Synthesis and biological evaluation of new 3-amino-2-azetidinone derivatives as anti-colorectal cancer agents

Article abstract of DOI:10.1039/c8md00147b

Several synthetic combretastatin A4 (CA-4) derivatives were recently prepared to increase the drug efficacy and stability of the natural product isolated from the South African tree Combretum caffrum. A group of ten 3-amino-2-azetidinone derivatives, as c

Full text of DOI:10.1039/c8md00147b

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Synthesis and Biological Evaluation of New 3-amino-2-azetidinone
Derivatives as anti-colorectal cancer agents
Farida Tripodia, Federico Dapiaggib, Fulvia Orsinib, Roberto Pagliarinb*, Guido Sellob* and Paola
Coccettia*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Several synthetic Combretastatin A4 (CA-4) derivatives were recently prepared to increase drug efficacy and stability of
the natural product isolated from South African tree Combretum caffrum. A group of ten 3-amino-2-azetidinone
derivatives, as Combretastatin A4 analogues, were selected through docking experiments, synthesized and tested for their
anti-proliferative activity against colon cancer SW48 cell line. These molecules, through the formation of amide bonds in
position 3, allow the synthesis of various derivatives that can modulate the activity with a great resistance to hydrolytic
conditions. The cyclization to obtain the 3-aminoazetidinone ring is highly diastereoselective and provides the trans
biologically active isomer under mild reaction conditions and with better yields than the 3-hydroxy-2-azetidinone
synthesis. All compounds showed IC50 values ranging between 14.0 and 564.2 nM, and the most active compound
showed inhibitory activity against tubulin polymerization in vitro, being a potential therapeutic agent against colon cancer.
www.rsc.org/
agents for a long time: the low water solubility and the
cis/trans CC double bound isomerization, which may occur
Introduction
Combretastatins are a group of polyhydroxylated stilbenes
isolated from the South African Combretum caffrum [1,2]. They
are natural occurring well known microtubule destabilizing
agents, that inhibit the polymerization of tubulin by binding to
colchicine binding site. Combretastatin A4 (CA-4, Fig. 1), the
main component of this family of natural products, is in
advanced clinical trials for cancer therapy because exhibits
potent anticancer activity against a panel of human cancer cells
including multi-drug resistant ones [3].
during storage and administration, causing a dramatic loss
of activity. Between the two stereoisomers, the cis form is
much more active than the trans one, due to the
pharmacophore of colchicine binding site, which
encompasses the trimethoxybenzene ring linked in cis to a
methoxy-containing arene [8]. The low solubility has been
overcome by the water-soluble phosphate prodrug [9] (Fig.
1), which is currently under investigation in human clinical
trials (phase III) as anticancer drug [4,9,10]. The low cis
isomer stability has been approached by designing a variety
of conformationally restricted cis-locked analogues. Many
structural modifications of Combretastatin A4 have been
proposed by incorporating the stilbene double bond into a
carbocyclic or heterocyclic moiety. Phenyl or triatomic and
pentaatomic carbocyclic derivatives have to be cited, as
well as nitrogen or oxygen containing heterocyclics [11],
Triazoles [12], Tetrazoles [13], Isooxazoles [14], Imidazoles
[15], Pyrazoles [16], Pyridines [17]. Other rings, such as
azetidinones, have been inserted between the two phenyl
groups of Combretastatin A4 [18,19]. In addition, trans-3,4-
disubstituted azetidinones show a strong anti-proliferative
activity against several cancer cell lines [20,21].
Combretastatin-like compounds bearing an azetidinone unit
between the A and B rings were presented by N.M. O'Boyle
and colleagues [22,23]. They firstly synthesized a few
Figure 1
CA-4 has antiangiogenic effects inducing apoptosis in
various human tumor cell lines [4–7], through a block of the
blood flow, vascular rupture and cellular necrosis. Two
problems, however, have limited their use as therapeutic
derivatives of trans 1,3,4 tri-aryl azetidinone 1 (Fig. 2) which
inhibited the polymerization of tubulin with a 6-fold rate of
inhibition in comparison with the natural compound CA-4
[22,23].
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We hypothesized the preparation of 10 derivatives
(compounds 11a-j) (Table 1), where the R residue deriving
from the acidic part of the amido group varies from the
acetic to the naphthoic acids. We included short chain,
aromatic, heteroaromatic, cyclic and polycyclic derivatives
with diverse functional groups.
Figure 2
We recently developed a trans-3-hydroxy-2-azetidinone,
bearing in position 1 the A ring and in position 4 the B ring
of CA-4 (compound 2) (Fig. 3) [24]. The racemic mixture of
this compound showed both high stability and solubility
(1700 µM) in aqueous media due to the presence of a
second hydroxyl group in position 3. It was synthetized
under thermodynamic control of the reaction at 100 °C with
Figure 4
First, we examined the docking of compounds 2a and 2b
showing anticancer activity in previous studies [24].
Molecular docking was performed generating 100 binding
poses for each compound. Considering the similarity of the
energies obtained from the molecular docking of these
compounds (Table S1 and S2), we performed a cluster
analysis on the 100 conformations obtained from each run
using an RMSD threshold of 2 Å. We considered the most
populated cluster of each compound as the best docked
conformation. Compound 2a (enantiomer SS) showed the
known interaction of trimethoxyphenyl group that made
contacts with Cysβ241 and Valβ238. In addition, it
interacted with Aspβ251 through hydrogen bonds.
Compound 2b (enantiomer RR) maintained the interaction
of the trimethoxyphenyl group with Cysβ241 and Valβ238.
In contrast, it did not interact with Aspβ251, suggesting that
the two enantiomers did not share the same docking
conformation.
Then, molecular docking studies were carried out on
compounds 11a-j in order to assess their ability to bind the
colchicine site of β tubulin.
RR enantiomers showed a low binding capability, displaying
a large number of clusters of different conformations.
Compound 11i resulted to be the most interesting
compound among the RR enantiomers, displaying a very
populated cluster containing 85% of the conformations.
Details regarding binding energies and clustering are
reported in Table S1.
a total yield of 33%. Compound
2 induced inhibition of
tubulin polymerization and subsequent G2/M arrest in
cancer cells [24]. At the same time, activation of AMP-
activated protein kinase (AMPK) and induction of apoptosis
were also reported [24]. Moreover, the drug displayed a
significant nanomolar cytotoxic activity against different
cancer cell lines, as well as a tumor growth delay in a mouse
xenograft model of colorectal cancer, confirming its
potential therapeutic action against colon cancer [25]. In
the present paper, we report the synthesis of compound
(Fig. 3), in which the hydroxyl group of compound was
substituted by an amino group, as well as a series of its
derivatives. Remarkably, the synthesis of compound was
easier than compound because in the Staudinger reaction
3
2
3
2
the trans closure of the azetidinone ring took place at room
temperature with a yield of 50% overall, with only few
traces of the cis isomer.
Much more interesting results were obtained from the
molecular docking of SS enantiomers. In this case most of
the docked compounds showed a higher populated cluster
with respect to RR compounds and binding energies around
-5 kcal mol-1 (Table S2). Compounds 11d
, 11f and 11g were
not suitable to bind within the colchicine site, due to the
steric hindrance of the R substituents of the amide group.
This was supported by highly diversified docked
Figure 3
In order to select derivatives of compound
3 which could
conformations. Compounds 11a, 11b, 11c, 11e, 11h and 11j
have a pronounced activity, we performed a preliminary
docking study before biological tests. The presence of the
amino group permits the easy preparation of a series of
derivatives by transforming the NH2 to a corresponding
amido group. The variability in size and functions of the
introduced substituents can be the source of valuable
insights into substrate binding site interactions.
shared the same orientation within the binding site (Fig. 5).
Like combretastatin compounds [8], the trimethoxyphenyl
group made contacts with Cysβ241 and Valβ238. Moreover,
amidic group in position 3 interacted with Metβ259
through a hydrogen bond (Fig. 6). The most interesting
compound, however, was 11i, showing a single cluster of
100 conformations.
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This compound showed a different docking pose with
respect to the aforementioned ones. It interacted with
Asnα101 and Aspβ251 through hydrogen bonds, while the
orientation of the trimethoxyphenyl group was towards
residues Cysβ241 and Valβ238 as in the previous cases (Fig.
7). Moreover, compound 11i showed a very similar docking
pose to compounds already studied by our group, namely
trans 3-Hydroxy-1,4-diaryl-2-azetidinone (2b) (Fig. 8)
In keeping with these results, the synthesis of all the
derivatives were performed.
observed between the amide group in position 3 and
Asnα101 as well as between the ketone oxygen of the
scaffold and Aspβ251. This interaction pattern was common
to compounds 11i and 2b
.
Figure 8 Superimposition of 11i (blue) and 2a (red) docking
poses.
Chemistry
In this work, we focused our attention on the preparation
of a small library of 3-amido-1,4-diaryl-2-azetidinones 11a-j
(Table 1), with the goal of improving the biological activity
and elucidating the biological effects by the introduction of
specific ligands useful to selectively target tumor cells.
Figure 5 Superimposition of docking poses of compounds
11a (blue), 11b (red), 11c (orange), 11e (yellow), 11h
(green), 11j (purple).
3,4-trans-3-Amino-4-(3-hydroxy-4-methoxyphenyl)-1-
(3,4,5-trimethoxyphenyl)-azetidin-2-one
as previously reported by the authors [24], via the
Staudinger approach between a properly selected imine
9 was synthesizes
6
and phthalylglycyl chloride as the ketene precursor
followed by hydrolysis with hydrazine dihydrochloride at 60
°C. The stereochemistry of the products obtained from the
Staudinger cycloaddition was affected by the experimental
conditions (such as solvent, temperature and order of
addition of the reagents) and by the type of the ketene
precursor: acetoxyacetyl chloride, or phthalylglycyl
chloride, used to introduce an OH group or a NH2 group,
respectively, at the position 3 of the azetidin-2-one. In the
latter case, the best results were obtained by dropwise
Figure 6 Interactions of the compound 11a. In the cartoon
tubulin α (orange) and β (cyan) subunits are represented.
Trimethoxyphenyl group made contacts with residues
Cysβ241 and Valβ238, as well as HB with residue Metβ259.
addition of a 4-fold excess of acid chloride
7 to a methylene
chloride solution of imine 6 and dry triethylamine at 0 °C,
followed by stirring at room temperature for twenty-four
hours. Hydrolysis of the Staudinger adduct with hydrazine
This interaction pattern was common to compounds 11a
,
,
11b, 11c, 11e 11h, 11j
.
afforded only trans–diastereoisomer
9 in 61% total yield
from the imine. A different approach [26], based on the use
of in-situ generated N-chloroamines as imines precursors,
was tested, without improving the yields of the reaction.
With respect to the previously reported 3-hydroxy-1,4-
diaryl-2-azetidinones, the 3-amino-1,4-diaryl-2-azetidinones
could a priori display a different and hopefully improved
activity, and the nitrogen atom at position 3 can be
derivatized to give stable amides, easily prepared by
reaction with a properly selected carboxylic acid, RCOOH. In
order to obtain a library of compounds able to interact with
the receptors in a variety of ways, R has been varied in term
of polarity, size, ability to act as a hydrogen bond acceptor
or donor and to give different types of interactions, namely
dipole-dipole, β-stacking and hydrophobic interactions.
Amides 11a-i were obtained by a five-steps synthesis. As
Figure 7 Interactions of the compound 11i. In the cartoon,
tubulin α (orange) and β (cyan) subunits were represented.
Trimethoxyphenyl group contacts with residues Cysβ241
and Valβ238. Moreover, two hydrogen bonds were
already described [24], imine
6
was obtained by reaction of
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compound
compound
4
6
and
was submitted to a Staudinger cyclization with
5
in anhydrous ethyl alcohol: the solid
obtained with total yields ranging from 60 to 81 depending
on the R substituents of the amide group. The synthesis of
the amides 11j required one additional step as tert-
butoxycarbonyl alanine was used as acylating reagent: the
removal of the protective tert-butoxycarbonyl moiety was
easily accomplished by treatment with trifluoroacetic acid
in methylene chloride.
phtalyl glycine chloride and was transformed in the β-
lactam , easily hydrolyzed to compound . 3,4-trans-3-
Amino-4-(3-hydroxy-4-methoxyphenyl)-1-(3,4,5-
trimethoxyphenyl)-azetidin-2-one was first added to an
8
9
9
acetonitrile solution of the selected carboxylic acid,
previously activated by reaction with N,N'-disuccinimidyl
carbonate (overnight at room temperature). A mixture of
the N,O-diacyl derivative 10 and amide 11 was obtained.
Pure samples of 11a-j were obtained by flash-
chromatograpy, whereas, the presence of N,O diacyl
derivatives 10a-j was recognized in the MS spectra of the
mixtures (10a-j and 11a-j) which were directly treated with
hydrazine hydrochloride at room temperature to hydrolyse
the ester moiety. The target amides 11a-j, were thus
Scheme 1. Reagents and conditions: a) anhydrous EtOH, b) Compounds 11a-j were tested against the SW48 human colon
Et3N/ CH2Cl2, c) NH2NH2.2HCl, Et3N, MeOH, 60 °C, d) N,N'- cancer cell line by testing their antiproliferative activity. They
Disuccinimidyl carbonate, Et3N, CH3CN, e) NH2NH2.2HCl, Et3N, retained nanomolar cytotoxic activity, and among them 11i,
MeOH r.t.
with a small alkyl group (methyl), showed the highest activity,
comparable to the activity of natural CA-4 and of the
previously reported 3-hydroxy azetidinone 2 (Table 1, Fig. S1).
Biological Results
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Activity was decreased by the introduction of a primary amino further reduced. The results of the activity tests were
group on the small alkyl chain in 11j, and, even more, by the consistent with the assessments from the docking study,
substitution of the methyl group with the more sterically where the compounds containing bulky aromatic residues did
demanding cyclohexyl group in 11h. Heteroaromatic rings, as not converge to a single structure.
in 11e and 11g, were approximately comparable to 11i. In the
presence of aromatic rings, as in 11a-11d, the activity was
CA-4 is known to act by inhibiting tubulin polymerization [1]. showed an IC50 value of 5.05 μM, higher than that of CA-4, but
Thus, we evaluated the effect of compound 11i, which showed indicative of the fact that one of the molecular targets of this
the highest activity against SW48 cells, on the in vitro active compound was tubulin, as reported for other
Entry
11a
Compound
IC50 (nM)
Entry
11f
Compound
IC50 (nM)
270.7±76.6
220.2±76.5
11b
384.2±117.3
11g
49.4±1.1
11c
108.4±11.6
11h
244.2±64.2
11d
564.2±61.7
11i
14.0±2.1
11e
18.6±1.7
11j
90.6±7.3
polymerization of purified tubulin, and CA-4 was used as a azetidinone compounds [24].
positive control (Table 2, Fig. S2). In the assembly assay, 11i
Table 1. Structures and IC50 of the indicated compounds in SW48 cell lines measured by MTT assay after 72 h of treatment. (0.1% DMSO
was
used
as
a
vehicle
control).
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irradiation (254 nm) or by exposition to Iodine vapors. 1H NMR
and 13C NMR spectra were recorded at 300 MHz and at 400
MHZ on Bruker Instruments (AC 300 UltrashieldTM 400), using
deuterochloroform solutions, unless otherwise specified and
Compound
11i
IC50 µM tubulin polimerization
5.05±1.2
1.32±0.2
CA-4
chemical shifts were represented as
δ-values relative to the
Table 2. In vitro inhibition of Tubulin Polymerization.
internal standard TMS. ESI-mass spectra were recorded on
Bruker Esquire 3000 Plus. The synthesized compounds
submitted to biological tests have a purity ≥95% determined by
HPLC (Phenomenex, Nucleosil 250x3.2 mm column, 5 µm, C18;
mobile phase 0.05 M phosphate buffer pH 7/acetonitrile, 7:3;
flow rate 1.5 mL/min) and they have been filtered in sterile
atmosphere with 0.20 µm filters.
Conclusions
Insertions of azetidinone rings into Combretastatin skeleton
have been valid modifications because they prevented the
isomerization of the cis double bond, while preserving in some
derivatives an activity comparable to the natural compound.
They made the natural compound more stable and the synthesis
provided good yields. In particular, the 3-aminoazetidinones,
compared to the 3-hydroxyazetidinones, which bear a hydroxyl
group in position 3 of the β-lactam ring, were synthesized with
higher yields since the Staudinger reaction almost exclusively
provided the 3,4-trans isomer. Furthermore, the derivatization
of the amino group furnished amide derivatives stable under
hydrolytic conditions. Remarkably, these new derivatives
showed a nanomolar anti-proliferative activity against SW48
cells, probably acting by inhibiting tubulin polymerization,
opening the route to a new class of potential therapeutic agents
against colon cancer.
3-Hydroxy-4-methoxybenzylidene-(3,4,5-
trimethoxyphenyl)amine (6). Under inert atmosphere,
compound 4 (1.9, 10.1 mmol) and compound 5 (1.5 g, 10.1
mmol) were dissolved together at room temperature with
stirring, in 10 mL Ethanol previously dried on molecular sieves.
The reaction was followed by TLC (pre-treatment of the plate
with CH2Cl2: Et3N 95: 5, then eluted with AcOEt: Esano 1: 1).
After 2 h, NMR analysis showed the formation of imine 6
observable also by the presence of a precipitate. The mixture
was filtered to collect the solid product. Filter was washed twice
with dichloromethane to dissolve the reaction product. The
solvent was removed and compound 6 was collected, pure
1
enough for the next step (yields = 93%). H-NMR (CDCl3): δ 8.41
Experimental Section
(1H, s), 7.63 (1H, s), 7.46 (1H, d, J = 7.2 Hz), 6.96 (1H, d, J = 7.2
Hz), 6.53 (2H, s), 5.77 (1H, s), 3.99 (3H, s), 3.92 (6H, s), 3.88 (3H,
s). EI-MS (m/z): 318 (M+).
Docking studies
Tubulin X-ray structure in complex with combretastatin was
obtained from the Protein Data Bank (PDB ID 5LYJ [8]). The
pocket analysis and the docking calculations were performed
only on one of the dimers in the asymmetric unit, labelled chain
A and B. Combretastatin was removed from the structure before
docking our compounds. Docking was carried out with Autodock
4.2 [27], employing a Lamarkian genetic algorithm [28]. 100
independent runs per molecule were performed. In each run, a
population of 50 individuals evolved along 27000 generations
and a maximum number of 25 million energy evaluations were
performed. The best fit (lowest docked energy) solutions of the
100 independent runs were stored for subsequent analysis. The
visual inspection of docked structures was carried out using
VMD [29].
(±)-3,4-trans-3-Amino-4-(3-hydroxy-4-methoxyphenyl)-1-(3,4,5-
trimethoxyphenyl)-azetidin-2-one ((±)-trans-9)
A solution of phthalylglycyl chloride (5.38 g, 24 mmol), in
anhydrous methylene chloride (7 mL) was added dropwise at 0
°C under nitrogen to a stirred solution of imine 6 (1.9 g, 6 mmol)
and TEA (8.3 mL, 60 mmol) in anhydrous methylene chloride (7
mL). The solution was maintained at 0 °C for 1 h and then
allowed to reach room temperature and stirred for 24 h. HCl (1
N) was added (40 mL), and the two phases were stirred for 40
min. The aqueuos phase was separated and extracted twice with
dichloromethane. Organic phases were washed with a saturated
solution of NaHCO3 dried with sodium sulfate, filtered, and
concentrated under reduced pressure, affording a brown oil
which was purified by flash column chromatography (silicagel;
eluent gradient of n-hexane/ethyl acetate from 7/3 to 4/6). The
isomer (±)-trans-8 only was isolated with 65% yield. The isomer
Chemistry
All commercially available reagents were purchased from Sigma-
Aldrich and used without further purification. Preparative
separations were usually performed by flash-column
chromatography on silica gel (Merck grade 9385). Thin-layer
chromatography were made on silica gel (Merck, 10x5 cm, Silica
gel 60 F254): zones were detected visually by ultraviolet
(±)-cis-8 was formed only in traces from the reaction. (±)-trans
-
8: 1H- NMR (CDCl3) δ 8−7.6 (8H, m), 7.29 (1H, dd, J1 = 8.4 Hz, J2
= 2.2 Hz), 7.19 (1H, d, J = 2.2 Hz), 7.00 (1H, d, J = 8.4 Hz), 6.57
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(2H, s), 5.28 (2H, s), 4.71 (2H, s), 3.85 (3H, s), 3.76 (3H, s), 3.70
(6H, s). 13C NMR (CDCl3): δ 167.46, 166.96, 165.41, 162.03,
140.09, 134.79, 134.53, 133.40, 132.15, 131.86, 128.37, 126.40,
125.08, 124.27, 123.87, 121.47, 113.64, 95.50, 67.78, 61.07,
56.24, 38.89. ESI-MS: m/z 691 (M+).
Hydrazine dihydrochloride (752.5 mg, 6.223 mmol) was added,
at 0 °C and under nitrogen to a stirred suspension of (±)-trans-8
(870 mg, 1.245 mmol) in methanol (10 mL). TEA (3.5 mL, 24.89
mmol) was then added dropwise. The mixture was allowed to
reach room temperature and then warmed at 50 °C for 5 h. The
solvent was removed at reduced pressure, and the residue was
anhydride under reduced pressure afforded diacyl derivative in
quantitative yields.
10i 1H NMR (300 MHz, CDCl3): δ ppm 7.19 (dd, 1H, J = 8.5, 1.7
Hz), 7.05 (d, 1H, J = 1.7 Hz), 6.95 (d, 1H, J = 8.5 Hz), 6.80 (d, 1H, J
=7.5 Hz), 6.48 (s, 2H), 4.94 (d, 1H, J = 1.7 Hz), 4.53 (dd, 1H, J
=7.4, 1.7 Hz), 3.81 (s, 3H), 3.75 (s, 3H), 3.69 (s, 6H), 2.28 (s, 3H),
2.06 (s, 3H). 13C (300 MHz, CDCl3): 171.45, 169.45, 164.88,
154.08, 152.16, 140.84, 135.12, 134.10, 129.46, 125.23, 121.82,
113.64, 95.73, 66.55, 63.27, 61.57, 56.67, 23.35, 21.25. MS (ESI):
m/z 481 (M+Na).
Spectroscopical data of compounds 11a-j
treated with
1
N
HCl (40 mL) and extracted with
N-(4-chlorobenzoil)-3,4-trans-3-amino-1-(3,4,5-
dichloromethane (3 × 10 mL). The aqueous phase was made
alkaline by 3 N NaOH and extracted with dichloromethane (3 ×
10 mL). The organic phase was dried (Na2SO4), and the solvent
was removed at reduced pressure. A solid was obtained, which
was purified by flash chromatography (silica gel; eluent n-
hexane/ethyl acetate, 2/8) to afford the stereoisomer (±)-trans-
9 (0.437 g, 1.17 mmol, 94% yield) as a yellow solid. 1H-NMR
(CDCl3): δ 6.92 (1H, s), 6.89 (2H, s), 6.54 (2H, s), 4.54 (1H, d, J =
2.2 Hz), 4.05 (1H, d, J = 2.2 Hz), 3.88 (3H, s), 3.76 (3H, s), 3.71
(6H, s). 13C NMR (CDCl3): δ 168.05, 153.65, 147.11, 146.51,
134.80, 133.80, 130.14, 118.14, 117.99, 112.33, 111.22, 95.38,
69.70, 66.70, 61.09, 56.22. ESI-MS: m/z 374 (M+).
trimethoxyphenyl)-4-(3-hydroxy-4-methoxyphenyl)-azetidin-2-
one.
11a (65 % yield). 1H NMR (400 MHz, CDCl3): δ ppm 7.79 (d, 2H,
J = 7.5 Hz), 7.43 (d, 2H, J =7.5 Hz), 7.23 (d, 1H, J = 6.5 Hz, NH),
6.98 (d, 1H, J = 2.5 Hz), 6.94 (dd, 1H, J = 7.5, 2.5 Hz), 6.88 (d, 1H,
J = 7.5 Hz), 6.55 (s, 2H), 5.00 (d, 1H, J = 2.0 Hz), 4.81 (dd, 1H, J =
7.5 Hz, 2.0 Hz), 3.92 (s, 3H), 3.77 (s, 3H), 3.72 (s, 6H). 13C (400
MHz, CDCl3): 168.13, 165.29, 154.31, 147.43, 146.57, 136.15,
135.06, 130.76, 130.86, 129.90, 129.15, 118.92, 116.33, 116.14,
112.57, 111.63, 95.72, 66.83, 63.93, 61.51, 56.25, 56.13, 51.44.
MS (ESI): m/z 535-537 (M+Na).
10a. MS (ESI): m/z 673-675 (M+Na).
General procedure for the preparation of compounds 11a-h and
11j
N-(4-fluorobenzoyl)-3,4-trans-3-amino-1-(3,4,5-
trimethoxyphenyl)-4-(3-hydroxy-4-methoxyphenyl)-azetidin-2-
one
To a stirred solution of the selected carboxylic acid RCOOH (0.88
mmols) in dry acetonitrile (6 mL), N,N′-Disuccinimidyl carbonate
DSC (0.88 mmols) and dry triethylamine ( 1.603 mmols) were
added at 0 °C, under nitrogen atmosphere. The stirring was
continued overnight and 3,4-trans-3-amino-4-(3-hydroxy-4-
11b (66 % yield). 1H NMR (400 MHz, CDCl3): δ ppm 7.90 (dd,
2H, J = 8.5, JH-F = 5.5 Hz), 7.82 (d, 1H, J = 7.4 Hz, NH), 7.08 (dd,
2H, J = 8.5, JH-F = 8.4 Hz), 6.95 (d, 1 H, J =1.5 Hz), 6.90 (dd, 1H, J
= 8.1, 1.5 Hz), 6.84 (d, 1H, J = 8.1 Hz), 6.52 (s, 2H), 5.01 (s, 1H),
4.85 (d, 1 H, J = 7.4 Hz), 3.89 (s, 3H), 3.74 (s, 3H), 3.68 (s, 6H).13C
(400 MHz, CDCl3): 168,05, 166,71 (JC-F = 253 Hz), 165,44,
154,95, 148,63, 147,87, 135,97,134,87,131,31 (JC-F = 9,1 Hz),
130,77, 130,44, 120,70, 116,98 (JC-F = 21,9 Hz),113,55, 112,41,
95,82, 66.73, 63.73, 61.58 , 56.65, 56.64, 51.45. MS (ESI): m/z
519 (M+Na).10b. MS (ESI): m/z 641 (M+Na).
methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-azetidin-2-one
9
(0.4 mmols) was added. The reaction was monitored by TLC
(silica gel, eluting with methylene chloride-methanol), treated
with a saturated solution of NaHCO3, extracted with methylene
chloride. The combined organic extracts were dried (Na2SO4)
and the solvent removed under reduced pressure. A mixture of
11a-i and 10a-i in varying amounts was obtained (the latter
generally detected in the MS and/or NMR spectrum of the crude
material), and directly hydrolyzed to 11a-i.
When Boc-N-methyl-L-alanine was used as acyl donor, a mixture
of Boc-protected N-acylated and N,O-di-acylated derivatives was
obtained, directly hydrolyzed to protected Boc-N-acyl derivative
and deprotected with trifluoroacetic acid to afford 11j.
N-(4-methoxybenzoyl)-3,4-trans-3-amino-1-(3,4,5-
trimethoxyphenyl)-4-(3-hydroxy-4-methoxyphenyl)-azetidin-2-
one.
11c (64 % yield). .1H NMR (400 MHz, CDCl3): δ ppm 7.82 (d, 2H,
J = 7.6 Hz), 6.98 (s, 1H), 6.93 (dd, 1H, J1 = 7.5 Hz), 6.86 (d, 1H, J =
7.5 Hz), 6.56 (s, 2H), 5.25 (s, 1H), 4.79 (d, 1H, J = 6.4 Hz), 5.85 (d,
1H, J= 6,4 Hz, NH), 3.89 (s, 3H), 3.87 (s, 3H), 3.77 (s, 3H), 3.70 (s,
Hydrolysis of the ester group was achieved by addition of
hydrazine di-chloride (0.174 mmols) and dry triethyl amine (0.34
mmols) to a stirred solution of the mixture 10a-j and 11a-j
6H).13C (300 MHz, CDCl3):
ppm 167.38, 164.82, 162.61,
153.34, 147.27, 146.37, 134.54, 133.43, 129.88, 129.2, 125.04,
118.17, 113.78, 112.49, 111.19, 95.24, 66.2, 63.17 60.88, 60.03,
55.95, 55.38, 50.55. MS (ESI): m/z 531 (M+Na).
(0.087 mmols) in methanol (4 mL), under
a nitrogen
atmosphere. The reaction was monitored by TLC analysis (Silica-
gel). The reaction was then treated with a KHSO4 solution (5 %)
and extracted with methylene chloride. The combined organic
extracts were dried and the solvent removed under reduced
pressure. The crude material was flash chromatographed on
10c. MS (ESI): m/z 665 (M+Na).
N-(3,4,5-trimethoxybenzoyl)-3,4-trans-3-amino-1-(3,4,5-
trimethoxyphenyl)-4-(3-hydroxy-4-methoxyphenyl)-azetidin-2-
one.
silica gel, eluting with
a gradient of methylene chloride-
methanol affording pure 11a-j.
11d (60 % yield). 1H NMR (400 MHz, CDCl3): δ ppm 8.34 (d, 1H,
J = 6.5 Hz), 7.11 (s, 2H), 6.87 (d, 1H, J 0 1.0 Hz), 6.82 (dd, 1H, J =
8.8, 1.0 Hz), 6.78 (d, 1H, J = 8.8 Hz), 6.50 (s, 2H), 5.02 (d, 1H, J =
2.2 Hz), 4.71 (dd, 1H, J = 6.5), 3. 82 (s, 3H), 3. 81 (s, 3H), 3.80 (s,
6H), 3.73 (s, 3H), 3.69 (s, 3H), 3.62 (s, 3H). 13C (300 MHz, CDCl3):
ppm 167.87, 165.82, 154.07, 153.99, 152.34, 148.09, 147.21,
135.08, 134.22, 129.99, 129.74, 128.58, 124.45, 118.75, 113.14,
N,O-(diacetyl)-3,4-trans-3-amino-1-(3,4,5-trimethoxyphenyl)-4-
(3-hydroxy-4-methoxyphenyl)-azetidin-2-one (10i). The title
compound was prepared as well by addition of acetic anhydride
(2 mL) to 3,4-trans-3-amino-4-(3-hydroxy-4-methoxyphenyl)-1-
(3,4,5-trimethoxyphenyl)-azetidin-2-one 1. Removal of acetic
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111.94, 108.12, 105.47, 95.74, 93.33, 66.88, 63.34, 61.55, 56.77,
56.67, 56.70, 51.00. MS (ESI): m/z 591 (M+Na).
10d. MS (ESI): m/z 785 (M+Na).
11i (80% yield). 1H NMR (400 MHz, CDCl3): δ ppm 6.95 (s, 1H),
6.89 and 6.87 (AB system, 2H, J = 7.4 Hz), 6.55 (s, 2H), 6.33 (d,
1H, J = 7.5 Hz, NH), 4.91 (d, 1H, J = 1.5 Hz), 4.62 (dd, 1H, J = 7.4,
1.5 Hz),3.91 (s, 3H), 3.86 (s, 3H), 3.72 (s, 6H), 2.10 (s, 3H). 13C
(400 MHz, CDCl3): 171.52, 165.24, 153.95, 153.93, 147.83,
147.02, 134.89, 134.18, 129.88, 118.79, 112.98, 111.80, 95.74,
66.18, 63.80, 61.57, 56.61, 23.36. MS (ESI): m/z : 439 (M+Na).
N-(2-furoyl)-3,4-trans-3-amino-1-(3,4,5-trimethoxyphenyl)-4-
(3-hydroxy-4-methoxiphenyl)-azetidin-2-one
11e (76 % yield). 1H NMR (400 MHz, CDCl3): δ ppm 8.07 (sdd,
1h, J =1.8, 0.9 Hz), 7.53 (dd, 1H, J =3.5, 0.9 Hz), 7.92 and 7.91
(AA’ system, 2H, J = 7.3 Hz), 6.90 (s, 1H), 6.79 (dd, 1H, J =3.5, 1.7
Hz), 6.61 s, 2H), 5.10 (s, 1H), 4.72 (s, 1H), 3.87 (s, CH3), 3.72 (s,
CH3), 3.70 (s, 2x CH3). 13C (300 MHz, CDCl3): δ ppm 165.52,
163.7, 153.91, 153.89, 147.87, 147.02, 146.41, 144.36, 135.02,
133.98, 129.76, 122,15, 118.89, 112.92, 111.83, 109.12, 95.76,
66.11, 63.92, 61.53, 56.59, 56.51. MS (ESI): m/z 491 (M+Na).
10e. MS (ESI): m/z 585 (M+Na).
N-(S-2-amino-propanoyl)-3,4-trans-3-amino-1-(3,4,5-
trimethoxyphenyl)-4-(3-hydroxy-4-methoxyphenyl)-azetidin-2-
one
11j (80% yield). 1H NMR (300 MHz, CDCl3/CD3OD): δ ppm 8.26
(bs, 2H), 6.91 (s, 1H), 6.84 and 6.79 (AB system, 2H, J = 8.2 Hz),
6.52 (s, 2H), 4.92 (s, 1H), 4.59 (s, 1H), 3.84 (s, 3H), 3.75 (s, 3H),
3.66 (s, 6H), 3.65 (m, 1H), 1.35 (d, 3H, J = 7.5 Hz). 13C (300 MHz,
CDCl3/CD3OD): 170.01, 168.29), 154.07, 147.79, 147.10, 135.31,
134.18, 130.32, 118.43, 112.94, 111.80, 96.06, 95.86, 69.87,
66.89, 61.54, 56.67, 56.64, 51.17, 49.99, 20.60. MS (ESI): m/z
446 (M+1).
N-Boc-11j. 1H NMR (300 MHz, CDCl3/CD3OD): 7.64 (s, 1H), 6.88
(s, 1H9, 6.88 and 6.86 (AB system, 2H, J = 7.4 Hz), 6.57 (s, 2H),
4.95 (s, 1H), 4.59 (s, 1H), 3.86 (s, 3H), 3.72 (s, 3H), 3.70 (s, 6H),
3.6 (q, 1H, J = 7.4 Hz), 1.45 (s, 9H), 1.34 (d, 3H, J = 7.4 Hz). MS
(ESI): m/z 568 (M+Na).
N-(2-naphthoyl)-3,4-trans-3-amino-1-(3,4,5-trimethoxyphenyl)-
4-(3-hydroxy-4-methoxyphenyl)-azetidin-2-one
11f (60 % yield). 1H NMR (400 MHz, CDCl3): δ ppm 8.39 (s, 1H),
8.08 (d, 1H, J = 5.9 Hz, NH), 7.90- 7.82 (4H), 7.55 (t, 1H, J = 7.4
Hz), 7.50 (t, 1H, J = 7.4 Hz),6.97 (s, 1H), 6.89 (d, 1H, J = 7.5 Hz),
6.81 (d, 1H, J = 7.5 Hz), 6.52 (s, 2H), 5.04 (s, 1H), 4.93 (d, 1H, J
=5.9 Hz), 3.85 (s, 3H), 3.73 (s, 3H), 3.65 (s, 6H). 13C (400 MHz,
CDCl3): 168.28, 165.35, 153.98, 147.81, 147.02, 135.58, 135.08,
134.17, 133.15, 130.69, 130.00, 129.79, 129.07, 128.94, 128.58,
128.34, 127.40, 124.25, 118.96, 113.02, 111.79, 95.81, 95.69,
66.87, 63.82, 61.59, 56.63, 56.59, 56.56. MS (ESI): m/z 551
(M+Na).
Biology
Reagents
RPMI-1640 medium, fetal bovine serum (FBS), l-glutamine,
penicillin, and streptomycin were obtained from Lonza (Basel,
Switzerland). All other reagents were purchased from Sigma-
Aldrich (St. Louis, MO).
10f. MS (ESI): m/z 705 (M+Na).
N-(indolo-3-acetyl)-3,4-trans-3-amino-1-(3,4,5-
Cell cultures
trimethoxyphenyl)-4-(3-hydroxy-4-methoxyphenyl)-azetidin-2-
one
Colorectal adenocarcinoma SW48 cells were purchased from
ATCC and were cultured using RPMI-1640 medium
supplemented with 10% (v/v) FBS, 2 mM l-glutamine, 100 U/mL
penicillin, 100 μg/mL streptomycin and were maintained at 37
°C in a humidified 5% CO2 incubator.
11g (75 % yield). 1H NMR (400 MHz, CDCl3): δ ppm 7.60 (d, 1H,
J = 7.34 Hz), 7.41 (d, 1H, J = 7.34 Hz), 7.28 (S, 1H), 7.26 (t, 1H, J =
7.34 Hz), 7.21 (t, 1H, J= 7.34 Hz), 7.16 (s, 1H, NH), 6.89 (s, 1H),
6.83 (s, 2H), 6.51 (s, 2H), 6.37 (d, 1H, J = 6.13 Hz, NH), 4.86 (s,
1H), 4.51 (d, 1H, J = 6.13 Hz), 3.89 (s, 3H), 3.80 (s, 2H), 3.76 (s,
3H), 3.69 (s, 6H). 13C (400 MHz, CDCl3): 172.69, 164.67, 154.07,
147.61, 146.69, 137.08, 135.3, 134.10, 130.12, 127.57, 123.44,
120.96, 119.24, 118.80, 112.87, 112.72, 112.27, 111.72, 108.87,
95.93, 66.60, 63.50, 61.58, 56.74, 56.70, 33.90. MS (ESI): m/z
554 (M+Na)
Growth inhibition assay
The antiproliferative activity of the azetidinone derivatives was
measured
by
the
3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay. 8000 cells were
seeded in 96-well plates and treated with different
concentrations of each compounds (dissolved in DMSO). After
72h, the MTT solution was added and then plates were
incubated for 2 h at 37 °C. The purple formazan crystals were
solubilized and the plates were read on a Model 550 Microplate
Reader (BioRad Laboratories, Hercules, CA) at 570 nm. Assays
were performed in triplicate in three independent experiments
and data were analyzed using the Sigma Plot software (using the
four parameter logistic equation) to estimate IC50 values,
defined as the concentration of drug causing a 50% inhibition in
absorbance compared to control cells (in which 0.1% DMSO was
used).
Tubulin Polymerization Assay. The effect of compounds on
tubulin polymerization was determined spectrophotometrically,
essentially as previously described [24]. Lyophilized purified
porcine brain tubulin (Cytoskeleton, Denver, CO) was
resuspended in assembly buffer (80 mM PIPES, pH 6.9, 1 mM
MgCl2, 2 mM EGTA, 10% glycerol) at 2.5 mg/mL and mixed with
10g. MS (ESI): m/z 711 (M+Na).
N-(Cyclohexanoyl)-3,4-trans-3-amino-1-(3,4,5-
trimethoxyphenyl)-4-(3-hydroxy-4-methoxyphenyl)-azetidin-2-
one
11h (69% yield). 1H NMR (400 MHz, CDCl3): δ ppm 6.92 (d, 1H, J
= 6.7 Hz, NH), 6.88 (s, 1 H), 6.83 (d, 1H, J = 8.4 Hz), 6,80 (d, 1H, J
=8.4 Hz), 6.44 (s, 2H), 4.87 (d, 1H, J = 1.5 Hz) 4.54 (dd, 1H, J = 6.7,
1.5 Hz), 3.84 (s, 3H), 3.76 (s, 3H), 3.66 (s, 6H), 2.15 (m, 1H), 1.86
(m, 2H), 1.75 (m, 2H), 1.64 (m, 2H), 1.43 (m, 2H), 1.25 (m,
2H).13C (400 MHz, CDCl3): 177.63, 165.29, 153.98, 147,85,
147.02, 135.06, 134.20, 130.06, 118.87, 113.04, 111.84, 95.83,
66.41, 63.79, 61.53, 56.74, 56.68, 56.61, 45.44, 41.17, 30.06,
26.29, 26.24. MS (ESI): m/z 507 (M+Na)
11h. MS (ESI): m/z 617 (M+Na).
1
mM GTP and varying concentrations of the indicated
N-(Acetyl)-3,4-trans-3-amino-1-(3,4,5-trimethoxyphenyl)-4-(3-
compounds. DMSO (0.2% v/v) was used as vehicle control.
Tubulin assembly was monitored at 340 nm at 37 °C for 15
hydroxy-4-methoxyphenyl)-azetidin-2-one
8 | J. Name., 2012, 00, 1-3
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ARTICLE
minutes in a Jasco V-530 spectrophotometer (Jasco Europe,
Italy). The IC50 values are the compound concentrations required
to inhibit tubulin polymerization by 50% and were estimated
using the Sigma Plot software (using the four parameter logistic
equation).
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