6-Arylmethylidene Penicillin-Based Sulfone Inhibitors for Repurposing Antibiotic Efficiency in Priority Pathogens

Article abstract of DOI:10.1021/acs.jmedchem.0c00127

The ability of 6-(aryl)methylidene penicillin-based sulfones 1-7 to repurpose β-lactam antibiotics activity with bacterial species that carry carbapenem-hydrolyzing class D β-lactamases (OXA-23, OXA-24/40 and OXA-48), as well as with class A (TEM-1, CTX-M-2) and class C (CMY-2, DHA-1) enzymes, is reported. The combinations imipenem/3 and imipenem/4 restored almost completely the antibiotic efficacy in OXA-23 and OXA-24/40 carbapenemase-producing A. baumannii strains (1 μg mL^-1) and also provided good results for OXA-48 carbapenemase-producing K. pneumoniae strains (4 μg mL^-1). Compounds 2-6 in combinations with ceftazidime and ampicillin were also efficient in restoring antibiotic efficacy in E. coli strains carrying class C (CMY-2 and DHA-1) and class A (TEM-1 and CTX-M-2) β-lactamase enzymes, respectively. Kinetic and inhibition studies with the OXA-24/40 enzyme, protein mass spectrometry analysis and docking studies allowed us to gain an insight into the inhibition mechanism and the experimentally observed differences between the ligands.

Full text of DOI:10.1021/acs.jmedchem.0c00127

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Article
6-Arylmethylidene Penicillin-based Sulfone Inhibitors for
Repurposing Antibiotic Efficiency in Priority Pathogens
Diana Rodríguez, Maria Maneiro, Juan C. Vázquez-
Ucha, Alejandro Beceiro, and Concepción González-Bello
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00127 • Publication Date (Web): 20 Mar 2020
Downloaded from pubs.acs.org on March 21, 2020
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Journal of Medicinal Chemistry
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6-Arylmethylidene Penicillin-based Sulfone Inhibitors for
Repurposing Antibiotic Efficiency in Priority Pathogens
a
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b
b
Diana Rodríguez, María Maneiro, Juan C. Vázquez-Ucha, Alejandro Beceiro, and Concepción González-
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Belloa,*
aCentro Singular de Investigación en Química Biolóxica e Materiais Moleculares (CiQUS), Departamento
de Química Orgánica, Universidade de Santiago de Compostela, Jenaro de la Fuente s/n, 15782 Santiago de
Compostela, Spain.
bServicio de Microbiología do Complexo Hospitalario Universitario da Coruña (CHUAC), Instituto de
Investigación Biomédica da Coruña (INIBIC), Xubias de Arriba, 84, 15006 A Coruña, Spain.
CORRESPONDING AUTHOR ADDRESS. Prof. Concepción González-Bello, Centro Singular de
Investigación en Química Biolóxica e Materiais Moleculares (CiQUS), Departamento de Química Orgánica,
Universidade de Santiago de Compostela, calle Jenaro de la Fuente s/n, 15782 Santiago de Compostela,
Spain. FAX: +34 881 815704; Phone: +34 881 815726.
KEYWORDS: antimicrobial resistance, β-lactamase inhibitors, carbapenem-hydrolyzing class D β-
lactamase enzymes, enzyme covalent adducts, proteomic analysis.
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ABSTRACT
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The ability of 6-(aryl)methylidene penicillin-based sulfones 1−7 to repurpose β-lactam antibiotics activity
with bacterial species that carry carbapenem-hydrolyzing class D β-lactamases (OXA-23, OXA-24/40 and
OXA-48), as well as with class A (TEM-1, CTX-M-2) and class C (CMY-2, DHA-1) enzymes, is reported.
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The combinations imipenem/3 and imipenem/4 restored almost completely the antibiotic efficacy in OXA-
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3 and OXA-24/40 carbapenemase-producing A. baumannii strains (1 µg mL ), and also provided good
−
1
results for OXA-48 carbapenemase-producing K. pneumoniae strains (4 µg mL ). Compounds 2−6 in
combinations with ceftazidime and ampicillin were also efficient in restoring antibiotic efficacy in E. coli
strains carrying class C (CMY-2 and DHA-1) and class A (TEM-1 and CTX-M-2) β-lactamase enzymes,
respectively. Kinetic and inhibition studies with the OXA-24/40 enzyme, protein mass spectrometry analysis
and docking studies allowed us to gain an insight into the inhibition mechanism and the experimentally
observed differences between the ligands.
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INTRODUCTION
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The growing appearance and dissemination worldwide of superbugs is increasingly limiting dangerously the
ability of antibiotics to cure bacterial infections.1,2,3,4 As noted by the World Health Organization (WHO) in
February 2017, there is a huge concern about the lack of effective therapies against the Gram-negative
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pathogens Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacteriaceae. In this respect,
great success has been achieved with combined therapy approaches that block the most relevant resistance
mechanism in Gram-negative bacteria, i.e., the enzymatic inactivation of β-lactams by β-
lactamases.6,7,8,9,10,11,12,13 These drugs are around 70% of commonly prescribed antibiotics. At the low
concentrations generally used, β-lactamase inhibitors do not prevent bacterial growth, but when co-
administered with the antibiotic they enhance the activity of the antibiotic, thus repurposing the existing life-
saving drugs in clinical use.14,15,16,17
There are four types of β-lactamases (classes A, B, C and D). Among them, the class D enzymes, which are
also known as ‘oxacillinases (OXA)’, are very relevant because they hydrolyze penicillins and narrow-
spectrum cephalosporins.18 Some class D β-lactamases have evolved to inactivate expanded-spectrum
cephalosporins and even carbapenems [carbapenem-hydrolyzing class D enzymes (CHDLs)].19,20 Infections
caused by bacteria that produce these last two groups of enzymes are of high concern, because they are
frequently found in deadly pathogens such as multidrug-resistant Acinetobacter baumannii (e.g. OXA-23,
OXA-24/40), or Enterobacteriaceae (e.g. OXA-48).21,22 The dissemination of these enzymes is seriously
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compromising the use of carbapenems, which are often considered as the antimicrobials of last resort. The
carbapenemase activity of OXA-23 and OXA-24/40 enzymes is improved by the presence of an uncommon
and highly hydrophobic “tunnel-like” entrance to the active site and this: (i) limits the entry to only certain
ligands; and (ii) fixes the antibiotic in the productive conformation for β-lactam ring opening for a longer
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time (Figure 1). This architecture of the active site, which is achieved by the side chains of a phenylalanine
(OXA-23) or a tyrosine (OXA-24/40) and a methionine, is very rigid and remains largely unchanged during
the catalysis. The OXA-48 enzyme, in which the hydrophobic bridge of OXA-23 and OXA-24/40 enzymes
is absent, has a similar carbapenemase efficiency to that of the OXA-24/40 enzyme. In this case, the
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enzymatic efficacy is achieved by the existence of a large hydrophobic region near the active site that allows
the carbapenem to be fixed into an efficient conformation for hydrolysis (Figure 1).24
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Figure 1. Overall structures of relevant CHDL enzymes: OXA-23 from A. baumannii (PDB ID 4K0X ),
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OXA-24/40 from A. baumannii (PDB ID 3G4P ), and OXA-48 from K. pneumoniae (PDB ID 4S2P ). The
tunnel-like entrance in OXA-23 and OXA-24/40 and the hydrophobic region in OXA-48 are highlighted in
green and yellow, respectively. The position of the catalytic serine is shown in red.
The β-lactamase inhibitors clavulanic acid and the penicillin-based sulfones sulbactam and tazobactam,
which are widely employed in clinic, are ineffective against the CHDLs enzymes. More recently developed
1,6-diazabicyclo[3,2,1]octane (DBO) β-lactamase inhibitors such as avibactam, which is used in
combination with ceftazidime to treat infections caused by strains carrying class A, C and some class D β-
lactamases (OXA-48), also proved to be inefficient against OXA-23 and OXA-24/40 (Figure 2A).28
Relebactam, another DBO inhibitor that was approved in 2019 in combination with imipenem-cilastatin,
shows a similar inhibition spectrum. More recently, Durand-Réville et al.29 demonstrated that the
rigidification of the six-membered ring of avibactam with a double bond, as well as the introduction of a
methyl group, provides a new DBO β-lactamase inhibitor, namely durlobactam (phase III clinical trials in
combination with sulbactam), with enhanced inhibitory capacity against bacteria that produce OXA-24/40.
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Journal of Medicinal Chemistry
3
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As revealed by the structure of OXA-24/40 inactivated by durlobactam (PDB ID 6MPQ, 1.95 Å), the
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methyl group of the ligand interacts with the hydrophobic bridge of the active site (Figure 2B). Moreover,
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the penicillin-based sulfone 1 (LN-1-255), which was developed by Buynak, proved to have a markedly
higher efficacy than tazobactam and avibactam against relevant CHDLs in A. baumannii (the plasmid
encoded OXA-23, OXA-24/40, OXA-58, OXA-143, and OXA-235, and the chromosomally encoded OXA-
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1) and K. pneumoniae OXA-48 (Figure 2C).28,32 This compound enhances the in vitro activity of imipenem
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by between 32- and 128-fold and also has good therapeutic efficacy in vivo. The effectiveness of LN-1-255
lies in its ability to form an indolizine adduct that is resistant to hydrolysis. This aromatic moiety would be
formed after dioxothiazolidine ring opening and by nucleophilic attack of the pyridine nitrogen atom on the
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conjugated initial imine adduct (Figure 2C). The available crystallographic structures of OXA-24/40 from
A. baumannii covalently modified by this type of (2-pyridyl)methylene penicillin-based sulfone ligand (PDB
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IDs 3MBZ, 3FYZ, 3FZC and 3FV7) shows numerous favorable lipophilic interactions of the enzyme
adduct with the tunnel and its entrance, which involves residues Trp115, Leu168, Met223 and Val130
(Figure 2D). These advantageous interactions would further increase the hydrophobicity of the tunnel-like
entrance, thus limiting the approach of the water molecule for the final hydrolysis.
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Figure 2. A. Chemical structures of avibactam, relebactam and durlobactam. These DBOs have been either
approved by the FDA or are currently under clinical studies in combination with an antibiotic, which is
indicated in parenthesis. B. Detailed view of the crystal structure of OXA-24/40 from A. baumannii
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covalently modified by durlobactam (PDB ID 6MPQ ). C. Mechanism of inhibition of 6-(2-
pyridyl)alkylidene penicillin-based sulfone 1 (LN-1-255). D. Detailed view of the enzyme adduct observed
in PDB ID 3FZC32 (OXA-24/40 from A. baumannii). The relevant side chain residues are shown and
labelled. E. Target derivatives 2−7.
Given the background outlined above, and in the search for new penicillin-based sulfone inhibitors, we
became interested in exploring the effect on the inhibitory potency of 1 against the CHDL enzymes of (i) the
incorporation of substituents in the pyridine ring that would change the electron density of this moiety, and
(ii) the use of larger pyridine-based heterocycles. To this end, we report here the synthesis of six 6-
arylmethylidene penicillin-based sulfones, compounds 2−7, which contain pyridine rings substituted with a
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Journal of Medicinal Chemistry
methylpyrazoyl group, a halogen (Br, F) or methoxy groups, a thieno[2,3-c]pyridine or an isoquinoline
Figure 2E). Microbiological studies on antimicrobial susceptibility with previously studied bacterial strains
of Acinetobacter baumannii harboring OXA-23 or OXA-24/40, and Klebsiella pneumoniae carrying OXA-
8 are reported. In addition, the capacity of compounds 2−7 to restore the antibiotic efficacy in widely
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(
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distributed bacterial strains of Escherichia coli producing β-lactamases of classes A (TEM-1, CTX-M-2) and
C (CMY-2, DHA-1) are also included. CMY-2 and DHA-1 are widely distributed plasmidic AmpC
enzymes, and TEM-1 and CTX-M-2 [Extended Spectrum Beta-lactamases (ESBLs)] are spread worldwide
in pathogenic Gram-negative bacteria, including Enterobacteriae and P. aeruginosa.18,35,36 The results of the
kinetic studies on the reported analogs against OXA-24/40, the detection by mass spectrometry of the
enzyme adducts, the monitoring of the β-lactam ring opening, and docking studies, allowed us to gain an
insight into the inhibition mechanism and the experimentally observed differences between the ligands.
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RESULTS AND DISCUSSION
Synthesis of Compounds 2−7 – The synthesis of the target compounds 2−7 involved the introduction of the
pyridine moiety by a Wittig reaction between the phosphonium salts 8−11 and previously reported 3-
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isoquinolylmethylphosphonium bromide and the ketone 12 as a key step (Scheme 1). The latter compound
was prepared according to modified reported protocols from commercially available (+)-6-aminopenicillanic
acid (13).38,39,40,41
R
O
H
Cl
O
O
S
27
O
N
+
N
2
CO CHPh2
PPh X
3
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11
12
H
N
H N
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O
CO H
2
13
Scheme 1. Synthetic approach for compounds 2−7.
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The synthesis of the required phosphonium salts 8, 9a−b, 10 and 11, was achieved from commercially
available pyridines 14, 18a−b, 19 and 21, respectively, as outlined in Scheme 2. Thus, phosphonium salt 8
was prepared in four steps from 2-methyl-4-bromopyridine (14) by Suzuki cross-coupling between the
bromide 14 and 1-methylpyrazole-4-boronic acid pinacol ester using Pd(PPh ) as catalyst. Oxidation of the
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methyl group at position C2 in the resulting pyridine 15 gave alcohol 16. Conversion of 16 into the chloride
7 by treatment with thionyl chloride and subsequent reaction with triphenylphosphine afforded the desired
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phosphonium salt 8. Compounds 9a-b were prepared by nucleophilic substitution of the halides 18a-b by
treatment with triphenylphosphine. Bromination of 2-methyl-5-fluoropyridine (19) with N-
bromosuccinimide in the presence of catalytic amounts of AIBN gave bromide 20, which was converted into
the phosphonium salt 10 in the same way as derivatives 9 from 18. Finally, phosphonium salt 11 was
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synthesized from previously described bromide 21 by treatment with triphenylphosphine.
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N
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N
Br
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quant.
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15 Z = H
b, 40%
c, 69%
d, 40%
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17 Z = Cl
8 Z = PPh Cl
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1R
2R
1R
2R
d
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X
N
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18a-b
9a-b
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a X = Cl; R = R = OMe, 82%
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b X = R = Br; R = H, 52%
F
F
e
N
50%
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20 Z = Br
10 Z = PPh Br
d, 89%
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d
N
N
56%
S
S
Br
PPh Br
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21
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Scheme 2. Synthesis of phosphonium salts 8−11. Reagents and conditions. (a) Pd(PPh ) , 1-methylpyrazole-
3 4
4
-boronic acid pinacol ester, dioxane, K PO (aq), 85 ºC. (b) 1. Ac O, 90 ºC; 2. KOH, MeOH, 0 ºC to RT.
3 4 2
(c) SOCl , DCM, 0 ºC to RT. (d) Ph P, PhMe, Δ. (e) NBS, AIBN, CCl , Δ.
2 3 4
The synthesis of target compounds 2−7 was carried out as outlined in Scheme 3. Firstly, sulfoxide 22 was
prepared in three steps from commercially available (+)-6-aminopenicillanic acid (13) following previously
reported protocols that involved: (i) esterification of the carboxylic acid in 13 by treatment with
diphenyldiazomethane;39,40 (ii) protection of the free amino group with allyl chloroformate in pyridine; and
3
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(iii) oxidation of the sulfide moiety with peracetic acid. Sulfoxide 22 was then transformed into disulfide
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3 following the strategy described by Kamiya et al. This transformation involves a thermal sigmatropic
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rearrangement of sulfoxide 22 and subsequent trapping of the sulfenic acid intermediate with 2-
mercaptobenzothiazole. However, when this reaction was carried out in toluene, as reported, in addition to
23 the formation of the undesired thermodynamic alkene 24 was also observed. Moreover, the use of other
solvents and mixtures thereof, as well as different reaction temperatures and reaction times, afforded
complex reaction mixtures that were tedious to purify. Fortunately, the formation of the thermodynamic
alkene 24 was avoided when the reaction was carried out in dioxane under reflux and in the presence of
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
anhydrous MgSO . Under these conditions, disulfide 23 was obtained in 91% yield as the only reaction
4
product and without the need for purification. Next, the cyclization of the disulfide 23 for the modification
of the pro-R methyl group with an ester group was carried out following previously reported protocols by
treatment with silver acetate and chloroacetic acid.38 This provided a chromatographically inseparable
mixture of the kinetic (five-membered, compound 25) and thermodynamic (six-membered, compound 26)
compounds in a 2:1 ratio. Deprotection of the allylcarbamate group in the resulting cyclic compounds 25
and 26 provided a chromatographically inseparable mixture of the amines 27 and 28 in a 2:1 ratio. Buynak
et al.38,44 described the transformation of amine 27 (and its six-membered isomer 28) into the corresponding
ketone 12 (and its six-membered isomer 29) through a sequence of two reactions: (i) the formation of the
corresponding diazonium salt by treatment with isopropyl nitrite and catalytic amounts of trifluoroacetic
acid; and (ii) oxidation of the crude reaction product with propylene oxide in the presence of catalytic
amounts of rhodium octanoate. However, in our hands this protocol was poorly reproducible and in many
cases involved the loss of large quantities of product. Fortunately, we found that treatment of the amine 27
(and its six-membered isomer 28) with triflic anhydride and triethylamine and subsequent hydrolysis by
treatment with dilute HCl provided ketone 12 (and its six-membered isomer 29) in a reproducible manner. In
addition, after the Wittig reaction between ketone 12 (and 29) and the phosphonium salts 8−11 and 3-
isoquinolylmethylphosphonium bromide it was possible to isolate the desired five-membered analogs 30
from their six-membered counterparts. Under these conditions – and in contrast to previously reported
protocols – the subsequent use of mixtures of compounds was avoided in the remaining steps of the
synthesis.
1
0
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Journal of Medicinal Chemistry
Oxidation of 30 with meta-chloroperbenzoic acid followed by deprotection of the chloroacetoxy group in the
resulting sulfone 31 by treatment with thiourea and pyridine led to alcohols 32. Esterification of 32 with the
protected acid 36 using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) as a coupling agent in the
presence of catalytic amounts of 4-N,N-dimethylaminopyridine provided esters 33 in good yields. Protected
acid 36 was prepared in two steps from commercially available 3,4-dihydroxyphenylacetic acid (35).
Finally, removal of the TBDPS protecting groups in 33 by treatment with TBAF/AcOH, followed by the
elimination of the benzhydryl protecting group in the resulting catechols 34 by reaction with m-cresol and
subsequent treatment with sodium bicarbonate, satisfactorily provided the target compounds 2−7.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
S
S
O
S
N
S
N
H
H
H
H
H N
AllocHN
O
AllocHN
O
AllocHN
O
2
S
S
S
S
ac
d
N
ref. 3235
N
N
N
O
CO H
CO CHPh
CO CHPh
CO CHPh2
2
2
2
2
2
2
13
22
23
24
e
O
H
N
O
H
N
H
Cl
H
H
Cl
H
O
O
O
O
O
O
Cl
O
S
Cl
S
S
S
g
G
G
O
O
+
+
N
N
N
N
O
O
f
O
2
CO CHPh2
CO CHPh2
CO CHPh
2 2
CO CHPh2
2
2
1
2
29
25 G = Alloc
7 G = H
26 G = Alloc
28 G = H
f
2
h
OG
OG
Ar
O
Ar
Ar
O
O
O
O
O
O
H
Cl
H
O
H
Z
S
S
S
i
k
m
O
O
O
2
7
N
N
N
O
CO CHPh2
CO2CHPh2
CO CHPh
2 2
2
30
3
1 Z = COCH Cl
33 G = TBDPS
34 G = H
2
j
l
32 Z = H
N
N
2R
OG
O
1R
MeO
HO
OG
N
N
N
N
N
Ar =
S
MeO
3
3
5 G = H
6 G = TBDPS
n
1
2
a
b R = Br; R = H
d
e
f
1
2
c R = H; R = F
Scheme 3. Synthesis of compounds 2−7. Reagents, conditions and yields. (a) Ph CN , CH Cl /MeOH, RT,
2
2
2
2
8
7%. (b) ClCO Allyl, Py, CH Cl , –10 °C, 93%. (c) MeCO H, CH Cl , –5 °C, 95%. (d) 2-
2 2 2 3 2 2
1
1
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mercaptobenzothiazole, MgSO , dioxane, ∆, 91%. (e) ClCH CO H, AgOAc, AcOEt, RT. (f) Bu SnH,
4
2
2
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Pd(PPh ) (cat.), AcOH, DCM, RT, 86% from 23. (g) 1. Tf O, Et N, DCM, –78 °C→0 °C. 2. Et N, –78 °C.
3
4
2
3
3
3
6
3. HCl (0.5 M). (h) 8, 9a, 9b, 10, 11 or (3-isoquinolyl)methyltriphenylphosphonium bromide , DCM, –78
C or RT, 20–49% from a mixture of amines 27 and 28. (i) MCPBA, DCM, RT, 70–81%. (j) Thiourea, Py,
DMF, 0 °C→RT. (k) 36, EDC, DMAP, DCM, –15 °C→RT, 37–73% from 31. (l) TBAF, AcOH, THF, 0 °C,
°
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
6–88%. (m) 1. m-cresol, 50 °C. 2. NaHCO or NH HCO , 27–66%. (n) 1. TBDPSCl, Im, DMF, RT. 2.
3 4 3
LiOH (aq), THF, RT. 3. HCl (aq), 64%.
Susceptibility Studies – Seven bacterial strains of A. baumannii, K. pneumoniae, and E. coli were
employed. These strains do not have a mechanism of resistance other than that caused by the enzymatic
hydrolysis of the β-lactam antibiotic and they were selected because they are widely distributed in priority
pathogens. Specifically, in this work three bacterial strains that produce CHDL enzymes (OXA-23, OXA-
24/40 and OXA-48), two strains that harbor class A enzymes (TEM-1 and CTX-M-2) and two strains that
carry class C enzymes (CMY-2 and DHA-1) were employed. All of the bacterial strains and plasmids used
in this study are summarized in Table 1. With the exception of E. coli TG1 bacterial strains that harbor
TEM-1 and CTX-M-2, which are described herein, all of the strains were coded in previous studies.
Table 1. Bacterial strains and plasmids used in this study
Reference or
Strains
Description
source
ATCC
a
A. baumannii ATCC 17978
A. baumannii reference strain completely
sequenced. Carbapenem-susceptible strain
(genbank code CP000521.1).
A. baumannii ATCC 17978
A. baumannii ATCC 17978 strain carrying pET-
28
28
(pET-RA-KmR+OXA-24/40) RA+KmR plasmid encoding blaOXA-24/40 gene.
Carbapenem-resistant strain.
A. baumannii ATCC 17978
A. baumannii ATCC 17978 strain carrying pET-
RA+KmR plasmid encoding blaOXA-23 gene.
(pET-RA-KmR+OXA-23)
1
2
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Journal of Medicinal Chemistry
Carbapenem-resistant strain.
1
2
K. pneumoniae ΔompK35/36
CSUB10R K. pneumoniae strain without porins
OmpK35 and OmpK36. β-Lactam-resistant strain.
55
56
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
K. pneumoniae ΔompK35/36
pBGS18+OXA-48)
K. pneumoniae ΔompK35/36 strain carrying
pBGS18 plasmid encoding blaOXA-48 gene.
Increased β-lactam resistance.
(
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
E. coli TG1
E. coli reference strain completely sequenced.
Protease deficient. It is suitable for transformation
and protein expression. Antimicrobial-susceptible
strain (genbank code U00096.3).
ATCCa
E. coli TG1 (pBGS18/TEM-1) E. coli TG1 strain with cloned pBGS18 plasmid
encoding blaTEM-1 gene. Penicillin-resistant strain.
This study
This study
E. coli TG1 (pBGS18/CTX-
M-2)
E. coli TG1 strain with cloned pBGS18 plasmid
encoding blaCTX-M-2 gene. β-Lactam-resistant
strain except to carbapenems.
E. coli TG1 (pBGS18+CMY-
E. coli TG1 strain with cloned pBGS18 plasmid
encoding blaCMY-2 gene. β-Lactam-resistant strain
except to carbapenems.
57
2
)
E. coli TG1 (pBGS18+DHA-
E. coli TG1 strain with cloned pBGS18 plasmid
encoding blaDHA-1 gene. β-Lactam-resistant strain
except to carbapenems.
58
1
)
E. coli BL21
E. coli reference strain completely sequenced.
Antimicrobial-susceptible strain (genbank code
CP001509.3).
ATCCa
28
E. coli BL21 (pGEX-6p-
E. coli BL21 strain carrying pGEX-6p-1 plasmid
encoding blaOXA-24/40 gene. Strain used for
expression and purification of OXA-24/40 β-
lactamase.
1
+OXA-24/40)
Reference or
source
59
Plasmids
pBGS18
Description
Plasmid carrying E. coli replication origin.
Encodes kanamycin resistance.
pET-RA-KmR
pGEX-6P-1
Plasmid carrying A. baumannii replication origin. 60
Encodes rifampicin and kanamycin resistance.
Plasmid carrying E. coli replication origin.
Bacterial expression plasmid. GST fusion vector.
Encodes ampicillin resistance.
GE Healthcare
aATCC = American Type Culture Collection
1
3
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The susceptibilities to ampicillin, ceftazidime and imipenem with class A, C and D β-lactamases,
respectively, in the absence of and in combination with compounds 2−7 at fixed concentrations of 16 µg
−
1
mL were determined. The enhancing effects of the reported inhibitors were also compared with that
obtained with compound 1 and two well-known non-penicillin-based sulfone inhibitors, namely the DBOs
avibactam and relebactam. The microdilution method was used to determine the Minimum Inhibitory
Concentration (MIC) of each antibiotic and antibiotic/inhibitor combination, with quoted results being the
mean of three independent replicates, following CLSI criteria.45 MICs were defined as the lowest
concentration of each combination that completely inhibited visible growth on plates. The results are
summarized in Table 2.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
4
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Journal of Medicinal Chemistry
−
1
Table 2. MIC values (µg mL ) for imipenem, ceftazidime and ampicillin with A. baumannii ATCC 17978, K. pneumoniae ΔompK 35/36 and E.
coli TG1 pBGS18 carrying representative β-lactamases of classes D (OXA-23, OXA-24/40 and OXA-48), A (TEM-1 and CTX-M-2), and C
(CMY-2 and DHA-1), in the presence and absence of β-lactamase inhibitors 1−7, avibactam and relebactam.a
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
Bacterial
strains
A. baumannii
A. baumannii
K. pneumoniae
E. coli TG1 +
E. coli TG1 +
E. coli TG1 +
E. coli TG1 +
pBGS18/
DHA-1
ATCC17978 + ATCC17978 + ΔompK 35/36 + pBGS18/TEM- pBGS18/CTX- pBGS18/CMY-
pET-RA-KmR pET-RA-KmR pBGS18/OXA-
1
M-2
2
/
OXA-23
/OXA-24/40
48
IMP
64
NA
8
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
Antibioticb
IMP
8
IMP
64
16
64
0.5
1
AMP
AMP
>1024
NA
16
CTZ
32
CTZ
32
No inhibitor
>1024
Avibactamc
4
NA
4
NA
NA
Relebactamc
8
 0.25
 0.25
 0.25
 0.25
 0.25
 0.25
 0.25
2
 0.25
 0.25
 0.25
 0.25
 0.25
 0.25
 0.25
2
1c
2c
3c
4c
5c
6c
7c
0.5
1
4
2
16
8
2
16
1
1
4
2
16
1
1
4
2
32
4
8
64
64
8
2
256
256
256
2
8
2
2
2
4
Wild-type
strains without
β-lactamase
0.5
0.5
0.5
2
2
 0.25
 0.25
1
5
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a
b
c
−1
Data represent the means of three independent experiments. IMP = imipenem; AMP = ampicillin; CTZ = ceftazidime. Inhibitor concentration = 16 µg mL .
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
NA = Not applicable (avibactam shows antimicrobial activity against Enterobacteriaceae).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
1
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Journal of Medicinal Chemistry
1
2
3
The results of these studies revealed that the combination ceftazidime/compounds 2−6 restored completely
the efficacy of ceftazidime to levels similar to that of the parent compound 1 and relebactam, and provided
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
−
1
MIC values of ≤ 0.25 µg mL in E. coli producing class C (CMY-2 and DHA-1). Under similar conditions
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
the combination ceftazidime/compound 7 proved to be much less efficient, with a MIC value of 2 µg mL−1.
A similar trend was observed with the combination ampicillin/compounds 2−6 in E. coli producing TEM-1,
−
1
which recovered completely the ampicillin efficacy (MIC = 2 µg mL ) to the level of compound 1, while a
two-fold decrease in the in vitro activity was observed with the combination ampicillin/compound 7 (MIC =
−
1
4
µg mL ). Moreover, the combinations ampicillin/2 and ampicillin/3 in E. coli strains producing the CTX-
M-2 enzyme provided a 64-fold decreases in the MIC value of the antibiotic, in a similar way to relebactam
and compound 1.
The results of the susceptibility studies against bacterial strains producing CHDLs enzymes (OXA-23,
OXA-24/40 and OXA-48) revealed that combinations of imipenem with the reported compounds were in
most cases more efficient than those with avibactam and relebactam, which generally did not show a
significant antibiotic enhancing effect. The results against A. baumannii strains carrying enzymes OXA-23
and OXA-24/40 revealed that the combinations of imipenem with compounds 2, 3 and 4 restored almost
−
1
completely the activity of the antibiotic, with MIC values in all cases of 1 µg mL . For OXA-48-producing
K. pneumoniae strains the reported compounds are less efficient than for the A. baumannii strains producing
OXA-23 and OXA-24/40. The best in vitro results were obtained with the combinations of imipenem with
−
1
compounds 3 and 4, with MIC values in both cases of 4 µg mL −i.e. similar to that of the parent compound
1
.
Kinetics and Inhibition Studies – The inhibitory properties of compounds 2–7 against OXA-24/40 from
2
8
the A. baumannii enzyme were tested as described previously. The inhibition data are provided in Table 3.
The best inhibitor in the series was compound 3, with an improved efficacy (kinact/K ) of approximately 1.6
I
times relative to the parent compound 1 (Table 3, entries 1 vs 3). Thus, compound 3 had the highest affinity
for the enzyme, which led to an almost two-fold decrease in the inhibition constant (K = 140 nM) relative to
I
1
7
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−
1
1
and the same kinact value as 1 (22 ms ). Methylpyrazole derivative 2 showed a slightly improved efficacy
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
−
1
and this was mainly due to its higher conversion (kinact = 24 ms ) by the enzyme (Table 3, entries 1 vs 2).
−
1
Compound 4 was the inhibitor that reacted most rapidly with the enzyme (kinact = 25 ms ) and it had the
lowest affinity of the series (K = 495 nM). However, compound 4 was also the worst recognized by OXA-
I
2
4/40. This situation meant that the efficiency of 4 was only half that of the parent compound. The efficacy
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
of compound 5 was approximately 15% lower than that of the parent compound 1, mainly due to its lower
−
1
affinity (K = 276 nM) for the enzyme but also because of its faster conversion (24 ms ) (Table 3, entries 1
I
vs 5). The ligands that contained large pyridine-based heterocycles (6 and 7) showed quite different behavior
(
Table 3, entries 6 and 7). For example, whereas compound 6 had good affinity for the enzyme, with a
reaction rate around 50% slower than the other analogs of the series, only slow reaction with the enzyme
was observed with compound 7.
Table 3. Inhibition kinetics of OXA-24/40 from A. baumannii by inhibitors 1−7
−
1
−1 −1
Entry
1
Inhibitor
Ar
K (nM)
kinact (ms )
kinact/K (M s )
I
I
1
N
234 ± 48
217 ± 19
22 ± 2
98225 ± 32157
109731 ± 27375
N
N
2
2
24 ± 6
N
Br
3
4
3
4
N
140 ± 26
495 ± 95
22 ± 4
159036 ± 1403
48903 ± 13560
F
25 ± 11
N
MeO
MeO
5
6
5
6
N
276 ± 120
247 ± 167
24 ± 13
11 ± 5
83233 ± 11298
47564 ± 15036
N
S
1
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1
2
3
4
5
7
7
ND
ND
ND
N
ND = not determined.
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Taken together, it seems that there is no correlation between the nucleophilicity of the pyridine nitrogen
atom and the inactivation rate, since either the introduction of electron-withdrawing (fluoro) or electron-
donating (methoxy, heterocycle) groups in the para- or meta-positions of the ring afforded in all cases
higher or equal kinact constants to the parent compound 1. In contrast, the substitution seems to have a more
important role in: (i) the affinity of the ligand for the enzyme, (ii) the formation of the enzyme/inhibitor
complex and/or (iii) the stability of the corresponding indolizine adduct. Moreover, comparison of the
results for the inhibitory efficacy of ligands 2−4 against the enzyme OXA-24/40 from A. baumannii with the
aforementioned in vitro data for the combinations imipenem/2−4, which afforded MIC values of 1 µg/mL in
all cases (64-fold decrease), suggests that the bacterial permeability of compounds 2 and 3 is lower than that
of compound 4. Incubation studies, mass spectrometry detection of the corresponding enzyme adducts and
docking studies were performed in order to gain an insight into the ligand mechanism of action and binding
interactions responsible for ligands efficiency. The results of these studies are discussed below.
UV-Vis Spectroscopy and Mass Spectrometry Studies – The formation of the indolizine derivatives of the
reported ligands was analyzed by monitoring the methanolysis of compounds 2−7 by UV-Vis spectroscopy
after treatment with 1 equivalent of NaOMe (1 M in MeOH) at 25 ºC for 15 min (Figure S1). At the
beginning, the formation of a band centered at 295 nm (for 3) in the UV spectra was observed and this
corresponds to the conjugated imine intermediate (Figure 2C). This band rapidly decreased as two new
bands centered at 253 nm and 408 nm (for 3) appeared due to the formation of the indolizine skeleton as
confirmed by the mass spectra of the reaction mixture.34
In order to find evidences for the covalent modification mechanism, compounds 2−7 were incubated with
OXA-48 and OXA-24/40 enzymes in 50 mM TRIS.HCl, 150 mM NaCl, 1 mM EDTA buffer at pH 7.0 and
1
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2
5 °C for 30 min in a 1:100 enzyme/ligand ratio. During this period, the activity of the enzyme was
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
progressively determined by UV-Vis spectroscopy using aliquots from the incubation samples and the
control using nitrocefin as substrate. After incubation, enzymatic activity was barely detected. The samples
were filtered, washed (5 mM ammonium bicarbonate) and concentrated using Amicon® centrifugal filters
prior to analysis by mass spectrometry (MALDI). The mass spectra revealed the formation of stable
inactivation adducts and each contained a peak for the covalently modified enzyme with an increase in mass
of between 360 and 502, which corresponds to the indolizine adduct with loss of the catechol group or SO2
depending on the particular case (Figure S2).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Binding mode of ligands 2−7 with OXA-24/40. Enzyme/ligand complexes – The binding modes of
compounds 2−7 in the active site of the β-lactamase OXA-24/40 from A. baumannii were analyzed by
docking (GOLD 5.2.2 program) using the available coordinates of the crystallographically determined
OXA-24/40 covalently modified by a derivative of 1 in which position 4 of the pyridine moiety is
2
6
substituted by the -NHCO Me group (PDB ID 3FV7, 2.0 Å). With the exception of compound 7, all of the
2
compounds were anchored to the enzyme active site in a similar arrangement for nucleophilic attack of the
catalytic serine. This anchoring involved a set of strong electrostatic and polar interactions similar to those
established by the reference compound 1 (Figure 3). In all cases, the catechol group seems to have great
flexibility and wide variability of arrangements, as identified by docking. For ligands 2−6, the carbonyl
group of the lactam ring is located very close to the catalytic serine, Ser81, and this group was in an
appropriate arrangement for nucleophilic attack, which triggers the enzymatic reaction (Figures 3A and 3C).
In these cases, the estimated distance was around 2.8 Å, as measured between heavy atoms (carbon atom of
the β-lactam carbonyl group and oxygen atom of the side chain of Ser81). In contrast, the β-lactam carbonyl
group of ligand 7 was located further from the catalytic serine (~3.4 Å) since its isoquinoline ring was
deeply embedded in the tunnel-like entrance of the OXA-24/40 active site − a situation that might explain its
lower activity (Figures 3B and 3C).
2
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Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
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4
4
4
4
4
4
5
5
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5
5
5
5
5
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5
6
0
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2
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5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Binding modes of ligands 1−7 obtained by docking in the active site of the OXA-24/40 from A.
baumannii enzyme. A. Comparison of the overall binding mode of ligands 1 (yellow), 2 (orange), 3 (green),
4
(violet), 5 (cyan) and 6 (pink). The pyridyl moieties are located at the entrance of the active center,
involving mainly apolar residues (green, Val130, Leu168, Gly224 and Trp115), and flanked by two polar
residues (red, Gln134 and Thr111). B. Comparison of the overall binding mode of ligands 1 (yellow) and 7
(dark cyan). C. Comparison of the relative position of ligands 2−7 to the catalytic serine (Ser81).
The pocket that forms the entrance to the active site, in which the pyridine-based moieties of ligands 2−7 are
located, is mainly apolar and specifically involves residues Val130, Leu168, Gly225, and Trp115 (green,
2
1
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Figures 3A−B). This pocket is flanked by two polar residues that are suitable for hydrogen bonding
interactions, namely residues Gln134 and Thr111 (red, Figures 3A−B). A hydrogen-bonding interaction
between the 1-methylpyrazole moiety in 2 and the amide side chain of residue Gln134, which is absent from
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
, seems to enhance the binding and fix the arrangement of the pyridine ring in the pocket – a disposition
that might explain the lower K and higher k for 3 than for the parent compound 1 (Figure S3). In
I
inact
0
1
2
3
4
5
6
7
8
9
0
1
2
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7
8
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0
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5
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
contrast, the close proximity of the fluoro-substituent in 4 to the hydroxyl group in the side chain of Thr111
would disfavor the binding, as reflected by the two-fold decrease in affinity of compound 4 vs 1 (Figure S3).
The binding mode of compound 3 revealed that the substituent of the pyridine ring (Br) does not appear to
interact with the enzyme since it is located in the position para to the nitrogen atom. However, this electron-
withdrawing group would reduce the electron density of the ring and this may enhance the CH-п interactions
with the side chains of the apolar residues that surround it, specifically Leu168, Val130, Trp115, Tyr112,
Met223 and the methyl group of Thr111 (Figures S3 and S4). This favorable interaction of the aromatic ring
with the pocket proved to be very relevant, since the introduction of electron-rich pyridine-based moieties,
as in compounds 5 and 6, led to a decrease in the inhibitor enzyme affinity of up to two-fold.
CONCLUSIONS
6
-Arylmethylidene penicillin-based sulfones 2−7, analogs of the known penicillin-based sulfone β-lactamase
inhibitor 1, were synthesized from commercially available (+)-6-aminopenicillanic acid (13) by a Wittig
reaction between the ketone 12 and arylphosphonium salts 8−12 as a key step. The results of the
susceptibility studies of ampicillin against E. coli strains producing class A β-lactamases (TEM-1, CTX-M-
2), ceftazidime against E. coli strains producing class C enzymes (CMY-2, DHA-1), imipenem against A.
baumannii strains harboring CHDL enzymes (OXA-23, OXA-24/40) and K. pneumoniae strains expressing
OXA-48, in the absence and in the presence of compounds 2−7 along with avibactam and relebactam,
revealed that: (i) the combinations ceftazidime/2-6 and ampicillin/2−6 restored completely the antibiotic
efficacy in strains harboring classes C (CMY-2, DHA-1) and A (TEM-1) enzymes to give similar results as
the parent compound 1; (ii) lower efficacy was obtained with the combination ampicillin/2−6 in strains
producing CTX-M-2, in particular, a 64-fold improvement in the antibiotic MIC value was achieved with
2
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Journal of Medicinal Chemistry
inhibitors 2 and 3, as found for relebactam and compound 1; (iii) the combination of imipenem and
compounds 2−7 for bacterial strains carrying the studied CHDL enzymes proved to be, in most cases, more
efficient than when using avibactam and relebactam, which in most cases did not show a significant
antibiotic enhancing effect. In this latter case, the best in vitro results were obtained using compounds 3 and
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
. Mass spectrometry (MALDI) studies carried out on the CHDL enzymes, OXA-24/40 and OXA-48,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
revealed that the reported penicillin-based sulfone inhibitors 2−7 form stable inactivation adducts with these
β-lactamase enzymes.
The inhibition data for compounds 2–7 with the A. baumannii OXA-24/40 enzyme revealed that compound
3
was the best inhibitor of the series, with an improved efficacy of approximately 1.6 times relative to the
−
1
parent compound 1, with a K of 140 nM and a k
of 22 ms . The kinetic results in combination with
I
inact
docking studies on the corresponding OXA-24/inhibitor binary complexes also suggested that the
substitution and the electron density of the pyridine moiety do not appear to have a significant effect on the
inactivation rate. On the contrary, the latter two factors would have a more important role in ligand affinity
and/or the stability of the corresponding indolizine adduct. Thus, the higher affinity of compound 3 is
thought to be due to the reduced electron density of its pyridine ring, which in turn would enhance CH–п
interactions with the side chains of the apolar residues of the tunnel-like entrance that surround it.
EXPERIMENTAL SECTION
General. All starting materials and reagents were commercially available and were used without further
1
13
31
purification. H NMR spectra (250, 300 and 500 MHz), C NMR spectra (63, 75 and 125 MHz), P NMR
1
9
spectra (202 MHz) and F NMR spectra (282 MHz) were measured in deuterated solvents. J values are
given in Hertz. NMR assignments were carried out by a combination of 1 D, COSY, and DEPT-135
experiments. FT-IR spectra were recorded in a PerkinElmer Two FTIR spectrometer with attenuated total
2
0
−1
−1
reference. [α] = values are given in deg mL g dm . Milli-Q deionized water was used in all the buffers.
D
Melting points were measured in a Büchi M-560 apparatus. The spectroscopic UV-Vis measurements were
made on a Varian Cary 100 UV-Vis spectrophotometer with a 1 cm pathlength cell fitted with a Peltier
temperature controller. Protein analysis was performed using a MALDI TOF/TOF Mass Spectromether
2
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(
4800 Analyzer, AbSciex). The ProteoMassTM MALDI calibration kit (Merck) was employed for calibration
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
and sinapic acid as a matrix. Protein spectra were analyzed using the Data ExplorerTM Software. The purity
of compounds 28 was analyzed by HPLC and by NMR. HPLC was performed on a Thermo Dionex
UltiMate 3000 apparatus having a Brucker amazon SL mass spectrometry detector, using a Phenomenex
kinetex XB-C18 column (particle size = 1.7 µm; dimensions: 50 mm × 2.1 mm, pore size = 100 Å), and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0

1
eluting at a flow rate of 0.35 mL min with a gradient of 5−75% B in 10 min [A = Milli-Q water + 0.1%
TFA; B = acetonitrile + 0.1% TFA]. All tested compounds have a purity ≥ 95%.
2
-Methyl-4-(1-methyl-1H-pyrazol-4-yl)pyridine (15)  A solution of 2-methyl-4-bromopyridine (14) (1 g,
.81 mmol) in dioxane (29 mL) under inert atmosphere, was treated with
5
tetrakis(triphenylphosphine)palladium(0) (134 mg, 0.12 mmol), aqueous solution of potassium phosphate
10.3 mL, 0.8 M), and 1-methylpyrazole-4-boronic acid pinacol ester (1.45 g, 7 mmol). The resulting
(
mixture was heated at 85 °C for 12 h. After cooling to room temperature, the reaction mixture was diluted
with a (1:1) dichloromethane/water, the organic layer was separated, dried (anh. Na SO ), filtered and
2
4
concentrated under reduced pressure. The resulting residue was purified by flash chromatography, eluting
with a gradient of methanol/ethyl acetate [1): (0:100); 2) (10:90)], to give the compound 15 (1.12 g, quant.)
1
as a white foam. H NMR (250 MHz, CDCl ) δ: 8.35 (d, J = 5.3 Hz, 1H, ArH), 7.76 (d, J = 0.7 Hz, 1H,
3
ArH), 7.65 (s, 1H, ArH), 7.14 (br s, 1H, ArH), 7.07 (dd, J = 1.8 and 5.3 Hz, 1H, ArH), 3.87 (s, 3H, NCH3)
1
3
and 2.48 (s, 3H, CH ) ppm. C NMR (75 MHz, CDCl ) δ: 158.7 (C), 149.5 (CH), 140.3 (C), 137.0 (CH),
3
3
1
27.9 (CH), 120.6 (C), 119.2 (CH), 117.0 (CH), 39.2 (CH ), and 24.4 (CH ) ppm. MS (ESI) m/z = 174
3 3
+
+
(MH ). HRMS calcd for C H N (MH ): 174.1026; found, 174.1025.
10 12 3
[4-(1-Methyl-1H-pyrazol-4-yl)pyridin-2-yl]methanol (16)  A solution of compound 15 (655 mg, 3.46
mmol) in acetic anhydride (6.6 mL) was heated at 90 ⁰C for 12 h. After cooling to room temperature, the
solvent was removed under reduced presure and the resulting residue was dissolved in methanol (4.3 mL).
The solution was cooled to 0 °C, treated with KOH (291 mg, 5.2 mmol) and stirred at room temperature for
1
h. Ethyl acetate and saturated solution of NaHCO was added to the reaction mixture. The organic layer
3
was separated, dried (Na SO anh.), and concentrated under reduced pressure. The resulting residue was
2
4
2
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Journal of Medicinal Chemistry
purified by flash chromatography on silica gel, eluting with a gradient of methanol/ethyl acetate/hexane: [1)
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
(
0:90:10); 2) (0:100:0); 3) (10:90:0)], to give the alcohol 16 (257 mg, 40%) as a white foam. H NMR (300
MHz, CDCl ) δ: 8.34 (d, J = 5.2 Hz, 1H, ArH), 7.76 (s, 1H, ArH), 7.67 (s, 1H, ArH), 7.37 (s, 1H, ArH), 7.14
3
13
(
d, J = 4.4 Hz, 1H, ArH), 5.12 (br s, 1H, OH), 4.71 (s, 2H, CH ) and 3.85 (s, 3H, CH ) ppm. C NMR (75
2
3
MHz, CDCl ) δ: 160.6 (C), 148.9 (CH), 141.0 (C), 137.1 (CH), 128.2 (CH), 120.4 (C), 118.4 (CH), 116.6
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
−
1
+
(
CH), 64.4 (CH ), and 39.2 (CH ) ppm. FTIR (ATR) ν: 3154 (OH) cm . MS (ESI) m/z = 190 (MH ).
2 3
+
HRMS calcd for C H N O (MH ): 190.0975; found, 190.0974.
10
12
3
2
-(Chloromethyl)-4-(1-methyl-1H-pyrazol-4-yl)pyridine (17)  A solution of the alcohol 16 (394 mg,
2.08 mmol) in dry dichloromethane (10.4 mL), under inert atmosphere and at 0 ⁰C, was treated with a
solution of freshly distilled thionyl chloride in dry dichloromethane (4.7 mL, 1.4 M). The ice bath was
removed, and the reaction mixture was stirred at room temperature for 12 h. Saturated aqueous solution of
Na CO was then added, the organic layer was separated, dried (Na SO anh.) and concentrated under
2
3
2
4
reduced pressure. The resulting residue was purified by flash chromatography on silica gel, eluting with
1
(
5:95) methanol/ethyl acetate, to afford the chloride 17 (297 mg, 69%) as a pink oil. H NMR (300 MHz,
CDCl ) δ: 8.42 (d, J = 5.2 Hz, 1H, ArH), 7.79 (s, 1H, ArH), 7.70 (s, 1H, ArH), 7.44 (s, 1H, ArH), 7.21 (d, J
3
1
3
= 5.2 Hz, 1H, ArH), 4.60 (s, 2H, CH ) and 3.87 (s, 3H, CH ) ppm. C NMR (75 MHz, CDCl ) δ: 156.9 (C),
2
3
3
1
49.8 (CH), 141.3 (C), 137.1 (CH), 128.1 (CH), 120.1 (C), 119.0 (CH), 118.7 (CH), 46.8 (CH ) and 39.3
2
+
+
(
CH ) ppm. MS (ESI) m/z = 208 (MH ). HRMS calcd for C H ClN (MH ): 208.0636; found, 208.0635.
3 10 11 3
4-[(1-Methyl-1H-pyrazol-4-yl)pyrid-2-yl]methyltriphenylphosphonium chloride (8)  A solution of the
chloride 17 (296 mg, 1.43 mmol) in toluene (6.5 mL) was treated with triphenylphosphine (449 mg, 1.71
mmol) and heated under reflux for 12 h. After cooling to room temperature, the solvent was concentrated
under reduced pressure, diethyl ether was added, and the resulting precipitate was filtered. The phosphonium
1
salt 8 (245 mg, 40%) was obtained as a pink solid. Mp: 297−298 ⁰C. H NMR (500 MHz, DMSO-d6) δ:
8
7
.27 (s, 1H, ArH), 8.24 (d, J = 5.3 Hz, 1H, ArH), 7.87−7.82 (m, 10H, 10×ArH), 7.73−7.69 (m, 6H, 6×ArH),
.60 (s, 1H, ArH), 7.40 (td, J = 1.4 and 5.3 Hz, 1H, ArH), 5.54 (d, J = 15.5 Hz, 2H, CH ) and 3.87 (s, 3H,
2
1
3
CH ) ppm. C NMR (125 MHz, DMSO-d6) δ: 150.9 (d, J = 8 Hz, C), 149.4 (CH), 141.0 (C), 136.7 (CH),
3
2
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1
34.6 (d, J = 2 Hz, 3×CH), 133.9 (d, J = 10 Hz, 6×CH), 129.8 (d, J = 13 Hz, 6×CH), 129.5 (CH), 121.1 (d, J
8 Hz, CH), 119.6 (C), 119.0 (C), 118.7 (CH), 118.4 (C), 39.4 (CH ) and 31.1 (d, J = 51 Hz, CH ) ppm.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
=
3
2
MS (ESI) m/z = 434 (MH−Cl). HRMS calcd for C H N P (MH−Cl): 434.1781; found, 434.1781.
2
8
25
3
(3,4-Dimethoxypyrid-2-yl)methyltriphenylphosphonium chloride (9a)  A solution of 2-chloromethyl-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
,4-dimethoxypyridine hydrochloride (18a) (1 g, 4.50 mmol) in distilled water (15 mL) was treated with
potassium carbonate (622 mg, 4.5 mmol) and then stirred at room temperature for 15 min. The aqueous
solution was extracted with diethyl ether (3). The combined organic extracts were dried (anh. Na SO ),
2
4
filtered and concentrated under reduced pressure. The resulting orange oil was dissolved, under inert
atmosphere and at room temperature, in dry toluene (20.5 mL) and treated with triphenylphosphine (1.43 g,
5
.4 mmol). The reaction mixture was heated under reflux 24 h. After cooling to room temperature, the
solvent was removed under reduced pressure and the resulting solid residue was suspended in diethyl ether
1
and filtered. The phosphonium salt 9a (1.53 g, 82%) was obtained as a white solid. Mp: 217 °C (dec.). H
NMR (500 MHz, DMSO-d6) δ: 7.86 (d, J = 5.5 Hz, 1H, ArH), 7.847.80 (m, 9H, 9ArH), 7.717.67 (m,
6
H, 6ArH), 7.02 (d, J = 5.5 Hz, 1H, ArH), 5.42 (d, J = 14.5 Hz, 2H, CH P), 3.84 (s, 3H, OCH ) and 3.75
2
3
1
3
(s, 3H, OCH ) ppm. C NMR (125 MHz, DMSO-d6) δ: 158.2 (C), 144.7 (CH), 143.7 (C), 143.6 (d, J = 7
3
CP
Hz, C), 134.3 (d, JCP = 3 Hz, 3CH), 133.7 (d, JCP = 10 Hz, 6CH), 129.7 (d, JCP = 13 Hz, 6CH), 119.9
3
1
(d, JCP = 88 Hz, 3C), 108.6 (CH), 60.5 (OCH ), 56.1 (OCH ) and 26.5 (d, J = 57 Hz, CH P) ppm. P
3
3
CP
2
NMR (202 MHz, DMSO-d6) δ: 26.6 (s, 1P) ppm. MS (ESI) m/z = 414 (MCl). HRMS calcd for
C H NO P (MCl): 414.1617; found: 414.1607.
2
6
25
2
44
(4-Bromopyrid-2-yl)methyltriphenylphosphonium bromide (9b)  A solution of bromide 18b (505 mg,
2
.02 mmol) in toluene (9.2 mL) was treated with triphenylphosphine (636 mg, 2.42 mmol) and heated under
reflux for 12 h. After cooling to room temperature, the solvent was concentrated under reduced pressure,
diethyl ether was added and the resulting precipitate was filtered. The phosphonium salt 9b (534 mg, 52%)
1
was obtained as a pink solid. Mp: 245−247 °C. H NMR (500 MHz, DMSO-d6) δ: 8.24 (d, J = 5.4 Hz, 1H,
ArH), 7.87−7.80 (m, 9H, 9×ArH), 7.74−7.70 (m, 6H, 6×ArH), 7.62 (br s, 1H, ArH), 7.54 (td, J = 1.7 and 5.4
2
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Journal of Medicinal Chemistry
1
3
Hz, 1H, ArH) and 5.55 (d, J = 15.5 Hz, 2H, CH Ar) ppm. C NMR (75 MHz, DMSO-d6) δ: 152.2 (d, J = 8
2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Hz, C), 150.3 (C), 134.8 (3×CH), 133.9 (d, J = 10 Hz, 6×CH), 132.8 (CH), 129.9 (d, J = 13 Hz, 6×CH),
128.6 (d, J = 8 Hz, CH), 126.0 (CH), 118.9 (d, J = 87 Hz, 3×C) and 30.6 (d, J = 51 Hz, CH ) ppm. MS (ESI)
2
79
m/z = 432 and 434 (M−Br). HRMS calcd for C H NP Br (M−Br): 432.0511; found, 432.0511.
2
4
20
2
-(Bromomethyl)-5-fluoropyridine (20)  A solution of 5-fluoro-2-methylpyridine (19) (150 mg, 1.35
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
mmol) in dry carbon tetrachloride (3.1 mL) and under inert atmosphere, was treated with N-
bromosuccinimide (385 mg, 2.16 mmol) and AIBN (22 mg, 0.14 mmol). The resulting mixture was heated
under reflux for 2 h. After cooling to room temperature, the reaction mixture was filtered and concentrated
under reduced pressure. The crude residue was purified by flash chromatography on silica gel, eluting with
1
(
10:90) diethyl ether/hexane, to give the bromide 20 (129 mg, 50%) as a pink oil. H NMR (300 MHz,
1
3
CDCl ) δ: 8.39 (d, J = 2.6 Hz, 1H, ArH), 7.45−7.34 (m, 2H, 2×ArH) and 4.52 (s, 2H, CH ) ppm. C NMR
3
2
(75 MHz, CDCl ) δ: 158.9 (d, J
= 265 Hz, C), 152.9 (d, JCF = 4 Hz, C), 138.0 (d, JCF = 24 Hz, CH),
CF
3
1
24.6 (d, JCF = 5 Hz, CH), 123.8 (d, JCF = 19 Hz, CH) and 32.7 (CH ) ppm. MS (CI) m/z = 190 and 192
2
+
79
+
(
M ). HRMS calcd for C H BrNF (M ): 189.9662; found, 189.9663.
6 5
(5-Fluoropyrid-2-yl)methyltriphenylphosphonium bromide (10)  A solution of bromide 20 (30 mg,
.16 mmol) in toluene (0.7 mL) was treated with triphenylphosphine (50 mg, 0.2 mmol) and heated under
0
reflux for 12 h. After cooling to room temperature, the solvent was concentrated under reduced pressure.
The resulting residue was suspended in diethyl ether and the resulting precipitate was filtered. The
1
phosphonium salt 10 (64 mg, 89%) was obtained as a white solid. H NMR (500 MHz, DMSO-d6) δ: 8.38
(d, J = 3.0 Hz, 1H, ArH), 7.87−7.66 (m, 16H, 16×ArH), 7.40 (dd, J = 4.2 and 8.5 Hz, 1H, ArH) and 5.51 (d,
1
3
J = 15.6 Hz, 2H, CH Ar) ppm. C NMR (125 MHz, DMSO-d6) δ: 158.4 (dd, J = 3 and 253 Hz, C), 146.7
2
(dd, J = 4 and 9 Hz, C), 137.4 (dd, J = 2 and 24 Hz, CH), 134.8 (d, J = 3 Hz, 3×CH), 133.9 (d, J = 10 Hz,
6
8
1
×CH), 129.9 (d, J = 13 Hz, 6×CH), 127.2 (dd, J = 5 and 8 Hz, CH), 124.5 (d, J = 19 Hz, CH), 118.9 (d, J =
1
9
7 Hz, 3×C) and 30.0 (d, J = 51 Hz, CH ) ppm. F NMR (282 MHz, DMSO-d6) δ: −128.0 (t, J
= 4 Hz,
FH
2
3
1
F) ppm. P NMR (202 MHz, DMSO-d6) δ: 27.0 (s, 1P) ppm. MS (ESI) m/z = 372 (M−Br). HRMS calcd
for C H NPF (M−Br): 372.1312; found, 372.1313.
2
4
20
2
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Journal of Medicinal Chemistry
Page 28 of 60
Triphenyl(thieno[2,3-c]pyridin-7-ylmethyl)phosphonium bromide (11)  A solution of the bromide 2142
272 mg, 1.2 mmol) in toluene (5.6 mL) was treated with triphenylphosphine (378 mg, 1.4 mmol) and then
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
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3
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3
3
3
3
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5
5
5
5
5
5
5
5
6
(
heated under reflux for 12 h. After cooling to room temperature, the solvent was concentrated under reduced
pressure and diethyl ether was added. The resulting white precipitate was filtered to give the phosphonium
1
salt 11 (327 mg, 56%) as a white solid. Mp: 277–278 ºC. H NMR (500 MHz, DMSO-d6) δ: 8.21 (d, J = 5.4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Hz, 1H, ArH), 8.09 (d, J = 5.5 Hz, 1H, ArH), 7.92 (dd, J = 7.1 and 14.6 Hz, 6H, 6×ArH), 7.81 (t, J = 6.6 Hz,
3
H, 3×ArH), 7.75 (d, J = 5.5 Hz, 1H, ArH), 7.69 (m, 6H, 6×ArH), 7.59 (d, J = 5.3 Hz, 1H, ArH), and 5.99
1
3
(
d, J = 14.8 Hz, 2H, CH Ar) ppm. C NMR (125 MHz, DMSO-d6) δ: 145.6 (C), 145.5 (d, J = 7 Hz, C),
2
C

P
1
41.5 (CH), 135.4 (d, JCP = 9 Hz, C), 134.3 (d, JCP = 2 Hz, 2×CH), 134.0 (CH), 133.8 (d, JCP = 10 Hz,
×CH), 129.7 (d, JCP = 13 Hz, 6×CH), 123.7 (CH), 120.0 (d, JCP = 88 Hz, 3×C), 117.5 (CH) and 30.3 (d,
6
3
1
JCP = 58 Hz, CH ) ppm. P NMR (202 MHz, DMSO-d6) δ: 26.56 (s, 1P) ppm. MS (ESI) m/z = 410
2
(
M−Br). HRMS calcd for C H NPS (M−Br): 410.1115; found, 410.1127.
26 21
Compound 23  A solution of sulfoxide 2238,39,40,41 (1 g, 2.07 mmol) in dry dioxane (8.3 mL) was treated
with anhydrous MgSO (1 g, 8.31 mmol) and 2-mercaptobenzothiazole (378 mg, 2.26 mmol). The resultant
4
yellow suspension was heated at 110 ºC for 14 h. After cooling to room temperature, the solid was removed
®
by filtration over Celita . The filtrate and the washings were concentrated under reduced pressure. The
resultant residue was dissolved in diethyl ether, treated with activated charcoal and stirred for 30 min. The
®
suspension was filtered through a plug of Celita . The filtrate and the washings (diethyl ether) were
concentrated under reduced pressure to give disulfide 2338,44 (1.2 g, 91%), as a pale yellow foam. Compound
2
3 was converted into amine 27 (and its six-membered analog 28) without further purification following
previously reported protocol.38,44
Compound 30a  A solution of the amines 27 and 28 (2:1 molar ratio, respectively) (621 mg, 1.31 mmol) in
dry dichloromethane (6.6 mL), under inert atmosphere and at –78 °C, was treated dropwise with dry
triethylamine (0.6 mL, 3.93 mmol). After 5 min stirring, trifluoromethanesulfonic anhydride was added
dropwise (0.7 mL, 3.93 mmol) during 5 min. The reaction mixture was warmed up to 0 °C during a 1 h
period, and then was cooled again to –78 °C. Dry triethylamine (0.6 mL, 3.93 mmol) was added dropwise
2
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Page 29 of 60
Journal of Medicinal Chemistry
during 10 min. The resulting mixture was stirred for 30 min and then treated with cold HCl (20 mL, 0.5 M).
After 10 min, the organic layer was separated, and the aqueous layer was extracted with dichloromethane.
The combined organic extracts were washed with cold HCl (3×10 mL, 0.5 M), dried (anh. Na SO ), filtered
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
4
and concentrated under reduced pressure to give the diketone 12 and its six-membered analog 29 (2:1 molar
ratio, respectively). The latter mixture of ketones was used for the subsequent Wittig reaction without
further purification. A solution of compound 8 (673 mg, 1.31 mmol) in dry THF (5.2 mL), under argon
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
t
atmosphere and at room temperature, was treated with KO Bu (146 mg, 1.31 mmol) and then stirred for 2 h.
The resultant solution, at room temperature and under inert atmosphere, was added via canula to a solution
of the previously obtained diketones 12 and 29 (2:1 molar ratio, respectively) (620 mg, 1.31 mmol) in dry
dichloromethane (10 mL). After 35 min stirring, saturated solution of NH Cl was added and the resultant
4
suspension was stirred for 10 min. The organic layer was separated, and the aqueous layer was extracted
with dichloromethane. The combined organic extracts were successively washed with water and brine, dried
(anh. Na SO ), filtered and concentrated under reduced pressure. The resulting residue was purified by flash
2
4
chromatography, eluting with ethyl acetate/hexane: 1) (55:45); 2) (70:30), to give the pyridine 30a (244 mg,
2
0
1
3
0%) as a yellow foam. [α] = +285.1 (c1.2, CHCl ). H NMR (300 MHz, CDCl ) δ: 8.56 (d, J = 5.0 Hz,
D
3
3
1H, ArH), 7.85 (s, 1H, ArH), 7.76 (s, 1H, ArH), 7.44−7.28 (m, 12H, 12×ArH), 6.98 (s, 1 H, CH=), 6.94 (s,
H CHPh ), 6.32 (s, 1H, H5), 4.99 (s, 1H, H2), 4.22 (d, J = 11.7 Hz, 1H, OCHH), 4.16 (d, J = 15.0 Hz, 1H,
1
2
CHHCl), 4.09 (d, J = 15.0 Hz, 1H, CHHCl), 3.97 (s, 3H, CH ), 3.93 (d, J = 11.7 Hz, 1H, OCHH), and 1.23
3
1
3
(s, 3H, CH ) ppm. C NMR (75 MHz, CDCl ) δ: 168.2 (C), 166.9 (C), 166.7 (C), 152.8 (C), 150.8 (CH),
3
3
1
1
7
45.4 (C), 141.2 (C), 139.2 (C), 139.2 (C), 137.2 (CH), 128.7 (4×CH), 128.4 (CH), 128.3 (CH), 128.2 (CH),
27.5 (2×CH), 127.2 (2×CH), 124.5 (CH), 122.8 (CH), 120.0 (C), 119.6 (CH), 78.5 (CH), 72.0 (OCH2),
0.6 (CH), 65.7 (CH), 64.1 (C), 40.8 (CH ), 39.4 (CH ) and 20.5 (CH ) ppm. FTIR (ATR) ν: 1756 (CO) and
2
3
3
−
1
+
+
1742 (CO) cm . MS (ESI) m/z = 629 (MH ). HRMS calcd for C H N ClO S (MH ): 629.1620; found,
3
3
30
4
5
6
29.1619.
Compound 30b − It was prepared according to the procedure described for compound 30a using: (i) for the
preparation of the diketone: amines 27 and 28 (2:1 molar ratio, respectively) (220 mg, 0.46 mmol) in dry
2
9
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