Synthesis of isotopically labeled versions of L-MTP-PE (mifamurtide) and MDP

Article abstract of DOI:10.1002/jlcr.3078

L-MTP-PE (1), an immunomodulator and its metabolite MDP (4) were synthesized from labeled L-alanine and its protected derivative, respectively. The key intermediate product for the labeled L-MTP-PE synthesis, [ ¹³C₃,D₄]-al

Full text of DOI:10.1002/jlcr.3078

Special Issue Article
Received 01 March 2013,
Revised 17 May 2013,
Accepted 21 May 2013
Published online 9 July 2013 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3078
Synthesis of isotopically labeled versions of
L-MTP-PE (mifamurtide) and MDP†
*
Yuexian Li, Mihaela Plesescu, and Shimoga R. Prakash
L-MTP-PE (1), an immunomodulator and its metabolite MDP (4) were synthesized from labeled L-alanine and its protected
derivative, respectively. The key intermediate product for the labeled L-MTP-PE synthesis, [13C3,D4]-alanyl-cephalin (2A),
was synthesized from [13C3,D4]-L-alanine (3A) in three steps. The key intermediate product for labeled MDP synthesis, amine
11, was prepared from [13C3,15N]-Boc-L-alanine (5A) in two steps.
Keywords: [13C3,D4]-L-alanine; [13C3,D4]-alanyl-cephalin; [13C3,15N]-Boc-L-alanine; [13C3,D4]-L-MTP-PE; [13C3,15N]-MDP
by deprotection of the amino group with deuterium gas in EtOD
Introduction
and CDCl3.3,4 Coupling of 2A and MDP (4) provided the labeled
L-MTP-PE.3,4 Although we did not anticipate any exchange of
L-MTP-PE (1, L-muramyl tripeptide phosphatidyl ethanolamine,
Figure 1) is an immunomodulator with antitumor effects that
deuterium, as a precautionary measure, deuterium labeled
appear to be mediated via activation of monocytes and
reagents (D2, EtOD, and CDCl3) were used in step 3.
macrophages.1L-MTP-PE, also known as MEPACTW (mifamurtide),
After the stable isotope-labeled version of L-MTP-PE was
is indicated in children, adolescents, and young adults for the
synthesized, the internal standard of the major metabolite
treatment of high-grade, resectable, non-metastatic osteosarcoma
(MDP) was required for the bioanalytical studies. To synthesize
after macroscopically complete surgical resection.1 Stable
the stable isotope-labeled version of MDP, commercially
isotopically labeled forms of L-MTP-PE and MDP (4, muramyl
available Boc-L-alanine (5A) labeled with C-13 and N-15 was
dipeptide), the major metabolite of L-MTP-PE, were prepared as
internal standards for LC/MS/MS bioanalytical assays.
chosen to obtain the appropriate increase in amu (Scheme 3).
Amidation of 5A with 9 using isobutyl chloroformate provided
10 in 86% yield.5 The removal of the BOC group of 10 with
1.6-M HCl yielded compound 11 in 88% yield.5 Coupling of 11
with acid 12 produced 13 by using DCC/HOSu in the yield of
98%.3,4 Deprotection of the acetonide on 13 with acetic acid in
water gave 14 in 99% yield.5 To minimize by-product formation,
the deprotection was conducted at a lower temperature (0 ꢀC
during day time and 5 ꢀC during overnight). [13C3,15N]-MDP
(4A) was synthesized via debenzylation of 14 in 67% yield.4,5
Results and discussion
The structure of L-MTP-PE (1) provides two potential units for
labeling: either alanyl-cephalin (2) or MDP (4) (Scheme 1).2
Experimental LC/MS/MS results indicated that labeling either
portion of the molecule would be adequate for the intended
bioanalytical studies. Initial requirement for bioanalytical
assays was labeled L-MTP-PE. To expedite the synthesis, we
chose labeling alanyl-cephalin (2) to prepare the labeled
version of L-MTP-PE. After completion of the synthesis of the Conclusions
labeled L-MTP-PE, we received a request for the labeled MDP, a
In summary, practical methods with excellent yields for the
preparation of stable isotope-labeled versions of L-MTP-PE and
MDP were developed. The key intermediate product for the
major metabolite of L-MTP-PE. Boc-L-alanine (5) was chosen for
the synthesis of the labeled version of MDP (4) (Scheme 1).
A labeled version of L-MTP-PE that is at least 3 amu higher
than the unlabeled version is required to ensure complete
separation of labeled and unlabeled molecular ion clusters
during mass spectrometric assays. To increase the molecular
weight, labeling alanyl-cephalin (2) with deuterium and C-13
was chosen.
Department of Drug Metabolism and Pharmacokinetics, Isotope Chemistry
Group, Millennium Pharmaceuticals, Inc., 40 Landsdowne Street, Cambridge,
MA 02139, USA
To prepare the key intermediate, [13C3,D4]-alanyl-cephalin
(2A), we chose commercially available [13C3,D4]-L-alanine (3A)
as the labeled precursor (Scheme 2). Standard amide coupling
reactions with appropriate protection-deprotection of functional
groups were utilized during the synthesis. Protection of [13C3,D4]-
L-alanine (3A) with benzyl chloroformate provided 6.2
Compound 6 was amidated with cephalin 7 to give 8 by DCC
*Correspondence to: Yuexian Li, Department of Drug Metabolism and
Pharmacokinetics, Isotope Chemistry Group, Millennium Pharmaceuticals, Inc.,
40 Landsdowne Street, Cambridge, MA 02139, USA.
E-mail: Yuexian.Li@mpi.com
† This article is published in Journal of Labelled Compounds and
Radiopharmaceuticals as a special issue on IIS 2012 Heidelberg Conference,
edited by Jens Atzrodt and Volker Derdau, Isotope Chemistry and Metabolite
Synthesis, DSAR-DD, Sanofi-Aventis Deutschland GmbH, Industriepark Höchst
and HOSu (N-hydroxysuccinimide) in DMF.2–4 2A was prepared G876, 65926 Frankfurt am Main, Germany.
J. Label Compd. Radiopharm 2013, 56 475–479
Copyright © 2013 John Wiley & Sons, Ltd.

Y. Li et al.
Stable Isotopes, USA. Compounds described in Scheme 2 were examined
by thin layer chromatography (TLC) on precoated plates of silica gel (J.T.Baker,
Austin, TX, USA) using the solvent systems and visualization method
indicated for each of them. Purities were determined by HPLC (Agilent,
Santa Clara, CA, USA) on Luna C18(2) column (5 mm, 4.6Â 150 mm) eluted
at 1mL/min with A (0.1% formic acid in 99% water and 1% CH3CN) and B
(0.1% formic acid in 5% water and 95% CH3CN). Elution was 0% B for 5 min,
0–100% B over 15 min, and 100% B holding for 5 min. UV detection was set
at 254 nm. NMR spectra were recorded on a Varian (Palo Alto, CA, USA)
300-MHz spectrometer. LC–MS analyses were performed on an Agilent
1100 Series spectrometer and AB Sciex (Framingham, MA, USA) API 4000
LC/MS/MS spectrometer. Column chromatography was carried out using
Teledyne Isco Combiflash Companion purification system (Charlotte, NC,
USA) and Biotage Flash system (Charlotte, NC, USA), and their respective
pre-packed silica gel columns. Preparative HPLC was performed on a Gilson
system (Middleton, WI, USA).
[13C3,D4]-(S)-2-(((Benzyloxy)carbonyl)amino)propanoic acid (6)2
Figure 1. Structures of L-MTP-PE and MDP.
A solution of [13C3,D4]-(S)-2-aminopropanoic acid (3A, 1 g, 10 mmol) in
water (11 mL) and 5-M NaOH (2.24 mL, 11.2 mmol, 1.1 eq) was cooled in
an ice bath. Benzyl chloroformate in toluene (50% technical solution,
3.2 mL, 22.4 mmol, 2 eq) was added slowly and portionwise while
maintaining the internal temperature at about 5 ꢀC. The solution turned
milky white, and it was treated with 2-M NaOH (6 mL, 12 mmol, 1 eq)
while maintaining the temperature below 10 ꢀC. After stirring at room
temperature for 30 min, the solution was made basic by adding 10-M
NaOH and extracted twice with 10 mL of ether. The aqueous solution
was then acidified to pH 3 with concentrated HCl. The product separated
as an oil, which was stored overnight in the refrigerator. The oil was still
present the following day, and it was placed in the freezer (À20 ꢀC) for
solidification. Upon transferring back into refrigerator, a white solid was
formed. After bringing to room temperature, the solid was collected on
a filter funnel, washed with additional water, and dried under high
labeled L-MTP-PE synthesis, [13C3,D4]-alanyl-cephalin (2A), was
synthesized from [13C3,D4]-L-alanine (3A) in three steps. The
key intermediate product for labeled MDP synthesis, amine 11,
was synthesized from [13C3,15N]-Boc-L-alanine (5A) in two steps.
[13C3,D4]-L-MTP-PE (1A) was prepared from [13C3,D4]-L-alanine
in an overall yield of 43%. [13C3,15N]-MDP (4A) was synthesized
from [13C3,15N]-Boc-L-alanine in an overall yield of 49%.
Experimental
General
All commercial reagents were used as supplied unless otherwise noted. vacuum at 50 ꢀC (1.92 g, 70%). 1H-NMR (300 MHz, [D6]-DMSO): d 10.1
[13C3,D4]-L-alanine and [13C3,15N]-Boc-L-alanine were purchased from Isotec
(1H, br), 9.86 (1H, br), 7.54 (5H, m), 5.00 (2H, s).
Scheme 1. Retro-synthesis of L-MTP-PE and MDP.
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J. Label Compd. Radiopharm 2013, 56 475–479

Y. Li et al.
Scheme 2. Synthesis of [13C3,D4]-L-MTP-PE.
Scheme 3. Synthesis of [13C3,15N]-MDP.
J. Label Compd. Radiopharm 2013, 56 475–479
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Y. Li et al.
13C3,D4]-3-(((2-((S)-2-(((Benzyloxy)carbonyl)amino)
dissolved in the solvent system of chloroform : methanol : water
(70:30:5, v/v) and subjected to column chromatography with the same
eluent mixture. The pure fractions were collected and concentrated to
dryness to obtain a white solid (125 mg). This material was redissolved
[
propanamido)ethoxy)(hydroxy)phosphoryl)oxy)propane-1,
2-diyl dipalmitate (8)2–4
[
13C3,D4]-(S)-2-(((Benzyloxy)carbonyl)amino)propanoic acid (6, 192 mg, in 10 mL of water and warmed up to 35 ꢀC. It was treated with 0.8%
0.83 mmol, 1.24 eq) and N-hydroxysuccinimide (109 mg, 0.95 mmol, aqueous sodium acetate solution (5 mL) for 10 min. The final product
1.4 eq) were dissolved in 2.8-mL DMF and chilled in an ice-salt bath. N, was isolated by lyophilization (162 mg, 70%). MRM (m/z (M + 1 + Na)):
N-dicyclohexylcarbodiimide (196 mg, 0.95 mmol, 1.4 eq) was added, and 1244.7. 1H-NMR ([D6]-DMSO, 300 MHz): d 8.30 (2H, d, J = 8.5 Hz), 8.10
the solution was stirred for 5 h during which time the reaction mixture (4H, m), 8.03 (2H, br), 7.97 (2H, d, J = 7.9 Hz), 7.83 (2H, d, J = 7.8 Hz), 7.46
was allowed to come to room temperature. A milky white mixture (2H, s), 6.98 (2H, m), 6.66 (1H, d, J = 7.7 Hz), 6.55 (1H, d, J = 7.6 Hz), 5.43
formed, and it was added dropwise to a solution of 3-(((2-aminoethoxy) (2H, m), 5.06 (2H, m), 4.95 (1H, t), 4.52 (1H, t), 4.27 (3H, t), 4.07 (2H, m),
(hydroxy)phosphoryl)oxy)propane-1,2-diyl dipalmitate (7, 465 mg, 3.61 (10H, m), 3.48 (4H, m), 3.03 (8H, m), 2.26 (4H, m), 1.75 (2H, s), 1.46
0.67 mmol, 1 eq) and triethylamine (0.28 mL, 2 mmol, 3 eq) in chloroform : (2H, br), 1.15 (39H, m), 0.83 (6H, t, J = 1.2 Hz).
methanol (10:4 (v/v)). The mixture was stirred overnight under an
atmosphere of nitrogen, and the progress was monitored by TLC using
[
13C3, 15N]-N-[(1,1-Dimethylethoxy)carbonyl]-L-alanyl-D-a-
glutamine phenylmethyl ester (10)5
the solvent system of chloroform : methanol : water (65:25:4, v/v) and
staining with sulfuric acid : 2.5% ammonium molibdate : 0.85% NaCl
(1:6:5, v/v) and heat. All solvent was removed under high vacuum, and
the residue was dissolved in 20% methanol in chloroform (5 mL) and
purified by column chromatography using a 3–10% gradient of methanol
in chloroform. The pure fractions detected using the TLC solvent system
described earlier were combined to give upon drying the desired
product (593 mg, 98%). 1H-NMR (300 MHz, [D6]-DMSO): d 9.88 (1H, br),
8.35 (1H, br), 7.32 (5H, m), 5.00 (1H, m), 4.98 (2H, d, J = 5.0 Hz), 4.26
(1H, d, J = 4.3 Hz), 4.11 (2H, m), 3.72 (2H, q), 3.62 (2H, m), 3.16 (2H, d,
J = 3.1 Hz), 3.06 (4H, m), 2.25 (4H, t, J = 2.3 Hz), 1.49 (4H, m), 1.17 (44H,
m), 0.85 (6H, t, J = 1.2 Hz).
To a solution of [13C3,15N]-2-[(tert-butoxycarbonyl)amino]propanoic acid
(5A, 1.06 g, 5.5 mmol) in DMF (1.7 mL) and ethyl acetate (3.8 mL) were
added N-methylmorpholine (0.6 mL, 5.5 mmol, 1.0 eq) and isobutyl
chloroformate (0.71 mL, 5.5 mmol, 1.0 eq) dropwise at À10 ꢀC. To the
resulting solution was added a solution of benzyl 4,5-diamino-5-
oxopentanoate. HCl (1.36 g, 5.0 mmol, 0.9 eq) in DMF (10 mL) and
N-methylmorpholine (0.6 mL, 5.5 mmol, 1.0 eq) were added dropwise
and stirred at À5 ꢀC for 30 min. The solvents were removed. To the
resulting residue were added H2O (15 mL) and ethyl acetate
(40 mL). The organic phase was separated, washed with saturated
NaHCO3 (1Â 20 mL) and H2O (2Â 20 mL), and dried with MgSO4.
Crystallization from ethyl acetate/hexanes (1/1, 42 mL) provided
1.95 g (86%) of product. LC–MS/MS (m/z (M À 1)): 410.4, 336.4,
302.4, 228.4. 1H-NMR ([D6]-DMSO, 300 MHz): d 7.92 (1H, m), 7.35
(5H, m), 7.18 (2H, s), 6.93 (1H, d, J = 13.8 Hz), 5.18 (2H, s), 4.18(1H,
m), 3.90 (1H, m), 2.34 (2H, t, J = 8.0 Hz), 2.10 (1H, m), 1.78 (1H, m),
1.35 (9H, s), 1.15 (3H, m).
[
13C3,D4]-3-(((2-((S)-2-Aminopropanamido)ethoxy)(hydroxy)
phosphoryl)oxy)propane-1,2-diyl dipalmitate (2A)3,4
To
a solution of [
13C3,D4]-3-(((2-((S)-2-(((benzyloxy)carbonyl)amino)
propanamido)ethoxy) (hydroxy)phosphoryl)oxy)propane-1,2-diyl dipalmitate
(8, 340 mg, 0.38 mmol) in a mixture of chloroform-d (10 mL) and ethanol-d6
(20 mL) was added 10% palladium on charcoal (300 mg, 0.3 mmol, 0.7 eq)
and platinum oxide (300mg, 1mmol, 3 eq). The mixture was placed under
vacuum, followed by repeated addition and degassing of deuterium gas for
three times. After 90min of stirring under a deuterated atmosphere, TLC
analysis (using the solvent system from previous step) indicated completion
of the reaction. The catalysts were removed by filtration. Upon solvent
removal by high vacuum, the wet product was subjected to freeze-dry
conditions overnight to recover a white solid that was stored at À20 ꢀC
(260mg, 89%). 1H-NMR (300 MHz, [D6]-DMSO): d 9.27 (1H, br), 8.10
(2H, br), 5.13 (1H, s), 4.27 (1H, d, J= 4.3Hz), 4.11 (2H, m), 3.72 (2H, q),
3.62 (2H, m), 3.17 (2H, d, J = 3.1Hz), 3.03 (4H, m), 2.25 (4H, t, J = 2.3Hz),
1.48 (4H, m), 1.21 (44H, m), 0.85 (6H, t, J= 1.2Hz).
[
13C3, 15N]-L-Alanyl-D-a-glutamine phenylmethyl ester
hydrochloride (11)5
To compound 10 (1.89 g, 4.6 mmol) was added 1.6-N HCl in diisopropyl
ether (75.4 mL). The resulting mixture was stirred at room temperature
overnight. The volatiles were removed. To the resulting residue was
added diisopropyl ether (10 mL), and the resulting solution was stirred
for 45 min. The solid was filtered and dried to yield the product (1.41 g,
88%). LC–MS/MS (m/z, (M + 1)): 312.4, 295.2, 267.2, 237.2, 192.0, 137.6.
[
13C3, 15N]-N2-[N-[N-acetyl-4,6-O-(1-Methylethylidene)-1-O-
(phenylmethyl)-a-muramoyl]-L-alanyl-D-a-glutamine
phenylmethyl ester (13)3–5
[
13C3,D4]-Sodium (5S,10S,16R)-16-(((3R,4R,5S,6R)-3-
To a mixture of compound 11 (1.39 g, 4.0 mmol) and compound
12 (1.88 g, 4.44 mmol, 1.11 eq) in ethyl acetate (29 mL) were added
N-hydroxysuccinimide (0.46g, 4.0mmol, 0.9eq, 1.0eq) and triethylamine
(0.59 mL, 4.2 mmol, 1.05 eq) dropwise. A solution of N,N-dicyclohexylcarbodiimide
(1.49 g, 7.2 mmol, 1.8 eq) in ethyl acetate (10 mL) was added dropwise.
The resulting mixture was stirred at room temperature for 2.5 h and
was filtered. The filtrate was washed with 20% citric acid aqueous
solution (20 mL), 5% NaCl aqueous solution (20 mL), 5% NaHCO3
aqueous solution (20 mL), and 20% NaCl aqueous solution (20 mL). The
solvent was removed to provide the product (1.12 g, 98%). LC–MS/MS
(m/z, (M À 1)): 715.2, 607.2, 350.4.
acetamido-2,5-dihydroxy-6hydroxymethyl)tetrahydro-2H-
pyran-4-yl)oxy)-10-carbamoyl-5,13-dimethyl-4,7,12,
15-tetraoxo-3,6,11,14-tetraazaheptadecyl (2,3-bis
(palmitoyloxy)propyl) phosphate (1A)3,4
A mixture of (4S)-4-(2-((R)-2-(((3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-
(hydroxymethyl) tetrahydro-2H-pyran-4-yl)oxy)propanamido)propanamido)-
5-amino-5-oxopentanoic acid (4, 120 mg, 0.24 mmol, 1.3 eq), N,
N-dicyclohexylcarbodiimide (85 mg, 0.41 mmol, 2.3 eq) and
N-hydroxysuccinimide (44 mg, 0.38mmol, 2.1eq) in DMF (8mL) was
stirred at room temperature for 2 h. A solution of [13C3,D4]-3-(((2-((S)-2-
aminopropanamido)ethoxy)(hydroxy) phosphoryl)oxy)propane-1,2-diyl
dipalmitate (2A, 140 mg, 0.18 mmol, 1 eq) and triethylamine (0.1 mL,
0.3 mmol, 4 eq) in t-butanol (22 mL) at 50 ꢀC was added, and the
resulting mixture was stirred at this temperature for 3 h. The reaction
was then cooled to room temperature and analyzed by TLC using the
solvent system of chloroform : methanol : water (70:30:5, v/v) (visualized
with sulfuric acid : 2.5% ammonium molibdate : 0.85% NaCl (1:6:5, v/v)
and heat). The solution was dried under high vacuum. The residue was
[
13C3, 1N]-N2-[N-[N-acetyl-1-O-(Phenylmethyl)-a-muramoyl]-
L-alanyl-D-a-glutamine phenylmethyl ester (14)5
Acetic acid (5.2 mL) was added dropwise to compound 13 (1.18 g,
1.65 mmol) at 0 ꢀC. H2O (5.2 mL) was added to the resulting mixture over
25 min. The reaction mixture was stirred during day time at 0 ꢀC and
allowed to stand overnight at 5 ꢀC for 5 days. The volatiles were removed.
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J. Label Compd. Radiopharm 2013, 56 475–479

Y. Li et al.
The resulting residue was crystallized from 1-butanol/ethanol (1/2, 8 mL) (1H, m), 4.98 (1H, s), 4.50 (2H, m), 4.35 (1H, m), 4.30 (1H, m), 4.18 (2H,
to give the product (1.11 g, 99%). LC–MS/MS (m/z, (M + 1)): 673.2, 595.2, m), 3.65 (3H, m), 3.48 (1H, m), 3.25 (1H, m), 2.24 (2H, t, J = 7.7 Hz), 1.95
437.2. 1H-NMR (300 MHz, [D6]-DMSO): d 8.35 (2H, m), 7.59 (1H, d,
J = 6.8 Hz), 7.35 (11H, m), 7.10 (1H, s), 5.30 (1H, d, J = 8.4 Hz), 5.10 (2H, s),
4.74 (1H, d, J = 3.5 Hz), 4.65 (2H, m), 4.44 (1H, d, J = 12.5 Hz), 4.25 (3H,
m), 3.80 (1H, m), 3.70 (1H, m), 3.50 (3H, m), 2.35 (2H, t, J = 7.9 Hz), 2.0
(2H, m), 1.80 (4H, s), 1.25 (6H, m).
(2H, m), 1.80 (3H, m), 1.70 (1H, m), 1.42 (1H, m), 1.28 (3H, d, J = 6.7 Hz),
1.00 (1H, m).
Acknowledgement
Thanks are due to the Pharmaceutical Operations Department of
Millennium Pharmaceuticals for the provision of unlabeled
intermediates and preparation information.
[
13C3, 15N]-N2-[N-[N-Acetyl-)-a-muramoyl]-L-alanyl-D-a-
glutamine (4A),4,5
To a solution of compound 14 (1.18 g, 1.65 mmol), THF (14.2 mL), and
H2O (4.5 mL) was added 10% Pd/C (0.21 g) at 55 ꢀC. The resulting mixture
was hydrogenated at 55 ꢀC by using a hydrogen-filled balloon for 3 h and
was then filtered through a celite cake. After evaporation, the residue
was purified by preparative HPLC on a C18(2) Luna column (5 mm,
250Â 21.2 mm) eluted at 14 mL/min with A (0.05% CH3COOH in H2O)
and B (0.05% CH3COOH in CH3CN). Elution was 1–10% B over 5 min,
10–100% B over 20 min, and 100% B holding for 30 min. The pure
fractions were combined and freeze-dried to provide the product
(0.55 g, 67%). LC–MS/MS (m/z, (M À 1)): 495.2, 292.5. 1H-NMR (300 MHz,
[D6]-DMSO): d 8.2 (1H, m), 8.08 (1H, m), 7.20 (2H, s), 6.80 (1H, m), 5.25
Conflict of Interest
The authors did not report any conflict of interest.
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