Analogue synthesis reveals decoupling of antibiofilm and β-lactam potentiation activities of a lead 2-aminoimidazole adjuvant against Mycobacterium smegmatis

Article abstract of DOI:10.1111/cbdd.13208

Biofilm formation is one of the many mechanisms bacteria utilize to survive antibiotic treatment. It has been demonstrated that when Mycobacterium tuberculosis exists in a biofilm in vitro, it expresses phenotypic resistance to antimicrobial drugs. As the in vivo survival of M.?tuberculosis following drug treatment is potentially linked to a biofilm-like expression of drug tolerance, it is hypothesized that biofilm dispersion should increase antibiotic susceptibility and reduce the duration of the current antibiotic treatment regimen. Previously, we have identified a 2-aminoimidazole (2-AI) compound capable of dispersing and inhibiting M.?tuberculosis and M.?smegmatis biofilms in vitro. Additionally, this compound potentiated the activity of carbenicillin against M.?tuberculosis and, to a lesser degree, M.?smegmatis. Here, we describe a SAR study on this compound evaluating each derivative for biofilm dispersion and β-lactam potentiation capabilities against M.?smegmatis. This study identified a compound that improved upon the biofilm dispersion capabilities of the lead compound. Interestingly, a different compound was identified with an increased ability to potentiate a subset of β-lactam antibiotics. These compounds indicate that biofilm dispersion and potentiation capabilities may not be associated.

Full text of DOI:10.1111/cbdd.13208

PROFESSOR CHRISTIAN MELANDER (Orcid ID : 0000-0001-8271-4696)
Article type : Research Article
Analogue Synthesis Reveals Decoupling of Antibiofilm and β-Lactam Potentiation
Activities of a Lead 2-Aminoimidazole Adjuvant Against Mycobacterium smegmatis.
Sara E. Martina, Catherine M. Nguyena, Randall J. Basarabab, and Christian Melandera*
Department of Chemistry, North Carolina State University, Raleigh, NC USA 27695a
Department of Microbiology, Immunology, and Pathology, Colorado State University, CO
USA 80525b
* to whom correspondence should be addressed: ccmeland@ncsu.edu
Abstract
Biofilm formation is one of the many mechanisms bacteria utilize to survive antibiotic
treatment. It has been demonstrated that when Mycobacterium tuberculosis exists in a
biofilm in vitro it expresses phenotypic resistance to antimicrobial drugs. Since the in vivo
survival of M. tuberculosis following drug treatment is potentially linked to a biofilm-like
expression of drug tolerance, it is hypothesized that biofilm dispersion should increase
antibiotic susceptibility and reduce the duration of the current antibiotic treatment regimen.
Previously, we have identified a 2-aminoimidazole (2-AI) compound capable of dispersing
and inhibiting M. tuberculosis and M. smegmatis biofilms in vitro. Additionally, this compound
potentiated the activity of carbenicillin against M. tuberculosis and, to a lesser degree, M.
smegmatis. Here, we describe a SAR study on this compound evaluating each derivative for
biofilm dispersion and β-lactam potentiation capabilities against M. smegmatis. This study
identified a compound that improved upon the biofilm dispersion capabilities of the lead
compound. Interestingly, a different compound was identified with an increased ability to
potentiate a subset of β-lactam antibiotics. These compounds indicate that biofilm dispersion
and potentiation capabilities may not be associated.
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13208
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Introduction
Antimicrobial resistance is one of the most pressing biomedical issues of the 21st
century [1]. Bacteria utilize many different mechanisms to evade the effects of antibiotics,
and one mechanism used by many species of bacteria is the formation of bacterial biofilms.
Biofilms are defined as a surface attached community of bacteria, which are embedded
within an extracellular polymeric matrix [2, 3]. The National Institutes of Health (NIH)
estimates that 80% of all bacterial infections in the human body are biofilm related, which
leads to approximately 550,000 annual fatalities [4]. Residing within a biofilm state confers a
large increase in the ability of bacteria to withstand antibiotics due to the extra protection
provided by the extracellular matrix, increased gene transfer between colonies, and
differential gene expression compared to planktonic bacteria [4, 5]. It has been established
that bacteria reside in a biofilm state to better protect themselves from environmental stress
or sources of eradication, which include antiseptics, antibiotics, and immune responses with
studies showing that biofilm associated bacteria can exhibit upwards of 1000-times less
sensitivity to conventional antibiotics [3, 4, 6].
Mycobacterium tuberculosis is the causative agent of tuberculosis (TB) and has
recently exceeded HIV as the number one cause of death of humans from an infectious
disease [7]. According to the CDC, TB accounted for approximately 1.7 million deaths
worldwide in 2016 [8], and it is believed that approximately one third of the world’s
population is currently infected with M. tuberculosis [8]. These statistics are alarming due to
the complexity and length of treatment along with the emergence of resistant strains [9]. The
contribution of M. tuberculosis biofilm formation on the pathogenesis of bacterial persistence
and antimicrobial drug resistance remains controversial; however, during the chronic stages
of a M. tuberculosis infection in humans and some animals, bacilli are released from dead
cells, and colonies survive by attaching to an extracellular matrix derived by the host and
pathogen. This process is similar to the survival techniques employed by biofilm forming
bacteria. Previously, we showed that in vitro culture of extracellular bacilli attached to an
extracellular matrix derived from lysed host cells expressed extreme phenotypic resistance
to first line anti-TB drugs alone or in combination. Therefore, a biofilm mode of growth is
thought to potentially play a role in the persistence of M. tuberculosis infections [10, 11].
Currently, the first-line treatment for drug-susceptible M. tuberculosis consists of a
minimum of two months of antibiotic combination therapy with isoniazid (INH), rifampin (RIF),
ethambutol (EMB), and pyrazinamide (PZA) followed by an additional 4-7 months of
combination therapy with INH and RIF [12, 13] to eliminate persistent reservoirs of M.
tuberculosis, which can lead to a rebound infection if not completely eradicated [11]. This
treatment regimen does, however, lead to patient non-compliance due to complications and
toxicity associated with long-term antibiotic therapy. Our previous work has shown that M.
tuberculosis biofilm communities are significantly more resistant than planktonic bacteria to
treatment with isoniazid [14]. Therefore, the development of small molecules that disrupt
mycobacterium biofilms may find use as adjuvants that, when paired with standard TB
antibiotics, could significantly reduce the TB treatment regimens and lead to better patient
compliance.
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Previously, we have shown that 2-aminoimidazole (2-AI) derivatives that disperse
Mycobacterium smegmatis biofilms will also disperse M. tuberculosis biofilms. These
compounds were shown to work synergistically with INH to effectively eradicate M.
tuberculosis biofilms in an in vitro model mimicking the lesions in tuberculosis patients’ lungs
by employing lysed human leukocytes as a biofilm growth surface [10]. One of these active
2-AI derivatives is compound 1 (Figure 1), which exhibited an EC50 value of 52.8 μM against
M. smegmatis [14]. Here, the EC50 value is defined as the concentration necessary to elicit
50% dispersion of a pre-formed biofilm as compared to media-only treated controls.
Interestingly, in addition to its biofilm dispersion activity, this compound also sensitized M.
tuberculosis
effecting a 16-fold
reduction in the minimum inhibitory concentration (MIC) of carbenicillin against the M.
tuberculosis H37Rv lab strain at 31.25 μM [15]. Compound 1
effects against M. smegmatis; however, they were substantially more moderate, achieving 2-
4 fold reductions. Due to the precedent of activity displayed by compound 1 and other 2-AI
containing molecules, this structure activity relationship (SAR) chose to keep the 2-AI portion
of the molecule consistent and use alterations of the alkyl tail portion in attempts to improve
activity. Additionally, recent literature has indicated that increasing the length of alkyl chain
within a pyrazinamide derivative increases potency against M. tuberculosis [16]. With this
precedent and the activity exhibited by compound 1, a SAR was performed to probe what
role, if any, alkyl chains play in anti-biofilm and potentiation activity. Interestingly, we find that
these activities are somewhat decoupled, as the most potent anti-biofilm compound and the
most potent β
or are distinct.
Materials and Methods
Broth Microdilution Method for MIC Determination of M. smegmatis
Cultures (48 h) were subcultured to 5 x 105 CFU/mL in 7H9 broth supplemented with ADC
enrichment. Aliquots (1 mL) were placed in culture tubes, and compound was added from
100 mM stock samples in DMSO, such that the compound concentration equaled the
highest concentration tested (200 μM). Samples were then aliquoted (200 μL) into the first
row of wells of a 96-well plate, with all remaining wells being filled with 100 μL of initial
bacterial subculture. Row one wells were mixed five times before 100 μL was transferred to
the following row (row two). Row two was then mixed five times, and 100 μL was transferred
to row three. This process provided a serial dilution of the compound and was continued until
the final row had been mixed. Plates were covered with Glad Press n’ Seal and were
incubated under stationary conditions at 37°C for 48 hours. After 48 hours, the wells were
stained with alamarBlue (Thermo- Fisher) and were incubated under stationary conditions at
37°C for 8 hours. After 8 hours, the wells with bacterial growth were visualized as pink and
the wells with no viable bacteria were visualized as blue. MIC values were recorded as the
lowest concentration at which no bacterial growth was observed.
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Broth Microdilution Method for Antibiotic Resensitization of M. smegmatis
Cultures (48 h) were subcultured to 5 x 105 CFU/mL in 7H9 broth supplemented with ADC
enrichment. Aliquots (4 mL) were placed in culture tubes, and compound was added from
100 mM stock samples in DMSO, such that the compound concentration was 30% of the
MIC of the compound against the particular bacterial strain. One milliliter of the resulting
solution was aliquoted into a separate culture tube and was dosed with antibiotic so that the
resulting concentration was the highest desirable concentration to be tested. Bacteria treated
with antibiotic alone were used as a control. Row one of a 96-well plate was filled with 200
μL of the antibiotic/ compound solution, and the remaining rows were filled with 100 μL per
well of the remaining 4 mL bacterial subculture. Row one wells were mixed five times before
100 μL was transferred to the following row (row two). Row two was then mixed five times,
and 100 μL was transferred to row three. This process was repeated until the second to last
row had been reached. The last row would have only compound and serve as a negative
control. The antibiotic only treated bacteria was plated by aliquoting 200 μL of the treated
bacteria into row one and filling the remaining rows with untreated bacteria from the original
subculture. The rows were mixed in the same way as described above. Plates were covered
with Glad Press n’ Seal and were incubated under stationary conditions at 37 °C for 48
hours. After 48 hours, the wells were stained with alamarBlue (Thermo-Fisher) and were
incubated under stationary conditions at 37 °C for 8 hours. After 8 hours, the wells with
bacterial growth were visualized as pink and the wells with no viable bacteria were visualized
as blue. MIC values were recorded as the lowest concentration at which no bacterial growth
was observed, which was determined by the blue well at the lowest concentration. Fold
reductions were determined by comparison of the compound treated wells with the antibiotic
only control well.
General static dispersion assay protocols for M. smegmatis
Cultures were incubated for 48 hours and then subcultured to 0.01 in Difco M9 minimal salts
media. 100 μL of the subculture was aliquoted into every well in columns 2-11 of a 96-well
PVC microtiter plate. Columns 1 and 12 were left empty to serve as control wells. Plates
were covered with Glad Press n’ Seal and were incubated under stationary conditions at 37
°C for 48 hours. After 48 hours, the media was discarded, and the plates were washed
thoroughly with water. 100 μL of fresh media containing the appropriate concentration of
compound was added to all of the wells in columns 2-4 and 9-11. 100 μL of sterile media
was added to all of the wells in columns 1 and 12 and columns 5-8. Plates were covered
with Glad Press n’ Seal and were incubated under stationary conditions at 37 °C for 24
hours. After 24 hours, media was discarded, and the plates were washed thoroughly with
water. 110 μL of a 0.1% aqueous solution of crystal violet was added to every well, and the
plates were left at ambient temperature for 30 minutes. After 30 minutes, the crystal violet
was disposed, and the plates were washed thoroughly with water. 200 μL of 95% ethanol
was added to each well, and the plates were left at ambient temperature for 10 minutes. 125
μL of the ethanol solution was transferred to a fresh polystyrene microtiter plate, and the
plate was quantified by measuring the OD540. The percent dispersion was calculated by
comparing the OD540 of the treated wells with the OD540 of the untreated wells, which
contained only media after biofilm growth. The first and last column, which had only sterile
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media, were used as blanks and those values were subtracted from the OD540 obtained in
the other columns.
Compound synthesis and characterization
4-(2-amino-1H-imidazol-5-yl)-N-propylbutanamide hydrochloride (9A)
Compound 3 (0.50 g, 1.1 mmol) was solubilized in anhydrous DCM (30 mL). Triethylamine
(3.5 eq) and propylamine (1.5 eq) were added to the solution followed by 2-(1H-benzotriazol-
1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) (3.5 eq). The solution was
allowed to stir at room temperature under nitrogen for 24 hours. After 24 hours, the reaction
was quenched with 100 mL ethyl acetate and was washed with 100 mL of water followed by
three washes with 100 mL of brine. The intermediate was submitted to flash column
chromatography with a 1% methanol saturated with ammonium and DCM solvent system to
separate excess tail and starting material from the desired product. The intermediate was
still impure after flash column chromatography so presence was confirmed by LC/MS. Due
to the presence of boc groups, the intermediate could not be fully characterized by NMR. To
create the final product, the intermediate was solubilized (0.29 g, 0.56 mmol) in anhydrous
dichloromethane (5.6 mL) and was cooled to 0°C. TFA (5.6 mL) was charged into the flask
and the reaction was stirred for 24 hours. After this time, the reaction was evaporated to
dryness and toluene (3.0 mL) was added. Again, the mixture was concentrated and the
process was repeated two additional times. The resulting TFA salt was dissolved in
methanol (2.0 mL) and concentrated HCl was added (1.5 mL). The mixture was then
evaporated to dryness. Once dry, cold ether (5.0 mL) was added to the mixture, and the
mixture was sonicated. The cold ether was pipetted off leaving the target compound 9A
1
(0.14 g, 0.56 mmol, 99 %) as a brown oil. H NMR (300 MHz, [D6] CD3OD): δ 6.52 (s, 1H),
3.14 (t, 2H, J=7.2 Hz), 2.52 (t, 2H, J=7.5 Hz), 2.27 (t, 2H, J=7.8 Hz), 1.89 (q, 2H, J=6.9 Hz),
1.53 (sex, 2H, J=7.2 Hz), 0.92 (t, 2H, J=7.2 Hz) ppm; 13C NMR (400 MHz, CD3OD): δ 175.7,
148.4, 128.1, 110.1, 42.5, 35.6, 25.6, 24.9, 23.4, 11.6 ppm; IR νmax (cm-1) 3255, 3141, 2963,
2935, 2876, 1674, 1628, 1550, 1458; λmax= 288 nm; HRMS (ESI) calculated for C10H18N4O
[M+H]+ 211.15534, found 211.15455.
4-(2-amino-1H-imidazol-5-yl)-N-butylbutanamide hydrochloride (9B)
Using the same general procedure used for 9A, Compound 3 (0.75 g, 1.6 mmol) was
solubilized in anhydrous DCM (30 mL). Triethylamine (3.5 eq) and butylamine (1.5 eq) were
added to the solution followed by HBTU (3.5 eq). This synthesis yielded the boc- protected
intermediate as a brown oil. The corresponding intermediate (0.39 g, 0.75 mmol) was then
1
converted to the title compound 9B (0.11 g, 0.41 mmol, 55 %) as a brown oil. H NMR (300
MHz, [D6] CD3OD): δ 6.49 (s, 1H), 3.13 (t, 2H, J=6.9 Hz), 2.48 (t, 2H, J=7.5 Hz), 2.22 (t, 2H,
J=7.5 Hz), 1.85 (q, 2H, 7.5 Hz), 1.44 (q, 2H, J=1.2 Hz), 1.31 (, 0.89 (t, 3H, J=7.5 Hz) ppm;
13C NMR (400 MHz, CD3OD): δ 175.2, 148.4, 128.1, 109.9, 40.1, 35.9, 32.4, 25.5, 24.9,
21.1, 14.1 ppm; IR νmax (cm-1) 3268, 3141, 2957, 2932, 2872, 1670, 1626, 1551, 1458,1438;
λmax= 286 nm; HRMS (ESI) calculated for C11H20N4O [M+H]+ 225.17099, found 225.17016.
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4-(2-amino-1H-imidazol-5-yl)-N-pentylbutanamide hydrochloride (9C)
Using the same general procedure used for 9A, Compound 3 (0.76 g, 1.6 mmol) was
solubilized in anhydrous DCM (30 mL). Triethylamine (3.5 eq) and amylamine (1.5 eq) were
added to the solution followed by HBTU (3.5 eq). This synthesis yielded the boc-protected
intermediate as a brown oil. The corresponding intermediate (0.47 g, 0.87 mmol) was
solubilized in anhydrous dichloromethane (8.7 mL) and was cooled to 0°C. TFA (8.7 mL)
was charged into the flask and the reaction was stirred for 24 hours. After this time, the
reaction was evaporated to dryness and toluene (3.0 mL) was added. Again the mixture was
concentrated and the process was repeated two additional times. The resulting TFA salt was
dissolved in methanol (2.0 mL) and concentrated HCl was added (1.5 mL). The mixture was
then evaporated to dryness. Once dry, cold acetone (5.0 mL) was added to the mixture, and
the mixture was sonicated. The cold acetone was pipetted off leaving the target compound
1
9C (0.11 g, 0.39 mmol, 44 %) as a tan oil. H NMR (300 MHz, [D6] CD3OD): δ 6.51 (s, 1H),
3.15 (t, 2H, J=6.9 Hz), 2.51 (t, 2H, J=7.2 Hz), 2.24 (t, 2H, J=7.5 Hz), 1.87 (q, 2H, J=7.8 Hz),
1.50 (q, 2H, 6.6 Hz), 1.32 (m, 4H), 0.91 (t, 3H, J=6.6 Hz) ppm; 13C NMR (400 MHz, CD3OD):
δ 175.1, 148.4, 128.0, 109.9, 40.3, 35.9, 30.2, 30.0, 25.5, 24.9, 23.3, 14.4 ppm; IR νmax (cm-
1) 3269, 3151, 2956, 2931, 1674, 1630, 1554; λmax= 286 nm; HRMS (ESI) calculated for
C12H22N4O [M+H]+ 239.18664, found 239.18565.
4-(2-amino-1H-imidazol-5-yl)-N-heptylbutanamide hydrochloride (9E)
Using the same general procedure used for 9A, Compound 3 (1.0 g, 2.1 mmol) was
solubilized in anhydrous DCM (30 mL). Triethylamine (3.5 eq) and heptylamine (1.5 eq) were
added to the solution followed by HBTU (3.5 eq). This synthesis yielded the boc-protected
intermediate as a brown oil. The corresponding intermediate (0.65 g, 1.1 mmol) was
converted using the same procedure described in the synthesis of 9A and yielded the title
compound 9E (0.34 g, 1.1 mmol, 98 %) as a white solid. 1H NMR (300 MHz, [D6] CD3OD): δ
6.51 (s, 1H), 3.16 (t, 2H, J=6.9 Hz), 2.51 (t, 2H, J=7.8 Hz), 2.25 (t, 2H, J=7.5 Hz), 1.88 (q,
2H, 7.2 Hz), 1.49 (m, 2H), 1.31 (m, 8H), 0.89 (t, 3H, J=6.9 Hz) ppm; 13C NMR (400 MHz,
CD3OD): δ 175.3, 148.2, 127.9, 109.9, 40.4, 35.8, 32.8, 30.2, 30.0, 27.9, 25.5, 24.9, 23.6,
14.5 ppm; IR νmax (cm-1) 3257, 3149, 2955, 2927, 2856, 1676, 1638; λmax= 286 nm; HRMS
(ESI) calculated for C14H26N4O [M+H]+ 267.21794, found 267.21675.
4-(2-amino-1H-imidazol-5-yl)-N-nonylbutanamide hydrochloride (9G)
Using the same general procedure used for 9A, Compound 3 (0.50 g, 1.1 mmol) was
solubilized in anhydrous DCM (30 mL). Triethylamine (3.5 eq) and nonylamine (1.5 eq) were
added to the solution followed by HBTU (3.5 eq). This synthesis yielded the boc-protected
intermediate as a brown oil. Using the same procedure described above, The boc-protected
intermediate (0.36 g, 0.60 mmol) was converted to the title compound 9G (0.074 g, 0.23
1
mmol, 38 %) as a white solid. H NMR (300 MHz, [D6] CD3OD): δ 6.51 (s, 1H), 3.16 (t, 2H,
J=6.6 Hz), 2.51 (t, 2H, J=8.1 Hz), 2.24 (t, 2H, J=7.2 Hz), 1.87 (q, 2H, J= 7.2 Hz), 1.49 (m,
2H), 1.29 (m, 12H), 0.89 (t, 3H, J=6.6 Hz) ppm; 13C NMR (400 MHz, CD3OD): δ 175.2,
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148.2, 128.1, 109.9, 40.4, 35.9, 33.0, 30.6, 30.4, 30.4, 30.4, 28.0, 25.6, 24.9, 23.7, 14.5
ppm; IR νmax (cm-1) 3269, 3148, 2955, 2923, 2854, 1674, 1633, 1552; λmax= 286 nm; HRMS
(ESI) calculated for C16H40N4O [M+H]+ 295.24924, found 295.24808.
4-(2-Amino-1H-imidazol-5-yl)-N-(pentan-3-yl) butanamide hydrochloride (10A)
Compound 3 (0.50 g, 1.1 mmol) was solubilized in anhydrous dichloromethane (DCM) (30
mL). Triethylamine (3.5 eq) and 1-ethylpropylamine (1.1 eq) were added to the solution
followed by 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
(HBTU) (3.5 eq). The solution was allowed to stir at room temperature under nitrogen for 24
hr. After 24 hours, the reaction was quenched with 100 mL ethyl acetate and was washed
with 100 mL of water followed by three washes with 100 mL of brine. The intermediate was
submitted to flash column chromatography with a 1% methanol saturated with ammonium
and DCM solvent system to separate starting material from the desired product. Due to the
presence of boc groups, the intermediate could not be fully characterized by NMR. The
intermediate (0.6 g, 1.1 mmol) was dissolved in anhydrous DCM (10.5 mL) and was cooled
to 0°C. TFA (10.5 mL) was charged into the flask and the reaction was stirred for 24 hours.
After this time, the reaction was evaporated to dryness and toluene (3 mL) was added. Again
the mixture was concentrated and the process was repeated two additional times. The
resulting TFA salt was dissolved in methanol (2 mL) and concentrated HCl was added (1.5
mL). The mixture was then evaporated to dryness. Once dry, cold ether (5.0 mL) was added
to the mixture, and the mixture was sonicated. The cold ether was pipetted off leaving the
target compound 10A (0.29 g, 1.1 mmol, 99 %) as a brown oil. 1H NMR (300 MHz, CD3OD):
δ 6.52 (s, 1H), 3.65 (m, 1H), 2.53 (t, 2H, J=7.5 Hz), 2.29 (t, 2H, J=6.9 Hz), 1.90 (quint. 2H,
J=7.5 Hz), 1.55 (m, 2H), 1.41(m, 2H), 0.89 (t, 6H, J=7.2 Hz) ppm; 13C NMR (400 MHz,
CD3OD) δ 175.4, 148.4, 128.0, 110.0, 53.9, 35.9, 28.3, 28.3, 25.8, 24.9, 10.9, 10.9 ppm; IR
νmax (cm-1) 3255, 3146, 2964, 2934, 1674, 1630, 1547; λmax = 286 nm; HRMS (ESI)
calculated for C12H22N4O [M+H]+ 239.18664, found 239.18570.
4-(2-Amino-1H-imidazol-5-yl)-N-(heptan-4-yl) butanamide hydrochloride (10B)
According to the procedure used for 10A, compound 3 (1.0 g, 2.1 mmol) was solubilized in
anhydrous DCM (30 mL). Triethylamine (3.5 eq) and 1-propylbutylamine (1.1 eq) were
added to the solution followed by HBTU (3.5 eq). This synthesis yielded boc-protected
intermediate as a colorless oil. Using the deprotection procedure described in the synthesis
of 10A, the corresponding intermediate (0.61 g, 1.1 mmol) gave the title compound 10B
1
(0.24 g, 0.78 mmol, 73 %) as a brown oil. H NMR (300 MHz, CD3OD): δ 6.52 (s, 1H), 3.84
(m, 1H), 2.52 (t, 2H, J=7.5 Hz), 2.26 (t, 2H, J=7.2 Hz), 1.88 (quint. 2H, J=7.5 Hz), 1.36 (m,
8H), 0.91 (t, 6H, J=6.9 Hz) ppm; 13C NMR (400 MHz, CD3OD) δ 174.9, 148.5, 128.1, 109.9,
50.0, 38.3, 38.3, 36.0, 25.7, 24.9, 20.3, 20.3, 14.3, 14.3 ppm; IR νmax (cm-1) 3256, 3152,
2957, 2933, 1673, 1629, 1548, 1459; λmax= 286 nm; HRMS (ESI) calculated for C14H26N4O
[M+H]+ 267.21794, found 267.21674.
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4-(2-Amino-1H-imidazol-5-yl)-N-(nonan-5-yl) butanamide hydrochloride (10C)
According to the procedure used for 10A, compound 3 (1.0 g, 2.1 mmol) was solubilized in
anhydrous DCM (30 mL). Triethylamine (3.5 eq) and 5-nonanamine (1.1 eq) were added to
the solution followed by HBTU (3.5 eq). This synthesis yielded the boc-protected
intermediate as a colorless oil. Using the deprotection procedure described in the synthesis
of 10A, the corresponding intermediate (0.73 g, 1.2 mmol) gave the title compound 10C
1
(0.39 g, 1.2 mmol, 96 %) as a brown oil. H NMR (300 MHz, CD3OD): δ 6.52 (s, 1H), 3.80
(m, 1H), 2.52 (t, 2H, J=7.8 Hz), 2.26 (t, 2H, J=7.5 Hz), 1.89 (quint. 2H, J=7.8 Hz), 1.38 (m,
12H), 0.90 (t, 6H, J=6.3 Hz) ppm; 13C NMR (400 MHz, CD3OD) δ 174.9, 148.4, 128.0, 109.9,
50.4, 36.0, 35.7, 35.7, 29.4, 29.4, 25.8, 24.9, 23.5, 23.5, 14.4, 14.4 ppm; IR νmax (cm-1) 3256,
2955, 2929, 2858, 1669, 1630, 1545, 1457; λmax= 286 nm; HRMS (ESI) calculated for
C16H30N4O [M+H]+ 295.24924, found 295.24817.
4-(2-Amino-1H-imidazol-5-yl)-N-(undecan-6-yl) butanamide hydrochloride (10D)
According to the procedure used for 10A, compound 3 (1.0 g, 2.1 mmol) was solubilized in
anhydrous DCM (30 mL). Triethylamine (3.5 eq) and 6-undecanamine (1.1 eq) were added
to the solution followed by HBTU (3.5 eq). This synthesis yielded the boc-protected
intermediate as a colorless oil. Using the deprotection procedure described in the synthesis
of 10A, the corresponding intermediate (0.95 g, 1.5 mmol) gave the title compound 10D
1
(0.48 g, 1.3 mmol, 87 %) as a brown oil. H NMR (300 MHz, CD3OD): δ 6.52 (s, 1H), 3.81
(m, 1H), 2.53 (t, 2H, J=7.2 Hz), 2.27 (t, 2H, J=7.5 Hz), 1.89 (quint. 2H, J=7.2 Hz), 1.32 (m,
16H), 0.89 (t, 6H, J=6 Hz) ppm; 13C NMR (400 MHz, CD3OD): δ 175.1, 148.5, 128.1, 109.9,
50.7, 36.0, 36.0, 36.0, 32.8, 32.8, 26.8, 26.8, 25.9, 24.9, 23.6, 23.6, 14.4, 14.4 ppm; IR νmax
(cm-1) 3269, 2928, 1678, 1639, 1549, 1458; λmax= 288 nm; HRMS (ESI) calculated for
C18H34N4O [M+H]+ 323.28054, found 323.27932.
4-(2-amino-1H-imidazol-5-yl)-N-(pentadecan-8-yl) butanamide hydrochloride (10E)
According to the procedure used for 10A, compound 3 (1.0 g, 2.1 mmol) was solubilized in
anhydrous DCM (30 mL). Triethylamine (3.5 eq) and 8-pentadecanamine (1.1 eq) were
added to the solution followed by HBTU (3.5 eq). This synthesis yielded the boc-protected
intermediate as a brown oil. Using the deprotection procedure described in the synthesis of
10A, the corresponding intermediate (0.76 g, 1.1 mmol) gave the title compound 10E (0.27
1
g, 0.64 mmol, 57 %) as a brown oil. H NMR (300 MHz, CD3OD): δ 6.50 (s, 1H), 3.80 (m,
1H), 2.52 (t, 2H, J=7.2 Hz), 2.26 (t, 2H, J=7.2 Hz), 1.88 (quint. 2H, J=7.2 Hz), 1.28 (m, 24H),
0.88 (t, 6H, J=6.6 Hz) ppm; 13C NMR (400 MHz, CD3OD): δ 174.2, 147.4, 128.3, 110.1, 50.4,
36.1, 36.1, 36.1, 33.0, 33.0, 30.5, 30.5, 30.4, 30.4, 27.2, 27.2, 25.9, 25.0, 23.7, 23.7, 14.4,
14.4 ppm; IR νmax (cm-1) 3289, 2921, 2851, 1686, 1639, 1549, 1456; λmax= 286 nm; HRMS
(ESI) calculated for C22H42N4O [M+H]+ 379.34314, found 379.34168.
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4-(2-amino-1H-imidazol-5-yl)-N-(heptadecan-9-yl) butanamide hydrochloride (10F)
According to the procedure used for 10A, compound 3 (1.0 g, 2.1 mmol) was solubilized in
anhydrous DCM (30 mL). Triethylamine (3.5 eq) and 9-heptadecanamine (1.1 eq) were
added to the solution followed by HBTU (3.5 eq). This synthesis yielded the boc-protected
intermediate as a brown oil. Using the deprotection procedure described in the synthesis of
10A, the corresponding intermediate (0.91 g, 1.3 mmol) gave the title compound 10F (0.44
1
g, 1.0 mmol, 78 %) as a tan solid. H NMR (300 MHz, CD3OD): δ= 6.52 (s, 1H), 3.80 (m,
1H), 2.52 (t, 2H, J=7.2 Hz), 2.26 (t, 2H, J=7.5 Hz), 1.89 (quint. 2H, J=7.8 Hz), 1.29 (m, 28H),
0.89 (t, 6H, J=4.5 Hz) ppm; 13C NMR (400 MHz, DMSO-d6): δ 171.0, 146.8, 125.8, 108.5,
47.5, 34.2, 34.2, 34.2, 31.0, 31.0, 28.7, 28.7, 28.7, 28.7, 28.4, 28.4, 25.2, 25.2, 23.9, 23.3,
21.8, 21.8, 13.7, 13.7 ppm; IR νmax (cm-1) 3290, 3151, 2921, 2850, 1690, 1641, 1551, 1457;
λmax= 286 nm; HRMS (ESI) calculated for C24H46N4O [M+H]+ 407.37444, found 407.37286.
4-(2-amino-1H-imidazol-5-yl)-N-(nonadecan-10-yl)butanamide hydrochloride (10G)
According to the procedure used for 10A, compound 3 (1.0 g, 2.1 mmol) was solubilized in
anhydrous DCM (30 mL). Triethylamine (3.5 eq) and 10-nonadecanamine (1.1 eq) were
added to the solution followed by HBTU (3.5 eq). This synthesis yielded the correspond
intermediate as a brown oil. Using the deprotection procedure described in the synthesis of
10A, the corresponding intermediate (1.0 g, 1.4 mmol) gave the title compound 10G (0.40 g,
1
0.86 mmol, 61 %) as a tan solid. H NMR (300 MHz, CD3OD): δ 6.51 (s, 1H), 3.3 (m, 1H),
2.52 (t, 2H, J=7.5 Hz), 2.26 (t, 2H, J=7.5 Hz), 1.88 (quint. 2H, J=7.5 Hz), 1.28 (m, 32H), 0.89
(t, 6H, J=6.9 Hz) ppm; 13C NMR (400 MHz, DMSO-d6): δ 180.5, 156.3, 135.7, 118.0, 57.3,
44.1, 44.0, 44.0, 40.8, 40.8, 38.5, 38.5, 38.5, 38.5, 38.4, 38.4, 38.2, 38.2, 35.0, 35.0, 33.7,
33.1, 31.6, 31.6, 23.4, 23.4 ppm; IR νmax (cm-1) 3268, 2922, 2853, 1677, 1637, 1546, 1458;
λmax= 286 nm; HRMS (ESI) calculated for C26H50N4O [M+H]+ 435.40574, found 435.40386.
4-(2-amino-1H-imidazol-5-yl)-N- (1-phenyloctyl) butanamide (11A)
Compound 3 (0.40 g, 0.85 mmol) was solubilized in anhydrous dichloromethane (DCM) (30
mL). Triethylamine (3.5 eq) and tail S4A (1.5 eq) were added to the solution followed by 2-
(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) (3.5 eq).
The solution was allowed to stir at room temperature under nitrogen for 18 hr. After 18
hours, the reaction was quenched with 100 mL ethyl acetate and was washed with 100 mL
of water followed by three washes with 100 mL of brine. The intermediate was submitted to
flash column chromatography with a 1.25% methanol saturated with ammonium and DCM
solvent system to separate starting material from the desired product. Due to the presence
of boc groups, the intermediate could not be fully characterized by NMR. The intermediate
(0.43 g, 0.66 mmol) was dissolved in anhydrous DCM (6.5 mL) and was cooled to 0°C. TFA
(6.5 mL) was charged into the flask and the reaction was stirred for 24 hours. After this time,
the reaction was evaporated to dryness and toluene (3 mL) was added. Again the mixture
was concentrated and the process was repeated two additional times. The resulting TFA salt
was dissolved in methanol (2 mL) and concentrated HCl was added (1.5 mL). The mixture
was then evaporated to dryness. Once dry, cold acetone (5.0 mL) was added to the mixture,
and the mixture was sonicated. The cold acetone was pipetted off leaving the target
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compound 11A (0.11 g, 0.29 mmol, 45%) as a brown oil. 1H NMR (400 MHz, [D6] CD3OD): δ
7.29(m, 5H), 6.44 (s, 1H), 4.81 (t, 1H, J=7.5 Hz), 2.46 (t, 2H, J= 7.2 Hz), 2.27 (dd, 2H, J= 7.2
Hz), 1.86 (q, 2H, J= 7.5Hz), 1.73 (m, 2H), 1.27 (m, 10H), 0.86 (t, 3H, J= 7 Hz) ppm; 13C NMR
(400 MHz, CD3OD): δ 174.5, 148.4, 144.5, 129.4, 129.4, 128.2, 128.0, 127.6, 127.6, 109.9,
54.8, 37.4, 35.6, 32.9, 30.3, 30.3, 27.5, 25.6, 24.9, 23.6, 14.4 ppm; IR νmax (cm-1) 2925,
1675, 1539, 1454, 699; λmax= 288 nm; HRMS (ESI) calculated for C21H32N4O [M+H]+
357.26489, found 357.26335.
4-(2-amino-1H-imidazol-5-yl)-N- (1-phenylnonyl) butanamide (11B)
Compound 3 (0.40 g, 0.85 mmol) was solubilized in anhydrous dichloromethane (DCM) (30
mL). Triethylamine (3.5 eq) and tail S4B (1.5 eq) were added to the solution followed by 2-
(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) (3.5 eq).
The solution was allowed to stir at room temperature under nitrogen for 18 hr. After 18
hours, the reaction was quenched with 100 mL ethyl acetate and was washed with 100 mL
of water followed by three washes with 100 mL of brine. The intermediate was submitted to
flash column chromatography with a 1.25% methanol saturated with ammonium and DCM
solvent system to separate starting material from the desired product. Due to the presence
of boc groups, the intermediate could not be fully characterized by NMR. The intermediate
(0.33 g, 0.49 mmol) was dissolved in anhydrous DCM (5 mL) and was cooled to 0°C. TFA (5
mL) was charged into the flask and the reaction was stirred for 24 hours. After this time, the
reaction was evaporated to dryness and toluene (3 mL) was added. Again the mixture was
concentrated and the process was repeated two additional times. The resulting TFA salt was
dissolved in methanol (2 mL) and concentrated HCl was added (1.5 mL). The mixture was
then evaporated to dryness. Once dry, cold acetone (5.0 mL) was added to the mixture, and
the mixture was sonicated. The cold acetone was pipetted off leaving the target compound
1
11B (0.11 g, 0.29 mmol, 57%) as a brown oil. H NMR (400 MHz, [D6] CD3OD): δ 7.29 (m,
5H), 6.45 (s, 1H), 4.81 (t, 1H, J= 6.6 Hz), 2.46 (t, 2H, J=7.6 Hz), 2.28 (dt, 2H, J= 7.6 Hz),
13
1.85 (q, 2H, J= 7.7 Hz), 1.73 (m, 2H), 1.26 (m, 12H), 0.86 (t, 3H, J= 7.1 Hz) ppm; C NMR
(400 MHz, CD3OD): δ 174.5, 148.4, 144.5, 129.5, 129.5, 128.2, 128.0, 127.6, 127.6, 109.9,
54.8, 37.4, 35.9, 32.9, 30.6, 30.3, 30.3, 27.5, 25.6, 24.9, 23.7, 14.4 ppm; IR νmax (cm-1) 2925,
2854, 1675, 1638, 1539, 1494, 699; λmax= 288 nm; HRMS (ESI) calculated for C22H34N4O
[M+H]+ 371.28054, found 371.27927.
4-(2-amino-1H-imidazol-5-yl)- N-(1-phenylundecan-5-yl)butanamide (12)
Compound 3 (0.25 g, 0.53 mmol) was solubilized in anhydrous dichloromethane (DCM) (30
mL). Triethylamine (3.5 eq) and tail S9 (1.5 eq) were added to the solution followed by 2-
(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) (3.5 eq).
The solution was allowed to stir at room temperature under nitrogen for 18 hr. After 18
hours, the reaction was quenched with 100 mL ethyl acetate and was washed with 100 mL
of water followed by three washes with 100 mL of brine. The intermediate was submitted to
flash column chromatography with a 1.25% methanol saturated with ammonium and DCM
solvent system to separate starting material from the desired product. Due to the presence
of boc groups, the intermediate could not be fully characterized by NMR. The intermediate
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(0.15 g, 0.21 mmol) was dissolved in anhydrous DCM (3 mL) and was cooled to 0°C. TFA (3
mL) was charged into the flask and the reaction was stirred for 24 hours. After this time, the
reaction was evaporated to dryness and toluene (3 mL) was added. Again the mixture was
concentrated and the process was repeated two additional times. The resulting TFA salt was
dissolved in methanol (2 mL) and concentrated HCl was added (1.5 mL). The mixture was
then evaporated to dryness. Once dry, cold acetone (5.0 mL) was added to the mixture, and
the mixture was sonicated. The cold acetone was pipetted off leaving the target compound
1
12 (0.07 g, 0.18 mmol, 83%) as a brown oil. H NMR (400 MHz, [D6] CD3OD): δ 7.20(t, 2H,
J= 8 Hz), 7.12 (m, 3H, J= 8 Hz), 6.49 (s, 1H), 3.80 (bs, 1H), 2.57 (t, 2H, J= 8 Hz), 2.49 (t, 2H,
J= 8 Hz), 2.26 (t, 2H, J= 8 Hz), 1.86 (m, 2H), 1.61 (m, 2H), 1.42 (m, 4H), 1.27 (m, 10H), 0.87
(t, 3H, J= 8 Hz) ppm; 13C NMR (400 MHz, CD3OD): δ 175.0, 148.5, 143.6, 129.4, 129.4,
129.2, 129.2, 128.1, 126.6, 110.0, 50.6, 36.7, 36.0, 36.0, 35.8, 32.9, 32.3, 30.2, 27.1, 26.6,
25.9, 24.9, 23.6, 14.8 ppm; IR νmax (cm-1) 2926, 2856, 1637, 1543, 1454, 1201, 697; λmax
292 nm; HRMS (ESI) calculated for C24H38N4O [M+H]+ 399.31184, found 399.31204.
=
4-(2-amino-1H-imidazol-5-yl)- N-(1-(4-hexylphenyl)heptyl)butanamide (13)
Compound 3 (0.465 g, 0.99 mmol) was solubilized in anhydrous dichloromethane (DCM) (30
mL). Triethylamine (3.5 eq) and tail S13 (1.5 eq) were added to the solution followed by 2-
(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) (3.5 eq).
The solution was allowed to stir at room temperature under nitrogen for 18 hr. After 18
hours, the reaction was quenched with 100 mL ethyl acetate and was washed with 100 mL
of water followed by three washes with 100 mL of brine. The intermediate was submitted to
flash column chromatography with a 1.25% methanol saturated with ammonium and DCM
solvent system to separate starting material from the desired product. Due to the presence
of boc groups, the intermediate could not be fully characterized by NMR. The intermediate
(0.45 g, 0.62 mmol) was dissolved in anhydrous DCM (6 mL) and was cooled to 0°C. TFA (6
mL) was charged into the flask and the reaction was stirred for 24 hours. After this time, the
reaction was evaporated to dryness and toluene (3 mL) was added. Again the mixture was
concentrated and the process was repeated two additional times. The resulting TFA salt was
dissolved in methanol (2 mL) and concentrated HCl was added (1.5 mL). The mixture was
then evaporated to dryness. Once dry, cold acetone (5.0 mL) was added to the mixture, and
the mixture was sonicated. The cold acetone was pipetted off leaving the target compound
1
13 (0.08 g, 0.18 mmol, 30%) as a brown oil. H NMR (400 MHz, [D6] CD3OD): δ 7.21(d, 2H,
J= 8 Hz), 7.11 (d, 2H, J= 8 Hz), 6.44 (s, 1H), 4.80 (t, 1H, J= 6.9 Hz), 2.56 (t, 2H, J= 7.6 Hz),
2.48 (t, 2H, J= 7.6 Hz), 2.29 (m, 2H), 1.87 (m, 2H), 1.74 (m, 2H), 1.56 (m, 2H), 1.28 (m,
13
14H), 0.88 (m, 6H) ppm; C NMR (400 MHz, CD3OD): δ 174.6, 148.4, 142.8, 141.5, 129.5,
129.5, 128.1, 127.5, 127.5, 110.0, 54.7, 37.4, 36.5, 35.8, 32.9, 32.8, 32.7, 30.0, 30.0, 27.5,
25.6, 24.8, 23.6, 23.6, 14.4, 14.4 ppm; IR νmax (cm-1) 3250, 2925, 2855, 1676, 1542, 1457,
564; λmax= 292 nm; HRMS (ESI) calculated for C26H42N4O [M+H]+ 427.34314, found
427.34363.
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1-phenylundecan-5-one (S8)
5-Phenylvaleric acid (S5) (2.5g, 14.02mmol) was purchased from a commercial source and
was converted to compound S6 using oxalyl chloride (5 eq.), anhydrous DCM (10mL), and
catalytic DMF (0.1mL) under nitrogen. After 16 hours, the reaction was evaporated to
dryness and placed under vacuum. The acid chloride S6 (3g, 15.25 mmol) was then
converted to the Weinreb amide (S7) using N,O dimethylhydroxylamine (1.1 eq), TEA (2.5
eq.) and anhydrous DCM (50mL). After 16 hours, 30mL of sodium bicarbonate was added to
the reaction and stirred for 10 minutes. After 10 minutes, the product was extracted with
DCM and flash column chromatography was performed using 1:3 ethyl acetate, petroleum
ether solvent system. To obtain compound S8, compound S7 was dissolved in anhydrous
THF (10mL) on ice and was then treated with hexylmagnesium bromide (2M) and allowed to
stir at room temperature for two hours. After two hours, ammonium chloride (10mL) was
added to the reaction mixture and was allowed to stir for 10 minutes. The product was
extracted using ethyl acetate and was evaporated to dryness. This yielded compound S8 as
a white solid in 49% yield. 1H NMR (400 MHz, [D6] CD3OD): δ 7.23(m, 2H, J=7.5 Hz), 7.15
(t, 3H, J=8.4 Hz), 2.81 (m, 1H), 2.61 (t, 2H, J=7.5 Hz), 1.57 (m, 4H), 1.33 (m, 12H), 0.90 (t,
3H, J= 7 Hz) ppm; 13C NMR (400 MHz, CD3OD): δ 213.6, 143.5, 129.3, 129.3, 129.2, 129.2,
126.7, 43.4, 43.2, 36.6, 32.7, 32.1, 29.9, 24.8, 24.4, 23.5, 14.4 ppm; IR νmax (cm-1) 2928,
2852, 1711, 1454, 746, 698; λmax= 284 nm; HRMS (ESI) calculated for C17H26O [M+H]+
247.20564, found 247.20553.
1-phenylundecan-5-amine (S9)
Compound S8 (0.75g, 3mmol) was dissolved in anhydrous methanol (30mL) and ammonium
acetate (10eq) was added to the reaction. The reaction mixture stirred for 90 minutes before
sodium cyanoborohydride (5eq) was added. The reaction stirred under nitrogen for 64 hours.
After 64 hours, concentrated HCl was added to the reaction mixture until the pH was 2. The
reaction mixture was then evaporated to dryness. Water (500mL) was added and solid KOH
was added until the pH reached 10. The product was extracted with DCM and was
evaporated to dryness. This yielded the desired product S9 as a colorless oil in 99% yield.
1H NMR (400 MHz, [D6] CD3OD): δ 7.23(m, 2H, J=7.5 Hz), 7.15 (t, 3H, J=8.4 Hz), 2.81 (m,
1H), 2.61 (t, 2H, J=7.5 Hz), 1.57 (m, 4H), 1.33 (m, 12H), 0.90 (t, 3H, J= 7 Hz) ppm; 13C NMR
(400 MHz, CD3OD): δ 143.6, 129.4, 129.4, 129.2, 129.2, 126.7, 52.3, 36.7, 36.6, 36.4, 32.9,
32.7, 30.5, 26.6, 26.2, 23.7, 14.4 ppm; IR νmax (cm-1) 2925, 2850, 1451, 732; λmax= 284 nm;
HRMS (ESI) calculated for C17H29N [M+H]+ 248.23728, found 248.23724.
1-(4-hexyl-henyl)heptan-1-one (S12)
4-Hexylbenzoyl chloride (S10) (2.5g, 11.12mmol) was purchased from a commercial source
and was converted to compound S11 using N,O dimethylhydroxylamine (1.1 eq), TEA (2.5
eq.) and anhydrous DCM (50mL). After 16 hours, 30mL of sodium bicarbonate was added to
the reaction and stirred for 10 minutes. After 10 minutes, the product was extracted with
DCM and flash column chromatography was performed using 1:3 ethyl acetate, petroleum
ether solvent system. This yielded compound S11 as a colorless oil in 98% yield. To obtain
compound S12, compound S11 was dissolved in anhydrous THF (10mL) on ice and was
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then treated with hexylmagnesium bromide (2M) and allowed to stir at room temperature for
two hours. After two hours, ammonium chloride (10mL) was added to the reaction mixture
and was allowed to stir for 10 minutes. The product was extracted using ethyl acetate and
was evaporated to dryness. This yielded compound S12 as a colorless oil in 99% yield. 1H
NMR (400 MHz, [D6] CD3OD): δ 7.87(d, 2H, J= 8 Hz), 7.27 (d, 2H, J=8 Hz), 2.96 (t, 2H, J=
7.4 Hz), 2.65 (t, 2H, J= 7.4 Hz), 1.65 (m, 4H), 1.33 (m, 12H), 0.89 (t, 6H, J= 5.6 Hz) ppm; 13C
NMR (400 MHz, CD3OD): δ 202.5, 150.1, 136.0, 129.7, 129.7, 129.4, 129.4, 39.4, 36.9,
32.9, 32.8, 32.3, 30.1, 30.0, 25.7, 23.6, 23.6, 14.4, 14.4 ppm; IR νmax (cm-1) 2927, 2856,
1683, 1607, 1465, 1180, 978; λmax= 286 nm; HRMS (ESI) calculated for C19H30O [M+H]+
275.23694, found 275.23717.
1-(4-hexylphenyl) heptan-1-amine (S13)
Compound S12 (1g, 3.64mmol) was dissolved in anhydrous methanol (120mL) and
ammonium acetate (10eq) was added to the reaction. The reaction mixture stirred for 90
minutes before sodium cyanoborohydride (5eq) was added. The reaction stirred under
nitrogen for 64 hours. After 64 hours, concentrated HCl was added to the reaction mixture
until the pH was 2. The reaction mixture was then evaporated to dryness. Water (500mL)
was added and solid KOH was added until the pH reached 10. The product was extracted
with DCM and was evaporated to dryness. This yielded the desired product S13 as a
colorless oil in 99% yield.1H NMR (400 MHz, [D6] CD3OD): δ 7.16(m, 4H), 3.81 (t, 1H, J= 6.7
Hz), 2.56 (m, 2H), 1.64 (m, 4H), 1.25 (m, 14H), 0.85 (m, 6H) ppm; 13C NMR (400 MHz,
CD3OD): δ 143.3, 142.3, 129.7, 129.7, 127.7, 127.7, 57.0, 39.1, 36.6, 32.9, 32.9, 32.7, 30.2,
30.0, 27.4, 23.7, 23.6, 14.4, 14.4 ppm; IR νmax (cm-1) 2924, 2854, 1458, 822, 558; λmax= 284
nm; HRMS (ESI) calculated for C19H33N [M+H]+ 276.26858, found 276.26807.
Results and Discussion
Analogue Synthesis
For this study, we elected to explore the SAR of the alkyl tail region of compound 1,
looking at linear chains as well as branched symmetric and asymmetric chains. Compounds
were synthesized utilizing the strategy outlined in Scheme 1, using the protected 2-AI 3 as
the common coupling partner.
The synthesis of 3 was initiated with the desymmerization of glutaric anhydride 5 to
produce the gamma-keto ester 6. This intermediate was treated with oxalyl chloride to form
the acid chloride, which was further reacted with diazomethane followed by the quenching
with hydrobromic acid (HBr) to afford the desired α-bromo ketone 4. The 2-AI ring was then
installed via Boc guanidine cyclization to yield the 2-AI 7. Finally, treatment with Boc
anhydride followed by hydrogenation to remove the benzyl group yielded 2-AI 3 (Scheme 2).
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With 3 in hand, all final 2-AI derivatives were synthesized by activating the carboxylic
acid with HBTU, coupling with the appropriate amine, removing the Boc-groups with TFA,
and performing counter ion exchange to generate the HCl salt of the desired product (9A-K,
10A-G, 11A-B, 12, 13)(Scheme 3). All the single carbon chain amines (as well as a select
number of the branched amines) were commercially available. The branched amines that
were not commercially available were synthesized from the corresponding ketones through a
reductive amination using ammonium acetate and sodium cyanoborohydride in anhydrous
methanol (Scheme S.1 and S.2). The asymmetric ketones necessary to access amines for
the synthesis of 12 and 13 were generated through the synthesis of the Weinreb amide
followed by a Grignard addition to produce the desired ketone. The previously described
reductive amination procedure was used to deliver the desired amines (Scheme S.3 and
S.4). Despite the addition of a chiral center, enantioselectivity was not controlled or resolved
during synthesis. Ultimately, this SAR study was to determine whether there was an
increase or decrease in activity with the absence of symmetry in the tail portion of the
molecule. If activity is observed, further studies to enhance activity by controlling
enantioselectivity will be performed.
Screening for biofilm dispersion
Initial screening focused on the ability of the compounds to induce dispersion of pre-
formed M. smegmatis biofilms as assessed using a crystal violet reporter assay (Table S.1)
[17]. Previously, we found compound 1 to have an EC50 of 52 μM. Upon evaluation of the
branched derivative library, it was revealed that lengthening the tail one carbon (10E)
increased activity, with this compound exhibiting an EC50 of 19.9 ± 5.3 μM (Figure S.1).
Shortening the tail by one carbon (10D), reduced activity to an EC50 of 86.3 ± 8.3 μM (Figure
S.2). Adding or removing two carbons to the tail resulted in a further decrease in activity with
compounds 10C (two carbons shorter) and 10F (two carbons longer) exhibiting EC50 values
of >200 μM and 150 ± 11 μM respectively. The single linear tails (9A-K), however,
possessed limited dispersion activity. Compound 9H displayed the best dispersion activity
with an EC50 of 89.9 ± 15 μM. None of the other branched derivatives induced dispersion at
200 μM (highest concentration tested). Next, we evaluated the asymmetric derivatives that
contained the most active branched tail lengths. Compound 11A, which has the same length
carbon tail (seven total carbons) as the most active compound 10E, exhibited an EC50 value
of 109 ± 13 μM. The addition of one carbon in compound 11B resulted in a decrease in EC50
value to 57.8 ± 4.6 μM, though this EC50 was still not comparable to lead compound 1 or 10E
(Figure S.3). With that, the synthesis efforts shifted to incorporating a phenalkyl chain to one
side of the branch while keeping the other branch consistent with a linear chain of six
carbons. Compounds 12 and 13 returned EC50 values of 66.9 ± 3.0 and 85.1 ± 13 μM
respectively, which were again less active than compounds 1 or 10E. The incorporation of
the aryl substituents did not improve upon the activity of 10E (Complete results can be found
in Table S.1).
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Screening for potentiation activity
We previously reported that compound 1 exhibits synergy with β-lactam antibiotics
against planktonic M. tuberculosis [18]. Based upon this observation, these newly
synthesized derivatives were screened for the ability to potentiate several cephalosporin and
penicillin antibiotics against planktonic M. smegmatis. In order to evaluate the potentiation
activity of these compounds against M. smegmatis, the MIC of antibiotics and the
compounds alone were first determined. Following this determination, the MIC of each
antibiotic was measured in the presence of each compound at 30% its MIC, which is the
typical concentration we employ for potentiation assays [18, 19]. Interestingly, none of the
symmetric branched tail compounds produced any notable reductions at this concentration.
When compound 1 was tested at 50% its MIC, results were modest producing at most a 16-
fold reduction of ceftazidime’s MIC. For the linear tail compounds at 30% the MIC, activity
increased slightly as the length of the tail increased, but remained modest with the largest
fold reduction observed being a four-fold reduction with cefotaxime and compound 9K.
Interestingly, when the asymmetric tails were analyzed, compound 11A showed no notable
activity at 30% its MIC for five of the six antibiotics, but effected a 32-fold reduction in the
cefotaxime MIC. Compound 11B, which was a more potent biofilm inhibitor than 11A,
showed only an eight fold reduction in the MIC of cefotaxime and again no notable reduction
in the MICs of the other antibiotics tested (Table 1). Additionally, compounds 12 and 13 were
screened and again, it was seen that the less active biofilm disruptor, compound 13, was
more active than the more potent antibiofilm derivative (12), but activity was limited to two
and four-fold reductions except again in the case of cefotaxime, where the reduction was 32-
fold (Complete results can be found in Tables S.2 and S.3). The selectivity of this series of
compounds for cefotaxime potentiation over other β-lactam antibiotics mirrors that observed
with a 2-aminobenzimidazole series previously reported, which possess a pattern of
selectivity distinct from direct β-lactamase inhibitors, such as clavulanic acid, and does not
directly inhibit β-lactamase activity [18].
Conclusion
In conclusion, the previously reported potentiation and biofilm dispersion activity of
compound 1 against M. smegmatis and M. tuberculosis led us to investigate the SAR of the
alkyl tail region of this compound. A library consisting of linear and branched symmetric and
asymmetric tails were synthesized and screened for their ability to disperse biofilms and
potentiate a subset of β-lactam antibiotics against M. smegmatis. This screen revealed that
compound 10E improved upon the previously reported EC50 value of compound 1, but did
not improve potentiation capabilities. Screening of the linear tails revealed little to no
dispersion or potentiation capabilities. The asymmetric tails containing alkyl chains with
lengths comparable to the most active branched compounds and phenyl rings added in
different orientations proved to be better than the linear compounds at biofilm dispersion, but
did not have EC50 values comparable to compound 1 or 10E. Despite the disappointing
antibiofilm activity, we found that the asymmetric tails possessed improved potentiation
capabilities, with compounds 11A and 13 reducing the MIC of cefotaxime from 64 to 2
μg/mL. These results show that biofilm dispersion capabilities may be decoupled from
antibiotic potentiation capabilities for this series of compounds.
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Acknowledgements
We would like to thank the NIH for their generous financial support on this project.
Conflict of Interest
Christian Melander is a co-founder of Agile Sciences, a biotechnology company
focused on commercial applications of nitrogen-rich small molecules, and currently sits on its
board-of-directors.
Figure legends
Figure 1- Structure of previously reported compound 1.
Figure S.1- Determination of EC50 for compound 10E.
Figure S.2- Determination of EC50 for compound 10D.
Figure S.3- Determination of EC50 for compound 11B.
Scheme 1- Retrosynthetic approach to the synthesis of the proposed 2-AI library.
Scheme 2- Synthesis of the 2-AI region of derivative library. a)BnOH, TEA, DMAP, DCM
(96%); b)i) (COCl)2, DMF, DCM; ii) CH2N2; iii) HBr (70%); c)Boc-guanidine, DMF (50%); d)
(Boc)2O, TEA, DMAP (90%); e) 10 wt% Pd, H2, THF (99%).
Scheme 3- Coupling amine tails to produce desired reverse amide compounds. a) H2N-R,
HBTU, TEA, DCM (40-70%); b) TFA, DCM; c) HCl, MeOH (50-99%).
Scheme S.1- Synthesis of the branched tails from the respective carbonyl derivatives. a) i)
NH4CH3CO2, Anhydrous CH3OH, 90 minutes; ii) NaBH3CN, 60 hours.
Scheme S.2- Synthesis of the asymmetric tails for compounds 11A-B from the respective
carbonyl derivatives. a) i) NH4CH3CO2, Anhydrous CH3OH, 90 minutes; ii) NaBH3CN, 60
hours.
Scheme S.3- Synthesis of asymmetric tail for compound 12 from the commercially available
carboxylic acid. a) Oxalyl chloride, Anhydrous DCM, catalytic DMF; b) N,O dimethyl
hydroxylamine, TEA, Anhydrous DCM; c) Hexylmagnesium bromide, THF; d) i) NH4CH3CO2,
Anhydrous CH3OH, 90 minutes; ii) NaBH3CN, 60 hours.
Scheme S.4- Synthesis of asymmetric tail for compound 13 from the commercially available
acid chloride. a) N,O dimethyl hydroxylamine, TEA, Anhydrous DCM; b) Hexylmagnesium
bromide, THF; c) i) NH4CH3CO2, Anhydrous CH3OH, 90 minutes; ii) NaBH3CN, 60 hours.
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Table 1- Biofilm dispersion and repotentiation activity of the most active derivatives (Fold
reductions in parenthesis).
Table S.1- EC50 values for derivative library obtained from biofilm dispersion assay with M.
smegmatis.
Table S.2- Potentiation of common penicillin β-lactam antibiotics against M. smegmatis
using 2-AI derivative library.
Table S.3- Potentiation of common cephalosporin β-lactam antibiotics against M. smegmatis
using 2-AI derivative library.
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Table 1
Compound EC50
MIC
Concentration
Penicillin Cefotaxime Ceftazidime
(μM)
(μM) Tested (μM)
MIC
MIC (μg/mL) MIC (μg/mL)
(μg/mL)
-
-
-
-
256
64
512
1
52
-
40
-
10
20
12.5
128 (2)
64 (4)
256(0)
64 (0)
8 (8)
64 (0)
512 (0)
32 (16)
512 (0)
10E
19.9 ±
5.3
50
11A
11B
12
109 ± 13 100
57.8±4.6 50
66.9±3.0 50
30
15
15
15
128 (2)
128 (2)
64 (4)
64(4)
2 (32)
8 (8)
128 (4)
128 (4)
512 (0)
512 (0)
8 (8)
13
85.1±13
50
2(32)
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This article is protected by copyright. All rights reserved.