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Abstract

i

The thesis entitled “Uses of Pyrrole Amino Acid based Scaffolds in Peptidomimetics

and Studies Directed Towards the Total Synthesis of Antascomicin A”, consists of

three chapters:

CHAPTER I: Deals with the synthesis and conformational studies of pyrrole amino acid

based peptidomimetics.

This chapter is divided into two parts:

Part A: Describes the synthesis and conformational studies of linear peptides of pyrrole

amino acid.

Part B: Describes the synthesis and structural studies of pyrrole amino acid containing

peptides.

CHAPTER II: Delineates the synthesis and DNA binding properties of pyrrole amino

acid containing peptides.

CHAPTER III: Describes the Studies Directed Towards the Total Synthesis of

Antascomicin A.

CHAPTER I

Part A

Synthesis and Conformational Studies of Linear Peptides of Pyrrole Amino Acid.

Biological activities of Peptides and proteins depend on their three dimensional

structures in the free state and when interacting with their receptors/acceptors. The

search for relationships between structure and activity has been the principal subject for

designing the selective drugs with no side effects. Initial efforts at finding a connection

Abstract

ii

between structure and activity equated structure with constitution, but it has soon become

evident that configuration was at least of equal importance.

Dehydro amino acids (∆Xaa) are found in many naturally occurring peptide

antibiotics of bacterial origin as well as in some proteins. They have also been used very

extensively as rigid scaffolds in structural studies of peptides. Synthesis of a novel

building block based on a pyrrole amino acid (Paa), 5-(aminomethyl)pyrrole-2-

carboxylic acid (1), its application in peptidomimetic studies as a conformationally

constrained surrogate of the Gly-∆Ala dipeptide residue (2) and the synthesis and

conformational studies of peptides 3 and 4 prepared from this template (Fig-1).

One of the most widely used protocols to restrict conformational degrees of freedom

in small peptides is by covalently stitching the “loose ends” together to fix various torsional

angles at values characteristic of the target structure. Bridging the terminal carbon of an

olefinic analogue of a Gly unit and the Cβ atom of an adjacent ∆Ala residue (2) leads to its

constrained version 1 with fixed ωi, ϕi+1 and probably, ψi+1 torsional angles; 1 was expected

to nucleate interesting structures in peptides 3 and 4.

The target peptidomimetics 3 and 4 were prepared from the pyrrole amino acid (Paa)

which in turn was prepared from pyrrole (Scheme 1)

3 & 4NH

COOMeBocHN

Scheme 1

5 6

NH

BocHNNH

HN

NH

HN

OMe

O

O

O

O

HN

NH

HN

NH

O

O

O

OMeBocHN

O

3

4

H2NN

HN

N

O

O

OH

OH

H

ωiφi+1

2

1

ψi+1

Gly ∆Ala

Figure 1

Abstract

iii

The synthesis of compound 5, i.e. the Boc-protected methyl ester of 1, was started

from pyrrole 6. The 5-formyl pyrrole-2-carboxylic acid methyl ester was prepared from 6 in

three steps following known procedures. A Vilsmeir-Haack reaction on pyrrole 6 furnished

2-formyl pyrrole 7, which was then subjected to Ag2O oxidation followed by treatment with

CH2N2 to give the 2-carbomethoxy group 9 (Scheme 2).

Second Vilsmeir-Haack reaction on pyrrole -2-carboxylic acid methyl ester 9

formylated 5-position 10 (49%) and 4-position 11 (14%). The compound methyl 5-formyl-

pyrrole-2-carboxylate 10 was reduced using NaBH4 in methanol to give an alcohol 12 in

92% yield (Scheme 3).

The alcohol 12 was then treated with CBr4, NaN3 & Ph3P in DMF at ambient

temperature to furnish the desired azido compound 13 in 60% yield. Our next objective

+NH

COOMeOHCNH

COOMe

OHC

POCl3, DMF,CH2-Cl

CH2-Cl

Reflux, 90 min

3.5 : 1

10 11

NaBH4, MeOH, 0 oC, 10 min

92%NH

COOMeHO

Scheme 3

9

10

12

POCl3, DMF,

Ag2O(Alkaline) MeOH:H2O(1:1) R.T, 1h

CH2N2, Ether, 0 oC

NH

NH

CHO NH

COOH

78%1h

67

10 min

85% in two stepsNH

COOMe

Scheme 2

8

9

CH2-Cl

8

CH2-Cl

Reflux

Abstract

iv

was conversion of azide group to primary amine. To achieve this, compound 13 was

treated with Ph3P in MeOH at room temperature. The resulting primary amine was in-

situ protected with Boc2O to furnish 5 in 60% yield (Scheme 4).

Compound 5 was subjected to alkaline hydrolysis using LiOH.H2O in THF-

MeOH-H2O (3:1:1) solution to furnish the carboxylic acid 14. The acid 14 was then

coupled with N-terminal free peptide TFA.H2N-Ala-Leu-Gly-OMe 16 under standard

solution phase peptide coupling using EDCI and HOBt as coupling agent and DIPEA as

base in dry CH2Cl2 solution to afford tripeptide Boc-Paa-Ala-Leu-Gly-OMe 3 in 65%

yield (Scheme 5).

TFA, CH2Cl2

NH

HN

O

COOMeTFA.H2N

O

i) EDCI, HOBt, CH2Cl2, 0 oC

then 16

DIPEA, CH2Cl2, 0 oC-R.TNH

HN

O

COOMeHN

O

3

NH

BocHN

O

5

LiOH.H2O

THF-MeOH-H2O

0 oC-rt, 48 h

NH

BocHN

O

OH

14

15

0 oC-R.T, 1 h

14

16

65%, 8 h

Scheme 5

i) Ph3P, MeOH, R.T, 3 h

ii) Boc2O, MeOH, R.T, 1 h

60%

NH

COOMeBocHN

12

Scheme 4

CBr4, NaN3, Ph3P

DMF, 0 oC-R.T, 1h

60%

NH

COOMeN3

13

13

5

Abstract

v

The acid Boc-Paa-OH 14 was coupled to TFA.H2N-Leu-Gly-OMe 17 under standard

peptide coupling conditions desired above to furnish the compound 18 Boc-Paa-Leu-Gly-

OMe in 73% yield. Boc deprotection of 18 using trifluoro acid (TFA) in CH2Cl2 solution

gave TFA.Paa-Leu-Gly-OMe 19 which was directly used for the peptide coupling reaction in

the next step. The Boc-Val-OH 20 was treated with TFA.Paa-Leu-Gly-OMe 19 to furnish

the peptidomimetic 4 in 75% yield (Scheme 6).

Detailed NMR studies in CDCl3 and DMSO-d6 revealed that pyrrole amino acid

based peptidomimetic 3 and 4 γ-turn type structure as shown in Figure 1 involving

intramolecular hydrogen bonding between the pyrrole NH and the carbonyl of the

previous residues, BocC=O (3) or ValC=O (4) (Fig-2).

14 17 EDCI, HOBt, CH2Cl2

DIPEA, 0 oC-R.T

73%, 12 h

HN

NH

COOMe

O

NH

BocHN

O

18

18 TFA, CH2Cl2

0 oC-R.T, 1 h

HN

NH

COOMe

O

NH

TFA.H2N

O

19

BocHNOH

O

20

EDCI, HOBt, CH2Cl2, 0 oC

then 19

DIPEA, CH2Cl2, 0 oC-R.T

75%, 12 h

4

Scheme 6

+

Abstract

vi

Figure 2. Schematic representation of the structures of 3 and 4 with the long range rOes seen in their

ROESY spectra.

In conclusion, the rigid scaffold of the pyrrole amino acid described here can serve as

a conformationally constrained template that may find useful application in developing novel

peptidomimetics with interesting structures and useful properties.

BocHNNH

HN

NH

HN

OMe

O

O

O

O

HN

NH

HN

NH

O

O

O

OMeBocHN

O

3 (in DMSO-d6)

4 (in DMSO-d6)

H H

H H

N

HN

NH

HN

OMe

O

O

O

O

3 (in CDCl3 with 12% DMSO-d6)

H H

HNO

OtBu

H

HH

N

HN

NH

OMe

O

O

O

4 (in CDCl3 with 12% DMSO-d6)

H H

HNO

H

HH

NH

H

H-Bond

rOeOtBu

O&

34

Abstract

vii

Part B

Synthesis and Structural Studies of Pyrrole Amino Acid Containing Peptides.

The recognition of β-sheet structures is important for protein-protein interactions.

Small organic molecules with hydrogen bonding pattern and structure complementary to

β-sheets are promising for interception of protein-protein interaction, for stopping protein

aggregation or stabilization or mimicking β-sheet structures. This has led to the design

and synthesis of a large number of conformationally constrained scaffolds, which help to

induce β-turns in short peptides, often a prerequisite to β-hairpin nucleation.

Here we describe the design, syntheses and detailed conformational studies of

three Paa-containing peptides Boc-Paa-Paa-L-Pro-Gly-Ala-Paa-Paa-OMe 2, Boc-Paa-

Paa-D-Pro-Gly-Xaa-Paa-Paa-OMe (3: Xaa = Ala; 4: Xaa = Val) with repeating units of

Paa dimers at both N- and C-termini (Fig-1).

Figure 1

H2NN

OH

OHωi

φi+1

1 (Paa)

ψi+1

1

2

34

5

6

HN

NH

N

O

BocHNNH

OO

NHHN

O

NH

HN

ONH

OHNMeO2C

2

Abstract

viii

It was envisaged that the D-Pro unit with a φ value of +60 ± 20° can induce a

reverse turn that can be further stabilized by noncovalent interactions facilitated by the

near planar disposition of the Paa-dimers at both ends with fixed ωi, ϕi+1 and probably,

ψi+1 torsional angles leading to the formation of hairpin architecture.

The target peptides 2, 3 and 4 were prepared from Boc-Paa-Paa-OMe 5 which in

turn was prepared from pyrrole amino acid (Paa) (Scheme 1).

The peptides 2, 3 and 4 were synthesized from pyrrole amino acid 6 (Paa). The

required dipeptide Boc-Paa-Paa-OMe 5 was prepared following solution phase peptide

coupling procedure. The coupling of Boc-Paa-OH 7 with TFA.H2N-Paa-OMe 8, using

HOBt and EDCI as coupling agents and DIPEA as base in CH2Cl2 gave the dipeptide

Boc-Paa-Paa-OMe 5 in 85% yield (Scheme 2).

NH

BocHNHN

NH

COOMe

O

2, 3 & 4

Scheme 1

5

6

HN

NH

N

O

BocHNNH

OO

NHHN

O

NH

HN

ONH

OHNMeO2C

R

3 [R = Me], 4 [R = (CH3)2CH]

LiOH.H2O

THF-MeOH-H2O

0 oC-rt, 48 h

NH

BocHN

O

OH

7

NH

BocHN

O

OMe

6

Abstract

ix

The carboxylic acid Boc-Paa-Paa-OH 9 was coupled with TFA.L-Pro-Gly-OMe

10 and TFA.D-Pro-Gly-OMe 11 using the same conditions desired above to furnish the

tetrapeptides 12 and 13 in 72% and 76% yields respectively (Scheme 3).

The other two tripeptides Boc-Ala-Paa-Paa-OMe 16, Boc-Val-Paa-Paa-OMe 18 were

prepared following the same protocol as described above (Scheme 4).

6TFA, CH2Cl2

NH

COOMeTFA.H2N

8

7 EDCI, HOBt, CH2Cl2, 0 oC

NH

BocHNHN

NH

COOMe

O

then 8


85%5

Scheme 2

NH

BocHNHN

NH

COOH

O

5LiOH.H2O

THF-MeOH-H2O0 oC-r.t, 48 h

9


then 10


72%

12

HN

NH

N

O

BocHNNH

OO

NHMeO

O

EDCI, HOBt, CH2Cl2, 0 oC

then 11


76%

13

HN

NH

N

O

BocHNNH

OO

NHMeO

O

9

Scheme 3

Abstract

x

Our next task was to couple the Boc-Paa-Paa-L-Pro-Gly-OMe 12, with the

tripeptide Boc-Ala-Paa-Paa-OMe 16, to get the target peptide 3. Accordingly, the

tetrapeptide 12 upon treatment with LiOH.H2O in THF-MeOH-H2O gave Boc-Paa-Paa-

L-Pro-Gly-OH 19, tripeptide Boc-Ala-Paa-Paa-OMe 16 was treated with TFA in DCM

to give TFA.H2N-Ala-Paa-Paa-OMe 20 which was subsequently coupled with Boc-Paa-

Paa-L-Pro-Gly-OH 19 under standard peptide coupling conditions as discussed above to

furnish Boc-Paa-L-Pro-Gly-Ala-Paa-Paa-OMe 2 in 65% yield (Scheme 5).

LiOH.H2O

THF-MeOH-H2O, 0 oC-rt NH

BocHNHN

NH

O O

HN

OH

O

O

N12

3 h19

16TFA, CH2Cl2

NH

HN

NH

COOMe

O

TFA.H2N

HN

O

20

BocHNOH

O

EDCI, HOBt, CH2Cl2, 0 oC,

then 14


15

16

12 h, 78%

BocHNOH

O

i) EDCI, HOBt, CH2Cl2, 0 oC,

then 14


1712 h, 73%

18

Scheme 4

TFA, CH2Cl2NH

TFA.H2NHN

NH

COOMe

O

5

14

Abstract

xi

The other two peptidomimetics 3 and 4 were synthesized by attaching TFA.H2N-

Ala-Paa-Paa-OMe 20 and TFA.H2N-Val-Paa-Paa-OMe 21 respectively, to the Boc-Paa-

Paa-D-Pro-Gly-OH 22 following the same reaction protocol, as described above for the

synthesis of 2 (Scheme 6).

18TFA, CH2Cl2

NH

HN

NH

COOMe

O

TFA.H2N

HN

O21

19HN

NH

N

O

BocHNNH

OO

NHHN

O

NH

HN

ONH

OHNMeO2C

2

i) EDCI, HOBt, CH2Cl2, 0 oC

then 20


8h, 65%

Scheme 5

13

LiOH.H2O

THF-MeOH-H2O, 0 oC-rt

3 h

HN

NH

N

O

BocHNNH

OO

NHHO

O

22


then 20


8 h, 65%

HN

NH

N

O

BocHNNH

OO

NHHN

O

NH

HN

ONH

OHNMeO2C

3

Abstract

xii

NO

NH

O

O NH

NH

NH

NH

NH

O

O

O

NH

O NH

O

NH

NH

O

H

H

HH

H

OMe

Detailed NMR studies in CDCl3 and DMSO-d6 and subsequent constrained MD

simulations revealed that in pyrrole amino acid based peptidomimetic 4, a type-II′ β-turn

is nucleating around the D-Pro-Gly residues. This leads to subsequent stabilization of β-

hairpin formation.

(a) (b)

Figure-2 (a) Schematic representation of the proposed structure of 4 with the long-range ROEs seen in the

ROESY spectrum. (b) One of the 50 energy-minimized structures of 4 sampled during the 300 ps

simulated annealing MD studies.


then 21


12 h, 65%

HN

NH

N

O

BocHNNH

OO

NHHN

O

NH

HN

ONH

OHNMeO2C

4

Scheme 6

Abstract

xiii

In this structure, the Val(5)NH is strongly hydrogen bonded to Paa(2)C=O

intramolecularly (i + 3 → i) leading to a 10-membered ring structure of type II′ β-turn,

supported by the φ, ψ angles of D-Pro and Gly (70°, −108° and −106°, 23°, respectively).

This induces nucleation of three additional hydrogen bonds, Paa(2)pyrroleNH →

Val(5)CO, Paa(6)PyrroleNH → Paa(1)CO and Paa(1)PyrroleNH → Paa(6)CO, leading

to the hairpin formation. Comparison of the structures of 3 and 4 makes it evident that

the replacement of Ala by Val in the 5-position helps compound 4 to adopt the hairpin

conformation due to the restricted rotation about χ1 of Val, 57° and 181° in Figure 2(b).

In conclusion, the known propensity of D-Pro-Gly unit to induce type II′ β-turn

coupled with the sheet forming tendency of pyrrole amino acid (Paa) can lead to a very

well defined β-hairpin conformation in short peptides.

CHAPTER II

Synthesis and DNA Binding Properties of Pyrrole amino acid containing peptides

Many research efforts have been aimed at targeting specific sequences in DNA

with synthetic ligands with the idea of designing both drugs and molecular probes for

DNA polymorphism. The minor groove of the double helical DNA is becoming a site of

great interest for developing new drugs since it is the site of non-covalent high sequence

specific interactions for a large number of small molecules. Minor groove binders are

one of the most widely studied class of agents characterized by high level of sequence

specificity and they are still an interesting class of DNA ligands which demonstrates to

possess several biological activities. In fact, they exhibit antiprotozoal, antibiotic,

antiviral properties. Furthermore, some of these have shown antitumor activity and can

be considered for therapeutical applications.

It was found that the flexibility of the ligand molecule allowed a substantial

resetting of the curvature of the ligand, both with respect to the twist along the groove

and also with respect to matching the bottom of the groove. Pyrrole amino acid (Paa),

5-(aminomethyl)pyrrole-2-carboxylic acid 1 is a new class of building block that has

been used in peptidomimetic studies as a structurally restricted surrogate of the Gly-∆Ala

dipeptide isostere (chapter 1). The flexible methylene spacer between the amino group

Abstract

xiv

and the pyrrole ring in 1 is expected to render in peptides derived from it the curvature

that is necessary to bind in the minor groove of DNA. Herein we describe the synthesis

and DNA binding properties of three Paa dimer-based polyamides 2-4 (Figure 1).

Figure1. Pyrrole amino acid (Paa) 1 Paa dimmer based polyamides 2-4.

The peptide 2 was synthesized from Pyrrole amino acid (paa), the required dipeptide

Boc-Paa-Paa-OMe 5 was prepared following solution phase peptide coupling procedure.

6

LiOH.H2O

THF-MeOH-H2O

0 oC-rt, 48 h

NH

BocHN

O

OH

7

NH

BocHN

O

OMe

6

TFA, CH2Cl2

NH

COOMeTFA.H2N

8

NH

H3NHN

NH

COOMe

O

NH

COOHH2N

1

+

2

NH

HN

HN

NH

COOMe

O

H3N

H3N

O

+

+

3

NH

H3NHN

NH

O

+ HN

NH3

+O

OMe

O

4

Abstract

xv

The compound 3 was prepared by coupling Boc-Lys(Boc)-OH 9 with TFA.H2N-Paa-

Paa-OMe 2 using 1-(3-dimethylaminopropyl)-3-ethylcarbodimide hydrochloride (EDCI)

and 1-hydroxybenzotriazole (HOBt) as coupling agents and DIPEA as base in dry

CH2Cl2 solution to furnish the peptide 10 in 65% yield. The Boc protection of 10 was

removed by treating it with trifluroacetic acid (TFA) in CH2Cl2 solution at 0oC to give

compound 3 (Scheme 2).

The other peptide 4 was synthesized by attaching Boc-Paa-Paa-OH 11 to the H2N-

Lys(Boc)-OMe 13 following the same reaction protocol, as described above for the

synthesis of 3 (Scheme 3).


NH

BocHNHN

NH

COOMe

O

then 8


85%5

NH

BocHNHN

NH

COOMe

O

5

TFA, CH2Cl2 2

Scheme 1

BocHNOH

O

NHBoc

EDCI, HOBt, CH2Cl2, 0oC

65%

then 2

DIPEA, CH2Cl2, 0oC-R.TNH

HN

NH

COOMe

O

BocHN

HN

O

NHBoc

10

NH

HN

NH

COOMe

O

BocHN

HN

O

NHBoc

10

TFA, CH2Cl23

9

Scheme 2

Abstract

xvi

The binding of these three newly synthesized polyamides containing pyrrole

amino acid (Paa), with duplex DNA was followed by UV melting, CD spectroscopy, an

ethidium bromide exclusion experiment followed by fluorescence spectroscopy along

with a gel electrophoretic mobility assay. Studies showed that all the ligands are able to

bind duplex DNA. In each of these ligands, the number of building blocks is only two

and yet they showed considerable binding affinities. It was found earlier that the bis-

pyrrole peptide did not exhibit any detectable binding with duplex DNA. From

comparative binding studies on bis-, tri-, tetra- and penta-pyrrole peptides of the

distamycin analogues with DNA it was concluded that the minimum number of pyrrole

carboxamide units for the onset of DNA binding, in the absence of the N-terminus amide

is three. In our study, we have observed that ligand 2 is able to bind to ds-DNA with a

considerable apparent binding affinity (Kapp= 2 × 104 M

−1) in spite of the fact that this

ligand contains only two pyrrole-based building blocks. The improvement in the binding

5

LiOH.H2O

THF-MeOH-H2O

0 oC-rt, 48 h

NH

BocHNHN

NH

COOH

O11

FmocHNOMe

O

NHBoc

20% Piperidine, DCMH2N

OMe

O

NHBoc

12

13


65%

then 13


NH

BocHNHN

NH

O

HN

NHBocO

OMe

O

14

TFA, CH2Cl2 4NH

BocHNHN

NH

O

HN

NHBocO

OMe

O

14

Scheme 3

Abstract

xvii

affinity may be due to the presence of the methylene spacer between the amino group

and the pyrrole ring in the ligand, which makes the ligand more flexible to fit in the

minor groove of the duplex DNA. Ligand 3 binds to duplex DNA with three times higher

affinity (Kapp= 6 × 104 M

−1) implying that the extra charge on the ligand improves the

binding ability of ligand. Ligand 4 with a positive charge placed at both ends of the

polyamide binds to duplex DNA with twice the affinity of 3 with two positive charges at

the same end. This result clearly indicates that the spatial distribution of the charge plays

a crucial role in the binding event of the ligand. In ligand 4 as the two charges are located

at either end of the polyamide, these charges can neutralize the negative charges of the

DNA phosphate backbone more efficiently giving increased electrostatic interactions.

In summary we have described the synthesis of polyamides using the monomeric

building block pyrrole amino acid (Paa), 5-(aminomethyl)pyrrole-2-carboxylic acid. The

flexible methylene spacer between the amino group and the pyrrole ring in this monomer

was expected to render the curvature in polyamides derived from it that is necessary to

bind in the minor groove of DNA. DNA binding studies revealed that polyamides with

two positive charges bind with affinities of the order of 105. We also observed that the

polyamide with a positive charge placed at either end of the polyamide binds to the DNA

duplex with twice the affinity compared to that with two positive charges at the same

end. These results will help in the more rational design of new minor grove binding

agents.

CHAPTER III

Studies Directed Towards the Total Synthesis of Antascomicin A

FK 506 (tacrolimus) and rapamycin (sirolimus) are potent immunosuppressive agents in

human clinical organ transplantation. The biological mode of action of these promising

natural products have been extensively investigated over the past decade. For example,

FK-506 is known to form an active complex with the cytosolic immunophilin FKBP12,

which subsequently binds to the secondary proteinous target calcenarium. The latter

interaction is responsible for potent immunosuppressive activity of FK-506. On the other

Abstract

xviii

hand, it has recently been discovered that FKBP is present in the brain at levels 10-40

times higher than in the immune system, suggesting the possibility of nervous system

roles for immunophilins. Subsequently, FK-506 has been reported to elicit neurite

outgrowth and neuroprotective effects in neuronal cultures at pico molar concentrations.

Although the mechanism of neuronal activity of FKBP has not yet been clarified, it has

been shown that the neurotrophic properties of FKBP ligands are independent of

calicineurin-mediated effects. Thus, FKBP ligands are now considered as potential lead

compounds for the development of drugs for treatment and cure of neurodegenerative

disorders such as Alzheimer's and Parkinson's diseases. With regard to clinical use,

efforts have been devoted to the design of neurotrophic FBBP ligands that are non-

immunosuppressive.

Macrolide antibiotics antascomicin A-E were found in a fermentation broth of a

strain of Micromonospora, which was isolated from a soil sample collected in China

(Fig-1). The molecular architecture of antascomicin A was determined by extensive

NMR studies. Antascomicin A exhibit potent binding affinity to FKBP12 (IC50 2.0 nM)

and antagonize the immunosuppressive effect of FK506 and rapamycin, but interestingly

it does not show immunosuppressive activity.

Antascomicin A 1 is an enticing target for the synthetic chemist as it has a

complex polyketide structure that includes both lactol and lactam functionalities, 11

stereogenic centers, and a masked 1,2,3-tricarbonyl unit. The wealth of biological

functions and chemical structures prompted us to initiate a program aimed at the total

synthesis of antascomicin A.

Abstract

xix

An RCM approach was contemplated to construct the macrocyclic ring of antascomicin A.

N

OO

O

HO

R2O

O

O

HO

O

n

R1 R3

ANTASCOMICINS

A: R1 = H, R2 = H, R3 = H, n = 2B: R1 = OH, R2 = H, R3 = H, n = 2C: R1 = OH, R2 = H, R3 = H, n = 2D: R1 = H, R2 = H, R3 = H, n = 1E: R1 = H, R2 = H, R3 = OH, n = 2

Figure 1

N

OO

O

HO

HO

O

O

HO

O

N

OO

O

TBSO

TBSO

O

O

TMSO

O

N

OO

O

OH

O

TMSO

O

OTBS

OTBSOH

RCM Reaction

1 2

3

4

+

Scheme 1. Retrosynthetic analysis of antascomicin A (1).

Abstract

xx

Retrosynthetic analysis (Scheme 1) reveals that an acyclic precursor like 2 was

ideally suited to carry out the planned RCM reaction. Triene 2 could be easily assembled

by coupling the C1-C21 unit 3 with the C22-C34 fragment 4.

Synthesis of C1-C21 unit

Our synthesis commenced with the mono-benzylation of commercially available

pentane-1,5-diol 5. Among two free hydroxyl groups of pentane-1,5-diol, one was

selectively protected using one equivalent each of sodium hydride and benzyl bromide in

THF to give mono-benzyl protected pentate-1,5-diol 6 in 68% yield. The Swern

oxidation of 6 provided aldehyde 7, in 92% yield. Asymmetric aldol addition of the

titanium enolate derived from the N-propanoyl oxazolidinethione 8 to aldehyde 7, gave

the ‘non-Evans’ syn aldol product 9 as the only isolable diastereomer in 78% yield.

Reduction of 9 with sodium borohydride in ethanol gave the diol 10 in 90% yield. Diol 10

was subjected to disilylation by using TBSCl, imidazole and catalytic amount of DMAP

in DMF to furnish the di-TBS ether 11 and finally debenzylation by hydrogenation gave

12 in 72% yield from 10 (Scheme 2).

OHHO OHBnO

HBnO

5 6

O

(COCl)2, DMSO, Et3N

CH2Cl2, -78 oC

NaH,BnBr,TBAI

THF, 0 oC-R.T

68%

12 h

7

O N

S O

Bn

BnO H

O

O N

S O

Bn

OBn

OH

TiCl4, DIPEA, CH2Cl2,

-78 oC, 15 min, then 7,

-78 oC to 0 oC, 3 h (78%)

8

79

NaBH4, EtOH,

0 oC, 10 min

90%

Abstract

xxi

Compound 12 was converted to acid 14 in a two-step oxidation protocol. First primary

alcohol 12 was converted to an aldehyde 13 in 94% yield, using Swern oxidation

procedures, which, without further purification and characterization was oxidized to acid

14 with 1.3 equivalents of NaClO2 and 1.3 equivalents of NaH2PO4 in t-BuOH and 2-

methyl-2-butene. Compound 14 was used to carry out N-acylation of the chiral

oxazolidinone 15, derived from D-Phe, under the mixed anhydride method to furnish 16

in 85% yield. Diastereoselective alkylation of the Na-enolate of 16 with MeI was

followed by reductive removal of the chiral auxiliary to give alcohol 18 as the only

isomer in 46% overall yield (Scheme 3).

OBn

OH

HO

10

OH

OTBS

TBSO

1. TBDMSCl, imidazole, DMAP (cat)

DMF, 0 oC to rt, 24 h

2. H2, Pd/C, EtOAc

72% in two steps

12

Scheme 2

12 OH

OTBS

TBSO

O

OTBS

TBSO OH

HN O

O

Bn

N

OTBS

TBSO

O

O

O

Bn

1. Swern oxidation

2. NaClO2, NaH2PO4,

2-methyl-2-butene, tBuOH

0 oC to rt

86% in two steps 14

Piv-Cl, Et3N, THF

then 15, LiCl, THF,

−20 oC to rt, 4 h

85%

15

16

1. NaHMDS, THF,

then MeI, −78 oC, 2 h

2. LiBH4, Et2O, H2O

0 oC, 10 min

46% in two steps 18

Scheme 3

Abstract

xxii

Swern oxidation of 18 gave an intermediate aldehyde 19, which was reacted with

the Li-enolate of ethyl acetate to give the β-hydroxy ester 20 as a mixture of isomers in

85% yield. Saponification of 20 and subsequent coupling with L-pipecolic acid methyl

ester furnished the amide 22 in 67% overall yield (Scheme 4).

Oxidation of the β-hydroxy amide 22 with Dess-Martin periodinane (DMP) gave

an ‘1,2,3-triketo’ intermediate, which was subjected to desilylation resulting in the

spontaneous formation of the hemiketal 24 in 60% yield. Disilylation of 24 gave a di-

TMS-ether intermediate 25. Brief exposure of this intermediate di-TMS-ether to mild

acid selectively deprotected the primary hydroxyl group to furnish the TMS-ether of the

hemiketal 26 in 70% yield. Finally oxidation of 26 gave the aldehyde 27 in 96% yield.

18

OTBS

TBSO

OH O

N

CO2Me

OTBS

TBSOCO2Et

OH

20

1. LiOH, THF-MeOH-H2O (3:1:1)

0 oC to rt, 4 h

2. L-Pip-OMe, EDCI, HOBt, DIPEA

CH2Cl2, 0 oC to rt, 3 h

67% in two steps 22

1. Swern oxidation

2. LDA, CH3CO2Et, THF

−78 to 0 oC, 30 min

85% in two steps

Scheme 4

22

O

OO

NOMe

O

HO

OH

1. DMP, py, CH2Cl2,

rt, 2 h

2. HF, CH3CN, 3 h

60% in two steps

1. TMSOTf, 2,6-lutidine,


2. 0.1(N) HCl, THF, rt,

15 min

70% in two steps

24

Abstract

xxiii

Synthesis of the C17-C21 (35) fragment

The synthesis of the C17-C21 (35) fragment started with the pentane-1,5-diol 5.

One of the hydroxyls of 5 was protected as tert-butyldimethylsilyl ether by using TBSCl

and NaH in THF at 0°C to room temperature for 3-4 h to afford compound 28 in 72%

yield. The aldehyde 29 was obtained by the oxidation of the primary hydroxyl group in

28 by employing the Swern oxidation condition. The aldehyde 29 was reacted with CHI3

in the presence of anhydrous CrCl2, generated in situ from CrCl3 and LAH, following the

Takai protocol to introduce an E-vinyl iodide moiety 30, in an overall yield of 73% after

two steps. Acid-catalyzed desilylation of 30 gave 31 in 81% yield. Tosylation of 31 was

followed by nucleophilic substitution of the tosylate group by PhSe−, generated in-situ by

sodium borohydride reduction of diphenyl diselenide, giving the phenylselenide 33, in

78% overall yield, which was then subjected to oxidation-elimination process. Oxidation

of selenide 33 using mCPBA and subsequent β-elimination of the resulting selenoxide

furnished the diene 35 in 71% yield (Scheme 6).

O

OO

NOMe

O

TMSO

OH

O

OO

NOMe

O

TMSO

H

O26

SO3-py, DMSO

Et3N, CH2Cl2,

0 oC, 1 h

96%

27

Scheme 5

TBSO OH

28

HO OH

TBS-Cl, NaH, THF

0 oC-r.t, 3h, 72%

5

Abstract

xxiv

Coupling of C1-C16 Moiety (27) and C17-C21 (35):

Coupling of C1-C16 (27) and C17-C21 (35) fragments to build the target C1-C21

moiety is shown in Scheme 7. A Nozaki-Hiyama-Kishi coupling was employed to carry

out the coupling, giving the coupled product 36 in 62% yield as a mixture of isomers.

Finally, Dess-Martin oxidation of 36 furnished dienone 37, the target C1-C21 fragment of

antascomicin A, in 85% yield.

1. Swern oxidation

2. CHI3, CrCl2, THF,

0 oC to rt, 1 h

73% in two steps

TBSOI

CSA, CH2Cl2, MeOH

0 oC, 30 min

81%

HOI

30

31

1. TsCl, Et3N, DMAP (cat),


2. PhSeSePh, NaBH4, EtOH,

THF, 0 oC to rt, 4 h

79% in two steps

PhSeI

33

1. mCPBA, CH2Cl2, −20 oC, 2 h

2. DIPA, CCl4, reflux, 4 h

71% in two steps

I

35

28

33

Scheme 6

27NOZAKI KISHI COUPLING

CrCl2, NiCl2 (0.1%), DMSO

N

OO

O

OMe

O

TMSO

HO

67%

R.T, 5 h

+ 35

36

Abstract

xxv

Synthesis of the C22-C34 fragment

For the synthesis of the C22-C34 fragment 4 of antascomicin A, D-quinic acid was

chosen as the starting material. Scheme 8 outlines the details of the synthesis. D-(-)-

Quinic acid 38 was transformed into the intermediate 6 in six steps and in 33% overall

yield following the procedure reported earlier.

OH

HO

HO

O

OH

D(-)QUINIC ACID

PhCHO, PTSA

Benzene, Reflux

OO

OH

OO

Ph

KH, CS2, MeI,

Bu3SnH, AIBN,

Toluene, Reflux, 3 h64% in two steps O

O

OO

Ph

NBS, AIBN (cat)

Benzene, Reflux, 2 h

Bu3SnH, AIBN

OO

BzO

K2CO3(Cat), MeOH

BzO

HO CO2Me

OH

0 oC, 10 min

70%

68% in two steps85%

12 h

40

38

39

THF, 0 oC - R.T, 2 h

OO

O

OO

Ph

40

CS-Me

S

41

Br

42

42

Toluene, Reflux, 3 h OO

BzO

43

Scheme 8

44

Scheme 7

DMP, DCM, R.T

N

OO

O

OMe

O

TMSO

O

85%

1 h36

37

Abstract

xxvi

Silylation of 44 was followed by reduction to furnish the diol 46 in 76% yield.

Selective acylation of the primary hydroxyl group, silylation of the secondary hydroxyl

and deprotection of the acetate gave the disilylated intermediate 49 in 78% yield in three

steps. Finally oxidation of 49 furnished the aldehyde 50 in 95% yield (Scheme 9).

Horner-Wadsworth-Emmons olefination of 50 with the (1S,2R)-(+)-

norephedrine-derived keto-phosphonate 51 using LiCl-DIPEA provided an α,β-

unsaturated amide intermediate, which was hydrogenated to furnish the N-acylated chiral

auxiliary 53 (Scheme 10).

46

HO

TBSOOAc

AcCl, 2,4,6,CollidineTBS-OTf, 2,6-lutidine

K2CO3, MeOH

TBSO

TBSOOH

DCM, -78 oC, 3 h

88% 47

DCM, 0oC, 5 min, 97%

TBSO

TBSOOAc

48

0 oC-R.T,1 h, 98%

49

TBSO

TBSOH

O

SO3-Py Complex

DCM, DMSO, NEt3

0 oC

95%

50Scheme 9

TBS-OTf

2, 6lutidine, DCM, 0 oC

94%

10 min

BzO

TBSO CO2Me

DIBAL, DCM, -78 oC-0 oC

HO

TBSOOH

86%

2 h

45

44

46

Abstract

xxvii

The two chiral centers in compound 61 were planned to be generated by an Evans

aldol reaction between 53 and the aldehyde 60, which in turn was prepared from

commercially available methyl (R)-(-)-3-hydroxy-2-methylpropionate 54. Acid catalyzed

benzylation of 54, in the presence of O-benzyltrichloroacetamidate and catalytic amount

of triflic acid in a mixture of cyclohexane and methylene chloride (2:1) at 0oC afforded

the benzyl ether 55 in 87% yield. Treatment of 55 with LiAlH4 in ether at 0oC gave the

alcohol 56 (98% yield). Compound 56 was then transformed into 60 in four steps, as

shown below, and in 65% overall yield (Scheme 11).

Ph

N

Me

O

O O

P(O)(OEt)2

Ph

N

Me

O

O O

OTBS

OTBS

TBSO

TBSOO

H50

1. 10, LiCl, DIPEA, CH3CN, rt, 6 h

2. H2, Pd-C, EtOAc, rt, 1 h71% in two steps

53

51

Scheme 10

OHMeO

O

Cl3C OBn

NH

Cyclohexane, DCM

0 oC-R.T, 5 h, 87%

LAH, Et2O, 0 oC

LiBH4, Et2OOBnHO

OBnH

O

i) Swern oxidation

Swern oxidation

ii) Ph3P=CH-CO2Et

80%

DCM, R.T, 12 h

54

TfOH (cat)

55

OBnMeO

O 30 min, 98%

56 OBnEtO

O

0 oC- R.T, 48 h

84%58 59

59

60

Scheme 11

56

Abstract

xxviii

Enolate of 53 was reacted with the aldehyde 60 to furnish the aldol product as a

single isomer 61. The stereochemistries of the newly generated chiral centers were

assigned on the basis of earlier reported work. The aldol adduct was then silylated and

the chiral auxiliary was removed by reduction with lithium triethylborohydride to furnish

63 in 40% yield in three steps. A two-step protocol was followed to convert the

hydroxymethyl of 63 to a methyl group – tosylation followed by nucleophilic

substitution of the tosylate group with hydride – to furnish 65 in 70% yield.

Debenzylation of 65 by catalytic hydrogenation, oxidation of the resulting primary

hydroxyl group, one-carbon Wittig olefination and finally an acid-catalyzed desilylation

step furnished the target fragment 4 in 75% yield (Scheme 12).

63

OTBS

OTBSOH

BnO

OTES

Ph

N

Me

O

O O

OTBS

OTBS

53

1. Bu2BOTf, Et3N, CH2Cl2, −78 oC

then 60, −78 oC to rt, 12 h

2. TESOTf, 2,6-lutidine, CH2Cl2,

0 oC, 5 min

3. LiEt3BH, Et2O, 0 oC, 30 min

40% in three steps

63

1. TsCl, Et3N, DMAP (cat)


2. LiEt3BH, THF, −20 to

0 oC, 5 h

70% in two steps

OTBS

OTBS

BnO

OTES

65

1. H2, Pd-C, EtOAc, rt, 1 h

2. SO3-py, Et3N, DMSO, CH2Cl2, 0 oC, 1 h

3. (Ph3P-CH3)I, NaNH2, Et2O, 0 oC, 10 min

4. CSA (cat), MeOH, CH2Cl2, 0 oC, 5 min

75% in four steps

OTBS

OTBS

OH

4

65

Scheme 12

Abstract

xxix

Coupling of C1-C21 Moiety (37) and C22-C34 Moiety (4):

Having synthesized both the fragments 37 and 4, the stage was know ready to

couple both the fragments to get compound 2. Initially our target was to deprotect the

methyl ester of compound 37 to get acid 3. However, all attempts to convert acid 3 from

the corresponding compound 37 by several methods did not give the corresponding

product. Our efforts to convert α,β-enone ester 37 to acid 3 by several methods like

LiOH, Ba(OH)2, TMSOK, TMSI are ended with decomposition of compound 37. Hence

we were forced to convert methyl ester 37 to an allyl ester by trans esterification method.

Next allyl ester 66 was successfully converted to acid 3 by using catalytic amount of

Pd(Ph3P)4 and morpholine in THF gave compound 3 in 60% yield (Scheme 13).

Compound 3 and 4 were successfully coupled by using 1 equivalent of DCC as a

coupling agent and catalytic amount of DMAP at -20oC in CH2Cl2 to furnish the coupled

product triene 2 in 60% yield. The triene 2 was now ready for the crucial Ring Closing

metathesis reaction (RCM). We thought that, the ring closing would give the desired

product, protected antascomicin A 67. But the RCM reaction method using 0.1 equivalents

of 2nd generation Grubbs catalyst in 10-2 M CH2Cl2 in dilution conditions failed to give the

N

OO

O

OMe

O

TMSO

O

Bu2SnO, Allyl alchohol

N

OO

O

O

O

TMSO

O

Reflux, 24 h

37

52%

66

66 Pd(PPh3)4, Morpholine, THF

60%

R. T, 3 h

N

OO

O

OH

O

TMSO

O

3Scheme 13

Abstract

xxx

desired product 67 and gave rise to an unwanted product (Scheme 14). Our efforts with other

solvents like benzene and toluene also could not provide the expected product. The synthesis

suffered a major setback at this point and our achievement became restricted up to the

synthesis of compound 2 leaving the total synthesis still elusive.

3DCC, DMAP, DCM, -20oC, 7 h

60%

N

OO

O

TBSO

TBSO

O

O

TMSO

O

4+

2

2

Scheme 14

N

OO

O

TBSO

TBSO

O

O

TMSO

O

DCM (10-2M), R.T, 24 h

2nd Generation Grubbs catalyst, 10 mol%

//

67

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