Exploration of Pyrrolobenzodiazepine (PBD)-Dimers Containing Disulfide-Based Prodrugs as Payloads for Antibody-Drug Conjugates

Article abstract of DOI:10.1021/acs.molpharmaceut.8b00431

A number of cytotoxic pyrrolobenzodiazepine (PBD) monomers containing various disulfide-based prodrugs were evaluated for their ability to undergo activation (disulfide cleavage) in vitro in the presence of either glutathione (GSH) or cysteine (Cys). A good correlation was observed between in vitro GSH stability and in vitro cytotoxicity toward tumor cell lines. The prodrug-containing compounds were typically more potent against cells with relatively high intracellular GSH levels (e.g., KPL-4 cells). Several antibody-drug conjugates (ADCs) were subsequently constructed from PBD dimers that incorporated selected disulfide-based prodrugs. Such HER2 conjugates exhibited potent antiproliferation activity against KPL-4 cells in vitro in an antigen-dependent manner. However, the disulfide prodrugs contained in the majority of such entities were surprisingly unstable toward whole blood from various species. One HER2-targeting conjugate that contained a thiophenol-derived disulfide prodrug was an exception to this stability trend. It exhibited potent activity in a KPL-4 in vivo efficacy model that was approximately three-fold weaker than that displayed by the corresponding parent ADC. The same prodrug-containing conjugate demonstrated a three-fold improvement in mouse tolerability properties in vivo relative to the parent ADC, which did not contain the prodrug.

Full text of DOI:10.1021/acs.molpharmaceut.8b00431

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Article
Exploration of Pyrrolobenzodiazepine(PBD)-dimers Containing
Disulfide-based Prodrugs as Payloads for Antibody-Drug Conjugates
Zhonghua Pei, Chunjiao Chen, Jinhua Chen, Josefa dela Cruz-Chuh, Reginald Delarosa, Yuzhong
Deng, Aimee Fourie-O’Donohue, Isabel Figueroa, Jun Guo, Weiwei Jin, Cyrus Khojasteh,
Katherine R Kozak, Brandon Latifi, James Lee, Guangmin Li, Eva Lin, Liling Liu, Jiawei Lu, Scott
Martin, Carl Ng, Trung Nguyen, Rachana Ohri, Gail Lewis Phillips, Thomas H Pillow, Rebecca
K Rowntree, Nicola J Stagg, David Stokoe, Sheila Ulufatu, Vishal A. Verma, John Wai, Jing
Wang, Keyang Xu, Zijin Xu, Hui Yao, Shang-Fan Yu, Donglu Zhang, and Peter S. Dragovich
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00431 • Publication Date (Web): 24 Jul 2018
Downloaded from http://pubs.acs.org on July 25, 2018
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Exploration of Pyrrolobenzodiazepine(PBD)-dimers Containing
Disulfide-based Prodrugs as Payloads for Antibody-Drug
Conjugates
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Zhonghua Pei,†,‡ Chunjiao Chen,║ Jinhua Chen,§ Josefa dela CruzꢀChuh,† Reginald Delarosa,†
Yuzhong Deng,† Aimee FourieꢀO’Donohue,† Isabel Figueroa,† Jun Guo,† Weiwei Jin,║ S.
Cyrus Khojasteh,† Katherine R. Kozak,† Brandon Latifi,† James Lee,† Guangmin Li,† Eva
Lin,† Liling Liu,† Jiawei Lu,║ Scott Martin,† Carl Ng,† Trung Nguyen,† Rachana Ohri,† Gail
Lewis Phillips,† Thomas H. Pillow,† Rebecca K. Rowntree,† Nicola J. Stagg,† David
Stokoe,†,┴ Sheila Ulufatu,† Vishal A. Verma,† John Wai,§ Jing Wang,§ Keyang Xu,† Zijin
Xu,§ Hui Yao,§ ShangꢀFan Yu,† Donglu Zhang,† Peter S. Dragovich*,†
†Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA
‡Present address: Ideaya Biosciences, 7000 Shoreline Court, South San Francisco, CA 94080,
USA
§Wuxi Apptec, 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai 200131, China
║WuXi Biologics, 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai 200131,
China
┴Present address: Calico Inc., 1170 Veterans Blvd, South San Francisco, CA, 94080, USA
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Abstract
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A number of cytotoxic pyrrolobenzodiazepine(PBD) monomers containing various
disulfideꢀbased prodrugs were evaluated for their ability to undergo activation (disulfide
cleavage) in vitro in the presence of either glutathione (GSH) or cysteine (Cys). A good
correlation was observed between in vitro GSH stability and in vitro cytotoxicity toward
tumor cell lines. The prodrugꢀcontaining compounds were typically more potent against cells
with relatively high intracellular GSH levels (e.g., KPLꢀ4 cells). Several antibodyꢀdrug
conjugates (ADCs) were subsequently constructed from pyrrolobenzodiazepine(PBD) dimers
that incorporated selected disulfideꢀbased prodrugs. Such HER2 conjugates exhibited potent
antiproliferation activity against KPLꢀ4 cells in vitro in an antigenꢀdependent manner.
However, the disulfide prodrugs contained in the majority of such entities were surprisingly
unstable toward whole blood from various species. One HER2ꢀtargeting conjugate that
contained a thiophenolꢀderived disulfide prodrug was an exception to this stability trend. It
exhibited potent activity in a KPLꢀ4 in vivo efficacy model that was approximately 3ꢀfold
weaker than that displayed by the corresponding parent ADC. The same prodrugꢀcontaining
conjugate demonstrated a 3ꢀfold improvement in mouse tolerability properties in vivo relative
to the parent ADC which did not contain the prodrug.
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Keywords: prodrug, disulfide, antibodyꢀdrug conjugate, pyrrolobenzodiazepine
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INTRODUCTION
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Tumors frequently experience high levels of oxidative stress and have evolved
various biological mechanisms to cope with and potentially exploit such conditions.1ꢀ3 For
example, the levels of reduced glutathione (GSH), a promiscuous, naturallyꢀoccurring antiꢀ
oxidant,4 are often elevated in tumors relative to nonꢀcancerous tissues.5 Numerous recent
drugꢀdelivery approaches have sought to utilize these differing GSH levels as a means to
selectively release various cytotoxic cargos in tumors without affecting nonꢀmalignant cells.6ꢀ
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As part of our onꢀgoing efforts to develop nextꢀgeneration antibodyꢀdrug conjugates
(ADCs),14ꢀ16 we were interested in similarly attempting to exploit high tumor GSH levels as a
means to prepare conjugates with improved therapeutic potential relative to entities that we
previously studied. Specifically, we sought to identify prodrugs of cytotoxic ADC payloads
that could be selectively activated by the high reducing potential present in many intratumor
environments following targeted, antibodyꢀmediated delivery. In this report, we describe our
efforts to design and synthesize such novel entities and also provide some initial details
regarding their in vitro and in vivo biological profiling.
We envisioned constructing ADCs derived from the highly potent cytotoxic
pyrrolobenzodiazepine(PBD)ꢀdimer 117 in which one of the compound’s reactive imine
moieties18 was masked as a disulfideꢀcontaining carbamate group and the other was utilized
for antibody attachment purposes via another carbamate and a cleavable linker (compound 2,
Scheme 1). Following antigen binding and internalization into a targeted tumor cell, the
linker portion of 2 was anticipated to undergo lysosomeꢀmediated cleavage and subsequent
selfꢀimmolation to unmask one of the imine moieties present in compound 1 (i.e., conversion
of 2 to 3, Scheme 1). Multiple ADCs containing similarly linked PBDꢀdimer payloads have
previously demonstrated strong in vivo efficacy,19,20 and these results provided high
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Scheme 1.
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confidence that the antibody attachment and release strategy described above would afford
robust intracellular levels of the released payload 3. However, in contrast to typical PBDꢀ
dimer payloads produced intracellularly following ADCꢀmediated delivery, intermediate 3 is
a PBDꢀdimer prodrug that is expected to exhibit attenuated cytotoxicity properties relative to
dimeric molecules such as 1.21 Consistent with this hypothesis, we previously demonstrated
that a compound related to structure 4 (R1ꢀR2 = cyclopropyl) exhibited significantly reduced
binding to DNA relative to the PBDꢀdimer 1.22 We also envisioned that the disulfide moiety
present in 3 would undergo reductive cleavage in the intracellular environment and ensuing
selfꢀimmolation to produce the corresponding PBDꢀdimer (i.e., the conversion of 3 to 4 to 1;
Scheme 1). Our prior studies of highly efficacious disulfideꢀlinked ADCs provided good
precedent that the described transformation could efficiently occur,23,24 although some
uncertainty existed regarding the exact cellular compartment in which disulfide reduction
took place.25ꢀ30 Ideally, high concentrations of the potent PBDꢀdimer 1 would only be
generated from 3 in strongly reducing intracellular environments (i.e., in tumor cells). This
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selective production of 1 was anticipated to help minimize damage caused to nonꢀcancerous
tissues by conjugates such as 2 in addition to any antigenꢀrelated selectivity benefits provided
by the targeted ADCꢀmediated delivery. Importantly, we expected that the steric and
electronic environments associated with the disulfide bonds present in compounds such as 2
and 3 would need to be carefully modulated so as to ensure (1) efficient cleavage in tumor
cells, (2) significantly attenuated cleavage in nonꢀtumor tissue, and (3) good blood/plasma
stability during ADC circulation in vivo. Accordingly, we began our exploration of these
prodrugs by seeking to identify disulfides that met these three criteria.
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METHODS
The majority of the disulfideꢀcontaining PBDꢀmonomer compounds described in this
work were prepared by the three related methods depicted in Scheme 2. These routes were
somewhat interchangeable and all worked relatively well. The particular synthetic route used
to make a given target compound was sometimes influenced by the sterics associated with the
various carbamate precursors and whether these entities contained a UVꢀactive chromophore.
In the first method, aniline 5031 was converted to a variety of carbamates containing various
disulfides derived from 5ꢀnitropyridineꢀ2ꢀthiol (52aꢀ52g). The disulfideꢀcontaining alcohols
required for these conversions (51aꢀ51g) were prepared as depicted in Scheme 3. Many of
these entities were known in the literature (51aꢀ51d),22,31,32 and the remainder were
synthesized from the corresponding aldehydes using the twoꢀstep procedure shown in
Scheme 3 (method A, compounds 51eꢀ51g).33 Removal of the silyl protecting groups present
in selected intermediates 52 under acidic conditions provided the corresponding primary
alcohols (53), and these entities were subsequently oxidized using the DessꢀMartin
periodinane34,35 to hemiꢀaminals 54 (Method 1). It was important to closely monitor the
progress of these oxidation reactions as prolonged exposure to the DessꢀMartin reagent led to
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overꢀoxidation of the carbamateꢀprotected hemiꢀaminal moieties present in the target
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compounds to give the corresponding carbamateꢀprotected amides.
Treatment of
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intermediates 54 with a variety of thiols (R3SH) then afforded the target compounds. The
final compounds that were prepared by this process are listed in Scheme 2, and the R1, R2,
and R3 groups associated with intermediates 52, 53, and 54 correspond to those listed in
Figure 1.
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Scheme 2. Preparation of target compounds 6ꢀ28.a
N
O2N
S
S
R1
S
R2
Cmpds 6, 7, 8, 9, 10, 11, 12, 13,
14, 17, 19, 20, 22, 23, 24, 25
R3S
OH
O
O
R1
R2
TBDMSO
H
TBDMSO
H
H2N
HN
O
O
O
O
Method 1
51a-51g
b
N
N
a
O
O
N
50
52a-52g
O2N
S
S
R1
R2
O
O
Cmpds 15, 16, 18
Method 2
S
R3S
OH
HO
H
HN
R1
R2
O
O
51j-51m
R3SH
N
d
a
O
N
Method 3
R3S
R1
O2N
S
S
53
S
Cmpds
21, 26, 27, 28
R1
R2
R2
O
O
O
O
RO
H
HO
H
HN
N
O
O
O
O
R3SH
c
c
6-28
d
N
N
O
O
54
55 R = TBDMS
56 R = H
b
aReagents and conditions: TBDMS = tBu(CH3)2Si. (a) triphosgene, Et3N or pyridine,
CH2Cl2, 0 °C; (b) HOAc/THF/H2O (3/2/1, v/v/v); (c): DessꢀMartin periodinane, CH2Cl2; (d):
R3SH, CH2Cl2.
Alternatively, several R3SH thiols were condensed with other selected intermediates
52 to provide the mixed disulfides 55 (Method 2). Removal of the silyl protecting groups
present in 55 followed by oxidation of the resulting primary alcohols afforded the target
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compounds. Final compounds 15, 16, and 18 were synthesized by this method and the R1,
R2, and R3 groups associated with intermediates 55 and 56 correspond to those depicted in
Figure 1.
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Scheme 3. Preparations of disulfides 51aꢀ51m.a
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R1
R2
R3
Preparation
N
a
H
H
H
Ref 31
O2N
b
c
d
e
f
(R)-CH3
Refs 22, 32
Ref 22
"
Cyclopropyl
"
"
"
"
Cyclobutyl
Cyclopentyl
Cyclohexyl
CH2OCH2CH2
Cyclobutyl
Ref 22
Method A
Method A
Method A
Method B
Method B
Method C
Method C
Method C
R3
S
S
OH
R1
R2
ga
h
i
"
Et
51
Et
Cyclopentyl
j
Cyclohexyl
Et
Et
k
CH3
CH3
CH3
CH3
CH3
CH3
iPr
l
tBu
m
Method C
aRacemic
N
N
O2N
S
S
NO2
O
a, b
HS
R1
OH
51e-51g
A
H
R1
R2
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c
R2
N
d
S
O2N
S
OH
51h 51i
-
B
C
R1
R2
52d-52f
e
HS
R1
OH
51j-51m
R2
aReagents and conditions: (a) S2Cl2, MTBE; (b) LiAlH4, THF; (c) CH2Cl2/CH3OH, 25 °C; (d)
EtSH, CH2Cl2; (e) I2, R3SH, CH3OH.
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In the final preparation method (Method 3), intermediate 5031 was transformed into
additional disulfide intermediates 55 using the disulfideꢀcontaining alcohols 51jꢀ51m. These
entities were prepared by oxidative condensation of the corresponding mercaptoꢀalcohols
with various R3 thiols (method C, Scheme 3). As described above for Method 2, the silyl
protecting groups were subsequently removed from the intermediates 55 to give the
corresponding alcohols, and these entities were then oxidized to afford the final products. As
shown in Scheme 2, final compounds 21 and 26ꢀ28 were prepared using Method 3, and the
R1, R2, and R3 groups associated with intermediates 55 and 56 correspond to those depicted
in Figure 1. Two additional disulfides (51h and 51i) employed in the synthesis of linkerꢀ
drugs 33 and 34 below were prepared by condensing the corresponding 2ꢀpyridylꢀ4ꢀnitroꢀ
disulfides 51d and 51e with ethane thiol (method B, Scheme 3).
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The two PBDꢀmonomer compounds containing thiophenolꢀderived disulfides were
synthesized by the method shown in Scheme 4. Coupling of aniline 5031 with alcohol 58a in
a manner related to that described above in Scheme 2 for the preparation of intermediates 52
and 55 provided the corresponding carbamate 59a. Subsequent removal of the silyl
protecting group present in 59a, oxidation, and disulfide exchange with either iPrSH or
tBuSH respectfully afforded the target compounds 29 and 30 via intermediates 60a and 61
(Scheme 4). Use of benzyl alcohol in lieu of alcohol 58a in the derivatization of aniline 50
gave carbamate 59b. This intermediate was transformed into target compound 31 via the
same deprotection/oxidation sequence used to convert 59a to 61. Disulfide 58a was prepared
in one step by condensation of (4ꢀmercaptophenyl)methanol with 1,2ꢀbis(5ꢀnitropyridinꢀ2ꢀ
yl)disulfane (Scheme 4, method D). A related disulfide (58b) required for the synthesis of
linkerꢀdrug 35 below was also generated from (4ꢀmercaptophenyl)methanol and tBuSH.
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Scheme 4. Preparation of target compounds 29ꢀ31 (Method 4).a
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R4
R4
O
O
O
O
TBDMSO
H
HO
H
HN
HN
O
O
O
O
58a or BnOH
b
50
N
N
a
O
O
59a R4 = SS(2-Pyridyl-4-NO2)
59b R4 = H
60a R4 = SS(2-Pyridyl-4-NO2)
60b R4 = H
c
c
R4
31
O
N
O
HO
H
N
O
O
iPrSH or tBuSH
d
29-30
O
61 R4 = SS(2-Pyridyl-4-NO2)
N
N
O2N
S S
NO2
HS
R4
d
58a R4 = SS(2-Pyridyl-4-NO2)
58b R4 = SStBu
D
or
OH
OH
tBuSH, e
aReagents and conditions: TBDMS = tBu(CH3)2Si. (a) triphosgene, Et3N, CH2Cl2; (b)
HOAc/THF/H2O (3/2/1, v/v/v); (c) DessꢀMartin periodinane, CH2Cl2; (d) CH2Cl2; (e) I2,
EtOH.
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Scheme 5. Preparations of linkerꢀdrugs 32ꢀ36.a
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TBDMSO
H
OTBDMS
H
H2N
O
NH2
O
O
O
O
O
N
N
O
O
NH2
NH
62
O
O
H
H
N
N
a
One of: 51a, 51h, 51i, 58b, or BnOH
N
N
H
O
OH
O
R
O
O
64
TBDMSO
H
OTBDMS
NH2
HN
O
O
O
H
N
N
O
O
O
O2N
N
63a R =
63b R =
O
O
NH2
S
O
S
NH
b
N
S
S
O
O
H
H
N
N
N
H
S
S
63c R =
63d R =
O
R
c
e
O
O
O
O
S
S
32-36
S
S
R1O
H
OR1
H
HN
NH
O
O
O
N
N
O
63e R =
63f R =
O
O
65b-65f R1 = TBDMS
66b-66f R1 = H
d
(R = same as 63b-f)
aReagents and conditions: TBDMS = tBu(CH3)2Si. (a) triphosgene, Et3N, CH2Cl2; (b) iPrSH,
CH2Cl2; (c) triphosgene, Et3N, CH2Cl2, then compound 64, Et3N, DMF; (d)
HOAc/THF/H2O(3/2/1, v/v/v); (e) IBX, DMSO, 40ꢀ50 °C.
The preparation of linkerꢀdrugs 32ꢀ36 is depicted in Scheme 5. In this method, one
aniline group present in the PBDꢀdimer 6236,37 was converted to the disulfideꢀcontaining
carbamate intermediates 63a and 63cꢀ63e via coupling with alcohols 51a, 51h, 51i, or 58b.
Disulfide exchange was subsequently performed with intermediate 63a and iPrSH to give
63b. The free aniline group present in intermediates 63bꢀ63e was then coupled with
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compound 6438ꢀ40 to give the bisꢀcarbamateꢀcontaining compounds 65bꢀ65e. Removal of
both silyl protecting groups present in these entities followed by oxidation of the resulting
primary alcohols (66bꢀ66e) afforded the desired linkerꢀdrugs 32ꢀ35. Use of benzyl alcohol in
lieu of alcohols 51a, 51h, 51i, or 58b in the derivatization of bisꢀaniline 62 afforded the
benzylꢀcarbamateꢀcontaining intermediate 63f. This entity was converted to linkerꢀdrug 36
via a sequence analogous to that used to prepare linkerꢀdrugs 32ꢀ35 from compounds 65bꢀ
65e. The preparation of linkerꢀdrug 37 is described in reference 31.
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General Experimental Criteria. All solutions were aqueous unless otherwise
indicated. The purity of all compounds submitted for biological assessment was determined
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to be ≥90% by HPLC. H NMR spectra were recorded on a Varian or Brucker spectrometer.
Chemical shifts are reported in ppm relative to tetramethylsilane as an internal standard.
Silica gel for column chromatography was typically (40−63 ꢁm, 60 Å). All solvents used
were reagentꢀgrade or better purity. The R3SH thiols employed in the Scheme 2 and Scheme
3 syntheses were commercially available. LCMS condition #1: mobile phase: 1.5 mL / 4 L
TFA in water (solvent A) and 0.75 mL / 4 L TFA in acetonitrile (solvent B), using the elution
gradient 5%ꢀ95% (solvent B) over 0.8 minutes and holding at 95% for 0.4 minutes at a flow
rate of 1.5 mL/min; column: MK RP18e 25ꢀ2 mm; wavelength: UV 220 nm and 254 nm;
column temperature: 50 °C; MS ionization: ESI. LCMS condition #2: experiments
performed on an Agilent 1290 UHPLC coupled with Agilent MSD (6140) mass spectrometer
using ESI as ionization source. The LC separation used a Phenomenex XBꢀC18, 1.7 ꢁm, 50 ×
2.1 mm column with a 0.4 mL/minute flow rate. Mobile phase A was water and mobile
phase B was acetonitrile (both with 0.1% formic acid). The gradient consisted of 2 ꢀ 98%
solvent B over 7 min then holding at 98% solvent B for 1.5 min followed by equilibration for
1.5 min. LC column temperature was 40 °C. UV absorbance was collected at 220 nm and
254 nm and mass spec full scan was applied to all experiments.
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Compound 6. Method 1. Representative example. A mixture of compound 5031
(70 mg, 0.17 mmol) and Et3N (88 mg, 0.86 mmol) in anhydrous CH2Cl2 (2.0 mL) was added
slowly to a solution of triphosgene (52 mg, 0.17 mmol) and 4 Å powdered molecular sieves
(150 mg) in CH2Cl2 (5.0 mL) at 0 °C. The reaction mixture was stirred at 20 °C for 1 h.
After the mixture was concentrated, it was dissolved in CH2Cl2 (5.0 mL) and a solution of
compound 51a31 (44 mg, 0.19 mmol) and Et3N (52 mg, 0.51 mmol) in CH2Cl2 (3.0 mL) was
added dropwise at 20 °C. The resulting mixture was stirred for 1 h at 20 °C, then the mixture
was concentrated under reduced pressure. The residue was purified by prepꢀTLC
(EtOAc:petroleum ether, 1:2, Rf = 0.5) to afford compound 52a (90 mg, 79%) as a yellow oil.
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LCMS #1: RT = 1.08 min, m/z = 665.1 [M+1]+. H NMR (400 MHz, CDCl3) δ 9.28 (d, J =
4.0 Hz, 1H), 9.15 (br s, 1H) 8.49 (dd, J = 8.4 Hz, 1H), 7.97 (d, J = 8.8 Hz, 1H), 7.82 (s, 1H),
6.85 (s, 1H), 5.00ꢀ4.92 (m, 2H), 4.70 (br s, 1H), 4.43ꢀ4.39 (m, 2H) 4.35ꢀ4.33 (m, 1H), 4.10ꢀ
4.00 (m, 1H), 3.94 (s, 3H), 3.85 (s, 3H), 3.83ꢀ3.75 (m, 1H), 3.72ꢀ3.56 (m, 1H), 3.14 (t, J = 6.0
Hz, 2H), 2.80ꢀ2.65 (m, 2H), 0.89 (s, 9H), 0.05 (s, 6H).
A solution of compound 52a (90 mg, 0.14 mmol) in THF (2.0 mL) was added to a
mixture of HOAc (2.0 mL) and water (2.0 mL) at 20 °C. The mixture was then stirred for 16
h at 40 °C. The reaction mixture was diluted with EtOAc (10 mL) and was washed with
water (5 mL x 2) and aq. NaHCO3 (5 mL x 2). The organic layer was dried over Na2SO4,
filtered, and was concentrated. The residue was purified by flash chromatography on silica
gel (0ꢀ10% CH3OH in CH2Cl2) to afford 53a (74 mg, 99%) as a yellow oil. LCMS #1: RT =
0.87 min, m/z = 573.0 [M+Na]+. 1H NMR (400 MHz, CDCl3) δ 9.27 (d, J = 2.8 Hz, 1H),
8.75 (br, 1H) 8.47 (dd, J = 8.8, 2.8 Hz, 1H), 7.95 (d, J = 8.8 Hz, 1H), 7.76 (s, 1H), 6.85 (s,
1H) 5.05ꢀ4.97 (m, 2H), 4.70 (br, 1H), 4.42ꢀ4.36 (m, 2H), 4.22 (d, J = 14.4 Hz, 1H), 4.15ꢀ4.04
(m, 1H), 3.94 (s, 3H) 3.88 (s, 3H), 3.82ꢀ3.70 (m, 2H), 3.42ꢀ3.27 (m, 1H), 3.15 (t, J = 6.4 Hz,
2H), 3.02 (t, J = 5.2 Hz, 1H), 2.85ꢀ2.78 (m, 1H), 2.52ꢀ2.44 (m, 1H).
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DessꢀMartin periodinane (69 mg, 0.16 mmol) and powdered 4Å molecular sieves
(150 mg) were added sequentially in portions to compound 53a (60 mg, 0.11 mmol) in
CH2Cl2 (5 mL) at 20 °C. The resulting mixture was stired for 1 h at 20 °C then was
quenched with saturated NaHCO3 and Na2SO3 solution (1:1, 6 mL). The mixture was then
extracted with EtOAc (5 mL x 3), and the combined organic layers were dried over Na2SO4,
filtered, and concentrated. The residue was purified by prepꢀTLC (8% CH3OH in CH2Cl2, Rf
= 0.6) to afford compound 54a (50 mg, 83%) as a yellow solid. LCMS #1: RT = 0.78 min,
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m/z = 549.2 [M+1]+. H NMR (400 MHz, CDCl3) δ 9.27 (s, 1H), 8.32 (dd, J = 8.8, 2.8 Hz,
1H), 7.65 (d, J = 8.8 Hz, 1H), 7.23 (s, 1H), 6.87 (s, 1H), 5.58 (d, J = 10.0 Hz, 1H), 5.20ꢀ5.14
(m, 2H), 4.44ꢀ4.39 (m, 1H), 4.30 (d, J = 16 Hz, 1H), 4.14ꢀ4.06 (m, 2H), 3.93 (s, 3H), 3.92 (s,
3H), 3.76ꢀ3.63 (m, 2H), 3.03ꢀ2.94 (m, 3H), 2.75ꢀ2.71 (m, 1H).
Ethanethiol (57 mg, 0.91 mmol) was added dropwise to a solution of 54a (50 mg,
0.09 mmol) in CH2Cl2 (10.0 mL) at 30 °C. The reaction mixture was stirred at 30 °C for 16
h, then MnO2 (100 mg) was added and the mixture stirred for another 20 min. MnO2 was
removed by filtration, and the filtrate was concentrated. The residue was purified by prepꢀ
TLC (8% CH3OH in CH2Cl2, Rf = 0.5) to afford compound 6 (35 mg, 84%) as a white solid.
LCMS #1: RT = 0.68 min, m/z = 477.0 [M+Na]+. 1H NMR (400 MHz, CDCl3) δ 7.22 (s,
1H), 6.74 (s, 1H), 5.59ꢀ5.57 (m, 1H), 5.18ꢀ5.11 (m, 2H), 4.57ꢀ4.45 (m, 1H), 4.30 (d, J = 16.0
Hz, 1H), 4.15 (m, J = 16.0 Hz, 2H), 3.94 (s, 6H), 3.64 (t, J = 10.1 Hz, 1H), 3.55 (d, J = 4.2
Hz, 1H), 2.97ꢀ2.83 (m, 2H), 2.81ꢀ2.61 (m, 4H), 1.28 (t, J = 7.6 Hz, 3H).
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Compound 7. Method 1. LCMS #2: RT = 1.72 min, m/z = 471 [M+1]+. H NMR
(400 MHz, DMSOꢀd6) δ 7.06 (s, 1H), 6.79 (d, J = 4.9 Hz, 1H), 6.62 (s, 1H), 5.37 (dd, J = 9.7,
5.8 Hz, 1H), 5.13 (d, J = 7.0 Hz, 2H), 4.88ꢀ4.80 (m, 1H), 4.45ꢀ4.34 (m, 1H), 4.14ꢀ4.05 (m,
1H), 4.02ꢀ3.93 (m, 1H), 3.80 (s, 6H), 3.79ꢀ3.78 (m, 1H), 3.60 (dq, J = 14.2, 6.5 Hz, 2H),
3.49ꢀ3.40 (m, 1H), 2.94ꢀ2.69 (m, 4H), 2.54 (s, 1H).
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Compound 8. Method 1. LCMS #2: RT = 3.05 min, m/z = 485 [M+1]+. H NMR
(400 MHz, DMSOꢀd6) δ 7.06 (s, 1H), 6.79 (s, 1H), 6.68ꢀ6.56 (m, 1H), 5.37 (dd, J = 9.7, 5.9
Hz, 1H), 5.13 (d, J = 7.2 Hz, 2H), 4.88 (t, J = 5.7 Hz, 1H), 4.37 (dd, J = 11.7, 6.0 Hz, 1H),
4.17ꢀ3.93 (m, 3H), 3.80 (s, 6H), 3.51ꢀ3.41 (m, 2H), 2.95ꢀ2.80 (m, 4H), 1.19 (d, J = 6.9 Hz,
3H).
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Compound 9. Method 1. LCMS #2: RT = 2.42 min, m/z = 469 [M+1]+. H NMR
(400 MHz, DMSOꢀd6) δ 7.06 (s, 1H), 6.80 (s, 1H), 6.62 (s, 1H), 5.37 (dd, J = 9.6, 5.4 Hz,
1H), 5.13 (d, J = 7.0 Hz, 2H), 4.37 (dt, J = 12.2, 6.3 Hz, 1H), 4.14ꢀ3.93 (m, 4H), 3.80 (s, 6H),
3.45 (t, J = 9.3 Hz, 1H), 3.01ꢀ2.83 (m, 3H), 1.23ꢀ1.16 (m, 6H).
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Compound 10. Method 1. LCMS #2: RT = 2.56 min, m/z = 483 [M+1]+. H NMR
(400 MHz, DMSOꢀd6) δ 7.06 (s, 1H), 6.79 (s, 1H), 6.62 (s, 1H), 5.37 (dd, J = 9.7, 5.9 Hz,
1H), 5.16ꢀ5.09 (m, 2H), 4.35 (dt, J = 12.2, 6.3 Hz, 1H), 4.10 (d, J = 16.0 Hz, 1H), 3.98 (d, J =
16.0 Hz, 2H), 3.80 (s, 6H), 3.44 (t, J = 9.3 Hz, 1H), 2.88 (t, J = 12.6 Hz, 3H), 2.54 (s, 1H),
1.25 (s, 9H), 0.08 (s, 1H).
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Compound 11. Method 1. LCMS #2: RT = 2.40 min, m/z = 469 [M+1]+. H NMR
(400 MHz, DMSOꢀd6) δ 7.07 (s, 1H), 6.82 (s, 1H), 6.63 (s, 1H), 5.38 (dd, J = 9.7, 5.9 Hz,
1H), 5.13 (d, J = 6.7 Hz, 2H), 4.21 (dd, J = 11.1, 6.2 Hz, 1H), 4.10 (d, J = 16.0 Hz, 1H), 3.97
(d, J = 16.1 Hz, 2H), 3.81 (d, J = 2.3 Hz, 6H), 3.45 (t, J = 9.3 Hz, 1H), 3.05 (s, 1H), 2.94ꢀ2.83
(m, 1H), 2.70ꢀ2.53 (m, 3H), 2.45 (p, J = 1.9 Hz, 1H), 1.16 (t, J = 7.2 Hz, 3H), 1.08ꢀ1.02 (m,
2H).
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Compound 12. Method 1. LCMS #2: RT = 2.16 min, m/z = 499 [M+1]+. H NMR
(400 MHz, DMSOꢀd6) δ 7.07 (s, 1H), 6.82 (s, 1H), 6.63 (br s, 1H), 5.38 (d, J = 9.7 Hz, 1H),
5.13 (d, J = 6.8 Hz, 2H), 4.22 (dd, J = 11.1, 6.2 Hz, 1H), 4.10 (d, J = 15.9 Hz, 1H), 3.97 (d, J
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= 15.4 Hz, 2H), 3.81 (s, 6H), 3.50ꢀ3.44 (m, 2H), 3.22 (s, 3H), 2.94ꢀ2.75 (m, 3H), 1.05 (d, J =
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Compound 13. Method 1. LCMS #2: RT = 2.08 min, m/z = 497 [M+1]+. H NMR
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(400 MHz, DMSOꢀd6) δ 7.08 (s, 1H), 6.82 (s, 1H), 6.65 (s, 1H), 5.38 (d, J = 9.7 Hz, 1H),
5.13 (d, J = 6.6 Hz, 2H), 4.77 (s, 3H), 4.45ꢀ4.34 (m, 1H), 4.26ꢀ4.06 (m, 3H), 4.02ꢀ3.93 (m,
1H), 3.81 (s, 6H), 3.45 (t, J = 9.3 Hz, 1H), 3.09 (s, 1H), 2.94ꢀ2.83 (m, 1H), 2.62ꢀ2.53 (m,
1H), 1.04 (d, J = 6.9 Hz, 2H), ꢀ0.03 to ꢀ0.13 (m, 1H).
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Compound 14. Method 1. LCMS #2: RT = 2.24 min, m/z = 525 [M+1]+. H NMR
(400 MHz, DMSOꢀd6) δ 7.07 (s, 1H), 6.82 (s, 1H), 6.61 (s, 1H), 5.37 (d, J = 9.6 Hz, 1H),
5.13 (d, J = 7.0 Hz, 2H), 4.23 (dd, J = 11.1, 6.0 Hz, 1H), 4.10 (d, J = 15.7 Hz, 1H), 4.02ꢀ3.83
(m, 4H), 3.81 (s, 6H), 3.45 (t, J = 9.3 Hz, 1H), 3.02 (dt, J = 11.1, 4.1 Hz, 1H), 2.94ꢀ2.83 (m,
2H), 2.59ꢀ2.52 (m, 2H), 1.95ꢀ1.76 (m, 2H), 1.55ꢀ1.37 (m, 2H), 1.27ꢀ1.21 (m, 1H), 1.05 (d, J
= 6.9 Hz, 3H).
Compound 15. Method 2. Representative example. A mixture of triphosgene (53.6
mg, 0.18 mmol) and powdered 4Å molecular sieves (150 mg) in CH2Cl2 (5 mL) was added to
a solution of compound 5031 (74 mg, 0.18 mmol) an
Et3N (92 mg, 0.90 mmol) in CH2Cl2
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(5.0 mL). After the reaction mixture was stirred for 1 h at 25 °C, it was concentrated and the
residue was dissolved in CH2Cl2 (5.0 mL). A solution of compound 51b32 (53 mg, 0.21
mmol) and Et3N (54 mg, 0.54 mmol) in CH2Cl2 (5 mL) was added dropwise at 25 °C, and the
reaction mixture was stirred for 1 h at 25 °C. The mixture was then concentrated, and the
residue was purified by prepꢀHPLC (Xtimate C18 150 x 25 mm x 5 ꢁm, acetonitrile 80ꢀ
100/0.23% formic acid in water) to afford 52b (79 mg, 65%) as a white solid. LCMS #1 RT
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= 1.17 min, m/z = 679.1 [M+1]+. H NMR (400 MHz, CDCl3) δ 9.24 (d, J = 2.8 Hz, 1H),
8.53 (d, J = 8.8, 2.0 Hz, 1H), 8.03 (d, J = 9.2 Hz, 1H), 7.82 (s, 1H), 6.85 (s, 1H), 4.99ꢀ4.92
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(m, 2H), 4.70 (brs, 1H), 4.28ꢀ4.24 (m, 2H), 4.13ꢀ4.08 (m, 2H), 3.93 (s, 4H), 3.85 (s, 3H),
3.74ꢀ3.67 (m, 1H), 3.36ꢀ3.28 (m, 1H), 2.73 (br, 2H), 1.41 (d, J = 6.8 Hz, 3H), 0.89 (s, 3H),
0.05 (s, 6H).
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To a mixture of 52b (78.0 mg, 0.1100 mmol) in CH2Cl2 (5.0 mL) was added 2ꢀ
propanethiol (88 mg, 1.15 mmol). The mixture was stirred under N2 at 25 °C for 16 h, then
MnO2 (100 mg) was added stirring was continued for another 20 min. MnO2 was removed by
filtration, and the filtrate was concentrated. The residue was purified by prepꢀTLC (5%
CH3OH in CH2Cl2, Rf = 0.6) to afford 55b (51 mg, 74%) as a white solid. LCMS #1: RT =
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1.21 min, m/z = 621.1 [M+Na]+. H NMR (400 MHz, CDCl3) δ 8.92 (br, 1H), 7.85 (s, 1H),
6.82 (s, 1H), 5.00ꢀ4.92 (m, 2H), 4.66 (br, 1H), 4.35ꢀ4.31 (m, 1H), 4.22 (d, J = 16 Hz, 1H),
4.14ꢀ4.09 (m, 2H), 3.94 (s, 3H), 3.85 (s, 4H), 3.66ꢀ3.63 (m, 1H), 3.13ꢀ2.95 (m, 2H), 2.71 (brs,
2H), 1.35ꢀ1.30 (m, 9H), 0.89 (s, 9H), 0.04 (s, 6H).
To a mixture of 55b (50 mg, 0.08 mmol) in water (1.0 mL) and THF (1.0 mL) was
added HOAc (1.5 mL) at 25 °C. After the reaction mixture was stirred at 40 °C for 15 h, it
was diluted with EtOAc (10 mL) and washed sequentially with water (5 mL × 2) and aq.
NaHCO3 (5 mL × 2). The organic layer was dried over Na2SO4, filtered, and was
concentrated. The residue was purified by flash chromatography on silica gel (0ꢀ10%
CH3OH in CH2Cl2) to afford 56b (32 mg, 77%) as a white solid. LCMS #1: RT = 0.91 min,
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m/z = 485.0 [M+1]+. H NMR (400 MHz, CDCl3) δ 8.54 (br, 1H), 7.75 (s, 1H), 6.83 (s, 1H),
5.03 (brs, 1H), 4.95 (br, 1H), 4.67 (br, 1H), 4.33ꢀ4.29 (m, 1H), 4.17ꢀ4.13 (m 3H), 3.93 (s,
3H), 3.87 (s, 3H), 3.75ꢀ3.63 (m, 1H), 3.44ꢀ3.35 (m, 1H), 3.12ꢀ2.97 (m, 2H), 2.78ꢀ2.76 (m,
1H), 2.49ꢀ2.44 (m, 1H), 1.35ꢀ1.30 (m, 9H).
To a solution of 56b (15 mg, 0.03 mmol) in CH2Cl2 (4.0 mL) was added DessꢀMartin
periodinane (18 mg, 0.04 mmol) and powdered 4Å molecular sieves (100 mg). The mixture
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was stirred at 25 °C for 1 h then was quenched with saturated NaHCO3 and Na2SO3 solution
(1:1, 3.0 mL). The mixture was subsequently extracted with EtOAc (5 mL x 3), and the
combined organic layers were dried over Na2SO4, filtered, and concentrated. The residue
was purified by prepꢀTLC (50% EtOAc in petroleum ether, Rf = 0.3) to afford compound 15
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(8.0 mg, 54%) as a white solid. LCMS #1: RT = 0.88 min, m/z = 483.2 [M+1]+. H NMR
(400 MHz, CDCl3) δ 7.22 (s, 1H), 6.77 (s, 1H), 5.59 (d, J = 8.8 Hz, 1H), 5.15 (br, 2H), 4.35ꢀ
4.27 (m, 2H), 4.16 (d, J = 16.4 Hz, 1H), 4.08ꢀ4.04 (m, 1H), 3.94 (s, 6H), 3.64 (t, J = 8.0 Hz,
1H), 3.51 (brs, 1H), 2.99ꢀ2.90 (m, 3H), 2.74ꢀ2.70 (m, 1H), 1.27ꢀ1.20 (m, 9H).
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Compound 16. Method 2. LCMS #2: RT = 2.70 min, m/z = 497 [M+1]+. H NMR
(400 MHz, DMSOꢀd6) δ 7.07 (s, 1H), 6.81 (s, 1H), 6.65 (d, J = 5.9 Hz, 1H), 5.38 (dd, J = 9.7,
5.9 Hz, 1H), 5.19ꢀ5.04 (m, 2H), 4.20 (dd, J = 11.2, 5.9 Hz, 1H), 4.10 (d, J = 15.8 Hz, 1H),
4.04ꢀ3.91 (m, 2H), 3.80 (s, 6H), 3.45 (t, J = 9.3 Hz, 1H), 3.04ꢀ2.82 (m, 2H), 2.58ꢀ2.53 (m,
1H), 1.23 (s, 9H), 1.03 (d, J = 6.9 Hz, 3H).
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Compound 17. Method 1. LCMS #2: RT = 1.57 min, m/z = 528 [M+1]+. H NMR
(400 MHz, DMSOꢀd6) δ 7.06 (s, 1H), 6.98 (s, 1H), 5.44 (d, J = 9.6 Hz, 1H), 5.13 (d, J = 4.6
Hz, 2H), 4.46 (s, 1H), 4.10 (d, J = 15.9 Hz, 2H), 3.97 (d, J = 15.7 Hz, 1H), 3.83 (s, 3H), 3.80
(s, 6H), 2.89 (s, 4H), 0.99 (d, J = 7.0 Hz, 3H).
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Compound 18. Method 2. LCMS #1: RT = 0.83 min, m/z = 481 [M+1]+. H NMR
(400 MHz, CDCl3) δ 7.21 (s, 1H), 6.80 (s, 1H), 5.57 (d, J = 9.6 Hz, 1H), 5.14 (s, 2H), 4.68 (d,
J = 11.2 Hz, 1H), 4.32ꢀ4.28 (m, 1H), 4.17ꢀ4.13 (m, 1H), 3.93 (s, 6H), 3.73ꢀ3.62 (m, 2H),
2.95ꢀ2.89 (m, 1H), 2.73ꢀ2.67 (m, 2H), 1.25ꢀ1.21 (m, 4H), 1.08ꢀ0.81 (m, 5H).
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Compound 19. Method 1. LCMS #2: RT = 2.61 min, m/z = 495 [M+1]+. H NMR
(400 MHz, DMSOꢀd6) δ 7.08 (s, 1H), 6.82 (s, 1H), 6.64 (d, J = 6.0 Hz, 1H), 5.45ꢀ5.36 (m,
1H), 5.13 (d, J = 7.2 Hz, 2H), 4.30 (d, J = 11.5 Hz, 1H), 4.10 (d, J = 15.8 Hz, 1H), 3.97 (d, J
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= 13.7 Hz, 2H), 3.80 (s, 6H), 3.46 (td, J = 9.5, 1.8 Hz, 1H), 2.89 (dd, J = 15.8, 9.2 Hz, 1H),
2.62ꢀ2.52 (m, 2H), 1.91 (s, 6H), 1.63 (s, 1H), 1.13 (t, J = 7.5 Hz, 3H).
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Compound 20. Method 1. LCMS #2: RT = 2.48 min, m/z = 509 [M+1]+. H NMR
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(400 MHz, DMSOꢀd6) δ 7.08 (s, 1H), 6.82 (s, 1H), 6.73ꢀ6.60 (m, 1H), 5.40 (d, J = 7.7 Hz,
1H), 5.13 (d, J = 7.2 Hz, 2H), 4.22 (d, J = 11.0 Hz, 1H), 4.10 (d, J = 15.9 Hz, 1H), 4.01ꢀ3.84
(m, 2H), 3.81 (s, 6H), 3.46 (t, J = 9.2 Hz, 1H), 2.88 (dd, J = 15.9, 9.3 Hz, 1H), 2.55 (dd, J =
4.1, 2.3 Hz, 2H), 1.81ꢀ1.39 (m, 10H), 1.12 (t, J = 7.3 Hz, 3H).
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Compound 21. Method 3. LCMS #1: RT = 0.89 min, m/z = 523 [M+1]+. H NMR
(400 MHz, CDCl3) δ 7.22 (s, 1H), 6.81 (s, 1H), 5.59 (d, J = 10.0 Hz, 1H), 5.15 (brs, 2H), 4.44
(d, J = 10.8 Hz, 1H), 4.33ꢀ4.28 (m, 1H), 4.17ꢀ4.14 (m, 1H), 3.94 (s, 6H), 3.75 (d, J = 10.4 Hz,
1H), 3.64 (t, J = 8.8 Hz, 1H), 3.50 (s, 1H), 2.97ꢀ2.88 (m, 1H), 7.74ꢀ2.72 (m, 1H), 2.72ꢀ2.59
(m, 1H), 1.61ꢀ0.85 (m, 13H).
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Compound 22. Method 1. LCMS #1: RT = 0.66 min, m/z = 525 [M+1]+. H NMR
(400 MHz, CDCl3) δ 7.23 (s, 1H), 6.85 (s, 1H), 5.65 (d, J = 9.6 Hz, 1H), 5.15 (s, 2H), 4.55 (d,
J = 11.2 Hz, 1H), 4.31ꢀ4.27 (m, 1H), 4.18ꢀ4.15 (m, 1H), 3.95 (s, 7H), 3.77ꢀ3.64 (m, 5H),
2.92ꢀ2.86 (m, 2H), 2.74ꢀ2.70 (m, 1H), 2.58ꢀ2.51 (m, 1H), 1.81ꢀ1.74 (m, 4H), 1.60ꢀ1.48 (m,
4H).
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Compound 23. Method 1. LCMS #2: RT = 1.95 min, m/z = 511 [M+1]+. H NMR
(400 MHz, DMSOꢀd6) δ 7.08 (s, 1H), 6.83 (s, 1H), 6.64 (s, 1H), 5.39 (d, J = 9.7 Hz, 1H),
5.13 (d, J = 7.1 Hz, 2H), 4.34 (d, J = 11.5 Hz, 1H), 4.10 (d, J = 16.0 Hz, 1H), 3.98 (t, J = 15.3
Hz, 2H), 3.81 (d, J = 2.4 Hz, 6H), 3.78ꢀ3.72 (m, 1H), 3.63 (d, J = 6.1 Hz, 1H), 3.54 (s, 1H),
3.46 (t, J = 9.2 Hz, 1H), 2.89 (dd, J = 15.7, 9.4 Hz, 1H), 1.96ꢀ1.72 (m, 3H), 1.20ꢀ1.06 (m,
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Compound 24. Method 1. LCMS #2: RT = 1.99 min, m/z = 511 [M+1]+. H NMR
(400 MHz, DMSOꢀd6) δ 7.08 (s, 1H), 6.83 (s, 1H), 6.64 (s, 1H), 5.39 (d, J = 9.7 Hz, 1H),
5.13 (d, J = 7.2 Hz, 2H), 4.34 (d, J = 11.3 Hz, 1H), 4.10 (d, J = 15.9 Hz, 1H), 3.98 (t, J = 15.5
Hz, 2H), 3.81 (d, J = 1.9 Hz, 7H), 3.63 (d, J = 6.1 Hz, 1H), 3.54 (s, 1H), 3.46 (t, J = 9.1 Hz,
1H), 2.89 (dd, J = 15.9, 9.4 Hz, 1H), 1.96ꢀ1.72 (m, 3H), 1.20 ꢀ1.06 (m, 3H).
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Compound 25. Method 1. LCMS #1: RT = 0.58 min, m/z = 527 [M+1]+. H NMR
(400 MHz, CDCl3) δ 7.23 (s, 1H), 6.79 (s, 1H), 5.64 (d, J = 10.0 Hz, 1H), 5.16 (s, 2H), 4.62ꢀ
4.60 (m, 1H), 4.31ꢀ4.28 (m, 1H), 4.18ꢀ4.10 (m, 1H), 3.95ꢀ3.85 (m, 11H), 3.71ꢀ3.62 (m, 5H),
2.92ꢀ2.86 (m, 2H), 2.74ꢀ2.67 (m, 1H), 2.61ꢀ2.52 (m, 1H), 2.03ꢀ1.84 (m, 2H).
Compound 26: Method 3. Representative example. A mixture of compound 5031
(50 mg, 0.12 mmol) and Et3N (37 mg, 0.37 mmol) in CH2Cl2 (3.0 mL) was added to a
solution of triphosgene (40 mg, 0.13 mmol) in CH2Cl2 (5.0 mL) at 25 °C. The reaction
mixture was stirred for 1 h at 25 °C then was concentrated. The residue was dissolved in
CH2Cl2 (5.0 mL) and a solution of compound 51k (41 mg, 0.25 mmol) in pyridine (29 mg,
0.25 mmol) and CH2Cl2 (5.0 mL) was added dropwise. The resulting mixture was stirred for
1 h at 25 °C then was concentrated under reduced pressure. The residue was purified by flash
column chromatography (0ꢀ50% EtOAc in petroleum ether, Rf = 0.5) to afford compound
55k (45 mg, 57%) as a colorless oil. LCMS #1: RT = 1.21 min, m/z = 621.2 [M+Na]+. H
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NMR (400 MHz, CDCl3) δ 8.87 (br, 1H), 7.84 (s, 1H), 6.81 (s, 1H), 4.99 (s, 1H), 4.92 (br,
1H), 4.64ꢀ4.57 (m, 1H), 4.22ꢀ4.07 (m, 4H), 3.93 (s, 3H), 3.84 (s, 3H), 3.75ꢀ3.55 (m, 1H),
2.73ꢀ2.67 (m, 4H), 1.34 (s, 6H), 1.28 (t, J = 7.2 Hz, 3H), 0.87 (s, 9H), 0.03 (s, 6H).
A solution of 55k (45 mg, 0.08 mmol), water (4.0 mL), and acetic acid (6.0 mL)was
stirred at 10 °Cfor 16 h. The mixture was concentrated to a residue, and the pH was adjusted
to about 8 with saturated NaHCO3 solution (10 mL). The resulting mixture was extracted
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with EtOAc (20 mL x 3), and the combined organic layers were washed sequentially with
water (20 mL) and saturated brine (20 mL x 2), then dried over Na2SO4, filtered and
concentrated. The residue was purified by column chromatography (0ꢀ10 % CH3OH in
CH2Cl2, Rf = 0.6) to afford 56k (20 mg, 55%) as a colorless oil. LCMS #1: RT = 0.93 min,
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m/z = 507.1 [M+Na]+. H NMR (400 MHz, CD3OD) δ 7.20 (s, 1H), 7.00 (s, 1H), 5.01ꢀ4.93
(m, 2H), 4.56 (s, 1H), 4.50 (br, 1H), 4.12ꢀ4.01 (m, 4H), 3.85 (s, 6H), 3.69ꢀ3.61 (m, 2H), 2.77ꢀ
2.67 (m, 4H), 1.32 (s, 6H), 1.28ꢀ1.25 (t, J = 7.6 Hz, 3H).
To a solution of 56k (20 mg, 0.04 mmol)in anhydrous CH2Cl2 (2.0 mL) was added
DessꢀMartin periodinane (26 mg, 0.06 mmol). The reaction mixture was stirred at 25 °C
for 1 h whereupon TLC (8% CH3OH in CH2Cl2, Rf = 0.6) showed that the desired product
was generated. The mixture was diluted with H2O (5.0 mL) followed by aq. Na2SO3 solution
(5.0 mL) and aq. NaHCO3 solution (5.0 mL). The resulting mixture was extracted with
EtOAc (20 mL × 3), and the combined organic layers were dried over Na2SO4, filtered, and
were concentrated. The residue was then purified by prepꢀTLC (8% CH3OH in CH2Cl2) to
give compound 26 (19 mg, 50%) as a white solid. LCMS #1: RT = 0.87 min, m/z = 505.1
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[M+Na]+. H NMR (400 MHz, CDCl3) δ 7.22 (s, 1H), 6.77 (s, 1H), 5.58 (d, J = 9.6 Hz, 1H),
5.14 (s, 2H), 4.38ꢀ4.28 (m, 2H), 4.14 (d, J = 16.4 Hz, 1H), 3.93 (s, 6H), 3.73 (d, J = 10.4 Hz,
1H), 3.66ꢀ3.61 (m, 1H), 3.47 (brs, 1H), 2.95ꢀ2.89 (m, 1H), 2.73ꢀ2.62 (m, 3H), 1.25 (t, J = 7.6
Hz, 3H), 1.15 (s, 3H), 1.08 (s, 3H).
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Compound 27. Method 3. LCMS #2: RT = 2.69 min, m/z = 497 [M+1]+. H NMR
(400 MHz, DMSOꢀd6) δ 7.08 (s, 1H), 6.82 (s, 1H), 6.64 (d, J = 6.0 Hz, 1H), 5.49ꢀ5.33 (m,
1H), 5.13 (d, J = 7.3 Hz, 2H), 4.21ꢀ4.05 (m, 2H), 4.04ꢀ3.91 (m, 1H), 3.81 (s, 4H), 3.79ꢀ3.72
(m, 1H), 3.46 (t, J = 9.3 Hz, 1H), 3.31 (s, 3H), 2.99ꢀ2.76 (m, 2H), 2.63ꢀ2.51 (m, 1H), 1.38ꢀ
0.94 (m, 12H).
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Compound 28. Method 3. LCMS #2: RT = 2.78 min, m/z = 511 [M+1]+. H NMR
(400 MHz, DMSOꢀd6) δ 7.08 (s, 1H), 6.81 (s, 1H), 6.63 (d, J = 5.9 Hz, 1H), 5.39 (dd, J = 9.7,
5.9 Hz, 1H), 5.13 (d, J = 5.7 Hz, 2H), 4.07 (dd, J = 19.9, 13.3 Hz, 2H), 3.96 (d, J = 15.9 Hz,
1H), 3.81 (d, J = 2.2 Hz, 5H), 3.77ꢀ3.70 (m, 1H), 3.50ꢀ3.43 (m, 1H), 2.89 (dd, J = 15.4, 9.5
Hz, 1H), 1.17 (s, 10H), 1.11ꢀ0.98 (m, 5H).
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Compound 29. Method 4. Representative example. A mixture of compound 5031
(300 mg, 0.74 mmol) and Et3N (75 mg, 0.74 mmol) in CH2Cl2 (10 mL) was added to a
solution of triphosgene (88 mg, 0.30 mmol) in CH2Cl2 (5 mL) at 0 °C over 15 min. The
reaction mixture was stirred for 1 h at 0 °C, and a solution of compound 58a (217 mg, 0.74
mmol) and Et3N (224 mg, 2.21 mmol) in CH2Cl2 (10 mL) was then added dropwise at 0 °C.
The resulting mixture was stirred for 1 h at 0 °C, then was concentrated under reduced
pressure. The residue was purified by prepꢀTLC (EtOAc:petroleum ether, 1:2, Rf = 0.5) to
afford compound 59a (286 mg, 50%) as a yellow oil. LCMS #1: RT = 1.01 min, m/z = 727.2
[M+1]+.
A solution of compound 59a (286 mg, 0.39 mmol) in THF (3 mL) was added to a
mixture of HOAc (5.0 mL) and water (2.0 mL) at 20 °C. The mixture was stirred for 16 h at
that temperature, then was heated to 40 °C for an additional 16 h. The mixture was
concentrated to a residue, and the pH was adjusted to about 8 with saturated NaHCO3
solution (10 mL). The mixture was then extracted with EtOAc (10 mL x 3) and the combined
extracts were washed with saturated brine (5 mL x 2), dried over Na2SO4, filtered and
concentrated. The residue was purified by prepꢀTLC (EtOAc:petroleum ether, 2:1, Rf = 0.4)
to afford 60a (206 mg, 80%) as yellow oil. LCMS #1: RT = 0.80 min, m/z = 613.1 [M+1]+.
DessꢀMartin periodinane (55 mg, 0.13 mmol) was added in portions to compound 60a
(40 mg, 0.070 mmol) in CH2Cl2 (5 mL) at 0 °C. After warning to 20 °C and stiring for 2 h,
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the mixture was quenched with saturated NaHCO3 and Na2SO3 solution (1:1, 6 mL). The
mixture was then extracted with EtOAc (5 mL x 3), and the combined organic layers were
dried over Na2SO4, filtered, and concentrated. The residue was purified by prepꢀTLC (5%
CH3OH in CH2Cl2, Rf = 0.6) to afford compound 61 (19 mg, 45%) as a yellow solid. LCMS
#1: RT =0.89 min, m/z = 611.4 [M+1]+. 1H NMR (400 MHz,CDCl3) δ 9.30 (s, 1H), 8.40
(dd, J = 2.4 Hz, 8.8 Hz, 1H), 7.83 (d, J = 8.8 Hz, 1H), 7.45 (d, J = 7.6 Hz, 2H), 7.17 (s, 1H),
7.15 (d, J = 7.6 Hz, 2H), 6.54 (s, 1H), 5.56 (d, J = 10 Hz, 1H), 5.23 (d, J = 12.8 Hz, 1H), 5.15
(s, 2H), 4.95ꢀ4.92 (m, 1H), 4.31ꢀ4.28 (m, 1H), 4.16ꢀ4.12 (m, 1H), 3.93 (s, 3H), 3.73 (s, 3H),
3.63 (t, J = 9.2 Hz, 1H), 2.96ꢀ2.89 (m, 1H), 2.73ꢀ2.69 (m, 1H).
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iPrSH (700 mg, 9.2 mmol) was added dropwise to a solution of compound 61 (40
mg, 0.070 mmol) in CH2Cl2 (5.0 mL) at 23 °C. The reaction mixture was stirred at that
temperature for 16 h, then MnO2(100 mg) was added and stirring was continued for another
20 min. MnO2 was removed by filtration, and the filtrate was concentrated. The residue was
purified by prepꢀTLC twice (5% CH3OH in CH2Cl2, Rf = 0.6) and lyophilized to afford 29
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(22 mg, 63%) as a white solid. LCMS #1: RT = 0.92 min, m/z = 531.1 [M+1]+. H NMR
(400 MHz, CDCl3) δ 7.47 (d, J = 8.0 Hz, 2H), 7.23 (s, 1H) , 7.17 (d, J = 7.6 Hz, 2H), 6.51 (s,
1H), 5.57 (d, J = 9.6 Hz, 1H), 5.32 (d, J = 12.8 Hz, 1H), 5.15ꢀ5.13 (m, 2H), 4.84 (d, J = 12.0
Hz, 1H), 4.33ꢀ4.29 (m, 1H), 4.18ꢀ4.14 (m, 1H), 3.94 (s, 3H), 3.70 (s, 3H), 3.67ꢀ3.62 (m, 1H),
3.08ꢀ3.04 (m, 1H), 2.98ꢀ2.88 (m, 1H), 2.72ꢀ2.68 (m, 1H), 1.29 (d, J = 6.4 Hz, 6H).
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NMR (400 MHz, CDCl3) δ 7.50 (d, J = 7.6 Hz, 2H), 7.22 (s, 1H) , 7.15 (d, J = 7.2 Hz, 2H),
6.51 (s, 1H), 5.59ꢀ5.57 (m, 1H), 5.38 (d, J = 12.0 Hz, 1H), 5.151 (brs, 2H), 4.83 (d, J = 12.4
Hz, 1H), 4.33ꢀ4.29 (m, 1H), 4.18ꢀ4.14 (m, 1H), 3.94 (s, 3H), 3.69 (s, 3H), 3.67ꢀ3.62 (m, 1H),
3.51 (brs, 1H), 2.96ꢀ2.90 (m, 1H), 2.74ꢀ2.70 (m, 1H), 1.31 (s, 9H).
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Compound 31. Method 4. LCMS #2: RT = 4.51 min, m/z = 425.2 [M+1]+. 1H
NMR (400 MHz, DMSOꢀd6) δ 7.46ꢀ7.15 (m, 5H), 7.06 (s, 1H), 6.76 (s, 1H), 6.64 (d, J = 6.0
Hz, 1H), 5.40 (dd, J = 9.7, 6.0 Hz, 1H), 5.22 (d, J = 12.6 Hz, 1H), 5.13 (d, J = 8.3 Hz, 2H),
4.89 (d, J = 12.6 Hz, 1H), 4.16ꢀ4.06 (m, 1H), 3.98 (d, J = 15.6 Hz, 1H), 3.79 (s, 4H), 3.70 (s,
3H), 3.51ꢀ3.42 (m, 1H), 2.89 (dd, J = 15.7, 9.4 Hz, 1H).
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Compound 51e. Method A. Representative example. To a solution of
cyclopentanecarbaldehyde (9.0 g, 92 mmol) in MTBE (30 mL) at 25 °C was added S2Cl2
(7.4 g, 55 mmol). The reaction mixture was then heated to 55 °C and was stirred at that
temperature for 16 h under nitrogen. After cooling to ambient temperature, the solvent was
removed under vacuum and the residue was purified via silica gel column chromatography
(EtOAc:petroleum ether, 1:80) to give 1,1'ꢀdisulfanediyldicyclopentanecarbaldehyde (5.5 g,
46%) as brown oil. 1H NMR (300 MHz, CDCl3) δ 9.23 (s, 2H), 2.41ꢀ1.51 (m, 16H).
To a solution of 1,1'ꢀdisulfanediyldicyclopentanecarbaldehyde (8.5 g, 32.9 mmol) in
THF (60 mL) at 25 °C was added LiAlH4 (2.5 g, 65.8 mmol) in portions. After completion
of the addition, the reaction mixture was stirred at ambient temperature for 2 h. The mixture
was subsequently acidified to pH = 6 by careful addition of 3 M HCl and was then extracted
with EtOAc (2 x 150 mL). The combined organic layers were dried over Na2SO4, filtered,
and concentrated. The residue was purified by silica gel column chromatography
(EtOAc:petroleum ether, 1:20) to give (1ꢀmercaptocyclopentyl)methanol (5.5 g, 63%) as
yellow oil. 1H NMR (300 MHz, CDCl3) δ 3.51 (s, 2H), 2.04 (s, 1H), 1.85ꢀ1.67 (m, 7H), 1.64
(s, 1H).
A mixture of (1ꢀmercaptocyclopentyl)methanol (3.5 g, 26.5 mmol) and 1,2ꢀbis(5ꢀ
nitropyridinꢀ2ꢀyl)disulfane (12.3 g, 39.8 mmol) in CH3OH (50 mL) was stirred at ambient
temperature for 16 h under nitrogen. After the reaction was completed, the mixture was
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concentrated under vacuum, and the residue was purified by silica gel column
chromatography (EtOAc:petroleum ether, 1:10) to give compound 51e (1.9 g, 25%) as a
yellow solid. 1H NMR (400 MHz, DMSOꢀd6): δ 9.24ꢀ9.20 (m, 1H), 8.58 (dd, J = 8.9, 2.7 Hz,
1H), 8.17 (dd, J = 8.9, 0.5 Hz, 1H), 5.18 (t, J = 5.5 Hz, 1H), 3.40 (d, J = 5.5 Hz, 2H), 1.63ꢀ
1.82 (m, 8H).
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Compound 51f. Method C. H NMR (400 MHz, CDCl3): δ 9.31 (s, 1H), 8.33 (dd, J
= 8.8, 2.4 Hz, 1H), 7.58 (d, J = 8.8 Hz, 1H), 3.34 (d, J = 5.6 Hz, 2H), 1.86ꢀ1.84 (m, 2H),
1.67ꢀ1.46 (m, 7H), 1.36ꢀ1.26 (m, 1H).
Compound 51g. To a solution of (tetrahydrofuranꢀ3ꢀyl)methanol (10 g, 98 mmol ) in
CH2Cl2 (300 mL) was added DessꢀMartin periodinane (46 g, 108 mmol) in portions. The
reaction mixture was stirred at 25 °C for 2 h then was filtered. The filtrate was concentrated
under vacuum, and the residue was purified by silica gel column chromatography
(EtOAc:petroleum ether, 1:7) to give tetrahydrofuranꢀ2ꢀcarbaldehyde (4.8 g, 49%) as a
colorless oil. 1H NMR (300 MHz, CDCl3) δ 9.65 (d, J = 2.4 Hz, 1H), 3.96ꢀ3.71 (m, 4H),
2.20 (dd, J = 12.3, 7.4 Hz, 2H). This material was converted to compound 51g using Method
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A. H NMR (400 MHz, DMSOꢀd6) δ 9.32 (d, J = 2.5 Hz, 1H), 8.37 (dd, J = 8.8, 2.6 Hz, 1H),
7.60 (d, J = 8.8 Hz, 1H), 4.12ꢀ3.84 (m, 4H), 3.56 (d, J = 1.5 Hz, 2H), 2.18ꢀ2.00 (m, 2H).
Compound 51h. Method B. Representative example. Ethanethiol (2.74 g, 44.1
mmol) was added to a mixture of 51d (400 mg, 1.47 mmol) in CH2Cl2 (10 mL) at 25 °C. The
reaction mixture was heated to 40 °C and was stirred at that temperature for 30 h. After
cooling to 25 °C, the reaction mixture was slurried with MnO2 (0.20 g) for 5 min and filtered.
The filtrate was concentrated under reduced pressure, and the residue was purified by prepꢀ
TLC (100% CH2Cl2, Rf = 0.5) to give compound 51h (110 mg, 42%) as a colorless oil. 1H
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NMR (400 MHz, CDCl3) δ 3.74 (s, 2H), 2.75ꢀ2.70 (m, 2H), 2.13ꢀ1.87 (m, 6H), 1.84 (s, 1H),
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1.30 (t, J = 7.6 Hz, 3H).
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Compound 51i. Method C. Representative example. I2 (2.33 g, 9.2 mmol) was
added slowly to a solution of (1ꢀmercaptocyclopentyl)methanol (500 mg, 3.78 mmol) and
ethanethiol (2.73 mL, 37.8 mmol) in CH3OH (30 mL) at 0 °C. The mixture was stirred at 8
°C for 1 h. then was concentrated under reduced pressure. The residue was dissolved in
CH2Cl2 (80 mL), and was washed with sat. Na2S2O3 solution (80 mL x 3). The organic layer
was dried over Na2SO4, filtered, and concentrated and the residue was purified by prepꢀTLC
(EtOAc:petroleum ether, 1:5, Rf = 0.4) to give the desired product (300 mg, 41%) as a
colorless oil. 1H NMR (400 MHz, CDCl3) δ 3.57 (d, J = 4.0 Hz, 2H), 2.77ꢀ2.72 (m, 2H),
1.79ꢀ1.68 (m, 8H), 1.31 (t, J = 7.2 Hz, 3H).
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Compound 51j. Method C. H NMR (400 MHz, CDCl3) δ 3.53 (d, J = 4.4 Hz, 2H),
2.73ꢀ2.67 (m, 2H), 1.76ꢀ1.63 (m, 2H), 1.62ꢀ1.55 (m, 8H), 1.31 (t, J = 6.8 Hz, 3H).
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Compound 51k. Method C. H NMR (400 MHz, CDCl3) δ 3.50 (s, 2H), 2.75ꢀ2.70
(m, 2H), 1.39ꢀ1.33 (m, 3H), 1.31 (s, 6H).
Compound 51l. A solution of propaneꢀ2ꢀthiol (2.5 g, 32.8 mmol) in CH2Cl2 (40 mL
was added dropwise with stirring to a solution of 1ꢀchloroꢀ1Hꢀ1,2,3ꢀbenzotriazole (7.26 g,
o
47.3 mmol) and 1Hꢀ1,2,3ꢀbenzotriazole (3.74 g, 31.4 mmol) in CH2Cl2 (200 mL) at ꢀ78 C.
The resulting solution was stirred at ꢀ78 oC for 1 h then was warmed to ꢀ20 oC. A solution of
2ꢀmethylꢀ2ꢀsulfanylpropanꢀ1ꢀol (5.0 g, 47.1 mmol) in CH2Cl2 (20 mL) was subsequently
o
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added dropwise with stirring at ꢀ20 C, and the resulting solution was warmed to 25 C and
stirred overnight. The reaction was quenched by the addition of saturated aqueous Na2S2O3
(200 mL) and the resulting mixture was sequentially with washed with 1 x 200 mL of
saturated aqueous NaHCO3 (1 x 200 mL) and brine (1 x 200 mL). The organic phase was
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dried over Na2SO4, filtered, and was concentrated under vacuum. The residue was purified
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by flashꢀPrepꢀHPLC to afford compound 51l (3.1 g) as light yellow oil. H NMR (300 MHz,
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CDCl3) δ 3.49 (s, 2H), 3.01ꢀ2.92 (m, 1H), 1.33ꢀ1.29 (m, 12H).
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Compound 51m. Prepared in a manner analogous to that described for compound
51l but using 2ꢀmethylpropaneꢀ2ꢀthiol in place of propaneꢀ2ꢀthiol. 1H NMR (300 MHz,
CDCl3) δ 3.43 (s, 2H), 1.34 (s, 9H), 1.29 (s, 6H).
Compound 58a. 1,2ꢀBis(5ꢀnitropyridinꢀ2ꢀyl)disulfane (2.2 g, 7.1 mmol) in CH2Cl2
(15 mL) was added to a solution of (4ꢀmercaptophenyl)methanol (940 mg, 6.7 mmol) in
CH2Cl2 (10 mL) dropwise under N2 over 15 min at 25 °C. The mixture was stirred for
another 1 h at 25 °C, whereupon TLC analysis showed a new product (33% EtOAc in
petroleum ether, Rf = 0.5). MnO2 (1.0 g, 11.5 mmol) was added, and the suspensions was
stirred for another 10 min at 25 °C. The yellow reaction mixture became colorless during this
time. Manganese dioxide was filtered off and filtrate was concentrated, and treated with
EtOH (5.0 mL). The resulting precipitate was filtered to remove the disulfide. The filtrate
was concentrated and the residue was purified by chromatography on silica (solvent gradient:
EtOAc:petroleum ether, gradient elution, 1:2 to 1:1) to afford compound 58a (1.1 g, 54.1%)
as a yellow solid. LCMS #1: RT = 0.73 min, m/z = 294.8 [M+1]+.
Compound 58b. 2ꢀMethylpropaneꢀ2ꢀthiol (2.01 mL, 17.85 mmol) was added to a
solution of (4ꢀmercaptophenyl)methanol (250 mg, 1.78 mmol) in 95% EtOH (10 mL) at 25
°C. The mixture was cooled to 0 °C, and a solution of iodine (200 mg, 0.79 mmol) in 95%
EtOH (10 mL) was added dropꢀwise until the color of the mixture changed from colorless to
brown. After the reaction mixture was stirred for 2 h at 0 °C, saturated NaHCO3 (2.0 mL)
was added until the pH was greater than 7. The mixture was then concentrated in vacuo. The
residue was dissolved in EtOAc (20 mL) and washed sequentially with 10% NaHCO3 (3 x 15
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mL) and brine. The organic layer was dried over Na2SO4, filtered, and was concentrated.
The residue was purified by flash column chromatography (EtOAc:petroleum ether, gradient
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elution, 0:100 to 1:2) to afford compound 58b (280 mg, 69%) as yellow oil. H NMR (400
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MHz, CDCl3) δ 7.51 (d, J = 8.4 Hz, 2H), 7.23 (d, J = 8.4 Hz, 2H), 4.57 (s, 2H), 2.40 (br, 1H),
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1.29 (s, 9H).
Linker-Drug 32. Representative example of linker-drug synthesis. To a solution
of triphosgene (192 mg, 0.65 mmol) in CH2Cl2 (5.0 mL) at 0 °C was added a solution of 51a
(300 mg, 1.29 mmol) and pyridine (306 mg, 3.87 mmol) in CH2Cl2 (5 mL). The mixture was
stirred at 8 °C for 10 min then was added dropwise to a solution of 62 (1.43 mg, 1.68 mmol)
and pyridine (306 mg, 3.87 mmol) in CH2Cl2 (5.0 mL) at 0 °C. The reaction mixture was
stirred at 8 °C for 30 min then was concentrated under reduced pressure. The residue was
purified by flash column chromatography (EtOAc:petroleum ether, 1:1) to give 63a (0.50 g,
33%) as a yellow oil. LCMS #1: RT =1.08 min, m/z = 1111.7 [M+1]+.
To a solution of 63a (300 mg, 0.27 mmol) in CH2Cl2 (10 mL) was added iPrSH (206
mg, 2.7 mmol) at 0 °C. The mixture was stirred at 8 °C for 10 min, then MnO2 (100 mg) was
added and the resulting suspension was stirred for 5 min and filtered. The filtrate was
concentrated to give the crude product, which was purified by flash column chromatography
(EtOAc:petroleum ether, 1:1) to give 63b (210 mg, 75%) as a yellow solid. LCMS #1: RT
=1.27 min, m/z = 1031.5 [M+1]+.
To a solution of triphosgene (30 mg, 0.10 mmol) in CH2Cl2 (5.0 mL) was added a
solution of 63b (210 mg, 0.20 mmol) and Et3N (62 mg, 0.61 mmol) in CH2Cl2 (5 mL) at 0
°C. The mixture was stirred at 8 °C for 30 min, then a solution of 64 (140 mg, 0.24 mmol)
and Et3N (62 mg, 0.61 mmol) in DMF (5 mL) was added dropwise. The mixture was stirred
at 8 °C for 12 h, then was concentrated under reduced pressure. Purification of the residue by
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flash column chromatography (8% CH3OH in CH2Cl2) gave 65b (140 mg, 42%) as a yellow
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oil. LCMS #1: RT =1.25 min, m/z = 816.1 [M/2+1]+.
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A solution of 65b (140 mg, 0.09 mmol) in acetic acid (3.0 mL), THF (2.0 mL) and
water (2.0 mL) was stirred at 8 °C for 8 h. The mixture was added to EtOAc (60 mL),
washed with water (50 mL x 3), sat. NaHCO3 (50 mL), brine (50 mL). The organic layer was
dried over Na2SO4, filtered, and was concentrated to give crude 66b (120 mg, 100%) as a
white solid. LCMS #1: RT =0.79 min, m/z =701.0 [M/2+1]+.
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To a solution of 66b (130 mg, 0.09 mmol) in DMSO (3.0 mL) was added IBX (130
mg, 0.46 mmol) at 10 °C. The reaction mixture was warmed to 50 °C and was maintained at
that temperature for 48 h. The mixture was purified by prepꢀHPLC (acetonitrile 35–65% /
0.225% FA in water) to give 32 (10 mg, 8%) as a white solid. LCMS #1: RT = 0.73 min, m/z
=1397.9 [M+1]+.
Linker-Drug 33. LCMS #1: RT = 0.76 min, m/z = 1423.4 [M+1]+.
Linker-Drug 34. LCMS #1: RT = 0.76 min, m/z = 1437.7 [M+1]+.
Linker-Drug 35. LCMS #1: RT = 0.81 min, m/z = 737.1 [M/2+1]+.
Linker-Drug 36. LCMS #1: RT = 0.73 min, m/z = 677.5 [M/2+1]+.
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