Anti-Cancer drugs -Telomeres and Cell Senescence

• Chromosomes within animal and human cells have a group of guanine groups (telomeres) at their 3’termini.

• These form a 4-membered structure or G-tetrads. Upon cell division a number of these guanine groups are lost and typically not replaced. Eventually the number of Guanine groups is reduced to the point that other cell-defence mechanisms destroy the cell (apoptosis) to reduce the risk of mutation of the gene sequence which may lead to disease, including certain cancers.

Unzipping of chromosonal DNA

Imperfect DNA replication mechanismsresults in loss of telomere G-tetrads shortening telomeres in daughter DNA strand

Cartoon representation of Guanine rich Hoogsteen base paired tetrads

G-Quadruplex Ligands• The natural shortening of telomeres in eukaryotic cells has been implicated in

senescence and subsequent apoptosis.• The competing action of telomerase is restricted to certain tumor and early

stem cells. • The genetic coding for the telomerase enzyme and the potential inhibition of

telomerase expression are considered important areas of research, not merely from the perspective of understanding the genetic dependency but, via a new fundamental maxim, will provide insight into prospective treatments.

N

NN

NN

O

NN

N

N

NH

O

N

N N

N N

O

N N

N

N

HN

O

H

H

H

H

H

H

H

H

H

H

Telomerase and telomere extension• Tumor cells are the exception to this rule,

in that an enzyme called Telomerase is produced and goes about replacing the G-quadruplex structure on the ends of the chromosomes making them theoretically immortal.

• A way of stopping the replenishment of the guanine groups would be to formulate a chemical that would adhere to the G-quadruplex in such a way as to prevent this telomeric elongation.

• This would cause cancer cells to lose their

telomeres as a normal healthy cell does. The fact that cancer cells replicate faster than normal healthy cells (whose telomeres are longer anyway) would serve to selectively diminish the length of telomeres on chromosones in cancerous cells and result in them dying (via apoptosis) faster than healthy cells.

Imperfect DNA replication mechanismsresults in loss of telomere G-tetrads shortening telomeres in daughter DNA strand

Telomerase protein comprising RNA and primer

Current thinking• Antisense oligonucleotides • Telomerase antagonists• Oligopeptide vaccines• G-Quadruplex Ligands

– Braco19 (Antisoma)– Xenograft tumour models clinical trials

Biomimetics• One of the strategies would be to design

a molecule capable of mimicking the G-quadruplex structure.

• High throughput screening using for example DOSY NMR would allow the formation of both static and dynamic combinatorial libraries of small organic species ultimately capable of stabilising the biological equivalent.

• Significant research could be carried out in the mode of drug action and the actions of the potential inhibitor within living systems.

• The investigation of potential G-quadruplex liganding agents would also involve consideration of physicochemical aspects of both the ligand library and the biological systems affected, inculding thermodynamics and chemical kinetics.OR

OR

ON

ON

RO

RO

O

O N

HN

N

NN

NHN

O

NN

NN

HN

O

N

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NH

O

G-quadruplex model

Linker Group

Calix foundation

Research Calix[4]arenes in host-guest chemistry

ROOR

OR RO

O

OHN

HN

OHN

O NH

HN

NH HN

NH

O O

OO

BuOOBu

OBu BuO

II

I I OHN

NH

O

Et3N, 15% Pd(OAc)2, DPPP,DMF, 100oC, 275hrs

On the scope and limitations of the Heck reaction of upper rim tetraiodocalix[4]arenes J. Chem. Soc. Perkin Trans. 1, 2001, 24, 3393-3398, Kuhnert N and Le Gresley A

Capusle formation and binding to Pesticide

SS S

N

S

N

Synthesis and capsule formation of upper rim substituted tetra acrylamidocalix[4]arenes Organic and Biomolecular Chemistry, 2005, 11, 2175-2182 N. Kuhnert and A. Le Gresley

8.1 Å

13.1 Å

Further corroborated by the ESI-MS showing a signal at m/z 2898

fungicide tetramethyl-thiuram-disulfide

Dynamic combinatorial libraries using calix[4]arenes

ROOR

OR RO

O

OO

O

OO

O O

N

N

NN

selection of aminesOR

OMe

ROOR

OR RO

O

OO

O

OO

O O

NN

NN

OMe

NHHN

S

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OH

Biotin

OR

OR

OO

O O

O

O

RORO

O

OO

O

OO

The synthesis of static and dynamic combinatorial libraries using deep cavity tetra-formyl calix[4]arenes N. Kuhnert and A. Le-Gresley, Tetrahedron Lett. 2005,46, 2059-2062.

DMF 100oC 24-150hrs

Et3N/ 10% Pd(OAc)2/ DPPP

O

R'

O

O O

R' =

2 3 4

5 6 7

ROOR

OR RO

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OR'OR'O

OR'O

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ROOR

OR RO

III

I

1

R = n-Bu

Heck Methodology

ROOROR RO

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R'R'R'

R'

ROOROR RO

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MS 4A

NH

OCH3

H3C (CH2)7

OH

(Et3O)SiHN

NO

N

N N

HNO

O

C O

O

C

O

O

O

R'NH2

a b c d e f g

h i j k l

m n o

R' =

8-225

Reversible Reactions

Kuhnert, N and Le-Gresley, A. Synthesis of upper rim calix[4]arene carcerands. Tetrahedron Letters, 98, 1274-1276. 2008

CHO CHO

ROOROR RO

OOOO

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N

ROOROR RO

OOOO

OOO O

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and

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O

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Barbituric Acid

Biotin

5cgg

5e

23

24

5ae

HN

OHCOHC

OHC

Imine Molecular Formula Mass Peak (m/z) Intensity

(relative %) Guest

5c C91H87O15N 1434 35 None

5ae C101H96O15N3 1590 15 -

5a C91H87O16N 1450 10 -

5e C94H90O15N2 1487 5 -

5e C84H90O15N2 1487 39 23

5cgg C99H105O13N3 1540 25 24

Combinatorial Library

Le Gresley, A The design and synthesis of deep cavity calix[4]arenes in the development of static and dynamic macrocyclic libraries. European Journal of Organic Chemistry, in press. 2009

Anthracene Diacrylamides

X

X

ONR2

X

ONR2ONR2

ONR2

O O

X=Cl X=Br X=CF3SO3

Pd(OAc)2

Ligand

DMF Et3N+

a. R= Meb. R= Hc. (CH2CH2)2O 1 R = H

3 R = Me

+

2 R = H4 R = Me

5 R = 6 R =

HETCOR & Activity

C3 C4 C6

C5

C2

C1

ONH2

ONH2

2

C2 C1 C4C3

C5

C6

Manuscript in preparation

G-Quadruplex Formation

Increase in G-quadruplex formation with time

•TTGGGGT forms parallel strand G – quadruplex

•Potassium ion stable

•Presence of anthracene acrylamide increases G-quadruplex component

•DOSY studies underway

Zhou, Q.; Lin, L.; Xiang, J.; Sun, H.; Tang, Y.; Fast screening and structural elucidation of G-quadruplex ligands from a mixture via G-quadruplex recognition and NMR methods. Biochimie, 2008, 1-5.

Acknowledgements

• Prof Nikolai Kuhnert, Jacobs Bremen• Dr Jean-Marie Peron, Kingston• Judith Peters, Surrey• RSC• Invitrogen

Compelling

• It has been approximately 15 years since telomerase was described as an almost universal marker for human cancer.

• Shortened telomeres undergo replicative senescence• TRAP Assay inappropriate for G-Quadruplex ligand

activity measurement Paper – De Clan et al. PNAS, 2008

• Grant applications currently pending, – Cancer Research UK– Royal Society

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