Anthranilic acid based CCK1 receptor antagonists: Blocking the receptor with the same 'words' of the endogenous ligand

Bioorganic & Medicinal Chemistry 17 (2009) 2336–2350

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Bioorganic & Medicinal Chemistry

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Anthranilic acid based CCK₁receptor antagonists: Blocking the receptor

with the same ‘words’ of the endogenous ligand

Lucia Lassiani ^a, Michela V. Pavan ^a, Federico Berti ^b, George Kokotos ^c, Theodoros Markidis ^c,

Laura Mennuni ^d, Francesco Makovec ^d, Antonio Varnavas ^a,

*

^aDepartment of Pharmaceutical Sciences, University of Trieste, P.le Europa 1, 34127 Trieste, Italy

^bDepartment of Chemical Sciences, University of Trieste, Via Giorgieri 1, 34127 Trieste, Italy

^cLaboratory of Organic Chemistry, Department of Chemistry, University of Athens, Panepistimiopolis, 15771 Athens, Greece

^dRottapharm S.p.A., Via Valosa di Sopra 7, 20052 Monza, Italy

a r t i c l e i n f o

a b s t r a c t

Article history:

The anthranilic acid diamides represent the more recent class of nonpeptide CCK₁receptor antagonists.

This class is characterized by the presence of anthranilic acid, used as a molecular scaffold, and two phar-

macophores selected from the C-terminal tetrapeptide of CCK. The lead compound coded VL-0395,

endowed with sub-micromolar afﬁnity towards CCK₁receptors, was characterized by the presence of

Phe and 2-indole moiety at the C- and N-termini of anthranilic acid, respectively. Herein we describe

the ﬁrst step of the anthranilic acid C-terminal optimization using, instead of Phe, aminoacids belonging

to the primary structure of CCK-8 and other not coded residues. Thus we demonstrate that the CCK₁

receptor afﬁnity depends on the nature of the aminoacidic side chain as well as that the free carboxy

group of the alpha-aminoacids is crucial for the binding. The R enantiomers of the most active com-

pounds represent the eutomers of this class of antagonists conﬁrming thus the stereo preference of

the receptor. Moreover this SAR study demonstrates that the receptor binding pocket, that host the

aminoacidic side chain, results much more tolerant respect to that accommodating the indole ring. As

a result, an appropriate variation of the aminoacidic side chain could provide a better CCK₁receptor afﬁn-

ity diorthosis.

Received 17 October 2008

Revised 3 February 2009

Accepted 8 February 2009

Available online 14 February 2009 14

February 2009

Keywords:

Cholecystokinin

CCK

Receptors

CCK₁-R

Ligands

Antagonists

Anthranilic acid

1. Introduction

op clinically useful small non-peptide molecules known as CCK

receptor antagonists.^10–15

Cholecystokinin (CCK) is a gut-brain peptide which acts as hor-

mone in the gastrointestinal (GI) tract and as neurotransmitter/

neuromodulator at the central nervous system (CNS).^1–4The entire

range of peripheral and central biological actions of CCK is medi-

ated by two distinct G-protein coupled receptor types: CCK₁and

CCK₂.^5–8Among the different molecular forms of CCK only the C-

Among the strategies adopted for the design of these antago-

nists, a particular consideration was given to the following four ap-

proaches: (a) The chemical simpliﬁcation and manipulation of

natural products, such as asperlicin^16,17(Fig. 1); (b) The dipeptoid

approach, in which the minimal structure responsible for the bind-

ing is a dipeptide derived from the C-terminal sequence of CCK; (c)

The key aminoacid derivatization, in which the receptor recogni-

tion is assigned to a single aminoacid;^18–20and (d) The use of

molecular scaffolds bearing suitable pharmacophoric groups.^21–24

All these strategies gave rise to a variety of CCK₁receptor antago-

nists characterized by different structure and structural complex-

ity, reﬂecting thus the philosophy of the designer, as well as by a

dissimilar biological proﬁle.

terminal

octapeptide

(Asp²⁶-Tyr(SO₃H)²⁷-Met²⁸-Gly²⁹-Trp³⁰

-

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

nanomolar afﬁnity. The smallest bioactive fragment of CCK

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

afﬁnity only the CCK₂receptor while its afﬁnity towards the

CCK₁receptor decreases to the millimolar range.⁹

In view of the importance of CCK in different physiopathological

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

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

etc., over the past 15 years, great efforts have been made to devel-

In an earlier report, we described the discovery of a new class of

CCK₁receptor ligands characterized by the presence of two phar-

macophores selected from the C-terminal tetrapeptide of CCK

(CCK-4) and by the anthranilic acid dimer used as molecular

scaffold.^23,24

The lead compound obtained from this part of our work coded

VL-0494 (Fig. 1) resembles some structural features of CCK-4

* Corresponding author. Tel.: +39 040 5587861; fax: +39 040 52572.

E-mail address: varnavas@units.it (A. Varnavas).

doi:10.1016/j.bmc.2009.02.012

L. Lassiani et al. / Bioorg. Med. Chem. 17 (2009) 2336–2350

2337

Asperlicin

CCK₁IC_{5 0}: 1.4 µM

CCK-4

CCK₁IC_{5 0}: 5.33 mM

group of CCK is crucial for the full biological activity and that

CCK analogs lacking the C-terminal primary amide group were able

to antagonize the contractile activity induced by CCK-7 or CCK-8.³⁰

Moreover, the same kind of modiﬁcations applied to the C-terminal

tetrapeptide of CCK, produced potent gastrin antagonists (e.g., Boc-

Trp-Leu-Asp-2-phenylethylamide, Boc-Trp-Leu-Asp-2-phenylethyl

ester).³¹

Finally, the pure enantiomers (compounds 22–25) of the most

active compounds have been synthesized and tested in order to

conﬁrm the stereo preference of the receptor.

O

N

HO

HN

N

H

N

O

NH

O

N

H₂N

H

N

Met

Asp

NH

NH₂

O

2. Chemistry

The procedure for the synthesis of aminoacidic derivatives 1–11

and 19–25 is depicted in Scheme 1.

DL-Aminoacids were protected by conversion to ethyl esters 1a–

5a, 7a–9a, 14a and 19a–21a. The optically active aminoacids were

purchased as ethyl esters hydrochlorides. For the tyrosine deriva-

tive 11a, both carboxylic and phenolic groups were protected by

benzylation at carboxy and hydroxy groups, following a described

procedure.³²Subsequently deprotection of the amino group by

treatment with gaseous HCl in ethyl acetate let us to obtain com-

pound 11a.

HN

O

NH

N

OH

NH

O

NH

H

O

H

N

OH

N

H

O

The o-aminobenzoylation of ^DL, L or D-aminoacid ethyl esters by

isatoic anhydride gave compounds 1b–5b, 7b–9b, 11b, 14b and

VL- 0494

CCK₁IC_{5 0}: 2.3 µM

VL- 0395

CCK₁IC_{5 0}: 0.20 µM

19b–25b, several of which previously described.²³

For compound 8b the synthesis of the o-nitrobenzoyl derivative

was preferred, followed by reduction of the nitro group.

The o-aminobenzoyl derivatives were converted to compounds

1c–9c, 11c, 14, and 19c–25c by reaction with indole-2-carboxylic

acid or N-methyl-indole-2-carboxylic acid activated by treatment

with PCl₅. The last step concerning the deprotection of the carbox-

ylic group of intermediates 1c–9c, 11c, and 19c–21c by base cata-

lyzed hydrolysis, gave ﬁnal compounds 1–9, 11 and 19–25.

The optical purity of the enantiomers (compounds 22–25) was

determined by analytical enantioselective HPLC (EHPLC) under re-

verse phase conditions.

Compound 11 was converted to derivative 10 by deprotection

of the phenolic group by catalytic hydrogenation.

The strategy for the synthesis of amine derivatives 16–18 is

identical, following the same procedures to convert amphetamine,

2-phenylethylamine and tryptamine to compounds 16–18 respec-

tively, without protection (Scheme 2).

The latter compounds (12, 13 and 15) were obtained with dif-

ferent procedures (Scheme 3).

The ethyl ester 14 was treated with a solution of methanol sat-

urated with gaseous ammonia to give the amide 12.

The lead compound VL-0395 was activated as mixed anhydride

with isobutyl chloroformate or ethyl chloroformate to give, by

treatment with dipentylamine, the amide derivative 13 and, by

reduction with NaBH₄, the corresponding alcohol 15.³³

Figure 1. Chemical structure and CCK₁receptor afﬁnity of Asperlicin, CCK-4, VL-

0494 and VL-0395.

having these two ‘tetrapeptides’ the same N- and C-terminal ami-

noacid side chains.²⁵

Further simpliﬁcation of anthranilic acid dimer scaffold to a

monomer led to a compound coded VL-0395 (Fig. 1) endowed with

submicromolar afﬁnity (IC₅₀= 197,5 nM) representing the new

starting point for the development of this innovative class of

CCK₁receptor antagonists.²⁶The antagonist nature of VL-0395

was conﬁrmed by an in vivo functional test. The compound inhib-

ited the guinea pig gallbladder contractions, induced by CCK-8,

with a potency comparable to that of the reference CCK₁selective

antagonist Loxiglumide.²⁶

It is interesting to notice that both the lead compounds VL-

0494 and VL-0395 share at the C-terminal site the Phe residue uti-

lized in our design strategy for its ‘address’ type contribute accord-

ing to the ‘message and address’ theory described by Schwyzer.²⁷

So, in order to initiate the second phase of the development of

this new class of CCK₁receptor antagonists, regarding the C-termi-

nal optimization of the anthranilic acid of VL-0395, we propose to

synthesize the compounds reported in Table 1 and test their afﬁn-

ity towards CCK receptors.

First of all, we select to introduce, on the C-terminus of

anthranilic acid, aminoacids belonging to CCK-8, that is, Gly,

Met, Asp, Trp, and Tyr (Compounds 1, 5, 7, 9 and 10, respectively).

In addition to the above single components of CCK-8 we select

other not coded aminoacids such as Ala, Val, Leu and Glu

(Compounds 2, 3, 4, and 8, respectively) for the following reasons.

Alanine, characterized by the simplest side chain, is selected in

order to ascertain the relative importance of the other aminoac-

ids. Valine and leucine are proposed in order to establish the

importance of branched aliphatic side chains while the glutamic

acid because has provided an important class of ligands for both

CCK receptors subtypes.^28,29

3. Biological activity

Binding data, along with those of the lead VL-0395, are reported

in Table 1. The obtained afﬁnities for CCK₁and CCK₂receptors of

the synthesised compounds 1–25 were evaluated according to

established protocols and are expressed as IC₅₀or as percentage

of inhibition (ISB%) determined at the highest used dose (1

lM,

3

l

M, 10 M or 30

l

M as indicated).²⁰When the ISB% values were

lower than 20% the compounds were considered inactive. Values

without standard errors were obtained from no more than two

experiments.

The present compounds can be divided into three groups (Table

1) and will be discussed separately according to the preﬁxed tar-

Secondly, compounds 12–21 are proposed in order to elucidate

the CCK₁receptor binding role of the C-terminal free carboxy

group of Phe. It is interesting to notice that the C-terminal amide

2338

L. Lassiani et al. / Bioorg. Med. Chem. 17 (2009) 2336–2350

Table 1

CCK receptors binding data

O

R1

N

NH

O

R

N

H

R2

Compd

R

R1

R2

Stereo

IC₅₀

(l

M)^a

Guinea pig brain cortex (CCK₂

f

Rat pancreatic acini (CCK₁)

)

VL-0395

1

2

3

4

5

6

7

8

–CH₂–C₆H₅

–H

–CH₃

–CH–(CH₃)₂

–CH₂–CH–(CH₃)₂

–(CH₂)₂–S–CH₃

–CH₂–COOH

–(CH₂)₂–COOH

–CH₂-3-indolyl

–CH₂–C₆H₄-4-OH

–CH₂–C₆H₄-4-OCH₂C₆H₅

H

CH₃

H

–COOH

RS

0.197 0.107

5.52 1.50

3.24 1.18

0.288 0.070

0.083 0.008

0.052 0.007

0.092 0.006

IN^d

16.40

IN^d

27.40

IN^d

36% ISB^b

IN^d

9

10

11

H

0.445 0.160

2.065 0.576

1.158 0.408

IN^d

12

13

14

15

16

17

18

19

20

21

–CH₂–C₆H₅

–CH₂-3-indolyl

–H

H

–CONH₂

–CON(C₅H₁₁

–COOCH₂CH₃

–CH₂OH

–CH₃

RS

—

RS

8.530 3.18

IN^c

IN^d

IN^e

NT

)

2

20% ISB^c

32% ISB^c

3.15 0.27

IN^c

IN^d

IN^e

16.90

–H

IN^c

–CH₂COOH

–(CH₂)₂COOH

–CH₂COOH

IN^c

–H

–CH₂–C₆H₅

IN^c

25% ISB^d

22

23

24

25

–CH₂–CH–(CH₃)₂

–(CH₂)₂–S–CH₃

H

–COOH

S

R

S

R

4.64 0.110

0.046 0.004

2.75 0.093

0.027 0.002

45% ISB^e

14.7 1.2

31% ISB^e

11.4 0.8

IN: inactive (ISB less than 20%). NT: not tested.

a

IC₅₀standard error (ALLFIT analysis); % ISB: percentage inhibition of speciﬁc binding of 25 pM [¹²⁵I]-(BH)–CCK8 at the maximal concentration tested.

b

c

d

e

f

1

3

10

30

l

M.

l

M.

Values without standard errors were obtained from not more than two experiments.

gets (importance of the aminoacid side chain, of C-terminal free

carboxy group and that of the chiral center conﬁguration).

methylene unit as in the Leu derivative (4). In this case the iso-pro-

pyl group of Leu can be viewed as bioisoster of the phenyl group of

Phe. Nevertheless, although the gain in afﬁnity observed with the

Leu derivative characterized by the presence of aliphatic branched

side chain, the best result in binding afﬁnity in this series was ob-

tained by the linear chain of Met (5). The four fold enhancement in

afﬁnity of the Met derivative respect to that of VL-0395 suggests

that a correct interaction with the hydrophobic binding pocket of

the receptor can be established not only by the aromatic ring of

Phe but even by linear aliphatic chains. Therefore, this receptor

pocket that host the aminoacidic side chain seems to be much

more tolerant respect to that accommodating the indole ring

which imposes a high degree of conformational restrictions.³⁴In-

deed, the introduction of a methyl group at the indole nitrogen

of 5 (6) produce a slight drop in afﬁnity similar to that observed

for the N-methyl indole derivative of VL-0395.³⁵This fact sug-

gests that the Met derivative interacts with the same receptor

binding pockets of VL-0395.

4. Results and discussion

4.1. SAR of the side chain

As previously observed,²⁶all the tested compounds showed low

afﬁnity towards the central CCK₂receptor subtype conﬁrming their

preference for the CCK₁receptors.

A macroscopic analysis of the obtained data shows that the

binding afﬁnity of compounds 1–11 appears clearly inﬂuenced by

the nature of the aminoacidic side chain. In fact, the lowest afﬁnity

observed was that of the Gly derivative (1) which is devoid of side

chain, thus offering a major conformational ﬂexibility to the C-ter-

minal carboxy group. The Ala derivative (2) resulted two fold more

potent than Gly and its afﬁnity value is useful as reference in this

SAR study dedicated to the side chain optimization. Substitution

of the methyl group of Ala (2) with the iso-propyl group of Val

(3) enhance ten fold the CCK₁receptor afﬁnity. This compound

showed an afﬁnity and apparent selectivity comparable to that of

the reference compound VL-0395 bearing the Phe residue. More-

over, further improvement in afﬁnity was obtained when the iso-

propyl group was distanced from the aminoacidic backbone by a

Furthermore, the loss in afﬁnity showed by the hydrophilic

chains of Asp and Glu conﬁrms the hydrophobic nature of the

receptor pocket.

Thelarge aminoacid Trp of 9 seems to reach thebinding pocket but

its lowered afﬁnity, possibly due to the steric hindrance of the indole

system, underlines a non optimal ﬁtting with the receptor sub-site.

L. Lassiani et al. / Bioorg. Med. Chem. 17 (2009) 2336–2350

2339

O

OH

R

O

R₂

H₂N

v, vi, vii

n

O

OH

H₂N

R₂= H

R₂= C₂H₅;

O

11a

n=0: 1a-5a, 7a, 9a,

14a

n=1: 19a, 21a

n=2: 20a

ii

O

R₁

NH₂

O

R

O

N

NH

O

R

O

ii

viii

N

H

O

R₂

n

N

H

O

R

n

iii, iv

n=2: 8a

n=0: 1b-5b, 7b-9b,

14b, 22b-25b

n=1: 19b, 21b

n=2: 20b

R₂= C₂H₅;

R₁= H; R₂= C₂H₅;

R₂= CH₂-C₆H₅; n=0: 11b

R = CH -C H ;

n=0: 11c

2 2 6 5

R₁= CH₃; R₂= C₂H₅; n=0: 6c

ix or x

or xi

xii

10

R₁= R₂= H; n=0: 1-5, 7-9, 11,

22-25

n=1: 19, 21

n=2: 20

R₁= CH₃; R₂= H; n=0: 6

Scheme 1. Synthesis of compounds 1–11, 19–25. Reagents and conditions: (i) HCl (g), EtOH abs.; (ii) Isatoic anhydride, TEA, EtOAc, reﬂux; (iii) o-nitrobenzoyl chloride, Et₃N,

CH₂Cl₂; (iv) Zn, AcOH, CH₂Cl₂; (v) (Boc)₂O, dioxane, NaOH 1 N, H₂O; (vi) benzyl bromide, K₂CO₃, acetone reﬂux; (vii) HCl (g), EtOAc; (viii) indole-2-carboxylic acid or N-

methyl-indol-2-carboxylic acid, PCl₅, dry CH₂Cl₂, pyridine or TEA; (ix) KOH, THF/H₂O 1:1; (x) KOH, MeOH, reﬂux; (xi) LiOH, THF/H₂O 1:1; (xii) H₂, 10% Pd/C, THF.

O

H

N

NH₂

O

R

NH

O

R

ii

i

N

R₂

H₂N

R₂

N

H

R₂= CH₃: 16

R₂= H: 17-18

R₂= CH₃: 16b

R₂= H: 17b-18b

Scheme 2. Synthesis of compounds 16–18. Reagents and conditions: (i) isatoic anhydride, EtOAc, reﬂux; (ii) indol-2-carboxylic acid, PCl₅, dry CH₂Cl₂, pyridine or TEA.

O

H

N

H

N

NH

O

NH

O

OH

N

H

i

iii

N

H

O

15

14

ii

VL-0395

O

H

N

H

N

NH

O

iv

NH

O

NH₂

N(C₅H₁₁)₂

N

H

N

H

O

13

12

Scheme 3. Synthesis of compounds 12, 13, 15. Reagents and conditions: (i) KOH, THF/H₂O 1:1; (ii) NH₃(g), MeOH; (iii) EtOCOCl, N-methylmorpholine, THF, DMF, NaBH₄,

i

MeOH; (iv) NH(C₅H₁₁)₂, BuOCOCl, TEA, THF.

2340

L. Lassiani et al. / Bioorg. Med. Chem. 17 (2009) 2336–2350

The Tyr derivative 10 deserves a particular attention because

bond at the central anthranilic core is conserved, and differing

only at the Leu side chain, which is turned towards the indole

ring, and at the indole system, which is also somewhat turned to-

wards the Leu chain. This leads to a loss of coplanarity between

the indole and anthranilic systems, but allows to establish hydro-

phobic interactions between the indole ring and the Leu methyls.

This tendency is enhanced in the Met derivative 5: the conforma-

tion reported in Figure 1c is 4 kcal/mol lower in enthalpy of for-

mation than that corresponding to VL-0395, and the geometry is

completely different. The central hydrogen bond is lost, and re-

placed by a new bond between the anthranil amide hydrogen

and the carboxylic end of the molecule. The amide groups of

the molecule are thus pointing to opposite sides, and there is a

strong interaction between the longer Met side chain and the in-

dole ring. These ‘closed’ conformations are likely to be even more

favoured in water, since they allow a minimization of the hydro-

phobic surface exposed to the solvent, and can be regarded as

rather realistic. However, since the afﬁnities of compounds 4

and 5 are similar, we have also considered the possibility that,

within low energy conformations of the two compounds, similar

families of conformations could be found. We have thus submit-

ted the two compounds, and VL-0395 as well, to a molecular

dynamics run at 300 K, and we have performed a population

analysis. Figure 3 reports the results of this analysis, taking as a

conformation descriptor the distance between the carboxylic car-

bon and the anthranil amide hydrogen. It can be clearly seen that

the minimum energy conformations are also the only ones popu-

lated at room temperature, at least within this model. Similar re-

represent a key aminoacid of CCK-8 in its recognition approach.³⁶

The afﬁnity observed for the Tyr derivative 10 was ten fold lower

respect VL-0395 and similar to that of Ala (2). Even the Tyr O-pro-

tected derivative (11) resulted more active than compound 10.

In any case, we are not surprised by this result since a similar

behavior was already observed in a previous SAR study concerning

the substitution of the Phe phenyl ring. In particular, all the four

substituted derivatives of Phe were from two to sixfold less active

than VL-0395 whereas the 2 and 3 substituted compounds were

active as the lead.³⁷

The antagonist nature of the most active compounds 4 and 5

was conﬁrmed by an in vitro functional test on isolated muscular

strips of guinea pig gallbladder stimulated by CCK-8. Both com-

pounds showed a 100% inhibition at the maximum concentration

tested of 1 lM.

Moreover, trying to correlate the observed afﬁnities of com-

pounds 1–11 to the hydrophobic parameter ClogP (calculated by

the ^{CHEMDRAW ULTRA}7.01 Software) it appears that the highest afﬁn-

ities are related to ClogP range of 2.5–4.

4.2. Molecular modeling

In order to evaluate the spatial arrangement of the pharmaco-

phoric groups of the most active compounds a conformational

analysis was carried out on compounds 4 and 5, and their geome-

tries were compared with that of the lead VL-0395. As to the struc-

ture of the lead, we started from the NMR-derived conformation

that has been recently identiﬁed.³⁶The structure of VL-0395 was

built manually in this conformation and then the system was opti-

mized by an AM1 calculation. We had already followed this proce-

dure in the past for the same compound in its neutral and

protonated forms.³⁵Here the optimization was repeated on the

carboxylic anion of VL-0395, as in the NMR derived structure

and a Monte Carlo conformational search was performed. The

search yielded 142 conformations for the anion of VL-0395, but

none was lower in energy than the NMR-derived structure

(Fig. 2a). The geometries of the anions 4 and 5 were then built over

the minimum energy conformation of VL-0395, and both the mol-

ecules were submitted to a conformational search. The searches

gave this time rather different results, and the geometry corre-

sponding to the NMR minimum of VL-0395 is not the absolute

minimum for both Leu and Met derivatives (Figs. 2b and c).

The enthalpy of formation for the minimum energy conforma-

tion of compound 4 is 2.1 kcal/mol more favorable than that with

the backbone in the same conformation of VL-0395. This geome-

try is by the way still similar to that of the lead, the hydrogen

sults are obtained considering other descriptors (torsion of

angle).

U

As a consequence, with this aliphatic-end compounds is difﬁ-

cult to ﬁnd a relationship between the activity and the distance be-

tween the ends of the molecules in their ground state. If we

consider the dependence of the activity upon the hydrophobic sur-

faces and volumes of the aminoacid side chains (VL-0395: area

98.4, volume 120.4; compound 4: area 75.8, volume 81.6; com-

pound 5: area 86.3, volume 91.4), we see that the best compound

in the series (5) bears a group of intermediate area and volume,

and that perhaps the benzyl group of the lead is somewhat too

large. On the other hand, the length of the aliphatic chains and

their mobility could play a major role in improving the activity.

4.3. SAR of the free carboxy group

In addition to the above described derivatives, compounds 12–

21 have been synthesized in order to elucidate the role of the

aminoacidic free carboxy group in the receptor binding.

Figure 2. NMR-derived conformation of VL-0395 (a) and lowest energy conformations of the higher afﬁnity compounds 4 (b) and 5 (c).

L. Lassiani et al. / Bioorg. Med. Chem. 17 (2009) 2336–2350

2341

respect to the side chain. It is interesting to notice that the b-Phe

residue confers the better afﬁnity in the series of the peptoid deriv-

atives like PD-140,548.¹⁹

All these ﬁndings suggests that in proximity of the hydrophobic

pocket that accommodates the aminoacidic side chains of the

above described anthranilic acid type ligands there is probably at

least one aminoacid of the receptor that is implicated in ionic bond

formation with the aminoacidic carboxy group.

4.4. Receptor stereoselectivity

Finally, compounds 22–25 were synthesized in order to validate

the inﬂuence of the stereochemistry of the aminoacidic chiral cen-

ter of the two more active compounds of this series (4 and 5) on

the receptor afﬁnity. Hence, we determined the eutomers and their

selectivity indices (SI) as well as the eudismic ratio (ER) of the pure

enantiomers of compounds 4 and 5.

The binding data reported in Table 1 show that the pharmaco-

logically active enantiomer is the one in which the absolute conﬁg-

uration of the only chiral center of the molecules is R. In fact,

compounds 23 and 25 are about 100-fold (ER ꢀ 100) more active

than their distomers 22 and 24, respectively.

Although the similar stereoselectivity (ER ꢀ 100) expressed by

the two eutomers the highest selectivity versus CCK₁receptors

(SI = 422) was observed for the eutomer of the Met derivative

(compound 25).

Figure 3. Population analysis of low energy conformations of VL-0395 and of

compounds 4 and 5 after molecular dynamics at 300 K.

The afﬁnity data obtained for compounds 12–18 demonstrate

clearly that the free carboxy group of the aminoacid is essential

for the receptor binding. In fact, the remarkable drop in receptor

afﬁnity of the primary amide (12) in addition to the inactivity of

the ethyl ester (14) conﬁrms that the free carboxy group is engaged

in the receptor binding through ionic or reinforced-ionic bond for-

mation rather than in a double hydrogen bond interaction. Never-

theless, since the double hydrogen bond formation can be

established even by the primary amide the 43-fold decrease of

its afﬁnity respect VL-0395 exclude this type of interaction. Still

the tertiary amide 13, bearing the same alkyl substituents on the

amido nitrogen of the known glutamic acid based CCK₁receptor

antagonist Lorglumide, was poorly tolerated in the receptor.

Reduction of the carboxylic acid group to alcohol or to methyl

group (15 and 16, respectively) conﬁrms the loss of the afﬁnity ob-

served for the amide and ester derivatives. On the other hand,

derivative 16, although its reasonably no drug-like feature due to

the presence of amphetamine, showed the better afﬁnity among

this subset of compounds. Deletion of the carboxylic acid func-

tional group of VL-0395 as well as that of compound 9 led to com-

pounds 17 and 18, respectively, lacking in afﬁnity towards CCK₁

receptors. Their inactivity seems plausible due to both effects, that

is, the deletion of the carboxylic group as well as to the abolish-

ment of the asymmetry center in alpha position that ensured the

correct spatial position of the chain toward the receptor binding

pocket.

5. Conclusion

In conclusion, by this SAR study dedicated to the C-terminal

optimization of the anthranilic acid derivatives, we have demon-

strate the following structural requirements:

ꢁ The receptor afﬁnity depends on the nature of the aminoacidic

side chain. Indeed, aminoacids of the endogenous ligand CCK-

8, offers a different CCK₁receptor ‘address’ type contribute

and the order of the relative importance is as follow:

Met > Phe > Trp > Tyr > Gly > Asp.

ꢁ The presence of an aromatic ring at the end of the aminoacidic

side chain is not mandatory since linear or branched aliphatic

chains produce a comparable or better afﬁnity.

ꢁ Hydrophilic side chains are not tolerated in the receptor pocket

conﬁrming thus its hydrophobic nature.

ꢁ The aminoacidic free carboxy group is engaged in the receptor

recognition through ionic bond formation and is essential for

the binding.

ꢁ The correct position of the aminoacidic free carboxy group is the

alpha-position respect to the side chain.

ꢁ The R conﬁguration of the aminoacidic chiral center is preferred

by the CCK1 receptor.

If all these ﬁndings are correct, an appropriate variation of the

aminoacidic side chain could provide a better CCK₁receptor afﬁn-

ity reﬁnement.

Having demonstrate that the carboxylic acid functional group is

essential for the receptor binding we focused our attention on the

correct position of this group in order to retain the receptor afﬁn-

ity. When the free carboxy group of the Gly derivative (1) was pro-

longed by one or two methylene groups the resulted compounds

19 and 20, respectively, were inactive at the maximum dose tested

6. Experimental

6.1. Chemical procedures

of 3 lM. Moreover, in order to exclude that the loss in afﬁnity ob-

All chemicals and solvents used in syntheses were reagent-

grade products and were used without additional puriﬁcation.

Reaction progress was monitored by ascending thin-layer chroma-

tography (TLC) using precoated silica gel plates (60F-254 Merck).

Visualization of the chromatograms was achieved by short wave

UV light (254 nm). Melting points were determined on a Büchi

served of compounds 19 and 20 was due to the lack of the amino-

acidic side chain we have also prolonged, by one more methylene

group, the position of the carboxylic acid of the reference com-

pound VL-0395. The failure in afﬁnity, even at the forced maxi-

mum dose of 10

lM, of the b-Phe derivative (21) conﬁrms that

the correct position of the free carboxy group is the alpha position

2342

L. Lassiani et al. / Bioorg. Med. Chem. 17 (2009) 2336–2350

510 melting point apparatus (Büchi, Flawil, Switzerland) and are

uncorrected. Preparative medium-pressure chromatography

(MPLC) was performed on a Büchi 688 apparatus using silica gel

6.3. General method for preparation of compound 1b–5b, 7b,

9b, 11b, 14b, 16b–25b

(Merck Kieselgel 60, 15–40

l

m). Proton (¹H NMR, 200 MHz) and

A suspension of 20.0 mmol of the aminoacid ethyl or benzyl es-

ter hydrochloride (1a–5a, 7a, 9a, 11a, 14a and 19a–25a) and TEA

(2.78 mL, 20.0 mmol) or a solution of the amine (amphetamine,

2-phenylethylamine, tryptamine for compounds 16b–18b, respec-

tively) in 150 mL of ethyl acetate was treated with isatoic anhy-

dride (3.26 g, 20.0 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 ꢃ 50 mL), water

(1 ꢃ 50 mL) and brine, dried over anhydrous sodium sulfate and

concentrated under vacuum. The residue was triturated with

petroleum ether 40–60° and the precipitate was collected by

ﬁltration.

carbon (¹³C NMR, 50 MHz) nuclear magnetic resonance spectra

were recorded on a Varian-Gemini 2000 Fourier Transform spec-

trometer using CDCl₃or (CD₃)₂SO as solvent. Chemical shifts were

reported as parts per million (ppm, d units) downﬁeld from an

internal Me₄Si standard. Splitting patterns are designated as s, sin-

glet; d, doublet; t, triplet; q, quartet; m, multiplet and b, broad. ¹³C

NMR spectra were determined using either the Attached Proton

Test (APT) or standard ¹³C pulse sequence parameters. Spectral

data are consistent with assigned structures. Mass spectra were re-

corded on an API-1 Perkin–Elmer SCIEX spectrometer by electro-

spray ionization (ES). Optical rotations were determined with a

Perkin–Elmer 241 polarimeter in a 1.0 dm tube. Determination of

enantiomeric excesses was run on a Jasco HPLC chromatographer

on CSP-TE-SP-100 (250 ꢂ 4 mm ID) at a ﬂow rate of 1.00 mL/min

of a mixture of ammonium acetate (20 mM) and methanol/water

85/15 (v/v), with an UV detection at 254 nm. Elemental analyses

were performed by the Microanalyses Laboratory of the Depart-

ment of Chemistry of the University of Trieste and were within

0.4% of the theoretical values calculated for C, H and N.

6.3.1. (2-Amino-benzoylamino)-acetic acid ethyl ester (1b)²³

The titled compound was obtained in 66% yield. TLC (EtOAc/

n-hexane 1:1). R_f0.52; mp 70–72 °C; ¹H NMR (CDCl₃) d 1.26 (t,

3H, –CH₃); 4.18 (m, 4H, –CH₂–O– and –CH₂–CO–); 5.41 (b, 2H,

–NH₂); 6.57–6.73 (m, 3H, –NH– and Ar); 7.17 (t, 1H, Ar); 7.38 (d,

1H, Ar); ¹³C NMR (CDCl₃) d 14.19, 41.62, 61.61, 115.18, 116.63,

117.30, 127.57, 132.61, 148.83, 169.35, 170.26.

6.2. General method for preparation of compound 1a–5a, 7a–

9a, 14a, 19a–21a

6.3.2. 2-(R,S)-(2-Amino-benzoylamino)-propionic acid ethyl

ester (2b)

The titled compound was obtained in 53% yield. TLC (EtOAc/

n-hexane 1:1). R_f0.63; mp 95–97 °C; ¹H NMR (CDCl₃) d 1.29 (t,

3H, –CH₂–CH₃); 1.48 (d, 3H, CH–CH₃); 4.22 (q, 2H, –CH₂–); 4.71

(m, 1H, –CH); 5.15 (b, 2H, –NH₂); 6.64 (m, 3H, –NH– and Ar);

7.19 (t, 1H, Ar); 7.38 (d, 1H, Ar); ¹³C NMR (CDCl₃) d 14.19, 18.66,

48.30, 61.59, 115.42, 116.63, 117.29, 127.47, 132.54, 148.84,

168.70, 173.32.

Gaseous HCl was bubbled for 30 min. into a stirred suspension

of 20.0 mmol of the aminoacid in 150 mL of abs. EtOH, cooled to 0

°C. The solvent was removed under vacuum and the residue was

triturated with cold diethyl ether and ﬁltered. The aminoacid ethyl

esters hydrochlorides were obtained in almost quantitative yield

and were used in the next step without further puriﬁcation.

6.2.1. 2-(R,S)-Amino-3-(4-benzyloxy-phenyl)-propionic acid

benzyl ester hydrochloride (11a)

6.3.3. 2-(R,S)-(2-Amino-benzoylamino)-3-methyl-butyric acid

ethyl ester (3b)²³

Benzyl bromide (10.21 mL, 85.7 mmol) was added to a suspen-

sion of N-Boc-^DL-tyrosine (6.00 g, 21.3 mmol) and K₂CO₃(8.85 g,

64.0 mmol) in 100 mL of acetone and the reaction mixture was re-

ﬂuxed overnight under stirring. The solvent was removed under

vacuum and the residue was partitioned between H₂O and diethyl

ether. The aqueous phase was extracted three times with ether and

the combined organic layers were washed with brine, dried over

anhydrous sodium sulfate and evaporated to dryness. The residue

was triturated with petroleum ether 40–60° and the precipitate

formed was collected by ﬁltration. O-Benzyl-N-Boc-^DL-tyrosine

benzyl ester (6.50 g, 66%) was obtained as a white powder. TLC

(EtOAc/n-hexane 1:1). R_f0.77; mp 88–89 °C; ¹H NMR (CDCl₃) d

1.41 (t, 9H, -(CH₃)₃); 3.03 (m, 2H, –CH₂–CH<); 4.58 (m, 1H,

–CH<); 5.02 (s, 2H, –CH₂–O–); 5.13 (s, 2H, –CH₂–O–); 6.63 (m,

2H, Ar); 6.94 (m, 2H, Ar); 7.34–7.41 (m, 10H, Ar); ¹³C NMR (CDCl₃)

d 28.32, 37.38, 54.50, 67.07, 69.94, 79.89, 114.76, 127.42, 127.92,

128.40, 128.53, 130.30, 135.10, 136.88, 154.98, 157.74, 171.69.

O-Benzyl-N-Boc-dl-tyrosine benzyl ester (6.25 g, 13.5 mmol)

was dissolved in 100 ml of ethyl acetate and gaseous HCl was bub-

bled at 0 °C for 5 min. After stirring at r.t. for 15 min, the solvent

was removed under vacuum and the residue was triturated with

cold diethyl ether and the precipitate formed was collected by ﬁl-

tration. O-Benzyl-dl-tyrosine benzyl ester hydrochloride (5.37 g,

99%) was obtained as a white solid. TLC (DCM/MeOH/H₂O/AcOH

9:1:0.1:0.1). R_f0.51; mp 208–209 °C; ¹H NMR (DMSO-d₆) d 3.07

(m, 2H, –CH₂–CH); 4.23 (m, 1H, –CH); 5.05 (s, 2H, –CH₂–O–);

5.11 (s, 2H, –CH₂–O–); 6.88 (m, 2H, Ar); 7.08 (m, 2H, Ar); 7.27–

7.41 (m, 10H, Ar); 8.75 (b, 3H, NH₃^þ); ¹³C NMR (DMSO-d₆) d

35.03, 53.27, 66.91, 69.08, 114.69, 126.42, 127.54, 127.73, 128.26,

128.33, 130.46, 134.73, 136.95, 157.43, 168.79.

Crystallization from EtOAc/n-hexane afforded the titled com-

pound in 65% yield. TLC (EtOAc/n-hexane 1:1). R_f0.70; mp 48 °C;

¹H NMR (CDCl₃) d 0.97 (m, 6H, –CH(CH₃)₂); 1.28 (t, 3H, –CH₂–

CH₃); 2.24 (m, 1H, –CH(CH₃)₂); 4.20 (m, 2H, –CH₂–O–); 4.68 (m,

1H, –NH–CH); 5.46 (b, 2H, –NH₂); 6.56 (d, 1H, –NH–); 6.64 (m,

2H, Ar); 7.19 (t, 1H, Ar); 7.39 (d, 1H, Ar); ¹³C NMR (CDCl₃) d

14.40, 18.10, 19.17, 31.70, 57.14, 61.46, 115.78, 116.70, 117.32,

127.46, 132.56, 148.77, 169.02, 172.23.

6.3.4. 2-(R,S)-(2-Amino-benzoylamino)-4-methyl-pentanoic

acid ethyl ester (4b)²³

The titled compound was obtained in 75% yield. TLC (EtOAc/n-

hexane 1:1). R_f0.73; mp 49–50 °C; ¹H NMR (CDCl₃) d 0.97 (m,

6H, –CH(CH₃)₂); 1.27 (t, 3H, –CH₂–CH₃); 1.69 (m, 3H, –CH₂–CH

and –CH(CH₃)₂); 4.19 (q, 2H, –CH₂–O–); 4.77 (m, 3H, –NH–CH

and –NH₂); 6.52 (d, 1H, –NH–); 6.59–6.66 (m, 2H, Ar); 7.18 (t,

1H, Ar); 7.38 (d, 1H, Ar); ¹³C NMR (CDCl₃) d 14.20, 22.15, 22.88,

25.03, 41.85, 50.93, 61.42, 115.86, 116.65, 117.29, 127.44, 132.52,

148.77, 168.94, 173.34.

6.3.5. 2-(R,S)-(2-Amino-benzoylamino)-4-methylsulfanyl-

butyric acid ethyl ester (5b)²³

The titled compound was obtained in 70% yield. TLC (EtOAc/n-

hexane 1:1). R_f0.63; mp 53–55 °C; ¹H NMR (CDCl₃) d 1.30 (t, 3H,

–CH₂–CH₃); 2.10 (s, 3H, –S–CH₃); 2.22 (m, 2H, –CH₂–CH); 2.57

(m, 2H, –CH₂–S–); 4.23 (q, 2H, –CH₂–O–); 4.84 (m, 3H, –CH and

–NH₂); 6.64 (m, 2H, Ar); 6.82 (d, 1H, –NH–); 7.20 (t, 1H, Ar); 7.40

(d, 1H, Ar); ¹³C NMR (CDCl₃) d 14.22, 15.58, 30.15, 31.86, 51.82,

61.77, 115.16, 116.65, 117.34, 127.46, 132.67, 148.94, 169.00,

172.20.

L. Lassiani et al. / Bioorg. Med. Chem. 17 (2009) 2336–2350

2343

6.3.6. 2-(R,S)-(2-Amino-benzoylamino)-succinic acid diethyl

ester (7b)²³

118.04, 118.14, 120.74, 122.38, 127.11, 127.82, 131.34, 136.05,

149.40, 168.65.

Puriﬁcation by ﬂash chromatography (CH₂Cl₂to EtOAc/CH₂Cl₂

1:4) afforded the titled compound in 59% yield. TLC (EtOAc/n-hex-

ane 1:1). R_f0.56; mp 89–90 °C; ¹H NMR (CDCl₃) d 1.27 (m, 6H,

–CH₃); 3.02 (m, 2H, –CH₂–CH); 4.16 (q, 2H, –CH₂–O–); 4.25 (q,

2H, –CH₂–O–); 5.00 (m, 1H, –CH); 5.52 (b, 2H, –NH₂); 6.67 (m,

2H, Ar); 7.15 (d, 1H, –NH–); 7.23 (t, 1H, Ar); 7.41 (d, 1H, Ar); ¹³C

NMR (CDCl₃) d 14.27, 36.57, 48.77, 61.20, 62.07, 115.16, 116.78,

117.32, 127.68, 132.74, 148.86, 168.69, 170.98, 171.20.

6.3.13. 3-(2-Amino-benzoylamino)-propionic acid ethyl ester

(19b)

Puriﬁcation by ﬂash chromatography (CH₂Cl₂to EtOAc/CH₂Cl₂

2:3) afforded the titled compound in 70% yield. TLC (EtOAc/n-hex-

ane 1:1). R_f0.35; mp 73 °C; ¹H NMR (CDCl₃) d 1.27 (t, 3H, –CH₃);

2.62 (t, 2H, –NH–CH₂–CH₂–); 3.67 (m, 2H, –NH–CH₂–CH₂-); 4.16

(q, 2H, –O–CH₂–); 5.54 (b, 2H, –NH₂); 6.60–6.69 (m, 2H, Ar); 6.77

(m, 1H, –NH–); 7.15–7.32 (m, 2H, Ar); ¹³C NMR (CDCl₃) d 14.34,

34.16, 35.05, 60.92, 115.84, 116.66, 117.31, 127.27, 132.37,

148.79, 169.18, 172.87.

6.3.7. 2-(R,S)-(2-Amino-benzoylamino)-3-(1H-indol-3-yl)-

propionic acid ethyl ester (9b)²³

The titled compound was obtained in 81% yield. TLC (EtOAc/

n-hexane 1:1). R_f0.49; mp 98–100 °C; ¹H NMR (CDCl₃) d 1.24 (t,

3H, –CH₃); 3.42 (m, 2H, –CH₂–CH); 4.16 (q, 2H, –CH₂–O–); 5.06

(m, 1H, –CH); 5.45 (b, 2H, –NH₂); 6.52–6.67 (m, 3H, Ar and

–NH–); 7.02–7.26 (m, 5H, Ar); 7.35 (d, 1H, Ar); 7.58 (d, 1H, Ar);

8.13 (b, 1H, –NH–); ¹³C NMR (CDCl₃) d 14.14, 27.71, 53.27, 61.61,

110.08, 111.32, 115.49, 116.67, 117.28, 118.69, 119.66, 122.24,

122.92, 127.60, 127.66, 132.53, 136.09, 148.79, 168.65, 172.17.

6.3.14. 4-(2-Amino-benzoylamino)-butyric acid ethyl ester

(20b)

Puriﬁcation by ﬂash chromatography (CH₂Cl₂to EtOAc/CH₂Cl₂

2:3) afforded the titled compound in 58% yield. TLC (EtOAc/n-hex-

ane 1:1). R_f0.37; mp 39–41 °C; ¹H NMR (CDCl₃) d 1.25 (t, 3H,

–CH₃); 1.95 (m, 2H, –NH–CH₂–CH₂–CH₂–); 2.43 (t, 2H, –NH–

CH₂–CH₂–CH₂–); 3.46 (m, 2H, –NH–CH₂–CH₂–CH₂–); 4.13 (q, 2H,

–O–CH₂–); 5.55 (b, 2H, –NH₂); 6.42 (m, 1H, –NH–); 6.61–6.69 (m,

2H, Ar); 7.16–7.34 (m, 2H, Ar); ¹³C NMR (CDCl₃) d 14.35, 24.60,

32.11, 39.40, 60.78, 115.94, 116.63, 117.35, 127.11, 132.29,

148.78, 169.41, 173.73.

6.3.8. 2-(R,S)-(2-Amino-benzoylamino)-3-(4-benzyloxy-

phenyl)-propionic acid benzyl ester (11b)

Puriﬁcation by ﬂash chromatography (CH₂Cl₂to 10% EtOAc/

CH₂Cl₂) followed by trituration with cold petroleum ether 40–

60° afforded the titled compound in 28% yield. TLC (EtOAc/n-hex-

ane 1:1). R_f0.62; mp 109–110 °C; ¹H NMR (CDCl₃) d 3.19 (m, 2H,

–CH₂–CH); 5.08 (m, 3H, –CH₂–O– and –CH); 5.21 (m, 2H,

–CH₂–O–); 5.49 (b, 2H,ANH₂); 6.53 (d, 1H, –NH–); 6.61–6.71

(m, 2H, Ar); 6.85 (d, 2H, Ar); 6.96 (d, 2H, Ar); 7.20–7.44 (m,

12H, Ar); ¹³C NMR (CDCl₃) d 37.02, 53.37, 67.29, 69.94, 114.85,

115.23, 116.60, 117.19, 127.33, 127.42, 127.86, 127.90, 128.50,

128.56, 128.62, 130.34, 132.51, 135.01, 136.85, 148.71, 157.79,

168.49, 171.50.

6.3.15. 3-(R,S)-(2-Amino-benzoylamino)-4-phenyl-butyric acid

ethyl ester (21b)

Puriﬁcation by ﬂash chromatography (CH₂Cl₂to EtOAc/CH₂Cl₂

1:4) afforded the titled compound in 48% yield. TLC (EtOAc/n-hex-

ane 1:1). R_f0.50; mp 90 °C; ¹H NMR (CDCl₃) d 1.28 (t, 3H, –CH₃);

2.57 (m, 2H, –CH₂–CO–); 2.91 (dd, 1H, –CH₂–C₆H₅); 3.07 (dd, 1H,

–CH₂–C₆H₅); 4.17 (q, 2H, –CH₂–O–); 4.65 (m, 1H, –CH); 5.49 (b,

2H, –NH₂); 6.64 (m, 2H, Ar); 6.87 (d, 1H, –NH); 7.15–7.35 (m, 7H,

Ar); ¹³C NMR (CDCl₃) d 14.25, 37.02, 40.06, 47.37, 60.79, 115.97,

116.65, 117.21, 126.67, 127.12, 128.56, 129.29, 132.22, 137.55,

148.63, 168.40, 171.97.

6.3.9. 2-(R,S)-(2-Amino-benzoylamino)-3-phenyl-propionic

acid ethyl ester (14b)

Obtained as previously described.²⁶

6.3.16. 2(S)-(2-Amino-benzoylamino)-4-methyl-pentanoic acid

ethyl ester (22b)

6.3.10. (1R,S)-2-Amino-N-(1-methyl-2-phenyl-ethyl)-

benzamide (16b)

The titled compound was obtained in 70% yield. TLC (EtOAc/n-

hexane 1:1). R_f0.73; ½a _D²⁵

ꢄ

= +17.3 (c 1.2, CH₂Cl₂); mp 53–54 °C;

Crystallization from EtOAc/n-hexane afforded the titled com-

pound in 78% yield. TLC (EtOAc/n-hexane 1:1). R_f0.61; mp 103

°C; ¹H NMR (CDCl₃) d 1.18 (d, 3H, –CH₃); 2.86 (m, 2H, –CH₂–);

4.41 (m, 1H, –CH), 5.42 (b, 2H, –NH₂); 5.94 (d, 1H, –NH–); 6.56–

6.65 (m, 2H, Ar); 7.13–7.33 (m, 7H, Ar); ¹³C NMR (CDCl₃) d 20.17,

42.54, 46.24, 116.58, 116.67, 117.31, 126.57, 127.10, 128.50,

129.60, 132.18, 137.98, 148.58, 168.64.

¹H NMR (CDCl₃) d 0.97 (m, 6H, –CH(CH₃)₂); 1.25 (t, 3H, –CH₂–

CH₃); 1.66 (m, 3H, –CH₂–CH and –CH(CH₃)₂); 4.21 (q, 2H, –CH₂–

O–); 4.73 (m, 3H, –NH–CH and –NH₂); 6.50 (d, 1H, –NH–);

6.56–6.64 (m, 2H, Ar); 7.20 (t, 1H, Ar); 7.35 (d, 1H, Ar); ¹³C NMR¹³

C

NMR (CDCl₃) d 14.20, 22.13, 22.85, 25.05, 41.80, 50.90, 61.39,

115.80, 116.60, 117.30, 127.40, 132.50, 148.80, 168.90, 173.30.

6.3.17. 2(R)-(2-Amino-benzoylamino)-4-methyl-pentanoic acid

ethyl ester (23b)

6.3.11. 2-Amino-N-phenethyl-benzamide (17b)²⁶

The titled compound was obtained as an oil in 82% yield. TLC

(EtOAc/n-hexane 1:1). R_f0.65; ¹H NMR (CDCl₃) d 2.91 (t, 2H,

–CH₂–C₆H₅); 3.65 (m, 2H, –CH₂–NH–); 5.40 (b, 2H, –NH₂); 6.19

(b, 1H, –NH–); 6.55–6.67 (m, 2H, Ar); 7.15–7.37 (m, 7H, Ar); ¹³C

NMR (CDCl₃) d 35.79, 40.88, 116.17, 116.63, 117.30, 126.59,

127.11, 128.73, 128.85, 132.23, 139.04, 148.66, 169.34.

The titled compound was obtained in 73% yield. TLC (EtOAc/n-

hexane 1:1). R_f0.73; ½a _D²⁵

ꢄ

= -16.1 (c 0.4, CH₂Cl₂); mp 50–51 °C;

¹H NMR (CDCl₃) d 0.97 (m, 6H, –CH(CH₃)₂); 1.26 (t, 3H, –CH₂–

CH₃); 1.65 (m, 3H, –CH₂–CH and –CH(CH₃)₂); 4.18 (q, 2H, –CH₂–

O–); 4.74 (m, 3H, –NH–CH and –NH₂); 6.49 (d, 1H, –NH–);

6.55–6.65 (m, 2H, Ar); 7.17 (t, 1H, Ar); 7.36 (d, 1H, Ar); ¹³C NMR

(CDCl₃) d 14.20, 22.14, 22.86, 25.03, 41.83, 50.92, 61.40, 115.82,

116.62, 117.31, 127.42, 132.54, 148.77, 168.92, 173.34.

6.3.12. 2-Amino-N-[2-(1H-indol-3-yl)-ethyl]-benzamide (18b)²³

Trituration with hot 95% EtOH afforded the titled compound in

83% yield. TLC (EtOAc/n-hexane 1:1). R_f0.43; mp 159–161 °C; ¹H

NMR (DMSO-d₆) d 2.96 (t, 2H, –CH₂–CH₂–NH–); 3.53 (m, 2H,

–CH₂–NH–); 6.45 (b, 2H, –NH₂); 6.52 (t, 1H, Ar); 6.72 (d, 1H, Ar);

6.97–7.21 (m, 4H, Ar); 7.37 (d, 1H, Ar); 7.49 (d, 1H, Ar); 7.62 (d,

1H, Ar); 8.37 (t, 1H, –NH–); 10.84 (s, 1H, –NH–); ¹³C NMR

(DMSO-d₆) d 25.05, 39.64, 111.19, 111.81, 114.36, 114.79, 116.14,

6.3.18. 2(S)-(2-Amino-benzoylamino)-4-methylsulfanyl-butyric

acid ethyl ester (24b)

The titled compound was obtained in 66% yield. TLC (EtOAc/n-

hexane 1:1). R_f0.63; ½a _D²⁵

ꢄ

= +35.3 (c 1.6, CH₂Cl₂); mp 55–57 °C;

¹H NMR (CDCl₃) d 1.30 (t, 3H, –CH₂–CH₃); 2.13 (s, 3H, –S–CH₃);

2.25 (m, 2H, –CH₂–CH); 2.60 (m, 2H, –CH₂–S–); 4.25 (q, 2H,

2344

L. Lassiani et al. / Bioorg. Med. Chem. 17 (2009) 2336–2350

–CH₂–O–); 4.80 (m, 3H, –CH and –NH₂); 6.60 (m, 2H, Ar); 6.79 (d,

1H, –NH–); 7.22 (t, 1H, Ar); 7.44 (d, 1H, Ar); ¹³C NMR (CDCl₃) d

14.20, 15.60, 30.18, 31.82, 51.88, 61.75, 115.10, 116.69, 117.30,

127.48, 132.70, 148.90, 169.20, 172.20.

7.49 (d, 1H, Ar); 7.63 (t, 1H, Ar); 7.71 (d, 1H, Ar); 7.90 (d, 1H,

Ar); 8.69 (d, 1H, Ar); 9.39 (t, 1H, –NH–CH₂–); 11.94 (s, 1H, –NH–

); 12.48 (s, 1H, –NH–); ¹³C NMR (DMSO-d₆) d 13.91, 41.26, 60.49,

102.45, 112.33, 118.76, 119.83, 120.03, 121.62, 122.48, 123.91,

126.79, 128.09, 131.42, 132.59, 136.95, 139.31, 159.01, 169.06,

169.26.

6.3.19. 2-(R)-(2-Amino-benzoylamino)-4-methylsulfanyl-

butyric acid ethyl ester (25b)

The titled compound was obtained in 72% yield. TLC (EtOAc/n-

6.4.2. 2-(R,S)-{2-[(1H-Indole-2-carbonyl)-amino]-benzoyl-

amino}-propionic acid ethyl ester (2c)

hexane 1:1). R_f0.63; ½a _D²⁵

ꢄ

= -33.7 (c 1.2, CH₂Cl₂); mp 51–53 °C;

¹H NMR (CDCl₃) d 1.28 (t, 3H, –CH₂–CH₃); 2.09 (s, 3H, –S–CH₃);

2.19 (m, 2H, –CH₂–CH); 2.52 (m, 2H, –CH₂–S–); 4.20 (q, 2H,

–CH₂–O–); 4.80 (m, 3H, –CH and –NH₂); 6.65 (m, 2H, Ar); 6.80

(d, 1H, –NH–); 7.24 (t, 1H, Ar); 7.46 (d, 1H, Ar); ¹³C NMR (CDCl₃)

d 14.23, 15.57, 30.14, 31.86, 51.85, 61.73, 115.15, 116.64, 117.35,

127.45, 132.65, 148.92, 169.00, 172.20.

Trituration with CHCl₃afforded the titled compound in 49%

yield. TLC (EtOAc/n-hexane 1:1). R_f0.57; mp 262–264 °C; ¹H

NMR (DMSO-d₆) d 1.20 (t, 3H, –CH₂–CH₃); 1.45 (d, 3H, CH–CH₃);

4.14 (q, 2H, –CH₂–); 4.58 (m, 1H, –CH); 7.09 (m, 2H, Ar); 7.24

(m, 2H, Ar); 7.49 (d, 1H, Ar); 7.62 (t, 1H, Ar); 7.71 (d, 1H, Ar);

7.93 (d, 1H, Ar); 8.66 (d, 1H, Ar); 9.23 (d, 1H, –NH–CH); 11.95

(s, 1H, –NH–); 12.32 (s, 1H, –NH–); ¹³C NMR (DMSO-d₆) d 13.88,

16.24, 48.42, 60.45, 102.41, 112.33, 119.23, 119.81, 120.03,

121.58, 122.38, 123.89, 126.78, 128.48, 131.42, 132.41, 136.96,

139.06, 158.99, 168.65, 172.08.

6.3.20. 2-(R,S)-(2-Amino-benzoylamino)-pentanedioic acid

diethyl ester (8b)

A solution of 4.80 g (20.0 mmol) of ^DL-glutamic acid diethyl es-

ter hydrochloride 8a and 2.78 mL (20.0 mmol) of TEA in 100 mL of

CH₂Cl₂was cooled to 0 °C, with stirring. o-Nitrobenzoyl chloride

(3.71 g, 20.0 mmol) was added and the mixture was stirred for 2

h at rt. The organic phase was washed with 0.1 N NaOH, H₂O, brine,

dried over anhydrous Na₂SO₄and evaporated. The crude product

was dissolved in 100 mL of CH₂Cl₂and 10 g of Zn were added.

The suspension was cooled to 0 °C and 12 mL of acetic acid were

added dropwise under stirring. The reaction mixture was stirred

for 1 h and ﬁltered. The organic phase was evaporated and the res-

idue was triturated with cold petroleum ether 40–60° to give 3.51

g (54%) of the titled compound. TLC (EtOAc/n-hexane 1:1). R_f0.57;

mp 78–80 °C; ¹H NMR (CDCl₃) d 1.22 (t, 3H, –CH₃); 1.29 (t, 3H,

–CH₃); 2.08–2.50 (m, 2H, –CH₂–CH); 2.46 (m, 2H, –CH₂–CO–);

4.10 (q, 2H, –CH₂–O–); 4.24 (q, 2H, –CH₂–O–); 4.74 (m, 1H,

–CH); 5.55 (b, 2H, –NH₂); 6.65 (m, 2H, Ar); 6.88 (d, 1H, –NH–);

7.20 (t, 1H, Ar); 7.40 (d, 1H, Ar); ¹³C NMR (CDCl₃) d 14.20, 27.32,

30.53, 52.05, 60.83, 61.72, 114.93, 116.58, 117.31, 127.49, 132.66,

149.03, 169.04, 172.12, 173.18.

6.4.3. 2-(R,S)-{2-[(1H-Indole-2-carbonyl)-amino]-benzoyl-

amino}-3-methyl-butyric acid ethyl ester (3c)

Trituration with hot MeOH followed by ﬂash chromatography

(CH₂Cl₂to EtOAc/CH₂Cl₂1:4) afforded the titled compound in

54% yield. TLC (EtOAc/n-hexane 1:1). R_f0.71; mp 211 °C; ¹H

NMR (DMSO-d₆) d 0.96 (m, 6H, –CH(CH₃)₂); 1.17 (t, 3H, –CH₂–

CH₃); 2.20 (m, 1H, –CH(CH₃)₂); 4.11 (q, 2H, –CH₂–); 4.31 (m, 1H,

–NH–CH); 7.00 (s, 1H, Ar); 7.07 (d, 1H, Ar); 7.20 (m, 2H, Ar);

7.44 (d, 1H, Ar); 7.58 (t, 1H, Ar); 7.66 (d, 1H, Ar); 7.89 (d, 1H,

Ar); 8.57 (d, 1H, Ar); 9.01 (d, 1H, –NH–CH); 11.90 (s, 1H, –NH–

); 12.06 (s, 1H, –NH–); ¹³C NMR (DMSO-d₆) d 14.94, 19.89, 30.10,

59.67, 61.22, 103.23, 113.23, 120.63, 120.74, 120.94, 122.47,

123.35, 124.82, 127.63, 129.79, 132.22, 133.25, 137.80, 139.57,

159.79, 170.01, 171.97.

6.4.4. 2-(R,S)-{2-[(1H-Indole-2-carbonyl)-amino]-benzoyl-

amino}-4-methyl-pentanoic acid ethyl ester (4c)

Trituration with hot MeOH afforded the titled compound in 67%

yield. TLC (EtOAc/n-hexane 1:1). R_f. R_f0.73; mp 202–203 °C; ¹H

NMR (CDCl₃) d 0.96 (dd, 6H, –CH(CH₃)₂); 1.34 (t, 3H, –CH₂–CH₃);

1.79 (m, 3H, –CH₂–CH(CH₃)₂); 4.28 (q, 2H, –CH₂–O–); 4.94 (m,

1H, –NH–CH); 7.05–7.31 (m, 5H, Ar and –NH–CH); 7.50 (m,

2H, Ar); 7.68 (m, 2H, Ar); 8.80 (d, 1H, Ar); 10.00 (s, 1H, –NH–);

12.22 (s, 1H, –NH–); ¹³C NMR (CDCl₃) d 14.26, 22.09, 22.97,

25.14, 41.59, 51.34, 61.80, 103.93, 112.11, 119.48, 120.61, 121.21,

122.33, 122.84, 124.69, 126.99, 127.86, 131.45, 133.04, 136.94,

139.90, 160.16, 168.99, 173.24.

6.4. General procedure for the synthesis of derivatives 1c–9c,

11c, 14, 16–18, 19c–25c

To a suspension of 10.0 mmol of indole-2-carboxylic acid (for

synthesis of compound 1c–5c, 7c–9c, 11c, 14, 16–18, 19c–25c) or

N-methylindole-2-carboxylic acid (for synthesis of compound 6c)

in 30 mL of acetyl chloride cooled in an ice-bath were added por-

tionwise, over a period of 0.5 h, 2.08 g (10.0 mmol) of PCl₅. 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 7.50 mmol of the deriv-

atives 1b–5b, 7b–9b, 14b, 16b–25b in 10 mL of pyridine. After the

addition was complete, the reaction mixture was stirred at room

temperature until completion (TLC monitoring). Compounds 1c,

2c and 5c precipitated from the reaction mixture and were ﬁltered.

For the others compound, CH₂Cl₂(150 mL) was added and the or-

ganic layer was washed twice with 40 mL of 1 N HCl, H₂O, 1 N

NaOH and brine. After drying over anhydrous Na₂SO₄, the organic

phase was rotary evaporated and the residue was puriﬁed as de-

scribed to yield the titled compounds.

6.4.5. 2-(R,S)-{2-[(1H-Indole-2-carbonyl)-amino]-benzoylamino}-

4-methylsulfanyl-butyric acid ethyl ester (5c)

Crystallization with MeOH afforded the titled compound in 85%

yield. TLC (EtOAc/n-hexane 1:1). R_f0.49; mp 216–218 °C; ¹H NMR

(DMSO-d₆) d 1.32 (t, 3H, –CH₂–CH₃); 2.07 (s, 3H, CH₃–S–); 2.14 (m,

2H, –CH₂–CH); 2.62 (m, 2H, –CH₂–S–); 4.15 (q, 2H, –CH₂–O–);

4.67 (m, 1H, –NH–CH); 7.09 (m, 2H, Ar); 7.25 (m, 2H, Ar); 7.49

(d, 1H, Ar); 7.59–7.73 (m, 2H, Ar); 7.94 (d, 1H, Ar); 8.65 (d, 1H,

Ar); 9.21 (d, 1H, –NH–CH); 11.95 (s, 1H, –NH–); 12.24 (s, 1H,

–NH–); ¹³C NMR (DMSO-d₆) d 13.89, 14.36, 29.62, 29.73, 51.76,

60.62, 102.40, 112.33, 119.34, 119.83, 120.04, 121.59, 122.44,

123.91, 126.77, 128.57, 131.38, 132.48, 136.95, 138.96, 158.97,

169.13, 171.35.

6.4.1. {2-[(1H-Indole-2-carbonyl)-amino]-benzoylamino}-

acetic acid ethyl ester (1c)

Trituration with cold petroleum ether 40–60° afforded the titled

compound in 76% yield. TLC (EtOAc/n-hexane 1:1). R_f0.52; mp

244–246 °C; ¹H NMR (DMSO-d₆) d 1.22 (t, 3H, –CH₃); 4.12 (m,

4H, –CH₂–CO– and –CH₂–O–); 7.07 (m, 2H, Ar); 7.24 (m, 2H, Ar);

6.4.6. 2-(R,S)-{2-[(1-Methyl-1H-indole-2-carbonyl)-amino]-

benzoylamino}-4-methylsulfanyl-butyric acid ethyl ester (6c)

The titled compound wasn’t isolated but used in the next step

without puriﬁcation and characterization.

L. Lassiani et al. / Bioorg. Med. Chem. 17 (2009) 2336–2350

2345

6.4.7. 2-(R,S)-{2-[(1H-Indole-2-carbonyl)-amino]-benzoylamino}-

succinic acid diethyl ester (7c)

¹H NMR¹H NMR (DMSO-d₆) d 1.23 (d, 3H, –CH₃); 2.87 (m, 2H,

–CH₂–); 4.40 (m, 1H, –CH); 7.03 (s, 1H, Ar); 7.07–7.28 (m,

8H, ar); 7.54 (m, 2H, Ar); 7.78 (m, 2H, Ar); 8.63 (d, 1H, Ar);

8.76 (d, 1H, –NH–CH); 11.93 (s, 1H, –NH–); 12.48 (s, 1H,

Trituration with hot MeOH afforded the titled compound in 90%

yield. TLC (EtOAc/n-hexane 1:1) R_f0.56; mp 199–200 °C; ¹H NMR

(DMSO-d₆) d 1.19 (m, 6H, –CH₃); 2.93 (m, 2H, –CH₂–CH); 4.10

(m, 4H, –CH₂–O–); 4.92 (m, 1H, –CH); 7.06 (s, 1H, Ar); 7.11 (d,

1H, Ar); 7.25 (m, 2H, Ar); 7.48 (d, 1H, Ar); 7.63 (t, 1H, Ar); 7.73

(d, 1H, Ar); 7.84 (d, 1H, Ar); 8.64 (d, 1H, Ar); 9.33 (d, 1H, –NH–

CH); 11.94 (s, 1H, –NH–); 12.24 (s, 1H, –NH–); ¹³C NMR

(DMSO-d₆) d 14.76, 14.81, 36.12, 50.12, 61.14, 61.88, 103.33,

113.22, 119.98, 120.83, 120.95, 122.56, 123.41, 124.83, 127.66,

129.19, 132.20, 133.52, 137.83, 139.92, 159.85, 169.39, 170.62,

170.98.

–NH–); ¹³C NMR (DMSO-d₆)

d 21.06, 42.38, 47.49, 103.33,

113.20, 120.57, 120.76, 120.88, 122.56, 123.16, 124.73, 126.76,

127.69, 128.78, 128.91, 129.81, 132.36, 132.86, 137.78, 139.75,

139.87, 159.79, 168.44. MS (ES) m/z 398.1 [MH]⁺; MW: 397.48

(calcd. for C₂₅H₂₃N₃O₂).

6.4.13. 1H-Indole-2-carboxylic acid (2-phenethylcarbamoyl-

phenyl)-amide (17)

Trituration with hot CHCl₃afforded the titled compound in 65%

yield. TLC (EtOAc/n-hexane 1:1). R_f0.63; mp 257–259 °C; ¹H NMR

(DMSO-d₆) d 2.92 (t, 2H, –CH₂–C₆H₅); 3.58 (m, 2H, –CH₂–NH–);

7.07 (s, 1H, Ar); 7.11–7.30 (m, 8H, Ar); 7.54 (m, 2H, Ar); 7.79 (m,

2H, Ar); 8.68 (d, 1H, Ar); 9.02 (t, 1H, –NH–CH₂-); 11.95 (s, 1H,

–NH–); 12.64 (s, 1H, –NH–); ¹³C NMR (DMSO-d₆) d 34.60, 40.59,

102.48, 112.32, 119.59, 119.76, 120.00, 121.68, 122.35, 123.86,

125.95, 126.86, 127.95, 128.11, 128.51, 131.52, 132.09, 136.94,

139.18, 159.00, 168.37. MS (ES) m/z 384.2 [MH]⁺; MW: 383.45

(calcd for C₂₄H₂₁N₃O₂).

6.4.8. 2-(R,S)-{2-[(1H-Indole-2-carbonyl)-amino]-benzoylamino}-

pentanedioic acid diethyl ester (8c)

Trituration with hot MeOH afforded the titled compound in 86%

yield. TLC (EtOAc/n-hexane 1:1). R_f0.56; mp 195–197 °C; ¹H NMR

(CDCl₃) d 1.21 (t, 3H, –CH₃); 1.32 (t, 3H, –CH₃); 2.19–2.60 (m, 2H,

–CH₂–CH); 2.54 (m, 2H, –CH₂–CO-); 4.12 (q, 2H, –CH₂–O–); 4.27

(q, 2H, –CH₂–O–); 4.86 (m, 1H, –CH); 7.07–7.32 (m, 4H, Ar);

7.49 (m, 3H, Ar and –NH–CH); 7.69 (m, 2H, Ar); 8.83 (d, 1H,

Ar); 9.86 (s, 1H, –NH–); 12.36 (s, 1H, –NH–); ¹³C NMR (CDCl₃) d

14.17, 14.21, 26.94, 30.51, 52.62, 61.06, 62.02, 103.90, 112.04,

118.89, 120.62, 121.19, 122.35, 122.84, 124.69, 127.07, 127.88,

131.50, 133.21, 136.87, 140.18, 160.11, 169.09, 171.77, 173.39.

6.4.14. 1H-Indole-2-carboxylic acid {2-[2-(1H-indol-3-yl)-

ethylcarbamoyl]-phenyl}-amide (18)

Trituration with EtOH and with hot isopropyl acetate affor-

ded the titled compound in 49% yield. TLC (EtOAc/n-hexane

1:1). R_f0.46; mp 254–256 °C; ¹H NMR (DMSO-d₆) d 3.05 (t,

2H, –CH₂–CH₂–NH–); 3.65 (m, 2H, –CH₂–NH–); 7.00–7.38 (m,

8H, Ar); 7.49–7.65 (m, 3H, Ar); 7.75 (d, 1H, Ar); 7.85 (d, 1H,

Ar); 8.70 (d, 1H, Ar); 9.08 (t, 1H, –CH₂–NH-); 10.87 (s, 1H,

6.4.9. 2-(R,S)-{2-[(1H-Indole-2-carbonyl)-amino]-benzoylamino}-

3-(1H-indol-3-yl)-propionic acid ethyl ester (9c)

Crystallization with MeOH afforded the titled compound in 85%

yield. TLC (EtOAc/n-hexane 1:1). R_f0.49; mp 216–218 °C; ¹H NMR

(DMSO-d₆) d 1.16 (t, 3H, –CH₃); 3.37 (m, 2H, –CH₂–CH); 4.13 (q,

2H, –CH₂–O–); 4.78 (m, 1H, –CH); 6.98–7.35 (m, 8H, Ar); 7.46–

7.69 (m, 4H, Ar); 7.84 (d, 1H, Ar); 8.67 (d, 1H, Ar); 9.23 (d, 1H,

–NH–CH); 10.85 (s, 1H, –NH–); 11.89 (s, 1H, –NH–); 12.28 (s, 1H,

–NH–); ¹³C NMR (DMSO-d₆) d 13.81, 26.25, 53.86, 60.47, 102.33,

109.60, 111.26, 112.28, 117.78, 118.20, 118.95, 119.66, 119.81,

120.78, 121.52, 122.14, 123.45, 123.75, 126.77, 126.85, 128.39,

131.40, 132.33, 135.96, 136.93, 139.19, 158.94, 168.79, 171.37.

–NH–); 11.95 (s, 1H, –NH–); ¹³C NMR (DMSO-d₆)

d 24.62,

40.13, 102.40, 111.22, 111.52, 112.32, 118.06, 119.55, 119.74,

120.00, 120.77, 121.68, 122.31, 122.52, 123.85, 126.86,

127.09, 128.02, 131.59, 132.09, 136.06, 136.93, 139.28,

159.01, 168.40. MS (ES) m/z 423.1 [MH]⁺; MW: 422.49 (calcd

for C₂₆H₂₂N₄O₂).

6.4.15. 3-{2-[(1H-Indole-2-carbonyl)-amino]-benzoylamino}-

propionic acid ethyl ester (19c)

6.4.10. (2R,S)-3-(4-Benzyloxy-phenyl)-2-{2-[(1H-indole-2-carbonyl)-

amino]-benzoylamino}-propionic acid benzyl ester (11c)

Trituration with MeOH, with cold diethyl ether followed by

ﬂash chromatography (petroleum ether 40–60°/CH₂Cl₂1:1 to

CH₂Cl₂to EtOAc/CH₂Cl₂1:9) afforded the titled compound in 26%

yield. TLC (EtOAc/n-hexane 1:1). R_f0.52; mp 179–180 °C; ¹H

NMR (DMSO-d₆) d 3.15 (m, 2H, –CH₂–CH); 4.84 (m, 1H, –CH);

4.94 (s, 2H, –CH₂–O–); 5.16 (s, 2H, –CH₂–O–); 6.87–6.98 (m, 3H,

Ar); 7.06–7.13 (m, 2H, Ar); 7.21–7.33 (m, 13H, Ar); 7.48 (d, 1H,

Ar); 7.61 (t, 1H, Ar); 7.70 (d, 1H, Ar); 7.80 (d, 1H, Ar); 8.64 (d,

1H, Ar); 9.32 (d, 1H, –NH–CH); 11.95 (s, 1H, –NH–); 12.21 (s,

1H, –NH–); ¹³C NMR (DMSO-d₆) d 35.16, 54.51, 66.08, 68.94,

102.47, 112.41, 114.39, 118.97, 119.77, 120.11, 121.73, 122.46,

124.01, 126.84, 127.53, 127.61, 127.87, 128.21, 128.37, 129.29,

130.05, 131.39, 132.64, 135.69, 136.90, 137.01, 139.11, 156.94,

158.96, 168.80, 171.03.

Trituration with hot MeOH followed by ﬂash chromatography

(CH₂Cl₂to EtOAc/CH₂Cl₂1:4) afforded the titled compound in

67% yield. TLC (EtOAc/n-hexane 1:1). R_f0.51; mp 204 °C; ¹H NMR

(DMSO-d₆) d1.13 (t, 3H, –CH₃); 2.61 (t, 2H, –NH–CH₂–CH₂-); 3.55

(m, 2H, –NH–CH₂–CH₂-); 4.03 (q, 2H, -O–CH₂-); 7.02–7.25 (m,

4H, Ar); 7.44 (d, 1H, Ar); 7.55 (t, 1H, Ar); 7.71 (d, 1H, Ar); 7.79

(d, 1H, Ar); 8.62 (d, 1H, Ar); 8.96 (t, 1H, –NH–CH₂–CH₂-); 11.89

(s, 1H, –NH–); 12.60 (s, 1H, –NH–); ¹³C NMR (DMSO-d₆) d 14.86,

34.29, 36.38, 60.77, 103.29, 113.20, 120.13, 120.65, 120.89,

122.58, 123.25, 124.76, 127.71, 128.95, 132.36, 133.15, 137.79,

140.11, 159.85, 169.37, 171.88.

6.4.16. 4-{2-[(1H-Indole-2-carbonyl)-amino]-benzoylamino}-

butyric acid ethyl ester (20c)

Trituration with hot MeOH followed by ﬂash chromatography

(CH₂Cl₂to EtOAc/CH₂Cl₂1:4) afforded the titled compound in

80% yield. TLC (EtOAc/n-hexane 1:1). R_f0.59; mp 210 °C; ¹H NMR

(DMSO-d₆) d 1.16 (t, 3H, –CH₃); 1.85 (m, 2H, –NH–CH₂–CH₂–CH₂-);

2.41 (t, 2H, –NH–CH₂–CH₂–CH₂–); 3.39 (m, 2H, –NH–CH₂–CH₂–

CH₂-); 4.04 (q, 2H, –O–CH₂-); 7.07–7.29 (m, 4H, Ar); 7.49 (d, 1H,

Ar); 7.60 (t, 1H, Ar); 7.74 (d, 1H, Ar); 7.88 (d, 1H, Ar); 8.67 (d,

1H, Ar); 8.97 (t, 1H, –NH–CH₂–); 11.95 (s, 1H, –NH–), 12.75 (s,

1H, –NH–); ¹³C NMR (DMSO-d₆) d 14.86, 24.97, 31.81, 60.53,

103.25, 113.20, 120.32, 120.64, 120.87, 122.52, 123.19, 124.71,

127.73, 128.94,132.46, 132.99, 137.82, 140.15, 159.86, 169.35,

173.26.

6.4.11. 2-(R,S)-{2-[(1H-Indole-2-carbonyl)-amino]-benzoylamino}-

3-phenyl-propionic acid ethyl ester (14)

Obtained as previously described.²⁶

6.4.12. (1R,S)-1H-Indole-2-carboxylic acid [2-(1-methyl-2-

phenyl-ethylcarbamoyl)-phenyl]-amide (16)

Trituration with hot MeOH followed by ﬂash chromatography

(CH₂Cl₂to EtOAc/CH₂Cl₂1:4) afforded the titled compound in

64% yield. TLC (EtOAc/n-hexane 1:1). R_f0.73; mp 248–249 °C;

2346

L. Lassiani et al. / Bioorg. Med. Chem. 17 (2009) 2336–2350

6.4.17. 3-(R,S)-{2-[(1H-Indole-2-carbonyl)-amino]-benzoyl-

amino}-4-phenyl-butyric acid ethyl ester (21c)

119.81, 120.03, 121.60, 122.46, 123.93, 126.74, 128.53, 131.32,

132.44, 136.96, 138.96, 158.91, 169.18, 171.32.

Puriﬁcation by ﬂash chromatography (CH₂Cl₂to EtOAc/CH₂Cl₂

1:4) afforded the titled compound in 74% yield. TLC (EtOAc/n-hex-

ane 1:1). R_f0.57; mp 211 °C; ¹H NMR (DMSO-d₆) d 1.09 (t, 3H

–CH₃); 2.64 (m, 2H, –CH₂–CO–); 2.92 (m, 2H, –CH₂–C₆H₅); 3.99

(q, 2H, –CH₂–O–); 4.68 (m, 1H, –CH); 6.95 (s, 1H, Ar); 7.07–7.27

(m, 8H, Ar); 7.48 (d, 1H, Ar); 7.56 (t, 1H, Ar); 7.72 (m, 2H, Ar);

8.60 (d, 1H, Ar); 8.81 (d, 1H, –NH–CH); 11.89 (s, 1H, –NH–);

12.18 (s, 1H, –NH–); ¹³C NMR (DMSO-d₆) d 14.18, 48.33, 60.13,

102.79, 112.60, 119.94, 120.22, 120.29, 121.91, 122.57, 124.13,

126.35, 127.07, 128.22, 129.28, 131.66, 132.32, 137.20, 138.41,

139.11, 159.16, 168.02, 170.72.

6.5. General procedure for the synthesis of compounds 1–9, 11,

19–21

A mixture of 4.0 mmol of the corresponding ethyl ester (com-

pounds 1c–9c, 11c and 19c–21c) in water (25 mL) and tetrahydro-

furan (25 mL) was stirred in the presence of KOH (0.22 g, 4.0

mmol) at room temperature (under reﬂux for compound 6) until

completion (TLC monitoring). The organic solvent was removed

under reduced pressure and, after cooling to 0 °C, the aqueous

solution was adjusted to pH 2–3 with diluted HCl to obtain the pre-

cipitation of the corresponding acid.

6.4.18. 2(S)-{2-[(1H-Indole-2-carbonyl)-amino]-benzoylamino}-

4-methyl-pentanoic acid ethyl ester (22c)

6.5.1. {2-[(1H-Indole-2-carbonyl)-amino]-benzoylamino}-

acetic acid (1)

Trituration with hot MeOH afforded the titled compound in 62%

yield. TLC (EtOAc/n-hexane 1:1). R_f0.73; ½a _D²⁵

ꢄ

= +26.5 (c 0.54,

d 0.96 (dd, 6H,

Trituration with hot MeOH afforded the titled compound in 47%

yield. TLC (EtOAc/MeOH 3:2). R_f0.40; mp 264–265 °C; ¹H NMR

(DMSO-d₆) d 4.05 (d, 2H, –CH₂-); 7.09 (m, 2H, Ar); 7.24 (m, 2H,

Ar); 7.49 (d, 1H, Ar); 7.63 (t, 1H, Ar); 7.75 (d, 1H, Ar); 7.93 (d,

1H, Ar); 8.72 (d, 1H, Ar); 9.32 (t, 1H, –NH–CH₂-); 11.95 (s, 1H,

–NH–); 12.62 (s, 1H, –NH–); ¹³C NMR (DMSO-d₆) d 41.12, 102.40,

112.32, 118.63, 118.76, 120.02, 121.71, 122.43, 123.91, 126.81,

128.09, 131.45, 132.56, 136.94, 139.44, 159.02, 168.92, 170.70.

MS (ES) m/z 336.1 [MH]⁺; MW: 337.34 (calcd for C₁₈H₁₅N₃O₄).

CH₂Cl₂); mp 205–206 °C; ¹H NMR (CDCl₃)

–CH(CH₃)₂); 1.32 (t, 3H, –CH₂–CH₃); 1.74 (m, 3H, –CH₂–CH(CH₃)₂);

4.20 (q, 2H, –CH₂–O–); 4.87 (m, 1H, –NH–CH); 7.01–7.35 (m, 5H,

Ar and –NH–CH); 7.50 (m, 2H, Ar); 7.67 (m, 2H, Ar); 8.80 (d, 1H,

Ar); 10.03 (s, 1H, –NH–); 12.20 (s, 1H, –NH–); ¹³C NMR (CDCl₃) d

14.24, 22.15, 23.01, 25.14, 41.65, 51.37, 61.80, 103.95, 112.15,

119.40, 120.50, 121.25, 122.30, 122.90, 124.75, 127.05, 127.97,

131.48, 133.10, 136.85, 139.90, 160.24, 169.00, 173.20.

6.4.19. 2(R)-{2-[(1H-Indole-2-carbonyl)-amino]-benzoylamino}-

4-methyl-pentanoic acid ethyl ester (23c)

6.5.2. 2-(R,S)-{2-[(1H-Indole-2-carbonyl)-amino]-benzoylamino}-

propionic acid (2)

Trituration with hot MeOH afforded the titled compound in 71%

Trituration with hot MeOH afforded the titled compound in 79%

yield. TLC (EtOAc/MeOH 2:1). R_f034; mp 274–276 °C; ¹H NMR

(DMSO-d₆) d 1.47 (d, 3H, –CH₃); 4.56 (m, 1H, –CH); 7.10 (m, 2H,

Ar); 7.24 (m, 2H, Ar); 7.50 (d, 1H, Ar); 7.62 (t, 1H, Ar); 7.75 (d,

1H, Ar); 7.97 (d, 1H, Ar); 8.69 (d, 1H, Ar); 9.13 (d, 1H, –NH–CH);

11.95 (s, 1H, –NH–); 12.49 (s, 1H, –NH–); ¹³C NMR (DMSO-d₆) d

16.46, 48.16, 102.42, 112.33, 119.14, 119.78, 120.02, 121.69,

122.33, 123.89, 126.83, 128.50, 131.48, 132.37, 136.96, 139.22,

159.03, 168.48, 173.62. MS (ES) m/z 350.2 [MꢅH]^ꢅ, 352.1 [MH]⁺;

MW: 351.37 (calcd for C₁₉H₁₇N₃O₄).

yield. TLC (EtOAc/n-hexane 1:1). R_f0.73; ½a _D²⁵

ꢄ

= -24.8 (c 0.15,

d 0.95 (dd, 6H,

CH₂Cl₂); mp 201–202 °C; ¹H NMR (CDCl₃)

–CH(CH₃)₂); 1.30 (t, 3H, –CH₂–CH₃); 1.75 (m, 3H, –CH₂–CH(CH₃)₂);

4.25 (q, 2H, –CH₂–O–); 4.90 (m, 1H, –NH–CH); 7.03–7.33 (m, 5H,

Ar and –NH–CH); 7.52 (m, 2H, Ar); 7.66 (m, 2H, Ar); 8.82 (d, 1H,

Ar); 10.05 (s, 1H, –NH–); 12.25 (s, 1H, –NH–); ¹³C NMR (CDCl₃) d

14.22, 22.12, 22.99, 25.18, 41.62, 51.38, 61.82, 103.98, 112.09,

119.44, 120.68, 121.28, 122.30, 122.86, 124.71, 126.92, 127.81,

131.40, 133.08, 136.90, 139.94, 160.13, 168.92, 173.25.

6.4.20. 2(S)-{2-[(1H-Indole-2-carbonyl)-amino]-benzoylamino}-

4-methylsulfanyl-butyric acid ethyl ester (24c)

6.5.3. 2-(R,S)-{2-[(1H-Indole-2-carbonyl)-amino]-benzoylamino}-

3-methyl-butyric acid (3)

Crystallization with MeOH afforded the titled compound in 76%

Trituration with hot abs. EtOH afforded the titled compound in

86% yield. TLC (EtOAc/MeOH 2:1). R_f0.58; mp 276 °C; ¹H NMR

(DMSO-d₆) d 0.99 (m, 6H, –CH(CH₃)₂); 2.25 (m, 1H, –CH(CH₃)₂);

4.38 (m, 1H, –NH–CH); 7.05 (s, 1H, Ar); 7.10 (d, 1H, Ar); 7.25

(m, 2H, Ar); 7.49 (d, 1H, Ar); 7.61 (t, 1H, Ar); 7.73 (d, 1H, Ar);

7.97 (d, 1H, Ar); 8.60 (d, 1H, Ar); 8.92 (d, 1H, –NH–CH); 11.90

(s, 1H, –NH–); 12.23 (s, 1H, –NH–); ¹³C NMR (DMSO-d₆) d 19.65,

20.08, 30.13, 59.25, 103.27, 113.21, 120.61, 120.70, 120.90,

122.57, 123.30, 124.79, 127.67, 129.75, 132.26, 133.18, 137.81,

139.69, 159.83, 169.79, 173.37. MS (ES) m/z 380.0 [MH]⁺; MW:

379.42 (calcd for C₂₁H₂₁N₃O₄).

yield. TLC (EtOAc/n-hexane 1:1). R_f0.49; ½a _D²⁵

ꢄ

= +48.7 (c 0.65,

CH₂Cl₂); mp 220–222 °C; ¹H NMR (DMSO-d₆) d 1,29 (t, 3H,

–CH₂–CH₃); 2.10 (s, 3H, CH₃–S–); 2.12 (m, 2H, –CH₂–CH); 2.68

(m, 2H, –CH₂–S–); 4.18 (q, 2H, –CH₂–O–); 4.70 (m, 1H, ACH);

7.10 (m, 2H, Ar); 7.30 (m, 2H, Ar); 7.50 (d, 1H, Ar); 7.60–7.75 (m,

2H, Ar); 7.98 (d, 1H, Ar); 8.63 (d, 1H, Ar); 9.25 (d, 1H, –NH–CH);

11.90 (s, 1H, –NH–); 12.21 (s, 1H, –NH–); ¹³C NMR (DMSO-d₆) d

13.84, 14.32, 29.60, 29.71, 51.73, 60.65, 102.39, 112.36, 119.31,

119.87, 120.08, 121.61, 122.42, 123.97, 126.72, 128.53, 131.41,

132.50, 136.90, 138.92, 158.93, 169.17, 171.30.

6.4.21. 2(R)-{2-[(1H-Indole-2-carbonyl)-amino]-benzoylamino}-

4-methylsulfanyl-butyric acid ethyl ester (25c)

6.5.4. 2-(R,S)-{2-[(1H-Indole-2-carbonyl)-amino]-benzoylamino}-

4-methyl-pentanoic acid (4)

Crystallization with MeOH afforded the titled compound in 80%

Crystallization with MeOH afforded the titled compound in 23%

yield. TLC (EtOAc/MeOH 3:1). R_f0.51; mp 257–259 °C; ¹H NMR

(DMSO-d₆) d (dd, 6H, –CH(CH₃)₂); 1.64–1.80 (m, 3H, –CH₂–

CH(CH₃)₂); 4.56 (m, 1H, –NH–CH); 7.09 (m, 2H, Ar); 7.25 (m,

2H, Ar); 7.49 (d, 1H, Ar); 7.62 (t, 1H, Ar); 7.74 (d, 1H, Ar); 7.96

(d, 1H, Ar); 8.67 (d, 1H, Ar); 9.07 (d, 1H, –NH–CH); 11.95 (s, 1H,

–NH–); 12.43 (s, 1H, –NH–); ¹³C NMR (DMSO-d₆) d 20.92, 22.78,

24.44, 50.83, 102.35, 112.32, 119.24, 119.79, 120.02, 121.69,

122.38, 124.00, 126.80, 128.61, 131.43, 132.41, 136.94, 139.11,

yield. TLC (EtOAc/n-hexane 1:1). R_f0.49; ½a _D²⁵

¼ ꢅ45:8 (c 0.45,

ꢄ

CH₂Cl₂); mp 214–215 °C; ¹H NMR (DMSO-d₆) d 1,30 (t, 3H,

–CH₂–CH₃); 2.06 (s, 3H, CH₃–S–); 2.10 (m, 2H, –CH₂–CH); 2.64

(m, 2H, –CH₂–S–); 4.14 (q, 2H, –CH₂–O–); 4.65 (m, 1H, ACH);

7.07 (m, 2H, Ar); 7.27 (m, 2H, Ar); 7.47 (d, 1H, Ar); 7.56–7.71 (m,

2H, Ar); 7.96 (d, 1H, Ar); 8.64 (d, 1H, Ar); 9.23 (d, 1H, –NH–CH);

11.93 (s, 1H, –NH–); 12.23 (s, 1H, –NH–); ¹³C NMR (DMSO-d₆) d

13.87, 14.34, 29.60, 29.75, 51.75, 60.62, 102.42, 112.30, 119.31,

L. Lassiani et al. / Bioorg. Med. Chem. 17 (2009) 2336–2350

2347

159.00, 168.80, 173.58. MS (ES) m/z 392.1 [MꢅH]^ꢅ; MW: 393.45

26.32, 53.78, 102.42, 110.19, 11.29, 112.32, 117.96, 118.22, 118.96,

119.65, 120.00, 120.80, 121.74, 122.28, 123.42, 123.89, 126.81,

126.95, 128.35, 131.42, 132.42, 135.94, 136.94, 139.23, 158.98,

168.60, 172.98. MS (ES) m/z 465.2 [MꢅH]^ꢅ; MW: 466.50 (calcd for

C₂₇H₂₂N₄O₄).

(calcd for C₂₂H₂₃N₃O₄).

6.5.5. 2-(R,S)-{2-[(1H-Indole-2-carbonyl)-amino]-benzoylamino}-

4-methylsulfanyl-butyric acid (5)

Trituration with MeOH afforded the titled compound in 59%

yield. TLC (EtOAc/MeOH 2:1). R_f0.38; mp 250–251 °C; ¹H NMR

(DMSO-d₆) d 2.07 (s, 3H, CH₃–S–); 2.14 (m, 2H, –CH₂–CH); 2.62

(m, 2H, –CH₂–S–); 4.67 (m, 1H, –CH); 7.09 (m, 2H, Ar); 7.26 (m,

2H, Ar); 7.50 (d, 1H, Ar); 7.63 (t, 1H, Ar); 7.75 (d, 1H, Ar); 7.98

(d, 1H, Ar); 8.69 (d, 1H, Ar); 9.11 (d, 1H, –NHACH); 11.95 (s, 1H,

–NH–); 12.42 (s, 1H, –NH–); ¹³C NMR (DMSO-d₆) d 14.37, 29.74,

29.97, 51.55, 102.42, 112.33, 119.23, 119.79, 120.03, 121.68,

122.37, 123.91, 126.81, 128.53, 131.44, 132.43, 136.96, 139.13,

159.01, 168.99, 172.89. MS (ES) m/z 410.2 [MꢅH]^ꢅ, 412.1 [MH]⁺;

MW: 411.48 (calcd for C₂₁H₂₁N₃O₄S).

6.5.10. (2R,S)-3-(4-Benzyloxy-phenyl)-2-{2-[(1H-indole-2-carbo-

nyl)-amino]-benzoylamino}-propionic acid (11)

Trituration with hot abs. EtOH afforded the titled compound in

76% yield. TLC (EtOAc/MeOH 2:1). R_f0.59; mp 263–264 °C; ¹H

NMR (DMSO-d₆) d 3.18 (m, 2H, –CH₂–CH); 4.73 (m, 1H, –CH);

4.91 (m, 2H, -O–CH₂-); 6.86–6.96 (m, 3H, Ar); 7.06–7.17 (m, 2H,

Ar); 7.21–7.31 (m, 8H, Ar); 7.47 (d, 1H, Ar); 7.59 (t, 1H, Ar); 7.74 (d,

1H, Ar); 7.83 (d, 1H, Ar); 8.65 (d, 1H, Ar); 9.14 (d, 1H, –NH–CH);

11.93 (s, 1H, –NH–); 12.26 (s, 1H, –NH–); 12.99 (b, 1H, -OH); ¹³C

NMR (DMSO-d₆) d 54.32, 68.94, 102.49, 112.42, 114.33, 119.03,

119.67, 120.12, 121.80, 122.38, 123.99, 126.85, 127.56, 128.21,

129.99, 131.42, 132.54, 137.01, 139.18, 152.57, 156.83, 158.96,

168.59, 172.66. MS (ES) m/z 532.1 [MꢅH]^ꢅ; MW: 533.59 (calcd for

C₃₂H₂₇N₃O₅).

6.5.6. 2-(R,S)-{2-[(1-Methyl-1H-indole-2-carbonyl)-amino]-

benzoylamino}-4-methylsulfanyl-butyric acid (6)

Crystallization with MeOH afforded the titled compound in 60%

yield. TLC (EtOAc/MeOH 2:1). R_f0.48; mp 205–206 °C; ¹H NMR

(DMSO-d₆) d 2.05 (s, 3H, CH₃–S–); 2.12 (m, 2H, –CH₂–CH); 2.60

(m, 2H, –CH₂–S–); 4.06 (s, 3H, N–CH₃); 4.61 (m, 1H, ACH); 7.12

(s, 1H, Ar); 7.16–7.30 (m, 2H, Ar); 7.34 (t, 1H, Ar); 7.61 (m, 2H,

Ar); 7.75 (d, 1H, Ar); 7.93 (d, 1H, Ar); 8.61 (d, 1H, Ar); 9.08 (d, 1H,

–NH–CH); 12.23 (s, 1H, –NH–); 12.90 (b, 1H, -OH); ¹³C NMR

(DMSO-d₆) d 14.35, 29.72, 29.91, 31.35, 51.49, 104.46, 110.54,

119.75, 119.89, 120.36, 121.75, 122.55, 124.12, 125.26, 128.49,

131.84, 132.29, 138.91, 159.65, 168.90, 172.85. MS (ES) m/z 424.2

[MꢅH]^ꢅ, 426.1 [MH]⁺; MW: 425.51 (calcd for C₂₂H₂₃N₃O₄S).

6.5.11. 3-{2-[(1H-Indole-2-carbonyl)-amino]-benzoylamino}-

propionic acid (19)

Trituration with hot ethyl acetate afforded the titled compound

in 65% yield. TLC (EtOAc/MeOH 2:1). R_f0.51; mp 277–278 °C; ¹H

NMR (DMSO-d₆) d 2.59 (t, 2H, –NH–CH₂–CH₂–); 3.55 (m, 2H, –NH–

CH₂–CH₂–); 7.06–7.28 (m, 4H, Ar); 7.48 (d, 1H, Ar); 7.59 (t, 1H, Ar);

7.76 (d, 1H, Ar); 7.85 (d, 1H, Ar); 8.67 (d, 1H, Ar); 8.99 (t, 1H, –NH–

CH₂); 11.92 (s, 1H, –NH–); 12.33 (b, 1H, –OH); 12.69 (s, 1H, –NH–);

¹³C NMR (DMSO-d₆) d 34.20, 36.45, 103.31, 113.20, 120.08, 120.61,

120.88, 122.63, 123.22, 124.76, 127.72, 128.99, 132.38, 133.13,

137.79, 140.16, 159.85, 169.34, 173.47. MS (ES) m/z 350.1 [MꢅH]^ꢅ,

352.0 [MH]⁺; MW: 351.37 (calcd for C₁₉H₁₇N₃O₄).

6.5.7. 2-(R,S)-{2-[(1H-Indole-2-carbonyl)-amino]-benzoylamino}-

succinic acid (7)

Crystallization with hot abs. EtOH afforded the titled compound

in 28% yield. TLC (EtOAc/MeOH 1:1). R_f0.28; mp 192 °C; ¹H NMR

(DMSO-d₆) d 2.82 (m, 2H, –CH₂-); 4.85 (m, 1H, –CH); 7.08 (m,

2H, Ar); 7.24 (m, 2H, Ar); 7.48 (d, 1H, Ar); 7.62 (t, 1H, Ar); 7.75

(d, 1H, Ar); 7.89 (d, 1H, Ar); 8.69 (d, 1H, Ar); 9.19 (d, 1H, –NH–

CH); 11.95 (s, 1H, –NH–); 12.49 (s, 1H, –NH–); 12.87 (b, 2H,

–OH); ¹³C NMR (DMSO-d₆) d 36.38, 50.13, 103.33, 113.21, 119.70,

120.64, 120.91, 122.64, 123.29, 124.82, 127.68, 129.16, 132.27,

133.46, 137.82, 140.16, 159.86, 169.24, 172.40, 172.85. MS (ES)

m/z 396.2 [MH]⁺; MW: 395.37 (calcd for C₂₀H₁₇N₃O₆).

6.5.12. 4-{2-[(1H-Indole-2-carbonyl)-amino]-benzoylamino}-

butyric acid (20)

Trituration with hot ethyl acetate afforded the titled compound

in 91% yield. TLC (EtOAc/MeOH 2:1). R_f0.71; mp 258 °C; ¹H NMR

(DMSO-d₆) d 1.80 (m, 2H, –NH–CH₂–CH₂–CH₂–); 2.32 (t, 2H,

–NH–CH₂–CH₂–CH₂–); 3.36 (m, 2H, –NH–CH₂–CH₂–CH₂–); 7.03–

7.26 (m, 4H, Ar); 7.45 (d, 1H, Ar); 7.56 (t, 1H, Ar); 7.72 (d, 1H,

Ar); 7.84 (d, 1H, Ar); 8.65 (d, 1H, Ar); 8.92 (t, 1H, –NH–CH₂–);

11.89 (s, 1H, –NH–); 12.10 (b, 1H, –OH); 12.68 (s, 1H, –NH–); ¹³C

NMR (DMSO-d₆) d 25.00, 31.88, 103.27, 113.20, 120.28, 120.57,

120.87, 122.61, 123.22, 124.74, 127.72, 128.99, 132.40, 133.03,

137.79, 140.12, 159.84, 169.34, 174.93. MS (ES) m/z 364.1 [MꢅH]^ꢅ,

366.1 [MH]⁺; MW: 365.39 (calcd for C₂₀H₁₉N₃O₄).

6.5.8. 2-(R,S)-{2-[(1H-Indole-2-carbonyl)-amino]-benzoylamino}-

pentanedioic acid (8)

Trituration with hot MeOH afforded the titled compound in 45%

yield. TLC (EtOAc/MeOH 1:1). R_f0.34; mp 239–241 °C; ¹H NMR

(DMSO-d₆) d 2.02–2.20 (m, 2H, –CH₂–CH); 2.42 (m, 2H, –CH₂–

CO–); 4.55 (m, 1H, –CH); 7.07 (m, 2H, Ar); 7.25 (m, 2H, Ar);

7.49 (d, 1H, Ar); 7.63 (t, 1H, Ar); 7.75 (d, 1H, Ar); 7.97 (d, 1H,

Ar); 8.70 (d, 1H, Ar); 9.10 (d, 1H, –NH–CH); 11.94 (s, 1H, –NH–);

12.44 (s, 1H, –NH–); 12.57 (b, 2H, –OH); ¹³C NMR (DMSO-d₆) d

25.52, 30.23, 51.90, 102.40, 112.32, 119.01, 120.01, 121.72, 122.34,

123.90, 126.81, 128.51, 131.43, 132.48, 136.95, 139.22, 159.01,

168.91, 172.78, 173.66. MS (ES) m/z 408.2 [MꢅH]^ꢅ; MW: 409.40

(calcd for C₂₁H₁₉N₃O₆).

6.5.13. 3-(R,S)-{2-[(1H-Indole-2-carbonyl)-amino]-benzoylamino}-

4-phenyl-butyric acid (21)

Trituration with hot ethyl acetate and then with hot EtOH affor-

ded the titled compound in 54% yield. TLC (EtOAc/MeOH 2:1). R_f

0.70; mp 274 °C; ¹H NMR (DMSO-d₆) d 2.60 (m, 2H, –CH₂–CO-);

2.90 (m, 2H, –CH₂–C₆H₅); 4.66 (m, 1H, –CH); 6.96 (s, 1H, Ar);

7.07–7.28 (m, 8H, Ar); 7.48 (d, 1H, Ar); 7.56 (t, 1H, Ar); 7.73 (m,

2H, Ar); 8.61 (d, 1H, Ar); 8.78 (d, 1H, –NH–CH); 11.89 (s, 1H,

–NH–); 12.23 (s, 1H, –NH–); 12.31 (b, 1H, –OH); ¹³C NMR (DMSO-

d₆) d 48.25, 102.90, 112.63, 119.90, 120.21, 120.33, 122.02, 122.58,

124.19, 126.34, 127.13, 128.24, 129.32, 131.72, 132.35, 137.23,

138.59, 139.20, 159.23, 168.01, 172.49. MS (ES) m/z 440.2

[MꢅH]^ꢅ,442.1 [MH]⁺; MW: 441.49 (calcd for C₂₆H₂₃N₃O₄).

6.5.9. 2-(R,S)-{2-[(1H-Indole-2-carbonyl)-amino]-benzoylamino}-

3-(1H-indol-3-yl)-propionic acid (9)

Crystallization with MeOH/1,4-dioxane afforded the titled com-

pound in 68% yield. TLC (EtOAc/MeOH 3:1). R_f0.44; mp 227–229

°C; ¹H NMR (DMSO-d₆) d 3.38 (m, 2H, –CH₂–CH); 4.80 (m, 1H,

–CH); 7.01–7.35 (m, 8H, Ar); 7.48–7.76 (m, 4H, Ar); 7.88 (d, 1H,

Ar); 8.68 (d, 1H, Ar); 9.13 (d, 1H, –NH–CH); 10.88 (s, 1H, –NH–);

11.94 (s, 1H, –NH–); 12.43 (s, 1H, –NH–); ¹³C NMR (DMSO-d₆) d

6.6. General procedure for the synthesis of compounds 22–25

A mixture of 0.60 mmol of the corresponding ethyl ester (com-

pounds 22c–25c) in water (25 mL) and tetrahydrofuran (25 mL)

2348

L. Lassiani et al. / Bioorg. Med. Chem. 17 (2009) 2336–2350

and in the presence of lithium hydroxide monohydrate (25 mg,

0.60 mmol) was stirred at room temperature for 24 h. The organic

solvent was removed under reduced pressure and 30 mL of satu-

rated NaHCO₃solution and 30 mL of AcOEt were added. After a vig-

orous stirring for few minutes the precipitated salt was collected

by ﬁltration. The obtained salt was taken up with 30 mL of tetrahy-

drofuran and the solution was acidiﬁed with 1 N HCl. The organic

phase was removed under reduced pressure and the product was

ﬁltered. Crystalizzation or trituration with MeOH afforded the ana-

lytically pure compounds 22–25.

172.87. MS (ES) m/z 412.1 [MH]⁺; MW: 411.48 (calcd for

C₂₁H₂₁N₃O₄S).

6.6.5. (2R,S)-3-(4-Hydroxy-phenyl)-2-{2-[(1H-indole-2-carbonyl)-

amino]-benzoylamino}-propionic acid (10)

To a solution of 0.400 g (0.641 mmol) of compound 11c in 150

ml of THF, 40 mg of 10% Pd/C were added and the reaction mixture

was stirred for 24 h (TLC control). The reaction mixture was ﬁltered

through Celite and evaporated. The residue was puriﬁed by ﬂash

chromatography (EtOAc to 15% MeOH) followed by trituration

with MeOH, obtaining 0.123 g (43%) of a white powder. TLC

(EtOAc/MeOH 2:1). R_f0.69; mp 251–252 °C; ¹H NMR (DMSO-d₆)

d 3.10 (m, 2H, –CH₂–); 4.64 (m, 1H, –CH); 6.65 (d, 2H, Ar); 6.99

(s, 1H, Ar); 7.05–7.28 (m, 5H, Ar); 7.46 (d, 1H, Ar); 7.59 (t, 1H,

Ar); 7.73 (d, 1H, Ar); 7.83 (d, 1H, Ar); 8.65 (d, 1H, Ar); 9.12 (d,

1H, –NH–CH); 9.22 (s, 1H, -OH); 11.94 (s, 1H, –NH–); 12.38 (s,

1H, –NH–); 12.95 (b, 1H, –OH); ¹³C NMR (DMSO-d₆) d 35.25,

54.51, 102.44, 112.39, 114.90, 118.90, 119.66, 120.07, 121.79,

122.39, 123.96, 126.85, 127.85, 128.34, 129.85, 131.44, 132.54,

136.99, 139.24, 155.71, 158.97, 168.60, 172.69. MS (ES) m/z 442.2

[MꢅH]^ꢅ, 444.2 [MH]⁺; MW 443.46 (calcd for C₂₅H₂₁N₃O₅).

6.6.1. 2-(S)-{2-[(1H-Indole-2-carbonyl)-amino]-benzoylamino}-

4-methyl-pentanoic acid (22)

Crystallization with MeOH afforded the titled compound in 32%

yield. TLC (EtOAc/MeOH 3:1). R_f0.51; ½a _D²⁵

= ꢅ33.2 (c 0.45, DMF);

ꢄ

ee > 99.4; mp 263–265 °C; ¹H NMR (DMSO-d₆) d 0.94 (dd, 6H,

–CH(CH₃)₂); 1.61–1.78 (m, 3H, –CH₂–CH(CH₃)₂); 4.51 (m, 1H,

–NH–CH); 7.12 (m, 2H, Ar); 7.28 (m, 2H, Ar); 7.51 (d, 1H, Ar);

7.65 (t, 1H, Ar); 7.78 (d, 1H, Ar); 8.01 (d, 1H, Ar); 8.62 (d, 1H,

Ar); 9.10 (d, 1H, –NH–CH); 11.90 (s, 1H, –NH–); 12.40 (s, 1H,

–NH–); ¹³C NMR (DMSO-d₆) d 20.89, 22.73, 24.47, 50.78, 102.31,

112.38, 119.19, 119.72, 120.08, 121.73, 122.32, 124.08, 126.84,

128.66, 131.48, 132.39, 136.90, 139.18, 159.08, 168.84, 173.61.

MS (ES) m/z 394.3 [MH]⁺; MW: 393.45 (calcd for C₂₂H₂₃N₃O₄).

6.6.6. (1R,S)-1H-Indole-2-carboxylic acid [2-(1-carbamoyl-2-

phenyl-ethylcarbamoyl)-phenyl]-amide (12)

Gaseous ammonia was bubbled for 20 min in a stirred suspen-

sion of 1.50 g (3.29 mmol) of 14 in 100 mL of MeOH. After stirring

for 7 days at rt the product was ﬁltered and puriﬁed by trituration

with hot EtOH to give 1.20 g (86%) of a white powder. TLC (EtOAc).

R_f0.74; mp > 300 °C; ¹H NMR (DMSO-d₆) d 3.11 (m, 2H, –CH₂–);

4.77 (m, 1H, –CH); 6.93 (s, 1H, Ar); 7.02–7.56 (m, 9H, Ar); 7.71

(m, 2H, Ar); 7.81 (d, 1H, Ar); 8.57 (d, 1H, Ar); 8.96 (d, 1H, –NH–

CH); 11.87 (s, 1H, –NH–); 12.24 (s, 1H, –NH–); ¹³C NMR

(DMSO-d₆) d 37.15, 54.74, 102.65, 112.51, 119.75, 119.85, 120.20,

121.81, 122.46, 124.05, 126.25, 126.96, 128.03, 128.55, 129.10,

131.64, 132.36, 137.10, 138.43, 139.19, 159.14, 168.57, 172.86.

MS (ES) m/z 427.2 [MH]⁺; MW: 426.48 (calcd for C₂₅H₂₂N₄O₃).

6.6.2. 2(R)-{2-[(1H-Indole-2-carbonyl)-amino]-benzoylamino}-

4-methyl-pentanoic acid (23)

Crystallization with MeOH afforded the titled compound in 38%

yield. TLC (EtOAc/MeOH 3:1). R_f0.51; ½a _D²⁵

¼ þ36:4 (c 1.2, DMF);

ꢄ

ee > 99.7; mp 259–260 °C; ¹H NMR (DMSO-d₆) d 0.96 (dd, 6H,

–CH(CH₃)₂); 1.62–1.77 (m, 3H, –CH₂–CH(CH₃)₂); 4.55 (m, 1H,

–NH–CH); 7.10 (m, 2H, Ar); 7.21 (m, 2H, Ar); 7.45 (d, 1H, Ar);

7.60 (t, 1H, Ar); 7.72 (d, 1H, Ar); 7.91 (d, 1H, Ar); 8.64 (d, 1H,

Ar); 9.03 (d, 1H, –NH–CH); 11.91 (s, 1H, –NH–); 12.40 (s, 1H,

–NH–); ¹³C NMR (DMSO-d₆) d 20.89, 22.71, 24.41, 50.86, 102.37,

112.39, 119.21, 119.72, 120.08, 121.63, 122.35, 124.04, 126.83,

128.67, 131.40, 132.44, 136.92, 139.18, 159.08, 168.85, 173.59.

MS (ES) m/z 394.2 [MH]⁺; MW: 393.45 (calcd for C₂₂H₂₃N₃O₄).

6.6.7. (1R,S)-1H-Indole-2-carboxylic acid [2-(1-dipentylcarbamoyl-

2-phenyl-ethylcarbamoyl)-phenyl]-amide (13)

6.6.3. 2(S)-{2-[(1H-Indole-2-carbonyl)-amino]-benzoylamino}-

4-methylsulfanyl-butyric acid (24)

To a solution of 1.00 g (2.34 mmol) of VL-0395 and 0.33 mL

(2.34 mmol) of TEA in 50 mL of THF cooled to ꢅ20 °C, 0.30 mL

(2.34 mmol) of isobutyl chloroformate were added dropwise under

stirring. After 30 min 0.96 mL (4.68 mmol) of dipentylamine were

added and the reaction mixture was stirred at rt for 18 h. The sol-

vent was evaporated in vacuo and 150 mL of ethyl acetate were

added. The organic phase was washed with 1 N NaOH, H₂O, 1 N

HCl, H₂O and 5% KCl. After drying on dry Na₂SO₄the solvent was

removed in vacuo and the residue was triturated with hot MeOH

and then puriﬁed by ﬂash chromatography (CH₂Cl₂to EtOAc/

CH₂Cl₂1:4). The product was triturated with hot EtOH to give

0.38 g (29%) of a white powder. TLC (EtOAc/n-hexane 1:1). R_f

0.26; mp 173 °C; ¹H NMR (DMSO-d₆) d 0.81 (m, 6H, –CH₃); 1.14–

1.70 (m, 12H, –CH₂–); 3.06–3.30 (m, 6H, –CH₂–CHe –CH₂–);

5.11 (m, 1H, –CH); 6.93 (s, 1H, Ar); 7.06–7.37 (m, 8H, Ar); 7.47

(d, 1H, Ar); 7.59 (m, 2H, Ar); 7.91 (d, 1H, Ar); 8.61 (d, 1H, Ar);

9.27 (d, 1H, –NH–CH); 11.94 (s, 1H, –NH–); 12.31 (s, 1H, –NH–);

¹³C NMR (DMSO-d₆) d 14.62, 22.60, 22.68, 27.53, 28.92, 29.22,

38.03, 46.26, 47.81, 52.09, 103.13, 113.30, 119.98, 120.47, 120.99,

122.11, 123.29, 124.83, 127.24, 127.55, 128.89, 129.29, 129.93,

132.25, 133.28, 137.74, 138.32, 139.73, 159.67, 168.98, 170.76.

MS (ES) m/z 567.2 [MH]⁺; MW: 566.75 (calcd for C₃₅H₄₂N₄O₃).

Trituration with MeOH afforded the titled compound in 48%

yield. TLC (EtOAc/MeOH 2:1). R_f0.38; ½a _D²⁵

¼ ꢅ55:3 (c 1.8, DMF);

ꢄ

ee > 98.9; mp 253–255 °C; ¹H NMR (DMSO-d₆) d 2.09 (s, 3H,

CH₃–S–); 2.17 (m, 2H, –CH₂–CH); 2.68 (m, 2H, –CH₂–S–); 4.65

(m, 1H, ACH); 7.11 (m, 2H, Ar); 7.30 (m, 2H, Ar); 7.54 (d, 1H,

Ar); 7.67 (t, 1H, Ar); 7.78 (d, 1H, Ar); 7.94 (d, 1H, Ar); 8.65 (d,

1H, Ar); 9.14 (d, 1H, –NH–CH); 11.98 (s, 1H, –NH–); 12.46 (s,

1H, –NH–); ¹³C NMR (DMSO-d₆) d 14.32, 29.71, 29.93, 51.52,

102.46, 112.30, 119.26, 119.81, 120.09, 121.64, 122.32, 123.96,

126.82, 128.58, 131.40, 132.48, 136.92, 139.17, 159.08, 168.93,

172.85. MS (ES) m/z 412.2 [MH]⁺; MW: 411.48 (calcd for

C₂₁H₂₁N₃O₄S).

6.6.4. 2(R)-{2-[(1H-Indole-2-carbonyl)-amino]-benzoylamino}-

4-methylsulfanyl-butyric acid (25)

Trituration with MeOH afforded the titled compound in 61%

yield. TLC (EtOAc/MeOH 2:1). R_f0.38; ½a _D²⁵

¼ þ54:4 (c 2.1, DMF);

ꢄ

ee > 99.6; mp 248–250 °C; ¹H NMR (DMSO-d₆) d 2.06 (s, 3H,

CH₃–S–); 2.16 (m, 2H, –CH₂–CH); 2.64 (m, 2H, –CH₂–S–); 4.66

(m, 1H, ACH); 7.10 (m, 2H, Ar); 7.24 (m, 2H, Ar); 7.52 (d, 1H,

Ar); 7.65 (t, 1H, Ar); 7.75 (d, 1H, Ar); 7.98 (d, 1H, Ar); 8.64 (d,

1H, Ar); 9.13 (d, 1H, –NH–CH); 11.96 (s, 1H, –NH–); 12.44 (s,

1H, –NH–); ¹³C NMR (DMSO-d₆) d 14.34, 29.73, 29.95, 51.53,

102.44, 112.31, 119.24, 119.80, 120.06, 121.66, 122.35, 123.94,

126.80, 128.55, 131.42, 132.45, 136.90, 139.14, 159.05, 168.96,

6.6.8. (1R,S)-1H-Indole-2-carboxylic acid [2-(1-hydroxymethyl-

2-phenyl-ethylcarbamoyl)-phenyl]-amide (15)

To a stirred solution of the acid VL-0395 (0.600 g, 1.40 mmol) in

THF/DMF 10/0.3 (10.3 mL) at ꢅ10 °C, N-methylmorpholine (0.15

L. Lassiani et al. / Bioorg. Med. Chem. 17 (2009) 2336–2350

2349

mL, 1.40 mmol) was added, followed by ethyl chloroformate (0.13

8.2. [¹²⁵I]BH–CCK-8. receptor binding assay in guinea pig

cerebral cortices

mL, 1.40 mmol). After 10 min, NaBH₄(0.159 g, 4.20 mmol) was

added in one portion. MeOH (15 mL) was then added dropwise

to the mixture over a period of 20 min at 0 °C. The solution was

stirred for additional 20 min, and then neutralized with 1 N HCl.

The organic solvents were removed and the product was extracted

with EtOAc (3 ꢃ 10 mL). The combined organic phases were

washed consecutively with 1 M HCl, H₂O, 5% aq NaHCO₃, H₂O,

dried (Na₂SO₄) and the solvent was evaporated. Crystallization

from EtOAc/petroleum ether 40–60° afforded 0.382 g (66%) of a

white powder. TLC (EtOAc/n-hexane 1:1). R_f0.23; mp 228 °C; ¹H

NMR (CDCl₃) d 2.91 (m, 2H, –CH₂–C₆H₅); 3.55 (m, 2H,–CH₂-OH);

4.34 (m, 1H, –CH); 4.96 (t, 1H, –OH); 6.97 (s, 1H, Ar); 7.04–7.32

(m, 8H, Ar); 7.47–7.60 (m, 2H, Ar); 7.74 (d, 1H, Ar); 7.82 (d, 1H,

Ar); 8.63 (m, 2H, –NH–CHe Ar); 11.91 (s, 1H, –NH–); 12.38 (s,

1H, –NH); ¹³C NMR (CDCl₃) d 37.23, 54.07, 63.66, 103.33, 113.21,

120.49, 120.88, 122.53, 123.13, 124.73, 126.65, 127.68, 128.75,

129.01, 129.76, 132.36, 132.83, 137.78, 139.78, 139.84, 159.77,

169.00. MS (ES) m/z 412.2 [M–H]^ꢅ; MW: 413.48 (calcd for

C₂₅H₂₃N₃O₃).

Membranes from guinea pig cerebral cortices, were prepared

as previously described.²⁰Protein concentration was determined

according to Bradford,⁴²using bovine serum albumin (BSA) as

standard. The binding experiments were performed in assay buf-

fer 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 la-

beled ligand and inhibitors was carried out in a microtiter 96-

wells 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]BH–CCK8 (25 pM), in a ﬁnal volume of 250

ciﬁc binding of iodinated peptide was deﬁned in the presence of

M CCK-8, accounting of 20% of total binding. Nonspeciﬁc

ll. The non-spe-

1

l

binding of [¹²⁵I]BH–CCK-8 to membrane ﬁlters (blank), measured

in wells containing an equal amount of labeled ligand, but no

membranes, was 0.2% of total radioligand added. After 120 min

at 25 °C, the 96-wells 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

7. Molecular modeling

polycarbonate tubes and counted in a COBRA-5002

(Packard Biosciences).

c-counter

AM1 and conformational search calculations were carried out

with the parallel version of the ^GAUSSIAN03 suite³⁸running on an In-

TM

tel Core 2 cpu 6600@2.40 GHz, 3.50 GB RAM, and with the MO-

8.3. Isolated muscular strips of guinea pig gallbladder

stimulated by CCK-8

^PAC2000 semiempirical package³⁹on a Kubuntu linux Intel Core

TM

2 cpu 6600@2.40 GHz, 3.50 GB RAM. The optimizations were car-

ried out at a gradient norm of 10^ꢅ3kcal/mol Å, with a tight SCF

convergence criterion on the RHF calculations, and using the

‘mmok’ keyword. The conformational searches were carried out

with the Tripos random search⁴⁰and using initially the Tripos

forceﬁeld; the geometries yielded by the search were then reop-

timized at the AM1 level.

The assay was based on the method described by Bishop

et al.⁴³Male Hartley guinea pigs weighting 400

450

-

g

(Charles River, Calco, Italy) were sacriﬁced by cervical disloca-

tion and exsanguination, after slight ethereal anesthesia. The

abdomen was opened, the gallbladder was removed and trans-

ferred to a Petri dish containing gassed Krebs–Henseleit solu-

tion of the following composition (mM): NaCl 118.9, KCl

4.69, CaCl₂ꢆ2H₂O 3.33, KH₂PO₄1.18, MgSO₄ꢆ7H₂O 1.2, NaHCO₃

25, 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 ꢃ 15 mm were obtained, containing mainly the cir-

cular smooth muscle ﬁbers. The strips were suspended with

silk thread in 20 ml organ baths containing Krebs–Henseleit

solution, maintained at 32°C, bubbled with 95% O₂–5% CO₂.

Contractions were measured with an isometric transducer

8. Biological evaluations

Male Hartley guinea pigs (300–350 g) and male Sprague Dawley

rats (250–300 g) were used. For binding assays to isolated rat pan-

creatic acini, animals were fasted, but allowed free access to water,

for 18–24 h prior to the experiment.

[

¹²⁵I]-BH–CCK-8 (CCK₈(sulphated), [¹²⁵I]Bolton and Hunter la-

beled-speciﬁc activity 2000 Ci/mol) was purchased from Amer-

sham Pharmacia Biotech (Buckinghamshire, UK). All other drugs

and reagents were obtained from commercial sources.

(mod. 7003—Basile U., Comerio, Italy).

A resting tension of

The binding parameters for the substances under investigation,

IC₅₀values and standard errors, were calculated from concentra-

tion–response curve analyzed by a computerized curve ﬁtting

technique (ALLFIT) using the four parameter logistic equation.⁴¹

0.5 g was applied. The isometric contraction was monitored

using a pen recorder (mod. 7070—Basile U., Comerio, Italy).

The strips were washed with fresh buffer every 15 min over

a 60 min equilibration period.

Submaximal-effect (70–80%) concentration of CCK-8 was cho-

sen to test the inhibitory effects of the substances under study.

Various concentrations of the antagonists or vehicles were added,

and allowed 5 min contact with the tissues before adding the ago-

nist to the bathing ﬂuid. The antagonist activity was expressed as

percentage of inhibition of the agonist contractions. The regression

line was calculated and the concentration effective in inhibiting by

50% the effect of the agonist (IC₅₀) were obtained from the regres-

sion line by using ^ALLFITprogram.

8.1. [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.²⁰Drug dis-

placing experiments were carried out by incubating acinar cells,

[

¹²⁵I]BH–CCK-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 Hepes-Ringer buffer (10 mM

Hepes, 118 mM NaCl, 1.13 mM MgCl₂, 1.28 mM CaCl₂, 1% BSA,

0.2 mg/mL Soybean trypsin inhibitor, pH 7.4) was added and the

tubes were centrifuged 5 min at 12,500g. The supernatant was

aspirated and the radioactivity associated to the pellet measured.

Acknowledgments

The corresponding author thanks the University of Trieste as

well MIUR for their consideration and their investment of 0,00 €

for his research group in the last eight years.

The non-speciﬁc binding was estimated in the presence of 1

CCK-8, accounting 15% of total binding.

lM

2350

L. Lassiani et al. / Bioorg. Med. Chem. 17 (2009) 2336–2350

23. Varnavas, A.; Lassiani, L.; Luxich, E.; Valenta, V. Farmaco 2000, 55, 293.

Supplementary data

24. Varnavas, A.; Lassiani, L.; Valenta, V. Farmaco 2000, 55, 369.

25. Varnavas, A.; Valenta, V.; Berti, F.; Lassiani, L. Farmaco 2001, 56, 555.

26. Varnavas, A.; Lassiani, L.; Valenta, V.; Berti, F.; Mennuni, L.; Makovec, F. Bioorg.

Med. Chem. 2003, 11, 741.

Supplementary data associated with this article can be found, in

the online version, at doi:10.1016/j.bmc.2009.02.012.

27. Schwyzer, R. Trends Pharmacol. Sci. 1980, 3, 327.

28. Makovec, F.; Chisté, R.; Bani, M.; Revel, L.; Setnikar, I.; Rovati, A. L. Eur. J. Med.

Chem. 1986, 21, 9.

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Anthranilic acid based CCK1 receptor antagonists: Blocking the receptor with the same 'words' of the endogenous ligand

DOI: 10.1016/j.bmc.2009.02.012

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