Characterizing the antimicrobial activity of N2,N4-disubstituted quinazoline-2,4-diamines toward multidrug-resistant Acinetobacter baumannii

Article abstract of DOI:10.1128/AAC.00059-17

We previously reported a series of N²,N⁴-disubstituted quinazoline-2,4- diamines as dihydrofolate reductase inhibitors with potent in vitro and in vivo antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) strains. In this work, we extended our previous study to the Gram-negative pathogen Acinetobacter baumannii. We determined that optimized N²,N⁴-disubstituted quinazoline-2,4-diamines are strongly antibacterial against multidrug-resistant A. baumannii strains when the 6-position is replaced with a halide or an alkyl substituent. Such agents display potent antibacterial activity, with MICs as low as 0.5 μM, while proving to be strongly bactericidal. Interestingly, these compounds also possess the potential for antibiofilm activity, eradicating 90% of cells within a biofilm at or near MICs. Using serial passage assays, we observed a limited capacity for the development of resistance toward these molecules (4-fold increase in MIC) compared to existing folic acid synthesis inhibitors, such as trimethoprim (64-fold increase) and sulfamethoxazole (128-fold increase). We also identified limited toxicity toward human cells, with 50% lethal doses (LD₅₀s) of ≤23 μM for lead agents 4 and 5. Finally, we demonstrated that our lead agents have excellent in vivo efficacy, with lead agent 5 proving more efficacious than tigecycline in a murine model of A. baumannii infection (90% survival versus 66%), despite being used at a lower dose (2 versus 30 mg kg^-1). Together, our results demonstrate that N²,N⁴-disubstituted quinazoline-2,4-diamines have strong antimicrobial and antibiofilm activities against both Gram-positive organisms and Gram-negative pathogens, suggesting strong potential for their development as antibacterial agents.

Full text of DOI:10.1128/AAC.00059-17

AAC Accepted Manuscript Posted Online 13 March 2017
Antimicrob. Agents Chemother. doi:10.1128/AAC.00059-17
Copyright © 2017 American Society for Microbiology. All Rights Reserved.
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Characterizing the Antimicrobial Activity of N2,N4-
Disubstituted Quinazoline-2,4-Diamines Towards
Multidrug Resistant Acinetobacter baumannii
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Renee Fleeman1,2#, Kurt S. Van Horn3#$, Megan M. Barber3^, Whittney N. Burda1&, David L. Flanigan3
Roman Manetsch4* and Lindsey N. Shaw1,2*
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1Department of Cell Biology, Microbiology and Molecular Biology, Center for Drug Discovery and
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Innovation, Department of Chemistry, University of South Florida, 4202 East Fowler Avenue, Tampa,
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FL 33620
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4Department of Chemistry and Chemical Biology and Department of Pharmaceutical Sciences
Northeastern University, 102 Hurtig Hall, 360 Huntington Avenue, Boston, MA 02115, USA.
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$ Avista Pharma Solutions, 3501 Tricenter Blvd, Durham NC 27713
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^Department of Pharmacy, Lake Erie College of Osteopathic Medicine, 1858 W Grandview Blvd, Erie,
Pennsylvania 16509
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&Department of Infectious Diseases & Pathology, College of Veterinary Medicine, University of Florida
2015 SW 16th Ave, Gainesville, FL 32608
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Author Contributions: #R. M. Fleeman and K. S. Van Horn contributed equally.
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Corresponding Author Information: *Phone: 813-974-2087; E-mail: shaw@usf.edu and Phone: 617-373-
6316; E-mail: r.manetsch@neu.edu
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Running Title: A. baumannii Quinazolines.

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Keywords: Acinetobacter baumannii; Quinazoline; Biofilm; Dihydrofolate reductase inhibitors; Gram
negative antibacterial.
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Abstract
We previously reported a series of N2,N4-disubstituted quinazoline-2,4-diamine as dihydrofolate reductase
inhibitors, with potent in vitro and in vivo antibacterial activity against MRSA strains. In this work we
extend our previous study to the Gram-negative pathogen Acinetobacter baumannii. We determined that
optimized N2,N4-disubstituted quinazoline-2,4-diamines are strongly antibacterial against multi-drug
resistant A. baumannii strains when the 6-position is substituted with a halide or an alkyl substituent.
Such agents display potent antibacterial activity, with MICs as low as 0.5 μM, whilst proving to be
strongly bactericidal. Interestingly, these compounds also possess the potential for anti-biofilm activity,
eradicating 90% of cells within a biofilm at or near MIC concentrations. Using serial passage assays we
observed a limited capacity for the development of resistance towards these molecules (4-fold increase in
MIC) compared to existing folic acid synthesis inhibitors, such as trimethoprim (64-fold increase) and
sulfamethoxazole (128-fold increase). We also identified limited toxicity towards human cells, with LD50
values of ≤ 23 µM for lead agents 4 and 5. Finally we demonstrate that our lead agents have excellent in
vivo efficacy, with lead agent 5 proving more efficacious than tigecycline in a murine model of A.
baumannii infection (90 % survival vs 66%), despite being used at a lower dose (2 mg kg-1 vs 30 mg kg-
1). As such, we demonstrate that N2,N4-disubstituted quinazoline-2,4-diamines have strong antimicrobial
and anti-biofilm activity, against both Gram-positive organisms and Gram-negative pathogens, suggesting
strong potential for their development as antibacterial agents.

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Introduction
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Acinetobacter baumannii is one of the most successful nosocomial pathogens, causing infections that
have over the past few decades become increasingly difficult to treat. The prolonged ability of A.
baumannii to survive on abiotic surfaces, alongside broad antimicrobial resistance, allows it not only to
survive, but thrive in hospital settings.(1) Consequently, there has been an alarming increase in mortality
associated with infections caused by this difficult to treat organism.(2) In addition to eliciting fatal
nosocomial infections, this pathogen is a primary agent of infections in military personnel, often resulting
from combat trauma or burns.(3, 4) These often result in chronic wound infections and biofilm-mediated
disease, with the latter resulting from surgery and implanted devices.(4) Such chronic A. baumannii
infections lead to complications, extended rehabilitation, increased use of hospital resources, and
considerably increased mortality.(4)
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Drug resistance in A. baumannii has resulted in few antibiotics left to eradicate the infections it causes,
with clinicians often turning to last resort, toxic treatment options.(1),(5, 6) The worldwide incidence of
pan drug resistant (PDR) A. baumannii has spread quickly, at least in part due to its naturally
transformable nature, leading to an increased capacity to acquire new determinants of resistance.(1), (6)
The occurrence of PDR isolates, with no effective treatment options, seemingly marks the beginning of a
post-antibiotic era for A. baumannii; thus, measures must be taken to identify effective therapeutic
options.(7)
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Quinazolines are an emerging class of compounds that have a broad range of biological activities ranging
from anti-cancer, anti-inflammatory, anti-psychotic, anti-diabetic, anti-leishmanial,(8, 9) and anti-
bacterial.(10-15) Kung et al. discovered a series of 2-substituted quinazolines with broad spectrum
antibacterial activity, inhibiting RNA synthesis and translation in a number of bacterial species.(16) More
relevant to this study, Harris et al. revealed 5-substituted 2,4-diaminoquinazolines that inhibited the
dihydrofolate reductase (DHFR) enzyme of Escherichia coli and S. aureus.(17) In so doing, they

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determined that the 5-substituted position of the 2,4-diaminoquinazolines was not as important for
enzyme binding affinity as the general structural type of the group. Unfortunately, these molecules were
not specific towards the bacterial DHFR enzyme, but also inhibited the bovine liver DHFR enzyme.(17)
Further analysis revealed that smaller substituents created greater activity in bacterial cells while larger
substituents were more active towards the bovine enzyme. However, unlike the quinazolines identified in
this study, the 5-substituted 2,4-diaminoquinazolines proved ineffective in animal models of
infection.(17)
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Our group has recently shown the utility of N2,N4-disubstituted quinazoline-2,4-diamines for the treatment
of S. aureus infections.(18) Specifically, we have shown them to be active against a library of MRSA
isolates, displaying strong bactericidal activities, with limited cytotoxic and hemolytic capacities towards
human cells. Mechanism of action profiling reveals that, much like other quinazoline compounds, they
appear to function by targeting bacterial dihydrofolate reductase.(18-21) We have also shown their
potential for anti-biofilm activity, low frequencies of mutation, and in vivo efficacy using murine models
of infection.(18)
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In this study, we have further explored the impact of N2,N4-disubstituted quinazoline-2,4-diamines as
antibacterial agents, focusing specifically on the Gram negative species A. baumannii. Using a library of
multi-drug resistant isolates, we reveal that these compounds are broadly bactericidal, dihydrofolate
reductase inhibitors. In addition, we observed that these compounds have low incidences of resistance and
possess the potential for anti-biofilm activity. Finally, we show the compounds are efficacious in vivo
using a murine model of A. baumannii infection. As such, we demonstrate for the first time the very real
potential of quinazoline derived compounds as antibacterial agents against the important human pathogen,
A. baumannii.
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Materials and Methods
General. All strains used in this study are listed in Table S1.
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Synthetic Protocols and Compound Characterization: Full details of compounds synthesis and
characterization can be found in the supplemental materials.
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Antibacterial activity assessment. Minimal inhibitory concentration (MIC) and minimal bactericidal
concentration (MBC) assays were performed in this study as documented by us previously.(18, 22-24)
Briefly, A. baumannii strains were grown in tryptic soy broth overnight cultures at 37 °C with shaking.
MIC determination was performed in a 96-well plate by diluting overnight cultures 1:1000 in Mueller
Hinton broth (MHB) and adding 195 µL to each well. Subsequently, 5 µL of quinazoline (or control
compounds) were added before incubation for 24 hours at 37 °C. Following, this MICs were determined
as the lowest concentration to produce a complete absence of growth. All compounds were diluted prior
to testing in DMSO to assess multiple concentrations with the addition of the same volume of solvent.
MBC assays were performed in an identical manner to MIC experiments, however after 24 hours
incubation, bacterial cells were serially diluted in phosphate buffered saline (PBS) and recovered on
antibiotic free tryptic soy agar (TSA) for 24 hours at 37 °C. MBC90 values were calculated using linear
regression of the percent recovery compared to no treatment controls.
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Biofilm eradication determination assay. These assays were performed as described by us
previously,(24, 25) as follows. Each of the A. baumannii strains were grown overnight in MHB. The next
day these were used to seed fresh MHB to an OD600 of 0.5, with 150 µL then added to the wells of a 96
well plate and grown for 24 hours at 37 °C. After 24 hours, the planktonic bacteria were carefully
removed and fresh MHB was added with increasing concentrations of lead quinazolines. After incubation
at 37 °C for 24 hours, planktonic cells were removed and biofilms were washed three times with PBS.
Biofilms were then resuspended in PBS and plated for cell viability on TSA. Biofilm recovery was
assessed compared to no drug controls, and determined as percent eradication. This was used to determine

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MBEC90 values (Minimal Biofilm Eradication Concentration), where the viability of cells within the
biofilm was reduced by 90%.
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Investigating the Mechanism of Action of Quinazoline-Based Compounds. To evaluate the effect
quinazolines have on DHFR reduction of dihydrofolic acid, a tetrahydrofolic acid rescue assay was
performed as described by us previously.(18) A. baumannii strain 1403 was grown overnight in LB, and
then diluted 1:1000 into fresh media. These cultures were then seeded into a sterile 96-well plate with
tetrahydrofolic acid added at concentrations ranging from 0 to 225 µM. Lead quinazoline 25 was then
added at 1X, 2X, and 5X the MIC and cultures were incubated at 37 °C for 18 hours. MICs were
determined and used to assess whether the addition of tetrahydrofolic acid rescued A. baumannii growth
from quinazoline inhibition. Assays were repeated in triplicate, alongside trimethoprim and
sulfamethoxazole controls.
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Serial Passage assay. In order to test potential resistance towards the quinazolines, a serial passage assay
was performed alongside control compounds (sulfamethoxazole and trimethoprim), as described by us
previously.(24) A. baumannii strain 1403 was grown overnight in LB media at 37 °C. The next day
cultures were diluted 1:100 in fresh media and seeded into a 96-well plate. Lead quinazolines or control
agents were added to respective wells at half MIC concentrations. Plates were then incubated for 24 hours
at 37 °C, followed by the removal of aliquots from these cultures to inoculate fresh media (1:100 dilution)
containing compounds at a 2-fold higher concentrations. These were then grown overnight, and the
procedure repeated for a total of eight days. The cultures were observed for a lack of growth, indicating
strains were no longer able to resist the action of a given compound. Each experiment was performed in
triplicate, yielding identical results.
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HepG2 cytotoxicity. Cytotoxicity assays were performed using human HepG2 cells (human liver
epithelial with hepatocellular carcinoma), as described by us previously.(18, 24) Cells were cultured in
Dulbecco’s modified eagle medium (DMEM), supplemented with 10% fetal bovine serum and 1%

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penicillin-streptomycin for 3 days at 37 °C and 5% CO2. Cells were then diluted to 1 x 105 ml-1 using
fresh DMEM and added to 96 well tissue culture plates at a volume of 100 μL. Plates were incubated for
24 hours at 37 °C and 5% CO2, allowing the cells to adhere to the plastic. After this time, media was
carefully removed and 200 μL fresh DMEM added with test compounds at concentrations: 0, 1, 2, 5, 10,
15, 30, and 50 μM. Plates were then incubated for 48 hours at 37 °C and 5% CO2. After 48 hours the
DMEM was removed and 100 μL of new media was added containing MTT (3-(4, 5-dimethylthiazol-2-
yl)-2, 5-diphenyltetrazolium bromide), followed by incubation for four hours at 37 °C and 5% CO2. After
4h, 75 μL of media was removed, replaced with 50 μL of 16% w/v SDS and DMSO followed by
incubation for ten minutes at 37 °C to solubilize any formazan produced. A Biotek plate reader was used
to measure the absorbance of formazan production at 540 nM. Lead compounds were solvated in 100%
DMSO for these studies, which served as the negative control. LD50 values were determined for each
compound by comparison to vehicle only controls.
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Hemolysis assay. A hemolysis assay was performed using whole human blood (Bioreclamation), as
described previously.(24) Briefly, human red blood cells (hRBCs) were resuspended in 20% v/v 1X HA
buffer (4.25 mL 10% NaCl; 1mL CaCl2 in 50 mL sterile water), before lead compounds were added at 2
µM, 10 µM and 20 µM, in a final volume of 100 µL. Cells were incubated for 15 minutes at 37 °C before
being centrifuged at 5,500 g for 1 minute to pellet non-lysed hRBCs. The supernatant was removed,
added to a 96-well Microtiter plate and the OD543 read using a BioTek Synergy2 plate reader. The
negative control was vehicle only (DMSO), and the positive control was 1% triton X-100. Assays were
performed in triplicate, with data displayed as percent hemolysis compared to controls, defined as:
Percent Hemolysis = (OD543 test sample – OD543 no drug control) / (OD543 triton X-100 – OD543 no drug
control) x 100.
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In vivo Efficacy Testing Using a Murine Model of Lethal Peritonitis. A murine model of lethal
peritonitis was used to demonstrate the effectiveness of the lead quinazolines to clear bacterial infections,
as described by us previously.(24) Six mice per group were infected via I.P. injection (right side) with 7.5

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x 108 CFU mL-1 of A. baumannii 1646 in PBS containing 5% mucin. After 1h, mice were inoculated by
I.P. injection to the left side of the abdomen with either 2 mg/kg of lead agent 25 (test group); 30 mg/kg
tigecycline (positive control); or vehicle alone (45% w/v (2-hydroxypropyl)-β-cyclodextrin in water
(negative control). Mice were monitored twice daily for five days to assess mortality. All animal studies
received written approval after review by the Institutional Animal Care & Use Committee in the Division
of Comparative Medicine & Division of Research Integrity & Compliance at the University of South
Florida. The clinical endpoint was reached for this study when the mice reached a pre-moribund state.
The number of mice surviving between control and treatment groups was compared and analyzed for
statistical significance using a log-rank (Mantel-Cox) test.
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Results and Discussion
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N2,N4-disubstituted quinazoline-2,4-diamines are active against multi-drug resistant A. baumannii
isolates. We have previously reported the activity of N2,N4-disubstituted quinazoline-2,4-diamines against
MRSA strains (18). To determine if our compounds have activity against any other bacterial species, we
screened them against the other ESKAPE pathogens. In so doing, we identified a number of analogues
that were effective against A. baumannii, but lacked activity towards other members of the ESKAPE
pathogen set. To explore these findings more broadly, we expanded our studies to include a clonally
diverse collection of A. baumannii isolates (Table 1). Strong activity was found against a number of
strains, with single digit micromolar MICs noted for three benzenoid substituted N2-benzyl-N4-
methylquinazolin-2,4-diamines 1, 2, and 3 against the 1646 strain.
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Following the identification of active quinazolines 1, 2, and 3, additional N2-benzyl-N4-methylquinazolin-
2,4-diamines were made with either 6- or 7- substitutions (Table 2). Substitution at the 6-position with a
bromo or a methyl group was found to be more beneficial for activity than substitution at the 7-position
when comparing 6-bromoquinazolin-2,4-diamine 4 with its 7-substituted counterpart 6 or the 6-methyl-
substituted quinazoline 5 with its 7-substituted analogue 7. Importantly, N2-benzyl-N4-methylquinazolin-
2,4-diamine analogue 8, which lacks any substitution at the benzenoid ring, was inactive with an MIC of
≥50 µM and therefore demonstrated the importance of a 6- or 7-substuent on the benzenoid ring.
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A similar trend was observed with N4-benzyl-N2-methylquinazolin-2,4-diamine analogues when
comparing 6-substituted compounds 10 and 11 with the 7-substituted analogues 14 and 15 (Table 3).
Furthermore, substitution in 6- or 7-position with an electron withdrawing chloro or a bromo moiety
yielded quinazolines 9, 10, 13, or 14 which were more potent than corresponding methyl- or methoxy-
substituted analogues 11, 12, or 15. Of all the quinazolines of the first two subseries tested, only
compound 5 was active against the clinically important 1403 strain with an MIC of 10 µM, leading us to
believe that continued work on the benzenoid ring would be highly beneficial.

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Extending the N2-benzyl chain to a N2-phenethyl was investigated to see if an increase in activity would
be found (Table 4). Compound 16 with no benzenoid substitution was 4-fold more active than the benzyl
analogue 8 (Table 1). Compounds 17 – 20 were also found to be slightly more potent than the benzyl
analogues 9 – 12 (Table 3) with MICs of 2 or 4 µM.
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With the importance of substitution at the 6-position identified, new analogues were evaluated with vinyl,
alkyl or aryl substitutions (Table 5) (Figure S1). While the MIC barrier of 2 µM was not broken against
the most susceptible strain (1646), major advances were seen in activity against the most resistant isolate
(1403). In particular, n-pentyl-, cyclohexenyl- and cyclohexyl-substituted quinazolines 27, 29 and 30 had
MICs of 2 µM against most isolates besides the 1652 strain for which they had MICs of 10 µM and 30
µM. These three compounds revealed that large, bulky and lipophilic groups at the 6-position are not only
tolerated but beneficial for inhibiting the growth of A. baumannii. Phenyl- and furanyl-substituted
quinazolines 31 and 32 were less active, as were the vinyl and ethyl analogues 22 and 23, the isopropenyl
and isopropyl analogues 24 and 25, and the cyclopentenyl-quinazoline 28.
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Lead Quinazolines are Bactericidal in Activity. Lead quinazolines 4, 5, 26, 29, 27, and 30 were
selected to be further evaluated for antimicrobial effects. The first assay utilized was a minimal
bactericidal concentration (MBC) assay, to assess whether leads compounds were bacteriostatic or
bactericidal. The six lead agents were screened to identify their MBC90 towards each of the six A.
baumannii isolates used in the SAR studies (Table S2), with data for isolate 1646 detailed in Table 6.
Lead agents were all found to be broadly bactericidal, with MBC90 values ranging from 0.77 µM to 1.8
µM. Compounds 4 and 5 were found to be the most efficacious at eliminating bacterial growth, with
MBC90 values of 0.81 µM and 0.77 µM respectively. Further to this, we were able to obtain complete
eradication of bacterial growth for these two compounds at 1 µM for 24 and 5 µM for 25. Although
marginally less effective, compounds 26, 29, 30, and 27, all still efficiently reduced bacterial viability,
with MBC90 values of 1.8 µM, 1.5 µM, 1.1 µM, and 1.1 µM respectively. Moreover, compound 26
resulted in complete bacterial eradication at 5 µM, which is only 5x its MIC.

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Front Runner Agents Impact the Viability of Cells Within a Biofilm. A. baumannii, like many
nosocomial pathogens, utilizes biofilm formation to increase persistence and decrease sensitivity to the
action of antibiotics. Accordingly, the ability to impact cell viability within a biofilm is an important
attribute for novel antimicrobial compounds. As such, we next tested our isolates for this activity, again
using our library of multi-drug resistant strains (Table S2), with data from strain 1646 shown in Table 6.
As with our bactericidal profiling, lead quinazolines 4 and 5 again had the most promising activity, with
90% biofilm eradication (MBEC90) seen at 3.3 µM and 2.8 µM, respectively (Table 6). Further to this,
analogue 5 was the most effective lead agent with a 3-log reduction in biofilm viability observed at 10
µM. Compound 4 reduced biofilm viability by 3.6-log, but not until a concentration of 50 µM. Lead
quinazoline 30 had biofilm eradication potential similar to 4, reducing viability by 4.2-log at 50 µM,
although its MBEC90 (1 log reduction) was found to be close to this value at 41 µM. Compounds 26 and
29 also had promising activity with both displaying MBEC90 at a concentration of 8.9 µM. Extended
testing with these two quinazolines revealed that compound 29 reduced biofilm viability by 1.6-log at 50
µM, while 26 resulted in a 1.4-log reduction in biofilm viability at the same concentration.
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Lead Quinazoline Derivatives Appear to Function by Targeting Dihydrofolate Reductase. To
determine if lead quinazolines inhibit the A. baumannii dihydrofolate reductase (DHFR) enzyme, similar
to that seen for sister compounds in our work with MRSA, an in vitro rescue assay was performed.(18)
Accordingly, the viability of A. baumannii cells was tested using lead quinazoline 5 in the presence or
absence of tetrahydrofolic acid (THF, 0 µM-225µM), the end product produced by DHFR. After 24 hours
incubation, we determined that only 10 µM of THF was sufficient to rescue bacterial growth from the
inhibitory effects of lead agent 25. This data suggests the potential mechanism of action for our
compounds is perhaps via inhibition of tetrahydrofolic acid production. Whilst this finding supports data
generated by ourselves and others regarding the impact of quinazoline molecules on bacterial cells,(18-
21) we cannot discount the possibility of other potential targets within for these compounds within A.
baumannii.

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Quinazolines Derived Compounds Induce Limited Capacity for Resistance. An important attribute of
novel antibiotics is the ability to fend off the development of resistance towards their effects. To assess
this capacity, A. baumannii strains were incubated overnight with 0.5x MIC of each of the frontrunners.
The next day cells were washed and used to inoculate fresh media that contained a two-fold increase in
drug. This was repeated for a total of 8 days, alongside sulfamethoxazole (SMX) and trimethoprim (TMP)
controls, both of which target the same pathway as our lead agents (Figure 1), as well as an unrelated
agents, tetracycline (TET).(26) Upon analysis, we determined that all of our frontrunner compounds out
performed SMX, TMP and TET, generating much lower incidences of resistance. Specifically, lead
agents 4 and 5 had the most striking effects with MICs increasing over the 8-day test period by only 4-
fold, compared to 64-fold (TMP) and 128-fold (SMX and TET) for the control agents. Each of the other 4
agents were similarly impressive in their ability to limited resistance, resulting in an increased MIC of
only 16-fold, which, whilst not as promising as 4 and 5, is still profoundly reduced compared to our
controls.
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Front Runner Quinazolines Have Limited Toxicity Towards Human Cells. In order to gain a sense of
the toxicity of lead quinazolines towards eukaryotic cells, we determined LD50 values for human HepG2
cells (Table 6, Figure 2A). Importantly, we observed >50% cell viability for all compounds at
concentrations up to 6 µM. Furthermore, 4 of our 6 leads returned >50% viability at 12 µM, whilst 26 and
29 were only marginally less promising, returning HepG2 cell viabilities of 49% and 42%, respectively, at
this concentration. When treated with 25 µM of each lead quinazoline or control antibiotics we observed
only fractionally less than 50% recovery. Importantly, lead agents 4 and 5 at 25 µM performed the best,
with 43% and 44% viability observed, respectively. Similarly, lead agents 26 and 30 allowed for 41% and
39% respective viability at this concentration, while treatment with 27 and 29 resulted in 31% and 32%
viability, respectively. The control compounds sulfamethoxazole and trimethoprim returned 46% and
45% viability at the highest concentration tested, which is in line with data generated from our front
runners. To place lead compound data in context, 4 and 5 have the greatest therapeutic window for

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infection treatment. Specifically, lead agent 4 possesses a 46-fold preference in specificity towards
bacteria, with an MIC (0.5 µM) much lower than the LD50 (23 µM) towards human liver cells. Similarly,
lead agent 5 displayed a 22-fold activity index (AI =LD50 / MIC), which is a measure of specificity
towards bacterial cells (Table 6). As an additional measure of toxicity, we next tested the hemolytic
capacity of the front runners using whole human blood (Figure 2B). Importantly, we observed negligible
capacity of our lead quinazolines to lyse human red blood cells when incubated for 1 hour at a
concentration of 10 µM. Specifically, we observed average hemolysis well below 1% (range = 0.24% –
0.47%), whilst the positive control (1% triton X-100) produced 100% lysis during a similar time frame.
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N2,N4-disubstituted quinazoline-2,4-diamines are efficacious in vivo. As a final assessment, we used a
murine model of lethal A. baumannii infection to determine the efficacy of quinazolines in vivo. This was
performed using frontrunner 5, which had the most promising properties from all of our biological testing.
Accordingly, mice were inoculated with a lethal dose of A. baumannii via intraperitoneal injection on the
right side of the abdomen. One hour post challenge, mice were treated with an intraperitoneal injection of
2 mg kg -1 of frontrunner 5 on the left side of the abdomen. As a control, we also performed similar testing
using 30 mg kg -1 of tigecycline, which we already know our test strain to be susceptible to in vitro. In so
doing, we determined that quinazoline 5 resulted in a statistically significant survival rate of 83% of
infected animals, compared to only 17% for vehicle only controls (Figure 3). We also saw significant
survival of animals injected with tigecycline, although this was at a rate of 66%, which is inferior to that
of our frontrunner agent. As such, this would suggest that our class of N2,N4-disubstitutedquinazoline-2,4-
diamines have excellent potential for development as antibacterial agents targeting multi-drug resistant A.
baumannii infections.
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Concluding Remarks: A library of N2,N4-disubstituted quinazoline-2,4-diamines, which was previously
shown to have antibacterial activity against MRSA,(18) was also found to have potent effects towards the
multi-drug resistant Gram negative species A. baumannii. We assessed seventy-three N2,N4-disubstituted
quinazoline-2,4-diamines and found that 6- or 7-substituted N2-benzyl-N4-methylquinazoline-2,4-

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302
303
304
305
306
307
308
309
310
311
312
313
diamines displayed promising activity, with MICs ranging from 0.5 to 30 μM against the six strains of A.
baumannii tested. Over thirty molecules were designed and synthesized to conduct a structure-activity
relationship study to systematically probe the substituents in the N2-, N4-, 6-, and 7-positions. The most
potent in vitro activities were obtained with quinazoline-2,4-diamines bearing a N2-benzyl moiety and a
N4-methyl group. Furthermore, quinazolines substituted in 6-position with a halide or alkyl group were
more potent compared to analogs substituted at the 7-position. 6-n-Pentyl- and 6-cyclohexyl-substituted
quinazolines 27 and 30 were among the most effective agents since they were equipotent with single-digit
µM MICs against the six tested A. baumannii strains. Following, frontrunner compounds 4, 5, 26, 29, 27,
and 30 were tested for bactericidal activities and biofilm eradication. We found the lead quinazolines 4
and 5 displayed the strongest bactericidal and biofilm activity towards A. baumannii, with MBC90 values <
1 μM and MBEC90 values < 4 μM. These compounds also allowed for limited resistance development,
displaying only a 4-fold increase in MIC against A. baumannii over an 8-day period, which was only a
fraction of that observed for control compounds. Using a murine model of infection, we determined that
lead agent 5 was more effective, and at lower concentrations, in rescuing mice from a lethal dose of A.
baumannii than our control agent tigecycline. Our results reveal the potent antibacterial activities of N2-
benzyl-N4-methylquinazoline-2,4-diamines against A. baumannii, and show their potential for
development to treat both Gram-positive and Gram-negative multidrug resistant infections.
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Funding information: This work was funded in part by the University of South Florida, Northeastern
University, and grants AI103715 and AI80626 (both L.N.S.) from the National Institutes of Health.
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21.
22.
23.
24.
25.
26.
396
397

398
399
400
401
402
Figure 1. Lead quinazolines generate limited resistance by A. baumannii isolates. The six lead
quinazolines were tested in a stepwise serial passage assay, alongside sulfamethoxazole (SMX),
trimethoprim (TMP) and tetracycline (TET) controls. The first passage began with 0.5 X MIC and with
each passage the concentration of all compounds was increased by 2-fold. The graph displays the fold
increase in the MIC over the course of 8 days. Representative data generated using strain 1403 is shown.
403
404
405
406
407
408
409
Figure 2. Cytotoxicity towards human cells. A: Lead quinazolines were tested at 25 µM, 12 µM, 6 µM,
and 3 µM, against human HepG2 cells compared to solvent only controls. The known antibiotics
sulfamethoxazole (SMX) and trimethoprim (TMP), which target the same pathway as our quinazolines,
were also tested in parallel. B: The six lead quinazolines were tested at 10 µM against whole human
blood for the ability to lyse erythrocytes. Shown is the percent lysis of human red blood cells compared to
a triton X-100 (T) control at a concentration of 1%.
410
411
412
413
414
Figure 3. Lead quinazolines are efficacious in vivo. Mice were administered with a lethal dose of A.
baumannii 1646. They were then give lead quinazoline 5 at 2 mg kg-1, tigecycline at 30 mg kg-1, or
vehicle. Survival was monitored over a 5 day period. Statistical significance was determined using a log-
rank (Mantel-Cox) test; *=p<0.05.
415

128
4
SMX
TMP
TET
5
26
29
27
30
64
32
16
8
2
0
2
4
6
8
Passage
Figure 1. Lead quinazolines generate limited resistance by
A. baumannii isolates. The six lead quinazolines were tested in
a stepwise serial passage assay, alongside sulfamethoxazole
(SMX), Trimethoprim (TMP) and Tetracycline (TET) controls. The
first passage began with 0.5 X MIC and with each passage the
concentration of all compounds was increased by 2-fold. The
graph displays the fold increase in the MIC over the course of 8
days. Representative data generated using strain 1403 is shown.

100
10
1
A
B
100
50
0
5
4
26
30
29
27
TMP
SMX
0.1
0.0
3.0
6.0
12.0
25.0
4
5
26 29 27 30
compound
T
m M
Figure 2. Cytotoxicity towards human cells. A: Lead quinazolines were tested at 25 µM, 12 µM, 6 µM, and 3
µM, against human HepG2 cells compared to solvent only controls. The known antibiotics sulfamethoxazole
(SMX) and trimethoprim (TMP), which target the same pathway as our quinazolines, were also tested in parallel.
B: The six lead quinazolines were tested at 10 µM against whole human blood for the ability to lyse erythrocytes.
Shown is the percent lysis of human red blood cells compared to a Triton X-100 (T) control at a concentration of
1%.

100
50
0
[5] 2 mg kg-1
*
*
[Tigecycline] 30 mg kg-1
Vehicle
0
1
2
3
4
5
Time / Days
Figure 3. Lead quinazolines are efficacious in vivo. Mice were administered with a lethal
dose of A. baumannii 1646. They were then give lead quinazoline 5 at 2 mg kg-1, tigecycline at
30 mg kg-1, or vehicle. Survival was monitored over a 5 day period. Statistical significance was
determined using a log-rank (Mantel-Cox) test; *=p<0.05.

a
Table 1. SAR focusing on benzenoid ring substitution of various quinazoline-2,4-diamines
1403
MIC
(µM)
1646
MIC
(µM)
1649
MIC
(µM)
1650
MIC
(µM)
1651
MIC
(µM
1652
MIC
(µM)
Compound
R
1
50
2
6
2
25
30
25
15
>50
50
6
20
50
30
2
3
>50
>50
10
10
a
Sulfamethoxazole (SMX) and trimethoprim (TMP) are internal controls for each in vitro MIC assay:
SMX, 138 µM 1403, 118 µM 1646, 118 µM 1649, 118 µM 1650, 118 µM 1651, and 118 µM 1652; TMP,
103 µM 1403, 34 µM 1646, 517 µM 1649, 120 µM 1650, 103 µM 1651, and 103µM 1652.

a
Table 2. Probing benzenoid substitution of N2-benzyl-N4-methylquinazolin-2,4-diamines
1403
MIC
(µM)
1646
MIC
(µM)
1649
MIC
(µM)
1650
MIC
(µM)
1651
MIC
(µM)
1652
MIC
(µM)
Compound
R
6-Br
6-Me
7-Br
7-Me
6-H, 7-H
4
5
6
7
8
50
10
2
2
12
25
15
50
8
10
20
20
>50
50
>50
6
12
50
25
25
>50
>50
50
>50
10
12
>50
>50
35
>50
a
Sulfamethoxazole (SMX) and trimethoprim (TMP) are internal controls for each in vitro MIC assay:
SMX, 138 µM 1403, 118 µM 1646, 118 µM 1649, 118 µM 1650, 118 µM 1651, and 118 µM 1652; TMP,
103 µM 1403, 34 µM 1646, 517 µM 1649, 120 µM 1650, 103 µM 1651, and 103µM 1652.

a
Table 3. Probing benzenoid ring substitution of N4-benzyl-N2-methylquinazolin-2,4-diamines
1403
MIC
(µM)
1646
MIC
(µM)
1649
MIC
(µM)
1650
MIC
(µM)
1651
MIC
(µM)
1652
MIC
(µM)
Compound
R
9
6-Cl
6-Br
6-Me
6-OMe
7-Cl
>50
>50
>50
>50
>50
>50
>50
4
2
6
6
4
50
50
50
6
>50
12
>50
>50
>50
>50
>50
>50
>50
10
11
12
13
14
15
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
8
>50
>50
>50
7-Br
7-Me
4
12
a
Sulfamethoxazole (SMX) and trimethoprim (TMP) are internal controls for each in vitro MIC assay:
SMX, 138 µM 1403, 118 µM 1646, 118 µM 1649, 118 µM 1650, 118 µM 1651, and 118 µM 1652; TMP,
103 µM 1403, 34 µM 1646, 517 µM 1649, 120 µM 1650, 103 µM 1651, and 103µM 1652.

a
Table 4. Benzenoid ring substitutions of N4-methyl-N2-phenethylquinazolin-2,4-diamines
1403
MIC
(µM)
1646
MIC
(µM)
1649
MIC
(µM)
1650
MIC
(µM)
1651
MIC
(µM)
1652
MIC
(µM)
Compound
R
16
17
18
19
20
6-H
6-Cl
6-OMe
6-Br
6-Me
>50
>50
>50
>50
>50
12
2
4
2
2
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
10
>50
>50
>50
>50
>50
10
a
Sulfamethoxazole (SMX) and trimethoprim (TMP) are internal controls for each in vitro MIC assay:
SMX, 138 µM 1403, 118 µM 1646, 118 µM 1649, 118 µM 1650, 118 µM 1651, and 118 µM 1652; TMP,
103 µM 1403, 34 µM 1646, 517 µM 1649, 120 µM 1650, 103 µM 1651, and 103µM 1652.

a
Table 5. Extension of the 6-position of N2-benzyl-N4-methylquinazolin-2,4-diamines
1403
MIC
(µM)
1646
MIC
(µM)
1649
MIC
(µM)
1650
MIC
(µM)
1651
MIC
(µM)
1652
MIC
(µM)
Compound
R
21
6-Me
10
25
30
>50
>50
10
2
2
2
25
10
10
25
35
10
2
50
20
10
25
2
20
25
22
23
24
25
26
27
28
29
30
31
32
2
30
30
2
>50
>50
25
10
35
2
>50
>50
30
25
2
2
2
2
10
>50
2
2
50
10
2
>50
10
30
2
>50
30
2
2
2
2
2
10
30
40
10
25
20
>50
20
2
50
>50
15
>50
a
Sulfamethoxazole (SMX) and trimethoprim (TMP) are internal controls for each in vitro MIC assay:
SMX, 138 µM 1403, 118 µM 1646, 118 µM 1649, 118 µM 1650, 118 µM 1651, and 118 µM 1652; TMP,
103 µM 1403, 34 µM 1646, 517 µM 1649, 120 µM 1650, 103 µM 1651, and 103µM 1652.

Table 6. In vitro antibacterial assessment of front runner quinazoline compounds against multi-drug
resistant A. baumannii. Activity Index = LD50 / MIC
Compound
4
5
26
1
29
2
27
1
30
2
MIC [µM]
0.5
0.8
3.3
23
1
MBC90[µM]
0.8
2.8
22
22
1.8
8.9
16
22
1.5
8.9
23
12
1.1
11.5
12
1.1
41.2
11
6
MBEC90 [µM]
LD50 (HepG2) [µM]
Activity Index
46
12