Synthesis directed towards antimicrobial purines

Lise-Lotte Gundersen

Department of Chemistry, University of Oslo, Norway

Nature 2004, 430, 242

>25% of all deaths worldwide direct result from infection

History of antimicrobial drugs

•Quinine (Cinchona pubescens; malaria) ca. 1825

•Salvarsan (syphilis) 1912

•Sulfa drugs (bacterial infections) ca. 1935

N

O

HO N

NN NH2

S

O

O

H2NH2N

NH2S

O

O

H2N

Prontocil

in vivo

Sulfanilamid

NH2S

O

O

HN

NO

Sulfametoksasol (Bactrim etc)

As

As

HO

OH

NH2

H2N

As As

As As

As As

AsAs

OH

NH2

OH

NH2

OHH2NHO

H2N

HO

H2N

OHNH2

HO

H2N

OH

NH2

•1928: Penicillum notatum (= P. chrysogenum) inhib. Staph. aureus growth

(Flemming)

•1938: Isolation “penicillin” (Florey, Chain)

•1941: Penicillin on humans

•1943: Big-scale production penicillin

•1943: Structure penicillin G proposed (Chain)

•1945: Nobelprize in medicine; Fleming, Chain, Florey

•1945: X-ray structure penicillin G (Hodgkin)

•1957: Synthesis penicillin G (Sheehan)

Penicillin

N

S

CO2H

H

O

NH

O

Penicillin G(Benzylpenicillin)

1944: Streptomycine (aminoglykoside)

1945: Cefalosporine C (cefalosporines)

1947: Chloramphenikol

1948: Chlorotetraycline (tetracyclines)

1950: Picromycine (makrolides)

1959: Rifamycine (ansamycines)

O

O

HO

O O

O O

NHO

O

OH

NH2

O

N

OHHOOOH

HOH H

Cl

O2

N

H

H

OH

NH

OH

O

Cl

Cl

HOO

NHMeHO

HOHO

O

HOOH

OH

HN

NH

NHH2N

NHH2N

OH OH

O

NH

O

OOHOH

HO

MeCO2

MeO

O

O

CO2H

US Surgeon General, 1967: “The war against infectious diseases has been won”

Bacitracine (peptides)

NH

HN

NH

HN

NH

HN

4

O

OHNNH2

O

HO2C

O NHN

O

HN

O

O

NH2

O

O

NHO

NHHO2CHNO

ON

S

NH2

Other classes of antibacterial antibiotics

N

S

OCO2H

HH

HN

HO

ONH2

O O

O

Nature 2004, 430, 242

Emerging and re-emerging infections ca. 1980 (AIDS)-date.

Emerging infections Re-emerging infections Deliberate emerging infections

Nature 2004, 430, 242

Nature 2004, 431, 892:

“Antibiotics are the worst

sort of pharmaceuticals

because they cure the

disease”

H

N

N N

N

NH2

Cl

Agelasine A

Agelasines Agelasimines Asmarines

N

N N

N

NHO

H

Asmarine A

N

N N

N

N

Agelasimine A

OH

H

Our Activities:•Purine-Containing Antimicrobial Natural Products from Marine Sponges

•Selective Antimycobacterial Purines N

N N

N

X

O

Marine Sponges?

Agelasines

•Isolated from marine sponges (Agelas sp.)

•11 comp. (Agelasine A - I, epiagelasine C and ageline B) known to date

•Total syntheses other groups: Agelasine A, B and C, and (±) agelasine F

•Bioactivities:

Cytotoxic

Antimicrobial

Inhibition of Na,K-ATPase

H

N

N N

N

NH2

Cl

Agelasine A

7,9-dialkylated Adeninium salt

Terpenoid side chain at N-7

Brown Tube Sponge: Agelas sp. 2.5 feet long

Synthesis of Trixagol and Agelasine E

N

N N

N

NH2

CH3

CO2H

OH

Agelasine E

Geraniol

Cl

N

N NH

N

NH2

Helv. Chim. Acta , 1995, 539

AdenineTrixagol

OH

Isolated from Bellardia trixagoTL, 1978, 3491Isolated from Agelas sp.

Tetrahedron Lett. 1984, 2989

Synthesis of Trixagol and ent-Trixagol

Bakkestuen et al., Tetrahedron 2003, 59, 115

HO2C

HO2C

HO2C

O

H3CO2C

OOOCH3

OCH3

O

O

4 steps

+c.f. lit.

Synth. rac. acid: Helv. Chim. Acta, 1952, 1752 Resolution:Helv. Chim. Acta. 1995, 539

c.f. lit.

Trixagol

ent-Trixagol

Agelasine E

Trixagol

OHOTHP

SO2Ph

PhSO2

OTs

OTHP

n-BuLiDMPUHO2C 4 steps 2 steps

OH

3 steps

Synthesis of Agelasine E and ent-Agelasine E

N

N N

N

NH2

R

N

N N

N

NH

R

R'N

N N

N

NH2 R

N

N N

N

NH R

R'

Regioselectivity in dialkylation of adenines

R'X R'X

N

N N

N

NH

R

CH3O

N

N N

N

NH

R

CH3OR'

X

R'X N

N N

N

N

R

CH3O R'

+

Bakkestuen et al., Org. Biomol. Chem. 2005, 3, 1025

N

N N

N

Cl

N

N N

N

NHMeO

N

N N

N

NMeO

N

N N

N

NMeO

+

MeONH3Cl, Et3N

n-BuOH, Δ63%

DMA, 50 oC

1) Zn, AcOHMeOH / H2O

2) NaCl(aq) Cl

(R) Enantiomer shown(S), Trixagol

OH

82% (R)96% (S)

BrPBr3

Et2O, 0 oC

N

N N

N

NH2

48% (R)44% (S)

32% (R)26% (S)

54% (R) Agelasine E89% (S)

Synthesis of Agelasine D

Agelasine D Manool

Sclareol

Salvia sclarea (Clary Sage)

Isolated from Agelas sp.Tetrahedron Lett. 1984, 2989

N

N N

N

NH2

H

H

X

H

HO

H

HO

OHCl

Synthesis of Agelasine D - Initial Approach

Drawbacks: Lack of selectivity

Utenova et al., Tetrahedron Lett. 2004, 45, 4233

H

Br

N

N N

N

NHMeO

N

N N

N

N

N

N N

N

NMeO

+

DMA, 50 oC

Zn, AcOH

MeOH, H2O

H

N

N N

N

NH2

H

MeO

H

60% E:Z 8:243% pure E after cryst.

27% E:Z 8:2

Cl

51%81% E:Z 8:2

PBr3, pyridine

Et2O, -35 oC

H

HO

(+) Manool

(+) Agelasine D

Improved Synthesis of Agelasine D

Synthesis of geometrically pure allyl bromide

H

Br

81% E:Z 8:2

PBr3, pyridine

Et2O, -35 oC

H

HO

(+) Manool

Vik et al., J. Nat. Prod. 2006, 69, 381

H

AcO

90% E:Z 92:8

Ac2O, DMAP, Et3N

THF, Δ

H

AcO

PdCl2(MeCN)2

THF, 0 oC

K2CO3

MeOH

H

HO

76% Pure E

PBr3, pyridine

Et2O, 0 oC

H

Br

76% Pure E

Anticopalol

Improved Synthesis of Agelasine D - Regioselective N-alkylation

Vik et al., J. Nat. Prod. 2006, 69, 381

H

Br

N

N N

N

NHO

N

N N

N

N

DMA, 50 oC

Zn, AcOH

MeOH, H2O

H

N

N N

N

NH2

H

O

Cl

49%

(+) Agelasine D

90%

Only isomer observed

N

N N

N

NHRO

DMA, 50 oC

PHCH2Br N

N N

N

NRO Ph

A

+ N

N N

N

NRO

Ph

B

R- Ratio A : B Yield (%) A Yield (%) B

CH3- 3 : 2 55 30

t-Bu- 9 : 1 86 9

PhCH2- 4 : 1 72 13

H

Br

N

N N

N

NHMeO

N

N N

N

N

N

N N

N

NMeO

+DMA, 50 oC

H

MeO

H

60% E:Z 8:2 27% E:Z 8:2

Synthesis directed towards Agelasine F

Isolated from Agelas sp.Tetrahedron Lett. 1984, 2989

N

N N

N

NH2

CH3

Agelasine F(Ageline A)

Cl

Isolated from pennyroyal

(Mentha Pulegium)

OH

Geraniol

N

N NH

N

NH2

Adenine

X(S)-Pulegone

O

(R)-Pulegone

O TMSO

TMSCl, Et3N

DMF

1) LDA2) MeI

THF- 78 oC

O

KOH (aq)

48% from pulegone

Cl SPh

Meth. A or B

O

PhS

A

O

PhS

B

+

Meth A: TiCl4, CH2Cl2, -23 oC

Yield(%) A Yield(%)B

57 33

Meth B: MeLi, DME, 0 oC 5 3

Proszenyak et al., Unpublished

TMSO

Cl SO2Ph

Meth. A or B

O

PhS

O

PhS

oxone

MeOH, H2OO

PhSO2

O

PhSO2

87%

85%

oxone

MeOH, H2O

BrO

(R)-Pulegone Side chain ent-Agelasine F

BrO

(R)-Pulegone Side chain ent-Agelasine F

Proszenyak et al., Unpublished

PhSO2

PhSO2

Agelasine E Agelasine F

More reactive

O

PhSO2 MeMgI

Et2O0 oC - rt

PhSO2

HO82%

HCO2H80 oC

PhSO2

1) BuLi

2) THPO

THF, 5 0 oC

THPO

SO2Ph

93% conversion

THPONa, NaHPO4

EtOH, THF

PPTs

EtOH, 55 oC

HO

I

Intermediate in synthesis of other natural products ?

O

(S)-Pulegone

O

O

O

OculatolideO

Subersin

S

NHH2N

HN

OO

(-) Agelasidine CAgelasidine D

OH

S

NHH2N

HN

O

O

PhSO2

O

(R)-Pulegone

O

O

O

Sollasin D

O

Sollasin A(Fulvanin 1)

O

S

NHH2N

HN

OO

S

HN

H2NNH

OO

(+) Agelasidine C

Agelasidine B

PhSO2

O

O

Microcionin 2

O

O

O

O

Striatol

OH

Synthetic intermediate - simpler side chain, Even better activities than naturally occuring agelasines!!

HN

N N

N

N

R''

RO

N

N N

N

N

R''

RO R'

N

N N

N

NH2

R''

R'

ClR'-CH2-Br

N

N N

N

N

CH3

MeO

HO

geranylgeraniol

Agelasine analog

Type A structure Type B structure

Structure - Activity Relationships

•Type A more active than type B structure

( otherwise same subst.)

•R’ must be relatively long and preferably

contain unsaturation(s)

N

N N

N

N

CH3

MeO

Agelasine analog Antimicrobial activities

Vik et al., Bioorg. Med. Chem. 2007, 15, 4016

Also activity against several cancer cell lines

Tuberculosis (TB)

Agelas dispar

WHO (1993): TB a “global emergency”

•AIDS

•Resistant strains

•Migration

•Powerty

Ca 3 mill deaths / year

•Bacterial infection (Mycobacterium tuberculosis)

•Inhalation

•Lung most commonly attacked

•All organs, incl. CNS, may be attacked

•Before 1950; 50% of all sick died

•First effective drug: Streptomycin (1943) - Toxic

•Several less toxic drugs developed ca 1950-1962

N

N N

N

N

CH3

MeO

Staphylococcus aureus

MIC (μg/mL)

Mycobacterium tuberculosisMIC (μg/mL)

HO

(Geranylgeraniol)

O

O

O

4.0 3.1

3.1n.d.

> 32 1.6

n.d. n.d.(0% inhib.

at 6.25 μg/mL)

Vik et al., Bioorg. Med. Chem. 2007, 15, 4016Planta Med. 2007, 73, 1410

Activity Patogenic Protozoa Tropical diseases

chloroquine 0.04 μg/mL

miltefosine 0.24 μg/mL

benznidazole 0.25 μg/mL

melarsoprol 0.005 μg/mL

N

N N

N

NO

N

N N

N

N

H

N

N N

N

NH2

H

O

Cl

(+) Agelasine D

MIC (μg/mL)P. falciparum(Malaria)

MIC (μg/mL)L.donovani(Viceral leichmaniasis / kala-azar)

MIC (μg/mL)T. cruzi(Chagas disease)

MIC (μg/mL)T. b. rhodesiense(African sleeping sickness)

0.10 <0.04 0.11 0.10

0.29 0.14 0.49 0.30

0.29 1.50 4.53 0.90

Vik et al., Unpublished

Viceral Leichmaniasis / Kala azar / Black Fewer

•Infection - Leichmania donovani s.l. (protozoa)

•Transmitted by sandflies

•Deathly if untreated; 59.000 deaths/year

•Treatment: Long time with antimon-compounds

(miltefosine, amfotericin B)

• > 90% i Bangladesh, Brasil, India, Sudan

Chagas disease / American Sleeping Sickness

•Infection - Trypanosoma cruzi (protozoa)

•Transmitted by insects (Teger)

•Deathly if not treated in time (Ofthen long period without symptoms);

13.000 deaths/year

•Treatment: Nifurtimox or benznidazol, in the early phase

•South- & Central America

H

N

N N

N

NH2

Cl

Agelasine A

Agelasines Agelasimines Asmarines

N

N N

N

NHO

H

Asmarine A

N

N N

N

N

Agelasimine A

OH

H

Our Activities:

Purine-Containing Marine Natural Productsfrom Marine Sponges

Compounds with Antimicrobial Activities

Asmarines

•Isolated from marine sponges (Raspailia sp.)

•11 comp. (Asmarine A - J)

•No total syntheses

•Bioctivities: Cytotox.

N

N N

N

NR

H

Asmarine A: R = OHAsmarine B: R = OH (epi at C'-5)Asmarine G: R = OMeAsmarine H: R = HAsmarine K: R = H (epi at C'-5)

N

N N

N

NR

H

O

* *

Asmarine D: R = OHAsmarine C: R = OH (epi at C'-5)Asmarine E: R = OMe (epi at C'-5)Asmarine F: R = OMe

N

N N

N

NR

H

Asmarine I: R = OHAsmarine J: R = H

Previous strategies for construction of the 7-membered ring

N

N

N

N

NR

R1R2

a

b cd

e

N

N

N

N

Cl

HNMOM

N

HN

N

N

NR3O

N

N

NH

N

NR

OH

N

N

N

N

NR

SOCl2Et3N, BuOH

R1Br

N

HN

N

N

NR3O

R1R2

R1 = R2 = H

R1

1. I2, NaHCO32. Bu3Sn

HBr, AcOH

No ex. of formation of bond c or d

Formation of bond d - Initial attempts

Vik et al.Tetrahedron. Lett. 2007, 48, 1931

Co-complex

NCo

N

N

N

OH2

O O

O

H

OH

N

N N

N

Cl

HXCH2CH=CH2

Pyridine, 100 oCN

N N

N

X

a: X = NH, 89%b: X = NMe, 92%c: X = Od: X = NBoc, 65%

(Boc)2ODMAP, CH2Cl2

N

N N

N

NBocGrubbs II or

Hoveyda-Grubbs II

DCE, Δ

54% (Grubbs II) 73% (How.-Grubbs II) Compds a-c: n.r.

N

N N

N

HN

HCl(aq)

MeOH81%

N

N N

N

HN

N

N NH

N

Cl I-CH2CH=CH2, K2CO3, Co-complex, CH3CN

(c.f. Lit.)

8

H2, Pd/C

H2SO2MeOH

85%

N

N N

N

NR

R'' R'

N

N N

N

NR

R'' R'

RCM ?

7

d

cat. Ru-complex+

Ring Closing Metathesis (RCM)

N

N N

N

NR

R'' R'

N

N N

N

NR

R'' R'

RCM ?

7

N

N N

N

NH

(Boc)2O, DMAP

MeCN

Hoveyda-Grubbs II

DCE, ΔN

N N

N

NH

N

N N

N

NBoc

68%

N

N N

N

NBoc7

95%

K2CO3

MeCN, Δ

100%

N

N N

N

HN

76%

1) H2, Pd/C, EtOAc

2) HCl, MeOH

Vik et al.Tetrahedron. Lett. 2007, 48, 1931

N

N N

N

HN

H

Asmarine H

H

N

N N

N

NH2

Cl

Agelasine A

Agelasines Agelasimines Asmarines

N

N N

N

NHO

H

Asmarine A

N

N N

N

N

Agelasimine A

OH

H

Our Activities:•Purine-Containing Antimicrobial Natural Products from Marine Sponges

•Selective Antimycobacterial Purines N

N N

N

X

O

MIC Mycobacterium avium: 25 μ /g mL

MIC Lactobacillus casei ( + ): >10 sensitive G bacteria μ / g mL

Active in vitro against . M tuberculosis infected macrophages

IC50 : 8.1 Vero cells μ / - (g mL SI IC50 / ): 10.4MIC

% . -562 10 Inhib K cells μ / : 27%g mL

. ( ) ≥ 500 /Max tollerated dose given IP no accute toxicity to rats mg Kg

High selectivity against. M tuberculosis

Acceptable toxicity

Activity against drug resistant strains

M. tub. H37RV

Isoniacid

Rifampin

Ethambutol

Kanamycin

Ciprofoxacin

0.78

3.13

1.56

<0.39

0.78

<0.39

MIC (μ / )g mL Strain . M tub

MIC M. Tub. 0.78 μg/mL

Rifampin:MIC M. Tub. 0.25 μg/mL

N

N N

N

Cl

Cl

cat. Pd N

N N

N

Cl

O SnBu3

O

N

N NH

N

Cl

Cl

Base, BrCH2Ph

Langli et al., Tetrahedron 1996, 52, 5625

Bakkestuen et al. Bioorg. Med. Chem. Lett. 2000, 10, 1207Gundersen et al. J. Med. Chem. 2002, 45, 1381

Antimycobacterial 6-(2-Furyl)purines: The N-9 Substituents

Gundersen et al. J. Med. Chem. 2002, 45, 1381

N

N N

N

O

R

R % Inhib. 6.25 μ /g mL MIC (μ / )g mL

6

0

-H

0-CH3

7O

OHO

OHHO

6

22

26

>90 3.13

13 Bakkestuen et al., Tetrahedron Lett., 2003, 44, 3359

B(OH)2

Cu(OAc)2Phenanthroline

Mol sieveCH2Cl2

N

N NH

N

Cl

N

N N

N

Cl

N

N N

N

O

"Stille"

71% 61%

+

Summary SAR knowledge

Gundersen et al. J. Med. Chem. 2002, 45, 1381Bakkestuen et al. J. Med. Chem. 2005, 48, 2710Brændvang et al. Bioorg. Med. Chem. 2005, 13, 6360Brændvang et al. Bioorg. Med. Chem. 2007, 15, 7144

N

N N

N

R6

R2

Aryl (electron rich)

EDG, lipophilic (o/p)

O

Smal, lipophilicMe > Cl = F > OMe = SMe > H = Et

* No subst

*

*

* *

*

but also non-purine analogs...

Acknowledgements

Synthesis•Dr. Anne Kristin Bakkestuen

•Dr. Morten Brændvang

•Abhijit K. Datta

•Veronika Ernst

•Anthony James

•Aisvareya Kulendern

•Dr. Geir Langli

•Dr. Pedro O. Miranda

•Dr. Edyta Nagrodzka

•Dr. Agnes Prozenyak

•Matthew L. Read

•Heidi Roggen

•Linda W. Tangen

•Dr. Bibigul T. Utenova

•Dr. Anders Vik

Bioactivities•Prof. Lars Bohlin & co-workers

•Dr. Colin Charnock•Tuberculosis Antimicrobial Acquisition

and Coordinating Facility (TAACF)•WHO - TDR

Financial Support•NFR (FRINAT & KOSK)

•UiO