β-Lactam analogues of combretastatin A-4 prevent metabolic inactivation by glucuronidation in chemoresistant HT-29 colon cancer cells

Article abstract of DOI:10.1016/j.ejmech.2017.02.049

Glucuronidation by uridine 5-diphosphoglucuronosyl transferase enzymes (UGTs) is a cause of intrinsic drug resistance in cancer cells. Glucuronidation of combretastatin A-4 (CA-4) was previously identified as a mechanism of resistance in hepatocellular ca

Full text of DOI:10.1016/j.ejmech.2017.02.049

European Journal of Medicinal Chemistry 130 (2017) 261e285
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
b-Lactam analogues of combretastatin A-4 prevent metabolic
inactivation by glucuronidation in chemoresistant HT-29 colon cancer
cells
,
Azizah M. Malebari a *, Lisa M. Greene b, Seema M. Nathwani b, Darren Fayne c,
Niamh M. O'Boyle b, Shu Wang a, Brendan Twamley d, Daniela M. Zisterer b,
Mary J. Meegan a
a School of Pharmacy and Pharmaceutical Sciences, Trinity Biomedical Sciences Institute, 152-160 Pearse Street, Trinity College Dublin, Dublin 2, Ireland
b School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, 152-160 Pearse Street, Trinity College Dublin, Dublin 2, Ireland
c Molecular Design Group, School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Ireland
d School of Chemistry, Trinity College Dublin, Dublin 2, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history:
Glucuronidation by uridine 5-diphosphoglucuronosyl transferase enzymes (UGTs) is a cause of intrinsic
Received 9 December 2016
Received in revised form
18 February 2017
Accepted 20 February 2017
Available online 24 February 2017
drug resistance in cancer cells. Glucuronidation of combretastatin A-4 (CA-4) was previously identified as
a mechanism of resistance in hepatocellular cancer cells. Herein, we propose chemical manipulation of b-
lactam bridged analogues of Combretastatin A-4 as a novel means of overcoming drug resistance
associated with glucuronidation due to the expression of UGTs in the CA-4 resistant human colon cancer
HT-29 cells. The alkene bridge of CA-4 is replaced with a b-lactam ring to circumvent potential iso-
merisation while the potential sites of glucuronate conjugation are deleted in the novel 3-substituted-
1,4-diaryl-2-azetidinone analogues of CA-4. We hypothesise that glucuronidation of CA-4 is the mech-
anism of drug resistance in HT-29 cells. Ring B thioether containing 2-azetidinone analogues of CA-4
such as 4-(4-(methylthio)phenyl)-3-phenyl-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (27) and 3-
hydroxy-4-(4-(methylthio)phenyl)-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (45) were identified as
the most potent inhibitors of tumour cell growth, independent of UGT status, displaying antiproliferative
activity in the low nanomolar range. These compounds also disrupted the microtubular structure in MCF-
7 and HT-29 cells, and caused G2/M arrest and apoptosis. Taken together, these findings highlight the
potential of chemical manipulation as a means of overcoming glucuronidation attributed drug resistance
in CA-4 resistant human colon cancer HT-29 cells, allowing the development of therapeutically superior
analogues.
Keywords:
Combretastatin A-4
2-azetidinone
b
-lactam
HT-29 colon cancer cells
Tubulin polymerisation inhibitor
© 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction
from the bark of the south African tree Combretuem caffrum, ex-
hibits strong antitubulin activity by binding to tubulin at the
Tubulin is the target of numerous small molecule anti-
proliferative ligands that act by interfering with microtubule dy-
namics [1]. These ligands are commonly classified into two major
categories: microtubule stabilizing drugs (e.g. taxanes, epothilones
and laulimalide) and microtubule destabilizing drugs (e.g. vinca
alkaloids, colchicine 1 (Fig. 1) and maytansine) [2e4]. Com-
bretastatin A-4 (CA-4) 2 (Fig. 1), a potent antimitotic agent isolated
colchicine binding site [5]. CA-4 shows potent cytotoxicity against a
range of human cancer cell lines, including those that are multi-
drug resistant [6,7]. To overcome the poor water solubility of CA-
4,
a
water-soluble
combretastatin
A4-phosphate
CA-
4P(fosbretabulin) (3, Fig. 1) was synthesized [5] and is currently
under investigation in combination with pazopanib in a Phase 1b
study for the treatment of advanced recurrent ovarian cancer [8].
Drugs undergo a series of phase I or phase II biotransformation
reactions in vivo, in which more water-soluble metabolites are
formed so that these xenobiotic substances can be excreted more
easily. Interestingly, these metabolites can result in
* Corresponding author.
E-mail address: melibaa@tcd.ie (A.M. Malebari).
http://dx.doi.org/10.1016/j.ejmech.2017.02.049
0223-5234/© 2017 Elsevier Masson SAS. All rights reserved.

262
A.M. Malebari et al. / European Journal of Medicinal Chemistry 130 (2017) 261e285
Fig. 1. Microtubule targeting agents colchicine (1), Combretastatin A-4 (2), Combretastatin A4-phosphate (3), and b-lactams 4 and 5.
pharmacological or toxicological activity or inactivity. The in vitro
phase I biotransformation of CA-4 in hepatic microsomal meta-
bolism involves two main metabolic pathways: O-demethylation
and aromatic hydroxylation [9]. Metabolites arising from aromatic
hydroxylation of ring B are readily oxidized into the reactive para-
quinone type metabolite which displays a significant cytotoxicity
independent of tubulin binding [10]. Phase II in vitro and in vivo
metabolic studies of CA-4 identified the formation of CA-4 glucu-
ronide and sulphate metabolites [11,12]. The formation of glucu-
ronide conjugates arising from phase I metabolism of CA-4 was also
observed [12]. Importantly, glucuronidation of CA-4 was associated
with its inactivation in resistant BEL-7402 and SMMC-7721 hepa-
tocellular carcinoma cells [13].
UDP-Glucuruonosyltransferases (UGTs) are the major phase II
drug metabolizing enzymes in the body, catalysing the conjugation
between UDP-glucuronic acid and numerous endogenous and
exogenous compounds [14]. It has been shown that overexpression
of UGTs in cells can contribute to carcinogenesis by promoting drug
resistance and this demonstrates the significant differences
observed in metabolism of drugs in cancer cells when compared to
normal cells [15]. Formation of the glucuronide metabolite is the
main metabolic pathway that contributes to the clearance of CA-4
and is mainly catalysed by UGT1A9 and UGT1A6 isoforms
[12,16,17]. In addition, glucuronidation was shown to be a mecha-
nism of intrinsic drug resistance in colon cancer cells, contributing
to the transport of drugs by proteins [18]. For example, the novel
topoisomerase I inhibitor NU/ICRF 505 was cleared after 24 h by
83% in chemoresistant HT-29 cells as the O-glucuronides while
maintained in HCT116 cells which do not overexpress UGTs [19].
Many conformationally restricted analogues of CA-4 have been
reported, in which the cis double bond is replaced by a heterocycle,
e.g. imidazole [20], tetrazole [21], triazole [22,23], pyridine [24],
benzoxepine [25], and thiophene [26] rings and others [27]. The
nanomolar range in combretastatin refractory HT-29 cells [36]. We
now hypothesise that 2 and its rigid analogues can undergo direct
glucuronidation at the meta-hydroxy position of ring B leading to
inactivation in HT-29 cells. The role of glucuronidation in reducing
the in vitro therapeutic potency of our b-lactam compound series is
a crucial modification to be analysed prior to further progressing
the lead compounds. Therefore, a strategy was investigated to
prevent this glucuronidation which leads to the resistance in HT-29
colon cancer cells: the first approach was to prevent direct glu-
curonidation by synthesising a series of novel analogues with
deletion of the meta-OH substituent in ring B of the b-lactam. We
also wished to include in this study compounds designed to pre-
vent phase I O-demethylation metabolism of Ring B leading to
phase II glucuronidation and subsequently to inactivation. The O-
demethylation metabolic pathway could be blocked by either
ethoxy or thioether substitution at the para position in Ring B.
These novel compounds reported herein contain aromatic, polar or
vinyl substituents at position 3 of the b-lactam ring designed to
examine the effect of the 3-substituent on enhanced efficacy in the
cancer cell lines. This study aims to investigate whether high
expression levels of UGT protein in chemoresistant HT-29 cells
compared to both human breast MCF-7 and human leukaemia HL-
60 cells could contribute to the resistance to CA-4 observed in the
combretastatin refractory HT-29 colon cancer cells.
2. Results and discussion
2.1. Chemistry
The b-lactam compounds to be investigated in the present study
are arranged in seven distinct structural groups, see Schemes 1-3
and Table 3.
introduction of the rigid
b-lactam ring scaffold allows a similar
Type 1: Structures containing p-methoxyaryl Ring B (15-20)
Type II: Structures containing p-ethoxyaryl Ring B (21-26)
Type III: Structures containing p-thiomethylaryl Ring B (27-32)
Type IV: Structures containing p-thioethylaryl Ring B (33-38)
Type V: 3-Unsubstituted b-lactams containing p-methoxyaryl,
p-ethoxyaryl, p-thiomethylaryl and p-thioethylaryl Ring B (39-
42)
structural arrangement between the two aromatic rings A and B as
observed in the non-planar cis-configuration of CA-4 while pre-
venting the undesired conversion to the inactive trans-configura-
tion [28e30]. We have previously designed and synthesised a series
of azetidinone (b-lactam) analogues of CA-4 and we demonstrated
the potent antiproliferative activity for series of 1,4-diarylazetidine-
2-ones and 1,3,4-triarylazetidine-2-ones in human breast cancer
cell lines [28,31e35]. Trans isomers of the phenolic 4 and amino
substituted compound 5 (Fig. 1) were identified as potent com-
pounds with IC50 values of 38 and 19 nM, respectively in MCF-
Type VI: 3-Hydroxy
b-lactams containing p-methoxyaryl, p-
ethoxyaryl, p-thiomethylaryl and p-thioethylaryl Ring B (43-46)
Type VII: Structures containing m-hydroxyyaryl Ring B (4, 47-
59) and p-hydroxyaryl Ring B (50)
7 cells [35]. However,
b-lactam compounds containing a B-ring
meta-hydroxy substituent group were not active in the com-
bretastatin refractory HT-29 colon cancer cells. However, we
observed that deletion of the ring B meta-hydroxy group or sub-
stitution of the ring B meta-hydroxy group with an amine conju-
gated amino acid significantly increased the activity with IC50 in the
In this study, we have focussed on replacing the 4-methoxyaryl
B ring of the selected series of b-lactams with a 4-thioarylether
substituent, (Types I, III and IV) The p-alkyl moiety was also var-
ied from methoxy to ethoxy (Type II) to attempt to minimize the
susceptibility of the compounds to Phase I O-demethylation and

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263
Scheme 1. Preparation of Benzaldehydes 7 and 8 and Imines 9-14. Reagents and conditions: (a) t-BuMe2SiCl, DBU, CH2Cl2, rt, until complete as indicated by tlc, 80e96%; (b) EtOH,
reflux, 3 h, 78e91%. R1 ¼ H unless otherwise indicated.
subsequently to the glucuronidation metabolic pathway. The
preparation of the target -lactam compounds is outlined in
Schemes 1 - 3. All -lactams identified for the synthesis contains a
3,4,5-trimethoxyaryl ring at N-1, as is present in ring A of CA-4. This
has previously been shown to be the optimal substitution pattern
phenoxy-2-azetidinones, we have previously observed that the
trans isomer of these -lactam products is significantly more
bioactive than the cis isomer [28,35,38,43]. Therefore it was
important that the synthetic approach employed produced the
optimal yield of the trans isomer by the choice of solvent, tem-
b
b
b
for anti-proliferative activity at this position of the
[30]. We have also included an aryl ring with 4-alkoxy or 4-
thioalkyl substitutions at the C4 position on the -lactam ring
(ring B of CA-4). The choice of the substituent at the C-3 position on
the panel of -lactam compounds to be synthesised included
b
-lactam ring
perature, and order of addition of the reagents [42]. b-Lactams 15-
38 (Types I-IV) were synthesized from the appropriate imine and
acid chloride, by this modified Staudinger route [38] (Scheme 2,
step a). The trans isomer was exclusively isolated in most cases as
evident from the representative 1H NMR spectrum of compound 27
b
b
phenoxy, aryl, hydroxy, chloro and alkenyl. We have previously
reported the synthesis and antifungal activity of a series of 3-
hydroxy-1,4-diaryl-2-azetidinones [37], while Tripodi et al. have
investigated the antitumour activity of 3-hydroxy-1,4-diaryl-2-
azetidinone compounds [38,39].
Imines 9-14 were obtained in high yields (78e91%) by
condensation of the appropriate amines and aldehydes under
reflux conditions in ethanol (Scheme 1). For the preparation of the
imine 12, 4-Fluorobenzaldehyde was treated with ethanthiol to
afford 4-(ethylthio)benzaldehyde. The phenolic groups present on
6a and 6b were protected as the tert-butyldimethylchlorosilane
(TBDMS) ether to yield benzaldehyde 7 and 8 respectively. Two
general routes for the azetidinone ring-forming reaction were
employed. The Staudinger reaction (for Type I-IV, VII compounds)
requires the cycloaddition reaction of appropriately substituted
acid chlorides and imines [40] (Scheme 2, routes a and c), while the
Reformatsky route (for Type V and VI compounds) requires reaction
(yield 55%) where the H3 and H4 were identified at d 4.25 and 4.85
respectively as a pair of coupled doublets, J3,4 ¼ 2.44 Hz. The
exception in this series was the isolation of the minor cis isomer for
3-phenoxy b-lactams 16, 22, 28 and 34 with trans/cis ratios deter-
mined as 4:1, 3:1, 5:1, and 4:1 respectively. The presence of struc-
tural isomers was confirmed by X-ray crystal structures of both
trans and cis isomers of compound 28 (Figs. 2 and 3 respectively),
(Table 1).
3-Unsubstituted
b
-lactams 39ꢀ42 (Type V) were obtained by a
Reformatsky-type reaction of a series of appropriately substituted
imines with ethyl bromoacetate under microwave conditions to
afford the 3-unsubstituted products, (Scheme 2, step b). The X-Ray
structure of the novel compound 41 is displayed in Fig. 4, (Table 1).
b-Lactams 43-46 (Type VI) containing the 3-hydroxy substituent at
C-3 position and the 3,4,5-trimethoxyphenyl at N-1 position were
prepared by the Staudinger reaction using the appropriate ketene
precursor acetoxyacetyl chloride; subsequently reaction with hy-
drazine dihydrochloride afforded the 3-hydroxy containing com-
pounds 43-46 (Scheme 2, steps a and c) and the trans isomers were
exclusively obtained with 20e41% yield. Hydrolysis of the acetate
ester was achieved with hydrazine dihydrochloride which was
used under milder conditions with higher yield compared with the
use of potassium carbonate for this step [37]. The X-Ray structure of
of an organozinc species (derived from an a-bromoester) and an
imine [33] (Scheme 2, route b).
The stereochemistry of the products from the Staudinger reac-
tion depends on many factors e.g. the reaction conditions, the order
of the addition of the reagents, and the substituents present on
both the imine and on acid chloride [38,41,42]. In the case of the 3-

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A.M. Malebari et al. / European Journal of Medicinal Chemistry 130 (2017) 261e285
Scheme 2. Synthesis of 2-azetidinones 15-46 (Types I- VI). Reagents and conditions: (a) Et3N, toluene, 100 ꢁC, 5 h then rt, 15 h, 10e70%; (b) Zn dust, (CH3)3SiCl, 40 ꢁC, 15 min, then
100 ꢁC, 2 min, microwave; BrCH2CO2Et, C6H6, 100 ꢁC, 30 min, microwave, 10e35%; (c) NH2NH2.2HCl, Et3N, MeOH, reflux, 4 h, 20e41%. Products obtained as a mixture of enan-
tiomers; one enantiomer represented. R1 ¼ H unless otherwise indicated.
the novel compound 44 is displayed in Fig. 5, (Table 1). In the case
of compounds containing phenolic hydroxyl substitutions in ring B
(compounds 47-50, Types VII), the appropriate benzaldehyde was
first protected using the TBDMS ether, and then used for the
preparation of Schiff bases 13 and 14 (Scheme 1). The silyl ether
the colchicine binding site of tubulin. The torsional angles (Ring A/
B) observed for compounds 28trans, 28cis, 41 and 44 were calcu-
lated to be 56.2ꢁ, 56.2ꢁ, 68.9ꢁ (ꢀ60.9ꢁ) and 51.5ꢁ respectively
compared to 53ꢁ and 55ꢁ for the corresponding rings in colchicine
[44] and CA-4 [45,46] respectively (Table 2). The greatest deviation
is observed for the 3-unsubstituted compound 41. (The numbers in
parentheses refer to the second crystallographically independent
molecule in the asymmetric unit for 41; each is a different enan-
tiomer C4 ¼ R and C28 ¼ S).
protecting group was removed with TBAF to give phenolic b-lac-
tams 47-50 in 9e30% yield (Scheme 3).
Preliminary stability studies of the representative
and 45 were carried out at acidic, neutral and basic conditions (pH
b
ꢀlactams 27
4, 7.4 and 9). Solutions of the
b
ꢀlactams (30
m
g/mL) were incubated
As expected in compound 28, the Ring B/C torsional angle value
was low for the cis isomer (-10.97ꢁ) compared to the trans isomer
(122.7ꢁ). Similarly, the torsional angle value between ring B and
hydroxyl group of 44 compound is (ꢀ114.7ꢁ) which is similar to the
trans isomer of 28 (ꢀ112.7ꢁ, Table 2). Interestingly, the torsional
angles (Ring A/B) for both trans and cis isomer of 28 are close in
value (56.2ꢁ), which indicates that a trans arrangement between
rings B and C facilitates a more favorable interaction of rings A and
in the appropriate phosphate buffer at 37 ꢁC and examined by
analytical HPLC at selected time intervals for 24 h. The half-life (t )
½
was determined to be greater than 24 h at pH 7.4 for compound 45.
Half-lives were shorter at pH 4 and 9 (16 h for both). Compound 27
was less stable than 45 at pH 4, 7 and 9 (t ¼ 10 h, 15 h and 8 h
½
respectively). Based on this stability study, both b-lactams 27 and
45 would be the suitable for further development.
B with the residues of the b-tubulin colchicine binding site. How-
ever, the unbound X-ray crystallographic structure of these 2-
azetidinones may not be the bioactive conformation in vivo.
2.1.1. X-ray structural study
An X-ray crystallography study confirmed the stereochemical
assignments for compounds 28trans, 28cis, 41 and 44 (Figs. 2e5
respectively). The crystal data for 28trans, 28cis, 41 and 44 are
displayed in Table 1. The X-ray structures demonstrated a config-
2.2. Biological results and discussion
uration for the
rings located at N-1 and C-4 of the
Thus, the -lactam ring forms a rigid scaffold for the planar hy-
drophobic aryl rings A and B, which is required for interaction with
b
-lactams where the rings A and B (i.e. aromatic
2.2.1. Expression level of UGT protein is significantly higher in CA-4
resistant HT-29 cells as compared to CA-4 sensitive MCF-7 and HL-
60 cells
b-lactam ring) are not coplanar.
b
Glucuronidation has been strongly implicated in the intrinsic

A.M. Malebari et al. / European Journal of Medicinal Chemistry 130 (2017) 261e285
265
Scheme 3. Synthesis of Phenolic 2-Azetidinones 47-50 (Type VII). Reagents and conditions: (a) Et3N, toluene, 100 ꢁC, 5 h then rt, 15 h, 30e55%; (b) Zn dust, (CH3)3SiCl, 40 ꢁC, 15 min,
then 100 ꢁC, 2 min, microwave; BrCH2CO2Et, C6H6, 100 ꢁC, 30 min, microwave, 12%; (c) NH2NH2.2HCl, Et3N, MeOH, reflux, 4 h, 70%; (d) TBAF, THF, 0 ꢁC, 15 min 9e30%. Products
obtained as a mixture of enantiomers; one enantiomer represented.
resistance to mycophenolic acid in HT-29 cells [47,48]. To investi-
gate the possibility of glucuronidation as a means of resistance to
Other independent researchers have noted a high UGT protein
expression in HT-29 and Lovo cells as compared with other sensi-
tive colon cell lines such as SW480, HCT116, and Colo320 [49].
CA-4 and selected
b-lactam derivatives in chemoresistant HT-
29 cells, the expression of UGT was first determined by western
blotting. As shown in Fig. 6, the endogenous level of UGT in CA-4
resistant HT-29 cells was significantly higher when compared to
UGT expression levels in CA-4 sensitive MCF-7 and HL-60 cells. The
apparent abundant expression of UGT in HT-29 cells would confer
resistance to CA-4 and derivatives of CA-4 that contain the required
phenolic functional groups which facilitate glucuronate conjuga-
tion and subsequent inactivation. UGT protein expression was
semi-quantified by densitometry analysis of band intensity corre-
sponding to the UGT in both HT-29 and MCF-7 cells. The significant
increase of UGT expression density in HT-29 cells (3.6 fold) was
observed compared to MCF-7 cells (**P < 0.01, data not shown).
2.2.2. U0126 inhibits CA-4 and its related
HT-29 cells
b-lactam inactivation in
U0126 (1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)
butadiene) is a selective inhibitor of MEK used for studies of MAPK/
ERK signalling. Recent studies demonstrated that U0126 enhanced
Combretastatin A-4 induced cytotoxicity by inhibiting glucur-
onidation of CA-4 [13]. These findings suggest that UDP-
glucuronosyltransferase may be a target of U0126 independent of
MEK inhibition. Further biochemical investigations were carried
out to investigate whether U0126 can reverse CA-4 resistance and
the related Ring B meta-hydroxylated b-lactam (47) resistance in

266
A.M. Malebari et al. / European Journal of Medicinal Chemistry 130 (2017) 261e285
Fig. 2. Molecular structure of 28trans (3R4R) with atomic displacement parameters shown at 50% probability. Partially occupied water molecule and hydrogen atoms omitted for
clarity. CCDC number: 1499366.
Fig. 3. Molecular structure of 28cis (3S4R) with atomic displacement parameters shown at 50% probability. Hydrogen atoms omitted for clarity. CCDC number: 1499367.
UGT expressing HT-29 cells. As shown in Fig. 7A, U0126 reversed
the resistance of CA-4 and also reversed the resistance of the Ring B
meta-hydroxylated b-lactam 47 in HT-29 cells. G2/M cell-cycle ar-
rest is a known marker of the activity of CA-4. Fig. 7B clearly
Table 1
Crystal data for 28trans, 28cis, 41 and 44.
Code
28trans
28cis
41
44
demonstrated a significant increase of G2/M cell-cycle induced by
Empirical formula C25H25.23NO5.11
S
C25H25NO5S C19H21NO4S C20H23NO6
fw
453.55
0.71073
Monoclinic
C2/c
19.9885(8)
13.2634(5)
17.5090(6)
90
107.753(1)
90
4420.9(3)
100
451.52
0.71073
359.43
0.71073
373.39
1.54178
CA-4 and its related Ring B meta-hydroxylated b-lactam 47 in the
presence of U0126 in HT-29 cells. We hypothesise that glucur-
onidation may represent a mechanism of intrinsic resistance to CA-
l
(Å)
Crystal System
SG
a (Å)
b (Å)
c (Å)
Monoclinic Monoclinic Monoclinic
P2(1)/c
11.4803(5)
11.7287(5)
16.6012(7)
90
92.061(1)
90
2233.89(17) 3509.75(17) 3686.2(3)
99.97
4
1.343
0.182
28701
4779
0.0684
1.007
0.0412
0.0855
1499367
P2(1)/c
10.1471(3)
12.0914(3)
28.6490(8)
90
C2/c
4 and its related Ring B meta-hydroxylated
b-lactam 47 in HT-
17.9743(8)
13.2393(6)
15.7125(8)
90
99.6430(15)
90
29 cells. Pharmacological inhibition of glucuronidation can thereby
restore sensitivity to Ring B hydroxylated combretastatins in UGT
expressing cells.
a
b
g
(ꢁ)
(ꢁ)
(ꢁ)
93.139(1)
90
V (Å3)
T (K)
Z
2.2.3. Anti-proliferative activities and structure-activity
relationships
100
8
99.99
8
1.346
0.827
25546
3401
0.0540
1.035
0.0539
0.1458
1499369
8
(Mg/cm3)
1.363
0.185
30528
4725
0.0393
1.024
0.0392
0.0938
1499366
1.360
0.208
99548
7205
0.0515
1.021
0.0339
0.0756
1499368
All
b-lactam compounds were next screened for their anti-
r
m
(mmꢀ1
)
proliferative activity against three representative cancer cell lines,
two CA-4-sensitive (breast cancer MCF-7 and leukemia HL-60) and
the CA-4-resistant UGT expressing human colon cancer HT-29 with
reference to the antiproliferative activity of CA-4 (Fig. 1). Treatment
at eight different concentrations was used in order to determine the
IC50 values for each compound in comparison to the control com-
Total reflns
Indep. reflns
R(int)
S
a
R1 [I > 2
s
(I)]
wR#2 [I > 2
s(I)]
CCDC number
pound CA-4 (Table 3). All the
b-lactams were evaluated as the trans
a
R1 ¼ SjjFoj ꢀ jFcjj/SjFoj, wR2 ¼ Sw(F2o ꢀ F2c)2/Sw(Fo2)2]1/2
.
isomer. It is evident that the majority of the target compounds 4,

A.M. Malebari et al. / European Journal of Medicinal Chemistry 130 (2017) 261e285
267
Fig. 4. Molecular structure of one of the independent molecules of 41 (4R) with atomic displacement parameters shown at 50% probability. Hydrogen atoms omitted for clarity.
CCDC number: 1499368.
Fig. 5. Molecular structure of the predominant disordered conformation of 44 (3S4S) with atomic displacement parameters shown at 50% probability. Hydrogen atoms omitted for
clarity. CCDC number: 1499369.
15-50 displayed moderate to potent anti-proliferative activity in
these cell lines. Compounds with meta-hydroxy substitution in
Ring B, e.g. compounds 47 [31], 4 [35] and 48 [33] showed potent
antiproliferative activity in the MCF-7 cancer cell line with IC50
values 4e38 nM, which reflects the importance of the meta-hy-
the meta-hydroxy substituent in ring B of the b-lactam for optimum
activity in MCF-7 and SW-480 cells.
Conversely, compounds containing the Ring B meta-hydroxy
group compounds 4, 47-49 displayed limited activity in the UGT
expressing chemoresistant colon HT-29 cells (IC50 values
droxy substituent of ring B of the
b
-lactam for optimum activity in
0.4e1 mM). This significant observation suggests that b-lactams
MCF-7 cells. In the present work, similar results were obtained for
compounds 47, 4 and 48 in the sensitive SW-480 colon cell line
with IC50 values of 4, 9 and 19 nM respectively. The absence of the
UGT protein band in SW-480 cells has been demonstrated by
westeren blot analysis compared to the high expression of this
protein in HT-29 cells [49]. These results reflect the importance of
with the meta-hydroxy group in ring B (e.g. compounds 4, 47-49)
may facilitate drug resistance to CA-4 and analogues. Improved
activity of oxazole bridged CA-4 analogues by substitution of the B
ring meta-hydroxy group with fluoro or amino substituent has been
reported [50]. Taken together, these results suggest a therapeutic
advantage in deleting/substituting the meta-hydroxy in ring B to

268
A.M. Malebari et al. / European Journal of Medicinal Chemistry 130 (2017) 261e285
Table 2
X-ray Crystallographic Data for b-lactams 28trans, 28cis, 41 and 44.
Compound Structure
Ring plane AB
angle(ꢁ)
Ring plane BC
angle(ꢁ)
Ring A to central Torsion
(ꢁ)a
Ring B to central Torsion
(ꢁ)b
RingAB Torsion
(ꢁ)c
RingBC Torsion
(ꢁ)d
28trans
86.7
71.5
10.1
ꢀ122.1
56.6
ꢀ122.7
28cis
76.4
77.6
5.7
ꢀ130.3
56.6
ꢀ10.97
41
89.9
86.2
e
10.7
ꢀ2.8
ꢀ144.7
68.9
ꢀ60.9
e
116.4
44
70.6
e
4.3
37.5
51.5
ꢀ114.7a
a
b
c
d
C18-C12-N1-C2
C18-C13-N1-C2
C17-C12-N1-C2
C41-C36-N25-C26
C19-C14-N1-C2
C10-C5-C4-N1
C10-C5-C4-N1
C10-C5-C4-N1
C34-C29-C28-N25
C10a-C5-C4-N1
C13-N1-C4-C5
C13-N1-C4-C5
C12-N1-C4-C5
C36-N25-C28-C29
C14-N1-C4-C5
C5-C4-C3-O26
C5-C4-C3-O26
e
e
C5eC4-C3-O27
a
Torsion angle for compound 44 refers to C-3 OH substituent.
introducing a number of different substituents at the C-3 position
(Table 3). Among the 4-methoxy containing compounds, com-
pound 15 displayed the most impressive potency in all the three
cell
lines
in
the
following
order
of
potencies
(15 > 19>16 > 39>17 > 20>18); IC50 values in MCF-7 cells were 15,
17, 23, 39, 56, 120 and 133 nM respectively. This result confirmed
that the presence of a variety of polar and smaller groups is
favourable on the 3-position of the b-lactams for antiproliferative
activity (e.g. phenyl 15, chloro 17, and vinyl 19 substituents). For
example, compound 17 bearing a 3-chloro substituent was twice as
potent as 3-dichloro compound 18 (IC50 values of 56 nM and
133 nM respectively). In contrast,
methoxy-meta-hydroxy substitution in ring B, (Type VII), having
the same substituents at the C-3-position of -lactams mentioned
above, demonstrated reduced activity in HT-29 cells and improved
activity in MCF-7 and HL-60 cells (compounds 4 and 47-49,
Table 3). For example, IC50 values for compound 47 are 4, 2, and
430 nM in MCF-7, HL-60 and HT-29 cells respectively. Additionally,
we synthesized a proposed metabolite of 15 that arises from O-
demethylation of 15 to evaluate the influence of the hydroxy group
on the activity. The presence of the para-hydroxy group in com-
b-lactam analogues with para-
Fig. 6. Western blot analysis of the expression of UGT protein levels in MCF-7, HT-29
and HL-60 cells using UGT antibody (sensitivity for UGT1A subfamily, Cell signalling).
Results are indicative of three separate experiments, performed independently. Each
b
lane was loaded with 20
mg of protein; to confirm equal protein loading, each mem-
brane was stripped and reproped with GAPDH antibody.
optimise activity of the b-lactams in chemoresistant HT-29 cells.
To establish a more detailed SAR and further examine the effects
on anti-proliferative activity in HT-29 cells of the deletion of the B
pound 50, in ring B of the b-lactam eliminated the activity (4000-
fold reduction) compared to its parent compound 15 in MCF-7,
ring meta-hydroxy group, a panel of b-lactams containing a variety
of polar and non-polar substituents at C-3 was examined, while
retaining the 3,4,5-trimethoxyphenyl substituent at N-1, (Types I-
VII). In the designed analogues, ring A possessed 3,4,5-trimethoxy
substitution, which was considered to be fundamental for the
tubulin binding activity, and the main modifications were made at
HL-60 and HT-29 cells.
A further series of 4-ethoxy Ring B substituent b-lactams was
synthesised to examine the effect of inhibiting O-demethylation of
the 4-methoxy group of ring B that leads to loss of activity in HT-
29 cells. Introduction of a 4-ethoxy substituent on ring B of b-lac-
tams, with a range of different substituents at 3-position e.g.
compounds 21-26 (Type II), and 40 (Type V), resulted in good
antiproliferative activity with only minor differences in IC50 values
compared to 4-methoxy series in all three cell lines (Table 3). The
4-position in the ring B and at 3-position of the
b-lactam ring.
The first series of the -lactam analogues 15-20 (Type I) and 39
b
(Type IV) examined contained a 4-methoxy substituent in Ring B,
but with deletion of meta-hydroxy substituent of ring B, and

A.M. Malebari et al. / European Journal of Medicinal Chemistry 130 (2017) 261e285
269
Fig. 7. (A) U0126 enhances CA-4 and
b-lactam 47 induced G2/M cell-cycle arrest in HT-29 cells. HT-29 cells were exposed to CA-4 and 47 (1 mM) alone or in the presence of 10 mM
U0126 for 24 h. Cells were collected, stained with PI, and analysed by flow cytometry. (B) Quantitative analysis of the percentage of G0/G1 and G2/M population. Statistical analysis
was performed using with Student's paired t-test in prism software. Data are presented as mean SEM of three independent experiments. *P < 0.05 vs control.
exception is that 22 (3-phenoxy-
against HL-60 and HT-29 cell with IC50 values of 0.494 and
0.388 M respectively but maintained excellent antiproliferative
activity in MCF-7 cells with IC50 value of 69 nM. It is of interest that
1,5-diarylsubstituted 1,2,3,4-tetrazole analogues of CA-4 which
contain the 4-ethoxyphenyl substitution on ring B, displayed
potent activity in HT-29 cells with an IC50 value below 10 nM [51].
In order to examine the significance of the 4-methoxy substit-
b
-lactam) was the least active
corresponding 4-methoxy containing compounds. However, it was
observed that the 4-thiomethyl ether compounds 27-32 and 41
exhibited better antiproliferative activity among the tested com-
pounds compared to 4-thioethyl containing compounds 33-38 and
42. For example, replacement of 4-S-CH3 in 27 with the 4-S-CH2-
CH3 substituent in 33 resulted in marked decrease in activity in all
the three cell lines: IC50 values for 27 were 65, 59, 56 nM and IC50
m
values for 33 were 0.380, 0.430, 3.142 mM in MCF-7, HL-60 and HT-
uent group in the ring B of
b
-lactam, a related series of thioether (S-
29 cells respectively. Generally, the nature of the substituents at the
para-position of the ring B significantly influenced the biological
activity [53]. The optimised trans stereochemistry for these com-
pounds was demonstrated in the case of compound 28, where the
CH3 and S-CH2-CH3) analogues were synthesized and evaluated for
anti-proliferative effects in MCF-7, HL-60 and HT-29 cancer cells
(compounds 27-38 (Type III and Type IV), 41 and 42 (Type V),
Table 3). A thiomethyl analogue of CA-4 with a 10-fold increase in
potency in HT-29 cells compared to the corresponding 4-methoxy
analogue is known [52]. The thiomethyl ether analogues 27-32
(Type III), 41 (Type V) demonstrated reasonable potency in all three
cell lines with 1e3 fold loss in potency compared to the
IC50 value for the cis compound was determined at 7.7
comparison to the trans isomer with IC50 value of 152 nM.
mM in
Interestingly, the presence of the 3-OH substituent at C3 of the
-lactam compounds having different substitution (e.g. OCH3,
b
OC2H5, SCH3, SC2H5) at C4 of ring B compounds 43, 44, 45 and 46

270
A.M. Malebari et al. / European Journal of Medicinal Chemistry 130 (2017) 261e285
Table 3
Antiproliferative Effects of b
-Lactams 4, 15-50 in MCF-7, HL-60 and HT-29 Cells.d
,b
IC50 value (m
M)a
Type
No.
R1
R2
R3
R4
MCF-7
HL-60
HT-29
I
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
4
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OC2H5
OC2H5
OC2H5
OC2H5
OC2H5
OC2H5
SCH3
C6H5
OC6H5
Cl
H
H
H
Cl
H
H
H
H
H
Cl
H
H
H
H
H
Cl
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0.015 0.002
0.023 0.090
0.056 0.007
0.133 0.020
0.017 0.003
0.120 0.020
0.050 0.005
0.069 0.002
0.087 0.004
0.270 0.030
0.014 0.001
0.131 0.030
0.065 0.007
0.152 0.010
0.073 0.004
0.291 0.030
0.051 0.001
0.131 0.040
0.380 0.090
0.210 0.030
0.251 0.060
0.660 0.030
0.181 0.010
0.606 0.070
0.039 0.006
0.064 0.009
0.067 0.009
0.141 0.020
0.004 0.0004
0.007 0.0009
0.005 0.0008
0.006 0.001
0.004 0.0006
0.023 0.005
0.006 0.0004
0.003 0.0005
>50
0.024 0.005
0.019 0.060
0.161 0.020
0.144 0.019
0.033 0.004
0.156 0.026
0.074 0.001
0.494 0.079
0.209 0.090
0.182 0.035
0.035 0.006
0.201 0.031
0.059 0.009
0.388 0.090
0.522 0.020
0.223 0.048
0.061 0.009
0.279 0.050
0.430 0.030
0.508 0.020
0.385 0.060
0.481 0.030
0.320 0.060
0.803 0.090
0.076 0.002
0.088 0.004
0.069 0.002
0.204 0.020
0.013 0.001
0.011 0.001
0.015 0.001
0.016 0.003
0.002 0.0006
0.007 0.0004
0.008 0.0005
0.005 0.0008
>50
0.022 0.003
0.052 0.090
0.135 0.060
0.271 0.060
0.010 0.006
0.107 0.030
0.075 0.001
0.388 0.010
0.203 0.040
0.325 0.020
0.097 0.003
0.063 0.008
0.056 0.003
0.336 0.030
0.137 0.030
0.306 0.020
0.113 0.060
0.204 0.090
3.142 0.900
0.632 0.060
0.238 0.020
0.554 0.080
0.257 0.060
1.558 0.700
0.065 0.003
0.065 0.004
0.108 0.010
0.311 0.060
0.012 0.002
0.015 0.001
0.015 0.002
0.012 0.001
0.430 0.060
0.692 0.030
0.521 0.070
0.463 0.010
>50
Cl
C2H3
C3H5
C6H5
OC6H5
Cl
II
Cl
C2H3
C3H5
C6H5
OC6H5
Cl
III
IV
SCH3
SCH3
SCH3
SCH3
Cl
C2H3
C3H5
C6H5
OC6H5
Cl
SCH3
SC2H5
SC2H5
SC2H5
SC2H5
SC2H5
SC2H5
OCH3
OC2H5
SCH3
SC2H5
OCH3
OC2H5
SCH3
SC2H5
OCH3
OCH3
OCH3
OCH3
OH
Cl
C2H3
C3H5
H
H
H
H
OH
OH
OH
V
VI
VII
OH
OH
OH
OH
OH
H
C6H5
OC6H5
H
OH
C6H5
48
49c
50
2d
0.0035 0.0005
0.0019 0.0005
4.165 0.100
a
IC50 values are half maximal inhibitory concentrations required to block the growth stimulation of cells. Values represent the mean for three experiments performed in
triplicate.
b
c
All compounds evaluated as trans isomers.
The IC50 value obtained for 49 (0.463
The IC50 value obtained for 2 (0.0035
m
m
M for HT-29) is in good agreement with reported values [39].
M for MCF-7 and 0.474 for HT-29 M) is in good agreement with reported values [39,51,66,67].
d
m
(Type VI) were the most active compounds in all the three cell lines
examined showing nanomolar activities, e.g. compound 43
demonstrated IC50 values of 4, 13 and 12 nM in MCF-7, HL-60 and
HT-29 cells respectively, (Table 3). In contrast, addition of a hy-
droxyl group at the meta-position of the B ring for compound 43 to
yield compound 49 selectively diminished the activity by 40-fold in
HT-29 cells with an IC50 value of 463 nM, while retaining nano-
molar activity in MCF-7 and HL-60 cells. These results suggest that
location of the phenolic OH at the meta-position of ring B is the not
favourable for cytotoxic activity of these compounds in HT-29 cells.
This could indicate that glucoronidation of the phenol at meta po-
The novel compounds 27, 29 and 45 were selected for further
development based on analysis of their drug-like (Lipinski) prop-
erties from a Tier-1 profiling screen, together with predictions of
permeability, metabolic stability, blood brain barrier partition,
plasma protein binding and human intestinal absorption properties
which indicated that they are moderately lipophilic-hydrophilic
drugs and are suitable candidates for further investigation
(Tables S1 and S2, Supporting Information).
Following the initial antiproliferative data obtained, the 3-
thiomethyl compounds 27, 29 and 45 were selected as represen-
tative examples for evaluation in the National Cancer Institute's
Division of Cancer Treatment and Diagnosis (DCTD) Development
Therapeutics Program (DTP) [54]. In the initial NCI one-dose
sition of ring B is favourable for metabolism and inactivation the b-
lactam analogues of CA-4. Taken together, these findings highlight
the potential of B-ring meta-hydroxy group deletion or substitution
in cis-stable analogues of CA-4 as a means of overcoming innate
resistance to CA-4 due to glucuronidation.
(10
mM) 60 cell line screen, using the sulforhodamine B (SRB)
protein assay, compounds 27, 29 and 45 displayed excellent activity
against all cell lines. Subsequent five-dose screening of compounds

A.M. Malebari et al. / European Journal of Medicinal Chemistry 130 (2017) 261e285
271
27, 29 and 45 to evaluate GI50 (50% growth inhibition) and LC50
(50% lethal concentration) was carried out in NCI 60 cell line panel
(Table S3, Supporting information). The GI50 measures the growth
inhibitory power of the test agent, while the LC50 value relates to
the cytotoxic effect. For compound 27, the mean GI50 value across
all cell lines tested was 40 nM. The GI50 values were in the nano-
molar range for all of the panel cell lines investigated, except 5 cell
lines including two of the melanoma cell lines (MALME-3M and
UACC-257 cells), one of the breast cancer cell lines (T-47D cells),
and two of renal cancer lines (TK-10 and UO-31 cells). Particularly of
interest was the activity of compound 27 against MCF-7
(GI50 ¼ 35 nM), HT-29 (GI50 ¼ 32 nM), and HL-60 (GI50 ¼ 23 nM)
cells which are in close agreement with the IC50 values of 65, 59 and
56 nM determined in-house respectively. The outstanding activity
of the 3-hydroxy compound 45 displayed in the one-dose screen
was also evident in excellent broad-spectrum antiproliferative ac-
tivity in the five-dose assay with GI50 values below 30 nM in all but
four of the panel cell lines tested. Activity was demonstrated
against all of the leukemia, prostate and renal cancer cell lines
tested. The mean GI50 value for compound 45 across the panel of
cell lines is 20 nM. The activity of the compound 45 against MCF-7,
HL-60 and HT-29 (GI50 < 10 nM) are in agreement with the IC50
values of 5, 15 and 15 nM respectively determined in-house. The
mean GI50 value for compound 29 across all cell lines tested was
18 nM. LC50 values for compound 27 were greater than 100
mM for
all the panel cell lines and for 45 were also greater than 100
m
M in
all but three cell lines indicating the potential use of this compound
for a wide range of therapeutic applications (Table S3, Supporting
information).
The NCI COMPARE algorithm was useful to compare the anti-
proliferative activities of compounds 27, 29 and 45 with com-
pounds of known mechanism of antiprolifertive action contained in
the NCI Standard Agents Database. The compounds 27, 29 and 45
showed high correlation to tubulin targeting agents such as may-
tansine, rhizoxin and the clinical anticancer drugs vincristine and
vinblastine, (see supporting Information, Table S4).
Fig. 8. Differential effects of (A) 27 and 45 on the cell cycle and apoptosis in MCF-7 cells and (B) 45, 15 and 47 in HT-29 cells. Cells were treated with either vehicle [0.1% ethanol (v/
v)], 15, 27, 45 (0.5 M) and 47 (1 M) for 24, 48 and 72 h. Cells were then fixed, stained with PI, and analyzed by flow cytometry. Cell cycle analysis was performed on histograms of
m
m
gated counts per DNA area (FL2-A). The number of cells with <2N (sub-G1), 2N (G0G1), and 4N (G2/M) DNA content was determined with CellQuest software. Values represent as the
mean for three separate experiments.

272
A.M. Malebari et al. / European Journal of Medicinal Chemistry 130 (2017) 261e285
2.2.4. Cell cycle analysis by flow cytometry
The effect of seleced the -lactam containing compounds on cell
cycle arrest in both sensitive and chemoresistant cell lines was next
investigated. First, we used flow cytometry to analyse the cell cycle
distribution of MCF-7 cells following treatment with the most
27 and 45 (both contain the 4-thiomethyl substituent in Ring B) on
the expression of the pro-apoptotic protein Bax and anti-apoptotic
protein Mcl-1 in HT-29 cells was examined. Western blotting of HT-
29 cell extracts treated with either 27 and 45 at 48 h showed a
decrease in the expression level of the anti-apoptotic protein Mcl-1
and correspondingly an up-regulation in the expression of the pro-
b
potent compounds 27 and 45 at 0.5
treatment. These compounds both contain the thiomethyl sub-
stituent at C4 of Ring B. The -lactams 27 and 45 were found to
mM after 24, 48 and 72 h of
apoptotic protein Bax at 48 h for both concentrations tested (0.1
and 1 M), Fig. 9A. Another marker of apoptosis was confirmed by
PARP cleavage of compounds 27 and 45 at 0.1 M in HT-29 cells
mM
b
m
arrest the cell cycle at G2/M phase compared to the untreated cell
(control) with 65%, 70% and 24% of cells in G2/M phase at 24 h
respectively (Fig. 8A). This was followed by a time-dependant in-
crease in the percentage of cells in the sub-G1 phase, indicative of
apoptosis, with 11% and 12% for 27 and 45 respectively comparing
to the control (4%) at 72 h.
m
(Fig. 9B). Taken together, in combination with cell cycle analysis,
this result indicates that compounds 27 and 45 induce apoptosis in
HT-29 cells.
2.2.6. Tubulin polymerisation studies
The effects of compounds 45 and 15 (compounds containing the
thiomethyl and methoxy substituent at C4 of Ring B respectively)
on cell cycle arrest in HT-29 cells were next investigated. At 0.5 mM
concentration, the two compounds 45 and 15 caused a significant
increase in the percentage of cells in G2/M arrest at 24 h with 68
and 50% of HT-29 cells respectively (p < 0.01) with a concomitant
decrease of cells in the other phases of the cell cycle (G0 and S
phases) (Fig. 8B). This result was similar to that obtained in the
MCF-7 cells. On the other hand, a significant difference was
The effects of representative 4-(4-thiomethylphenyl) b-lactam
CA-4 analogues (compounds 27, 29, 31 and 45) which demon-
strated potent antiproliferative effects in vitro was assessed on the
assembly of purified bovine tubulin. The ability of CA-4 to effec-
tively inhibit the assembly of tubulin was assessed as a positive
control. Tubulin polymerisation was determined by measuring the
increase in absorbance over time at 340 nm. The Vmax value offers
the most sensitive indicator of tubulin/ligand interactions and
hence fold-changes in Vmax values for polymerisation curves of the
compound with reference to ethanol control were calculated.
Tubulin polymerisation studies on 45 showed a 3.5-fold reduction
in the Vmax at 10 mM compared to a 3.4-fold reduction for b-lactams
27 and 31. Compound 29 and CA-4 showed the same inhibition of
tubulin polymerisation by two fold reduction (Fig. 10). This con-
firms that the molecular target of these antiproliferative b-lactams
is tubulin. These binding parallels may rationalise the cytotoxic
potency observed for 27 and 45 in its tubulin effects.
observed when HT-29 cells were treated with b-lactam 47 (com-
pound containing thiomethyl substituent at C4 and OH substituent
at C3 of Ring B). The cells did not result in accumulation in the G2/M
phase. At 72 h there was an increase in apoptosis as determined by
quantification of the sub-G1 cells for 45 and 15 (15% and 19%
respectively) compared to 5% and 6% for 47 and for untreated cells.
The effect of compounds 45 and 15 on the cell cycle resulting in G2/
M arrest followed by apoptosis is typical of tubulin targeting
compounds.
2.2.7. Confocal microscopy
2.2.5. Induction of apoptosis by westeren blots analysis
To further investigate the effects of the novel compounds on
apoptosis in HT-29 cells, the effects of treatment with compounds
To determine the microtubule disrupting effects of the CA-4
analogues in MCF-7 cells, an immunofluoresence assay was per-
formed to study the effects of b-lactams 27 and 45 (both contain the
Fig. 9. (A) b-Lactams 27 and 45 decrease the expression of anti-apoptotic protein Mcl-1 and increase the expression of pro-apoptotic protein Bax in HT-29 cells; (B) Effect of 27 and
45 on PARP cleavage as indicative of apoptosis in HT-29 cells. HT-29 cells were treated with 27 or 45 at the indicated concentrations for 48 h or left untreated (U). Then, the cells
were harvested for western blot analysis to detect the level of the apoptosis related proteins. Results are indicative of three separate experiments, performed independently. To
confirm equal protein loading, each membrane was stripped and reprobed with GAPDH antibody.

A.M. Malebari et al. / European Journal of Medicinal Chemistry 130 (2017) 261e285
273
Fig. 10. Inhibition of tubulin polymerisation by b-lactam analogues. Effect of compounds 27, 29, 31 and 45 on in vitro tubulin polymerisation. Purified bovine tubulin and GTP were
mixed in a 96-well plate. The reaction was started by warming the solution from 4 to 37 ꢁC. Ethanol (1%v/v) was used as a vehicle control. The effect on tubulin assembly was
monitored in a Spectramax 340 PC spectrophotometer at 340 nm at 30 s intervals for 30 min at 37 ꢁC. The graph shows one representative experiment. Each experiment was
performed in triplicate.
Fig. 11. CA-4 (2) and
(0.5 M), or 45 (0.5
dye, and counterstained with DAPI (blue). Images were captured by Leica SP8 confocal microscopy with Leica application suite X software. Representative confocal micrographs of
three separate experiments are shown. Scale bar: 25 M. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
b-lactams 27 and 45 depolymerize the microtubule network of MCF-7 cells. MCF-7 cells were treated with vehicle control [ 1% ethanol (v/v)], 2 (0.1
mM), 27
m
m
M) for 16 h. Cells were fixed in 4% paraformaldehyde and stained with mouse monoclonal anti- -tubulin-FITC antibody (clone DM1A) (green), Alexa Flour 488
a
m
4-thiomethyl substituent in Ring B) on the microtubule structure in
cells. As shown in Fig. 11, cells exposed to 0.1 M of CA-4, or 0.5 mM
of 27 and 45 for 16 h exhibited complete inhibition of microtubule
formation and the microtubule spindles significantly shrunk
around the center of the cells, consistent with the presence of
microtubules. In addition they contained multiple micronuclei, a
phenomenon described as mitotic catastrophe [55]. In contrast,
microtubule structures in HT-29 cells treated with CA-4 and meta-
hydroxylated b-lactam 47 were homogenous and well organized,
similar to the vehicle control, indicating that these compounds
produce minimal effects on the microtubule structure.
m
depolymerized microtubules. In addition,
b-lactam 27 (0.5 mM)
demonstrated less distinct abnormalities of the spindle formation
compared to CA-4 and 45 which indicated a higher concentration is
required for disruption of the microtubules.
To confirm our finding that (i) 15 and 45 (both containing the 4-
thiomethyl substituent in Ring B) strongly inhibited tubulin poly-
merization in HT-29 cells and (ii) CA-4 and the structurally related
2.2.8. Colchicine binding assay
To confirm the binding of our compounds to the colchicine
binding site of tubulin, we carried out a whole cell based assay
using N,N0-ethylene-bis(iodoacetamide) (EBI) as an alkylating agent
that cross-links cysteine residues at positions 239 and 354 in the
Ring B meta-hydroxylated
b-lactam 47 do not inhibit tubulin
colchicine-binding site, forming
adduct is conveniently detectable by Western blotting as an
immunoreactive band that migrates faster than -tubulin [56,57].
Generally, when cells are pre-treated with a colchicine-site inhib-
itor such as Colchicine 1 and CA-4 2, the formation of the -tubulin-
EBI adduct is prevented. MCF-7 cells were initially treated with CA-
4 or a selected -lactam 45 (10 M) for 2 h, followed by addition of
EBI for an additional 1.5 h (Fig. 13A). Control samples (no drug)
show the presence of the -tubulin-EBI adduct at a lower position
a b-tubulin-EBI adduct. This
polymerization in HT-29 cells, the effect of 15 and 45 on cellular
network was examined using immunofluorescence. Colchicine (1)
and CA-4 (2) were also tested for comparison. Confocal analysis of
b
HT-29 cells stained with
a-tubulin mAb demonstrated a well-
b
organized microtubular network in cells treated with vehicle con-
trol (Fig. 12) and in untreated cells (data not shown). Cells exposed
to 1, 15 or 45 for 16 h were characterized by complete loss of
b
m
microtubule
structure
consistent
with
depolymerized
b

274
A.M. Malebari et al. / European Journal of Medicinal Chemistry 130 (2017) 261e285
Fig. 12. Colchicine (1), CA-4 (2) and
b-lactams 27 and 45 depolymerize the microtubule network of HT-29 cells. HT-29 cells were treated with vehicle control [ 1% ethanol (v/v)],
Colchicine (0.1 mM), CA-4 (1 mM), 27 (0.5 mM), 45 (0.5 mM) or 47 (1 mM) for 16 h cells were fixed in 4% paraformaldehyde and stained with mouse monoclonal anti-a-tubulin-FITC
antibody (clone DM1A) (green), Alexa Flour 488 dye, and counterstained with DAPI (blue). Images were captured by Leica SP8 confocal microscopy with Leica application suite X
software. Representative confocal micrographs of three separate experiments are shown. Scale bar: 25
reader is referred to the web version of this article.)
mM. (For interpretation of the references to colour in this figure legend, the
on the gel, indicating that EBI has cross-linked Cys239 and Cys354
on -tubulin. In cells treated with CA-4 and -lactam 45 adduct
ray structure was utilised for the study [44]. It was proposed that 27
and 45 bind to tubulin at the colchicine binding site as we have
b
b
formation is inhibited, indicating that 45 is binding at the colchi-
cine site of tubulin. Similar results were obtained when HT-29 cells
demonstrated that CA-4 and
binding site on -tubulin in the colchicine binding experiment.
The colchicine-binding site in tubulin is mainly buried in the
subunit while maintaining few interactions with the -subunit;
there is one such binding site on each tubulin heterodimer. Two
important residues for binding of colchicine type ligands to tubulin
b-lactam 45 interact with colchicine-
b
were treated with Colchicine and
b-lactam 45, indicating that these
b-
compounds bind at the colchicine-binding site in HT-29 cells
(Fig. 13B).
a
have been identified as Val
enantiomers of 27 (3-S/4-R and 3-R/4-S) and 45 (3-S/4-S and 3-R/4-
R) were considered in the docking calculations.
b318 and Cys b241 [44]. Both trans
2.3. Molecular modelling
Docking approaches have been utilised to postulate on binding
The four stereoisomers were separately docked, 100 binding
poses generated for each conformer and the highest ranked 50
poses for each were retained. 27 (3-S/4-R) was the ranked 1 (green)
and the 3-R/4-S enantiomer (light blue) was ranked 7 out of the 200
ranked poses which are illustrated in Fig. 14. Both enantiomers are
capable of overlaying on the DAMA-colchicine A- and C-rings
thereby forming similar interactions with the surrounding binding
modes of many CA-4 analogues into the colchicine binding site of
and subunits of tubulin and demonstrated a favourable overlap of
the co-crystallised DAMA-colchicine domain [4,58e60]. In order to
investigate the recognition process of our -lactam CA-4 analogues
at the colchicine-binding site of tubulin, a docking study was
retrospectively performed on the most potent -lactam compounds
27 and 45 with the MOE 2015 software package [61] in order to
rationalise possible binding modes and compare to the X-ray
crystal structure of N-deacetyl-N-(2-mercaptoacetyl) colchicine
(DAMA-colchicine). Analogous to other studies, the 1SA0 tubulin X-
a
b
b
b
pocket amino acids. By flipping the orientation of the
b-lactam
carbonyl group by 180ꢁ, the 3-R/4-S enantiomer is able to position
the 3-phenyl and the 4, 4-(methylthiophenyl) rings in the same

A.M. Malebari et al. / European Journal of Medicinal Chemistry 130 (2017) 261e285
275
Fig. 13. Effects of colchicine (1), CA-4 (2) or 45 on the inhibition of the bisthioalkylation of Cys239 and Cys354 of
(A) or HT-29 cells (B). Cells were treated with vehicle control [ethanol 0.1% (v/v)] (U), colchicine (1), CA-4 (2) or 45 (all 10
(100 M) for an additional 1.5 h. Cells were harvested, lysed and analysed using sedimentation and Western blotting for -tubulin and b
b
-tubulin by N,N’-ethylene-bis(iodoacetamide)(EBI) in MCF-7 cells
M) for 2 h; selected samples were then treated with EBI
-tubulin-EBI adduct. Results are indicative of
m
m
b
three separate experiments, performed independently. To confirm equal protein loading, each memberane was stripped and reprobed with GAPDH antibody.
Fig. 14. Overlay of the X-ray structure of tubulin cocrystallised with DAMA-colchicine (PDB entry 1SA0) [44] on the best ranked docked poses of 27 (3-S/4-R) and 27 (3-R/4-S). The
overlays distinctly illustrate the similarities in positioning of the three compounds. Tubulin and DAMA-colchicine are coloured by atom type, 27 (3-S/4-R) is green and 27 (3-R/4-S)
in light blue. The atoms are coloured by element type, carbon ¼ grey, hydrogen ¼ white, oxygen ¼ red, nitrogen ¼ blue, sulphur ¼ yellow. Key amino acid residues are labelled. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
positions as in the 3-S/4-R enantiomer. The carbonyl group is
therefore directed outside the binding pocket and unable to
unable to locate the 4-thiomethylphenyl ring deeper within the
pocket so this is directed outside towards the -tubulin chain. The
a
become involved in possible stabilising interactions with Ala
b
250,
poor ranking score and relative paucity of binding site interactions
as is the case for the 3-S/4-R enantiomer. 45 (3-S/4-S) (green) was
ranked 23 while the best overlaying (3-R/4-R) (light blue) was
ranked 153 as shown in Fig. 15. The binding pose of the S/S enan-
tiomer maps very well to that of the crystallised DAMA-colchicine
e recapitulating the C-ring overlay including the methyl hetero-
atom position and the 3,4,5-trimethoxyphenyl groups which are
co-located. This ring makes multiple hydrophobic contacts within
suggests that the R/R enantiomer is a minor contributor to tubulin
inhibition. Additionally, the S/S
hydrogen bond acceptor interaction with Ala
the compound in the site and the -lactam ring is adjacent to a
small hydrophobic region bordering Lys 254. The protein ligand
b
-lactam carbonyl makes
a
b
250 helping to lock
b
b
interaction for compounds 27 and 45 are displayed in Figs. S5 and
S6 respectively (Supporting information). Docking analysis sug-
gests that both trans isomer enantiomers of 27 and 45 are able to
recapitulate the colchicine binding mode and therefore contribute
to inhibition of tubulin polymerisation. The X-ray structure of a 1,4-
the subpocket delineated by Val
group hydrogen bonds with Cys
displays the trimethoxy phenyl mapping onto the A-ring but is
b
318 at the bottom and methoxy
b241. The R/R enantiomer also

276
A.M. Malebari et al. / European Journal of Medicinal Chemistry 130 (2017) 261e285
Fig. 15. Overlay of the X-ray structure of tubulin cocrystallised with DAMA-colchicine (PDB entry 1SA0) [44] on the best ranked docked poses of 45 (3-S/4-S) and 45 (3-R/4-R). The
overlays distinctly illustrate the similarities in positioning of the 3,4,5-trimethoxyphenyl moieties of three compounds. Tubulin and DAMA-colchicine are coloured by atom type, 45
(3-S/4-S) is green and 45 (3-R/4-R) in light blue. The atoms are coloured by element type, carbon ¼ grey, hydrogen ¼ white, oxygen ¼ red, nitrogen ¼ blue, sulphur ¼ yellow. Key
amino acid residues are labelled. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
diaryl-2-azetidinone co-crystallised in the colchicine binding site of
tubulin has been recently reported [29]. The enantioselective syn-
thesis of 27 and 45 are in progress which will allow determination
of the optimum configuration of these compounds for biochemical
activity.
4. Experimental section
4.1. Chemistry
All reagents were commercially available and were used
without further purification unless otherwise indicated. Anhydrous
solvents were obtained by distillation from the indicated systems
immediately prior to use: Dichloromethane from calcium hydride
and toluene from sodium. Uncorrected melting points were
measured on a Gallenkamp apparatus. Infra-red (IR) spectra were
recorded on a Perkin Elmer FT-IR Paragon 1000 spectrometer. 1H
and 13C nuclear magnetic resonance spectra (NMR) were recorded
at 27 ꢁC on a Brucker DPX 400 spectrometer (400.13 MHz, 1H;
100.61 MHz, 13C) in CDCl3 (internal standard tetramethylsilane
(TMS)) For CDCl3, 1H NMR spectra were assigned relative to the TMS
peak at 0.00 ppm and 13C NMR spectra were assigned relative to the
middle CDCl3 peak at 77.0 ppm. Electrospray ionisation mass
spectrometry (ESI-MS) was performed in the positive ion mode on
3. Conclusion
We suggest that increased expression of UGTs in the chemo-
resistant HT-29 colon cancer cell line leads to glucuronidation of
CA-4 at the meta-hydroxy group of Ring B, and subsequently to CA-
4 resistance in this cell line. We now report the rational modifica-
tion of CA-4 to prevent inactivation by either isomerisation or
glucuronidation leading to the identification of a panel
analogue with improved activity over CA-4 in CA-4 resistant HT-
29 cells. We have successfully demonstrated a novel series of
b-lactam
b
-
lactam analogues that are characterized by greater improvement in
metabolic stability towards the glucuronidation pathway in HT-
a
liquid chromatography time-of-flight mass spectrometer
29 cells. This was achieved by designing a panel of
logues with deletion of standard CA-4 Ring B meta-OH substituent
in these -lactams to prevent direct metabolic glucuronidation.
Introduction of ethoxy or thiomethyl substitution in place of the
methoxy group at C-4 of ring B of these -lactams was demon-
strated to be effective, possibly preventing indirect glucuronidation
by blocking the sites of metabolism via O-demethylation. The
lactam compounds 27 and 45, both containing the 4-thiomethyl
substituent in Ring B exhibited IC50 values of 56 nM and 15 nM
respectively in the chemoresistant HT-29 colon cancer cell line,
while retaining impressive antiproliferative activity at nanomolar
levels against a range of susceptible human cancer cell lines. These
compounds were also shown to initiate apoptosis selectively via a
mechanism involving inhibition of tubulin polymerization. These
data provide important structureeactivity information that may be
used in the design of superior combretastatin analogues that may
advance the clinical development of CA-4 in the treatment of CA-4
resistant cell lines. Taken together, these findings highlight the
potential of chemical manipulation as a means of overcoming drug
resistance in colon cancer mediated by glucuronidation. Further-
more, the data obtained may not only influence the design of CA-4
derivatives but also other molecules prone to inactivation by means
of glucuronidation.
b-lactam ana-
(Micromass LCT, Waters Ltd., Manchester, UK). The samples were
introduced to the ion source by an LC system (Waters Alliance 2795,
Waters Corporation, USA) in acetonitrile: water (60:40 %v/v) at
b
200 mL/min. The capillary voltage of the mass spectrometer was at
b
3 kV. The sample cone (de-clustering) voltage was set at 40 V. For
exact mass determination, the instrument was externally calibrated
for the mass range m/z 100 to m/z 1000. A lock (reference) mass (m/
z 556.2771) was used. TLC was performed using Merck Silica gel 60
TLC aluminium sheets with fluorescent indicator visualizing with
UV light at 254 nm. Flash chromatography was carried out using
standard silica gel 60 (230e400 mesh) obtained from Merck. All
products isolated were homogenous on TLC. The purity of the
tested compounds was determined by HPLC, the purity level was
ꢂ95%. Analytical high-performance liquid chromatography (HPLC)
was performed using a Waters 2487 Dual Wavelength Absorbance
detector, a Waters 1525 binary HPLC pump and a Waters 717 plus
Autosampler. The column used was a Varian Pursuit XRs C18
reverse phase 150 ꢃ 4.6 mm chromatography column. Samples
were detected using a wavelength of 254 nm. All samples were
analyzed using acetonitrile (70%): water (30%) over 10 min and a
flow rate of 1 mL/min. Microwave experiments were carried using a
Biotage Discover CEM microwave synthesiser on standard power
b
-

A.M. Malebari et al. / European Journal of Medicinal Chemistry 130 (2017) 261e285
277
setting (maximum power supplied is 300 watts) unless otherwise
stated.
4.1.3.2. (E)-1-(4-Ethoxyphenyl)-N-(3,4,5-trimethoxyphenyl)meth-
animine (10).
(E)-1-(4-Ethoxyphenyl)-N-(3,4,5-trimethoxyphenyl)methanimine
(10) was synthesized using the general method II above and was
obtained from 4-ethoxybenzaldehyde and 3,4,5-trimethoxyaniline
as yellow crystals, Yield 78%, Mp 99 ꢁC. Purity: (HPLC) 98.5%. IRnmax
4.1.1. 4-(Ethylthio)benzaldehyde
4-(Ethylthio)benzaldehyde was prepared from ethanthiol
(10 mmol) and 4-flourobenzaldehyde (15 mmol) in the presence of
potassium carbonate (5 mmol) in DMF (10 mL). The reaction
mixture was heated to 200 ꢁC and stirred for 16 h. The compound
was purified by column chromatography over silica gel (eluent:
hexane: ethyl acetate, 6:1) to afford the product as brown oil in 96%
1739.5 (C¼N) cmꢀ1. 1H NMR (400 MHz, CDCl3):
1.43 (t, J ¼ 7.02 Hz,
d
3 H), 3.84 (s, 3 H), 3.88 (s, 6 H), 4.09 (q, J ¼ 6.71 Hz, 2 H), 6.45 (s, 2 H),
6.95 (d, J ¼ 8.55 Hz, 2 H), 7.80 (d, J ¼ 8.54 Hz, 2 H), 8.37 (s, 1 H). 13
C
NMR (100 MHz, CDCl3):
d 14.71, 56.09, 60.98, 63.64, 76.67, 76.99,
yield. Purity (HPLC): 99%. IR nmax: 1672.0 (C¼O), 3426.0 (OH) cmꢀ1
.
77.30, 98.11, 114.66, 130.43, 153.52, 159.11, 161.68. HRMS: calculated
for C18H22NO4 [MþþH] 316.1543; found 316.1530.
1H NMR (400 MHz, CDCl3):
d
ppm 1.36 (t, J ¼ 7.32 Hz, 2 H), 3.01 (q,
J ¼ 7.32 Hz, 3 H), 7.32 (m, J ¼ 8.54 Hz, 2 H), 7.71e7.76 (m, 2 H), 9.89
(s, 1 H); 13C NMR (100 MHz, CDCl3):
d 14.12, 25.22, 130.35, 131.84,
4.1.3.3. (E)-1-(4-(Methylthio)phenyl)-N-(3,4,5-trimethoxyphenyl)
methanimine (11).
161.44, 190.79. HRMS: calculated for C9H10OS [MþþH] 166.0452;
(E)-1-(4-(Methylthio)phenyl)-N-(3,4,5-trimethoxyphenyl)meth-
animine (11) was obtained using the general method II above from
4-methylthiobenzaldehyde and 3,4,5-trimethoxyaniline as yellow
crystals, yield 91%, Mp 110 ꢁC, purity (HPLC) 98.2%. IR nmax: 1584.5
found 166.0432.
4.1.2. General method I: silylation of phenols
To a solution of the appropriately substituted phenolic benzal-
dehyde (10 mmol) and tert-butyl-dimethylsilylchloride (12 mmol)
in dry DCM (30 mL) under a nitrogen atmosphere, 1,8-diazobicyclo
[5.4.0]undec-7-ene (DBU) (17 mmol) was added dropwise via a
syringe. The resulting mixture was stirred at room temperature
under a nitrogen atmosphere until reaction was complete by thin
layer chromatography. The solution was then diluted with DCM
(10 mL) and washed successfully with water (30 mL), 0.1 M HCl
(20 mL) and saturated aqueous.
NaHCO3 (30 mL), retaining the organic each time, before drying
over anhydrous sodium sulfate. The solvent was removed by
evaporation under reduced pressure to yield the protected benz-
aldehyde, which was used immediately without further purifica-
tion as starting material for the synthesis of the following protected
Schiff bases. Aldehyde 8 was prepared and characterised as
described previously by us [31].
(C¼N) cmꢀ1. 1H NMR (400 MHz, CDCl3):
d 2.52 (s, 3H), 3.81 (s, 3 H),
3.91 (s, 6 H), 6.51 (s, 2H), 7.29 (d, J ¼ 8.54 Hz, 2 H), 7.82 (d, J ¼ 8.54,
2 H), 8.40 (s, 1 H). 13C NMR (100 MHz, CDCl3):
d 15.07, 56.11, 60.99,
98.17, 125.68, 129.01, 137.03, 147.95, 153.55, 158.93. HRMS: calcu-
lated for C17H19NNaO3S [MþþNa] 340.0983; found 340.0993.
4.1.3.4. (E)-1-(4-(Ethylthio)phenyl)-N-(3,4,5-trimethoxyphenyl)
methanimine (12).
(E)-1-(4-(Ethylthio)phenyl)-N-(3,4,5-trimethoxyphenyl)meth-
animine (12) was synthesized using the general method II above
and was obtained from 4-ethylthiobenzaldehyde and 3,4,5-
trimethoxyaniline as yellow crystals, yield 80%, Mp 122 ꢁC, purity
(HPLC) 100%. IR nmax 1675.5 (C¼N) cmꢀ1. 1H NMR (400 MHz, CDCl3):
d
ppm 1.36 (t, J ¼ 7.32 Hz, 3 H), 3.01 (q, J ¼ 7.32 Hz, 2 H), 3.83 (s, 6 H),
3.91 (s, 3 H), 6.46 (s, 2 H), 7.33 (m, J ¼ 7.93 Hz, 2 H), 7.77 (m,
J ¼ 8.54 Hz, 2 H), 8.40 (s, 1 H). 13C NMR (100 MHz, CDCl3):
d 14.03,
26.52, 56.11, 60.98, 98.17, 127.34, 129.02, 133.07, 136.37, 141.85,
4.1.2.1. 4-((Tert-butyldimethylsilyl)oxy)benzaldehyde)
4-((Tert-butyldimethylsilyl)oxy)benzaldehyde) (7) was prepared as
(7).
147.92, 153.55, 158.91. HRMS: calculated for
C18H21NNaO3S
[MþþNa] 354.1140; found 354.1154.
described above from 4-hydroxybenzaldehyde and was obtained as
a
colourless oil; Yield: 80%. Purity (HPLC): 99.1%. 1H NMR
4.1.3.5. (E)-1-(4-((Tert-butyldimethylsilyl)oxy)phenyl)-N-(3,4,5-
trimethoxyphenyl)methanimine (13).
(400 MHz,CDCl3):
d
0.20 (s, 6 H), 0.95 (s, 9 H), 6.90 (d, J ¼ 8.54 Hz,
2 H), 7.74 (d, J ¼ 8.54 Hz, 2 H), 9.83 (s, 1 H). 13C NMR (100 MHz,
(E)-1-(4-((Tert-butyldimethylsilyl)oxy)phenyl)-N-(3,4,5-
trimethoxyphenyl)methanimine (13) was synthesized as described
above using the general Method II from 4-((tert-butyldimethylsilyl)
oxy)benzaldehyde) 7 and 3,4,5-trimethoxyaniline as a brown oil,
CDCl3):
d
ꢀ4.42, 18.18, 25.50, 130.35, 131.84, 161.44, 190.79; HRMS:
calculated for C13H21OSi [MþH]þ 237.1311; found 237.1315.
yield 87%, Purity (HPLC) 97.5%. 1H NMR (400 MHz, CDCl3):
d ppm
4.1.3. General method II: preparation of imines
The appropriately substituted benzaldehyde (10 mmol) and
substituted aniline (10 mmol) were heated together at reflux in
ethanol (40 mL) for 4 h. The reaction mixture was reduced by
evaporation in vacuo and the resulting solid product was recrys-
tallised from ethanol. Imine 14 was prepared and characterised as
described previously by us [31].
0.19e0.25 (m, 6 H), 0.95e0.99 (m, 9 H), 3.77 (s, 3 H), 3.80 (s, 6 H),
6.44 (s, 2 H), 6.83e7.01 (m, 2 H), 7.76 (d, J ¼ 8.54 Hz, 2 H), 8.37 (s,
1 H). 13C NMR (100 MHz, CDCl3):
d
ꢀ4.40, 18.37, 25.52, 55.88, 56.07,
60.96, 92.65, 98.12, 120.44, 120.48, 129.55, 131.88, 148.24, 153.51,
159.26. HRMS: calculated for C22H31NNaO4Si [MþþNa] 424.1920;
found 424.1929.
4.1.3.1. (E)-1-(4-Methoxyphenyl)-N-(3,4,5-trimethoxyphenyl)meth-
animine (9).
4.1.4. General method III: preparation of 2-azetidinones (staudinger
reaction)
(E)-1-(4-Methoxyphenyl)-N-(3,4,5-trimethoxyphenyl)meth-
animine (9) was synthesized using the general method II above and
was obtained from 4-methoxybenzaldehyde and 3,4,5 trimethox-
yaniline as pale yellow crystals, Yield: 89%, Mp 120 ꢁC [31], Purity
The appropriate imine (5 mmol) and the appropriate acid
chloride (7 mmol) were dissolved in dry toluene (50 mL), under
nitrogen atmosphere with stirring at 0 ꢁC. The solution was allowed
to reach room temperature and then warmed to 100 ꢁC. Dry trie-
thylamine TEA (9 mmol) was added dropwise. The mixture was
warmed at 100 ꢁC for 5 h and stirred at room temperature over-
night until the starting material had disappeared as monitored by
TLC. The reaction mixture washed with water (2 ꢃ 100 mL), dried
over anhydrous Na2SO4 and solvent was removed by evaporation in
vacuo. The crude product was purified by flash chromatography
(HPLC) 97.2%. IRnmax: 1604.3 (C¼N) cmꢀ1
.
1H NMR (400 MHz,
CDCl3):
d
3.84 (s, 6 H), 3.97 (s, 6 H), 6.50 (s, 2H), 7.87 (d, J ¼ 8.03 Hz,
2 H), 7.00 (d, J ¼ 8.53 Hz, 3 H), 8.42 (s, 1H). 13C NMR (100 MHz,
CDCl3): d 55.65, 60.57, 97.6, 113.8, 128.5, 135.6, 147.7, 153.08, 158.69,
161.9. HRMS: calculated for C17H19NNaO4 [MþNa]þ 324.1212; found
324.1205.

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A.M. Malebari et al. / European Journal of Medicinal Chemistry 130 (2017) 261e285
over silica gel (eluent: 5:1; n-hexane: ethyl acetate). Compounds 4,
47, 48 were prepared and characterised as described previously by
us [31e33,35].
from imine 9 and dichloroacetyl chloride, to afford the desired
product as a yellow powder, yield 63%, Mp 119e120 ꢁC, Purity
(HPLC): 99.4%.
IR nmax: 1759.8 (C¼O) cmꢀ1, 1H NMR (400 MHz, CDCl3):
d ppm
4.1.4.1. 4-(4-Methoxyphenyl)-3-phenyl-1-(3,4,5-trimethoxyphenyl)
azetidin-2-one (15).
3.69 (s, 6 H), 3.76 (s, 3 H), 3.81 (s, 3 H), 5.40 (s,1 H), 6.52 (s, 2 H), 6.93
(d, J ¼ 8.54 Hz, 2 H), 7.21e7.28 (m, 2 H). 13C NMR (100 MHz, CDCl3):
4-(4-Methoxyphenyl)-3-phenyl-1-(3,4,5-trimethoxyphenyl)azeti-
din-2-one (15) was synthesized using the general method III above
from imine 9 and phenylacetyl chloride to afford the product as
pale yellow crystals, yield: 56%, Mp 113e114 ꢁC [31], Purity (HPLC):
d 55.31, 56.11, 60.94, 74.06, 83.44, 95.89, 114.32, 123.36, 129.20,
131.87, 135.69, 153.61, 158.36, 160.79. HRMS: calculated for
C
19H19Cl2NNaO5 [MþNa]þ, 434.0538; found 434.0523.
97%. IR nmax: 1744.4 (C¼O) cmꢀ1
.
1H NMR (400 MHz, CDCl3):
4.1.4.6. 4-(4-Methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-3-
vinylazetidin-2-one (19).
d
ppm 3.70 (s, 6 H), 3.75 (s, 3 H), 3.81 (s, 3 H), 4.26 (d, J ¼ 1.83 Hz,
1 H), 4.84 (d, J ¼ 1.83 Hz, 1 H), 6.58 (s, 2 H), 6.92 (d, J ¼ 8.54 Hz, 2 H),
4-(4-Methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-3-
vinylazetidin-2-one (19) was synthesized using the general method
III above from imine 9 and crotonyl chloride to afford the desired
product as a yellow powder, yield 46%, Mp 95e96 ꢁC, purity (HPLC):
7.29e7.39 (m, 7 H). 13C NMR (100 MHz, CDCl3):
d
55.33, 56.00, 60.91,
63.81, 65.01, 94.86, 114.66, 127.30, 127.38, 128.99, 165.39. HRMS:
calculated for C25H25NNaO5 [MþþNa], 442.1630; found 442.1642.
98.4%. IR nmax: 1736.1 (C¼O) cmꢀ1. 1H NMR (400 MHz, CDCl3):
d 3.68
4.1.4.2. Trans-4-(4-Methoxyphenyl)-3-phenoxy-1-(3,4,5-
trimethoxyphenyl)azetidin-2-one (16trans).
Trans-4-(4-Methoxyphenyl)-3-phenoxy-1-(3,4,5-
(s, 6 H), 3.71e3.73 (m, 1 H), 3.74 (s, 3 H), 3.79 (s, 3 H), 4.70 (d,
J ¼ 2.44 Hz, 1 H), 5.27e5.39 (m, 2 H), 5.98 (dd, J ¼ 9.77, 7.32 Hz, 1 H),
6.53 (s, 2 H), 6.90 (d, J ¼ 8.54 Hz, 2 H), 7.28 (d, J ¼ 8.54 Hz, 2 H). 13
C
trimethoxyphenyl)azetidin-2-one (16trans) was synthesized
following the general method III above from imine 9 and phenox-
yacetyl chloride to afford the product as a cream powder, yield 56%,
Mp 161e163 ꢁC [35], Purity (HPLC): 100%. IR nmax: 1740.4 (C¼O)
NMR (100 MHz, CDCl3): d 55.32, 56.00, 60.91, 61.35, 63.87, 94.73,
114.58, 119.80, 127.25, 129.14, 130.55, 133.81, 153.45, 159.87, 165.31.
HRMS: calculated for
392.1459.
C
21H23NNaO5[MþþNa], 392.1474; found
cmꢀ1. 1H NMR (400 MHz, CDCl3):
d ppm 3.73 (s, 6 H), 3.79 (s, 3 H),
3.87 (s, 3 H), 4.96 (d, J ¼ 2.11 Hz, 1 H), 5.14 (d, J ¼ 2.11 Hz, 1 H), 6.59
4.1.4.7. 4-(4-Methoxyphenyl)-3-(prop-1-en-2-yl)-1-(3,4,5-
trimethoxyphenyl)azetidin-2-one (20).
4-(4-Methoxyphenyl)-3-(prop-1-en-2-yl)-1-(3,4,5-
(s, 2 H), 6.89 (d, J ¼ 8.03 Hz, 2 H), 6.97e7.07 (m, 3 H), 7.24e7.32 (m,
4 H). 13C NMR (100 MHz, CDCl3):
d
55.18, 56.04, 60.91, 62.06, 81.11,
95.32, 113.90, 115.69, 122.18, 124.40, 129.44, 133.08, 153.49, 164.3.
HRMS: calculated for C25H25NNaO6 [MþþNa], 458.1580; found
458.1568.
trimethoxyphenyl)azetidin-2-one (20) was synthesized using the
general method III above from imine 9 and dimethylacryloyl
chloride to afford the desired product as a yellow powder, yield
52%, Mp 101e102 ꢁC, purity (HPLC): 95.5%. IR nmax: 1735.3 (C¼O)
4.1.4.3. Cis-4-(4-Methoxyphenyl)-3-phenoxy-1-(3,4,5-
trimethoxyphenyl)azetidin-2-one (16cis).
cmꢀ1. 1H NMR (400 MHz, CDCl3):
d ppm 1.85 (s, 3 H), 3.69 (s, 6 H),
3.74 (s, 3 H), 3.79 (s, 3 H), 3.80 (br. s, 1 H), 4.73 (d, J ¼ 2.44 Hz, 1 H),
Cis-4-(4-Methoxyphenyl)-3-phenoxy-1-(3,4,5-trimethoxyphenyl)
azetidin-2-one (16cis) was synthesized following the general
method III above from imine 9 and phenoxyacetyl chloride to afford
the product as a creamy powder, yield 14%, Mp 170e180 ꢁC, Purity
4.98 (s, 1 H), 5.04 (s, 1 H), 6.54 (s, 2 H), 6.90 (d, J ¼ 8.55 Hz, 2 H), 7.28
(d, J ¼ 8.55 Hz, 2 H). 13C NMR (100 MHz, CDCl3):
d 20.15, 54.91,
55.56, 59.87, 60.51, 66.48, 94.22, 113.97, 114.16, 126.82, 129.08,
133.35, 133.89, 137.73, 153.03, 159.39, 164.89. HRMS: calculated for
(HPLC): 95.3%. IR nmax: 1751.4 (C¼O) cmꢀ1
.
1H NMR (400 MHz,
C
22H25NNaO5 [MþþNa], 406.1630; found 406.1627.
CDCl3):
d ppm 3.71 (s, 6 H), 3.75 (s, 3 H), 3.80 (s, 3 H), 5.56 (d,
J ¼ 4.52 Hz, 1 H), 5.35 (d, J ¼ 4.52 Hz, 1 H), 6.64 (s, 2 H), 6.89 (d,
4.1.4.8. 4-(4-Ethoxyphenyl)-3-phenyl-1-(3,4,5-trimethoxyphenyl)
azetidin-2-one (21).
J ¼ 8.03 Hz, 2 H), 6.95e6.71 (m, 3 H), 7.20 (d, J ¼ 7.78 Hz 2 H), 7.36
(d, J ¼ 8.53 Hz, 2 H). 13C NMR (100 MHz, CDCl3):
d
55.42, 56.19,
4-(4-Ethoxyphenyl)-3-phenyl-1-(3,4,5-trimethoxyphenyl)azeti-
din-2-one (21) was synthesized using the general method III above
from imine 10 and phenylacetyl chloride to afford the desired
product as a yellow powder, yield 51%, Mp 113e114 ꢁC [32], purity.
(HPLC): 98.3%. IR nmax: 1736.4 (C¼O) cmꢀ1. 1H NMR (400 MHz,
60.79, 62.25, 81.01, 105.25, 114.39, 115.66, 122.19, 128.15, 129.22,
130.46, 153.15, 162.39. HRMS: calculated for
C25H25NNaO6
[MþþNa], 458.1580; found 458.1587.
4.1.4.4. 3-Chloro-4-(4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)
azetidin-2-one (17).
CDCl3):
d
ppm 1.40 (t, J ¼ 6.71 Hz, 3 H), 3.70 (s, 6 H), 3.75 (s, 3 H),
4.02 (q, J ¼ 6.71 Hz, 2 H), 4.26 (d, J ¼ 2.44 Hz, 1 H), 4.84 (d,
3-Chloro-4-(4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)azeti-
din-2-one (17) was synthesized following the general method III
above from imine 9 and chloroacetyl chloride to afford the desired
product as a cream powder, yield 44%, Mp 117e118 ꢁC, purity
J ¼ 2.44 Hz, 1 H), 6.59 (s, 2 H), 6.91 (d, J ¼ 8.54 Hz, 2 H), 7.29e7.39
(m, 7 H). 13C NMR (100 MHz, CDCl3):
d 14.76, 55.99, 60.91, 63.55,
63.85, 64.99, 94.85, 115.17, 127.28, 127.39, 127.83, 128.98, 153.47,
162.12. HRMS: calculated for C26H26NO5 [Mþ-H] 432.1811; found
432.1822.
(HPLC): 99.1%. IR nmax: 1746.5 (C¼O) cmꢀ1
.
1H NMR (400 MHz,
CDCl3): d 3.69 (s, 6 H), 3.75 (s, 3 H), 3.80 (s, 3 H), 4.56e4.60 (m, 1 H),
4.88e4.93 (m, 1 H), 6.51 (s, 2 H), 6.92 (d, J ¼ 8.54 Hz, 2 H), 7.29 (d,
4.1.4.9. Trans-4-(4-Ethoxyphenyl)-3-phenoxy-1-(3,4,5-
trimethoxyphenyl)azetidin-2-one (22trans).
J ¼ 8.54 Hz, 2 H). 13C NMR (100 MHz, CDCl3):
d ppm 55.36, 56.04,
60.92, 63.18, 66.11, 95.29, 109.99, 114.80, 126.79, 127.50, 132.92,
Trans-4-(4-Ethoxyphenyl)-3-phenoxy-1-(3,4,5-trimethoxyphenyl)
azetidin-2-one (22trans) was synthesized using the general method
III above from imine 10 and phenoxyacetyl chloride to afford the
desired product as a yellow powder, yield 52%, Mp 148e149 ꢁC,
153.52,
C
159.95,
160.68.
HRMS:
calculated
for
19H20ClNNaO5[MþþNa], 400.0928; found 400.0930.
4.1. 4. 5 . 3, 3 - Dich l oro - 4 - ( 4 - me t h oxy p h e nyl) - 1 - ( 3 , 4, 5 -
trimethoxyphenyl)azetidin-2-one (18).
3,3-Dichloro-4-(4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)
azetidin-2-one (18) was synthesized using the general method III
purity (HPLC): 100%. IR nmax
(400 MHz, CDCl3):
:
1750.4 (C¼O) cmꢀ1
.
1H NMR
d
ppm 1.41 (t, J ¼ 7.02 Hz, 3 H), 3.67 (s, 6 H), 3.74
(s, 3 H), 4.03 (q, J ¼ 6.71 Hz, 2 H), 4.90 (d, J ¼ 2.14 Hz, 1 H), 5.12 (d,
J ¼ 2.14 Hz, 1 H), 6.54 (s, 2 H), 6.84 (m, J ¼ 7.93 Hz, 2 H), 6.90e7.01

A.M. Malebari et al. / European Journal of Medicinal Chemistry 130 (2017) 261e285
279
(m, 3 H), 7.22 (d, J ¼ 7.32 Hz, 2 H), 7.31 (d, J ¼ 8.54 Hz, 2 H). 13C NMR
4.1.4.14. 4-(4-Ethoxyphenyl)-3-(prop-1-en-2-yl)-1-(3,4,5-
trimethoxyphenyl)azetidin-2-one (26).
(100 MHz, CDCl3):
d 14.74, 55.98, 60.90, 63.59, 64.09, 87.21, 95.32,
115.28, 122.23, 127.19, 129.61, 133.11, 134.88, 153.44, 157.00, 159.65,
162.57. HRMS: calculated for C26H27NNaO6 [MþþNa] 472.1736;
found 472.1733.
4-(4-Ethoxyphenyl)-3-(prop-1-en-2-yl)-1-(3,4,5-
trimethoxyphenyl)azetidin-2-one (26) was synthesized using the
general method III above from imine 10 and dimethylacryloyl
chloride to afford the title product as a yellow powder, yield 46%,
Mp 102 ꢁC, purity (HPLC): 98.4%. IR nmax: 1735.2 (C¼O) cmꢀ1
.
1H
4.1.4.10. Cis-4-(4-Ethoxyphenyl)-3-phenoxy-1-(3,4,5-
trimethoxyphenyl)azetidin-2-one (22cis).
Cis-4-(4-Ethoxyphenyl)-3-phenoxy-1-(3,4,5-trimethoxyphenyl)
azetidin-2-one (22cis) was synthesized using the general method
III above from imine 10 and phenoxyacetyl chloride to afford the
NMR (400 MHz, CDCl3):
d
ppm 1.39 (t, J ¼ 7.02 Hz, 3 H), 1.85 (s, 3 H),
3.69 (s, 7 H), 3.74 (s, 3 H), 4.01 (q, J ¼ 7.32 Hz, 2 H), 4.72 (d,
J ¼ 1.83 Hz, 1 H), 4.98 (s, 1 H), 5.04 (s, 1 H), 6.54 (s, 2 H), 6.88 (d,
J ¼ 8.54 Hz, 2 H), 7.27 (d, J ¼ 8.54 Hz, 2 H). 13C NMR (100 MHz,
CDCl3):
d 14.62, 55.80, 60.43, 61.28, 63.68, 102.03, 117.17, 119.78,
desired product as a oil, yield 12%, purity (HPLC): 96.5%. IR nmax
:
120.65, 127.43, 129.98, 132.38, 133.11, 134.72, 138.07, 153.53, 165.16.
HRMS: calculated for C23H27NNaO5 [MþþNa] 420.1787; found
420.1787.
1755.1 (C¼O) cmꢀ1
. d ppm 1.37 (t,
1H NMR (400 MHz, CDCl3):
J ¼ 7.05 Hz, 3 H), 3.71 (s, 6 H), 3.76 (s, 3 H), 3.90 (q, J ¼ 6.60 Hz, 2 H),
5.30 (d, J ¼ 4.98 Hz, 1 H), 5.51 (d, J ¼ 4.98 Hz 1 H), 6.61 (s, 2 H), 6.80
(dd, J ¼ 8.09, J ¼ 4.35 Hz, 2 H), 6.89e7.00 (m, 3 H), 7.10 (d, J ¼ 7.11 Hz,
4.1.4.15. 4-(4-(Methylthio)phenyl)-3-phenyl-1-(3,4, 5-
trimethoxyphenyl)azetidin-2-one (27).
2 H), 7.30 (d, J ¼ 8.71 Hz, 2 H). 13C NMR (100 MHz, CDCl3):
d 14.72,
56.04, 60.91, 62.10, 63.38, 81.11, 95.33,114.42,122.16,124.22,129.25,
133.11, 134.96, 153.49, 159.98, 159.32, 162.99. HRMS: calculated for
4-(4-(Methylthio)phenyl)-3-phenyl-1-(3,4,5-trimethoxyphenyl)
azetidin-2-one (27) was synthesized following the general method
III above from imine 11 and phenylacetyl chloride to afford the title
product as a yellow powder, yield 55%, Mp 114 ꢁC, purity (HPLC):
C
26H27NNaO6 [MþþNa] 472.1736; found 472.1730.
4.1.4.11. 3-Chloro-4-(4-ethoxyphenyl)-1-(3,4,5-trimethoxyphenyl)
azetidin-2-one (23).
99.4%. IR nmax: 1741.8 (C¼O) cmꢀ1. 1H NMR (400 MHz, CDCl3):
d ppm
2.48 (s, 3 H), 3.71 (s, 6 H), 3.76 (s, 3 H), 4.25 (d, J ¼ 2.44 Hz, 1 H), 4.85
(d, J ¼ 2.44 Hz, 1 H), 6.58 (s, 2 H), 7.29e7.34 (m, 5 H), 7.34e7.39 (m,
3-Chloro-4-(4-ethoxyphenyl)-1-(3,4,5-trimethoxyphenyl)azetidin-
2-one (23) was synthesized using the general method III above
from imine 10 and chloroacetyl chloride to afford the desired
product as a yellow powder, yield 46%, Mp 99e100 ꢁC, purity
4 H). 13C NMR (100 MHz, CDCl3):
d 15.52, 55.41, 56.04, 63.74, 64.45,
94.86, 126.43, 126.95, 127.38, 127.94, 129.04, 153.54, 163.12. HRMS:
calculated for C25H24NO4S [M - H]þ 434.1426; found 434.1421.
(HPLC): 100%. IR nmax: 1756.1 (C¼O) cmꢀ1
.
1H NMR (400 MHz,
ppm 1.36e1.42 (m, 3 H), 3.71 (s, 6 H), 3.74 (s, 3 H),
CDCl3):
d
4.1.4.16. Trans-4-(4-(Methylthio)phenyl)-3-phenoxy-1-(3,4,5-
trimethoxyphenyl)azetidin-2-one (28trans).
Trans-4-(4-(Methylthio)phenyl)-3-phenoxy-1-(3,4,5-
3.97e4.05 (m, 2 H), 4.58 (d, J ¼ 2.22 Hz, 1 H), 4.89 (d, J ¼ 2.22 Hz,
1 H), 6.50 (s, 2 H), 6.85e6.94 (m, 2 H), 7.21e7.30 (m, 2 H). 13C NMR
(100 MHz, CDCl3):
d
14.69, 56.03, 60.91, 63.17, 63.61, 66.15, 95.28,
trimethoxyphenyl)azetidin-2-one (28trans) was synthesized
following the general method III above from imine 11 and phe-
noxyacetyl chloride to afford the title product as yellow crystals,
yield 35%, Mp 129e130 ꢁC, purity (HPLC): 98.5%. IR nmax: 1737.1
115.30, 126.59, 127.49, 132.94, 135.10, 153.51, 159.87, 160.71. HRMS:
calculated for C20H22ClNNaO5 [MþNa]þ 414.1084; found 414.1084.
(C¼O) cmꢀ1. 1H NMR (400 MHz, CDCl3):
d 2.49 (s, 3 H), 3.69 (s, 6 H),
4 .1. 4 .12 . 3 , 3 - D i ch l o ro - 4 - ( 4 - e t h o x y p h e n yl ) - 1 - ( 3 , 4 , 5 -
trimethoxyphenyl)azetidin-2-one (24).
3.75 (s, 3 H), 4.92 (d, J ¼ 2.14 Hz, 1 H), 5.09 (d, J ¼ 2.14 Hz, 1 H), 6.54
(s, 2 H), 6.85 (d, J ¼ 8.54 Hz, 2 H), 7.00 (d, J ¼ 7.32 Hz, 3 H), 7.29e7.35
3,3-Dichloro-4-(4-ethoxyphenyl)-1-(3,4,5-trimethoxyphenyl)aze-
tidin-2-one (24) was synthesized using the general method III
above from imine 10 and dichloroacetyl chloride to afford the
desired product as a dark brown powder, yield 64%, Mp 115e116 ꢁC,
(m, 4 H). 13C NMR (100 MHz, CDCl3):
d 15.33, 56.02, 60.91, 63.99,
87.11, 95.33, 115.38, 122.36, 126.83, 129.65, 131.97, 135.01, 140.30,
153.49, 156.92, 162.40. HRMS: calculated for
C25H25NNaO5S
[MþþNa] 474.1351; found 474.1369.
purity (HPLC): 98.0%. IR nmax: 1770.2 (C¼O) cmꢀ1
.
1H NMR
(400 MHz, CDCl3):
d
ppm 1.37 (t, J ¼ 7.02 Hz, 3 H), 3.78 (s, 6 H), 3.85
4.1.4.17. Cis-4-(4-(Methylthio)phenyl)-3-phenoxy-1-(3,4,5-
trimethoxyphenyl)azetidin-2-one (28cis).
Cis-4-(4-(Methylthio)phenyl)-3-phenoxy-1-(3,4,5-
(s, 3 H), 3.97 (q, J ¼ 7.12 Hz, 2 H), 5.34 (s,1 H), 6.47 (s, 2 H), 6.76e6.84
(m, 2 H), 7.22e7.24 (m, 2 H). 13C NMR (100 MHz, CDCl3):
d
14.71,
56.25, 60.88, 63.70, 74.30, 83.80, 104.77, 115.07, 119.39, 127.03,
129.01, 135.15, 153.61, 157.90, 159.65. HRMS: calculated for
trimethoxyphenyl)azetidin-2-one
(28cis)
was
synthesized
following the general method III above from imine 11 and phe-
C
20H21Cl2NNaO5 [MþNa]þ 448.0695; found 448.0679.
noxyacetyl chloride to afford the title product as oil, yield 7%, purity
(HPLC): 97.3%. IR nmax: 1757.0 (C¼O) cmꢀ1
.
1H NMR (400 MHz,
2.43 (s, 3 H), 3.72 (s, 6 H), 3.76 (s, 3 H), 5.31 (d, J ¼ 4.56 Hz,
4.1.4.13. 4-(4-Ethoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-3-
vinylazetidin-2-one (25).
CDCl3):
d
1 H), 5.53 (d, J ¼ 4.56 Hz,1 H), 6.59 (s, 2 H), 6.80 (d, J ¼ 7.88 Hz, 2 H),
4-(4-Ethoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-3-vinylazetidin-
2-one (25) was synthesized using the general method III above
from imine 10 and crotonyl chloride to afford the title product as a
white powder, yield 56%, Mp 97e98 ꢁC, purity (HPLC): 98.4%. IR
6.92 (d, J ¼ 7.46 Hz, 2 H), 7.27e7.84 (m, 1 H), 7.30 (d, J ¼ 3.05 Hz,
4 H). 13C NMR (100 MHz, CDCl3):
d 15.35, 56.07, 60.91, 62.02, 81.12,
95.30, 114.88, 122.31, 126.04, 129.16, 132.95, 135.03, 139.52, 153.54,
156.92, 162.89. HRMS: calculated for C25H25NNaO5S [MþþNa]
474.1351; found 474.1350.
nmax: 1735.4 (C¼O) cmꢀ1
. d ppm
1H NMR (400 MHz, CDCl3):
1.36e1.43 (m, 3 H), 3.69 (s, 6 H), 3.70e3.73 (m, 1 H), 3.74 (s, 3 H),
4.01 (q, J ¼ 7.32 Hz, 2 H), 4.69 (d, J ¼ 2.44 Hz, 1 H), 5.27e5.39 (m,
2 H), 5.94e6.05 (m, 1 H), 6.53 (s, 2 H), 6.89 (d, J ¼ 8.54 Hz, 2 H),
4.1.4.18. 3-Chloro-4-(4-(methylthio)phenyl)-1-(3,4,5-
trimethoxyphenyl)azetidin-2-one (29).
3-Chloro-4-(4-(methylthio)phenyl)-1-(3,4,5-trimethoxyphenyl)
azetidin-2-one (29) was synthesized following the general method
III above from imine 11 and chloroacetyl chloride to afford the
desired product as an orange powder, yield 57%, Mp 102e104 ꢁC,
7.25e7.29 (m, 2 H). 13C NMR (100 MHz, CDCl3):
d
14.74, 55.99, 60.91,
61.38, 63.54, 63.85, 94.72, 115.09, 119.76, 127.24, 128.94, 130.57,
153.45, 164.69. HRMS: calculated for C22H24NO5 [M - H]þ 382.1654;
found 382.1653.

280
A.M. Malebari et al. / European Journal of Medicinal Chemistry 130 (2017) 261e285
purity (HPLC): 97.6%. IR nmax
(400 MHz, CDCl3): d 2.47 (s, 3 H), 3.69 (s, 6 H), 3.75 (s, 3 H), 4.58 (d,
:
1747.2 (C¼O) cmꢀ1
.
1H NMR
4.1.4.23. Trans-4-(4-(Ethylthio)phenyl)-3-phenoxy-1-(3,4,5-
trimethoxyphenyl)azetidin-2-one (34trans).
J ¼ 1.22 Hz,1 H), 4.91 (d, J ¼ 1.22 Hz,1 H), 6.50 (s, 2 H), 7.27e7.82 (m,
Trans-4-(4-(Ethylthio)phenyl)-3-phenoxy-1-(3,4,5-
4 H). 13C NMR (100 MHz, CDCl3):
d
15.35, 56.07, 60.92, 63.07, 66.01,
trimethoxyphenyl)azetidin-2-one (34trans) was synthesized
following the general method III above from imine 12 and phe-
noxyacetyl chloride to afford the product as a cream powder, yield
55%, Mp 130e131 ꢁC, purity (HPLC): 94.8%,. IR nmax: 1751.3 (C¼O)
95.28, 126.54, 126.84, 131.34, 132.79, 135.24, 140.77, 153.57, 160.50.
HRMS: calculated for C19H20ClNNaSO4 [Mþ þNa] 416.0699; found
416.0688.
cmꢀ1, 1H NMR (400 MHz, CDCl3):
d
1.33 (t, J ¼ 7.32 Hz, 3 H), 2.98 (q,
J ¼ 7.32 Hz, 2 H), 3.69 (s, 6 H), 3.76 (s, 3 H), 4.91 (d, J ¼ 2.22 Hz, 1 H),
5.11 (d, J ¼ 2.22 Hz, 1 H), 6.54 (s, 2 H), 6.87 (d, J ¼ 7.93 Hz, 2 H), 7.01
4.1.4.19. 3,3-Dichloro-4-(4-(methylthio)phenyl)-1-(3,4,5-
trimethoxyphenyl)azetidin-2-one (30).
3,3-Dichloro-4-(4-(methylthio)phenyl)-1-(3,4,5-
(t, J ¼ 7.32 Hz, 2 H), 7.24 (s, 1 H), 7.28 (s, 1 H), 7.30e7.37 (m, 4 H). 13
C
NMR (100 MHz, CDCl3):
d 14.23, 26.34, 55.62, 60.12, 63.55, 64.12,
trimethoxyphenyl)azetidin-2-one (30) was synthesized following
the general method III above from imine 11 and dichloroacetyl
chloride to afford the product as a brown powder, yield 63%, Mp
94.67, 115.67, 127.26, 127.98, 127.19, 128.10, 154.47, 163.32. HRMS:
[MþþNa] calculated for C26H27NNaO5S, 488.1508; found 488.1531.
118e119 ꢁC, purity (HPLC): 98.9%. IR nmax: 1770.2 (C¼O) cmꢀ1
.
1H
4.1.4.24. Cis-4-(4-(Ethylthio)phenyl)-3-phenoxy-1-(3,4,5-
trimethoxyphenyl)azetidin-2-one (34cis).
Cis-4-(4-(Ethylthio)phenyl)-3-phenoxy-1-(3,4,5-
NMR (400 MHz, CDCl3):
d 2.48 (s, 3 H), 3.70 (s, 6 H), 3.77 (s, 3 H),
5.28 (s, 1 H), 6.51 (s, 2 H), 7.20e7.23 (m, 2 H), 7.24e7.28 (m, 2 H). 13
NMR (100 MHz, CDCl3): 15.11, 56.16, 60.95, 73.97, 83.78, 95.87,
C
d
trimethoxyphenyl)azetidin-2-one
(34cis)
was
synthesized
114.74, 126.06, 128.13, 135.63, 153.66, 157.41, 158.91. HRMS: calcu-
lated for C19H19Cl2NNaO4S [MþNa]þ 450.0310; found 450.0320.
following the general method III above from imine 12 and phe-
noxyacetyl chloride to afford the product as an oil, yield 13%, purity
(HPLC): 95.1%. IR nmax: 1752.4 (C¼O) cmꢀ1
;
1H NMR (400 MHz,
1.25 (t, J ¼ 7.26 Hz, 3 H), 2.97 (q, J ¼ 7.46 Hz, 2 H), 3.68 (s,
4.1.4.20. 4-(4-(Methylthio)phenyl)-1-(3,4,5-trimethoxyphenyl)-3-
vinylazetidin-2-one (31).
CDCl3):
d
6 H), 3.75 (s, 3 H), 5.01 (d, J ¼ 4.51 Hz, 1 H), 5.62 (d, J ¼ 4.51 Hz, 1 H),
6.53 (s, 2 H), 6.80 (d, J ¼ 7.88 Hz, 2 H), 6.86 (d, J ¼ 7.88 Hz, 2 H), 7.10
(d, J ¼ 8.29 Hz, 1 H), 7.27e7.37 (m, 4 H). 13C NMR (100 MHz, CDCl3):
4-(4-(Methylthio)phenyl)-1-(3,4,5-trimethoxyphenyl)-3-
vinylazetidin-2-one (31) was synthesized following the general
method III above from imine 11 and crotonyl chloride to afford the
product as an oil, yield 72%, purity (HPLC): 98.8%. IR nmax: 1742.0
d
14.11, 27.02, 55.00, 60.91, 64.01, 64.08, 95.31, 114.72, 127.19, 128.11,
129.39, 132.95, 156.93, 162.13. HRMS: [MþþNa] calculated for
(C¼O) cmꢀ1. 1H NMR (400 MHz, CDCl3):
d 2.46 (s, 3 H), 3.70 (s, 6 H),
3.71 (d, J ¼ 2.44, 1 H), 3.74 (s, 3 H), 4.71 (d, J ¼ 2.44 Hz, 1 H),
C
26H27NNaO5S, 488.1508; found 488.1500.
5.27e5.39 (m, 2 H), 5.98 (d, J ¼ 17.09 Hz, 1 H), 6.52 (s, 2 H),
4.1. 4. 2 5 . 3 - C h l o ro - 4 - ( 4 - ( e t hyl t h i o )p h e nyl ) - 1 - ( 3 , 4 , 5-
trimethoxyphenyl)azetidin-2-one (35).
7.22e7.29 (m, 4 H). 13C NMR (100 MHz, CDCl3):
d 15.53, 56.04, 60.91,
61.26, 63.84, 94.73, 119.99, 126.39, 126.90, 130.38, 133.68, 133.86,
134.55, 139.44, 153.51, 165.11. HRMS: calculated for C21H23NNaO4S
[MþNa]þ 408.1246; found 408.1234.
3-Chloro-4-(4-(ethylthio)phenyl)-1-(3,4,5-trimethoxyphenyl)aze-
tidin-2-one (35) was synthesized following the general method III
above from imine 12 and chloroacetyl chloride to afford the product
as a brown powder, yield 31%, Mp 97e98 ꢁC, purity (HPLC): 96.2%.
IR nmax: 1757.2 (C¼O) cmꢀ1
. d 1.32 (t,
1H NMR (400 MHz, CDCl3):
J ¼ 7.32 Hz, 3 H), 2.95 (q, J ¼ 7.32 Hz, 2 H), 3.70 (s, 6 H), 3.76 (s, 3 H),
4.60 (d, J ¼ 1.83 Hz, 1 H), 4.93 (d, J ¼ 1.83 Hz, 1 H), 6.51 (s, 2 H),
4.1.4.21. 4-(4-(Methylthio)phenyl)-3-(prop-1-en-2-yl)-1-(3,4,5-
trimethoxyphenyl)azetidin-2-one (32).
4-(4-(Methylthio)phenyl)-3-(prop-1-en-2-yl)-1-(3,4,5-
trimethoxyphenyl)azetidin-2-one (32) was synthesized following
the general method III above from imine 11 and dimethylacryloyl
chloride to afford the product as a yellow powder, yield 56%, Mp
7.25e7.31 (m, 4 H). 13C NMR (100 MHz, CDCl3):
d 14.11, 27.01, 56.07,
60.93, 63.05, 66.04, 95.28, 126.59, 128.88, 131.98, 132.82, 135.24,
139.19, 153.58, 160.50. HRMS: [MþþNa] calculated for
99e100 ꢁC, purity (HPLC): 99.1%. IR nmax: 1741.7 (C¼O) cmꢀ1
.
1H
C20H22ClNNaSO4, 430.0856; found 430.0842.
NMR (400 MHz, CDCl3):
d 1.85 (s, 3 H), 2.46 (s, 3 H), 3.66e3.69 (m,
1 H), 3.69 (s, 6 H), 3.75 (s, 3 H), 4.74 (d, J ¼ 2.44 Hz, 1 H), 4.99e5.07
(m, 2 H), 5.60e5.91 (m, 1 H), 6.53 (s, 2 H), 7.24e7.29 (m, 4 H). 13
NMR (100 MHz, CDCl3): 15.09, 20.13, 55.60, 59.78, 60.51, 66.44,
4.1.4.26. 3,3-Dichloro-4-(4-(ethylthio)phenyl)-1-(3,4,5-
trimethoxyphenyl)azetidin-2-one (36).
C
d
3,3-Dichloro-4-(4-(ethylthio)phenyl)-1-(3,4,5-trimethoxyphenyl)
azetidin-2-one (36) was synthesized following the general method
III above from imine 12 and dichloroacetyl chloride to afford the
product as a grey powder, yield 45%, purity (HPLC): 95.5%, Mp
114e115 ꢁC. IR nmax: 1772.3 (C¼O) cmꢀ1. 1H NMR (400 MHz, CDCl3):
94.22, 114.16, 125.44, 126.47, 133.81, 137.56, 138.93, 153.07, 164.68.
HRMS: calculated for
438.1141.
C
22H25KNO4S [MþþK] 438.1141; found
4.1. 4 . 22. 4- (4-(Ethylthio) phenyl)- 3-p henyl-1 -(3, 4 , 5 -
trimethoxyphenyl)azetidin-2-one (33).
4-(4-(Ethylthio)phenyl)-3-phenyl-1-(3,4,5-trimethoxyphenyl)aze-
tidin-2-one (33) was synthesized using the general method III
above from imine 12 and phenylacetyl chloride to afford the
desired product as a brown oil, yield 28%, purity (HPLC): 98.4%. IR
d
1.33 (t, J ¼ 7.32 Hz, 3 H), 2.98 (q, J ¼ 7.32 Hz, 2 H), 3.71 (s, 6 H), 3.78
(s, 3 H), 5.29 (s, 1 H), 6.52 (s, 2 H), 7.23 (d, J ¼ 8.54 Hz, 2 H), 7.33 (d,
J ¼ 8.54 Hz, 2 H). 13C NMR (100 MHz, CDCl3):
d 14.03, 26.76, 56.15,
60.96, 73.99, 83.97, 95.87, 127.95, 128.19, 128.40, 131.77, 139.95,
153.67, 159.59. HRMS: calculated for C20H21Cl2NNaO4S [MþþNa]
464.0466; found 464.0457.
nmax: 1753.3 (C¼O) cmꢀ1
. d 1.32 (t,
1H NMR (400 MHz, CDCl3):
J ¼ 7.32 Hz, 3 H), 2.93e3.00 (m, 2 H), 3.71 (s, 6 H), 3.77 (s, 3 H), 4.27
(d, J ¼ 2.44 Hz,1 H), 4.86 (d, J ¼ 2.44 Hz,1 H), 6.58 (s, 2 H), 7.00e7.07
(m, 4 H), 7.20 (d, J ¼ 8.54 Hz, 1 H), 7.37 (d, J ¼ 6.71 Hz, 2 H), 7.39 (d,
4.1.4.27. 4-(4-(Ethylthio)phenyl)-1-(3,4,5-trimethoxyphenyl)-3-
vinylazetidin-2-one (37).
4-(4-(Ethylthio)phenyl)-1-(3,4,5-trimethoxyphenyl)-3-
vinylazetidin-2-one (37) was synthesized following the general
method III above from imine 12 and crotonyl chloride to afford the
desired product as a colourless oil, yield 42%, purity (HPLC): 95.7%.
J ¼ 7.27 Hz, 2 H). 13C NMR (100 MHz, CDCl3):
d 14.03, 26.76, 55.99,
60.91, 63.85, 64.99, 94.85, 115.17, 127.28, 127.39, 127.83, 128.98,
153.47, 162.11. HRMS: calculated for
472.1559; found 472.1571.
C
26H27NO4S [MþNa]þ
. d 1.31 (t,
IR nmax: 1749.9 (C¼O) cmꢀ1 1H NMR (400 MHz, CDCl3):

A.M. Malebari et al. / European Journal of Medicinal Chemistry 130 (2017) 261e285
281
J ¼ 7.32 Hz, 3 H), 2.95 (q, J ¼ 7.32 Hz, 2 H), 3.71 (s, 6 H), 3.71e3.75
(m, 1 H), 3.76 (s, 3 H), 4.72 (d, J ¼ 2.44 Hz, 1 H), 5.30e5.41 (m, 2 H),
evaporation in vacuo. The crude product was isolated by flash col-
umn chromatography over silica gel (eluent: hexane: ethyl acetate
gradient).
5.99 (dd, J ¼ 10.07, 7.63 Hz,1 H), 6.52 (s, 2 H), 7.23e7.30 (m, 4 H). 13
C
NMR (100 MHz, CDCl3):
d 14.20, 27.26, 56.03, 60.93, 61.30, 63.81,
94.73, 120.02, 126.43, 129.09, 130.39, 133.70, 134.58, 137.79, 153.52,
165.09. HRMS: calculated for C22H25NNaO4S [MþNa]þ 422.1402;
found 422.1391.
4.1.6.1. 4-(4-Methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)azetidin-2-
one (39).
4-(4-Methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)azetidin-2-one
(39) was prepared following the general Reformatsky method V
above from imine 9, and the product was obtained as a brown solid,
yield: 35%, Mp: 86e87 ꢁC [31], purity (HPLC): 98.2%. IR nmax: 1751.7
4.1.4.28. 4-(4-(Ethylthio)phenyl)-3-(prop-1-en-2-yl)-1-(3,4,5-
trimethoxyphenyl)azetidin-2-one (38).
4-(4-(Ethylthio)phenyl)-3-(prop-1-en-2-yl)-1-(3,4,5-
(C¼O) cmꢀ1
;
1H NMR (400 MHz, CDCl3):
d
2.91 (dd, J ¼ 15.26,
trimethoxyphenyl)azetidin-2-one (38) was synthesized following
the general method III above from imine 12 and dimethylacryloyl
chloride to afford the title product as a colourless oil, yield 21%,
2.44 Hz, 1 H), 3.49 (dd, J ¼ 15.26, 5.49 Hz, 1 H), 3.68 (s, 6 H), 3.73 (s,
3 H), 3.78 (s, 3 H), 4.89 (dd, J ¼ 5.49, 2.44 Hz, 1 H), 6.51 (s, 2 H), 6.88
(m, J ¼ 8.54 Hz, 2 H), 7.29 (d, J ¼ 8.54 Hz, 2 H). 13C NMR (100 MHz,
purity (HPLC): 95.2%. IR nmax: 1736.2 (C¼O) cmꢀ1
.
1H NMR
CDCl3): d 46.89, 54.08, 55.29, 55.97, 60.88, 94.47, 114.51, 127.25,
(400 MHz, CDCl3):
d
1.37e1.43 (m, 3 H), 1.85 (s, 3 H), 2.91e2.98 (m,
129.98, 134.04, 134.27, 153.42, 159.78, 164.54. HRMS: calculated for
2 H), 3.71 (s, 7 H), 3.76 (s, 3 H), 4.75 (d, J ¼ 2.44 Hz, 1 H), 5.01e5.06
C
19H21NNaO5[MþþNa], 366.1317; found 366.1318.
(m, 2 H), 6.53 (s, 2 H), 7.22e7.30 (m, 4 H). 13C NMR (100 MHz,
CDCl3):
d
14.03, 26.76, 56.15, 60.96, 73.99, 95.87, 114.23, 115.11,
4.1.6.2. 4-(4-Ethoxyphenyl)-1-(3,4,5-trimethoxyphenyl)azetidin-2-
one (40).
127.95, 128.19, 128.40, 131.77,139.95, 153.67, 165.32. HRMS: calcu-
lated for C23H27NNaO4S [MþNa]þ 436.1559; found 436.1550.
4-(4-Ethoxyphenyl)-1-(3,4,5-trimethoxyphenyl)azetidin-2-one
(40) was prepared following the general Reformatsky method V
above from imine 10 to afford the title product as a colourless oil.
yield 21%, purity (HPLC): 96.5%. IR nmax: 1751.7 (C¼O) cmꢀ1. 1H NMR
4.1.5. General method IV: desilylation of
TBAF (1M solution in hexanes) (2 mmol) was added dropwise to
a solution of the appropriate -lactam silyl ether (2 mmol) in
b-lactams
b
(400 MHz, CDCl3):
d
1.39 (t, J ¼ 7.02 Hz, 3 H), 2.93 (dd, J ¼ 15.26,
ꢁ
anhydrous THF under nitrogen at 0 C. The reaction mixture was
stirred at 0 ꢁC until complete as indicated by TLC. Ethyl acetate
(50 mL) was added and the mixture was washed with 0.1 M HCl
(100 mL). The aqueous layer was extracted with ethyl acetate
(25 mL ꢃ 2). The combined organic layers were washed with brine
(100 mL), water (100 mL) and dried over anhydrous Na2SO4. The
solvent was removed by evaporation in vacuo and the crude reac-
tion mixture was purified by flash chromatography over silica gel
(eluent: hexane: ethyl acetate gradient).
2.44 Hz, 1 H), 3.50 (dd, J ¼ 15.26, 6.10 Hz, 1 H), 3.69 (s, 6 H), 3.74 (s,
3 H), 3.96e4.03 (m, 2 H), 4.90 (dd, J ¼ 5.49, 2.44 Hz, 1 H), 6.52 (s,
2 H), 6.88 (d, J ¼ 8.54 Hz, 2 H), 7.28 (d, J ¼ 8.54 Hz, 2 H). 13C NMR
(100 MHz, CDCl3):
d 14.74, 46.89, 54.13, 55.97, 60.89, 63.52, 94.47,
115.05, 127.23, 129.80, 134.07, 153.42, 164.12. HRMS: calculated for
C
20H23NNaO5 [MþþNa] 380.1474; found 380.1471.
4.1.6.3. 4-(4-(Methylthio)phenyl)-1-(3,4,5-trimethoxyphenyl)azeti-
din-2-one (41).
4-(4-(Methylthio)phenyl)-1-(3,4,5-trimethoxyphenyl)azetidin-2-
one (41) was prepared following the general Reformatsky method
V from imine 11 to afford the desired product as a yellow powder,
yield 11%, Mp 95e96 ꢁC, Purity (HPLC): 97.1%. IR nmax: 1751.03
4.1.5.1. 4-(4-Hydroxyphenyl)-3-phenyl-1-(3,4,5-trimethoxyphenyl)
azetidin-2-one (50).
4-(4-Hydroxyphenyl)-3-phenyl-1-(3,4,5-trimethoxyphenyl)azeti-
din-2-one (50) was synthesized using the general method III from
(C¼O) cmꢀ1
. d 2.46 (s, 3 H), 2.91 (dd,
1H NMR (400 MHz, CDCl3):
imine 13 and phenylacetyl chloride to afford the protected
b
-lactam
J ¼ 14.95, 2.75 Hz, 1 H), 3.52 (dd, J ¼ 15.26, 5.49 Hz, 1 H), 3.70 (s,
6 H), 3.74 (s, 3 H), 4.91 (dd, J ¼ 5.49, 2.44 Hz, 1 H), 6.51 (s, 2 H),
7.22e7.25 (m, 2 H), 7.26e7.31 (m, 2 H). 13C NMR (100 MHz, CDCl3):
intermediate, which was then treated with TBAF as described above
in General method IV to afford the desired product as a colourless
oil, yield 9%, purity (HPLC): 85.2%. IR nmax: 1740.1 (C¼O) cmꢀ1. 1H
d
15.54, 46.82, 54.02, 56.01, 60.90, 94.48, 126.43, 126.87, 133.91,
NMR (400 MHz, CDCl3)
d
3.69 (s, 6 H), 3.76 (s, 3 H), 3.79 (s, 1 H), 4.25
134.40, 134.74, 139.30, 153.47, 164.31. HRMS: calculated for
(d, J ¼ 2.49 Hz, 1 H), 4.84 (d, J ¼ 2.49 Hz, 1 H), 6.58 (s, 2 H), 6.85 (d,
C
19H21NNaO4S [MþþNa] 382.1089; found 382.1089.
J ¼ 8.71 Hz, 2 H), 7.27 (d, J ¼ 8.71 Hz, 2 H), 7.29e7.33 (m, 3 H),
7.33e7.38 (m, 2 H). 13C NMR (100 MHz, CDCl3)
d
55.99, 60.93, 63.84,
4.1.6.4. 4-(4-(Ethylthio)phenyl)-1-(3,4,5-trimethoxyphenyl)azetidin-
2-one (42).
64.97, 94.91, 116.17, 127.38, 129.01, 133.64, 134.67, 153.48, 156.18,
165.73. HRMS: calculated for C24H23NO5[M]þ 405.1576; found
405.1520.
4-(4-(Ethylthio)phenyl)-1-(3,4,5-trimethoxyphenyl)azetidin-2-
one (42) was prepared following the general Reformatsky method
V from imine 12 to afford the desired product as a grey solid, yield
26%, Mp 89e90 ꢁC, purity (HPLC): 96.5%. IR nmax: 1751.4 (C¼O)
4.1.6. General method V: preparation of 2-azetidinones
(reformatsky reaction)
cmꢀ1
.
1H NMR (400 MHz, CDCl3):
d
1.29 (t, J ¼ 7.32 Hz, 3 H),
Zinc powder (9 mmol) was activated using trimethyl-
chlorosilane (7 mmol) in anhydrous benzene (4 mL) by heating for
15 min at 40 ꢁC and subsequently for 2 min at 100 ꢁC with micro-
wave irradiation. After cooling, the appropriate imine (2 mmol) and
ethyl 2-bromoacetate (5 mmol) were added to the reaction vessel
and the mixture was placed in the microwave reactor for 30 min at
100 ꢁC. The reaction mixture was filtered through Celite to remove
the zinc catalyst and then diluted with CH2Cl2 (30 mL). This solu-
tion was washed with saturated ammonium chloride solution
(20 mL) and 25% ammonium hydroxide (20 mL), and then with
dilute HCl (40 mL), followed by water (40 mL). The organic phase
was dried over anhydrous Na2SO4 and the solvent was removed by
2.87e2.96 (m, 3 H), 3.52 (dd, J ¼ 15.26, 6.10 Hz, 1 H), 3.69 (s, 6 H),
3.74 (s, 3 H), 4.91 (dd, J ¼ 5.49, 2.44 Hz, 1 H), 6.50 (s, 2 H), 7.25e7.33
(m, 4 H). 13C NMR (100 MHz, CDCl3):
d 14.18, 27.27, 46.80, 54.06,
55.99, 60.90, 94.47, 126.45, 129.09, 133.92, 134.39, 135.46, 137.62,
153.47, 164.28. HRMS: calculated for C20H23NNaO4S [MþþNa]
396.1245; found 396.1246.
4.1.7. General method VI: preparation of 3-hydroxyazetidin-2-ones
The imine (5 mmol) and 2-acetoxyacetyl chloride (7 mmol)
were dissolved in dry toluene (50 mL), under nitrogen atmosphere
with stirring at 0 ꢁC. The solution was allowed to reach room
temperature and then warmed to 100 ꢁC. Dry TEA (9 mmol) was

282
A.M. Malebari et al. / European Journal of Medicinal Chemistry 130 (2017) 261e285
added dropwise for 10 min and keep it at 100 ꢁC for 5 h. The re-
action mixture was washed with water (50 mL ꢃ 2), dried over
anhydrous Na2SO4 and solvent was removed in vacuo. The crude
product was purified by flash chromatography over silica gel
(eluent: n-hexane: ethyl acetate, gradient). Hydrazine dichloride
(5 mmol) was added to a stirred solution of the crude product in
methanol (30 mL) at 0 ꢁC and under nitrogen, then dry TEA
(9 mmol) was added dropwise. The mixture was allowed to reach
room temperature and then heated at reflux at for 4 h. The solvent
was removed by evaporation at reduced pressure, and the residue
was treated with a saturated solution of KHSO4 and extracted with
ethyl acetate (2 ꢃ 50 mL). The organic phase was dried over Na2SO4,
and the solvent after filtration, was removed by evaporation at
reduced pressure. The crude residue was purified by flash chro-
matography over silica gel (eluent: n-hexane: ethyl acetate, 2:1) to
afford the desired product.
6.47 (s, 2 H), 7.27e7.32 (m, 4H). 13C NMR (100 MHz, CDCl3):
d 14.15,
27.22, 55.99, 60.91, 65.50, 95.29, 110.00, 116.02, 126.62, 129.01,
133.03, 133.22, 138.03, 153.45, 162.45. HRMS: calculated for
C
20H23NNaO5S [MþþNa], 412.1195; found 412.1190.
4.1.7.5. 3-Hydroxy-4-(3-hydroxy-4-methoxyphenyl)-1-(3,4,5-
trimethoxyphenyl)azetidin-2-one (49).
3-Hydroxy-4-(3-hydroxy-4-methoxyphenyl)-1-(3,4,5-
trimethoxyphenyl)azetidin-2-one (49) was synthesized using the
general procedure VI above and followed by procedure IV from
imine 14, and afforded the product as a white powder, yield 30%,
Mp: 140 ꢁC [38], purity (HPLC): 97.3%. IR nmax: 3411.0 (C¼O) cmꢀ1
.
1H NMR (400 MHz, CDCl3)
d 1.58 (s, 1 H), 3.69 (s, 6 H), 3.75 (s, 3 H),
3.87 (s, 3 H), 4.79 (d, J ¼ 1.66 Hz, 1 H), 5.37 (d, J ¼ 1.66 Hz, 1 H), 5.68
(s, 1 H), 6.53 (s, 2 H), 6.82e6.87 (m, 2 H), 6.90 (s, 1 H). 13C NMR
(100 MHz, CDCl3):
d 55.11, 56.32, 61.21, 64.22, 83.51, 94.65, 125.53,
133.44, 135.35, 139.18, 155.11, 165.12. HRMS: calculated for
4.1.7.1. 3-Hydroxy-4-(4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)
azetidin-2-one (43).
C
19H21NNaO7 [MþþNa], 398.1216; found 398.1213.
3-Hydroxy-4-(4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)aze-
tidin-2-one (43) was obtained following the general procedure VI
above from imine 9 and afforded a white powder, yield 30%, Mp
4.1.8. Stability study of compounds 27 and 45
Analytical high-performance liquid chromatography (HPLC)
stability studies were performed using a Symmetry® column (C18
,
135e136 ꢁC [30], purity (HPLC): 97.5%. IR nmax: 3412.4 (C¼O) cmꢀ1
;
5
mm, 4.6 ꢃ 150 mm), a Waters 2487 Dual Wavelength Absorbance
1H NMR (400 MHz, CDCl3):
d
3.80 (s, 6 H), 3.89 (s, 3 H), 3.94 (s, 3 H),
detector, a Waters 1525 binary HPLC pump and a Waters 717 plus
Autosampler (Waters Corporation, Milford, MA, USA). Samples
were detected at wavelength of 254 nm. All samples were analysed
using acetonitrile (70%): water (20%) as the mobile phase over
15 min and a flow rate of 1 mL/min. Stock solutions are prepared by
dissolving 10 mg of compounds 27 and 45 in 10 mL of mobile phase
(1 mg/mL). Phosphate buffers at the desired pH values (4, 7.4 and 9)
were prepared in accordance with the British Pharmacopoeia
4.88 (s, 1 H), 4.93 (s, 1 H), 5.23 (br. s, 1 H), 6.61 (s, 2 H), 7.03 (d,
J ¼ 8.54 Hz, 2 H), 7.39 (d, J ¼ 8.55 Hz, 2 H). 13C NMR (100 MHz,
CDCl3):
d 55.77, 56.20, 60.10, 65.23, 83.56, 95.78, 126.44, 132.48,
134.44, 139.17, 153.58, 166.20. HRMS: calculated for C19H21NNaO6
[MþþNa], 382.1268; found 382.1261.
4.1.7.2. 4-(4-Ethoxyphenyl)-3-hydroxy-1-(3,4,5-trimethoxyphenyl)
azetidin-2-one (44).
monograph 2015. 30 mL of stock solution was diluted with 1 mL of
4-(4-Ethoxyphenyl)-3-hydroxy-1-(3,4,5-trimethoxyphenyl)azeti-
din-2-one (44) was synthesized using the general procedure VI
above from imine 10 and afforded the product as a white solid, yield
20%, Mp 128e129 ꢁC, purity (HPLC): 100%. IR nmax: 3434.6 (OH),
appropriate buffer, shaken and injected immediately. Samples were
withdrawn and analysed at time intervals of t ¼ 0 min, 5 min,
30 min, 60 min and subsequently every hour for 24 h.
1731.9 (C¼O) cmꢀ1. 1H NMR (400 MHz, CDCl3):
d
1.39 (t, J ¼ 7.02 Hz,
4.2. Biochemical evaluation of activity
3 H), 3.65 (s, 6 H), 3.73 (s, 3 H), 3.99 (q, J ¼ 6.72 Hz, 2H), 4.23 (br. s,
1 H), 4.72 (s, 1 H), 4.75 (s, 1 H), 6.47 (s, 2 H), 6.87 (d, J ¼ 8.54 Hz, 2 H),
All biochemical assays were performed in triplicate on at least
three independent occasions for the determination of mean values
reported.
7.19e7.26 (m, 2 H). 13C NMR (100 MHz, CDCl3):
d 14.72, 55.94, 60.89,
63.54, 65.66, 83.53, 95.31, 115.05, 127.46, 127.69, 133.13, 134.66,
153.36, 159.33, 166.82. HRMS: calculated for
C20H23NNaO6
[MþþNa] 396.1423; found 396.1420;
4.2.1. Cell culture
The human breast tumour cell line MCF-7 was cultured in Eagles
4.1.7.3. 3-Hydroxy-4-(4-(methylthio)phenyl)-1-(3,4,5-
trimethoxyphenyl)azetidin-2-one (45).
minimum essential medium with 10% fetal bovine serum, 2 mM L-
glutamine and 100 lg/mL penicillin/streptomycin. The medium was
supplemented with 1% non-essential amino acids. HT-29 cells
originate from a human adenocarcinoma of the colon and were
originally obtained from the European Collection of Cell Cultures
and were grown in DMEM Glutamax media. HT-29 media were
supplemented with 10% foetal bovine serum (FBS). HL-60 was
cultured in RPMI-1640 with GlutaMax I and supplemented with
10% foetal bovine serum. All media contained 100 U/ml penicillin
3-Hydroxy-4-(4-(methylthio)phenyl)-1-(3,4,5-trimethoxyphenyl)
azetidin-2-one (45) was synthesized using the general procedure
VI above from imine 11 and afforded the product as a white powder,
yield 41%, Mp 150e151 ꢁC, purity (HPLC): 100%. IR nmax: 3391.9
(OH), 1748.1 cmꢀ1 (C¼O). 1H NMR (400 MHz, CDCl3):
d 2.60 (s, 3 H),
3.80 (s, 6 H), 3.88 (s, 3 H), 4.85 (s, 2 H), 4.92 (s, 1 H), 6.59 (s, 2 H),
7.34e7.41 (m, 4 H). 13C NMR (100 MHz, CDCl3):
d 15.46, 55.97, 60.91,
65.61, 83.47, 95.32, 126.59, 126.76, 132.52, 132.94, 134.71, 139.59,
and 100 m
g/mL streptomycin. Cells were maintained at 37 ꢁC in 5%
CO2 in a humidified incubator. All cells were sub-cultured 3 times/
week by trypsinisation.
153.37, 167.0. HRMS: calculated for
398.1038; found 398.1036.
C
19H21NNaO5S [MþNa]þ
4.1. 7. 4 . 4-(4-(Ethylthio )phenyl) -3 -hy droxy-1-(3, 4, 5-
trimethoxyphenyl)azetidin-2-one (46).
4.2.2. Cell viability assay
Cells were seeded at a density of 5 ꢃ 103 cells/well (MCF-7),
1 ꢃ 104 cells/well (HT-29) and 4 ꢃ 104 cells/well (HL-60) in 96-well
plates. After 24 h, cells were then treated with either medium
alone, vehicle [1% ethanol (v/v)] or with serial dilutions of CA-4 or
4-(4-(Ethylthio)phenyl)-3-hydroxy-1-(3,4,5-trimethoxyphenyl)
azetidin-2-one (46) was synthesized using the general procedure
VI above from imine 12, and afforded the product as a brown oil,
yield 32%, Purity (HPLC): 100%. IR nmax: 3412.8 (OH), 1747.9 (C¼O)
b-lactam analogue in triplicate. Cell proliferation for MCF-7 and HL-
cmꢀ1. 1H NMR (400 MHz, CDCl3):
J ¼ 7.32 Hz, 2 H), 3.67 (s, 6 H), 3.74 (s, 3 H), 4.72 (s, 1 H), 4.78 (s, 1 H),
d
1.29 (t, J ¼ 7.02 Hz, 3 H), 2.93 (d,
60 was analysed using the Alamar Blue assay (Invitrogen Corp.)
according to the manufacturer's instructions. After 72 h, Alamar

A.M. Malebari et al. / European Journal of Medicinal Chemistry 130 (2017) 261e285
283
Blue [10% (v/v)] was added to each well and plates were incubated
for 3e5 h at 37 ꢁC in the dark. Fluorescence was read using a 96-
well fluorimeter with excitation at 530 nm and emission at
590 nm. For HT-29 cells, the assay determined according to the
sulforhodamine B (SRB) protocol [62]. Results were expressed as
percentage viability relative to vehicle control (100%). Dose
response curves were plotted and IC50 values (concentration of
drug resulting in 50% reduction in cell survival) were obtained
using the commercial software package Prism (GraphPad Software,
Inc., La Jolla, CA, USA).
performed as described above using antibodies directed against
Mcl-1 [1:1000] (Millipore), PARP [1:1000] (Millipore) and Bax
[1:1000] (Millipore) followed by incubation with a horseradish
peroxidase-conjugated anti-mouse antibody [1:2000] (Promega,
Madison, WI, USA). All blots were probed with anti-GAPDH anti-
body [1:5000] (Millipore) to confirm equal loading. Proteins were
detected using chemiluminescent Western blot detection (Clarity
Western ECL substrate) (Bio Rad) on the ChemiDoc MP System (Bio
Rad). Experiments were performed on three independent
occasions.
4.2.3. Cell cycle analysis
4.2.7. Tubulin polymerization assay
Adherent and detached cells were collected by trypsinization
and centrifuged at 800x g for 15 min. Cells were washed twice with
ice-cold PBS and fixed in ice-cold 70% ethanol overnight at ꢀ20 ꢁC.
Fixed cells were centrifuged at 800x g for 15 min and stained with
The assembly of purified bovine tubulin was monitored using a
kit, BK006, purchased from Cytoskeleton Inc., (Denver, CO, USA).
The assay was carried out in accordance with the manufacturer's
instructions using the standard assay conditions [63]. Briefly, pu-
rified (>99%) bovine brain tubulin (3 mg/mL) in a buffer consisting
of 80 mM PIPES (pH 6.9), 0.5 mM EGTA, 2 mM MgCl2,1 mM GTP and
10% glycerol was incubated at 37 ꢁC in the presence of either vehicle
50 mg/mL of PI, containing 50 m
g/mL of DNase-free RNase A, at 37 ꢁC
for 30 min. The DNA content of cells (10,000 cells/experimental
group) was analysed by flow cytometer at 488 nm using a FACS-
Calibur flow cytometer (BD Biosciences, San Jose, CA) and all data
were recorded and analysed using the CellQuest Software (Becton-
Dickinson).
(2% (v/v) ddH2O) or compounds 2, 27, 29, 31, 45 (10 mM). Light is
scattered proportionally to the concentration of polymerised mi-
crotubules in the assay. Therefore, tubulin assembly was monitored
turbidimetrically at 340 nm in a Spectramax 340 PC spectropho-
tometer (Molecular Devices, Sunnyvale, CA, USA). The absorbance
was measured at 30 s intervals for 60 min.
4.2.4. Immunofluorescence microscopy
Confocal microscopy was used to study the effects of drug
treatment on MCF-7 and HT-29 cytoskeleton. For immunofluores-
cence, MCF-7 cells and HT-29 cells were seeded at 1 ꢃ 105 cells/mL
on eight chamber glass slides (BD Biosciences). Cells were either
4.2.8. Colchicine-binding site assay
N,N0-Ethylene-bis(iodoacetamide) (EBI) (Santa Cruz Biotech-
untreated or treated with vehicle [1% ethanol (v/v)], 1 (0.1
m
M), 2
nology) dissolved in ethanol (100 mM). MCF-7 or HT-29 cells were
(0.1 M in MCF-7,1 M in HT-29 cells), 27, 45 (0.5 M) and 47 (1
m
m
m
mM)
seeded at a density of 5 ꢃ 104 cells/well in 6-well plates and
for 16 h. Following treatment cells were gently washed in PBS, fixed
for 20 min with 4% paraformaldehyde in PBS and permeabilised in
0.5% Triton X-100. Following washes in PBS containing 0.1% Tween
(PBST), cells were blocked in 5% bovine serum albumin diluted in
incubated overnight. Cells were treated with vehicle control
[ethanol (0.1% v/v)], 1 or 2 and compound 45 (all 10 mM) for 2 h.
After this time, selected wells were treated with EBI for 1.5 h.
Following treatment, cells were twice washed with ice-cold PBS
and lysed by addition of Laemmli buffer. Samples were separated by
SDS-PAGE, transferred to polyvinylidenedifluride membranes, and
PBST. Cells were then incubated with mouse monoclonal anti-a-
tubulinꢀFITC antibody (clone DM1A) (Sigma) (1:100) for 2 h at rt.
Following washes in PBST, cells were incubated with Alexa Fluor
488 dye (1:500) for 1 h at rt. Following washes in PBST, the cells
were mounted in Ultra Cruz Mounting Media (Santa Cruz
Biotechnology, Santa Cruz, CA) containing 4,6-diamino-2-
phenolindol dihydrochloride (DAPI). Images were captured by
Leica SP8 confocal microscopy with Leica application suite X soft-
ware. All images in each experiment were collected on the same
day using identical parameters. Experiments were performed on
three independent occasions.
probed with b-tubulin antibodies (Sigma Aldrish) [56].
4.3. Computational procedure
The 1SA0 X-ray structure of bovine tubulin co-crystallised with
N-deacetyl-N-(2-mercaptoacetyl)-colchicine
(DAMA-colchicine)
was downloaded from the PDB website (entry 1SA0) [44]. A UniProt
Align analysis confirmed a 100% sequence identity between human
and bovine beta tubulin. The crystal structure was prepared using
QuickPrep (minimised to a gradient of 0.001 kcal/mol/Å), Protonate
3D, Residue pKa and Partial Charges protocols in MOE 2015 with
the MMFF94x force field. Compounds 27 and 45 were drawn in
ChemBioDraw 13.0, saved as mol files and opened in MOE. For both
enantiomers of each compound, MMFF94x partial charges were
calculated and each was minimised to a gradient of 0.001 kcal/mol/
Å. Default parameters were used for Docking except that 100 poses
were sampled for each enantiomer and the top 50 docked poses
were retained for subsequent analysis [44].
4.2.5. Western blot
Cells were harvested in RIPA buffer supplemented with protease
inhibitors (Roche Diagnostics), phosphatase inhibitor cocktail 2
(Sigma-Aldrich), and phosphatase inhibitor cocktail 3 (Sigma-
Aldrich). Equal quantities of protein (as determined by a BCA assay)
were resolved by SDS-PAGE (12%) followed by transfer to PVDF
membranes. Membranes were blocked in 5% bovine serum albu-
min/TBST for 1 h. Membranes were incubated in the relevant pri-
mary antibody UGT [1:1000] (cell signaling) at 4 ꢁC overnight,
washed, and incubated in horseradish peroxidase conjugated sec-
ondary antibody for 1 h at rt and washed again. Enhanced chem-
iluminescence was used for detection of protein expression.
4.4. X-ray crystallography
Data for samples 28trans(3R4R) and 28cis(3S4R) were collected
on a Bruker D8 QUEST Eco using Mo K
and samples 41(4R) and 44(3S4S) were collected on a Bruker Apex
DUO using Mo K and Cu K radiation (
Mitegen cryoloop and at 100(2) K (Oxford Cryostream, Oxford Co-
bra Cryosystem respectively). Bruker APEX [64] software was used
to collect, correct (Lorentz and polarization) and reduce data,
determine the space group, solve and refine the structure.
a
radiation (
l
¼ 0.71073 Å)
4.2.6. Evaluation of expression levels of anti-apoptotic proteins
Mcl-1, pro-apoptotic proteins Bax and PARP cleavage
a
a
l
¼ 1.54178 Å) using a
HT-29 cells were seeded at a density of 1 ꢃ 105 cells/flask in T25
flasks. After 48 h, whole cell lysates were prepared from untreated
cells or cells treated with vehicle control (EtOH, 0.1% v/v) or com-
pound 27 (0.1, 1 mM) or 45 (0.1, 1 mM). Western blot analysis was

284
A.M. Malebari et al. / European Journal of Medicinal Chemistry 130 (2017) 261e285
Absorption corrections were applied using SADABS 2014 [65].
Bruker APEX software was used to determine the space group, solve
and refine the structure.
UGT
UV
Uridine 5'-diphosphoglucuronosyltransferase (UDP-
glucuronosyltransferase)
Ultraviolet
All non-hydrogen atoms were refined anisotropically. Hydrogen
atoms were assigned to calculated positions using a riding model
with appropriately fixed isotropic thermal parameters. In 28trans, a
partial occupancy H2O (50% occupied) on a special position was
refined using restraints (DFIX). The phenylethoxy group in 44 was
disordered in two positions with 93//7% occupancy and was
modelled with constraints (EADP) and restraints (SADI).
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AMM) is gratefully acknowledged. This work was also supported by
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Abbreviations
AML
CA-4
DBU
DCM
DCTD
DMF
DTP
EBI
Acute myeloid leukemia
Combretastatin A-4
1,8-Diazabicyclo[5.4.0]undec-7-ene
Dichoromethane
Division of Cancer Treatment and Diagnosis
N,N-Dimethylformamide
Development Therapeutics Program
N,N0-ethylene-bis(iodoacetamide)
Electron Impact
EI
GTP
HRMS
IC
Guanidine triphosphate
High resolution molecular mass spectrometry
Inhibitory concentration
IR
Infra-Red
MAPK
MDS
MEK
MS
Mitogen-activated protein kinases
Myelodysplastic syndrome
Mitogen-activated protein kinase kinase
Mass spectrometry
MTD
NCI
Maximum tolerated dose
National Cancer Institute
NIH
NMR
PBS
National Institute of Health
Nuclear Magnetic Resonance
Phosphate buffer saline
SAR
TBAF
Structure-activity relationship
Tetrabutylammonium fluoride
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TBDMS tert-Butyldimethylchlorosilane
TEA
Triethylamine
TMS
TMCS
Tetramethylsilane
Tetramethylchlorosilane

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