ACETOHEXAMIDE

Abdullah A. Al-Badr and Humeida A. El-Obeid

Pharmaceutical Chemistry Department

College of Pharmacy

King Saud University

Riyadh, Saudi Arabia

ANALYTICAL PROFILES OF DRUG SUBSTANCES Copyright 0 1992 by Academic Press, Inc.

-
AND EXCIPIENTS VOLUME 21 1 All rights of reproduction reserved in any form
2 ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID

C O N T E N T S

1. DESCRIPTION
1 . 1 Nomenclature
1.1.1 Chemical Names
1.1.2 Genermic Names
1.1.3 Trade Names
1 . 2 Formulae
1.2.1 Empirical
1.2.2 Structural
1.2.3 GAS No.
1.3 Molecular Weight
1 . 4 Elemental Composition
1 . 5 Appearance

2. PHYSICOCHEMICAL PROPERTIES
2 . 1 Melting Range
2.2 S o l u b i l i t y
2 . 3 Polymorphism
2 . 4 Thermal Analysis
2 . 5 X-ray Powder D i f f r a c t i o n
2.6 Spectral Properties
2.6.1 U l t r a v i o l e t Spectrum
2.6.2 I n f r a r e d Spectrum
2.6.3 Proton Nuclear Magnetic Resonance (PMR)
Spectrum
2.6.4 lac-Nuclear Magnetic Resonance (‘SC-NMR)
Spectrum
2.6.5 Mass Spectra

3. SYNTHESIS

4. METHODS OF ANALYSIS
4 . 1 T i t r i m e t r i c Methods
4.1.1 Nonaqueous
4.1.2 Gravimetric
4.1.3 Campleximetric
4.2 Spectrometric Methods
4.2.1 Colorimetric
4.2.2 U1t r a v i o l e t
4.2.3 Infrared
4.2.4 Fluormetric
4.2.5 Proton Magnetic Resonance
 ACETOHEXAMIDE 3

4.3 Chromatographic Methods

4.3.1 Thin-Layer Chromatography (TLC)
4.3.2 Gas-Liquid Chromatography (GLC)
4.3.3 High-Performance Liquid Chromatography
(HPLC)

5. PHARMACOKINETICS
5 . 1 Introduction
5.2 Mechanism o f Action
5.3 Onset and Duration o f Action
5.4 Absorption
5.5 Distribution
5.6 Metabol ism
5.7 Excretion
5.8 Half-Life

ACKNOWLEDGEMENT

REFERENCES
4 ABDULLAH A. AL-BADR AND HUMEIDA A . EL-OBEID

ACETOHEXAMIDE

1. DESCRIPTION

-
1 1 Nomenclature

1.1.1 Chemical Names

4-Acetyl-N-[(cyclohexylamino)carbonyl]benzenesul-
fonamide
l-[(pAcetylphenyl)sulfonyl]-3-cyclohexylurea.
3-Cyclohexyl-l-(pacetylphenylsulfonyl)urea.
N-(pAcetyl benzyl sul fonyl l-N -cyclohexyl urea.

1.1.2 Generic Names

Acetohexamide, Acetohexamidum

-
1 1.3 Trade Names

Cycl am1de , Dime 1in , Dime1o r , Dyme 1o r , Gamadiabet,

Metaglucina, Ordimel, Tsiklamid.

1.2 Formulae

1.2.1. EmDlriCal

Ct sHzoNz04S

1.2.2 Structural

1.2.3 CAS No.

[968-81-01

1.3 Molecular Weight

324.42 (1)

1.4 Elemental ComDosltion

C 55.54%, H 6.21%, N 8.64%, 0 19.73%, S 9.89% (1).

 ACETOHEXAMIDE 5

1.5 Armearance

A w h i t e , c r y s t a l l i n e powder; o d o r l e s s o r almost
odorless (2).

2. PHYSICOCHEMICAL PROPERTIES

2.1 M e l t i n g Range

C r y s t a l s from 90% aqueous e t h a n o l m e l t between

188-190" ( 3 ) . Crystals from d i l u t e ethanol m e l t between
175-177 (4).

2.2 Solubility

Soluble i n p y r i d i n e , s l i g h t l y soluble i n alcohol

and chloroform. I n s o l u b l e i n water and ether ( 1 ) .

2.3 P01YmOrDh'ism

The l i t e r a t u r e r e p o r t s i n d i c a t e t h a t acetohexamide
e x i s t s as more t h a n one polymorphic forms ( 5 - 1 5 ) .
G i rgis-Takla and Chroneos (5) prepared acetohexamide
polymorphs A and B by h e a t i n g t h e drug ( 1 gm) w i t h
g l a c i a l a c e t i c a c i d o r chloroform respectively, before
c r y s t a l l i z a t i o n a t 1 0 5 ' and room t e m p e r a t u r e
respectively. While acetohexamide polymorph A showed a
m e l t i n g range o f 180"-183', t h e acetohexamide polymorph
B melted a t 183'-185". D i f f e r e n t i a l scanning
calorimetry and I R spectroscopy showed t h a t c r y s t a l s o f
polymorph B were converted t o polymorph A by grinding.
A c c o r d i n g l y , t h e s e r e s u l t s i n d i c a t e t h a t any
i d e n t i f i c a t i o n t e s t u t i l i z i n g g r i n d i n g may f a i l to
i d e n t i f y t h e two polymorphs. I n t h e i r phystco-chemical
studies on t h e polymorphism o f acetohexamide, Kuroda e t
a7 (6) obtained t h r e e polymorphs o f acetohexamide by
r e c r y s t a l l i z a t i o n from d i f f e r e n t solvents. These are
f o r m I,f o r m I 1 and CHC13-11. A l t h o u g h t h e X-ray
d i f f r a c t i o n p a t t e r n s , I R s p e c t r a and d i f f e r e n t i a l
scanning calorimeter curves o f t h e CHC13-I1 polymorph
were i d e n t i c a l w i t h those o f polymorph 11, t h e CHC13-I1
t y p e c o n t a i n e d a C H C l j molecule which c o u l d n o t be
removed by normal d r y i n g condition. Polymorph CHC13-I1
seemed t o be unsuitable f o r medicinal use. Form I 1 i s
1.2 times more soluble than form I.
6 ABDULLAH A. AL-BADR AND HUMElDA A. EL-OBEID

Burger ( 7 ) c h a r a c t e r i z e d t h e t h r e e p o l y m o r p h i c
m o d i f i c a t i o n s o f acetohexamide by thermomicroscopy,
d i f f e r e n t i a l scanning calorimetry and I R spectroscopy.
The s o l u b i l i t y behavior o f the three modifications o f
the drug i n butanol and buffer solutions i s described
and d i s c u s s e d i n r e l a t i o n t o thermodynamics and
pharmacological parameters such as b i o a v a i l a b i l i t y from
t a b l e t s and USP X I X d i s s o c i a t i o n t e s t . M u e l l e r and
L a g a s ( 8 ) h a v e c o n f i r m e d t h e e x i s t e n c e and
characterized two polymorphic forms o f acetohexamide
using d i f f e r e n t i a l scanning calorimetry, thermogravi-
metric analysis, scanning e l e c t r o n microscopy as we1 1
as I R , NMR and X-ray analysis. The study has pointed t o
the u n s u i t a b i l i t y o f phosphate b u f f e r s o l u t i o n which i s
sometimes prescribed f o r use i n the d i s s o l u t i o n t e s t s
o f the drug since the s a l t o f the drug c r y s t a l l i z e s out
during the t e s t . I n another study (9) the same authors
reported t h a t form Idecomposed during melting and form
I1 melted a t 180" and then r e c r y s t a l l i z e d t o form I.A t
a heating r a t e o f lO'/minute melting points o f 193.6"
and 180.5" were found f o r forms Iand 11, respectively.
No morphological differences were observed between the
two forms. I n s o l u b i l i t y and d i s s o l u t i o n r a t e studies
i n sodium potassium b u f f e r , potassium acetohexamide
c r y s t a l l i z e d e x h i b i t i n g a lower s o l u b i l i t y than
acetohexamide. I n t h i s respect, form I 1 was transferred
t o potassium acetohexamide more quickly than form I.

Yokoyama e t a7 (10) calculated the thermodynamic values

o f forms I and I 1 o f acetohexamide from s o l u b i l i t y
measurements. The t r a n s i t l o n temperature and the heat
o f t r a n s i t i o n were 154" and 230 cal/mole, respectively.
I t i s found t h a t the polymorphic forms o f acetohexamide
d i d n o t a f f e c t i t s b i o a v a i l a b i l i t y when i n v i v o
absorption studies o f form I & I 1 were c a r r i e d out i n
b e a g l e dogs. The p r e p a r a t i o n o f f o u r c r y s t a l l i n e
modifications o f acetohexamide was reported (11). Their
thermograms, I R s p e c t r a , X-ray d i f f r a c t i o n and
s o l u b i l i t y are also reported. Two o f the forms reverted
t o the most stable form on storage i n solution.

S o l i d dispersion o f acetohexamide was studied by Graf

e t a7 (12-14) u s i n g d i f f e r e n t polymers and v a r i o u s
ratios. C o p r e c i p i t a t e s o f acetohexamide w i t h
polyethylene g l y c o l (PEG 6000) were prepared by t h e
s o l v e n t method w i t h ethanol ( c r y s t a l l i n e form I)or
with chloroform ( c r y s t a l 1ine form 111). Phase diagrams
 ACETOHEXAMIDE I

o f form I-PEG and form 111-PEG coprecipitates were o f

the p e r i t e c t i c type and the molecular compounds were
formed i n the r a t i o o f 1 mole o f acetohexamide t o 4
moles o f PEG. The e u t e c t l c t e m p e r a t u r e , e u t e c t i c
composition and the end o f melting o f the two binary
system were, however, d i f f e r e n t ( 1 2 ) . Both the
s o l u b i l i t y and t h e s o l u t i o n r a t e were increased by PEG.
S i m i l a r r e s u l t s were o b t a i n e d by s u b s t i t u t i n g
p o l y ( v i n y l p y r r o 1 i d o n e ) (PVP) f o r PEG ( 1 3 ) . Also,
c o p r e c i p i t a t e s o f acetohexamide-PVP ( i n e t h a n o l )
containing drug concentrations o f 60% o r more showed
the same X-ray d i f f r a c t i o n pattern as t h a t o f form I.
Increasing the PVP concentration ( > 55%) d i d n o t show
any c r y s t a l behavior i n the X-ray analysis. I n another
r e p o r t Graf e t a7 ( 1 4 ) d e s c r i b e d t h e methods o f
p r e p a r a t i o n and t h e e f f e c t o f t h e s o l v e n t s on t h e
acetohexamide-PVP coprecipi tates. They were obtained
from ethanol o r chloroform by evaporating the solvent
a t room temperature, under vacuum or by spray drying.
Changing t h e s o l v e n t and/or i t s e v a p o r a t i o n r a t e
affected the polymorphic form, the c r y s t a l l i n i t y and
the s o l u t i o n r a t e o f acetohexamide i n coprecipitates
containing less than 70% PVP.

Kassem e t a7 (15) studied the enhancement o f the r a t e

o f release o f acetohexamide from i t s t a b l e t s by t h e
f o r m a t i o n o f s o l i d d i s p e r s i o n s w i t h each o f f o u r
water-sol uble pol ymers prepared in d i f f e r e n t r a t i0s.
The polymers were r a t e d i n t h e o r d e r o f decreasing
r e l e a s e r a t e s as f o l l o w s : PEG 6 0 0 0 , PVP,
hydroxypropylmethylcellulose, methylcellulose.
2.4 Thermal Analysis

The h e a t o f f u s i o n and m e l t i n g p o i n t o f
acetohexamide were done u s i n g DuPont TA 9900 on t h e
DSC- u n i t a t a temperature range i n d i c a t e d i n t h e
thermogram (Figure 1). Sample i s done i n duplicate and
the average o f t h e value i s reported as follows:

AHf = 63.7 kJ/mOle Purity = 99.82% Tm = 187.45 C

2.5 X-ray Powder D i f f r a c t i o n

The X-ray powder d i f f r a c t i o n p a t t e r n o f acetohexa-

mide was determined using P h i l i p s f u l l automated X-ray
d i f f r a c t i o n spectrogoniometer equipped w i t h PW1730/10
 PURITY v l . l A

F i g u r e 1. Thermal cu rve o f acetohexamide.

 ACETOHEXAMIDE 9

Figure 2 . X-Ray powder d i f f r a c t i o n p a t t e r n of acetohexamide.

10 ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID

generator. Radiation was provided by a copper t a r g e t

(Cu anode 2000W, X = 1.5480 A), high I n t e n s i t y X-ray
tube operated a t 40 kV and 35 mA. The monochromator was
a curved s i n g l e c r y s t a l one (PW1752/00). Divergance
s l i t and t h e r e c e i v i n g s l i t were 1 and 0.1
r e s p e c t i v e l y . The scanning speed o f t h e gonlometer
(PW1050/81) used was 0.02 2 8 p e r second. The
instrument i s combined w i t h P h i l i p s PM8210 p r i n t i n g
r e c o r d e r w i t h b o t h analogue r e c o r d e r and d i g i t a l
p r i n t e r . The goniometer was a l i g n e d using s i l i c o n
sample before use.

The X-ray pattern o f acetohexamlde I s presented i n

Figure 2. The interplanar distance d(A) and r e l a t i v e
i n t e n s i t i e s 1/10 are shown i n Table 1.

2.6 Spectral ProDerties

2.6.1 U l t r a v i o l e t Spectrum

The u l t r a v i o l e t a b s o r p t i o n s p e c t r u m o f
acetohexamide i n methanol was obtained on a Cary 219
spectrophotometer. The spectrum, shown i n Figure 3, i s
characterized by two maxima. The one w i t h a Xmax a t 247
nm i s t y p i c a l o f s u b s t i t u t e d acetophenones. The
absorption a t X m a x 283 nm represents a conjugated
aromatic r i n g system.

2.6.2 I n f r a r e d SDectrum

The i n f r a r e d absorption spectrum o f acetohexamide,

obtained from a potassium bromide d i s p e r s i o n , was
recorded on a Pye Unicam SP 1025 spectrometer and i s
shown i n Figure 4. The assignment o f the c h a r a c t e r i s t l c
bands are shown i n Table 2.
 t I 1 1 i

220 XO nm 300 3 50 400 450

Figure 3 . U l t r a v i o l e t spectrum o f acetohexamide in methanol.
v,.
m.
c
N.
C V
cn
c -I-
I-- U
6,. L
m
Y
al. "
Q.
z!
W
E
N
Y
W
c
t-. 0
c,
W
V
tcl
'*-
0
f
L
c,
V
W
0
cn
*I
v), U
E
fu
I
U
 ACETOHEXAMIDE 13

Table 1: X-ray d i f f r a c t i o n p a t t e r n o f acetohexamfde

d(A) 1/10 d(A) 1/10

15.74 31.25 2.55 1.82

9.47 30.04 2.49 1.75
7.85 6.89 2.40 6.19
7.21 2.25 2.36 4.44
5.30 100.00 2.31 5.26
4.99 8.28 2.29 2.20
4.93 10.95 2.27 1.81
4.55 4.71 2.24 2.54
4.30 5.30 2.18 2.04
4.19 15.19 2.15 2.38
4.08 23.07 2.13 1.20
3.92 5.44 2.09 2.56
3.78 2.82 2.04 4.51
3.60 24.35 1.99 1.69
3.50 4.52 1.95 2.91
3.28 23.29 1.94 4.16
3.26 9.83 1.89 1.50
3.15 5.72 1.81 1.48
3.07 9.36 1.77 1.29
3.01 1.26 1.72 1.77
2.91 7.99 1.66 1 .oo
2.88 2.79 1.64 1.18
2.74 4.08 1.61 1.16
2.65 1.51 1.57 1.32
2.61 2.15 1.47 0.85
2.58 1.80 1.35 0.80

d = Interplanner distance
1/10 = r e l a t i v e i n t e n s i t y (based on highest i n t e n s i t y of
100).
14 ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID

Table 2: I n f r a r e d c h a r a c t e r i s t i c bands and t h e i r

assignments.

Frequency (cm- Assignment

3340, 3270 Amide N-H s t r e t c h

2980, 2940 Aromatic C-H s t r e t c h

0

1710, 1680 Conjugated - E-

1602 , 1600 Aromatic C s t r e t c h

1455 C - CH3 bending

0

1345

Aromatic C-H out o f plane

780, 760
bending .

2.6.3 Proton Nuclear Magnetic Resonance ( W R l

Spectrum

Acetohexamide s o l u t i o n i n DMSO-de was used t o

obtain the PMR spectrum on a Varian XL 200 MHr FT NWR
spectrometer u s i n g TMS as i n t e r n a l reference. The
spectrum i s shown i n Figure 5. The number o f protons i s
established by both i n t e g r a t i o n o f the area under the
curve and t h e m u l t i p l i c i t i e s o f t h e peaks. Table 3
a s s i g n s t h e chemical s h i f t s t o t h e i r r e s p e c t i v e
protons. F u r t h e r evidence f o r p r o t o n assignment i s
obtained from the HETCOR pulse sequence (Figure 9).
Figure 5. PMR spectrum of acetohexamide i n DMSO-dG using TMS
as internal reference.
F i g u r e 6. 1 3 C NMR spectrum o f acetohexzmide i n DMSO-ds using
TMS as internal reference.
Figure 7. 1 3 C NMR spectrum o f acetohexarnide u s i n g DEPT
.
ex P e r iment
F i g u r e 8. 1 3 C I M R sDectrum of acetohexanide u s i n g APT
expe r irnent .
Figure 9. 13C N M R spectrum of acetohexamide using HETCOR
experiment.
20 ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID

Table 3: Assignment o f the NMR chemical s h i f t s t o the

d i f f e r e n t protons

Chemical s h i f t Multiplicity Proton No. o f

(6) assignment protons

1.09 - 1.71 mu1ti p l e t Cyclohexyl 11

ring3

2.66 singlet -
CH3-0 3

6.45 doublet -
CH-NH 1

8.06 - 8.19 mu1t ip l e t Aromat ic d 4

2.6.4 13C-Nuclear Magnetic Resonance (13C NHR)

SDect rum

The 1 3 C NMR spectra o f acetohexamide i n DMSO-ds

using TMS as i n t e r n a l reference are obtained using a
V a r i a n XL 200 MHz p u l s e FT s p e c t r o m e t e r and a r e
p r e s e n t e d i n F i g u r e s 6-9. The assignment o f t h e
chemical s h i f t s and the degree o f carbon protonation,
presented i n Table 4, are achieved u s i n g t h e DEPT
(Figure 7) and APT (Figure 8) experiments as well as
t h e HETCOR p u l s e sequence ( F i g u r e 9 ) and t h e
approximate a d d i t i v e e f f e c t s o f substituents.

2.6.5 Mass SDectra

The 70 eV e l e c t r o n impact mass spectrum o f

acetohexamide, presented i n Figure 10, was obtained on
Varian MAT 311 mass spectrometer u s i n g i o n source
pressure o f 10-0 Torr, i o n source temperature o f 180'C
and an emission current o f 300 M. The molecular i o n i s
detectable a t m/e 324 and the base peak a t m/e 56. A
proposed fragmentation p a t t e r n and t h e mass/charge
r a t i o s o f the major fragments are shown I n Scheme 1.
 ACETOHEXAMIDE 21

Table 4: Assignment o f the carbon chemical s h i f t s .

Chemical s h i f t Carbon assignment Number o f Protons

(PHI attached

24.26 d 2

25.07 C 2

26.99 e 3

32.33 b 2

48.30 a 1

127.73 i 1

128.73 j 1

140.00 k 0

143.93 h 0

150.45 9 0

197.30 f 0

The chemical i o n i z a t i o n spectrum, shown i n Figure 11,

was obtained on Finnigan 4000 mass spectrometer using
methane gas as a reagent with ion electron energy o f
100 eV, ion source pressure o f 0 . 3 T o r r , i o n source
temperature o f 150’C and emission current o f 300 pA.
The spectrum i s dominated by a quasimolecular i o n (M +
1 ) . Two peaks appearing a t m/e 353 and m/e 365 are
a t t r i b u t a b l e t o the t r a n s f e r o f carbocations from the
c a r r i e r gas. The mass s p e c t r a l assignment o f t h e
N
N

F i g u r e 10. Electron impact mass spectrum o f acetohexamide.

Figure 11. Chemlcal ionization mass spectrum o f
acetohexarnide.
24 ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID

prominent ions under the chemical i o n i z a t i o n conditions

are presented i n Table 5.

Table 5: Mass spectral assignment o f acetohexamide

using chemical ionization.

M/e Species

365 [M t C3H5]+

353 [M t CzHs]+

325 [M t H (MH)1+

324 [MI+

3. SYNTHESIS

Marshall e t a7 ( 4 ) reported a method o f synthesis

o f acetohexamide which i n v o l v e s t h e r e a c t i o n o f t h e
diazonium s a l t from paminoacetophenone w i t h s u l f u r
dioxide t o a f f o r d the sulfonyl chloride which i s then
converted t o the sulfonamide by reaction w i t h a m n i a .
E l a b o r a t i o n v i a t h e carbamate w i t h cyclohexylamine
a f f o r d s acetohexamide. Another r e p o r t e d method ( 1 6 )
uses p-chloroacetophenone as t h e s t a r t i n g m a t e r i a l .
Both methods are o u t l i n e d i n Scheme 2.
 ACETOHEXAMIDE 25

Scheme 1: Proposed mass fragmentation pattern o f

acetohexamide

n
0 0
W-Q-

I
O H
mle 3 2 4

+
0-H NH
I1

m/e 243
26 ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID

Scheme 1 Continued ...

mle183
m l e 324
I -CH,-CO

m /el41

mle 324 m l e 200

I
- YN0,S

0
C H3I; 0 -i

mle76 m l e 104 mle 119

 ACETOHEXAMIDE 21

Scheme 1 Continued ...

1
[ O N H I +

m/e 324

i
0
II

2 68
+
28 ABDULLAH A . AL-BADR A N D HUMEIDA A. EL-OBEID

Scheme 3: Synthesis o f acetohexamide

Method 1 (4)

SO,-NH-C-NH

Method 2 (16)

0 0
CH,t@ S03Na p0c13*

c H 3 - ! G so, CI SO,NH,-
 ACETOHEXAMIDE 29

4. METHODS OF ANALYSIS

4.1 T i t r i m e t r i c Methods

4.1.1 Nonaaueous

A non-aqueous t i t r a t i o n method f o r t h e d r u g and

other hypoglycemic and d i u r e t i c agents was reported by
Agarwal and Walash (17). The drug i n t a b l e t o r pure
form was d i s s o l v e d i n t e t r a m e t h y l urea and t i t r a t e d
w i t h 0.1 N l i t h i u m m e t h o x i d e i n benzene-methanol
medium. The end p o i n t was determined u s i n g 0.2% azo
v i o l e t i n toluene as i n d i c a t o r . Recovery ranged from
98.8% t o 101.6%.

A n o t h e r non-aqueous t i t r a t i o n p r o c e d u r e , f o r t h e
q u a n t i t a t i v e a n a l y s i s o f t h e d r u g and o t h e r
h y p o g l y c e m i c s u l f o n y l u r e a s u s i n g HC104 t i t r a t i o n
method, was a l s o reported (18).

4.1.2 Gravimetric

Amer and Walash (19, 20a) r e p o r t e d a method f o r

t h e g r a v i m e t r i c d e t e r m i n a t i o n o f acetohexamide by
treatment w i t h 2,4-dinitrophenylhydrazine t o
p r e c i p i t a t e t h e h y d r a z o n e (19). A m i x t u r e o f
acetohexamide, tolbutamide and chlorpropamide was a l s o
determined g r a v i m e t r i c a l l y (20a).

4.1.3 Compleximetric

G u e r e l l o and Dobrecky (21) have d e s c r i b e d a

procedure f o r t h e compleximetric e v a l u a t i o n o f
medications w i t h hyoglycemic a c t i o n i n c l u d i n g
acetohexamide. The procedure permits the determination
o f t h e hypoglycemic sulphonylureas. A weighed amount o f
drug was h y d r o l y s e d by h e a t i n g f o r 30 minutes w i t h
d i l u t e aqueous sodium h y d r o x i d e and t h e s o l u t i o n
n e u t r a l i z e d w i t h 0.1 N HC1, t r e a t e d w i t h 0.1 M CuSO4,
then w i t h b u f f e r s o l u t i o n t o pH 6, and f i l t e r e d . The
excess C U + ~ i n t h e f i l t r a t e was d e t e r m i n e d by
complexometric t i t r a t i o n w i t h 0.02 M EDTA disodium s a l t
u s i n g 1-(2-pyridylazo)-2-naphthol as i n d i c a t o r . The
method i s applicable t o evaluate drugs i n t a b l e t .
30 ARDUI.1.AH A. AL-BADR AND HUMEIDA A. EL-OBEID

4.2 SDect romet r i c

4.2.1 Colorimetric

Reaction o f acetohexamide w i t h 2,4-dinitropheny’l-

hydrazine t o produce t h e colored hydrazone was used by
Amer and Walash (19) t o determine t h e drug c o l o r i m e t r i -
c a l l y . The c o l o r e d p r o d u c t was d i s s o l v e d i n KOH and
determined a t 480 nm. The accuracy o f the method was
claimed t o be 100%. A n i n h y d r i n c o l o r i m e t r i c method f o r
some o r a l hypoglycemic agents was a l s o reported (20b).

Meier e t a7 ( 2 2 ) analysed acetohexamide and o t h e r

h y p o g l y c e m i c a g e n t s by d i s s o l v i n g t h e d r u g i n
chloroform, adding calcium acetate (1% i n methanol),
propylamine (5% i n methanol), d i l u t i n g w i t h chloroform
and reading the absorbance a t 565 nm a f t e r 15 minutes.
Pharmaceutical preparations may be estimated s i m i l a r l y .

4.2.2 U l t r a v i o l e t (UV)

Solomonova and D v o r n i t s k a y a ( 2 3 ) d e t e r m i n e d
acetohexamide by measuring t h e absorbance a t 229 nm i n
ethanol or 0.1 M sodium hydroxide. Other UV t e s t s f o r
t h e drug are a l s o reported (24, 25).

4.2.3 Infrared (IR)

Acetohexamide and o t h e r s u l p h o n y l u r e a s were

analysed by IR (22). A t e s t have a l s o been described
(24). Lazaryan (26) determined t h e d r u g and o t h e r
h y p o g l y c e m i c a g e n t s by i n f r a - r e d a b s o r p t i o m e t r i c
determination. A sample i s t r e a t e d with chloroform and
t h e s o l u t i o n from t h e t a b l e t sample i s f i l t e r e d . A
p o r t i o n o f s o l u t i o n i s d i l u t e d w i t h chloroform and t h e
absorbance i s measured a t 1722 t o 1715 cm-1 i n 0.25 m
NaCl c e l l against chloroform.

4.2.4 F1uoromet r ic

G i r g i s - T a k l a and Chroneos ( 2 7 ) d e s c r i b e d a
s e n s i t i v e method f o r t h e f l u o r o m e t r i c determination o f
t h e d r u g i n plasma o r i n t a b l e t s by means of i t s
r e a c t i o n w i t h 1 - m e t h y l n l c o t l n a m i d e . The l i m i t o f
d e t e c t i o n was approximately 0.2 Mg o f t h e drug/mL and
t h e r e l a t i v e standard d e v i a t i o n was 31% f o r 2 Ng/ml i n
 ACETOHEXAMIDE 31

plasma. The method i s s u i t a b l e f o r plasma samples

containing 0.5-2.5 Mg o f the drug/ml.

4.2.5 Proton Magnetic Resonance

Al-Badr and Ibrahim (28) described a simple, r a p i d

and accurate method f o r t h e assay o f the drug and other
hypoglycemic agents u s i n g p r o t o n magnetic resonance
spectrometry. The pure drug o r i n t a b l e t form, can be
determined using DMSO-ds as solvent and maleic a c i d as
i n t e r n a l standard.

The reported recovery i s 100 f 1.5% f o r pure drug and

98 t o 99.6 f 1.4% f o r t a b l e t s .

4.3 ChromatonraDhic Methods

4.3.1 Thin-Layer ChromatonraDhY (TLC]

Gergis-Takla and Josh1 (29) reported a TLC method

f o r the i d e n t i f i c a t i o n , assay and p u r i t y determination
o f t h e drug and other hypoglycemic agents i n powder and
i n t a b l e t f o r m u l a t i o n . The d r u g was d e t e c t e d by
d i s s o l v i n g powdered t a b l e t s o r powder i n
dichloromethane-acetone m i x t u r e (2: 1) and chromato-
graphing t h e s o l u t i o n on s i l i c a gel F 2 5 4 p l a t e s with
cyclohexane-chloroform-acetic a c i d and e t h a n o l
(10:7:2:1). For q u a n t i t a t i v e determination, t h e spots
were s e p a r a t e d , e l u t e d w i t h m e t h a n o l i c sodium
hydroxide, d i l u t e d w i t h m e t h a n o l i c HC1 and t h e
absorbance was measured.

Surborg and Roeder (30) have recommended c o n s t a n t -

b o i l i n g s o l v e n t m i x t u r e s f o r t h e development o f
chromatograms on s i l i c a gel f o r acetohexamide and o t h e r
a n t i d i a b e t i c drugs: propanol-cyclohexane (37:163),
propanol-benzene-cyclohexene (9:14:27), and cyclohexane
- isopropanol (177:23). The R f values o f t h e drugs a r e
t a b u l a t e d , s p o t s were l o c a t e d by v i e w i n g i n 254 nm
radiation.

4.3.2 Gas L i a u i d ChromatonraDhY (GLC)

Kleber e t a l . (31) determined acetohexamide and

hydroxyhexamide i n b i o l o g i c a l f l u i d s u s i n g GLC.
Tolbutamide was used as an i n t e r n a l standard and M-HC1
was added t o the sample o f plasma o r urine, the m i x t u r e
32 ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID

was shaken w i t h t o l u e n e and was c e n t r i f u g e d . The

separated organic phase was shaken w i t h 7.5% KzC03
s o l u t i o n and centrifuged again. The aqueous phase was
heated a t 6 0 " f o r 1 0 m i n u t e s w i t h methanol and
dimethylsulphate, cooled and M-acetate b u f f e r s o l u t i o n
was added t o a d j u s t t o pH 5.2. The m e t h y l a t e d
sulphonylureas were e x t r a c t e d w i t h hexane and t h e
e x t r a c t was evaporated t o dryness a t 50' i n a stream o f
nitrogen. The residue was dissolved i n CS2-CHC13 (l:l),
25 u l and 2 p l was submitted t o GLC on a glass column
(61 cm X 3 mn) containing 0.5% o f PEG 20 M on Gas-Chrm
Q (80 t o 100 mesh) and the temperature was programed
f o r 190 t o 240' a t 5 min-1, w i t h helium as c a r r i e r gas
(90 m l min-1) and flame i o n i s a t i o n d e t e c t i o n . Peak
heights were compared. A t concentrations o f 10 t o 40 ug
m l - 1 i n plasma. The mean recoveries (8 determination)
were : f o r acetohexamide 9.9 and 39.4 c(g m l - 1 ; f o r the
metabolite hydroxyhexamide 14.1 and 40 c(g m l - 1 .

Fricke (32) presented a GLC method f o r t h e analysis o f

t h e drug and o t h e r drugs i n pharmaceuticals, u s i n g
s i m p l e e x t r a c t i o n s and semiautomated g a s - l i q u i d
chromatography, using Ddxil 300 as the l i q u i d phase and
an automatic sample i n j e c t o r . Results by t h i s method
and t h e o f f i c i a l and o t h e r a p p l i c a b l e methods a r e
compared. Content uniformity analysis can be made by
u s i n g t h i s procedure. The e x t r a c t i o n and chromato-
graphic conditions were standardized t o make possible a
successful interlaboratory study.

4.3.3 Hinh-Performance L i a u i d ChrmatoqraDhy

( HPLCl

A simple HPLC assay o f t h e drug i n plasma was

developed by T a k a g i s h i e t a7 ( 3 3 ) . A sample was
extracted w i t h a mixture o f benzene and e t h y l acetate
a t pH 5 and t h e organic phase was evaporated. A 50%
s o l u t i o n i n CH3CN o f the residue was chromatographed
u s i n g a Lichrosorb RP-8 reversed-phase column and a
mobile phase composed o f 0.2% a c e t i c a c i d - methyl
c y a n i d e ( 1 : l ) . The m e t h o d c a n be u s e d f o r
b i o a v a i l a b i l i t y and c l i n i c a l pharmacokinetic studies o f
acetohexamide.

Beyer (34) used high speed l i q u i d chromatography f o r

analysis o f the drug and other a n t i d i a b e t i c agents. The
reocvery o f the drug from i n e r t t a b l e t ingredients by
 ACETOHEX AM I DE 33

t h i s method was near 100%. A column (100 cm X 2 . 1 mm)

packed w i t h 1% o f ethylene-propene copolymer on Zipax
was used w i t h mobile phase o f 0.01 M disodium hydrogen
c i t r a t e containing 15% o f methanol (pH 4 . 4 ) . Detection
was c a r r i e d o u t a t 254 nm and pack a r e a s were
integrated.

Testosterone, chlorpropamide i n 95% ethanol were used

i n t e r n a l s t a n d a r d s . The p r o c e d u r e was a p p l i e d t o
compressed t a b l e t s , t h e powdered sample being e x t r a c t e d
w i t h t h e i n t e r n a l standard s o l u t i o n . Recoveries o f
added sulphonylurea were 98.9% t o 100.2%.

5. PHARMACOKINETICS

5.1 Introduction

Acetohexamide i s used as an o r a l a n t i d i a b e t i c
agent f o r t h e t r e a t m e n t o f k e t o a c i d o s i s - r e s i s t a n t
d i a b e t e s . I t i s an i n t e r m e d i a t e a c t i n g s u l f o n y l u r e a
d e r i v a t i v e . The c l i n i c a l e f f e c t s o f lowering elevated
blood glucose l e v e l s i s s i m i l a r f o r a l l o f t h e
sulfonylurea d e r i v a t i v e s . Acetohexamide, however, i s
t h e only one t o a l s o possess u r i c o s u r i c a c t i v i t y and
t h e r e f o r e i s a p r e f e r a b l e agent t o t r e a t d i a b e t i c
p a t i e n t s w i t h gout.

The d u r a t i o n o f a c t i o n o f acetohexamide (12-24 hours)

permits once o r t w i c e d a i l y dosage. The crossover study
o f Fox e t a7. (35) conducted i n 36 p a t i e n t s w i t h
m a t u r i t y onset diabetes m e l l i t u s i n d i c a t e d t h a t both
chlorpropamide and acetohexamide gave s i m i l a r responses
based on f a s t i n g blood sugar. Acetohexamide was used i n
a dose range o f 500-3,000 mg/day and i t i s i n d i c a t e d
t h a t primary f a i l u r e on acetohexamide i s more l i k e l y t o
respond t o chlorpropamide and v i c e versa. Appropriate
dosing r e q u i r e i n d i v i d u a l i z a t i o n o f therapy t i t r a t e d t o
t h e d e s i r e d t h e r a p e u t i c e f f e c t . The usual PO dosage
range i s 250-1500 mg/day i n s i n g l e o r d i v i d e d doses
(36,37), w i t h a maximum recommended dose o f 1500
mg/day. The 250 mg dose o f acetohexamide i s equivalent
t o 500 mg t o l b u t a m l d e , 100 mg tolazamide, o r 100 mg
chlorpropamide (36). The o r a l a n t i d i a b e t i c agents prove
more u s e f u l when d i e t a r y r e s t r i c t i o n and w e i g h t
reduction accompany t h e i r use.
34 ABDULLAH A. AL-EADK AND HUMEIDA A. EL-OBEID

Acetohexamide i s l a r g e l y metabolized t o an a c t i v e
metabolite which is excreted i n t h e u r i n e (see below).
Therefore, dosage adjustment o r t o t a l avoidance i s
necessary i n c e r t a i n cases. One such case i s t h e renal
f a i l u r e . Azotenic p a t i e n t s may experience prolonged
hypoglycemia. A t w i c e d a i l y dose i s recommended f o r
p a t i e n t s w i t h m i l d r e n a l f a i l u r e and p a t i e n t s w i t h
moderate t o severe renal f a i l u r e should not receive t h e
drug (38,39).

Dosage adjustment may a l s o be required i n p a t i e n t s with

1i v e r i n s u f f i c i e n c y since acetohexamide i s e x t e n s i v e l y
metabolized i n t h e l i v e r . Prolonged hypoglycemia may
r e s u l t i n p a t i e n t s w i t h severe l i v e r impairment (36).
Dosage r e d u c t i o n may be r e q u i r e d i n e l d e r l y o r
d e b i l i t a t e d p a t i e n t s , due t o renal o r l i v e r impairment
o r hyperresponsiveness (36).

I t i s recommended by Bennett e t a7. (39) t h a t no dosage

supplementatlon i s r e q u i r e d i n p a t i e n t s f o l l o w i n g
.
p e r i t o n e a l d i a1y s i s

L i k e other o r a l a n t i d i a b e t i c agents, acetohexamide may

be used i n combination w i t h i n s u l i n t o reduce i n s u l i n
r e q u i r e m e n t s i n i n s u l i n dependent m a t u r i t y o n s e t
d i a b e t i c s and t o r e d u c e t h e p o t e n t i a l f o r a
hypoglycemic reaction.

5.2 Mechanism o f Action

Acetohexamide i s a sulfonylurea d e r i v a t i v e , t h a t
produces i t s hypoglycemic e f f e c t by s t i m u l a t i n g t h e
i s l e t t i s s u e t o s y n t h e s i z e and r e l e a s e endogenous
i n s u l i n ( 4 0 ) . The h y p o g l y c e m i c e f f e c t s a r e a l s o
a t t r i b u t e d t o an increased s e n s i t i v i t y o f i n s u l i n
receptors as w e l l as improved peripheral u t i l i z a t i o n o f
i n s u l i n (37).

A r e p o r t by Lebowitz and Feinglos (41) i n d i c a t e s t h a t ,

d u r i n g chronic administration, p a r t o f t h e hypoglycemic
action o f the sulfonylureas i s e x t r a pancreatic.
Peripheral t i s s u e s may become more s e n s i t i v e t o a f i x e d
dose o f an a d m i n i s t e r e d hormone p o s s i b l y due t o an
increase i n the number o f i n s u l i n receptors.

A study on the mode o f a c t i o n o f t h e sulfonylureas (42)

has shown t h a t acetohexamide increased glucose uptake
 ACETOHEXAMIDE 35

by r a t diaphragm, i n h i b i t e d the a c t i v i t y o f glucose-6-

phosphatase, triosephosphate isomerase and l i p o p r o t e i n
1ipase .
5.3 Onset and Duration o f Action

B r e i d a h l e t a 7 . ( 4 3 , 4 4 1 r e p o r t e d a peak
hypoglycemic e f f e c t t o occur between 8 t o 10 hour post
ingestion o f acetohexamide.

A duration o f action o f 12 t o 24 hours i s reported by

Breidahl et a7. (43,44) and Galloway et a7. (45) which
i s s i m i l a r t o t h a t o f tolazamide (up t o 24 hours), less
than t h a t o f chlorpropamide (60 hour) and greater than
t h a t o f tolbutamide (6 t o 12 hours) (37).

The serum c o n c e n t r a t i o n s i n d i a b e t i c p a t i e n t s
responding w e l l t o t h e drug had mean acetohexamide
l e v e l s o f 3.7 mg/dL w i t h a ragne f o 2.5 t o 4 . 9 mg/dL
f o l l o w i n g dosage regimens o f 0.5 t o 3 g/day (46). No
good c o r r e l a t i o n between b l o o d c o n c e n t r a t i o n s o f
acetohexamide and therapeutic e f f e c t i s established.
However, f a s t i n g blood glucose concentrations a r e
decreased i n a dose-dependent f a s h i o n i n t h e dosage
range between 250 mg t o 1,000 mg (47).

5.4 AbsorDtion

O r a l l y a d m i n i s t e r e d acetohexamide i s almost
completely absorbed (47). I t i s reported t o appear i n
the blood w i t h i n 30 minutes a f t e r PO administration and
peak l e v e l s occur a f t e r 3 t o 5 hours (43,44). Galloway
et a7. (45) reported t h a t , f o l l o w i n g single PO doses o f
1 g o f acetohexamide, mean peak blood l e v e l s o f t h e
drug t o be 47 mcg/ml and f o r hydroxyhexamide mean
l e v e l s o f 60.3 mcg/ml were achieved. These peak l e v e l s
occurred w i t h i n 1.5 t o 2 hours f o r the parent compound
versus 2 t o 6 hours f o r th e a c t i v e metabolite,
hydroxyhexamide.

5.5 Distribution

J u d i s ( 4 8 , 4 9 ) r e p o r t e d t h a t acetohexamide
extensively binds t o plasma proteins t o the extent o f
65 t o 90%.
36 ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID

5.6 Hetabol ism

A c e t o h e x a m i d e is mainly metabolized b y
hydroxylation reactions in the liver to inactive and
active metabolites. The primary metabolite (47 to 60%)
is hydroxyhexamide (47,501. It is an active metabolite
and is reported (45,50) to be excreted unchanged in the
urine, as well as metabolized to the inactive
dihydroxyhexamide (38).
Hydroxyhexamide, like acetohexamide, possesses both
hypoglycemic and uricosuric properties (51,52), but it
is 2.5 times as potent as its parent d r u g (36).
Impairment of hydroxyhexamide’s el imination has been
reported (51) to result in severe hypoglycemia.
Kojima et a7. (53) investigated the effect of various
drugs on the i n v i v o metabolic reduction of
acetohexamide. Most of the nonsteroidal anti-
inflammatory drugs inhibited the acetohexamide
reduction in liver, kidney and heart cytosol from
rabbits. Ketone-containing drugs including warfarin
also inhibited the reduction reaction in both the liver
and the kidney; in the heart, acetohexamide reduction
was inhibited only by warfarin.
Species differences in the i n v i t r o metabolic reduction
of acetohexamide were studied (54) in rabbit, guinea
pig, hamster, rat and mouse. The rabbit exhibited the
highest acetohexamide reductase activity in the cytosol
of the liver and kidney among the species tested. The
sensitivity to specific inhibitors of cytosolic
acetohexamide reductase in the liver and kidney of the
rabbit were different from those of the rat. Only rats
and guinea pigs showed significant activity of
acetohexamide reductase activity in the microsomes of
the liver and kidney.
Nagamine e t a7 (55) estimated the rates of available
fraction for 4-acetamidoacetophenone, 4-acetylbenzene-
sulfonamide, and acetohexamide and their respective
reduced compounds, 4-substituted a-hydroxyethylphenyl
derivatives, in rats. The study indicated that the
compounds are i n a reversible drug-metabolite
relationship. The pharmacokinetic profiles o f the
agents were studied after an intraportal administration
 ACETOHEXAMIDE 37

i n comparison w i t h those a f t e r I . V . administration

using an interconversion model.

5.7 Excretion

Acetohexamide and i t s m e t a b o l i t e s a r e m a i n l y
e x c r e t e d b y t h e k i d n e y s . The u r i n a r y r e c o v e r y o f
radioactivity a f t e r the administration o f oral
14C-labeled acetohexamide averaged 71.6% i n 24 hours
(45). Approximatley one-half t o two-third o f t h e drug
was r e p o r t e d t o be excreted i n u r i n e as t h e a c t i v e
metabolite, hydroxyhexamide (45,501. Fecal excretion o f
r a d i o a c t i v i t y f o l l o w i n g o r a l administration o f t h e drug
i n one p a t i e n t was 15%. Even a f t e r 1 g I . V . dose
u r i n a r y recovery was o n l y 85% ( 4 5 1 , suggesting t h a t
b i l i a r y e x c r e t i o n represents a secondary r o u t e o f
e l i m i n a t i o n o f acetohexamide and/or i t s metabolites.
However, more data are needed t o confirm the occurrence
o f b l l i a r y excretion.

5.8 Half-Life

F o l l o w i n g o r a l a d m i n i s t r a t i o n o f 14C-labeled
acetohexamide t o human subjects, a mean blood h a l f - l i f e
o f t h e drug o f 1 . 6 hours was determined, using isotope
d i l u t i o n a n a l y s i s , w i t h a range o f 0 . 8 - 2 . 4 hours
(45,56).

F i e l d e t a l . ( 5 1 ) , however, reported a range o f 21-70

minutes averaging t o a value o f 55.8 minutes. The
combined h a l f - l l f e o f t h e parent compound and i t s
a c t i v e metabolite, hydroxyhexamide, i s reported t o be
5.3 hours (43-45). The h a l f - l i f e o f acetohexamide i s
reported be prolonged i n renal f a i l u r e ( 3 8 ) .

The a c t i v e m e t a b o l i t e , hydroxyhexamide i s r e p o r t e d
(45,561 t o have a mean h a l f - l i f e o f 5.3 hours with a
range o f 3.7-6.4 hours. The average value o f 5.3 hours
agrees w i t h t h e f i n d i n g o f F i e l d e t a l . ( 5 1 ) who
reported a range o f 3.2-7.6 hours.

The blood and u r i n e data reported by Galloway et a l .

(45) agree w i t h those reported by Scheldon et a l . (46)
and confirm the report by Smith e t a l . ( 5 6 ) , t h a t the
combined half-1 i f e o f acetohexamide and hydroxyhexamide
i s comparable w i t h t h a t o f tolbutamide.
38 ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID

ACKNOWLEGEMENT

The authors would l i k e t o thank M r . Tanvir A. B u t t

f o r t y p i n g t h i s manuscript.

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11. E . G r a f , C. Beyer and 0. Abdallah, A c t a Pharm.

Technol., 5 , 9 (1979).

12. E. Graf, C. Beyer and 0. Abdallah, @id, 73 (1982).

28,

13. E. Graf, C. Beyer and 0. A b d a l l a h , ibid, a,131

(1982).

14. E. G r a f , C. Beyer and 0. A b d a l l a h , m, 28, 225

(1982).
 ACETOHEXAMIDE 39

15. A.A. Kassem, A.M. F o u l i , S. Said and E. Shehata, B u l l .

Fac. Pharm. (Cairo Univ.), 19, 309 (1982).

16. Mfg. Chem., 34, 454, 467 (1963).

17. S u r a j P. Agarwal and Mohammed I.Wa ash; I n d i a n J.

Pharm., 3 4 ( 5 ) , 109-111 (1972).

18. Jose Dobrecky and Rogelio J . C a l l e j a ; Rev. Fac. Quim

Farm., [Univ. Cent. Ecuador], El(16); 44 49 (1969).

19. M.M. Amer and M . I . Walash; B u l l . Fac. Pharm.. Cairo

Univ., l 2 ( 2 ) , 199-209 (1973).

20a. M.M. Amer and M.I. Walash: W, l2(2),223-233 (1973).

20b. M.H. h e r and M.I. Walash; m, l 2 ( 2 ) , 189-198 (1973)
(Pub. 1975).

21. L i l o 0. Guerello and Jose Dobrecky; Rev. Asoc. Bioauim.

Argent. 33(178-1791, 185-8 (1968).

22. N.G. Meier, S.O. Kohor, O.F. P i e r a r t , S . S . J . Cortes;

Rev. Real. Acad. Cience. Exactas. Fis. Natur. Madrid,
-
6 5 ( 3 ) , 653-674 (1971).

23. S.G. Solomonova and L . Z . Dvornitskaya; Farm. Zh.

(Kiev), 2, 80-82 (1977).

24. Edward F. Salem and W.W. H i l t y ; J. Pharm. Sci., 56(3),

385-386 (1967).

25. M a r i a K u h n e r t - B r a n d s t a e t t e r , Adel h e i d K o f l e r , A.
Vlachopoulas and A. Lobenwein; S c i . Pharm., 38(3)
154-163 (1970).

26. D.S. Lazaryan, Farmatsiya (Moscow), 29(2) , 36-38,

(1980).

27. Pamela Gergis-Takla and Ioannis Chroneos; Analyst, 104

(1235) 117-123 (1979).

28. A.A. Al-Badr and S.E. Ibrahim; Pharmazie; 37(5), 378

(1982).

29. Pamela Gergls-Takla and Shanta Raj Joshi; J. Biomed.

Anal. 1 ( 2 ) , 189-193 (1983).
40 ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID

30. K.H. Surborg and E. Roeder; Pharmazie, =(7), 485-486,

(1 97.3).

31. J.W. Kleber, J.A. Galloway and B.E. Rodda; J. Pharm.

u,
m(5), 635-638 (1977).

32. Fred L. F r i c k e , J. Ass. Off. Anal. Chem., 55(6),

1162-1 167 (1972).

33. Yasushi Takagishi; K o j i Sato; Keizo Tomita and Teruo

Sakamoto; Yakusaku Zasshi, B ( 9 ) , 961-963 (1979).

34. W i l l i a m F. Beyer; Anal. Chem., 44(7), 1312-1314 (1972).

35. O.J. Fox e t a l , J. Med. Assoc. Alabama, 31, 1155

(1968).

36. Product Information: Acetohexamide, E l i L i l l y & Co.,

Indianapolis, 1N: 1983.

37. AMA Department o f Drugs: AMA Drug Evaluations, 4 t h ed.

American Medical Association, Chicago, I L , 1980.

38. B.D. Cohen, J.A. Galloway, R.E. McMahon et a l , Am. J.

Med. Sci., 254, 608 (1967).

39. W.M. Bennett, G.R. Aronoff, G. Morrison et a l , Am. J.

Kidney Dis., 3, 155 (1983).

40. A.G. Gillman, L.S. Goodman and A. Gillman; Goodman and

Gillman’s The Pharmacological Basis o f TheraDeutics,
MacMillan & Co. New York. 1980.

41. H.E. Lebowitz and M.N. Feinglos, Diabetes Care, 1, 189

(1978).

42. K.T. Augusti and P.A. Kurup, Indian J. Biochem., 6(1),

36 (1969).

43. H.D. Breidahl, G.C. Ennis, F . I . Martin et a l , Drugs, 3,

79 (1972).

44. H.D. Breldahl, G.C. Ennis, F.I. Martin et a l , M, 3 ,

204 (1972).

45. J.A. Galloway, R.E. McMahon, H.W. Culp, F.J. Marshall

and E.C. Young , Diabetes, 16, 118 (1967).
 ACETOHEXAMIDE 41

46. J. Scheldon, J. Anderson and L. Stoner, m, l4, 362

(1965).

47. R.E. Ferner and S. Chaplin, Clin. Pharmacokinet., 2,

379 (1987).
48. J. Judis, J. Pharm. Sci., 6 l , 89 (1972).

49. J. Judis, w, 62, 232 (1973).

50. R . E . McMahon, F . J . M a r s h a l l and H.W. Culp, J.

Pharmacol. EXD. Ther., 149, 272 (1965).

51. J.B. Field, M. Ohta, C. Boyle e t a l , N. Ensl. J. Med.,

277, 889 (1967).

52. T.F. Yu, L. Berger and A.B. Gutman, Metabolism, 17, 309
(1968).

53. Y. Kojima, Y. Imamura and M. Otagiri, Yakusaku Zasshi,

108(1), 66 (1988).

54. Y. Imamura, Y. Kojima and M. Otagiri, Chem. Pharm.

Bull., 36(1), 4199 (1988).

55. S. Nagamine, T . Otawa, H. Nakae and S. Asada, M,

36(11), 4612 (1988).

56. D.L. Smith, T.J. Vecchio and A.A. Forist, Metabolism,

-
14, 229 (1965).
ACETOHEXAMIDE

C. E. Shafer
 C. E. SHAFER

CONTENTS

1. Description
1.1 Name, Formula, Molecular Weight
2. Physicai Properties
2.1 Infrared Spectrum
2.2 Nuclear Magnetic Resonance Spectrum
2.3 Ultraviolet Spectrum
2.4 Melting Range
2.5 Differential Thermal Analysis
2.6 Thermogravimetric Analysis
2.7 Solubility
3. Synthesis
3.1 First Example
3.2 Second Example
4. Stability
4.1 Infrared Analysis
4.2 Solubility Analysis
5. Drug Metabolic Products
6. Methods of Analysis
6.1 Titrimetric
6.2 Ultraviolet Spectrophotometric (Alkali)
6.3 Ultraviolet Spectrophotometric
(Alcohol)
7. Pharmacokinetics
8. Identification
9. References
 ACETOHEXAMI DE

1. Description
1.1 Name, Formula, Molecular Weight
Acetohexamide is N- (p-acetylphenylsul-
fony1)-N'-cyclohexylureal, and is also known as
1-[ (pacetylpheny1)sulfonyl]-3-cyclohexylurea2~4.

CH 3 8 0 SO -N H ! N H o

C15H20N204S M1. wt. = 324.40

2. Physical Properties
2.1 Infrared Spectrum
The infrared absorption spectrum of
acetohexamide (Lilly Reference Standard, Lot
No. 2KT47) is presented in Figure No. 1. The
spectrum was taken in a KBr pellet with a
Perkin-Elmer 221 Infrared S ectrophotometer.
-
The band at 3300, 3200 cm.-' is tyqical of an
N-H stretch; the band at 1680 cm. of a
conjugated ketone, and the band at 1445 cm.-l
of a C-CH3 group.
2.2 Nuclear Magnetic Resonance Spectrum
The NMR spectrum of acetohexamide
(Lilly Reference Standard, Lot No. 2KT47) is
presented in Figure 2. The spectrum was
produced using a Varian A60 NMR Instrument.
The quartet at low field (<86) is an A2B2
pattern that is typical of para substitution
and the singlet at 2.526 is typical of a methyl
group adjacent to a carbonyl function.
2.3 Ultraviolet Spectrum
The UV absorption spectrum of aceto-
hexamide (Lilly Reference Standard, Lot No.
2KT47) is presented in Figure 3. The spectrum
was produced using a Cary 14 instrument. The
sample was dissolved in 95% ethanol using
8.06 mg. per 25 ml. of solution. The X max.
at 247 nm is typical of substituted aceto-

3
 FR E 0 UE N C Y ( C M - l I
10
0.0

0.2
z
4
m
w
2m 0.4
a
0.6
0.8
1.0
1.5
2 3 4 5 6 7 8 9 10 11 12 13 14 1s
W A V E L E N G T H (MICRONS)

Fig. 1. Infrared spectrum

 ACETOHEXAMIDE

PPM (d)

Fig. 2 . NMR spectrum (sweep o f f s e t 000,200.0 Hz)

0.9 -

0.8 -

Y
0
z
2a
0
UY
m
a

Fig. 3 . U l t r a v i o l e t spectrum

5
 C. E. SHAFER

phenone, and the A max. at 284 nm is typical

of a conjugated aromatic ring.
2.4 Melting Range4
Between 184" and 189" (Class Ia NF).
2.5 Differential Thermal Analysis
The curve in Figure 4 is a DTA spectrum
of acetohexamide (Lilly Reference Standard, Lot
No. 2KT47) as produced by a DuPont 900 D.T.
analyzer, The spectrum shows a sharp phase
transition at 192°C.
2.6 Thermogravimetric Analysis
The TGA spectrum of acetohexamide in
Figure 5 (Lilly Reference Standard, Lot No.
2KT47) was produced with a DuPont 950 T.G.
Analyzer. It indicates less than 1% weight
loss to 191"C., approximately 5% weight loss
at 198"C., and approximately 20% weight loss
at 210 "c.
2.7 Solubility4
Practically insoluble in water.
Practically insoluble in ether.
Slightly soluble in alcohol.
Slightly soluble in chloroform.
Soluble in pyridine.
Soluble in dilute solutions of
alkali hydroxides.
3. Synthesis
Two examples for the preparation of aceto-
hexamide are listed.
3.1

CH 3CO- oNH2
First Examnle5

NaNO2
HC1, AcOH >

6
 ACETOHEXAMI DE

217 236 256

TEMPERATURE ,'C

Fig. 4 . DTA spectrum

I I I I I I I I I
1 40 59 79 98 117 137 157 177 197
197 217 236 256 276
T I O C (CHROMEL: ALUMEL)

Fig. 5. TGA spectrum

 C. E . SHAFER

SO 2 , AcOH
CH3CO-f -\)N:HCl CuCl2.2H20 '
CH3CO 4 - 3 S O 2 C l NH ,OH >

CHsCO+ 9 - S 0 2 N H 2 K2C03

C H 3 C-
O ~ S 0 2 N H C O N H ~

3.2 Second E x a m p l e '

Na2S03
CH3CO

CH3CO 0 S 0 3 N a POC13 >

CH 3 CO O s o 2 c 1 NH40H >

CH 3 CO 0 SO 2NH 2
C 1 C O 2C z H 5
K2C03

CHBCO SO 2NHC02C 2H 5
2
,
CH 3 C O -
- 0 S 0 2NHCC?NH-c>

8
 ACETOHEXAMI DE

4. Stabilitv
Acetohexamide is stable under all normal
storage conditions. Temperatures above 80°C.
were required to produce measurable degradation7
as indicated by solubility analysis, or by
spectrophotometric assay at 249 nm8. Irradiation
under a Hanovia lamp for one, and for three
hours, showed about 25%, and 50%, decomposition
by the spectrophotometer assay7. The compound
p-acetylbenzenesulfonamide was detected as a
degradation product using thin layer chroma-
tography with several combinations of plates,
solvents and visualization techniques7.
4.1 Infrared Analysis7
Weigh 250 mg. of acetohexamide. Trans-
fer to a 100-ml. volumetric flask. Add 10 ml.
of 0.5 N sodium hydroxide solution and about
20 ml. of water. Shake for 30 minutes on an
automated shaker, dilute to volume with water
and mix well.
Transfer a 20-ml. aliquot of the above
solution to a 125-ml. separatory funnel and
heat on a steam bath for 10 minutes. Allow to
cool, and make strongly acid with several drops
of concentrated hydrochloric acid.
Extract the sample with three 25-ml.
portions of chloroform. Drain the extract
through anhydrous sodium sulfate and collect it
in a 150-ml. beaker. Thoroughly wash the sodium
sulfate with chloroform, and collect the wash
with the extract.
Evaporate the sample, using a mild
temperature and a stream of air, to a suitable
volume f o r quantitative transfer to a 25-ml.
volumetric flask. Use chloroform to transfer
the sample, dilute to volume with chloroform,
and mix well.
Using a suitable spectrophotometer,
determine the absorbance of the final solution
at the maximum at about 5.90 p in 1.0 mm. sodium
chloride cells using chloroform as the blank.

9
 C . E. SHAFER

In the same manner determine the

absorbance of 250 mg. of NF Acetohexamide
Reference Standard.
(Sa. 4bs./Std. Abs.) x (mg. Std./mg.
Sa.) x 100% = percent acetohexamide.
4.2 Solubility Analysis8
Thermally degraded acetohexamide was
evaluated using solubility analysis. Because
of the high cost due to the large amount of
time involved, and because the method is
applicable only to the drug substance free of
excipients, the use of solubility analysis as a
routine procedure is limited.
5. Drug Metabolic Products
The principal (urine) metabolite in man
was found to be 1-(p-hydroxyethylbenzene-
sulfonyl)-3-cyclohexylurea (hydroxyhexamide)
by Welles, Root and Anderson2. Later, McMahon,
Marshall and Culpg discovered other (urine)
metabolites in man in which the cyclohexane
ring was hydroxylated to form hydroxyaceto-
hexamide and hydroxyhydroxyhexamide. (See the
following structures with names.)
0 0
SO 2NHCNH Acetohexa-
mide
H
Hydroxy-

t
OH hexamide

CH3C
' e S O 2 N H ! N H e Hydroxy-
OH

- t
aceto-
hexam ide
CH 3 1 0 S O2 N H ! N H e OH
Hydroxy-
/
OH - aceto-
hexamide

10
 ACETOHEXAMI DE

The metabolites of radioactive acetohexamide

found in the urine of man by Galloway, McMahon,
Culp, Marshall, and Young" were hydroxyhexamide,
smaller amounts of hydroxyacetohexamide and
hydroxyhydroxyhexamide, and small quantities of
other hydroxylated isomers. Smith, Vecchio and
Foristll determined the average biological half-
lives of acetohexamide and its major metabolite
hydroxyhexamide as 1.3 hours and 4.6 hours,
respectively. The hydroxy metabolites also have
hypoglycemic activity.
6. Methods of Analvsis
6.1 Titrimetric4
This method is described in detail.
6.2 Ultraviolet Spectrophotometric'
( alkali)
Transfer 50.0 mg. of acetohexamide to
a 100-ml. volumetric flask. Add 15 ml. of
purified water and 5.0 ml. of 0.5 N sodium
hydroxide. Shake mechanically for at least 30
minutes. Dilute to volume with purified water
and mix well. Transfer 2.0 ml. of the solution
to a 100-ml. volumetric flask. Dilute to volume
with purified water and mix well.
Weigh 50.0 mg. of NF Acetohexamide Reference
Standard, transfer to a 100-ml. volumetric flask.
Dissolve and dilute just like the sample.
Concomitantly determine the absorbance of
the sample solution and of the standard solution
in 1-cm. silica cells, at the maximum at about
249 nm, with a suitable spectrophotometer, using
purified water as the blank.
(Sa. abs./Std. abs.) x 100% = percent
acetohexamide.
6.3 Ultraviolet Spectrophotometric
(alcohol)
This method12 uses absolute alcohol to
dissolve 25.0 mg. of the acetohexamide and 25.0
mg. of the acetohexamide reference standard,

11
 C. E. SHAFER

is diluted to 5 0 . 0 ml.; then 2 . 0

ml. of each are diluted to 100.0 ml. with
absolute alcohol. The absorbances of the
solutions are determined at the maximum at
about 248 nm, using absolute alcohol as the
blank. Calculation: (Sample abs./Standard
abs.) x 100% P percent acetohexamide.
7. Pharmacokinetics
No pharmacokinetic studies have been
reported in the literature. However, seventy
to eighty percent of a single oral dose of 1
gram of acetohexamide was recovered as a
metabolite within 24 hours in the urine of four
human volunteers. It is suggested that aceto-
hexamide is converted to hydroxyhexamide in the
liver. Acetohexamide and hydroxyhexamide are
probably converted to hydroxyacetohexamide and
hydroxyhydroxyhexamide in both the liver and
kidneysg.
A multiple compartment system would probably
be necessary to obtain a rate constant f o r
metabolism, distribution rate constant, rate
constant for absorption, etc. Sulfonylurea
drugs may lower blood sugar by stimulating the
beta cells of the pancreatic islets to release
endogenous insulin, Also, it has been reported
that the sulfonylureas block the degradation
of insulin by the enzyme insulinasel3.
8. Identification
A. The X-ray diffraction pattern of aceto-
hexamide conforms to the pattern stated in the
NF4. No polymorphs have been observed and
documented.7 Infrared and a chemical method
are listed for identification purposes4.
B. A 1 in 100,000 solution of acetohexamide
in 0.01 N sodium hydroxide exhibits an ultra-
violet absorbance maximum at about 249 nm. Two
other sulfonylurea compounds, chlorpropamide
and tolbutamide, exhibit a maximum absorptivity

12
 ACETOHEXAMI DE

at about 230 and about 228 nm, respectively, in

the same medium3.
C. Strickland14 described a method for the
separation and detection of four of the more
important oral hypoglycemic agents using thin-
layer chromatography. It was used for the
identification of acetohexamide, chlorpropamide,
tolbutamide, and phenformin hydrochloride.

13
 C. E. SHAFER

References

1. USAN, J.A.M.A., -
180, 232 (1962).
2. Welles, J. S., Root, M. A., Anderson,
R. C., Proc. SOC. Exp. Biol. and Med.,
107, 583-5 (1961).
3. m i m , E. F. and Hilty, W. W , , J. Pharm.
Sci., 56, 385-6 (1967).
4. h e r . Pharm. Ass., National Formulary
XIII, 19-21 (1970).
5. U.S. Patent 3,320,312(Patented May 16,
1967).
6. Mfg. Chem., - 34, 454-6, 467 (1963).
7. Comer, J. P., The Lilly Research
Laboratories, unpublished data.
8. Comer, J. P. and Howell, L. D., J. Pharm.
Sci., 53, 335-7 (1964).
9. McMahoc R. E., Marshall, F. J., and
Culp, H.W., Pharmacol. Exptl. Therap.,
-
149, 272-9 (1965).
10. Galloway, J.A., McMahon, R. E., Culp,
H. W., Marshall, F. J., and Young, E. C.,
Diabetes, 16, 118 (1967).
11. Smith, D. c, Vecchio, T. J., and
Forist, A. A., Metab., Clin. Exptl.,
-
14, 229-40 (1965).
12. Baltazar, J. and Ferreira Braga, M. M.,
Revista Portuguesa de Farmacia, - 16, 169-74
(1966).
13. Mirsky, I. A , , Perisutti, G., and Diengott,
D., Metab. Clin. Exptl., 5, 156-61 (1956).
14. Strickland, R. D., J. Chromatog. - 24,
455-8 ( 1966).
The author expresses appreciation to Mr. C.
D. Underbrink and Dr. A. D. Kossoy of the
Analytical Development Department at Eli Lilly
and Company for assistance in preparing and
interpreting data in this profile.

14