Ci a ion: Ramos, N.L.P.; Oli ei a, R.;
Cos a, S.P.G.; Raposo, M.M.M.
Syn hesis, Cha ac e iza ion and
P elimina y An ibac e ial E alua ion
agains S aphylococcus au eus o a
New 2,4,5-T i(he e o)a ylimidazole
De i a i e Based on Azaindole
He e ocycle. Chem. P oc. 2022,8, 104.
h ps://doi.o g/10.3390/ecsoc-25-
11781
Academic Edi o : Julio A. Seijas
Published: 14 No embe 2021
Publishe ’s No e: MDPI s ays neu al
wi h ega d o ju isdic ional claims in
published maps and ins i u ional a il-
ia ions.
Copy igh : © 2021 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
P oceeding Pape
Syn hesis, Cha ac e iza ion and P elimina y An ibac e ial
E alua ion agains S aphylococcus au eus o a New
2,4,5-T i(he e o)a ylimidazole De i a i e Based on
Azaindole He e ocycle †
Nuna L. P. Ramos 1, Rui Oli ei a 2, Susana P. G. Cos a 1and Ma ia Manuela M. Raposo 1,*
1Cen e o Chemis y, Campus de Gual a , Uni e si y o Minho, 4710-057 B aga, Po ugal;
[email p o ec ed] (N.L.P.R.); [email p o ec ed] (S.P.G.C.)
2Cen e o Molecula and En i onmen al Biology (CBMA), Depa men o Biology, Campus de Gual a ,
Uni e si y o Minho, 4710-057 B aga, Po ugal; [email p o ec ed]
*Co espondence: [email p o ec ed]
†
P esen ed a 25 h In e na ional Elec onic Con e ence on Syn he ic O ganic Chemis y, 15–30 No embe 2021;
A ailable online: h ps://ecsoc-25.sci o um.ne .
Abs ac :
Imidazole de i a i es a e known o hei a ied biological applica ions, such as an ibac-
e ial, an i ungal, an ioxidan , an i i al, an ipa asi ic and an icance compounds, among o he s.
The e o e, nume ous imidazole de i a i es ha e been syn hesized and de eloped in ecen yea s
as po en ial d ugs in he ea men o se e al diseases. In his communica ion, we epo he syn-
hesis o a new imidazole de i a i e, subs i u ed a posi ions 2, 4 and 5 wi h he e ocyclic g oups,
using a simple syn he ic me hodology and an easy pu i ica ion p ocedu e. The new compound was
cha ac e ized by he usual spec oscopic echniques (NMR, UV-Vis abso p ion and emission). The
e alua ion o he no el imidazole de i a i e as a po en ial an ibio ic d ug was ca ied ou agains
he G am-posi i e bac e ium S aphylococcus au eus, using he disk es di usion me hod. The esul s
showed a dose– esponse e ec agains he bac e ium unde s udy, e ealing ha he a ional design
o his imidazole de i a i e is qui e p omising o imp o e he an ibac e ial ac i i y o imidazole
de i a i es.
Keywo ds: imidazole; syn hesis; an ibac e ial ac i i y; S aphylococcus au eus
1. In oduc ion
The imidazole ing shows a ious in e es ing p ope ies and is one o he mos p omi-
nen s uc u es in nume ous na u al p oduc s, such as his idine, his amine and i amin
B
12
and is a componen o DNA bases. Due o he cha ac e is ics o his he e ocyclic ing
and he binding p ope ies o se e al analy es and biological s uc u es [
1
–
5
], his co e
is also combined wi h se e al syn he ic d ugs wi h he apeu ic use in medicine, such as
omep azole, ep osa an and me onidazole [6–8].
O e he pas yea s, chemis s ha e syn he ized a di e se ange o new imidazole
de i a i es as possible chemo he apeu ic agen s o he ea men o se e al diseases, such
as an ibac e ial, an i ungal, an i-in lamma o y, an i i al, an ipa asi ic and an icance [
8
–
11
].
This ac can be explained by aking in o accoun ha imidazole de i a i es a e pola
and ionizable and can, he e o e, be used o op imize he solubili y and bioa ailabili y
pa ame e s o exis ing molecules. On he o he hand, he s uc u e o he imidazole ing
allows binding o nume ous enzymes and ecep o s a he biological le el h ough a ious
ypes o hyd ogen bonds and
π
-
π
s acking in e ac ions, leading o a wide ange o biological
ac i i ies [7,12–14].
One o he majo p oblems ha humani y aces is he inc ease o mul id ug- esis an
bac e ia, which is why he cons an disco e y o new po en ial d ugs is impo an o comba
Chem. P oc. 2022,8, 104. h ps://doi.o g/10.3390/ecsoc-25-11781 h ps://www.mdpi.com/jou nal/chemp oc
Chem. P oc. 2022,8, 104 2 o 6
hese mic oo ganisms. The mos epo ed example in he li e a u e is he esis ance o
S aphylococcus au eus. Soon a e he widesp ead use o penicillin, esis ance o penicillin
apidly appea ed in he clinic. Yea s la e , a new an ibio ic, me hicillin, was de eloped [
15
],
which e ec i ely con olled in ec ions caused by penicillin- esis an S. au eus. Howe e ,
wo yea s a e he use o me hicillin, he i s s ain o me hicillin- esis an S. au eus (MRSA)
was isola ed [13,15].
In addi ion o he a ied an ibac e ial ac i i ies [
16
–
18
], imidazoles also display in e -
es ing pho ophysical p ope ies, namely unable abso p ion and luo escence by he ca e ul
choice and placemen o elec on dono s and/o accep o subs i uen s a he imidazole co e
o he de elopmen o luo escen bioimaging p obes [13,16,19].
In his communica ion, we epo he syn hesis and he spec oscopic cha ac e iza ion
o a new 2,4,5- i(he e o)a ylimidazole de i a i e based on azaindole he e ocycle wi h
po en ial an ibio ic ac i i y agains S. au eus bac e ia.
2. Ma e ials and Me hods
Comme cial eagen s we e supplied by Sigma-Ald ich (S . Louis, MO, USA), Ac os,
Fluka, Pan eac, Lio ilchem and we e used as ecei ed. Thin laye ch oma og aphy (TLC)
was pe o med on silica gel 60 pla es wi h luo escence indica o F254 (Mache ey-Nagel,
Dü en, Ge many). The
1
H and
13
C nuclea magne ic esonance (NMR) spec a we e
eco ded using a B uke A ance III de ice a 400 MHz and 100.6 MHz, espec i ely, using
he sol en peak as an in e nal e e ence. The assignmen o he
1
H and
13
C signals
was pe o med using wo-dimensional he e onuclea co ela ion echniques, using as
sol en DMSO-d
6
wi h a 99.9% deu e a ion deg ee, con aining 0.1% / e ame hylsilane
om Sigma-Ald ich. The UV- isible abso p ion spec a we e made using a Shimadzu
UV/2501PC spec opho ome e , and he luo escence emission spec a using a Fluo oMax-
4 (HORIBA) spec o luo ime e using 9,10-diphenylan h acene as luo escence s anda d.
Fo he s udy o an ibac e ial ac i i y, he bac e ial s ain o he bac e ia S. au eus ATCC
6538 was used. Compound
3
was applied in s e ilized Wha man No. 1 il e pape , 6 mm
diame e disks.
2.1. Syn hesis and Spec oscopic Cha ac e iza ion o Imidazole De i a i e 3
The 7-azaindole-3-ca boxaldehyde
1
(1 mmol), u il
2
(1 mmol), NH
4
OAc (20 mmol)
and I
2
(5 mol %) we e dissol ed in e hanol (5 mL), ollowed by s i ing and hea ing a e lux
o 2 h. The eac ion was moni o ed by TLC, using a mix u e o dichlo ome hane/me hanol
(9:1) as eluen . Then, he eac ion mix u e was dilu ed wi h wa e (15 mL), ha ing a small
amoun o Na
2
S
2
O
3
, and was cooled in an ice ba h. The esul ing c ude p oduc , which
p ecipi a ed, was pu i ied by ec ys alliza ion om e hanol o gi e he pu e imidazole
de i a i e 3as a b own solid in 16% yield, m.p. = [238–240] ◦C (Figu e 1).
Chem. P oc. 2022, 8, 104 2 o 7
One o he majo p oblems ha humani y aces is he inc ease o mul id ug- esis an
bac e ia, which is why he cons an disco e y o new po en ial d ugs is impo an o
comba hese mic oo ganisms. The mos epo ed example in he li e a u e is he e-
sis ance o S aphylococcus au eus. Soon a e he widesp ead use o penicillin, esis ance o
penicillin apidly appea ed in he clinic. Yea s la e , a new an ibio ic, me hicillin, was
de eloped [15], which e ec i ely con olled in ec ions caused by penicillin- esis an S.
au eus. Howe e , wo yea s a e he use o me hicillin, he i s s ain o me hicil-
lin- esis an S. au eus (MRSA) was isola ed [13,15].
In addi ion o he a ied an ibac e ial ac i i ies [16–18], imidazoles also display in-
e es ing pho ophysical p ope ies, namely unable abso p ion and luo escence by he
ca e ul choice and placemen o elec on dono s and/o accep o subs i uen s a he im-
idazole co e o he de elopmen o luo escen bioimaging p obes [13,16,19].
In his communica ion, we epo he syn hesis and he spec oscopic cha ac e iza-
ion o a new 2,4,5- i(he e o)a ylimidazole de i a i e based on azaindole he e ocycle
wi h po en ial an ibio ic ac i i y agains S. au eus bac e ia.
2. Ma e ials and Me hods
Comme cial eagen s we e supplied by Sigma-Ald ich (S . Louis, MO, USA), Ac os,
Fluka, Pan eac, Lio ilchem and we e used as ecei ed. Thin laye ch oma og aphy (TLC)
was pe o med on silica gel 60 pla es wi h luo escence indica o F254 (Mache ey-Nagel,
Dü en, Ge many). The 1H and 13C nuclea magne ic esonance (NMR) spec a we e eco ded
using a B uke A ance III de ice a 400 MHz and 100.6 MHz, espec i ely, using he sol en
peak as an in e nal e e ence. The assignmen o he 1H and 13C signals was pe o med using
wo-dimensional he e onuclea co ela ion echniques, using as sol en DMSO-d6 wi h a
99.9% deu e a ion deg ee, con aining 0.1% / e ame hylsilane om Sigma-Ald ich. The
UV- isible abso p ion spec a we e made using a Shimadzu UV/2501PC spec opho ome e ,
and he luo escence emission spec a using a Fluo oMax-4 (HORIBA) spec o luo ime e
using 9,10-diphenylan h acene as luo escence s anda d.
Fo he s udy o an ibac e ial ac i i y, he bac e ial s ain o he bac e ia S. au eus
ATCC 6538 was used. Compound 3 was applied in s e ilized Wha man No. 1 il e pape ,
6 mm diame e disks.
2.1. Syn hesis and Spec oscopic Cha ac e iza ion o Imidazole De i a i e 3
The 7-azaindole-3-ca boxaldehyde 1 (1 mmol), u il 2 (1 mmol), NH4OAc (20 mmol)
and I2 (5 mol %) we e dissol ed in e hanol (5 mL), ollowed by s i ing and hea ing a e-
lux o 2 h. The eac ion was moni o ed by TLC, using a mix u e o dichlo o-
me hane/me hanol (9:1) as eluen . Then, he eac ion mix u e was dilu ed wi h wa e (15
mL), ha ing a small amoun o Na2S2O3, and was cooled in an ice ba h. The esul ing c ude
p oduc , which p ecipi a ed, was pu i ied by ec ys alliza ion om e hanol o gi e he pu e
imidazole de i a i e 3 as a b own solid in 16% yield, m.p. = [238–240] °C (Figu e 1).
Figu e 1. S uc u e o imidazole de i a i e 3.
1H NMR (400 MHz, DMSO-d6): δ = 6.62–6.63 (m, 2H, H4″ and H4″′), 6.87 (d, J = 3.2
Hz, 2H, H3″ and H3″′), 7.20–7.23 (q ap, J = 4.8 and 8.0 Hz, 1H, H5′), 7.76 (d, J = 0.8 Hz, 2H,
Figu e 1. S uc u e o imidazole de i a i e 3.
1
H NMR (400 MHz, DMSO-d
6
):
δ
= 6.62–6.63 (m, 2H, H4
00
and H4
000
), 6.87 (d, J = 3.2
Hz, 2H, H3
00
and H3
000
), 7.20–7.23 (q ap, J = 4.8 and 8.0 Hz, 1H, H5
0
), 7.76 (d, J = 0.8 Hz, 2H,
H5
00
and H5
000
), 8.19 (d, J = 2.8 Hz, 1H, H2
0
), 8.29 (dd, J= 1.2 and 4.4 Hz, 1H, H6
0
), 8.69 (dd,
J = 1.6 and 8.0 Hz, 1H, H4
0
), 11.96 (s, 1H, H
imid
) ppm. The indole NH was no isible in
hese condi ions.
Chem. P oc. 2022,8, 104 3 o 6
13
C NMR (100.6 MHz, DMSO-d
6
):
δ
= 104.94 (C3
0
), 107.34 (C3
00
and C3
000
), 111.83 (C4
00
and C4
000
), 116.63 (C5
0
), 117.44 (C3a
0
), 124.90 (C2
0
), 129.78 (C4
0
), 142.10 (C2
00
+ C2
000
+ C5
000
+ C5
000
), 143.71 (C2 and C6
0
), 148.58 (C6a
0
) ppm. The signals o ca bons 4 and 5 we e no
isible, as al eady epo ed in he li e a u e o simila sys ems [19].
UV/Vis (ace oni ile, nm): λmax (log ε) = 321 (4.3); λem (ΦF) = 455 (0.14).
2.2. An ibac e ial Ac i i y o Imidazole De i a i e 3
The inoculum suspension was p epa ed by selec ing wo mo phologically simila
colonies o S. au eus om o e nigh g ow h a 37
◦
C on solid Lu ia-Be ani aga medium
(LB aga ; 1% w/ yp one, 0.5% w/ yeas ex ac , 1% w/ NaCl, 2% w/ aga ) wi h a
s e ile loop. These colonies we e suspended in LB liquid medium ( he same composi ion
as LB aga bu wi hou aga ) and we e incuba ed o 4–6 h a 37
◦
C wi h 200 pm. The
u bidi y o he cul u e was isually adjus ed o 0.5 o he McFa land scale wi h s e ilized
deionized wa e .
The es solu ions we e p epa ed by se ial dilu ions o he imidazole de i a i e
3
, in
DMSO, in o de o p epa e concen a ions o 0.5, 1.5, and 3.0
µ
g/
µ
L. Ampicillin (0.3
µ
g/
µ
L)
was used as a s anda d d ug o an ibac e ial ac i i y. Fo each Pe i dish, p e iously
p epa ed wi h LB aga medium, he op aga was p epa ed wi h 100
µ
L bac e ial suspension
and 5 mL o LB aga (wi h 1.5% o aga ) a 50
◦
C. This mix u e was pou ed on he pla es
(app oxima ely 5 mL) and cooled down o 15 min. S e ilized disks we e placed i mly on o
he medium wi h he op aga . In each disk, 10
µ
L o he solu ions o con ols we e applied
and we e allowed o di use a oom empe a u e o 1–2 h be o e incuba ion a 37
◦
C o
18 h. These assays we e pe o med in iplica e. A e incuba ion, inhibi ion zones we e
measu ed a he poin whe e no ob ious g ow h o bac e ia is de ec ed by he unaided eye.
3. Resul s and Discussion
3.1. Syn hesis and Spec oscopic Cha ac e iza ion o he Imidazole De i a i e 3
Imidazole de i a i e
3
was syn hesized om 7-azaindole-3-ca boxaldehyde (
1
) and
u il (
2
), in he p esence o ammonium ace a e, using e hanol as sol en and iodine as
he ca alys , o 2 h, gi ing he pu e compound as a b own solid wi h 16% yield a e
ec ys alliza ion om e hanol (Scheme 1). Iodine as a ca alys in o ganic eac ions ac s as a
mild Lewis acid and has he abili y o bonding wi h he ca bonyl oxygen, inc easing he
eac i i y o he ca bonyl compound [20].
Chem. P oc. 2022, 8, 104 3 o 7
H5″ and H5″′), 8.19 (d, J = 2.8 Hz, 1H, H2′), 8.29 (dd, J= 1.2 and 4.4 Hz, 1H, H6′), 8.69 (dd, J
= 1.6 and 8.0 Hz, 1H, H4′), 11.96 (s, 1H, Himid) ppm. The indole NH was no isible in hese
condi ions.
13C NMR (100.6 MHz, DMSO-d6): δ = 104.94 (C3′), 107.34 (C3″ and C3″′), 111.83 (C4″
and C4″′), 116.63 (C5′), 117.44 (C3a′), 124.90 (C2′), 129.78 (C4′), 142.10 (C2″ + C2″′ + C5″’ +
C5″′), 143.71 (C2 and C6′), 148.58 (C6a′) ppm. The signals o ca bons 4 and 5 we e no
isible, as al eady epo ed in he li e a u e o simila sys ems [19].
UV/Vis (ace oni ile, nm): λmax (log ε) = 321 (4.3); λem (ΦF) = 455 (0.14).
2.2. An ibac e ial Ac i i y o Imidazole De i a i e 3
The inoculum suspension was p epa ed by selec ing wo mo phologically simila
colonies o S. au eus om o e nigh g ow h a 37 °C on solid Lu ia-Be ani aga medium
(LB aga ; 1% w/ yp one, 0.5% w/ yeas ex ac , 1% w/ NaCl, 2% w/ aga ) wi h a
s e ile loop. These colonies we e suspended in LB liquid medium ( he same composi ion
as LB aga bu wi hou aga ) and we e incuba ed o 4–6 h a 37 °C wi h 200 pm. The
u bidi y o he cul u e was isually adjus ed o 0.5 o he McFa land scale wi h s e ilized
deionized wa e .
The es solu ions we e p epa ed by se ial dilu ions o he imidazole de i a i e 3, in
DMSO, in o de o p epa e concen a ions o 0.5, 1.5, and 3.0 μg/μL. Ampicillin (0.3
μg/μL) was used as a s anda d d ug o an ibac e ial ac i i y. Fo each Pe i dish, p e-
iously p epa ed wi h LB aga medium, he op aga was p epa ed wi h 100 μL bac e ial
suspension and 5 mL o LB aga (wi h 1.5% o aga ) a 50 °C. This mix u e was pou ed on
he pla es (app oxima ely 5 mL) and cooled down o 15 min. S e ilized disks we e
placed i mly on o he medium wi h he op aga . In each disk, 10 μL o he solu ions o
con ols we e applied and we e allowed o di use a oom empe a u e o 1–2 h be o e
incuba ion a 37 °C o 18 h. These assays we e pe o med in iplica e. A e incuba ion,
inhibi ion zones we e measu ed a he poin whe e no ob ious g ow h o bac e ia is de-
ec ed by he unaided eye.
3. Resul s and Discussion
3.1. Syn hesis and Spec oscopic Cha ac e iza ion o he Imidazole De i a i e 3
Imidazole de i a i e 3 was syn hesized om 7-azaindole-3-ca boxaldehyde (1) and
u il (2), in he p esence o ammonium ace a e, using e hanol as sol en and iodine as he
ca alys , o 2 h, gi ing he pu e compound as a b own solid wi h 16% yield a e e-
c ys alliza ion om e hanol (Scheme 1). Iodine as a ca alys in o ganic eac ions ac s as a
mild Lewis acid and has he abili y o bonding wi h he ca bonyl oxygen, inc easing he
eac i i y o he ca bonyl compound [20].
Scheme 1. Syn hesis o imidazole de i a i e 3.
Pu e imidazole de i a i e 3 was isola ed in low yield (16%), p obably due o losses
du ing he ec ys alliza ion p ocess, as well as he low eac i i y o he he e ocyclic alde-
hyde bea ing a py ole elec on- ich moie y which will enhance he elec onic densi y on
he ca bonyl g oup, and, he e o e, will induce a lowe eac i i y o his compound. A e
syn hesis, imidazole de i a i e 3 was cha ac e ized by NMR spec oscopy (Figu e 2).
Scheme 1. Syn hesis o imidazole de i a i e 3.
Pu e imidazole de i a i e
3
was isola ed in low yield (16%), p obably due o losses
du ing he ec ys alliza ion p ocess, as well as he low eac i i y o he he e ocyclic aldehyde
bea ing a py ole elec on- ich moie y which will enhance he elec onic densi y on he
ca bonyl g oup, and, he e o e, will induce a lowe eac i i y o his compound. A e
syn hesis, imidazole de i a i e 3was cha ac e ized by NMR spec oscopy (Figu e 2).
Chem. P oc. 2022,8, 104 4 o 6
Chem. P oc. 2022, 8, 104 4 o 7
Figu e 2. 1H NMR (A) and 13C NMR (B) spec a o imidazole de i a i e 3 ( he colo s co espond o
he di e en moie ies, as shown in Scheme 1).
S anda d spec oscopic cha ac e iza ion o compound 3 was pe o med in ace oni-
ile (ACN) solu ions wi h concen a ions 1 × 10−5 and 1 × 10−6 mol/dm3 (Figu e 3). The
ela i e quan um luo escence yield o he compound was calcula ed using he luo es-
cence s anda d 9,10-diphenylan h acene in e hanol, whose absolu e luo escence quan-
um yield is 0.95 [21].
Figu e 3. No malized abso p ion and emission spec a o imidazole de i a i e 3.
Compound 3 exhibi ed a maximum wa eleng h o abso p ion a 321 nm, showing
an in ense abso p ion band. Rega ding he emission p ope ies, his imidazole showed a
maximum emission wa eleng h a 455 nm, wi h a la ge S okes’ shi alue o (134 nm),
meaning i is possible o conclude ha his compound has he po en ial o be used as a
luo escence p obe in biological media since a la ge shi allows an imp o ed sepa a ion
o ligh om he ma ix and he inhe en ligh sca e ed by he sample [1,22]. Finally, he
imidazole de i a i e showed a ela i e luo escence quan um yield o 0.14.
3.2. An ibac e ial Ac i i y o Imidazole De i a i e 3
In i o an ibac e ial s udies o imidazole de i a i es we e pe o med agains he
G am-posi i e bac e ium S. au eus by disk es di usion me hod (Figu e 4). The com-
pound inhibi ion halo alues we e measu ed in iplica e (Table 1), ampicillin was used
as he s anda d an ibio ic d ug o he posi i e con ol, and DMSO was used as he neg-
a i e con ol.
0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0
200 300 400 500 600
I/a.u.
Abs
Wa eleng h (nm)
Abs
Emi
Figu e 2. 1
H NMR (
A
) and
13
C NMR (
B
) spec a o imidazole de i a i e
3
( he colo s co espond o
he di e en moie ies, as shown in Scheme 1).
S anda d spec oscopic cha ac e iza ion o compound
3
was pe o med in ace oni ile
(ACN) solu ions wi h concen a ions 1
×
10
−5
and 1
×
10
−6
mol/dm
3
(Figu e 3). The
ela i e quan um luo escence yield o he compound was calcula ed using he luo escence
s anda d 9,10-diphenylan h acene in e hanol, whose absolu e luo escence quan um yield
is 0.95 [21].
Chem. P oc. 2022, 8, 104 4 o 7
Figu e 2. 1H NMR (A) and 13C NMR (B) spec a o imidazole de i a i e 3 ( he colo s co espond o
he di e en moie ies, as shown in Scheme 1).
S anda d spec oscopic cha ac e iza ion o compound 3 was pe o med in ace oni-
ile (ACN) solu ions wi h concen a ions 1 × 10−5 and 1 × 10−6 mol/dm3 (Figu e 3). The
ela i e quan um luo escence yield o he compound was calcula ed using he luo es-
cence s anda d 9,10-diphenylan h acene in e hanol, whose absolu e luo escence quan-
um yield is 0.95 [21].
Figu e 3. No malized abso p ion and emission spec a o imidazole de i a i e 3.
Compound 3 exhibi ed a maximum wa eleng h o abso p ion a 321 nm, showing
an in ense abso p ion band. Rega ding he emission p ope ies, his imidazole showed a
maximum emission wa eleng h a 455 nm, wi h a la ge S okes’ shi alue o (134 nm),
meaning i is possible o conclude ha his compound has he po en ial o be used as a
luo escence p obe in biological media since a la ge shi allows an imp o ed sepa a ion
o ligh om he ma ix and he inhe en ligh sca e ed by he sample [1,22]. Finally, he
imidazole de i a i e showed a ela i e luo escence quan um yield o 0.14.
3.2. An ibac e ial Ac i i y o Imidazole De i a i e 3
In i o an ibac e ial s udies o imidazole de i a i es we e pe o med agains he
G am-posi i e bac e ium S. au eus by disk es di usion me hod (Figu e 4). The com-
pound inhibi ion halo alues we e measu ed in iplica e (Table 1), ampicillin was used
as he s anda d an ibio ic d ug o he posi i e con ol, and DMSO was used as he neg-
a i e con ol.
0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0
200 300 400 500 600
I/a.u.
Abs
Wa eleng h (nm)
Abs
Emi
Figu e 3. No malized abso p ion and emission spec a o imidazole de i a i e 3.
Compound
3
exhibi ed a maximum wa eleng h o abso p ion a 321 nm, showing
an in ense abso p ion band. Rega ding he emission p ope ies, his imidazole showed
a maximum emission wa eleng h a 455 nm, wi h a la ge S okes’ shi alue o (134 nm),
meaning i is possible o conclude ha his compound has he po en ial o be used as a
luo escence p obe in biological media since a la ge shi allows an imp o ed sepa a ion
o ligh om he ma ix and he inhe en ligh sca e ed by he sample [
1
,
22
]. Finally, he
imidazole de i a i e showed a ela i e luo escence quan um yield o 0.14.
3.2. An ibac e ial Ac i i y o Imidazole De i a i e 3
In i o an ibac e ial s udies o imidazole de i a i es we e pe o med agains he
G am-posi i e bac e ium S. au eus by disk es di usion me hod (Figu e 4). The compound
inhibi ion halo alues we e measu ed in iplica e (Table 1), ampicillin was used as he
s anda d an ibio ic d ug o he posi i e con ol, and DMSO was used as he nega i e
con ol.
Chem. P oc. 2022,8, 104 5 o 6
Chem. P oc. 2022, 8, 104 5 o 7
Figu e 4. Disk es di usion me hod o an ibac e ial ac i i y o imidazole 3 agains S. au eus ATCC
6538: (A) wi h 10 µL o DMSO, (B) 5 µg/disk, (C) 15 µg/disk and (D) 30 μg/disk.
Table 1. Inhibi ion halos and s anda d de ia ion o imidazole de i a i e 3 agains S. au eus ATCC
6538 in he disk di usion assay.
Compound
3 μg/disk
5 μg/disk
15 μg/disk
30 μg/disk
3
-
ND
ND
(8 ± 1)
Ampicillin
(23.0 ± 0.1)
-
-
-
ND: no de ec ed; -: no de e mined.
Bea ing in mind he esul s o he an ibac e ial ac i i y, a clea inhibi ion zone is
p esen a ound he es disk, whose diame e is la ge when a highe concen a ion (30
μg/disk) is applied. Howe e , in Figu e 4C i is possible o isualize an inhibi ion o
g ow h close o he disk ma kedly smalle han 30 µg/disk (Figu e 4D), which was no
measu able, hus sugges ing ha he an ibac e ial e ec is dependen on he concen a-
ion ha is applied o he disk. As expec ed, he b oad-spec um an ibio ic ampicillin
displayed conside ably highe ac i i y han imidazole 3. Ampicillin is a semi-syn he ic
an ibio ic, which is op imized o maximal ac i i y and imp o ed pha macokine ic pa-
ame e s. Based on p e ious wo k by his esea ch g oup [23] and he a ional design o
he imidazole de i a i e 3, i is possible o conclude ha imidazole 3 is a new
2,4,5- i(he e o)a ylimidazole wi h he po en ial o be he basis o he de elopmen o
no el d ugs wi h an ibio ic ac i i y in medicinal chemis y.
4. Conclusions
Imidazole de i a i e 3 was syn hesized using a simple syn he ic me hodology and
an easy pu i ica ion p ocedu e and was cha ac e ized by he usual NMR and UV-Vis
abso p ion and emission spec oscopies.
Compound 3 showed an ibac e ial ac i i y agains S. au eus, which can be consid-
e ed a po en ial d ug in he ea men o diseases caused by pa hogenic bac e ia.
Au ho Con ibu ions: Concep ualiza ion, M.M.M.R.; me hodology, M.M.M.R., S.P.G.C. and R.O.;
alida ion, N.L.P.R., M.M.M.R. and S.P.G.C.; o mal analysis, N.L.P.R., M.M.M.R., S.P.G.C. and
R.O.; in es iga ion, N.L.P.R.; esou ces, M.M.M.R., S.P.G.C. and R.O.; w i ing—o iginal d a
p epa a ion, N.L.P.R.; w i ing— e iew and edi ing, N.L.P.R., M.M.M.R., S.P.G.C. and R.O.; supe -
ision, M.M.M.R., S.P.G.C. and R.O.; unding acquisi ion, M.M.M.R., S.P.G.C. and R.O. All au ho s
ha e ead and ag eed o he published e sion o he manusc ip .
Funding: This esea ch was unded by he Founda ion o Science and Technology (FCT) h ough
CQ/UM (UIDB/00686/2020) and CBMA/UM “Con a o-P og ama” UIDB/04050/2020 unded by
na ional unds. Thanks a e also due o FCT o inancial suppo o he Po uguese NMR Ne wo k
(PTNMR, B uke A ance III 400-Uni . Minho).
Ins i u ional Re iew Boa d S a emen : No applicable.
In o med Consen S a emen : No applicable.
Da a A ailabili y S a emen : No applicable.
Con lic s o In e es : The au ho s decla e no con lic o in e es .
Figu e 4.
Disk es di usion me hod o an ibac e ial ac i i y o imidazole
3
agains S. au eus ATCC
6538: (A) wi h 10 µL o DMSO, (B) 5 µg/disk, (C) 15 µg/disk and (D) 30 µg/disk.
Table 1.
Inhibi ion halos and s anda d de ia ion o imidazole de i a i e
3
agains S. au eus ATCC
6538 in he disk di usion assay.
Compound 3 µg/disk 5 µg/disk 15 µg/disk 30 µg/disk
3- ND ND (8 ±1)
Ampicillin (23.0 ±0.1) - - -
ND: no de ec ed; -: no de e mined.
Bea ing in mind he esul s o he an ibac e ial ac i i y, a clea inhibi ion zone is p esen
a ound he es disk, whose diame e is la ge when a highe concen a ion (30
µ
g/disk) is
applied. Howe e , in Figu e 4C i is possible o isualize an inhibi ion o g ow h close o
he disk ma kedly smalle han 30
µ
g/disk (Figu e 4D), which was no measu able, hus
sugges ing ha he an ibac e ial e ec is dependen on he concen a ion ha is applied
o he disk. As expec ed, he b oad-spec um an ibio ic ampicillin displayed conside ably
highe ac i i y han imidazole
3
. Ampicillin is a semi-syn he ic an ibio ic, which is op i-
mized o maximal ac i i y and imp o ed pha macokine ic pa ame e s. Based on p e ious
wo k by his esea ch g oup [
23
] and he a ional design o he imidazole de i a i e
3
, i
is possible o conclude ha imidazole
3
is a new 2,4,5- i(he e o)a ylimidazole wi h he
po en ial o be he basis o he de elopmen o no el d ugs wi h an ibio ic ac i i y in
medicinal chemis y.
4. Conclusions
Imidazole de i a i e
3
was syn hesized using a simple syn he ic me hodology and
an easy pu i ica ion p ocedu e and was cha ac e ized by he usual NMR and UV-Vis
abso p ion and emission spec oscopies.
Compound
3
showed an ibac e ial ac i i y agains S. au eus, which can be conside ed
a po en ial d ug in he ea men o diseases caused by pa hogenic bac e ia.
Au ho Con ibu ions:
Concep ualiza ion, M.M.M.R.; me hodology, M.M.M.R., S.P.G.C. and R.O.;
alida ion, N.L.P.R., M.M.M.R. and S.P.G.C.; o mal analysis, N.L.P.R., M.M.M.R., S.P.G.C. and R.O.;
in es iga ion, N.L.P.R.; esou ces, M.M.M.R., S.P.G.C. and R.O.; w i ing—o iginal d a p epa a ion,
N.L.P.R.; w i ing— e iew and edi ing, N.L.P.R., M.M.M.R., S.P.G.C. and R.O.; supe ision, M.M.M.R.,
S.P.G.C. and R.O.; unding acquisi ion, M.M.M.R., S.P.G.C. and R.O. All au ho s ha e ead and ag eed
o he published e sion o he manusc ip .
Funding:
This esea ch was unded by he Founda ion o Science and Technology (FCT) h ough
CQ/UM (UIDB/00686/2020) and CBMA/UM “Con a o-P og ama” UIDB/04050/2020 unded by
na ional unds. Thanks a e also due o FCT o inancial suppo o he Po uguese NMR Ne wo k
(PTNMR, B uke A ance III 400-Uni . Minho).
Ins i u ional Re iew Boa d S a emen : No applicable.
In o med Consen S a emen : No applicable.
Da a A ailabili y S a emen : No applicable.
Con lic s o In e es : The au ho s decla e no con lic o in e es .

Chem. P oc. 2022,8, 104 6 o 6
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