Research Statement

Dr. Gururaj M. Shivashimpi

Research Assistant at NCL-Pune. India

Resolution of (R)-Ondansetron.HCl

[Emcure Pharmaceuticals, India]

For treating nausea and vomiting

Preparation of Roflumilast

[Cipla Pharmaceutical Co., India]

Anti-inflammatory drug

Preparation of Nevirapine intermediates

[Emcure Pharmaceuticals, India]

For treating HIV-I infection

Preparation of Galanthamine Intermediates

[Emcure Pharmaceuticals, India]

For treating Alzheimer's disease

Process Development of Generic Drugs [Industry sponsored projects]

Nishino Laboratory

Thesis Title: “Molecular Design of Histone Deacetylase Inhibitors

by Aromatic Ring Arrangement”

Doctoral Study (PhD) in Japan

Graduate School of Life Science and Systems Engineering

Kyushu Institute of Technology, Kitakyushu. JAPAN

Major post translational

modifications

Mai, A., Massa, S., et al. Med. Res. Rev. 2005, 25, 261–309.

• The chromatin structure consists of a histone octamer wrapped with 146 base pairs of DNA.

• The histone octamer composed each of the four core histones and basic N-terminal histone tails

protrude from the core nucleosome.

• The Epigenetic changes modulate the gene expressions and are recognized as integral to

the pathogenesis of many diseases like cancer.

A

M

P

Ub

Acetylation

Methylation

Ubiquitination

Phosphorylation

Histone Deacetyalses and Epigenetic Regulation

DNA

Histone 8-mer

N-terminal region

Histone

acetyltransferase

(HAT)

Histone

deacetylase

(HDAC) Deacetylated histone

Transcriptional deactivation

of pre-programmed set of genes

Cell growth

Tumor growth

Hyper acetylated histone

Transcriptional activation

of pre-programmed set of genes

Cell growth arrest, differentiation

And/or apoptosis

Tumor suppression

Histone Deacetyalses and Epigenetic Regulation

Structure of histone deacetylase-like protein (HDLP) co-crystallized with TSA *

*Finnin, M. S., Donigian, J. R., et al.

Nature 1999, 401, 188-193.

Three major features - terminal group to bind the zinc in the active

site of enzymes - linker unit, residing in the channel

- capping group that principally occupies the

external entrance to the channel of the

enzyme

How to Design and HDAC inhibitor??

Trapoxin A (n = 2),

Trapoxin B (n = 1)

Chlamydocin

Tan-1746

Apicidin

FR235222

Naturally Occurring Cyclictetrapeptide HDAC Inhibitors

Chlamydocin Scaffold and Research Focus

cyclo(-L-Am7(S2Py)-A2in-L-Ala-D-Pro-) cyclo(-L-Am7(S2Py)-D-A1in-L-Ala-D-Pro-) cyclo(-L-Am7(S2Py)-L-A1in-L-Ala-D-Pro-)

cyclo(-L-Am7(S2Py)-D-2MePhe-L-Ala-D-Pro-) cyclo(-L-Am7(S2Py)-L-2MePhe-L-Ala-D-Pro-) cyclo(-L-Am7(S2Py)-Aib-L-Ala-D-Tic-)

Aromatic Ring Shifting in Chlamydocin Framework

b) Library of cyclic tetrapeptides

a) Aromatic amino isobutyric acid

analogs

cyclo(-L-Am7(S2Py)-Aib-L-Phg-D-Pro-) cyclo(-L-Am7(S2Py)-Aib-L-Ph4-D-Pro-) cyclo(-L-Am7(S2Py)-Aib-L-Ph5-D-Pro-)

cyclo(-L-Am7(S2Py)-Aib-L-Ser(Bzl)-D-Pro-) cyclo(-L-Am7(S2Py)-Aib-L-Ser-D-Pro-)

Aromatic Ring Shifting in Chlamydocin Framework

b) Library of cyclic tetrapeptides

a) Phenyl alanine analogs

Scheme 1. Reagents and conditions: (a) AcSK, DMF, r.t.; (b) 2,2’-dipyridyl disulphide, MeNH2/MeOH

Synthesis of Chlamydocin Analogs as HDAC Inhibitors

a) General scheme: Synthesis of cyclic tetrapepdie framework

b) Functional group modification

*column: Chromolith performance RP-18e (100 x 4.6 mm). Eluent: 10-100% CH3CN gradient containing 0.1% TFA over 15 min.

† Selectivity among HDAC1 and HDAC4

(With 0.1 m M DTT)

Enzyme Inhibition Data and SAR Study

No. Compounds IC50 (μM) p21 promoter assay.

EC1000 (μM) HDAC1 HDAC4 HDAC6

4 cyclo(-L-Am7(S2Py)-D-A1in-L-Ala-D-Pro-) 0.0027 0.0024 0.0120 0.055

5 cyclo(-L-Am7(S2Py)-L-A1in-L-Ala-D-Pro-) 0.0360 0.0250 0.0329 2.0

6 cyclo(-L-Am7(S2Py)-D-2MePhe-L-Ala-D-Pro-) 0.1700 0.0700 0.0710 25.6

7 cyclo(-L-Am7(S2Py)-L-2MePhe-L-Ala-D-Pro-) 0.0037 0.0022 0.0560 0.55

1. Conformational analysis of diastereomers by CD spectra:

2. cyclo(-L-Am7(S2Py)-Aib-L-Phe-D-Pro-)

4. cyclo(-L-Am7(S2Py)-D-A1in-L-Ala-D-Pro-)

5. cyclo(-L-Am7(S2Py)-L-A1in-L-Ala-D-Pro-)

6. cyclo(-L-Am7(S2Py)-D-2MePhe-L-Ala-D-Pro-)

7. cyclo(-L-Am7(S2Py)-L-2MePhe-L-Ala-D-Pro-)

• At 220 nm region, compounds 4 and 7 show

-ve ellipticity, but 5 and 6 show +ve ellipticity.

• Compounds with –ve ellipticity show good

biological activity.

(With 0.1 mM DTT)

Enzyme Inhibition and Biological Activity of Diastereomers

Compound 4 Compound 5

Conformational analysis of diastereomers by NMR spectrometry

Why Non-peptides small molecules??

• Cyclic tetrapeptides although being potent HDAC inhibitors, need laborious and expensive

synthesis process.

• As per Lipinski rule in medicinal chemistry, a molecule to becomes orally active drug, if its

molecular mass should is below 500 daltons.

Non-Peptide HDAC inhibitors

1. Trichostatin and SAHA analogs 2. Diketopiperazine HDAC inhibitors

Reagents and conditions: (a) AcSK, DMF, r.t.; (b) MeNH2/MeOH, R-Br, Et3N

Reagents and conditions: (a) AcSK, DMF, r.t.; (b) MeNH2/MeOH, R-Br, Et3N

Non-Peptide HDAC inhibitors

a) Scheme for Synthesis of SAHA analogs

b) Scheme for synthesis of Trichostatin-A analogs

HN

NH

O

O

O

O

HN

NH

O

O

R1

OH

O

HN

NH

O

O

R1

NH

O

HN

NH

O

O

R1

NH

O

OHO

R1

a b

c

Scheme 1: Reagents and conditions (a) Pd-C, MeOH, H2. (b) HCl.H2N-OBzl, DCC, HOBt.H2O, Et3N

(c) Pd-BaSO4, AcOH, H2

R = -CH3, -Bzl

AA = DL-Pro, DL-Tic, DL-MePhe and

BzlGly

R1 = DL-Pro, DL-Tic, DL-MePhe and BzlGly

Synthesis of Diketopiperazine Hydroxamic Acids

Conclusions

• Focused on arrangement of aromatic ring on cap group region, a

library of cyclic tetrapeptides, non-peptides and diketopiperazine

based HDAC inhibitors were designed and synthesized.

• In-vitro and In-vivo assay studies done and some of the compounds

showed exciting results and some were disappointing.

• Cyclic tetrapeptides with proper orientation of aromatic ring on their

macrocyclic cap group, showed better interaction with surface of

HDACs by inhibiting potently, as compared to non-peptides and cyclic

dipeptides.

• Therefore, cyclic tetrapeptide based HDAC inhibitors can be the

challenging antitumor agents.

Postdoctoral Research Experience in USA

Postdoctoral Researcher at Holton Laboratory

[2008 to 2010]

Postdoctoral Research Experience in USA

What is Cancer??

Mutated normal cells when undergo uncontrolled proliferation lead to

malignant tumors called Cancer.

Cancer eventually spreads throughout the body and causing inevitable death.

About 13% of all human death is reported due to Cancer.

A chemical compound that is selectively toxic to these dividing cells has

potential as anti-cancer drugs.

Taxol is natural terpene that stabilizes microtubules, thus interferes with

the normal breakdown of microtubules during Mitosis, leading to tumor

growth suppression.

Semi synthesis of Taxol (Holton and et al)

β-lactams are protected using Enol ethers (Ex. 2-methoxy propene Or 3-methoxy pentene).

Introduction to Taxol:

Isolated from bark of Taxus brevifolia plant

(1967) M. E. Wall and M. C. Wani

First time synthesized by Holton, et. al (1994).

Taxol is used to treat Lung, Ovary and Breast

Cancers.

Postdoctoral Research Experience in USA

OH

O

O

HOBzlOAcO

OAcO

O

OH

Ph

NH

O

Ph

Taxol (Pacilitaxel)

Enol ethers versatile hydroxy

protecting groups,

Proposing to prepare enol ethers by

either-

i) O-alkylation of enolates, OR

ii) On pot synthesis from ketones

New Methodology for Enol-ethers Synthesis;

Selective O-/C-alkylation

Enolates:- Enolates are ambident Nucleophiles.

May attack the alkylating agent via either the oxygen or a-carbon atoms.

Controlling the ratio O-/C-alkylation can be difficult and depends on several factors.

What factors influence the O-/C-alkylation ratio?

O-alkylation is favoured by:

hard electrophiles (Tosylate, triflate LGs).

large counter-cations.

dipolar aprotic solvents (To solvated M+ ion).

C-alkylation is favoured by:

soft electrophiles (Iodide is best LG).

small counter-cations (Eg.: Li+).

protic solvents.

Base Methylating

agent Solvent Temperature

KHMDS CH3I THF -780 C

NaHMDS CH3OTs THF:HMPA 00 C

KH CH3OTf HMPA RT

NaH DMPU 500 C

New Methodology for Enol-ethers Synthesis;

Selective O-/C-alkylation

Remarks:- Methylating agent like CH3OTf, CH3OTs in dipolar

aprotic solvents gave O-methylated products.

Methyl iodide gave only C-methylated product.

Using CH3OTf and KH base in DMPU

combination at RT gave comparably good

O-methylation (Ration = 1.1:1.0).

Ulternate method: One pot synthesis of

Enol ether (Ref:Synthesis, 1994, 38)

Scheme: Alkylation of ketone

Table 1. Reaction conditions

OH

O

O

HOBzlOAcO

OAcO

O

OH

Ph

NH

O

PhOH

O

HO

BzlOAcO

OAcO

Baccatin-III 1-Deoxy Baccatin-III

(Ananog)

Process for Hydroxylation of Enone in the Taxol AB-Ring Synthesis

Taxol Analog Synthesis

Hydroxylation of Enone in AB-ring synthesis

Remarks-

• This position gave enough exposure to taxol chemistry.

• Got trained in writing research proposals, reports and varied literature search methods.

titution.

α-hydroxylation of enones by

oxidation of enolates

[By nascent oxygen, triethyl

phosphite]

α-hydroxylation of enones by

oxidation of enolates

[By nascent oxygen, NaI, 0 0C]

Postdoctoral Research Experience in JAPAN

Postdoctoral Researcher at Hayase Laboratory

[2011 to 2014]

Curtsey, Dr. Nate Lewis, Caltech website

Prospective Renewable Energy Sources

Wind

14 TW

Geothermal

1.9 TW

Biomass

5-7 TW

Hydroelectric

1.2 TW

Energy Gap ~ 14 TW by 2050

~ 33 TW by 2100

Current Energy Use

About 14 TW !!!

Tide/Ocean Currents

0.7 TW

Energy Demand and Supply

(Active area more than 1cm2 recorded by authorized institutions. M. A. GREEN et al., Progress in Photovoltaic Research & Applications; (Ver. 42); 2013, 21, 827-837)

Organic Solar Cells

0

10

20

Effi

cie

ncy（％）

c-Si

25.0%

p-Si

20.4%

a-Si DSSC CIGS

19.6%

11.9%

10.1 %

CdTe

19.6%

Polymer

10.7 %

Commercialized

Under Commercialization

Perovskite

14.1% Sharp Mitsubishi

EPFL

Certified Efficiencies of Solar Cells

SnO2/F

3 I-

I3-

Pt/SnO2/F TiO2 layer: 10-20 μm

Electrolyte layer: 30 μm

TiO2

10-20nm

TiO2

Ru N

N

C O

C O O H

O

Ti

N

N

C O

H O O C

O

Ti

S C N N C S OHP257

e

e

e e

e

e

e

e

e

e

e

e

e

Efficiency > 10 %

Working Principle of Dye Sensitized Solar Cells

Spectral Response in High Efficiency DSSCs

Dye YD2-O-C8

Efficiency = 11.9 %

Ru-dye-N719

Efficiency = 11 %

Dye Perovskite

Efficiency = 15 %

R & D of Novel

Sensitizers

M. Gratzel , et al., J. Am. Chem. Soc. (2005) 127 16835 Ashwini Yella , et al., Science.

(2011), 334, 629

J. Burschka, et al., Nature. (2013), 499, 316

Potentiality of NIR Dyes

Well-known NIR Dyes: Phthalocyanine, Squaraine and

Cyanine sensitizers.

Characteristic features,

1. Sharp, narrow and Intense light absorption.

2. High molar extinction coefficient.

3. Capability of sensitization of wide band gap semiconductors.

4. Possibility to tailor the optical absorption window from visible to

NIR wavelength region.

SQ-8 SQ-84 SQ-83

Dyes Jsc (mA/cm2) Voc (V) FF Efficiency

SQ-8 6.71 0.58 0.72 2.82 %

SQ-83 11.53 0.63 0.69 5.03 %

SQ-84 6.93 0.58 0.66 2.67 %

Cu

rren

t d

ensi

ty (

mA

/cm

2)

Voltage (V)

COOH

Cyanoacrylate

Thiophene-cyanoacrylate

0 0.1 0.2 0.3 0.4 0.5 0.6

2

4

6

8

10

12

IPC

E

Wavelength (nm)

COOH

Cyanoacrylate

Thiophene-Cyanoacrylate

300 400 500 600 700 8000

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Wavelength (nm)

Ab

sorb

an

ce (

No

rm.)

SQ-83 [653 nm]

SQ-84 [663 nm]

SQ-8 [636 nm]

300 400 500 600 7000

0.4

0.8

1.2

Effect of Anchoring on Photosensitization Behavior

Structure of Squaraine dyes

UV spectra SQ-Dyes in Ethanol IV-curve of SQ-dyes Photocurrent action spectra

General Scheme: Synthesis of Squaraine sensitizers

b) Thiophene linked SQ-CA Dye

a) Synthesis of SQ-CA dye

Yanagisawa, et al;

J. Porp. Phthal. 6 (2002) 217-224.

Efficiency = 0.61%

DSSCs Based on Phthalocyanine Dyes !

Efficiency = 3.52%

Cid, J.-J. et al;

Angew. Chem., Int. Ed. Engl., 46

(2007) 8358–8362.

Efficiency = 0.11% Yanagisawa, et al;

J. Porp. Phthal. 6 (2002) 217-224.

Efficiency = 6.49%

Ragoussi. et al;

ChemPhysChem (2014) Early

Park, S. Hayase et al,;

ECS JSS, 2 (2012) Q6-Q11.

Efficiency = 0.5% (On SnO2)

Phthalocyanine dyes –

• Macrocyclic pi-extended framework.

• Thermal, Chemical and photochemical

stability.

• Sharp and intense absorption with high

molar extinction co-efficient.

• Poor performance due to aggregation and

lack of solubility in common solvents.

Zinc Phthalocyanine Dyes as NIR Sensitizers for DSSC

Remarks-

• Succeeded in widening the absorption window (>700 nm) by a, a-dithiopnene

substitution.

• Poor photovoltaic performance (= <1% to 1.0%), probably due to dye aggregation.

NC

NC

TIPS

i) TBAF

CN

O

OH

Br

NC

NC

SPh

SPh

N

N

N

N Zn

PhS SPh

SPh

SPh

PhS SPh COOH

CN

NC

NC

CN

O

OH

ii) Pd2(dba3), AsPh3

Zn(OAc)2DBU, Pentanol

General Scheme for ZnPc synthesis:

Zinc Phthalocyanines

Phosphorous Phthalocyanine Dyes as Sensitizers for DSSC

Electronic absorption spectra

Aggregation studies

Synthetic scheme

λmax = 720 nm

η = 2.7%,

IPCE=25%

Thank you very much for your kind attention !