Synthesis of anticancer β-lactams: Mechanism of action

Article abstract of DOI:10.1016/j.bmc.2004.03.033

Synthesis of the trans 1-N-chrysenyl and 1-N-phenanthrenyl 3-acetoxy-4-phenyl-2-azetidinones has been achieved. Microwave-assisted reaction has proved useful in the synthesis of these compounds. Cell growth inhibition study has indicated selective anticancer activity against two leukemia and colon carcinoma cell lines. A mechanistic correlation of their anticancer activity has been described. Striking G₂ blockade that is clearly distinct in cell cycle analysis and demonstrated only in sensitive cell lines has been observed. They do not induce apoptosis in sensitive or resistant lines. They also do not inhibit topoisomerases. Ames test has shown they are nonmutagenic.

Full text of DOI:10.1016/j.bmc.2004.03.033

Bioorganic & Medicinal Chemistry 12 (2004) 2523–2528
Synthesis of anticancer b-lactams: mechanism of action
Bimal K. Banik,* Frederick F. Becker and Indrani Banik
The University of Texas M.D. Anderson Cancer Center, Department of Molecular Pathology, Unit 89, 1515 Holcombe Boulevard,
Houston, TX 77030, USA
Received 22 November 2003; revised 8 March 2004; accepted 16 March 2004
Available online 10 April 2004
Abstract—Synthesis of the trans 1-N-chrysenyl and 1-N-phenanthrenyl 3-acetoxy-4-phenyl-2-azetidinones has been achieved.
Microwave-assisted reaction has proved useful in the synthesis of these compounds. Cell growth inhibition study has indicated
selective anticancer activity against two leukemia and colon carcinoma cell lines. A mechanistic correlation of their anticancer
activity has been described. Striking G2 blockade that is clearly distinct in cell cycle analysis and demonstrated only in sensitive cell
lines has been observed. They do not induce apoptosis in sensitive or resistant lines. They also do not inhibit topoisomerases. Ames
test has shown they are nonmutagenic.
Ó 2004 Published by Elsevier Ltd.
1. Introduction
our open-chain diamides (1 and 2) may increase
potency. Structures 1 and 2 suggest that a ring forma-
tion using N1 and C4 would result in b-lactam 3. Our
research in this field has culminated in the synthesis of a
In previous publications, we demonstrated synthesis and
biological evaluation of some derivatives of polyaro-
matic amines, which were open-chain amides to which
the polycyclic residue was bound (e.g., 1 and 2, Fig. 1).1
From the literature, we are aware that conformationally
constrained molecules often have a greater effect on
biological properties when compared to the relatively
flexible open-chain compounds.2 Therefore, we antici-
pated that conformationally constrained analogues of
few b-lactams derived from polyaromatic imines.3
A
preliminary structure–activity study has revealed that
trans 1-N-chrysenyl and 1-N-phenanthrenyl 3-acetoxy-
4-aryl-2-azetidinones have potent, selective anticancer
activity. Because b-lactams are widely and safely used as
antibacterials without significant toxicity, studies of
these molecules as new and novel anticancer agents
would be timely. In this paper, we describe mechanistic
correlation of their anticancer activity for the first time
and demonstrate a remarkable G2 blockade that is
clearly distinct in cell cycle analysis. In addition, syn-
thesis of these agents using microwave irradiation is also
described here.
1.1. Synthesis of b-lactams
The two b-lactams 7 and 8 were prepared by following
Staudinger reaction. Cycloaddition of imines 5 and 6
with acetoxy acetyl chloride 4 in the presence of trieth-
ylamine afforded trans-b-lactams 7 and 8, respectively
(Scheme 1). Although formation of b-lactams using the
Staudinger reaction was discovered more than 90 years
ago, surprisingly, there has not been a precedent
recorded in the literature regarding the use of tetracyclic
or pentacyclic aromatic systems in imine components.
Interestingly, the formation of trans-b-lactams as seen in
Figure 1.
Keywords: b-Lactams; Anticancer agents; Cell cycle; Apoptosis.
* Corresponding author. Tel.: +1-713-792-7749; fax: +1-713-792-5940;
e-mail: bbanik@mail.mdanderson.org
0968-0896/$ - see front matter Ó 2004 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2004.03.033

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B. K. Banik et al. / Bioorg. Med. Chem. 12 (2004) 2523–2528
domestic microwave oven.5c;d Polar solvents like chloro-
benzene and DMF can be used with success. However,
chlorobenzene proved to be the preferred solvent in this
case. The boiling point of these solvents is much higher
than the projected reaction temperature. b-Lactams 7
and 8 was obtained after 4–5 min of irradiation in the
microwave when chlorobenzene was the solvent. The
temperature of the reaction vessel was maintained at
100–110 °C by a proper adjustment of the ‘on–off’ cycle
and a ‘heat sink’.5c;d Microwave energy is absorbed by
all of the polar molecules at the same time effectively,
therefore no stirrers was needed. Reflux condenser was
not required since liquids were maintained below their
boiling points under this condition. Therefore, an easy
access of these b-lactams was realized using microwave-
induced method.
Scheme 1.
The mechanism of b-lactam formation has been inves-
tigated extensively. However, the rationale for the
observed diastereoselectivity and enantioselectivity
remains unknown. It has been shown that the stereo-
selectivity depends on a number of factors: the structure
of the imine, acid-chloride (equivalent) sequence of
reagent addition, solvent, temperature, and bases. It is
conceivable that the mechanism of b-lactam formation
reaction by Staudinger reaction is not only very com-
plex, but also unique since it varies considerably with
minor structural changes and conditions. Certainly, any
of the postulated mechanisms may be operative, and
there may be a continuum of mechanisms acting in
concert to promote the reaction depending on the par-
ticular method employed.7
Scheme 2.
the present investigation also has not been described in
the literature. Some previous studies were directed at the
formation of trans-b-lactams. However, the experimen-
tal conditions in those investigations were clearly dif-
ferent from those in the present one. For example,
synthesis of some trans-b-lactams was achieved using
high-power microwave irradiation and changing the
order in which the reagents were added.4c;d Further-
more, in two cases, trans-b-lactams were formed in low
yield as the only isolated products using cyclic imines,
but they were not derived from an aromatic amine.4a;b
1.2. In vitro cytotoxicity of the b-lactams
These b-lactams 7 and 8 were tested using nine human
cancer cell lines with cisplatin and diamide compounds
1b and 2b as controls; results are given in Table 1.
The use of domestic microwave in organic synthesis is
well known.5 In conformity with earlier hypothesis,
irradiation of a solution of imines 5 and 6 in chloro-
benzene in the presence of acetoxy acetyl chloride 4 and
triethylamine using a domestic microwave oven afforded
trans-b-lactams 7 and 8, respectively, in comparable
yield (Scheme 2).6 trans-b-Lactams 7 and 8 were also
obtained when the above reaction mixtures were heated
in an oil-bath (120 °C) for 30 min using a stirrer and
reflux condenser.
2. Mechanism of action
Because of the presence of the cyclic four-membered
amide connected to the multicyclic ring systems, an
effort to define their mechanism of action would be
highly important. Despite a number of synthetic efforts,
a relatively small amount of research has been focused
on the use of compounds related to PAHs as anticancer
agents. For example, Bair et al.8 reported a close cor-
relation between antitumor activity and the shape of the
polyaromatic system. However, his group did not make
An unconventional experimental setup for conducting
this reaction to take special advantages of the specific
nature of microwave energy was used. A large size
Erlenmeyer flask was the reaction vessels in unmodified
Table 1. In vitro cytotoxicity of b-lactam compounds on human cancer cell lines (lM)
Compounds
BRO
MCF-7
MDA-231
OVCAR-3
SKOV-3
PC-3
HL-60
K-562
HT-29
Cisplatin
7.66
33.64
3.9
10.05
40.0
4.3
12.33
12.23
5.1
3.99
18.11
4.1
5.99
11.05
3.9
4.66
27.29
3.5
1.66
9.41
2.1
2.33
12.70
1.9
16.99
16.70
3.9
1b
2b
7
10.48
10.84
10.09
9.81
12.49
11.98
18.0
4.17
18.00
6.88
9.3
16.32
5.21
3.64
4.0
4.33
10.49
5.66
8
All of the in vitro cytotoxicity assays were performed in the Pharmacology and Analytic Core Laboratory of our Cancer Center as described
previously.1 In summary, an MTT assay was carried out using the nine human cancer cell lines.

B. K. Banik et al. / Bioorg. Med. Chem. 12 (2004) 2523–2528
2525
a definitive correlation between the ability of these
compounds to bind to DNA and their cytotoxic activity.
Bair’s group developed benzylic aminopropanediols
from a structure–activity relationship study. These
amino propanediols are believed to interact with DNA
via intercalation and to be topoisomerase II inhibitors.
The antitumor activity of two of the most active naph-
thalimides amonafide and mitonafide, has been shown
to have to be due to intercalation.9 To improve its
potency, naphthalene was modified to anthracene to
serve as the chromophore. The resulting analogue,
azonafide showed increased cytotoxic potency over
naphthalene-based compounds.10 Not all azonafide
derivatives, which have characteristic mechanistic
properties when compared with existing classes of DNA
intercalators, localize in the nucleus. Other differences
with existing DNA intercalators, such as mitoxan-
trone,11 include a lack of inhibition of topoisomerase II
enzymes at equicytotoxic concentrations. Another
approach to improving cellular cytotoxic potency has
been adopted by linking two naphthalimide or anthral-
imide groups with a polyamine bridge. Other studies
concerning the target for several newly synthesized
agents have been published by Denny and co-workers12
The synthesis and mechanism of action of b-lactams as
different types of biologically active compounds have
been investigated.13;14 However, studies directed to
define the mechanism of action of b-lactams as anticancer
agents has not been explored. The active compounds 7
and 8 may follow one of these mechanisms as described
above or it may act on cancer cells through a novel
mechanism. To define the mechanism of action of our
two potent compounds, the studies described below
were performed.
article. Data sets for tester strains TA98 and TA100
were judged to be positive if the increase in mean rever-
tants at the peak of the dose response was equal to or
greater than twice the mean vehicle control value.
The results of the mutagenicity assay indicated that
under the conditions of this study, neither 7 nor 8
demonstrated a positive response with these tester
strains at any concentration in either the presence or
absence of Aroclor-induced rat liver S9, indicating that
neither of these compounds demonstrate any mutage-
nicity. These findings support the potential use of these
compounds as antitumor agents and eliminate potential
concerns arising from the presence of the phenanthrene
and chrysene nuclei.
2.2. Apoptosis
To determine whether exposure to 7 and/or 8 induced
DNA cleavage in sensitive and/or resistant tumor cell
lines, the APO-BRDU assay was carried out. As shown
in Tables 2 and 3, 8 demonstrated no significant increase
in BRDU insertion into the DNA of HL-60 cells ex-
posed for 24 h. In two similar runs 7 also failed to
demonstrate an increase in BRDU insertion. In all of
these experiments, concentrations approximately twice
that required for the IC50 of 7 and three times greater
than that for 8 were used. In one of these experiments,
MDA-231, a human breast cancer line that was resistant
in vitro to the effects of 8, was tested for BRDU inser-
tion. As expected, a significant increase was not de-
tected. These results confirmed scanning electron
microscopy studies of exposed cells (data not presented)
that confirmed the lack of alterations in these cell lines,
which are associated with the induction of apoptosis in
the target cells.
2.1. Mutagenicity assays
Mutagenicity assays were performed by BioReliance
Corporation (Rockville, MD) as study AA65FL-
FM.501.BTA. Compounds 7 and 8 were examined using
the Salmonella Plate Incorporation, Mutagenicity
Assay. The tester strains used were Salmonella typimu-
rium histidine auxotrophs TA98 and TA100 as described
by Ames et al.15 Tester strain TA98 was reverted from
auxotrophy to prototrophy by frame shift mutagens.
Tester strain TA100 is reverted by mutagens that cause
both frame shift and base pair substitution mutations.
2.3. Interactions with topoisomerases
For the purposes of these analytic procedures, we
used the assay kits provided by TOPOGEN, Inc.
Table 2. BRDU insertion in sensitive tumor cellsa
Negative cells (kit)
Positive cells (kit)
(2)
(2)
1.1%
38.5%
Cell lines
0 h
6 h
24 h
Compounds 7 and 8 were exposed via the plate incor-
poration methodology originally described by Ames
et al. and updated by Maron and Ames.16 Each one of
the compound was tested at 10 dose levels along with
appropriate vehicle control and positive controls with
tester strains TA98 and TA100 in the presence and
absence of Aroclor-induced rat liver S9. All dose levels
of test compounds, vehicle control and positive controls,
were plated in duplicate.
HL-60 (untreated) (2)
HL-60 (8, 10 lg/mL) (2)
HL-60 (7, 10 lg/mL) (2)
2.9%
2.7%
3.2%
2.9%
2.4%
2.9%
2.3%
4.2%
3.5%
a Percentage of cells demonstrating BRDU insertion.
Table 3. BRDU insertion in sensitive/resistant tumor cells
Negative cells (kit)
Positive cells (kit)
(1)
(1)
0.9%
46%
Cell lines
0 h
6 h
24 h
For each test compound to be evaluated positive, it must
have caused a dose-related increase in the mean rever-
tants per plate for at least one tester strain over a min-
imum of two increasing concentrations of the test
HL-60 (8, 10 lg/mL) (1)
MDA-231 (no treatment) (1)
MDA-231 (7, 10 lg/mL) (1)
6.6%
6.7%
5.4%
6.6%
6.6%
8.0%
4.7%
4.7%
10.0%

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B. K. Banik et al. / Bioorg. Med. Chem. 12 (2004) 2523–2528
Table 5. Analysis of cell cycle studies
(Columbus, Ohio). These kits contained all of the
reagents required for routine detection of topoisomerase
I and II with DNA markers for detection of enzymatic
action and standard topoisomerase inhibitors. The
activity of 7 and 8 and was determined using these
systems.
Cell lines
Time of
exposure (h)
G1
S
G2
HL-60
(8, 10 lg/mL)
0
6
38.0%
26.8%
11.0%
48.7%
50.4%
54.9%
13.3%
22.5%
34.1%
24
MDA-231
(no compound)
0
6
58.2%
58.0%
59.4%
28.1%
29.2%
26.2%
13.7%
12.9%
14.4%
Using approximately three times the concentration of 8
and two times the concentration of 7 produced IC50 in the
HL-60 line; no inhibition of either topoisomerase I or
topoisomerase II was detected. Therefore, there was no
evidence that the active b-lactams 8 and 7 had their
cytotoxic activity in sensitive cell lines through interac-
tion with DNA or DNA-related enzyme systems. These
compounds failed to increase BRDU insertion into DNA
and display any inhibitory action toward the two topo-
isomerase systems examined. In a broad sense therefore,
the failure to induce apoptosis was also consistent with
these findings because in many instances, this phenome-
non was the putative result of such DNA interactions.
24
MDA-231
(7, 10 lg/mL)
0
6
60.9%
49.9%
48.5%
26.7%
32.2%
37.6%
12.4%
17.1%
13.9%
24
3. Conclusion
Although the mechanism of action of the lead compounds
has not been totally established, our research on cell cycle
analysis offers intriguing possibilities. In the absence of
effect upon DNA or DNA enzyme systems they produced
a striking G2 blockade. This ‘check point’ in the cell cycle
has attracted a considerable amount of attention
recently.17 Studies are now underway to determine the
exact site of this action and to utilize it in an effort to
modify our b-lactams to obtain more potent compounds.
2.4. Cell cycle activity
In parallel with the apoptosis studies described above,
we utilized insertion of propidium iodine (the marker for
DNA content) as a marker of the cell cycle. This cyto-
metric determination was calibrated separately and
calculated for a related program to determine the per-
centage of cells in each phase of the cell cycle. In each
instance the treated cells were compared with that of
control HL-60 cells for the percentage of cells in each
cell cycle compartment over a period of 24 h. HL-60 cells
treated with 10 lg of 8 demonstrated a consistent
increase in the percentage of cells in the G2 phase that
was particularly striking at 24 h. While the average
percentage of untreated cells in G2 was 17.2%, that of
cells treated with 8 and 7 was 38.4% and 23.6%,
respectively (Table 4). To determine whether alteration
of the cell cycle occurred in a cell line that had repeat-
edly demonstrated resistance to the in vitro effect of 8,
we examined the human breast cancer cell line MDA-
231 (Table 5). While HL-60 cells treated with 8 (10 lg)
again demonstrated a striking increase in the G2 phase
at 24 h (34.1%), no increase was detected in MDA-231,
which was in effect identical to that in untreated MDA-
231 (approximately 14%).
4. Experimental
4.1. General methods
All of the solvents and reagents were obtained from
commercial sources and used without purification.
Reactions were monitored by TLC using pre-coated
silica gel aluminum plates containing a fluorescence
indicator. Chemical shifts of 1H NMR spectra were
given in parts per million with respect to TMS, and the
coupling constant J was measured in Hz. The data are
reported as follows: chemical shifts, multiplicity
(s ¼ singlet,
d ¼ doublet,
t ¼ triplet,
q ¼ quartet,
m ¼ multiplet). 1H NMR spectra were recorded in
CDCl3 using tetramethylsilane as an internal standard.
IR spectra were expressed as wave numbers (cmꢀ1).
4.2. Preparation of imine (general procedure)
To a solution of the amine (10 mmol) in toluene (40 mL)
was added benzaldehyde (12 mmol) and the mixture was
refluxed overnight using a Dean–Stark water separator
(monitored by TLC). When the reaction was over, tol-
uene was evaporated under reduced pressure, and the
crude product was used as such for the next reaction.
Table 4. Analysis of cell cycle studiesa
Cell lines
Time of
exposure (h)
G1
S
G2
HL-60
(no treatment)
0
6
43.3%
41.6%
45.0%
42.8%
44.2%
37.9%
13.9%
16.2%
17.2%
24
HL-60
(8, 10 lg/mL)
0
6
44.8%
27.1%
8.9%
44.6%
57.8%
52.4%
13.2%
16.9%
38.4%
4.3. Preparation of b-lactam (general procedure)
24
A
solution consisting of acetoxy acetyl chloride
HL-60
(7, 10 lg/mL)
0
6
45.3%
31.4%
18.1%
43.2%
51.3%
58.4%
11.5%
17.4%
23.6%
(1.5 mmol) in dichloromethane (10 mL) was added drop
wise to a stirred solution containing imine (1 mmol) and
distilled triethylamine (3 mmol) in dry dichloromethane
(10 mL) at ꢀ78 °C. The reaction mixture was then stirred
24
a The results represent the average of two separate runs.

B. K. Banik et al. / Bioorg. Med. Chem. 12 (2004) 2523–2528
2527
overnight at room temperature, washed with saturated
References and notes
sodium bicarbonate solution (10 mL), dilute hydrochlo-
ric acid (10%, 10 mL), brine (10 mL), dried with anhy-
drous sodium sulfate, and evaporated to obtain the crude
product. Proton NMR was performed to calculate the
ratio of the isomeric b-lactams. The pure product (70–
80%) was then isolated via column chromatography over
silica gel using ethyl acetate–hexanes (1:4) as the solvent.
1. (a) Becker, F. F.; Banik, B. K. Bioorg. Med. Chem. Lett.
1998, 8, 2877–2880; (b) Becker, F. F.; Mukhopadhyay, C.;
Hackfeld, L.; Banik, I.; Banik, B. K. Bioorg. Med. Chem.
2000, 8, 2693–2699; (c) Banik, B. K.; Becker, F. F. Bioorg.
Med. Chem. 2001, 9, 593–605; (d) Banik, B. K.; Becker,
F. F. Curr. Med. Chem. 2001, 8, 1513–1533; (e) Banik,
B. K., Ed.; Curr. Med. Chem. 2001, 8, 1343–1553; (a) Also
see: Becker, F. F.; Banik, B. K. U.S. Patent 6,015,811,
2000; (b) Becker, F. F.; Banik, B. K. U.S. Patent
6,184,224, 2001; (c) Becker, F. F.; Banik, B. K. U.S.
Patent 6,362,200, 2002.
4.4. Microwave-assisted preparation of the b-lactam
(general procedure)
2. (a) Lin, T. H.; Rogers, T. S.; Hill, D. L.; Simpson-Herren,
L.; Farnell, D. R.; Kochhar, D. M.; Alam, M.; Brouillette,
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Sugden, D. Bioorg. Med. Chem. Lett. 1994, 4, 1559–1564;
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Karlsson, A.; Camarasa, M. J.; Perez, M. J. J. Med. Chem.
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A.; Das, J.; Iqbal, J.; Rajagopalan, R. J. Med. Chem. 2002,
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3. Banik, I.; Becker, F. F.; Banik, B. K. J. Med. Chem. 2003,
46, 12–15; For some of our research on b-lactams, see: (a)
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6554; (d) Ng, S.; Banik, I.; Okawa, A.; Becker, F. F.;
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The same amount of imine, acid chloride, triethylamine
along with chlorobenzene (5 mL) was placed in an
Erlenmeyer flask (125 mL capacity). The flask was then
capped with a glass funnel and placed in a microwave
oven (G. E. Model, 1450 W). A 500 mL beaker con-
taining 200 mL of water was placed in the oven next to
the reaction flask to serve as a heat sink. The mixture
was irradiated for 3 min at intervals of 1 min each. After
the usual work up as described above, the b-lactam was
isolated (80% yield).
4.5. trans-N-(9-Phenanthrenyl)-3-acetoxy-4-phenyl-2-
azetidine-2-one (7)
Mp 152–154 °C; IR cmꢀ1 (CH2Cl2) 1754, 1625, 1599,
1528, 1498, 1455, 1401, 1369, 1280, 1219; 1H NMR
(CDCl3) d (ppm) 2.27 (s, 3H), 5.43–5.44 (d, J ¼ 1:92 Hz,
1H), 5.70–5.71 (d, J ¼ 1:96 Hz, 1H), 7.27–7.29 (m, 3H),
7.40–7.42 (m, 2H), 7.52–7.63 (m, 3H), 7.73–7.78 (m,
3H), 8.29–8.32 (m, 1H), 8.62–8.64 (d, 1H), 8.70–8.73 (m,
1H); Anal. Calcd for C25H19NO3: C, 78.72; H, 5.02; N,
3.67. Found: C, 78.69; H, 5.17; N, 3.70.
4.6. trans-N-(6-Chrysenyl)-3-acetoxy-4-phenyl-2-azeti-
dine-2-one (8)
Mp 174–176 °C; IR cmꢀ1 (CH2Cl2) 1755, 1595, 1515,
1486, 1456, 1440, 1394, 1373, 1314, 1283, 1219; 1H
NMR (CDCl3) d (ppm) 2.36 (s, 3H), 5.54–5.55 (d,
J ¼ 1:91 Hz, 1H), 5.77–5.78 (d, J ¼ 1:94 Hz, 1H), 7.26–
7.31 (m, 3H), 7.46–7.49 (m, 2H), 7.60–7.71 (m, 2H),
7.77–7.80 (m, 2H), 7.95–7.99 (m, 2H), 8.38–8.41 (m,
1H), 8.45 (s, 1H), 8.50–8.53 (d, 1H), 8.63–8.66 (d, 1H),
8.79–8.82 (m, 1H); Anal. Calcd for C29H21NO3: C,
80.72; H, 4.91; N, 3.25.12. Found: C, 80.69; H, 4.89; N,
3.19.
4. (a) Alcaide, B.; Vicente-Rodriguez, A. Tetrahedron Lett.
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Acknowledgements
We gratefully acknowledge the partial financial support
for this research project from The University of Texas
M. D. Anderson Cancer Center. We are also thankful
for NIH Cancer Center Support Grant 5-P30-CA16672-
25, in particular, the shared resources of the Pharma-
cology and Analytical Center Facility.

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B. K. Banik et al. / Bioorg. Med. Chem. 12 (2004) 2523–2528
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