New anthranilic acid based antagonists with high affinity and selectivity for the human cholecystokinin receptor 1 (hCCK1-R)

ARTICLE

pubs.acs.org/jmc

New Anthranilic Acid Based Antagonists with High Affinity and

Selectivity for the Human Cholecystokinin Receptor 1 (hCCK₁-R)

Michela V. Pavan,^†Lucia Lassiani,^†Federico Berti,^‡Giorgio Stefancich,^†Alessia Ciogli,^§

Francesco Gasparrini,^§Laura Mennuni,^||Flora Ferrari,^||Chantal Escrieut,^{^}Esther Marco,^{^}

Francesco Makovec,^||Daniel Fourmy,^{^}and Antonio Varnavas*^,†

^†Department of Chemical and Pharmaceutical Sciences, University of Trieste, P.le Europa 1, 34127 Trieste, Italy

^‡Department of Chemical and Pharmaceutical Sciences, University of Trieste, Via Giorgieri 1, 34127 Trieste, Italy

^§Department of Chemistry and Technology of Biologically Active Substances, University “La Sapienza”,

P.le Aldo Moro 5, 00185 Rome, Italy

Rottapharm—Madaus SpA, Via Valosa di Sopra 7, 20052 Monza, Italy

^{^}Universitꢀe de Toulouse 3, EA 4552, I2MC, 1 Avenue Jean Poulhꢁes, 31432 Toulouse, France

S Supporting Information

b

ABSTRACT:

The anthranilic acid diamides represent the most recent class of nonpeptide CCK₁receptor (CCK₁-R) antagonists. Herein we

describe the second phase of the anthranilic acid C-terminal optimization using nonproteinogenic amino acids containing a phenyl

ring in their side chain. The Homo-Phe derivative 2 (VL-0797) enhanced 12-fold the aﬃnity for the rat CCK₁-R aﬃnity and 15-fold

for the human CCK₁-R relative to the reference compound 12 (VL-0395). The eutomer of 2 (6) exhibited a nanomolar range

aﬃnity toward the human CCK₁-R and was at least 400-fold selective for the CCK₁-R over the CCK₂-R. Molecular docking in the

modeled CCK₁-R and its validation by site-directed mutagenesis experiments showed that the 6 binding site overlaps that occupied

by the C-terminal bioactive region of the natural agonist CCK. Owing to their interesting properties, new compounds provided by

this study represent a solid basis for further advances aimed at synthesis of clinically valuable CCK₁-R antagonists.

’ INTRODUCTION

disorders etc., over the past 15 years, great eﬀorts have been made

to develop clinically useful small nonpeptide molecules known as

CCK receptor antagonists.^10ꢀ15

In an earlier report, we described an innovative class of non-

peptide CCK₁-R antagonists keeping appropriate pharmacopho-

ric groups on the anthranilic acid unit employed as a molecular

scaﬀold.¹⁶The lead compound obtained 2(R,S)-{2-[(1H-indole-

2-carbonyl)-amino]-benzoylamino}-3-phenyl-propionic acid (VL-

0395) (Figure 1) is characterized by the presence of Phe and

2-indole moiety at the C- and N-termini of anthranilic acid,

Cholecystokinin (CCK) is a gutꢀbrain peptide engaged as

endocrine messenger in the gastrointestinal (GI) tract and as

neurotransmitter/neuromodulator at the central nervous system

(CNS).^1ꢀ4The biological actions of CCK are mediated by two

distinct G-protein coupled receptors: CCK₁and CCK₂.^5ꢀ8CCK

circulates in the blood in diﬀerent molecular forms, and only the

C-terminal octapeptide (Asp²⁶-Tyr(SO₃H)²⁷-Met²⁸-Gly²⁹-Trp³⁰-

Met³¹-Asp³²-Phe³³-NH₂or CCK-8S) binds both receptors with

subnanomolar aﬃnity. The smallest bioactive fragment of CCK

(Trp³⁰-Met³¹-Asp³²-Phe³³-NH₂or CCK-4) binds with nanomolar

aﬃnity only the CCK₂receptor (CCK₂-R), while its aﬃnity toward

the CCK₁receptor (CCK₁-R) decreases to the micromolar range.⁹

In view of the importance of CCK in diﬀerent physiopatho-

logical processes as irritable bowel syndrome (IBS), chronic and

acute pancreatitis, gastric ulcer, anxiety, panic disorder, eating

respectively, and is endowed with submicromolar aﬃnity (IC₅₀

=

197.5 nM) toward CCK₁-R, demonstrating also an antagonist

nature similar to that exhibited by the reference CCK₁selective

Received: April 12, 2011

Published: July 05, 2011

r

2011 American Chemical Society

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dx.doi.org/10.1021/jm200438b J. Med. Chem. 2011, 54, 5769–5785

|

Journal of Medicinal Chemistry

ARTICLE

antagonist Loxiglumide.¹⁶A possible mode of interaction of

compound 12 (VL-0395) with the CCK₁-R has already been

proposed and involves at least two hydrophobic pockets in order

to accommodate the main pharmacophoric groups (2-indole ring

and Phe side chain).

Previously, a structureꢀaﬃnity relationship study (SAfR) en-

abled us to demonstrate that the N-terminal substituent highly

modulates the aﬃnity and that the 2-indole moiety behaves as a

“needle” because any substitution of the indole group with at

least 50 diﬀerent other residues produces a loss in aﬃnity.^17ꢀ19

The only modiﬁcation allowed in this ﬁrst optimization step is

the substitution on the indole ring with groups characterized by

minimal steric hindrance, a parameter that deeply inﬂuences the

aﬃnity. In addition, the replacement of the phenyl ring of the

indole moiety with a saturated one (cyclohexyl or cyclopentyl) is

detrimental for the binding aﬃnity, indicating the essential role

of the planar aromatic ring.¹⁸

On the contrary, the results obtained from a preliminary SAR

study regarding the C-terminal optimization of the anthranilic

acid of compound 12 suggested that the hypothesized receptor

pocket, which accommodates the aminoacidic residue, allows many

more degrees of freedom than those imposed to the N-terminal

group.²⁰In fact, the substitution on the phenyl ring of Phe or its

saturation was tolerated by the receptor pocket, as well as re-

placement of the Phe residue of 12 by other proteinogenic amino

acids of the C-terminal sequences of CCK led to compounds

endowed with comparable receptor aﬃnity and selectivity as the

lead one.²¹

So, to optimize compound 12, we synthesized compounds

reported in Table 1 and tested their aﬃnity and activity toward

CCK receptors.

First, to investigate the tolerance of the hydrophobic pocket

engaged by the Phe side chain of 12, we introduced, on the

C-terminus of anthranilic acid, nonproteinogenic amino acids

with diﬀerent degrees of homology to Phe (compounds 1ꢀ4).

Then, the amino acidic side chain which confers the higher aﬃnity

has been utilized in order to explore the topographic tolerance of

the receptor binding subsite associated to the amino acidic

C-terminal free carboxy group (compounds 7ꢀ10). In addition

to the above aims, compound 11 was designed in order to high-

light the importance of the anthranilic acid scaﬀold for the correct

spatial orientation of the two main pharmacophoric groups (indole

Figure 1. Structure of the anthranilic acid based lead antagonist 12.

Table 1. CCK Receptors Binding Data

IC₅₀(μM)^a

rat pancreatic

acini (CCK₁)

guinea pig brain

e

compd

X

Y

n

R

stereo *

cortex (CCK₂)

SI

1/ER

12

1

CO

NH

1

0

2

3

4

ꢀCOOH

RS

0.197 ( 0.107

0.317 ( 0.066

0.0157 ( 0.0008

0.107 ( 0.048

39% ISB ^c

16.40

83^f

NT

2

5.1 ( 0.3

0.6 ( 0.1

NT

324^f

6^f

3

4

5

6

CO

NH

2

ꢀCOOH

S

0.313 ( 0.100

0.0095 ( 0.001

7.9 ( 1.5

3.8 ( 0.6

25

33

R

400

7

CO

NH

2

ꢀCOOC₂H₅

ꢀCONH₂

ꢀCH₃

RS

24% ISB ^b

IN ^b

36% ISB ^c

NT

IN ^c

8

9

NT

37% ISB ^d

10

ꢀCONHOH

0.419 ( 0.026

11

NH

CO

2

ꢀCOOH

RS

0.717 ( 0.110

6.2 ( 0.4

8^f

^aIC₅₀( standard error (ALLFIT analysis); % ISB, percentage inhibition of speciﬁc binding of 25 pM [¹²⁵I]-(BH)-CCK-8 at the maximal concentration

tested. ^b3 μM. ^c10 μM. ^d30 μM. ^eValues without standard errors were obtained from not more than two experiments. IN: inactive (ISB less than 20%).

NT: not tested. SI: selectivity index. ^fApparent selectivity index. ER: eudismic ratio.

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dx.doi.org/10.1021/jm200438b |J. Med. Chem. 2011, 54, 5769–5785

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ARTICLE

Scheme 1. Synthesis of Compounds 1ꢀ10^a

^aReagents and conditions: (i) ((a) MsCl, TEA, CH₂Cl₂, b) NaI, acetone; (ii) EtONa, abs EtOH, reﬂux; (iii) 6N HCl, 1,4-dioxane, reﬂux; (iv) HCl (g),

abs EtOH; (v) isatoic anhydride, TEA, AcOEt, reﬂux; (vi) indole-2-carboxylic acid, PCl₅, dry CH₂Cl₂, pyridine; (vii) KOH, THF/H₂O 1:1; (viii)

LiOH H₂O, THF/H₂O 1:1; (ix) hydroxylamine hydrochloride, MeONa, MeOH; (x) TEA, PyBOP, NH₄Cl, DMF.

3

and amino acidic side chain) of this class of CCK₁antagonists. In

the ﬁnal step, we extended the study on the binding eﬀect in

relation to the receptor subsite stereospeciﬁcity, determining the

eutomer and the eudismic ratio (ER) of the two pure enantiomers

of the most active compound in this new series (compounds 5, 6).

intermediates 1f, 3f, and 4f, using a KOH aqueous solution,

aﬀorded the ﬁnal compounds 2, 1, 3, and 4 respectively as

racemates.

The optical purity of the enantiomers 5 and 6 was determined

by analytical reverse phase enantioselective HPLC (RP-eHPLC)

using a chiral stationary phase (CSP) containing native teicopla-

nin chemically bonded on silica matrix according to a recently

published procedure.^22,23

’ CHEMISTRY

All analytical data were obtained by using UV, chiro-optical

(circular dichroism, CD), and evaporative light scattering (ELSD)

detectors.

Ethyl ester 7 was treated with MeONa in MeOH followed by

hydroxylamine hydrochloride, yielding hydroxamic acid 10 as

mixture of E/Z isomers while treatment of the ﬁnal compound 2

with PyBop and ammonium chloride in DMF provided primary

amide 8.

Derivatives 1ꢀ10 were synthesized according to the synthetic

pathway reported in Scheme 1. The ethyl esters of the racemic

phenyl-glycine (1d) and those of the pure enantiomers of Homo-

Phe (5d, 6d) were commercially available, while the ethyl esters

of Homo-Phe (7d) and those of its higher homologues, intro-

duced in compounds 3 and 4 (3d, 4d), were obtained in three

subsequent synthetic steps as follows. Alkylation of diethyl acet-

amidomalonate with the corresponding alkyl halide aﬀorded the

intermediates 3b, 4b, and 7b. In the case of intermediate 4b, the

alkyl halide used (4a) was obtained starting from the correspond-

ing alcohol, whereas the other two alkyl halides (3a and 7a) were

commercially available. Acidic hydrolysis in HCl 6N of 3b, 4b,

and 7b aﬀorded the unnatural amino acids 3c, 4c, and 7c as

racemates, which were converted successively, by treatment with

gaseous HCl in absolute EtOH, in the corresponding ethyl esters

hydrochloride 3d, 4d, and 7d.

Compound 11 was synthesized according to the synthetic

route reported in Scheme 2.

Alkylation of diethyl malonate with 2-bromo-ethyl benzene

aﬀorded the intermediate 11b, which was converted successively

to 11c by base catalyzed monohydrolysis. The o-phenylendia-

mine derivative 11d was obtained by TBTU mediated condensa-

tion of 11c with diamine. Indolation of 11d and successive base

catalyzed hydrolysis of 11e using the same protocols of the an-

thranilic acid derivatives of Scheme 1 aﬀorded the ﬁnal o-pheny-

lendiamine based compound 11.

o-Aminobenzoylation of 2-methyl-3-phenyl propylamine and

of the C-protected aminoacids including the commercially avail-

able ones (1d, 5d, 6d) with isatoic anhydride in ethyl acetate af-

forded the anthranilic intermediates 9e, 1e, and3eꢀ7e, respectively.

Further coupling with indole-2-carboxylic acid, via acyl chlor-

ide formation using PCl₅in dry CH₂Cl₂, provided the ﬁnal

compounds 7 and 9 as well as the indole intermediates 1f, and

3fꢀ6f.

’ RESULTS AND DISCUSSION

The binding aﬃnities of the synthesized compounds 1ꢀ11 for

CCK₁and CCK₂-Rs were evaluated according to established

protocols and are expressed as IC₅₀or as percentage inhibition of

speciﬁc binding of 25 pM [¹²⁵I]-(BH)-CCK-8 (ISB%) deter-

mined at the highest used dose (3, 10, and 30 μM as indicated).²⁴

When the ISB% values were lower than 20%, the compounds

The optically active ﬁnal compounds 5 and 6 were obtained by

base catalyzed hydrolysis with LiOH of the corresponding esters

5f and 6f. Base catalyzed hydrolysis of the ethyl ester 7 and of the

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Scheme 2. Synthesis of Compound 11^a

Table 2. CCK Receptors Binding Data from Human CCK₁

and CCK₂Receptors Expressed on Cos-7 Cells

human CCK₁-R human CCK₂-R

compd n stereo * K_i(nM, SEM) K_i(μM, SEM)

SI^a

368 ^a

3900 ^a

1/ER

423

12

2

1

2

RS

S

59.8 ( 4.7

3.9 ( 0.2

973 ( 196

2.3 ( 0.2

22.0 ( 3.6

15.3 ( 1.1

1ꢀ10

5

1ꢀ10

435ꢀ4348

6

R

1ꢀ10

^aApparent selectivity index; ER: eudismic ratio.

^aReagents and conditions: (i) EtONa, abs EtOH, reﬂux; (ii) KOH, abs

EtOH; (iii) TEA, TBTU, o-phenylenediamine, CH₂Cl₂; (iv) indole-2-

carboxylic acid, PCl₅, dry CH₂Cl₂, pyridine; (v) KOH, THF/H₂O 1:1.

stereochemistry of the amino acidic chiral center of the more

active racemate of this series (compound 2) on the receptor aﬃnity.

Accordingly, we determined the eutomer and the distomer of 2

and their selectivity indices (SI) as well as the eudismic ratio

(ER) of these two pure enantiomers. The binding data reported

in Table 1 establish that the pharmacologically active enantiomer

is that in which the absolute conﬁguration of the single chiral

center is R (rectus). In fact, compound 6 exhibited a nanomolar

range aﬃnity (IC₅₀= 9.5 ( 1.0 nM) and good selectivity (SI =

400), resulting in about 33-fold (1/ER ≈ 33) increase of activity

relative to the distomer (compound 5).

In view to interspecies diﬀerence polymorphisms in G-protein-

coupled receptors that can alter drug aﬃnity and/or activity, a

binding and activity studies were also carried out on cells ex-

pressing human CCK receptors. For this purpose, Cos-7 cells

were transiently transfected with plasmids containing cDNA

encoding either human CCK₂or CCK₁-R cloned from human

pancreas and gallbladder, respectively.^25,26

were considered inactive. Values without standard errors are those

obtained from no more than two experiments.

Binding data, selectivity indices (SI, expressed as CCK₂/

CCK₁ratio), and the eudismic ratio (ER) for the optically active

compounds, along with those of the lead 12, all useful for the

following discussion of SAR, are reported in Table 1.

The synthesized compounds were divided into four groups

(Table 1) and will be discussed separately according to the pre-

ﬁxed targets (importance of the amino acid side chain, of the chiral

center conﬁguration, of C-terminal free carboxy group and that of

anthranilic acid scaﬀold).

1. SAR Study. As expected, all the tested compounds showed

low affinity toward the CCK₂-R, confirming their preference for

the CCK₁-Rs.

The analysis of the obtained data shows that the binding af-

ﬁnity of compounds 1ꢀ4 appears clearly inﬂuenced by the number

of the methylene units of the amino acidic side chain. In fact, the

receptor aﬃnity displays a biphasic proﬁle in relation to the

number of the methylene units which are interposed between the

amino acidic backbone and the phenyl ring. In the absence of

methylene units, as in the case of the phenylglycine derivative

(compound 1), the receptor aﬃnity resulted approximately

2-fold lower than that of the lead compound and comparable

to that of compound 3 characterized by the presence of 3

methylene units. The smallest binding aﬃnity for the CCK₁-R

was achieved with 4 methylene units, while the optimum was

presented by the Homo-Phe side chain (compound 2) bearing 2

methylene units.

The Homo-Phe derivative (compound 2), coded VL-0797,

presented a 12-fold higher aﬃnity for the CCK₁-R and 4-fold

higher apparent selectivity than the reference compound 12. Thus, a

simple elongation, by one methylene unit, of the Phe side chain

in compound 12 yielded the most potent anthranilic acid based

CCK₁-R ligand 2, which displays a comparable aﬃnity to that of

the best benzodiazepine derivative Devazepide ((()-L 364,718)

in its racemic form (IC₅₀, 10.4 nM ( 1.5 and 169 nM ( 41.7 for

the CCK₁and CCK₂-R, respectively).

As illustrated in Table 2, all derivatives inhibited the binding of

[

¹²⁵I]BH-(Thr,-Nle)-CCK-9 to the CCK₁-R with higher aﬃnity

than they did to the CCK₂-R. The lead compound, 12, and the

new one, 2, both in their racemic form, showed near 3- or 4-fold

increased aﬃnity for the human CCK₁-R in comparison to that

for the rat receptors and 4- or 12-fold enhancement, respectively,

of their apparent indices of selectivity, thus denoting a deteriorate

human CCK₂-R recognition. Accordingly, the eutomer (compound 6)

showed a nanomolar range aﬃnity (IC₅₀of 2.3 nM) with 400-

fold enhancement of potency relative to the distomer (com-

pound 5) and at least 400-fold improvement of selectivity toward

the human CCK₁over the CCK₂-R.

In addition to the above derivatives, compounds 7ꢀ10 were

synthesized in order to elucidate the role of the amino acidic free

carboxy group in receptor recognition. The binding aﬃnity data

for compounds 7ꢀ10 demonstrate clearly that the free carboxy

group of the amino acid is essential for the receptor binding. In

fact, the remarkable drop in receptor aﬃnity of the ethyl ester

derivative (compound 7) in addition to the inactivity of the

primary amide (compound 8) supports the view that the free

carboxy group is engaged in the receptor binding through ionic

or reinforced ionic bond formation rather than in a double hy-

drogen bond interaction. Nevertheless, because the double hydro-

gen bond formation can be established even by the primary amide,

Compounds 5 and 6, coded VL-0797L and VL-0797D, re-

spectively, were synthesized in order to check the inﬂuence of the

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Table 3. Functional Test: Inhibition of Guinea Pig Gall-

bladder CCK-8-Induced Contractions in Vitro and in Vivo

The lead compound 12 was included as reference in the modeling

study, and its minimum energy conformation resulting from the

present analysis is closely similar to that derived from NOE NMR

data recently described by ourselves (Table 4, Figure 2).

The molecule is U-shaped, and the two aromatic indole and

phenylalanine rings are found on the same side at a centroid dis-

tance of 7.6 Å. The indole and the anthranilic acid rings lie almost

on the same plane, while the plane angle between the aromatic

ends of the molecules is 141ꢀ. The centroid distance between the

anthranilic and the phenylalanine rings is 8.1 Å. This conforma-

tion is stabilized also by two intramolecular hydrogen bonds at

the core anthranilic residue and between the carboxylate group

and hydrogen 15 (Scheme 3a).

in vitro IC₅₀

(nM, SEM)

in vivo ID₅₀

compd

n

stereo *

(μmol/kg, SEM)

A similar conformation is shared by all the compounds con-

taining the anthranilic core, with the exception of the hydroxamic

derivative, as it can be seen from the overlay in Figure 3.

For this reason, the distances between the pharmacophoric

groups and the surface and volume of the phenylalanine one are

mainly dependent upon the length of the phenylalanine side

chain and are optimal in compound 2 (Table 4).

The presence of the negatively charged carboxy group plays a

key role for the activity. This is clearly seen within the series of

C2 side chain compounds, which show very similar surfaces and

volumes but very large diﬀerence in activities. This is most likely

due to a ﬁeld eﬀect, but also the centroid distance between the

ends is somewhat aﬀected by the lack of the negative charge and

is higher in the neutral compounds. This is due to the fact that in

the negatively charged compounds, torsion angle H₁₅N₁₄C₁₆C₁₇

is constrained also by the hydrogen bond interaction between

H₁₅and one of the oxygens of the carboxy group.

2

5

6

2

RS

S

48.9 ( 10.4

420.3 ( 2.6

43.9 ( 1.7

NT^a

NT

R

0.35 ( 0.13

^aNT: not tested.

the dramatic decrease of the binding aﬃnity of substituted pri-

mary amide compound relative to compound 2 excludes this type

of interaction. Further investigations in order to replace the hy-

pothesized ionic bond between compound 2 and the receptor

binding pocket with a double hydrogen bond interaction was

accomplished by designing the hydroxamate derivative 10, which

is considered as alternative bioisoster of the carboxylic acid func-

tionality. The compound having an hydroxamic acid moiety (com-

pound 10) showed, as expected, a 27-fold lower aﬃnity relative

to compound 2, thus conﬁrming the crucial importance of the

ionic interaction.

The reduction of the free carboxylic group of 2 to a methyl

group (compound 9) conﬁrms the losses of the aﬃnity observed

for the ester and amide derivatives.

When the carboxylic group is replaced by the hydroxamic acid

moiety, the ground state conformation is diﬀerent from that of

the other compounds. The hydroxamic group is stabilized by a

strong intramolecular hydrogen bond between the carbonyl

oxygen and the terminal hydrogen (Scheme 3b, Figure 4). The

formation of this 5-membered pseudocycle is well-known and

makes the terminal hydrogen less acidic than the N-hydrogen.^27,28

In our case, also the N-hydrogen is involved in a hydrogen bond

with the central anthranilic core, and for this reason the molecule

adopts a conformation that moves both the hydroxamic group

itself and the phenyl ring away from the positions adopted by the

other ligands (Figure 4). The distance between the aromatic ends

of the molecule is thus the largest of the series.

Moreover, the phenylenediamine derivative 11 shows a dif-

ferent conformation (Scheme 3c, Figure 5), and the amino acidic

side chain is turned in order to allow the carboxylate group to

establish strong interactions with both the core N-hydrogens.

The distance between the ends of the molecule is thus somewhat

higher, and the carboxylate seems not available for intermolecular

interactions, at least if the molecule is bound in its ground state

conformation.

Dynamic Behavior of Compounds 2 and 12. The longer

chain connecting the phenyl ring with the core of the molecule in

compound 2 allows a higher conformational freedom to this mol-

ecule in comparison with the phenylalanine derivative 12. This effect

could also give a contribution to the better activity of 2 because the

ligand could reach also conformational states that are less populated

in the shorter lead compound. To investigate this possibility, we have

submitted the two molecules to several molecular dynamics runs.

The centroid distance between the indole and the phenyl ring

was monitored during the simulations, and Figure 6 reports the

typical behavior of the two compounds.

Finally, compound 11 was designed and synthesized in order

to highlight the importance of the anthranilic acid scaﬀold for the

correct spatial orientation of the main pharmacophoric groups

(indole and amino acidic side chain) during the ligandꢀreceptor

interactions. The 45-fold lower aﬃnity of compound 11 toward

CCK₁-Rs reinforces the role of the anthranilic acid template for

the receptor subtype recognition. In fact, compound 11 exhibited

a binding aﬃnity to the CCK₂-R similar to that of 2, conﬁrming

the critical function of the anthranilic acid for the CCK₁-R selectivity.

2. In Vitro and in Vivo Functional Studies. The antagonist

nature of compounds reported in Table 3 was checked by in vitro

and/or in vivo functional tests according to established protocols

reported in Experimental Section. The compounds inhibited the

(Thr,Nle)-CCK-9-induced production of inositol phosphate,

with potencies in close agreement with their affinity values. More-

over, isolated guinea pig gallbladder was used to verify antagon-

ism of these compounds toward CCK₁-R both on in vitro and

in vivo models. None of these compounds showed any intrinsic

contractile effect in the gallbladder preparations, while in both

models compound 6 was a potent inhibitor of CCK-8 induced

contraction, with an IC₅₀value around 40 nM in vitro and an

ID₅₀of 0.35 μmol/kg in vivo. In comparison, compound 6 in

vitro was about 10-fold more potent than distomer 5 and 30

times more active relative to Dexloxiglumide (IC₅₀= 1230 ( 20)

used as the reference compound.

3. Molecular Modeling Study. Ground States. We have

carried out first a conformational analysis on the ligands, and the

main structural parameters for their ground state conformations

are reported in Table 4.

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Table 4

Pl.

angle^d

deg

A^a

Å

B^b

Å

C^c

Å

H₉N₉C_9aC_13a

deg

C_9aC_13aC₁₄O₁₄

deg

H

₁₅N₁₅C₁₆C₁₇

deg

S_phe

Å²

V_phe

Å³

e

f

compd

n

1

0

1

2

3

4

7.4

7.6

6.9

8.1

7.5

7.4

7.6

92.0

141.4

84.8

85.0

87.9

88.6

86.9

110.9

9.4

347.4

346.9

346.2

340.5

340.1

340.6

340.3

347.4

357.8

357.6

44.3

43.5

43.6

44.7

44.6

44.8

45.6

57.5^h

7.7

95.6

114.9

99.2

89.0

98.4

84.0

120.4

125.5

128.2

129.0

125.8

127.1

129.6

136.9

151.9

12

2

8.8

9.5

123.4

125.3

126.3

121.4

125.3

123.1

143.1

161.1

10

7

12.1

10.0

11.5

9.4

9.6

24.2

9.5

62.7

8

9.6

28.6

9

11^g

9.2

62.3

9.8

9.9

21.0ⁱ

111.0

110.4

3

10.8

11.5

11.0

11.8

4

63.9

8.0

^aDistance between centroids deﬁned on the aromatic systems of indole and phenylalanine. ^bDistance between centroids deﬁned on the aromatic

systems of phenylalanine and anthranilic acid. ^cDistance between centroids deﬁned on the aromatic systems of anthranilic acid and indole. ^dPlane angle

between plane 1, containing the indole ring, and plane 2, containing the phenylalanine ring. ^eConnoly surface calculated with a spherical probe of 0.51 Å

radius on all the atoms from C₁₆to the end of the phenylalanine side chain. ^fVolume under surface S. ^gPhenylelediamine derivative (compd 11), the

positions of O₁₄ꢀH₁₅and C₁₄ꢀN₁₅are inverted. ^hC_9aC_13aN₁₅H₁₅. ⁱO₁₄C₁₄C₁₆C₁₇.

Scheme 3. Schematic Representation of the Ground State

Minimum Energy Conformation of Compound 12 (a), Com-

pound 10 (b), and Compound 11 (c)

second populated conformation at a distance of about 11 Å, with

loss of the hydrogen bond between H15 and the carboxylate

residue. Similar but less populated conformations are found in

Figure 2

compound 12 at a distance of about 9.5 Å.

4. Molecular Docking of Compounds 2 and 12 in the

Modeled CCK₁-R. To identify the key amino acids involved in

It can be seen that compound 2 can reach during the simulation

both larger and shorter distances than the equilibrium one much

more frequently than 12. A population analysis of such distances

the CCK₁receptor recognition process, a molecular docking

is reported in Figure 7.

study and its validation by site-directed mutagenesis experiments

The distances available span was in the range 4.5ꢀ12.5 Å for

has been accomplished. In fact, we previously mapped the CCK₁-R

binding site for CCK using site-directed mutagenesis and molec-

ular modeling and found that the binding sites of nonpeptide

agonists and antagonists most likely occupy a region of CCK₁-R,

which interacts with the C-terminal amidated tripeptide of CCK,

i.e. Met-Asp-Phe-NH₂.²⁹

compound 12 and between 3.5 and 14.5 Å for compound 2.

During the runs, the central hydrogen bond at the anthranilic

residue has never been lost by both the molecules. Compound 12

is found in its ground state conformation in about the 80% of the

population, while only a 40% of the population of compound 2

conformers is represented by its ground state, and there is a

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Figure 3. Overlay of the ligands optimized structures: compd 12, white;

compd 2, yellow; compd 3, cyano; compd 1, green; compd 8, magenta;

compd 7, orange; compd 9, purple; compd 4, red.

Figure 6. Compound 2 (black) and compound 12 (red).

Figure 4. Overlay of the optimized structures of 2 (green) and of its

hydroxamic acid derivative 10.

Figure 7. Population analysis of low energy conformations of com-

pound 12 (red) and of compound 2 (black) after molecular dynamics

at 300 K.

in helix IV, Met195 and Arg197 in the second extracellular loop,

Thr117 and Met121 in helix III, Arg336 in helix VI, and Ile352,

Leu356, and Tyr360 in helix VII (Figure 9). In this orientation,

the indole moiety of the ligand occupies the same region as that

occupied by the Trp indole ring of CCK-9 between helices II, III,

and VII. The phenyl moiety of the ligand approximates Trp39 in

helix I, Phe107 in the ﬁrst extracellular loop and Met195 in the

second extracellular loop, mimicking in this way the sulfated

tyrosine of CCK. The carboxylate group of the ligand can interact

with two arginines, Arg197 in the second extracellular loop and

Arg336 in the helix VI. The same interactions were observed in

the inactive state of CCK₁-R.

Figure 5. Overlay of the optimized structures of compound 2 (green)

and of compound 11.

Initially, two docking orientations of compound 12 in the

binding pocket of the CCK₁-R were generated. In orientation A,

compound 12 overlapped with the N-terminal moiety of CCK,

Asp-Tyr-Thr-Gly-Trp (Figure 8a), whereas in orientation B, the

antagonist overlapped with the amidated C-terminus of CCK,

namely Trp-Met-Asp-Phe-NH₂(Figure 8b).

In orientation A, the binding site is situated between Trp39

and Tyr48 in helix I, Phe107 in the ﬁrst extracellular loop, Tyr176

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Figure 8. Superposition of CCK-9 (yellow) and compound 12 (blue) in orientations (a) and (b).

Table 5. Eﬀects of CCK₁Receptor Mutations on the Recep-

tor Binding

b

CCK₁K_d³H-SR-27897 K_d¹²⁵I-BH-JMV-179 K_icompd 6

F_mut

receptor

(nM)^a

(nM) ^a

(nM) ^a(compd 6)

WT

21.9 ( 1.2

0.32 ( 0.04

5.8 ( 0.3

3.7 ( 0.4

3.8 ( 0.8

7.6 ( 0.7

19.6 ( 2.8

522 ( 25

4.9 ( 0.1

39.1 ( 5.3

5.0 ( 0.6

1

WT

3.4 ( 0.1

2.4 ( 0.2

5.0 ( 0.3

2.9 ( 0.1

5.6 ( 0.7

47.1 ( 4.5

13.2 ( 1.9

6.0 ( 0.4

W39A

F107A

M195A

R197A

R336A

F330A

I352A

N333A

0.6

0.8

1.3

3.8

101.6

1.1

7.4

16.2

Figure 9. Serpentine representation of the CCK₁receptor. Numbered

amino acids are those which are part of the binding site of CCK and/or

compound 12.

23.7 ( 2.8

^aResults are the mean ( SEM from at least three experiments

performed in duplicate. ^bThe mutation factors (F_mut) were calculated

as K_i(mutated receptor)/K_i(wild-type-CCK₁R).

In orientation B, the binding site for the ligand is formed by

Thr117, Thr118, and Met121 in helix III, Tyr176 in helix IV,

Met195 in the second extracellular loop, Asn333 and Arg336 in

helix VI, Ile352, Leu356, and Tyr360 in helix VII (Figure 9). In

this case, the indole ring of the ligand engages the same region

occupied by the Trp indole ring of CCK. The carboxylate group

accepts two hydrogen bonds one from Arg336 and the other

from the amide of Asn330 in helix VI. The phenylalanine aromatic

ring is sited between helix III and helix VI. This positioning is

observed in the inactive state of CCK₁-R.

5. Validation of the Docking Orientation through Site-

Directed Mutagenesis. To discriminate between both orienta-

tions we have performed site directed mutagenesis and competition

binding assays in the presence of two known CCK₁-R antago-

nists: SR-27897 and JMV-179. We have focused our attention on

the residues Trp39, Phe107, Met195, Arg197, Asn333, and Arg336

(Table 5).

The two most important residues for the binding of com-

pound 6 are Arg336 and Asn333 because Arg336A and Asn333A

mutants bind the ligand with 101.6, 16.2-fold lower aﬃnity than

the wild-type receptor, respectively. The Arg197Ala mutant also

shows lower aﬃnity, but its F_mutis only 3. Met195Ala and

Trp39Ala mutations do not aﬀect ligand binding, indicating that

eventual interactions with these residues are not signiﬁcant.

In this manner, we have three important interactions Arg336 >

Asn333 > Arg197. Interactions with Arg336 and Asn333 are

present in orientation B of docking and interactions with Arg197,

and Arg336 are present in orientation A.

Because the initial docking was performed in the CCK₁-R

taken from the CCK₁-R/CCK complex and the binding experi-

ments have been performed in the presence of antagonist which

bind to the inactive state of the CCK₁-R, we decided to carry out

the docking in the inactive conformation of the receptor. In this

case, orientation B is not found but instead, an orientation very

similar to A was observed, with a small displacement of the ligand

toward Asn333 in a manner that its amide can interact with the

indole-2-carbonyl group of the ligand.

In this disposition, the carboxylate group of 6 interacts with

Arg197 and Arg336 and the indole carbonyl interacts with Asn333.

The interactions of 2 with the receptor amino acids aﬀect only to

a minor extent its conformation when compared with the ground

state one. The indole and anthranyl sides of the molecule are

almost perfectly superimposable in the free and bound mol-

ecules. On the other side, the intramolecular hydrogen bond be-

tween H15 and the carboxylate end is lost in favor of the

interactions with Arg197 and Arg336. This leads also to a slight

rotation of the phenyl end of the molecule, and the bound con-

formation is somewhat intermediate between the two popula-

tions of conformers found by MD. The slight movement of the

phenyl ring might improve the interactions of this aromatic end

with Phe107, Met195, and Ile 352.

Moreover, we have studied the stability of the complex by

means of molecular dynamics simulations in an explicit lipid

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Table 6. Calculated van der Waals and Electrostatic Interac-

tion Energies between Compound 6 and CCK₁Receptor^a

Ile352Ala mutant binds 6 with 7.4 lower aﬃnity that the WT

receptor, but the Phe330Ala mutation does not aﬀect the binding

(F_mut: 1.1). This last result rules out orientation B.

CCK₁receptor

van der Waals

electrostatic

CCK₂receptor

In summary, both site-directed mutagenesis ﬁndings and in

silico docking showed that the binding site of compound 6

overlaps that occupied by the C-terminal bioactive region of the

natural agonist CCK as well as that of other nonpeptide antagonists

as the benzodiazepine derivative Devazepide; the thiazole deri-

vative SR-27897 and of the pyridopyrimidine-based ligands.²⁹

Description of the Binding Site after Molecular Dynamics

Simulations. The binding site for compound 6 comprises Phe107

in the first extracellular loop, Gln194 and Met195 of the second

extracellular loop, Thr117, Thr118, and Met121 in helix III,

His210 in helix V, Asn333 and Arg336 in helix VI, and in helix

VII, Ile352, Leu356, and Tyr360.

The indole-2-carbonyl group is located between helices II, III,

and VII and occupies the same region that is occupied by the Trp

indole ring of CCK-9. Asn333 donates a hydrogen bond to the

indole-2-carbonyl oxygen. The phenylalanine ring interacts with

Phe107 in the ﬁrst extracellular loop, Met195 in the second extra-

cellular loop, and Ile352 in helix VII. The carboxylate group inter-

acts with Arg336 side chain and possibly with Gln194. In the

ester and amide derivatives (compounds 7 and 8), it is not

possible to have these interactions (Figure 10).

Trp39

ꢀ0.2

ꢀ0.8

ꢀ1.1

ꢀ0.4

ꢀ1.2

ꢀ2.4

ꢀ3.3

ꢀ0.1

ꢀ3.6

ꢀ0.2

ꢀ2.0

ꢀ3.8

ꢀ1.7

ꢀ3.2

ꢀ0.7

Glu

Phe107

Thr117

Thr118

Met121

Gln194

Met195

Arg197

His210

Phe330

Asn333

Arg336

Ile352

Phe120

Val130

Ser131

Met134

Leu203^b

Gln204^b

Val206^b

Ser219^b

Try350

Asn353

Arg356

Ile372

ꢀ11.4

ꢀ1.3

ꢀ2.1

ꢀ5.8

ꢀ21.8

Leu356

Tyr360

His376^b

Tyr380

^aCalculated with ANAL program of AMBER. ^bResidues marked in bold

are those important for the binding, as revealed by energetic analysis

decomposition, and also diﬀerent between CCK₁and CCK₂receptors.

The selectivity can be explained by these interactions.

’ CONCLUSION

In this second phase of the development of this new class of

CCK₁-R antagonists dedicated to the C-terminal optimization of

the anthranilic acid of 12, we synthesized new compounds bearing

unnatural amino acids instead of Phe present in the lead. The

Homo-Phe derivative (compound 2) showed a 12-fold increased

binding aﬃnity for the CCK₁-R and 4-fold increased apparent

selectivity over the CCK₂-R relative to the reference compound

12. We conﬁrm the receptor subsite stereospeciﬁcity deﬁning the

eutomer of 2 (compound 6) in which the absolute conﬁguration

of the single chiral center is R (rectus). Compound 2 and its

eutomer exhibited a nanomolar range aﬃnity toward the human

CCK₁-R, and compound 6 was at least 400-fold more selective

for the human CCK₁over the CCK₂-Rs. The antagonist nature

of 6 was validated by an in vitro and in vivo functional test using

guinea pig gallbladder preparations. In both models, compound 6

conﬁrmed to be a potent inhibitor of CCK-8-induced contrac-

tion of gallbladder and was 30 times more active than reference

compound Dexloxiglumide in vitro.

The lower aﬃnity observed for compounds 7ꢀ10 ascertains

that the free carboxy group of the Homo-Phe is essential for the

receptor recognition, suggesting its engagement through ionic bond

formation rather than in a double hydrogen bond interaction.

Moreover, data with compound 11 underlines the importance

of the anthranilic acid scaﬀold for the correct spatial orientation

adopted by the main pharmacophoric groups of 2 in receptor

subtype recognition.

The molecular modeling study elucidate that the higher receptor

aﬃnity of 2 is due to the higher conformational freedom of the

Homo-Phe side chain in comparison with the Phe derivative 12.

Finally, identiﬁcation of key amino acids of CCK₁-R involved

in the receptor desmodynamic process of 6 has been accom-

plished via molecular docking study and successive validation by

site-directed mutagenesis experiments.

Figure 10. Docking of compound 6 to CCK₁receptor in the inactive

conformation after molecular dynamic simulation in an explicit mem-

brane. Residues relevant for compound 6 binding are displayed as sticks.

environment. The interaction with Arg197 is lost, but an elec-

trostatic analysis revealed that there is still an electrostatic inter-

action (Table 6). A long-range scale electrostatic interaction

between the carboxylate group of 6 and Arg197 may be the re-

ason of the observed loss of aﬃnity with Arg197Ala mutant.

Nevertheless, as the interaction with Ag197 is very small and

site directed mutagenesis does not reveal contacts with Met195

and Trp39, we constructed additional mutants to better discri-

minate A and B. To this purpose, we mutated Ile352 and Phe330.

Such results provide suitable information for our research project,

and we are convinced that further eﬀorts should be accomplished

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in order to establish with precision the receptor subsite tolerance

in relation to the variation of the aminoacidic side chain.

Nevertheless, compound 6, owing to its chemical features

(simple structure, low molecular weight, number of synthetic

steps, overall yield and low cost synthesis), represents an attractive

opportunity for the pharmaceutical industry. Thus, the anthra-

nilic acid based derivative 6 is actually under further biological

investigation in order to establish its drug-like properties.

dropwise and the reflux was continued overnight. The reaction mixture

was then poured into 500 mL of 0.05 M KHSO₄and 250 mL of n-hexane

under vigorous stirring at 0 ꢀC. The product precipitated at the interface

was filtered and recrystallized from AcOEt/n-hexane.

2-Acetylamino-2-(3-phenyl-propyl)-malonic Acid Diethyl Ester

(3b). The product was obtained in 64% yield. TLC (AcOEt/n-hexane

1:1); R_f0.45; mp 85 ꢀC [lit. 86ꢀ87 ꢀC].^{32 1}H NMR (CDCl₃) δ 1.21 (t,

6H, ꢀCH₂-CH₃), 1.43 (m, 2H, ꢀCH₂-CH₂-CH₂ꢀ), 2.02 (s, 3H,

ꢀCO-CH₃), 2.43 (m, 2H, -CH₂-(CH₂)₂-C₆H₅), 2.60 (t, 2H, ꢀCH₂-

C₆H₅), 4.21 (m, 4H, ꢀCH₂-Oꢀ), 6.79 (s, 1H, ꢀNHꢀ), 7.11ꢀ7.26 (m,

5H, Ar). ¹³C NMR (CDCl₃) δ 14.06, 23.15, 25.78, 31.92, 35.52, 62.60,

66.53, 125.98, 128.34, 128.42, 141.82, 168.16, 169.08.

’ EXPERIMENTAL SECTION

Chemistry. All chemicals and solvents used in the syntheses were

reagent grade and were used without additional purification. The pro-

gress of all reactions was monitored by thin-layer chromatography (TLC)

using precoated silica gel plates (60F-254 Merck) and visualized by UV

light (254 nm). Melting points were determined on a B€uchi 510 melting

point apparatus (B€uchi, Flawil, Switzerland) and are uncorrected. Silica

gel (Merck Kieselgel 60, 40ꢀ63 μm) was used for flash chromatography.

Specific rotations were measured with a Perkin-Elmer 241 polarimeter at

589 nm (Na D-line) and 25 ꢀC. Enantiomeric excess determinations

were performed on a Jasco HPLC chromatograph on CSP-TE-SP-100

(250 mm ꢁ 4.0 mm ID) column by using an ammonium acetate

solution (20 mM) in methanol/water 85/15 (v/v) as the mobile phase

at flow rate of 1.00 mL/min with UV detection at 254 nm. Proton (¹H

NMR, 200 MHz) and carbon (¹³C NMR, 50 MHz) nuclear magnetic

resonance spectra were recorded on a Varian-Gemini 2000 Fourier

transform spectrometer for CDCl₃or (CD₃)₂SO solutions, using Me₄Si

as internal standard. Splitting patterns are designated as: s, singlet; d,

doublet; t, triplet; q, quartet; m, multiplet; b, broad. Coupling constants

(J values) are given in hertz (Hz). Spectral data are consistent with

assigned structures. Mass spectra were recorded an API-1 Perkin-Elmer

SCIEX spectrometer by electrospray ionization (ES). The purities

(>95%) of all compounds tested in biological systems were established

by elemental analyses. Elemental analyses were performed by the

Microanalyses Laboratory of the Department of Chemical and Pharma-

ceutical Sciences of the University of Trieste and were within (0.4% of

the theoretical values calculated for C, H and N.

The following abbreviations are used: TEA, triethylamine; MeOH,

methanol; MeONa, sodium methoxide; EtOH, ethanol; AcOEt, ethyl

acetate; AcOH, acetic acid; n-BuOH, 1-butanol; THF, tetrahydrofuran;

DMF, dimethylformamide; MsCl, methanesulfonyl chloride; TBTU,

O-benzotriazol-1-yl-N,N,N⁰,N⁰-tetramethyluronium tetraﬂuoroborate;

PyBOP, benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium hexaﬂuo-

rophosphate.

4-Phenyl-1-iodobutane (4a). To a 4-phenyl-butan-1-ol (10.00 g,

66.60 mmol) and TEA (14.10 mL, 101.33 mmol) solution in CH₂Cl₂

(150 mL), MsCl (5.84 mL, 75.20 mmol) was added dropwise at 0 ꢀC.

After 2 h stirring at room temperature, the solution was washed with

iceꢀwater (2 ꢁ 50 mL), cold 5N HCl (2 ꢁ 50 mL), saturated aqueous

NaHCO₃(2 ꢁ 50 mL), and brine (2 ꢁ 50 mL), dried over anhydrous

Na₂SO₄, and concentrated. The mesylate product was mixed with NaI

(43.30 g, 283.05 mmol) in acetone (250 mL), and the mixture was

refluxed until TLC indicated full conversion of the starting material.

Water was added (200 mL), and the solution was extracted with diethyl

ether (3 ꢁ 100 mL). The combined organic layers were washed with

water (2 ꢁ 50 mL) and brine (2 ꢁ 50 mL) and concentrated.³⁰The

product was purified by flash chromatography (pentane) to yield (4a) as

a colorless oil. Spectroscopic data were consistent with data described

elsewhere.³¹

2-Acetylamino-2-(4-phenyl-butyl)-malonic Acid Diethyl Ester (4b).

The product was obtained in 80% yield. TLC (AcOEt/n-hexane 1:1); R_f

0.52; mp 56ꢀ57 ꢀC [lit. 83ꢀ84 ꢀC].^{33 1}H NMR (CDCl₃) δ 1.10ꢀ1.29

(m, 8H, ꢀCH₂-CH₃and ꢀCH₂-(CH₂)₂-C₆H₅), 1.64 (m, 2H, -CH₂-

CH₂-C₆H₅), 2.01 (s, 3H, ꢀCO-CH₃), 2.32ꢀ2.41 (m, 2H, ꢀCH₂-

(CH₂)₃-C₆H₅), 2.60 (t, 2H, ꢀCH₂-C₆H₅), 4.22 (m, 4H, ꢀCH₂-Oꢀ),

6.82 (s, 1H, ꢀNHꢀ), 7.12ꢀ7.30 (m, 5H, Ar). ¹³C NMR (CDCl₃)

δ 14.09, 23.02, 23.13, 30.93, 31.97, 35.43, 62.53, 66.58, 125.73, 128.27,

128.39, 142.07, 168.14, 168.95.

2-Acetylamino-2-phenylethyl-malonic Acid Diethyl Ester (7b). The

product was obtained in 75% yield. TLC (AcOEt/n-hexane 1:1); R_f

0.55; mp 116 ꢀC (lit. 111ꢀ114 ꢀC).^{34 1}H NMR (CDCl₃) δ 1.24 (t, 6H,

ꢀCH₂-CH₃), 1.97 (s, 3H, ꢀCO-CH₃), 2.45ꢀ2.75 (m, 4H, ꢀ(CH₂)₂ꢀ),

4.17 (m, 4H, ꢀCH₂-Oꢀ), 6.79 (s, 1H, ꢀNHꢀ), 7.12ꢀ7.26 (m, 5H,

Ar). ¹³C NMR (CDCl₃) δ 14.08, 23.05, 30.23, 33.40, 62.65, 66.45,

126.16, 128.44, 128.51, 140.61, 168.10, 169.14.

General Procedure for the Synthesis of Amino Acids

Hydrochloride (3c, 4c, 7c). A suspension of 25.00 mmol of the

corresponding diethyl R-alkyl-acetamidomalonic derivative 3b, 4b, or

7b in 150 mL of a solution 1:1 of 6N HCl and 1,4-dioxane was stirred

under reflux. After completion (TLC monitoring), the solvent was removed

in vacuo and the residue triturated with cold diethyl ether. The product

was collected by filtration.

2-(R,S)-Amino-5-phenyl-pentanoic Acid Hydrochloride (3c). The

product was obtained in 77% yield. TLC (n-BuOH/AcOH/H₂O 3:1:1);

R_f0.66; mp 228 ꢀC. ¹H NMR (DMSO-d₆) δ 1.60ꢀ1.85 (m, 4H,

ꢀ(CH₂)₂ꢀ), 2.55 (m, 2H, ꢀCH₂-C₆H₅), 3.84 (m, 1H, ꢀCH<),

+

7.15ꢀ7.30 (m, 5H, Ar), 8.57 (b, 3H, NH₃). ¹³C NMR (DMSO-d₆)

δ 26.06, 29.47, 34.42, 51.81, 125.62, 128.08, 141.22, 170.67.

2-(R,S)-Amino-6-phenyl-hexanoic Acid Hydrochloride (4c). The

product was obtained in 95% yield. TLC (n-BuOH/AcOH/H₂O

3:1:1); R_f0.66; mp 168ꢀ170 ꢀC. ¹H NMR (DMSO-d₆) δ 1.32ꢀ1.61

(m, 4H, ꢀ(CH₂)₂-CH₂-C₆H₅), 1.84 (m, 2H, ꢀCH₂-CH<), 2.54 (m,

2H, ꢀCH₂-C₆H₅), 3.84 (m, 1H, ꢀCH<), 7.13ꢀ7.31 (m, 5H, Ar), 8.50

+

(b, 3H, NH₃). ¹³C NMR (DMSO-d₆) δ 23.89, 29.89, 30.50, 34.78,

51.77, 125.57, 128.13, 128.16, 141.79, 170.78.

2-(R,S)-Amino-4-phenyl-butyric Acid Hydrochloride (7c). The pro-

duct was obtained in 53% yield. TLC (n-BuOH/AcOH/H₂O 3:1:1); R_f

0.64; mp 272 ꢀC [lit. 260ꢀ272 ꢀC]. ¹H NMR (DMSO-d₆) δ 2.10 (m,

2H, ꢀCH₂-CH<), 2.68 (m, 2H, ꢀCH₂-C₆H₅), 3.87 (m, 1H, ꢀCH<),

+

7.22ꢀ7.35 (m, 5H, Ar), 8.66 (b, 3H, NH₃). ¹³C NMR (DMSO-d₆)

δ 30.23, 31.70, 51.41, 125.97, 128.08, 128.28, 140.24, 170.55.

General Procedure for the Synthesis of Amino Acids Ethyl

Esters Hydrochloride (3d, 4d, 7d). Gaseous HCl is bubbled for

30 min into a stirred suspension of 20.00 mmol of the corresponding

aminoacid hydrochloride 3c, 4c, or 7c in 150 mL of abs EtOH and

cooled to 0 ꢀC. After solvent removal, the residue is triturated with cold

diethyl ether and filtered.

General Procedure for Synthesis of Diethyl r-Alkyl-acet-

amidomalonic Derivatives (3b, 4b, 7b). To a solution of 20.00 g

(92.00 mmol) of diethyl acetamidomalonate in 90 mL of abs EtOH,

EtONa 96% (6.94 g, 98.00 mmol) was added. After stirring under reflux

for 30 min, 90.0 mmol of the corresponding alkyl halide was added

2-(R,S)-Amino-5-phenyl-pentanoic Acid Ethyl Ester Hydrochloride

(3d). The product was obtained in 88% yield. TLC (AcOEt/MeOH

1:1); R_f0.73; mp 126 ꢀC. ¹H NMR (DMSO-d₆) δ 1.20 (m, 3H, ꢀCH₃),

1.62ꢀ1.82 (m, 4H, ꢀ(CH₂)₂ꢀ), 2.56 (m, 2H, ꢀCH₂-C₆H₅), 3.97

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(m, 1H, ꢀCH<), 4.18 (m, 2H, ꢀO-CH₂ꢀ), 7.18ꢀ7.28 (m, 5H, Ar),

anhydrous Na₂SO₄, and concentrated in vacuo. The residue was purified

as described to yield the titled compounds.

+

8.73 (b, 3H, NH₃). ¹³C NMR (DMSO-d₆) δ 13.78, 25.85, 29.37, 34.27,

51.55, 61.43, 125.66, 128.09, 141.11, 169.20.

2-(R,S)-Amino-6-phenyl-hexanoic Acid Ethyl Ester Hydrochloride

Compound (9e) was obtained in the same way as described above,

except no TEA was added to the reaction.

(4d). The product was obtained in 66% yield. TLC (AcOEt/MeOH

(R,S)-(2-Amino-benzoylamino)-phenyl Acetic Acid Ethyl Ester (1e).

Trituration with cold petroleum ether 40ꢀ60ꢀ afforded the titled com-

pound in 42% yield. TLC (AcOEt/n-hexane 1:1); R_f0.71; mp 81ꢀ83 ꢀC.

¹H NMR (CDCl₃) δ 1.23 (m, 3H, ꢀCH₃), 4.20 (m, 2H, ꢀO-CH₂ꢀ),

5.03 (b, 2H, ꢀNH₂), 5.70 (m, 1H, >CH-C₆H₅), 6.64 (m, 2H, >CH-

NHꢀ and Ar), 7.00ꢀ7.43 (m, 8H, Ar). ¹³C NMR (CDCl₃) δ 14.07,

56.71, 62.07, 116.61, 117.36, 127.24, 127.56, 128.49, 128.85, 128.99,

132.75, 136.83, 149.06, 168.44, 171.11.

1

1:1); R_f0.79; mp 126ꢀ27 ꢀC. H NMR (DMSO-d₆) δ 1.16 (t, 3H,

ꢀCH₃). 1.22ꢀ1.46 (m, 4H, ꢀ(CH₂)₂-CH₂-C₆H₅), 1.58 (m, 2H, ꢀCH₂-

CH<), 2.54 (m, 2H, ꢀCH₂-C₆H₅), 3.89 (m, 1H, ꢀCH<), 4.14 (q, 2H,

+

ꢀO-CH₂ꢀ), 7.14ꢀ7.27 (m, 5H, Ar), 8.67 (b, 3H, NH₃). ¹³C NMR

(DMSO-d₆) δ 13.88, 23.69, 29.72, 30.30, 34.64, 51.68, 61.52, 125.55,

128.09, 128.14, 141.67, 169.30.

2-(R,S)-Amino-4-phenyl-butyric Acid Ethyl Ester Hydrochloride

(7d). The product was obtained in 84% yield. TLC (AcOEt/MeOH

2-(R,S)-(2-Amino-benzoylamino)-5-phenyl-pentanoic Acid Ethyl

Ester (3e). Purification by flash chromatography (AcOEt/n-hexane)

afforded the titled compound in 60% yield. TLC (AcOEt/n-hexane 1:1);

R_f0.81; mp 53 ꢀC. ¹H NMR (CDCl₃) δ 1.29 (t, 3H, ꢀCH₃). 1.32ꢀ2.00

(m, 4H, ꢀ(CH₂)₂ꢀ), 2.67 (m, 2H, ꢀCH₂-C₆H₅), 4.22 (q, 2H, ꢀ

O-CH₂ꢀ), 4.80 (m, 1H, ꢀCH<), 5.49 (b, 2H, ꢀNH₂), 6.66 (m, 3H,

>CH-NHꢀ and Ar), 7.16ꢀ7.33 (m, 6H, Ar), 7.40 (d, 1H, Ar). ¹³C NMR

(CDCl₃) δ 14.32, 27.20, 32.29, 35.46, 52.21, 61.63, 115.45, 116.70, 117.38,

126.02, 127.55, 128.48, 132.66, 141.74, 148.92, 168.95, 172.80.

2-(R,S)-(2-Amino-benzoylamino)-6-phenyl-hexanoic Acid Ethyl Es-

ter (4e). Purification by flash chromatography (CH₂Cl₂/AcOEt) af-

forded the titled compound in 50% yield. TLC (AcOEt/n-hexane 1:1);

R_f0.86; mp 63ꢀ64 ꢀC. ¹H NMR (CDCl₃) δ 1.27 (t, 3H, ꢀCH₃), 1.42

(m, 2H, ꢀCH₂ꢀ), 1.46ꢀ2.02 (m, 4H, ꢀ(CH₂)₂ꢀ), 2.61 (m, 2H,

ꢀCH₂-C₆H₅), 4.21 (q, 2H, ꢀO-CH₂ꢀ), 4.74 (m, 1H, ꢀCH<), 5.46 (b,

2H, ꢀNH₂), 6.66 (m, 3H, >CH-NHꢀ and Ar), 7.13ꢀ7.38 (m, 7H, Ar).

¹³C NMR (CDCl₃) δ 14.21, 24.73, 30.97, 32.45, 35.55, 52.16, 61.50,

115.35, 116.56, 117.19, 125.65, 127.36, 128.21, 128.29, 132.47, 142.06,

148.69, 168.68, 172.64.

1

1:1); R_f0.65; mp 137 ꢀC [lit. 135ꢀ136 ꢀC]. H NMR (DMSO-d₆)

δ 1.23 (t, 3H, ꢀCH₃), 2.14 (m, 2H, ꢀCH₂-CH<), 2.71 (m, 2H, ꢀCH₂-

C₆H₅), 3.96 (m, 1H, ꢀCH<), 4.18 (q, 2H, ꢀO-CH₂ꢀ), 7.21ꢀ7.31 (m,

+

5H, Ar), 8.65 (b, 3H, ꢀNH₃). ¹³C NMR (DMSO-d₆) δ 13.78, 30.07,

31.67, 51.32, 61.54, 125.98, 128.11, 128.24, 140.09, 169.06.

2-Phenethyl-malonic Acid Diethyl Ester (11b). To a solution

of EtONa (2.30 g, 32.90 mmol) in abs EtOH (150 mL), diethyl malonate

was added dropwise during 10 min. After stirring under reflux for 45 min,

4.50 mL (32.90 mmol) of (2-bromo-ethyl)-benzene were added drop-

wise and the reaction mixture was refluxed overnight. The solution was

concentrated under reduced pressure, and the residue, taken up in

100 mL of water, was extracted with diethyl ether (3 ꢁ 50 mL). The

combined organic layers were washed with water (3 ꢁ 25 mL) and brine

(25 mL), dried over anhydrous Na₂SO₄, and concentrated. Distillation

(60ꢀ80 ꢀC and 0.04 mmHg) of the residual reactants afforded a pale-

yellow oil in 42% yield. TLC (AcOEt/n-hexane 1:3); R_f0.67. ¹H NMR

(CDCl₃) δ 1.27 (t, 6H, ꢀCH₃), 2.22 (m, 2H, ꢀCH₂-CH<), 2.66 (t, 2H,

ꢀCH₂-C₆H₅), 3.34 (t, 1H, ꢀCH<), 4.19 (q, 4H, ꢀO-CH₂ꢀ), 7.17ꢀ

7.32 (m, 5H, Ar). ¹³C NMR (CDCl₃) δ 14.25, 30.50, 33.45, 51.37, 61.47,

126.24, 128.50, 128.59, 140.69, 169.35.

2-(R,S)-Phenethyl-malonic Acid Monoethyl Ester (11c). A

mixture of 11b (2.53 g, 9.57 mmol) and KOH (0.54 g, 9.57 mmol) in abs

EtOH was stirred overnight at room temperature. After solvent removal,

the residue was dissolved in H₂O and extracted with diethyl ether (3 ꢁ

50 mL). Aqueous layer was adjusted to pH 2ꢀ3 with 1N HCl and

extracted with diethyl ether (3 ꢁ 50 mL). The combined organic layers

were washed with water and brine, dried over anhydrous Na₂SO₄, and

concentrated in vacuo. The crude residue was used without further

purification in the next step.

(+)-2-(S)-(2-Amino-benzoylamino)-4-phenyl-butyric Acid Ethyl Es-

ter (5e). Purification by flash chromatography (CH₂Cl₂/AcOEt) fol-

lowed by trituration with cold n-hexane afforded the titled compound in

62% yield. TLC (AcOEt/n-hexane 1:1); R_f0.64; [R]_D²⁵= +40.6 (c = 2.0,

CH₂Cl₂); mp 95 ꢀC. ¹H NMR (CDCl₃) δ 1.31 (t, 3H, ꢀCH₃),

2.06ꢀ2.40 (m, 2H, ꢀCH₂-CH<), 2.73 (m, 2H, ꢀCH₂-C₆H₅), 4.22

(q, 2H, ꢀO-CH₂ꢀ), 4.82 (m, 1H, ꢀCH<), 5.50 (b, 2H, ꢀNH₂),

6.56ꢀ6.69 (m, 3H, >CH-NHꢀ and Ar), 7.18ꢀ7.33 (m, 7H, Ar). ¹³

C

NMR (CDCl₃) δ 14.37, 31.85, 34.24, 52.35, 61.70, 115.36, 116.64,

117.31, 126.24, 127.47, 128.47, 128.60, 132.62, 140.85, 148.87, 168.83,

172.49.

2-(R,S)-(2-Amino-phenylcarbamoyl)-4-phenyl-butyric Acid

Ethyl Ester (11d). To a solution of 11c (1.75 g, 7.41 mmol), TEA

(1.15 mL, 8.15 mmol), and TBTU (2.62 g, 8.15 mmol) in CH₂Cl₂, o-

phenylenediamine (3.06 g, 14.82 mmol) was added.³⁷The resulting

mixture was stirred at room temperature overnight and then concen-

trated in vacuo. The residue, dissolved in 100 mL of AcOEt, was washed

twice with 1N HCl, 1N NaOH, water, and brine, dried over anhydrous

Na₂SO₄, and rotary evaporated. Trituration with a solution CH₂Cl₂/

n-hexane 1:1 afforded a white solid in 79% yield. TLC (AcOEt/n-hexane

1:1); R_f0.39; mp 110 ꢀC. ¹H NMR (CDCl₃) δ 1.30 (t, 3H, ꢀCH₃), 2.32

(m, 2H, ꢀCH₂-CH<), 2.72 (m, 2H, ꢀCH₂-C₆H₅), 3.39 (m, 1H, ꢀCH<),

3.82 (b, 2H, ꢀNH₂), 4.21 (q, 2H, ꢀO-CH₂ꢀ), 6.74ꢀ7.33 (m, 9H, Ar),

8.30 (s, 1H, ꢀNHꢀ). ¹³C NMR (CDCl₃) δ 14.29, 32.34, 33.48, 52.60,

62.00, 117.84, 119.36, 123.69, 125.36, 126.36, 127.38, 128.60, 140.54,

140.76, 166.93, 171.97.

General Procedure for the Synthesis of Anthranoyl Deri-

vatives (1e, 3eꢀ7e, 9e). A suspension of 20.00 mmol of the amino

acid ethyl ester hydrochloride in 100 mL of AcOEt was treated with TEA

(2.78 mL, 20.00 mmol) followed by isatoic anhydride (3.26 g, 20.00

mmol). The resulting mixture was refluxed under stirring for 4 h, cooled

to room temperature, and filtered. The organic phase was washed with 1

N NaOH (3 ꢁ 50 mL), water (1 ꢁ 50 mL), and brine, dried over

(ꢀ)-2-(R)-(2-Amino-benzoylamino)-4-phenyl-butyric Acid Ethyl Es-

ter (6e). Purification by flash chromatography (CH₂Cl₂/AcOEt) fol-

lowed by trituration with cold n-hexane afforded the titled compound in

58% yield. TLC (AcOEt/n-hexane 1:1); R_f0.64; [R]_D²⁵= ꢀ40.8 (c =

1

2.0, CH₂Cl₂); mp 95 ꢀC. H NMR (CDCl₃) δ 1.29 (t, 3H, ꢀCH₃),

2.05ꢀ2.34 (m, 2H, ꢀCH₂-CH<), 2.72 (m, 2H, ꢀCH₂-C₆H₅), 4.21 (q,

2H, ꢀO-CH₂ꢀ), 4.81 (m, 1H, ꢀCH<), 5.50 (b, 2H, ꢀNH₂),

6.59ꢀ6.67 (m, 3H, >CH-NHꢀ and Ar), 7.17ꢀ7.32 (m, 7H, Ar). ¹³

C

NMR (CDCl₃) δ 14.38, 31.88, 34.21, 52.37, 61.69, 115.36, 116.61,

117.32, 126.25, 127.50, 128.48, 128.61, 132.61, 140.87, 148.90, 168.88,

172.51.

2-(R,S)-(2-Amino-benzoylamino)-4-phenyl-butyric Acid Ethyl Ester

(7e). Trituration with cold petroleum ether 40ꢀ60ꢀ followed by crys-

tallization from MeOH afforded the titled compound in 56% yield. TLC

(AcOEt/n-hexane 1:1); R_f0.64; mp 97 ꢀC. ¹H NMR (CDCl₃) δ 1.29 (t,

3H, ꢀCH₃), 2.05ꢀ2.38 (m, 2H, ꢀCH₂-CH<), 2.72 (m, 2H, ꢀCH₂-

C₆H₅), 4.21 (q, 2H, ꢀO-CH₂ꢀ), 4.81 (m, 1H, ꢀCH<), 5.14 (b, 2H,

ꢀNH₂), 6.59ꢀ6.67 (m, 3H, >CH-NHꢀ and Ar), 7.17ꢀ7.28 (m, 7H, Ar).

¹³C NMR (CDCl₃) δ 14.34, 31.84, 34.16, 52.35, 61.70, 115.37, 116.65,

117.35, 126.28, 127.54, 128.51, 128.65, 132.66, 140.90, 148.92, 168.95, 172.60.

2-(R,S)-Amino-N-(1-methyl-3-phenyl-propyl)-benzamide (9e). Pur-

ification by trituration with cold petroleum ether 40ꢀ60ꢀ afforded the

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dx.doi.org/10.1021/jm200438b |J. Med. Chem. 2011, 54, 5769–5785

Journal of Medicinal Chemistry

ARTICLE

titled compound in 62% yield. TLC (AcOEt/n-hexane 1:1); R_f0.71; mp

100 ꢀC. ¹H NMR (CDCl₃) δ 1.27 (d, 3H, ꢀCH₃), 1.87 (m, 2H, ꢀCH₂-

CH<), 2.72 (m, 2H, ꢀCH₂-C₆H₅), 4.23 (m, 1H, ꢀCH<), 5.81 (b, 2H,

ꢀNH₂), 5.90 (m, 1H, ꢀNHꢀ), 6.62ꢀ7.33 (m, 9H, Ar). ¹³C NMR

(CDCl₃) δ 21.14, 32.59, 38.69, 45.28, 116.54, 117.26, 125.95, 127.02,

128.38, 128.52, 132.12, 141.80, 148.66, 168.72.

afforded the titled compound in 82% yield. TLC (AcOEt/n-hexane 1:1); R_f

0.63; [R]_D²⁵= +54.5 (c = 0.51, CH₂Cl₂); mp 243ꢀ244 ꢀC. ¹H NMR

(DMSO-d₆) δ 1.19 (t, J = 6.8 Hz, 3H, ꢀCH₃); 2.15 (m, 2H, ꢀCH₂-

CH<), 2.75 (m, 2H, ꢀCH₂-C₆H₅), 4.13 (q, J = 6.8 Hz,2H, ꢀO-CH₂ꢀ),

4.45 (m, 1H, ꢀCH<), 7.03ꢀ7.97 (m, 13H, Ar), 8.64 (d, J = 8.1 Hz, 1H,

Ar), 9.25 (d, J = 6.6 Hz,1H, >CH-NHꢀ), 11.94 (s, 1H, ꢀNHꢀ), 12.18

(s, 1H, ꢀNHꢀ). ¹³C NMR (DMSO-d₆) δ 14.00, 31.53, 31.81, 52.27,

60.58, 102.42, 112.35, 119.59, 119.88, 120.05, 121.56, 122.49, 123.92,

125.81, 126.77, 128.16, 128.23, 128.59, 131.37, 132.43, 136.94, 138.87,

140.58, 158.94, 169.05, 171.53.

(ꢀ)-2-(R)-{2-[(1H-Indole-2-carbonyl)-amino]-benzoylamino}-4-phe-

nyl-butyric Acid Ethyl Ester (6f). Trituration with hot MeOH followed

by flash chromatography (from CH₂Cl₂to CH₂Cl₂/AcOEt 4:1) af-

forded the titled compound in 81% yield. TLC (AcOEt/n-hexane 1:1);

R_f0.63; [R]_D²⁵= ꢀ54.4 (c = 0.54, CH₂Cl₂); mp 243ꢀ244 ꢀC. ¹H NMR

(DMSO-d₆) δ 1.19 (t, J = 6.7 Hz, 3H, ꢀCH₃), 2.15 (m, 2H, ꢀCH₂-

CH<), 2.75 (m, 2H, ꢀCH₂-C₆H₅), 4.13 (q, J = 6.7 Hz, 2H, ꢀ

O-CH₂ꢀ), 4.45 (m, 1H, ꢀCH<), 7.03ꢀ7.97 (m, 13H, Ar), 8.64 (d,

J = 8.0 Hz, 1H, Ar), 9.25 (d, J = 6.5 Hz, 1H, >CH-NHꢀ), 11.93 (s, 1H,

ꢀNHꢀ), 12.18 (s, 1H, ꢀNHꢀ), ¹³C NMR (DMSO-d₆) δ 14.00, 31.53,

31.82, 52.27, 60.59, 102.42, 112.35, 119.59, 119.88, 120.06, 121.56,

122.50, 123.92, 125.81, 126.77, 128.17, 128.23, 128.59, 131.37, 132.44,

136.94, 138.87, 140.58, 158.94, 169.05, 171.53.

2-(R,S)-{2-[(1H-Indole-2-carbonyl)-amino]-benzoylamino}-4-phe-

nyl-butyric Acid Ethyl Ester (7). Trituration with hot MeOH afforded

the titled compound in 36% yield. TLC (AcOEt/n-hexane 1:1); R_f0.63;

mp 214 ꢀC. ¹H NMR (DMSO-d₆) δ 1.19 (t, J = 6.9 Hz, 3H, ꢀCH₃),

2.17 (m, 2H, ꢀCH₂-CH<), 2.75 (m, 2H, ꢀCH₂-C₆H₅), 4.14 (q, J = 6.9

Hz, 2H, ꢀO-CH₂ꢀ), 4.45 (m, 1H, ꢀCH<), 7.04ꢀ7.98 (m, 13H, Ar),

8.65 (d, J = 8.1 Hz, 1H, Ar), 9.27 (d, J = 6.7 Hz, 1H, >CH-NHꢀ), 11.96

(s, 1H, ꢀNHꢀ), 12.21 (s, 1H, ꢀNHꢀ). ¹³C NMR (DMSO-d₆) δ 13.91,

31.46, 31.74, 52.21, 60.53, 102.40, 112.34, 119.56, 119.87, 120.05,

121.56, 122.49, 123.91, 125.81, 126.77, 128.16, 128.23, 128.60, 131.38,

132.45, 136.96, 138.89, 140.59, 158.97, 169.10, 171.57. MS (ES) m/z 470.2

[MH]⁺; MW 469.53 (calcd for C₂₈H₂₇N₃O₄).

General Procedure for the Synthesis of Derivatives (1f,

3fꢀ6f, 7, 9, and 11e). To a suspension of 0.81 g (5.00 mmol) of

indole-2-carboxylic acid in 20 mL of acetyl chloride, 1.04 g (5.00 mmol)

of PCl₅was added in portions at 0 ꢀC over a period of 0.5 h. After the

mixture turned into a clear solution, stirring was continued at room

temperature for 3 h. The solution was concentrated under reduced

pressure, and the residue, taken up in 5 mL of dry CH₂Cl₂, was added

dropwise at 0 ꢀC to a solution of 4.00 mmol of the corresponding

derivatives (1e, 3eꢀ7e, 9e, and 11d) in 4 mL of pyridine. After the ad-

dition was completed, the reaction mixture was stirred at room temperature

overnight. Then 150 mL of CH₂Cl₂were added and the organic layer

washed twice with 40 mL of 1N HCl, 1N NaOH, H₂O, and brine. The

organic layer was dried over anhydrous Na₂SO₄and concentrated, and

the mixture was purified as described to yield the titled compounds.

Compound (1f) was collected by filtration from the reaction mixture.

(R,S)-{2-[(1H-Indole-2-carbonyl)-amino]-benzoylamino}-phenyl

Acetic Acid Ethyl Ester (1f). Trituration with cold petroleum ether

40ꢀ60ꢀ afforded the titled compound in 61% yield. TLC (AcOEt/

n-hexane 1:1); R_f0.72; mp 264ꢀ266 ꢀC. ¹H NMR (DMSO-d₆) δ 1.17

(t, J = 6.8 Hz, 3H, ꢀCH₃), 4.17 (m, 2H, ꢀO-CH₂ꢀ), 5.74 (d, J = 6.3 Hz,

1H, >CH-C₆H₅), 7.07ꢀ7.26 (m, 4H, Ar), 7.39ꢀ7.62 (m, 7H, Ar), 7.71

(m, 1H, Ar), 7.97 (d, J = 7.8 Hz, 1H, Ar), 8.62 (d, J = 8.1 Hz, 1H, Ar),

9.57 (d, J = 6.2 Hz, 1H, >CH-NHꢀ), 11.95 (s, 1H, ꢀNHꢀ), 12.15 (s,

1H, ꢀNHꢀ). ¹³C NMR (DMSO-d₆) δ 13.82, 57.01, 60.92, 102.47,

112.35, 119.43, 119.89, 120.05, 121.57, 122.40, 123.92, 126.78, 128.21,

128.37, 129.02, 131.39, 132.47, 135.32, 136.97, 138.87, 159.00, 168.73,

170.02.

2-(R,S)-{2-[(1H-Indole-2-carbonyl)-amino]-benzoylamino}-5-phe-

nyl-pentanoic Acid Ethyl Ester (3f). Trituration with hot MeOH af-

forded the titled compound in 28% yield. TLC (AcOEt/n-hexane 1:1);

R_f0.75; mp 173 ꢀC. ¹H NMR (DMSO-d₆) δ 1.19 (t, J = 7.1 Hz, 3H,

ꢀCH₃), 1.74ꢀ1.90 (m, 4H, ꢀ(CH₂)₂ꢀ), 2.63 (m, 2H, ꢀCH₂-C₆H₅),

4.15 (q, J = 7.0 Hz,2H, ꢀO-CH₂ꢀ), 4.45 (m, 1H, ꢀCH<), 7.06 (s, 1H,

Ar), 7.11ꢀ7.28 (m, 8H, Ar), 7.50 (d, J = 8.1 Hz, 1H, Ar), 7.63ꢀ7.71 (m,

2H, Ar), 7.94 (d, J = 7.8 Hz, 1H, Ar), 8.65 (d, J = 8.1 Hz, 1H, Ar), 9.20 (d,

J = 6.7 Hz,1H, >CH-NHꢀ), 11.96 (s, 1H, ꢀNHꢀ), 12.27 (s, 1H,

ꢀNHꢀ). ¹³C NMR (DMSO-d₆) δ 13.93, 27.35, 29.68, 34.43, 52.71,

60.44, 102.39, 112.33, 119.38, 119.84, 120.03, 121.58, 122.44, 123.90,

125.53, 126.77, 128.05, 128.57, 131.39, 132.42, 136.96, 138.94, 141.47,

158.97, 168.97, 171.59.

(R,S)-1H-Indole-2-carboxylic Acid [2-(1-Methyl-3-phenyl-propylcar-

bamoyl)-phenyl]-amide (9). Trituration with hot CH₂Cl₂followed by

crystallization from EtOH afforded titled compound in 50% yield. TLC

1

(AcOEt/n-hexane 1:1); R_f0.68; mp 242 ꢀC. H NMR (DMSO-d₆)

δ 1.21 (d, J = 6.2 Hz, 3H, ꢀCH₃), 1.87 (m, 2H, ꢀCH₂-CH<), 2.64 (t,

J = 6.4 Hz, 2H, ꢀCH₂-C₆H₅), 4.14 (m, 1H, ꢀCH<), 7.06ꢀ7.89 (m,

13H, Ar), 8.62ꢀ8.73 (m, 2H, >CH-NHꢀ and Ar), 11.93 (s, 1H,

ꢀNHꢀ), 12.61 (s, 1H, ꢀNHꢀ). ¹³C NMR (DMSO-d₆) δ 20.42,

31.87, 37.20, 44.62, 102.35, 112.33, 119.81, 120.02, 120.16, 121.62,

122.35, 123.85, 125.47, 126.84, 128.03, 128.08, 128.17, 131.55, 131.95,

136.94, 139.00, 141.57, 159.00, 167.85. MS (ES) m/z 412.2 [MH]⁺;

MW 411.50 (calcd for C₂₆H₂₅N₃O₂).

2-(R,S)-{2-[(1H-Indole-2-carbonyl)-amino]-benzoylamino}-6-phenyl-

hexanoic Acid Ethyl Ester (4f). Trituration with hot MeOH followed

by flash chromatography (from CH₂Cl₂/petroleum ether 40ꢀ60ꢀ 1:1

to CH₂Cl₂to CH₂Cl₂/AcOEt 20:1) afforded the titled compound in

91% yield. TLC (AcOEt/n-hexane 1:1); R_f0.69; mp 167ꢀ168 ꢀC. ¹H

NMR (DMSO-d₆) δ 1.17 (t, J = 7.1 Hz, 3H, ꢀCH₃), 1.44 (m, 2H,

ꢀCH₂-CH₂ꢀCH<), 1.61 (m, 2H, ꢀCH₂-CH₂-C₆H₅), 1.86 (m, 2H,

ꢀCH₂-CH<), 2.56 (m, 2H, ꢀCH₂-C₆H₅), 4.11 (q, J = 7.1 Hz, 2H, ꢀ

O-CH₂ꢀ), 4.47 (m, 1H, ꢀCH<), 7.03ꢀ7.30 (m, 9H, Ar), 7.47 (m, 1H,

Ar), 7.57ꢀ7.70 (m, 2H, Ar), 7.88 (d, J = 7.6 Hz,1H, Ar), 8.62 (d, J =

8.1 Hz, 1H, Ar), 9.09 (m, 1H, >CH-NHꢀ), 11.91 (s, 1H, ꢀNHꢀ),

12.20 (s, 1H, ꢀNHꢀ). ¹³C NMR (DMSO-d₆) δ 14.06, 25.27, 29.98,

30.40, 34.86, 52.81, 60.54, 102.46, 112.40, 119.49, 119.91, 120.11, 121.66,

122.51, 123.98, 125.48, 126.82, 128.06, 128.09, 128.62, 131.42, 132.49,

136.99, 138.84, 141.87, 159.00, 168.97, 171.69.

2-(R,S)-{2-[(1H-Indole-2-carbonyl)-amino]-phenylcarbamoyl}-4-phe-

nyl-butyric Acid Ethyl Ester (11e). Trituration with hot MeOH followed

by flash chromatography (from CH₂Cl₂to CH₂Cl₂/AcOEt 4:1) af-

forded titled compound in 71% yield. TLC (AcOEt/n-hexane 1:1); R_f

1

0.53; mp 191 ꢀC. H NMR (DMSO-d₆) δ 1.05 (t, J = 6.8 Hz, 3H,

ꢀCH₃), 2.14 (m, 2H, ꢀCH₂-CH<), 2.54 (m, 2H, ꢀCH₂-C₆H₅), 3.60

(m, 1H, ꢀCH<), 4.02 (m, 2H, ꢀO-CH₂ꢀ), 7.05ꢀ7.77 (m, 14H, Ar),

9.78 (s, 1H, ꢀNHꢀ), 10.15 (s, 1H, ꢀNHꢀ), 11.84 (s, 1H, ꢀNHꢀ),

¹³C NMR (DMSO-d₆) δ 14.62, 31.21, 33.46, 52.64, 61.62, 104.02,

113.17, 120.75, 122.37, 124.58, 125.90, 126.13, 126.29, 126.61, 127.70,

128.90, 129.00, 131.09, 131.47, 131.83, 137.61, 141.53, 160.26, 168.44,

170.12.

General Procedure for the Synthesis of Compounds (1ꢀ4,

11). A mixture of the corresponding ethyl ester 1f, 3fꢀ6f, 7, and 11e

(4.0 mmol) and KOH (0.22 g, 4.00 mmol) in water (25 mL) and THF

(25 mL) was stirred at room temperature until hydrolysis completion

(TLC monitoring). After organic solvent removal, the aqueous solution

(+)-2-(S)-{2-[(1H-Indole-2-carbonyl)-amino]-benzoylamino}-4-phen-

yl-butyric Acid Ethyl Ester (5f). Trituration with hot MeOH followed

by flash chromatography (from CH₂Cl₂to CH₂Cl₂/AcOEt 4:1)

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was adjusted to pH 2ꢀ3 with diluted HCl at 0 ꢀC, yielding precipitation

of the titled compound.

in 50 mL of a solution of H₂O/THF 1:1, 25 mg (0.60 mmol) of

LiOH H₂O were added. After stirring 24 h at room temperature, the

3

(R,S)-{2-[(1H-Indole-2-carbonyl)-amino]-benzoylamino}-phenyl

Acetic Acid (1). Trituration with hot MeOH afforded the titled com-

pound in 77% yield. TLC (AcOEt/MeOH 3:1); R_f0.34; mp 290ꢀ

organic solvent was evaporated and 30 mL of NaHCO₃satd and AcOEt

were added. The mixture was then vigorously stirred; the salt formed was

filtrated and taken up in THF.

1

291 ꢀC. H NMR (DMSO-d₆) δ 5.64 (d, J = 5.4 Hz, 1H, ꢀCH<),

The pH of the solution was adjusted to 2ꢀ3 with 1N HCl, the organic

solvent evaporated under reduced pressure, and the white solid collected

by ﬁltration. Trituration with hot AcOEt and then with abs EtOH af-

forded the titled compounds.

7.07ꢀ7.40 (m, 7H, Ar), 7.48ꢀ7.64 (m, 4H, Ar), 7.73 (m, 1H, Ar), 8.00

(d, J = 7.6 Hz, 1H, Ar), 8.64 (d, J = 8.1 Hz, 1H, Ar), 9.35 (d, J = 6.4 Hz,

1H, >CH-NHꢀ), 11.96 (s, 1H, ꢀNHꢀ), 12.29 (s, 1H, ꢀNHꢀ), 12.80

(b, 1H, ꢀOH). ¹³C NMR (DMSO-d₆) δ 57.09, 102.53, 112.33, 119.60,

119.91, 120.01, 121.68, 122.46, 123.89, 126.80, 127.57, 127.91, 128.13,

128.82, 131.42, 132.35, 136.96, 137.04, 138.96, 159.04, 168.19, 171.29.

MS (ES) m/z 414.2 [MH]⁺; MW 413.43 (calcd for C₂₄H₁₉N₃O₄).

2-(R,S)-{2-[(1H-Indole-2-carbonyl) -amino]-benzoylamino}-4-phe-

nyl-butyric Acid (2). The target compound was obtained by hydrolysis

of 7. Trituration with hot MeOH afforded the titled compound in 75%

yield. TLC (AcOEt/MeOH 2:1); R_f0.48; mp 259ꢀ260 ꢀC. ¹H NMR

(DMSO-d₆) δ 2.17 (m, 2H, ꢀCH₂-CH<), 2.75 (m, 2H, ꢀCH₂-C₆H₅),

4.42 (m, 1H, ꢀCH<), 7.07ꢀ8.01 (m, 13H, Ar), 8.68 (d, J = 8.1 Hz, 1H,

Ar), 9.16 (d, J = 7.0 Hz, 1H, >CH-NHꢀ), 11.98 (s, 1H, ꢀNHꢀ), 12.39

(s, 1H, ꢀNHꢀ), 12.83 (b, 1H, ꢀOH). ¹³C NMR (DMSO-d₆) δ 31.65,

31.88, 52.05, 102.41, 112.33, 119.50, 119.80, 120.03, 121.65, 122.43,

123.89, 125.74, 126.79, 128.13, 128.23, 128.55, 131.42, 132.36, 136.95,

139.03, 140.76, 158.99, 168.89, 173.16. MS (ES) m/z 442.2 [MH]⁺;

MW 441.48 (calcd for C₂₆H₂₃N₃O₄)

(ꢀ)-2-(S)-{2-[(1H-Indole-2-carbonyl)-amino]-benzoylamino}-4-phe-

nyl-butyric Acid (5). The product was obtained in 66% yield. TLC

(AcOEt/MeOH 2:1); R_f0.48; [R]_D²⁵= ꢀ34.8 (c = 0.65, DMF); ee >

99.9%, mp 267ꢀ268 ꢀC. ¹H NMR (DMSO-d₆) δ 2.16 (m, 2H, ꢀCH₂-

CH<), 2.74 (m, 2H, ꢀCH₂-C₆H₅), 4.42 (m, 1H, ꢀCH<), 7.05ꢀ8.00

(m, 13H, Ar), 8.66 (d, J = 8.0 Hz, 1H, Ar), 9.14 (d, J = 7.1 Hz, 1H, >CH-

NHꢀ), 11.94 (s, 1H, ꢀNHꢀ), 12.35 (s, 1H, ꢀNHꢀ), 12.83 (b, 1H,

ꢀOH). ¹³C NMR (DMSO-d₆) δ 31.77, 31.94, 52.05, 102.48, 112.42,

119.52, 119.89, 120.14, 121.74, 122.55, 124.01, 125.85, 126.86, 128.24,

128.32, 128.66, 131.47, 132.50, 137.01, 139.08, 140.77, 159.04, 168.99,

173.18. MS (ES) m/z 442.2 [MH]⁺; MW 441.48 (calcd for C₂₆H₂₃N₃O₄).

(+)-2-(R)-{2-[(1H-Indole-2-carbonyl)-amino]-benzoylamino}-4-

phenyl-butyric Acid (6). The product was obtained in 60% yield. TLC

(AcOEt/MeOH 2:1); R_f0.48; [R]_D²⁵= +35.4 (c = 0.65, DMF); ee >

99.7%, mp 267ꢀ268 ꢀC. ¹H NMR (DMSO-d₆) δ 2.16 (m, 2H, ꢀCH₂-

CH<), 2.74 (m, 2H, ꢀCH₂-C₆H₅), 4.42 (m, 1H, ꢀCH<), 6.95 (s, 1H,

Ar), 7.06ꢀ7.83 (m, 12H, Ar), 8.63 (d, J = 8.1 Hz, 1H, Ar), 9.17 (d, J = 7.0

Hz, 1H, >CH-NHꢀ), 11.92 (s, 1H, ꢀNHꢀ), 12.25 (s, 1H, ꢀNHꢀ),

12.81 (b, 1H, ꢀOH). ¹³C NMR (DMSO-d₆) δ 31.74, 31.93, 52.04,

102.44, 112.38, 119.48, 119.85, 120.09, 121.71, 122.50, 123.95, 125.81,

126.82, 128.19, 128.29, 128.61, 131.44, 132.45, 136.97, 139.05, 140.74,

159.00, 168.94, 173.13. MS (ES) m/z 442.2 [MH]⁺; MW 441.48 (calcd

for C₂₆H₂₃N₃O₄).

(R,S)-1H-Indol-2-carboxylic Acid 2-(1-Hydroxycarbamoyl-

3-phenyl-propylcarbamoyl)-phenyl-amide (10). Ethyl ester 7

(0.30 g, 0.64 mmol) was dissolved in MeOH (15 mL). To the solution

hydroxylamine hydrochloride (0.09 g, 1.3 mmol) was added, followed by

MeONa, freshly prepared from sodium (0.10 g, 3.19 mmol) dissolved in

MeOH (2 mL). The reaction mixture was stirred for 36 h at room

temperature and then worked up by partitioning between diluted hy-

drochloric acid and AcOEt. The aqueous phase was extracted with

AcOEt, the combined organic layers were dried on anhydrous Na₂SO₄,

and the solvent was evaporated.³⁸The product was crystallized from

diethyl ether/MeOH and collected by filtration (0.14 g, 48% yield).

TLC (AcOEt); R_f0.66; mp 218ꢀ219 ꢀC (dec). ¹H NMR (DMSO-d₆)

δ 2.07 (m, 2H, ꢀCH₂-CH<), 2.62 (m, 2H, ꢀCH₂-C₆H₅), 4.41 (m, 1H,

ꢀCH<), 7.04ꢀ7.26 (m, 9H, Ar), 7.46 (d, J = 8.2 Hz, 1H, Ar), 7.58 (m,

1H, Ar), 7.68 (d, J = 7.9 Hz, 1H, Ar), 7.98 (d, J = 7.7 Hz, 1H, Ar), 8.60 (d,

J = 8.2 Hz, 1H, Ar), 8.90, 9.35 (2 s, 1H, ꢀOH), 9.01 (d, J = 7.8 Hz, 1H,

>CH-NHꢀ), 10.86, 11.17 (2 s, 1H, ꢀNH-OH), 11.91 (s, 1H, ꢀNH),

12.34, 12.36 (2 s, 1H, ꢀNH) (doubling of resonance is due to Z- and E-

amide conformers). ¹³C NMR (DMSO-d₆) δ 31.69, 33.06, 51.09, 102.52,

112.39, 119.72, 119.84, 120.07, 121.63, 122.41, 123.93, 125.71, 126.81,

128.16, 128.82, 131.52, 132.29, 136.97, 139.03, 140.90, 159.02, 167.86,

168.62. MS (ES) m/z 457.3 [MH]⁺; MW 456.49 (calcd for C₂₆H₂₄N₄O₄).

(R,S)-1H-Indole-2-carboxylic Acid [2-(1-Carbamoyl-3-phe-

nyl-propylcarbamoyl)-phenyl]-amide (8). To a solution of 2

(0.88 g, 2.00 mmol), TEA (1.10 mL, 7.90 mmol), and PyBOP (1.54 g,

3.00 mmol) in 150 mL of DMF, 0.21 g (4.00 mmol) of NH₄Cl were

added. After overnight mixture stirring, DMF was evaporated in vacuo

and the residue dissolved in 50 mL of AcOEt. The organic layer was

washed twice with 50 mL of 1N HCl, 1N NaOH, water, and brine and

then dried over anhydrous Na₂SO₄.³⁹After solvent removal, the resulting

residue was purified by flash chromatography (CH₂Cl₂/AcOEt 3:2)

and triturated with n-hexane to give the titled compound in 50%

2-(R,S)-{2-[(1H-Indole-2-carbonyl)-amino]-benzoylamino}-5-phe-

nyl-pentanoic Acid (3). Trituration with hot MeOH afforded the titled

compound in 50% yield. TLC (AcOEt/MeOH 2:1); R_f0.54; mp 256 ꢀC.

¹H NMR (DMSO-d₆) δ 1.72ꢀ1.91 (m, 4H, ꢀ(CH₂)₂ꢀ), 2.63 (t, J =

7.0 Hz, 2H, ꢀCH₂-C₆H₅), 4.53 (m, 1H, ꢀCH<), 7.08 (s, 1H, Ar),

7.10ꢀ7.29 (m, 8H, Ar), 7.50 (d, J = 8.1 Hz, 1H, Ar), 7.62 (t, J = 7.7 Hz,

1H, Ar), 7.74 (d, J = 7.9 Hz, 1H, Ar), 7.97 (d, J = 7.6 Hz, 1H, Ar), 8.68 (d,

J = 8.3 Hz, 1H, Ar), 9.10 (d, J = 7.3 Hz, 1H, >CH-NHꢀ), 11.96 (s, 1H,

ꢀNHꢀ), 12.43 (s, 1H, ꢀNHꢀ). 12.82 (s, 1H, ꢀOH). ¹³C NMR (DM-

SO-d₆) δ 27.54, 29.81, 34.47, 52.43, 102.39, 112.32, 119.27, 119.80, 120.01,

121.68, 122.38, 123.89, 125.51, 126.80, 128.05, 128.55, 131.43, 132.38,

136.95, 139.09, 141.54, 159.00, 168.81, 173.12. MS (ES) m/z 456.2

[MH]⁺; MW 455.51 (calcd for C₂₇H₂₅N₃O₄).

2-(R,S)-{2-[(1H-Indole-2-carbonyl)-amino]-benzoylamino}-6-phe-

nyl-hexanoic Acid (4). Trituration with hot MeOH afforded the titled

compound in 74% yield. TLC (AcOEt/MeOH 1:1); R_f0.86; mp

237ꢀ238 ꢀC. ¹H NMR (DMSO-d₆) δ 1.45 (m, 2H, ꢀCH₂-CH₂-CH<),

1.58 (m, 2H, ꢀCH₂-CH₂-C₆H₅), 1.87 (m, 2H, ꢀCH₂-CH<), 2.59 (m,

2H, ꢀCH₂-C₆H₅), 4.47 (m, 1H, ꢀCH<), 7.06ꢀ7.30 (m, 9H, Ar),

7.46ꢀ7.74 (m, 3H, Ar), 7.92 (d, J = 7.9 Hz, 1H, Ar), 8.65 (d, J = 8.3 Hz,

1H, Ar), 9.05 (d, J = 7.3 Hz, 1H, >CH-NHꢀ), 11.86 (s, 1H, ꢀNHꢀ),

12.41 (s, 1H, ꢀNHꢀ), 12.81 (b, 1H, ꢀOH). ¹³C NMR (DMSO-d₆)

δ 25.46, 30.10, 30.47, 34.90, 52.55, 102.45, 112.40, 119.38, 119.85,

120.08, 121.76, 122.46, 123.97, 125.46, 126.85, 128.06, 128.09, 128.61,

131.48, 132.46, 137.00, 139.11, 141.93, 159.04, 168.80, 173.23. MS (ES)

m/z 470.2 [MH]⁺; MW 469.53 (calcd for C₂₈H₂₇N₃O₄).

2-(R,S)-{2-[(1H-Indole-2-carbonyl)-amino]-phenylcarbamoyl}-4-phe-

nyl-butyric Acid (11). Trituration with hot MeOH followed by AcOEt

afforded the titled compound in 26% yield. TLC (AcOEt/MeOH 2:1);

R_f0.57; mp 196 ꢀC. ¹H NMR (DMSO-d₆) δ 2.11 (m, 2H, ꢀCH₂-CH<),

2.55 (m, 2H, ꢀCH₂-C₆H₅), 3.50 (m, 1H, ꢀCH<), 7.03ꢀ7.81 (m, 14H,

Ar), 9.79 (s, 1H, ꢀNHꢀ), 10.15 (s, 1H, ꢀNHꢀ), 11.85 (s, 1H,

ꢀNHꢀ), 12.90 (b, 1H, ꢀOH). ¹³C NMR (DMSO-d₆) δ 31.31, 33.59,

52.79, 104.10, 113.14, 120.67, 122.51, 124.55, 125.78, 126.02, 126.54,

127.75, 128.88, 128.96, 131.03, 131.43, 131.83, 137.59, 141.67, 160.27,

169.08, 171.83. MS (ES) m/z 442.2 [MH]⁺; MW 441.48 (calcd for

C₂₆H₂₃N₃O₄).

General Procedure for the Synthesis of Compounds (5, 6).

To 0.60 mmol of the corresponding ethyl ester (compounds 5f and 6f)

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yield. TLC (AcOEt/MeOH 1:1); R_f0.40; mp 295ꢀ296 ꢀC. ¹H NMR

(DMSO-d₆) δ 2.09 (m, 2H, ꢀCH₂-CH<), 2.63 (m, 2H, ꢀCH₂-C₆H₅),

4.43 (m, 1H, ꢀCH<), 7.06ꢀ7.14 (m, 8H, Ar), 7.21ꢀ7.73 (m, 4H, Ar),

7.99 (d, J = 7.8 Hz, 1H, Ar), 8.61 (d, J = 8.3 Hz, 1H, Ar), 8.92 (d, J = 7.8

Hz, 1H, >CH-NHꢀ), 11.91 (s, 1H, ꢀNHꢀ), 12.38 (s, 1H, ꢀNHꢀ).

¹³C NMR (DMSO-d₆) δ 31.84, 32.91, 53.052 102.45, 112.34, 119.86,

120.02, 121.62, 122.41, 123.87, 125.62, 126.80, 128.09, 128.70, 131.48,

132.15, 136.91, 138.92, 141.06, 158.99, 168.61, 173.04. MS (ES) m/z

441.0 [MH]⁺; MW 440.49 (calcd for C₂₆H₂₄N₄O₃).

Molecular Modeling and Docking. The geometries of all the

ligands were optimized at different levels of theory, including MM cal-

culations carried out with the AMBER 8 force field 99, and AM1 and

DFT calculations carried out with the parallel version of the Gaussian 03

suite⁴⁰running on an Intel Core 2 CPU 6600@2.40 GHz, 3.50 GB

RAM. All the ab initio and semiempirical calculations on ligands were

carried using the Berny algorithm for full optimizations with the default

Gaussian SCF and convergence criteria.

means of molecular dynamics simulations in an explicit lipid bilayer. The

simulation was carried out at neutral pH, and the positive charges inside

the receptor structure were neutralized by the addition of 15 chloride

ions. The receptor model was placed in a rectangular box (78 ꢁ 80 ꢁ 78

in size) containing a lipid bilayer (153 molecules of palmitoyl-oleoyl-

phosphatidylcholine and 8708 molecules of water in addition to the re-

ceptor structure), resulting in a final density of 1.0 g/cm³. The initial

steric clashes were eliminated by means of 1000 steps of steepest descent

energy minimization, followed by 2000 steps of conjugate gradient energy

minimization of all the system with the exception of CR carbons of

helices I, II, and III that were harmonically restrained to their initial

positions. Molecular dynamic (MD) simulations were then run at 300 K

and constant pressure (1 atm) with anisotropic scaling were then run for

1 ns using the SANDER module in AMBER 8. The conditions of the

MDs and the simulation protocol were the same as previously described.⁴⁹

The resulting complexes were then studied by MDs in an explicit lipid

bilayer under the same conditions as those described above.

Conformational analysis: our previous work on similar molecules

ensured us that, for anthranilic acid derivatives, a Monte Carlo search

carried out with the AM1 semiempirical Hamiltonian⁴¹yields results

comparable with those obtained at higher ab initio levels.^16,20All the

optimizations and searches were thus obtained from AM1 calculations. The

only exception is represented by the hydroxamic derivative 10 (Table 4):

the AM1 method is in fact unable to model the structural features of the

hydroxamic group, and the optimizations were carried out using the

multilayer ONIOM method⁴²which has proven by Remko to peform

well with hydroxamic acids.²⁷According to this protocol, the high layer

was represented by the hydroxamic group and by carbon 16 (Table 4),

and its geometry was optimized with a DFT calculation at the B3LYP/

6.311+G (d,p)^43ꢀ45level, while the low layer, containing all the re-

maining atoms of the molecule, was optimized at the AM1 level. All the

molecules containing a carboxylic group at carbon 16 were considered as

carboxylate anions, while the hydroxamic acid derivatives were kept in

their neutral protonated form, because the pK_aexperimental values for

such acids are found in the 8.5ꢀ8.7 range.²⁸

Biological Evaluation. Male Hartley guinea pigs (300ꢀ350 g) and

male SpragueꢀDawley rats (250ꢀ300 g) were used. For binding assays

to isolated rat pancreatic acini, animals were fasted, but allowed free

access to water, for 18ꢀ24 h prior to the experiment.

[

¹²⁵I]-BHꢀCCK-8 (CCK₈(sulphated), [¹²⁵I]Bolton and Hunter

labeled-speciﬁc activity 2000 Ci/mol) was purchased from Amersham

Pharmacia Biothech (Buckinghamshire, UK). All other drugs and reagents

were obtained from commercial sources.

Cumulative curves analysis (log nM or log μM concentration of test

compound vs % speciﬁc residual binding B/Bo) was performed by using

the Allﬁt program, which calculates lower and upper plateau, slope, and

antagonist IC₅₀.⁵⁰

[

¹²⁵I]-BH-CCK-8 Receptor Binding Assay in Isolated Rat

Pancreatic Acinar Cells. Isolated pancreatic acini were prepared by

enzymatic digestion of pancreas as previously described by Makovec

et al.⁵¹Drug competition experiments were carried out incubating acinar

cells, [¹²⁵I]BHꢀCCK-8 (25 pM final concentration), and competitors

in 0.5 mL total volume at 37 ꢀC for 30 min, in shaking bath. At the end of

incubation 1 mL of ice-cold HepesꢀRinger buffer (10 mM Hepes,

118 mM NaCl, 1.13 mM MgCl₂, 1.28 CaCl₂, 1% BSA, 0.2 mg/mL

Soybean trypsin inhibitor, pH 7.4) was added and the tubes were

centrifuged 5 min at 12500g. The supernatant was aspirated and the

radioactivity associated to the pellet measured. The nonspecific binding

was estimated in the presence of 1 μM CCK-8, accounting 15% of total

binding.

The molecular dynamics runs were carried out with the SANDER

module of AMBER 8 and the Sybyl7.3 suite running on SGI Octane2-

MIPS R12000 workstations or on a RedHat linux Intel Pentium 4 2.53

GHz machine. After relaxing the complexes, the structures were allowed

to reach thermal equilibrium at 300 K by a multistep molecular dynamic

run in the NTV ensemble (5000 fs at 100 K and 200 K and 5000 fs at

300 K). The behavior of the two ligands was analyzed at 300 K, after a

slow equilibration, by collecting dynamics in the NTV ensemble for 1 μs

at 0.2 fs steps. The Tripos forceﬁeld was used to run such simulations.⁴⁶

Docking of Compound 2 inside the Homology Model of

CCK₁R. Because antagonists are able to bind both the inactive and the

active conformations of the receptor, we have selected the solutions that

satisfy this assumption and thus performed docking on both the inactive

and active conformations of CCK₁R. A previously described model of

CCK1R*²⁹was used as starting point to dock compound 2. The starting

three-dimensional conformation of the ligands was obtained using Corina

software.⁴⁷Electrostatic potential derived charges were obtained with

the restrained electric potential methodology⁴⁸using a 6-31G* basis set

as implemented in the Gaussian 98 program. The Lamarckian genetic

algorithm77 implemented in Auto-Dock 3.0 was used to generate

docked conformations of 2 in a putative pocket within the transmem-

brane helices of CCK₁R by randomly changing the overall orientation of

the molecules as well as torsion angles. Default settings were used, except

for the number of runs, population size, and maximum number of energy

evaluations, which were fixed at 100, 100, and 250000, respectively.

Rapid intra- and intermolecular energy evaluation of each configuration

was achieved by having the receptor atomic affinity potentials for carbon,

oxygen, and hydrogen atoms precalculated in a three-dimensional grid

with a spacing of 0.375 Å. The selected docking solutions were refined by

[

¹²⁵I]-BH-CCK-8 Receptor Binding Assay in Guinea Pig

Cerebral Cortices. Membranes from guinea pig cerebral cortices

were prepared as previously described.⁵¹Protein concentration was

determined according to Bradford, using bovine serum albumin (BSA)

as standard.⁵²

The binding experiments were performed in assay buﬀer containing

10 mM Hepes, 118 mM NaCl, 4.7 mM KCl, 5.0 mM MgCl₂, 1.0 mM

EGTA, pH 6.5, and supplemented with 0.2 mg/mL bacitracin. The

incubation of membranes suspension with labeled ligand and inhibitors

was carried out in a microtiter 96-well ﬁlter plate (Multiscreen, Millipore

Inc., Bedford, MA) with integral Whatman GF/B membrane ﬁlters.

Aliquot of membranes (0.5 mg of protein/ml) were added to each well,

containing [¹²⁵I]BHꢀCCK-8 (25 pM), in a ﬁnal volume of 250 μL. The

nonspeciﬁc binding of iodinated peptide was deﬁned in the presence of

1 μM CCK-8, accounting of 20% of total binding. Nonspeciﬁc binding of

[

¹²⁵I]BH-CCK-8 to membrane ﬁlters (blank), measured in wells con-

taining an equal amount of labeled ligand, but no membranes, was 0.2%

of total radioligand added. After 120 min at 25 ꢀC, the 96-well plate was

placed on the vacuum ﬁltration apparatus (Millipore Inc.). The integral

membrane ﬁlters were rinsed with 0.25 mL of ice cold assay buﬀer, dried,

punched into polycarbonate tubes, and counted in a COBRA-5002

γ-counter (Canberra Packard) with 85% eﬃciency.

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Functional Studies. Isolated Muscular Strips of Guinea Pig

Gallbladder Stimulated by CCK-8. The assay was based on the method

described by Bishop et al.⁵³Male Hartley guinea pigs weighing 400ꢀ450 g

(Charles River, Calco, Italy) were sacrificed by cervical dislocation and

exsanguination after slight ethereal anesthesia. The abdomen was opened,

and the gallbladder was removed and transferred to a Petri dish con-

taining gassed KrebsꢀHenseleit solution of the following composition

(mM): NaCl 118.9, KCl 4.69, CaCl₂.2H₂O 3.33, KH₂PO₄1.18,

MgSO₄.7H₂O 1.2, NaHCO₃25, glucose 11.1. The neck and the fundus

of the gallbladder was cut away; then the tissue was cut transversally,

obtaining two rings, each about 3 mm high. From these rings two strips

of about 3 mm ꢁ 15 mm were obtained, containing mainly the circular

smooth muscle fibres. The strips were suspended with silk thread in

20 mL organ baths containing KrebsꢀHenseleit solution, maintained at

32 ꢀC, bubbled with 95% O₂ꢀ5% CO₂. Contractions were measured

with an isometric transducer (model 7003; Basile U, Comerio, Italy). A

resting tension of 0.5 g was applied. The isometric contraction was

monitored using a pen recorder (model 7070; Basile U, Comerio, Italy).

The strips were washed with fresh buffer every 15 min over a 60 min

equilibration period.

Submaximal-eﬀect (70ꢀ80%) concentration of CCK-8 was chosen

to test the inhibitory eﬀects of the substances under study. Various con-

centrations of the antagonists or vehicles were added and allowed 5 min

contact with the tissues before adding the agonist to the bathing ﬂuid.

The antagonist activity was expressed as percentage of inhibition of the

agonist contractions. The regression line was calculated and the con-

centration eﬀective in inhibiting by 50% the eﬀect of the agonist (IC₅₀)

were obtained from the regression line by using Allﬁt program.

In Vivo Guinea Pig Gallbladder Contractions Induced by CCK-8. The

assay was based on the method described by Chang and Lotti.⁵⁴Male

Hartley guinea pigs weighting 400ꢀ 450 g (Charles River, Calco, Italy)

fasted for 48 h were anesthetized with urethane 1.5 g/kg ip (6 mL/kg

of 25% w/v solution in NaCl 0.9%). The external jugular vein was

exposed and cannulated with a PE 50 polyethylene tubing, filled with

saline for iv drug administration. The trachea was exposed and cannulated

with a PE 240 polyethylene tubing to facilitate breathing. Then 10 ng/kg

(0.0087 nmol/kg) of CCK-8 was administered iv to induce gallbladder

emptying. A midline incision 2 cm long was made in the superior part of

the abdomen, the gallbladder was gently separated from the adhering

liver tissue and was lifted cautiously, and the fundus was tied with a silk

thread. The thread was passed in a caudal direction at about 45ꢀ over a

freely movable pulley and was connected to an isotonic transducer

(model 7006; Basile U, Comerio, Italy). To minimize the possible inter-

ference of the respiratory movements, the abdomen was kept open using

an adjustable diverter; the peritoneal cavity and the gallbladder were

covered with paraffin oil. A resting tension of 1 g was applied and the

gallbladder contractions were recorded using a pen recorder (model

Unirecord 7050; Basile U, Comerio, Italy). An equilibration period of

15 min was allowed before the administration of the substances. CCK-8

10 ng/kg caused contractions that developed slowly and reached their

peak after 2 min. The relaxation was slowed, and a 30 min delay period

was allowed to recover the basal tension. CCK-8 was administered

repeatedly until reproducible contractions were obtained.

France) using the human CCK₁R cDNAs cloned into pRFENeo vector

as template. The presence of the desired and the absence of undesired

mutations were confirmed by automated sequencing of both cDNA strands

(Applied Biosystem).

COS-7 cells (1.5 ꢁ 10⁶) were plated onto 10 cm culture dishes and

grown in Dulbecco’s Modiﬁed Eagle’s Medium containing 5% fetal calf

serum (complete medium) in a 5% CO₂atmosphere at 37 ꢀC. After

overnight incubation, cells were transfected with 2 μg/plate of pRFE-

Neo vectors containing the cDNA for the wild-type or mutated CCK

receptors, using a modiﬁed DEAE-dextran method. Cells were trans-

ferred to 24-well plates at a density of 20000ꢀ80000 cells/well 24 h after

transfection, depending on transfected mutant and experiment to be

performed.

Receptor Binding Assay. Approximately 24 h after the transfer of

transfected cells to 24-well plates, the cells were washed with phosphate-

buffered saline pH 6.95, 0.1% BSA, and then incubated for 60 min at

37 ꢀC in 0.5 mL of Dublecco’s Modified Eagle’s Medium, 0.1% BSA with

either 71 pM ¹²⁵I-BH-(Thr, Nle)CCK-9,⁵⁵or 1.83 nM [³H]SR-27,897

(tritiated 1-[2-(4-(2-chlorophenyl)thiazol-2-yl)aminocarbonyl-indoyl]ace-

tic acid, specific activity 31 Ci/mmol, Sanofi-Synthelabo, Toulouse, France)

in the presence or in the absence of competing agonists or antagonists.

The cells were washed twice with cold phosphate-buffered saline pH

6.95 containing 2% BSA, and cell associated radioligand was collected

with NaOH 0.1N added to each well. The radioactivity was directly

counted in a γ counter (Auto-Gamma, Packard, Downers Grove, IL) or

added to scintillant and counted for the tritiated radioligand.

Inositol Phosphate Assay. Approximately 24 h after the transfer

to 24-well plates and following overnight incubation in complete medium

containing 2 μCi/ml of myo-2-[³H]inositol (Amersham biosciences,

Les Ulis, France), the transfected cells were washed with Dubecco’s

Modified Eagle’s Medium and then incubated for 30 min in 1 mL/well

Dubecco’s Modified Eagle’s Medium containing 20 mM LiCl at 37 ꢀC.

The cells were washed with PI buffer at pH 7.45: phosphate-buffered

saline containing 135 mM NaCl, 20 mM HEPES, 2 mM CaCl₂, 1.2 mM

MgSO₄, 1 mM EGTA, 10 mM LiCl, 11.1 mM glucose, and 0.5% BSA.

The cells were then incubated for 60 min at 37 ꢀC in 0.3 mL of PI buffer

with or without ligands at various concentrations. The reaction was

stopped by adding 1 mL of methanol/hydrochloric acid to each well, and

the content was transferred to a column (Dowex AG 1-X8, formate form,

Bio-Rad, Hercules, CA) for the determination of inositol phosphates.

The columns were washed twice with 3 mL of distilled water and twice

more with 2 mL of 5 mM sodium tetraborate/60 mM sodium formate.

The content of each column was eluted by addition of 2.5 mL of 1 M

ammonium formate/100 mM formic acid. Then 0.5 mL of the eluted

fraction was added to scintillant and β radioactivity was counted.

’ ASSOCIATED CONTENT

S

Supporting Information. Elemental analysis results for

b

compounds 1ꢀ11 and enantiomeric excess determinations for

compounds 5 and 6. This material is available free of charge via

the Internet at http://pubs.acs.org.

The substances under study were administered iv at diﬀerent doses

2 min before CCK-8 in order to test their capacity to antagonize CCK-8

induced contractions. The antagonizing eﬀects were expressed as per-

centage of reduction of the area of the CCK-8 contractions. The per-

centages of inhibition were plotted against the logarithm of the antagonists

doses. The regression line was calculated and the concentration eﬀective

in inhibiting by 50% the eﬀect of the agonist (IC₅₀) were obtained from

the regression line by using Allﬁt program.

’ AUTHOR INFORMATION

Corresponding Author

*Phone: +39-040-5587861. Fax: +39-040-52572. E-mail: varnavas@

units.it.

’ ACKNOWLEDGMENT

Site-Directed Mutagenesis and Transfection of COS-7 Cells.

All mutant receptor cDNAs were constructed by oligonucleotide-directed

mutagenesis (Quick-Change site-directed mutagenesis kit, Stratagene,

The corresponding author thanks Dr. Maria Gryllaki of

NADA, Greece, for her special contribution.

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dx.doi.org/10.1021/jm200438b |J. Med. Chem. 2011, 54, 5769–5785

Journal of Medicinal Chemistry

ARTICLE

’ ABBREVIATIONS USED

Derivatives as CCK₁Receptor Antagonists: The Final approach. Med.

Chem. 2005, 1, 501–517.

CCK, cholecystokinin; CCK₁-R, cholecystokinin receptor 1;

CCK₂-R, cholecystokinin receptor 2; GI, gastrointestinal; CNS,

central nervous system; SAfR, structureꢀaﬃnity relationship;

SAR, structureꢀactivity relationship; ER, eudismic ratio; CSP,

chiral stationary phase; HPLC, high performance liquid chroma-

tography;RP-eHPLC, reverse phase enantioselective HPLC; CD,

circular dichroism; ELSD, evaporative light scattering detector;

SI, selectivity indices; MD, molecular dynamics; WT, wild type

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Varnavas, A. 2D-QSAR and 3D-QSAR/CoMFA analyses of the N-term-

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Rhodus, hic saltus”. Bioorg. Med. Chem. 2009, 17, 5198–5206.

(20) Varnavas, A.; Lassiani, l.; Valenta, V.; Mennuni, L.; Makovec, F.;

Hadjipavlou-Litina, D. Anthranilic Acid Based CCK₁Receptor Antagonists:

Preliminary Investigation On Their Second “Touch point”. Eur.

J. Med. Chem. 2005, 40, 563–581.

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Mennuni, L.; Makovec, F.; Varnavas, A. Anthranilic acid based CCK1

receptor antagonists: blocking the receptor with the same “words” of the

endogenous ligand. Bioorg. Med. Chem. 2009, 17, 2336–2350.

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5785

dx.doi.org/10.1021/jm200438b |J. Med. Chem. 2011, 54, 5769–5785

Article Doi

New anthranilic acid based antagonists with high affinity and selectivity for the human cholecystokinin receptor 1 (hCCK1-R)

DOI: 10.1021/jm200438b

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