Synthesis

NH2

MeO

MeO

NH

O

MeNH

MeO

MeO

NH

O

Me

O

CbzHN COOMe

H

N

MeO

MeO O

NH

O

Me

H1

NHCbz

5% TFA, DCM 1.Toluene, Reflux

2. Chromatographic Separation

1. H2, Pd/C

2. Ac2O, py

(35%) (86%)

1

N

MeO

MeO O

NH

O

Me

H1

NHCbz(38%) (82%)

H3

H3

NHCbz

COOMe

N

MeO

MeO O

NH

O

Me

H1

NHAc

N

MeO

MeO O

NH

O

Me

H1

NHAc

H3

H3

H10

H10

TETRAHYDROISOQUINOLINE-BASED PEPTIDOMIMETICS MIMICKING REVERSE TURN SECONDARY STRUCTURES

Nicola Landoni, Giordano Lesma, Alessandro Sacchetti and Alessandra Silvani

Dipartimento di Chimica Organica e Industriale, Università degli Studi di Milano, via Venezian 21 – 20133 Milano, Italy E-mail: nicola.landoni@unimi.it

The Tic heterocyclic frame (Tic = 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) is present in many biologically active natural alkaloids and in a great number of pharmaceutical compounds. Particularly, the presence of the Tic nucleus, which is a conformationally constrained Phe analogue, seems to be essential in many synthetic peptidomimetics showing agonistic and antagonistic activity towards many G protein-coupled receptors.

This project is aimed to identify new Tic-based privileged structures having the capability to mimic reverse turn secondary motifs.

MeO

MeON

O

NH

Me

O

HN

H

MeO

MeON

O

NH

Me

O

HN

H

O

O

N

NO

O

H

H

O

NH

Me

N

OH

O

R1

R2

R3

R4

R5

Conformational analysis (intramolecolar hydrogen bond evaluation):

- 1H NMR;- IR;- CD.

Peptidomimetics synthesis:

- Control of stereochemistry .

Peptidomimetics design through computational chemistry:

- Conformational analysis (MM / MC);- Analysis of geometrical parameters of low energy conformers.

Pyrrolo-tetrahydroisoquinoline (AHPIC)(AHPIC = 2-amino-8,9-dimethoxy-3-oxo-1,2,3,5,6,10b-hexahydro-pyrrolo[2,1-a]isoquinoline-5-carboxylic acid)

Conformational analysis of the structures AHPIC (Spartan ‘06, MC search, MMFF94 force field):

No. of conf. < 6

kcal/mol

% dα < 7Å

% β < 30°

% HA bond % HB bond

1 50 64 (32) 52 (26) 12 (6) 34 (17)

2 47 45 (21) 83 (39) 4 (2) 38 (18)Results expressed as % of the conformers that meet the requirement. In parentheses, the number of conformers.

MeO

MeON

O

N

1HN

O

3

HO

H

A

BMeO

MeON

O

N

1HN

O

3

HO

H

A

B

-turn typeConf. n° I I’ II II’ III III’

1 1 0.88 0.84 0.82 0.97 0.86 0.83

21 0.72 0.66 0.71 0.75 0.71 0.67

15 0.86 0.85 0.78 0.97 0.86 0.82

Similarity analysis of Ac-AHPIC-NHMe 1 and 2 with standard type β-turns.

The score is defined as [(1-R2)/N], where R2 is the r.m.s. of the distances between points of similarity of structures and N is the number of centres.

δ (ppm)b Δδ/ΔT (ppb/K)c IR (cm-1)

1NHMeNHAc

7.516.52

- 4.5- 5.5

3453, 3352

2NHMeNHAc

6.906.29

- 4.4- 4.0

3458, 3370

a All analyses were performed on 3.0 mM CDCl3 solutions. b At 298 K. c Determined between 298 and 328 K.

•NMR and IR spectroscopical data. a

MeO

MeON

O

N

1

HN

O

3

HO

H

A

BMeO

MeON

O

N

1

HN

O

3

HO

H

A

B

The presence of a reverse turn was observed in both the structures, with the C1 stereochemistry playing a central role in determining stable conformations. In particular, all the analyses led to the conclusion that a type II’ -turn is mostly stabilized in tetrapeptide mimic 1, while a typical inverse γ-turn geometry is revealed for the diastereoisomer 2.

Synthesis of a II’-turn mimic fragment of HOE 140.

BRADIKININ (BK): an endogenous ligand for G protein-coupled receptors (GPCRs):

GPCRs: membrane receptors involved in signal transduction;Control of different aspects of cell function, through the

mediation of the response to different extracellular stimuli;Adjust many biological processes, including sensory (smell,

taste, sight) and not sensory (appetite, digestion, blood pressure, reproduction, inflammation) activities;

Approximately 50% of drugs on the market today exerts its therapeutic function through interaction with the GPCRs.

NH

ON

HON

COOH

NH

NH

H2N

O

H-(D)-Arg-Arg-Pro-Hyp-Gly-Thi

H

O

N

H H

Ser6

D-Tic7

Oic8

Arg9

BRADIKININ (BK): H-Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9-OH

NH

ON

HO

O HN

N OH

O

HN NH

NH2

O

Ser6

Pro7Phe8

Arg9

H -turn (type II’)

Receptors (GCPRs) FunctionConcerned Pathologies

B2 receptors

(physiologically expressed)

Mediators of physiological action of BK (eg vasodilatation)

Bronchial asthma, allergic rhinitis, hypertension

B1 receptors

(overexpressed in case of trauma or infection)

Mediators of BK action during pathologies (eg

prostaglandin synthesis)

Chronic pain, inflammation

Spyro-pyrrolo-tetrahydroisoquinoline (SIPP)(SIPP = 2-(2'-oxo-2,4-dihydro-1H-spiro[isoquinoline-3,3'-pyrrolidine]-1'-yl)propanoic acid)

HOE 140: A powerful antagonist for the bradykinin B2 receptor

Type II’ -turn with D-Tic in the i+1 position.

(1) Boc-O-benzyl-L-serine,BOP-Cl, CH2Cl2, TEA

NH

N

CO2MeO

N

N

CO2MeO

OOBn

NHBoc

N

NO

OOBn

NHBoc

NH

O COOMe

(1) LiOH, THF, 0°C(2) N-Nitro-L-arginine methyl ester hydrochloride,BOP-Cl, CH2Cl2, TEA

MeNH2 8M in EtOH

33%

35% 97%

HN N

NH2

NO2

N

NO

OOBn

NHBoc

NH

O CONHMe HN N

NH2

NO2

δ (ppm) Δδ/ΔT (ppb/K) IR (cm-1)

NHMe n.d. n.d. 3323, 3435

•NMR and IR spectroscopical data.

CD (MeOH 0.2 mM)

Type II’ -turn

Biochemistry, 1978, 17, 4951.

Synthesis

N O

NO NHMe

O

n

n = 0, 1, 2, 3

% of conformers which meet the requirements.

NH2

COOH

(1) NaOH, H2O, EtOH(2) t-BuCHO, CH2Cl2, Reflux

(3) CbzCl, CH2Cl2, DMAP, 0°C

NO

Cbz

H O

NO

Cbz

O

KHMDS -78°C

Br

(45%) (86%) (99%)(4) F.C.

NO

Cbz

O

O

HO3, CH2Cl2/MeOH

Me2S, -78 °C+ H2N

OMe

O(1) NaCNBH4, MeOH NaOAc, 4 Å MS

(2) HOBt,Toluene, Reflux HN

N

MeO2C

Cbz

O

MeNH2, MeOH

(67%)

H2, Pd/C

DioxaneNH2

N

MeO2C

O(HCHO)n

20% TFA, CH2Cl2 NH

N

CO2MeO

N

NO

O

(95%) (48%)

Ac2O, Py

N

N

CO2MeO

(98%)

Ac

O

NH

Me

H

(90%)

Synthesis

δ (ppm)b Δδ/ΔT (ppb/K)c IR (cm-1)

NHMe 7.79 - 2.75 3351

•NMR e IR spectroscopical data. a

7,5

7,7

7,9

8,1

8,3

0 10 20 30

ppm

% DMSO-d6

Titration with DMSO-d6

a All analyses were performed on 3.0 mM CDCl3 solutions. b At 298 K. c Determined between 298 and 328 K.

X-Ray Structure (from isopropanol)

dα (Å) β (o) Φ2 (o) Ψ2 (o) Φ3 (o) Ψ3 (o)

Type II’ -turn 4.75 1.05 60 -120 -80 0

X-Ray 5.467 -11.98 48.86 -133.68 -89.44 10.79

MM 5.586 -19.54 51.69 -136.43 -103.77 41.15

HF (6-31G*) 5.942 -28.10 48.44 -136.52 -108.51 26.97

• Analysis of geometrical parameters of 3.

Application of the SIPP scaffold to mimic a bioactive peptide

N

NO

O

OBnBocHN

N

O CONHMe

H

HN N

NH2

NO2

1

2

3

1

2

1 2

3

3

4

4

1

2

1 2

1

G. Lesma, E. Meschini, T. Recca, A. Sacchetti, A. Silvani, Tetrahedron 2007, 63, 5567-5578;N. Landoni, G. Lesma, A. Sacchetti, A. Silvani, J. Org. Chem. 2007, 72, 9765-9768.