A versatile strategy for the synthesis of 4,5-dihydroxy-2,3-pentanedione (DPD) and related compounds as potential modulators of bacterial quorum sensing

Article abstract of DOI:10.3390/molecules23102545

Resistance to antibiotics is an increasingly serious threat to global public health and its management translates to significant health care costs. The validation of new Gram-negative antibacterial targets as sources for potential new antibiotics remains a challenge for all the scientists working in this field. The interference with bacterial Quorum Sensing (QS) mechanisms represents a potentially interesting approach to control bacterial growth and pursue the next generation of antimicrobials. In this context, our research is focused on the discovery of novel compounds structurally related to (S)-4,5-dihydroxy-2,3-pentanedione, commonly known as (S)-DPD, a small signaling molecule able to modulate bacterial QS in both Gram-negative and Gram-positive bacteria. In this study, a practical and versatile synthesis of racemic DPD is presented. Compared to previously reported syntheses, the proposed strategy is short and robust: it requires only one purification step and avoids the use of expensive or hazardous starting materials as well as the use of specific equipment. It is therefore well suited to the synthesis of derivatives for pharmaceutical research, as demonstrated by four series of novel DPD-related compounds described herein.

Full text of DOI:10.3390/molecules23102545

molecules
Article
A Versatile Strategy for the Synthesis of
4,5-Dihydroxy-2,3-Pentanedione (DPD) and Related
Compounds as Potential Modulators of Bacterial
Quorum Sensing
Silvia Stotani 1,2, Viviana Gatta 3, Federico Medda 1,†, Mohan Padmanaban 1,
Anna Karawajczyk 1,‡, Päivi Tammela 3 , Fabrizio Giordanetto 1,§, Dimitrios Tzalis 1
and Simona Collina 2,
*
1
Medicinal Chemistry, Taros Chemicals GmbH & Co. KG, Emil-Figge-Straße 76a, 44227 Dortmund, Germany;
silviastotani@hotmail.it (S.S.); fmedda@centurionbiopharma.com (F.M.); mpadmanaban@taros.de (M.P.);
akarawajczyk@gmail.com (A.K.); Fabrizio.Giordanetto@deshawresearch.com (F.G.); dtzalis@taros.de (D.T.)
Department of Drug Sciences, Medicinal Chemistry and Pharmaceutical Technology Section, University of
Pavia, Viale Taramelli 12, 27100 Pavia, Italy
Drug Research Program, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of
Helsinki, FI-00014 Helsinki, Finland; viviana.gatta@helsinki.fi (V.G.); paivi.tammela@helsinki.fi (P.T.)
Correspondence: simona.collina@unipv.it; Tel.: +39-0382-987379
2
3
*
†
Current address: Centurion Biopharma Corporation, Engesserstraße 4, 79108 Freiburg im
Breisgau, Germany.
‡
Current address: Selvita S.A., Park Life Science, Bobrzynˇskiego 14, 30-348 Krakow, Poland.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 16 September 2018; Accepted: 3 October 2018; Published: 6 October 2018
Abstract: Resistance to antibiotics is an increasingly serious threat to global public health and
its management translates to significant health care costs. The validation of new Gram-negative
antibacterial targets as sources for potential new antibiotics remains a challenge for all the scientists
working in this field. The interference with bacterial Quorum Sensing (QS) mechanisms represents a
potentially interesting approach to control bacterial growth and pursue the next generation of
antimicrobials. In this context, our research is focused on the discovery of novel compounds
structurally related to (S)-4,5-dihydroxy-2,3-pentanedione, commonly known as (S)-DPD, a small
signaling molecule able to modulate bacterial QS in both Gram-negative and Gram-positive bacteria.
In this study, a practical and versatile synthesis of racemic DPD is presented. Compared to previously
reported syntheses, the proposed strategy is short and robust: it requires only one purification
step and avoids the use of expensive or hazardous starting materials as well as the use of specific
equipment. It is therefore well suited to the synthesis of derivatives for pharmaceutical research,
as demonstrated by four series of novel DPD-related compounds described herein.
Keywords: antibiotic resistance; quorum sensing; DPD; DPD-related compounds
1. Introduction
Bacterial chemical communication (i.e., quorum sensing, QS) allows bacteria to coordinate their
gene expression and act as a population [
QS regulates pathogenic processes such as the virulence factor production [
antibiotics [ ] and biofilm formation [ 11]. In recent decades, the modulation of QS has therefore
1–
5]. This phenomenon is detrimental for humans as
6,7
], susceptibility to
8
9–
emerged as a potential therapeutic approach to fight bacterial infections [12–17].
QS is mediated by production and release of and response to small molecules called
autoinducers (AIs). Among these AIs, Autoinducer-2 (AI-2) is responsible for intra- and interspecies
Molecules 2018, 23, 2545; doi:10.3390/molecules23102545
www.mdpi.com/journal/molecules

Molecules 2018, 23, 2545
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bacterial communication and, as a consequence, it has been termed the “universal autoinducer”.
The development of small molecules able to modulate the AI-2-mediated signaling would possibly
result in broad-spectrum antimicrobial activity. However, targeting the AI-2-based QS remains
challenging mostly because of the rapid interconversion of the AI-2 precursor (S)-DPD (Figure 1)
to several linear and cyclic forms recognized by different bacteria [18] (Figure 1). In aqueous solutions,
(S)-DPD is in equilibrium with its two cyclic stereoisomers (S-DHMF and R-DHMF; Figure 1) [19].
Hydration of the C3 carbonyl group of both the cyclic and linear structures was confirmed by X-ray
crystallography. In the presence of boric acid, S-THMF (Figure 1) forms a borate ester (S-THMF-borate;
Figure 1) which is recognized by LuxP in V. harveyi (PDB ID: 1JX6) [20]. R-THMF instead (Figure 1) does
not coordinate boron and binds to the transporter LsrB which is responsible for its internalization and
acts as the active species in S. thyphimurium AI-2-mediated QS (PDB ID: 1TJY) [21]. The hydrated form
of linear (S)-DPD (S-THP, Figure 1) is phosphorylated by LsrK, resulting in phospho-DPD (P-DPD,
Figure 1) [22] recognized by the transcriptional repressor LsrR (PDB ID: 4L4Z) [23] and responsible for
E coli and S. typhimurium AI-2-mediated signaling.
Figure 1. (S)-DPD in an aqueous medium: all species in equilibrium. (2S,4S)-2,4-dihydroxy-2-
methyldihydrofuran-3-one (S-DHMF); (S)-4,5-dihydroxy-2,3-pentanedione (DPD); (2R,4S)-2,4-
dihydroxy-2-methyldihydrofuran-3-one
tetrahydroxytetrahydrofuran (S
(S-THP); (2R,4S)-2-methyl-2,3,3,4-
methyl-2,3,3,4-tetrahydroxytetrahydrofuranborate
tetrahydroxy-2-pentanone-5-phosphate (P-DPD).
(R-DHMF);
THMF); (S)-3,3,4,5-tetrahydroxy-2-pentanone
tetrahydroxytetrahydrofuran
(2S,4S)-2-methyl-2,3,3,4-
(R-THMF);
(S-THMF-borate);
(2S,4S)-2-
(S)-3,3,4,5-
Modulation/inhibition of QS can control several bacterial virulence factors (e.g., biofilm formation)
that facilitate human infections and reduce their negative effects, including mortality [24]. Quorum
Sensing Inhibitors (QSI) therefore represent interesting tools to use in combination with “conventional”
antibiotic therapies against antimicrobial resistance (AMR) [25,26].
In this work, we describe the set-up of a new protocol for the synthesis of racemic DPD and
its application to the synthesis of four novel small libraries of DPD-related compounds (Figure 2),
designed to target LsrK kinase, a key mediator in AI-2-mediated QS in enteric bacteria. The essential
role of the enzyme has been demonstrated by LsrK gene deletion in E. coli, generating a mutant strain
unable to activate AI-2-mediated QS [27]. Therefore, we believe that the generation of DPD-related

Molecules 2018, 23, 2545
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compounds for the inhibition of LsrK may be the starting point for the development of new QSI that
will serve as potential tools for overcoming antimicrobial resistance.
Figure 2. The DPD-related compounds presented in this work.
2. Results and Discussion
Since 2004, much effort has been devoted to the study of synthetic pathways for the
preparation of DPD and analogs in both racemic and enantiomeric forms. Literature analysis
revealed that the synthesis of homochiral DPD requires the use of expensive (i.e.,
(S)-1,4-dioxaspiro[4.5]decane-2-carboxaldehyde) or unstable (i.e., (S)-glyceraldehyde acetonide)
chiral starting materials and of further time-consuming purification steps [18,28–33]. Conversely,
the synthetic procedures published so far to obtain racemic DPD proceed smoothly but suffer from
hazardous chemical steps (i.e., reductive ozonolysis or the use of diazomethane) [34,35].
Starting from these considerations and keeping in mind that in the initial phase of the drug
discovery process racemic compounds are usually evaluated and only once the most active ones have
been identified both enantiomers must be prepared for biological testing [36], herein we studied a novel
versatile strategy for the synthesis of racemic DPD suitable for readily supporting practical chemical
diversification. The proposed synthetic strategy leading to DPD could be useful for the preparation of
C1 DPD-analogs and for the synthesis of DPD structurally related compounds, where the two carbonyl
groups of DPD at C2 and C3 are embedded in heteroaromatic rings (Figure 2). To the best of our
knowledge, no modification at C2 have been reported and position C3 has been barely explored and
no heteroaromatic substituents (except for a furan at C1) were previously described.
2.1. Synthesis of DPD and Ph-DPD
The synthetic strategies originally evaluated are outlined in Scheme 1.

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Scheme 1. The synthesis of racemic DPD, Ph-DPD, quinoxaline-DPD and quinoxaline-Ph-DPD.
◦
Reagents and conditions: (a) 1-propynylmagnesium bromide (0.5 M in THF, 1.3 eq), THF, 0 C to rt, 3 h;
0
◦
(a ) phenylethynylmagnesium bromide (1.0 M in THF, 1.3 eq), THF, 0 C to rt, 3 h; (b) TBDMSCl (1.2 eq),
0
NaH (2.0 eq), THF, rt, 3 h; (b ) TMSCl (1.2 eq), NaH (2.0 eq), THF, rt, 3 h; (
c
) NaIO4 (4.4 eq), Ru2O H2O
·
(2.5% mol), CHCl3/ACN/H2O (1:1:1), rt, 1 h; (d) see Table S1; (e) Dowex50WX8 100–200 mesh, MeOH,
rt, overnight; (f) cyclohexanone dimethyl ketal (3.0 eq), p-TSA (cat.), rt, overnight; (g) Dowex50WX8
100–200 mesh, D2O (10 mM), rt, overnight; (h) o-phenylendiamine (2.0 eq), rt, overnight.
Briefly,
(t-butyldimethylsilyloxy)acetaldehyde [37] (
resulting secondary alcohol with TBDMSCl or TMSCl afforded compounds
(Scheme 1). The subsequent oxidation of the internal alkyne to yield diketone
the
addition
of
1
1-propynylmagnesium
, Scheme 1), followed by the protection of the
or , respectively
or was performed
bromide
to
3
4
5
6
under optimized RuO2*H2O/NaIO4-catalyzed conditions (Table 1, entry 5) using CHCl3/ACN/H2O
(1:1:1) as the solvent.
Table 1. The optimization of the conditions for the oxidation of compound 3. All the reactions were
performed at room temperature.
Entry
Solvent
Oxidant and eq
Time
Yield (%)
1
2
Acetone
Acetone
KMnO4/NaHCO3/MgSO4 3.8/0.6/2.0
KMnO4/NaHCO3/MgSO4 3.9/0.6/4.2
NaIO4/RuO2·H2O
Overnight
Overnight
No reaction
Traces
3
4
5
CCl4/ACN (1:1)
CCl4/ACN (1:1)
3 h
3 h
3 h
Traces
23
2.2 eq/2.5% mol
NaIO4/RuO2·H2O
4.4 eq/2.5% mol
NaIO4/RuO2·H2O
CHCl3/ACN/H2O (1:1:1)
52
4.4 eq/2.5% mol
The final acidic removal of the two TBDMS groups of compound 5 was performed under different
conditions, but resulted in being unsuccessful (Table S1). Particularly, decomposition was observed
when H2SO4 (or D2SO4) and TBAF were employed (Table S1). The partial removal of the two protecting
groups (up to a maximum of 30% in total) was achieved with the use of acetic acid or Dowex50WX8
(Table S1). When the bulky protecting TBDMS group was replaced with TMS, (Scheme 1), similar results
were obtained and a maximum of 40% cleavage was achieved using Dowex50WX8 in ACN-d3.
A different approach was then investigated: compound
2
and the analogous
7 were deprotected
in acidic conditions (Dowex50WX8), affording diols and
8
9, respectively. These intermediates were

Molecules 2018, 23, 2545
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then protected with a cyclohexyliden group and oxidized under the previously described conditions
(Table 1). The oxidation of 10 and 11 was followed by the Dowex50WX8-mediated removal of the
1
protecting group. H-NMR analysis of the crude products was consistent with the literature-reported
data and revealed the presence of a mixture of structures in equilibrium with each other (see SI
for additional details). To further confirm the success of our procedure, the mixtures were treated
with o-phenilendiamine to form, respectively, quinoxaline-DPD and quinoxaline-Ph-DPD (Scheme 1),
which were isolated and fully characterized.
To sum up, the approach described above allows for the rapid production of racemic DPD
in five steps and it does not require the use of dangerous or expensive reagents nor of particular
equipment (i.e., ozonolysator); furthermore, only one purification step via column chromatography is
necessary. Not less important, this procedure is suitable for the synthesis of C1-DPD analogs (as long
as the corresponding Grignard reagent can be purchased or produced) as the synthesis of Ph-DPD
demonstrated. Additionally, the ethyne function introduced in the first step is a practical synthetic
handle for further chemical derivatization, as demonstrated by the four small series of derivatives
described below.
2.2. Synthesis of DPD-Related Compounds
As anticipated, we designed novel DPD-related compounds in which the carbonyl groups
at C2 and C3 are embedded in heteroaromatic moiety to obtain compounds stable in solution,
thus avoiding the open/closed equilibrium typical of the majority of the DPD-analogs reported
so far (Figure 1). As heteroaromatic rings, we selected 1,2,3-triazole and isoxazole, two scaffolds
common in medicinal chemistry present in several natural and synthetic drugs including antimicrobial,
anticancer, anti-inflammatory and antireumatic drugs [38–43].
The newly designed compounds can be obtained starting from the two common intermediates
15 and 16 (Scheme 2) strictly related to
of the two building blocks necessary to start the synthesis of all the analogs presented in this work
was produced by the Grignard addition of ethynylmagnesium bromide to aldehyde , followed by
2 and 7 (Scheme 1). In details, as in the case of DPD, the first
1
acidic removal of the TBDMS protecting group. Further protection of the resulting diol 15 as acetal,
using cyclohexanone dimethyl ketal, afforded the second building block compound 16 (Scheme 2).
Scheme 2. The synthesis of intermediates 15 and 16. Reagents and conditions: (a) ethynylmagnesium
bromide (0.5 M in THF, 1.3 eq), THF, 0 ◦C to rt, 3h; (b) Dowex50WX8 100–200 mesh, MeOH, rt,
overnight; (c) cyclohexanone dimethyl ketal (3.0 eq), p-TSA (cat.), rt, overnight.
2.2.1. 1,4- and 1,5-Disubstituted 1,2,3-Triazoles DPD-Derivatives (Series I and II)
1,2,3-triazoles (both 1,4- and 1,5-disubstituted) can be synthesized applying azide-alkyne Huisgen
cycloaddition conditions where an azide is reacted with an alkyne in a 1,3-dipolar cycloaddition
reaction. At first, we tested three different Copper-Catalyzed Azide-Alkyne Cycloaddition (CuAAC)
conditions to select the best procedure for the synthesis of the planned compounds. For this purpose,
(2-azidoethyl)benzene (17a) was chosen as the reference azide (Table 2). First, we used CuI (10% mol)
and DIPEA (15% mol) in nonaqueous, nonprotic THF to afford the desired product with 58% isolated
yield (Table 2, entry 1) [44]. As the addition of AcOH was found to accelerate the protonation of
the Cu-C bond [45–47] (thus facilitating the formation of the product), a catalytic amount of AcOH
was added to the mixture (containing 2% mol CuI and 4% mol DIPEA). This acid-base system jointly
promoted CuAAC and resulted in a 14% increase of the isolated yield (Table 2, entry 2) when compared

Molecules 2018, 23, 2545
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to the previous conditions (Table 2, entry 1) [48]. It is known that the use of ligands is beneficial for the
reaction as it prevents Cu(I) oxidation and avoids the use of a base. Therefore, it is not surprising that
the in situ generation of Cu(I) by the reduction of CuSO4*5H2O from sodium ascorbate together with
the formation of L-ascorbic acid (that acts both as a ligand and as acidic source) raised the yield up
to 89% (Table 2, entry 3) [49]. The 1,4-disubstitution was confirmed by the HMBC of compound 18a
(see Supporting Information).
Table 2. The reaction conditions to obtain 18a
room temperature except for entry 4 where the mixture was heated at 60 C.
–
f
and 19a. All reactions were performed overnight at
◦
Entry
R1
Azide, eq
Solvent
Catalyst
Product Yield (%) a
Ref.
CuI (10% mol)
DIPEA (15% mol)
CuI (2% mol)
1
(CH2)2-Ph
17a, 1.1
THF
18a
18a
58
72
[44]
2
(CH2)2-Ph
17a, 1.05
DCM
DIPEA (4% mol)
[48]
AcOH (cat)
t-BuOH/H2O
(1:1)
1,4-dioxane
t-BuOH/H2O
(1:1)
CuSO4·5H2O (5% mol)
Na Ascorbate (0.5 eq)
(Cp*RuCl(PPh3)2) (2% mol)
CuSO4·5H2O (5% mol)
Na Ascorbate (0.5 eq)
CuSO4·5H2O (5% mol)
Na Ascorbate (0.5 eq)
CuSO4·5H2O (5% mol)
Na Ascorbate (0.5 eq)
CuSO4·5H2O (5% mol)
Na Ascorbate (0.5 eq)
CuSO4·5H2O (5% mol)
Na Ascorbate (0.5 eq)
3
4
5
(CH2)2-Ph
(CH2)2-Ph
(CH2)-Ph
17a, 1.0
17a, 1.0
17b, 1.0
18a
19a
18b
89
87
60
[49]
[50]
[49]
t-BuOH/H2O
6
7
8
9
(CH2)2-o-F-Ph
(CH2)2-m-Pyr
(CH2)5-CN
17c, 1.0
17d, 1.0
17e, 1.0
17f, 1.0
18c
18d
18e
18f
62
88
72
73
[49]
[49]
[49]
[49]
(1:1)
t-BuOH/H2O
(1:1)
t-BuOH/H2O
(1:1)
t-BuOH/H2O
(1:1)
(CH2)2-CyH
a
Isolated yield.
The corresponding 1,5-disubstituted 1,2,3-triazole 19a was also synthesized by varying
the experimental conditions: the regioselective synthesis was achieved with the use
of Ruthenium-catalyzed Azide-Alkyne Cycloaddition (RuAAC) conditions. Azide 17a was
reacted with terminal alkyne 15 in the presence of 2% mol pentamethylcyclopentadienylbis
(triphenylphosphine)ruthenium(II) chloride (Cp*RuCl(PPh3)2) regioselectively yielding, after stirring
overnight the mixture in refluxing 1,4-dioxane, the corresponding 1,5-disubstituted 1,2,3-triazole 19a
1
(Table 2, entry 4). H, 13C, TLC, UHPLC, and HMBC unambiguously confirmed the different nature of
the two compounds (see Supporting Information) [50].
Once optimal conditions for the regioselective synthesis of 1,4-disubstituted 1,2,3-triazoles were
established, we synthesized five azides of different chemical nature including aromatic, heteroaromatic
and aliphatic elements (17b
–f). This was achieved by stirring overnight at room temperature the
corresponding bromo compounds with an excess (1.5 eq) of sodium azide. The five azides were reacted
with alkyne 15 applying the previously found conditions and products 18b–f were isolated in good to
excellent yields (60–88%, Table 2, entry 5–9).
As the synthesis of triazoles substituted with short alkyl chains (e.g., methyl, butyl) was
unattainable by this route because of safety issues related to the explosive and unstable nature
of the required azides, we installed the desired substituents on the triazole scaffold via alkylation.
We elected to use a single, small and dangerous azide (i.e., TMSN3) over the use of four different
ones. The acetal protected terminal alkyne 16 was carefully reacted with an excess (10.0 eq) of TMSN3
under previously established CuAAC conditions. The resulting unsubstituted triazole (20, Scheme 3)

Molecules 2018, 23, 2545
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was both deprotected under acidic conditions (18g, Scheme 3) and, to install the desired substituents,
alkylated with four different (i.e., methyl, cyclopropylmethyl, butyl, ethoxyethyl) bromides (Scheme 3).
Scheme 3. The synthesis of 1,4-disubstituted 1,2,3-triazoles 18g
–k
and 1,5-disubstituted 1,2,3-triazoles
19h
–
k
. Reagents and conditions: (a) TMSN3 (10.0 eq), CuSO4
·
5H2O (5% mol), Na ascorbate (0.5 eq),
t-BuOH/H2O (1:1), rt, overnight; (b) 12M HCl (cat.), 1,4-dioxane, 0 ◦C to rt, 1–3 h; (c) R1Br (1.5 eq),
◦
K2CO3 (2.0 eq), THF, 40 C, overnight; preparative HPLC.
As expected, no regioselectivity was observed and both the 1,4- and the 1,5-disubstituted
1,2,3-triazoles formed. Experimenting with base (i.e, 1.1 eq, 1.3 eq and 1.5 eq of K2CO3) and/or
the alkylbromides (i.e., 0.8 eq and 0.9 eq of R1Br) stoichiometry did not consistently changed
the ratio of the two regioisomers (data not shown). For each substituent, the two corresponding
regioisomers were isolated by preparative HPLC. The resulting eight products (21h–k and 22h–k,
Scheme 3) were lastly deprotected with a catalytic amount of concentrated hydrochloric acid. The ratio
of the two regioisomers was determined by crude NMR. For all of the four regioisomeric pairs,
the 1,4-dibustituted 1,2,3-triazoles formed in excess when compared to the respective 1,5-regioisomers
and, as predictable, the ratio decreased as the sterical hindrance of the R1 substituent increased
(Scheme 3). Concentrated HCl was preferred over Dowex 50WX8 for the removal of the acetal
protecting group due to the shorter reaction time (1–3 h vs. overnight) and shorter workup (no
filtration to remove the acidic resin required).
2.2.2. 3,5-Disubstituted Isoxazoles DPD-Derivatives (Series III and IV)
Compound 15 (Scheme 2) is also the key intermediate for the synthesis of 3,5-disubstituded DPD
related compounds 26l
oximes 24l using NH2OH
reaction with N-chlorosuccinimide (NCS). According to Himo et al. [49], the addition of CuSO4
Na ascorbate, and KHCO3 in t-BuOH/H2O (1:1) to the isolated chloro-oximes allowed them to
form the nitrile oxide which reacted by 1,3-dipolar cycloaddition with 15. After preparative HPLC
–
r
(Scheme 4). Briefly, aldehydes 23l
HCl. The resulting crude compounds were directly chlorinated by a
5H2O,
–r were converted into their corresponding
–
r
·
·
purification, the targeted isoxazoles 26l–r were, therefore, obtained in good to excellent yields (i.e.,
63–89%, Scheme 4).

Molecules 2018, 23, 2545
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Scheme 4. The synthesis of 3,5-disubstituted isoxazoles 26l
(a) NH2OH HCl (3.0 eq), Et3N (1.5 eq), H2O/EtOH (1:1), rt, 1–3 h; (b) NCS (1.0 eq), DMF, rt, 1–2 h;
(c) 15 (1.0 eq), CuSO4 5H2O (5% mol), Na ascorbate (0.5 eq), KHCO3, t-BuOH/H2O (1:1), rt, overnight.
–r
.
Reagents and conditions:
·
·
The same procedure was attempted to obtain 3,5-disubstituted isoxazoles of DPD-analogs bearing
an amide moiety at position 3, but starting from the protected precursor 16 instead of 15 due to the
cross-reactivity between the 1,3-diol and the reagents necessary in the following steps (e.g., NaOH,
DIPEA, Scheme 5). Formation of the nitrile oxide for the cycloaddition was attempted using the
dehydration of ethyl nitroacetate with several bases (i.e., DABCO, DMAP, DBU, NMI, Scheme S1,
conditions a) and also with a combination of PhNCO/Et3N (Scheme S1, conditions b), commonly used
to activate nitro groups. All of the aforementioned methods resulted in a mixture of unreacted starting
materials [51,52].
Scheme 5. The synthesis of 3,5-disubstituted isoxazoles (with an amide moiety at position 3) 33b, 33s–z.
Reagents and conditions: (a) 27 (2.0 eq), NaOCl (40.0 eq), THF, rt, 12 h; (b) 12M HCl (cat.), 1,4-dioxane,
◦
0 C to rt, 1–3 h; (c) NaOH 1M (3.0 eq), THF, rt, overnight; (d) amine (2.0 eq), HOBt (2.0 eq), EDC*HCl
(2.0 eq), Et3N, DCM, rt, overnight.
The 1,3-dipole species was then changed to the chloro-oxime of ethyl glyoxalate (50% solution in
toluene) but the employment of the same conditions as above (CuSO4 5H2O (5% mol), Na ascorbate
·
(0.5 eq), KHCO3, t-BuOH/H2O (1:1), Scheme 4) did not yield the desired product while the simple use
of an equimolar amount of Et3N gave only traces of 28 (Scheme 5) [49,53].
We then change our strategy and employed the oxime of ethyl glyoxalate 27 together with
an excess (40.0 eq) of sodium hypochlorite, both as a chlorinating agent and as a base to form the
corresponding nitrile oxide, following the procedure already described by Quan et al. [54]. Compound
28 was successfully obtained, even if with a low yield (16%). Different reaction times, as well as ratios
of dipolarophile 16 and 1,3-dipole 28, were then tested (Table S2) in order to improve the initially poor
yield (i.e., 16%, Table S2). Increasing the concentration of 1,3-dipole 27 enhanced the formation of

Molecules 2018, 23, 2545
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intermediate 28 up to a maximum of 36% isolated yield (Table S2) with the complete consumption of
the dipolarophile 16, followed by removal of the excess of 27 by column chromatography.
Once a solution for the key 1,3-dipolar cycloaddition step was found, the rest of the synthetic
pathway proceeded smoothly (Scheme 5). Saponification of the ethylic ester was followed by the
amidification of the resulting carboxylic acid moiety using HOBt as the coupling agent and employing
both primary and secondary amines (aromatic, heteroaromatic, aliphatic). The final acidic removal of
the acetal protecting group afforded six 3,5-disubstituted isoxazoles (with an amide moiety at position
3) 33b, 33s–z in moderate to excellent yields (i.e., 37–79%, Scheme 5). Two more products were isolated
after the acidic deprotection of intermediates 28 and 30 (i.e., 29 and 31, respectively, Scheme 5).
2.3. Biological Evaluation of Synthetized Compounds
The activity of the synthetized compounds was evaluated with a bioluminescence-based assay
against the target enzyme. Our results clearly highlight that racemic DPD prepared using our procedure
is efficiently phosphorylated by LsrK (see Supporting Information, Figure S1). In fact, the level of ATP
is significantly reduced by the addition of racemic DPD, resulting in a light emission lower than the
sample including only LsrK and ATP.
These results confirmed the validity of the approach adopted. Indeed, in this initial phase of the
drug discovery process, we prepared racemic DPD and studied a versatile synthesis suitable for readily
supporting practical chemical diversification racemic compounds. Only once the most active ones
have been identified will both enantiomers be prepared for biological testing. Accordingly, the activity
of racemic DPD is essential for demonstrating that our approach has a valid basis. Regarding the
DPD-derivatives, unfortunately, they did not show any activity (the data are reported in Supporting
Information, Table S3).
3. Experimental
3.1. Chemistry
Chemicals and solvents were obtained from commercial suppliers and were used without further
purification. All dry reactions were performed under a nitrogen atmosphere using commercial dry
solvents. Flash column chromatography was performed on a silica column using 230-400 mesh silica
gel or the Grace Reveleris X2 flash chromatography system using silica gel packed Macherey Nagel
Chromabond Flash BT cartridges (60 Å, 45
µm) and Grace Reveleris flash Cartridges (60 Å, 40 µm).
Thin layer chromatography was performed on Macherey Nagel precoated TLC aluminum sheets with
silica gel 60 UV254 (5–17
(254 nm) and/or staining with KMnO4 solutions. H-NMR spectra were recorded at room temperature
on a Bruker Avance spectrometer operating at 300 MHz (Hamburg, Germany). Chemical shifts are
µ
m). TLC visualization was accomplished by irradiation with a UV lamp
1
given in ppm (δ) from tetramethylsilane as an internal standard or residual solvent peak. Significant
1H-NMR data are tabulated in the following order: multiplicity (s, singlet; d, doublet; t, triplet; q,
quartet; m, multiplet; dd, doublet of doublets; dt, doublet of triplets; td, triplet of doublets; br, broad),
coupling constant(s) in hertz, number of protons. Proton decoupled 13C-NMR data were acquired
at 100 MHz. 13C chemical shifts are reported in parts per million (
δ, ppm). All NMR data were
collected at room temperature (25 ◦C). Analytical, preparative HPLC and Electron Spray Ionization
(ESI) mass spectra were performed on an Agilent UHPLC (1290 Infinity, Santa Clara, CA, USA) and an
Agilent Prep-HPLC (1260 Infinity), both equipped with a Diode Array Detector and a Quadrupole MS
using mixture gradients of formic acid/water/acetonitrile as solvents. High-resolution electrospray
ionization mass spectra (ESI-FTMS) were recorded on a Thermo LTQ Orbitrap (Thermo Electron,
Dreieich, Germany) coupled to an ‘Accela’ HPLC system supplied with a ‘Hypersil GOLD’ column
(Termo Electron).

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3.2. Synthesis of DPD and Ph-DPD
Synthesis of 2 and 7: to a stirred solution of (t-butyldimethylsilyloxy)acetaldehyde (1.0 eq)
in dry THF, 1-propynylmagnesium bromide was added (over 15 min; 0.5 M in THF, 1.3 eq)
(or phenylethynylmagnesium bromide (1.0 M in THF, 1.3 eq) at 0 ◦C). After the addition, the reaction
was allowed to reach room temperature and stirred for 3 h. The solvent was removed under reduced
pressure, the residue was poured into a cold saturated solution of NH4Cl and extracted three times
with Et2O. The organic layer was washed twice with water and once with brine, dried over MgSO4,
filtered and concentrated in vacuo to yield 2 as a yellowish oil (96%) or 7 as a yellow oil (98%).
1
1-[(t-Butyldimethylsilyl)oxy]pent-3-yn-2-ol (
(300 MHz, CDCl3)
2
): yellowish oil, 96%, Rf = 0.20 (CyH/EtOAc 9:1). H-NMR
δ
4.36–4.34 (m, 1H), 3.73 (dd, J = 3.6 Hz, J = 10.0 Hz, 1H), 3.59 (dd, J = 7.7 Hz,
J = 10.0 Hz,1H), 2.57 (s br, 1H), 1.83 (d, J = 1.9 Hz, 3H), 0.91 (s, 9H), 0.08 (d, J = 1.3 Hz, 6H) ppm;
13C-NMR (100 MHz, CDCl3) δ 81.8, 79.6, 67.3, 66.3, 25.8, 18.3, 3.5, −5.4 ppm [55].
1-[(t-Butyldimethylsilyl)oxy]-4-phenylbut-3-yn-2-ol (
7): yellow oil, 98%, Rf = 0.72 (CyH/EtOAc 9:1).
1H-NMR (300 MHz, CDCl3)
δ
7.45–7.42 (m, 2H), 7.32–7.29 (m, 3H), 4.65–4.60 (m, 1H), 3.87 (dd,
J = 3.8 Hz, J = 10.0 Hz, 1H), 3.75 (dd, J = 6.9 Hz, J = 10.0 Hz, 1H), 2.71 (d, J = 4.9 Hz, 1H), 0.93 (s, 9H),
0.13 (d, J = 3.1 Hz, 6H) ppm; 13C-NMR (100 MHz, CDCl3)
δ 131.8, 128.4, 128.2, 122.5, 87.0, 85.3, 67.0,
63.6, 25.9, 18.4, 5.3 ppm [56].
Synthesis of 8 and 9: to a stirred solution of
2 (or 7) (1.0 eq) in MeOH, Dowex50WX8 100–200 mesh
(100 mg/1 mL) was added. The reaction was stirred at room temperature overnight. The mixture was
filtered through paper and the solvent was evaporated under reduced pressure to yield
8 as an orange
oil (98%) or 9 as an orange oil (97%).
1
Pent-3-yne-1,2-diol (
8
): orange oil, 98%, Rf = 0.38 (CHCl3/MeOH 9:1). H-NMR (300 MHz, CDCl3)
δ
4.44–4.39 (m, 1H), 3.70 (dd, J = 3.8 Hz, J = 11.3 Hz, 1H), 3.62 (dd, J = 6.6 Hz, J = 11.3 Hz, 1H), 2.41 (s br,
2H), 1.85 (d, J = 2.1 Hz, 3H) ppm; 13C-NMR (100 MHz, CDCl3) δ 82.8, 79.7, 66.8, 63.4, 3.5 ppm [32].
4-Phenylbut-3-yne-1,2-diol (
CDCl3)
9
): orange oil, 97%, Rf = 0.44 (CHCl3/MeOH 9:1). 1H-NMR (300 MHz,
δ
7.46–7.42 (m, 2H), 7.36–7.29 (m, 3H), 4.69 (dd, J = 3.9 Hz, J = 6.5 Hz, 1H), 3.87–3.74 (m,
2H), 2.23 (s br, 2H) ppm; 13C-NMR (100 MHz, CDCl3)
δ 131.8, 128.7, 128.3, 122.0, 86.5, 86.3, 66.6,
63.7 ppm [56].
Synthesis of 10 and 11: to 8 (or 9) (1.0 eq) cyclohexanone dimethyl ketal (3.0 eq) and a catalytic
amount of p-TSA was added. The reaction was stirred at room temperature overnight. The solvent
was removed under reduced pressure and the crude was re-dissolved in Et2O and washed three times
with NaHCO3. The organic layer was dried over MgSO4, filtered and concentrated in vacuo to yield
10 as a yellow oil (64%) or 11 as a yellow oil (72%).
1
2-(Prop-1-yn-1-yl)-1,4-dioxaspiro[4.5]decane (10): yellow oil, 64%, Rf = 0.50 (CyH/EtOAc 9:1). H-NMR
(300 MHz, CDCl3)
δ 4.70–4.64 (m, 1H), 4.11 (dd, J = 6.2 Hz, J = 7.9 Hz, 1H), 3.81 (t, J = 7.5 Hz, 1H), 1.85
(d, J = 2.1 Hz, 3H), 1.74–1.70 (m, 2H), 1.65–1.57 (m, 6H), 1.43–1.38 (m, 2H) ppm; 13C-NMR (100 MHz,
CDCl3) δ 110.5, 85.8, 82.3, 69.7, 65.5, 35.8, 25.1, 23.9, 3.7 ppm [32].
1
2-(2-Phenylethynyl)-1,4-dioxaspiro[4.5]decane (11): yellow oil, 72%, Rf = 0.60 (CyH/EtOAc 9:1). H-NMR
(300 MHz, CDCl3) δ 7.46–7.42 (m, 2H), 7.33–7.28 (m, 3H), 4.95 (t, J = 6.4 Hz, 1H), 4.23 (dd, J = 6.3 Hz,
J = 7.9 Hz, 1H), 4.01 (dd, J = 6.5 Hz, J = 7.9 Hz, 1H), 1.81–1.77 (m, 2H), 1.68–1.56 (m, 6H), 1.44–1.41 (m,
2H), ppm; 13C-NMR (100 MHz, CDCl3)
23.9 ppm.
δ 131.8, 128.5, 128.2, 122.4, 111.0, 86.6, 85.6, 69.7, 65.7, 35.5, 25.1,

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Synthesis of 12 and 13: to a stirred solution of 10 (or 11) (1.0 eq) in a 1:1:1 mixture of
CHCl3/ACN/H2O, NaIO4 (4.4 eq) and RuO2 H2O (2.5% mol) were added. The mixture was vigorously
·
stirred at room temperature overnight. The solvent was evaporated under reduced pressure and the
crude was re-dissolved in CHCl3 and filtered through a silica pad. The eluate was washed three times
with water, dried over MgSO4, filtered and concentrated in vacuo to yield 12 as a yellow oil (54%) or
13 as yellow oil (47%).
1-{1,4-Dioxaspiro[4.5]decan-2-yl}propane-1,2-dione (12): yellow oil, 54%, Rf = 0.42 (CyH/EtOAc 3:1).
1H-NMR (300 MHz, CDCl3)
δ 5.14 (dd, J = 5.3 Hz, J = 7.9 Hz, 1H), 4.35 (dd, J = 8.0 Hz, J = 8.9 Hz, 1H),
3.99 (dd, J = 5.3 Hz, J = 8.9 Hz, 1H), 2.39 (s, 3H), 1.66–1.57 (m, 8H), 1.45–1.42 (m, 2H) ppm; 13C-NMR
(100 MHz, CDCl3) δ 197.5, 190.0, 109.2, 75.9, 66.9, 36.4, 35.6, 25.9, 24.8, 24.0 ppm [28].
1-{1,4-Dioxaspiro[4.5]decan-2-yl}-2-phenylethane-1,2-dione (13): yellow oil, 47%, Rf = 0.46 (CyH/EtOAc
1
9:1). H-NMR (300 MHz, CDCl3),
δ
7.98 (d, J = 7.1 Hz, 2H), 7.66 (d, J = 7.4 Hz, 1H), 7.51 (t, J = 7.7 Hz
,
2H), 5.12 (t, J = 6.2 Hz, 1H), 4.34 (d, J = 6.4 Hz, 2H), 1.65–1.49 (m, 8H), 1.39–1.32 (m, 2H) ppm; 13C-NMR
(100 MHz, CDCl3)
23.0 ppm.
δ 200.5, 193.0, 134.9, 132.2, 129.9, 128.9, 112.2, 77.9, 65.9, 35.4, 34.6, 24.9, 23.8,
Synthesis of DPD and Ph-DPD: to a stirred solution of 12 (or 13) (10 mM) in D2O, Dowex 50WX8
resin was added (100 mg/1 mL). The mixture was stirred at room temperature overnight. The mixture
was filtered to remove the resin and extracted with CDCl3 to remove the released cyclohexanone.
4,5-Dihydroxy-2,3-pentanedione (DPD): 1H-NMR (300 MHz, D2O)
δ 4.41–4.37 (m, 1H), 4.21–4.14 (m, 2H),
4.07 (dd, J = 3.2 Hz, J = 6.0 Hz, 1H), 3.99 (dd, J = 3.8 Hz, J = 7.4 Hz, 1H), 3.86–3.78 (m, 2H), 3.69–3.65
(m, 1H), 3.59 (dd, J = 5.6 Hz, J = 9.4 Hz, 1H), 2.39 (s, 3H), 1.46 (s, 3H), 1.43 (s, 3H) ppm [34]. The NMR
shows that some cyclohexanone is left as two multiplets at 1.88–1.86 and 1.75–1.74 ppm.
1
3,4-Dihydroxy-1-phenylbutane-1,2-dione (Ph-DPD): H-NMR (300 MHz, D2O)
δ 8.25–8.15 (m, 2H),
8.07–7.92 (m, 2H), 7.73–7.68 (m, 1H), 7.62–7.59 (m, 5H), 7.48–7.46 (m, 5H), 4.49–4.42 (m, 1H), 4.40–4.36
(m, 1H), 4.13 (dd, J = 2.7 Hz, J = 5.6 Hz, 1H), 4.09 (d, J = 2.8 Hz, 1H), 4.06 (d, J = 2.6 Hz, 1H), 3.88 (d,
J = 4.0 Hz, 1H), 3.85–3.79 (m, 1H), 3.73–3.66 (m, 1H) ppm [30].
Synthesis of quinoxaline-DPD and quinoxaline-Ph-DPD: to a stirred solution of DPD
(or Ph-DPD) in D2O, o-phenylendiamine (2.0 eq) was added. The reaction was stirred at room
temperature overnight. The solvent was evaporated under reduced pressure, the crude was
re-dissolved in ACN (1 mL), filtered and purified by preparative HPLC.
1-(3-Methylquinoxalin-2-yl)ethane-1,2-diol (Quinoxaline-DPD): orange solid, Rf = 0.52 (CHCl3/MeOH
1
9:1). H-NMR (700 MHz, MeOD)
δ 8.09–8.07 (m, 1H), 7.98–7.97 (m, 1H), 7.76 (pd, J = 7.0 Hz, J = 1.6 Hz,
2H), 5.15–5.13 (m, 1H), 4.02 (dd, J = 11.4 Hz, J = 5.4 Hz, 1H), 3.96 (dd, J = 11.4 Hz, J = 6.3 Hz, 1H),
2.84 (s, 3H) ppm; 13C-NMR (176 MHz, MeOD)
δ 156.6, 154.7, 142.3, 141. 8, 131.2, 130.5, 129.9, 128.8,
72.9, 66.3, 22.3 ppm; HRMS (ESI-MS) calcd. for C11H12N2O2 [M + H]+ = 205.0899. Found: 205.0972.
The NMR was consistent with previously reported data [18]. The NMR was measured with a Bruker
DRX700 (700 MHz).
1-(3-Phenylquinoxalin-2-yl)ethane-1,2-diol
(
Quinoxaline-Ph-DPD): orange solid, Rf
=
0.48
(CHCl3/MeOH 9:1). 1H-NMR (700 MHz, CDCl3)
δ
8.18 (dd, J = 6.4 Hz, J = 3.3 Hz, 1H),
8.13 (dd, J = 6.1 Hz, J = 3.6 Hz, 1H), 7.82 (dd, J = 6.4 Hz, J = 3.4 Hz, 2H), 7.68 (dd, J = 7.8 Hz, J = 1.3
Hz, 2H), 7.57–7.53 (m, 3H), 5.30 (dd, J = 4.9 Hz, J = 3.6 Hz, 1H), 3.74 (dd, J = 11.7 Hz, J = 3.4 Hz, 1H),
3.54 (dd, J = 11.7 Hz, 5.1 Hz, 1H) ppm; 13C-NMR (176 MHz, CDCl3)
δ
153.7, 152.5, 141.8, 139.6, 137.6,
130.5, 130.4, 129.6, 129.4, 129.0, 128.8, 128.4, 70.6, 65.7 ppm. HRMS (ESI-MS) calcd. for C16H14N2O2

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[M + H]+ = 267.1055. Found: 267.1129. The NMR was consistent with previously reported data [57].
The NMR was measured with a Bruker DRX700 (700 MHz).
3.3. General Procedures for the Synthesis of 1,4- and 1,5-Disubstituted Triazoles DPD-Derivatives (Series I
and II)
Synthesis of 1-[(t-butyldimethylsilyl)oxy]but-3-yn-2-ol (14)
:
to a stirred solution of
(t-butyldimethylsilyloxy)acetaldehyde (1.0 eq) in dry THF, ethynylmagnesium bromide (0.50 M in THF,
1.3 eq) was added over 15 minutes at 0 ◦C. After the addition, the reaction was allowed to reach room
temperature and stirred for 3 hours. The solvent was removed under reduced pressure, the residue
was poured into a cold saturated solution of NH4Cl and extracted three times with Et2O. The organic
layer was washed twice with water and once with brine, dried over MgSO4, filtered and concentrated
1
in vacuo to yield 14 as a yellow oil, 99%, Rf = 0.55 (CyH/EtOAc 3:1). H-NMR (300 MHz, CDCl3)
δ
4.40–4.37 (m, 1H), 3.79 (dd, J = 3.8 Hz, J = 10.1 Hz, 1H), 3.66 (dd, J = 6.8 Hz, J = 10.0 Hz, 1H), 2.62 (d,
J = 5.1 Hz, 1H), 2.42 (d, J = 2.2 Hz, 1H), 0.91 (s, 9H), 0.10 (d, J = 1.5 Hz, 6H) ppm; 13C-NMR (100 MHz,
CDCl3) δ 81.9, 73.4, 66.8, 62.9, 25.8, 18.3, −5.4 ppm [58].
Synthesis of but-3-yne-1,2-diol (15): to a stirred solution of 14 in MeOH, a Dowex50WX8
100–200 mesh (100 mg/1 mL) was added. The reaction was stirred at room temperature overnight.
The mixture was filtered through paper and the solvent was evaporated under reduced pressure to
1
yield 15 as an orange oil, 99%, Rf = 0.50 (CHCl3/MeOH 9:1). H-NMR (300 MHz, CDCl3)
δ
4.49–4.45
(m, 1H), 3.80–3.68 (m, 1H), 2.51 (d, J = 2.2 Hz, 1H), 2.31 (s, 2H) ppm; 13C-NMR (100 MHz, CDCl3)
δ
81.5, 74.3, 66.3, 63.0 ppm [59].
Synthesis of 2-ethynyl-1,4-dioxaspiro[4.5]decane (16): to 15 (1.0 eq) cyclohexanone dimethyl
ketal (10.0 eq) and a catalytic amount of p-TSA were added. The reaction was stirred at room
temperature overnight. The solvent was removed under reduced pressure and the crude was
re-dissolved in Et2O and washed three times with NaHCO3. The organic layer was dried over
MgSO4, filtered and concentrated in vacuo to yield 16 as a yellow oil, 57%, Rf = 0.42 (CyH/EtOAc
1
9:1). H-NMR (300 MHz, CDCl3)
δ 4.71 (dt, J = 2.0 Hz, J = 6.3 Hz, 1H), 4.16 (dd, J = 6.4 Hz, J = 8.0 Hz,
1H), 3.94 (dd, J = 6.3 Hz, J = 8.0 Hz, 1H), 2.48 (d, J = 2.0 Hz, 1H), 1.77–1.72 (m, 2H), 1.65–1.59 (m,
6H), 1.42–1.39 (m, 2H) ppm; 13C-NMR (100 MHz, CDCl3)
23.8 ppm [30].
δ 111.2, 81.6, 73.7, 69.5, 64.9, 35.6, 25.0,
General procedure for the synthesis of 17a–f: to a stirred suspension of NaN3 (1.5 eq) in DMSO
(5 mL), the corresponding bromo compound (1.0 eq) was added. The reaction was stirred at room
temperature overnight. The mixture was diluted with diethyl ether and extracted five times with water
and once with brine, dried over MgSO4, filtered and concentrated in vacuo to yield the desired azide
as a colorless/yellowish oil.
General procedure for the synthesis of 18a–f: to a stirred solution of 15 (1.0 eq) in a 1:1 mixture
of H2O/t-BuOH, the corresponding azide (1.0 eq), sodium ascorbate (0.5 eq) and CuSO4 5H2O
·
(5% mol) were added. The reaction was stirred at room temperature overnight. The solvent was
evaporated under reduced pressure, the crude was redissolved in ACN (1 mL), filtered and purified
by preparative HPLC [49].
1-[1-(2-Phenylethyl)-1H-1,2,3-triazol-4-yl]ethane-1,2-diol (18a): orange oil, 89%, Rf = 0.24 (CHCl3/MeOH
1
9:1), UHPLC-ESI-MS: Rt = 1.80, m/z = 234.2 [M + H]+. H-NMR (300 MHz, CD3CN)
δ 7.56 (s, 1H),
7.31–7.20 (m, 3H), 7.15 (d, J = 6.7 Hz, 2H), 4.75 (dd, J = 4.3 Hz, J = 6.7 Hz, 1H), 4.57 (t, J = 7.3 Hz, 2H),
3.72 (dd, J = 4.2 Hz, J = 11.2 Hz, 1H), 3.60 (dd, J = 6.9 Hz, J = 11.2 Hz, 1H), 3.17 (t, J = 7.2 Hz, 2H), 2.23
(s br, 1H) ppm; 13C-NMR (100 MHz, CD3CN)
36.9 ppm.
δ 149.4, 138.8, 129.7, 129.4, 127.6, 122.8, 68.5, 66.8, 51.9,
1-(1-Benzyl-1H-1,2,3-triazol-4-yl)ethane-1,2-diol (18b): yellowish oil, 60%, Rf = 0.24 (CHCl3/MeOH 9:1),
1
UHPLC-ESI-MS: Rt = 1.70, m/z = 220.2 [M + H]+. H-NMR (300 MHz, CD3CN)
δ
7.72 (s, 1H), 7.38–7.29

Molecules 2018, 23, 2545
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(m, 5H), 5.51 (s, 2H), 4.79 (dd, J = 4.4 Hz, J = 6.3 Hz, 1H), 3.75 (dd, J = 4.2 Hz, J = 11.2 Hz, 1H), 3.64 (dd,
J = 6.7 Hz, J = 11.2 Hz, 1H) ppm; 13C-NMR (100 MHz, CD3CN)
δ 150.1, 136.9, 129.8, 129.3, 128.9, 123.0,
68.5, 66.7, 54.4 ppm.
1-{1-[.2-(2-Fluorophenyl)ethyl]-1H-1,2,3-triazol-4-yl}ethane-1,2-diol (18c): colorless oil, 62%, Rf = 0.49
1
(CHCl3/MeOH 9:1), UHPLC-ESI-MS: Rt = 1.82, m/z = 252.2 [M + H]+. H-NMR (300 MHz, CD3CN)
δ
7.58 (s, 1H), 7.30–7.23 (m, 1H), 7.14–7.04(m, 3H), 4.75(dd, J = 4.3 Hz, J = 6.8 Hz, 1H), 4.58 (t, J = 7.1 Hz,
2H), 3.71 (dd, J = 4.2 Hz, J = 11.2 Hz, 1H), 3.59 (dd, J = 6.9 Hz, J = 11.2 Hz, 1H), 3.21 (t, J = 7.1 Hz, 2H),
2.23 (s br, 1H) ppm; 13C-NMR (100 MHz, CD3CN)
δ 162.8 (d, J = 243.8 Hz), 150.2, 132.9 (d, J = 4.6 Hz),
130.6 (d, J = 8.2 Hz), 126.2, 126.0 (d, J = 3.5 Hz), 123.6, 116.8 (d, J = 22.0 Hz), 69.2, 67.5, 51.2, 31.3 (d,
J = 2.4 Hz) ppm.
1-{1-[2-(Pyridin-2-yl)ethyl]-1H-1,2,3-triazol-4-yl}ethane-1,2-diol (18d): yellow oil, 88%, Rf = 0.28
1
(CHCl3/MeOH 9:1), UHPLC-ESI-MS: Rt = 0.38, m/z = 232.2 [M + H]+. H-NMR (300 MHz, CD3CN)
δ
8.51 (s, 1H), 7.66–7.59 (m, 2H), 7.21–7.12 (m, 2H), 4.75 (t, J = 7.1 Hz, 3H), 3.71 (dd, J = 4.1 Hz, J = 11.2 Hz,
1H), 3.59 (dd, J = 6.8 Hz, J = 11.1 Hz, 1H), 3.33 (t, J = 7.1 Hz, 2H) ppm; 13C-NMR (100 MHz, CD3CN)
δ
158.5, 150.3, 143.3, 137.5, 124.4, 123.2, 122.8, 68.5, 66.8, 50.0, 38.8 ppm.
6-[4-(1,2-Dihydroxyethyl)-1H-1,2,3-triazol-1-yl]hexanenitrile (18e): orange oil, 72%, Rf = 0.54
1
(CHCl3/MeOH 9:1), UHPLC-ESI-MS: Rt = 1.39, m/z = 225.2 [M + H]+. H-NMR (300 MHz, CD3CN)
δ
7.68 (s, 1H), 4.78 (s, 1H), 4.34 (dt, J = 1.9 Hz, J = 7.1 Hz, 2H), 3.76–3.73 (m, 1H), 3.66–3.57 (m, 1H), 3.08 (s
br, 1H), 2.37 (dt, J = 1.9 Hz, J = 7.1 Hz, 2H), 1.88–1.83 (m, 2H), 1.68–1.58 (m, 2H), 1.44–1.34 (m, 2H) ppm;
13C-NMR (100 MHz, CD3CN) δ 148.4, 121.5, 119.8, 67.3, 65.6, 49.2, 28.8, 24.9, 24.2, 16.0 ppm.
1-[1-(2-Cyclohexylethyl)-1H-1,2,3-triazol-4-yl]ethane-1,2-diol (18f): orange oil, 73%, Rf = 0.43
1
(CHCl3/MeOH 9:1), UHPLC-ESI-MS: Rt = 2.20, m/z = 240.2 [M + H]+. H-NMR (300 MHz, CD3CN)
δ
7.69 (s, 1H), 4.78 (t, J = 5.3 Hz, 1H), 4.35 (t, J = 7.5 Hz, 2H), 3.74 (s, 1H), 3.67–3.62 (m, 1H), 2.21 (s br,
1H), 1.78–1.63 (m, 7H), 1.24–1.17 (m, 4H), 1.0–0.94 (m, 2H) ppm; 13C-NMR (100 MHz, CD3CN)
123.5, 69.2, 67.5, 49.4, 39.0, 36.4, 34.2, 27.8, 27.5 ppm.
δ 150.4,
Synthesis of 1-[1-(2-phenylethyl)-1H-1,2,3-triazol-5-yl]ethane-1,2-diol (19a)
:
to a stirred
solution of 15 (1.0 eq) in 1,4-dioxane, (2-azidoethyl)benzene (17a) (1.0 eq) and Cp*RuCl(PPh3)2 (2%
mol) were added. The reaction was stirred at reflux overnight. The solvent was evaporated under
reduced pressure, the crude was re-dissolved in ACN (1 mL), filtered and purified by preparative
HPLC to yield 19a as a yellow solid, 87%, Rf = 0.17 (CHCl3/MeOH 9:1), UHPLC-ESI-MS: Rt = 1.75,
1
m/z = 234.2 [M + H] +. H-NMR (300 MHz, CD3CN)
δ 7.51 (s, 1H), 7.28 (t, J = 7.3 Hz, 2H), 7.23 (t,
J = 7.3 Hz, 1H), 7.15 (d, J = 7.1 Hz, 2H), 4.62 (d, J = 6.3 Hz, 1H), 4.61–4.58 (m, 2H), 3.70 (s br, 1H),
3.62 (dd, J = 6.6 Hz, J = 11.3 Hz, 1H), 3.56 (dd, J = 4.9 Hz, J = 11.3 Hz, 1H), 3.21 (t, J = 7.4 Hz, 2H), 2.21 (s,
1H) ppm; 13C-NMR (100 MHz, CD3CN)
50.4, 37.0 ppm.
δ 139.0, 138.5, 132.1, 129.7, 129.4, 127.6, 65.5 (d, J = 7.6 Hz),
Synthesis of 4-{1,4-dioxaspiro[4.5]decan-2-yl}-1H-1,2,3-triazole (20): to a stirred solution of 16
(1.0 eq) in a 1:1 mixture of H2O/t-BuOH, trimethylsilyl azide (10.0 eq), sodium ascorbate (0.5 eq)
and CuSO4 5H2O (5% mol) were added. The reaction was stirred at room temperature overnight.
·
The solvent was evaporated under reduced pressure, the crude was re-dissolved in EtOAc and
extracted three times with water. The organic layer was dried over MgSO4, filtered and concentrated in
vacuo. The crude was purified using CyH/TBME (3:1) as an eluent to yield 20 as a yellowish oil, 36%,
1
Rf = 0.61 (CHCl3/MeOH 9:1), UHPLC-ESI-MS: Rt = 2.13, m/z = 210.2 [M + H] +. H-NMR (300 MHz,
CD3CN)
δ 7.72 (s, 1H), 5.25 (t, J = 6.6 Hz, 1H), 4.31–4.26 (m, 1H), 4.00–3.95 (m, 1H), 2.37 (s br, 1H),
1.64–1.58 (m, 8H), 1.43–1.40 (m, 2H) ppm; 13C-NMR (100 MHz, CD3CN)
36.8, 35.9, 25.8, 24.7, 24.6 ppm.
δ 147.4, 130.0, 111.1, 70.9, 69.8,

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General procedure for the synthesis of 21h–k and 22h–k: to a stirred solution of 20 (1.0 eq)
in dry THF, K2CO3 (2.0 eq) and the corresponding alkyl halide (bromide or iodide) were added.
The reaction was stirred at reflux overnight. The solvent was evaporated under reduced pressure,
the crude was re-dissolved in ACN (1 mL), filtered and purified by preparative HPLC.
For each compound, two different fractions were isolated corresponding to the 1,4- and
1,5-disubstitued products. The different substitution was determined by HMBC of two representative
samples (21i, 22i).
General procedure for the synthesis of 18g–k and 19h–k: a stirred solution of 20 (or 21h
–k,
or 22h
–
k
) in 1,4-dioxane was cooled to 0 ◦C using an ice bath. A catalytic amount of 12 M HCl was
added. The reaction was stirred at room temperature overnight. The solvent was evaporated under
reduced pressure; the crude was re-dissolved in Et2O and extracted with water. The aqueous layer
was extracted three times with Et2O and dried in vacuo to yield the corresponding products 18g
and 19h–k.
–k
1-(1H-1,2,3-Triazol-4-yl)ethane-1,2-diol (18g): yellowish oil, 98%, Rf = 0.13 (CHCl3/MeOH 9:1),
1
UHPLC-ESI-MS: Rt = 0.32, m/z = 130.3 [M + H]+. H-NMR (300 MHz, MeOD)
δ
8.38 (s, 1H), 5.05 (t,
J = 5.5 Hz, 1H), 3.86–3.74 (m, 2H) ppm; 13C-NMR (100 MHz, MeOD) δ 146.0, 126.4, 67.0, 66.2 ppm.
1-(1-Methyl-1H-1,2,3-triazol-4-yl)ethane-1,2-diol (18h): colorless oil, 66%, Rf = 0.29 (CHCl3/MeOH 9:1),
1
UHPLC-ESI-MS: Rt = 0.45, m/z = 144.2 [M + H]+. H-NMR (300 MHz, MeOD)
δ
8.40 (s, 1H), 5.04 (t,
151.9, 133.5, 70.0,
J = 5.5 Hz, 1H), 4.35 (s, 3H), 3.94–3.80 (m, 2H) ppm; 13C-NMR (100 MHz, MeOD)
δ
68.2, 42.9 ppm.
1-(1-Methyl-1H-1,2,3-triazol-5-yl)ethane-1,2-diol (19h): colorless oil, 65%, Rf = 0.32 (CHCl3/MeOH 9:1),
1
UHPLC-ESI-MS: Rt = 0.65, m/z = 144.1 [M + H]+. H-NMR (300 MHz, MeOD)
δ 7.60 (s, 1H), 4.81 (dd,
J = 4.8 Hz, J = 6.8 Hz, 1H), 4.13 (s, 3H), 3.81–3.68 (m, 2H) ppm; 13C-NMR (100 MHz, MeOD)
δ 150.6,
133.4, 68.7, 67.0, 41.7 ppm.
1-[1-(Cyclopropylmethyl)-1H-1,2,3-triazol-4-yl]ethane-1,2-diol (18i): colorless oil, 73%, Rf = 0.32
1
(CHCl3/MeOH 9:1), UHPLC-ESI-MS: Rt = 1.25, m/z = 184.2 [M + H]+. H-NMR (300 MHz, CD3CN)
δ
8.29 (d, J = 9.1 Hz, 1H), 6.21 (s br, 2H), 5.02 (dd, J = 3.9 Hz, J = 6.4 Hz, 1H), 4.36 (dd, J = 2.5 Hz,
J = 7.4 Hz, 2H), 3.80 (d, J = 5.3 Hz, 2H), 1.40–1.35 (m, 1H), 0.70–0.64 (m, 2H), 0.52–0.47 (m, 2H) ppm;
13C-NMR (100 MHz, CD3CN) δ 146.8, 125.8, 66.9, 65.7, 57.8, 11.1, 4.5 ppm.
1-[1-(Cyclopropylmethyl)-1H-1,2,3-triazol-5-yl]ethane-1,2-diol (19i): colorless oil, 77%, Rf = 0.35
1
(CHCl3/MeOH 9:1), UHPLC-ESI-MS: Rt = 1.31, m/z = 184.2 [M + H]+. H-NMR (300 MHz, CD3CN)
δ
7.87 (s, 1H), 4.91 (t, J = 5.5 Hz, 1H), 4.34 (d, J = 7.3 Hz, 2H), 3.76 (d, J = 5.5 Hz, 2H), 1.43–1.38 (m, 1H),
0.64–0.58 (m, 2H), 0.49–0.46 (m, 2H) ppm; 13C-NMR (100 MHz, CD3CN)
11.7, 4.6 ppm.
δ 140.3, 130.7, 65.5, 65.4, 55.2,
1-(1-Butyl-1H-1,2,3-triazol-4-yl)ethane-1,2-diol (18j): colorless oil, 85%, Rf = 0.37 (CHCl3/MeOH 9:1),
1
UHPLC-ESI-MS: Rt = 1.45, m/z = 186.2 [M + H]+. H-NMR (300 MHz, MeOD)
δ
8.28 (s, 1H), 4.77 (t,
J = 5.5 Hz, 1H), 4.39 (t, J = 7.2 Hz, 2H), 3.63–3.49 (m, 2H), 1.81–1.71(m, 2H), 1.23–1.11 (m, 2H), 0.76 (t,
J = 7.4 Hz, 3H) ppm; 13C-NMR (100 MHz, MeOD) δ 147.5, 126.7, 67.2, 66.1, 53.8, 32.6, 20.5, 13.7 ppm.
1-(1-Butyl-1H-1,2,3-triazol-5-yl)ethane-1,2-diol (19j): colorless oil, 95%, Rf = 0.39 (CHCl3/MeOH 9:1),
1
UHPLC-ESI-MS: Rt = 1.50, m/z = 186.2 [M + H]+. H-NMR (300 MHz, MeOD)
δ
8.21 (s, 1H), 4.98 (t,
J = 5.7 Hz, 1H), 4.62 (dd, J = 6.5 Hz, J = 8.4 Hz, 2H), 3.91–3.78 (m, 2H), 2.04–1.94 (m, 2H), 1.49–1.37 (m,
2H), 1.00 (t, J = 7.3 Hz, 3H) ppm; 13C-NMR (100 MHz, MeOD)
δ 142.7, 130.1, 65.9, 65.8, 51.5, 32.8, 20.7,
13.8 ppm.

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1-[1-(2-Ethoxyethyl)-1H-1,2,3-triazol-4-yl]ethane-1,2-diol (18k):
colorless oil, 91%, Rf = 0.31
1
(CHCl3/MeOH 9:1), UHPLC-ESI-MS: Rt = 1.21, m/z = 202.2 [M + H]+. H-NMR (300 MHz, CD3CN)
δ
7.92 (s, 1H), 4.89 (t, J = 5.1 Hz, 1H), 4.54 (t, J = 5.1 Hz, 2H), 3.83–3.68 (m, 4H), 3.47 (q, J = 7.0 Hz, 2H),
1.10 (t, J = 7.0 Hz, 3H) ppm; 13C-NMR (100 MHz, CD3CN)
δ
148.3, 124.9, 68.8, 67.7, 66.9, 66.3, 52.1,
15.2 ppm.
1-[1-(2-Ethoxyethyl)-1H-1,2,3-triazol-4-yl]ethane-1,2-diol (19k):
colorless oil, 82%, Rf = 0.37
1
(CHCl3/MeOH 9:1), UHPLC-ESI-MS: Rt = 1.23, m/z = 202.2 [M + H]+. H-NMR (300 MHz, MeOD)
δ
8.10 (s, 1H), 5.00 (t, J = 5.5 Hz, 1H), 4.74 (dd, J = 3.7 Hz, J = 5.3 Hz, 2H), 3.84 (t, J = 5.2 Hz, 2H), 3.77 (t,
J = 5.8 Hz, 2H), 3.46–3.38 (m, 2H), 1.05 (t, J = 7.0 Hz, 3H) ppm; 13C-NMR (100 MHz, MeOD)
δ 143.4,
130.3, 69.6, 67.7, 66.0, 65.9, 51.7, 15.3 ppm.
3.4. General Procedures for the Synthesis of 3,5-Disubstituted Isoxazoles DPD Derivatives (Series III and IV)
General procedure for the synthesis of 24l–r: to a stirred solution of the corresponding aldehyde
(1.0 eq) in EtOH (10 mL), Et3N (1.5 eq) and NH2OH*HCl (1.5 eq) dissolved in water (10 mL) were
added. The reaction was stirred at room temperature for 1–3 hours (monitored by TLC). The solvent
was evaporated under reduced pressure; the crude was re-dissolved in EtOAc and extracted three
times with water. The organic layer was dried over MgSO4, filtered and concentrated in vacuo to
yield the corresponding oxime. All the resulting compounds were used in the next step without
being purified.
General procedure for the synthesis of 25l–r: to a stirred solution of the corresponding oxime
(1.0 eq) in DMF, N-chlorosuccinimide (1.0 eq) was added in two portions. The reaction was stirred
at room temperature for 1–2 h (monitored by TLC). The crude was diluted with Et2O and extracted
five times with water and once with brine. The organic layer was dried over MgSO4, filtered and
concentrated in vacuo to yield the corresponding chloro-oxime. All the resulting compounds were
used in the next step without being purified.
General procedure for the synthesis of 26l–r: to a stirred solution of 15 (1.0 eq) in a 1:1 mixture
of H2O/t-BuOH, the corresponding chloro-oxime (1.0 eq), sodium ascorbate (0.5 eq), CuSO4 5H2O
·
(5% mol) and KHCO3 (4.3 eq) were added. The mixture was stirred at room temperature overnight.
The solvent was evaporated under reduced pressure; the crude was redissolved in ACN (1 mL),
filtered and purified by preparative HPLC [49].
1-[3-(4-Methylphenyl)-1,2-oxazol-5-yl]ethane-1,2-diol (26l): white solid, 82%, Rf = 0.38 (CHCl3/MeOH 9:1),
1
UHPLC-ESI-MS: Rt = 2.15, m/z = 220.1 [M + H]+. H-NMR (300 MHz, CD3CN)
δ
7.73 (d, J = 8.0 Hz
,
2H), 7.31 (d, J = 7.9 Hz, 2H), 6.67 (s, 1H), 4.82 (d, J = 5.2 Hz, 1H), 3.92 (s br, 1H), 3.83–3.71 (m, 2H),
3.14 (s br, 1H), 2.38 (s, 3H) ppm; 13C-NMR (100 MHz, CD3CN)
101.2, 69.2, 66.0, 22.0 ppm.
δ 175.3, 163.7, 142.0, 131.3, 128.2, 127.9,
1-[3-(3-Chlorophenyl)-1,2-oxazol-5-yl]ethane-1,2-diol (26m): white solid, 87%, Rf = 0.51 (CHCl3/MeOH
1
9:1), UHPLC-ESI-MS: Rt = 2.24, m/z = 240.0 [M + H]+. H-NMR (300 MHz, CD3CN)
δ 7.87 (s, 1H),
7.77 (dd, J = 5.4 Hz, J = 6.8 Hz, 1H), 7.48 (d, J = 5.8 Hz, 2H), 6.74 (s, 1H), 4.84 (t, J = 5.3 Hz, 1H), 3.78 (dq,
J = 5.3 Hz, J = 11.4 Hz, 2H) ppm; 13C-NMR (100 MHz, CD3CN)
δ 175.2, 162.0, 135.4, 132.0, 131.6, 130.9,
127.5, 126.1, 100.8, 68.5, 65.2 ppm.
1-[3-(2,4-Difluorophenyl)-1,2-oxazol-5-yl]ethane-1,2-diol (26n): white solid, 78%, Rf = 0.49 (CHCl3/MeOH
1
9:1), UHPLC-ESI-MS: Rt = 2.06, m/z = 242.2 [M + H]+. H-NMR (300 MHz, CD3CN)
δ
7.97–7.89 (m,
1H), 7.14–7.06 (m, 2H), 6.69 (d, J = 3.3 Hz, 1H), 4.86 (t, J = 5.3 Hz, 1H), 3.85–3.73 (m, 2H) ppm; 13C-NMR
(100 MHz, CD3CN) 174.8, 164.9 (dd, J = 8.5 Hz, J = 246.4 Hz), 161.4 (dd, J = 8.5 Hz, J = 249.4 Hz), 157.9,
δ
131.6 (dd, J = 4.6 Hz, J = 10.1 Hz), 114.7 (dd, J = 3.9 Hz, J = 12.6 Hz), 113.2 (dd, J = 3.6 Hz, J = 21.9 Hz),
105.6 (t, J = 26.1 Hz), 102.7 (d, J = 7.4 Hz), 68.5, 65.3 ppm.

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1-[3-(Pyridin-3-yl)-1,2-oxazol-5-yl]ethane-1,2-diol (26o): orange solid, 71%, Rf = 0.22 (CHCl3/MeOH 9:1),
1
UHPLC-ESI-MS: Rt = 1.14, m/z = 207.2 [M + H]+. H-NMR (300 MHz, CD3CN)
δ
9.04 (s, 1H), 8.67 (s,
1H), 8.18 (d, J = 7.9 Hz, 1H), 7.46 (dd, J = 5.3 Hz, J = 7.2 Hz, 1H), 6.79 (s, 1H), 4.87 (t, J = 5.3 Hz, 1H),
3.85–3.73 (m, 2H) ppm; 13C-NMR (100 MHz, CD3CN)
100.7, 68.5, 65.3 ppm.
δ 175.4, 160.9, 151.9, 148.7, 135.0, 126.2, 124.9,
1-(3-Cyclopropyl-1,2-oxazol-5-yl)ethane-1,2-diol (26p): yellow oil, 63%, Rf = 0.12 (CHCl3/MeOH 9:1),
1
UHPLC-ESI-MS: Rt = 1.91, m/z = 170.2 [M + H]+. H-NMR (300 MHz, CD3CN)
δ
5.98 (s, 1H), 4.71 (ddd,
J = 0.6 Hz, J = 4.6 Hz, J = 6.1 Hz, 1H), 3.74–3.62 (m, 2H), 2.00–1.91 (m, 1H), 1.03–0.97 (m, 2H), 0.78–0.72
(m, 2H) ppm; 13C-NMR (100 MHz, CD3CN) δ 173.5, 167.3, 99.7, 68.3, 65.2, 8.3, 7.8 ppm.
1-[3-(Oxolan-3-yl)-1,2-oxazol-5-yl]ethane-1,2-diol (26q): yellow oil, 77%, Rf = 0.50 (CHCl3/MeOH 9:1),
1
UHPLC-ESI-MS: Rt = 1.53, m/z = 200.2 [M + H]+. H-NMR (300 MHz, CD3CN)
δ
6.22 (s, 1H), 4.77–4.73
(m, 1H), 4.03–3.98 (m, 1H), 3.94–3.85 (m, 1H), 3.82–3.76 (m, 1H), 3.74–3.65 (m, 2H), 3.55–3.47 (m, 1H),
2.37–2.25 (m, 2H), 2.09–2.00 (m, 1H) ppm; 13C-NMR (100 MHz, CD3CN)
65.3, 63.7, 37.4, 32.5 ppm.
δ 174.1, 166.1, 101.2, 72.7, 68.5,
1-(3-Cyclohexyl-1,2-oxazol-5-yl)ethane-1,2-diol (26r): yellow oil, 89%, Rf = 0.31 (CHCl3/MeOH 9:1),
1
UHPLC-ESI-MS: Rt = 2.13, m/z = 212.2 [M + H]+. H-NMR (300 MHz, CD3CN)
δ
6.18 (s, 1H), 4.73 (t,
J = 5.4 Hz, 1H), 3.71 (dq, J = 5.4 Hz, J = 11.3 Hz, 2H), 2.70 (dt, J = 3.3 Hz, J = 10.7 Hz, 1H), 2.21 (s br,
2H), 1.90–1.69 (m, 5H), 1.50–1.25 (m, 5H) ppm; 13C-NMR (100 MHz, CD3CN)
66.0, 37.3, 33.4, 27.3, 27.2 ppm.
δ 173.9, 169.8, 101.5, 69.2,
Synthesis of ethyl (2E)-2-(hydroxyimino)acetate (27): to a stirred solution of ethyl glyoxalate
(50% solution in toluene, 1.0 eq) in EtOH, Et3N (1.5 eq) and NH2OH HCl (1.5 eq) dissolved in water
·
(10 mL) were added. The reaction was stirred at room temperature for 2 hours (monitored by TLC).
The solvent was evaporated under reduced pressure; the crude was re-dissolved in Et2O and extracted
three times with water. The organic layer was dried over MgSO4, filtered and concentrated in vacuo to
1
yield 27 as a colorless oil, 84%, Rf = 0.64 (CHCl3/MeOH 9:1). H-NMR (300 MHz, CDCl3)
δ 9.83 (s br,
1H), 7.56 (s, 1H), 4.32 (q, J = 7.1 Hz, 2H), 1.34 (t, J = 7.1 Hz, 3H) ppm; 13C-NMR (100 MHz, CDCl3)
δ
162.4, 141.6, 61.8, 13.8 ppm [60].
Synthesis of ethyl 5-{1,4-dioxaspiro[4.5]decan-2-yl}-1,2-oxazole-3-carboxylate (28): to a stirred
solution of 16 (1.0 eq) in THF, 27 (2.0 eq) and NaOCl (40.0 eq portion wise over 12 hours) were added.
The reaction was stirred at room temperature for 12 hours. The solvent was evaporated under reduced
pressure, the crude was re-dissolved in DCM and washed three times with water. The organic layer
was dried over MgSO4, filtered and concentrated in vacuo. The crude was re-dissolved in ACN (1 mL),
filtered and purified by preparative HPLC to yield 28 as a yellowish oil, 36%, Rf = 0.57 (CyH/EtOAC
1
3:1), UHPLC-ESI-MS: Rt = 3.04, m/z = 282.2 [M + H]+. H-NMR (300 MHz, CDCl3)
δ 6.67 (s, 1H),
5.23 (t, J = 6.0 Hz, 1H), 4.43 (q, J = 7.2 Hz, 2H), 4.35 (dd, J = 6.7 Hz, J = 8.6 Hz, 1H), 4.09 (dd, J = 5.4 Hz,
J = 8.6 Hz, 1H), 1.71–1.62 (m, 9H), 1.41 (t, J = 7.1 Hz, 4H) ppm; 13C-NMR (100 MHz, CDCl3)
δ
173.5,
159.8, 156.3, 111.9, 102.5, 69.8, 68.1, 62.2, 35.8, 34.9, 25.0, 23.9, 23.8, 14.1 ppm.
Synthesis of 5-{1,4-dioxaspiro[4.5]decan-2-yl}-1,2-oxazole-3-carboxylic acid (30)
: a stirred
◦
solution of 28 in THF was cooled to 0 C using an ice bath. A solution of 10 M NaOH (5.0 eq) was added
dropwise and the reaction was stirred at room temperature overnight. The solvent was evaporated
under reduced pressure; the crude was re-dissolved in DCM and extracted with water. The aqueous
layer was acidified with 1M HCl until pH = 1 and extracted three times with CHCl3/i-PrOH (7:3).
The organic layer was dried over MgSO4, filtered and concentrated in vacuo to yield 30 as a white
solid, 99%, Rf = 0.17(CHCl3/MeOH 5:1), UHPLC-ESI-MS: Rt = 2.37, m/z = 254.2 [M + H] +. H-NMR
1
(300 MHz, CDCl3) δ 6.74 (s, 1H), 5.26 (t, J = 5.9 Hz, 1H), 4.38 (dd, J = 6.6 Hz, J = 8.7 Hz, 1H), 4.12 (dd,
J = 5.3 Hz, J = 8.7 Hz, 1H), 1.73–1.60 (m, 8H), 1.47–1.43 (m, 2H) ppm; 13C-NMR (100 MHz, CDCl3)
δ
174.2, 162.4, 155.6, 112.1, 102.8, 69.8, 68.1, 35.9, 34.9, 24.9, 23.9, 23.8 ppm.

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General procedure for the synthesis of 32b, 32s–z: the reactions were performed in parallel
in 15 mL reaction tubes in a 24 position Mettler-Toledo Miniblock equipped with a heat transfer
block and inert gas manifold. Each reaction tube was loaded with a previously prepared solution
of 30 mg of 28 (1.0 eq) in 2 mL of DMF, DIPEA (5.0 eq), HOBt (2.0 eq), EDC HCl (2.5 eq). Then the
®
·
corresponding amine was added (2.0 eq). The reaction mixtures were stirred at room temperature
overnight. The reaction conversion was confirmed through a UHPLC check of some representative
samples. The mixtures were evaporated until dryness. The crudes were re-dissolved in 1.0 mL of
ACN, filtered and purified with preparative HPLC (gradient acetonitrile/water with 0.1% formic acid,
2–98%).
General procedure for the synthesis of 29, 31, 33b, 33s–z: a stirred solution of 28 (or 30, or 32b
,
or 32s
–
z
) was cooled to 0 ◦C using an ice bath. A catalytic amount of concentrated HCl was added.
The reactions were stirred at room temperature overnight. The solvent was evaporated under reduced
pressure, the crudes were re-dissolved in ACN (1 mL), filtered and purified by preparative HPLC.
Ethyl 5-(1,2-dihydroxyethyl)-1,2-oxazole-3-carboxylate (29): colorless oil, 46%, Rf = 0.44 (CHCl3/MeOH
1
9:1), UHPLC-ESI-MS: Rt = 1.51, m/z = 202.2 [M + H]+. H-NMR (300 MHz, CD3CN)
δ 6.65 (s, 1H),
4.84 (t, J = 5.3 Hz, 1H), 4.37 (q, J = 7.1 Hz, 2H), 3.76 (dd, J = 4.2 Hz, J = 5.2 Hz, 2H), 2.18 (s br, 1H),
1.35 (t, J = 7.1 Hz, 3H) ppm; 13C-NMR (100 MHz, CD3CN)
δ 176.2, 160.8, 157.4, 103.0, 68.3, 65.1, 62.9,
14.3 ppm.
5-(1,2-Dihydroxyethyl)-1,2-oxazole-3-carboxylic acid (31): colorless oil, 55%, Rf = 0.11 (CHCl3/MeOH 9:1),
1
UHPLC-ESI-MS: Rt = 0.42, m/z = 174.2 [M + H]+. H-NMR (300 MHz, CD3CN)
δ
6.65 (s, 1H), 4.84 (t,
J = 5.1 Hz, 1H), 3.81–3.70 (m, 2H) ppm; 13C-NMR (100 MHz, CD3CN)
δ 176.2, 161.0, 157.2, 103.2, 68.3,
65.1 ppm.
N-Benzyl-5-(1,2-dihydroxyethyl)-1,2-oxazole-3-carboxamide (33b): white solid, 58%, Rf = 0.25
1
(CHCl3/MeOH 9:1), UHPLC-ESI-MS: Rt = 1.89, m/z = 263.2 [M + H]+. H-NMR (300 MHz, Acetone-d6)
δ
7.40–7.27 (m, 4H), 7.26–7.22 (m, 1H), 6.68 (s, 1H), 4.91 (t, J = 5.4 Hz, 1H), 4.59 (s, 2H), 3.89–3.78 (m,
2H) ppm; 13C-NMR (100 MHz, Acetone-d6)
δ 177.2, 160.6, 141.0, 137.3, 130.2, 129.4, 128.9, 102.9, 69.5,
66.4, 44.4 ppm.
5-(1,2-Dihydroxyethyl)-N-(4-fluorophenyl)-1,2-oxazole-3-carboxamide (33s): white solid, 58%, Rf = 0.25
1
(CHCl3/MeOH 9:1), UHPLC-ESI-MS: Rt = 1.97, m/z = 267.2 [M + H]+. H-NMR (300 MHz, Acetone-d6)
δ
7.92–7.88 (m, 2H), 7.16 (t, J = 8.8 Hz, 2H), 6.76 (s, 1H), 4.95 (t, J = 5.4 Hz, 1H), 3.92–3.81 (m, 2H) ppm;
13C-NMR (100 MHz, Acetone-d6)
δ
177.6, 162.8, 159.8 (d, J = 133.8 Hz), 159.6, 136.3 (d, J = 2.7 Hz),
124.1 (d, J = 7.7 Hz), 117.2 (d, J = 22.6 Hz), 103.1, 69.5, 66.4 ppm.
5-(1,2-Dihydroxyethyl)-N-[(thiophen-2-yl)methyl]-1,2-oxazole-3-carboxamide (33t): white solid, 65%,
1
Rf = 0.34 (CHCl3/MeOH 9:1), UHPLC-ESI-MS: Rt = 1.79, m/z = 269.2 [M + H]+. H-NMR (300 MHz,
Acetone-d6)
δ 8.43 (s br, 0.5H), 7.32 (dd, J = 1.3 Hz, J = 5.1 Hz, 1H), 7.06 (dd, J = 1.1 Hz, J = 3.4 Hz, 1H),
6.95 (dd, J = 3.5 Hz, J = 5.1 Hz, 1H), 6.68 (s, 1H), 4.99 (s br, 0.5H), 4.90 (t, J = 5.1 Hz, 1H), 4.77–4.75 (m,
2H), 4.15 (s br, 0.5H), 3.84–3.81 (m, 2H), 2.87 (s br, 0.5H) ppm; 13C-NMR (100 MHz, Acetone-d6)
δ 177.3,
160.4, 143.6, 141.4, 128.5, 127.8, 126.8, 102.9, 69.6, 66.5, 39.3 ppm.
5-(1,2-Dihydroxyethyl)-N-[(pyridin-3-yl)methyl]-1,2-oxazole-3-carboxamide (33u): yellow oil, 37%, Rf = 0.33
1
(CHCl3/MeOH 9:1), UHPLC-ESI-MS: Rt = 0.38, m/z = 264.2 [M + H]+. H-NMR (300 MHz, MeOD)
δ
8.56 (s, 1H), 8.44 (s br, 1H), 7.86 (d, J = 7.9 Hz, 1H), 7.43 (dd, J = 4.9 Hz, J = 7.8 Hz, 1H), 6.70 (s, 1H),
4.84 (d, J = 5.8 Hz, 1H), 4.59 (s, 1H), 3.80 (dd, J = 3.3 Hz, J = 5.6 Hz, 2H) ppm; 13C-NMR (100 MHz,
MeOD) δ 176.6, 161.6, 159.6, 149.6, 149.0, 137.8, 136.5, 125.3, 102.1, 68.8, 65.6, 41.6 ppm.

Molecules 2018, 23, 2545
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5-(1,2-Dihydroxyethyl)-N-(2-methoxyethyl)-1,2-oxazole-3-carboxamide (33v): yellow oil, 78%, Rf = 0.38
1
(CHCl3/MeOH 9:1), UHPLC-ESI-MS: Rt = 1.23, m/z = 231.2 [M + H]+. H-NMR (300 MHz, Acetone-d6)
δ
6.64 (s, 1H), 4.90 (t, J = 5.4 Hz, 1H), 3.83 (dd, J = 3.6 Hz, J = 5.4 Hz, 2H), 3.58–3.50 (m, 4H), 3.32 (s, 3H),
2.85 (s br, 2H) ppm; 13C-NMR (100 MHz, Acetone-d6)
δ 177.3, 160.5, 147.7, 102.8, 72.4, 69.6, 66.5, 59.6,
40.5 ppm.
1-[3-(Pyrrolidine-1-carbonyl)-1,2-oxazol-5-yl]ethane-1,2-diol (33z): yellow oil, 79%, Rf = 0.30
1
(CHCl3/MeOH 9:1), UHPLC-ESI-MS: Rt = 1.51, m/z = 227.2 [M + H] +. H-NMR (300 MHz, Acetone-d6)
δ
6.58 (s, 1H), 4.90 (t, J = 5.3 Hz, 1H), 3.83 (t, J = 5.1 Hz, 2H), 3.76 (t, J = 6.6 Hz, 2H), 3.54 (t, J = 6.6 Hz,
2H), 2.84 (s br, 2H), 1.99–1.89 (m, 4H) ppm; 13C-NMR (100 MHz, Acetone-d6)
δ 175.8, 161.7, 149.6, 104.2,
69.6, 66.6, 50.0, 48.2, 27.8, 25.5 ppm.
3.5. Biology
All chemicals were purchased from Sigma (Hamburg, Germany) if not otherwise stated. (S)-DPD
was purchased from OMM Scientific (Dallas, TX, USA). The ATP Bioluminescence kit CLS II and
Kinase Glo Luminescence assay kit were respectively purchased from Roche Scientific (Manheim,
Germany) and Promega (Madison, WI, USA).
3.5.1. LsrK Overexpression and Purification
E. coli MET1158 (E. coli, amp resistance, BL21 (DE3) luxS-, with pMET1144 (lsrK-His in pET21b)),
kindly donated by Prof. Karina Xavier (Instituto Gulbenkian de Ciência, Portugal) [61], was used for
the overexpression of LsrK from S. typhimurium. The bacteria were grown overnight in 2
(yeast, tryptone, phosphate buffer and glucose) mediums supplemented with 100 g/mL ampicillin.
-D-1
×
YPTG
µ
At the exponential phase, protein expression was induced by the addition of 0.1 mM isopropyl
β
thiogalactopyranoside for 9 h at 22 ◦C (250 rpm). Cells were harvested and frozen overnight before
proceeding with lysis and purification, according to the literature [62].
3.5.2. DPD Activity Evaluation
Phosphorylation of DPD by LsrK was evaluated with a bioluminescence-based assay,
ATP Bioluminescence kit CLSII (Roche) as previously described in Reference [61]. DPD was plated
at 200
buffer (25 mM triethanolamine, pH 7.4, 200
for comparison at 200 M. The level of ATP was monitored by the ATP Bioluminescence kit CLSII
µM and 400
µM and a reaction mixture containing 200 nM Lsrk and 20
µM ATP in assay
µM MgCl2). Commercially available DPD was tested
µ
following the manufacturer’s instructions. The experiment was performed in the kinetic-mode,
monitoring the luminescence every 2 min within a time window of 30 min at the Varioskan LUX plate
reader (Thermo Fisher Scientific, Vantaa, Finland).
3.5.3. Screening of DPD-Related Compounds
The activity of DPD-related compounds was evaluated in an LsrK inhibition assay. Compounds
were plated in a 384 well-plate to a final concentration of 200
300 M DPD diluted in an assay buffer (25 mM triethanolamine, pH 7.4, 200
BSA) were added to the plate followed by 100 M ATP to start the reaction. After 15 min of reaction,
µM in triplicate. A 300 nM LsrK and
µ
µM MgCl2, 0.1 mg/mL
µ
the Kinase Glo Luminescence assay reagent was added according to the manufacturer’s instructions.
The experiment was carried on in end-point mode and the luminescence was recorded at the Varioskan
LUX plate reader.
4. Conclusions
Resistance to antibiotics poses a continuous threat to public health. In the last few decades,
receptors able to modulate QS started to be considered interesting targets for anti-infective therapy
and the modulation/inhibition of QS has become an appealing strategy against bacterial resistance.

Molecules 2018, 23, 2545
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Several studies have already shown that interference with QS affects biofilm formation and biofilm
properties (e.g., thickness, mass). Particularly, DPD, the key compound in the biosynthesis of AI-2,
is able to modulate QS in both Gram-negative and Gram-positive bacteria. Accordingly, DPD-analogs
may have great potential as QSI and, therefore, as antimicrobial drugs. Of note, two different
DPD-related compounds (i.e. isobutyl-DPD and phenyl-DPD) in combination with gentamicin have
almost completely cleared the pre-existing biofilms in E. coli and P. aeruginosa, respectively [63].
In this work, we successfully developed a new short and robust strategy for the synthesis of DPD
which requires only one purification step. Ph-DPD was also synthesized to show the applicability of
our protocol to the production of different C1-DPD analogs. The new strategy inspired the synthesis
of 30 novel DPD-related compounds: the cycloaddition to two common precursors was employed
to produce (in maximum four steps) four different small libraries where the diketo moiety of DPD
was embedded in heteroaromatic rings. All the designed compounds were purified and characterized
by 1H-NMR, 13C-NMR, and UHPLC-MS (purity > 90%). It is worth noting that in these compounds
the open/closed equilibrium (typical of the majority of the DPD-analogs reported so far, Figure 1) is
not possible. The so-obtained more stable compounds were easily purify by column chromatography.
Moreover, the presence of heteroaromatic groups increases the UV absorbance and MW, rendering the
compound detection by the classical analytical method (e.g., LC-MS) easier compared to previously
reported analogs (e.g., ethyl-DPD).
Our new synthetic approach allowed us to synthetize a small set of racemic DPD-related
compounds in a relatively easy and fast way. We demonstrated that racemic DPD is efficiently
phosphorylated by LsrK, corroborating the validity of our approach. On the other hand, all compounds
of our library of DPD-related did not show any activity on LsrK. Nevertheless, the synthetic procedure
herein proposed might lead to the preparation of a wider compound library, thus, allowing for the
discovery of a new class of LsrK inhibitors as potential antivirulence agents. Moreover, we decided
to add these products to the library of MuTaLig, an innovative ligand identification platform for the
drug-discovery process.
Supplementary Materials: Supplementary materials are available online.
Author Contributions: Conceptualization, S.S., F.G. and S.C.; Methodology, S.S., F.G.; Formal Analysis, F.M., M.P.;
Investigation, S.S., V.G., M.P., F.G., S.C., P.T.; Resources, D.T., P.T.; Writing-Original Draft Preparation, S.S., V.G.,
P.T.; Writing-Review & Editing, S.S., F.G., P.T., S.C.; Supervision, S.C.; Project Administration, A.K., D.T.; Funding
Acquisition, F.G., D.T.
Funding: This research was funded by the European Union’s Horizon 2020 research and innovation programme
for funding INTEGRATE under the Marie Sklodowska Curie grant agreement N◦. 642620 and for the Marie
Skłodowska-Curie Actions Individual Fellowship to SS.
Acknowledgments: Martyna Bielska, Xenia Iwanova, Virginia Llemos and Eduard Ackerman are acknowledged
for technical assistance.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are all available from the authors.
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