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Synthesis directed towards antimicrobial purines. Lise-Lotte Gundersen Department of Chemistry, University of Oslo, Norway. >25% of all deaths worldwide direct result from infection. Nature 2004 , 430 , 242. History of antimicrobial drugs Quinine ( Cinchona pubescens ; malaria) ca. 1825 - PowerPoint PPT Presentation
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Synthesis directed towards antimicrobial purines
Lise-Lotte GundersenDepartment 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
SO
OH2N
H2N
NH2SO
OH2N
Prontocil
in vivo
Sulfanilamid
NH2SO
OHN
NO
Sulfametoksasol (Bactrim etc)
AsAs
HOOH
NH2
H2N
As As
As As
As As
AsAs
OHNH2
OH
NH2
OHH2NHO
H2N
HO
H2N
OHNH2
HO
H2N
OHNH2
•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
OCO2H
US Surgeon General, 1967: “The war against infectious diseases has been won”
Bacitracine (peptides)
NH
HN
NH
HN
NH
HN
4O
OHNNH2
OHO2C
O NHN
O
HN
O
O
NH2
O
ONHO
NHHO2CHNO
ON
S
NH2
Other classes of antibacterial antibiotics
N
S
OCO2H
HH
HN
HOONH2
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:CytotoxicAntimicrobialInhibition 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
OHOTHPSO2Ph
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%
ΔMA, 50 oC
1) Zn, AcOHMeOH / H2O
2) NaCl(aq) Cl
(R ) Enantiom er 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 SPhMeth. 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 SO2PhMeth. 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
OO
PhSO2O
(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 (mg/m L)
Mycobacterium tuberculosisMIC (mg/m L)
HO
(G eranylgeraniol)
O
O
O
4.0 3.1
3.1n.d .
> 32 1.6
n.d . n.d .(0% inhib.
at 6.25 mg/m L)
Vik et al., Bioorg. Med. Chem. 2007, 15, 4016Planta Med. 2007, 73, 1410
Activity Patogenic Protozoa Tropical diseases
chloroquine 0.04 mg/mL
miltefosine 0.24 mg/mL
benznidazole 0.25 mg/mL
melarsoprol 0.005 mg/mL
N
N N
N
NO
N
N N
N
N
H
N
N N
N
NH2
H
O
Cl
(+) Agelasine D
MIC (mg/m L)P. falciparum(Malaria)
MIC (mg/m L)L.donovani(V iceral le ichm aniasis / kala-azar)
MIC (mg/m L)T. cruzi(Chagas disease )
MIC (mg/m L)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
NNR
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
NNR
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. Bu3SnHBr, 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) Com pds 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-com plex, CH3CN
(c.f. Lit.)
8
H2, Pd/C
H2SO2MeOH85%
N
N N
NNR
R'' R'
N
N N
N
NR
R'' R'
RCM ?
7
dcat. 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 mg/mL
MIC Lactobacillus casei (sensitive G+ bacteria): >10 mg/mL
Active in vitro against M. tuberculosis infected macrophages
IC50 Vero cells: 8.1 mg/mL - SI (IC50 / MIC): 10.4
%Inhib. K-562 cells 10 mg/mL: 27%
Max. tollerated dose given IP (no accute toxicity) to rats ≥ 500 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 (mg/mL)Strain M. tub
MIC M. Tub. 0.78 mg/mL
Rifampin:MIC M. Tub. 0.25 mg/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 mg/mL MIC (mg/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
