox-
idase inhibitors for treatment of gout. Compounds with a thia-
zole nucleus that have potential for the treatment of tumors and
as anti-inflammatory agents (Fig. 1) are also documented in lit-
erature [1-5]. Approximately 85% of all biologically active com-
pounds contain a heterocyclic unit that can affect polarity, solu-
bility, lipophilicity and ability to bind to target proteins by forming
hydrogen bonds to active sites [6].
2. Results and Discussion
2.1. Chemistry
In this work several classes of thiazole derivatives have been
synthesized due to the evidenced great potential of biological ac-
tivity of this heterocyclic core. New amides 1-10, imines 11-15,
amines 16-20, and methylated amides 21-23 and amines 24-26
have been successfully synthesized, spectroscopically characterized
and their biological activity was investigated in potential anti-
inflammatory activity by testing of compounds on lipopolysaccha-
ride (LPS)-stimulated tumor necrosis factor alpha (TNFα) and inter-
leukin 8 (IL-8) production in human peripheral blood mononuclear
cells (PBMCs) (Schemes 1-4). Although some of these derivatives
were mentioned in the literature earlier [9-19], they were obtained
and isolated by different procedures and only some of them were
surveyed for testing biological activity.
Taking into account a number of already available medicines
[7,8] and novel compounds in the research phase that all contain
a thiazole unit, it can be concluded that compounds with such an
important unit still have a great potential in various applications.
In our current study series of thiazole derivatives were designed
and synthesized as a starting fragments to be further developed in
∗
Corresponding authors:
The amides 1-10 were synthesized from 2-amino thi-
azole and various carboxylic acids (Scheme 1) activated
E-mail addresses: Marina.Modric@fidelta.eu (M. Modric´), iskoric@fkit.hr (I.
Škoric´).
https://doi.org/10.1016/j.molstruc.2021.130526
0022-2860/© 2021 Elsevier B.V. All rights reserved.

M. Modri´c, M. Božicˇevi´c, I. Faraho et al.
Journal of Molecular Structure 1239 (2021) 130526
Fig. 1. Examples of thiazole bearing anti-inflammatory drugs. [3].
washed with the mixture of methanol and ammonium hydroxide
for successfully isolation of very little amount of 9. All synthesized
amides 1-10 were characterized by NMR, UPLC and MS analyses
(See SI) and the characteristic signals in the 1H NMR spectra for
products 7 and 8 are also shown on Fig. 2.
All imines 11-15 (Scheme 2) and amines 16-20 (Scheme 3) were
synthesized by reductive amination reaction starting from 2-amino
thiazole [9]. Three test reactions were performed, two Buchwald-
Hartwig reactions with BrettPhos Pd G3 as a catalyst, and potas-
sium tert-butoxide as a base, one of which was carried out with
heating at 80°C in the conventional manner, on a magnetic stirrer
overnight and was further heated at 100°C for 3 hours, and the
second reaction was heated at 100°C by microwave irradiation for
30 minutes. The third test reaction, which proved to be the best for
several reasons, was the reductive amination, carried out at 80°C
on a stirrer with a heating element overnight.
Better conversion itself was one of the reasons why reductive
amination was chosen for further reactions. Another very impor-
tant reason is that reductive amination is a reaction in 2 steps
where in the first step imines are formed which can be isolated
as separate products (See Experimental). By such a reaction, it was
achieved that 2 types of product were isolated, imines 11-15 from
half of the reaction mixture, while the other part of the reac-
tion mixture was reduced to the corresponding amines 16-19 with
NaBH4 as reducing agent (Scheme 3), only amine 20 has not been
successfully synthesized using NaBH4.
Scheme 1. Reaction pathway for obtaining amide derivatives 1-10.
with
1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-
Although reductive amination has proven to be a good way of
parallel synthesis of imines and corresponding amines, this ap-
proach makes it difficult to determine the exact yields because tak-
ing aliquots of the reaction mixture after imine formation, which is
further used to reduce to amines, introduces a certain calculation
error. All synthesized imines 11-15 and amines 16-19 were charac-
terized by NMR, UPLC and MS analyses (See SI) and the character-
istic signals in the 1H NMR spectra for imine 15 and amine 18 are
also shown on Figs. 3 and 4a.
b]pyridinium-3-oxide-hexa-fluorophosphate (HATU), along with
diisopropylethylamine (DIPEA) [19]. The activation of carboxylic
acid takes place in 2 steps, of which in the first step the base,
in this case DIPEA, first deprotonates the carboxylic acid, and the
resulting carboxylate anion attacks the positively charged carbon
atom in HATU. In the second step, the newly formed OAt anion
reacts with the newly formed activated carboxylic acid to form an
activated ester. After the formation of the activated ester, a further,
third step is the formation of the desired amides 1-10 (Scheme 1)
by reacting the amine deprotonated with DIPEA and the activated
ester.
To assess the influence of the NH group on biological activity,
three amides 1-3 and their analogous three amines 17-19 were ad-
ditionally methylated (Scheme 4). For this purpose, iodomethane
with NaH was used as the base.
The relevant reactant in the synthesis of amides 1-5 was 2-
aminothiazole, and the acid was added in excess, resulting in a lag
of the HATU-activated acid complex that made it difficult to iso-
late the compounds by column chromatography. This was a direct
cause of smaller amounts of isolated desired products. By reverse
approach to the synthesis of further amides where the reactions
for the preparation of compounds 6-10 were carried out with an
excess of 2-aminothiazole, and the relevant reactant was a certain
acid, the lag of the resulting complex was avoided and thus the
isolation of these compounds was simpler. Therefore, desired prod-
uct and higher yields were achieved as can be seen in Scheme 1.
Amides 7 and 9 deviate from such a trend. In the isolation of prod-
uct 7, column chromatography was not required because it precip-
itated during the synthesis, but the yield was much lower than in
other products. On the other hand, amide 9 bound to the silica gel
column during column chromatography and the system used was
not strong enough to elute from the column and the column was
All methylated thiazole amides 21-23 and amines 24-26 were
successfully isolated and characterized by NMR, UPLC and MS anal-
yses (See SI) and additionally the characteristic signals in the 1H
NMR spectra methylated amine 25 are also shown on Fig. 4b along
with the spectrum of the corresponding unmethylated amine 18
(Fig. 4a).
2.2. Biology
Biological activity of 24 succesfully synthesized thiazole deriva-
tives (1-12, 14-19 and 21-26) was evaluated in vitro. To assess
potential anti-inflammatory activity, the effect of compounds on
lipopolysaccharide (LPS)-stimulated tumor necrosis factor alpha
(TNFα) and interleukin 8 (IL-8) production in human peripheral
blood mononuclear cells (PBMCs) was tested. Out of 24 tested
2

M. Modri´c, M. Božicˇevi´c, I. Faraho et al.
Journal of Molecular Structure 1239 (2021) 130526
Fig. 2. 1H NMR spectra (DMSO) of the thiazole derivatives 7 (a) and 9 (b).
Scheme 2. Synthesis of imines 11-15 by reductive amination reaction.
Scheme 3. Reduction reaction of imines to amines 16-19 and the planned amine 20.
3

M. Modri´c, M. Božicˇevi´c, I. Faraho et al.
Journal of Molecular Structure 1239 (2021) 130526
Fig. 3. 1H NMR spectrum (DMSO) of thiazole imine derivative 15.
Scheme 4. Products 21-26 obtained by methylation of the corresponding amides 1-3 and amines 17-19.
compounds, 12 compounds inhibited LPS-induced TNFα release
rings (cPr, cyclohexyl) as well as saturated rings with basic centre
with IC50 values in μM range (Table 1 and Fig. 5). On the other
hand, only compound 26, at the highest concentration tested, in-
hibited IL-8 release (Scheme 5, Fig. 6).
(piperidone).
Analysing of activity on TNFα for the set of compounds with
amide bond, compound 7 with fused quinoxaline system showed
best activity, although with low inhibition plateau (47%). In com-
parison with compound 8 bearing pyridine, activity was lost 4
folds, not completely. Also, compounds with para-phenyl -EDGs
such as methoxy (compound 2) are beneficial for TNFα inhibition.
In addition to explore importance of –CONH amide potential hy-
drogen bond for activity was explored by introducing of methyl
group. Interestingly, by methylating compound 2 to obtain com-
pound 22, activity was completely lost showing importance of H-
bond for activity. On the other hand, compound 3 that was inac-
tive, by methylating amide, compound 23 showed activity.
For imine containing linker, compound 15 with quinoxaline unit
was active, losing 2 folds activity in comparison to amide bond.
Due to unsuccessful reduction of imine 15 to corresponding amine,
library is lacking information about the activity of amine. More-
over, by reducing –C=O to obtain library of benzyl amine ana-
logues, all compounds with variously substituted benzyl units were
active leading to conclusion of importance of rotatable bonds for
activity.
In addition, the effect of compounds on viability of ovarian
adenocarcinoma cell line SK-OV-3 was assessed. Briefly, cells were
treated with compounds for 24 h and intracellular ATP levels were
measured. Since ATP is produced only by live cells, the signals ob-
tained were proportional to number of cells in wells. In this assay
only compound 10 had some effect on viability of SK-OV-3 (Fig. 7).
In both of these assays reference compounds were also tested.
In LPS stimulated PBMC assay corticosteroid dexamethasone was
used, due to its known anti-inflammatory effects. The activity on
both TNFα and IL-8 release was reproducible, with IC50 values of
7.5 nM and 3.6 nM, respectively. Staurosporine, a pan-kinase in-
hibitor, was used as reference compound in cytotoxicity assay, and
it concentration-dependently reduced number of live cells, with
IC50 value 421 nM. Activities of both dexamethasone and stau-
rosporine in corresponding assays are shown on Fig. 8.
Obtained results were summarized in structure-activity rela-
tionship overview (Fig. 9). In library design, 2-amino-1,3-thiazole
was considered as starting point in synthesis to be further cou-
pled by amide, imine and amine linkers with diversely substi-
tuted phenyl, pyridine, fused system such as quinoxaline, saturated
Compound 26 was the only one showing moderate activity on
IL-8 inhibition in the range of activity for TNFα. Evaluation of com-
4

M. Modri´c, M. Božicˇevi´c, I. Faraho et al.
Journal of Molecular Structure 1239 (2021) 130526
Fig. 4. Aromatic part of the 1H NMR spectra (DMSO) of the unmethylated thiazole amine 15 (a) and the corresponding methylated amine 25 (b).
pounds by screening in in vitro cell viability assays on SK-OV-3
cells did not show cytotoxic effect. Encouraged by promising data
obtained on small fragments a new series of thiazole derivatives
are in plan to be developed in drug like molecules with anti-
inflammatory activity aiming IC50 values in nM range with accept-
able ADME-Tox properties.
Table 1
Activities of 1,3-thiazole derivatives tested in two in vitro assays.
IC50 value (μM)
Compound
PBMC-LPS/ TNFα
PBMC-LPS/ IL-8
SK-OV-3 viability
1
>100
24
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
47
>100
>100
>100
>100
>100
>100
>100
>100
>100
>33
2
3
>100
>100
>100
>100
8a
4
3. Experimental Section
5
6
3.1. General
7
8
>33
22b
The amidation test reactions and the reductive amination reac-
tions were carried out on a magnetic stirrer with a heating body,
while part of the amidation reactions were carried out on a shaker
with a heating body. Chromatographic separations were performed
on a Biotage instrument using puriFlash silica gel-filled columns
(4g, 15 μm and 12 g, 15 μm).
All products obtained were identified and characterized by
ultra high efficiency liquid chromatography (UPLC) with mass
and UV detectors and nuclear magnetic resonance (NMR). 1H
NMR and 13C NMR spectra were recorded on Bruker Ultra Shield
300, Bruker Ultra Shield 400, Bruker Ultra Shield 500 and Bruker
Ultra Shield 600 depending on the amount of compound and
the desired spectra. The 2D-HH-COSY technique was also used
to assign signals and determine interactions in the molecule.
Ultra-high performance liquid chromatography monitoring the
range of the reaction and determining the purity of the final
9
10
11
12
14
15
16
17
18
19
21
22
23
24
25
26
>100
>100
>100
>100
17
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>33
>33
10
>100
>100
40
>33
25
56
a
– low inhibition plateau (47%)
b
– compound was cytotoxic in PBMC assay at 100 μM
MW= 258.20
Scheme 5. Simplified demonstration of 4-fluorobenzoic acid activation with HATU.
5

M. Modri´c, M. Božicˇevi´c, I. Faraho et al.
Journal of Molecular Structure 1239 (2021) 130526
Fig. 5. Effect of compounds 2, 7, 8, 9, 15, 17-19 and 23-26 on LPS-stimulated TNFα production in hPBMCs.
Fig. 6. Effect of compound 26 on IL-8 production in LPS stimulated PBMCs assay.
Fig. 7. Effect of compound 10 on SK-OV-3 cell viability.
product was performed on a Waters Acquity Ultra Performance LC
with an SQD mass spectrometer (UPLC-MS/UV). Depending on the
structure of the compound, two methods were used to monitor
the range of the reaction: Method A - method duration = 2 min,
acidic conditions (0.1% HCOOH in H2O / 0.1% HCOOH in MeCN);
Method B - method duration = 2 min, basic conditions (0.05%
NH3 in H2O / 0.05% NH3 in MeCN). The following abbreviations
were used: s, singlet; d, doublet; t, triplet, dd, doublet of doublet;
m, multiplet; DMF-dimethylformamide, DCM-dichloromethane,
6

M. Modri´c, M. Božicˇevi´c, I. Faraho et al.
Journal of Molecular Structure 1239 (2021) 130526
Fig. 8. Activity of reference compounds dexamethasone (A) and staurosporine (B) in LPS stimulated PBMCs and SK-OV-3 cytotoxicity assays, respectively.
Fig. 9. SAR of thiazole fragment based library concerning their anti-inflammatory activity.
MeOH-methanol, MeCN-acetonitrile, DIPEA-diisopropylethylamine,
HATU-1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-
b]pyridinium-3-oxide hexa-fluorophosphate.
ditionally dried through a phase separator, through which the or-
ganic layer passes, and any water present remains in the separator.
The reaction mixture thus treated was evaporated to dryness on a
rotary evaporator.
The evaporated products were dissolved in a small amount of
dichloromethane and applied to TLC plates developed in various
solvent systems in order to find the best chromatographic separa-
tion system. Silica gel was then added to the solution, the mixture
was evaporated to dryness and tested in a pre-column. Compounds
were isolated by column chromatography on a Biotage device with
puriFlash columns. Certain solvent systems, ie eluents, flow rates,
columns used as well as elution time are shown in Table 2.
In previous procedures, the presence of HATU-activated acid
complexes often meant difficult isolation of the resulting products.
It was therefore decided to approach the following reactions get-
ting products 6-10 in another way in which the relevant reactant
was set up and 2-amino thiazole was added to the reaction in ex-
cess to ensure that all the resulting complex would be consumed
in the reaction.
3.2. Amidation reactions
Procedure for compounds 1-5: 1.2 equivalents of a certain car-
boxylic acid was dissolved in 1 mL of DMF in a round bottom flask
and 1.2 equivalents of HATU and 1.2 equivalents of DIPEA were
added to activate the acid present for subsequent reactions. After
15 minutes, 1 equivalent of 2-amino thiazole (50 mg, 0.499 mmol)
was added to the reaction mixture, and the reaction mixture was
stirred on a magnetic stirrer overnight at 50°C. The extent of the
reaction was monitored by UPLC-MS/UV (Method A). The presence
of HATU-activated acid intermediates was observed in all reaction
mixtures (Fig. 10). The reaction was then stopped and extraction
was performed. The reaction mixture was transferred to a separa-
tory funnel and 10 mL of saturated aqueous ammonium chloride
solution (NH4Cl) and 10 mL of ethyl acetate were added. When
the layers were separated, the aqueous layer was drained from the
funnel, and the residual organic layer was further washed with 10
mL of saturated aqueous ammonium chloride solution and 2 × 10
mL of distilled water. After the extraction, the organic layer is ad-
Procedure for compounds 6-10: 1 equivalent of a certain car-
boxylic acid was dissolved in 2 mL of DMF in an 8 mL vial followed
by the addition of 1 equivalent of HATU and 1 equivalent of DIPEA
to activate the acid present. The vial was then placed on a shaker.
7

M. Modri´c, M. Božicˇevi´c, I. Faraho et al.
Journal of Molecular Structure 1239 (2021) 130526
Fig. 10. Representation of the mass spectrum and TIC spectrum of the reaction range of the synthesis of 4-fluoro-N-thiazol-2-yl-benzamide (1).
Table 2
Conditions for chromatographic separation of products 1-5.
Isolated
Product
Eluent and flow rate
Column
Elution time
quantity/mg
1
2
3
4
5
DCM:(DCM:MeOH=100:1) 0-100% 6 mL/min
DCM:(DCM:MeOH=100:1) 0-100% 6 mL/min
DCM:(DCM:MeOH:NH4OH = 90:9:1.5) 0-100%
DCM:(DCM:MeOH=100:1) 0-100% 6 mL/min
DCM:(DCM:MeCN=10:1) 0-100% 6 mL/min
puriFlash 4g, 15μm
puriFlash 4g, 15μm
puriFlash 4g, 15μm
puriFlash 4g, 15μm
puriFlash 4g, 15μm
30% DCM:MeOH= 100:1
30% DCM:MeOH= 100:1
5% DCM:MeOH:NH4OH = 90:9:1.5
40% DCM:MeOH= 100:1
35% DCM:MeCN= 10:1
30.6
45.7
68.99
53.3
127.3
After 15 minutes, 1.1 equivalent of 2-amino thiazole and 1.1 equiva-
lent of DIPEA were added to the reaction mixture, and the reaction
mixture was heated to 50°C and stirred on a shaker overnight. The
extent of the reaction was monitored by UPLC-MS/UV (method A).
By changing the ratio of reactants, the lag of the resulting HATU-
activated acid complex was successfully avoided. The reaction mix-
ture was then transferred to a separatory funnel after which 10 mL
of distilled water and 10 mL of ethyl acetate were added. The aque-
ous layer was then drained from the funnel, the organic layer was
transferred to a round bottom flask, and the aqueous layer was re-
turned to the separatory funnel and extracted once more with 10
mL of ethyl acetate. The aqueous layer was again drained through
a funnel, and the organic layer joined the previous one in a round-
bottomed flask. The amount of product remaining in the aqueous
layer was monitored by UPLC-MS/UV (method A) and it was ob-
served that the product was present in traces in the aqueous layers
and the aqueous layer was discarded, except for product 8 where
the pH was adjusted to 6.5 with 1M aqueous HCl and product 9
in which the pH was adjusted to 9.1 with 1M aqueous NaOH to
allow most of the resulting products to pass into the organic layer.
The pH value at which the desired product has net charge zero,
therefore higher solubility in the organic layer was determined us-
ing the ACD/Percepta software. The organic layer was then washed
with 10 mL of aqueous lithium chloride solution, 10 mL of distilled
water and 10 mL of aqueous sodium chloride solution, then dried
over Na2SO4, filtered and evaporated to dryness on a rotary evap-
orator. The evaporated products were dissolved in a small amount
of dichloromethane and applied to TLC plates developed in various
solvent systems in order to find the best chromatographic separa-
tion system. Silica gel was then added to the solution, the mixture
was evaporated to dryness and added to the pre-column. Chro-
matographic separation of products 1-10 was performed on a Bio-
tage instrument on 4 g and 12 g puriFlash columns, depending on
the amount of product obtained, in solvent systems at flow rates
that proved to be best for individual products (Table 3). The only
product that did not need to be isolated by column chromatogra-
phy was product 7 because it precipitated during the synthesis in
an amount sufficient for further testing.
After chromatographic separation, fractions were plotted on TLC
plates to determine which fractions contained the desired prod-
uct and whether any impurities were present. After selection, the
fractions were transferred to a round bottom flask and evaporated
to dryness on a rotary evaporator and dried in a vacuum oven
overnight at 40°C.
The final products 1-10 were characterized by NMR and their
purity was determined by UPLC-MS/UV with a blank.
4-fluoro-N-thiazol-2-yl-benzamide (1) [9]: white powder, m.p.
165 °C; Rf (DCM:MeOH=100:1) = 0.46, UPLC-MS/UV (method A)
rt=0.87 min, m/z=223.00 ES+, purity (DAD)= 97.99%, 1H NMR (400
MHz, DMSO-d6) δ/ppm 12.66 (br s, 1H), 8.19-8.14 (m, 2H), 7.55 (d,
J = 3.6 Hz, 1H), 7.41-7.34 (m, 2H), 7.28 (d, J = 3.6 Hz, 1H), 13C NMR
8

M. Modri´c, M. Božicˇevi´c, I. Faraho et al.
Journal of Molecular Structure 1239 (2021) 130526
Table 3
Conditions for chromatographic separation of products 6-10.
Product
Eluent and flow rate
Column
Elution time
Isolated quantity/ mg
6
DCM:(DCM:MeOH=100:1) 0-100% 6 mL/min
/
puriFlash 4g, 50μm
/
40% DCM:MeOH= 100:1
/
132.64
37.33
7
8
DCM:(DCM:MeOHl:NH3 = 90:4:1) 0-100%, 8 mL/min
MeOH, 8 mL/min
puriFlash12g, 15μm
puriFlash 12g, 15μm
puriFlash 4g, 15μm
40-45% DCM:MeOH:NH3 = 90:4:1
40% MeOH
116.77
13.50
9
10
DCM:(DCM:MeCN=10:0.5) 0-100% 6mL/min
5%DCM:MeCN=10:0.5
110.32
rity (DAD) = 95.80%, 1H NMR (600 MHz, CDCl3) δ/ppm 11.46 (br
s, 1H), 7.47 (d, J = 3.5 Hz, 1H), 7.04 (d, J = 3.5 Hz, 1H), 2.98 (d,
J = 11.2 Hz, 2H), 2.51-2.40 (m, 1H), 2.33 (s, 3H), 2.05 (br s, 2H),
2.03-1.94 (m, 4H), 13C NMR (400 MHz, DMSO-d6) δ/ppm 173.5,
158.0, 137.5, 113.2, 54.5, 46.1, 40.9, 28.1;
(400 MHz, DMSO-d6) δ/ppm 165.8, 163.4, 131.1, 131.0, 129.0, 115.7,
115.5, 113.9;
4-methoxy-N-thiazol-2-yl-benzamide (2) [10]: white powder,
m.p. 158 °C; Rf (DCM:MeOH=100:1) = 0.30, UPLC-MS/UV (method
A), rt=0.86 min, m/z=235.04 ES+, purity (DAD)= 98.43%, 1H NMR
(400 MHz, DMSO-d6) δ/ppm: 12.45 (br s, 1H); 8.12-8.06 (m, 2H);
7.53 (d, J = 3.6 Hz, 1H); 7.24 (d, J = 3.6 Hz, 1H); 7.09-7.03 (m, 2H);
3.84 (s, 3H), 13C NMR (400 MHz, DMSO-d6) δ/ppm 166.0, 162.6,
137.0, 130.1, 124.0, 113.8, 113.7, 113.6, 55.9;
2-phenoxy-N-thiazol-2-yl-acetamide (10): white powder, m.p.
174 °C; Rf (DCM:MeCN=10:0.5) = 0.66, UPLC/MS-UV (method A),
rt=0.87 min, m/z=235.06 ES+, 1H NMR (300 MHz, DMSO-d6)
δ/ppm 12.34 (br s, 1H), 7.49 (br s, 1H), 7.38-7.17 (m, 3H), 6.95 (d,
J = 7.5 Hz, 3H), 4.84 (s, 2H), 13C NMR (400 MHz, DMSO-d6) δ/ppm
166.8, 157.8, 157.0, 137.8, 129.6, 121.3, 114.5, 113.9;
N-thiazol-2-yl-2-(trifluoromethyl)benzamide (3): white pow-
der, m.p. 171 °C; Rf (DCM:MeOH:NH4OH
=
90:9:1.5)
=
0.80,
UPLC/MS-UV (method A), rt=0.91 min, m/z =273.34 ES+, purity
(DAD)= 99.82%, 1H NMR (300 MHz, DMSO-d6) δ/ppm: 12.78 (br
s, 1H), 7.89-7.71 (m, 4H), 7.53 (d, J = 3.5 Hz, 1H), 7.32 (d, J = 3.4
Hz, 1H); 13C NMR (400 MHz, DMSO-d6) δ/ppm 165.5, 137.8, 133.7,
132.6, 130.8, 128.9, 126.4, 126.0, 125.0, 122.3, 114.2;
3.3. Reductive amination reactions
All imines and amines in this work were synthesized by re-
ductive amination reaction. It is a two-step reaction where in the
first step an imine is formed and in the second step the reduction
of the imine to the desired amine is continued, with a reducing
agent such as NaBH4. The fact that imines and amines can be iso-
lated by this reaction was the main reason why reductive amina-
tion was chosen before the Buchwald-Hartwig reaction which was
also carried out. Although the Buchwald Hartwig reaction is very
widespread as a way of amine synthesis, in this paper it proved
to be a less interesting approach and the reaction profile looked
worse.
N-thiazol-2-ylcyclopropane carboxamide (4): red-brown pow-
der, m.p. 139 °C; Rf (DCM:MeOH=100:1) = 0.19, UPLC/MS-UV
(method A), rt=0.63 min, m/z=168.98 ES+, purity (DAD)= 96.33%,
1H NMR (400 MHz, DMSO-d6) δ/ppm: 12.34 (s, 1H), 7.44 (d, J = 3.7
Hz, 1H); 7.15 (d, J = 3.7 Hz, 1H); 1.97-1.90 (m, 1H); 0.91-0.84 (m,
4H), 13C NMR (400 MHz, DMSO-d6) δ/ppm 171.8, 158.1, 137.6, 113.3,
13.5, 8.2;
4,4-difluoro-N-thiazol-2-yl-cyclohexane carboxamide (5):
white powder, m.p. 162 °C; Rf (DCM:MeCN =10:1)
=
0.49,
UPLC/MS-UV (method A), rt=0.63min, m/z = 247.09 ES+, purity
(DAD) 99.83%, 1H NMR (400 MHz, DMSO-d6) δ/ppm 12.16 (br s,
1H), 7.45 (d, J = 3.5 Hz, 1H), 7.19 (d, J = 3.5 Hz, 1H), 2.14-2.01
(m, 2H), 1.98-1.74 (m, 5H), 1.72-1.56 (m, 2H), 13C NMR (400 MHz,
DMSO-d6) δ/ppm 172.7, 157.9, 137.6, 113.4, 32.4, 32.0, 25.2, 24.9;
N-thiazol-2-yl-benzamide (6) [11]: white powder, m.p. 152 °C;
Rf (DCM:MeOH=100:1) = 0.45, UPLC/MS-UV (method A), rt=0.82
min, m/z=205.04 ES+, purity (DAD) = 99.29%, 1H NMR (500 MHz,
DMSO-d6) δ/ppm 12.64 (br s, 1H), 8.09 (d, J = 7.3 Hz, 2H), 7.67-7.59
(m, 1H), 7.59-7.52 (m, 3H), 7.29 (d, J = 3.4 Hz, 1H), 13C NMR (400
MHz, DMSO-d6) δ/ppm 164.0, 156.0, 132.5, 128.6, 128.5, 128.2,
128.1, 113.9;
3.3.1. Imine synthesis
Procedure for compounds 11-15: 1 equivalent (200 mg, 1.997
mmol) of 2-amino thiazole was dissolved in 7 mL of absolute
ethanol followed by the addition of 1 equivalent of the desired
aldehyde. The reaction mixture was heated to 80°C in a sealed
Wheaton glass vial to prevent evaporation of the solvent. The reac-
tion was performed for 3 hours after which was checked by UPLC-
MS/UV (method B). It was observed that starting reagents were
still present. The product 11 was then evaporated to dryness, while
the other reactions were carried out overnight to improve the con-
version followed by UPLC-MS/UV (method B). 3.5 mL of all reaction
mixtures except product 1 which was weighed 150 mg, dissolved
in 3-5 mL of absolute ethanol and separated in a round bottom
flask to reduce the imines to the amine as described in section
4.3.2. The remaining reaction mixture was evaporated to dryness
and triturated with diethyl ether to remove residual aldehyde af-
ter which the residual product precipitated. The product thus ob-
tained was dissolved in dichloromethane, silica gel was added, the
mixture was evaporated to dryness and added to the pre-column,
and chromatographic separation was carried out as previously de-
scribed in the amide synthesis. The solvent systems and chromato-
graphic separation conditions are shown in Table 4. The amount
of isolated products 12 and 13 after column chromatography was
insufficient and their synthesis was repeated without the step of
separating aliquots for the imine to amine reduction reaction.
After chromatographic separation, the fractions in which the
desired product is present were collected in a round-bottomed
flask and evaporated to dryness. Due to the instability of the
imine, aldehyde is often present in all fractions, even after chro-
matographic separation, which is why the evaporated products are
N-thiazol-2-yl-quinoxaline 2-carboxamide (7) [12]: yelow
powder, m.p. 148 °C; Rf (DCM:MeOH = 100:1) = 0.56, UPLC/MS-UV
(method A), rt=0,90 min, m/z=257.07 ES+, purity (DAD)=99.79%,
1H NMR (300 MHz, DMSO-d6) δ/ppm 12.63 (s, 1H), 9.55 (s, 1H),
8.33-8.20 (m, 2H), 8.08-7.99 (m, 2H), 7.62 (d, J = 3.5 Hz, 1H), 7.40
(d, J = 3.5 Hz, 1H), 13C NMR (400 MHz, DMSO-d6) δ/ppm 174.0,
154.0, 144.0, 143.7, 143.2, 140.0, 132.8, 131.7, 130.0, 129.6, 129.2,
114.8;
N-thiazol-2-yl pyridine 3-carboxamide (8) [13]: white pow-
der, m.p. 143 °C; Rf (DCM:MeOH:NH3/MeOH = 90:4:1) = 0.26,
UPLC/MS-UV (method A), rt=0.50 min, m/z=206.03 ES+, purity
(DAD) = 100%, 1H NMR (300 MHz, DMSO-d6) δ/ppm 12.86 (br s,
1H), 9.19 (d, J = 2.1 Hz, 1H), 8.78 (dd, J = 1.6, 4.9 Hz, 1H), 8.40 (m,
J = 1.9, 8.0 Hz, 1H), 7.60-7.54 (m, 2H), 7.31 (d, J = 3.5 Hz, 1H), 13C
NMR (400 MHz, DMSO-d6) δ/ppm 162.0, 156.0, 152.8, 149.2, 136.1,
135.9, 135.8, 123.6, 114.1;
1-methyl-N-thiazol-2-yl-piperidine
4-carboxamide
(9):
brown-yellow powder, m.p. 155 °C; Rf (MeOH)
UPLC/MS-UV (method A), rt=0.37 min, m/z=226.11 ES+, pu-
=
0.29,
9

M. Modri´c, M. Božicˇevi´c, I. Faraho et al.
Journal of Molecular Structure 1239 (2021) 130526
Table 4
Conditions for chromatographic separation of products 11-15
Product
Eluent and flow rate
Column
Elution time
Isolated quantity/mg
11
12
13
14
15
DCM:(DCM:MeCN=10:0.5) 0-100% 6mL/min
DCM:(DCM:MeCN=10:0.5) 0-100% 8mL/min
DCM:(DCM:MeCN=10:0.5) 0-100% 8mL/min
DCM:(DCM:MeCN=10:0.5) 0-100% 8mL/min
DCM:(DCM:MeCN=10:1) 0-100% 7mL/min
puriFlash 4g, 15μm
puriFlash 12g, 15μm
puriFlash 12g, 15μm
puriFlash 12g, 15μm
puriFlash 12g, 15μm
20% DCM:MeCN=10:0.5
20-30% DCM:MeCN=10:0.5
40-50% DCM:MeCN=10:0.5
20-30% DCM:MeCN=10:0.5
40-60% DCM:MeCN=10:1
20.55
23.67
23.01
17.60
27.91
re-triturated with diethyl ether, except for products 12 and 13
which are triturated with pentane because they are soluble in di-
ethyl ether. After trituration, the resulting mixture was centrifuged,
the liquid portion was discarded, and the precipitate was dried in
a vacuum oven at room temperature.
was then stirred on a magnetic stirrer overnight and the range
was monitored by UPLC-MS/UV (method B). It was noticed that
the range of reactions was quite low, which may be the result of
the age of NaBH4 and loss of its activity, so another 0.5 equiva-
lents of NaBH4 was added to the reaction mixture and the reaction
mixture was heated to 70°C and stirred for another 3 hours. The
range was re-checked by UPLC-MS/UV (method B) and it was ob-
served that significantly more of the desired product was formed
than before, although the starting reagents for the synthesis of
the desired imines as well as the imine itself were still present.
Despite the presence of starting reagents, the range of the reac-
tion was satisfactory and the reactions were stopped. The reac-
tion mixture was then transferred to a separatory funnel and 10
mL of ethyl acetate and 10 mL of brine were added. The aqueous
layer was then drained and the organic layer was further washed
with 10 mL of saturated aqueous NaCl solution and distilled water.
The aqueous layers were all collected and checked by UPLC-MS/UV
(Method B) to determine if the desired products were present.
When it was concluded that there were no products in the aque-
ous layers or they were visible in traces, the aqueous layer was
discarded. The organic layer was dried over NaSO4, filtered and
evaporated to dryness on a rotary evaporator. The product thus
obtained was dissolved in dichloromethane, silica gel was added,
the mixture was evaporated to dryness and the dry product on sil-
ica gel was transferred to a pre-column. Product 16, unlike other
products, was dissolved in a minimal amount of DCM and applied
directly to the conditioned column. The obtained products were
isolated by column chromatography on a Biotage instrument with
4g, and 12g puriFlash columns in different solvent systems seen in
Table 5.
The final products 11-15 were characterized by NMR, but their
purity could not be accurately determined by UPLC-MS/UV system
due to the already mentioned instability and tendency to return
to starting reagents while performing ultra high efficiency liquid
chromatography system in water/acetonitrile system. The data be-
low is given for the main isomer in the mixture.
1-phenyl-N-thiazol-2-yl-metanimine (11) [14-16]: yellow pow-
der, m.p. 112 °C; Rf (DCM:MeCN = 10:0.5) = 0.76, UPLC/MS-UV
(method B), rt=1.04 min, m/z=189.05 MS ES+, 1H NMR (600 MHz,
DMSO-d6) δ/ppm 8.34 (d, J = 7.2 Hz, 1H), 7.49 (t, J = 7.2 Hz, 2H),
7.37 (m, 2H), 7.31 (t, J = 7.2 Hz, 2H), 7.02 (d, J = 3.7 Hz, 2H), 6.66
(d, J = 3.7 Hz, 2H), 13C NMR (400 MHz, DMSO-d6) δ/ppm 167.5,
139.0, 129.6, 128.8, 128.3, 127.2, 127.1;
1-(4-fluorophenyl)-N-thiazol-2-yl-metanimine (12): white-
yellow powder, m.p. 107 °C; Rf (DCM:MeCN = 10:0.5) = 0.82,
UPLC/MS-UV (method B), rt=1.03 min, m/z=207.02 MS ES+, purity
(NMR) 95%, 1H NMR (300 MHz, DMSO-d6) δ/ppm 9.08 (s, 1H), 8.11
(t, J = 6.5 Hz, 2H), 7.72 (d, J = 3.8 Hz, 2H), 7.65 (d, J = 3.8 Hz, 2H),
7.39 (t, J = 8.4 Hz, 2H);
1-(4-methoxyphenyl)-N-thiazol-2-yl-metanimine (13): yellow
powder, m.p. 97 °C; Rf (DCM:MeCN = 10:0.5) = 0.74, UPLC/MS-
UV (method B), rt=1.01 min, m/z=219.06 MS ES+, 1H NMR (600
MHz, DMSO-d6) δ/ppm 9.11 (s, 1H), 7.11 (d, J = 7.8 Hz, 2H), 6.37 (d,
J = 7.8 Hz, 2H), 6.22 (d, J = 3.5 Hz, 2H), 5.82 (d, J = 3.5 Hz, 2H),
2.98 (s, 3H); 13C NMR (400 MHz, DMSO-d6) δ/ppm 167.0, 139.5,
139.0, 132.1, 128.5, 115.5, 114.6, 107.3, 56.0;
As in previous procedures after chromatographic separation,
fractions were plotted on TLC plates to determine which fractions
contained the desired product and whether any impurities were
present. After selection, the fractions were transferred to a round
bottom flask of known mass and evaporated to dryness and dried
in a vacuum oven overnight at 40°C. The obtained final products
were characterized by NMR and their purity was determined by
UPLC-MS/UV using a blank.
N-thiazol-2-yl-1-[2-(trifluoroethyl)phenyl]metanimine (14):
yellow powder, m.p. 115 °C; Rf (DCM:MeCN =10:0.5) = 0.83,
UPLC/MS-UV (method B), rt=1.91 min and 2.31 min, m/z=257.01
MS ES+, purity (NMR) 95%, 1H NMR (300 MHz, DMSO-d6) δ/ppm
9.38 (d, J = 2.3 Hz, 1H), 8.42 (d, J = 7.1 Hz, 1H), 7.95-7.77 (m, 5H),
13C NMR (400 MHz, DMSO-d6) δ/ppm 171.0, 159.0, 157.8, 141.9,
133.3, 132.9, 132.0, 128.7, 126.5, 121.5, 106.5;
N-benzylthiazole 2-amine (16): white powder, m.p. 153 °C; Rf
(DCM:MeCN=10:1) = 0.51, UPLC/MS-UV (method B) rt=0.98 min,
m/z=191.06 ES+, purity (DAD)= 99.38%, 1H NMR (300 MHz, DMSO-
d6) δ/ppm 8.08-7.96 (m, 1H), 7.36-7.21 (m, 5H), 6.98 (d, J = 3.7 Hz,
1H), 6.59 (d, J = 3.7 Hz, 1H), 4.42 (d, J = 5.9 Hz, 2H), 13C NMR
(400 MHz, DMSO-d6) δ/ppm 169.0, 139.3, 138.6, 128.3, 127.4, 126.8,
106.2, 47.7;
1-quinoxalin-2-yl-N-thiazol-2-yl-metanimine (15): red-brown
powder, m.p. 95 °C; Rf (DCM:MeCN =10:1) = 0.72, UPLC/MS-
UV (metoda B), rt=1.04 min, m/z=241.05 MS ES+, cˇistoc´a
(NMR)=95+%, 1H NMR (300 MHz, CDCl3) δ/ppm 9.63 (s, 1H), 9.21
(s, 1H), 8.27-8.16 (m, 2H), 7.91-7.84 (m, 2H), 7.36 (d, J = 3.5 Hz,
1H), 13C NMR (400 MHz, DMSO-d6) δ/ppm 192.9, 159.0, 145.5,
144.3, 142.8, 135.1, 133.2, 133.1, 131.6, 130.2, 129.2, 128.9;
N-[(4-fluorophenyl)methyl]thiazole 2-amine (17): white pow-
der, m.p. 169 °C; Rf (DCM:MeCN = 10:1) = 0.50, UPLC/MS-UV
(method B) rt=1.02 min, m/z=209.05 ES+, purity (DAD) 99.12%, 1H
NMR (300 MHz, DMSO-d6) δ/ppm 8.03 (t, J = 5.5 Hz, 1H), 7.36
(dd, J = 5.5, 8.6 Hz, 2H), 7.13 (t, J = 8.9 Hz, 2H), 6.99 (d, J = 3.7
Hz, 1H), 6.60 (d, J = 3.7 Hz, 1H), 4.40 (d, J = 5.7 Hz, 2H), 13C NMR
3.3.2. Amine synthesis
Procedure for compounds 16-20: To the reaction mixture from
the previous imine synthesis was added 1 equivalent of NaBH4
relative to the presumed amount of product present when the
conversion of the previous reaction would be 100%. The reaction
10

M. Modri´c, M. Božicˇevi´c, I. Faraho et al.
Journal of Molecular Structure 1239 (2021) 130526
Table 5
Conditions for chromatographic separation of products 16-20.
Product
Eluent and flow rate
Column
Elution time
Isolated quantity/mg
16
17
18
19
20
DCM:(DCM:MeCN=10:1) 0-100% 6 mL/min
DCM:(DCM:MeCN=10:1) 0-100% 6 mL/min
DCM:(DCM:MeCN=10:1) 0-100% 8 mL/min
DCM:(DCM:MeCN=10:1) 0-100% 8 mL/min
DCM:(DCM:MeOHl:NH3=90:4:1) 0-100%, 8 mL/min
puriFlash 4g, 15μm
puriFlash 12g, 15μm
puriFlash 12g, 15μm
puriFlash 12g, 15μm
puriFlash12g, 15μm
15-25% DCM:MeCN=10:1
20-30% DCM:MeCN=10:1
50-60% DCM:MeCN=10:1
20-30% DCM:MeCN=10:1
60% DCM:MeOH:NH3=90:4:1
35.64
62.76
40.33
66.50
27.91
(400 MHz, DMSO-d6) δ/ppm 168.9, 162.4, 138.6, 135.5, 129.3, 114.9,
106.4, 46.9;
products 21-26 were characterized by NMR and their purity was
determined by UPLC-MS/UV using blank sample.
N-[(4-methoxyphenyl)methyl]thiazole 2-amine (18) [17,18]:
white powder, m.p. 149 °C; Rf (DCM:MeCN
=
10:1)
=
0.43,
UPLC/MS-UV (method B) rt=0.90 min, m/z=221.43 ES+, purity
(DAD) = 98.61%, 1H NMR (300 MHz, DMSO-d6) δ/ppm 7.94 (t,
J = 5.3 Hz, 1H), 7.31-7.19 (m, J = 8.7 Hz, 2H), 6.98 (d, J = 3.5 Hz,
1H), 6.91-6.83 (m, J = 8.5 Hz, 2H), 6.57 (d, J = 3.7 Hz, 1H), 4.33 (d,
J = 5.7 Hz, 2H), 3.71 (s, 3H), 13C NMR (400 MHz, DMSO-d6) δ/ppm
169.0, 158.0, 138.6, 131.1, 128.7, 113.6, 106.1, 55.0, 47.2;
N-[[2-(trifluorophenyl)phenyl]methyl]thiazole 2-amine (19):
white powder, m.p. 160 °C; Rf (DCM:MeCN
=
10:1)
=
0.68,
UPLC/MS-UV(method B) rt=1.20 min, m/z=259.04 ES+, purity
(DAD) 99.50%, 1H NMR (300 MHz, DMSO-d6) δ/ppm 8.12 (t, J = 5.7
Hz, 1H), 7.73-7.57 (m, 3H), 7.50-7.43 (m, 1H), 6.99 (d, J = 3.5 Hz,
1H), 6.63 (d, J = 3.7 Hz, 1H), 4.63 (d, J = 5.9 Hz, 2H), 13C NMR
(400 MHz, DMSO-d6) δ/ppm 168.0, 152.0, 138.6, 133.0, 132.6, 128.6,
127.4, 125.8, 125.7, 106.8, 43.9;
N-methyl-N-thiazol-2-yl-2-(trifluoromethyl)benzamide
(21)
[9]: white powder, m.p. 114 °C; Rf (DCM:MeCN = 10:0.5) = 0.67,
UPLC/MS-UV (method A) rt=1.04 min, m/z=287.02 ES+, purity
(DAD) = 99.87%, 1H NMR (600 MHz, DMSO-d6) δ/ppm 7.95 (d,
J = 8.1 Hz, 1H), 7.89-7.84 (m, 1H), 7.82-7.77 (m, 2H), 7.65 (d,
J = 3.5 Hz, 1H), 7.45 (d, J = 3.5 Hz, 1H), 3.38 (s, 3H);
3.4. Synthesis of methylated amine derivatives 21-26
4-fluoro-N-methyl-N-thiazol-2-yl-benzamide (22): white pow-
der, m.p. 127 °C; Rf (DCM:MeCN = 10:0.5) = 0.48, UPLC/MS-UV
(method A) rt=0.92 min, m/z=237.04 ES+, purity (DAD) = 100%,
1H NMR (600 MHz, DMSO-d6) δ/ppm 8.28 (t, J = 6.7 Hz, 2H), 7.58
(d, J = 4.6 Hz, 1H), 7.30 (t, J = 8.4 Hz, 2H), 7.07 (d, J = 4.6 Hz,
1H), 3.82 (s, 3H), 13C NMR (400 MHz, DMSO-d6) δ/ppm (1 s miss-
ing because of the small quantity), 158.0, 131.2, 131.1, 129.0, 113.8,
113.7, 108.0, 55.8, 35.9;
To determine the importance of the NH bond for the biological
activity, some products were further methylated. Three amides and
three similar matched pairs of amines were selected for methyla-
tion. The synthesis was performed under the same conditions and
with the same reagents for all compounds. A certain amount of the
corresponding amide or amine (10-20 mg) was dissolved in 0.5 mL
of anhydrous THF in an 8 mL vial, and the reaction mixture was
cooled to 0°C in an ice bath with stirring on a magnetic stirrer. Af-
ter 15 minutes, 2 equivalents of NaH were added to the reaction
mixture and the reaction mixture was cooled for a further 15 min-
utes. After 15 minutes, 2 equivalents of iodomethane were added
to the reaction mixture, after which the reaction mixture was fur-
ther cooled for 10 minutes and then removed from the ice and
carried out at room temperature overnight. The extent of the re-
action was monitored by UPLC-MS/UV (method A for methylated
amides, method B for methylated amines). The reaction was then
quenched and extracted in a vial. Then 0.5 mL of water and 2 mL
of dichloromethane were added with stirring. The reaction mixture
is then transferred to a phase separator, which passes the organic,
in this case the lower layer, while the aqueous layer remains in
the separator. The organic layer was then evaporated to dryness
and weighed to determine the amount of product obtained. In all
cases, it is seen that the desired product is not left in the aqueous
layer and the aqueous layers are discarded. The resulting precipi-
tates and oily products were then dissolved in a minimal amount
of dichloromethane and applied directly to a conditioned puriFlash
column followed by chromatographic separation. All products 21-
26 were isolated by column chromatography on a Biotage instru-
ment on a 4g, 15 μm puriFlash column with DCM: (DCM: ACN 10:
0.5) 1-100% system as eluent (Table 6).
4-methoxy-N-methyl-N-thiazol-2-yl-benzamide (23): white
powder, m.p. 119 °C; Rf (DCM:MeCN = 10:0.5) = 0.37, UPLC/MS-UV
(method A) rt=0.85 min, m/z=249.05 ES+, purity (DAD) = 100%,
1H NMR (600 MHz, DMSO-d6) δ/ppm 8.18 (d, J = 8.2 Hz, 2H), 7.53
(d, J = 4.6 Hz, 1H), 7.02-7.00 (m, 3H), 3.83 (s, 3H), 3.80 (s, 3H);
N-[(4-fluorophenyl)methyl]-N-methyl-thiazole 2-amine (24):
colourless oil, Rf (DCM:MeCN
=
10:0.5)
=
0.56, UPLC/MS-UV
(method B) rt=1.07 min, m/z=223.05 ES+, purity (DAD)=98.86%,
1H NMR (500 MHz, DMSO-d6) δ/ppm 7.32 (t, J = 6.6 Hz, 2H), 7.19-
7.12 (m, 3H), 6.75 (d, J = 3.4 Hz, 1H), 4.67 (s, 2H), 3.00 (s, 3H);
N-[(4-methoxyphenyl)methyl]-N-methyl-thiazole
2-amine
(25): colourless oil, Rf (DCM:MeCN = 10:0.5)= 0.50, UPLC/MS-UV
(method B) rt=1.04 min, m/z=235,08 min, purity (DAD) = 99.15%,
1H NMR (500 MHz, DMSO-d6) δ/ppm 7.23-7.19 (m, J = 8.5 Hz, 2H),
7.14 (d, J = 3.7 Hz, 1H), 6.91-6.87 (m, 2H), 6.73 (d, J = 3.7 Hz, 1H),
4.60 (s, 2H), 3.72 (s, 3H), 2.97 (s, 3H);
N-methyl-N-[[2-(trifluoromethyl)phenyl]methyl]thiazole
2-amine (26): colourless oil, Rf (DCM:MeCN
= 10:0.5)= 0.65,
UPLC/MS-UV (method B) rt=1.21 min, m/z= 273.05 ES+, purity
(DAD)= 99.53%, 1H NMR (500 MHz, DMSO-d6) δ/ppm 7.77 (d,
J = 7.6 Hz, 1H), 7.64 (t, J = 7.6 Hz, 1H), 7.49 (t, J = 7.6 Hz, 1H),
7.29 (d, J = 7.6 Hz, 1H), 7.12 (d, J = 3.7 Hz, 1H), 6.78 (d, J = 3.7
Hz, 1H), 4.90 (s, 2H), 3.11 (s, 3H).
After chromatographic separation, fractions were plotted on TLC
plates to determine which fractions contained the desired prod-
uct and whether impurities were present. After selection, the frac-
tions were transferred to a round bottom flask and evaporated to
dryness and dried in a vacuum oven overnight at 40°C. The final
4. Conclusion
New 1,3-thiazole derivatives 1-19 and 21-26 were synthesized
with one unsuccessful synthesis of product 20. Ten amides were
obtained by the reaction of 2-aminothiazole and HATU activated
11

M. Modri´c, M. Božicˇevi´c, I. Faraho et al.
Journal of Molecular Structure 1239 (2021) 130526
Table 6
Conditions for chromatographic separation of products 21-26.
Product
Eluent and flow rate
Column
Elution time
Isolated quantity/mg
21
22
23
24
25
26
DCM:(DCM:MeCN=10:0.5) 0-100% 6 mL/min
DCM:(DCM:MeCN=10:0.5) 0-100% 6 mL/min
DCM:(DCM:MeCN=10:0.5) 0-100% 6 mL/min
DCM:(DCM:MeCN=10:0.5) 0-100% 6 mL/min
DCM:(DCM:MeCN=10:0.5) 0-100% 6 mL/min
DCM:(DCM:MeCN=10:0.5) 0-100% 6 mL/min
puriFlash 4g, 15μm
puriFlash 4g, 15μm
puriFlash 4g, 15μm
puriFlash 4g, 15μm
puriFlash 4g, 15μm
puriFlash 4g, 15μm
20-30% DCM:MeCN=10:0.5
30-40% DCM:MeCN=10:0.5
5-10% DCM:MeCN=10:0.5
15% DCM:MeCN=10:0.5
3.35
11.11
7.42
8.56
30-40% DCM:MeCN=10:0.5
10-15% DCM:MeCN=10:0.5
12.13
12.88
carboxylic acid and very good yields were observed in these re-
actions. After the synthesis of the first amides 1-5, the reactions
were optimized and the additional amides 6-10 were synthesized
in parallel, in which an increase in yield was observed and a larger
amount of compounds was isolated. The highest isolated yield of
79% was obtained in the case of N-thiazol-2-ylbenzamide (6). The
purity of all synthesized amides is above 95% according to the
UPLC analyses which was made them suitable for biological tests.
In addition, five imines 11-15 were synthesized by the reductive
amination reaction, part of which was used for reduction to the
amines. Of the five planned amines 16-20, four of them were suc-
cessfully synthesized. Although for imines 11-15 the purity was es-
timated from 1H NMR spectra, all synthesized amines 16-19 show
purities above 95% according to DAD detector. The limiting factor
for biological activity of imines is their tendency to hydrolyze and
decompose in contact with water, which is why it is necessary
to apply reaction optimization and translate such products into
a more suitable form. Additionally, three analogous amides and
amines were subjected to the methylation reaction to get methy-
lated products 21-26. The yields in these reactions range from 31%
in the case of 22 to 70% for 23.
title Preparation of new annelated heteropolycyclic systems is also
gratefully acknowledged.
Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.molstruc.2021.130526.
References
[1] P. Someshwar, Significance of Thiazole-based Heterocycles for Bioactive Sys-
tems, in: R. Varala (Ed.), Scope of Selective Heterocycles from Organic and
Pharmaceutical Perspective, Intech Open, 2016, doi:10.5772/60890.
[2] A.J. Kadhim, J.H. Mohammed, N.M. Aljamali, Thiazole Amide Derivatives. Syn-
thesis, Spectral Investigation, Chemical Properties, Antifungal Assay, Neuro-
Quantology 28 (2020) 16–25, doi:10.14704/nq.2020.18.1.NQ20102.
[3] A. Ayati, S. Emami, A. Asadipour, A. Shafiee, A. Foroumadi, Recent Applica-
tions of 1,3-thiazole Core Structure in the Identification of New Lead Com-
pounds and Drug Discovery, Eur. J. Med. Chem. 97 (2015) 699–718, doi:10.1016/
j.ejmech.2015.04.015.
[4] M.T. Chhabria, S. Patel, P. Modi, P.S. Bramkshatriya, Thiazole:
A Review on
Chemistry, Synthesis and Therapeutic Importance of its Derivatives, Curr. Top.
Med. Chem 16 (2016) 2841–2862, doi:10.2174/1568026616666160506130731.
[5] S. Cascioferro, B. Parrino, D. Carbone, D. Schillaci, E. Giovannetti, G. Cirrincione,
P. Diana, Thiazoles, their benzofused systems and thiazolidinone derivatives:
Versatile and promising tools to combat antibiotic resistance, J. Med. Chem. 63
(2020) 7923–7956, doi:10.1021/acs.jmedchem.9b01245.
Concerning the anti-inflammatory activity of synthesized 1,3-
thiazoles, amide 7 with fused quinoxaline system showed the best
activity. In comparison with its pyridine analogue 8, activity was
lost 4 times, but not completely. Compounds with para-phenyl -
EDGs such as methoxy (compound 2) are beneficial for TNFα in-
hibition. Methylating compound 2 to obtain analogue 22, activity
was completely lost showing the great importance of the hydrogen
bond for the activity. On the other hand, compound 3 that was
inactive, by methylating gave amide 23 with higher activity. For
imine containing linker, compound 15 with quinoxaline unit was
active, but weaker than its amide analogue. By reducing the car-
bonyl group to obtain library of benzyl amine analogues, all com-
pounds with variously substituted benzyl units were active leading
to conclusion of importance of rotatable bonds for activity. Methy-
lated amine 26 was the only one showing moderate activity on IL-
8 inhibition in the range of activity for TNFα. Evaluation of com-
pounds by screening in in vitro cell viability assays did not show
cytotoxic effect. Encouraged by promising data obtained on small
fragments a new series of 1,3-thiazole derivatives are in plan to be
developed in drug like molecules with anti-inflammatory activity
aiming IC50 values in nM range with acceptable ADME-Tox proper-
ties.
[6] J. Jampilek, Heterocycles in Medicinal Chemistry, Molecules 24 (2019) 3839,
doi:10.3390/molecules24213839.
[7] T. Ge, J. Yang, S. Zhou, Y. Wang, Y. Li, X. Tong, The Role of Pentose Phosphate
Pathway in Diabetes and Cancer, Frontiers in Endocrinology 11 (2020) 365,
doi:10.3389/fendo.2020.00365.
[8] Farrington, M., Antibacterial Drugs, in Bennett, P. N., Brown, M. J., Sharma, P.
(Eds.), Clinical Pharmacology, 11ed., 2012, Ch. 13, 173-190.
[9] N.J. Taylor, E. Emer, S. Preshlock, M. Schedler, M. Tredwell, S. Verhoog,
J. Mercier, Ch. Genicot, V. Gouverneur, Derisking the Cu-Mediated F-
Fluorination of Heterocyclic Positron Emission Tomography Radioligands, J. Am.
Chem. Soc. 139 (2017) 8267–8276, doi:10.1021/jacs.7b03131.
[10] O. Hiroaki, et al., Structure-activity relationship study of 4-(thiazol-5-
yl)benzoic acid derivatives as potent protein kinase CK2 inhibitors, Bioorg.
Med. Chem. 24 (2016) 1136–1141, doi:10.1016/j.bmc.2016.01.043.
[11] K. Fang, et al., Electrochemical N-acylation synthesis of amides under aqueous
conditions, Green Chemistry 21 (2019) 4329–4333, doi:10.1039/C9GC01391A.
[12] C.B. Andersen, et al., Discovery of Selective Aminothiazole Aurora Kinase In-
hibitors, ACS Chemical Biology 3 (2008) 180–192, doi:10.1021/cb700200w.
[13] L. Qun-Li, et al., Inhibitors of type I MetAPs containing pyridine-2-carboxylic
acid thiazol-2-ylamide. Part 1: SAR studies on the determination of the key
scaffold, Bioorg. Med. Chem. Lett. 15 (2005) 635–638, doi:10.1016/j.bmcl.2004.
11.034.
[14] P. Vicini, et al., Synthesis and biological evaluation of benzo[d]isothiazole, ben-
zothiazole and thiazole Schiff bases, Bioorg. Med. Chem. 11 (2003) 4785–4789,
doi:10.1016/s0968-0896(03)00493-0.
[15] A. Parvez, J. Meshram, V. Tiwari, Microwave mediated cyclocondensation of
2-aminothiazole into β-lactam derivatives: virtual screening and in vitro an-
timicrobial activity with various microorganisms, Int. J. Chem. Tech. Research
2 (2010) 956–964.
Declaration of Competing Interest
[16] A. Parvez, et al., Theoretical Calculations and Experimental Verification
of the Antibacterial Potential of Some Monocyclic β-Lactams Containing
two Synergetic Buried Antibacterial Pharmacophore Sites, Phosphorus, Sul-
fur and Silicon and the Related Elements 185 (2010) 1500–1510, doi:10.1080/
10426500903095556.
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared to
influence the work reported in this paper.
[17] S.L. Daniel, et al., The discovery of benzoxazine sulfonamide inhibitors of Na
1.7: Tools that bridge efficacy and target engagement, Bioorg. Med. Chem. Lett.
27 (2017) 3477–3485, doi:10.1016/j.bmcl.2017.05.070.
Acknowledgement
[18] J.-F. Cheng, et al., Heteroaryl substituted bis-trifluoromethyl carbinols as
malonyl-CoA decarboxylase inhibitors, Bioorg. Med. Chem. Lett. 16 (2006)
3484–3488, doi:10.1016/j.bmcl.2006.03.100.
Presented work has been performed at Fidelta Ltd., Zagreb. The
University of Zagreb short term scientific support (2020) under the
12