Synthesis of a new hapten for generating catalytic antibodies that activate doxorubicin prodrugs

Article abstract of DOI:10.1016/S0040-4039(01)01681-1

In order to assess a novel strategy for catalytic activation of a new prodrug of doxorubicin, a new hapten has been designed and prepared, which can be used to induce an immune response from which catalytic antibodies (cAbs) may be produced.

Full text of DOI:10.1016/S0040-4039(01)01681-1

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 7819–7822
Synthesis of a new hapten for generating catalytic antibodies
that activate doxorubicin prodrugs
Carlos Jime´neza and Alfonso Tramontanob,*
aDepartamento de Qu´ımica Fundamental, Facultade de Ciencias, Universidade de A Corun˜a, 15071 A Corun˜a, Spain
bDepartment of Pathology and Laboratory Medicine, University of Texas Houston Medical School, Houston, TX 77030, USA
Received 6 September 2001; accepted 7 September 2001
Abstract—In order to assess a novel strategy for catalytic activation of a new prodrug of doxorubicin, a new hapten has been
designed and prepared, which can be used to induce an immune response from which catalytic antibodies (cAbs) may be
produced. © 2001 Elsevier Science Ltd. All rights reserved.
Doxorubicin (adryamycin®) (1)1 and its derivatives,
such as daunomycin (2), within the family of the
anthracyclines glycosides, are among the most potent
and clinically useful of all the anticancer agents. The
activity is due to inhibition of DNA and RNA synthe-
sis.2 Effective dosages in animal models are in the 1–10
mg/kg range, but prolonged treatment can result in
serious cardiotoxicity3 and so, its clinical efficacy is
limited.
drug. Examples of antibody-mediated prodrug activa-
tion by hydrolytic or retro-aldol reactions have been
demonstrated.7,8 The antibody can also be humanized
to render it nonimmunogenic. A hybrid antibody or
immune complex between the cAb and a tumor specific
antibody could serve as a well-tolerated pre-targeting
agent in ADEPT.
O
O
OH
R1
OH
Targeting the drug to the affected tissue by means of a
specific anti-tumor monoclonal antibody has been
explored as a means to mitigate toxicity against normal
tissue, but this approach has some limitations on the
effective dose of drug that can be delivered per anti-
body molecule.4 An interesting solution to this problem
and the prevailing targeting approach is to chemically
modify the drug into a less toxic derivative (prodrug)
that can be converted back to the active form by the
action of a specific enzyme delivered to the tumor cell,
a process termed antibody-directed enzyme prodrug
therapy (ADEPT).5 The ideal enzyme for ADEPT
should have high specific activity for the prodrug,
should not elicit an immune response and should not be
widely distributed in the body.
O
OH
O
HO
NHR2
HO
1 Doxorubicin R1 = CH2OH, R2 = H
2 Daunomycin R1 = Me , R2 = H
3 Prodoxorubicin R1 = CH2OH, R2 =
CO
NO2
Here we describe the design and synthesis of a doxo-
rubicin prodrug and the respective hapten that could be
used to develop a custom cAb for the hydrolytic pro-
drug activation strategy.
In principle, a catalytic antibody (cAb)6 offers many
advantages. The cAb could be selected for binding
specificity or chemical specificity that identifies the pro-
Design and synthesis of the prodrug
The design of a prodrug required a simple, enzymati-
cally reversible chemical modification, which masks the
toxicity of the parent drug. The amino group within the
sugar moiety of doxorubicin is important for activity of
this drug because it is postulated to interact strongly
Keywords: anthracyclinone; antitumour compound; antibodies; cataly-
sis; sulfonamide.
* Corresponding author. Tel.: 713 500-5318; fax: 713 500 0730;
e-mail: alfonso.tramontano@uth.tmc.edu
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)01681-1

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C. Jime´nez, A. Tramontano / Tetrahedron Letters 42 (2001) 7819–7822
with the phosphate backbone of nucleic acids. This
functionality served as a convenient site for modification
(mainly acylation) to find a derivative to be tested for
reduced toxicity in cytotoxicity assays.9 Among several
amide derivatives tested we found that compound 3 with
a N-p-nitrobenzamide moiety at C-2 of the daunosamine
sugar was 30 times less cytotoxic than doxorubicin itself.
This compound was easily synthesized in high yield by
nitrobenzoylation of doxorubicin with succinimidyl p-
nitrobenzoate.10
The hapten 4 was prepared by coupling of the
chlorosugar 9 and the methylester alcohol 12. The
chlorosugar 9 was prepared from daunosamine (6),
available in one-step from the commercially available
b-methyl daunosaminide (5). The amine group of
daunosamine was converted to the N-p-nitroben-
zenesulphonyl derivative 7 in 64% yield from 5.12
This was subsequently acetylated and transformed to
the chlorosugar 9 by treatment with dry HCl (Scheme
1).
Chemical synthesis of the hapten 4
On the other hand, the aglycon alcohol 12 was obtained
from the commercially available tetrahydronaphthalene
propionic acid 10 by esterification in methanolic HCl,
followed by ketone reduction with NaBH4 (78% yield
overall). Glycosidation of alcohol 12 with the chloro-
sugar 9 by a Koneigs–Knorr procedure afforded com-
pound 13 in a 27% yield from 8.13 Finally, hydrolysis of
13 with 0.5N NaOH removed both the acetyl group
and the methyl ester to give 4 in a quantitative yield
(Scheme 2).14
In the design of a hapten for eliciting cAbs to catalyze
hydrolysis of 3, we proposed a sulfonamide group as the
mimic for the transition state for amide hydrolysis.
Compared to other derivatives used as hydrolytic transi-
tions state analogues, the sulfonamide group lacks a net
charge. However, the tetrahedral structure, chemical
stability and SꢀO bond polarity are favorable features.
Antibodies obtained against a sulfonamide hapten have
been reported to accelerate hydrolysis of a heterocyclic
amide.11Additional design concerns include the presenta-
tion of structural features of the substrate that are remote
from the site of chemical activity in order to obtain cAbs
with desirable substrate specificity. Consequently, we
designed compound 4, containing appropriate function-
ality to satisfy these requirements. In addition to the
sulfonylated daunosamine and glycosidic group approx-
imating the anthracyclin unit, a carboxylic group in the
glycoside provides a site for covalent coupling for prepar-
ing an immunogenic conjugate.
Chemical synthesis of substrates 18 and 19
Corresponding substrates 3-[4-O-(2,3,6-trideoxy-3-N-p-
nitrobenzoyl - a -
hydronaphthalen-2-yl]-propionic
1-O-(2,3,6-trideoxy-3-N-p-nitrobenzoyl-a-
L
- lyxohexopyranosyl) - 1,2,3,4 - tetra-
acid (18) and
-lyxohexo-
L
pyranosyl)-1,2,3,4-tetrahydronaphthalene (19) were pre-
pared in order to facilitate screening assays for the
identification of hydrolytic antibodies elicited by 4.
Following a similar strategy used in the synthesis of
hapten 4, compounds 18 and 19 were prepared from 10
and 1,2,3,4-tetrahydro-1-naphthol, respectively, as
shown in the Scheme 3. The use of the p-nitrobenzoyl
group for protection of both hydroxyl and acetal groups
afforded the a isomer in both cases.15
COOH
O
Link to BSA
O
The sulfonamide hapten 4 was readily conjugated with
carrier proteins using DCC/NHS in 4:1 water/DMF to
obtain an immunogen for mouse immunization. The
preparation and assay of monoclonal antibodies will be
NH S
NO2
OH
O
Hapten Structure (4)
OH
OH
b
O
O
a
c
5
NH2
HCl
O
HO
HO
HN
S
NO2
O
6
7
Cl
OAc
O
O
d
O
S
O
AcO
HN
AcO HN
NO2
S
NO2
O
O
8
9
Scheme 1. Reagents and conditions: (a) 1% HCl aqueous, reflux 2 h; (b) p-nitrosulfonyl chloride, 2:1 CH3CN/0.1N CO3Na2, 2 h,
64% from 5; (c) Ac2O/Py, 65%; (d) bubbling HCl gas, 10 min in dry benzene.

C. Jime´nez, A. Tramontano / Tetrahedron Letters 42 (2001) 7819–7822
7821
(MAR99-0287) and Xunta de Galicia (PGIDT00-
MAR10301PN).
COOR
b
COOMe
O
References
OH
12
10 R = H
a
1. Arcamone, F. Doxorubicin Anticancer Antibiotics; Aca-
demic Press: New York, 1981.
2. Gewirtz, D. A. Biochem. Pharmacol. 1999, 57, 727–741.
3. De Beer, E. L.; Bottone, A. E.; Voest, E. E. Eur. J.
Pharmacol. 2001, 415, 1–11.
11 R = Me
COOR1
c
12 + 9
4. Soyez, H.; Schacht, E.; Vanderkerken, S. Adv. Drug
Deliv. Rev. 1996, 21, 81–106.
O
O
5. For a recent review, see: Michel, S.; Desbene, S.; Gesson,
J.-P.; Monneret, C.; Tillequin, F. Stud. Nat. Prod. Chem.
2000, 21, 157–180.
6. Stevenson, J. D.; Thomas, N. R. Nat. Prod. Rep. 2000,
17, 535–577.
O
HN
R2O
S
NO2
O
7. Shabat, D.; Rader, C.; List, B.; Lerner, R. A.; Barbas,
III, C. F. Proc. Natl. Acad. Sci. USA 1999, 96, 6925–
6930.
13 R1 = Me, R2 = Ac
d
4 R1 = R2 = H
8. Miyashita, H.; Karaki, Y.; Kikuchi, M.; Fujii, I. Proc.
Natl. Acad. Sci. USA 1993, 90, 5337–5340.
Scheme 2. Reagents and conditions: (a) satd HCl in dry
MeOH, reflux at 100°C, 48 h, 95%; (b) NaBH4 in MeOH,
82%; (c) silver triflate in dry DMF, rt, 3 h, 27% from 8; (d)
0.5N NaOH in THF, 94%.
9. Chakravarty, P. K.; Carl, P. L.; Weber, M. J.; Katzenel-
lenbogen, J. A. J. Med. Chem. 1983, 26, 638–644.
10. Preparation of 3: To a solution of 5 mg (0.009 mmol) of
doxorubicin (1) in 0.5 mL of CH3CN/0.1N Na2CO3 (3:1)
was added 10 mg of succinimidyl p-nitro benzoate and
stirred at rt for 1 h. The mixture was evaporated under
reduced pressure, dissolved in H2O, and extracted with
CHCl3. The organic extract was then chromatographed
by preparative TLC (20% MeOH in CH2Cl2) to give 5 mg
of N-p-nitrobenzoyldoxorubicin (3) in a 84% yield as an
amorphous red powder; 1H NMR (CDCl3) l: 8.25 and
7.89 (2H each, d, J=8.6 Hz, AB system of p-nitrobenzoyl
moiety), 8.05 (1H, d, J=7.6 Hz) and 7.80 (1H, dd, J=7.6
and 8.3 Hz), and 7.39 (1H, d, J=8.3 Hz) corresponding
to ABX system of D ring, 6.62 (1H, J=8.0 Hz, -NH-),
5.55 (1H, brd, J=3.7 Hz, H-1%), 1.33 (3H, d, J=6.4 Hz,
Me-5%); IR (CDCl3) wmax 1780, 1750, 1645 (amide), 1535
cm−1; (+)-LRFABMS m/z 677 (M+H)+.
OR1
Cl
b
O
NHR1
14
O
a
6
N
R1O
R1O
H
15
c
R
1
R2
R2
d
O
O
O
O
NHR1
NHR1
HO
R1O
11. Benedetti, F.; Berti, F.; Colombatti, A.; Ebert, C.; Linda,
P.; Tonizzo, F. Chem. Commun. 1996, 1417–1418.
12. Preparation of 7: To a solution of daunosamine hydro-
chloride (6) in 3 mL of a 2:1 CH3CN/0.1N Na2CO3
solution was added 300 mg of p-nitrobenzenesulphonyl
chloride and the mixture was stirred at rt for 1 h, keeping
the pH above 8.0 by adding several drops of a concen-
trated Na2CO3 solution. After 1 h, further 120 mg of
p-nitrobenzenesulphonyl chloride was added to the reac-
tion mixture and stirred for an additional 30 min. Then,
20 mL of water was added, and the solution was
extracted first with CHCl3 and then with n-BuOH satu-
rated with water. The butanolic extract was concentrated
under reduced pressure and separated by flash column
chromatography (5% MeOH in CH2Cl2) to give 268 mg
16 R2 = - (CH2)2-COOMe
17 R2 = H
18 R2 = - (CH2)2-COOH
19 R2 = H
R1 =
CO
NO2
Scheme 3. Reagents and conditions: (a) p-Nitrobenzoyl chlo-
ride in pyridine, 45%; (b) bubbling HCl anhyd. in dry CH2Cl2
for 10 min; (c) 12 or 1,2,3,4-tetrahydro-1-naphtol, silver tri-
flate in dry DMF, rt, 3 h, 15% for 16, 8% for 17 both from
14; (d) 0.1N NaOH in THF, 52% for 18, 77% for 19.
performed by the standard methods.16,17 The studies
will establish whether the expected amidase cAbs can
work as efficient reagents for an ADEPT strategy.
1
of pure compound 7 (64% yield from compound 5). H
NMR (MeOH-d4) l: 8.30 and 8.04 (4H each, d, J=6.9
Hz, AB aromatic system), 5.01 (1H, brd, J=2.9 Hz,
H-1a), 4.52 (1H, dd, J=9.2 and 2.6 Hz, H-1b), 3.96 (2H,
q, H-5), 3.66 (2H, m, H-4), 1.73 and 1.42 (4H, m, H-2).
(+)-LRFABMS m/z 333 (M+H)+.
Acknowledgements
This work was supported by Grants from CICYT

7822
C. Jime´nez, A. Tramontano / Tetrahedron Letters 42 (2001) 7819–7822
13. Preparation of 13: To a stirred mixture of 247 mg of the
H-1b), 4.80 and 4.67 (1H each, m, H-4), 4.11 (2H, m,
H-5%), 3.80 (2H, q, H-3%), 3.59 and 3.49 (1H each, br d,
H-4%), 1.20 (6H, J=6.6 Hz, Me-5%). 13C NMR (CDCl3) l:
,
alcohol 12 (1.055 mmol) and powered 4 A molecular
sieves in 1 mL of dry methylene chloride were added the
previously obtained chlorosugar 9 (aprox. 200 mg) in 3
mL of dry methylene chloride and a solution of 166 mg
of silver trifluormethanesulfonate in 1 mL of dry DMF
under dry argon in the dark at 0°C. The ice-water bath
was removed and the mixture was stirred at room tem-
perature for 2 h. Then, the mixture was poured into a
cooled brine solution, filtered, and the organic layer was
separated, washed with water, dried (MgSO4), and evapo-
rated to give a residue which was flash chromatographed
in a silica gel (18 g) column with EtOAc/hexanes (2:3) to
yield 110 mg of a mixture of the a,b-glycoside 13 (27% of
177.2 (C6 OOH), 149.6–124.0 (aromatics), 98.8 (C-1a), 92.9
(C-1b), 72.1, 70.7, 66.7 (C-4%, C-5%, C-4), 49.5 (C-3%), 16.6
(Me-5). (+)-LRFABMS m/z 667 [M+Cs]+; 799 [M−H+
2Cs]+. UV (MeOH) umax 268 nm. The a and b isomers
can be separated by reverse phase HPLC using 60%
CH3CN/40% H2O with 0.01 M TEA as eluent.
15. Selected data for substrates: 18: 1H NMR (C5D5N) l:
8.40 and 8.23 (2H each, d, J=8.8 Hz, AB aromatic
system), 7.32–7.19 (4H, aromatics), 5.45 (1H, brd, H-1a),
4.33 (1H, m, H-4), 3.73 (3H, s, OMe), 2.83 (2H, m, H-1),
1.52 (3H, J=5.9 Hz, Me-5%). 13C NMR (C5D5N) l: 174.7
(CO), 141.7–123.8 (aromatics), 100.6 (C-1a), 78.1 (C-4%),
69.6 (C-5%), 68.1 (C-4), 52.1 (OMe), 48.3 (C-3%), 18.0
1
yield from compound 8). H NMR (CDCl3) l: 8.35 and
8.04 (4H each, d, J=8.9 Hz, AB aromatic system),
7.26–7.21 (8H, aromatic protons), 5.20 (1H, brd, H-1a),
5.13 (1H, brd, H-1b), 5.04 and 5.01 (1H each, d, J=8.6
Hz each, -NH-), 4.94 and 4.87 (1H each, m, H-4%), 4.79
and 4.67 (1H each, m, H-4), 4.12 (2H, m, H-5%), 4.00 (2H,
q, H-3%), 3.68 (6H, OMe), 2.80 (4H, m, H-1), 1.06 (6H,
J=6.6 Hz, Me-5%). 13C NMR (CDCl3) l: 173.5
1
(Me-5). (+)-LRFABMS m/z 645 [M+Cs]+. 19: H NMR
(CDCl3) l: 8.27 and 7.92 (2H each, d, J=8.8 Hz, AB
aromatic system), 7.22–7.14 (4H, aromatics), 6.62 (1H, d,
J=8.2 Hz, -NH-), 5.22 (1H, brd, H-1a), 4.75 (1H, m,
H-4), 4.58 (1H, m, H-4%), 3.69 (1H, m, H-3%), 4.25 (1H,
brq, J=6.7 Hz, H-5%), 2.81 (2H, m, H-1), 1.28 (3H,
J=6.7 Hz, Me-5%). (+)-LRFABMS m/z 559 [M+Cs]+.
16. Tramontano, A.; Schloeder, D. Methods Enzymol. 1989,
178, 531–550.
(C6 OOMe), 170.2 (OC6 OMe), 149.6–124.0 (aromatics),
98.1 (C-1a), 92.6 (C-1b), 72.1, 70.7, 65.4 (C-4%, C-5%, C-4),
51.1 (OMe), 47.9 (C-3%), 20.2 (MeCO), 16.3 (Me-5). (+)-
LRFABMS m/z 723 [M+Cs]+; UV (MeOH) umax 268 nm.
1
17. Riva, S.; Mendozza, M.; Carrea, G.; Chattopadhyay, P.;
Tramontano, A. Appl. Biochem. Biotechnol. 1998, 75,
33–44.
14. Selected data for 4: H NMR (CDCl3) l: 8.32 and 8.04
(4H each, d, J=8.8 Hz, AB aromatic system), 7.30–7.19
(8H, aromatics), 5.14 (1H, brd, H-1a), 5.07 (1H, brd,