A flexible approach to 1,4-di-substituted 2-aminoimidazoles that inhibit and disperse biofilms and potentiate the effects of β-lactams against multi-drug resistant bacteria

Article abstract of DOI:10.1016/j.ejmech.2012.12.005

The pyrrole-imidazole alkaloids are a 2-aminoimidazoles containing family of natural products that possess anti-biofilm activity. A library of 1,4-di-substituted 2-aminoimidazole/triazoles (2-AITs) was synthesized, and its anti-biofilm activity as well as oxacillin resensitization efficacy toward methicillin resistant Staphylococcus aureus (MRSA) was investigated. These 2-AITs were found to inhibit biofilm formation by MRSA with low micromolar IC₅₀ values. Additionally, the most active compound acted synergistically with oxacillin against MRSA lowering the minimum inhibitory concentration (MIC) 4-fold.

Full text of DOI:10.1016/j.ejmech.2012.12.005

Accepted Manuscript
A Flexible Approach to 1, 4-Disubstituted 2-Aminoimidazoles that Inhibit and Disperse
Biofilms and Potentiate the Effects of β-Lactams against Multi-Drug Resistant
Bacteria
Robert E. Furlani, Andrew A. Yeagley, Christian Melander
PII:
S0223-5234(12)00727-1
10.1016/j.ejmech.2012.12.005
EJMECH 5876
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 August 2012
Revised Date: 26 November 2012
Accepted Date: 5 December 2012
Please cite this article as: R.E. Furlani, A.A. Yeagley, C. Melander, A Flexible Approach to 1, 4-
Disubstituted 2-Aminoimidazoles that Inhibit and Disperse Biofilms and Potentiate the Effects of β-
Lactams against Multi-Drug Resistant Bacteria, European Journal of Medicinal Chemistry (2013), doi:
10.1016/j.ejmech.2012.12.005.
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*Graphical Abstract (for review)
Click here to download Graphical Abstract (for review): Graphical Abstract.docx
ACCEPTED MANUSCRIPT
Graphical Abstract:
Compounds are described that inhibit and disperse MRSA biofilms, with the most potent
compound displaying an IC50 of 4.14 M. 2-Aminoimidazoles also suppress the MIC of
oxacillin upwards of 4-fold against MRSA.

*Highlights (for review)
ACCEPTED MANUSCRIPT
Highlights:
 Development of a synthetic route to 1,4-substituted 2-aminoimidazoles
 Lead compounds inhibit both MRSA and multidrug resistant Acinetobacter
baumanii biofilm formation
 Lead compounds disperse MRSA biofilm formation
 Lead compound suppresses the oxacillin MIC against MRSA 4-fold

*Revised Manuscript
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ACCEPTED MANUSCRIPT
A Flexible Approach to 1, 4-Disubstituted 2-Aminoimidazoles that Inhibit and Disperse
Biofilms and Potentiate the Effects of β-Lactams against Multi-Drug Resistant Bacteria
Robert E. Furlani, Andrew A. Yeagley, and Christian Melander*
Abstract:
The pyrrole-imidazole alkaloids are a 2-aminoimidazoles containing family of natural products
that possess anti-biofilm activity. A library of 1,4-disubstituted 2-aminoimidazole/triazoles (2-
AITs) was synthesized, and its anti-biofilm activity as well as oxacillin resensitization efficacy
towards methicillin resistant Staphylococcus aureus (MRSA) was investigated. These 2-AITs
were found to inhibit biofilm formation by MRSA with low micromolar IC50 values.
Additionally, the most active compound acted synergistically with oxacillin against MRSA
lowering the minimum inhibitory concentration (MIC) four-fold.
1. Introduction:
A biofilm can be defined as a matrix enclosed population of microorganisms that adhere to a
biological or non-biological surface.[1] Bacteria within a biofilm are upwards of 1000-fold more
resistant towards antibiotics than their planktonic counterparts.[2] The National Institutes of
Health have estimated that over 80% of microbial infections in the human body are a result of
biofilms.[3] As a result, bacterial biofilms can represent a significant impediment to the treatment
of bacterial infections as they are less susceptible to antibiotic therapy.
Outside of the phenotypic defense against antibiotics that biofilms provide, bacteria have
a number of additional defense mechanisms against an antibiotic threat. Chief amongst these are
genotypic responses that render bacteria multi-drug resistant. An example of this phenomenon
includes resistance to β-lactam antibiotics that have arisen as a result of mutation, selection, and
horizontal gene transfer.[4] The rise of drug resistant bacteria, coupled with the diminished
interest in antibiotic research and development from pharmaceutical companies poses a very real
threat to modern health care. This epidemic has become so serious that the Infectious Disease
Society of American has recently issued a call to action from the medical community.[5] 2-
Aminoimidazole (2-AI) derivatives have recently been synthesized in our lab and have been
shown to be effective toward biofilm inhibition and dispersion.[6] The 2-aminoimidazole
functionality is essentially a guanidine mimetic with a highly modulated pKa and is found in
numerous marine natural products. Prototypical examples in this class include the natural
products such as ageleferin and oroidin, isolated from the sponge Agelas conifera (Figure 1).[7]
Given the high efficacy of the 2-AI moiety toward modulating bacterial behavior, it was posited
that these compounds might be able to be utilized as adjuvants to existing antimicrobial drugs
such as β-Lactams by resensitizing the bacteria to the effects of the antibiotic.

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2-Aminoimidazole/triazole (2-AIT) compounds have been previously investigated in our lab
and have proved to be very active towards inhibiting and dispersing biofilms of a number of
strains of pathogenic bacteria. We recently documented that compound 3 also possesses the
ability to lower the MIC of oxacillin against an Iberon clone of MRSA (ATCC BAA-44) four
fold.[8] Previous studies in our group have focused on synthetic approaches to generate
analogues of the 2-AI tail region „tail„ region of 2-AIT compound and the biological impact
these modifications have upon biofilm inhibition and dispersion.[6e, 9] The next logical step in
our medicinal chemistry program is to develop synthetic approaches to access defined
substitution patterns on the 2-aminoimidazole (2-AI) head region while maintaining the triazole
containing tail intact (figure 2), and studying the impact that these substitution patterns have
upon biofilm inhibition/dispersion, suppression of antibiotic resistance in multi-drug resistant
bacterial strains, and the inherent microbicidal activity of the 2-AI compounds.[6e, 9a, 10]
Herein we report a versatile synthetic approach to 1,4 substituted 2-AITs, the use of this
approach to the assembly of a pilot library of 2-AIT analogues, and the biological activity of this
library against methicillin resistant staphylococcus aureus (MRSA) and multi-drug resistant
Acinetobacter baumannii (MDRAB).
2. Results and Discussion
2.1. Chemistry
It was initially believed that the synthesis of the 1, 4-substituted 2-AIT could be accomplished by
cyclization of an N-substituted amino ketone with cyanamide, as similar experiments in our lab
have produced 4,5-substituted 2-AIs from unsubstituted α-amino ketones.[6f] We envisioned
the relatively unstable intermediary amino ketone could be synthesized from the nucleophilic
ring opening of an epoxide with a primary amine(Figure 3).
The synthesis of epoxide 10 began by isomerizing internal alkyne 5 via the alkyne “zipper”
reaction to give terminal alkyne 6 (Scheme 1).[11] An azide-alkyne Huisgen cycloaddition
(“click reaction“) was then performed on terminal alkyne 6 and azide 7 to provide alcohol 8 in
quantitiative yield.[12] The alcohol was then subjected to a combined Swern/Wittig protocol, to
deliver alkene 9 in 66% yield over 2 steps.[13] The alkene was then selextively epoxidized with
m-chloroperoxybenzoic acid, to yield the key epoxide intermediate 10 in an 80% yield.
We initially hoped to directly open the epoxide with a primary amine substrate at the less
hindered carbon that following oxidation of the resultant alcohol would yield α-amino ketone 11.
Pyrimidine and aniline were initially chosen as nucleophiles to open the epoxide, but in each
o
attempt there was no reaction, even upon heating to 60 C (Table 1). It was thought that a more
nucleophillic primary amine would better serve to open the epoxide. Product was formed upon
reaction of the epoxide with 8 equivalents of decylamine, in a DMSO:H2O solvent system at 60

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oC Although this procedure delivered the amino alcohol in a 55% yield, the reactants suffered
from poor solubility in the DMSO:H2O solution and the product contained a considerable
amount of impurites that persisted even after multiple attempts at purification. The amino
alcohol was also formed upon reaction with undecylamine as the nucleophile; however the use of
different conditions (DMF, Montmorillonite K10, 60 oC) chosen to improve solubility still led to
impurites that could not be separated from the product even after multiple attempts at
purification. Given the inability to separate the amino alcohol from the impurities associated
with the reaction, we subjected each mixture to the Swern oxidation, in an attempt to produce
the amino ketone; however in each case produced no product. Smaller molecular weight
primary amines, hexyl- and heptyl-, as well as benzylamine, were also used as nucleophiles,
under similar conditions, and in every case no product was obtained.
Given the failure of this route to generate the target substitution pattern, we explored the use
of a reductive aza-wittig reaction.[14] Secondary amines have been prepared from azides
previously by use of this one-pot method by use of a Staudinger reduction followed by imine
formation with the corresponding aldehyde, and in situ reduction of the crude imine. This was
explored by opening epoxide 10 with sodium azide under reflux, yielding the azido-alcohol 12
in 87% yield (Scheme 2). The oxidation of the alcohol was initially attempted with IBX as well
as DMP; however neither oxidant produced the corresponding azido amine. The Swern
oxidation was found to be an ideal oxidation, producing azido ketone 13 in 94% yield. Next we
wanted to perform an aza-wittig reaction on the azido group, followed by a direct reduction of
the intermediate imine functionality. This method would be an effective way to produce alkyl
derivatives at the N1 position on the 2-AI ring. The azide was reduced with PPh3 and in the
presence of an excess of a corresponding aldehyde, imine formation was observed after two
hours in refluxing toluene, however the imine could not be isolated, and all efforts towards in
situ imine reduction using Na(CN)BH3 and acetic acid proved fruitless, as decomposition was
observed in each case.
With the failure of this second route to access the N-substituted amino-ketone intermediate,
it became necessary to explore a third route toward synthesizing the key intermediate 11.
Recently, Sorrell and coworkers observed that the reaction of an α-bromo ketone with a primary
amine produced the desired N-substituted α-amino ketone, which in their hands was followed by
direct reaction with formamide to yield 1,4 substituted imidazoles.[15] Based upon this
precedent, it was reasoned that using cyanamide rather than formamide would yield the desired
1,4 substituted 2-AI, which necessitated to synthesis of the α-bromo ketone 15 (Scheme 3). This
was accomplished by performing a Jones oxidation on the previously synthesized triazole-
alcohol 8, producing carboxylic acid 14 in 70% yield after purifaction. The carboxylic acid was
then reacted with oxalyl chloride producing the intermediate acid-chloride, followed by
treatment with diazomethane, which after treatment with HBr to yield α-Bromo ketone 14 in
84% yield over the three steps.

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Numerous attempts to react α-bromo ketone 15 with a variety of primary amines failed to
1
provide the desired amino ketone. Upon monitoring the reaction by H NMR, it was observed
that the bromide displacement was rapid, giving almost full conversion to the amino ketone
within 10 minutes evidenced by the shift in the methylene singlets from the starting material to
the product (-Δppm 0.4). It was observed that the amino-ketone singlet converted into a
multiplet after one hour, corresponding to an undesired side product (most likely self
condensation dimer). Based upon this observation, we developed a one-pot tandem procedure
for the bromide displacement and subsequent cyanamide cyclization to deliver the target 2-AI
derivatives (Scheme 4). This was accomplished by reacting the α-bromo ketone 15 with 0.95
equivalents of the primary amine for 30 minutes in ethanol that was followed by addition of
cyanamide. The pH of the reaction was then adjusted to 4.3 by adition of 0.1N HClaq and the
resulting solution was refluxed for three hours to deliver the targeted 1,4 substituted 2AITs as
their free bases after chromatography with CH2Cl2/MeOH saturated with NH3. Treatment with
HCl provided the final HCl salts for biological testing in yields up to 36% over the 3 steps
(Scheme 4, table 2).
2.2. MRSA and MDRAB biofilm modulation
The 2-aminoimidazole moiety has been observed in nature to possess antibiofouling
properties[16] and has provided a priveledged scaffold for the development of anti-biofilm
compounds.[6a, 6b, 6e, 6f, 9b, 10, 17] Most these 2-AI compounds appear to effect biofilms at
both the beginning and final stages of a biofilm development cycle, leading to compounds that
both inhibit and disperse bacterial biofilms. Therefore we were initially interested in testing the
effect that the 1,4-substitution pattern has upon the 2-AITs ability to inhibit of biofilm formation,
as well as induce dispersion of preformed biofilms.
As 1,4 di-substituted 2-AITs have previously not been investigated for antibiofilm
activity, we proceeded to evaluate their ability to inhibit biofilm formation by a representative
Gram-positive bacterial strain, MRSA (ATCC BAA-44), and a representative Gram-negative
bacterial strain, MDRAB (ATCC BAA-1605) We also assesed their ability to disperse
preexisting biofilms. Here, we define the 2-AIT concentration required to inhibit 50% of
biofilm growth, relative to an untreated control, as the IC50 value, while the concentration
required to disperse 50% of a preformed biofilm is defined as the EC50 value. Finally, we also
assayed each compound for their inherent antibiotic activity by determining the MIC of each
compound under CLSI conditions using the microdilution protocol.
The ability of each compound to inhibit biofilm development was assessed using the
crystal violet reporter assay.[18] The most potent MRSA biofilm inhibitor of the initial library,
4h, contained an n-pentyl substitutent (Table 3). Compound 4h exhibited an IC50 of 4.14µM
against MRSA, which was slightly lower than the MIC of our lead compound 3, and was also
shown to act in a non-microbicidal fashion according to growth curve analysis. In an attempt to

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increase activity, analogues of 4h were synthesized in which the alkyl chain was either shortened
to n-butyl, 4g, and lengthened to n-hexyl, 4i. However in both cases activity decreased as we
‟
observed IC50 s of 10.8, and 6.2 µM respectively. The benzyl substituted 2-AI, 4k, gave a
promising IC50 of 7.16 µM. Again, we attempted to augment activity by synthesizing analogues
in which we inserted a methylene unit, 4l, or removed a methylene unit, 4j; however both
analogues had dimineshed anti-biofilm activity, returning IC50 values of 25.1 and 9.9 µM
respectively. The cyclopentyl derivative, 4b, gave an IC50 of 9.01 µM, but again as in the
previous cases, decreasing or increasing the ring size to a cyclopropy, 4a, or cyclohexyl, 4c,
‟
delivered compounds with decreased activity (IC50 s of >100 µM and 18.7 µM respectively).
We were also interested determining the activity of our 2-AITs against a multi-drug
resistant Gram-negative bacterium. To this end, we investigated MDRAB. A. baumannii is
nosocomial pathogen that survives in hospital settings based, in part, by its ability to form robust
biofilms.[19] The antimicrobial activity and biofilm inhibition activity were determined for all
compounds (table 3). When compared to MRSA, these compounds were less active towards
MDRAB displaying much higher MIC and IC50 values. The leading biofilm inhibitor was hexyl
derivative 4i, with an IC50 of 26.2 µM, followed closely by the n-pentyl 4h, cyclopentyl 4b, and
benzyl derivative 4k, with IC50 values of 31.4, 33.7, and 33.9 µM respectively. Furthermore,
four of the compounds, 4a, 4c, 4d and 4f, showed sharp drops in inhibition over a very narrow
range in concentrations, usually indicative of toxic mechanisms.
Finally, we were interested in the dispersion potential of our 2-AITs with a preformed
biofilm. Dispersion of a preformed biofilm is a very desirable property as compounds which
simply inhibit biofilm formation will mostly likely find utility as prophylactic agents, while
compounds that disperse pre-formed biofilms have the potential to treat an established disease
state. The 5 most potent biofilm inhibitors, 4b, 4h, 4i, 4k and 4l were selected for analysis, and
their EC50s were determined against MRSA. All compounds exhibited similar biofilm dispersion
activity against MRSA, and returned EC50 values of 33.0 – 45.1 μM (Table 4).
2.3. Resensitization of MRSA
Due to the growing problem of multi-drug resistant bacteria, our group has been
exploring the ability of suitably derived 2-AIs to suppress antibiotic resistance in multi-drug
resistant bacteria.[17d, 20] To this end, we were interested in testing the potential of our 1,4
substituted 2-AITs to resensitize MDR bacteria towards oxacillin, since 2-AIT 3 has been
documented to reduce the MIC of oxacillin four-fold at 25% of its MIC (25 µM).[8] To study the
resensitization effects, the minimum inhibitory concentration (MIC) of each 2-AIT against
MRSA (ATCC BAA-44) was initially determined by using a microdilution protocol. To probe
suppression of antibiotic resistance, the MIC of oxacillin was determined in the presence of 25%
the MIC of each 2-AIT compound. In our experience, 2-AI compounds themselves at 25% their
MIC value display limited toxicity to bacteria. Mueller Hinton Broth was inoculated with BAA
44, and oxacillin was serially diluted in both the inoculated media alone to serve as a control, as

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well as inoculated media supplemented with 2-AITs. The MICs of the oxacillin in the presence
of the 2-AITs were compared with that of oxacillin alone, and a fold-reduction was noted by
dividing the oxacillin MIC by the MIC of oxacillin with the 2-AITs (Table 5). The most active
compound was 4e, which has an isobutyl substituent at the 1‟ nitrogen. Compound 4e elicited a
four-fold reduction in the MIC of oxacillin. Unfortunately no other compounds tested were able
to match the resensitization ability of the lead compound 4e or the parent compound 3.
Finally, the checkerboard assay was used to determine if there was any synergism
between the 2-AIT compounds and oxacillin. [21] The assay is performed by serially diluting
one antibiotic vertically down a 96-well plate, followed by the second antibiotic horizontally
across the plate. The fractal inhibitory concentration (FIC) is then calculated by dividing the
MIC of each antibiotic in the combinations, by that of the antibiotic alone. An FIC index (ΣFIC)
is than determined for each well by adding each of the individual antibiotic FICs corresponding
to that well. A ΣFIC of ≤ 0.5 is indicative of synergistic effects between the two antibiotics. The
lead compound in our library gave a ΣFIC of 0.5 suggesting synergism between the compound
and oxacillin. However all other 1, 4 substituted 2-AIT tested gave a ΣFIC was higher than 0.5
(Table 5), further confirming the lack of synergy between the 1,4-substituted 2-AITs and
oxacillin.
3. Conclusion
We have successfully developed an efficient approach to the synthesis of 1,4-substituted-
2-aminoimidazole/triazole conjugates via a key one-pot N-alkylation/cyanimide cyclization
procedure, and have employed this strategy to assemble a pilot library of 2-AIT derivatives.
This synthesis requires the use of no protecting groups, and can be further used for the
construction of a library of general 1,4-substituted 2-AIs. The compounds were found to be
active biofilm inhibitors, with the lead compound being n-pentyl derivative 4h. Compound 4h, as
well as 4g (n-butyl) and 4f (allyl) inhibited biofilm formation through a non-microbicidal
mechanism. Compounds 4b, 4i, 4k, and 4l were also active; however growth curve analysis
showed that these compounds acted in a microbicidal fashion at their IC50 concentration.
Combination studies with these compounds demonstrated that certain 2-AIT analogues
suppressed oxacillin resistance in the MRSA strain, with the lead compound, 4e, effecting a four-
fold reduction in the MIC of oxacillin, as well as a ΣFIC of 0.500 indicating a synergistic
behaviour between compound 4e and oxacillin. Given the potential of the 2-AIT scaffold to
serve a platform to design molecules that both inhibit/disperse biofilms and suppress antibiotic
resistance in MDR bacteria, we are currently exploring the potential of lead 2-AIT derivatives
and other 2-AI compounds in animal models of MDR bacterial infection. These studies will be
reported in due course.
4. Experimental Section

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4.1. Chemistry
All reagents used for chemical synthesis were purchased from commercially available sources,
and required no further purification. Chromatography was performed with 60 Å mesh standard
grade silica gel from Sorbtech.
Infrared spectra were obtained on an FT/IR-4100
spectrophotometer (ν max in cm-1). UV absorbance was recorded on a Genesys 10 scanning
UV/Vis spectrophotometer (max in nm). NMR solvents were obtained from Cambridge Isotope
1
13
Labs. H NMR (400MHz and 300 MHz) and C NMR (100 MHz) spectra were recorded at
25oC on Varian Mercury spectrometers. Chemical shifts () are given in ppm relative to their
respective solvent. Coupling constants (J) are in Hertz (Hz). Abbreviations are as followed: s =
singlet, d = doublet, t = triplet, dt = doublet of triplets, brs = broad singlet, and m = multiplet.
Mass spectra were obtained at the NCSU Department of Chemistry Mass Spectrometry Facility.
MRSA (ATCC # BAA-44), and MDRAB (ATCC # BAA-1605) were obtained from the
American Type Culture Collection. Mechanically defribulated sheep blood (DSB100) was
obtained from Hemostat Labs. Oxacillin sodium salt was purchased from Fluka.
Synthesis of N-(2-(4-(6-hydroxyhexyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-pentylbenzamide (8)
1-Octyn-ol (10 g, 79.2 mmol) was dissolved in 150 mL of tBuOH/H2O/DCM(2/2/1), and at
room temperature was added N-(2-azidoethyl)-4-pentylbenzamide (xx)(22.7 g, 87.16 mmol),
CuSO4 (1.89 g, 12 mmol) and sodium ascorbate (6.27 g, 31.7 mmol), and the reaction was stirred
for 16 hours. Reaction was quenched with water and extracted with a 10% MeOH in DCM
solution 2 times, and once with DCM. The organic layers were combined and washed with
brine, dried with magnesium sulfate, filtered, and concentrated in vacuo. Crude mixture was
purified by flash chromatography (2.5% to 10% gradient MeOH/DCM) to provide N-(2-(4-(6-
hydroxyhexyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-pentylbenzamide (30.154 g, 99% yield) as a white
1
solid. H NMR (CDCl3, 400 MHz)  7.68 (d, J = 8.4 Hz, 2H), 7.31 (s, 2H), 7.18 (d, J = 8 Hz,
2H), 4.54 (t, J = 5.8 Hz, 2H), 3.91 (dt, J = 5.6, 5.6, 2H), 3.58 (t, J = 6.4 Hz, 2H), 2.66-2.58 (m,
4H), 2.32(brs, 1H), 1.62-1.52(m, 4H), 1.52-1.47(m, 2H), 1.33-1.26(m, 8H), 0.86 (t, J = 6.8 Hz,
3H); 13C NMR (CDCl3, 100 MHz)  168.2, 148.3, 147.4, 131.4, 128.8, 127.4, 122.2, 62.8, 49.6,
40.1, 36.0, 32.7, 31.6, 31.1, 29.4, 28.9, 25.5, 25.5, 22.7, 14.2; IR (CDCl3) 3356, 3297, 2927,
2853, 2359, 2338, 1641, 1540, 1303, 1055; UV (max nm) 238; observed melting point: 94oC;
+
HRMS (ESI+) m/z 387.2746 [(M+H)+; calculated mass for C22H34N4O2 : 387.2755 amu].
N-(2-(4-(hept-6-enyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-pentylbenzamide (9). To a solution of
oxalyl chloride ( 13.8 mL, 156 mmol) in 340 mL of DCM at -78oC was added DMSO (27.7 mL,
390 mmol) dropwise. After 10 minutes, N-(2-(4-(6-hydroxyhexyl)-1H-1,2,3-triazol-1-yl)ethyl)-
4-pentylbenzamide was added dropwise over 10 minutes. This solution was then stirred for 30
minutes, until triethyl amine (75.7 mL, 546 mmol) was added dropwise, and stirring continued.
After 30 minutes the cold bath was removed and the solution was allowed to warm to room
temperature. Reaction was diluted with DCM then washed with water (3x500mL), and brine
(1x300mL). The organic layer was dried with magnesium sulfate, filtered and concentrated in

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vacuo, and the resultant N-(2-(4-(6-oxohexyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-pentylbenzamide
was kept under high vacuum until subsequent use.
To a slight slurry of
methyltriphenylphosphonium bromide( 30 g, 78 mmol) in 390 mL of toluene at -30oC was added
0.91 molar potassium bis(trimethyl)silyl amide(214.3 mL, 195 mmol) dropwise over 30 minutes,
and the solution was allowed to stir for 30 minutes at room temperature. The reaction vessel was
cooled back down to -30oC whereupon N-(2-(4-(6-oxohexyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-
pentylbenzamide was added dropwise dissolved in 100 mL of THF. Cold bath was removed
after addition. After 20 minutes 300 mL of brine was poured into the reaction vessel, and the
resultant mixture was extracted with DCM ( 3 x 300 mL), and the organic layer was dried with
magnesium sulfate, filtered and concentrated in vacuo. Crude mixture was purified by flash
chromatography ( 50%-100% Ethyl Acetate in Hexanes) to provide N-(2-(4-(hept-6-enyl)-1H-
1
1,2,3-triazol-1-yl)ethyl)-4-pentylbenzamide (19.67 g, 66%), as a white solid. H NMR (CDCl3,
400 MHz)  7.85 (t, J = 5.6 Hz, 1H), 7.71 (d, J = 8.4 Hz, 2H), 7.26 (s, 1H), 7.132 (d, J = 8 Hz,
2H), 5.76-5.66 (m, 1H), 4.93-4.85 (m, 2H), 4.50 (t, J = 5.4 Hz, 2H), 3.87 (dt, J = 5.2, 6 Hz, 2H),
2.58-2.52 (m, 4H), 1.97-1.92 (m, 2H), 1.59-1.51 (m, 4H), 1.34-1.21 (m, 8H), 0.83 (t, J = 6.8 Hz,
3H); 13C NMR (CDCl3, 100 MHz)  168.3, 148.2, 147.2, 139.0, 131.5, 128.7, 127.5,
122.1,114.6, 49.5, 40.3, 36.0, 33.8, 31.6, 31.1, 29.5, 28.9, 28.8, 25.7, 22.7, 14.2; IR (CDCl3)
3308, 3144, 3078, 2959, 2927, 2854, 1647, 1556, 1506, 1434,1333, 1304,1053, 909; UV (max
nm) 238; observed melting point: 87oC; HRMS (ESI+) m/z 383.2802 [(M+H)+; calculated mass
+
for C22H34N4O2 : 383.2805].
N-(2-(4-(5-(oxiran-2-yl)pentyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-pentylbenzamide (10). To a
solution of N-(2-(4-(hept-6-enyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-pentylbenzamide (16.7 g, 43.6
mmol) dissolved in 300 mL of DCM at 0oC was added mCPBA(24.5 g, 109 mmol). The
reaction vessel was then removed from cold bath and extracted with DCM (3x200 mL), and
organic layers were combined and washed with aq. NaHCO3(2x100 mL), and brine (1x100 mL).
The solution was dried with magnesium sulfate, filtered and concentrated in vacuo. Crude
mixture was purified by flash chromatography ( 100% EtOAc ) to afford N-(2-(4-(5-(oxiran-2-
1
yl)pentyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-pentylbenzamide (13.92 g, 80%) as a white solid. H
NMR (CDCl3, 400 MHz)  7.66 (d, J = 8 Hz, 2H), 7.30 (s, 1H), 7.22 (d, J= 8Hz, 2H), 6.81 (brs,
1H), 4.56 (t, J = 6 Hz, 2H), 3.96 (dt, J = 6 Hz, 5.6 Hz, 2H) 2.88 (m, 1H), 2.74-2.68 (m, 2H), 2.63
13
(t, J = 7.8 Hz, 2H), 2.45-2.43 (m, 1H), 1.69-1.28 (m, 15H), 0.88 (t, J = 7 Hz, 3H); C NMR
(CDCl3, 100 MHz)  168.1, 148.3, 147.4, 131.4, 128.8, 127.3, 122.1, 52.5,49.5, 47.2, 40.1, 36.0,
32.5, 31.6, 31.1, 29.5, 29.1, 25.9, 25.6, 22.7, 14.2; IR (CDCl3) 3313, 3295, 3146, 2928, 2851,
2365, 2330, 1643, 1549, 1301, 1058; (max nm) 238; observed melting point: 90oC; HRMS
+
(ESI+) m/z 399.2751 [(M+H)+; calculated mass for C22H34N4O2 : 399.2755].
N-(2-(4-(7-azido-6-hydroxyheptyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-pentylbenzamide (11). N-
(2-(4-(5-(oxiran-2-yl)pentyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-pentylbenzamide(1.17 g, 2.94 mmol)
was dissolved in 30 mL of 2-methoxyethanol/water(8:1). To this solution was added sodium
azide ( 0.5734 g, 8.82 mmol) and ammonium chloride ( 0.173 g, 3.23 mmol). The reaction was

ACCEPTED MANUSCRIPT
stirred at reflux (150oC) for 3 hours, at which point the reaction was allowed to cool back down
to room temperature, and then quenched with water and diluted with EtOAc. Resultant mixture
was washed with water (3x50mL) and brine (1x 50mL) and then dried with magnesium sulfate,
filtered, concentrated in vacuo, and placed under high vacuum overnight. Product obtained was
pure N-(2-(4-(7-azido-6-hydroxyheptyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-pentylbenzamide ( 1.13g,
1
87%) as a white solid, and needed no further purification. H NMR (CDCl3, 400 MHz)  7.67 (d,
J = 8.4 Hz, 2H), 7.45 (s, 1H), 7.32 (s, 1H), 7.16 (d, J = 8.4 Hz, 2H), 4.52 (t, J = 5.4 Hz, 2H),
3.88 (dt, J = 6 Hz, 5.2 Hz, 2H) 3.73-3.70 (m, 1H), 3.27-3.16 (m, 3H), 2.63-2.56 (m, 4H), 1.60-
1.54 (m, 4H), 1.42-1.24 (m, 10H), 0.85 (t, J = 7Hz, 3H); 13C NMR (CDCl3, 100 MHz)  168.3,
147.5, 132.3, 132.2, 128.8, 127.4, 122.2, 70.7, 57.2, 49.5, 40.1, 36.0, 34.4, 31.6, 31.1, 29.3, 28.9,
25.4, 25.2, 22.7, 14.2; IR (CDCl3) 3351, 2928, 2855, 2102, 1643, 1540, 1504, 1437, 1302, 1054;
(max nm) 238; observed melting point: 84oC; HRMS (ESI+) m/z 442.2922 [(M+H)+; calculated
+
mass for C23H36N7O2 : 442.2930].
N-(2-(4-(7-azido-6-oxoheptyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-pentylbenzamide (12). To a
solution of oxalyl chloride( .31 mL mL, 3.5 mmol) in 7 mL of DCM at -78oC was added DMSO
(.062 mL, 8.73 mmol) dropwise. After 10 minutes, N-(2-(4-(7-azido-6-hydroxyheptyl)-1H-
1,2,3-triazol-1-yl)ethyl)-4-pentylbenzamide(0.77 mg, 1.75 mmol) was added dropwise over 10
minutes. This solution was then stirred for 30 minutes, until triethyl amine (1.7 mL, 12.25
mmol) was added dropwise, and stirring continued. After 30 minutes the cold bath was removed
and the solution was allowed to warm to room temperature. Reaction was diluted with DCM
then washed with water ( 3x30mL ), and brine (1x30mL). The organic layer was dried with
magnesium sulfate, filtered and concentrated in vacuo, affording pure N-(2-(4-(7-azido-6-
1
oxoheptyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-pentylbenzamide (0.719 g, 94%) as a white solid . H
NMR (CDCl3, 400 MHz)  7.68 (d, J = 8 Hz, 2H), 7.47 (s, 1H), 7.32 (s, 1H), 7.14 (d, J = 8Hz,
2H), 4.52 (t, J = 5.8 Hz, 2 H), 3.86 (dt, J = 6 Hz, 6 Hz, 2H), 3.08-3.01 (m, 2H), 2.62-2.54 (m,
4H), 2.36 (t, J = 7.4, 2H), 1.60-1.51 (m, 4H), 1.33 (t, J = 7.4 Hz, 2H), 1.281-1.20 (m, 6H),
0.824 (t, J = 7Hz, 3H); 13C NMR (CDCl3, 100 MHz)  204.7, 168.1, 147.3, 132.3, 132.2, 129.0,
127.4, 122.2, 57.5, 49.5, 46.1, 39.9, 36.0, 31.6, 31.1, 29.1, 28.5, 25.4, 23.1, 22.6, 14.2; IR
(CDCl3) 3307, 3121, 3070, 2930, 2854, 2738, 2677, 2491, 2104, 1717, 1638, 1541, 1291; (max
nm) 238; observed melting point: 84oC; HRMS (ESI+) m/z 440.2763 [(M+H)+; calculated mass
+
for C23H34N7O2 : 440.2774].
6-(1-(2-(4-pentylbenzamido)ethyl)-1H-1,2,3-triazol-4-yl)hexanoic acid (14). To a stirred
solution of N-(2-(4-(6-hydroxyhexyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-pentylbenzamide( 8.94 g,
23.13 mmol) dissolved in 30 mL of acetone at 0oC was added freshly made 2 mM Jones
reagent(20mL) dropwise until titrated solution persisted green color indication oxidation had
completed. Reaction mixture then allowed to stir at room temperature for an additional 50
minutes, at which point 75 mL of water was added. Aqueous layer was extracted with ethyl
ether (2x60 mL) , and the ether layers were combined, and back extracted with 1M aq. NaOH
(2x60 mL). The combined aqueous layers were combined and acidified with concentrated HCl

ACCEPTED MANUSCRIPT
until pH indicated a pH of 1-2, and aqueous layer was extracted with ether (2x60mL). Ether
extracts were combined and washed with brine, dried with magnesium sulfate, filtered, and
concentrated in vacuo. Product afforded was pure 6-(1-(2-(4-pentylbenzamido)ethyl)-1H-1,2,3-
1
triazol-4-yl)hexanoic acid (6.436 g, 70 %) . H NMR (CDCl3, 400 MHz)  7.68 (d, J = 8 Hz,
2H), 7.37 (s, 1H), 7.31 (brs, 1H), 7.19 (d, J = 8.4 Hz, 2H), 4.55 (t, J = 5.6 Hz, 2H), 3.91 (dt, J =
5.6 Hz, 5.6 Hz, 2H), 2.66 (t, J = 7.4 Hz, 4H), 2.61 (t, J = 8 Hz, 2H), 1.67-1.55 (m, 6H), 1.38-
13
1.25 (m, 6H), 0.87 (t, J = 6.8 Hz, 3H); C NMR (CDCl3, 100 MHz)  178.0, 168.4, 148.0,
147.6, 131.2, 128.8, 127.4, 122.5, 49.7, 40.1, 36.0, 34.0, 31.6, 31.1, 28.9, 28.4, 25.2, 24.5, 22.7,
14.2; IR (CDCl3) 3735, 3435, 2935, 2849, 2362, 2008, 1637, 1426, 1057; (max nm) 238;
observed melting point: 100oC; HRMS (ESI+) m/z 401.2547 [(M+H)+; calculated mass for
+
C22H34N4O2 : 401.2550].
N-(2-(4-(7-bromo-6-oxoheptyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-pentylbenzamide (15). To a
solution of 6-(1-(2-(4-pentylbenzamido)ethyl)-1H-1,2,3-triazol-4-yl)hexanoic acid (2.8 g, 7
mmol) dissolved in 40 mL DCM, to which was added 5-10 drops of DMF at 0oC. At 0oC was
added oxalyl chloride (2 mL, 23 mmol) dropwise. The reaction continued stirring for 15 minutes
at 0oC, and then allowed to warm to room temperature, and stirring continued for an additional
hour. The reaction was then concentrated in vacuo, and placed under high vacuum for 2 hours.
The crude mixture was then dissolved in 20 mL of DCM, and placed in an ice bath. At 0oC the
crude mixture dissolved in DCM was added dropwise to a freshly made diazomethane (35 mmol)
in 80 mL of ethyl ether. The reaction was allowed to stir for 1 hour. At 0oC was added 4 mL of
HBr to the mixture, and the reaction was stirred for an additional 30 minutes. Reaction was
quenched with 100 mL of aqueous NaHCO3, and stirred for 10 minutes. 200 additional mL was
added to the reaction flask, and reaction was washed with aqueous NaHCO3 (3x100) and brine
(1x100), then organic layer was dried with magnesium sulfate, filtered and concentrated in vacuo
to afford pure N-(2-(4-(7-bromo-6-oxoheptyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-pentylbenzamide (
1
2.8 g, 84%); H NMR (CDCl3, 300 MHz)  7.67 (d, J = 8.1 Hz, 2H), 7.32 (s, 1H), 7.20 (d, J =
8.1 Hz, 2H), 7.08 (s, 1H), 4.56 (t, J = 5.4 Hz, 2H), 3.94 (dt, J = 5.6 Hz, 5.6 Hz, 2H), 3.85 (s,
13
2H), 2.70-2.59 (m, 6H), 1.67-1.57 (m, 6H), 1.34-1.29(m, 6H), 0.87 (t, J = 6.6 Hz, 3H); C
NMR (CDCl3, 100 MHz)  202.3, 168.2, 147.3, 131.4, 128.7, 127.4, 122.2, 60.4, 49.5, 48.5,
40.2, 36.0, 34.3, 31.6, 31.1, 28.7, 28.5, 25.4, 24.8, 22.7, 14.2; IR (CDCl3) 3311, 3120, 3070,
2929, 2854, 1719, 1637, 1540, 1295, 1051; (max nm) 238; observed melting point: 89oC; HRMS
+
(ESI+) m/z 477.1854 [(M+H)+; calculated mass for C23H33BrN4O2 : 477.1860 amu].
General procedure for synthesis of 1,4-2 AIs:
N-(2-(4-(7-bromo-6-oxoheptyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-pentylbenzamide (100 mg, 0.21
mmol) was dissolved in 95% ethanol. At room temperature R-NH2 (20 mmol, 0.95 eq) was
added dropwise to solution, and reaction was allowed to stir for 30 minutes. Cyanamide ( 0.264
g, 6.3 mmol) was added to the reaction vessel, and the pH was adjust to 4.3, using .1M HCl.
Reaction was refluxed at 95oC for 3 hours, then allowed to cool to room temperature, and

ACCEPTED MANUSCRIPT
concentrated in vacuo. Crude mixture was purified by flash chromatography (1%-7% MeOH
sat. with NH3 in DCM) to afford the free base. Free base put under high vacuum for 3 hours to
remove any excess NH3, then diluted with DCM, and treated with 5 drops of concentrated HCl.
Mixture concentrated in vacuo, and then placed under high vacuum overnight to afford pure
product as the HCl salt.
N-(2-(4-(5-(2-amino-1-cyclopropyl-1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-
pentylbenzamide hydrochloride (4a). Following the general procedure for they synthesis of
1,4-subsituted 2-aminoimidazoles, N-(2-(4-(7-bromo-6-oxoheptyl)-1H-1,2,3-triazol-1-yl)ethyl)-
4-pentylbenzamide was reacted with cyclopropylamine to provide N-(2-(4-(5-(2-amino-1-
cyclopropyl-1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-pentylbenzamide
1
hydrochloride ( 17 mg, 16%) as a yellow oil.; H NMR (CD3OD, 300 MHz)  8.61(s, 1H), 7.68
(d, J = 8.4 Hz, 2H), 7.26 (d, J = 7.8 Hz, 2H), 6.54 (s,1H), 4.84 (t, J = 5.3 Hz, 2H), 3.94 (t, J = 5.0
Hz, 2H), 3.08-3.02 (m, 1H), 2.88 (t, J = 7.4 Hz, 2H), 2.64 (t, J = 7.5 Hz, 2H), 2.43 (t, J = 7.5 Hz,
2H), 1.78-1.56 (m, 5H), 1.45-1.30 (m, 6H), 1.11 (dt, J = 7.8 Hz, 6.6 Hz, 2H), 0.97-0.87 (m, 6H);
13C NMR (CD3OD, 100 MHz)  169.3, 147.6, 144.3, 131.0, 128.5, 127.6, 127.2, 126.5, 111.8,
53.2, 39.2, 35.5, 31.3, 31.0. 27.8, 27.6, 27.5, 25.9, 24.0, 22.7, 22.4, 13.2, 6.0; IR (CDCl3) 3326,
2928, 2855, 2240, 1686, 1541, 1457, 1056, 975, 817; (max nm) 238; HRMS (ESI+) m/z
478.3290 [(M+H)+; calculated mass for C27H40ClN7O+: 478.3289 amu].
N-(2-(4-(5-(2-amino-1-cyclopentyl-1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-
pentylbenzamide hydrochloride (4b). Following the general procedure for the synthesis of 1,4-
subsituted 2-aminoimidazoles, N-(2-(4-(7-bromo-6-oxoheptyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-
pentylbenzamide was reacted with cyclopentylamine to provide N-(2-(4-(5-(2-amino-1-
cyclopentyl-1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-pentylbenzamide
1
hydrochloride ( 18 mg, 16%) as a yellow oil. H NMR (CD3OD, 300 MHz)  8.61(s, 1H), 7.69
(d, J = 8 Hz, 2H), 7.26 (d, J =8.4 Hz, 2H), 6.69 (s, 1H), 4.85 (t, J = 5.2 Hz, 2 H), 4.44-4.41(m,
1H), 3.94 (t, J = 5.2 Hz, 2H), 2.89 (t, J = 5.2 Hz, 2H), 2.63 (t, J = 7.6 Hz, 2H), 2.46 (t, J = 7.6
13
Hz, 2H), 2.14 (m, 2H), 1.87-1.61 (m, 10H), 1.42-1.32 (m, 8H), 0.89 (t, J = 6.8 Hz, 3H); C
NMR (CD3OD, 100 MHz)  169.3, 147.6, 145.9, 144.3, 131.0, 128.5, 127.6, 127.3, 108.5, 56.2,
53.2, 39.2, 35.5, 31.5, 31.4, 31.0, 27.9, 27.6, 24.2, 23.5, 22.7, 22.4, 13.2; IR (CDCl3) 3294, 3140,
2930, 2859, 1655, 1539, 1503, 1455, 1302, 1057, 856; (max nm) 238; HRMS (ESI+) m/z
506.3592 [(M+H)+; calculated mass for C29H44ClN7O+: 506.3607 amu].
N-(2-(4-(5-(2-amino-1-benzyl-1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-
pentylbenzamide hydrochloride (4k). Following the general procedure for the synthesis of
1,4-subsituted 2-aminoimidazoles, N-(2-(4-(7-bromo-6-oxoheptyl)-1H-1,2,3-triazol-1-yl)ethyl)-
4-pentylbenzamide was reacted with benzylamine to provide N-(2-(4-(5-(2-amino-1-benzyl-1H-
imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-pentylbenzamide hydrochloride ( 27 mg,
1
23%) as a yellow oil. H NMR (CD3OD, 300 MHz)  8.62(s, 1H), 7.69 (d, J = 8 Hz, 2H), 7.41-
7.27 (m, 5H), 7.24 (d, J = 8 Hz, 2H), 6.56 (s, 1H), 5.04 (s, 2H), 4.85 (t, J = 5.2 Hz, 2H), 3.94 (t, J

ACCEPTED MANUSCRIPT
= 5.2 Hz, 2H), 2.87 (t, J = 7.6 Hz, 2H), 2.61 (t, J = 7.6 Hz, 2H), 2.45 (t, J = 7.4 Hz, 2H), 1.75-
1.56 (m, 6H), 1.43-1.26 (m, 6H), 0.88 (t, J = 7 Hz, 3H); 13C NMR (CD3OD, 100 MHz)  169.3,
147.6, 146.4, 144.2, 134.9, 131.0, 129.0, 128.5, 128.4, 127.6, 127.5, 127.3, 112.0, 53.2. 39.2,
35.5, 31.3, 31.0, 27.8, 27.5, 27.4, 24.0, 22.7, 22.3, 13.2; IR (CDCl3) 3281, 3128, 2930, 2866,
1663, 1540, 1501, 1455, 1305, 1188, 1054, 975; (max nm) 238; HRMS (ESI+) m/z 528.3462
[(M+H)+; calculated mass for C31H41N7O+: 528.3445 amu].
N-(2-(4-(5-(2-amino-1-hexyl-1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-
pentylbenzamide hydrochloride (4i). Following the general procedure for the synthesis of 1,4-
subsituted 2-aminoimidazoles, N-(2-(4-(7-bromo-6-oxoheptyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-
pentylbenzamide was reacted with hexylamine to provide N-(2-(4-(5-(2-amino-1-hexyl-1H-
imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-pentylbenzamide hydrochloride ( 29 mg,
1
25%) as a yellow oil. H NMR (CD3OD, 300 MHz)  8.24 (s, 1H), 7.66 (d, J = 7.8 Hz, 2H),
7.26 (d, J = 8.1 Hz, 2H), 6.59 (s, 1H), 4.74 (t, J = 5.2 Hz, 2H), 3.89 (t, J = 5.2 Hz, 2H), 3.77 (t, J
= 7.1 Hz, 2H), 2.80 (t, J =6.9 Hz, 2H), 2.64 (t, J = 7.5 Hz, 2H), 2.44 (t, J = 7.5 Hz, 2H), 1.17 (m,
4H), 1.61 (m, 4H), 1.33 (m, 12H), 0.914-0.870 (m, 6H); 13C NMR (CD3OD, 100 MHz)  169.3,
147.47, 146.1, 145.5, 131.2, 128.8, 127.2, 126.1, 112.0, 52.0, 45.1, 39.45, 35.5, 31.4, 31.0, 28.7,
28.1, 28.0, 27.6, 25.9, 24.1, 23.5, 22.4, 13.2; IR (CDCl3) 3112, 2929, 2857, 2361, 1660, 1540,
1503, 1439, 1053, 973; (max nm) 238; HRMS (ESI+) m/z 522.3906 [(M+H)+; calculated mass
for C30H47N7O+: 522.3915 amu].
N-(2-(4-(5-(2-amino-1-phenethyl-1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-
pentylbenzamide hydrochloride (4l). Following the general procedure for the synthesis of 1,4-
subsituted 2-aminoimidazoles, N-(2-(4-(7-bromo-6-oxoheptyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-
pentylbenzamide was reacted with phenethylamine to provide N-(2-(4-(5-(2-amino-1-phenethyl-
1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-pentylbenzamide hydrochloride ( 12
1
mg, 10%) as a yellow oil. H NMR (CD3OD, 300 MHz)  8.31 (s, 1H), 7.66 (d, J = 8.1 Hz,
2H), 7.30-7.15 (m, 7H), 6.39 (s, 1H), 4.75 (t, J = 5.7 Hz, 2H), 4.04 (t, J = 7.1 Hz, 2H), 3.90 (t, J
= 5.4 Hz, 2H), 3.02 (t, J = 6.9 Hz, 2H), 2.8 (t, J = 7.5 Hz, 2H), 2.64 (t, J = 7.7 Hz, 2H), 2.39 (t, J
= 7.5 Hz, 2H), 1.72-1.53 (m, 6H), 1.34-1.29 (m, 10H), 0.88 (t, J = 6.9 Hz, 3H); 13C NMR (CD3-
OD, 100 MHz)  169.3, 147.5, 137.2, 131.2, 128.8, 128.6, 128.5, 127.2, 126.9, 125.6, 112.0,
51.7. 46.2, 39.4, 35.5, 34.5, 31.4, 31.0, 29.5, 28.1, 27.8, 27.5, 23.9, 23.5, 22.3, 13.2; IR (CDCl3)
3121, 2927, 2855, 1660, 1540, 1499, 1454, 1180, 1056, 973; (max nm) 238; HRMS (ESI+) m/z
542.3600 [(M+H)+; calculated mass for C32H43N7O+: 542.3602 amu].
N-(2-(4-(5-(2-amino-1-butyl-1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-
pentylbenzamide hydrochloride (4g). Following the general procedure for the synthesis of 1,4-
subsituted 2-aminoimidazoles, N-(2-(4-(7-bromo-6-oxoheptyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-
pentylbenzamide was reacted with butylamine to provide N-(2-(4-(5-(2-amino-1-butyl-1H-
imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-pentylbenzamide hydrochloride ( 40 mg,
36%) as a yellow oil. 1H NMR (CD3OD, 400 MHz)  8.59 (s, 1H), 7.68 (d, J = 8.4 Hz, 2H), 7.26

ACCEPTED MANUSCRIPT
(d, J = 8 Hz, 2H), 6.61 (s, 1H), 4.83 (t, J = 5.6 Hz, 2H), 3.94 (t, J = 5.6 Hz, 2H), 3.78 (t, J = 7.4
Hz, 2H), 2.88 (t, J = 7.8 Hz, 2H), 2.64 (t, J = 7.6 Hz, 2H), 2.46 (t, J = 7.8, 2H), 1.76-1.69 (m,
13
4H), 1.62 (m, 4H), 1.43-1.28 (m, 8H), 0.97 (t, J = 7.2 Hz, 3H), 0.89 (t, J = 7.2 Hz, 3H); C
NMR (CD3OD, 100 MHz)  162.3, 147.5, 146.1, 144.8, 131.2, 128.5, 127.3, 127.0, 112.0, 52.7,
44.9, 39.3, 35.5, 31.4, 31.0, 30.8, 27.9, 27.8, 27.6, 24.1, 23.1, 22.4, 19.5, 13.2, 12.8; IR (CDCl3)
3280, 3128, 2931, 2857, 1725, 1661, 1538, 1468, 1376, 1288, 1162, 1058; (max nm) 238;
HRMS (ESI+) m/z 494.3602 [(M+H)+; calculated mass for C28H44N7O+: 494.3624 amu].
N-(2-(4-(5-(2-amino-1-isobutyl-1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-
pentylbenzamide hydrochloride (4e). Following the general procedure for the synthesis of 1,4-
subsituted 2-aminoimidazoles, N-(2-(4-(7-bromo-6-oxoheptyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-
pentylbenzamide was reacted with isobutylamine to provide N-(2-(4-(5-(2-amino-1-isobutyl-1H-
imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-pentylbenzamide hydrochloride ( 15 mg,
1
14%) as a yellow oil. H NMR (CD3OD, 300 MHz)  8.42 (s, 1H), 7.67 (d, J = 8.1 Hz, 2H),
7.26 (d, 8.1 Hz, 2H), 6.58 (s, 1H), 7.48 (t, J = 4.2 Hz, 2H), 3.91 (t, J = 5.4 Hz, 2H), 3.60 (d, J =
7.5 Hz, 2H), 2.84 (t, J = 7.2 Hz, 2H), 2.64 (t, J = 7.5 Hz, 2H), 2.46 (t, J = 7.5 Hz, 2H), 2.07 (m,
1H), 1.73-1.59 (m, 5H), 1.40-1.28 (m, 6H), 0.95-0.87 (m, 9H); 13C NMR (CD3OD, 100 MHz) 
169.3, 147.6, 144.5, 131.1, 128.5, 127.3, 127.2, 127.0, 112.5, 53.1, 51.8, 39.2, 35.5, 31.4, 31.0,
28.4, 27.9, 27.6, 27.5, 24.0, 22.8, 22.4, 18.9, 18.8, 18.6, 13.2; IR (CDCl3) 3274, 3134, 2946,
2931, 2884, 2855, 1678, 1655, 1544, 1456, 1299; (max nm) 238; HRMS (ESI+) m/z 494.3589
[(M+H)+; calculated mass for C28H43N7O+: 494.3602 amu].
N-(2-(4-(5-(2-amino-1-phenyl-1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-
pentylbenzamide hydrochloride (4j). Following the general procedure for the synthesis of 1,4-
subsituted 2-aminoimidazoles, N-(2-(4-(7-bromo-6-oxoheptyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-
pentylbenzamide was reacted with aniline to provide N-(2-(4-(5-(2-amino-1-phenyl-1H-
imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-pentylbenzamide hydrochloride ( 30 mg,
1
26%) as a yellow oil. H NMR (CD3OD, 300 MHz)  8.60 (s, 1H), 7.68 (d, J = 6.6 Hz, 2H),
7.66-7.49 (m, 5H), 7.26 (d J = 8.4 Hz, 2H), 6.76 (s, 1H), 4.83 (t, J = 5.4 Hz, 2H), 3.94 (t, J = 5.4
Hz, 2H), 2.90 (t, J = 7.5 Hz, 2H), 2.66 (t, J = 7.7 Hz, 2H), 2.55 (t, J = 7.4 Hz, 2H), 1.79-1.58 (m,
6H), 1.47 (m, 2H), 1.32 (m, 4H), 0.89 (t, J = 6.9 Hz, 3H); 13C NMR (CD3OD, 100 MHz)  169.3,
147.5, 146.3, 144.7, 134.3, 131.1, 129.8, 128.5, 128.0, 127.3, 127.1, 125.5, 113.2, 52.8, 39.3,
35.5, 31.4, 31.0, 27.9, 27.7, 27.4, 24.0, 22.9, 22.4, 13.2; IR (CDCl3) 3266, 3111, 2930, 2857,
2363, 1658, 1537, 1503, 1456, 1302, 1190, 1052; (max nm) 238; HRMS (ESI+) m/z 514.3279
[(M+H)+; calculated mass for C30H39N7O+: 514.3289 amu].
N-(2-(4-(5-(1-allyl-2-amino-1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-
pentylbenzamide hydrochloride (4f). Following the general procedure for the synthesis of 1,4-
subsituted 2-aminoimidazoles, N-(2-(4-(7-bromo-6-oxoheptyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-
pentylbenzamide was reacted with allylamine to provide N-(2-(4-(5-(1-allyl-2-amino-1H-
imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-pentylbenzamide hydrochloride ( 27 mg,

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1
25%) as a yellow oil. H NMR (CD3OD, 400 MHz)  8.58 (s, 1H), 7.68 (d, J = 8 Hz, 2H), 7.26
(d, J = 8.4 Hz, 2H), 6.56 (s, 1H), 5.78-5.90 (s, 1H), 5.34-5.29 (s, 1H), 5.19-5.13 (s, 1H), 4.83 (t, J
= 5.4 Hz, 2H), 4.43 (m, 2H), 3.94 (t, J = 5.4 Hz, 2H), 2.88 (t, J = 7.6 Hz, 2H), 2.64 (t, J = 7.6 Hz,
2H), 2.47 (t, J = 7.4 Hz, 2H), 1.76-1.70 (m, 2H), 1.62 (m, 4H), 1.43-1.39 (m, 2H), 1.33-1.28 (m,
4H), 0.89 (t, J = 7 Hz, 3H); 13C NMR (CD3OD, 100 MHz)  169.3, 147.6, 144.4, 131.0,
128.5,127.4, 127.2, 117.8, 111.9, 53.1, 47.0, 39.2, 35.5, 31.4, 30.9, 27.8, 27.6, 27.5, 24.0, 22.7,
22.4, 13.2; IR (CDCl3) 3320, 2926, 2855, 1665, 1539, 1503, 1455, 1302, 1160; (max nm) 238;
HRMS (ESI+) m/z 478.3283 [(M+H)+; calculated mass for C27H40N7O+: 478.3289 amu].
N-(2-(4-(5-(2-amino-1-pentyl-1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-
pentylbenzamide hydrochloride (4h). Following the general procedure for the synthesis of
1,4-subsituted 2-aminoimidazoles, N-(2-(4-(7-bromo-6-oxoheptyl)-1H-1,2,3-triazol-1-yl)ethyl)-
4-pentylbenzamide was reacted with pentylamine to provide N-(2-(4-(5-(2-amino-1-pentyl-1H-
imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-pentylbenzamide hydrochloride ( 30 mg,
1
26%) as a yellow oil. H NMR (CD3OD, 400 MHz)  8.50 (s, 1H), 7.67 (d, J = 8 Hz, 2H), 7.26
(d, J = 8 Hz, 2H), 6.60 (s, 1H), 4.81 (t, J = 5.2 Hz, 2H), 3.93 (t, J = 5.2 Hz, 2H), 3.77 (t, J = 7
Hz, 2H), 2.86 (t, J = 7.4 Hz, 2H), 2.45 (t, J = 7 Hz, 2H), 1.75-1.70 (m, 4H), 1.61 (m, 4H), 1.42-
1.29 (m, 10H), 0.95-0.87 (m, 6H); 13C NMR (CD3OD, 100 MHz)  169.3, 147.6, 146.1, 144.3,
131.0, 128.5, 127.5, 127.2, 111.9, 53.2, 45.0, 39.2, 35.5, 31.3, 30.9, 28.4, 28.4, 27.8, 27.6, 27.5,
24.0, 22.7, 22.3, 22.1, 13.2, 13.1; IR (CDCl3) 3317, 2930, 2857, 2202, 1664, 1539, 1503, 1457,
1377, 1304, 1163, 1084; (max nm) 238; HRMS (ESI+) m/z 508.3742 [(M+H)+; calculated mass
for C29H45N7O+: 508.3758 amu].
N-(2-(4-(5-(2-amino-1-cyclohexyl-1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-
pentylbenzamide hydrochloride (4c). Following the general procedure for the synthesis of 1,4-
subsituted 2-aminoimidazoles, N-(2-(4-(7-bromo-6-oxoheptyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-
pentylbenzamide was reacted with cyclohexylamine to provide N-(2-(4-(5-(2-amino-1-
cyclohexyl-1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-pentylbenzamide
1
hydrochloride ( 33 mg, 28%) as a yellow oil. H NMR (CD3OD, 400 MHz)  8.62 (s, 1H), 7.69
(d, J = 8 Hz, 2H), 7.26 (d, J = 8.4 Hz, 2H), 6.73 (s, 1H), 4.85 (t, J = 5.2 Hz, 2H), 4.35-4.24 (m,
1H), 3.94 (t, J = 5.2 Hz, 2H), 2.89 (t, J = 7.4 Hz, 2H), 2.63 (t, J = 7.6 Hz, 2H), 2.46 (t, J = 7.6
13
Hz, 2H), 2.02-1.23 (m, 22H), 0.89 (t, J = 7 Hz, 3H); C NMR (CD3OD, 100 MHz)  147.5,
144.3, 131.2, 131.0, 128.7, 128.5, 127.4, 127.2, 108.4, 54.5, 53.2, 50.4, 39.2, 39.5, 32.0, 31.4,
31.0, 30.8, 27.9, 27.6, 25.1, 24.8, 24.2, 22.7, 22.4, 13.2; IR (CDCl3)3318, 2930, 2859, 2343,
1727, 1689, 1541, 1457, 1338, 1062; HRMS (ESI+) m/z 520.3752 [(M+H)+; calculated mass for
C30H45N7O+: 520.3758 amu].
N-(2-(4-(5-(2-amino-1-isopropyl-1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-
pentylbenzamide hydrochloride (4d). Following the general procedure for the synthesis of 1,4-
subsituted 2-aminoimidazoles, N-(2-(4-(7-bromo-6-oxoheptyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-
pentylbenzamide was reacted with isopropylamine to provide N-(2-(4-(5-(2-amino-1-isopropyl-

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1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-yl)ethyl)-4-pentylbenzamide hydrochloride ( 36
1
mg, 33%) as a yellow oil. H NMR (CD3OD, 300 MHz)  8.42 (s, 1H), 7.68 (d, J = 8.1 Hz,
2H), 7.26 (d, J =8.4 HZ. 2H), 6.73 (s, 1H), 4.79 (t, J = 5.7 Hz, 2H), 4.35-4.30 (m, 1H), 3.92 (t, J
= 5.3 Hz, 2H), 2.85 (t, J = 7.5 Hz, 2H), 2.64 (t, J = 7.4 Hz, 2H), 2.46 (t, J = 7.7 Hz, 2H), 1.75-
1.67 (m, 3H), 1.66-1.56 (m, 3H), 1.39 (d, J = 6.6 Hz, 6H), 1.32-1.29 (m, 6H), 0.89 (t, J = 6.8 Hz,
3H); 13C NMR (CD3OD, 100 MHz)  161.8, 147.5, 145.3, 131.2, 128.5, 127.7, 127.2,
125.8,107.7, 51.5, 39. 5, 35.5, 31.4, 30.9, 28.1, 28.0, 27.7, 23.6, 22.7, 20.7, 13.2; IR (CDCl3)
3344, 2937, 2860, 2542, 1696, 1652, 1546, 1457, 1335, 1159, 1056; (max nm) 238; HRMS
(ESI+) m/z 480.3443 [(M+H)+; calculated mass for C27H41N7O+: 480.3445 amu].
4.2. Biological Screening
Inhibition assays of selected compounds against MRSA and MDRAB: Inhibition Assays
were performed by overnight culturing the desired bacteria, MRSA or MDRAB, and
subculturing at an OD600 of 0.01 into tryptic soy broth with a 0.5% glucose supplement (TSBG)
for MRSA, or Luria-Bertani (LB) media for MDRAB. Stock solutions of a predetermined
concentration of the selected compounds were prepared in the resulting bacterial suspension.
Aliquots of 100 µL were distributed to the wells of a 96-well plate. Plates were covered and
sealed with GLAD Press‟n Seal, then incubated under stationary conditions at 37oC for 24 hours.
The media was then discarded, and the plates were washed thoroughly with water to remove any
loosely adherent bacteria. Each well was then stained with 110 µL of crystal violet (0.1%
solution), and allowed to sit for 30 minutes. The crystal violet was then discarded, and the wells
were thoroughly washed with water again. The remaining stain was then dissolved in 95%
ethanol (200 µL) and 125 µL was transferred to corresponding wells of a polystyrene microtiter
dish. Biofilm inhibition was quantified by measuring the OD540 value of each well. Blank wells
were employed as background check.
Dispersion assays of selected compounds against MRSA: Dispersion assays were performed
by taking overnight cultures of MRSA and subculturing at an OD600 of 0.1 into TSBG media.
100 µL aliquots of the resulting bacterial suspension were distributed to the wells of a 96 well
plate. Plates were then covered and sealed with GLAD Press‟n Seal followed by incubation
under stationary conditions at 37oC for 24h. The plates were unwrapped and the media was
discarded, and the plates were washed thoroughly with water to remove any loosely adherent
bacteria. Stock solutions of predetermined concentrations of the selected compounds were
prepared in TSBG, and 100 µL was transferred from stock solutions into the 96-well plate.
Media alone was added as a control. Plates were then rewrapped with GLAD Press‟n Seal, and
incubated under stationary conditions for 24 hours. The media was discarded from the plates and
the wells were washed thoroughly with water. Each well was then stained with 110 µL of crystal
violet (0.1% solution) at room temperature, and allowed to sit for 30 minutes. The crystal violet
was then discarded and the wells were thoroughly washed with water again. The remaining stain
was then dissolved in 95% ethanol (200 µL) and 125 µL was transferred to corresponding wells

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of a polystyrene microtiter dish. Biofilm inhibition was quantified by measuring the OD540 value
of each well. Blank wells were employed as background check.
Broth microdilution method for antibiotic resensitization: Mueller-Hinton Broth (MHB) was
inoculated (5x105 CFU mL-1) with MRSA. Aliquots (5 mL) of the resulting bacterial suspension
were distributed to culture tubes, and compound from a 100 mM stock solution was added to
give the final desired concentration. Bacteria not treated with the tested 2-AIT served as a
control. After sitting at room temperature for 30 minutes, 1 mL of each sample was transferred
to a culture tube, and oxacillin sodium salt was added from a 128 mg mL-1 water stock solution
to give a final concentration of 128 µg mL-1. Rows 2-12 of a 96-well plate were filled (110 µL
per well) from the remaining 4 mL bacterial subculture. After standing for 10 minutes, aliquots
(200 µL) of the samples containing antibiotic were distributed to the corresponding first row
wells of the microtiter plate. Row 1 was mixed 6-8 times, and then 100 µL was transferred from
row 1 to row 2. This procedure was then repeated to dilute the rest of the wells, to which no
antibiotic was added, to check for bacterial growth in the presence of compound alone. The plate
was then covered and sealed with GLAD Press‟n Seal, and incubated under stationary conditions
o
at 37 C. MIC values were then recorded as the lowest concentrations at which no bacterial
growth was recorded, and a fold reduction was determined by comparison with control lane.
Checkerboard assay: MHB was inoculated with MRSA (5 x 105 CFU ml-1) and 100 µL
aliquots were distributed to all wells of a 96-well plate except for well 1a. Inoculated MHB (200
µL) containing a selected compound (at a concentration for 2x the highest concentration being
tested) was added to well 1a, and 100 µL of the same sample was added to wells 2a-12a.
Column A cells were mixed 6-8 times, and then 100 µL was withdrawn and transferred to
column B. This process was repeated up to column G (column H was not mixed to determine
the MIC of the antibiotic alone. Inoculated media (100 µL) containing antibiotic at 2x the
highest concentration being tested was placed in wells A1-H1 and serially diluted, all the way
until row 11 (row 12 was not mixed to determine the MIC of the compound alone). The plates
were covered and sealed with GLAD Press‟n Seal, and incubated under stationary conditions at
37oC. After 16h the MIC values of both compound and antibiotic were recorded, as well as
combination. The ΣFIC values were calculated as follows. ΣFIC = FICcmpd + FICantibiotic, where
FICcmpd = [MICcmpd in combination]/[MICcmpd alone], and FICantibiotic
[MICantibiotic in
=
combination]/[MICantibiotic alone]. The combination is considered synergistic if ΣFIC ≤ 0.5,
indifferent if 0.5 < ΣFIC < 2, and antagonistic if ΣFIC > 2.
5. Acknowledgements
The authors thank the DOD DMRDP program (W81XWH-11-2-0115) for support of this work.
The DMRDP program is administered by the Department of Army; The U.S. Army Medical
Research Acquisition Activity, 820 Chandler Street, Fort Detrick, MD 21702-5014 is the
awarding and administering office. The content of this manuscript does not necessarily reflect

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the position or the policy of the Government, and no official endorsement should be inferred.
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Figure 1

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Figure 2

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Figure 3

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Scheme 1

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Scheme 2

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Scheme 3

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Scheme 4

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Tables:
Table 1. Attempts at nucleophilic epoxide opening with primary amines.
Amine
Conditions
a
Equivalents
1
Yield
NR
b
b
c
d
e
f
1
1
8
8
8
8
NR
NR
NR
55a
35a
NR
d
e
8
8
NR
NR
Conditions: a) toluene, reflux. b) Montmorillonite (K10), DCM, rt. c) DMF: H2O (1:1), rt. d) DMSO: H2O (1:1), 60 oC. e) DMF,
Montmorillonite K 10, 60 oC. e) DMF, 60 oC. f) DMF: DMSO/H2O (4:1), Montmorillonite (K10), 60 oC.
[a] contained impurities after isolation

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Table 2. Primary amines used for one pot 2-AI ring formation
Amine
Product
4a
Yield (%)
16
Amine
Product
4g
Yield
36
4b
16
4h
26
4c
4d
28
33
4i
4j
25
26
4e
4f
14
25
4k
4l
26
10