S. Andjouh, Y. Blache
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Psammaplin A
structure–activity relationship (SAR) could be highlighted at this stage:
all compounds possessing an alkyl-type linker (series a, b, c) were in-
active (less than 50% of inhibition of the adhesion). Replacement of the
carbon in the series a by an oxygen (series d) or nitrogen (series e)
enhanced the activity. Considering aromatic linkers (series f and g),
only the compounds connected in the 1,4-positions were founded to be
active (series g). In order to precise structure-activity relationships,
effective concentrations to inhibit 50% of the bacterial adhesion (ex-
pressed as EC50) were determined for compounds 4d-g, 5d-e, 6d-g, 7d-
g which inhibited > 50% of adhesion at 200 μM. Results of this screen
are outlined in Table 2. In this way, first observation was to note that
globally the TC14 strain was more sensitive to this class of bis-triazoles
than TC8 and 4 M6 strains. Among the three classes tested, series g
possessing an 1,4-linked aromatic ring as central part (4g, 6g, 7g) were
the more potent compounds especially 6g an 7g with EC50 closed to
ampicillin and tributyltin oxide (TBTO). In term of SAR, it is interesting
to note that the dimethylaminoethyl chain (6g) as well as the di-
methylaminopropyl chain (7g) are common natural framework found
in bromotyrosine alkaloïds possessing antifouling properties, and that
this class of substituents afforded a beneficial aspect when compared to
simple hydroxyl or methyl groups found in alkaloids extracted from
sponges such as aplysamines or hemibastadins or simple 2-(3′-Bromo-
4′-hydroxyphenol)ethanamine.33
HO
Br
OH
N
O
H
N
S
S
Br
N
H
O
N
N
HO
OH
Targeted library
N
N
N
N
N
OR
RO
Br
Linker
Br
Figure 1. Structure of psammaplin A and of targeted library.
3,4 and 6 carbons, two heteroatomoic dialkynes (oxygen and nitrogen),
and finally two aromatic systems. The formation of the bis-triazole
analogues was then achieved by performing the copper(I)-catalyzed
1,3-dipolar cycloaddition of the organic azides with appropriate dia-
lkynes resulting in the formation of two 1,2,3-triazoles. In general,
these reactions usually proceed to completion in 6–36 h at room tem-
perature in water with a variety of organic co-solvents, such as tert-
butanol, ethanol, DMF, DMSO, THF or CH3CN.28,29 Ethanol was usually
chosen rather than DMF to allow an easier workup and a better purity
of products as described in our previous work but in this case DMF was
used because of the poor solubility of the resulting bis-triazoles in
ethanol. Practically, 1 equivalent of dialkyne was added to a solution of
appropriate azide (3a-d, 2.6 equivalents), CuSO4/sodium ascorbate in a
water/DMF mixture (50/50) and the reaction time was optimized at
24 h at room temperature. Results reported in Table 1 show that all
compounds were obtained in excellent yields (> 77%), but it is notable
that compound 5g bearing an 1,4-linked aromatic ring could not be
isolated and purified for further biological tests.
In order to determine if these compounds exhibited a specific anti-
biofilm activity or if this observation was simply related to a general
toxic effect on the bacteria, a growth inhibition and viability assay was
performed. Active compounds 6g and 7g were tested for their capacity
to inhibit the growth of the three strains TC14, TC8 and 4M6.
Experiments were performed at the high concentration of 100 μM
static effects on the three bacterial strains. A slight effect was observed
for compound 7g while compound 6g exhibited effects much more
closed to ampicillin especially on the TC8 and 4M6 strains which seems
to be more sensitive than TC14.
For viability, the same methodology used for antiadhesion assay
with Syto®61 was applied using resazurin test at the concentrations of
5, 10, 20, 50, 100, 200 μM. Results at 5 and 100 μM are reported in
Fig. 3 (see supplementary materials for detailed results). At a low
concentration of 5 μM, concentration closed to their EC50 as antibiofilm
compounds, compounds 6g and 7g were not lethal to bacteria. This
suggested that their anti-biofilm activities were not directly connected
to antibacterial effect in contrast to ampicillin which is toxic especially
to TC14 and TC8. Furthermore, at the high concentrations (100 μM),
compounds 6g and 7g presented a slight bactericidal effect on the three
In order to assess anti-biofilm activity of these compounds against
representative Gram-negative bacterial biofilms, three strains were
chosen for their capacity to form biofilms: Pseudoalteromonas lipolytica
(4 M6).30 In an initial screening process, all compounds were tested for
their ability to modulate biofilm formation at concentration of 200 μM
by using our previous method adapted from Leroy et al. using the
specific fluorophore Syto®6131,32
.
Partial information about
N3
Cl
Cl
,HCl
N3
Cl
N
n
( )
RO
(i)
(ii)
(iii)
Br
Br
Br
Br
OMe
OH
OH
2 (97%)
3a (96%)
3c R = -(CH2)2-N(CH3)2 (99%)
3d R = -(CH2)3-N(CH3)2 (98%)
1
(ii)
N3
(i) : BBr3(1.5eq.)/CH2Cl2, 0°C, rt, 4h
(ii) : NaN3/DMF, 5h, 90°C
RO
Br
(iii) : K2CO3 ,18-Crown-6, acetone,reflux
3b R = Me
Scheme 1. Preparation of starting azides 3a-d.
2
Andjouh, Sofyane
