Arturo San Feliciano · CARDIAC INSUFFICIENCY PRE CHARGE POST- CHARGE COMPENSATING MECHANISMS ......

3. Pharmacophore Identification in

Natural Cardenolides

Identificación del Farmacóforo en Cardenolidas Naturales

Arturo San Feliciano

Department of Pharmaceutical Chemistry Faculty of Pharmacy, CIETUS, IBSAL.

University of Salamanca. Salamanca. Spain

1 - Antecedents

2 - Design and Synthesis of

Cardenolide Analogues

3 - Bioevaluation

4 - Discussion and conclusions

Contents

TYPES

· Acute

· Chronic

· Right

· Left

· Sistolic

· Diastolic

· Congestive

CONTRACTION

VOLUME

EJECTED

VOLUME

MINUTE

FRECUENCY

CARDIAC INSUFFICIENCY

POST-CHARGE PRE-CHARGE

COMPENSATING MECHANISMS

· Miocardic hypertrophia

· hydrosaline retention

· Neurohormonal activation

· DIURETICS · VASODILATORS · POSITIVE INOTROPICS

· b- BLOCKERS · OTHER

TREATMENT OF CARDIAC INSUFFICIENCY

· DIURETICS · VASODILATORS · POSITIVE INOTROPICS increase intracelular cAMP

Increase intracelular Ca 2+

Modulate ionic pumps / channels

Multiple mechanism · b- BLOCKERS

· OTHER


· DIURETICS · VASODILATORS · POSITIVE INOTROPICS increase intracelular cAMP

b1-Agonists

Phosphodiesterase Inhibitors Increase intracelular Ca 2+

Increase Ca 2+ liberation

Sensitisers to Ca 2+ action

Modulate ionic pumps / channels

Digitalis and related Glycosides

Multiple mechanism · b- BLOCKERS

· OTHER


ESTRUCTURA DE LA ATPasa-Na+,K+

ACTIVIDAD TRANSPORTADORA DE LA ATPasa-Na+,K+

E 1

E 1 P ( 3 N a + ) E 2 ( 2 K

+ )

E 2 P

3 N a +

A T P

A D P

3 N a +

2 K +

P i

2 K +

o u a b a í n a

E x t e r i o r

C i t o p l a s m a c e l u l a r

EFFECTS AND TROUBLES

OF DIGITALIS CARDIOTONICS

PHYSIOLOGIC Positive Inotropism ( POTENCY + )

Negative Chronotropism ( RATE - ) TOXIC / ADVERSE


Insufficiency Negative Chronotropism ( RATE - )

Tachycardia, atrial fibrilation TOXIC / ADVERSE




Insufficiency Negative Chronotropism ( RATE - )

Tachycardia, atrial fibrilation TOXIC / ADVERSE Low Therapeutic Index Hipokalemia Bradicardia Ventricular extrasistolia Digitalisation (accumulation) Death … Nausea, vomiting, diarrhoea



PHYTOCHEMICAL ANTECEDENTS Studies on Digitalis sp. endemic to the West of Spain Studies on native Spanish Conifers (Juniperus)

Digitalis thapsi

(Scrophulariaceae)

O O

OH

gli-O

CARDENOLIDES

DIGITANOLS

OTHER

Phytochemical studies at the USAL:

digitoxin

O

O O O

O

O

HO OH

HO

HO

O

O

OH

Molecules 5, 51- 81 (2000)

( b -D-DIG) 3 - O

O O

OH

OH

DIGOXIN

OH

COMMUNIC ACIDS ,

PIMARIC ACIDS,

OTHER TERPENOIDS

LIGNANS

Junipers Juniperus

(sect. Oxycedrus)

Sabines Juniperus phoenicea

( sect. sabina ) HOOC

Sandaracopimaric acid

Phytochemical studies at the USAL:

HOOC

Sandaracopimaric Acid

O O

OH

( b -D-DIG) 3 - O

DIGITOXIN

DIGITALIS - LIKE

CARDIOTONICS

?

OBJECTIVE

1 - Antecedents



3 - Bioevaluation


Contents

Natural and Natural-Product Related Cardiotonics: Cardenolides

O

OH

(b-D-DIG) 3-O

O

DIGOXIN

OH

O

OH

-L-RHAMN-O

O

HO

HO

HO

OUABAIN

O

OOO

O

O

HOOH

HO

HO

O

O

OH

OH

DIGOXIN

OH

H

O

O

O

OO

OHOH

H

H

GOMPHOSIDE

UZARIGENIN


R1

OH

R2

O

IC50 (nM)

-COCH2OH

-CH2OH

-CH2NH2.HCl

-CH2NO2

-CH2CH2NO2

-CH2NH2·HCl

-CH2NO2

1070

360

99

12

45

270

88

R1

R2

-L-rhamnose

-L-rhamnose

-L-rhamnose

-L-rhamnose

-L-rhamnose

b-D-digitoxose

b-D-digitoxose

O

N

NH

NH2

NH


OH

MeOOC

ONH

ERYTHROPHLEINE

O

Natural and Natural-Product Related Cardiotonics: Terpenoidal alkaloids

Main Objectives

1. Synthesis of diterpenic analogues of

cardenolides, using natural pimarane

derivatives as starting materials.

2. Search for the minimum structural

requirements for cardiotonic activity.

3. Total synthesis of simpler inotropic

substances.

PIMARANES

natural CYCLOHEXANES

synthesis

INDANES

synthesis

OO

OH

OO

OHR

1

R2

OO

Z

OH

OO

OHX

Z

Y

H

Objectives


Pimaranes

digitoxigenin Cyclohexanes

Indanes

Dinor-seco-steroids

D

D

B

A,B A,B

D C

B A

The Pimarane Strategy

OH

O O

Gly-O

C D

glycoside

B A

steroidal A-D rings

The Pimarane Strategy

glycoside

OH

O O

Gly-O

C D

B A

steroidal A-D rings

O

OO

Z

A B

C

OH

glucose (sugar) solubilizer

A - C rings, 5-H

lack of Ring D

The Cyclohexane Strategy

OH

OO

Gly-O

C D

steroidal A-D rings

glycoside

A B

butenolide

The Cyclohexane Strategy

OO

OHY

Z

C

espacer solubilizer lack of Rings

A-B and D

OH

OO

Gly-O

C D

steroidal A-D rings

glycoside

A B

butenolide

The Indanol strategy

OH

C D

OO

Gly-O

butenolide

Cardenolide C-D rings

A-B rings and glycoside

The Indanol strategy

OH

C D

OO

Gly-O

butenolide


A-B rings and glycoside

Y OH

R

X

D C

Butenolide or equivalent


Z

spacer (Y) and solubilizer (Z) fragments

Butenolide or

equivalent

CD + A rings

X,Y = C, N

Z Solubilizer Lack of ring B

X

OHX

YZ

The di-nor-seco-steroid strategy

The Pimarane-butenolide Synthesis.

OO

Z

OH

HOOC

FROM Juniperus phoenicea or Tetraclinis articulata


1. The side Chain

OO

Z

OH

R = H, Ac

HOOC

FROM Juniperus phoenicea or Tetraclinis articulata

R1

R1

OR

BBN, H2O2


2. The 14-hydroxyl function

R1

OR

BH3-THF

OR

OH

H

R1

OR

BH3-THF

OR

OH

H



OR

OH

H



R1

OR

BH3-THF

OR

OH

H

R = H, Ac, THP, TBDMS, TBDPS

OR

OH

H



R1

OR

BH3-THF

OR

OH

H

R = H, Ac, THP, TBDMS, TBDPS

R

O

Si No association O - B

face protected

OR

OH

H


3. The Butenolide

OH

OAc

H

OAc

H

OAcO

Jones; SOCl 2; CH2N2

HCl; NaOAc/Ac2O


3. The Butenolide

OH

OAc

H

OAc

H

OAcO


HCl; NaOAc/Ac2O

OH

H

O

OH

H

OOO

OH

+

Reformatsky


3. The Butenolide

OO

R1

OH

OH

OAc

H

OAc

H

OAcO


HCl; NaOAc/Ac2O

OH

H

O

OH

H

OOO

OH

+

Reformatsky

The Pimarane-Butenolide Synthesis.

4. The End Products

R = COOH, CH 2 OH, O

O HO

HO

OH

OH

O O

R 1

OH

O O

R

OH

The Synthesis of

Cyclohexylmethylbutenolides R

1

OO

OHR

2

R1= H, Me

R2= H, Me

OCOOH

O Cl4 steps

Mn(OAc)3

HOAc, KOAc

a:(COCl) 2/Benz

b:CH2N2; HCl

Horner

The Synthesis of

Cyclohexylmethylbutenolides R

1

OO

OHR

2

R1= H, Me

R2= H, Me

OO

OH

OCOOH

O Cl

OO

OO

4 steps

OH

Mn(OAc)3

Br

HOAc, KOAc

a:(COCl) 2/Benz

b:CH2N2; HCl

Horner

NBS,HClO 4 n-Bu3Sn +

The Enantioselective Synthesis of Indanols

RR

OO O

O O

O

(R = H, Me)

The Enantioselective Synthesis of Indanols

OH

RR

OO O

O O

O

R

O

O

OOH

(R = H, Me)

(R = H) (R = Me)

S-(-)-proline

piperidine, DMFO

O O

O

Tetrahedron Asymm. 8, 2075 (1997)

HAJOS-PARRISH ketones ( ee 95-100% )

The Indanol Synthesis: C-1-elongation

OH

O

O

OH

O

OH

O

S

SOH

HS(CH 2)3SH

BF3.Et2O

PH 3P=CH 2 HgO, THF/H 2O

BF3.Et2O

I


OH

O

O

OH

O

OH

O

O

O

S

SOH

BF3.Et2O

O

O

PH 3P=CH 2S

S

HS(CH 2)3SH

HS(CH 2)3SH

BF3.Et2O

PH 3P=CH 2

p-TsOH

HgO, THF/H 2O

HgO, THF/H 2O

BF3.Et2O

HS(CH 2)3SH TMSCl

BF3.Et2O


OH OH OH OH

CHO

OH

CHOOH OH

CHO CHOOH OH

I

BH3.THF

H2O2

PCC

PCC

1:1

1:1

BH3.THF

H2O2

II

III


OH OH OH OH

CHO

OH

CHO

I

OH OH

BH3.THF

H2O2

CHO CHOOH OH

BH3.THF

H2O2

II

PCC

PCC

1:1

1:1

OH

CHO

OH

COOMe

Ph 3P=CHCOOMe

III

III OH

COOMe

O

deprotection

The Indanol Synthesis: Stereochemical Control

OH

CHO

OH

CHO

Y Y

X X

?


X,Y H =O H, OH OH, H -O-(CH2)2-O- -O-(CH2)3-O- -S(CH2)3S-

DH b -0.02 +0.12 -1.64 +1.39 -1.09 -0.85 -2.98

(Kcal/mole)

OH

CHO

OH

CHO

Y Y

X X

?


OH

CHO

OH

CHO

S

S

S

S

5% KOH / MeOH

b = 1 / 9

OH

CHO

OH

CHO

Y Y

X X

?

X,Y H =O H, OH OH, H -O-(CH2)2-O- -O-(CH2)3-O- -S(CH2)3S-

DH b -0.02 +0.12 -1.64 +1.39 -1.09 -0.85 -2.98

(Kcal/mole)

The Indanol Synthesis: Final Products

R

H, Me

X,Y

H,H; =O; H,OH;

H,OAc;

-O-(CH2)2-O-; -O-(CH2)3-O-;

-S-(CH2)3-S-; =N-N=C(NH2)2

Z

COMe; COOMe; SMe, NO2

G H,OH; CHNO2;

N-N=C(NH2)2

OH

Z

Y

Z

Y

XX

R R

OHY

X

R

Y

X

R

G G

The Synthesis of Ent-Indanols

O

O

O

+

O

O

N

N

NNH2N

NH2

NH2

NH2

30

R-(+)-proline

43

OTMS

O

O

OTMS

O

H2N

OTMS

O

N

NC

OTMS

O

HO

OTMS

O

NOH

O

O

5

6a

7a

8a

9a1

a

b

c

a) TMSTf, Et3N, CH2Cl2

b) NH4OAc, NaBH3CN, MeOH

c) Piperidine, HCl/MeOH, NaBH3CN (r.t.)

OO

OHX

Z

Y

H

Synthesis of B,B-dinor-B-seco-steroids

a) TMSTf, Et3N, CH2Cl2

c) Piperidine, HCl/MeOH, NaBH3CN (r.t.)

OO

OHX

Z

Y

H


OH

O

OOTMS

O

1 11

ca

+

OTMSN

NC

OTMSN

N

NCN

12a 13a

13b12b

+

OH

O

O

OH

O

NC

CNOH

O

NC

CNOH

O

NC

CN

+

OH

O

NO2

OH

O

NO2

OH

O

OH

O

OH

O

NCOH

O

NC

1

14 15a 15b

16a 16b 17a 17b

18a 18b

+

+

a

b

c d

e

OO

OHX

Z

Y

H

a) malononitrile, b-alanine,

HOAc / EtOH, r.t.

b) DIBAL-H 1M, hex, THF, 0ºC

c) MeNO2, EDA, D

d) H2, Pd/C (10%), EtOH, r.t.

e) TosMIC, KOtBu, EtOH,

1,2-dimethoxyethane, r.t.


OH

O

O

OH

O

COOEt

OH

O

EtOOC

OH

O

COOEt

OH

O

COOEt

OH

O

COOEt

OH

O

COOEt

1

19

20

21a

21b

22a

22b

a b c

a) Ph3P=CHCOOEt, benz., D

b) H2, Pd/C (10%), EtOH, r.t.

c) I) LDA, HMPTA, THF, -78ºC,

ii) H2C=NMe2I, THF, - 42ºC to r.t.

iii) MeI, dioxane, 90ºC, iv) NaHCO3, H2O/EtOAc, r.t.

OO

OHX

Z

Y

H


b) i) Me3SiOC(=CH2)CH=CHOMe, benz.

ii) HCl (c), CHCl3, r.t

c) H2, Pd/C (10%), EtOH, r.t.

OO

OHX

Z

Y

H


OH

O

COOEt

OH

O

COOEt

EtOOC

O

EtOOC

O

EtOOC

EtO

OEt22a,22b

23 24 27

+

b

c

EtOOC

O

EtOOC

O

EtOOC

EtO

OEt25 26 28

+ c

OO

OHX

Z

Y

H


a) LAH, Et2O, r.t.

b) TBDMSTf, Et3N, CH2Cl2

EtOOC

EtO

OEt

EtO

OEt

OH

OH

HO

EtO

OEt

OH

OH

TBDMSO

2732 35

a b

EtO

OEt

OH

OH

TBDMSO

EtO

OEt

OH

O

TBDMSO

EtO

OEt

OH

OH

TBDMSO

OH

O

TBDMSO

O

OH

O

TBDMSO

HO

OH

TBDMSO

O

HO

33 36

3537

38

39

+

a

ab

OO

OHX

Z

Y

H


a) PCC, Py, CH2Cl2,

b) NaBH4, MeOH, -15ºC

1 - Antecedents



3 - Bioevaluation


Contents

Bioevaluation Methods:

Spontaneously bating right atria (rat)

Electrically stimulated left atria (rat)



contraction and rate


contraction



contraction and rate


contraction

Na,K – ATPase (rat)

Na,K - ATPase isozymes (human)

Bioevaluation results:

Cyclohexanebutenolides:

inactive Pimaranebutenolides: inactive // negative inotropic

Indanederivatives:

positive inotropic

Dinorsecosteroids: pending

-50

0

50

100

150

200

250

300

% O

F C

ON

TR

OL

-8 -7 -6 -5 -4 -3

Concentration (Log M)

36b

17a

17b

CONTRACTION FORCE

RIGHT ATRIA

The Indanol Evaluation

-50

0

50

100

150

200 %

OF

CO

NT

RO

L

-8 -7 -6 -5 -4 -3

Concentration (Log M)

36b

Digoxin

CONTRACTION FORCE

LEFT ATRIA

The Indanol Evaluation

Indane Positive Inotropics

NO 2

N

N H 2 N

NH 2

N

N

N H 2 N

NH 2

N

NH 2

NH 2

36b

20

The Indanol Evaluation Rat Na+,K+ - ATPase inhibition

[20 ]/ M ; [ouabain] / M

50

10 -8 10 -7 10 -6 10 -5 0

100

130

resi

du

al N

KA

act

ivit

y /

%

J. Med. Chem. 45, 127 (2002)

IC50 (M)

Compound 20 Ouabain

2.10-6 3.10-7

5.10-4 5.10-5

The Indanol Evaluation Rat Na+,K+ - ATPase inhibition

Human Na,K-ATPase isoenzymes activities

in absence (0) or presence of 36b

0

50

100

150

200

250

0 a

1b

1

1m

M a

1b

1

0a1

b1

1m

Ma1

b1

0a2

b1

1m

M a

2b

1

0a2

b1

1m

M a

2b

1

0 a

2b

1

5m

M a

2b

1

5m

M a

2b

1

0a3

b1

1m

M a

3b

1

0a3

b1

1m

Ma3

b1

Data #CAR 10 pmol

0

1b

1

0

1b

1

1m

M

1b

1

1m

M

1b

1

1m

M

b

1

1m

M

b

1

5m

M

b

1

5m

M

b

1

1m

M

b

1

1m

M

b

1

0

b

1

0

b

1

0

b

1

0

b

1

0

b

1

1b1

2b1

3b1

1 - Antecedents



3 - Bioevaluation


Contents

The reason for the inactivity of pimarane-lactones The Reason for the Inactivity of Pimarane-butenolides?

The Reason for the Inactivity of pimarane-butenolides?

The Reason for the Inactivity of pimarane-butenolides?

D H = 14 kJ / mole

The Reason for the Activity of Guanylhydrazones?

superimposition guanylhydrazone / digitoxigenin

*


superimposition guanylhydrazone/erythrophleine

*



*

*

The Ent-Indanol Evaluation

- 100

- 50

0

50

100

150

200

250

1,E - 09 1,E - 07 1,E - 05 1,E - 03

Concentration (M)

Inotr

op

ic R

esp

on

se

(% o

f C

on

tro

l)

11

15

16

19

20

22

- 100

- 50

0

50

100

150

200

250

1,E - 09 1,E - 07 1,E - 05 1,E - 03

Concentration (M)

Inotr

op

ic R

esp

on

se

(% o

f C

on

tro

l)

11

15

16

19

20

22

11 11

15 15

16 16

19 19

20 20

22 22

43

CONTRACTION FORCE

RIGHT ATRIA


Inotropic activity of guanylhydrazones (left atria)

and effect on the cardiac frequency (right atria)

compound Emax(1) Emax(2) Emax(3)

22 (norm) 129 6.5 33.7±6.3 0.3

43 (ent) 170 66.5 - 5.1±7.4 0.3

Digoxin 103 14.3 - 28.3±3.7 10-3

Ouabain 122 58.4 80.4±39.4 10-3

(1) % of basal force, (2) % of basal frequency, (3) mM

superimposition of bishydrazone enantiomers?

N

N

NNH2N

NH2

NH2

NH2N

N

NNH2N

NH2

NH2

NH2

n e

* *

* *

superimposition of the enantiomers?

n e

* *

*

*

superimposition of the enantiomers

n e

* *

superimposition of the enantiomers

n e

*

*

n e

* *

superimposition of the enantiomers?

n e

* *

2b1 isoenzyme activity in absence (0) or

presence of 36b (n), 43 (ent) and ouabain

0

1000

2000

3000

4000

5000

6000

dp

m/o

oci

to

0 a

2b

1

33

a2

b1

43

a2

b1

Ou

abaí

na

a2b

1

n.i

.

0

b

1

33

b1

43

b1

Ou

ab

.

b

1

0 n

.i.

enantio (43)

normal (36b)

ouabain

(0)

Rb

Uptake

(%)

100

Column 1

compound 36b normal 43 enantio

3b11b1 2b1

50

86

isozyme b b 1 3 11 1 2b


Conclusions: - We have developed a synthetic methodology

for the preparation of a variety of cardioactive

hydroindene derivatives.

- Those belonging to the normal steroidal

series, act through inhibition of Na,K-ATPase

- Those belonging to the enantio series, being

more potent, does not act by this mechanism

- They constitute ones of the simplest

homocyclic structures reported as

cardiotonics and negative inotropics.

Chemistry QF-USAL

Dr. Manuel Medarde

Dr. José Luis López

Dr. Fernando Tomé

Dr. Belen Hebrero

Dr. Melchor Boya

Dr. Concepción P. Melero

Dr. Luis G. Sevillano

Pharmacology - USAL

Dr. María José Montero

Dr. Rosalía Carrón

Dr. Pilar Prieto

Molecules 5, 51

Tetrahedron 45, 1815

Tetrahedron Letters 35, 8683

An. Quim 91, 89,

Synt. Commun. 25, 1377

Eur J Med Chem 26, 799

Bioorg Med Chem Lett 8, 3217

Eur J Med Chem 28, 887

Bioorg Med Chem 7, 2991

Tetrahedron 58, 1266

J. Med. Chem. 45, 127

Biochemical Assays

Drs. Lionel G. Lelièvre, Concepción P. Melero

University of Paris 7. France

Drs. Käthi Geering, Gilles Crambert, Luis G. Sevillano

University of Lausanne. Switzerland.

Financing:

DGICYT, CICYT, JCyL, (SPAIN)

Muito obrigado!!

UNIVERSITY OF SALAMANCA

1218



Erythrophleine conformers

1 + 2 + 3

Superposición de secoesteroide con digitoxigenina y eritrofleína

1 + 2 2 + 3

Digitoxigenin dinorsecoesteroid erythrophleine 1 2 3

superimposition dinorsteroid, digitoxigenin, erythrophleine

UNIVERSITY OF SALAMANCA

1218

Universidad 2oo2 Universidad 2oo2

Arturo San Feliciano · CARDIAC INSUFFICIENCY PRE CHARGE POST- CHARGE COMPENSATING MECHANISMS ......

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