nptel phase ii (syllabus template) course title: bio ... · mechanism of action of anticancer drugs...

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

Joint initiative of IITs and IISc – Funded by MHRD of 70

NPTEL Phase – II (Syllabus Template)

Course Title: Bio-Organic Chemistry of Natural Enediyne

Anticancer Antibiotics

Module 1: Introduction to Enediyne Class of Natural Products: History of Discovery of Enediynes;

Isolation of Enediynes; Molecular Structures of Enediynes; Biological Properties and

Mechanisms of Action of Naturally Occurring Enediynes; The Bergman Cyclisation Reaction;

The Myers-Saito Cyclisation Reaction.

1. 1. History of Discovery of Enediynes

1.1.1. Introduction

Deoxyribonucleic Acid (DNA), discovered in cell nuclei in 1868 by Fritz Meischer a Swiss

Physicist, holds all the information of life within biological systems from which the blueprint of

life is built by mother nature similar to the information hidden in a brick that determine the full

blueprint of the entire building. Discovering the modern structure of DNA, in 1953, Watson and

Crick were subsequently awarded the Nobel Prize in 1962. DNA is involved in storage and

transmission of genetic information. Everything about us including our hair/eye color, body type,

talent/intelligence, prone to diseases, and response to a particular medication, etc. is contained

within this incredible creation by the ingenious job of Mother Nature, the DNA. The blueprint or

genetic code is stored in our DNA that replicates at each cell division. Therefore, every cell

contains information about the entire organism. DNA is a double stranded helix made up of

millions of base fragments. A single DNA chain may be up to 12 centimeters in length and

contain 250 million base pairs.



Cell division is essential for an organism to grow. During this cell division process, DNA

makes its own copy. So two daughter cells will have the same genetic information as their

parent. This process of making perfect copy of DNA by DNA polymerase enzyme is known as

DNA replication. In brief, DNA replication occurs as per the scheme 1. Thus, in solution, DNA

unwinds to give single strands, which bond with free bases in solution, to form complimentary

strands, thereby reproducing another DNA molecule. Following are the main steps involved in

DNA replication carried out by DNA polymerase (Scheme 1).

Scheme 1. DNA replication within our cells.

1. Here, the two strands of DNA are separated in solution. The complementary sequences of

each separated strands are recreated by an enzyme, called DNA polymerase.

2. This enzyme makes the complementary strand by finding the correct base through

complementary base pairing, and bonding it onto the original strand.

3. DNA polymerases extend a DNA strand in a 5′ to 3′ direction. Different mechanisms are

used to copy the antiparallel strands of the double helix.

4. Thus, base on the old strand dictates the bases to appear in the new strand, and the cell

ends up with a perfect copy of its DNA.

Despite the enormous size of DNA molecules, the replication sequence is carried out in

relative harmony with errors occurring only about once in every 10-100 billion base pairs.

Although errors do not occur often in a numerical sense; however, any error may interrupt the

genetic code. In such a case, if left unrepaired the error by cell’s repairing machinery an incorrect

transmission of genetic information by DNA in replication of these 250 million may lead to a

mutation. Such mutations are also caused upon exposure to light, radiation, viruses, transposons

and mutagenic chemicals.



Many mutations are harmless and do not affect our blueprint. However, some mutations may

lead to uncontrolled replication. In most DNA sequences, our body knows when replication

should be controlled, but when mutations occur, the body is overridden, and replication does not

stop. The process of cell division is a very tightly controlled process and occurs in the body only

to the extent needed in any particular situation. During the process of cell division if nncontrolled

replication of the mutated DNA occurs, the daughter cell may acquire some genetic mutation that

would alter the cell division control mechanisms of that cell. This altered cell no longer listens to

the control signals for cell division and may continue to divide and multiply. The cells replicate

so rapidly and continuously that they will have a very high error rate in DNA replication. The

population of oncogenic cells is highly varied and some are able to avoid normal tumor necrosis

factors and T cell mediated destruction. This differential survival from a varied population of

replicating cells makes for a rapid evolution, resulting in a tumor with abnormal numbers of

chromosomes, an over expression of telomerase to resist cell death, and a lack of response to

normal growth regulating factors. The cells then continue to replicate without recognition of

normal tissue cell-to-cell boundaries so they become invasive. This uncontrolled cell division

and growth ultimately results in malignant tumors, and cancer. Multiple genetic events are

involved in the development of most malignancies.

In an effort to fight this uncontrolled growth of cells, chemists have been trying to find

molecules to arrest formation of the malignant tumors. These molecules are known as antitumor

antibiotics.

1.2. History of Cancer and Treatment Regimen

(a) Long history of treating cancer, but did not successfully begin until the invention of the

microscope.

(b) Early 20th - surgery and radiation

(c) World Wars began chemical warfare, and thus began chemotherapy - nitrogen mustards

(d) Currently, targeted cancer therapy

1.2.1. Common Treatments of Cancer

(a) Surgery (before 1955)

Direct removal of tumor

(b) Radiotherapy (1955~1965)

Using ionizing radiation to control malignant cells

(c) Chemotherapy (after 1965)

Using chemicals to kill actively dividing cells

(d) Immunotherapy and Gene therapy



1.2.2. Cancer Chemotherapy (After 1965)

Chemotherapy is usually the first choice for the treatment of many cancer types.

(a) Injection - Intrathecal, Intramuscular, Intravenous, Intra-arterial

(b) Orally

(c) Topically

1.2.3. The Classification of Anticancer Drugs and Drug Targets The drugs are classified as below:

(a) According to chemical structure and resource of the drug:

Alkylating Agents,

Antimetabolite,

Antibiotics,

Plant Extracts，

Hormones，

Others

(b) According to biochemistry mechanisms of anticancer action:

Block nucleic acid biosynthesis

Direct influence the structure and function of DNA

Interfere transcription and block RNA synthesis

Interfere protein synthesis and function

Influence hormone homeostasis

Others

(c) According to the cycle or phase specificity of the drug

Cell cycle nonspecific agents (CCNSA): drugs that are active throughout the

cell cycle.

Alkylating Agents

Platinum Compounds

Antibiotics

Cell cycle specific agents (CCSA): drugs that act during a specific phase of

the cell cycle.

S Phase Specific Drug:- Aantimetabolites, Topoisomerase Inhabitors

M Phase Specific Drug:- Vinca Alkaloids, Taxanes

G2 Phase Specific Drug:- Bbleomycin



1.2.4. The Anticancer Drug’s Targets

(a) Enzymes - Antimetabolites

(b) Hormones - Androgens, Oestrogens, Progestins, LHRH agonists, Antioestrogens,

Antiandrogens

(c) Nucleic Acids - Intercalating agents, alkylating agents, chain cutters

(d) Structural proteins

(e) Signaling pathways

1.2.5. Mechanism of Action of Anticancer Drugs

The majority of anticancer drugs affect cell division or DNA synthesis and function. Many of

them also work on interacting with RNA or protein. The DNA interacting drugs can be classified

as alkylating agents, antimetabolites, anticancer antibiotics, mitotic inhibitors and plant alkaloids.

Some chemotherapeutics do not directly interact with DNA, but target molecular abnormality in

certain types of cancer. Furthermore, some drugs are used to modulate cancer cell behavior,

without directly attacking, like in the hormone treatment.

Development of new efficient anticancer drugs needs a detailed knowledge of the mechanism

of drug action at the cellular and molecular levels. Since most anticancer drugs shows their

activity through the interaction with DNA, extensive research efforts in this field have

culminated in useful information about DNA structure, dynamics and DNA-drug interactions.

There are several ways of interaction between DNA/RNA/protein and a drug and they can be

classified as follows

(a) Block nucleic acid (DNA, RNA) biosynthesis: Antimetabolites

Folic Acid Antagonist: inhibit dihydrofolate reductase (methotrexate)

Pyrimidine Antagonist: inhibit thymidylate synthetase (fluorouracil) ; inhibit

DNA polymerase (cytarabine)

Purine Antagonist: inhibit interconversion of purine nucleotide (mercaptopurine)

Ribonucleoside Diphosphate Reductase Antagonist: (hydroxyurea)

(b) Directly destroy DNA and inhibit DNA reproduction

Intercalating agents, alkylating agents, chain cutters-Antibiotics

(c) Interfere transcription and block RNA synthesis

Bind with DNA to block RNA production: doxorubicin



(d) Interfere protein synthesis and function

Antitubulin: vinca alkaloids and taxanes;

Interfere the function of ribosome: harringtonines

Influence amino acid supply: L-asparaginase

Bind tubulin, destroy spindle to produce mitotic arrest.

(e) Influence the Structure and Function of DNA

Alkylating Agent: mechlorethamine, cyclophosphamide and thiotepa

Platinum: cis-platinium

Antibiotic: bleomycin and mitomycin C

Topoismerase inhibitor: camptothecine and podophyllotoxin

(f) Influence hormone homeostasis: These drugs bind to hormone receptors to block

the actions of the sex hormones which results in inhibition of tumor growth.

Estrogens and estrogen antagonistic drug

Androgens and androgen antagonistic drug

Progestogen drug

Glucocorticoid drug

gonadotropin-releasing hormone inhibitor: leuprolide, goserelin

aromatase inhibitor: aminoglutethimide, anastrazole

1.2.6. The Anticancer Antibiotics

Analysis of the number and sources of anticancer and antiinfective agents, indicates that over

60% of the approved drugs and pre-approved candidates developed in the area of these diseases

are of natural origin. Drugs of natural origin have been classified as original natural products,

products derived semisynthetically from natural products, or synthetic products based on natural

product models. Anticancer antibiotics are drugs derived from microbial sources. Data showed

that of the new approved drugs reported between 1983 and 1994 antibacterial drugs of natural

origin is 78% while 61% of the 31 anticancer drugs are naturally-derived or are modeled on a

parent natural product. Of the 87 approved anticancer drugs, 62% are of natural origin or are

modeled on natural parent products-fifteen are the original natural product, and 25 are

semisynthetic derivatives including steroids, nucleosides, microbial- and plant-origin, choline

and peptide derivative. Approved rest 14 anticancer drugs may be classified natural product

models. Of the 299 pre-approved anticancer drug candidates which were in preclinical or clinical

development for the period 1989-1995, 50 are the original natural product of marine-,

microbial-, and plant source and 48 are semisynthetic derivatives of nucleosides, aniimal-,

microbial-, and plant origin, while 30 are based on natural product models and 88 are biologics.

Among them enediyne class of natural products are newer anticancer antibiotics.

Over the past ten years there has been a rapid escalation in the discovery of molecular targets

that may be applied to the discovery of novel tools for the diagnosis, prevention, and treatment of

human diseases. With the sequencing of the human genome, there has been an explosion in the



knowledge of combinatorial synthesis and screening to select the active drug candidates

including anticancer agents.Therefore now a day many new drugs are coming up which are

already approved or in clinical or pre-clinical trial. Some important examples of anticancer drugs

are given below.



(a) Anthracyclines

First anthracycline antibiotics were isolated from Streptomyces peucetius in 1958

Mechanisms of action: Disrupt DNA

Intercalate into the base pairs in DNA minor grooves

Inhibits topoiosomerase II enzyme, preventing the relaxing of supercoiled

DNA, thus blocking DNA transcription and replication

Cause free radical damage of ribose in the DNA

Some of the most effective cancer drugs available

Adramycin is used to treat acute leukemias, lymphoma, and a number of

solid tumors

Common Anthracyclines (Figure 1)

Daunorubicin (Cerubidine, DaunoXome)

Doxorubicin (Adriamycin, Rubex, Doxil)

Epirubicin (Ellence, Pharmorubicin)

Idarubicin (Idamycin)

Daunomycin (DNR) for acute lymphocytic and myeloid leukenmia

Doxorubicin (DOX) for chemotherapy for solid tumors including breast cancer,

soft tissue sarcomes, and aggressive lymphomas

Daunorubicin was isolated from Streptomyces coeruleorubidus and S. peucetius.

This significant discovery was made independently in France and Italy in 1963.

The most important member of the anthracycline, doxorubicin (adriamycin) was

isolated from S. peucetius var. caesius in 1969 in Italy.

Figure 1. Chemical structures of anthracyclin class of anticancer drugs.



(b) Dactinomycin (Cosmegen)

Actinomycin D intercalates DNA and thereby prevents DNA transcription

and messenger RNA synthesis.

The drug is given intravenously, and its clinical use is limited to the

treatment of trophoblastic (gestational) tumors and the treatment of

pediatric tumors, such as Wilms’ tumor and Ewing’s sarcoma (Figure 2).

(c) Plicamycin (Mithramycin)

(d) Mitomycin (Mutamycin)

Figure 2. Chemical structures of some anticancer agents.



(e) Platinum Compound

Cisplatin:Mechanism of Action:

Cisplatin binds to guanine in DNA and RNA, and the interaction is

stabilized by hydrogen bonding. The molecular mechanism of

action is unwinding and shortening of the DNA helix (Figure 2).

(c) Bleomycin (Blenoxane)

Natural glycopeptidic antibiotics produced by bacterium Streptomyces verticillus.

Efficacy against tumors

Mainly used in therapy in a combination with radiotherapy or chemotherapy

Bleomycin refers to a family of structurally related compounds. Commonly

administered as Blenoxane, a drug that includes both bleomycin A2 and B2.

Bleomycin was first discovered in 1962 when the Japanese scientist Hamao

Umezawa found anti-cancer activity while screening culture filtrates of S.

verticullus. Umezawa published his discovery in 1966. The drug was launched in

Japan by Nippon Kayaku in 1969. In the US bleomycin gained Food and Drug

Administration (FDA) approval in July 1973 (Figure 3).

Initially marketed by Bristol-Myers Squibb under brand name Blenoxance.

Mechanism:

Induction of DNA strand breaks

Medicate DNA strand scission of single and double strand breaks

dependent on metal ions and oxygen

It is believed that bleomycin chelates metal ions (primarily iron)

producing a pseudoenzyme that reacts with oxygen to produce superoxide

and hydroxide free radicals that cleave DNA.

Figure 3. Chemical structure of Bleomycin.



(g) Enediynes

Enediyne anticancer antibiotics belong to the family of highly potent anticancer

agents that bind to specific DNA sequences and cause double-stranded DNA

lesions (Figure 4).

Their positioning within the minor groove is such that the active form of a drug

can abstract two hydrogen atoms from the sugars of opposite strands.

Figure 4. Chemical structure of Enediyne-Calicheamicin.



Below is a schematic presentation of the A-T and G-C base pairs in the minor groove with

designated sites of action of various anticancer drugs (Figure 5).

Figure 5. Schematic presentation of the A-T and G-C base pairs in the minor groove with designated sites of anticancer drug action.

N

NN

N

HN

N

NH2

O

O

O

O

P

O

OO

O

PO

O

O

O

N

NHN

N NH2

O

1'

2'3'4'

1'2'3'

4'

5'

HO5'

O

O

O

O

P

O

ON

NO

NH2

AT

G C

O

P

O

O

O

O

1'

2 ' 3'

4'

5'

1'

2 ' 3'

4'

5'

Cisplatin

DuocarmycinBLM/PEP

DNR/DOX

Enediyneantibiotics

Duocarmycin



1.3. Microbial Drug Discovery

1.3.1. Introduction

According to a scientific-meets-mythic detective study it is rumored that Alexander the Great

(356 - 323 B.C.) more than 2,000 years ago died because of an extraordinarily toxic bacterium

harbored by the "infernal" Styx River. According to the study, calicheamicin, a secondary

metabolite of Micromonospora echinospora, is what gave the river its toxic reputation. The

researchers believe this mythic poison must be calicheamicin. "This is an extremely toxic, gram-

positive soil bacterium and has only recently come to the attention of modern science. It was

discovered in the 1980s in caliche, crusty deposits of calcium carbonate that form on limestone

and is common in Greece," author Antoinette Hayes, toxicologist at Pfizer Research, told

Discovery News. What ever may be, it is believed that the naturally occurring calicheamicin,

which is extremely cytotoxic, could still be the culprit for the death of Alexander the Great.

As was discussed earlier, natural products continue to be an unparalleled resource for drug

discovery. 63% of all small molecule drugs launched between 1981 and 2006, are natural

products. In antitumor, antimicrobial, and antihypertensive drug discovery, in particular, natural

products have played a large role. Of all drugs derived from natural products macro and

microorganisms have certainly hold a tremendous reservoir of bioactive constituents.

Microorganisms produce some of the most important medicines ever developed. They are the

source of lifesaving treatments for bacterial and fungal infections (e.g., penicillin, erythromycin,

streptomycin, tetracycline, vancomycin, amphotericin), cancer (e.g., daunorubicin, doxorubicin,

mitomycin, bleomycin), transplant rejection (e.g., cyclosporin, FK-506, rapamycin), and high

cholesterol (e.g., statins such as lovastatin and mevastatin). Microbial natural products are

remarkable not only for their potent therapeutic activities, but also for the fact that they

frequently possess the desirable pharmacokinetic properties required for clinical development.

Traditionally, the search for new natural products has started by growing microorganisms in

the laboratory and testing the fermentation broths for bioactivity. Natural fermentation products

have long been studied as attractive targets for drug discovery due to their amazing diverse,

complex chemical structures and biological activities. As such, a number of revolutionary drugs

developed from natural fermentation products have contributed to global human health. To

commercialize a drug derived from natural fermentation products, an effective chemical entity

must be identified and thoroughly researched, and an effective manufacturing process to prepare

a commercial supply must be developed. All the aforementioned drugs were derived from

fermentation broths of microorganism.

Naturally occurring antibiotics are no exception. These are also produced by fermentation

process that is an 8000 years old technique initially developed for beverages and food

production. Beer is one of the world’s oldest beverages which was produced from barley by

fermentation since sixth millennium BC which is the written history of ancient Egypt and

Mesopotamia. Use of Penicillium roqueforti during the past 4000 years for cheese production,

for the past 3000 years soy sauce in Asia and bread in Egypt are the representative examples of

traditional fermentations process.Natural products of industrial importance are produced from

primary or secondary metabolism of living organisms such as plants, animals or microorganisms.



Advancement of separation, and isolation and screening process, millions of natural products

have been discovered today by using fermentation process. Among them, 50–60% are produced

by plants which includes alkaloids, flavonoids, terpenoids, steroids, carbohydrates, etc. and 5%

have a microbial origin. Of all the reported natural products, approximately 20–25% show

biological activity. Of all the active natural products approximately 10% have been obtained

from microbes. Furthermore, from the 22 500 biologically active compounds that have been

obtained so far from microbes, 45% are produced by actinomycetes, 38% by fungi and 17% by

unicellular bacteria. The increasing role of microorganisms in the production of antibiotics and

other drugs for treatment of serious diseases has been dramatic.

The story of fermentation derived drug discovery goes back to 1928, when Alexander

Fleming1 began the microbial drug era. He discovered in a Petri dish seeded with

Staphylococcus aureus that a compound produced by a mold killed the bacteria. The mold that

was identified as Penicillium notatum produced an active agent. This active agent was named

penicillin. Later on penicillin was isolated as a yellow powder and used as a potent antibacterial

compound during World War II. Till the day it is being widely used antibiotics. Since the

discovery of penicillin, other naturally occurring substances, such as chloramphenicol and

streptomycin, were isolated by using Fleming’s method.

1.3.2. Antitumor Drugs From Microbes

Approximately 10 million new cases of cancer were diagnosed in the world in 2000 out of

which 6 million deaths are cancer-related. The tumor types with the highest incidence were lung

(12.3%), breast (10.4%) and colorectal (9.4%). Metabolites of microorganisms are among the

most important of the cancer chemotherapeutic agents and they started to appear around 1940

with the discovery of actinomycin. Just after the discovery of actinomycin, many compounds

with anticancer activities have been isolated from natural sources. More than 60% of the current

compounds with antineoplasic properties were isolated from natural products. Among the

approved products as drug candidates, actinomycin D, anthracyclines (daunorubicin,

doxorubicin, epirubicin, pirirubicin and valrubicin), bleomycin, mitosanes (mitomycin C),

anthracenones (mithramycin, streptozotocin and pentostatin), enediynes (calcheamycin), taxol

and epothilones deserve special interest.

Actinomycin D is the oldest microbial metabolite used in cancer therapy. Its relative,

actinomycin A, was the first antibiotic isolated from actinomycetes. The latter was obtained from

Actinomyces antibioticus which is now called as Streptomyces antibioticus by Waksman and

Woodruff. As it binds DNA at the transcription initiation complex, it prevents elongation by

RNA polymerase.

The anthracyclines are some of the most effective antitumor compounds developed, and are

effective against more types of cancer than any other class of chemotherapy agents.The first

anthracycline discovered was daunorubicin (daunomycin) in 1966, which is produced naturally

by Streptomyces peucetius. Doxorubicin (adriamycin) was developed in 1967. Another

anthracycline is epirubicin. This compound, approved by the FDA in 1999, is favored over

doxorubicin in some chemotherapy regimens as it appears to cause fewer side effects.



Bleomycin is a non-ribosomal glycopeptide microbial metabolite produced as a family of

structurally related compounds by the bacterium Streptomyces verticillus. First reported by

Umezawa et al. in 1966. Bleomycin obtained FDA approval in 1973. When used as an anticancer

agent (inducing DNA strand breaks), the chemotherapeutic forms are primarily bleomycins A2

and B2.

Calicheamicins are highly potent antitumor microbial metabolites of the ENEDIYNE family

produced by Micromonospora echinospora. Their antitumor activity is apparently due to the

cleavage of double- stranded DNA. They are highly toxic. However, the ingenious idea of the

Wyeth Laboratories to make Antibody- Calicheamicin conjugate avoided the side effects of

calicheamicin. Thus, the drug GEMTUZUMAB is effective against acute myelogenous leukemia

(AML) that was approved by the FDA for use in patients over the age of 60 years with relapsed

AML who are not considered candidates for standard chemotherapy.

A successful non-actinomycete molecule is taxol (paclitaxel), which was first isolated from

the Pacific YEW tree, Taxus brevifolia. This is also produced by the endophytic fungi

Taxomyces andreanae and Nodulisporium sylviforme. This compound inhibits rapidly dividing

mammalian cancer cells by promoting tubulin polymerization and interfering with normal

microtubule breakdown during cell division. The drug also inhibits several fungi (Pythium,

Phytophthora and Aphanomyces) by the same mechanism. In 1992, taxol was approved for

refractory ovarian cancer, and today it is used against breast and advanced forms of Kaposi’s

sarcoma. A new formulation is available in which paclitaxel is bound to albumin. Taxol is the

third largest selling drug in 2006 from Bristol Myers-Squibb. Currently, taxol production uses

plant cell fermentation technology.

The epothilones, the name derived from its molecular features containing epoxide, thiazole

and ketone, are macrolides originally isolated from the broth of the soil myxobacterium

Sorangium cellulosum as weak agents against rust fungi. They were identified as

microtubulestabilizing drugs, acting in a similar manner to taxol. However, they are generally 5–

25 times more potent than taxol in inhibiting cell growth in cultures. Five analogs are now under

investigation as anticancer drugs. Their preclinical studies have indicated a broad spectrum of

antitumor activity, including taxol-resistant tumor cells. One epothilone, ixabepilone (16-

membered polyketide macrolactone with a methylthiazole group connected to the macrocycle by

an olefinic bond) was approved in October 2007 by the FDA for use in the treatment of

aggressive metastatic or locally advanced breast cancer no longer responding to currently

available chemotherapies.

Testicular cancer is the most common cancer diagnosis in men between the ages of 15 and 35

years. The majority (95%) of testicular neoplasms are germ cell tumors, which are relatively

uncommon carcinomas, accounting for only 1% of all male malignancies. Two chemotherapy

regimens are effective for testicular germ cell tumor prognosis: four cycles of etoposide and

cisplatin (introduction of cisplatin was in the mid-1970s) or three cycles of bleomycin, etoposide

and cisplatin. Of the drugs used to treat testicular cancer, bleomycin and etoposide are natural

products.



1.4. Isolation of Enediynes

Enediynes are relatively new class of natural anticancer agents isolated from microorganism.

Soon after the discovery of naturally occurring enediynes, in the mid 1980’s, an unprecedented

flurry of activities had started which is still continuing in various domains of chemical and

biological research. The enediyne antitumor antibiotics are a small family of natural compounds

with potent antitumor activity, comprising only nine members to date with the youngest member

uncialamicin discovered only recently. Although neocarzinostatin, the first member of enediyne

class of natural products, was isolated in the sixties, story on the enediyne antibiotics began to

unfold only around 1985-1987, when the structures of neocarzinostatin and calicheamicin were

uncovered. The novel molecular architecture of these compounds was rather surprising since

they were quite unexpected in nature. The interest of the scientific community on these

compounds arose not only because of their unusual but unique molecular architechture but also

because of their potent cytotoxicity, striking mode of action, high potency, and an exceptional

biological profile. Their unprecedented mode of action opened the door for the development of

several artificial enediynes endowed with interesting biological properties (Figure 6-7).

Neocarzinostatin (NCS): The first member of this class, Neocarzinostatin (NCS)

chromophore, first reported by Ishida et al. in 1965, and was isolated from Streptomyces

carzinostaticus Var. F-41. It is a 1:1 noncovalently associated mixture of a protein component

(NCS apoprotein) and a chromophoric molecule (NCS chromophore). The mixture was separated

somewhat later into its component parts and eventually characterized structurally. The

chromophoric component was shown in 1985 to have the novel bicyclic polyeneyne skeleton by

Edo et al., the apoprotein has been characterized as a 113 amino acid polypeptide based upon the

gene base sequence and apoprotein crystal structure, the three dimensional solution structure of

intact neocarzinostatin has been determined by Hirama et al. using 2 D NMR techniques, and the

crystal structure of holo-NCS has been reported by the groups of Myers and Rees.

Calicheamicins: One year later, in 1986, the first member of the calicheamicin family was

isolated. The calicheamicins are a family of enediyne antibiotics isolated from Micromonospora

echinospora spp. calichensis. They were identified through a research program that identified

microbial fermentation products active in the biochemical induction assay. Calicheamicin 1I is

the most distinguished member of this class of compounds. The calicheamicins are extremely

active against both Gram-positive and Gram-negative bacteria. Moreover, they exhibit

extraordinary activity against murine tumors, such as leukemia and solid neoplasms. The

calicheamicins are profoundly potent sequence specific, double strand DNA-cleaving agents.

Esperamicins: Esperamicins are a subgroup of naturally occurring enediyne antibiotics.

They are broad-spectrum antibiotics and antitumor agents. Esperamicins were isolated from the

fermentation broth of Actinomadura verrucosospora. The name of this enediyne group was

derived from the place where the producing organism was collected, Pto Esperanza in Argentina.

The molecular structure of the esperamicin family was disclosed clearly in 1987. The chemical

structure of this class of enediynes is closer to the structure of calicheamicins having an

additional hydroxyl group in the bicyclic core and unusual structural motifs in sugar appendages.

Other than similarity in structure, they also have similarities in biological activity and mode of

action. Like calicheamicins, esperamicins exert their biological activity by damaging the DNA.

The dynemicin was next isolated and characterized in 1989.



Figure 6. The structures of some naturally occurring enediyne antibiotics (the 9-membered family).

Figure 7. The structures of some naturally occurring enediyne antibiotics (the 10-membered family).

O

O

OH

Cl

NH2

OO

OH C O

(H3C)2N

H3C

OHCH3

O

O

NH

OH3CO CH2

O

Lidamycin Chromophore

OMe

Me

OH

C

O

O

O

O

O

O

O

OMeHO

HO

MeHN

Neocarzinostatin Chromophore

Cl

OMe

OH

O

O

HO

OH

HO

O

N1999A2

N

O

O

MeO

Cl

OH

HO

O

OMe

OHOH

HN

O

Me

Me

OH

Maduropeptin Chromophore

NH

O

MeO

OMe

O

MeMe

NCl

O O O

H

O

OOH

NMe

Me Me

O O

H

OMe

HO

OH

Me

Kedarcidin Chromophore

Natural enediyne antitumor antibiotics: the 9-membered family



Below is an example of process for the isolation of calicheamicins from the fermentation

broth of NRRL15839 (Scheme 2).

Scheme 2. Schematic presentation of process for the isolation of calicheamicins pxBv and Ti31 from the fermentation of NRRL15839.



1.5. Molecular Structure of Enediynes

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 8).

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 8. 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. 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 (Table 1, Figure 9).

Table 1. 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 9. Structural features of Calicheamicin.



1.6. Biological Properties and Mechanisms of Action of Naturally Occurring

Enediynes

The ene-diyne antibiotics are known to arrest formation of malignant tumors by "tying" the

two strands of DNA together. By tying the two DNA strands together, ene-diynes prevent them

from unraveling and thus arresting the replication process. One of the ene-diynes, Dynemicin A

(Figure 7), has been particularly effective. As shown in Scheme 3, the ene-diyne portion of this

molecule closes to form a cyclic structure. This cyclic structure has two radical centers. These

radical centers interact with DNA, forming labile centers, which then form a covalent bond

across the two strands, rendering it unable to unravel or replicate. Mutated or cancerous cells

replicate faster than normal cells, so ene-diynes will have more of an effect on the mutated DNA,

helping to prevent the spread of tumorous tissue. The major problem, however, is that

Dynemicin A, and other ene-diynes, also react with healthy DNA, stopping all replication

processes. As a result, these potential antibiotics are presently too toxic for widespread use in

cancer therapy.

The enediyne antitumor antibiotics contain either DNA intercalating groups (such as DYN)

or DNA minor groove binding function (such as CAL and NCS). The biological actions of these

molecules are a result of three important functional domains. Each molecule contains an

assemblage that consists of (a) an enediyne moiety; (b) a delivery system that communicates the

enediyne moiety to its DNA target; and (b) a triggering device that, when activated, initiates the

cascade of reactions that leads to generation of the reactive chemical species.

Thus the Common modes of action of enediyne class of natural anitibiotics:

• intercalation into minor groove

• reaction (activation) with either a thiol of NADPH - generates radical

• radical cleavage of DNA



The enediyne antibiotics share a common mechanism for producing radical cleavage of

DNA: First, the enediynes undergo cycloaromatization reactions resulting in formation of highly

reactive diradical intermediates. Second, these highly reactive radicals are capable of abstracting

hydrogen atoms from the DNA backbond to trigger DNA damage. The key transformation of 3-

ene-1,5-diynes is a thermal rearrangement that was disclosed in the early 1970 by Masamune and

Bergman that is commonly known as Bergman cyclization. The classical Bergman reaction is

believed to precede through a reactive diradical benzenoid species (a p-benzyne) which cleaves

the DNA by abstracting H-atom from sugar-phosphate backbone of DNA (Scheme 3).

Scheme 3. Mechanism of action of enediyne anticancer antibiotics: DNA cleavage initiated by C4' or C5' hydrogen atom abstraction.



Scheme 4. Mechanism of action of enediyne anticancer antibiotics: DNA cleavage initiated by (a) C4' or (b) C5' hydrogen atom

abstraction.

Less than 20% of the strand breaks result from hydrogen atom abstraction at C(4

(Scheme 4) -stranded DNA cuts by the

(Scheme 4) of deoxyribose, whereas those double stranded lesions which

are observed involve additional hydrogen abstraction by the C(2) radica (Scheme

5)

Scheme 5. Mechanism of action of enediyne anticancer antibiotics: DNA cleavage initiated by C1' hydrogen atom abstraction.



Below are few examples of DNA cleavage shown by natural enediynes (Scheme 6-11).

Scheme 6. Mechanism of DNA cleavage by Calicheamicin

Trisulfide reductioninitiates the activation

Intercalatesinto DNA

Responsible for DNA strand scission

Mechanism of DNA Cleavage by Calicheamicins

Calicheamicins

Scheme 7. Mechanism of DNA cleavage by Calicheamicin



Scheme 8. Mechanism of biological action of dynemicin A.

Scheme 9. Mechanism of DNA cleavage by Neocarzinostatin (zinostatin).



NH

O

MeO

OMe

O

MeMe

NCl

O O O

H

O

OOH

NMe

Me Me

O O

H

OMe

HO

OH

Me

Kedarcidin Chromophore

O

OMe

HO

OH

Me

Nu

Nucleophilic Attack Cycloaromatization

RO

Nu

R

O

o

Me

OH

NMe2

HOOH

O

Nu

R

RO

O

OMe

OH

NMe2

OMe

HO

OH

Me

OHO

Nu

R

RO

O

OMe

OH

NMe2

OMe

HO

OH

Me

DNA

DNA Radical DNA Single Strand CleavageO2

Proposed mechanism of action of the kedarcidin chromophore

+

Scheme 10. Proposed mechanism of action of the kedarcidin chromophore.



HNOH

OH OH

CO2H

OMe

O

O

Me

O

H

H2O or Nu

HNOH

OH OH

CO2H

OMe

OH

OH

Me

O

H

HNOH

OH OH

CO2H

OMe

O

OH

Me

HO

H HNOH

OH OH

CO2H

OMe

O

OH

Me

HO

H

HNOH

OH OH

CO2H

OMe

OH

OH

Me

HO

H

OH

HNOH

OH OH

CO2H

OMe

O

O

Me

HO

H

H

H+

HNOH

OH OH

CO2H

OMe

O

O

Me

HO

H

HHNOH

OH OH

CO2H

OMe

OH

OH

Me

HO

H

OH

Nucleophilic attack

Proton Transfer

Cycloaromatization

Cycloaromatization

HNOH

OH OH

CO2H

OMe

OH

OH

Me

HO

H

OH

Path BPath A

HNOH

OH OH

CO2H

OMe

O

O

Me

HO

H

H

DNA

DNA Diradical

DNA Double Strand Cleavage

O2

HNOH

OH OH

CO2H

OMe

O

O

Me

HO

H

OH

DNA

DNA Diradical

DNA Double Strand Cleavage

O2

O2

Dynemicin

Mechanism of Biological Action of Dynemicin A

Scheme 11. Mechanism of biological action of dynemicin A.



1.7. The Bergman Cyclisation Reaction

1.7.1. Introduction

The enediynes can be synthesized via Sonogashira coupling protocol. The enediynes

undergoes a key cycloaromatization reaction which is the heart at the chemistry of enediyne

molecules. The Bergman cyclization or Bergman reaction or Bergman cycloaromatization is

an organic reaction and more specifically a rearrangement reaction taking place when an

enediyne is heated in presence of a suitable hydrogen donor (Scheme 12). It is named for the

American chemist Robert George Bergman. The reaction product is a derivative of benzene.

Scheme 12. Bergman cyclization or cycloaromatization

The Cope rearrangement of hex-3-ene-1,5-diyne (A), which results in concerted cyclization

via transition state B to p-benzyne (C), was first reported by Bergman in 1972, and has become

known as the Bergman cyclization. Included in Bergman’s report were instances of intra- and

intermolecular trapping of p-benzynes to give “cycloaromatized” products (D, Scheme 12).

Masamune et al. had described the conversion of cyclic enediynes into the benzenoid systems

(Scheme 13), prior to the studies by Bergman. When the enyne moiety is incorporated into a 10-

membered hydrocarbon ring (e.g. cyclodeca-3-ene-1,5-diyne in Scheme 13) the reaction, taking

advantage of increased ring strain in the reactant, is possible at the much lower temperature of

37°C. However, the involvement of a diradical species was not mentioned in this paper.

Scheme 13. Observation by Masamune et al. (1971).



In 1980, Wong and Sondheimer observed a cycloaromatization from in situ generated cyclic

enediynes e.g. (K) and postulated the intermediacy of a 1,4-diradical (Scheme 14).

Scheme 14. Pioneering work by Wong and Sondheimer (1980)

1.7.2. Importance of BC in Biology

The BC is perhaps one of the few reactions that was discovered but did not get much

attention before their counterpart was discovered in Nature. The reason for the nonexploitation of

the reaction is probably the necessity of high temperature. No one could foresee that the same

reaction would be possible under ambient conditions until Mother Nature showed the way to do

it through the chemistry of the natural enediynes. In the mid to late 1980s, it became clear that an

emerging series of naturally occurring antibiotics, including calicheamicin, esperamicin, and

dynemicin, all operated via Bergman cyclization to a p-benzyne derivative, followed by H atom

abstraction, especially from DNA. As a consequence of the antibiotics becoming

cycloaromatized, the cell under chemical attack suffered DNA cleavage, ultimately leading to

cell death. Therefore, the BC is at the heart of the chemistry of enediynes and is primarily

responsible for their biological activities. Understanding the parameters controlling BC kinetics

is of paramount importance for the design of any new enediyne.

The strong DNA-cleaving activity of these molecules led to the synthesis of many nonnatural

targets containing the active enediyne “warhead” of the antibiotics. All the natural antibiotics, as

well as the synthetic mimics, possess an enediyne unit within a medium ring of 9-10 atoms, thus

incorporating the strain necessary to enable the cyclization to occur at biologically relevant

temperatures. Most of these systems are polycyclic, and contain other adjustable strain-inducing

elements, as well as triggering devices that can release a more reactive form of the enediyne

upon activation. The utility of this strategy lies in retaining the enediyne in prodrug form until it

reaches its biological target, following which the active drug is unveiled. Although several of the

naturally occurring enediynes are undergoing clinical evaluation, efforts to produce comparable

designed enediynes remain a formidable challenge because of problem in controlling reactivity

of enediynes. It is therefore highly desirable to determine the factors that govern the cyclization

step.



1.7.3. Parameters that Control the Kinetics of BC

Research in the fifteen years or so has enabled us to understand some of the controlling

parameters for BC kinetics. From the very inception of its discovery, BC is known to have high

activation barriers for acyclic enediynes (Scheme 15). Cyclic enediynes, on the other hand,

generally have much lower activation energy so that the same reaction can take place at a lower

temperature. For example, a 10-membered carbocyclic enediyne (P) or a heterocyclic enediyne

(N or O analogue, structure R and T respectively) undergoes cyclization at ambient temperatures

with fairly decent half-lives (except the sulfur analogue V which is stable at room temperature).

Fusion of strained rings on to the cyclic enediynes (examples X-Z) brings back the stability

(Scheme 15). Incorporation of strain raises the energy of the transition state more than that in the

ground state thus elevating the activation barrier for BC.

Scheme 15. Some examples of stable and unstable enediynes.

To ascertain what exactly prompted these molecules to readily undergo BC, studies were

undertaken in various laboratories. Several theories have been put forward which are

described here.



1.7.3.1. Nicolaou’s Distance Theory

On the basis of his extensive studies, Nicolaou et al., in 1988, proposed that the distance

between the terminal acetylenic carbons of the enediyne group (d) is a major determinant of

reactivity, and the values of d between 3.20 and 3.31 Å would be necessary for their biologically

relevant reactivity. Nicolaou et al. also found that while the cyclic enediynes (n = 3-8;

Figure 10) were stable at room temperature, the 10-membered analogue (n = 2) having an

c,d-distance of 3.25 Å undergoes cyclization at 37 oC with a t1/2 of 18 h (Table 1).

Figure 10. Cyclic enediynes by Nicolaou et al.

Table 1. Calculated c,d-distances and stability of conjugated enediynes

Entry (n = x)

Ring Size

c,d-distance

(Å)

Stability

1

2

3

4

5

6

7

8

(n = 1) 9

(n = 2) 10

(n = 3) 11

(n = 4) 12

(n = 5) 13

(n = 6) 14

(n = 7) 15

(n = 8) 16

2.84

3.25

3.61

3.90

4.14

4.15

4.33

4.20

Unknown

Cyclization at 25 oC

Stable at 25 oC

Stable at 25 oC

Stable at 25 oC

Stable at 25 oC

Stable at 25 oC

Stable at 25 oC

Recently, Schreiner using the Density Functional Theory (DFT) estimated the activation

enthalpies for the BC of a series of cyclic hydrocarbon enediynes and extended the “critical

range” of 3.31-3.20 Å for spontaneous cyclization to 3.40-1.90 Å.

d



1.7.3.2. The Molecular Strain Theory

In contrast, to Nicolaou’s hypothesis, both Magnus et al. and Snyder argued that differential

molecular strain between the endiyne and transition states is the commanding element for ring

closure. It also considered the transition state for the BC as product like. This was substantiated

by experimental observations (activation energies and X-ray structures of a few C9-C12 cyclic

enediynes) and by empirical computations. As for example the enediyne A with a greater c,d-

distance undergoes faster cyclization compared to B with a smaller c,d-distance (Scheme 16).

Although the molecular strain theory seems more quantitative, Nicolaou’s theory has been

used more frequently because of its simplicity. In recent years, DFT-based calculations lent

support to the distance theory; only the range of critical c, d-distance has been modified and

calculated to be 3.40-2.90 Ǻ.

Scheme 16. Role of strain energy on the kinetics of BC



1.7.3.3. The Role of Conformational Changes upon Metal ion Co-ordination on

The Kinetics of Bergman Cyclization

Metal coordination by ligands attached to enediyne framework has significant effect on the

kinetics of BC. It is interesting to note that there is no precedence of metal ion triggering of

enediynes in Nature so far and hence this idea is quite novel in the true sense. Considering

Nicolaou’s distance theory, one can think that, an effective method to temporarily shorten the “c-

d” distance, the use of transition metal complexation can serve a unique mechanism. Thus the

rate of BC may be perturbed by the presence of metal ions or organometallic reagents. The basic

principle behind such metal ion mediated BC is quite simple. The enediyne having ligating

system in the two acetylenic arms chelates a metal ion thus forming a cyclic network or a

metallocycle. This, in turn, is expected to lower the activation barrier for BC. The situation may,

however, be much more complex and in many cases, metal ion complexation has a detrimental

effect on the cyclization kinetics because of certain configurational restrictions.



The use of metal ions to control the kinetics of BC was first demonstrated by Konig et al. to

be followed by Buchwald and his coworkers. The former studied the effect of complexation of

adjacent crown ether moieties in an acyclic enediyne with different metal ions. The thermal

properties of the bis-benzo crown ether and its complexes were investigated by Differential

Scanning Calorimetry (DSC). Judging from the various onset temperatures for BC, the effect of

metal ion complexation appears to be marginal. However, this work did set the tune for one of

the future directions the enediyne chemistry was going to take.

Although formation of a cyclic network upon complexation is expected to bring down the

c,d-distance between the reacting acetylenic carbon atoms, a change in conformation is also

critical at the same time in deciding the activation barrier for BC. Konig et al. have elegantly

demonstrated a significant lowering of activation barrier for BC by a subtle change in the

conformation. They have shown that the induced conformational change of bipyridyl based

macrocyclic enediyne shown in Scheme 17 brought about by metal ion coordination results in a

drop of cyclization temperature by about 100 degrees.

Scheme 17. Metal coordination bring the conformational changes.



Sometimes, complexation leads to the removal of transannuler repulsion between the lone pair of

electrons on the nitrogen atoms thus bringing them closure together to form the metallocycle. As

a result, the distance between the two reacting acetylenic carbons is reduced, thereby lowering

the activation barrier to BC. Such shortening of N-N-distance upon complexation is depicted

below in Scheme 18.

Scheme 18. Trans annular repulsion of “N” loan pairs and the BC



1.6.3.4. Conformation and Reactivity in Enediynes: Role of Steric Interactions Conformational changes brought about various ways can bring significant changes in the

kinetics of BC. The conformation that a molecule will adopt in the ground state (G. S) as well at

the transition state (TS) depends upon several factors e.g., substituents attached to enediyne

system, their steric bulk and electronic characteristics as well as the stereochemistry. Discussed

below are some examples where substituent effects are quite strong.

Schreiber et al. have reported that the stereochemistry at C-10a dictated the thermal reactivity

of dynemycin models (A) and (B) equipped with an exo epoxide. Structure (B) with an

substituent is quite unreactive whereas that with a -substituent (A) undergoes cyclization at

80 oC. MM calculations indicate that this reactivity difference is due to a steric interaction that

develops as the substituted molecules undergo a conformational change for BC (Figure 11).

Figure 11. Role of Steric Interaction on the Kinetics of BC



To demonstrate the effect of steric interactions on the kinetics of Bergman cyclisation,

Zaleski et al. reported the syntheses and thermal reactivities of two symmetric and two

asymmetric enediyne chelates of the form 1,8-bis (R, R1) oct- 4-ene-2,6-diyne where R and R1 =

dimethylamino, amino, or 3-hydroxypyridine (Figure 12). The asymmetric compounds are

synthetically unique, and exhibit cyclization temperatures between the bis(dimethylamino) B and

the novel diamino A compound, the latter of which is substantially more reactive. The thermal

reactivities of these enediynes systematically illustrate the importance of intraligand steric

hindrance in influencing Bergman cyclization temperatures.

Figure 12. Role of Steric Interaction on the Kinetics of BC by Zaleski et al.

Compound B is a thermally stable that exhibits a Bergman cyclization temperature of 186 °C.

Substitution of one dimethylamino group with 3-hydroxypyridine (E) dramatically reduces the

Bergman cyclization temperature to 149 °C. The result derives primarily from two sources. First,

the ability of the pyridine ring to rotate out of the enediyne plane about the oxygen bond relieves

steric clashes with the opposing substituent. Secondly, the addition of the sp3 oxygen between

the pyridine ring and the alkyne termini effectively distances the pyridine substituent from the

alkyne termini by another atom, further reducing the interaction between substituents.

A more pronounced trend is observed between the three compounds in the

bis(dimethylamino) to diamino enediyne series (A, B, F). Monosubstitution of amino for

dimethylamino (F) yields a dramatic (47 °C) decrease in the Bergman cyclization temperature (F

= 139 °C). Further substitution to form the diamino compound A produces an additional 33 °C

decrease in the Bergman cyclization temperature (A = 106 °C, onset: 55 °C) indicating that A

has one of the most facile thermal reactivities of an acyclic enediyne reported to date. The

enhanced thermal reactivity of A results from a combination of the reduced steric hindrance of

the primary amine functionalities, as well as an additional contribution from intramolecular

hydrogen bonding (Figure 12).



1.7.3.5. Role of Electronic Nature of Substituents

Although the most studied factors which affect the Bergman cyclization of many natural and

synthetic enediynes are molecular strain energy and the distance separating the acetylenic

termini, electronic characteristics of substituent effects also influence this process. Electron-

withdrawing substituents either attached to triple-bond termini or associated with the double

bond of enediynes can lower the activation enthalpy of the Bergman reaction by decreasing the

degree of repulsion of the in-plane orbitals in the transition state (Scheme 19). There are a

number of reports of substituent effects in the Bergman cyclization.

Scheme 19. Possible substitutions on to enediyne framework



1.7.3.6. Role of Terminal Acetylene Substituents Grissom et al. studied the effect of terminal acetylene substituents in aromatic acyclic

enediynes on the kinetics of BC. Incorporation of one acetylene substituent on the enediyne

caused a small decrease in the rate. However, addition of a second acetylene tether had a

substantial effect (Scheme 20). Bergman noted a similar effect in the enediyne with two

acetylene substituents. Thus, steric interactions between the acetylene substituents can either

push the acetylenes apart or distort the enediyne from planarity.

Scheme 20. Effect of terminal acetylene substitution on the kinetics of BC

Wittman et al. reported that substituents at the propargylic position have significant effect on

the BC of simple bicyclic enediynes (Figure 13). Thus cycloaromatization of (A) occurs at a much

lower temperature than that required for (B) or (C).

Figure 13. Effects of propargylic substitution on the kinetics of BC



1.6.3.7. Role of Vinylic Substituents Maier and Greiner have reported a pronounced retardation of the Bergman cyclization of the

bicycle [7.3.1] enediyne A (Figure 14) when the double bond is substituted with an electron-

donating group (R = p-C6H4OMe). The cause of retardation was explained by this way that

subtle conformational effects may be at play here, perhaps including interaction between the

OTBDMS group at the bridgehead of A with the electron rich anisyl group which might cause

the anisyl to adopt a dihedral angle relationship with the enediyne unit that is not ideal for

cyclization assistance and thereby lead to the observed retardation.

Figure 14. Vinylic substituted enediynes from various group

Jones and Plourde have reported the retardation of the Bergman cyclization of a series of

cyclic enediynes B-D (Figure 14) when the double bond is substituted with chlorine. A

systematic study of substituent effects in the Bergman cyclization of 1,2-dialkynylbenzene

derivatives E-J (Figure 14), by Russell and co-workers, demonstrated a linear free energy

relationship between the cyclization rate and the Hammett m substituent coefficient.

Recently, Kerwin and coworkers have reported the synthesis and the kinetic studies of the

aza-Bergman reactions of a series of 6-triisopropylsilyl (P-R) (Scheme 21) and 6-unsubstituted

1-phenyl-4-aryl-3-aza-3-ene-1,5-diynes (S-U) (Scheme 21) in which the aryl group is phenyl, o-

(methoxy)phenyl, or p-(methoxy)phenyl. These aza-enediynes undergo aza-Bergman reaction

followed by a rapid retroaza-Bergman cyclization to afford alkynyl acrylonitrile products. In

no case products corresponding to trapping the intermediate 2,5-didehydropyridine diradicals (V)

(Scheme 21) were isolated. They have shown that while rate of aza-Bergman cyclization is not

greatly affected by the nature of the 4-aryl substituent, the rate is very dependent on the nature of

the 6-substituent. Thus the aza-Bergman reaction of U that lack a 6-substituent undergo aza-

Bergman cyclization spontaneously at 20 °C with first-order half lives of 36-78 min. and the rate

is approximately two-times faster than that of either S or T. They stated that the origin of this

modest substituent effect is not known; however, it is likely that the steric interaction between

the o-methoxy group and the adjacent alkyne moiety in U plays a role. The lack of an observable

difference in the rate of aza-Bergman reaction of S and T (Scheme 21) was surported in light of

the theoretical studies by Kraka et al. Calculations of a variety of aza-enediynes in which the

imine double bond is replaced with an amide, amidine, or amidinium group predict that the



barrier to aza-Bergman cyclization is relatively insensitive to these changes as was done by

Kraka and coworker.

Scheme 21. Aza-Bergman cyclisation and effect of vinylic substituent on BC

1.7.3.8. Geometrically Constricting- Influence of the Tetrahydrofuranyl Ring on

the Kinetics of BC

Nantz et al. have shown that the presence of multiple fused ring systems exerts geometrical

constraints that prevent cycloaromatization of the otherwise facile cyclization previously observed

for the core system (Scheme 22).

Scheme 22. Geometrically constricting influence of the tetrahydrofuranyl ring on the kinetics of BC.



1.7.3.9. Role of Conformational Motifs

Subtle changes in conformation can have significant effect on the kinetics of BC of

enediynes. Conformational changes can be brought about in various ways. For example,

Semmelhack has recently reported considerable difference in BC rates for cyclohexane-fused

enediynes depending upon whether the cyclohexane is in the chair or in the boat conformation.

In the bicyclo[7.3.1]tridec-4-ene-2,6-diyne framework (Scheme 23), characteristic of

calicheamicin, DFT calculations predict that the chair conformer should be much more reactive

toward cycloaromatization compared to the boat form.

Scheme 23. DFT calculated activation energies of calicheamicin framework.



A functionalized derivative (A) of this framework with an added two-atom bridge to enforce

the boat conformation was synthesized by Semmelhack and coworkers and shown to be stable at

23 oC (Scheme 24). Cleavage of the bridge releases the conformational lock (B) and

cycloaromatization proceeds with t1/2 40.5 min at 24.5 oC, presumably through the chair

conformation. This confirms the prediction based on computation and points to a new principle

for triggering the enediyne toxins.

Scheme 24. Chair and boat conformation dependent BC



1.7.3.10. The Role of Solvent Although cyclization step had been reported to be the rate determining step in the

cycloaromatization of aliphatic enediynes, cyclic systems have been known whose

cycloaromatizations are dependent upon the presence of solvents which act as H-donors. In the

recent example, Kim and Russell studied the cyclization behavior of quinoxaline enediynes (A)

in various solvents (Scheme 25). The cyclization rate was surprisingly found to be solvent

dependent. The half-lives varied from 361 min in CH3CN to 16 min in THF. The half-life data

show a good correlation with the solvents' spectroscopic E(30) values which is the empirical

solvent polarity parameter and the value reflects all the non-specific intermolecular forces

between solvent and solute molecules and dielectric constants.

Scheme 25. The effect of solvent on the thermocyclization of A

The only other example of an enediyne reported in the literature, which shows significant

solvent dependent thermal cyclization, is the C-1027 chromophore (Figure 15). Yoshida tested

the reactivity of the C-1027 chromophore in ethanol, ethyl acetate, DMSO, DMF and THF. The

molecule showed the shortest half-life in THF.

Figure 15. The natural enediyne “C-1027”



Table 4. Kinetic data of Thermal Cyclization of A in Various Solvents at 168 oC.

Solvent Rate

constant

(kobs)

Correlation

coefficient

Half

life

(t1/2;min)

Product

Yield

(%)a,b,c

Dielectric

Constant

()

ET(30)

(Kcal/

mol)

CH3CN 3.3 x 10-5 0.986 361 C (7) 36.64 45.6

Methanol 3.7 x 10-5 0.994 312 C (2) 33.00 55.4

Benzene 1.2 x 10-4 0.990 96 C (12)

D (58)

2.38 36.0

Dioxane 1.9 x 10-4 0.990 60 C (15) 2.21 36.0

CCl4 2.6 x 10-4 0.994 44 E (81) 2.23 39.0

THF 7.1 x 10-4 0.989 16 C (36)

F (9)

7.52 37.4

aBulk cyclization was performed under the same condition as kinetics; bSolvent

adducts gave expected spectral data; cIsolated yields; ET = Empirical solvent

polarity parameter. This value reflects all the non-specific intermolecular forces

between solvent and solute molecules.

1.7.3.11. The Role of Electrostatic and H-bonding Interaction on the Kinetics of

Bergman Cyclization (BC)

Through extensive research inputs over the past years, it became clear that cyclic enediynes are

more reactive than their acyclic counterparts. Thus, apart from directly starting with a cyclic

framework; activation of enediynes towards Bergman cyclization (BC)2 can be achieved in principle,

via the formation of an in situ cyclic moiety.



Vasella and coworkers attempted to study the thermolysis of the 1,2-

bis(glucosylethynyl)benzenes A and H to evaluate the effects of intramolecular H-bonding on

the activation energy of the Bergman cycloaromatization, and to evaluate the use of the

cycloaromatization for the synthesis of diglycosylated naphthalenes. They have synthesized the

corresponding enediynes both in protected (O-benzylated) and unprotected forms. Thermolysis

of A in PhCl gave the naphthylglucitols D and E, irrespective of whether 1,4-cyclohexadiene

(1,4-CHD) was present or not. Formation of D is rationalized by a Bergman cyclization to a

diradical, followed by regioselective abstraction of a H atom from the O-benzyl group, and

diastereoselective combination of the doubly benzylic diradical (Scheme 26).

Scheme 26. Bergman cyclisation of sugar anchored enediyne



Thermolysis of the mono-C-glycosidic dialkyne F occures at 185 oC in PhCl in presence of

1,4-CHD gave G in 39% yield. The di- C-glycosidic enediyne A cyclises at higher temperature

i.e. at 230 oC in the presence or absence of 1,4-CHD to give D (47-55%) and E (6-17%). The

thermolysis of A follows the established mechanism of Bergman Cyclisation leading to the

naphthalene diradical B (Scheme 26) that is sufficiently long lived to adopt a conformation

allowing the regioselective intramolecular H-abstraction from the O-benzyl group at C(2) to

give C. External H-donors such as 1,4-CHD, even when used as solvent, do not compete with

this abstraction. Disrotatory movement of the glucosyl residues around the C(2")-C(1) and

C(3")- C(1') bonds is necessary before the two PhCH radicals can recombine to give D.

Prolonged thermolysis of A increased the amount of E suggesting that it is formed via D.

Thermolysis of D under same conditions, indeed, provided 17% of E. While thermolysis of the

corresponding di-C-glycosidic enediyne A in EtOH sets in at approximately 140 oC, H did not

react at 160 oC and decomposed at 180-220 oC.



Scheme 27. Thermolysis of enediyne F and the enediyne H

Intramolecular H-bonds may shorten the bonding distance between the alkenyl groups in H

as compared to A, and lower the activation energy for the cycloaramatisation. Not expectedly,

however, H proved only soluble in polar solvents. Macromodel calculations (MM3* force field,

gas phage) showed that a H-bond between HO-C(2) and O-C(2’) is feasible, but that it should be

weak, and may hardly influence the distance between the alkynyl groups. Indeed, no significance

difference was observed between the/ T values of the OH signals of H which falls in the

range of 5-6 ppb/K in d6-DMSO. Thus, there are at best very weak intramolecular H-bonds

(Scheme 27).

The thermolysis of A in ethanol for 24 h yielded D (33% at 180 oC; 6% at 160 oC and 1% at

140 oC) and a number of undesired products, while the thermolysis of H in ethanol and in

presence of 1,4-CHD, however, did not proceed at 160 oC and led to decomposition between 180

and 220 oC. The hypothetical cycloaromatisation of H appears to require a higher temperature

than the one of A. This observation lead the authors to conclude that the solvation of H and less

efficient H-abstraction of the intermediate diradical play an important role in determining the

inefficiency of H to undergo Bergman cyclisation, rather decomposition occurs (Scheme 27).

Alabugin et al. in his theoretical calculation showed that sometimes the rate of BC is

enhanced and sometimes decreased due to H-bonding and electrostatic interactions between the

ortho substituents of aromatic enediynes and the in plane acetylenic orbital. They have shown

that CH3, NH2, and syn-OH increase the activation energy because they stabilize the starting

material by a hydrogen bond between the X-H moiety and in-plane acetylenic orbital. The

strength of the hydrogen bond increases with the electronegativity of X (C, N, O).5 This

interaction results in an increase of the C1-C6 distance and outward bending of the acetylene

group. As the reaction proceeds, the stabilizing interaction disappears since the overlap of *X-H

orbital and the in-plane bond decreases as a result of inward bending of the acetylene moiety

away from the X-H group. Thus a retardation of rate was observed (Scheme 28).

In contrast, in the case of a positively charged functional group, NH3+, the strength of

hydrogen bonding increases at the beginning of the cyclization step, thus providing extra

stabilization to the TS. This accelerating effect can be attributed to a larger electrostatic

component in the H-bonding in the case of a positively charged group and to a concomitant

strong through-space electron transfer from the adjacent in-plane bond of acetylene moiety to



the ammonium group. As a result, population of this bond is markedly decreased, thus

decreasing electron repulsion in the transition state and hence a rate enhancement observed.

Scheme 28. Effect of ortho substituents and electrostatic interactions on BC by Alabugin et al.

The large decrease in the activation energy upon protonation is important because cancer

cells are more acidic (pH 5.5) than normal cells. At this pH, anilines are protonated noticeably.

Hence, the fact that protonation of the ortho-amino group decreases the activation energy by 3.9

kcal/mol (and thus can speed up the reaction by the factor of 600 at 37 oC) can be used in the

design of tumor-specific DNA cleaving agents.

Basak et al. have studied the importance of H-bonding and electrostatic interactions on the

kinetics of BC of various enediynyl amino acid and peptides (Scheme 29).

Scheme 29. Thermal behavior of enediynyl amino acids and peptides.



Through their study of differential scanning calorimetric and variable temperature NMR

experiment with their designed enediynyl aminoacid and peptides they have been able to

demonstrate that the peptides or the amino acids that forms relatively stronger H-bond cyclized

at comparatively lower temperature.

The same group have been able to demonstrate the role of weak H-bonding and electrostatic

interactions in lowering the activation energy of BC. The enediynyl amino acids form pseudo

cyclic framework via intramolecular H-bond or electrostatic interactions between the terminal

zwitter ions thereby lowering the c,d-distance and hence the activation barrier for BC (Figure

16).

Figure 16. The enediynyl amino acids and peptides



1.7.3.12. Role of Hybridization on the Kinetics of Bergman Cyclization (BC)

1.7.3.12.1. Role of Hybridization in Natural Enediynes

The first enediyne natural products are the calicheamicins and esperamicins. They belong to

separate family of enediynes but share the same enediyne-containing bicyclic core [7.3.1] (Figure

17) as well as similar mode of biological action.

Extensive in vitro studies suggest that the biological action of these molecules starts with the

reorganization and site specific binding to the DNA followed by the attack of a nucleophile (e.g.

glutathione) at the central sulfur atom of the trisulfide moiety to generate the thiolate (Scheme

30). The geometry of the molecule enables this thiolate to undergo Michael addition to the enone

producing a dihydrothiophene (C). As a consequence, hybridization of the bridgehead carbon

changes from sp2 to sp3 which allows the enediyne to undergo BC with concomitant generation

of diradical as a rate-determining step.

Figure 17. The natural enediyne Calicheamicin 1

1

O

O

O

OO

O

NHEt

OMe

Me

NH

O

OHMe

OH

ONHCOOMe

MeSSS

OHS

O

O

Me

I

OMe

OMe

OH

OHMe

MeO



The reactive diradical (C) is capable, and well positioned, to abstract two hydrogen atoms,

one from the C5 position of deoxycytidine and the other from a ribose position of the opposing

strand. The DNA radicals so generated then proceed to react with molecular oxygen leading to

double strand cleavages. Townsend et al. have demonstrated the existence of the

dihydrothiophene intermediate by 1H-NMR. They have also measured the half-life of this

intermediate which came out to be about 4.5 s at 37 oC (Scheme 30).

Scheme 30. The role of hybridization and Mechanism of DNA-Cleavage by Calicheamicin 1

1.

Like calicheamicins, the esperamicins (E) also exerts their biological action by damaging

DNA. The mechanism of the DNA-cleavage by Esperamicins A1 is identical to that of the

cleavage by calicheamicin 1. Esperamicin A1, however, exhibits less sequence selectivity than

calicheamicin 1 and shows preference in the order T > C > A > G and results in both single and

double strand cuts.

Figure 18. The natural enediyne Esperamicin A1 (E).



1.7.3.12.2. Role of Hybridization in Unnatural Enediynes

Magnus and co-workers were the first to study the dependence of reactivity of enediynes

with the state of hybridization. They had prepared bicycle [7.3.1] enediyne analogues and

showed that the enediyne ring of A (Scheme 30) was resistant to cycloaromatization at ambient

temperature, but reacted slowly at 82 °C to form the corresponding benzenoid product. A change

in hybridization of the one-carbon bridge (C-13) from sp2 to sp3 (A to B) was shown to effect a

dramatic increase in the rate of cycloaromatization. Thus, enediyne B undergoes complete

cycloaromatization at 20 °C within 30 min. Hence, it is evident that changing C-13 from trigonal

to tetrahedral geometry considerably lowers the activation barrier for BC.

Scheme 30. Effect of hybridization on the rate of BC (Magnus et al.).

Magnus et al. have also introduce a bridgehead double bond (C-1,2) to see the effect on the

rate of diyl formation. Thus they have synthesized C from A and got a mixture of C and

unreacted A. Though they could not be separated by chromatography, merely heating the

mixture of C and A at 80 oC, in presence of 1,4-cyclohexadiene converted A into the less polar

benzenoid adduct D while C was recovered unchanged. This study reveals that changes in

hybridization at the bridging carbon (C-1) dramatically change the rate of diyl formation and also

able to show that the bridgehead sp2 carbon retard the rate of cyclization dramatically.



In 1989, Magnus et al. have shown that the comparatively unreactive enediyne C undergoes

cyclization after removal of the C1-C2 double bond. Thus heating C at 110 oC in 1,4-CHD in the

presence of 4-chlorothiophenol and N-morpholine gave the aromatized product G (Scheme 31).

This result convincingly demonstrate that the formation of putative 1,4-diyl can be triggered

intermolecularly by thiol addition to C-2 and provides an alternative triggering device that may

be exploited in the design of so called rational analogs and also the importance of double bond

on the rate of cyclisation.

Scheme 31. Effect of hybridization on the rate of BC (Magnus et al.).



During the study of relative rates of cycloaromatization of Dynemicine

azabicyclo[7.3.1]enediyne core structures, Magnus et al. showed an interesting role of

hybridization of bridging carbon atom on to the cyclisation barrier. They have synthesized and

studied the rates of BC of several dynemicine core structures. Thus, while distance between the

bonding acetylenic carbon atoms in H and L is virtually the same, and the hybridization at the

bridging carbon atoms is trigonal in both compounds, L cyclises 500 times faster than H at 37 oC. They have found an even more dramatic change in rate when the bridging trigonal carbon

atom is made tetrahedral. Thus reduction of H with sodium borohydride in methanol at 25 oC

gave directly the cycloaromatized alcohol K. Conservative estimation showed that the alcohol J

cycloaromatizes 106 faster than H at 37 oC (Scheme 32).

Scheme 32. Effect of hybridization on the rate of BC Magnus et al.



Annelation of a bicyclo [7.3.1] enediyne core has been shown by Nantz et al. to alter the

cycloaromatization reactivity. The enediyne (O) containing a sp3-hybridized bridging carbon atom

is resistant to cycloaromatization whereas the immediate sp2-hybridized precursor enediyne (N)

readily undergoes transformation to its benzenoid counterpart (Scheme 33). This observation is

just opposite to the study of Magnus but give the light about the role of hybridization on the rate of

BC.

Scheme 33. Role of annelation and hybridization on BC

1.7.3.12.3. Role of Hybridization of Ring Nitrogen on the Reactivity of N-Containing Cyclic

Enediynes

In recent years, various cyclic enediynes with a nitrogen atom replacing a non-enediynyl

carbon have been prepared in our laboratory and their thermal reactivities were studied (Scheme

34). The nitrogen atom in all these enediynes was equipped with aryl sulfonamido groups. These

groups were so chosen as to perturb the extent of pyramidalization of the ring nitrogen atom. The

enediyne, with an electron withdrawing nitro group, was found to undergo cyclization at a faster

rate as compared to other enediynes bearing phenyl sulfonyl or para-toluene sulfonyl group. The

results were explained on the basis of degree of pyramidalisation of the ring nitrogen atom. A

similar effect was also observed in case of amides (Scheme 35).

Scheme 34. Extent of pyramidalization of the amide nitrogen atom on the kinetics of BC.



Scheme 35. Extent of pyramidalization of the enediynyl sulphonamides and amides nitrogen atom on the kinetics of BC.

1.7.3.13. Role of Remote Hybridization on the Kinetics of BC Basak et al. designed the following pyridazinedione-based enediynes containing remote sp2

hybridized “C” to demonstrate the role of remote hybridization on the kinetics of BC (Scheme

36).

Scheme 36. Formation of Bergman cyclised products from A and B



By studying the DSC onset temperature that is the signature of temperature of BC, solution

phase kinetics and semiemperical PM5 calculation they observed that saturation of the double

bond speeds up the reaction of BC that can be exploited to use this as a triggering mechanism.

1.7.3.14. Effect of -Stacking and Charge Transfer Interaction on the Kinetics of

BC

Intermolecular charge transfer (CT) complexes arising from the interaction of aromatic

donor and acceptor molecules have been studied extensively. However, fewer intramolecular

CT analogues have been reported, primarily of the cyclophane type, in which the donor and

acceptor portions are locked together in a rather rigid arrangement. More flexible intramolecular

CT complexes have been reported in a recent communication, whereby a cyclohexane skeleton is

substituted at adjacent trans positions with aromatic donor and acceptor groups. Besides charge

transfer interactions, attractive nonbonded interactions between aromatic units (-stacking) play

a central role in many areas of chemistry and biochemistry as discussed earlier. The activity

profiles of the well-known medicinally important enediynes are greatly perturbed by weak

interactions. Jones et al. have shown that the strong electron-withdrawing groups increase the

barrier for Bergman cyclization, while -donating groups decrease it while conjugation,

especially, donation, has little effect. Alabugin in a recent paper evaluated the stereoelectronic

effects in cyclohexane, 1,3-dioxane, 1,3-oxathiane and 1,3-dithiane based enediynes. Zaleski et

al. have shown how dramatically the steric influences of the functional groups at the termini of

acyclic enediynes can affect the Bergman cyclization temperatures of the resulting compounds.

That the perturbation can be a cause of metal ligand charge transfer transition as was shown by

Zaleski et al., may be extended to the charge transfer between the organic donor and the acceptor

moiety hooked into the two arms of the acyclic enediyne or possible stacking interactions between

the two donor moieties in both the arms. As the charge transfer or stacking interaction occurs, the

two arms may possibly come closer. This should lower the distance between two reactive acetylenic

bonds and thus elevation rates of BC can be expected. With these ideas in mind we framed our

objectives as stated below.

To elaborate the concept of weak interactions and their effect on Bergman Cyclization (BC),

Basak et al. synthesized several enediyne compounds of general structures, as in the Figure 19,

incorporating Donor and Acceptor units in the two arms of enediynes and followed their charge

transfer/stacking interactions by studying UV/VIS spectroscopy.

Figure 19. Representation of donor-acceptor containing enediynyl compounds.



Specifically, their intention was to investigate the possibility of encouraging Bergman

Cyclization (BC) by pulling the reactive centers together with, if possible, CT / stacking

interactions between the two arms of the enediynes. As was discussed earlier, among the various

parameters that control the kinetics of BC, the distance between the acetylenic carbon atoms

undergoing covalent connection (c,d-distance), has become extremely useful in spite of some

limitations. As compared to the cyclic ones, acyclic enediynes have a comparatively high c,d-

distance, which is much above the critical distance range required for spontaneous cyclization as

proposed by Nicolaou and others. However, it is not unreasonable to think that similar to

enediynyl amino acids and peptides (Scheme 29, Figure 16), acyclic enediynyl compounds

containing the donor and acceptor moieties in the two arms, may involve in through-space

intramolecular charge transfer (ICT) interaction and the stacking interaction between them.

This should lower the c,d-distance and hence the activation barrier for cycloaromatization. Thus,

they have synthesized the following compounds shown in Figure 20. As the donor units,

electron rich aromatic compounds like naphthalene or derivative of naphthalene (containing

electron donating substituent) and anthracene derivatives were employed. Benzene derivatives

with strong electron withdrawing groups like -NO2, -CN, -CF3, were used as acceptor moieties.

Figure 20. D/D, D/A and A/A containing enediynes



They have demonstrated that charge transfer or -stacking interactions can enhance the

cyclization kinetics. Repulsion between electron deficient partners raises the activation energy.

While the D/A enediynes become activated because of CT-interactions, the enediynes with D/D

arms also show greater reactivity than the corresponding A/A counterparts possibly because of

stacking interaction.

1.8. The Myers-Saito Cyclization Reaction

1.8.1. Introduction Although enediynes have stimulated considerable synthetic interest, their clinical use has

been limited because of their modest selectivity for cancer cells.

The biological activity of these

enediyne compounds is dependent on mainly the ease of Bergman cyclization. The discovery of

the biradical mechanism in neocarzinostatin eventually leading to an effective DNA cleavage

emphasized the importance of the enyne [3] cumulene core A in the reactive form of this natural

antitumor antibiotic. This discovery prompted Myers et al., and Saito et al., separately into

further investigations into the aromatization of enyne-allenes. The first order thermal

cycloaromatization of (Z)-1,2,4-heptatriene-6-yne produced an intermediate that could be

represented as α,3-dehydrotoluene biradical through a C2-C7 cyclization. The reaction, known as

Myers−Saito cyclization (MSC), which is depicted in Scheme 37. Neocarzinostatin shows its

biological activity through the involvement of such a reaction (Scheme 38).

HH

H

H

H

HMyers-Saito cyclization

12

3

4

56

71

2

3

4

5

6

7

(Z)-1,2,4-heptatriene-6-yne

[ H ]

Scheme 37. Myers-Saito cyclization.



Neocarzinostatin is a bacterial antibiotic that also shows antitumor activity. Here, the

occurrence of a Myers-Saito Cyclization sets the stage for the cleavage of DNA:

Neocarsinostatin (A) represents the class of enediyne natural products that operates via a Myers-

Saito pathway (Scheme 37). In A, 1,8-addition of a thiol to the unsaturated epoxide produces the

highly reactive cumulene intermediate B. Compound B undergoes a Myers-Saito cyclization to

the biradical species C, which in turn abstracts two hydrogen atoms from DNA, again resulting

in double-strand cleavage (Scheme 38).

Scheme 38. Myres-Saito cyclization and the DNA cleavage by Neocarzinostatin chomophore.



1.8.2. Comparison between BC and M-S Cyclization

Contrary to BC, acyclic eneyne allene systems undergo MSC under ambient conditions.

Cyclic eneyne allenes are also extremely reactive like their acyclic counterparts. For synthetic

purposes, organometallic reagents can be used to generate a precursor to the Bergman

Cyclization in which the metal center forms a part of the cumulated unsaturated system; these

cyclizations occur at relatively low temperatures, as shown in the example reported by Finn et al.

(J. Am. Chem. Soc. 1995, 117, 8045). Here the cyclization can be viewed as a Myers-Saito

Cyclization that gives rise to a metal-centered radical (Scheme 39)

Scheme 39. Metal-centered radical and Myers-Saito cyclization.



Comparison of the Bergman and Myers-Saito mechanisms accounts for the source of

differing reactivity (Scheme 40A-B). For instance, the Bergman cyclization proceeds from an

enediyne structure, so its reactivity depends on conformation and electronic effects inherent in

the molecule. In contrast, Myers-Saito cyclization requires formation of the highly reactive

allene; simply accessing this intermediate is paramount to controlling its reactivity.

Scheme 40. Comparison between Myers-Saito and Bergman cyclization.

A key difference between the two mechanisms is the nature of the biradical species formed.

The Bergman cyclization forms the p-benzyne σ,σ biradical intermediate U. The putative singlet

biradical U is thought to be in equibilbrium with a triplet state biradical.

This exchange is

translated via field effects and through-bond effects. Because singlet biradicals are less reactive

than triplet species, the expectation of intersystem crossing is that the rate of hydrogen atom

abstraction increases (Scheme 40).

In contrast to the Bergman reaction, the Myers-Saito reaction proceeds through a 1,4-toluene

σ,π biradical intermediate Q.

Theoretical and experimental evidence supports the existence of an

equilibrium between Q and the zwitterion S.

The increase in ionic character is believed to

decrease the efficiency of DNA cleavage through the established biradical mechanism (Scheme

40).



1.8.3. Controlling the Reactivity of Myers-Saioto Cyclisation

Research into the Myers-Saito mechanism has focused on accessing the highly reactive

enyne allene intermediate. In many systems its formation is sufficient to drive cyclization, as it

releases approximately 15 kcal/mol upon conversion to the σ,π biradical.

As a consequence of its

great reactivity, several methods to mask and unveil the allene have been developed.

1.8.3.1. pH Dependent Myers-Saito Cylization

One promising result was observed upon incubating enediyne systems A and B at 37 oC for

24h with supercoiled DNA (Scheme 41).

In this reaction, the hydroxyl group is believed to

induce allene formation via acid-mediated elimination of the methoxymethyl (MOM) ether. No

rationale for the differing reactivities between A and B was provided. Particularly pH range of 5-

6. Since cancer cells (pH 5.5) are more acidic than normal cells (physiological pH 7.4), this

compound may prove to be of practical importance.

Scheme 41. pH dependent Myers-Saito cylization.

pH Dependent Myers-Saito Cylization

OH

OMOM

Ph Ph

OH

OMOM

PhPh

O

A BIncubation at 37 oC for 24h

with supercoiled DNA

-OH group assisted allene formation via acid-mediated elimination

of the methoxymethyl (MOM) ether

Compound B shows selective reactivity in thepH range of 5-6. Since cancer cells (pH 5.5) are more acidic thannormal cells (physiological pH 7.4), this compoundmight find practical application.



1.8.3.2. Decarboxylation as a Trigger for Myers-Saito Cyclization Decarboxylation of A under basic conditions triggers formation of the allene B. In protic

solvent such as methanol, an ionic pathway prevails (Case 1). The reaction path switches from

radical chemistry likely because of the ability of a protic solvent to stabilize ionic species like C;

methanol then traps the zwitterion to yield F. In benzene, the radical pathway dominates the

reaction as evidenced by the formation of G and H, with no evidence of F (Case 2). Under these

conditions, decarboxylation to afford B followed by Myers-Saito cylization gives D and 1,4-

cyclohexadiene traps the biradical, providing G directly. Use of an ortho methoxy methyl

substituent adds an element of complexity to the reaction pathway, because the biradical D may

undergo intramolecular 1,5 hydrogen abstraction to form the oxygen stabilized radical E leading

to H. Interestingly, when DMF is employed as solvent, the radical pathway proceeds even

without 1,4-cyclohexadiene; however, even 10% methanol directs the reaction along the ionic

pathway (Case 4). The authors suggest that the ionic pathway renders the abstraction of

hydrogen atoms from DNA less likely, thus highlighting the importance of reaction environment

on the Myers-Saito cyclization (Scheme 42).

Scheme 42. Myers-Saito cyclization triggered by decarboxylation.

MeO COOH

OMe

F

F

Ph

OMe

PhAr

OMeBase, Additive

Solvent

Ar

OMe

OMe

Ph

Ar

OMe

OMe

Ph

Ar

OMe

O

Ph[1,5]-H

Shift

Ar

OMe

OMe

Ph

Case 1: Base = Et3N; Additive = 1,4-CHD/O2; Solvent = MeOH; F = 77%; G = 0%; H= 0%.Case 2: Base = No.; Additive = No.; Solvent = DMF/MeOH(9/1); F = 58%; G = 0%; H= 0%.

Case 3: Base = Et3N; Additive = 1,4-CHD; Solvent = Benzene; F = 0%; G = 23%; H= 10%.

Case 4: Base = No.; Additive = No.; Solvent = DMF; F = 0%; G = 0%; H= 31%.

1,4-CHDMeOH

1,4-CHD

Ar

OMe

OMe

Ph

MeO

HH

ArMeO

OPh

A B

C D E

F G H

Myers-Saito Cyclization

Myers-Saito CyclizationTriggerred by Decarboxylation



1.8.3.3. Effect of Steric Hindrance on Mode of Cyclization

The synthetic potential of thermal enyne-allenes reactions was extended by Schmittel and co-

workers. During the course of their study oon eneyne-allenes they found quite unexpected

product which was different from Myers-Saito cyclised product when the the terminus of the

enyne-allenes was attached of an aryl group or sterically bulky groups (e.g, tBu, SiMe3). The

steric bulk of the terminus led to complete switch from the Myers-Saito C2 - C7 cyclization to a

C2 - C6 cyclization, giving rise to a formal ene and Diels-Alder products. This C2 - C6

cyclization reaction is now popularly known as Schmittel cyclization (Scheme 43). Thus, simple

replacement of the H atom at the acetylene end of ene-yne-allene with a phenyl group in

switches the reaction (Scheme 43) from Myers-Saito to Schmittel cyclization.

Scheme 43. Myers-Saito vs. Schmittel cyclization.

In the following example, replacement of –Me group by a tolyl group in acetylenic arm lead

to switch over from Myers-Saito to Schimittel cyclization. This is again attributed to the steric

bulk of the tolyl group.

Scheme 43. Role of steric effect in switching the reaction from Myers-Saito to Schmittel cyclization.

R1R2

R1

R2

H

H

Myers-Saito cyclization

12

3

45 6

712

3

4

56

7

(Z)-1,2,4-heptatriene-6-yne derivative

[ H ]

,biradical intermediate

R1

R2R

C2-C7C2-C6

R RR1

R2

R

123

4

6

7

5R1

R2

R

123

4

6

7

5

,biradical intermediate

H

H

Schmittel cyclization

[ H ]

Switching from Myers-Saito to Schmittel Cyclization: Effect of Steric Hindrence

POPh2

C4H9

Me

H

C4H9

Me

OH

C4H9

POPh2

Me

C4H9

POPh2

H

H

[ H ]Me

POPh2

C4H9

H

C4H9

OH

Me Me

C4H9

POPh2

Me

C4H9

POPh2

Me

H

H

76%

57%

PClPh2/NEt3,

-78 oC, 1,4 CHD

[ H ]PClPh2/NEt3,

-78 oC, 1,4 CHD


Schmittel Cyclization

50 oC ,

(t½ = 1 h)

84 oC ,

(t½ = 1 h)



It is well established that concerted Diels – Alder reactions are prevented by ortho-alkyl

substituents because of steric hinderance, therefore the only option is a stepwise formal Diels-

Alder cycloaddition. During the course of the synthesis of benzocarbazole, transformation of the

–ph linked eneyne-allene to the Schmittel product gave strong evidence for the existence of the

Schmittel biradical intermediate. However, Removal of the bulky phenyl on the acetylene

terminus gave the C2 – C7 type cyclization product in 31% yield. The extra product formed

(15% yield) is a product of addition of biradical intermediate to the hydrogen atom donor present

(1,4-cyclohexadiene, 1,4-CHD) (Scheme 44).

Scheme 44. Role of steric effect in switching the reaction from Myers-Saito to Schmittel cyclization.

Initially it was thought that the switch in the mode of the reaction from Myers-Saito to

Schimittel cyclization or vice versa is as a result of the stabilising effect of the aryl group on the

vinyl radicals. However, further investigation clearly shows that replacement of the hydrogen at

the acetylene unit by a phenyl group raises the barrier of the Myers cyclization significantly,

presumably by steric hindrance and ground state stabilization of the acetylene moiety; therefore

the reaction follows Schimittel cyclization pathway.

NMes

Mes

Ph

Me

N

Me

PhMe

Me

MeMes

N

Me

Ph

Me

MeMesH

N

Me

PhMe

Me

MeMes

N

Ph

Me

H

O

Mes

Mes

Florisil, P2O5, 1,4-CHD,

pyridine, reflux, 30 h

61% recovered

NMes

Ph

H

Me Me

N

H

Me

H

O

Mes

Ph

Florisil, P2O5, 1,4-CHD,

pyridine, reflux, 3 h N

Me

Ph

Me

N

Me

Ph

Me

NMe Ph

15%

31%

+


Schmittel Cyclization



1.9. Selected References

1. (a) Nicolaou, K. C.; Dai, W. M. Angew. Chem. Int. Ed. Engl. 1991, 30, 1387. (b)

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