Synthesis and biological activity of 2-aminoimidazole triazoles accessed by Suzuki-Miyaura cross-coupling

Article abstract of DOI:10.1039/c0ob00925c

A pilot library of 2-aminoimidazole triazoles (2-AITs) was synthesized and assayed against Acinetobacter baumannii and methicillin-resistant Staphylococus aureus (MRSA). Results from these studies show that these new derivatives have improved biofilm dispersal activities as well as antibacterial properties against A. baumannii. With MRSA biofilms they are found to possess biofilm inhibition capabilities at low micromolar concentrations.

Full text of DOI:10.1039/c0ob00925c

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PAPER
Synthesis and biological activity of 2-aminoimidazole triazoles accessed by
Suzuki–Miyaura cross-coupling†
Samuel Reyes, Robert W. Huigens III, Zhaoming Su, Michel L. Simon and Christian Melander*
Received 23rd October 2010, Accepted 3rd February 2011
DOI: 10.1039/c0ob00925c
A pilot library of 2-aminoimidazole triazoles (2-AITs) was synthesized and assayed against
Acinetobacter baumannii and methicillin-resistant Staphylococus aureus (MRSA). Results from these
studies show that these new derivatives have improved biofilm dispersal activities as well as antibacterial
properties against A. baumannii. With MRSA biofilms they are found to possess biofilm inhibition
capabilities at low micromolar concentrations.
While the threat of bacterial biofilms is alarming,19 it has been
only within the last decade that compounds have been developed
Introduction
Biofilms are ubiquitous in nature.1 They are formed when
planktonic bacteria attach onto a surface, and create an encased
bacterial community with an organized structure at an extracel-
lular polymeric interface.2 Biofilm formation is a serious medical
concern as biofilm-associated infections account for nearly 80% of
microbial infections in humans.3 Biofilms are implicated in several
diseases that range from cystic fibrosis4,5 to tooth decay6 and
nosocomial infections.7 Outside of medicine, bacteria enclosed
within biofilms are the underlying cause of biofouling which is
a serious problem for the shipping industry.8 Biofilms also pose
severe problems that are detrimental in food processing plants and
agriculture products.9
to combat surface-associated bacteria.20,21 Part of the difficulty in
treating biofilm-associated infections is caused by the protective
lifestyle that biofilms confer to the bacteria which is absent when
bacteria are in planktonic form.22,23
Quorom sensing (QS) has been shown to be involved in biofilm
development.24 Baceterial cells communicate through this QS
prcoess which is mediated by organic compounds secreted by
the bacteria.25 Most biofilm-modulating small molecules that
have been reported have targeted quorum sensing machinery
(Fig. 1).26–28 Examples of such compounds include acylated
homoserine lactones (AHL) and AHL mimics,29,30 halogenated
furanones,31 and cyclic peptide derivatives.32
Acinetobacter baumannii is a gram-negative bacterium that has
been recognized as a human pathogen since the 1970s.10 Recent
reports show that there is an increasing number of infections
attributed to A. baumannii, most notably in intensive care units.11,12
A. baumannii infections also occur in victims of traumatic disasters
such as earthquakes13 and war.14 The survival capability of this
bacterium in hospital settings and other sterile environment is
attributed to the ability of A. baumannii to form tenaciously
persistent biofilms.15
Methicillin-resistant Staphylococus aureus (MRSA) infections,
which are traditionally acquired from hospital settings, have
been emerging outside long-term care facilities and have become
common within society.16 More of a concern is that risk factors
that are typically associated with MRSA colonization are notice-
ably absent from recent reports of community-acquired MRSA
infections.17 The disturbing rate at which antibiotic-resistant
strains are emerging necessitates the need for the development
of new therapeutic strategies.18
Fig. 1 Examples of compounds that interfere with quorum sensing.
Our group has turned to marine alkaloids for inspiration in de-
signing compounds that have potential in modulating biofilms.33,34
More specifically, we have focused on developing compound
libraries that resemble bromoageliferin and oroidin, sponge
metabolites that have been shown to inhibit biofilm formation
and bacterial growth of the marine bacterium, Rhodospirillum
salexigens (Fig. 2a).20,35 We have shown that by retaining the
2-aminoimidazole (2-AI) moiety, simple synthetic derivatives
are effective at inhibiting and dispersing bacterial and fungal
biofilms.36–39
North Carolina State University, Department of Chemistry, Raleigh, North
Carolina, 27695-8204, USA. E-mail: Christian_Melander@ncsu.edu
† Electronic supplementary information (ESI) available: 1H and 13C NMR
scans and biofilm inhibition and dispersion and growth curves analyses.
See DOI: 10.1039/c0ob00925c
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Fig. 3 Retrosynthetic approach to derivatizing the tail end of the lead
2-AIT via Suzuki–Miyaura cross-coupling.
Scheme 1 (a) Synthesis of allylic azide 4; (b) synthesis of vinyl bromide
2.
Fig. 2 (a) Anti-biofilm compounds from the oroidin family of alkaloids;
(b) examples of 2-AIT analogues synthesized in our labs.
compound, this methodology, avoided both the use of Na/Hg and
saturated NH3–MeOH column chromatography40 Cycloaddition
of 7 with 4, however, resulted in, disappointingly, low yields. This
was circumvented by exhaustive Boc-protection of the exocyclic
amine (Scheme 1b). The resulting tri-Boc-protected 2-AI-alkyne 3
enabled us to conduct the copper-catalyzed [3 + 2] cycloaddition
on a larger scale, with higher yields, to furnish the corresponding
vinyl bromide 2 needed for Suzuki–Miyaura couplings.
Compound 2 was reacted with various boronic acids using
standard Suzuki–Miyaura conditions to furnish Boc-protected 2-
AITs (Scheme 2). In general, most electron-rich boronic acids gave
superior yields than those that are either electron-deficient or are
sterically congested. Deprotection of the cross-coupling products
In our efforts to identify and develop novel 2-AI structures
with antibiofilm activity, we have developed several classes of 2-
aminoimidazole/triazole (2-AIT) libraries (Fig. 2b).40,41 The lead
2-AIT from these libraries demonstrated biofilm inhibition and
dispersal activities across bacterial order, class and phylum.40
In another application, the lead 2-AIT was shown to work in
concert with photodynamic inactivation to reduce biofilm mass
population of A. baumannii,42 as well as suppress antibiotic
resistance in planktonic bacterial cultures.43 Encouraged by the
improved anti-pathogenic properties of these 2-AIT scaffolds,
we have sought to synthesize 2-AIT analogues based on this
lead 2-AIT structure and evaluate them for biofilm modulatory
properties. In this paper, we report the chemical synthesis of such
analogues and their antibiofilm and antibacterial activities against
A. baumannii and MRSA.
Results and Discussion
Our design of 2-AIT analogues was focused on the structural
modification of the aromatic appendage of the 2-AIT lead
compound. This approach was driven by previous results from
our studies suggesting that: (i) the 2-AI moiety is, at least in part,
responsible for the observed antibiofilm activity; and (ii) a five
carbon spacer for the linker is optimum for antibiofilm activity.
Consequently, we have employed Suzuki–Miyaura coupling to
introduce diversity in these structures.44
The requisite vinyl halide 2 was prepared via [3+2] cycloaddition
of 2-AI-alkyne 3 and allyic azide 4 (Fig. 3). Compound 4 was
accessed from allylic alcohol 5 (Scheme 1a).45 The 2-AI precursor
7 was constructed via cyclization of a-bromoketone 6 and Boc-
guanidine.46–48 In contrast with the original synthesis of the lead
Scheme 2 Suzuki–Miyaura cross-couplings of vinyl bromide 2 and
various boronic acids. Yields shown are after step (i). Yields after step
(ii) are generally >95%.
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Table 1 Biological activities of the most active compounds against
A. baumannii and MRSA
that 8g, 8i and 8j were non-microbicidal at their respective
IC50 concentrations. Compound 8h, however, was found to be
microbicidal and was determined to have an MIC of 6.25 mM
(2.77 mg ml-1) against MRSA.
MIC vs A.
baumannii
EC50/mM vs
A. baumannii
Compound
mM
mg ml-1
IC50/mM vs. MRSA
Conclusions
8g
8h
8i
25
10.92
2.77
5.74
6.95
11.78
59.61 7.28
53.90 3.39
44.70 2.25
49.38 4.83
9.86 2.85
—
In summary, we have reported an improved synthesis of the lead
2-AIT compound which is amenable to synthesizing a series of
derivatives with antibiofilm as well as antibacterial properties.
Derivatizing the aromatic appendage of the 2-AIT lead compound
increases biofilm dispersal activities against A. baumannii. These
results further support our recent report that subtle changes in
the triazole appendage can have siginificant changes in biological
activity of 2-AI-based antibiofilm agents.55 These modifications
also impart antibacterial capabilities to some of these 2-AITs.
Analogues possessing a hydrophobic chain at the tail region
proved to be the most active, with the n-butyl showing better
bioactivities compared to the n-hexyl and n-heptyl. Compounds
8g–j were shown to possess such antibacterial activities with
biofilm dispersal capabilities against A. baumannii. Moreover,
three (8g, 8i and 8j) of the aforementioned compounds were
found to inhibit MRSA biofilm formation at low micromolar
concentrations. These compounds demonstrate the ability of 2-
AITs to control bacterial behavior and could potentially lead to
new therapeutic strategies against bacterial infection.
a
6.25
12.5
12.5
25
8.55 1.06
4.50 0.53
8j
b
b
8n
—
—
a Microbicidal. b Not active.
followed by salt exchange afforded the 2-AIT derivatives 8 as HCl
salts.
To assess the biofilm modulating capabilities of the newly
synthesized compounds, A. baumannii (ATCC 19606) was chosen
as the initial bacteria for these studies. The impetus for chosing A.
baumannii is that: (i) this strain forms robust biofilms on synthetic
surfaces such as glass and polystyrene, and such synthetic surfaces
are commonly used in production of medical devices;15 and (ii) A.
baumannii belongs to a cluster of Acinetobacter strains that are
more prone to multi-drug resistance.49 The compounds were first
tested for biofilm inhibition at 100 mM using crystal violet (CV)
reporter assay.50 Compounds 8g–j and 8n were found to inhibit
biofilm formation upwards of 94% at the initial concentration
of 100 mM. Each of these compounds were then subjected to
dose-response assays to determine IC50 values. Examining the
data revealed an abrupt drop in activity at lower concentrations,
indicating that the compounds may be acting as microbicides.
Accordingly, the five compounds were subjected to microdilution
susceptibility assays to find the minimum inhibitory concentration
(MIC).51 A 96-well microtiter plate with the top row having an
initial concentration of 400 mM was subjected to 12 two-fold serial
dilutions that resulted in a concentration range of 400–0.395 mM
which were used for MIC determination.52 After incubation at
Experimental
Chemistry
All chemicals and solvents used were purchased from commercial
suppliers and used without further purification. Silica gel was used
˚
for column chromatography and was performed with 60-A mesh
standard grade silica gel from Sorbtech. 1H and 13C NMR spectra
were performed using Varian Mercury 300 MHz and 400 MHz
spectrometers. Compounds8a–8n are amorphous solids. Chemical
shifts are reported in parts per million relative to CDCl3 (d 7.27),
◦
37 C for 16 h, the plates were visually inspected and the MICs
1
and DMSO-d6 (d 2.50) for H NMR and relative to CDCl3 (d
determined as the lowest concentrations were no growth are
observed. Compounds 8h, 8i and 8j were found to have the most
potent antibacterial activities from this library of 2-AITs (Table 1).
We then subjected these compounds to biofilm dispersion
assays. Here, biofilms are allowed to form first before treatment
with the compound. Initial biofilm dispersal activities at 200 mM
revealed four compounds (8g–j) possessed the highest biofilm
dispersal acitivities. Each of these compounds were then sub-
jected to dose-response assays to determine the EC50 (effective
concentration at which 50% of established biofilms are dispersed).
Compound 8i was found to be the most effective at dispersing
preformed A. baumannii biofilms with an EC50 of 44.70 mM
(Table 1). It is noteworthy to mention that compounds 8g–j
displayed better dispersal properties compared with the lead 2-
AIT (EC50 = 130 mM).
77.0), and DMSO-d6 (d 39.99) for 13C NMR. Abbreviations used
for 1H NMR splitting are as follows: s = singlet, bs = broad singlet,
d = doublet, dd = doublet of doublets, dt = doublet of triplets, t =
triplet, q = quartet, m = multiplet. High-resolution mass spectra
were obtained at the North Carolina State Mass Spectrometry
Laboratory for Biotechnology.
3-Bromo-2-methyl-prop-2-en-1-ol (5). Following literature
procedure,45 the title compound was obtained as colorless oil
1
(8.71 g, 65%). H NMR (300 MHz, CDCl3) d 6.23 (q, J = 1.38,
1H), 4.07 (s, 2H), 1.81 (d, J = 1.38, 3H). The proton NMR matched
the one that was reported.
3-Azido-1-bromo-2-methyl-propene (4). A solution of 5 (3.02 g,
20 mmol) in 40 mL anhydrous Et2O was cooled to 0 ◦C (ice bath).
To this solution was added slowly, via syringe, PBr3 (1.0 mL,
All compounds were then tested against MRSA (ATCC BAA-
44) biofilms in biofilm inhibition assays. As with A. baumannii,
the same four 2-AITs were most active against MRSA. Follow
up dose-response studies revealed IC50 values of 9.86, 8.55 and
4.50 mM for compounds 8g, 8i and 8j, respectively. Subsequent
analysis of both growth curves and colony counts indicated
◦
10 mmol). The colorless solution was stirred at 0 C for 30 min,
then at 25 ◦C for 4 h. At this time, the solution has turned yellow
orange and the starting material has been consumed (TLC). The
reaction was then quenched by slowly adding 70 mL aqueous
K2CO3 solution (1 g/50 mL water). The resulting mixture was
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extracted with 50 mL Et2O. The layers were then separated and
the organic layer dried over Na2SO4. Evaporation of the solvent
under reduced pressure gave the allylic bromide intermediate as
light yellow oil.
The reaction was stirred at 0 ◦C for 1 h then concentrated under
reduced pressure. The residue was taken up in CH2Cl2/hexanes
and the solvent removed under reduced pressure. This was done
3x before drying the acid chloride under high vacuum for 2 h.
The foregoing acid chloride was dissolved in 40 mL CH2Cl2
and the resulting solution was added dropwise, over 10 min,
to a cooled solution of CH2N2 (66 mmol, 2.77 g) in 200 mL
anhydrous Et2O. The yellow orange turbid reaction was stirred
at 0 ◦C for 1 h. After this time, 9 mL concentrated HBr was
added dropwise (evolution of gas was observed) and stirring was
continued for another 25 min. The reaction was quenched by
adding 100 mL saturated NaHCO3. The layers were separated and
the organic layer was washed with 50 mL saturated NaHCO3 (2¥),
50 mL brine, before drying over Na2SO4. Evaporation of solvent
under reduced pressure followed by column chromatography (10%
EtOAc/hexanes) furnished the title compound as colorless oil
(4.512 g, 92%). 1H NMR (400 MHz, CDCl3) d 3.88 (s, 2H), 2.66
(t, J = 7.41, 2H), 2.18 (dt, J = 6.97, 2.71, 2H), 1.93 (t, J = 2.71,
The foregoing allylic bromide intermediate (20 mmol, theo-
retical) was dissolved in 30 mL acetone. The resulting solution
was then added to a stirring mixture of NaN3 (6.5 g, 100 mmol)
in 50 mL acetone and the mixture was refluxed under nitrogen
atmosphere for 16 h. After this time, the mixture was allowed to
cool to room temperature then 100 mL Et2O and 80 mL water were
added. The layers were separated and the water layer extracted
with 40 mL Et2O. The combined Et2O layers were concentrated
under reduced pressure and the residual mixture was taken in
100 mL CH2Cl2. The resulting solution was washed with brine and
dried over Na2SO4. Removal of the solvent under reduced pressure
gave the title compound as light yellow orange oil (2.188 g, 62%,
1
2 steps). H NMR (300 MHz, CDCl3) d 6.28 (q, J = 1.39, 1H),
3.79 (s, 2H), 1.87 (d, J = 1.39, 3H); 13C NMR (75 MHz, CDCl3) d
136.4, 106.7, 56.8, 17.6. NMR data matched the one reported.53
1H), 1.68–1.57 (m, 2H), 1.56–1.49 (m, 2H), 1.45–1.39 (m, 2H); 13
C
NMR (100 MHz, CDCl3) d 201.9, 84.2, 68.4, 39.5, 34.2, 28.0, 27.9,
23.2, 18.1; HRMS (ESI) calcd for C9H13BrO (M + H) 217.0223,
found 217.0228.
1-Bromo-non-8-yn-2-one (6). A 200 mL ethylenediamine in a
dry, 1 liter round bottom flask was cooled to 0 ◦C (ice bath).
Sodium hydride (60% dispersion in mineral oil) (16 g, 397 mmol)
was then added portion-wise into the flask. The white mixture was
stirred under nitrogen atmosphere at 0 ◦C for 15 min, then at 25 ◦C
for 1h. After this time, the mixture has turned to brownish purple.
The mixture was then heated to 60 ◦C and was stirred at this
temperature for 1 h. The temperature was then reduced to 40 ◦C
before adding 3-octyn-1-ol (11.4 mL, 79.5 mmol). The mixture
was stirred at 40 ◦C for 1.5 h before heating to 65 ◦C. The ◦mixture
was stirred at this temperature for 2 h then cooled to 0 C. The
reaction was quenched by adding 150 mL water and 150 mL 1
N HCl. The resulting mixture was extracted with 100 mL ether
(3¥) and 100 mL EtOAc (2¥). The combined organic layers were
washed with 200 mL brine and dried over Na2SO4. After removal
of the solvent under reduced pressure, the isomerized alkyne was
purified via silica plug using 10% EtOAc/hexanes as solvent to
give a colorless oil (8.62 g, 86%). 1H NMR (400 MHz, CDCl3) d
3.65 (t, J = 6.50, 2H), 2.20 (dt, J = 7.08, 2.65, 1H), 1.95 (dt, J =
2.65, 0.68, 2H), 1.62–1.51 (m, 4H), 1.48–1.38 (m, 4H). The proton
NMR data matched the one reported from the literature.54
Jones reagent was prepared by adding 10 mL concentrated
H2SO4 to a cold mixture of CrO3 (3.1 g, 31.3 mmol) in 50 mL water.
The reagent was cooled to 0 ◦C then a solution of the isomerized
alkyne (3.3 g, 26.1 mmol) in 15 mL acetone was added dropwise via
separatory funnel for 10 min. The reaction mixture was stirred at
0–25 ◦C for 18 h before diluting with 50 mL EtOAc. The layers were
separated and the aqueous layer extracted with 20 mL EtOAc (3¥).
The combined organic layers were washed with brine and dried
over Na2SO4. The solvent was removed under reduced pressure
and the residue purified via column chromatography using 20%
EtOAc/hexanes to give the acid as light yellow oil (2.22 g, 64%).
1H NMR (300 MHz, CDCl3) d 10.70 (s, 1H), 2.35 (t, J = 7.48, 2H),
2.18 (dt, J = 6.82, 2.80, 2H), 1.93 (t, J = 2.80, 1H), 1.64 (quintet,
J = 2.80, 2H), 1.57–1.41 (m, 4H); HRMS (ESI) calcd for C8H12O2
(M + H) 141.0910, found 141.0906.
2-Amino-4-hept-6-ynyl-imidazole-1-carboxylic acid tert-butyl es-
ter (7). To a solution of alpha-bromo ketone 6 (1.128 g,
5.2 mmol) in 12 mL DMF was added Boc-guanidine (2.5 g,
15.6 mmol) and NaI (0.78 g, 5.2 mmol). The reaction mixture was
stirred for 3 days. The solvent was removed under reduced pressure
and to the residue was added 100 mL EtOAc and 30 mL water. The
layers were separated and the organic layer was washed with 30 mL
water (3¥). The combined aqueous washes were back extracted
with 30 mL EtOAc. The combined organic extracts were washed
with brine and dried over Na2SO4. Evaporation of the solvent
under reduced pressure followed by column chromatography with
5% MeOH/CH2Cl2 gave the title compound as light yellow solid
(0.619 g, 43%). NMR (300 MHz, CDCl3) d 6.51(s, 1H), 5.62 (s,
1H), 2.37 (t, J = 7.70, 2H), 2.20 (dt, J = 6.88, 2.76, 2H), 1.94 (t, J =
2.76), 1.65–1.60 (m, 2H), 1.56 (s, 9H), 1.54–1.42 (m, 4H); HRMS
(ESI) calcd for C15H23N3O2 (M + H) 278.1863, found 278.1856.
2-[Bis(tert-butoxycarbonyl)amino]-4-(5-{1-[2-bromo-1-methyl-
vinyl]-1H-1,2,3-triazol-4-yl}pentyl-1Himidazole-1-carboxylic acid
methyl ester (2). To a solution of mono Boc-protected 2-AI 7
(0.750 g, 2.71 mmol) in 100 mL anhydrous THF was added DMAP
(0.033 g, 0.271 mmol) and Boc anhydride (1.77 g, 8.13 mmol). The
reaction was stirred under N2 atmosphere at 25 ◦C for 48 h. After
this time, TLC has indicated consumption of starting material.
The reaction was diluted with 200 mL EtOAc and was washed
with 50 mL 10% citric acid solution (2x). The organic layer was
then dried over Na2SO4, concentrated under reduced pressure and
dried under high vacuum to give the crude tri Boc-protected
compound 3 which was carried over to the next step without
further purification. Compound 3 (0.477 g, 1 mmol) and allylic
azide 4 (0.180 g, 1 mmol) were dissolved in 5 mL CH2Cl2, 5 mL
tert-BuOH and 5 mL water. The mixture was stirred vigorously
then CuSO4 (0.024 g, 0.15 mmol) was added followed by sodium
ascorbate (0.059 g, 0.30 mmol). The reaction mixture was stirred
at high speed for 5 h. After this time TLC indicated persistence
of compound 3. Additional compound 4 (0.090 g, 0.5 mmol) was
introduced and the reaction stirred for another 12 h. TLC after
A solution of the acid-alkyne (3.2 g, 22.9 mmol) in anhydrous
CH2Cl2 was cooled to 0 ◦C. Five drops of DMF were then added
followed by dropwise addition of oxalyl chloride (6 mL, 71 mmol).
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this time indicated completion of the reaction. Ethyl acetate (50
mL) and water (50 mL) were added to the reaction mixture and
the layers were separated. The organic layer was washed with
20 mL brine and dried over Na2SO4. Evaporation of the solvent
under reduced pressure, followed by purification via column
chromatography afforded the title compound as thick viscous light
yellow oil (0.481 g, 74%). 1H NMR (300 MHz, CDCl3) d 7.25 (s,
1H), 7.06 (s, 1H), 6.27 (q, J = 1.2, 1H), 4.90 (s, 2H), 2.68 (t, J =
7.8, 2H), 2.49 (t, J = 7.6, 2H), 1.73 (d, J = 1.2, 3H), 1.71–160 (m,
6H), 1.55 (s, 9H), 1.39 (s, 18H); 13C NMR (75 MHz, CDCl3) d
149.7, 149.1, 146.7, 140.5, 137.8, 136.5, 120.6, 114.1, 108.1, 85.7,
83.5, 55.9, 29.4, 28.8, 28.6, 28.4, 28.09, 28.07, 25.8, 17.4; HRMS
(ESI) calcd for C29H45BrN6O6 (M + H) 653.2657, found 653.2638.
4-(3-{4-[5-(2-Amino-1H-imidazol-4-yl)-pentyl]-[1,2,3]triazol-1-
yl}-2-methyl-propenyl)-benzamide hydrochloride (8b). Following
the procedure for 8a, tri Boc-protected compound 2 was coupled
with 4-aminocarbonylphenylboronic acid to furnish the protected
precursor as yellow oil (0.231 g, 64%).1H NMR (400 MHz, CDCl3)
d 7.81 (d, J = 8.1, 2H), 7.344 (d, J = 8.1, 2H), 7.34 (s, 1H), 7.08 (s,
1H), 6.46 (s, 1H), 6.20 (bs, 1H), 5.67 (bs, 1H), 5.03, (s, 2H), 2.73 (t,
J = 7.7, 2H), 2.51 (t, J = 7.4, 2H), 1.83 (s, 3H), 1.76–1.64 (m, 6H),
1.57 (s, 9H), 1.41 (s, 18H); 13C NMR (75 MHz, CDCl3) d 168.8,
149.5, 148.8, 146.5, 140.3, 140.2, 137.5, 134.3, 131.8, 129.1, 128.4,
127.4, 120.6, 113.9, 85.5, 83.3, 58.1, 29.2, 28.6, 28.4, 27.9, 27.86,
27.8, 25.6, 15.8.
After deprotection and salt exchange, the title compound was
obtained as viscous yellow oil (0.142 g, 99%). 1H NMR (400 MHz,
DMSO-d6) d 12.15 (s, 1H), 11.67 (s, 1H), 7.91 (s, 1H), 7.87 (d, J =
8.1, 2H), 7.35 (d, J = 8.1, 2H), 8.01 (bs, 2H), 6.54 (s, 1H), 6.48 (s,
1H), 5.07 (s, 1H), 2.63 (t, J = 7.6, 2H), 2.39 (d, J = 7.4, 2H),1.75 (s,
3H), 1.62 (quintet, J = 7.0, 2H), 1.55 (quintet, J = 7.4, 2H), 1.35–
1.27 (m, 2H); 13C NMR (100 MHz, DMSO-d6) d 168.2, 147.6,
147.4, 140.0, 135.2, 133.2, 129.2, 128.24, 128.2, 127.4, 1, 123.0,
109.1, 57.8, 29.2, 28.5, 28.0, 25.5, 24.6, 16.3; HRMS (ESI) calcd
for C21H28N7O2 (M + H)+ 394.235, found 394.2357.
General Procedure for Suzuki-Miyaura Couplings and Deprotec-
tion. 4-(3-{4-[5-(2-Amino-1H-imidazol-4-yl)-pentyl]-[1,2,3]triazol-
1-yl}-2-methyl-propenyl)-benzoic acid methyl ester hydrochloride
(8a). A 20 mL vial was charged with vinyl bromide 2 (0.314 g,
0.480 mmol) and 4-methoxycarbonylphenylboronic acid (0.112 g,
0.62 mmol). The vial was flushed with nitrogen and 5 mL THF
was added. PdCl2(PPh3)2 (0.017 g, 0.024 mmol) and 0.72 mL 2 M
Na2CO3 (1.44 mmol) were then added and the vial sealed with
rubber septum. The reaction mixture was sonicated for 10 s and
then refluxed for 2 h. TLC after this time indicated consumption
of starting material. The reaction mixture was transferred into
a separatory funnel and 30 mL EtOAc was added. The reaction
was washed with 10 mL water and the organic layer was dried
over Na2SO4. Evaporation of the solvent under reduced pressure
followed by column chromatography (40% EtOAc/hexanes) gave
4-(3-{4-[5-(2-Amino-1H-imidazol-4-yl)-pentyl]-[1,2,3]triazol-1-
yl}-2-methyl-propenyl)-benzaldehyde hydrochloride (8c). Follow-
ing the procedure for 8a, tri Boc-protected compound 2 was
coupled with 4-formylphenylboronic acid to furnish the protected
precursor as yellow oil(0.219 g, 60%). 1HNMR(400MHz, CDCl3)
d 9.95 (s, 1H), 7.82 (d, J = 8.0, 2H), 7.38 (d, J = 8.3, 2H), 7.33
(s, 1H), 7.04 (s, 1H), 6.43 (s, 1H), 5.00 (s, 2H), 2.68 (t, J = 7.8,
2H), 2.48 (t, J = 7.5, 2H), 1.80 (s, 3H), 1.71–1.58 (m, 6H), 1.53 (s,
9H), 1.36 (s, 18H); 13C NMR (100 MHz, CDCl3) d 191.5, 149.3,
148.7, 146.3, 142.6, 140.1, 137.4, 1335.3, 134.8, 129.5, 129.3, 128.1,
120.6, 113.7, 85.4, 83.1, 57.9, 29.0, 28.4, 28.3, 27.8, 27.72, 27.69,
25.2, 15.8.
1
the protected precursor as yellow oil (0.184 g, 54%). H NMR
(400 MHz, CDCl3) d 8.01 (d, J = 8.4, 2H), 7.33 (s, 1H), 7.3 (d, J =
8.4, 2H), 7.08 (s, 1H), 6.47 (s, 1H), 5.03 (s, 2H), 3.92 (s, 3H), 2.92
(t, J = 7.6, 2H) 2.52 (t, J = 7.4, 2H), 1.83 (d, J = 1.3, 3H), 1.75–1.64
(m, 6H), 1.57 (s, 9H), 1.41 (s, 18H); 13C NMR (75 MHz, CDCl3) d
167.0, 149.8, 149.1, 146.7, 141.3, 140.5, 137.8, 134.8, 129.8, 129.1,
129.0, 128.8, 120.7, 114.1, 85.7, 83.5, 58.4, 52.3, 29.4, 28.9, 28.7,
28.4, 28.2, 28.1, 28.0, 27.9, 25.9, 16.0.
After deprotection and salt exchange, the title compound was
1
obtained as viscous yellow brown oil (0.133 g, 99%). H NMR
(400 MHz, DMSO-d6) d 12.21 (s, 1H), 11.73 (s, 1H), 7.92 (s, 1H),
7.29–7.72 (m, 4H), 6.53 (s, 1H), 6.45 (s, 1H), 5.05 (s, 2H), 2.63 (t,
J = 7.6, 2H), 2.38 (t, J = 7.4, 2H), 1.72 (s, 3H), 1.61 (quintet, J =
7.3, 2H), 1.54 (quintet, J = 7.6, 2H), 1.33–1.27 (m, 2H); 13C NMR
(100 MHz, DMSO-d6) d 147.5, 1474, 142.0, 133.1, 129.4, 129.2,
129.0, 128.2, 127.4, 127.1, 123.0, 109.1, 58.1, 29.2, 28.5, 28.0, 25.4,
24.6, 19.2; HRMS (ESI) calcd for C21H26N6O (M + H) 379.2241,
found 379.2245
The protected cross-coupling product was taken up in 4.2 mL
CH2Cl2 and then the solution was cooled to 0 ◦C. TFA (1.8 mL)
was then added dropwise and the reaction was stirred from 0–25 ◦C
for 18 h. After this time the solvent was evaporated under reduced
pressure and the residue was taken up in CH2Cl2/hexanes and was
concentrated under reduced pressure. This was done three times,
after which the residue was dried under vacuum for 2 h. The
foregoing TFA salt was removed from vacuum then 5 mL MeOH
and a drop of concentrated HCl were added. The solution was
concentrated under reduced pressure and then redissolved in 5 mL
MeOH. The resulting solution was passed through a Whatman 0.2
micron polypropylene filter, concentrated under reduced pressure
thendried under high vacuumto give the title compound as viscous
yellow oil (0.110 g, 95%). 1H NMR (400 MHz, DMSO-d6) d 12.18
(s, 1H), 11.69 (s, 1H), 7.90 (d, J = 8.1, 2H), 7.91, (s, 1H), 7.40 (d,
J = 8.1, 2H), 6.83 (bs, 2H),6.50 (s, 1H), 6.47 (s, 1H), 5.06 (s, 2H),
3.81 (s, 3H), 2.60 (t, J = 7.6, 2H), 2.36 (t, J = 7.4, 2H), 1.73 (s,
3H), 1.59 (quintet, J = 7.2, 2H), 1.52 (quintet, J = 7.4, 2H), 1.31–
1.24 (m, 2H); 13C NMR (100 MHz, DMSO-d6) d 166.6, 147.6,
147.4, 142.0, 136.3, 129.9, 129.7, 128.5, 127.9, 127.4, 123.1, 109.1,
57.8, 52.8, 29.2, 28.5, 28.0, 25.4, 24.5, 16.3; HRMS (ESI) calcd for
C22H29N6O2 (M + H)+ 409.2347, found 409.2346.
4-(5-{1-[2-Methyl-3-(3-nitro-phenyl)-allyl]-1H-[1,2,3]triazol-4-
yl}-pentyl)-1H-imidazol-2-ylamine hydrocholoride (8d). Follow-
ing the procedure for 8a, tri Boc-protected compound 2 was
coupled with 3-nitrophenylboronic acid to furnish the protected
precursor as yellow oil(0.157 g, 58%). 1HNMR(300MHz, CDCl3)
d 8.14–8.11 (m, 2H) 7.59–7.51 (m, 2H), 7.35 (s, 1H), 7.09 (s, 1H),
6.45 (s, 1H), 5.05 (s, 2H), 2.74 (t, J = 7.8, 2H), 2.52 (t, J = 7.6, 2H),
1.85 (d, J = 1.3, 3H), 1.77–1.65 (m, 6H), 1.58 (s, 9H), 1.42 (s, 18H);
13C NMR (100 MHz, CDCl3) d 149.5, 148.9, 148.2, 146.5, 140.3,
138.1, 137.5, 135.6, 134.8, 129.3, 126.9, 123.6, 122.0, 120.7, 113.9,
85.5, 83.3, 57.8, 29.2, 28.6, 28.4, 27.9, 27.88, 27.85, 25.6, 15.7
After deprotection and salt exchange, the title compound was
obtained as viscous yellow oil (0.97 g, 99%). 1H NMR (400 MHz,
DMSO-d6) d 12.20 (s, 1H), 11.71 (s, 1H), 8.11 (d, J = 8.0, 1H),
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8.07 (s, 1H), 7.98 (s, 1H), 7.74 (d, J = 7.8, 1H), 7.66 (t, J = 8.0,
1H), 7.25 (bs, 2H), 6.57 (s, 1H), 6.52 (s, 1H), 5.11 (s, 2H), 2.64
(t, J = 7.4, 2H), 2.39 (t, J = 7.2, 2H), 1.75 (s, 3H), 1.62 (quintet,
J = 7.3, 2H), 1.55 (quintet, J = 7.4, 2H), 1.34–1.29 (m, 2H); 13C
NMR (100 MHz, DMSO-d6) d 148.5, 147.5, 147.4, 138.8, 136.6,
135.9, 130.6, 127.4, 127.0, 123.7, 123.2, 122.4, 109.0, 57.7, 29.1,
28.5, 28.0, 25.4, 24.5, 16.1; HRMS (ESI) calcd for C20H26N7O2
(M + H)+ 396.2142, found 396.2139.
8.5, 1.7, 1H), 7.37 (s, 1H), 7.08 (s, 1H), 6.64 (s, 1H), 5.06 (s, 2H),
2.73 (t, J = 7.9, 2H), 2.52 (t, J = 7.2, 2H), 1.88 (d, J = 1.3, 3H),
1.77–1.63 (m, 6H), 1.57 (s, 6H), 1.41 (s, 18H); 13C NMR (100 MHz,
CDCl3) d 149.7, 149.0, 146.7, 140.5, 137.8, 134.2, 133.4, 132.9,
132.6, 130.0, 128.2, 128.13, 128.07, 127.8, 127.2, 126.5, 126.3,
120.7, 114.1, 85.7, 83.5, 58.8, 29.5, 28.9, 28.7, 28.2, 28.11, 28.09,
25.9, 16.0.
After deprotection and salt exchange, the title compound was
obtained as viscous yellow oil (0.109 g, 99%). 1H NMR (400 MHz,
DMSO-d6) d 12.18 (s, 1H), 11.70 (s, 1H), 7.96 (s, 1H), 7.92–7.88
(m, 3H), 7.81 (s, 1H), 7.50–7.48 (m, 2H), 7.44, (d, J = 8.4, 1H),
7.33(bs, 2H), 6.62 (s, 1H), 6.53 (s, 1H), 5.12 (s, 2H), 2.654 (t, J =
7.4, 2H), 2.39 (t, J = 7.4, 2H), 1.82 (s, 3H), 1.63 (quintet, J = 7.0,
2H), 1.55 (quintet, J = 7.2, 2H), 1.35–1.30 (m, 2H); 13C NMR
(100 MHz, DMSO-d6) d 147.6, 147.4, 134.7, 134.2, 133.5, 132.5,
129.0, 128.6, 128.4, 128.14, 128.1, 127.7, 127.4, 127.0, 126.8, 123.1,
109.1, 58.1, 29.2, 28.5, 28.0, 25.4, 24.6, 16.4; HRMS (ESI) calcd
for C24H29N6 (M + H)+ 401.2448, found 401.2447.
1-[4-(3-{4-[5-(2-Amino-1H-imidazol-4-yl)-pentyl]-[1,2,3]triazol-
1-yl}-2-methyl-propenyl)-phenyl]-ethanone hydrochloride (8e).
Following the procedure for 8a, tri Boc-protected compound
2 was coupled with 4-acetylphenylboronic acid to furnish the
protected precursor as yellow oil (0.070 g, 27%). 1H NMR
(400 MHz, CDCl3) d 7.95 (d, J = 8.4, 2H), 7.36 (d, J = 8.1, 2H),
7.34 (s, 1H), 7.08 (s, 1H), 6.47 (s, 1H), 5.03 (s, 2H), 2.73 (t, J = 7.7,
2H), 2.52 (t, J = 7.0, 2H), 1.84 (s, 3H), 1.76–1.64 (m, 6H), 1.57 (s,
9H), 1.42 (s, 18H);13C NMR (100 MHz, CDCl3) d 197.5, 149.5,
148.8, 146.5, 141.3, 140.3, 137.5, 135.6, 134.7, 129.0, 128.4, 128.3,
120.6, 113.9, 85.5, 83.3, 58.2, 29.2, 28.6, 28.4, 27.9, 27.87, 27.8,
26.6, 25.6, 15.8.
After deprotection and salt exchange, the title compound was
obtained as viscous yellow oil (0.043 g, 99%). 1H NMR (400 MHz,
DMSO-d6) d 12.11 (s, 1H), 11.64 (s, 1H), 7.95 (d, J = 8.2, 2H),
7.92 (s, 1H), 7.42 (d, J = 8.2, 2H), 7.31 (bs, 2H), 6.54 (s, 1H), 6.50
(s, 1H), 5.01 (s, 2H), 2.63 (t, J = 7.6, 2H), 2.34 (t, J = 7.4, 2H),
1.76 (s, 3H), 1.62 (quintet, J = 7.6, 2H), 1.55 (quintet, J = 7.8,
2H), 1.35–1.29 (m, 2H); 13C NMR (100 MHz, DMSO-d6) d 198.0,
147.7, 147.4, 141.9, 136.3, 135.8, 129.6, 129.0, 127.9, 127.5, 123.0,
109.1, 57.7, 29.2, 28.5, 28.0, 27.4, 25.5, 24.6, 16.4; HRMS (ESI)
calcd for C22H29N6O (M + H)+ 393.2397, found 393.2401.
4-(5-{1-[3-(4-Butyl-phenyl)-2-methyl-allyl]-1H-[1,2,3] triazol-4-
yl}-pentyl)-1H-imidazol-2-ylamine hydrochloride 8h. Following
the procedure for 8a, tri Boc-protected compound 2 was coupled
with 4-butylphenylboronic acid to furnish the protected precursor
as yellow oil (0.062 g, 60%). 1H NMR (400 MHz, CDCl3) d 7.31
(s, 1H), 7.22–7.16 (m, 4H), 7.08 (s, 1H), 6.49 (s, 1H), 5.00 (s, 2H),
2.72 (t, J = 8.0, 2H), 2.61 (t, J = 7.6, 2H), 2.52 (t, J = 7.4, 2H),
1.81 (d, J = 1.2, 3H), 1.75–1.66 (m, 6H), 1.58 (s, 9H), 1.42 (s, 18H),
1.40–1.33 (m, 4 H), 0.93 (t, J = 7.3, 3H);13C NMR (100 MHz,
CDCl3) d 149.5, 148.7, 146.5, 142.1, 140.3, 137.5, 133.7, 131.4,
129.8, 128.8, 128.3, 120.3, 113.9, 85.4, 83.2, 58.7, 35.3, 33.5, 29.2,
28.6, 28.4, 27.9, 27.86, 27.8, 25.7, 22.3, 15.7, 13.9.
After deprotection and salt exchange, the title compound was
obtained as viscous yellow oil (0.101 g, 99%). 1H NMR (400 MHz,
DMSO-d6) d 12.27 (s, 1H), 11.76 (s, 1H), 9.60 (bs, 2H), 8.0, 7.18–
7.13 (m, 4H), 6.51 (s, 1H), 6.46 (s, 1H), 2.63 (t, J = 7.4, 2H), 2.52
(t, J = 7.5, 2H), 2.57 (t, J = 7.2, 2H), 1.71 (s, 3H), 1.61 (quintet,
J = 7.3, 2H), 1.57–1.46 (m, 4H), 1.31–1.22 (m, 4H), 0.84 (t, J =
7.3, 3H); 13C NMR (100 MHz, DMSO-d6) d 147.3, 146.9, 141.7,
134.2, 132.2, 129.2, 129.17, 128.7, 127.1, 123.3, 108.8, 58.3, 35.0,
33.5, 28.3, 28.2, 27.7, 25.0, 24.3, 22.2, 16.0, 14.2; HRMS (ESI)
calcd for C24H35N6 (M + H)+ 407.2918, found 407.2918.
4-{5-[1-(3-Furan-3-yl-2-methyl-allyl)-1H-[1,2,3]triazol-4-yl]-
pentyl}-1H-imidazol-2-ylamine hydrochloride (8f). Following the
procedure for 8a, tri Boc-protected compound 2 was coupled with
3-furanylboronic acid to furnish the protected precursor as yellow
1
oil (0.139 g, 75%). H NMR (300 MHz, CDCl3) d 7.49 (s, 1H),
7.42 (t, J = 1.7, 1H), 7.29 (s, 1H), 7.08 (s, 1H), 6.48 (dd, J = 1.7,
0.8, 1H), 6.26 (s 1H), 4.98 (s, 2H), 2.71 (t, J = 8.0, 2H), 2.52 (t,
J = 7.5, 2H), 1.81 (d, J = 1.3, 3H), 1.75–1.62 (m, 6H), 1.57 (s, 6H),
1.41 (s, 18H); 13C NMR (100 MHz, CDCl3) d 149.5, 148.8, 146.5,
143.0, 141.5, 140.3, 137.5, 131.0, 121.8, 120.3, 120.1, 113.9, 110.6,
85.5, 83.3, 58.5, 29.2, 28.6, 28.4, 27.9, 27.87, 27.86, 25.7, 16.0.
After deprotection and salt exchange, the title compound was
obtained as viscous yellow oil (0.081 g, 99%). 1H NMR (400 MHz,
DMSO-d6) d 12.15 (s, 1H), 11.68 (s, 1H), 7.87, (s, 1H), 7.78 (s, 1H),
7.68(s, 1H), 7.34 (bs, 2H), 6.60 (s, 1H), 6.54(s, 1H), 6.23 (s, 1H),
5.00 (s, 1H), 2.62 (t, J = 7.6, 2H), 2.38 (t J = 7.4, 2H), 1.71 (s, 3H),
4-(5-{1-[3-(4-Butoxy-phenyl)-2-methyl-allyl]-[1,2,3] triazol-4-
yl}-pentyl)-1H-imidazol-2-ylamine hydrochloride (8i). Following
the procedure for 8a, tri Boc-protected compound 2 was coupled
with 4-butoxyphenylboronic acid to furnish the protected
precursor as yellow oil (0.091 g, 41%). 1H NMR (400 MHz,
CDCl3) d 7.32 (s, 1H), 7.27–7.23 (m, 2H), 7.08 (s, 1H), 6.85 (d,
J = 7.1, 1H), 6.81–6.80 (m, 2H), 6.46 (s, 1H), 5.00 (s, 2H), 3.96(t,
J = 6.5, 2), 2.72 (t, J = 7.8, 2H), 2.52 (t, J = 7.6, 2H), 1.81 (d, J =
1.3, 3H), 1.79–1.64 (m, 6H), 1.58 (s, 9H), 1.52–1.47 (m, 4H), 1.41
(s, 18H), 0.98 (t, J = 7.5, 3H); 13C NMR (100 MHz, CDCl3) d
159.0, 149.5, 148.8, 140.3, 137.7, 132.4, 129.7, 129.2, 121.2, 120.4,
115.1, 113.9, 113.2, 85.5, 83.3, 67.6, 58.5, 31.3, 29.2, 28.6, 28.4,
27.9, 27.86, 27.8, 25.7, 19.2, 15.7, 13.8.
1.61 (quintet, J = 8.0, 2H), 1.56–1.50 (m, 2H), 1.32–1.26 (2H); 13
C
NMR (100 MHz, DMSO-d6) d 147.5, 147.4, 144.1, 142.4, 132.2,
127.4, 122.9, 122.4, 119.6, 111.5, 109.1, 58.0, 29.2, 28.5, 28.0, 25.4,
24.5, 16.7; HRMS (ESI) calcd for C18H25N6O (M + H)+ 341.2084,
found 341.2093.
4-{5-[1-(2-Methyl-3-naphthalen-1-yl-allyl)-1H-[1,2,3]triazol-4-
yl]-pentyl}-1H-imidazol-2-ylamine hydrochloride (8g). Follow-
ing the procedure for 8a, tri Boc-protected compound 2 was
coupled with 1-napthylboronic acid to furnish the protected
precursor as yellow oil (0.177g, 82%). 1H NMR (300MHz, CDCl3)
d 7.83–7.80 (m, 3H), 7.73 (s, 1H), 7.49–7.46 (m, 2H), 7.40 (dd, J =
After deprotection and salt exchange, the title compound was
obtained as viscous yellow oil (0.057 g, 99%). 1H NMR (400 MHz,
DMSO-d6) d 12.24 (s, 1H), 11.74 (s, 1H), 7.97 (s, 1H), 7.26–7.22
(m, 2H), 6.92 (bs, 2H), 6.84–6.79 (m, 2H), 6,52 (s, 1H), 6.44 (s,
1H), 5.05 (s, 2H), 3.94 (t, J = 6.2, 2H), 2.63 (t, J = 7.2, 2H), 2.38
3046 | Org. Biomol. Chem., 2011, 9, 3041–3049
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(t, J = 7.0, 2H), 1.72 (s, 3H), 1.69–1.60 (m, 4H), 1.57–1.51 (m,
2H), 1.40 (q, J = 7.2, 2H), 1.34–1.26 (m, 2H), 0.90 (t, J = 7.2, 3H);
13C NMR (100 MHz, DMSO-d6) d 147.4, 147.3, 133.6, 130.0,
129.2, 127.3, 123.3, 121.7, 115.4, 113.8, 109.0, 67.7, 58.2, 31.4,
29.11, 28.5, 28.0, 25.3, 24.6, 19.4, 16.3, 14.4; HRMS (ESI) calcd
for C24H35N6O (M + H)+ 423.2867, found 423.2868.
pound 2 was coupled with 4-{[N-(tert-butoxycarbonyl)amino]-
methyl}phenylboronic acid to furnish the protected precursor as
yellow oil (0.163 g, 81%). 1H NMR (400 MHz, CDCl3) d 7.32 (s,
1H), 7.29–7.23 (m, 4H), 7.08 (s, 1H), 6.41 (s, 1H), 5.00 (s, 2H),
4.87 (bs, 1H), 4.32 (d, J = 5.6, 2H), 2.72 (t, J = 7.6, 2H), 2.52 (t,
J = 7.4, 2H), 1.80 (d, J = 1.2, 3H), 1.71–1.64 (m, 6H), 1.59 (s, 9H),
1.49 (s, 9H), 1.41 (s, 18H); 13C NMR (100 MHz, CDCl3) d 149.5,
148.8, 146.5, 140.3, 137.9, 137.5, 135.5, 132.3, 129.3, 129.1, 127.4,
127.1, 120.4, 113.9, 85,5, 83.3, 83.2, 58.5, 44.3, 29.7, 29.2, 28.7,
28.4, 27.9, 27.8, 25.7, 15.6, 14.2.
After deprotection and salt exchange, the title compound was
obtained as viscous yellow oil (0.099 g, 99%). 1H NMR (400 MHz,
DMSO-d6) d 12.17 (s, 1H), 11.69 (s1H), 8.53 (bs, 2H), 7.91 (s, 1H),
7.47 (d, J = 8.1, 2H), 7.31 (d, J = 8.1, 2H), 6.53 (s, 1H), 6.46 (s,
1H), 5.06 (s, 2H), 4.02 (d, J = 6.0, 2H), 2.63 (t, J = 7.5, 2H), 2.39
(t, J = 7.5, 2H), 1.73 (s, 3H), 1.62 (quintet, J = 7.6, 2H), 1.55
(quintet, J = 7.4, 2H), 1.34–1.29 (m, 2H); 13C NMR (100 MHz,
DMSO-d6) d 147.3, 147.1, 136.9, 134.0, 133.1, 129.3, 129.2, 128.0,
127.1, 122.7, 108.8, 57.5, 42.2, 28.9, 28.2, 27.7, 25.2, 24.3, 15.9;
HRMS (ESI) calcd for C21H30N7 (M + H)+ 380.2557, found
380.2558.
4-(5-{1-[3-(2-Bromo-4-butoxy-6-fluoro-phenyl)-2-methyl-allyl]-
1H-[1,2,3]triazol-4-yl}-pentyl)-1H-imidazol-2-ylamine
hydro-
chloride (8j). Following the procedure for 8a, tri Boc-
protected compound 2 was coupled with 2-bromo-4-butoxy-5-
fluorophenylboronic acid to furnish the protected precursor as
yellow oil (0.068 g, 24%). 1H NMR (400 MHz, CDCl3) d 7.39 (s,
1H), 7.26 (dd, J = 8.8, 1.7, 1H), 7.07 (s, 1H), 6.80 (t, J = 8.8, 1H),
6.16 (s, 1H), 5.05 (s, 2H), 4.00 (t, J = 6.7, 2H), 2.70 (t, J = 7.6,
2H), 2.50 (t, J = 7.3, 2H), 1.82–1.75 (m, 2H), 1.73–1.56 (m, 4H),
1.56 (s, 9H), 1.53 (s, 3H), 1.51–1.43 (4H), 1.40 (s, 18H), 0.96 (t,
J = 7.4, 3H);13C NMR (100 MHz, CDCl3) d 151.2, 149.7, 149.1,
148.7, 147.2, 146.7, 140.5, 137.8, 137.4, 127.5, 126.0, 123.2, 120.6,
114.8, 114.1, 85.7, 83.5, 69.6, 57.4, 31.3, 29.5, 28.8, 28.6, 28.2,
28.1, 25.9, 19.3, 16.4, 16.36, 14.0.
After deprotection and salt exchange, the title compound was
obtained as viscous yellow oil (0.046 g, 99%). 1H NMR (400 MHz,
DMSO-d6) d 12.17 (s, 1H), 11.70 (s, 1H), 7.85 (s, 1H), 7.4 (d, J =
8.5, 1H), 7.31 (bs, 2H), 7.09 (t, J = 8.5, 1H), 6.52 (s, 1H), 6.05
(s, 1H), 5.14, (s, 2H), 4.02 (t, J = 6.0, 2H), 2.64 (t, J = 7.2, 2H),
2.38 (t, J = 7.0, 2H), 1.68 (quintet, J = 7.1, 2H), 1.65–1.53 (m,
4H), 1.42 (s, 3H), 1.41–1.38 (m, 2H), 1.35–1.26 (m, 2H), 0.90 (t,
J = 7.2, 3H); 13C NMR (100 MHz, DMSO-d6) d 147.6, 147.4,
147.1, 138.7, 128.3, 128.7, 127.4, 126.0, 122.9, 121.5, 115.8, 113.9,
109.1, 69.3, 56.3, 31.2, 29.2, 28.5, 28.0, 25.4, 24.6, 19.3, 16.8, 14.3;
HRMS (ESI) calcd for C24H33BrFN6O (M + H)+ 519.1878, found
519.1881.
4-(5-{1-[3-(4-Heptyl-phenyl)-2-methyl-allyl]-1H-[1,2,3]triazol-
4-yl}-pentyl)-1H-imidazol-2-ylamine hydrochloride (8m). Fol-
lowing the procedure for 8a, tri Boc-protected compound 2 was
coupled with 4-heptylphenylboronic acid to furnish the protected
precursor as yellow oil(0.058 g, 55%). 1HNMR(400MHz, CDCl3)
d 7.32 (s, 1H), 7.22–7.14 (m, 4H), 7.08 (s, 1H), 6.48 (s, 1H), 5.00 (s,
2H), 2.71 (t, J = 7.7, 2H), 2.59 (t, J = 7.8, 2H), 2.51 (t, J = 7.5, 2H),
1.81 (d, J = 1.3, 3H), 1.75–1.60 (m, 6H), 1.57 (s, 9H), 1.41 (s, 18H),
1.36–1.19 (m, 10H), 0.87 (t, J = 7.0, 3H); 13C NMR (100 MHz,
CDCl3) d 149.7, 148.9, 146.7, 142.4, 140.5, 137.8, 133.9, 131.5,
130.2, 129.1, 128.6, 120.7, 114.1, 85.8, 83.6, 59.0, 35.9, 32.0, 31.6,
29.5, 29.4, 29.38, 28.8, 28.6, 28.1, 28.08, 28.07, 25.8, 22.9, 15.9,
14.3.
After deprotection and salt exchange, the title compound was
obtained as viscous yellow oil (0.037 g, 99%). 1H NMR (400 MHz,
DMSO-d6) d 12.11 (s, 1H), 11.64 (s1H), 7.88 (s, 1H), 7.31 (bs, 2H),
7.20–7.16 (m, 4H), 6.54 (s, 1H), 6.43 (s, 1H), 5.03 (s, 2H), 2.62 (t,
J = 7.3, 2H), 2.56–2.52 (m, 2H), 2.39 (t, J = 6.9, 2H), 1.72 (s, 3H),
1.61 (quintet, J = 7.4, 2H), 1.58–1.51 (m, 4H), 1.32–1.23 (m, 8H),
0.84 (t, J = 6.7,3H); 13C NMR (100 MHz, DMSO-d6) d 147.6,
147.4, 141.8, 134.5, 132.8, 129.4, 129.0, 128.9, 127.5, 122.8, 109.1,
58.1, 35.5, 31.9, 31.6, 29.3, 29.23, 29.2, 28.5, 28.0, 25.5, 24.6, 22.8,
16.2, 14.6; HRMS (ESI) calcd for C27H41N6 (M + H)+ 449.3387,
found 449.3398.
4-(5-{1-[3-(2,4-Dimethoxy-pyrimidin-5-yl)-2-methyl-allyl]-1H-
[1,2,3]triazol-4-yl}-pentyl)-1H-imidazol-2 -ylamine hydrochloride
(8k). Following the procedure for 8a, tri Boc-protected com-
pound 2 was coupled with 2,4-dimethoxy-5-pyrimidinylboronic
acid to furnish the protected precursor as yellow oil (0.148 g,
1
74%). H NMR (400 MHz, CDCl3) d 7.39 (s, 1H), 8.07 (s, 1H),
7.28 (s, 1H), 7.01 (s, 1H), 6.22 (s, 1H), 4.95 (2, H), 3.93 (s, 3H),
3.927 (s, 3H), 2.65 (t, J = 7.9, 2H), 2.45 (t, J = 7.5, 2H), 1.67
(d, J = 1.3, 3H), 1.65–157 (m, 6H), 1.50 (s, 9H), 1.34 (s, 18H);
13C NMR (100 MHz, CDCl3) d 168.4, 164.1, 157.2, 149.3, 148.5,
146.3, 140.1, 137.4, 133.7, 120.3, 120.0, 113.7, 111.1, 85.3, 83.1,
57.8, 54.6, 53.9, 29.0, 28.4, 28.2, 28.0, 27.7, 27.65, 25.5, 15.6.
After deprotection and salt exchange, the title compound was
obtained as viscous yellow oil (0.092 g, 99%). 1H NMR (400 MHz,
DMSO-d6) d 12.13 (s, 1H), 11.66 (s1H), 8.23 (s, 1H), 7.88 (s, 1H),
7.31 (bs, 2H), 6.34 (s, 1H), 6.17 (s, 1H), 5.07 (s, 2H), 3.92 (s, 3H),
3.91 (s, 3H), 2.63 (t, J = 7.2, 2H), 2.39 (t, J = 7.2, 2H), 1.67 (s,
3H), 1.65–1.58 (m, 2H), 1.55 (quintet, J = 7.4, 2H), 1.34–1.27
(m, 2H); 13C NMR (100 MHz, DMSO-d6) d 159.2, 158.8, 157.6,
147.5, 147.4, 135.6, 127.5, 123.1, 119.1, 111.8, 109.1, 57.3, 55.4,
54.8, 29.2, 28.5, 28.0, 25.4, 24.5, 16.3; HRMS (ESI) calcd for
C20H29N8O2 (M + H)+ 413.2408, found 413.2412.
4-(5-{1-[3-(4-Hexyl-phenyl)-2-methyl-allyl]-1H-[1,2,3]triazol-4-
yl}-pentyl)-1H-imidazol-2-ylamine hydrochloride (8n). Follo-
wing the procedure for 8a, tri Boc-protected compound 2 was
coupled with 4-hexylphenylboronic acid to furnish the protected
precursor as yellow oil(0.209 g, 79%). 1HNMR(400MHz, CDCl3)
d 7.32 (s, 1H), 7.22–7.16 (m, 4H), 7.08 (s, 1H), 6.49 (s, 1H), 5.00 (s,
2H), 2.71 (t, J = 7.6, 2H), 2.60 (t, J = 7.8, 2H), 2.52 (t, J = 7.4, 2H),
1.81 (d, J = 1.3), 1.74–1.64 (m, 6H), 1.58 (s, 9H), 1.41 (s, 18H),
1.39–1.26 (m, 8H), 0.88 (t, J = 6.9, 3H); 13C NMR (100 MHz,
CDCl3) d 149.7, 149.0, 146.7, 142.4, 140.5, 134.0, 131.6, 130.1,
129.5, 129.1, 128.6, 120.6, 114.1, 85.7, 83.5, 59.0, 35.9, 32.0, 31.9,
31.6, 29.5, 28,9, 28.7, 28.2, 28.11, 28.0, 25.9, 22.8, 15.9, 14.3.
4-(5-{1-[3-(4-Aminomethyl-phenyl)-2-methyl-allyl]-1H-[1,2,3]-
triazol-4-yl}-pentyl)-1H-imidazol-2-ylamine hydrochloride (8l).
Following the procedure for 8a, tri Boc-protected com-
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After deprotection and salt exchange, the title compound was
obtained as viscous yellow oil (0.132 g, 99%). 1H NMR (400 MHz,
DMSO-d6) d 12.14 (s, 1H), 11.67 (s, 1H), 7.89 (s, 1H), 7.31 (bs,
2H), 7.20–7.16 (m, 4H), 6.53 (s, 1H), 6.43(s, 1H), 5.03 (s, 2H), 2.62
(t, J = 7.6, 2H), 2.54 (t, J = 7.6, 2H), 2.38 (t, J = 7.4, 2H), 1.72 (s,
3H), 1.61 (quintet, J = 7.3, 2H), 1.58–1.51 (m, 4H), 1.36–1.22, (m,
6H), 0.84 (t, J = 6.7, 3H); 13C NMR (100 MHz, DMSO-d6) d 147.6,
147.4 141.9, 134.5, 132.8, 129.4, 129.0, 128.9, 127.4, 122.9,109.1,
58.1, 35.5, 31.8, 31.5, 29.2, 29.0, 28.5, 28.0, 25.5, 24.6, 22.7, 16.2,
14.6; HRMS (ESI) calcd for C26H39N6 (M + H)+ 435.231, found
435.3227.
strain at an OD600 of 0.05. A 96-well microtiter plate was filled
with 100 mL of inoculated medium and was incubated at 25 ◦C
for 24 h. After this time, the medium was discarded and the plates
were thoroughly washed two or three times with water. The plates
were shaken off gently to remove excess water and then refilled
with 110 mL of media with test compound at a pre-determined
concentration. The plates were then covered with the lid and
wrapped in SaranTM wrap and incubated at 37 ◦C for 24 h. After
this time, the plate’s contents were discarded, washed and stained
with crystal violet as before (see biofilm inhibition assay above).
Each concentration reported during the course of this study was
repeated two to four times with each biofilm dispersion assay being
done in 6 or 8 replicates each. The dispersion effectiveness was
plotted to generate a 5 to 7 point curve of the percent inhibition
of biofilm formation versus the concentration of test compound.
This curve was used to determine EC50 values.
Biology
Biofilm Screening. Compounds 8a–n were dissolved in
DMSO, filtered through Whatman 13 mm polypropylene case
filter with 0.2 micron pore sized and stored 100 mM solutions
at -20 ◦C until needed for biological study. The DMSO used
in these biofilm screens did not exceed 1% by volume and had
no effect on bacterial growth or biofilm formation. Acinetobacter
baumannii (ATCC 19606) and MRSA (BAA-44) were purchased
from ATCC. A. baumannii was grown in LB media during the
course of this study for inhibition assay and dispersion assays.
MRSA was grown in Tryptic Soy Broth supplemented with 0.5%
glucose for biofilm inhibition assays.
Microdilution susceptibility testing. All antibiotic susceptibil-
ity testing was completed with a starting inoculum of 5 ¥ 105
CFU mL-1 according to NCCLS standards and incubated for
16–20 h at 37 ◦C. After this time bacterial growth was visually
inspected and the lowest concentration at which no observable
bacterial growth or turbidity was observed was considered to
be the MIC value. Pellets that formed at the bottom of 96 well
microtiter plates were considered growth and although there was
essentially no turbidity, the pellet was considered bacterial growth
in these studies. Microdilutions were made in the 96-well microtiter
plates from a starting concentration of 400 mM with the addition of
200 mL to a predetermined well. From this, two-fold serial dilutions
were made by transferring 100 mL of the initially treated well (of
400 mM) into the next well and mixing once using a multichannel
pipette, this next well then contained 200 mL of bacterial, media
and test 2-AIT compound at a concentration of 200 mM. The
transfer was done in succession for a total of 12 two-fold serial
dilutions giving a range of 400–3.91 mM for tested 2-AIT in 96
well microtiter plates. After the final dilution, the wells in this row
then had 200 mL (of the 3.91 mM wells), 100 mL of which werer
pipetted out and thrown away. The plates were then covered by a
plastic lid and then were wrapped in SaranTM wrap and placed in
a humidified chamber and incubated for 16–20 h at 37 ◦C. After
the incubation time, the wells were observed for bacterial growth
and MICs were determined accordingly (described above).
Biofilm Inhibition Assay. An overnight culture of bacterial
strain was subcultured at an OD600 of 0.01 into the media used
depending on bacterial strain. This was pipetted into test tubes
along with a predetermined concentration of test compound.
The contents of the test tubes were then poured into tilted
Petri dishes and 100 mL of medium, bacteria and compound
were then transferred into 96-well PVC microtiter plates. These
microtiter plates were then covered with a plastic lid, wrapped
in SaranTM wrap and incubated at either room temperature or
37 ◦C for 24 h. After incubation, the medium was discarded
and the plates were thoroughly washed two or three times with
water. The remaining biofilm was stained with 100 mL of a 0.1%
crystal violet solution and allowed to sit at room temperature for
30 min. After 30 min, the crystal violet was discarded and washed
thoroughly again with water. The remaining crystal violet which
stained the biofilm on the inside of the wells was solubilized with
200 mL of 95% ethanol. The quantitation of biofilm formation was
accomplished by transferring 125 mL of the ethanol solution into a
polystyrene microtiter plate and reading the absorbance at 540 nm
(A540) using a Biotek ELX808 plate reader After subtracting
the background from each well, a percent inhibition could be
calculated by subtracting from a hundred, the ratio of the amount
of crystal violet stain in the wells that contained compound by
the amount of crystal violet stain in wells that contained bacteria
only. Each concentration reported during the course of this study
was repeated two to four times with each biofilm inhibition assay
being done in 6 or 8 replicates each. The inhibition effectiveness
was plotted to generate a 5 to 9 point curve of the percent inhibition
of biofilm formation versus the concentration of compound tested.
This curve was used to determine IC50 values.
Growth Curves. An overnight culture of bacterial strain was
subcultured into its respective media at an OD600 of 0.01. The
inoculated media (3 ml) was added to two test tubes, one that
would not be treated, to serve as a control for bacterial growth,
and one that would be treated with 8 g, 8i or 8j at the respective
IC50 value determined in biofilm inhibition assays. The test tubes
were shaken while incubated and optical densities were taken after
2, 4, 6, 8 and 24 h. These experiments were done from two different
overnight cultures from two different bacterial colonies in single
replicate.
Acknowledgements
Biofilm Dispersion Assay. Biofilm dispersion is similar to
biofilm inhibition assay, but here bacterial biofilms are allowed
to first form in 96-well PVC plates using a sub-cultured bacterial
The authors would like to thank the North Carolina Biotechnol-
ogy Center for funding this work.
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