1,2,3-Triazole-based inhibitors of Porphyromonas gingivalis adherence to oral streptococci and biofilm formation

Article abstract of DOI:10.1016/j.bmc.2016.08.059

The development and use of small-molecule inhibitors of the adherence of Porphyromonas gingivalis to oral streptococci represents a potential therapy for the treatment of periodontal disease as these organisms work in tandem to colonize the oral cavity. Earlier work from these laboratories demonstrated that a small synthetic peptide was an effective inhibitor of the interaction between P. gingivalis and Streptococcus gordonii and that a small-molecule peptidomimetic would provide a more stable, less expensive and more effective inhibitor. An array of 2-(azidomethyl)- and 2-(azidophenyl)-4,5-diaryloxazoles having a full range of hydrophobic groups were prepared and reacted with substituted arylacetylenes to afford the corresponding ‘click’ products. The title compounds were evaluated for their ability to inhibit P. gingivalis’ adherence to oral streptococci and several were found to be inhibitory in the range of (IC₅₀) 5.3–67?μM.

Full text of DOI:10.1016/j.bmc.2016.08.059

Bioorganic & Medicinal Chemistry 24 (2016) 5410–5417
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
1,2,3-Triazole-based inhibitors of Porphyromonas gingivalis
adherence to oral streptococci and biofilm formation
a,
Pravin C. Patil a, Jinlian Tan b, Donald R. Demuth b, , Frederick A. Luzzio
⇑
⇑
a Department of Chemistry, University of Louisville, 2320 South Brook Street, Louisville, KY 40292, USA
b Department of Oral Immunology and Infectious Diseases, University of Louisville School of Dentistry, 501 S. Preston St., Louisville, KY 40292, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The development and use of small-molecule inhibitors of the adherence of Porphyromonas gingivalis to
oral streptococci represents a potential therapy for the treatment of periodontal disease as these organ-
isms work in tandem to colonize the oral cavity. Earlier work from these laboratories demonstrated that a
small synthetic peptide was an effective inhibitor of the interaction between P. gingivalis and
Streptococcus gordonii and that a small-molecule peptidomimetic would provide a more stable, less
expensive and more effective inhibitor. An array of 2-(azidomethyl)- and 2-(azidophenyl)-4,5-diaryloxa-
zoles having a full range of hydrophobic groups were prepared and reacted with substituted arylacetyle-
nes to afford the corresponding ‘click’ products. The title compounds were evaluated for their ability to
inhibit P. gingivalis’ adherence to oral streptococci and several were found to be inhibitory in the range of
Received 15 July 2016
Revised 26 August 2016
Accepted 29 August 2016
Available online 7 September 2016
Keywords:
Azides
Biofilms
Click chemistry
Oxazoles
(IC50) 5.3–67 lM.
Ó 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
Peptidomimetics
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction
response in P. gingivalis that may be involved in its adaptation to
biofilm growth in the oral cavity.8 Daep et al. also identified a dis-
Periodontal disease is caused by a consortium of anaerobic bac-
teria including Porphyromonas gingivalis, Tannerella forsythsis and
Treponema denticola.1 These organisms have been designated as
the ‘red complex’ and are strongly associated with chronic adult
periodontitis. Current methods to treat periodontitis involves scal-
ing and root planing and in more severe cases, surgery may be
required to reduce pocket depth. In general, therapeutic
approaches are lacking that specifically target periodontal patho-
gens like P. gingivalis and furthermore, treatments that prevent
or limit the re-colonization of the oral cavity by P. gingivalis after
treatment procedures are not available. Although the primary
niche for P. gingivalis is in a mixed community of bacterial species
in the subgingival pocket, upon initial entry into the oral cavity it
must first colonize supragingival plaque.2 Our previous results sug-
gest that the interaction of P. gingivalis with oral streptococci is
important for this early colonization event.3,4 Thus, this interaction
represents an ideal point for therapeutic intervention to control
colonization (or re-colonization) of oral tissues by P. gingivalis.
Adherence of P. gingivalis to streptococci is species specific and is
driven by a protein–protein interaction that occurs between the
minor fimbrial antigen (Mfa) of P. gingivalis and the antigen I/II
(Ag I/II) polypeptide of streptococci.5–7 This interaction induces a
crete domain in Ag I/II protein that mediates its interaction with
Mfa and showed that this region resembles the eukaryotic nuclear
receptor (NR) box protein–protein interaction domain.5,6 Further-
more, variation in the sequence and structure of the NR box in dif-
ferent members of the Ag I/II family of proteins accounted for the
species selectivity of P. gingivalis adherence to oral streptococci.5
We also showed that the NR box is comprised of two functional
peptide motifs, VXXLL and NITVK, and that 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.7 Together, these results suggest that
P. gingivalis colonization of the oral cavity can be controlled by pre-
venting its initial association with streptococci and that inhibitors
of the Mfa-AgI/II interaction represent potential therapeutic agents
to control periodontal disease. However, the use of peptides as
therapeutic agents has limitations arising from the relatively high
cost of peptide synthesis and their susceptibility to degradation by
proteases expressed by oral organisms, including P. gingivalis itself.
To overcome these limitations, we sought to produce a potent and
stable inhibitor that is not based on a peptide backbone but mimics
the natural peptide substrate recognized by Mfa. Since the
streptococcal NR box-like sequence contains two functional motifs,
we envisioned that the design and study of small-molecule, non-
peptide based inhibitors of the Mfa/AgI/II interaction encompassed
the employment of two synthetic small-molecule scaffolds joined
⇑
Corresponding authors.
http://dx.doi.org/10.1016/j.bmc.2016.08.059
0968-0896/Ó 2016 The Authors. Published by Elsevier Ltd.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

P. C. Patil et al. / Bioorg. Med. Chem. 24 (2016) 5410–5417
5411
together via the ‘click’ reaction.9,10 Within the expansive area of
nitrogen/oxygen heterocycles, we have identified the 2,4,5-trisub-
stituted oxazole framework as a starting point for the NITVK-asso-
ciated inhibitors of Mfa/AgI/II interaction11–13 and show here that
these compounds potently block P. gingivalis adherence to strepto-
cocci in vitro when clicked with substituted arylalkynes. As click
chemistry involves the classical cycloaddition reaction between
an alkyl or aryl azide and an alkyl or aryl alkyne, the oxazoles were
functionalized with the azide moiety and an array of arylalkynyl
click partners containing hydrophobic groups were chosen for both
evaluating the click reactions and exploring and evaluating the
minimal structural requirements for the VXXLL mimic. The
newly-formed 1,2,3-triazole resulting from the click reaction may
function as a polar, slightly basic backbone of the complete pep-
tidomimetic and depending on the spacer group between the tria-
zole and the oxazole, may affect the topology of the entire
molecule. It may also be noted that triazole rings can function as
the oxazole. So the structural and substituent differences of the
Group I, II and III azides offer a great breath of molecular diversity
for the NITVK mimic. Our initial design of the VXXLL-mimic
required an alkynyl reacting group and encompassed a scaffold
of lesser structural complexity, namely a carbocyclic aromatic ring
bearing hydrophobic groups (Table 1).15 While, previous workers
have identified di- and trialkylaminotriazines as VXXLL mimics in
research involving the nuclear receptor boxes,16,17 we first opted
for the albeit simpler and planar aromatic analogue bearing the
characteristic symmetrically-opposed hydrophobic groups. The
alkylamino-triazine scaffold (Table 1) possesses non-polar
hydrophobic N-alkyl groups which mimic the one valine and two
leucine groups in the helical peptide. Given the requirement of
the alkynyl reacting group at position 1 of the aryl ring, the only
requirement remaining was two hydrophobic substituents, each
at positions 3 and 5. For the hydrophobic substituents, we chose
the halogens (F, Cl, Br), methoxy (OCH3) and trifluoromethyl
(CF3) groups, all of which were situated on aromatic rings. In terms
of hydrophobicity trends, F > Cl > Br with the methoxy group being
more polar and less hydrophobic than bromine and the trifluo-
romethyl group being more hydrophobic than a single fluorine.
Given that the naphthalene core has been implicated in mimicking
the double leucine motif in earlier studies,16 our selection included
an alkynylnaphthalene bearing a hydrophobic methoxy group
(Table 1). It should be noted that our initial selections of simple
aryl-substituted VXXLL mimics constituted only aryl rings which
were singly-substituted with hydrophobic groups. The singly-sub-
stituted aryl components are functionalized with a terminal acety-
lene moiety to facilitate the 3+2 cycloaddition or ‘click’ reaction
with the azide-functionalized NITVK mimics.18–20 Consequently,
lead optimization may occur through adjustment of the hydropho-
bic groups on both the mono-aryl and diaryloxazole scaffolds as
well as the topology and size of the alkyl-triazole or aryl-triazole
linker.
mild hydrogen bond acceptors as well as structures with
p-stack-
ing potential.14
2. Design of the target compounds
Our initial approach toward the design of the peptidomimetics
was focused on the installation of three dimensional hydrophobic
moieties that will mimic both the VXXLL and NITVK motifs of the
helical peptide. The 2,4,5-trisubstituted oxazole scaffold was cho-
sen as the peptidomimetic model of the NITVK motif. The oxazole
ring comprises a central torus whereby two aromatic rings, each
located at the 4 and 5 positions of the heterocycle, bear hydropho-
bic residues (Fig. 1). The positions of the hydrophobic residues may
be placed ortho, meta or para on each ring at the 4- or 5-position of
the oxazole. The hydrophobic residues may take the form of halo-
gens, alkoxy groups or alkyl groups. The planarity of the substi-
tuted oxazole ring excludes any stereochemical considerations
thereby simplifying the initial design of the inhibitory NITVK scaf-
folds. 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 located at
the 2-position of the oxazole. The azides take the form of three
general groups which vary at the 2-position of the 4,5-diaryloxa-
zole. The Group I azide precursors 10–13 possess aryl groups situ-
ated at the 4- and 5-positions of the oxazole ring. Both aryl groups
are substituted at the para positions with fluorine, chlorine, bro-
mine or methoxy groups and have the azidomethyl reacting group
at the 2-position of the oxazole. Similarly, the Group II azide pre-
cursors 18–21 possess substituted aryl groups situated at the 4-
and 5-positions of the oxazole and bear fluorine, chlorine, bromine
and methoxy groups at the para positions. In contrast to the Group
I candidates, compounds 18–21 possess a meta-azidophenyl group
at the 2-position of the oxazole. The Group III targets 26–29 have
the same para aryl substituent pattern at oxazole positions 4 and
5, but employ a para-azidophenyl reacting group at position 2 of
2.1. Synthesis
Our syntheses of the Group I compounds begins with the prepa-
ration of 2-(azidomethyl)-4,5-diaryloxazoles 10–13 from the cor-
responding unsubstituted or substituted benzoins (Scheme 1).
Benzoin or the substituted benzoins 1–4 are esterified with chlor-
oacetyl chloride in the presence of pyridine to give the aryl-substi-
tuted chloroacetyl esters 6–9 (43–85%). The chloroacetyl (benzoin)
esters 6–9 are then cyclized to the corresponding 2-chloromethy-
loxazoles with ammonium acetate in acetic acid followed by
immediate treatment with sodium azide in N,N-dimethylfor-
mamide (DMF) to provide the Group I azidomethyloxazoles 10–
13. The progress of the cyclization reactions were monitored by
thin-layer chromatography, normally take 1–3 h and afforded the
product chloromethyloxazoles in 71–80% yield. The air-stable
and non-hygroscopic products (10–13) obtained from the room-
temperature azide displacement reaction are purified using grav-
ity-column silica gel chromatography with mixtures of hexanes/
ethyl acetate as the mobile phase. Our synthesis of the Group II
3-azidophenyl precursors 18–21 begins with the esterification of
benzoin or substituted benzoins 2–5 with 3-azidobenzoyl chlo-
ride/4-dimethylaminopyridine (48–93%, Scheme 2). The resulting
azidobenzoyl esters 14–17 were cyclized to the corresponding
2-(3-azidophenyl) oxazoles 18–21 using ammonium acetate in
acetic acid (115 °C/3 h/44–63%). The synthesis of the Group III 2
(4-azidophenyl)-4,5-diaryloxazoles paralled that of the Group II
compounds (Scheme 3). Benzoins 2–5 were esterified with
4-azidobenzoyl chloride/DMAP which afforded the 4-azido ben-
zoyl esters 22–25 (72–93%). The esters were then cyclized to the
oxazoles 26–29 with ammonium acetate in acetic acid (47–69%).
The chromatographic conditions for purification of Group II and
Triazole linker:
R=methylene, meta phenyl
or para phenyl 'spacer'
hydrophobic
substitution
hydrophobic
substitution
N
R1
R2
N
N
R3
N
N
N3
R
R
O
O
hydrophobic
substitution
Figure 1. General azide/alkyne scaffold construction.

5412
P. C. Patil et al. / Bioorg. Med. Chem. 24 (2016) 5410–5417
Table 1
Group III azides were similar to that used for the Group I com-
Aryl alkynyl click partners 30–40a
pounds. Both the Group I, Group II and Group II azides were
reacted with arylalkynes (30–40, Table 1) using a system com-
posed of aqueous tetrahydrofuran (THF), copper sulfate and
sodium ascorbate over a reaction period of sixteen hours at room
temperature (Tables 2–4). After workup of the reaction mixtures,
the product triazoles (41–90, Tables 2–4) were purified by grav-
ity-column chromatography on silica gel using hexanes/ethyl acet-
ate or chloroform/methanol as the mobile phase. The pure triazoles
were isolated as crystalline compounds whose composition and
structures were confirmed by NMR and HRMS (See Section 4).
The product yields for the click reactions of azidomethyl oxazoles
10–13 and arylalkyne reactants 30–40 are detailed in Table 2
(Compounds 41–66). The product yields for the click reactions of
the meta-substituted azidophenyloxazoles 20–21 and arylalkyne
reactants selected from 30–40 are detailed in Table 3 (Compounds
67–80) and the product yields for the click reactions of the para-
substituted azidophenyloxazoles 26–27 and arylalkyne reactants
selected from 30–40 are detailed in Table 4 (Compounds 81–90).
The regiochemistry of the dipolar cycloaddition ‘click’ reaction
involves products in which the substitution could be 1,4- or 1,5-
on the triazole ring depending on the reaction conditions. All of
the click products described herein (Tables 2–4) were formed as
a result of copper(I) catalysis which gives exclusively the 1,4- or
‘anti’-substituted 1,2,3-triazole.18 While the acetylenes 30–39 were
used to explore both the reactivity demand of the click reaction
and evaluate the hydrophobic nature of the substituent groups
which mimic the VXXLL motif, we sought a more specifically-
designed scaffold which utilized hydrophobic groups attached to
a more hydrophilic scaffold. An interesting scaffold which would
accommodate both a charge and a wide range of hydrophobic
groups is acetylenic triazine 40 (Table 1). Using the 1,3,5-triazine
as the central torus, a wide range of hydrophobic groups may be
accommodated, and in the case of 40, the isopentyl(3-methylbutyl)
groups were chosen for the synthesis (Scheme 4). Lithiation of
ethynyltrimethylsilane at 0 °C followed by addition of the organo-
lithium reagent to commercially-available cyanuric chloride gives
the mono-(trimethylsilylethynyl) dichlorotriazine 91. Direct addi-
tion of excess isopentylamine gave the acetylenic diaminotriazine
92 (51%) which was purified by column chromatography. Desilyla-
tion of 92 with tetra-n-butylammonium fluoride (TBAF) in THF
afforded the ethynyl diaminotriazine coupling partner 40 (84%,
Scheme 4).
OCH3
N
C6H5
O
30
34
38
F3C
CH3
CF3
31
35
CF3
F
39
H
N
N
32
36
N
N
NH
H3CO
F
40b
triazine
33
37
a
Arranged in order of increasing molecular weight.
See Section 4 for preparation.
b
R1
R1
O
O
a
O
Cl
OH
O
R2
R2
R1
6-9
1-4
1
2
1
2
10
11
1
2
, R , R =H
, R , R =H
b, c
1
2
1
2
, R , R =F
, R , R =F
12, R1, R2=Cl
13, R1, R2=Br
3, R1, R2=Cl
4, R1, R2=Br
5, R1, R2=OCH3
6, R1, R2=H
N3
N
O
1
2
7
8
9
, R , R =F
1
2
, R , R =Cl
R2
1
2
10-13
, R , R =Br
Scheme 1. Synthesis of Group I azide precursors, 2-(azido-methyl)-4,5,-dipheny-
loxazoles 10–13. Reagents/conditions: (a) chloroacetyl chloride/DMAP/CH2Cl2/5 °C
to rt/16 h; (b) NH4OAc/HOAc/115 °C/3 h; (c) NaN3/DMF/rt/ 3 h.
N3
N3
R1
R1
R1
R1
O
O
O
O
O
O
COCl
a
COCl
O
O
N3
a
OH
OH
R2
R2
R1
N3
R2
R2
R1
14-17
2-5
2-5
22-25
b
b
1
2
1
2
1
2
1
2
22
23
24
26
27
28
, R , R =F
, R , R =F
14
, R , R =F
18
19
, R , R =F
N3
1
2
1
2
1
2
1
2
, R , R =Cl
, R , R =Cl
15
, R , R =Cl
16, R1, R2=Br
17, R1, R2=OCH3
, R , R =Cl
N
O
N
O
1
2
1
2
20, R1, R2=Br
N3
, R , R =Br
, R , R =Br
25, R1, R2=OCH3 29, R1, R2=OCH3
21, R1, R2=OCH3
R2
R2
18-21
26-29
Scheme 2. Synthesis of Group II azide precursors, 2-(3-azido-phenyl)-4,5,-
diphenyloxazoles 18–21. Reagents/conditions: (a) DMAP/CH2Cl2/5 °C to rt/16 h;
(b) NH4OAc/HOAc/115 °C/3 h.
Scheme 3. Synthesis of Group III azide precursors, 2-(4-azido-phenyl)-4,5,-
diphenyloxazoles 26–29. Reagents/conditions: (a) DMAP/CH2Cl2/5 °C to rt/16 h;
(b) NH4OAc/HOAc/115 °C/3 h.

P. C. Patil et al. / Bioorg. Med. Chem. 24 (2016) 5410–5417
5413
Table 2
Activities of 2-(triazolylmethyl)-4,5-diphenyloxazole click products 41–66
R1
R2
N
O
R1
N
30-40
+
N
N
R2
O
N3
R3
N
10-13
41-66
Compound (Yield %)a
IC50
>60
(
lM)
S. gordonii growth
inhibition (%)
P. gingivalis growth
inhibition (%)
Concentration
(lM)
41, R1, R2 = Ph, R3 = 3-Pyridyl (99)
42, R1, R2 = Ph, R3 = 2-Methylphenyl (90.5)
43, R1, R2 = Ph, R3 = 3-Fluorophenyl (99)
44, R1, R2 = Ph, R3 = 4-Fluorophenyl (98)
45, R1, R2 = Ph, R3 = 2-Methoxyphenyl (86)
>56.3
>60
>60
>60
>60
35.5
>60
>60
>60
36.9
59.8
>60
>60
>60
29.3
>60
>60
56.8
56.7
5.3
46, R1, R2 = Ph, R3 = 2-(Trifluoromethyl)phenyl (89)
47, R1, R2 = Ph, R3 = 4-(n-Pentyl)phenyl (94)
6.6
6.8
À14.5
60
60
48, R1, R2 = Ph, R3 = 6-Methoxynaphthalene-2-yl (99)
49, R1, R2 = Ph, R3 = 4-(Phenoxy)phenyl (86)
50, R1, R2 = Ph, R3 = 3,5-Di-(trifluoromethyl)phenyl (97)
51, R1, R2 = Ph, R3 = 6-(N2,N4-Diisopentyl-1,3,5-triazine)-2,4-diamine (90)
52, R1, R2 = 4-Fluorophenyl, R3 = 3-Pyridyl (66)
72.8
53, R1, R2 = 4-Fluorophenyl, R3 = 3-Fluorophenyl (72)
54, R1, R2 = 4-Fluorophenyl, R3 = 2-Methoxyphenyl (92)
55, R1, R2 = 4-Fluorophenyl, R3 = 2-(Trifluoromethyl)phenyl (98)
56, R1, R2 = 4-Fluorophenyl, R3 = 6-Methoxynaphthalene-2-yl (97)
57, R1, R2 = 4-Chlorophenyl, R3 = 3-Pyridyl (99)
0.2
À3.3
60
58, R1, R2 = 4-Chlorophenyl, R3 = 3-Fluorophenyl (89)
59, R1, R2 = 4-Chlorophenyl, R3 = 2-Methoxyphenyl (93)
60, R1, R2 = 4-Chlorophenyl, R3 = 2-(Trifluoromethyl)phenyl (89)
61, R1, R2 = 4-Chlorophenyl, R3 = 6-Methoxynaphthalene-2-yl (95)
62, R1, R2 = 4-Bromophenyl, R3 = 3-Pyridyl (85)
6.6
À19.6
60
60
À3.8
0.3
50.9
>60
43.0
>60
33.4
63, R1, R2 = 4-Bromophenyl, R3 = 3-Fluorophenyl (95)
64, R1, R2 = 4-Bromophenyl, R3 = 2-Methoxyphenyl (96)
65, R1, R2 = 4-Bromophenyl, R3 = 2-(Trifluoromethyl)phenyl (96)
66, R1, R2 = 4-Bromophenyl, R3 = 6-Methoxynaphthalene-2-yl (92)
11.1
À12.4
À9.9
60
60
À8.6
a
Yields are for isolated pure compounds.
Table 3
Activities of 2-(3-triazolylphenyl)-4,5-diphenyloxazole click products 67–80
R1
R2
N
O
R1
N
+
30-40
R2
O
N
N
N
N3
R3
20-21
67-80
Compound (Yield %)a
IC50
(lM)
S. gordonii growth
inhibition (%)
P. gingivalis growth
inhibition (%)
Concentration
(lM)
67, R1, R2 = 4-Methoxyphenyl, R3 = 3-Pyridyl (89)
57.7
5.9
68, R1, R2 = 4-Methoxyphenyl, R3 = 3-Fluorophenyl (97)
69, R1, R2 = 4-Methoxyphenyl, R3 = 4-Fluorophenyl (86)
70, R1, R2 = 4-Methoxyphenyl, R3 = 2-Methoxyphenyl (83)
À9.1
À22.1
0.8
À9.7
À2.6
À14.6
À6.2
40
40
40
40
53.0
15.4
27.0
>60
13.0
>60
47.4
31.0
5.0
71, R1, R2 = 4-Methoxyphenyl, R3 = 2-(Trifluoromethyl)phenyl (95)
72, R1, R2 = 4-Methoxyphenyl, R3 = 4-(n-Pentyl)phenyl (99)
73, R1, R2 = 4-Methoxyphenyl, R3 = 6-Methoxynaphthalene-2-yl (77)
74, R1, R2 = 4-Methoxyphenyl, R3 = 4-Phenoxyphenyl (89)
75, R1, R2 = 4-Methoxyphenyl, R3 = 3,5-Di-(trifluoromethyl)phenyl (95)
76, R1, R2 = 4-Bromophenyl, R3 = 3-Pyridyl (72)
À1.6
À1.3
3.0
20
77, R1, R2 = 4-Bromophenyl, R3 = 3-Fluorophenyl (85)
À5.7
10.5
2.0
À4.3
4.3
À0.6
5
40
40
78, R1, R2 = 4-Bromophenyl, R3 = 2-Methoxyphenyl (71)
79, R1, R2 = 4-Bromophenyl, R3 = 2-(Trifluoromethyl)phenyl (90)
80, R1, R2 = 4-Bromophenyl, R3 = 6-Methoxynaphthalene-2-yl (91)
15.0
15.2
20.3
a
Yields are for isolated pure compounds.
To assess the functional activity of the click products, com-
of inhibition of P. gingivalis adherence is shown in Figure 2. For
each compound tested, an IC50 value for adherence inhibition
was approximated after quantifying the ratio of green to red fluo-
rescence and these values are shown in Tables 2–4. Compounds
pounds 41–90 were examined for inhibition of P. gingivalis adher-
ence to S. gordonii using an established biofilm model system8 as
described in the Section 4. A representative dose response series

5414
P. C. Patil et al. / Bioorg. Med. Chem. 24 (2016) 5410–5417
Table 4
Activities of 2-(4-triazolylphenyl)-4,5-diphenyloxazole click products 81–90
R1
R2
R1
N
N
O
N
N
N
N3
+
30-40
R3
R2
O
26-27
81-90
Compound (Yield %)a
IC50
(lM)
S. gordonii growth
inhibition (%)
P. gingivalis growth
inhibition (%)
Concentration
(lM)
81, R1, R2 = 4-Fluorophenyl, R3 = 3-Pyridyl (78)
25.6
51.5
7.7
12.8
52.2
40.9
15.7
37.3
25.1
32.4
82, R1, R2 = 4-Fluorophenyl, R3 = 3-Fluorophenyl (92)
83, R1, R2 = 4-Fluorophenyl, R3 = 2-Methoxyphenyl (81)
À7.8
À0.5
20
40
84, R1, R2 = 4-Fluorophenyl, R3 = 2-(Trifluoromethyl)phenyl (83)
85, R1, R2 = 4-Fluorophenyl, R3 = 6-Methoxynaphthalene-2-yl (83)
86, R1, R2 = 4-Chlorophenyl, R3 = 3-Pyridyl (100)
À11.0
0.1
87, R1, R2 = 4-Chlorophenyl, R3 = 3-Fluorophenyl (33)
8.7
11.6
40
88, R1, R2 = 4-Chlorophenyl, R3 = 2-Methoxyphenyl (100)
89, R1, R2 = 4-Chlorophenyl, R3 = 2-(Trifluoromethyl)phenyl (94)
90, R1, R2 = 4-Chlorophenyl, R3 = 6-Methoxynaphthalene-2-yl (74)
a
Yields are for isolated pure compounds.
were then classified into three groups; strong inhibitors of adher-
ence where IC50 was <10 M (compounds 61, 68, 77 and 83), mod-
erate inhibitors of adherence where IC50 was <20 M (compounds
70, 73, 78, 79, 84 and 87) and the remainder were weak inhibitors
of adherence (IC50 > 20 M). In surveying the structural relation-
Within the same group, 68 and 70 bear the para-methoxy combi-
nation and with meta-fluoro (68) and ortho-methoxy (70) VXXLL
mimics. In terms of molecular shape, the click compounds derived
from the Group II azides have a characteristic ‘L’ shape which is
imparted by the meta-juxtaposition of the 2-oxazole group and
the newly-formed triazole group on the phenyl ‘spacer’ ring. The
shape adopted by the para- or 1,4-alignment of the 2-oxazole
group and triazole groups on the phenyl ‘spacer’ ring is more linear
by comparison. In contrast, compound 61 exhibits a more flexible
topology whereby the methylene group or spacer conjoining the
oxazole and triazole rings allows for several degrees of rotation
and multiple binding modes to the helical peptide. Linear or
L-shaped scaffolds are common in the design of small-molecule
inhibitors, especially when combined with the multi-pronged,
hydrophobic functionality found in all three groups of the
inhibitors described herein.24 To confirm that the reduction of
P. gingivalis levels resulted from inhibition of its adherence to
S. gordonii rather than a general toxic effect of the compounds,
active compounds from all groups were tested for inhibition of
P. gingivalis and S. gordonii planktonic growth at the concentration
listed in Tables 2–4. As shown, few of the compounds had a signif-
icant effect on planktonic growth, suggesting that they are not
functioning as antibiotics but rather as inhibitors of P. gingivalis
adherence. In addition, our preliminary results suggest that the
l
l
l
ships among the most active compounds listed above, click com-
pound 61, bearing the triazolylmethyl ‘linker’ is derived from the
Group I 2-(azidomethyl)oxazoles. The Group II oxazoles give rise
to compounds 68, 70, 73, 77, 78 and 79 and possess a meta-
triazolyl linker (Table 3). In contrast, the Group III oxazoles are pre-
cursors for compounds 83, 84 and 87 which have a para-triazolyl
linker (Table 4). Of all the most active compounds mentioned
above, save for 68, 70 and 73, every compound bears the full range
of halogen substituents at both para-positions of the 4,5-dipheny-
loxazole.21,22 The most active compounds derived from Group III
(83, 84, 87) bear para-fluorines on the phenyl rings of the NITVK-
mimic and render that section of the molecule the most hydropho-
bic.23 Compounds 61 and 83 bear arylmethoxy groups while 68
and 77 bear meta-substituted fluorines derived from the VXXLL
mimic. As a group, the most active compounds (68, 70, 73, 77, 78
and 79) were derived from the meta-substituted azidophenyl
precursors whereby the click reaction gave the meta-substituted
triazole linker between the oxazole scaffold and VXXLL mimic.
For example, 73 possesses the combination of two 4,5-para-meth-
oxy groups and the highly hydrophobic naphthalene ring which
allows the optimal interaction within the VXXLL binding motif.
compounds have little cytotoxic activity against
epithelial cell line (not shown).
a gingival
3. Conclusions
TMS
Cl
N
Cl
An array of small-molecule peptidomimetics which carry the
core interacting mimics of the biofilm-inhibitory BAR peptide have
been prepared by click reactions in moderate to high yield. Using a
simple two-species biofilm model, several synthetic targets proved
to inhibit the adherence of P. gingivalis to oral streptococci by inter-
fering with the interaction of Mfa-AgI/II. While this model does not
represent the complexity of the oral biofilm in vivo, it does reflect
an initial interaction that may be essential for P. gingivalis coloniza-
tion of the oral cavity. Therefore, our results suggest that these
compounds represent potential targeted therapeutics to limit
P. gingivalis colonization. Both the azide and the alkynyl reacting
components contain moderate to strongly-acting hydrophobic
groups such as halogens and alkoxy groups which are situated on
aromatic rings. These compounds are more compact, possess
increased stability and can be synthesized and purified at a much
lower expense than the polypeptides they mimic. The key
Cl
N
a
N
N
N
N
Cl
Cl
91
b
R=isopentyl
H
N
TMS
N
R
c
N
H
N
R
40
N
92
Scheme 4. Synthesis of acetylenic triazine precursor 40. Reagents/conditions: (a)
ethynyltrimethylsilane/n-Bu-Li/THF/0 °C/1 h; (b) ispopentylamine/48 h/0 °C to rt;
(C) TBAF/THF/rt.

P. C. Patil et al. / Bioorg. Med. Chem. 24 (2016) 5410–5417
5415
Figure 2. Dose dependent inhibition of P. gingivalis adherence to S. gordonii by compound 78. Two species biofilms comprising S. gordonii (red) and P. gingivalis (green) were
formed as described in Section 4 in the absence (control) and presence of 5–40 M of compound 78.
l
adjustment parameters are the inclusion of suitable hydrophobic
groups on both the NITVK and VXXLL mimics. The inhibitory
results indicated that the interactions were predominantly through
hydrophobic forces and that suitable substitutions in the aryl rings
could increase those interactions as part of the optimization pro-
cess. As such, there is a strong indication that affinity of the
small-molecule scaffolds increases with both molecular weight
and hydrophobicity and even includes the aromatic nature of the
parent structures.25 Given the relatively high numbers of halogens
in the active compounds, one cannot exclude the possibility of sec-
ondary interactions which involve electrostatic influences such as
halogen bonding.26,27 The lead candidates in the Mfa/AgI/II inhibi-
tor evaluation exemplify a number of features common to many
protein–protein inhibitor interaction problems. The most active
candidates and their development involve leads that have charac-
teristic shapes or topologies, significant hydrophobicity and the
conjunction of several low to high affinity pharmacophores.28
The optimization process involving many of the compounds
described in this communication is ongoing and will further reveal
many of the properties and interactions responsible for inhibitory
activity. As newer compounds are involving in this Mfa/AgI/II ‘click’
study, they will be the subject of future communications from
these laboratories.
4.1.2. 2-Oxo-1,2-diphenylethyl-2-chloroacetates (6–9)
The acyloins 1–5 (1.3–2 mmol, 1.0 equiv) were dissolved in
dichloromethane (40 mL) at room temperature followed by the
addition of 4-dimethylaminopyridine (2–3 mmol, 1.5 equiv) under
nitrogen. The resultant solution was cooled (0–5 °C) and chloroace-
tyl chloride (1.7–2.6 mmol, 1.3 equiv) was added by syringe. The
reaction mixture was warmed gradually to room temperature
and then stirred (16 h) under nitrogen. The reaction mixture was
then washed with water (30 mL), 5% aqueous HCl (30 mL), and
5% aqueous sodium bicarbonate (30 mL). The organic layer was
separated, dried over anhydrous sodium sulfate, filtered and con-
centrated. The crude residues was purified by gravity-column
chromatography (hexane/ethyl acetate, 9:1) to provide the pure
chloroacetyl esters 6–9.
4.1.3. 2-(Azidomethyl)-4,5-diphenyloxazoles (10–13)
The chloro-acetyl esters 6–9 (1.1–1.7 mmol, 1.0 equiv) were dis-
solved in glacial acetic acid (50 mL) and to the clear solution was
added ammonium acetate (17–27 mmol, 15 equiv). The reaction
mixture was then heated at 115 °C (oil bath, 3 h) under a nitrogen
atmosphere. Upon completion of the reaction as indicated by TLC
(hexane/ethyl acetate, 9:1), the mixture was allowed to cool to
room temperature. The reaction mixture was then poured into cold
water (40 mL), slowly neutralized with saturated aqueous
sodium bicarbonate and then extracted with dichloromethane
(3 Â 25 mL). The organic extracts were combined, dried over anhy-
drous sodium sulfate and concentrated to obtain the crude residues
which were purified by gravity-column chromatography (hexane/
ethyl acetate, 9:1) to afford the intermediate chloromethyloxazoles
which were used directly in the next step. Each intermediate chlor-
omethyl oxazole (0.6–0.9 mmol, 1.0 equiv) was dissolved in DMF
(10 mL) at room temperature. To the clear solution was added
sodium azide (0.6–1.0 mmol, 1.1 equiv) and the mixture was stirred
at room temperature (3 h) under a nitrogen atmosphere. The reac-
tion progress was monitored by TLC (hexane/ethyl acetate, 9:1) and
when completed the DMF was removed under high vacuum. The
residual crude oil was then purified by gravity-column chromatog-
raphy (hexane/ethyl acetate, 9:1) to obtain the corresponding
2-azidomethyl-(4,5)-diphenyloxazoles 10–13.
4. Experimental
4.1. Chemistry
4.1.1. General methods
Unless otherwise specified, all solvents and reagents were ACS
grade and were used as supplied. Alkynes 30–39 were commer-
cially available and were used as supplied. 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 at (500 MHz, 125 MHz for 13C). High
resolution mass spectra (HRMS) were performed using electro-
spray ionization (ESI). All air and moisture-sensitive reactions 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 thickness) and visualized using 2% anisalde-
hyde in ethanol, 2.5% phosphomolybdic acid in ethanol or 10%
sulfuric acid in ethanol.
4.1.4. General procedure for the synthesis of the substituted 2-
oxo-1,2-diphenylethyl-3-azidobenzoates (14–17) and the 2-
oxo-1,2-diphenylethyl-4-azidobenzoates (22–25)
The substituted benzoin 2–5 (1.3–2.0 mmol, 1.0 equiv.) was dis-
solved in dichloromethane (50 mL) at room temperature. The
resulting clear solution was cooled (0–5 °C) followed by addition
of DMAP (2.0–3.0 mmol, 1.5 equiv). 3- or 4-Azidobenzoyl chloride

5416
P. C. Patil et al. / Bioorg. Med. Chem. 24 (2016) 5410–5417
(prepared by treating the corresponding carboxylic acids (1.7–
2.6 mmol, 1.3 equiv) with thionyl chloride), dissolved in dichloro-
methane (20 mL) was added dropwise to the cooled solution while
stirring under nitrogen. After the reaction was complete, the reac-
tion mixture was washed with 5% aqueous HCl (2 Â 50 mL) fol-
lowed by 5% aqueous sodium bicarbonate. The organic layer was
separated, and dried over anhydrous sodium sulfate and filtered.
Concentration of the filtrate gave a crude residue which was chro-
matographed on silica gel (hexane/ethylacetate, 9:1) to afford the
pure 3-azidobenzoyl esters 14–17 or 4-azidobenzoyl esters 22–25.
added alkynes 30–40 (0.11–0.2 mmol) and solid copper sulfate
pentahydrate (0.1–0.18 mmol) at room temperature. To this mix-
ture was then added
a solution of sodium ascorbate (0.5–
0.09 mmol) in water (1 mL). The resulting reaction mixture was
stirred at room temperature (16 h). The progress of reaction, as
indicated by formation of the less-mobile product, was monitored
by TLC. After completion of reaction as indicated by TLC, the THF
was removed under vacuum and the residue was partitioned
between dichloromethane (15 mL) and water (10 mL). The organic
layer was separated, dried over anhydrous sodium sulfate and fil-
tered. Concentration of the filtrate afforded the usually solid crude
residues which was purified by column chromatography (hexanes/
EtOAc, 7.5:2.5; or chloroform/methanol, 9:1). to give the corre-
sponding pure triazoles 41–90 (See Supporting information).
4.1.5. General procedure for the synthesis of 2-(3-azidophenyl)
4,5-diphenyloxazoles (18–21) or 2-(4-azidophenyl)-4,5-diphenyl-
oxazoles oxazoles (26–29)
The 3-azidobenzoyl esters 14–17 or the 4-azidobenzoyl esters
22–25 (0.4–0.5 mmol, 1.0 equiv) were dissolved in glacial acetic
acid (50 mL) at room temperature. To the clear solution was added
ammonium acetate (6–8 mmol, 15 equiv) under a nitrogen atmo-
sphere and the resulting solution was heated at 115 °C (oil bath,
3 h). After completion of the reaction as indicated by TLC (hex-
anes/ethyl acetate, 9:1), the reaction mixture was allowed to cool
to room temperature, poured into cold water (30 mL) and slowly
neutralized with saturated aqueous sodium bicarbonate. The mix-
ture was then extracted with dichloromethane (3 Â 25 mL) and the
organic extracts were combined and dried over anhydrous sodium
sulfate. Removal of the drying agent and concentration gave the
corresponding crude 2-(3-azidophenyl)-4,5-disubstituted oxazoles
18–21 or the 2-(4-azidophenyl0-4,5-diphenyloxazoles 26–29. Both
series of compounds were further purified by gravity-column chro-
matography on silica gel (hexane/ethyl acetate, 9:1).
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
lg/
mL menadione, and 5 g/mL hemin. Twenty five milliliters of
l
media were reduced for 24 h under anaerobic conditions by equi-
librating in an atmosphere consisting of 10% CO2, 10% H2, and 80%
N2. Following equilibration, P. gingivalis was inoculated in the
media and grown for 48 h at 37 °C under anaerobic conditions. S.
gordonii DL-1 (1) was cultured aerobically 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 adherence to S. gordonii, we determined their affect
the planktonic growth of P. gingivalis and S. gordonii. Broth cultures
were grown as described above and compared to cultures grown in
the absence of the compound. The concentration of each com-
pound used in the growth inhibition experiments is shown in
Tables 2–4. Cell density of each culture was determined by mea-
suring the optical density at 600 nm (O.D.600nm) after 24 h of
growth. 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) À 1] Â 100.
4.1.6. N2,N4-Diisopentyl-6-(2-(trimethylsilyl)ethynyl)-1,3,5-
triazine-2,4-diamine (92)
To
a solution of ethynyltrimethylsilane (0.53 g, 0.76 mL,
5.4 mmol) in dry THF (5.0 mL) was added n-butyllithium (1.6 M
solution in hexane, 3.37 mL, 5.4 mmol) by syringe at 0 °C under
argon while stirring. After one hour, the resultant solution of
lithioethynyltrimethylsilane was canulated dropwise onto a solu-
tion of cyanuric chloride (1.0 g, 5.4 mmol) in dry THF (5.0 mL)
while stirring. The addition of the lithiated acetylene resulted in
4.2.2. Biofilm model for in vitro analysis of P. gingivalis
adherence to S. gordonii
a
viscous red-brown suspension. Stirring was continued
(2 h/0 °C), then isopentylamine (1.89 g, 2.51 mL, 10.8 mmol) was
added dropwise by syringe at 0 °C. The reaction mixture was
allowed to come to room temperature and stirring was continued
(48 h). Column chromatography of the crude mixture on silica gel
provided pure triazine 91 (0.96 g, 51.1%).
To prepare bacterial cells for biofilm culture, 10 ml of an over-
night S. gordonii culture was centrifuged at 5600 rpm for 5 min
and the cell pellet was suspended in 1 mL sterile PBS. Subse-
quently, 20 lL of 5 mg/ml 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 buf-
fered 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 pel-
4.1.7. 6-Ethynyl-N2,N4-diisopentyl-1,3,5-triazine-2,4-diamine
(40)
To
a
prechilled (0 °C) solution of N2,N4-diisopentyl-
(500 mg,
6-((trimethylsilyl)ethynyl)-1,3,5-triazine-2,4-diamine
1.4 mmol) in dry THF (25 mL) was slowly added tetra-n-butylam-
monium fluoride (TBAF, 0.72 mL, 0.72 mmol) under a nitrogen
atmosphere. The resulting solution was stirred at 5–10 °C (1 h).
After the reaction was complete as confirmed by TLC, cold water
(25 mL) was added to the reaction mixture followed by extraction
with dichloromethane (2 Â 25 mL). The combined organic layers
were dried over anhydrous sodium sulfate and concentrated to
obtain crude residue. The residue was purified by column chro-
matography (hexane/EtOAc, 7/3 ? hexane/EtOAc, 3/7) to give pure
40 (331 mg, 84%) as light yellow solid: Rf. 0.48 (CHCl3/MeOH, 9:1).
let was suspended in 1 mL PBS and 20 ll of 5(6)-carboxyfluores-
cein N-hydroxysuccinimide ester (4 mg/ml, 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 described 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 concentration of
test compound was added and incubated under anaerobic condi-
tions for 22 h at 37 °C. Test compounds were dissolved in
dimethylsulfoxide (DMSO) to generate 1000Â stock solutions and
4.1.8. General procedure for the synthesis of click compounds
(41–90)
To a solution of Group I (41–66), Group II (20–21) or Group III
(26–27) azides (0.1–0.18 mmol) in anhydrous THF (1.5 mL) were

P. C. Patil et al. / Bioorg. Med. Chem. 24 (2016) 5410–5417
5417
were routinely tested over a final concentration range of 0–60
l
M.
40–90 and 92 along with experimental procedures) associated
with this article can be found, in the online version, at http://
dx.doi.org/10.1016/j.bmc.2016.08.059.
1
l
L of the appropriate stock solution was added to each 1 mL ali-
quot of labeled P. gingivalis cells and incubated for 30 min at room
temperature prior to adding the suspension to the microtiter plate
wells. For control biofilms, 1
lL of DMSO was added to 1 mL of
References and notes
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To visualize P. gingivalis/S. gordonii biofilm formation, unbound
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of 25
l
m in depth were collected at three randomly chosen frames
m. Background
on each coverslip using a z-step thickness of 0.7
l
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 was 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 tripli-
cate for each concentration of test compound and three indepen-
dent experiments were conducted for each compound. Graphpad
InStat3 software was used for data analysis and statistical signifi-
cance was defined as p <0.05.
Acknowledgments
The measurement of high resolution mass spectra by the Texas
A&M University Laboratory for Biological Mass Spectrometry, Dr.
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support from the NIH/NIDCR through grant 1RO1DE023206 is
gratefully acknowledged.
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A. Supplementary data
Supplementary data (the 1H, 13C, FTIR) for compounds 6–29,
40–90 and 92; and HRMS data for new compounds 7–9, 11–29,