Synthesis and antibiofilm activity of a second-generation reverse-amide oroidin library: A structure-activity relationship study

FULL PAPER

DOI: 10.1002/chem.200801419

Synthesis and Antibiofilm Activity of a Second-Generation Reverse-Amide

Oroidin Library: A Structure–Activity Relationship Study

T. Eric Ballard, Justin J. Richards, Amanda L. Wolfe, and Christian Melander*^[a]

Abstract: A second-generation library

of 2-aminoimidazole-based derivatives

incorporating a “reversed amide” (RA)

motif in comparison to the marine nat-

ural product oroidin were synthesized

and subsequently assayed for antibio-

film activity against the medically rele-

vant Gram-negative proteobacteria P.

aeruginosa and A. baumannii. Most no-

tably, an in-depth activity profile is re-

ported for the most active subclass of

derivatives that bear linear aliphatic

chains off the amide bond. Additional-

ly, further structural modifications of

the core template, such as removal of

the amide bond or substitution with a

triazole isostere, resulted in the discov-

ery of analogues with antibiofilm activ-

ities that varied with respect to their

inhibition and dispersal properties of P.

aeruginosa and A. baumannii biofilms.

Keywords: amino acids · biological

activity · drug design · medicinal

chemistry

·

structure–activity

relationships

Introduction

are much less susceptible to traditional means of eradication

including antiseptics and antibiotic therapy.^[5,6]Additionally,

given the combination of high morbidity and mortality rates

of infectious diseases due to biofilm virulence, there is a sig-

nificant need for new antibiofilm modulators.

It has been estimated that approximately 80% of the

worldꢀs microbial biomass resides in the biofilm state and

yet there are relatively few known naturally occurring mo-

lecular scaffolds that possess the ability to disrupt biofilm

development and maintenance (e.g., 1–3).^[1–4]Bacterial bio-

Antibiotic drug resistance is a matter of survival for bac-

teria and has been problematic since the beginning of heavy

administration of antibiotics in the mid 1900s. With the in-

troduction of penicillin, only several years were needed for

resistant bacterial strains to appear.^[7,8]Today, multidrug re-

sistant bacteria are becoming increasingly commonplace and

various antibiotics are being exhausted in attempts to

combat particularly aggressive infections.^[8–10]Additionally,

only two new classes of antibiotics (oxazolidinones and lipo-

peptides) have been discovered in the last 40 years.^[11]Van-

comycin and its congeners remain at the forefront of antibi-

otic therapy, but even vancomycin-resistant strains are be-

ginning to appear.^[8,12]

As most pathogenic bacteria exist as biofilm communities,

which hinder antibiotic effectiveness and facilitate the evo-

lution of resistant phenotypes,^[5,6]the discovery and develop-

ment of antibiofilm-modulating compounds should have an

impact on human medicine.^[13]Given that antibiofilm modu-

lators inhibit and/or disperse bacterial biofilms in a nontoxic

manner, there is significant potential for co-dosing antibio-

film compounds with an antibiotic to eliminate biofilm in-

fections. This approach may be particularly important to

treat cystic fibrosis (CF) patients, infection of indwelling

medical devices, and the remediation of hospital facilities.

Furthermore, the lack of induced microbicidal activity

films are often described as surface-associated complex com-

munities of bacteria encased in a protective extracellular

matrix.^[5]Pathogenic infections commonly persist due to the

respective bacteriaꢀs ability to form robust biofilms, which

[a] Dr. T. E. Ballard,⁺J. J. Richards,⁺A. L. Wolfe, Prof. Dr. C. Melander

Department of Chemistry

North Carolina State University

Raleigh, NC 27695-8204 (USA)

Fax : (+1)919-515-5079

E-mail: christian_melander@ncsu.edu

[⁺] Both authors contributed equally to this work.

Supporting information for this article is available on the WWW

under http://dx.doi.org/10.1002/chem200801419.

Chem. Eur. J. 2008, 14, 10745 – 10761

ꢁ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

10745

should limit or mitigate development of resistance to the an-

tibiofilm molecule.

cation especially problematic.^[30]Approximately 25% of all

hospital swabs are positive for A. baumannii.^[31]Reports of

antibiotic resistance to carbapenems^[32]and polymyxins^[33]

are becoming increasingly frequent. It is believed that this is

attributed to changes in porin proteins, development of

efflux pumps, and production of b-lactamases in the bacte-

ria.^[26,32]Biofilms easily facilitate these adaptations for sur-

vival through the selection and reproduction of bacteria that

can withstand harsh environmental pressures. Only a few

small molecules have been documented to modulate the

biofilm activity of A. baumannii and these have all originat-

ed from the oroidin scaffold.^[14,18–20]Therefore, screening our

second-generation RA library for inhibition and dispersion

activity against A. baumannii biofilms was also pursued.

Herein, we report the synthesis of a diverse 24-compound

second-generation RA library and its subsequent antibiofilm

properties against Pseudomonas aeruginosa and Acineto-

bacter baumannii. Analysis of the activity trends points to

important structural features that augment antibiofilm prop-

erties.

Our research^[14–20]has focused on the generation of small-

molecule libraries that borrow structural inspiration from

the oroidin family^[21]of marine natural products. We have

been able to identify effective antibiofilm modulators by ex-

ploiting the oroidin scaffold against a number of medically

relevant biofilm-forming bacteria (Scheme 1).^[14–20]Most no-

Results and Discussion

Design: The first-generation RA library identified the dode-

cyl-based RA analogue 7 (Table 1) as being both a potent

inhibitor and disruptor of P. aeruginosa biofilms (PA14 bio-

film inhibition IC₅₀=(2.3Æ0.83) mm, PA14 biofilm dispersion

EC₅₀=(21Æ3.9) mm).^[17]However, a more comprehensive

study was initiated to identify the optimal chain length for

antibiofilm potency (Table 1).

Scheme 1. Oroidin as inspiration for novel classes of biofilm modulators.

tably, the reverse-amide (RA) motif has been one of the

most potent structure classes formulated to date.^[17]After

the preliminary success of our pilot RA library, delineation

of a detailed structure–activity relationship (SAR) study

was deemed as an attractive approach to generate antibio-

film compounds with superior activity.

Table 1. Examining the aliphatic chain length.

Previously, the first-generation RA library was screened

against PAO1 and PA14, two of the most commonly studied

strains of the bacterium Pseudomonas aeruginosa. P. aerugi-

nosa is an opportunistic Gram-negative g-proteobacteria

that is relatively innocuous to healthy individuals and is

ubiquitous throughout the environment.^[22]However, P. aer-

uginosa is the second most common pathogenic bacteria in

hospital-acquired pneumonia and is a serious threat to cystic

fibrosis (CF) patients.^[23–27]A combination of genetic disposi-

tion and the abnormal composition of respiratory airways in

CF patients in conjunction with the virulence of P. aerugino-

sa commonly leads to chronic infections that drive increased

morbidity and mortality rates.^[28]The inability to treat CF

patients with chronic P. aeruginosa infections has been di-

rectly correlated to the virulence of P. aeruginosa bio-

films.^[24,26]

Another opportunistic g-proteobacterium that has

become a serious threat over the last decade due to its mul-

tidrug resistance is Acinetobacter baumannii.^[29]This Gram-

negative bacterium is also benign to healthy individuals, but

onset of an A. baumannii infection can be life-threatening.

A. baumannii biofilms are particularly hardy, being able to

survive for weeks on inanimate objects, thus making eradi-

1st generation

Proposed 2nd generation

4 (n=5)

5 (n=7)

6 (n=9)

7 (n=11)

8 (n=10)

9 (n=12)

10 (n=13)

11 (n=15)

12 (n=17)

Following the completion of the chain-length study, our

design for a second-generation RA library would be roughly

based on derivatization around the hexyl RA analogue 4,

the synthesis and activity of which was also previously dis-

closed.^[17]This analogue was chosen as a template for addi-

tional SAR analysis due to its moderate activity profile

(PA14 IC₅₀=(40Æ13) mm), availability, and overall ease of

synthesis across the desired scaffolds for SAR consideration.

The SAR for 4 was divided into six separate themes: dele-

tion of the amide bond, linker chain modification, sliding of

the amide bond, increased substitution of the amide bond,

reversal of the amide bond directionality to mimic oroidin,

and examination of a triazole isostere (Scheme 2).

10746

www.chemeurj.org

ꢁ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Chem. Eur. J. 2008, 14, 10745 – 10761

A Second-Generation Reverse-Amide Oroidin Library

FULL PAPER

to ensure that the antibiofilm

activity was not a result of a

bactericidal effect. Bacterial-

cell densities remained the

same throughout a 24 h time

period (Supporting Informa-

tion).

These results show that by

incrementally increasing the

aliphatic chain length for the

RA scaffold, we were able to

identify one of the few nano-

molar inhibitors of biofilm for-

mation that have been dis-

closed.^[19,34]It is also worth

noting the correlation between

the aliphatic chain length and

Scheme 2. Core approaches to tuning the RA scaffold for antibiofilm activity.

Initially, it was planned to evaluate all of the target ana-

logues against Pseudomonas aeruginosa PA14 biofilms.

When active compounds were identified, IC₅₀values would

then be determined. Additionally, the most active analogues

in each subclass would then be tested for antibiofilm inhibi-

tion activity against Acinetobacter baumannii biofilms. Pend-

ing the identification of active compounds in these screens,

A. baumannii IC₅₀values would be determined. Finally, the

most active analogues would then be assayed for their abili-

ty to disperse established PA14 and/or A. baumannii bio-

films.

Table 2. Synthesis and antibiofilm activity of second-generation aliphatic

RA analogues.^[a]

Optimizing the aliphatic RA chain length: The first goal of

the study was to further investigate the full extent of anti-

biofilm activity attainable by tuning the length of the ali-

phatic side chain. To this end, we synthesized compounds

10–12 as their HCl salts following our previously established

chemistry^[17](Table 2).

Amine

Coupled product

(yield [%])

Target

ACHTUNGTRENUN(NG yield [%])

PA14 IC₅₀[mm]

U

The hexadecyl- (11) and octadecyl-based (12) analogues

displayed modest antibiofilm activity (PA14 IC₅₀values of

50 and 110 mm, respectively). Tetradecyl RA analogue 10

was slightly less active than 7 with a PA14 IC₅₀=(2.9Æ

1.2) mm, from which can be concluded that the highest activi-

ty for the RA scaffold should be a tridecyl RA analogue.

With that hypothesis, the tridecyl RA analogue 9 was syn-

thesized and assayed for its ability to inhibit PA14 biofilm

formation (Table 2). As anticipated, the tridecyl RA ana-

logue 9 was a potent antibiofilm modulator with an IC₅₀

value of (729Æ85) nm against PA14. This makes 9 over

threefold more active than 7 in reference to inhibition of

PA14 biofilms and brings the activity profile of this class of

compounds into the high nanomolar region. Analogue 8 was

then synthesized to complete the aliphatic trend and corre-

sponded well with the previously reported aliphatic data

(PA14 IC₅₀=(4.3Æ0.67) mm) as it was slightly less active

than the dodecyl RA analogue 7, but slightly more active

than the decyl RA analogue 6. This structure–activity rela-

tionship is graphically depicted in Figure 1. Growth curves

were performed in the presence and absence of 9 with PA14

tetradecylamine

hexadecylamine

octadecylamine

undecylamine

tridecylamine

13 (24)

14 (19)

15 (13)

16 (31)

17 (25)

10 (92)

11 (92)

12 (94)

8 (98)

2.9Æ1.2

50

110

4.3Æ0.67

9 (99)

0.73Æ0.08

[a] Reaction conditions: a) EDC, HOBt, DMF; b) TFA, CH₂Cl₂; c) 2m

HCl/Et₂O.

the activity profile of the aliphatic RA analogues (Figure 1).

Based upon this data, it would seem probable that these ali-

phatic RA analogues are eliciting their activity through

binding a hydrophobic pocket. Surpassing the optimum

chain length displays a rapid loss of activity as the analogues

would loose their affinity for the binding pocket.

Lastly, it is important to point out that all of the identified

aliphatic RA derivatives are still more active than the

parent natural product oroidin.^[16,17]Having now identified

the most active aliphatic RA side chain and the break-point

in activity, interest turned to how modifications of the core

scaffold would tune antibiofilm activity with respect to the

derivatization of the hexyl RA analogue 4.

Chem. Eur. J. 2008, 14, 10745 – 10761

ꢁ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

10747

C. Melander et al.

Specifically, deletion of the amide bond in the hexyl RA an-

alogue 4 would yield undecyl fatty acid analogue 19

(Scheme 3). Several unsaturated analogues were also pre-

pared. The 2-AI targets were quickly accessed as outlined in

Scheme 3. Briefly, the acid chlorides were homologated with

diazomethane followed by quenching with concentrated

HBr to afford the requisite a-bromoketones. The a-bromo-

ketones were then condensed with Boc-guanidine in the

presence of NaI followed by deprotection with acid and salt

exchange to afford the target compounds.

Antibiofilm assessment of these analogues lacking the

amide bond exhibited a range of potencies. The isopropyl-

based fatty acid 25 exhibited modest activity, inhibiting bio-

film formation 48% at 100 mm. The most active analogue

obtained was the nonyl 2-AI fatty acid 18 with a PA14 IC₅₀

value of (14Æ2.2) mm (Table 3). This was followed closely by

Figure 1. Correlation of aliphatic RA analogues and PA14 biofilm inhibi-

tion.

Deletion of the amide bond: From a synthetic standpoint,

removal of the amide bond has a great advantage in simpli-

fying the synthetic sequence for this class of compounds,

most notably circumventing the low-yielding amide bond

formation step. A quick search of commercially available

acid chlorides revealed a large number of building blocks

that, when derivatized, would be directly comparable to the

RA scaffold. It also allowed for additional functionality,

such as points of unsaturation along the chains, to be incor-

porated into the molecules. Ultimately, if these compounds

were to show promise, additional analogue development

could easily be executed through further manipulation of

the double bond.^[35,36]

Table 3. Antibiofilm activity of an amide deletion subclass against PA14.

18 (n=8)

19 (n=10)

20 (n=12)

IC₅₀=(14Æ2.2) mm

IC₅₀=(18Æ0.70) mm

IC₅₀=(18Æ1.3) mm

the undecyl and tridecyl analogues (IC₅₀values of (19Æ

0.70) and (18Æ1.3) mm, respectively). With the success of

the shorter-chain derivatives (18–20), longer analogues were

analyzed (21 and 22), including several unsaturated deriva-

tives (23 and 24), but these analogues were completely inac-

tive toward inhibiting PA14 biofilms at 100 mm. Analogue 18

was chosen for PA14 growth-curve analysis to represent this

class and in the presence or ab-

Starting with a sampling of commercially available acid

chlorides, we elected to synthesize several analogues that

would mimic the aliphatic RA derivatives in overall length.

sence of 18, PA14 cellular den-

sities remained the same

throughout a 24 h time period

(Supporting Information).

In summary, removal of the

amide bond from the aliphatic

chain of the RA scaffold is not

entirely detrimental to antibio-

film activity. The undecyl 2-AI

fatty acid 19 is directly compa-

rable to the hexyl RA deriva-

tiveꢀs overall chain length, thus

showing that removal of the

amide bond led to activity that

was well over twofold more

active than hexyl analogue 4.

Two additional derivatives

were also shown to be potent

inhibitors of PA14 biofilm for-

mation. Although these com-

pounds are significantly less

active then tridecyl RA ana-

logue 9, simplification of the

Scheme 3. Deletion of the amide bond: a) i) CH₂N₂, Et₂O/CH₂Cl₂, 08C; ii) conc. HBr; b) Boc-guanidine, NaI,

DMF; c) TFA, CH₂Cl₂; d) 2m HCl/Et₂O. Boc: tert-butoxycarbonyl; TFA: trifluoroacetic acid.

10748

www.chemeurj.org

ꢁ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Chem. Eur. J. 2008, 14, 10745 – 10761

A Second-Generation Reverse-Amide Oroidin Library

FULL PAPER

Table 4. Synthesis and antibiofilm activity of modified linker analogues.^[a]

synthetic approach allows rapid access to compounds of this

class.

Linker modification: Examination of the majority of com-

pounds comprising the oroidin class of natural products

often reveals a three-carbon methylene linker between the

2-aminoimidazole (2-AI) head group and the pyrrole car-

boxamide group.^[16]This three-carbon linker was previously

confirmed to be required for maximum biological activity by

a thorough oroidin template SAR in the context of P. aeru-

ginosa antibiofilm activity.^[16]Although this aspect was not

detailed in our previous RA work (which was solely based

on a three-carbon linker), we felt it necessary to revisit this

structural modification and investigate the spacer effect in

the RA class of molecules. Therefore, the two- and four-

carbon Boc-2-AI carboxylic acid homologues necessary for

amide bond coupling were synthesized (Scheme 4). Briefly,

succinic anhydride was opened with benzyl alcohol followed

by diazomethane/a-bromo homologation of the acid chlo-

ride to afford the a-bromo succinic benzyl ester. The a-bro-

moketone was then condensed with Boc-guanidine followed

by hydrogenolysis of the benzyl ester to afford the two-

carbon Boc-2-AI carboxylic acid 26. Similarly, the four-

carbon homologue was synthesized from the known mono-

benzyl ester of dipentanoic acid following the same proce-

dure to deliver the intermediate acid 27 (Scheme 4).^[37]

With these scaffolds in hand, coupling to hexylamine

under carbodiimide conditions afforded derivatives compa-

rable to the hexyl RA analogue 4 in that they were either a

carbon atom shorter (28) or longer (29) in overall length

from the 2-AI motif (Table 4). The two-methylene-unit ana-

logue 28 had much lower activity than the parent three-

carbon linker, displaying only 32% inhibition at 100 mm.

Similarly, the four-methylene-unit linker 29 only inhibited

PA14 biofilm formation 28% at 100 mm (Table 4).

Following the use of hexylamine as the coupling partner

and lack of observed potencies with both linker homologues,

we elected to investigate a few additional amines employing

the two-carbon scaffold due to availability (Table 5). The

two compounds prepared were chosen to probe whether a

phenyl ring could mimic the activity of the aliphatic chains

and recapitulate potency. It was anticipated that the p-

bromo phenethyl RA analogue 30 would follow the same

trend observed in other studies with increasing antibiofilm

Target

Biofilm inhibition at 100 mm vs PA14 [%]

28

29

32Æ5

28Æ5

[a] Reaction conditions: a) hexylamine, EDC, HOBt, DMF; b) TFA,

CH₂Cl₂; c) 2m HCl/Et₂O.

Table 5. Synthesis and antibiofilm activity of phenylamine analogues.^[a]

Target

PA14 IC₅₀[mm]

30

31

15Æ3.9

59Æ15

[a] Reaction conditions: a) EDC, HOBt, DMF; b) TFA, CH₂Cl₂; c) 2m

HCl/Et₂O.

activity correlating to an increased degree of bromina-

tion.^[14,16]Analogue 30 exhibited potent activity with an IC₅₀

value of (15Æ3.9) mm against PA14, whereas the phenbutyl

RA analogue 31 was less active with an IC₅₀value of (59Æ

15) mm. Again, to demonstrate the nontoxic nature of these

analogues on planktonic bacterial growth, growth-curve ex-

periments with the p-bromo analogue 30 were performed.

Both the treated and untreated cell densities remained iden-

tical during a 24 h time period (Supporting Information).

Ultimately either the addition or removal of a single

methylene unit to the linker of the RA scaffold with the

hexylamide drastically reduced antibiofilm potency. Howev-

er, it was shown that activity could be regained with the ad-

dition of a phenyl ring. Com-

pounds possessing increasing

degrees of bromination similar

to 30 along with other various-

ly substituted ring systems are

currently being formulated and

evaluated for antibiofilm prop-

erties to further probe this ob-

servation.

Sliding the amide bond: We

envisioned using the hexyl RA

analogue as our starting point

Scheme 4. Synthesis of Boc-2-AI linker acids: a) BnOH, TEA, DMAP, CH₂Cl₂(87%); b) i) (COCl)₂, DMF

(cat.), CH₂Cl₂; ii) CH₂N₂, Et₂O/CH₂Cl₂, 08C; iii) conc. HBr; c) Boc-guanidine, DMF; d) H₂(1 atm), 10% Pd/C,

THF. DMAP: 4-dimethylaminopyridine; TEA: triethylamine.

Chem. Eur. J. 2008, 14, 10745 – 10761

ꢁ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

10749

C. Melander et al.

and sliding the amide bond one carbon atom in each direc-

tion along the chain (Scheme 5). This would be complemen-

tary to the linker-chain modification study detailed above.

By using the aforementioned two- and four-carbon homo-

al aliphatic chain to the amide moiety in anticipation that it

would be able to further enhance activity.

Therefore, both a secondary and tertiary dihexyl RA de-

rivative were synthesized to simultaneously test for the

affect of additional aliphatic chains and the necessity of the

NH amide proton for antibiofilm activity. The synthesis of

the secondary dihexyl derivatives are outlined in Scheme 6.

Scheme 5. Sliding the amide bond.

logue carboxylic acids, 26 was coupled to heptylamine

whereas 27 was coupled to pentylamine to afford the two

analogues both with identical total chain lengths in refer-

ence to the parent hexyl RA analogue 4 (Table 6). Evalua-

Scheme 6. Synthesis of additionally substituted analogues: a) EDC,

HOBt, DMF; b) TFA, CH₂Cl₂; c) 2m HCl/Et₂O; d) dihexylamine, TEA,

DMAP, CH₂Cl₂(87%); e) i) (COCl)₂, DMF (cat.), CH₂Cl₂; ii) CH₂N₂,

Et₂O/CH₂Cl₂, 08C; iii) conc. HBr; f) Boc-guanidine, DMF (27% over

2 steps); g) TFA, CH₂Cl₂; h) 2m HCl/Et₂O (94%). EDC: N’-(3-dimethyl-

AHCTUNGERTGaNNUN minopropyl)-N-ethylcarbodiimide; HOBt: 1-hydroxybenzotriazole.

Table 6. Synthesis and antibiofilm activity of slider analogues.^[a]

Coupling the three- and two-carbon carboxylic acids with

the known branched secondary hexaheptylamine^[38]under

EDC conditions followed by deprotection and salt exchange

afforded the target secondary dihexyl RA analogues 34 and

35. Synthesis of the tertiary dihexyl RA analogue 36 was

executed through opening of glutaric anhydride with dihex-

ylamine and transforming the resulting acid intermediate

into the final 2-AI target.

Upon biological evaluation of these analogues, the terti-

ary derivative 36 displayed moderate inhibitory activity

against PA14 biofilms with an IC₅₀value of (60Æ12) mm

(Table 7). It is notable that within the experimental errors

Target

Biofilm inhibition at 100 mm vs PA14 [%]

PA14 IC₅₀[mm]

32

33

74Æ2

24Æ5

43Æ8

n.d.

[a] Reaction conditions: a) EDC, HOBt, DMF; b) TFA, CH₂Cl₂; c) 2m

HCl/Et₂O. n.d.=not determined.

Table 7. Antibiofilm activity of additionally substituted RA analogues.

tion of these two analogues for biofilm inhibitory activity

against PA14 showed that the two-carbon analogue 32

(PA14 IC₅₀=(43Æ8) mm) was only slightly less active than

the parent three-carbon hexyl RA 4, but this difference was

negligible due to associated error (Table 6). The four-carbon

analogue 33 was significantly less active, only being able to

inhibit PA14 biofilm formation 24% at 100 mm.

Increased substitution: Long aliphatic amides on a RA 2-AI

scaffold afforded compounds that were extremely active at

inhibiting PA14 biofilm formation.^[17]Increased molecular

diversity can be accessed through installation of an addition-

PA14 IC₅₀[mm]

34: 18Æ1.3

35: 16Æ1.8

36: 60Æ12

10750

www.chemeurj.org

ꢁ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Chem. Eur. J. 2008, 14, 10745 – 10761

A Second-Generation Reverse-Amide Oroidin Library

FULL PAPER

reported, this activity is almost identical to the monohexyl

RA analogue 4. Evaluation of the three-carbon secondary

dihexyl RA analogue 35 against inhibiting PA14 biofilms,

however, gave an IC₅₀value of (16Æ1.8) mm that was over

twofold more active than the monohexyl RA analogue. The

two-carbon secondary dihexyl RA analogue 34 was also

active at inhibiting PA14 biofilms with an IC₅₀=(18Æ

1.3) mm, essentially identical to the three-carbon homologue.

Growth curve experiments with compound 35 validated that

cellular densities remained identical over a 24 h time period

(Supporting Information). Clearly, activity increases with

the increased substitution alpha to the amide, and activity is

still retained when the NH amide proton is replaced with an

aliphatic chain in this subset of compounds. Furthermore, it

is anticipated that future analogues, such as those with

longer disubstituted aliphatic chains (i.e., di-tridecyl), will

also display increased potency in comparison to those con-

taining just a single aliphatic chain.

a hydrogen atmosphere followed by in situ acylation em-

ploying heptanoic anhydride to afford the native amide ana-

logue 39 in a high yield over two steps (70%). This pathway

also allows access to a Boc-protected derivative of 37, which

is not accessible through the established routes to this inter-

mediate. Deprotection afforded 39, the native amide hexyl

analogue of 4, thus producing a compound that contains the

amide directionality seen in the natural products.

Assays performed by investigating this analogue against

PA14 proved very interesting as it was completely inactive

within the concentrations tested, yielding <5% biofilm in-

hibition at 100 mm. This result is in stark contrast to the ac-

tivity reported for 4 and is very instructive in understanding

the impact of amide directionality coupled with an appropri-

ate aliphatic chain on antibiofilm properties. Further devel-

opment and tuning of this methodology is currently under

investigation to increase chemical diversity and antibiofilm

properties around this portion of the molecule.

A native amide approach: Both amide bond deletion and

additional substitution of the amide functionality were ef-

forts to elucidate if the aforementioned region of the mole-

cule was critical to eliciting antibiofilm activity. Some modi-

fications in structure are well tolerated within the context of

antibiofilm activity. Throughout the synthetic efforts on this

project, one aspect that we had not addressed was the con-

struction of dihydrooroidin analogues with aliphatic side

chains that contained the amide in the native orientation.

Evaluation of these derivatives is complicated by the lack of

synthetic methods to rapidly acylate a core 2-AI derivative

to generate molecular diversity. Typical methods to acylate

core 2-AI derivatives, such as 37, rely on the use of trichlor-

oesters as coupling partners.^[21]The use of 4-(3-aminoprop-

yl)-2-aminoimidazole 37 in acylation

Examination of a triazole isostere: En route to synthesizing

the natural amide hexyl analogue, it was also observed that

construction of a triazole isostere was possible through the

utilization of the pendant azide functionality.^[40]The overall

dipolar moment of a triazole system is larger than that of an

amide bond, thus making its hydrogen-bonding donor and

acceptor properties more pronounced.^[41,42]Due to the

recent success of a small library of 2-AI/triazole conjugates

in inhibiting and dispersing various medically relevant bio-

films,^[19]an analogue of this nature would be a valuable ad-

dition to the current SAR study. To this end, the Boc-2-AI

azide 38 was reacted with 1-octyne in a [3+2] copper-cata-

lyzed click reaction followed by deprotection to smoothly

afford the triazole hexyl isostere 40 (Table 8). The triazole

reactions involving the oroidin class

of molecules leaves much to be de-

Table 8. Synthesis and antibiofilm activity of a triazole isostere.^[a]

sired in terms of yields and ease of

purification.^[16]Upon close examina-

tion of the literature, a new approach was developed to cir-

cumvent these problems while also again taking advantage

of the ability to synthesize a large number of chemically

unique derivatives. The idea was to develop acylation

chemistry around a masked amine relative of our estab-

lished Boc-2-AI carboxylic acid intermediates (i.e., 26 and

27).

Target

PA14 IC₅₀[mm]

27Æ4

40

[a] Reaction conditions: a) 1-octyne, CuSO₄·5H₂O (20 mol%), Na Ascor-

bate (10 mol%), H₂O/EtOH 1:1 (55%); b) TFA, CH₂Cl₂; c) 2m HCl/

Et₂O (99%).

Advancement of the known^[39]4-azido-butyric acid pro-

ceeded smoothly to afford the Boc-2-AI azide 38

(Scheme 7). This intermediate was reduced with Pd/C under

analogue 40 was observed to possess an IC₅₀value of (27Æ

4) mm, allowing for retainment of activity in comparison with

4. A PA14 growth curve was

also performed in the presence

and absence of 40. Over a 24 h

time period, both the treated

and untreated cell densities re-

mained identical (Supporting

Information). Additional libra-

ries taking advantage of both

the speed and diversity of the

Scheme 7. A native amide bond approach: a) i) (COCl)₂, DMF (cat.), CH₂Cl₂; ii) CH₂N₂, Et₂O/CH₂Cl₂, 08C;

iii) conc. HBr (90%); b) Boc-guanidine, DMF (63%); c) i) H₂(1 atm), 10% Pd/C, THF; ii) heptanoic anhy-

dride (70%); d) TFA, CH₂Cl₂; e) 2m HCl/Et₂O (99%).

Chem. Eur. J. 2008, 14, 10745 – 10761

ꢁ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

10751

C. Melander et al.

click reaction with the newly developed scaffold 38 are cur-

rently being generated.

logues were less active than the parent three-carbon hexyl

RA analogue.

While sliding the amide bond and changing the chain

length tended to reduce activity, increasing the substitution

adjacent to the amide bond tended to cause an increase in

activity. Synthesis of the secondary dihexyl derivative 35 in-

creased the PA14 biofilm inhibition activity twofold. While

this may be attributable to an

PA14 biofilm inhibition: Activity profile comparisons: A

summary of the results obtained via amide bond modifica-

tions is depicted in Scheme 8. The most revealing observa-

tion about the current RA SAR study (Scheme 8) was the

increase in the longest chain (7

C atoms for 35, 6 C atoms for

4), the activity corresponds

closely to that of the octyl RA

analogue, which indicates that

the increased substitution is

modulating the activity.^[17]The

tertiary-amide derivative 36 is

only slightly less active then

the monohexyl RA analogue.

Deletion of the amide bond

provided several active fatty

acid derived 2-AI analogues

(i.e., 19), which displayed in-

creased activity in comparison

to their RA relatives. There

was, however, a clear cut-off

point for the potency of this

class of molecules as only the

Scheme 8. PA14 inhibition IC₅₀values from selected SAR analogues.

shorter-chain saturated deriva-

tives were active.

lack of activity exhibited by the native amide hexyl conge-

ner 39 relative to the RA derivative 4. The native amide 39

was completely inactive, and unable to inhibit PA14 biofilm

formation even at the highest concentration tested (100 mm).

This result confirms that the directionality of the amide

bond can have a significant impact on antibiofilm activities.

Interestingly, 32 (which bears the amide carbonyl towards

the side of the 2-AI motif) was an active biofilm modulator,

in stark contrast to 39 (Scheme 9).

Additionally, exchange of the amide bond with a triazole

surrogate to produce 40 showed that activity could be slight-

ly enhanced with this substitution. These observations again,

may be hinting towards a crucial placement of the amide

carbonyl/triazole moiety to elicit a maximum biological

effect through possible intramolecular or intermolecular in-

teractions. Both the two- (32) and four-carbon (33) ana-

Acinetobacter baumannii biological evaluation: To this

point, we have exclusively employed PA14 to evaluate the

current library as antibiofilm agents. To determine the

broad spectrum of activity of the compounds, it was deemed

necessary to assay our most active and structurally diverse

analogues against the Gram-negative g-proteobacterium

Acinetobacter baumannii.

Nine of the most active analogues obtained from the

Pseudomonas biofilm inhibition assays underwent a prelimi-

nary screen for biofilm inhibition activity against A. bau-

mannii (Table 9). Three of these derivatives (30, 36, and 40)

showed only marginal activity at 100 mm, which was opposed

to the potency observed for these analogues against PA14.

Tridecyl RA analogue 9, the most active compound with ac-

tivity in the high nanomolar range against PA14, was also

noticeably less potent against A. baumannii (IC₅₀=(26Æ

3) mm). The analogues derived from the subclass of com-

pounds in which the amide bond had been removed (18–20)

displayed the greatest activity out of any of the compounds

assayed against Acinetobacter. It was observed that as the

chain length increased, potency increased culminating in the

most potent derivative 20 (IC₅₀=(13Æ0.70) mm). This trend

is opposite to that observed for PA14 inhibition activity, as

the shortest-chain fatty acid derivative 18 was the most

potent.

Growth curves were then performed with several of the

active compounds (9, 19, 20, and 35) at their respective IC₅₀

Scheme 9. Amide orientation comparison.

10752

www.chemeurj.org

ꢁ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Chem. Eur. J. 2008, 14, 10745 – 10761

A Second-Generation Reverse-Amide Oroidin Library

FULL PAPER

Table 9. Antibiofilm activity against A. baumannii.

A. baumannii antibiofilm-modulating properties of the RA

class of compounds may be due, in part, to bacteriostatic ef-

fects, their activity lends hope to the RA libraryꢀs ability to

possibly inhibit other biofilms formed by medically relevant

bacteria, thus solidifying their importance as novel small

molecules in the antibiofilm arena.

Dispersion of established biofilms: Perhaps a more impor-

tant characteristic of small molecules known to inhibit bio-

film formation is their ability to disperse pre-existing bio-

films (Table 10). This is significant from a biomedical stand-

Table 10. Selected compounds with biofilm dispersing properties.

Analogue

Biofilm inhibition at 100 mm

vs A. baumannii [%]

A. baumannii IC₅₀[mm]

9

ꢀ95

26Æ3

43Æ1

13Æ0.70

8.0Æ0.32

n.d.

51Æ1

38Æ1

n.d.

18

19

20

30

34

35

36

40

ꢀ95

18Æ3

ꢀ95

34Æ4

22Æ4

n.d

Analogue P. aeruginosa: PA14 dispersion

A. baumannii dispersion

100 mm [%]

EC₅₀[mm]

100 mm [%]

EC₅₀[mm]

values. Surprisingly, the compounds seemed to be eliciting

some of their antibiofilm properties through a bacteriostatic

effect, as evidenced by the lack of bacterial growth for the

bacteria that were dosed with compound. A. baumannii

growth was suppressed by the analogues for at least 9 h, but

by the 24 h time-point, the treated cell densities were nearly

the same as the untreated cell densities. This led to growth

curve experiments performed at the analogues respective

IC₃₀values (9: 15, 19: 10, 20: 6.3, 35: 37 mm) as a means to

determine the toxicity of these compounds at lower concen-

trations. At the IC₃₀value, growth was evident after only

3 h, but was still slower relative to untreated samples. After

24 h at the IC₃₀, the cell densities were, again, relatively

identical to the untreated cell densities. This appears to indi-

cate that these compounds have a very small window be-

tween inhibiting biofilm formation and inhibiting planktonic

A. baumannii growth. Overall, these compounds are still

eliciting a biofilm inhibitory effect that is unique to this

class of compounds. Their A. baumannii biofilm inhibitory

activity may be modulated by slight bacteriostatic effects,

but during the course of the assay (24 h), the treated bacte-

ria have enough time to become homeostatic with respect to

the untreated controls as evidenced by the growth curve.

Despite the overall decrease in the general ability to in-

hibit Acinetobacter biofilm formation relative to PA14, the

inhibition data presented is still noteworthy. Although the

9

73Æ2

ꢀ95

54Æ6

64Æ3

67Æ5

n.d.

38Æ1

39Æ3

72Æ1

37Æ3

<5

77Æ1

65Æ3

n.d.

75Æ13

131Æ10

68Æ2

121Æ9

n.d.

60Æ2

61Æ3

n.d.

18

19

20

30

34

35

40

ꢀ95

24Æ4

18Æ3

n.d.

ꢀ95

26Æ2

33Æ6

n.d

point as treatment for infections usually begins after biofilm

formation has been initiated and current therapies may be

of no use. It was shown previously that our most active RA

derivative 7 (obtained from the first-generation library)

acted as both a superior inhibitor and dispersal agent against

Pseudomonas PAO1 and PA14 biofilms. With the discovery

of more potent biofilm inhibitors, it was investigated as to

whether this success would also translate into enhanced bio-

film dispersal activity. To test this, PA14 and A. baumannii

biofilms were first allowed to form in the absence of com-

pound before being dosed with our most active derivatives

that encompassed various structural characteristics and po-

tencies against both strains of bacteria.

The stringency applied to the dispersion screens was simi-

lar to that used in the inhibition assays. Dispersal activity

was first assessed at 100 mm to determine which compounds

would then be moved on further for EC₅₀value determina-

Chem. Eur. J. 2008, 14, 10745 – 10761

ꢁ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

10753

C. Melander et al.

tion. One general trend observed for dispersion activity was

that all biofilm dispersion EC₅₀values were higher than the

respective compounds biofilm inhibition IC₅₀values regard-

less of the bacterial strain employed.

biofilm modulators. The fatty acid 2-AI analogues were also

shown to possess some bacteriostatic properties during the

early stages of A. baumannii biofilm development.

Despite the inability of our most active PA14 biofilm in-

hibitor (tridecyl RA analogue 9) to become the most potent

PA14 biofilm dispersal agent, the dispersal activity of select-

ed members from the library is noteworthy due to the po-

tency of analogue 35 in being able to disperse both PA14

and A. baumannii biofilms. It is also interesting that a few

analogues that were active in the inhibition assays lacked

significant dispersal activity (i.e., compounds 30 and 40).

This trend is not without precedence as other active antibio-

film molecules have demonstrated similar activity profiles

against various bacterial biofilms.^[14,18,34]

In conclusion, we have demonstrated that the RA class of

2-aminoimidazoles has provided a fertile avenue for the ex-

ploration and development of numerous biofilm modulators.

Information obtained with the SAR profile from this class

of compounds should allow for further analogue tuning and

ultimately facilitate the construction of even more potent 2-

AI antibiofilm derivatives. With the lack of new classes of

antibiotics and increased multidrug resistance, the need for

alternative approaches to mitigate infectious diseases is

sorely needed.

The hope of duplicating the dispersal activity of 7 with

the newly identified most active PA14 biofilm inhibitor 9

was not realized. This compound only showed an average

dispersal activity against PA14 with an EC₅₀value of (54Æ

6) mm. The only other compound of exceptional note was

the secondary dihexyl derivative 35, the EC₅₀dispersal activ-

ity of which is, within error, just as potent as 7. Two of the

shorter-chain fatty acid analogues 18 and 19 also showed

moderate biofilm dispersing properties, whereas the triazole

analogue 40, which was a potent PA14 biofilm inhibitor, did

not posses the same dispersal activity.

The most active small molecules identified in the dispersal

of existing Acinetobacter baumannii biofilms were those

bearing the dihexyl side chains on the two- (34) or three-

carbon (35) amide bond linker (EC₅₀values of (60Æ2) and

(61Æ3) mm, respectively). Closely following in potency was

the fatty acid derivative 19 and the tridecyl reverse amide

analogue 9.

Conclusions

Overall, the most insightful information obtained from this

SAR study involved the activities of the analogues bearing

the native oroidin amide directionality and its replacement

with a triazole isostere. Intriguingly, no antibiofilm activity

was observed for the natural amide congener 39, which is in

contrast to the parent hexyl RA 4. Also, a direct amide

bond directionality comparison can be made with analogues

32 and 39 (Scheme 9). Analogue 32 was shown to be nearly

as active as the hexyl RA 4, whereas 39, with the native

amide orientation, displayed no activity. Additionally, the

triazole isostere 40 recapitulated the activity of RA 4, which

will allow for further SAR analysis.

It remains to be seen whether other derivatives bearing

the amide orientation present in the natural products have

the ability to be as potent as those within the RA scaffold.

Exploitation of the click reaction and reductive acylation

methodology outlined above will ultimately allow this point

to be addressed head-on and is currently under intensive in-

vestigation.

Further SAR information indicates that increased substi-

tution directly adjacent to the amide bond is an effective

means of increasing activity. Applying this to the newly

identified tridecyl RA analogue 9, it may be possible to fur-

ther enhance its activity and efforts to address this issue are

also being formulated.

Additionally, removal of the amide bond entirely allowed

for the facile multigram generation of several fatty acid 2-

AI derivatives (18–20) that were shown to possess antibio-

film activity. However, the range of active analogues does

not mirror that of the RA aliphatic chain subclass, but the

three active fatty acid analogues identified were active anti-

Experimental Section

General: Stock solutions (100, 50, 10, 1 mm) of all compounds assayed

for biological activity were prepared in DMSO and stored at room tem-

perature. The amount of DMSO used in both inhibition and dispersion

screens did not exceed 1% (by volume). P. aeruginosa PA14 was gra-

ciously supplied by the Wozniak group at Wake Forest University School

of Medicine and by the OꢀToole Group at Dartmouth Medical School. A.

baumannii was purchased from ATCC (ATCC 19606).

General static inhibition assay protocol for Pseudomonas aeruginosa and

Acinetobacter baumannii: An overnight culture of the wild-type strain

was subcultured at an OD₆₀₀of 0.01 into LBNS (PA14) or LB (A. bau-

mannii) along with a predetermined concentration of the small molecule

to be tested for biofilm inhibition. Samples were then aliquoted (100 mL)

into the wells of a 96-well PVC microtiter plate. The microtiter dishes

were covered and sealed before incubation under stationary conditions at

378C for 24 h. 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 mL) and allowed to stand at

ambient temperature for 30 min. Following another thorough washing

with water, the remaining stain was solubilized with 200 mL of 95% etha-

nol. Biofilm inhibition was quantitated by measuring the OD₅₄₀for each

well by transferring 125 mL of the ethanol solution into a fresh polystyr-

ene microtiter dish for analysis.

General static dispersion assay protocols for Pseudomonas aeruginosa

and Acinetobacter baumannii: An overnight culture of the wild-type

strain was subcultured at an OD₆₀₀of 0.05 into LBNS (PA14) or LB (A.

baumannii) and then aliquoted (100 mL) into the wells of a 96-well PVC

microtiter plate. The microtiter dishes were covered and sealed before in-

cubation under stationary conditions at room temperature to allow for-

mation of the biofilms. After 24 h, the medium was discarded and the

plates thoroughly washed with water. Fresh medium containing the ap-

propriate concentration of compound was then added to the wells. The

plates were again sealed and this time incubated under stationary condi-

tions at 378C. After 24 h, the media was discarded from the wells and the

plates washed thoroughly with water. The wells were inoculated with a

10754

www.chemeurj.org

ꢁ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Chem. Eur. J. 2008, 14, 10745 – 10761

A Second-Generation Reverse-Amide Oroidin Library

FULL PAPER

0.1% aqueous solution of crystal violet (100 mL) and allowed to stand at

ambient temperature for 30 min. Following another thorough washing

with water, the remaining stain was solubilized with 200 mL of 95% etha-

nol. Biofilm dispersion was quantitated by measuring the OD₅₄₀for each

well by transferring 125 mL of the ethanol solution into a fresh polystyr-

ene microtiter dish for analysis. Percent dispersion was calculated by

comparison of the OD₅₄₀for established biofilm (untreated) versus treat-

ed established biofilm under identical conditions.

2-Amino-4-(3-undecylcarbamoyl-propyl)imidazole-1-carboxylic acid tert-

butyl ester (16): By using the general procedure, 2-amino-4-(3-carboxy-

propyl)imidazole-1-carboxylic acid tert-butyl ester (0.150 g, 0.58 mmol)

gave the title compound 16 (0.073 g, 31%) as

a tan-colored solid.

¹H NMR (300 MHz, [D₆]DMSO): d=7.72 (t, 1H, J=7.2 Hz), 6.50 (s,

1H), 6.37 (s, 2H), 3.00 (q, 2H, J=6.9 Hz), 2.21 (t, 2H, J=6.9 Hz), 2.04

(t, 2H, J=7.2 Hz), 1.70 (quint., 2H, J=7.5 Hz), 1.53 (s, 9H), 1.36 (m,

2H), 1.23 (brs, 16H), 0.85 ppm (t, 3H, J=6.9 Hz); ¹³C NMR (75 MHz,

[D₆]DMSO): d=171.69, 149.90, 148.93, 138.38, 105.81, 84.05, 38.33, 34.92,

31.30, 29.15, 29.01, 28.34, 27.52, 27.21, 26.39, 24.09, 22.11, 13.97 ppm;

HRMS (ESI): m/z: calcd for C₂₃H₄₂N₄O₃: 422.3257 [M]⁺; found:

422.3257.

Chemistry: All reagents including anhydrous solvents used for the chemi-

cal synthesis of the library were purchased from commercially available

sources and were used without further purification unless otherwise

noted. All reactions were run under either a nitrogen or argon atmos-

phere. Flash silica-gel chromatography was performed with 60 ꢂ mesh

standard grade silica gel from Sorbtech. ¹H and ¹³C NMR spectra were

obtained by using Varian 300 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 [D₆]DMSO (d=2.50 ppm),

CD₃OD (d=3.34 ppm), and CDCl₃(d=7.27 ppm) for proton spectra and

relative to [D₆]DMSO (d=39.51 ppm), CD₃OD (d=49.86 ppm), and

CDCl₃(d=77.21 ppm) for carbon spectra with an internal TMS standard.

HRMS were obtained at the North Carolina State Mass Spectrometry

Laboratory for Biotechnology. ESI experiments were carried out on an

Agilent LC-TOF mass spectrometer.

2-Amino-4-(3-tridecylcarbamoylpropyl)imidazole-1-carboxylic acid tert-

butyl ester (17): By using the general procedure, 2-amino-4-(3-carboxy-

propyl)imidazole-1-carboxylic acid tert-butyl ester (0.150 g, 0.580 mmol)

gave the title compound 17 (0.062 g, 25%) as

a tan-colored solid.

¹H NMR (300 MHz, [D₆]DMSO). d=7.72 (t, 1H, J=7.2 Hz), 6.50 (s,

1H), 6.37 (s, 2H), 3.00 (q, 2H, J=6.9 Hz), 2.21 (t, 2H, J=6.9 Hz), 2.04

(t, 2H, J=7.2 Hz), 1.70 (quint., 2H, J=7.5 Hz), 1.53 (s, 9H), 1.36 (m,

2H), 1.23 (brs, 20H), 0.85 ppm (t, 3H, J=6.9 Hz); ¹³C NMR (75 MHz,

[D₆]DMSO): d=171.70, 149.92, 148.96, 138.39, 105.81, 84.05, 38.38, 34.92,

31.32, 29.16, 29.03, 28.73, 27.53, 27.22, 26.40, 24.09, 22.12, 13.97 ppm;

HRMS (ESI): m/z: calcd for C₂₅H₄₆N₄O₃: 450.3567 [M]⁺; found:

450.3576.

Optimizing the RA chain length: General EDC/HOBt procedure: 2-

Amino-4-(3-carboxy-propyl)imidazole-1-carboxylic acid tert-butyl ester^[17]

(0.100 g, 0.371 mmol), HOBt (0.100 g, 0.742 mmol), EDC (0.142 g,

0.742 mmol), and diisopropylethylamine (0.260 mL, 1.48 mmol) were dis-

solved in anhydrous DMF (3 mL) and allowed to stir for 15 min. The ap-

propriate amine coupling partner (0.780 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 (3ꢃ

10 mL), 1m HCl (3ꢃ10 mL), sat. NaHCO₃(2ꢃ10 mL), and brine

(10 mL) before being dried (Na₂SO₄) and evaporated to dryness. The

crude product was purified by flash column chromatography (2–10%

MeOH/CH₂Cl₂) to afford the target compounds.

4-(2-Amino-1H-imidazol-4-yl)-N-undecyl-butyramide hydrochloride (8):

A solution of 16 (0.027 g, 0.064 mmol) in anhydrous dichloromethane

(1 mL) was cooled to 08C. TFA (1.0 mL) was charged into the flask and

the reaction was stirred for 6 h. After this time, the reaction was evapo-

rated to dryness and toluene (2 mL) was added. Again the mixture was

concentrated and the process repeated. The resulting TFA salt was dis-

solved in dichloromethane (1 mL), and 2m HCl in diethyl ether (0.5 mL)

was added followed by cold diethyl ether (8 mL). The precipitate was col-

lected by filtration and washed with diethyl ether (3 mL) to yield the

target compound 8 (0.023 g, 99%) as a white amorphous solid. ¹H NMR

(300 MHz, [D₆]DMSO): d=12.09 (s, 1H), 11.65 (s, 1H), 7.85 (m, 1H),

7.30 (s, 2H), 6.55 (s, 1H), 3.00 (q, 2H, J=5.7 Hz), 2.38 (t, 2H, J=

7.5 Hz), 2.07 (t, 2H, J=7.5 Hz), 1.73 (quint., 2H, J=7.5 Hz), 1.36 (m,

2H), 1.23 (brs, 16H), 0.85 ppm (t, 3H, J=6.9 Hz); ¹³C NMR (75 MHz,

[D₆]DMSO): d=171.29, 146.74, 126.34, 108.64, 38.40, 34.38, 31.27, 29.12,

29.01, 28.98, 28.72, 28.68, 26.41, 23.86, 23.60, 22.07, 13.93 ppm; HRMS

(ESI): m/z: calcd for C₁₈H₃₄N₄O: 322.2733 [M]⁺; found: 322.2744.

2-Amino-4-(3-tetradecylcarbamoyl-propyl)imidazole-1-carboxylic

acid

tert-butyl ester (13): By using the general procedure, the title compound

1

13 (0.042 g, 24%) was formed as a tan-colored solid. H NMR (400 MHz,

[D₆]DMSO):d=7.72 (m, 1H), 6.50 (s, 1H), 6.37 (s, 2H), 3.00 (q, 2H, J=

6.4 Hz), 2.21 (t, 2H, J=6.8 Hz), 2.04 (t, 2H, J=7.6 Hz), 1.71 (m, 2H),

1.53 (s, 9H), 1.36 (m, 2H), 1.23 (brs, 22H), 0.85 ppm (t, 3H, J=5.2 Hz);

¹³C NMR (100 MHz, [D₆]DMSO): d=171.64, 149.91, 148.92, 138.26,

105.79, 84.03, 38.32, 34.90, 31.32, 29.16, 29.07, 29.04, 28.74, 28.02, 27.87,

27.51, 27.18, 26.41, 24.07, 22.12, 13.96; HRMS (ESI): m/z: calcd for

C₂₆H₄₈N₄O₃: 464.3726 [M]⁺; found: 464.3742.

4-(2-Amino-1H-imidazol-4-yl)-N-tridecyl-butyramide hydrochloride (9):

By using the same general procedure as that used for the synthesis of 8,

17 (0.028 g, 0.062 mmol) gave the title compound 9 (0.024 g, 99%) as a

white amorphous solid. ¹H NMR (300 MHz, [D₆]DMSO): d=12.08 (s,

1H), 11.66 (s, 1H), 7.85 (m, 1H), 7.30 (s, 2H), 6.55 (s, 1H), 3.00 (q, 2H,

J=5.7 Hz), 2.38 (t, 2H, J=7.5 Hz), 2.07 (t, 2H, J=7.5 Hz), 1.73 (quint.,

2H, J=7.5 Hz), 1.36 (m, 2H), 1.23 (brs, 20H), 0.85 ppm (t, 3H, J=

6.9 Hz); ¹³C NMR (75 MHz, [D₆]DMSO): d=171.29, 146.74, 126.34,

108.64, 38.40, 34.38, 31.27, 29.12, 29.01, 28.98, 28.72, 28.68, 26.41, 23.86,

23.60, 22.07, 13.93 ppm; HRMS (ESI): m/z: calcd for C₂₀H₃₈N₄O:

350.3046 [M]⁺; found: 350.3052.

2-Amino-4-(3-hexadecylcarbamoyl-propyl)imidazole-1-carboxylic

acid

tert-butyl ester (14): By using the general procedure, the title compound

1

14 (0.034 g, 19%) was formed as a tan-colored solid. H NMR (400 MHz,

[D₆]DMSO): d=7.72 (m, 1H), 6.50 (s, 1H), 6.39 (s, 2H), 3.00 (q, 2H, J=

6.4 Hz), 2.21 (t, 2H, J=7.2 Hz), 2.04 (t, 2H, J=7.2 Hz), 1.71 (m, 2H),

1.53 (s, 9H), 1.36 (m, 2H), 1.23 (brs, 26H), 0.85 ppm (t, 3H, J=5.2 Hz);

¹³C NMR (100 MHz, [D₆]DMSO): d=171.63, 149.89, 148.93, 138.33,

105.79, 84.01, 38.30, 34.89, 31.30, 29.14, 29.03, 28.71, 27.51, 27.19, 26.38,

24.06, 22.10, 13.96 ppm; HRMS (ESI): m/z: calcd for C₂₈H₅₂N₄O₃:

492.4039 [M]⁺; found: 492.4033.

4-(2-Amino-1H-imidazol-4-yl)-N-tetradecyl-butyramide

hydrochloride

(10): By using the same general procedure as that used for the synthesis

of 8, 13 (0.030 g, 0.064 mmol) gave the title compound 10 (0.024 g, 92%)

as a white solid. ¹H NMR (400 MHz, [D₆]DMSO): d=12.05 (s, 1H),

11.62 (s, 1H), 7.83 (m, 1H), 7.31 (s, 2H), 6.55 (s, 1H), 3.00 (q, 2H, J=

6.4 Hz), 2.37 (t, 2H, J=7.2 Hz), 2.07 (t, 2H, J=7.2 Hz), 1.73 (m, 2H),

1.35 (m, 2H), 1.23 (brs, 22H), 0.85 ppm (t, 3H, J=6.4 Hz); ¹³C NMR

(75 MHz, [D₆]DMSO): d=171.30, 146.72, 126.38, 108.67, 38.41, 34.39,

31.28, 29.13, 29.03, 28.99, 28.73, 28.69, 26.42, 23.86, 23.61, 22.08,

13.94 ppm; HRMS (ESI): m/z: calcd for C₂₁H₄₀N₄O: 364.3203 [M]⁺;

found: 364.3197.

2-Amino-4-(3-octadecylcarbamoyl-propyl)imidazole-1-carboxylic

acid

tert-butyl ester (15): By using the general procedure, the title compound

1

15 (0.025 g, 13%) was formed as a tan-colored solid. H NMR (400 MHz,

[D₆]DMSO): d=7.73 (m, 1H), 6.50 (s, 1H), 6.38 (s, 2H), 3.00 (q, 2H, J=

6.4 Hz), 2.21 (t, 2H, J=7.2 Hz), 2.03 (t, 2H, J=7.2 Hz), 1.70 (m, 2H),

1.53 (s, 9H), 1.35 (m, 2H), 1.22 (brs, 30H), 0.85 ppm (t, 3H, J=6.0 Hz);

¹³C NMR (75 MHz, [D₆]DMSO): d=171.61, 149.84, 148.90, 138.38,

105.76, 83.99, 38.28, 24.88, 31.24, 29.10, 28.96, 28.64, 27.85, 27.48, 27.18,

26.33, 24.04, 22.04, 13.90 ppm; HRMS (ESI): m/z: calcd for C₃₀H₅₆N₄O₃:

520.4352 [M]⁺; found: 520.4355.

4-(2-Amino-1H-imidazol-4-yl)-N-hexadecyl-butyramide

hydrochloride

(11): By using the same general procedure as that used for the synthesis

of 8, 14 (0.029 g, 0.059 mmol) gave the title compound 11 (0.023 g, 92%)

as a white solid. ¹H NMR (400 MHz, [D₆]DMSO): d=12.02 (s, 1H),

11.59 (s, 1H), 7.82 (m, 1H), 7.31 (s, 2H), 6.55 (s, 1H), 3.00 (q, 2H, J=

Chem. Eur. J. 2008, 14, 10745 – 10761

ꢁ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

10755

C. Melander et al.

6.4 Hz), 2.37 (t, 2H, J=7.2 Hz), 2.07 (t, 2H, J=7.2 Hz), 1.73 (m, 2H),

1.35 (m, 2H), 1.23 (brs, 26H), 0.85 ppm (t, 3H, J=6.0 Hz); ¹³C NMR

(75 MHz, [D₆]DMSO): d=171.27, 146.67, 126.38, 108.67, 38.38, 34.36,

31.25, 29.10, 28.99, 28.96, 28.70, 28.66, 26.39, 23.83, 23.58, 22.05,

13.91 ppm; HRMS (ESI): m/z: calcd for C₂₃H₄₄N₄O: 392.3515 [M]⁺;

found: 392.3514.

36.06, 31.02, 28.76, 28.58, 28.47, 28.39, 28.19, 23.03, 21.78, 13.58 ppm;

HRMS (ESI): m/z: calcd for C₁₉H₃₇BrO: 360.2028 [M]⁺; found: 360.2036.

AHCTUNGERTG(NNUN Z,Z)-1-Bromo-nonadeca-10,13-dien-2-one: By using the same general

procedure as that used for 1-bromo-undecan-2-one, octadecanoyl chlo-

ride (1.00 g, 3.34 mmol) gave a crude oil that was purified by flash

column chromatography (0–8% Et₂O/hexanes) to give the title com-

pound (0.940 g, 78%) as

a

colorless oil. ¹H NMR (300 MHz,

4-(2-Amino-1H-imidazol-4-yl)-N-octadecyl-butyramide

hydrochloride

[D₆]DMSO): d=5.32 (m, 4H), 4.31 (s, 2H), 2.74 (t, 2H, J=5.4 Hz), 2.57

(t, 2H, J=4.2 Hz), 2.02 (q, 4H, J=6.6 Hz), 1.48 (quint., 2H, J=6.6 Hz),

1.27 (brs, 14H), 0.86 ppm (t, 3H, J=6.6 Hz); ¹³C NMR (75 MHz,

[D₆]DMSO): d=201.62, 129.70, 129.67, 127.74, 127.72, 39.05, 36.72, 30.89,

28.99, 28.72, 28.63, 28.50, 28.40, 26.60, 25.20, 23.18, 21.96, 13.89 ppm;

HRMS (ESI): m/z: calcd for C₁₉H₃₃BrO: 356.1715 [M]⁺; found: 356.1716.

(12): By using the same general procedure as that used for the synthesis

of 8, 15 (0.028 g, 0.054 mmol) gave the title compound 12 (0.023 g, 94%)

as a white solid. ¹H NMR (300 MHz, [D₆]DMSO): d=12.02 (s, 1H),

11.60 (s, 1H), 7.82 (s, 1H), 7.31 (s, 2H), 6.55 (s, 1H), 3.00 (q, 2H, J=

6.6 Hz), 2.37 (t, 2H, J=6.6 Hz), 2.06 (t, 2H, J=7.2 Hz), 1.73 (m, 2H),

1.35 (m, 2H), 1.23 (brs, 30H), 0.85 ppm (t, 3H, J=6.0 Hz); ¹³C NMR

(100 MHz, [D₆]DMSO): d=171.29, 146.68, 126.38, 108.70, 38.40, 34.38,

31.29, 29.14, 29.02, 28.74, 28.70, 26.42, 23.85, 23.60, 22.09, 13.96 ppm;

HRMS (ESI): m/z: calcd for C₂₅H₄₈N₄O: 420.3828 [M]⁺; found: 420.3839.

(Z)-1-Bromo-octadec-10-en-2-one: By using the same general procedure

as that used for 1-bromo-undecan-2-one, octadecanoyl chloride (3.30 g,

11.0 mmol) gave a crude oil that was purified by flash column chromatog-

raphy (0–20% Et₂O/hexanes) to give the title compound (3.89 g, 98%)

Deletion of the amide bond

as

a

colorless oil. The product solidified upon standing. ¹H NMR

1-Bromo-undecan-2-one: Decanoyl chloride (2.25 mL, 11.0 mmol) was

dissolved into anhydrous dichloromethane (10 mL) and added dropwise

to a 08C solution of CH₂N₂(33.0 mmol generated from Diazald/KOH) in

diethyl ether (100 mL). This solution was stirred at 08C for 1.5 h, after

which time, the reaction was quenched by the dropwise addition of 48%

HBr (4.0 mL). The reaction mixture was diluted with dichloromethane

(25 mL) and immediately washed with sat. NaHCO₃(3ꢃ25 mL) and

brine (2ꢃ25 mL) before being dried (MgSO₄), filtered, and concentrated.

The crude oil was purified by flash column chromatography (0–8%

Et₂O/hexanes) to give the title compound (2.31 g, 84%) as a white solid.

¹H NMR (400 MHz, [D₆]DMSO): d=4.32 (s, 2H), 2.56 (t, 2H, J=

7.2 Hz), 1.47 (m, 2H), 1.23 (brs, 12H), 0.85 ppm (t, 3H, J=6.0 Hz);

¹³C NMR (100 MHz, [D₆]DMSO): d=201.70, 39.08, 36.86, 31.29, 28.86,

28.80, 28.67, 28.43, 23.21, 22.12, 13.96 ppm; HRMS (ESI): m/z: calcd for

C₁₁H₂₁BrO: 248.0776 [M]⁺; found: 248.0787.

(300 MHz, [D₆]DMSO): d=5.31 (t, 2H, J=4.2 Hz), 4.29 (s, 2H), 2.56 (t,

2H, J=6.9 Hz), 1.97 (q, 4H, J=6.0 Hz), 1.47 (t, 2H, J=6.6 Hz), 1.24

(brs, 20H), 0.85 ppm (t, 3H, J=6.6 Hz); ¹³C NMR (75 MHz,

[D₆]DMSO): d=201.53, 129.56, 129.53, 38.95, 38.66, 36.69, 31.32, 29.14,

29.12, 28.89, 28.74, 28.70, 28.65, 28.52, 28.44, 26.59, 23.20, 22.12,

13.91 ppm; HRMS (ESI): m/z: calcd for C₁₉H₃₅BrO: 358.1871 [M]⁺;

found: 358.1866.

(E)-1-Bromo-9-methyl-dec-7-en-2-one: By using the same general proce-

dure as that used for 1-bromo-undecan-2-one, octadecanoyl chloride

(1.50 g, 7.95 mmol) gave a crude oil that was purified by flash column

chromatography (0–10% Et₂O/hexanes) to give the title compound

1

(1.92 g, 98%) as an amber oil. H NMR (300 MHz, [D₆]DMSO): d=5.34

(m, 2H), 4.31 (s, 2H), 2.57 (t, 2H, J=7.2 Hz), 2.20 (m, 1H), 1.93 (q, 2H,

J=6.0 Hz), 1.48 (m, 2H), 1.28 (m, 2H), 0.92 ppm (d, 6H, J=6.6 Hz);

¹³C NMR (75 MHz, [D₆]DMSO): d=201.56, 137.41, 126.44, 38.89, 36.75,

31.63, 30.38, 28.37, 22.69, 22.49 ppm; HRMS (ESI): m/z: calcd for

C₁₁H₁₉BrO: 246.0619 [M]⁺; found: 246.0614.

1-Bromo-tridecan-2-one: By using the same general procedure as that

used for 1-bromo-undecan-2-one, dodecanoyl chloride (4.72 mL,

19.9 mmol) gave a crude oil that was purified by flash column chromatog-

raphy (0–10% Et₂O/hexanes) to give the title compound (4.59 g, 83%)

as a white solid. ¹H NMR (300 MHz, [D₆]DMSO): d=4.32 (s, 2H), 2.56

(t, 2H, J=7.2 Hz), 1.47 (m, 2H), 1.24 (brs, 16H), 0.85 ppm (t, 3H, J=

6.6 Hz); ¹³C NMR (75 MHz, [D₆]DMSO): d=201.71, 38.96, 36.88, 31.32,

29.02, 28.90, 28.79, 28.73, 28.43, 23.21, 22.12, 13.97 ppm; HRMS (ESI):

m/z: calcd for C₁₃H₂₅BrO: 276.1089 [M]⁺; found: 276.1098.

2-Amino-4-nonyl-imidazole-1-carboxylic acid tert-butyl ester: 1-Bromo-

undecan-2-one (0.748 g, 3.00 mmol), Boc-guanidine (1.40 g, 9.00 mmol),

and NaI (0.900 g, 6.00 mmol) were dissolved in DMF (15 mL) and al-

lowed to stir at room temperature. After 72 h, the DMF was removed

under reduced pressure and the residue was taken up in ethyl acetate

(50 mL) and washed with water (3ꢃ25 mL) and brine (25 mL) before

being dried (Na₂SO₄), filtered, and evaporated to dryness. The resulting

oil was purified by flash column chromatography (0–50% EtOAc/hex-

anes) to give the title compound (0.408 g, 44%) as a tan-colored solid.

¹H NMR (400 MHz, [D₆]DMSO): d=6.47 (s, 1H), 6.41 (s, 2H), 2.21 (t,

2H, J=7.2 Hz), 1.51 (s, 9H), 1.47 (m, 2H), 1.22 (brs, 12H), 0.84 ppm (t,

3H, J=6.0 Hz); ¹³C NMR (100 MHz, [D₆]DMSO): d=149.93, 148.98,

138.91, 105.44, 83.88, 31.32, 28.99, 28.92, 28.74, 27.83, 27.72, 27.48, 22.13,

13.93 ppm; HRMS (ESI): m/z: calcd for C₁₇H₂₃N₃O₂: 309.2416 [M]⁺;

found: 309.2426.

1-Bromo-pentadecan-2-one: By using the same general procedure as that

used for 1-bromo-undecan-2-one, tetradecanoyl chloride (2.97 mL,

11.0 mmol) gave a crude oil that was purified by flash column chromatog-

raphy (0–8% Et₂O/hexanes) to give the title compound (3.08 g, 92%) as

a white solid. ¹H NMR (400 MHz, [D₆]DMSO, 608C): d=4.27 (s, 2H),

2.57 (t, 2H, J=7.2 Hz), 1.50 (m, 2H), 1.25 (brs, 20H), 0.86 ppm (t, 3H,

J=6.0 Hz); ¹³C NMR (100 MHz, [D₆]DMSO, 608C): d=201.43, 38.93,

36.10, 31.03, 28.77, 28.74, 28.71, 28.60, 28.49, 28.42, 28.20, 23.04, 21.80,

13.60 ppm; HRMS (ESI): m/z: calcd for C₁₅H₂₉BrO: 304.1402 [M]⁺;

found: 304.1415.

2-Amino-4-undecyl-imidazole-1-carboxylic acid tert-butyl ester: By using

the same general procedure as that used for 2-amino-4-nonyl-imidazole-

1-carboxylic acid tert-butyl ester, 1-bromo-tridecan-2-one (4.00 g,

14.4 mmol) gave a crude oil that was purified by flash column chromatog-

raphy (0–30% EtOAc/hexanes) to give the title compound (2.23 g, 46%)

as a tan-colored solid. ¹H NMR (400 MHz, [D₆]DMSO): d=6.49 (s, 1H),

6.37 (s, 2H), 2.22 (brs, 2H), 1.52 (s, 9H), 1.48 (m, 2H), 1.23 (brs, 16H),

0.86 ppm (t, 3H, J=6.0 Hz); ¹³C NMR (75 MHz, [D₆]DMSO): d=149.87,

148.97, 138.94, 105.52, 83.95, 31.31, 29.01, 28.87, 28.72, 27.80, 27.70, 27.51,

22.11, 13.96 ppm; HRMS (ESI): m/z: calcd for C₁₉H₃₅N₃O₂: 337.2729

[M]⁺; found: 337.2730.

1-Bromo-heptadecan-2-one: By using the same general procedure as that

used for 1-bromo-undecan-2-one, hexadecanoyl chloride (3.33 mL,

11.0 mmol) gave a crude oil that was purified by flash column chromatog-

raphy (0–8% Et₂O/hexanes) to give the title compound (3.18 g, 87%) as

a white solid. ¹H NMR (400 MHz, [D₆]DMSO, 758C): d=4.25 (s, 2H),

2.58 (t, 2H, J=7.2 Hz), 1.51 (m, 2H), 1.26 (brs, 24H), 0.87 ppm (t, 3H,

J=6.0 Hz); ¹³C NMR (100 MHz, [D₆]DMSO, 758C): d=201.34, 38.87,

35.83, 30.92, 28.64, 28.48, 28.36, 28.29, 28.11, 22.97, 21.68, 13.46 ppm;

HRMS (ESI): m/z: calcd for C₁₇H₃₃BrO: 332.1715 [M]⁺; found: 332.1725.

1-Bromo-nonadecan-2-one: By using the same general procedure as that

used for 1-bromo-undecan-2-one, octadecanoyl chloride (3.30 g,

11.0 mmol) gave a crude solid that was triturated with a 1:1 EtOAc/

CH₂Cl₂mixture and filtered to give the title compound (3.61 g, 91%) as

a white solid. ¹H NMR (300 MHz, [D₆]DMSO, 808C): d=4.22 (s, 2H),

2.58 (t, 2H, J=7.2 Hz), 1.52 (m, 2H), 1.26 (brs, 28H), 0.87 ppm (t, 3H,

J=6.6 Hz); ¹³C NMR (100 MHz, [D₆]DMSO, 618C): d=201.41, 38.91,

2-Amino-4-tridecyl-imidazole-1-carboxylic acid tert-butyl ester: By using

the same general procedure as that used for 2-amino-4-nonyl-imidazole-

1-carboxylic acid tert-butyl ester, 1-bromo-pentadecan-2-one (0.916 g,

3.00 mmol) gave a crude oil that was purified by flash column chromatog-

raphy (0–60% EtOAc/hexanes) to give the title compound (0.473 g,

43%) as a tan-colored solid. ¹H NMR (400 MHz, [D₆]DMSO, 758C): d=

6.48 (s, 1H), 6.22 (s, 2H), 2.24 (t, 2H, J=7.2 Hz), 1.54 (s, 9H), 1.50 (m,

10756

www.chemeurj.org

ꢁ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Chem. Eur. J. 2008, 14, 10745 – 10761

A Second-Generation Reverse-Amide Oroidin Library

FULL PAPER

2H), 1.25 (brs, 20H), 0.86 ppm (t, 3H, J=6.0 Hz); ¹³C NMR (100 MHz,

[D₆]DMSO, 758C): d=149.45, 148.69, 138.82, 105.28, 83.71, 30.92, 28.64,

28.59, 28.48, 28.36, 28.30, 27.51, 27.43, 27.27, 21.67, 13.44 ppm; HRMS

(ESI): m/z: calcd for C₂₁H₃₉N₃O₂: 365.3042 [M]⁺; found: 365.3048.

0.650 mmol) in anhydrous dichloromethane (6 mL) was cooled to 08C.

TFA (6 mL) was charged into the flask and the reaction stirred for 6 h.

After this 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 was dissolved in dichloromethane

(1 mL) and 2m HCl in diethyl ether (1.0 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 18

2-Amino-4-pentadecyl-imidazole-1-carboxylic acid tert-butyl ester: By

using the same general procedure as that used for 2-amino-4-nonyl-imi-

dazole-1-carboxylic acid tert-butyl ester, 1-bromo-heptadecan-2-one

(1.00 g, 3.00 mmol) gave a crude oil that was purified by flash column

chromatography (0–60% EtOAc/hexanes) to give the title compound

(0.628 g, 53%) as a tan-colored solid. ¹H NMR (400 MHz, [D₆]DMSO,

758C): d=6.49 (s, 1H), 6.20 (s, 2H), 2.24 (t, 2H, J=7.2 Hz), 1.54 (s, 9H),

1.50 (m, 2H), 1.25 (brs, 24H), 0.86 ppm (t, 3H, J=6.4 Hz); ¹³C NMR

(100 MHz, [D₆]DMSO, 758C): d=149.42, 148.67, 138.82, 105.31, 83.73,

30.90, 28.61, 28.56, 28.45, 28.34, 28.27, 27.49, 27.42, 27.28, 21.65,

13.43 ppm; HRMS (ESI): m/z: calcd for C₂₃H₄₃N₃O₂: 393.3355 [M]⁺;

found: 393.3362.

(0.159 g, 99%) as

a

brown amorphous solid. ¹H NMR (400 MHz,

[D₆]DMSO): d=12.19 (s, 1H), 11.76 (s, 1H), 7.35 (s, 2H), 6.54 (s, 1H),

2.37 (t, 2H, J=7.2 Hz), 1.49 (m, 2H), 1.22 (brs, 12H), 0.83 ppm (t, 3H,

J=6.0 Hz); ¹³C NMR (100 MHz, [D₆]DMSO): d=146.80, 126.85, 108.42,

31.29, 28.91, 28.69, 28.35, 27.61, 23.99, 22.12, 13.97 ppm; HRMS (ESI):

m/z: calcd for C₂₁H₂₃N₃: 209.1892 [M]⁺; found: 209.1901.

4-Undecyl-1H-imidazol-2-ylamine hydrochloride (19): By using the same

general procedure as that used for the synthesis of 18, 2-amino-4-undec-

yl-imidazole-1-carboxylic acid tert-butyl ester (2.23 g, 6.60 mmol) gave

2-Amino-4-heptadecyl-imidazole-1-carboxylic acid tert-butyl ester: By

using the same general procedure as that used for 2-amino-4-nonyl-imi-

dazole-1-carboxylic acid tert-butyl ester, 1-bromo-nonadecan-2-one

(1.14 g, 3.15 mmol), with additional DMF (25 mL) for solubility, gave a

crude oil that was purified by trituration (MeOH/H₂O 1:20) to give the

title compound (1.10 g, 83%) as a white solid. ¹H NMR (400 MHz,

[D₆]DMSO, 808C): d=6.49 (s, 1H), 6.19 (s, 2H), 2.24 (t, 2H, J=7.2 Hz),

1.54 (s, 9H), 1.50 (m, 2H), 1.25 (brs, 28H), 0.87 ppm (t, 3H, J=6.8 Hz);

¹³C NMR (100 MHz, [D₆]DMSO, 808C): d=149.45, 148.71, 138.85,

105.33, 83.76, 30.93, 28.64, 28.49, 28.38, 28.30, 27.62, 27.52, 27.45, 27.30,

21.68, 13.44 ppm; HRMS (ESI): m/z: calcd for C₂₅H₄₇N₃O₂: 421.3668

[M]⁺; found: 421.3678.

the title compound 19 (1.80 g, 99%) as

a brown amorphous solid.

¹H NMR (400 MHz, [D₆]DMSO): d=12.19 (s, 1H), 11.72 (s, 1H), 7.34 (s,

2H), 6.54 (s, 1H), 2.37 (d, 2H, J=5.6 Hz), 1.50 (m, 2H), 1.24 (brs, 16H),

0.85 ppm (t, 3H, J=6.0 Hz); ¹³C NMR (100 MHz, [D₆]DMSO): d=

146.76, 126.81, 108.39, 31.32, 29.05, 29.02, 28.96, 28.76, 28.69, 28.36, 27.61,

27.61, 23.99, 22.12, 13.98 ppm; HRMS (ESI): m/z: calcd for C₁₄H₂₈N₃:

237.2205 [M]⁺; found: 237.2209.

4-Tridecyl-1H-imidazol-2-ylamine hydrochloride (20): By using the same

general procedure as that used for the synthesis of 18, 2-amino-4-tridec-

yl-imidazole-1-carboxylic acid tert-butyl ester (0.200 g, 0.550 mmol) gave

the title compound 20 (0.158 g, 96%) as a brown amorphous solid.

¹H NMR (400 MHz, [D₆]DMSO): d=12.19 (s, 1H), 11.68 (s, 1H), 7.34 (s,

2H), 6.53 (s, 1H), 2.38 (t, 2H, J=7.2 Hz), 1.50 (m, 2H), 1.23 (brs, 20H),

0.84 ppm (t, 3H, J=7.2 Hz); ¹³C NMR (100 MHz, [D₆]DMSO): d=

146.77, 126.80, 108.36, 31.31, 29.08, 29.05, 28.96, 28.73, 28.69, 28.36, 27.61,

23.98, 22.11, 13.96 ppm; HRMS (ESI): m/z: calcd for C₁₆H₃₁N₃: 265.2518

[M]⁺; found: 265.2529.

ACHTUNGTRENNUNG(Z,Z)-2-Amino-4-heptadeca-8,11-dienyl-imidazole-1-carboxylic acid tert-

butyl ester: By using the same general procedure as that used for 2-

amino-4-nonyl-imidazole-1-carboxylic acid tert-butyl ester, 1-bromo-non-

adeca-10,13-dien-2-one (0.660 g, 1.85 mmol) gave a crude oil that was pu-

rified by flash column chromatography (0–30% EtOAc/hexanes) to give

the title compound (0.382 g, 50%) as

a

dark-brown oil; ¹H NMR

(400 MHz, [D₆]DMSO): d=6.47 (s, 1H), 6.37 (s, 2H), 5.31 (m, 4H), 2.72,

(t, 2H, J=6.0 Hz), 2.21 (t, 2H, J=7.2 Hz), 2.00 (q, 4H, J=6.8 Hz), 1.52

(s, 9H), 1.48 (m, 2H), 1.25 (brs, 14H), 0.84 ppm (t, 3H, J=6.4 Hz);

¹³C NMR (100 MHz, [D₆]DMSO): d=149.88, 148.96, 138.92, 129.69,

127.73, 105.45, 83.92, 30.92, 29.04, 28.74, 28.71, 28.61, 27.80, 27.71, 27.49,

26.62, 25.21, 21.98, 13.90 ppm; HRMS (ESI): m/z: calcd for C₂₅H₄₃N₃O₂:

417.3355 [M]⁺; found: 417.3360.

4-Pentadecyl-1H-imidazol-2-ylamine hydrochloride (21): By using the

same general procedure as that used for the synthesis of 18, 2-amino-4-

pentadecyl-imidazole-1-carboxylic acid tert-butyl ester (0.200 g,

0.510 mmol) gave the title compound 21 (0.167 g, 99%) as a tan-colored

solid. ¹H NMR (400 MHz, [D₆]DMSO): d=12.18 (s, 1H), 11.65 (s, 1H),

7.32 (s, 2H), 6.53 (s, 1H), 2.38 (t, 2H, J=7.2 Hz), 1.50 (m, 2H), 1.23

(brs, 24H), 0.84 ppm (t, 3H, J=7.6 Hz); ¹³C NMR (100 MHz,

[D₆]DMSO): d=146.73, 126.77, 108.33, 31.31, 29.08, 29.03, 28.98, 28.74,

28.71, 28.39, 27.63, 23.99, 22.12, 13.96 ppm; HRMS (ESI): m/z: calcd for

C₁₈H₃₅N₃: 293.2831 [M]⁺; found: 293.2844.

(Z)-2-Amino-4-heptadec-8-enyl-imidazole-1-carboxylic acid tert-butyl

ester: By using the same general procedure as that used for 2-amino-4-

nonyl-imidazole-1-carboxylic acid tert-butyl ester, 1-bromo-octadec-10-

en-2-one (1.00 g, 2.78 mmol) gave a crude oil that was purified by flash

column chromatography (20–100% EtOAc/hexanes) to give the title

compound (0.578 g, 50%) as pale-yellow oil. ¹H NMR (400 MHz,

[D₆]DMSO): d=6.48 (s, 1H), 6.36 (s, 2H), 5.31 (t, 2H, J=4.4 Hz), 2.21

(t, 2H, J=7.2 Hz), 1.97 (m, 4H), 1.52 (s, 9H), 1.48 (m, 2H), 1.25 (brs,

10H), 1.23 (brs, 10H), 0.84 ppm (t, 3H, J=6.0 Hz); ¹³C NMR (100 MHz,

[D₆]DMSO): d=149.88, 148.96, 138.92, 129.65, 105.49, 83.96, 31.30, 29.11,

28.85, 28.72, 28.60, 28.55, 27.80, 27.72, 27.51, 26.57, 22.12, 13.96 ppm;

HRMS (ESI): m/z: calcd for C₂₅H₄₅N₃O₂: 419.3512 [M]⁺; found:

419.3515.

4-Heptadecyl-1H-imidazol-2-ylamine hydrochloride (22): By using the

same general procedure as that used for the synthesis of 18, 2-amino-4-

heptadecyl-imidazole-1-carboxylic acid tert-butyl ester (0.400 g,

0.950 mmol) gave the title compound 22 (0.320 g, 94%) as a white solid.

¹H NMR (400 MHz, [D₆]DMSO): d=12.10 (brs, 1H), 11.70 (brs, 1H),

7.33 (s, 2H), 6.54 (s, 1H), 2.37 (t, 2H, J=7.6 Hz), 1.50 (m, 2H), 1.23

(brs, 28H), 0.84 ppm (t, 3H, J=6.0 Hz); ¹³C NMR (75 MHz,

[D₆]DMSO): d=146.75, 126.80, 108.36, 31.31, 29.06, 28.76, 28.39, 27.62,

24.00, 22.12, 13.96 ppm; HRMS (ESI): m/z: calcd for C₂₀H₃₉N₃: 321.3144

[M]⁺; found: 321.3146.

(E)-2-Amino-4-(7-methyl-oct-5-enyl)imidazole-1-carboxylic acid tert-

butyl ester: By using the same general procedure as that used for 2-

amino-4-nonyl-imidazole-1-carboxylic acid tert-butyl ester, 1-bromo-9-

methyl-dec-7-en-2-one (1.00 g, 4.05 mmol) gave a crude oil that was puri-

fied by flash column chromatography (0–80% EtOAc/hexanes) to give

the title compound (0.594 g, 48%) as a yellow oil. ¹H NMR (400 MHz,

[D₆]DMSO): d=6.49 (s, 1H), 6.38 (s, 2H), 5.34 (m, 2H), 2.22 (m, 3H),

1.94 (q, 2H, J=6.4 Hz), 1.52 (s, 9H), 1.48 (m, 2H), 1.31 (m, 2H),

0.92 ppm (d, 6H, J=6.8 Hz); ¹³C NMR (100 MHz, [D₆]DMSO): d=

149.89, 148.95, 138.88, 137.27, 126.76, 105.55, 83.98, 31.80, 30.40, 28.76,

27.51, 27.36, 22.56 ppm; HRMS (ESI): m/z: calcd for C₁₇H₂₉N₃O₂:

307.2260 [M]⁺; found: 307.2262.

G

hydrochloride

4-Nonyl-1H-imidazol-2-ylamine hydrochloride (18):

A solution of 2-

amino-4-nonyl-imidazole-1-carboxylic acid tert-butyl ester (0.200 g,

Chem. Eur. J. 2008, 14, 10745 – 10761

ꢁ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

10757

C. Melander et al.

ester (0.528 g, 1.26 mmol) gave the title compound 24 (0.447 g, 100%) as

a brown oil. H NMR (400 MHz, [D₆]DMSO): d=12.10 (s, 1H), 11.75 (s,

127.89, 127.74, 105.92, 84.12, 65.33, 32.31, 27.48, 23.31 ppm; HRMS

(ESI): m/z: calcd for C₁₈H₂₃N₃O₄: 345.1689 [M]⁺; found: 345.1689.

1

1H), 7.35 (s, 2H), 6.52 (s, 1H), 5.31 (m, 2H), 2.37 (t, 2H, J=7.2 Hz),

1.97 (m, 4H), 1.50 (m, 2H), 1.25 (brs, 10H), 1.22 (brs, 10H), 0.84 ppm (t,

3H, J=6.0 Hz); ¹³C NMR (100 MHz, [D₆]DMSO): d=146.82, 129.64,

129.61, 126.79, 108.35, 31.31, 29.14, 29.12, 28.86, 28.72, 28.61, 28.58, 28.39,

27.62, 26.62, 26.59, 24.00, 22.12, 13.95 ppm; HRMS (ESI): m/z: calcd for

C₂₀H₃₇N₃: 319.2988 [M]⁺; found: 319.3009.

2-Amino-4-(2-carboxy-ethyl)imidazole-1-carboxylic acid tert-butyl ester

(26): 2-Amino-4-(2-benzyloxycarbonyl-ethyl)imidazole-1-carboxylic acid

tert-butyl ester (2.00 g, 5.79 mmol) was added to a solution of anhydrous

THF (50 mL) and 10% Pd/C (0.200 g). Air was removed from the system

and the reaction was back flushed with hydrogen. This process was re-

peated three times before setting the reaction under a hydrogen balloon

at atmospheric pressure and temperature for 1 h. After this time, the re-

action was filtered through a Celite pad and the filter cake was washed

with THF (10 mL). The filtrate was concentrated under reduced pressure

to afford the title compound 26 (1.35 g, 92%) as a white solid. ¹H NMR

(400 MHz, [D₆]DMSO): d=6.54 (s, 1H), 6.44 (s, 2H), 2.46 (m, 4H),

1.53 ppm (s, 9H); ¹³C NMR (100 MHz, [D₆]DMSO): d=173.98, 149.96,

148.89, 137.55, 105.77, 84.19, 32.51, 27.52, 23.30 ppm; HRMS (ESI): m/z:

calcd for C₁₁H₁₇N₃O₄: 255.1219 [M]⁺; found: 255.1219.

(E)-4-(7-Methyl-oct-5-enyl)-1H-imidazol-2-ylamine hydrochloride (24):

By using the same general procedure as that used for the synthesis of 18,

(E)-2-amino-4-(7-methyl-oct-5-enyl)-imidazole-1-carboxylic acid tert-

butyl ester (0.479 g, 1.56 mmol) gave the title compound 24 (0.350 g,

1

93%) as a brown oil. H NMR (400 MHz, [D₆]DMSO): d=12.18 (s, 1H),

11.71 (s, 1H), 7.34 (s, 2H), 6.54 (s, 1H), 5.35 (m, 2H), 2.39 (t, 2H, J=

7.6 Hz), 2.20 (m, 1H), 1.94 (q, 2H, J=6.4 Hz), 1.50 (quint., 2H, J=

7.6 Hz), 0.92 ppm (d, 6H, J=6.8 Hz); ¹³C NMR (100 MHz, [D₆]DMSO):

d=146.77, 137.46, 126.74, 126.53, 108.44, 31.53, 30.38, 28.33, 27.13, 23.81,

22.53 ppm; HRMS (ESI): m/z: calcd for C₁₂H₂₁N₃: 207.1736 [M]⁺; found:

207.1738.

7-Bromo-6-oxo-heptanoic acid benzyl ester: By using the same general

procedure as that used for 5-bromo-4-oxo-pentanoic acid benzyl ester,

hexanedioic acid monobenzyl ester^[37](2.60 g, 11.0 mmol) gave a crude

amber oil (2.65 g, 77%) that was used in the next steps without further

purification. ¹H NMR (300 MHz, CDCl₃): d=7.27 (m, 5H), 5.03 (s, 2H),

3.77 (s, 2H), 2.58 (m, 2H), 2.30 (m, 2H), 1.58 ppm (q, 4H, J=3.6 Hz);

¹³C NMR (75 MHz, CDCl₃): d=201.80, 173.23, 136.14, 128.75, 128.41,

77.66, 77.23, 76.81, 66.41, 39.48, 34.30, 34.08, 24.35, 23.36 ppm; HRMS

(ESI): m/z: calcd for C₁₄H₁₇BrO₃: 312.0361 [M]⁺; found: 312.0367.

Linker modification

Succinic acid monobenzyl ester: Succinic anhydride (5.00 g, 50.0 mmol)

was dissolved in anhydrous dichloromethane (40 mL). Benzyl alcohol

(5.69 mL, 55.0 mmol), triethylamine (7.50 mL, 55.0 mmol), and a catalytic

amount of DMAP were added to this solution. The resulting clear solu-

tion was stirred at ambient temperature for 15 h, after which time, all vol-

atiles were removed by rotary evaporation. The crude residue was taken

up in diethyl ether (200 mL) and was washed with 2n NaOH (2ꢃ75 mL).

The aqueous extracts were carefully acidified to pH 2 with concentrated

HCl and then extracted with diethyl ether (2ꢃ100 mL) and subsequently

dried (Mg₂SO₄), filtered, and concentrated to afford the title compound

(9.06 g, 87%) as a white solid with no further purification needed.

¹H NMR (400 MHz, [D₆]DMSO): d=12.26 (s, 1H), 7.32 (m, 5H), 5.08 (s,

2H), 2.56 (m, 2H), 2.49 ppm (m, 2H); ¹³C NMR (100 MHz, [D₆]DMSO):

d=173.53, 172.13, 136.26, 128.48, 128.02, 127.88, 65.56, 28.80, 28.73 ppm;

HRMS (ESI): m/z: calcd for C₁₁H₁₂O₄: 208.0736 [M]⁺; found: 208.0736.

2-Amino-4-(4-benzyloxycarbonyl-butyl)imidazole-1-carboxylic acid tert-

butyl ester: By using the same general procedure as that used for 2-

amino-4-(2-benzyloxycarbonyl-ethyl)imidazole-1-carboxylic acid tert-

butyl ester, 7-bromo-6-oxo-heptanoic acid benzyl ester (1.50 g,

4.79 mmol) gave a crude oil that was purified by flash column chromatog-

raphy (40–100% EtOAc/hexanes) to give the title compound (0.896 g,

50%) as an amber oil. ¹H NMR (300 MHz, [D₆]DMSO): d=7.35 (m,

5H), 6.51 (s, 1H), 6.38 (s, 2H), 5.07 (s, 2H), 2.36 (t, 2H, J=6.9 Hz), 2.24

(t, 2H, J=6.9 Hz), 1.52 ppm (m, 13H); ¹³C NMR (75 MHz, [D₆]DMSO):

d=172.73, 149.84, 148.92, 138.54, 136.27, 128.38, 127.91, 127.85, 105.64,

84.00, 65.25, 33.26, 27.48, 27.26, 27.18, 24.08 ppm; HRMS (ESI): m/z:

calcd for C₂₀H₂₇N₃O₄: 373.2002 [M]⁺; found: 373.2006.

5-Bromo-4-oxo-pentanoic acid benzyl ester: Succinic acid monobenzyl

ester (2.00 g, 9.60 mmol) was dissolved in anhydrous dichloromethane

(30 mL) at 08C and a catalytic amount of DMF was added. Oxalyl chlo-

ride (2.50 mL, 28.8 mmol) was added dropwise to this solution, and the

resulting mixture 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 dropwise to a 08C solution of CH₂N₂(28.8 mmol gen-

erated from Diazald/KOH) in diethyl ether (75 mL). This solution was

stirred at 08C for 1.5 h, after which time, the reaction was quenched by

the dropwise addition of 48% HBr (3.5 mL). The reaction mixture was

diluted with dichloromethane (25 mL) and immediately washed with sat.

NaHCO₃(3ꢃ25 mL) and brine (2ꢃ25 mL) before being dried (MgSO₄),

filtered, and concentrated. The resulting yellow oil (2.59 g, 95%) was car-

ried onto the next step without further purification. ¹H NMR (400 MHz,

[D₆]DMSO): d=7.36 (m, 5H), 5.08 (s, 2H), 4.39 (s, 2H), 2.88 (t, 2H, J=

6.0 Hz), 2.60 ppm (t, 2H, J=6.0 Hz); ¹³C NMR (75 MHz, [D₆]DMSO):

d=200.35, 171.83, 136.06, 128.36, 127.94, 127.77, 65.54, 36.33, 34.20,

27.77 ppm; HRMS (ESI): m/z: calcd for C₁₂H₁₃BrO₃: 284.0048 [M]⁺;

found: 284.0056.

2-Amino-4-(4-carboxy-butyl)imidazole-1-carboxylic acid tert-butyl ester

(27): By using the same general procedure as that used for 26, 2-amino-4-

(4-benzyloxycarbonyl-butyl)imidazole-1-carboxylic acid tert-butyl ester

(0.840 g, 2.25 mmol) gave the title compound 27 (0.538 g, 84%) as a

white solid. ¹H NMR (300 MHz, [D₆]DMSO): d=6.52 (s, 1H), 6.40 (s,

2H), 2.20 (m, 4H), 1.50 ppm (m, 13H); ¹³C NMR (75 MHz, [D₆]DMSO):

d=174.56, 149.88, 148.92, 138.56, 105.68, 84.03, 33.59, 27.50, 27.32,

24.19 ppm; HRMS (ESI): m/z: calcd for C₁₃H₂₁N₃O₄: 283.1532 [M]⁺;

found: 283.1532.

2-Amino-4-(2-hexylcarbamoyl-ethyl)imidazole-1-carboxylic acid tert-

butyl ester: By using the general EDC/HOBt procedure, the title com-

pound was formed as a yellow foam (0.021 g, 8%). ¹H NMR (300 MHz,

CDCl₃): d=6.57 (s, 1H), 6.06 (s, 1H), 5.67 (brs, 2H), 3.20 (q, 2H, J=

6.9 Hz), 2.69 (t, 2H, J=7.2 Hz), 2.47 (t, 2H, J=7.2 Hz), 1.57 (s, 9H), 1.43

(m, 2H), 1.25 (brs, 6H), 0.87 ppm (t, 3H, J=6.6 Hz); ¹³C NMR

(75 MHz, CDCl₃): d=172.58, 150.20, 149.58, 137.38, 107.53, 85.10, 39.63,

35.87, 31.69, 29.69, 28.15, 26.73, 24.37, 22.75, 14.23 ppm; HRMS (ESI):

m/z: calcd for C₁₇H₃₁N₄O₃: 338.2318 [M]⁺; found: 338.2323.

2-Amino-4-(4-hexylcarbamoyl-butyl)imidazole-1-carboxylic acid tert-

butyl ester: By using the general EDC/HOBt procedure, the title com-

pound was formed as a colorless oil (0.044 g, 17%). ¹H NMR (300 MHz,

[D₆]DMSO): d=7.73 (t, 1H, J=5.4 Hz), 6.50 (s, 1H), 6.38 (s, 2H), 3.00

(q, 2H, J=6.6 Hz), 2.23 (m, 2H), 2.03 (m, 2H), 1.53 (s, 9H), 1.47 (m,

4H), 1.35 (m, 2H), 1.23 (m, 4H), 0.84 ppm (t, 3H, J=6.6 Hz); ¹³C NMR

(75 MHz, [D₆]DMSO): d=171.78, 149.81, 148.92, 138.70, 105.60, 84.00,

38.31, 35.25, 30.95, 29.09, 27.49, 27.39, 26.03, 25.03, 22.01, 13.85 ppm;

HRMS (ESI): m/z: calcd for C₁₉H₃₄N₄O₃: 366.2631 [M]⁺; found:

366.2632.

2-Amino-4-(2-benzyloxycarbonyl-ethyl)imidazole-1-carboxylic acid tert-

butyl ester: 5-bromo-4-oxo-pentanoic acid benzyl ester (1.00 g,

3.51 mmol) and Boc-guanidine (1.66 g, 10.5 mmol) were dissolved in

DMF (10 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 (50 mL) and washed with water (3ꢃ25 mL) and brine

(25 mL) before being dried (Na₂SO₄), filtered, and evaporated to dryness.

The resulting oil was purified by flash column chromatography (20–

100% EtOAc/hexanes) to give the title compound (0.725 g, 60%) as a

yellow solid. ¹H NMR (400 MHz, [D₆]DMSO): d=7.32 (m, 5H), 6.54 (s,

1H), 6.44 (s, 2H), 5.09 (s, 2H), 2.58 (m, 4H), 1.52 ppm (s, 9H); ¹³C NMR

(100 MHz, [D₆]DMSO): d=172.23, 150.00, 148.88, 137.21, 136.27, 128.36,

2-Amino-4-{2-[2-(4-bromo-phenyl)ethylcarbamoyl]ethyl}imidazole-1-car-

boxylic acid tert-butyl ester: By using the general EDC/HOBt procedure,

the title compound was formed as an off-white solid (0.019 g, 6%).

10758

www.chemeurj.org

ꢁ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Chem. Eur. J. 2008, 14, 10745 – 10761

A Second-Generation Reverse-Amide Oroidin Library

FULL PAPER

¹H NMR (300 MHz, CDCl₃): d=7.39 (d, 2H, J=8.1 Hz), 7.03 (d, 2H, J=

8.4 Hz), 6.58 (s, 1H), 6.41 (m, 1H), 3.45 (q, 2H, J=6.3 Hz), 2.72 (q, 2H,

J=6.9 Hz), 2.47 (t, 4H, J=6.9 Hz), 1.59 ppm (s, 9H); ¹³C NMR (75 MHz,

CDCl₃): d=172.19, 150.03, 148.93, 138.20, 133.98, 131.78, 130.66, 120.44,

107.50, 86.48, 40.53, 35.27, 35.04, 28.12, 23.12 ppm; HRMS (ESI): m/z:

calcd for C₁₉H₂₅BrN₄O₃: 436.1110 [M]⁺; found: 436.1112.

105.63, 84.05, 38.37, 33.99, 31.23, 29.16, 28.42, 27.51, 26.32, 24.04, 22.06,

13.95 ppm; HRMS (ESI): m/z: calcd for C₁₈H₃₂N₄O₃: 352.2474 [M]⁺;

found: 352.2489.

2-Amino-4-(4-pentylcarbamoyl-butyl)imidazole-1-carboxylic acid tert-

butyl ester: By using the general EDC/HOBt procedure, the title com-

pound was formed as an amber oil (0.012 g, 5%). ¹H NMR (300 MHz,

CDCl₃): d=6.52 (s, 1H), 5.66 (s, 2H), 5.55 (m, 1H), 3.23 (q, 2H, J=

6.0 Hz), 2.38 (t, 2H, J=6.6 Hz), 2.18 (t, 2H, J=7.2 Hz), 1.66 (m, 4H),

1.59 (s, 9H), 1.50 (m, 2H), 1.29 (m, 4H), 0.89 ppm (t, 3H, J=6.9 Hz);

¹³C NMR (75 MHz, CDCl₃): d=173.07, 150.12, 149.63, 138.74, 106.99,

84.91, 39.70, 36.85, 29.91, 29.58, 29.30, 28.20, 28.14, 27.94, 25.55, 22.56,

14.18 ppm; HRMS (ESI): m/z: calcd for C₁₈H₃₂N₄O₃: 352.2474 [M]⁺;

found: 352.2482.

2-Amino-4-[2-(4-phenyl-butylcarbamoyl)ethyl]imidazole-1-carboxylic

acid tert-butyl ester: By using the general EDC/HOBt procedure, the

title compound was formed as a yellow foam (0.081 g, 27%). ¹H NMR

(400 MHz, CDCl₃): d=7.26 (t, 2H, J=7.6 Hz), 7.15 (m, 2H), 6.55 (s,

1H), 6.28 (brs, 1H), 5.75 (brs, 2H), 3.22 (q, 2H, J=6.4 Hz), 2.66 (t, 2H,

J=7.2 Hz), 2.59 (t, 2H, J=7.6 Hz), 2.45 (t, 2H, J=7.2 Hz), 1.55 (m,

11H), 1.47 ppm (m, 3H); ¹³C NMR (75 MHz, [D₆]DMSO): d=171.19,

149.79, 148.86, 142.08, 138.12, 128.21, 128.17, 125.58, 105.58, 84.01, 38.09,

34.73, 33.98, 28.77, 28.27, 27.47, 24.01 ppm; HRMS (ESI): m/z: calcd for

C₂₁H₃₁N₄O₃: 386.2318 [M]⁺; found: 386.2319.

3-(2-Amino-1H-imidazol-4-yl)-N-heptyl-propionamide

hydrochloride

(32): By using the same general procedure as that used for the synthesis

of 8, 2-amino-4-(2-heptylcarbamoyl-ethyl)imidazole-1-carboxylic acid

tert-butyl ester (0.031 g, 0.088 mmol) gave the title compound 32 (0.025 g,

3-(2-Amino-1H-imidazol-4-yl)-N-hexyl-propionamide hydrochloride (28):

By using the same general procedure as that used for the synthesis of 8,

2-amino-4-(2-hexylcarbamoyl-ethyl)imidazole-1-carboxylic acid tert-butyl

ester (0.020 g, 0.062 mmol) gave the title compound 28 (0.017 g, 100%)

as a tan-colored foam. ¹H NMR (300 MHz, CD₃OD): d=6.38 (s, 1H),

3.05 (t, 2H, J=6.6 Hz), 2.66 (t, 2H, J=6.6 Hz), 2.37 (t, 2H, J=6.6 Hz),

1.36 (m, 2H), 1.19 (brs, 6H), 0.79 ppm (m, 3H); ¹³C NMR (75 MHz,

CD₃OD): d=174.12, 148.60, 128.03, 110.15, 40.57, 35.27, 32.75, 30.48,

27.77, 23.73, 21.72, 14.45 ppm; HRMS (ESI): m/z: calcd for C₁₂H₂₂N₄O:

238.1794 [M]⁺; found: 238.1795.

99%) as

a

tan-colored amorphous solid. ¹H NMR (400 MHz,

[D₆]DMSO): d=11.88 (s, 1H), 11.56 (s, 1H), 7.93 (m, 1H), 7.33 (s, 2H),

6.51 (s, 1H), 3.02 (q, 2H, J=6.4 Hz), 2.62 (t, 2H, J=7.2 Hz), 2.35 (t, 2H,

J=7.2 Hz), 1.36 (m, 2H), 1.23 (brs, 8H), 0.86 ppm (t, 3H, J=6.4 Hz);

¹³C NMR (75 MHz, [D₆]DMSO): d=170.61, 146.56, 126.24, 108.55, 38.48,

33.48, 31.20, 29.06, 28.37, 26.33, 22.02, 20.20, 13.93 ppm; HRMS (ESI):

m/z: calcd for C₁₃H₂₄N₄O: 252.1950 [M]⁺; found: 252.1959.

5-(2-Amino-1H-imidazol-4-yl)pentanoic acid pentylamide hydrochloride

(33): By using the same general procedure as that used for the synthesis

of 8, 2-amino-4-(4-pentylcarbamoyl-butyl)imidazole-1-carboxylic acid

tert-butyl ester (0.012 g, 0.034 mmol) gave the title compound 33 (0.009 g,

100%) as an amber oil. ¹H NMR (300 MHz, CD₃OD): d=6.50 (s, 1H),

3.15 (t, 2H, J=6.9 Hz), 2.51 (t, 2H, J=6.6 Hz), 2.22 (t, 2H, J=6.9 Hz),

1.63 (m, 4H), 1.49 (m, 2H), 1.32 (m, 4H), 0.91 ppm (t, 3H, J=6.9 Hz);

¹³C NMR (75 MHz, CD₃OD): d=176.25, 149.01, 129.29, 110.34, 40.89,

37.04, 30.72, 30.61, 29.28, 26.69, 25.65, 23.89, 14.82 ppm; HRMS (ESI):

m/z: calcd for C₁₃H₂₄N₄O: 252.1950 [M]⁺; found: 252.1951.

5-(2-Amino-1H-imidazol-4-yl)pentanoic acid hexylamide hydrochloride

(29): By using the same general procedure as that used for the synthesis

of 8, 2-amino-4-(4-hexylcarbamoyl-butyl)imidazole-1-carboxylic acid tert-

butyl ester (0.036 g, 0.098 mmol) gave the title compound 29 (0.029 g,

100%) as a colorless oil. ¹H NMR (300 MHz, [D₆]DMSO): d=11.99 (s,

1H), 11.55 (s, 1H), 7.78 (m, 1H), 7.29 (s, 2H), 6.55 (s, 1H), 3.00 (q, 2H,

J=6.9 Hz), 2.39 (brs, 2H), 2.06 (brs, 2H), 1.48 (brs, 4H), 1.36 (m, 2H),

1.28 (brs, 4H), 0.85 ppm (m, 3H); ¹³C NMR (75 MHz, [D₆]DMSO): d=

171.62, 146.61, 126.73, 108.51, 38.34, 34.92, 30.93, 29.07, 27.23, 26.03,

24.55, 23.71, 22.00, 13.85 ppm; HRMS (ESI): m/z: calcd for C₁₄H₂₆N₄O:

266.2107 [M]⁺; found: 266.2109.

Increased substitution

2-Amino-4-[2-(1-hexyl-heptylcarbamoyl)ethyl]imidazole-1-carboxylic

acid tert-butyl ester: By using the general EDC/HOBt procedure, 26 af-

forded an off-white solid (0.176 g, 51%). ¹H NMR (300 MHz,

[D₆]DMSO): d=7.46 (d, 1H, J=8.7 Hz), 6.50 (s, 1H), 6.36 (s, 2H), 3.63

(m, 1H), 2.47 (t, 2H, J=7.2 Hz), 2.28 (t, 2H, J=7.2 Hz), 1.52 (s, 9H),

1.20 (m, 20H), 0.84 ppm (t, 6H, J=7.2 Hz); ¹³C NMR (75 MHz,

[D₆]DMSO): d=170.79, 149.80, 148.93, 138.07, 105.66, 83.95, 47.74, 34.65,

33.98, 31.25, 28.63, 27.50, 25.44, 24.24, 22.06, 13.93 ppm; HRMS (ESI):

m/z: calcd for C₂₄H₄₄N₄O₃: 436.3413 [M]⁺; found: 436.3414.

3-(2-Amino-1H-imidazol-4-yl)-N-[2-(4-bromo-phenyl)ethyl]propionamide

hydrochloride (30): By using the same general procedure as that used for

the synthesis of 8, 2-amino-4-{2-[2-(4-bromo-phenyl)ethylcarbamoyl]ethy-

l}imidazole-1-carboxylic acid tert-butyl ester (0.019 g, 0.043 mmol) gave

the title compound 30 (0.016 g, 100%) as a brown foam. ¹H NMR

(300 MHz, CD₃OD): d=7.29 (d, 2H, J=7.8 Hz), 6.99 (d, 2H, J=8.1 Hz),

6.33 (s, 1H), 3.27 (t, 2H, J=7.2 Hz), 2.61 (m, 4H), 2.33 ppm (t, 2H, J=

6.6 Hz); ¹³C NMR (75 MHz, CD₃OD): d=174.20, 139.89, 132.63, 131.92,

127.98, 121.19, 110.14, 41.72, 35.95, 35.18, 21.62 ppm; HRMS (ESI): m/z:

calcd for C₁₄H₁₇BrN₄O: 336.0586 [M]⁺; found: 336.0591.

2-Amino-4-[3-(1-hexyl-heptylcarbamoyl)propyl]imidazole-1-carboxylic

acid tert-butyl ester: By using the general EDC/HOBt procedure, the

title compound was formed as

a tan-colored solid (0.035 g, 21%).

¹H NMR (400 MHz, [D₆]DMSO): d=7.43 (d, 1H, J=8.8 Hz), 6.48 (s,

1H), 6.38 (s, 2H), 3.66 (m, 1H), 2.21 (t, 2H, J=7.2 Hz), 2.04 (t, 2H, J=

7.2 Hz), 1.71 (m, 2H), 1.52 (s, 9H), 1.23 (m, 20H), 0.84 ppm (m, 6H);

¹³C NMR (100 MHz, [D₆]DMSO): d=171.31, 149.94, 148.94, 138.41,

105.72, 84.01, 47.68, 35.06, 34.67, 31.30, 31.11, 28.63, 27.51, 27.18, 25.52,

24.23, 22.06, 22.03, 13.95 ppm; HRMS (ESI): m/z: calcd for C₂₅H₄₆N₄O₃:

450.3570 [M]⁺; found: 450.3562.

3-(2-Amino-1H-imidazol-4-yl)-N-(4-phenyl-butyl)propionamide

hydro-

chloride (31): By using the same general procedure as that used for the

synthesis of 8, 2-amino-4-[2-(4-phenyl-butylcarbamoyl)ethyl]imidazole-1-

carboxylic acid tert-butyl ester (0.080 g, 0.206 mmol) gave the title com-

pound 31 (0.066 g, 100%) as a tan-colored foam. ¹H NMR (300 MHz,

[D₆]DMSO): d=11.91 (s, 1H), 11.59 (s, 1H), 7.97 (m, 1H), 7.34 (s, 2H),

7.25 (m, 2H), 7.17 (d, 3H, J=7.2 Hz), 6.50 (s, 1H), 3.05 (q, 2H, J=

6.0 Hz), 2.58 (m, 4H), 2.35 (t, 2H, J=7.2 Hz), 1.53 (quint., 2H, J=

7.2 Hz), 1.38 ppm (quint., 2H, J=7.2 Hz); ¹³C NMR (75 MHz,

[D₆]DMSO): d=170.62, 146.57, 142.06, 128.24, 128.19, 126.21, 125.61,

108.51, 38.24, 34.73, 33.46, 28.72, 28.30, 20.19 ppm; HRMS (ESI): m/z:

calcd for C₁₆H₂₂N₄O: 286.1794 [M]⁺; found: 286.1799.

4-Dihexylcarbamoyl-butyric acid: Glutaric anhydride (1.00 g, 8.76 mmol)

was dissolved in anhydrous dichloromethane (12 mL). Dihexylamine

(2.27 mL, 9.64 mmol), triethylamine (1.30 mL, 9.64 mmol), and a catalytic

amount of DMAP were added to this solution and the resulting clear so-

lution was stirred at ambient temperature for 15 h, after which time, all

volatiles were removed by rotary evaporation. The crude residue was par-

titioned between ethyl acetate (150 mL) and a 1n HCl aqueous solution

(100 mL). The organic layer was subsequently washed with 1n HCl (3ꢃ

50 mL), saturated NaHCO₃(2ꢃ75 mL), and brine (1ꢃ50 mL). The or-

ganics were then dried over anhydrous Na₂SO₄, filtered, and evaporated

to dryness to afford the title compound (2.44 g, 93%) as a viscous oil

that required no further purification. ¹H NMR (300 MHz, CDCl₃): d=

3.28 (t, 2H, J=7.5 Hz), 3.19 (t, 2H, J=7.5 Hz), 2.41 (m, 4H), 1.95 (t,

2H, J=6.9 Hz), 1.51 (m, 4H), 1.27 (brs, 12H), 0.88 ppm (m, 6H);

Sliding the amide bond

2-Amino-4-(2-heptylcarbamoyl-ethyl)imidazole-1-carboxylic acid tert-

butyl ester: By using the general EDC/HOBt procedure, 2-amino-4-(3-

carboxy-propyl)imidazole-1-carboxylic acid tert-butyl ester (0.115 g,

0.450 mmol) gave the title compound (0.044 g, 27%) as a tan-colored

solid. ¹H NMR (400 MHz, [D₆]DMSO): d=7.78 (m, 1H), 6.50 (s, 1H),

6.38 (s, 2H), 3.00 (q, 2H, J=5.6 Hz), 2.46 (t, 2H, J=8.0 Hz), 2.28 (t, 2H,

J=7.2 Hz), 1.52 (s, 9H), 1.35 (m, 2H), 1.22 (brs, 8H), 0.85 ppm (m, 3H);

¹³C NMR (100 MHz, [D₆]DMSO): d=171.18, 149.82, 148.91, 138.09,

Chem. Eur. J. 2008, 14, 10745 – 10761

ꢁ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

10759

C. Melander et al.

¹³C NMR (75 MHz, CDCl₃): d=177.62, 172.32, 48.32, 46.34, 33.65, 32.08,

31.78, 31.69, 29.25, 27.89, 26.89, 26.69, 22.70, 20.74, 14.19, 14.14 ppm;

HRMS (ESI): m/z: calcd for C₁₇H₃₃NO₃: 299.2460 [M]⁺; found: 299.2462.

A native amide bond

5-Azido-1-bromo-pentan-2-one:

4-Azido-butyric

acid^[39]

(2.15 g,

16.7 mmol) was dissolved in anhydrous dichloromethane (80 mL) at 08C

and a catalytic amount of DMF was added. Oxalyl chloride (4.4 mL,

50 mmol) was added dropwise to this solution and the resulting mixture

was then warmed to room temperature. After 1 h, the solvent and excess

oxalyl chloride were removed under reduced pressure. The resulting oil

was dissolved into anhydrous dichloromethane (10 mL) and added drop-

wise to a 08C solution of CH₂N₂(50 mmol generated from Diazald/

KOH) in diethyl ether (125 mL). This solution was stirred at 08C for

1.5 h, after which time, the reaction was quenched by the dropwise addi-

tion of 48% HBr (6.0 mL). The reaction mixture was diluted with di-

chloromethane (25 mL) and immediately washed with sat. NaHCO₃(3ꢃ

25 mL) and brine (2ꢃ25 mL) before being dried (MgSO₄), filtered, and

concentrated. The amber oil obtained (3.10 g, 90%) upon concentration

was pure and was used in the following steps without further purification.

¹H NMR (300 MHz, [D₆]DMSO): d=4.35 (s, 2H), 3.33 (t, 2H, J=

6.9 Hz), 2.66 (t, 2H, J=7.2 Hz), 1.75 ppm (quint., 2H, J=6.9 Hz);

¹³C NMR (100 MHz, CDCl₃): d=201.33, 50.62, 36.63, 34.26, 23.25 ppm;

HRMS (ESI): m/z: calcd for C₅H₈BrN₃O: 204.9851 [M]⁺; found:

204.9850.

6-Bromo-5-oxo-hexanoic acid dihexylamide: 4-Dihexylcarbamoyl-butyric

acid (1.61 g, 5.40 mmol) was dissolved in anhydrous dichloromethane

(15 mL) at 08C and a catalytic amount of DMF was added. Oxalyl chlo-

ride was added (1.41 mL, 16.2 mmol) dropwise to this solution and the

solution was then warmed to room temperature. After 1 h, the solvent

and excess oxalyl chloride were removed under reduced pressure. The re-

sulting oil was dissolved into anhydrous dichloromethane (10 mL) and

added dropwise to a 08C solution of CH₂N₂(16.20 mmol generated from

Diazald/KOH) in diethyl ether (50 mL). This solution was stirred at 08C

for 1.5 h, after which time, the reaction was quenched by the dropwise

addition of 48% HBr (2.50 mL). The reaction mixture was diluted with

dichloromethane (25 mL) and immediately washed with sat. NaHCO₃

(3ꢃ25 mL) and brine (2ꢃ25 mL) before being dried (MgSO₄), filtered,

and concentrated. The crude yellow oil (0.780 g, 38%) was subsequently

used in the next step without any further purification. ¹H NMR

(400 MHz, [D₆]DMSO): d=4.33 (s, 2H), 2.45 (t, 2H, J=8.8 Hz), 2.75 (t,

2H, J=7.6 Hz), 2.41 (t, 2H, J=7.6 Hz), 1.59 (m, 4H), 1.27 (brs, 12H),

0.86 ppm (m, 6H).

2-Amino-4-(3-dihexylcarbamoyl-propyl)imidazole-1-carboxylic acid tert-

butyl ester: 6-Bromo-5-oxo-hexanoic acid dihexylamide (0.780 g,

2.07 mmol) and Boc-guanidine (0.988 g, 6.21 mmol) were dissolved in

DMF (8 mL) and allowed to stir at room temperature. After 72 h, the

DMF was removed under reduced pressure and the residue was taken up

in ethyl acetate (100 mL) and washed with water (3ꢃ50 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/CH₂Cl₂) to give the title compound (0.643 g, 71%) as a

white foam. ¹H NMR (400 MHz, [D₆]DMSO): d=6.47 (s, 1H), 6.41 (s,

2H), 3.42 (t, 4H, J=7.6 Hz), 2.36 (m, 4H), 1.51 (brs, 13H), 1.25 (brs,

14H), 0.84 ppm (t, 6H, J=6.4 Hz); ¹³C NMR (100 MHz, [D₆]DMSO):

d=183.99, 168.98, 149.95, 148.84, 136.98, 106.15, 103.11, 84.86, 84.06,

48.93, 30.75, 28.05, 27.71, 27.46, 25.35, 21.99, 20.89, 13.81 ppm; HRMS

(ESI): m/z: calcd for C₂₄H₄₄N₄O₃: 436.3413 [M]⁺; found: 436.3412.

2-Amino-4-(3-azido-propyl)imidazole-1-carboxylic acid tert-butyl ester

(38): 5-Azido-1-bromo-pentan-2-one (0.870 g, 4.22 mmol) and Boc-guani-

dine (2.00 g, 12.7 mmol) were dissolved in DMF (15 mL) and allowed to

stir at room temperature. After 24 h, the DMF was removed under re-

duced pressure and the residue was taken up in ethyl acetate (50 mL)

and washed with water (3ꢃ25 mL) and brine (25 mL) before being dried

(Na₂SO₄), filtered, and evaporated to dryness. The resulting oil was puri-

fied by flash column chromatography (10–100% EtOAc/hexanes) to give

the title compound 38 (0.700 g, 63%) as

a

white solid. ¹H NMR

(300 MHz, CDCl₃): d=6.53 (s, 1H), 5.98 (s, 2H), 3.30 (t, 2H, J=6.9 Hz),

2.43 (t, 2H), J=7.2 Hz), 1.87 ppm (quint., 2H, J=6.9 Hz); ¹³C NMR

(75 MHz, CDCl₃): d=150.56, 149.56, 137.64, 107.09, 84.82, 50.89, 28.13,

27.79, 25.26 ppm; HRMS (ESI): m/z: calcd for C₁₁H₁₈N₆O₂: 266.1491

[M]⁺; found: 266.1499.

2-Amino-4-(3-heptanoylamino-propyl)imidazole-1-carboxylic acid tert-

butyl ester: 2-Amino-4-(3-azido-propyl)imidazole-1-carboxylic acid tert-

butyl ester 38 (0.100 g, 0.380 mmol) was added to a solution of anhydrous

THF (4 mL) and 10% Pd/C (0.010 g). Air was removed from the system

and the reaction was back flushed with hydrogen. This process was re-

peated three times before setting the reaction under a hydrogen balloon

at atmospheric pressure and temperature for 8 h. After that time, hepta-

noic anhydride (0.104 mL, 0.390 mmol) was added dropwise to the reac-

tion, which was then allowed to stir overnight. After this time, the reac-

tion was filtered through a Celite pad and the filter cake was washed

with THF (20 mL). The filtrate was concentrated under reduced pressure

to afford the crude product, which was subsequently purified by flash

column chromatography (0–10% MeOH/CH₂Cl₂) to give the title com-

pound (0.093 g, 70%) as a white solid. ¹H NMR (300 MHz, [D₆]DMSO):

d=7.76 (m, 1H), 6.52 (s, 1H), 6.38 (s, 2H), 3.02 (q, 2H, J=6.9 Hz), 2.35

(t, 2H, J=7.5 Hz), 2.03 (t, 2H, J=7.5 Hz), 1.60 (quint., 2H, J=7.2 Hz),

1.53 (s, 9H), 1.46 (m, 2H), 1.24 (m, 6H), 0.85 ppm (t, 3H, J=6.6 Hz);

¹³C NMR (75 MHz, [D₆]DMSO): d=171.89, 149.86, 148.90, 138.36,

105.71, 84.02, 38.10, 35.41, 30.98, 28.28, 27.87, 27.48, 25.27, 25.20, 21.97,

13.88 ppm; HRMS (ESI): m/z: calcd for C₁₈H₃₂N₄O₃: 352.2474 [M]⁺;

found: 352.2488.

3-(2-Amino-1H-imidazol-4-yl)-N-(1-hexyl-heptyl)propionamide

hydro-

chloride (34): By using the same general procedure as that used for the

synthesis of 8, 2-amino-4-[2-(1-hexyl-heptylcarbamoyl)ethyl]imidazole-1-

carboxylic acid tert-butyl ester (0.130 g, 0.297 mmol) gave the title com-

pound 34 (0.107 g, 97%) as an amber oil. ¹H NMR (300 MHz,

[D₆]DMSO): d=11.92 (s, 1H), 11.62 (s, 1H), 7.65 (d, 1H, J=8.7 Hz),

7.36 (s, 2H), 6.51 (s, 1H), 3.65 (m, 1H), 2.63 (t, 2H, J=7.2 Hz), 2.36 (t,

2H, J=7.2 Hz), 1.25 (m, 20H), 0.84 ppm (t, 6H, J=7.2 Hz); ¹³C NMR

(75 MHz, [D₆]DMSO): d=170.24, 146.57, 126.18, 108.50, 47.99, 34.51,

33.54, 31.17, 28.53, 25.39, 21.98, 20.37, 13.87 ppm; HRMS (ESI): m/z:

calcd for C₁₉H₃₆N₄O: 336.2889 [M]⁺; found: 336.2894.

4-(2-Amino-1H-imidazol-4-yl)-N-(1-hexyl-heptyl)butyramide hydrochlo-

ride (35): By using the same general procedure as that used for the syn-

thesis of 8, 2-amino-4-[3-(1-hexyl-heptylcarbamoyl)propyl]imidazole-1-

carboxylic acid tert-butyl ester (0.032 g, 0.071 mmol) gave the title com-

pound 35 (0.027 g, 99%) as

a

tan-colored oil. ¹H NMR (300 MHz,

[D₆]DMSO): d=11.99 (s, 1H), 11.57 (s, 1H), 7.52 (d, 1H, J=8.7 Hz),

7.31 (s, 2H), 6.55 (s, 1H), 3.70 (m, 1H), 2.38 (t, 2H, J=7.2 Hz), 2.08 (t,

2H, J=7.2 Hz), 1.73 (m, 2H), 1.24 (m, 20H), 0.84 ppm (m, 6H);

¹³C NMR (75 MHz, [D₆]DMSO): d=171.00, 146.67, 126.46, 108.70, 47.80,

38.41, 34.59, 31.27, 31.07, 28.61, 25.50, 24.15, 23.60, 22.05, 13.96 ppm;

HRMS (ESI): m/z: calcd for C₂₀H₃₈N₄O: 350.3046 [M]⁺; found: 350.3033.

Heptanoic acid [3-(2-amino-1H-imidazol-4-yl)propyl]amide hydrochlo-

ride (39): By using the same general procedure as that used for the syn-

thesis of 8, 2-amino-4-(3-heptanoylamino-propyl)imidazole-1-carboxylic

acid tert-butyl ester (0.041 g, 0.120 mmol) gave the target compound 39

(0.033 g, 99%) as a tan-colored amorphous solid. ¹H NMR (400 MHz,

[D₆]DMSO): d=12.08 (s, 1H), 11.61 (s, 1H), 7.91 (m, 1H), 7.33 (s, 2H),

6.57 (s, 1H), 3.03 (q, 2H, J=6.8 Hz), 2.39 (t, 2H, J=6.8 Hz), 2.05 (t, 2H,

J=6.4 Hz), 1.62 (t, 2H, J=6.4 Hz), 1.47 (m, 2H), 1.23 (brs, 6H),

0.85 ppm (t, 3H, J=6.4 Hz); ¹³C NMR (75 MHz, [D₆]DMSO): d=172.21,

146.67, 126.34, 108.65, 37.49, 35.42, 30.99, 28.32, 27.84, 25.25, 21.98, 21.44,

13.90 ppm; HRMS (ESI): m/z: calcd for C₁₃H₂₄N₄O: 252.1950 [M]⁺;

found: 252.1957.

4-(2-Amino-1H-imidazol-4-yl)-N,N-dihexyl-butyramide

hydrochloride

(36): By using the same general procedure as that used for the synthesis

of 8, 2-amino-4-(3-dihexylcarbamoyl-propyl)imidazole-1-carboxylic acid

tert-butyl ester (0.562 g, 1.29 mmol) gave the title compound 36 (0.451 g,

94%) as a brown foam. ¹H NMR (300 MHz, [D₆]DMSO): d=12.21 (s,

1H), 11.67 (s, 1H), 7.39 (s, 2H), 6.50 (s, 1H), 3.43 (t, 4H, J=7.5 Hz),

2.45 (m, 4H), 1.57 (m, 4H), 1.25 (brs, 14H), 0.85 ppm (t, 6H, J=6.6 Hz);

¹³C NMR (100 MHz, [D₆]DMSO): d=146.78, 125.40, 108.98, 83.99, 49.07,

30.79, 28.11, 25.41, 24.33, 21.99, 20.63, 13.85 ppm; HRMS (ESI): m/z:

calcd for C₁₉H₃₆N₄O: 336.2889 [M]⁺; found: 336.2897.

10760

www.chemeurj.org

ꢁ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Chem. Eur. J. 2008, 14, 10745 – 10761

A Second-Generation Reverse-Amide Oroidin Library

FULL PAPER

Examination of a triazole isostere

[10] B. R. Bloom, C. J. L. Murray, Science 1992 , 257 , 1055.

[11] J. M. Conly, B. L. Johnston, Can. J. Infect. Dis. Med. Microbiol.

2005, 16, 159.

[12] A. P. Johnson, A. H. C. Uttley, N. Woodford, R. C. George, Clin. Mi-

crobiol. Rev. 1990, 3, 280.

[13] D. J. Musk Jr, P. J. Hergenrother, Curr. Med. Chem. 2006, 13, 2163.

[14] R. W. Huigens III, L. Y. Ma, C. Gambino, P. D. R. Moeller, A.

Basso, J. Cavanagh, D. J. Wozniak, C. Melander, Mol. BioSyst. 2008 ,

4 , 614.

[15] R. W. Huigens, J. J. Richards, G. Parise, T. E. Ballard, W. Zeng, R.

Deora, C. Melander, J. Am. Chem. Soc. 2007 , 129 , 6966.

[16] J. J. Richards, T. E. Ballard, R. W. Huigens, C. Melander, ChemBio-

Chem 2008 , 9 , 1267.

[17] J. J. Richards, T. E. Ballard, C. Melander, Org. Biomol. Chem. 2008 ,

6 , 1356.

[18] J. J. Richards, R. W. Huigens, T. E. Ballard, A. Basso, J. Cavanagh,

C. Melander, Chem. Commun. 2008 , 1698.

[19] S. A. Rogers, C. Melander, Angew. Chem. 2008 , 120 , 5307; Angew.

Chem. Int. Ed. 2008 , 47 , 5229.

[20] J. J. Richards, C. S. Reed, C. Melander, Bioorg. Med. Chem. Lett.

2008 , 18 , 4325.

2-Amino-4-[3-(4-hexylACHTUNGTRENNUNG[1,2,3]triazol-1-yl)propyl]imidazole-1-carboxylic

acid tert-butyl ester: Azide 38 (0.108 g, 0.406 mmol) was dissolved in a

1:1 mixture of ethanol (2 mL) and water (2 mL). Aodium-l-ascorbate

(0.017 g, 0.081 mmol), CuSO₄·5H₂O (0.010 g, 0.041 mmol), and 1-octyne

(0.054 g, 0.487 mmol) were added to this solution. The reaction was

stirred at room temperature and monitored by TLC analysis. When com-

pletion of the reaction was evident, the ethanol was removed by rotary

evaporation. The aqueous residue was diluted with water (50 mL) and

EtOAc (100 mL). The organic layer was washed with sat. NaHCO₃(3ꢃ

50 mL) and brine (50 mL), before being dried (Na₂SO₄), filtered, and

evaporated to dryness. Purification of the crude product by flash column

chromatography (0–10% MeOH/CH₂Cl₂) afforded the desired product

(0.084 g, 55%) as an amber oil. ¹H NMR (300 MHz, [D₆]DMSO): d=

7.84 (s, 1H), 6.55 (s, 1H), 6.41 (s, 2H), 4.30 (t, 2H, J=6.9 Hz), 2.58 (t,

2H, J=7.5 Hz), 2.22 (t, 2H, J=6.9 Hz), 2.02 (quint., 2H, J=6.9 Hz),

1.55 (m, 11H), 1.27 (m, 6H), 0.85 ppm (t, 3H, J=6.9 Hz); ¹³C NMR

(75 MHz, [D₆]DMSO): d=149.93, 148.86, 146.77, 137.46, 121.61, 106.02,

84.10, 48.68, 30.95, 28.89, 28.54, 28.19, 27.48, 24.99, 24.56, 21.96,

13.86 ppm; HRMS (ESI): m/z: calcd for C₁₉H₃₂N₆O₂: 376.2587 [M]⁺;

found: 376.2595.

[21] H. Hoffmann, T. Lindel, Synthesis 2003, 1753.

[22] I. A. Holder, Vaccine 2004 , 22 , 831.

4-[3-(4-Hexyl

chloride (40): By using the same general procedure as that used for the

synthesis of 8, 2-amino-4-[3-(4-hexyl[1,2,3]triazol-1-yl)propyl]imidazole-1-

A

hydro-

[23] R. C. Boucher, Eur. Respir. J. 2004 , 23 , 146.

[24] J. W. Costerton, Trends Microbiol. 2001 , 9 , 50.

[25] J. A. Driscoll, S. L. Brody, M. H. Kollef, Drugs 2007 , 67 , 351.

[26] T. F. C. Mah, G. A. OꢀToole, Trends Microbiol. 2001 , 9 , 34.

[27] R. B. Moss, Clin. Infect. Dis. 1995, 21, 839.

[28] J. B. Lyczak, C. L. Cannon, G. B. Pier, Clin. Microbiol. Rev. 2002 , 15 ,

194.

[29] M. E. Falagas, E. A. Karveli, Clin. Microbiol. Infect. Clin. Microbiol.

Infec. 2007, 13, 117.

[30] A. P. Tomaras, C. W. Dorsey, R. E. Edelmann, L. A. Actis, Microbi-

ology 2003 , 149 , 3473.

AHCTUNGTRENNUNG

carboxylic acid tert-butyl ester (0.054 g, 1.43 mmol) gave the title com-

pound 40 (0.045 g, 100%) as an amber oil. ¹H NMR (300 MHz,

[D₆]DMSO): d=12.12 (s, 1H), 11.68 (s, 1H), 7.88 (s, 1H), 7.37 (s, 2H),

6.59 (s, 1H), 4.32 (t, 2H, J=6.9 Hz), 2.59 (t, 2H, J=7.5 Hz), 2.38 (t, 2H,

J=7.2 Hz), 2.06 (quint., 2H, J=7.5 Hz), 1.57 (m, 2H), 1.27 (m, 6H),

0.85 ppm (t, 3H, J=6.9 Hz); ¹³C NMR (75 MHz, [D₆]DMSO): d=146.83,

146.78, 125.47, 121.83, 108.91, 48.33, 30.97, 28.90, 28.24, 28.01, 24.96,

21.99, 21.20, 13.91 ppm; HRMS (ESI): m/z: calcd for C₁₄H₂₄N₆: 276.2062

[M]⁺; found: 276.2066.

[31] D. H. Forster, F. D. Daschner, Eur. J. Clin. Microbiol. 1998, 17, 73.

[32] C. Qi, M. Malczynski, M. Parker, M. H. Scheetz, J. Clin. Microbiol.

2008 , 46 , 1106.

[33] A. O. Reis, D. A. M. Luz, M. C. B. Tognim, H. S. Sader, A. C. Gales,

Emerging Infect. Dis. 2003, 9, 1025.

Acknowledgements

Financial support is gratefully acknowledged from the NCSU, Agile Sci-

ences, the University of North Carolina Competitiveness Research Fund,

and the Robert Proctor Undergraduate Research Scholarship (ALW).

[34] L. M. Junker, J. Clardy, Antimicrob. Agents Chemother. 2007 , 51 ,

3582.

[35] S. J. Connon, S. Blechert, Angew. Chem. 2003 , 115 , 1944; Angew.

Chem. Int. Ed. 2003 , 42 , 1900.

[36] M. Schuster, S. Blechert, Angew. Chem. 1997 , 109 , 2124; 1997, 36,

2036.

[37] A. R. English, D. Girard, V. J. Jasys, R. J. Martingano, M. S. Kellogg,

J. Med. Chem. 1990 , 33 , 344.

[38] Y. K. Che, A. Datar, K. Balakrishnan, L. Zang, J. Am. Chem. Soc.

2007 , 129 , 7234.

[39] N. Khoukhi, M. Vaultier, R. Carrie, Tetrahedron 1987 , 43 , 1811.

[40] H. C. Kolb, K. B. Sharpless, Drug Discovery Today Drug Discov.

Today 2003 , 8 , 1128.

[41] V. D. Bock, H. Hiemstra, J. H. van Maarseveen, Eur. J. Org. Chem.

2006 , 51.

[42] G. C. Tron, T. Pirali, R. A. Billington, P. L. Canonico, G. Sorba,

A. A. Genazzani, Med. Res. Rev. 2008 , 28 , 278.

[1] G. D. Geske, R. J. Wezeman, A. P. Siegel, H. E. Blackwell, J. Am.

Chem. Soc. 2005 , 127 , 12762.

[2] J. F. Hu, E. Garo, M. G. Goering, M. Pasmore, H. D. Yoo, T. Esser,

J. Sestrich, P. A. Cremin, G. W. Hough, P. Perrone, Y. S. L. Lee, N. T.

Le, M. OꢀNeil-Johnson, J. W. Costerton, G. R. Eldridge, J. Nat.

Prod. 2006 , 69 , 118.

[3] M. Manefield, R. de Nys, N. Kumar, R. Read, M. Givskov, P. Stein-

berg, S. A. Kjelleberg, Microbiology 1999, 145, 283.

[4] M. Manefield, T. B. Rasmussen, M. Henzter, J. B. Andersen, P.

Steinberg, S. Kjelleberg, M. Givskov, Microbiology 2002, 148, 1119.

[5] J. W. Costerton, P. S. Stewart, E. P. Greenberg, Science 1999 , 284 ,

1318.

[6] D. Davies, Nat. Rev. Drug Discovery 2003, 2, 114.

[7] C. M. McKee, C. L. Houck, Proc. Soc. Exp. Biol. Med. 1943, 53, 33.

[8] H. C. Neu, Science 1992 , 257 , 1064.

Received: July 13, 2008

[9] B. R. Bloom, Nature 1992 , 358 , 538.

Published online: October 22, 2008

Chem. Eur. J. 2008, 14, 10745 – 10761

ꢁ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

10761

Article Doi

Synthesis and antibiofilm activity of a second-generation reverse-amide oroidin library: A structure-activity relationship study

DOI: 10.1002/chem.200801419

Source and publish data:

Authors:

Article abstract of DOI:10.1002/chem.200801419

Full text of DOI:10.1002/chem.200801419

Products guided by the article

R&D Labs maybe for 1110786-37-2

Relevant to this article

Hot Product