Synthesis of antitumor 6-alkylidenepenicillanate sulfones and related 3-alkylidene-2-azetidinones

Article abstract of DOI:10.1016/j.bmcl.2003.09.078

6-Alkylidenepenicillanate sulfoxides and sulfones were synthesized on the base of 6-oxopenicillanate esters. The targeted splitting of their thiazolidine ring led to the formation of 3-alkylidene substituted 4-heteryldithio and 4-methylsulfonyl azetidin-2-ones. Some of mono and bicyclic β-lactams revealed potent cytotoxic properties towards monolayer tumor cells in <10-μM concentrations.

Full text of DOI:10.1016/j.bmcl.2003.09.078

Bioorganic & Medicinal Chemistry Letters 14 (2004) 147–150
Synthesis of antitumor 6-alkylidenepenicillanate sulfones
and related 3-alkylidene-2-azetidinones
Grigory Veinberg,* Irina Shestakova, Maxim Vorona, Iveta Kanepe
and Edmunds Lukevics
Latvian Institute of Organic Synthesis, 21 Aizkraukles Street, Riga, LV 1006, Latvia
Received 15 May 2003; revised 11 August 2003; accepted 30 September 2003
Abstract—6-Alkylidenepenicillanate sulfoxides and sulfones were synthesized on the base of 6-oxopenicillanate esters. The targeted
splitting of their thiazolidine ring led to the formation of 3-alkylidene substituted 4-heteryldithio and 4-methylsulfonyl azetidin-2-
ones. Some of mono and bicyclic b-lactams revealed potent cytotoxic properties towards monolayer tumor cells in <10-mM con-
centrations.
# 2003 Elsevier Ltd. All rights reserved.
Recently discovered antitumor monocyclic and bicyclic
b-lactam systems1 in general are in a good agreement
with the phenomenon of azetidin-2-one pharmacophore
inexhaustible pharmacological potential due to specific
ability of its numerous derivatives to inhibit not only
bacterial transpeptidase but also mammalian serin and
cystein proteases.2
same reaction of 3a–e with 3-fold excess of m-CPBA
resulted in the formation of sulfones 5a–e, 7d,e. All Z and
E isomeric sulfoxides and sulfones were successfully sepa-
rated by column chromatography with the only exception
in the case of 5d and 7d mixture (Scheme 1).
Reaction of Z and E isomeric sulfoxides 4a–e and 6d,e
with 2-mercaptobenzothiazole (8) according to well
known procedure6 led to the splitting of thiazolidine
ring in penicillin nucleus and to the formation of 3(Z)
and 3(E)-alkylidene-4-heteroaryldithioazetidin-2-ones
9a–e and 10d,e (Scheme 2).
Potent inhibiting properties exhibited by 7-alkylidene
substituted cephalosporanate sulfones against tumor
strains both in vitro and vivo1c encouraged us to subject
penicillanate sulfones and 4-heteroaryldithio and 4-
methylsulfonyl azetidin-2-ones containing alkylidene
side chain at positions 6 and 3 to similar biological
investigation.
The treatment of 6-(Z)-alkylidenepenicillanate sulfones
5a,b,e with DBU and alkylation of intermediate sulfinic
anion 11 with methyliodide according to Lukic proce-
dure7 resulted in the formation of 3(Z)-alkylidene-4-
methylsulfonylazetidin-2-ones 12a,b. In the case of 6(Z)-
4-nitrobenzylidenepenicillanate 5e parallel to mentioned
reaction there was observed side-chain transformation
into the equilibrium mixture of 3(Z) and 3(E) isomers
12e (Scheme 3).
The condensation of 6-oxopenicillanates obtained by
two alternative methods3,4 with phosporanes 2 accord-
ing to published methodology1c resulted in the prepara-
tion of pure Z isomer 3a and Z/E mixtures of 6-
alkylidenepenicillanates 3b–e. Oxidation of penicilla-
nates 3b–e with equivalent of m-CPBA due to the specific
structure of the side chain similar to those described in
Buynak work5 produced sterically more preferable R
sulfoxides 4a–e, 6d,e. However in the case of 4a accord-
ing to PMR and HPLC data there was obtained unse-
parable mixture of R and S sulfoxides in 86:14 ratio. The
For the purpose of SAR investigation, there were also
synthesized mono and bicyclic b-lactams 13, 14 and 16
by the deprotection of carboxyl and hydrogenation of
double bond in 5e and by the splitting of 4b with 2-
mercaptobenzimidazole (15) (Scheme 4).8
Cytotoxic properties of synthesized compounds were
tested in vitro on standard monolayer tumor cell lines:
* Corresponding author. Tel.: +371-755-5946; fax: +371-755-0338;
e-mail: veinberg@osi.lv
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.09.078

148
G. Veinberg et al. /Bioorg. Med. Chem. Lett. 14 (2004) 147–150
Scheme 1. 3a R=t-Bu, R1=MeCO (E/Z=0/100), 80%; 3b R=t-Bu, R1=t-BuOCO (E/Z=8/92), 15%; 3c R=Bh, R1=t-BuOCO (E/Z=4/96),
36%; 3d R=t-Bu, R1=Ph (E/Z=16/84), 49%; 3e R=t-Bu, R1=4-O2N–C6H4 (E/Z=40/60), 42%; 4a R=t-Bu, R1=MeCO (R/S=84/16), 85%; 4b
R=t-Bu, R1=t-BuOCO, 57%; 4c R=Bh, R1=t-BuOCO, 48%; 4d R=t-Bu, R1=Ph, 28%; 4e R=t-Bu, R1=4-O2N–C6H4, 13%; 5a R=t-Bu,
R1=MeCO, 70%; 5b R=t-Bu, R1=t-BuOCO, 27%; 5c R=Bh, R1=t-BuOCO, 13%; 5e R=t-Bu, R1=4-O2N–C6H4, 37%; 6d R=t-Bu, R1=Ph,
21%; 6e R=t-Bu, R1=4-O2N–C6H4, 19%; 7e R=t-Bu, R1=4-O2N–C6H4, 37%; 5d/7d R=t-Bu, R1=Ph (Z/E=80/20), 32%.
Scheme 2. 9a R=t-Bu, R1=MeCO, 75%; 9b R=t-Bu, R1=t-BuOCO, 42%; 9c R=Bh, R1=t-BuOCO, 36%; 9d R=t-Bu, R1=Ph, 9%; 9e
R=t-Bu, R1=4-O2N–C6H4, 60%; 10d R=t-Bu, R1=Ph, 14%; 10e R=t-Bu, R1=4-O2N–C6H4, 45%.
Scheme 3. 12a R=MeCO (Z/E=100/0), 18%; 12b R=t-BuOCO,
(Z/E=100/0), 23%; 12e R=4-O2N–C6H4, (Z/E=55/45), 23%.
MG-22A (mouse hepatoma), HT-1080 (human fibro-
sarcoma), B16 (mouse melanoma), Neuro 2A (mouse
neuroblastoma) and on normal cell lines: 3T3 (mouse
fibroblasts) and BHK (baby hamster kidney) using 96
well plates, CV, MTT and NR coloration.9,10 Concen-
tration of NO in supernatant generated by cells in the
presence of tested compounds was determined by
Greyss method.9 Extrapolation of obtained values for
Scheme 4.
100% live cells allowed to calculate comparable Total
Generation ability parameter (TG100) for tested com-
pounds.1c
tumor and normal cells. Isomeric mixture 5d/7d inhib-
ited tumor cells growth at six time lower concentrations
than normal 3T3 cells only in the case of HT-1080. The
same selectivity was observed for azedidinone 12e
towards MG-22A. The overwhelming majority of tested
compounds with intensive antitumor properties caused
similar NO generation in appropriate cells lines and vice
versa.
IC50-cytotoxic concentrations providing 50% cell inhi-
biting effect and TG100 data for synthesized compounds
are presented in Table 1. Their analysis evidenced that
the incorporation of tert-butoxycarbonylmethylene,
benzylidene and 4-nitrobenzylidene structures at the C6
position of penicillanate sulfoxides and sulfones the
same as at C3 positions of 4-heteroaryldithio and 4-
methylsulfonyl azetidin-2-ones in many cases provided
antitumor effect in <10 mM concentrations. However,
only 5c and 9c demonstrated good selectivity towards
Azetidinone 9e with 3(Z)-4-nitrobenzylidene side chain
turned out to be more active than its E-isomeric coun-
terpart 10e towards all tested tumor cell lines. Similar
leadership of Z-isomer for the 5e and 7e pair was lim-
ited by three tumor species, but isomeric couple 9d and

Table 1. In vitro cytotoxic and NO generating activity of 6-alkylidenepenicillanate sulfones and 3-alkylidene-2-azetidinones
Compd
Cell lines
B16
HT-1080
MG-22A
Neuro 2A
3T3
BHK 21
d
IC50 (CV)a IC50 (MTT)b TG100 IC50 (CV) IC50 (MTT) TG100 IC50 (CV) IC50 (MTT) TG100 IC50 (CV) IC50 (MTT) TG100 IC50 (CV) IC50 (MTT) IC50 (NR)c IC50 (CV) IC50 (MTT)
4a
4c
5a
5b
6.7
24.1
93.3
1.2
10.5
2.6
2.4
90.0
0.9
6.3
64.6
9.8
0.4
4.13.2
9.8
9.9
7.2
3.7
15.3
20.2
189
<1.2
10.5
2.6
n.t.
750
33
640
800
700
250
73
300
350
1100
750
1500
61.2
12.1
>200
79.5
16.1
>200
n.t.
750
5
389
533
800
400
58
300
300
1050
139
440
0.7
900
450
300
450
13
101
12.1
101
20.2
31
566
113
24.2
79.5
>200
71
31
14.4
16.1
12.2
18.5
9.1
n.t.
3.3
n.t.
2.5
7.8
6.4
1.9
2.5
7.4
12.2
32.5
16.2
3.9
7.9
2.8
19.9
3.9
15.9
2.1
100
800
800
400
16.2
>20
12.2
2.4
17.5
>200
600
18
800
400
5.0
5.8
n.t.
n.t.
4.0
3.9
n.t.
n.t.
5.2
5c
>200
>200
5d/7d
5e
6d
7e
9a
9b
9d
9c
9e
10d
10e
12b
12e
14
15.9
1.6
18.6
2.8
21.2
2.4
3.8
106
0.9
4.6
116
154
2.3
29.3
6.7
5.1
0.8
32.2
32.5
6.7
10.8
0.7
300
9.8
15.2
4.8
4.7
9.8
6.7
11.8
0.5
6.2
22.4
6.7
10.8
0.5
300
10.6
9.3
34.2
8.0
350
300
800
1000
375
4.3
1000
600
300
63
3.8
58.7
71.4
11.8
3.8
30.4
62.5
13.7
400
83
850
750
27
3.3
31.4
2.4
33.1
3.3
28.4
6.7
n.t.
n.t.
2.4
51.3
4.5
n.t.
n.t.
8.90
17.9
9.8
0.4
350
8.8
9.3
3.4
2.7
11.8
12.4
5.7
15.7
n.t.
19.6
15.5
>200
>200
3.4
4.5
3.6
2.5
300
72.5
10.4
1.9
4.8
4.8
2.1
700
400
350
450
9
10.6
9.9
3.6
1.6
13.7
8.3
1.4
4.6
51.0
12.2
4.8
850
350
250
250
19.6
n.t.
5.3
16.7
n.t.
n.t.
6.0
n.t.
n.t.
1.9
5.8
11.0
2.3
13.8
8.5
13.1
32.1
>200
>200
>200
23.2
134
>200
39.4
134
16
13
23.2
17.7
39.4
18.6
1400
200
1100
200
25.5
34.8
400
62.6
37.1
500
30.2
7.0
7.0
n.t., not tested; IC50, concentration (mM) providing 50% cell killing effect.
a CV, coloration.
b MTT, coloration.
c NR, coloration.
d Extrapolated total NO radicals generation ability at 100 mM concentration of tested compound.

150
G. Veinberg et al. /Bioorg. Med. Chem. Lett. 14 (2004) 147–150
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and azetidinones with the same side chain did not allow
to make definite judgement about the preference of
closed or opened thiazolidine ring in b-lactam contain-
ing structures. The IC50 data obtained for 13 an 14 gave
clear evidence about the inefficiency of the deprotection
of carboxyl and of the side-chain hydrogenation.
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