Enzymatic regioselective production of chloramphenicol esters

Article abstract of DOI:10.1016/j.tet.2011.02.070

An enzymatic study has been performed in the search for synthetic routes to produce chloramphenicol derivatives through regioselective processes using lipases. Complementary transesterification and hydrolytic reactions have been carried to synthesize chloramphenicol regioisomers. Reaction parameters, such as biocatalyst, solvent, acyl donor, and temperature have been optimised in order to obtain chloramphenicol esters with high yields through acylation processes. Scale-up of the enzymatic reactions (1 g-scale at 0.25 M) and catalyst recycling (up to 10 cycles) have been successfully achieved. Furthermore, monoacylated derivatives at the more hindered secondary position could also be obtained employing hydrolysis processes.

Full text of DOI:10.1016/j.tet.2011.02.070

Tetrahedron 67 (2011) 2858e2862
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Tetrahedron
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Enzymatic regioselective production of chloramphenicol esters
,
,
,
b
Ayla M.C. Bizerra a b, Tasso G.C. Montenegro a b, Telma L.G. Lemos b , Maria C.F. de Oliveira ,
*
b
a
a
a
ꢀ
ꢀ
Marcos C. de Mattos , Ivan Lavandera , Vicente Gotor-Fernandez , Gonzalo de Gonzalo ,
,
Vicente Gotor a
*
a
ꢀ
ꢀ
ꢀ
Departamento de Química Orgaˇ nica e Inorgaˇ nica, Instituto Universitario de Biotecnología de Asturias, Universidad de Oviedo, c/Julian Clavería 8, 33006 Oviedo, Spain
Departamento de Química Organica e Inorganica, Universidade Federal do Ceara, 60451-970 Fortaleza, Ceara, Brazil
b
ꢀ
ꢀ
a r t i c l e i n f o
a b s t r a c t
Article history:
An enzymatic study has been performed in the search for synthetic routes to produce chloramphenicol
derivatives through regioselective processes using lipases. Complementary transesterification and hy-
drolytic reactions have been carried to synthesize chloramphenicol regioisomers. Reaction parameters,
such as biocatalyst, solvent, acyl donor, and temperature have been optimised in order to obtain
chloramphenicol esters with high yields through acylation processes. Scale-up of the enzymatic reactions
(1 g-scale at 0.25 M) and catalyst recycling (up to 10 cycles) have been successfully achieved. Further-
more, monoacylated derivatives at the more hindered secondary position could also be obtained em-
ploying hydrolysis processes.
Received 19 January 2011
Received in revised form 21 February 2011
Accepted 22 February 2011
Available online 1 March 2011
Keywords:
Biocatalysis
Chloramphenicol
Lipases
Ó 2011 Elsevier Ltd. All rights reserved.
Regioselective processes
Medium engineering
1. Introduction
HO OH
OH
O
CH3
( )
O
14
The selective modification of one alcohol group in a poly-
hydroxylated compound is an important issue for organic chemists
since usually it requires time-consuming protection and depro-
tection steps that enhance costs and by-products lowering the final
yields. In this sense, biocatalytic transformations have become
standard procedures applied to the regioselective acylation of
polyfunctionalised derivatives maximising the efficiency of these
processes.1 Furthermore, in many cases the acylated derivatives
obtained present better biological or availability properties than
the parent substrates.1,2
This is also the case of chloramphenicol, (1R,2R)-2-dichlor-
oacetamido-1-p-nitrophenyl-1,3-propanediol (1, Fig. 1),3 a bacte-
riostatic antimicrobial, which acts as an effective agent against
a broad spectra of gram-positive and gram-negative bacteria. It was
introduced for clinical treatment in human and animals around the
mid 20th century, and its first indication was in the treatment of
typhoid. Furthermore, its efficiency in the treatment of tetracycline-
resistant cholera, staphylococcal brain abscesses, meningitis, in-
fluenza, pneumonia, and several infections has effectively been
demonstrated over the years.4 Unfortunately, it can also produce
HN
O
HN
O
Cl
Cl
Cl
Cl
O2N
O2N
1
2a
Fig. 1. Structures of chloramphenicol (1) and chloramphenicol palmitate ester (2a).
adverse effects, such as aplastic anaemia, bone marrow suppression,
childhood leukaemia, and grey baby syndrome, so this compound
requires medical prescription. Therefore, for the administration of
this drug there are currently several ways, for instance capsules, oily
or liquid form, but the bitter taste of the pharmaceutical has led to
the production of different alternatives, such as chloramphenicol
succinate or chloramphenicol palmitate (2a, Fig. 1) esters by selec-
tive modification of the primary hydroxyl group.
Originally, chloramphenicol was isolated from the bacterium
Streptomyces venezulae,5 but the production of this drug and its
derivatives have attracted very much attention by means of regio-
or stereoselective chemical6 and enzymatic7 methods. Among all
biocatalytic processes, the use of lipases presents many advantages
in comparison with other synthetic transformations due to the mild
reaction conditions and the high level of selectivity displayed by
this type of catalysts. In this manner, Ottolina et al. reported the
lipase-mediated regioselective esterification of chloramphenicol
for the synthesis of several derivatives in anhydrous acetone
* Corresponding authors. Tel./fax: þ34 985 103448 (V.G.); tel.: þ55 85 33669366;
fax: þ55 85 33669782 (T.L.G.L); e-mail addresses: tlemos@ufc.br (T.L.G. Lemos),
vgs@fq.uniovi.es (V. Gotor).
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.02.070

A.M.C. Bizerra et al. / Tetrahedron 67 (2011) 2858e2862
2859
Table 2
exploring the influence of different trifluoroethyl esters. Chromo-
bacterium viscosum lipase (CVL) or lipase G were the best bio-
catalytic agents, obtaining the corresponding esters after 24e72 h
of reaction at 45 ꢀC in moderate to excellent yields.8 Later, Lin et al.
demonstrated the versatility of hydrolases, such as Lipozyme or the
protease from Bactillus subtilis in the synthesis of chloramphenicol
vinyl esters.9
Herein we develop the synthesis of a wide set of chloram-
phenicol esters by using complementary synthetic approaches,
such as acylation and hydrolysis processes. We have especially fo-
cused on the optimisation of the reaction conditions including the
scaling-up and the enzyme recycling outcome.
Lipase-mediated reaction of 1 with 5 equiv of vinyl decanoate (3b) in different or-
ganic solvents at 30 ꢀC
Entry
Enzyme
Solvent
t (h)
2ba (%)
1
2
3
4
5
6
CAL-B
CAL-B
CAL-B
CAL-B
CAL-B
CAL-B
THF
THF
MeCN
MeCN
TBME
TBME
24
48
24
48
24
48
31
37
74
80
27
29
7
8
9
10
11
12
PSL-C Amano
PSL-C Amano
PSL-C Amano
PSL-C Amano
PSL-C Amano
PSL-C Amano
THF
THF
MeCN
MeCN
TBME
TBME
24
48
24
48
24
48
15
21
51
62
42
51
2. Results and discussion
13
14
15
16
17
18
PSL-C I
PSL-C I
PSL-C I
PSL-C I
PSL-C I
PSL-C I
THF
THF
MeCN
MeCN
TBME
TBME
24
48
24
48
24
48
12
17
33
50
38
43
First of all, different commercially available lipases were tested10
in order to find the highest activity in the esterification of chlor-
amphenicol with a half-size chain activated ester, such as vinyl
decanoate (3b). From previous studies developed in our research
group, 1,4-dioxane was selected as a suitable solvent using 5 equiv
of the acyl donor (Scheme 1, Table 1).
a
Percentage of monoester 2b determined by HPLC.
OR'
O
influence of tetrahydrofuran (THF), acetonitrile (MeCN), and tert-
butyl methyl ether (TBME) was initially examined with CAL-B
(entries 1e6), where the highest conversions were with MeCN
(entry 4), which allowed the formation of 2b in 80% yield after 48 h.
It should be noted that the diacylated compound 4b was not
detected in any case. Different results were attained when using
PSL as a biocatalyst (entries 7e18). None of the three solvents im-
proved the high performance obtained in 1,4-dioxane (87 and 94%
in entries 3 and 4 of Table 1, respectively) although with acetonitrile
higher conversions than THF and TBME were achieved.
The effect of the temperature was analysed when employing the
best conditions previously found: (a) CAL-B and MeCN; or (b) PSL-C
I and 1,4-dioxane (Table 3).11 Surprisingly, CAL-B led to a higher
conversion into 2b at 20 ꢀC rather than at 30 ꢀC (entries 1 and 2),
although this enzyme usually presents an optimum temperature
between 30 and 40 ꢀC.12 When using PSL-C I, a remarkable decrease
of the conversion occurred when going from 30 to 40 ꢀC (entries 4
and 5), maybe caused by deactivation of the biocatalyst, meanwhile
the reaction at 20 ꢀC (entry 3) led to a similar value than the one
obtained at 40 ꢀC.
CAL-B or PSL
3'
O
1'
2'
1
+
O
R
O
R
MeCN or Dioxane
20 or 30 ºC
HN Cl
Cl
O2N
O
250 rpm
3a, R= (CH2)14CH3
3b, R= (CH2)8CH3
3c, R= (CH2)10CH3
3d, R= CH2CH3
3e, R= CH3
2a, R= (CH2)14CH3, R'= H;
2b, R= (CH2)8CH3, R'= H;
2c, R= (CH2)10CH3, R'= H;
2d, R= CH2CH3, R'= H;
2e, R= CH3, R'= H;
3f, R= Ph
2f, R= Ph, R'= H
4b, R= CO(CH2)8CH3, R'= CO(CH2)8CH3
Scheme 1. Lipase-mediated esterification of chloramphenicol (1) using vinyl esters
3aef in organic solvents.
Table 1
Lipase-mediated reaction of 1 with 5 equiv of vinyl decanoate (3b) in 1,4-dioxane at
30 ꢀC after 48 h
Entry
Enzyme
2ba (%)
4ba (%)
1
2
3
4
CAL-A
CAL-B
PSL-C Amano
PSL-C I
22
72
87
94
11
5
Table 3
d
Temperature effect in the lipase-catalysed acylation of 1 with 5 equiv of vinyl dec-
anoate (3b) after 48 h
d
a
Percentage of compounds determined by HPLC.
Entry
Enzyme
Solvent
T (ꢀC)
2ba (%)
1
2
3
4
5
CAL-B
CAL-B
PSL-C I
PSL-C I
PSL-C I
MeCN
MeCN
1,4-Dioxane
1,4-Dioxane
1,4-Dioxane
20
30
20
30
40
94
80
61
94
58
After 48 h at 30 ꢀC and 250 rpm, Candida antarctica lipase A
(CAL-A, entry 1) gave low conversion and poor selectivity, affording
the formation of the 30-acylated compound (2b) and the 10,30-
diacylated product (4b). C. antarctica lipase B (CAL-B) catalysed the
acylation reaction with good selectivity and reaction rate, yielding
72% of the desired 30-monoacylated derivative (entry 2). Mean-
while, as shown in entries 3 and 4, two different preparations of
Pseudomonas cepacia lipase from Amano (PSL-C Amano, also known
as Burkholderia cepacia lipase) and type I from Aldrich (PSL-C I),
produced with complete selectivity the monoester 2b in 87% and
94% conversion, respectively, as the sole product. These achieve-
ments were ideal starting points for further optimisation of the
reaction conditions.
a
Percentage of compounds determined by HPLC.
After optimisation of the experimental conditions for the acyl-
ation reaction, we explored the possibilities to regioselectively
produce several chloramphenicol ester derivatives of different
length chain using CAL-B or both preparations of PSL-C as bio-
catalysts in MeCN or 1,4-dioxane as solvent, respectively. The best
results have been summarised in Table 4. Chloramphenicol palmi-
tate ester (2a) was selected as the first candidate due to its ther-
apeutical uses13 and results were in accordance with the ones
obtained when using vinyl decanoate as acyl donor. Both CAL-B and
PSL-C I led to nearly complete conversions after 24 h of reaction
(entries 1 and 2).
Due to the results obtained for these two biocatalysts, a search
for a suitable solvent was carried out taking CAL-B and PSL as
catalysts applied to the regioselective esterification of chloram-
phenicol. Reactions were performed for 24 and 48 h (Table 2). The

2860
A.M.C. Bizerra et al. / Tetrahedron 67 (2011) 2858e2862
Table 4
In all cases the reaction times diminished in comparison with
the more diluted one (see Table 4), finding that for CAL-B only 3 or
4 h were enough to afford total conversions into the corresponding
monoesters 2aee. Among all the enzymatic processes carried out
with PSL, PSL-C I showed better reaction rates than PSL-C Amano.
Finally, the biotransformations with vinyl benzoate (entries 16e18)
led also to higher conversions at 150 mM concentration rather than
15 mM, obtaining 45% isolated yield of monoester 2f with PSL-C I
(entry 17).
The next step was to study the recycling of the enzyme by
performing the acylation at 0.15 M concentration of 1 and vinyl
palmitate with CAL-B in MeCN at 20 ꢀC. The biocatalyst performed
the regioselective synthesis of 2a with total conversion after 3 h
during 10 cycles of reutilisation.
After having found excellent conversion in the biocatalysed
regioselective acylation of diol 1, we set up a preparative experi-
ment at 1 g-scale. For that, the biocatalytic processes to obtain
derivatives 2aee were performed using the best conditions pre-
viously found for the acylation at 0.15 M concentration of 1: CAL-B
as biocatalyst, 5 equiv of the corresponding ester and MeCN as
solvent at 20 ꢀC. All the reactions proceeded until complete con-
version to isolate 2a,b,dee in 3 h, requiring 4 h for the laurate ester
2c. Additionally, the palmitate derivative 2a was obtained in 98%
conversion when the reaction was repeated at 0.25 M concentra-
tion of chloramphenicol. Further increase of substrate concentra-
tion led to solubility problems.
Previous experiments have demonstrated that the acylated de-
rivatives of chloramphenicol at the primary alcohol can be obtained
with high yields, but we were also interested in the obtaining of
analogues acylated at the more hindered secondary position. The
chemical modification of a secondary alcohol in the presence of
a primary one is a complicated feature, which has scarcely been
observed.15 Herein, taking advantage of the chemical complemen-
tarity shown by lipases in stereo- and regioselective processes,16 we
focused on the preparation of monoesters 5aee by firstly synthe-
sising the diacylated derivatives 4aee, and later performing the
regioselective lipase-catalysed hydrolysis at the primary position
(Scheme 2).
Enzymatic acylation of 1 with 5 equiv of the vinyl ester 3a,cef at different tem-
peratures in a 15 mM substrate concentration
Entry Enzyme
T (ꢀC) Solvent
Final product t (h) Conva (%)
1
2
CAL-B
PSL-C I
20
30
MeCN
1,4-Dioxane 2a
2a
24
24
>99
95
3
4
CAL-B
PSL-C I
20
30
MeCN
1,4-Dioxane 2c
2c
24
24
>99
91
5
6
7
CAL-B
20
MeCN 2d
24
24
24
>99
99
95
PSL-C Amano 30
1,4-Dioxane 2d
1,4-Dioxane 2d
PSL-C I
30
8
9
CAL-B
20
MeCN
2e
40
40
>99
97
PSL-C Amano 30
CAL-B 20
1,4-Dioxane 2e
10
11
MeCN 2f
40
40
4
17
PSL-C Amano 30
1,4-Dioxane 2f
a
Percentage of compounds determined by HPLC.
Next, vinyl laurate (3c) was reacted with 1 also observing an
excellent reactivity (entries 3 and 4), especially in the case of CAL-B,
which led to quantitative conversion of the monoester 2c. This
trend was also observed when the chain length of the vinyl ester
decreased, obtaining excellent results when vinyl propanoate (3d)
was employed as acyl donor with all the biocatalysts tested (entries
5e7). Surprisingly, longer reaction times were needed to reach high
conversion values using vinyl acetate (3e, entries 8 and 9). Vinyl
benzoate (3f) led to the lowest conversion values (entries 10 and
11) due to the poorer reactivity of this acyl donor. These results
present lipases as ideal catalysts for the regioselective acylation of
chloramphenicol, showing an excellent selectivity towards the ac-
ylation of the primary alcohol. It is important to remark that in no
case was the secondary hydroxyl group acylated to form the cor-
responding 10-monoester or 10,30-diester derivatives, even after
longer reaction times (data not shown).
From a preparative point of view, both higher substrate con-
centrations and recycling of the catalyst lead to more economical
and green processes.14 Therefore, after testing the solubility of all
products involved in these processes, the reactions to achieve 2aef
were carried out at a higher concentration of chloramphenicol
(0.15 M), as summarised in Table 5.
O
O
R
O
O
R
O OH
CAL-B
Table 5
O
R
Lipase-mediated reaction of 1 with 5 equiv of the vinyl ester 3aef at different
temperatures and 250 rpm at 0.15 M substrate concentration
HN Cl
Cl
HN Cl
Cl
Buffer/Org. solvent
20 or 30 ºC
O2N
O2N
O
O
Entry Enzyme
T (ꢀC) Solvent
Final
product
t (h) Conva (%) Isolated
250 rpm
yieldb (%)
4a, R= (CH2)14CH3
4b, R= (CH2)8CH3
4c, R= (CH2)10CH3
4d, R= CH2CH3
4e, R=CH3
5a, R= (CH2)14CH3
5b, R= (CH2)8CH3
5c, R= (CH2)10CH3
5d, R= CH2CH3
5e, R=CH3
1
2
3
CAL-B
PSL-C I
20
30
MeCN
1,4-Dioxane 2a
1,4-Dioxane 2a
2a
3
3
48
>99
>99
79
91
75
78
PSL-C Amano 30
4
5
6
CAL-B
PSL-C I
20
30
MeCN
1,4-Dioxane 2b
1,4-Dioxane 2b
2b
3
4
22
>99
>99
>99
92
84
91
Scheme 2. Lipase-catalysed hydrolysis of diacylated chloramphenicol derivatives
4aee in aqueous media.
PSL-C Amano 30
7
8
9
CAL-B
PSL-C I
PSL-C Amano 30
20
30
MeCN
1,4-Dioxane 2c
1,4-Dioxane 2c
2c
4
7
10
>99
>99
>99
91
88
88
Enzymatic hydrolyses were initially performed with the diac-
etylated compound 4e by employing a mixture of phosphate buffer
100 mM pH 7.0 and an organic co-solvent (80:20 v vꢁ1), such as
MeCN, 1,4-dioxane or THF with the previous lipases, but only CAL-B
showed activity (Table 6). Using this biocatalyst the 30-acylated
derivative 2e was not observed in any case and the desired product
5e was mainly achieved in MeCN at 20 ꢀC with high conversion
(78%), giving substrate 4e and chloramphenicol 1 as minor com-
pounds (entry 3). Percentage of MeCN in the reaction medium
(5e80%) and the pH of the reaction (range 5.5e8.5) were also
studied (see Supplementary data for more information), but no
further improvement of the conversion value was achieved.
10
11
12
CAL-B
PSL-C I
PSL-C Amano 30
20
30
MeCN
1,4-Dioxane 2d
1,4-Dioxane 2d
2d
3
6
9
>99
98
>99
89
90
82
13
14
15
CAL-B
PSL-C I
PSL-C Amano 30
20
30
MeCN
1,4-Dioxane 2e
1,4-Dioxane 2e
2e
3
6
9
>99
99
98
90
90
79
16
17
18
CAL-B
PSL-C I
PSL-C Amano 30
20
30
MeCN
1,4-Dioxane 2f
1,4-Dioxane 2f
2f
48
48
48
31
53
13
25
45
8
a
Percentage of compounds determined by HPLC.
Isolated yields after purification by flash chromatography.
b

A.M.C. Bizerra et al. / Tetrahedron 67 (2011) 2858e2862
2861
Table 6
respectively. C. antarctica lipase A (CAL-A) was purchased from
Codexis (2.6 U/mg of solid). All other reagents were purchased from
different commercial sources and used without further purifica-
tion. Solvents were distilled over an adequate desiccant under ni-
trogen. Flash chromatographies were performed using silica gel 60
(230e240 mesh).
Lipase-mediated hydrolysis of 4e with CAL-B in a buffer/organic solvent system
(80:20 v vꢁ1
) at different temperatures and 250 rpm at 0.15 M concentration
(t¼24 h)
Entry
T (ꢀC)
Solvent
1a (%)
4ea (%)
5ea (%)
1
2
3
4
5
6
20
30
20
30
20
30
1,4-Dioxane
1,4-Dioxane
MeCN
MeCN
THF
23
18
14
8
12
16
63
73
8
18
21
54
14
9
78
74
67
30
4.2. General procedure for the esterification reaction of (L)-
chloramphenicol at 0.25 M substrate concentration using
vinyl palmitate
THF
a
Percentage of compounds determined by HPLC and 1H NMR.
To a suspension of (ꢁ)-chloramphenicol (1, 1 g, 3.1 mmol) and
CAL-B (1 g) in dry MeCN (12.5 mL) under nitrogen atmosphere,
vinyl palmitate (3a, 4.26 g, 15.0 mmol) was added, and the reaction
was shaken at 20 ꢀC and 250 rpm. Aliquots were regularly analysed
by HPLC and the reaction was stopped after complete consumption
of the starting material after 1 h. Finally the enzyme was filtered off
and washed with EtOAc (3ꢂ10 mL). The solvent was evaporated
under reduced pressure. The reaction crude was purified by flash
chromatography on silica gel (30% EtOAc/hexane) affording the
corresponding ester 2a (87% isolated yield). Characterizations of
monoester 2a and all novel compounds are given in the
Supplementary data file.
The hydrolyses of diacylated derivatives 4aed were then carried
out under the optimal conditions depicted in entry 3 of Table 6,
observing a correlation between the chain length of the ester moiety
and the enzymatic conversion (Fig. 2). Thus, when longer ester
moieties were employed, lower conversions were reached. While
the dipropionate compound 4d afforded 74% of 5d,17 didecanoate 4b
provided only a 10% of 5b, dilaurate 4c a 4% of 5c and dipalmitate 4a
did not react. This effect can be explained because bulkier diacylated
products could not correctly fit in the active site of the enzyme.
Acknowledgements
The authors thank to the Brazilian and Spanish agencies CAPES-
DGU (Process: 149/07), CNPq, FUNCAP, PRONEX, Ministerio de
ꢀ
Ciencia e Innovacion (MICINN, Project CTQ 2007-61126) for fel-
ꢀ
lowships and financial support. I.L. and V.G.-F. (Ramon y Cajal
program) thank MICINN for personal grants.
Supplementary data
Supplementary data associated with this article can be found in
online version at doi:10.1016/j.tet.2011.02.070. These data include
MOL files and InChIKeys of the most important compounds de-
scribed in this article.
Fig. 2. Effect of the chain length in the CAL-B-catalysed hydrolysis of diacylated
chloramphenicol derivatives.
References and notes
3. Conclusions
1. (a) Danieli, B.; Riva, S. Pure Appl. Chem. 1994, 66, 2215e2218; (b) Ferrero, M.;
Gotor, V. Chem. Rev. 2000, 100, 4319e4347; (c) Riva, S. J. Mol. Catal. B: Enzym.
2002, 19e20, 43e54; (d) Stolz, T.; Gu, J.; Wilk, B.; Olsen, E. Tetrahedron Lett.
2010, 51, 5511e5515; (e) Magrone, P.; Cavallo, F.; Panzeri, W.; Passarella, D.;
Riva, S. Org. Biomol. Chem. 2010, 8, 5583e5590.
2. A nice example is the regioselective enzymatic acylation of rapamycin. See, for
instance: Gu, J.; Ruppen, M. E.; Cai, P. Org. Lett. 2005, 7, 3945e3948.
3. Rebstock, M. C.; Crooks, H. M., Jr.; Controulis, J.; Bartz, Q. R. J. Am. Chem. Soc.
1949, 71, 2458e2462.
In summary, we have successfully demonstrated the versatility
of lipases, especially CAL-B, applied to the regioselective production
of chloramphenicol esters, acylating or hydrolysing the primary
position in short reaction times. Industrial application of the
transesterification reaction has been considered showing the pos-
sibility to carry out the experiments at high substrate concentra-
tions (0.25 M) and recycling the enzyme for a large number of
cycles (up to 10), thus obtaining interesting compounds, such as the
palmitate derivative with excellent yields. Furthermore, mono-
acylated compounds on the secondary alcohol group, difficult to
achieve by conventional methodologies, were obtained with good
to moderate yields through biocatalysed regioselective hydrolytic
processes.
4. El-Wahed, M. G. A.; Refat, M. S.; El-Megharbel, S. M. J. Mol. Struct. 2008, 892,
402e413.
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2463e2468; (b) Long, L. M.; Troutman, H. D. J. Am. Chem. Soc. 1949, 71,
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17. 30-Acylated compound 3d (3%) and chloramphenicol (4%) were also
detected.