Click Chemistry in Lead Optimization of Boronic Acids as β-Lactamase Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.5b00341

Boronic acid transition-state inhibitors (BATSIs) represent one of the most promising classes of β-lactamase inhibitors. Here we describe a new class of BATSIs, namely, 1-amido-2-triazolylethaneboronic acids, which were synthesized by combining the asymme

Full text of DOI:10.1021/acs.jmedchem.5b00341

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Article
Click Chemistry in Lead Optimization of
Boronic Acids as ß-Lactamase Inhibitors
Fabio Prati, Emilia Caselli, CHIARA ROMAGNOLI, Roza
Vahabi, Magdalena A. Taracila, and Robert A. Bonomo
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00341 • Publication Date (Web): 23 Jun 2015
Downloaded from http://pubs.acs.org on July 3, 2015
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Click Chemistry in Lead Optimization of
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Boronic Acids as βꢀLactamase Inhibitors
Emilia Caselli†, Chiara Romagnoli†, Roza Vahabi†#, Magdalena A. Taracila‡,
Robert A. Bonomo∞,‡, Fabio Prati†*
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†University of Modena and Reggio Emilia, Department of Life Science, via Campi 103, 41125,
Modena, Italy
∞Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Research Service,
Cleveland, OH
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‡Case Western Reserve University, Departments of Medicine, Pharmacology, Biochemistry and
Molecular Biology and Microbiology, Cleveland, OH
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#Present Address: Institut für Bioorganische Chemie der HeinrichꢀHeine Universität Düsseldorf im
Forschungszentrum Jülich, Stetternicher Forst,
Geb. 15.8, 52426 Jülich, Germany
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*Corresponding Author: Eꢀmail: fabio.prati@unimore.it. Phone: (+39)059ꢀ2055056
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KEYWORDS Boronic acid transition state inhibitor, βꢀlactamase, click chemistry,
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triazole, cephalosporinase, penicillinase
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ABSTRACT
Boronic acid transition state inhibitors (BATSIs) represent one of the most promising class
of βꢀlactamase inhibitors. Here we describe a new class of BATSIs, namely 1ꢀamidoꢀ2ꢀ
triazolylethaneboronic acids, which were synthesized combining the asymmetric homologation of
boronates with CopperꢀCatalyzed AzideꢀAlkyne Cycloaddition (CuAAC) for the stereoselective
insertion of the amido group and the regioselective formation of the 1,4ꢀdisubstituted triazole,
respectively. This synthetic pathway, which avoids intermediate purifications, proved to be flexible
and efficient, affording in good yields a panel of fourteen BATSIs bearing three different R1 amide
side chains (acetamido, benzylamido and 2ꢀthienylacetamido) and several R substituents on the
triazole. This small library was tested against two clinically relevant class C βꢀlactamases from
Enterobacter spp. and Pseudomonas aeruginosa. The Ki value of the best compound (13a) was as
low as 4 nM with significant reduction of bacterial resistance to the combination of cefotaxime/13a.
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Funding
The Cleveland Department of Veterans Affairs, the Veterans Affairs Merit Review Program award
number 1I01BX001974, and the Geriatric Research Education and Clinical Center VISN 10
supported RAB. This work was also supported by funds from: National Institute of Allergy and
Infectious Diseases of the National Institutes of Health under award numbers R01AI063517 and
R01AI072219 (RAB, FP and EC) and the Harrington Discovery Institute. The content is solely the
responsibility of the authors and does not necessarily represent the official views of the National
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Department
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INTRODUCTION
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The World Health Organization recently declared that antimicrobial resistance is one of the
three greatest threats to human health. This current alarming public health crisis is driven by two
key factors: the emergence of antibacterial resistance in important pathogens and a lack of
development of new antibacterial agents.1 Commercial, developmental, and regulatory hurdles
challenge industry in identifying new antibacterial agents. Since antibiotics are usually prescribed
as shortꢀduration therapies (as brief as a single dose), the emergence of resistance compromises
their commercial life span, and the cost of carrying out the large studies that are required for
approval are significant.
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Until now, the main approach to discovering new antibacterial agents has been to modify
existing classes of drugs that inhibit validated bacterial targets. Yet, how many more generations of
"modified molecules" can be obtained on known scaffolds that will avert resistance? In addition,
circumventing preꢀexisting resistance mechanisms that have arisen in the clinic also challenges us.
Recently, efforts are underway to develop drug libraries based on novel chemistries. A premier
example of this is the emerging interest in boron chemistry.2 Boronꢀbased compounds demonstrate
ideal drugꢀlike properties and possess specific geometries and reactivities that enhance their
capacity to interact with select biological targets. Presently, boron containing molecules are directed
against new targets, such bacterial leucyl tRNA synthetase inhibitors (GSK2251052, under Clinical
Phase II), or as ‘potentiators’ that when administered together with antibiotics enhance their effect.
In the latter case the development of effluxꢀpump3 and βꢀlactamase inhibitors (BLIs)4 are the main
thrusts of this research.
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Bacterial production of βꢀlactamases represents the most clinically concerning mechanism
of resistance to βꢀlactam antibiotics in Gram negative bacteria. The emergence of many recent βꢀ
lactamase variants (> 1600 new enzymes) jeopardizes the efficacy of both the latest developed
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antibiotic and the combination of βꢀlactamꢀBLI (e.g., the formulation ampicillin and clavulanic acid
are ineffective against bacteria expressing inhibitor resistant TEM and SHV βꢀlactamases). At the
moment, boronic acids transition state inhibitors (BATSIs) represent one of the most promising and
exciting class of BLIs in development, as testified by the advancement of a combination of a
boronic acid inhibitor (RPX7009 currently developed by the Medicines Company) with meropenem
(a carbapenem), in clinical trials (Clinical Trials Phase 1 registration number NCT01897779,
RPX7009 is joined with RPX2014). Carbavance™ (meropenem/RPX7009) is particularly targeted
against KPC (Klebsiella pneumoniae carbapenemase)ꢀproducing carbapenemꢀresistant
Enterobacteriaceae (CRE).
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Independently, we5aꢀc,6,7 and others5d previously demonstrated that the design and synthesis
of several new BATSIs, whose structures resembled more and more the parental βꢀlactams, is an
innovative strategy in novel BLI discovery.5,6,7 The rationale behind the design of these potent
BATSIs relies on the bioisoster identification of the boronic moiety replacing the highly reactive βꢀ
lactam ring, and on the mimicking of the natural substrates. The more sophisticated structures, in
particular the 1ꢀamidoꢀ2ꢀ(metaꢀcarboxyphenyl)ethaneboronic acids, incorporate different features
that make them similar to the parental βꢀlactam during the hydrolytic process: a R1 amide group on
the boronꢀbearing carbon atom C1 mimicking the βꢀlactams R1 side chain, an aromatic ring
stereoselectively inserted in C2 to resemble the ring adjacent to the βꢀlactam, and the presence of a
carboxylate on the phenyl important for recognition and cell penetration (Figure 1).6
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R2
X
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S
H
N
H
R1
R1
N
S
N
COOH
H
O
O
N
-O
O
X
O
HO
Ser
COOH
βL
βL-Ser-OH
high energy
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deacylation intermediate
H
R2
H
R1
N
COOH
R1
N
COOH
O
B
O
HO
B
HO
OH
O
HO
βL-Ser-OH
Ser
βL
1-amido-2-(meta-carboxyphenyl)
ethaneboronic acids
Figure 1. Mechanism of action of a βꢀlactamase (βL) with a βꢀlactam and with a boronic acid.
This strategy has been very successful, since the activities of 1ꢀamidoꢀ2ꢀ
phenylethaneboronic acids against different enzymes (as measured by Kis) and against specific
strains (minimum inhibitory concentrations, MICs) are encouraging. Nevertheless the synthetic
pathway leading to these structures is substantially rigid, allowing the insertion of different R1 side
chains, but forcing the choice of the R2 group from the beginning with precise constraints given by
the conditions of the homologation reaction that stereospecifically introduces a halogenated carbon
atom. (Scheme 1, A.).
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TMS
H
H
A.
COOtBu
COOtBu
N
COOtBu
R1
N
Cl
R1
N
COOH
COOtBu
TMS
B
O
B
B
O
B
B
O
O
O
O
O
O
HO OH
O
O
I
TMS
H
H
N
CLICK
REACTION
N3
Cl
R1
N
N
N
B.
R1
N
N
N
N
N
N3
TMS
N3
+
B
O
B
R
O
B
B
B
O
O
HO OH
O
O
O
O
O
O
R
II
Scheme 1. Stereosective synthesis of 1ꢀamidoꢀ2ꢀphenylethaneboronic acids I and of the newly
designed αꢀamidoꢀβꢀtriazolylethanboronic acids II.
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Here we report our attempts to simplify this synthesis and improve the lead optimization
process in order to obtain a new class of BATSIs with more potent biochemical affinity, increased
accessibility to the target, and chemical diversity. The approach undertaken (Scheme 1, B) relies on
the “click chemistry” reaction, in particular the wellꢀknown Copper Catalyzed AzideꢀAlkyne
Cycloaddition (CuAAC).8 This can be performed in the last step on a chiral boronic scaffold
bearing both the R1 amide side chain and a properly inserted azido group with monoꢀsubstituted
alkynes, thus affording highly substituted αꢀamidoꢀβꢀtriazolylethanboronic acids II. Whereas the
1,4ꢀdisubstituted 1,2,3ꢀtriazole ring replaces the mꢀcarboxybenzyl group preserving its aromatic
character, it exploits a stronger dipole moment, thus potentially acting as an active linker, and
allows a large and easy variability of the R groups (Scheme 1, B).9 The novel synthetic pathway
proves to be more flexible, efficient, with higher yields and avoids the laborious process required to
purify intermediates. The development of this original class of potent BLIs is based both on
bioisoster identification, and on rational scaffold hopping of the R2 ring.10
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In this report a panel of fourteen 1ꢀamidoꢀ2ꢀtriazolylethaneboronic acids compounds bearing
three different R1 amide side chains (acetamido, benzylamido and 2ꢀthienylacetamido) and at
least three R substituents for each series, was synthesized and tested against two clinically
relevant class C βꢀlactamases, P99 and PDCꢀ3. To assess the activity of our compounds, we
targeted representative class C cephalosporinases that are often responsible for highꢀlevel
resistance to βꢀlactam antibiotics among lactose fermenting (e.g., Enterobacter cloacae) and nonꢀ
lactose fermenting (Pseudomonas aeruginosa) strains.11ꢀ13 The atomic structures of these βꢀ
lactamases were previously determined (4HEF and 1XX2 respectively) and served as models to
help us understand why each compound was so effective. As expected, the BATSIs synthesized
by click chemistry showed potent activity against these enzymes, spanning from low micromolar
to low nanomolar range inhibition constants.
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We advance that this activity was primarily modulated by the identity of the R1 acetylamino
side chain, while the possibility of using the click chemistry to introduce different R chemical
groups on the triazole ring allowed a first exploration of the recognition of the R2 side of the
molecules. Comparison of the Kis permitted an evaluation of the energetic contribution to
molecular recognition due to the best R substituents and to the triazole ring by itself. The
synergistic effect with βꢀlactams to reverse antibiotic resistance in cell culture assays was
explored.
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RESULTS
Design
The previous studies that focused on the activity of a series of 1ꢀamidoꢀ2ꢀ
phenylethaneboronic acids I (Scheme 1) against class C (AmpC) βꢀlactamases, demonstrated that
the presence of a phenyl side chain on the stereogenic carbon atom improved activity when
compared to the achiral compounds, in particular with small R1, such as for side chains typical of
penicillin G.6 Since P99 and PDCꢀ3 βꢀlactamases are structurally similar to AmpC βꢀlactamase
(Figure 2) and share 62.4% similarity (Fig S1, supporting materials), we chose the acetyl,
phenylacetyl and 2ꢀthienylꢀ2ꢀacetyl side chains as R1 groups.
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Figure 2. Superimposition of the active site of class C βꢀlactamases: PDCꢀ3 (PDB_4HEF) colored by atoms,
P99 (PDB_1XX2) carbons magenta, and AmpC active site (yellow) in complex with a boronic inhibitor,
(PDB_1MXO).
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We reasoned that the triazole ring could replace the aromatic phenyl ring, allowing the
introduction of different R groups to explore the topology of the βꢀlactamase active site. As shown
in Figure 2 the active site pocket is very similar in all three βꢀlactamases; the only notable
differences are at position 343 where 1) asparagine is replaced by serine for P99 enzyme and 2) the
oxyanion hole Ser318 in P99 and PDCꢀ3 is replaced by alanine in AmpC enzyme. The Arg residue
at position 349 and Asn343 and 346 respectively are conserved as well.
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As a first approach, we chose to use simple alkynes to assess the best conditions for the
click reaction, bearing different polarities and hydrophilicities which might take advantage of the
active site electrostatic potential (Figure S2 supporting materials).
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Synthesis of α-amido-β-triazolylethaneboronic acids.
The synthesis of the new BATSIs is depicted in Scheme 2; the stereoselective synthesis of
these inhibitors is based on Matteson’ homologation of (+)ꢀpinanediol azidomethaneboronate 2,14 a
strategy that allow to introduce a stereogenic carbon in α position of boronic esters.15
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Cl
O
TMS
a
b
c
d
COOH
OH
N
Cl
O
N3
O
B
TMS
N3
N3
LiN(TMS)2
81%
O
NaN3
95%
LiCHCl2
82%
B
B
B
O
O
O
O
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7
8
9
1
2
3
4
R =
COOH
NH2
1. MeOH
2.R1COCl
62-90%
e
f
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H
H
H
H
R1
N
R1
N
N
N
R
R1
N
N
Deprotection
98%
N
N
R
N3
N
R
O
B
O
B
O
B
80-98%
O
O
HO
OH
O
O
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9
a-c R1= Me
a-e R1= Bn
10 a-f R1= ThMe
5
6
7
R1= Me
R1= Bn
R1= ThMe
11 a-c R1= Me
12 a-e R1= Bn
13 a-f R1= ThMe
ThMe =
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S
Scheme 2. General synthesis of αꢀamidoꢀβꢀtriazolylethaneboronic acids
Compound 2 was obtained (97%) through chlorine substitution of 1 with sodium azide in a
biphasic system (water/ethyl acetate), in the presence of tetrabutylammonium iodide as phase
transfer catalyst.14 Subsequent treatment of 2 with in situ generated dichloromethyl lithium at –100
°C allowed the insertion of an halogenated carbon on the carbonꢀboron bond; the use of (+)ꢀ
pinanediol as chiral auxiliary agent afforded the expected αꢀchloroboronate (S)ꢀ3 with high
diastereoselectivity (d.e.>98%, 96% yield). Treatment with lithium bis(trimethylsilyl)amide,
performed at –100 °C to minimize the elimination reaction, produced the αꢀaminoboronate 4
(80%), the key intermediate for the synthesis of all BATSIs. In situ deprotection of the aminoꢀgroup
with a stoichiometric amount of methanol followed by coupling with the selected acylꢀchloride
afforded compounds 5-7, which were recovered as pure solids (62ꢀ90% yield) by fractional
crystallization of the crude. These compounds were stable at 4 °C for months without any
appreciable degradation.
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With the αꢀacylamidoꢀβꢀazido derivatives 5-7 in hand for the assembling of the right part of the
inhibitors we resorted to the Huisgen’s 1,3ꢀdipolar azideꢀalkyne cycloaddition exploiting copper
catalysis (CuAAC) to gain 1,4ꢀregioselectivity in the formation of the triazole. Among the wide
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variety of conditions described in the literature for this robust reaction, we adopted the most
common one, employing water/t.butanol as solvent and generating the Cu(I) catalyst in situ from
CuSO4 in the presence of an excess of ascorbate as reducing agent (5% catalyst, 20% ascorbate).8.
In these conditions the three azides 5-7 were allowed to react at 60 °C with the terminal alkynes a-f;
complete conversions were reached in 0.5ꢀ18 hours and the expected 1,4ꢀdisubstituted triazoles 8-
10 were recovered in good to excellent yields (80ꢀ98%) by extraction with ethyl acetate and
crystallization of the crude. The formation of triazoles 8ꢀ10 was confirmed by the presence in the
1H NMR spectra of a singlet downfield at 7.6ꢀ8.7 ppm which correlates (HSQC experiment) with a
signal at 119ꢀ130 ppm of the 13CꢀNMR spectra, as expected for a triazolic CH. Moreover, the 1,4ꢀ
disubstitution was confirmed by the longꢀrange correlation (HMBC experiment). The 4ꢀ
unsubstituted triazole 10f was obtained using trimethylsilylacetylene as alkyne counterpart; in this
case, the 4ꢀtrimethylsilylsubstitued triazole formed by CuCAAC undergoes spontaneous protoꢀ
desilylation to 10f in the reaction conditions partially and was isolated by chromatography. Finally,
deprotection of the boronic moiety was accomplished by transesterification with phenylboronic acid
in a biphasic system acetonitrile/nꢀhexane,16 allowing the removal of the chiral auxiliary (+)ꢀ
pinanediol and to obtain the final products 11a-c, 12a-e, 13a-f (80ꢀ100% yield). Except for the
starting compound 1, purified by chromatography, intermediates 2, 3 and 4 didn’t required any
purification, while intermediate 8a-c, 9a-e,10a-e, as well as the final product 11a-c, 12a-e, 13a-f
could be purified by crystallization of the crude material. NMR spectra of all BATSIs are reported
in the Supporting Information.
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Inhibition Kinetics.
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As a result of the synthesis above, fourteen new chiral boronic acid inhibitors were tested
against the representative class C βꢀlactamases, P99 and PDCꢀ3. All compounds were first screened
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with a 5 min preꢀincubation between the βꢀlactamase and BATSI, and the reaction initiated with
nitrocefin (NCF). Activity was also measured at different time points (0 to 10 min) to assess the
preꢀincubation effect on inhibitor affinity.
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As expected, a timeꢀdependence effect on inhibition was observed, in particular, the most
potent inhibitor, compound 13a showing the greater inhibition (Figure S3, supporting materials).
Three compounds 11a-c bearing the R1 acetamido side chain demonstrated an IC50 in the
micromolar range (5ꢀ27 ꢀM), while activity significantly improved for inhibitors bearing the
phenylacetyl or 2ꢀthienylꢀacetyl side chains the (IC50s reduced to the nanomolar range). These
results are consistent with previous data obtained for achiral αꢀacylaminoboronic acids against class
C AmpC betaꢀlactamase, where the simplest acetamido side chain performed worse than more
elaborate side chains.7
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
210
215
220
Consequently, five alkynes in both series 12 and 13 were used to further investigate
recognition for R1 amide side chain and R2 groups. We determined that IC50s (after 5 minute
incubation) were in general better for BATSI bearing the R1 typical of cephalothin (series 13) than
of penicillin G (series 12), with the most notable difference being compounds 12c vs compound 13c
against P99. Amongst the R groups introduced through click chemistry, very low IC50s were
obtained for boronic acids bearing the R carboxy sidechain as in 13a (35 ± 2 nM vs. PDCꢀ3 and
140 ± 20 nM vs. P99) and the 3ꢀcarboxyphenyl sidechain as in 13d (67 ± 2 nM vs. PDCꢀ3 and 84 ±
10 nM vs. P99), supporting our early findings that a carboxylate on the R2 side chain improves
recognition.6
Table 1: IC50s values of compounds 11ꢀ13 versus class C βꢀlactamases PDCꢀ3 and P99.
H
R1
N
N
N
N
R
O
B
HO
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IC50[µM]
Entries
Structures
Cmp
R
5
6
PDC-3
P99
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5±1
27.1±3
COOH
OH
1
2
11a
11b
H
N
N
N
18.4±0.3
71±5
N
R
O
B
HO
OH
9.3±1
13.2±2
3
11c
0.23±0.01
1.18±0.2
0.11±0.01
0.66±0.01
COOH
OH
4
5
12a
12b
H
N
6
7
N
12c
12d
0.3±0.02
0.15±0.02
0.11±0.01
N
N
R
O
B
HO
OH
COOH
0.12±0.01
NH2
8
12e
0.4±0.05
0.6±0.02
9
13a
13b
COOH
0.035±0.002
0.25±0.05
0.14±0.02
0.42±0.01
OH
10
H
N
S
N
11
12
13
13c
13d
13e
0.3±0.02
0.067±0.002
0.28±0.02
0.32±0.02
0.084±0.01
0.36±0.02
N
N
R
O
B
OH OH
COOH
NH2
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H
14
15
13f
A
1.3±0.2
3.3±0.2
0.8±0.02
2.7±0.2
H
N
S
O
B
HO OH
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
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50
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60
H
N
COOH
S
16
B
0.15±0.01
0.1±0.02
O
B
HO OH
225
230
235
Antimicrobial Activity and Binding Energies.
MIC values (ꢁg/mL) of E. coli DH10B expressing PDCꢀ3 and P99 class C βꢀlactamases
were determined by the agar dilution method to assess potency in whole cell assays. Based upon
previous studies, we chose cefotaxime (TAX) as a partner βꢀlactam. These results are summarized
in Table 2. The best compounds of the newly synthesized series, 13a and 13d, were compared to
previously described BATSIs: the achiral thienylacetamidomethane boronic acid (reference
compound A) and the correspondent chiral metaꢀcarboxybenzyl (reference compound B). Boronic
acids bearing the triazole mojety show ability to penetrate E. coli DH10B and inhibit both P99 and
PDCꢀ3 in whole cell assays with MICs lowered into the susceptible range.
Table 2: Synergy of compounds 13a, 13d and reference compounds A and B with cefotaxime
against E.coli bacteria producing PDCꢀ3 and P99. The boronate were tested at a constant concentration of
4µg mLꢀ1
H
H
N
COOH
N
S
S
13a
13d
O
B
O
B
HO OH
HO OH
A
B
β-lactamase
Strain
TAX
+ TAX
+ TAX
+ TAX
+ TAX
E.coli
DH10B
E.coli
<0.12
5
none
<0.125
0.5
<0.125
1
<0.125
2
<0.125
PDCꢀ3
16
4-8
DH10B
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pBCSKꢀ
4
5
6
7
8
E.coli
DH10B
Pcs900
P99
16
2
4
2
4
9
10
11
12
13
14
15
To evaluate the energetic contribution of different groups to molecular recognition in the newly
synthesized inhibitors, Kis of the best inhibitors as well as of the two reference compounds A and B
were determined taking in consideration the substrate nitrocefin contribution (Table 3).
16
240
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 3: Kis (ꢀM) values of compounds 13a, 13d, 13f and of reference compounds A and B.
K
i (ꢀM) vs.
K
i (ꢀM) vs.
Cmp
13a
Structure
PDC-3
P99
H
N
N
S
N
N
0.004±0.001
0.008±0.001
0.023±0.002
0.014±0.001
O
B
HO OH
COOH
H
N
S
N
N
N
O
B
13d
HO OH
COOH
H
N
S
N
N
N
0.164±0.008
0.411±0.001
0.013±0.006
0.134±0.006
0.451±0.002
0.015±0.005
13f
O
B
HO OH
H
N
S
Ref cmp A
Ref cmp B
O
B
HO OH
H
N
COOH
S
O
B
HO OH
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Boronates 13a and 13d bearing a negative charge on the triazole substituent, have Kis of 4 ± 1
and 8 ± 1 nM respectively against PDCꢀ3 and of 23 ± 2 and 14 ± 1 nM against P99. These results
are amongst the best obtained against class C βꢀlactamases. Since these compounds bind covalently
and reversibly to the enzyme, differential binding energies can be calculated directly from Ki
values. For example, the contribution to binding of the simple carboxylate in 13a against PDCꢀ3,
can be calculated by comparing the affinities of compound 13f (Ki = 164 ± 8 nM) and compound
13a (Ki = 4 ± 1 nM), thus obtaining an energetic contribution of 2.2 Kcal/mol (using ꢁꢁGbind = ꢀ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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60
RTln Ki
/Ki), a value consistent with the 2.1 Kcal/mol previously calculated for chiral
ref
acylaminophenylmethaneboronic acids vs. AmpC.
DISCUSSION AND CONCLUSIONS
In this study we developed a flexible synthetic pathway to obtain a series of chiral boronic
acids that exhibit potent activity against representative class C serine βꢀlactamases, P99 and PDCꢀ3.
The BATSIs designed were highly active microbiologically against E. coli harboring the tested
cephalosporinases and biochemically as they mimicked the natural substrate of these enzymes, the
βꢀlactams, and exploited the chemical properties that facilitate cell entry.
260
The synthetic pathway depicted in Scheme 2 illustrates that the key intermediate 4, the (+)ꢀ
pinanediol 1ꢀ(N,Nꢀbisꢀtrimethylsilyl)ꢀaminoꢀ2ꢀazidoethaneboronate, is able to exploit the presence
of two orthogonally reactive groups for in parallel synthesis: the bisꢀtrimethylsilylamino group as
precursor for amide formation in R1, and the azido group for triazole formation in R2 through the
click reaction (CuAAC). Three acylamido groups are introduced on 4 as R1 amide side chains: the
acetamido as reference compound (compound 5), the benzylacetamido typical of the antibiotic
penicillin G (compound 6) and the 2ꢀthienylacetamido typical of cephalothin (compound 7).
Three terminal alkynes bearing a carboxy, a hydroxymethyl, and a phenyl group are next
used in click reaction with each βꢀazidoboronate 5-7. Triazole formation is obtained through the
265
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wellꢀknown click reaction catalyzed by Copper (I), that represent a rapid, selective and efficient
strategy to synthesize a library of compounds either during early stage drug discovery and lead
optimization process. The strengths of this intrinsically convergent reaction resides on wide
functional groups compatibility, mild reaction conditions, regioselective formation of the 1,4ꢀ
disubstituted isomer, high yields and ease of product purification.8,17 Indeed click reaction on
compounds 5-7 proceeds easily in each case, employing water/t.butanol as solvent and generating
the Cu(I) catalyst in situ by reduction of copper sulfate. Products 8a-c, 9a-c, 10a-c are obtained in
high yields (80ꢀ98%) after simple crystallization of the crude material. Upon removal of the
pinanediol chiral auxiliary, nine boronic acids 11a-c, 12a-c and 13a-c are obtained and tested
against two class C betaꢀlactamases, PDCꢀ3 and P99, two common cephalosporinases responsible
for resistance against the most potent cephalosporin (eg. cefotaxime).
10
11
12
13
14
15
16
17
18
19
20
21
22
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41
42
43
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45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Boronates 11a-c bearing the acetamido side chain exhibit IC50 values in the micromolar
range (entries 1ꢀ3, 5ꢀ71 ꢀM), proving that the simple methyl group does not enhance active site
recognition. In contrast, introduction of the PenG side chain as in 12a-c improves IC50s up to two
orders of magnitude (entries 4ꢀ6, 0.11ꢀ1.18 ꢀM). As expected, the cephalothin side chain
demonstrates the highest inhibition, with IC50 values that are reduced of one more orders of
magnitude for compound 13a compared to 12a against PDCꢀ3 (entry 9, 0.035 ± 0.002 ꢀM vs. entry
285
4, 0.23 ± 0.01 ꢀM). This trend closely resembles data obtained with chiral acylaminoboronic acids
vs AmpC,6 thus suggesting a similar spatial arrangement in the catalytic pocket.
To help understand the activity of these compounds we resorted to molecular models
(Figure 2). Our analysis shows that most of the residues presented in the active site of AmpC are
preserved in P99 and PDCꢀ3 and maintain similar conformations (less than 1Å difference in side
chain conformation). We observe that the flexibility of Q120 (showed to have multiple
conformations in different crystallographic structures of class C beta lactamases) along with N152
allows Hꢀbond interactions with acylamino part of the R1 side of new BATSI series. The other
residues which can be part of the inhibitorꢀenzyme interaction (N346, R349, S318, K315, and Y150
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or K67) are preserved. Furthermore, the minimized structure of PDCꢀ3 and P99 in complex with
13a (Figure 3a and b) suggest that boronic moiety hydroxyl group makes Hꢀbonds with S318, Y150
and possible K67. The R1 amide recognition pattern is highly conserved (Q120 and N152) for both
class C enzymes.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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41
42
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44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
300
Figure 3a. PDCꢀ3 and b. P99 with 13a docked in the active site. The molecular docking revealed
key interactions between conserved residues in class C βꢀlactamase and R1 and R2 groups of
BATSI.
305
To assess the potential of the click chemistry approach to better explore the R2 binding site
of the catalytic pocket, we focused our attention on the R triazole substituent, obtaining two more
compounds of series 12, inserting a metaꢀcarboxyꢀ and a metaꢀaminoꢀphenyl group (compounds
12d and 12e), and three more compounds of series 13, inserting the same groups (compounds 13d
and 13e), as well as the simple hydrogen atom (compound 13f). All these compounds prove to be
very good βꢀlactamase inhibitors with IC50s in the low micromolar and nanomolar range (entries 7ꢀ
14, 1.3 ꢀM to 67 nM), affirming the best inhibitors as those bearing the R1 amide side chain typical
of cephalothin vs that of Penicillin G (series 13 vs 12), with the exception of compound 13c vs 12c
in the assays against P99. Furthermore compounds bearing a carboxylate in the triazole R
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substituent exhibit the best inhibition values. In particular compound 13d bearing the metaꢀ
carboxyphenyl R group, has IC50 slightly worse than compound 13a against PDCꢀ3 (entry 12 vs 9,
67 vs 35 nM), but even better against P99 (entry 12 vs 9, 84 vs 140 nM), suggesting of quite flexible
pocket able to accommodate differently hindered R groups bearing a negative charge.18 Lastly, our
models show that the carboxyl of the BATSI is positioned in a favorable conformation to make Hꢀ
bonds with N343, and a longer R2 substituent of 13a introduced by the methyltriazole can facilitate
the interaction with R349 and/or N346 in PDCꢀ3 and P99 (Figure 3a and b).
10
11
12
13
14
15
16
17
18
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25
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41
42
43
44
45
46
47
48
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50
51
52
53
54
55
56
57
58
59
60
We next explored the contribution of the triazole ring to active site binding. Comparing the
affinity of the unsubstituted triazole 13f (Ki = 164 ± 8 nM vs PDCꢀ3) to the achiral cephalothin
analogue Ref Compound A (Ki = 411 ± 0.001nM) suggests that the methyltriazole itself contributes
0.5 Kcal/mol to binding. Even if at a first examination this might not appear a significant interaction
energy (especially when compared to the contribution of 1.3 Kcal/mol of the simple phenyl ring for
chiral acylaminophenylmethaneboronic acids)6 we stress that the methylene bridge between the
triazole and the α carbon atom increases the entropic factor negatively affecting the overall free
energy contribution of the triazole (ꢁꢁG). Another comparison that might further assist in
evaluating the contribution of the triazole to binding is when Reference compound B, bearing the
metaꢀcarboxybenzyl moiety, is compared to 13a, bearing the 4ꢀcarboxytriazolylmethyl moiety, and
an improvement of 0.7 Kcal/mol in recognition caused by ring hopping is obtained. Thus, the
triazole not only reveals to be a phenyl bioisoster and a valuable solution for an easier synthetic
feasibility, but also behaves as an active linker partly for its high dipole moment (about 5 D).19
Another avenue explored is the role of the triazole in cell penetration. It is recognized that
1,4ꢀdisubstituted triazoles are not a novelty in medicinal chemistry. Indeed, more than seven
thousand 1,4ꢀdisubstituted 1ꢀHꢀ1,2,3ꢀtriazole compounds had been reported before the discovery
of the copper catalyzed click reaction. Triazoles are in fact known to possess a number of
desirable features in the context of medicinal chemistry. For example, triazoles are stable to acid
and base hydrolysis and reductive and oxidative conditions, as well as metabolic degradation.9
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One of the three commercially available βꢀlactamase inhibitors, tazobactam, is among the bestꢀ
known examples of triazoleꢀcontaining pharmaceutically active compounds. Clinical experience
has shown that tazobactam coupled to piperacillin is a potent combination with greater potency
and more broad spectrum activity than amoxicillinꢀclavulanic acid or ampicillinꢀsulbactam, and
part of this potency is ascribed to the presence of the triazole ring.20 In order to evaluate the role of
the triazole in the new BATSI we tested the best inhibitors 13a and 13d and reference compounds
A and B as well against E. coli strains expressing either PDCꢀ3 and P99 (Table 2).
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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25
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31
32
33
34
35
36
37
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40
41
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45
46
47
48
49
50
51
52
53
54
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56
57
58
59
60
345
350
355
360
The addition of each of these BATSI (13a, 13d, A and B) results in a significant lowering of
MICs when coupled to cefotaxime. Without a BATSI, MICs are in the resistant range. In the best
case, the addition of the 13a BATSI results in a five dilution (2 log) reduction in MIC against E.
coli possessing the P. aeruginosa AmpC and 13d results in a similar lowering. Since 13a differs
from B only for the phenylꢀtriazole ring hopping, the significant improvement in biological
activity can be ascribed to the triazole ring.
In conclusion, this work describes the design, synthesis and biological properties of a new
class of chiral boronic acids that effectively target representative βꢀlactamase that are found in
problematic hospital pathogens. Most notably, two out of fourteen compounds (13a and 13d)
demonstrate a βꢀlactamase inhibitory profile (Kis) similar to a chiral boronate previously
synthesized (reference compound B), with improved in vitro activity (MICs, see Table 2).
Furthermore, the improved synthetic accessibility of these boronic acids, which relies on the click
chemistry reaction (CuAAC), opens up the possibility of obtaining a large number of compounds
with a wider range of substituents that could better explore the βꢀlactamase binding site. This
synthetic approach promises to further expand the repertoire of compounds to be explored against
βꢀlactamases that have defied inhibition and have contributed to the current crisis in antimicrobial
chemotherapy. Extension of the studies herein against different microbiological targets of
importance and diverse βꢀlactamases are in progress.
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METHODS
Synthesis
All reactions were performed under argon using ovenꢀdried glassware and dry solvents. Dry
tetrahydrofuran (THF) and diethyl ether were obtained by standard methods and freshly distilled
under argon from sodium benzophenone ketyl prior to use. The –100 °C bath was prepared by
addition of liquid nitrogen to a preꢀcooled (–78 °C) mixture of ethanol/methanol (1:1). Preloaded
(0.25 mm) glass supported silica gel plates (Kieselgel 60, Merck) were used for TLC analysis, and
compounds were visualized by exposure to UV light and by dipping the plates in 1 %
Ce(SO4)·4H2O, 2.5% (NH4)6Mo7O24·4H2O in 10% sulfuric acid followed by heating on a hot plate.
Chromatographic purification of the compounds was performed on silica gel (particle size 0.05ꢀ0.20
mm). Melting points were measured in open capillary tubes on a Stuart SMP30 Melting Point
apparatus. Optical rotations were determined at +20 °C on a PerkinꢀElmer 241 polarimeter and are
10
11
370
12
13
14
15
16
17
18
19
20
21
375
22
23
24
25
26
27
28
29
30
31
32
expressed in 10ꢀ1 deg cm2 gꢀ1. H and 13C NMR spectra were recorded on a Bruker Avanceꢀ400
1
380
385
390
MHz spectrometer. Chemical shifts (δ) are reported in ppm and were calibrated to the residual
signals of the deuterated solvent.21 Multiplicity is given as s = singlet, d = doublet, t = triplet, q =
quartet, m = multiplet, br = broad signal; coupling constants (J) are given in Hz. Twoꢀdimensional
33
34
35
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39
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41
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58
59
60
1
NMR techniques (COSY, HMBC, HSQC) were used to aid in the assignment of signals in H and
13C spectra. Particularly, in the 13C spectra, the signal of the boronꢀbearing carbon atom, which tend
to be broadened, and the signal of the quaternary triazole carbon are often beyond the detection
limit, but their resonances were unambiguously determined by HSQC. Mass spectra were
determined on an Agilent Technologies LCꢀMS (n) Ion Trap 6310A (ESI, 70 eV). Highꢀresolution
mass spectra were recorded on an Agilent Technologies 6520 AccurateꢀMass QꢀTOF LC/MS.
The purity of all tested compounds was above 95%, determined by elemental analysis performed
on a Carlo Erba elemental analyzer 1110; results from elemental analyses for the compounds were
within (0.3% of the theoretical values). For these BATSIs the presence of residual traces of copper
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was checked on a Quadrupole Inductively Coupled Plasma Mass Spectrometer ICPꢀMS X Series II,
indicating the presence of copper ≤ 0.5 ppm. Data from MS fragmentation and elemental analyses
of free boronic acids were not obtainable because of the formation of dehydration products.
Nevertheless, these boronic acids could be converted into analytically pure pinacol/pinanediol
esters by exposure to an equimolar amount of pinacol/pinanediol in anhydrous THF.
395
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(+)ꢀpinanediol chloromethaneboronate 1 was synthesized according to the literature.22
(+)ꢀpinanediol azidomethaneboronate 2: was synthesized according to the literature.14
400
405
410
(+)-Pinanediol (1S)-chloro-2-azidoethaneboronate (3): was synthesized according to the
literature.14
(+)-pinanediol (1R)-2-azido-1-(N-bis(trimethylsilyl)amino)ethaneboronate (4): Lithium
bis(trimethylsilyl)amide (1 M solution in THF, 9.87 mL, 9.87 mmol) was added dropwise to a
solution of 3 (2.80 g, 9.87 mmol) in anhydrous THF (25 mL) at –100 °C under argon flow. The
mixture was allowed to warm to room temperature overnight. The resulting solution was
concentrated under reduced pressure, and the crude was treated with light petroleum (150 mL). The
white inorganic precipitate (LiCl) was filtered off on a MgSO4 pad and washed with abundant light
petroleum. The solvent was evaporated in vacuo to afford 4 as a pale yellow oil (3.23 g, 80%), used
1
as such for the subsequent reaction. [α]D = +4.2 (c 1.6, CHCl3). H NMR (400 MHz, CDCl3):
δ
0.15 (18H, s, [Si(CH)3]2), 0.83 (3H, s, pynanyl CH3), 1.10 (1H, d, J = 10.9, pinanyl Hendo), 1.28
(3H, s, pinanyl CH3), 1.39 (3H, s, pinanyl CH3), 1.84ꢀ 2.31 (5H, m, pinanyl protons), 2.85 (1H, dd,
J = 8.5, 5.9, BCH), 3.29 (1H, dd, J = 12.3, 8.5, BCHꢀCH2), 3.37 (1H, dd, J = 12.3, 5.9, BCHꢀCH2),
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4.31 (1H, dd, J = 8.7, 1.7, CHOB). 13C NMR (100 MHz, CDCl3):
35.2, 38.2, 39.4, 43.4 (br, CB), 51.4, 56.1, 78.6, 86.1.
δ 2.5, 2.7, 24.0, 26.3, 27.0, 28.3,
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(+)-Pinanediol (1
R
)-acetylamino-2-azidoethaneboronate (5). A solution of dried methanol (0.05
mL, 1.2 mmol) in anhydrous THF (1.5 mL) was added to a solution of 4 (0.5 g, 1.2 mmol) in
anhydrous THF (4 mL) at ꢀ10 °C under argon flow. After being stirred for 10 min at ꢀ10 °C, the
cooling bath was removed. The reaction mixture was stirred for 1 hr at rt. Thereafter the reaction
mixture was cooled again at ꢀ10 °C and a solution of acetylchloride (0.09 mL, 1.2 mmol) was
slowly added. The resulting mixture was allowed to react for one hour, and thereafter partitioned
between EtOAc (80 mL) and H2O (20 mL). The aqueous phase was extracted with EtOAc (2 x 25
mL) and the combined organic phases were washed with saturated NaHCO3 and concentrated
under vacuum to afford 5 as a beige solid (0.337, 90.1%), mp 44ꢀ46 °C. [α]D – 66.9 (c, 2.2 ,
420
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CHCl3). 1H NMR (400 MHz, CDCl3):
δ 0.88 (3H, s, pynanyl CH3), 1.30 (3 H, s, pinanyl CH3), 1.40
(3H, s, pinanyl CH3), 1.42 (1H, d, J = 7.1, pinanyl H endo), 1.77ꢀ2.37 (5H, m, pinanyl protons), 2.15
(3H, s, CH3CONH), 2.81 (1H, d, J = 9.5, BCH), 3.35 (1H, dd, J = 13.0,11.0, BCHꢀCH2), 3.67 (1H,
dd, J = 13.0, 3.6, BCHꢀCH2), 4.21 (1H, dd, J = 8.6, 2.0, CHOB), 8.43 (1H, b, NH). 13C NMR (100
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430
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36
37
38
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MHz, CDCl3):
δ 18.1, 24.2, 26.7, 27.4, 29.3, 36.8, 38.1, 40.1, 43.5 (CB), 52.4, 54.2, 76.4, 83.6,
175.8. MS (ESI, Ion Trap): 307 [M + H]+; MS/MS 307, m/z (%): 279 (4), 263 (10) , 250 (59), 237
(7), 208 (100), 155 (61), 130 (3), 100 (8). Anal. Calcd for C14H23BN4O3: C, 54,92; H, 7,57; N,
18,30. Found: C, 54,73; H, 7,88; N, 18,050
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(+)-Pinanediol (1R)-phenylacetylamino-2-azidoethaneboronate (6). According to the procedure
described above, phenylacetylchloride was added to a solution of deprotected 4 (2.10 g, 5.14 mmol)
to afford 6 as a brownish solid, which was crystallized from nꢀhexane and diethylether (75% yield),
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mp 130ꢀ132°C. [α]D – 64.9 (c, 1.2 , CHCl3) . H NMR (400 MHz, CDCl3):
δ 0.89 (3H, s, pynanyl
440
CH3), 1.31 (3 H, s, pinanyl CH3), 1.40 (1H, d, J = 10.4, pinanyl Hendo), 1.43 (3H, s, pinanyl CH3),
1.78ꢀ 2.39 (5H, m, pinanyl protons), 2.88 (1H, m, BCHCH2), 3.41 (1H, dd, J = 13.0, 10.7,
BCHCH2), 3.64 (1H, dd, J = 13.0, 3.6, BCHCH2), 3.75 (2H, s, CH2CO), 4.26 (1H, dd, J = 8.7, 2.2,
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CHOB), 6.65 (1H, b, NH), 7.26ꢀ7.45 (5H, m, Har). 13C NMR (100 MHz, CDCl3):
δ 24.2, 26.6,
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6
7
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27.3, 29.1, 36.5, 38.2, 39.5, 40.0, 42.5 (CB), 52.2, 54.2, 76.7, 84.1, 128.2, 129.4, 129.5, 132.1,
176.3. MS (ESI, Ion Trap): 383 [M+ + H]+ ; MS/MS 383, m/z (%): 355 (5), 326 (100), 231 (45),
208 (33), 176 (5), 159 (10). Anal. Calcd for C20H27BN4O3: C, 62,84; H, 7,12; N, 14,66. Found: C,
62,50; H, 7,36; N, 14,43.
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(+)-Pinanediol (1R)-2-azido-1-(2-thienylacetylamino)ethaneboronate (7). According to the
procedure described above, 2ꢀthienylacetylchloride was added to a solution of deprotected 4 (3.32
g, 7.91 mmol) to afford 7 as a brownish solid, which was crystallized from diethyl ether and nꢀ
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hexane (62% yield), mp 139ꢀ140°C. [α]D – 61.5 (c, 1.0 , CHCl3). H NMR (400 MHz, CDCl3):
δ
0.85 (3H, s, pynanyl CH3), 1.28 (3 H, s, pinanyl CH3), 1.34 (1H, d, J = 10.5, pinanyl Hendo), 1.39
(3H, s, pinanyl CH3), 1.77ꢀ2.36 (5H, m, pinanyl protons), 2.95 (1H, dt, J = 10.1, 3.4, BCH), 3.42
(1H, dd, J = 12.9, 10.1, BCHCH2), 3.60 (1H, dd, J = 12.9, 3.4, BCHCH2), 3.91 (2H, s, CH2CO),
4.24 (1H, dd, J = 8.7, 2.1, CHOB), 6.59 (1H, br, NH), 6.96ꢀ6.97 (1H, m, Har), 7.02 (1H, dd, J = 5.2,
3.5, Har), 7.30 (1H, dd, J = 5.2, 1.1, Har). 13C NMR (100 MHz, CDCl3):
δ 24.2, 26.6, 27.3, 28.9,
34.0, 36.3, 38.2, 39.9, 41.4 (br, CB), 52.0, 53.9, 77.3, 84.6, 126.3, 127.6, 128.2, 133.4, 174.4. MS
(ESI, Ion Trap): 389 [M+ + H]+ ; MS/MS 389, m/z (%): 355 (5), 326 (100), 231 (45), 208 (33), 176
(5), 159 (10). Anal. Calcd for C18H25BN4O3S: C, 55,68; H, 6,49; N, 14,43; S, 8,26. Found: C,
55,59; H, 6,41; N, 14,28; S, 8,02.
General Procedure for Cu/C-catalyzed “click” reaction:
In a glass vial the starting compound (0.6 mmol) and the proper alkyne (1.5 mmol) were dissolved
in tertꢀBuOH (3 mL) and CuSO4 (0.06 mmol), sodium ascorbate (0.12 mmol) and water (3 mL)
were added. The vessel was sealed and temperature raised at 60 °C. The reaction was monitored
through TLC until disappearance of the azidoboronate. After the proper reaction time, that spanned
from 0.5 hour for more activated alkynes to 2 hours for less activated alkynes, the reaction mixture
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was partitioned between EtOAc (30 mL), H2O (12 mL) and saturated NaCl (7 mL). The aqueous
phase was extracted with EtOAc (2 x 20 mL) and the combined organic phases were washed with
saturated NaCl, dried over Na2SO4, and concentrated under vacuum to afford the crude product
which was thereafter purified as described.
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(+)-Pinanediol (1R)-2-(4-carboxy-[1,2,3]triazol-1-yl)-1-acetylaminoethaneboronate (8a). The
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reaction was terminated after 30 min. The crude residue was crystallized from CH2Cl2 and nꢀhexane
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to afford 5a as a beige solid (173 mg, 80% yield), mp 177ꢀ179°C. [α]D – 71.9 (c 1.3 , DMSO). H
NMR (400 MHz, DMSO):
δ 0.82 (3H, s, pynanyl CH3), 1.23 (3H, s, pynanyl CH3), 1.24 (3H, s,
pynanyl CH3), 1.39 (1H, d, J = 9.7, pynanyl Hendo), 1.63ꢀ2.21 (5H, m, pynanyl protons), 2.05 (3H, s,
CH3CONH), 2.95ꢀ2.98 (1H, m, BCHCH2), 4.00 (1H, d, J = 7.1, CHOB), 4.30ꢀ4.38 (2H, m,
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485
490
BCHCH2), 8.70 (1H, s, CHtriazole), 9.81 (1H, s, NH). 13C NMR (100 MHz, DMSO):
δ 17.4, 24.5,
26.8, 27.8, 30.0, 37.4, 38.1, 40.0, 44.2 (br, CB), 52.7, 53.3, 75.8, 82.3, 129.7 (CHtriazole), 140.0,
162.4,177.0. MS (ESI, Ion Trap): 377 [M + H]+, MS/MS 377, m/z (%): 359 (3), 264 (96), 225
(100),140 (4%), 130 (4%). Anal. Calcd for C17H25BN4O5: C, 54,27; H, 6,70; N, 14,89. Found: C,
53,99; H, 6,91; N, 14,53.
(+)-Pinanediol
(1R)-2-[4-(1-hydroxy-1-methylethyl)-[1,2,3]triazol-1-yl)-1-
acetylaminoethaneboronate (8b). The reaction was terminated after 30 min. The crude residue
was triturated from nꢀhexane to afford 8b as a whitish solid (172 mg, 79% yield), mp 75ꢀ77°C. [α]D
– 58.5 (c, 1.3 , CHCl3).
1H NMR (400 MHz, CDCl3 ):
δ 0.91 (3H, s, pynanyl CH3), 1.32 (3H, s, pynanyl CH3), 1.44 (3H, s,
pynanyl CH3), 1.48 (1H, d, J = 10.1, pynanyl Hendo), 1.53 (6H, s, (CH3)2COH), 1.81ꢀ2.42 (5H, m,
pynanyl protons), 2.20 (3H, s, CH3CONH), 3.15ꢀ3.18 (1H, m, BCH), 4.24 (1H, d, J = 6.9, CHOB),
4.43ꢀ4.51 (2H, m, BCHCH2), 7.65 (1H, s, CHtriazole), 9.25 (1H, br, NH). 13C NMR (100 MHz,
CDCl3 ):
δ
17.6, 24.3, 27.0, 27.5, 29.4, 29.7, 37.1, 38.2, 40.2, 45.5 (br, CB), 52.5, 53.3, 76.3, 83.2,
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119.6 (CHtriazole), 176.8, Cꢀ4 triazole ring not seen. MS (ESI, Ion Trap): 391.3 [M + H]+, MS/MS
391, m/z (%): 373 (67), 345 (23), 317 (10), 264 (100), 222 (7), 193 (10), 135 (9), 130 (7), 112 (6).
Anal. Calcd for C19H31BN4O4: C, 58,47; H, 8,01; N, 14,36. Found: C, 58,18; H, 7.75; N, 14,08.
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(+)-Pinanediol (1R)-2-[4phenyl-[1,2,3]triazol-1-yl)-1-acetylaminoethaneboronate (8c). The
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reaction was terminated after 2 hours. The crude residue was triturated from nꢀhexane to afford 8c
1
as a whitish solid (117 mg, 86% yield), mp 77ꢀ77°C. [α]D – 60.3 (c 1.0, CHCl3). H NMR (400
MHz, CDCl3):
δ 0.93 (3H, s, pynanyl CH3), 1.34 (3H, s, pynanyl CH3), 1.47 (3H, s, pynanyl CH3),
1.54 (1H, d, J = 10.2, pynanyl Hendo), 1.85 – 2.45 (5H, m, pynanyl protons), 2.26 (3H, s,
CH3CONH), 3.29 (1H, d, J = 7.3, BCH), 4.27 (1H, d, J =7.4, CHOB), 4.45 (1H, dd, J = 14.7, 2.0,
BCHCH2), 4.62 (1H, dd, J = 14.7, 12.4, BCHCH2), 7.30 – 7.45 (5H, m, Har), 7.69 (1H, s, CHtriazole),
505
510
515
9.43 (1H, br, NH). 13C NMR (100 MHz, CDCl3 ):
δ 17.8, 24.3, 27.0, 27.5, 29.4, 37.1, 38.2, 40.2,
45.0 (br, CB), 52.6, 53.4, 76.4, 83.2, 119.4 (CHtriazole), 125.2, 128.2, 128.8, 129.8, 147.6, 176.9. MS
(ESI, Ion Trap): 409 [M + H]+, MS/MS 409, m/z (%): 324 (26), 264 (100), (75), 187 (7), 135 (5).
Anal. Calcd for C22H29BN4O3: C, 64,72; H, 7,16; N, 13,72. Found: C, 64,58; H, 7,28; N, 13,54.
(+)-Pinanediol (1R)-2-(4-carboxy-[1,2,3]triazol-1-yl)-1-phenylacetylaminoethaneboronate (9a).
The reaction was terminated after 30 min. The crude residue was crystallized from CH2Cl2 and nꢀ
hexane to afford 9a as a beige solid (277 mg, 80% yield), mp 130ꢀ132°C. [α]D – 87.5 (c 1.1,
1
DMSO). H NMR (400 MHz, DMSO):
δ 0.81 (3H, s, pynanyl CH3), 1.21 (3H, s, pynanyl CH3),
1.22 (3H, s, pynanyl CH3), 1.34 (1H, d, J = 10.0, Hendo), 1.62ꢀ2.22 (5H, m, pynanyl protons), 3.01ꢀ
3.03 (1H, m, BCH), 3.667 (1H, d, J = 14.8, PhCH2), 3.675 (1H, d, J = 14.8, PhCH2), 4.01 (1H, d, J
=6.8, CHOB), 4.35 (1H, dd, J = 14.4, 10.5, BCHCH2), 4.43 (1H, dd, J = 14.4, 3.9, BCHCH2) 7.23ꢀ
13
7.35 (5H, m, Har), 8.71 (1H, s, CHtriazole), 9.89 (1H, s, NH). C NMR (100 MHz, DMSO):
δ 24.5,
26.7, 27.7, 29.8, 37.2, 37.1, 38.0, 40.3, 43.4 (br, CB), 52.5, 52.9, 75.9, 82.6, 127.6, 129.0, 129.4,
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129.8 (CHtriazole), 134.1, 140.0, 162.3,177.0. MS (ESI, Ion Trap): 453 [M + H]+, MS/MS 453, m/z
(%): 340 (75), 301 (100), 206 (8). Anal. Calcd for C23H29BN4O5: C, 61,07; H, 6,46; N, 12,39.
Found: C, 60,86; H, 6,63; N, 12,10.
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(+)-Pinanediol
(1R)-2-[4-(1-hydroxy-1-methylethyl)-[1,2,3]triazol-1-yl)-1-
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phenylacetylaminoethaneboronate (9b). The reaction was terminated after 30 min. The crude
residue was triturated from nꢀhexane to afford 9b as a beige solid (288 mg, 98% yield), mp 78ꢀ
80°C. [α]D – 73.9 (c 1.1, CHCl3). 1H NMR (400 MHz, CDCl3):
δ 0.91 (3H, s, pynanyl CH3), 1.32
(3H, s, pynanyl CH3), 1.44 (3H, s, pynanyl CH3), 1.45 (1H, d, J = 9.5, Hendo), 1.49 (6H, s,
(CH3)2COH), 1.82ꢀ2.53 (5H, m, pynanyl protons), 3.14ꢀ3.17 (1H, m, BCH), 3.77 (2H, s, PhCH2),
4.25 (1H, dd, J = 6.6, 1.9, CHOB), 4.41ꢀ4.49 (2H, m, BCHCH2), 7.25ꢀ7.35 (5H, m, Har), 7.65 (1H,
s, CHtriazole), 9.16 (1H, br, NH). 13C NMR (100 MHz, CDCl3 ):
δ 24.3, 26.9, 27.4, 29.3, 30.9, 31.3,
37.0, 38.1, 38.2, 40.2, 44.9 (br, CB), 52.4, 53.0, 76.4, 83.4, 119.8 (CHtriazole), 127.8, 129.0, 129.3,
132.3, 155.5, 177.4. MS (ESI, Ion Trap): 467 [M + H]+, MS/MS 467, m/z (%): 449 (53), 421 (23),
340 (100), 297 (16), 269 (7). Anal. Calcd for C25H35BN4O4: C, 64,38; H, 7,56; N, 12,01. Found: C,
64,30; H, 7,65; N, 11,93.
(+)-Pinanediol (1R)-2-(4-phenyl-[1,2,3]triazol-1-yl)-1-phenylacetylaminoethaneboronate (9c).
The reaction was terminated after 2 hours. The crude residue was triturated from nꢀhexane to afford
1
9c as a beige solid (303 mg, 85% yield), mp 94ꢀ96 °C. [α]D – 68.9 (c 1.0 , CHCl3). H NMR (400
MHz, CDCl3):
δ 0.93 (3H, s, pynanyl CH3), 1.33 (3H, s, pynanyl CH3), 1.46 (3H, s, pynanyl CH3),
1.52 (1H, d, J = 10.1, Hendo), 1.85 – 2.41 (5H, m, pynanyl protons), 3.26ꢀ3.29 (1H, m, BCH), 3.80
(2H, s, PhCH2), 4.29 (1H, d, J = 8.1, CHOB), 4.46ꢀ4.60 (2H, m, BCHCH2), 7.29ꢀ7.48 (10H, m,
Har), 7.67 (1H, s, CHtriazole), 8.89 (1H, br, NH). 13C NMR (100 MHz, CDCl3 ):
δ 24.3, 26.9, 27.4,
29.3, 37.0, 38.2, 38.5, 40.2, 44.2 (br, CB), 52.4, 52.9, 76.6, 83.6, 119.8 (CHtriazole), 125.3, 127.9,
128.2, 128.8, 129.1, 129.4, 130.0, 132.1, 147.5, 177.4. MS (ESI, Ion Trap): 485 [M + H ]+, MS/MS
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485, m/z (%): 340 (100), 333 (65), 324 (10), 188 (3.5). Anal. Calcd for C28H33BN4O3: C, 69,43; H,
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6,87; N, 11,57. Found: C, 69,59; H, 6,70; N, 11,31.
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(+)-Pinanediol
(1R)-2-[4-(3-carboxy-phenyl)-[1,2,3]triazol-1-yl]-1-
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phenylacetylaminoethaneboronate (9d). The reaction was terminated after 2.5 hours. The crude
residue was repeatedly washed with diethyl ether to afford 9d as a white solid (248 mg, 89% yield),
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mp 202ꢀ205 °C (dec). [α]D – 94.3 (c 1.0 , CH3OH). H NMR (400 MHz, CD3OD):
δ 0.88 (3H, s,
pynanyl CH3), 1.28 (3H, s, pynanyl CH3), 1.35 (3H, s, pynanyl CH3), 1.42 (1H, d, J = 10.4, Hendo),
1.70 – 2.40 (5H, m, pynanyl protons), 3.21 (1H, dd, J = 4.1, 10.5, BCH), 3.31 (2H, s, PhCH2), 4.12
(1H, dd, J = 2.3, 8.6, CHOB), 4.22 (1H, dd, J = 14.5, 10.5, BCHCH2), 4.50 (1H, dd, J = 4.2, 14.5,
BCHCH2), 7.20ꢀ7.35 (5H, m, Har), 7.56 (1H, t, J = 7.7, Har), 8.03 (2H, m, Har) 8.45 (1H, s,
CHtriazole), 8.48 (1H, s, Har). 13C NMR (100 MHz, CD3OD ):
δ 24.6, 27.6, 27.8, 29.7, 37.8, 38.1,
39.1, 41.4, 46.1 (br, CB), 53.7, 53.8, 77.5, 84.4, 123.2 (CHtriazole), 127.9, 128.7, 129.9, 130.2, 130.3,
130.4, 131.0, 132.4,134.1, 173.0 179.9. MS (ESI, Ion Trap): 529 [M + H]+, MSMS 529, m/z (%):
377 (26), 340 (100), 222 (8), 188 (14). Anal. Calcd for C29H33BN4O5: C, 65,92; H, 6,29; N, 10,60.
Found: C, 65,77; H, 6,50; N, 10,43.
(+)-Pinanediol
(1R)-2-[4-(3-amino-phenyl)-[1,2,3]triazol-1-yl]-1-(2-phenylacetylamino)
ethaneboronate (9e). The reaction was terminated after 2 hours. The crude residue was triturated
from diethylether/n.hexane (1 : 1) to afford 9e as light brown solid (250 mg, 96% yield), mp 125ꢀ
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128 °C. [α]D – 83.9 (c 0.45 , MeOH). H NMR (400 MHz, CD3OD):
δ 0.81 (3H, s, pynanyl CH3),
1.21 (3 H, s, pinanyl CH3), 1.28 (3H, s, pinanyl CH3), 1.34 (1H, d, J = 10.3, pinanyl Hendo), 1.85ꢀ
2.35 (5H, m, pinanyl protons), 3.14 (1H, dd, J = 4.2, 10.5, BCH), 3.67 (2H, s, CH2CO), 4.12 (1H,
dd, J = 2.3, 8.7, CHOB), 4.32 (1H, dd, J = 14.5, 10.5 , BCHCH2), 4. 46 (1H, dd, J = 14.5, 4.2,
BCHCH2), 6.64 (1H, d, J = 6.6, Har), 7.00ꢀ7.29 (8H, m, Har), 8.20 (1H, s, CHtriazole). 13C NMR (100
570
MHz, CD3OD):
δ
24.5, 27.6, 27.8, 29.8, 32.8, 37.8, 38.0, 39.2, 41.4 (CB), 53.69, 53.74, 77.4, 84.4,
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113.4, 116.6, 115.9, 122.5 (CHtriazole), 128.7, 129.9, 130.3, 130.7, 132.4, 134.1, , 149.3(CHtriazole
)
179.8. MS (ESI, Ion Trap): 500 [M + H]+, MSMS 500, m/z (%): 347 (21), 340 (100), 222 (7), 206
(11), 188 (19), 144 (23). Anal. Calcd for C28H34BN5O3: C, 67,34; H, 6,86; N, 14,02. Found: C, C,
67,18; H, 6,71; N, 13,89.
575
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(+)-Pinanediol (1R)-2-(4-carboxy-[1,2,3]triazol-1-yl)-1-(2-thienylacetylamino)ethaneboronate
(10a). The reaction was terminated after 30 min. The crude residue was crystallized from diethyl
ether and nꢀhexane to afford 10a as a beige solid (150 mg, 82% yield), mp 112ꢀ114°C. [α]D –71.6
580
(c 1.3, MeOH). 1H NMR (400 MHz, DMSOꢀd6):
δ 0.82 (3H, s, pinanyl CH3), 1.23 (3H, s, pinanyl
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CH3), 1.24 (3H, s, pinanyl CH3), 1.37 (1H, d, J = 9.9, Hendo), 1.64ꢀ2.23 (5H, m, pinanyl protons),
3.05 (1H, d, J = 8.6, BCH), 3.89 (2H, s, CH2CO), 4.06 (1H, d, J = 7.1, CHOB), 4.35 (1H, dd, J =
14.4, 10.6, BCHCH2), 4.43 (1H, dd, J = 14.4, 3.9, BCHCH2), 6.95ꢀ6.99 (2H, m, Har), 7.43 (1H, d, J
= 4.9, Har), 8.69 (1H, s, CHtriazole), 9.76 (1H, s, NH), 13.06 (1H, br, COOH). 13C NMR (100 MHz,
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DMSOꢀd6): δ 24.0, 26.2, 27.2, 29.2, 31.6, 36.5, 37.6, 39.3, 42.5 (br, CB), 52.0, 52.3, 75.5, 82.4,
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125.7, 126.8, 126.9, 129.3 (CHtriazole), 134.6, 139.5, 161.9, 175.1. MS (ESI, Ion Trap): 471 [M +
Na]+, 459 [M + H]+, MS/MS 459, m/z (%): 346 (44), 307 (100), 212 (6). HRMS (ESIꢀTOF) m/z:
[M–H]– Calcd for C21H26BN4O5S 457.1726; Found 457.1747. Anal. Calcd for C21H27BN4O5S: C,
55,03; H, 5,94; N, 12,22; S, 7,00. Found: C, 54.92; H, 6,05; N, 12,06; S, 6,81.
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(+)-Pinanediol
(1R)-2-[4-(1-hydroxy-1-methylethyl)-[1,2,3]triazol-1-yl)-1-(2-
thienylacetylamino)ethaneboronate (10b). The reaction was terminated after 30 min. The crude
residue was triturated from nꢀhexane to afford the compound 10b as pale yellow solid (217 mg,
92% yield), mp 76ꢀ78 °C. [α]D – 54.9 (c, 0.90 , MeOH). 1H NMR (400 MHz, CDCl3):
δ 0.90 (3H, s,
595
pinanyl CH3), 1.32 (3 H, s, pinanyl CH3), 1.43 (6H, s, (CH3)2COH), 1.47 (1H, d, J = 10.0, Hendo),
1.49 (3H, s, pinanyl CH3), 1.82ꢀ 2.41 (5H, m, pinanyl protons), 3.18ꢀ3.200 (1H, m, BCH), 3.95 (2H,
s, CH2CO), 4.26 (1H, dd, J = 8.6, 2.1, CHOB), 4.43 (1H, dd, J = 14.6, 3.4, BCHCH2), 4.51 (1H, dd,
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