Structure–Activity Relationships of 2-Sufonylpyrimidines as Quorum-Sensing Inhibitors to Tackle Biofilm Formation and eDNA Release of Pseudomonas aeruginosa

Article abstract of DOI:10.1002/cmdc.201600419

Drug-resistant Pseudomonas aeruginosa (PA) strains are on the rise, making treatment with current antibiotics ineffective. Hence, circumventing resistance or restoring the activity of antibiotics by novel approaches is of high demand. Targeting the Pseudomonas quinolone signal quorum sensing (PQS-QS) system is an intriguing strategy to abolish PA pathogenicity without affecting the viability of the pathogen. Herein we report the structure–activity relationships of 2-sulfonylpyrimidines, which were previously identified as dual-target inhibitors of the PQS receptor PqsR and the PQS synthase PqsD. The SAR elucidation was guided by a combined approach using ligand efficiency and ligand lipophilicity efficiency to select the most promising compounds. In addition, the most effective inhibitors were rationally modified by the guidance of QSAR using Hansch analyses. Finally, these inhibitors showed the capacity to decrease biofilm mass and extracellular DNA, which are important determinants for antibiotic resistance.

Full text of DOI:10.1002/cmdc.201600419

DOI: 10.1002/cmdc.201600419
Full Papers
Structure–Activity Relationships of 2-Sufonylpyrimidines
as Quorum-Sensing Inhibitors to Tackle Biofilm Formation
and eDNA Release of Pseudomonas aeruginosa
Andreas Thomann,[a] Christian Brengel,[a] Carsten Bçrger,[b] Dagmar Kail,[b] Anke Steinbach,[a]
Martin Empting,*[a] and Rolf W. Hartmann*[a, c]
Drug-resistant Pseudomonas aeruginosa (PA) strains are on the
rise, making treatment with current antibiotics ineffective.
Hence, circumventing resistance or restoring the activity of an-
tibiotics by novel approaches is of high demand. Targeting the
Pseudomonas quinolone signal quorum sensing (PQS-QS)
system is an intriguing strategy to abolish PA pathogenicity
without affecting the viability of the pathogen. Herein we
report the structure–activity relationships of 2-sulfonylpyrimi-
dines, which were previously identified as dual-target inhibitors
of the PQS receptor PqsR and the PQS synthase PqsD. The SAR
elucidation was guided by a combined approach using ligand
efficiency and ligand lipophilicity efficiency to select the most
promising compounds. In addition, the most effective inhibi-
tors were rationally modified by the guidance of QSAR using
Hansch analyses. Finally, these inhibitors showed the capacity
to decrease biofilm mass and extracellular DNA, which are im-
portant determinants for antibiotic resistance.
Introduction
Bacterial resistance is one of the major challenges in drug dis-
covery today, as increasingly more pathogens develop strat-
egies to evade therapy and immune responses. Recently, the
most challenging bacteria have been referred to as the ESKAPE
pathogens: Enterococcus faecium, Staphylococcus aureus, Kleb-
siella pneumoniae, Acinetobacter baumanii, Pseudomonas aeru-
ginosa, and Enterobacter spp.[1] They can “escape” antibiotic
treatment through intrinsic tolerance and/or acquired multi-
drug resistance. Therefore, novel drug targets and new modes
of action need to be discovered to provide a basis for the ra-
tional development of novel anti-infective agents.
gen is able to embed itself into a heterogeneous hydrogel-like
structure known as a biofilm.[8] This matrix consists of various
components such as extracellular DNA (eDNA), polysaccha-
rides, and proteins which have been shown to be important
concealing the pathogen from host defense mechanisms[9] and
antibiotics.[10] In particular, eDNA has recently been discussed
as one major factor that prevents the antibiotic activity of ami-
noglycosides[11] and fluoroquinolones,[12,13] which are the first
choice to be combined with b-lactam antibiotics in the treat-
ment of PA infections. Moreover, eDNA is heavily involved in
resistance against antimicrobial peptides of the host’s innate
immune system.[13,14]
Pseudomonas aeruginosa (PA) is a ubiquitously present
Gram-negative opportunistic bacterium that predominantly in-
fects immunocompromised patients suffering from cystic fibro-
sis,[3] burn wounds[4] or HIV.[5] To establish an infection, PA has
developed various virulence factors that damage epithelial
cells[6] and impair the immune system.[7] In addition, the patho-
Therefore, agents that decrease biofilm and eDNA hold
great potential not only to restore antibiotic efficacy, but also
to enable the human immune system to clear the infection.
Biofilm formation and eDNA release are both controlled by
cell-density-dependent communication systems in PA.[17] This
inter-bacterial signaling network uses small molecules to sense
the presence of other bacteria and is referred to as quorum
sensing (QS). Importantly, interference with QS does not affect
the viability of PA.[18] Hence, the disturbance of QS is a new ap-
proach, which has been shown to decrease pathogenicity
in vivo with a lower potential for resistance development by
circumventing selection pressure.[19] One PA-specific QS signal
is the Pseudomonas quinolone signal (PQS), which is a 2-alkyl-
quinolone that regulates the production of virulence factors
and biofilms.[17,20] Addressing the PQS-QS system has the
advantage of not interfering with the QS systems of other
bacteria (e.g., targeting the widespread las and rhl systems)[21]
that are essential to human health (i.e., the gut microbiome);
therefore, such a strategy should minimize potential side ef-
fects.
[a] A. Thomann, C. Brengel, Dr. A. Steinbach, Dr. M. Empting,
Prof. Dr. R. W. Hartmann
Helmholtz Institute for Pharmaceutical Research Saarland,
Department of Drug Design and Optimization,
Campus E8.1, 66123 Saarbrꢀcken (Germany)
E-mail: rolf.hartmann@helmholtz-hzi.de
martin.empting@helmholtz-hzi.de
[b] Dr. C. Bçrger, Dr. D. Kail
PharmBioTec GmbH, Science Park 1, 66123 Saarbrꢀcken (Germany)
[c] Prof. Dr. R. W. Hartmann
Saarland University,
Department of Pharmacy, Pharmaceutical and Medicinal Chemistry
Campus C2.3, 66123 Saarbrꢀcken (Germany)
Supporting information and the ORCID identification number(s) for the
author(s) of this article can be found under http://dx.doi.org/10.1002/
cmdc.201600419.
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PQS is produced by the synthases of the pqsA–E operon and
PqsH.[22] After the concentration of PQS reaches a certain
threshold, the transcriptional regulator PqsR, also referred to as
MvfR, is activated and induces the production of virulence fac-
tors like pyocyanin[23] and biofilm[24] formation, and drives for-
ward the expression of the pqsA–E operon in a positive feed-
back auto-loop mechanism (Figure 1).[23,25] Moreover, PQS-defi-
cient mutants were shown to have strongly decreased patho-
genicity in nematode models.[26] These biological studies pro-
vided the basis for drug discovery campaigns targeting the
synthases of PQS[27–29] and its receptor.[18,30,31] The first discov-
ered inhibitors showed anti-virulence as well as anti-biofilm ac-
tivity and were effective in vivo.[18,28,31]
but also showed pronounced activity against biofilm formation
and eDNA release in cellular assays using Pseudomonas aerugi-
nosa PA14. Furthermore, we found that interference with PQS-
QS and eDNA release increased the activity of ciprofloxacin
under biofilm conditions. Additionally, we could decrease the
pathogenicity of PA in an in vivo model by treating Galleria
mellonella larvae with compound 1. These promising results
encouraged us to further modify the structure of 1 toward
higher activity for its respective targets.
Herein we report the exploration of the chemical space of
compounds 1 and 2, as well as the quantitative structure–ac-
tivity relationship (QSAR) through a Hansch analysis.[32] Inspired
by in silico flexible alignments with a substrate of PqsD (b-ke-
todecanoic acid)[33] and the natural ligand of PqsR (HHQ)[25]
compounds were designed under the guidance of ligand effi-
ciency (LE) and ligand lipophilicity efficiency Astex (LLEAT)
scores.[34] The latter metric is exceptionally useful during the
drug development process, as it not only evaluates compound
activities based on molecular weight, but also on lipophilicity.
Hence, this guidepost helps medicinal chemists circumvent the
pitfall of an activity increase being based solely on a gain in
hydrophobicity.[35] A combination of both metrics and rational
design strategies led to the discovery of potent, ligand-effi-
cient and drug-like inhibitors of biofilm formation and eDNA
release.
Results and Discussion
Figure 1. Schematic representation of the Pseudomonas quinolone signal
quorum sensing system (PQS-QS). Interference with the PQS/HHQ receptor
PqsR and a synthase (PqsD), critical for PQS and HHQ production, leads to
more highly efficient attenuation of PA pathogenicity than single-target ap-
proaches.[2] AA=anthranilic acid, 2-ABACoA=2-aminobenzoyl acetate coen-
zyme A, HHQ=2-heptyl-4-hydroxyquinoline, PQS=Pseudomonas quinolone
signal.
The synthesis of compounds listed in Table 1 was carried out
by either SNAr, cycloaddition of sodium azide, or Suzuki cou-
pling and subsequent oxidation of the thioether. Triazole com-
pounds listed in Table 2 were accessed by standard copper(I)-
mediated azide–alkyne click reactions followed by oxidation.
Tetrazole derivatives 23–26 could be obtained by a micro-
wave-assisted SNAr method coupled with an oxidation step.
Dual inhibitors in Table 4 below were synthesized by replacing
the sulfomethyl group of 1 or 2 by the corresponding thiol re-
actant and subsequent treatment with Oxoneꢁ (Scheme 1).
Using the initial hits 1 and 2 as starting points, we wanted
to further improve the activity of the compounds by structural
modifications. In a first step, we explored the chemical space
We recently published a study on the rational discovery of
the first dual-active compounds that simultaneously target
PQS synthase (PqsD) and PqsR.[2] The most effective com-
pounds from this study, 1 and 2, were found to be active
against virulence factor production (pyocyanin, pyoverdine),
Table 1. Inhibition of PqsD and PqsR by compounds 1–9 and calculated LE and LLEAT for the respective target.
PqsD
PqsR
Compd
R1
IC50 [mm]
LE/LLEAT [kcal/HA]
IC50 [mm]
LE/LLEAT [kcal/HA]
1[2]
2[2]
3
4
5
6
7
8
9
tetrazol-1-yl
1,2,3-triazol-1-yl
tetrazol-5-yl
1,2,4-triazol-1-yl
pyrazol-1-yl
imidazol-1-yl
thiophen-3-yl
benzimidazol-1-yl
benzotriazol-1-yl
21ꢀ2
0.44/0.66
0.43/0.58
15ꢀ3
25ꢀ8
0.45/0.67
0.43/0.58
–
0.43/0.61
0.35/0.45
0.38/0.52
–
23ꢀ1
>50; 35%ꢀ15
>25; 21%ꢀ8
>50; 41%ꢀ3
50ꢀ7
–
–
–
>200; 11%ꢀ9
25ꢀ3
175ꢀ39
0.40/0.54
–
0.33/0.39
0.35/0.41
75ꢀ26
>50; 3%ꢀ1
33ꢀ6
>200; 33%ꢀ0
146ꢀ34
0.28/0.34
0.29/0.35
17ꢀ4
107ꢀ25
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Scheme 1. Synthesis of compounds 1–44: a) 1a, N-heterocycle, TEA, DMF, microwave;[15] b) thioether, Oxoneꢁ or mCPBA, EtOAc/H2O or CH2Cl2; c) 1 or 2, thiol,
DMF, ꢁ208C; d) 1a, NaN3, DMF;[15] e) 10b, tBuOH/H2O, CuSO4, sodium ascorbate;[2] f) 1a, TMS-acetylene, TEA, CuI, (PPh3)2PdCl2, 608C then TBAF/THF; g) 22b,
benzylazide, tBuOH/H2O, CuSO4, sodium ascorbate; h) 23a, 1H-tetrazole, TEA, DMF, microwave;[16] i) 23b, TEA, DMF, microwave;[16] j) 1a, HI then CuCN, pyri-
dine, reflux; k) 3b, NaN3, NH4Cl, DMF, 808C; l) 24a, (AcO)2O, reflux; m) 1a, TMS-Br, MeCN, 408C; n) 7a, H2O2, ammonium molybdate tetrahydrate, EtOH, 08C;
o) 7b, Pd2dba3, PCy3, K3PO4, 3-thiopheneboronic acid, dioxane/H2O, 1008C.
at the 4-position of pyrimidine and introduced several hetero-
cycles (Scheme 1, Table 1). Regarding tetrazole substitution,
compound 3, a regioisomer of 1, was only weakly active on
PqsD and almost inactive on PqsR. This might arise either from
the acidic character of the tetrazole, resulting in a negative
charge (when deprotonated) or from the hydrogen bond
donor properties (when protonated). These presumably un-
compensated hydrogen bonds and/or ionic interactions might
therefore be the root cause for the activity decrease due to de-
solvation penalties.[36,37] With regard to other pentacycles (4–6)
of the subset, azoles lacking a nitrogen atom at the 3-position
(4 and 5) were less active on both targets than compounds
1 and 2, while only 4 was equally active against PqsR. Interest-
ingly, when switching from azoles to thiophene (7), activity
was strongly impaired on both targets. In a next step, we en-
larged the azole motif and introduced benzimidazole 8 and
benzotriazole 9. In comparison with 1, we observed a loss of
activity on PqsR for both compounds, whereas activity on
PqsD could be maintained.
tion penalties.[36,37] Thus, for physicochemical reasons we kept
the hydroxy group and varied substituents at the methylene
unit. Enlargement of cyclopentyl (10) to cyclohexyl (11) result-
ed in a decrease of activity on PqsR without any beneficial
effect on PqsD. Introduction of phenyl (12) increased activity
on PqsD but was not very well tolerated by PqsR. Thus, we de-
cided to decrease the substituent size toward an unsubstituted
hydroxy (13) and dimethyl-substituted derivative (14). Regard-
ing PqsD, we could observe no restoration of activity upon de-
creasing size while keeping the hydroxy functionality in place
(compare 2 with 10–14). For PqsR we maintained activity rela-
tive to 2. Next, the hydroxy group was exchanged for a methyl
function (15), yielding a compound similar in size to 14 but
lacking the issue of desolvation penalties (see above). The de-
creased activity of 15 on PqsR revealed the hydroxy group to
be beneficial over methyl. For PqsD, a slight increase in activity
(compare 14 and 15) was observed, implying that the hydro-
philicity might be more detrimental for activity than steric
demand. From this series we concluded that the hydroxy func-
tion does not impair activity on PqsR, but improves LLEAT due
to its hydrophilicity (compare 2 and 13). Although we could
not increase activity on both targets relative to 2, knowledge
of the hydroxy group being in principal tolerated by PqsR
could be valuable information for later-stage drug develop-
ment if solubility or lipophilicity issues might arise. To further
explore the applicability of a fragment-growing strategy start-
ing at the 4-position of the triazole of 2, we broadened the
scope of substituents and introduced cyclopropyl (16) and
phenyl groups (17). Although 16 did not show increased activi-
ty on both targets, 17 was about five times more active than
its parent compound 2 regarding inhibition of PqsD. Hence, 17
was the most promising hit from this series, with ligand effi-
Analysis of ligand efficiency and ligand lipophilicity efficiency
of this subset (LE and LLEAT) still rendered compounds 1 and 2
to be the most promising scaffolds. Consequently, for all sub-
sequent structure modifications, either triazole 2 or tetrazole
1 served as the core structure. Therefore, we introduced sub-
stituents at the 4-position of the triazolyl substituent of 2
(Table 2). Compound 10, which we recently reported as a pure
PqsR antagonist, is almost equally active on PqsR as 2.[2] Re-
garding its structure and activity, the hydroxy functionality of
10 does not seem to be beneficial for antagonism on PqsR,
but is at least tolerated. This observation is quite unusual, as
the introduction of non-interacting hydrophilic groups (e.g.,
hydroxy, amino) can lead to decreased activity due to desolva-
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Table 2. Inhibition of PqsD and PqsR by compounds 10–22 and calculated LE and LLEAT for the respective target.
PqsD
PqsR
Compd
R2
IC50 [mm]
LE/LLEAT [kcal/HA]
–
IC50 [mm]
LE/LLEAT [kcal/HA]
0.30/0.39
10[2]
>50; 12%ꢀ2
26ꢀ9
11
>50; 14%ꢀ4
–
~50; 53%ꢀ12
0.27/0.34
0.27/0.37
12
13
~50; 52%ꢀ1
>50; 24%ꢀ5
0.27/0.37
–
<50; 74%ꢀ16
22ꢀ4
0.38/0.62
0.34/0.51
14
15
16
>50; 15%ꢀ3
>50; 44%ꢀ2
29ꢀ5
–
–
27ꢀ12
>50; 40%ꢀ3
25ꢀ8
0.35/0.44
0.36/0.44
–
17
18
19
20
4ꢀ0.3
0.36/0.41
<200; 66%ꢀ8
>10; 18%ꢀ10
>10; 21%ꢀ6
>10; 30%ꢀ13
>10; 35%ꢀ8
>10; 33%ꢀ3
>10; 41%ꢀ1
–
–
–
–
–
–
21
>25; 37%ꢀ0
>50; 8%ꢀ7
–
–
>50; 0%ꢀ0
>50; 22%ꢀ3
–
–
ciencies in the preferable range of 0.3 kcal per heavy atom
(HA) regarding activity on PqsD.[34] Consequently, we substitut-
ed the phenyl ring with a methyl group at the 3- and 4-posi-
tions to further explore the size of the binding pocket. Besides
the dramatic decrease in solubility (Supporting Information),
from 200 mm determined for 17 to 10 mm for 18 and 19, the
potency was also decreased on PqsD. Additionally, only weak
antagonism of PqsR was found for these compounds. Hence,
we decided to introduce less hydrophobic substituents. The in-
troduction of fluorine into the 4-position of phenyl (20) de-
creased solubility without increasing activity. For the methoxy
substituent (21), we found a less dramatic loss in solubility
(50 mm) but lower potency than 17 on both targets. To investi-
gate whether a more flexible moiety increases solubility and
activity, we introduced a benzyl substituent. As copper(I)-medi-
ated cycloaddition of benzylacetylene and 10b (Scheme 1)
was not successful, we decided to bioisosterically replace the
1,2,3-triazol-1-yl with 1,2,3-triazol-5-yl regarding the bonding
to the 4-position of pyrimidine (22). Unfortunately, activity
dropped toward PqsD and PqsR as well as solubility. Regarding
activity and solubility, we conclude that an introduction of this
subset of functional groups is not beneficial for the develop-
ment of an efficient drug-like compound. Thus, further modifi-
cations of the structure of 1 were carried out at the 6-position
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Table 3. Inhibition of PqsD and PqsR by compounds 23–26 and calculated LE and LLEAT for the respective target.
PqsD
PqsR
Compd
R3
IC50 [mm]
LE/LLEAT [kcal/HA]
–
IC50 [mm]
LE/LLEAT [kcal/HA]
–
23
>50; 0%ꢀ0
>50; 23%ꢀ6
~50; 49%ꢀ4
24
~50; 50%ꢀ11
0.32/0.51
0.32/0.51
25
26
>50; 36%ꢀ8
3.2ꢀ0.0
–
>50; 27%ꢀ4
~50; 48%ꢀ0
–
0.35/0.43
0.27/0.36
of pyrimidine. We chose four substituents differing in size and
functionality to be introduced (Table 3). For N-methylamine
(23) we found an almost complete loss of activity on both tar-
gets. The same was true for acetylamide (24). The enlarged
flexible hydrophobic residue benzylamine (25) was also neither
tolerated by PqsR nor PqsD. Introduction of phenoxy to the 6-
position of pyrimidine (26) lead to an increase in potency on
PqsD by about fourfold relative to 1 with an IC50 value of
3.2 mm, but for PqsR a decrease was observed. Regarding
ligand efficiencies, 26 is below the minimum LE score of 0.3
for PqsR, rendering this compound a suboptimal choice for fur-
ther optimization. To derive a rational basis for further modifi-
cations to the structure of 2 we conducted flexible alignment
experiments of 2 with the described substrate (b-ketodecanoic
acid) of PqsD and the natural ligand (HHQ) of PqsR (Figure 2).
In detail, the (1,2,3-triazol-1-yl)pyrimidine part of 2 matches the
quinolone core of HHQ, and the pyrimidine N1 atom of 2 over-
laps with N1 of PqsR’s ligand HHQ. For b-ketodecanoic acid we
observed a very good match of both hydrogen bond acceptor
features of the carboxylic acid moiety with N1 and N2 of the
pyrimidine of 2. Furthermore, the ketone in the b-position of
PqsD’s substrate overlaps perfectly with an oxygen atom of
the sulfone group of 2. Thus, both alignments suggested the
sulfomethyl group to be an ideal starting point for the intro-
duction of an enlarged alkyl chain being potentially beneficial
for activity on both targets. To investigate the steric dimen-
sions of the pocket we synthesized compounds bearing iso-
propyl (27) and tert-butyl (28) at R4 (Table 4). For these com-
pounds a loss in activity was observed on both targets. These
results shed light on the architecture of the binding sites of
PqsD and PqsR, as bulky substituents at R4 are not tolerated.
Thus, we decided to synthesize unbranched derivatives with
chain lengths ranging from 1 to 7 carbons (2, 29–34). Notably,
we achieved an increase in activity regarding PqsD inhibition
when increasing chain length combined only slight losses in
PqsR antagonism. Interestingly, a pronounced potency increase
on PqsD was observed at a minimum chain length of 4 (com-
Figure 2. Best-scored flexible alignment (MOE 2014.09) of HHQ (green car-
bons, B) and 2 (grey carbons, B). Based on these results, an alignment of
PqsD’s artificial substrate b-ketodecanoic acid (green carbons, A) and 2 (grey
carbons, A) was obtained, resulting in a related arrangement, providing
a starting point for ligand-based optimization.
pare 29–31 with 2), but activity leveled out at IC50 =1.7 mm re-
garding hexyl (33) and heptyl (34). To investigate the relation-
ship of the length of the alkyl chain and activity on PqsD we
correlated both parameters and retrieved a Hansch equation
of sigmoidal shape (Figure 3B). The function showed a very
high correlation of experimental versus calculated pIC50 values
(r=0.9984, Figure 3C).
Regarding this correlation, the hexyl chain seems to be of
ideal length to fill the hydrophobic channel possibly occupied
by PqsD’s substrate b-ketodecanoic acid being in good agree-
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Table 4. Inhibition of PqsD and PqsR by compounds 27–44 and calculated LE and LLEAT for the respective target.
PqsD
PqsR
Compd
R4
X
IC50 [mm]
LE/LLEAT [kcal/HA]
IC50 [mm]
LE/LLEAT [kcal/HA]
27
28
29
30
31
32
33
34[2]
iPr
tBu
Et
nPr
nBu
nPent
nHex
nHept
CH
CH
CH
CH
CH
CH
CH
CH
>50; 23%ꢀ7
>50; 27%ꢀ0
26ꢀ1
–
–
~50; 49%ꢀ13
>50; 24%ꢀ10
14ꢀ2
0.35/0.49
–
0.42/0.54
0.39/0.47
0.35/0.40
–
0.40/0.52
0.39/0.47
0.39/0.43
0.41/0.41
0.40/0.38
0.38/0.34
19ꢀ3
19ꢀ5
9.9ꢀ1.5
30ꢀ15
2.9ꢀ0.5
>50; 31%ꢀ0
~50; 47%ꢀ22
>50; 17%ꢀ2
1.7ꢀ0.0
0.30/0.28
–
1.7ꢀ0.4
35
CH
14ꢀ1
0.34/0.47
18ꢀ7
0.33/0.46
36
37
38
39
40
41
42
43
44
Ph
Ph
4-Cl-Ph
4-OMe-Ph
4-F-Ph
3-F-Ph
3-Cl-Ph
3-OMe-Ph
3,4-di-Cl-Ph
CH
N
N
N
N
N
N
N
N
1.8ꢀ0.1
1.7ꢀ0.2
0.9ꢀ0.1
2.5ꢀ0.6
2.0ꢀ0.3
0.7ꢀ0.1
0.6ꢀ0.1
1.6ꢀ0.0
0.4ꢀ0.1
0.40/0.44
0.40/0.49
0.40/0.44
0.36/0.44
0.38/0.47
0.41/0.49
0.41/0.45
0.37/0.44
0.41/0.41
30ꢀ17
16ꢀ5
0.32/0.35
0.34/0.42
0.29/0.36
–
0.31/0.40
0.31/0.38
0.32/0.35
–
<50; 74%ꢀ3
>50; 42%ꢀ31
21ꢀ12
26ꢀ5
17ꢀ5
>50; 43%ꢀ27
24ꢀ5
0.29/0.30
ment with the flexible alignment (Figure 2A). The IC50 toward
PqsR was ~50 mm for 33, but only residual activity was found
when heptyl was introduced (34). Interestingly, relationships
between alkyl chain length and activity were also observed for
PQS and HHQ derivatives published by Hodgkinson, Lu, and
Ilangovan corroborating our findings.[31,38]
Regarding ligand efficiency metrics, compound 33 was still
above the suggested score of 0.3 on both targets, with the
LLEAT value for PqsR being the exception (0.28). Thus, we intro-
duced an ester functionality into the alkyl chain to achieve
a more hydrophilic compound, thereby increasing LLEAT (35).
Interestingly, activity was restored on PqsR, but was impaired
on PqsD. With regard to lipophilicity, represented by the LLEAT
score, we could improve the compound’s properties, making it
more efficient than its alkyl congener (compare 32 and 35).
In a next step, we investigated whether aromatic substitu-
ents are tolerated at R4. Thus, we introduced phenyl to 1 and
2 at R4. The resulting compounds (36 and 37) showed in-
creased activity on PqsD and no loss of activity on PqsR. Inter-
estingly, 37 had a better performance than 36 regarding activi-
ty on PqsR and LLEAT. Hence, we chose 37 for further modifica-
tions at the phenyl ring. To rationalize the choice of substitu-
ents to be introduced, we followed the Topliss scheme and in-
troduced methoxy and chlorine to the 4-position.[39] Activity
increased for the introduction of chlorine (38) and dropped for
methoxy (39) regarding PqsD, highlighting the importance of
electronic properties for potency. To investigate this relation-
ship, we synthesized compounds 40–43. For these compounds
a general trend could be observed: electron-withdrawing
groups (EWGs) increase potency, while electron-donating
groups (EDGs) result in decreased potency. Thus, we synthe-
Figure 3. Hansch analysis of alkyl-substituted compounds 2 and 29–34 (A)
covering chain lengths from 1 to 7 carbon atoms. Carbon atom count of the
alkyl chain and the biological activity on PqsD showed a very high degree of
correlation, resulting in a sigmoidal shape function (B), which was also true
for the plot of calculated versus experimentally determined pIC50 values (C).
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sized 44 bearing two chlorine atoms at positions 2 and 4 of
the sulfophenyl substituent. Corroborating our hypothesis, we
achieved higher potency on PqsD with IC50 =0.4 mm. To further
rationalize our observation we set up a Hansch equation corre-
lating s Hammet parameters[40] of the substituents (Figure 4B)
with the corresponding biological activities (Figure 4C). The re-
sulting equation showed very good correlation between the
parameter and the biological activity; moreover, the predicta-
bility of the model could be verified by plotting calculated
versus observed pIC50 values (r=0.9574, Figure 4D). The plot
of pIC50 versus lipophilic parameters p and cLogP showed
a lower degree of correlation (r=0.8416 and 0.8348), rendering
the electronic parameter to be the variable of choice for QSAR
(Supporting Information).
Compound 44, as well as most derivatives from this subset,
are almost as active on PqsR as 1 (Table 3). Notably, LE/LLEAT
metrics calculated for compound 44 are in the desirable range
for both targets. The PQS-QS is largely involved in the regula-
tion of biofilm formation and eDNA release, which are two
highly relevant biological determinants for PA resistance to an-
tibiotics and immune response. To test whether our com-
pounds are able to decrease the production of biofilm and
eDNA, we conducted experiments using Pseudomonas aerugi-
nosa PA14.[41] Storz et al. have shown that the relationship be-
tween in vitro potency and in cellulo effectivity is highly de-
pendent on structural modifications and cannot be easily ex-
plained by focusing only on physicochemical properties.[27]
Thus, we chose compounds 33 and 44, both with promising
activity in vitro, but reflecting a larger chemical space and
tested them at a concentration of 100 mm for their ability to
decrease biofilm formation and eDNA release by PA14. Indeed,
both compounds significantly decreased biofilm volume and
eDNA production (Figure 5) without impairing bacterial
Figure 5. Effect of compounds 1, 33, and 44 at 100 mm on Pseudomonas aer-
uginosa PA14 biofilm formation (Biofilm) and release of extracellular DNA
(eDNA) versus 1% DMSO solvent control, which was set to 100%.
growth. Although less potent at the targets, compound 33
was slightly more efficient in decreasing biofilm and eDNA
than 44. In addition, 33 showed similar inhibition of the two
pathogenicity traits as compound 1, suggesting that the im-
proved PqsD efficacy compensates the losses of PqsR antago-
nism (Figure 5). One possible explanation might be differences
in permeation through the Gram-negative cell envelope or in-
teraction with the biofilm matrix. Therefore, the concentration
of drug in the media might be higher than inside the cell, re-
sulting in lower amounts of drug at the site of action. To cor-
roborate these findings, we investigated whether 44 can inhib-
it pyocyanin production (see Supporting Information), a PQS-
dependent virulence factor. Indeed, compound 44 showed
lower efficacy (14% at 100 mm) than compound 1 (IC50 =
86 mm) in a cellular assay which supports our latter hypothesis.
Figure 4. Hansch analysis of compounds 37–44 (A) substituted with
a phenyl group functionalized with EWGs and EDGs (B). The Hammet param-
eter and biological activity versus PqsD showed a very high degree of corre-
lation, resulting in a linear function (C). Very good predictivity of the model
was found by correlating calculated versus experimentally determined pIC50
values (D).
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tion pIC50-derived LLEAT values by Equations (9)–(11), published by
Thomann et al.[35,43]
Conclusions
In this study, we report the ligand-based design and structural
modification of dual-inhibitor compounds targeting two key
players in the Pseudomonas aeruginosa virulence and biofilm
machinery, namely PqsD and PqsR. Based on flexible alignment
approaches and subsequent Hansch analyses we were able to
discover potent and ligand-efficient PqsD inhibitors while
keeping activity on the secondary target PqsR. The two investi-
gated candidates from these approaches displayed a strong
decrease in biofilm mass and eDNA release, two major causes
of antibiotic treatment failure, as well as reduced host immune
clearance. However, increasing activity on both targets at the
same time is a difficult task. Nevertheless, we were successful
in identifying hotspots at the ligands’ structure, facilitating op-
timization toward one target (PqsD) while keeping activity on
the second (PqsR). In general, polypharmacology is an intrigu-
ing approach to combat bacterial strains highly resistant to ex-
isting antibiotics, as multitarget drugs lower the potential of
drug–drug interactions relative to combination strategies.
Therefore, the compounds reported herein are promising can-
didates for further in vivo studies using, for example, an acute
arthropod infection model.
Synthesis and analytical characterization. NMR spectra were re-
corded on a Bruker Avance AV300 or a Bruker DRX500. The residual
1H or 13C resonances of the >99% deuterated solvents were used
1
for internal reference of all spectra acquired (CDCl3: H 7.260 ppm,
13C 77.16 ppm; [D6]DMSO: 1H 2.500 ppm, 13C 39.52 ppm). Electro-
spray ionization (ESI) mass spectrometric data were recorded
with either a Surveyor LC system MSQ electrospray mass spectrom-
eter LC–MS couple (ThermoFisher, Dreieich, Germany) using an
MeCN/H2O gradient in positive mode (+), if not otherwise indicat-
ed 0.1% TFA was added if necessary, or by a Waters instrument
containing a 2767 sample manager, a 2545 binary gradient pump,
a 2998 PDA detector, and a 3100 electrospray mass spectrometer
by use of an MeCN/H2O gradient in positive mode (+); if not indi-
cated otherwise 0.1% formic acid was added if necessary. Purity of
final compounds was determined by the UV254 trace of the LC
chromatogram. Compounds 1, 2, 10, 34, 10a, and 23a were syn-
thesized as previously described.[2,16,43] Analytical and experimental
details of intermediates can be found in the Supporting Informa-
tion.
General procedure (b) for the oxidation of thioethers. Thioether
(1 equiv) was dissolved in EtOAc and an aqueous solution of
Oxoneꢁ (3 equiv) was added. The biphasic mixture was stirred vig-
orously at room temperature until TLC showed completion. The
final products were purified as indicated.
2-(methylsulfonyl)-4-(1H-tetrazol-5-yl)pyrimidine (3). Compound
1a (1 equiv) was suspended in 28% hydriodic acid and stirred at
room temperature for 16 h. The suspension was extracted using
CH2Cl2, washed with sat. NaHCO3(aq) and dried over Na2SO4. Solvent
was removed under reduced pressure to give 3a as a yellow oil
(yield 98%). The crude product was dissolved in pyridine and cop-
per(I) cyanide was added (1.1 equiv). The mixture was held at
reflux for 4 h and then acidified with 2m HCl. The aqueous layer
was extracted with EtOAc. The organic layer was dried over
Na2SO4, filtered over a pad of silica and solvent was removed
under reduced pressure to give 3b as a brown oil (yield 99%). 3b
(1 equiv), NaN3 (1.4 equiv), NH4Cl (1.4 equiv) were dissolved in DMF
and stirred at 808C under an inert atmosphere for 23 h. The reac-
tion was acidified to pH 1 using 1m HCl and the aqueous layer
was extracted with EtOAc. The organic layer was dried over Na2SO4
and the solvent was removed under reduced pressure to give 3c
as a white solid (pure, 74%). 3 was synthesized according to gener-
al procedure b using 3c. The mixture was extracted with EtOAc
and solvent was removed under reduced pressure to give 3 as
Experimental Section
Determination of ligand efficiency (LE) and ligand lipophilicity effi-
ciency Astex (LLEAT) were carried out with Equations (1)–(11):
DG
HAC
ð1Þ
ð2Þ
ð3Þ
ð4Þ
LE ¼
LE ꢂ
LE ꢂ
*
*
ꢁ R
T
lnðIC50Þ
HAC
*
*
*
R
T
lnð10Þ pIC50
HAC
*
1:4 pIC50
LE ꢂ
HAC
*
DG ¼ DG ꢁ DGlipo
ð5Þ
ð6Þ
*
* *
*
DG ꢂ R T lnð10Þ ðpIC50 ꢁ LogPÞ
*
DG
1
ð7Þ
ð8Þ
a white solid (28%). H NMR ([D6]DMSO, 300 MHz): d=3.5 (s, 3H),
LLEAT ¼ 0:11 ꢁ
HAC
8.5 (d, J=5.1 Hz, 1H), 9.3 ppm (d, J=5.1 Hz, 1H); 13C NMR
([D6]DMSO, 126 MHz): d=39.1, 121.5, 153.5, 154.7, 161.1,
165.9 ppm; ESI-MS(+): m/z 226.7 [M+H]+; purity 99%.
*
*
*
R
T
lnð10Þ ðpIC50 ꢁ LogPÞ
LLEAT ꢂ 0:11 ꢁ
LLEAT ꢂ 0:11 ꢁ
LLEAT ꢂ 0:11 ꢁ
HAC
General procedure for the synthesis of 4, 5, 6, 8 and 9. The com-
pounds were synthesized as previously described for 1.[2] First the
N-heterocycle (1 equiv) was quickly added to 1a (1 equiv) followed
by Et3N (1 equiv). The mixture was stirred in a microwave for 10–
20 min at 60–1208C. Except if otherwise noted, thioethers (4a–6a,
8a and 9a) were oxidized according to procedure b.
*
*
*
ꢁ R
T
lnð10Þ ðLogP ꢁ pIC50Þ
ð9Þ
HAC
*
ꢁ 1:4 ðLogP ꢁ pIC50Þ
ð10Þ
ð11Þ
HAC
LogP ꢁ pIC50
*
LLEAT ꢂ 0:11 þ 1:4
HAC
2-(methylsulfonyl)-4-(1H-1,2,4-triazol-1-yl)pyrimidine (4). Com-
pound 4a (1 equiv) was oxidized using mCPBA (2.25 equiv) in
CH2Cl2 at 08C. The crude product was purified by flash chromatog-
raphy (EtOAc/hexane, 2:1) to give a white solid (39%). 1H NMR
(CDCl3, 500 MHz): d=3.41 (s, 4H), 8.08 (d, J=5.7 Hz, 1H), 8.19 (s,
1H), 9.02 (d, J=5.4 Hz, 1H), 9.33 ppm (s, 1H); 13C NMR (CDCl3,
in which HAC=heavy atom count.
The calculation of LE by Equations (1)–(4) was based on the find-
ings of Shulz.[42] The calculation of LLEAT with Equations (5)–(8) was
based on the work of Mortenson and Murray used for the calcula-
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126 MHz): d=39.2, 112.0, 143.1, 154.5, 156.3, 161.0, 165.8 ppm; ESI-
MS(+): m/z 226.0 [M+H]+; purity 96%.
(1-(2-(methylsulfonyl)pyrimidin-4-yl)-1H-1,2,3-triazol-4-yl)(phe-
nyl)methanol (12). The crude product was purified by flash chro-
matography (EtOAc/hexane, 6:4) to give a white solid (54%).
1H NMR (CDCl3, 300 MHz): d=3.11 (d, J=3.6 Hz, 1H), 3.40 (s, 3H),
6.13 (d, J=2.61 Hz, 1H), 7.30–7.44 (m, 3H), 7.45–7.54 (m, 2H), 8.36
(d, J=5.6 Hz, 1H), 8.55 (s, 1H), 9.03 ppm (d, J=5.5 Hz, 1H);
13C NMR (CDCl3, 75 MHz): d=39.2, 69.3, 112.5, 119.3, 126.4, 128.5,
128.9, 141.1, 152.9, 156.4, 160.7, 166.1 ppm; ESI-MS(+): m/z 332.1
[M+H]+; purity 99%.
2-(methylsulfonyl)-4-(1H-pyrazol-1-yl)pyrimidine (5). Compound
5a (1 equiv) was oxidized using mCPBA (2.25 equiv) in CH2Cl2 at
08C. The crude product was purified by flash chromatography
(EtOAc/hexane, 1:1) to give a white solid (65%). 1H NMR (CDCl3,
500 MHz): d=3.40 ppm (s, 4H) 6.57 (dd, J=2.8, 1.6 Hz, 1H) 7.85
(dd, J=1.6, 0.6 Hz, 1H), 8.11 (d, J=5.4 Hz, 1H); 8.66 (dd, J=2.8,
0.6 Hz, 1H), 8.87 ppm (d, J=5.4 Hz, 1H); 13C NMR (CDCl3, 126 MHz):
d=39.1, 110.3, 111.3, 128.5, 145.2, 158.2, 159.6, 165.6 ppm; ESI-
MS(+): m/z 225.0 [M+H]+; purity 99%.
(1-(2-(methylsulfonyl)pyrimidin-4-yl)-1H-1,2,3-triazol-4-yl)metha-
nol (13). The crude product was purified by extraction with EtOAc
to yield a white solid (74%). 1H NMR (CDCl3, 300 MHz): d=2.40
(brs, 1H) 3.32–3.53 (s, 3H), 4.94 (s, 2H), 8.39 (d, J=5.5 Hz, 1H),
8.73 (s, 1H), 9.08 ppm (d, J=5.5 Hz, 1H); 13C NMR ([D6]DMSO,
75 MHz): d=39.7, 55.1, 113.2, 121.0, 150.5, 155.8, 162.6, 165.9 ppm;
ESI-MS(+): m/z=256.1 [M+H]+; purity 99%.
4-(1H-imidazol-1-yl)-2-(methylsulfonyl)pyrimidine (6). Compound
6a (1 equiv) was oxidized using mCPBA (2.25 equiv) in CH2Cl2 at
08C. The crude product was purified by flash chromatography
(EtOAc/MeOH, 9:1) to give a white solid (36%). 1H NMR (CD3OD,
500 MHz): d=3.45 (s, 3H), 7.24 (s, 1H), 8.03 (d, J=5.8 Hz, 1H), 8.08
(s, 1H), 8.80 (s, 1H), 9.05 ppm (d, J=5.5 Hz, 1H); 13C NMR (CD3OD,
126 MHz): d=39.5, 113.1, 118.0, 132.0, 157.6, 162.4, 167.3 ppm; ESI-
MS(+): m/z 225.0 [M+H]+; purity 99%.
2-(1-(2-(methylsulfonyl)pyrimidin-4-yl)-1H-1,2,3-triazol-4-yl)pro-
pan-2-ol (14). The crude product was purified by flash chromatog-
raphy (EtOAc/hexane, 8:2) to yield a colorless oil (45%). 1H NMR
(CDCl3, 300 MHz): d=1.72 (s, 6H), 2.20–2.80 (brs, 1H), 3.44 (s, 3H),
8.38 (d, J=5.49 Hz, 1H), 8.62 (s, 1H), 9.05 ppm (d, J=5.49 Hz, 1H);
13C NMR (CDCl3, 75 MHz): d=30.3, 39.2, 68.6, 117.4, 112.5, 156.5,
157.3, 160.7, 166.2 ppm; ESI-MS(+): m/z 284.0 [M+H]+; purity
99%.
2-(methylsulfonyl)-4-(thiophen-3-yl)pyrimidine (7). To a mixture
of 7b (1 equiv) and PCy3 (0.09 equiv) in dioxane was given Pd2dba3
(0.03 equiv), K3PO4(aq) (2 equiv) and 3-thiopheneboronic acid
(1.2 equiv). The reaction was stirred in a sealed tube for 1 h at
1008C. The reaction was extracted three times with EtOAc and the
solvent was removed under reduced pressure. The crude product
was purified by preparative HPLC to give a white solid (22%).
1H NMR (CD3OD, 300 MHz): d=3.43 (s, 3H), 7.61 (dd, J=5.0, 3.0 Hz,
1H), 7.87 (d, J=6.1 Hz, 1H), 8.04 (d, J=5.4 Hz, 1H), 8.54 (d, J=
3.0 Hz, 1H), 8.90 ppm (d, J=5.2 Hz, 1H); 13C NMR (CD3OD, 75 MHz):
d=39.4, 120.5, 127.3, 128.9, 131.1, 139.6, 160.4, 162.9, 167.5 ppm;
ESI-MS(+): m/z 241.0 [M+H]+; purity 96%.
4-(4-(tert-butyl)-1H-1,2,3-triazol-1-yl)-2-(methylsulfonyl)pyrimi-
dine (15). The crude product was purified by flash chromatogra-
phy (EtOAc/hexane, 7:3) to yield a white solid (22%). 1H NMR
(CDCl3, 300 MHz): d=1.44 (s, 9H), 3.44 (s, 3H), 8.37 (d, J=5.6 Hz,
1H), 8.39 (s, 1H), 9.01 ppm (d, J=5.5 Hz, 1H); 13C NMR (CDCl3,
75 MHz): d=30.0, 30.0, 31.0, 39.2, 112.4, 116.6, 156.6, 159.5, 160.3,
166.2 ppm; ESI-MS(+): m/z 282.0 [M+H]+; purity 99%.
1-(2-(methylsulfonyl)pyrimidin-4-yl)-1H-benzo[d]imidazole
(8).
4-(4-cyclopropyl-1H-1,2,3-triazol-1-yl)-2-(methylsulfonyl)pyrimi-
dine (16). The crude product was filtered over a pad of Celiteꢁ/
silica to yield a white solid (83%). 1H NMR (CDCl3, 300 MHz): d=
0.81–1.01 (m, 2H), 1.02–1.16 (m, 2H), 1.96–2.21 (m, 1H), 3.42 (s,
3H), 8.34 (d, J=5.5 Hz, 1H), 8.37 (s, 1H), 9.01 ppm (d, J=5.49 Hz,
1H); 13C NMR (CDCl3, 75 MHz): d=6.6, 8.2, 39.2, 112.3, 117.3, 152.3,
156.3, 160.4, 166.1 ppm; ESI-MS(+): m/z 266.0 [M+H]+; purity
99%.
The crude product was purified by flash chromatography (EtOAc/
MeOH, 98:2) to give a white solid (61%). 1H NMR ([D6]DMSO,
300 MHz): d=3.52 (s, 3H), 7.35–7.57 (m, 2H), 7.82 (d, J=7.9 Hz,
1H), 8.37 (d, J=5.8 Hz, 1H), 8.57 (d, J=8.2 Hz, 1H), 9.16 (d, J=
5.9 Hz, 1H), 9.27 ppm (s, 1H); 13C NMR ([D6]DMSO, 75 MHz): d=
112.6, 115.7, 120.3, 124.6, 125.2, 131.2, 142.5, 144.4, 157.1, 160.3,
165.3 ppm; ESI-MS(+): m/z 274.8 [M+H]+; purity 96%.
1-(2-(methylsulfonyl)pyrimidin-4-yl)-1H-benzo[d][1,2,3]triazole
(9). Purified by flash chromatography (EtOAc/hexane, 9:1) to give
a white solid (99%). H NMR (CDCl3, 300 MHz): d=3.47 (s, 3H), 7.58
(t, J=8.4 Hz, 1H), 7.77 (t, J=8.4 Hz, 1H), 8.20 (d, J=8.3 Hz, 1H),
8.50 (d, J=5.7 Hz, 1H), 8.75 (d, J=8.3 Hz, 1H), 9.04 ppm (d, J=
5.7 Hz, 1H); 13C NMR (CDCl3, 126 MHz): d=39.4, 112.4, 115.3, 120.5,
126.5, 130.9, 131.1, 147.0, 158.5, 159.7, 165.8 ppm; ESI-MS(+): m/z
338.8 [M+MeCN+Na]+; purity 95%.
2-(methylsulfonyl)-4-(4-phenyl-1H-1,2,3-triazol-1-yl)pyrimidine
(17). The crude product was filtered over a pad of Celiteꢁ/silica to
yield an off-white solid (87%). 1H NMR ([D6]DMSO, 300 MHz): d=
3.60 (s, 3H), 7.43 (t, J=7.1 Hz, 1H), 7.52 (t, J=7.0 Hz, 2H), 8.09 (d,
J=8.4 Hz, 2H), 8.47 (d, J=5.6 Hz, 1H), 9.29 (d, J=5.6 Hz, 1H),
9.60 ppm (s, 1H); 13C NMR ([D6]DMSO, 75 MHz): d=39.5, 113.4,
119.3, 126.3, 129.4, 129.5, 129.7, 148.4, 155.8, 162.6, 165.9 ppm; ESI-
MS(+): m/z 302.0 [M+H]+; purity 99%.
1
General procedure (d) for the synthesis of 11–21. The com-
pounds were synthesized using the same procedure as reported
for compound 10.[2] 10a (1 equiv) was quickly dissolved in tBuOH/
H2O (1:1) and the alkyne (1 equiv), CuSO4 (0.02 equiv) and sodium
ascorbate (0.1 equiv) was added and stirred at room temperature
for 16 h.
2-(methylsulfonyl)-4-(4-(m-tolyl)-1H-1,2,3-triazol-1-yl)pyrimidine
(18). The crude product was filtered over a pad of Celiteꢁ/silica to
yield a white solid (62%). 1H NMR (CDCl3, 300 MHz): d=2.45 (s,
3H), 3.46 (s, 3H), 7.21 (d, J=7.6 Hz, 1H), 7.39 (t, J=7.6 Hz, 7H),
7.75 (d, J=7.7 Hz, 1H), 7.80 (s, 1H), 8.43 (d, J=5.5 Hz, 1H), 8.90 (s,
1H), 9.06 ppm (d, J=5.5 Hz, 1H); 13C NMR (CDCl3, 75 MHz): d=
21.4, 21.4, 39.3, 112.5, 116.9, 123.3, 126.8, 128.8, 129.0, 130.1, 138.9,
149.4, 156.4, 160.6, 166.2 ppm; ESI-MS(+): m/z 316.0 [M+H]+;
purity 99%.
cyclohexyl(1-(2-(methylsulfonyl)pyrimidin-4-yl)-1H-1,2,3-triazol-
4-yl)methanol (11). The crude product was purified by flash chro-
matography (EtOAc/hexane, 7:3) to give a white solid (48%).
1H NMR ([D6]DMSO, 300 MHz): d=1.21–2.10 (m, 11H), 3.47–3.67 (m,
3H), 5.17 (s, 1H), 8.42 (d, J=5.5 Hz, 1H), 8.81 (s, 1H), 9.26 ppm (d,
J=5.5 Hz, 1H); 13C NMR (75 MHz, [D6]DMSO): d=22.0, 25.6, 37.9,
39.5, 68.5, 113.2, 119.2, 155.9, 158.3, 162.5, 165.9 ppm; purity 96%.
2-(methylsulfonyl)-4-(4-(p-tolyl)-1H-1,2,3-triazol-1-yl)pyrimidine
(19). The crude product was filtered over a pad of Celiteꢁ/silica to
yield a white solid (61%). 1H NMR (CDCl3, 300 MHz): d=2.43 (s,
3H), 3.46 (s, 3H), 7.32 (d, J=8.01 Hz, 2H), 7.86 (d, J=8.2 Hz, 2H),
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8.43 (d, J=5.5 Hz, 1H), 8.87 (s, 1H), 9.06 ppm (d, J=5.6 Hz, 1H);
13C NMR ([D6]DMSO, 126 MHz): d=21.4, 39.2, 112.5, 116.5, 126.1,
129.8, 139.4, 149.3, 156.4, 160.5, 166.2 ppm; ESI-MS(+): m/z 316.0
[M+H]+; purity 99%.
General procedure for the synthesis of 23, 25, 26. Synthesis of
the thioethers was reported before.[16] Oxidation was carried out
following procedure b.
N-methyl-2-(methylsulfonyl)-6-(1H-tetrazol-1-yl)pyrimidin-4-
amine (23). Purified by flash chromatography (EtOAc/hexane, 8:2)
to yield a white solid (58%). 1H NMR ([D6]DMSO, 300 MHz): d=
2.78–3.16 (m, 3H), 3.41–3.54 (m, 3H), 6.97–7.32 (m, 1H), 8.55–9.06
(m, 1H), 10.06–10.45 ppm (m, 1H) (rotamers); 13C NMR ([D6]DMSO,
75 MHz): d=28.1, 96.3, 142.5, 151.2, 165.2, 165.8, 166.5 ppm; ESI-
MS(+): m/z 227.8 [MꢁN2 +H]+; purity 95%.
N-benzyl-2-(methylsulfonyl)-6-(1H-tetrazol-1-yl)pyrimidin-4-
amine (25). The crude product was purified by flash chromatogra-
phy (EtOAc/hexane, 1:1) to yield a white solid (37%). 1H NMR
([D6]DMSO, 300 MHz): d=3.40 (s, 3H), 4.69 (d, J=5.7 Hz, 2H), 7.27
(s, 1H), 7.33–7.45 (m, 5H), 9.22 (t, J=5.7 Hz, 1H), 10.23 ppm (s, 1H)
(rotamers); 13C NMR ([D6]DMSO,126 MHz): d=44.2, 95.7, 127.1,
127.3, 127.8, 128.5, 128.7, 137.9, 142.0, 151.1, 164.1, 165.9 ppm;
ESI-MS(+): m/z 331.9 [M+H]+, 303.9 [MꢁN2 +H]+; purity 99%.
4-(4-(4-fluorophenyl)-1H-1,2,3-triazol-1-yl)-2-(methylsulfonyl)pyr-
imidine (20). The crude product was filtered over a pad of Celiteꢁ/
silica to yield a white solid (40%). 1H NMR (CDCl3, 300 MHz): d=
3.46 (s, 3H), 7.20 (t, J=8.6 Hz, 2H), 7.93 (d, J=5.3 Hz, 1H), 7.96 (d,
J=5.4 Hz, 1H), 8.43 (d, J=5.5 Hz, 1H), 8.88 (s, 1H), 9.07 ppm (d,
J=5.5 Hz, 1H); 13C NMR (CDCl3, 75 MHz): d=39.2, 112.5, 116.4,
116.8, 125.1, 128.0, 128.1, 148.4, 156.4, 160.6, 161.7, 164.9,
166.3 ppm; ESI-MS(+): m/z 319.8 [M+H]+; purity 99%.
4-(4-(4-methoxyphenyl)-1H-1,2,3-triazol-1-yl)-2-(methylsulfonyl)-
pyrimidine (21). The crude product was filtered over a pad of
Celiteꢁ/silica to yield a white solid (60%). 1H NMR ([D6]DMSO,
300 MHz): d=3.59 (s, 3H), 3.83 (s, 3H), 7.09 (d, J=8.9 Hz, 2H), 8.02
(d, J=8.8 Hz, 2H), 8.47 (d, J=5.5 Hz, 1H), 9.28 (d, J=5.6 Hz, 1H),
9.50 ppm (s, 1H); 13C NMR ([D6]DMSO, 126 MHz): d=39.0, 55.2,
112.8, 114.5, 117.7, 121.7, 127.2, 147.9, 155.4, 159.8, 162.0,
165.3 ppm; ESI-MS(+): m/z 331.8 [M+H]+; purity 96%.
2-(methylsulfonyl)-4-phenoxy-6-(1H-tetrazol-1-yl)pyrimidine (26).
The crude product was purified by flash chromatography (EtOAc/
1
4-ethynyl-2-(methylthio)pyrimidine
(22b).
Compound
1a
hexane, 1:1) to yield a white solid (38%). H NMR (CDCl3, 300 MHz):
(1 equiv) was dissolved in DMF and NEt3 (3 equiv), TMS-acetylene
(2 equiv), (PPh3)2PdCl2 (0.03 equiv), and CuI (0.1 equiv) were added.
The reaction was stirred under inert atmosphere for 2 h at 508C.
Brine was added and the aqueous layer was extracted with EtOAc.
The combined organic layers were dried over Na2SO4 and the sol-
vent was removed under reduced pressure. The crude material
was purified by flash chromatography (EtOAc/petroleum ether,
2:98) to yield 22a as a colorless oil.[44] The TMS-protected product
was deprotected with 1.1 equiv of TBAF·3H2O in THF for 2 h at
room temperature. The reaction was filtered over a pad of silica
and solvent was removed under reduced pressure to yield 22b as
a white solid in quantitative yield. 1H NMR (CDCl3, 500 MHz): d=
2.57 (s, 3H), 3.34 (s, 1H), 7.06 (d, J=5.0 Hz, 1H), 8.51 ppm (d, J=
4.7 Hz, 1H); 13C NMR (CDCl3, 126 MHz): d=14.1, 80.7, 81.4, 118.7,
149.8, 157.2, 173.4 ppm.
d=3.19 (s, 3H), 7.22 (d, J=8.1 Hz, 2H), 7.38 (t, J=7.5 Hz, 1H), 7.51
(t, J=7.5 Hz, 2H), 7.71 (s, 1H), 9.65 ppm (s, 1H) 13C NMR (CDCl3,
75 MHz): d=38.7, 99.1, 99.2, 120.9, 127.2, 130.3, 140.8, 151.4, 155.5,
166.2, 172.4 ppm; purity 95%.
General procedure for the synthesis of 27–44. The compounds
were synthesized using the same route as reported for compound
34,[2] 1 (1 equiv for 37a–44a), or 2 (1 equiv for compounds 27a–
36a) was quickly dissolved in DMF and K2CO3 (3 equiv) was added.
The mixture was cooled to ꢁ208C and the thiol (1 equiv) was
added at once. The reaction was allowed to continue until TLC
showed full conversion. Resulting thioethers were oxidized using
procedure b.
2-(isopropylsulfonyl)-4-(1H-1,2,3-triazol-1-yl)pyrimidine (27). The
crude product was purified by flash chromatography (EtOAc/
1
hexane, 1:1) to yield a white solid (63%). H NMR (CDCl3, 300 MHz):
4-(1-benzyl-1H-1,2,3-triazol-4-yl)-2-(methylsulfonyl)pyrimidine
(22). Compound 22b (1 equiv) was dissolved in tBuOH/H2O (1:1)
and benzylazide[45] (1 equiv), sodium ascorbate (0.1 equiv), and
CuSO4 (0.02 equiv) were added. The mixture was stirred at room
temperature for 16 h and then extracted with EtOAc. The com-
bined organic layers were dried over Na2SO4 and purified by flash
chromatography (EtOAc/hexane, 3:7) to yield 22c as a white solid
(60%). 22c was oxidized using procedure b, and 22 was purified
by flash chromatography (EtOAc/hexane, 1:1) to give a white solid
d=1.54 (s, 9H), 7.92 (d, J=1.3 Hz, 1H), 8.41 (d, J=5.4 Hz, 1H), 8.73
(d, J=1.3 Hz, 1H), 9.16 ppm (d, J=5.4 Hz, 1H); 13C NMR (CDCl3,
75 MHz): d=23.9, 61.2, 112.3, 121.8, 135.2, 156.1, 160.9, 164.6 ppm;
ESI-MS(+): m/z 254.0 [M+H]+; purity 95%.
2-(tert-butylsulfonyl)-4-(1H-1,2,3-triazol-1-yl)pyrimidine (28). The
crude product was purified by flash chromatography (EtOAc/
1
hexane, 1:1) to yield a white solid (54%). H NMR (CDCl3, 300 MHz):
d=1.47 (d, J=6.9 Hz, 6H), 4.01 (spt, J=6.9 Hz, 1H), 7.92 (d, J=
1.4 Hz, 1H), 8.41 (d, J=5.5 Hz, 1H), 8.75 (d, J=1.4 Hz, 1H),
9.11 ppm (d, J=5.5 Hz, 1H); 13C NMR (CDCl3, 75 MHz): d=15.0,
51.6, 112.5, 121.9, 135.2, 156.4, 160.9, 165.3 ppm; ESI-MS(+): m/z
268.0 [M+H]+; purity 95%.
1
(53%). H NMR (CDCl3, 300 MHz): d=3.36 (s, 3H), 5.62 (s, 2H), 7.28–
7.49 (m, 5H), 8.25–8.40 (m, 2H), 8.90 ppm (d, J=5.2 Hz, 1H);
13C NMR (CDCl3, 75 MHz): d=39.1, 54.7, 118.7, 125.1, 128.3, 129.2,
129.3, 133.6, 145.1, 158.7, 159.3, 166.1 ppm; ESI-MS(+): m/z 268
[M+H]+; purity 99%.
2-(ethylsulfonyl)-4-(1H-1,2,3-triazol-1-yl)pyrimidine (29). The
N-(2-(methylsulfonyl)-6-(1H-tetrazol-1-yl)pyrimidin-4-yl)aceta-
mide (24). Compound 24a[16] was dissolved in acetic anhydride
and stirred for 2 h at 1408C. Solvent was removed under reduced
pressure and the crude material was purified by flash chromatogra-
phy (EtOAc/hexane, 7:3) to yield 24b as a white solid (47%). 24b
was oxidized using procedure b. The crude material was purified
by flash chromatography (EtOAc/hexane+formic acid, 7:3+0.1%)
to give 24 as a white solid (70%). 1H NMR ([D6]DMSO, 300 MHz):
d=2.24 (s, 3H), 3.54 (s, 3H), 8.80 (s, 1H), 10.46 (s, 1H), 11.98 ppm
(s, 1H); 13C NMR ([D6]DMSO, 75 MHz): d=24.2, 24.4, 100.0, 142.2,
154.0, 161.4, 164.8, 171.6 ppm; purity 96%.
crude product was purified by flash chromatography (EtOAc/
hexane, 8:2) to yield a white solid (73%). H NMR (CDCl3, 300 MHz):
1
d=1.49 (t, J=7.5 Hz, 3H), 3.63 (q, J=7.5 Hz, 2H), 7.92 (d, J=
1.3 Hz, 1H), 8.42 (d, J=5.5 Hz, 1H), 8.75 (d, J=1.3 Hz, 1H),
9.09 ppm (d, J=5.5 Hz, 1H); 13C NMR (CDCl3, 75 MHz): d=6.9, 45.9,
112.6, 121.9, 135.2, 156.5, 160.9, 165.7 ppm; ESI-MS(+): m/z 240.0
[M+H]+; purity 95%.
2-(propylsulfonyl)-4-(1H-1,2,3-triazol-1-yl)pyrimidine (30). The
crude product was purified by flash chromatography (EtOAc/
1
hexane, 3:7) to yield a white solid (88%). H NMR (CDCl3; 300 MHz):
&
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Full Papers
d=1.00–1.21 (m, 3H), 1.80–2.07 (m, 2H), 3.49–3.66 (m, 2H), 7.92
(d, J=1.3 Hz, 1H), 8.41 (d, J=5.5 Hz, 1H), 8.75 (d, J=1.3 Hz, 1H),
9.09 ppm (d, J=5.5 Hz, 1H); 13C NMR (CDCl3, 75 MHz): d=13.2,
16.0, 53.0, 112.6, 121.9, 135.2, 156.4, 160.9, 166.0 ppm; ESI-MS(+):
m/z 254.0 [M+H]+; purity 97.0%.
13C NMR (CDCl3, 126 MHz): d=166.9, 162.2, 154.1, 142.0, 140.5,
135.0, 131.3, 129.8, 112.7 ppm; purity 99%.
2-((4-methoxyphenyl)sulfonyl)-4-(1H-tetrazol-1-yl)pyrimidine
(39). The crude product was purified by flash chromatography
(EtOAc/hexane, 1:1) to yield a white solid (90%). 1H NMR (CDCl3,
300 MHz): d=3.90 (s, 3H), 6.98–7.14 (m, 2H), 7.98–8.12 (m, 2H),
8.18 (d, J=5.4 Hz, 1H), 9.14 (d, J=5.4 Hz, 1H), 9.61 ppm (s, 1H);
13C NMR (CDCl3, 126 MHz): d=55.8, 112.3, 114.8, 127.4, 132.2, 140.5,
154.0, 162.2, 164.9, 167.6 ppm; ESI-MS(+): m/z 285.2 [M+H]+;
purity 98%.
2-(butylsulfonyl)-4-(1H-1,2,3-triazol-1-yl)pyrimidine (31). The
crude product was purified by flash chromatography (EtOAc/
1
hexane, 8:2) to yield a white solid (76%). H NMR (CDCl3, 300 MHz):
d=0.98 (t, J=7.4 Hz, 3H), 1.54 (dq, J=14.9, 7.4 Hz, 2H), 1.83–1.97
(m, 2H), 3.40–3.76 (m, 2H), 7.92 (d, J=1.3 Hz, 1H), 8.41 (d, J=
5.5 Hz, 1H), 8.75 (d, J=1.4 Hz, 1H), 9.09 ppm (d, J=5.4 Hz, 1H);
13C NMR (CDCl3, 75 MHz): d=13.5, 21.7, 24.0, 51.1, 112.6, 121.9,
135.2, 156.4, 160.9, 166.0 ppm; purity 99%.
2-((4-fluorophenyl)sulfonyl)-4-(1H-tetrazol-1-yl)pyrimidine (40).
The crude product was purified by flash chromatography (EtOAc/
1
hexane, 1:1) to yield a white solid (74%). H NMR (CDCl3, 300 MHz):
2-(pentylsulfonyl)-4-(1H-1,2,3-triazol-1-yl)pyrimidine (32). The
d=7.27–7.40 (m, 2H), 8.04–8.28 (m, 3H), 9.14 (d, J=5.4 Hz, 1H),
9.63 ppm (s, 1H); 13C NMR (CDCl3, 75 MHz): d=112.7, 116.9, 132.3,
132.9, 140.5, 154.1, 162.2, 166.8, 167.1 ppm; ESI-MS(+): m/z 279.0
[MꢁN2 +H]+; purity 99%.
crude product was purified by flash chromatography (EtOAc/
1
hexane, 4:6) to yield a white solid (79%). H NMR (CDCl3, 300 MHz):
d=0.78–1.04 (m, 3H), 1.25–1.57 (m, 4H), 1.92 (quin, J=7.7 Hz,
2H), 3.51–3.72 (m, 2H), 7.92 (d, J=0.8 Hz, 1H), 8.41 (d, J=5.5 Hz,
1H), 8.65–8.83 (m, 1H), 9.09 ppm (d, J=5.5 Hz, 1H); 13C NMR
(CDCl3, 75 MHz): d=13.6, 21.7, 22.1, 30.5, 51.3, 112.5, 121.9, 135.2,
156.4, 160.8, 165.9 ppm; ESI-MS(+): m/z 282.0 [M+H]+; purity
96%.
2-((3-fluorophenyl)sulfonyl)-4-(1H-tetrazol-1-yl)pyrimidine (41).
The crude product was purified by flash chromatography (EtOAc/
1
hexane, 1:1) to yield a white solid (52%). H NMR (CDCl3, 300 MHz):
d=7.45 (tdd, J=8.3, 8.3, 2.5, 1.0 Hz, 1H), 7.64 (td, J=8.1, 5.2 Hz,
1H), 7.85 (ddd, J=7.7, 1.9 Hz, 1H), 7.95 (ddd, J=7.8, 1.0 Hz, 1H),
8.25 (d, J=5.5 Hz, 1H), 9.16 (d, J=5.4 Hz, 1H), 9.63 ppm (s, 1H);
13C NMR (CDCl3, 75 MHz): d=166.7, 162.3, 162.5, 154.1, 140.5,
138.4, 131.2, 125.7, 122.3, 117.2, 112.8 ppm; ESI-MS(+): m/z 279.0
[MꢁN2 +H]+; purity 99%.
2-(hexylsulfonyl)-4-(1H-1,2,3-triazol-1-yl)pyrimidine (33). Purified
by flash chromatography (EtOAc/hexane, 1:9) to yield a white solid
1
(71%). H NMR (CDCl3, 300 MHz): d=0.89 (t, J=7.2 Hz, 3H), 1.26–
1.41 (m, 4H), 1.50 (quin, J=7.8 Hz, 2H), 1.91 (quin, J=7.5 Hz, 2H),
3.58 (t, J=8.0 Hz, 2H), 7.92 (d, J=1.0 Hz, 1H), 8.41 (d, J=5.5 Hz,
1H), 8.75 (d, J=1.3 Hz, 1H), 9.09 ppm (d, J=5.5 Hz, 1H); 13C NMR
(CDCl3, 75 MHz): d=13.9, 22.0, 22.2, 28.1, 31.1, 51.3, 112.5, 121.9,
135.2, 156.4, 160.8, 166.0 ppm; ESI-MS(+): m/z 296.0 [M+H]+;
Purity 99%.
2-((3-chlorophenyl)sulfonyl)-4-(1H-tetrazol-1-yl)pyrimidine (42).
The crude product was purified by flash chromatography (EtOAc/
1
hexane, 1:1) to yield a white solid (39%). H NMR (CDCl3, 300 MHz):
d=7.59 (t, J=7.8 Hz, 1H), 7.71 (ddd, J=1.0, 2.1, 8.0 Hz, 1H), 8.04
(ddd, J=1.0, 1.8, 7.8 Hz, 1H), 8.13 (t, J=1.9 Hz, 1H), 8.24 (d, J=
5.4 Hz, 1H), 9.15 (d, J=5.4 Hz, 1H), 9.62 ppm (s, 1H); 13C NMR
(CDCl3, 126 MHz): d=113.5, 128.6, 130.5, 131.3, 135.8, 136.4, 180.7,
138.8, 141.2, 154.8, 163.0, 167.4 ppm; purity 99%.
2-((4-(1H-1,2,3-triazol-1-yl)pyrimidin-2-yl)sulfonyl)ethyl acetate
(35). The crude product was purified by flash chromatography
(EtOAc/hexane, 1:1) to yield a white solid (42%). 1H NMR (CDCl3,
300 MHz): d=3.00 (t, J=7.5 Hz, 2H), 3.71 (s, 3H), 3.94 (t, J=7.3 Hz,
2H), 7.92 (d, J=1.3 Hz, 1H), 8.42 (d, J=5.5 Hz, 1H), 8.75 (d, J=
1.3 Hz, 1H), 9.09 ppm (d, J=5.5 Hz, 1H); 13C NMR (CDCl3, 75 MHz):
d=27.5, 46.9, 52.4, 112.8, 121.9, 135.3, 156.4, 160.9, 165.5,
170.4 ppm; ESI-MS(+): m/z 298.0 [M+H]+; purity 95%.
2-((3-methoxyphenyl)sulfonyl)-4-(1H-tetrazol-1-yl)pyrimidine
(43). The crude product was purified by flash chromatography
(EtOAc/hexane, 1:1) to yield a white solid (75%). 1H NMR (CDCl3,
300 MHz): d=3.89 (s, 3H), 7.19–7.27 (m, 1H), 7.53 (t, J=8.1 Hz,
1H), 7.59–7.67 (m, 1H), 7.73 (dt, J=7.7, 1.2 Hz, 1H), 8.21 (d, J=
5.4 Hz, 1H), 9.15 (d, J=5.4 Hz, 1H), 9.60 ppm (s, 1H); 13C NMR
(CDCl3, 75 MHz): d=55.9, 112.6, 114.2, 121.5, 122.1, 130.5, 137.5,
140.6, 154.1, 160.2, 162.3, 167.2 ppm; ESI-MS(+): m/z 285.2 [M+
H]+; purity 99%.
2-(phenylsulfonyl)-4-(1H-1,2,3-triazol-1-yl)pyrimidine (36). The
crude product was purified by flash chromatography (EtOAc/
1
hexane, 6:4) to yield a white solid (23%). H NMR (CDCl3, 300 MHz):
d=7.59 (t, J=7.2 Hz, 2H), 7.69 (t, J=7.2 Hz, 1H), 1H), 8.01 (s, 2H),
8.13 (d, J=5.5 Hz), 8.22 (d, J=7.5 Hz, 2H), 8.97 ppm (d, J=5.4 Hz,
1H); 13C NMR (CDCl3, 75 MHz): d=111.8, 129.2, 129.9, 134.4, 137.3,
139.1, 157.4, 160.5, 167.0 ppm; ESI-MS(+): m/z 288.0 [M+H]+;
purity 99%.
2-((3,4-dichlorophenyl)sulfonyl)-4-(1H-tetrazol-1-yl)pyrimidine
(44). The crude product was purified by flash chromatography
(EtOAc/hexane, 1:1) to yield
a
white solid (36%). 1H NMR
([D6]DMSO, 300 MHz): d=7.94–8.04 (m, 1H), 8.06–8.17 (m, 1H),
8.28 (d, J=2.0 Hz, 1H), 8.40 (d, J=5.5 Hz, 1H), 9.31 (d, J=5.5 Hz,
1H), 10.34 ppm (s, 1H); 13C NMR ([D6]DMSO, 75 MHz): d=130.1,
131.6, 132.5, 133.2, 137.2, 139.0, 143.0, 154.6, 163.0,
163.5, 165.5 ppm; ESI-MS(+): m/z 331.9 [MꢁN2 +H]+, 303.9
[Mꢁ2N4 +H]+; purity 99%.
2-(phenylsulfonyl)-4-(1H-tetrazol-1-yl)pyrimidine (37). The crude
product was purified by flash chromatography (EtOAc/hexane, 1:1)
1
to yield a white solid (91%). H NMR (CDCl3, 300 MHz): d=7.64 (t,
J=8.1 Hz, 2H), 7.75 (t, J=7.4 Hz, 1H), 8.15 (d, J=8.5 Hz, 2H), 8.21
(d, J=5.4 Hz, 1H), 9.15 (d, J=5.4 Hz, 1H), 9.58 ppm (s, 1H);
13C NMR (CDCl3, 75 MHz): d=112.5, 129.4, 129.9, 135.0, 136.4,
140.5, 154.0, 162.3, 167.2 ppm; ESI-MS(+): m/z 261 [MꢁN2 +H]+;
purity 99%.
Acknowledgements
2-((4-chlorophenyl)sulfonyl)-4-(1H-tetrazol-1-yl)pyrimidine (38).
We thank Carina Scheidt and Simone Amann (Helmholtz Institute
for Pharmaceutical Research Saarland) for biological testing and
Nadja Klippel (Saarland University) for supporting compound
synthesis. C.B. and D.K. are grateful to the European Regional De-
The crude product was purified by flash chromatography (EtOAc/
hexane, 1:1) to yield a white solid (58%). H NMR (CDCl3, 300 MHz):
d=7.62 (dt, J=8.8, 2.3 Hz, 2H), 8.09 (dt, J=8.8, 2.3 Hz, 2H), 8.23
(d, J=5.4 Hz, 1H), 9.13 (d, J=5.4 Hz, 1H), 9.62 ppm (s, 1H);
1
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Keywords: biofilms · drug design · Hansch analysis · medicinal
chemistry · nitrogen heterocycles · P. aeruginosa
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Received: August 19, 2016
Revised: September 27, 2016
Published online on && &&, 0000
&
ChemMedChem 2016, 11, 1 – 13
www.chemmedchem.org
12
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

FULL PAPERS
Dual-target SAR: a challenging task!
With the use of flexible alignment and
Hansch analysis, drug-like dual inhibi-
tors of the Pseudomonas quinolone
signal (PQS) synthase (PqsD) and the
PQS receptor (PqsR) were developed.
These compounds have high ligand effi-
ciencies and can be used to combat ex-
tracellular DNA and biofilm formation
by the drug-resistant and pathogenic
bacteria Pseudomonas aeruginosa.
A. Thomann, C. Brengel, C. Bçrger, D. Kail,
A. Steinbach, M. Empting,*
R. W. Hartmann*
&& – &&
Structure–Activity Relationships of 2-
Sufonylpyrimidines as Quorum-
Sensing Inhibitors to Tackle Biofilm
Formation and eDNA Release of
Pseudomonas aeruginosa
ChemMedChem 2016, 11, 1 – 13
www.chemmedchem.org
13
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ