Synthesis, evaluation and structural studies of antiproliferative tubulin-targeting azetidin-2-ones

Bioorganic & Medicinal Chemistry 19 (2011) 2306–2325

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry

journal homepage: w ww.elsevier.com/locate/bmc

Synthesis, evaluation and structural studies of antiproliferative

tubulin-targeting azetidin-2-ones

Niamh M. O’Boyle ^a,, Lisa M. Greene ^b, Orla Bergin ^c, Jean-Baptiste Fichet ^a,

⇑

Thomas McCabe ^d, David G. Lloyd ^e, Daniela M. Zisterer ^b, Mary J. Meegan ^a,

⇑

^aSchool of Pharmacy and Pharmaceutical Sciences, Centre for Synthesis and Chemical Biology, Trinity College Dublin, Dublin 2, Ireland

^bSchool of Biochemistry & Immunology, Trinity College Dublin, Dublin 2, Ireland

^cUCD Conway Institute and School of Biomolecular and Biomedical Science, University College Dublin, Belﬁeld, Dublin 4, Ireland

^dSchool of Chemistry, Trinity College Dublin, Dublin 2, Ireland

^eMolecular Design Group, School of Biochemistry & Immunology, 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:

A series of azetidin-2-ones substituted at positions 1, 3 and 4 of the azetidinone ring scaffold were syn-

thesised and evaluated for antiproliferative, cytotoxic and tubulin-binding activity. In these compounds,

the cis double bond of the vascular targeting agent combretastatin A-4 is replaced with the azetidinone

ring in order to enhance the antiproliferative effects displayed by combretastatin A-4 and prevent the cis/

trans isomerisation that is associated with inactivation of combretastatin A-4. The series of azetidinones

was synthetically accessible via the Staudinger and Reformatsky reactions. Of a diverse range of hetero-

cyclic derivatives, 3-(2-thienyl) analogue 28 and 3-(3-thienyl) analogue 29 displayed the highest potency

in human MCF-7 breast cancer cells with IC₅₀values of 7 nM and 10 nM, respectively, comparable to

combretastatin A-4. Compounds from this series also exhibited potent activity in MDA-MB-231 breast

cancer cells and in the NCI60 cell line panel. No signiﬁcant toxicity was observed in normal murine breast

epithelial cells. The presence of larger, bulkier groups at the 3-position, for example, 3-naphthyl deriva-

tive 21 and 3-benzothienyl derivative 26, resulted in relatively lower antiproliferative activity in the

Received 3 January 2011

Revised 11 February 2011

Accepted 13 February 2011

Available online 17 February 2011

Keywords:

Combretastatin A-4 analogues

Colchicine

b-Lactam

Azetidinone

Antiproliferative

Cytotoxicity

Tubulin

Structure–activity

Staudinger reaction

Reformatsky reaction

micromolar range. Tubulin-binding studies of 28 (IC₅₀= 1.37 lM) conﬁrmed that the molecular target

of this series of compounds is tubulin. These novel 3-(thienyl) b-lactam antiproliferative agents are useful

scaffolds for the development of tubulin-targeting drugs.

1. Introduction

a highly-validated target in cancer therapy and a large number

of chemically diverse substances bind to tubulin and alter

The mitotic phase of cell division relies on assembly of the mi-

totic spindle. Microtubules are the main constituent of the mitotic

microtubule polymerisation and dynamics in diverse ways.³These

ligands can be broadly divided into two categories: those that

inhibit the formation of the mitotic spindle, for example, colchicine

(1, Fig. 1) and the vinca alkaloids, and those that inhibit the

disassembly of the mitotic spindle once it has formed, for example,

paclitaxel and epothilone.⁴Tubulin-binding compounds, such as

paclitaxel and vinblastine, are in widespread clinical use for vari-

ous types of cancer.³

spindle and are composed of the a–b heterodimeric protein tubu-

lin.¹They are highly dynamic structures that alternate between

periods of growing and shrinking through the addition or removal

of tubulin subunits at the ends of microtubules.²Microtubules are

Abbreviations: CA-4, combretastatin A-4; CA-4P, combretastatin A-4 phosphate;

DAMA-colchicine, N-deacetyl-N-(2-mercaptoacetyl)-colchicine; DCM, dichloro-

methane; GTP, guanidine triphosphate; HRMS, high resolution molecular ion

determination; IR, infra red; LDA, lithium diisopropylamide; MTT, 3-(4,5-dimeth-

ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NCI, National Cancer Institute;

NMR, nuclear magnetic resonance; PBS, phosphate buffered saline; SAR, structure–

activity relationship; TBAF, tetrabutylammonium ﬂuoride; TBDMS, tert-butyldim-

ethylchlorosilane; TLC, thin layer chromatography; THF, tetrahydrofuran; TMCS,

trimethylchlorosilane.

The combretastatins are a group of tubulin-binding diaryl

stilbenes isolated from the stem wood of the South African tree

Combretum Caffrum.⁵There is no written evidence of use of the

plant for treating cancer amongst the indigenous people of Africa.⁶

A number of constituent stilbenes were found to inhibit the growth

of colon cancer cells and were strong inhibitors of tubulin polymer-

isation.⁵Combretastatin A-4 (2a, Fig. 1) and combretastatin A-1

(2b, Fig. 1) exhibited potent anticancer activity against a panel of

human cancer cell lines from diverse origins, including leukaemic,

breast and multi-drug resistant cancers.⁴Stilbenes 2a and 2b

⇑

Corresponding authors. Tel.: +353 1 896 2798; fax: +353 1 896 2793.

E-mail addresses: oboyleni@tcd.ie (N.M. O’Boyle), mmeegan@tcd.ie (M.J.

Meegan).

doi:10.1016/j.bmc.2011.02.022

N. M. O’Boyle et al. / Bioorg. Med. Chem. 19 (2011) 2306–2325

2307

R₁

OCH₃

R₂

R₁

OCH₃

H₃CO

O

R₂

B

OCH₃

N

O

OCH₃

A

N

H

O

OCH₃

H₃CO

1

3a

R₁=R₂= H

Colchicine

2a

R₁=OH, R₂=H[CA-4]

R₁=R₂=OH[CA-1]

3b

2b

R₁=H; R₂=OH

3c R₁=R₂=OH

2c R₁=OPO₃Na₂, R₂=H[CA-4P]

Figure 1. Structures of small molecule tubulin-binding agents.

inhibit the formation of the mitotic spindle by binding to the

colchicine-binding site of tubulin and were also shown to exhibit

anti-vascular properties in vivo, probably by increasing tumor-

vessel permeability.^7,8However, 2a and 2b display poor water

solubility rendering them unsuitable for clinical use and water-

soluble prodrugs, including combretastatin A-4-phosphate (2c,

Fig. 1), are in clinical trials, for example evaluation of 2c for

advanced anaplastic thyroid cancer and in combination with

chemotherapy for advanced solid tumours.^9–112c displays excel-

lent water solubility, good stability and cell growth inhibitory

activity comparable to that of 2a.¹¹

and also induced apoptosis in ex vivo samples from patients with

chronic myeloid leukaemia, including those positive for the T315I

mutation displaying resistance to imatinib mesylate and dasati-

nib.²⁵Due to the potency of the 3-phenyl substituted compounds

3a and 3b, a series of novel analogues with diverse carbocycles

and heterocycles to replace the phenyl ring were developed and

evaluated for their antiproliferative activity and tubulin effects.

These novel compounds reported herein contain carbocyclic, het-

erocyclic or modiﬁed aryl substituents at position 3 of the b-lactam

ring while the aryl rings A and B present in 2a are retained at posi-

tions 1 and 4 of the azetidinone scaffold. The rigid b-lactam ring

structure facilitates a similar spatial arrangement between the

two aryl rings at N-1 and C-4 as is observed for the cis conﬁgura-

tion of 2a.

However, the biological activity of 2a is lost if isomerisation to

the inactive trans form occurs, for example during storage.^12,13

Many conformationally restricted analogues of 2a are known, in

which the cis double bond is replaced by a heterocycle, thereby

locking the two aryl rings in a cis-like conﬁguration relative to each

other. A diverse range of heterocycles as replacements for the

double bond have been reported including benzoxepins,¹⁴oxadi-

azolines,¹⁵imidazoles,¹⁶combretoxazolones,¹⁷combretocyclopen-

tenones¹³and thiophenes,¹⁸and are the subject of many

reviews.^4,12,19Previously, b-lactam containing compounds were

reported to have anticancer activity^20,21and the b-lactam ring scaf-

fold has been investigated as a template for analogues of 2a.^22–24

We have recently reported a series of antiproliferative, tubulin-

binding b-lactam compounds, where 3a, 3b and 3c (Fig. 1) emerged

2. Results and discussion

2.1. Chemistry

The synthetic routes for target b-lactam preparation are illus-

trated in Schemes 1–6. The compounds chosen for initial investiga-

tion all contained the 3,4,5-trimethoxyphenyl (mimicking ring A of

2a) as the b-lactam N-1 substituent, together with the 4-methoxy-

phenyl ring as the b-lactam C-4 substituent. Two routes for the

b-lactam ring-forming reaction were employed. The Staudinger

reaction requires appropriately substituted acetic acids or acid

chlorides and imines²⁶(Scheme 3; routes I–III), while the Refor-

matsky reaction requires an organozinc species (derived from an

as the most potent agents with activity comparable to 2a.²⁴

A

3-phenyl ring substituent improved the potency of this series of

b-lactam compounds compared to either 3-methyl or 3,3-dimethyl

substitution or b-lactams unsubstituted at C-3. b-Lactam 3b was

shown to induce rapid apoptosis in vitro in leukaemic HL-60 cells

a

-bromoester) and an imine (Scheme 6).²⁷In two cases, the

desired acetic acid precursors for b-lactam preparation were not

O

Cl

H

OC₂H₅

O

R₁

R₂

O

H

P

a

b

O

OC₂H₅

O

P

N⁺

Cl^-

Cl

+

N

+

S

O

OC₂H₅

O

N

4

R₁

R₂

R₁

O

P

OC₂H₅

d

c

S

OC₂H₅

OH

R₂

N

O

5a: R₁=H, R₂=CH₃

5b

: R₁=R₂=C₄H₄

Scheme 1. Synthesis of substituted acetic acids 5a and 5b. Reagents and conditions: (a) diethyl ether, 0 °C, 1 h; (b) 20 °C; (c) NaH, toluene, 50 °C, 1 h; (d) 10 M HCl, 50 °C,

30 min.

2308

N. M. O’Boyle et al. / Bioorg. Med. Chem. 19 (2011) 2306–2325

OCH₃

8a

: R₁=H

N

H₃CO

8b: R₁=OSi(CH₃)₂C(CH₃)₃

: R₁=NO₂

R₁

8c

OCH₃

R₂

R₃

8d:

R₂=R₃=-C₄H₄-; R₄=H

8e: R₂=H; R₃=R₄=-C₄H₄-

N

H₃CO

NH₂

H₃CO

O

a

+

R₄

R

H

OCH₃

S

N

H₃CO

8f

OCH₃

Scheme 2. Synthesis of imines 8a–8f. Reagents: EtOH, reﬂux, 3 h.

R₁

Cl

OH

a

R₂

R₁

O

6a

: R₁=CH₃; R₂=C₆H₅

6b: R₁=H; R₂=CH₂C₃H₃S

OCH₃

R₁

H₃CO

10 - 18

b: Route I:

Cl

c: Route II: 19

OCH₃

R₃

R₂

+

N

R

O

R₁

R₂

8a 8f

-

R₁

O

S

N

O

OCH₃

R₁

H₃CO

OH

R₂

OCH₃

d: Route III:

20 - 27

H₃CO

10 - 15

H₃CO

N

8a 8f

20 - 27

-

and

16 - 19

R₂=H unless otherwise indicated

10: R₁=C₆H₁₁

19: R₁=(3-NO₂,4-OCH₃)C₆H₃

20: R₁=C₁₀H₇(1-naphthyl)

11: R₁=CH₃; R₂=C₆H₅

12

21

: R₁=C₁₀H₇(2-naphthyl)

: R₁=R₂=C₆H₅

13: R₁= -SC₄H₃-

22: R₁=C₈H₅NCH₃

23: R₁=C₄H₃O

14: R₁= -SC₄H₃-; R₂=OTBDMS

15

24

: R₁= -C₂HSC₂H₂-

: R₁=CH₂-SC₄H₃-

16: R₁=C₁₀H₇(1-naphthyl)

17: R₁=C₁₀H₇(2-naphthyl)

25: R₁= -SC(CH₃)C₃H₂-

26: R₁=C₈H₅S (benzothienyl)

18

27

: R₁= -C₂HSC₂H₂-; R₂=OTBDMS

: R₁= -SC₄H₃-

Scheme 3. Synthesis of b-lactams 10–27. Reagents and conditions: (a) SOCl₂, CHCl₃, reﬂux, 3 h; (b) (route I) triethylamine, CH₂Cl₂, reﬂux, 3 h; (c) (route II) triethylamine,

CH₂Cl₂, 18 h; (d) (route III) triphosgene, triethylamine, anhydrous CH₂Cl₂, reﬂux, 5 h, 18 h; TBMDS = tert-butyldimethylchlorosilyl.

commercially available. To prepare selected 2-thienyl containing

derivatives, the appropriately substituted 2-thienyl aldehydes

were converted to the corresponding acetic acids through the use

of tetraethyl dimethylaminomethylenediphosphonate (4, Scheme

1).²⁸It was previously reported that the most convenient and efﬁ-

cient method to produce this aminodiphosphonate reagent was the

reaction of dimethylchloroformiminium chloride with 2.2 equiva-

lents of triethyl phosphite.²⁹The in situ generated iminium ion re-

acts with triethyl phosphite to generate 4 in high yields of up to

76% (Scheme 1). This reagent reacts with the aldehyde of interest

to form an enamine phosphonate, which is hydrolysed with strong

acid to produce the substituted acetic acids 5a and 5b (Scheme 1).

Substituted acetic acids were required in the Staudinger reac-

tion for the preparation of b-lactams in two procedures. Firstly,

the acid chloride could be generated from the acid for use in a

traditional Staudinger reaction. Alternatively, direct preparation

of b-lactams from substituted acetic acids by the Staudinger route

using an acid-activating agent is possible (see below). In the ﬁrst

N. M. O’Boyle et al. / Bioorg. Med. Chem. 19 (2011) 2306–2325

2309

HO

O₂N

H₂N

TBDMSO

OCH₃

R

O

R

S

a

N

O

OCH₃

O

OCH₃

H₃CO

19

30

14: R= 2-thienyl

27

28: R= 2-thienyl

29

: R= 3-thienyl

Scheme 5. Synthesis of amino-substituted azetidinone 30. Reagents and condtion:

(a) Zn, CH₃CO₂H, 7 days.

Scheme 4. Synthesis of phenolic azetidinones 28 and 29. Reagents and conditions:

(a) TBAF, THF, 0 °C, 15 min; TBMDS = tert-butyldimethylchlorosilyl.

immediately used in the following b-lactam forming reaction with-

out further puriﬁcation (Scheme 3).

The preparation of imine precursors 8a–8f is achieved in high

yield by condensation of the appropriately substituted aldehydes

and anilines (Scheme 2).³⁰In the case of 3-hydroxy-4-methoxy-

benzaldehyde, the hydroxyl group was ﬁrst protected using the

TBDMS silyl ether group and then used for the preparation of Schiff

base 8b.³¹

option, generation of the acid chloride (6a and 6b) from the corre-

sponding substituted acetic acid was achieved by chlorination with

thionyl chloride. The chlorination reactions were monitored by IR

until absorption was observed between

m m

1780 cm^À1and

1815 cm^À1, due to carbonyl stretching in the acid chloride. Acid

chlorides 6a and 6b were synthesised in high yield and were

OCH₃

O

OCH₃

Br

OH

O

CH₃

R

a

b

+

OCH₃

N

H₃CO

O

OCH₃

O

OCH₃

R

H

N

H₃CO

OCH₃

8a

9

31: R=

32: R=

33: R=

O

S

34: R=

H₃C

S

OCH₃

O

R

N

O

35: R=

36: R=

OCH₃

S

c

OH

OCH₃

H₃CO

R

S

35, 36

N

O

OCH₃

37

: R=

d

S

R

H₃CO

N

O

38: R=

OCH₃

H₃C

H₃CO

37, 38

Scheme 6. Synthesis of azetidinones 9 and 31–33. Reagents and conditions: (a) Zn, TMCS, benzene, microwave; (b) LDA, dry THF, À78 °C; (c) pyridinium chlorochromate,

CH₂Cl₂, 2 h; (d) tosyl chloride, pyridine, reﬂux, 5 h.

2310

N. M. O’Boyle et al. / Bioorg. Med. Chem. 19 (2011) 2306–2325

Figure 2. Ortep representation of the X-ray crystal structure of b-lactam 11 drawn with 50% thermal ellipsoids:(a) cis isomer and (b) trans isomer; 2 enantiomers shown with

relative stereochemistry.

N. M. O’Boyle et al. / Bioorg. Med. Chem. 19 (2011) 2306–2325

2311

Figure 3. Ortep representation of the X-ray crystal structure of b-lactam 12 drawn with 50% thermal ellipsoids.

The preparation of target b-lactams 10–27 is illustrated in

Scheme 3. b-Lactam synthesis was primarily carried out using

the Staudinger reaction, which is a cycloaddition reaction between

a ketene and an imine under basic conditions, where the ketene

can be generated from an acid chloride.³²b-Lactams 10–18 were

prepared by this method (Scheme 3, route I). A modiﬁed procedure

for preparation of the 3-thienyl compound 19 was employed, as

the standard Staudinger reaction conditions were unsuccessful.

b-Lactam 19 was obtained in 48% yield using milder conditions

with overnight stirring at room temperature (Scheme 3, route

II).³³The b-lactam ring scaffold could also be generated directly

from the appropriately substituted acetic acid and imine precur-

sors using an acid-activating agent in a one-step reaction, without

generation and isolation of the acid chloride (Scheme 3, route III).

Many acid-activating agents are known in literature, for example,

Mukaiyama’s reagent, p-toluene-sulfonyl chloride and various

phosphorous derived reagents.³²Triphosgene has been reported

for the synthesis of b-lactams and was employed as an acid-acti-

vating agent in synthesis of compounds 20–27.^34,35

The stereochemistry of products from the Staudinger reaction

depends on numerous factors, including the reaction conditions,

the order of addition of the reagents and the substituents present

on both the imine and on the acid chloride.^20,32,36The trans prod-

ucts were isolated exclusively in all but one case, as evident from

the representative ¹H NMR spectrum of compound 13 where the

H-3 and H-4 were identiﬁed at d 4.47 ppm and d 4.90 ppm respec-

tively as a pair of coupled doublets, J_3,4= 2.5 Hz. The formation of

the trans isomer is likely due to steric hindrance when two aryl

rings present in the b-lactam structure at C-3 and C-4. The sole

exception in this series of compounds was the 3-methyl-3-phenyl

substituted b-lactam 11, which was obtained as a mixture of cis/

trans isomers (ratio 1:1.13 cis:trans) and separated by crystallisa-

tion from ethanol. The presence of structural isomers was con-

ﬁrmed by X-ray crystal structures of both isomers of 11 (Fig. 2).

Both enantiomers of 11 can be seen in the crystal structure of

the trans isomer (Fig. 2b). The X-ray crystal structure of 3,3-diphe-

nyl b-lactam 12 is illustrated in Figure 3

The phenolic products 28 and 29 were obtained on treatment of

the silyl ethers 14 and 27 respectively with tetrabutylammonium

ﬂuoride at 0 °C (Scheme 4).³¹Separation of the silylated b-lactams

14 and 27 from the silylated imine in the ﬁnal reaction mixture

was difﬁcult and hence removal of the silyl protecting group was

carried out before subsequent puriﬁcation. Reduction of the nitro

group in compound 19 to the corresponding amine 30 was achieved

by treatment with zinc dust and glacial acetic acid (Scheme 5).³⁷

Preliminary biochemical assessments of b-lactams 10–30 in

MCF-7 human breast cancer cells revealed potent antiproliferative

activity for thiophene containing compounds 13, 24, 28, 29 and 30

(Table 2). On the basis of these results, further sulfur-containing

b-lactam analogues were prepared. 3-Unsubstituted b-lactam 9

was used as a precursor for a variety of substitution reactions at

C-3. Compound 9 was obtained by Reformatsky reaction of imine

8a with ethylbromoacetate and zinc using microwave technology

and TMCS as the zinc-activating agent as we have previously re-

ported (Scheme 6).^24,27Deprotonation of 9 with LDA at À78 °C fol-

lowed by reaction with aldehydes^32,38was successful for the

preparation of secondary alcoholic derivatives 31–34 (Scheme 6).

Further treatment of compounds 32 and 33 by oxidation with

pyridinium chlorochromate³⁹yielded ketone analogues 35 and

36 (Scheme 6). Transformation of alcoholic derivatives 32 and 34

by dehydration with tosyl chloride in pyridine³⁸delivered corre-

sponding vinylogous analogues 37 and 38 (Scheme 6). Although

formation of E/Z isomers at the 3-position double bond is possible

for 37 and 38, only one isomer was obtained in each case, possibly

due to steric hindrance between the thiophene ring and aryl sub-

stituents at positions 3 and 4 of the azetidinone ring. The products

37 and 38 were assigned the Z conﬁguration, by comparison of the

signal for H-

for compounds 37 and 38, respectively) with reported values for

in related E and Z 3-methylenesubstituted azetidin-2-ones.²¹

a

in the ¹H NMR spectrum (d 6.45 ppm and d 6.38 ppm

Ha

2312

N. M. O’Boyle et al. / Bioorg. Med. Chem. 19 (2011) 2306–2325

Table 1

Table 1 (continued)

Azetidin-2-one combretastatin A-4 analogues^a

Compound No.

R₁

R₂

H

R₂

OCH₃

22

N

R₁

CH₃

N

O

OCH₃

23

24

H

O

H₃CO

H

S

Compound No.

R₁

R₂

H

25

26

27

H₃C

S

10

H

S

H

CH₃

11

12

OTDBMS^b

S

a

Routes for synthesis were: compounds 10–18: route I; compound 19: route II;

compounds 20–27: route III; route I: triethylamine, CH₂Cl₂, reﬂux, 3 h; route II:

triethylamine, CH₂Cl₂, 18 h; route III: triphosgene, triethylamine, anhydrous CH₂Cl₂,

reﬂux, 5 h, 18 h.

H

b

13

14

TBMDS = tert-butyldimethylchlorosilyl.

S

OTBDMS^b

Table 2

Antiproliferative activities of b-lactams in human MCF-7 breast cancer cells

H

15

Compound number

Antiproliferative activity IC₅₀(l

M)^a

S

10

11

12

13

15

16

17

18

19

20

21

22

23

24

25

26

28

29

30

31

32

33

34

35

36

37

38

3a²⁴

2a^b

0.58 0.4

43.5 15

43.2 26

S

0.064 0.02

1.64 1.9

0.12 0.07

0.62 0.3

0.91 0.3

0.37 0.2

11.3 7.0

2.5 0.9

N

16

O

OCH₃

H₃CO

6.6 4.1

0.15 0.1

0.06 0.03

0.25 0.2

0.85 0.5

0.007 0.002

0.01 0.007

0.042 0.02

1.1 0.7

S

N

17

O

OCH₃

H₃CO

1.2 0.8

0.99 0.8

1.26 1.4

0.95 0.5

0.47 0.07

4.05 2.5

0.58 0.2

0.034 0.02

0.0052 0.002

S

N

18

OCH₃

H₃CO

a

IC₅₀values are half maximal inhibitory concentrations required to block the

growth stimulation of MCF-7 cells. Values represent the mean SEM (error val-

NO₂

H

ues Â 10^À6) for at least three experiments performed in triplicate.

19

20

21

S

b

The IC₅₀value obtained for 2a in this assay is 0.0052 lM for MCF-7 which is in

good agreement with the reported values for 2a using the MTT assay on human

MCF-7 breast cancer cell line.^18,47,48,67

All b-lactam compounds 10–27 were obtained as enantiomeric

mixtures and separation by chiral liquid chromatography was

demonstrated for selected compounds 13 and 33, indicating a

H

N. M. O’Boyle et al. / Bioorg. Med. Chem. 19 (2011) 2306–2325

2313

Table 3

1:1 mixture of the two enantiomers for both compounds (Fig. 10,

Antiproliferative activities of b-lactams in human MDA-MB-231 breast cancer cells

Supplementary data). We have previously demonstrated stability

of 3-phenyl b-lactams over the pH range 4–9.²⁴Preliminary stabil-

ity studies of 3-(2-thienyl) b-lactams with aryl, naphthyl and thie-

nyl substituents at C-4 (compounds 13, 16, 17 and 18) were carried

out in acidic, neutral and basic pH conditions. The half-lives for

these compounds were determined to be greater than 24 h at pH

values of 4, 7.4 and 9, with the compounds being least stable at

pH 4 for all four analogues assessed.

Compound number

Antiproliferative activity IC₅₀(l

M)^a

13

28

29

0.19 0.2

0.18 0.2

0.049 0.04

0.11 0.07

0.08 0.03

0.043

30

3a²⁴

2a^b

a

IC₅₀values are half maximal inhibitory concentrations required to block the

growth stimulation of MDA-MB-231 cells. Values represent the mean SEM (error

2.2. Biological results and discussion

values Â 10^À6) for at least three experiments performed in triplicate.

b

The IC₅₀value obtained for 2a in this assay is 0.043 lM for MDA-MB-231 which

2.2.1. Antiproliferative effects

is in good agreement with the reported values for 2a using the MTT assay on the

human MDA-MB-231 breast cancer cell line.^68,69

The series of b-lactam analogues of 2a were initially evaluated

for their antiproliferative activity in human MCF-7 breast cancer

cells using the MTT cell viability assay.⁴⁰The previously reported

lead compound, 3a, showed potent activity in this cancer cell line

MCF-7 cells, 3-methyl-3-phenyl substituted 11 (IC₅₀of 43.45

evaluated as a mixture of cis/trans isomers) and 3,3-diphenyl

substituted 12 (IC₅₀of 43.17 M). From these results, it can be

lM;

with an IC₅₀of 0.034 lM and further investigation established 3a

and 3b as potential lead development candidates for the treatment

of leukaemia.^24,25To establish a more detailed SAR and further

examine the effects of 3-substitution on antiproliferative activity,

the phenyl ring at the 3-position of 3a was replaced with a wide

variety of carbocyclic and heterocyclic substituents, while retain-

ing the N-1, C-4 substituents of 3a (Table 1).

The most potent b-lactams in MCF-7 cells were those with 3-(2-

thienyl) (13) and 3-(3-thienyl) (24) substituents with IC₅₀values of

64 and 60 nM, respectively. Replacement of the sulfur atom of 24

with oxygen (furan analogue 23) led to a twofold decrease in

activity. Introduction of multiple and/or larger substituents at this

position led to substantial decrease in activity compared to substi-

tution with a phenyl ring, for example diphenyl 12 (IC₅₀of

l

deduced that substituents larger than a phenyl ring at the 3-posi-

tion are detrimental to the antiproliferative activity of this series of

compounds.

Compounds 28 (IC₅₀= 7 nM) and 29 (IC₅₀= 10 nM) with addi-

tional hydroxyl groups at the 3-position of the 4-phenyl ring, anal-

ogous to 2a, displayed increased potency in MCF-7 cells over their

respective parent compounds, 13 and 24, of 9- and 10-fold, respec-

tively. A dose response graph for 13, 29 and 2a is shown in Figure

4. b-Lactam 30, in which the phenolic moiety of 28 is replaced with

an amino substituent, was marginally more potent than 13 but less

active than 28 (IC₅₀values of 42, 64 and 7 nM, respectively). Both

28 and 30 offer the possibility of further modiﬁcation to form ester

or amide prodrugs via their phenolic and amino groups.

The effect of introduction of a carbon spacing atom between the

thiophene ring and C-3 of the b-lactam ring was investigated (com-

pounds 15, 32–37). Secondary alcohols 32 and 33 and ketone

43.17

and methyl indole 22 (6.59

logue 26 has a much greater IC₅₀value of 0.85

sponding thiophene derivative 13 (IC₅₀value of 0.064

l

M), 1-naphthyl 20 (11.32

M). The bulky benzothiophene ana-

M than the corre-

M). This is

lM), 2-naphthyl 21 (2.47 lM)

l

in-line with previous observations that poly-substitution of the C-3

phenyl ring led to decreased activity.²⁴A cyclohexane ring at

position 3 of the b-lactam (10) showed decreased activity of over

100-fold compared to the 3-phenyl substituted compound 3a.

Cyclohexane-substituted 10 has a higher c Log P value of 4.87 com-

pared to 3.88 for 3a, indicating a marked increase in hydrophobic-

ity. It is possible that this property contributes to its relative lack of

antiproliferative activity. Disubstitution at the 3-position yielded

the two compounds with the least antiproliferative activity in

derivatives 35 and 36 (IC₅₀values = 1.17, 0.99, 0.95 and 0.47

respectively) were over 16-fold less potent than 13 and 24. Meth-

ylene 15 and alkene 37 have IC₅₀values of 1.64 and 4.05 M,

respectively, conﬁrming that extension of the distance between

the thiophene and b-lactam ring has a detrimental effect on the po-

tency of this series.

Analogues of 2a with a thiophene ring at the 3-position and

naphthyl substituents at the 4-position (16 and 17) in place of

the 3-hydroxy-4-methoxyphenyl moiety were assessed for anti-

proliferative activity as the naphthyl moiety has been previously

shown to be a good replacement for the B-ring of 2a.³⁷However,

both 4-(2-naphthyl) b-lactam 16 and 4-(1-naphthyl) b-lactam 17

lM,

l

displayed decreased IC₅₀values of 0.12 and 0.62

0.007 M for 28. Replacement of the 4-position substituted phenyl

ring with a thiophene ring (18) led to decreased activity (IC₅₀va-

lue = 0.91 M) indicating that, while a C-3 thiophene ring is advan-

lM compared to

l

tageous for activity, such a substitution is not tolerated at C-4.

The most active analogues in the MCF-7 antiproliferative stud-

ies (13, 28, 29 and 30) were subsequently evaluated against human

MDA-MB-231 breast cancer cells and exhibited submicromolar

IC₅₀values. Of the four compounds tested, 3-(3-thienyl) b-lactam

29 was the most potent (IC₅₀= 49 nM) and showed improved

activity compared to the lead compound 3a (IC₅₀= 78 nM)

(Table 3).

Figure 4. Antiproliferative effect of 2a and 3-(3-thienyl)-b-lactams 13 and 29 in

MCF-7 human breast cancer cells. MCF-7 cells were seeded at

a density of

2.5 Â 10⁴cells per well in 96-well plates and left for 24 h to allow the cells to

2.2.2. Further biochemical assessment: NCI60 cell line screen,

cytotoxicity and tubulin polymerisation

Compounds 13, 28 and 30 were chosen for speciﬁc analysis and

further development (screening in the National Cancer Institute

(NCI) 60-cell line panel, determination of cytotoxicity, tubulin-bind-

ing and molecularmodelling) basedon theanalysis of their drug-like

adhere to the surface of the wells. A range of concentrations (0.01 nM–50 lM) of

the compound were added in triplicate and the cells left for 72 h. Control wells

contained the equivalent volume of the vehicle ethanol/DMSO (70:30%) (1% v/v). An

MTT assay was performed to determine the level of anti-proliferation. The values

represent the mean SEM (error values) for three experiments performed in

triplicate.

2314

N. M. O’Boyle et al. / Bioorg. Med. Chem. 19 (2011) 2306–2325

properties from a Tier-1 proﬁling screen (based on experimentally

determined solubility and chemical stability together with predic-

tions of permeability, metabolic stability, Pgp substrate status,

blood–brain barrier partition, plasma protein binding and human

intestinal absorption properties which indicated the suitability of

these compounds for further development). These compounds

satisfy Lipinski’s ‘rule of ﬁve’ for drug-like properties, for example,

molecular weights of 13, 28 and 30 are less than 500, the number

of oxygen/nitrogen atoms is less than 10, the number of hydrogen

bond donors is less than 5 and the c Log P values are 2.78, 1.88

and 1.95, respectively (<5), implying that they are moderate

lipophilic–hydrophobic drugs and are suitable candidates for

further investigation.

than 51 nM in 47 of the cell lines. The mean GI₅₀value for 13 across

all cell lines is 27.54 nM [log GI₅₀= (À7.56 M)]. The antiprolifera-

tive activity of 13 was particularly potent for all three leukaemic

cell lines (<10 nM) and for CNS, melanoma and breast cell lines,

indicating a wide-range of potential therapeutic applications. The

mean LC₅₀for 13 across the range of cell lines is >100 lM indicat-

ing minimal cytotoxicity. In addition, matrix ^COMPAREanalysis^42,43

(measuring the correlation between two compounds with respect

to their differential antiproliferative activity) demonstrated good

correlation between 13, 3b and 2a (r = 0.76 and 0.61, respectively).

However, this algorithm does not distinguish between different

tubulin-based mechanisms of action.⁴⁴The COMPARE algorithm was

also used to compare the differential antiproliferative activities of

13 to compounds with known mechanisms of action in the NCI

Standard Agent Database⁴and showed correlations to vincristine,

paclitaxel, maytansine and rhizoxin, all of which affect microtu-

bule polymerisation (Table 4).

3-Thienyl b-lactam 13 was screened using the NCI60 panel of

cell lines (Table 5, Supplementary data)⁴¹and exhibited IC₅₀values

of less than 10 nM in 25 of the 56 cell lines, and IC₅₀values of less

Table 4

2.2.3. Evaluation of toxicity in normal murine mammary

epithelial cells

Standard ^COMPAREanalysis of b-lactam 13^a

Further toxicity measurements were carried out on 3-(2-thie-

nyl) b-lactam 28, the most potent antiproliferative b-lactam in

antiproliferative assessment with MCF-7 cells. Toxicity studies in

healthy mouse mammary epithelial cells at two different cell con-

centrations were carried out (25,000 and 50,000 cells/mL har-

vested from mid- to late-pregnant CD-1 mice and cultured as

described previously^45,46). These results indicate a favourable tox-

icity proﬁle for 28 in comparison to 2a. The IC₅₀value for both

Rank

Compound

r

Based on GI₅₀mean graph

1

2

3

4

5

Rhizoxin

Maytansine

0.55

0.531

0.495

0.457

0.443

Vincristine sulfate (hiConc = 10^À3M)

Vincristine sulfate (hiConc = 10^À5M)

Trimetrexate

Based on TGI mean graph

1

2

3

4

5

Vincristine sulfate

0.583

0.555

0.529

0.52

Rhizoxin (hiConc = 10^À4.3M)

Maytansine

compounds was greater than 10 lM indicating minimal toxicity

for this compound (Fig. 5) (Table 7 and Fig. 12, Supplementary

data).

Rhizoxin (hiConc = 10^À9M)

Rhizoxin (hiConc = 10^À4M)

0.507

Based on LC₅₀mean graph

2.2.4. Tubulin polymerisation studies

1

2

3

Thioguanine

Paclitaxel

-aspiriginase

0.915

0.854

0.84

The effects of representative b-lactam CA-4 analogue (com-

pound 28) which demonstrated potent antiproliferative effects

in vitro was assessed on the assembly of puriﬁed bovine tubulin.

The ability of 2a to effectively 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 V_maxvalue offers the most sensitive indicator of

tubulin/ligand interactions and hence fold-changes in V_maxvalues

for polymerisation curves of the compound with reference to eth-

L

4

5

Morpholino-ADR

Mitramycin

0.736

0.733

a

The target set was the standard agent database and the target set endpoints

were selected to be equal to the seed end points. Standard ^COMPAREanalysis was

performed. Correlation values are Pearson correlation coefﬁcients. Vincristine sul-

fate and rhizoxin appear at different concentrations as they have been tested by the

NCI at multiple concentration ranges.

Figure 5. Cell viability in healthy murine epithelial cells. Mouse mammary epithelial cells were harvested from mid- to late- pregnant CD-1 mice and cultured. The isolated

mammary epithelial cells were seeded at two concentrations. After 24 h, they were treated with 2 L volumes of test compound which had been pre-prepared as stock

solutions in ethanol to furnish the concentration range of study, 1 nM–100 M, and re-incubated for a further 72 h. Control wells contained the equivalent volume of the

vehicle ethanol (1% v/v). The cytotoxicity was assessed using alamar blue dye.

l

N. M. O’Boyle et al. / Bioorg. Med. Chem. 19 (2011) 2306–2325

2315

the binding site for these compounds is most likely to be the

colchicine site, as it has been demonstrated that 2a and many

reported examples of the structurally related conformationally

constrained 2a analogues bind at the colchicine site.^47–49The col-

chicine-binding site in tubulin is mainly buried in the b-subunit

of tubulin, whilst maintaining some limited interactions with the

a-subunit. The H7 and H8 a-helices, the T7 loop and the S8 and

S9 b-strands contribute to the binding site and interact with the

colchicine-site ligand.⁵⁰Two of the most important residues for

colchicine-binding are Val318 and Cys241. Val318 tubulin variants

have reduced sensitivity to 1, and 1 substituted with more reactive

groups instead of the methoxys can be crosslinked with

Cys241.^51,52The Thr179 residue has also been highlighted as being

important, though not critical, for binding.⁵³Previously reported b-

lactams 3a and 3b both show interactions with Val318 and Cys241,

while 3b has an additional hydrogen-bonding interaction with

Thr179.²⁴

Figure 6. Inhibition of tubulin polymerisation for b-lactam 28. Effects of compound

28 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 340PC spectrophotometer at 340 nm at 30 s

intervals for 60 min at 37 °C. The graph shows one representative experiment. Each

experiment was performed in triplicate.

The antiproliferative assessment of the b-lactam compounds

10–38 (Table 2) established a clear trend, where 2- and 3-thio-

phene substituents at C-3 proved extremely potent (e.g., com-

pounds 13, 28, 29 and 30). In contrast, bulkier substituents at the

3-position of the azetidinone ring (e.g., 3,3-diphenyl substituted

analogue 12) led to a substantial decrease in activity, even though

the required substitution pattern of rings A and B were preserved.

To rationalise this observation, molecular structures of compounds

11 and 12, determined by single-crystal X-ray crystallography,

were examined (Figs. 2 and 3). The structures revealed a conforma-

tion for the azetidinones 11 and 12 in which the two aromatic rings

located at N-1 and C-4 are not coplanar. The observed dihedral an-

gle between ring A and ring B in the X-ray crystal structures of

these analogues is À61.7° for compound 12 (Fig. 3). For compound

11, a dihedral angle of 73.4° is observed for the cis isomer while

values of 62.7° and À66.1° are calculated for the two enantiomers

anol control were calculated. Tubulin polymerisation studies on 28

showed a 3.2-fold reduction in the V_maxat 10

sixfold reduction for 2a tested as a control. The IC₅₀value for 28

for the inhibition of V_maxwas calculated to be 1.37 0.85 M,

while an IC₅₀value of 6.25 2.53 M was obtained for the effect

lM compared to a

l

in overall polymer mass (calculated from area under the polymer-

isation curve) (Fig. 6). This conﬁrms that the molecular target of

these antiproliferative b-lactams is tubulin.

2.3. Structural studies, molecular modelling and rationalisation

of biochemical activity

Based on the 3D structural similarity between the ligands 1, 2a

and the b-lactam analogues reported in this study, we propose that

Figure 7. Comparison of the docked conformations of b-lactams 3a, 12 and DAMA-colchicine. Compound 12 is shown in green; 3a (left) and DAMA-colchicine (right) are

coloured by atom with oxygen red, nitrogen blue, carbon grey and sulfur yellow. Protein residues are not shown for clarity.

2316

N. M. O’Boyle et al. / Bioorg. Med. Chem. 19 (2011) 2306–2325

of the trans isomer (Fig. 2). These values are very different to the

dihedral angle previously observed for 3a of 46.9°,²⁴and are also

higher than the values for 1 (55°)⁵¹and 2a (53°).⁵⁴It is possible

that this difference in orientation between the two rings is one

of the factors leading to the decreased antiproliferative activity ob-

served for 11 and 12. When these compounds 11 and 12 are

docked computationally in the colchicine-binding site of tubulin,

the reason for the decreased biochemical activity in vitro becomes

apparent. The docked conformations of both 3a and 12 and of 1

and 12 (Fig. 7) reveal that 12 is predicted to be orientated differ-

ently to both 3a and 1 within the binding site. The N-1 trimethoxy-

phenyl rings of 3a and 12 adopt similar positions in the binding

site but the C-4 4-methoxyphenyl ring lies in a different plane, pro-

jecting backwards for 1 and 3a, but forward for 12. The 3,3-diphe-

nyl rings of analogue 12 occupy the same part of the binding site as

the 4-(4-methoxyphenyl) ring of 3a, indicating that this part of the

colchicine-binding site of tubulin can accommodate a larger

volume and explains the relative switch in orientation for 12 com-

pared to 1 and 3a. This may account for the loss in antiproliferative

activity seen with 12 and any other related analogues with a bulky

substitution pattern at this position (e.g., compounds 11, 20, 21

and 22), as the potential for forming a binding interaction with

Thr179 is lost and microtubule dynamics may not be as dramati-

cally affected. However, interactions between the N-1 trimethoxy-

phenyl ring of 12 and both Val318 and Cys241 are maintained,

accounting for the residual antiproliferative activity of this

compound.

interact with the trimethoxyphenyl ring of 28. Hydrogen bonding

of the phenolic group of ring B to Thr179 and Lys352 contributes

to the strong tubulin-binding of this compound observed in vitro

and is suggested to account for the increased antiproliferative activ-

ity seen with the phenolic compounds 28 and 29. Further hydro-

phobic interactions between the 3-(2-thienyl) ring and the

colchicine site residues (Fig. 9) reinforce the binding to the protein,

for example with Val181, Leu248 and Ala250. These interactions, in

contrast to those of b-lactam 12, may stabilise the binding of 28 and

provide a rational basis for the potent antiproliferative and tubulin-

binding activity displayed by these compounds.

3. Conclusions

Building on previous work where the b-lactam ring scaffold was

utilised to replace the isomerisable double-bond of 2a, further

investigations to determine a comprehensive SAR of antiprolifera-

tive b-lactams has led to the new discovery of novel analogues

with signiﬁcant antiproliferative and tubulin-binding activity. A

trend for small ring heterocyclic systems at the 3-position ring

leading to increased potency was determined, with larger ring sys-

tems such as naphthyl, indole and benzothiophene leading to sig-

niﬁcantly less potent antiproliferative activities. 3-(2-Thienyl) and

3-(3-thienyl) derivatives 28 and 29 displayed the most potent anti-

proliferative activity in MCF-7 and MDA-MB-231 breast cancer cell

lines with low nanomolar antiproliferative IC₅₀values, and 28 was

shown to be minimally toxic to normal murine epithelial cells. The

molecular target of b-lactam 28 was conﬁrmed to be tubulin, and

In contrast to 3,3-diphenyl b-lactam 12, virtual molecular dock-

ing of the most potent b-lactam from this series, 3-(2-thienyl) b-lac-

tam 28, predicts a docked conformation similar to that previously

predicted for 3b (Fig. 8, also Fig. 11 in Supplementary data).²⁴The

dihedral angle between the N-1 and C-4 phenyl rings of 28, calcu-

lated from the energy minimised structure rather than a crystal

structure, is 61.8°. The predicted 2D interactions of the ligand with

the protein are shown in Figure 9.⁵⁵Residues Cys241 and Val318

this compound displayed an IC₅₀value of 1.37 lM for inhibition

of tubulin polymerisation. The 2-thienyl and 3-thienyl containing

compounds reported herein will be evaluated in further in vitro

and in vivo studies to develop their potential vascular targeting

and antiangiogenic applications.

4. Experimental section

4.1. Chemistry: experimental methods

All reagents were commercially available and were used

without further puriﬁcation unless otherwise indicated. Tetra-

hydrofuran (THF) was distilled immediately prior to use from

Na/benzophenone under a slight positive pressure of nitrogen,

toluene was dried by distillation from sodium and stored on acti-

vated molecular sieves (4 Å) and dichloromethane was dried by

distillation from calcium hydride prior to use. IR spectra were

recorded as thin ﬁlms on NaCl plates or as KBr discs on a Perkin-

Elmer Paragon 100 FT-IR spectrometer. ¹H and ¹³C NMR spectra

were obtained on a Bruker Avance DPX 400 instrument at 20 °C,

400.13 MHz for ¹H spectra, 100.61 MHz for ¹³C spectra, in CDCl₃

(internal standard tetramethylsilane) by Dr. John O’Brien and Dr.

Manuel Ruether in the School of Chemistry, Trinity College Dublin.

Low resolution mass spectra were run on a Hewlett–Packard 5973

MSD GC–MS system in an electron impact mode, while high reso-

lution accurate mass determinations for all ﬁnal target compounds

were obtained on a Micromass Time of Flight mass spectrometer

(TOF) equipped with electrospray ionisation (ES) interface oper-

ated in the positive ion mode at the High Resolution Mass Spec-

trometry Laboratory by Dr. Martin Feeney in the School of

Chemistry, Trinity College Dublin. Elemental analysis was carried

out in the microanalytical laboratory, University College Dublin,

Belﬁeld, Dublin 4. Thin layer chromatography was performed using

Merck Silica Gel 60 TLC aluminium sheets with ﬂuorescent indica-

tor visualising with UV light at 254 nm. Flash chromatography was

carried out using standard Silica Gel 60 (230–400 mesh) obtained

from Merck. All products isolated were homogenous on TLC.

Figure 8. Docked pose of b-lactam 28 in the colchicine-binding site of tubulin.

Docked pose of b-lactam 28 in the colchicine-binding site of tubulin (PDB entry

1SA0). Signiﬁcant binding residues Thr179, Cys241, Val318 and Lys352 are

indicated. Hydrogens are not shown for clarity. Coloured by atom: grey (carbon);

red (oxygen); blue (nitrogen); yellow (sulfur). Residue numbers are those used by

Ravelli et al.⁵¹

.

N. M. O’Boyle et al. / Bioorg. Med. Chem. 19 (2011) 2306–2325

2317

Figure 9. 2D representation of binding interactions of b-lactam 28 in the colchicine-binding site of tubulin. 2-D rendering of ligand–protein interactions using LigX module of

MOE used to create docked structures of 28 in the colchicine-binding site of tubulin.⁵⁵Residue numbers are those used by Ravelli et al.⁵¹

.

Analytical high-performance liquid chromatography (HPLC) to

determine the purity of the ﬁnal compounds was performed using

a Waters 2487 Dual Wavelength Absorbance detector, a Waters

1525 binary HPLC pump, a Waters In-Line Degasser AF and a

Waters 717plus Autosampler. The column used was a Varian Pur-

suit XRs C18 reverse phase 150 Â 4.6 mm chromatography col-

umn. Samples were detected using a wavelength of 254 nm. All

samples were analysed using acetonitrile (70%): water (30%) over

10 min and a ﬂow rate of 1 mL/min. Unless otherwise indicated,

the purity of the ﬁnal products was P 95% (see Table 6, Supple-

mentary data). Chiral liquid chromatography was carried out on

selected compounds using a Chiral-AGP™ 150 Â 4.0 mm column

supplied by ChromTech Ltd (now supplied by Chiral Technologies

Europe) with a Chiral-AGP™ guard column and the same Waters

hardware as used above for purity testing. Gradient elution was

used beginning with 10% of organic phase and ﬁnishing with 90%

of organic phase over a period of 20 min. The organic mobile phase

was 2-propanol and the aqueous phase was a sodium phosphate

buffer. The sodium phosphate buffer, consisting of 10 mM sodium

dihydrogen orthophosphate dihydrate (NaH₂PO₄) in HPLC-grade

water, was made up to pH 7.0 using sodium hydroxide. The ﬂow

rate was 0.5 mL/min and detection was carried out at 225 nm.

2318

N. M. O’Boyle et al. / Bioorg. Med. Chem. 19 (2011) 2306–2325

4.1.1. 3-(tert-Butyldimethylsilanyloxy)-4-methoxybenz-aldehyde

To a solution of 3-hydroxy-4-methoxybenzaldehyde (0.02 mol)

and dimethyl-tert-butylchlorosilane (0.024 mol) in dry CH₂Cl₂

(60 mL) was added 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)

(0.032 mol). The resulting mixture was stirred at room tempera-

ture under a nitrogen atmosphere until complete on thin layer

chromatography. The solution was then diluted with CH₂Cl₂

(80 mL) and washed successively with water (60 mL), 0.1 M HCl

(60 mL) and saturated aqueous NaHCO₃(60 mL). The organic layer

was removed and dried by ﬁltration through anhydrous sodium

sulfate, Na₂SO₄. 3-(tert-Butyldimethylsilanyloxy)-4-methoxybenz-

organic extracts were dried over anhydrous Na₂SO₄and concen-

trated to give the desired product.

4.1.4.1. Benzo[b]thiophen-2-yl-acetic acid (5a). This was ob-

tained from (2-benzo[b]thiophen-2-yl-1-dimethylamino-vinyl)-

phosphonic acid diethyl ester as a light brown powder (61.2%

yield); mp: 130 °C (lit. 140–142 °C²⁹); IR (KBr disk) m_max

:

1715.55 cm^À1(–C@O); ¹H NMR (400 MHz, CDCl₃) d 3.99 (s, 2H,

CH₂), 7.24–7.37 (m, 3H, ArH), 7.74–7.76 (m, 1H, ArH), 7.81–7.83

(m, 1H, ArH); ¹³C NMR (100 MHz, CDCl₃) d 35.39 (CH₂), 122.18,

123.36, 124.06, 124.19, 124.39, 135.03, 139.60, 140.03 (ArC),

175.87 (C@O).

aldehyde was isolated as a brown oil (yield 93.8%)⁵⁶¹H NMR

;

(400 MHz, CDCl₃) d 0.19 (s, 6H, –SiCH₃), 1.02 (s, 9H, –CH₃), 3.91

(s, 3H, OCH₃), 6.97 (d, 1H, J = 8.52 Hz, ArH), 7.39 (s, 1H, ArH),

7.48–7.50 (m, 1H, ArH), 9.83 (s, 1H, –CHO); ¹³C NMR (100 MHz,

CDCl₃) d 18.43 (–SiCH₃), 25.65 (–CH₃), 55.58 (OCH₃), 111.19,

120.05, 126.39, 130.17, 145.57, 156.65 (ArC), 191.01 (CHO); HRMS

(M⁺+H): C₁₄H₂₂O₃S requires 266.1338; found: 266.1349.

4.1.4.2. (5-Methylthiophen-2-yl)acetic acid (5b). This was ob-

tained as a brown oil from [1-dimethylamino-2-(5-methylthio-

phen-2-yl)-vinyl]-phosphonic acid diethyl ester (1% yield) and

was used immediately in the subsequent reaction without further

puriﬁcation;²⁹IR (KBr disk) _max: 1705.90 cm^À1(–C@O); ¹H NMR

m

(400 MHz, CDCl₃) d 2.49 (s, 3H, CH₃), 3.83 (s, 2H, CH₂), 6.65 (d,

1H, J = 2.52, ArH), 6.77 (d, 1H, J = 2.52, ArH); ¹³C NMR (100 MHz,

CDCl₃) d 15.29 (CH₃), 35.25 (CH₂), 124.99, 127.19, 131.63, 139.95,

177.04 (C@O).

4.1.2. Tetraethyl dimethylaminomethylenediphosphonate (4)

To a chilled solution of dimethylformamide (97.9 mmol) in

diethyl ether (150 mL) was added dropwise with stirring a solution

of oxalyl chloride (97.9 mmol) in diethyl ether (20 mL). Following

addition, the mixture was allowed to warm to room temperature

and stirred for 1 h. Triethyl phosphite (215 mmol) was then added

dropwise with stirring. After one hour the mixture was concen-

trated under reduced pressure. The product was obtained as a yel-

low oil in 75.5% yield.²⁸; d 0.92 (m, 12H, 4 Â CH₃), 2.19 (s, 6H,

2 Â CH₃), 2.90 (t, 1H, 4 Â CH), 3.68–3.80 (m, 8H, CH₂); HRMS

(M⁺+Na): C₁₁H₂₇NNaO₆P₂requires 354.1211; found: 354.1218.

4.1.5. General method for chlorination of acetic acid derivatives

The appropriate acetic acid (10 mmol) was brought to reﬂux

with thionyl chloride (12 mmol) in chloroform (30 mL). The reac-

tion was monitored by IR until absorption appeared between

1780 cm^À1and 1815 cm^À1. The solvent was evaporated under re-

duced pressure.

4.1.5.1. 2-Phenylpropionyl chloride (6a)⁵⁷

. This was prepared

4.1.3. General method for preparation of enamine phosphonate

To a suspension of NaH (33 mmol) in dry toluene (20 mL) was

added dropwise with stirring a solution of 4 (16.7 mmol) in dry tol-

uene (20 mL). After 1 h, a solution of the appropriate aldehyde

(16.7 mmol) in dry toluene (20 mL) was added. The mixture was

stirred at 50 °C for one hour and then concentrated. The residue

was partitioned between ethyl acetate and water and the aqueous

layer was extracted with ethyl acetate three times. The residue was

puriﬁed by column chromatography (hexane/ethyl acetate gradi-

ent) to afford the clean product.

from phenylpropionic acid in 92.2% yield as a pale yellow oil and

was used immediately in the subsequent reaction without further

puriﬁcation; IR (KBr)

m

_max: 1784.17 cm^À1(–C@O, acid chloride).

4.1.5.2. 3-Thiophen-2-yl-propionyl chloride (6b)⁵⁸

.

This was

prepared from 3-thiophen-2-yl-propionic acid (10 mmol) and

was used immediately in the subsequent reaction without further

puriﬁcation (pale yellow oil, 90.9% yield); IR (KBr) m_max

:

1782.84 cm^À1(–C@O, acid chloride).

4.1.6. General method for imine formation

4.1.3.1. (2-Benzo[b]thiophen-2-yl-1-dimethylaminovinyl)phos-

phonic acid diethyl ester. This was obtained by reaction of

benzo[b]thiophene-2-carbaldehyde with tetraethyl dimethyl-

aminomethylenediphosphonate (4). The product was obtained as

a dark orange oil (56.7% yield).^28,29¹H NMR (400 MHz, CDCl₃) d

1.36 (t, 6H, 2 Â CH₃), 2.69 (s, 6H, 2 Â CH₃), 4.12–4.22 (m, 4H,

2 Â CH₂), 7.31–7.40 (m, 3H, ArH), 7.73–7.78 (m, 2H, ArH); HRMS

(M⁺+Na): C₁₆H₂₂NNaO₃PS requires 362.0956; found: 362.0946.

The appropriate amine (10 mmol) was heated at reﬂux with the

appropriate aldehyde (10 mmol) in ethanol (50 mL) for 3 h. The

reaction mixture was reduced in vacuo and the resulting solution

was left to stand until solid product crystallised. The resulting

imine was recrystallised from ethanol.

4.1.6.1.

N-(4-Methoxybenzylidene)-3,4,5-trimethoxybenzen-

amine (8a). This was synthesised by reacting 3,4,5-trimethoxy-

benzenamine with 4-methoxybenzaldehyde. The product was

obtained as pale yellow crystals (yield 87%); mp: 120 °C;²⁴IR

4.1.3.2.

phosphonic acid diethyl ester. This was obtained by reaction of

5-methyl-thiophene-2-carbaldehyde with tetraethyl dim-

[1-Dimethylamino-2-(5-methylthiophen-2-yl)vinyl]-

(KBr disk) m

_max: 1604.66 cm^À1(C@N); ¹H NMR (400 MHz, CDCl₃)

d 3.86 (s, 3H, OCH₃), 3.87 (s, 3H, OCH₃), 3.90 (s, 6H, 2 Â OCH₃)

6.47 (s, 2H, ArH) 6.98 (d, 2H, J = 9.2 Hz, ArH) 7.84 (d, 2H,

J = 9.2 Hz, ArH) 8.40 (s, 1H, –CH@N); ¹³C NMR (100 MHz, CDCl₃) d

55.35 (OCH₃), 56.00 (OCH₃), 60.94 (OCH₃), 98.00, 114.13, 128.97,

130.39, 135.97, 148.22, 153.45, 159.03(ArC), 162.20 (CH@N); Ele-

mental analysis: found: C, 67.73; H, 6.35; N, 4.63; C₁₇H₁₉NO₄re-

quires C, 67.76; H, 6.36; N, 4.65.

ethylaminomethylenediphosphonate (4). The product was ob-

tained as a dark orange oil (22.5% yield); ¹H NMR (400 MHz,

CDCl₃) d 1.32 (t, 6H, 2 Â CH₃), 2.45 (s, 3H, CH₃), 2.60 (s, 6H,

2 Â CH₃), 4.06–4.13 (m, 4H, 2 Â CH₂), 6.63 (s, 1H, CH), 6.98 (d,

1H, J = 3.52, ArH), 7.18 (d, 1H, J = 12.04, ArH); HRMS (M⁺+Na):

C₁₃H₂₂NNaO₃PS requires 326.0956; found: 326.0972.

4.1.4. General procedure for hydrolysis of enamine phos-

phonates

The appropriate enamine phosphonate was reﬂuxed in 10 M

HCl (50 mL) for 30 min. The mixture was poured onto ice water

(200 mL) and extracted with ethyl acetate (twice). The combined

4.1.6.2.

dene](3,4,5-trimethoxyphenyl)amine (8b). This was synthes-

ised by reaction of 3-(tert-butyldimethylsilanyloxy)-4-

[3-(tert-Butyldimethylsilanyloxy)-4-methoxybenzyli-

methoxybenzaldehyde with 3,4,5-trimethoxybenzenamine. The

product was obtained as a yellow solid. Yield 64%, mp: 105 °C;²⁴

N. M. O’Boyle et al. / Bioorg. Med. Chem. 19 (2011) 2306–2325

2319

IR (KBr disk) m_max1619.77 cm^À1, 1579.73 cm^À1(C@N); ¹H NMR

(400 MHz, CDCl₃) d 0.20 (s, 6H, 2 Â CH₃), 1.03 (s, 9H, C(CH₃)₃),

3.87–3.91 (m, 12H, 4 Â OCH₃), 6.48 (s, 2H, ArH), 6.93 (d, 2H,

J = 8.04 Hz, ArH), 7.43–7.47 (m, 1H, ArH), 8.35 (s, 1H, CH@N); ¹³C

NMR (100 MHz, CDCl₃) d À5.04 (CH₃–Si–CH₃), 18.03 (CH₃–C–

CH₃), 25.27 (C(CH₃)₃), 54.98 (OCH₃), 55.63 (OCH₃), 97.62, 110.94,

119.71, 123.48, 128.95, 135.53, 144.87, 147.94, 153.05, 153.59

(ArC), 158.84 (C@N); HRMS (M⁺+H): C₂₃H₃₄NO₅Si requires

432.2206; found: 432.2213.

nium chloride solution (20 mL) and 25% ammonium hydroxide

(20 mL), and then with dilute HCl (40 mL), followed by water

(40 mL). 4-(4-Methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)azeti-

din-2-one (9) was obtained as green crystals (yield 43%); mp:

70–71 °C; IR (NaCl ﬁlm) m

_max: 1747.5 cm^À1(C@O, b-lactam); ¹H

NMR (400 MHz, CDCl₃): d 2.85 (dd, 1H, J = 2.48 Hz, 12.56 Hz, H-

3), 3.48 (dd, 1H, J = 5.52 Hz, J = 9.56 Hz, H-4), 3.65 (s, 6H,

2 Â OCH₃), 3.70 (s, 3H, OCH₃), 3.73 (s, 3H, OCH₃), 4.88 (d, 1H,

J = 2.76 Hz, H-4), 6.53 (s, 2H, ArH), 6.86 (d, 2H, J = 8.56 Hz, ArH),

7.26 (d, 2H, J = 8.56 Hz, ArH); ¹³C NMR (100 MHz, CDCl₃): d 46.36

(C-3), 53.56 (OCH₃), 54.78 (OCH₃), 55.23 (OCH₃), 55.49 (OCH₃),

60.36 (C-4), 93.92, 113.58, 126.83, 129.48, 133.62, 133.68,

152.94, 159.29 (ArC), 164.14 (C@O); HRMS (M⁺+Na). Found

366.1330; C₁₉H₂₁NO₅Na requires 366.1317.

4.1.6.3. (4-Methoxy-3-nitrobenzylidene)(3,4,5-trimethoxyphen-

yl)amine (8c). This was synthesised by reaction of 3,4,5-trimeth-

oxyphenylamine and 4-methoxy-3-nitrobenzaldehyde. The product

was obtained as a yellow powder (yield 88%); mp: 162–163 °C;²⁴IR

(KBr disk) m_max1616.90 cm^À1, 1580.79 cm^À1(C@N); ¹H NMR

(400 MHz, CDCl₃) d 3.89 (s, 3H, OCH₃), 3.93 (s, 6H, 2 Â OCH₃), 4.06

(s, 3H, OCH₃), 6.52 (s, 2H, ArH), 7.21 (d, 1H, J = 8.52 Hz, ArH), 8.13

(dd, 1H, J = 8.52 Hz, J = 2.48 Hz, ArH), 7.39 (d, 1H, J = 2.48 Hz, ArH),

8.45 (s, 1H, (C@N); ¹³C NMR (100 MHz, CDCl₃) d 55.70 (OCH₃),

56.40 (OCH₃), 60.59 (OCH₃), 97.75, 113.20, 125.59, 128.52, 133.29,

146.59, 153.19, 154.41 (ArC), 155.66 (C@N). Elemental analysis:

found: C, 58.91; H, 5.25; N, 7.95; C₁₇H₁₈N₂O₆requires C, 58.96; H,

5.24; N, 8.09%.

4.1.8. General method I for b-lactam preparation

The appropriate imine (5 mmol) and triethylamine (15 mmol)

were added to dry CH₂Cl₂(50 mL) and the mixture was brought

to reﬂux at 60 °C. Once reﬂuxing, the appropriately substituted

acid chloride (7.5 mmol) was injected dropwise through a rubber

stopper. This mixture was reﬂuxed for 3 h. The mixture was

washed ﬁrstly with distilled water (50 mL) (twice) and then with

saturated aqueous sodium bicarbonate solution (50 mL). The or-

ganic layer was dried by ﬁltration through anhydrous sodium sul-

fate. The organic layer containing the product was reduced in

vacuo. The pure product was isolated by ﬂash column chromatog-

raphy over silica gel (hexane/ethyl acetate gradient).

4.1.6.4. 3,4,5-Trimethoxy-N-(naphthalen-2-ylmethylene)aniline

(8d). This was synthesised using 3,4,5-trimethoxyphenylamine

and 2-naphthaldehyde as a yellow solid (78% yield); mp: 132–

136 °C; IR (KBr disk) m

_max: 1626.22 and 1581.26 cm^À1(C@N); ¹H

NMR (400 MHz, CDCl₃): d 3.91 (s, 3H, OCH₃), 3.95 (s, 6H, 2 Â OCH₃),

6.29 (s, 2H, ArH), 7.58 (m, 2H, ArH), 7.94–7.96 (m, 3H, ArH), 8.84

(m, 2H, ArH), 8.67 (s, 1H, HC@N). Elemental analysis: Found: C,

74.68; H: 6.02; N: 4.31; C₂₀H₁₉NO₉requires C, 74.65, H, 5.98, N,

4.26.

4.1.9. 3-Cyclohexyl-4-(4-methoxyphenyl)-1-(3,4,5-trimethoxy-

phenyl)azetidin-2-one (10)

was obtained from 2-cyclohexylacetyl chloride and N-(4-

methoxybenzylidene)-3,4,5-trimethoxybenzenamine (8a) as

a

:

white powder (15.0% yield); mp: 144 °C; IR (NaCl ﬁlm) m_max

1744.47 cm^À1(C@O, b-lactam); ¹H NMR (400 MHz, CDCl₃) d 1.14–

1.32 (m, 5H, CH₂), 1.68–1.78 (m, 3H, CH₂), 1.82–1.90 (m, 2H, CH₂),

2.05–2.09 (m, 1H, CH), 2.96 (m, 1H, H-3), 3.71 (s, 6H, 2 Â OCH₃),

3.77 (s, 3H, OCH₃), 3.81 (s, 3H, OCH₃), 4.69 (d, 1H, J = 2.52 Hz, H-4),

6.54 (s, 2H, ArH), 6.89–6.93 (m, 2H, ArH), 7.28–7.31 (m, 2H, ArH);

¹³C NMR (100 MHz, CDCl₃) d 25.77 (CH₂), 25.92 (CH₂), 26.22 (CH₂),

30.71 (CH₂), 30.92 (CH₂), 38.28 (CH), 55.31 (OCH₃), 55.94 (OCH₃),

58.93 (C-3), 60.93 (OCH₃), 66.11 (C-4), 94.45, 114.51, 127.23,

130.28, 134.05, 134.08, 153.42, 159.57 (ArC), 167.47 (C@O); HRMS

(M⁺+Na): C₂₅H₃₁NO₅Na requires 448.2100; found 448.2101.

4.1.6.5. 3,4,5-Trimethoxy-N-(naphthalen-1-ylmethylene)aniline

(8e). This was synthesised using 3,4,5-trimethoxyphenylamine

and 2-naphthaldehyde as a yellow solid (77% yield); mp: 108–

116 °C; IR (KBr disk)

m

_max: 1625.74, 1610.62 and 1583.40 cm^À1

(C@N); ¹H NMR (400 MHz, CDCl₃): d 3.92 (s, 3H, OCH₃), 3.96 (s,

6H, 2 Â OCH₃), 6.61 (s, 2H, ArH), 7.59–7.67 (m, 3H, ArH), 7.96 (d,

1H, J = 8.52 Hz, ArH), 8.02 (d, 1H, J = 8 Hz, ArH), 8.12 (m, 1H,

ArH), 9.05 (d, 1H, J = 8.52 Hz, ArH), 9.15 (s, 1H, HC@N). Elemental

analysis: found: C, 74.67, H, 5.97, N, 4.30; C₂₀H₁₉NO₉requires C,

74.65, H, 5.98, N, 4.26.

4.1.9.1.

4-(4-Methoxyphenyl)-3-methyl-3-phenyl-1-(3,4,5-tri-

4.1.6.6.

3,4,5-Trimethoxy-N-(thiophen-2-ylmethylene)aniline

methoxyphenyl)azetidin-2-one (11). This was obtained from 2-

phenylpropionyl chloride (6a) and N-(4-methoxybenzylidene)-

3,4,5-trimethoxybenzenamine (8a) as a white powder (29.4%

(8f). This was synthesised from 3,4,5-trimethoxyphenylamine

and thiophene-2-carbaldehyde as a yellow solid (81% yield); mp:

92–98 °C; IR (KBr disk)

m

_max: 1617.78 and 1584.53 cm^À1(C@N);

yield); mp: 183 °C; IR (NaCl ﬁlm) m

_max: 1737.24 cm^À1(C@O, b-lac-

¹H NMR (400 MHz, CDCl₃): d 3.88 (s, 3H, OCH₃), 3.92 (s, 6H,

2 Â OCH₃), 6.52 (s, 2H, ArH), 7.16–7.18 (m, 1H, ArH), 7.52–7.55

(m, 2H, ArH), 8.61 (s, 1H, HC@N); Elemental analysis: found: C,

60.62; H, 5.44; N, 5.01; C₁₄H₁₅NO₃S requires C, 60.63; H, 5.45; N,

5.05.

tam); ¹H NMR (400 MHz, CDCl₃) (cis isomer) d 1.91 (s, 3H, CH₃),

3.72–3.73 (m, 9H, 3 Â OCH₃), 3.79 (s, 3H, OCH₃), 5.00 (s, 1H, H-

4), 6.66 (d, 2H, J = 7 Hz, ArH), 6.94 (d, 2H, J = 8 Hz, ArH), 7.09–

7.13 (m, 5H, ArH); ¹³C NMR (100 MHz, CDCl₃) (cis isomer) d

24.34 (CH₃), 54.68 (OCH₃), 55.52 (OCH₃), 60.50 (OCH₃), 64.01 (C-

3), 68.32 (C-4), 94.53, 113.23, 126.28, 126.81, 127.59, 128.10,

133.60, 133.87, 137.32, 152.96, 158.84 (ArC), 168.80 (C@O); ¹H

NMR (400 MHz, CDCl₃) (trans isomer) d 1.91 (s, 3H, CH₃), 3.73 (s,

6H, 2 Â OCH₃), 3.80 (s, 3H, OCH₃), 3.85 (s, 3H, OCH₃), 5.19 (s, 1H,

H-4), 6.64 (d, 2H, J = 7 Hz, ArH), 6.97 (d, 2H, J = 8 Hz, ArH), 7.12

(m, 1H, ArH), 7.28–7.34 (m, 3H, ArH), 7.40–7.44 (t, 2H, ArH),

7.55–7.56 (m, 2H, ArH); ¹³C NMR (100 MHz, CDCl₃) (trans isomer)

d 19.20 (CH₃), 54.86 (OCH₃), 55.52 (OCH₃), 55.58 (OCH₃), 60.53

(OCH₃), 62.13 (C-3), 66.56 (C-4), 94.68, 113.23, 113.80, 125.43,

126.08, 126.81, 126.90, 127.59, 127.82, 128.10, 128.47, 133.24,

141.43, 153.01, 159.13 (ArC), 168.77 (C@O); HRMS (M⁺+H):

4.1.7. 4-(4-Methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-

azetidin-2-one (9)

Zinc powder (0.927 g, 15 mmol) was activated using trimethyl-

chlorosilane (0.65 mL, 5 mmol) in anhydrous benzene (5 mL), by

heating for 15 min at 40 °C and subsequently for 2 min at 100 °C

in a microwave. After cooling, N-(4-methoxybenzylidene)-3,4,5-

trimethoxybenzenamine (8a) (10 mmol) and ethyl 2-bromoacetate

(12 mmol) were added to the reaction vessel and the mixture was

placed in the microwave for 30 min at 100 °C. The reaction mixture

was ﬁltered through Celite to remove zinc, then diluted with

CH₂Cl₂(50 mL). This solution was washed with saturated ammo-

C₂₆H₂₈NO₅requires 434.1967; found 434.1953.

2320

N. M. O’Boyle et al. / Bioorg. Med. Chem. 19 (2011) 2306–2325

4.1.9.2. 4-(4-Methoxyphenyl)-3,3-diphenyl-1-(3,4,5-trimethoxy-

phenyl)azetidin-2-one (12). This was obtained 2,2-diphenylace

tyl chloride and N-(4-methoxybenzylidene)-3,4,5-trimethoxy-

benzenamine (8a) as a white crystalline material (70.3% yield);

129.17, 132.89, 133.06, 133.25, 133.94, 135.55, 153.13 (ArC),

163.88 (C@O); HRMS (M⁺+Na): C₂₃H₂₃NO₅NaS requires 468.1245;

found 468.1227.

Mp: 167 °C; IR (KBr disk)

m

_max: 1729.20 cm^À1(C@O, b-lactam);

4.1.9.7. 1-(3,4,5-Trimethoxyphenyl)-4-(naphthalen-1-yl)-3-(thio

phen-2-yl)azetidin-2-one (17). This was obtained from 2-(thio-

phen-2-yl)acetyl chloride and 3,4,5-trimethoxy-N-(naphthalen-1-

ylmethylene)aniline (8e) as a brown oil (15.2% yield); IR (KBr disk)

¹H NMR (400 MHz, CDCl₃) d 3.72–3.74 (m, 9H, 3 Â OCH₃), 3.78 (s,

3H, OCH₃), 5.74 (s, 1H, H-4), 6.68–6.72 (t, 4H, ArH), 7.06–7.09

(m, 5H, ArH), 7.16–7.18 (m, 2H, ArH), 7.29–7.32 (t, 1H, ArH),

7.39–7.43 (m, 2H, ArH), 7.67 (d, 2H, J = 7.52 Hz, ArH); ¹³C NMR

(100 MHz, CDCl₃) d 54.74 (OCH₃), 55.53 (OCH₃), 58.06 (OCH₃),

60.50 (OCH₃), 66.94 (C-4), 71.53 (C-3), 94.70, 113.35, 126.33,

126.38, 126.78, 127.00, 127.54, 127.91, 128.35, 128.44, 133.26,

134.02, 136.77, 140.44, 152.94, 159.00 (ArC), 166.70 (C@O); HRMS

(M⁺+Na): C₃₁H₂₉NO₅Na requires 518.1943; found 518.1962.

m

_max: 1755.27 cm^À1(C@O); ¹H NMR (400 MHz, CDCl₃): d 3.71 (s,

6H, 2 Â OCH₃), 3.83 (s, 3H, OCH₃), 4.54 (d, 1H, J = 2.5 Hz), 5.77 (d,

1H, J = 2.5 Hz), 6.69 (s, 2H, ArH), 7.07 (m, 1H, ArH), 7.14 (s, 1H,

ArH), 7.38–7.40 (m, 1H, ArH), 7.49–7.59 (m, 4H, ArH), 7.88–7.98

(m, 3H, ArH); ¹³C NMR (100 MHz, CDCl₃): d 55.70 (OCH₃), 55.77

(OCH₃), 59.51 (OCH₃), 60.55 (C-3), 61.86 (C-4), 94.71, 122.39,

125.12, 125.22, 125.81, 126.29, 126.37, 127.06, 128.50, 128.73,

130.01, 131.89, 133.33, 133.49, 134.30, 135.96, 153.21 (ArC),

164.17 (C@O); HRMS (M⁺+Na): C₂₆H₂₃NO₄NaS requires 468.1245;

found 468.1269.

4.1.9.3. 4-(4-Methoxyphenyl)-3-thiophen-2-yl-1-(3,4,5-trime-

thoxyphenyl)azetidin-2-one (13). This was obtained from 2-

(thiophen-2-yl)acetyl chloride and N-(4-methoxybenzylidene)-

3,4,5-trimethoxybenzenamine (8a) as a white powder (4.6% yield);

mp: 115 °C; IR (NaCl ﬁlm)

m

_max: 1756.78 cm^À1(C@O, b-lactam); ¹H

4.1.9.8. 1-(3,4,5-Trimethoxyphenyl)-3,4-di(thiophen-2-yl)azeti-

din-2-one (18). This was obtained from 2-(thiophen-2-yl)acetyl

chloride and 3,4,5-trimethoxy-N-(thiophen-2-ylmethylene)aniline

NMR (400 MHz, CDCl₃) d 3.72 (s, 6H, 2 Â OCH₃), 3.77 (s, 3H, OCH₃),

3.82 (s, 3H, OCH₃), 4.47 (d, 1H, J = 2.5 Hz, H-3), 4.90 (d, 1H,

J = 2.5 Hz, H-4), 6.59 (s, 2H, ArH), 6.95 (d, 2H, J = 8.56 Hz, ArH),

7.01–7.03 (t, 1H, ArH), 7.08 (d, 1H, J = 3.48 Hz, ArH), 7.26 (d, 1H,

ArH, J = 5 Hz), 7.36–7.38 (d, 2H, ArH, J = 8.52 Hz); ¹³C NMR

(100 MHz, CDCl₃) d 54.93 (OCH₃), 55.58 (OCH₃), 59.78 (C-3),

60.52 (OCH₃), 64.12 (C-4), 94.46, 113.86, 114.27, 124.43, 124.87,

125.29, 126.82, 126.90, 128.29, 133.19, 134.11, 135.70, 148.98,

153.06, 159.60 (ArC), 163.98 (C@O); HRMS (M⁺+Na): C₂₃H₂₃NO_5-

NaS requires 448.1195; found 448.1186.

(8f) as a brown oil (6.7% yield); IR (KBr disk) m

_max: 1755.61 cm^À1

(C@O); ¹H NMR (400 MHz, CDCl₃): d 3.78 (s, 6H, 2 Â OCH₃), 3.81

(s, 3H, OCH₃), 4.68 (d, 1H, J = 2 Hz), 5.23 (d, 1H, J = 2 Hz), 6.67 (s,

2H, ArH), 7.06–7.09 (m, 1H, ArH), 7.08 (d, 1H, J = 3.52 Hz, ArH),

7.23–7.24 (m, 1H, ArH), 7.33 (m, 1H, ArH), 7.42 (m, 2H, ArH); ¹³C

NMR (100 MHz, CDCl₃): d 56.07 (OCH₃), 60.98 (C-3), 61.07 (C-4),

94.9, 98.33, 125.58, 125.97, 126.20, 126.37, 127.41, 127.48,

133.44, 135.60, 140.69, 153.57 (ArC), 163.96 (C@O); HRMS

(M⁺+Na): C₂₆H₂₃NO₄NaS requires 424.4889; found 424.0628.

4.1.9.4. 4-(3-((tert-Butyldimethylsilyl)oxy)-4-methoxyphenyl)-

3-(thiophen-2-yl)-1-(3,4,5-trimethoxyphenyl)azetidin-2-one

(14). This was obtained from 2-(thiophen-2-yl)acetyl chloride

4.1.10. General method II for b-lactam preparation

The appropriate imine (10 mmol) and acetyl chloride (10 mmol)

were added to CH₂Cl₂(50 mL) under nitrogen and the mixture was

left stirring for 2 h. Triethylamine (10 mmol) was added dropwise.

The mixture was left to stir overnight. The mixture was washed

ﬁrstly with distilled water (50 mL) (twice) and then with saturated

aqueous sodium bicarbonate solution (50 mL). The organic layer

was dried by ﬁltration through anhydrous sodium sulfate. The or-

ganic layer containing the product was collected and reduced in

vacuo. The pure product was isolated by ﬂash column chromatog-

raphy over silica gel (hexane/ethyl acetate gradient).

and

[3-(tert-butyldimethylsilanyloxy)-4-methoxybenzyli-

dene](3,4,5-trimethoxyphenyl)amine (8b) as a brown oil and was

desilylated to form 33 without further puriﬁcation (crude yield:

4.3%).

4.1.9.5. 4-(4-Methoxyphenyl)-3-(thiophen-2-ylmethyl)-1-(3,4,5-

trimethoxyphenyl)azetidin-2-one (15). This was obtained from

3-thiophen-2-yl-propionyl chloride (6b) and N-(4-methoxybenzyl-

idene)-3,4,5-trimethoxybenzenamine (8a) and isolated as a yellow

oil in 0.6% yield; IR (NaCl ﬁlm) m

_max: 1746.67 cm^À1(C@O, b-lac-

tam); ¹H NMR (400 MHz, CDCl₃) d 3.30–3.36 (m, 1H, CH₂), 3.40–

3.44 (m, 1H, CH₂), 3.49–3.54 (m, 1H, H-3), 3.72 (s, 6H, 2 Â OCH₃),

3.78 (s, 3H, OCH₃), 3.81 (s, 3H, OCH₃), 4.71 (d, 1H, J = 2.5 Hz, H-

4), 6.54 (s, 2H, ArH), 6.86–6.89 (m, 3H, ArH), 6.94–6.97 (m, 1H,

ArH), 7.16–7.19 (m, 3H, ArH); ¹³C NMR (100 MHz, CDCl₃) d 28.32

(CH₂), 54.84 (OCH₃), 55.56 (OCH₃), 60.25 (C-3), 60.49 (C-4),

94.33, 113.99, 123.71, 125.32, 126.63, 126.85, 128.89, 133.37,

139.45, 153.01, 159.23 (ArC), 165.95 (C@O); HRMS (M⁺+Na):

4.1.10.1. 4-(4-Methoxy-3-nitrophenyl)-3-thiophen-2-yl-1-(3,4,5

-trimethoxyphenyl)azetidin-2-one (19). This was obtained from

2-(thiophen-2-yl)acetyl chloride and (4-methoxy-3-nitrobenzyli-

dene)(3,4,5-trimethoxyphenyl)amine (8c) as

(48.4% yield); mp: 123 °C; IR (KBr disk)

(C@O, b-lactam); ¹H NMR (400 MHz, CDCl₃)

a

brown powder

_max: 1742.06 cm^À1

3.76 (s, 6H,

m

d

2 Â OCH₃), 3.79 (s, 3H, OCH₃), 4.00 (s, 3H, OCH₃), 4.49 (d, 1H,

J = 2.5 Hz, H-3), 4.98 (d, 1H, J = 2.5 Hz, H-4), 6.57 (s, 2H, ArH),

7.03–7.05 (m, 1H, ArH), 7.10 (d, 1H, J = 3 Hz, ArH), 7.18 (d, 1H,

J = 8.52 Hz, ArH), 7.33 (d, 1H, J = 5 Hz, ArH), 7.60–7.63 (dd, 1H,

ArH), 7.94 (s, 1H, ArH); ¹³C NMR (100 MHz, CDCl₃) d 55.78

(OCH₃), 56.31 (OCH₃), 59.75 (C-3), 60.52 (OCH₃), 62.92 (C-4),

94.48, 114.25, 123.13, 125.28, 125.66, 127.07, 128.82, 130.78,

132.59, 134.56, 134.77, 139.45, 152.80 (ArC), 163.33 (C@O); HRMS

(M⁺+Na): C₂₃H₂₂N₂O₇NaS requires 493.1045; found 493.1047.

C₂₄H₂₅NO₅NaS requires 462.1351; found 462.1333.

4.1.9.6. 1-(3,4,5-Trimethoxyphenyl)-4-(naphthalen-2-yl)-3-(thio

phen-2-yl)azetidin-2-one (16). This was obtained from

2-(thiophen-2-yl)acetyl chloride and 3,4,5-trimethoxy-N-(naph-

thalen-2-ylmethylene)aniline (8d) as a brown oil (9.1% yield); IR

(KBr disk) m

_max: 1754.84 cm^À1(C@O); ¹H NMR (400 MHz, CDCl₃):

d

3.70 (s, 6H, 2 Â OCH₃), 3.78 (s, 3H, OCH₃), 4.59 (d, 1H,

J = 2.0 Hz, H-3), 5.15 (d, 1H, J = 2.0 Hz, H-4), 6.66 (s, 2H, ArH),

7.07–7.08 (m, 1H, ArH), 7.13–7.14 (m, 1H, ArH), 7.29 (s, 1H, ArH),

7.34–7.35 (m, 1H, ArH), 7.54–7.57 (m, 2H, ArH), 7.88–8.03 (m,

4H, ArH); ¹³C NMR (400 MHz, CDCl₃): d 55.59 (OCH₃), 59.75

(OCH₃), 60.51 (OCH₃), 64.12 (C-3), 64.64 (C-4), 94.46, 114.27,

122.35, 125.02, 125.07, 125.47, 126.26, 126.41, 126.96, 127.46,

4.1.11. General method III for b-lactam preparation

The appropriate acetic acid (15 mmol) was reﬂuxed for 30 min

with triphosgene [bis(trichloromethyl) carbonate] (5 mmol) in

dry CH₂Cl₂(50 mL). A solution of the appropriately substituted

imine (10 mmol) in dry CH₂Cl₂(10 mL) was added dropwise to

the reﬂuxing solution. Triethylamine (30 mmol) was added. The

N. M. O’Boyle et al. / Bioorg. Med. Chem. 19 (2011) 2306–2325

2321

reaction mixture was heated at reﬂux for 5 h and stirred at room

temperature overnight. The mixture was washed ﬁrstly with dis-

tilled water (twice) (50 mL) and then with saturated aqueous so-

dium bicarbonate solution (50 mL). The organic layer was dried

over anhydrous sodium sulphate. The pure product was isolated

by ﬂash column chromatography over silica gel (hexane/ethyl ace-

tate gradient).

4), 94.93, 108.75, 110.69, 114.68, 127.37, 128.82, 133.75, 134.58,

142.86, 147.50, 153.53, 160.03 (ArC), 163.38 (C@O); HRMS

(M⁺+H): C₂₃H₂₄NO₆requires 410.1604; found 410.1605.

4.1.11.5. 4-(4-Methoxyphenyl)-3-thiophen-3-yl-1-(3,4,5-trime-

thoxyphenyl)azetidin-2-one (24). was obtained from 2-(thio-

phen-3-yl)acetic acid and N-(4-methoxybenzylidene)-3,4,5-tri

methoxybenzenamine (8a) as a off-white powder (19.2% yield);

4.1.11.1. 4-(4-Methoxyphenyl)-3-naphthalen-1-yl-1-(3,4,5-tri-

methoxyphenyl)azetidin-2-one (20). was obtained from 2-(naph

thalen-1-yl)acetic acid and N-(4-methoxybenzylidene)-3,4,5-tri-

methoxybenzenamine (8a) as a pale yellow crystalline powder

mp: 130 °C; IR (KBr disk) m

_max: 1750.82 cm^À1(C@O, b-lactam);

¹H NMR (400 MHz, CDCl₃) d 3.74 (s, 6H, 2 Â OCH₃), 3.79 (s, 3H,

OCH₃), 3.85 (s, 3H, OCH₃), 4.35 (d, 1H, J = 2.52 Hz, H-3), 4.87 (d,

1H, J = 2.52 Hz, H-4), 6.61 (s, 2H, ArH), 6.96–6.98 (m, 2H, ArH),

7.09–7.11 (m, 1H, ArH), 7.29–7.30 (m, 1H, ArH), 7.36–7.40 (m,

3H, ArH); ¹³C NMR (100 MHz, CDCl₃) d 54.93 (OCH₃), 55.56

(OCH₃), 60.09 (OCH₃), 60.52 (C-3), 62.89 (C-4), 94.34, 114.25,

122.01, 125.85, 126.42, 126.85, 133.33, 134.20, 153.05, 159.52

(ArC), 164.89 (C@O); HRMS (M⁺+Na): C₂₃H₂₃NO₅NaS requires

448.1195; found 448.1189.

(6.9% yield); Mp: 164 °C; IR (NaCl ﬁlm) m

_max: 1741.44 cm^À1(C@O,

b-lactam); ¹H NMR (400 MHz, CDCl₃) d 3.72 (s, 6H, 2 Â OCH₃), 3.77

(s, 3H, OCH₃), 3.87 (s, 3H, OCH₃), 4.78 (d, 1H, J = 2.4 Hz, H-3), 4.98

(d, 1H, J = 2.4 Hz, H-4), 6.63 (s, 2H, ArH), 7.00 (d, 2H, J = 8.52 Hz,

ArH), 7.36–7.46 (m, 6H, ArH), 7.77 (d, 1H, J = 7.04 Hz, ArH), 7.84 (d,

1H, J = 8.52 Hz, ArH), 7.89 (d, 1H, J = 8.04 Hz, ArH); ¹³C NMR

(100 MHz, CDCl₃) d 54.94 (OCH₃), 55.54 (OCH₃), 60.51 (OCH₃),

62.00 (C-3), 63.33 (C-4), 94.39, 114.30, 123.29, 123.82, 125.28,

125.58, 125.96, 127.47, 128.00, 128.46, 128.97, 131.16, 131.21,

133.29, 133.44, 134.01, 153.05, 159.65 (ArC), 165.27 (C@O); HRMS

(M⁺+Na): C₂₉H₂₇NO₅Na requires 492.1787; found 492.1774.

4.1.11.6. 4-(4-Methoxyphenyl)-3-(5-methylthiophen-2-yl)-1-(3,4,

5-trimethoxyphenyl)azetidin-2-one (25). This was obtained from

2-(5-methylthiophen-2-yl)acetic acid (5b) and N-(4-methoxybenzyli-

dene)-3,4,5-trimethoxybenzenamine (8a) as a brown oil (3.1% yield);

IR (NaCl ﬁlm) m_max

:

1736.68 cm^À1(C@O, b-lactam); ¹H NMR

4.1.11.2. 4-(4-Methoxyphenyl)-3-naphthalen-2-yl-1-(3,4,5-trime-

thoxyphenyl)azetidin-2-one (21). was obtained from 2-(naphtha-

len-2-yl)acetic acid and N-(4-methoxybenzylidene)-3,4,5-trimeth

oxybenzenamine (8a) as a white solid (2.5% yield); Mp: 150 °C; IR

(400 MHz, CDCl₃) d 2.50 (s, 3H, CH₃), 3.74 (s, 6H, 2 Â OCH₃), 3.84–

3.87 (m, 6H, 2 Â OCH₃), 4.41 (d, 1H, J = 2.5 Hz, H-3), 4.89 (d, 1H,

J = 2.5 Hz, H-4), 6.60 (s, 2H, ArH), 6.67–6.71 (m, 3H, ArH), 6.86 (d, 1H,

ArH, J = 3.52 Hz), 6.96 (d, 2H, ArH, J = 8.76 Hz); ¹³C NMR (100 MHz,

CDCl₃) d 15.36 (CH₃), 55.38 (OCH₃), 56.04 (OCH₃), 60.51 (OCH₃),

60.97 (C-3), 61.00 (OCH₃), 64.62 (C-4), 94.93, 114.70, 125.30, 125.73,

127.26, 128.89, 133.73, 140.03, 153.45, 153.52, 160.02 (ArC), 164.71

(C@O); HRMS (M⁺+H): C₂₄H₂₆NO₅S requires 440.1532; found

440.1535.

(NaCl ﬁlm) m_max

:

1739.65 cm^À1(C@O, b-lactam); ¹H NMR

(600 MHz, CDCl₃) d 3.73 (s, 6H, 2 Â OCH₃), 3.78 (s, 3H, OCH₃), 3.84

(s, 3H, OCH₃), 4.45 (d, 1H, J = 2.52 Hz, H-3), 4.94 (d, 1H, J = 2.48 Hz,

H-4), 6.64 (s, 2H, ArH), 6.95–6.97 (m, 2H, ArH), 7.36–7.42 (m, 3H,

ArH), 7.48–7.50 (m, 2H, ArH), 7.80–7.88 (m, 4H, ArH); ¹³C NMR

(100 MHz, CDCl₃) d 55.41, 56.06 (OCH₃), 61.00 (OCH₃), 63.94 (C-3),

65.27 (C-4), 94.89, 114.75, 125.04, 126.23, 126.52, 127.39, 127.74,

127.88, 128.97, 129.30, 132.14, 132.87, 133.48, 133.76, 134.51,

153.55, 160.02 (ArC), 165.65 (C@O); HRMS (M⁺+Na): C₂₉H₂₇NO₅Na

requires 492.1787; found 492.1790.

4.1.11.7. 3-Benzo[b]thiophen-2-yl-4-(4-methoxyphenyl)-1-(3,4,

5-trimethoxyphenyl)azetidin-2-one (26). was obtained from 2-

(benzo[b]thiophen-2-yl)acetic acid (5a) and N-(4-methoxybenzyli-

dene)-3,4,5-trimethoxybenzenamine (8a) as a white solid (5.6%

yield); mp: 118 °C; IR (NaCl ﬁlm) m

_max: 1747.58 cm^À1(C@O, b-lac-

4.1.11.3. 4-(4-Methoxyphenyl)-3-(1-methyl-1H-indol-2-yl)-1-(3,

4,5-trimethoxyphenyl)azetidin-2-one (22). This was obtained

from 2-(1-methyl-1H-indol-2-yl)acetic acid and N-(4-methoxy-

benzylidene)-3,4,5-trimethoxybenzenamine (8a) as a yellow solid

tam); ¹H NMR (400 MHz, CDCl₃) d 3.75 (s, 6H, 2 Â OCH₃), 3.81 (s,

3H, OCH₃), 3.86 (s, 3H, OCH₃), 4.56 (d, 1H, J = 2.0 Hz, H-3), 5.02

(d, 1H, J = 2.0 Hz, H-4), 6.63 (s, 2H, ArH), 6.99 (d, 2H, J = 8.52 Hz,

ArH), 7.35–7.41 (m, 5H, ArH), 7.76 (d, 1H, J = 7.04 Hz, ArH), 7.83

(d, 1H, J = 7.52, ArH); ¹³C NMR (100 MHz, CDCl₃) d 54.95 (OCH₃),

55.60 (OCH₃), 60.35 (OCH₃), 60.53 (C-3), 63.60 (C-4), 94.48,

114.33, 121.82, 121.99, 123.16, 124.10, 124.17, 126.84, 128.16,

133.13, 134.20, 136.49, 139.11, 153.10, 159.68 (ArC), 163.36

(C@O); HRMS (M⁺+H): C₂₇H₂₆NO₅S requires 476.1532; found

476.1537.

(11.9% yield); mp: 77–78 °C; IR (NaCl ﬁlm)

m

_max: 1747.66 cm^À1

(C@O, b-lactam); ¹H NMR (400 MHz, CDCl₃) d 3.75 (s, 6H, NCH₃,

OCH₃), 3.80 (s, 3H, OCH₃), 3.81 (s, 3H, OCH₃), 3.87 (s, 3H, OCH₃),

4.52 (d, 1H, J = 2.0 Hz, H-3), 4.94 (d, 1H, J = 2.0 Hz, H-4), 6.66 (s,

2H, ArH), 6.99 (d, 2H, J = 8.52 Hz, ArH), 7.17 (m, 2H, ArH), 7.29

(m, 1H, ArH), 7.40–7.42 (m, 4H, ArH); ¹³C NMR (100 MHz, CDCl₃)

d 32.34 (NCH₃), 54.92 (OCH₃), 55.56 (OCH₃), 57.49 (C-3), 60.52

(OCH₃), 63.22 (C-4), 94.32, 107.51, 109.67, 114.21, 118.51,

119.16, 121.72, 126.33, 126.59, 126.92, 129.36, 133.61, 133.90,

136.84, 153.07, 159.39 (ArC), 166.09 (C@O); HRMS (M⁺+H):

4.1.11.8. 4-(3-((tert-Butyldimethylsilyl)oxy)-4-methoxyphenyl)-3-(t

hiophen-3-yl)-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (27).

This was obtained from 2-(thiophen-3-yl)acetic acid and [3-(tert-

butyldimethylsilanyloxy)-4-methoxybenzylidene](3,4,5-trime-

thoxyphenyl)amine (8b) as a brown oil and was desilylated to form

29 without further puriﬁcation (crude yield: 79.5%).

C₂₈H₂₉N₂O₅requires 473.2076; found 473.2075.

4.1.11.4. 3-Furan-3-yl-4-(4-methoxyphenyl)-1-(3,4,5-trimethox

yphenyl)azetidin-2-one (23). This was obtained from 2-(furan-

3-yl)acetic acid and N-(4-methoxybenzylidene)-3,4,5-trimethoxy-

benzenamine (8a) as brown crystals (4.9% yield); mp: 127 °C; IR

4.1.12. General method IV for preparation of b-lactams 28–29

To a solution of the appropriately protected phenol (10 mmol)

in THF (50 mL) was added 1.5 equiv of 1 M tetrabutylammonium

ﬂuoride. The solution was stirred in an ice-bath for 15 min. The

reaction mixture was diluted with ethyl acetate (100 mL) and

quenched with 10% HCl (100 mL). The layers were separated and

the aqueous layer was extracted with ethyl acetate (2 Â 50 mL).

The organic layer was then washed with water (100 mL) and brine

(100 mL) and was dried with sodium sulphate. The pure product

(NaCl ﬁlm) m_max

:

1743.69 cm^À1(C@O, b-lactam); ¹H NMR

(400 MHz, CDCl₃) d 3.75 (s, 6H, 2 Â OCH₃), 3.80 (s, 3H, OCH₃),

3.85 (s, 3H, OCH₃), 4.34 (d, 1H, J = 2.5 Hz, H-3), 5.06 (d, 1H,

J = 2.5 Hz H-4), 6.35 (d, 1H, J = 3.28 Hz, ArH), 6.41 (t, 1H, ArH),

6.62 (s, 2H, ArH), 6.96 (d, 2H, J = 4.52 Hz, ArH), 7.36–7.38 (m, 2H,

ArH), 7.45 (d, 1H, J = 0.76 H, ArH); ¹³C NMR (100 MHz, CDCl₃) d

55.38 (OCH₃), 56.05 (OCH₃), 58.67 (C-3), 60.97 (OCH₃), 61.43 (C-

2322

N. M. O’Boyle et al. / Bioorg. Med. Chem. 19 (2011) 2306–2325

was isolated by ﬂash column chromatography over silica gel (hex-

ane/ethyl acetate gradient).

after which the reaction mixture was allowed to heat up to room

temperature. It was poured into a saturated sodium chloride solu-

tion (50 mL). This solution was extracted with ethyl acetate, the or-

ganic layer was separated and was dried over anhydrous sodium

sulphate. The pure product was isolated by ﬂash column chroma-

tography over silica gel (hexane/ethyl acetate gradient).

4.1.12.1. 4-(3-Hydroxy-4-methoxyphenyl)-3-thiophen-2-yl-1-(3,

4,5-trimethoxyphenyl)azetidin-2-one (28). This was obtained

from 4-(3-((tert-butyldimethylsilyl)oxy)-4-methoxyphenyl)-3-(th

iophen-2-yl)-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (14) as

brown crystals (1.3% overall yield); Mp: 113–114 °C; IR (KBr disk)

4.1.14.1. 3-(Furan-3-yl-hydroxymethyl)-4-(4-methoxyphenyl)-1

-(3,4,5-trimethoxyphenyl)azetidin-2-one (31). This was ob-

tained as a yellow oil by reaction of furan-3-carbaldehyde and 4-

(4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (9)

m

_max: 1721.07 cm^À1(C@O, b-lactam); ¹H NMR (400 MHz, CDCl₃) d

3.76 (s, 6H, 2 Â OCH₃), 3.80 (s, 3H, OCH₃), 3.94 (s, 3H, OCH₃),

4.48 (d, 1H, J = 2.5 Hz, H-3), 4.87 (d, 1H, J = 2.5 Hz, H-4), 5.75 (s,

1H, OH), 6.62 (s, 2H, ArH), 6.89–6.95 (m, 2H, ArH), 7.01–7.03 (m,

3H, ArH), 7.31–7.32 (m, 1H, ArH); ¹³C NMR (100 MHz, CDCl₃) d

55.58 (OCH₃), 55.61 (OCH₃), 59.70 (OCH₃), 60.51 (C-3), 64.07 (C-

4), 94.50, 110.60, 111.46, 117.36, 124.86, 125.28, 126.87, 129.54,

133.18, 134.15, 135.68, 145.93, 146.53, 149.32, 153.06 (ArC),

163.90 (C@O); HRMS (M⁺+Na): C₂₃H₂₃NO₆SNa requires 464.1144;

found 464.1124.

in 50.2% yield; IR (NaCl ﬁlm) m

_max: 1740.12 cm^À1(C@O, b-lactam),

3453.40 cm^À1(–OH); ¹H NMR (400 MHz, CDCl₃) d 3.44–3.49 (m,

1H, H-3), 3.77 (s, 6H, 2 Â OCH₃), 3.81 (s, 3H, OCH₃), 3.82 (s, 3H,

OCH₃), 4.84 (d, 0.6H, J = 2.52 Hz, H-3), 5.08 (d, 0.4H, J = 2 Hz, H-

4), 5.14 (t, 0.6H), 6.55 (m, 3H, ArH), 6.86–6.91 (m, 2H, ArH),

7.18–7.25 (m, 2H, ArH), 7.39–7.41 (m, 1.3H, ArH); ¹³C NMR

(100 MHz, CDCl₃) d 53.00 (OCH₃), 54.83 (C-4), 54.88 (OCH₃),

55.51 (OCH₃), 55.59 (OCH₃), 55.83 (OCH₃), 57.32 (C-3), 59.98

(OCH₃), 60.48 (OCH₃), 63.31, 64.52, 64.79, 65.14 (CH), 94.26,

94.32, 108.16, 108.81, 114.02, 114.12, 125.25, 125.83, 126.97,

127.00, 128.48, 128.81, 133.15, 133.97, 139.00, 139.81, 143.19,

143.23, 153.00, 159.17, 159.39 (ArC), 164.79 (C@O), 164.81

(C@O); HRMS (M⁺+Na): C₂₄H₂₅NO₇Na requires 462.1529; found

462.1509.

4.1.12.2. 4-(3-Hydroxy-4-methoxyphenyl)-3-thiophen-3-yl-1-(3,

4,5-trimethoxyphenyl)azetidin-2-one (29). This was obtained

from 4-(3-((tert-butyldimethylsilyl)oxy)-4-methoxyphenyl)-3-(th

iophen-3-yl)-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (27) as a

pale pink solid (18.6% yield); mp: 151–152 °C; IR (KBr disk) m_max

:

1739.65 cm^À1(C@O, b-lactam), 3187.91 cm^À1(–OH); ¹H NMR

(400 MHz, CDCl₃) d 3.76 (s, 6H, 2 Â OCH₃), 3.80 (s, 3H, OCH₃),

3.93 (s, 3H, OCH₃), 4.34 (d, 1H, J = 2.2, H-3), 4.82 (d, 1H,

J = 2.2 Hz, H-4), 5.77 (s, 1H, OH), 6.63 (s, 2H, ArH), 6.89–6.96 (m,

2H, ArH), 7.02 (m, 1H, ArH), 7.09–7.11 (m, 1H, ArH), 7.29–7.31

(m, 1H, ArH), 7.39–7.41 (m, 1H, ArH); ¹³C NMR (100 MHz, CDCl₃)

d 56.05 (OCH₃), 56.09 (OCH₃), 60.48 (C-3), 60.98 (OCH₃), 63.32

(C-4), 94.87, 111.06, 111.98, 117.81, 122.44, 126.33, 126.85,

130.43, 133.78, 134.65, 146.39, 146.93, 153.53 (ArC), 165.28

(C@O); HRMS (M⁺+Na): C₂₃H₂₃NO₆SNa requires 464.1144; found

464.1153.

4.1.14.2. 3-(Hydroxythiophen-2-yl-methyl)-4-(4-methoxyphen

yl)-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (32). This was

obtained as a light yellow powder from thiophene-2-carbaldehyde

and 4-(4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)azetidin-2-

one (9) in 12.4% yield by the above method; Mp: 96 °C; IR (KBr

disk) m

_max: 1745.90 cm^À1(C@O, b-lactam), 3436.73 cm^À1(–OH);

¹H NMR (400 MHz, CDCl₃) d 3.53–3.56 (m, 1H, H-3), 3.71 (s, 6H,

2 Â OCH₃), 3.77–3.81 (m, 6H, 2 Â OCH₃), 4.83 (d, 0.6H, J = 2.5 Hz,

H-4), 5.20 (d, 0.4 Hz, J = 2.5 Hz, CH), 5.41 (d, 0.6H, J =

6.52 Hz, CH), 5.61 (d, 0.4H, J = 3.92 Hz, CH), 6.55 (d, 2H, J = 6 Hz,

ArH), 6.83–6.89 (m, 2H, ArH), 6.92–6.96 (m, 1H, ArH), 7.00–7.02

(m, 0.6H, ArH), 7.14–7.17 (m, 2.5H, ArH), 7.26–7.29 (m, 1H, ArH);

¹³C NMR (100 MHz, CDCl₃) d 54.81 (OCH₃), 54.86 (OCH₃), 55.51

(C-4), 55.54, 55.59, 55.63, 57.54 (C-3), 60.49 (OCH₃), 65.50, 65.94,

66.04, 68.02 (CH), 94.31, 94.39, 94.63, 113.58, 113.91, 114.04,

123.55, 124.55, 124.91, 125.34, 126.42, 126.44, 126.85, 127.05,

128.26, 128.80, 133.09, 133.12, 133.92, 134.01, 143.48, 144.59,

152.96, 153.00, 153.05, 159.11, 159.33 (ArC), 164.64 (C@O); HRMS

(M⁺+Na): C₂₄H₂₅NO₆NaS requires 478.1300; found 478.1292.

4.1.13. 4-(3-Amino-4-methoxyphenyl)-3-thiophen-2-yl-1-(3,4,5

-trimethoxyphenyl)azetidin-2-one (30)

To 4-(4-methoxy-3-nitrophenyl)-3-thiophen-2-yl-1-(3,4,5-tri-

methoxyphenyl)azetidin-2-one (19) (10 mmol) in glacial AcOH

(5 mL) was added metallic zinc dust (10 equiv). The mixture was

stirred for 6 days at room temperature under nitrogen until TLC in-

dicted formation of product. The residue was ﬁltered through Cel-

ite and was extracted with dichloromethane. The amino compound

was isolated using a hexane and ethyl acetate gradient column.

and was obtained as a brown residue (48.5% yield); IR (NaCl ﬁlm)

m

_max: 1749.94 cm^À1(C@O, b-lactam); ¹H NMR (400 MHz, CDCl₃) d

4.1.14.3. 3-(Hydroxythiophen-3-yl-methyl)-4-(4-methoxyphen-

yl)-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (33). This was

obtained from thiophene-3-carbaldehyde and 4-(4-methoxy-

phenyl)-1-(3,4,5-trimethoxy-phenyl)azetidin-2-one (9) as a yellow

powder (43.6% yield) by the above method; Mp: 69 °C; IR (NaCl

3.76 (s, 6H, 2 Â OCH₃), 3.80 (s, 3H, OCH₃), 3.89 (s, 3H, OCH₃),

4.49 (d, 1H, J = 2 Hz, H-3), 4.83 (d, 1H, J = 2.52 Hz, H-4), 6.64 (s,

2H, ArH), 6.78–6.81 (m, 3H, ArH), 7.03–7.05 (m, 1H, ArH), 7.08–

7.09 (m, 1H, ArH), 7.29–7.31 (m, 1H, ArH); ¹³C NMR (100 MHz,

CDCl₃) d 55.59 (OCH₃), 56.07 (OCH₃), 60.13 (C-3), 60.98 (OCH₃),

64.84 (C-4), 94.93, 110.53, 111.56, 116.43, 125.25, 125.70,

127.31, 129.34, 133.81, 134.50, 136.36, 136.96, 147.76, 153.50

(ArC), 164.60 (C@O); HRMS (M⁺+H): C₂₃H₂₅N₂O₅S requires

441.1484; found 441.1471.

ﬁlm) m

_max: 1745.08 cm^À1(C@O, b-lactam), 3438.10 cm^À1(–OH);

¹H NMR (400 MHz, CDCl₃) d 2.85 (br s, 0.4H), 3.01 (br s, 0.4H),

3.48–3.52 (m, 1H, H-3), 3.71 (s, 6H, 2 Â OCH₃), 3.77 (m, 6H,

2 Â OCH₃), 4.82 (d, 0.6H, J = 2 Hz, H-4), 5.08 (d, 0.6H, J = 2 Hz, H-

4), 5.25 (d, 0.5H, J = 6 Hz), 5.45 (s, 0.5H), 6.55 (s, 2H, ArH), 6.81–

7.02 (m, 2H, ArH), 7.02 (m, 1H, ArH), 7.15–7.17 (m, 2H, ArH),

7.28–7.41 (m, 2H, ArH); ¹³C NMR (100 MHz, CDCl₃) d 54.80

(OCH₃), 54.87 (OCH₃), 55.56 (C-4), 55.59, 57.44 (C-3), 59.22,

60.49, 65.12, 65.64, 66.34, 66.45, 67.96 (CH), 76.81, 94.28, 94.34,

94.60, 113.59, 113.91, 114.07, 120.70, 122.41, 125.00, 125.97,

126.14, 126.19, 126.72, 126.84, 128.48, 128.89, 133.20, 133.91,

133.96, 141.61, 142.25, 152.99, 153.05, 159.01, 159.32 (ArC),

164.97 (C@O), 165.53 (C@O); HRMS (M⁺+Na): C₂₄H₂₅NO₆NaS re-

quires 478.1300; found 478.1282.

4.1.14. General procedure for synthesis of b-lactams 31–34

A

solution of 4-(4-methoxyphenyl)-1-(3,4,5-trimethoxy-

phenyl)azetidin-2-one 9 (2.5 mmol) in dry THF (20 mL) was stirred

at À78 °C under a nitrogen atmosphere. A 2 M lithium diisopropyl-

amide (5 mmol) solution was added quickly and the mixture was

stirred for 5 min at À78 °C. A solution of the appropriate aldehyde

(3.75 mmol) in dry tetrahydrofuran (5 mL) was added slowly to the

reaction mixture. The reaction was stirred at À78 °C for 30 min

N. M. O’Boyle et al. / Bioorg. Med. Chem. 19 (2011) 2306–2325

2323

4.1.14.4. 3-[Hydroxy-(5-methylthiophen-2-yl)-methyl]-4-(4-me

thoxyphenyl)-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (34).

This was obtained by reaction of 5-methylthiophene-2-

carbaldehyde and 4-(4-methoxyphenyl)-1-(3,4,5-trimethoxy-

phenyl)azetidin-2-one (9) as a white powder (16.5% yield) by the

for 5 h under a nitrogen atmosphere. After cooling, ice/water

(50 mL) was added and the mixture was extracted twice with chlo-

roform (50 mL). The combined organic extracts were washed twice

with dilute hydrochloric acid (50 mL) and once with water (50 mL),

dried with anhydrous sodium sulfate and solvent evaporated in va-

cuo. The pure product was isolated by ﬂash column chromatogra-

phy over silica gel (hexane/ethyl acetate gradient).

above method; Mp: 186 °C; IR (KBr disk)

m

_max: 1736.68 cm^À1

(C@O, b-lactam), 3500 cm^À1(–OH); ¹H NMR (400 MHz, CDCl₃) d

2.48 (s, 3H, CH₃), 2.79 (s, 1H, OH), 3.53–3.55 (dd, 1H, H-3), 3.73

(s, 6H, 2 Â OCH₃), 3.78 (s, 3H, OCH₃), 3.82 (s, 3H, OCH₃), 4.83 (d,

0.8H, J = 2.52 Hz, H-4), 5.19 (d, 0.2H, J = 2 Hz, CH), 5.31 (d, 0.8 Hz,

J = 6.56 Hz, CH), 5.50 (s, 0.2H), 6.56 (s, 2H, ArH), 6.65 (m, 1H,

ArH), 6.87–6.95 (m, 3H, ArH), 7.17–7.20 (m, 1H, ArH), 7.29 (s, 1H,

ArH); ¹³C NMR (100 MHz, CDCl₃) d 14.95 (CH₃), 54.86 (OCH₃),

55.54 (OCH₃), 57.61 (C-3), 60.50 (OCH₃), 65.34 (C-4), 68.21 (CH),

94.30, 113.94, 114.04, 124.41, 125.02, 126.90, 128.38, 133.20,

140.24, 140.82, 153.01 (ArC), 163.54 (C@O); HRMS (M⁺+Na):

C₂₅H₂₇NO₆NaS requires 492.1457; found 492.1473.

4.1.16.1. (Z)-4-(4-Methoxyphenyl)-3-thiophen-2-ylmethylene-

1-(3,4,5-trimethoxyphenyl)azetidin-2-one (37). This was pre-

pared from 3-(hydroxythiophen-2-yl-methyl)-4-(4-methoxyphenyl)

-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (32) in 30.0% yield as a yel-

low oil; IR (KBr disk) m

_max: 1721.18 cm^À1(–C@O); ¹H NMR (400 MHz,

CDCl₃) d 3.77 (s, 6H, 2 Â OCH₃), 3.80 (s, 3H, OCH₃), 3.84 (s, 3H, OCH₃),

5.37 (s, 1H, H-4), 6.45 (s, 1H, CH), 6.68 (s, 2H, ArH), 6.95 (d, 2H,

J = 9.04 Hz, ArH), 7.08–7.10 (m, 1H, ArH), 7.39–7.42 (m, 2H, ArH),

7.45 (d, 1H, J = 4.52 Hz, ArH), 7.71 (d, 1H, J = 3.52 Hz, ArH); ¹³C NMR

(100 MHz, CDCl₃) d 54.90 (OCH₃), 55.54 (OCH₃), 60.53 (OCH₃), 62.01

(C-4), 93.96, 114.09, 120.91, 127.51, 127.93, 128.23, 128.96, 131.16,

133.70, 136.99, 137.60, 153.06 (ArC), 159.67 (C@O), 159.82 (C@O);

HRMS (M⁺+Na): C₂₄H₂₃NO₅NaS requires 460.1195; found 460.1189.

4.1.15. General procedure for oxidation of alcohols 32 and 33

Pyridinium chlorochromate (10 mmol) was suspended in anhy-

drous dichloromethane (15 mL). The appropriate alcohol (32, 33)

(15 mmol, 1.5 equiv) was dissolved in anhydrous dichloromethane

(20 mL) and was added to the pyridinium chlorochromate suspen-

sion. The solution became brieﬂy homogenous before depositing

the black insoluble reduced reagent and was stirred for a further

2 h. The reaction mixture was then diluted with ﬁve volumes of

anhydrous ether. The solvent was decanted and the black residue

was further washed with ether until the entire oxidised product

was removed. The solvent was removed in vacuo and the product

was isolated by ﬂash column chromatography over silica gel using

a hexane: ethyl acetate gradient elution.

4.1.16.2. (Z)-4-(4-Methoxyphenyl)-3-(5-methylthiophen-2-yl-

methylene)-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (38)..This

was prepared from 3-[hydroxy-(5-methylthiophen-2-yl)-methyl]-

4-(4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)azetidin-2-one

(34) as a brown oil (9.0% yield); IR (KBr disk) m

_max: 1725.19 cm^À1(–

C@O); ¹H NMR (400 MHz, CDCl₃) d 2.54 (s, 3H, CH₃), 3.77–3.84 (m,

12H, 4xOCH₃), 5.35 (s, 1H, H-4), 6.38 (s, 1H, CH), 6.68–6.74 (m, 2H,

ArH), 6.94–6.96 (m, 2H, ArH), 7.35–7.41 (m, 3H, ArH); ¹³C NMR

(100 MHz, CDCl₃) d 15.69 (CH₃), 55.35 (OCH₃), 56.05 (OCH₃),

56.17 (OCH₃), 58.67 (OCH₃), 62.43 (C-4), 94.33, 94.93, 114.34,

114.52, 114.67, 118.58, 121.85 (–CH–), 126.19, 127.37, 128.39,

128.89, 132.28, 134.29, 135.59, 136.49, 145.21, 153.51 (ArC),

160.08 (C@O), 160.51 (C@O); HRMS (M⁺+H): C₂₅H₂₆NO₅S requires

452.1532; found 452.1527.

4.1.15.1. 4-(4-Methoxyphenyl)-3-(thiophene-2-carbonyl)-1-(3,4,

5-trimethoxyphenyl)azetidin-2-one (35). This was prepared

from 3-(hydroxythiophen-2-yl-methyl)-4-(4-methoxyphenyl)-1-

(3,4,5-trimethoxyphenyl)azetidin-2-one (32) as a yellow powder

(27.3% yield); mp: 123 °C; IR (KBr disk) m

_max: 1751.87 cm^À1(b-lac-

tam –C@O), 1655.95 cm^À1(C@O); ¹H NMR (400 MHz, CDCl₃) d 3.73

(s, 6H, 2 Â OCH₃), 3.78 (s, 3H, OCH₃), 3.83 (s, 3H, OCH₃), 4.71 (d, 1H,

J = 2.52 Hz, H-3), 5.65 (d, 1H, J = 2.52 Hz, H-4), 6.57 (s, 2H, ArH),

6.96 (d, 2H, J = 8.56, ArH), 7.21–7.23 (t, 1H, ArH), 7.41 (d, 2H,

J = 8.52 Hz, ArH), 7.76 (d, 1H, J = 4.52 Hz, ArH), 8.01 (d, 1H,

J = 4 Hz, ArH); ¹³C NMR (100 MHz, CDCl₃) d 54.92 (OCH₃), 55.41

(OCH₃), 55.54 (C-4), 60.51 (OCH₃), 68.20 (C-3), 94.39, 114.24,

127.20, 127.87, 128.19, 132.94, 134.21, 134.58, 135.02, 142.35,

153.03 (ArC), 159.51 (C₂= O), 159.61 (C₂= O), 182.86 (C@O); HRMS

(M⁺+Na): C₂₄H₂₃NO₆NaS requires 476.1144; found 476.1141.

4.2. Biochemistry: experimental methods

4.2.1. MTT assay procedure

All assays were performed in triplicate for the determination of

mean values reported. Compounds were assayed as the free bases

isolated from reaction. The human breast tumour cell line MCF-7

was cultured in Eagles minimum essential medium in a 95%O₂/

5% CO₂atmosphere with 10% fetal bovine serum, 2 mM

L-gluta-

mine and 100 g/mL penicillin/streptomycin. The medium was

l

supplemented with 1% non-essential amino acids. MDA-MB-231

cells were maintained in Dulbecco’s Modiﬁed Eagle’s medium

(DMEM), supplemented with 10% (v/v) Fetal bovine serum, 2 mM

4.1.15.2. 4-(4-Methoxyphenyl)-3-(thiophene-3-carbonyl)-1-(3,4,

5-trimethoxyphenyl)azetidin-2-one (36). This was prepared from

3-(hydroxythiophen-3-yl-methyl)-4-(4-methoxyphenyl)-1-(3,4,5-tri-

methoxyphenyl)azetidin-2-one (33) as a white solid (18.6% yield); mp:

L-glutamine and 100 lg/mL penicillin/streptomycin (complete

medium). Cells were trypsinised and seeded at a density of

2.5 Â 10⁴cells/mL in a 96-well plate and incubated at 37 °C,

95%O₂/5% CO₂atmosphere for 24 h. After this time they were trea-

143–144 °C; IR (KBr disk)

m

_max: 1735.14 cm^À1(b-lactam –C@O),

1673.80 cm^À1(C@O); ¹H NMR (400 MHz, CDCl₃) d 3.74 (s, 6H,

2 Â OCH₃), 3.79 (s, 3H, OCH₃), 3.84 (s, 3H, OCH₃), 4.68 (d, 1H,

J = 2.44 Hz, H-3), 5.67 (d, 1H, J = 2.44 Hz, H-4), 6.58 (s, 2H, ArH), 6.96

(d, 2H, J = 8.32, ArH), 7.37–7.43 (m, 3H, ArH), 7.70 (d, 1H, J = 1 Hz,

ArH), 8.43–8.44 (m, 1H, ArH); ¹³C NMR (100 MHz, CDCl₃) d 55.38

(OCH₃), 55.69 (OCH₃), 56.00 (C-4), 60.98 (OCH₃), 69.45 (C-3), 94.85,

114.70, 126.64, 127.12, 127.64, 128.45, 133.45, 134.69, 135.34, 140.91,

153.51 (ArC), 160.05 (–C₂@O), 160.25 (–C₂@O), 184.60 (–C@O); HRMS

(M⁺+Na): C₂₄H₂₃NO₆NaS requires 476.1144; found 476.1124.

ted with 2

pared as stock solutions in ethanol to furnish the concentration

range of study, 1 nM–100 M, and re-incubated for a further

lL volumes of test compound which had been pre-pre-

l

72 h. Control wells contained the equivalent volume of the vehicle

ethanol (1% v/v). The culture medium was then removed and the

cells washed twice with 100

prior to the addition of 50

PBS. Cells were incubated for 2 h in darkness at 37 °C. At this point

solubilisation was begun through the addition of 200 L DMSO and

lL phosphate buffered saline (PBS)

lL of a 1 mg/mL solution of MTT in

l

the cells maintained at room temperature in darkness for 20 min to

ensure thorough colour diffusion before reading the absorbance.

The absorbance value of control cells (no added compound) was

set to 100% cell viability and from this graphs of absorbance versus

4.1.16. General procedure for dehydration of alcohols 32 and 34

A solution of appropriate alcohol (32, 34) (10 mmol) and tosyl

chloride (20 mmol) in dry pyridine (50 mL) was heated at reﬂux

2324

N. M. O’Boyle et al. / Bioorg. Med. Chem. 19 (2011) 2306–2325

cell density per well were prepared to assess cell viability using

GraphPad Prism software.⁵⁹

outlined previously.¹⁴The top binding poses were reﬁned using

the LigX procedure (MOE–Chemical Computing Group)⁶⁵together

with Postdock analysis (SVL script; MOE) of the docked ligand

poses.

.

4.2.2. Cytotoxicity assay using murine mammary epithelial cells

Mammary glands from 14 to 18 day pregnant CD-1 mice were

used as source and primary mammary epithelial cell cultures were

prepared from these. Mammary epithelial cells were isolated as

described by us previously.²⁴The isolated mammary epithelial

cells were seeded at two concentrations. After 24 h, they were trea-

4.5. X-ray crystallography

The X-ray crystallography data for crystals was collected on a

Rigaku Saturn 724 CCD Diffractometer. A suitable crystal was se-

lected and mounted on a glass ﬁbre tip and placed on the goniom-

eter head in a 123K N2 gas stream. The data set was collected using

Crystalclear-SM 1.4.0 software and 1680 diffraction images, of 0.5°

per image, were recorded. Data integration, reduction and correc-

tion for absorption and polarisation effects were all performed

using Crystalclear-SM 1.4.0 software. Space group determination,

structure solution and reﬁnement were obtained using Crystal-

structure ver. 3.8 and Bruker Shelxtl Ver. 6.14 software.⁶⁶

ted with 2

pared as stock solutions in ethanol to furnish the concentration

range of study, 1 nM–100 M, and re-incubated for a further

lL volumes of test compound which had been pre-pre-

l

72 h. Control wells contained the equivalent volume of the vehicle

ethanol (1% v/v). The cytotoxicity was assessed using alamar blue

dye as reported previously.⁶⁰

4.2.3. Tubulin polymerisation

Tubulin polymerisation was carried out using a kit supplied by

Cytoskeleton. It is based on the principal that light is scattered by

microtubules to an extent that is proportional to the concentration

of the microtubule polymer. Compounds that interact with tubulin

will alter the polymerisation of tubulin, and this can be detected

using a spectrophotometer. The absorbance at 340 nm at 37 °C is

monitored. The experimental procedure of the assay was per-

formed as described in version 8.2 of the tubulin polymerisation

assay kit manual.⁶¹

4.5.1. Crystal data for 11 (cis)

C

₁₀₄H₁₀₈N₄O₂₀, M_w1733.94 (4 molecules). Monoclinic, space

ꢀ

group P1; a = 12.364(4), b = 13.084(4), c = 14.956(4) Å,

a

= 82.867

(11)^o, b = 72.242(7)^o,

c

= 84.107(12)°; U = 2280.9) Å; Z = 1; D_c=

1.262 Mg m^À3; m = 0.087 mm^À1; Range for data collection = 1.12–

25.00; Reﬂections collected 35,367, unique reﬂections 8025

[R_int= 0.0486]; data/restraints/parameters 8025/0/587; Goodness-

of-ﬁt on F₂1215; R indices (all data) = R₁= 0.0728, wR₂= 0.1442;

Final R indices [I > 2s(I)] = R₁= 0.0642, wR₂= 0.1393. CCDC deposi-

tion No. 778106.

4.3. Stability studies for compounds 13, 16, 17 and 18

Analytical high-performance liquid chromatography (HPLC)

stability studies were performed using a Symmetry^Òcolumn

4.5.2. Crystal data for 11 (trans)

C₁₀₄H₁₀₈N₄O₂₀, M_w1733.94 (4 molecules). Monoclinic, space

(C₁₈, 5

l

m, 4.6 Â 150 mm), a Waters 2487 Dual Wavelength Absor-

group P21/c; a = 11.538(3), b = 12.295(3), c = 18.953(4) Å,

a = c =

bance detector, a Waters 1525 binary HPLC pump and a Waters

717plus Autosampler. Samples were detected at wavelength of

254 nm. All samples were analysed using acetonitrile (80%)/water

(20%) as the mobile phase over 10 min and a ﬂow rate of 1 mL/min.

Stock solutions are prepared by dissolving 5 mg of compound in

10 mL of mobile phase. Phosphate buffers at the desired pH values

(4, 7.4, and 9) were prepared in accordance with the British Phar-

macopoeia monograph 2010. Thirty microlitres of stock solution

were diluted with 1 mL of appropriate buffer, shaken and injected

immediately. Samples were withdrawn and analysed at time inter-

vals of t = 0, 5, 30, 60, 90, 120 min and 21 h. Retention times were

13: 2.70 min; 16: 3.75 min; 17: 3.74 min; 18: 3.75 min.

90°, b = 123.08(12)°; U = 2252.8(9) Å; Z = 1; D_c= 1.278 Mg m^À3

;

m = 0.088 mm^À1; range for data collection = 1.12–25.00; reﬂec-

tions collected 17,982, unique reﬂections 3955 [R_int= 0.0441];

data/restraints/parameters 3955/0/295; Goodness-of-ﬁt on F₂

1244; R indices (all data) = R₁= 0.0674, wR₂= 0.1195; ﬁnal R indi-

ces [I > 2s(I)] = R₁= 0.0616, wR₂= 0.1195. CCDC deposition No.

778108.

4.5.3. Crystal data for 12

C₁₂₄H₁₁₆N₄O₂₀, M_W1982.21 (4 molecules). Monoclinic, space

group P21/c; a = 10.349(3), b = 9.828(3), c = 26.547(8) Å,

a = c =

90°, b = 110.277(10)°; U = 2532.8(13) Å; Z = 1; D_c= 1.300 Mg m^À3

;

m = 0.088 mm^À1; range for data collection = 1.12–25.00; reﬂec-

tions collected 30,080, unique reﬂections 4457 [R_int= 0.0652];

data/restraints/parameters 4457/0/338; Goodness-of-ﬁt on F₂

1222; R indices (all data) = R₁= 0.0789, wR₂= 0.1389; ﬁnal R indi-

ces [I > 2s(I)] = R₁= 0.0835, wR₂= 0.1410. CCDC deposition No.

778107.

4.4. Computational procedures

For ligand preparation, all compounds were built using ACD/

Chemsketch v10 to generate ^SMILES. A single conformer from each

string was generated using Corina v3.4 and ensuring Omega

v2.2.1 was subsequently employed to generate a maximum of 50

conformations of each compound. For the receptor preparation,

the PDB entries 1SA0 were downloaded from the Protein Data Bank

(PDB). All waters were retained in both isoforms. Addition and

optimisation of hydrogen positions for these waters was carried

out using MOE v2009.10 ensuring all other atom positions

remained ﬁxed. Using the reported X-ray structure of tubulin

Acknowledgements

We thank Dr. Niall Keely for the kind gift of combretastatin A-4.

A Trinity College Dublin postgraduate research studentship (Code

1252) and a one-year postgraduate research award for continuing

students (Code 7017) are gratefully acknowledged. Support from

the Health Research Board is also gratefully acknowledged.

co-crystallised with

a colchicine derivative, DAMA-colchicine

(PDB entry: 1SA0),⁵¹possible binding orientations of the b-lactam

ligands were probed with the docking programme FREDv2.2.3

(Openeye Scientiﬁc Software).⁶²Docking was carried out using

FREDv2.2.3 in conjunction with the PLP scoring function. 3D ligand

conformations were enumerated using ^CORINAv3.4 (Molecular

Networks GMBH)⁶³followed by generation of multiple conforma-

tions using ^OMEGAv2.2.1 (Openeye Scientiﬁc Software).⁶⁴Each

conformation was subsequently docked and scored with PLP as

Supplementary data

NCI60 cell-line data for b-lactam 13, HPLC purity data for target

azetidinone compounds, chiral separation chromatograms for

b-lactams 13 and 28, additional molecular modelling for b-lactam

28 and cytotoxicity data for 2a and 28 in murine epithelial cells at

N. M. O’Boyle et al. / Bioorg. Med. Chem. 19 (2011) 2306–2325

2325

35. Cotarca, L.; Delogu, P.; Nardelli, A.; Sunjic, V. Synthesis 1996, 553.

36. Singh, S. B.; Pettit, G. R. Synth. Commun. 1987, 17, 877.

37. Maya, A. B. S.; Perez-Melero, C.; Mateo, C.; Alonso, D.; Fernandez, J. L.; Gajate,

C.; Mollinedo, F.; Pelaez, R.; Caballero, E.; Medarde, M. J. Med. Chem. 2005, 48,

556.

50,000 cells/mL. Supplementary data associated with this article

can be found, in the online version, at doi:10.1016/j.bmc.2011.

02.022.

38. Mayrhofer, R.; Otto, H.-H. Synthesis 1980, 247.

39. Corey, E. J.; Suggs, J. W. Tetrahedron Lett. 1975, 31, 2647.

40. Mosmann, T. J. Immunol. Methods 1983, 65, 55.

41. National Cancer Institute (NCI)/Division of Cancer Treatment and Diagnosis

(DCTD)/Developmental Therapeutics Program (DTP), 2008.

42. Paull, K. D.; Shoemaker, R. H.; Hodes, L.; Monks, A.; Scudiero, D. A.; Rubinstein,

L.; Plowman, J.; Boyd, M. R. J. Natl. Cancer. Inst. 1989, 81, 1088.

43. DTP Human Tumour Cell Line Screen Standard Agent Database. http://

dtp.nci.nih.gov/docs/cancer/searches/standard_agent.html (11 May).

44. Paull, K. D.; Lin, C. M.; Malspeis, L.; Hamel, E. Cancer Res. 1992, 52, 3892.

45. Furlong, E. E.; Keon, N. K.; Thornton, F. D.; Rein, T.; Martin, F. J. Biol. Chem. 1996,

271, 29688.

References and notes

1. Downing, K. H. Annu. Rev. Cell Dev. Biol. 2000, 16, 89.

2. Kavallaris, M. Nat. Rev. Cancer 2010, 10, 194.

3. Dumontet, C.; Jordan, M. A. Nat. Rev. Drug Discovery 2010, 9, 790.

4. Cragg, G. M.; Kingston, D. G.; Newman, D. J. Anticancer Agents from Natural

Products; CRC press: Florida, 2005.

5. Pettit, G. R.; Singh, S. B.; Hamel, E.; Lin, C. M.; Alberts, D. S.; Garcia-Kendall, D.

Experientia 1989, 45, 209.

6. Watt, J. M.; Gerdina, M. The Medicinal and Poisonous Plants of Southern and

Eastern Africa; E&S Livingstone Ltd: Edinburgh and London, 1962.

7. Hamel, E.; Lin, C. M. Biochem. Pharmacol. 1983, 32, 3864.

8. Tozer, G. M.; Kanthou, C.; Parkins, C. S.; Hill, S. A. Int. J. Exp. Pathol. 2002, 83, 21.

9. Pettit, G. R.; Lippert, J. W. Anticancer Drug Des. 2000, 15, 203.

10. Strebhardt, K.; Ullrich, A. Nat. Rev. Cancer 2008, 8, 473.

11. Pettit, G. R.; Temple, C., Jr.; Narayanan, V. L.; Varma, R.; Simpson, M. J.; Boyd, M.

R.; Rener, G. A.; Bansal, N. Anticancer Drug Des. 1995, 10, 299.

12. Nam, N. H. Curr. Med. Chem. 2003, 10, 1697.

46. Murtagh, J.; McArdle, E.; Gilligan, E.; Thornton, L.; Furlong, F.; Martin, F. J. Cell

Biol. 2004, 166, 133.

47. Simoni, D.; Romagnoli, R.; Baruchello, R.; Rondanin, R.; Rizzi, M.; Pavani, M. G.;

Alloatti, D.; Giannini, G.; Marcellini, M.; Riccioni, T.; Castorina, M.; Guglielmi,

M. B.; Bucci, F.; Carminati, P.; Pisano, C. J. Med. Chem. 2006, 49, 3143.

48. Liou, J. P.; Chang, Y. L.; Kuo, F. M.; Chang, C. W.; Tseng, H. Y.; Wang, C. C.; Yang,

Y. N.; Chang, J. Y.; Lee, S. J.; Hsieh, H. P. J. Med. Chem. 2004, 47, 4247.

49. Gastpar, R.; Goldbrunner, M.; Marko, D.; von Angerer, E. J. Med. Chem. 1998, 41,

4965.

13. Nam, N.-H.; Kim, Y.; You, Y.-J.; Hong, D.-H.; Kim, H.-M.; Ahn, B.-Z. Bioorg. Med.

Chem. Lett. 2002, 12, 1955.

14. Barrett, I.; Carr, M.; O’Boyle, N.; Greene, L. M.; Knox, A.; Lloyd, D. G.; Zisterer, D.

M.; Meegan, M. J. J. Enzyme Inhib. Med. Chem. 2010, 25, 180.

15. Lee, L.; Robb, L. M.; Lee, M.; Davis, R.; Mackay, H.; Chavda, S.; Babu, B.; O’Brien,

E. L.; Risinger, A. L.; Mooberry, S. L.; Lee, M. J. Med. Chem. 2010, 53, 325.

16. Wang, L.; Woods, K. W.; Li, Q.; Barr, K. J.; McCroskey, R. W.; Hannick, S. M.;

Gherke, L.; Credo, R. B.; Hui, Y. H.; Marsh, K.; Warner, R.; Lee, J. Y.; Zielinski-

Mozng, N.; Frost, D.; Rosenberg, S. H.; Sham, H. L. J. Med. Chem. 2002, 45, 1697.

17. Nam, N.-H.; Kim, Y.; You, Y.-J.; Hong, D.-H.; Kim, H.-M.; Ahn, B.-Z. Bioorg. Med.

Chem. Lett. 2001, 11, 3073.

18. Flynn, B. L.; Flynn, G. P.; Hamel, E.; Jung, M. K. Bioorg. Med. Chem. Lett. 2001, 11,

2341.

19. Tron, G. C.; Pirali, T.; Sorba, G.; Pagliai, F.; Busacca, S.; Genazzani, A. A. J. Med.

Chem. 2006, 49, 3033.

50. Dorleans, A.; Gigant, B.; Ravelli, R. B. G.; Maillet, P.; Mikol, V.; Knossow, M. Proc.

Natl. Acad. Sci. U.S.A. 2009, 106, 13775.

51. Ravelli, R. B. G.; Gigant, B.; Curmi, P. A.; Jourdain, I.; Lachkar, S.; Sobel, A.;

Knossow, M. Nature 2004, 428, 198.

52. Andreu, J. M.; Perez-Ramirez, B.; Gorbunoff, M. J.; Ayala, D.; Timasheff, S. N.

Biochemistry 1998, 37, 8356.

53. Nguyen, T. L.; McGrath, C.; Hermone, A. R.; Burnett, J. C.; Zaharevitz, D. W.; Day,

B. W.; Wipf, P.; Hamel, E.; Gussio, R. J. Med. Chem. 2005, 48, 6107.

54. Lara-Ochoa, F.; Espinosa-Pérez, G. Tetrahedron Lett. 2007, 48, 7007.

55. Clark, A. M.; Labute, P. J. Chem. Inf. Model. 2007, 47, 1933.

56. Pettit, G. R.; Singh, S. B.; Cragg, G. M. J. Org. Chem. 1985, 66, 406.

57. Darvesh, S.; McDonald, R. S.; Darvesh, K. V.; Mataija, D.; Conrad, S.; Gomez, G.;

Walsh, R.; Martin, E. Bioorg. Med. Chem. 2007, 15, 6367.

58. Poirier, Y.; Legrand, L.; Lozac’h, N. Bull. Soc. Chim. Fr. 1966, 1054.

59. D’hooghe, M.; Dekeukeleire, S.; Mollet, K.; Lategan, C.; Smith, P. J.; Chibale, K.;

De Kimpe, N. J. Med. Chem. 2009, 52, 4058.

60. Yang, Y.; Carta, G.; Peters, M. B.; Price, T.; O’Boyle, N.; Knox, A. J. S.; Fayne, D.;

Williams, D. C.; Meegan, M. J.; Lloyd, D. G. Mol. Inf. 2010, 29, 421.

61. Cytoskeleton, Tubulin Polymerization Assay Kit Manual (Cat. # CDS03 and

BK006); 2009, pp. 1–18.

62. Schulz-Gasch, T.; Stahl, M. J. Mol. Model. 2003, 9, 47.

63. Feher, M.; Williams, C. I. J. Chem. Inf. Model. 2009.

64. Takagi, T.; Amano, M.; Tomimoto, M. J. Chem. Inf. Model. 2009, 49, 1377.

65. MOEv2009.10, developed and distributed by Chemical Computing Group.

http://www.chemcomp.com.

66. Software Reference Manual, ^SHELXTL, An Integrated System for Data Collection,

Processing, Structure Solution and Reﬁnement; Bruker Analytical X-ray Systems:

Madison, WI, 2001.

67. La Regina, G.; Sarkar, T.; Bai, R.; Edler, M. C.; Saletti, R.; Coluccia, A.; Piscitelli, F.;

Minelli, L.; Gatti, V.; Mazzoccoli, C.; Palermo, V.; Mazzoni, C.; Falcone, C.;

Scovassi, A. I.; Giansanti, V.; Campiglia, P.; Porta, A.; Maresca, B.; Hamel, E.;

Brancale, A.; Novellino, E.; Silvestri, R. J. Med. Chem. 2009, 52, 7512.

68. Messaoudi, S.; Treguier, B.; Hamze, A.; Provot, O.; Peyrat, J.-F.; De Losada, J. R.;

Liu, J.-M.; Bignon, J.; Wdzieczak-Bakala, J.; Thoret, S.; Dubois, J.; Brion, J.-D.;

Alami, M. J. Med. Chem. 2009, 52, 4538.

20. Banik, I.; Becker, F. F.; Banik, B. K. J. Med. Chem. 2003, 46, 12.

21. Ruf, S.; Neudert, G.; Gürtler, S.; Grünert, R.; Bednarski, P. J.; Otto, H.-H. Monatsh.

Chem. 2008, 139, 847.

22. Sun, L.; Vasilevich, N. I.; Fuselier, J. A.; Hocart, S. J.; Coy, D. H. Bioorg. Med. Chem.

Lett. 2004, 14, 2041.

23. Carr, M.; Greene, L. M.; Knox, A. J. S.; Lloyd, D. G.; Zisterer, D. M.; Meegan, M. J.

Eur. J. Med. Chem. 2010, 45, 5752.

24. O’Boyle, N. M.; Carr, M.; Greene, L. M.; Bergin, O.; Nathwani, S. M.; McCabe, T.;

Lloyd, D. G.; Zisterer, D. M.; Meegan, M. J. J. Med. Chem. 2010, 53, 8569.

25. Greene, L. M.; Nathwani, S. M.; Bright, S. A.; Fayne, D.; Croke, A.; Gagliardi, M.;

McElligott, A. M.; O’Connor, L.; Carr, M.; Keely, N. O.; O’Boyle, N. M.; Carroll, P.;

Sarkadi, B.; Conneally, E.; Lloyd, D. G.; Lawler, M.; Meegan, M. J.; Zisterer, D. M.

J. Pharmacol. Exp. Therap. 2010, 335, 302.

26. Banik, B. K.; Banik, I.; Becker, F. F. Bioorg. Med. Chem. 2005, 13, 3611.

27. Ocampo, R.; Dolbier, J. W. R. Tetrahedron 2004, 60, 9325.

28. Gross, H.; Costisella, B. Angew. Chem., Int. Ed. Engl. 1968, 7, 391.

29. Degenhardt, C. R. Synth. Commun. 1982, 12, 415.

30. Cushman, M.; He, H. M.; Lin, C. M.; Hamel, E. J. Med. Chem. 1993, 36, 2817.

31. Wuts, P. G. M.; Greene, T. W. Greene’s Protective Groups in Organic Synthesis, 4th

ed.; John Wiley & Sons: Hoboken, New Jersey, 2007.

32. Georg, G. I. The Organic Chemistry of b-Lactams; VCH: New York and Cambridge,

1992.

33. Bose, A. K.; Spiegelman, G.; Manhas, M. S. Tetrahedron Lett. 1971, 12, 3167.

34. Krishnaswamy, D.; Govande, V. V.; Gumaste, V. K.; Bhawal, B. M.; Deshmukh, A.

R. A. S. Tetrahedron 2002, 58, 2215.

69. Mousset, C.; Giraud, A.; Provot, O.; Hamze, A.; Bignon, J.; Liu, J.-M.; Thoret, S.;

Dubois, J.; Brion, J.-D.; Alami, M. Bioorg. Med. Chem. Lett. 2008, 18, 3266.

Article Doi

Synthesis, evaluation and structural studies of antiproliferative tubulin-targeting azetidin-2-ones

DOI: 10.1016/j.bmc.2011.02.022

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.bmc.2011.02.022

Full text of DOI:10.1016/j.bmc.2011.02.022

Products guided by the article

R&D Labs maybe for 1312122-24-9

Relevant to this article

Hot Product