Optically active 1,3,4,4-tetrasubstituted β-lactams: Synthesis and evaluation as tumor cell growth inhibitors

Article abstract of DOI:10.1016/j.ejmech.2011.08.025

The in vitro cytotoxicity assays of several enantiopure (3S,4S)- and (3R,4R)-1,3,4,4-tetrasubstituted β-lactams derived from amino acids have shown that the (3S,4S)-4-benzyl-1-p-methoxybenzyl-3-methyl-4-methoxycarbonyl derivative 2a, obtained from Phe, displays significant activity, which is comparable to that of the anticancer drug Doxorubicin against HT29 cell lines. Modifications at positions 1 and 4 of the β-lactam ring led to identify the Tyr(2,6-ClBz) analogu 26d with similar activity data to those of 2a. The synthesis and SAR of all these tetrasubstituted β-lactams are reported here.

Full text of DOI:10.1016/j.ejmech.2011.08.025

European Journal of Medicinal Chemistry 46 (2011) 5108e5119
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Optically active 1,3,4,4-tetrasubstituted
tumor cell growth inhibitors
b-lactams: Synthesis and evaluation as
Paula Pérez-Faginas a, M. Teresa Aranda a, M. Teresa García-López a, Andrés Francesch b, Carmen Cuevas b,
,
Rosario González-Muñiz a
*
a Instituto de Química Médica (IQM-CSIC), Juan de la Cierva 3, 28006 Madrid, Spain
b PharmaMar S.A., Avda. de los Reyes 1, 28770 Colmenar Viejo, Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
The in vitro cytotoxicity assays of several enantiopure (3S,4S)- and (3R,4R)-1,3,4,4-tetrasubstituted
Received 9 June 2011
Accepted 16 August 2011
Available online 23 August 2011
b
-lactams derived from amino acids have shown that the (3S,4S)-4-benzyl-1-p-methoxybenzyl-3-
methyl-4-methoxycarbonyl derivative 2a, obtained from Phe, displays significant activity, which is
comparable to that of the anticancer drug Doxorubicin against HT29 cell lines. Modifications at positions
1 and 4 of the
b
-lactam ring led to identify the Tyr(2,6-ClBz) analogu 26d with similar activity data to
Keywords:
those of 2a. The synthesis and SAR of all these tetrasubstituted
b
-lactams are reported here.
Beta-lactams
Amino acids
Cytotoxicity
Ó 2011 Elsevier Masson SAS. All rights reserved.
Solid-phase synthesis
SAR studies
1. Introduction
derivatives that act as histone deacetylase (HDAC) inhibitors [22].
In our case, the screening of an in house library of structurally
diverse compounds, resulted in the identification of the racemic
2-azetidinone 1 [23] as a moderate inhibitor of cell growth in
different tumor human cell lines (Fig. 1). This fact and the
mentioned precedents led us to devise an enantioselective
In recent years, b-lactam derivatives have found various medical
applications beyond their use as antibiotics, due to their low
toxicity [1,2]. They have been successfully exploited as anti-
inflammatory and cardiovascular agents, and in the treatment of
cancer [2e4]. Among the several 2-azetidinones showing potent
cytotoxic activity in a variety of tumor cell lines [5e14], some of
them have been described to activate apoptosis in tumor cells
[10e14]. Because of the remarkable G2 blockade against sensitive
approach for the synthesis of 1,3,4-trisubstituted-4-carboxy-b-
lactams 2 and 3 from amino acids [24]. Now, to get some insight
into the structural requirements for antitumor activity, the enan-
tiopure (3S,4S)- and (3R,4R)-azetidinones 2aed and 3aed have
been firstly evaluated and, then, different modifications of the best
compound have been achieved in an attempt to optimization. The
synthesis and in vitro cytotoxicity of these new modified analogs
are also reported here.
tumor cell lines of certain racemic b-lactams derived from poly-
aromatic amines [15e17], Banik and coworkers have recently
developed a method for the asymmetric synthesis of optically
active 1-chrysenyl-2-azetidinone derivatives [18,19]. The signifi-
cant differences in the in vitro cytotoxicity between the two
enantiomers reported against several cell lines suggested that the
target of action of these
[17e19]. Additional enantiopure
the anticancer therapy include a series of cyclic RGD-
2. Results and discussion
b
-lactams should be highly selective
-lactams of potential interest in
-lactam
b
Enantiopure
b-lactams 2aed and their enantiomers 3aed were
b
tested against three human cancer cell lines using racemic
b
-lactam
peptide derivatives, reported by Aizpurua and coworkers, which
bind to different integrins and induce differential gene expression
in human endothelial cells [20,21], and some bicyclic penicillin
1 and Doxorubicin for comparison purposes (Table 1). As shown in
Table 1, the antitumor activity of the enantiomerically pure Phe
derivative 2a is increased compared to the initial racemic prototype
1 in the three tumor cell lines, and is equipotent to Doxorubicin in
HT29 colon cells. The (3S,4S) configuration seems to be crucial for
the cytotoxic effect of 2a, since its (3R,4R) enantiomer 3a was
devoid of activity. The presence of the Phe side chain in 2a also
* Corresponding author. Tel.: þ34 915622900; fax: þ34 915485300.
E-mail address: iqmg313@iqm.csic.es (R. González-Muñiz).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.08.025

P. Pérez-Faginas et al. / European Journal of Medicinal Chemistry 46 (2011) 5108e5119
5109
resin and subsequent acidic detachment with TFA. Unexpectedly,
the reductive amination to the corresponding intermediate 4 did
not work in the case of aldehydes f and j. Subsequently, compound
7f was prepared by saponification of its methyl ester 13f, which was
prepared in solution following our reported method (Scheme 2)
[23]. However, this conventional reductive amination in solution
also failed with aldehyde j. To solve this problem, the incorporation
of the 2ephenylethyl group was achieved through reaction
between NenosylePheeOMe and 2-phenylethanol under Mitsu-
nobu conditions, followed by removal of the nosyl group [26].
Ulterior acylation of intermediate 11j with 2S-chloropropionic acid,
cyclization to the corresponding 2-azetidinone and hydrolysis of
the methyl ester afforded the desired derivative 7j (Scheme 2).
Inhibition of cell proliferation by the
b-lactam derivatives 7aem
is recorded in Table 2, along with 2a, used for comparative
purposes. Concerning the p-substituent of the 1-benzyl group,
none of the replacements of the O-methoxy group led to more
potent compounds (7ae7i and 13f) than the prototype 2a. Never-
theless, the antiproliferative activities of the unsubstituted and p-
fluor benzyl derivatives 7a and 7b were also in the micromolar
range, while the data of the O-propyloxy analogu 13f were only
slightly decreased compared to this prototype. The other 1-benzyl
Fig. 1. Racemic hit b-lactam 1 and first optically pure analogs 2aed and 3aed assayed.
Table 1
In vitro cytotoxicity (GI50, mM) of compounds 2 and 3 on human cell cancer lines.
Compd.
R
MDA-MB-231
A549
5.38
HT29
1.58
b-lactams 7ce7i did not show cytotoxicity except for 7f that dis-
2a
2b
2c
2d
3a
3b
3c
3d
CH2Ph
CH3
7.07
>36.1
>23.1
>23.0
>28.3
>36.1
18.2
played some inhibition in the HT29 colon cell line, but lower than
its 4-methyl ester 13f. This fact suggests that the presence of a free
4-carboxylic acid group is not suitable for the cell growth inhibition
>36.1
>23.1
>23.0
>28.3
>36.1
>23
>36.1
>23.1
>23.0
>28.3
>36.1
21.0
(CH2)2COt2Bu
(CH2)4NHBoc
CH2Ph
in this series of b-lactams. The total loss of cytotoxicity resulting
CH3
either from lengthening the alkyl chain of the 1-benzyl group in 7a
(homologs 7j and 7k) or from replacing this group with alkyl
moieties (compounds 7l and 7m) seems to indicate that the pres-
ence of this 1-aralkyl group, either with no substituents or with
certain small substituents at p-position, is required for activity.
Series 2.To study the influence of the Phe side chain at position 4
of the azetidinone ring on the cytotoxic activity of 2a, analogs
incorporating Tyr residues at this position were prepared in solu-
tion, as indicated in Scheme 3. As shown there, treatment of
Tyr(Bzl)eOMe and HeTyr(2,6-ClBzl)eOMe with p-methox-
ybenzaldehyde under reductive conditions, followed by acylation
with 2-(S)-chloropropionyl chloride, and subsequent intra-
(CH2)2COt2Bu
(CH2)4NHBoc
CH2Ph
>23.0
15.9
>23.0
24.8
>23.0
6.2
1
Doxorubicin
_
0.49
0.66
1.13
seems to be essential for cell growth inhibition, because analogous
-lactams derived from Ala (2b), Glu (2c) and Lys (2d) were
inactive.
In view of these results, we decided to carry out various struc-
b
tural modifications on 2a to improve its cytotoxic activity. As shown
in Fig. 2, these modifications include (a) replacement of the
p-methoxybenzyl group at position 1 with different substituents
(Series 1); (b) replacement of the Phe residue at position 4 with
other aromatic side-chains (Tyr and O-benzyl Tyr derivatives, Series
2), and (c) formation of 4-carboxamides from selected compounds
resulting from modifications a and b (Series 3).
molecular cyclization afforded the expected b-lactams 18 and 19.
Compound 18 was converted to the Tyr containing derivative 20 by
hydrogenolysis to remove the O-benzyl protecting group, while the
analogu 19 was saponified to the corresponding carboxylic acid 21.
This series of tetrasubstituted b-lactams was tested against the
same three cancer cell lines as Series 1 and, in any case, none of
them was more potent than the prototype 2a (Table 3). However, it
is interesting to note the total lack of activity, in the three cell lines,
of compound 20 resulting from replacing the Phe residue of 2a with
the Tyr moiety. However, derivatives 18 and 19 having Tyr(Bzl) and
Tyr(2,6-ClBzl) residues displayed cell growth inhibition in two
Series 1. Compounds with different substitutions at the 1-benzyl
group (aei), and analogs with aralkyl (j, k), and alkyl groups (l, m)
at that position were prepared by solid-phase methodologies
(Scheme 1). Thus, following the synthetic protocol previously
described [25], 1-substituted-4-carboxy-2-azetidinone derivatives
7aem were synthesized in three steps from Phe-linked to a Wang
Fig. 2. Modifications of prototype 2a.

5110
P. Pérez-Faginas et al. / European Journal of Medicinal Chemistry 46 (2011) 5108e5119
Scheme 1. Solid-phase synthesis of compounds 7aem (Series 1).
(A549 and HT29) and one (HT29) of the cell lines, respectively.
These results seem to indicate that the presence of a polar OH
substituent on the 4-benzyl group is completely detrimental for
activity, but the incorporation of O-aralkyl groups at that position
could contribute to design new compounds with higher selectivity
for specific tumor cell lines. Again, a loss of cytotoxicity is observed
when the 4-methyl carboxylate derivative is compared to the cor-
responding free carboxylic acid. This is the case of the b-lactam 19
Scheme 2. Synthesis of compounds 7f and 7j in solution.

P. Pérez-Faginas et al. / European Journal of Medicinal Chemistry 46 (2011) 5108e5119
5111
Table 2
4-carboxylic acids, a third series of analogs of the Phe derivative 2a
was designed to explore the influence on the antitumor activity of
different substituents at the 4-carboxyl group. Thus, the methyl
ester of the prototype 2a was transformed to amides of different
chemical nature (carbocyclic, benzylic or heterocyclic) and able to
endow the final compounds with different solubility. When this
amidation of compound 2a led to active compounds, a similar
transformation of the Tyr derivative 19 was achieved.
The preparation of the designed compounds, 22aed and 23a,d,
was carried out by saponification of methyl esters 2a and 19, fol-
lowed by coupling of the resulting carboxylic acids with the cor-
responding amine, using BOP as coupling agent (Scheme 4).
In vitro cytotoxicity (GI50, mM) of compounds 7aem on human cell cancer lines.
Compd.
CH2R1
4-
MDA-
A549 HT29
Carboxylate MB-231
7a
7b
CO2H
CO2H
14.9
18.3
25.5
22.9
9.70
6.42
F
7c
CO2H
CO2H
>30.9
>26.5
>30.9 >30.9
>26.5 >26.5
As shown in Table 4, b-lactams derived from Phe 22a and 22d,
Me
CF3
incorporating cyclohexyl and 2- (piperidin-1-yl)ethyl groups at the
4-amide moiety, respectively, showed cytotoxic activities in the
three cell lines tested, only slightly lower than that of the prototype
methyl ester 2a. On the contrary, the benzyl and morpholine
analogs 22b and 22c were inactive. It is worth mentioning that the
incorporation of amines a and d at the 4-carboxylate moiety of the
7d
7e
CO2H
>28.2
>28.2 >28.2
b
-lactams derived from Tyr 19 resulted in a substantial increase of
antitumor activity in both cases (compare GIs50 of 23a and 23d to
those of 19). Within this series of 4-carboxamide substituted
NO2
b
-
lactams, the best compound is the Tyr derivative 23d, with cell
growth inhibition data similar to those of the prototype methyl
ester 2a in the three tumor lines. These results open the possibility
of preparing new substituted amides to improve the inhibition of
tumor cell proliferation.
7f
CO2H
CO2H
>27.2
>22.8
>27.2
22.0
O
O
O
7g
>22.8 >22.8
>25.9 >25.9
N
3. Conclusions
7h
7i
CO2H
CO2H
>25.9
>27.8
The in vitro cytotoxicity evaluation of a series of (3S,4S)- and
(3R,4R)-1,3,4,4,-tetrasubstituted
b-lactams derived from amino
acids has revealed that the (3S,4S)-enantiomer derived from Phe,
2a, shows antiproliferative activity on MDA-MBA-231, A549 and
HT29 cell cancer lines. The cell growth inhibition values show that
this Phe derivative is approximately one order of magnitude less
potent than Doxorubicin against MDA-MBA-231, A549 cell lines
and is virtually equipotent to this anticancer drug against HT29
>27.8 >27.8
7j
CO2H
CO2H
>30.9
>29.6
>30.9 >30.9
>29.6 >29.6
colon cells. Several structural modifications of this b-lactam at 1
and 4 positions led to the Tyr derivative analogu 23d with similar
in vitro cytotoxicity data to those of 2a in the three cell lines.
These results together with the structure-activity studies, here
7k
reported, can be of interest in the search of innovative and safe
lactams with anticancer properties.
b-
7l
CO2H
CO2H
>36.3
>36.3
>36.3 >36.3
>36.3 >36.3
4. Experimental section
7m
4.1. General methods
All reagents were of commercial quality. Solvents were dried
and purified by standard methods. 1H NMR spectra were recorded
at 300 MHz in CDCl3, using TMS as internal standard. 13C NMR
spectra were registered at 75 MHz. Electrospray mass spectra
(positive mode) were also recorded. Analytical TLC was performed
on aluminum sheets with a 0.2 mm layer of silica gel F254. Silica gel
60 (230e400 mesh) was used for column chromatography. Silica
gel or reverse phase C18 SPE cartridges (Supelco) were also used for
compound purification. Analytical HPLC was performed on
13f
13j
2a
CO2Me
CO2Me
CO2Me
9.18
18.4
12.1
O
>29.6
>29.6 >29.6
7.07
5.38
1.58
a
Novapak C18 (3.9
ꢀ
150 mm, 0.004 mm), Deltapak C18
M)
(3.9 ꢀ 150 mm, 0.004 mm) or on a Sunfire C18 (3.9 ꢀ 150 mm, 4
m
column, with a flow rate of 1 mL/min, using a tuneable UV detector
set at 214 nm. Mixtures of MeCN (solvent A) and 0.05% TFA in H2O
(solvent B) were used in the mobile phase. The solvent mixtures are
specified in each case. Compounds 7ke7m were prepared as
described [24].
when compared to its ineffective analog 21 against HT29 colon
cells.
Series 3. In view of the differences in cytotoxicity between the
4-carboxymethyl b-lactam derivatives and the corresponding free

5112
P. Pérez-Faginas et al. / European Journal of Medicinal Chemistry 46 (2011) 5108e5119
Scheme 3. Synthesis of compounds of series 2.
4.2. Solid-phase synthesis of quaternary
acids
b-lactams from amino
4.2.1. Reductive amination (resins 4aem)
FmocePheeWang resin (0.2 mmol) was washed with DCM/
DMF/DCM/DMF (5 ꢀ 0.5 min) and piperidine/DMF (1:4 v/v,
1 ꢀ1 min, 3 ꢀ 10 min). After removal of the Fmoc protecting group,
the resins were washed with DMF/DCM/DMF/DCM (5 ꢀ 0.5 min)
and TMOF (2 ꢀ 0.5 min). Then, TMOF and the corresponding
aldehyde (1.2 mmol) were successively added. In the case of
Table 3
In vitro cytotoxicity (GI50, mM) of compounds 18e21 on human cell cancer lines.
Compd.
R1
MDA-MB-231
A549
10.4
HT29
14.6
18
>21.8
Cl
Cl
19
>18.5
>18.9
15.7
Cl
20
21
H
>16.8
>19.4
>16.8
>19.4
>16.8
>19.4
Cl
2a
_
7.07
5.38
1.58
Scheme 4. Synthesis of compounds of series 3 in solution.

P. Pérez-Faginas et al. / European Journal of Medicinal Chemistry 46 (2011) 5108e5119
5113
Table 4
In vitro cytotoxicity (GI50
,
m
M) of compounds 22e23 on human cell cancer lines.
R1 R2
Compd.
MDA-
MB-231
A549
14.7
HT29
22a
13.3
8.56
HN
HN
22b
225c
22d
>23.4
>24.5
12.9
>23.4
>24.5
19.6
>23.4
>24.5
N
O
9.56
N
HN
HN
Cl
Cl
23a
23d
>16.8
13.4
11.4
O
Cl
6.24
4.21
3.12
O
N
HN
Cl
2a
19
OMe
7.07
5.38
1.58
Cl
OMe
>18.5
>18.9
15.7
O
Cl
aliphatic aldehydes an additional 1% of MeOH was also added to the
reaction mixture. Formation of the imine overnight at room
temperature was followed by filtration, and washing with TMOF
(1 ꢀ 0.5 min). To the resulting resin-bound imines were added
TMOF, NaBH3CN (0.6 mmol), and AcOH (1%), and the reaction
continued for 24 h. The obtained resins were filtered, washed
swollen in DMF and the reaction was repeated again. Finally, resin-
bound -lactams 6aem were filtered, washed with DMF/DCM/
DMF/DCM (5 ꢀ 0.5 min), and dried under vacuum.
b
4.2.4. Cleavage from the resin to 1,3,4,4-tetrasubstituted b-lactams
7aem
consecutively with DMF (5
5 ꢀ 0.5 min) and DCM (5 ꢀ 0.5 min), and dried under vacuum.
ꢀ
0.5 min), DMF/MeOH (1:1,
Resins 6aem (0.2 mmol) were treated with TFA/H2O (95:5,
1.5 ml) at room temperature for 4 h. After filtration and washing
with DCM, the combined filtrates were concentrated to give
a residue which was dissolved in mixtures of MeCN/H2O and
lyophilized. The resulting residues were purified on silica gel
cartridges or reverse-phase cartridges (as indicated in each case),
evaporated and lyophilized from H2O or MeCN/H2O mixtures.
4.2.2. Incorporation of the chloropropionyl group (resins 5aem)
Resins 4aem (0.2 mmol) were swollen in DCM (5 ꢀ 0.5 min) and
THF (2 ꢀ 0.5 min). Then, THF was added and the resin was treated
with (S)-2-chloropropionic acid (1 mmol), Cl3CCN (2.25 mmol) and
propylene oxide (3 mmol), and cooled to 0 ꢁC. Then, Ph3P
(2.4 mmol) was added and the reaction was continued at room
temperature for 5 h. The resin was filtered and washed consecu-
tively with THF/DMF:MeOH (1:1)/DMF/DCM/MeOH (1:1)/DCM/
THF (5 ꢀ 0.5 min). The reaction was repeated once and left over-
night. Finally, resins 5aem were filtered, washed with THF/
DMF:MeOH(1:1)/DMF/DCM:MeOH(1:1)/DCM (5 ꢀ 0.5 min), and
dried under vacuum.
4.2.4.1. (3S,4S)-4-Carboxy-1,4-dibenzyl-3-methyl-2-azetidinone (7a).
Syrup. Yield: 23%. Silica-gel cartridge. Eluent: DCM:MeOH (Gradient
from 100:1 to 90:1). [
a
]
¼ þ10.1 (c 1.05, CHCl3). HPLC (Sunfire):
D
tR ¼ 8.07 min (Gradient from 0 to 100% A in 15 min). 1H NMR
(300 MHz, CDCl3):
d
7.30e7.12 (m, 10H, Ph), 4.61 (d, 1H, J ¼ 15.6, 1-
CH2), 4.09 (d, 1H, J ¼ 15.6, 1-CH2), 3.36 (d, 1H, J ¼ 14.5, 4-CH2),
3.25 (q, 1H, J ¼ 7.5, 3-H), 3.11 (d, 1H, J ¼ 14.5, 4-CH2), 1.26 (d, 3H,
J ¼ 7.5, 3-CH3). 13C NMR (75 MHz, CDCl3):
d 175.4, 170.4 (CO), 136.8,
4.2.3. Base-promoted cyclization to resin-bound
6aem)
b
-lactams (resins
136.4, 130.2, 128.7, 127.4, 126.8 (CAr), 69.4 (4-C), 53.5 (3-C), 46.2 (1-
CH2), 35.6 (4-CH2), 10.1 (3-CH3). MS (ES positive mode): 310.3
[Mþ1]þ. Anal (%) Calc. for C19H19NO3: C 73.77, H 6.19, N 4.53. Found
(%): C 73.82, H 6.23, N 4.19.
The corresponding resins 5aem (0.2 mmol) were swollen with
DCM/DMF/DCM/DMF (5 ꢀ 0.5 min), then suspended in DMF and
treated at room temperature with BTPP (1 mmol), under an argon
atmosphere. Reactions were continued overnight, filtered and
washed with DMF/DCM/DMF/DCM (5 ꢀ 0.5 min). The resins were
4.2.4.2. (3S,4S)-4-Benzyl-4-carboxy-1-p-fluorobenzyl-3-methyl-2-az-
etidinone (7b). Solid. Yield: 59%. Silica-gel cartridge. Eluent:

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P. Pérez-Faginas et al. / European Journal of Medicinal Chemistry 46 (2011) 5108e5119
DCM:MeOH (90:1).M.p.: 108e110 ꢁC [
HPLC (Sunfire): tR ¼ 8.42 min (Gradient from 0 to 100% A in 15 min).
1H NMR (300 MHz, CDCl3):
9.50 (broad s, 1H, CO2H), 7.26e6.94 (m,
a]
D
¼ þ3.1 (c 1.12, CHCl3).
C), 63.7 (10-C), 56.5 (20-C), 52.8 (200-C), 52.5 (3-C), 45.4 (1-CH2), 40.8 (4-
CH2), 10.5 (3-CH3). MS (ES positive mode): 439.5 [Mþ1]þ.
d
9H, Ar), 4.97 (d,1H, J ¼ 15.6, 1-CH2), 4.12 (d, 1H, J ¼ 15.6, 1-CH2), 3.44
4.2.4.7. (3S,4S)-4-Benzyl-4-carboxy-3-methyl-1-p-phenylmethyl-2-
(d, 1H, J ¼ 14.4, 4-CH2), 3.83 (q, 1H, J ¼ 7.5, 3-H), 3.09 (d, 1H, J ¼ 14.4,
azetidinone (7h). Solid. Yield: 35%. Silica-gel cartridge. Eluent:
4-CH2), 1.26 (d, 3H, J ¼ 7.5, 3-CH3). 13C NMR (75 MHz, CDCl3):
d
175.1,
DCM:MeOH (90:1). M.p.: 158e160 ꢁC [
a]
¼ þ7.7 (c 1.06, CHCl3).
D
170.5 (CO), 162.1 (d, 1C, JC-F ¼ 245.9, C4 Ar), 134.6 (C Ar), 132.3 (d, 1C,
JC-F ¼ 3.3, C1 Ar),130.0 (d, 2C, JC-F ¼ 7.7, C2,C6 Ar),129.8,128.5,127.3 (C
Ar), 115.2 (d, 2C, JC-F ¼ 21.9, C3,C5 Ar), 69.3 (4-C), 53.5 (3-C), 45.7 (1-
CH2), 40.5 (4-CH2), 10.1 (3-CH3). MS (ES positive mode): 328.3
[Mþ1]þ. Anal (%) Calc. for C19H18FNO3: C 69.71, H 5.54, N 4.28. Found
(%): C 69.78, H 5.59, N 4.01.
HPLC (Sunfire): tR ¼ 10.46 min (Gradient from 0 to 100% A in
15 min). 1H NMR (300 MHz, CDCl3):
d
7.60e7.12 (m, 8H, Ph), 6.90
(m, 6H, Ar), 4.63 (d, 1H, J ¼ 15.6, 1-CH2), 4.12 (d, 1H, J ¼ 15.6, 1-CH2),
3.41 (d, 1H, J ¼ 14.5, 4-CH2), 3.28 (q, 1H, J ¼ 7.5, 3-H), 3.13 (d, 1H,
J ¼ 14.5, 4-CH2), 1.28 (d, 3H, J ¼ 7.5, 3-CH3). 13C NMR (75 MHz,
CDCl3):
d 175.5, 170.6 (CO), 140.6, 140.3, 135.5, 134.6, 129.9, 129.4,
128.8,127.3,127.0 (C Ar), 69.3 (4-C), 53.3 (3-C), 46.1 (1-CH2), 40.4 (4-
CH2), 10.2 (3-CH3). MS (ES positive mode): 386.2 [Mþ1]þ. Anal (%)
Calc. for C25H23NO3: C 77.90, H 6.01, N 3.63. Found (%): C 77.95, H
6.08, N 3.37.
4.2.4.3. (3S,4S)-4-Benzyl-4-carboxy-3-methyl-1-p-methylbenzyl-2-
azetidinone (7c). Solid. Yield: 62%. Silica-gel cartridge. Eluent:
DCM:MeOH (Gradient from 100:1 to 80:1). M.p.: 103e105 ꢁC
[
a]
¼ þ13.9 (c 1.05, CHCl3).HPLC (Sunfire): tR ¼ 9.79 min (Gradient
D
from 0 to 100% A in 15 min). 1H NMR (300 MHz, CDCl3):
d
7.79
4.2.4.8. (3S,4S)-4-Benzyl-4-carboxy-3-methyl-1-(2-naphtylmethyl)-
2-azetidinone (7i). Syrup. Yield: 32%. Silica-gel cartridge. Eluent:
DCM:MeOH (90:1). HPLC (Sunfire): tR ¼ 10.4 min (Gradient from
(broad s, 1H, CO2H), 7.18e6.91 (m, 9H, Ph), 4.57 (d, 1H, J ¼ 15.5, 1-
CH2), 4.08 (d, 1H, J ¼ 15.5, 1-CH2), 3.31 (d, 1H, J ¼ 14.4, 4-CH2),
3.22 (q, 1H, J ¼ 7.6, 3-H), 3.11 (d, 1H, J ¼ 14.4, 4-CH2), 2.33 (s, 3H,
0 to 100% A in 15 min). 1H NMR (300 MHz, CDCl3):
d 7.83e6.87 (m,
CH3), 1.24 (d, 3H, J ¼ 7.6, 3-CH3). 13C NMR (75 MHz, CDCl3):
d
175.0,
12H, Ph), 4.75 (d, 1H, J ¼ 15.7, 1-CH2), 4.23 (d, 1H, J ¼ 15.7, 1-CH2),
3.38 (d, 1H, J ¼ 14.5, 4-CH2), 3.26 (q, 1H, J ¼ 7.5, 3-H), 3.08 (d, 1H,
J ¼ 14.5, 4-CH2), 1.91 (d, 3H, J ¼ 7.5, 3-CH3). 13C NMR (75 MHz,
170.7 (CO), 137.0, 134.7, 133.3, 129.9, 128.4, 127.2 (C Ar), 69.2 (4-C),
52.9 (3-C), 46.1 (1-CH2), 40.2 (4-CH2), 21.1 (CH3), 10.1 (3-CH3). MS
(ES positive mode): 324.2 [Mþ1]þ. Anal (%) Calc. for C20H21NO3: C
74.28, H 6.55, N 4.33. Found (%): C 74.32, H 6.63, N 4.27.
CDCl3):
d 176.1, 168.2 (CO), 138.1, 137.3, 134.6, 133.9, 131.7, 129.4,
128.7, 128.5, 128.3, 127.6, 127.1, 128.8, 126.2, 125.8 (C Ar), 69.3 (4-C),
52.3 (3-C), 47.6 (1-CH2), 41.3 (4-CH2), 11.2 (3-CH3). MS (ES positive
mode): 360.3 [Mþ1]þ.
4.2.4.4. (3S,4S)-4-Benzyl-4-carboxy-3-methyl-1-p-trifluoromethyl-
benzyl-2-azetidinone (7d). Syrup. Yield: 28%. Silica-gel cartridge.
Eluent: DCM:MeOH (100:1). HPLC (Sunfire): tR
¼
10.6 min
4.3. Solution synthesis of N-benzyl amino acid derivatives
(Gradient from 0 to 100% A in 15 min). 1H NMR (300 MHz, CDCl3):
d
7.52 (d, 2H, J ¼ 8.1, 2-H and 6-H Ar), 7.30e7.12 (m, 5H, Ph), 6.95 (d,
A solution of the corresponding amino acid derivative
2H, J ¼ 8.1, 3-H and 5-H Ar), 4.51 (d, 1H, J ¼ 16.0, 1-CH2), 4.08 (d, 1H,
J ¼ 16.0, 1-CH2), 3.53 (d, 1H, J ¼ 14.4, 4-CH2), 3.30 (q, 1H, J ¼ 7.5, 3-
H), 3.07 (d, 1H, J ¼ 14.4, 4-CH2), 1.28 (d, 3H, J ¼ 7.5, 3-CH3). 13C NMR
(19.40 mmol) in MeOH (30 ml) was treated with TEA (2.7 ml,
19.40 mmol) and the corresponding aldehyde (29.09 mmol). The
reaction was stirred at room temperature for 1.5 h. The reaction
was cooled to 0 ꢁC and NaBH4 (1.47 g, 38.79 mmol) was added,
then left stirring at room temperature for 30 min. The solvent
was evaporated to dryness and the remaining residue was par-
titioned between H2O and EtOAc. The organic layer was sepa-
rated and washed with brine, dried over Na2SO4 and, after
evaporation, the residue was purified on a silica gel column as
specified in each case.
(75 MHz, CDCl3): d 175.2, 170.5 (CO), 140.7, 134.5, 129.7 (C Ar), 129.2
(d, 1C, JC-F ¼ 34.3, C4 Ar), 128.6, 128.4, 127.7 (C Ar), 127.8 (c, 1C, JC-
¼ 262.8,), 69.5 (4-C), 54.0 (3-C), 46.0 (1-CH2), 40.9 (4-CH2), 10.2 (3-
F
CH3). MS (ES positive mode): 378.3 [Mþ1]þ.
4.2.4.5. (3S,4S)-4-Benzyl-4-carboxy-3-methyl-1-p-nitrobenzyl-2-az-
etidinone (7e). Syrup. Yield: 32%. Silica-gel cartridge. Eluent:
DCM:MeOH (Gradient from 100:1 to 80:1). HPLC (Sunfire):
tR ¼ 0.89 min (Gradient from 0 to 100% A in 5 min). 1H NMR
4.3.1. N-(p-propoxy)benzyl-
Syrup. Yield: 78%. Eluent: EtOAc:hexane (1:5). [
1.02, CHCl3). HPLC (Sunfire): tR ¼ 4.07 min (Gradient from 20 to
100% A in 15 min). 1H NMR (300 MHz, CDCl3):
7.20e7.07 (m, 5H,
L
-Phe-OMe (8f)
(300 MHz, Acetone-d6):
d
8.15 (d, 2H, J ¼ 8.6, 3-H and 5-H Ar), 7.62
a
]
¼ ꢂ4.8 (c
D
(d, 2H, J ¼ 8.6, 2-H and 6-H Ar), 7.14e7.03 (m, 5H, Ph), 5.32 (s, 2H,
1-CH2), 2.85 (d, 1H, J ¼ 14.3, 4-CH2), 2.79 (q, 1H, J ¼ 7.2, 3-H), 2.68
(d, 1H, J ¼ 14.3, 4-CH2), 0.95 (d, 3H, J ¼ 7.2, 3-CH3). 13C NMR
d
Ph), 7.04 (d, 2H, J ¼ 8.7, 2-H and 6-H Ar), 6.73 (d, 2H, J ¼ 8.7, 3-H
(75 MHz, Acetone-d6):
d 178.6, 175.8 (CO), 149.5, 148.6, 139.2,
and 5-H Ar), 3.84 (t, 2H, J ¼ 7.3, 10-H), 3.66 (d, 1H, J ¼ 12.9, N-CH2),
131.7, 131.2, 129.7, 128.2, 124.9 (C Ar), 63.2 (1-CH2), 59.2 (4-C), 44.8
(3-C), 41.6 (4-CH2), 13.1 (3-CH3). MS (ES positive mode): 355.2
[Mþ1]þ.
3.56 (s, 3H, OMe), 3.49 (d, 1H, J ¼ 12.9, N-CH2), 3.46 (m, 1H,
a-H
Phe), 2.87 (d, 2H, J ¼ 6.8,
b
-H Phe), 1.72 (m, 2H, 20-H), 0.95 (t, 3H,
J ¼ 7.5, 30-H). 13C NMR (75 MHz, CDCl3):
d
175.1 (CO), 158.2, 137.3,
-C
Phe), 51.6 (OMe), 51.4 (N-CH2), 39.7 (b
-C Phe), 22.6 (20-C), 10.5 (30-
131.4, 129.3, 129.2, 128.3, 126.6, 114.3 (C Ar), 69.5 (10-C), 61.9 (
a
4.2.4.6. (3S,4S)-4-Benzyl-4-carboxy-3-methyl-1-p-[20-(morpholin-400-il)
ethoxy]benzyl-2-azetidinone trifluoroacetate (7g). Syrup. Yield: 42%.
Reverse-phase cartridge. Eluent: H2O:MeCN (Gradient from 0 to 5%
C). MS (ES positive mode): 328.3 [Mþ1]þ. Anal. (%) Calc. for
C20H25NO3: C, 73.37; H, 7.70; N, 4.28. Found (%): C, 73.28; H, 7.65;
N, 4.14.
MeCN). [
a]
D
¼ þ7.3 (c 0.98, CHCl3). HPLC (Sunfire): tR ¼ 5.36 min
1
(Gradient from 0 to 100% A in 15 min). H NMR (300 MHz, CDCl3):
d
10.52 (broad s, 1H, CO2H), 7.22 (d, 2H, J ¼ 7.6, 2-H and 6-H Ar),
4.3.2. N-p-Methoxybenzyl-L-Tyr(Bzl)-OMe (14)
7.20e7.04 (m, 5H, Ph), 6.86 (d, 2H, J ¼ 7.6, 3-H and 5-H Ar), 4.44 (d, 1H,
J ¼ 15.4, 1-CH2), 4.19 (m, 2H, 10-H), 4.03 (d, 1H, J ¼ 15.4, 1-CH2), 3.68 (m,
4H, 200-H), 3.37 (d, 1H, J ¼ 14.2, 4-CH2), 3.18 (d, 1H, J ¼ 14.2, 4-CH2), 3.08
(q, 1H, J ¼ 7.6, 3-H), 2.91 (m, 2H, 20-H), 2.71 (m, 4H, 300-H), 1.14 (d, 3H,
Syrup. Yield: 66%. Eluent: CH2Cl2:Et2O:hexane (1:1:1).
[
a
]
¼ þ3.1 (c 1.12, CHCl3). HPLC (Sunfire): tR ¼ 7.00 min (Gradient
D
from 2 to 100% A in 15 min). 1H NMR (300 MHz, CDCl3): 7.45e7.29
(m, 5H, Ph), 7.14 (d, 2H, J ¼ 8.7, 2-H and 6-H Pmb), 7.07 (d, 2H,
J ¼ 8.9, 2-H and 6-H Ar Tyr), 6.88 (d, 2H, J ¼ 8.9, 3-H and 5-H Ar Tyr),
6.81 (d, 2H, J ¼ 8.7, 3-H and 5-H Pmb), 5.04 (broad s, 2H, OCH2), 3.79
J ¼ 7.6, 3-CH3). 13C NMR (75 MHz, CDCl3):
d 175.1, 170.1 (CO), 156.8,
136.1, 130.4, 130.1, 129.9, 128.2, 126.8, 114.2 (C Ar), 69.5 (4-C), 64.7 (100-

P. Pérez-Faginas et al. / European Journal of Medicinal Chemistry 46 (2011) 5108e5119
5115
(s, 3H, OMe), 3.74 (d, 1H, J ¼ 12.9, N-CH2), 3.57 (d, 1H, J ¼ 12.9, N-
C24H24N2O6S: C, 61.52; H, 5.16; N, 5.98. Found (%): C, 61.61; H, 5.09;
N, 5.64.
CH2), 3.64 (s, 3H, OMe), 3.49 (m, 1H,
a
-H Tyr), 2.90 (d, 2H, J ¼ 6.8,
b-
H Tyr), 1.74 (broad s, 1H, NH). 13C NMR (75 MHz, CDCl3): 175.0 (CO),
158.6, 157.6137.0, 131.7, 130.1, 129.6, 129.3, 128.5, 127.8, 127.4, 114.7,
4.6. Synthesis of N-phenetyl- -Phe-OMe (11j)
L
113.7 (C Ar), 69.9 (OCH2), 62.0 (a-C Tyr), 55.2, 51.5 (OMe), 51.4 (N-
CH2), 38.8 (
b
-C Tyr). MS (ES positive mode): 406.5 [Mþ1]þ. Anal. (%)
A solution of 10j (0.70 g, 1.5 mmol) in MeCN (20 ml) was treated
with K2CO3 (0.62 g, 4.5 mmol) and thiophenol (0.31 ml, 3 mmol).
The reaction was then stirred at room temperature for 20 h. The
solvent was evaporated and the residue was extracted with EtOAc
and washed with H2O and brine. The organic layer was dried over
Na2SO4 and the solvent evaporated. The residue was purified by
column chromatography (EtOAc:hexane 1:1). Syrup. Yield: 0.36 g,
Calc. for C25H27NO4: C, 74.05; H, 6.71; N, 3.45. Found (%): C, 74.18; H,
6.68; N, 3.22.
4.3.3. N-p-Methoxybenzyl-
Solid. Yield: 76%. Eluent: CH2Cl2:Et2O:Hexane (1:1:1). M.p.
82e84 ꢁC [
L
-Tyr(2,6-dichlorobenzyl)-OMe (15)
a
]
¼ þ9.4 (c 1.02, CHCl3). HPLC (Sunfire): tR ¼ 7.69 min
D
(Gradient from 2 to 100% A in 15 min). 1H NMR (300 MHz, CDCl3):
7.42e7.30 (m, 3H, Cl2C6H3), 7.20 (d, 2H, J ¼ 8.7, 2-H and 6-H Pmb),
7.15 (d, 2H, J ¼ 8.8, 2-H and 6-H Ar Tyr), 6.99 (d, 2H, J ¼ 8.8, 3-H and
5-H Ar Tyr), 6.87 (d, 2H, J ¼ 8.7, 3-H and 5-H Pmb), 5.30 (broad s, 2H,
OCH2), 3.84 (s, 3H, OMe), 3.80 (d, 1H, J ¼ 12.9, N-CH2), 3.72 (s, 3H,
85%. [
(Gradient from 20 to 100% A in 15 min). 1H NMR (300 MHz, CDCl3):
7.19e7.05 (m, 10H, Ph), 3.55 (s, 3H, OMe), 3.47 (m, 1H, -H Phe),
2.86 (d, 2H, J ¼ 6.9,
-H Phe), 2.82e2.62 (m, 4H, 10-H, 20-H). 13C NMR
(75 MHz, CDCl3): 174.6 (CO), 139.4, 136.9, 128.8, 128.4, 128.2, 127.8,
126.2, 125.9 (C Ar), 62.8 (
a
]
¼ þ9.4 (c 0.95, CHCl3). HPLC (Sunfire): tR ¼ 7.14 min
D
d
a
b
d
OMe), 3.63 (d, 1H, J ¼ 12.9, N-CH2), 3.56 (m, 1H,
2H,
(CO), 158.6, 157.7, 136.9, 132.2, 131.7, 130.3, 130.2, 130.0, 129.3, 128.4,
a
-H Tyr), 2.97 (m,
a b-C
-C Phe), 51.3 (OMe), 49.1 (10-C), 39.3 (
b
-H Tyr), 1.84 (broad s, 1H, NH). 13C NMR (75 MHz, CDCl3): 175.0
Phe), 36.1 (20-C). MS (ES positive mode): 284.2 [Mþ1]þ. Anal. (%)
Calc. for C18H21NO2: C, 76.29; H, 7.47; N, 4.94. Found (%): C, 76.34; H,
7.21; N, 4.84.
114.9, 113.7 (C Ar), 65.2 (OCH2), 62.0 (a-C Tyr), 55.2, 51.6 (OMe), 51.4
(N-CH2), 38.8 (
(%) Calc. for C25H25Cl2NO4: C, 63.30; H, 5.31; N, 2.95. Found (%): C,
63.39; H, 5.22; N, 2.76.
b
-C Tyr). MS (ES positive mode): 475.4 [Mþ1]þ. Anal.
4.7. Solution synthesis of N-substituted N-chloropropionyl amino
acid derivatives
4.4. Synthesis of N-(2-nitrophenyl)sulfonyl-
L
-PheeOMe (9)
To a solution of 2(S)-chloropropionic acid (2.87 mmol) in THF
(15 ml) was added trichloroacetonitrile (3.8 mmol). The reaction
was cooled to 0 ꢁC and treated with Ph3P (1 g, 3.8 mmol), and
stirring continued at room temperature for 1 h. Then, a solution of
the N-alkyl amino ester (1.92 mmol) and propilene oxide
(28.6 mmol) in THF (10 ml) was added. The reaction was stirred
overnight. The solvent was evaporated to dryness and the resulting
syrup was extracted with EtOAc and washed with citric acid (10%),
HNaCO3 (10%) and brine. The organic layer was dried over Na2SO4
and, after evaporation of the solvent, the residue was purified on
a silica gel column as specified in each case.
A solution of HePheeOMe HCl (1.08 g, 5.0 mmol)) in DCM
(40 ml) was treated with TEA (0.69 ml, 5 mmol). After 15 min, the
reaction was cooled to 0 ꢁC and TEA (0.91 ml 6.5 mmol) and 2-
nitrobenzenesulfonyl chloride (1.44 g, 6.5 mmol) were added.
The reaction was then stirred at room temperature for 12 h. The
solvent was evaporated and the residue was extracted with
EtOAc, and washed with citric acid (10%), HNaCO3 (10%) and brine.
The organic layer was dried over Na2SO4 and the solvent evapo-
rated to give quantitatively to the title compounds as a syrup,
which was used in the next synthetic step without further puri-
fication. HPLC (Sunfire): tR ¼ 2.62 min (Gradient from 50 to 100%
4.7.1. N-[(S)-2-Chloropropionyl]-N-(p-propoxy)benzyl-
L
-Phe-OMe
A in 5 min). 1H NMR (300 MHz, CDCl3):
d
7.95 (m, 1H, 3-H Ar), 7.84
(m, 1H, 6-H Ar), 7.67 (m, 2H, 4-H, 5-H Ar), 7.25e7.09 (m, 5H, Ph),
6.01 (broad s, 1H, J ¼ 8.7, NH), 4.46 (m, 1H, -H Phe), 3.52 (s, 3H,
OMe), 3.16 (dd, 1H, J ¼ 5.6, 13.8, -H Phe), 3.07 (dd, 1H, J ¼ 6.9,
13.8, 170.3 (CO), 147.3, 134.7,
-H Phe). 13C NMR (75 MHz, CDCl3):
134.0, 133.4, 132.9, 130.2, 129.2, 128.6, 127.4, 125.5 (C Ar), 57.8
(12f)
Syrup. Yield: 87%. Eluent: EtOAc:hexane (1:4). [
a
]
¼ ꢂ82.9 (c
D
a
0.79, CHCl3). HPLC (Sunfire): tR ¼ 7.63 min (Gradient from 20 to
b
100% A in 15 min). 1H NMR(300 MHz, CDCl3):
d main rotamer
b
d
7.29e7.07 (m, 5H, Ph), 6.98 (d, 2H, J ¼ 8.7, 2-H and 6-H Ar), 6.79 (d,
2H, J ¼ 8.7, 3-H and 5-H Ar), 4.49 (d, 1H, J ¼ 15.7, N-CH2), 4.48 (q, 1H,
(OMe), 52.4 (
a
-C Phe), 39.1 (
b
-C Phe). MS (ES positive mode):
J ¼ 6.4, CH-Cl), 4.39 (dd, 1H, J ¼ 6.2, 8.9,
a-H Phe), 3.99 (d, 1H,
387.4 [MþNa]þ.
J ¼ 15.7, N-CH2), 3.89 (t, 2H, J ¼ 7.1, 10-H), 3.60 (s, 3H, OMe), 3.35 (dd,
1H, J ¼ 6.2, 14.2,
b
-H Phe), 3.20 (dd, 1H, J ¼ 8.9, 14.2,
b-H Phe), 1.80
4.5. Synthesis of N-(2-nitrophenyl)sulfonyl-N-phenetyl-
(10j)
L
-PheeOMe
(m, 2H, 20-H), 1.67 (d, 3H, J ¼ 6.4, Me), 1.03 (t, 3H, J ¼ 7.5, 30-H). 13C
NMR (75 MHz, CDCl3): d main rotamer 170.3, 169.5 (CO), 158.8,
137.6, 132.9, 130.3, 129.3, 128.7, 127.2, 114.6 (C Ar), 69.5 (10-C), 60.2
A solution of the nosyl-derivative 9 (1.82 g, 5 mmol) in THF
(30 ml) was treated with 2-phenylethanol (0.41 ml, 3.33 mmol) and
Ph3P (1.31 g, 5 mmol) under argon atmosphere and cooled at 0 ꢁC.
Then DIAD (0.98 ml, 5 mmol) was added. After 18 h of stirring at
room temperature, the solvent was evaporated to dryness and the
residue was purified by column chromatography (EtOAc:hexane,
(a-C Phe), 52.1 (OMe), 51.3 (N-CH2), 49.8 (CH-Cl) 35.0 (b-C Phe),
22.9 (20-C), 22.5 (Me), 10.5 (30-C). MS (ES positive mode): 440.2
[M þ Na]þ. Anal. (%) Calc. for C23H28ClNO4: C, 66.10; H, 6.75; N, 3.35.
Found (%): C, 66.23; H, 6.68; N, 3.29.
4.7.2. N-[(S)-2-Chloropropionyl]-N-phenethyl-
Syrup. Yield: 85%. Eluent: EtOAc:hexane (1:8). [
0.89, CHCl3). HPLC (Sunfire): tR ¼ 5.98 min (Gradient from 50 to
100% A in 15 min). 1H NMR (300 MHz, CDCl3):
main rotamer
7.31e7.03 (m, 10H, Ph), 4.31 (q, 1H, J ¼ 6.6, CHCl), 4.28 (m, 1H, -H
L
-Phe-OMe (12j)
1:4). Syrup. Yield: 1.35 g, 95%. [
a
]
¼ ꢂ21.5 (c 0.99, CHCl3). HPLC
a]
D
¼ ꢂ92.4 (c
D
(Sunfire): tR ¼ 7.14 min (Gradient from 50 to 100% A in 15 min). 1H
NMR (300 MHz, CDCl3):
d
7.82 (m, 1H, 3-H Ar), 7.63 (m, 1H, 6-H Ar),
d
7.56 (m, 2H, 4-H, 5-H Ar), 7.30e7.20 (m, 10H, Ph), 3.70e3.56 (m, 4H,
10-H, -H Phe), 3.54 (s, 3H, OMe), 3.08e2.86 (m, 3H, 20-H,
-H Phe,
-H Phe). 13C NMR (75 MHz, CDCl3):
170.7 (CO), 156.3, 148.2, 138.3,
136.1,133.5,132.9,131.5, 130.7, 129.1,128.8,128.6, 128.3, 127.0,126.7,
a
a
b
Phe), 3.77 (s, 3H, OMe), 3.47e3.29 (m, 3H, b
-H Phe, 10-H), 3.08 (m,
b
d
1H, 10-H), 2.64 (m, 2H, 20-H), 1.60 (d, 3H, J ¼ 6.6, Me). 13C NMR
(75 MHz, CDCl3):
d main rotamer 170.5, 169.1 (CO), 137.8, 129.2,
124.0 (C Ar), 52.3 (OMe), 52.1 (
a
-C Phe), 41.5 (10-C), 36.6 (20-C), 36.6
129.0, 128.7, 128.6, 128.5, 127.4, 126.7 (C Ar), 62.6 (
a-C Phe), 52.4
(b
-C Phe). MS (ES positive mode): 469.2 [Mþ1]þ. Anal. (%) Calc. for
(OMe), 51.1 (10-C), 49.5 (CHCl), 35.1 ( -C Phe), 34.4 (20-C), 21.0 (Me).
b

5116
P. Pérez-Faginas et al. / European Journal of Medicinal Chemistry 46 (2011) 5108e5119
MS (ES positive mode): 374.2 [Mþ1]þ. Anal. (%) Calc. for
C21H24ClNO3: C, 67.46; H, 6.47; N, 3.75. Found (%): C, 67.23; H, 6.39;
N, 3.77.
(OMe), 45.4 (1-CH2), 40.3 (4-CH2), 22.5 (20-C), 10.4 (30-C), 10.0 (3-
CH3). MS (ES positive mode): 382.3 [Mþ1]þ. Anal (%) Calc. for
C23H27NO4: C 72.42, H 7.13, N 3.67. Found (%): C 72.51, H 7.03, N 3.58.
4.7.3. N-[(S)-2-Chloropropionyl]-N-p-methoxybenzyl-
L
-Tyr(Bzl)-
4.8.2. (3S,4S)-4-Benzyl-4-methoxycarbonyl-3-methyl-1-phenethyl-
OMe (16)
2-azetidinone (13j)
Syrup. Yield: 88%. Eluent: EtOAc:hexane (1:7). [
1.04, CHCl3). HPLC (Sunfire): tR ¼ 12.33 min (Gradient from 10 to
100% A in 15 min). 1H NMR (300 MHz, CDCl3):
main rotamer
a
]
¼ ꢂ55.3 (c
Syrup.Yield:75%.Eluent:EtOAc:hexane(1:4). [
a
]D ¼ꢂ21.9(c0.93,
D
CHCl3). HPLC (Sunfire): tR ¼ 4.34 min (Gradient from 50 to 100% A in
d
15 min). 1H NMR (300 MHz, CDCl3):
d 7.30e7.07 (m, 10H, Ph), 3.80 (s,
7.45e7.32 (m, 5H, Ph), 7.00 (d, 2H, J ¼ 8.7, 2-H and 6-H Pmb), 6.98
(d, 2H, J ¼ 8.9, 2-H and 6-H Ar Tyr), 6.87 (d, 2H, J ¼ 8.9, 3-H and 5-H
Ar Tyr), 6.80 (d, 2H, J ¼ 8.7, 3-H and 5-H Pmb), 5.06 (broad s, 2H,
OCH2), 4.50 (d, 1H, J ¼ 16.1, N-CH2), 4.47 (m, 1H, CHCl), 4.35 (dd, 1H,
3H, OMe), 3.51 (d, 1H, J ¼ 14.3, 4-CH2), 3.23 (m, 1H, 10-H), 3.16e2.93
(m, 4H,10-H, 20-H, 3-H), 3.08 (d,1H, J ¼ 14.3, 4-CH2),1.17 (d, 3H, J ¼ 7.5,
3-CH3). 13C NMR (75 MHz, CDCl3):
d 171.8, 169.5 (CO), 138.9, 135.1,
129.7,128,7,128.6,128.4,127.4,126.3 (C Ar), 68.9 (4-C), 53.8 (3-C), 52.2
(OMe), 44.9 (10-C), 40.8 (4-CH2), 34.4 (20-C), 10.2 (3-CH3). MS (ES
positive mode): 338.3 [Mþ1]þ. Anal. (%) Calc. for C21H23NO3: C, 74.75;
H, 6.87; N, 4.15. Found (%): C, 74.86; H, 6.68; N, 4.00.
J ¼ 6.2, 9.0, -H Tyr), 4.01 (d, 1H, J ¼ 16.1, N-CH2), 3.79 (s, 3H, OMe),
a
3.60 (s, 3H, OMe), 3.28 (dd, 1H, J ¼ 6.2, 14.1,
b-H Tyr), 3.14 (dd, 1H,
J ¼ 9.0, 14.1,
CDCl3):
b
-H Tyr), 1.67 (d, 3H, J ¼ 6.5, Me). 13C NMR (75 MHz,
d
main rotamer 170.3, 169.4 (CO), 159.2, 157.6, 136.9, 133.1,
130.2, 129.8, 128.7, 128.5, 127.9, 127.4, 114.9, 114.0 (C Ar), 70.0
4.8.3. (3S,4S)-4-p-Benzyloxybenzyl-1-p-methoxybenzyl-4-methoxy-
(OCH2), 60.3 (
a
-C Tyr), 55.2, 52.0 (OMe), 51.2 (N-CH2), 49.8 (CCl),
carbonyl-3-methyl-2-azetidinone (18)
34.1 (
b
-C Tyr), 21.0 (CH3). MS (ES positive mode): 496.8 [Mþ1]þ.
Syrup. Yield: 73%. Eluent: EtOAc:hexane (1:5).[
a
]
¼ ꢂ55.3 (c
D
Anal. (%) Calc. for : C28H30ClNO5: C, 67.80; H, 6.10; N, 2.82. Found
(%): C, 67.87; H, 6.04; N, 2.79.
1.04, CHCl3). HPLC (Sunfire): tR ¼ 11.63 min (Gradient from 10 to
100% A in 15 min). 1H NMR (300 MHz, CDCl3): 7.44e7.30 (m, 7H, Ar),
6.86e6.76 (m, 6H, Ar), 5.02 (broad s, 2H, OCH2), 4.41 (d, 1H, J ¼ 15.4,
1-CH2), 4.11 (d, 1H, J ¼ 15.4, 1-CH2), 3.79 (s, 3H, OMe), 3.70 (s, 3H,
OMe), 3.27 (d, 1H, J ¼ 14.5, 4-CH2), 3.14 (q, 1H, J ¼ 7.5, 3-H), 3.03 (d,
1H, J ¼ 14.5, 4-CH2), 1.14 (d, 3H, J ¼ 7.5, 3-CH3). 13C NMR (75 MHz,
CDCl3): 171.8, 169.7 (CO), 158.8, 157.9, 136.9, 131.0, 129.8, 129.0,
128.6, 127.9, 127.4, 127.3, 114.8, 113.7 (C Ar), 70.0 (OCH2), 69.1 (4-C),
55.2 (OMe), 53.4 (3-C), 52.0 (OMe), 45.5 (1-CH2), 39.6 (4-CH2), 10.1
(3-CH3). MS (ES positive mode): 460.5 [Mþ1]þ. Anal. (%) Calc. for:
C28H29NO5: C, 73.18; H, 6.36; N, 3.05. Found (%): C, 73.22; H, 6.42; N,
2.71.
4.7.4. N-[(S)-2-Chloropropionyl]-N-p-methoxybenzyl-L-Tyr(2,6-
dichlorobenzyl)-OMe (17)
Syrup. Yield: 93%. Eluent: gradient from 12 to 16% of EtOAc in
hexane. [
¼ ꢂ46.4 (c 1.01, CHCl3). HPLC (Sunfire): tR ¼ 13.13 min
(Gradient from 19 to 100% A in 15 min). 1H NMR (300 MHz, CDCl3):
a]
D
d
main rotamer 7.42e7.30 (m, 1H, Cl2C6H3), 7.07 (d, 2H, J ¼ 8.4, 2-H
and 6-H Pmb), 7.04 (d, 2H, J ¼ 8.7, 2-H and 6-H Ar Tyr), 6.97 (d, 2H,
J ¼ 8.7, 3-H and 5-H Ar Tyr), 6.88 (d, 2H, J ¼ 8.4, 3-H and 5-H Pmb),
5.30 (broad s, 2H, OCH2), 4.54 (d, 1H, J ¼ 16.2, N-CH2), 4.53 (m, 1H,
CHCl), 4.40 (dd, 1H, J ¼ 6.2, 8.9, -H Tyr), 4.13 (d, 1H, J ¼ 16.2, N-
a
CH2), 3.83 (s, 3H, OMe), 3.66 (s, 3H, OMe), 3.35 (dd, 1H, J ¼ 6.2, 14.1,
-H Tyr), 3.21 (dd, 1H, J ¼ 8.9, 14.1, -H Tyr), 1.72 (d, 3H, J ¼ 6.5, Me).
13C NMR (75 MHz, CDCl3):
main rotamer 170.3, 169.5 (CO), 159.2,
157.7, 136.9, 132.1, 130.4, 130.3, 130.2, 128.7, 128.4, 127.2, 115.1, 114.0
(C Ar), 75.3 (OCH2), 60.4 ( -C Tyr), 55.3, 52.1 (OMe), 51.3 (N-CH2),
4.8.4. (3S,4S)-4-p-(2,6-Dichlorobenzyloxi)benzyl-1-p-methoxybenzyl-
4-methoxycarbonyl-3-methyl-2-azetidinone (19)
b
b
d
Syrup. Yield: 89%. Eluent: gradient from 17 to 33% of EtOAc in
hexane. [
a
]
¼ ꢂ5.5 (c 1.05, CHCl3). HPLC (Sunfire): tR ¼ 12.46 min
D
a
(Gradient from 10 to 100% A in 15 min). 1H NMR (300 MHz, CDCl3):
7.38e7.25 (m, 3H, Cl2C6H3), 7.15e6.81 (m, 8H, Ar), 5.22 (broad s, 2H,
OCH2), 4.44 (d, 1H, J ¼ 15.4, 1-CH2), 4.13 (d, 1H, J ¼ 15.4, 1-CH2), 3.80
(s, 3H, OMe), 3.72 (s, 3H, OMe), 3.29 (d, 1H, J ¼ 14.5, 4-CH2), 3.06 (d,
1H, J ¼ 14.5, 4-CH2), 3.16 (c, 1H, J ¼ 7.5, 3-H), 1.15 (d, 3H, J ¼ 7.5, 3-
CH3). 13C NMR (75 MHz, CDCl3): 171.8, 169.7 (CO), 158.9, 157.9, 137.0,
132.0, 131.0, 130.4, 129.8, 129.0, 128.5, 127.7, 114.9, 113.7 (C Ar), 69.2
(OCH2), 65.2 (4-C), 55.3 (OMe), 53.4 (3-C), 52.1 (OMe), 45.5 (1-CH2),
39.6 (4-CH2), 10.0 (3-CH3). MS (ES positive mode): 529.4 [Mþ1]þ.
Anal. (%) Calc. for C28H27Cl2NO5: C, 63.64; H, 5.15; N, 2.65. Found
(%): C, 63.31; H, 5.23; N, 2.58.
49.8 (CCl), 34.1 (b-C Tyr), 21.1 (CH3). MS (ES positive mode): 565.8
[Mþ1]þ. Anal. (%) Calc. for C28H28Cl3NO5: C, 59.53; H, 5.00; N, 2.48.
Found (%): C, 59.66; H, 5.02; N, 2.23.
4.8. Solution synthesis of 2-azetidinone derivatives
A
solution of the corresponding N-substituted N-chlor-
opropionyl derivative (1 mmol) in dry MeCN (12 ml) was treated
with BTPP (1.5 mmol) and stirred at room temperature until
disappearance of the starting material. The solvent was then
evaporated to dryness, and the residue was partitioned between
EtOAc and H2O. The organic layer was separated, dried over Na2SO4
and, after evaporation, the residue was purified on a silica gel
column as specified in each case.
4.9. Solution synthesis of 4-carboxy-2-azetidinones
A
solution of the corresponding 4-methoxycarbonyl-2-
azetidinone (0.45 mmol) in MeOH (10 ml) was treated with 2M
NaOH (0.46 ml, 1.12 mmol) and stirred overnight at room temper-
ature. The solvent was evaporated and the residue was dissolved in
H2O (8 mL) and acidified with 1M HCl to pH ¼ 3. The aqueous layer
was extracted with EtOAc, the phases were separated and the
organic layer was dried over Na2SO4, and the solvent evaporated.
4.8.1. (3S,4S)-4-Benzyl-4-methoxycarbonyl-3-methyl-1-p-propoxy-
benzyl-2-azetidinone (13f)
Syrup. Yield: 70%. Eluent: EtOAc:hexane (1:2). [
1.05, CHCl3). HPLC (Sunfire): tR ¼ 5.90 min (Gradient from 50 to 100%
A in 15 min). 1H NMR (300 MHz, CDCl3):
7.20 (m, 3H, Ph), 7.13 (d,
a]
D
¼ ꢂ1.26 (c
d
2H, J ¼ 8.5, 2-H and 6-H Ar), 6.94 (m, 2H, Ph), 6.82 (d, 2H, J ¼ 8.5, 3-H
and 5-H Ar), 4.41 (d, 1H, J ¼ 15.3, 1-CH2), 4.12 (d, 1H, J ¼ 15.3, 1-CH2),
3.90 (t, 2H, J ¼ 6.9, 10-H), 3.70 (s, 3H, OMe), 3.31 (d, 1H, J ¼ 14.4, 4-
CH2), 3.15 (q, 1H, J ¼ 7.5, 3-H), 3.10 (d, 1H, J ¼ 14.4, 4-CH2), 1.79 (m,
2H, 20-H), 1.14 (d, 3H, J ¼ 7.5, 3-CH3), 1.03 (t, 3H, J ¼ 7.4, 30-H). 13C
4.9.1. (3S,4S)-4-Benzyl-4-carboxy-3-methyl-1-p-propoxybenzyl-2-
azetidinone (7f)
Syrup. Yield: 83%. HPLC (Sunfire): tR ¼ 9.87 min (Gradient from
0 to 100% A in 15 min). 1H NMR (300 MHz, CDCl3):
d 9.14 (broad s,
NMR (75 MHz, CDCl3):
d
171.6, 169.6 (CO), 158.4, 135.0, 129.9, 129.7,
1H, CO2H), 7.17 (m, 3H, Ph), 7.16 (d, 2H, J ¼ 8.7, 2-H and 6-H Ar), 6.92
128.6,128.4, 127.1, 114.3 (C Ar), 69.4 (10-C), 68.9 (4-C), 53.4 (3-C), 52.0
(m, 2H, Ph), 6.83 (d, 2H, J ¼ 8.7, 3-H and 5-H Ar), 4.57 (d,1H, J ¼ 15.4,

P. Pérez-Faginas et al. / European Journal of Medicinal Chemistry 46 (2011) 5108e5119
5117
1-CH2), 4.06 (d,1H, J ¼ 15.4,1-CH2), 3.90 (t, 2H, J ¼ 6.9,10-H), 3.30 (d,
4.11.1. (3S,4S)-4-Benzyl-4-ciclohexylcarbamoyl-1-p-
1H, J ¼ 14.3, 4-CH2), 3.21 (q, 1H, J ¼ 7.3, 3-H), 3.11 (d, 1H, J ¼ 14.3, 4-
CH2), 1.79 (m, 2H, 20-H), 1.24 (d, 3H, J ¼ 7.3, 3-CH3), 1.03 (t, 3H,
methoxybenzyl-3-methyl-2-azetidinone (22a)
Solid. Yield: 87%. Eluent: EtOAc:hexane (1:5). M.p.: 147e149 ꢁC
¼ ꢂ65.5 (c 1.08, CHCl3). HPLC (Sunfire): tR ¼ 4.81 min (Gradient
J ¼ 7.4, 30-H). 13C NMR (75 MHz, CDCl3):
d
175.1, 170.6 (CO), 158.5,
[a]
D
134.7, 129.9, 129.8, 128.4, 128.3, 127.2, 114.4 (C Ar), 69.5 (10-C), 69.1
(4-C), 52.9 (3-C), 45.8 (1-CH2), 40.1 (4-CH2), 22.5 (20-C), 10.5 (30-C),
10.1 (3-CH3). EM (ES positive mode): 368.3 [Mþ1]þ.
from 50 to 100% A in 15 min). 1H NMR (300 MHz, CDCl3):
d
7.27e7.19 (m, 5H, Ph), 6.85 (d, 2H, J ¼ 8.7, 2H and 6H Pmb), 6.75 (d,
2H, J ¼ 8.7, 3H and 5H Pmb), 5.29 (d, 1H, J ¼ 8.3, CONH), 4.68 (d, 1H,
J ¼ 15.3, 1-CH2), 3.83 (d, 1H, J ¼ 14.3, 4-CH2), 3.71 (s, 3H, OMe), 3.53
(d, 1H, J ¼ 15.3, 1-CH2), 3.47 (m, 1H, 10-H Chx), 3.07 (q, 1H, J ¼ 7.5, 3-
H), 3.04 (d, 1H, J ¼ 14.3, 4-CH2), 1.44 (m, 5H, 20-H, 30-H, 40-H Chx),
1.14 (m, 2H, 30-H Chx), 1.09 (d, 3H, J ¼ 7.5, 3-CH3), 0.79 (m, 1H, 30-H
Chx), 0.38 (m, 1H, 20-H Chx), 0.20 (m, 1H, 20-H Chx). 13C NMR
4.9.2. (3S,4S)-4-Benzyl-4-carboxy-3-methyl-1-phenethyl-2-
azetidinone (7j)
Solid. Yield: 95%. M.p.: 110e112 ꢁC. HPLC (Sunfire): tR ¼ 9.23 min
(Gradient from 0 to 100% A in 15 min). 1H NMR (300 MHz, CDCl3):
d
8.47 (broad s, 1H, CO2H), 7.27e7.04 (m, 10H, Ph), 3.51 (d, 1H,
(75 MHz, CDCl3): d 171.2, 168.7 (CO),159.6, 130.2, 130.0, 128.9, 128.6,
J ¼ 14.3, 4-CH2), 3.31e2.92 (m, 5H, 10-H, 20-H, 3-H), 3.10 (d, 1H,
127.1, 114.6 (C Ar), 70.6 (4-C), 55.2 (OMe), 54.9 (3-C), 47.9 (10-C Chx),
43.9 (1-CH2), 40.2 (4-CH2), 32.4, 32.9 (20-C Chx), 25.2 (40-C Chx),
24.7, 24.6 (30-C Chx), 9.9 (3-CH3). MS (ES positive mode): 421.5
[Mþ1]þ. Anal (%) Calc. for C26H32N2O3: C 74.26, H 7.67, N 6.66.
Found (%): C 74.32, H 7.70, N 6.27.
J ¼ 14.3, 4-CH2), 1.25 (d, 3H, J ¼ 7.5, 3-CH3). 13C NMR (75 MHz,
CDCl3):
d 175.2, 170.4 (CO), 138.9, 134.9, 129.8, 128,7, 128.6, 128.4,
127.5, 126.4 (C Ar), 68.8 (4-C), 53.4 (3-C), 45.1 (10-C), 40.5 (4-CH2),
34.3 (20-C), 10.2 (3-CH3). MS (ES positive mode): 324.2 [Mþ1]þ.
4.9.3. (3S,4S)-4-Carboxy-4-p-(2,6-dichlorobenzyloxy)benzyl-1-p-
methoxybenzyl-3-methyl-2-azetidinone (21)
4.11.2. (3S,4S)-4-Benzyl-4-benzylcarbamoyl-1-p-methoxybenzyl-3-
methyl-2-azetidinone (22b)
Syrup. Yield: 91%. HPLC (Sunfire): tR ¼ 10.35 min (Gradient from
15 to 100% A in 15 min).1H NMR (300 MHz, CDCl3): 7.37e7.25 (m, 3H,
Cl2C6H3), 7.18 (d, 2H, J ¼ 8.6, 2-H and 6-H Pmb), 6.89e6.81 (m, 6H,
Ar), 5.21 (broad s, 2H, OCH2), 4.56 (d,1H, J ¼ 15.4,1-CH2), 4.09 (d,1H,
J ¼ 15.4,1-CH2), 3.79 (s, 3H, OMe), 3.30 (d,1H, J ¼ 14.6, 4-CH2), 3.21 (q,
1H, J ¼ 7.5, 3-H), 3.07 (d, 1H, J ¼ 14.6, 4-CH2), 1.25 (d, 3H, J ¼ 7.5, 3-
CH3). 13C NMR (75 MHz, CDCl3): 175.4, 170.7 (CO), 159.0, 157.9,
137.0, 132.1, 131.1, 130.4, 129.9, 128.8, 128.4, 127.6, 114.9, 113.9 (C Ar),
69.5 (OCH2), 65.2 (4-C), 55.2 (OMe), 52.8 (3-C), 45.7 (1-CH2), 39.2 (4-
CH2), 10.0 (3-CH3). MS (ES positive mode): 515.4 [Mþ1]þ.
Syrup. Yield: 98%. Eluent: EtOAc:hexane (1:10
a
1:2).
[a]
D
¼ ꢂ52.8 (c 1.15, CHCl3). HPLC (Sunfire): tR ¼ 3.98 min
(Gradient from 50 to 100% A in 15 min). 1H NMR (300 MHz,
CDCl3):
d
7.26e6.81 (m, 10H, Ar), 6.75 (d, 2H, J ¼ 8.6, 2H and 6H
Pmb), 6.52 (d, 2H, J ¼ 8.6, 3H and 5H Pmb), 5.70 (m, 1H, CO-NH),
4.66 (d, 1H, J ¼ 15.1, 1-CH2), 4.11 (dd, 1H, J ¼ 14.3, 6.2, NHCH2Ph),
3.89 (dd, 1H, J ¼ 14.3, 5.1, NHCH2Ph), 3.81 (d, 1H, J ¼ 14.5, 4-CH2),
3.64 (s, 3H, OMe), 3.53 (d, 1H, J ¼ 15.1, 1-CH2), 3.09 (d, 1H, J ¼ 14.5,
4-CH2), 3.08 (q, 1H, J ¼ 7.5, 3-H), 1.07 d, 3H, J ¼ 7.5, 3-CH3). 13C
NMR (75 MHz, CDCl3):
d 170.9, 169.8 (CO), 159.2, 136.9, 135.9,
130.1, 129.8, 128.7, 128.3, 127.9, 127.3, 127.2, 114.4 (C Ar), 70.7 (4-
C), 54.9 (OMe), 53.3 (3-C), 44.2 (1-CH2), 43.9 (NHCH2Ph), 40.2 (4-
CH2), 10.2 (3-CH3). MS (ES positive mode): 429.5 [Mþ1]þ. Anal (%)
Calc. for C27H28N2O3: C 75.68, H 6.59, N 6.54. Found (%): C 75.43, H
6.61, N 6.26.
4.10. Synthesis of (3S,4S)-4-p-hydroxybenzyl-1-p-methoxybenzyl-
4-methoxycarbonyl-3-methyl-2-azetidinone (20)
A solution of the 2-azetidinone 18 (46 mg, 0.1 mmol) in MeOH
(15 mL) was treated with Pd-C (0.05 mg, 10%) and hydrogenated at
30 psi of pressure and room temperature for 4 h. After filtration of
the catalyst and evaporation to dryness, the title compound was
4.11.3. (3S,4S)-4-Benzyl-1-p-methoxybenzyl-3-methyl-4-(4-
morpholine)carbonyl-2-azetidinone (22c)
obtained as a syrup (37 mg, 98%).[
a
]
¼ ꢂ4.1 (c 1.10, CHCl3). HPLC
Syrup. Yield: 62%. Eluent: DCM:MeOH (20:1). [
0.94, CHCl3). HPLC (Sunfire): tR ¼ 1.48 min (Gradient from 50 to
100% A in 15 min). 1H NMR (300 MHz, CDCl3):
a
]
¼ ꢂ2.87 (c
D
D
(Sunfire): tR ¼ 7.21 min (Gradient from 15 to 100% A in 15 min). 1H
NMR (300 MHz, CDCl3): 7.18 (d, 2H, J ¼ 8.8, 2-H and 6-H Pmb), 6.83
(d, 2H, J ¼ 8.8, 3-H and 5-H Pmb), 6.78 (d, 2H, J ¼ 8.6, 2-H and 6-H
4-Bn), 6.63 (d, 2H, J ¼ 8.6, 3-H and 5-H 4-Bn), 4.41 (d, 1H, J ¼ 15.3,
1-CH2), 4.21 (d, 1H, J ¼ 15.3, 1-CH2), 3.79 (s, 3H, OMe), 3.69 (s, 3H,
OMe), 3.22 (d, 1H, J ¼ 14.2, 4-CH2), 3.12 (q, 1H, J ¼ 7.5, 3-H), 3.05 (d,
1H, J ¼ 14.2, 4-CH2), 1.13 (d, 3H, J ¼ 7.5, 3-CH3). 13C NMR (75 MHz,
CDCl3): 171.7, 170.2 (CO), 158.9, 155.2, 131.1, 129.9, 128.7, 126.4,
115.4, 113.8 (C Ar), 69.2 (4-C), 55.3 (OMe), 52.9 (3-C), 52.1 (OMe),
45.5 (1-CH2), 39.1 (4-CH2), 10.1 (3-CH3). MS (ES positive mode):
370.4 [Mþ1]þ. Anal. (%) Calc. for : C21H23NO5: C, 68.28; H, 6.28; N,
3.79. Found (%): C, 68.32; H, 6.31; N, 3.44.
d
7.38 (d, 2H, J ¼ 8.7,
2H and 6H Pmb), 7.23 (m, 3H, Ph), 6.91 (m, 2H, Ph), 6.84 (d, 2H,
J ¼ 8.7, 3H and 5H Pmb), 4.55 (d, 1H, J ¼ 15.2, 1-CH2), 4.48 (d, 1H,
J ¼ 15.2, 1-CH2), 3.90e3.40 (m, 8H, CH2 morpholine), 3.77 (s, 3H,
OMe), 3.34 (d, 1H, J ¼ 14.9, 4-CH2), 2.97 (d, 1H, J ¼ 14.9, 4-CH2),
2.84 (q, 1H, J ¼ 7.3, 3-H), 1.24 (d, 3H, J ¼ 7.3, 3-CH3). 13C NMR
(75 MHz, CDCl3):
d 169.7, 168.9 (CO), 158.7, 133.9, 130.0, 129.6,
129.5, 128.3, 127.5, 113.7 (C Ar), 69.3 (4-C), 66.9, 66.2 (CH2 mor-
pholine), 55.1 (OMe), 51.0 (3-C), 45.6 (1-CH2), 39.9 (4-CH2), 12.1 (3-
CH3). MS (ES positive mode): 409.5 [Mþ1]þ. Anal (%) Calc. for
C24H28N2O4: C 70.57, H 6.91, N 6.86. Found (%): C 70.33, H 7.16, N
6.51.
4.11. General procedure for the preparation of 4-carboxamide
derivatives
4.11.4. (3S,4S)-4-Benzyl-1-p-methoxybenzyl-3-methyl-4-[(20-
piperidin-100-il)ethyl]carbamoyl-2-azetidinone (22d)
A
solution of the corresponding 4-carboxy-2-azetidinone
Syrup. Yield: 48%. Eluent: DCM:MeOH (20:1). [
0.81, CHCl3). HPLC (Sunfire): tR ¼ 3.54 min (Gradient from 20 to
100% A in 15 min). 1H NMR (300 MHz, CDCl3):
7.31e7.18 (m, 5H,
Ph), 7.04 (d, 2H, J ¼ 8.6, 2H and 6H Pmb), 6.82 (d, 2H, J ¼ 8.6, 3H and
5H Pmb), 5.84 (broad s, 1H, CONH), 4.59 (d, 1H, J ¼ 15.2, 1-CH2), 3.87
(d, 1H, J ¼ 15.2, 1-CH2), 3.78 (s, 3H, OMe), 3.69 (d, 1H, J ¼ 14.8, 4-
CH2), 3.24 (m, 1H, 10-H), 3.17 (d, 1H, J ¼ 14.8, 4-CH2), 3.13 (q, 1H,
J ¼ 7.3, 3-H), 2.92 (m,1H,10-H), 2.21 (m, 4H, 200-H, 600-H), 2.12 (m,1H,
20-H),1.99 (m,1H, 20-H), 1.46 (m, 4H, 300-H, 500-H), 1.39 (m, 2H, 400-H),
a
]
¼ ꢂ36.0 (c
D
(0.18 mmol) was dissolved in DCM (5 ml), and then treated with
the corresponding amine (0.36 mmol), BOP (0.36 mmol) and TEA
(0.36 mmol), in this order. The solution was stirred for 12 h at room
temperature. The solvent was evaporated and the residue was
extracted with EtOAc, and washed with citric acid (10%), HNaCO3
(10%) and brine. The organic layer was dried over Na2SO4 and, after
evaporation of the solvent, the residue was purified on a silica gel
column as specified in each case.
d

5118
P. Pérez-Faginas et al. / European Journal of Medicinal Chemistry 46 (2011) 5108e5119
1.16 (d, 3H, J ¼ 7.3, 3-CH3). 13C NMR (75 MHz, CDCl3):
d
170.6, 169.9
4.12.3. Cytotoxicity assay
Triplicate cultures were incubated for 72 h in the presence or
absence of test compounds in doseeresponse curves (10 concen-
(CO), 159.1, 135.7, 129.9, 129.8, 128.7, 128.4, 126.9, 114.1 (C Ar), 70.1
(4-C), 56.5 (20-C), 55.0 (OMe), 54.1 (3-C), 53.8 (200-C, 600-C), 44.4 (1-
CH2), 39.6 (4-CH2), 35.6 (10-C), 25.3 (300-C, 500-C), 23.7 (400-C), 9.71
(3-CH3). MS (ES positive mode): 450.5 [Mþ1]þ. Anal (%) Calc. for
C27H35N3O3: C 72.13, H 7.85, N 9.35. Found (%): C 72.03, H 7.92, N
9.28.
trations, typically ranging from 10 to 0.0026 mg/mL). A colorimetric
assay using sulforhodamine B (SRB) was adapted for quantitative
measurement of cell growth and cytotoxicity [27]. Briefly, cells were
seeded in 96-well microtiter plates and allowed to settle for 24 h in
drug-free medium before treatment with vehicle alone or test
compounds for 72 h. Forquantification, cells werewashedtwicewith
phosphate buffered saline (PBS), fixed for 15 min in 1% glutaralde-
hyde solution, rinsed twice with PBS, stained in 0.4% SRB-1% acetic
acid solution for 30 min, rinsed several times with 1% acetic acid
solution and air-dried. SRB was then extracted in 10 mM trizma base
solution and the optical density measured at 490 nm in a microplate
spectrophotometer. Cytotoxicity was estimated applying the
National Cancer Institute (NCI) algorithm [28]. Being Tz ¼ number of
control cells at time zero, C ¼ number of cells in control wells at 72 h
and T ¼ number of cells in the test wells at 72 h, then if Tz < T < C (no
effect or growth inhibition), cell survival ¼ 100*([T-Tz]/[C-Tz]) and if
T < Tz (net cell killing), cell survival ¼ 100*([T-Tz]/Tz). Three dos-
eeresponse parameters are calculated for each experimental agent.
GI50, compound concentration that produces 50% inhibition on cell
growth as compared to control cells. TGI, compound concentration
that produces total growth inhibition as compared to control cells.
LC50, compound concentration that produces 50% net cell killing.
4.11.5. (3S,4S)-4-Cyclohexylcarbamoyl-4-p-(2,6-dichlorobenzyloxy)
benzyl-1-p-methoxybenzyl-3-methyl-2-azetidinone (23a)
Syrup. Yield: 67%. Eluent: EtOAc:hexane (1:4). [
0.85, CHCl3). HPLC (Sunfire): tR ¼ 12.82 min (Gradient from 15 to
100% A in 15 min). 1H NMR (300 MHz, CDCl3):
7.39e7.27 (m, 3H,
a
]
¼ ꢂ12.9 (c
D
d
Cl2C6H3), 7.23 (d, 2H, J ¼ 8.7, 2-H and 6-H Ar Tyr), 7.00 (d, 2H,
J ¼ 8.4, 2-H and 6-H Pmb), 6.96 (d, 2H, J ¼ 8.7, 3-H and 5-H Ar Tyr),
6.83 (d, 2H, J ¼ 8.4, 3-H and 5-H Pmb), 5.37 (broad s, 1H, J ¼ 8.3,
NH), 5.29 (broad s, 2H, OCH2), 4.77 (d, 1H, J ¼ 15.1, 1-CH2), 3.83 (d,
1H, J ¼ 14.4, 4-CH2), 3.79 (s, 3H, OMe), 3.66 (d, 1H, J ¼ 15.1, 1-CH2),
3.55 (m, 1H, 10-H Chx), 3.21 (q, 1H, J ¼ 7.5, 3-H), 3.08 (d, 1H,
J ¼ 14.4, 4-CH2), 1.62e1.42 (m, 5H, 20-H, 30-H Chx), 1.21 (m, 1H, 30-H
Chx), 1.17 (d, 3H, J ¼ 7.5, 3-CH3), 0.87 (m, 2H, 40-H Chx), 0.48 (m,
1H, 30-H Chx), 0.28 (m, 1H, 30-H Chx). 13C NMR (75 MHz, CDCl3):
d
171.4, 168.9 (CO), 159.7, 158.2, 137.0, 132.1, 131.2, 130.5, 130.3,
129.1, 128.9, 128.5, 115.2, 114.8 (C Ar), 70.8 (OCH2), 65.4 (4-C), 55.4
(OMe), 54.9 (3-C), 48.0 (10-C Chx), 44.2 (1-CH2), 39.5 (4-CH2), 32.6,
32.2 (20-C Chx), 25.4 (40-C Chx), 24.9, 24.8 (30-C Chx), 10.0 (3-CH3).
MS (ES positive mode): 597.5 [Mþ1]þ. Anal (%) Calc. for
C33H36Cl2N2O4: C 66.55, H 6.09, N 4.70. Found (%): C 66.98, H 6.13,
N 4.35.
Acknowledgments
Supported by the Spanish Ministry of Science and Innovation
(SAF2009-09323). We thank the CSIC for a predoctoral I3P fellow-
ship to P.P.-F., and a postdoctoral I3P contract to M.T.A. This work
has been honored with the FAES S.A. award (within the XV edition
of the SEQT awards for young scientists) to P.P.-F.
4.11.6. (3S,4S)-4-p-(2,6-Dichlorobenzyloxy)benzyl-1-p-
methoxybenzyl-3-methyl-4-[(20-piperidin-100-il)ethyl]carbamoyl-2-
azetidinone (23d)
Syrup. Yield: 71%. Eluent: MeOH:CH2Cl2 (1:20).[
0.97, CHCl3). HPLC (Sunfire): tR ¼ 7.68 min (Gradient from 15 to
100% A in 15 min). 1H NMR (300 MHz, CDCl3):
7.38e7.25 (m, 3H,
a
]
¼ ꢂ24.6 (c
D
References
d
Cl2C6H3), 7.13 (d, 2H, J ¼ 8.5, 2-H and 6-H Ar Tyr), 7.08 (d, 2H, J ¼ 8.2,
2-H and 6-H Pmb), 6.95 (d, 2H, J ¼ 8.5, 3-H and 5-H Ar Tyr), 6.84 (d,
2H, J ¼ 8.2, 3-H and 5-H Pmb), 5.96 (broad s, 1H, NH), 5.26 (broad s,
2H, OCH2), 4.59 (d, 1H, J ¼ 15.1, 1-CH2), 3.97 (d, 1H, J ¼ 15.1, 1-CH2),
3.79 (s, 3H, OMe), 3.63 (d, 1H, J ¼ 14.6, 4-CH2), 3.27 (m, 1H, 10-H),
3.13 (d, 1H, J ¼ 14.6, 4-CH2), 3.13 (m, 1H, 3-H), 3.00 (m, 1H, 10-H),
2.31 (m, 4H, 200-H, 600-H), 2.22 (m, 1H, 20-H), 2.09 (m, 1H, 20-H), 1.52
(m, 4H, 300-H, 500-H), 1.42 (m, 2H, 400-H), 1.16 (d, 3H, J ¼ 7.5, 3-CH3).
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