Anti-biofilm activity of quinazoline derivatives against: Mycobacterium smegmatis

Article abstract of DOI:10.1039/c9md00156e

Bacteria employ a number of mechanisms to resist the effects of antibiotics, including the formation of biofilms. We explored the anti-biofilm capabilities of a library of compounds based upon a 2-aminoquinazoline (2-AQ) scaffold against Mycobacterium sme

Full text of DOI:10.1039/c9md00156e

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DOI: 10.1039/C9MD00156E
Journal Name
COMMUNICATION
Anti-biofilm Activity of Quinazoline Derivatives against
Mycobacterium smegmatis
Karlie E. Coxa and Christian Melander a*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Bacteria employ a number of mechanisms to resist the effects of prevent disease relapse. Challenges with this regimen include
antibiotics, including the formation of biofilms. We explored the ensuring patient completion and substantial drug toxicity due
anti-biofilm capabilities of a library of compounds based upon a 2- to extended antibiotic therapy.7
aminoquinazoline (2-AQ) scaffold against Mycobacterium
Recently it has been shown that M. tuberculosis readily
smegmatis. This study resulted in the identification of 2-AQ forms biofilm-like communities on cellular debris in vitro, and in
derivatives with biofilm inhibition activity against M. smegmatis.
these communities the bacteria are more resistant to treatment
with isoniazid.8 M. tuberculosis biofilms have also been shown
to survive treatment with isoniazid and rifampin while
maintaining a minimum inhibitory concentration (MIC) 50 times
greater than planktonic bacteria.9 It has been hypothesized that
these biofilm-like communities may be the source of persistent
TB reservoirs within an infected host.10 Therefore, the
development of small molecules with anti-biofilm properties
against mycobacteria could potentially be employed as
adjuvants in combination with standard TB antibiotics, leading
to a reduction of treatment time and increased patient
compliance.
M. smegmatis is used as a model bacterium for M.
tuberculosis since it is a non-pathogenic, faster growing
bacterium. Furthermore, M. smegmatis shares over 2000
genetic homologs with M. tuberculosis including cell wall
structure and resistance mechanisms.11 Previously we have
shown that molecules identified as anti-biofilm molecules
against M. smegmatis, showed anti-biofilm properties against
M. tuberculosis, thus validating the use of M. smegmatis as a
model organism for the discovery of anti-biofilm agents against
M. tuberculosis. Additionally, these anti-biofilm compounds
effectively reversed biofilm-based antibiotic tolerance when
paired with isoniazid. The lead compound from these initial
studies was the 2-aminoimidazole (2-AI) derivative 1 (Figure 1),
which was found to inhibit M. smegmatis biofilm formation with
an IC50 value of 5.3 μM (where IC50 is defined as the
concentration at which the compounds inhibits 50% biofilm
formation in comparison to untreated control).8
Biofilms are an assembly of surface associated bacteria that
are encased in an extracellular polymeric substance (EPS).1 The
EPS is typically composed predominantly of polysaccharides,
and shields bacteria embedded within the biofilm from some
microbicides and the host immune response.2 Within a biofilm,
bacteria are upwards of 1000-fold more resistant to antibiotics
when compared to their planktonic (free-floating) brethren,
which often renders antibiotic treatments ineffective against
biofilm-based infections.3 Given the central importance of
biofilms within certain diseases, small molecules that control
biofilm formation may serve as key adjuvants that, when paired
with conventional antibiotics, lead to combinational therapies
for the effective eradication of bacteria within a biofilm state.4
Mycobacterium tuberculosis is a slow growing pathogen
that causes tuberculosis (TB). TB is predominantly an infection
of the lungs and can be classified as a latent infection or active
disease. Active TB disease patients present symptoms including
chest pain, cough, fatigue, weight loss, and fever.5 TB now ranks
above HIV/AIDS as the leading cause of death from a single
infectious agent.6 Globally, it is estimated that one-third of the
population is infected with TB.6
Current treatment for TB is a long process that lasts a
minimum of six months. Generally, a patient is treated with
isoniazid, rifampin and pyrazinamide for two months followed
by isoniazid and rifampin for four months, with the overall goal
of eradicating persistent TB reservoirs within the body to
a. Department of Chemistry and Biochemistry, University of Notre Dame, Notre
Dame, IN 46556.
b. *ccmeland@nd.edu
†Electronic Supplementary Information (ESI) available: Experimental procedures
and characterization data. See DOI: 10.1039/x0xx00000x
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The necessity of the amide linker wasViepwrAorbticeledOnlbinye
DOI: 10.1039/C9MD00156E
synthesizing a limited number of sulfonamide derivatives. These
derivatives
(5a-f)
were
synthesized
from
2,6-
diaminoquinazoline by reaction with the appropriate sulfonyl
chloride, followed by generation of the HCl salt (Scheme 2). All
these compounds were less active than the corresponding
amide derivatives (Table 2).
Figure 1. Active compound 1 and proposed structural modifications.
After establishing that the 2-AQ core motif was in fact a
viable scaffold for the synthesis of anti-biofilm agents, we next
turned to varying the identity of the alkyl chain. The first set of
derivatives accessed, was a conservative change from alkyl to
phenalkyl derivatives. Using the synthetic approach outlined in
Scheme 1, we accessed derivatives 4i-p (Figure 2). These
As part of a greater program directed towards the
identification of anti- biofilm agents, we have established that
both 2-aminopyrimidines and 2-aminobenzimidazoles have
anti-biofilm properties.12,13 Given the structural relationship
between our 2-AI derivatives and the 2-aminobenzimidazole
ring (essentially a 2-AI ring appended to a benzene ring), we
hypothesized that performing a similar structural modification
of the 2-aminopyrimidine ring, to yield 2-aminoquinazolines (2-
AQ), would also deliver active anti-biofilm agents (Figure 1).
Furthermore, given the success of quinazoline-based drugs, 2-
AQs with antibiofilm properties may be suitable for future in
vivo studies.14,15 Herein we report the synthesis of a pilot library
of 2-AQs and establish, for the first time, that this heterocycle
has potent antibiofilm activity against M. smegmatis.
This study was initiated with the synthesis of 2,6-
diaminoquinazoline 3 (Scheme 1), that we envisioned would
subsequently be employed for selective acylation at N-6, to
deliver the pilot library for screening. 2,6-Diaminoquinazoline 3
was synthesized by cyclizing commercially available 2-fluoro-5-
nitrobenzaldehyde with guanidine carbonate salt to yield 6-
nitro-2-aminoquinazoline 2, which, following hydrogenation,
delivered 3.16 This intermediate was then acylated with alkyl
acid chlorides (to initially mimic 1) and then was treated with
HCl and methanol to generate the HCl salts 4a-h (Scheme 1).
With these initial eight compounds in hand, we tested their
ability to inhibit the formation of M. smegmatis biofilms (Table
1) using a crystal violet reporter assay.12 We noted a trend
where activity correlated to alkyl chain length, as activity
increased from methyl to pentyl, with activity then dropping off.
Of these initial compounds, the pentyl derivative 4e was the
most active, returning an IC50 of 23 μM.
Scheme 1. Synthesis of sulfonamides.a,b apyridine, DCM, rt, 10 hr; bHCl/MeOH
Table 2. IC50 values of sulfonamides 5a-f.
compounds also demonstrated a dependence on chain length
but were generally more active than our initial set of alkyl
derivatives. The most active compound was the 4-propyl
derivative 4l, which returned an IC50 of 16 μM (Table 3).
Table 3. IC50 values of phenalkyl derivatives 4i-p.
Table 1. IC50 values of initial compounds 4a-h.
Figure 2. Structure of phenalkyl and halide containing derivatives, 4i-x.
Previous studies in our group on 2-AI derivatives have
shown that replacing phenalkyl motifs with halogenated
benzene motifs can deliver compounds with equipotent, if not
more potent activity.17 Based on this precedent, we posited that
the same general trend would be observed on the 2-AQ
scaffold. The 4-chloro derivative 4q was initially synthesized to
test the hypothesis, again using the approach outlined in
Scheme 1. 4q was essentially equipotent to 4l and delivered an
Scheme 2. Synthetic route to afford 6 substituted 2-aminoquinazoline analogues.a-d
aK2CO3, ACN, molecular sieves, 72°C, 16 hr; bH2, 10% Pd/Carbon, rt, 8 hr; cK3PO4, dry
THF, 0°C to rt,10 hr; dHCl/MeOH
2 | J. Name., 2012, 00, 1-3
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IC50 value of 15 μM. We then varied the identity of the halide
and found that replacement of the chlorine with either fluorine
(4s) or iodine (4t) abolished activity, while activity was
diminished by substitution with bromine (4r), which returned
an IC50 value of 44 μM. Di-substituted derivatives, 4u and 4v,
were active, albeit less than 4q, and returned IC50 values of 42
and 46 μM respectively. Finally, we tested whether chlorination
on the alkyl derivatives would generate compounds with
equivalent or improved activity by synthesizing and testing
compounds 4w and 4x. Both compounds were slightly more
active than the corresponding methyl derivative 4a, with IC50
values of 43 μM and 50 μM respectively.
Conflicts of interest
Dr. Melander is co-founder of Agile Sciences, a biotechnology
company seeking to commercialize small molecules with
antibiofilm activity.
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DOI: 10.1039/C9MD00156E
Notes and references
1
2
3
4
5
T. F. Mah and G. A. O’Toole, Trends Microbiol, 2001, 9 (1),
34-39.
R. J. Worthington and C. Melander, Anti-Infective agents,
2014, 12 (1), 120-138.
K. Lewis, Antimicrob Agents Chemother, 2001, 45 (4), 999-
1007.
R.M. Donlan and J.W. Costerton, Clin Microbiol Rev, 2002, 15
(2), 167-193.
L. Rook, Tuberculosis in Priority Medicines for Europe and
the World "A Public Health Approach to Innovation”, World
Health Organization, Geneva, Switzerland 2013.
World Health Organization and Global Tuberculosis
Programme, Global tuberculosis control, WHO report. Global
Tuberculosis Programme, Geneva, Switzerland 2017, 15.
C.R. Horsburgh Jr., C.E. Barry 3rd, and C. Lange, N Engl J Med,
2015, 373 (22), 2149-60.
Finally, we tested the toxicity of lead 2-AQ derivatives
Table 4. IC50 values of halide containing derivatives 4q-x.
6
7
8
9
against both planktonic M. smegmatis cells as well as eukaryotic
red blood cells (a standard test for antibiotic toxicity). M.
smegmatis planktonic toxicity was quantified to determine if
D.F. Ackart, E.A. Lindsey, B.K. Podell, R.J. Melander, R.J.
Basaraba, and C. Melander, Pathog Dis, 2014, 70 (3), 370-8.
K. Kulka, G. Hatfull, and A.K. Ojha, J Vis Exp, 2012, (60),
10 I.M. Orme, Tuberculosis (Edinb), 2014, 94, 11.
11 M. Altaf, C.H. Miller, D.S. Bellows, and R. O’Toole,
Tuberculosis (Edinb), 2010, 90 (6), 333-7.
12 E.A. Lindsey, R.J. Worthington, C. Alcaraz, and C. Melander,
Org Biomol Chem, 2012, 10 (13), 2552-61.
13 E.A. Lindsey, C.M. Brackett, T. Mullikin, C. Alcaraz, and C.
Melander, Medchemcomm, 2012, 3 (11), 1462-1465.
14 T. P. Selvam and P. V. Kumar, Res Pharm, 2011, 1, 1.
15 Shagufta and I. Ahmad, Medchemcomm, 2017, 8, 871.
16 M.M. Chen, M. Zhang, B. Xiong, Z.D. Tan, W. Lv, and H.F.
Jiang, Organic Letters, 2014, 16 (22), 6028-6031.
17 C. M. Brackett, R. J. Melander, I.H. An, A. Krishnamurthy, R. J.
Thompson, J. Cavanagh, and C. Melander, J. Med. Chem.,
2014, 57, 7450.
biofilm
inhibition
was
occurring
through
an
antibiotic/microbicidal mechanism or through a non-toxic
mechanism and was established by comparing the growth
curves of bacteria grown in the absence and presence of
compound. The growth curves of bacteria treated with either 4l
or 4q at 60 μM indicate that biofilm inhibition occurs through a
non-toxic mechanism (Figure S1). Red blood cell toxicity was
measured by quantifying hemolysis of lead compounds 4l and
4q as well as inactive compound 4p. At 500 μM (highest
concentration tested), 4l, 4p and 4q caused 25%, 57%, and 24%
lysis respectively compared to 1% Triton-X. At 200 μM, 4l, 4p
and 4q effected 14%, 31% and 4% lysis respectively. Both active
compounds, 4l and 4q, return a hemolytic index (defined here
as concentration that causes hemolysis/IC50) of >33, indicating
their relative non-toxic nature.
In conclusion, we have established for the first time that the
2-AQ heterocycle can form the basis for the construction of anti-
biofilm molecules. In a proof of concept study, we have
established that lead compounds 4l and 4q are low micromolar
inhibitors of M. smegmatis biofilm formation, making them
some of the most potent compounds reported for this
application. These compounds modulate biofilms in a non-toxic
manner and possess a relatively low hemolytic index. Given
these properties, the 2-AQ heterocycle may form the basis for
future in vivo experiments to study the impact inhibition of
biofilm formation has upon TB treatment.
This work was funded by the National Institutes of Health
(AI106733).
We purchased the red blood cells from Hemostat
laboraties https://hemostat.com/
This journal is © The Royal Society of Chemistry 20xx
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