Inhibition and dispersion of Pseudomonas aeruginosa biofilms with reverse amide 2-aminoimidazole oroidin analogues

Article abstract of DOI:10.1039/b719082d

The marine alkaloid oroidin along with a small library of reverse amide (RA) 2-aminoimidazoles were synthesized and assayed for anti-biofilm activity against PAO1 and PA14, two strains of the medically relevant γ-proteobacterium Pseudomonas aeruginosa. Analogues that contained a long, linear alkyl chain were more potent inhibitors than the natural product at preventing the formation of PAO1 and PA14 biofilms. The most active compound in the series was also shown to disperse established PAO1 and PA14 biofilms at low micromolar concentrations. The Royal Society of Chemistry.

Full text of DOI:10.1039/b719082d

www.rsc.org/obc
Volume 6 | Number 8 | 21 April 2008 | Pages 1301–1512
ISSN 1477-0520
FULL PAPER
Christian Melander et al.
Inhibition and dispersion of
Pseudomonas aeruginosa biofilms
EMERGING AREA
Takanori Shibata and Kyoji Tsuchikama
with reverse amide 2-aminoimidazole Recent advances in enantioselective
oroidin analogues [2 + 2 + 2] cycloaddition

PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Inhibition and dispersion of Pseudomonas aeruginosa biofilms with reverse
amide 2-aminoimidazole oroidin analogues†
Justin J. Richards,‡ T. Eric Ballard‡ and Christian Melander*
Received 11th December 2007, Accepted 31st January 2008
First published as an Advance Article on the web 5th March 2008
DOI: 10.1039/b719082d
The marine alkaloid oroidin along with a small library of reverse amide (RA) 2-aminoimidazoles were
synthesized and assayed for anti-biofilm activity against PAO1 and PA14, two strains of the medically
relevant c-proteobacterium Pseudomonas aeruginosa. Analogues that contained a long, linear alkyl
chain were more potent inhibitors than the natural product at preventing the formation of PAO1 and
PA14 biofilms. The most active compound in the series was also shown to disperse established PAO1
and PA14 biofilms at low micromolar concentrations.
Our research group has become interested in exploiting the
oroidin class of marine alkaloids as molecular inspiration for de-
Introduction
veloping novel compounds that possess anti-biofilm properties.10
The activity of oroidin has been documented in a limited number
of studies involving bacterial attachment and colonization.11,12
Oroidin has also been shown to inhibit biofouling driven by the
marine a-proteobacterium R. salexigens.13 Due to this activity and
chemical simplicity, oroidin was selected as a lead compound
for structure activity relationship (SAR) studies in hopes of
discovering a diverse range of compounds that possess anti-biofilm
properties. One intriguing approach involved reversal of the amide
bond highlighted in Fig. 2 which connects the bromopyrrole tail
of oroidin 4 to the 2-aminoimidazole (2-AI) head.
Recent developments in the fields of bacteriology and infectious
disease have revealed that bacteria often exist as intertwined
communities rather than as independent microorganisms.1 These
surface associated microcolonies have come to be known as
biofilms and are ubiquitous in nature.2 Biofilms underpin a signif-
icant list of problems encountered in the agricultural, engineering,
and medical sectors of the global economy. The NIH estimates
that approximately 3 out of 4 microbial infections that occur
in the body are biofilm mediated.3 Biofilms also underlie high
morbidity rates in patients who suffer from cystic fibrosis. Bacteria
that reside within the biofilm state display different phenotypes
than their planktonic brethren and become more resistant to
many antibiotics and biocides that would often lead to their
eradication.4,5
Despite the prevalence of biofilms in our society, examples of
molecular scaffolds that inhibit biofilm formation are scarce. These
limited examples include the homoserine lactones (1) which are
naturally occurring signaling molecules that elicit their activity
through disruption of bacterial communication,6 brominated
furanones (2) which were originally isolated from the macroalga
Delisea pulchra,7,8 and ursene triterpenes (3) from the plant
Diospyros dendo (Fig. 1).9
Fig. 2 Retrosynthetic analysis of oroidin and the RA scaffold.
One of the best methods for large scale preparation of the 2-AI
scaffold en route to prepare oroidin and other family members
involves Akabori reduction (Na–Hg) of ornithine methyl ester 7
followed by condensation with cyanamide under pH controlled
conditions.14–16 Derivatization can then be achieved via acylation
of the alkyl amine off the carbon tail with variously substituted
trichloroacetyl pyrroles. However, this chemistry is plagued by
severe limitations, most notably the overall lack of compatibility
of this system with other trichloroacetyl esters. In addition,
solubility issues of the parent 2-AI leaves much to be desired. Many
attempts by our group in developing other acylation conditions
that would allow for the generation of greater diversity have
proven unfruitful. From a practical standpoint, purifications of
intermediates bearing an unprotected 2-AI often require large
amounts of methanol saturated with ammonia (MeOH–NH3),
Fig. 1 Molecules known to inhibit biofilm formation.
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 NMR spectra
for representative compounds in the RA library, representative dose–
response curves and planktonic growth curves for PAO1 and PA14 in
the presence and absence of 12 and oroidin 4 are provided. See DOI:
10.1039/b719082d
‡ Both authors contributed equally to this work
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which is cumbersome to prepare and can be difficult to remove
from the pure sample after column chromatography.
The first approach to the RA scaffold relied heavily on
the aminolysis of intermediate 18 since this would afford the
Boc-protected RA precursors in a single synthetic step. After
deprotection with TFA and HCl salt exchange, isolation of the
targets would require only filtration with no need for further
purification. Based upon the seminal paper published by Weinreb
and co-workers on the transformation, trimethylaluminium was
used as the Lewis acid to affect the direct aminolysis reaction.20
Numerous reaction factors were taken into account such as choice
of solvent, equivalents of aluminium–amine complex, reagent
order of addition, time, and temperature. Despite all of the
conditions scanned (data not shown), the highest yielding reaction
occurred in only 55% yield when aniline was used as the amine
partner. Triazabicyclo[4.4.0]dec-5-ene (TBD) was also examined
as a potential catalyst to promote the direct aminolysis of ester
18.21 Heating both starting materials in the presence of 30 mol%
of TBD in toluene at elevated temperatures for extended periods
of time failed to produce any desired product as evident by TLC
analysis (data not shown).
Implementation of a reverse amide approach, coupled with a
practical protecting group strategy, would effectively eliminate
many of the aforementioned handicaps with current methods.
Installation of the reverse amide bond could be obtained by direct
aminolysis of an intermediate Boc-2AI alkyl ester or through
couplings of a carboxylic acid (Fig. 2). These intermediates
could be accessed through a-bromoketones which are obtained by
diazomethane homologation with the proper acyl chloride. Addi-
tionally, significant diversity can be achieved by incorporating any
commercially available amine with a common RA intermediate.
Herein we report the synthesis of a focused reverse amide (RA)
library (Fig. 3) and subsequent biological evaluation of the library
in comparison to the natural product oroidin in the context of
anti-biofilm activity of biofilms formed by the medically relevant
c-proteobacterium Pseudomonas aeruginosa.
Due to the problems encountered utilizing aminolysis, we opted
for a more conventional route to access the RA scaffold through
the intermediacy of an activated carboxylic acid. Unfortunately,
saponification of the methyl ester 18 proved problematic on
this system as cleavage of the Boc group was observed under
the basic conditions of both LiOH–MeOH–THF–H2O or LiI–
pyridine. Decomposition of the methyl ester was also observed
when TMSOK in methylene chloride or (Bu3Sn)2O in toluene
at either ambient temperature or reflux were employed as the
saponification agents (data not shown).
Fig. 3 Members of the RA pilot library.
Persuaded by these results that the current route required
revision, we began a second generation approach to our core
scaffold (Scheme 2). This approach relied on a different protecting
group strategy, substituting the methyl ester for a benzyl ester
which, in the case of another failed attempt at aminolysis, would
undergo hydrogenolysis under mild conditions to deliver the
corresponding Boc-protected acid 22. Synthesis began with the
known monobenzyl ester acid22 20 which was transformed into the
benzyl protected a-bromoketone by conversion to its acid chloride
followed by diazomethane homologation and concomitant quench
with concentrated HBr. Cyclization of this intermediate afforded
the Boc-protected 2-AI 21 in 66% yield. All attempts at direct
aminolysis of benzyl ester 21 resulted in sluggish reactions that
were plagued by the formation of multiple side products.
Results and discussion
Synthesis of RA Library
Scaffold synthesis began with treatment of the commercially avail-
able acid chloride 17 with diazomethane (Scheme 1).17 Quenching
with concentrated HCl or HBr delivered the corresponding a-
haloketones in excellent yields which were isolable by column
chromatography. Installation of the protected 2-aminoimidazole
moiety was achieved through a Boc-guanidine condensation in
DMF at ambient temperature to yield 18. Significantly higher
yields for this step were obtained when two equivalents of sodium
iodide were added to the reaction mixture and represents a
significant improvement over previous reports.18,19 It was also
observed during this sequence that the a-bromoketone afforded
higher yields than its a-chloro counterpart in the cyclization
reaction.
Scheme 2 2nd Generation synthesis of the RA scaffold. Reaction
conditions: (a) i. (COCl)2, DMF (cat.), CH2Cl2 ii. CH2N2, Et2O–CH2Cl2,
0
◦C iii. conc. HBr, 88% (b) Boc-guanidine, DMF, 66% (c) H2 (1 atm),
10% Pd/C, THF, 98% (d) EDC, HOBt, NR1R2, DMF (e) TFA, CH2Cl2
(f ) 2 M HCl in Et2O.
Given the failure of the direct aminolysis conversion, the two-
step approach to the RA scaffold was investigated. Deprotection
proceeded as planned and was accomplished by subjecting 21 to a
hydrogen atmosphere at balloon pressure which cleanly afforded
pure Boc-protected acid 22 in near quantitative yield (98%). With
Scheme 1 1st Generation synthesis of the RA scaffold. Reaction condi-
tions: (a) i. CH2N2, Et2O–CH2Cl2, 0 ◦C ii. conc. HCl (90%) or conc. HBr
(93%) (b) Boc-guanidine. NaI, DMF, 65% (c) LiOH, MeOH–THF–H2O
(3 : 1 : 1) then 1 N HCl to pH = 5, 94% (d) AlMe3, NR1R2, DCE, 0 ◦C to
60 ◦C (e) TFA, CH2Cl2, (f ) 2 M HCl in Et2O.
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the acid now in hand and available on a multi-gram scale, attempts
to install the key amide bond were assessed. A number of activating
agents were scanned including DCC, EDC, HCTU, CDI, and
cyanuric chloride to affect the transformation. Of those listed
only EDC and HCTU were able to give consistent and tangible
results. EDC was chosen over HCTU due to ease of purification
in separating side products during column chromatography. It
was during this optimization that the limitation of the synthetic
route was identified to be the reactivity of the Boc group. A
significant quantity of a Boc-protected starting amine was isolated
and characterized, signifying the lability of the Boc-group due to
Boc-transfer under the reaction conditions regardless of which
activating agent was used.
Members of the reverse amide library along with oroidin16
were initially screened at 500 lM in a 96-well format using a
crystal violet reporter assay to assess each compound’s ability
to inhibit the formation of PAO1 or PA14 biofilms (Fig. 4).27
Compounds 5, 19, and 32 were used as controls in the assays
and all showed only marginal inhibition. There was a remarkable
range of activities among the RA compounds analyzed in the
inhibition assay. Similar activities were observed between PAO1
and PA14, although most compounds were slightly more potent
against PA14. Interestingly, this trend is opposite to our previ-
ously reported bromoageliferin analogues.10 These screens also
suggested that the aliphatic chain derivatives (9–12) and oroidin 4
were very potent inhibitors of P. aeruginosa biofilms.
With two routes in hand to generate the RA scaffold, we
assembled the focused library outlined in Table 1. EDC–HOBt
couplings of acid 22 were used to generate most of the linear alkyl
chain analogues (28–34%) while aminolysis of the methyl ester
intermediate 18 furnished the remaining compounds (11–55%)
in the library (Table 1). The final step of the synthetic approach
required removal of the Boc group, which proceeded at room
temperature in TFA–DCM. The resulting trifluoroacetate salts of
each target were then traded out for their HCl counterparts before
characterization and assessment of their biological activity.
Biological evaluation of RA library
Nosocomial infections are driven by a persistent bacterial colo-
nization of hospital facilities, wherein the bacteria are extremely
resistant to eradication because they exist in a biofilm state. P.
aeruginosa is an opportunistic c-proteobacterium that is a serious
threat to immunocompromised patients and is frequently isolated
from patients found in intensive care units suffering from severe
burns or other traumas. It is the second most common pathogen
in hospital-acquired pneumonia behind Staphylococcus aureus.23
For cystic fibrosis patients, the onset of colonization by this
bacterium is of great concern. Morbidity rates of patients who
suffer from the disease are directly correlated to the virulence of
P. aeruginosa biofilms.24,25 The speed and prevalence with which
multidrug resistant (MDR) strains are appearing puts pressure
on the medical community to find ways to combat the aggressive
nature of this bacterium.26
Fig. 4 Preliminary inhibition data at 500 lM for the RA library. All
values are averages of at least three experiments.
Table 1 Completion of the RA library
Subsequently the aliphatic derivatives (9–12) and oroidin 4 were
selected for IC50 value determination against PAO1 and PA14
(Table 2). The generation of dose–response curves for compounds
9–12 revealed a correlation between the length of the carbon chain
and the potency of the compound (supporting information). This
trend is apparent when the IC50 values are plotted as a function
of chain length in both PAO1 and PA14 (Fig. 5). Increasing the
Amine
Conditions
Coupled product
Target
Isobutylamine
Hexylamine
Octylamine
a
b
b
a
b
a
a
a
a
23
24
25
26
27
28
29
30
31
8
9
10
11
12
13
14
15
16
Table 2 PAO1 and PA14 IC50 values
Decylamine
Dododecylamine
Cyclopentylamine
Morpholine
Aniline
2-Aminopyrimidine
Compound
PAO1 IC50/lM
190
32.7 6.5
18.4 2.3
PA14 IC50/lM
Oroidin (4)
9 (n = 5)
9
166 23
39.9 13.0
13.3 1.8
10 (n = 7)
11 (n = 9)
12 (n = 11)
Reaction conditions: (a) AlMe3, 18, DCE, 0 ◦C to 60 ◦C (b) 22, EDC,
HOBt, DMF (c) TFA, CH2Cl2, (d) 2 M HCl in Et2O
8
1
6
1
2.84 0.93
2.26 0.83
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Fig. 6 Dispersion of established P. aeruginosa with 12.
derivatives allows for rapid synthesis and simplified purification
of all library members. Clearly, the most potent derivatives were
those that contained linear carbon chains of various lengths from
the amide nitrogen. The most active of these compounds, 12, has
also been shown to disperse established P. aeruginosa biofilms at
low micromolar concentrations, making it a highly noteworthy
addition to the limited number of small molecules known to
possess such characteristics.29–31 Current efforts are under way to
develop a more versatile route which leads to improved yields
during the amide bond formation step. It also remains a goal to
identify even more potent compounds with structures similar to
the most active analogues identified in this study while gaining
mechanistic insight into how these compounds elicit their anti-
biofilm effects.
Fig. 5 Structure activity relationship of aliphatic chain RA analogues.
chain length from six to twelve carbons effectively increased the
inhibition activity over a full order of magnitude in both strains.
All linear carbon chain analogues were significantly more potent
than oroidin (PAO1 IC50 = 190 lM, PA14 IC50 = 166 lM).
The most active RA analogue identified was 12 (PAO1 IC50
=
2.84 lM, PA14 IC50 = 2.26 lM), effectively demonstrating that
changes to certain portions of the natural product have the ability
to dramatically increase biological activity.
To validate that our compounds were true inhibitors of biofilm
formation and not acting as bactericidal agents, growth curves
were performed at the determined IC50 values for PAO1 and PA14
with the dodecyl-based analogue 12 and oroidin 4. Bacterial cell
densities for both strains remained unchanged when grown in
the presence or absence of either the natural product 4 or 12
throughout a 24 hour time period (supporting information).
While the focus has predominantly been on designing small
molecules that inhibit the formation of bacterial biofilms, the more
significant challenge is the development of a small molecule that
will disperse established biofilms. Treatment of chronic infections
is commonly hindered by the presence of established biofilms that
impart increased resistance to conventional antibiotics.28 Small
molecules able to disperse established biofilms are, therefore, of
great interest to the medical community. To test for the ability
to disperse established biofilms, PAO1 and PA14 were allowed to
form biofilms for 24 hours in the absence of compound. After this
time the media was discarded. The wells were washed and fresh
media was added containing varying concentrations of 12 and
then incubated at 37 ◦C for 24 hours. RA analogue 12 displayed
significant anti-biofilm activity, dispersing established PAO1 and
Experimental
Stock solutions (100, 10, 1 mM) of all compounds assayed for
biological activity were prepared in DMSO and stored at room
temperature. The amount of DMSO used in both inhibition and
dispersion screens did not exceed 1% (by volume). P. aeruginosa
strains PAO1 and PA14 were graciously supplied by the Wozniak
group at Wake Forest University School of Medicine.
General static inhibition assay protocol for Pseudomonas
aeruginosa.
An overnight culture of the wild type strain was subcultured
at an OD600 of 0.10 into LBNS along with a predetermined
concentration of the small molecule to be tested for biofilm
inhibition. Samples were then aliquoted (100 lL) into the wells
of a 96-well PVC microtiter plate. The microtiter dishes were
covered and sealed before incubation under stationary conditions
at 37 ◦C for 24 hours. After that time, the medium was discarded
and the plates thoroughly washed with water. The wells were then
inoculated with a 0.1% aqueous solution of crystal violet (100 lL)
and allowed to stand at ambient temperature for 30 minutes.
Following another thorough washing with water the remaining
stain was solubilized with 200 lL of 95% ethanol. Biofilm
inhibition was quantitated by measuring the OD540 for each well by
transferring 125 lL of the ethanol solution into a fresh polystyrene
microtiter dish for analysis.
PA14 biofilms with EC50 values of 32.8
4.7 lM and 21.3
3.9 lM respectively (Fig. 6).
Conclusion
We have identified several reverse amide (RA) analogues that
possess potent anti-biofilm properties. These compounds are
based on a reverse amide scaffold which switches the directionality
of the amide bond frequently found in many members of the
oroidin class of marine alkaloids. The path taken to access these
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General static dispersion assay protocols for Pseudomonas
aeruginosa.
6-Chloro-5-oxo-hexanoic acid methyl ester. Using the same
general procedure as used above but instead quenching with conc.
HCl afforded the chloro derivative (2.93 g, 90%) as a colorless oil.
1H NMR (400 MHz, CDCl3) d 4.13 (s, 2H), 3.67 (s, 3H), 2.69 (t, 2H,
J = 7.2 Hz), 2.38 (t, 2H, J = 7.2 Hz), 1.94 (quint., 2H, J = 7.2 Hz);
13C NMR (100 MHz, CDCl3) d 201.85, 173.33, 51.57, 48.22, 38.47,
32.66, 18.61; HRMS (ESI) calcd for C7H12O3Cl (MH)+ 179.0469,
found 179.0476.
An overnight culture of the wild type strain was subcultured at
an OD600 of 0.50 into LBNS and then aliquoted (100 lL) into the
wells of a 96-well PVC microtiter plate. The microtiter dishes were
covered and sealed before incubation under stationary conditions
at room temperature to allow formation of the biofilms. After
24 hours the medium was discarded and the plates thoroughly
washed with water. Fresh medium containing the appropriate
concentration of compound was then added to the wells. The
plates were again sealed and this time incubated under stationary
conditions at 37 ◦C. After 24 hours, the media was discarded
from the wells and the plates washed thoroughly with water. The
wells were inoculated with a 0.1% aqueous solution of crystal
violet (100 lL) and allowed to stand at ambient temperature for
30 minutes. Following another thorough washing with water the
remaining stain was solubilized with 200 lL of 95% ethanol.
Biofilm dispersion was quantitated by measuring the OD540 for
each well by transferring 125 lL of the ethanol solution into a
fresh polystyrene microtiter dish for analysis. Percent dispersion
was calculated by comparison of the OD540 for established biofilm
(untreated) versus treated established biofilm under identical
conditions.
6-Bromo-5-oxo-hexanoic acid benzyl ester. Butanoic acid
monobenzyl ester 20 (3.00 g, 13.6 mmol) was dissolved in an-
hydrous dichloromethane (70 mL) at 0 ◦C and a catalytic amount
of DMF was added. To this solution was added oxalyl chloride
(3.60 mL, 41.3 mmol) drop-wise and the solution was then warmed
to room temperature. After 1 h, the solvent and excess oxalyl
chloride were removed under reduced pressure. The resulting solid
was dissolved into anhydrous dichloromethane (10 mL) and added
drop-wise to a 0 ◦C solution of CH2N2 (42.0 mmol generated
R
from Diazaldꢀ–KOH) in diethyl ether (120 mL). This solution
was stirred at 0 ◦C for 1.5 h at which time the reaction was
quenched via the drop-wise addition of 48% HBr (4.7 mL). The
reaction mixture was diluted with dichloromethane (25 mL) and
immediately washed with sat. NaHCO3 (3 × 25 mL) and brine (2 ×
25 mL) before being dried (MgSO4), filtered and concentrated. The
crude oil was purified by flash column chromatography (0–30%
EtOAc–hexanes) to obtain the title compound (3.57 g, 88%) as a
colorless oil. 1H NMR (400 MHz, CDCl3) d 7.35 (m, 5H), 5.12 (s,
2H), 3.85 (s, 2H), 2.73 (t, 2H, J = 6.8 Hz), 2.42 (t, 2H, J = 6.8 Hz),
1.96 (quint., 2H, J = 6.8 Hz); 13C NMR (75 MHz, CDCl3) d 201.43,
172.88, 136.15, 128.79, 128.48, 128.45, 66.52, 38.74, 34.07, 33.20,
19.23; HRMS (ESI) calcd for C13H16O3Br (MH)+ 299.0277, found
299.0279.
Chemistry
All reagents including anhydrous solvents used for the chemical
synthesis of the library were purchased from commercially avail-
able sources and used without further purification unless otherwise
noted. All reactions were run under either a nitrogen or argon
atmosphere. Flash silica gel chromatography was performed with
1
60 A mesh standard grade silica gel from Sorbtech. H and 13C
˚
2-Amino-4-(3-methoxycarbonyl-propyl)-imidazole-1-carboxylic
acid tert-butyl ester (18). 6-Bromo-5-oxo-hexanoic acid methyl
ester (2.30 g, 10.3 mmol), Boc-guanidine (4.92 g, 30.9 mmol),32
and NaI (3.07 g, 20.6 mmol) were dissolved in DMF (30 mL) and
allowed to stir at room temperature. After 24 h the DMF was
removed under reduced pressure and the residue was taken up
in ethyl acetate (100 mL) and washed with water (3 × 50 mL)
and brine (50 mL) before being dried (Na2SO4), filtered and
evaporated to dryness. The resulting oil was purified by flash
column chromatography (50–100% EtOAc–hexanes) to obtain a
yellow oil. Trituration of the viscous oil with cold hexanes (20 mL)
produced a precipitate, which upon filtration yielded 18 (1.89 g,
NMR spectra were obtained using Varian 300 MHz or 400 MHz
spectrometers. NMR solvents were purchased from Cambridge
Isotope Labs and used as is. Chemical shifts are given in parts per
million relative to DMSO-d6 (d 2.50) and CDCl3 (d 7.27) for proton
spectra and relative to DMSO-d6 (d 39.51) and CDCl3 (d 77.21) for
carbon spectra with an internal TMS standard. High-resolution
mass spectra were obtained at the North Carolina State Mass
Spectrometry Laboratory for Biotechnology. ESI experiments
were carried out on an Agilent LC-TOF mass spectrometer.
6-Bromo-5-oxo-hexanoic acid methyl ester. Methyl glutaryl
chloride (2.5 mL, 18.23 mmol) was dissolved into anhydrous
dichloromethane (10 mL) and added drop-wise to a 0 ◦C solution
1
65%) as a pale yellow solid. H NMR (400 MHz, CDCl3) d 6.53
R
of CH2N2 (55.0 mmol generated from Diazaldꢀ–KOH) in diethyl
(s, 1H), 5.6 (br s, 2H), 2.41 (t, 2H, J = 7.2 Hz), 2.37 (t, 2H,
J = 7.2 Hz), 1.93 (quint., 2H, J = 7.2 Hz), 1.58 (s, 9H); 13C
NMR (75 MHz, CDCl3) d 174.09, 150.11, 149.61, 138.39, 107.15,
84.81, 51.56, 33.65, 28.18, 27.68, 23.82; HRMS (ESI) calcd for
C13H22N3O4 (MH)+ 284.1604, found 284.1606.
ether (150 mL). This solution was stirred at 0 ◦C for 1.5 h at
which time the reaction was quenched via the drop-wise addition
of 48% HBr (7.5 mL). The reaction mixture was diluted with
dichloromethane (25 mL) and immediately washed with sat.
NaHCO3 (3 × 25 mL) and brine (2 × 25 mL) before being dried
(MgSO4), filtered and concentrated. The crude oil was purified
via flash column chromatography (10–30% EtOAc–hexanes) to
4-(2-Amino-1H-imidazol-4-yl)butyric acid hydrochloride (19).
To 2-amino-4-(3-methoxycarbonyl-propyl)-imidazole-1-carbo-
xylic acid tert-butyl ester 18 (50 mg, 0.176 mmol) was added
methanol (0.60 mL), tetrahydrofuran (0.20 mL), and water
(0.20 mL). Lithium hydroxide (9 mg, 0.352 mmol) was then added
and the reaction was stirred at room temperature for 30 min. The
pH of the solution was carefully adjusted to pH = 5 with a 1
N aqueous solution of HCl before being evaporated to dryness.
1
obtain the title compound (3.76 g, 93%) as a colorless oil. H
NMR (400 MHz, CDCl3) d 3.91 (s, 2H), 3.68 (s, 3H), 2.76 (t, 2H,
J = 7.2 Hz), 2.38 (t, 2H, J = 7.2 Hz), 1.95 (quint., 2H, J = 7.2 Hz);
13C NMR (75 MHz, CDCl3) d 201.36, 173.41, 51.67, 38.74, 34.16,
32.87, 19.13; HRMS (ESI) calcd for C7H12O3Br (MH)+ 222.9964,
found 222.9964.
1360 | Org. Biomol. Chem., 2008, 6, 1356–1363
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R
The crude product was purified via a silica gel plug (100% MeOH
sat. NH3) to deliver the product as its corresponding free base.
The hydrochloride salt was obtained through addition of a single
drop of concentrated HCl to a methanolic solution (2 mL) of the
free base. Rotary evaporation of this solution afforded 19 (34 mg,
94%) as a white solid. 1H NMR (300 MHz, DMSO-d6) d 12.25 (s,
1H), 12.13 (br s, 1H), 11.77 (s, 1H), 7.33 (s, 2H), 6.54 (s, 1H), 2.43
(t, 2H, J = 7.2 Hz), 2.21 (t, 2H, J = 7.2 Hz), 1.73 (m, 2H); 13C
NMR (100 MHz, DMSO-d6) d 174.04, 146.92, 126.01, 108.62,
32.81, 23.47, 23.05; HRMS (ESI) calcd for C7H12N3O2 (MH)+
170.0924, found 170.0927.
was warmed to ambient temperature and Celiteꢀ was added. After
stirring for 5 min, the mixture was filtered and the filtrate washed
with brine (2 × 3 mL), dried (Na2SO4), and evaporated to dryness.
The crude product was purified via flash column chromatography
(2–10% MeOH–CH2Cl2) to afford pure product.
2-Amino-4-(3-isobutylcarbamoyl-propyl)-imidazole-1-carboxy-
lic acid tert-butyl ester (23). White solid (46 mg, 40%). 1H NMR
(400 MHz, DMSO-d6) d 7.75 (m, 1H), 6.51 (s, 1H), 6.37 (br s,
2H), 2.85 (t, 2H, J = 6.4 Hz), 2.22 (t, 2H, J = 6.8 Hz), 2.07 (t,
2H, J = 7.2 Hz), 1.61–1.73 (m, 3H), 1.53 (s, 9H), 0.81 (d, 6H,
J = 6.4 Hz); 13C NMR (100 MHz, DMSO-d6) d 171.82, 149.92,
148.95, 138.39, 105.84, 84.04, 45.94, 34.88, 28.09, 27.52, 27.23,
24.10, 20.14; HRMS (ESI) calcd for C16H29N4O3 (MH)+ 325.2234,
found 325.2238.
2-Amino-4-(3-benzyloxycarbonyl-propyl)-imidazole-1-carboxy-
lic acid tert-butyl ester (21). 6-Bromo-5-oxo-hexanoic acid ben-
zyl ester (3.42 g, 11.99 mmol) and Boc-guanidine (5.73 g,
35.97 mmol) were dissolved in DMF (35 mL) and allowed to
stir at room temperature. After 48 h the DMF was removed under
reduced pressure and the residue was taken up in ethyl acetate
(100 mL) and washed with water (3 × 50 mL) and brine (50 mL)
before being dried (Na2SO4), filtered and evaporated to dryness.
The resulting oil was purified by flash column chromatography
(30–100% EtOAc–hexanes) to obtain the title compound (2.79 g,
66%) as a colorless oil which solidified upon prolonged standing.
1H NMR (400 MHz, CDCl3) d 7.35 (m, 5H), 6.51 (s, 1H), 5.91
(s, 2H), 5.12 (s, 2H), 2.41 (m, 4H), 1.94 (quint., 2H, J = 7.2 Hz),
1.57 (s, 9H); 13C NMR (75 MHz, CDCl3) d 173.45, 150.31, 149.59,
138.27, 136.36, 128.67, 128.29, 128.27, 107.05, 84.73, 66.23, 33.82,
28.16, 27.62, 23.79; HRMS (ESI) calcd for C19H26N3O4 (MH)+
360.1917, found 360.1919.
2-Amino-4-(3-decylcarbamoyl-propyl)-imidazole-1-carboxylic
acid tert-butyl ester (26). Tan solid (24 mg, 16%). 1H NMR
(400 MHz, DMSO-d6) d 7.74 (m, 1H), 6.52 (s, 1H), 6.49 (br s,
2H), 3.00 (q, 2H, J = 6.8 Hz), 2.22 (t, 2H, J = 6.8 Hz), 2.04
(t, 2H, J = 6.8 Hz), 1.71 (quint., 2H, J = 6.8 Hz), 1.53 (s, 9H),
1.36 (m, 2H), 1.23 (s, 14H), 0.85 (t, 3H, J = 6.8 Hz); 13C NMR
(100 MHz, DMSO-d6) d 171.64, 149.82, 148.86, 137.90, 105.90,
84.16, 38.33, 34.88, 31.31, 29.16, 29.03, 28.97, 28.75, 28.73, 28.02,
27.51, 27.05, 26.40, 24.04, 22.12, 13.97; HRMS (ESI) calcd for
C22H41N4O3 (MH)+ 409.3173, found 409.3175.
2-Amino-4-(3-cyclopentylcarbamoyl-propyl)-imidazole-1-car-
boxylic acid tert-butyl ester (28). White solid (54 mg, 45%). H
1
NMR (400 MHz, DMSO-d6) d 7.72 (d, 1H, J = 6.4 Hz), 6.5 (s,
1H), 6.38 (s, 2H), 3.97 (m, 1H), 2.21 (t, 2H, J = 7.2 Hz), 2.02 (t, 2H,
J = 7.2 Hz), 1.73 (m, 4H), 1.61 (m, 2H), 1.53 (s, 9H), 1.47 (m, 2H),
1.32 (m, 2H); 13C NMR (75 MHz, DMSO-d6) d 171.27, 149.92,
148.96, 138.37, 105.88, 84.02, 50.04, 34.79, 32.32, 28.05, 27.53,
27.17, 24.00, 23.43; HRMS (ESI) calcd for C17H29N4O3 (MH)+
337.2234, found 337.2235.
2-Amino-4-(3-carboxy-propyl)-imidazole-1-carboxylic acid tert-
butyl ester (22). To a solution of anhydrous THF (2 mL) and
10% Pd/C (12 mg) was charged 2-amino-4-(3-benzyloxycarbonyl-
propyl)-imidazole-1-carboxylic acid tert-butyl ester 21 (101 mg,
0.281 mmol). Air was removed from the system and the reaction
was back flushed with hydrogen. This process was repeated
three times before setting the reaction under a hydrogen balloon
at atmospheric pressure and temperature for 1 h. After that time
2-Amino-4-(4-morpholin-4-yl-4-oxo-butyl)-imidazole-1-carbox-
ylic acid tert-butyl ester (29). Tan solid (33 mg, 27%). 1H NMR
(400 MHz, DMSO-d6) d 6.52 (s, 1H), 6.39 (s, 2H), 3.52 (m,
4H), 3.41 (m, 4H), 2.28 (m, 4H), 1.42 (quint., 2H, J = 7.2 Hz),
1.53 (s, 9H); 13C NMR (75 MHz, DMSO-d6) d 171.43, 150.56,
149.64, 139.17, 106.58, 84.78, 66.85, 46.14, 32.23, 28.23, 27.89,
24.19; HRMS (ESI) calcd for C16H27N4O4 (MH)+ 339.2026, found
339.2027.
R
the reaction was filtered through a Celiteꢀ pad and the filter cake
was washed with THF (8 mL). The filtrate was concentrated under
reduced pressure to afford the title compound 21 (75 mg, 98%) as
a white solid. 1H NMR (400 MHz, DMSO-d6) d 6.52 (s, 1H), 6.42
(br s, 2H), 2.52 (t, 2H, J = 5.4 Hz), 2.18 (t, 2H, J = 5.4 Hz), 1.71
(m, 2H), 1.53 (s, 9H); 13C NMR (100 MHz, DMSO-d6) d 175.00,
149.99, 148.95, 138.28, 105.86, 84.09, 39.24, 38.85, 33.70, 27.52,
27.08, 23.52; HRMS (ESI) calcd for C12H20N3O4 (MH)+ 270.1448,
found 270.1452.
2-Amino-4-(3-phenylcarbamoyl-propyl)-imidazole-1-carboxylic
acid tert-butyl ester (30). White solid (66 mg, 55%). H NMR
1
(300 MHz, DMSO-d6) d 9.88 (s, 1H), 7.59 (d, 2H, J = 8.1 Hz),
7.27 (t, 2H, J = 7.5 Hz), 7.00 (t, 1H, J = 7.2 Hz), 6.55 (s, 1H),
6.44 (br s, 2H), 2.97 (m, 4H), 1.82 (m, 2H), 1.53 (s, 9H); 13C NMR
(75 MHz, DMSO-d6) d 170.95, 149.71, 148.86, 139.25, 138.16,
128.47, 122.78, 118.98, 105.92, 84.00, 38.42, 35.71, 27.44, 27.01,
23.72; HRMS (ESI) calcd for C18H25N4O3 (MH)+ 345.1921, found
345.1920.
General aminolysis procedure
◦
To a stirring 0 C solution of amine (0.704 mmol) in anhydrous
1,2-dichloroethane (1 mL) was added drop-wise a 2 M solution of
AlMe3 in PhCH3 (0.351 mL, 0.704 mmol). The solution was stirred
for 10 min before the addition of 2-amino-4-(3-methoxycarbonyl-
propyl)-imidazole-1-carboxylic acid tert-butyl ester 18 (100 mg,
0.352 mmol) in several portions. Once dissolution was complete,
the reaction was warmed to 60 ◦C and stirred until completion
as evident by TLC analysis. The reaction was then cooled back
down to 0 ◦C before being diluted with dichloromethane (5 mL)
and quenched with water (1 mL). The resulting viscous solution
2-Amino-4-[3-(pyrimidin-2-ylcarbamoyl)-propyl]-imidazole-1-
carboxylic acid tert-butyl ester (31). Tan solid (19 mg, 11%). 1H
NMR (400 MHz, DMSO-d6) d 10.51 (s, 1H), 8.63 (d, 2H, J =
4.8 Hz), 7.15 (t, 1H, J = 4.8 Hz), 6.54 (s, 1H), 6.40 (s, 2H), 2.49 (t,
2H, J = 7.2 Hz), 2.29 (t, 2H, J = 7.2 Hz), 1.80 (quint., 2H, J =
7.2 Hz), 1.53 (s, 9H); 13C NMR (75 MHz, DMSO-d6) d 171.34,
This journal is
The Royal Society of Chemistry 2008
Org. Biomol. Chem., 2008, 6, 1356–1363 | 1361
©

158.11, 157.63, 149.76, 148.85, 138.31, 116.37, 105.81, 83.98,
35.75, 27.44, 27.05, 23.37; HRMS (ESI) calcd for C16H23N6O3
(MH)+ 347.1826, found 347.1827.
was dissolved in dichloromethane (1 mL) and 2 M HCl in diethyl
ether (0.50 mL) was added followed by cold diethyl ether (8 mL).
The precipitate was collected by filtration and washed with diethyl
ether (3 mL) to yield the target compound 8 (59 mg, 97%) as a
tan solid. 1H NMR (300 MHz, DMSO-d6) d 12.14 (s, 1H), 11.70
(s, 1H), 7.89 (m, 1H), 7.34 (br s, 2H), 6.55 (s, 1H), 2.84 (t, 2H,
J = 6.6 Hz), 2.38 (t, 2H, J = 7.5 Hz), 2.10 (t, 2H, J = 7.5 Hz),
1.60–1.79 (m, 3H), 0.82 (d, 6H, J = 6.3 Hz); 13C NMR (75 MHz,
DMSO-d6) d 171.52, 146.78, 126.31, 108.68, 46.00, 34.41, 28.09,
23.94, 23.64, 20.18; HRMS (ESI) calcd for C11H21N4O (MH)+
225.1709, found 225.1711.
General EDC–HOBt procedure
2-Amino-4-(3-carboxy-propyl)-imidazole-1-carboxylic acid tert-
butyl ester 22 (100 mg, 0.371 mmol), 1-hydroxybenzotriazole
(100 mg, 0.742 mmol) and N-(3-dimethylaminopropyl)-Nꢀ-
ethylcarbodiimide hydrochloride (142 mg, 0.742 mmol) were
dissolved in anhydrous DMF (3 mL). The appropriate amine
coupling partner (1.48 mmol) was then added and the solution
was stirred at ambient temperature until completion was evident
by TLC analysis. The reaction was concentrated under reduced
pressure and the residue partitioned between ethyl acetate (20 mL)
and water (10 mL). The organic layer was successively washed
with water (3 × 10 mL), a 10% aqueous solution of citric acid (2 ×
10 mL), sat. NaHCO3 (2 × 10 mL), and brine (10 mL) before being
dried (Na2SO4) and evaporated to dryness. The crude product
was purified via flash column chromatography (2–10% MeOH–
CH2Cl2) to afford the target compound.
4-(2-Amino-1H-imidazol-4-yl)-N-hexyl-butyramide hydrochlo-
ride (9). Using the same general procedure as used for the
synthesis of 8, 2-amino-4-(3-hexylcarbamoyl-propyl)-imidazole-
1-carboxylic acid tert-butyl ester 24 (90 mg, 0.255 mmol) gave 9
(70 mg, 96%) as a pale yellow foam. 1H NMR (300 MHz, DMSO-
d6) d 11.96 (s, 1H), 11.54 (s, 1H), 7.81 (m, 1H), 7.29 (br s, 2H), 6.56
(s, 1H), 3.01 (m, 2H), 2.40 (t, 2H, J = 7.8 Hz), 2.07 (t, 2H, J =
7.2 Hz), 1.73 (m, 2H), 1.23–1.36 (m, 8H), 0.85 (m, 3H); 13C NMR
(100 MHz, DMSO-d6) d 171.35, 146.72, 126.37, 108.71, 38.43,
34.41, 31.00, 29.12, 26.12, 23.87, 23.62, 22.09, 13.96; HRMS (ESI)
calcd for C13H25N4O (MH)+ 253.2022, found 253.2025.
2-Amino-4-(3-hexylcarbamoyl-propyl)-imidazole-1-carboxylic
1
acid tert-butyl ester (24). Pale yellow solid (41 mg, 32%). H
4-(2-Amino-1H-imidazol-4-yl)-N-octyl-butyramide hydrochlo-
ride (10). Using the same general procedure as used for the
synthesis of 8, 2-amino-4-(3-octylcarbamoyl-propyl)-imidazole-
1-carboxylic acid tert-butyl ester 25 (50 mg, 0.131 mmol) gave
NMR (300 MHz, DMSO-d6) d 7.73 (m, 1H), 6.50 (s, 1H), 6.39 (s,
2H), 2.99 (q, 2H, J = 6.3 Hz), 2.21 (t, 2H, J = 7.5 Hz), 2.04 (t, 2H,
J = 7.2 Hz), 1.70 (m, 2H), 1.53 (s, 9H), 1.31 (m, 3H), 1.23 (br s,
7H), 0.85 (t, 3H, J = 5.1 Hz); 13C NMR (75 MHz, DMSO-d6) d
171.68, 149.93, 148.95, 138.37, 105.81, 84.04, 38.35, 34.91, 31.01,
29.14, 27.52, 27.22, 26.10, 24.06, 22.09, 13.93; HRMS (ESI) calcd
for C18H33N4O3 (MH)+ 353.2547, found 353.2549.
1
10 (39 mg, 93%) as a white solid. H NMR (300 MHz, DMSO-
d6) d 12.13 (s, 1H), 11.69 (s, 1H), 7.87 (m, 1H), 7.33 (br s, 2H),
6.55 (s, 1H), 2.99 (q, 2H, J = 6.3 Hz), 2.38 (t, 2H, J = 7.5 Hz),
2.07 (t, 2H, J = 7.5 Hz), 1.73 (m, 2H), 1.35 (m, 2H), 1.23 (m,
10H), 0.85 (t, 3H, J = 6.3 Hz); 13C NMR (75 MHz, DMSO-d6)
d 171.30, 146.80, 126.32, 108.57, 38.40, 34.39, 31.15, 29.06, 28.62,
28.56, 26.37, 23.86, 23.57, 21.99, 13.85; HRMS (ESI) calcd for
C15H29N4O (MH)+ 281.2335, found 281.2339.
2-Amino-4-(3-octylcarbamoyl-propyl)-imidazole-1-carboxylic
1
acid tert-butyl ester (25). White solid (48 mg, 34%). H NMR
(300 MHz, DMSO-d6) d 7.73 (m, 1H), 6.50 (s, 1H), 6.38 (s, 2H),
2.99 (q, 2H, J = 5.4 Hz), 2.21 (t, 2H, J = 7.5 Hz), 2.04 (t, 2H, J =
7.2 Hz), 1.73 (m, 2H), 1.53 (s, 9H), 1.36 (m, 4H), 1.23 (br s, 10H),
0.85 (t, 3H, J = 5.1 Hz); 13C NMR (75 MHz, DMSO-d6) d 171.72,
149.95, 148.95, 138.43, 105.83, 84.06, 38.35, 34.92, 31.26, 29.16,
28.71, 27.53, 27.22, 26.43, 24.10, 22.12, 13.98; HRMS (ESI) calcd
for C20H37N4O3 (MH)+ 381.2860, found 381.2861.
4-(2-Amino-1H-imidazol-4-yl)-N-decyl-butyramide hydrochlo-
ride (11). Using the same general procedure as used for the
synthesis of 8, 2-amino-4-(3-decylcarbamoyl-propyl)-imidazole-
1-carboxylic acid tert-butyl ester 26 (32 mg, 0.078 mmol) gave 11
(27 mg, 99%) as a white solid. 1H NMR (400 MHz, DMSO-d6) d
12.07 (s, 1H), 11.64 (s, 1H), 7.85 (s, 1H), 7.32 (br s, 2H), 6.56 (s,
1H), 3.00 (q, 2H, J = 6.4 Hz), 2.38 (t, 2H, J = 7.2 Hz), 2.07 (t, 2H,
J = 7.2 Hz), 1.73 (quint., 2H, J = 7.2 Hz), 1.36 (m, 2H), 1.23 (s,
14H), 0.85 (t, 3H, J = 7.2 Hz); 13C NMR (100 MHz, DMSO-d6)
d 171.33, 146.72, 126.36, 108.70, 38.42, 34.41, 31.32, 29.15, 29.04,
28.99, 28.77, 28.73, 26.45, 23.89, 23.62, 22.12, 13.99; HRMS (ESI)
calcd for C17H33N4O (MH)+ 309.2648, found 309.2647.
2-Amino-4-(3-dodecylcarbamoyl-propyl)-imidazole-1-carboxylic
1
acid tert-butyl ester (27). White solid (44 mg, 28%). H NMR
(400 MHz, DMSO-d6) d 7.73 (t, 1H, J = 5.6 Hz), 6.50 (s, 1H),
6.38 (s, 2H), 3.00 (q, 2H, J = 5.6 Hz), 2.21 (t, 2H, J = 7.6 Hz),
2.04 (t, 2H, J = 7.6 Hz), 1.71 (quint., 2H, J = 7.6 Hz), 1.53 (s,
9H), 1.36 (m, 2H), 1.23 (s, 18H), 0.85 (t, 3H, J = 6.0 Hz); 13C
NMR (75 MHz, DMSO-d6) d 171.58, 149.77, 148.88, 138.42,
105.76, 83.98, 38.28, 34.90, 31.18, 29.06, 28.88, 28.58, 27.47,
27.18, 26.29, 24.04, 21.96, 13.81, 13.27; HRMS (ESI) calcd for
C24H45N4O3 (MH)+ 437.3486, found 437.3487.
4-(2-Amino-1H-imidazol-4-yl)-N-dodecyl-butyramide
hydro-
chloride (12). Using the same general procedure as used for
the synthesis of 8, 2-amino-4-(3-dodecylcarbamoyl-propyl)-
imidazole-1-carboxylic acid tert-butyl ester 27 (20 mg,
0.046 mmol) gave 12 (16 mg, 94%) as a white solid. 1H
NMR (400 MHz, DMSO-d6) d 12.03 (s, 1H), 11.60 (s, 1H), 7.83
(t, 1H, J = 6.4 Hz), 7.31 (s, 2H), 6.56 (s, 1H), 3.00 (q, 2H, J =
6.4 Hz), 2.38 (t, 2H, J = 7.2 Hz), 2.07 (t, 2H, J = 7.2 Hz), 1.73
(quint., 2H J = 7.2 Hz), 1.36 (m, 2H), 1.23 (s, 18H), 0.85 (t, 2H,
J = 6.4 Hz); 13C NMR (75 MHz, DMSO-d6) d 171.20, 146.66,
126.34, 108.59, 38.34, 34.32, 34.32, 31.15, 29.02, 28.86, 28.6,
4-(2-Amino-1H-imidazol-4-yl)-N-isobutyl-butyramide hydro-
chloride (8). A solution of 2-amino-4-(3-isobutylcarbamoyl-
propyl)-imidazole-1-carboxylic acid tert-butyl ester 23 (76 mg,
0.234 mmol) in anhydrous dichloromethane (1 mL) was cooled
to 0 ◦C. TFA (1 mL) was charged into the flask and the reaction
stirred for 5 h. After that time the reaction was evaporated to
dryness and toluene (2 mL) was added. Again the mixture was
concentrated and the process repeated. The resulting TFA salt
1362 | Org. Biomol. Chem., 2008, 6, 1356–1363
This journal is
The Royal Society of Chemistry 2008
©

28.55, 26.31, 23.78, 23.52, 21.94, 13.79; HRMS (ESI) calcd for
C19H37N4O (MH)+ 337.2961, found 337.2964.
(pre-doctoral fellowship to T.E.B.). We thank Dr Reza Ghiladi
(NCSU) for spectroscopic assistance.
4-(2-Amino-1H-imidazol-4-yl)-N-cyclopentyl-butyramide hy-
drochloride (13). Using the same general procedure as used
for the synthesis of 8, 2-amino-4-(3-cyclopentylcarbamoyl-
propyl)-imidazole-1-carboxylic acid tert-butyl ester 28 (100 mg,
0.297 mmol) gave 13 (78 mg, 96%) as a pale yellow foam. 1H NMR
(400 MHz, DMSO-d6) d 11.99 (s, 1H), 11.57 (s, 1H), 7.79 (d, 1H,
J = 7.2 Hz), 7.30 (s, 2H), 6.56 (s, 1H), 3.97 (m, 1H), 2.37 (t, 2H,
J = 7.2 Hz), 2.05 (t, 2H, J = 7.2 Hz), 1.69–1.79 (m, 4H), 1.59 (m,
2H), 1.47 (m, 2H), 1.31 (m, 2H); 13C NMR (100 MHz, DMSO-d6)
d 170.97, 146.74, 126.38, 108.72, 50.11, 34.36, 32.31, 23.81, 23.45,
23.62; HRMS (ESI) calcd for C12H21N4O (MH)+ 237.1709, found
237.1711.
References
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4-(2-Amino-1H-imidazol-4-yl)-1-morpholin-4-yl-butan-1-one
hydrochloride (14). Using the same general procedure as
used for the synthesis of 8, 2-amino-4-(4-morpholin-4-yl-4-oxo-
butyl)-imidazole-1-carboxylic acid tert-butyl ester 29 (44 mg,
0.133 mmol) gave 14 (25 mg, 70%) as a tan solid. 1H NMR
(400 MHz, DMSO-d6) d 12.1 (s, 1H), 11.64 (s, 1H), 7.33 (s, 2H),
6.58 (s, 1H), 3.54 (m, 4H), 3.42 (m, 4H), 2.43 (t, 2H, J = 7.2 Hz),
2.33 (t, 2H, J = 7.2 Hz), 1.75 (quint., 2H, J = 7.2 Hz); 13C NMR
(75 MHz, DMSO-d6) d 170.27, 146.72, 126.39, 108.61, 66.04,
45.28, 31.05, 23.56, 23.17; HRMS (ESI) calcd for C11H19N4O2
(MH)+ 239.1502, found 239.1503.
11 S. R. Kelly, E. Garo, P. R. Jensen, W. Fenical and J. R. Pawlik, Aquat.
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4-(2-Amino-1H-imidazol-4-yl)-N-phenyl-butyramide hydrochlo-
ride (15). Using the same general procedure as used for the
synthesis of 8, 2-amino-4-(3-phenylcarbamoyl-propyl)-imidazole-
1-carboxylic acid tert-butyl ester 30 (80 mg, 0.232 mmol) gave 15
1
(64 mg, 99%) as a tan solid. H NMR (400 MHz, DMSO-d6) d
12.03 (s, 1H), 11.60 (s, 1H), 9.98 (s, 1H), 7.59 (d, 2H, J = 8.0 Hz),
7.33 (br s, 2H), 7.28 (t, 2H, J = 8.0 Hz), 7.02 (t, 1H, J = 7.6 Hz),
6.61 (s, 1H), 2.44 (m, 2H), 2.32 (t, 2H, J = 6.8 Hz), 1.85 (m, 2H);
13C NMR (75 MHz, DMSO-d6) d 170.65, 146.77, 139.25, 137.24,
128.59, 126.40, 123.00, 119.13, 108.83, 35.33, 23.60, 23.51; HRMS
(ESI) calcd for C13H17N4O (MH)+ 245.1396, found 245.1401.
4-(2-Amino-1H-imidazol-4-yl)-N -pyrimidin-2-yl-butyramide
hydrochloride (16). Using the same general procedure as used
for the synthesis of 8, 2-amino-4-[3-(pyrimidin-2-ylcarbamoyl)-
propyl]-imidazole-1-carboxylic acid tert-butyl ester 31 (50 mg,
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1
0.144 mmol) gave 16 (41 mg, 99%) as a white solid. H NMR
(400 MHz, DMSO-d6) d 12.06 (s, 1H), 11.64 (s, 1H), 10.59 (s, 1H),
8.64 (d, 2H, J = 4.8 Hz), 7.34 (s, 2H), 7.17 (t, 1H, J = 4.8 Hz),
6.60 (s, 1H), 2.51 (m, 2H), 2.46 (t, 2H, J = 7.2 Hz), 1.83 (quint.,
2H, J = 7.2 Hz); 13C NMR (75 MHz, DMSO-d6) d 158.17, 157.54,
146.73, 129.02, 126.33, 116.5, 108.71, 35.35, 23.48, 23.09; HRMS
(ESI) calcd for C11H15N6O (MH)+ 247.1301, found 247.1304.
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Acknowledgements
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1210–1223.
Financial support is gratefully acknowledged from NCSU,
Burroughs-Wellcome (pre-doctoral fellowship to J.J.R.) and GSK
32 C. W. Zapf, C. J. Creighton, M. Tomioka and M. Goodman, Org. Lett.,
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