August 2012 931Chem. Pharm. Bull. 60(8) 931–944 (2012)

© 2012 The Pharmaceutical Society of Japan

An Overview on Chemical Derivatization and Stability Aspects of Selected Avermectin Derivatives

Atul Awasthi,a,b Majid Razzak,b Raida Al-Kassas,a Joanne Harvey,c and Sanjay Garg*,a,d

a School of Pharmacy, The University of Auckland; Private Bag 92019, Auckland, New Zealand:

b Ancare Scientific Ltd.; Auckland, P.O. Box 36240, Northcote, Auckland, New Zealand: c School of Chemical and Physical Sciences, Victoria University of Wellington;

P.O. Box 600, Wellington, New Zealand: and d School of Pharmacy and Medical Sciences, University of South Australia;

P.O. Box 2471, Adelaide, Australia.Received March 16, 2012; accepted April 20, 2012

Naturally occurring avermectins (AVMs) and its derivatives are potent endectocide compounds, well-known for their novel mode of action against a broad range of nematode and anthropod animal parasites. In this review, chemical and pharmaceutical aspects of AVM derivatives are described including stability, syn-thetic and purification processes, impurities and degradation pathways, and subsequent suggestions are made to improve the chemical stability. It has been found out that unique structure of AVM molecules and pres-ence of labile groups facilitated the derivatization of AVM into various compounds showing strong anthel-mintic activity. However, the same unique structure is also responsible for labile nature related to sensitive stability profile of molecules. AVMs are found to be unstable in acidic and alkaline conditions. In addition, these compounds are sensitive to strong light, and subsequently presence of photo-isomer in animals treated topically with AVM product is well known. The pharmacoepial recommendations for addition of antioxidant into drug substance, as well as its products, arises from the fact that AVM are very sensitive to oxidation. Formations of solvates, salts, epoxides, reduction of double bonds and developing liquid formulation around pH 6.2, were some chemical approaches used to retard the degradation in AVM. This coherent review will contribute towards the better understanding of the correlation of chemical processes, stability profile and biological activity; therefore, it will help to design the shelf-life stable formulations containing AVMs.

Key words avermectin; chemical stability; natural product; degradation mechanism; related substance; mac-rocyclic lactone

1. IntroductionThe term avermectin (AVM) stands for closely and struc-

turally related 16 membered compounds known as macrocy-clic lactones (ML). Mishima et al. from Sankyo, Japan, have discovered naturally occurring compounds, commonly known as B41 from culture of the Streptomyces.1,2) The mixture B41 was used as insecticidal preparations, and later these com-pounds were evaluated analytically as 16 member macrocyclic ring compounds (aglycones) as A1, A2, A3, A4, B1, B2, B3, C1 and C2. These compounds led the discovery of currently used AVMs as well as new milbemycins (MBN) like milbe-mycin D, milbemycin-5-oxime and moxidectin. AVM and MBN are commonly known as ML, and it is interesting to note that MBN (1976) and their potent miticidal and acaricidal properties were discovered shortly before the AVM, their an-thelmintic properties become known only after the discovery of AVM (1979).3)

A new strain of bacterium Steptomyces discovered in a golf field of Japan, by local scientists led by Prof. S. Omura of Kitasato Institute, was fully investigated by scientist at Merck and Co., U.S.A.4) The compounds isolated from the fermenta-tion broth after controlled growth, were coded as C-076 or AVM.3,4) Four major and four minor compounds of C-076, as four pairs of homologs, were identified as C-076 A1a, A1b, A2a, A2b, B1a, B1b, B2a, and B2b. A detailed identification and characterization study disclosed these as disaccharide (α-L-oleandrosyl-α-L-oleandroside) derivatives of previously discovered MBN. The detailed structures of these AVM ho-mologs and their substitutions that distinguish them from each other, is demonstrated by Fig. 1 and Table 1.4) Component A and B are distinguished by substitution at C5 as methoxy (A) or hydroxyl (B) group, along with whether C22–23 bond is a single bond (1) or double bond (2), as well as C23 has either s-butyl (a) or a n-butyl (b) group.5)

The above compounds are not produced equally by the fer-mentation process and each homolog pairs were present about

* To whom correspondence should be addressed. e-mail: sanjay.garg@unisa.edu.au

The authors declare no conflict of interest.

Review

932 Vol. 60, No. 8

20–30% of total content.5) Purification and isolation of these structurally similar compounds at production scales was a ma-jor challenge for the scientists. A series of attempts involved Sephadex column chromatography, hydroxyalkylated dextran gel separation, non-aqueous solvent extraction method and HPLC separations.6)

Specifically, AVMs are obtained as mixture of the ‘a’ as well as ‘b’ units (typically ≥90% ‘a’ and ≤10% ‘b’).7,8) As shown in Table 1, these components varies in the nature of R2 substituent, furthermore, these small differences in structure has little effect on chemical or biological activity.8) Despite this, component ‘a’ and ‘b’ are separated by chromatographic techniques, however, this not a usual manufacturing practice and commercially available AVMs such as ivermectin (IVM), eprinomectin (EPM) and abamectin (ABM) are available as mixture of one minor (B1b) and one major (B1a) homologs.7,8) Thus, presence of component ‘a’ and ‘b’ in a mixture is dem-onstrated by leaving out the ‘a’ or ‘b’ from the designated compounds, i.e., a mixture of AVM B1a and B1b is referred as AVM B1.6)

Given the structural differences, these compounds can be easily identified by liquid chromatography. A mixture of all eight C-076 compounds was analyzed by HPLC using a µ-Bondapack, C18 column (300×4 mm, 10 µm) at 40°C, eluted with mobile phase (methanol–water, 85 : 15, v/v) at flow 1.2 mL/min, and these compounds were well separated

as shown by their retention times (RT) presented in Table 2.6)

Commercially AVM hold a major share in animal health market. IVM (22,23-didehydro-AVM) is one of the most wide-ly used antiparasitic agent having annual sales >$ 1 billion,9) and has applications to animal as well as human health.10) IVM has been recognized for treatment of the Onchocerciasis (River Blindness), a chronic human filarial disease caused by infection with Onchocerca volvulus worms, as well as lymphatic filariasis, also known as ‘Elephantiasis’ caused by Wuchereria bancrofti, Brugia malayi or Brugia timori.9,10) These two parasitic infections affecting over 1 billion people in more than 80 countries. The initiatives of Professor Omura, Merck and World Bank, led the free distribution of ivermectin tablets in African countries to save the lives of underprivi-leged people suffering from such parasitic infections.9)

IVM is officially presented in various pharmacopeial mono-graphs.7) Similarly EPM also has a compendial monograph in United States Pharmacopeia (USP),8) however, there is no

Atul Awasthi is a Research Scientist at Ancare Scientific, New Zealand and pursuing macrocyclic lactone research (Ph.D.) with the University of Auckland, New Zealand. He was born in Madhya Pradesh, India in 1972 and received his MS and MPhil degree (1995) in chemistry. He has worked with many multinational pharmaceutical/animal health companies in and currently associated with Ancare Scientific and principally supervised by Professor Sanjay Garg and Dr. Majid Razzak for his research work. His recent published work involved evaluation of stability and chemical degradation of avermectin using innovative approaches and sophisticated instruments like high resolution MS and NMR. He is experienced in chemical semi-synthesis, regulatory stability studies, modern pharmaceu-tical analysis, use of degradation mechanism to stabilize compositions, isolation and characteriza-tion of degradents, and GLP/GMP compliance. He has been awarded with Technology New Zealand Scholarship. His research interests include drug degradation pathways and stabilization techniques, new method devel-opments and regulatory compliance.

Atul Awasthi

Fig. 1. General Structure of Avermectin, Where Dotted Line Shows a Single or Double Bond, R1 Is –OH Only When Dotted Line Is a Single Bond, R2 Is Propyl or Butyl and R3 Is –OH or Methoxy

Table 1. Structural Differences and Similarities of Four Avermectin Pair Compounds4–6)

C-076 components R1 R2 R3

A1a Double bond s-Butyl –OCH3

A1b Double bond n-Propyl –OCH3

A2a –OH s-Butyl –OCH3

A2b –OH n-Propyl –OCH3

B1a Double bond s-Butyl –OHB1b Double bond n-Propyl –OHB2a –OH s-Butyl –OHB2b –OH n-Propyl –OH

Table 2. HPLC Analysis of Avermectin Homologs: Identification by Retention Times (RT)6)

C-076 components Retention time

B2b 6.14B2a 6.74A2b 7.22A2a 8.00B1b 8.32B1a 9.70A1b 11.06A1a 11.78

August 2012 933

pharmacopeial monograph available for ABM.Notably, in the past a significant attention has been fo-

cused on main avermectin compounds such as IVM, EPM, and ABM, however, there are other AVM derivatives having potential biological anthelmintic activities, looks ideal candi-dates for commercial formulations, needs further research and discussion. Furthermore, majority of the scientific develop-ment work has been carried out by Merck and its subsidiaries, whereas some other groups like Pfizer, Novartis, Bayer etc., are also involved in AVM research and development. Hence, there is an opportunity to correlate the findings on chemistry and stability aspects of AVM from these groups together. In addition, there is also a need to review the development work carried out by exploiting the reactive centers of molecules, since these groups in turn are totally responsible for stability profile as well as anthelmintic activities of AVM compounds. In addition, the information collected from chemical de-rivatization can be used to understand the stability profile un-der various stressors such as acid, alkali, oxidizing agents and lights, and envisage the ways to stabilize these compounds and their formulated products.

Hence, this review work is aimed at elaborative understand-ing of chemical derivatization of AVMs using reactive centers at molecules, stability profile, degradation pathways and ways to address the stability issues.

2. Isolation and Chemical Semi-Synthesis of Avermec-tin

The AVMs or C-076 were initially screened in a study where thousands of microbial fermentation products were tested in animals for activity against the nematode Nematospi-roidesdubius.11) Among the few compounds showing activity in this test, a sample from soil microorganism was isolated by researchers at the Kitasato Institute. The microorganism was classified as a new species of actinomycete, Streptomyces avermetilis.11)

On industrial scale, AVM are manufactured by aerobic fer-mentation of broth containing producing strain of Streptomy-ces avermetilis. Such nutrient media (pH about 6.0–7.5, tem-perature 27–28°C) contains carbohydrates such as sugars like dextrose, sucrose, oleandrose etc., and nitrogen sources yeast hydrolysates, soybean meal etc., as suitable sources of as-similable carbon and nitrogen in nutrition media. In addition,

media also contains low level of inorganic salts yielding sodium, potassium, chloride, sulfate ions, as well as trace amount of elements, such as cobalt, manganese, iron etc., for the growth of microorganism.4)

The current strain of Streptomyces avermetilis used for fermentation process, i.e. MA-4848, is basically a mutant of originally discovered MA-4680. It was obtained by exposing the clear glass vials containing lyophilized strain to the UV irradiation.4)

A typical scale-up fermentation process involved selec-tive propagation method starting from inoculation of frozen MA-4848 strain of bacterium in a 50 mL media.4) ca. 10 mL of this media is inoculated with 500 mL media followed by 467 L media. Half of this media is finally inoculated with 4310 L of media for final fermentation process. After 6 d (144 h), the me-dia is filtered, and resultant cake is washed up with water and then extracted with acetone. The crude mixture goes through various steps of purification and isolation processes involving solvent extraction, chromatographic separations etc.

Following sections provide a detailed information on bio-logical and chemical semi-synthesis of available AVMs, how-ever ABM and its derivatives are the only commercially used AVMs in animal health industry.

2.1 Abamectin ABM is a mixture of AVM B1a and B1b homologs (B1a ≥90%, and B1b ≤10%) obtained from strain MA-4848 after subsequent purification and isolations steps.4,6) It is also used as a precursor for the synthesis of very impor-tant drug of this group like IVM, emamectin (EMM) and EPM. The structure of ABM is shown in Fig. 2.

2.2 Derivatives of Abamectin 2.2.1 Ivermectin The C-076 compounds with the

C22,23-unsaturations or IVM are ‘1-series’ compounds, obtained by the reduction of the C22,23-double bond of AVM B1.12) The hydrogenation process is catalyzed by tris-(triphenylphosphine)-rhodium(I)-chloride in benzene, also known as Wilkinson’s homogeneous catalyst. The mixture of AVM B1, catalyst and benzene is subjected to hydrogenation at room temperature for 21 h, and then pure compounds were isolated by techniques described in section 2.6) The manufac-turing process of IVM is schematically demonstrated by Fig. 2.

2.2.2 Emamectin and Eprinomectin 4″-Deoxy-4″-amino-AVM Bl or emamectin (EMM) and 4″-deoxy-4″-

Fig. 2. Chemical Reaction Representing Conversion of Abamectin into Ivermectin

934 Vol. 60, No. 8

acetylamino-AVM Bl or eprinomectin (EPM), are derivatives of ABM B1 wherein the 4″-hydroxy group is selectively oxidized to ketone and then replaced by an amino or acetyl amino group.13) The procedure is best described by Fig. 3.

2.3 Other Avermectin Derivatives Given the unique molecular structure and higher chemical reactivity of AVMs, many derivatives were obtained by manipulating the substitu-tions at known reactions centers in molecule.

ABM and its derivates have hydroxyl (–OH) groups at C5, C23 (not present in ABM and IVM), C7 and C4″ (not present in EPM) positions, and with the use of certain pro-tecting techniques, all possible combinations of alkylated, acylated and glycosylated C-076 compounds can be prepared. The –OH groups at the C5 and C23 positions are much more reactive than the C4″ (disaccharide), C4′ (monosaccharide) and C13 position (aglycone). C7–OH being shielded shows

least reactivity and does not react except under more vigorous conditions.11,12) Hence reactivity of various –OH groups can be demonstrated by Fig. 4.

Thus, where C5 substituted compounds are desired, no protection is necessary for the C4,″ C4′, C23 or C13 positions, and if substitution is aimed at C23, only C5–OH group needs to be protection.14–20) In general, tert-butyldimethylsilyl (TB-DMS) chloride or acetate is used as protective reagent, and once the substitution is over, the TBDSM moiety is removed by p-toluenesulphonic acid (p-TSA) to achieve the alkyl, acyl, acetyl amino, glycosyl etc. derivatives.14,15)

There are also reports available describing substitutions at glycosyl unit/s at positions other than C4″–OH or C4′–OH groups.21,22) In addition, the rearrangements involving double bonds and positions of atoms or groups in molecule, are also used as basis for substitutions.23,24) On the other hand, the use

Fig. 3. Substitution of 4″ -OH Group for Synthesis of Emamectin and Eprinomectin

Fig. 4. Relative Reactivity of Various –OH Groups of Avermectin Molecule, C5–OH Being Most Reactive and C7–OH Is Least Reactive

August 2012 935

Tabl

e 3.

A

verm

ectin

Der

ivat

ives

Syn

thes

ized

by

Man

ipul

atin

g Ta

rget

Fun

ctio

nal G

roup

s

Targ

et g

roup

s/po

sitio

nD

eriv

ativ

esPr

oced

ure

outli

neR

efer

ence

sR

emar

ksA

ssig

nee

–OH

gro

up/s

23-K

eto

deriv

ativ

es o

f A

VM

1. P

rote

ctio

n of

C4″

and

C5–

OH

gro

up u

sing

TB

DM

S2.

Oxi

datio

n of

C23

–OH

gro

up u

sing

PC

3. D

e-pr

otec

tion

of C

4″ a

nd C

5-O

H

Gul

lo e

t al.26

)Sh

own

anth

elm

intic

act

ivity

Mer

ck

Alk

yl d

eriv

ativ

es o

f A

VM

1. A

lkyl

hal

ide

(e.g

. met

hyl i

odid

e) re

acts

with

ave

rmec

tin

to f

orm

ally

l der

ivat

ives

2. R

eact

ion

is c

atal

yzed

by

solv

er o

xide

Fish

er e

t al.14

)1.

Sub

stitu

tion

can

also

occ

ur C

7–O

H g

roup

2. S

how

n an

thel

min

tic a

ctiv

ity

Mer

ck

Acy

l der

ivat

ives

of

AV

MA

cyl o

r be

nzoy

l anh

ydrid

e re

acts

with

ave

rmec

tin to

for

m

acyl

der

ivat

ives

Mro

zik

et a

l.15)

Show

n an

thel

min

tic a

ctiv

ityM

erck

4″-K

eto,

and

4″-

amin

o-de

oxy,

an

d su

bstit

uted

am

ino

AV

M1.

C4″

–OH

oxi

dize

d to

ket

o w

hile

pro

tect

ing

C5–

OH

2. C

4″-K

eto

com

poun

d fr

om a

bove

ste

p is

rea

cted

with

am

mon

ium

ace

tate

to g

ive

4″-a

min

o A

VM

3. C

4″-A

min

o de

rivat

ive

is tr

eate

d w

ith a

cetic

anh

ydrid

e to

giv

e C

4″-a

cety

l am

ine

deriv

ativ

e

Mro

zik

et a

l.27)

1. P

roce

dure

for

em

amec

tin a

nd

Eprin

omec

tin s

ynth

esis

2. A

min

o de

rivat

ives

sho

wn

to p

os-

ses

parti

al a

nti-b

acte

rial a

ctiv

ity

Mer

ck

13-K

eto/

imin

o/am

ino

deriv

ates

1. C

13–O

H o

f A

VM

agl

ycon

e or

MB

N (

e.g.

nem

adec

tin)

can

be o

xidi

zed

to g

et C

13-k

eto

deriv

ativ

e2.

The

ket

o gr

oup

can

be f

urth

er s

ubst

itute

d to

for

m

13-a

min

o or

13-

imin

o de

rivat

es

Linn

et a

l.28)

Show

s lin

k be

twee

n A

VM

and

m

ilbem

-5-o

xim

e, a

nd m

oxid

ectin

Mer

ck

13-H

alo,

and

13-

deox

y A

VM

1. C

13-H

ydro

xy a

re p

repa

red

by a

cid

hydr

olys

is o

f A

VM

2. C

ompo

unds

fro

m a

bove

ste

p re

acts

with

alk

ylam

ine

and

met

hyle

ne c

hlor

ide

to g

ive

13 h

alo

agly

cone

of

AV

M

Mro

zik

et a

l.18)

Show

n an

thel

min

tic a

ctiv

ityM

erck

Suga

r de

rivat

ive

of A

VM

Extra

sug

ar s

ubst

ituen

t can

be

obta

ined

by

repl

acin

g C

5,

C4″

(A

VM

B1)

, C5,

C4′

(M

ono

B1)

, C13

(A

G),

C23

(A

VM

B2)

Fish

er e

t al.29

)Sh

own

anth

elm

intic

act

ivity

Mer

ck

Difl

ouro

der

ivat

ives

of

AV

M1.

=C

F2 g

roup

is o

btai

ned

at C

4,″

C23

, C4′

(MS)

, C13

, C

23(A

G)

2. P

rote

ctio

n/de

prot

ectio

n m

odel

3. F

luor

ina t

ion

is u

sual

ly a

chie

ved

by H

F·py

ridin

e m

ix-

ture

Mei

nke

et a

l.30)

1. S

how

n an

thel

min

tic a

ctiv

ity2.

Can

be

used

as

prem

ix f

or a

ni-

mal

s an

d pl

ant p

est c

ontro

l

Mer

ck

5-K

etox

ime

deriv

ativ

e of

AV

M1.

Oxi

datio

n of

C5–

OH

gro

up u

sing

MnO

2, us

ing

prot

ec-

tion

depr

otec

tion

mod

el2.

Con

vers

ion

of C

5-ke

to g

roup

to k

etox

ime

deriv

ativ

e us

ing

hydr

oxyl

am

ine

Cve

tovi

ch e

t al.31

)1.

Pro

cedu

re e

stab

lish

the

clos

e lin

k w

ith m

ilbem

ycin

5-o

xim

e, a

nd

mox

idec

tin2.

Sho

wn

anth

elm

intic

act

ivity

Mer

ck

Acy

l or

benz

oyl a

min

o de

riva-

tive

of a

min

o-av

erm

ectin

Furth

er s

ubst

itutio

n of

am

ino

grou

p at

C4″

pos

ition

usi

ng

alky

lam

ine

give

am

ine

deriv

e of

am

ino

com

poun

dLi

nn e

t al.17

)Su

bstit

utio

n ai

med

for

EMM

and

EP

MM

erck

936 Vol. 60, No. 8Ta

ble

3.

Ave

rmec

tin D

eriv

ativ

es S

ynth

esiz

ed b

y M

anip

ulat

ing

Targ

et F

unct

iona

l Gro

ups

Targ

et g

roup

s/po

sitio

nD

eriv

ativ

esPr

oced

ure

outli

neR

efer

ence

sR

emar

ksA

ssig

nee

8,9-

or

10,1

1, o

r 22

,23

Dou

ble

bond

der

ivat

ives

8,9,

22,2

3-C

yclo

prop

yl c

om-

poun

ds o

f A

VM

1. T

he r

educ

tions

of

C8,

9/10

,11/

22,2

3 do

uble

bon

ds w

hile

pr

otec

ting

C5–

OH

.2.

Com

poun

d fr

om s

tep

1 re

acts

with

, ido

met

hane

, p-T

SA

and

DM

C to

giv

e m

etha

no d

e riv

ativ

es

Wyv

ratt

et a

l.23)

Show

n an

thel

min

tic a

ctiv

ityM

erck

23 o

r 24

Thi

ol d

eriv

ativ

es o

f A

VM

1. P

rote

ctio

n of

C5–

OH

2. R

eact

ion

of th

iol d

eriv

ed a

lum

inum

sulfid

es to

get

C

23–2

4 ep

oxy

com

poun

d3.

Rea

ctio

n of

dim

ethy

lalu

min

ium

sulfid

e w

ith A

VM

to

get 2

3 th

iol d

eriv

ativ

es

New

bold

et a

l.32)

Show

n an

thel

min

tic a

ctiv

ityM

erck

Δ26,

27-A

VM

1. T

he s

ingl

e bo

nd a

t C26

–27

is s

elec

tivel

y ca

taly

tical

ly

oxid

ized

to v

ario

us p

ositi

on2.

Fur

ther

sub

stitu

tion

coul

d be

an

epox

ided

at 8

,9-p

osi-

tion

Chr

iste

nsen

et a

l.33)

1. A

pplic

able

to A

BM

, IV

M, a

nd

EMM

.2.

Sho

wn

anth

elm

intic

act

ivity

Mer

ck

Δ23,

24-A

VM

1. A

VM

A2

or B

2 (–

OH

gro

up a

t C23

) is

trea

ted

with

D

AST

at l

ow te

mpe

ratu

re.

2. R

emov

al u

npro

tect

ed C

23–O

H is

indi

catio

n of

com

ple-

tion

of r

eact

ion.

Shih

et a

l.24)

The

com

poun

ds h

ave

bette

r bio

logi

-ca

l act

ivity

than

par

ent A

2 or

B2

AV

Ms

Mer

ck

4a-S

ubst

itute

d A

VM

1. M

ethy

l gro

up a

t C4

is s

elec

tivel

y ox

idiz

ed (w

hile

pro

-te

ctin

g ot

her

OH

gro

up)

to a

dd –

OH

gro

up2.

Thi

s –O

H g

roup

can

be

used

for

fur

ther

sub

stitu

tion,

ac

yl, b

enzo

yl o

f su

bstit

uted

der

ivat

ive

ther

eof

Jone

s et

al.34

)C

ompo

unds

sho

wn

anth

elm

intic

ac

tivity

, and

can

be

used

as

anti-

para

sitic

s fo

r an

imal

s an

d pl

ants

Mer

ck

C-5

Sub

stitu

ted

AV

M1.

–O

H g

roup

at C

-5 p

ositi

on is

sel

ectiv

ely

rem

oved

2. A

Cl o

r B

r su

bstit

utio

n at

the

sam

e po

sitio

n us

ing

spec

ific

reag

ents

Rob

en e

t al.35

)C

ompo

unds

sho

wn

anth

elm

intic

ac

tivity

Bay

er

Subs

titut

ion

at o

lean

dros

yl u

nit

Mon

osac

char

ide

and

agly

cone

de

rivat

ives

AV

M A

or

B in

sol

vent

s w

ith m

isci

ble

with

wat

er, a

re

treat

ed w

ith s

ulfu

ric a

cid

1. A

cid

conc

entra

tion

0.01

–0.1

% r

esul

ts in

mon

osac

cha-

ride

2. A

cid

conc

entra

tion

1–10

% r

esul

ts in

agl

ycon

e

Mro

zik

et a

l.36)

Com

poun

ds s

how

n an

thel

min

tic

activ

ity, b

ut le

ss a

ctiv

e th

an p

aren

t co

mpo

unds

, but

has

a b

ette

r saf

ety

regi

me

Mer

ck

13-e

pi A

VM

der

ivat

ives

1. A

dditi

on o

f C

5 pr

otec

ted

IVM

agl

ycon

e w

ith T

BA

I an

d ot

her

reag

ent g

ive

C13

-bet

a io

dide

agl

ycon

e.2.

Rea

ctio

n of

C13

-bet

a co

mpo

und

with

silv

er T

FMSA

gi

ves

C13

-epi

com

poun

d.3.

Dis

acch

arid

e ca

n be

add

ed to

13

posi

tion

to a

ffor

d 13

-ep

i ave

rmec

tin a

nd d

eriv

a tiv

es li

ke E

MM

, EPM

Bliz

zard

et a

l.22)

Com

poun

ds s

how

n an

thel

min

tic

activ

ityM

erck

4′ o

r 4″

Sub

stitu

ted

oxid

ized

A

VM

1. C

4′ o

r C

4″ o

xida

tion

of C

5 pr

otec

ted

AV

M o

r m

ono-

sacc

harid

e2.

Con

vers

ion

of -

oxo

in to

-ox

ime

usin

g hy

drox

yl a

min

e hy

droc

hlor

ide

3. F

urth

er -

oxim

e gr

oup

subs

titut

ion

to g

et -

sulfo

der

iva-

tives

(e.

g. s

ulfe

nim

ine

deriv

ativ

es)

Pitte

rna

et a

l.20)

1. E

or

Z is

omer

s of

ther

eof

2. M

etho

d fo

r co

n tro

lling

pes

t ad

habi

tat

Sing

enta

4′ o

r 4″

Sub

stitu

ted

amin

oxy

AV

MSy

nthe

sis

of C

4′ o

r C

4″-a

min

oxy

or s

ubst

itute

d-am

inox

y de

rivat

ive

Pitte

rna

et a

l.20)

1. E

or

Z is

omer

s of

ther

eof

2. M

etho

d fo

r co

ntro

lling

pes

t ad

habi

tat

Mer

ial

Ant

ipar

asiti

c ag

ents

Var

ious

C3

(hal

o, m

erca

pto

etc.

), an

d C

5 (o

xo, o

xim

e,

hydr

azon

es e

tc.)

subs

titut

ed c

ompo

unds

Bis

hop

et a

l.37)

For

the

cont

rol o

f fle

a an

d ot

her

para

sitic

infe

cti o

ns in

ani

mal

sPfi

zer

Inc.

August 2012 937

Tabl

e 3.

A

verm

ectin

Der

ivat

ives

Syn

thes

ized

by

Man

ipul

atin

g Ta

rget

Fun

ctio

nal G

roup

s

Targ

et g

roup

s/po

sitio

nD

eriv

ativ

esPr

oced

ure

outli

neR

efer

ence

sR

emar

ksA

ssig

nee

AV

M d

eriv

ativ

e w

ith s

pace

r be

twee

n ag

lyco

ne a

nd

disa

ccha

ride

Synt

hesi

s of

hyd

roxy

alk

yl C

13-e

pi o

r -O

der

ivat

ives

us

ing

silv

er te

traflu

robo

rate

and

oth

er r

eage

nts

Bliz

zard

et a

l.38)

Use

ful f

or c

ontro

lling

pes

t of

plan

ts

and

anim

als

Mer

ck

Spiro

epo

xide

AV

M d

eriv

ativ

eA

dditi

on o

f di

azom

etha

ne w

ith C

4′ o

r C

4″ o

xo A

VM

de

rivat

ive

to a

ffor

d C

4′ o

r C

4″ s

piro

epo

xide

Mei

nke

et a

l.39)

Use

ful f

or c

ontro

lling

par

asite

s of

pl

ants

and

ani

mal

sM

erck

Inte

rcon

vers

ions

Inte

rcon

vers

ion

of a

verm

ectin

Rea

ctio

n of

C4″

and

C5

prot

ecte

d A

2 an

d B

2 (le

ss b

io-

logi

cally

act

ive)

to r

emov

e C

23–O

H g

roup

to a

ffor

d A

1 an

d B

1 (m

ore

activ

e) h

omol

ogs

usin

g su

bstit

uted

th

ioca

rbon

yl h

alid

e

Mro

zik

et a

l.40)

—M

erck

Der

ivat

ives

from

fer

men

tatio

n pr

oces

sN

ovel

der

ivat

ives

of

AV

M1.

Thr

ee n

ew c

ompo

unds

isol

ated

afte

r fe

rmen

tatio

n of

m

utat

ed S

. ave

rmet

ilis

MA

4818

stra

in2.

The

se a

glyc

one

com

poun

ds a

re v

ery

sim

ilar t

o M

BN

B

with

som

e ad

ditio

nal g

roup

s an

d bo

nds

Aris

on e

t al.41

)Th

ese

com

poun

ds r

etai

ns th

e ty

pica

l bi

olog

ical

act

ivity

of

AV

MM

erck

Proc

ess

for

glyc

osyl

atio

n of

A

VM

com

poun

dFe

rme n

tatio

n of

AV

M a

glyc

one

with

Sac

char

opol

yspo

ra

eryt

hrea

res

ults

in a

dditi

on o

f m

ono

or d

isac

char

ide

at

C13

pos

ition

Schu

lman

et a

l.42)

Ferm

enta

tions

als

o ex

ert h

e co

ndi-

tions

of

typi

cal a

lkal

ine

reac

tion

i.e. f

orm

atio

n of

2-e

pi a

nd Δ

2,3

isom

ers

Mer

ck

Ant

ipar

asiti

c m

acro

lide

anti-

biot

ics

1. T

wel

ve n

ew a

verm

ectin

s gr

oup

com

poun

d is

olat

ed

from

fer

men

tatio

n br

oth

of S

trep

tom

yces

hyg

rosc

opic

us2.

The

se c

ompo

unds

hav

e a

typi

cal C

22–O

H g

roup

, and

is

o-pr

opio

nate

or

buta

noat

e gr

oups

at e

ither

C13

or

C23

Hax

ell e

t al.43

)Th

ese

com

poun

ds r

etai

ns th

e ty

pica

l bi

olog

ical

act

ivity

of

AV

MPfi

zer

938 Vol. 60, No. 8

of mutant bacterium was also employed for back engineered fermentation for some known molecules.25) Table 3 shows a summary of procedures for some strategically important AVM derivatives categorized by reaction centers.26–43)

3. Chemical Stability of AvermectinAVM, like other ML family member compounds, tend to

be unstable under normal conditions of preparation, use, stor-age, and therefore it is often recommended that these material as active pharmaceutical ingredients (API) be refrigerated, protected from moisture, kept away from light, limit exposure to extreme pH conditions and may contain a small amount of antioxidant to avoid oxidation at atmospheric conditions.44)

As explained in section 2, AVM B1 and its derivatives contains very reactive –OH groups, labile/conjugated double bonds, acidic hydrogen prone to epimerization and glycosidic linkage, present a very complex stability scenario having im-plications on obtaining a stable API for formulation prepara-tions.

Stabilization of the AVM class of compounds depends on the compound of interest and the method of stabilization, e.g. some AVM compounds require the addition of antioxidants, such as butylated hydroxy anisole (BHA), butylated hydroxy toluene (BHT), monothioglycerol, propyl gallate vitamin E etc., to the bulk product for inhibiting degradations, whereas some other AVMs have been stabilized by the formation of benzoate salts such as EMM·benzoate. On the other hands, stability of AVMs can be significantly increased by

recrystallization of drug substances with a sterically ma-nipulated alcohol, that results in a new form of AVM molecule where a spatial arrangement of the alcohol in the crystal leads to enhanced thermal stability.45)

3.1 Effect of Acidic Conditions Under mild acidic condition, selective removal of carbohydrate side chain at the 4′-position of the parent C-076 compounds, i.e., 4′-(α-L-oleandrosyl)-α-L-oleandrose group, results in C-076 monosac-charide while prolonged or strong acidic conditions lead to the formation of a terminal aglycone, is one of the most widely discussed issues of AVM chemistry.36)

Furthermore, monosaccharide and aglycone preparation involves dissolving the C-076 compound in water miscible solvent, and then an acid is added under stirring at room tem-perature to trigger the reaction.36) The lower concentrations of acid (0.01 to 0.1%) used in chemical reaction give rise to mainly monosaccharide and higher acid concentrations from 1 to 10% results in aglycone formation. In addition, interme-diate acid concentrations will generally produce mixtures of monosaccharide and aglycone. In general, sulfuric acid is sued to carryout above reactions.36)

Although, presence of monosaccharide and/or aglycone in AVMs drug substance or finished product thereof is consid-ered as degradation products or related substances, therefore, it is controlled by using an appropriate acceptance criteria. In contrast, these two derivatives are also shown to be effective against the control of parasites and thoroughly investigated for further derivatization and clinical applications.36) The reaction

Fig. 5. Acid Hydrolysis of AVMs

August 2012 939

mechanism of a typical glycolysis is shown in Fig. 5.The most appropriate way to avoid acid hydrolysis driven

degradation in AVM is to ignore developing formulated prepa-ration at pH 4.0 or below. However, aglycone and monosac-charide are known to carry forward characteristic ML anti-parasitic actions, but their presence as degradation product is avoided to prevent the chemical purity of specified molecule, because their biology efficacy is inferior to parent AVM mol-ecules.36)

3.2 Effect of Alkaline Conditions The biological activ-ity of AVM is greatly affected by minor molecular modifica-tions, in particular stereochemical changes at C2 (epimeriza-tion) position of oxahydrindene (hexahydrobenzofuran) unit results in substantial decreases of the biological activity.46,47) An investigation demonstrating hydroxide ion mediated isom-erization was previously reported for ivermectin,48) where DP was used as an internal standard for HPLC analysis.

Kinetic evidence suggested that base catalyzed conversion of AVM to conjugated Δ2,3 isomer, proceeded through the 2-epi isomer as an intermediate,48) although there is a pos-sibility of parallel conversion to both geometric forms to the conjugated isomer, as shown in Fig. 6 for AVM B1.47,48) Recent interest in total synthesis of the AVM B1 has revolved around the issue of deconjugating a Δ2,3 precursor with simultane-ous establishment of the proper stereochemistry at the C2 position.46–48) The equilibrium nature of the epimerization, as well as the total absence of deconjugation under equilibrium conditions, establish structural proof for both isomers 2-epi and Δ2,3-isomer.46)

Furthermore, AVMs are known to be most stable at pH 6.2–6.3,49) hence, there is possibility to avoid based catalyzed degradation when formulations are designed at/around the pH 6.25. In addition, there are also detailed reports avail-able on C2 epimerization of AVM in presence of methanolic potassium hydroxide (KOH) solution.48) Pivnichny and co-workers have shown that treatment of AVM B with methano-lic KOH solution affords a mixture of 2-epi isomer and the Δ2,3-isomer.47) Whereas, a subsequent study also showed the

formation of 2-epi- (25%) and Δ2,3- (70%) from parent com-pound using methanolic aqueous sodium hydroxide (NaOH) solution.46) It was also concluded that partial epimerization of 2-epi to parent compound was possible in a protic medium only if the polarity of the molecule are appropriate. Therefore, establishing the condition that epimerize the 2-epi isomer back to the desired natural isomer without concomitant con-jugation and/or aromatization, presents a major challenge and would be key to solve the stability issues of avermectin for-mulations. After investigating a number of reagents and condi-tions, it was found that imidazole in benzene was an effective reagent for the partial conversion of the 2-epi isomers in to the parent compound at about 40% isolated yields.48)

3.3 Effect of Oxidation AVMs, like their counterparts milbemycin, are prone to radical-induced oxidation at the C2–C8 oxahydrindene positions.50) Catagerocially, depending on the position and chemical reactions, oxidation can take place

Fig. 6. Abamectin Reactivity in Alkaline Conditions

Fig. 7. Scheme Showing Radical-Induced Oxidation of the Oxahydrindene Portion of Ivermectin B1, Where: 1) Ivermectin B1, 2) 8a-oxo Derivative, 3) 5-oxo Derivative, 4) 5,8a-Bisoxo Derivative

940 Vol. 60, No. 8

at either on hydrocarbon position (e.g. C8a) or involve substi-tution of existing –OH groups (e.g. C5), and forms derivatives such as 5-oxo-, 8a-oxo, 23-oxo, 13-oxo (aglyone), 4″-oxo (in disaccharide) and 4′-oxo (in monosaccharide).

Studies of oxidation in AVM have included synthesis of the 5-oxo derivatives by oxidation of the allylic C5 hydroxyl with MnO2.51) Oxidation of the C23–OH group of AVM B to the corresponding ketone is affected by a more complex method, while hydroxylation of the 4-methyl group with selenium oxide (SeO) and tert-butylhydroperoxide (t-BuOOH) is also reported. Furthermore, treatment of AVM with m-chloroper-benzoic acid (mCPBA) produce a number of epoxides includ-ing 8,9-oxide as major product.51)

In general, auto-oxidation requires the presence of an ini-tiator that provides sufficiently reactive free radicals to begin the chain reaction. Potential initiators in dosage forms would arise from peroxides and hydroperoxides in common formula-tion solvents, such as ethers, secondary alcohols, and esters. A reasonable model for auto-oxidation is Co2+ catalyzed decom-position of t-BuOOH, which produces tert-butylperoxy and tert-butoxy radicals. When AVM H2B1 (IVM) is allowed to react with O2 in the presence of these initiators, at least three oxahydrindene oxidation products can be formed, such as 8a-oxo-, 5-oxo derivatives and 5,8a-bisoxo-H2B1.

50) The mecha-nism and pathway of -oxo derivative formation is explained by scheme in Fig. 7.

Compounds 2, 3, and 4 possess a unique absorption spec-trum that characteristically sets it apart from the parent com-pounds as well as its acid- and base-catalyzed degradents. Further studies have also reported a highly specific and sensi-tive method for the determination of 2.52)

Although oxo derivatives of AVM are treated as degrada-tion products of drug substance and formulations [IVM USP monograph], but it still retains the characteristic anthelmintic activity of AVM, however, these are not superior to the parent molecules.53) On such instance, the 8a-oxo derivatives of AVM was synthetically prepared and evaluated for anthelmintic activity.53) It was also discovered that C5 carbon of AVM and milbemycins can be oxidized using pyridynium dichromate to the keto compounds.53) Given the high reactivity of –OH groups, it is essential to follow protection/de-protection model to facilitate desired oxidation. C5-oxo derivates are also used for the determination of parent compound52); it was reported that reaction of ammonium acetate produce a very strong florescent adduct which is used as key component for the analysis of the plasma sample for identification of trace levels of AVM residues.52)

As discussed in above sections, oxidation process takes place mainly at oxahydrindene unit attached to macrocycle, which is a common feature for AVM and MBN (e.g. nemadec-tin). A pioneer study by Eastlick et al. in 1989, at American Cyanamid, demonstrate that “these compounds are unstable under normal conditions of preparation, use and storage, and stability can be considerably improved by the addition of an-tioxidant, therefore, quality can be controlled in advance well before using for formulation.”54) The antioxidant can be pres-ent from 0.02 to 0.3% in samples. The experiment involved dissolving the nemadectin powder in acetone as control and duplication of same preparation with addition of antioxidants. In addition, other experiments involved omission of solvent from the preparations, where dry powders in sealed vials

were stressed for two weeks at 50°C. The results are shown in Table 4.

Addition of antioxidant has resulted in significant improve-ment in stability of the dry powder, where BHT found to be most effective at 250 ppm. In addition, presence of BHT in the also protects the compounds from photo-degradation as show in Table 5.

Light exposure experiments clearly demonstrated the advan-tage of having BHT in sample blend, resulting in remarkable difference of ca. 8% potency for the sample with and without BHT.

3.4 Effect of Light AVM shows sensitivity towards the strong light.55) The UV spectra of AVM demonstrating strong absorption below 300 nm, makes it prone to degrade under light exposure. UV light below 280 nm rapidly isomerizes the E (trans) 8,9 and 10,11 double bonds to the 8,9- and 10,11-Z isomers, and prolonged irradiation leads to a large number of unidentified decomposition products which lack any UV absorption.55)

Irradiation of methanolic AVM solution in a photo-chamber at maximum emission of 300 nm, produces a new compound, which was isolated from the reaction mixture and shown as double bond isomer giving a distinct peak in HPLC chro-matogram.55) The untreated solution showed two peaks with retention times (RT) of 6.1 and 7.3 min in a ratio of 6 to 94 for AVM B1b and B1a, respectively. After 20 min of irradiation, three peaks were observed at 6.0, 7.3 and 8.6 min RT, and the peak at 8.6 min was later identified as 8,9-Z isomer. Interest-ingly, the formation of 8,9 and a minor 10,11 isomer occurs via a reversible reaction, hence photo irradiation of pure compounds of these two isomers resulted in parent AVM com-pounds as shown in mechanism in Fig. 8.55)

The main photo isomer of ABM (8,9-Z) is also found in bi-ological samples of animals treated by topical preparations.56) A thin film of ABM B1a degrades with half-life of 4–6 h in sunlight, producing two optical isomers (mainly 8,9-Z) and several de-alkylated derivatives.57)

Therefore, use of light resistant container for storage of avermectin drugs and drug products, as well as utilizing low actinic glassware during the analysis, are considered as the best approach to avoid the photo-degradation.

However, on a different approach, elimination of double bonds responsible for light absorption can be considered as an option to stabilize avermectins too. AVM typically contains 5 double bonds (3= 4, 8= 9, 10= 11, 14= 15 and 22= 23), which can be reduced to respective dihydro derivatives. AVM double

Table 4. Effect of BHT on Stability of Nemadectin54)

Composition Antioxidant% Change in po-tency control (no

antioxidant)

% Change in potency (with antioxidant)

Preparation 1a) BHT (25 ppm) −26.3 −1.1Preparation 2a) BHT (250 ppm) −36.8 No changePreparation 3a) PG (250 ppm) −27.3 −8.8Preparation 4a) TBHQ

(250 ppm)−27.3 −8.8

Preparation 5b) BHT (250 ppm) −23.0 −10.0

Where a) Solution of nemadectin and BHT in acetone, b) BHT was triturated with dry nemadectin powder, BHT: butylated hydroxytoluene, PG: propyl gallate, TBHQ: t-butyl hydroquinone.

August 2012 941

bonds are also sensitive enough to undergo epoxide forma-tion under mild oxidizing condition; Mrozik et al. have found out that oxidizing agent can form epoxide from the C8,9 and C14,15 double bonds, when used in mild strength to protect other bonds and functional groups.58) Oxidizing agents such as mCPBA, alkyl hydroperoxides catalyzed with vanadyl acetylacetonates etc. were found suitable for epoxide forma-tion. When it is desired to prepare the 8,9-epoxide, a slight excess (10–30%) of the oxidizing agent is employed, and if 14,15 or both epoxides are desired, an amount of oxidizing agent equivalent to a slight excess of the two moles is em-ployed.58) Figure 9 shows a general illustration of the epoxide compounds.

In addition, novel derivatives of AVM are also prepared where one or all of the 3,4, 8,9 or 10,11 double bonds are reduced.59) The distortion in conjugated diene found to have a considerable impact on the light (UV) absorption charac-teristics of the molecule. As shown in Table 6, reduction of 10,11 double bond of IVM has resulted in significant increase in photo-stability of the molecules exposed under UV light, therefore, making them an ideal candidate for formulations where photo stability is a requirement for optimum perfor-mance of active compound.60) Figure 10 shows a typical struc-ture of a 3,4,8,9,10,11,22,23-octahydro-AVM B1a.59)

3.5 Solvent Adducts The novel form of compounds are also reported, where the AVM are crystallized as alcohol solvates to greatly enhance the stability of the conjugate drug during long-term storage. AVM conjugate compounds and their compositions were found to be highly potent antipara-sitic, insecticidal and anthelmentics.45)

Isopropanol (IPA) adduct of L-648,548 [13-O-MEM (methoxyethoxymethyl)-AVM B1 aglycone] is prepared by heating the compound with IPA, followed by crystallization using water. The resultant mixture was 1 : 1, IPA : avermectin adduct with a structure shown in Fig. 11.45)

During the study, IPA solvate were subjected to accelerated condition at room temperature and 40°C for 28 weeks, while taking a simple solvate (ethanol) as control. An HPLC result showed that ethanol solvate exhibited 12% degradation during storage even at room temperature while novel IPA solvate did not decompose significantly as shown in Table 7.45)

Table 5. Effect of BHT on Photolysis of Nemadectin54)

Nemadectin powder Storage conditionMeasured potency (%)

Start 3 d 7 d 10 d

No BHT 2–8°C 100.0 100.0 101.2 98.8Under UV light 100.0 92.8 83.5 84.3

BHT (250 ppm) 2–8°C 100.0 100.0 100.5 100.1Under UV light 100.0 100.8 92.5 92.5

Fig. 8. Photo-Degradation of Avermectin

Fig. 9. Epoxides of Avermectins

942 Vol. 60, No. 8

Table 6. Effect of UV Light Exposure (Maximum 300 nm) on Ivermectin and Its Reduced Derivative Solutions60)

Compound% Remaining

40 min 6 h 20 h 40 h

22,23-Dihydro-avermectin (ivermectin) 100 56 36 <510,11,22,23-Tetrahydro-avermectin 104 87 75 36

Fig. 10. Reduced Avermectin B1a (Where A, B, C and D Represents Single Bonds)

Fig. 11. IPA: 22,23-Dihydro Avermectin Aglycone Solvate45)

Table 7. Stability Results of IPA:22,23-Dihydro Avermectin Aglycone Solvate45)

Storage condition Solvate Initial 28 weeks % Degradation

Room temperature Ethanol 92.5 80.6 12IPA 94.9 95.6 0

40°C Ethanol NA NA NAIPA 94.9 91.4 4

Table 8. Efficacy Data of Prominent Avermectin and Derivatives11)

Avermectin component Dose (mg/kg)

Efficacya)

H.c.c) O.c.c) T.a.c) T.c.c) C.spp.c) O.c.c)

A1 0.1 2 2 0 0 2 0A2 0.1 3 3 3 3 0 3B1 0.05 3 3 3 3 3 3B2 0.1 0 3 3 3 3 3H2A1b) 0.3 3 2 0 1 0 3H2B1b) 0.1 3 3 3 3 3 3B1MSb) 0.15 2 2 3 3 3 0B2MSb) 0.2 1 1 3 3 3 3H2B1MSb) 0.3 3 3 3 3 2 3H2B1AGb) 3.0 1 2 3 3 1 3H4B1b) 0.2 0 0 1 0 0 3

Where a) 0=<50%, 1=50–74%, 2=75–90%, 3=>90% efficacy; b) MS=monosaccharide, AG= aglycone, H2=22,23 dihydro derivative, H4=3,4,22,23-tetrahydro derivative; c) H.c.=Haemonchus contortus, O.c. Ostertagia circumcinta, T.a.=Trichostrongylus axei, T.c.=Trichostrongylus colubriformis, C.spp.=Cooperia spp., O.c.=Oesophagostomum columbianum.

Table 9. Biological Activity of Avermectin B1 and Its Based Catalyzed Degradents47)

CompoundMortality %

6.25 ppm 1.25 ppm 0.25 ppm 0.05 ppm 0.01 ppm LC90 ppm

Avermectin B1 — — — 100 66 0.0382-epi-Avermectin B1 100 78 17 3 — 4.0Δ2,3-Avermectin B1 100 100 97 35 — 0.23

August 2012 943

4. Effect of Stability and Chemical Modifications on Biological Activity of AVM

It has been shown in above sections that stability of AVM is mainly compromised by the lability of –OH groups, shift/reduction of double bond/s, reactive disaccharide unit/s and reasonably acidic proton at C2 position of oxahydrindene unit. It has been demonstrated that removal of either one or both saccharide rings has resulted in loss of efficacy,36) whereas reduction of double bond resulted in decline of overall efficacy too.11) In general, compounds of B series containing –OH are most potent than A series compounds with –OCH3 group.3) Although 22,23-AVM B1 has shown slight deficiency than parent AVM B1, but conferred sufficient overall efficacy and safety. In contrast, its monosaccharide is 2–4 folds and agly-cone was 30 fold less potent than parent compound (see Table 8).

On other hand, activity of AVM B and its base catalyzed degradation products 2-epimer and conjugatively stable Δ-2,3 isomer were compared against two-spotted spider mite.47) The data in Table 9 shows that 2-epimer is approximately 1/100 potent as AVM B1 while Δ2,3 isomer is 1/5 potent.

5. ConclusionABM and its major derivatives like IVM and EPM has

been researched in details. It has been found out that unique structure of AVM molecules and presence of labile groups facilitated the derivatization of AVM into various compounds showing strong anthelmintic activity. However, the same unique structure is also responsible for labile nature related to sensitive stability profile of molecules. AVMs are found to be unstable in acidic and alkaline conditions, and to avoid their degradation in formulated products, a pH close to 6.25 is rec-ommended. On other hand, these compounds are sensitive to strong light, and presence of photo-isomer in animals treated topically with AVM product, is well known. The pharmaco-epial recommendation for addition of antioxidant into drug substance, as well its product, arises from the fact that AVM are very sensitive to oxidation. Formations of solvates, salts, epoxides, reduction of double bonds etc., were some chemi-cal approaches used to retard the degradation in AVM. This coherent review of the AVM drug substances and derivatives candidate showing potential as an anthelmintic, will help to establish the correlation of chemical process, relevant stability profile and biological activity. Therefore, it will help to design the shelf-life stable formulations containing AVMs.

Acknowledgements This research work was supported by Technology New Zealand (TechNZ). The authors would like to take this opportunity to thank Mr. Robert Holmes and Ancare Scientific Ltd., New Zealand, for their continuous support.

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