Metabolites of antibody-maytansinoid conjugates: Characteristics and in vitro potencies

Article abstract of DOI:10.1021/mp5007757

Several antibody-maytansinoid conjugates (AMCs) are in clinical trials for the treatment of various cancers. Each of these conjugates can be metabolized by tumor cells to give cytotoxic maytansinoid metabolites that can kill targeted cells. In preclinical

Full text of DOI:10.1021/mp5007757

Article
pubs.acs.org/molecularpharmaceutics
Metabolites of Antibody−Maytansinoid Conjugates: Characteristics
and in Vitro Potencies
Wayne Widdison,* Sharon Wilhelm, Karen Veale, Juliet Costoplus, Gregory Jones, Charlene Audette,
†
Barbara Leece, Laura Bartle, Yelena Kovtun, and Ravi Chari
ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451, United States
ABSTRACT: Several antibody−maytansinoid conjugates
(
AMCs) are in clinical trials for the treatment of various
cancers. Each of these conjugates can be metabolized by tumor
cells to give cytotoxic maytansinoid metabolites that can kill
targeted cells. In preclinical studies in mice, the cytotoxic
metabolites initially formed in vivo are further processed in the
mouse liver to give several oxidized metabolic species. In this
work, the primary AMC metabolites were synthesized and
incubated with human liver microsomes (HLMs) to determine
if human liver would likely give the same metabolites as those formed in mouse liver. The results of these HLM metabolism
studies as well as the subsequent syntheses of the resulting HLM oxidation products are presented. Syntheses of the minor
impurities formed during the conjugation of AMCs were also conducted to determine their cytotoxicities and to establish how
these impurities would be metabolized by HLM.
KEYWORDS: ADC, antibody, conjugate, DM1, DM4, drug, liver, maytansinoid, metabolism, microsome
INTRODUCTION
maytansine possessing a C3 ester side chain bearing a
nonhindered thiol or a dimethyl hindered thiol substituent
respectively, Figure 1. DM1 (2a) and DM4 (2b) have been
conjugated to antibodies (mAbs) via thioether bonds or
disulfide bonds to give antibody−maytansinoid conjugates
■
An antibody−drug conjugate (ADCs) is composed of a
monoclonal antibody that is conjugated to a cytotoxic
compound via a linker.
to an antigen found on the surface of a cancer cell.
Internalization of the ADC−antigen immune complex then
enables the intracellular release of cytotoxic metabolites that kill
the cell. ADCs typically utilize highly cytotoxic compounds
because very little of the administered conjugate reaches its
1
−4
The antibody of the ADC binds
1
1,14,15
(
AMCs).
5
targeted tumor cells in vivo. Antibodies are known to be
6
,7
degraded by cells of the reticuloendothelial system (RES). It
is therefore likely that RES cells degrade the majority of an
administered ADC over time to release cytotoxic metabolites
throughout the body. The cytotoxic metabolites of ADCs
which utilize maytansinoid or auristatin payloads are ultimately
8,9
Figure 1. Structures of maytansine, DM1 and DM4.
excreted primarily by hepatic clearance.
The types of
metabolites released and the reactions that can take place
during liver metabolism can depend on the linkage system
utilized by the ADC.
Several AMCs with the mAb-SPP-DM1 (3a) design were
evaluated in the clinic, and several AMCs with the generalized
structures mAb-SPDB-DM4 (3b), mAb-sulfo-SPDB-DM4
1
0,11
As part of understanding the properties of an ADC, it is
important to determine both the cytotoxicities of metabolites
generated from an ADC and the metabolic fate of any highly
(
3c), and mAb-SMCC-DM1 (4) are currently in clinical trials
16,17
for the treatment of various cancer indications, Figure 2.
1
2
potent impurities that might be present in the ADC.
These AMCs are prepared by attaching DM1 or DM4 to lysine
residues of an antibody via the cleavable disulfide linkers SPP
Maytansinoids, of which maytansine (1) is the parent
compound, are a class of antimitotic agents that are 100- to
1
000-fold more cytotoxic than conventional cancer chemo-
Special Issue: Antibody-Drug Conjugates
therapeutic agents such as methotrexate, daunorubicin, or
1
3
2
vincristine.2 The semisynthetic maytansinoid compounds N ′-
deacetyl-N ′-(3-mercapto-1-oxopropyl)maytansine (2a, DM1)
and N ′-deacetyl-N ′-(4-mercapto-4-methyl-1-oxopentyl)-
maytansine (2b, DM4) are semisynthetic analogues of
Received: November 19, 2014
Revised: March 20, 2015
Accepted: March 31, 2015
2
2
©
XXXX American Chemical Society
A
DOI: 10.1021/mp5007757
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Article
Figure 2. Antibody−maytansinoid conjugates (A) and their utilized linkers (B).
Figure 3. Antibody conjugation to form AMCs and the minor side products produced using the (A) SPP, SPDB, sulfo-SPDB, or (B) SMCC linkers.
(
5a), SPDB (5b), or sulfo-SPDB (5c) or via the noncleavable
the DM1 disulfide dimer side product, DM1 dimer (7), can be
formed. In contrast, DM4 (2b), presumably because of its
hindered thiol, does not generate an appreciable amount of
disulfide dimer during conjugation. These side products and
unreacted components are then removed to varying degrees
during the conjugate purification process, which often involves
size exclusion chromatography (SEC) or tangential flow
filtration (TFF).
linker SMCC (6) to give a thioether-linked conjugate, Figure 3.
The AMC, ado-trastuzumab emtansine (Kadcyla), in which
DM1 is linked to the trastuzumab antibody via the SMCC
linker has been approved for marketing for the treatment of
metastatic HER2-positive breast cancer in the US and other
countries.
The conjugation process can produce small amounts of side
products, such as maytansinoid linked to a hydrolyzed linker,
DM1-TPA (8a), DM1-TBA (8b), DM1-sulfo-TBA (8c), or
DM1-MCC (9). When DM1 (2a) is used to prepare AMCs,
AMCs are administered intravenously and function by
binding to the targeted surface antigen on a cancer cell after
which the AMCs are internalized and processed to release one
B
DOI: 10.1021/mp5007757
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Molecular Pharmaceutics
Article
Figure 4. AMC metabolites generated in tumor or RES cells (primary metabolites).
Figure 5. Mouse liver metabolism of AMC primary metabolites.
or more metabolites which can kill the cell. The initial AMC
processing step has been shown in vitro to involve proteolytic
degradation of the antibody component leaving a lysine residue
of the antibody attached to the linker−maytansinoid moiety as
the RES metabolize AMCs to give the same metabolites as
produced in targeted cancer cells. AMC metabolites formed in
targeted cancer cells or from RES metabolism will be referred
to as primary metabolites. Experiments in mice also indicated
that each of the primary AMC metabolites described is
ultimately routed to the liver where S-methylation of DM1
1
8
shown in Figure 4. Thus, lysosomal degradation of the AMCs
3
a, 3b, and 3c by target cells initially gives the metabolites 10a,
(
2a) or DM4 (2b), or oxidative phase I metabolism to convert
1
0b, and 10c respectively, while degradation of AMC 4 bearing
ε
S-Me DM1 (12a) or S-Me DM4 (12b) to the corresponding
sulfones (14a) and (14b) or sulfoxides (13a) and (13b),
the noncleavable linker gave the Lys-N -SMCC-DM1 (11)
metabolite. The metabolite Lys-N -SPP-DM1 (10a) undergoes
ε
9
respectively, can occur, Figure 5. Disulfide reduction of
further processing via reduction of its disulfide bond,
presumably by intracellular glutathione, to release DM1 (2a).
primary metabolites also appears to occur, but no liver phase II
metabolism other than S-methylation was detected in any of the
AMC-treated mice.
The syntheses and in vitro cytotoxicities of primary AMC
metabolites and results of their incubation with human liver
microsomes (HLMs) are reported herein. Syntheses and
cytotoxicities for both HLM-derived metabolites and the
known conjugation side products produced during the
preparation of the described AMCs are also presented.
ε
The disulfide bonds of the Lys-N -SPDB-DM4 (10b) and Lys-
ε
N -sulfo-SPDB-DM4 (10c) metabolites are reduced in cells to
give DM4 (2b). Intracellular enzymes then S-methylate a
portion of the DM1 or DM4 to give metabolites S-Me-DM1
(
12a) or S-Me-DM4 (12b) with formation of 12a being less
ε
favorable. The Lys-N -SMCC-DM1 (11) metabolite was not
metabolized further. All of the AMC metabolites that have been
described appear to be highly potent against the cells that
produced them. The polar lysine-bearing metabolites 10a, 10b,
EXPERIMENTAL SECTION
1
0c, and 11 are less cytotoxic when added exogenously to cells.
In contrast, the noncharged hydrophobic metabolites DM1
2a), DM4 (2b), S-Me-DM1 (12a), and S-Me-DM4 (12b),
generated in the parent cell, are highly cytotoxic and have the
■
N-4-(Maleimidomethyl)cyclohexane carboxylic acid (9) was
purchased from Speed Chemicals. All other reagents were
obtained from Sigma-Aldrich Chemical Co. or Chem Impex,
unless otherwise stated. All synthetic reactions were conducted
under an argon atmosphere with magnetic stirring. The
compounds SPDB (5b), SPP (5a), DM1 (2a), DM4 (2b),
DM1-TPA (8a), DM4-TBA (8b), and DM1 dimer (7) were
(
19
potential to diffuse into, and kill, neighboring cells.
Plasma samples taken from mice injected with AMCs have
been found to contain the same metabolites as those formed in
9
vitro in tumor cells exposed to AMCs, indicating that cells of
C
DOI: 10.1021/mp5007757
Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics
prepared as previously described. The sulfo-SPDB linker (8c)
Article
15
2
2
Syntheses of Test Compounds. N ′-Deacetyl-N ′-[4-
methyl-4-(3-carboxy-3-sulfopropyldithio)-1-oxopentyl]-
maytansine (DM4-sulfo-TBA, 8c). To a solution of DM4 (2b,
37 mg, 0.047 mmol) in a 2:1 dimethyl sulfoxide:100 mM
potassium phosphate pH 8 buffer (1.2 mL) was added sulfo-
SPDB (5c, 19.2 mg, 0.047 mmol), and the reaction mixture was
stirred for 36 h. Product was purified by preparative HPLC, and
fractions containing desired product (retention time 23 min)
were combined, frozen, and lyophilized to give 5 mg (11%
20
was prepared as previously described. Human liver micro-
somes derived from a pool of ten human liver preparations
were purchased from XenoTech. In vitro cytotoxicities were
determined by clonogenic assays using KB cells purchased from
21
ATCC (ATCC CCl-17) as previously described. Proton
1
magnetic resonance ( H NMR) and carbon magnetic
1
3
resonance spectra ( C NMR) were obtained on a Bruker
Avance 400 spectrometer operating at 400 and 100 MHz,
respectively. The NMR chemical shifts are reported in δ values
relative to the utilized NMR solvent. Figures showing HPLC
traces were acquired with detection at 252 nm.
1
yield) of product as a white solid. H NMR (400 MHz, 2:1
methanol-d :D O): δ 8.21 (s, 1H), 7.17 (dd, J = 5.0, 1.8 Hz,
H), 6.74 (d, J = 11.2 Hz, 1H), 6.69−6.55 (m, 3H), 5.74 (dd, J
15.1, 9.1 Hz, 1H), 5.51 (q, J = 6.7 Hz, 1H), 4.69 (dd, J = 12.0,
.0 Hz, 1H), 4.21 (td, J = 10.6, 8.8, 5.8 Hz, 2H), 4.00 (d, J = 2.3
Hz, 3H), 3.65 (d, J = 12.6 Hz, 1H), 3.59 (d, J = 9.0 Hz, 1H),
4
2
1
=
3
Preparative and Analytical HPLC. Unless otherwise
stated, preparative high performance liquid chromatography
was performed on a Varian Prostar instrument using a Kromasil
C8 (length, 250 mm; i.d., 20 mm) 10 μm column. The column
was eluted at 21.0 mL/min with deionized water containing
3
2
.39 (s, 3H), 3.28−3.23 (m, 3H), 2.97 (d, J = 9.7 Hz, 1H),
.90−2.83 (m, 3H), 2.78−2.67 (m, 3H), 2.56 (dt, J = 11.8, 6.7
Hz, 2H), 2.51−2.39 (m, 2H), 2.33−2.21 (m, 2H), 2.15 (dd, J =
0.1% formic acid and acetonitrile using programmed linear
1
(
4.5, 3.0 Hz, 1H), 2.03−1.87 (m, 3H), 1.70 (s, 3H), 1.61−1.47
changes in mobile phase composition as follows: 15% to 32%
acetonitrile from 0 to 15 min, 32% to 48% acetonitrile from 15
to 25 min, 48 to 58% acetonitrile from 25 to 35 min, 55% to
m, 3H), 1.34−1.27 (m, 6H), 1.27−1.19 (m, 5H), 0.87 (s, 3H).
+
HRMS: calcd for C H ClN O S [M − H + 2Na]
4
2
60
3
15 3
1
022.2592; found 1022.2574.
90% acetonitrile from 35 to 55 min.
2
2
N ′-Deacetyl-N ′-[3-[[1-[(4-carboxycyclohexyl)methyl]-2,5-
Synthesized metabolites and compounds generated from
dioxo-3-pyrrolidinyl]thio]-1-oxopropyl]maytansine (DM1-
MCC Both Isomers, 9). DM1 (2a, 100 mg, 0.135 mmol) was
dissolved in a solution of DMA (6.7 mL) and 100 mM
potassium phosphate buffer pH 7.5 (2 mL). Diisopropyl ethyl
amine (35 μL, 0.203 mmol) and N-4-(maleimidomethyl)-
cyclohexanecarboxylic acid (17, 48 mg, 0.203 mmol) were then
added, and the reaction mixture was stirred for 1 h. The
mixture was adjusted to pH 6 by the addition of dilute HCl, and
approximately 1/2 of the volume was evaporated under
vacuum. The desired isomer mixture was purified by
preparative C18 HPLC, and fractions containing desired
product (retention time 31 min) were combined, frozen, and
incubation of compounds with human liver microsomes were
analyzed using a system composed of an Agilent 1100 HPLC
equipped with an analytical Kromasil C8 (length, 150 mm; i.d.,
2.1 mm) 5 μm column in series with a Bruker Esquire 3000 ion
trap MS operating in alternating positive/negative ion mode or
2
in a positive MS/MS mode. The column was eluted at a flow
rate of 0.22 mL/min with deionized water containing 0.1%
formic acid and acetonitrile using programmed linear changes
in mobile phase composition as follows: 15% to 32%
acetonitrile from 0 to 15 min, 32% to 48% acetonitrile from
15 to 25 min, 48% to 58% acetonitrile from 25 to 35 min, 58%
to 90% acetonitrile from 35 to 55 min, decreasing to 15%
acetonitrile from 55 to 56 min, and holding at 15% acetonitrile
from 56 to 64 min. HPLC/MS data were processed using
Agilent Chemstation and Bruker Daltonic Data Analysis version
lyophilized to give 80 mg (60% yield) of both isomeric product
1
forms of 9 as a white solid. H NMR (400 MHz, CDCl ): δ
3
6
4
2
6
.98 (s, 1H), 6.88 (s, 1H), 6.86−6.78 (m, 2H), 6.73−6.59 (m,
H), 6.47−6.33 (m, 2H), 5.72−5.59 (m, 2H), 5.46−5.34 (m,
H), 4.84−4.72 (m, 2H), 4.26 (t, J = 11.1 Hz, 2H), 3.98 (s,
H), 3.82 (dt, J = 27.5, 13.8 Hz, 3H), 3.71−3.59 (m, 3H), 3.45
3.3 software.
Incubation of Primary Metabolites with Human Liver
Microsomes. A nicotinamide adenine dinucleotide phosphate
reduced form (NADPH) regeneration solution was prepared by
dissolving NADPH (17 mg, 0.023 mmol), glucose-6-phosphate
sodium salt (78 mg, 0.28 mmol), and glucose-6-phosphate
dehydrogenase (100 units) in 2.0% aqueous sodium
bicarbonate (8.6 mL). A human liver microsome stock solution
(
2
2
t, J = 9.3 Hz, 2H), 3.34 (s, 4H), 3.33 (s, 3H), 3.31−3.22 (m,
H), 3.19 (s, 6H), 3.16−2.92 (m, 9H), 2.86−2.80 (m, 7H),
.67−2.53 (m, 4H), 2.39 (dd, J = 18.7, 3.6 Hz, 2H), 2.29−2.12
(
(
m, 4H), 2.06−1.95 (m, 4H), 1.75−1.65 (m, 5H), 1.64−1.61
m, 7H), 1.59−1.49 (m, 2H), 1.49−1.40 (m, 3H), 1.37 (d, J =
1
0
1
1
1
8
6
4
3
0.6 Hz, 3H), 1.33−1.24 (m, 12H), 1.24−1.16 (m, 2H), 1.07−
(
HLM stock) was prepared by suspending human liver
13
.90 (m, 4H), 0.79 (s, 6H). C NMR (101 MHz, CDCl ): δ
3
microsomes (10 mg) in 50 mM tris-hydroxymethylamino-
methane (TRIS)·HCl buffer, pH 7.4 (1.5 mL). Compounds to
be incubated with microsomes were first dissolved in 9:1 (v/v)
acetonitrile:deionized water to give a 2−3 mM stock solution,
an aliquot (10 μL) of which was added to a mixture of TRIS·
HCl buffer, pH 7.4 (640 μL), NADPH regeneration solution
80.21, 180.16, 177.09, 176.94, 174.82, 174.73, 170.84, 170.81,
70.72, 170.59, 168.88, 156.08, 153.48, 153.28, 142.25, 141.17,
39.07, 133.09, 128.17, 125.61, 122.22, 118.89, 113.25, 88.68,
8.60, 80.86, 80.82, 78.23, 78.15, 74.34, 67.32, 67.22, 60.08,
0.04, 56.87, 56.82, 56.72, 52.67, 52.48, 46.78, 44.78, 44.60,
2.70, 42.61, 40.02, 39.82, 38.87, 36.27, 35.91, 35.79, 35.65,
5.61, 34.27, 34.03, 32.52, 30.92, 30.81, 29.75, 29.67, 28.28,
(250 μL), and HLM stock solution (100 μL), and then placed
in a 37 °C water bath for 45 min. Samples were removed from
the heating bath and diluted with acetonitrile (2.0 mL),
vortexed for 10 s, and centrifuged in a SpeedVac without
vacuum for 5 min. Supernatants (1.0 mL) were transferred to
autosampler vials for HPLC analysis. Control incubations were
conducted similarly except 2% aqueous sodium bicarbonate
28.22, 27.69, 27.16, 15.61, 14.64, 13.63, 13.45, 12.29, 12.24.
+
HRMS: calcd for C H ClN O S [M + Na] 997.3566; found
47
63
4
14
997.3647.
2
2
N ′-Deacetyl-N ′-[4-[[4-[[(1S)-2-amino-1-carboxypentyl]-
amino]-3-oxopropyl]dithio]-1-oxopropyl]maytansine (Lys-
ε
N -SPP-DM1 Both Isomers, 10a). Lysine (92 mg, 0.63
(250 μL) was used instead of the NAPDH regeneration
mmol) and DM1 (2a, 48 mg, 0.065 mmol) were suspended
solution.
in 100 mM potassium phosphate buffer pH 7.4 (1.5 mL) and
D
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Molecular Pharmaceutics
Article
DMA (4 mL) with stirring at ambient temperature. After 10
min, SPP (5a, 23 mg, 0.065 mmol) was added. The reaction
was run at ambient temperature for 2 h and purified by
preparative C18 HPLC. Fractions containing pure desired
product (retention time 25 min) were combined, frozen, and
= 12.1, 2.8 Hz, 2H), 4.06 (t, J = 11.2 Hz, 2H), 3.93 (s, 6H),
3.74 (q, J = 6.0 Hz, 2H), 3.50−3.46 (m, 8H), 3.25 (s, 6H), 3.10
(s, 6H), 3.04−2.88 (m, 4H), 2.80 (d, J = 9.6 Hz, 2H), 2.71 (s,
6H), 2.65−2.55 (m, 4H), 2.08−1.98 (m, 6H), 1.86 (t, J = 12.4
Hz, 2H), 1.79−1.68 (m, 6H), 1.58 (s, 6H), 1.50−1.36 (m,
12H), 1.30−1.21 (m, 4H), 1.16 (m, 12H), 1.13−1.08 (m,
lyophilized to give 25 mg (38% yield) of product as a white
1
13
solid. H NMR (400 MHz, CD CN): δ 8.38 (s, 1H), 7.14−7.01
12H), 0.78 (s, 6H). C NMR (101 MHz, DMSO): δ 171.35,
3
(
m, 1H), 6.69−6.44 (m, 3H), 5.59 (dd, J = 14.2, 9.1 Hz, 1H),
170.98, 170.89, 170.74, 168.15, 167.84, 167.77, 155.29, 151.23,
141.28, 141.25, 138.21, 138.18, 132.58, 128.54, 125.28, 121.55,
117.06, 117.04, 113.95, 88.19, 79.99, 77.66, 73.17, 66.77, 64.48,
64.45, 60.04, 56.56, 56.11, 52.06, 51.98, 51.59, 50.03, 45.53,
37.68, 37.56, 37.51, 36.34, 35.80, 35.28, 31.93, 29.68, 29.38,
29.11, 28.56, 28.38, 28.00, 27.84, 27.69, 27.60, 26.92, 26.83,
21.29, 20.63, 15.01, 14.40, 13.01, 11.40. HRMS: calcd for
5
=
3
.37−5.26 (m, 1H), 4.55 (dd, J = 12.1, 2.9 Hz, 1H), 4.14 (dd, J
16.3, 5.8 Hz, 1H), 3.99−3.86 (m, 3H), 3.55−3.59 (m, 6H),
.51 (dd, J = 10.7, 5.4 Hz, 3H), 3.28 (s, 3H), 3.16 (d, J = 3.0
Hz, 3H), 3.14−3.00 (m, 2H), 2.97−2.80 (m, 4H), 2.77 (s, 3H),
2
1
.75−2.63 (m, 2H), 2.63−2.51 (m, 1H), 2.12 (m, 3H), 1.91−
.69 (m, 2H), 1.69−1.55 (m, 4H), 1.54−1.29 (m, 6H), 1.22 (d,
+
J = 6.8 Hz, 3H), 1.15 (d, J = 6.4 Hz, 3H), 1.10 (dd, J = 9.6, 6.8
C H ClN O S [M + H] 1106.3897; found 1106.3910.
48
72
5
16 3
2
2
Hz, 3H), 0.75 (s, 3H). HRMS: calcd for C H ClN O S [M
N ′-Deacetyl-N ′-[3-[[1-[[4-[[[(1S)-5-amino-1-
4
6
68
5
13 2
+
+
Na] 1020.3841; found 1020.3770.
carboxypentyl]amino]carbonyl] cyclohexyl]methyl]-2,5-
2
2
ε
N ′-Deacetyl N ′-[4-[[4-[[(1S)-2-amino-1-carboxypentyl]-
amino]-4-oxobutyl]dithio]-4-methyl-1-oxopentyl]-
maytansine (Lys-N -SPDB-DM4, 10b). Lysine hydrochloride
dioxo-3-pyrrolidinyl]thio]-1-oxopropyl]maytansine (Lys-N -
SMCC-DM1 Both Isomers, 11). Lysine (104 mg, 0.71 mmol)
and DM1 (2a, 48 mg, 0.065 mmol) were suspended in a
solution of 100 mM potassium phosphate buffer pH 7.4 (2 mL)
and DMA (4 mL). After 1 min a solution of SMCC (7, 24 mg,
0.072 mmol) in DMA (0.5 mL) was added and stirred at
ambient temperature for 1 h. The mixture was purified by
preparative C18 HPLC. Fractions containing the coeluting
isomers of desired product (retention time 24 min) were
ε
salt (38 mg, 0.173 mmol) and DM4 (2b, 20 mg, 0.025 mmol)
were weighed into a 2 mL vial to which was added 100 mM
potassium phosphate buffer pH 7.4 (200 μL) and DMA (200
μL). The vial was capped and vortexed for 1 min, and SPDB
(5b, 12 mg, 0.037 mmol) was then added. The vial was capped
and vortexed, a magnetic stir bar was added, and the reaction
mixture was stirred at room temperature for 2 h. Product was
purified by preparative HPLC, and fractions containing desired
product (retention time 24 min) were combined, frozen, and
lyophilized to give 7.3 mg (29% yield) of 10b as a white solid.
combined, frozen, and lyophilized to give 41 mg (57% yield) of
1
compound 11 as a white solid. H NMR (400 MHz, CD CN):
3
δ 7.04 (dd, J = 7.0, 5.6 Hz, 2H), 6.62−6.55 (m, 2H), 6.49 (dd, J
= 10.2, 3.0 Hz, 4H), 5.63−5.50 (m, 2H), 5.35−5.25 (m, J = 6.8,
4.3 Hz, 2H), 4.53 (d, J = 9.4 Hz, 2H), 4.13 (t, J = 11.2 Hz, 3H),
4.03 (s, 7H), 3.90 (s, 6H), 3.84−3.76 (m, 1H), 3.76−3.68 (m,
1H), 3.59−3.45 (m, 6H), 3.26 (s, 6H), 3.22−2.77 (m, 26H),
2.73 (d, J = 1.0 Hz, 5H), 2.69−2.49 (m, 4H), 2.36−1.98 (m,
6H), 1.71 (s, 7H), 1.57 (s, 10H), 1.53−1.37 (m, 10H), 1.36−
1.08 (m, 21H), 0.97−0.79 (m, 3H), 0.74 (s, 3H), 0.73 (s, 3H).
1
H NMR (CD CN:D O 9:1): δ 7.05 (d, J = 1.6 Hz, 1H), 6.58
3
2
(
=
d, J = 1.6 Hz, 1H), 6.45−6.56 (m, 2H), 5.61 (dd, J = 9.2 Hz, J
14.8, 1H), 5.33 (q, J = 6.8 Hz, 1H), 4.55 (dd, J = 2.8 Hz, J =
1
3
2 Hz, 1H), 4.13 (td, J = 10.0 Hz, J = 2.8 Hz, 1H), 3.92 (s, 3H),
.46−3.54 (m, 4H), 3.38−3.42 (m, 1H), 3.27 (s, 3H), 3.22 (d, J
=
=
10.1 Hz, 1H), 3.18 (s, 3H), 3.11 (t, J = 7.2 Hz, 2H), 2.93 (d, J
9.6 Hz, 1H), 2.79 (s, 3H), 2.58−2.63 (m, 3H), 2.42−2.53 (m,
13
C NMR (101 MHz, CD CN): δ 179.31, 178.89, 177.70,
3
1
H), 2.28−2.37 (m, 1H), 2.17 (t, J = 10.1 Hz, 2H), 2.07−2.13
172.92, 172.77, 172.39, 172.35, 170.87, 156.85, 156.81, 154.81,
142.67, 142.65, 142.33, 142.30, 140.98, 140.94, 134.28, 128.83,
125.86, 125.83, 122.94, 118.86, 118.84, 115.15, 115.10, 100.99,
89.08, 81.45, 78.88, 75.13, 68.44, 61.57, 57.54, 57.19, 53.68,
53.58, 46.87, 45.54, 40.88, 40.66, 39.62, 39.07, 37.21, 36.56,
36.49, 36.45, 36.37, 34.95, 34.68, 33.28, 31.31, 31.25, 30.41,
29.46, 27.71, 27.52, 23.05, 15.66, 14.63, 13.68, 12.28. HRMS:
(
m, 1H), 1.99 (m, 1H), 1.89 (m, 2H), 1.62−1.83 (m, 6H), 1.59
s, 3H), 1.22−1.54 (m, 8H), 1.16−1.22 (m, 12H), 0.80 (s, 3H).
C NMR (CD CN:D O 9:1): δ 173.03, 172.45, 171.06,
(
1
3
3
2
1
1
5
3
2
69.10, 155.66, 152.58, 141.33, 139.22, 132.88, 127.86, 124.84,
21.63, 113.70, 87.93, 80.20, 77.69, 73.73, 66.87, 60.07, 56.19,
5.81, 52.12, 49.79, 45.67, 38.96, 38.24, 37.92, 35.93, 35.67,
5.03, 33.99, 32.02, 29.96, 28.77, 28.34, 26.94, 26.34, 24.72,
+
calcd for C H ClN O S [M + Na] 1125.4597; found
53
75
6
15
1.91, 14.26, 13.38, 12.39, 11.13. HRMS: calcd for
1125.4506.
+
2
2
C H ClN O S [M + H] 1026.4335; found 1026.4355.
N ′-Deacetyl-N ′-(3-methylthio-1-oxopropyl)maytansine
(S-Me-DM1, 12a). Methyl iodide (34 μL, 0.64 mmol) and
diisopropyl ethyl amine (144 μL, 0.82 mmol) were added to a
solution of DM1 (2a, 405 mg, 0.549 mmol) in DMF (45 mL)
at ambient temperature. After 1 h dithiothreitol (50 mg, 0.32
mmol) was added to destroy excess methyl iodide. After an
additional 1 h the mixture was purified by preparative HPLC
using a Kromasil cyano column (length, 250 mm; i.d., 21 mm)
and 252 nm detection with 20 mL/min isocratic elution using
hexanes:2-propanol:ethyl acetate 68:8:24. Fractions containing
desired product (retention time 11 min) were combined.
4
8
72
5
13 2
2
2
N ′-Deacetyl N ′-[4-[[4-[[(1S)-2-Amino-1-carboxypentyl]-
amino]-4-oxo-3-sulfonate-butyl]dithio]-4-methyl-1-
oxopentyl]maytansine (Lys-N -sulfo-SPDB-DM4 Both Iso-
ε
mers, 10c). Lysine (37.5 mg, 0.256 mmol) dissolved in 1/2
saturated aqueous sodium bicarbonate (4 mL) was added to a
stirring solution of DM4 (2b, 200 mg, 0.256 mmol) in DMA (4
mL); sulfo-SPDB (5c, 125 mg, 0.308 mmol) was then added.
Some precipitate quickly formed, and DMA (2 mL) was added
followed by deionized water (1 mL) and stirred for 45 min.
Product was purified by preparative HPLC, and fractions
containing desired product (retention time 21 min) were
Solvent was removed under vacuum to give 139 mg (72%
1
combined, frozen, and lyophilized to give 71 mg (29% yield) of
yield) of 12a as a white solid. H NMR (400 MHz, CDCl ): δ
3
1
1
0c as a white solid. H NMR (400 MHz, DMSO-d ): δ 7.72
6.80 (s, 1H), 6.69 (d, J = 10.9 Hz, 1H), 6.62 (s, 1H), 6.40 (dd, J
= 15.2, 11.2 Hz, 1H), 6.32 (s, 1H), 5.63 (dd, J = 15.3, 9.0 Hz,
1H), 5.38 (dd, J = 13.3, 6.5 Hz, 1H), 4.74 (dd, J = 11.9, 2.8 Hz,
1H), 4.24 (t, J = 10.6 Hz, 1H), 3.95 (s, 3H), 3.63 (d, J = 12.7
6
(dt, J = 18.6, 5.8 Hz, 2H), 7.19 (dd, J = 6.1, 1.9 Hz, 2H), 6.87
(s, 2H), 6.60 (t, J = 11.5 Hz, 4H), 6.53 (d, J = 7.9 Hz, 4H), 5.59
(dd, J = 14.7, 9.0 Hz, 2H), 5.32 (q, J = 6.7 Hz, 2H), 4.53 (dd, J
E
DOI: 10.1021/mp5007757
Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics
Article
Hz, 1H), 3.57 (s, 1H), 3.47 (d, J = 8.9 Hz, 1H), 3.33 (s, 3H),
0.77(s, 3H), 0.76 (s, 3H). 13C NMR (101 MHz, CDCl ): δ
3
3
1
2
.17 (s, 3H), 3.08 (d, J = 12.5 Hz, 1H), 2.99 (d, J = 9.6 Hz,
H), 2.82 (s, 4H), 2.74−2.62 (m, 2H), 2.62−2.41 (m, 2H),
.14 (dd, J = 14.3, 2.7 Hz, 1H), 2.02 (s, 3H), 1.61 (s, 3H), 1.53
170.59, 170.22, 170.08, 168.84, 156.24, 156.10, 152.40, 142.37,
142.31, 140.96, 140.91, 139.70, 139.38, 133.43, 133.29, 127.98,
127.73, 125.39, 125.19, 122.29, 121.98, 119.15, 119.07, 113.30,
100.16, 88.74, 88.67, 80.95, 78.32, 74.23, 67.21, 60.11, 56.82,
56.75, 56.70, 52.93, 52.76, 50.10, 49.41, 46.78, 46.72, 39.36,
39.19, 38.99, 36.41, 35.65, 35.54, 32.57, 31.06, 30.99, 26.94,
26.41, 15.71, 15.65, 14.72, 13.56, 12.31, 12.26. HRMS: calcd for
(d, J = 13.4 Hz, 1H), 1.44 (dt, J = 16.3, 8.2 Hz, 1H), 1.26 (dd, J
13
=
8.9, 6.7 Hz, 6H), 1.20 (d, J = 13.1 Hz, 1H), 0.77 (s, 3H). C
NMR (101 MHz, CDCl ): δ 171.13, 170.88, 168.88, 156.09,
3
1
1
5
2
52.44, 142.25, 141.21, 139.33, 133.41, 127.84, 125.50, 122.26,
18.87, 113.31, 88.64, 80.95, 78.26, 74.25, 67.34, 60.10, 56.76,
6.72, 52.46, 46.70, 38.97, 36.32, 35.63, 33.95, 32.53, 30.83,
+
C H ClN O S [M + Na] 790.2752; found 790.2751.
3
6
50
3
11
2
2
N ′-Deacetyl-N ′-(4-methyl-4-methylsulfoxy-1-oxopentyl)-
9.53, 16.05, 15.63, 14.71, 13.46, 12.24. HRMS: calcd for
maytansine (S-Me-DM4 Sulfoxide Both Isomers, 13b). To a
solution of S-Me-DM4 (12b, 20 mg, 0.025 mmol) in ethanol
(300 μL) were added vanadyl acetylacetonate (2.0 mg, 0.0075
mmol) and a solution of 5.5 M tert-butyl hydrogen peroxide in
decane (5.0 μL, 0.025 mmol) at ambient temperature. The
reaction mixture was stirred for 1 h and then purified by
preparative HPLC. Fractions containing coeluting desired
products (retention time 26 min) were combined, frozen,
+
C H ClN O S [M + Na] 774.2803; found 774.2735.
3
6
50
3
10
2
2
N ′-Deacetyl-N ′-(4-methyl-4-methythio-1-oxopentyl)-
maytansine (S-Me-DM4, 12b). Methyl iodide (91 mg, 0.64
mmol) and diisopropyl ethyl amine (112 μL, 0.64 mmol) were
added to a solution of DM4 (2b, 200 mg, 0.256 mmol) in DMF
(1.3 mL) at ambient temperature. After 7 h dithiothreitol (20
mg, 0.13 mmol) was added. After an additional hour of stirring
the mixture was purified by preparative HPLC using a Kromasil
cyano column (length, 250 mm; i.d., 21 mm), with 252 nm
detection with 20 mL/min isocratic elution using hexanes:2-
propanol:ethyl acetate 68:8:24. Fractions containing the desired
product (retention time 12 min) were combined and solvent
and lyophilized to give 13 mg (65% yield) of a mixture of
1
desired product isomers as a white solid. H NMR (CDCl ): δ
3
6.82 (d, J = 1.2 Hz, 1H), 6.815 (d, J = 1.2 Hz, 1H,), 6.69 (d, J =
11.2 Hz, 2H), 6.64 (d, J = 1.2 Hz, 1H,), 6.62 (d, J = 1.2 Hz,
2H), 6.41 (dd, J = 11.2 Hz, J = 15.2 Hz, 2H,), 6.28 (s, 2H), 5.66
(dd, J = 8.8 Hz, J = 15.2 Hz, 1H,), 5.38 (q, J = 6.8 Hz, 2H,),
4.78 (dt, J = 2.4 Hz, J = 12.0 Hz, 2H,), 4.27 (dd, J = 10.8 Hz, J
= 10.8 Hz, 2H), 3.98 (s, 3H), 3.97 (s, 3H,), 3.61 (d, J = 12.4
Hz, 2H), 3.49 (d, J = 9.2 Hz, 2H), 3.38−3.43 (m, 2H), 3.35 (s,
6H), 3.21 (d, J = 3.6 Hz, 6H), 3.11 (d, J = 12.4 Hz, 2H), 3.02
(d, J = 2.4 Hz, 1H), 3.00 (d, J = 2.4 Hz, 1H), 2.86 (s, 6H), 2.33
(s, 6H), 2.32−2.68 (m, 6H), 2.18 (dd, J = 2.8 Hz, J = 14.4 Hz,
2H), 2.05−2.25 (m, 1H), 1.95−2.02 (m, 2H), 1.77−1.87 (m,
1H), 1.64 (s, 6H), 1.57 (d, J = 13.6 Hz, 2H), 1.40−1.49 (m,
2H), 1.30 (d, J = 3.6 Hz, 6H), 1.28 (d, J = 2.8 Hz, 6H), 1.90−
1.28 (m, 2H), 1.177 (d, J = 1.6 Hz, 6H), 1.11 (s, 6H), 0.80 (s,
was rotary evaporated under vacuum to give 137 mg of 12b
1
(68% yield) as a white solid. H NMR (CDCl ): δ 6.813 (d, J =
3
1
1
.6 Hz, 1H), 6.76 (d, J = 11.2 Hz, 1H), 6.65 (d, J = 1.6 Hz,
H), 6.42 (dd, J = 4.4 and 11.2 Hz, 1H), 6.21 (s, 1H), 5.67 (dd,
J = 6 and 9.2 Hz, 1H), 5.43 (m, 1H), 4.77 (dd, J = 3.2 and 8.8
Hz, 1H), 4.27 (t, 1H), 3.98 (s, 3H), 3.65 (d, J = 12.8 Hz, 1H),
3
1
.50 (d, J = 8.8 Hz, 1H), 3.35 (s, 3H), 3.23 (s, 3H), 3.10 (d, J =
2.4 Hz, 1H), 3.04 (d, J = 9.6 Hz, 1H), 2.86 (s, 3H), 2.60 (dd, J
=
2.4 and 12 Hz, 1H), 2.350−2.58 (m, 2H), 2.18 (dd, J = 3.2
and 11.2 Hz, 1H), 1.71−1.91 (m, 2H), 1.78 (s, 3H), 1.64 (s,
3
1
3
1
1
6
3
1
H), 1.57 (d, J = 13.6 Hz, 1H), 1.43−1.49 (m, 1H), 1.30 (d, J =
.2 Hz, 3H), 1.28 (d, J = 1.6 Hz, 3H), 1.24 (m, 2H), 1.21 (s,
6H). 13C NMR (CDCl ): δ 171.64, 170.69, 170.66, 168.70,
3
1
3
H), 1.19 (s, 3H), 0.80 (s, 3H). C NMR (CDCl ): δ 172.73,
156.05, 156.01, 152.25, 142.27, 142.22, 140.92, 139.24, 139.08,
133.25, 133.18, 127.82, 127.69, 125.41, 125.30, 121.95, 118.91,
118.87, 113.09, 88.48, 88.44, 80.87, 78.02, 77.99, 74.11, 66.99,
66.95, 59.99, 59.96, 56.64, 56.57, 54.74, 54.66, 52.53, 52.43,
46.63, 38.89, 36.20, 35.56, 35.50, 32.48, 32.38, 31.86, 31.31,
31.27, 30.88, 28.00, 27.82, 20.53, 20.24, 19.30, 19.19, 15.46,
14.56, 13.35, 12.19. HRMS: calcd for C H ClN O S [M +
3
71.18, 168.92, 156.17, 152.37, 142.50, 141.17, 139.28, 133.51,
27.92, 125.71, 122.39, 113.27, 88.66, 81.11, 78.41, 74.33,
7.48, 60.14, 56.81, 56.77, 52.54, 46.80, 43.58, 39.10, 36.32,
6.07, 35.74, 32.60, 30.96, 29.50, 28.74, 28.33, 15.67, 14.77,
3.45, 12.31, 10.72. HRMS: calcd for C H ClN O S [M +
3
9
56
3
10
+
Na] 816.3273; found 816.3284.
39
56
3
11
2
2
+
N ′-Deacetyl-N ′-(3-methylsulfoxy-1-oxopropyl)-
maytansine (S-Me-DM1 Sulfoxide Both Isomers, 13a). To a
solution of S-Me-DM1 (12a, 100 mg, 0.133 mmol) in ethanol
Na] 832.3222; found 832.3229.
2
2
N ′-Deacetyl-N ′-(3-methylsulfonyl-1-oxopropyl)-
maytansine (S-Me-DM1 sulfone, 14a). S-Me-DM1 sulfoxide
(13a, 35 mg, 0.046 mmol) was dissolved in methanol (1.0 mL)
to which vanadium acetylacetonate (8.0 mg, 0.03 mmol) and
5.5 M tert-butyl hydrogen peroxide in decane (41 μL, 0.23
mmol) were added. After 2 h the mixture was purified by
preparative HPLC. Fractions containing desired product
(retention time 28 min) were combined, frozen, and
(1.0 mL) were added vanadyl acetylacetonate (11 mg, 0.041
mmol) and a solution of 5.5 M tert-butyl hydrogen peroxide in
decane (24 μL, 0.13 mmol) at ambient temperature. The
reaction mixture was stirred for 1 h and was purified by
preparative HPLC. Fractions containing the coeluting desired
products (retention time 26 min) were combined, frozen, and
lyophilized to give 60 mg (59% yield) of a mixture of desired
lyophilized to give 20 mg (56% yield) of 14a as a white
1
1
product isomers as a white solid. H NMR (400 MHz, CDCl ):
solid. H NMR (400 MHz, CDCl ): δ 6.82 (d, J = 1.7 Hz, 1H),
3
3
δ 6.83−6.78 (m, 2H),6.66−6.58 (m, 4H), 6.41 (dd, J = 14.2,
6.67−6.57 (m, 2H), 6.43 (dd, J = 15.3, 11.1 Hz, 1H), 6.23 (s,
1H), 5.61 (dd, J = 15.3, 9.0 Hz, 1H), 5.37 (q, J = 6.8 Hz, 1H),
4.77 (dd, J = 12.0, 3.0 Hz, 1H), 4.26 (t, J = 10.4 Hz, 1H), 3.98
(s, 3H), 3.57 (d, J = 12.7 Hz, 1H), 3.53−3.42 (m, 2H), 3.36 (s,
3H), 3.29 (d, J = 1.8 Hz, 1H), 3.20 (s, 3H), 3.18−3.07 (m,
1H), 3.00 (dd, J = 10.3, 7.5 Hz, 2H), 2.93 (s, 3H), 2.89 (s, 3H),
2.85−2.72 (m, 1H), 2.59 (dd, J = 14.4, 12.1 Hz, 1H), 2.19 (dd,
J = 14.4, 3.0 Hz, 1H), 1.65 (s, 3H), 1.55 (d, J = 13.3 Hz, 1H),
1.51−1.40 (m, 1H), 1.32 (d, J = 6.9 Hz, 3H), 1.29 (d, J = 6.3
1
4
3
2.2 Hz,2H), 6.30 (s, 2H), 5.69−5.55 (m, 2H), 5.35 (m, 2H),
.75 (d, J = 11.7 Hz, 2H), 4.24 (m, 2H), 3.96 (s, 3H),3.95 (s,
H), 3.68 (s, 2H), 3.58 (m, 2H), 3.47 (d, J = 9.0 Hz, 2H), 3.33
(
s, 6H), 3.19 (d, J = 9.9 Hz, 6H), 3.15−3.03 (m, 4H), 3.02−
2
2
2
.96(m, 2H), 2.96−2.90 (m, 2H), 2.88 (s, 3H), 2.87 (s, 3H),
.86 (s, 2H), 2.82−2.61 (m, 4H), 2.57 (s, 3H), 2.55 (s, 3H),
.16 (dd, J = 14.3, 2.8 Hz,2H), 1.62 (s, 6H), 1.53 (d, J = 13.3
Hz, 2H), 1.49−1.37 (m, 2H), 1.30 (d, J = 3.6 Hz, 3H), 1.29 (d,
J = 3.6 Hz, 3H), 1.27 (s, 2H), 1.25 (s, 2H), 1.24−1.17 (m, 2H),
13
Hz, 2H), 1.27−1.19 (m, 1H), 0.79 (s, 3H). C NMR (101
F
DOI: 10.1021/mp5007757
Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics
Article
Figure 6. Synthesis of S-Me-DM1, S-Me-DM4, and the oxidized metabolites of S-Me-DM1, S-Me-DM4, DM1, and DM4.
MHz, CDCl ): δ 170.48, 169.13, 168.85, 156.33, 152.30,
2H), 1.15 (d, J = 6.4 Hz, 3H), 0.74 (s, 3H). HRMS: calcd for
3
+
1
1
5
1
42.47, 140.92, 139.70, 133.45, 127.83, 125.30, 121.94, 119.28,
13.35, 88.65, 81.09, 78.45, 74.27, 67.23, 60.10, 56.89, 56.81,
2.86, 50.58, 46.87, 41.76, 39.08, 36.33, 32.68, 31.09, 26.56,
5.74, 14.79, 13.65, 12.40. HRMS: calcd for C H ClN O S
C H ClN O S [M − H + 2Na] 830.2314; found 830.2247.
3
5
48
3
13
2
2
N ′-Deacetyl-N ′-(4-sulfonate-4-methyl-1-oxopentyl)-
maytansine (DM4 Sulfonic Acid, 15b). To a solution of DM4
(2b, 25 mg, 0.032 mmol) in methanol (2.0 mL) were added
vanadyl acetylacetonate (10 mg, 0.038 mmol) and a solution of
5.5 M tert-butyl hydrogen peroxide in decane (29 μL, 0.16
mmol) at ambient temperature. The reaction mixture was
stirred for 30 min and then purified by preparative C18 HPLC.
Fractions containing desired product (retention time 24 min)
3
6
50
3
12
+
[
M + Na] 806.2701; found 806.2702.
2
2
N ′-Deacetyl-N ′-(4-methyl-4-methysulfonyl-1-oxopentyl)-
maytansine (S-Me-DM4 Sulfone, 14b). S-Me-DM4 (12b, 20
mg, 0.025 mmol) was dissolved in ethanol (300 μL) to which
vanadium acetylacetonate (2.0 mg, 0.0075 mmol) and 5.5 M
tert-butyl hydrogen peroxide in decane (10 μL, 0.05 mmol)
were added. After 1 h the mixture was purified by preparative
HPLC. Fractions containing desired product (retention time 28
were combined, frozen, and lyophilized to give 11 mg (41%
1
yield) of 15b as a white solid. H NMR (400 MHz, CD CN): δ
3
8.37 (s, 1H), 7.06 (d, J = 1.6 Hz, 1H), 6.61 (d, J = 1.6 Hz, 1H),
6.55−6.45 (m, 1H), 5.67−5.53 (m, 1H), 5.33−5.25 (m, 1H),
4.55 (dd, J = 12.1, 3.0 Hz, 1H), 4.20−4.09 (m, 1H), 3.91 (s,
3H), 3.54−3.42 (m, 2H), 3.28 (s, 3H), 3.19 (d, J = 12.6 Hz,
1H), 3.13 (s, 3H), 2.89 (d, J = 9.7 Hz, 1H), 2.79 (s, 3H), 2.72−
2.51 (m, 2H), 2.37−2.27 (m, 2H), 2.12 (dd, J = 14.6, 2.4 Hz,
1H), 1.85−1.75 (m, 2H), 1.58 (s, 3H), 1.54−1.28 (m, 2H),
1.22 (d, J = 6.9 Hz, 3H), 1.15 (d, J = 6.4 Hz, 3H), 1.11 (m,
min) were combined, frozen, and lyophilized to give 15 mg
1
(
(
70% yield) of 14b as a white solid. H NMR (CDCl ): δ 6.82
3
d, J = 1.6 Hz, 1H), 6.69 (d, J = 9.2 Hz, 1H), 6.61 (d, J = 1.6
Hz, 1H), 6.42 (dd, J = 10.8 and 15.2 Hz, 1H), 6.22 (s, 1H),
.68 (dd, J = 9.2 and 15.2 Hz, 1H), 5.38 (q, J = 6.8, 1H), 4.79
dd, J = 3.2 and 12.4 Hz, 1H), 4.26 (t, J = 10.4, 1H), 3.98 (s,
H), 3.61 (d, J = 12.4 Hz, 1H), 3.50 (d, J = 8.8 Hz, 1H), 3.35
s, 3H), 3.28 (s, 1H), 3.21 (s, 3H), 3.12 (d, J = 9.6 Hz, 1H),
.02 (d, J = 9.6 Hz, 1H), 2.86 (s, 3H), 2.72 (s, 3H), 2.14−2.23
m, 2H), 2.03−2.12 (m, 1H), 1.64 (s, 3H), 1.57 (d, J=13.6,
H), 1.42−1.49 (m, 2H), 1.34 (s, 3H), 1.34 (s, 3H), 1.29 (d, J
5
(
3
(
3
(
1
6H), 0.75 (s, 3H). HRMS: calcd for C38
+ 2Na] 872.2783; found 872.2714.
H
54ClN
O13S [M − H
3
+
RESULTS
■
=
1.2 Hz, 3H), 1.27 (d, J = 1.6 Hz, 3H), 1.23 (m, 2H), 0.80 (s,
The primary- and HLM-derived metabolites from the AMCs
3a, 3b, 3c, and 4, as well as side products from AMC
preparation, were required for these studies and were prepared
by the following methods. Reaction of DM1 (2a) or DM4 (2b)
with methyl iodide in the presence of N,N-diisopropylethyl-
amine (DIPEA) gave the corresponding S-methylation
products S-Me-DM1 (12a) and S-Me-DM4 (12b), Figure 6.
Oxidation of S-Me-DM1 (12a) with tert-butyl peroxide in the
presence of a catalytic amount of vanadyl acetylacetonate gave
the two corresponding isomers of S-Me-DM1 sulfoxide (13a).
Similarly reaction of S-Me-DM4 with tert-butyl peroxide in the
presence of a catalytic amount of vanadyl acetylacetonate gave
the two isomers of S-Me-DM4 sulfoxide (13b) as a mixture of
two isomers. Oxidation of S-Me-DM1 (12a) or S-Me-DM4
(12b) with excess tert-butyl peroxide and vanadyl acetylacet-
onate gave the corresponding S-Me-DM1 sulfone (14a) or S-
Me-DM4 sulfone (14b) repectively, via initial formation of the
corresponding sulfoxides. Oxidation of DM1 (2a) or DM4
(2b) with tert-butyl peroxide in the presence of a catalytic
amount of vanadyl acetylacetonate gave the sulfonic acid of
DM1 (15a) or the sulfonic acid of DM4 (15b) respectively.
Sulfinic acid derivatives of DM1 (15c) or DM4 (15d) were not
detected by HPLC analysis of either reaction mixture.
3
1
1
5
3
H). 13C NMR (CDCl ): δ 171.31, 170.72, 168.66, 156.63,
3
52.12, 142.30, 140.83, 139.16, 133.26, 127.73, 125.37, 121.96,
18.96, 113.26, 88.47, 80.90, 78.18, 74.09, 67.09, 60.55, 59.92,
6.63, 56.58, 52.56, 46.65, 38.87, 36.14, 35.52, 34.82, 32.41,
1.51, 30.84, 28.46, 22.23, 21.47, 15.47, 14.56, 13.34, 12.18.
HRMS: calcd for C H ClN O S [M + Na] 848.3175; found
+
39
56
3
12
8
48.3165.
2
2
N ′-Deacetyl-N ′-(3-sulfonate-1-oxopropyl)maytansine
(
DM1 Sulfonic Acid, 15a). To a solution of DM1 (2a, 48 mg,
.065 mmol) in methanol (4.0 mL) were added vanadyl
0
acetylacetonate (20 mg, 0.075 mmol) and a solution of 5.5 M
tert-butyl hydrogen peroxide in decane (59 μL, 0.33 mmol) at
ambient temperature. The reaction mixture was stirred for 1 h
and then concentrated to approximately 2 mL in vacuo. The
product was purified by preparative C18 HPLC, and fractions
containing desired product (retention time 23 min) were
combined, frozen, and lyophilized to give 22 mg (43% yield) of
1
1
7
5
1
3
(
2
5a as a white solid. H NMR (400 MHz, CD CN): δ 7.12−
3
.03 (m, 1H), 6.67−6.59 (m, 1H), 6.53−6.44 (m, 2H), 5.65−
.50 (m, 1H), 5.33−5.21 (m, 1H), 4.53 (dd, J = 12.1, 3.1 Hz,
H), 4.21−4.08 (m, 1H), 3.92 (s, 3H), 3.54−3.42 (m, 2H),
.27 (s, 3H), 3.19 (d, J = 12.3 Hz, 1H), 3.13 (s, 3H), 3.10−2.93
m, 3H), 2.93−2.83 (m, 4H), 2.81 (s, 3H), 2.76−2.49 (m, 3H),
.19−2.08 (m, 1H), 1.59 (s, 3H), 1.52−1.31 (m, 2H), 1.24 (m,
Lysine-bearing primary metabolites were prepared as shown
in Figure 7. Reaction of DM1 (2a) with SPP (5a) and lysine in
G
DOI: 10.1021/mp5007757
Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics
Article
Figure 7. Synthesis of lysine-maytansinoid cellular metabolites.
Figure 8. Synthesis of hydrolyzed linker−maytansinoid adducts.
ε
aqueous sodium bicarbonate gave Lys-N -SPP-DM1 (10a).
DM1 (2a) with commercially available 17 in the presence of
base, Figure 8. All of the synthesized compounds had the same
HPLC retention times and mass spectral ion patterns as the
corresponding metabolites produced by incubation of AMCs
with target cells in vitro or from in vivo mouse experi-
ε
The two lysine linked DM4 metabolites, Lys-N -SPDB-DM4
10b) and Lys-N -sulfo-SPDB-DM4 (10c), were similarly
prepared by reacting DM4 (2b) and lysine with SPDB (5b)
or sulfo-SPDB (5c), respectively, in the presence of aqueous
sodium bicarbonate. Reaction of a thiol, such as that of DM1
ε
(
9,16,22
ments.
(
2a), with a maleimide moiety occurs at either face of the
We have previously shown that mouse liver metabolizes
several primary AMC metabolites to give less potent
compounds, and we wished to determine if such detoxification
would be likely to occur in humans during liver metabolism.
For these studies, the maytansinoids of interest were treated
with human liver microsomes, both with and without active
NADPH. The samples were analyzed by HPLC/MS, and the
identities of eluting compounds were established by comparing
their retention times and mass spectral profiles to synthetic
standards.
maleimide, resulting in two isomeric products. Thus, reaction of
DM1 (2a) with SMCC (6) and lysine gave Lys-N -SMCC-
DM1(11) as a 1:1 mixture of diastereomers.
ε
Preparation of the side products from the conjugation
reactions, DM1-TPA (8a) and DM1 dimer (7) and DM4-TBA
11
(8b), have been described previously. The side product DM4-
sulfo-TBA (8c) produced in the mAb-sulfo-SPDB-DM4
conjugation process was prepared by reacting DM4 (2b) with
sulfo-SPDB (5c) at an alkaline pH and allowing the N-
hydroxsuccinimidyl ester to hydrolyze in solution. A diastereo-
meric mixture of DM1-MCC (9) was prepared by reacting
HPLC traces from the incubation of human liver microsomes
with a small amount of acetonitrile (control) or a solution of
H
DOI: 10.1021/mp5007757
Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics
Article
Figure 9. HPLC traces from the incubations of HLM and activated NADPH with (a) no added compound, used as a control to identify HPLC
control debris (CD) peaks that are not of interest, (b) S-Me-DM1, (c) S-Me-DM4, (d) DM1, (e) DM4, and (f) DM4-SMe.
test compound in acetonitrile are shown in Figure 9. The
control HPLC trace identifies HPLC peaks that are due to
control debris (CD). Incubation of S-Me-DM1 (12a) with
human liver microsomes (HLM) and active NADPH gave both
isomers of the corresponding sulfoxides (13a and 13a′) in a 1:1
ratio as the major products along with S-Me-DM1 sulfone
designated 13b and 13b′ and the S-Me-DM4 sulfone 14b.
Incubation of DM1 (2a) with HLM and active NADPH gave
DM1 disulfide dimer (7) as the major product, with the
sulfonic acid of DM1 (15a) as a minor product. An HPLC peak
at 26 min had a negative MS ion of 768 m/z, which was
tentatively identified as the M − 1 ion of DM1 sulfinic acid
(15c). DM1 (2a) incubated with HLM in the absence of active
NADPH was also converted to DM1 disulfide dimer (7),
however the sulfonic and sulfinic acids of DM1 (15a and 15c)
respectively were not detected. No metabolites were formed
(14a) as a minor product. Incubation of S-Me-DM4 (12b) with
HLM and active NADPH gave a single broad HPLC peak for
the sulfoxide (13b) as the major product, which could be due
to one or both possible isomeric forms of the sulfoxide,
I
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Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics
Article
a
Table 1. AMC Metabolite in Vitro Cytotoxicities toward KB Cells
b
primary metabolite
S-Me-DM1 (12a)
IC50 (M)
HLM metabolite(s)
IC50 (M)
2.3 x10−8
fold decrease in potency
2.2 × 10−
11
S-Me-DM1 sulfoxide (13a)
1050
160
77
31
ND
40
ND
90
−
−
−
−
−
3.5 × 10−
9
S-Me-DM1 sulfone (14a)
2.6 × 10−
11
S-Me-DM4 sulfoxide (13b)
2.0 × 10
−9
S-Me-DM4 (12b)
DM1 (2a)
8.0 × 10−
10
S-Me-DM4 sulfone (14b)
1 × 10−
3 × 10−
9
c
DM1 sulfinic acid (15c)
ND
4.0 × 10−
8
DM1 sulfonic acid (15a)
10
c
DM4 (2b)
DM4 sulfinic acid (15d)
ND
2.7 × 10−
8
DM4 sulfonic acid (15b)
ε
−8
−8
−9
−8
8
Lys-N -SPP-DM1 (10a)
1.0 × 10
3.3 × 10
3.0 × 10
9.7 × 10
none
none
none
none
none
none
none
none
none
−
−
−
−
−
−
−
−
−
ε
Lys-N -SPDB-DM4 (10b)
ε
Lys-N -sulfo-SPDB-DM4 (10c)
ε
Lys-N -SMCC-DM1 (11)
DM1-TPA (8a)
DM4-TBA (8b)
DM4-sulfo-TBA (8c)
DM1-MCC (9)
DM1 dimer (7)
1.8 × 10−
−8
2.3 × 10
−
−
−
−
4.0 × 10−
9
−8
2.9 × 10
1.4 × 10−
10
a
ND (not determined), the compound was not yet synthesized. − indicates no value because parent compounds were not metabolized. Sulfoxides
b
c
were assayed as the mixture of isomers. Fold decrease in potency = (IC50 HLM metabolite)/(IC primary metabolite). In vitro cytotoxicity varies
50
due to thiol reactivity with cystine and other disulfide containing components present in cell media.
from the incubation of DM1 dimer (7) with HLM and active
NADPH. Incubation of DM4 (2b) with HLM gave small
amounts of metabolites, including DM4 sulfonic acid (15b) as
well as a product with a major MS ion of 834 m/z in positive
charged maytansinoids were less potent with IC values mostly
50
in the 2−20 nM range.
DISCUSSION
■
+
ion mode, presumably the (M + Na) ion of DM4 sulfinic acid
All primary maytansinoid metabolites derived from the four
AMCs, mAb-SPP-DM1 (3a), mAb-SPDB-DM4 (3b), mAb-
sulfo-SPDB-DM4 (3c), and mAb-SMCC-DM1 (4), were
synthesized and their cytotoxicities were determined on KB
cancer cells. These metabolites were also incubated with HLM,
and the identities of the resulting products, with the exception
of the sulfinic acids, were confirmed by independent synthesis
and their cytotoxicities were determined. Analysis of the
primary or HLM metabolites showed that the maytansinoid
macrocycle was untouched. Alterations were only observed on
the maytansinoid C3 ester side chain. As expected noncharged,
relatively hydrophobic maytansinoids were the most cytotoxic,
while charged or more polar maytansinoids were several fold
less potent. As we have discussed in previous publications, we
believe that this is because charged metabolites poorly diffuse
into cells. However, such metabolites may be highly potent if
1
5d. The HPLC peak at 26.3 min was identified as the DM4−
cysteine adduct because reaction of cystine with DM4 (2b)
gave one product with the same HPLC retention time and MS
ion pattern (data not shown). In order to understand why
HLM metabolism did not occur with DM1 dimer (7) and to
determine if other maytansinoids bearing disulfide bonds would
be metabolized, the methyl disulfide of DM4, DM4-SMe (18),
was incubated with HLM and active NADPH, Figure 9f. Several
major products were formed including DM4 sulfonic acid
(15b), a compound tentitively identified as DM4 sulfinic acid
(
8
15d), and one or more compounds having a major MS ion of
64 m/z in positive ion mode, presumably the M + Na ion of
+
one or more mono-oxidized DM4-SMe species, data not
shown. No metabolism occurred when DM4-SMe (18) was
incubated with HLM in the absence of active NADPH.
22
ε
generated inside a cell, as occurs during ADC delivery.
Each of the lysine bearing metabolites Lys-N -SPP-DM1
ε
ε
Incubation of DM1 (2a) with HLM in vitro formed the DM1
disulfide dimer (7). However, in vivo concentrations of DM1
(
(
10a), Lys-N -SPDB-DM4 (10b), Lys-N -sulfo-SPDB-DM4
ε
10c), and Lys-N -SMCC-DM1 (11) were incubated with
(
2a) derived by slow release from mAb-SPP-DM1 are expected
HLM, however no microsomal metabolites were detected (data
not shown). The maytansinoid−linker conjugation side
products 8a, 8b, 8c, and 9 were also incubated with HLM,
and again no microsomal metabolites were detected.
The synthesized primary and HLM metabolites as well as
synthesized side products from conjugation reactions were
evaluated for their ability to suppress the proliferation of KB
to be much lower than those used in the in vitro assay with
HLM. Thus, mAb-SPP-DM1 would not be expected to
generate a high enough concentration of DM1 (2a) to promote
DM1 disulfide (7) formation in vivo. The DM1 dimer (7) was
not significantly metabolized by HLM in the presence of active
NADPH while the lower molecular weight, disulfide-bearing
compound DM4-SMe (18) was readily oxidized. This indicates
that a disulfide moiety of a maytansinoid is not resistant to
oxidation. Many P-450 enzymes present in HLM however are
known to have limitations on the size of substrate they can
(human epidermoid carcinoma) cell lines in vitro. Cells were
exposed to the compounds for 72 h, and the surviving fractions
of cells were measured by using a clonogenic plating efficiency
assay to determine IC50 values. The cytotoxic potency of these
compounds is shown in Table 1, along with the fold decrease in
potency that arises from the HLM transformation. All of the
noncharged maytansinoids were highly cytotoxic against KB
cells, with IC50 values in the sub-nanomolar range, while the
23
accommodate in their active site. It is therefore possible that
the DM1 dimer may be too large for these oxidative enzymes to
process.
The most cytotoxic AMC metabolites, S-Me-DM1(12a) and
S-Me-DM4 (12b), were oxidatively detoxified by HLM, giving
J
DOI: 10.1021/mp5007757
Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics
Article
the respective sulfoxides and sulfones. The P-450 enzymes
carboxypentyl]amino]-4-oxo-3-sulfonate-butyl]dithio]-4-meth-
ε
2
responsible for S-Me-DM4 (12b) oxidation have been
yl-1-oxopentyl]maytansine; Lys-N -SPP-DM1, N ′-deacetyl-
24
2
identified by Davis et al. Maytansine is a highly potent
compound, but interestingly Liu et al. found that its major
HLM metabolite is 2′-N-des-methylmaytansine, which is only
N ′-[4-[[4-[[(1S)-2-amino-1-carboxypentyl]amino]-3-
oxopropyl]dithio]-1-oxopropyl]maytansine; NADPH, nicotina-
mide adenine dinucleotide phosphate reduced form; RES,
reticuloendothelial system; SEC, size exclusion chromatogra-
25
slightly less cytotoxic than maytansine. Unlike maytansine, S-
Me-DM1 (12a) and S-Me-DM4 (12b) contain a readily
oxidized sulfide moiety which may be essential for liver
detoxification of these metabolites. Microsome preparations can
be used to model liver oxidation of compounds, but unlike liver
itself, microsomes cannot induce phase II metabolism such as
S-methylation. The thiol-bearing compounds DM1 (2a) and
DM4 (2b) were not efficiently oxidized by HLM to give their
sulfinic or sulfonic acids, which indicates that S-methylation
would likely be an important first step for liver detoxification of
DM1 (2a) or DM4 (2b). All of the metabolites or conjugation
2
2
phy; S-Me-DM1, N ′-deacetyl-N ′-(3-methylthio-1-oxopropyl)-
2
2
maytansine; S-Me-DM1 sulfone, N ′-deacetyl-N ′-(3-methyl-
2
sulfonyl-1-oxopropyl)maytansine; DM1 sulfonic acid, N ′-
2
deacetyl-N ′-(3-sulfonate-1-oxopropyl)maytansine; S-Me-DM1
2
2
sulfoxide, N ′-deacetyl-N ′-(3-methylsulfoxy-1-oxopropyl)-
2
2
maytansine; S-Me-DM4, N ′-deacetyl-N ′-(4-methyl-4-methy-
2
thio-1-oxopentyl)maytansine; S-Me-DM4 sulfone, N ′-deace-
2
tyl-N ′-(4-methyl-4-methysulfonyl-1-oxopentyl)maytansine; S-
2
2
Me-DM4 sulfoxide, N ′-deacetyl-N ′-(4-methyl-4-methylsul-
foxy-1-oxopentyl)maytansine; SMCC, N-succinimidyl 4-(N-
maleimidomethyl) trans-cyclohexane 1-carboxylate; SPDB, N-
succinimidyl 4-(2-pyridyldithio)butyrate; SPP, N-succinimidyl
4-(2-pyridyldithio)pentanoate; sulfo-SPDB, N-succinimidyl 4-
(2-pyridyldithio)-2-sulfopentanoate; TFF, tangential flow filtra-
tion
ε
side products bearing a carboxylic acid such as Lys-N -SMCC-
DM1 (11) or DM1-MCC (9) had relatively poor cytotoxicities
and were not metabolized by HLM. This is consistent with our
previous work showing that mouse liver does not metabolize
ε
Lys-N -SMCC-DM1 to give oxidized compounds. Perfused rat
liver and rat liver in vivo have been shown to reduce disulfide
26
bonds while microsomes do not. The human liver would
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