Synthesis and biological evaluation of new dipicolylamine zinc chelators as metallo-β-lactamase inhibitors

Article abstract of DOI:10.1016/j.tet.2019.02.004

Antibiotics are key drugs in modern healthcare, especially in hospitals, where multiresistant bacteria resides and is a potential threat to human health. In the present work, a new series of adjuvants working synergistically with the carbapenem meropenem, in which a selective zinc-chelating agent was covalently linked to the small bacterial peptide D-Ala-D-Ala, was synthesized and tested against VIM-2 and NDM-1 metallo-β-lactamases (MBLs). The nature of the linker was modified in a structure-activity relationship study. Compound 1i, having an ethyl piperidine linker, lowered the MIC of meropenem from 32 to 64 mg/L to 2 and 1–2 mg/L against VIM-2- and NDM-1-producing clinical isolates, respectively. The IC₅₀ value of 1i against VIM-2 was 9.8 and 2.2 μM after 5 and 20 min, respectively. Compound 1i also showed intrinsic toxicity against three eukaryotic human tumoral cell lines between 50 and 120 μM.

Full text of DOI:10.1016/j.tet.2019.02.004

Tetrahedron xxx (xxxx) xxx
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis and biological evaluation of new dipicolylamine zinc
chelators as metallo-b-lactamase inhibitors
a
,
b
b
b
c
Anthony Prandina , Sylvie Radix , Marc Le Borgne , Lars Petter Jordheim ,
Zineb Bousfiha , Christopher Fr o€ hlich , Hanna-Kirsti S. Leiros , Ørjan Samuelsen f,
Espen Frøvold , Pål Rongved , Ove Alexander Høgmoen Åstrand
Department of Pharmaceutical Chemistry, School of Pharmacy, University of Oslo, PO Box 1068 Blindern, 0316, Oslo, Norway
EA 4446 Bioactive Molecules and Medicinal Chemistry, Facult ꢀe de Pharmacie e ISPB, Universit ꢀe de Lyon, Universite Lyon 1, SFR Sant ꢀe Lyon-Est CNRS
c
d,
e
e
d,
a
a
a, *
a
b
UMS3453 - INSERM US7, F-69373, Lyon cedex 08, France
c
Centre de Recherche en Canc ꢀe rologie de Lyon, INSERM 1052, CNRS UMR5286, Centre L ꢀe on B ꢀe rard Universit ꢀe de Lyon, Universit ꢀe Claude Bernard Lyon 1, 8
avenue Rockefeller, 69373, Lyon cedex 8, France
d
Norwegian National Advisory Unit on Detection of Antimicrobial Resistance, Department of Microbiology and Infection Control, University Hospital of
North Norway, 9038, Tromsø, Norway
e
The Norwegian Structural Biology Centre (NorStruct), Department of Chemistry, UiT The Arctic University of Norway, 9037 Tromsø, Norway
Department of Pharmacy, UiT The Arctic University of Norway, 9037, Tromsø, Norway
f
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 October 2018
Received in revised form
Antibiotics are key drugs in modern healthcare, especially in hospitals, where multiresistant bacteria
resides and is a potential threat to human health. In the present work, a new series of adjuvants working
synergistically with the carbapenem meropenem, in which a selective zinc-chelating agent was covalently
linked to the small bacterial peptide D-Ala-D-Ala, was synthesized and tested against VIM-2 and NDM-1
metallo-b-lactamases (MBLs). The nature of the linker was modified in a structure-activity relationship
study. Compound 1i, having an ethyl piperidine linker, lowered the MIC of meropenem from 32 to 64 mg/L
to 2 and 1e2 mg/L against VIM-2- and NDM-1-producing clinical isolates, respectively. The IC50 value of 1i
29 January 2019
Accepted 1 February 2019
Available online xxx
Keywords:
against VIM-2 was 9.8 and 2.2
toxicity against three eukaryotic human tumoral cell lines between 50 and 120
© 2019 Published by Elsevier Ltd.
m
M after 5 and 20 min, respectively. Compound 1i also showed intrinsic
MBL-producing gram negative bacteria
Multidrug resistant bacteria
Zinc chelators
Dipicolylamine derivatives
Antibiotic adjuvant
mM.
́
1
. Introduction
antibiotic era in which common infections could once again
become life threatening [1,2]. The increase in mortality with
bloodstream infections caused by methicillin-resistant S. aureus
(MRSA) and third-generation cephalosporin-resistant E. coli is sig-
nificant, and the prolongation of hospital stay imposes a consid-
erable burden on health care systems [3,4]. The introduction of
more potent alternatives of existing antibiotics provides only
temporary solutions, since existing resistance mechanisms rapidly
adapt to accommodate these new drugs [5,6].
Antimicrobial resistance (AMR), and in particular antibacterial
resistance, is an increasingly serious threat to global public health.
AMR develops when a microorganism (bacteria, fungus, virus or
parasites) no longer responds to a drug to which it was originally
sensitive [1,2]. Antibiotics have an enormous impact on modern
medicine. They are essential in the treatment of many human
diseases such as urinary tract infections, wound infections, blood-
stream infections, pneumonia, tuberculosis and they are a prereq-
uisite for chemotherapy or surgery. Without harmonized and
immediate worldwide action to develop agents countering highly
resistant bacteria (e.g. Escherichia coli, Klebsiella pneumoniae and
Staphylococcus aureus), the world is heading towards a post-
b
-lactam antibiotics have been the largest and most important
group of antimicrobial drugs since the discovery of penicillins. The
most important antibiotic resistance mechanism against -lactam
antibiotics, in terms of distribution and clinical relevance, are
lactamases [7]. These enzymes hydrolyze -lactam antibiotics
compromising their efficacy [8,9]. -lactamases are structurally
grouped into two super families, the serine -lactamases (SBLs) and
the metallo- -lactamases (MBLs), which hydrolyze -lactams by
b
b-
b
b
b
*
Corresponding author.
b
b
E-mail address: o.a.h.astrand@farmasi.uio.no (O.A.H. Åstrand).
https://doi.org/10.1016/j.tet.2019.02.004
040-4020/© 2019 Published by Elsevier Ltd.
0
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b-lactamase

2
A. Prandina et al. / Tetrahedron xxx (xxxx) xxx
two conceptually different mechanisms [10,11]. The SBLs utilize an
active site serine while MBLs require divalent cations, always zinc,
to present simultaneously the chemical diversity of the linkers used
and their impact on inhibition of MBLs and potentiation of carba-
penem activity in MBL-producing bacteria.
As shown in Fig. 2, D-Ala-D-Ala was used as the peptide and DPA
as the zinc chelator, respectively, and they were connected via ten
different linkers in this study. A non-chelating negative control 14
was included together with compound 10 without linker and with
the chelator directly attached to D-Ala-D-Ala.
for the hydrolysis of
of inhibitors in combination with
success and extended the therapeutic life of
15]. Although evolution of -lactamases also has counteracted this
b
-lactams [12e14]. Inhibition of SBLs by the use
-lactams has been a therapeutic
-lactam antibiotics
b
b
[
b
approach, the recent development and introduction of new SBL
inhibitors such as avibactam show the viability of this approach [16].
However, there are no clinically available MBL inhibitors and an
analysis of reported MBL inhibitors [12,17,18] makes it clear that the
clinical need for a MBL inhibitor is more than ever a priority for
medicinal chemists and drug developers. Since some studies
revealed the importance of zinc homeostasis for the regulation of
MBLs and generally bacterial pathogens, [19,20] zinc chelation has
become an exciting strategy to overcome bacterial resistance.
Recent works [21e24] validated this approach by using small
molecule zinc chelating agents to potentiate the activity of mer-
2. Results and discussion
2.1. Synthesis of bivalent hybrids 1a-j
All bivalent hybrids 1a-j were obtained following a simple and
straightforward chemical pathway, which mainly involved amide
bond formation reactions between linker and the D-Ala-D-Ala
dipeptide or linker and the zinc chelator DPA. Hence, mono Boc-
protected diamines 2a-j were used as linker precursors since they
permit the coupling of two different fragments (Fig. 3).
Most of the mono Boc-protected diamines were commercially
available except 2g and 2h which were obtained with good yields
by regioselective protection from 4-(2-aminoethyl)aniline and di-
tert-butyl dicarbonate (Scheme 1). Actually, carrying out the pro-
tection reaction under classical conditions allowed the synthesis of
openem (MEM), a carbapenem b-lactam susceptible to inactivation
by MBLs. New small molecules have also emerged very recently on
the basis of 2,6-dipicolinic acid which displayed a propensity to
chelate zinc [25,26]. We have earlier studied TPA as a zinc chelator,
[
27] however, we now wanted to study dipicolylamine (DPA) as the
chelating moiety [28,29]. As metal chelators are generally toxic, we
need a chelator that is selective for zinc, and by selective we mean
that they bind weaker to iron, manganese, sodium, potassium,
calcium and other relevant biological cations than to zinc. The DPA
based zinc chelators we use fit this criterion and that is why, we
think, we are able to get a decent toxicity profile. In parallel, we
chose to connect the bacterial dipeptide D-Ala-D-Ala to the linker-
chelator construct in hope it would modulate the lipophilicity of
2g [31] while the use of 10% aqueous acetic acid facilitated selective
introduction of the Boc group on the aromatic amine [32].
The dipeptide Boc-D-Ala-D-Ala-OH (4) used as the peptide
precursor was prepared according to a two-step sequence
described in Scheme 2. The synthesis started with the coupling
reaction between the amino and carboxyl protected alanines under
standard peptide synthetic procedure using HATU [33] (O-(7-
azabenzotriazolazabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate) as the coupling reagent and N-methyl-
morpholine (NMM) as a base. The fully protected dipeptide 3 was
then hydrolyzed to produce 4 in a good yield [34].
the construct in addition to mimicking the b-lactams [30]. In order
to study such bivalent hybrids, a series of linkers was selected to
link the zinc chelator to the peptide (Fig. 1). The aim of the study is
At this point, the synthesis of bivalent hybrids 1a-j was achieved
in five steps from the mono Boc-protected diamines 2a-j used as
starting materials (Scheme 3). First, compounds 2a-j were reacted
Fig. 1. General structure of a bivalent hybrid which consists of a lipophilic chelator
selective for zinc linked to a vector with selective affinity for bacterial cell structure.
ꢁ
with chloroacetyl chloride, at ꢀ78 C, to give the appropriate
a-
Fig. 2. Structure of putative MBL inhibitors 1a-j.
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b-lactamase

A. Prandina et al. / Tetrahedron xxx (xxxx) xxx
3
chloroamide derivatives 5a-j in excellent yield. DPA was then easily
introduced by nucleophilic substitution on chlorides 5a-j under
basic conditions and in presence of catalytic amounts of potassium
iodide to facilitate the reaction [35]. It is noteworthy that de-
rivatives 6a-j were obtained with a satisfactory purity level without
the need for chromatography if one equivalent of DPA was strictly
used. After deprotection of N-Boc amines by trifluoroacetic acid
(TFA) combined with freebase generation under basic conditions,
the vector, as the Boc-D-Ala-D-Ala-OH dipeptide 4, was coupled to
the free N-terminal amine of derivatives 7a-j according to the same
procedure described above to afford N-Boc-protected bivalent hy-
brids 8a-j in low to excellent yields. These latter compounds were
purified on C-18 reverse phase column chromatography to remove
the common HATU by-product N,N,N,N-tetramethylurea. Three
derivatives 8a, 8i and 8j were obtained with much lower yields.
Coupling of the ethylene derivative 8a led to the formation of
numerous side-products and problems were encountered during
the purification process of compounds 8i and 8j. A final Boc-
deprotection step readily afforded ZnChel compounds 1a-j with
good yields. In addition, it should be noted that all the piperidinyl
linker derivatives were shown to exist as a mixture of amide
rotamers by NMR spectroscopy.
Fig. 3. General structure of linker precursors.
Two control compounds 10 and 14 were synthesized to compare
their antibacterial activities to those obtained for bivalent hybrids
1a-j and thus to examine the influence of the linker fragment and
the relevance of the DPA moiety, respectively. Therefore, the
construct DPA-D-Ala-D-Ala 10 was designed without any linker to
conjugate the zinc chelator fragment and the vector moiety. Com-
pound 10 was readily synthesized in two steps (59% overall yield)
from dipeptide 4 by coupling reaction with DPA following by a N-
Boc deprotection/neutralization sequence (Scheme 4).
To study the relevance of the zinc chelator, we designed a sec-
ond construct, compound 14, in which DPA fragment was replaced
by dibenzylamine moiety. Therefore, the synthetic pathway to
prepare 14 described in Scheme 5 was analogous to that performed
for 1g. A nucleophilic substitution was carried out between
a-
chloroacetamide 5g and dibenzylamine in presence of DIPEA (N,N-
diisopropylethylamine) and a catalytic amount of KI in refluxed
acetonitrile to give 11 with 68% yield. After the same N-Boc
deprotection/neutralization sequence as described above, the Boc-
D-Ala-D-Ala-OH dipeptide 4 was coupled to primary amine 12 to
afford the N-Boc-protected derivative 13 which was finally depro-
tected in acidic conditions to give the construct 14 as a hydro-
chloride salt.
Scheme 1. Syntheses of mono-Boc-protected diamines 2g and 2h.
3. Biological activities
3.1. Microbrothdilution MIC assay
To investigate the synergistic potential of the newly synthesized
compounds in combination with MEM, they were subjected to
in vitro testing against two clinical Gram-negative strains of
P. aeruginosa [36] and K. pneumoniae [37] each harboring the
metallo
Both strains were resistant to MEM alone with MIC values of
2e64 mg/L. Addition of 125 M of the strong chelator TPEN
(N,N,N ,N -tetrakis (2-pyridinylmethyl)-1,2-ethanediamine) low-
ered the MIC values of MEM to 1 mg/L (P. aeruginosa, VIM-2) and ꢂ
.5 mg/L (K. pneumoniae, NDM-1). When evaluated alone, the
compounds 1a-j and 10 did not present any antibacterial activity up
to 1000 M (data not shown). However, synergistically all of our
b-lactamases VIM-2 and NDM-1, respectively (Table 1).
3
(
m
0
0
0
Scheme 2. Synthesis of the Boc-D-Ala-D-Ala-OH dipeptide 4 as vector precursor. Re-
m
ꢁ
agents and conditions: a) HATU, NMM, DMF, 0 C for 1 h then RT, overnight; b) i) NaOH
compounds, with the exception of 1g and 1h, were able to lower
the MIC of MEM. Compound 1i even demonstrated comparable MIC
results to TPEN and reduced the MIC of MEM to 2 mg/L
ꢁ
6
M, 0 C for 30 min then RT for 2 h; ii) HCl 2 M (pH 7), Et
2
O.
Please cite this article as: A. Prandina et al., Synthesis and biological evaluation of new dipicolylamine zinc chelators as metallo-
inhibitors, Tetrahedron, https://doi.org/10.1016/j.tet.2019.02.004
b-lactamase

4
A. Prandina et al. / Tetrahedron xxx (xxxx) xxx
ꢁ
Scheme 3. General synthetic pathways to ZinChel compounds 1a-j. Reagents and conditions: a) chloroacetyl chloride, NEt
3
, CH
2
Cl
2
, -78 C for 30e60 min then RT for 24 h; b) DPA
ꢁ
ꢁ
(
1.0 equiv.), KI (cat.), DIPEA, CH
3
CN, reflux, 16 h; c) i) TFA, CH
2
Cl
2
, 0 C to RT, 3 h; ii) aq. K
2
CO
3
(1 M); d) Boc-D-Ala-D-Ala-OH 4, HATU, NMM, CH
2
Cl
2
, 0 C to RT, 4.5 h.
(
P. aeruginosa, VIM-2) and 1e2 mg/L (K. pneumoniae, NDM-1)
Ala-D-Ala was directly bound to the chelator via an amide bond did
not lower the MIC value of meropenem despite having the same
chelator structure as compound 1a-j. This could be due to metal
which is below the clinical breakpoint according to EUCAST
Version 8.0, 2018. http://www.eucast.org. Compound 10, where D-
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A. Prandina et al. / Tetrahedron xxx (xxxx) xxx
5
Scheme 4. Synthesis of the control compound 10. Reagents and conditions: a) DPA (1.0
ꢁ ꢁ
2 2 2 2
Cl , 0 C to RT, 4 h; b) i) TFA, CH Cl , 0 C to RT, 3 h; ii) aq.
equiv.), HATU, NMM, CH
NaOH (1 M).
assisted hydrolysis of the amide in the presence of zinc or other
metal ions [38]. Compound 14, where the 2-pyridine rings were
replaced with phenyl groups, showed no synergistic properties
with MEM, as was expected since it lacks zinc-chelating properties.
3.2. Enzyme inhibition assay
The synthesized compounds were preincubated for 5 and
0 min with purified VIM-2 and residual enzymatic activity was
determined (Table 1). All compounds, except 1h, demonstrated
time-dependent inhibition as the IC50 values dropped considerably,
Scheme 5. Synthesis of the reference compound 14. Reagents and conditions: a)
3
9
ꢁ
2
dibenzylamine, KI (cat.), DIPEA, CH CN, reflux, 17 h; b) i) TFA, CH Cl , 0 C to RT, 1 h; ii)
3
2
2
ꢁ
aq. K
2
CO
3
(1 M); c) Boc-D-Ala-D-Ala-OH 4, HBTU, NMM, CH
, 0 C to RT, 1 h; ii) HCl (2 M), CH
2
Cl
2
, 0 C to RT, 3 h; d) i)
ꢁ
TFA, CH
2
Cl
2
2
Cl
2 2
, Et O.
going for example with compound 1d from 14.7 to 2.7
pound 1i, which had the best synergistic effect in combination with
MEM in the MIC assays, had also a low IC50 value (2.8 M at 20 min).
Interestingly, compounds 1b and 1g demonstrated enzyme inhi-
M at 20 min,
respectively). However, no synergistic activity in combination with
MEM in the MIC assay (Table 1) could be shown. This could indicate
permeability differences across the bacterial cell wall between the
compounds.
mM. Com-
compounds 1a-j. IC50 values were determined after 72 h exposure
to compounds 1a-j or other metal chelators as TPEN or DPA as
described in the procedures. As expected, the strong chelator, TPEN,
m
bition comparable to compound 1i (IC50 ¼ 2.5 and 1.4
m
ꢀ15
ꢀ16
M,
which have a dissociation constant (K
41] was more than one order of magnitude more toxic than the
other compounds, with IC50 values between 3 and 5 M, which is
far below the 125 M concentration used in the MIC assays. On the
other hand, DPA had IC50 values ranging from 57.2 to 104.3 M. The
new compounds generally showed high IC50 values between 100
and 200 M against the MDA-MB-231 and Mia-PaCa2 cell lines, and
slightly lower for Colo-357 (50e175 M). This is within the con-
d
) of 10
M ꢀ 2.6 ꢃ 10
[
m
m
m
3.3. Cell sensitivity assay
m
m
A common challenge with metal chelators is their unwanted
centration range used in the synergistic MIC assays with MEM and
points towards a possible selectivity challenge. Compound 14,
which is not a zinc chelator and did not show any synergistic ac-
tivity with MEM in the MIC assays, was only slightly less toxic than
TPEN. This was unexpected, and we can only speculate on why this
eukaryotic toxicity. In this work, the well-described colorimetric
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide) assay [40] was used to evaluate the sensitivity of human
breast cancer cells (MDA-MB-231) and pancreatic cancer cells
(
MIA-PaCa2 and Colo-357) towards all synthesized hybrid
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b-lactamase

6
A. Prandina et al. / Tetrahedron xxx (xxxx) xxx
Table 1
Effect of compounds 1a-j on the potentiation of MEM against different clinical strains of MBL-producing P. aeruginosa and K. pneumoniae (MIC values), enzymatic inhibition of
the compound 1a-j (IC50) and the MDA-MB-231, Mia-PaCa2 and Colo-357 cell lines sensitivity (IC50).
Cpd
Y
MIC (mg/L)a
Pure enzyme inhibition
Intrinsic Toxicity IC50 (mM)
VIM-2 IC50
time (min)
(
mM) Incubation
P. aeruginosa
VIM-2
K. pneumoniae
NDM-1
5
20
MDA-MB
231
Mia-PaCa2
Colo-357
MEM
TPEN
DPA
32e64
1
32e64
ꢂ0.5
ND
ND
ND
ND
30.7
ND
ND
ND
6.3
ND
ND
ND
e
e
e
4.8 ± 1.2
104.3 ± 47.8
152.5 ± 48.1
3.7 ± 1.2
38.7 ± 12.2
137.6 ± 26.6
5.0 ± 0.8
57.2 ± 22.5
78.8 ± 6.2
ND
32
1
1
1
1
1
a
b
c
8
16
16
8
4
4
4
2
6.1
2.5
210.3 ± 50.7
121.0 ± 4.2
168.6 ± 77.5
164.8 ± 63.0
134.2 ± 32.5
117.5 ± 13.6
117.9 ± 33.0
119.5 ± 17.4
99.4 ± 17.3
175.8 ± 86.1
105.9 ± 20.5
85.2 ± 11.3
16.8
14.7
31.4
14.2
2.7
d
e
8
17.9
1
1
f
8
8
14.2
2.3
3.4
1.4
193.2 ± 66.1
127.2 ± 91.6
121.6 ± 28.0
118.4 ± 31.5
100.3 ± 17.5
37.9 ± 19.0
g
32
16
1h
32
16
76.0
75.8
178.0 ± 41.2
130.1 ± 70.8
140.4 ± 50.7
1
i
j
2
8
1e2
9.8
2.8
6.9
116.6 ± 53.4
148.7 ± 18.3
56.0 ± 11.3
50.1 ± 13.1
92.6 ± 28.6
1
4
24.7
142.4 ± 53.1
1
0
4
32
64
32
64
>125
>125
>125
>125
ND
ND
ND
e
e
1
15.4 ± 10.0
12.7 ± 4.3
20.4 ± 5.1
ND: not determined. MIC assay performed as one biological replicate and two technical replicates. For assays on human cells, all experiments were performed in triplicate and
at least three times. All values are expressed as the mean ± SD.
a
For MIC determination, all compounds were tested at 125 mM in co-administration with MEM.
is the case. In conclusion, the three human cancer cell lines showed
a lower sensitivity to all the tripartite compounds, including the
most active compound 1i, than to TPEN or DPA. However, the
intrinsic toxicity of the new compounds are too high to be used in
the present form and must be optimized further.
nature of the linker plays a crucial role in the ability of these
bivalent hybrids 1a-j to potentiate the activity of MEM since
compound 10 did not show any activity.
Of all the new hybrids studied in this article, the compound 1i
with a 4-substituted piperidine linker chain showed the best syn-
ergistic activity in combination with MEM in the MIC assays against
clinical isolates of P. aeruginosa and K. pneumoniae harboring VIM-2
and NDM-1, respectively. 1i demonstrated a potent inhibitory ac-
4
. Conclusions
tivity against purified VIM-2 enzyme (IC50 ¼ 2.8
mM after 20 min).
In this work, we described the straightforward synthesis of ten
new DPA zinc chelators 1a-j as MBL inhibitors. Two negative con-
trols without linker (compound 10) or without zinc chelator
Moreover, breast cancer cell line (MDA-MB231) and pancreatic
cancer cell lines (Mia-PaCa2 and Colo-357) showed a lower sensi-
tivity to all the bivalent hybrids, including the most active
(
compound 14) were added to complete our study. As expected, the
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A. Prandina et al. / Tetrahedron xxx (xxxx) xxx
7
compound 1i, than to TPEN or DPA alone. However, 1i, is still only
(pentet, J ¼ 7.3 Hz, 1H), 4.17 (s, 1H), 3.73 (s, 3H, COOCH
3
), 1.43 (s, 9H,
effective at concentrations close to the eukaryotic IC50 values and
must be improved further in order to be safe and effective.
However, the moderate toxicity towards three human cell lines
C(CH
3
)
3
), 1.39 (d, J ¼ 7.2 Hz, 3H, CH
3
), 1.35 (d, J ¼ 7.1 Hz, 3H, CH
3
).
5.2.2. Synthesis of Boc-D-alanyl-D-alanine-OH (4)
To a 6 M NaOH solution (30 mL, 180 mmol, 10 equiv.) cooled to
(
all IC50 values > 50
m
M) was equal or lower than the dose used in
M). The ability of hybrid 1i
ꢁ
the co-administration MIC assay (125
m
0 C was added Boc-D-alanyl- -alanine methyl ester (4.94 g,
D
ꢁ
to potentiate the activity of MEM could be explained by the fact that
a zinc-atom could potentially bind to one more nitrogen piperidinyl
atom in 1i than in the other derivatives and possibly be removed
from the active site in the enzyme. Finally, it is worth noting that
hybrids 1b and 1g displayed an even higher inhibitory activity
against the purified VIM-2 than compound 1i while the former
ones did not potentiate the activity of MEM. This potential differ-
ence of mechanism of action should be considered in further
studies. However, more potent compounds with lower eukaryotic
toxicity should be explored by changing the vector and chelator
moiety in a multifactorial experimental design to optimize the MIC
performance further towards a potential drug against antimicrobial
resistance.
18 mmol, 1 equiv.). The mixture was left to stir at 0 C for 30 min
and for 2 more h at room temperature. HCl was added to adjust the
pH to 7. The compound was extracted using EtOAc (3 ꢃ 100 mL) and
dried over MgSO . Filtration and solvent removal under reduced
4
pressure gave title compound 4 as a white solid (3.375 g, 72% yield):
1
H NMR (400 MHz, CDCl ):
3
d
¼ 7.96 (s,1H, COOH), 7.04 (d, J ¼ 7.5 Hz,
), 4.26 (m,
), 1.46e1.41 (m, 12H, C(CH ) , CH ), 1.35 (d, J ¼ 7.0 Hz, 3H,
1H, NH), 5.35 (s, 1H, NH), 4.56 (pentet, J ¼ 7.2 Hz, 1H, CH
a
1H, CH
CH3).
a
3
3
3
5.2.3. General procedure for synthesis of 2-chloroacetamide
derivatives (5a-j) from mono-Boc protected diamines (2a-j)
A solution of the corresponding mono-Boc-protected diamine
ꢁ
2
a-j (1.0 equiv.) in CH
dry ice). NEt
chloroacetyl chloride (1.2 equiv.) in CH
2
Cl
2
(0.5 M) was cooled to ꢀ78 C (acetone/
5
. Experimental section
3
(1.5 equiv.) was rapidly added before a solution of
Cl (0.5 M) was added
2
2
5.1. General
dropwise for 30e60 min. The resulting white slurry mixture was
left to warm up to room temperature to become dark brown, and
then was stirred at room temperature for 24 h. The resulting
mixture was washed with 0.5 M citric acid (3 times) and then with
water (twice). The organic layer was extracted, dried over K CO
2 3
and the solvent removed under reduced pressure to give the title
compound as a dark brown oil.
All reagents and solvents were of analytical grade and were used
as received, without further purification. Compounds 2g [31] and
1
2
h [32] were synthesized according to literature procedures. H
spectra were recorded with Bruker DRX400 or DRX300 Fourier
transform spectrometers, using an internal deuterium lock, oper-
13
ating at 400 MHz or 300 MHz. C NMR spectra were recorded with
a Bruker DRX400 or DRX300 Fourier transform spectrometers,
using an internal deuterium lock, operating at 100 MHz or 75 MHz.
5
.2.3.1. Tert-butyl (2-(2-chloroacetamido)ethyl)carbamate (5a) [42].
1
Yield (6.298 g, 85%): H NMR (300 MHz, CDCl
NH), 4.89 (bs, 1H, NH), 4.03 (s, 2H, CH Cl), 3.40 (m, 2H), 3.31 (m,
3
):
d
¼ 7.18 (bs, 1H,
ꢁ
All spectra were recorded at 25 C. Chemical shifts are reported in
2
parts per million (ppm) relative to residual protons or carbons
2
H), 1.44 (s, 9H, C(CH
3 3
) ).
1
thirteen of deuterated solvent (
d
¼ 2.50 ppm for H NMR and
13
1
d
¼ 39.52 ppm for C NMR for DMSO‑d
6
,
d
d
¼ 7.26 ppm for H NMR
5
.2.3.2. Tert-butyl (4-(2-chloroacetamido)butyl)carbamate (5b) [42].
13
1
and
and
NMR and
d
¼ 77.16 ppm for C NMR for CDCl
3
,
¼ 3.31 ppm for H NMR
1
Yield (1.166 g, 83%): H NMR (400 MHz, CDCl
NH), 4.60 (bs, 1H, NH), 4.04 (s, 2H, CH
Cl), 3.32 (quartet, J ¼ 6.5 Hz,
2H, CH NH), 1.63e1.47 (m,
NH), 3.14 (quartet, J ¼ 6.5 Hz, 2H, CH
H), 1.43 (s, 9H, C(CH ); C NMR (100 MHz, CDCl ):
¼ 166.0
(CO), 156.1 (NCOOtBu), 79.4 (C(CH ), 42.8 (CH ), 40.2 (CH ), 39.6
(CH ), 28.5 (C(CH ) ), 27.6 (CH ), 26.7 (CH ); HRMS-ESI m/z
3
):
d
¼ 6.68 (bs, 1H,
13
1
d
¼ 49.00 ppm for C NMR for CD
3
OD,
d
¼ 1.94 ppm for
H
1
3
2
d
¼ 1.32 and 118.26 ppm for C NMR for CD
3
CN and
1
2
2
d
¼ 4.79 ppm for H NMR for D
2
O). Carbon multiplicity was deter-
13
4
3
)
3
3
d
mined by DEPT experiments. Electron-Spray low-resolution mass
spectra were recorded on a Thermo ALCQ Advantage spectrometer
or Agilent 6120 spectrometer. High-resolution mass spectra were
recorded on a Bruker MicroTOF Q or ThermoQuest FINNIGAN MAT
3
)
3
2
2
2
3 3
2
2
þ
[MþNa] calculated for C H ClNaN O : 287.1133, found: 287.1133.
11
21
2 3
9
3
5 XL apparatus operating at 70eV (for compounds 5a-j, chlorine-
5 isotope was the only isotope to be analyzed in mass spectro-
5
.2.3.3. Tert-butyl (6-(2-chloroacetamido)hexyl)carbamate (5c).
metric measurements). TLC analyses were carried out using Meck
Aluminum Oxide 60 F256 pates visualized by UV light.
1
Yield (730 mg, 93%): H NMR (400 MHz, CDCl
NH), 4.52 (bs, 1H, NH), 4.04 (s, 2H, CH
H, CH
NH), 3.10 (t, J ¼ 7.0 Hz, 2H, CH
J ¼ 6.8 Hz, 2H), 1.43 (s, 9H, C(CH
100 MHz, CDCl ):
2.8 (CH ), 39.8 (CH
(CH ), 26.4 (CH
3
):
d
¼ 6.63 (bs, 1H,
2
Cl), 3.29 (td, J ¼ 7.2, 6.0 Hz,
NH), 1.54 (m, 2H), 1.47 (t,
2
2
2
5
5
.2. Chemistry
13
3
)
3
), 1.37e1.31 (m, 4H); C NMR
(
3
d
¼ 165.9 (CO), 156.2 (NCOOtBu), 79.4 (C(CH
2 2 2 3 3
3
)
3
),
.2.1. Synthesis of methyl Boc-D-alanyl-D-alaninate (3)
4
2
), 30.1 (CH ), 29.4 (CH ), 28.6 (C(CH ) ), 26.5
D-Ala-OMe HCl (3.688 g, 26.4 mmol, 1 equiv.), Boc-D-Ala-OH
); HRMS-ESI m/z [MþNa]þ calculated for
(
5.00 g, 26.4 mmol, 1 equiv.), and HATU (10.048 g, 26.4 mmol, 1
2
2
ꢁ
13 25 2 3
C H ClNaN O : 315.1445, found: 315.1451.
equiv.) were dissolved in DMF (25 mL) and cooled to 0 C in an ice
bath. N-methylmorpholine (28.14 mL, 52.53 mmol, 2 equiv.) was
ꢁ
slowly added and the mixture was left to stir at 0 C for 1 h before
5.2.3.4. Tert-butyl (1-chloro-2-oxo-7,10,13-trioxa-3-azahexadecan-
16-yl)carbamate (5d). Yield (801 mg, 72%): H NMR (400 MHz,
1
warming up to room temperature. The reaction was left stirring
overnight before it was diluted with 350 mL of water and extracted
with small portions of EtOAc (3e10 ꢃ 20e40 mL). Combined
organic extracts were washed with 0.1 M HCl (2 ꢃ 50 mL), 0.5 M
CDCl
3
):
d
¼ 4.95 (bs, 1H, NH), 4.01 (bs, 2H, CH
2
Cl), 3.66e3.55 (m,
NH),
NH), 1.92 (s, 1H), 1.81 (pentet,
), 1.74 (pentet, J ¼ 6.3 Hz, 2H,
1
0H), 3.52 (t, J ¼ 6.0 Hz, 2H), 3.42 (quartet, J ¼ 5.9 Hz, 2H, CH
2
3
.21 (quartet, J ¼ 6.4 Hz, 2H, CH
CH CH
2
NaHCO
phase was dried over MgSO
title compound 3 as a white powder (5.432 g, 75% yield): H NMR
400 MHz, CDCl ):
¼ 6.9 (bs, 1H, NH), 5.06 (s, 1H, NH), 4.56
3
(2 ꢃ 50 mL), and with brine (50 mL). Resulting organic
J ¼ 5.9 Hz, 2H, CH
2
2
2
4
, filtered and evaporated to give the
13
1
CH CH CH ), 1.42 (s, 9H, C(CH ) ); C NMR (100 MHz, CDCl ):
2
2
2
3 3
3
(
3
d
d
¼ 166.0 (CO), 156.1 (NCOOtBu), 79.0 (C(CH
3
)
3
), 70.69 (CH
2
), 70.67
Please cite this article as: A. Prandina et al., Synthesis and biological evaluation of new dipicolylamine zinc chelators as metallo-
inhibitors, Tetrahedron, https://doi.org/10.1016/j.tet.2019.02.004
b-lactamase

8
A. Prandina et al. / Tetrahedron xxx (xxxx) xxx
(
CH
2
), 70.60 (CH
2
), 70.57 (CH
2
), 70.3 (CH
2
), 69.7 (CH
), 28.7 (CH ), 28.6 (C(CH
33ClNaN : 419.1921,
2
), 42.8 (CH
2
),
2H), 3.23 (quartet, J ¼ 6.0 Hz, 2H), 2.54e2.37 (m, 6H), 1.44 (s, 9H,
13
C(CH
3
)
3
);
C NMR (100 MHz, CDCl
), 57.2 (CH ), 52.9 (CH
), 37.1 (CH ), 28.5 (C(CH
25ClN 306.1580, found:
3
):
d
¼ 165.1 (CO), 156.0
), 52.5 (CH ), 46.4
); HRMS-ESI
3
8.9 (CH ), 38.7 (CH
2
2
), 29.8 (CH
2
2
3 3
)
);
þ
HRMS-ESI m/z [MþNa] calculated for C17
H
2
O
6
(NCOOtBu), 79.4 (C(CH
3
)
3
2
2
2
found: 419.1924.
(
CH
2
), 42.2 (CH
2
), 41.0 (CH
2
2
3 3
)
m/z [MþH]þ calculated for
C
13
H
3 3
O :
5
.2.3.5. Tert-butyl (cis-4-(2-chloroacetamido)cyclohexyl)carbamate
306.1579.
1
(
5e). Yield (875 mg, 80%): H NMR (300 MHz, CDCl
J ¼ 7.9 Hz, 1H, NH), 4.77 (s, 1H, NH), 3.94 (s, 2H, CH
J ¼ 7.7, 4.1 Hz, 1H), 3.53 (m, 1H), 1.73e1.59 (m, 4H), 1.58e1.43 (m,
3
):
d
¼ 6.61 (d,
2
Cl), 3.83 (tt,
5.2.4. General procedure for nucleophilic substitution by
dipicolylamine [14]
13
4
H), 1.35 (s, 9H, C(CH
55.1 (NCOOtBu), 79.1 (C(CH
8.6 (CH ), 28.4 (C(CH ), 27.8 (CH
2 3
calculated for C13 23ClNaN O : 313.1292, found: 313.1294.
3
)
3
); C NMR (75 MHz, CDCl
3
):
d
¼ 165.0 (CO),
The corresponding 2-chloroacetamide 5a-j (1.0 equiv.) and KI
(0.6 equiv.) were dissolved in MeCN (0.05 M) and DPA (1.0 equiv.)
was added to the stirring mixture. DIPEA (9.7 equiv.) was then
added and the mixture was heated to reflux for 16 h. After cooling
to room temperature, the mixture was concentrated under reduced
1
3
)
3
), 46.5 (CH), 46.2 (CH), 42.7 (CH
2
Cl),
2
2
3
)
3
2
); HRMS-ESI m/z [MþNa]þ
H
2 2
pressure, dissolved in CH Cl , and washed with a 1 M NaOH solu-
5
.2.3.6. Tert-butyl
mate (5f). Reaction solvent THF, yield (1.032 g, 76%): H NMR
400 MHz, CDCl ):
¼ 6.39 (m, 1H, NH), 4.44 (s, 1H, NH), 4.01 (s, 2H,
CH Cl), 3.74 (m, 1H), 3.42 (m, 1H), 2.08-1.96 (m, 4H), 1.43 (s, 9H,
C(CH
(trans-4-(2-chloroacetamido)cyclohexyl)carba-
1
tion, to give title compounds 6a-j as orange sticky oils generally
employed without further purification in the following step. In the
case of trace amounts of DPA, the compound was purified by
neutral alumina column chromatography (1e2% MeOH in CH Cl ).
(
3
d
2
1
3
2
2
3
)
3
), 1.35e1.13 (m, 4H); C NMR (100 MHz, CDCl
CO), 155.3 (NCOOtBu), 79.5 (C(CH ), 48.9 (CH), 48.3 (CH), 42.8
), 28.5 (C(CH ); HRMS-ESI m/z
23ClNaN 313.1292, found:
3
):
d
¼ 165.3
(
3 3
)
5
.2.4.1. Tert-butyl (2-(di-2-picolylaminoacetamido)ethyl)carbamate
(
[
CH
2
Cl), 32.0 (CH
2
), 31.5 (CH
2
3 3
)
1
3
(6a). Yield (3.37 g, quantitative yield): H NMR (400 MHz, CDCl ):
þ
MþNa]
calculated for
C
13
H
2 3
O :
d
¼ 8.82 (t, J ¼ 6.3 Hz, 1H, NH), 8.62 (d, J ¼ 4.9 Hz, 2H), 7.67 (t, J ¼ 7.7,
313.1289.
1
.8 Hz, 2H), 7.30 (d, J ¼ 7.7 Hz, 2H), 7.21 (m, 2H), 5.97 (t, J ¼ 5.7 Hz,
1
H), 3.85 (s, 4H, 2 ꢃ CH
2 2
Pyr), 3.43 (m, 2H, CH NH), 3.35e3.23 (m,
5.2.3.7. Tert-butyl
(4-(3-chloro-2-oxopropyl)phenethyl)carbamate
4H, CH NH, CH CO), 1.42 (s, 9H C(CH ) ); 13C NMR (100 MHz,
1
(5g). Yield (7.46 g, 75%): H NMR (400 MHz, DMSO‑d
6
):
d
¼ 10.21 (s,
2 2 3 3
CDCl
2 ꢃ CH Pyr), 137.3 (2 ꢃ CH Pyr), 123.9 (2 ꢃ CH Pyr), 122.8 (2 ꢃ CH
Pyr), 79.1 (C(CH Pyr), 58.2 (NCH CO), 40.9 (CH ),
9.3 (CH ), 28.5 (C(CH
3
):
d
¼ 171.1 (CO), 157.3 (2 ꢃ Cquat Pyr), 156.1 (NCOOtBu), 149.2
1
H, NH), 7.49 (d, J ¼ 8.4 Hz, 2H), 7.14 (d, J ¼ 8.4 Hz, 2H), 6.84 (t,
(
J ¼ 5.6 Hz, 1H, NH), 4.23 (s, 2H, CH
2
Cl), 3.08 (m, 2H), 2.65 (m, 2H),
):
¼ 164.4 (CO),
13
1.36 (s, 9H, C(CH
3
)
3
); C NMR (100 MHz, DMSO‑d
6
d
3
)
3
), 59.9 (2 ꢃ CH
2
2
2
); HRMS-ESI m/z [MþH]þ calculated for
1
55.5 (NCOOtBu), 136.5 (Cquat arom), 134.9 (Cquat arom), 128.9
2 ꢃ CH arom), 119.4 (2 ꢃ CHarom), 77.4 (C(CH ), 43.5 (CH ), 41.5
CH ), 34.9 (CH ), 28.2 (C(CH ).
3
2
3 3
)
(
(
3
)
3
2
21 30 5 3
C H N O : 400.2343, found: 400.2348.
2
2
3 3
)
5
.2.4.2. Tert-butyl (4-(di-2-picolylaminoacetamido)butyl)carbamate
1
3
(6b). Yield (1.611 g, quantitative): H NMR (400 MHz, CDCl ):
5
.2.3.8. Tert-butyl (4-(2-(2-chloroacetamido)ethyl)phenyl)carbamate
1
d
¼ 8.77 (t, J ¼ 5.6 Hz, 1H, NH), 8.54 (ddd, J ¼ 4.9, 1.8, 0.9 Hz, 2H),
(
5h). Yield (1.44 g, 99%): H NMR (400 MHz, CDCl
J ¼ 8.5 Hz, 2H), 7.11 (d, J ¼ 8.5 Hz, 2H), 6.59 (s, 1H, NH), 6.52 (s, 1H,
NH), 4.01 (s, 2H, CH NH), 2.79 (t,
Cl), 3.52 (td, J ¼ 6.9, 5.8 Hz, 2H, CH
J ¼ 7.0 Hz, 2H, ArCH ); C NMR (100 MHz,
CDCl ):
¼ 165.9 (CO), 152.9 (NCOOtBu), 137.1 (Cquat arom), 133.0
Cquat arom), 129.4 (2 ꢃ CHarom), 119.0 (2 ꢃ CHarom), 80.7
C(CH ), 42.8 (CH ), 41.1 (CH ), 34.9 (CH ), 28.5 (C(CH ); HRMS-
21ClNaN : 335.1128, found:
3
):
d
¼ 7.31 (d,
7
.60 (m, 2H), 7.24 (dt, J ¼ 7.7,1.0 Hz, 2H), 7.15 (ddd, J ¼ 7.5, 4.8,1.2 Hz,
2
2
H), 4.65 (s, 1H, NH), 3.82 (s, 4H, 2 ꢃ CH Pyr), 3.32e3.23 (m, 4H),
2
2
13
2
), 1.51 (s, 9H, C(CH
3
)
3
3.11 (quartet, J ¼ 6.5 Hz, 2H, CH NH), 1.62e1.45 (m, 4H), 1.41 (s, 9H,
2
13
3
d
C(CH ) );
3
3
C NMR (100 MHz, CDCl ):
3
d
¼ 171.3 (CO), 158.3
(
(
(2 ꢃ Cquat Pyr), 156.1 (NCOOtBu), 149.5 (2 ꢃ CH Pyr), 136.6 (2 ꢃ CH
Pyr), 123.3 (2 ꢃ CH Pyr), 122.5 (2 ꢃ CH Pyr), 79.1 (C(CH ) ), 60.6
3
)
3
2
2
2
3 3
)
3
3
þ
ESI m/z [MþNa] calculated for C15
H
2 3
O
(
2
2 ꢃ CH
2
Pyr), 58.1 (NCH
), 26.9 (CH
); HRMS-ESI m/z [MþH] calculated for
23 34 5 3
C H N O : 428.2639, found: 428.2656.
2 2 2 3 3
CO), 40.4 (CH ), 38.8 (CH ), 28.5 (C(CH ) ),
3
35.1133.
þ
7.6 (CH
2
2
5.2.3.9. Tert-butyl (2-(1-(2-chloroacetyl)piperidin-4-yl)ethyl)carba-
mate (5i). The title compound was isolated as a mixture of amide
5
.2.4.3. Tert-butyl (6-(di-2-picolylaminoacetamido)hexyl)carbamate
1
rotamers (519 mg, 78% yield):
¼ 4.62e4.43 (m, 2H), 4.07 (d, J ¼ 12 Hz, AB system, 1H), 4.02 (d,
J ¼ 12 Hz, AB system, 1H), 3.88e3.73 (m, 1H), 3.21e2.89 (m, 3H),
.59 and 2.50 (td, J ¼ 12.9, 2.9 Hz, 1H), 1.88e1.65 (m, 2H), 1.63e1.48
H
NMR (400 MHz, CDCl
3
):
1
(6c). Yield (675 mg, 87%): H NMR (400 MHz, CDCl
3
):
d
¼ 8.70 (t,
d
J ¼ 5.7 Hz, 1H, NH), 8.55 (m, 2H), 7.63 (td, J ¼ 7.7, 1.7 Hz, 2H), 7.30 (d,
J ¼ 7.8 Hz, 2H), 7.18 (dd, J ¼ 7.5, 5.0 Hz, 2H), 4.55 (m, 1H, NH), 3.88 (s,
2
4
H, 2 ꢃ CH
CH
NH), 3.06 (quartet, J ¼ 6.8 Hz, 2H, CH
.48e1.38 (m, 11H, CH , C(CH ), 1.36e1.26 (m, 4H); C NMR
100 MHz, CDCl ):
¼ 170.7 (CO), 157.8 (2 ꢃ Cquat Pyr), 156.0
NCOOtBu), 149.0 (2 ꢃ CH Pyr), 136.9 (2 ꢃ CH Pyr), 123.4 (2 ꢃ CH
Pyr), 58.1
2
Pyr), 3.34 (s, 2H, NCH
2
CO), 3.26 (quartet, J ¼ 6.7 Hz, 2H,
1
3
(
m, 1H), 1.49e1.36 (s, 11H), 1.30e1.05 (m, 2H); C NMR (100 MHz,
CDCl ): ),
¼ 168.9 and 165.0 (CO), 156.1 (NCOOtBu), 79.4 (C(CH
), 33.7 and
); HRMS-
: 327.1456, found:
2
2
NH), 1.61e1.47 (m, 2H),
3
d
3 3
)
13
1
(
(
2
3 3
)
5
3.5, 46.7, 42.7, 41.8, 41.3, 38.1, 36.74 and 36.68 (5 ꢃ CH
2
3
d
3
3.5 (CH), 32.6, 32.5, 31.8 and 31.7 (2 ꢃ CH
2 3 3
), 28.5 (C(CH )
H25ClNaN O
2 3
þ
ESI m/z [MþNa] calculated for C14
Pyr), 122.6 (2 ꢃ CH Pyr), 79.0 (C(CH
3
)
3
), 60.1 (2 ꢃ CH
2
3
27.1446.
(NCH
2
CO), 40.5 (CH
), 26.6 (CH ), 26.5 (CH
: 456.2982, found: 456.2974.
2
), 39.0 (CH
2
), 30.0 (CH
2
), 29.4 (CH
2
), 28.4
þ
(C(CH
3
)
3
2
2
); HRMS-ESI m/z [MþH] calculated
5.2.3.10. Tert-butyl (2-(piperazin-1-yl)ethyl)carbamate (5j). pH of
38 5 3
for C25H N O
the mixture was adjusted to 10e11 with 0.5 M K
2
3
CO before
extraction.
1
Yield (617 mg, 93%): H NMR (400 MHz, CDCl
3
):
d
¼ 4.91 (bs, 1H,
5.2.4.4. Tert-butyl (4-oxo-1-(pyridin-2-yl)-2-(pyridin-2-ylmethyl)-
NH), 4.05 (s, 2H, CH
2
Cl), 3.62 (t, J ¼ 5.1 Hz, 2H), 3.51 (t, J ¼ 5.0 Hz,
9,12,15-trioxa-2,5-diazaoctadecan-18-yl)carbamate
(6d). Yield
Please cite this article as: A. Prandina et al., Synthesis and biological evaluation of new dipicolylamine zinc chelators as metallo-
inhibitors, Tetrahedron, https://doi.org/10.1016/j.tet.2019.02.004
b
-lactamase

A. Prandina et al. / Tetrahedron xxx (xxxx) xxx
9
(
705 mg, quantitative): 1H NMR (300 MHz, CDCl
3
):
d
¼ 8.70 (t,
CDCl
3
):
d
¼ 171.3 (CO), 158.3 (2 ꢃ Cquat Pyr), 152.8 (NCOOtBu),
J ¼ 5.7 Hz,1H, NH), 8.54 (m, 2H), 7.59 (td, J ¼ 7.7,1.8 Hz, 2H), 7.26 (m,
149.4 (2 ꢃ CH Pyr), 136.7 (Cquat Phenyl), 136.6 (2 ꢃ CH Pyr), 134.0
2
H), 7.15 (ddd, J ¼ 7.6, 4.9, 1.3 Hz, 2H), 5.01 (s, 1H, NH), 3.83 (s, 4H,
ꢃ CH Pyr), 3.65e3.46 (m, 12H), 3.35 (quartet, J ¼ 6.6 Hz, 2H,
NH), 3.29 (s, 2H, NCH NH),
CO), 3.20 (quartet, J ¼ 6.3 Hz, 2H, CH
.84 (pentet, J ¼ 6.7 Hz, 2H, CH CH CH ), 1.74 (pentet, J ¼ 6.2 Hz, 2H,
CH CH CH ), 1.41 (s, 9H, C(CH
¼ 171.3 (CO), 158.4 (2 ꢃ Cquat Pyr), 156.1 (NCOOtBu), 149.5
2 ꢃ CH Pyr), 136.6 (2 ꢃ CH Pyr), 123.3 (2 ꢃ CH Pyr), 122.5 (2 ꢃ CH
Pyr), 79.0 (C(CH ), 70.4 (CH ), 70.3 (CH ), 69.7
Pyr), 58.2 (CH ), 54.9 (CH ), 38.7
(Cquat Phenyl), 129.3 (2 ꢃ CH Pyr), 123.2 (2 ꢃ CH Phenyl), 122.4
2
2
(2 ꢃ CH Pyr), 118.6 (2 ꢃ CH Phenyl), 80.5 (C(CH
3
)
3
), 60.6
), 28.5 (C(CH );
34 5 3
H N O : 476.2673, found:
CH
2
2
2
(2 ꢃ CH
2
Pyr), 58.1 (NCH
2
CO), 40.4 (CH
2
), 35.0 (CH
2
3 3
)
þ
HRMS-ESI m/z [MþH] calculated for C27
1
2
2
2
3 3 3
) ):
); 1 C NMR (75 MHz, CDCl
3
476.2656.
2
2
2
d
5.2.4.9. Tert-butyl (2-(1-(bis(pyridin-2-ylmethyl)glycyl)piperidin-4-
(
yl)ethyl)carbamate (6i). The title compound was isolated as a
mixture of amide rotamers (758 mg, quantitative yield): H NMR
3
)
3
), 70.7 (2 ꢃ CH
), 60.6 (2 ꢃ CH
), 29.8 (CH ), 28.6 (C(CH
2
2
2
1
(
CH
CH
2
2
), 69.2 (CH
), 36.5 (CH
2
2
2
2
(400 MHz, CDCl
3
):
d
¼ 8.57e8.46 (m, 2H), 7.68e7.59 (m, 2H), 7.53
(
2
2
3
)
3
); HRMS-ESI m/z [MþH]þ
and 7.34 (d, J ¼ 7.8 Hz, 2H), 7.14 (ddd, J ¼ 7.5, 4.9, 1.2 Hz, 2H),
46 5 6
calculated for C29H N O : 560.3455, found: 560.3448.
4
.63e4.44 (m, 3H), 3.91 (d, J ¼ 14 Hz, AB system, 2H), 3.86 (d,
J ¼ 14 Hz, AB system, 2H), 3.75 (m, 1H), 3.42 (d, J ¼ 14.7 Hz, AB
system, 1H), 3.37 (d, J ¼ 14.7 Hz, AB system, 1H), 3.21e3.05 (m, 3H),
2.80 (td, J ¼ 12.8, 2.7 Hz, 1H), 2.46 (m, 2H), 1.79e1.55 (m, 2H),
5
.2.4.5. Cis tert-butyl (4-(di-2-picolylaminoacetamido)cyclohexyl)
1
carbamate (6e). Yield (1.272 g, 98%): H NMR (300 MHz, CDCl
¼ 8.65 (d, J ¼ 7.8 Hz, 1H, NH), 8.57 (ddd, J ¼ 4.9, 1.8, 0.9 Hz, 2H),
.62 (td, J ¼ 7.6, 1.9 Hz, 2H), 7.28 (m, 2H), 7.18 (ddd, J ¼ 7.6, 4.9,
3
):
13
d
1.55e1.32 (m, 11H); C NMR (100 MHz, CDCl ):
d
¼ 168.9 and 168.6
3
7
(CO), 159.9 and 159.1 (2 ꢃ Cquat Pyr), 156.0 (NCOOtBu), 149.4 and
1
.2 Hz, 2H), 4.50 (s, 1H, NH), 3.92 (m, 1H, CHNH), 3.84 (s, 4H,
149.1 (2 ꢃ CH Pyr), 136.6 and 136.5 (2 ꢃ CH Pyr), 123.8 and 122.4
2
(
(
ꢃ NCH
2
Py), 3.60 (m, 1H, CHNH), 3.29 (s, 2H, NCH
2
CO), 1.80e1.59
); C NMR (75 MHz, CDCl ):
¼ 170.7
CO), 158.7 (2 ꢃ Cquat Pyr), 155.5 (NCOOtBu), 149.8 (2 ꢃ CH Pyr),
36.9 (2 ꢃ CH Pyr), 123.7 (2 ꢃ CH Pyr), 122.8 (2 ꢃ CH Pyr), 78.9
(2 ꢃ CH Pyr), 122.2 and 122.0 (2 ꢃ CH Pyr), 79.3 (C(CH
3 3
) ), 60.5
13
m, 8H), 1.47 (s, 9H, C(CH
3
)
3
3
d
(2 ꢃ CH Pyr), 56.1, 54.9, 46.7, 45.5, 42.2, 41.8, 38.1 and 36.8
2
(5 ꢃ CH
2
), 33.6 (CH), 32.6, 32.0 and 31.8 (2 ꢃ CH
2
), 28.5 (C(CH
3 3
) );
1
þ
HRMS-ESI m/z [MþH] calculated for C26
H
38
N
5
O
3
: 468.2973,
(
C(CH
3
)
3
), 60.7 (2 ꢃ CH
), 28.7 (C(CH
2
Pyr), 58.4 (NCH
2
CO), 45.4 (2 ꢃ CH), 28.9
found: 468.2969.
(
C
4 ꢃ CH
2
3
)
3
); HRMS-ESI m/z [MþH]þ calculated for
25
H
36
N
5
O
3
: 454.2827, found: 454.2818.
5.2.4.10. Tert-butyl (2-(4-(bis(pyridin-2-ylmethyl)glycyl)piperazin-1-
yl)ethyl)carbamate (6j). Yield (1.05 g, 75%): H NMR (400 MHz,
1
5
.2.4.6. Trans tert-butyl (4-(di-2-picolylaminoacetamido)cyclohexyl)
CDCl
J ¼ 7.8, 1.1 Hz, 2H), 7.16 (dtd, J ¼ 7.5, 4.3, 3.8, 1.2 Hz, 2H), 4.92 (bs, 1H,
NH), 3.89 (s, 4H, 2 ꢃ NCH Py), 3.57 (t, J ¼ 4.9 Hz, 2H), 3.42 (s, 2H,
NCH
CO), 3.38 (t, J ¼ 5.0 Hz, 2H), 3.22 (m, 2H), 2.43 (t, J ¼ 6.0 Hz,
2H), 2.38 (t, J ¼ 5.1 Hz, 2H), 2.33 (t, J ¼ 5.1 Hz, 2H), 1.45 (s, 9H,
3
):
d
¼ 8.53 (ddd, J ¼ 4.9, 1.8, 0.9 Hz, 2H), 7.65 (m, 2H), 7.52 (dt,
1
carbamate (6f). Yield (1.048 g, 75%): H NMR (400 MHz, CDCl
d
3
):
¼ 8.88 (d, J ¼ 8.4 Hz, 1H, NH), 8.54 (tdd, J ¼ 4.9, 1.9, 0.9 Hz, 2H),
2
7
.58 (td, J ¼ 7.7, 1.8 Hz, 2H), 7.22 (dt, J ¼ 7.8, 1.1 Hz, 2H), 7.15 (m, 2H),
2
4
.46 (d, J ¼ 8.3 Hz, 1H, NH), 3.82 (s, 4H, 2 ꢃ NCH
CO), 2.06e1.89 (m,
), 1.36 (td, J ¼ 12.4, 3.3 Hz, 2H), 1.26 (m, 2H);
C NMR (100 MHz, CDCl ):
¼ 170.7 (CO), 158.4 (2 ꢃ Cquat Pyr),
55.4 (NCOOtBu), 149.4 (2 ꢃ CH Pyr), 136.6 (2 ꢃ CH Pyr), 123.2
2 ꢃ CH Pyr), 122.5 (2 ꢃ CH Pyr), 79.3 (C(CH ), 60.5 (2 ꢃ CH Pyr),
), 31.7 (2 ꢃ CH ),
2
Py), 3.72 (m, 1H,
13
C(CH
2 ꢃ Cquat Pyr), 156.0 (NCOOtBu), 149.2 (2 ꢃ CH Pyr), 136.6 (2 ꢃ CH
Pyr), 123.8 (2 ꢃ CH Pyr), 122.3 (2 ꢃ CH Pyr), 79.4 (C(CH ), 60.5
2 ꢃ CH Pyr), 56.2 (NCH CO), 55.0 (CH ), 53.1 (CH ), 52.8 (CH ),
5.2 (CH ), 41.7 (CH ), 37.2 (CH ), 28.6 (C(CH ); HRMS-ESI m/z
[MþH] calculated for C25 : 469.2913, found: 469.2922.
3
)
3
);
C NMR (100 MHz, CDCl
3
):
d
¼ 168.9 (CO), 159.0
CHNH), 3.45 (m, 1H, CHNH), 3.29 (s, 2H, NCH
H), 1.43 (s, 9H, C(CH
2
(
4
3 3
)
1
3
d
3 3
)
3
1
(
2
2
2
2
2
(
3
)
3
2
4
2
2
2
3 3
)
þ
5
8.2 (NCH
2
CO), 49.2 (CH), 47.3 (CH), 32.2 (2 ꢃ CH
2
2
37 6 3
H N O
2
4
8.5 (C(CH
54.2798, found: 454.2813.
3
)
3
); HRMS-ESI m/z [MþH]þ calculated for C25
36 5 3
H N O :
5.2.5. General procedure for N-Boc deprotection of compounds 6a-j
The corresponding N-Boc-protected amine 6a-j (1.0 equiv.) was
ꢁ
5
.2.4.7. Tert-butyl (4-(di-2-picolylaminoacetamido)phenethyl)carba-
dissolved in CH
Trifluoroacetic acid (60.0 equiv.) in CH
2
Cl
2
(0.25 M) and cooled to 0 C in an ice-water bath.
1
mate (6g). Yield (11.3 g, quantitative yield): H NMR (400 MHz,
CDCl ):
¼ 10.86 (s, 1H, NH), 8.61 (d, J ¼ 4.1 Hz, 2H), 7.70 (d,
2
Cl
2
(13 M) was then slowly
ꢁ
3
d
added to the stirring mixture. The reaction was left at 0 C for
20 min before warming up to room temperature. The mixture was
stirred for an additional 3 h at room temperature, until TLC (2%
MeOH in CH Cl , alumina plates) indicated consumption of the
carbamate. After solvent removal under reduced pressure, excess
M aqueous K CO (50 mL) was added to the mixture, and the
compound was extracted with CH Cl
(3e20 ꢃ 20 mL). The com-
bined organic layers were washed with fresh 0.5 M K CO
3 ꢃ 50 mL), dried on K CO , filtered, and the solvent removed
under reduced pressure, to give title compound 7a-j as a brown oil.
J ¼ 4.1 Hz, 2H), 7.61 (td, J ¼ 7.7, 1.7 Hz, 2H), 7.27 (m, 2H), 7.22e7.11
(
m, 4H), 4.54 (s, 1H, NH), 3.93 (s, 4H, 2 ꢃ NCH
2
Py), 3.42-3.25 (m,
13
2
2
2
H), 2.76 (t, J ¼ 6.8 Hz, 2H),1.42 (s, 9H, C(CH
):
¼ 169.8 (CO), 158.2 (2 ꢃ Cquat Pyr), 155.9 (NCOOtBu),
49.5 (2 ꢃ CH Pyr), 137.1 (Cquat Phenyl), 136.7 (2 ꢃ CH Pyr), 134.4
Cquat Phenyl), 129.2 (2 ꢃ CH Phenyl), 123.3 (2 ꢃ CH Pyr), 122.6
2 ꢃ CH Pyr), 112.0 (2 ꢃ CH Phenyl), 79.2 (C(CH ), 60.4
2 ꢃ CH Pyr), 58.8 (NCH CO), 41.9 (CH ), 35.7 (CH ), 28.5 (C(CH ).
3 3
) ); C NMR (100 MHz,
CDCl
1
3
d
1
2
3
2
2
(
(
(
2
3
3 3
)
(
2
3
2
2
2
2
3 3
)
5
.2.4.8. Tert-butyl (4-(2-(di-2-picolylaminoacetamido)ethyl)phenyl)
5
.2.5.1. N-(2-aminoethyl)-2-(di-2-picolylamino)acetamide
(7a).
¼ 8.81 (t, J ¼ 5.8 Hz,
H, NH), 8.50 (m, 2H), 7.55 (td, J ¼ 7.6, 1.8 Hz, 2H), 7.20 (dt, J ¼ 7.7,
.1 Hz, 2H), 7.10 (m, 2H), 3.78 (s, 4H, 2 ꢃ NCH Py), 3.33 (quartet,
J ¼ 5.8 Hz, 2H), 3.28 (s, 2H, NCH CO), 2.83 (dd, J ¼ 6.5, 5.2 Hz, 2H),
); C NMR (75 MHz, CDCl ):
¼ 171.6 (CO), 158.1
(2 ꢃ Cquat Pyr), 149.4 (2 ꢃ CH Pyr), 136.6 (2 ꢃ CH Pyr), 123.4
1
carbamate (6h). Yield (2149 g, quantitative yield):
400 MHz, CDCl ):
¼ 8.66 (t, J ¼ 5.8 Hz, 1H, NH), 8.49 (ddd, J ¼ 4.7,
.8, 1.1 Hz, 2H), 7.56 (td, J ¼ 7.7, 1.8 Hz, 2H), 7.22 (d, J ¼ 8.0 Hz, 2H),
.17e7.07 (m, 6H), 6.47 (bs, 1H, NH), 3.76 (s, 4H, 2 ꢃ NCH Py), 3.54
td, J ¼ 7.0, 5.7 Hz, 2H, CH NH), 3.27 (s, 2H, NCH CO), 2.81 (t,
J ¼ 7.0 Hz, 2H, PhCH ), 1.51 (s, 9H, C(CH
); 13C NMR (100 MHz,
H
NMR
1
3
Yield (1.52 g, 60%): H NMR (300 MHz, CDCl ): d
1
(
1
3
d
1
2
7
2
2
(
2
2
13
2
.59 (s, 2H, NH
2
3
d
2
3 3
)
Please cite this article as: A. Prandina et al., Synthesis and biological evaluation of new dipicolylamine zinc chelators as metallo-
inhibitors, Tetrahedron, https://doi.org/10.1016/j.tet.2019.02.004
b-lactamase

10
A. Prandina et al. / Tetrahedron xxx (xxxx) xxx
(
4
C
2 ꢃ CH Pyr), 122.5 (2 ꢃ CH Pyr), 60.4 (2 ꢃ CH
2
Pyr), 58.2 (NCH
); HRMS-ESI m/z [MþH] calculated for
2
CO),
Pyr), 60.5 (2 ꢃ CH
(2 ꢃ CH ), 31.8 (2 ꢃ CH
O: 354.2276, found: 354.2288.
2
Pyr), 58.2 (NCH
2
CO), 50.2 (CH), 47.6 (CH), 35.5
þ
þ
2.1 (CH
2
), 41.6 (CH
2
2
2
); HRMS-ESI m/z [MþH] calculated for
H
16 22
N
5
O: 300.1831, found: 300.1824.
20 28 5
C H N
5
.2.5.2. N-(4-aminobutyl)-2-(di-2-picolylamino)acetamide
(7b).
5.2.5.7. N-(4-(2-aminoethyl)phenyl)-2-(di-2-picolylamino)acet-
1
1
Yield (910 mg, 74%): H NMR (400 MHz, CDCl
3
):
d
¼ 8.74 (t,
amide (7g). Yield (155 mg, 98%): H NMR (400 MHz, DMSO‑d
6
):
J ¼ 6.1 Hz,1H, NH), 8.53 (ddd, J ¼ 4.9,1.8, 0.9 Hz, 2H), 7.58 (td, J ¼ 7.6,
d
¼ 10.53 (s, 1H, NH), 8.52 (m, 2H), 7.75 (td, J ¼ 7.6, 1.8 Hz, 2H), 7.58
1
.8 Hz, 2H), 7.24 (dt, J ¼ 7.8, 1.1 Hz, 2H), 7.14 (ddd, J ¼ 7.6, 4.9, 1.2 Hz,
(d, J ¼ 8.4 Hz, 2H), 7.45 (d, J ¼ 7.8 Hz, 2H), 7.27 (ddd, J ¼ 7.5, 4.9,
1.0 Hz, 2H), 7.15 (d, J ¼ 8.5 Hz, 2H), 3.90 (m, 4H, 2 ꢃ NCH
(m, 2H, NCH
NMR (100 MHz, CDCl
2
H), 3.81 (s, 4H, 2 ꢃ NCH
H), 1.62e1.53 (m, 2H), 1.51e1.41 (m, 2H), 1.38 (br s, 2H, NH
):
¼ 171.2 (CO), 158.4 (2 ꢃ Cquat Pyr), 149.4
2
Py), 3.32e3.23 (m, 4H), 2.67 (t, J ¼ 6.9 Hz,
2
Py), 3.41
13
13
2
2
);
C
2
CO), 2.73 (t, J ¼ 7.1 Hz, 2H), 2.58 (t, J ¼ 7.1 Hz, 2H);
C
NMR (100 MHz, CDCl
3
d
3
):
d
¼ 169.0 (CO), 158.4 (2 ꢃ Cquat Pyr), 149.0
(
2 ꢃ CH Pyr), 136.6 (2 ꢃ CH Pyr), 123.3 (2 ꢃ CH Pyr), 122.5 (2 ꢃ CH
(2 ꢃ CH Pyr), 136.7 (Cquat Phenyl), 136.6 (2 ꢃ CH Pyr), 135.3 (Cquat
Phenyl), 128.9 (2 ꢃ CH Phenyl), 123.0 (2 ꢃ CH Pyr), 122.4 (2 ꢃ CH
Pyr), 60.5 (2 ꢃ CH
2
Pyr), 58.1 (NCH
2
CO), 42.0 (CH
2
), 39.0 (CH
2
), 31.3
O:
þ
(
CH
2
), 26.9 (CH
2
); HRMS-ESI m/z [MþH] calculated for C18
H
26
N
5
Pyr), 119.0 (2 ꢃ CH Phenyl), 59.4 (2 ꢃ CH
2
Pyr), 57.8 (NCH
2
CO), 43.7
(CH
2
); HRMS-ESI m/z [MþH]þ calculated for C22
26 5
H N O: 376.2059,
3
28.2116, found: 328.2132.
found: 376.2133.
5
.2.5.3. N-(6-aminohexyl)-2-(di-2-picolylamino)acetamide
(7c).
¼ 8.78 (ddd,
J ¼ 5.6, 1.6, 0.8 Hz, 2H), 8.29 (td, J ¼ 7.8, 1.6 Hz, 2H), 7.83 (dt, J ¼ 8.0,
.0 Hz, 2H), 7.77 (ddd, J ¼ 7.7, 5.6, 1.2 Hz, 2H), 4.39 (s, 4H,
ꢃ NCH Py), 3.63 (s, 2H, NCH CO), 3.17 (t, J ¼ 7.2 Hz, 2H), 2.91 (t,
1
5
.2.5.8. N-(4-aminophenethyl)-2-(di-2-picolylamino)acetamide
3
Yield (47 mg, 60%): H NMR (400 MHz, CD OD): d
1
(7h). Yield (1.42 g, 90%): H NMR (400 MHz, CDCl
3
):
d
¼ 8.54 (m,
1
2
H, NH), 8.50 (ddd, J ¼ 4.8, 1.8, 1.0 Hz, 2H), 7.58 (td, J ¼ 7.6, 1.8 Hz,
1
H), 7.18e7.10 (m, 4H), 6.99 (m, 2H), 6.58 (m, 2H), 3.77 (s, 4H,
2
2
2
1
3
2
NCH
(
(
ꢃ NCH
CO), 2.75 (t, J ¼ 7.1 Hz, 2H, PhCH
100 MHz, CDCl ):
2 ꢃ CH Pyr), 144.8 (Cquat Phenyl), 136.6 (2 ꢃ CH Pyr), 129.6 (2 ꢃ CH
2
Py), 3.50 (td, J ¼ 7.1, 5.7 Hz, 2H, CH
2
NH), 3.26 (s, 2H,
), 1.04 (bs, 2H, NH ); C NMR
2
J ¼ 7.7 Hz, 2H), 1.70e1.28 (m, 10H, 4 ꢃ CH
2
, NH
2
);
C NMR
13
(
(
3
100 MHz, CD OD): d
2 ꢃ CH Pyr), 144.9 (2 ꢃ CH Pyr), 127.1 (2 ꢃ CH Pyr), 126.4 (2 ꢃ CH
¼ 172.1 (CO), 155.7 (2 ꢃ Cquat Pyr), 145.3
2
2
3
d
¼ 171.1 (CO), 158.3 (2 ꢃ Cquat Pyr), 149.4
Pyr), 58.4 (2 ꢃ CH
CH ), 28.5 (CH ), 27.5 (CH
calculated for C20 O: 356.2443, found: 356.2450.
2
Pyr), 58.0 (NCH
2
CO), 40.6 (CH
2
), 40.3 (CH
2
), 30.1
þ
Phenyl), 129.1 (Cquat Phenyl), 123.2 (2 ꢃ CH Pyr), 122.4 (2 ꢃ CH
Pyr), 115.3 (2 ꢃ CH Phenyl), 60.6 (2 ꢃ CH Pyr), 58.1 (NCH CO), 40.5
). HRMS-ESI m/z [MþH] calculated for C22 O:
(
2
2
2
), 27.1 (CH
2
); HRMS-ESI m/z [MþH]
30 5
H N
2
2
þ
(CH
2
), 34.8 (CH
2
26 5
H N
376.2133, found: 376.2132.
5
2
.2.5.4. N-(3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)-2-(di-
1
-picolylamino)acetamide (7d). Yield (362 mg, 89%): H NMR
):
¼ 8.71 (t, J ¼ 5.8 Hz, 1H, NH), 8.54 (ddd, J ¼ 5.2,
.0, 0.9 Hz, 2H), 7.60 (m, 2H), 7.26 (m, 2H), 7.14 (m, 2H), 3.82 (s, 4H,
ꢃ NCH Py), 3.70e3.45 (m, 12H), 3.35 (quartet, J ¼ 6.6 Hz, 2H), 3.29
CO), 2.76 (t, J ¼ 6.7 Hz, 2H), 1.84 (pentet, J ¼ 6.7 Hz, 2H),
5
.2.5.9. 1-(4-(2-Aminoethyl)piperidin-1-yl)-2-(di-2-picolylamino)
(
400 MHz, CDCl
3
d
ethan-1-one (7i). The title compound was isolated as a mixture of
amide rotamers (465 mg, 81% yield): H NMR (400 MHz, CDCl
d
2
1
3
):
2
2
¼ 8.57e8.47 (m, 2H), 7.69e7.59 (m, 2H), 7.53 and 7.34 (dt, J ¼ 7.9,
(
s, 2H, NCH
.70 (pentet, J ¼ 6.5 Hz, 2H), 1.54 (bs, 2H, NH
CDCl ):
¼ 171.3 (CO), 158.4 (2 ꢃ Cquat Pyr), 149.5 (2 ꢃ CH Pyr),
2
13
1.1 Hz, 2H), 7.18e7.09 (m, 2H), 4.52 (ddt, J ¼ 13.3, 4.2, 2.4 Hz, 1H),
1
2
); C NMR (100 MHz,
3
2
.91 (d, J ¼ 14.1 Hz, AB system, 2H), 3.86 (d, J ¼ 14.1 Hz, AB system,
H), 3.74 (m, 1H), 3.42 (d, J ¼ 14.7 Hz, AB system, 1H), 3.38 (d,
3
d
1
36.6 (2 ꢃ CH Pyr), 123.3 (2 ꢃ CH Pyr), 122.5 (2 ꢃ CH Pyr), 70.72
J ¼ 14.7 Hz, AB system, 1H), 2.82 (m, 1H), 2.71 (m, 2H), 2.48 (td,
(
CH
2
), 70.69 (CH
0.5 (2 ꢃ CH Pyr), 58.1 (NCH
9.7 (CH
60.2927, found: 460.2923.
2
), 70.4 (CH
2
), 70.3 (CH
2
), 69.5 (CH
2
), 69.2 (CH
), 33.5 (CH
2
),
),
J ¼ 13.0, 3.0 Hz, 1H), 1.74e1.46 (m, 5H), 1.37 (q, J ¼ 6.9 Hz, 2H), 1.05
6
2
4
2
2
CO), 39.7 (CH
2
), 36.5 (CH
2
2
13
(m, 2H); C NMR (100 MHz, CDCl
3
):
d
¼ 168.6 (CO), 159.8 and 159.1
þ
2
); HRMS-ESI m/z [MþH] calculated for C24
38 5 4
H N O :
(
1
2 ꢃ Cquat Pyr), 149.3 and 149.1 (2 ꢃ CH Pyr), 136.5 (2 ꢃ CH Pyr),
23.8 and 122.4 (2 ꢃ CH Pyr), 122.2 and 122.0 (2 ꢃ CH Pyr), 60.5
Pyr), 56.0 and 54.9 (NCH CO), 45.5 (CH ), 42.2 (CH ), 40.2
), 33.7 (CH), 32.8 (CH ), 32.1 (CH ); HRMS-ESI m/z
O: 368.2449, found: 368.2445.
(
(
[
2 ꢃ CH
2
2
2
2
5.2.5.5. Cis N-(4-aminocyclohexyl)-2-(di-2-picolylamino)acetamide
CH
2
), 39.4 (CH
2
2
2
1
(
(
7e). Yield (608 mg, 78%): H NMR (300 MHz, DMSO‑d
m, 2H), 8.34 (d, J ¼ 7.8 Hz, 1H, NH), 7.75 (m, 2H), 7.42 (m, 2H), 7.27
m, 2H), 3.80 (s, 4H, 2 ꢃ NCH Py), 3.70 (m, 1H, CH), 3.16 (s, 2H,
CO), 2.73 (m, 1H, CH), 1.70e1.31 (m, 10H, 4 ꢃ CH
NMR (75 MHz, DMSO‑d ):
¼ 169.0 (CO), 158.4 (2 ꢃ Cquat Pyr),
6
):
d
¼ 8.55
þ
MþH] calculated for C21
30 5
H N
(
2
5
.2.5.10. 1-(4-(2-Aminoethyl)piperazin-1-yl)-2-(di-2-picolylamino)
13
NCH
2
2
, NH
2
);
C
1
ethan-1-one (7j). Yield (500 mg, 79%): H NMR (400 MHz, CDCl
d
3
):
6
d
¼ 8.52 (ddd, J ¼ 4.9, 1.8, 0.9 Hz, 2H), 7.64 (td, J ¼ 7.6, 1.8 Hz, 2H),
1
49.1 (2 ꢃ CH Pyr), 136.6 (2 ꢃ CH Pyr), 123.0 (2 ꢃ CH Pyr), 122.3
7
.51 (dt, J ¼ 7.8, 1.1 Hz, 2H), 7.15 (ddd, J ¼ 7.5, 4.9, 1.3 Hz, 2H), 3.89 (s,
(
3
C
2 ꢃ CH Pyr), 59.6 (2 ꢃ CH
1.1 (2 ꢃ CH ), 27.8 (2 ꢃ CH
O: 354.2300, found: 354.2293.
2
Pyr), 57.4 (NCH
2
CO), 47.6 (CH), 44.6 (CH),
4
H, 2 ꢃ NCH
2
Py), 3.56 (t, J ¼ 5.2 Hz, 2H), 3.41 (s, 2H, NCH
2
CO), 3.37
þ
2
2
); HRMS-ESI m/z [MþH] calculated for
(
m, 2H), 2.76 (t, J ¼ 6.1 Hz, 2H), 2.49e2.28 (m, 6H), 1.78 (bs, 2H,
H
20 28
N
5
13
NH
2
); C NMR (100 MHz, CDCl
3
):
d
¼ 168.8 (CO), 158.9 (2 ꢃ Cquat
Pyr),149.1 (2 ꢃ CH Pyr),136.6 (2 ꢃ CH Pyr),123.8 (2 ꢃ CH Pyr),122.3
5
.2.5.6. Trans
N-(4-aminocyclohexyl)-2-(di-2-picolylamino)acet-
(
(
[
2 ꢃ CH Pyr), 61.1 (CH
2
), 60.5 (2 ꢃ CH
), 45.3 (CH ), 41.8 (CH ), 38.7 (CH
O: 369.2387, found: 369.2397.
2
Pyr), 56.1 (NCH
2
CO), 53.5
1
amide (7f). Yield (631 mg, 83%): H NMR (400 MHz, CDCl
d
3
):
¼ 8.67 (d, J ¼ 8.3 Hz, 1H, NH), 8.53 (ddd, J ¼ 4.9, 1.8, 0.9 Hz, 2H),
CH
2
), 53.0 (CH
2
2
2
2
); HRMS-ESI m/z
þ
MþH] calculated for C20
29 6
H N
7
3
3
1
(
(
.59 (td, J ¼ 7.6, 1.8 Hz, 2H), 7.23 (dt, J ¼ 7.8, 1.1 Hz, 2H), 7.14 (m, 2H),
.81 (s, 4H, 2 ꢃ NCH
.27 (s, 2H, NCH
CO), 2.67 (tt, J ¼ 10.6, 3.9 Hz, 1H, CHNH
.96e1.82 (m, 4H), 1.43e1.12 (m, 6H, 2 ꢃ CH
100 MHz, CDCl ):
¼ 170.5 (CO), 158.4 (2 ꢃ Cquat Pyr), 149.4
2 ꢃ CH Pyr), 136.6 (2 ꢃ CH Pyr), 123.3 (2 ꢃ CH Pyr), 122.5 (2 ꢃ CH
2
Py), 3.70 (tdt, J ¼ 11.8, 8.3, 4.0 Hz, 1H, CHNH),
),
); C NMR
5
.2.6. General procedure for peptide coupling reaction
2
2
The corresponding primary amine 7a-j (1.0 equiv.) was dis-
13
ꢁ
2
, NH
2
solved in CH
2
Cl
2
(0.25 M) and cooled to 0 C in an ice-water bath. N-
3
d
Boc-D-Ala-D-Ala-OH 4 (1.0 equiv.) and HATU (1.0 equiv.) were
added, before Ne methylmorpholine (2 equiv.) was added to the
Please cite this article as: A. Prandina et al., Synthesis and biological evaluation of new dipicolylamine zinc chelators as metallo-
inhibitors, Tetrahedron, https://doi.org/10.1016/j.tet.2019.02.004
b-lactamase

A. Prandina et al. / Tetrahedron xxx (xxxx) xxx
11
stirring mixture. The mixture was stirred in the ice-water bath for
5 min before slowly warming up to room temperature. After
.5 h at room temperature, the mixture was washed with
.5 M K CO (3 times), dried over K CO , filtered and concentrated
Cl
and purified by C-18 reverse phase column chromatography
20e75% MeOH in water) to afford title compounds 8a-j as brown
oils.
0.9 Hz, 2H), 7.60 (td, J ¼ 7.6, 1.8 Hz, 2H), 7.26 (dt, J ¼ 7.8, 1.1 Hz, 2H),
7.15 (ddd, J ¼ 7.5, 4.8, 1.2 Hz, 2H), 6.90 (t, J ¼ 5.6 Hz, 1H, NH), 6.85 (d,
J ¼ 7.5 Hz, 1H, NH), 5.14 (m, 1H, NH), 4.38 (pentet, J ¼ 7.1 Hz, 1H,
1
4
0
2
3
2
3
CHa
), 4.12 (t, J ¼ 7.5 Hz, 1H, CH
.65e3.47 (m, 12H), 3.37 (m, 2H), 3.30 (s, 2H, NCH
H), 1.84 (pentet, J ¼ 6.7 Hz, 2H), 1.76 (pentet, J ¼ 6.2 Hz, 2H), 1.42 (s,
a
), 3.83 (s, 4H, 2 ꢃ NCH
2
Py),
under reduced pressure. The oily residue was dissolved in CH
2
2
3
2
9
2
CO), 2.76 (m,
(
13
H, C(CH
):
55.6 (NCOOtBu), 149.5 (2 ꢃ CH Pyr), 136.7 (2 ꢃ CH Pyr), 123.4
2 ꢃ CH Pyr), 122.5 (2 ꢃ CH Pyr), 80.2 (C(CH ), 70.7 (CH ), 70.6
CH ), 70.3 (CH ), 70.2 (CH ), 69.7 (CH ), 69.1 (CH ), 60.5
2 ꢃ CH Pyr), 58.1 (NCH CO), 50.5 (CH ), 49.0 (CH ), 37.8 (CH ),
6.5 (CH ), 28.4 (C(CH ), 18.7 (CH ), 18.6
CH : 702.4211,
found: 702.4190.
3
)
3
), 1.35 (s, 3H, CH
3 3
), 1.33 (s, 3H, CH ); C NMR (100 MHz,
CDCl
3
d
¼ 172.4 (CO), 172.0 (CO), 171.4 (CO), 158.3 (2 ꢃ Cquat Pyr),
1
5
.2.6.1. Tert-butyl ((10R,13R)-10-methyl-4,9,12-trioxo-1-(pyridin-2-
(
(
3
)
3
2
yl)-2-(pyridin-2-ylmethyl)-2,5,8,11-tetraazatetradecan-13-yl)carba-
2
2
2
2
2
1
mate (8a). Yield (250 mg, 28%): H NMR (400 MHz, CDCl
(
(
3
):
d
¼ 8.90
(
2
2
a
a
2
t, J ¼ 5.2 Hz, 1H, NH), 8.48 (ddd, J ¼ 4.9, 1.8, 0.9 Hz, 2H), 7.59e7.48
3
2
), 29.7 (CH
); HRMS-ESI m/z [MþH] calculated for C35
2
), 29.0 (CH
2
3
)
3
3
m, 3H, 2*CH Pyr, NH), 7.20 (dt, J ¼ 7.7, 1.2 Hz, 2H), 7.1e7.06 (m, 3H,
þ
(
3
56 7 8
H N O
2
*CH Pyr, NH), 5.49 (d, J ¼ 7.2 Hz, 1H, NH), 4.32 (pentet, J ¼ 7.1 Hz,
H, CH ), 4.09 (m, 1H, CH Py), 3.42e3.27 (m,
), 3.76 (s, 4H, 2 ꢃ NCH
H), 3.24 (s, 2H, NCH CO), 1.34 (s, 9H, C(CH
), 1.25 (d, J ¼ 7.1 Hz,
H, CH ); C NMR (100 MHz, CDCl ):
), 1.21 (d, J ¼ 7.0 Hz, 3H, CH
¼ 172.5 (2 ꢃ CO), 172.2 (CO), 157.9 (2 ꢃ Cquat Pyr), 155.6
1
4
3
d
a
a
2
2
3 3
)
13
5.2.6.5. Tert-butyl ((R)-1-(((R)-1-((cis-4-(2-(bis(pyridin-2-ylmethyl)
amino)acetamido)cyclohexyl)amino)-1-oxopropan-2-yl)amino)-1-
3
3
3
1
oxopropan-2-yl)carbamate (8e). Yield (660 mg, 78%): H NMR
(
NCOOtBu), 149.3 (2 ꢃ CH Pyr), 136.7 (2 ꢃ CH Pyr), 123.4 (2 ꢃ CH
Pyr), 122.5 (2 ꢃ CH Pyr), 79.9 (C(CH ), 60.3 (2 ꢃ CH Pyr), 58.0
), 39.9 (CH ), 38.6 (CH ), 28.3
(
1
2
300 MHz, CDCl
3
):
d
¼ 8.66 (d, J ¼ 7.6 Hz, 1H, NH), 8.53 (ddd, J ¼ 4.9,
3
)
3
2
.9, 0.9 Hz, 2H), 7.58 (td, J ¼ 7.7, 1.8 Hz, 2H), 7.23 (dt, J ¼ 7.8, 1.2 Hz,
(
NCH
2
CO), 50.2 (CH
a
), 48.8 (CH
a
2
2
H), 7.17e7.04 (m, 3H, 2H Pyr, NH), 6.64 (d, J ¼ 7.4 Hz, 1H, NH), 5.35
), 4.10 (m, 1H,
Py), 3.23 (s, 2H, NCH CO),
), 1.32 (d, J ¼ 7.0 Hz, 3H, CH ), 1.27
); C NMR (75 MHz, CDCl ):
¼ 173.0 (CO),
(
C(CH
3
)
3
), 18.6 (CH ), 18.3 (CH
3
3
); HRMS-ESI m/z [MþH]þ calculated
(d, J ¼ 7.1 Hz, 1H, NH), 4.41 (pentet, J ¼ 7.0 Hz, 1H, CH
CH
1
a
40 7 5
for C27H N O : 542.3061, found: 542.3085.
a
), 3.84 (m, 2H), 3.78 (s, 4H, 2 ꢃ NCH
2
2
.64 (m, 8H), 1.35 (s, 9H, C(CH
3
)
3
3
13
5.2.6.2. Tert-butyl ((12R,15R)-12-methyl-4,11,14-trioxo-1-(pyridin-2-
(d, J ¼ 7.1 Hz, 3H, CH
3
3
d
yl)-2-(pyridin-2-ylmethyl)-2,5,10,13-tetraazahexadecan-15-yl)carba-
171.2 (CO), 170.4 (CO), 158.2 (2 ꢃ Cquat Pyr), 155.5 (NCOOtBu), 149.4
1
mate (8b). Yield (800 mg, 92%): H NMR (400 MHz, CDCl
t, J ¼ 5.8 Hz, 1H, NH), 8.54 (ddd, J ¼ 4.9, 1.8, 0.9 Hz, 2H), 7.60 (td,
J ¼ 7.7, 1.8 Hz, 2H), 7.24 (dt, J ¼ 7.9, 1.1 Hz, 2H), 7.16 (ddd, J ¼ 7.6, 4.9,
.2 Hz, 2H), 6.96 (t, J ¼ 5.6 Hz, 1H, NH), 6.87 (d, J ¼ 7.7 Hz, 1H, NH),
.22 (d, J ¼ 6.1 Hz, 1H, NH), 4.43 (pentet, J ¼ 7.1 Hz, 1H, CH ), 4.11
m, 1H, CH Py), 3.34e3.17 (m, 6H), 1.56 (m,
), 3.82 (s, 4H, 2 ꢃ NCH
H), 1.41 (s, 9H, C(CH ), 1.32 (d,
), 1.34 (d, J ¼ 7.1 Hz, 3H, CH
); C NMR (100 MHz, CDCl ):
¼ 172.6 (CO),
3
):
d
¼ 8.82
(2 ꢃ CH Pyr), 136.5 (2 ꢃ CH Pyr), 123.3 (2 ꢃ CH Pyr), 122.5 (2 ꢃ CH
(
Pyr), 80.0 (C(CH
8.9 (CH ), 46.3 (CH
), 18.5 (CH ), 17.8 (CH
: 596.3572, found: 596.3560.
3
)
3
), 60.3 (2 ꢃ CH
2
Pyr), 58.0 (NCH
2
CO), 50.3 (CH
a),
4
a
2
), 45.3 (CH
2
), 38.6 (CH
2
), 28.3 (C(CH
3
)
3
), 28.0
1
5
þ
(CH
2
3
3
). HRMS-ESI m/z [MþH] calculated for
a
C
H
31 46
N
7
O
5
(
4
a
2
3
)
3
3
5
.2.6.6. Tert-butyl ((R)-1-(((R)-1-((trans-4-(2-(bis(pyridin-2-
1
3
J ¼ 6.1 Hz, 3H, CH
3
3
d
ylmethyl)amino)acetamido)cyclohexyl)amino)-1-oxopropan-2-yl)
1
(
72.1 (CO), 171.5 (CO), 158.2 (2 ꢃ Cquat Pyr), 155.8 (NCOOtBu), 149.5
1
amino)-1-oxopropan-2-yl)carbamate (8f). Yield (800 mg, 95%):
NMR (400 MHz, CDCl ):
H
2 ꢃ CH Pyr), 136.7 (2 ꢃ CH Pyr), 123.4 (2 ꢃ CH Pyr), 122.6 (2 ꢃ CH
Pyr), 80.4 (C(CH Pyr), 58.1 (NCH CO), 50.7 (CH ),
9.0 (CH ), 39.4 (CH ), 26.4
CH ), 18.4 (CH ), 18.3 (CH
: 570.3388, found: 570.3398.
3
d
¼ 8.90 (d, J ¼ 8.3 Hz, 1H, NH), 8.54 (ddd,
3
)
3
), 60.6 (2 ꢃ CH
), 38.7 (CH
2
2
a
J ¼ 4.9, 1.8, 0.9 Hz, 2H), 7.58 (td, J ¼ 7.7, 1.8 Hz, 2H), 7.22 (dt, J ¼ 7.8,
1.1 Hz, 2H), 7.15 (ddd, J ¼ 7.6, 4.8, 1.2 Hz, 2H), 6.72 (d, J ¼ 7.5 Hz, 1H,
NH), 6.60 (d, J ¼ 8.2 Hz, 1H, NH), 5.08 (m, 1H, NH), 4.40 (pentet,
4
(
C
a
2
2
), 28.4 (C(CH
3
)
3
), 27.1 (CH
2
þ
2
H
3
3
); HRMS-ESI m/z [MþH] calculated for
29
44
N
7
O
5
J ¼ 7.1 Hz, 1H, CH
ꢃ NCH
(s, 9H, C(CH
¼ 172.5 (CO), 171.5 (CO), 170.7 (CO), 158.4 (2 ꢃ Cquat Pyr), 155.8
(NCOOtBu), 149.4 (2 ꢃ CH Pyr), 136.6 (2 ꢃ CH Pyr), 123.2 (2 ꢃ CH
Pyr), 122.5 (2 ꢃ CH Pyr), 80.6 (C(CH ), 60.4 (2 ꢃ CH Pyr), 58.2
NCH CO), 50.8 (CH ), 49.2 (CH ), 47.9 (CH), 47.3 (CH), 31.62 (CH ),
1.60 (CH ), 31.5 (CH ), 31.4 (CH ), 28.4 (C(CH ), 18.4 (CH ), 18.3
CH : 596.3542,
); HRMS-ESI m/z [MþH] calculated for C31
found: 596.3555.
a
), 4.10 (pentet, J ¼ 6.7 Hz, 1H, CH
a
), 3.81 (s, 4H,
CO), 1.95 (bs, 4H), 1.43
), 1.40e1.23 (m, 10H); C NMR (100 MHz, CDCl ):
2
2 2
Py), 3.73 (m, 2H), 3.29 (s, 2H, NCH
13
5.2.6.3. Tert-butyl ((14R,17R)-14-methyl-4,13,16-trioxo-1-(pyridin-2-
3
)
3
3
yl)-2-(pyridin-2-ylmethyl)-2,5,12,15-tetraazaoctadecan-17-yl)carba-
d
1
mate (8c). Yield (40 mg, 58%): H NMR (300 MHz, CDCl
(
3
):
d
¼ 8.73
bs, 1H, NH), 8.53 (m, 2H), 7.59 (td, J ¼ 7.6, 1.9 Hz, 2H), 7.25 (d,
3
)
3
2
J ¼ 9.0 Hz, 2H), 7.16 (dt, J ¼ 7.4, 4.6 Hz, 2H), 6.89 (d, J ¼ 7.6 Hz, 1H,
(
2
a
a
2
NH), 6.69 (d, J ¼ 5.8 Hz, 1H, NH), 5.22 (bs, 1H, NH), 4.44 (quartet,
3
(
2
2
2
3
)
3
3
J ¼ 7.0 Hz, 1H, CH
a
), 4.11 (m, 1H, CH
a), 3.83 (s, 4H, 2 ꢃ NCH
2
Py),
þ
3
46 7 5
H N O
3
.35e2.92 (m, 6H), 1.53 (m, 2H), 1.47e1.39 (m, 11H), 1.38e1.22 (m,
0H); 13C NMR (75 MHz, CDCl
):
¼ 172.6 (CO), 172.0 (CO), 171.3
CO), 158.4 (2 ꢃ Cquat Pyr), 155.8 (NCOOtBu), 149.5 (2 ꢃ CH Pyr),
36.6 (2 ꢃ CH Pyr), 123.3 (2 ꢃ CH Pyr), 122.5 (2 ꢃ CH Pyr), 80.4
C(CH Pyr), 58.2 (NCH CO), 50.7 (CH ), 49.0 (CH ),
), 29.4 (CH ), 29.3 (CH ), 28.4 (C(CH
), 18.4 (CH ), 18.3 (CH
calculated for C31 : 598.3726, found: 598.3716.
1
3
d
(
1
5.2.6.7. Tert-butyl
((R)-1-(((R)-1-((4-(2-(bis(pyridin-2-ylmethyl)
amino)acetamido)phenethyl)amino)-1-oxopropan-2-yl)amino)-1-
(
3
)
3
), 60.5 (2 ꢃ CH
9.3 (CH ), 38.8 (CH
), 26.2 (CH
2
2
a
a
1
oxopropan-2-yl)carbamate (8g). Yield (3.65 g, 77%):
400 MHz, DMSO‑d ):
¼ 10.53 (s, 1H, NH), 8.57 (ddd, J ¼ 4.9, 1.8,
.9 Hz, 2H), 7.90 (d, J ¼ 5.6 Hz, 1H, NH), 7.82-7.67 (m, 3H, 2H arom,
NH), 7.58 (m, 2H), 7.43 (m, 2H), 7.28 (m, 2H), 7.14 (m, 2H), 6.98 (d,
J ¼ 7.3 Hz, 1H, NH), 4.20 (m, 1H, CH ), 3.90 (m, 5H), 3.41 (s, 2H,
NCH CO), 3.21 (m, 2H), 2.63 (m, 2H), 1.37 (s, 9H, C(CH ), 1.15 (d,
J ¼ 7.0 Hz, 6H, 2 ꢃ CH ¼ 171.8
H NMR
3
(
2
2
2
2
3
)
3
), 26.3
(
6
d
þ
CH
2
2
3
3
); HRMS-ESI m/z [MþH]
0
48 7 5
H N O
a
5
2
.2.6.4. Tert-butyl ((21R,24R)-21-methyl-4,20,23-trioxo-1-(pyridin-
-yl)-2-(pyridin-2-ylmethyl)-9,12,15-trioxa-2,5,19,22-
2
3 3
)
13
3
6
); C NMR (100 MHz, DMSO‑d ): d
1
tetraazapentacosan-24-yl)carbamate (8d). Yield (264 mg, 87%): H
NMR (400 MHz, CDCl ):
¼ 8.77 (m, 1H, NH), 8.55 (ddd, J ¼ 5.0, 1.9,
(CO), 169.0 (CO), 158.4 (2 ꢃ Cquat Pyr), 156.1 (NCOOtBu), 149.0
3
d
(2 ꢃ CH Pyr), 136.9 (Cquat Phenyl), 136.7 (2 ꢃ CH Pyr), 134.2 (Cquat
Please cite this article as: A. Prandina et al., Synthesis and biological evaluation of new dipicolylamine zinc chelators as metallo-
inhibitors, Tetrahedron, https://doi.org/10.1016/j.tet.2019.02.004
b-lactamase

12
A. Prandina et al. / Tetrahedron xxx (xxxx) xxx
Phenyl), 128.9 (2 ꢃ CH Phenyl), 123.0 (2 ꢃ CH Pyr), 122.4 (2 ꢃ CH
Pyr), 57.8
), 34.4 (CH ), 28.1
47 8 5
for C31H N O : 611.3659, found: 611.3664.
Pyr), 119.0 (2 ꢃ CH Phenyl), 78.1 (C(CH
3
)
3
), 59.4 (2 ꢃ CH
2
5.2.7. General procedure for N-Boc deprotection of compounds 8a-j
(
NCH
2
CO), 49.8 (CH
a
), 48.0 (CH
a
), 40.2 (CH
2
2
The compounds 1a-j were synthesized and purified according
(
3
C(CH )
3
), 17.9 (2 ꢃ CH
3
).
the previously described method (see general procedure for N-Boc
deprotection of compounds 6a-j). All the compounds, except 1g,
were obtained as oils.
5.2.6.8. Tert-butyl ((R)-1-(((R)-1-((4-(2-(2-(bis(pyridin-2-ylmethyl)
amino)acetamido)ethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-
1
oxopropan-2-yl)carbamate (8h). Yield (2.15 g, 94%):
H
NMR
¼ 8.80 (s, 1H, NH), 8.73 (t, J ¼ 5.9 Hz, 1H, NH),
.47 (m, 2H), 7.56 (td, J ¼ 7.7, 1.8 Hz, 2H), 7.47 (d, J ¼ 8.0 Hz, 2H),
5.2.7.1. (R)-2-Amino-N-((R)-1-((2-(2-(di-2-picolylamino)acetamido)
(
3
400 MHz, CDCl ): d
ethyl)amino)-1-oxopropan-2-yl)propanamide (1a). Yield (80 mg,
8
7
1
5
4%): H NMR (400 MHz, CD
3
CN):
d
¼ 8.53 (m, 2H), 8.28 (bs, 1H,
.15e7.07 (m, 6H), 6.95 (d, J ¼ 7.5 Hz, 1H, NH), 5.21 (d, J ¼ 5.8 Hz, 1H,
NH), 4.58 (quartet, J ¼ 7.1 Hz, 1H, CH ), 4.16 (bs, 1H, CH ), 3.74 (s,
H, 2 ꢃ NCH Py), 3.53 (quartet, J ¼ 6.7 Hz, 2H, CH NH), 3.26 (s, 2H,
NCH ), 1.43 (d, J ¼ 7.1 Hz, 3H, CH ),
CO), 2.80 (t, J ¼ 6.9 Hz, 2H, PhCH
.40 (s, 9H, C(CH ); C NMR
), 1.37 (d, J ¼ 7.1 Hz, 3H, CH
100 MHz, CDCl ):
¼ 173.0 (CO), 171.4 (CO), 170.3 (CO), 158.2
NH), 8.23 (bs, 1H, NH), 7.69 (m, 2H), 7.51 (m, 1H, NH), 7.32 (dt,
a
a
J ¼ 7.8, 1.1 Hz, 2H), 7.22 (ddd, J ¼ 7.7, 4.8, 1.2 Hz, 2H), 4.17 (pentet,
4
2
2
J ¼ 7.2 Hz, 1H, CH
a
), 4.00 (t, J ¼ 7.2 Hz, 1H, CH
a), 3.74 (s, 4H,
2
2
3
2
ꢃ NCH
.34e1.25 (m, 4H), 1.22 (d, J ¼ 7.2 Hz, 3H, CH
CD CN):
¼ 173.3 (CO), 171.4 (CO), 161.5 (CO), 159.0 (2 ꢃ Cquat Pyr),
50.3 (2 ꢃ CH Pyr), 137.9 (2 ꢃ CH Pyr), 124.8 (2 ꢃ CH Pyr), 123.5
2 ꢃ CH Pyr), 60.9 (2 ꢃ CH Pyr), 58.7 (NCH CO), 54.6 (CH ), 50.7
and 50.6 (CH ), 40.6 (CH ), 39.4 (CH ), 17.73 (CH ), 17.66 (CH );
: 464.2372,
Py), 3.34e3.14 (m, 4H), 1.47 (d, J ¼ 7.0 Hz, 3H, CH
),
2
3
13
1
3
)
3
3
13
1
3
); C NMR (100 MHz,
(
(
3
d
3
d
2 ꢃ Cquat Pyr), 156.1 (NCOOtBu), 149.4 (2 ꢃ CH Pyr), 136.7 (2 ꢃ CH
1
Phenyl), 136.5 (Cquat Phenyl), 135.1 (Cquat Phenyl), 129.1 (2 ꢃ CH
(
2
2
a
Pyr), 123.2 (2 ꢃ CH Pyr), 122.5 (2 ꢃ CH Pyr), 120.0 (2 ꢃ CH Phenyl),
a
2
2
3
3
8
0.8 (C(CH
3
)
3
), 60.5 (2 ꢃ CH
2
Pyr), 58.1 (NCH
), 28.4 (C(CH ), 18.1 (CH
: 618.3385, found:
2
CO), 51.0 (CH
a
), 49.7
þ
HRMS-ESI m/z [MþNa] calculated for C22
7 3
H31NaN O
(
CH ), 40.4 (CH
a
2
), 35.1 (CH
2
3
)
3
3
), 18.0 (CH
3
);
found: 464.2381.
þ
HRMS-ESI m/z [MþH] calculated for C33
44 7 5
H N O
618.3398.
5.2.7.2. (R)-2-Amino-N-((R)-1-((4-(2-(bis(pyridin-2-ylmethyl)
amino)acetamido)butyl)amino)-1-oxopropan-2-yl)propanamide
1
5
.2.6.9. Tert-butyl
((R)-1-(((R)-1-((2-(1-(bis(pyridin-2-ylmethyl)
(1b). Yield (94 mg, 57%): H NMR (400 MHz, CDCl ):
d
¼ 8.78 (t,
3
glycyl)piperidin-4-yl)ethyl)amino)-1-oxopropan-2-yl)amino)-1-
oxopropan-2-yl)carbamate (8i). The title compound was isolated as
a mixture of amide rotamers (200 mg, 27% yield): H NMR
J ¼ 5.9 Hz, 1H, NH), 8.54 (ddd, J ¼ 4.9, 1.8, 0.9 Hz, 2H), 7.76 (d,
J ¼ 8.0 Hz, 1H, NH), 7.60 (td, J ¼ 7.7, 1.8 Hz, 2H), 7.23 (dt, J ¼ 7.8,
1.1 Hz, 2H), 7.16 (ddd, J ¼ 7.6, 4.9, 1.2 Hz, 2H), 7.03 (bt, J ¼ 5.5 Hz, 1H,
1
(
400 MHz, CDCl
J ¼ 7.7, 1.8 Hz, 2H), 7.53 (d, J ¼ 7.8 Hz, 2H), 7.15 (ddd, J ¼ 7.5, 4.9,
.2 Hz, 2H), 6.68e6.53 (m, 2H, 2*NH), 5.00 (s, 1H, NH), 4.51 (d,
J ¼ 13.3 Hz, 1H, NH), 4.41 (pentet, J ¼ 7.2 Hz, 1H, CH ), 4.07 (m, 1H,
CH
), 3.91 (d, J ¼ 14.1 Hz, AB system, 2H), 3.86 (d, J ¼ 14.1 Hz, AB
system, 2H), 3.75 (m, 1H), 3.40 (s, 2H, NCH CO), 3.29 (m, 1H), 3.16
m, 1H), 2.80 (m, 1H), 2.47 (m, 1H), 2.04 (bs, 1H), 1.76e1.56 (m, 2H),
.55e1.39 (m, 11H, C(CH , CH ), 1.36
), 1.37 (d, J ¼ 3.0 Hz, 3H, CH
d, J ¼ 3.1 Hz, 3H, CH ), 1.11e0.94 (m, 2H); C NMR (100 MHz,
CDCl ):
¼ 172.6 (CO), 171.9 (CO), 168.7 (CO), 159.1 (2 ꢃ Cquat Pyr),
56.1 (NCOOtBu), 149.1 (2 ꢃ CH Pyr), 136.6 (2 ꢃ CH Pyr), 123.8
2 ꢃ CH Pyr), 122.3 (2 ꢃ CH Pyr), 81.0 (C(CH ), 60.5 (2 ꢃ CH Pyr),
6.1 (NCH CO), 51.2 (CH ), 49.1 (CH ), 45.49 and 45.44 (CH ), 42.2
CH ), 37.1 and 37.0 (CH ), 36.4, 36.0 and 35.9 (CH ), 33.7 and 33.5
CH), 32.6 (CH ), 28.4 (C(CH ), 18.0
3
):
d
¼ 8.52 (ddd, J ¼ 4.9, 2.0, 1.1 Hz, 2H), 7.65 (td,
NH), 4.47e4.33 (pentet, J ¼ 7.2 Hz, 1H, CH
a
), 3.81 (s, 4H,
), 3.29e3.18 (m, 6H),
),1.29 (d, J ¼ 7.0 Hz, 3H,
¼ 176.0 (CO), 172.4 (CO), 171.5
CO), 158.1 (2 ꢃ Cquat Pyr), 149.5 (2 ꢃ CH Pyr), 136.8 (2 ꢃ CH Pyr),
Pyr), 58.1
2
1
ꢃ NCH
2
Py), 3.45 (quartet, J ¼ 7.0 Hz, 1H, CH
a
1
.63e1.38 (m, 6H),1.34 (d, J ¼ 7.0 Hz, 3H, CH
3
a
13
3 3
CH ); C NMR (100 MHz, CDCl ): d
a
(
2
1
23.5 (2 ꢃ CH Pyr), 122.6 (2 ꢃ CH Pyr), 60.6 (2 ꢃ CH
2
(
1
(
(
NCH
2
CO), 50.7 (CH
a
), 48.5 (CH
a
), 39.2 (CH
2
), 38.7 (CH
2
), 27.1 (CH
2
),
3
)
3
2
3
þ
13
26.4 (CH ), 21.6 (CH ), 18.2 (CH ); HRMS-ESI m/z [MþH] calculated
2
3
3
3
for C24
36 7 3
H N O : 470.2871, found: 470.2874.
3
d
1
5
.2.7.3. (R)-2-Amino-N-((R)-1-((6-(2-(di-2-picolylamino)acetamido)
hexyl)amino)-1-oxopropan-2-yl)propanamide (1c). Yield (10 mg,
1%): H NMR (600 MHz, CDCl
(
3
)
3
2
5
(
2
a
a
2
1
5
3
):
d
¼ 8.69 (m, 1H, NH), 8.55 (ddd,
2
2
2
J ¼ 5.2, 1.7, 0.9 Hz, 2H), 7.71 (dd, J ¼ 17.4, 7.7 Hz, 1H, NH), 7.61 (td,
(
(
6
2
), 31.94 and 31.87 (CH
2
3 3
)
H N O :
48 7 5
J ¼ 7.6, 1.9 Hz, 2H), 7.26 (m, 2H), 7.16 (m, 2H), 6.51 (t, J ¼ 5.5 Hz, 1H,
þ
2 ꢃ CH
3
); HRMS-ESI m/z [MþH] calculated for C32
NH), 4.39 (m, 1H, CH
.30 (s, 2H, NCH
m, 2H),1.43 (m, 3H),1.36 (d, J ¼ 7.0 Hz, 3H, CH
a
), 3.84 (s, 4H, 2 ꢃ NCH
CO), 3.26 (m, 2H), 3.19 (m, 2H), 2.08 (m, 2H), 1.53
), 1.34e1.29 (m, 6H);
¼ 176.1 (CO), 172.2 (CO), 171.3 (CO),
58.4 (2 ꢃ Cquat Pyr), 149.5 (2 ꢃ CH Pyr), 136.7 (2 ꢃ CH Pyr), 136.6
2
Py), 3.48 (m, 1H, CHa),
10.3700, found: 610.3711.
3
2
(
3
5.2.6.10. Tert-butyl
((R)-1-(((R)-1-((2-(4-(bis(pyridin-2-ylmethyl)
13
3
C NMR (151 MHz, CDCl ): d
glycyl)piperazin-1-yl)ethyl)amino)-1-oxopropan-2-yl)amino)-1-
oxopropan-2-yl)carbamate (8j). Yield (200 mg, 25%): H NMR
1
(
1
2 ꢃ CH Pyr), 123.3 (2 ꢃ CH Pyr), 122.5 (2 ꢃ CH Pyr), 60.6
(
400 MHz, CDCl
J ¼ 7.6, 1.8 Hz, 2H), 7.51 (dt, J ¼ 7.8, 1.2 Hz, 2H), 7.16 (ddd, J ¼ 7.5, 4.9,
.3 Hz, 2H), 6.73 (d, J ¼ 7.6 Hz, 1H, NH), 6.65 (bs, 1H, NH), 5.04 (d,
J ¼ 6.4 Hz, 1H, NH), 4.42 (pentet, J ¼ 7.2 Hz, 1H, CH ), 4.12 (m, 1H,
CH Py), 3.57 (m, 2H), 3.45e3.20 (m, 6H),
), 3.88 (s, 4H, 2 ꢃ NCH
.46 (t, J ¼ 6.6 Hz, 2H), 2.43e2.29 (m, 4H), 1.43 (s, 9H, C(CH ), 1.36
3
):
d
¼ 8.52 (ddd, J ¼ 5.0, 1.8, 0.9 Hz, 2H), 7.65 (td,
(
3
1
2 ꢃ CH
8.8 (CH
7.8 (CH
2
Pyr), 58.2 (NCH
2
CO), 50.7 (CH
), 29.5 (CH ), 29.4 (CH
a
), 48.6 (CH
a
), 39.3 (CH
2
),
),
2
2
2
), 26.4 (CH
2
), 26.3 (CH ), 21.7 (CH
2
3
1
þ
3
); HRMS-ESI m/z [MþH] calculated for C26
40 7 3
H N O :
a
4
98.3198, found: 498.3192.
a
2
2
3 3
)
5
2
.2.7.4. (R)-2-Amino-N-((R)-4,20-dioxo-1-(pyridin-2-yl)-2-(pyridin-
1
3
(
(
(
d, J ¼ 6.3 Hz, 3H, CH
100 MHz, CDCl ):
¼ 172.5 (CO), 172.0 (CO), 168.8 (CO), 158.9
2 ꢃ Cquat Pyr), 155.8 (NCOOtBu), 149.2 (2 ꢃ CH Pyr), 136.6 (2 ꢃ CH
), 60.4
), 52.8 (CH ),
), 36.2 (CH ), 28.4
3
), 1.34 (d, J ¼ 6.2 Hz, 3H, CH
3
); C NMR
-ylmethyl)-9,12,15-trioxa-2,5,19-triazadocosan-21-yl)propanamide
3
d
1
(
1d). Yield (34 mg, 94%): H NMR (400 MHz, CDCl
3
):
d
¼ 8.74 (t,
J ¼ 6.0 Hz, 1H, NH), 8.53 (ddd, J ¼ 4.9, 1.7, 0.9 Hz, 2H), 7.74 (d,
J ¼ 7.8 Hz, 1H, NH), 7.59 (td, J ¼ 7.7, 1.8 Hz, 2H), 7.25 (m, 2H), 7.14
(ddd, J ¼ 7.3, 4.9,1.2 Hz, 2H), 7.02 (t, J ¼ 5.3 Hz,1H, NH), 4.36 (pentet,
Pyr), 123.8 (2 ꢃ CH Pyr), 122.3 (2 ꢃ CH Pyr), 80.6 (C(CH
2 ꢃ CH Pyr), 56.6 (CH ), 56.2 (NCH CO), 53.1 (CH
0.6 (CH ), 49.0 (CH ), 45.1 (CH ), 41.7 (CH
C(CH ), 18.4 (CH ), 18.2 (CH
3 3
)
(
5
2
2
2
2
2
a
a
2
2
2
J ¼ 7.1 Hz, 1H, CH
a
), 3.80 (s, 4H, 2 ꢃ NCH
2
Py), 3.65e3.41 (m, 13H),
CO), 2.83 (bs, 2H), 1.81 (pentet,
(
3
)
3
3
3
); HRMS-ESI m/z [MþH]þ calculated
3.39e3.28 (m, 4H), 3.26 (s, 2H, NCH
2
Please cite this article as: A. Prandina et al., Synthesis and biological evaluation of new dipicolylamine zinc chelators as metallo-
inhibitors, Tetrahedron, https://doi.org/10.1016/j.tet.2019.02.004
b-lactamase

A. Prandina et al. / Tetrahedron xxx (xxxx) xxx
13
J ¼ 6.7 Hz, 2H), 1.74 (pentet, J ¼ 6.1 Hz, 2H), 1.32 (d, J ¼ 7.0, 3H, CH
3
),
¼ 175.8
CO), 172.3 (CO), 171.3 (CO), 158.2 (2 ꢃ Cquat Pyr), 149.4 (2 ꢃ CH
(bs, 2H, NH
2
), 1.40 (d, J ¼ 6.9 Hz, 3H, CH
3
), 1.29 (d, J ¼ 6.9 Hz, 3H,
13
13
1
.28 (d, J ¼ 6.9 Hz, 3H, CH
3
); C NMR (100 MHz, CDCl
3
):
d
CH
3
); C NMR (100 MHz, CDCl ):
3
d
¼ 176.3 (CO), 171.2 (CO), 170.5
(
(CO), 158.1 (2 ꢃ Cquat Pyr), 149.2 (2 ꢃ CH Pyr), 136.6 (Cquat Phenyl),
136.5 (2 ꢃ CH Pyr), 134.8 (Cquat Phenyl), 129.0 (2 ꢃ CH Phenyl),
123.1 (2 ꢃ CH Pyr), 122.3 (2 ꢃ CH Pyr), 119.8 (2 ꢃ CH Phenyl), 60.4
Pyr), 136.7 (2 ꢃ CH Pyr), 123.4 (2 ꢃ CH Pyr), 122.5 (2 ꢃ CH Pyr), 70.6
(
CH
2 ꢃ CH
6.5 (CH
2
), 70.5 (CH
2
), 70.3 (CH
CO), 50.7 (CH
), 28.9 (CH ), 21.6 (CH
: 602.3658, found: 602.3661.
2
), 70.2 (CH
2
), 69.6 (CH
), 48.5 (CH
), 18.5 (CH
2
), 69.0 (CH
), 37.7 (CH
); HRMS-ESI
2
), 60.5
(
3
2
Pyr), 58.1 (NCH
2
a
a
2
),
(2 ꢃ CH Pyr), 58.0 (NCH
34.9 (CH ), 21.5 (CH ), 18.1(CH
36 7 3
for C28H N O : 518.2864, found: 518.2874.
2
2
CO), 50.6 (CH
a), 49.2 (CH
a
), 40.2 (CH
2
),
þ
2
), 29.6 (CH
2
2
3
3
2
3
3
); HRMS-ESI m/z [MþH] calculated
þ
m/z [MþH] calculated for C30
H
48
N
7
O
6
5
.2.7.5. (R)-2-amino-N-((R)-1-((4-(2-(di-2-picolylamino)acetamido)
cyclohexyl)amino)-1-oxopropan-2-yl)propanamide (1e). Yield
¼ 8.60 (d, J ¼ 7.9 Hz, 1H,
NH), 8.57 (m, 2H), 7.82 (d, J ¼ 7.8 Hz, 1H, NH), 7.62 (td, J ¼ 7.7, 1.9 Hz,
H), 7.27 (m, 2H), 7.17 (ddd, J ¼ 7.5, 4.8, 1.2 Hz, 2H), 6.66 (d,
), 3.87 (m, 2H),
Py), 3.52 (quartet, J ¼ 6.9 Hz, 1H, CH ), 3.26 (s,
5
.2.7.9. (R)-2-Amino-N-((R)-1-((2-(1-(bis(pyridin-2-ylmethyl)glycyl)
piperidin-4-yl)ethyl)amino)-1-oxopropan-2-yl)propanamide (1i).
The title compound was isolated as a mixture of amide rotamers
1
(
3
115 mg, 87%): H NMR (400 MHz, CDCl ): d
1
(
92 mg, 61% yield): H NMR (400 MHz, CDCl
J ¼ 4.9, 1.8, 0.9 Hz, 2H), 7.76 (d, J ¼ 7.8 Hz, 1H, NH), 7.65 (td, J ¼ 7.6,
.8 Hz, 2H), 7.53 (dt, J ¼ 7.9, 1.1 Hz, 2H), 7.15 (ddd, J ¼ 7.4, 4.9, 1.3 Hz,
2H), 6.52 (t, J ¼ 5.8 Hz, 1H, NH), 4.52 (d, J ¼ 13.2 Hz, 1H), 4.36
(pentet, J ¼ 7.2 Hz, 1H, CH ), 3.91 (d, J ¼ 14.1 Hz, AB system, 2H),
3.86 (d, J ¼ 14.1 Hz, AB system, 2H), 3.76 (m, 1H), 3.49 (quartet,
J ¼ 7.0 Hz,1H, CH ), 3.40 (s, 2H, NCH CO), 3.24 (m, 2H), 2.79 (m,1H),
2.47 (tt, J ¼ 12.8, 3.3 Hz, 1H), 1.83 (bs, 2H, NH ), 1.72e1.55 (m, 3H),
3
):
d
¼ 8.52 (ddd,
2
J ¼ 7.6 Hz, 1H, NH), 4.41 (pentet, J ¼ 7.2 Hz, 1H, CH
3
2
CH
d
a
1
.82 (s, 4H, 2 ꢃ NCH
2
a
H, NCH
2
CO), 2.51 (bs, 2H),1.75e1.60 (m, 8H),1.38 (d, J ¼ 6.9 Hz, 3H,
a
1
3
3
), 1.33 (d, J ¼ 6.9 Hz, 3H, CH
3 3
); C NMR (100 MHz, CDCl ):
¼ 175.8 (CO), 171.5 (CO), 170.4 (CO), 158.3 (2 ꢃ Cquat Pyr), 149.5
a
2
(
2 ꢃ CH Pyr), 136.7 (2 ꢃ CH Pyr), 123.5 (2 ꢃ CH Pyr), 122.6 (2 ꢃ CH
Pyr), 58.1 (NCH CO), 50.6 (CH ), 48.8 (CH ), 46.2
), 28.17 (CH ), 21.5 (CH ),
2
1
1
1
(
.33 (m, 2H),1.36 (d, J ¼ 7.0 Hz, 3H, CH
3
), 1.33 (d, J ¼ 7.0 Hz, 3H, CH
.04 (m, 2H); C NMR (100 MHz, CDCl ):
¼ 176.1, 172.2, 170.2 and
68.7 (3 ꢃ CO), 159.1 (2 ꢃ Cquat Pyr), 149.1 (2 ꢃ CH Pyr), 136.6
2 ꢃ CH Pyr), 123.8 (2 ꢃ CH Pyr), 122.3 (2 ꢃ CH Pyr), 60.5
2 ꢃ CH Pyr), 56.1 (NCH CO), 50.6 (CH ), 48.6 (CH ), 45.4 (CH ),
), 37.2 and 37.1 (CH ), 36.11 and 36.05 (CH ), 33.9 and 33.8
(CH), 32.6 and 32.5 (CH ), 32.0 and 31.9 (CH ), 21.7 (CH ),17.5 (CH );
39NaN : 532.2990,
3
),
Pyr), 60.6 (2 ꢃ CH
CH), 45.3 (CH), 28.4 (2*CH
7.8 (CH
2
2
a
a
13
3
d
(
1
2
), 28.20 (CH
); HRMS-ESI m/z [MþH] calculated for C26
2
2
3
þ
3
38 7 3
H N O :
4
96.3026, found: 496.3031.
(
2
2
a
a
2
5.2.7.6. Trans (R)-2-amino-N-((R)-1-((4-(2-(di-2-picolylamino)acet-
42.1 (CH
2
2
2
amido)cyclohexyl)amino)-1-oxopropan-2-yl)propanamide
Yield (45 mg, 55%): H NMR (400 MHz, CDCl
(1f).
¼ 8.88 (d,
2
2
3
3
1
þ
3
):
d
HRMS-ESI m/z [MþNa] calculated for C27
H
7 3
O
J ¼ 8.3 Hz, 1H, NH), 8.52 (ddd, J ¼ 4.8, 1.8, 0.9 Hz, 2H), 7.78 (d,
J ¼ 7.9 Hz,1H, NH), 7.57 (td, J ¼ 7.7,1.8 Hz, 2H), 7.21 (dt, J ¼ 7.7,1.1 Hz,
found: 532.3007.
2
H), 7.14 (ddd, J ¼ 7.6, 4.9, 1.2 Hz, 2H), 6.80 (d, J ¼ 8.1 Hz, 1H, NH),
5
.2.7.10. (R)-2-Amino-N-((R)-1-((2-(4-(bis(pyridin-2-ylmethyl)
4
2
2
d
.40 (pentet, J ¼ 7.1 Hz, 1H, CH
a
), 3.80 (s, 4H, 2 ꢃ NCH
2
Py), 3.71 (m,
CO),
.02e1.86 (m, 4H), 1.46e1.19 (m, 12H); C NMR (100 MHz, CDCl ):
¼ 175.8 (CO), 171.8 (CO), 170.7 (CO), 158.3 (2 ꢃ Cquat Pyr), 149.4
glycyl)piperazin-1-yl)ethyl)amino)-1-oxopropan-2-yl)propanamide
(1j). Yield (80 mg, 53%): H NMR (400 MHz, CDCl ):
1
H), 3.45 (quartet, J ¼ 6.9 Hz, 1H, CH
a
), 3.27 (s, 2H, NCH
2
d
¼ 8.52 (ddd,
3
13
3
J ¼ 4.9, 1.8, 0.9 Hz, 2H), 7.72 (d, J ¼ 7.9 Hz, 1H, NH), 7.64 (td, J ¼ 7.6,
1.8 Hz, 2H), 7.50 (dt, J ¼ 7.8, 1.1 Hz, 2H), 7.15 (ddd, J ¼ 7.5, 4.9, 1.3 Hz,
(
2 ꢃ CH Pyr), 136.6 (2 ꢃ CH Pyr), 123.2 (2 ꢃ CH Pyr), 122.5 (2 ꢃ CH
Pyr), 58.2 (NCH CO), 50.7 (CH ), 48.6 (CH ), 47.8
CH), 47.3 (CH), 31.49 (2 ꢃ CH ), 21.7 (CH ), 18.5
: 496.3023,
2H), 6.62 (t, J ¼ 5.2 Hz, 1H, NH), 4.38 (pentet, J ¼ 7.1 Hz, 1H, CH
a),
Pyr), 60.4 (2 ꢃ CH
2
2
a
a
3.87 (s, 4H, 2 ꢃ NCH Py), 3.54 (m, 2H), 3.46 (quartet, J ¼ 7.0 Hz, 1H,
2
(
(
2
), 31.46 (2 ꢃ CH
2
H
3
CH
a
), 3.43e3.21 (m, 6H), 2.44 (t, J ¼ 6.1 Hz, 2H), 2.34 (m, 4H), 1.66
þ
13
CH
3
); HRMS-ESI m/z [MþH] calculated for C26
38
N
7
O
3
(s, 2H), 1.36 (d, J ¼ 7.0 Hz, 3H, CH ), 1.30 (d, J ¼ 7.0 Hz, 3H, CH );
C
3
3
found: 496.3031.
NMR (100 MHz, CDCl ):
d
¼ 175.8 (CO), 172.3 (CO), 168.8 (CO), 158.9
3
(
2 ꢃ Cquat Pyr),149.1 (2 ꢃ CH Pyr),136.6 (2 ꢃ CH Pyr),123.8 (2 ꢃ CH
5
.2.7.7. (R)-1-(((R)-1-((4-(2-(Di-2-picolylamino)acetamido)phe-
Pyr), 122.3 (2 ꢃ CH Pyr), 60.4 (2 ꢃ CH Pyr), 56.7 (CH ), 56.5 (CH ),
5
2
2
2
nethyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-aminium
chloride (1g). Yield (2.60 g, 83%): H NMR (400 MHz, D
6.1 (NCH
2
CO), 53.0 (CH
2
), 52.7 (CH
2
), 50.7 (CH
a), 48.5 (CH
a), 45.2
1
2
O):
d
¼ 8.77
(
[
CH
2
), 41.7 (CH
2
), 36.1 (CH
2
), 21.7 (CH ), 18.1 (CH
3
3
); HRMS-ESI m/z
(
d, J ¼ 5.4 Hz, 2H), 8.53 (td, J ¼ 8.0, 1.4 Hz, 2H), 8.07 (d, J ¼ 8.0 Hz,
þ
MþNa] calculated for C26
H
38NaN O : 533.2940, found: 533.2959.
8 3
2
4
H), 7.96 (t, J ¼ 6.8 Hz, 2H), 7.27 (m, 4H), 4.51 (s, 4H, 2 ꢃ NCH
.29-4.02 (m, 2H), 3.78 (s, 2H, NCH CO), 3.65e3.32 (m, 2H), 2.84 (q,
), 1.27 (d, J ¼ 7.2 Hz, 3H,
¼ 174.8 (CO), 170.9 (CO), 170.8
CO), 152.7 (2 ꢃ Cquat Pyr), 147.3 (2 ꢃ CH Pyr), 142.0 (Cquat Phenyl),
37.1 (Cquat Phenyl), 134.7 (2 ꢃ CH Pyr), 129.9 (2 ꢃ CH Phenyl),
2
Py),
2
5
.2.8. Synthesis of tert-butyl ((R)-1-(((R)-1-(di-2-picolylamino)-1-
oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate (9)
N-Boc-D-alanyl- -alanioic acid 4 (100 mg, 0.384 mmol, 1.0
equiv.), DPA (0.07 mL, 0.384 mmol, 1.0 equiv.) and HATU (144.5 mg,
J ¼ 6.7 Hz, 2H), 1.51 (d, J ¼ 7.1 Hz, 3H, CH
3
13
3 2
CH ); C NMR (100 MHz, D O): d
D
(
1
1
0
0
0
.384 mmol, 1.0 equiv.) dissolved in CH
2
Cl
2
(4 mL) and cooled to
27.5 (2 ꢃ CH Phenyl), 126.7 (2 ꢃ CH Pyr), 122.1 (2 ꢃ CH Pyr), 58.3
ꢁ
C
in an ice-water bath. N-methylmorpholine (0.085 mL,
(
3
2 ꢃ CH
4.4 (CH
calculated for C26
2
Pyr), 57.1 (NCH
), 17.1 (CH ), 16.9 (CH
37NaN : 518.2880, found: 518.2879.
2
CO), 50.3 (CH
a
), 49.2 (CH
a
), 40.7 (CH
2
),
.768 mmol, 2 equiv.) was added and the stirring mixture was left
þ
2
3
3
); HRMS-ESI m/z [M þ H þ Na]
at 0 C for 15 min before slowly warming up to room temperature.
After 4 h at room temperature, the mixture was washed with 1 M
ꢁ
H
7 3
O
NaOH (4 ꢃ 20 mL), dried over K
under reduced pressure. The yellow oily residue was dissolved in
CH Cl and purified by column chromatography on neutral alumina
0e5% MeOH in CH Cl ) to afford the title compound 9 as a mixture
of amide rotamers which is a clear yellow oil. Yield (150 mg, 91%):
2 3
CO , filtered and concentrated
5
.2.7.8. (R)-2-Amino-N-((R)-1-((4-(2-(2-(di-2-picolylamino)acet-
amido)ethyl)phenyl)amino)-1-oxopropan-2-yl)propanamide (1h).
¼ 9.34 (s, 1H, NH),
.70 (t, J ¼ 5.7 Hz, 1H, NH), 8.43 (ddd, J ¼ 4.8, 1.9, 1.0 Hz, 2H), 7.99 (d,
J ¼ 7.8 Hz, 1H, NH), 7.51 (td, J ¼ 7.7, 1.8 Hz, 2H), 7.41 (m, 2H),
.12e7.00 (m, 6H), 4.63 (pentet, J ¼ 7.1 Hz, 1H, CH ), 3.70 (s, 4H,
ꢃ NCH Py), 3.47 (m, 3H, CH , CH NH), 3.22 (s, 2H, NCH CO), 1.46
2
2
1
3
Yield (1.111 g, 88%): H NMR (400 MHz, CDCl ): d
8
(
2
2
1
H NMR (400 MHz, CDCl
m, 4H), 7.03 (t, J ¼ 8.5 Hz, 1H, NH), 5.08 (bs, 1H, NH), 4.99 (m, 1H,
CH ), 4.89e4.63 (m, 4H), 4.16 (bs, 1H, CH ), 1.43 and 1.42 (s, 9H,
3
):
d
¼ 8.50 (m, 2H), 7.62 (m, 2H), 7.23e7.11
7
a
(
2
2
a
2
2
a
a
Please cite this article as: A. Prandina et al., Synthesis and biological evaluation of new dipicolylamine zinc chelators as metallo-
inhibitors, Tetrahedron, https://doi.org/10.1016/j.tet.2019.02.004
b-lactamase

14
A. Prandina et al. / Tetrahedron xxx (xxxx) xxx
C(CH
3
)
3
), 1.38 (d, J ¼ 6.7 Hz, 3H, CH
3
), 1.32 (d, J ¼ 7.0 Hz, 3H, CH
3
);
Trifluoroacetic acid (6 mL, 78.35 mmol) was then added dropwise
to the stirring solution over the course of 5 min. The reaction was
left for 1 h at room temperature, before it was concentrated under
reduced pressure to a light brown oil. The crude material was
dissolved in 25 mL ethyl acetate and washed with 50 mL 0.5 M
13
C NMR (100 MHz, CDCl ):
Pyr), 155.8 (Cquat Pyr), 155.3 (NCOOtBu), 150.0 (CH Pyr), 149.4 (CH
Pyr), 137.0 (CH Pyr), 136.9 (CH Pyr), 122.8 (CH Pyr), 122.5 (CH Pyr),
3
d
¼ 173.5 (CO), 171.9 (CO), 156.9 (Cquat
122.2 (CH Pyr), 121.6 (CH Pyr), 121.5 (CH Pyr), 80.1 (C(CH
3
)
3
), 51.4
aqueous K
ethyl acetate. The combined organic phases were pooled and
washed with 25 mL 0.5 M aqueous K CO and then dried over
CO , filtered and concentrated under reduced pressure to give
the title compound 12 (0.404 g, 93%) as a pale light oil: H NMR
400 MHz, DMSO‑d ):
¼ 9.52 (s, 1H, NH), 7.46 (m, 2H), 7.41 (d,
2
CO
3
. The water phase was extracted with 3 ꢃ 20 mL
(
NCH
Py), 50.2 (CHa), 45.9 and 45.8 (CHa), 28.4 (C(CH ) ), 19.1, 19.0
2 3 3
); HRMS-ESI m/z [MþH]þ calculated for
and 18.8 (2*CH
C H N O : 442.2442, found: 442.2449.
23 32 5 4
3
2
3
K
2
3
1
5.2.9. Synthesis of (R)-2-((R)-2-aminopropanamido)-N,N-
(
6
d
bis(pyridine-2-ylmethyl)propanamide (10)
The N-Boc-protected amine 9 (60 mg, 0.136 mmol, 1.0 equiv.)
was dissolved in CH
J ¼ 7.0 Hz, 4H), 7.34 (t, J ¼ 7.5 Hz, 4H), 7.29-7.21 (m, 2H), 7.11 (m, 2H),
3
2
.75 (s, 4H, 2 ꢃ NCH
H), 2.57 (t, J ¼ 7.2 Hz, 2H), 1.34 (bs, 2H, NH
):
2
Ph), 3.20 (s, 2H, NCH
2
CO), 2.72 (t, J ¼ 7.2 Hz,
ꢁ
2
Cl
2
(2 mL) and cooled to 0 C in an ice-water
13
2
); C NMR (100 MHz,
bath. Trifluoroacetic acid (0.84 mL, 6.8 mmol, 60.0 equiv.) in
DMSO‑d
6
d
¼ 168.65 (CO), 138.5 (2 ꢃ Cquat Phenyl), 136.3 (Cquat
CH
2
Cl
2
(2 mL) was then slowly added to the stirring mixture. The
Phenyl),135.4 (Cquat Phenyl),128.75 (4 ꢃ CH Phenyl), 128.3 (4 ꢃ CH
ꢁ
reaction was left at 0 C for 20 min before warming up to room
temperature for an additional 3 h. After solvent removal under
reduced pressure,1 M NaOH (20 mL) was added to the mixture, and
Phenyl), 128.05 (CH Phenyl), 127.85 (CH Phenyl), 127.1 (2 ꢃ CH
Phenyl), 119.3 (2 ꢃ CH Phenyl), 57.5 (2 ꢃ NCH
2.1 (CH ), 43.7 (CH
calculated for C24
2
Ph), 56.1 (NCH
2
CO),
þ
5
2
2
); HRMS-TOF MS ES m/z [MþH] calculated for
the compound was extracted with CH
organic layers were washed with fresh 1 M NaOH (2 ꢃ 20 mL), dried
on K CO , filtered, and the solvent removed under reduced pres-
sure, to give the title compound 10 as a mixture of rotamers which
2
Cl
2
(6 ꢃ 5 mL). The combined
H
N
27 3
O: 374.2232, found: 374.2234.
2
3
5.2.12. Synthesis of tert-butyl ((R)-1-((R)-1-((4-(2-(dibenylamino)
acetamido)phenethyl)amino)-1-oxopropan-2-yl)amino)-1-
oxopropan-2-yl)carbamate (13)
1
is a clear yellow oil. Yield (30 mg, 65%): H NMR (400 MHz, CDCl
¼ 8.50 (dd, J ¼ 4.9, 1.9 Hz, 2H), 7.87 (d, J ¼ 7.9 Hz, 1H, NH), 7.62 (td,
J ¼ 7.7, 1.8 Hz, 2H), 7.23 (dt, J ¼ 8.1, 1.1 Hz, 2H), 7.14 (m, 2H), 4.99 (dq,
J ¼ 8.2, 6.8 Hz, 1H, CH ), 4.94e4.59 (m, 4H, 2 ꢃ NCH Py), 3.43 (m,
H, CH ), 1.68 (s, 2H, NH ), 1.30 (d,
), 1.38 (d, J ¼ 6.7 Hz, 3H, CH
); C NMR (100 MHz, CDCl ):
¼ 175.3 and 175.2
CO), 173.84 and 173.77 (CO), 157.0 (Cquat Pyr), 156.04 and 156.01
Cquat Pyr), 149.93 and 149.91 (CH Pyr), 149.4 and 149.3 (CH Pyr),
36.94 (CHPyr), 136.89 and 136.86 (CH Pyr), 122.7 (CH Pyr), 122.5
and 122.4 (CH Pyr), 122.19 and 122.15 (CH Pyr), 121.54 and 121.52
CH Pyr), 52.8 and 52.6 (CH ), 51.44 and 51.39 (CH ), 50.82 and
0.80 (CH ), 45.4 and 45.3 (CH ), 21.7 (CH ), 19.0 (CH ); HRMS-ESI
: 342.1930, found: 342.1925.
3
):
d
Amine 12 (0.404 g, 1.017 mmol) was dissolved in 4 mL DMF. Boc-
a
2
D-alanyl- -alanine-OH hydrochloride 4 (0.285 g, 1.095 mmol) and
D
1
a
2
3
HBTU (0.413 g, 1.090 mmol) were added to the stirring solution and
13
ꢁ
J ¼ 7.0 Hz, 3H, CH
3
3
d
cooled to 0 C in an ice-water bath. NMM (0.115 mL, 1044 mmol)
ꢁ
(
(
1
was added and the solution was left 30 min at 0 C and then 3 h in
room temperature. The mixture was diluted with 200 mL water and
extracted with 3*50 mL ethyl acetate. The combined organic phases
were pooled and washed with 25 mL 0.5 M aqueous NaHCO
then dried over K CO , filtered and concentrated under reduced
pressure to give the title compound 13 (0.498 g, 80%) as a pale light
3
and
(
5
2
2
2
3
a
a
3
3
þ
1
m/z [MþH] calculated for C18
H
24
N
5
O
2
oil: H NMR (400 MHz, DMSO‑d
6
):
d
¼ 9.57 (s, 1H, NH), 7.91 (t,
J ¼ 5.7 Hz, 1H, NH), 7.78 (d, J ¼ 7.5 Hz, 1H, NH), 7.51-7.30 (m, 10H),
7.30-7.21 (m, 2H), 7.15-7.08 (m, 2H), 7.01 (d, J ¼ 7.4 Hz, 1H, NH), 4.19
5.2.10. Synthesis of tert-butyl (4-(2-dibenzylamino)acetamido)
phenethyl)carbamate (11)
(pentet, J ¼ 7.1 Hz, 1H, CH
a
), 3.93 (pentet, J ¼ 7.3 Hz, 1H, CH
a), 3.75
Chloroacetamide 5g (1.002 g, 3.20 mmol) and KI (0.597 g,
(s, 4H, 2 ꢃ NCH
1.38 (s, 9H, C(CH
(100 MHz, DMSO‑d
2
Ph), 3.24 (m, 2H, NCH
), 1.15 (d, J ¼ 7.2 Hz, 6H, 2 ꢃ CH
):
2
CO), 2.64 (t, J ¼ 7.1 Hz, 2H),
13
3
.60 mmol) were dissolved in 350 mL MeCN and dibenzylamine
3
)
3
3
); C NMR
(
(
0.74 mL, 3.85 mmol) was added to the stirring mixture. DIPEA
5.5 mL, 31.6 mmol) was then added and the mixture was heated to
6
d
¼ 172.15 (CO), 171.8 (CO), 168.7 (CO), 155.1
(NCOOtBu), 138.5 (2 ꢃ Cquat Phenyl), 136.6 (Cquat Phenyl), 134.2
(Cquat Phenyl), 128.79 (CH Phenyl), 128.76 (4 ꢃ CH Phenyl), 128.3
(4 ꢃ CH Phenyl), 127.1 (2 ꢃ CH Phenyl), 119.2 (2 ꢃ CH Phenyl), 78.1
reflux and left for 17 h. After cooling to room temperature, the
mixture was concentrated under reduced pressure and purified
using colum chromatography on silica. The product was eluted
using 20e50% ethyl acetate in heptane, yielding 1.03 g (68%) of the
title compound 11 as a pale light oil: H NMR (400 MHz, DMSO‑d
(C(CH ) ), 57.5 (2 ꢃ NCH Ph), 56.0 (NCH CO), 49.7 (CH
3
3
2
2
a
), 47.95
(CH
a
), 40.1 (CH
2
), 34.4 (CH
2
), 28.1 (C(CH
3
)
3
), 18.5 (CH ), 17.9
3
1
):
þ
6
(
2 ꢃ CH
3
); HRMS-TOF MS ES m/z [MþH] calculated for calculated
d
¼ 9.54 (s, 1H, NH), 7.48 (d, J ¼ 8.1 Hz, 2H), 7.42 (d, J ¼ 7.0 Hz, 4H),
.34 (t, J ¼ 7.2 Hz, 4H), 7.30-7.19 (m, 2H), 7.11 (d, J ¼ 8.2 Hz, 2H), 6.83
Ph), 3.21 (s, 2H,
CO), 3.09 (t, J ¼ 8.0 Hz, 2H), 2.63 (t, J ¼ 8.4 Hz, 2H), 1.37 (s, 9H,
45 5 5
for C35H N O : 616.3498, found: 616.3511.
7
(
t, J ¼ 5.7 Hz, 1H, NH), 3.76 (s, 4H, 2 ꢃ NCH
2
5.2.13. Synthesis of (R)-2-amino-N-((R)-1-((4-(2-(dibenylamino)
NCH
2
acetamido)phenethyl)amino)-1-oxopropan-2-yl)propanamide
hydrochloride (14)
13
C(CH
3
)
3
); C NMR (100 MHz, DMSO‑d
6
):
d
¼ 168.7 (CO), 155.45
(
(
(
NCOOtBu), 138.5 (2 ꢃ Cquat Phenyl), 136.55 (Cquat Phenyl), 134.35
The N-Boc-protected amine 13 (0.254 g, 0.413 mmol) was dis-
Cquat Phenyl),128.7 (4 ꢃ CH Phenyl),128.3 (4 ꢃ CH Phenyl),128.04
CH Phenyl), 127.85 (2 ꢃ CH Phenyl), 127.1 (CH Phenyl), 119.3
ꢁ
solved in 25 mL CH
2
Cl
2
and cooled to 0 C in an ice-water bath.
Trifluoroacetic acid (2 mL, 26.12 mmol) was dissolved in 25 mL
CH Cl and added dropwise to the stirring solution over the course
(
2 ꢃ CH Phenyl), 77.4 (C(CH
3
)
3
), 57.5 (2 ꢃ NCH
), 28.2 (C(CH ); HRMS-TOF MS ES m/z
: 474.2756, found:
2 2
Ph), 56.1 (NCH CO),
2
2
4
0.2 (CH
2
), 34.9 (CH
2
3
)
3
of 5 min. The reaction was left at room temperature for 1 h. The
solution was concentrated under reduced pressure. The residue
was diluted in 5 mL dry CH Cl and 1 mL 2 M HCl in diethyl ether
þ
[
4
MþH] calculated for calculated for C29
25 3 3
H N O
74.2763.
2
2
was added to get a precipitate which was filtered, washed with
5
.2.11. Synthesis of N-(4-(2-aminoethyl)phenyl)-2-(dibenzylamino)
diethyl ether and dried under reduced pressure to obtain the
1
acetamide (12)
compound 14 as a powder: H NMR (400 MHz, D
J ¼ 6.8, 2.9 Hz, 4H), 7.42-7.29 (m, 6H), 7.07 (m, 2H), 6.92 (m, 2H),
2
O):
d
¼ 7.46 (dd,
The N-Boc-protected amine 11 (550 mg, 1.16 mmol) was dis-
ꢁ
þ
solved in 25 mL CH
2
Cl
2
and cooled to 0 C in an ice-water bath.
4.50 (s, 3H, NH
3
), 4.06 (q, J ¼ 7.2 Hz, 1H, CH ), 3.93 (q, J ¼ 7.1 Hz,
a
Please cite this article as: A. Prandina et al., Synthesis and biological evaluation of new dipicolylamine zinc chelators as metallo-
inhibitors, Tetrahedron, https://doi.org/10.1016/j.tet.2019.02.004
b-lactamase

A. Prandina et al. / Tetrahedron xxx (xxxx) xxx
15
1
2
H, CH
H), 1.36 (d, J ¼ 7.2, 3H, CH
O):
¼ 175.04 (CO), 171.09 (CO), 164.46 (CO), 137.52,
34.34, 132.09, 131.21, 130.16, 130.13, 130.01, 129.05, 122.49, 60.54
2 ꢃ NCH Ph), 53.02 (NCH CO), 50.56 (CH ), 49.47 (CH ), 40.89
CH ), 34.64 (CH ), 17.33 (CH ), 17.14 (CH ); HRMS-TOF MS ES m/z
M] calculated for calculated for C30 : 516.2974, found:
16.2985.
a
), 3.87 (s, 4H), 3.49-3.37 (m, 5H), 3.22 (m, 1H), 2.74-2.58 (m,
Acknowledgements
13
3
), 1.11 (d, J ¼ 7.2, 3H, CH
3
); C NMR
(
100 MHz, D
2
d
We thank Anne Baudouin from NMR Centre of University of
Lyon 1 for her help. O.A.H.Å is grateful for funding from The
Research Council of Norway (NFR) mobility grant (grant number:
240215). G.K-A and C.S are grateful for a NFR Biotek2021 grant
(244219) and a NFR FORNY grant (273430). We also thank NOVO
Pre Seed (NOVO Nordisk Fonden ByGS/SSch) for a generous grant as
well as funding from the University of Oslo.
1
(
(
[
2
2
a
a
2
2
3
3
þ
38 5 3
H N O
5
5
5
.3. Biological studies
References
.3.1. Microdilution MIC assay
The MIC values of MEM alone or in combination with the
[1] World Health Organization, Global Report on Surveillance, 2014. http://www.
who.int/antimicrobial-resistance/publications/surveillancereport/en/.
compounds were determined using pre-made broth microdilution
plates containing MEM in 2-fold dilution steps ranging from 0.03 to
[2] World Health Organization, Global Action Plan on Antimicrobial Resistance,
015. http://www.who.int/antimicrobial-resistance/global-action-plan/en/.
2
[
3] M.E.A. de Kraker, P.G. Davey, H. Grundmann, on behalf of the, B. s. g., Mortality
and Hospital Stay Associated with Resistant Staphylococcus aureus and
Escherichia coli Bacteremia: estimating the Burden of Antibiotic Resistance in
Europe, PLoS Med. 8 (10) (2011) e1001104.
6
4 mg/L (TREK Diagnostic Systems/Thermo Fisher Scientific, East
Grinstead, UK). The compounds were tested at a final concentration
of 125 M. The MIC assays were performed using two MBL-
m
[4] S.H. Park, Third-generation cephalosporin resistance in gram-negative bac-
teria in the community: a growing public health concern, Kor. J. Intern. Med.
producing MEM resistant strains, one NDM-1-producing
K. pneumoniae [43,44] and one VIM-2-producing P. aeruginosa
29 (1) (2014) 27e30.
[
45]. In brief, the compounds were diluted in MH-broth (Becton
[5] R. Bax, N. Mullan, J. Verhoef, The millennium bugs e the need for and
development of new antibacterials, Int. J. Antimicrob. Agents 16 (1) (2000)
Dickinson, Franklin Lakes, NJ USA) and 50 L of the suspension were
added to each well of the MEM plate. Bacterial colonies were sus-
pended in 0.9% saline buffer to 0.5 McFarland and further diluted
1
5
m
51e59.
[
[
6] N. Høiby, T. Bjarnsholt, M. Givskov, S. Molin, O. Ciofu, Antibiotic resistance of
bacterial biofilms, Int. J. Antimicrob. Agents 35 (4) (2010) 322e332.
7] K. Bush, J.F. Fisher, Epidemiological expansion, structural studies, and clinical
:100 in MH broth (Thermo Fisher Scientific, East Grinstead, UK).
L of the diluted bacterial suspension were added to the MEM
challenges of new
b-lactamases from gram-negative bacteria, Annu. Rev.
0
m
Microbiol. 65 (1) (2011) 455e478.
plates to a final volume of 100
m
L. The plates were incubated for
[8] A.M. Queenan, K. Bush, Carbapenemases: the versatile
b
-lactamases, Clin.
ꢁ
Microbiol. Rev. 20 (3) (2007) 440e458.
2
0 h at 37 C. Each MIC was determined in duplicate. To investigate
[9] J.D.D. Pitout, Infections with extended-spectrum b-lactamase-producing
any intrinsic antibacterial activity of the compounds the MIC of
each compound were determined in a 2-fold serial dilution series in
MH broth (Becton Dickinson, Franklin Lakes, NJ USA) ranging from
enterobacteriaceae, Drugs 70 (3) (2010) 313e333.
[10] R.P. Ambler, A.F. Coulson, J.M. Fr eꢁ re, J.M. Ghuysen, B. Joris, M. Forsman,
R.C. Levesque, G. Tiraby, S.G. Waley, A standard numbering scheme for the
class A
b-lactamases, Biochem. J. 276 (Pt 1) (1991) 269e270.
2
to 1000 mM 50 mL of each dilution step was then mixed with 50 mL
[
11] K. Bush, G.A. Jacoby, A.A. Medeiros, A functional classification scheme for b-
of a bacterial inoculum, prepared as described above, in 96 well
microtiter plates (Thermo Fisher Scientific, Roskilde, Denmark). The
plates were incubated, and MIC determined as described above.
lactamases and its correlation with molecular structure, Antimicrob. Agents
Chemother. 39 (6) (1995) 1211e1233.
[
[
12] S.M. Drawz, R.A. Bonomo, Three decades of
Microbiol. Rev. 23 (1) (2010) 160e201.
13] T.R. Walsh, M.A. Toleman, L. Poirel, P. Nordmann, Metallo-
b
-lactamase inhibitors, Clin.
-Lactamases: the
b
quiet before the storm? Clin. Microbiol. Rev. 18 (2) (2005) 306e325.
5
.3.2. Enzyme inhibition assay
For the determination of the inhibitor concentration 50% (IC50),
[14] C. Bebrone, H. Delbrück, M.B. Kupper, P. Schl o€ mer, C. Willmann, J.-M. Fr eꢁ re,
R. Fischer, M. Galleni, K.M.V. Hoffmann, The structure of the dizinc subclass B2
metallo-
the histidine site, Antimicrob. Agents Chemother. 53 (10) (2009) 4464e4471.
[15] J.D. Buynak, Understanding the longevity of the -lactam antibiotics and of
antibiotic/ -lactamase inhibitor combinations, Biochem. Pharmacol. 71 (7)
2006) 930e940.
b-lactamase CphA reveals that the second inhibitory zinc ion binds in
we preincubated purified VIM-2 (1 nM) [39] at inhibitor concen-
trations ranging from 0 to 250 M. The incubation was carried out
in 50 mM HEPES buffer pH 7.5 containing BSA (2
m
b
b
m
g/mL) and ZnSO
4
(
ꢁ
(
1
m
M) at 25 C for 5 and 20 min. The initial reaction velocity was
[
16] K.M. Papp-Wallace, S.A. Becka, E.T. Zeiser, N. Ohuchi, M.F. Mojica, J.A. Gatta,
M. Falleni, D. Tosi, E. Borghi, M.L. Winkler, B.M. Wilson, J.J. LiPuma, M. Nukaga,
R.A. Bonomo, Overcoming an extremely drug resistant (XDR) pathogen: avi-
bactam restores susceptibility to ceftazidime for burkholderia cepacia com-
plex isolates from cystic fibrosis patients, ACS Infect. Dis. 3 (7) (2017)
502e511.
measured by adding nitrocefin (50 mM) as a reporter substrate. The
kinetics were recorded at 482 nm under stable temperature con-
ditions (25 C) using a SpectraMax Plus plate reader. Data evalua-
tion as well as the IC50 calculation of at least two independent
measurements was done by GraphPad Prism 4.0. All reported
concentrations are final values for 100 L assay volume.
ꢁ
(
R)
[17] W. Fast, L.D. Sutton, Metallo-
Biochim. Biophys. Acta Protein Proteonomics 1834 (8) (2013) 1648e1659.
18] S.M. Drawz, K.M. Papp-Wallace, R.A. Bonomo, New -lactamase inhibitors: a
therapeutic renaissance in an MDR world, Antimicrob. Agents Chemother. 58
4) (2014) 1835e1846.
b-lactamase: inhibitors and reporter substrates,
m
[
b
(
5
.3.3. Cell sensitivity assay
Human breast cancer cells (MDA-MB-231) and pancreatic can-
[19] J.J. Braymer, D.P. Giedroc, Recent developments in copper and zinc homeo-
stasis in bacterial pathogens, Curr. Opin. Chem. Biol. 19 (2014) 59e66.
[20] A.I. Karsisiotis, C.F. Damblon, G.C.K. Roberts, A variety of roles for versatile zinc
ꢁ
cer cells (MIA-PaCa2 and Colo-357) were cultured at 37 C with 5%
in metallo- -lactamases, Metallomics 6 (7) (2014) 1181e1197.
b
CO
biotics. For cell survival assays, cells were seeded in 96 well plates
3000 cells per well, 100 L medium), adhered for 24 h and exposed
to different concentrations (from 300 M to 0.3 M) of indicated
2
in DMEM media containing 10% fetal bovine serum and anti-
[21] S.B. Falconer, S.A. Reid-Yu, A.M. King, S.S. Gehrke, W. Wang, J.F. Britten,
B.K. Coombes, G.D. Wright, E.D. Brown, Zinc chelation by a small-molecule
adjuvant potentiates meropenem activity in vivo against NDM-1-producing
Klebsiella pneumoniae, ACS Infect. Dis. 1 (11) (2015) 533e543.
(
m
m
m
[22] K.H.M.E. Tehrani, N.I. Martin, Thiol-containing metallo-
resensitize resistant gram-negative bacteria to meropenem, ACS Infect. Dis. 3
10) (2017) 711e717.
b-lactamase inhibitors
compounds for an additional 72 h. Then, metabolically active cells
were quantified by the MTT assay [40] by adding 100 ng MTT per
well, incubating cells for 2 h, replacing media with 100
propanol/HCl/H 0 (90/1/9, v/v/v) and determining optical density
at 540 nm. IC50 values were determined using the CompuSyn
(
[
23] D. Büttner, J.S. Kramer, F.-M. Klingler, S.K. Wittmann, M.R. Hartmann,
mL iso-
€
C.G. Kurz, D. Kohnhauser, L. Weizel, A. Brüggerhoff, D. Frank, D. Steinhilber,
T.A. Wichelhaus, D. Pogoryelov, E. Proschak, Challenges in the development of
2
a thiol-based broad-spectrum inhibitor for metallo-b-lactamases, ACS Infect.
Dis. 4 (3) (2018) 360e372, https://doi.org/10.1021/acsinfecdis.7b00129.
24] A.M. King, S.A. Reid-Yu, W. Wang, D.T. King, G. De Pascale, N.C. Strynadka,
T.R. Walsh, B.K. Coombes, G.D. Wright, Aspergillomarasmine A overcomes
software. Results were expressed as mean values of IC50 ± SD (mM)
of three independent experiments performed in triplicate.
[
Please cite this article as: A. Prandina et al., Synthesis and biological evaluation of new dipicolylamine zinc chelators as metallo-
inhibitors, Tetrahedron, https://doi.org/10.1016/j.tet.2019.02.004
b-lactamase

16
A. Prandina et al. / Tetrahedron xxx (xxxx) xxx
metallo-
b
-lactamase antibiotic resistance, Nature 510 (2014) 503.
[36] Ø. Samuelsen, M.A. Toleman, A. Sundsfjord, J. Rydberg, T.M. Leegaard,
M. Walder, A. Lia, T.E. Ranheim, Y. Rajendra, N.O. Hermansen, T.R. Walsh,
C.G. Giske, Molecular epidemiology of metallo- -lactamase-producing Pseu-
[
25] A. Allam, L. Maigre, R. Alves de Sousa, E. Dumont, J. Vergalli, J.-M. Pag eꢁ s,
I. Artaud, New amphiphilic neamine conjugates bearing a metal binding motif
active against MDR E. aerogenes Gram-negative bacteria, Eur. J. Med. Chem.
b
domonas aeruginosa isolates from Norway and Sweden shows import of in-
ternational clones and local clonal expansion, Antimicrob. Agents Chemother.
54 (1) (2010) 346e352.
127 (2017) 748e756.
[
26] A.Y. Chen, P.W. Thomas, A.C. Stewart, A. Bergstrom, Z. Cheng, C. Miller,
C.R. Bethel, S.H. Marshall, C.V. Credille, C.L. Riley, R.C. Page, R.A. Bonomo,
M.W. Crowder, D.L. Tierney, W. Fast, S.M. Cohen, Dipicolinic acid derivatives
[37] A. Heikal, Ø. Samuelsen, T. Kristensen, O.A. Økstad, Complete genome
sequence of a multidrug-resistant, bla(NDM-1)-expressing Klebsiella pneumo-
niae K66-45 clinical isolate from Norway, Genome Announc. 5 (27) (2017)
e00601ee00617.
as inhibitors of New Delhi metallo-
b-lactamase-1, J. Med. Chem. 60 (17)
(
2017) 7267e7283.
[
27] C. Schnaars, G. Kildahl-Andersen, A. Prandina, R. Popal, S. Radix, M. Le Borgne,
[38] J. Uwe, M. Stephan, B. Willi, Efficient transfer of chelating amides into
different types of esters and lactones, Eur. J. Org. Chem. 2014 (31) (2014)
6963e6974.
T. Gjøen, A.M.S. Andresen, A. Heikal, O.A. Økstad, C. Fr o€ hlich, Ø. Samuelsen,
S. Lauksund, L.P. Jordheim, P. Rongved, O.A.H. Åstrand, Synthesis and pre-
clinical evaluation of TPA-based zinc chelators as metallo-
b
-lactamase in-
[39] T. Christopeit, K.-W. Yang, S.-K. Yang, H.-K.S. Leiros, The structure of the
hibitors, ACS Infect. Dis. 4 (9) (2018) 1407e1422.
metallo-b-lactamase VIM-2 in complex with a triazolylthioacetamide inhibi-
[
28] O.A.H. Åstrand, L.P.E. Austdal, R.E. Paulsen, T.V. Hansen, P. Rongved, Synthesis
tor, Acta Crystallogr. F 72 (11) (2016) 813e819.
2þ
and characterization of new selective Zn fluorescent probes for function-
alization: in vitro Cell imaging applications, Tetrahedron 69 (40) (2013)
[40] T. Mosmann, Rapid colorimetric assay for cellular growth and survival:
application to proliferation and cytotoxicity assays, J. Immunol. Methods 65
(1) (1983) 55e63.
8645e8654.
[
29] O.A.H. Åstrand, G. Aziz, S.F. Ali, R.E. Paulsen, T.V. Hansen, P. Rongved, Syn-
thesis and initial in vitro biological evaluation of two new zinc-chelating
compounds: comparison with TPEN and PAC-1, Bioorg. Med. Chem. 21 (17)
[41] A.E. Martell, R.M. Smith, second supplement, Critical Stability Constants, 1 ed.,
vol. 6, Springer ScienceþBusiness Media New York, New York, 1989, p. 643.
[42] A.-M. Fanning, S.E. Plush, T. Gunnlaugsson, Tuning the properties of cyclen
based lanthanide complexes for phosphodiester hydrolysis; the role of basic
cofactors, Chem. Commun. (36) (2006) 3791e3793.
[43] A. Heikal, Ø. Samuelsen, T. Kristensen, O.A. Økstad, Complete genome
sequence of a multidrug-resistant, bla(NDM-1)-expressing Klebsiella pneumo-
niae K66-45 clinical isolate from Norway, Genome Announc. 5 (27) (2017)
e00601ee00617.
(
2013) 5175e5181.
[30] R.F. Pratt, Substrate specificity of bacterial DD-peptidases (penicillin-binding
proteins), Cell. Mol. Life Sci. 65 (14) (2008) 2138e2155.
[
31] J.-M. Hah, P. Mart ꢀa sek, L.J. Roman, R.B. Silverman, Aromatic reduced amide
bond peptidomimetics as selective inhibitors of neuronal nitric Oxide syn-
thase, J. Med. Chem. 46 (9) (2003) 1661e1669.
[
32] V. Perron, S. Abbott, N. Moreau, D. Lee, C. Penney, B. Zacharie, A method for
the selective protection of aromatic amines in the presence of aliphatic
amines, Synthesis 2009 (02) (2009) 283e289.
[44] Ø. Samuelsen, M.A. Toleman, V. Hasseltvedt, K. Fuursted, T.M. Leegaard,
T.R. Walsh, A. Sundsfjord, C.G. Giske, Molecular characterization of VIM-
producing Klebsiella pneumoniae from Scandinavia reveals genetic related-
ness with international clonal complexes encoding transferable multidrug
resistance, Clin. Microbiol. Infect. 17 (12) (2011) 1811e1816.
[
[
[
33] L.A. Carpino, 1-Hydroxy-7-azabenzotriazole. An efficient peptide coupling
additive, J. Am. Chem. Soc. 115 (10) (1993) 4397e4398.
34] Y. Xiao, K. Lee, P. Liu, Syntheses of the P-methylase substrates of the bialaphos
biosynthetic pathway, Org. Lett. 10 (24) (2008) 5521e5524.
35] Z. Xu, K.-H. Baek, H.N. Kim, J. Cui, X. Qian, D.R. Spring, I. Shin, J. Yoon, Zn
[45] Ø. Samuelsen, L. Buarø, M.A. Toleman, C.G. Giske, N.O. Hermansen, T.R. Walsh,
A. Sundsfjord, The first metallo-b-lactamase identified in Norway is associated
2þ
-
with a TniC-like transposon in a Pseudomonas aeruginosa isolate of sequence
type 233 imported from Ghana, Antimicrob. Agents Chemother. 53 (1) (2009)
331e332.
2þ
Triggered amide tautomerization produces a highly Zn -selective, cell-
permeable, and ratiometric fluorescent sensor, J. Am. Chem. Soc. 132 (2)
(
2010) 601e610.
Please cite this article as: A. Prandina et al., Synthesis and biological evaluation of new dipicolylamine zinc chelators as metallo-
inhibitors, Tetrahedron, https://doi.org/10.1016/j.tet.2019.02.004
b-lactamase