Structure?Activity Relationships of Melanocortin Agonists Containing the Benzimidazole Scaffold

Structure–Activity Relationships of MelanocortinAgonists Containing the Benzimidazole Scaffold

Aleksandar Todorovic, Christine G.Joseph, Nicholas B. Sorensen, Michael S.Wood and Carrie Haskell-Luevano*

Department of Medicinal Chemistry, College of Pharmacy, Universityof Florida, Gainesville, FL 32610, USA*Corresponding author: Carrie Haskell-Luevano, [email protected]

The melanocortin system has been implicated inregulating various physiological processes inclu-ding pigmentation, energy homeostasis, obesity,steroidogenesis cardiovascular, and exocrinegland function. The five melanocortin receptorsthat belong to the super family of G protein-coupled receptors are stimulated by naturallyoccurring agonists. The aim of this research wasfocused on the design, synthesis, and pharmacolo-gical characterization of melanocortin ligands thatcontain the 1,2,5-trisubstituted benzimidazolescaffold. A series of benzimidazole analogues,with three points of diversity at positions 1, 2, and5, were designed, synthesized, pharmacologicallyassayed at the mouse melanocortin receptorsMC1R, MC3R, MC4R, and MC5R and resulted in lig-ands possessing a range of agonist activity fromnM to no stimulation at up to 100 lM concentra-tions. This study demonstrates that the benzimi-dazole structure template can be appended withkey melanocortin agonist amino acids for thedesign melanocortin receptor agonist ligands.

Key words: benzimidazole, melanocortin, obesity, solid-phase,synthesis

Received 22 March 2007, revised and accepted for publication 16 April2007

The melanocortin system has been identified as an important regu-lating factor implicated in a diverse number of physiological path-ways including pigmentation, energy homeostasis, obesity,cardiovascular, sexual function, and exocrine gland function. Themelanocortin system consists of endogenous agonists, antagonists,and receptors. Five melanocortin receptors have been identified todate (MC1R-MC5R) (1–6), that belong to the G protein-coupledreceptor (GPCR) super family and stimulate the cyclic adenosine-5¢-monophosphate (cAMP) signal transduction pathway via the Gs

protein and adenylyl cyclase. The MC1R is primarily expressed inmelanocytes where it is involved in the regulation of pigmentation

(4,7,8). The MC2R is expressed in the adrenal gland where itregulates lipogenesis, glucocorticoid and aldosterone production(4,9,10). The MC3R and MC4R are located in the central nervoussystem (CNS) (11–13). The MC3R is involved in the regulation ofenergy homeostasis, while the MC4R has been reported to have animportant role in obesity, appetite control and sexual function (14–17). The MC5R is expressed both centrally and in the periphery,and has been implicated in the regulation of exocrine gland func-tion in mice (18,19).

All naturally occurring melanocortin agonists are derived from theproopiomelanocortin (POMC) prohormone by prohormone convertas-es (PC) (20,21). The POMC processed melanocortin agonists includemelanocyte stimulating hormones, a-MSH, b-MSH, c-MSH and theadrenocorticotropic hormone (ACTH). Natural melanocortin agonistsdiffer in the length and the amino acid sequence. Some of whichare processed at the termini (a-MSH is acylated at the N-terminusand carboxamidated at the C-terminus), while the others, i.e.,ACTH, possess free carboxylic acid group at the C-terminus. Inter-estingly, a-MSH corresponds to the first 13 amino acid residues ofthe ACTH molecule. All natural agonist ligands possess the com-mon structural motif, His–Phe–Arg–Trp that has been identified asan important factor in eliciting pharmacological response at theMC1R, MC3R, MC4R, and MC5R. It appears that MC2R can only bestimulated by the ACTH molecule (4,5). The Ac–His–DPhe–Arg–Trp–NH2 sequence has been subjected to the extensive structure activ-ity relationship studies (SAR). The positional scanning approach(PSA), (one amino acid position is targeted with different encodedand non-encoded amino acids, while the other three were kept con-stant), has resulted in ligands with altered potency, selectivity andconversion from agonist into antagonist activity (22–25). The PSAfocused on the acyl or capping region of the Ac–His–DPhe–Arg–Trp–NH2 tetrapeptide, has been shown to be an excellent tool forimproving MC1R potency and selectivity (26–29). As it is wellrecognized that peptides generally possess low bioavailability pro-files and fast metabolism rates, a goal in the discovery of novelmelanocortin ligands is the design and synthesis of small moleculesand ligands with increased enzymatic stability and in vivo half lives.The ideal candidate would possess high receptor selectivity, nM

potency, low toxicity and side effects, and appropriate metabolicprofile.

The important physiological implications of the melanocortin systemin the regulation of obesity and energy homeostasis have resultedin the discovery of small molecule ligands. Several publicationshave revealed the synthesis and pharmacological characterization ofsmall molecule melanocortin ligands (based on the piperidine and

338

Chem Biol Drug Des 2007; 69: 338–349

Research Article

ª 2007 The AuthorsJournal compilation ª 2007 Blackwell Munksgaard

doi: 10.1111/j.1747-0285.2007.00511.x

piperazine scaffolds) (30–34) that may be related to the privilegedstructure and drug design approach. A privileged structure can bedefined as the series of molecules with capacity to bind to multiplereceptors with high affinity (35). A large set of molecular scaffoldshas been identified as privileged structures and reviewed (36,37).The benzimidazole structure has been reported in commerciallyavailable drugs, including antihistamine Astemizole, and the antiul-cerative drug Omeprazole (38,39). Benzimidazoles have also beenreported as inhibitors of phosphodiesterase IV (40), antagonists ofangiotensin II receptors (41,42), and neuropeptide Y binding (43,44).Additionally, benzimidazole structures have been identified as anti-arrhythmics (45) and antivirals (46,47). More recently, it has beenreported that benzimidazole small molecules are ligands for sero-tonin receptors (5-HT) (48–50), histamine (H4) receptors (51), bradyk-inin (B2) receptors (52), and dopamine (D4) receptors (53). The aimof this study was to investigate whether the 1,2,5-trisubstitutedbenzimidazole template may be used as a scaffold for the design ofmelanocortin ligands. Concurrently with our design and synthesis,1,2,6-trisubstituted benzimidazoles and analogous compounds havebeen reported in the patent literature by Poitout et al. to be potentMC4R agonists (54).

Materials and Methods

Benzimidazole synthesisThe amino acids fluorenylmethyloxycarbonyl (Fmoc)-Trp(Boc), Fmoc-Arg(Pbf), Fmoc-DPhe, and Fmoc-His(Trt) were purchased fromPeptides International (Louisville, KY, USA). The rink-amide p-methyl-benzylhydrylamine (MBHA) resin (0.40 mEq/g substitution) and WangResin (0.90 mEq/g substitution) were purchased from Peptides Inter-national. The coupling reagents, 1-hydroxybenzotriazole (HOBt) andbenzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluoro-phosphate (BOP) were purchased from Peptides International. Glacialacetic acid (HOAc), dichloromethane (DCM), methanol (MeOH), aceto-nitrile (ACN), and anhydrous ethyl ether were purchased from Fisher(Fair Lawn, NJ, USA). N,N-dimethylformamide (DMF) was purchasedfrom Burdick and Jackson (McGaw Park, IL, USA). Trifluoroacetic acid(TFA), N,N'- diisopropylcarbodiimide (DIC), pyridine, piperidine and4-dimethylaminopyridine (DMAP) were purchased from Sigma (StLouis, MO, USA). N,N-diisopropylethylamine (DIEA), triisoprpylsilane(TIS), 4-fluoro-3-nitrobenzoic acid, N-methyl-2-pyrrolidone, 3,3-diphe-nylpropylamine, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ),2-amino-1-phenylethanol, 2-(4-chlorophenyl)ethylamine, 4(5)-imidazol-

Scheme 1: Synthesis of benzimidazoles on solid support. (a) 3.0 equivalents 4-fluoro-3-nitrobenzoic acid, 1.5 eq. DIC in DMF 3 h, 23 �C;(b) 5.0 eq. R1NH2, 10% v/v DIEA in NMP 18 h 23 �C; (c) 3.0 M SnCl2 in NMP 16 h 23 �C; (d) 5.0 eq. of R2CHO, 2.0 eq of DDQ, DMF 18 h23 �C; (e) cleavage (see Materials and Methods for details); DIC ¼ 1,3-diisopropylcarbodiimide, DIEA ¼ N,N-diisopropylethylamine, NMP ¼1-methyl-2-pyrrolidinone, DDQ ¼ 2,3-dichloro-5,6-dicyano-1,4-benzoquinone.

Structure–activity relationships of melanocortin agonists

Chem Biol Drug Des 2007; 69: 338–349 339

ecarboxaldehyde, 2-phenylethylamine, tryptamine, 1-naphtalenemeth-ylamine, n-octanal, tyramine, histamine, 2-naphtaldehyde, 1-(2-ami-noethylpiperidine), and indole-3-carbaldehyde were purchased fromAldrich (Milwaukee, WI, USA). The stannous chloride dihydrate(SnCl2 · 2H2O) was purchased from Acros Organics (Moris Plains,NJ, USA). The 3-phenylpropionaldehyde was purchased from Fluka(Buchs SG, Switzerland). All reagents and chemicals were ACS gradeor better and were used without further purification.

The synthesis of benzimidazoles on solid support is based on theprevious reports (55,56), and is summarized in Scheme 1. The initialbenzimidazole compounds (1,2) were synthesized on the either RinkAmide or Wang Resin. Synthesis was performed on a 0.2 mmolescale in a manual reaction vessel (Peptides International). The resinwas allowed to swell in DMF at least 2 h before the synthesis.Three equivalents of 4-fluoro-3-nitrobenzoic were coupled to theFmoc-deprotected Rink Amide MBHA resin in the presence of 1.5equivalents of DIC in the DMF for 3 h. The Wang Resin was trea-ted with 4 equivalents of 4-fluoro-3-nitrobenzoic acid, 2 equivalentsof DIC, and 0.1 equivalent of DMAP in DMF at room temperaturefor 16 h. The successful attachment of 4-fluoro-3-nitrobenzoic acidonto the resin yielded the bright yellow color of the resin (in thecase of Rink Amide linker, Ninhydrin test may be performed to mon-itor complete coupling) (57). The resin was washed extensively withDMF (3·) and NMP (5·). Ipso substitution of fluorine was achievedby employing 5 equivalents of amine in the 10% v/v DIEA in NMP,for 18 h at room temperature. The resin was washed 5· with NMPthe following day. The aromatic nitro group was reduced with tin(II) chloride. The resin was treated with 3.0 M solution of stannouschloride in NMP at room temperature for 16 h. After the nitrogroup has been reduced to the primary aromatic amine, the brightyellow color of the resin disappeared. The resin was washed exten-sively with NMP, DMF, DCM, and DMF. The benzimidazole ring clo-sure was achieved by treating the resin with 5 equivalents ofaldehyde, 2 equivalents of DDQ in DMF for 18 h at room tempera-ture. The resulting resin was washed with DMF (3·), DCM (5·) anddried in vacuo before the cleavage. Cleavage from the solid supportwas performed with 50% of TFA in DCM (keep the volume of clea-vage cocktail �5 mL) for 30 min (Wang Resin) or 1 h (Rink AmideResin). The resin was washed with 50% of TFA in DCM (2 mL). Col-leted samples were mixed together and the solvents were evapor-ated (Nitrogen gas stream). The samples were dried in vacuo for48 h. Crude analogues were purified by RP-HPLC using a Shimadzuchromatography system with a photodiode array detector and asemipreparative RP-HPLC C18-bonded silica column (Vydac218TP1010, 1.0 · 25 cm; Hesperia, CA, USA) and lyophilized. Thepurity was >95% pure as determined by analytical RP-HPLC (k ¼280 or 214 nm), and had the correct molecular mass (University ofFlorida protein chemistry core facility) (Table 1).

The amino acid building blocks were introduced through the stand-ard Fmoc coupling strategy (58–60) in the position 5 of benzimid-azoles. The Rink Amide MBHA resin was allowed to swell for 2 hin DMF followed by Na deprotection of the Fmoc group using 20%piperidine in DMF for 2 min followed by another 20-min 20% pip-eridine treatment. A positive Kaiser test was used to indicate thepresence of free amine groups on the resin (57). The growing pep-tide chain was synthesized using the general amino acid coupling

cycle: addition of threefold excess amino acid, addition of threefoldexcess BOP, and HOBt and addition of sixfold excess of DIEA inDMF to the manual reaction vessel. The coupling reaction is mixedby bubbling with nitrogen (N2) gas for 2 h, followed by emptyingthe reaction vessel under vacuum. The resin-Na-protected peptideis first washed with DMF (5 · 1 min) to remove the excess rea-gents, and subsequently Na-Fmoc deprotection is performed using20% piperidine in DMF as described above. The peptide resin iswashed with DMF to remove the piperidine and the remainingamino acids are coupled to the growing peptide chain as describedabove, until amino acid elongation has been completed. The benz-imidazole synthesis was performed as described above. The clea-vage of benzimidazoles with amino acids present in the position 5was achieved by resin treatment with the cleavage cocktail (95%TFA, 2.5% water and 2.5% TIS) for 3 h in the manual reaction ves-sel. The cleavage product was collected into pre-weighted 50 mLconical tube. The resin was washed with 1.5 mL of cleavage cock-tail for 5 min and drained to the collection vial. The crude com-pound was then precipitated with cold (4 �C) anhydrous ethyl ether(up to 50 mL). The flocculent compounds were pelleted by centrifu-gation (Sorval Super T21 high speed centrifuge using the swingingbucket rotor) at 3313 ·g for 5 min. The ether was decanted off,the product was washed once again with cold anhydrous ethylether and pelleted. The crude benzimidazoles were dried in vacuuofor 48 h. Crude analogues were purified by RP-HPLC using a Shim-adzu chromatography system with a photodiode array detector anda semipreparative RP-HPLC C18-bonded silica column (Vydac218TP1010, 1.0 · 25 cm; Hesperia, CA, USA) and lyophilized.

Table 1: Analytical data for the benzimidazoles synthesized inthis study

CompoundHPLC k '(System 1)

HPLC k '(System 2)

Purity(%)

Calculatedmass

Mass spectralanalysis (M+1)

1 9.2 16.4 >99 460.0 462.02 9.3 16.4 >98 461.0 463.03 3.2 6.6 >99 347.4 348.64 5.0 9.2 >99 365.8 366.75 3.6 7.0 >99 487.6 488.66 3.9 6.7 >99 485.6 486.47 3.3 5.3 >99 515.6 516.58 2.8 5.5 >98 503.6 504.69 2.8 5.6 >98 537.6 538.7

10 3.5 6.9 >99 526.6 527.511 7.8 13.3 >98 555.8 557.612 4.5 8.6 >98 523.6 525.013 4.0 7.0 >99 810.9 811.914 6.0 11.0 >99 860.0 861.215 4.3 8.6 >99 673.8 674.616 5.0 9.5 >99 673.8 674.7

HPLC k' ¼ [(peptide retention time – solvent retention time) / solventretention time] in system 1 (10% acetonitrile in 0.1% triflouroacetic acid/water and a gradient to 90% acetonitrile over 35 min) or solvent system 2(10% methanol in 0.1% triflouroacetic acid/water and a gradient to 90%methanol over 30 min. The analytical column Vydac C18 (Vydac 218TP104)was used, in both cases with flow of 1.5 mL/min. The percentage peptidepurity is determined by HPLC at a wavelength of k. ¼ 214 or 280 nm. MassSpectral Analysis was performed in the Protein Chemistry Core Facility, Uni-versity of Florida, using MALDI-TOF (Voyager DE-Pro, Foster City, CA, USA).

Todorovic et al.

340 Chem Biol Drug Des 2007; 69: 338–349

The purity was >95% pure as determined by analytical RP-HPLC(k ¼214 nm), and had the correct molecular mass (University ofFlorida protein chemistry core facility) (Table 1).

Cell culture and transfectionBriefly, HEK-293 cells were maintained in Dulbecco's modifiedEagle's medium (DMEM) with 10% fetal calf serum and seeded1 day prior to transfection at 1 to 2 · 106 cell/100-mm dish. Mel-anocortin receptor DNA in the pCDNA3 expression vector (20 lg)were transfected using calcium phosphate method. Stable receptorpopulations were generated using G418 selection (1 mg/mL) forsubsequent bioassay analysis.

Functional bioassayHEK 293 cells stably expressing the melanocortin receptors weretransfected with 4 lg CRE/b-galactosidase reporter gene as previ-ously described (24,25,61). In brief, 5000 to 15 000 posttransfectioncells were planted into 96-well Primera plates (Falcon, FranklinLakes, CA, USA) and incubated overnight. Forty eight hours post-transfection, the cells were stimulated with 100 lL peptide (10)4–10)12

M) or forskolin (10)4M) control in assay medium (DMEM

containing 0.1 mg/mL BSA and 0.1 nM isobutylmethylxanthine) for6 h. The assay media was aspirated and 50 lL of lysis buffer(250 mM Tris–HCl pH ¼ 8.0 and 0.1% Triton X-100) was added.The plates were stored and )80 �C overnight. The plates contain-ing the cell lysates were thawed the following day. Aliquots of10 lL were taken from each well and transferred to another 96-well plate for relative protein determination. To the cell lysateplates, 40 lL phosphate- buffered saline with 0.5% BSA wasadded to each well. Subsequently, 150 lL substrate buffer (60 mM

sodium phosphate, 1 mM MgCl2, 10 mM KCl, 5 mM b-mercaptoeth-anol, 2 mg/mL ONPG) was added to each well and the plates wereincubated at 37 �C. The sample absorbance OD405, was measuredusing a 96-well plate reader (Molecular Devices, Sunnyvale, CA,USA). The relative protein was determined by adding 200 lL 1:5dilution Bio-Rad G250 protein dye:water to the 10 lL cell lysatesample taken previously, and the OD595 was measured on a95-well plate reader (Molecular Devices). Data points were normal-ized both to the relative protein content and non-receptor depend-ant forskolin stimulation.

Data analysisEC50 values represent the mean of duplicate experiments performedin quadruplet or more independent experiments. EC50 estimates andtheir associated standard errors were determined by fitting the datato a nonlinear least-squares analysis using the PRISM program(v4.0, GraphPad Inc., San Diego, CA, USA).

Results

Chemical synthesis and characterizationThe compounds reported herein were synthesized in the manualreaction vessel (Peptides International) using standard fluorenyl-methyloxycarbonyl (Fmoc) chemistry (58–60) based upon the

previously reported strategy for the synthesis of benzimidazoles onthe solid support (55,56), summarized in Scheme 1. The compoundswere purified to homogeneity using semipreparative reversed-phasehigh-pressure liquid chromatography (RP-HPLC). All compounds pos-sessed the correct molecular weights, as determined by MALDI-TOFmass spectrometry (Table 1). These compounds were characterizedby analytical RP-HPLC in two diverse solvent systems (Table 1) and1H NMR.

Biological resultsTables 2 and 3 and Figure 1 represent the pharmacological sum-mary obtained in this study. The benzimidazole scaffold utilized inthis study possessed three positions for diversification (positions 1,2 and 5). Compounds presented in Table 2 (1–5) were devoid ofany melanocortin receptor functional activity, at up to 100 lM con-centrations. Compounds 6–9 were devoid of any functional activityat the mouse MC3R, MC4R, and MC5R, up to 100 lM concentra-tions. However, some stimulatory activity was observed at themMC1R, but not sufficient enough to determine EC50 values. Com-pounds 6 and 7 possessed almost identical pharmacological pro-file at the mMC1R, with 62% and 60% maximal observablestimulation at 100 lM concentrations, respectively. Benzimidazole8 possessed the capability to stimulate the MC1R up to 70% at100 lM, whereas compound 9 caused 52% stimulation at the100 lM concentration (Table 2). The derivative 10, with 3-indolyl-ethyl moiety at position 1 of the benzimidazole scaffold wasdevoid of functional activity at the MC3R. Compound 10, pos-sessed low lM EC50 values at the mMC1R (EC50 ¼ 5.9 lM) anddecreased potency at the MC4R and MC5R (EC50 ¼ 41 lM andEC50 ¼ 30 lM, respectively) but lacked stimulatory activity at theMC3R. Benzimidazole 11, with the n-heptyl chain at the 2 posi-tion, possessed equipotent EC50 values at the peripherallyexpressed MC1R and MC5R, within the threefold experimentalerror, while being completely devoid of any stimulatory activity atthe MC3 and MC4 receptors at up to 100 lM concentrations.Compound 12, with 1-naphtylmethyl moiety at the position 1 and5-imidazolyl moiety at the position 2 of the benzimidazole scaffold,possessed slightly higher potency at the MC1R versus MC5R,albeit in the lM range, and some stimulatory activity for com-pound 12 was observed at the MC3R and MC4R (Table 2). Thecompounds 13–16 possessed more then one amino acid at posi-tion 5 of the benzimidazole scaffold (Table 3). Compound 13, withHis–DPhe–Arg sequence, was the most potent compound designedin this study and possessed EC50 values in the nM range: 48, 640,610, and 440 nM at the mouse MC1R, MC3R, MC4R, and MC5R,respectively. Compound 14 that possessed Trp instead of His inthe tripeptidyl sequence (position 5 of the benzimidazole) wasdevoid of any stimulatory activity at the MC3R up to 100 lM con-centrations, while being only slight active at the MC4R (44%activity at 100 lM) (Table 3). Compound 14 possessed EC50 valuesof 650 nM at the MC1R and 5 lM at the MC5R. Similarly, the dip-eptidyl DPhe–Arg benzimidazole 15, possessed no functional activ-ity at the MC3R, and only some stimulatory activity at thecentrally expressed MC4R. Analogue 16, possesses the Arg–DPhecombination at the 5 position, resulted in with EC50 values of 2.6,21.5, 15.5, and 5.7 lM at the mouse MC1R, MC3R, MC4R, andMC5R, respectively.



Table

2:

Func

tiona

lact

ivity

ofth

ebe

nzim

idaz

ole-

base

dco

mpo

unds

atth

em

ouse

mel

anoc

ortin

rece

ptor

sa

R2

NN

O

R3

R1

Com

poun

dR 1

R 2R 3

mM

C1R

EC50

(lM

)m

MC3

REC

50(l

M)

mM

C4R

EC50

(lM

)m

MC5

REC

50(l

M)

1)

NH 2

>100

000

>100

000

>100

000

>100

000

2)

OH>1

0000

0>1

0000

0>1

0000

0>1

0000

0

3O

HN

N H

)N

H 2>1

0000

0>1

0000

0>1

0000

0>1

0000

0

4

Cl

N

N H

)N

H 2>1

0000

0>1

0000

0>1

0000

0>1

0000

0

5N

N H

NH

NH2

O

HN

NH2

NH

>100

000

>100

000

>100

000

>100

000

Todorovic et al.


Table

2:

Cont

inue

d

Com

poun

dR 1

R 2R 3

mM

C1R

EC50

(lM

)m

MC3

REC

50(l

M)

mM

C4R

EC50

(lM

)m

MC5

REC

50(l

M)

6N

N

N H

62%

@10

0lM

>100

000

>100

000

>100

000

7

N

N H

NH

NH

2

O

HN

NH

2

NH

60%

@10

0lM

>100

000

>100

000

>100

000

8

HO

N

N H

NH

NH

2

O

HN

NH

2

NH

70%

@10

0lM

>100

000

>100

000

>100

000

9

N

N H

NH

NH2

O

HN

NH2

NH

52%

@10

0lM

>100

000

>100

000

>100

000

10

N H

N

N H

NH

NH

2

O

HN

NH

2

NH

5.9

€1.

8>1

0000

041

.3€

24.8

30€

0.9

11

n-He

ptyl

NH

NH

2

O

HN

NH

2

NH

14.0

€6.

5>1

0000

0>1

0000

025

€3.

0



Discussion

The melanocortin system has been implicated in the regulation of var-ious physiological functions including pigmentation, steroidogenesis,energy homeostasis, food intake and control, inflammation, erectileand sexual activity and exocrine gland function (7–10,14,15,17,18,62).During this past decade, extensive SAR studies of melanocortin lig-ands have been performed by both academic and industrial researchgroups. An approach utilized by our laboratory, as well as others,focused upon SPPS and positional scanning approaches in attemptsto optimize the His-Phe–Arg–Trp 'core' melanocortin agonist tetrapep-tide sequence in terms of potency and receptor subtype selectivity.With a similar objective in mind, we utilized peptide backbone modifi-cations of the tetrapeptide (63), applied peptoid,(64), and urea based(65) synthesis in order to design analogues with altered melanocortinreceptor selectivity and/or potency. Herein, we focus on the design,synthesis and pharmacological characterization of 1,2,5-trisubstitutedbenzimidazole ligands targeting MC1R, MC3R-MC5R.

The synthesis of benzimidazole molecules on solid support may beachieved using different synthetic routes. The most common precur-sor in the synthesis is 4-fluoro-3-nitrobenzoic acid (55,56), or otheraromatic system that contains flouro and nitro groups which areadjacent to each other (66). In such case, nucleophilic aromatic dis-placement (SNAr) of the fluoride at the ipso position is achieved bycommercially available amines (Scheme 1) (67,68). Upon successfulfluoride replacement, the aromatic nitro group is reduced to theprimary aromatic amine. It appears that the nitro reduction onsolid support may be performed with variety of reducing reagents(55,69–71), but we have elected to utilize SnCl2 · 2H2O in NMP(2.0 M) strategy (56,70,71). Upon the aromatic nitro reduction,o-phenylendiamine on the solid support was subjected to theon-resin cyclocondensation that is performed with variety of com-mercially available aldehydes in DMF. Initial conditions for thecyclocondensation included DDQ reagent (compounds 1–4), but laterit was not used in the synthesis as it has been reported that over-night exposure to the air is enough to catalyze the oxidative cyclo-condensation (56,72–74). Upon completion of the ligand synthesis,final cleavage and side chain deprotection was achieved using TFA.

Apart from the SAR studies based on the melanocortin peptides,diverse non-peptide templates have been reported that may beused as leads in the discovery of novel melanocortin ligands. Mel-anocortin selective agonists would be of a great interest in treatingobese individuals or regulating skin pigmentation, while the discov-ery of the centrally selective MCR antagonists in treating cachexiaand involuntary weight loss as a result of cancer would also benoteworthy. Initial piperazine based analogues attached to theDTic-(p-Cl)DPhe dipeptide resulted in the potent and selective MC4Ranalogues in the nM range (30). Additionally, similar piperazine andpiperidine-based melanocortin analogues have been reported (31–33). More rigid templates included piridazinone scaffold (34), 1,2,4-thiadiazole (75), and cyclocondensed imidazoles (76) have also beenreported. The partially reduced imidazoles have been identified aspotent MC4R antagonists that effectively reduce tumor-inducedweight loss in mouse model (77). To the best of our knowledge, the1,2,5-trisubstituted benzimidazole scaffold has not yet been reported

Table

2:

Cont

inue

d

Com

poun

dR 1

R 2R 3

mM

C1R

EC50

(lM

)m

MC3

REC

50(l

M)

mM

C4R

EC50

(lM

)m

MC5

REC

50(l

M)

12

N

N H

NH

NH

2

O

HN

NH

2

NH

5.1

€2.

470

%@

100l

M63

%@

100l

M18

.7€

8.9

a The

indi

cate

der

rors

repr

esen

tth

est

anda

rder

ror

ofth

em

ean

dete

rmin

edfro

mat

leas

tth

ree

inde

pend

ent

expe

rimen

ts.>

100

000

indi

cate

sth

atan

EC50

valu

ew

asno

tre

porta

ble

atup

to10

0lM

ligan

dco

ncen

tra-

tions

.Ape

rcen

tage

valu

ein

dica

tes

that

som

est

imul

ator

yag

onis

tph

arm

acol

ogy

resu

lted

atup

to10

0l

Mco

ncen

tratio

ns,b

utth

em

axim

alst

imul

atio

nle

vels

wer

ele

ssth

enth

eno

n-re

cept

or-d

epen

dent

fors

kolin

con-

troll

evel

.

Todorovic et al.


as a template used to target melanocortin system, although thesynthesis and its application in studying other GPCR and non-GPCRsystems has been extensive, vide supra. Our rationale for the benz-imidazole selection as the template is based on its inclusion in thefamily of substances refereed to as privileged structures. Ourtemplate consisted of series of analogues with substitutions atpositions 1, 2, and 5. The compounds 1–5 were designed in orderto investigate the robustness of the synthetic method. 3,3-diphenyl-propyl moiety in compounds 1 and 2 (R1) was chosen based on theprevious identification of biphenyl scaffold as privileged structure(37). Limited SAR studies performed in this project have identifiedthe benzimidazoles with limited or no detectable activity at theMCRs (Table 2), and improved pharmacological profile (Table 3) thatis in the range from lM to nM EC50 values. It appears that introduc-tion of the basic guanidino-group belonging to the Arg residue at

the position 5 of benzimidazole, results in ligands that are able toelicit pharmacological response at the MCRs examined in this study(Tables 2 and 3). Compounds 7–9, although containing an Arg resi-due in the 5 position, were devoid of any potency at the MC3R–MC5R. Moreover, no preference for the MC1R was observed,despite the fact that some agonist stimulatory activity was elicitedin the lM range (Table 3). Compound 10 was selected as our 'lead'molecule for the subsequent design and characterization of com-pounds 13–16 (Table 3). The benzimidazole 10 was capable of eli-citing pharmacological response at three MCR subtypes, includingMC1R, MC4R and MC5R. Compounds 11 and 12 possessed agonistactivity at the MC1R and MC5R in the lM range. Compounds 10–

13 possessed fused rings (either naphtyl or indolyl) connected withmethylene or ethylene units to the N1 of the benzimidazole. Itappears that additional aromatic fused ring in this position favors

Table 3: Functional activity of the benzimidazole based compounds at the mouse melanocortin receptors. The R3 substituent was diversi-fied with amino acids at the position 5 of benzimidazolea

NH

N

N

N

O

R3

HN

Compound R3

mMC1REC50 (nM)

mMC3REC50 (nM)

mMC4REC50 (nM)

mMC5REC50 (nM)

NDP–MSH 0.04 € 0.02 0.27 € 0.04 0.12 € 0.04 0.48 € 0.1013. –Arg–DPhe–His–NH2 48 € 14 670 € 130 610 € 280 440 € 26014. –Arg–DPhe–Trp–NH2 650 € 290 >100 000 44%@100 lM 5000 € 130015. –Arg–DPhe–NH2 1100 € 500 >100 000 38%@100 lM 46 000 € 10 20016. –DPhe–Arg–NH2 2600 € 2100 21 500 € 2700 11 800 € 7500 5100 € 1900

aThe indicated errors represent the standard error of the mean determined from at least three independent experiments. >100 000 indicates that an EC50 valuewas not reportable at up to 100 lM ligand concentrations. A percentage value indicates that some stimulatory agonist pharmacology resulted at up to 100 lM

concentrations, but the maximal stimulation levels were less then the non-receptor dependent forskolin control level.

Figure 1: Graphical representa-tion summarizing the effect of lig-and potency [agonist EC50 values(Z-axis)] at the mouse melanocortinreceptors (Y-axis).



the response for at least some of the MCR (as opposed to the sin-gle aromatic system). When the non-aromatic ring (compound 6) ormono-aromatic 5-imidazolylethyl (compounds 7, 9) or 4-hydroxyphe-nylethyl (compound 8) moieties are present at N1 of the benzimi-dazole structure, the resulting compounds were not able to fullyelicit agonist activity at neither MCR examined in this study. Inter-estingly, compound 11 with n-heptyl chain in the position 2,possessed full agonist profile at the MC1R and MC5R that is inaccordance with previously reported data that introduction of longaliphatic chains in the melanocortin peptides' template may be usedas a tool to improve MCR potency (26,29). Compounds 11 and 12

possessed no selectivity preference between MC1R and MC5R,within the threefold experimental error. On the other hand, com-pound 10, possessed slightly preference for the MC1R versusMC5R (sixfold), albeit in the lM range.

Compounds 13–16 were designed with the objective to improvemelanocortin receptor agonist potency of benzimidazole 10. Themodifications incorporated into the design strategy were based ona retro-approach in which amino acids are placed retro as com-pared to the parent compound (78). The benzimidazole R3 positionconceptually represents the retro-design of the Ac–His–DPhe–Arg–Trp–NH2 melanocortin ligand, and was utilized to examine theeffect upon addition of key melanocortin agonist amino acids to thebenzimidazole template. Compounds 13–16, presented in Table 3,possessed 3-indolylethyl at the R1 position and the 5-imidazolylmoiety at the R2 position of the benzimidazole template. ExtensiveSAR studies on the truncated forms of a-MSH (79–81) and NDP-MSH (82–84) have been performed that resulted in the melanocor-tin tetrapeptides Ac–Phe–Arg–Trp–NH2 and Ac–DPhe–Arg–Trp–NH2

as possessing lM full agonist activity at one or more of the mousemelanocortin receptors (Table 4) (84). Unexpectantly, when the coreHis–Phe–Arg–Trp tetrapeptide is not modified at either the N- or C-terminus, a peptide results that is unable to stimulate the mMC1,mMC3, and mMC4 receptors at up to 100 lM concentrations and isonly able to stimulate the mMC5R to 50% of the maximal stimula-tion observed for the non-receptor-dependent cAMP control, forsko-lin (Table 4). Thus, based upon these data and the results obtainedfor the benzimidazole 10 compound, we decided to append thistemplate at the C-terminal position with amino acids that havebeen previously demonstrated to increase receptor agonist potency.Based upon the MC4R preference for benzimidazole 10, we decidedto append the DPhe–His residues to the Arg moiety (retro design)that resulted in compound 13. While this resulted in the most

potent mMC1R and mMC4R ligand discovered from this study, inter-estingly, it is more potent than the His–Phe–Arg–Trp, Ac–His–Phe–Arg–Trp–NH2, and Ac–DPhe–Arg–Trp–NH2 peptides demonstratingthe proof-of-concept that inclusion of the benzimidazole privilegestructure into melanocortin agonist templates can be a viableapproach for the design of melanocortin based agonist ligands aswell as other peptide GPCRs.

Conclusion

The study presented herein reports benzimidazole containing ligandsare capable of stimulating the melanocortin receptors. Through avariety of structural changes at three points of diversity, we wereable to design and pharmacologically characterize agonists withEC50 values from lM to nM range. Improved potency was obtainedfor the analogues 13–16, that consisted of different amino acidsat position 5 (R3).

Acknowledgments

This work has been supported by NIH Grant RO1-DK57080. Aleksan-dar Todorovic is a recipient of the American Heart Association Pre-doctoral Fellowship.

References

1. Chhajlani V., Wikberg J.E. (1992) Molecular cloning and expres-sion of the human melanocyte stimulating hormone receptorcdna. FEBS Lett;309:417–420.

2. Chhajlani V., Muceniece R., Wikberg J.E. (1993) Molecular clo-ning of a novel human melanocortin receptor. Biochem BiophysRes Commun;195:866–873.

3. Chhajlani V. (1996) Distribution of cdna for melanocortin receptorsubtypes in human tissues. Biochem Mol Biol Int;38:73–80.

4. Mountjoy K.G., Robbins L.S., Mortrud M.T., Cone R.D. (1992) Thecloning of a family of genes that encode the melanocortinreceptors. Science;257:1248–1251.

5. Schioth H.B., Chhajlani V., Muceniece R., Klusa V., Wikberg J.E.(1996) Major pharmacological distinction of the acth receptorfrom other melanocortin receptors. Life Sci;59:797–801.

Table 4: Functional activity of the His–Phe–Arg–Trp tetrapeptide analoguesa

CompoundmMC1REC50 (nM)

mMC3REC50 (nM)

MMC4REC50 (nM)

mMC5REC50 (nM)

NDP–MSH 0.04 € 0.02 0.27 € 0.04 0.12 € 0.04 0.48 € 0.10Ac–His–DPhe–Arg–Trp–NH2 20.1 € 0.57 156 € 9.2 17.2 € 2.78 3.96 € 0.94Ac–His–Phe–Arg–Trp–NH2 7690 € 3585 4370 € 535 2110 € 243 103 € 28Ac–DPhe–Arg–Trp–NH2 4540 € 454 25%@100 lM 2320 € 227 1660 € 257Ac–Phe–Arg–Trp–NH2 5870 € 2040 >100 000 >100 000 25%@100 lM

His–Phe–Arg–Trp >100 000 >100 000 >100 000 50%@100 lM

aThe indicated errors represent the standard error of the mean determined from at least three independent experiments. >100 000 indicates that an EC50 valuewas not reportable at up to 100 lM ligand concentrations. A percentage value indicates that some stimulatory agonist pharmacology resulted at up to 100 lM

concentrations, but the maximal stimulation levels were less then the non-receptor dependent forskolin control level.

Todorovic et al.


6. Weber A., Kapas S., Hinson J., Grant D.B., Grossman A., ClarkA.J. (1993) Functional characterization of the cloned human acthreceptor: Impaired responsiveness of a mutant receptor infamilial glucocorticoid deficiency. Biochem Biophys ResCommun;197:172–178.

7. Robbins L.S., Nadeau J.H., Johnson K.R., Kelly M.A., Roselli-Rehfuss L., Baack E., Mountjoy K.G., Cone R.D. (1993) Pigmenta-tion phenotypes of variant extension locus alleles result frompoint mutations that alter msh receptor function. Cell;72:827–834.

8. Valverde P., Healy E., Jackson I., Rees J.L., Thody A.J. (1995)Variants of the melanocyte-stimulating hormone receptor geneare associated with red hair and fair skin in humans. NatGenet;11:328–330.

9. Beuschlein F., Fassnacht M., Klink A., Allolio B., Reincke M.(2001) Acth-receptor expression, regulation and role in adreno-cortial tumor formation. Eur J Endocrinol;144:199–206.

10. Hofmann K., Stehle C.J., Finn F.M. (1988) Identification of aprotein in adrenal particulates that binds adrenocorticotropinspecifically and with high affinity. Endocrinology;123:1355–1363.

11. Gantz I., Konda Y., Tashiro T., Shimoto Y., Miwa H., Munzert G.,Watson S.J., DelValle J., Yamada T. (1993) Molecular cloning ofa novel melanocortin receptor. J Biol Chem;268:8246–8250.

12. Gantz I., Miwa H., Konda Y., Shimoto Y., Tashiro T., Watson S.J.,DelValle J., Yamada T. (1993) Molecular cloning, expression, andgene localization of a fourth melanocortin receptor. J BiolChem;268:15174–15179.

13. Roselli-Rehfuss L., Mountjoy K.G., Robbins L.S., Mortrud M.T.,Low M.J., Tatro J.B., Entwistle M.L., Simerly R.B., Cone R.D.(1993) Identification of a receptor for gamma melanotropin andother proopiomelanocortin peptides in the hypothalamus andlimbic system. Proc Natl Acad Sci U S A;90:8856–8860.

14. Butler A.A., Kesterson R.A., Khong K., Cullen M.J., Pelleymount-er M.A., Dekoning J., Baetscher M., Cone R.D. (2000) A uniquemetabolic syndrome causes obesity in the melanocortin-3 recep-tor-deficient mouse. Endocrinol;141:3518–3521.

15. Chen A.S., Marsh D.J., Trumbauer M.E., Frazier E.G., Guan X.M.,Yu H., Rosenblum C.I. et al. (2000) Inactivation of the mousemelanocortin-3 receptor results in increased fat mass andreduced lean body mass. Nat Genet;26:97–102.

16. Huszar D., Lynch C.A., Fairchild-Huntress V., Dunmore J.H., FangQ., Berkemeier L.R., Gu W., et al. (1997) Targeted disruption ofthe melanocortin-4 receptor results in obesity in mice.Cell;88:131–141.

17. Van der Ploeg L.H., Martin W.J., Howard A.D., Nargund R.P.,Austin C.P., Guan X., Drisko J. et al. (2002) A role for the mel-anocortin 4 receptor in sexual function. Proc Natl Acad Sci U SA;99:11381–11386.

18. Chen W., Kelly M.A., Opitz-Araya X., Thomas R.E., Low M.J.,Cone R.D. (1997) Exocrine gland dysfunction in mc5-r-deficientmice: Evidence for coordinated regulation of exocrine glandfunction by melanocortin peptides. Cell;91:789–798.

19. Gantz I., Shimoto Y., Konda Y., Miwa H., Dickinson C.J., YamadaT. (1994) Molecular cloning, expression, and characterization ofa fifth melanocortin receptor. Biochem Biophys Res Com-mun;200:1214–1220.

20. Benjannet S., Rondeau N., Day R., Chretien M., Seidah N.G.(1991) Pc1 and pc2 are proprotein convertases capable of clea-

ving proopiomelanocortin at distinct pairs of basic residues. ProcNatl Acad Sci U S A;88:3564–3568.

21. Zhou A., Bloomquist B.T., Mains R.E. (1993) The prohormoneconvertases pc1 and pc2 mediate distinct endoproteolytic cleav-ages in a strict temporal order during proopiomelanocortin bio-synthetic processing. J Biol Chem;268:1763–1769.

22. Holder J.R., Xiang Z., Bauzo R.M., Haskell-Luevano C. (2003)Structure–activity relationships of the melanocortin tetrapeptideAc–His–Dphe–Arg–Trp–NH2 at the mouse melanocortin recep-tors. Part 3: Modifications at the arg position. Peptides;24:73–82.

23. Holder J.R., Xiang Z., Bauzo R.M., Haskell-Luevano C. (2002)Structure–activity relationships of the melanocortin tetrapeptideAc–His–DPhe–Arg–Trp–NH2 at the mouse melanocortin recep-tors. 4. Modifications at the trp position. J Med Chem;45:5736–5744.

24. Holder J.R., Bauzo R.M., Xiang Z., Haskell-Luevano C. (2002)Structure–activity relationships of the melanocortin tetrapeptideac–his–d-phe–arg–trp–nh(2) at the mouse melanocortin recep-tors. 1. Modifications at the his position. J Med Chem;45:2801–2810.

25. Holder J.R., Bauzo R.M., Xiang Z., Haskell-Luevano C. (2002)Structure–activity relationships of the melanocortin tetrapeptideac–his–d-phe–arg–trp–nh(2) at the mouse melanocortin recep-tors: Part 2 modifications at the phe position. J MedChem;45:3073–3081.

26. Holder J.R., Marques F.F., Xiang Z., Bauzo R.M., Haskell-LuevanoC. (2003) Characterization of aliphatic, cyclic, and aromatic n-ter-minally 'Capped' His–d-phe–arg–trp–nh2 tetrapeptides at themelanocortin receptors. Eur J Pharmacol;462:41–52.

27. Koikov L.N., Ebetino F.H., Solinsky M.G., Cross-Doersen D., Knit-tel J.J. (2003) Sub-nanomolar hmc1r agonists by end-capping ofthe melanocortin tetrapeptide his–d-phe–arg–trp–nh(2). BioorgMed Chem Lett;13:2647–2650.

28. Koikov L.N., Ebetino F.H., Solinsky M.G., Cross-Doersen D., Knit-tel J.J. (2004) Analogs of sub-nanomolar hmc1r agonist lk-184[ph(ch2)3co-his–d-phe–arg–trp–nh2]. An additional binding sitewithin the human melanocortin receptor 1? Bioorg Med ChemLett;14:3997–4000.

29. Todorovic A., Holder J.R., Bauzo R.M., Scott J.W., Kavanagh R.,Abdel-Malek Z., Haskell-Luevano C. (2005) N-terminal fatty acylat-ed his–dphe–arg–trp–nh(2) tetrapeptides: influence of fatty acidchain length on potency and selectivity at the mouse melanocor-tin receptors and human melanocytes. J Med Chem;48:3328–3336.

30. Dyck B., Parker J., Phillips T., Carter L., Murphy B., Summers R.,Hermann J., Baker T., Cismowski M., Saunders J., Goodfellow V.(2003) Aryl piperazine melanocortin mc4 receptor agonists. Bio-org Med Chem Lett;13:3793–3796.

31. Richardson T.I., Ornstein P.L., Briner K., Fisher M.J., Backer R.T.,Biggers C.K., Clay M.P. et al. (2004) Synthesis and structure–activity relationships of novel arylpiperazines as potent andselective agonists of the melanocortin subtype-4 receptor.J Med Chem;47:744–755.

32. Arasasingham P.N., Fotsch C., Ouyang X., Norman M.H., KellyM.G., Stark K.L., Karbon B., Hale C., Baumgartner J.W., Zambr-ano M., Cheetham J., Tamayo N.A. (2003) Structure–activityrelationship of (1-aryl-2-piperazinylethyl)piperazines: antagonists



for the agrp/melanocortin receptor binding. J Med Chem;46:9–11.

33. Sebhat I.K., Martin W.J., Ye Z., Barakat K., Mosley R.T.,Johnston D.B., Bakshi R. et al. (2002) Design and pharmacologyof n-[(3r)-1,2,3,4-tetrahydroisoquinolinium- 3-ylcarbonyl]-(1r)-1-(4-chlorobenzyl)- 2-[4-cyclohexyl-4-(1h-1,2,4-triazol- 1-yl-methyl)piperidin-1-yl]-2-oxoethylamine (1), a potent, selective,melanocortin subtype-4 receptor agonist. J Med Chem;45:4589–4593.

34. Ujjainwalla F., Warner D., Walsh T.F., Wyvratt M.J., Zhou C.,Yang L., Kalyani R.N., MacNeil T., Van der Ploeg L.H., Rosenb-lum C.I., Tang R., Vongs A., Weinberg D.H., Goulet M.T. (2003)Design and syntheses of melanocortin subtype-4 receptor agon-ists: Evolution of the pyridazinone archetype. Bioorg Med ChemLett;13:4431–4435.

35. Evans B.E., Rittle K.E., Bock M.G., DiPardo R.M., Freidinger R.M.,Whitter W.L., Lundell G.F. et al. (1988) Methods for drug discov-ery: development of potent, selective, orally effective chole-cystokinin antagonists. J Med Chem;31:2235–2246.

36. Todorovic A., Haskell-Luevano C. (2005) A review of melanocor-tin receptor small molecule ligands. Peptides;26:2026–2036.

37. Horton D.A., Bourne G.T., Smythe M.L. (2003) The combinatorialsynthesis of bicyclic privileged structures or privileged substruc-tures. Chem Rev;103:893–930.

38. Al-Muhaimeed H. (1997) A parallel-group comparison of astemiz-ole and loratadine for the treatment of perennial allergic rhini-tis. J Int Med Res;25:175–181.

39. Richter J.E. (1997) Long-term management of gastroesophagealreflux disease and its complications. Am J Gastroenter-ol;92:30S–34S discussion 34S–35S.

40. Regan J., Bruno J., McGarry D., Poli G., Hanney B., Bower S.,Travis J., Sweeney D., Miller B., Souness J., Djuric S. (1998) 2-substituted-4-methoxybenzimidazole-based pde4 inhibitors. Bio-org Med Chem Lett;8:2737–2742.

41. Thomas A.P., Allott C.P., Gibson K.H., Major J.S., Masek B.B.,Oldham A.A., Ratcliffe A.H., Roberts D.A., Russell S.T., ThomasonD.A. (1992) New nonpeptide angiotensin ii receptor antagonists.1. Synthesis, biological properties, and structure–activity relation-ships of 2-alkyl benzimidazole derivatives. J Med Chem;35:877–885.

42. Kubo K., Inada Y., Kohara Y., Sugiura Y., Ojima M., Itoh K., Fur-ukawa Y., Nishikawa K., Naka T. (1993) Nonpeptide angiotensinii receptor antagonists. Synthesis and biological activity of benz-imidazoles. J Med Chem;36:1772–1784.

43. Zarrinmayeh H., Nunes A.M., Ornstein P.L., Zimmerman D.M.,Arnold M.B., Schober D.A., Gackenheimer S.L., Bruns R.F., Hips-kind P.A., Britton T.C., Cantrell B.E., Gehlert D.R. (1998) Synthesisand evaluation of a series of novel 2-[(4-chlorophenoxy)methyl]benzimidazoles as selective neuropeptide y y1 receptor antago-nists. J Med Chem;41:2709–2719.

44. Daniels A.J., Grizzle M.K., Wiard R.P., Matthews J.E., Heyer D.(2002) Food intake inhibition and reduction in body weight gainin lean and obese rodents treated with gw438014a, a potentand selective npy-y5 receptor antagonist. Regul Pept;106:47–54.

45. Ellingboe J.W., Spinelli W., Winkley M.W., Nguyen T.T., ParsonsR.W., Moubarak I.F., Kitzen J.M., Von Engen D., Bagli J.F.(1992) Class iii antiarrhythmic activity of novel substituted

4-[(methylsulfonyl) amino]benzamides and sulfonamides. J MedChem;35:705–716.

46. Holmes D.S., Bethell R.C., Cammack N., Clemens I.R.,Kitchin J., McMeekin P., Mo C.L., Orr D.C., Patel B., Paternos-ter I.L., et al. (1993) Synthesis and structure–activity relation-ships of a series of penicillin-derived hiv proteinase inhibitorscontaining a stereochemically unique peptide isostere. J MedChem;36:3129–3136.

47. Zarrinmayeh H., Zimmerman D.M., Cantrell B.E., Schober D.A.,Bruns R.F., Gackenheimer S.L., Ornstein P.L., Hipskind P.A., BrittonT.C., Gehlert D.R. (1999) Structure–activity relationship of aseries of diaminoalkyl substituted benzimidazole as neuro-peptide y y1 receptor antagonists. Bioorg Med Chem Lett;9:647–652.

48. Lopez-Rodriguez M.L., Benhamu B., Morcillo M.J., Tejada I., Avi-la D., Marco I., Schiapparelli L., Frechilla D., Del Rio J. (2004)Benzimidazole derivatives. Part 5: Design and synthesis of newbenzimidazole-arylpiperazine derivatives acting as mixed 5-ht1a/5-ht3 ligands. Bioorg Med Chem;12:5181–5191.

49. Lopez-Rodriguez M.L., Benhamu B., Murcia M., Alvaro E., Cam-pillo M., Pardo L. (2003) Benzimidazole derivatives. 4. The recog-nition of the voluminous substituent attached to the basicamino group of 5-ht4 receptor antagonists. J Comput Aided MolDes;17:515–524.

50. Lopez-Rodriguez M.L., Benhamu B., Morcillo M.J., Tejada I., Avi-la D., Marco I., Schiapparelli L., Frechilla D., Del Rio J. (2003)Design and synthesis of new benzimidazole-arylpiperazine deriv-atives acting as mixed 5-ht1a/5-ht3 ligands. Bioorg Med ChemLett;13:3177–3180.

51. Terzioglu N., van Rijn R.M., Bakker R.A., De Esch I.J., Leurs R.(2004) Synthesis and structure–activity relationships of indoleand benzimidazole piperazines as histamine h(4) receptor antag-onists. Bioorg Med Chem Lett;14:5251–5256.

52. Sawada Y., Kayakiri H., Abe Y., Mizutani T., Inamura N.,Asano M., Hatori C., Aramori I., Oku T., Tanaka H. (2004)Discovery of the first non-peptide full agonists for the humanbradykinin b(2) receptor incorporating 4-(2-picolyl-oxy)quinoline and 1-(2-picolyl)benzimidazole frameworks. J MedChem;47:2853–2863.

53. Stewart A.O., Cowart M.D., Moreland R.B., Latshaw S.P.,Matulenko M.A., Bhatia P.A., Wang X. et al. (2004) Dopamined4 ligands and models of receptor activation: 2-(4-pyridin-2-ylpi-perazin-1-ylmethyl)-1h-benzimidazole and related heteroarylmeth-ylarylpiperazines exhibit a substituent effect responsible foradditional efficacy tuning. J Med Chem;47:2348–2355.

54. Poitout L., Brault V., Sackur C., Roubert P., Plas P. (2005) Prepar-taiton of benzimidazoles and imidazopyridines having affinity formelanocorrtin (mc), in paticular mc4 receptors (Fr Demande).

55. Mayer J.P., Lewis G.S., McGee C., Bankaitis-Davis D. (1998)Solid-phase synthesis of benzimidazoles. TetrahedronLett;39:6655–6658.

56. Vourloumis D., Takahashi M., Simonsen K.B., Ayida B.K., Barlu-enga S., Winters G.C., Hermann T. (2003) Solid-phase synthesisof benzimidazole libraries biased for rna targets. TetrahedronLett;44:2807–2811.

57. Kaiser E., Colescot R.L., Bossinge C.D., Cook P.I. (1970) Color testfor detection of free terminal amino groups in solid-phase syn-thesis of peptides. Anal Biochem;34:595–598.

Todorovic et al.


58. Chang C.D., Meienhofer J. (1978) Solid-phase peptide synthesisusing mild base cleavage of n alpha-fluorenylmethyloxycarbonyl-amino acids, exemplified by a synthesis of dihydrosomatostatin.Int J Pept Protein Res;11:246–249.

59. Carpino L.A., Han G.Y. (1970) 9-fluorenylmethoxycarbonyl func-tion, a new base-sensitive amino-protecting group. J Am ChemSoc;92:5748–5749.

60. Carpino L.A., Han G.Y. (1972) 9-fluorenylmethoxycarbonyl amino-protecting group. J Org Chem;37:3404–3409.

61. Chen W., Shields T.S., Stork P.J., Cone R.D. (1995) A colorimetricassay for measuring activation of gs- and gq-coupled signalingpathways. Anal Biochem;226:349–354.

62. Catania A., Gatti S., Colombo G., Lipton J.M. (2004) Targetingmelanocortin receptors as a novel strategy to control inflamma-tion. Pharmacol Rev;56:1–29.

63. Todorovic A., Holder J.R., Scott J.W., Haskell-Luevano C. (2004)Synthesis and activity of the melanocortin Xaa–D–Phe–Arg–Trp–NH tetrapeptides with amide bond modifications. J PeptideRes;63:270–278.

64. Holder J.R., Bauzo R.M., Xiang Z., Scott J., Haskell-Luevano C.(2003) Design and pharmacology of peptoids and peptide-pep-toid hybrids based on the melanocortin agonists core tetrapep-tide sequence. Bioorg Med Chem Lett;13:4505–4509.

65. Joseph C.G., Bauzo R.M., Xiang Z., Haskell-Luevano C. (2003)Urea small molecule agonists on mouse melanocortin receptors.Bioorg Med Chem Lett;13:2079–2082.

66. Tumelty D., Schwarz M.K., Cao K., Needels M.C. (1999) Solid-phase synthesis of substituted benzimidazoles. TetrahedronLett;40:6185–6188.

67. Pan P.C., Sun C.M. (1998) Soluble polymer-supported synthesisof arylpiperazines. Tetrahedron Lett;39:9505–9508.

68. Dankwardt S.M., Newman S.R., Krstenansky J.L. (1995) Solid-phase synthesis of aryl and benzylpiperazines and their applica-tion in combinatorial chemistry. Tetrahedron Lett;36:4923–4926.

69. Phillips G.B., Wei G.P. (1996) Solid phase synthesis of benzimi-dazoles. Tetrahedron Lett;37:4887–4890.

70. Kilburn J.P., Lau J., Jones R.C.F. (2000) Solid-phase synthesis ofsubstituted 2-aminomethylbenzimidazoles. TetrahedronLett;41:5419–5421.

71. Tumelty D., Schwarz M.K., Needels M.C. (1998) Solid-phasesynthesis of substituted 1-phenyl-2-aminomethyl-benzimidazolesand 1-phenyl-2-thiomethyl-benzimidazoles. Tetrahedron Lett;39:7467–7470.

72. Farrant E., Rahman S.S. (2000) A solid-phase synthetic route tosubstituted 7-azabenzimidazoles suitable for combinatorial librarysynthesis. Tetrahedron Lett;41:5383–5386.

73. Smith J.M., Krchnak V. (1999) A solid phase traceless synthesisof benzimidazoles with three combinatorial steps. TetrahedronLett;40:7633–7636.

74. Vandeneynde J.J., Delfosse F., Lor P., Vanhaverbeke Y. (1995)2,3-dichloro-5,6-dicyano-1,4-benzoquinone, a mild catalyst forthe formation of carbon–nitrogen bonds. Tetrahedron;51:5813–5818.

75. Pan K., Scott M.K., Lee D.H., Fitzpatrick L.J., Crooke J.J., RiveroR.A., Rosenthal D.I., Vaidya A.H., Zhao B., Reitz A.B. (2003) 2,3-diaryl-5-anilino[1,2,4]thiadiazoles as melanocortin mc4 receptoragonists and their effects on feeding behavior in rats. BioorgMed Chem;11:185–192.

76. Marsilje T.H., Roses J.B., Calderwood E.F., Stroud S.G., ForsythN.E., Blackburn C., Yowe D.L., Miao W., Drabic S.V., BohaneM.D., Scott Daniels J., Li P., Wu L., Patane M.A., Claiborne C.F.(2004) Synthesis and biological evaluation of imidazole-basedsmall molecule antagonists of the melanocortin 4 receptor (mc4-r). Bioorg Med Chem Lett;14:3721–3725.

77. Vos T.J., Caracoti A., Che J.L., Dai M., Farrer C.A., Forsyth N.E.,Drabic S.V. et al. (2004) Identification of 2-[2-[2-(5-bromo-2-methoxyphenyl)-ethyl]-3-fluorophenyl]-4,5-dihydro-1h-imidazole(ml00253764), a small molecule melanocortin 4 receptor antag-onist that effectively reduces tumor-induced weight loss in amouse model. J Med Chem;47:1602–1604.

78. Chorev M., Goodman M. (1995) Recent developments in retropeptides and proteins–an ongoing topochemical exploration.Trends Biotechnol;13:438–445.

79. Castrucci A.M., Hadley M.E., Sawyer T.K., Wilkes B.C., al-ObeidiF., Staples D.J., de Vaux A.E., Dym O., Hintz M.F., Riehm J.P., etal. (1989) Alpha-melanotropin: the minimal active sequence inthe lizard skin bioassay. Gen Comp Endocrinol;73:157–163.

80. Hruby V.J., Wilkes B.C., Hadley M.E., Al-Obeidi F., Sawyer T.K.,Staples D.J., de Vaux A.E., Dym O., Castrucci A.M., Hintz M.F.,et al. (1987) Alpha-melanotropin: the minimal active sequence inthe frog skin bioassay. J Med Chem;30:2126–2130.

81. Eberle A.N. (1988) The melanotropins: Chemistry, physiology andmechanisms of action. Basel: Karger.

82. Haskell-Luevano C., Sawyer T.K., Hendrata S., North C., Panahi-nia L., Stum M., Staples D.J., Castrucci A.M., Hadley M.F., HrubyV.J. (1996) Truncation studies of alpha-melanotropin peptidesidentify tripeptide analogues exhibiting prolonged agonist bioac-tivity. Peptides;17:995–1002.

83. Sawyer T.K., Castrucci A.M., Staples D.J., Affholter J.A., DeVaux A., Hruby V.J., Hadley M.E. (1993) Structure–activity rela-tionships of [nle4, d-phe7]alpha-msh. Discovery of a tripeptidylagonist exhibiting sustained bioactivity. Ann NY AcadSci;680:597–599.

84. Haskell-Luevano C., Holder J.R., Monck E.K., Bauzo R.M. (2001)Characterization of melanocortin ndp-msh agonist peptide frag-ments at the mouse central and peripheral melanocortin recep-tors. J Med Chem;44:2247–2252.



Structure?Activity Relationships of Melanocortin Agonists Containing the Benzimidazole Scaffold

Documents

Transcript of Structure?Activity Relationships of Melanocortin Agonists Containing the Benzimidazole Scaffold