Synthesis of camptothecin melantotransferrin (p97) conjugate through a carbamate bond

Article abstract of DOI:10.1081/SCC-120039495

Through its 20-hydroxy group and a carbamate bond, 20(S)-camptothecin (1) is chemically linked to melanotransferrin (p97). The key step to modify the steric hindered hydroxy group of camptothecin is achieved by using triphosgene/DMAP in DMF and immediately reacting with tert-butyl 6-aminohexanoate (8). The novel camptothecin conjugate 4 is a potential agent for chemotherapy of brain tumor.

Full text of DOI:10.1081/SCC-120039495

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Synthesis of Camptothecin
Melantotransferrin (p97)
Conjugate Through a
Carbamate Bond
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): Synthesis of
Camptothecin Melantotransferrin (p97) Conjugate Through a Carbamate Bond,
Synthetic Communications: An International Journal for Rapid Communication of
Synthetic Organic Chemistry, 34:13, 2415-2424
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SYNTHETIC COMMUNICATIONSw
Vol. 34, No. 13, pp. 2415–2424, 2004
Synthesis of Camptothecin
Melantotransferrin (p97) Conjugate
Through a Carbamate Bond
#
*
Qingqi Chen and Reinhard Gabathuler
Biomarin Pharmaceutical Inc., Novato, California, USA
ABSTRACT
Through its 20-hydroxy group and a carbamate bond, 20(S)-camptothecin
(1) is chemically linked to melanotransferrin (p97). The key step to
modify the steric hindered hydroxy group of camptothecin is achieved
by using triphosgene/DMAP in DMF and immediately reacting with
tert-butyl 6-aminohexanoate (8). The novel camptothecin conjugate 4 is
a potential agent for chemotherapy of brain tumor.
Key Words: Camptothecin; Melanotransferrin; Brain tumor; Blood–
brain barrier; Drug delivery; Bioconjugate.
*
Correspondence: Qingqi Chen, Biomarin Pharmaceutical Inc., 46, Galli Drive,
Novato, CA 94949, USA; Fax: 415-382-0113; E-mail: qchen@bmrn.com.
#Fax: 415-382-7427; E-mail: rgabathuler@bmrn.com
2415
DOI: 10.1081/SCC-120039495
0039-7911 (Print); 1532-2432 (Online)
www.dekker.com
Copyright # 2004 by Marcel Dekker, Inc.

2416
Chen and Gabathuler
INTRODUCTION
Drug delivery technologies, which target reducing the toxicity of drugs,
increasing their absorption, and improving their release profile and efficiency,
are receiving increasing attentions during recent years. To deliver drugs across
the blood–brain barrier (BBB) is one of the most challenging problems in
drug delivery research.[1–3] Melanotransferrin (p97) has been found to loca-
lize in capillary endothelial cells of the human brain and to play an important
role in the transport of iron across the BBB.[4] This provides a base for a
vector-mediated approach to delivery of therapeutics to the brain. We previ-
ously reported the synthesis of doxorubicin-p97 conjugates and their use for
brain drug delivery.[5] To explore the application of p97, we are interested
in using it as a carrier to deliver camptothecin into the brain so as to target
tumors in central nervous system.
20(S)-Camptothecin (1), first isolated by Wall et al. in 1966 from the
Chinese tree Camptotheca acuminata,[6] was the first member of a group of
pentacyclic alkaloid anticancer agents, and is still the research area receiving
intensive attention because of their ability to selectively inhibit DNA topo-
isomerase I and because of their broad range of clinical activity against
solid tumors.[7] However, the therapeutic efficacy of 20(S)-camptothecin is
limited in the treatment of human tumors by the instability of the active
lactone form due to preferential binding of the carboxylate to serum albumin
and by difficulty in formulation due to the sparing aqueous solubility. One
approach to circumvent those problems is to covalently link the 20-hydroxy
group of 20(S)-camptothecin via different spacer arms to polymers such as
poly-(L-glutamic acid),[8] PEG,[9] and polyamide.[10] However, those biocon-
jugates have not shown the capacity to cross the BBB. In this paper, we report
the synthesis of camptothecin p97 conjugate through the 20-hydroxy group
and a carbamate bond, which is a hydrolysable linkage.
RESULTS AND DISCUSSIONS
The hydroxy group of camptothecin is the only functional group that can
be used for the bioconjugation synthesis. However, this hydroxyl group is
steric hindered, less active, and usually gives poor yields upon esterification.
We use triphosgene to activate the hydroxy group, a procedure reported by
Greenwald et al.,[11] in which 1 is dissolved in anhydrous DMF and treated
with triphosgene and N,N0-dimethylaminopyridine (Sch. 1). The phosgene-
activated camptothecin intermediate is then treated in situ with tert-butyl
6-aminohexanoate to give the corresponding ester 2 in 87% yield. Deprotec-
tion of tert-butyl ester of 2 affords the expected acid 3 in 97% yield. The

Synthesis of Camptothecin p97 Conjugate
2417
Scheme 1. Reagents and conditions: a—DMF/DMAP, triphosgene, tert-butyl 6-
aminohexanoate (8), 87%; b—TFA, 97%; c—DMF, BTTU, room temperature, 2 hr,
then p97 in PBS, pH ¼ 7.4, room temperature, 12 hr. Molecules substituted ratio
(MSR): 8.5; protein recovery: 93%.
carboxy group in compound 3 is activated via a reaction with O-benzotriazol-
yl-N,N,N0,N0-tetramethyl-uronium tetrafluoroborate (BTTU) in DMF, and
then coupled with protein p97 in PBS buffer pH ¼ 7.4. The reaction was
monitored by fast protein liquid chromatography (FPLC) using a MonoQ
10TM ion exchange column and was found to be complete after 6 hr at room
temperature. The conjugate was purified by dialysis using SnakeSkinTM
tube against PBS buffer pH 7.4.
The molar ratio of camptothecin to p97 was measured by UV-VIS as
described previously.[4] The conjugate 4 is also analyzed by SDS–PAGE on
reduced and nonreduced gel; and Western blotting using L235 antibody on
reduced and nonreduced gel, which showed 4 as a pure compound with mole-
cular weight of ꢀ88,000 D, and the conformation of p97 is being preserved.
The protected amino acid 8 was synthesized as shown in Sch. 2. The reac-
tion of 6-aminohexanoic acid with benzyl chloroformate in a aqueous sodium
hydroxide gave the corresponding N-benzoylcarbonyl protected amino acid 6
in 95% yield.[12] Esterification of 6 with tert-butyl alcohol using diisopropyl-
carbodiimide (DIPC) as catalyst yielded the expected tert-butyl ester 7, which
is converted to the target compound 8 in 95% yield under catalytic hydrogen-
ation using 10% Pd–C in THF.

2418
Chen and Gabathuler
Scheme 2. Reagents and conditions: a—4 N NaOH, benzyl chloroformate, room temp-
erature, 20 min, 95%. b—CH2Cl2/DIPC, tert-butyl alcohol, room temperature, 12hr,
89%. c—Pd–C/H2, THF, room temperature, 12 hr, 95%.
EXPERIMENTAL SECTION
Camptothecin was purchased from Qventas, Inc. Dialysis Cassettes Slide-
A-Lyzer (10 K, 10,000 MWCO), Dialysis Tubing SnakeSkinTM (10,000
MWCO), were purchased from Pierce, Inc. All other reagents and solvents
were purchased from Aldrich, Sigma, and VWR, and used as received. The
silica gel used in flash chromatography was Merck silica gel 60, 230–
400 mesh, whilst Rf values were measured on Merck silica TLC aluminum
sheets (silica gel 60F254). Melting points were determined on a Thomas hot
1
stage or Buchi apparatus and are uncorrected. H and 13C NMR spectra
were recorded on Bruker AC-200, AMX-400, or AMX-500 MHz instruments.
APT was recorded on AMX-400 instruments. 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, Amersham Pharmacia Bio-
tech) using Mono QR HR 10/10 ion exchange column (from Pharmacia Bio-
tech 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 size exclusion column (from
Phenomenex, Inc.), and 0.01 M PBS buffer (pH ¼ 6.80). Elemental analyses,
high and low resolution mass spectra were obtained from Department of
Chemistry, University of British Columbia.
N-Benzyloxylcarbonyl-6-aminocaproic acid [6, C14H19NO4, FW ¼
265]. In a 1-L three-necked round-bottomed flask, equipped with two
addition funnels, magnetic stirrer, 6-aminocaproic acid (5, 60 g, 0.435 mol),
and sodium hydroxide (2 M, 300 mL) were added. The mixture was stirred

Synthesis of Camptothecin p97 Conjugate
2419
and the solution was cooled to 08C using an ice–salt bath. Benzyl chlorofor-
mate (95%, 65 mL ¼ 78 g, 0.435 mol, 1.0 equiv.) and sodium hydroxide (4 M,
112.5 mL, 0.435 mol, 1.0 equiv.) were placed in two addition funnels, respect-
ively, and added dropwise at the same speed to the vigorously stirred solution
over a period of 30 min. The mixture was stirred for an additional 20 min and
then extracted with ether (3 ꢀ 100 mL). The aqueous phase was acidified to
pH ¼ 3.0 using conc. HCl between 08C and 108C. The precipitate was filtered,
washed with small portions of cold water, and dried in the air, then under
vacuum overnight to yield the expected product 6 as white powder (109.5 g,
95%). M.P. 78–808C. IR: n ¼ 3330, 2943, 2874, 2607, 1722, 1391, 1531,
1483, 1454, 1415, 1342, 1257, 1225, 1163, 1141, 1105, 1035, 1006, 947,
908, 840, 783, 750, 727, 638 cm21 1H NMR (DMSO-d6, 200 MHz),
.
d ¼ 1.25 (m, 6H, 3CH2), 2.05 (t, J ¼ 7.0 Hz, 2H, CH2), 3.05 (t, J ¼ 7.0 Hz,
2H, CH2), 5.20 (s, 2H, OCH2), 7.25 (m, 5H, 5 phenyl-H), 12.05 (brs 1H,
COOH) ppm. 13C NMR (DMSO-d6, 50 MHz), d ¼ 24.5 (CH2), 26.6 (CH2),
30.3 (CH2), 35.5 (CH2), 44.7 (CH2), 67.8 (OCH2), 127.5, 127.8, 128.5,
140.2, 157.6 (COO), 176.5 (COO) ppm. MS (EI), m/z ¼ 266 (Mþ þ 1).
Anal. calcd for C14H19NO4 (265.13): C, 63.38; H, 7.22; N, 5.28. Found: C,
63.12; H, 7.32; N, 5.07.
tert-Butyl N-benzyloxylcarbonyl-6-aminocaproate [7, C18H27NO4,
FW ¼ 321]. In a 250-mL round-bottomed flask, equipped with a magnetic
stirrer, N-benzyloxycarbonyl-6-aminocaproic acid (6, 30 g, 113 mmol),
dichloromethane (150 mL), N,N0-dimethylaminopyridine (2.80 g, 23.6 mmol,
0.2 equiv.), and dry tert-butanol (21 mL, 226 mmol, 2.0 equiv.) were added.
The mixture was stirred and cooled to 08C by an ice–salt bath. Then, a
solution of DIPC (20 mL, 124 mmol, 1.1 equiv.) in dichoromethane (50 mL)
was added dropwise over 5 min. White solid (N,N0-diisopropylurine) was
observed after 10 min. The mixture was stirred at room temperature for
12 hr. The solid was removed by suction filtration. The filtrate was taken
by dichloromethane (300 mL), and was partitioned by 0.1 M aqueous
hydrochloric acid (100 mL). The organic phase was separated and was washed
successively with 0.1 M aqueous hydrochloric acid (100 mL ꢀ 3) and brine
(100 mL). After drying over anhydrous sodium sulfate and removal of the
solvent, the oily residue was purified by a short silica gel column
(3.5 cm ꢀ 7.5 cm) and eluated with dichloromethane. After removal of the
solvent and drying under vacuum, compound 7 was obtained as an oily
light yellow product (32.50 g, 89%). IR: n ¼ 3310, 2870, 1718, 1648, 1591,
1421, 1397, 1274, 1201, 1128, 1080, 1024, 995, 955, 882, 761, 740 cm21
.
1H NMR (CDCl3, 200 MHz), d ¼ 1.50 (s, 9H, 3CH3), 1.60 (m, 6H, 3CH2),
2.20 (t, J ¼ 7 Hz, 2H, CH2), 3.20 (t, J ¼ 7 Hz, 2H, CH2), 4.95 (brs, 1H,
NH), 5.05 (s, 2H, CH2), 7.25 (s, 5H, 5-phenyl-H) ppm. 13C NMR (CDCl3,
50 MHz), d ¼ 22.5 (CH2), 27.6 (CH2), 29.9 (CH3), 31.3 (CH2), 35.2 (CH2),

2420
Chen and Gabathuler
43.7 (CH2), 66.8 (OCH2), 80.3 (OC(CH3)3), 126.5, 127.5, 129.5, 139.2, 156.6
(COO), 173.5 (COO) ppm. MS(EI), m/z ¼ 322 (Mþ þ 1). Anal. calcd for
C18H27NO4 (321.41): C, 67.26; H, 8.47; N, 4.36. Found: C, 67.12; H, 8.22;
N, 4.57.
tert-Butyl 6-aminocaproate [8, C10H21NO2, FW ¼ 187]. In a 500-mL
three-necked round-bottomed flask, equipped with a hydrogen balloon, a
vacuum adaptor, a magnetic stirrer, tert-butyl N-benzyloxycarbonyl-6-amino-
caproate (7, 11.87 g, 37 mmol) and palladium on active carbon (10%, 0.7 g)
were added and followed by anhydrous THF (250 mL) and triethylamine
(1 mL). The mixture was stirred. The air in the flask was removed by water-
aspirator pump, and the flask was then filled with hydrogen from the balloon.
This procedure was repeated three times to make sure that the flask was com-
pletely filled with hydrogen. The mixture was stirred at room temperature for
12 hr. The hydrogen balloon was re-filled several times when it became small.
The hydrogen balloon and vacuum adaptor were removed from the flask. The
catalyst was removed by filtration. The filtrate was evaporated under vacuum to
dryness. The oily residue was then dried under high vacuum to yield compound
8 as a light yellow oily product (6.5 g, 95%). IR: n ¼ 3320, 2929, 2860, 1726,
1
1568, 1458, 1365, 1253, 1149, 846, 817, 734, 584 cm21. H NMR (CDCl3,
200 MHz), d ¼ 1.50 (s, 9H, 3CH3), 1.60 (m, 6H, 3CH2), 2.20 (t, J ¼ 7 Hz,
2H, CH2), 2.80 (t, J ¼ 7 Hz, 2H, CH2), 3.20 (brs, 2H, NH2) ppm. 13C NMR
(CDCl3, 50 MHz), d ¼ 23.5 (CH2), 26.8 (CH2), 29.7 (CH3), 30.3 (CH2), 35.7
(CH2), 44.5 (CH2), 67.8 (OCH2), 80.1 (OC(CH3)3), 172.4 (COO) ppm. MS
(EI), m/z ¼ 188 (Mþ þ1). Anal. calcd for C10H21NO2 (187.15): C, 64.13;
H, 11.30; N, 7.48. Found: C, 63.95; H, 11.02; N, 7.57.
tert-Butyl N-carbonyl-(20-(S)-O-camptothecin)-6-aminocaproate [2,
C31H35N3O7, FW ¼ 561]. In a 500-mL round-bottomed flask, equipped
with a magnetic stirrer and reflux condenser with a anhydrous calcium chlor-
ide drying tube at the top, 20-(S)-camptothecin (1.392 g, 4.0 mmol), triphos-
gene (450 mg, 1.486 mmol, 0.37 equiv.), and dichloromethane (new opened
bottle, 150 mL) were added. The mixture was stirred, and then a solution
of 4-N,N0-dimethylaminopyridine (1.56 g, 12.78 mmol, 3.2 equiv.) in dry
dichloromethane (50 mL) was added dropwise over a period of 10 min. The
suspension was observed to form a yellow color at first, then pale, and finally
slightly red. The solid was gradually dissolved after the addition is com-
pleted. The reaction was monitored by TLC (MeOH/DCM, 10/90,
Rf ¼ 0.7 for the product, and Rf ¼ 0.55 for the starting material), and was
confirmed to finish after 30 min. To this mixture, a solution of tert-butyl
6-aminocaproate (8, 1.20 g, 6 mmol, 1.50 equiv.) was added at once. The mix-
ture was allowed to stir overnight at room temperature. The solution was
mixed with dichloromethane (100 mL) and washed with 0.1 M HCl
(6 ꢀ 150 mL), brine (200 mL). After drying over anhydrous sodium sulfate,

Synthesis of Camptothecin p97 Conjugate
2421
the solvent was removed under vacuum. The yellow residue was taken
up by ether (10 mL), and then precipitated by dropwise adding
hexane (ꢀ100 mL). The solid product was collected by suction filtration
and washed by cold hexane (2 ꢀ 20 mL) to yield the expected product 2
(1.91 g, 87%). M.P. 98–1028C. IR: n ¼ 3310, 2937, 2868, 1816, 1735,
1657, 1502, 1444, 1411, 1367, 1352, 1248, 1155, 1083, 1018, 952, 916,
848, 763, 788, 725, 594 cm21. UV-Vis (DMSO), lmax (1) ¼ 370 (18
900) nm. 1H NMR (CDCl3, 400 MHz), d ¼ 1.05 (t, J ¼ 7 Hz, 3H, CH3),
1.45 (m, 11H, CH2, 3CH3 from tert-butyl), 1.6–1.7 (m, 4H, 2CH2), 2.25
(m, 2H, CH2), 2.45 (m, 2H, CH2), 3.60 (t, J ¼ 7 Hz, 2H, CH2), 5.00–5.40
(m, 4H, CH2, OCH2), 7.65 (m, 1H), 7.78 (s, 1H, NH), 7.84 (t, J ¼ 1 Hz,
1H), 7.95 (t, J ¼ 7 Hz, 1H), 8.20 (d, J ¼ 8 Hz, 1H), 8.40 (d, J ¼ 6 Hz,
1H) ppm. 13C NMR (CDCl3, 100 MHz), d ¼ 7.8 (CH3), 25.7 (CH2), 25.6
(CH2), 26.3 (CH2), 29.1 (CH3), 29.5 (CH2), 34.5 (CH2), 42.5 (CH2N), 50.2
(CH2N), 65.2 (OCH2), 72.5 (20-C), 79.1 (OC(CH3)3), 96.4, 118.5, 119.1,
126.2, 128.3, 130.1, 130.9, 145.4, 146.4, 149.7, 150.1, 152.0, 153.5
(C55N), 155.5 (C55O), 156.6 (C55O), 172.4 (C55O), 173.5 (C55O) ppm.
LSIMS (3-NBA), m/e ¼ 562 (Mþ), 506 (Mþ-butyl), 488, 331. Anal. calcd
.
for C31H35N3O7 1/2H2O (571): C, 65.21; H, 6.36, N, 7.36. Found: C,
65.01; H, 6.02; N, 7.07.
N-Carbonyl-(20-(S)-O-camptothecin)-6-aminocaproic acid trifluoro-
.
.
acetic acid salt [3 TFA, C27H27N3O7 CF3CO2H, FW ¼ 619.54]. (20-(S)-
O-camptothecin)-6-aminocaproate (2, 1.50 g, 2.67 mmol) was stirred with TFA
(10mL)atroomtemperaturefor20 min. Solventwasthenremovedundervacuum
at room temperature. The yellow residue was taken up by dichloromethane (5 mL)
and then precipitated by dropwise adding hexane (ꢀ100 mL). The solid product
was collected by suction filtration and washed by cold hexane (2 ꢀ 20 mL) to
yield the expected product 3 as yellow powder (1.60 g, 97%). M.P. 123–1258C.
IR: n ¼ 3320, 2840, 1728, 1618, 1581, 1411, 1393, 1284, 1207, 1118, 1070,
1014, 985, 955, 761, 740 cm21. UV-Vis (DMSO), lmax (1) ¼ 370 (17,500)nm.
1H NMR (CDCl3, 400MHz), d ¼ 1.05 (t, J ¼ 7.20 Hz, 3H, CH3), 1.35 (m, 2H,
CH2), 1.5–1.7 (m, 4H, 2CH2), 2.25 (m, 2H, CH2), 2.40 (m, 2H, CH2), 3.20
(m, 2H, CH2), 5.35 (s, 2H, CH2), 5.40, 5.65 (dd, J ¼ 4.77 Hz, 17 Hz, 2H,
OCH2), 7.45 (d, J ¼ 8 Hz, 1H), 7.70 (t, J ¼ 7 Hz, 1H), 7.85 (t, J ¼ 7 Hz, 1H),
7.95 (d, J ¼ 8 Hz, 1H), 8.33 (d, J ¼ 8 Hz, 1H), 8.50 (s, 1H, NH) ppm. 13C
NMR (CDCl3, 100MHz), d ¼ 7.5 (CH3), 25.2 (CH2), 26.1 (CH2), 26.5 (CH2),
29.3 (CH3), 34.1 (CH2), 42.7 (CH2N), 51.1 (CH2N), 65.1 (OCH2), 71.9 (20-C),
97.2, 119.1, 120.1, 127.2, 129.1, 130.2, 131.1, 146.2, 147.1, 148.9, 150.2,
151.9, 153.1 (C55N), 155.4 (C55O), 157.1 (C55O), 172.6 (C55O), 176.5
(C55O) ppm. LSIMS (3-NBA), m/e ¼ 506 (Mþ þ 1), 488, 331. Anal. calcd for
.
C27H27N3O7 CF3CO2H (619.54): C, 56.22; H, 4.56; N, 6.78. Found: C, 56.01;
H, 4.22; N, 6.57.

2422
Chen and Gabathuler
Synthesis of camptotehtcin-p97 conjugate 4. A mixture of N-carbonyl-
(20-(S)-O-camptothecin)-6-aminocaproic acid (3, 39.5 mg, 0.078 mmol,
50 equiv.), BTTU (37.57 mg, 0.117 mmol, 1.50 equiv. of compound 3), triethyl-
amine (0.04 mL), and DMF (10 mL) were stirred at room temperature for
30 min and monitored by TLC (MeOH/dichloromethane, v/v, 5/95). The
solution was saved for the following reaction.
Melanotransferin (p97) (conc. 1.23 mg/mL, 120 mL, 1.56 ꢀ 1023 mM)
was placed in a 250-mL round-bottomed flask. To this solution, the above pre-
pared camptothecin solution (10 mL, 0.078 mmol, 50equiv. plus 38 mL DMF)
was added under vigorous stirring. The mixture was then gently stirred at room
temperature. The reaction was monitored by FPLC (Mono Q 10/10 column).
It was found that after 30 min, all starting p97 was converted to the product.
The reaction was monitored by FPLC every 60 min, which showed several
peaks during the first few hours, which were protein p97 fractions with differ-
ent amount of compound 3 attached. After 16 hr, the peaks were diminished
and the major one grew long and changes with reaction time, which indicated
the completion of the reaction. The product was purified by dialysis using
SnakeSkinTM tube (MWCO ¼ 10 K) against phosphate buffer solution
(PBS, 10 mM, pH ¼ 7.4). Molar ratio of camptothecin to p97 was 8.2 as
measured by UV-Vis, protein recovery 85%.
ACKNOWLEDGMENTS
The authors are grateful to Ms. Grace Laliberte, Dr. Pascale Tiger,
Dr. Malcolm Kennard, and Dr. Gavin Arthur for help discussions and part
of analytic work.
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Received in the USA March 3, 2004