NPTEL Phase II (Syllabus Template) Course Title: Bio ... · NPTEL – Chemistry – Bio-Organic...

NPTEL – Chemistry – Bio-Organic Chemistry of Natural Enediyne Anticancer Antibiotics

Joint initiative of IITs and IISc – Funded by MHRD of 115

NPTEL Phase – II (Syllabus Template)

Course Title: Bio-Organic Chemistry of Natural Enediyne

Anticancer Antibiotics

Module 3: Designed Enediyne Model Systems: Introduction to Structural Features of Enedynes; Factors

Affecting the Reactivity of Enediynes; Molecular Design of Enediyne Models; Various Synthetic

Approaches to Acyclic/Cyclic Enediynes; Various Synthetic Aprroaches to Cyclic Enediynes;

Synthesis of Dienediyne Core of NCS chromophore; β-Lactam as a Molecular Lock of

Enediyne: Synthesis of β-Lactam Fused Enediynes; Enediynes with pH-Based Triggering

Devices; Photoswitchable Enediynes; Biological Actions of Some Synthetic Models; Enediyne

as a Scaffold for Peptidomimetics; Enediyne as Peptide Cleaving Agent.

3.1. Introduction to Structural Features of Enedynes

The common structural motif among enediyne antibiotics is an enediyne moiety (Z-hexa-1,5-

diyn-3-ene), the conjugated system, found embedded within a 9- or 10-membered cyclic

framework. The enediyne antibiotics have been divided into two subfamilies, including 9-

membered cyclic enediynes such as NCS, kedarcidin, LDM, maduropeptin and N1999A2 and

10-membered cyclic enediynes such as CAL, ESP, DYN, and shishijimicins A-C (Figure 1).

9-membered cyclic enediynes contain the chromophore containing the enediyne core and an

apoprotein unit with noncovalent binding. The enediyne core of the chromophore is located in

the center of the pocket and other substituents are arranged around the core. The enediyne core

of the chromophore is the anticancer part, but the free chromophore is labile. The apoprotein is

inactive in cleavage of DNA; however it plays an important role in drug action by stabilizing the

labile chromophore. The apoprotein is believed to be resistant to proteases, protect the

chromophore from deactivation and to deliver the enediyne to intracellular target DNA. Only

N1999A2 is a non-protein 9-membered cyclic enediyne antibiotic and is stable in nature.

The structures of 10-membered cyclic enediynes do not contain an apoprotein and are more

stable than those of 9-membered cyclic enediynes.

Figure 1. Presentation of three types of enediynes.



In calicheamicins and dynemicins, a 3-ene-1, 5-diyne system is embedded in a 10-membered

ring. These compounds belong to Type I enediynes.

In Type II enediynes, the 3-ene-1, 5-diyne system is included in a 9-membered ring as in

kedarcidin.

There is another class of enediyne (Type III), in which a 9-membered cyclic dienediyne is

present as in neocarzinostatin.

The enediynes represent an ingenuity of nature’s work. It has been compared to a smart

bomb equipped with: a) a delivery system which is responsible for a strong and specific

complexation with DNA. This system is represented by the oligosaccharide unit as in

calicheamicin and esperamicin; b) a warhead (the enediyne moiety) that is able to attack

simultaneously the two complementary DNA strands, causing the lethal double strand cut; c) a

safety catch or a locking device that prevents the enediyne from undergoing the diradical

formation, by imposing a structural restraint to its reaction. In this way the warhead does not

explode until a particular chemical event takes place (Table 1). In calichamicin this is

represented by the enamine double bond; d) finally, a chemical trigger that mediates the removal

of the safety catch and therefore unleashes the high reactivity of the enediyne. In calicheamicins

the trigger is the trisulfide group (Figure 2). Table 1. General structural features of enediynes

Structural

Units

Structural Features/Functions Pictorial presentation

Warhead: The Enediyne.

Locking

Device

Stabilizes the enediyne from

undergoing rearrangement

Triggering

Device

It offers a mechanism by which

locking is removed and enediynes

become reactive

Binding

Device

Gives Specificity



Figure 2. Structural features of Calichiamycin.

3.2. Factors Affecting the Reactivity of Enediynes

The process of generating benzenoid diradical in enedynes via the well known Bergman

cyclization (BC), and/ or Myers−Saito cyclization (MSC) reaction is primarily responsible for

the antitumor activity of naturally occurring enediynes, such as calicheamicins. Since the

discovery, these two reactions are believed to be at the heart of antitumor activity naturally

occurring enediynes. Therefore the factors that affect the kinetics of these reactions are mostly

responsible for biological activity of enediynes. It is the inherent property of 9- or 10-membered

monocyclic natural enediynyl systems to cyclize spontaneously at physiological temperature,

while the acyclic enediynes undergo thermal cycloaromatization at elevated temperatures only

(≥200 °C).

To ensure the safe delivery of the enediyne molecule to the target before the enediyne

functionality is activated toward diradical generation DNA cleavage by abstraction of H-atom

from the sugar−phosphate backbone of DNA via a triggering mechanism Nature has

incorporated locking devices into these systems. The triggering or activation process for the

natural enediynes primarily involves a change of hybridization at a carbon center encompassing

the enediyne moiety or an opening of the epoxide ring fused onto the enediyne in bi- or tricyclic

fashion. This structural change is believed to lower the activation barrier for the

cycloaromatization process either by bringing the terminal acetylenic carbon atoms (the c and d

distance) closer or by minimizing the overall conformational restrictions. Both the factors

(distance and strain) often act synergistically to drive the process of diradical generation (BC) at

a physiologically relevant temperature.



Mainly the following factors play the important role in affecting/predicting the kinetics of

BC.

1. Ring Size or c, d Distance (Distance theory)

2. State of Hybridization (Strain Theory)

3. Incorporation of Hetero Atom or Strained Ring System

4. Metal ion Complexation

5. Weak non-covalent Interactions

The details of all the factors are described in module 1. Here the summary of the most

important two factors are given below in Table 2.

Table 2. Factors affecting kinetics of BC.

Distance theory 1. Rate is proportional to the c,d-distance

3.20 Å -3.31 Å = Critical Distance.

2. It mainly relies on the ground-state

configuration of the enediynes.

3. For Acyclic or Simple monocyclic systems,

distance theory may be applied.

4. For bicyclic or strained systems it needs

caution to apply the theory.

Strain Theory 1. Rate is proportional to the strain energy

difference between the TS and GS.

2. Strain theory is more general but difficult to

estimate.

3. Other steric and stereoelectronic and factors

also affects the rate.

4. The strain theory thus supports the

consideration of the stereo-electronic factor

for the design, synthesis, and study of the

chemical and biological reactivity of

synthetic mimics.



DFT-based calculations on monocyclic enediynes showed that there is no predictive

relationship between the alkyne carbon distance and the cyclization activation enthalpy.

However, for similar monocyclic systems, a crude relationship does exist between the c

and d distance and the kinetics of BC (Nicolaou's empirical critical distance of 3.31−3.20

Å for spontaneous cyclization at room temperature was extended to 3.40−2.90 Å).

These calculations were in agreement with the argument that ring-strain effects may

become more important in strained systems.

It may be mentioned that BC leading to the formation of a diradical is an endothermic

process. Dimethyl substitution at both of the alkyne termini increases the endothermicity

of the reaction by 12 kcal mol-1. The greater the endothermicity, the slower is the kinetics

of BC.

In BC the orthogonal relationship is obeyed between the diradical and the aromatic π

system. This ruled out the influence of a mesomeric effect upon the stability of the radical

and hence the cyclization rate.

Ortho effect on BC: Substituents in ortho position in benzannulatedenediynes exert a

significant influence on BC kinetics (ortho effect) (Scheme 1). This interaction can be either

destabilizing through steric or stabilizing through hydrogen-bonding/hyperconjugation/electron

transfer. As for example acceleration of the BC rate is observed for X = OMe, NH3+, NO2, and

CF3. DFT-based calculations have shown that the ortho effect operates through the σ framework

of the resulting neutral diradical intermediate.

Scheme 1. Stabilizing/destabilizing effects of ortho-substituents on the kinetics of BC.



3.3. Molecular Design of Enediyne Models The cycloaromatization reaction of enediynes has created the door of never-ending

opportunities for synthetic organic chemists for the development of strategies to synthesize

model enediyne architectures. That is why field of enediyne chemistry is rapidly growing toward

the design of novel anticancer antibiotic model systems with suitable triggering devices.

Research in this field is mainly aim to enhanc the reactivity of designed enediyne toward BC as

well as to lower the toxicity under suitable triggering conditions. As a result of tremendous

research efforts, during the past 20 years, various enediyne models have been designed and

reported that showed potent DNA cleaving activity. The basis of all of these designs can be

classified into the following:

1. Consideration of Two Theory: Both the “distance theory” as well as “molecular

strain theory” must be considered to design new enediynes as possible therapeutic

agents.

2. Stability of Designed Enediyne: The designed enediyne should be sufficiently stable

at biological temperature at which the enediyne must possess sufficient half-life

(“decent half-life”) that enables full characterization of the molecule. The intrinsically

reactive enediynes synthesized must have half-lives ranging from 10 to 36 h at the

biological temperature of 37 °C.

3. Designing Acyclic Enediyne: Generally the acyclic enediynes are stable and do not

undergo facile BC at biological temperature required to become an efficiently

bioactive enediynes. Therefore the designed acyclic enediynes of sufficient stability

should be converted to a cyclic framework which would undergo cycloaromatization

(BC) at a lower temperature as compared to their progenitor. There are various

strategies available exploiting which the stable acyclic enediynes can be converted to

a comparatively more reactive cyclic enediynes. These are as follows:

(a) Formation of a cyclic framework.

(b) Conversion into a metal coordinated cyclic network (metallocycle)

(c) Generation of an H-bonded cyclic architecture.

(d) Creation of a pseudocyclic network via (i) electrostatic interaction, (ii) charge

transfer interaction, (iii) - stacked cycle.

(e) Attaching electron withdrawing groups might also lead to enhance the rate of

BC.

(f) Alternatively, isomerization of acyclic enediyne to a more reactive

eneyne−allene system might allow the comparatively unreactive acyclic

enediyne to undergo spontaneous MSC.



4. Designing Cyclic Enediyne: In the case of design of cyclic enediyne the attractive

strategy that is generally adopted is the generation of reactive cyclic enediynes from

precursors, which is made ambiently stable by incorporation of small, strained ring

systems. The activation/triggering process then operates for the removal of strain via

the opening of small rings under various conditions leading to the generation of

reactive cyclic enediyne that undergo facile cycloaromatisation to show potent DNA

cleaving activity. There are several activation strategies which includes:

(a) Oxidative activation of stable cyclic enediynes.

(b) Organometal-mediated activation.

(c) pH based triggering.

(d) Changing the state of hybridization as is shown in Calicheamicin.

(e) Photo chemical activation

5. Designing Macrocyclic Enediyne: For design of macrocyclicenediynes one should

have option to convert macrocycle into systems of appropriate smaller size (e.g., 10-

membered ring via transanular reaction) that could react under ambient conditions.

6. DNA Binding Appendage: To design any enediyne capable of showing DNA

cleavage activity, the enediyne must possess DNA binding appendage.

All of these approaches are summarized in Scheme 2 and 5.



3.3.1. Designing Strategy for Acyclic Enediynes

Scheme 3.2: Design strategy of acyclic enediynes.

Scheme 2. Design strategy for acyclic enediynes.



3.3.1.1. Structural Representation of Various Strategically Designed Acyclic

Enediynes

3.3.1.1. 1. (a) Organometal/(b) Coenzyme/(c) Base mediated rearrangement

Scheme 3. Structural representations of (a) Organometal/(b) Coenzyme/(c) Base mediated rearrangement in acyclic enediynes.

3.3.1.1. 2. (d) Acid mediated rearrangement/(e) photochemical/(f) metal ligation

Scheme 4. Structural representations of various designs (d-acid mediated rearrangement; e- photochemical reaction; f-bidentate ligation

to metal ion) of acyclic enediynes.



3.3.1.1. 3. (g, h) photochemical activation

Scheme 5. Structural representations of various designs (g, h-photochemical activation) of acyclic enediynes.

3.3.1.2. Representative Examples of Various Strategically Designed Acyclic

Enediynes

3.3.1.2.1. Examples of Metal-Ion-Induced Bergman Cyclization (Pathway “a”)

Scheme 6. Metal-ion-induced BC of biscrown ether.



Scheme 7. Metal-ion-induced BC of bisphosphinoenediyne.

Scheme 8. Hg-ion induced of BC bipyridylenediyne.



Scheme 9. Metal-ion-induced BC of bis-sulfonamido and bis-aminoenediynes.

Scheme 10. Metal-ion-induced BC of tetraamino enediynes.



Scheme 11. Metal-ion-induced BC of bissalicylaldimino enediynes.

Scheme 12. Pd-mediated BC of Tetrahedral Pd-phosphino-enediyne complex.



Scheme 13. BC of Cu(I) and Cu(II) complexes of enediynes.

3.3.1.2.2. Examples of Pathway “b” Coenzyme mediated BC

Scheme 14. Pyridoxal co-enzyme-mediated BC of acyclic enediyne.

3.3.1.2.3. Examples of Pathway “c”-Base mediated BC

Scheme 15. Representative examples of various designs (base mediated BC).



3.3.1.2.4. Examples of Pathway “d”-Acid Mediated BC

Scheme 16. Representative examples of various designs (acid mediated BC).

3.3.1.2.5. Examples of Pathway “e”-Photochemical Mediated BC

Scheme 17. Representative examples of various designs (Photochemical BC).



3.3.1.2.6. Examples of Pathway “f”-Enediyne Cyclization Mediated by Organometallic Reagents

Scheme 18. MSC mediated by formation of organometallic complex.

Scheme 19. BC of acyclic aliphatic enediynes mediated by Rh-metal complexation and the mechanism.



Scheme 20. Organometallic reagent mediated BC.

3.3.1.2.7. Examples of Pathway “g”-Phochemical Activation Mediated BC

Scheme 21. Phochemical activation mediated BC.

3.3.1.2.8. Examples of Pathway “h”-Photochemical mediated BC

Scheme 22. Phochemical activation mediated BC.



3.3.2. Designing Strategy for Cyclic Enediynes

Scheme 23. Design strategy of cyclic enediynes.



3.3.2.1. Structural Representation of Various Strategically Designed Cyclic

Enediynes: 3.3.2.1. 1. (i-n) Small ring fused cyclic/stable cyclic enediyne/cyclic-1,4-diyne

Scheme 24. Structural representations of various designs (i-photochermical, j-base mediated, k-hydride mediated) of cyclic enediynes.

Scheme 25. Structural representations of various designs (l- Small ring cleavage by nucleophile, m- Oxidative activation, n-Reductive

formation of enediyne) of cyclic enediynes.

3.3.2.1. 2. (o-q) Hybridisation/allylic rearrangement/organometallic activation



Scheme 26. Structural representations of various designs (o-Change of hybridization, p- Allylic rearrangement, q-Organometallic

reagent induced activation) of cyclic enediynes.

3.3.2.1. 2. (r) Enediyne Macrocyle to reactive cyclic enediyne via trans annular reaction

Scheme 27. Design strategy of macrocyclic enediyne.



3.3.2.3.3. Representative Examples of Various Strategically Designed Cyclic Enediynes:

3.3.2.3.3.1. Examples of Pathway “i”

Scheme 28. Representative examples of various designs (i-Photochemical removal of protecting group) of cyclic enediynes.



3.3.2.3.3.2. Examples of Pathway “j” and k

Scheme 29. Representative examples of various designs (j-Base induced removal of protecting group, k -Hydride mediated removal of

protecting group) of cyclic enediynes.

3.3.2.3.3.3. Examples of Pathway “l”

Scheme 30. Representative examples of various designs (l-Small ring cleavage by nucleophile,) of cyclic enediynes.



3.3.2.3.3.3. Examples of Pathway “m”

Scheme 31. Representative examples of various designs (m-Oxidative activation) of cyclic enediynes.

3.3.2.3.3.3. Examples of Pathway “n”

Scheme 32. Representative examples of various designs (n-Reductive formation of enediyne,) of cyclic enediynes.



3.3.2.3.3.3. Examples of Pathway “o”

Scheme 33. Representative examples of various designs (o-Change of hybridization) of cyclic enediynes.

3.3.2.3.3.3. Examples of Pathway “p”

Scheme 34. Representative examples of various designs (p-Allylic rearrangement) of cyclic enediynes.



3.3.2.3.3.3. Examples of Pathway “q”

Scheme 35. BC Mediated by Pentamethyl cyclopentadienyl ruthenium cation (A).

3.3.2.3.3.3. Examples of Pathway “r”

Scheme 36. Representative example of (r)-transannular reaction of cyclic enediynes.



3.4. Various Synthetic Approaches to Acyclic/Cyclic Enediynes

3.4.1. Basic Strategies for Enediyne Synthesis

Scheme 37. Basic retrosynthetic strategies for enediyne synthesis.

3.4.2. Methodologies Involved for the Synthesis of Enediyne

Scheme 38. Various methodologies for the synthesis of enediyne.

R1

R2Y1

Y2

ZRLarge

X1 X2R X3

OR

+

(Z)

Basic Strategies for Enediyne Synthesis

Z

Bu3Sn Cu(CN)Li

Bu3Sn

R1 R3

I

R1 R3R1 R3

R1 R2

Bu3Sn

R2 R1

R2 R1

R2R2

3 Steps I2, THF

0 oC- r.t

R1

ZnCl2, Et2O, -45 oC

R4 ZnCl

Pd(PPh3)4, THF

80%

R2 =45-75%

R1 = (CH3)2CO2Me

R1 X , -50 oC2.

1

X= Br, I

Magriotis, P. A.; Scott, M. E.; Kim, K. D. Tetrahedron Lett. 1991, 32, 6085.

R2 X

I2, CH2Cl2, 0 oC

Pd(PPh3)4, CuI,

BuNH2,Benzene, r.t

1.

2.

SPhMe OH

OTBS

Stracker, E. C.; Zweifel, G. Tetrahedron Lett. 1991, 32, 3329.

R1, R3 = H, R2 = Sliyl, R4 = H, Alkyl, Aryl, Silyl

R1 X

R2X

HR

R1

R2

R

R

Pd-Cat, Cu-Cat

Base

+

Various Methodologies for the Synthesis of Enediynes: Pd-Mediated Coupling Reactions




ZZ

C

H

TBS TMS

R

R

B

R TMSR TMS

H

HO TBS

H

PPh3

I+Ph2TfO-

OPMB

RR

ODMST

OPMB

RR

ODMST

R=H, Me, Ph

R= n-Bu, Ph

cis : trans > 99 :1 to 96 : 4

2. HO(CH2)2NH2

1. RCHO

KH,Et2O, r.t

Shibuya, M.; Sakai, Y.; Naoe, Y. Tetrahedron Lett. 1995, 36, 897

94~81%

MsCl, Et3N, DCM, 0 oC

HO

Wang, K. K.; Wang, Z.; Gu, Y. G. Tetrahedron Lett. 1993, 34, 8391

R H n-BuLi

CuCN,DCM, -78 ~25 oC

40-69% Z= CH2, O; R=TMS, t-Bu, Bu, Ph

Stang, P. J.; Blume, T.; Zhdankin, V. V. Synthesis 1993, 35.

Hypervalent iodine

Olefination:

Elimination Reaction

Various Methodologies for the Synthesis of Enediynes




Cl

Cl

R

R

R

R

O

O

O

OH

O

KH, THF, 25 oC

90%

Malaic Anhydride,

Toluene,

36%

R = nPr

NiCl2(dppp), THF,

-78 to 20 oC

Hopf, H.; Theurig, M. Angew. Chem., Int. Ed. Engl. 1994, 33, 1099.

Bunnage, M. E.; Nicolaou, K. C. Angew. Chem., Int. Ed. Engl. 1996, 35, 4986.

Ph

OO

O

O

O

O

O

Nuss, J. M.; Murphy, M. M. Tetrahedron Lett. 1994, 35, 37.

h, Benzene, Pyrex

98%

28 29

Diels Alder reaction:

Retro-Diels Alder reaction:

Norrish type II fragmentation:


+



Scheme 41. Various methodologies for the synthesis of enediyne

Ramberg Backlund reaction:

Carbenoid coupling elimination:

a) Jones, G. B.; Huber, R. S.; Mathews, J. E. J. Chem. Soc., Chem. Commun. 1995, 1791.b) Huber, R. S.; Jones, G. B. Tetrahedron Lett. 1994, 35, 2655.

Nicolaou, K. C.; Zuccarello, G.; Riemer, C.; Estevez, V. A.; Dai, W. M. J. Am. Chem. Soc. 1992, 114, 7360.

Br

Br

Ph2t-Bu- SiO

Ph2t-Bu- SiO

OH

OH

SO2

Cl Ph2t-Bu- SiO

Ph2t-Bu- SiO

Co2(CO)8

Co2(CO)8

LiHMDS, Co2(CO)8,

HMPA/THF,- 450C

1)Na2S.Al2O3, DCM, EtOH2) mCPBA, Et2O, -30°C

3) SO2Cl2, pyr, DCM, -78°C4) mCPBA, Et2O, 25°C%

1) MeLi, THF, -78 °C

2) TBAF, THF, 0 oC

92%

HO

HO

OH

OH

OH

OH

SO

O

n n

NP

NR

Me

Me

Ar

X

Y

TBSO

Ar

X

Y

TBSO

R =Me

(-20 to -5 oC)

R = Ph

(20 to 25 oC)

7 steps

Semmelhack, M. F.; Gallagher, J. Tetrahedron Lett. 1993, 34, 4121

n = 2 (8%)n = 3 (48%)

Maier, M. E.; Greiner, B. Liebigs Ann. Chem. 1992, 855.

DDQ, Benzene

Ar = p-MeOC6H4

n = 2, 3

Corey-Winter reaction:

Oxidation reaction:




3.5. Various Synthetic Aprroaches to Cyclic Enediynes

3.5.1. Synthesis of 9-Membered Enediynes

For the synthesis of the enediyne core analogue of 9-membered enediynes, various carbon

carbon bond forming reactions are needed to be carried out. Because of the instinct instability of

9-membered ring enediyne, it is always necessary to prepare them in a suitably protected form

like (a) the incorporation of epoxide as in found in kedarcidin chromophore or (b) the protection

of one of the triple bond of the enediyne via complexation with metal or (c) incorporation of a

strained bicyclic core.

The general synthetic approaches to the bicyclic system represented by the following figure

can be summarized as follows:

i) Sonogashira coupling to form the bonds a, b and d.

ii) Bond “e” can be formed by intramolecular aldol type condensation followed by -

elimination.

iii) For the generation of NCS core, the double bond “c” is usually converted to epoxide

before carrying out the intramolecular aldol reaction for ring closing.

iv) The ring closing is preceded by complexation with cobalt.

d

b

c

e

a

General structure of 9-membered enediynes with a bicyclic core



3.5.2. Magnus’ Approach

Scheme 42. Magnus’ approach to the synthesis of 9-membered ring enediyne.

OH

OO

O

OO

OR

EtOOEt

TBSO

OO

R = H

R = TBS

H Li

THF

-78 oC to

rt

Pd(PPh3)4,

CuI, n-BuNH2

EtO

EtOCHO

EtO

EtO

I

EtO

EtOI

CO2Et

EtO2C

IPPh3

i) DIBAL

ii) TBSOTf, Et3N

OR

EtOOEt

TBSO

OO

Co(CO)3

Co(CO)3

OR

H

O

HO

Co(CO)3

Co(CO)3

O

OR

OH

OBBu2

Co(CO)3Co(CO)3

O H

OR

OH

OBBu2

O H

Co2(CO)8H3O+

NMMOOBBu2

OHH

OR

O

OBBu2

OHH

OR

O

H

H

Bu2BOTf

Et3N

1.2.14

Synthesis of A

OTBS

.

.

1,4-CHD

A

A

Magnus' Approach to the Synthesis of 9-Membered Ring Enediyne



3.5.3. Grierson’s Approach

Scheme 43. Grierson’s approach to the synthesis of 9-membered ring enediyne.

(OC)3CoCo(CO)3

OTBDPS

(OC)3Co Co(CO)3

OTBDPS

CO2Me

OHH

O

H

CO2Me

OTBDPS

CO2Me

OHH

+

OTBDPS

CO2Me

OTHPH

Br

OTHP

HBr

(OC)3Co Co(CO)3

OTBDPS

OHH

OH

Br

OH

HBr

S

OHH

OTHPH

Br

OTHPH

OTHPH

f

Reagents and conditions: a) (Meo)MeAlCl, CH2Cl2, 40 oC, (91%); b) CAN, MeOH, 0 oC,

(84%); c) DHP, (TMSO)2SO2, CH2Cl2, 0 oC, (87%); d) i. DIBAL-H/THF, 0 oC; ii. TBAF/THF,

0 oC; iii. CBr4, PPh3, 2,6-lutidine, MeCN, 0 oC, (3 steps, 83%); e) DIBAL-H/THF, 0 oC, (81%);

f) Na2S / Al2O3, EtOH-CH2Cl2 (2:5), 0 oC, (90%); g) LIHMDS, HMPA, THF, -40 oC, (10%).

a

b

c

d

e

g

Grierson's Approach to the Synthesis of 9-Membered Ring Enediyne



3.5.4. Caddik’s Approach

Michael addition of enediyne acetylide cyclopentenone .cobalt complexation acetal

hydrolysis and aldol condensation the 9-membered framework (Scheme 44).

Scheme 44. Caddik’s approach to the synthesis of 9-membered ring enediyne.

O

TBSOPO

MeO

MeO

OTBDPSHH

O

TBSOOH

O

TBSO

PhCO2

R3

OTBDPS

HH

OTBDPS

HH

OHHO

TBSO

PhCO2

H

H

b

c

HH

H CO2PhH

H

O

TBSO

OHOTBDPS

Co2(CO)6

Co2(CO)6Co2(CO)6

HOTBDPS

OMe

OMe

Br

OTBDPS

H

BrHO

TMS

Br

Br

EtO

O

a

d

Reagents and conditions: a) i. A, n- BuLi, THF, 0 oC, - 78 oC, 30 min., ii. Et2AlCl, rt, 1

h, iii. 1.2.27, (51%); b) PhCOCL, Pyr, DMAP, CH2Cl2, (89%); c) Co2(CO)8, CH2Cl2, 0 oC,

(84%); d) TFA, CH2Cl2, (96%); e) n-Bu2OTf, Et3N, CH2Cl2, (68-75%).

+

e

P = H

P = COPh

R3 = CH(OMe)2

R3 = CHO

1.2.32

Caddik's Approach to the Synthesis of 9-Membered Ring Enediyne

A



3.5.5. Hirama’s Approach

Scheme 45. Hirama’s approach to the synthesis of 9-membered ring enediyne.

OMOMMOMO

OI

OO

I

TBSO

O

NCl

OO

NHBoc

OTES

OMOM

TES

OO

TBSO

O

NCl

OO

NHBoc

OTESOMOM

TESO OTBSO

O

NCl O

O

NH

OTES

OMOM

TES

OMeMeO

i-PrO

OAllyl

O

O

OMOMMOMO

IHO

MOMO

MOMO

MOMO

MOMO

HO I

HO

OO

O I

OO

I

TBSO

O

NCl

OMeO

NHBoc

OO

I

TBSO

TBSO

NCl

OHO

NHBoc

HO OMeMeO

i-PrO OAllyl

O

OH

HO

OH

OMOM

TES

a, b c de-g

h-k l, m n-p

q r-t

Reagents and conditions: a) TPAP/NMO; b) I2/Py. (89%, in 2 steps); c) i-PrMgCl/CH2Cl2, -

78 to 0 oC, THF, 6 h (69-71%); d) HClO4, THF(aq) (61%); e) (Cl3C)2C=O/Py; f) PPTS/2-

Butanone; g) TBSCl, imidazole. (77% in 3 steps); h) DIBAL-H, -80 oC; i)

(MeO)MeCH=CH2; j) TBAF, 0 oC; k) DIAD/PPh3 (72%, in 4 steps); l)A, CsF, DMF, 60 oC; m)

TBSCl. (90%, in 2 steps); n) 0.3 M KOH (aq); o) B, EDC.HCl, DMAP; p) TESCl (89%, in 3

steps); q) CuI, Pd2(dba)3.CHCl3, i-Pr2NEt, DMF, rt, 1 h (88 - 90%); r) TBSOTf; s) SiO2; t) C,

HOAT, EDC.HCl (87%, in 3 steps).

1.2.41

4/4

2

8

1

A B C

Hirama 's Approach to the Synthesis of 9-Membered Ring Enediyne: Synthesis of seco analogues of 9-membered systems present in kedarcidin



3.5.6. Basak’s Approach

Scheme 46. Basak’s approach to the synthesis of 9-membered ring enediyne.

,

OMs

NH

Ts

OMs

NH

Ts

(OC)3CoCo(CO)3

NH

OMs(OC)3CoCo(CO)3

Ts

N Ts

(OC)3Co Co(CO)3

OH

NH

Ts

a

Reagents and conditions: a) MsCl, NEt3, DCM, 0 oC; b) Co2(CO)8 (2.2 eq),

DCM, 0 oC, 30 min; c) K2CO3, DMF, rt, 40 min; d) I2, THF, 0 oC, 1 h; e) Co2(CO)8

(1.1 eq), DCM, 0 oC, 5 min

+

b

c, d

c

N Ts

I

I

N Ts

H

H

+ N Ts

H

I

+

N Ts N Tsd

OMs(OC)3Co

Co(CO)3

HN

Ts

(OC)3CoCo(CO)3

CH3

NH

Ts

c, d

e

+

(89%)

(63%)

(55%)

(40%)

(50%)

(8%) (64%) (13%)

.

.

A

A

Basak 's Approach to the Synthesis of 9-Membered Ring Enediyne



3.6. Synthesis of Dienediyne Core of NCS chromophore

The synthesis of parent dienediyne unit of NCS chromophore has been approached from

three different angles looking at the simplicicity and feasibility of the reactions. The synthesis of

actual NCS chromophore core unit has been reported by Myers and subsequently by Kobayashi.

The targets are divided into the following categories:

a) Category A: In this category the bicyclic dienediyne lacked the epoxy unit that is

replaced by two saturated carbons.

b) Category B: In category B one of the epoxy carbons in NCS chromophore has a hydroxy

functionality which can be converted into an epoxy unit.

c) Category C: In this category C, the dienediyne unit has the epoxy unit like the natural

product.

Below are the presentations of various synthetic approaches to these molecules.

3.6.1. Synthesis of Category A molecules: Wender’s Approach

The strategy relied on the following steps

a) addition of an acetylide to a halo cyclopentenone,

b) a Sonogashira coupling to generate the diyne functionality and

c) ring formation to generate the bicyclic system.

d) The final cyclic network formation was carried out by a photochemical extrusion of SO2

from a cyclic sulphone.

e) After then β-elimination generated the target framework.



The sequence of reactions is represented in Scheme 47.

Scheme 47. Wender’s approach to the synthesis of dienediyne core (category A).

1. Sonogashira coupling

2. Homologation of terminal alkyne

Y

YOH

X

OH

X

O

R

M

Target A

Y +

Elimination

Organometallic

addition to a carbonyl

+

O

Br

HO

Br

OHHO

OH

OH

HO

S

O

O HO

Reagents and conditions: a) HCCCH2MgBr, Et2O, rt; b) EtMgBr, HMPA, Et2O,

50 oC; CH2O, Et2O, rt; c) PdCl2(PPh3)2, CuI, (i-Pr)2NH, HCCCH2OTBDPS, THF,

rt; d) n-Bu4NF, THF, -50 oC to rt; e) MsCl, Et3N, CH2Cl2, -20 oC; f) Na2S, aq.

EtOH, rt; g) m-CPBA, NaHCO3, CH2Cl2, 0 oC to rt; h) PhCOPh, MeCN/PhH

(1:1), hn, rt; i) MsCl, DMAP, CH2Cl2.

a, b c, d

e-g h i

Wender's Retrosynthetic Approach to Dienediyne core (Category A)

Wender's Synthetic Approach to Dienediyne core (Category A)



3.6.2. Synthetic Approaches to category B molecules

3.6.2.1. Wender’s Approach

In Wender’s appraoch to category A molecules the saturated carbons may not serve any

useful purpose for the enediyne to show MSC and hence the biological activity. Thus this was

modified wherein the earlier saturated carbon bears hydroxy group that might allow potential

creation of the epoxide as in NCS. The molecules belonging to this category are designated by

structure target A in the following scheme.

Scheme 48. Wender’s approach to the synthesis of dienediyne core (category B).

This approach follows the similar steps to the ones described for the synthesis of category A

molecules. The final ring closure was carried out using a Nozaki-Hayashi reaction mediated by

CrCl2 which worked nicely to afford the target molecule in high yield (Scheme 48).

OHHOHO OH

OH

HOOH

IOH

O

X M++

HOOTBS

OH

HOOTBS

Br

HO CHO

Br

OHHO

Reagents and conditions: a) Et3N, MsCl, -78 oC; LiBr, (CH3)2CO,

rt; b) 3:l:l HOAc/THF/H2O, rt; c) MnO2, rt; d) CrCl2, THF.

a

b, c d

Target A

Wender's Retrosynthetic Approach to Dienediyne core (Category B)

Wender's Synthetic Approach to Dienediyne core (Category B)



3.6.2.2. Myers’ Approach

Myers adopted a different strategy for the synthesis of the bis-oxygenated dienediyne system

(Scheme 49). The key step was the transannular reductive cyclization of the potassium salt of the

tetrayne alcohol with Red-Al that afforded the bicyclic product in good yield (50-54%). It is

important to mention that the proximal transannular cyclization was favoured over the distal

mode.

Scheme 49. Myers’ approach to the synthesis of dienediyne core (category B).

(Proximal)

Red-Al

OTIPS

HO

TIPSO

OTIPS

TIPSO

TIPSO

HO

TIPSO OSPIT

OSPIT

OTIPS

HO

TIPSO

OTIPS

(Distal)

1. Cu(OAC)2,

DBU, O2

2. Et3N.HF

(Not obtained)Target

Myers' Synthetic Approach to Dienediyne core (Category B)



3.6.2.3. Magnus’ Approach

In their synthesis of NCS chromophore analogue, Magnus et al. introduced an epoxide

moiety on the ene part of acyclic enediyne. This was followed by cobalt complexation and final

intramolecular aldol condensation (Scheme 50).

Scheme 50. Magnus’ approach to the synthesis of dienediyne core (category B).

O O

TBSO

EtO OEt

OH

O O

TBSO

EtO OEt

OCOBut

O

O

TBSO

EtO OEt

OCOBut

O

O

TBSO

OCOBut

O

Co(CO)3

CoCO3

OHH O

TBSO

H

OCOBut

O

Co(CO)3

Co(CO)3

O

O

TBSO

EtO OEt

OCOBut

O

Co(CO)3

Co(CO)3

O

TBSO

OCOBut

O

Co(CO)3

Co(CO)3OH

H O

TBSO

OCOBut

O

OHH O

TBSO

OCOBut

O

OHH

Reagents and conditions: a) i. (-)DET/3Å MS/Ti(OPri)4/t-BuOOH/CH2Cl2; ii. t-

BuCOCl/Et3N/DMAP/CH2Cl2 (67% overall, 1:1 diastereoisomers); b)

CF3CO2H/THF/H2O, 5 oC (99%); c) Co2CO8/n-heptane, 25 oC (80%); d)

CF3CO2H/CHCl3 (56%); e) n-Bu2BOTf/Et3N/CH2Cl2, -78 o to 0 oC (57%, ca. 1:1

diastereomars); f) I2/PhH, 25 oC (69% and 75%).

+

+

a b

c

de

f

Magnus' Synthetic Approach to Dienediyne core (Category B)



3.6.3. Synthetic Approaches to category C molecules

The synthesis of the actual core of NCS chromophore containing the epoxy functionality was

done following two different strategies which are given below.

3.6.3.1. Myers’ Approach

Myers et al. reported the synthesis of the target aglycon. The target was achieved from an

epoxy alcohol (Scheme 51).

Scheme 51. Myers’ approach to the synthesis of dienediyne core (category C).

O

H

TBSO

HR1

CHO

TMSO

O

H

TBSO

HR1

TMSO

OH

O

H

TBSO

HR1

TMSO

CHO

O

O

TBSO

HR1

TMSO

OOH

O

HO

HR1

HO

OO Cl

O

R2

HO

OOH

O

H

R1

d

O

R2

H

HO

OOR

R1

R = TES

R = H

O

R2

H

HO

HO

HO

OOH

R = H

R = TESj

O

O

O

R2

H

R1

O OR

O

R2

H

R1

OH

OO

Me

MeO OH

ab, c

e, f

Reagents and conditions: a) DIBAL, CH2Cl2, -78 oC (94%); b) (+) DET, Ti(Oi-Pr)4, TBHP,

3A MS, CH2Cl2, -30 oC, (98%); c) Dess-Martin, pyr, CH2Cl2, (97%); d) LiN(SiPhMe2)3,

LiCl, THF, -78 oC, (85%); e) (ClCH2CO)2O, pyr, CH2Cl2, 0o C, (89%); f) Et3N.3HF, THF, 23oC, (100%); g) 1.3.48, DCC, THF & n-PrNH2, -10 oC, (80%, two steps) h) p-TsOH, MeOH,

23 oC, (81%); i) CDI, THF, 0 to 23 oC; then (+)-CSA, CH3CN/H2O, (89%); j) TESOTf, 2,6-

lutidine, CH2Cl2, -78oC, (100%); k) Martine Sulfurane, CH2Cl2, 23 oC; l) Et3N.3 HF, THF,

0 oC, (78%), (two steps); m) PPh3, I2, imidazole, CH2Cl2, -10 oC, (71%).

R1 =

i

k m

g h

l

R2 =

Myers' Synthetic Approach to Dienediyne core (Category C)



3.6.3.2. Kobayashi’s Approach

In Kobayashi’s approach the steps involved:

(a) stereoselective intramolecular acetylide-aldehyde cyclization to form the C5-C6

bond.

(b) installation of the epoxide.

(c) Then the formation of naphthoate and carbonate.

(d) ultimately the C8-C9 olefin was introduced by using the Martin sulfurane

dehydration reaction to furnish the target aglycon.

Scheme 52. Kobayashi’s approach to the synthesis of dienediyne core (category C).

O

OOH

PivO

O

O

I

MPMO

TBDPSO

O

+

MPMO

TBDPSO

O

R1

OTES

PivO

MPMO

TESO R1

OTES

OPivTESO

MPMO

TESO R1

OTES

OTESO

MPMO

TESO R1

OTES

OHTESO

MPMO

HO R1

OH

OMsTESO

MPMO

HO R1

O

TESO

MPMO

O

O

OSET

OHHOO

OH

OMeMPMO

O

O

OH

OOH

OMe

O O

O

TESO

O

O

OH

OOTES

OMe

O O

O

HO

O

O

OOH

OMe

O O

O

R1 =

a, b

c-e

Reagents and conditions: a) (Ph3P)4Pd, CuI, iPr2NEt, DMF, (97%); b) TESOTf, 2,6-

lutidine, CH2Cl2, -70 oC, (100%); c) HCCCH2MgBr, toluene, -78 oC; d) TBAF, THF,

(74%); e) TESOTf, 2,6-lutidine, CH2Cl2, -50 oC, (90%); f) DIBAL, Et2O, -78 oC, (89%);

g) Dess-Martin periodinane, pyridine, CH2Cl2, (90%); h) LiN(TMS)2, CeCl3, THF, 35

to 0 oC, (73%); i) MsCl, Et3N, CH2Cl2, -20 oC; j) TBAF, THF, -35 oC; k) K2CO3, EtOH,

(52%); l) 1.3.48, EDC.HCl, DMAP, CH2Cl2, 0 oC, (64%); m) 0.5% HF in CH3CN, 0 oC;

n) carbonyldiimidazole, THF, (78%); o) TBAF, THF, 0 oC, (84%); p) DDQ, CH2Cl2-

H2O (20:1), (81%); q) TESOTf, 2,6-lutidine, CH2Cl2, -90 oC, (71%); r) Martin

sulfurane dehydrating agent, CH2Cl2; s) TFA-THF-H2O (1:10:5), (61%).

f, g

hi, j

56

+

15% 58%

k

l, m n, o

p, q r, s

89

Kobayashi's Synthetic Approach to Dienediyne core (Category C)



3.7. β-Lactam as a Molecular Lock of Enediyne: Synthesis of β-Lactam fused

Enediynes

3.7.1. β-Lactam as Molecular Lock

The first use of -lactam as a molecular lock was reported by Basak et al. and is

demonstrated in -lactam fused bispropargyl sulphones. The bis-propargyl sulphones are found

to be good DNA-cleaving agents. Bis-propargyl sulphones on isomerization afforded allenic

sulphone which is a good Michael acceptor. Thus, DNA base attack it in a conjugate fashion.

The positive charge developed on the DNA base ultimately resulting in a Maxam-Gilbert type

cleavage (Scheme 53). An alternative mechanism, involving formation of diradicals via Garatt-

Braverman rearrangement might also operate to show DNA cleavage potential. Similar catechol

or alizarin based bis-propargyl sulphones readily isomerized to the allenic sulphones under the

same conditions (Scheme 53).

Scheme 53. Bis-propargyl sulphones’s isomerization to allenic sulphone and the DNA cleavage activity.

SR

R

O

O

pH>7S

R

R

O

O

Nu-DNA

SR

R

O

O

Nu-DNA

S

R

R

O

O

O

S

R

R

O

OS

R

R

O

O

DNA cleavage

DNA cleavage

1.H2O2. pH>7

Garratt-Braverman mechanism

Maxam-Gilbert mechanism

Mechanism of DNA cleavage by bis-propargyl sulphones



Basak et al. was successful in arresting the spontaneous isomerization of bis-propargyl

sulphones to afforded allenic sulphone by fusing a -lactam ring onto its’ skeleton. Thus, the -

lactam ring acts as a molecular lock in stabilizing the bispropargyl sulphone even under basic

conditions (Scheme 54).

Scheme 54. Areested isomerization of bis-propargyl sulphones to allenic sulphone by fusing a -lactam ring.

Banfi and Guanti have demonstrated that the -lactam ring can act as a locking device to

stabilize the otherwise unstable 10-membered enediyne system. Opening of the -lactam ring

unlock the system which enabled it to undergo facile BC. They have further developed a -

lactam fused system with a nucleophilic handle that under acidic conditions opens up the -

lactam ring to produce bicyclic reactive enediyne which ultimately undergoes facile BC. These

examples demonstrats the ability of the azetidinone ring to act as a molecular lock (Scheme 55).

Scheme 55. -lactam as a locking device to stabilize unstable 10-membered enediyne.

NO

H O S

O

O

NO

H O S

O

O

NO

H O S

O

O S

O

O

O

O S

O

O

O

O S

(Not Obtained)

O

O

O

O

SO

O

O

O

S

O

O O

O

O

O

S

O

O

1. mCPBA

2. NaHCO3

CH2Cl2

1. mCPBA

2. NaHCO3

CH2Cl2

1. mCPBA

2. NaHCO3

CH2Cl2

+

+

+

Arrested isomerization in -lactam fused bis-propargyl sulphones

Spontaneous isomerization in -lactam fused bis-propargyl sulphones

N

OMe

OR

O

OH

N

OMe

NHBoc

O

OH

N

OMeO

OH

NH2

OMe

OH

NH

OR = OTBS

R = NHBoc

H+

BC

-lactam as a molecular lock of reactive enediyne



3.7.2. Strategies for the Synthesis of β-Lactam Fused Enediynes

There are three possible strategies for the synthesis of -lactam fused enediynes which are

presented in Scheme 56.

(i). Pathway a and b: In these two pathways the formation of enediyne takes place on to

a -lactam ring.

(ii). Pathway c: It involves the formation of -lactam on to an enediyne.

(iii). The third alternative pathway involves (path d) involves the concerted formation of

both the enediyne and -lactam rings.

Scheme 56. Strategy for the design and synthesis of β-lactam fused enediynes.

NO

R

N

R

ON

O

R

N

O

R

N

O

RN

O

R

a

b

c

d

1.4.24

Strategy for the Synthesis of -Lactam Fused Enediynes



3.7.2.1. Guanti’s Approach for β-Lactam Fused Enediyne

Scheme 57. Guanti’s approach for β-lactam fused enediyne.

NMeO

OH

OTBSO

N

OMe

OHOTBS

O

TMS

N

OMe

OOTBS

O

I

HN

OMe

OTBS

O

OH

NH

OMe

OTBS

O

OH

O -

NH

OMe

OTBS

O

O -

OH

..

a

b-d

e

Reagents and conditios: a) Pd(PhCN)2Cl2,

CuI, piperidine/THF; b) AgNO3, KCN,

(83%); c) I2-morpholine, benzne, rt (95%);

d) (COCl)2, DMSO, Et3N, CH2Cl2, (96%); e)

CrCl2, NiCl2 cat, THF, rt (64%).

.

.

N

OMe

OTBS

O

OH

N

OMe

NHBoc

O

OAc

N

OMeO

OH

NH2

OMe

OH

NH

O

NH

OMeO

OH

NH

OMeO

OHH

H

..pH > 7

..

DNA

DNACleaved

Reagents and conditions: a) Ac2O,

pyridine; b) HF, CH3CN/H2O, (96%); c) i.

MsCl, Et3N, CH2Cl2, -30 oC; ii. NaN3, DMF,

60oC, (87%); iii. Ph3P, THF/H2O, 8 h, rt; iv)

Boc2O, Et3N, (85%); d) MeONa, MeOH, 0 oC;

e) CF3CO2H, CH2Cl2.

a-c

d, e

.

.

pH > 7

Guanti's Approach for -Lactam Fused Enediyne



3.7.2.2. Basak’s Approach for β-Lactam Fused Enediyne

Synthetic strategy involves:

(a) an intramolecular N-alkylation (Scheme 58)

(b) an intramolecular carbene insertion (Scheme 59).

These two methods started with either the sensitive -lactam or the sensitive cyclic enediyne.

(c) To bypass the problem associated with the instability of the starting core an

intramolecular Kinugasa reaction was adopted by the same group in which

process the two rings, -lactam and the cyclic enediyne are formed in a single

step. (Scheme 60).

Scheme 58. Intramolecular N-alkylation route to β-Lactam fused enediyne.

Scheme 59. Intramolecular carbine insertion route to β-Lactam fused enediyne.

NHO

NHO

OH

NHO

Cl

NO

Reagents and conditions: a) Pd(PPh3)4, CuI, n-BuNH2, C6H6 (61%); b) TsCl,

DMAP, CH2Cl2 (60%); c) K2CO3, KI, DMF, rt (40%).

OHCl

+a b c

Intramolecular N-alkylation Route to -Lactam Fused Enediyne

NH N

COOEt

OCOOEt

COCl

N

COOEt

O

N2

PTSN3, K2CO3

NO

COOEt

Et3N, CH2Cl2

Rh2(OAC)4/CH2Cl2, rt.

Intramolecular Carbene Insertion Route to -Lactam Fused Enediyne



Scheme 60. Intramolecular Kinugasa reaction route to β-Lactam fused enediyne.

O

N Ph

O

H

H

n

OH

N

Ph

O

O

N

H

Ph

O

n O

N Ph

O

H

H

n

Ph

H

N

O

Ph

OH

H

N

O

H

n = 0 n = 0 n = 0

n = 1 n = 1 n = 1

+

+Cu(I)

NEt3

Cu(I)

NEt3

Intramolecular Kinugasa Reaction Route to -Lactam Fused Enediyne



3.8. Enediynes with pH-Based Triggering Devises

3.8. 1. Introduction

Tumor cells have acidic pH. The pH of these cells again becomes low when they are

administered hyperglycaemic agents while the normal cells remain unaffected. Thus, greater

selectivity against tumor cells is expected with enediynes triggered by acidic pH. This is the most

striking phenomenon that led the scientists to design and synthesize tumor specific enediyne

warheads. Although there is no apparent advantage if the triggering happens physiological pH

(higher that the pH of tumor cell), several designed enediyneprodrugs were activated under mild

basic condition. Some of these prodrugs showed excellent IC50 values against a number of cancer

cell lines. Many of these showed selectivity against cancer cells.

3.8.2. Approaches for Enediynes with pH-Based Triggering

Various strategies are adapted for pH-based triggering of enediynes which can be classified

into the following five categories (Scheme 61). Examples of each category are subsequently

described.

Scheme 61. Strategies for designing enediynes with pH-based triggering devices.



3.8.2.1. Category 1: Activation through Enediyne to Eneyne−Cumulene

Conversion in Altered pH

This strategy is one of the widely exploited strategies in pH-based triggering of enediynes. In

their quest to develop new DNA-cleaving agents related to the neocarzinostatinchromophore,

Toshima and co-workers have synthesized the cyclic sulfide A which upon oxidation with

mCPBA produced the allenicsulfone B. Compound B when treated with a base, such as 1,8-

diazabicyclo[5.4.0]undec-7-ene (DBU), isomerized to the eneynecumulene C. The cumulene,

being extremely reactive, underwent spontaneous MSC under ambient conditions to the diradical

that was able to cause damage to double-stranded (ds) DNA (Scheme 62).

Scheme 62. Base catalysed activation of cyclic sulfones via eneyne−cumulene conversion.

They have also reported that the thia, oxa, or azaenediyne undergo MSC when subjected to

weakly acidic or basic conditions. Under such pH values, the compound first isomerizes to the

eneyne−allene and subsequently undergoes MSC to generate the toluene diradicals which have

been shown to cleave ds DNA.



Shibuya et al. exploited the approach depicted under this category. They synthesized

enediyne model compounds represented by A. Compound A produced the eneyne−allene B and

ultimately generated toluene diradicals C via a reaction cascade triggered by hydrolysis of the

malonyl ester group under basic conditions (Scheme 63).

Scheme 63. Activation through eneyne−allene via decarboxylation.

Enediyne models having electron-withdrawing groups were also designed and subsequently

synthesized. These molecules, represented by F, upon treatment with TFA in the presence of 1,4-

cyclohexadiene (1,4-CHD) in benzene or MeOH at 37 °C afforded the phenol J as the only

isolable product, thus indicating that cycloaromatization proceeded via a diradical pathway as

shown in Scheme 64.

Scheme 64. Activation through acid catalysed eneyne−allene conversion by lactonization.



Kerwin's group has prepared 4-aza-3-ene-1,6-diyne systems represented by structure A and

demonstrated that these compounds possess powerful pH-dependent DNA-cleavage activity with

some degree of cytosine specificity. The probable mechanism involves isomerization to the

azaeneyne−allene system, which undergoes aza MSC to generate methyl pyridiniumdiradicals C

(Scheme 65). The latter then cleaves the ds DNA, producing mainly single-strand cuts at a

concentration of 100 μM. Another possible mechanism of DNA cleavage involves the alkylation

pathway through the intermediacy of D or through the formation of carbene intermediate F via

the ylide E (Scheme 65).

Scheme 65. pH-dependent activation and DNA cleavage of Azaenediynes.



3.8.2.2. Category 2: Activation through Acid- or Base-Catalyzed Ring Opening

In this strategy, the designed molecules have one special structural feature in common. The

common feature is the cyclic unstable enediyne which has been made stable by the fusion of a

locking device. The locking device is usually a small ring, such as an epoxide, a β-lactam, or

even an isooxazoline ring. All of these molecules are equipped with pH-based triggering devices,

which unlock them by opening the small ring. With the removal of strain, the molecules become

active under ambient conditions.

Inspired by the chemistry of the natural dynemicins, Nicolaouet et al. first reported the

synthesis of a series of analogues in which the flow of electrons to open the epoxide ring was

blocked by the incorporation of protecting groups in potential donors. In one such design, the

pivaloyl group was used to protect the free phenolic -OH group to generate the enediyne A.

Base-promoted hydrolysis produced the free phenolic form B, which is capable of promoting the

epoxide ring opening via the flow of electrons followed by BC to give diradical D which can

cleave DNA (Scheme 66).

Scheme 66. Base-mediated deprotection and activation of dynemicin model enediynes.



Similar to dynemicin, electrons can also flow from the ring nitrogen if its lone pair is free.

On that basis, the enediyne F was synthesized by Nicolaou and co-workers. The interesting

feature of this molecule is that the nucleophilic N atom is made non-nucleophilic by protection

with a 2-(phenylsulphonyl) ethoxy carbonyl group. The group falls off upon treatment with mild

bases, thus leading to the generation of free amine G the lone pair of which is being free flows

toward the direction of epoxide, which then opens up. With the release of strain, the resulting

compound I shows BC under ambient conditions (Scheme 67). The produced diradical J have

DNA-cleaving activity at micromolar concentrations, resulting in the formation of both relaxed

and linear forms of DNA.

Scheme 67. Base-catalyzed β-elimination and activation of Dynemicin model enediyne.



The sensitivity of epoxide rings under acidic conditions was exploited by Unno and co-

workers in their designed dynemicin analogues. Thus, several novel analogues designated by

structure K were made, and their DNA-cleaving potential under acidic conditions was evaluated.

It was demonstrated that the size and electronic character of the substituents (R1 and R2) at the

C9 position critically influenced the DNA-cleaving ability of the synthesized enediynes. The

compounds represented by K were shown to undergo BC under acidic conditions (Scheme 68).

A similar cascade of reactions involving ring opening and BC may operate in cancer cells

because they are sufficiently acidic with pH less than 7.

Scheme 68. Acid-catalyzed ring opening of epoxide and activation of enediyne.

From previous discussions, it is clear that, among the various small ring systems, Nature has

picked up the epoxide ring to lock the unstable enediynes, with the reason being the easy

unlocking of these systems by opening of the strained epoxide ring by an acid-catalyzed process

or opening because of an inward flow of electrons.



3.8.2.3. Category 3: Activation through Salt Formation

The rationale behind the activation upon protonation (salt formation) of an enediyne with a

basic functionality near the enediyne moiety lies in the fact that the electron-withdrawing effect

(−I or electron transfer) of the protonated species lowers the repulsion between the in-plane

alkyne π orbitals (Koga−Morokuma hypothesis).

By electron withdrawal the protonated enediyne lowers the singlet−triplet gap and thus

favors the triplet state. The triplet state is proven to be a better hydrogen abstractor than a singlet.

Thus, the triplet state produces the cleavage of DNA. Computational analysis supports this which

revealed that a singlet diradical abstract hydrogen much in a slower rate compared to the triplet

diradical. The prediction has been confirmed for the singlet didehydroanthracenediradical, for

which the hydrogen-abstraction rate from 2-propanol was found to be reduced by 2−3 orders of

magnitude relative to phenyl or 9-anthryl radicals. For a singlet ground-state biradical to show

radical-like chemistry, e.g., hydrogen abstraction, it must add extra energy to scale up the

singlet−triplet gap. The singlet lies below the triplet because it is stabilized and, accordingly, one

has to pay back the stabilization energy to reach a TS where the two electrons in the nonbonded

molecular orbitals (NBMOs) are uncoupled to reach the triplet state.

It can also be summarized that an increase in the electron density in the intervening σ bonds

can increase the through-bond coupling and hence increase the singlet−triplet splitting.

Conversely, a decrease in electron density will decrease the coupling and hence decrease the

singlet−triplet gap. Thus, the in-plane lone pair of the nitrogen atom in the 2,5-didehydropyridine

diradical lies antiperiplanar to the σ bonds coupling the NBMOs and therefore could donate

electron density. However, when the nitrogen is protonated, the effect is reversed. This has been

confirmed by ab initio computed singlet−triplet gaps, where the didehydropyridine with its lone

pair shows a much larger singlet−triplet gap in comparison to the protonated form. The

protonated azaenediyne liesg mostly in the triplet state and hence should be a better hydrogen-

atom abstractor.

The first strategy is reflected in the works from the research group of Alabugin et al., as well

as from Basak’s group. Alabugin et al. has reported that the rate of BC in

benzannulatedenediynes can be tuned by varying the electronic nature of the ortho substituents.

The most striking of them is the acceleratory effect on BC kinetics when an amino group at the

ortho position is protonated B (Scheme 69) (The ortho effects on BC has been described in

Module 1).

Scheme 69. Ortho effect on protonation on the rate of BC of benzannulated enediynes.



Such acceleration of the BC rate upon protonation has also been reported by Basak et al. in

the case of 2,6-diamino pyridine-based enediynes E. The latter was prepared by double N-

alkylation of the bis-sulfonamide B. Deprotection using PhSH under basic conditions (K2CO3)

gave the free amine E. The reactivity of the enediyne E and its salts F(X), F(Y), F(Z) with acids

of various pKa values (X-Z) was studied by DSC, which indicated the lowering of the onset

temperature for BC upon salt formation. Interestingly, the extent of lowering was shown to

depend upon the degree of salt formation, which was monitored by 1H nuclear magnetic

resonance (NMR) studies. The greater the degree of salt formation, higher the lowering of the

onset temperature for BC (Scheme 70).

Scheme 70. Reactivity of pyridine diamine based enediyne upon salt formation.

Another set of example of salt formation and enhancement of rate of BC is shown by the 10-

membered azaenediyne shown in Scheme 71.

Scheme 71. Synthesis and reactivity of free amine from 10-membered azaenediyne.

OH

HN S

O

O

NO2

OMs

HN S

O

O

NO2MsCl, Et3N, 0 °C K2CO3, DMF, r.t

PhSH, K2CO3,

DMFN S

O

O

NO2NH N

pH = 8

NH

H

H

H

r.t.

BC

Reactivity of Free Amine Salt from 10-Membered Azaenediyne



The cyclization chemistry of C, N-dialkynyl imine azaenediyne A in which one of the ene

carbons is replaced by nitrogen was first reported by Kerwin's group. They have reported that the

ultimate product of BC, (G), could only be isolated if there was a small amount of picric acid.

Computational studies have shown that the singlet diradical (F), which is stabilized by the

pyridine N lone pair, is a poor hydrogen-atom abstractor as compared to the triplet diradical (C),

which is mainly the species generated from the protonated form of azaenediyne E (Scheme 72).

Scheme 72. Reactivity of azaenediyne under acidic pH.



3.8.2.4. Category 4: In Situ Generation of Enediynes through Allylic

Rearrangement/β-Elimination

The drive for this type of design was derived from the chemistry of an artifact of the natural

enediyne maduropeptin chromophore. The compound A was obtained during isolation of the

parent enediyne using methanol. An intramolecular nucleophilic attack in an SN2' fashion

generates the reactive enediyne functionality B from compound A. Compound B then undergoes

BC and shows DNA-damaging property (Scheme 73). This overall cascade of reactions

represents an emerging strategy for the design of the enediyne prodrug.

Scheme 73. Triggering of Maduropeptin artifact under acidic pH.

An intermolecular allylic rearrangement strategy for enediyne generation from the prodrug

was adopted by Dai et al. They designed and synthesized the alcohol A, which, upon treatment

with nucleophiles, such as ethanol or water, in acidic medium, underwent a SN2' reaction to

generate the reactive enediyne system B. Compound B then underwent BC under ambient

conditions, and the resulting diradical C was found to cleave ds DNA (Scheme 74) at millimolar

concentrations, producing relaxed DNA.

Scheme 74. In situ generation of enediyne by allylic rearrangement under acidic pH.

OR

HO

Ph

OR

H2O

PhROH

OR

Ph

EtO

DNA

Cleaved DNA

OR

Ph

EtO

H

H

Dai, W. M.; Fong, K. C.; Lau, C. W.; Zhau, L.; Hamaguchi, W.; Nishimoto, S-i. J. Org. Chem. 1999, 64, 682.

CSAEtOH

OR

Ph

EtO

BC

In Situ Generation of Enediyne by Allylic Rearrangement Under Acidic pH

A B C D E



The synthesis of a number of enediyne prodrugs possessing free hydroxymethyl groups on

the exocyclic double bond has been reported by Dai et al. These compounds as represented by A

cause single-strand cleavage in circular supercoiled DNA at a pH of 8.5. A mechanism based on

allylic rearrangement to form a putative epoxyenediyne C has been proposed. Cleavage of DNA

may have taken place via both BC pathways, involving hydrogen abstraction by the diradical and

alkylation of the DNA base followed by a Maxam−Gilbert-type reaction (Scheme 75).

Scheme 75. In situ generation of enediyne by allylic rearrangement under acidic pH.

Below is an example of base mediated MSC (Scheme 76).

Scheme 76. Activation of enediyne toward MSC under basic pH.

OR

X

Ph

OSi

OR

Ph

OSi

OR

Ph

O

-X

Allyl Cation Formation

OR

Ph

HO

X

Ph

O

OR

Ph

HO DNA

OR

Ph

HO

DNA

pH = 8.5

pH = 8.5 pH = 8.5pH = 8.5, DNA

Cleaved DNA

DNA

Cleaved DNA Cleaved DNA

X = OH, OAc, OCH2OMe

Maxam-Gilbert-type

In Situ Generation of Enediyne via Allyllic Rearrangement and DNA Cleavage under Basic pH

BC

ArOMOM

CO2Et

OMeCO2Et

Ar

OMe

CO2Et

OMOM

C

Ar

CO2Et

OMe

OMOMAr

CO2Et

OMe

OMOM

OEt

OTDS

Ar=

KOH

Myers-SaitoCyclizaiton EtOH

Shibuya, M.; Naoe, Y.; Bando, M.; Nemoto, H. Tetrahedron Lett. 1998, 39, 2361.

Activation of Enediyne under Basic pH



Takahashi et al. prepared the cyclic 1,5-diyne derivative A equipped with a good leaving

group in one of the two propargylic carbons separating the alkynes. Treatment with a base-like

DBU converted B via β-elimination to the 10-membered enediyne C, which then smoothly

underwent BC under ambient conditions (Scheme 77).

Scheme 77. In situ generation of enediyne via β-elimination under basic pH.

3.8.2.5. Category 5: Activation through Acid-Catalyzed Enol to Keto

Tautomerism

On the basis of the way of activation of calicheamicins and esperamicins via conjugate

addition that converts bridgehead sp3 carbon into a sp2 carbon, Semmelhack and co-workers104

have designed an enediyne system (A-B, F) in which a double bond is present at the bridgehead

in the form of an enol. Conversion of the enol into the keto form (C, G) under acidic conditions

removes the bridgehead double bond and triggers the molecule toward undergoing BC. It is

interesting to note that similar acid treatment of the enolic enediyne system (J-K) generated the

ketone L, which is, however, stable under ambient conditions (Scheme 78). The reason for this

anomalous behavior (less reactivity) of the substituted ketone is likely to be steric effect exerted

by the two –R groups.

Scheme 78. Triggering enedyne via acid-catalyzed enol−keto tautomerization.



3.9. Photoswitchable Enediynes

3.9.1. Introduction Recently photodynamic therapy (PDT) has become attracted clinical attention for selective

targeting and damaging of tumor cells that are quite localized and yet metastasized. The

photodynamic therapy (PDT) combines a drug and a light of particular wavelength. The light

used for PDT can come from a laser or other source. In this process the light is directed through

an optical fiber to deliver light at the appropriate localized area. One important issue in PDT is

the penetration ability of the light through the tissue. That is why PDT is less effective toward

large tumors. The use of light of a longer wavelength increases the penetration ability. Photo

triggering method is not only interesting but important because this would allow one to develop

target-specific chemotherapeutic agents. There is no precedence of this type of triggering of

enediynes in Nature. Therefore researchers of the field of enediyne have taken the advantage of

light triggering concept to design and synthesized photo-triggerable enediynes which can be

activated by light of a higher wavelength. Few such examples are given in the following sections

to address the efforts made to generate light-mediated triggering devices in enediyne chemistry.



3.9.2. Strategies for Designing Enediynes with Photo-Triggering Devices Literature reports revealed a wide variety of available strategies to trigger enediynes by

photochemical means. Some of these strategies involve Bergman Cyclization induced by

irradiation. Other strategies use photoirradiation wherein a structural change occurs in the

prodrug. Upon change in structure the enediyne is activated toward thermal BC. All of these

strategies can be classified according to the flow chart shown below in Scheme 79.

Scheme 79. Strategies for photo-activation of enediynes.



3.9.2.1. Category 1: Use of Enediynes Activated toward Photo-Bergman

Cyclization

Turro, Evanzahav, and Nicolaou were the first to show that enediynes also undergo

cycloaromatization upon photo-irradiation and they were able to produce products similar to that

obtained in a thermal BC. The process is now popularly known as Photo-Bergman cyclization

(photo-BC). They have reported the formation of naphthalene derivatives B when an

isopropanol solution of n-propyl- or n-phenyl-substituted enediyne A was irradiated. However,

in addition, products resulting from photoreduction of one of the alkynes were also formed

(compounds C and D) (Scheme 80).

Scheme 80. Photo-induced BC.

Unlike the thermal counterpart, photo-BC is not as versatile and the quantum yield as well as

the actual isolated yield of the cyclized product are usually low. However, certain enediynes can

show facile photo-BC, depending upon the nature of substituents at the alkyne termini or the

electronic nature of the ring fused onto the enediyne. The use of different modes of energy

transfer is another approach to improve the efficiency of photo-BC. As a result of tremendous

research efforts, several novel enediynes were synthesized that could be activated toward BC

upon irradiation.

The photo-BC is an efficient and symmetry-allowed process when the photochemical

excitation involves the in-plane p-orbitals (Figure 3). This is likely to be the case, because such

excitation promotes an electron from the molecular orbital (MO) that is C1−C6-antibonding to a

MO, which is C1−C6-bonding. Thus, the excitation should increase the extent of C1−C6 bonding



at a relatively early reaction stage. However, the in-plane excitation requires much more energy

than the excitation of the out-of-plane orbitals and is thus difficult to access experimentally.

But, the efficiency of the photochemical BC can be increased by decreasing the energy gap

between the in-plane frontier orbitals. This can be achieved by putting the enediyne framework

in a cyclic cage. Decreasing the C1−C6 distance destabilizes the occupied MO in which the

interaction between the end orbitals is antibonding. At the same time decrease in the C1−C6

distance the empty MO has got stabilization which has favorable interactions between the end

orbitals. With a decrease in the energy gap between the highest occupied molecular orbital

(HOMO) and lowest unoccupied molecular orbital (LUMO), photo-BC is favored.

Figure 3. (a) Frontier molecular orbital diagrams involved in photo-BC. (b) Comparison between the antiaromatic π−π* interaction

pattern and the antiaromatic TS for the [2s + 2s] cycloaddition involved in photo-BC. (c) Effect of locking the enediyne moiety in a cycle

on the energy gap between the frontier in-plane MOs.



Funk and Williams have designed the pyrene-based enediyne which was tethered to an

aminoalkyl side chain to provide affinity for DNA (Scheme 43). This, upon irradiation in the

presence of pBR 322 DNA, caused cleavage of the DNA. In this process both forms II and III of

DNA at micromolar concentrations were observed. This is the first example of an enediyne-

based DNA photocleaver.

Scheme 81. Photo-activated pyrenyl enediyne.

Hirama et al. have reported the photo-BC of several non-benzenoid enediynes. They have

reported the photo-BC of 1,2-diakynyl cyclopentene. Usually, the yield of cyclized product was

low ( 3%), except for the dipropynyl derivative in which case the cyclized product was isolated

in yields up to 71%. No photoreduction products were observed unlike in the case of the

benzenoid counterpart (Scheme 82). The 10-membered ring enediyne upon similar irradiation

with a low-pressure Hg-lamp produced the cycloaromatized product along with the acyclic

enediyne. The latter was generated via retro-BC (Scheme 82). It may be noted that such a retro-

BC product was not isolated under the thermal conditions.

Scheme 82. Photo-BC of non-benzenoid cyclic enediynes.



Russell et al. have reported facile photo-BC of pyrimidine-based enediynes A in isopropanol

solution to quinazoline (X = H or OH) (Scheme 83). It is interesting to note that the

corresponding ketone C failed to undergo photo-BC. However, compound C give cyclization

products only under thermal conditions. This clearly demonstrated different activation

parameters controlling the kinetics of BC under thermal and photochemical conditions.

Scheme 83. Pho-induced reactivity of pyrimidine-based enediynes.



Apart from their DNA-cleaving property, the enediynes have been shown to possess protein-

cleavage ability. This is one of the mechanism via which microorganisms producing enediynes

protect themselves through the sacrifice of a protein that is secreted by the organism itself. Jones

et al. have showed the likely mechanism of protein cleavage and synthesized photo-activated

enediynes to study their protein-cleaving ability. The mechanism of protein cleavage involved

the formation of radical B at the captodatively stabilized α carbon. This radical then reacts with

molecular oxygen to form the peroxo radical E. This can undergo strand scission or cross-linking

to provide G and H, the cleaved protein (Scheme 84).

Scheme 84. Protein degradation pathways enediynes.



Support for the proposed mechanism depicted in Scheme 84 came from the

cycloaromatization reaction of an enediyne in the presence of labeled amino acids, such as

dideuteriated glycine (A) (Scheme 85). When the diradical is formed upon BC, the deuterium

got abstracted by the diradical to form compound D. The isolation of the dimerization product F

and the amide H together with the deuteriated aldehyde I could be explained on the basis of the

formation of glycyl radical E.

Scheme 85. Fate of the diradical produced after BC in the presence of glycine-d2.

Conjugates of photo-activated enediynes and amino acids were also synthesized by Jones's

group. These conjugated assemblies readily underwent photo-BC upon irradiation, thus making

them potential agents for photodynamic therapy. The photo-activated enediyne bearing amino

acid upon irradiation was found to cause degradation of bovine serum albumin, histone, and an

estrogen receptor (Scheme 86). These observations on protein degradation by enediynes open up

a new application of enediynes as chemical proteases.

Scheme 86. Photo-activation of enediyne−amino acid conjugates.



Peterson et al. has reported the synthesis and reactivity of imidazole-fused cyclic enediynes

toward photo-induced BC. The more conformationally rigid analogues gave higher yields of

cycloaromatized products upon irradiation at ambient temperature. The bicyclic analogue shown

in Scheme 87 was shown to undergo photo-BC to produce the cycloaromatized product and

consequently to induce single-strand breaks in supercoiled DNA at micromolar concentrations.

Scheme 87. Photo-activation of imidazole-fused enediynes.

Photo-irradiation of bis(phenylethynyl) sulfide in hexane in the presence of 1,4-CHD

produced 3,4-diphenyl-thiophene through the presumed intermediacy of the 2,5-

didehydrothiophene diradical (Scheme 88). This constitutes the first example of a 5-membered

ring cycloaromatization exploiting the aromaticity of heterocyclic rings, such as thiophene.

Scheme 88. Photo-BC of diethynyl sulfide.



3.9.2.2. Category 2: Activation of Enediynes via Photo-induced Electron Transfer

Alabugin et al. have shown that bistetrafluoropyridinyl ethynyl benzenes I A-D, a class of

photo-activated benzenes, smoothly undergo a novel C1−C5 photochemical cyclization to provide

isomeric indenes II A-D and III A-D (Scheme 89). In this process the cycloaromatization

follows a different mechanism than that operating in normal BC. The key step in this is the

photo-induced electron transfer from 1,4-CHD to the singlet excited state of the enediyne. The

presence of strongly electron-withdrawing tetrafluoropyridinyl (TFP) substituents renders the

photo-induced electron transfer from 1,4-CHD to the singlet state of enediyne highly exothermic.

Unlike the cyclization of neutral enediynes, the C1−C5 cyclization of enediyne radical anions H

leads to an intermediate L stabilized by resonance involving cyclopentadienyl anions, which

makes the cyclization mode possible (Scheme 89). This is inferred after calculating the energies

of the starting enediyne, the TS, and the radical product formed by photochemical cyclization. It

is also to be noted that the enediyne−lysine conjugate E has been shown to possess some degree

of sequence-selective binding and cleavage of DNA.

Scheme 89. Photo-activation of fluoropyridinyl enediyne and the mechanism for the formation of indenes.



3.9.2.3. Category 3: Photo-excitation of Enediyne Metal Complexes via Ligand-

to-Metal or Metal-to-Ligand Charge Transfer

Photodynamic therapy relies on the use of longer excitation wavelengths for the drugs to be

used. This will ensure enhanced tissue penetration by near-infrared (IR) photons. Therefore, two

possible approaches can be adopted to shift the excitation wavelength to a longer region (beyond

λ > 600 nm) required for photo-BC. The first choice of design involves the synthesis of

enediynes with extended π conjugation. However this is synthetically challenging and may suffer

from solubility problems. The alternative strategy is the use of long-wavelength electronic

transition with considerable absorptivity that can be achieved via metal-to-ligand charge transfer

(MLCT) process within compounds where both the metal oxidation state and donor/acceptor

redox potentials have been properly chosen.

This idea was utilized demonstrated with design of a novel vanadium (V) metalloenediyne

compound A of a 4,5-bis(phenylethynyl) benzene 1,2-diol ligand by Zaleski et al. The

metalloenediyne exhibited strong ligand-to-metal charge transfer (LMCT) transitions in the near-

IR region because of low redox potentials of the high valent vanadium center and the easily

oxidizable metal-binding motif. Differential Scanning Calorimetry (DSC) and resonance Raman

spectroscopy showed that these LMCTs can be successfully used to photothermally activate the

metalloenediyne toward BC. Thus, upon laser excitation at 785 or 1064 nm compound A in the

solid state become photothermally activated toward BC to produce the insoluble polymeric

material C. The compound was inert to BC upon electronic excitation in the UV spectral region

(Scheme 90) suggesting the necessity of excitation at very long wave length.

Scheme 90. Photo-excitation of enediyne via ligand-to-metal charge transfer (LMCT).



Photo-BC can also be prompted by MLCT. Zaleski et al. have reported that photolysis of

copper complexes of Cu(bpod)2PF6 and/or Cu(bpod)2(NO3)2 (A) (bpod = cis-1,8-bis(pyridine-3-

oxy)oct-4-ene-2,6-diyne) yielded BC of bound ligands. In contrast, the uncomplexed ligand and

Zn(bpod)2(CH3COO)2 compound (E) were photochemically inert under the same conditions

(Scheme 91). The observed BC of the compounds A has been ascribed because of MLCT.

Moreover, the intermediates produced upon photolysis are capable of degrading both pUC19

plasmid DNA as well as a 25 base pair double-stranded oligonucleotide via C-4' hydrogen-atom

abstraction in micromolar concentrations.

Scheme 91. Photo-excitation of enediyne via metal-to-ligand charge transfer (MLCT).

O

O

N

N

O

O

N

NCu

O

O

N

N

O

O

N

NCu

O

O

N

N

O

O

N

N

O

O

N

NCu

S

S

> 395 nm

1,4-CHD or 2-propanol,

CH3CN

EDTA,

DMF·H2O, CH2Cl2.

> 395 nm,1,4-CHD

or 2-propanol, CH3CN

EDTA, DMF·H2O,

CH2Cl2.

(a)

(b)

O

O

N

N

O

O

N

NZn

OCOMe

OCOMe

A B

C D

E

S = Solvent

Photo-excitation of Enediyne via Metal-to-Ligand Charge Transfer



The above example depicted nicely how a metal ion can facilitate photo-BC (Scheme 91).

An interesting variation whereby the photo-BC is completely shut down upon metal

complexation is provided in the next example (Scheme 92). Thus, the parent benzene-fused

enediyne A undergoes photo-BC upon irradiation to produce the cycloaromatized product C via

the diradical B. However, the ruthenium complex of A (D) did not produce any cyclized product

upon similar photo-irradiation. This fact thus, highlights the importance of the electronic effect

in controlling the BC kinetics (Scheme 92). The reluctance of compound D to undergo

cycloaromatization (relative to A) is most likely due to decreased aromaticity in the incipient

1,4-diradical, which would be generated from D.

Scheme 92. Shut down of photo-BC of benzene-fused enediyne upon complexation with Ru- metal.



3.9.2.4. Category 4: Photo-activation of Locked or Acyclic Enediyne

The basis of this design strategy relies on the use of photo-cleavable protecting groups. This

group masks the nucleophilic character of an amine or a phenolic hydroxyl group. The protecting

group falls off upon photolysis and then liberates the free amine or phenol. Then, the free amine

or the phenol allows flow of electrons which results in the opening of a strained ring-like

epoxide. Thus, the strain is released. As a result, the enediynes get activated toward BC under

ambient conditions.

This idea was best demonstrated in the design of a model compound reported by Nicolaou et

al. (Scheme 93). Thus, the compound A under photolytic conditions got converted into the diol

D via the intermediacy of the quinone−methide C formed by epoxide ring opening. With the

release of strain, the compound D underwent BC. The addition of an external nucleophile, such

as EtOH or EtSH, led to the isolation of the cycloaromatized product.

Scheme 93. Photodeprotection and activation of the dynemicin model.



Another design based on similar idea was devised by Wender et al. who synthesized a 5-

nitroveratryloxy (N-Voc)-protected dynemicin analogue E (Scheme 94). Compound E is

photochemically activable dynemicin analogue that underwent cycloaromatization upon

irradiation with wavelengths greater than 300 nm. The ability of this compound to DNA

cleavage was also demonstrated. It was also observed that the enediyne E is also activated

toward thermal BC by treatment with acid, which opens up the epoxide. Thus, the molecule is

equipped with a dual triggering mechanism, pH as well as light.

Scheme 94. Photodeprotection and activation of the dynemicin model.

The design of molecules with an acyclic framework to be activated in a similar way is based

on the fact that cyclic 10-membered azaenediyne spontaneously cyclizes under ambient

conditions with a decent half-life (t1/2 = 36 h at 30 °C). Taking the idea from this fact, a new

design strategy has been adopted in which an acyclic enediyne having an amino group in one

arm of the enediyne is protected in the form of N-Voc or β-lactam. Cleavage of N-Voc or

opening of the β-lactam ring releases the nucleophilic nitrogen free which then undergoes

intramolecular attack to close the cycle. The latter with appropriate size then undergoes BC. The

resulting diradical in this process shows DNA cleavage or antibacterial property.

Thus, Basak et al. designed an acyclic enediyne molecule with a photo-cleavable amine

protecting group that is stable at biological temperatures but is convertible to a cyclic 10-

membered enediyne after a triggering reaction. This in situ generated cyclic enediyne could then

be capable of showing DNA cleavage activity upon BC (Scheme 95). Thus, they have

synthesized the enediyne A, where the photocleavable protecting group satisfies two criteria: (a)

it suppresses the nucleophilicity of the nitrogen, and (b) it is removed by photolysis. The

compound A upon irradiation at 365 nm was able to induce single-strand cleavage of plasmid

DNA. It is also to be mentioned that the ketone A also caused partial DNA damage under non-

irradiating conditions. However, the efficiency of cleavage was 2.5 times less compared to that

observed for the in situ generated cyclic enediyne C which could be produced after deprotection

N

HO H

H

O

OO

NO2MeO

MeO

N

HO H

H

O

H

N

HO H

H

HO

MeOH

HN

HO H

H

HO

MeO

HN

HO H

H

HO

MeO

HN

HO H

H

HO

MeO

H

H

N

HO H

H

HO

Cl

OO

NO2MeO

MeO

Activation of the Dynemicin Model by Photodeprotection

hn (365 nm)

THF-MeOH

AcCl, THF, MeOH

DNA

CleavedDNA

E F G

HIJK

N

HO H

H

O

OO

NO2MeO

MeO

N

HO H

H

O

H

N

HO H

H

HO

MeOH

HN

HO H

H

HO

MeO

HN

HO H

H

HO

MeO

HN

HO H

H

HO

MeO

H

H

N

HO H

H

HO

Cl

OO

NO2MeO

MeO

Activation of the Dynemicin Model by Photodeprotection

hn (365 nm)

THF-MeOH

AcCl, THF, MeOH

DNA

CleavedDNA

E F G

HIJK



of the protecting group of A and an intramolecular ring closure by nucleophilic attack by free –

NH2 to the ketone. This ruled out the cleavage via the Maxam−Gilbert mechanism as the major

pathway.

Scheme 95. Photodeprotection and the mechanism of DNA cleavage of enediyne containing heteroatom “N”. Lane 1: DNA; lane 2: DNA

+ 5.082 (10 μM) (24 h) + hν (365 nm); lane 3: DNA + 5.082 (10 μM) (24 h).



3.9.2.5. Category 5: Activation of the Prodrug via Photolysis of

Cyclopropenone/Diazoketone

In this design strategy, a reactive enediyne system is protected with a group that arrests the

spontaneous BC to occur at ambient conditions of a monocyclic enediyne. Although cobalt

carbonyl complexation of the acetylenic moiety is a commonly used protecting group, the

difficulty in the removal of such a group under photochemical conditions prohibited its use.

Popik et al. have used cyclopropenone as a photocleavable protecting group to lock a benzene-

fused enediyne system A. Irradiation led to cheletropic removal of carbon monoxide with

consequent generation of enediyne B, which then underwent BC. It may be noted that the

acetylene-protected enediyne A is thermally stable, showing no sign of decomposition even after

heating at 84 °C for 7 days. They have also demonstrated that the p-quinonoid cyclopropenone-

containing enediyne prodrug A can be activated by photolysis via a single- or two-photon

transfer to the enediyne. The latter undergoes BC at 40 °C with a half-life of 88 h (Scheme 96).

Scheme 96. Photochemical in situ generation of enediyne.



Similar to the MSC, eneyne−ketones can also undergo similar cyclization chemistry to form

a phenoxy diradical. The latter reaction also takes place under ambient conditions similar to the

MSC. On the basis of this Saito et al. synthesized the diazoketone B via Sonogashira coupling

followed by the reaction with diazomethane or methyldiazomethane with the acid chloride A.

The resulting diazoketone, upon photo-irradiation (high-pressure Hg-lamp), rearranges into the

ketene D. The ketene is conjugated to the eneyne system. Therefore it undergoes

cycloaromatization to produce the phenoxy diradical E which was shown to induce cleavage of

ds plasmid DNA (pBR322) (Scheme 97).

Scheme 97. Photo-activation through conversion of eneyne−ketene.



3.9.2.6. Category 6: Activation via Photo-isomerization of Azobenzene-Based

Enediynes and Sulfones

Conformational changes can bring about significant perturbation in the kinetics of BC.

Previously, König et al. have shown that (for a bipyridyl enediyne) a decrease in the distance

between the two acetylenic arms undergoing covalent connection (c and d distance) upon

complexation to mercury(II) brings about a remarkable increase in the reactivity toward BC.

It is thus reasonable to think that a similar conformational change might be achieved if a

group capable of switching between E and Z configurations is incorporated in an enediyne

moiety (Scheme 98). Thus, Basak et al. designed azo-based enediyne systems represented by the

general structure A. These molecules should exist in the thermally stable E isomer. Photo-

isomerization to the Z isomer B is expected to bring down the c and d distance (Scheme 98),

which should lead to an increase in reactivity.

Scheme 98. Rationale behind triggering through E−Z isomerization and phototriggering of azo enediynes.



Scheme 99. Synthesis of nonaromatic azo enediyne.



With this idea, the cyclic enediyne A, containing a stable E-azo moiety (azoenediynes) was

synthesized and reported by Basak et al. The key step is the double N-alkylation to form the

cyclic network (Scheme 99). The stable E-azoenediynes A and G upon irradiation with long-

wavelength UV isomerize to the Z compounds F and H respectively, which can be thermally re-

isomerized to the corresponding E compounds (Scheme 100). Reactivity studies toward BC

using DSC predictably indicated higher reactivity for the Z isomers. These studies provide a

novel way to modulate the reactivity of enediynes under thermal or photochemical conditions.

With an appropriately sized enediyne, there could be a possibility of inducing BC upon

irradiation under ambient conditions.

Scheme 100. Photochemical trans-cis isomerization of azo enediynes and thermal reactivity thereof.



Encouraged by the above trans-cis isomerization of azo enediynes in effecting a change in

BC kinetics, a novel cyclic bispropargyl sulfone A containing stable E-azo moiety has also been

synthesized by Basak et al. (Scheme 101).

Scheme 101. Synthesis of azo bispropargyl sulfones.



The compound A upon irradiation with long UV (350 nm) isomerized to the Z compounds B,

which can be thermally re-isomerized to the E compounds. The E isomer A upon treatment with

Et3N equilibrates to the mono-allenic sulfone C. The formation of bisallene E was not seen

during the base treatment. On the other hand, a similar treatment of the corresponding Z isomer

forms the monoallene D first, which subsequently further isomerized to the unstable bisallene F

which finally underwent decomposition presumably via the Garratt−Braverman pathway

(Scheme 102). Incubation with plasmid DNA also indicated higher DNA-cleavage efficiency

( 2.5 times) for the Z isomer.

Scheme 102. Photochemical trans-cis isomerization of azo bis-propargyl sulphones their reactivity thereof.



A similar increase in DNA-cleavage activity of a related sulfone with an extra conjugation

was also observed for the Z isomer Q as compared to the E analogue P (Schemes 103).

Scheme 103. Synthesis of unsaturated bispropargyl sulfones.



Dai et al. have earlier reported efficient photo-inducible DNA-cleaving ability of propargylic

sulfone conjugated to the anthraquinone moiety (Scheme 104). From DNA-cleavage studies

using different anthraquinone-based sulfones, it was concluded that appropriate spatial

arrangement between the activated allenic sulfone and nucleobase, which is derived from an

efficient intercalation, leads to a substantial amount of DNA cleavage via alkylation of the

nucleobase and photo-induced one-electron oxidation of guanine bases.

Scheme 104. Anthraquinone-based propargyl sulfone.



3.10. Biological Actions of Some Synthetic Models

In general two types of DNA cleavages occur- (a) the single- and (b) double-strand cuts

(Figure 4a). In a typical experiment to check the DNA-cleaving activity a supercoiled plasmid

DNA is used. Single-strand cleavage means the breakage or nicking of only one of the two

strands, which enables the DNA to relax. On the other hand, a linear form will result if cuts are

produced on complimentary sites (or close to complimentary sites) of both of the strands. The

mobilities of all three forms in a gel under electric field are different and hence can be easily

identified via gel electrophoresis technique (Figure 4d). Calicheamicin show its potential DNA

cleaving activity at nanomolar concentrations (1 pg μL-1). However, all the reported synthetic

enediynes show lower DNA cleavage activity compared to natural calichiamicin. These synthetic

enediynes show their potency at micromolar concentrations. Thus, in comparison to the natural

enediynes, the cleavage efficiency of designed enediynes is a moderate.

Figure 4. (a) Single- and double-strand DNA cuts. (b) Supercoiled plasmid DNA. (c) Relaxed plasmid DNA. (d) Gel electrophoresis

pattern of various forms of plasmid DNA [form I: supercoiled; form II: nicked, single-stranded (ss) cleavage; form III: linear, double-

stranded (ds) cleavage].



3.10. 1. Biological Activity of Basak’s β-Lactam Fused Enediyne

-lactam fused enediyne as is shown in Figure 5

showed cleavage of supercoiled plasmid DNA (pBR 322) at concentrations of 5 . No such

cleavage was observed under neutral conditions. In -lactam hydrolyses

to produce the amine, which is predominantly present in the protonated form and is the actual

DNA cleaving agent. The result clearly showed the p -lactam fused enediyne as a

cytotoxic agent.

Figure 5. Interaction of supercoiled DNA (in Tris-acetate buffer, pH 8.0) and enediyne in acetonitrile. Lanes 1: DNA; Lanes 2: DNA +

enediyne 1.4.46 (50 mmol); Lanes 3: DNA.

N

O

COOEt

H

NH2

EtOOC

H

CO2

BC and DNA Cleavage

Form I

1 2 3

Form II

Interaction of Supercoiled DNA and -Lactam Fused Enediyne



3.10. 2. Biological Activity of Basak’s Azo Bispropargyl Sulfones

The amino enediyne shown in the figure below which is an intermediate for the synthesis of

Basak’s β-lactam-fused enediyne by the carbene insertion route, is able to cleave double strand

DNA at a pH of 8.0. Both the double-strand cut (linear form) as well as the nicked form was seen

using pBR 322 plasmid in micromolar concentrations range (Figure 6A). Only the nicked form

of cleaved DNA was seen at similar concentrations while pBlueScript SK+ plasmid DNA was

used (Figure 6B). The protonated form was proposed as the DNA-damaging agent. The

positively charged nitrogen is able to withdraw electrons from the enediyne π- framework

leading to lowering the activation barrier for the generation of benzene diradical via BC. It is

also important to note that the perturbation of the singlet−triplet barrier in the diradical via

through-bond or through-space could also be responsible for such DNA cleavage activity. It was

also demonstrated that the protonated amine in the form of tosylate salt also undergoes BC at 30

°C with a half-life of 30 days.

NH NH2

BC and DNA Cleavage

Form I

1 2

Form II

Interaction of Supercoiled DNA and the Precursor Amino Enediyne of -Lactam Fused Enediyne

pH = 7.8

Form III

Form I

Form II

1 2

With Supercoiled Plasmid DNA pBlueScript SK+

With Supercoiled Plasmid DNA(pBR 322)

(A) (B)

Figure 6. Interaction of supercoiled plasmid DNA (A) pBR 322 and (B) pBlueScript SK+ in Tris-acetate buffer at pH 8.0 and enediyne

shown in acetonitrile. Agarose (0.7%) gel electrophoresis using ethidium bromide stain-Lane 1: DNA; Lane 2: DNA + enediyne (50

μmol).



3.10.2. Biological Activity of Basak’s Azo Bispropargyl Sulfones

The cyclic Z-azo-bispropargyl sulfone showed higher DNA-cleavage efficiency ( 2.5 times)

than the corresponding E isomer presumably via the Garratt−Braverman pathway (Figure 7).

O

O

N NOO

NN

h 350 nm

Ktrans Et3N

SSO

C

O

N N

O

C

O

NN

SS

O O

OO

O O

OO

C

O

C

O

N N

C

O

C

O

NN

SS

OO

O O

Et3N

X

DNA Cleavage viaGarratt-Braverman

DNA Cleavage

Interaction of Supercoiled DNA with Azo Bis-Propargyl Sulphones

Form I

Form II

1 2 3

With Supercoiled Plasmid DNA pBlueScript SK+

Kcis

A B

C D

E F

Figure 7. DNA-cleavage experiment of compounds A and B in TAE buffer (pH 8.5) with 7 μL DNA of 0.4 μm/bp concentration. Lane 1:

control DNA + CH3CN (10 μL); Lane 2: DNA + Z-sulfone B (0.02 mM) in 5 μL CH3CN; Lane 3: DNA + E-sulfone A (0.02 mM) in 5 μL

CH3CN.



3.10.3. Biological Activity of Basak’s Unsaturated Azo Bispropargyl Sulfones

A similar increase in DNA-cleavage activity of a related sulfone with an extra conjugation

was also observed for the Z isomer as compared to the E analogue (Figure 8).

O

O

N N

S

O

O

O

N N

O

S

OO

h 350 nm

DNA Cleavage

Interaction of Supercoiled DNA with Unsaturated Azo Bis-Propargyl Sulphones

Trans azo- Cis azo-

Form I

Form II

1 2 3

With Supercoiled Plasmid DNA

Figure 8. DNA-cleavage experiment of trans- and cis- unsaturated azo-bis-propargyl sulphone in TAE buffer (pH 8.5), and 7 μL of

plasmid DNA (0.4 μm/bp). Lane 1: control DNA + CH3CN (10 μL); Lane 2: DNA + E-sulfone (0.02 mM) in 5 μL CH3CN; Lane 3: DNA +

Z-sulfone (0.02 mM) in 5 μL CH3CN.



3.10.4. Biological Activity of Popik’s In-situ Generated Enediyne

An evaluation of the photogenerated nine-membered ring enediyne B, towards nuclease

activity was carried out using supercoiled plasmid DNA cleavage assays. Three forms of this

DNA: native (RF I), circular relaxed (RF II, produced by single-strand cleavage), and linear (RF

III, formed by scission of both strand in close proximity) are readily separated by the agarose gel

electrophoresis. Aqueous solution (5 mM) of cyclopropenone A was irradiated with low-pressure

mercury lamp to produce reactive enediyne B. A solution of φX174 supercoiled circular DNA

(10 ng/μL) in TE buffer was added to photolysate and incubated for 16 h at 25 °C.

O

h (300 nm)

-CO

Photo Bergman

Cyclization

Photochemical in Situ Generation of Enediyne and the DNA cleavage

OH OH OH

DNA Cleavage

1 2 3 4 5

Form I

Form II

Form III

DNAA B C

Figure 9. Cleavage of φX174 plasmid DNA by the photogenerated enediyne B. Lane 1: DNA alone; Lane 2 and 4: DNA + cyclopropenone

precursors A in Dark; Lane 3 and 5: DNA + irradiated solution of A. (Pandithavidana, D. R.; Poloukhtine, A.; Popik, V. V. J. Am. Chem.

Soc. 2009, 131, 351.).

Incubation of the DNA with cyclopropenone precursor A (lanes 2 and 4, Figure 9) does not

induce any detectable DNA cleavage. The photogenerated enediyne B, on the other hand,

induces substantial single strand cleavage of φX174 DNA (RF II), while linearized form (RF III)

becomes prominent only at higher (5 mM) concentration of the cleaving agent (lanes 3 and 5,

Figure 9). Integration of fluorescence of bands on the gel shown in Figure 9, allowed the authors

to evaluate the relative abundance of the native, circular, and linearized forms of φX174 DNA.

Thus, incubation of the DNA with the 1 mM of enediyne B produces 45% of single strand

cleavage (RF II) and less than 5% of the double strand cleavage (RF III, lane 3, Figure 9). At 5

mM concentration of B, DNA-cleavage efficiency increases to 67% and 10%, respectively (lane

5, Figure 9).



3.10.5. Biological Activity of Rashel’s Pyrimidine Based Enediyne

Compounds A and B cleave double-stranded DNA which is shown in Figure 10 Both of

them showed this property when incubating with ΦX174 dsDNA for 70 h at 40 °C. The ketone B

showed significant DNA nicking (Form II) at concentrations as low as 40 μM and nearly

complete nicking at 4 mM. The alcohol A showed almost no reactivity at 40 μM but was able to

nick DNA at 4 mM. While no double-strand (ds) cleavage (Form III) was observed at the lower

concentrations, both A and B show slight ds cleavage at 4 mM.

N

N

N

N

Photo BC

Thermal BC

DNA Cleavage Activity of Pyrimidine Based Enediyne

OCH3

H3CO

OCH3

H3CO

O

OHOH

O

DNA

DNA Cleavage

1 2 3 4 5 6 7 8

Form II

Form III

Form I

A

B

Figure 10. Supercoiled DNA interaction ΦX174. DNA was incubated for 70 h at 40 °C with compounds A and B in buffer (TE, pH 7.6)

and analyzed by electrophoresis (1% agarose gel, ethinium bromide stain). Lanes 1−3: A (4000, 400, and 40 μM); Lane 4: DNA control;

Lane 5: DNA control + restriction enzyme, Dra I; Lanes 6−8: B (4000, 400, and 40 μM).

Photochemical DNA cleavage ability was also demonstrated for A and B (hν, 40 °C, 3 h). In

this case, compound A showed superior DNA cleavage ability. At 40 μM, compound B showed

significant DNA single-strand cleavage. But, compound B showed no discernible activity. At

higher concentrations (4000 μM), both compounds showed signs of double-strand scission, again

with A giving the more complete reaction.

Compounds A and B both showed anticancer activity against human leukemic lymphoblasts

of the CCRF-CEM cell line (log-phase cultures) (IC50 values of ≈1.25 μM).



3.11. Enediyne as a Scaffold for Peptidomimetics

3.11.1. Introduction: Z-Enediyne as Scaffold for Peptidomimetics Design

During the past decade, the construction and investigation of expanded acetylenic -

chromophores has become a central area of chemical research. It has been fueled by the

availability of new synthetic methods, in particular Pd(0)-catalyzed cross-coupling reactions, the

discovery of the antitumor activity of a series of natural compounds possessing reactive Z-

-chromophores, and the need for new nanoscale molecules and polymeric materials

that exhibit unusual electronic and optical functions and properties. The enediyne antitumor

antibiotics are appreciated for their novel molecular architecture, their remarkable biological

activity and their fascinating mode of action and many have spawned considerable interest as

anticancer agents in the pharmaceutical industry. Of equal importance to these astonishing

properties, the enediynes also offer a distinct opportunity to study the unparalleled biosyntheses

of their unique molecular scaffolds and what promises to be unprecedented modes of self-

resistance to highly reactive natural products. Elucidation of these aspects should unveil novel

mechanistic enzymology, and may provide access to the rational biosynthetic modification of

enediyne structure for new drug leads, the construction of enediyne overproducing strains and

eventually lead to an enediyne combinatorial biosynthesis program.



3.11.2. Enediynyl Amino Acid AS β-Sheet Nucleator

As already mentioned, Enediynes have drawn unprecedented interest amongst the scientific

community because of their cytotoxic activity and possible use as anticancer drug. All studies so

far have been concentrated on their synthesis and evaluation of chemical as well as biological

activity. The special structural feature of Z-enediynes is the type of reverse-turn associated with

the two acetylenic arms. One can consider making enediynyl amino acid containing peptides,

which may be forced to adopt typical conformational motifs.

Figure 11. The solution conformation of the enediynyl peptides

This structural motif, for the first time in enediyne chemistry, was used, in our laboratory, as

a possible scafolld for peptide secondary structure mimetic. Specially, we were interested in

designing and synthesis of the -sheet mimetic by incorporating the enediynyl amino acid

into a peptide chain. We thought that this enediynyl motif can act as nucleator and thus may

induce in adopting the sheet which constitutes a well-studied subset of the reverse turn and is

a common feature in biologically active peptides and globular proteins. The sheet capping turns

are widely believed to act as a molecular recognition site for many biological processes.

Bag and Basak et al. have incorporated the enediynyl amino acid A into peptides B-D

(Figure 11) and then found out the conformational preferences by NMR and CD-measurements.

Circular Dichroism (CD) spectra of the fully protected peptides and the generally higher

(T) values for the chemical shifts of and N-H’s reveales that the peptides adapt a

significant proportion of -sheet like conformation. However, the results also indicated the

presence of other conformations as well, specially the -NH as being intramolecularly H-

bonded. The variable temperature NMR experiments indicate that the conformation resembling

-sheet capping type motif is more predominant (Figure 11).



3.11.3. Enediynyl Pentapeptide AS β-Turn Peptidomimetics

3.11.3.1. Introduction

An enediynyl pentapeptide A (Figure 12) in which a novel enediynyl -amino acid acts as

fluorophoric reporter was designed and synthesized by Basak et al. The reason behind the design

was two fold: a) to use the Z-enediyne moiety as a nucleator for turn peptidomimetic and b) to

exploit and elaborate further, the intrinsic florophoric properties of this moiety simultaneaously.

This was the first report of application of the intrinsic fluorophoric property of Z-enediyne in

monitoring binding process of metal ions and colloidal gold nano-particles.

Figure 12. The enediynyl pentapeptide.



3.11.3.2. Synthesis of the Target Enediynyl Pentapeptide

A proline residue was chosen because prolines and its derivatives are extremely important in

synthetic as well as biological chemistry and its known ability to induce turns. The chiral

auxiliary oxazaborolidine and the many of the protease inhibitors required for management of

AIDS are modeled after proline. The various structural motifs like -turn or -bend are often

found where a polypeptide chain often abruptly reverses its direction as were revealed from the

discussions above. Proline residues are frequently found in -turns because the peptide bonds

involving the imino nitrogen of proline readily assume the cis-configuration, a form that is

particularly amenable to a tight turn. One extremely important enzyme that cleaves the peptide

bond involving proline [actually Tyr (Phe)-Proline] is HIV-protease, which cleaves the long

polypeptide chain into the functional oligomers. Various proline containing peptidomimetics

have been made that are promising candidates as anti AIDS-drugs. Another class of drugs where

proline plays an important role is in the case of management of hypertension (ACE inhibitors).

Proline or modified Proline containing natural products with interesting biological activities are

also well known. These include Kainic acid and (-) Detoxinine. The synthesis of the enediynyl

pentapeptide in the protected form is given below (Scheme 105).

Scheme 105. Synthesis of Novel enediynyl pentapeptide A.



3.11.3.3. Evidence of β-Turn Structural Motif

The pentapetide A exists predominantly in -turn structural motif as revealed by variable

temperature. NMR and CD spectroscopy. In the presence of transition metal ions and gold

nanoparticles, the fluorescence intensity of the peptide got enhanced with remarkable quantum

yield with the Z-enediynyl -amino acid acting as a fluorophoric reporter. The interesting

photophysical behaviors with alkali and alkaline earth metal ions are also reported.

Figure 13. The enediynyl pentapeptide and its CD spectra.

The secondary structure of peptide A was estimated by recording its CD spectrum in

methanol, which showed a strong maximum at ∼198 nm followed by several broad minima at

∼205, 212, and 222 nm, indicating that the peptide predominantly adopts a β-turn like structure

(Figure 13) at least in the solvent used for the study. The peptide secondary structure estimation

using CD estima program shows a 60% turn like structure, the existence of which implies the

possible presence of intramolecular H-bond between the peptide strands on the two arms of the

enediyne framework. This could be assessed by determining the variation of chemical shifts of

the various NHs with temperature in DMSO-d6 in which all the four NHs exhibited different

chemical shifts. Interestingly, the turn like structure is more or less maintained in the presence of

Ca2+ ions.

Of the four amide NH’s, one alanine NH and the NH belonging to the enediynyl amino acid

exhibited Δδ/ΔT values that are within the Kessler limit of −3 ppb/K, indicating strong

intramolecular H-bonding and supported the predominant turn like structure of the peptide. The

appearance of a crosspeak for the hydrogens attached to C-2 and C-11 in NOESY spectrum also

provided further evidence for the turn like conformation of the two peptide arms of the enediyne

backbone. The H-bonded conformation was also supported by the semi-empirical AM1 geometry

optimization.



3.11.3.4. β-Turn Mimitic Enediyne as A Fluorescence Chemosensor

Ever since the discovery of naturally occurring enediynes, all efforts have been drawn

towards the finding analogous rationally designed molecules having excellent antitumor

activities with less toxicity as well as evaluation of the parameters controlling the Bergman

cyclization (BC). The photophysical properties of this class of molecules have so far been

restricted to the study of BC under photoirradiation. Because of the presence of special structural

features, in which a double bond is flanked between two acetylenes in a cis fashion, it occurred

to us that, the enediynes might serve as a probe for fluorescent spectroscopy, for example to

study their binding with DNA or sensing of metal ions. As a matter of fact, the photo physical

properties of this class of molecules have not been explored to a large extent except for the

photo-Bergman Cyclization.

Enediynes with metal coordinated architectures might show interesting photophysical

properties, esspecially upon complexation a change in the absorption / fluorescence behaviour of

the enediynes might occur. This change might allow one to use the enediyne scaffold in

fluorescence based sensing of ions that is of great interest as sensors in biomedical research and

in molecular information processing.

3.11.3.5. Fluorescence Photophysical Behavior in Bare and Complexed form of the

Enediynyl Pentapeptide

The peptide itself in TFE showed emission at max 380 nm when excited at 320 nm with a

large Stoke shift (60 nm), a characteristic emission of enediyne moiety. A significant changes in

fluorescence intensity was observed upon addition of metal ions. Transition metal ions all caused

enhancement of fluorescence intensity with the extent of enhancement depending upon the

nature of the metal ions added. Although the precise nature of interactions between the peptide

and the metal ions are not known, it is possible that the intrinsic turn like structure allows the

accommodation of the metal ion for binding. Generally, transition metal ions are strong

quenchers of fluorescence. However, in this case the enhancement of fluorescence demonstrated

the ability of a enediynyl peptide to show fluorescence enhancement upon complexation to

various transition metal ions similar to cryptand-based fluorophores. One possible explanation

behind the enhancement of fluorescence may be that the distance and orientation of the metal ion

entering the cavity, in relation to the -system is such that the spin-orbit coupling which

facilitates the S-T intersystem crossing is minimum. In other words the metal ion-fluorophore

communication is much less than the metal-receptor interaction.



A good correlation between fluorescence quantum yield and their complex formation

constants was observed (Table 3). The order of K and is Co(II) Zn(II) > Cu(II)> Ni(II).

Fluorescence titration experiment with Cu2+ ion showed that the peptide binds more than one

Cu2+ ion. For the other metal ions, a linear plot revealed 1:1 complex formation.

Table 3. Summary of complex formation constants and fluorescence Quantum Yields in TFE at

298K

The pentapeptide also showed an enhancement of fluorescence intensity upon complexation

with Au(0)-nano particle in dry THF solvent. nano particle was increased. The gold

nanoparticles are nonfluorescent and enediyne in THF is moderately fluorescent with an

emission maximum at 372 nm. The peptide bound to gold nanoparticles exhibit strong emission

bands at 375 nm and the intensity increases as the concentration of nano particles increases. The

red-shift in the emission peaks parallels the shift in absorption bands. These new electronic

transitions of the enediyne chromophore become allowed as the N-of amide functional groups

binds strongly to the gold particle. No such spectral shifts or enhanced emission could be seen

when we added a THF solution of enediynyl peptide containing tetraoctyl ammonium bromide

and treated with NaBH4. The fluorescence quantum yield of surface-bound peptide is as high as

0.32 at a gold concentration of 30 x 10-5 mM. On the basis of the absorption, steady-state

emission, it is concluded that surface binding has a significant effect on the fluorescence yield,

but it has no observable effect on the intersystem crossing efficiency and thus the enhancement

of fluorescence was observed (Figure 14).

Figure 14. Probable mode of fluorescence enhancement by gold nano particle



The observed increase in the fluorescence yield reflects the suppression of the nonradiative

decay processes upon adsorption of peptide on to the surface of gold nanoparticles. The

photoinduced electron transfer between the lone pair of N-of amide functional groups and the

enediyne moiety competes with the radiative and nonradiative decay of the singlet excited state.

Upon binding of the N-lone pairs to the gold surface, the electron-donating ability of the N is

decreased and this in turn suppresses the electron transfer from its lone pair to the enediyne

moiety (Figure 14). A similar chelation-enhanced fluorescence has been reported earlier by

Czarnick and co-workers and de Silva and co-workers. By binding metal cations to amine

functional groups of probes (e.g., anthracene), they were able to demonstrate the suppression of

intramolecular quenching and thereby enhancement of fluorescence was observed.

3.11.4. Cyclic Enediyne Amino Acids as β-Turn Peptidomimetic

3.11.4.1. Introduction

Looking at constrained peptidomimetics and their structure-function relationships, it is

reasonable to think that incorporation of an enediyne framework into a cyclic peptide scaffold

might offer useful structures with defined and characteristic secondary structures such as the β-

turn mimetics. Moreover, cyclic enediynyl amino acids may have potential affinity for biological

targets such as receptors or transcriptional regulators thereby promoting nicking and degradation.

Such interactions could also provide information as to the location of ligand binding by

inspection of where the target is cleaved. With this idea in mind, Allen B. Reitz et al. have

investigated the incorporation of benzofused enediynes into 10- and 12-membered cyclic α-

amino acids 1 and dipeptide 2, respectively, and examined their reactivity and conformational

preferences (Figure 15).

Figure 15. Structures of cyclic enediynyl amino acids.



3.11.4.2. Synthesis of 10-Membered Cyclic Enediynyl Amino Acids

The synthesis of 10-membered cyclic enediynyl amino acid 1 was started with Sonogashira

coupling of propargyl glycine (2) to 1,2-diiodobenzene (3) in DME solvent using ammonia as

base. The produced alkyne 4 was coupled in a second Sonogashira reaction with propargyl

alcohol to form 1,2-diynylbenzene 6 in 94% yield (Scheme 106). Intramolecular Mitsunobu

reaction of 6 at 0 °C afforded the final enediyne 1 in 90% yield. Compound 4 and 1,4-

cyclohexadiene (100 mol equiv) were subjected to microwave irradiation in DMF when the

cyclized product 7 was produced in 84% yield after 10 min at 120 °C (Scheme 106 ).

Scheme 106. Synthesis of 10-membered cyclic enediyne amino acid 1.

The cyclic 10-membered ring enediynyl amino acid 1 is stable for several months at room

temperature in the solid state. The single-crystal X-ray structure analysis revealed that the tosyl

group adopts an endo-orientation placing it above the enediyne core which was also observed in

solution as was revealed from a NOESY NMR experiment in CDCl3.

The amino acid 1 was also found to cleave double-stranded DNA when incubated with

supercoiled DNA at 37 °C for a period of 24 h at concentrations 10 μM and higher, with many

fragments being observed at the highest concentrations.



3.11.4.3. Synthesis of 12-Membered Cyclic Enediynyl Amino Acids/Peptide

The synthesis of dipeptide 2 is depicted below in Scheme 107.

Scheme 107. Synthesis of 12-membered ring enediynyl peptide 2.

The cyclic enediynyl dipeptide 2 did not undergo the Bergman cyclization possibly because

the terminal alkynes are too far apart (3.86 Å contrary to the distance required for spontaneous

Bergman cyclization of 2.9–3.3 Å.) (Scheme 107).

Upon irradiation with light, regioselective reduction of peptide 2 provided Z-olefin 13 instead

of expected product 12.

In the photoreduction process generation of radicals were proposed. Thus, it was observed

that the enediynyl peptide 2 lead to non-specific protein degradation when incubated with bovine

serum albumin (BSA) and irradiated the mixture at ambient temperature for a period of 1 h (at

1.0 μM).



3.11.4.4. Peptide β-Turn Conformation Adopted by 12-Membered Cyclic Enediynyl Peptide 2

Single crystal X-ray structure analysis of cyclic enediynyl dipeptide 2 revealed a reversed

turn structure with hydrogen bonding between the carbonyl on the benzyloxycarbonyl protecting

(-Cbz) group and -NHs on both the N-methyl amide terminus and the bridging internal amide

(Figure 16). A β-turn will fall into a particular defined class if three of the four backbone

torsional angles do not deviate more than 30 °C and the other not more than 45 °C from ideal

values. Evaluation of the torsional angles of 2 indicated that it adopts a Type II β-turn

conformation in the solid state. The Type II β-turn comprises of about 13% of β-turns among

structures in the Protein Data Bank.

Figure 16. Structural adoption (Similar to its X-ray structure of enediyne 2), Various torsional parameters revealed a Type II β-turn

conformation in the solid state possessed by enediyne 2.

Dihedral Angles Ideal Type II b-Turn Enediyne 2

i + 1 -60 o -50.4o

i + 1 120 o 132.6o

i + 2 80 o 58o

i + 2 0 o 29o



3.12. Enediyne as Peptide Cleaving Agent

3.12.1. Introduction: Self-Decomposition of C-1027 Chromophore

Some naturally occurring enediynes were found to have proteolytic activity. As for example

both the naturally occurring kedarcidin and the related enediyne neocarzinostatin (NCS) are

chromoproteins and capable of causing damage to histones its enediyne warhead. Esperamicin

also has proteolytic and antitumour activity via the involvement of its enediyne moiety. It is

believed that the diradicals generated from the reactive enediyne core via Bergman

cycloaromatization abstract hydrogen from proteins producing a C-centered radical intermediate.

The generated C-centered radical intermediate, in aerobic condition, react with molecular

oxygen. The resulting peroxy radical ultimately is responsible to cleave the peptide.

Alternatively, the peptide radical may cross-link or form an adduct with another radical source.

The apoprotein class of enediyne antibiotics, such as neocarzinostatin, kedarcidin, and

maduropeptin, C-1027 are composed of a highly reactive enediyne chromophore in complex

with an apoprotein. The apoprotein (10489 Da) is a single polypeptide chain of 110 amino acid

residues cross-linked by two disulfide bonds. The enediyne chrophores are bound noncovalently

in a cleft of the apoprotein via hydrophobic interaction force. Studies revealed that the

apoprotein stabilizes the radical-generating enediyne chromophore in C-1027 by tight binding.

Though there involved tight binding, yet the chromophore is released to react upon reaching its

target DNA in the cell nucleus. However, the ways in which apoprotein modulates and deliver

the highly reactive enediyne chromophore in biological target was unclear.

Considering the fact that the C-1027 constantly generates the benzene diradical even when

bound to the apoprotein, hydrogen abstraction from the apoprotein by the diradical then is

expected to lead to its facile decomposition before reaching its biological target. However the

studies showed that the hydrogen abstraction is geometry unfavorable. Therefore, the apoprotein

kinetically stabilizes the enediyne moiety by positioning the diradical with low accessibility to

hydrogen of apoprotein. Thus, self-decomposition of the chromophore−protein complex is

halted. Thus, the apoprotein appears to function both as a stabilizer and as an effective carrier/

delivery system. Although, the apoprotein stabilizes the enediyne core from BC to occure, C-

1027 is known to undergo aging i.e. the apoprotein is unable to completely inhibit the radical to

abstract proton from its backbone leading to decomposition of apoprotein. Several studies thus

showed that suppressing the self-decomposition pathway provides a basis for enhanced

properties for C-1027.

The self-decomposition pathway of C-1027 was studied in detail. It is now well accepted that

the p-benzyne B is in equilibrium with chromophore A and abstracts the hydrogen of Gly96 to

generate the Gly radical C in the first step. Then, the Gly96 radical (C) produces the peroxy

radical D upon reacting with oxygen. This is that peroxy radical which is responsible for peptide

cleavage via dioxetane intermediate E. The whole process is shown below in Scheme 108.



Scheme 108. The mechanism of self-decomposition pathway of chromoprotein of C-1027. Mass spectroscopy data of the peptide

fragments.



3.12.2. Mechanism of Peptide Cleavage by Enediyne

Figure 17. Mechanism of peptide cleavage by enediyne and the generation and fate of C-centered peptide radicals.

3.12.3. Basak’s Design Strategy of Peptide Cleaving Enediyne

Inspired by the self-decomposition pathway of C-1027 several research groups come forward

for the synthesis of peptide/protein cleaving enediynes. Basak et al. synthesized a novel enediyne

–peptide hybrid with peptide cleavage activity at tailor-made positions. In their design, they used

a molecular scaffold where the basic template contains two long parallel chains in the cisoid

form. One of these is a polypeptide chain while the other carries the enediyne frame connected to

the template via a suitable linker (Figure 18). The cisoid conformation of these two chains is

essential to bring the enediyne moiety closer to the peptide chain. In order to achieve such an

arrangement, they selected linkers that can participate in hydrogen bonding with the peptide

chain. The purpose is to cut the peptide chain selectively via the aid of the 1,4-diyl radicals.

Figure 18. Design strategy of peptide cleaving enediyne.

HN

O R H

OHN

O R

O

HN

O R

O

O O

HN

O R

O

O O

HN

O

R

O

O

HO

HN

O R

O

O

R

O

O

NH2

O

Enediyne

Protein Cleavage by Hirama's Pathway

Protein Cleavage by Jones Pathway

+

+

Aerobic3O2

3O2

Cleavageof Peptide by enediyne: Generation and Fate of C-Centered Peptide Radicals



3.12.3.1. Example of Basak’s Peptide Cleaving Enediynes

Based upon consideration of H-bonded enediyne containing scaffolds Basak et al. have

designed and synthesized a novel class of peptide–enediyne conjugate A and B (Figure 19) and

demonstrated their selective cleavage ability of the peptide moiety maintaining selectivity in the

degradation pathway by mass spectrometric studies.

Figure 19. Examples of enediyne–peptide hybrids for peptide cleavage.

NH

HN

NH

HN

ON

O

O

O

O

O

NH

NHBoc

O

O

NH

HN

NH

HN

ON

O

O

O

O

O

NH

NHBoc

O

OPh

O

O

N N

O

O

HN

NH

HN

OCH2Ph

O

NH

ON

O

O

OPh

O

O

O

N N

O

O

HN

NH

HN

OCH2Ph

O

NH

ON

O

O

OPh

O

A B

C D

Enediyne–Peptide Hybrid with Peptide Cleavage Activity



3.12.3.2. Synthesis of a Representative Peptide Cleaving Enediynes A and B

Scheme 109. Synthesis of a peptide cleaving enediynes A and B.

I

I

NHBoc

OH

NHBoc

OMs

NHBoc

N3

NHBoc

NH2

BrN

O

ON

O

OCHPh2

O

O

ON

O

OH

O

O

NHBoc

NH

ON

O

O

O

NH2.TFA

NH

ON

O

O

O

NH

HN

NH

HN

ON

O

O

O

O

O

NH

NHBoc

O

O

A

HONH

HN

O

O

NH

NHBoc

O

OO

O

O OH

1 2 3 4 5

6 8 9

7

10 11

12

Reagents and conditions: (a) MsCl, Et3N, DCM, 0 oC, 15 min, 96%; (b) NaN3, DMF, rt. 7 h, 67%; (c) PPh3, THF, H2O, rt, 10 h,

70%; (d) 7, Cs2CO3, DMF, rt, 10 h, 59%; (e) TFA, anisole, DCM,0 oC to rt, 1 h; (f) 5, EDC.HCl, DMAP, DCM, rt, 10 h, 50%; (g) TFA,

DCM, 0 oC to rt, 1.5 h; (h) 12, EDC.HCl, DMAP, DCM, 0 oC to rt, 10 h, 41%; (i) 13, EDC.HCl, DMAP, DCM, 0 oC to rt, 12 h, 39%.

(a) (b) (c)

(d)

(f)

(e)

(g)

(h)NH

HN

NH

HN

ON

O

O

O

O

O

NH

NHBoc

O

OPh

(i)

HONH

HN

O

O

NH

NHBoc

O

OPh

13

B



3.12.3.3. Study of Peptide Cleavage by Basak’s Designed Enediynes

Scheme 110. Mass fragmentation pattern for α-H-abstraction at C-3 by enediyne-peptide hybrid A.



Scheme 111. Possible fragmentation (that was not observed) for α-H-abstraction at C-2 by enediyne-peptide hybrid A.

Scheme 112. Abstraction of α-H from third glycine unit of the pentapeptide chain of by enediyne-peptide hybrid B.



3.12.4. Protein Degradation by Jones’ Photoactivated Enediynes (Jones, G. B.

et al. J. Org. Chem. 2005, 70, 9789.)

3.12.4.1. Design Strategy

In their design Jones et al. considered the following criteria:

(a) the atom-transfer chemistry of enediyne-derived diyls with peptide under both thermal

and photochemical activation conditions

(b) the impact that hydrophilic functionality on abstraction efficiency

(c) considerable flexibility would need to be built into the system for application to protein

targets.

(d) Their design involves the derivatization of the alkyne termini of the enediyne with

appropriate protein recognition functions

(e) The derivatization also takes care the tuning of photoactivity of the Bergman

cycloaromatization.

(f) Most photoactivated enediynes studied have the vinyl moiety embedded in an arene. In

their design they concentrated on the photochemical activation to as a function of ring-

strain and electronic effects. (Figure 20).

Figure 20. Structures of Jones’ photoactivable enediynes.



3.12.4.2. Synthesis of Jones’ photoactivable enediynes

Scheme 113. Synthesis and photo-Bergman cyclization of enediyne-peptide hybrids.

TIPS

O

O

PhPh

H

Ph

OH

O

Ph

HN

O

Ph

COOH

HN

O

Ph

COOH

I

OH

O

HN

O

R

COOH

Ph

QI R = Ph,QII R = COOH

QIII R = CH2COOH

P

1 2 4

3

6

(a) (b)

(c) (d)

Reagents and Conditions: (a) (i) NaBH4, EtOH, (ii) PPh3, Br2, DCM, (iii) LiHMDS, HMPA, THF, (iv) TBAF, THF; (b) (i) 3, Pd(PPh3)4, CuI, Et3N, (ii) LiOH, THF, H2O; (c) (i) 5, EDCI, DMF, (ii) LiOH, THF, H2O; (d) h, 450W, 3h, i-PrOH

ClH.H2N

Ph

COOMe

5



3.12.4.3. Applications (Protein Degradation) of Jones’ photoactivable enediynes

A. Interaction with Bovine Serum Albumin (BSA)

Photochemical activation (450 W, 3 h) of the enediyne-peptide hybrid P (Figure 20)

gave arene product 6 in appreciable yield, together with unreacted starting material. They

have also studied the photochemically induced modification of BSA and lysozyme by

photolyzing compound P in aqueous buffer and using 6 and enediynes 4 as controls. In

the case of 66 kD protein BSA, subsequent analysis (SDS−PAGE) showed some

evidence of enediyne-induced degradation with one principal fragment ( 40 kDa) visible

(<5% relative to parent) when a 5:1 ratio of enediyne P/BSA was employed.

Encouraged by this initial finding, they have also synthesized enedynes QI-QIII and

studied their protein degradation ability. Compound QII and QIII showed enhanced

affinity for BSA when photolysis was conducted. Significant cleavage into two principal

fragments (31 and 35 kDa) was observed.

In the case of the 14 kDa protein lysozyme, dimerization to form a species with m/z 28.6

kDa was observed when a mixture of enediyne P and lysozyme were irradiated that

suggests the possibility of intermolecular diyl-mediated cross-coupling.

B. Interaction with Histone H1

Naturally occurring chromoproteins kedarcidin, C-1027, and neocarzinostatin, were able

to show proteolytic activity.

In the case of kedarcidin, studies suggested that the highly acidic apoprotein was capable

of inducing selective degradation of histone H1.

Following these findings, Jones et al. have prepared an acidic enediyne−peptide

conjugate to determine if enediyne-mediated histone degradation could be achieved.

To investigate this, enediyne containing the triaspartic acid derivative R was also

synthesized (R, Figure 20) studied for their interaction with Histone H1. Binding of this

agent to a variety of histones revealed that affinity, as expected, was greater for H1

versus others. Irradiation of triaspartate-enediyne hybrid in the presence of histone H1

(21.5 kDa) led to degradation of the protein into principal components in the 8−11 kDa

range.

The application of a designed photoactivated enediyne with near micromolar affinity for

a biological target is highly interesting and suggests even greater affinity might be

attainable by coupling the photowarhead to the ligand with a complimentary protein

receptor.



C. Interaction with Human Estrogen Receptor α

Jones et al. have also synthesized enediyne-lipophilic steroidal ligands hybrid molecule

(S, Figure 20) to study the capability of degrading the human estrogen receptor (hER). In

their design, they have utilized estrone wherein the enediyne functionality was introduced

at the 17α position of estrone. The hybrid underwent smooth photocycloaromatization to

give complete conversion to cycloaromatized product within 12 h. Irradiation in the

presence of recombinant hER (66 kDa) resulted in marked degradation to produce two

discrete fragments within 12 h (31 and 35 kDa) .

The ability to induce on demand degradation of a nuclear receptor using a lipophilic

warhead is of significance and underscores the potential use of such entities as molecular

reagents.

4.13. Selected References

1. Basak, A.; Mandal, S.; Bag, S. S. Chem. Rev. 2003, 10, 4077 and references therein.

2. Kar, M.; Basak, A. Chem. Rev. 2007, 107, 2861–2890 and references therein.

3. Basak, A.; Roy, S.; Roy, B.; Basak, A. Curr. Top. Med. Chem. 2008, 8, 487 and

references therein.

4. (a)Basak, A.; Mitra, D.; Kar, M.; Biradha, K. Chem.Commun. 2008, 3067-9. (b)

Mitra, D.; Banerjee, D. R.; Das, A. K.; Basak, A. Bioor. Med. Chem. Lett. 2010, 20,

6831. (c) Mitra, D.; Kar, M.; Pal, R.; Basak, A. Bioor. Med. Chem. Lett. 2007, 1007.

(d) Basak, A.; Roy, S.; Das, S.; Hazra, A.; Ghosh, S.; Jha, S. Chem.Commun. 2007,

622. (e) Roy, S. K.; Basak, A. Chem. Commun. 2006, 1646. (f) Basak, A.; Mandal,

S. Tetrahedron Lett. 2002, 43, 4241. (g) Basak, A.; Rudra, K.; Bag, S. S.; Basak, A.

J. Chem. Soc. Perkin Trans. 1 2002,1805. (h) Basak, A.; Mandal, S.; Das, A. K.;

Bertolosi, V. Med. Chem. Lett. 2002, 12, 873. (i) Basak, A.; Bag, S. S.; Rudra, K.;

Burman, J.; Dutta, S. Chem. Lett. 2002, 710. (j) Basak, A.; Rudra, K.; Bag, S. S.; J.

Chem. Soc. Perkin Trans 2002, 1, 1805. (k) Pal, R.; Basak, A. Chem. Commun.2006,

2992.

5. (a) Biggins, J. B.; Onwueme, K. C.; Thorson, J. S. Science 2003, 301, 1537. (b)

Singh, S.; Hager, M. H.; Zhang, C.; Griffith, B. R.; Lee, M. S.; Hallenga, K.;

Markley, J. L.; Thorson, J. S. Acs. Chem. Biol. 2006, 1, 451–460.

6. Hirama, M.; Akiyama, K.; Tanaka, T.; Noda, T.; Iida, K.-i.; Sato, I.; Hanaishi, R.;

Fukuda-Ishisaka, S.; Ishiguro, M.; Otani, T.; Leet, J. E. J. Am. Chem. Soc. 2000,

122, 720-721.

7. Usuki, T.; Inoue, M.; Hirama, M.; Tanaka, T. J. Am. Chem. Soc. 2004, 126, 3022.

8. Roy, S.; Basak, A. Chem. Commun. 2010, 2283.

9. Basak, A.; Bag, S.S.; Bdour, H. M. M. Chem. Commun. 2003, 1, 2614–2615.

10. Fouad, F. S.; Wright, J. M.; Plourde II, G.; Purohit, A. D.; Wyatt, J. K.; El-Shafey,

A.; Hynd, G.; Crasto, C. F. ; Lin, Y.; Jones, G. B. J. Org. Chem. 2005, 70, 9789-

9797.

11. Du,Y.; Creighton, C. J.; Yan, Z.; Gauthier, D. A.; Dahl, J. P.; Zhao, B.; Belkowski,

S. M.; Reitz, A. B. Bioorg. Med. Chem. 2005, 13, 5936–5948

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