Synthesis of new 2-aminoimidazolones with antiproliferative activity via base promoted amino-β-lactam rearrangement

Article abstract of DOI:10.1016/j.tet.2015.10.048

A facile and efficient transformation of amino-β-lactam guanidines to 2-aminoimidazolones is described. The base-promoted transformation proceeds in two steps, with the rearrangement of four-membered β-lactam ring to five-membered imidazolone and subsequent E1cB elimination and formation of double bond at the 4-position of imidazolone ring, which is supported with quantum chemical calculations. The benzoylaminoimidazolone and 2-aminoimidazolone products are obtained in high yields. The benzoylaminoimidazolone products show antiproliferative activity in HCT116 (colon carcinoma) and H460 (lung carcinoma) cell lines.

Full text of DOI:10.1016/j.tet.2015.10.048

Accepted Manuscript
Synthesis of new 2-aminoimidazolones with antiproliferative activity via base
promoted amino-β-lactam rearrangement
Tonko Dražić, Katarina Vazdar, Mario Vazdar, Marijana Đaković, Ana-Matea Mikecin,
Marijeta Kralj, Martina Malnar, Silva Hećimović, Ivan Habuš
PII:
S0040-4020(15)30148-4
10.1016/j.tet.2015.10.048
TET 27220
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 24 July 2015
Revised Date: 6 October 2015
Accepted Date: 16 October 2015
Please cite this article as: Dražić T, Vazdar K, Vazdar M, Đaković M, Mikecin A-M, Kralj M, Malnar M,
Hećimović S, Habuš I, Synthesis of new 2-aminoimidazolones with antiproliferative activity via base
promoted amino-β-lactam rearrangement, Tetrahedron (2015), doi: 10.1016/j.tet.2015.10.048.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis of new 2-aminoimidazolones with antiproliferative activity via
base promoted amino-β-lactam rearrangement
a,
a
a
b
Tonko Dražić, * Katarina Vazdar, Mario Vazdar, Marijana Đaković, Ana-Matea
a
a
a
a
a
Mikecin, Marijeta Kralj, Martina Malnar, Silva Hećimović, Ivan Habuš
a
Ruđer Bošković Institute, Bijenička c. 54, HR-10002 Zagreb, Croatia
b
Faculty of Science, Horvatovac 102a, HR-10002 Zagreb, Croatia
Corresponding author:
*
E-mail: tdrazic@irb.hr
Keywords: β-lactam, guanidine, 2-aminoimidazolone, rearrangement, antiproliferative
activity
ABSTRACT
A facile and efficient transformation of amino-β-lactam guanidines to 2-
aminoimidazolones is described. The base-promoted transformation proceeds in two
steps, with the rearrangement of four-membered β-lactam ring to five membered
imidazolone and subsequent E1cB elimination and formation of double bond at the 4-
position of imidazolone ring, which is supported with quantum chemical calculations.
The benzoylaminoimidazolone and 2-aminoimidazolone products are obtained in high
yields. The benzoylaminoimidazolone products show antiproliferative activity in
HCT116 (colon carcinoma) and H460 (lung carcinoma) cell lines.
1

ACCEPTED MANUSCRIPT
1
. Introdiction
β-Lactams are useful building blocks for the synthesis of a large number of
organic molecules, such as α-amino acids, β-amino acids, their derived peptides, and
1
other nitrogen containing heterocycles and open chain molecules. Nucleophilic cleavage
of the 2-azetidinone ring at the N1-C2 bond (Figure 1) usually leads to the formation of
2
β-amino acids. However, it has been shown that methanolysis of an appropriately
substituted β-lactam derivative with sodium or potassium methoxide results in the direct
formation of a five membered ring from the azetidinone ring. Using this approach,
3
4
Alcaide et al. prepared cyclic enaminones, as well as pyrrole derivatives. On the other
hand, Mehra et al. employed sodium methoxide for the preparation of
5
,6
thioxoimidazolidines, imidazolidin-2-ones and 4,5-dihydroimidazoles.
3
4
N
O 2 1 R
Figure 1. β-Lactam ring N1-C2 bond cleavage.
In our investigation we introduced a guanidine group at the C3 position of the
7
-9
azetidinone ring due to its high nucleophilicity, in order to promote the transformation
from the four membered azetidinone ring to a five membered 2-aminoimidazolone ring.
2
-Aminoimidazoles are an important group of heterocyclic compounds and their
structural motif is present in many natural alkaloids isolated mainly from marine
1
0-13
organisms.
Naturally occuring aminoimidazoles, as well as their synthetic analogues,
possess a wide range of biological activity. For example, marine natural products isolated
2

ACCEPTED MANUSCRIPT
1
4
from sponges have anti HIV, antifungal and antimicrobial activity. 2-Aminoimidazole
1
5-18
derivatives are inhibitors of β-secretase,
a key enzyme in the pathogenesis of
Alzheimer's disease. 2-Aminoimidazole amino acids are inhibitors of the Binuclear
Manganese Metalloenzyme Human Arginase I, an enzyme that catalyzes the hydrolysis
1
9
of L-arginine to L-ornithine and urea. 2-Aminoimidazolones, an important group of 2-
aminoimidazoles, together with 2-aminothiazolone derivatives possess anticancer
2
0
activity.
Methods for the synthesis of 2-aminoimidazoles, including 2-aminoimidazolones,
th
21,22
were described in the middle of the 20 century,
however the most progress was
achieved after the 1980s, when the 2-aminoimidazole and 2-aminoimidazolone
containing alkaloids from marine sponges were first discovered and isolated. Most of
these methods have been developed and applied in the total synthesis of marine
2
3-33
alkaloids.
Some of the methods include cyclocondensation of α-halocarbonyl
2
3-25
compounds with N-protected guanidine,
α-aminocarbonyl derivatives with
2
6
27
cyanamide,
or S-methylisothiourea with β-aminodiacetals.
Furthermore, 2-
2
0,29,32,33
aminoimidazolones can be obtained from 2-thiohydantoin.
Despite the existence
of numerous methods in literature for the synthesis of 2-aminoimidazoles and 2-
aminoimidazolones, direct preparation from a four membered ring has not been reported
yet.
In this paper we describe a novel method for the preparation of benzoyl-2-
aminoimidazolone and 2-aminoimidazolone derivatives (Figure 2) directly from
guanidine-β-lactams. We propose the reaction mechanism based on experimental
evidence and support it with quantum-chemical calculations. Furthermore, we determined
3

ACCEPTED MANUSCRIPT
antiproliferative activity of new compounds on two human tumor cell lines and tested a
potential inhibitory activity of new compounds against β-secretase.
H CO
H CO
3
3
O
HN
N
N
H N
2
N
R
O
N
O
R
16-20, 27, 30
21-25, 28
Figure 2. Novel benzoylaminoimidazolones 16-20, 27 and 30 and 2-aminoimidazolones 21-25
and 28.
2
2
1
. Results and discussion
.1. Synthesis. Enantiomerically pure trans-3-amino-β-lactams (3R,4R)-1a and (3S,4S)-
b (Figure 3) were synthesized applying the chiral ester enolate-imine condensation
34
35
according to the procedure described by Ojima et al. and Poljak et al. (1R,2S,5R)-(-)-
menthol was used as chiral auxiliary for the synthesis of (3R,4R)-1a enantiomer, whereas
for the synthesis of (3S,4S)-1b enantiomer (1S,2R,5S)-(+)-menthol was used.
OCH3
OCH3
H N
H N
2
2
N
N
O
O
F
F
1
a
1b
Figure 3. Structure of trans-3-amino-
β
-lactams (3R,4R)-1a and (3S,4S)-1b.
4

ACCEPTED MANUSCRIPT
Thioureas 2-8 were obtained from benzoyl isothiocyanate and corresponding amines in
6
0-90% yield. β-Lactam guanidines 9a-13 were synthesized in high yields (60-87%) from
amino-β-lactam 1a and thioreas 2-6 by applying HgCl -promoted guanylation described
2
3
6
by Cunha et.al. (Scheme 1), when 1a was reacted with thioureas 7 and 8, the expected
guanidines 14 and 15 were not detected.
OCH3
OCH3
O
H
N
H
N
H
N
O
N
H N
2
R
HgCl , Et N
2
3
+
S
HN
R
N
N
DMF, r.t., overnight
O
O
F
F
1
a
2-8
9-15
yield
2
3
4
5
6
7
8
, R = Ph
, R = 4-F-C H4
9a, R = Ph
10, R = 4-F-C H4
60%
77%
6
6
, R = 4-CH O-C H
11, R = 4-CH O-C H 78%
3
6
4
3 6 4
, R = 4-CH -C H
12, R = 4-CH -C H
4
78%
87%
/
/
3
6
4
3
6
, R = cyclohexyl
13, R = cyclohexyl
, R = 4-NC-C H4
14, R = 4-NC-C H4
6
6
, R = 4-O N-C H
15, R = 4-O N-C H
2
6
4
2 6 4
Scheme 1. Synthesis of guanidines 9-13 from β-lactam 1a and thioureas 2-8.
The treatment of guanidines 9-13 with 2.0 equivalents of K CO in methanol at room
2
3
temperature resulted in almost quantitative formation of benzoylaminoimidazolones 16-
2
0 and 2-aminoimidazolones 21-25 (Scheme 2).
5

ACCEPTED MANUSCRIPT
H CO
H CO
3
3
OCH3
O
H
N
N
R
K CO3
O
2
HN
N
+
NH
O
N
MeOH, r.t., overnight
N
H N
2
N
O
N
O
R
R
F
9
-13
16-20
yield
21-25
yield
9
a, R = Ph
0, R = 4-F-C H4
1, R = 4-CH O-C H
2, R = 4-CH -C H
16, R = Ph
17, R = 4-F-C H
18, R = 4-CH O-C H 92%
19, R = 4-CH -C H
82%
83%
21, R = Ph
22, R = 4-F-C H
23, R = 4-CH
24, R = 4-CH
25, R = cyclohexyl
17%
17%
8%
1
1
1
1
6 4
6
6
4
O-C
-C
H
6 4
3
6
4
3
6
4
3
69%
66%
3
6
H
4
1
2
5%
6%
3
6
4
3
6
4
3, R = cyclohexyl
20, R = cyclohexyl
Scheme 2. Synthesis of benzoylaminoimidazolones 16-20 and 2-aminoimidazolones 21-25.
In order to confirm the structure of obtained products and determine the stereochemistry
around the newly formed double bond, as well as the position of the benzoyl group, we
prepared single crystals of 2-aminoimidazolone 22 (Figure 4) and intermediate 26 (Figure
5
, vide infra). The newly formed double bond in 22 was of Z-configuration, whereas the
configuration around the guanidine double bond in 26 was E. Furthermore, only one
isomer can be detected in the NMR spectra of all compounds.
Figure 4. Molecular structure of 22 with the atom numbering scheme
.
6

ACCEPTED MANUSCRIPT
The formation of imidazolone derivatives by the treatment of the corresponding
guanidines with K CO in methanol occurs via N1-C2 amidolysis of β-lactam ring and
2
3
rearrangement to five-membered imidazolone ring. 2-Aminoimidazolones are formed as
a consequence of benzoyl group cleavage in the presence of base.
When thioureas 7 and 8, containing electron withdrawing groups (i.e. cyano and
nitro) were subjected to guanylation with β-lactam 1a, the expected guanidines 14 and 15
could not be isolated. In an attempt to obtain β-lactam guanidine 14, imidazolone
intermediate 26 was isolated (Scheme 3). The structure of intermediate 26 was confirmed
by X-ray crystallography (Figure 5). Guanidine 14 undergoes the rearrangement to
intermediate 26 in the given reaction conditions presumably due to electron withdrawing
effect of cyano group on the side chain phenyl. However, in the guanylation reaction
conditions, the presumed imidazolone product 27 was not formed. When intermediate 26
was treated with K CO in methanol, final products 27 and 28 were obtained (90% and
2
3
~1%, respectively). Subjecting intermediate 26 to purification by silica gel column
chromatography also led to the formation of product 27, but then no product 28 was
detected.
7

ACCEPTED MANUSCRIPT
3
H CO
OCH
3
OCH
3
H
H
N
H
N
H
N
O
H
O
O
HN
H
2
N
O
H
HgCl
2
, Et
3
N
N
N
N
F
+
S
N
O
N
CN
DMF, r.t., overnight
NH
O
N
NC
F
F
NC
26 (27%)
1
a
7
14
H
3
CO
H CO
3
H CO
3
H
H
N
O
O
HN
HN
N
K
2
CO
3
H
F
+
N
N
2
H N
N
O
N
O
N
O
MeOH, r.t., overnight
NC
NC
27 (85%)
NC
28 (∼ 1%)
2
6
Scheme 3. Formation of intermediate 26 from amino-β-lactam 1a and thiourea 7 and its
subsequent transformation to final products 27 and 28.
Figure 5. Molecular structure of 26 with the atom numbering scheme.
Guanylation reaction of thiourea 8 and β-lactam 1a afforded a mixture of several
compounds including the final product 24 and presumably five-membered ring
1
intermediate 29 (detected by H NMR spectroscopy) (Scheme 4). Guanidine 15 was not
8

ACCEPTED MANUSCRIPT
detected upon the reaction completion and pure imidazolone intermediate 29 could not be
isolated and fully characterized. The treatment of the mixture obtained in the guanylation
reaction with K CO resulted in the formation of benzoylaminoimidazolone 30 in 62%
2
3
yield. However, the expected corresponding deprotected 2-aminoimidazolone was not
detected.
OCH3
OCH3
H
N
H
N
O
O
H N
H
N
2
HgCl , Et N
N
2
3
+
S
N
NO
2
DMF, r.t., overnight
NH
O
N
O
O N
2
F
F
1
a
8
15
H CO
H CO
3
3
H
O
O
N
HN
N
HN
N
H H
2
K CO3
F
N
N
O
O
MeOH, r.t., overnight
O N
O N
2
2
2
9
30 (62%)
Scheme 4. Synthesis of benzoylaminoimidazolone 30.
The influence of K CO ratio on the formation of benzoylaminoimidazolone and
2
3
2
-aminoimidazolone products was further investigated. We wanted to determine if
reaction required stoichiometric base, as well as if the amount of base influences the ratio
of benzoyl and hydrolyzed product. For this purpose guanidine 9a was treated with
2 3
different amounts of K CO (0.1-20 equiv) in MeOH overnight at room temperature
(
Table 1). When the amount of K CO was 0.5 equivalent or higher, the product ratio and
2
3
9

ACCEPTED MANUSCRIPT
2 3
the reaction time remained unchanged. However, when 0.1 equivalent of K CO was
used, the reaction proceeded much slower giving benzoylaminoimidazolone 16 in lower
yield (75%) and 2-aminoimidazolone 21 was not detected (Table 1). Despite the lower
reaction rate when 0.1 equivalent of K CO was applied, intermediate 31 (Scheme 5, vide
2
3
infra) could not be detected. These results show that K CO can act in a catalytic as well
2
3
as stoichiometric quantity and the ratio of final products remains unchanged when the
amount of K CO is above 0.1 equivalent.
2
3
a
2 3
Table 1. The influence of K CO amount on the transformation reaction of guanidine 9a
OCH
3
OCH
3
O
H
N
N
N
N
2
H N
N
O
O
K CO3
2
16
21
(
equiv)
yield (%)
yield (%)
b
0
.1
75
88
92
90
‐
10
7
0
.5
1
2
0
8
a
The transformation reaction of guanidine 9a (30 mg, 0.06 mmol) proceeded in the presence of indicated
amount of K CO in MeOH overnight at r.t.
2
3
b
reaction proceeded for 4 days
2 3
In order to further elucidate the role of K CO , we used acetonitrile as a solvent
instead of methanol to exclude formation of potassium methoxide. The reaction was
carried out with β-lactam 1a and thiourea 2 in acetonitrile following the general
procedure for the preparation of benzoylaminoimidazolones and gave a mixture of
1
0

ACCEPTED MANUSCRIPT
intermediate 31 (22%) and product 16 (41%) (Scheme 5). Intermediate 31 was isolated as
a mixture of two isomers which could not be separated and their configuration could not
be unequivocally determined.
H CO
3
H CO
3
OCH
3
O
N
H
N
O
O
NH
HN
K CO
HN
N
2
3
HN
F
+
N
N
N
CH
3
CN, r.t., overnight
O
N
O
O
F
9a
31 (22%)
16 (41%)
Scheme 5. Formation of intermediate 31 and final product 16 in CH CN.
3
To exclude the possibility that the stereochemistry of the starting β-lactam
guanidine influences the reaction course, or intermediate and product structures, we
synthesized guanidine 9b from (3S,4S)- enantiomer of amino-β-lactam (1b) (Figure 3)
2 3
and subjected it to K CO promoted transformation. The reaction resulted in the
formation of benzoylaminoimidazolone 16 and 2-aminoimidazolone 21 with identical
yields as in the reaction with 9a (82% and 17%, respectively). The above results strongly
suggest that the transformation reaction of β-lactam guanidine to five-membered
imidazolones is independent of β-lactam, imidazolone or side chain stereochemistry.
2
.2. Reaction Mechanism. In order to further understand the β-lactam reactivity and to
elucidate the formation of described products, we propose the reaction mechanism shown
in the Scheme 6. To support the idea, we performed quantum chemical calculations with
model compounds denoted as M-1-M-7, M-3’-M-7’, M-TS , M-TS-isomerization, M-
1
1
1

ACCEPTED MANUSCRIPT
TS
2
and M-TS
2
’ (see Computational Details). Gibbs free energy differences, along with
all calculated energies and Gibbs free energy data, are given in Table 2. All data
correspond to experimental conditions where reactions are performed in methanol at 298
K.
F
H
3
C
H3C
O
O
H
3
C H
N
H
N
NH
O
CH
3
N
HN
CH
O
O
N
3
HN
CH
HN
3
H
O
F
NH
N-
H
3
C
N
H
H
3
C
N
N
N
Ph
Ph
N
O
N
O
Ph
Ph
base
M3'
F
F
M3
M1
M2
H+
+H+
+
F
F
F
base
HN
H
2
N
3
H C H
HN
H
N
O
CH
O
CH
3
H
O
O
HN
HN
3
HN
HN
CH
3
H
O
H
H
F
H
3
C
N
H
3
C
N
H
H
3
C
N
N
O
N
O
3
H C
N
N
N
O
Ph
Ph
Ph
base
Ph
M6
M5
M4
M4'
NH
F
2
O
CH
3
HN
+
H
3
C
N
N
O
H
3
C H
O
H
3
C H
O
Ph
O
N
O
N
HN
HN
H
H
M7
F
F
H
3
C
N
H
3
C
N
N
N
Ph
Ph
M6'
M5'
H
3
C
NH
F
2
O
HN
+
H
3
C
N
N
O
Ph
M7'
Scheme 6. Proposed transformation of model β-lactam ring into a five-membered ring and
subsequent elimination of aryl-amine group.
1
2

ACCEPTED MANUSCRIPT
Table 2. Calculated total electronic energies, correction to Gibbs free energies, Gibbs
free energies (in hartrees), Gibbs free energy differences and energy barriers (in
-1
kcal mol ) at 298 K for compounds M-1-M-7, M-3’-M-7’, M-TS , M-
1
TSisomerization, M-TS , M-TS ’ along the proposed reaction path.
2
2
Energy
a
a
b,c
B3LYP/6-31+G(d) MP2/6-311++G(d,p)
Gcorr
G
∆G
c
Barrier
0.0
M-1
M-2
−1204.859021
−1204.383361
−1204.349293
−1204.370246
−1204.359731
−1204.370532
−1204.880454
−1204.877966
−1204.394982
−1204.395988
−1204.854220
−1204.856162
−1204.851183
−1204.853322
−1204.871200
−1204.874244
−1201.794121
−1201.319888
−1201.295367
−1201.311973
−1201.299531
−1201.311115
−1201.823550
−1201.818676
−1201.334172
−1201.335011
−1201.794624
−1201.796656
−1201.784193
−1201.786708
−1201.795221
−1201.798497
0.298585 −1201.495536
0.0
0.284546 −1201.035342 288.8 288.8
0.286919 −1201.008448 305.7 16.9
0.285712 −1201.026261 294.5 −11.2
M-TS1
M-3
M-TSisomerisation
M-3’
0.287750 −1201.011781 303.6
0.285285 −1201.025831 294.7
9.1
−8.8
M-4
0.300704 −1201.522846 −17.1 −311.6
0.300380 −1201.518296 −14.3 −309.0
0.286199 −1201.047973 280.9 298.0
0.286580 −1201.048431 280.6 294.8
M-4’
M-5
M-5’
M-6
0.302231 −1201.492393
0.302440 −1201.494216
0.298953 −1201.485240
0.299327 −1201.487381
2.0
0.8
6.5
5.1
−278.9
−279.7
4.5
M-6’
M-TS2
M-TS2’
M-7
4.3
0.285881 −1201.509340 −8.7 −15.1
0.286803 −1201.511694 −10.1 −15.3
M-7’
a
Calculated at the B3LYP/6-31+G(d) optimized geometry
b
c
With respect to reactant 1.
Calculated as the difference between Gibbs free energies for each step along the reaction path.
In the first step, model guanidine β-lactam M-1 is deprotonated by a base forming anion
M-2. The anion M-2 undergoes an intramolecular nucleophilic attack of the guanidinium
nitrogen atom toward carbonyl group of the β-lactam ring forming five-membered ring in
1
3

ACCEPTED MANUSCRIPT
imidazolone anion M-3. Model quantum chemical calculations suggest that the reaction
occurs via a bicyclic transition state M-TS presented in Figure 6.
1
Figure 6. Optimized structures of anion M-2, transition state M-TS and imidazolone anion M-3
1
obtained at the SMD/B3LYP/6-31+G(d) level of theory with methanol as solvent.
Figure 6 shows formation of the five-membered ring and simultaneous cleavage of the
1
four-membered β-lactam ring via the transition state M-TS . Inspection of the selected C-
N bonds reveals a shortening of the distance between the guanidinium nitrogen atom and
the carbon atom of the β-lactam carbonyl group from 2.928 Å to 1.386 Å and a
simultaneous cleavage of the corresponding C-N bond of the β-lactam ring from 1.368 Å
to 2.881 Å via the transition state M-TS . The free energy activation barrier for the
1
-1
-1
reaction is 16.9 kcal mol whereas the barrier for the reverse reaction is 11.2 kcal mol .
It has been shown that β-lactam ring could also be specifically opened with methoxide
2 3
(formed in the mixture of K CO and methanol) to give methyl ester prior to the ring
5
,6
rearrangement. However, this pathway could not be confirmed in our model conditions
and seems unlikely. This is further corroborated by experimental modifications which
demonstrate the formation of the same product regardless of the base used (i.e. Et N and
3
K CO ).
2
3
1
4

ACCEPTED MANUSCRIPT
Figure 7. Optimized structures of imidazolone anion M-3, transition state M-TSisomerization and
isomeric imidazolone anion M-3’ obtained at the SMD/B3LYP/6-31+G(d) level of theory with
methanol as solvent.
By monitoring the rotation around the C2-C3 bond of the imidazolone anion M-3 (C1-C4
dihedral angle −78.1˚) it is possible to obtain a stable isomeric structure of imidazolone
M-3’ (C1-C4 dihedral angle 54.7˚), Figure 7. The isomerization occurs via the transition
state M-TSisomerization. The free energy activation barrier for the isomerization reaction is
-1
-1
9
.1 kcal mol whereas the barrier for the reverse reaction is 8.8 kcal mol . The
-1
imidazolone M-3 is only 0.2 kcal mol more stable than the isomeric imidazolone M-
3
’and so is safe to assume that both of the structures are involved in the reaction pathway.
The second step of the reaction can proceed from both imidazolone anion M-3 as well as
the isomeric form M-3’. The imidazolone anions M-3 and M-3’ can be protonated to
form intermediate M-4 and M-4’, respectively. The intermediate M-4 is by -17.1 kcal
-1
-1
mol more stable than the reactant M-1 while M-4’ is by 2.8 kcal mol less stable than
M-4. Deprotonation of intermediate M-4 and isomer M-4’ at carbon atom adjacent to the
carbonyl group (Scheme 6) leads to formation of anion M-5 and M-5’, respectively. In
experimental conditions when the reaction is carried out in the presence of strong base,
1
5

ACCEPTED MANUSCRIPT
such as methoxide, deprotonation of intermediate compound (corresponding to model
intermediate M-4 and M-4’) immediately follows the first step and the five-membered
ring intermediate cannot be isolated. However, in the presence of a weaker base, such as
K CO in acetonitrile or Et N, the reaction equilibrium is shifted toward the intermediate
2
3
3
(corresponding to model intermediate M-4 and M-4’) and resulting in the lower amount
of required anion (corresponding to model anion M-5 and M-5’). As an experimental
confirmation compound 26 (corresponding to model intermediate M-4’) was isolated
from the reaction mixture and the structure confirmed by X-ray crystallography (Figure
5
).
3
7,38
Following the E1cB elimination mechanism
we expected the direct
subsequent elimination of the aryl amine group from model anion M-5 and anion M-5’.
However, this step is computationally not observed and elimination of aryl amine group
occurs only after protonation of anion M-5 and anion M-5’ at the nitrogen atom of the
amine group through intermediate M-6 and M-6’, respectively. The elimination reaction
then proceeds via the transition state M-TS
(Figure 8) in case of anion M-5 and via the transition state M-TS2’ giving product
complex M-7’ (Figure 9) in case of anion M5’.
2
leading to formation of product complex M-
7
1
6

ACCEPTED MANUSCRIPT
2
Figure 8. Optimized structures of intermediate M-6, transition state M-TS and product complex
M-7 obtained at the SMD/B3LYP/6-31+G(d) level of theory with methanol as solvent
.
Figure 9. Optimized structures of intermediate M-6, transition state M-TS
2
’ and product complex
M-7 obtained at the SMD/B3LYP/6-31+G(d) level of theory with methanol as solvent
.
Figure 8 shows cleavage of C-N bond of the leaving aryl amine group by lengthening of
C-N bond from 1.601 Å in intermediate M-6 to 4.402 Å in the final product complex M-
7
via transition state M-TS . Product complex M-7 is stabilized by weak van der Waals
2
interactions (as indicated by a very long C-N distance) and it readily dissociates giving
final reaction products. The same process can be observed in the case of intermediate M-
6
’ where the cleavage of C-N bond of the leaving aryl amine group occurs by lengthening
of the bond from 1.603 Å in intermediate M-6’ to 4.841 Å in the final product complex
M-7’ via transition state M-TS ’, Figure 9. The final products are isomeric forms which
2
differ in the orientation of substituents around the newly formed double bond. The
1
7

ACCEPTED MANUSCRIPT
-1
product M-7’ is by 1.4 kcal mol more stable then isomer M-7 which can account for the
fact that only one isomer can be detected in the reaction mixture. The overall schematic
representation of the reaction mechanism is shown in Figure 10.
-1
Figure 10. Relative Gibbs free energy differences in kcal mol obtained at the SMD/MP2/6-
3
11++G(d,p)//B3LYP/6-31+G(d) level of theory with methanol as solvent.
2
.3. Antiproliferative activity.
Seven novel benzoylaminoimidazolones and six 2-
aminoimidazolones were tested for their antiproliferative activity on HCT116 (colon
carcinoma) and H460 (lung carcinoma) cell lines (Table 3). Some of the tested
benzoylaminoimidazolones (27, 30) were poorly soluble in DMSO and therefore, their
results should be interpreted with caution. Most of the benzoylaminoimidazolones
exhibited high activity with IC values in the micromolar range, which is comparable to
5
0
IC values of cisplatin (IC 10 and 1 ꢀM, respectively) and sorafenib (IC 3 and 3 ꢀM,
5
0
50
50
respectively), drugs commonly used in clinics. The most active compound was 17 with
IC50 1 and 2 ꢀM in HCT116 and H460 cell line, respectively. Interestingly,
1
8

ACCEPTED MANUSCRIPT
benzoylaminoimidazolone 20 with cyclohexyl group at the 1- position of the imidazolone
ring exhibited lower activity (IC50 19 and 32 ꢀM, respectively) than
benzoylaminoimidazolones containing aryl substituents. On the other hand, 2-
aminoimidazolones had on average 10-fold higher IC values. 2-Aminoimidazolone 24
5
0
was the most active with IC value 26 ꢀM. Compounds 22, 23 and 28 had IC values
5
0
50
higher than 100 ꢀM in H460 cells and were considered inactive. Therefore, it seems that
benzoyl substituent at 2-amino group is neccessary for high antiproliferative activity of
tested aminoimidazolone compounds.
Table 3. IC50 values (ꢀM) of benzoylaminoimidazolones 16-20 and 2-
aminoimidazolones 21-25.
IC50 (ꢀM)
Compound
HCT116
H460
1
1
1
1
2
6
7
8
9
0
4 ± 2.6
1 ± 0.9
8 ± 2.3
3 ± 0.5
19 ± 12.6
7 ± 1.9
2 ± 0.0
6 ± 0.9
6 ± 2.1
32 ± 7.8
a
a
2
7
13 ± 8.5
17 ± 8.8
a
a
3
2
2
2
2
2
2
0
1
2
3
4
5
8
4 ± 23.1
32 ± 3.1
30 ± 7.3
35 ± 10.5
57 ± 16.0
26 ± 4.8
35± 5.5
41 ± 23.1
10 ± 2.0
3 ± 0.8
62 ± 4.8
>100
>100
60 ± 23.6
26 ± 2.0
>100
Cisplatin
Sorafenib
1 ± 0.1
3 ± 0.8
IC : the concentration that causes 50% growth inhibition.
5
0
a
The compounds precipitated in DMSO
1
9

ACCEPTED MANUSCRIPT
2
.4. In vitro inhibition of β-secretase activity.
Since 2-aminoimidazoles are known
1
5-18
β-secretase inhibitors,
we tested in vitro β-secretase inhibitory activity of three novel
2
-aminoimidazolones 21, 22 and 23. Chinese hamster ovary (CHO) cell line transiently
transfected with amyloid precursor protein (APP) was used as a cell model. APP and
sAPPβ levels were monitored after 24 h treatment by western blot analysis. While the
known β–secretase inhibitor IV (C3) caused a complete loss in sAPPβ generation (due to
β-secretase inhibition), tested compounds failed to show β-secretase inhibitory activity
(
Figure S3, Supporting material). Therefore, other 2-aminoimidazolones were not further
tested. Benzoylaminoimidazolones were also not tested due to their high antiproliferative
activity.
3
. Conclusions
In conclusion, we have developed a facile and practical method for the synthesis of
biologically active aminoimidazolones via the direct base-promoted transformation of β-
lactam to imidazole ring. By using quantum chemical calculations, we showed that the
reaction involves rearrangement of β-lactam guanidines followed by E1cB elimination at
the 4- position of imidazole. New aminoimidazolones show strong antiproliferative
activity, which is highly structure dependent.
4
4
. Experimental section
.1. General methods. Melting points were determined on a Reichert Thermovar 7905
apparatus and are uncorrected. The IR spectra were recorded on a PerkinElmer Spectrum
RX I FT-IR System spectrometer (KBr pellets technique) (PerkinElmer Instruments,
2
0

ACCEPTED MANUSCRIPT
1
13
Norwalk, CT, USA). The H and C NMR spectra (in CDCl
3
and DMSO-d
6
at RT) were
recorded on a Bruker AV 300 and/or AV 600 spectrometer (Bruker BioSpin GmbH.,
Rheinstetten, Germany), δ is expressed in ppm relative to tetramethylsilane as an internal
reference. Microanalysis was performed on a PE 2400 Series II CHNS/O Analyzer
(PerkinElmer Instruments, Shelton, CT, USA). Samples for HR-MS analysis were
suspended in 5 µl of THAP/DAC matrix and 1 µl was spotted onto a MALDI plate. Mass
spectra were obtained on a matrix-assisted laser desorption/ionization-time-of-flight
MALDI-TOF/TOF mass spectrometer (4800 Plus MALDI-TOF/TOF Analyzer, Applied
Biosystems, Foster City, CA, USA) equipped with Nd:YAG laser operating at 355 nm
with firing rate 200 Hz in the positive ion reflector mode. 1600 shots per spectrum were
taken covering mass range 100 – 1000 Da, focus mass 500 Da and delay time 100 ns.
CCDC 1045578-1045579 contain the supplementary crystallographic data for
this paper, which can be obtained free of charge via www.ccdc.cam.uk/data_request/cif
[Cambridge Crystallographic Data Centre (CCDC), 12 Union Road, Cambridge CB2
1
EZ, UK; Fax: ++44 1223 336033; email: data_request@ccdc.cam.ac.uk]. Structure
factor tables are available from the authors. Selected bond distances, angles, and details
on hydrogen bond geometry are presented in tables S2, S3, respectively (Supporting
material).
4
4
.2. Synthesis of β-lactams 1a and 1b.
.2.1. (3R,4R)-3-Amino-1-(4-fluorophenyl)-4-(4-methoxyphenyl)-2-azetidinone (1a).
Amino-β-lactam 1a was synthesized according to the procedure described by Ojima et
34
35
al. and Poljak et al. (1R,2S,5R)-(−)-menthol was used as chiral auxiliary. Crude
2
1

ACCEPTED MANUSCRIPT
product was purified by a silica gel column chromatography (EtOAc) and obtained as
2
0
white crystals (1.09 g, 53%). [α]D −20 (c 10 mg/mL EtOAc); mp 78-79 °C; FT-IR
-1
1
(
KBr) cm : 2959, 2930, 1734, 1609, 1508, 1388, 1246, 1027, 835; H NMR (300 MHz,
CDCl ): 7.22-7.27 (m, 4H, Ar-H), 6.88-6.95 (m, 4H, Ar-H), 4.59 (d, J = 2.1 Hz, 1H, C3,
3
β-lactam), 4.04 (d, J = 2.1 Hz, 1H, C4, β-lactam), 3.80 (s, 3H, OCH ), 1.76 (s, 2H, NH );
3
2
1
3
C NMR (75 MHz, CDCl ): 168.0 (C=O, β-lactam); 160.0 (4-OCH -C H ), 159.2 (d, J =
3
3
6
4
2
43.9 Hz, 4-F-C
OCH -C
4-OCH
MALDI-TOF/TOF) m/z: [M + H ] Anal. Calcd for C H FN O 287.1190; Found
6
H
4
), 133.8 (d, J = 2.7 Hz, 4-F-C
6
H
4
), 128.5 (4-OCH
3
-C
6
H
4
), 127.3 (4-
), 114.7
); HRMS
3
6
H
4
), 119.0 (d, J = 7.8 Hz, 4-F-C
6
H
4
), 116.0 (d, J = 22.8 Hz, 4-F-C
6
H
4
(
3
-C
6
H
4
), 70.2 (C3, β-lactam), 66.6 (C4, β-lactam), 55.5 (OCH
3
+
(
1
6
16
2
2
2
4
87.1199.
.2.2. (3S,4S)-3-amino-1-(4-fluorophenyl)-4-(4-methoxyphenyl)-2-azetidinone (1b).
Amino-β-lactam 1b was synthesized according to the procedure for 1a. (1S,2R,5S)-(+)-
menthol was used as chiral auxiliary. The product was obtained as white crystals (779
2
D
0
-1
mg, 61%). [α]
+20 (c 10 mg/mL EtOAc); mp 78-79 °C; FT-IR (KBr) cm : 2959,
1
2
930, 1734, 1609, 1508, 1388, 1246, 1027, 835; H NMR (300 MHz, CDCl
3
): 7.22-7.27
(m, 4H, Ar-H), 6.88-6.95 (m, 4H, Ar-H), 4.59 (d, J = 2.1 Hz, 1H, C3, β-lactam), 4.04 (d,
1
3
J = 2.1 Hz, 1H, C4, β-lactam), 3.80 (s, 3H, OCH ), 1.76 (s, 2H, NH ); C NMR (75
3
2
MHz, CDCl ): 168.0 (C=O, β-lactam), 160.0 (4-OCH -C H ), 159.2 (d, J = 243.9 Hz, 4-
3
3
6
4
F-C H ), 133.8 (d, J = 2.7 Hz, 4-F-C H ), 128.5 (4-OCH -C H ), 127.3 (4-OCH -C H ),
6
4
6
4
3
6
4
3
6
4
1
7
19.0 (d, J = 7.8 Hz, 4-F-C H ), 116.0 (d, J = 22.8 Hz, 4-F-C H ), 114.7 (4-OCH -C H ),
6 4 6 4 3 6 4
3
0.2 (C3, β-lactam), 66.6 (C4, β-lactam), 55.5 (OCH ); HRMS (MALDI-TOF/TOF) m/z:
+
[
M + H ] Anal. Calcd for C16
H16FN
2
O
2
287.1190; Found 287.1199.
2
2

ACCEPTED MANUSCRIPT
4
.3. General procedure for the preparation of thioureas 2-8. Benzoyl isothiocyanate
(1.0 equiv) was added to a solution of a corresponding amine (1.0 equiv) in acetonitrile (6
mL). The reaction proceeded overnight with stirring at room temperature, after which the
reaction mixture was evaporated to dryness and the crude product purified by silica gel
column chromatography or recrystallization.
4
.3.1. N-(Phenylcarbamothioyl)benzamide (2). Isolated as white crystals (489 mg,
7
3%) after silica gel column chromatography (hexane/EtOAc 5:1). mp 140-141 °C; FT-
-
1
1
IR (KBr) cm : 1670, 1606, 1560, 1540, 1361, 1256, 1146; H NMR (300 MHz, CDCl
2.57 (bs, 1H, NH), 9.08 (bs, 1H, NH), 7.87-7.90 (m, 2H, Ar-H), 7.72 (d, J = 8.0 Hz, 2H,
Ar-H), 7.62-7.67 (m, 1H, Ar-H), 7.53 (t, J = 7.4 Hz, 2H, Ar-H), 7.42 (t, J = 7.8 Hz, 2H,
3
):
1
1
3
Ar-H), 7.28 (t, J = 7.7 Hz, 1H, Ar-H); C NMR (75 MHz, CDCl ): 178.5 (C=S), 167.1
3
(
C=O), 137. 8 (C H ), 133.9 (C H ), 131.8 (C H ), 129.4 (C H ), 129.0 (C H ), 127.6
6 5 6 5 6 5 6 5 6 5
(
C H ), 127.0 (C H ), 124.2 (C H ); Anal. Calcd for C H N OS: C, 65.60; H, 4.72; N,
6 5 6 5 6 5 14 12 2
1
4
0.93. Found: C, 65.23; H, 4.90; N, 10.92.
.3.2. N-(4-Fluorophenylcarbamothioyl)benzamide (3). Isolated as yellow crystals
(500 mg, 70%) after silica gel column chromatography (hexane/EtOAc 5:1). mp 120-121
-1
1
°
C; FT-IR (KBr) cm : 3225, 3133, 3035, 1670, 1508, 1329, 1260, 1153, 838, 805; H
NMR (300 MHz, CDCl ): 12.50 (bs, 1H, NH), 9.07 (bs, 1H, NH), 7.87-7.90 (m, 2H, Ar-
3
H), 7.64-7.68 (m, 3H, Ar-H), 7.55 (t, J = 7.5 Hz, 2H, Ar-H), 7.11 (t, J = 8.6 Hz, 2H, Ar-
1
3
H); C NMR (75 MHz, CDCl ): 178.9 (C=S), 167.1 (C=O), 161.0 (d, J = 247.1 Hz, 4-F-
3
C H ), 133.8 (C H ), 133.7 (d, J = 3.0 Hz, 4-F-C H ), 131.6 (C H ), 129.2 (C H ), 127.6
6
4
6
5
6
4
6
5
6
5
(C
6
H
5
), 126.3 (d, J = 8.4 Hz, 4-F-C
6 4 6 4
H ), 115.8 (d, J = 22.9 Hz, 4-F-C H ); Anal. Calcd
for C14H11FN
2
OS: C, 61.30; H, 4.04; N, 10.21. Found: C, 61.12; H, 4.42; N, 10.22.
2
3

ACCEPTED MANUSCRIPT
4
.3.3. N-(4-Methoxyphenylcarbamothioyl)benzamide (4). Isolated as white crystals
(452 mg, 60%) after silica gel column chromatography (hexane/acetone 2:1). mp 148-149
-1
°
C; FT-IR (KBr) cm : 3229, 3032, 1668, 1535, 1510, 1338, 1270, 1248, 1154, 1027,
1
8
2
34; H NMR (300 MHz, CDCl ): 12.39 (bs, 1H, NH), 9.07 (bs, 1H, NH), 7.88-7.89 (m,
3
H, Ar-H), 7.51-7.67 (m, 5H, Ar-H), 6.93 (d, J = 8.8 Hz, 2H, Ar-H), 3.82 (s, 3H, OCH3);
1
3
C NMR (75 MHz, CDCl ): 178.7 (C=S), 167.0 (C=O), 158.4 (4-OCH -C H ), 133.8
3
3
6
4
(
C
6
H
5
), 131.9 (C
-C
2.92; H, 4.93; N, 9.78. Found: C, 62.71; H, 5.23; N, 9.99.
.3.4. N-(4-Methylphenylcarbamothioyl)benzamide (5). Isolated as white crystals (632
6
H
5
), 130.7 (4-OCH
3
-C
6
H
4
), 129.4 (C
6
H
5
), 127.6 (C
6
H
5
), 125.9 (4-
OCH
3
6
H
4
), 114.3 (4-OCH
3
-C
6
H
4
), 55.6 (OCH
3
); Anal. Calcd for C15
H
14
N
2
O
2
S: C,
6
4
mg, 90%) after silica gel column chromatography (hexane/EtOAc/CH Cl 5:1:1). mp
2
2
-1
1
1
46-147 °C; FT-IR (KBr) cm : 3467, 1670, 1599, 1508, 1354, 1255, 1147, 816; H NMR
(
300 MHz, CDCl ): 12.46 (bs, 1H, NH), 9.05 (bs, 1H, NH), 7.87-7.90 (m, 2H, Ar-H),
3
7
.62-7.67 (m, 1H, Ar-H), 7.51-7.58 (m, 4H, Ar-H), 7.20-7.25 (m, 2H, Ar-H), 2.36 (s, 3H,
1
3
CH
3
); C NMR (75 MHz, CDCl
3
): 178.6 (C=S), 167.0 (C=O), 137.0 (4-CH
3
-C
6
H
4
5
),
),
1
1
35.2 (4-CH
3
-C ), 133.9 (C ), 131.9 (C
6
H
4
6
H
5
6
H
5
), 129.7 (4-CH -C ), 129.4 (C
3
6 4
H
6
H
27.6 (C H ), 124.3 (4-CH -C H ), 21.2 (CH ); Anal. Calcd for C H N OS: C, 66.64;
6
5
3
6
4
3
15 14
2
H, 5.22; N, 10.36. Found: C, 66.58; H, 5.62; N, 10.53.
4
.3.5. N-(Cyclohexylcarbamothioyl)benzamide (6). Isolated as white crystals (579 mg,
8
5%) after silica gel column chromatography (hexane/EtOAc 5:1). mp 61-62 °C; FT-IR
-
1
1
(
KBr) cm : 3170, 2927, 2853, 1672, 1544, 1341, 1262, 1171; H NMR (300 MHz,
CDCl ): 10.71 (bs, 1H, NH), 8.91 (bs, 1H, NH), 7.81-7.84 (m, 2H, Ar-H), 7.59-7.64 (m,
H, Ar-H), 7.48-7.53 (m, 2H, Ar-H), 4.25-4.34 (m, 1H, cyclohexyl), 2.03-2.11 (m, 2H,
3
1
2
4

ACCEPTED MANUSCRIPT
cyclohexyl), 1.73-1.77 (m, 2H, cyclohexyl), 1.58-1.66 (m, 1H, cyclohexyl), 1.23-1.52 (m,
1
3
5
1
H, cyclohexyl); C NMR (75 MHz, CDCl ): 178.4 (C=S), 166.9 (C=O), 133.6 (C H ),
3 6 5
32.0 (C H ), 129.3 (C H ), 127.5 (C H ), 54.5 (cyclohexyl), 31.7 (cyclohexyl), 25.6
6
5
6
5
6
5
+
(
cyclohexyl), 24.5 (cyclohexyl); HRMS (MALDI-TOF/TOF) m/z: [M + H ] Calcd for
C H N OS 263.1212; Found 263.1221.
14
19
2
4
.3.6. N-(4-Cyanophenylcarbamothioyl)benzamide (7). Isolated as light yellow
crystals (582 mg, 79%) after silica gel column chromatography (hexane/EtOAc 5:1). mp
-
1
1
1
66-167 °C; FT-IR (KBr) cm : 3481, 3236, 1664, 1598, 1522, 1341, 1266, 1165, 836; H
NMR (600 MHz, CDCl ): 12.96 (bs, 1H, NH), 9.09 (bs, 1H, NH), 7.99 (d, J = 8.7 Hz,
H, Ar-H), 7.90 (d, J = 7.3 Hz, 2H, Ar-H), 7.67-7.71 (m, 3H, Ar-H), 7.57 (t, J = 7.8 Hz,
3
2
2
1
3
H, Ar-H); C NMR (150 MHz, CDCl ): 178.3 (C=S), 167.3 (C=O), 141.7 (4-CN-
3
C H ), 134.3 (4-CN-C H ), 133.1 (C H ), 131.4 (C H ), 129.5 (C H ), 127.7 (C H ),
6
4
6
4
6
5
6
5
6
5
6
5
1
6
4
23.7 (4-CN-C H ), 118.6 (4-CN-C H ), 109.9 (C≡N); Anal. Calcd for C H N OS: C,
6 4 6 4 15 11 3
4.04; H, 3.94; N, 14.94. Found: C, 64.16; H, 3.84; N, 15.01.
.3.7. N-(4-Nitrophenylcarbamothioyl)benzamide (8). Isolated as yellow crystals (691
-1
mg, 88%) after recrystallization in acetonitrile. mp 175-176 °C; FT-IR (KBr) cm : 3249,
1
1
667, 1577, 1515, 1316, 1267, 1166, 850; H NMR (300 MHz, CDCl ): 13.07 (bs, 1H,
3
NH), 9.12 (bs, 1H, NH), 8.29 (d, J = 9.1 Hz, 2H, Ar-H), 8.06 (d, J = 9.1 Hz, 2H, Ar-H),
1
3
7
1
1
.91 (d, J = 7.3 Hz, 2H, Ar-H), 7.54-7.71 (m, 3H, Ar-H); C NMR (75 MHz, CDCl ):
3
78.3 (C=S), 167.3 (C=O), 145.4 (4-NO -C H ), 143.4 (4-NO -C H ), 134.3 (C H ),
2
6
4
2
6
4
6
5
31.4 (C H ), 129.5 (C H ), 127.7 (C H ), 124.8 (4-NO -C H ), 123.3 (4-NO -C H );
6
5
6
5
6
5
2
6
4
2
6
4
Anal. Calcd for C14
3.94.
11 3 3
H N O S: C, 55.80; H, 3.68; N, 13.95. Found: C, 55.83; H, 3.84; N,
1
2
5

ACCEPTED MANUSCRIPT
4
.4. General procedure for the preparation of guanidines 9-13. Guanidines 9-13 were
36
synthesized according to the procedure described by Cunha et al. To a solution of
corresponding thiourea 2-6 (1.0 equiv) in DMF (5 mL) β-lactam 1a or 1b (1.0 equiv) and
Et N (2.0 equiv) was added, after which the reaction mixture was cooled to 0 °C and
3
HgCl (1.0 equiv) added. The reaction mixture was stirred vigorously overnight, filtered
2
and the filtrate evaporated to dryness. Deionised water was added to the filtrate and the
resulting mixture extracted with ethyl acetate (3 × 20 mL). Combined organic layers were
dried on Na
silica gel column chromatography (hexane/EtOAc 2:1) or recrystallization.
.4.1. N-((((3R,4R)-1-(4-Fluorophenyl)-4-(4-methoxyphenyl)-2-oxoazetidin-3-
yl)amino)(phenylamino)methylene)benzamide (9a). Isolated as white crystals (183 mg,
2 4
SO , filtered and evaporated. Crude products 9-13 were purified by either
4
2
0
6
0%) after recrystallization from diethy ether. [α]D +21 (c 10 mg/mL EtOAc); mp 165-
-1
1
66 °C; FT-IR (KBr) cm : 3468, 3271, 1742, 1601, 1570, 1508, 1354, 1229, 1024, 830;
1
H NMR (300 MHz, CDCl ): 12.22 (bs, 1H, NH), 7.77 (d, J = 7.4 Hz, 2H, Ar-H), 7.40-
3
7
.46 (m, 2H, Ar-H), 7.26-7.32 (m, 8H, Ar-H), 6.87-6.98 (m, 6H, Ar-H), 5.54 (bs, 1H,
NH), 5.25 (d, J = 2.1 Hz, 1H, C4, β-lactam), 4.65 (dd, J = 6.7 Hz, 2.2 Hz, 1H, C3, β-
13
lactam), 3.80 (s, 3H, OCH ); C NMR (75 MHz, CDCl ): 178.4 (N-(C=O)C H ), 164.2
3
3
6
5
(
C=O, β-lactam), 160.3 (4-OCH -C H ), 159.3 (d, J = 243.9 Hz, 4-F-C H ), 158.0 (C=N,
3 6 4 6 4
guanidine), 137.6 (C H ), 135.5 (C H ), 133.8 (d, J = 2.8 Hz, 4-F-C H ), 131.4 (C H ),
6
5
6
5
6
4
6
5
1
30.4 (C H ), 129.3 (C H ), 128.3 (4-OCH -C H ), 127.9 (4-OCH -C H ), 127.72
6 5 6 5 3 6 4 3 6 4
(
C H ), 127.65 (C H ), 125.9 (C H ), 119.2 (d, J = 7.8 Hz, 4-F-C H ), 116.1 (d, J = 22.6
6
5
6
5
6
5
6
4
Hz, 4-F-C
6
H
4
), 114.9 (4-OCH
3
-C
6 4
H ), 67.8 (C3, β-lactam), 63.4 (C4, β-lactam), 55.5
2
6

ACCEPTED MANUSCRIPT
(
OCH
3
); Anal. Calcd for C30
.15; N, 10.57.
.4.2.
4 3
H25FN O : C, 70.85; H, 4.95; N, 11.02. Found: C, 70.51; H,
5
4
N-((((3S,4S)-1-(4-Fluorophenyl)-4-(4-methoxyphenyl)-2-oxoazetidin-3-
yl)amino)(phenylamino)methylene)benzamide (9b). Isolated as white crystals (115
2
0
mg, 38%) after recrystallization from diethy ether. [α]D −21 (c 10 mg/mL EtOAc); mp
-
1
1
8
7
1
65-166 °C; FT-IR (KBr) cm : 3468, 3271, 1742, 1601, 1570, 1508, 1354, 1229, 1024,
1
30; H NMR (300 MHz, CDCl
3
): 12.22 (bs, 1H, NH), 7.77 (d, J = 7.4 Hz, 2H, Ar-H),
.40-7.46 (m, 2H, Ar-H), 7.26-7.32 (m, 8H, Ar-H), 6.87-6.98 (m, 6H, Ar-H), 5.54 (bs,
H, NH), 5.25 (d, J = 2.1 Hz, 1H, C4, β-lactam), 4.65 (dd, J = 6.7 Hz, 2.2 Hz, 1H, C3, β-
13
lactam), 3.80 (s, 3H, OCH ); C NMR (75 MHz, CDCl ): 178.4 (N-(C=O)C H ), 164.2
3
3
6
5
(
C=O, β-lactam), 160.3 (4-OCH -C H ), 159.3 (d, J = 243.9 Hz, 4-F-C H ), 158.0 (C=N,
3 6 4 6 4
guanidine), 137.6 (C H ), 135.5 (C H ), 133.8 (d, J = 2.8 Hz, 4-F-C H ), 131.4 (C H ),
6
5
6
5
6
4
6
5
1
30.4 (C H ), 129.3 (C H ), 128.3 (4-OCH -C H ), 127.9 (4-OCH -C H ), 127.72
6 5 6 5 3 6 4 3 6 4
(
C H ), 127.65 (C H ), 125.9 (C H ), 119.2 (d, J = 7.8 Hz, 4-F-C H ), 116.1 (d, J = 22.6
6
5
6
5
6
5
6
4
Hz, 4-F-C
OCH ); Anal. Calcd for C30
.15; N, 10.57.
.4.3. N-((((3R,4R)-1-(4-Fluorophenyl)-4-(4-methoxyphenyl)-2-oxoazetidin-3-
6
H
4
), 114.9 (4-OCH
3
-C
6
H
4
), 67.8 (C3, β-lactam), 63.4 (C4, β-lactam), 55.5
(
3
H25FN
4
O
3
: C, 70.85; H, 4.95; N, 11.02. Found: C, 70.51; H,
5
4
yl)amino)(4-fluorophenylamino)methylene)benzamide (10). Isolated as white crystals
2
0
(
284 mg, 77%) after silica gel column chromatography. [α]D +40 (c 10 mg/mL EtOAc);
-1
1
mp 156-157 °C; FT-IR (KBr) cm : 3468, 1740, 1560, 1508, 1355, 1227, 830; H NMR
300 MHz, CDCl ): 12.14 (bs, 1H, NH), 7.76 (d, J = 7.4 Hz, 2H, Ar-H), 7.25-7.32 (m,
H, Ar-H), 7.12 (t, J = 8.5 Hz; 2H, Ar-H), 6.88-6.98 (m, 6H, Ar-H), 5.40 (bs, 1H, NH),
(
3
7
2
7

ACCEPTED MANUSCRIPT
5
3
.24 (d, J = 2.2 Hz, 1H, C4, β-lactam), 4.65 (dd, J = 6.7 Hz, 2.3 Hz, 1H, C3, β-lactam),
1
3
.80 (s, 3H, OCH ); C NMR (75 MHz, CDCl ): 178.4 (N-(C=O)C H ), 164.3 (C=O, β-
3
3
6
5
lactam), 163.5 (d, J = 254.9 Hz, 4-F-C H ), 160.3 (4-OCH -C H ), 159.3 (d, J = 244.5
6
4
3
6
4
Hz, 4-F-C H ), 158.3 (C=N, guanidine), 137.4 (C H ), 133.7 (4-F-C H ), 131.5 (C H ),
6
4
6
5
6
4
6
5
1
31.3 (4-F-C H ), 129.3 (C H ), 128.3 (d, J = 7.1 Hz, 4-F-C H ), 128.2 (4-OCH -C H ),
6 4 6 5 6 4 3 6 4
1
27.8 (4-OCH -C H ), 127.7 (C H ), 119.1 (d, J = 7.9 Hz, 4-F-C H ), 117.3 (d, J = 23.0
3
6
4
6
5
6
4
Hz, 4-F-C
6
H
4
), 116.1 (d, J = 22.6 Hz, 4-F-C
6
H
4
), 114.9 (4-OCH
3
-C
6
H
4
), 67.7 (C3, β-
: C, 68.43; H,
lactam), 63.5 (C4, β-lactam), 55.5 (OCH
3
); Anal. Calcd for C30
.59; N, 10.64. Found: C, 68.25; H, 4.68; N, 10.86.
.4.4. N-((((3R,4R)-1-(4-Fluorophenyl)-4-(4-methoxyphenyl)-2-oxoazetidin-3-
H
24
F
2
N
4
O
3
4
4
yl)amino)(4-methoxyphenylamino)methylene)benzamide (11). Isolated as white
2
0
crystals (294 mg, 78%) after silica gel column chromatography. [α]D +34 (c 10 mg/mL
-1
EtOAc); mp 160-161 °C; FT-IR (KBr) cm : 3468, 3265, 1742, 1600, 1560, 1508, 1357,
1
1
248, 1150, 1034, 831; H NMR (300 MHz, CDCl ): 11.96 (bs, 1H, NH), 7.73 (d, J = 7.5
3
Hz, 2H, Ar-H), 7.20-7.31 (m, 7H, Ar-H), 6.86-6.98 (m, 8H, Ar-H), 5.70 (d, J = 6.0 Hz,
H, NH), 5.19 (d, J = 1.9 Hz, 1H, C4, β-lactam), 4.65 (dd, J = 7.0 Hz, 1.8 Hz, 1H, C3, β-
1
1
3
lactam), 3.80 (s, 3H, OCH ), 3.72 (s, 3H, OCH ); C NMR (75 MHz, CDCl ): 178.1 (N-
3
3
3
(
C=O)C H ), 164.5 (C=O, β-lactam), 160.2 (4-OCH -C H ), 159.3 (d, J = 244.1 Hz, 4-F-
6 5 3 6 4
C H ), 159.1 (4-OCH -C H ), 158.7 (C=N, guanidine), 137.7 (C H ), 133.7 (d, J = 2.7
6
4
3
6
4
6
5
Hz, 4-F-C H ), 131.3 (C H ), 129.2 (C H ), 128.3 (4-OCH -C H ), 127.9 (C H ), 127.8
6
4
6
5
6
5
3
6
4
6
5
(
4-OCH -C H ), 127.7 (4-OCH -C H ), 127.6 (4-OCH -C H ), 119.0 (d, J = 7.9 Hz, 4-F-
3
6
4
3
6
4
3
6
4
C
6
H
4
), 116.0 (d, J = 22.7 Hz, 4-F-C
6
H
4
3 6 4 3 6 4
), 115.5 (4-OCH -C H ), 114.8 (4-OCH -C H ),
2
8