'Second-generation' 1,2,3-triazole-based inhibitors of: Porphyromonas gingivalis adherence to oral streptococci and biofilm formation

Article abstract of DOI:10.1039/c8md00405f

Several 'second-generation' click inhibitors of the multi-species biofilm propagated by the adherence of the oral pathogen Porphyromonas gingivalis to Streptococcus gordonii were synthesized and evaluated. The design of the structures was based on the results obtained with the first-generation diphenyloxazole 'click' inhibitors which bear suitable hydrophobic and polar groups within a dual scaffold molecule bearing a 1,2,3-triazole spacer. The structures of the synthetic targets reported herein now consist of a triazolyl(phenylsulfonylmethyl) and a triazolyl(phenylsulfinylmethyl) spacer which joins a 4,5-diphenyloxazole with both phenyl rings bearing lipophilic substituents. The triazolyl "linker" group is formed by a click reaction between the 4-azido(phenylsulfonyl/sulfinylmethyl) oxazoles and acetylenic components having aryl groups bearing hydrophobic substituents. The 1,3,5-trisubstituted-2,4,6-triazine scaffold of the most active click compounds were modeled after the structural motif termed the VXXLL nuclear receptor (NR) box. When substituted at the 3- and 5-positions with 2- and 4-fluorophenylamino and N,N-diethylamino units, the candidates bearing the 1,3,5-trisubstituted-2,4,6-triazine scaffold formed a substantial subset of the second-generation click candidates. Four of the click products, compounds 95, 111, 115 and 122 showed inhibition of the adherence of P. gingivalis to S. gordonii with an IC₅₀ range of 2.3-4.3 μM and only 111 exhibited cytotoxic activity against telomerase immortalized gingival keratinocytes at 60 μM. These results suggest that compounds 95, 115, 122, and possibly 111 represent the most suitable compounds to evaluate for activity in vivo.

Full text of DOI:10.1039/c8md00405f

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RESEARCH ARTICLE
‘Second-generation’ 1,2,3-triazole-based
inhibitors of Porphyromonas gingivalis adherence
Cite this: DOI: 10.1039/c8md00405f
to oral streptococci and biofilm formation†
a
Pravin C. Patil,a Jinlian Tan,b Donald R. Demuth
b and Frederick A. Luzzio
*
*
Several ‘second-generation’ click inhibitors of the multi-species biofilm propagated by the adherence of
the oral pathogen Porphyromonas gingivalis to Streptococcus gordonii were synthesized and evaluated.
The design of the structures was based on the results obtained with the first-generation diphenyloxazole
‘click’ inhibitors which bear suitable hydrophobic and polar groups within a dual scaffold molecule bearing
a 1,2,3-triazole spacer. The structures of the synthetic targets reported herein now consist of a
triazolylIJphenylsulfonylmethyl) and
a
triazolylIJphenylsulfinylmethyl) spacer which joins
a
4,5-
diphenyloxazole with both phenyl rings bearing lipophilic substituents. The triazolyl “linker” group is formed
by a click reaction between the 4-azidoIJphenylsulfonyl/sulfinylmethyl) oxazoles and acetylenic compo-
nents having aryl groups bearing hydrophobic substituents. The 1,3,5-trisubstituted-2,4,6-triazine scaffold
of the most active click compounds were modeled after the structural motif termed the VXXLL nuclear re-
ceptor (NR) box. When substituted at the 3- and 5-positions with 2- and 4-fluorophenylamino and N,N-
diethylamino units, the candidates bearing the 1,3,5-trisubstituted-2,4,6-triazine scaffold formed a substan-
tial subset of the second-generation click candidates. Four of the click products, compounds 95, 111, 115
and 122 showed inhibition of the adherence of P. gingivalis to S. gordonii with an IC50 range of 2.3–4.3 μM
and only 111 exhibited cytotoxic activity against telomerase immortalized gingival keratinocytes at 60 μM.
These results suggest that compounds 95, 115, 122, and possibly 111 represent the most suitable com-
pounds to evaluate for activity in vivo.
Received 17th August 2018,
Accepted 18th November 2018
DOI: 10.1039/c8md00405f
rsc.li/medchemcomm
able. Although the primary niche for P. gingivalis is a mixed
community of bacterial species in the subgingival pocket,
1. Introduction
In the human oral cavity, a consortium of anaerobic bacteria
including Porphyromonas gingivalis, Tannerella forsythensis
and Treponema denticola colonizes the subgingival pocket and
has been designated as the ‘red complex’ that is strongly as-
sociated with chronic adult periodontitis.1 P. gingivalis is con-
sidered a key periodontal pathogen that may function to
shape the overall microbial community leading to dysbiosis
and tissue damage.2–5 Current methods to treat periodontitis
involves scaling and root planing and surgery may be re-
quired to reduce subgingival pocket depth in more severe
cases. Therapeutic approaches that specifically target peri-
odontal pathogens like P. gingivalis are lacking. In addition,
therapies that prevent or limit re-colonization of the oral cav-
ity by P. gingivalis after treatment procedures are not avail-
upon initial entry into the oral cavity it first colonizes supra-
gingival plaque6 and the interaction of P. gingivalis with oral
streptococci is important for this early colonization event.7,8
Thus, adherence of P. gingivalis with commensal streptococci
represents an ideal point for therapeutic intervention to control
colonization (or re-colonization) of the oral cavity by P.
gingivalis. Adherence to streptococci is mediated by a protein–
protein interaction that occurs between the minor fimbrial
antigen (Mfa) of P. gingivalis and the antigen I/II (Ag I/II) poly-
peptide of streptococci.9–11 Daep et al. identified a discrete
domain in Ag I/II protein that is required for interaction with
Mfa and showed that this region resembles the eukaryotic nu-
clear receptor (NR) box protein–protein interaction do-
main.9,10 Daep et al. also showed that the NR box is com-
posed of two functional peptide motifs, VXXLL and NITVK,
and a synthetic peptide encompassing both motifs functioned
as a potent inhibitor of P. gingivalis adherence to streptococci
and significantly reduced P. gingivalis virulence in vivo.11
These studies suggested that P. gingivalis colonization of the
oral cavity can be controlled by preventing its initial association
with streptococci and that inhibitors of the Mfa–Ag I/II
a Department of Chemistry, University of Louisville, 2320 South Brook Street,
Louisville, Kentucky 40292, USA
b Department of Oral Immunology and Infectious Diseases, University of
Louisville, School of Dentistry, 501 S. Preston St., Louisville, Kentucky 40292,
USA. E-mail: drdemu01@louisville.edu
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c8md00405f
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interaction may represent potential therapeutic agents to con-
trol periodontal disease. However, the use of peptides as ther-
apeutic agents has limitations arising from the relatively high
cost of peptide synthesis and their susceptibility to degrada-
tion by proteases expressed by oral organisms, including P.
gingivalis itself. The development of small-molecule
peptidomimetics is one approach to generate hydrolytically
stable inhibitory analogues of the inhibitory peptide and at a
decreased cost of synthesis. To this end, we previously
reported the synthesis and testing of small-molecule inhibi-
tors based on a non-peptide backbone that mimic the natural
peptide substrate recognized by Mfa.12,13 A 2,4,5-trisubstituted
oxazole framework was utilized to mimic the NITVK motif,
and mono-, di- and trisubstituted aryl rings having hydropho-
bic substituents represented VXXLL mimics. These two syn-
thetic small-molecule scaffolds were subsequently joined via
the ‘click’ reaction.14–22 In this report, we describe the synthe-
ses and click reactions of similar 2,4,5-trisubstituted oxazole
NITVK mimics.23–26 These reacting components are now used
in conjunction with sulfinylaryl- and sulfonylaryl-1,2,3-triazole
linkers, albeit with the mono- and disubstituted aryl groups
acting as the VXXLL structural mimic. Similar to the previous
‘first-generation’ compounds, the newly formed 1,2,3-triazole
linker arising from the click reaction functions as the polar,
slightly basic section of the entire peptidomimetic with the
phenylsulfinyl or phenylsulfonyl portion allowing several
more degrees of conformational freedom in the molecule's
overall topology. These second-generation compounds are di-
vided into three groups (Groups I, II, and III) based on the
linker motifs joining the two scaffolds, the substitutions on
the phenyl rings of the 4,5-diaryloxazole, the hydrophobic sub-
stituents on the distal (VXXLL) aryl ring and those in which
the 3,5-(arylamino)-substituted 2,4,6-triazine unit represents
the distal VXXLL locus (Fig. 1). Further, the 2,4,6-triazines all
Fig.
1
General azide/alkyne scaffold construction of second-
generation inhibitors via a ‘click’ reaction. The entire linker modality is
a sulfinyl- or sulfonylphenyltriazole. NITVK locus = green; VXXLL locus
= blue; yellow R = sulfinyl/sulfonyl.
sesses nonpolar hydrophobic N-alkyl groups which mimic the
one valine and two leucine groups in the inhibitory helical
peptide. For the hydrophobic substituents on the benzenoid
rings of the VXXLL locus, we chose the halogens (F, Cl, Br),
methoxy (OCH3) and trifluoromethyl (CF3) groups, all of
which were situated on aromatic rings. In terms of hydropho-
bicity trends, F > Cl > Br, with the methoxy group being
more polar and less hydrophobic than bromine and the
trifluoromethyl group being more hydrophobic than a single
fluorine. The planarity of the substituted oxazole ring ex-
cludes any stereochemical considerations thereby simplifying
the initial design of the inhibitory NITVK scaffolds. The
NITVK scaffolds were designed with the azide function of the
click coupling partner positioned with at least a one-carbon
spacer or a phenyl-ring spacer between the azide moiety and
the oxazole torus with the azidoalkyl or azidoaryl group lo-
cated at the 2-position of the oxazole. Given that the naphtha-
lene core has been implicated in mimicking the double leu-
cine motif in earlier studies, our selection included an
alkynylnaphthalene bearing a hydrophobic methoxy group
(Table 1). It should be noted that our initial selections of sim-
ple aryl-substituted VXXLL mimics constituted only aryl rings
which were singly substituted with hydrophobic groups.
Moreover, the newly formed 1,2,3-triazole or otherwise
“linker” ring resulting from the click reaction may function
bear
hydrophobic
substituents
such
as
2-
and
4-fluorophenylamino and required installation of the acetylenic
component so that the resulting 6-acetylenic-2,4-di-N-
substituted 1,3,5-triazines could react with the NITVK-mimic
co-reactant azides in the click reaction.
2. Results and discussion
2.1. Design and chemistry
Our approach toward the design of the second-generation in-
hibitors is modelled after the previously reported individual
VXXLL and NITVK-peptidomimetic motifs which encom-
passes both the trisubstituted oxazole (NITVK) and mono-
and disubstituted aryl (VXXLL) segments of the original in-
hibitory polypeptide designed in these laboratories.12 The
separate motifs were each functionalized with azide and acet-
ylene reactant groups to facilitate the formation of the “click”
triazole linker which provided the completed scaffold. Within
the substituted aryl groups of the VXXLL segment bearing
acetylenic reacting components is the subgroup of
meta-disposed trisubstituted triazines which bear two fluoro-
phenylamino groups. The alkyldiaminotriazine scaffold pos-
as
a polar, slightly basic backbone of the complete
peptidomimetic. It may also be noted that triazole rings can
function as mild hydrogen bond acceptors as well as
π-stacking structures. We chose a sulfinyl/sulfonyl group in
tandem with a para-substituted azidophenyl group to now
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Table 1 Arylalkynyl click partners 16–27a
chloromethyl oxazoles 1–3 are reacted with 4-amino-
thiophenol in the presence of sodium hydride in THF to af-
ford the 4-(aminothiophenyl)oxazoles 4–6 in yields ranging
from 66% to 85%. Treatment of the (aminothiophenyl) oxa-
zoles 4–6 with sodium nitrite in 10% aqueous HCl and so-
dium azide (5 °C to rt, 16 h) gave the corresponding
4-azidophenylIJoxazolyl)sulfides 7–9 (61–98%) with no interfer-
ence from the sulfide group. Oxidation of the sulfide moiety
of 7–9 could provide either the corresponding sulfoxides
10–12 or the corresponding sulfones 13–15 depending on the
equivalents of oxidant employed. Thus, treatment of the sul-
phides 7–9 with m-chloroperbenzoic acid (1.2 equivalents) in
dichloromethane (16 h, rt) gave the corresponding sulfoxides
10–12 (86–95%), while treating 7–9 with three equivalents of
the oxidant provided the corresponding sulfones 13–15 (86–
93%) in excellent yield. Both the oxidations of the sulfides to
the sulfoxides and sulfones were optimized and these com-
pounds were obtained as crystalline solids which were easily
purified by column chromatography on silica gel. Interest-
16
18
17
19
21
20
22
23
24
25
27
26
Arranged in order of increasing molecular weight.
1
ingly, H NMR spectra revealed a distinct non-equivalence be-
a
tween the enantiotopic methylene protons at the 2-position
of the oxazole ring in sulfoxides 10–12, while the same set of
protons in the corresponding sulfones 13–15 were equivalent.
Using the percarboxylic acid oxidation protocol, the sulfoxide
products are presumed to be racemic at sulfur and were bio-
assayed as the racemic mixtures. The click reactions of
azidophenylsulfoxides 10–12 and azidophenylsulfones 13–15
with the acetylenic partners 16–24 from Table 1 to give the
click triazole products 28–45 (Scheme 2) were conducted un-
der standard conditions (CuSO4/sodium ascorbate/THF/H2O)
developed in these laboratories for our previously reported
candidates. The yields of the triazole click products 28–45 are
presented in Table 2.
The 2-substituted-4,5-diphenyloxazole azide units containing
the 4,5-di-(4-chlorophenyl) and 4,5-di-(4-fluorophenyl) groups
(11, 14 and 12, 15) offered hydrophobic interactions at both
aryl positions in the NITVK-trisubstituted oxazole-associated
locus and comprised the next group (Group II) of triazole-
linked phenyl sulfoxides and sulfones (Scheme 3). Therefore,
the combination of azides 11, 12, 14, 15 and arylacetylenes
17–19 (Table 1) gave click products which carried the com-
plete array of halogenated (F, Cl) substituents on both the
aryl rings of the 4,5-diaryloxazole (NITVK) and the aryltriazole
(VXXLL) loci. The 4,5-bisIJ4-fluorophenyl)oxazole sulfoxide 11
and the azido 4,5-bisIJfluorophenyl) oxazole sulfone 14
provide an extended linker which should have unique inter-
action properties. There is a dual character of the weakly po-
lar sulfonyl group as a hydrogen bond acceptor and as a hy-
drophobic group.27 Furthermore, the sulfonyl group is
capable of forming van der Waals interactions with nonpolar
atoms together with weak hydrogen bonds involving hydro-
gens α to electron-withdrawing groups. A very similar issue
characterizes the sulfinyl group whereby the sulfoxide (SO)
itself is also a very weak hydrogen bond acceptor.28 The
Group I inhibitor synthesis begins with the preparation of
the individual azide and acetylene click reacting partners
(Scheme 1). 2-Chloromethyl-4,5-diphenyloxazoles 1–3, versatile
synthetic intermediates prepared in multi-gram quantities
and in modest to good yields by methods previously reported
from these laboratories, are the starting points.23–26 The
Scheme 1 Synthesis of azidophenylsulfinyl and azidophenylsulfonyl
click
partners
10–15.
Reagents/conditions:
(a)
NaH/4-
Scheme
2 Click reactions of azidophenylsulfoxides 10 and
aminothiophenol/THF/0–5 °C to rt/16 h (66–85%). (b) NaNO2/10% HCl/
NaN3/0–5 °C to rt/16 h (61–98%). (c) MCPBA (1.2 equiv.)/DCM/rt/16 h
(86–95%). (d) MCPBA (3 eq.)/DCM/rt/16 h (86–93%).
azidophenyl-sulfones 13 with selected arylacetylenes 16–24 to give
Group I triazoles 28–45. aReagents/conditions: CuSO4·5H2O/Na ascor-
bate/THF–H2O (2 : 1).
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Table Group
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2
I
click products of azidophenylsulfoxides 10 and
Table 3 Group II click products 46–57 of halogenated azidomethyl-
sulfoxides 11, 12 and azidophenyl sulfones 14, 15 with selected arylacetyl-
enes 17–19
azidophenyl sulfones 13 with selected arylacetylenes 16–24
Compound
Yielda (%)
28, R1 = Ph, R2 = 3-pyridyl, R3 = SO
46
86
97
95
60
58
39
79
89
73
63
69
98
54
97
77
97
63
Compound
Yielda (%)
29, R1 = Ph, R2 = 4-fluorophenyl, R3 = SO
46, R1 = 4-fluorophenyl, R2 = 3-fluorophenyl, R3 = SO
47, R1 = 4-fluorophenyl, R2 = 4-fluorophenyl, R3 = SO
48, R1 = 4-fluorophenyl, R2 = 2-methoxypheny, R3 = SO
49, R1 = 4-chlororophenyl, R2 = 3-fluorophenyl, R3 = SO
50, R1 = 4-chlorophenyl, R2 = 4-fluorophenyl, R3 = SO
51, R1 = 4-chlorophenyl, R2 = 2-methoxyphenyl, R3 = SO
52, R1 = 4-fluorophenyl, R2 = 3-fluorophenyl, R3 = SO2
53, R1 = 4-fluorophenyl, R2 = 4-fluorophenyl, R3 = SO2
54, R1 = 4-fluorophenyl, R2 = 2-methoxyphenyl, R3 = SO2
55, R1 = 4-chlororophenyl, R2 = 3-fluorophenyl, R3 = SO2
56, R1 = 4-chlorophenyl, R2 = 4-fluorophenyl, R3 = SO2
57, R1 = 4-chlorophenyl, R2 = 2-methoxyphenyl, R3 = SO2
78
83
92
52
93
76
79
83
95
84
93
81
30, R1 = Ph, R2 = 3-fluorophenyl, R3 = SO
31, R1 = Ph, R2 = 2-methoxyphenyl, R3 = SO
32, R1 = Ph, R2 = 2-(trifluoromethyl)phenyl, R3 = SO
33, R1 = Ph, R2 = 6-methoxynaphthalene-2-yl, R3 = SO
34, R1 = Ph, R2 = 4-(n-pentyl)phenyl, R3 = SO
35, R1 = Ph, R2 = 4-(phenoxy)phenyl, R3 = SO
36, R1 = Ph, R2 = 3,5-di-(trifluoromethyl)phenyl, R3 = SO
37, R1 = Ph, R2 = 3-pyridyl, R3 = SO2
38, R1 = Ph, R2 = 4-fluorophenyl, R3 = SO2
39, R1 = Ph, R2 = 3-fluorophenyl, R3 = SO2
40, R1 = Ph, R2 = 2-methoxyphenyl, R3 = SO2
41, R1 = Ph, R2 = 2-(trifluoromethyl)phenyl, R3 = SO2
42, R1 = Ph, R2 = 6-methoxynaphthalene-2-yl, R3 = SO2
43, R1 = Ph, R2 = 4-(n-pentyl)phenyl, R3 = SO2
44, R1 = Ph, R2 = 4-(phenoxy)phenyl, R3 = SO2
45, R1 = Ph, R2 = 3,5-di-(trifluoromethyl)phenyl, R3 = SO2
a
Yields are for isolated pure compounds.
of the 2,4,6-triazine.30–33 Furthermore, the utilization of halo-
gens and halogen isosteres as substituents on the aryl rings
of the NITVK/oxazole framework has been commented on in
our previous in vitro and in vivo studies,12,13 and as a result
we decided on the array of azide coupling partners bearing
halogenated and methoxy substitution 73–87 (Scheme 4) to
be coupled with the acetylenic triazines 25–27. The prepara-
tion of the azidophenyloxazole-based click partners 73–87
which were reacted with the acetylenic triazines 25–27 is de-
a
Yields are for isolated pure compounds.
together with the corresponding azido bis-(4-chlorophenyl)
sulfoxide and sulfone 12, 15 (from Scheme 1) were reacted
with arylacetylenes 17–19 to afford the sulfinyl and sulfonyl-
linked triazole click products 46–57 in yields ranging from
52% to 93% (Scheme 3). The click reaction between azides
11, 12, 14 and 15 and the group of arylacetylenes 17–19 were
conducted under the same conditions as those detailed in
Scheme 2. The yields of the triazole click products 46–57 are
presented in Table 3.
The third group (Group III) of second-generation inhibi-
tors includes a more varied array of the VXXLL sectors which
are based on the 1,3,5-trisubstituted-2,4,6-triazine motif put
forth by Katzenellenbogen and others29 in their work on
mimicking helical peptide NR boxes containing suitably jux-
taposed leucine residues. Thus, the acetylenic triazine click
partners 25–27 (from Table 1) are constructs which have the
moderately hydrophobic dialkylamino as well as the highly
hydrophobic fluorophenylamino groups at positions 3 and 5
tailed in Scheme 4. The appropriate benzoin, where R1
=
phenyl, 4-fluorophenyl, 4-chlorophenyl, 4-methoxyphenyl or
2-furyl, was reacted with 2-azido-, 3-azido- or 4-azidobenzoyl
chloride in the presence of 4-dimethylaminopyridine to give
the corresponding benzoin esters 58–72. The yields ranged
from 29% to 99% after purification by column chromatogra-
phy on silica gel. Cyclization of the benzoin esters 58–72 to
the corresponding 4,5-diaryl-2-azidophenyloxazoles 73–87 was
accomplished by heating the benzoin esters with ammonium
acetate in acetic acid (115–120 °C). The 4,5-diaryloxazoles
73–87 having 2-azidophenyl, 3-azidophenyl or 4-azidophenyl
substituents at the 2-position of the oxazole ring were puri-
fied by silica gel column chromatography and provided crys-
talline products in yields ranging from 13% to 94% with the
cyclization of the 2-furyl esters giving the lowest yields. The
acetylenic triazine click partners 25–27 which comprised the
VXXLL scaffold portion of click products 90–124 (Table 4)
were prepared from 2,4,6-trichloro-1,3,5-triazine (cyanuric
chloride) (Scheme 5). Lithioethynyltrimethylsilane, formed
from ethynyltrimethylsilane and n-butyllithium (THF/0–5 °C),
was added to cyanuric chloride to give 2,4-dichloro-6-((tri-
methylsilyl)ethynyl)-1,3,5-triazine 88 (0 °C → rt/3 h). Direct
addition of excess amine (diethylamine, 2-fluoroaniline or
4-fluoroaniline) to 88 followed by stirring (0 °C → rt/16 h)
gave the TMS-acetylenic 3,5-diaminotriazines 89a–c which
Scheme 3 Click reactions of halogenated azidomethylsulfoxides 11
and 12 and azidophenylsulfones 14 and 15 with selected arylacetylenes
17–19 to give Group II click products 46–57. aReagents/conditions:
CuSO4·5H2O/Na ascorbate/THF–H2O (2 : 1).
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dues.37 All of the click products described herein (Tables 2–4)
were formed as a result of copperIJI) catalysis which gave ex-
clusively the 1,4- or “anti”-substituted 1,2,3-triazole.21
2.2. Bioassay results and discussion
To assess the functional activity of the click products, com-
pounds 28–57 (Groups I and II) and 90–124 (Group III) were
examined for inhibition of P. gingivalis adherence to S.
gordonii using an established biofilm model system as de-
scribed in section 4.2.1.38 A representative dose response se-
ries of inhibition of P. gingivalis adherence for compound
111 is shown in Fig. 2. For each compound tested, an IC50
value for adherence inhibition was approximated after quan-
tifying the ratio of green to red fluorescence and these values
are shown in Table 5. Eleven compounds exhibited IC50
values <10 μM and the most potent of these exhibited IC50
values that were approximately 2.5-fold lower than the most
potent of the previously reported generation
1 com-
pounds.12,13 Compounds 54, 56, 93, 95, 102, 103, 111, 115
and 122 were also tested to determine if they affect the plank-
tonic growth of P. gingivalis or S. gordonii. As shown in
Table 6, little effect on planktonic growth of P. gingivalis or S.
gordonii cultures was observed for any of the compounds
tested when cultures were incubated overnight in the pres-
ence of 40 μM compound. For the four most potent com-
pounds (95, 111, 115 and 122), this concentration is approxi-
mately 10- to 20-fold greater than the IC50 values determined
for inhibition of adherence. This indicates that the com-
pounds do not function as antibiotics but rather specifically
inhibit P. gingivalis adherence to streptococci. Indeed, our
goal is to develop targeted approaches to prevent colonization
of the oral cavity by the periodontal pathogen P. gingivalis.
The limited distribution of the Mfa protein in other oral bac-
teria will prevent these compounds from acting on other
commensal oral bacteria and compounds that target Mfa
function will likely be highly selective agents. In comparing
and contrasting the six compounds which exhibit the highest
inhibitory activity, five (95, 102, 111, 115, and 122) are in
Scheme 4 Synthesis of 2-(2-azidophenyl)-, 2-(3-azidophenyl)- and
2-(4-azidophenyl)-4,5-diaryloxazole click partners 73–87 through
benzoin esters 58–72. Reagents/conditions: (a) DMAP/DCM/16 h. (b)
NH4OAc/HOAc/115–120 °C/3–4 h. Yields are in parentheses.
were purified by column chromatography. Removal of the tri-
methylsilyl group was accomplished with tetra-n-
butylammonium fluoride (TBAF/THF/0 °C
→ rt) which
afforded the acetylenic triazines 25–27. The click reactions of
azides 73–87 with the arylacetylenic substrates 25–27 were
conducted using the same copperIJII) sulfate/sodium ascorbate
protocol with tetrahydrofuran/water as a solvent system
(Scheme 6). The regiochemistry of the dipolar cycloaddition
‘click’ reaction affords products in which the substitution
could be 1,4- or 1,5- on the triazole ring depending on the re-
agents and the conditions used. The click reactions afforded
the corresponding 4,5-diaryloxazolyl-1,2,3-triazoles 90–124 as
solid compounds with the expected 1,4-regiochemistry. In-
cluded with our Group III candidates are a new set of com-
pounds 98, 99, 108, 109, and 122–124 (Table 4) where both
phenyl rings at the 4,5-positions of the oxazole were
substituted with furan rings. Replacement of benzene with
furan is not an uncommon example of effective bioisosteric
replacement in medicinal chemistry.34 The furan system is
not only π-electron rich but also carries a mild hydrogen-
bonding component along with its aromatic structure. The
yields of the triazole click products 90–124 are listed in
Table 4. The oxazole and triazole motifs in the backbone of
our peptidomimetic inhibitor candidates offer rigidity as well
as the potential for hydrogen bonding. Since the introduction
of click chemistry, the 1,2,3-triazole has been effectively
deployed as an easily formed peptide bond isostere thereby
linking complex polypeptides as well as sectors of less com-
plex peptidomimetics.35,36 A similar case is made for the oxa-
zole/NITVK scaffold whereby its conferred rigidity influences
the disposition of the distal 4,5-(halogenated) diphenyl resi-
Group III and one (46) is in Group II. Compound 46 (IC50
,
5.3 μM) is the only candidate which possesses a sulfoxide
group as part of the backbone as well as an m-fluorophenyl
group on the triazole linker which mimics the VXXLL motif.
Similar to two other highly active compounds 102 (5.0 μM)
and 115 (2.3 μM), the NITVK-mimic sector is the 4,5-di-(4-
fluorophenyl)oxazole. The sulfoxide group does impart asym-
metry to the molecule; however, the mixture of enantiomers,
assuming the peracid oxidation was racemic, was not sepa-
rated and bioassayed. The Group III active compounds
(95–117) all contain the 1,3,5-trisubstituted-2,4,6-triazine
modeled after the Rodriguez et al. rendering of an active
motif whereby the key leucine residues which mimicked the
VXXLL receptor box lie in a triangular arrangement.29 While
the original iteration of the Katzenellenbogen VXXLL mimics
were the hydrophobic 3-disubstituted-di-N-alkylamino-triazine
motifs, we introduced the hydrophobic 2- and
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Table 4 Group III click products of azides 73–87 and acetylenes 25–27
Compound
Yielda (%)
90, R1 = phenyl, R2 = 3-triazolyl, R3, R4 = ethyl
76
90
48
45
52
57
53
63
52
46
80
65
95
77
70
69
66
75
69
83
30
82
48
77
87
26
83
55
51
43
77
67
49
69
68
91, R1 = phenyl, R2 = 4-triazolyl, R3, R4 = ethyl
92, R1 = 4-fluorophenyl, R2 = 3-triazolyl, R3, R4 = ethyl
93, R1 = 4-fluorophenyl, R2 = 4-triazolyl, R3, R4 = ethyl
94, R1 = 4-chlorophenyl, R2 = 3-triazolyl, R3, R4 = ethyl
95, R1 = 4-chlorophenyl, R2 = 4-triazolyl, R3, R4 = ethyl
96, R1 = 4-methoxyphenyl, R2 = 3-triazolyl, R3, R4 = ethyl
97, R1 = 4-methoxyphenyl, R2 = 4-triazolyl, R3, R4 = ethyl
98, R1 = 2-furyl, R2 = 3-triazolyl, R3, R4 = ethyl
99, R1 = 2-furyl, R2 = 4-triazolyl, R3, R4 = ethyl
100, R1 = phenyl, R2 = 3-triazolyl, R3 = 2-fluorophenyl, R4 = H
101, R1 = phenyl, R2 = 4-triazolyl, R3 = 2-fluorophenyl, R4 = H
102, R1 = 4-fluorophenyl, R2 = 3-triazolyl, R3 = 2-fluorophenyl, R4 = H
103, R1 = 4-fluorophenyl, R2 = 4-triazolyl, R3 = 2-fluorophenyl, R4 = H
104, R1 = 4-chlorophenyl, R2 = 3-triazolyl, R3 = 2-fluorophenyl, R4 = H
105, R1 = 4-chlorophenyl, R2 = 4-triazolyl, R3 = 2-fluorophenyl, R4 = H
106, R1 = 4-methoxyphenyl, R2 = 3-triazolyl, R3 = 2-fluorophenyl, R4 = H
107, R1 = 4-methoxyphenyl, R2 = 4-triazolyl, R3 = 2-fluorophenyl, R4 = H
108, R1 = 2-furyl, R2 = 3-triazolyl, R3 = 2-fluorophenyl, R4 = H
109, R1 = 2-furyl, R2 = 4-triazolyl, R3 = 2-fluorophenyl, R4 = H
110, R1 = phenyl, R2 = 2-triazolyl, R3 = 4-fluorophenyl, R4 = H
111, R1 = phenyl, R2 = 3-triazolyl, R3 = 4-fluorophenyl, R4 = H
112, R1 = phenyl, R2 = 4-triazolyl, R3 = 4-fluorophenyl, R4 = H
113, R1 = 4-fluorophenyl, R2 = 2-triazolyl, R3 = 4-fluorophenyl, R4 = H
114, R1 = 4-fluorophenyl, R2 = 3-triazolyl, R3 = 4-fluorophenyl, R4 = H
115, R1 = 4-fluorophenyl, R2 = 4-triazolyl, R3 = 4-fluorophenyl, R4 = H
116, R1 = 4-chlorophenyl, R2 = 2-triazolyl, R3 = 4-fluorophenyl, R4 = H
117, R1 = 4-chlorophenyl, R2 = 3-triazolyl, R3 = 4-fluorophenyl, R4 = H
118, R1 = 4-chlorophenyl, R2 = 4-triazolyl, R3 = 4-fluorophenyl, R4 = H
119, R1 = 4-methoxyphenyl, R2 = 2-triazolyl, R3 = 4-fluorophenyl, R4 = H
120, R1 = 4-methoxyphenyl, R2 = 3-triazolyl, R3 = 4-fluorophenyl, R4 = H
121, R1 = 4-methoxyphenyl, R2 = 4-triazolyl, R3 = 4-fluorophenyl, R4 = H
122, R1 = 2-furyl, R2 = 2-triazolyl, R3 = 4-fluorophenyl, R4 = H
123, R1 = 2-furyl, R2 = 3-triazolyl, R3 = 4-fluorophenyl, R4 = H
124, R1 = 2-furyl, R2 = 4-triazolyl, R3 = 4-fluorophenyl, R4 = H
a
Yields are for isolated purified compounds.
4-(fluorophenyl)amino groups at positions 3 and 5 of the
2,4,6-triazine (see compounds 102, 111, 115, and 122). Com-
pound 95, however (IC50 = 3.7 μM), contains the more closely
related, nonaromatic lipid-like diethylamino groups at posi-
tions 3 and 5 of the 2,4,6-triazine motif. The deployment of
furan rings at the 4,5-positions of the oxazole in compounds
98, 99, 108, 109, and 122–124 led to only one compound 122
Scheme 5 Synthesis of the acetylenic triazine click partners 25–27.
Reagents/conditions: (a) ethynyltrimethylsilane/n-BuLi/THF/0 °C/1 h;
(b) 4-fluoroaniline, 2-fluoroaniline or diethylamine/THF/0 °C to rt/16 h;
(c) TBAF/THF/0 °C to rt/2 h.
Scheme 6 Click reactions of azides 73–87 with acetylenes 25–27 to
give Group III triazole products 90–124. aConditions/reagents: CuSO4
·5H2O/Na ascorbate/THF–H2O (2 : 1).
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Table 6 Inhibition of planktonic growth
Percent inhibition of:
IC50
Compound
(μM)
P. gingivalis
S. gordonii
Teta
95
111
115
122
54
56
93
102
103
95.7 1.2
0.4 6.1
8.3 3.6
94.2 1.7
−0.8 2.8
2.9 3.9
−0.6 5.8
−0.6 3.9
−3.3 6.0
6.2 8.8
−2.0 8.9
−0.6 0.8
−8.6 2.0
3.7
4.3
2.3
2.4
21.2
6.3
9.4
5.0
12.3
−2.8 6.4
2.5 5.4
−3.4 10.4
−2.7 6.6
2.0 6.1
−8.3 5.2
−1.3 14.2
Fig. 2 Inhibition of P. gingivalis (green) adherence to S. gordonii (red)
by compound 111. Biofilms were formed in the presence of PBS/0.1%
DMSO (A), or 5 μM (B), 20 μM (C), or 40 μM (D) compound 111.
a
Inhibition of planktonic growth by 10 μg ml−1 tetracycline.
2.2.1. Cytotoxicity of compounds 95, 111, 115 and 122. To
initially assess the cytotoxicity of the most active compounds,
telomerase immortalized gingival keratinocytes (TIGK cells)
were incubated with each compound and the release of lac-
tate dehydrogenase (LDH) into the culture medium was
followed as a measure of cell lysis. In addition, the level of
cellular metabolism was determined by measuring ATP
(IC50 2.4 μM) of significant activity. Interestingly, along with
the di-furyloxazole arrangement, candidate 122 possessed the
3,5-di-(4-fluorophenyl)aminotriazine, and was also the com-
pound which exhibited the most bent conformation by na-
ture of the ortho disposition of the oxazole to the triazole
linker.
Table 5 IC50 values for Group I compounds (28–45), Group II compounds (46–57) and Group III compounds (90–124)
a
a
Group I
IC50 (μM)
Group II
IC50 (μM)
Group III
IC50a (μM)
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
NA
NA
47.3
NA
NA
NA
NA
NA
NA
46
47
48
49
50
51
52
53
54
55
56
57
5.30
35.9
7.20
15.1
39.8
10.2
40.0
40.0
21.2
18.2
6.3
90
91
92
93
94
95
96
97
98
20.7
NA
NA
9.4
16.0
3.7
NA
36.6
NA
NA
9.6
NA
99
15.7
33.6
13.1
NA
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
20.0
NA
5.00
12.3
33.8
NA
NA
40.0
NA
NA
NA
4.3
NA
49.2
NA
2.3
>40.0
27.8
13.3
NA
NA
NA
2.4
8.00
7.8
NA
36.6
16.5
38.5
a
NA = not active.
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levels. As shown in Fig. 3A and B, lysis of TIGK cells results 3. Conclusions
in a significant increase in LDH release relative to the me-
We have prepared a number of newer “second-generation”
click compounds which are effective inhibitors of P. gingivalis
adherence to oral streptococci. While we noted previously
that lead optimization of our first-generation inhibitors could
occur through adjustment/positioning of the hydrophobic
groups of the mono-aryl and diaryloxazole scaffolds, we in-
stead opted to change the nature of the linkers conjoining
the two scaffolds and keep the aryl substitution constant.
The new linkers comprised both sulfonyl and sulfoxide
groups. Through exploring the diaminotriazine motif,
changes were also made to optimize the VXXLL sectors of the
general inhibitor scaffold. Four of these “second-generation”
compounds (95, 111, 115 and 122) were shown to be more
potent inhibitors of P. gingivalis adherence than our “first-
generation” compounds. Of these, only compound 111
exhibited cytotoxicity against TIGK cells and this was ob-
served only at the highest concentration tested. As in the de-
velopment of the first-generation compounds, the evaluation
of this new series of candidates in pertinent in vivo animal
studies will be reported in due course.
dium only or medium/DMSO controls. None of the com-
pounds induced a significant increase in LDH release at any
of the tested concentrations. Lysis of TIGK cells also resulted
in a significant decrease in cellular ATP levels (Fig. 3B). Incu-
bation of cells with compounds 95, 115 or 122 did not signifi-
cantly reduce the levels of ATP. In contrast, incubation of
TIGK cells with compound 111 appeared to result in a dose-
dependent reduction of ATP levels; however, only the reduc-
tion observed at 60 μM was statistically significant. These re-
sults suggest that with the possible exception of compound
111, the four most potent inhibitors of P. gingivalis adherence
to oral streptococci exhibit little cytotoxic activity against gin-
gival epithelial cells. It should also be noted that the cytotoxic
activity of compound 111 was only observed at a concentra-
tion that is approximately 15-fold greater than its IC50 for in-
hibition of P. gingivalis adherence.
4. Experimental
4.1. Chemistry
Unless otherwise specified, all solvents and reagents were
ACS grade and were used as supplied. Solvents were removed
from reaction mixtures and extracts using standard Büchi ro-
tary evaporators under a water aspirator vacuum. Alkynes
16–24 were commercially available and were used as sup-
plied. All melting points (mp) were determined using a
Thomas-Hoover apparatus. Infrared spectra were recorded on
a Perkin-Elmer Spectrum Version 10.02.00 instrument and
absorptions are reported as reciprocal centimeters (cm−1).
Proton (1H) and carbon (13C) NMR spectra were recorded on
a Varian INOVA instrument (400 MHz, 100 MHz for 13C) or a
Bruker instrument (500 MHz, 125 MHz for 13C). High-
resolution mass spectra (HRMS) were recorded using electro-
spray ionization (ESI). All air- and moisture-sensitive reac-
tions were run in oven-dried glassware under an atmosphere
of dry nitrogen. Gravity-column chromatography was
performed on silica gel 60 (E. Merck, 7734, 70–230 mesh).
Thin-layer chromatography (TLC) was performed with glass-
backed plates (E. Merck, 5715, silica gel 60 F254 2.5 mm thick-
ness) and visualized using 2% anisaldehyde in ethanol, 2.5%
phosphomolybdic acid in ethanol or 10% sulfuric acid in
ethanol.
4.1.1. General procedure for the synthesis of amino-
phenylsulfides 4–6. To a suspension of freshly washed so-
dium hydride (1.2 equiv.) in dry THF (15 mL) was added
4-aminothiophenol (1.0 equiv.) under a nitrogen atmosphere
at 0–5 °C followed by stirring for 30 min. To this mixture was
added dropwise a solution of compounds 1–3 (1.1 equiv.)
dissolved in THF (15 mL) at 0–5 °C. After the addition was
complete, the reaction temperature was gradually raised to
Fig.
3 Release of lactate dehydrogenase (LDH) activity (A) and
measurement of ATP levels (B) in TIGK cells after 18 h exposure to
peptidomimetic compounds. Concentrations of compounds used are
as follows: 95 (5, 20 and 40 μM), 111 (5, 20, 60 μM), 115 (5, 10, 20 μM)
and 122 (5, 20, 60 μM). The asterisk indicates a significant decrease in
ATP relative to cells exposed to medium containing 0.1% DMSO; *p <
0.05.
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room temperature and allowed to stir (16 h). After comple-
tion of the reaction, as indicated by TLC (hexane/ethyl ace-
tate, 3 : 1), the reaction mixture was quenched with cold water
(25 mL). The resulting reaction mixture was extracted into
dichloromethane (2 × 25 mL) and the organic layers were
combined, dried over anhydrous sodium sulfate, filtered and
evaporated. The obtained off-white crude residue was submit-
ted to silica gel column chromatography (hexanes/ethyl ace-
tate, 3 : 1) to give aminophenylsulfides 4–6 as pure off-white
products.
saturated aqueous NaHCO3 solution and the organic layer
was separated and dried over anhydrous sodium sulfate. Con-
centration of the organic layer provided an off-white residue
of the corresponding sulfoxides and sulfones which were sub-
mitted to column chromatography (hexane/ethyl acetate) to
afford the pure azidophenyl sulfoxides and sulfones 10–15.
4.1.3.1. 2-(((4-Azidophenyl)sulfinyl)methyl)-4,5-diphenyloxa-
zole (10). Colorless oil; yield 86%; Rf = 0.1 (hexane/ethyl
acetate, 3 : 1); FT-IR: 3055, 2125, 2090, 1586, 1051 cm−1; 1H
NMR (400 MHz, CDCl3): δ 7.60 (d, J = 8.4 Hz, 2H), 7.57–7.55
(m, 2H), 7.47–7.45 (m, 2H), 7.39–7.34 (m, 6H), 7.14 (d, J = 8.4
Hz, 2H), 4.41 (d, J = 13.2 Hz, 1H), 4.21 (d, J = 13.2 Hz, 1H)
ppm; 13C NMR (175 MHz, CDCl3): δ 153.1, 147.1, 144.0,
138.8, 136.1, 131.7, 129.0, 128.7, 128.6, 128.4, 126.1, 127.8,
126.5, 126.1, 119.8, 56.2 ppm; HRMS (+ESI) m/z calcd for
[C22H16N4O2S]+ 401.1072, found 401.1119 ([M + H]+).
4.1.1.1. 4-(((4,5-Diphenyloxazol-2-yl)methyl)thio)aniline (4).
Light yellow solid; yield 55%; mp = 131–133 °C; Rf = 0.17
(hexane/ethyl acetate, 7 : 3); FT-IR: 2125, 2090, 1591, 1487,
1
1291 cm−1; H NMR (500 MHz, CDCl3): δ 7.61 (dd, J = 1.5 Hz,
3.5 Hz, 2H), 7.53 (dd, J = 2.0 Hz, 4.0 Hz, 2H), 7.37–7.29 (m,
8H), 6.60 (dd, J = 2.0 Hz, 6.5 Hz, 2H), 4.07 (s, 2H), 3.75 (s,
2H) ppm; 13C NMR (125 MHz, CDCl3): δ 160.1, 147.0, 145.8,
135.5 (overlap), 132.3, 128.5 (overlap), 128.0 (overlap), 127.9
(overlap), 126.4 (overlap), 121.2, 115.4 (overlap), 33.7 ppm;
HRMS (+ESI): calcd for [C22H18N2O5] 359.1218, found
359.1267 ([M + H]+).
4.1.4. General procedure for the synthesis of acetylenic tri-
azines 25–27. To a solution of ethynyltrimethylsilane (1.0
equiv.) in dry THF (5.0 mL) was added n-butyllithium (1.6 M
solution in hexane, 1.0 equiv.) by syringe at 0 °C under argon
while stirring. After stirring for one hour at 0 °C, the resulting
solution of lithioethynyltrimethylsilane was cannulated
dropwise onto a solution of cyanuric chloride (1.0 equiv.) in
dry THF. The resulting viscous red-brown suspension was
stirred at 0 °C (2 h) before the corresponding amines
(diethylamine, 2-fluoroaniline or 4-fluoroaniline, 2.0 equiv.)
were added dropwise at 0 °C. The reaction temperature was
gradually increased to room temperature and the mixture was
allowed to stir for 48 h. After completion of the reaction, as
monitored by TLC, the reaction mixture was quenched with
cold water and extracted into dichloromethane (2 × 25 mL).
The organic layers were combined, dried over anhydrous so-
dium sulfate and evaporated to give a crude brownish residue
which was further purified by using column chromatography
to obtain the pure TMS acetylenic triazines 89a–89c. To a
prechilled solution of the corresponding trimethylsilyl triazines
89a–89c (1.0 equiv.) in dry THF (25 mL) was slowly added
tetra-n-butylammonium fluoride (TBAF, 1.0 equiv.) under a ni-
trogen atmosphere. The resulting brown solution was stirred
at 5–10 °C (1 h). The progress of the reaction was monitored
by TLC (hexane/ethyl acetate, 9 : 1). After completion of the re-
action, cold water (25 mL) was added to the reaction mixture
followed by extraction into dichloromethane (2 × 25 mL). The
combined organic layers were dried over anhydrous sodium
sulfate, filtered and evaporated to obtain a crude residue
which was submitted to silica gel column chromatography
(hexane/ethyl acetate, 9 : 1) to give the pure acetylenic diamino-
triazines 25–27 as solid materials.
4.1.2. General procedure for the synthesis of
azidophenylsulfides 7–9. To a pre-chilled solution of com-
pounds 4–6 (1.0 equiv.) in aqueous HCl (4 N) was added
dropwise a chilled aqueous solution of sodium nitrite (1.1
equiv./10 mL/H2O). The resulting yellowish reaction mixture
was allowed to stir at 0–5 °C (1 h). To this clear solution, was
added dropwise an aqueous solution of sodium azide (1.05
equiv./10 mL/H2O) and stirring was continued (16 h) at room
temperature. The progress of the reaction was monitored by
TLC (hexane/ethyl acetate). After completion of the reaction,
the reaction mixture was extracted into dichloromethane (2 ×
25 mL) and organic layers were combined. The organic layer
was washed with saturated NaHCO3 solution (30 mL), sepa-
rated, dried over anhydrous sodium sulfate and filtered. Con-
centration of the filtrate gave the crude residue of the corre-
sponding products which was submitted to column
chromatography (hexane/ethyl acetate, 3 : 1) to give the pure
azidophenyl sulfides 7–9.
4.1.2.1. 2-(((4-Azidophenyl)thio)methyl)-4,5-diphenyloxazole
(7). Light brown solid; yield 87%; mp = 64–65 °C; Rf = 0.5
(hexane/ethyl acetate, 7.5 : 2.5); FT-IR: 2125, 2090, 1591, 1487,
1
1291 cm−1; H NMR (400 MHz, CDCl3): δ 7.59 (d, J = 8.0 Hz,
2H), 7.53–7.50 (m, 3H), 7.48 (s, 1H), 7.38–7.34 (m, 6H), 6.98
(d, J = 8.4 Hz, 2H), 4.19 (s, 2H) ppm; 13C NMR (125 MHz,
CDCl3): δ 159.3, 145.9, 139.6, 135.3, 133.3, 132.0, 130.4, 128.5,
128.4, 128.1, 127.8, 126.3, 119.5, 31.9 ppm; HRMS (+ESI) m/z
calcd for [C22H16N4OS]+ 385.1123, found 385.1133 ([M + H]+).
4.1.3. General procedure for the synthesis of azidophenyl
sulfoxides and sulfones 10–15. To a clear solution of com-
pounds 7–9 (1.0 equiv.) in dichloromethane (30 mL) was
added m-CPBA (1.2 equiv. for synthesis of sulfoxides 10–12
and 3.0 equiv. for sulfones 13–15) and the resulting solution
was stirred for 16 h at room temperature. The reaction prog-
ress was monitored by TLC (hexane/ethyl acetate). After com-
pletion of the reaction, the reaction mixture was washed with
4.1.4.1. N2,N2,N4,N4-Tetraethyl-6-((trimethylsilyl)ethynyl)-
1,3,5-triazine-2,4-diamine (89a). Pale yellow solid; 50% yield;
mp = 102–103 °C; Rf = 0.56 (hexane/ethyl acetate, 3 : 1); FT-IR:
1
2981, 2964, 2932, 1535, 1493, 1359, 1249, 1079 cm−1; H NMR
(400 MHz, CDCl3): δ 3.60 (s, 4H), 3.53 (d, J = 6.4 Hz, 4H), 1.15
(t, J = 7.2 Hz, 12H), 0.26 (s, 9H) ppm; 13C NMR (125 MHz,
CDCl3): δ 163.7, 158.3, 110.0, 103.6, 90.6, 40.9, 13.5, 12.8,
−0.28 ppm; LRMS (+ESI) for [C16H29N5Si], found 319.
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4.1.4.2. N2,N2,N4,N4-Tetraethyl-6-ethynyl-1,3,5-triazine-2,4-
diamine (25). Off-white solid; 59% yield; mp = 47–49 °C; Rf =
0.33 (hexane/ethyl acetate, 9 : 1); FT-IR: 3228, 2977, 2933,
2112, 1539, 1492, 1355, 1084 cm−1; 1H NMR (400 MHz,
CDCl3): δ 3.59 (d, J = 6.0 Hz, 4H), 3.53 (d, J = 6.0 Hz, 4H),
2.87 (s, 1H), 1.15 (t, J = 7.2 Hz, 12H) ppm; 13C NMR (125
MHz, CDCl3): δ 163.6, 157.9, 82.7, 72.9, 41.1, 13.5, 12.8 ppm;
LRMS (+ESI) for [C13H21N5], found 247.
4.1.5. General procedure for the synthesis of benzoin es-
ters 58–72. To a clear pre-chilled solution of benzoin, the
halogenated benzoins or furoin (1.0 equiv.) in
dichloromethane (50 mL) was added DMAP (1.5 equiv.) and
stirring was continued (30 min). To the reaction mixture was
added dropwise a solution of 2-, 3- or 4-azidobenzoyl chloride
(prepared by reacting the corresponding azidocarboxylic acids
(1.3 equiv.) with thionyl chloride at 85–90 °C for 2 h)
dissolved in dichloromethane (20 mL) under a nitrogen at-
mosphere. The progress of the reaction was monitored by
TLC (hexane/ethyl acetate). After completion of the reaction,
the reaction mixture was washed with aqueous HCl (5%, 2 ×
50 mL) followed by aqueous NaHCO3 (5%). The organic layer
was separated, dried over anhydrous sodium sulfate, filtered
and evaporated to obtain the crude residue of corresponding
azidobenzoyl esters which were purified by column chroma-
tography on gravity silica gel (hexane/ethyl acetate) to give
the pure 2-, 3-, 4-azidobenzoyl esters 58–72.
4.1.5.1. 2-Oxo-1,2-diphenylethyl 2-azidobenzoate (58).
Colorless oil; yield 95%; Rf = 0.4 (hexane/ethyl acetate, 3 : 1);
FT-IR: 3065, 2118, 2090, 1714, 1686, 1596, 1487, 1235 cm−1;
1H NMR (400 MHz, CDCl3) δ 8.08 (dd, J = 1.2 Hz, 7.6 Hz, 1H),
7.98 (dd, J = 1.2 Hz, 8.2 Hz, 2H), 7.57–7.51 (m, 4H), 7.44–7.35
(m, 5H), 7.24–7.17 (m, 1H), 7.08 (s, 1H) ppm; 13C NMR (100
MHz, CDCl3): δ 193.6, 164.3, 140.6, 134.7, 133.7, 133.5, 133.4,
132.4, 129.4, 129.2, 128.9, 128.7, 128.6, 124.5, 121.4, 119.8,
78.1 ppm; LRMS for [C21H15N3O5], found 358 (M + H).
4.1.6. General procedure for the synthesis of 4,5-diaryl-2-
azidophenyloxazoles 73–87. The azidobenzoyl esters 58–72
(1.0 equiv.) were dissolved in glacial acetic acid (100 mL) at
room temperature. To the clear solution was added ammo-
nium acetate (15 equiv.) under a nitrogen atmosphere. The
resulting reaction mixture was heated at 115 °C (oil bath tem-
perature) and the temperature was maintained for 3 h. After
completion of the reaction, as indicated by TLC, the reaction
mixture was cooled to room temperature. Cold water (150
mL) was added to the reaction mixture which was then slowly
neutralized with saturated NaHCO3 solution. The crude prod-
uct was extracted with dichloromethane (2 × 50 mL) and the
organic extracts were combined, dried over anhydrous so-
dium sulfate and filtered. Concentration of the dried extracts
provided the corresponding crude azido oxazoles which were
submitted to silica gel column chromatography (hexane/ethyl
acetate) and afforded the pure 4,5-diaryl-2-azidophenyloxa-
zoles 73–87.
NMR (400 MHz, CDCl3) δ 8.32 (d, J = 7.6 Hz, 1H), 7.94 (dd, J
= 8.0 Hz, 18.4 Hz, 4H), 7.70 (t, J = 7.6 Hz, 1H), 7.65–7.52 (m,
7H), 7.47 (t, J = 8.0 Hz, 1H) ppm; 13C NMR (100 MHz, CDCl3)
δ 157.8, 145.9, 138.2, 136.7, 134.9, 132.5, 131.4, 130.7, 129.9,
129.0, 128.9, 128.7, 128.69, 128.64, 128.3, 126.6, 124.9, 119.8,
119.2 ppm; HRMS (ESI) m/z calcd for [C21H15N4O] 339.1240,
found 339.1242.
4.1.7. General procedure for the synthesis of the click
triazole products 28–57 and 90–124. To a solution of the
group of the azidophenylsulfoxide and sulfone click partners
10–15 and the azidophenyl click partners 73–87 (1.0 equiv.)
in anhydrous THF (2.0 mL) were added the requisite acetyle-
nic click partners 16–27 (1.1 equiv.) followed by addition of
solid copper sulfate pentahydrate (0.1 equiv.) at room tem-
perature. To the reaction mixture was then added a freshly
prepared clear solution of sodium ascorbate (0.5 equiv.) in
water (1 mL). The resulting reaction mixture was stirred at
room temperature (5–16 h). The progress of the reaction was
monitored by TLC using the mobile phases hexane/ethyl ace-
tate and/or chloroform/methanol. After completion of the re-
action, the reaction mixture was concentrated and the crude
residue was partitioned between dichloromethane (15 mL)
and water (10 mL). The organic layer was separated, dried
over anhydrous sodium sulfate and concentrated to obtain
the crude residue of the click products which were submitted
to silica gel column chromatography (hexanes/ethyl acetate,
or chloroform/methanol) to give the corresponding pure click
product triazoles 28–57 and 90–124.
4.1.7.1. 4,5-Diphenyl-2-(((4-(4-(pyridin-3-yl)-1H-1,2,3-triazol-
1-yl)phenyl)sulfinyl)methyl) oxazole (28). Light yellow solid;
yield 48%; mp = 193–195 °C; Rf = 0.26 (methanol/chloroform,
1 : 9); FT-IR (neat): 3085, 3038, 2986, 2929, 1593, 1507, 1404,
1238, 1049, 687 cm−1; 1H NMR (400 MHz, CDCl3): δ 9.07 (s,
1H), 8.65 (s, 1H), 8.29 (d, J = 8.4 Hz, 1H), 8.19 (s, 1H), 7.96
(d, J = 8.4 Hz, 2H), 7.82 (d, J = 8.0 Hz, 2H), 7.57–7.55 (m, 1H),
7.47–7.42 (m, 3H), 7.37–7.29 (m, 6H), 4.40 (dd, J = 14.0 Hz,
68.4 Hz, 2H) ppm; 13C NMR (175 MHz, CDCl3): δ 152.7,
149.8, 147.2, 147.1, 145.8, 143.7, 139.2, 136.2, 133.2, 131.6,
129.0, 128.72, 128.66, 128.51, 128.0, 127.8, 126.5, 126.1,
126.0, 123.9, 121.1, 117.7, 56.1 ppm; HRMS (+ESI) m/z calcd
for [C29H21N5O2S]+ 504.1494, found 504.1516 ([M + H]+).
4.2. Biology
4.2.1. Bacterial strains and culture conditions. P. gingivalis
ATCC33277 was grown in reduced trypticase soy broth (Difco)
supplemented with 0.5 percent yeast extract, 1 μg mL−1 men-
adione, and 5 μg mL−1 hemin. Twenty five milliliters of me-
dium were reduced for 24 h under anaerobic conditions by
equilibrating in an atmosphere consisting of 10% CO2, 10%
H2, and 80% N2. Following equilibration, P. gingivalis was in-
oculated in the medium and grown for 48 h at 37 °C under
anaerobic conditions. S. gordonii DL-1 (1) was cultured aero-
bically without shaking in brain heart infusion (BHI) broth
supplemented with 1 percent yeast extract for 16 hours at
37 °C. For some compounds that inhibited P. gingivalis
4.1.6.1. 2-(2-Azidophenyl)-4,5-diphenyloxazole (73). Pale
yellow solid; 84% yield; Rf = 0.22 (hexane/ethyl acetate, 9 : 1);
FT-IR: 3059, 2121, 2089, 1581, 1501, 1291, 1070 cm−1; 1H
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adherence to S. gordonii, we determined their effect on plank-
tonic growth of P. gingivalis and S. gordonii. The appropriate
growth medium (10 ml) was supplemented with 10 μl of a 40
mM compound stock solution in DMSO. After inoculation,
broth cultures were incubated as described above and com-
pared to cultures grown in medium that was supplemented
only with 0.1% DMSO. The cell density of each culture was
determined by measuring the optical density at 600 nm (O.
D.600nm) and the percent growth inhibition/stimulation was
calculated using the following equation: [(O.D.600nm of the
control/O.D.600nm of the culture grown in the presence of
compound) − 1] × 100.
nail polish. Visualization of biofilms was carried out by laser
scanning confocal microscopy using a Leica SP8 confocal
microscope (Leica Microsystems Inc., Buffalo Grove, IL) using
a 488 nm laser to detect labeled P. gingivalis and a 552 nm la-
ser to detect S. gordonii. Z-plane scans of 25 μm in depth
were collected at three randomly chosen frames on each cov-
erslip using a z-step thickness of 0.7 μm. Background noise
was minimized using software provided with the Leica SP8
and three-dimensional reconstruction of the Z-plane scans
and quantification of total green and red fluorescence was
conducted using Volocity 6.3 image analysis software (Perkin
Elmer, Akron, Ohio). Data were expressed as the ratio of total
green (P. gingivalis) to red (S. gordonii) fluorescence and the
IC50 for each compound was defined as the concentration
that reduced the ratio of green to red fluorescence by 50%.
Experiments were carried out in triplicate for each concentra-
tion of test compound and three independent experiments
were conducted for each compound. GraphPad InStat3 soft-
ware was used for data analysis and statistical significance
was defined as p < 0.05.
4.2.4. Cell culture. Human telomerase immortalized gingi-
val keratinocytes (TIGKs) were provided by Dr. Richard
Lamont (University of Louisville) and were authenticated by
comparison to primary gingival epithelial cells for cell mor-
phology, growth, cytokeratin expression and the expression
of toll-like receptors. TIGKs were cultured at 37 °C in an at-
mosphere of 5% CO2 in Basal Medium Dermalife K complete
kit with Supplements (LifeLine, Frederick, MD). Cultures
were incubated for 5 days and attained >95% confluence.
4.2.5. Measurement of lactate dehydrogenase (LDH) activ-
ity. Lactate dehydrogenase (LDH) activity was determined
using the CytoTox 96 non-radioactive Cytotoxicity Assay
(Promega). TIGK cells were inoculated in a 96-well microtiter
plate at a density of 4000 cells per well and grown for 24 h.
The medium was then removed and replaced with fresh me-
4.2.2. Biofilm model for in vitro analysis of P. gingivalis
adherence to S. gordonii. Two species biofilms were formed
essentially as previously described.39 To prepare bacterial
cells for biofilm culture, 10 ml of an overnight S. gordonii cul-
ture was centrifuged at 5600 rpm for 5 min and the cell pellet
was suspended in 1 mL sterile PBS. Subsequently, 20 μL of 5
mg ml−1 hexidium iodide (Molecular Probes) was added to
the cell suspension and incubated for 15 min with gentle
shaking at room temperature in the dark. The labeled cells
were centrifuged as described above, washed with phosphate-
buffered saline (10 mM Na2HPO4, 18 mM KH2PO4, 1.37 M
NaCl, and 2.7 mM KCl, pH 7.2; [PBS]) and the cell pellet was
suspended in PBS at a final O.D.600nm of 0.8. Similarly, 10 mL
of a 48 h culture of P. gingivalis was centrifuged at 5600 rpm
for 15 min, the cell pellet was suspended in 1 mL PBS and 20
μL of 5IJ6)-carboxyfluorescein N-hydroxysuccinimide ester (4
mg ml−1, Molecular Probes, Inc.) was added. After incubation
for 30 min with gentle shaking at room temperature in the
dark, the suspension was centrifuged and washed as de-
scribed above and suspended in PBS at a final O.D.600nm of
0.4. For biofilm cultures, 1 mL of labeled S. gordonii cells was
added to each well of a 12-well microtiter plate (Greiner Bio-
one) containing a circular coverslip (Fisher brand) and incu-
bated in an anaerobic chamber with rotary shaking for 24 h
at 37 °C. Unbound cells were removed by aspiration and 1 ml
of labeled P. gingivalis cells containing the desired concentra-
tion of test compound was added and incubated under an-
aerobic conditions for 22 h at 37 °C. Test compounds were
dissolved in dimethyl sulfoxide (DMSO) to generate 1000×
stock solutions and were routinely tested over a final concen-
tration range of 0–60 μM. 1 μL of the appropriate stock solu-
tion was added to each 1 mL aliquot of labeled P. gingivalis
cells and incubated for 30 min at room temperature prior to
adding the suspension to the microtiter plate wells. For con-
trol biofilms, 1 μL of DMSO was added to 1 mL of labeled P.
gingivalis and incubated as described above.
dium
containing
the
desired
concentration
of
peptidomimetic compound. The cells were further cultured
for 18 h in the presence of the peptidomimetic compounds,
centrifuged for 4 min at 250g and 50 μl of supernatant was
transferred to a fresh 96-well microtiter plate. Subsequently,
50 μl of LDH substrate was added per well and plates were in-
cubated at room temperature for 30 min. Reactions were ter-
minated by the addition of 50 μl of stop solution provided in
the CytoTox 96 kit. LDH activity was determined by measur-
ing the optical density at a wavelength of 490 nm. For posi-
tive control reactions, 15 μl of lysis buffer provided in the
CytoTox 96 kit was added to the cells and incubated for 1 h.
Negative control reactions comprised cells that were incu-
bated with medium alone. All samples were assayed in
triplicate.
4.2.6. Measurement of cellular ATP levels. Cell metabolic
activity was assessed by quantifying total ATP levels in cell
culture samples using CellTiterGlo reagent (Promega). TIGK
and J774A.1 cells were cultured and incubated with com-
pounds as described above, washed three times with sterile
PBS and incubated with 100 μl of CellTiterGlo substrate for 2
4.2.3. Visualization of two species biofilms. To visualize P.
gingivalis/S. gordonii biofilms, unbound P. gingivalis cells
were removed by aspiration and coverslips were washed once
with PBS. Biofilms were fixed by incubating the coverslips
with 1 mL of 4% paraformaldehyde for 5 min followed by
two washes with PBS. The coverslips were then removed,
placed face down on a glass microscope slide containing a
drop of antifade reagent (Life Technology) and sealed with
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min with shaking and for an additional 10 min without shak-
ing. Total light production was measured using a Victor 3
multi-label plate reader (PerkinElmer) in luminometer mode.
All samples were assayed in triplicate.
15 S. Borman, Chem. Eng. News, 2002, 80, 29.
16 M. Gehringer and S. A. Laufer, Angew. Chem., Int. Ed.,
2017, 56, 2–4.
17 M. S. Singh, S. Chowdury and S. Koley, Tetrahedron,
2016, 72, 5257–5383.
Conflicts of interest
18 J. Totobenazara and A. J. Burke, Tetrahedron Lett., 2015, 56,
2853–2859.
19 K. C. Majumdar and K. Ray, Synthesis, 2011, 23,
3767–3783.
20 C. D. Hein, X.-M. Liu and D. Wang, Pharm. Res., 2008, 25,
2216–2230.
21 M. Meldal and C. W. Tornøe, Chem. Rev., 2008, 108,
2952–3015.
22 J. E. Moses and A. D. Moorhouse, Chem. Soc. Rev., 2007, 37,
1249–1262.
23 C. M. Loner, F. A. Luzzio and D. R. Demuth, Tetrahedron
Lett., 2012, 53, 5641–5644.
There are no conflicts to declare.
Acknowledgements
The measurement of high- and low-resolution mass spectra
by Dr. Michael Walla of the Mass Spectrometry Laboratory,
Department of Chemistry and Biochemistry, University of
South Carolina, is acknowledged. Financial support from the
NIH/NIDCR through grant 1R01DE023206 is gratefully
acknowledged.
24 P. C. Patil, F. A. Luzzio and D. R. Demuth, Tetrahedron Lett.,
2015, 56, 3039–3041.
Notes and references
1 S. S. Socransky, A. D. Haffajee, M. A. Cugini, C. Smith and
R. L. Kent, Jr., J. Clin. Periodontol., 1998, 25, 134–144.
2 G. Hajishengallis, S. Liang, M. A. Payne, A. Hashim, R.
Jotwani, M. A. Eskan, M. L. McIntosh, A. Alsam, K. L.
Kirkwood, J. D. Lambris, R. P. Darveau and M. A. Curtis, Cell
Host Microbe, 2011, 10, 497–506.
25 P. C. Patil and F. A. Luzzio, Tetrahedron Lett., 2016, 57,
757–759.
26 P. C. Patil and F. A. Luzzio, J. Org. Chem., 2017, 81,
10521–10526.
27 C. Bissantz, B. Kuhn and M. Stahl, J. Med. Chem., 2010, 53,
5061–5084.
3 R. J. Lamont and G. Hajishengallis, Trends Mol. Med.,
2015, 21, 172–183.
28 A. Luzar and D. Chandler, J. Chem. Phys., 1993, 98,
8160–8173.
4 G. Hajishengallis and R. J. Lamont, Trends Microbiol.,
2016, 24, 477–489.
5 G. Hajishengallis, R. P. Darveau and M. A. Curtiss, Nat. Rev.
Microbiol., 2012, 10, 717–725.
6 C. J. Wright, L. H. Burns, A. A. Jack, C. R. Back, L. C. Dutton,
A. H. Nobbs, R. J. Lamont and H. F. Jenkinson, Mol. Oral
Microbiol., 2013, 28, 83–101.
29 A. L. Rodriguez, A. Tamrazi, M. L. Collins and J. A.
Katzenellenbogen, J. Med. Chem., 2004, 47, 600–611.
30 N. A. Meanwell, J. Med. Chem., 2018, 61, 5822–5880.
31 E. P. Gillis, K. J. Eastman, M. C. Hill, D. J. Connelly and
N. A. Meanwell, J. Med. Chem., 2015, 58, 8315–8359.
32 P. Shah and A. D. Westwell, J. Enzyme Inhib. Med. Chem.,
2007, 22, 527–540.
7 R. J. Lamont, A. El-Sabaeny, Y. Park, G. S. Cook, J. W. Costerton
and D. R. Demuth, Microbiology, 2002, 148, 1627–1636.
8 Y. Park, M. R. Simionato, K. Sekiya, Y. Murakami, D. James,
W. Chen, M. Hackett, D. R. Demuth and R. J. Lamont, Infect.
Immun., 2005, 73, 3983–3989.
33 H.-J. Böhm, D. Banner, S. Bensels, M. Kansy, B. Kuhn, K.
Müller, U. Obst-Sander and M. Stahl, ChemBioChem, 2004, 5,
637–643.
34 J. B. Sperry and D. L. Wright, Curr. Opin. Drug Discovery
Dev., 2005, 8, 723–740.
9 C. A. Daep, D. M. James, R. J. Lamont and D. R. Demuth,
Infect. Immun., 2006, 74, 5756–5762.
10 C. A. Daep, R. J. Lamont and D. R. Demuth, Infect. Immun.,
2008, 76, 3272–3280.
35 V. D. Bock, D. Speijer, H. Hiemstra and J. H. van
Maarseveen, Org. Biomol. Chem., 2007, 5, 971–975.
36 D. S. Pedersen and A. Abell, Eur. J. Org. Chem.,
2011, 2399–2411.
11 C. A. Daep, E. A. Novak, R. J. Lamont and D. R. Demuth,
Infect. Immun., 2011, 79, 67–74.
37 M. Falorni, G. Giacomelli, A. Porcheddu and G. Dettori, Eur.
J. Org. Chem., 2000, 3217–3222.
12 P. C. Patil, J. Tan, D. R. Demuth and F. A. Luzzio, Bioorg.
Med. Chem., 2016, 24, 5410–5417.
13 J. Tan, P. C. Patil, F. A. Luzzio and D. R. Demuth, Antimicrob.
Agents Chemother., 2018, 62(7), e00400-18, DOI: 10.1128/
AAC.00400-18.
14 V. P. Mocharla, B. Colasson, L. V. Lee, S. Roper, K. B.
Sharpless, C. H. Wong and H. C. Kolb, Angew. Chem., Int.
Ed., 2005, 44, 116–120.
38 K. Maeda, G. T. Tribble, C. M. Tucker, C. Anaya, S.
Shizukuishi, J. P. Lewis, D. R. Demuth and R. J. Lamont,
Mol. Microbiol., 2008, 69, 1153–1166.
39 J. Becerril, J. M. Rodriguez, P. N. Wyrembak and A. D.
Hamilton, in Protein Surface Recognition: Approaches for
Drug Discovery, ed. E. Giralt, M. W. Peczuh and X.
Salvatella, John Wiley and Sons, Ltd., New York, 2011, pp.
105–131.
Med. Chem. Commun.
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