Synthesis and evaluation of biological activities of tripodal imines and β-lactams attached to the 1,3,5-triazine nucleus

Article abstract of DOI:10.1007/s00706-020-02592-8

Abstract: A novel collection of tripodal β-lactam derivatives with 1,3,5-triazine as the central core is presented for the first time. The diastereoselective synthesis of tris-β-lactams was achieved via a [2 + 2] cycloaddition reaction between s-triazine-

Full text of DOI:10.1007/s00706-020-02592-8

Monatshefte für Chemie - Chemical Monthly
https://doi.org/10.1007/s00706-020-02592-8
ORIGINAL PAPER
Synthesis and evaluation of biological activities of tripodal imines
and β‑lactams attached to the 1,3,5‑triazine nucleus
1
1
2
3
4
Mohammad Bashiri · Aliasghar Jarrahpour · Banafsheh Rastegari · Aida Iraji · Cambyz Irajie ·
Zahra Amirghofran5 · Saeed Malek‑Hosseini · Mohammad Motamedifar · Mahtab Haddadi ·
,6
6
7,8
8
9
9
10
Kamiar Zomorodian · Zahra Zareshahrabadi · Edward Turos
Received: 5 January 2020 / Accepted: 27 March 2020
©
Springer-Verlag GmbH Austria, part of Springer Nature 2020
Abstract
A novel collection of tripodal β-lactam derivatives with 1,3,5-triazine as the central core is presented for the ꢀrst time. The
diastereoselective synthesis of tris-β-lactams was achieved via a [2+2] cycloaddition reaction between s-triazine-based
1
tris-imines and activated aryloxyacetic acid derivatives. All the s-triazine hybrids were identiꢀed by FT-IR, H NMR,
1
3
C NMR spectroscopies and elemental analysis, and found to have all-cis relative stereochemistry of the three β-lactams
rings. The intermediate imines displayed moderate in vitro inhibitory activity against MCF-7 and HeLa cancer cell lines.
The tris-β-lactams were tested against four kinds of bacteria including Gram-negative bacteria, E. coli and P. aeruginosa, a
Gram-positive S. aureus and one capsulated Gram-negative K. pneumonia by the Kirby–Bauer disc diꢁusion method. Two
compounds have shown moderate antibacterial activity against S. aureus, but not against the other bacteria, or fungal isolates
C. albicans and A. fumigatus. The tris-β-lactams displayed good inhibitory behavior against the K562 human leukemia cell
line, and antioxidant properties as radical scavengers.
Graphic abstract
OAr
Z
Y
O
Z
Y
N
N
X
Z
N
X
Z
Y
X
O
N
Y
O
N
O
N
N
N
N
N
X
OAr
O
O
O
O
O
N
Z
X
Y
Z
N
OAr
2
ArOCH COOH
Y
X
TsCl / Et
CH Cl
3
N
2
2
Keywords 1,3,5-Triazine · β-Lactam · Tripodal · Hybrid · Organic synthesis
Introduction
with industrial and biological applications [1–3]. Benzene
and s-triazine are the most common central cores for the
synthesis of these star-shaped molecules. Numerous inves-
tigations have likewise been carried out to synthesize com-
pounds containing the s-triazine core as antibacterial [4],
antiviral [5], antimalarial [6], and antitumor [7] agents
(Fig. 1) [8]. These eꢁorts have led to the expansion of syn-
thesis of s-triazine heterocycles [9] and modiꢀcation of drug
molecules [10].
A class of branched star-shaped molecules having a general
structure bearing three or more chains connected symmetri-
cally to a single multifunctional core have been synthesized
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s00706-020-02592-8) contains
supplementary material, which is available to authorized users.
s-Triazine has threefold symmetry and can be prepared
with up to three substituents [11]. The trisubstituted analogs
are easily obtained by reacting cyanuric chloride with three
*
Aliasghar Jarrahpour
aliasghar6683@yahoo.com; jarahpor@shirazu.ac.ir
Extended author information available on the last page of the article
Vol.:(0
1
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3

M. Bashiri et al.
Fig. 1 Compounds with biological activities possessing the s-triazine nucleus
molar equivalents of the desired nucleophilic compounds
has led to new synthetic methods and strategies to enhance
the biological properties of medicinal agents. Hybridization
of bioactive compounds into a single molecule may help
increase the biological function and reduce toxicity [21, 22].
Our laboratories have reported a number of diꢁerent cat-
egories of bioactive hybrid molecules, and in this study we
set out to explore the assembly of s-triazine–β-lactam con-
jugates to investigate their potential biological properties.
[
12]. Precisely controlling the reaction conditions such as
temperature and reaction times, a selection of mono-, bi-,
and tri-substituted compounds can be prepared selectively.
Melamine, altretamine, and triethylenemelamine are exam-
ples of s-triazines with three identical substituents (Fig. 2).
Because of the branching capabilities of these derivatized
systems, s-triazine serves as a central core structure in den-
drimers for drug and gene delivery [13].
Imines are among the most important classes of organic
molecules, serving as intermediates for organic synthesis
Results and discussion
[
14] and for various biological applications [15]. Perhaps
most notably, imines serve as starting compounds for the
preparation of β-lactam compounds having antibacterial [16,
The Staudinger ketene–imine cycloaddition reaction is the
most common method for the construction of β-lactam
[23]. In our current study, a selection of novel s-tria-
zine-based β-lactams has been synthesized as shown in
Scheme 1. In the ꢀrst step, s-triazine tris-aldehyde was
1
7], anticancer [18], antimalarial [19], and antiinꢂammatory
activity [20]. One of the most important concerns in medici-
nal chemistry is the continuing rise in drug resistance that
Fig. 2 Drugs containing the
s-triazine nucleus with three
identical substituents
1
3

Synthesis and evaluation of biological activities of tripodal imines and β‑lactams attached…
Scheme 1
synthesized from cyanuric chloride (1) and p-hydroxyben-
zaldehyde (2) according to a reported procedure [24]. The
condensation of tris-aldehyde 3 with substituted anilines
IR spectroscopy
All synthesized compounds 3, 5a–5i, 8a–8g, and 9a–9g
were analyzed by infrared spectroscopy. The aldehyde car-
bonyl groups of the tris-aldehyde 3, which were observed at
4
a–4i in the presence of a catalytic amount of glacial ace-
tic acid in reꢂuxing ethanol aꢁorded nine s-triazine-based
tris-imines 5a–5i in excellent yields.
−
1
1705 cm , disappeared after the Schiꢁ base formation. The
C=N bond stretching frequency of the imines appeared at
The tris-imines 5a–5g were then treated with O-ary-
lacetic acid derivatives 6 and 7 in the presence of p-tol-
−
1
1627 cm . The presence of a strong C=O absorption bands
−
1
uensulfonyl chloride and dry tertiary amine (Et N) in
for the β-lactam carbonyls are at 1751, 1759, or 1766 cm .
The C–O–Ar absorption was observed as a separate band
3
dry dichloromethane, and stirred at room temperature
−
1
overnight.
at 1365 cm . The imines and β-lactams each showed an
−
1
The desired β-lactams 8a–8g and 9a–9g were aꢁorded
in yields ranging from 50 to 70%. Similar eꢁorts to obtain
β-lactam adducts from imines 5h and 5i were unsuccessful.
The tris-β-lactams bearing the phenoxy group (8a–8g) were
obtained in slightly higher yields than those with the 2-naph-
thoxy group (9a–9g). Infrared and NMR spectroscopy were
used to assign the structure of each product.
absorption band at 1566 cm for the C=N stretching vibra-
tion of the s-triazine ring.
NMR spectroscopy
The proton NMR spectral data of the imines and the ꢀnal
β-lactam adducts indicate that all of the s-triazine derivatives
1
3

M. Bashiri et al.
5
a–5i, 8a–8g, and 9a–9g have trifold symmetry. The
the s-triazine core. Elemental analyses are in accord with the
structural assignments.
appearance of a singlet at 8.5 ppm for the azomethine pro-
ton (HC=N) instead of an aldehydic proton resonance at
1
0.0 ppm conꢀrms the formation of one geometric isomer
Pharmacology
of each imine, which we attribute to the E-isomer based on
the extensive literature reports [25–29]. For the β-lactam
adducts, two new resonances at 5.5 ppm and 6.1 ppm are
observed, indicative of the vicinal H-3 and H-4 protons of
the β-lactam ring. The important issue to consider in the
proton spectrum of the β-lactam ring is the stereochemistry
of this ring, which is related to J (coupling constant) of
The tris-imine s-triazines 5a–5i were screened for in vitro
cytotoxicity against a cervical cancer cell line (HeLa) and
breast cancer cell line (MCF-7). All tris-β-lactam 8a–8g
and 9a–9g were likewise tested for antibacterial properties
against four bacterial species including E. coli, P. aerugi-
nosa, S. aureus, and K. pneumonia. Tris-β-lactam–s-triazine
adducts were examined for potential inhibitory activity
toward the growth of a human leukemia tumor cell line
3
,4
the protons H-3 and H-4. J > 4.0 is characteristic of the
3
,4
cis stereoisomer and J < 3.0 accounts for the trans ste-
3
,4
reoisomer [30–34]. From this, we can conꢀrm that all of
s-triazine–β-lactams are formed with exclusive cis stereo-
chemistry (Fig. 3).
(
K562) as well as for antioxidant activity.
While we can clearly identify the "all-cis" disubstituted
β-lactam rings in these adducts, based on their NMR spectra,
we cannot deꢀnitively determine which of the three possible
Investigation of anticancer activities of tris‑imines
5a–5i
"
all-cis" diastereomers are formed or if they are all obtained
The anticancer activity of tris-imine s-triazines 5a–5i were
screened against cervical cancer (HeLa) and breast can-
cer (MCF-7) cell lines, at 25, 50, 75, and 100 µM for 24 h
versus 72 h. The results are shown in Tables 1 and 2.
These data indicate that the tris-imines all have moderate
activity against MCF-7 and HeLa cell lines. Among these,
5g showed the best activity against MCF-7, since at 100 µM
only 60% of the cells survived after 24 h. The presence of
the tree chlorines in 5g thus appear to exert a role in this
activity. The second best compound is tris-imine 5d, with
64% MCF-7 cell viability at 100 µM after 72 h. Tris-imines
5f and 5i also exhibited similar anticancer activities against
the cervical cancer cell (HeLa). The data for other com-
pounds are visible on the graphs in Figs. 5 and 6.
as an indistiguishable mixture (Fig. 4). While each of the
adducts 8a–8g and 9a–9g are powdery solids, and none have
yielded crystals suitable for single-crystal X-ray analysis.
The chemical shifts of the aliphatic and aromatic protons
1
3
appear within the expected regions. The C NMR spectra
likewise corroborate the symmetry of these adducts. For the
tris-imines and tris-β-lactams, one absorption at 172.0 ppm
conꢀrms the equivalency of the C-2, C-4, and C-6 carbons
of the s-triazine ring. We also note the appearance of two
1
3
new signals from 60.0 or 80.0 ppm in the C NMR spectra
of the tris-β-lactam ring adducts, for the C-3 and C-4 car-
bons of the β-lactam rings. Each signal appears as a singlet,
indicative of the equivalency of the three β-lactam rings on
Fig. 3 Example of expanded β-lactam region
1
3

Synthesis and evaluation of biological activities of tripodal imines and β‑lactams attached…
Fig. 4 Three possible diastereomers of the all-cis tris-β-lactam products
Evaluation of biological activities of tris‑β‑lactams
a–8g and 9a–9g: antibacterial activity
Antifungal assay
8
The synthesized compounds 8a–8g and 9a–9g were evalu-
ated for antifungal activity against Candida albicans (ATCC
10261) and Aspergillus fumigatus (ATCC 14110). These
experiments were done by serial broth dilution, up to 256 µg/
Antibacterial activity of the synthesized tris-β-lactams
a–8g and 9a–9g was assessed against four kinds of bac-
8
teria including the Gram-negative microbes E. coli and P.
aeruginosa, the Gram-positive S. aureus and one capsu-
lated Gram-negative K. pneumonia, using the Kirby–Bauer
disk diꢁusion method, at a constant mass amount of 10 μg.
Oxacillin, gentamicin, amikacin, and streptomycin were
used as positive control agents (Table 3). Our aim was to
investigate the eꢁect of the various aryl ring substituents
on N-1 as well as the phenoxy versus naphthoxy ring on
C-3 of the β-lactam rings. Only compounds 8a and 8c were
moderately active against S. aureus.
3
cm . Unfortunately, none of the tested compounds exhibited
3
antifungal activities at concentrations up to 256 µg/cm .
Antioxidant activity
An antioxidant assay was performed to determine the
potency of the tris-β-lactams 8a–8g and 9a–9g as free radi-
cal reducing agents. In this case, the oxidant is 2,2-diphenyl-
1-picrylhydrazyl (DPPH) radical, which undergoes a change
1
3

M. Bashiri et al.
Table 2 Cell viability of HeLa cell treated with tris-imines 5a–5i
Table 1 Cell viability of MCF-7 cells treated with tris-imines 5a–5i
Imines
Concentration/ Cell viabil-
Cell viabil-
ity/%± STDEV
72 h
Imines
Concentration/ Cell viabil-
Cell viabil-
ity/%± STDEV
72 h
µM
ity/%± STDEV
uM
ity/%± STDEV
2
4 h
24 h
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
25
98.42± 2.21
94.07± 7.01
98.80± 2.63
95.50± 5.47
84.16± 3.71
100.65± 1.43
93.37± 7.26
89.14± 7.36
79.58± 1.93
100.57± 3.87
100.38± 2.44
95.45± 5.37
87.04± 2.40
94.68± 6.15
81.28± 6.89
74.13± 10.25
63.89± 14.40
98.29± 4.30
100.64± 1.48
93.80± 3.18
88.52± 4.59
99.48± 1.50
90.54± 7.50
74.10± 2.64
64.25± 9.40
99.80± 1.20
100.78± 2.94
88.43± 7.85
79.10± 7.32
99.97± 6.40
99.45± 5.99
84.46± 5.37
72.94± 12.90
99.51± 0.92
98.47± 6.89
88.27± 2.73
65.40± 4.54
5a
25
50
75
100
25
50
75
100
25
50
75
100
25
50
75
100
25
50
75
100
25
50
75
100
25
50
75
100
25
50
75
100
25
101.74± 1.57
103.71± 3.55
96.13± 13.81
86.70± 15.69
100.13± 2.74
100.95± 2.59
101.62± 1.30
93.96± 4.45
99.24± 2.33
102.59± 3.60
85.12± 13.97
83.73± 4.15
96.56± 1.06
98.05± 3.26
78.68± 9.93
72.34± 6.21
99.14± 3.26
97.30± 4.47
93.44± 6.74
99.14± 3.26
100.31± 2.95
99.70± 1.09
90.07± 6.13
73.39± 6.90
99.43± 1.52
99.11± 3.67
96.85± 5.95
97.32± 4.86
97.72± 2.58
102.29± 3.27
95.14± 3.69
89.93± 4.43
99.69± 3.10
99.69± 2.29
93.55± 6.74
68.73± 3.65
101.12± 3.06
100.12± 2.06
96.56± 1.78
76.75± 14.43
102.12± 7.14
94.20± 4.64
99.66± 3.80
102.12± 3.05
101.02± 0.64
94.69± 12.48
82.72± 6.24
81.97± 2.79
99.25± 1.15
98.29± 1.90
84.77± 11.23
68.84± 4.30
99.25± 1.15
102.14± 2.66
100.60± 5.74
85.79± 5.25
98.48± 4.23
103.44± 1.65
79.25± 5.92
64.09± 4.33
105.73± 4.53
98.52± 3.49
102.23± 1.75
99.92± 2.90
105.45± 4.11
97.13± 2.01
95.74± 4.68
86.50± 3.46
100.01± 1.98
92.97± 8.19
96.41± 5.95
71.68± 9.34
5
7
0
5
100.48± 0.64
95.13± 0.44
93.19± 2.95
99.29± 1.76
92.75± 1.70
89.47± 7.41
85. 30± 6.24
101.20± 0.20
99.13± 0.17
93.77± 1.97
89.31± 2.57
100.55± 0.33
100.79± 1.97
91.74± 2.35
79.45± 1.25
99.67± 0.25
98.45± 0.62
95.30± 2.40
92.25± 3.30
99.97± 0.57
98.28± 5.86
91.80± 3.32
88.24± 2.21
99.03± 1.58
103.97± 2.81
96.34± 2.95
94.14± 2.18
101.23± 1.04
98.74± 3.84
96.25± 9.26
59.66± 12.87
98.93± 3.82
95.34± 0.25
92.97± 1.38
87.58± 4.29
1
1
1
1
1
1
1
1
1
00
25
5b
5c
5d
5e
5f
5
7
0
5
00
25
5
7
0
5
00
25
5
7
0
5
00
25
5
7
0
5
00
25
5
7
0
5
00
25
g
h
i
5g
5h
5i
5
7
0
5
00
25
5
7
0
5
00
25
5
7
0
5
50
75
100
00
in color from violet to light yellow upon reduction. The
results of the DPPH assay are presented in Table 4. Com-
pound 9d bearing the electron-releasing N,N-diethylamino
group on the N-1 phenyl ring as well as the naphthoxy group
at C-3 of the β-lactam ring oꢁers the best radical scavenging
potential (IC =430.35 µM) of all the compounds tested. As
with their phenoxy counterparts (8a–8g). The other potent
compound in this series is 9c with an IC of 490 µM.
5
0
Antiproliferative activity
50
expected, replacement of the naphthoxy substituent at C-3
of the β-lactam ring with the smaller phenoxy group (com-
pound 9d versus 8d) resulted in a marked decrease in anti-
oxidant activity. Similar results were observed in the case
of the other naphthoxy derivatives (9a–9g) in comparison
The antiproliferative activity of tris-β-lactams 8a–8g and
9a–9g was examined against a standard human leukemia
tumor cell line (K562) by 3-(4,5-dimethylthiazol-2-yl)-2,5-di-
phenyltetrazolium bromide (MTT) assay in serial dilutions,
ranging in concentrations of 200, 100, 50, 10, 1, and 0.1 µg/
1
3

Synthesis and evaluation of biological activities of tripodal imines and β‑lactams attached…
4 h
2
2 4 h
150
1
50
5
5
5
5
5
5
5
5
i
5
5
i
h
g
f
h
100
5 g
1
00
5
5
f
e
d
c
b
e
50
5 d
5
0
0
5
5
c
b
0
5 a
5 a
25
50
75
100
0
25
50
75
Concentration/μM
10
Concentration/μM
72 h
7
2 h
1
1
50
00
5
5
5
5
5
5
5
5
i
1
1
50
00
h
g
f
5
i
5 h
5
5
g
f
e
d
c
b
50
5 e
5
5
5
d
c
b
50
0
5 a
2
5
50
75
100
0
5 a
Concentration/μM
0
0
25
5
75
10
Concentration/μM
Fig. 5 Anticancer activity pattern of tris-imines 5a–5i against MCF-7
Fig. 6 Anticancer activity pattern of tris-imines 5a–5i against HeLa
3
cm . Cisplatin was used as the reference drug and DMSO was
the negative control (blank) at concentrations equal to those of
the test wells. The results are shown in Table 5. The inhibitory
eꢁects of the s-triazine derivatives 8a–8g and 9a–9g on K562
tumor cell line after 48 h are graphed in Fig. 7. Compounds
core. The tris-imines were converted to tris-β-lactams 8a–8g
and 9a–9g by cycloaddition reaction with an aryloxyacetic
acid in the presence of the Et N and tosyl chloride, using dry
3
CH Cl as the solvent at room temperature overnight. The tris-
2
2
8a–8e displayed good activity against this cell line. The struc-
imines and tris-β-lactams were examined further to determine
their in vitro biological behavior. The tris-imines were found
to exert moderate anticancer activity against the MCF-7 and
HeLa cell lines. Analogously, the antibacterial activity was
examined for the β-lactams against four kinds of bacteria by
agar well diꢁusion. β-Lactams 8a and 8c showed moderate
antibacterial activity against Gram-positive bacteria S. aureus,
while all the other derivatives were inactive. Also, two species
of fungi were tested, but showed no sensitivity to any of these
tris-β-lactams. Antioxidant activity of the β-lactam derivatives
were measured and the results showed that the naphthoxy con-
taining derivatives at the C-3 β-lactam position (9a–9g) were
more potent than their phenoxy counterparts (8a–8g) in all
cases. The growth inhibitory eꢁect of the 8a–8g and 9a–9g
were performed at human leukemia tumor cell line (K562) and
the data were displayed that the compounds with the phenoxy
group at C-3 of the β-lactam ring have good activity toward
this cell line.
ture–activity relationship shows that the presence of the N,N-
diethylamino group at the para position of the N-1 phenyl of
the β-lactam ring 8d maximizes the antiproliferative activity.
Compound 8e, having two methoxy groups at the 2,4 positions
of the N-1 phenyl ring, provides the second best activity, with
the activity order being 4-(N,N-diethylamino)>2,4-dimeth-
oxy>4-CH >4-ethoxy>4-methoxy. In addition, replacement
3
of the C-3 phenoxy group of the β-lactam ring with a naphth-
oxy signiꢀcantly reduces the antiproliferative activity in this
assay. The data represent mean± standard deviation (SD) of
two independent experiments in triplicate.
Conclusion
s-Triazine trichloride was used as a cheap commercially acces-
sible starting material to prepare a range of tripodal tris-imines
and, subsequently, tris-β-lactams oꢁ a central 1,3,5-triazine
1
3

M. Bashiri et al.
Table 3 Antibacterial activities
of s-triazine β-lactams 8a–8g
and 9a–9g
Test material
S. aureus (ATCC
E. coli (ATCC
K. pneumonia (ATCC P. aerugi-
2
5,923)
25,922)
700,881)
nosa (ATCC
2
7,853)
DMSO
Oxacillin
Gentamicin
Amikacin
Streptomycin
–
–
–
–
23 mm
19 mm
NA
NA
NA
≥17
≥15
–
–
–
NA
NA
≥17
≥15
–
–
–
NA
NA
≥17
≥15
–
–
–
NA
8
8
a
c
18 mm
12 mm
–
Other lactams
Table 4 Radical scavenging potential of compounds 8a–8g and 9a–
g versus 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical
48 h
100
9
8
g
Compounds Inhibition at
00 µM/%
± SE)
Inhibition at
600 µM/%
(± SE)
IC /µM
8 f
5
0
80
3
8
e
(
6
4
2
0
0
0
0
8 d
8 c
8 b
8 a
8
8
8
8
8
8
8
9
9
9
9
9
9
9
a
b
c
d
e
f
23.30 (± 0.64)
27.63 (± 1.53)
13.16 (± 1.29)
33.08 (± 2.75)
14.59 (± 1.42)
32.30 (± 1.93)
21.07 (± 4.70)
32.08 (± 1.48)
31.70 (± 1.48)
31.55 (± 2.13)
36.65 (± 0.59)
28.59 (± 3.08)
37.64 (± 0.78)
31.63 (± 0.52)
34.81 (± 5.15)
30.74 (± 1.67)
25.26 (± 2.09)
47.44 (± 3.57)
31.42 (± 1.64)
45.65 (± 0.69)
38.20 (± 2.02)
54.20 (± 1.28)
53.26 (± 1.66)
60.25 (± 1.65)
65.06 (± 4.81)
53.85 (± 1.78)
52.15 (± 0.75)
54.90 (± 3.81)
>600
>600
>600
>600
>600
>600
>600
574.24 (± 11.90)
549.68 (± 12.65)
489.91 (± 11.28)
430.35 (± 16.34)
582.14 (± 6.70)
537.17 (± 12.37)
518.83 (± 5.97)
4.62 (± 2.26)
1
1
0
0
0.
10
50
20
10
g
a
b
c
d
e
f
Concentration/μg cm-3
48 h
1
00
9
g
f
9
9
8
6
0
0
e
9 d
g
9
9
9
c
b
a
Quercetin
40
2
0
0
1
1
0
20
0.
1
0
50
00
1
Table 5 IC50 values for 8a–8g and 9a–9g against human leukemia
tumor cell line (K562)
Concentration/μg cm-3
IC50 ± SD/µg cm−3
IC50 ± SD/µg cm−3
β-lactam
Fig. 7 % Inhibition for tris-β-lactams 8a–8g and 9a–9g against
β-lactam
After 48 h
After 48 h
human leukemia tumor cell line (K562)
8
8
8
8
8
8
8
a
b
c
d
e
f
50.04± 2.0
68.7± 2.6
50.8± 1.3
48.4± 1.5
49.04± 2.1
103.1± 2.4
>200
9a
9b
9c
9d
9e
9f
>200
197.4± 2.9
>200
>200
>200
>200
155.7± 4.4
Experimental
All starting materials and reagents commercially available
were used without further purification. They were pro-
vided from Merck (Darmstadt, Germany), Fluka (Buchs,
Switzerland) and Sigma-Aldrich (St. Louis, Missouri,
g
9g
Cisplatin
1.3
1
3

Synthesis and evaluation of biological activities of tripodal imines and β‑lactams attached…
USA) chemical companies. All reagents and solvents such
as EtOH and AcOH were anhydrous and CH Cl and Et N
(n-hexane/ EtOAc 1:3). After ending the reaction, the mix-
ture was cooled to room temperature, the precipitate was
ꢀltered, washed with ethanol and air-dried to give tris-imines
5a–5i.
2
2
3
were dried before use by distillation over CaH . The struc-
2
tures of all compounds were determined by examining
1
13
their FT-IR, H NMR, and C NMR spectroscopic data,
and elemental analysis. Infrared absorption spectra were
obtained with a Shimadzu FT-IR 8300 spectrometer using
KBr discs on a beam splitter. The absorption frequencies
1 , 1 ′ , 1 ″ ‑ [ [ ( 1 , 3 , 5 ‑Tr i a z i n e ‑ 2 , 4 , 6 ‑ t r i y l ) t r i s ( ox y ) ] ‑
tris(benzene‑1,4‑diyl)]tris[N‑(p‑tolyl)methanimine] (5a,
C H N O ) White powder; yield 90%; m.p.: 229–231 °C;
4
5 36 6 3
−
1
1
were reported in reciprocal centimetres (cm ). H and
FT-IR (KBr): v̄=1627 (HC=N imine), 1566 (C=N triazine),
1
3
−1 1
C NMR spectra were recorded on a Bruker Ascend or
1365 (C–O–Ar triazine) cm ; H NMR (300 MHz, DMSO-
Bruker Avance III spectrometer, using DMSO-d as the
d ): δ=8.61 (3H, s, HC=N), 7.97 (6H, d, J=7.4 Hz, ArH),
6
6
solvent and tetramethylsilane as the internal standard. The
7.40 (6H, d, J = 7.4 Hz, ArH), 7.16 (12H, d, J = 7.0 Hz,
1
3
chemical shift of DMSO-d relative to tetramethylsilane
ArH), 2.33 (9H, s, CH ) ppm; C NMR (100 MHz, DMSO-
6
6
3
1
13
(
TMS) in H NMR is reported at 2.50 ppm and in
C
d ): δ = 172.9 (C−O triazine), 158.5 (C=N imine), 148.5,
NMR at 39.52 ppm. Chemical shifts are reported in δ
135.4, 134.0, 129.6, 129.2, 121.9, 120.9, 114.0 (aromatic
n
(
parts per million) values, and coupling constants ( J) are
carbons), 20.5 (CH ) ppm.
3
reported in Hertz. The following abbreviations are used
for the multiplicities: s = singlet, d = doublet, dd = doublet
of doublet, t = triplet, q = quartet, m = multiplet, signal
for proton spectra. Elemental analyses were measured on
Thermo Finnigan Flash EA-1112 series. Melting points
were specified on a Büchi B-545 oil-heated melting point
system (Büchi Labortechnik, Flawil, Switzerland). Ana-
lytical thin-layer chromatography (TLC) was done using
1 , 1 ′ , 1 ″ ‑ [ [ ( 1 , 3 , 5 ‑Tr i a z i n e ‑ 2 , 4 , 6 ‑ t r i y l ) t r i s ( ox y ) ] ‑
tris(benzene‑1,4‑diyl)]tris[N‑(4‑methoxyphenyl)methan‑
imine] (5b, C H N O ) White powder; yield 95%; m.p.:
4
5 36 6 6
225–227 °C; FT-IR (KBr): v̄ = 1627 (C=N imine), 1566
−
1 1
(C=N triazine), 1365 (C–O–Ar triazine) cm ; H NMR
(300 MHz, DMSO-d ): δ=8.61 (3H, s, HC=N), 7.95 (6H,
6
d, J=7.9 Hz, ArH), 7.37 (6H, d, J=7.9 Hz, ArH), 7.24 (6H,
silica gel 60 F
(aluminum sheets). TLC plates were
50
d, J=5.4 Hz, ArH), 6.93 (6H, d, J=7.9 Hz, ArH), 3.78 (9H,
2
1
3
analyzed by an exposure to ultraviolet (UV) light or/and
s, OCH ) ppm; C NMR (100 MHz, DMSO-d ): δ=172.9
3 6
in I vapor and spots were visualized with ultraviolet
(C−O triazine), 157.9 (C=N imine), 157.1, 153.2, 143.9,
134.2, 129.5, 122.4, 121.9, 114.3 (aromatic carbons), 55.2
2
light. Also, graphics were illustrated by GraphPad Prism.
(
OCH ) ppm.
3
4
,4′,4″‑[(1,3,5‑Triazine‑2,4,6‑triyl)tris(oxy)]tribenzaldehyde
(
3, C H N O ) 4-Hydroxybenzaldehyde (3.00 mmol) was
1 , 1 ′ , 1 ″ ‑ [ [ ( 1 , 3 , 5 ‑Tr i a z i n e ‑ 2 , 4 , 6 ‑ t r i y l ) t r i s ( ox y ) ] ‑
tris(benzene‑1,4‑diyl)]tris[N‑(4‑ethoxyphenyl)methan‑
imine] (5c, C H N O ) White powder; yield 95%; m.p.:
2
4 15 3 6
3
3
weighed into a round-bottom ꢂask (100 cm ), and 30 cm
water and solid NaOH (3.00 mmol) were then added. A solu-
4
8 42 6 6
3
tion of trichlorotriazine (1.00 mmol) in 30 cm acetone was
206–208 °C; FT-IR (KBr): v̄ = 1627 (C=N imine), 1566
−
1 1
prepared and added slowly into a solution of the 4-hydroxy-
benzaldehyde, then the resulting solution was stirred at room
temperature for 3 h. The reaction mixture was ꢀltered and
the resulting white solid was washed several times with
water and recrystallized from ethanol to aꢁord pure alde-
hyde 3. White powder; yield 92%; m.p.: 173–175 °C; FT-IR
(C=N triazine), 1365 (C–O–Ar triazine) cm ; H NMR
(300 MHz, DMSO-d ): δ=8.61 (3H, s, HC=N), 7.94 (6H,
6
d, J=7.8 Hz, ArH), 7.37 (6H, d, J=7.6 Hz, ArH), 7.22 (6H,
d, J=7.8 Hz, ArH), 6.91 (6H, d, J=7.7 Hz, ArH), 4.03 (6H,
q, J = 5.8 Hz, OCH ), 1.36 (9H, t, J = 6.8 Hz, CH ) ppm;
2
3
1
3
C NMR (100 MHz, DMSO-d ): δ=172.9 (C−O triazine),
6
(
KBr): v
̄
=1705 (C=O aldehyde), 1566 (C=N triazine), 1365
157.2 (C=N imine), 157.0, 153.2, 143.7, 134.2, 129.5,
−
1 1
(
C–O–Ar triazine) cm ; H NMR (400 MHz, DMSO-d ):
122.4, 121.9, 114.8 (aromatic carbons), 63.2 (OCH ), 14.6
6
2
δ=9.99 (3H, s, HC=O aldehyde), 7.98 (6H, d, J=8.6 Hz,
(CH ) ppm.
3
ArH), 7.49 (6H, d, J=8.5 Hz, ArH) ppm.
1
, 1 ′ , 1 ″ ‑ [ [ ( 1 , 3 , 5 ‑Tr i a z i n e ‑ 2 , 4 , 6 ‑ t r i y l ) t r i s ( ox y ) ] ‑
tris(benzene ‑1, 4‑ diyl)]tris(methaneylylidene)]‑
tris(azaneylylidene)]tris(N,N‑diethylaniline) (5d,
C H N O ) Green powder; yield 97%; m.p.: 226–228 °C;
General experimental method
for the preparation of tris‑imines 5a–5i
5
4 57 9 3
FT-IR (KBr): v̄=1627 (C=N imine), 1566 (C=N triazine),
−1 13
A mixture of compound 3 (1.00 mmol), aniline derivatives
1365 (C–O–Ar triazine) cm ; C NMR (100 MHz, DMSO-
4
a–4i (3.00 mmol), and two to three drops of glacial AcOH
d ): δ = 172.3 (C–O-triazine), 163.7 (C=N imine), 132.6,
6
3
was stirred under reꢂux in 30 cm ethanol for 12–24 h. The
reaction progress was checked by thin-layer chromatography
129.5, 123.2, 122.9, 116.3, 112.2, 111.6 (aromatic carbons),
44.3 (NCH ), 12.9 (CH ) ppm.
2
3
1
3

M. Bashiri et al.
1
, 1 ′ , 1 ″ ‑ [ [ ( 1 , 3 , 5 ‑Tr i a z i n e ‑ 2 , 4 , 6 ‑ t r i y l ) t r i s ( ox y ) ] ‑
(6H, d, J = 7.0 Hz, ArH), 7.54 (6H, d, J = 7.8 Hz, ArH),
7.40 (6H, d, J=7.2 Hz, ArH), 7.17 (6H, d, J=7.1 Hz, ArH)
tris(benzene‑1,4‑diyl)]tris[N‑(2,4‑dimethoxyphenyl)metha‑
nimine] (5e, C H N O ) White powder; yield 98%; m.p.:
1
3
ppm; C NMR (75 MHz, DMSO-d ): δ=176.4 (C−O tria-
4
8
42
6
9
6
2
30–232 °C; FT-IR (KBr): v
̄
= 1627 (C=N imine), 1573
zine), 161.5 (C=N imine), 148.5, 134.5, 133.4, 131.9, 129.3,
−
1 1
(
(
C=N triazine), 1380 (C–O–Ar triazine) cm ; H NMR
124.1, 123.8, 122.3 (aromatic carbons) ppm.
300 MHz, DMSO-d ): δ=8.53 (3H, s, HC=N), 7.92 (6H,
6
d, J=8.5 Hz, ArH), 7.38 (6H, d, J=8.4 Hz, ArH), 7.00 (3H,
d, J = 8.6 Hz, ArH), 6.62 (3H, d, J = 2.2 Hz, ArH), 6.42–
General experimental procedure for the synthesis
of tris β‑lactams 8a–8g and 9a–9g
1
3
6
.51 (3H, m, ArH), 3.77 (18H, s, OCH ) ppm; C NMR
3
(
(
100 MHz, DMSO-d ): δ = 172.9 (C–O triazine), 158.6
Imines 5a–5g (1.00 mmol), dry triethylamine (15.00 mmol),
diꢁerent acetic acid derivatives (5.00 mmol), and p-tosyl
chloride (5.00 mmol) were put into a dried round-bottom
6
C=N imine), 157.7, 153.3, 153.1, 134.4, 134.0, 129.5,
1
5
21.8, 120.7, 104.7, 99.5 (aromatic carbons), 55.6 (OCH ),
3
3
3
5.3 (OCH ) ppm.
ꢂask (25 cm ) ꢀtted with a magnetic bar and 15 cm dry
CH Cl was added and the mixture was stirred overnight at
3
2
2
1
, 1 ′ , 1 ″ ‑ [ [ ( 1 , 3 , 5 ‑Tr i a z i n e ‑ 2 , 4 , 6 ‑ t r i y l ) t r i s ( ox y ) ] ‑
room temperature. The progress of the reaction was moni-
tored by thin-layer chromatography (n-hexane/EtOAc 1:2).
After the reaction completion, the mixture was washed with
tris(benzene‑1,4‑diyl)]tris(N‑phenylmethanimine) (5f,
C H N O ) Light yellow powder; yield 85%; m.p.:
4
2 30 6 3
3
3
1
64–166 °C; FT-IR (KBr): v
̄
= 1627 (C=N imine), 1566
20 cm 1 N aqueous HCl solution, 20 cm saturated aque-
−
1
1
3
(
(
(
C=N triazine), 1365 (C–O–Ar triazine) cm ; H NMR
ous NaHCO solution, and 20 cm salt water. The organic
3
300 MHz, DMSO-d ): δ=8.61 (3H, s, HC=N imine), 7.99
layer was dried over Na SO , ꢀltered, and evaporated under
6
2
4
6H, d, J=6.8 Hz, ArH), 7.33 7.44 (12H, m, ArH), 7.22 (9H,
reduced pressure. Finally, the tris-β-lactams 8a–8g and
d, J=7.2 Hz, ArH) ppm.
9a–9g were obtained by recrystallization from n-hexane.
1
, 1 ′ , 1 ″ ‑ [ [ ( 1 , 3 , 5 ‑Tr i a z i n e ‑ 2 , 4 , 6 ‑ t r i y l ) t r i s ( ox y ) ] ‑
4 , 4 ′ , 4 ″ ‑ [ [ ( 1 , 3 , 5 ‑Tr i a z i n e ‑ 2 , 4 , 6 ‑ t r i y l ) t r i s ( ox y ) ] ‑
tris(benzene‑1,4‑diyl)]tris[3‑phenoxy‑1‑(p‑tolyl)azeti‑
din‑2‑one] (8a, C H N O ) White powder; yield 70%;
tris(benzene‑1,4‑diyl)]tris[N‑(naphthalen‑1‑yl)methan‑
imine] (5g, C H N O ) White powder; yield 70%; m.p.:
5
4
36
6
3
69 54 6 9
1
94–196 °C; FT-IR (KBr): v
̄
= 1620 (C=N imine), 1566
m.p.: 129–131 °C; FT-IR (KBr): v̄=1759 (C=O β-lactam),
−
1
1
−1 1
(
(
(
C=N triazine), 1365 (C–O–Ar triazine) cm ; H NMR
1566 (C=N triazine), 1373 (C–O–Ar triazine) cm ; H
300 MHz, DMSO-d ): δ=8.70 (3H, s, HC=N imine), 8.28
NMR (300 MHz, DMSO-d ): δ = 7.35 (6H, d, J = 7.3 Hz,
6
6
2H, d, J = 7.2 Hz, ArH), 8.14 (6H, d, J = 7.0 Hz, ArH),
ArH), 7.13–7.21 (21H, m, ArH), 6.85–6.95 (6H, m, ArH),
6.81 (6H, d, J=6.6 Hz, ArH), 5.89 (3H, d, J=4.5 Hz, H-3
β-lactam), 5.71 (3H, d, J=4.5 Hz, H-4 β-lactam), 2.22 (9H,
7
.92 (2H, d, J=6.9 Hz, ArH), 7.78 (2H, d, J=7.5 Hz, ArH),
7
.47–7.53 (9H, m, ArH), 7.25–7.44 (9H, m, ArH), 7.12 (3H,
1
3
d, J=5.7 Hz, ArH) ppm.
s, CH ) ppm; C NMR (100 MHz, DMSO-d ): δ = 172.7
3 6
(
C−O triazine), 162.5 (C=O β-lactam), 156.3, 151.1, 134.2,
1
, 1 ′ , 1 ″ ‑ [ [ ( 1 , 3 , 5 ‑Tr i a z i n e ‑ 2 , 4 , 6 ‑ t r i y l ) t r i s ( ox y ) ] ‑
133.7, 131.0, 129.3, 121.9, 121.2, 119.7, 117.1, 117.0,
115.3 (aromatic carbons), 80.6 (C-3 β-lactam), 60.0 (C-4
tris(benzene‑1,4‑diyl)]tris[N‑(4‑chlorophenyl)methan‑
imine] (5h, C H Cl N O ) White powder; yield 82%; m.p.:
β-lactam), 20.4 (CH ) ppm.
4
2
27
3
6
3
3
1
96–198 °C; FT-IR (KBr): v̄ = 1627 (C=N imine), 1566
−
1 1
(
(
C=N triazine), 1365 (C–O–Ar triazine) cm ; H NMR
4 , 4 ′ , 4 ″ ‑ [ [ ( 1 , 3 , 5 ‑Tr i a z i n e ‑ 2 , 4 , 6 ‑ t r i y l ) t r i s ( ox y ) ] ‑
tris(benzene‑1,4‑diyl)]tris[1‑(4‑methoxyphenyl)‑3‑phe‑
noxyazetidin‑2‑one] (8b, C H N O ) White powder;
300 MHz, DMSO-d ): δ=8.61 (3H, s, HC=N), 7.97 (6H,
6
d, J = 6.7 Hz, ArH), 7.40 (12H, d, J = 5.3 Hz, ArH), 7.24
6
9 54 6 12
1
3
(
6H, d, J=6.9 Hz, ArH) ppm; C NMR (75 MHz, DMSO-
yield 71%; m.p.: 125–127 °C; FT-IR (KBr): v̄ = 1759
d ): δ = 172.4 (C–O triazine), 160.8 (C=N imine), 132.5,
(C=O β-lactam), 1566 (C=N triazine), 1365 (C–O–Ar
6
−
1 1
1
31.8, 130.5, 123.7, 122.8, 122.5, 119.1, 116.2 (aromatic
triazine) cm ; H NMR (300 MHz, DMSO-d ): δ=7.33–
6
carbons) ppm.
7.38 (6H, m, ArH), 7.25–7.29 (6H, m, ArH), 7.16 (6H,
d, J = 4.4 Hz, ArH), 7.11 (6H, d, J = 7.5 Hz, ArH), 6.90–
6.95 (9H, m, ArH), 6.77–6.81 (6H, m, ArH), 5.87 (3H,
d, J = 4.8 Hz, H-3 β-lactam), 5.69 (3H, d, J= 4.8 Hz, H-4
1
, 1 ′ , 1 ″ ‑ [ [ ( 1 , 3 , 5 ‑Tr i a z i n e ‑ 2 , 4 , 6 ‑ t r i y l ) t r i s ( ox y ) ] ‑
tris(benzene‑1,4‑diyl)]tris[N‑(4‑bromophenyl)methan‑
imine] (5i, C H Br N O ) White powder; yield 80%; m.p.:
1
3
β-lactam), 3.69 (9H, s, OCH ) ppm; C NMR (100 MHz,
4
2
27
3
6
3
3
2
18–220 °C; FT-IR (KBr): v
̄
= 1627 (C=N imine), 1566
DMSO-d ): δ = 172.7 (C−O triazine), 162.1 (C=O
6
−
1 1
(
(
C=N triazine), 1365 (C–O–Ar triazine) cm ; H NMR
β-lactam), 156.3, 156.0, 151.1, 131.1, 129.9, 129.3, 129.2,
300 MHz, DMSO-d ): δ=8.61 (3H, s, HC=N imine), 7.97
6
1
3

Synthesis and evaluation of biological activities of tripodal imines and β‑lactams attached…
1
21.9, 121.2, 118.5, 115.3, 114.6 (aromatic carbons), 80.6
4 , 4 ′ , 4 ″ ‑ [ [ ( 1 , 3 , 5 ‑Tr i a z i n e ‑ 2 , 4 , 6 ‑ t r i y l ) t r i s ( ox y ) ] ‑
tris(benzene‑1,4‑diyl)]tris(3‑phenoxy‑1‑phenylazeti‑
din‑2‑one) (8f, C H N O ) White powder; yield 55%; m.p.:
(
C-3 β-lactam), 60.1 (C-4 β-lactam), 55.2 (OCH ) ppm.
3
6
6 48 6 9
4
, 4 ′ , 4 ″ ‑ [ [ ( 1 , 3 , 5 ‑Tr i a z i n e ‑ 2 , 4 , 6 ‑ t r i y l ) t r i s ( ox y ) ] ‑
121–123 °C; FT-IR (KBr): v̄=1759 (C=O β-lactam), 1566
−
1 1
tris(benzene‑1,4‑diyl)]tris[1‑(4‑ethoxyphenyl)‑3‑phe‑
(C=N triazine), 1373 (C–O–Ar triazine) cm ; H NMR
noxyazetidin‑2‑one] (8c, C H N O ) White powder;
(300 MHz, DMSO-d ): δ = 7.83–7.93 (12 H, m, ArH),
7
2
60
6
12
6
yield 65%; m.p.: 132–134 °C; FT-IR (KBr): v
̄
= 1759
7.44–7.51 (10H, m, ArH), 7.24–7.34 (16H, m, ArH), 6.87–
(
C=O β-lactam), 1566 (C=N triazine), 1365 (C–O–Ar tria-
7.00 (4H, m, ArH), 5.86 (3H, d, J=4.5 Hz, H-3 β-lactam),
−
1
1
13
zine) cm ; H NMR (300 MHz, DMSO-d ): δ=7.35 (6H,
5.80 (3H, d, J = 4.5 Hz, H-4 β-lactam) ppm; C NMR
6
d, J=7.5 Hz, ArH), 7.26 (6H, d, J=7.6 Hz, ArH), 7.14–7.21
(100 MHz, DMSO-d ): δ = 173.0 (C−O triazine), 163.2
6
(
9H, m, ArH), 7.11 (6H, d, J=7.9 Hz, ArH), 6.90–6.94 (6H,
(C=O β-lactam), 154.5, 151.6, 133.6, 131.4, 129.8, 129.0,
128.1, 126.6, 125.5, 119.2, 118.2, 108.5 (aromatic carbons),
84.9 (C-3 β-lactam), 62.8 (C-4 β-lactam) ppm.
m, ArH), 6.77–6.82 (6H, m, ArH), 5.87 (3H, d, J=5.4 Hz,
H-3 β-lactam), 5.67 (3H, d, J=5.4 Hz, H-4 β-lactam), 3.90–
3
.98 (m, 6H, OCH ), 1.25 (9H, t, J = 6.8 Hz, CH ) ppm;
2
3
1
3
C NMR (101 MHz, DMSO-d ): δ=172.7 (C−O triazine),
4 , 4 ′ , 4 ″ ‑ [ [ ( 1 , 3 , 5 ‑Tr i a z i n e ‑ 2 , 4 , 6 ‑ t r i y l ) t r i s ( ox y ) ] ‑
tris(benzene‑1,4‑diyl)]tris[1‑(naphthalen‑1‑yl)‑3‑phe‑
noxyazetidin‑2‑one] (8g, C H N O ) White powder; yield
6
1
1
62.1 (C=O β-lactam), 156.3, 155.3, 151.1, 131.1, 129.8,
29.3, 129.2, 121.9, 121.2, 118.5, 115.3, 115. (aromatic
7
8 54 6 9
carbons), 80.6 (C-3 β-lactam), 63.2 (OCH ), 60.1 (C-4
60%; m.p.: 130–132 °C; FT-IR (KBr): v̄ = 1766 (C=O
2
β-lactam), 14.5 (CH ) ppm.
β-lactam), 1566 (C=N triazine), 1365 (C–O–Ar tria-
3
−
1 1
zine) cm ; H NMR (300 MHz, DMSO-d ): δ=8.25 (3H,
6
4
, 4 ′ , 4 ″ ‑ [ [ ( 1 , 3 , 5 ‑Tr i a z i n e ‑ 2 , 4 , 6 ‑ t r i y l ) t r i s ( ox y ) ] ‑
tris(benzene‑1,4‑diyl)]tris[1‑[4‑(N,N‑diethylamino)phenyl]‑
‑phenoxyazetidin‑2‑one] (8d, C H N O ) Gray pow-
d, J = 6.8 Hz, ArH), 7.91–7.96 (3H, m, ArH), 7.78–7.84
(3H, m, ArH), 7.64 (12H, d, J = 8.4 Hz, ArH), 7.43–7.48
(6H, m, ArH), 7.26–7.32 (6H, m, ArH), 7.15–7.21 (6H,
m, ArH), 6.92–7.02 (9H, m, ArH), 5.73 (3H, d, J=4.8 Hz,
3
7
8 75 9 9
der; yield 60%; m.p. 121–123 °C; FT-IR (KBr): v
̄
= 1751
(
C=O β-lactam), 1566 (C=N triazine), 1365 (C–O–Ar tria-
H-3 β-lactam), 5.62 (3H, d, J=4.8 Hz, H-4 β-lactam) ppm;
−1
1
13
zine) cm ; H NMR (300 MHz, DMSO-d ): δ=7.31–7.39
C NMR (100 MHz, DMSO-d ): δ=172.7 (C–O triazine),
6
6
(
6H, m, ArH), 7.08–7.17 (15H, m, ArH), 6.83–6.97 (6H, m,
ArH), 6.75–6.82 (6H, m, ArH), 6.56–6.64 (6H, m, ArH), 5.82
3H, d, J=4.5 Hz, H-3 β-lactam), 5.62 (3H, d, J=4.5 Hz,
H-4 β-lactam), 3.18–3.27 (12H, m, NCH ), 0.94–1.03 (18H,
163.5 (C=O β-lactam), 156.7, 151.4, 133.7, 131.4, 131.0,
129.7, 128.7, 128.1, 127.1, 126.6, 126.4, 125.5, 124.2,
122.2, 121.9, 119.2, 115.3, 114.5 (aromatic carbons), 84.8
(C-3 β-lactam), 62.8 (C-4 β-lactam) ppm.
(
2
1
3
m, CH ) ppm; C NMR (100 MHz, DMSO-d ): δ=172.7
3
6
(
C=O triazine), 161.4 (C=O β-lactam), 156.4, 151.0, 144.6,
4 , 4 ′ , 4 ″ ‑ [ [ ( 1 , 3 , 5 ‑Tr i a z i n e ‑ 2 , 4 , 6 ‑ t r i y l ) t r i s ( ox y ) ] ‑
tris(benzene‑1,4‑diyl)]tris[3‑(naphthalen‑2‑yloxy)‑1‑(p‑tolyl)‑
azetidin‑2‑one] (9a, C H N O ) White powder; yield 65%;
1
31.4, 129.3, 125.3, 121.8, 121.2, 118.7, 115.2, 111.8, 111.7
(aromatic carbons), 80.5 (C-3 β-lactam), 60.0 (C-4 β-lactam),
8
1 60 6 9
4
3.6 (NCH ), 12.3 (CH ) ppm.
m.p.: 139–141 °C; FT-IR (KBr): v̄=1759 (C=O β-lactam),
2
3
−
1 1
1
566 (C=N triazine), 1365 (C–O–Ar triazine) cm ; H
4
, 4 ′ , 4 ″ ‑ [ [ ( 1 , 3 , 5 ‑Tr i a z i n e ‑ 2 , 4 , 6 ‑ t r i y l ) t r i s ( ox y ) ] ‑
NMR (300 MHz, DMSO-d ): δ = 7.76 (3H, d, J = 8.0 Hz,
6
tris(benzene‑1,4‑diyl)]tris[1‑(2,4‑dimethoxyphenyl)‑
‑phenoxyazetidin‑2‑one] (8e, C H N O ) White pow-
ArH), 7.68 (6H, dd, J=12.5, 6.7 Hz, ArH), 7.37–7.40 (7H,
m, ArH), 7.29–7.33 (5H, m, ArH), 7.22–7.27 (8H, m, ArH),
7.14–7.18 (6H, m, ArH), 7.01–7.07 (6H, m, ArH), 6.88–6.96
(4H, m, ArH), 6.03 (3H, d, J=4.5 Hz, H-3 β-lactam), 5.81
3
7
2 60 6 15
der; yield 61%; m.p.: 107–109 °C; FT-IR (KBr): v̄ =1759
(
C=O β-lactam), 1566 (C=N triazine), 1365 (C–O–Ar tria-
−
1 1
zine) cm ; H NMR (300 MHz, DMSO-d ): δ=7.63 (4H,
(3H, d, J=4.5 Hz, H-4 β-lactam), 2.22 (9H, s, CH ) ppm;
6
3
1
3
d, J = 6.6 Hz, ArH), 7.42–7.49 (3H, m, ArH), 7.33–7.37
C NMR (100 MHz, DMSO-d ): δ=172.6 (C−O triazine),
6
(
5H, m, ArH), 7.11–7.16 (6H, m, ArH), 7.02–7.09 (6H, m,
162.3 (C=O β-lactam), 154.0, 151.0, 134.1, 133.7, 133.6,
131.0, 129.7, 129.3, 129.1, 128.9, 127.4, 126.7, 126.5,
124.1, 121.2, 117.8, 117.1, 109.0 (aromatic carbons), 80.6
ArH), 6.86–6.90 (3H, m, ArH), 6.81 (4H, d, J=7.4 Hz, ArH),
.55 (6H, d, J=8.7 Hz, ArH), 5.86 (3H, d, J=4.5 Hz, H-3
β-lactam), 5.81 (3H, d, J=4.5 Hz, H-4 β-lactam), 3.71 (9H,
6
(C-3 β-lactam), 60.0 (C-4 β-lactam), 20.4 (CH ) ppm.
3
1
3
s, OCH ), 3.70 (9H, s, OCH ) ppm; C NMR (100 MHz,
3
3
DMSO-d ): δ=172.7 (C=O triazine), 163.7 (C=O β-lactam),
4 , 4 ′ , 4 ″ ‑ [ [ ( 1 , 3 , 5 ‑Tr i a z i n e ‑ 2 , 4 , 6 ‑ t r i y l ) t r i s ( ox y ) ] ‑
tris(benzene‑1,4‑diyl)]tris[1‑(4‑methoxyphenyl)‑3‑(naph‑
thalen‑2‑yloxy)azetidin‑2‑one] (9b, C H N O ) White
6
1
58.4, 156.3, 152.6, 150.8, 132.2, 131.1, 129.3, 124.8, 121.7,
1
20.9, 117.0, 115.2, 105.1, 99.6 (aromatic carbons), 81.6
8
1 60 6 12
(
(
C-3 β-lactam), 63.4 (C-4 β-lactam), 55.7 (OCH ), 55.3
powder; yield 60%; m.p.: 135–137 °C; FT-IR (KBr):
3
OCH ) ppm.
v̄ = 1759 (C=O β-lactam), 1566 (C=N triazine), 1365
3
1
3

M. Bashiri et al.
−
1 1
(
C–O–Ar triazine) cm ; H NMR (300 MHz, DMSO-d ):
FT-IR (KBr): v
̄
= 1759 (C=O β-lactam), 1566 (C=N tria-
6
−
1 1
δ=7.76 (3H, d, J=7.9 Hz, ArH), 7.60–7.70 (6H, m, ArH),
zine), 1365 (C–O–Ar triazine) cm ; H NMR (300 MHz,
7
.38 (12H, d, J = 8.3 Hz, ArH), 7.29 (12H, d, J = 9.3 Hz,
DMSO-d ): δ = 7.66 (9H, d, J = 8.0 Hz, ArH), 7.36–7.42
6
ArH), 7.02–7.10 (6H, m, ArH), 6.94 (6H, d, J = 7.5 Hz,
ArH), 6.03 (3H, d, J=4.8 Hz, H-3 β-lactam), 5.79 (3H, d,
(9H, m, ArH), 7.34 (9H, d, J =5.7 Hz, ArH), 7.03 (3H, d,
J=6.7 Hz, ArH), 6.89–6.98 (6H, m, ArH), 6.57–6.69 (6H,
m, ArH), 6.01 (3H, d, J=5.1 Hz, H-3 β-lactam), 5.91 (3H,
1
3
J = 4.8 Hz, H-4 β-lactam), 3.68 (9H, s, OCH ) ppm;
C
3
1
3
NMR (100 MHz, DMSO-d ): δ = 172.6 (C−O triazine),
d, J=5.1 Hz, H-4 β-lactam), 3.71 (18H, s, CH ) ppm;
C
6
3
1
1
1
61.9 (C=O β-lactam), 156.0, 154.0, 151.0, 133.6, 131.0,
29.9, 129.3, 129.1, 128.9, 127.4, 126.7, 126.5, 124.1,
21.2, 118.5, 117.8, 114.6, 109.0 (aromatic carbons), 80.6
NMR (100 MHz, DMSO-d ): δ = 172.6 (C−O triazine),
6
163.5 (C=O β-lactam), 158.5, 154.0, 152.6, 150.7, 133.6,
133.6, 131.0, 129.3, 128.8, 127.4, 126.7, 126.4, 124.8,
124.0, 122.3, 120.9, 117.9, 108.8, 105.1, 99.6 (aromatic
carbons), 81.6 (C-3 β-lactam), 63.4 (C-4 β-lactam), 55.8
(OCH ), 55.3 (OCH ) ppm.
(
C-3 β-lactam), 60.1 (C-4 β- lactam), 55.2 (OCH ) ppm.
3
4
, 4 ′ , 4 ″ ‑ [ [ ( 1 , 3 , 5 ‑Tr i a z i n e ‑ 2 , 4 , 6 ‑ t r i y l ) t r i s ( ox y ) ] ‑
tris(benzene‑1,4‑diyl)]tris[1‑(4‑ethoxyphenyl)‑3‑(naphtha‑
3
3
len‑2‑yloxy)azetidin‑2‑one] (9c, C H N O ) White powder;
4 , 4 ′ , 4 ″ ‑ [ [ ( 1 , 3 , 5 ‑Tr i a z i n e ‑ 2 , 4 , 6 ‑ t r i y l ) t r i s ( ox y ) ] ‑
tris(benzene‑1,4‑diyl)]tris[3‑(naphthalen‑2‑yloxy)‑1‑phe‑
nylazetidin‑2‑one] (9f, C H N O ) White powder; yield
8
4 66 6 12
yield 50%; m.p.: 115–117 °C; FT-IR (KBr): v̄=1759 (C=O
β-lactam), 1566 (C=N triazine), 1365 (C–O–Ar triazine)
7
8 54 6 9
−
1 1
cm ; H NMR (300 MHz, DMSO-d ): δ = 7.80 (6H, d,
50%; m.p.: 148–150 °C; FT-IR (KBr): v̄ = 1759 (C=O
6
J=7.3 Hz, ArH), 7.64–7.72 (6H, m, ArH), 7.39–7.45 (9H,
m, ArH), 7.29–7.37 (12H, m, ArH), 7.08 (6H, d, J=7.1 Hz,
ArH), 6.91–6.96 (6H, m, ArH), 6.04 (3H, d, J=4.8 Hz, H-3
β-lactam), 5.79 (3H, d, J=4.8 Hz, H-4 β-lactam), 3.87–3.97
β-lactam), 1566 (C=N triazine), 1373 (C–O–Ar triazine)
−
1 1
cm ; H NMR (300 MHz, DMSO-d ): δ = 7.77 (5H, d,
6
J=6.6 Hz, ArH), 7.64–7.71 (6H, m, ArH), 7.39–7.45 (10H,
m, ArH), 7.32–7.37 (13H, m, ArH), 7.03–7.14 (10H, m,
ArH), 6.87–6.97 (4H, m, ArH), 6.06 (3H, d, J = 4.5 Hz,
H-3 β-lactam), 5.83 (3H, d, J=4.5 Hz, H-4 β-lactam) ppm;
1
3
(
(
(
6H, m, OCH ), 1.22–1.29 (9H, m, CH ) ppm; C NMR
2
3
100 MHz, DMSO-d ): δ = 172.7 (C−O triazine), 161.9
6
1
3
C=O β-lactam), 155.3, 154.1, 151.1, 133.6, 131.0, 129.8,
C NMR (100 MHz, DMSO-d ): δ=172.6 (C–O triazine),
6
1
1
29.3, 129.2, 129.0, 127.4, 126.8, 126.5, 124.1, 121.2,
18.6, 117.9, 115.1, 109.0 (aromatic carbons), 80.7 (C-3
162.7 (C=O β-lactam), 154.0, 151.1, 136.6, 133.6, 130.9,
129.4, 129.1, 128.9, 127.4, 126.7, 126.5, 124.5, 124.1,
122.3, 121.2, 117.9, 117.1, 109.1 (aromatic carbons), 80.6
(C-3 β-lactam), 60.1 (C-4 β-lactam) ppm.
β-lactam), 63.2 (OCH ), 60.2 (C-4 β-lactam), 14.5 (CH )
2
3
ppm.
4
, 4 ′ , 4 ″ ‑ [ [ ( 1 , 3 , 5 ‑Tr i a z i n e ‑ 2 , 4 , 6 ‑ t r i y l ) t r i s ( ox y ) ] ‑
4 , 4 ′ , 4 ″ ‑ [ [ ( 1 , 3 , 5 ‑Tr i a z i n e ‑ 2 , 4 , 6 ‑ t r i y l ) t r i s ( ox y ) ] ‑
tris(benzene‑1,4‑diyl)]tris[1‑(naphthalen‑1‑yl)‑3‑(naphtha‑
len‑2‑yloxy)azetidin‑2‑one] (9g, C H N O ) White pow-
tris(benzene‑1,4‑diyl)]tris[1‑[4‑(N,N‑diethylamino)‑
phenyl]‑3‑(naphthalen‑2‑yloxy)azetidin‑2‑one] (9d,
C H N O ) White powder; yield 55%; m.p.: 112–114 °C;
9
0 60 6 9
der; yield 53%; m.p.: 143–145 °C; FT-IR (KBr): v̄ =1766
9
0 81 9 9
FT-IR (KBr): v
̄
= 1766 (C=O β-lactam), 1566 (C=N tria-
(C=O β-lactam), 1566 (C=N triazine), 1365 (C–O–Ar tria-
−
1
1
−1 1
zine), 1365 (C–O–Ar triazine) cm ; H NMR (300 MHz,
zine) cm ; H NMR (300 MHz, DMSO-d ): δ=7.30–7.39
6
DMSO-d ): δ = 7.76 (3H, d, J = 6.4 Hz, ArH), 7.63–7.71
(25H, m, ArH), 7.20 (6H, d, J = 7.1 Hz, ArH), 7.10–7.17
(12H, m, ArH), 6.88–6.94 (6H, m, ArH), 6.81 (5H, d,
J = 6.7 Hz, ArH), 5.91 (3H, d, J = 5.1 Hz, H-3 β-lactam),
6
(
6H, m, ArH), 7.37–7.43 (9H, m, ArH), 7.27–7.33 (6H, m,
ArH), 7.12–7.18 (6H, m, ArH), 6.99–7.08 (6H, m, ArH),
1
3
6
.87–6.95 (3H, m, ArH), 6.56–6.65 (6H, m, ArH), 5.96 (3H,
5.73 (3H, d, J = 5.1 Hz, H-4 β-lactam) ppm; C NMR
d, J =4.8 Hz, H-3 β-lactam), 5.72 (3H, d, J= 4.8 Hz, H-4
(101 MHz, DMSO-d ): δ = 173.0 (C−O triazine), 163.3
6
β-lactam), 3.14–3.26 (12H, m, NCH ), 0.94–0.98 (18H,
(C=O β-lactam), 154.5, 151.7, 136.6, 133.9, 133.8, 133.6,
131.5, 129.9, 129.4, 129.0, 128.1, 127.6, 127.1, 126.6,
126.4, 125.5, 124.2, 122.1, 121.2, 119.2, 118.2, 117.1,
115.3, 108.6 (aromatic carbons), 80.6 (C-3 β-lactam), 60.1
(C-4 β-lactam) ppm.
2
1
3
m, CH ) ppm; C NMR (100 MHz, DMSO-d ): δ=172.7
3
6
(
C=O triazine), 161.2 (C=O β-lactam), 154.1, 150.9, 144.6,
1
1
33.6, 131.4, 131.4, 129.3, 128.8, 127.4, 126.7, 126.4,
25.3, 124.0, 121.2, 118.7, 117.9, 111.8, 108.9 (aromatic
carbons), 80.5 (C-3 β-lactam), 60.0 (C-4 β-lactam), 43.6
(
NCH ), 12.2 (CH ) ppm.
2 3
Cytotoxicity assay method of tris‑imines 5a–5i
4
, 4 ′ , 4 ″ ‑ [ [ ( 1 , 3 , 5 ‑Tr i a z i n e ‑ 2 , 4 , 6 ‑ t r i y l ) t r i s ( ox y ) ] ‑
tris(benzene‑1,4‑diyl)]tris[1‑(2,4‑dimethoxyphenyl)‑3‑
n a p h t h a l e n ‑ 2 ‑ y l o x y ) a z e t i d i n ‑ 2 ‑ o n e ] ( 9 e ,
Cytotoxicity was evaluated using HeLa and MCF-7 cell
lines, respectively. For this propose, cell lines were sub-
cultured in complete media containing high glucose
(
C H N O ) White powder; yield 55%; m.p.: 123–125 °C;
8
4 66 6 15
1
3

Synthesis and evaluation of biological activities of tripodal imines and β‑lactams attached…
Dulbecco’s modiꢀed Eagle’s medium (DMEM, Sigma-
Aldrich) supplemented with 10% heat-inactivated fetal
bovine serum (FBS, Gibco, Thermoꢀsher Scientiꢀc) in the
presence of 1% penicillin/streptomycin (Gibco, Thermo
l-glutamine and phenol red, without bicarbonate) (Sigma,
USA) was prepared and buꢁered at pH 7.0 with 0.165 mol
of 3-(N-morpholino)propanesulfonic acid (MOPS) (Sigma-
Aldrich, Steinheim, Germany). Serial dilutions of the com-
3
ꢀ
sher Scientiꢀc) at 37.0 °C under a humidiꢀed atmosphere
pounds (0.25–256 µg/cm ) were prepared in 96-well micro-
of 5% CO . Then, cells were trypsinized with 0.25% pre-
titre trays using RPMI-1640 media (Sigma, St. Louis, USA)
buꢁered with MOPS (Sigma, St. Louis, USA). Double dilu-
tions of ꢂuconazole were also prepared for each of the tested
2
warmed trypsin (Gibco, Thermo Fisher Scientiꢀc), fol-
4
lowed by seeding 1.5 and 1.0 × 10 cells/well for HeLa
3
and MCF-7 in to 96-well plates overnight in the above
fungi with the ꢀnal concentration of 0.125–64 µg/cm . Stock
condition. In the next day, the medium was replaced by
inoculums were prepared by suspending three colonies of
3
3
1
1
00 mm fresh culture medium containing 25, 50, 75, and
the examined C. albicans in 5 cm sterile 0.85% NaCl, and
00 µM 5a–5i. After 24 and 72 h of incubation, cells were
adjusting the turbidity of the inoculums to 0.5 McFarland
standard at 630 nm wavelength (this yields stock suspen-
washed once with PBS and incubated in fresh medium
3
6
3
containing 0.5 mg/cm of MTT solution. The plates were
sion of 1–5×10 cells/cm ). For A. fumigatus, conidia were
recovered from the 7-day-old cultures grown on potato dex-
trose agar by a wetting loop with Tween 20. The collected
conidia were transferred in sterile saline and their turbid-
ity was adjusted to optical density of 0.09–0.11 that yields
covered by aluminum foil and incubated in incubator under
CO pressure for 4 h. Thereafter, the culture medium was
2
removed, and the formazan crystals were dissolved with
3
1
00 mm solubilizing solution (40.0% (v/v) DMF, 16.0%
6
3
(
w/v) SDS, pH ∼ 4.7). Finally, the absorbance of the solu-
tion was recorded in wavelength of 570 nm via plate reader
Polar Star Omega, BMG LABTECh, Germany). The cell
viability was determined as follows:
0.4–5 × 10 conidia/cm . Initial fungal suspensions were
prepared by making a 1/50 and 1/1000 dilution with RPMI
of the stock suspension for molds and yeasts, respectively.
(
3
After addition of 0.1 cm of the inoculums to the wells, the
trays were incubated at 30 °C for 24–48 h in a humid atmos-
A Sample − A Blank
A Negative control − A Blank
3
Cell viability (%) =
× 100
phere. 200 mm of the uninoculated medium was included
as a sterility control (blank). In addition, growth controls
(medium with inoculums and 5% (v/v) without the synthetic
compounds or ꢂuconazole) were also included. The growth
in each well was compared with that of the growth control
well. MICs were visually determined and deꢀned as the low-
est concentration of the compounds that produced no visible
growth. Each experiment was performed in triplicate.
Antibacterial activity test method for tris β‑lactams
a–8g and 9a–9g
8
Sterile blank disks (Padtanteb, 6.4 mm diameter) each
containing 10 μg of the test compound in DMSO solution
was placed on the surface of streaked Muller–Hinton agar
plates (Merck, German) inoculated with a Gram-positive
bacterium (Staphylococcus aureus ATCC 25923), two
genera of Gram-negative bacteria (Escherichia coli ATCC
Determination of the reducing activity of the stable
radical DPPH
The free radical-scavenging capacity of the synthesized
compounds was evaluated by measuring the bleaching of the
2
5922, Pseudomonas aeruginosa ATCC 27,853), and one
3
stable radical DPPH. In this method, 180 mm of a metha-
capsulated Gram-negative bacterium (Klebsiella pneumo-
nia ATCC 700881). Amikacin (10 μg), oxacillin (10 μg),
gentamicin (10 μg), and streptomycin (10 μg) were used
as the reference antibiotics. The plates were incubated at
3
nolic solution of DPPH (110 μM) was added to 20 mm of
tested compounds (900, 600, 300, 150, 100, 50, and 25 µM
dissolved in DMSO) in a 96-well plate. The plate was incu-
bated for 20 min at room temperature. The absorbance was
assessed spectrophotometrically at 517 nm and the percent-
age of activity was calculated. The IC (inhibition concen-
3
7 ℃ for 24 h and inhibition zones of bacterial growth
produced by the various agents were then measured (mm).
5
0
tration 50—the concentration of antioxidant which reduces
reactive species intensity by about 50%) was determined
using Curve Expert and the results were compared with
quercetin as the standard [35].
Antifungal testing of tris β‑lactams 8a–8g
and 9a–9g
Minimal inhibitory concentrations of the synthetic com-
pounds against standard species of the fungi were deter-
mined by the broth microdilution method as recommended
by the clinical and laboratory standards institute (CLSI),
with some modiꢀcations. Brieꢂy, the RPMI-1640 (with
1
3

M. Bashiri et al.
7
8
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a–8g and 9a–9g
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K562 myelogenous leukemia cells were grown in RPMI
640 medium supplemented with 10% fetal bovine serum,
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Gibco, Ashland, KY) at 37 °C incubator with 5% CO and
(
2
9
5% humidity. Conꢂuent cells with viability more than 95%
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bromide (MTT) colorimetric assay. Brieꢂy, 15×10 cells/
1
well in 96-well culture plates were seeded in triplicate and
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then treated with 10 mm of each compound at a ꢀnal con-
1
1
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centration of 0.1–200 µg/cm . The negative control was
cells treated with the solvent (dimethylsulfoxide, DMSO) at
concentrations equal to test wells. Cells treated with cispl-
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1
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atin were used as positive control. After incubation of cells
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(2005) Bioorg Med Chem Lett 15:1371
3
at 37 °C in a humidiꢀed CO incubator for 48 h, 10 mm
2
3
of 5 mg/cm of MTT solution (Sigma, St Louis, MO) was
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added to each well and incubation continued for another
2
1. Gao F, Wang T, Gao M, Zhang X, Liu Z, Zhao S, Xiao J (2019)
Eur J Med Chem 165:323
4
h. The plates were then centrifuged and after removing
3
the supernatant, 150 mm DMSO was added to dissolve
the formazan crystals. The absorbance was then read by
a microplate reader (BioTek, Winooski, VT) at 570 nm
with a reference wavelength of 630 nm. The percentage of
cell growth inhibition was calculated as follows: % Inhi-
bition = 100 − [(test absorbance/negative control absorb-
ance) × 100]. The half maximal inhibitory concentration
2
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Synthesis and evaluation of biological activities of tripodal imines and β‑lactams attached…
Aꢀliations
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Mohammad Bashiri · Aliasghar Jarrahpour · Banafsheh Rastegari · Aida Iraji · Cambyz Irajie ·
Zahra Amirghofran5 · Saeed Malek‑Hosseini · Mohammad Motamedifar · Mahtab Haddadi ·
,6
6
7,8
8
9
9
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Kamiar Zomorodian · Zahra Zareshahrabadi · Edward Turos
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1
Department of Chemistry, College of Sciences, Shiraz
University, 71946-84795 Shiraz, Iran
Shiraz HIV/AIDS Research, Center, Institute of Health,
Shiraz University of Medical Sciences, 71348-14336 Shiraz,
Iran
2
Diagnostic Laboratory Science and Technology Research
Center, School of Paramedical Science, Shiraz University
of Medical Sciences, 71348-14336 Shiraz, Iran
Department of Bacteriology and Virology, School
of Medicine, Shiraz University of Medical Sciences,
7
1348-14336 Shiraz, Iran
3
4
Central Research Laboratory, Shiraz University of Medical
Sciences, 71348-14336 Shiraz, Iran
Department of Medical Mycology and Parasitology, School
of Medicine, Shiraz University of Medical Sciences,
Department of Medical Biotechnology, School of Advanced
Medical Sciences and Technologies, Shiraz University
of Medical Sciences, 71348-14336 Shiraz, Iran
7
1348-14336 Shiraz, Iran
0
Department of Chemistry, Center for Molecular Diversity
in Drug Design, Discovery, and Delivery, University
of South Florida, 4202 E. Fowler Avenue, CHE 205, Tampa,
FL 33620, USA
5
6
Autoimmune Diseases Research Center, Shiraz University
of Medical Sciences, 71348-14336 Shiraz, Iran
Department of Immunology, Shiraz University of Medical
Sciences, 71348-14336 Shiraz, Iran
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3