Synthesis, isolation, and purification of benzylpenicillin β-diethylaminoethyl ester hydroiodide

Article abstract of DOI:10.1134/S1070427210070128

Processes of synthesis, isolation, and purification of benzylpenicillin β-diethylaminoethyl ester hydroiodide were studied. The optimal conditions of benzylpenicillin esterification with chloroethyldiethylamine in dimetylformamide and acetonitrile were found. Processes for isolation of the target product were developed and its physicochemical properties were studied. Product stabilization conditions providing a stable pH value in solutions of benzylpenicillin β-diethylaminoethyl ester hydroiodide were found.

Full text of DOI:10.1134/S1070427210070128

ISSN 1070-4272, Russian Journal of Applied Chemistry, 2010, Vol. 83, No. 7, pp. 1230−1237. © Pleiades Publishing, Ltd., 2010.
Original Russian Text © E.A. Savel’ev, I.M. Volkova, A.L. Kovalevskaya, 2010, published in Zhurnal Prikladnoi Khimii, 2010, Vol. 83, No. 7, pp. 1124−1131.
ORGANIC SYNTHESIS
AND INDUSTRIAL ORGANIC CHEMISTRY
Synthesis, Isolation, and Purification of Benzylpenicillin
β-Diethylaminoethyl Ester Hydroiodide
E. A. Savel’ev, I. M. Volkova, and A. L. Kovalevskaya
Fine Chemicals, Limited-Liability Research-and-Production Company, St. Petersburg, Russia
Received March 12, 2010
Abstract—Processes of synthesis, isolation, and purification of benzylpenicillin β-diethylaminoethyl ester
hydroiodide were studied. The optimal conditions of benzylpenicillin esterification with chloroethyldiethylamine
in dimetylformamide and acetonitrile were found. Processes for isolation of the target product were developed
and its physicochemical properties were studied. Product stabilization conditions providing a stable pH value in
solutions of benzylpenicillin β-diethylaminoethyl ester hydroiodide were found.
DOI: 10.1134/S1070427210070128
Benzylpenicillin β-diethylaminoethyl ester hydro-
iodide (penetamate hydroiodide) is a semisynthetic
antibioticexhibitinghighactivityincuringofanumberof
diseases caused by penicillin-sensitive microorganisms.
A specific feature of this preparation, due to the cationic
nature of the active component of the molecule, consists
in its capacity to concentrate in lactation organs and in
lungs. As a result, penetamate hydroiodide has recently
found wide use as a means for curing mastites and lung
infections, primarily in veterinary medicine.
to several patents [1–3] whose use on the industrial scale
fails to provide the required quality of the target product
or involves gross technological expenditures because of
the low yield and numerous stages of the process.
The goal of our study was to obtain data on
technological specific features of synthesis of
benzylpenicillin β-diethylaminoethyl ester and on
processes of its isolation from the reaction mass and
purification.
Benzylpenicillin β-diethylaminoethyl ester hydro-
EXPERIMENTAL
iodide
is
[2s-(2α,5α,6β)]-3,3-dimethyl-7-oxo-6-
[(phenylacetyl)amino]-4-thia-1-azabiscyclo-[3,2,0]-
heptane-2-carboxylic acid 2-(diethylamino)ethyl ester
monohydroiodide (Scheme 1).
We synthesized penetamate hydroiodide from
a sodium salt of benzylpenicillin, which satisfied the
requirements of FSP (Pharmacopoeia Standard of
Manufacturer) 42-2804–07 and a mass fraction of water
not exceeding 0.5%.
The evidence about methods for synthesis of
benzylpenicillin diethylaminoethyl ester salts are limited
Scheme 1.
S
CH2CONH
O
CH3
CH3
C2H5
C2H5
COOCH2CH2N
HI
1230

SYNTHESIS, ISOLATION, AND PURIFICATION OF BENZYLPENICILLIN
1231
Table 1. Specific rotation of 1% penetamate hydroiodide
solutions
The esterification was made with N-(2-chloroethyl)-
diethylamine hydrochloride (diethylaminoethyl
chloride) manufactured by Medilux laboratories Pvt.
Ltd (India), as the iodine-containing component served
sodium iodide dihydrate [GOST (State Standard) 8422–
76], with a 99.0% mass fraction of the main substance.
Sample no.
αD20, deg
151
Activity A, U mg–1
Reference sample
1005
990
1
2
3
4
5
147
148
995
The mass fraction of the main substance in the
product was quantitatively determined by iodometric
titration and high-performance liquid chromatography
(HPLC) on a Waters chromatograph, with a sample
from Schweizerhall Pharma GmbH as reference.
156
1045
1030
1020
152
150
Both the methods furnished identical results.
technical solutions can be determined by polarimetry
with a comparatively small error. We found that,
on passing from dimethylformamide solutions to
solutions in other polar solvents, the specific rotation
of penetamate hydroiodide changes only slightly.
Thus, rough technological calculations in synthesis and
isolation of the product can be based on the results of a
polarimetric determination of the concentration:
We studied the polarimetric characteristics of the
preparation in various solvents: in water, acetonitrile,
and dimethylformamide. This was done using an A1-
EPO automated photoelectric polarimeter at a working
wavelength of 589.3 nm, with an admissible error of
±0.01°. It was found that, because of the poor solubility
of penetamate hydroiodide in water, the specific rotation
in this solvent can be determined with a gross error
and difficulties in preparing a sample of the aqueous
solution. The optimal way to determine the specific
rotation of the penetamate is to use a 1% solution of
the preparation in dimethylformamide. Table 1 lists
the results of measurements of the optical rotation of
a 1% solution of penetamate hydroiodide samples in
dimethylformamide. It follows from the data in Table
1 that the specific rotation of penetamate hydroiodide
is linearly related to the activity of the preparation. In
accordance with data on the theoretical activity of the
preparation (1058 U mg–1) and the experimentally
measured values of the specific rotation, the specific
rotation was found to be 160°C. The specific rotation of
the preparation was determined inconformity with the
requirements of GF (State Pharmacopoeia) XI [4].
1058 aP
A =
160
where A is the activity of a preparation sample under
study (U mg–1); α, optical rotation of a solution of
the preparation (deg); 1058, theoretical activity of the
penetamate (U mg–1); and 160, specific rotation of the
penetamate (deg).
When developing a technological process for pro-
duction of benzylpenicillin diethylaminoethyl ester,
we considered as the main variant of synthesis the
esterification of N-benzylpenicillin with N-(2-chloro-
ethyl)-diethylamine
hydrochloride
in
dimethyl-
formamide or acetonitrile (Scheme 2).
With the specific rotation of penetamate hydroiodide
determined, the concentration of the preparation in
Alternative variants, synthesis of diethylaminoethyl
ester from benzylpenicillin chloroanhydride [1] or from
Scheme 2.
S
CH3
CH2CONH
.
HCl
HCl
+ ClCH2CH2N(C2H5)2
N
CH3
O
COONa
S
CH2CONH
CH3
CH3
+ NaCl
N
O
.
HCl
COOCH2CH2N(C2H5)2 HCl
RUSSIAN JOURNAL OF APPLIED CHEMISTRY Vol. 83 No. 7 2010

1232
SAVEL’EV et al.
a mixed anhydride of benzylpenicillin and aliphatic
carboxylic acid [2], gave the product in a substantially
lower yield, compared with Scheme 2.
esterification reaction is complete in 30–0 min.
Because the subsequent isolation of the target
product includes evaporation of the solvent from the
reaction mass, we considered the possibility of replacing
dimethylformamide with a solvent with a lower boiling
point and, specifically, acetonitrile.
Benzylpenicillin diethylaminoethyl ester was
synthesized by the following method [5]: a 1000-
ml three-necked flask was charged with 200–300 ml
of dimethylformamide and 20 g of a sodium salt of
benzylpenicillin (56.1 mmol). The suspension was
agitated at room temperature for 30 min, and then
7.8–14.5 g of chloroethyldiethylamine hydrochloride
(44.8–84.15 mmol) preliminarily dissolved in 200 ml
of dimethylformamide at a temperature 45–50°C was
added in the course of 3–3.5 h. Upon introduction of
chloroethyldiethylamine hydrochloride, the mixture
was additionally kept under agitation at 20–80°C for
20–60 min and a sample was taken to determine the
degree of conversion. The degree of conversion was
found by determining the ratio between the amount of
benzylpenicillin diethylaminoethyl ester formed and
the corresponding theoretical value. The concentration
of the target product was determined by HPLC after
a preliminary filtration of an aliquot and its dilution with
dimethylformamide and then with water. The results of
the synthesis are listed in Table 2.
The method for esterification in acetonitrile is
similar to that described above for dimethylformamide.
The relative amounts of the components of the reaction
mixture and the temperature-and-time parameters of
the process of esterification in acetonitrile correspond
to the optimal range found for the esterification in
dimethylformamide. The degree of conversion in
acetonitrile was 75–80%.
The benzylpenicillin diethylaminoethyl ester
hydrochloride formed in the esterification reaction
should be converted to a hydroiodide salt. This was
done by taking advantage of the different solubilities
of chlorides and iodides in organic solvents [6, 7]. It
follows from the results of [6, 7] that the solubility of
iodides in organic solvents substantially exceeds that of
chlorides. This property was used to separate chlorides
from the reaction mass obtained.
On charging chloroethyldiethylamine hydrochloride
and agitating the reaction mass for 30–45 min,
we introduced sodium iodide into the solution in
a stoichiometric amount with respect to benzylpenicillin.
After sodium iodide was dissolved, an exchange reaction
occurred by Scheme 3.
It follows from the data in Table 2 that the optimal
ratio between the components entering into the
reaction is the stoichiometric ratio or a small excess
of chloroethyldiethylamine hydrochloride, and the
optimal reaction temperature is 40–60°C. At higher
temperatures, thermal destruction of benzylpenicillin
presumably occurs. At the optimal parameters, the
Introduction of iodide ions into the reaction mass
Table 2. Esterification of a sodium salt of benzylpenicillin with diethylaminoethyl chloride hydrochloride
Benzylpenicillin :
chloroethyldiethylamine
molar ratio
Volume ratio of DMFA and charged
Na-salt of benzylpenicillin,
ml : g
Т, °С
τ, min
Conversion, %
1 : 1
200 : 1
200 : 1
200 : 1
300 : 1
200 : 1
250 : 1
250 : 1
250 : 1
250 : 1
20
20
60
50
40
50
80
50
30
30
60
60
45
45
60
50
45
60
12
14
75
70
60
58
70
80
45
1 : 0.8
1 : 1.5
RUSSIAN JOURNAL OF APPLIED CHEMISTRY Vol. 83 No. 7 2010

SYNTHESIS, ISOLATION, AND PURIFICATION OF BENZYLPENICILLIN
1233
Scheme 3.
S
CH2CONH
CH3
CH3
NaI
+
N
O
C2H5
C2H5
HCl
COOCH2CH2N
S
CH2CONH
CH3
CH3
C2H5
C2H5
+ NaCl
O
COOCH2CH2N
HI
simultaneously promotes a more complete esterification
because of the catalytic effect of I– anions in substitution
reactions [8, 9].
then acetone was introduced into the distillation residue
and the product was crystallized in a mixture of acetone
and acetonitrile (or acetone and dimethylformamide).
The separation completeness of chlorides was
monitored by determining the amount and composition
of the precipitate obtained upon filtration of the reaction
mass (Table 3).
Processes for isolation of penetamate hydroiodide
were for the most part tested for solutions obtained in
esterification in acetonitrile. Commonly the evaporation
of acetonitrile begins at a vapor temperature of about
20°C. and an insignificant overheating of the reaction
mass. As the solvent is evaporated and the concentration
of benzylpenicillin diethylaminoethyl ester hydroiodide
in the reaction mass increases, the degree of overheating
grows.As the volume of the distillation residue decreases
to 25–30% relative to the initial value, the reaction mass
temperature increases to 35–40°C. Because of this
circumstance, the degree of evaporation of the reaction
mass is limited by the maximum possible temperature
of the distillation residue (40°C), commonly being 65–
70%.
The target product was isolated from the reaction
mass by the method of salting-out crystallization. This
method includes crystallization of the target product
from solutions by diminishing the solvent activity
in solution to a value lower than that in a saturated
solution. In this case, the required effect is achieved by
introducing into the solution an auxiliary solvent and,
in particular, acetone, in which the target product is
poorly soluble. This procedure is similar to the “cold”
recrystallization technique [10, 11].
On performing the esterification process and
separating sodium chloride, we partly evaporated
from the resulting solution the solvent (acetonitrile
or dimethylformamide), in which benzylpenicillin
diethylaminoethyl ester hydroiodide is well soluble, and
When studying the solubility of benzylpenicillin
diethylaminoethyl ester hydroiodide in acetonitrile,
we found that the high solubility of benzylpenicillin
diethylaminoethyl ester in acetonitrile gives no way of
Table 3. Characterization of the sediment formed upon filtration of the reaction mass of benzylpenicillin diethylaminoethyl ester
hydroiodide
Charged, mg-mol
Mass fraction
of NaCl, %
Molar ratio between NaCl
formed and NaI charged
Sediment mass, g
chloroethyldiethylamine
hydrochloride
NaI
56.1
56.1
56.1
56.8
55.9
56.0
8.2
81
78
74
2 : 1
8.29
8.5
1.99 : 1
1.92 : 1
RUSSIAN JOURNAL OF APPLIED CHEMISTRY Vol. 83 No. 7 2010

1234
SAVEL’EV et al.
isolating the product from the reaction mass even on
evaporating the solvent to 10–15% relative to the initial
volume of the reaction mass. Isolation of the product
from the reaction mass concentrated by evaporation is
hindered not only by the high solubility, but also by the
formation of penetamate hydroiodide solvates with the
solvent, which results in that solutions of penetamate
hydroiodide in acetonitrile are formed as a thick syrupy
fluid. In the method we developed, the solvation shell is
disintegrated, with the product solubility in the solution
simultaneously lowered, by introducing acetone into the
concentrated reaction mass.
hydroiodide changes because of the oxidation of iodide
ions: the white or nearly white powder becomes yellow.
The optimal mode of drying of the preparation is that in
a vacuum at a residual pressure not exceeding 50 mm
Hg at temperatures of 25–35°C. It was found that, after
1.5–2 h of drying in this mode, the content of volatiles
in the product does not exceed 1.5%.
We studied on model solutions the possibility of
sorption recovery of benzylpenicillin diethylaminoethyl
ester from acetonitrile–acetone and acetone mother
liquorswithanKB-4P-2carboxycation-exchangerresin.
The high sorption selectivity of organic compounds of
this kind from aqueous solutions was noted in [12, 13].
The kinetics of the crystallization process is
shown in Fig. 1, whence follows that evaporation
of the acetonitrile solution of the penetamate to 30–
35% relative to the initial volume and a 2–2.5-fold
dilution of the distillation residue enable isolation of
the product in a 50–55% yield. The crystallization
of penetamate hydroiodide is complete in 2–2.5 h;
to achieve a more complete recovery of the product
from solution, the suspension is cooled to 8–10°C.The
precipitated benzylpenicillin diethylaminoethyl ester
hydroiodide is a crystalline product of white color
or white with a slight yellowish tint. The penetamate
hydroiodide paste produced on washing with acetone
and squeezing to remove the solvent contains no more
than 10% volatiles.
Figure 2 shows outlet elution curves for the
benzylpenicillin diethylaminoethyl ester cation
sorbed on the Na-form of the KB-4P-2 carboxy cation
exchanger. It can be seen that capacity of KB-4P-2 to
penetamate breakthrough is 150 g l–1 in sorption from
an acetonitrile–acetone solution and about 300 g l–1 in
sorption from an acetone solution. These values of the
sorption capacity are sufficient for solving the practical
problem of the product recovery from mother liquors.
Because there are no competing cations in the solution,
the value of the selectivity coefficient is unimportant in
this case.
The desorption of penetamate from the carboxy
cation-exchanger phase was studied using a 0.2 M
solution of hydroiodic acid in acetone as eluent. It was
shown that using an acetone solution of hydroiodic acid
as eluent provides a product yield of about 75–80%
in the active fraction of the eluate. We recommend
The drying mode of benzylpenicillin diethyl-
aminoethylesterhydroiodidewasstudiedattemperatures
of 25 to 110°C. It was found that, at temperatures higher
than 40°C, the outward appearance of penetamate
V, ml
τ, h
Fig. 2. Elution curves for penetamate hydroiodide sorbed
by the sodium form of KB-4P-2. Sorbent volume in the
column 10 ml. (c) Penetamate hydroiodide concentration at
the column outlet and (V) filtrate volume. Sorption: (1) from
a 1 : 1 acetonitrile–acetone solution and (2) from an acetone
solution.
Fig. 1. Kinetics of penetamate hydroiodide crystallization in a
1 : 1 acetonitrile–acetone system. (c) Penetamate hydroiodide
concentration and (τ) crystallization duration. T (°C): (1) 8–10
and (2) 25–30.
RUSSIAN JOURNAL OF APPLIED CHEMISTRY Vol. 83 No. 7 2010

SYNTHESIS, ISOLATION, AND PURIFICATION OF BENZYLPENICILLIN
Table 4. Quality characteristics of penetamate hydroiodide samplesa
1235
Test results for samples
Characteristic
Outward appearance
Property and standard
no. 1
+
no. 2
+
no. 3
+
no. 4
+
no. 5
Crystalline powder of
white color or white with
a yellowish tint
+
Content of the main substance, U mg–1
mp, °C
950–1110
990
1020
1050
990
1010
173–179,
within 2°C
174–174.5 174.5–175 175–176 173.5–174 174.5–
175
Specific rotation, deg
145–160
1.5
149
154
158
150
152
0.3
Mass fraction of water, %, not more
0.40
0.20
0.15
0.35
pH value of the aqueous suspension,
106 U in 30 ml of water
at the instant of suspension preparation
in 72 h
5.0–7.0
no less than 3.5
6.25
3.8
6.0
3.6
6.5
4.05
6.0
3.7
5.9
3.7
Identity reaction for iodides
Positive for iodide ions
0.5
+
+
+
+
+
Mass fraction of benzylpenicillin, %, not
more
0.15
0.18
0.27
0.31
0.36
Mass fraction of solvents, %, not more,
including acetonitrile
0.1
0.005
0.082
0.002
0
0
0.041
0.001
0.052
0.002
0
0
Solubility
Poorly soluble in water,
readily soluble in
+
+
+
+
+
dimethylformamide
Permeability of a preparation suspension
through a needle
No hindrance
+
+
+
+
+
+
+
+
+
+
Identity
Agreement with PMR
spectra of a standard
sample of the preparation
Stability of the suspension during 72 h at
room temperature
Stable
+
+
+
+
+
a “+” corresponds to the standard.
to perform the following procedures for recovery
of penetamate hydroiodide from the eluate: (1)
neutralization of the excess amount of hydroiodic acid
in the eluate with a 20% solution of sodium hydroxide
(with the solution cooled to 2–5°C under agitation) until
the pH of the medium reaches a values of 6.5–7.0; (2)
evaporation of acetone from the neutralized eluate to
a distillation residue constituting 1/4–1/5 of the initial
volume; (3) crystallization of the product from the
resulting distillation residue upon its cooling to 2–5°C
and agitation for 2 h; (4) filtration and washing of the
resulting precipitate of penetamate hydroiodide; and (5)
recrystallization of the product obtained in acetonitrile
in case of its insufficient quality.
Table 4 lists the rated quality parameters of
benzylpenicillin diethylaminoethyl ester hydroiodide
RUSSIAN JOURNAL OF APPLIED CHEMISTRY Vol. 83 No. 7 2010

1236
SAVEL’EV et al.
and results of tests of preparation samples produced by
the technique described above.
The neutralization is necessary because chloroethyl-
diethylamine hydrochloride used in synthesis contains
a certain excess amount of the hydrochloride with
respect to the stoichiometry. Introduction of phosphoric
acid salts as a neutralizing agent does not contaminate
the product with toxic substances and is not prohibited
by regulations [15].
Benzylpenicillindiethylaminoethylesterhydroiodide
is unstable in aqueous solutions. Hydrolysis of the
preparation leads to a decrease in the pH of the medium.
The hydrolysis rate strongly depends on specific features
of how the product is obtained, especially in final stages.
It was found that the purer the product, the higher its
stability in aqueous solutions and suspensions. A rated
parameter for penetamate hydroiodide is a pH value of
no less than 3.5 after 72 h of storage of the suspension.
In the course of storage, the suspension is periodically
shaken.
Neutralization of the suspension of penetamate
hydroiodide in the course of its recovery from the
reaction mass provides a higher pH value both at the
instant of sample preparation and during the time of
observation.
CONCLUSIONS
Figure 3 shows how the pH value varies in storage
of the penetamate suspension in water in the course of
48–96 h. It can be seen that an unstabilized penetamate
hydroiodide sample has a lower pH value both at the
instant of suspension preparation and in the course of
its exposure. After 48 h of storage, the suspension of
unstabilized penetamate hydroiodide has a pH value of
2.8, which is lower than the rated value.
(1) Benzylpenicillin diethylaminoethyl ester
hydroiodide was synthesized with a degree of
conversion of up to 80% by esterification of a sodium
salt of benzylpenicillin with chloroethyldiethylamine
hydrochloride at the stoichiometric ratio between the
components.
(2) The conditions of isolation of the target product
from the reaction mass by salting-out crystallization
from an acetone–acetonitrile solution were determined.
To obtain satisfactory results for the “pH of the
aqueous suspension, ” a procedure for neutralization
of the excess amount of the acid with phosphoric acid
salts has been introduced [14]. The optimal way is to
introduce a mixture of KH2PO4 and Na2HPO4 at a 15 : 85
mass ratio of the components. The stabilizing additives
are introduced into the reaction mass after a vacuum-
evaporation of acetonitrile, and its dilution with acetone.
The amount of phosphates introduced into the reaction
mass is 0.75% relative to the amount of the sodium salt
of benzylpenicillin, taken for synthesis of penetamate
hydroiodide.
(3) It was found that the optimal variant of product
desorption from the KB-4P-2 carboxy cation exchanger
is elution with an acetone solution of hydroiodic acid.
REFERENCES
1. Spanish Patent 432241.
2. GB Patent 759603.
3. GB Patent 675422.
4. Gosudarstvennaya
farmakopeya
SSSR
(State
Pharmacopoeia of the USSR), Moscow: Meditsina, 1990,
11th ed., p. 30.
5. RF Patent 2167162.
6. Kogan, V.B., Fridman, V.M., and Kafarov, V.V.,
Spravochnik po rastvorimosti (Reference Book of
Solubilities), Moscow: Akad. Nauk SSSR, 1961, vol. 1,
pp. 655–663.
7. Mishchenko, K.P. and Poltoratskii, G.M., Voprosy
termodinamiki i stroeniya vodnykh i nevodnykh rastvorov
elektrolitov (Problems of Thermodynamics and Structure
of Aqueous and Nonaqueous Electrolyte Solutions),
Leningrad: Khimiya, 1968.
τ, h
Fig. 3. Variation of the pH value of an aqueous suspension of
penetamate hydroiodide. (τ) Exposure duration. Suspension:
(1, 2) penetamate stabilized in the course of isolation and (3)
unstabilized penetamate.
8. Moelwyn-Hughes, E.A., The Chemical Statics and
Kinetics of Solutions, London: Academic, 1971.
RUSSIAN JOURNAL OF APPLIED CHEMISTRY Vol. 83 No. 7 2010

SYNTHESIS, ISOLATION, AND PURIFICATION OF BENZYLPENICILLIN
1237
9.Beronius, P. and Pataki, L., Acta Chem. Scand., 1971, vol.
25, pp. 3705–3709.
Ionnyi obmen (Ion Exchange), Leningrad: Nauka, 1969.
13. Feitelson, J., Ion exchange: A Series of Advances, vol. 2,
Marinsky, J.A., Ed., New York: Dekker, 1969, pp. 135–
166.
10. Reichardt, C., Losungmittel-Effekte in der organischen
Chemie, Verlag Chemie, 1969.
11. Reynolds, W.F. and Priller, U.R., Canad. J. Chem., 1968,
14. RF Patent 2305100.
vol. 46, pp. 2787–2791.
15. European Drag Master File, Supplement to Directive
75/318/EEC, section C, parts I and II.
12. Samsonov, G.V., Trostyanskaya, E.B., and El’kin, G.E.,
RUSSIAN JOURNAL OF APPLIED CHEMISTRY Vol. 83 No. 7 2010