Synthesis and total 1H- and 13C-NMR assignment of cephem derivatives for use in ADEPT approaches

Article abstract of DOI:10.3390/molecules13040841

We report the synthesis and total NMR characterization of 5-thia-1-azabicyclo-[4.2.0]oct-2-ene-2-carboxylic acid-3-[[[(4″- nitrophenoxy)carbonyl]oxy]-methyl]-8-oxo-7-[(2-thienyloxoacetyl)amino] -diphenylmethyl ester-5-dioxide (5), a new cephalosporin derivative. This compound can be used as the carrier of a wide range of drugs containing an amino group. The preparation of the intermediate product, 5-thia-1-azabicyclo[4.2.0] oct-2-ene-2-carboxylic acid-3-[methyl 4-(6-methoxyquinolin-8-ylamino) pentylcarbamate]-8-oxo-7-[(2-thienyloxoacetyl)amino]-diphenylmethyl ester-5-dioxide (6), as well as the synthesis of the antimalarial primaquine prodrug 5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid-3-[methyl 4-(6-methoxyquinolin-8-ylamino)pentylcarbamate]-8-oxo-7-[(2-thienyloxoacetyl) amino]- 5-dioxide (7) are also described, together with their total ¹H- and ¹³C-NMR assignments.

Full text of DOI:10.3390/molecules13040841

Molecules 2008, 13, 841-854
molecules
ISSN 1420-3049
© 2008 by MDPI
www.mdpi.org/molecules
Full Paper
Synthesis and Total 1H- and 13C-NMR Assignment of Cephem
Derivatives for Use in ADEPT Approaches
Lorena Blau 1, Renato Farina Menegon 1, Elizabeth Igne Ferreira 2, Antonio Gilberto Ferreira 3,
Elisangela Fabiana Boffo 3, Leila Aley Tavares 3, Vladimir Constantino Gomes Heleno 3,4,* and
Man-Chin Chung 1,*
1
Departamento de Fármacos e Medicamentos, Faculdade de Ciências Farmacêuticas, Universidade
Estadual Paulista, CP 502 – 14801-902, Araraquara, SP, Brazil; E-mails:
lorenblau@universiabrasil.net; renato.farina@universiabrasil.net
2
Departamento de Farmácia, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, CP
66083 – 05389-970, São Paulo, SP, Brazil; E-mail: hajudan@usp.br
3
Departamento de Química, Centro de Ciências Exatas e Tecnológicas, Universidade Federal de São
Carlos, CP 676 – 13565-905, São Carlos, SP, Brazil; E-mails: giba_04@yahoo.com.br;
efboffo@yahoo.com.br; leilaley@yahoo.com
4
Núcleo de Pesquisas em Ciências Exatas e Tecnológicas – Universidade de Franca, CP 82 – 14404-
600, Franca, SP, Brazil
* Authors to whom correspondence should be addressed; E-mail for V.C.G.H.:
heleno.vcg@unifran.br; Phone: +55 16 37118871; Fax: +55 16 37118878; E-mail for M-C.C.:
chungmc@fcfar.unesp.br; Phone: +55 16 33016962; Fax: +55 16 33016960.
Received: 12 March 2008; in revised form: 6 April 2008 / Accepted: 7 April 2008 / Published: 10
April 2008
Abstract: We report the synthesis and total NMR characterization of 5-thia-1-azabicyclo-
[4.2.0]oct-2-ene-2-carboxylic acid-3-[[[(4’’-nitrophenoxy)carbonyl]oxy]-methyl]-8-oxo-7-
[(2-thienyloxoacetyl)amino]-diphenylmethyl ester-5-dioxide (5), a new cephalosporin
derivative. This compound can be used as the carrier of a wide range of drugs containing
an amino group. The preparation of the intermediate product, 5-thia-1-
azabicyclo[4.2.0]oct-2-ene-2-carboxylic
acid-3-[methyl
4-(6-methoxyquinolin-8-
ylamino)pentylcarbamate]-8-oxo-7-[(2-thienyloxoacetyl)amino]-diphenylmethyl ester-5-
dioxide (6), as well as the synthesis of the antimalarial primaquine prodrug 5-thia-1-

Molecules 2008, 13
azabicyclo[4.2.0]oct-2-ene-2-carboxylic
842
acid-3-[methyl
4-(6-methoxyquinolin-8-
ylamino)pentylcarbamate]-8-oxo-7-[(2-thienyloxoacetyl)amino]- 5-dioxide (7) are also
described, together with their total 1H- and 13C-NMR assignments.
Keywords: ADEPT, prodrug, cancer, cephalosporin, 1H, 13C and 2D NMR.
Introduction
About one third of the world’s population is currently at serious risk of contracting malaria. It is
estimated that approximately 2-3 million people die from malaria every year, and in the sub-Saharan
part of Africa alone about half million children below the age of five die of malaria in this same time
period [1]. Treating malaria is becoming more difficult because of the spreading resistance of the
parasite to standard antimalarial drugs, especially to chloroquine [2]. Most of the drugs currently
employed in antimalarial chemotherapy are particularly active against the asexual blood forms of the
parasite, responsible for the clinical symptoms of the disease [3]. With the rapid spread of drug-
resistant Plasmodium falciparum strains, the development of safe and effective antimalarial drugs that
prevent transmission and cure patients has become an important strategy toward an effective control of
the disease [3].
In contrast with the asexual blood forms of Plasmodium, the sexual form of the parasite is a much
less explored life-cycle target. Nowadays, primaquine is the only available transmission-blocking
antimalarial drug that displays marked activity against gametocytes of all species of the parasite
responsible for human malaria, including chloroquine-resistant P. falciparum. However, the use of
primaquine is limited by its extensive metabolic conversion into carboxyprimaquine and its toxic
effects, which include hemolytic anemia, particularly in the case of patients deficient in glucose-6-
phosphate dehydrogenase [4,5].
As a way to solve problems such as low drug concentration in target cells, systemic toxicity [6],
lack of selectivity for target cells over normal cells, and the appearance of drug-resistant cells, new
drug delivery approaches have been the focus of much research in recent years [7], particularly for the
cancer chemotherapy. One such approach is known as ADEPT (antibody-directed enzyme-prodrug
therapy), and it consists of a promising two-step strategy, which should increase drug selectivity
toward the target cells.
Cephalosporins are highly versatile carriers in the construction of enzyme-activated prodrugs, and
they find potential application in ADEPT approaches. The prodrugs, which consist of cephalosporins
substituted at the C-3’ position, are activated after antibody-beta-lactamase conjugate hydrolysis,
which in turn releases the active drug and drives it toward the target [8]. This approach has been tested
for anticancer therapy and is being currently applied to African trypanosomiasis [9].
The first ADEPT step consists in the administration of a monoclonal antibody-enzyme conjugate
with high affinity for a specific antigen of tumor cells (Figure 1). The antibody-enzyme conjugate is
responsible for the transformation of the prodrug into the active form.

Molecules 2008, 13
843
Figure 1. Cephalosporin carrier for the ADEPT approach.
The second step involves administration of a prodrug with a specific substrate of the conjugated
enzyme (carrier) covalently attached to a cytotoxic drug, so that the prodrug will be selectively
activated and released on tumor cells. Cephalosporins were chosen as candidate prodrug carriers
because of the readily controlled release of cytotoxic agents covalently attached to these compounds
(Figure 1).
In this work, we present the synthesis of a cephalosporin-derivative carrier for use in ADEPT, to
which any drug with a terminal amino group could be added. The synthesis of a prodrug from the
cephalosporin derivative containing the atimalarial drug primaquine attached to it is also described.
This prodrug could be used in malaria therapy, thus reducing the toxic effect associated with
primaquine.
Detailed NMR data for the carriers and prodrugs used in the ADEPT approaches are considerably
scarce in the literature, but particularly useful for this kind of research. Understanding what occur in
each step of the ADEPT process is only possible if elucidation of the substances involved in these
steps, namely the carrier, the drugs, the prodrugs and even the generated metabolites, is carried out.
Therefore, a detailed analysis of the carrier 5, the intermediate 6 and the prodrug 7 was performed as
1
13
part of our interest in structural elucidation by NMR techniques [10,11], and the total H- and C-
NMR assignments for 5, 6 and 7, including 1H-NMR, 13C {1H}-NMR and 2D NMR (gCOSY, gHSQC
and gHMBC) experiments, are presented.
Results and Discussion
Synthesis
The cephalosporin intermediate was synthesized according to Scheme 1. Isomer 2a was employed
as the hydroxymethylcephalothin precursor, as opposed to the isomer 2b used in the synthesis of
Jungheim (Figure 2) [12].

Molecules 2008, 13
844
Scheme 1. Synthesis of the cephalothin derivative 5.
H
H
N
S
N
S
O
S
S
O
O
N
O
OH
N
O
O
O
O
1) NaOH, -20ºC
2) HCl
OH
O
2a
Na
1
N N
EtOAc
NO2
O
H
N
H
N
S
O
S
S
S
O
O
O
THF, DMAP
2,6-lutidine
O
OH
N
O
N
O
O
O
O
4
3
p-O2NC6H4OC(O)Cl
m-CPBA 70-75%
CH2Cl2
NO2
O
O
O
H
N
S
O
S
O
N
O
O
O
O
O
5
Figure 2. Hydroxymethylcephalothin isomers.
O
O
S
HN
O
S
HN
O
S
S
N
O
OH
N
OH
OH
O
OH
2b
2a
A strategic yet difficult step in the synthesis of cephalosporin-based prodrugs is a deacetylation
process for the further drug attachment. Cephalosporin deacetylation by acid hydrolysis gives a γ-
lactone compound, whereas alkaline hydrolysis results in opening of the lactam ring (Scheme 2). Both
methods are unable to yield the prodrug that is employed in ADEPT [13].
This deacetylation can be achieved by enzymatic cleavage of the immobilized esterase [14], but
this method is very difficult. Therefore, we carried out deacetylation by a convenient method
developed by Mobashery for sodium cephalothin (1) [15], in which the special conditions of
temperature (-20 ºC) and reaction time (135 seconds) are very important for the success of this
reaction. We observed that when the reaction product is frozen and lyophilized, the degradation of 2a
does not occur.
To protect the cephalosporin carboxyl group, such as the one forund in hydroxymethylcephalothin
(2a), it is necessary to avoid the formation of the corresponding lactone (Scheme 2). Esterification

Molecules 2008, 13
845
reagents or methods which lead to lactonization should be avoided, so the preferred esterification
reagents are aryldiazoalkanes, particularly aryldiazomethanes. Alternatively, the ester may be prepared
by reacting an aralkyl chloride or bromide with an alkali metal salt; e.g., the sodium salt, of the 3-
hydroxymethylcephemoic acid analogue. Examples of esters that may be prepared by this latter
method are the 4-triphenylmethyl esters and 4-benzyl esters [16].
Scheme 2. Cephalothin degradation.
O
S HN
O
S
N
O
O
+ O
O-
Na
(1)
HCl, 0 ºC
NaOH, 0 ºC
O
O
S HN
S
S HN
S
-
+ O
Na
N
N
O
O
O-
Na
+
O
O
O
β-lactam ring opened
γ-lactone
The protection of the carboxylic group of cephalosporins is necessary for subsequent drug
attachment to these compounds. Jungheim performed isomerization of hydroxymethylcephalothin (2a)
to isomer 2b (Figure 2), a more soluble product, before attaching the protection [12]. We carried out
the protection of hydroxymethylcephalothin (2a) in good yield without this isomerization, so
precautions against isomerization are not necessary and one step can be avoided.
Compound 3 was then reacted with p-nitrophenylchloroformate, leading to the attachment of a
good leaving group under basic conditions, which should facilitate drug attachment [17]. Moreover,
the resulting carbamate moiety, to which the drug can be attached, should provide an appropriate and
efficient environment for drug release from the cephem nucleus during the ADEPT process [18].
Finally, the oxidation of the sulfur atom in the thiazolinic ring (step 4, Scheme 1) was necessary
because acylation of the hydroxymethyl group in cephalosporins gives a mixture of the isomers with
the double bond in the C-2 and C-3 positions [18]. This oxidation converts the C-3 double bond isomer
into the C-2 double bond isomer. This process is based on the fact that β,γ-unsaturated sulfoxides are
thermodynamically more stable than the corresponding α,β-unsaturated ones [19]. This oxidation
process gave rise to a new cephalothin derivative 5, which can be used to attach a variety of
compounds containing an amino group. The cephalothin sulfoxide had been the original target, but
only the sulfone 5 was obtained. The most important advantage of this result is that compound 5 can
be easily purified by washing with saturated aqueous sodium bicarbonate solution and triturated with
methanol, thus avoiding a column chromatography step.

Molecules 2008, 13
846
The antimalarial drug primaquine was then successfully attached to carrier 5, leading to the
cephalothin-primaquine prodrug 7, as shown in Scheme 3.
Scheme 3. Preparation of the cephalothin-primaquine prodrug (7).
O O
H
N
S
S
O
N
O
O
O
O
O
O
O
NO2
primaquine, THF
room temperature, 24h
5
O O
S
H
N
N
H
S
O
O
N
O
N
N
H
O
O
O
O
O
TFA, CH2Cl2, anisole
0 ºC
6
O O
S
H
N
N
H
S
O
N
O
N
N
H
O
O
O
O
O
OH
7
The condensation of 5 with primaquine in THF provided the desired carbamate 6 in good yield,
and the final compound 7 was easily obtained through deprotection with trifluoroacetic acid (TFA) and
anisole in dichloromethane.
NMR Assignments
Literature NMR data on substances like 5 and 7 are rare, incomplete and little detailed. However,
total characterization cannot be completely achieved without the complete assignment of NMR
signals. Therefore, we decided to carry out the total NMR characterization of these compounds so as to
provide enough data for their prompt structural determination in further works.
Compound 5 was analyzed by the 1H-NMR, 13C-NMR{1H}, and gHMBC techniques. The 1H- and
1
13C-NMR data assignments were carefully undertaken by detailed analysis of the H-NMR spectrum.
Expansions were made for all signals, which allowed the measurement of several J values. Analysis of
1
the multiplicities and their coupling constants was useful to verify and confirm some H/1H
correlations.
Next, the 13C-NMR spectrum analysis, combined with the information from 1H-NMR and gHMBC
experiments, led to the total assignment of the 13C-NMR signals. A careful observation of the gHMBC
results shows the perfect agreement of the data with the structure of 5. Thus, these data (shown in
Table 1) can be considered enough to guide future synthetic work with this carrier.

Molecules 2008, 13
847
Table 1. 1H- and 13C-NMR chemical shifts for compound 5.
H
O
13
12
10
N
S
14
15
7
6
2
3
9
11
1
'4
8
S
2'
3'
O
N
O
O
5'
5
16
4
1''
O
1'
O
6'
O
O
NO2
4''a
3''
5''
6''
2''
4''b
Position
δ
13C (ppm)
δ 1H (ppm); (int., mult.); J (Hz)
a – 4.08; (1H, d); J=18.6
b – 3.71; (1H, d); J=18.6
---
gHMBC
2
45.4
C3; C4; C6
3
4
6
7
8
120.1
125.3
66.6
58.2
164.5
---
---
---
---
4.99; (1H, d); J=4.5
5.98; (1H, dd); J=8.3; J=4.5
---
C8
C6; C8; C10
---
C7; C8; C10
---
9
10
8.47; (1H, d); J=8.3
---
170.2
a – 3.92; (1H, d); J=15.5
b – 3.83; (1H, d); J=15.5
---
7.29; (1H, d); J=5.5
7.28; (1H, dd); J=6.7; J=5.5
7.54; (1H, d); J=6.7
a – 5.30 ; (1H, d); J=13,0
b – 4.91; (1H, d); J=13,0
---
11
35.7
C10; C12; C13
12
13
14
15
136.7
126.9
126.7
125.1
---
---
---
---
1’
67.4
C2; C3; C4; C2’
2’
3’
4’
5’
6’
1”
2”
3”
151.5
155.1
122.4
125.4
145.2
159.7
79.3
---
---
---
7.52; (2H, d); J=9.0
8.31; (2H, d); J=9.0
---
C3’; C5’; C6’
C3’; C5’; C6’
---
---
---
C1’’; C3’’; C4’’
---
6.96; (1H, s)
139.6
---
a – 7.44; (2H, d); J=7.4
b – 6.98; (2H, d); J=3.5
7.34; (4H, dd); J=7.4; J=6.7
7.38; (2H, dd); J=6.7; J=3.5
4”
126.6
C2’’
5”
6”
128.5
128.4
C3’’; C6’’
---
Attachment of a primaquine molecule to carrier 5 led to compound 6, which was also submitted to
1
a detailed NMR analysis. Expansion of the H-NMR signals allowed the measurement of some J
values and led to the assignment of all the 1H-NMR data. With those results and with the information
13
from the gHSQC experiment, almost all C-NMR signals could be unequivocally assigned. For the
quaternary carbons, the gHMBC experiment was essential. Again, the gHMBC experiment data was in
perfect agreement with the structure. In this case, working with a larger number of signals and signal
overlapping due to the presence of additional hydrogen and carbon atoms in compound 6, compared
with structure 5, was the most important difficulty. All the NMR data obtained for compound 6 are
presented in Table 2.

Molecules 2008, 13
848
Table 2. 1H- and 13C-NMR chemical shifts for compound 6.
16'
15'
14'
17'
H
O O
S
13
18'N
12
10
N
14
15
7
6
2
3
13'
12'
9
H
11
1
8'
19'
5'
8
20'
9'
S
2'
O
N
O
N
16
7'
10'
5
N
O
3'
4
O
O
1'
11'
4'
6'
1''
O
H
O
O
4''a
3''
5''
6''
2''
4''b
δ 1H (ppm), (int., mult.), J
Position
δ
13C (ppm)
gHSQC
gHMBC
(Hz)
a – 3.62; (1H, d); J=18.8
2
45.6
C2
C3; C4; C6; C1’
b – 2.94; (1H, d); J=18.8
3
4
6
7
8
---
---
123.4
124.6
66.7
59.0
164.5
---
170.5
37.2
135.0
127.2
127.4
125.9
---
---
C6
C7
---
---
---
C11
---
---
---
4.17 – 4.26; (1H, m)
5.81 – 5.96; (1H, m)
C8
C6; C8; C10
---
C7; C10
---
---
9
6.99; (1H, d); J=9.8
10
11
12
13
14
15
---
3.72; (2H, s)
---
C10; C11; C12
---
6.83 – 6.92; (2H, m)
C13; C14
C15
C12; C15
---
7.08 – 7.40; (1H, m)
a – 5.05; (1H, d); J=14.3
b – 4.52; (1H, d); J=14.3
---
2.99 – 3.13; (2H, m)
1.38 – 1.66; (4H, m)
3.43 – 3.55; (1H, m)
1.18; (3H, d); J=6.0
---
1’
63.4
C1’
C2; C3; C4; C2’
2’
4’
5’, 6’
7’
156.0
41.2
26.6; 33.9
47.9
20.6
144.9
92.0
159.6
97.1
130.1
135.1
122.1
144.4
135.3
55.5
160.1
---
C4’
---
---
C8’
---
C11’
---
C13’
---
---
---
---
---
C4’; C5’; C6’; C7’; C8’
C5’; C6’
C6’; C7’
8’
10’
11’
12’
13’
14’
15’
16’
17’
19’
20’
1’’
---
6.25; (1H, brs)
---
6.19; (1H, brs)
---
C12’; C13’; C19’
---
C11’; C12’; C19’
---
---
---
7.83; (1H, d); J=8.1
7.07 – 7.41; (1H, m)
8.43; (1H, dd); J=5.8; J=1.6
---
C17’
---
C20’
C16’; C19’
---
3.79; (3H, s)
C12’; C20’
---
---
---
2’’
3’’
6.81; (1H, s)
80.4
139.2
C2’’
---
C1’’; C3’’; C4’’a; C4’’b
---
---
4’’a,
4’’b
5’’, 6’’
127.7; 127.8
128.6; 128.8;
128.3; 128.4
7.08 – 7.40; (10H, m)
---
---
For the final product 7, 1H-NMR, 13C-NMR{1H}, gCOSY, gHSQC and gHMBC experiments were
performed. Table 3 shows the spectral information obtained for this compound.

Molecules 2008, 13
849
Table 3. 1H- and 13C-NMR chemical shifts for compound 7.
16'
15'
14'
17'
H
O O
S
13
18'N
12
10
N
14
15
7
6
2
3
13'
12'
9
H
11
1
8'
19'
5'
8
20'
9'
S
2'
O
N
O
N
16
7'
10'
5
N
O
3'
4
O
1'
11'
O
4'
6'
1''
O
H
O
OH
Position δ 1H (ppm); (int., mult.); J (Hz)
δ
13C (ppm)
gCOSY
---
gHSQC
C2
gHMBC
C3; C4; C6
2
3
4
3.75 – 3.94; (2H, m)
45.2
---
---
119.7
125.3
66.2
---
---
H7
---
---
C6
---
---
6
4.88; (1H, d); J=4.8
C8
C6; C8; C10
---
C7; C8; C10
---
7
8
9
10
11
12
5.80; (1H, dd); J=8.4; J=4.8
58.0
164.1
---
170.0
35.6
136.8
H6; H9
---
H7
---
---
C7
---
---
---
C11
---
---
8.42; (1H, d); J=8.4
---
3.75 – 3.94; (2H, m)
---
C10; C12; C13
---
---
13; 14
15
6.91 – 6.97; (2H, m)
126.4; 126.7
125.1
H15
C13; C14
C15
C12; C15
C12; C13
7.36; (1H, dd); J=4.8; J=1.8
a – 5.11; (1H, d); J=13.1
b – 4.52; (1H, d); J=13.1
---
H13; H14
1’
62.7
H1’a; H1’b
C1’
C2; C3; C4; C2’
2’
3’
4’
5’; 6’
7’
8’
10’
11’
12’
13’
14’
15’
16’
17’
19’
20’
1’’
155.8
---
40.3
26.1; 33.2
47.0
20.1
144.5
91.7
159.0
96.3
129.6
135.0
122.1
144.1
134.3
55.0
---
H4’
H3’; H5’; H6’
---
---
C4’
---
---
---
C8’
7.29; (1H, t); J=5.9
2.98; (2H, d); J=5.9
1.41 – 1.70; (4H, m)
3.55 – 3.66; (1H, m)
1.19; (3H, d); J=6.3
---
6.48; (1H, d); J=2.6
---
6.25; (1H, d); J=2.6
---
H4’; H7’
H8’
C5’; C6’
C7’
---
C6’; C7’
---
H7’
---
H13’
---
H11’
---
C8’
---
C11’
---
C13’
---
C15’
C16’
C17’
---
C20’
---
C12’; C13’; C15’
---
C12’; C15’
---
C11’; C15’; C17’
C14’; C17’
C15’; C16’
---
8.08; (1H, dd); J=8.4; J=1.5
7.43; (1H, dd); J=8.4; J=4.2
8.53; (1H, dd); J=4.2; J=1.5
---
H16’
H15’; H17’
H16’
---
3.75 – 3.94; (3H, m)
---
---
---
C12’; C20’
---
162.0
1
Once more the detailed H-NMR spectrum analysis enabled the measurement of several
homonuclear hydrogen coupling constant values. This allowed verification of some hydrogen
1
correlations and assignment of most of the H NMR signals. The information from the gCOSY
experiment was used to achieve and confirm all assignments of the hydrogen signals. The assignment
1
13
of H- and C-NMR data for 7 was carried out in a similar way to the work done with compound 6,
using information from gHSQC and gHMBC experiments. A detailed 2D data analysis was
undertaken, which allowed total structure confirmation.

Molecules 2008, 13
850
Conclusions
A new cephalosporin carrier and a new correlated prodrug were successfully synthesized. The
carrier itself is now available for use with a wide range of drugs. The preparation of the primaquine
prodrug shows that several prodrugs can be developed. The NMR study undertaken for the carrier, the
prodrug and the intermediate product furnished a considerable amount of new NMR data. These data
can be used as a tool to verify and prove structural changes in future works.
Experimental
General
Petroleum ether, ethyl acetate, dichloromethane and THF were dried with anhydrous sodium
sulfate and distilled at atmospheric pressure. Flash chromatography was carried out on Merck
Kieselgel 60 silica gel (230-400 mesh). Melting points were measured in an Eletrothermal 9200
apparatus and are uncorrected. All glassware was oven-dried, assembled hot, and cooled under a
stream of dry nitrogen before use. Infrared spectra were recorded with a Shimadzu FTIR-8300
spectrophotometer. Elemental analysis was performed in a Perkin-Elmer 240 A analyzer.
NMR Experiments
All NMR experiments were performed on a Bruker DRX400 instrument (9.4 T) operating at
1
13
1
400.13 MHz for H and at 100.58 MHz for C. The H-NMR and 2D-NMR (gCOSY, gHSQC and
gHMBC) experiments were undertaken with an inverse 5 mm probehead (BBI), and the 13C {1H}-
NMR experiments were carried out with a direct 5 mm probehead (BBO). The temperature of the
sample was always 298 K and the concentrations ranged from 15 to 35 mg·mL-1 in DMSO-d6. TMS
was used as internal reference.
Synthesis
Cephalothin derivative 5
Cephalothin sodium salt (1) was provided by EMS/Sigma Pharma Group and was readily
deacylated by basic hydrolysis at – 20 ºC [6]. Hydroxymethylcephalothin (2a) was reacted with
diphenyldiazomethane, a carboxylic blocking agent that was prepared by oxidation of benzophenone
hydrazone (see below), to give compound 3. The latter was substituted at the C-3 hydroxyl group with
p-nitrophenylchloroformate, using 4-DMAP and 2,6-lutidine as catalysts, to give a mixture of isomers
4. Oxidation of 4 with m-chloroperbenzoic acid (m-CPBA) provided the derivative cephalothin 5
(Scheme 1).

Molecules 2008, 13
851
Diphenyldiazomethane
A mixture of benzophenone hydrazone (9.8 g; 0.05 mol) and yellow mercuric oxide (11 g; 0.05
mol) in dry petroleum ether (50 mL) was placed in a pressure bottle at room temperature for six hours.
This mixture was filtered and some insoluble material was removed. The filtrate was evaporated,
giving a dark red oil at room temperature. Freezing this oil with dry ice and then allowing it to warm to
room temperature gave dark red crystals. M.p. 29-30 ºC; IR (KBr, cm-1): 2037 (diazo) and 1593, 1495
(aromatic ring).
3-Hydroxymethyl-8-oxo-7-[(2-thienylacetyl)-amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic
acid (2a)
An aqueous solution (15 mL) of sodium cephalothin (1, 3.0 g, 7.48 mmol) was added at 0 ºC to
NaOH 19% (30 mL) that had been cooled to –20 ºC. The reaction mixture was stirred at –10 ºC for
135 seconds and then quenched by the rapid addition of glacial acetic acid (10 mL) at room
temperature. The reaction vessel was then transferred to an ice-water bath and the pH of the mixture
was immediately lowered to 1.5 by adding concentrated HCl, which had previously been cooled to –20
ºC. The white solid was filtered, washed twice with cold water, suspended in water, frozen and
lyophilized to give 2a as a white powder (2.07 g, 80%); m.p. 210 ºC (dec.); IR (KBr, cm-1): 3413
(OH), 1760 (β-lactam ring), 1719 (COOH) and 1655 (amide).
3-Hydroxymethyl-8-oxo-7-[(2-thienylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic
aciddiphenylmethyl ester (3)
A solution of diphenyldiazomethane (3.24g, 16.68 mmol) in dry ethyl acetate (50 mL) was added
dropwise to a mixture of hydroxymethylcephalothin (2a; 6.0g, 16.92 mmol) in dry ethyl acetate (250
mL). The reaction mixture was stirred at room temperature for four hours and then filtered. The
organic layer was washed with cold aqueous saturated NaHCO3 (3 x 200 mL) and cold water (1 x 200
mL), and dried over anhydrous Na2SO4. The organic layer was concentrated in vacuum to give 3 as a
white powder (10.0 g, 52.44%); m.p. 165 – 168 ºC; IR (KBr, cm-1): 1755 (β-lactam ring), 1724 (ester),
and 1666 (amide).
5-Thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid-3-[[[(4’’-nitrophenoxy)carbonyl]oxy]methyl]-
8-oxo-7-[(2-thienylacetyl)amino]-diphenylmethyl ester and 5-thia-1-azabicyclo[4.2.0]oct-3-ene-2-
carboxylic acid-3-[[[(4’’-nitrophenoxy)carbonyl]oxy]methyl]-8-oxo-7-[(2-thienylacetyl)amino]-
diphenylmethyl ester (4)
DMAP (100mg; 0.8 mmol) was added to a mixture of 3 (10.0 g; 19.23 mmol) at 0 ºC in dry THF
(50 mL), followed by p-nitrophenylchloroformate (5.8 g; 28.85 mmol). Finally 2,6-lutidine (3.34 mL,
28.85 mmol) was added dropwise at 0 ºC. After 16 hours at room temperature, additional THF (200
mL) was added and the reaction mixture was stirred for another 8 hours at room temperature. The
resulting mixture was filtered and some insoluble material was removed. The filtrate was concentrated

Molecules 2008, 13
852
under vacuum, to give an oil. This oil was dissolved in dichloromethane (100 mL), washed with cold
0.2 N HCl (3 x 50 mL) and cold water (1 x 50 mL), dried over anhydrous Na2SO4, and purified by
flash column chromatography using 5% ethyl acetate in dichloromethane as eluent to give 9.0 g of 4
(69% yield). M.p. 69-71 ºC; IR (CHCl3, cm-1): 1780 (β-lactam ring), 1751 (carbonate), 1684 (amide)
and 1529 (NO2).
5-Thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid-3-[[[(4’’-nitrophenoxy)carbonyl]oxy]methyl]-
8-oxo-7-[(2-thienyloxoacetyl)amino]-diphenylmethyl ester-5-dioxide (5)
m-Chloroperbenzoic acid (70-75%; 0.65 g, 2.75 mmol) in dichloromethane (20 mL) was added
dropwise to a solution of 4 (0.75 g, 1.1 mmol) in dichloromethane (20 mL) at 0 ºC, over a period of 15
min. The reaction mixture was stirred for 4 hours at 0 ºC and then washed with cold saturated aqueous
NaHCO3 (4 x 50 mL) and cold saturated aqueous NaCl (1 x 50 mL). The organic layer was dried over
anhydrous Na2SO4 and concentrated under vacuum. The residue was triturated with methanol, to give
pure 5 (0.46g, 60%) as a white powder. M.p. 164-168 ºC; IR (KBr, cm-1): 1786 (β-lactam ring), 1767
(carbonate), 1655 (amide) and 1720 (ester); Anal. Calcd. for C34H27N3O12S2: C, 55.66; H, 3.71; N,
1
13
5.73. Found: C, 55.70; H, 3.72; N, 5.71. The H- and C-NMR chemical shifts were assigned and all
the values are listed in Table 1.
5-Thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid-3-[methyl 4-(6-methoxyquinolin-8-ylamino)-
pentylcarbamate]-8-oxo-7-[(2-thienyloxoacetyl)amino]-diphenylmethyl ester-5-dioxide (6)
Primaquine diphosphate (500 mg, 1.1 mmol) water solution (30 mL) was neutralized with 5%
K2CO3 and extracted with ethyl ether (50 mL). The organic phase was evaporated and the residue was
dissolved in THF (10 mL). The obtained solution was added to compound 5 (220 mg, 0.3 mmol). After
stirring for 24 h at room temperature, the target compound 6 was isolated by column chromatography
on silica gel employing 30 to 50% ethyl acetate in dichloromethane as eluent. This procedure yielded
166 mg of 6 (195 mmol; 65%). M.p. 162 – 164 ºC; IR (KBr, cm-1): 1794 (β-lactam ring), 1718 (ester),
1
13
1695 (carbamate) and 1659 (amide). The H- and C-NMR chemical shifts were assigned and all the
values are listed in Table 2.
5-Thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, 3-[methyl 4-(6-methoxyquinolin-8-ylamino)-
pentylcarbamate]-8-oxo-7-[(2-thienyloxoacetyl)amino]- 5-dioxide (7)
Distilled anisole (1.0 mL) and freshly distilled CF3CO2H (TFA, 1.0 mL) were added to a solution
of 6 (200 mg, 0.23 mmol) in dry CH2Cl2 (10 mL) at -20 ºC. After 15 minutes, the reaction was warmed
up to 0 ºC and, after a further 30 minutes, 10 mL of ethyl acetate and 10 mL of cold water was added.
The organic layer was dried over anhydrous sodium sulfate and evaporated, yielding 110 mg (0.16
mmol; 70%) of the title compound. M.p. 166-168 ºC; IR (KBr, cm-1): 1765 (β-lactam ring), 1718
1
(carbamate), 1701 (carboxylic acid) and 1657 (amide). The H- and 13C-NMR chemical shifts were
assigned and all the values are listed in Table 3.

Molecules 2008, 13
853
Acknowledgements
The authors are grateful to FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo),
CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) and CNPq (Conselho
Nacional de Desenvolvimento Científico e Tecnológico) for financial support.
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Sample Availability: No compound samples are available.
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