Efficient synthesis of doxorubicin melanotransferrin p97 conjugates through SMCC linker

Article abstract of DOI:10.1081/SCC-120039494

Doxorubicin-succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC) 3, prepared by treating doxorubicin (1) with SMCC 2, is treated with 2-mercaptoacetic acid to give doxorubicin-SMCC-sulfo-acetic acid 5. Treating with benzotriazol-1-yl-N,N,N′,

Full text of DOI:10.1081/SCC-120039494

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Efficient Synthesis of
Doxorubicin Melanotransferrin
p97 Conjugates Through SMCC
Linker
Qingqi Chen a & Reinhard Gabathuler a
a Biomarin Pharmaceutical Inc., 46 Galli Drive,
Novato, California, 94949, USA
Available online: 10 Jan 2011
To cite this article: Qingqi Chen & Reinhard Gabathuler (2004): Efficient Synthesis
of Doxorubicin Melanotransferrin p97 Conjugates Through SMCC Linker, Synthetic
Communications: An International Journal for Rapid Communication of Synthetic
Organic Chemistry, 34:13, 2407-2414
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SYNTHETIC COMMUNICATIONSw
Vol. 34, No. 13, pp. 2407–2414, 2004
Efficient Synthesis of Doxorubicin
Melanotransferrin p97 Conjugates
Through SMCC Linker
*
Qingqi Chen and Reinhard Gabathuler
Biomarin Pharmaceutical Inc., Novato, California, USA
ABSTRACT
Doxorubicin–succinimidyl
4-[N-maleimidomethyl]cyclohexane-1-
carboxylate (SMCC) 3, prepared by treating doxorubicin (1) with
SMCC 2, is treated with 2-mercaptoacetic acid to give doxorubicin–
SMCC–sulfo-acetic acid 5. Treating with benzotriazol-1-yl-N,N,N0,N-
tetra-methyluronium tetrafluoroborate (BTTU), the carboxy group of 5
is activated, and reacts efficiently with the amino group of melanotrans-
ferrin p97 to afford the expected doxorubicin-p97 conjugate 6, which is a
potential agent capable to cross the blood–brain barrier (BBB) to treat
brain tumors.
Key Words: Doxorubicin; Blood–brain barrier; Melanotransferrin;
Vector-mediated approach; SMCC linker; Bioconjugate.
*
Correspondence: Qingqi Chen, Biomarin Pharmaceutical Inc., 46 Galli Drive,
Novato, CA 94949, USA; E-mail: qchen@bmrn.com.
2407
DOI: 10.1081/SCC-120039494
0039-7911 (Print); 1532-2432 (Online)
www.dekker.com
Copyright # 2004 by Marcel Dekker, Inc.

2408
Chen and Gabathuler
INTRODUCTION
Doxorubicin (1, dox) is a widely used anti-cancer drug for the treatment
of human cancers such as leukemia, breast carcinoma, etc.[1] However, a
serious problem in using this drug in cancer chemotherapy is its toxicity to
normal tissues, especially its toxic dose-related side effects, such as cumula-
tive cardiotoxicity, myelosuppression, nephrotoxicity, and extravasation.[1b,1c]
Moreover, dox is known to be excluded from the brain by the blood–brain
barrier (BBB), and therefore could not be directly used to treat brain
cancers. Melanotransferrin (p97) has been found to localize in capillary
endothelial cells of the human brain and to play an important role in the
transport of iron across the BBB.[2] This provides a base for a vector-mediated
approach in delivery of therapeutics to the brain. We previously synthesized
dox-p97 conjugates through its 30-amino, 13-ketone, and 14-hydrxoxy
group, and used them for brain drug delivery study.[3] In this paper,
we reported the novel synthesis of doxorubicin-p97 conjugate through
a succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC)
linker.
RESULTS AND DISCUSSIONS
SMCC, a heterobifunctional cross-linker, is widely used for the synthesis
of bioconjuates.[4] In a typical procedure, the N-succinimidyl ester of SMCC 2
is first coupled with the amino group of a drug to afford the corresponding pro-
duct with a maleimide group. The SMCC-modified drugs is then treated with
the sulfuryl-modified proteins through Michael-type conjugation addition
reaction to give the expected bioconjugates. Consequently, proteins used for
such purpose have to be pre-modified by sulfuryl agents such
as N-succinimidyl S-acetylthioacetate (SATA) or 2-iminothiolane (2-IT).
To avoid the protein pre-modified step, a process which usually causes
about 10% loss of the protein, we are interested in developing so-called
“one-step” synthesis, i.e., the expected bioconjugates are synthesized by coup-
ling the modified drug directly with p97 so as to reduce the loss of protein
during the reaction and purification. Thus, our novel procedure, as shown
in Sch. 1, consists of the preparation of dox–SMCC, the reaction with
2-mercaptoacetic acid, and the direct coupling reaction with p97.
Dox (1) is well-known for its sparse solubility in a variety of organic
solvents. We discovered that, after stirring with Hu¨nig’s base (diisopropyl-
ethylamine) in DMF at ambient temperature for about 15–30 min, the
original suspension of dox (1) in DMF became a clear solution, which was
coupled with the SMCC 2 in DMF at room temperature. After stirring in

Synthesis of Dox-p97 Conjugates Through SMCC Linker
2409
Scheme 1. Reagents and conditions: (a) diisopropylethylamine/DMF, r.t. 12 hr, 96%;
(b) diisopropylethylamine/DMF, 2-mercaptoacetic acid, r.t. 2 hr, 96%; (c) H2O,
ion-exchange resin, 71%; (d) diisopropylethylamine/DMF, benzotriazol-1-yl-
N,N,N0,N-tetramethyluronium tetrafluoroborate (BTTU), r.t. 2 hr, then p97 in PBS,
pH ¼ 7.4, r.t. 12 hr. MSR ¼ 8.2, protein recovery 91%.
the dark overnight, TLC indicated that the reaction was completed (spot-to-
spot conversion). The solvent was then removed at reduced pressure (30–
40 mmHg) under 35–408C. The residue was taken up in dichloromethane
and purified by column chromatography to afford the expected dox–SMCC
3 in 96%.
Upon treatment with 2-mercaptoacetic acid in a basic solution (2 N
NaOH) at room temperature for 30 min, dox–SMCC 3 was converted to
product 5 in about 30% yield. Seeking for a more efficient method for the
synthesis of 5, we interested a variety of bases, and found that the best
yield of 4 (96%) was obtained with diisopropylethylamine. Thus, compound
3 was dissolved in methylene chloride and treated with mercaptoacetic acid
and diisopropylethylamine. The solution was stirred at ambient temperature
in the dark for 2 hr and monitored by TLC (dichloromethane/methanol/acetic

2410
Chen and Gabathuler
acid, 7/2/1, v/v/v). The reaction mixture was concentrated under vacuum
and the residue was taken up in methanol. Precipitation occurred when the
resulting methanol solution was concentrated. The solid was collected by
filtration and identified as the corresponding ammonium salt 4. The free
acid was subsequently regenerated by passing a solution of the ammonium
salt in a small amount of methanol through a short pad of weakly acidic
ion-exchange resin eluting with water. The free acid 5 was obtained in 71%
yield. Upon treatment with O-benzotriazol-1-yl-N,N,N0,N0-tetramethyluronium
tetrafluoroborate (BTTU) in DMF, the carboxy group of 5 was activated
and could be directly coupled with the amino groups of p97 in phosphate
buffer solution (PBS), pH 7.4 room temperature 12 hr. The dox-p97 conjugate
6 is purified by dialysis against PBS, pH ¼ 7.4. Molecule substituted ratio
(MSR) is to be 8.2 as measured by UV-VIS method as described previously.[3]
The protein recovery is 91%. The conjugate is characterized by FPLC, SDS–
PAGE, and Western-blotting, which demonstrated that the conformation of
p97 is preserved. In vitro and in vivo analyses are currently under study.
EXPERIMENTAL SECTION
Reagents were purchased or obtained from commercial sources and used as
received. Proton and carbon nuclear magnetic resource spectra were obtained on
a Bruker AC 300 spectrometer at 300 MHz, using tetramethylsilane as the
internal standard. Infrared spectra were run on a Perkin Elmer Spectrum 1000
spectrometer. UV-VIS spectra were recorded on a Beckman DU640 photo
diode array spectrophotometer (instrumental precision +2 nm) in the solvents
indicated. IR spectra were recorded on a Shimadzu FTIR 8400. FPLC was
run in a AKTA PurifierTM FPLC (using UNICORNTM version 3.10 as operation
system, obtained from Amersham Pharmacia Biotech) using Mono Qw HR
10/10 ion-exchange column (from Pharmacia Biotech Inc.) with PBS buffer
(0.01 M, pH ¼ 7.4) and 1 M NaCl–0.001 M PBS buffers as the mobile phases
or using BIOSEPTM 300 size exclusion column (from Phenomenex, Inc) and
0.01M PBS buffer (pH ¼ 6.80). Elemental analyses, high and low resolution
mass spectra were obtained from Department of Chemistry, the University of
British Columbia. Thin-layer chromatographic (TLC) analysis was performed
on 1 ꢀ 3 in. Whatman Silica Gel GE plates (250m thick). The TLC plate was
visualized by UV light (254nm) and/or staining with 20% phosphomolybdic
acid reagent. Doxorubicin HCl salt was purchased from Albany Molecule
Research Inc, New York. Dialysis Cassettes Slide-A-Lyzer (10K, 10,000
MWCO), Dialysis Tubing SnakSkinTM (10,000 MWCO), and SMCC were
purchased from Biolynx Inc., Brockville, Ontario.

Synthesis of Dox-p97 Conjugates Through SMCC Linker
2411
Dox–SMCC (3). In a 250-mL round-bottomed flask containing a mag-
netic stirrer bar, anhydrous DMF (150 mL), doxorubicin HCl salt (1, 795 mg,
1.371 mmol), SMCC (2, 550 mg, 1.645 mmol), and diisopropylethylamine
(0.36 mL, 2.057 mmol) were added. The reaction flask was wrapped with
aluminum foil and the mixture was stirred at room temperature overnight
under nitrogen. The solvent was removed under vacuum at 35–408C. The resi-
due was taken up by dichloromethane (200 mL) and washed with brine
(50 mL), followed by drying over anhydrous magnesium sulfate. The solids
were filtered, the filter cake was washed with dichloromethane (3 ꢀ 50 mL),
and the filtrate and washings were combined and concentrated under vacuum
until viscous oil was obtained. The residue was purified by silica gel chroma-
tography eluting with 0–5% methanol/methylene chloride to yield a red solid
(998 mg, 96%): m.p. 162–1728C; [a]2D5 þ 189.208 (c 0.12, CH2CI2); ESI MS
m/z 761 [C39H42N2O14 2 H]2; Rf 0.31 (94/6 CH2CI2/MeOH); UV (CH2CI2)
lmax 202, 233, 252, 288, 478, 495 nm. 1H NMR (300 MHz, DMSO-d6)
d ¼ 0.83 (m, 2H), 1.19 (m, 4H), 1.63 (m, 4H), 1.84 (m, 1H), 2.18 (m, 3H),
2.92 (d, J ¼ 12 Hz, 2H), 3.19 (d, J ¼ 6.8 Hz, 2H), 3.92 (m, 1H), 3.97
(s, 3H), 4.14 (q, J ¼ 6.6 Hz, 1H), 4.57 (s, 2H), 4.77 (d, J ¼ 5.6 Hz, 1H),
4.91 (m, 1H), 5.21 (s, 1H), 5.44 (s, 1H), 5.77 (s, 1H), 6.99 (s, 2H), 7.40
(d, J ¼ 8.0 Hz, 1H), 7.62 (dd, J ¼ 3.3, 3.4 Hz, 1H), 7.90 (m, 2H), 13.26
(s, 1H), 13.99 (s, 1H) ppm; 13C NMR (75 MHz, DMSO-d6) d 16.9, 28.3,
28.5, 29.3, 29.6, 31.9, 36.0, 36.3, 43.0, 43.4, 44.6, 54.8, 56.5, 63.6, 66.7,
68.0, 69.9, 74.9, 100.4, 110.5, 110.7, 118.9, 119.6, 119.9, 133.9, 134.2,
134.6, 135.4, 136.1, 154.4, 156.0, 160.7, 171.1, 174.2, 186.4, 213.6 ppm.
.
Anal. Calcd for C39H42N2O14 0.5CH2C12: C, 58.92; H, 5.38; N, 3.48.
Found: C, 59.14; H, 5.49; N, 3.26.
Dox–SMCC–S-acetic acid ammonium salt 4. To a 500-mL round-bot-
tom flask containing a magnetic stirrer bar was added CH2Cl2 (300 mL), dox–
SMCC (3, 980 mg, 1.285 mmol), Hu¨nig’s base (0.34 mL, 1.927 mmol), and
2-mercaptoacetic acid (0.14 mL, 1.927 mmol). The reaction flask was wrapped
with aluminum foil and the mixture was stirred at room temperature for 2 hr
under nitrogen, then concentrated under vacuum. The residue was taken up
in methanol (30 mL) and the resulting methanol solution was re-concentrated.
Precipitation occurred during the concentration. However, the slurry was
further concentrated until about 3 mL of total volume remained. The solid
was collected by vacuum filtration and washed with i-PrOH/EtOAc
(3 ꢀ 3 mL) to yield 1.178 g (96%) of the ammonium salt as a red solid:
m.p. 141–1508C; [a]2D5 þ 2l8.98 (c, 0.25, MeOH/EtOAc, 2/1 v/v); ESI MS
m/z ¼ 853 [C49H65N3O16S–C8H19N 2 H]2; Rf 0.43 (88/10/2 CH2CI2/
1
MeOH/AcOH); UV (CH2Cl2) lmax 234, 251, 288, 400, 477 nm; H NMR
(300 MHz, DMSO-d6) d ¼ 0.87 (m, 2H), 1.11–1.23 (m, 19H), 1.37–1.65
(m, 6H), 1.84 (m, 1H), 2.05–2.25 (m, 3H), 2.60 (d, J ¼ 3.5 Hz, 1H),

2412
Chen and Gabathuler
2.84–3.05 (m, 3H), 3.10–3.20 (m, 3H), 3.35–3.55 (m, 4H), 3.95 (m, 4H),
4.05 (m, 1H), 4.16 (q, J ¼ 6.6 Hz, 1H), 4.57 (s, 2H), 4.85 (m, 1H), 5.21
(s, 1H), 5.45 (br, 1H), 7.40 (d, J ¼ 8.0 Hz, 1H), 7.62 (dd, J ¼ 3.3, 3.4 Hz,
1H), 7.85 (m, 2H), 13.25 (br, 1H), 13.99 (s, 1H) ppm. 13CNMR (75 MHz,
DMSO-d6) d 17.4, 18.6, 28.8, 28.9, 29.7, 33.8, 35.7, 43.8, 44.4, 45.1, 48.9,
56.9, 64.0, 67.1, 68.6, 70.3, 75.3, 100.8. 100.9, 111.1, 119.3, 120.1, 120.3,
134.9, 135.8, 136.5, 154.8, 156.4, 161.1, 171.0, 174.6, 175.7, 177.1, 186.7,
.
186.8, 214.1 ppm. Anal. Calcd for C49H65N3O6S 3H2O: C, 56.69; H,
6.89; N, 4.05. Found: C, 56.62; H, 6.86; N, 4.17.
Dox–SMCC–S-acetic acid 5. A sample of the dox–SMCC–S-acetic
acid ammonium salt (4, 242 mg) was dissolved in MeOH (2 mL). The solution
was loaded onto Dowex weakly acidic ion-exchange resin (8.34 g), pre-
packed in a column (1.6 ꢀ 32 cm2) and eluted with water. The red aqueous
solution was extracted with CH2C12 (3 ꢀ 60 mL). The extracts were com-
bined and dried over Na2SO4. The solids were vacuum filtered, and the filter
cake was washed with CH2Cl2 (3 ꢀ 10 mL). The filtrate and washings
were combined and the solvent was completely removed under vacuum to
afford 150 mg (71%) of the free acid as a red solid: m.p. l43–1508C;
[a]2D5 þ 202.508 (c, 0.12, MeOH/CH2C12, 1/1, v/v); ESI MS m/z ¼ 877
[C41H46N2O6S þ Na]; Rf 0.29 (88/10/2 CH2Cl2/MeOH/AcOH); UV
1
(MeOH/CH2CI2, 1/1, v/v) lmax 203, 234, 252, 286, 478, 495, 529 nm; H
NMR (300 MHz, DMSO-d6) d ¼ 0.86 (m, 2H), 1.18 (m, 5H), 1.35–1.65
(m, 1H), 1.61–1.67 (m, 3H), 1.85 (m, 1H), 2.00–2.25 (m, 3H), 2.50
(m, 2H), 2.90 (q, J ¼ 12.5 Hz, 2H), 3.17–3.20 (m, 3H), 3.38–3.46 (m, 3H),
3.62 (d, J ¼ 15 Hz, 1H), 3.96 (s, 4H), 4.05 (s, 1H), 4.14 (s, 1H), 4.58
(s, 2H), 4.62 (s, 1H), 4.75 (s, 1H), 4.91 (s, 1H), 5.21 (s, 1H), 5.35 (s, 1H),
7.28 (d, J ¼ 10 Hz, 1H), 7.58 (d, J ¼ 5 Hz, 3.4 Hz, 1H), 7.85 (m, 2H), 12.69
(br, 1H), 13.19 (s, 1H), 13.90 (s, 1H) ppm; 13C NMR (75 MHz, DMSO-d6)
d ¼ 16.9, 28.4 28.5, 29.2, 32.6, 35.2, 35.3, 43.4, 44.0, 44.7, 56.5, 63.6,
66.7, 68.1, 69.8, 74.9, 100.3, 110.5, 110.6, 119.6, 119.9, 133.9, 134.5, 135.4,
154.4, 155.9, 160.7, 170.5, 174.2, 175.0, 176.4, 186.2, 186.3, 213.4 ppm;
Anal. Calcd for C41H46N2O16S: C, 57.60; H, 5.42; N, 3.28. Found: C, 57.87;
H. 5.59; N, 2.97.
Synthesis of Dox-p97 Conjugate 6
A mixture of dox–SMCC–S-acetic acid 5 (6.9 mg, 7.826 mmol, 50 equiv.
of p97), BTTU (6.3 mg, 0.01956 mmol, 2.50 equiv. of 5), triethylamine
(0.04 mL), DMF (1 mL) was stirred at room temperature for 60 min. TLC
confirmed the reaction completed (MeOH/CH2Cl2, v/v, 5/95). The solution
is saved for the following reaction.

Synthesis of Dox-p97 Conjugates Through SMCC Linker
2413
p97 (c ¼ 1.23 mg/mL, 12 mL, 1.56 ꢀ 1024 mM) was added to a 50 mL
flask. To this solution, BTTU-activated doxorubicin compounds 5 prepared
from the above (1 mL, 7.8 mmol, 50 equiv. of p97) mixed with 3.7 mL
DMF (ꢀ28% total concentration) was added over a period of 2 min under
vigorous stirring. The mixture was then stirred at room temperature and moni-
tored by FPLC (Mono Q column, wavelength: 280 and 480 nm). It was found
that after 20 hr at room temperature all starting p97 was converted to the
conjugate. During the first few hours, FPLC showed several peaks that
were assigned to the starting p97, and conjugates with different ratio of doxo-
rubicin conjugated to p97. The conjugate peaks finally became to one
major peak after 20 hr at room temperature. The product was then purified
by dialysis using SnakeSkinTM tubing (MWCO ¼ 10 K) against PBS
(10 mM, pH ¼ 7.4) to give the expected conjugate 6 with MSR 8.2, protein
recovery 91%.
ACKNOWLEDGMENTS
We are grateful to Albany Molecular Research Inc for the preparation
of some of the starting materials, Ms. Grace Laliberte, Dr. Pascale Tiger,
Dr. Malcolm Kennard, Dr. Gavin Arthur for help discussions and part of
analytic work.
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