Design, Synthesis, and Structure-Activity Relationships of Novel Tetrahydroisoquinolino Benzodiazepine Dimer Antitumor Agents and Their Application in Antibody-Drug Conjugates

Article abstract of DOI:10.1021/acs.jmedchem.0c01385

A series of tetrahydroisoquinoline-based benzodiazepine dimers were synthesized and tested for in vitro cytotoxicity against a panel of cancer cell lines. Structure-activity relationship investigation of various spacers guided by molecular modeling studie

Full text of DOI:10.1021/acs.jmedchem.0c01385

pubs.acs.org/jmc
Article
Design, Synthesis, and Structure−Activity Relationships of Novel
Tetrahydroisoquinolino Benzodiazepine Dimer Antitumor Agents
and Their Application in Antibody−Drug Conjugates
Naidu S. Chowdari,* Yong Zhang, Ivar McDonald, Walter Johnson, David R. Langley,
Prasanna Sivaprakasam, Robert Mate, Tram Huynh, Srikanth Kotapati, Madhura Deshpande, Chin Pan,
Daniel Menezes, Yichong Wang, Chetana Rao, Ganapathy Sarma, Bethanne M. Warrack,
Vangipuram S. Rangan, Sung Mei-Chen, Pina Cardarelli, Shrikant Deshpande, David Passmore,
Richard Rampulla, Arvind Mathur, Robert Borzilleri, Arvind Rajpal, Gregory Vite, and Sanjeev Gangwar
Cite This: https://dx.doi.org/10.1021/acs.jmedchem.0c01385
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: A series of tetrahydroisoquinoline-based benzodiazepine dimers were synthesized and tested for in vitro cytotoxicity
against a panel of cancer cell lines. Structure−activity relationship investigation of various spacers guided by molecular modeling
studies helped to identify compounds with picomolar activity. Payload 17 was conjugated to anti-mesothelin and anti-fucosylated
monosialotetrahexosylganglioside (FucGM1) antibodies using lysosome-cleavable valine−citrulline dipeptide linkers via
heterogeneous lysine conjugation and bacterial transglutaminase-mediated site-specific conjugation. In vitro, these antibody drug
conjugates (ADCs) exhibited significant cytotoxic and target-mediated selectivity on human cancer cell lines. The pharmacokinetics
and efficacy of these ADCs were further evaluated in gastric and lung cancer xenograft models in mice. Consistent pharmacokinetic
profiles, high target specificity, and robust antitumor activity were observed in these models after a single dose of the ADC-46 (0.02
μmol/kg).
the increasing prevalence of drug-resistant cancers.10 Pyrrolo-
[2,1-c][1,4]benzodiazepines (PBDs) are naturally occurring
INTRODUCTION
■
Antibody drug conjugates (ADCs) are gaining momentum in
drug discovery for cancer therapy.1 FDA approval of
compounds isolated from Streptomyces species and are known
for their antitumor activity.11 PBDs have been shown to bind
brentuximab vedotin,2 trastuzumab emtansine,3 and gemtuzu-
mab ozogamicin4 for various cancers largely fueled research in
in the minor groove of DNA, where they covalently cross-link
exocyclic amino groups of guanine. PBD dimers with increased
this area in both academic laboratories and the pharmaceutical
industry. The first two drugs contain tubulin inhibitor payloads
auristatin and maytansinoid, while gemtuzumab ozogamicin
contains a DNA cleaving payload, calicheamicin. Subsequently,
potency and selective DNA interstrand cross-linking have
subsequently been reported.12 Recently, PBDs 1 and 2 were
employed as potent payloads in several ADCs currently being
four more ADCs were approved based on these payloads for
other cancer indications.5−7 Most recently, camptothecin-
Received: August 27, 2020
based ADCs, sacituzumab govitecan,8 and trastuzumab
deruxtecan9 were approved for the treatment of metastatic
breast cancer.
Identification of novel and potent payloads with different
mechanisms of action is becoming increasingly important with
https://dx.doi.org/10.1021/acs.jmedchem.0c01385
© XXXX American Chemical Society
J. Med. Chem. XXXX, XXX, XXX−XXX
A

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
evaluated in clinical trials.13,14 An indolinobenzodiazepinene
monomer 3 using a five-carbon spacer, we obtained compound
4, which exhibited subnanomolar potency across different cell
lines (Table 1). We maintained the S stereochemistry on the
new monomer, which should facilitate tight binding in the
minor groove, consistent with reported data for PBDs.20
Addition of a dimethyl amino group resulted in compound 5
with reduced potency. Introduction of a double bond into a
five-membered ring provided compound 6 with improved
potency. Replacement of the methyl group on an endocyclic
double bond with aryl groups followed by decreasing the
spacer length to three-carbon provided compounds 7−9 that
gave a three- to sixfold reduction in potency. Addition of a
methoxy group resulted in compounds 8 and 9 with twofold
improvement in potency. Swapping an NH2 group from the
pyrrolidine monomer to the THIQ monomer did not alter the
activity (8 vs 9). In general, THIQ-based compounds had IC50
values comparable to traditional PBD dimers.
dimer-based ADC is also in development.15
Based on molecular modeling studies, we envisioned that
replacement of the pyrrolidine ring in PBD, DC-81, with
tetrahydroisoquinoline (THIQ) would result in compound 3
with additional DNA binding to provide improved activity
(Figure 1).16−19 We report here our structure−activity
Table 2 summarizes our efforts on different aliphatic spacers.
We prepared payloads with spacer lengths of three and five
carbons. Compound 10, with a three-carbon spacer showed
subnanomolar potency. Introduction of an NH2 group
provided compounds 11 and 12 with twofold loss in potency.
Compound 13 is a THIQ-benzodiazepine version of
compound 2. As originally hypothesized, the THIQ-
benzodiazepine monomeric units in compound 13 showed
some additional van der Waals contacts and CH−π
interactions with the sugar-phosphate backbone in our DNA
binding models compared to compound 2, which has the
classical PBD units. Compound 13 with a five-carbon spacer
was found to be twofold more potent compared to compound
10 with the three-carbon spacer. The higher potency of
compound 13 was attributed to better binding with the DNA
(Figure 2). Unlike the three-carbon spacer analogue 10, the
five-carbon spacer analogue 13 exhibited a fully extended
conformation of the alkyl spacer and presented the THIQ-
benzodiazepine monomeric units slightly differently in the
minor groove. This may have impacted favorably the covalent
linking with the guanine base and/or maximized the van der
Figure 1. Structures of benzodiazepine-based dimers and monomers.
relationship (SAR) findings for various THIQ-based dimers
with different spacer lengths and functional groups. Finally,
compound 17 was conjugated to anti-mesothelin and anti-
FucGM1 antibodies using a lysosome-cleavable valine−citrul-
line dipeptide linker, and the ADCs were evaluated for
antitumor activity in mouse xenograft models.
RESULTS AND DISCUSSION
■
To understand the SAR of THIQ-based benzodiazepine
dimers, we prepared a series of compounds with pyrrolidine
and THIQ rings with different spacer lengths. These dimers
were evaluated in human cellular proliferation assays using
lung (H226), gastric (N87), ovarian (OVCAR3), and colon
(HCT116) carcinoma cell lines. By coupling DC-81 with
a
Table 1. Cytotoxicity Data for Representative Benzodiazepine Dimers (IC50 in nM)
a
Cancer cell lines were incubated with compounds for 72 h. IC50 values were determined by quantitating viable cells using a CellTiter-Glo
luminescent assay. NA = not available.
B
https://dx.doi.org/10.1021/acs.jmedchem.0c01385
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
a
Table 2. Cytotoxicity Data for the Dimers with Aliphatic Spacers (IC50 in nM)
a
Cancer cell lines were incubated with compounds for 72 h. IC50 values were determined by quantitating viable cells using a CellTiter-Glo
luminescent assay.
Waals contacts with the sugar-phosphate backbone in
compound 13 relative to compound 10. This phenomenon
of higher potency for five-carbon spacers versus three-carbon
spacers is in agreement with reported data for PBDs.21 Unlike
compound 11, introduction of NH2 groups for compounds
with five-carbon spacers increased potency sixfold as shown in
the case of 14 and 15. Spacers with ether (16), amino (17),
and amide (18) groups showed reduced activity. Hydroxy and
methoxy substituents on pendent phenyl ring were well
tolerated with retention of high potency. Ethylene glycol
monomer (21) and dimer (22) substitution on the aryl moiety
were also well tolerated. Glycine amide 23 exhibited 18-fold
reduction in potency compared to 14.
Table 3 summarizes dimers with aryl spacers. Introduction
of a benzene ring into the spacer with metasubstitution
provided compound 24 with more than threefold improve-
ment in potency compared to its aliphatic sibling 13 with a
pentyl spacer. This improvement in activity is likely attributed
to the CH−π interactions exhibited by the phenyl spacer
acceptor of 24 with the aliphatic CH donors from the sugar
moieties in the DNA. para and ortho substitution resulted in
compounds 25 and 26 with substantially reduced activity.
Molecular modeling studies highlighted the high complemen-
tarity of the angle of curvature in the meta-phenyl spacer
compound 24 with the DNA minor groove curvature (Figure
2C). The para-phenyl spacer compound 25 loses its ideal
geometry for the minor groove binding, and this may in part
explain the reduced potency. The ortho-phenyl spacer
compound 26 deviates the most from its ideal geometry to
complement the minor groove resulted in significant loss of
potency. One of the two payload units is precluded from
binding in the minor groove because of the least optimal
geometrical separation of the monomeric payload units in
compound 26 relative to compounds 24 and 25. Amino (27),
azido (28), and azido methyl (29) groups are well tolerated on
the benzene ring. These groups can provide another viable
option for appending linkers through amide bond and click
chemistry. We incubated compound 27 with a double-stranded
DNA at 37 °C for 4 h and identified DNA a cross-linking
adduct as confirmed by liquid chromatography−mass spec-
trometry (LCMS) (see the Supporting Information). This is
similar to the findings reported by others for PBDs.12
We then moved to introducing heterocyclic spacers (Table
4), which could potentially help to improve aqueous solubility.
Compound 30, with pyridine in the spacer, exhibited activity
similar to phenyl-containing compound 24. Walking the
nitrogen to other carbons on the ring did not impact the
activity (31 and 32). Introduction of an amino group on one
monomer (33) also did not alter the activity. Introduction of
CO2Me (34), Cl (35), and OMe (36) groups at the para-
position on the pyridine spacer was also well tolerated.
Hydroxy (37) and hydroxymethyl (38) substituents reduced
activity more than 100-fold. Methoxy substitution at the meta-
position (39) also lost 40-fold activity compared to the para-
position (36). Compound 40 with a pyrazine spacer showed
excellent activity similar to pyridine analogues 30−32. Pyrazine
C
https://dx.doi.org/10.1021/acs.jmedchem.0c01385
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Figure 2. Putative binding model of compound 13 illustrating the formation of interstrand covalent cross-linking in an 8-mer duplex DNA and
ligand overlay of compounds 24, 25, and 26. (A) Surface representation of compound 13 (green carbon) bound to DNA. Compound 13 fits snugly
in the DNA minor groove because of its high shape complementarity. (B) Specific interactions exhibited by compound 13 are highlighted.
Compound 13 is hypothesized to make covalent bonds with the exocyclic N2 amino groups of guanines on both strands through its C11/C11′
reactive centers. The hydrogen bonds between the N10/N10′ of compound 13 and carbonyl oxygens on C2 of cytosines (adjacent to the
covalently modified guanines) are denoted as yellow dotted lines. The inset in B shows the nucleotide sequence of the 8-mer duplex DNA and the
putative cross-linking and hydrogen bonding sites of compound 13 (magenta guanines and cyan cytosines, respectively). (C) Ligand overlay of
optimized structures of compounds 24 (green), 25 (magenta), and 26 (blue). The green arc shows the angle of curvature provided by the central
phenyl spacer to the two monomeric payload units in compound 24. The magenta curve shows the slightly flattened corresponding angle of
curvature in compound 25. The blue curve shows the highly contracted angle of curvature in compound 26. The 1,3-, 1,4-, and 1,2-attachment
points in the central phenyl spacer for the two monomeric payload units create moderate to significant differences in the overall angle of curvatures
in compounds 24, 25, and 26 to complement the curvature of the DNA minor groove. The figure was prepared using Pymol (The PyMOL
̈
Molecular Graphics System, Versions 1.6.1.0 and 2.0 Schrodinger, LLC).
41 with a para-linkage showed reduced activity compared to
the meta-linkage, a trend similar to the aryl spacers observed
for compounds 24−26 in Table 3. Furan (42) and thiophene
(43) containing spacers resulted in significant loss in activity.
Antibody Drug Conjugates. In the literature, PBD
payloads 1 and 2 were conjugated to antibodies using
maleimide chemistry through reduced interchain disulfide
bonds and engineered cysteines. Payload 1 was coupled with a
protease cleavable valine−alanine dipeptide linker using C-2
aniline,13 whereas 2 was linked through the N10 position.14
We preferred valine−citrulline over valine−alanine because of
more efficient enzymatic cleavage of the former dipeptide
linker. From our SAR studies, we have chosen highly potent
payload 14 and moderately potent payload 17 to append
linkers for making conjugates. To evaluate these two payloads,
we prepared lysosome cleavable valine−citrulline dipeptide
linker-payload compounds 44 and 45 (Figure 3) and
conjugated to an antibody which targets the mesothelin
antigen22 using heterogeneous thiolated lysine conjugation.
Mesothelin is a 40 kDa protein present on normal mesothelial
cells and overexpressed in several human cancer types,
including mesothelioma, ovarian, nonsmall cell lung, gastric,
and pancreatic adenocarcinoma. Some of the lysines present
on the antibody were modified with 2-iminothiolane (2-IT) to
D
https://dx.doi.org/10.1021/acs.jmedchem.0c01385
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Table 3. Cytotoxicity Data for the Dimers with Aromatic
Spacers (IC50 in nM)
Table 4. Cytotoxicity Data for the Dimers with Heterocyclic
Spacers (IC50 in nM)
a
a
a
Cancer cell lines were incubated with compounds for 72 h. IC50
values were determined by quantitating viable cells using a CellTiter-
Glo luminescent assay.
introduce thiol groups,23 which were then used to conjugate
linker-payloads using a Michael reaction through the
maleimide group. These mesothelin-targeted ADCs 44 and
45 were tested for in vitro cytotoxic activity on N87 cell lines
and found to exhibit IC50 of 0.01 and 0.04 nM, respectively.
We have also prepared ADC-45 with PEG 8, and in vitro
cytotoxicity was found to be similar to ADC-45 without PEG.
The IC50 value of ADC-45 was 32-fold lower than the
corresponding payload alone (1.28 nM), while ADC-44 and its
payload 14 (0.03 nM) did not show much difference between
their in vitro activity profiles. To understand the discrepancy of
ADC-45 and its payload activity, we measured the permeability
of payloads 14 and 17 using a parallel artificial membrane
permeability assay and found them to be 335 and 0 nM/s,
respectively. The moderate activity of payload 17 alone in cell
lines is attributed to the poor permeability, whereas ADC-45
exhibited higher potency by overcoming the permeability issue.
We envisioned that ADCs derived from poorly permeable
payloads might be beneficial in preventing the leakage of the
payloads from the cancer cells to the neighboring healthy
tissue, thereby avoiding bystander killing. In addition, any
premature release of payload in the systemic circulation might
cause less harm to healthy tissue. This prompted us to choose
ADC-45 for further evaluation in tumor xenograft models.
We were interested in site-specific conjugation to take
advantage of any benefits that this might offer. In this regard,
linker-payloads 46−48 with an amino handle were prepared
for bacterial transglutaminase (BTGase)-mediated site-specific
conjugation24 with glutamine at the Q295 position on
mesothelin antibody. Antibodies with a glutamine handle
a
Cancer cell lines were incubated with compounds for 72 h. IC50
values were determined by quantitating viable cells using a CellTiter-
Glo luminescent assay.
were generated by insertion of an EEQYASTY tag at the Q295
position through N297A mutation.25 Site-specific conjugation
was then achieved using a BTGase enzyme-mediated trans-
E
https://dx.doi.org/10.1021/acs.jmedchem.0c01385
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Figure 3. Structures of ADCs 44−48.
amidation with amine containing linker-payloads, producing
ammonia as a byproduct. Linker-payloads 45−48 were
conjugated to anti-mesothelin, anti-CD70,26 and anti-
FucGM1 antibodies,27 which specifically bind to target
antigens expressed on N87 gastric cancer (mesothelin+),
786-O renal cell carcinoma (CD70+), and H187 (FucGM1+)
lung carcinoma cell lines, respectively. Having the payload
conjugated to three different antibodies, tools have been used
to evaluate cytotoxicity in multiple tumor models and to
examine the specificity and selectivity of this targeting
approach (i.e., ADC with targeted antibody vs nontargeting
antibody and assessments in cell lines with and without the
respective target antigens). Lysine conjugation provided
heterogeneous ADCs with an average drug to antibody ratio
(DAR) of 1 for ADC-44 and 3 for ADC-45, whereas site-
specific conjugation provided homogeneous ADCs with a DAR
of 2. First, we tested the mesothelin-targeted ADCs 45−48 for
cytotoxic activity on N87 cell lines with IC50 in the range of
0.02−0.08 nM (Table 5). We did not observe significant
differences in in vitro potencies between the heterogeneous
conjugate 45 and homogeneous site-specific conjugates 46−
48. Nontargeting anti-CD70-45 and anti-FucGM1-45 showed
significantly lower activity on the N87 cell line with IC50 of 9.9
and 9.2 nM, respectively, confirming more than 200-fold
specificity of the anti-mesothelin ADCs. Next, to confirm if
these ADCs could be cleaved enzymatically using a lysosomal
protease cathepsin B, ADC incubations were performed with
cathepsin B in the 2-morpholinoethanesulfonic acid (MES)
buffer (pH 5.5) at 37 °C for 4 h. These data revealed that
>70% of payload was cleaved within 4 h, indicating that they
are good substrates for enzyme binding at the cleavage site. To
determine stability in vitro, ADCs were incubated in mouse
serum at 37 °C for 24 h and found to be generally stable with
Table 5. In Vitro Stability and Cytotoxicity Data for the
Anti-mesothelin ADCs
% payload released
cathepsin B
b
mouse
c
IC50 for N87 cells
a
d
ADC
PEG DAR
3
digestion
serum
(nM)
meso-45
meso-46
meso-47
meso-48
83
73
85
3
1.9
1
0.04
0.02
0.08
0.08
2
4
8
1.9
1.9
1.8
100
1.5
a
DAR = drug−antibody ratio was measured by UV spectroscopy and
b
LC−MS methods. ADCs were incubated with cathepsin B enzyme in
MES buffer (pH 5.5) at 37 °C for 4 h and the cleaved payload was
quantified by the LC−MS/MS MRM method. ADCs incubated in
mouse serum at 37 °C for 24 h and the released payload was
c
d
quantified using the LC−MS/MS MRM method. Cancer cell lines
were incubated with compounds for 72 h. IC50 values were
determined by quantitating viable cells using a CellTiter-Glo
luminescent assay.
minimal loss of payload (<3%). Site-specific conjugates with
deeply embedded payloads are relatively more stable in serum
compared to the heterogeneous conjugates, with their greater
surface exposure of payloads.
We then evaluated these ADCs in preclinical studies to
characterize their pharmacokinetics (PKs), tolerability, and
efficacy profiles. Unconjugated anti-mesothelin antibody (1
mg/kg) and its conjugates ADC-45 and ADC-46 (0.02 μmol/
kg) were administered to mice intraperitoneally (IP). Plasma
samples were collected at various time points post single-dose
administration and subject to quantitative ELISA and mass
spectrophotometry analyses. Unconjugated antibody in circu-
lation was assessed using an antihuman IgG reagent. ADC-
conjugated payload was measured using 19H9 and 20F7
F
https://dx.doi.org/10.1021/acs.jmedchem.0c01385
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Figure 4. Plasma PK disposition of heterogeneous ADC-45 (A) and site-specific ADC-46 (B). Concentration of total ADC and unconjugated
antibodies in plasma was determined with an ELISA-based quantitative assay.
Figure 5. (A) Percent body weight change from randomization of wild-type BALB/c mice treated with 0.005, 0.02, and 0.05 μmol/kg single dose
of anti-Meso-45. (B) In vivo antitumor activity of the anti-Meso-45 in N87 gastric xenograft cancer model in SCID mice.
G
https://dx.doi.org/10.1021/acs.jmedchem.0c01385
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Figure 6. (A) Percent body weight change from randomization after a single administration of 0.05 μmol/kg of site-specific anti-mesothelin ADCs
with varying PEG lengths in the N87 gastric tumor xenograft model in SCID mice. (B) In vivo antitumor activity of site-specific anti-mesothelin
ADC-46 in the N87 xenograft model.
antibodies that recognized the drug-linker component of ADC.
ADC-45 prepared through heterogeneous lysine conjugation
has a plasma half-life of 6 days compared to its unconjugated
antibody counterpart, which has a half-life of 10 days (Figure
4). Site-specific ADCs have shown a longer half-life of 16 days.
We did not observe a difference between the half-life of ADC-
46 and its corresponding unconjugated antibody. In general,
heterogeneous conjugates were cleared almost threefold faster
than the corresponding site-specific conjugates.
47), and 8 (ADC-48). To examine the impact of varying ADC
PEG length on tolerability, mice were then administered a
single IP dose of 0.05 μmol/kg. Mice treated with PEG 2 and 4
ADCs showed <10% body weight loss, while those treated with
the PEG 8 ADC exhibited >10% body weight loss (Figure 6).
Although we did not see the effect of PEG length on mouse
serum stability in vitro over 24 h (Table 5), PEG 8 ADC lost
its payload overtime in circulation, causing associated toxicity.
Thus, we selected PEG 2-incorporated anti-mesothelin ADC-
46 for further efficacy evaluation. In the N87 gastric tumor
xenograft model, mice were administered a single IP dose of
either 0.005, 0.02, or 0.05 μmol/kg of meso-46 ADC, and
tumor volume measurements were performed over the 60 day
study duration (Figure 6B). Dose-responsive antitumor activity
in vivo was observed with meso-46 ADC against N87 tumors.
Mice that received doses of 0.02 and 0.05 μmol/kg showed
complete tumor regression, whereas those that had received
the 0.005 μmol/kg dose showed a partial response. From a
comparison across studies, the site-specific ADCs appeared
twofold more potent when compared with their heterogeneous
ADC counterparts at similar doses. The higher efficacy
observed with site-specific conjugates may be attributed to
the higher stability and drug exposure because of lack of
competitive inhibition of antigen resulting from DAR 1 and
unconjugated antibody species, which are known to negatively
impact heterogeneous conjugates.24,25
We next focused on evaluating these ADCs for toxicity and
efficacy in preclinical models in vivo (Figure 5). A single IP
dose of ADC-45 to wild-type BALB/c mice demonstrated that
ADCs were well tolerated up to the maximal dose tested of
0.05 μmol/kg corresponding to 2.5 mg/kg (Figure 5A). No
significant body weight loss was observed relative to vehicle-
treated mice. In addition, no clinical symptoms of adverse
tolerability were reported in mice. In vivo efficacy studies were
conducted in a mesothelin-expressing N87 human gastric
tumor xenograft model in SCID mice. Targeted anti-
mesothelin ADC-45 was administered at doses of 0.005,
0.02, and 0.05 μmol/kg. Anti-FucGM1 ADC-45 was
administered with 0.02 μmol/kg dose and served as a
nontargeted, isotype-ADC control. Treatment with anti-
mesothelin ADC-45 resulted in a dose-dependent antitumor
activity against N87 tumors (Figure 5B). The mice that
received an ADC dose of 0.005 μmol/kg elicited a partial
tumor response, whereas doses of 0.05 μmol/kg resulted in
complete tumor regression. While the anti-mesothelin ADC-45
significantly inhibited tumor growth, the nontargeted anti-
FucGM1 ADC-45 demonstrated partial tumor growth
inhibition at a dose of 0.02 μmol/kg, thus confirming
immunological specificity. In the case of site-specific ADCs,
we prepared ADCs with PEG lengths of 2 (ADC-46), 4 (ADC-
To demonstrate the broader scope of the ADC activity
across tumor models, efficacy was explored in a SCLC tumor
model. FucGM1 is highly expressed in a large subset of SCLC
cancers,27 wherein therapeutic antibodies were previously
developed against this target. To further improve the
therapeutic benefit of these anti-FucGM1 antibodies, linker-
payloads 45−48 were conjugated to generate anti-FucGM1-
H
https://dx.doi.org/10.1021/acs.jmedchem.0c01385
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
ADCs. FucGM1 ADCs were tested for cellular activity on the
FucGM1-expressing H187 SCLC cell line in vitro. H187 cells
do not express mesothelin and CD70, and hence, anti-
mesothelin and anti-CD70 ADCs were used as nonbinding,
isotype specific controls. FucGM1 ADCs 45−48 showed good
in vitro potency (IC50 = 1−3.5 nM) compared to isotype
control ADCs (IC50 = 10−100 nM) (Table 6). The potency of
and 0.06 μmol/kg demonstrated strong and durable antitumor
activity (seen as complete tumor regressions). Nontargeting
anti-CD70-45 ADC administered at 0.01 μmol/kg, although
exhibiting some partial inhibition of tumor growth, induced
less activity than an equivalent dose of FucGM1ADC-45. To
compare the efficacy of heterogeneous lysine conjugates of
FucGM1 ADC-45 to the site-specific FucGM1 ADC-46, H187
tumor-bearing mice were treated with 0.02 and 0.005 μmol/kg
of each ADC (Figure 7B). At each of the dose levels tested, the
site-specific ADCs were more efficacious compared to the
heterogeneous lysine conjugates.
Table 6. In Vitro Antitumor Activity of ADCs on H187
Cells
a
b
ADC
PEG
DAR
IC50 (nM)
FucGM1-45
CD70-45
FucGM1-46
meso-46
FucGM1-47
meso-47
2.5
2.8
1.9
1.9
1.9
1.9
1.9
1.8
1
CHEMISTRY
■
10.6
2.2
>100
3.5
37.8
1.9
26
Synthesis of Payloads. Payloads 4−43 were prepared as
depicted in Scheme 1 using three common intermediates 49−
51 and various alkyl, aryl, and heteroaromatic spacers (Figure
8). SEM and Alloc protecting groups were chosen because of
their robustness in later stage transformations for the synthesis
of payloads and linker-payloads. Payloads were assembled from
monomers through alkylation or a Mitsunobu reaction using
various spacers. Dimers with SEM-protecting groups were
reduced with LiBH4 or LiEt3BH (superhydride), followed by
mild acid treatment (silica gel or formic acid) to obtain bis-
imine compounds. In the case of Alloc-protected compounds,
Pd(PPh3)4 in the presence of pyrrolidine or morpholine was
used for deprotection to prepare bis-imine compounds.
Monomer 49 was prepared in five steps from nitro ester
derivative 52 (Scheme 2). Compound 52 was treated with
aqueous NaOH at 50 °C for 5 h to provide acid 53, which was
reacted with oxalyl chloride, followed by quinoline derivative
54 to obtain amide 55 in 98% yield. The latter was treated with
Zn/NH4Cl to provide reductive cyclization product 56 in 96%
yield. The SEM-protecting group was installed using NaH as
the base to provide 58, which was subjected to debenzylation
under palladium-catalyzed hydrogenation to obtain monomer
49 in 83% yield.
2
2
4
4
8
8
FucGM1-48
meso-48
a
DAR = drug−antibody ratio was measured by UV spectroscopy and
b
LC−MS methods. Cancer cell lines were incubated with compounds
for 72 h. IC50 values were determined by quantitating viable cells
using a CellTiter-Glo luminescent assay.
FucGM1 ADCs against H187 SCLC cells was more than 10-
fold lower compared to anti-mesothelin ADCs against N87
gastric cells (the IC50 value ranged from 0.02 to 0.08 nM,
Table 5). This may be attributed to the lower expression
density and kinetics of receptor internalization of FucGM1 on
H187 SCLC cells.
To examine the efficacy of anti-FucGM1 ADC-45, mice
were dosed with a single IP administration of 0.01, 0.03, or
0.06 μmol/kg, and tumor growth was monitored for 55 days
(Figure 7A). While ADC-45 dosed at 0.01 μmol/kg elicited
significant tumor growth inhibition, the higher doses of 0.03
Figure 7. (A) In vivo antitumor activity of anti-FucGM1-45 ADC in the H187 SCLC xenograft model. (B) In vivo antitumor activity of anti-
FucGM1-45 ADC vs site-specific anti-FucGM1-46 ADC in the H187 SCLC xenograft model in SCID mice.
I
https://dx.doi.org/10.1021/acs.jmedchem.0c01385
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Scheme 1. Retrosynthetic Analysis of Benzodiazepine Dimers
Figure 8. Monomers and spacers employed in this work.
a
Scheme 2. Synthesis of SEM Monomer 49
a
Reagents and conditions: (a) NaOH, THF−water, 50 °C, 5 h, 100%; (b) (1) (COCl)2, THF, DMF, rt, 2 h; (2) 54, Et3N, THF, 4 h, 98%; (c) Zn,
NH4Cl, MeOH, 50 °C, 16 h, 96%; (d) 57, NaH, DMF, rt, 1 h, 78%; (d) 10% Pd/C, H2, EtOH, rt, 3 h, 83%.
Monomer 50 was prepared as shown in Scheme 3.
Quinoline derivative 59 was protected with TBS to furnish
60, which was reacted with benzoic acid derivative 61 using
HATU coupling to give amide 62 in 66% yield. The nitro
group was reduced with Zn/NH4Cl and protected with Alloc
to provide compound 64. The TBS-protecting group was
removed by treatment with methanolic HCl to obtain 65,
which was subjected to Swern oxidation to obtain compound
66 in 92% yield. The hydroxyl group was protected with TBS
using the TBS-OTf reagent in the presence of 2,6-lutidine to
give fully protected compound 67 in 97% yield. Selective TIPS
deprotection in the presence of TBS was accomplished with
lithium acetate in DMF−water to provide Alloc monomer 50
in 71% yield.
Compound 53 was converted to acid chloride and coupled
with 70 to provide amide 71, which was subjected to Zn-
promoted reduction of both nitro groups to give cyclization
product 72 in 82% yield. Trityl protection provided 73, which
was subsequently protected with SEM to obtain 74 in 80%
yield. Trityl and benzyl protecting groups were removed using
palladium-catalyzed hydrogenation to obtain 75. Bis Alloc
protection followed by selective hydrolysis of phenolic
carbonate with LiOH provided monomer 51 with 75% yield.
Payload 4 was synthesized as shown in Scheme 5. Monomer
49 was treated with 1,5-diiodopentane 4a (5 equiv) in the
presence of K2CO3 at room temperature for 2 h to obtain
monoalkylated compound 4b in 77% yield. Compound 4b was
coupled with a known lactam 4c29 in the presence of K2CO3
and treated with SEM-Cl to obtain compound 4e in 20% yield
over two steps. Dimer 4e was subjected to LiBH4 reduction,
followed by silica gel treatment to obtain payload 4.
Monomer 51 was prepared as shown in Scheme 4.
Commercially available amino acid 68 was esterified with
ethanol in the presence of sulfuric acid to provide 69, which
was subjected to nitration to furnish 70 in 91% yield.
J
https://dx.doi.org/10.1021/acs.jmedchem.0c01385
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
a
Scheme 3. Synthesis of Alloc Monomer 50
a
Reagents and conditions: (a) TBSCl, imidazole, CH3CN, rt, 1.5 h, 68%; (b) 61, HATU, DCM, i-Pr2NEt, rt, 24 h, 66%; (c) Zn, NH4Cl, MeOH, rt,
24 h, 83%; (d) allyl chloroformate, pyridine, DCM, −78 °C to rt, 1 h, 88%; (e) HCl, MeOH, 20 min, rt, 84%; (f) (COCl)2, DMSO, DCM, −78 °C
to rt, 30 min, 92%; (g) TBS-OTf, 2,6-lutidine, 0 °C, 30 min, 97%; (h) LiOAc, DMF−water, rt, 16 h, 71%.
a
Scheme 4. Synthesis of Monomer 51
a
Reagents and conditions: (a) H2SO4, EtOH, 80 °C, 20 h, 83%; (b) H2SO4, HNO3, −5 °C, 30 min, 91%; (c) (1) 53, (COCl)2, THF, DMF, rt, 2 h;
(2) 70, Et3N, THF, rt, 30 min, 78%; (d) Zn, NH4Cl, MeOH, 50 °C, 16 h, 82%; (e) Trityl-Cl, Et3N, DCM, rt, 3 h, 88%; (f) SEM-Cl, NaH, DMF, 0
°C to rt, 2 h, 80%; (g) 10% Pd/C, H2, EtOAc, rt, 2 days, 90%; (h) (1) allyl chloroformate, Et3N, 0 °C, 2 h; (2) LiOH, MeOH−water, rt, 16 h, 75%.
Payload 5 was prepared as described in Scheme 6.
Compound 72 was subjected to reductive amination with
formaldehyde in the presence of sodium cyanoborohydride to
obtain dimethylamino compound 5a in 98% yield. This was
treated with SEM-Cl in the presence of NaH followed by
debenzylation using Pd/C to provide compound 5c in 76%
yield. This was reacted with 5d in the presence of K2CO3 to
obtain SEM-dimer 5e in 82% yield. LiBH4 reduction followed
by silica gel treatment provided payload 5 in 44% yield.
K
https://dx.doi.org/10.1021/acs.jmedchem.0c01385
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
a
Scheme 5. Synthesis of Payload 4
a
Reagents and conditions: (a) 49, K2CO3, DMSO, rt, 2 h, 77%; (b) 4c, K2CO3, DMF, 50 °C, 2 h; (c) SEM-Cl, NaH, DMF, 0 °C to rt, 2 h, 20%;
(d) (1) LiBH4, THF, −78 °C, 1 h; (2) Silica gel, CHCl3−EtOH−water, 24 h, 9%.
a
Scheme 6. Synthesis of Payload 5
a
Reagents and conditions: (a) 72, NaBH3CN, formaldehyde, AcOH, THF, rt, 2 h, 98%; (b) SEM-Cl, NaH, DMF, rt, 16 h; (c) 10% Pd/C, H2,
EtOH/EtOAc, rt, 4 h, 76%; (d) 5d, K2CO3, DMSO, rt, 14 h, 82%; (e) (1) LiBH4, THF, −78 °C, 2 h; (2) Silica gel, CHCl3−EtOH−water, 24 h,
44%.
a
Scheme 7. Synthesis of Payload 6
a
Reagents and conditions: (a) 1,5-diiodopentane, K2CO3, DMF, rt, 40 min, 88%; (b) 6b, K2CO3, DMF, 60 °C, 3 h, 66%; (c) Pd(PPh3)4,
pyrrolidine, rt, 12 h, 41%.
a
Scheme 8. Synthesis of Payload 7
a
Reagents and conditions: (a) 7a, K2CO3, DMSO, rt, 3 h, 83%; (b) 49, K2CO3, DMSO, rt, 4 h, 80%; (c) (COCl)2, DMSO, Et3N, DCM, −78 to 0
°C, 15 min, 88%; (d) Tf2O, 2,6-lutidine, DCM, −78 to −30 °C, 3 h, 45%; (e) 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline, PdCl2(dppf),
35 °C, 1 h, 69%; (f) (1) LiEt3BH, THF, −78 °C, 1 h; (2) HCO2H, THF−EtOH−water, rt, 6 h, 37%.
Payload 6 was synthesized as shown in Scheme 7. Monomer
50 was reacted with 1,5-diiodopentane in the presence of
K2CO3 to provide 6a in 88% yield. This was coupled with PBD
monomer 6b30 in the presence of K2CO3 in DMF at 60 °C for
3 h to obtain dimer 6c in 66% yield which was treated with
Pd(PPh3)4 and pyrrolidine to give payload 6 in 41% yield.
Payload 7 was prepared as depicted in Scheme 8. Monomer
7b was reacted with 1,3-dibromopropane 7a in the presence of
K2CO3 to provide 7c in 83% yield. The latter was reacted with
monomer 49 in the presence of K2CO3 to obtain alcohol 7d in
80% yield. Swern oxidation provided ketone 7e in 88% yield
which was treated with trifluoromethanesulfonic anhydride in
L
https://dx.doi.org/10.1021/acs.jmedchem.0c01385
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
a
Scheme 9. Synthesis of Payload 8
a
Reagents and conditions: (a) (1) 53, (COCl)2, THF, rt, 2 h; (2) 8a, Et3N, 0 °C, 2 h, 68%; (b) Zn, NH4Cl, MeOH, 50 °C, 16 h, 82%; (c) THP,
PPTS, THF, rt, 16 h, 77%; (d) SEM-Cl, NaH, DMF, rt, 1 h, 58%; (e) 10% Pd/C, H2, EtOAc, rt, 2 h, 64%; (f) 7c, K2CO3, DMSO, rt, 16 h, 91%;
(g) MeI, K2CO3, DMSO, rt, 16 h; (h) Pyr-SO3, DCM, rt, 16 h, 52%; (i) Tf2O, 2,6-lutidine, DCM, −78 to 0 °C, 2 h, 63%; (j) 4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)aniline, PdCl2(dppf), K3PO4, THF, 45 °C, 2 h, 63%; (f) (1) LiEt3BH, THF, −78 °C, 1 h; (2) HCO2H, THF−
EtOH−water, rt, 2 h, 38%.
a
Scheme 10. Synthesis of Payload 9
a
Reagents and conditions: (a) 7c, K2CO3, DMSO, rt, 5 h, 91%; (b) (COCl)2, DMSO, Et3N, DCM, −78 to 0 °C, 15 min, 79%; (c) Tf2O, 2,6-
lutidine, DCM, −78 to −10 °C, 20 min, 60%; (d) (4-methoxyphenyl)boronic acid, PdCl2(dppf), K3PO4, THF, 35 °C, 2 h, 93%; (e) (1) LiEt3BH,
THF, −78 °C, 1 h; (2) Silica gel, CHCl3−EtOH−water, 3 days, 39%.
the presence of 2,6-lutidine to give triflate 7f in 45% yield. This
was subjected to Suzuki coupling with 4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)aniline in the presence of the
PdCl2(dppf) catalyst to provide 7g in 69% yield. Superhydride
reduction followed by formic acid treatment provided payload
7 in 37% yield.
Payload 8 was synthesized as shown in Scheme 9. Acid 53
was treated with oxalyl chloride and coupled with amine 8a to
obtain amide 8b in 68% yield, which was subjected to Zn/
NH4Cl reduction to provide lactam 8c in 82% yield. Protection
of the phenolic group with tetrahydropyran in the presence of
pyridinium p-toluenesulfonate (PPTS) in DCM provided 8d in
77% yield. The latter was reacted with SEM-Cl in the presence
of NaH to provide 8e in 58% yield, which was subjected to
debenzylation with Pd/C in the presence of hydrogen to give
8f in 64% yield. This was treated with 7c in the presence of
K2CO3 followed by THP deprotection with PPTS in methanol
that provided 8g in 91% yield. Thus, reaction of 8g with
iodomethane in the presence of K2CO3 followed by oxidation
with sulfur trioxide−pyridine provided 8i in 52% yield. This
was treated with trifluoromethanesulfonic anhydride in the
presence of 2,6-lutidine to provide triflate 8j in 63% yield,
which was subjected to Suzuki coupling with 4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)aniline in the presence of
the PdCl2(dppf) catalyst to give 8k in 63% yield. Superhydride
reduction followed by formic acid treatment afforded payload
8 in 38% yield.
Payload 9 was prepared as described in Scheme 10.
Monomer 51 was treated with 7c in the presence of K2CO3
in DMSO to provide alcohol 9a in 91% yield, which was
M
https://dx.doi.org/10.1021/acs.jmedchem.0c01385
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
a
Scheme 11. Synthesis of Payloads 10, 13, 16, and 17
a
Reagents and conditions: (a) 49, K2CO3; (b) Pd(PPh3)4, morpholine, THF, rt, 2 h, 70%; (c) (1) LiBH4 or LiEt3BH; (2) Silica gel, CHCl3−
EtOH−water.
a
Scheme 12. Synthesis of Payloads 11, 14, and 19−23
a
Reagents and conditions: (a) 49, K2CO3, DMSO, rt, 1−2 h; (b) 51, K2CO3, DMSO, rt, 1−5 h; 19a: (1) 8f, K2CO3, DMSO, rt, 5 h (2) PPTS,
MeOH, 40 °C, 1 h, 72%; (c) Pd(PPh3)4, morpholine, DCM, 2 h; (d) MeI, K2CO3, DMSO, rt, 3 h; (e) 2-bromoethanol, K2CO3, DMF, 80 °C, 4 h;
(f) 1-bromo-2-(2-methoxyethoxy)ethane, K2CO3, DMSO, rt, 3 h; (g) (1) LiEt3BH, THF, −78 °C, 1 h; (2) silica gel, CHCl3−EtOH−water, 24 h.
(h) (1) Fmoc-Gly-OH, HATU, i-Pr2NEt, DMF, rt, 4 h; (2) piperidine, DMF, rt, 1 h.
a
Scheme 13. Synthesis of Payloads 12, 15, and 18
a
Reagents and conditions: (a) 49, K2CO3, DMF, rt, 2 h; (b) 10% Pd/C, H2, MeOH, rt, 5 h, 86%; (c) ClCH2COCl, Et3N, DCM, rt, 1 h; (d) 49,
K2CO3, DMSO, 50 °C, 4 h, 73%; (e) (1) 51, K2CO3, rt, 1−6 h; (2) Pd(PPh3)4, morpholine, DCM; (f) (1) Alloc-Cl, Et3N, THF, rt, 3 h, 85%; (2)
NaBH3CN, formaldehyde, AcOH, MeOH/THF, rt, 15 h, 85%; (3) Pd(PPh3)4, pyrrolidine, DCM, 15 h, 86%; (g) (1) LiEt3BH, THF, −78 °C, 1 h;
(2) silica gel, CHCl3−EtOH−water, rt, 24 h.
subjected to Swern oxidation to give ketone 9b in 79% yield.
The latter was treated with trifluoromethanesulfonic anhydride
in the presence of 2,6-lutidine to provide triflate 9c in 60%
yield. Suzuki coupling of 9c with (4-methoxyphenyl)boronic
acid in the presence of PdCl2(dppf) catalyst provided 9d in
93% yield. Superhydride reduction followed by silica gel
treatment gave payload 9 in 39% yield.
Scheme 11 shows a general synthesis of symmetric dimer
payloads from aliphatic spacers and monomer 49. Thus, spacer
7a was treated with monomer 49 in the presence of K2CO3 at
room temperature to obtain SEM-protected dimer 10b.
Reduction with LiBH4 and subsequent treatment with silica
gel provided payload 10 in 50% yield. In a similar fashion,
payloads 13, 16, and 17 were prepared from the corresponding
spacers 4a, 16a, and 17a.
A typical synthesis of nonsymmetric dimer payloads was
described in Scheme 12. Monomer 49 was reacted with 1,3-
dibromopropane in the presence of K2CO3 to obtain
monoalkylated compound 11a, which was further reacted
with monomer 51 to obtain fully protected dimer 11b in 92%
yield. The Alloc group was removed using Pd(PPh3)4 and
morpholine to provide 11c. The latter was reduced with
superhydride, and subsequent treatment with silica gel
provided payload 11 in 27% yield. Payloads 14 and 19 were
prepared in a similar fashion. Phenol containing dimer 19a was
prepared from 4b by reaction with monomer 8f and
subsequent deprotection of the THP group with PPTS in
MeOH. Alkylation of 19a with MeI, 2-bromoethanol, and 1-
bromo-2-(2-methoxyethoxy)ethane followed by reduction
provided payloads 20−22, respectively. Payload 14 was
N
https://dx.doi.org/10.1021/acs.jmedchem.0c01385
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
a
Scheme 14. Synthesis of Payloads 24, 28, and 29
a
Reagents and conditions: (a) LiOAc, DMF/water, rt, 15 h, 94%; (b) 29a, Cs2CO3, DMF, rt, 2 h; (c) 50, Cs2CO3, acetone, 40 °C, 1 h; (d) NaN3,
DMF, rt, 15 h; (e) Pd(PPh3)4, pyrrolidine, DCM, rt, 30−90 min.
a
Scheme 15. Synthesis of Payloads 25 and 41
a
Reagents and conditions: (a) 50, K2CO3, DMF, rt; (b) Pd(PPh3)4, pyrrolidine, DCM, rt, 1.5 h.
a
Scheme 16. Synthesis of Payload 26
a
Reagents and conditions: (a) 50, Cs2CO3, DMF, rt, 1 h; 88% (b) Pd(PPh3)4, pyrrolidine, DCM, rt, 1.5 h, 39%.
a
Scheme 17. Synthesis of Payloads 27
a
Reagents and conditions: (a) (1) 10% Pd/C, H2, MeOH, rt, 5 h; (2) Alloc-Cl, K2CO3, THF, rt, 3 h, 22%; (b) mesyl chloride, Et3N, −10 °C, 1 h,
95%; (c) 49, K2CO3, DMF, rt, 2 h; 66% (d) Pd(PPh3)4, morpholine, DCM, 0 °C, 2 h, 87%; (e) (1) LiBH4, THF/EtOH, rt, 2 h; (2) silica gel,
CHCl3−EtOH−water, rt, 3 days, 46%.
coupled with Fmoc-glycine in the presence of HATU and
deprotected with piperidine to give payload 23.
Payloads 12, 15, and 18 were prepared as shown in Scheme
13. Dimerization of monomer 51 using spacers 4a and 7a in
the presence of K2CO3 followed by Alloc deprotection
provided 12a and 15a. Thus, 15a was converted to
dimethylamino intermediate 15b through mono Alloc
protection/reductive amination/Alloc deprotection sequence.
The dimer 18e with the amide spacer was assembled from 18c
by chloroacetylation, followed by alkylation of monomer 51.
The monomer 18c was constructed from tosylate 18a through
O
https://dx.doi.org/10.1021/acs.jmedchem.0c01385
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
a
Scheme 18. Synthesis of Payloads 30 and 34−38
a
Reagents and conditions: (a) 50, Cs2CO3, DMF, rt, 1 h (b) 29b, Cs2CO3, DMF, rt, 1−2.5 h (c) TBAF, THF, rt, 30 min; (d) Pd(PPh3)4,
pyrrolidine, DCM, rt, 30 min.
a
Scheme 19. Synthesis of Payloads 31, 32, 39, and 40
a
Reagents and conditions: (a) 50, DIAD, PPh3, THF, rt, 1.5 h, 15%; (b) 29b, Cs2CO3, DMF, rt, 1 h, 73%; (c) 50, Cs2CO3, DMF, rt; (d)
Pd(PPh3)4, pyrrolidine, DCM, rt, 30 min.
a
Scheme 20. Synthesis of Payload 33
a
Reagents and conditions: (a) 49, K2CO3, DMF, rt, 16 h, 89%; (b) 50, Cs2CO3, DMF, rt, 16 h, 79%; (c) Pd(PPh3)4, pyrrolidine, DCM, rt, 1 h,
64%; (2) silica gel, CHCl3−EtOH−water, rt, 3 days, 82%.
alkylation of 49, followed by Cbz-protecting group removal
with Pd/C.
Payload 27 with an aniline spacer was synthesized as shown
in Scheme 17. Nitro compound 27a was reduced with 10%
Pd/C and protected with Alloc−Cl to provide compound 27b.
This was reacted with mesyl chloride to obtain bis-mesylate
27c, which was subjected to alkylation with monomer 49 in
the presence of K2CO3 at room temperature for 2 h to give
SEM-protected dimer 27d. The Alloc group was removed with
Pd(PPh3)4 in the presence of morpholine to provide 27e in
87% yield. Reduction with LiBH4 and subsequent treatment
with silica gel provided payload 27 in 46% yield.
Payloads with aryl spacers were assembled, as described in
Scheme 14. Monomer 29b was prepared from 66 by TIPS
deprotection with LiOAc in DMF/water. The reaction of 29b
with spacer 29a in the presence of Cs2CO3 gave dimer 29c,
whereas dimers 24b and 28b were prepared from the
corresponding spacers 24a and 28a by reaction with monomer
50. Bromide 29c was converted to azide 29d, followed by
treatment of these dimers with Pd(PPh3)4/pyrrolidine, which
provided payloads 24, 28, and 29.
Payloads with para-substituted aryl spacers were synthesized
as shown in Scheme 15. Spacers 25a and 41a were treated with
monomer 50 in the presence of K2CO3 to provide protected
dimers 25b and 41b, respectively. Alloc and TBS groups were
removed using Pd(PPh3)4 in the presence of pyrrolidine to
give payloads 25 and 41.
Payloads with pyridyl spacers were assembled as described in
Scheme 18. Spacers 30a and 38a were treated with monomer
50 in the presence of Cs2CO3 to provide TBS-protected
dimers 30b and 38b. Whereas dimers 34b−37b were
synthesized from corresponding spacers 34a−37a and
monomer 29b. The phenolic TBS group in 38b was removed
by treatment with TBAF to give 38c. Imine groups were
unmasked by treatment with Pd(PPh3)4/pyrrolidine to provide
payloads 30 and 34−38.
Payload 26 with an ortho-substituted aryl spacer was
prepared as described in Scheme 16. Spacer 26a was treated
with monomer 50 in the presence of Cs2CO3 to provide
protected dimer 26b, which was subjected to deprotection
with Pd(PPh3)4 and pyrrolidine to give payload 26.
Spacers with pyridyl isomers were fused as shown in Scheme
19. The reaction of spacer 31a with monomer 50 under
Mitsunobu conditions (DIAD/PPh3) provided dimer 31b.
P
https://dx.doi.org/10.1021/acs.jmedchem.0c01385
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
a
Scheme 21. Synthesis of Payloads 42 and 43
a
Reagents and conditions: (a) 50, DIAD, PPh3, THF, rt, 1.5 h, 8%; (b) 50, K2CO3, DMF, 40 °C, 3 h, 42%; (c) Pd(PPh3)4, pyrrolidine, DCM, rt, 1
h.
a
Scheme 22. Synthesis of Linker-Payload 44
a
Reagents and conditions: (a) 14b, HATU, DMF, 2,6-lutidine, rt, 1 h, 71%; (b) piperidine, THF, rt, 2 h, 65%; (c) (1) LiEt3BH, THF, −78 °C, 1 h;
(2) silica gel, CHCl3−EtOH−water, rt, 36 h, 55%; (d) MAL-dPEG8-NHS ester, 2,6-lutidine, DMSO, rt, 1 h, 55%.
a
Scheme 23. Synthesis of Linker-Payload 45
a
Reagents and conditions: (a) 17, i-Pr2NEt, DMSO, rt, 16 h, 32%.
Dimer 32b was prepared from spacer 32a and monomer 29b
under Cs2CO3/DMF conditions, while 39b and 40b were
prepared by reaction of the corresponding spacers 39a and 40a
with monomer 50. These dimers were treated with Pd(PPh3)4
Q
https://dx.doi.org/10.1021/acs.jmedchem.0c01385
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
a
Scheme 24. Synthesis of Linker-Payload 46
a
Reagents and conditions: (a) 17c, i-Pr2NEt, NMP, rt, 16 h, 57%; (b) piperidine, THF, rt, 30 min, 79%; (c) (1) LiEt3BH, THF, −78 °C, 1 h; (2)
silica gel, DCM−EtOH−water, rt, 3 days, 55%; (d) (1) Fmoc-PEGn-acid, HATU, 2,6-lutidine, DMF, rt, 2 h; (2) Et2NH, DMF, rt, 1 h, 22%. (e)
Fmoc-PEG4-OSu, THF, rt, 1 h, 48%; (f) LiEt3BH, THF, −78 °C, 1 h, 30%.
in the presence of pyrrolidine to provide payloads 31, 32, 39,
and 40.
Payloads with furan and thiophene spacers were synthesized
as shown in Scheme 21. Spacer 42a was subjected to
Mitsunobu reaction with 50 to provide protected dimer 42b,
whereas thiophene spacer 43a was treated with monomer 50 in
the presence of K2CO3 to provide dimer 43b in 42% yield.
Alloc and TBS groups in dimers 42b and 43b were removed
using Pd(PPh3)4 in the presence of pyrrolidine to provide
payloads 42 and 43.
Payload 33 with the pendant aniline moiety was prepared as
described in Scheme 20. Spacer 30a was treated with
monomer 49 in the presence of K2CO3 to provide compound
33a, which was further reacted with monomer 51 in the
presence of K2CO3 to obtain compound 33b. The Alloc group
was removed with Pd(PPh3)4 and morpholine to provide 33c.
Reduction with superhydride and subsequent treatment with
silica gel gave payload 33 in 50% yield over three steps.
Synthesis of Linker-Payloads. We prepared linker-
payloads with a maleimide handle for heterogeneous lysine
R
https://dx.doi.org/10.1021/acs.jmedchem.0c01385
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
1
Chemicals, Gibbstown, NJ). H NMR spectra were recorded on a
Bruker 400 MHz spectrometer. All spectra were determined in the
solvents indicated. Multiplicity patterns are designated as follows: s,
singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad; dd,
doublet of doublets; dt, doublet of triplets. Preparative HPLC was
performed on the Waters instrument using the following conditions:
column: XBridge C18, 200 mm × 19 mm, 5 μm particles; mobile
phase A: 5:95 acetonitrile: water with 0.05% formic acid; mobile
phase B: 95:5 acetonitrile: water with 0.05% formic acid; gradient: a 0
min hold at 5% B, 5−45% B over 20 min, then a 0 min hold at 100%
B; flow rate: 20 mL/min; column temperature: 25 °C. HPLC
fractions with desired product mass were filtered under gravity
through a cartridge containing PL-HCO3 MP-resin (Agilent), washed
with acetonitrile, and lyophilized. Liquid chromatography (LC) data
were recorded on a Shimadzu LC-10AS liquid chromatograph using
an SPD-10AV UV−VIS detector with mass spectrometry (MS) data
determined using a Micromass Platform for LC in electrospray mode.
Purities of final compounds were determined by HPLC/UPLC and
were greater than 95%.
2-IT conjugations and amino handles for BTGase-mediated
site-specific conjugations. Intermediate 14b was reacted with
Fmoc-protected valine−citrulline28 dipeptide 76 using HATU
coupling to provide 77 in 71% yield (Scheme 22). Fmoc
deprotection was achieved with piperidine in THF, followed by
superhydride reduction and treatment with silica gel in
CHCl3−EtOH−water giving 79 in 36% yield over three
steps. Mal-dPEG8-NHS ester was reacted with 79 in the
presence of 2,6-lutidine to provide linker payload 44 in 55%
yield. Payload 17 was treated with maleimide-containing linker
carbonate 80 in the presence of Hunig’s base to provide linker-
payload 45 in 32% yield (Scheme 23). Linker payloads for site-
specific conjugations were synthesized as shown in Scheme 24.
Fmoc-Val-Cit-PABC derivative 81 was reacted with 17c in the
presence of Hunig’s base to provide 82 in 57% yield. The
Fmoc-protecting group was removed using piperidine in DMF
to provide 83 in 79% yield. Reduction of SEM amide 83 with
superhydride and subsequent treatment with silica gel provided
bis-imine 84 in 55% yield. This was treated with the activated
ester of Fmoc-protected PEG2 acid and subsequently the
Fmoc group was removed using piperidine to provide linker-
payload 46 with an amino handle suitable for BTGase-
mediated site-specific conjugations. Linker-payloads with
PEG4 (47) and PEG8 (48) were prepared using similar
methods. We also explored an alternative route for making 47
through intermediate 85, where PEG was introduced prior to
superhydride reduction.
(S)-3-((5-Iodopentyl)oxy)-2-methoxy-5-((2-(trimethylsilyl)-
ethoxy)methyl)-7,12-dihydrobenzo[5,6][1,4]diazepino[1,2-b]-
isoquinoline-6,14(5H,6aH)-dione (4b). To a suspension of 49
(250 mg, 0.55 mmol) and potassium carbonate (304 mg, 2.2 mmol)
in DMSO (5 mL) was added 1,5-diiodopentane (891 mg, 2.75
mmol). The reaction was then stirred at room temperature for 2 h
before it was diluted with EtOAc and washed with brine. The organic
layer was separated, dried over sodium sulfate, and concentrated in
vacuo. The crude product was purified by silica gel chromatography
(CombiFlash, 12 g column, 0−10% MeOH/DCM) to give the title
compound 4b as a yellow oil (275 mg, 0.423 mmol, 77% yield). MS
(C29H40IN2O5Si) m/z: 651 (M + H)+. 1H NMR (400 MHz, CDCl3):
δ 7.33−7.24 (m, 5H), 7.21 (s, 1H), 5.50 (d, J = 9.9 Hz, 1H), 5.15 (d,
J = 15.4 Hz, 1H), 4.66 (d, J = 9.9 Hz, 1H), 4.41 (d, J = 15.4 Hz, 1H),
4.33−4.25 (m, 1H), 4.05 (d, J = 8.6 Hz, 2H), 3.89 (s, 3H), 3.80 (td, J
= 9.6, 6.9 Hz, 1H), 3.72−3.62 (m, 1H), 3.56 (dd, J = 15.5, 7.4 Hz,
1H), 3.22 (t, J = 6.9 Hz, 2H), 3.00 (dd, J = 15.6, 6.4 Hz, 1H), 1.98−
1.80 (m, 4H), 1.67−1.54 (m, 2H), 0.98 (m, 2H), 0.04 (s, 9H).
(S)-2-Methoxy-3-((5-(((S)-7-methoxy-5-oxo-2,3,5,11a-tetra-
hydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)-
pentyl)oxy)-6a,7-dihydrobenzo[5,6][1,4]diazepino[1,2-b]-
isoquinolin-14(12H)-one (4). A suspension of 4b (50 mg, 0.077
mmol), potassium carbonate (31.9 mg, 0.231 mmol), and 4c (40.3
mg, 0.154 mmol) in DMF (1.5 mL) was heated at 50 °C for 2 h. The
reaction was then cooled to room temperature and quenched with
water. The resulting mixture was extracted with EtOAc (3×). The
combined organic layers were washed with brine, dried over sodium
sulfate, and concentrated in vacuo. The crude material was purified by
silica gel chromatography (24 g column, 0−10% MeOH/DCM) to
give the dimer 4d. MS (C42H52N4O9Si) m/z: 785 (M + H)+. This was
dissolved in DMF (0.5 mL) and cooled to 0 °C before sodium
hydride (60% dispersion in mineral oil, 3.07 mg, 0.077 mmol) and (2-
(chloromethoxy)ethyl)trimethylsilane (0.014 mL, 0.077 mmol) were
added sequentially. The reaction was warmed to room temperature
and stirred for 2 h. The reaction was then quenched with water,
extracted with EtOAc, dried over sodium sulfate, and concentrated in
vacuo to give 4e as a white solid (14 mg, 0.015 mmol, 20% yield). MS
(C48H67N4O10Si2) m/z: 915 (M + H)+.
CONCLUSIONS
■
Novel benzodiazepine dimers with aliphatic, aromatic, and
heteroaromatic spacers were prepared. Molecular models of
select novel benzodiazepine dimers in an 8-mer GC-rich
sequence-based-duplex DNA helped explain some of the
observed SAR. Bis alkylation and Mitsunobu reactions were
utilized to prepare dimers in a convergent fashion. Various
substitutions on the spacer as well as the pendant aryl moiety
in benzodiazepine dimers were well tolerated. Spacers with
different lengths were explored and five-carbon spacers were
found to be optimal for high cellular potency. Aryl spacers
provided payloads with further improvement in potency, likely
because of the additional interactions exhibited in the DNA
minor groove as discerned by the molecular modeling studies.
Payload 17 was conjugated to anti-mesothelin and anti-
FucGM1 antibodies using a valine−citrulline dipeptide linker.
A maleimide handle was used for heterogeneous conjugation
with thiol-modified lysines present on the antibody. An amino
handle was used for site-specific conjugation with glutamine at
the Q295 position using the BTGase enzyme. The resulting
ADCs showed picomolar activity in cytotoxic experiments
using multiple cancer cell lines. Site-specific ADCs had better
PK profiles and twofold greater efficacy compared to
heterogeneous ADCs at similar doses. Mesothelin-positive
N87 mouse xenograft models exhibited robust antitumor
activity after a single dose of anti-mesothelin ADC-46 (0.02
μmol/kg). Anti-FucGM1 ADC-46 induced complete tumor
growth inhibition at a 0.03 μmol/kg dose in an H187 mouse
xenograft model.
To a solution of compound 4e (14 mg, 0.015 mmol) in THF/
EtOH (1:1, 1 mL) at 0 °C was added a solution of lithium
borohydride (101 μL, 0.202 mmol, 2 M in THF). The reaction was
slowly warmed to room temperature and stirred for 15 min before it
was quenched with brine. The resulting mixture was extracted with
chloroform (3×). The organic layer was separated, dried over sodium
sulfate, and concentrated in vacuo. The residue was then taken up in
EtOH/chloroform (1:1, 2 mL). Silica gel (700 mg) was added,
followed by water (0.6 mL). The resulting suspension was stirred at
room temperature for 1 day and then filtered and washed with 10%
MeOH/chloroform. The filtrate was concentrated and purified by
preparative HPLC to give the title compound 4 as a white solid (0.92
mg, 1.330 μmol, 8.69% yield). MS (C36H39N4O6) m/z: 623 (M +
EXPERIMENTAL SECTION
■
All reagents and solvents were purchased from commercial sources
and used without further purification. All anhydrous reactions were
performed under a N2 atmosphere. Analytical thin-layer chromatog-
raphy was performed on silica gel 60 F254 aluminum sheets (EMD
S
https://dx.doi.org/10.1021/acs.jmedchem.0c01385
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
H)+. 1H NMR (400 MHz, CDCl3): δ 7.69 (d, J = 4.4 Hz, 1H), 7.57−
7.53 (m, 2H), 7.50 (d, J = 5.1 Hz, 1H), 7.41−7.32 (m, 4H), 6.82 (d, J
= 1.5 Hz, 2H), 5.03 (d, J = 15.6 Hz, 1H), 4.59 (d, J = 15.6 Hz, 1H),
4.18−4.06 (m, 4H), 3.97 (s, 3H), 3.96 (s, 3H), 3.75 (d, J = 7.3 Hz,
4H), 3.61 (dt, J = 11.8, 7.8 Hz, 1H), 3.34−3.26 (m, 1H), 3.22−3.15
(m, 1H), 2.34 (br s, 2H), 2.11−2.06 (m, 1H), 2.01−1.94 (m, 4H),
1.71−1.66 (m, 2H).
6H), 2.34 (td, J = 6.7, 3.0 Hz, 2H), 2.16−1.90 (m, 6H), 1.78−1.62
(m, 3H).
(S)-2-Methoxy-3-((5-(((S)-7-methoxy-2-methyl-5-oxo-5,11a-
dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)-
pentyl)oxy)-7,12-dihydrobenzo[5,6][1,4]diazepino[1,2-b]-
isoquinolin-14(6aH)-one (6). To a solution of 50 (200 mg, 0.381
mmol) and 1,5-diiodopentane (0.284 mL, 1.906 mmol) in DMF (2
mL) was added potassium carbonate (70 mg, 0.506 mmol) and
stirred at room temperature for 40 min. The reaction mixture was
purified by silica gel chromatography (40 g column, 30−100%
EtOAc/hexane) to afford 6a as a white solid (247 mg, 0.336 mmol,
88% yield). MS (C33H46IN2O6Si) m/z: 721 (M + H)+.
To a solution of 6a (116 mg, 0.161 mmol) and 6b (66 mg, 0.139
mmol) in DMF (1.5 mL) was added potassium carbonate (40 mg,
0.289 mmol) and stirred at 60 °C for 3 h. The reaction mixture was
diluted with EtOAc and washed with water and brine. Organics were
dried over Na2SO4, concentrated, and purified by silica gel
chromatography (40 g column, 0−100% EtOAc/hexane) to afford
6c as a white solid (100 mg, 0.092 mmol, 66% yield). MS
(C57H79N4O12Si2) m/z: 1067 (M + H)+.
To a solution of 6c (100 mg, 0.094 mmol) and pyrrolidine (0.027
mL, 0.328 mmol) in DCM (1.3 mL) was added Pd(PPh3)4 (13.53
mg, 0.012 mmol) and allowed to stir at room temperature for 12 h.
The reaction mixture was diluted with DCM, washed twice with
saturated aqueous NH4Cl, and water. The organics were dried
(Na2SO4) and concentrated, and the residue was purified by
preparative HPLC to obtain 6 as a white solid (25 mg, 0.038
mmol, 40.8% yield). MS (C37H39N4O6) m/z: 635 (M + H)+.
(S)-3-(3-(((2R,11aS)-2-Hydroxy-7-methoxy-5,11-dioxo-10-
((2-(trimethylsilyl)ethoxy)methyl)-2,3,5,10,11,11a-hexahydro-
1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-2-
methoxy-5-((2-(trimethylsilyl)ethoxy)methyl)-7,12-
dihydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinoline-6,14-
(5H,6aH)-dione (7d). To a solution of 1,3-dibromopropane 7a
(1.881 mL, 18.36 mmol) and (2R,11aS)-2,8-dihydroxy-7-methoxy-10-
((2-(trimethylsilyl)ethoxy)methyl)-2,3-dihydro-1H-benzo[e]pyrrolo-
[1,2-a][1,4]diazepine-5,11(10H,11aH)-dione 7b (1.5 g, 3.67 mmol)
in DMSO (10 mL) was added K2CO3 (1.522 g, 11.02 mmol). The
resulting suspension was stirred at room temperature for 3 h. The
reaction was then quenched with brine and extracted with EtOAc
(3×). The combined organic layers were washed with brine, dried
over sodium sulfate, and concentrated. The crude product was
purified by silica gel chromatography (40 g column, 0−100% EtOAc/
hexane) to give compound 7c as a white solid (1.62 g, 3.06 mmol,
83% yield). MS (C22H34BrN2O6Si) m/z: 531 (M + H)+.
(S)-10-(Dimethylamino)-3-hydroxy-2-methoxy-5-((2-
(trimethylsilyl)ethoxy)methyl)-6a,7-dihydrobenzo[5,6][1,4]-
diazepino[1,2-b]isoquinoline-6,14(5H,12H)-dione (5c). To a
solution of 72 (220 mg, 0.512 mmol) in MeOH (3 mL) and THF
(3 mL) were added formaldehyde (37% aqueous solution, 0.572 mL,
7.68 mmol) and a few drops of acetic acid. The solution was stirred at
room temperature for 10 min before sodium cyanoborohydride (129
mg, 2.049 mmol) was added. The reaction was then stirred at room
temperature for 2 h before it was concentrated in vacuo. The crude
material was purified by silica gel chromatography (24 g column, 0−
10% MeOH/DCM) to give the dimethylamino compound 5a (230
mg, 0.503 mmol, 98% yield). MS (C27H28N3O4) m/z: 458 (M + H)+.
This was taken in DMF (5 mL), and sodium hydride was added (60%
dispersion in mineral oil, 25.1 mg, 0.628 mmol) at 0 °C. The mixture
was stirred for 15 min before (2-(chloromethoxy)ethyl)-
trimethylsilane (0.111 mL, 0.628 mmol) was added. The reaction
was slowly warmed to room temperature and stirred overnight. The
reaction was then quenched with water and extracted with EtOAc
(3×). The combined organic layers were dried over sodium sulfate
and concentrated in vacuo. The crude material was purified by silica
gel chromatography (0−10% MeOH/DCM) to give the SEM-
protected compound 5b. MS (C33H42N3O5Si) m/z: 588 (M + H)+.
This was suspended in EtOH/EtOAc (1:1, 10 mL) and combined
with 10% Pd/C (20 mg). The mixture was purged with N2 and then
stirred under a balloon of H2 for 4 h. The reaction was then filtered
through a pad of Celite and concentrated in vacuo. The crude material
was purified by silica gel chromatography (40 g column, 0−10%
MeOH/DCM) to give compound 5c (190 mg, 0.382 mmol, 76%
1
yield). MS (C26H36N3O5Si) m/z: 498 (M + H)+. H NMR (400
MHz, CDCl3): δ 7.45 (s, 1H), 7.21 (s, 1H), 7.18−7.10 (m, 1H), 6.69
(m, 2H), 5.51 (d, J = 9.9 Hz, 1H), 5.12 (d, J = 15.2 Hz, 1H), 4.65 (d,
J = 10.1 Hz, 1H), 4.33 (d, J = 15.2 Hz, 1H), 4.24 (m, 1H), 3.91 (s,
3H), 3.79 (s, 1H), 3.72−3.63 (m, 1H), 3.52−3.40 (m, 1H), 2.93 (s,
6H), 1.05−0.89 (m, 2H), 0.03 (s, 9H).
(S)-10-(Dimethylamino)-2-methoxy-3-((5-(((S)-7-methoxy-5-
oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]-
diazepin-8-yl)oxy)pentyl)oxy)-6a,7-dihydrobenzo[5,6][1,4]-
diazepino[1,2-b]isoquinolin-14(12H)-one (5). To a solution of 5c
(17 mg, 0.034 mmol) and 5d (22.12 mg, 0.038 mmol) in DMSO (1
mL) was added potassium carbonate (9.44 mg, 0.068 mmol) and
stirred at room temperature for 14 h. The reaction was then diluted
with EtOAc, washed with water and then brine, dried, and
concentrated. The residue was purified by silica gel chromatography
(24 g column, 0−10% MeOH/DCM) to give dimer 5e (27 mg, 0.028
mmol, 82% yield). MS (C48H67N4O10Si2) m/z: 915 (M + H)+. This
was dissolved in THF/EtOH (1:1, 1 mL) and cooled to 0 °C. A
solution of LiBH4 (0.342 mL, 0.683 mmol) in THF (2 M) was added
dropwise. The resulting solution was stirred for 2 h and then
quenched with water (1 mL). The reaction was then extracted with
chloroform, dried over sodium sulfate, filtered, and concentrated. The
residue was then taken up in chloroform/EtOH (1:1, 2 mL). Silica gel
(0.8 g) was added, followed by water (0.6 mL) and stirred at room
temperature for 1 day and then filtered, washing with 10% MeOH/
chloroform. The filtrate was concentrated and purified by preparative
HPLC to give 5 as a white solid (11 mg, 0.015 mmol, 43.5% yield).
MS (C38H44N5O6) m/z: 666 (M + H)+. 1H NMR (400 MHz,
CDCl3): δ 7.68 (d, J = 4.4 Hz, 1H), 7.54 (d, J = 1.8 Hz, 2H), 7.52 (d,
J = 5.3 Hz, 1H), 7.23 (d, J = 8.8 Hz, 1H), 6.82 (d, J = 1.8 Hz, 2H),
6.73−6.65 (m, 2H), 4.97 (d, J = 15.4 Hz, 1H), 4.53 (d, J = 15.4 Hz,
1H), 4.29−4.03 (m, 4H), 3.96 (s, 3H), 3.92 (s, 3H), 3.84 (ddd, J =
11.7, 7.1, 4.5 Hz, 1H), 3.78−3.72 (m, 1H), 3.66−3.56 (m, 1H),
3.28−3.17 (m, 1H), 3.06 (dd, J = 15.4, 4.2 Hz, 1H), 3.01−2.95 (m,
To a solution of 7c (1 g, 1.889 mmol) and 49 (0.859 g, 1.889
mmol) in DMSO (10 mL) was added K2CO3 (0.522 g, 3.78 mmol).
The resulting suspension was stirred at room temperature for 4 h
before it was quenched with water. The mixture was extracted with
EtOAc (3×). The combined organic layers were washed with brine,
dried over sodium sulfate, and concentrated in vacuo. The crude
product was purified by silica gel chromatography (0−6% MeOH/
DCM, 40 g column) to give 7d as a white solid (1.6 g, 1.506 mmol,
1
80% yield). MS (C46H63N4O11Si2) m/z: 903 (M + H)+. H NMR
(400 MHz, CDCl3): δ 7.43−7.19 (m, 8H), 5.56−5.41 (m, 2H), 5.30
(s, 1H), 5.15 (d, J = 15.3 Hz, 1H), 4.79−4.66 (m, 2H), 4.69−4.60
(m, 2H), 4.42 (d, J = 15.3 Hz, 1H), 4.35−4.22 (m, 6H), 3.98−3.84
(m, 7H), 3.84−3.48 (m, 6H), 3.10−2.89 (m, 2H), 2.84 (dd, J = 19.0,
3.7 Hz, 1H), 2.42 (p, J = 6.0 Hz, 2H), 2.11 (m, 1H), 1.11−0.89 (m,
4H), 0.12 to −0.04 (m, 18H).
(S)-7-Methoxy-8-(3-(((S)-2-methoxy-6,14-dioxo-5-((2-
( t r i m e t h y l s i l y l ) e t h o x y ) m e t h y l ) - 5 , 6 , 6 a , 7 , 1 2 , 1 4 -
hexahydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinolin-3-yl)-
oxy)propoxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1,11a-di-
hydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-2,5,11-
(3H,10H)-trione (7e). To a solution of oxalyl chloride (0.233 mL,
2.66 mmol) in DCM (15 mL) at −78 °C was added DMSO (0.377
mL, 5.31 mmol) dropwise. Stirred for 15 min, then a solution of 7d
(1.6 g, 1.772 mmol) in DCM (15 mL) was added dropwise. Stirred
for 10 min before a solution of triethylamine (1.481 mL, 10.63 mmol)
in DCM (10 mL) was added dropwise. The resulting solution was
T
https://dx.doi.org/10.1021/acs.jmedchem.0c01385
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
stirred at −78 °C for 15 min and then slowly warmed to 0 °C. The
reaction was then quenched with ice water and extracted with DCM.
The organic layers were washed with brine, dried, concentrated, and
purified by silica gel chromatography (0−8% MeOH/DCM, 40 g
column) to give 7e as a white solid (1.41 g, 1.565 mmol, 88% yield).
to give the title compound 8b (1.75 g, 68% yield). MS (C26H25N2O8)
m/z: 493 (M + H)+.
A suspension of 8b (1.75 g, 3.55 mmol), zinc (1.394 g, 21.32
mmol), and ammonium chloride (2.281 g, 42.6 mmol) in MeOH (10
mL) was heated at 50 °C overnight. The reaction was then filtered
through a pad of Celite, washing with 20% MeOH/DCM. The filtrate
was concentrated to give the title compound 8c (1.25 g, 2.90 mmol,
1
MS (C46H61N4O11Si2) m/z: 901 (M + H)+. H NMR (400 MHz,
CDCl3): δ 7.43−7.19 (m, 8H), 5.53 (td, J = 11.5, 9.9 Hz, 2H), 5.32
(s, 1H), 5.17 (d, J = 15.3 Hz, 1H), 4.85−4.67 (m, 2H), 4.64 (dd, J =
9.9, 3.1 Hz, 3H), 4.43 (d, J = 15.3 Hz, 1H), 4.36−4.21 (m, 5H), 3.93
(s, 3H), 3.89 (s, 3H), 3.82−3.47 (m, 5H), 3.01 (dd, J = 15.5, 6.4 Hz,
1H), 2.91−2.73 (m, 1H), 2.44 (t, J = 6.0 Hz, 2H), 1.11−0.88 (m,
4H), 0.06 (s, 18H).
(S)-3-(3-(((S)-2-(4-Aminophenyl)-7-methoxy-5-oxo-5,11a-di-
hydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)-
propoxy)-2-methoxy-7,12-dihydrobenzo[5,6][1,4]diazepino-
[1,2-b]isoquinolin-14(6aH)-one (7). To a solution of 7e (1.4 g,
1.554 mmol) in CH2Cl2 (30 mL) was added 2,6-lutidine (0.362 mL,
3.11 mmol). The reaction was then cooled to −78 °C before
trifluoromethanesulfonic anhydride (0.526 g, 1.864 mmol) was added
dropwise. The reaction was stirred at −78 °C for 30 min before it was
allowed to warm to 0 °C and stirred at that temperature for 3 h. The
reaction was then quenched with brine and extracted with DCM
(3×). The combined organic layers were dried over sodium sulfate
and concentrated. The crude product was purified by silica gel
chromatography (0−100% EtOAc/hexane, 40 g column) to give 7f
(0.72 g, 0.697 mmol, 44.9% yield). MS (C47H60F3N4O13SSi2) m/z:
1033 (M + H)+.
1
82% yield). MS (C25H23N2O5) m/z: 431 (M + H)+. H NMR (400
MHz, DMSO-d6): δ 10.25 (br s, 1H), 9.26 (s, 1H), 7.53−7.32 (m,
5H), 7.24 (s, 1H), 7.07 (d, J = 8.1 Hz, 1H), 6.79 (s, 1H), 6.71−6.60
(m, 2H), 5.08 (d, J = 4.0 Hz, 2H), 4.83 (d, J = 15.2 Hz, 1H), 4.22 (d,
J = 15.4 Hz, 1H), 4.08 (t, J = 6.7 Hz, 1H), 3.76 (s, 3H), 3.16 (dd, J =
15.4, 6.8 Hz, 1H), 2.85 (dd, J = 15.1, 6.3 Hz, 1H).
(6aS)-3-(Benzyloxy)-2-methoxy-10-((tetrahydro-2H-pyran-
2-yl)oxy)-5-((2-(trimethylsilyl)ethoxy)methyl)-7,12-
dihydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinoline-6,14-
(5H,6aH)-dione (8e). To a solution of 8c (1.25 g, 2.9 mmol) in
DCM (40 mL) were added PPTS (0.073 g, 0.29 mmol) and
tetrahydropyran (2.84 mL, 29 mmol). The reaction was stirred at
room temperature overnight and then quenched with saturated
aqueous NaHCO3 solution. The resulting mixture was extracted with
DCM (3×). The combined organic layers were dried over sodium
sulfate and concentrated in vacuo. The crude product mixture was
purified by silica gel chromatography (40 g column 0−100% EtOAc/
hexane) to give compound 8d as a white solid (1.15 g, 2.235 mmol,
77% yield). MS (C30H31N2O6) m/z: 515 (M + H)+.
To an ice-cooled solution of 8d (1.2 g, 2.332 mmol) in DMF (10
mL) was added sodium hydride (60% dispersion in mineral oil, 0.168
g, 2.80 mmol). The mixture was stirred at 0 °C for 15 min and then
warmed to room temperature and stirred for 10 min before it was
cooled back to 0 °C. (2-(Chloromethoxy)ethyl)trimethylsilane (0.496
mL, 2.80 mmol) was then added. The reaction was stirred at 0 °C for
30 min before it was then allowed to warm to room temperature and
stirred for 1 h. The reaction was then quenched with brine. The
resulting mixture was extracted with EtOAc (3×). The combined
organic layers were dried over sodium sulfate and concentrated in
vacuo. The crude product mixture was purified by silica gel
chromatography (40 g column, 0−50% EtOAc/hexane) to give the
title compound 8e (875 mg, 1.357 mmol, 58.2%). MS
(C36H45N2O7Si) m/z: 645 (M + H)+. 1H NMR (400 MHz,
CDCl3): δ 7.48−7.41 (m, 2H), 7.39−7.28 (m, 4H), 7.24 (s, 1H),
7.19 (d, J = 8.1 Hz, 1H), 7.02−6.92 (m, 2H), 5.51−5.32 (m, 2H),
5.20 (s, 2H), 5.12 (dd, J = 15.3, 3.2 Hz, 1H), 4.50 (dd, J = 9.8, 1.7 Hz,
1H), 4.33 (dd, J = 15.3, 5.2 Hz, 1H), 4.22 (ddd, J = 7.4, 6.5, 4.3 Hz,
1H), 3.91 (s, 4H), 3.76−3.56 (m, 3H), 3.47 (dd, J = 15.4, 7.7 Hz,
1H), 2.92 (dd, J = 15.4, 6.4 Hz, 1H), 2.03−1.92 (m, 1H), 1.88−1.81
(m, 2H), 1.74−1.57 (m, 3H), 0.97 (m, 2H), 0.09−0.01 (m, 9H).
(S)-8-(3-(((S)-2,10-Dimethoxy-6,14-dioxo-5-((2-
( t r i m e t h y l s i l y l ) e t h o x y ) m e t h y l ) - 5 , 6 , 6 a , 7 , 1 2 , 1 4 -
hexahydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinolin-3-yl)-
oxy)propoxy)-7-methoxy-10-((2-(trimethylsilyl)ethoxy)-
methyl)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]-
diazepine-2,5,11(3H,10H)-trione (8i). A suspension of 8e (875
mg, 1.357 mmol) and Pd/C (10%, 87 mg) in EtOAc (10 mL) was
stirred under a balloon of H2 at room temperature for 2 h. The
reaction was then filtered through a pad of Celite and concentrated in
vacuo. The crude product mixture was purified by silica gel
chromatography (40 g column, 0−100% EtOAc/hexane) to give
compound 8f as a white solid (485 mg, 0.874 mmol, 64.4% yield). MS
(C29H39N2O7Si) m/z: 555 (M + H)+. 1H NMR (400 MHz, CDCl3):
δ 7.33 (d, J = 0.7 Hz, 1H), 7.27 (s, 1H), 7.22 (d, J = 8.1 Hz, 1H),
7.04−6.95 (m, 2H), 6.01 (s, 1H), 5.46 (d, J = 9.7 Hz, 1H), 5.44−5.34
(m, 1H), 5.16 (dd, J = 15.3, 2.6 Hz, 1H), 4.71 (dd, J = 9.7, 1.8 Hz,
1H), 4.35 (dd, J = 15.3, 4.5 Hz, 1H), 4.25 (ddd, J = 7.7, 6.4, 4.0 Hz,
1H), 4.01−3.87 (m, 4H), 3.80−3.59 (m, 3H), 3.52 (dd, J = 15.5, 7.8
Hz, 1H), 2.95 (dd, J = 15.5, 6.4 Hz, 1H), 2.05−1.96 (m, 1H), 1.92−
1.82 (m, 2H), 1.77−1.62 (m, 3H), 1.06−0.97 (m, 2H), 0.05 (s, 9H).
To a solution of 8f (100 mg, 0.180 mmol) and 7c (117 mg, 0.198
mmol) in DMSO (2 mL) was added potassium carbonate (62.3 mg,
0.451 mmol). The mixture was stirred at room temperature for 16 h.
The reaction was then diluted with EtOAc and washed sequentially
To a degassed solution of 7f (78 mg, 0.075 mmol), PdCl2(dppf)
(2.76 mg, 3.77 μmol), and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)aniline (19.85 mg, 0.091 mmol) in THF (2 mL) was added
aqueous solution of tripotassium phosphate (0.377 mL, 0.377 mmol).
The reaction was stirred under N2 at 35 °C for 1 h. The reaction was
then diluted with EtOAc, washed with brine, concentrated, and
purified by silica gel chromatography (12 g column, 0−100% EtOAc/
hexane) to give 7g as a white solid (51 mg, 0.052 mmol, 69.2% yield).
MS (C52H66N5O10Si2) m/z: 976 (M + H)+.
To a solution of 7g (18 mg, 0.018 mmol) in THF (1 mL) at −78
°C was added lithium triethylhydroborate (0.184 mL, 0.184 mmol) (1
M in THF). The reaction was stirred at −78 °C for 1 h and then
quenched with water. The mixture was warmed to room temperature
and extracted with EtOAc. The crude product was then taken up in
THF/EtOH (10 mL, 1:1), and aqueous HCOOH (0.05%, 8 mL) was
added. The mixture was stirred at room temperature for 6 h,
monitoring with neutral LCMS for disappearance of the SEM-hemi
aminal. The mixture was then concentrated, neutralized with solid
NaHCO3, and then extracted with CHCl3/MeOH (10:1, 3×). The
combined organic layers were concentrated and purified by silica gel
chromatography (24 g column, 0−10% MeOH/DCM) to give 7 as a
white solid (5.2 mg, 6.84 μmol, 37.1% yield). MS (C40H38N5O6) m/z:
684 (M + H)+.
(S)-3-(Benzyloxy)-10-hydroxy-2-methoxy-7,12-
dihydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinoline-6,14-
(5H,6aH)-dione (8c). To a solution of 4-(benzyloxy)-5-methoxy-2-
nitrobenzoic acid 53 (1.97 g, 6.51 mmol) in THF (30 mL) was added
dropwise oxalyl chloride (1.09 mL, 12.5 mmol), followed by DMF
(30 μL). The resulting solution was stirred at room temperature for 2
h before it was concentrated in vacuo to give the acid chloride as a
yellow solid. The acid chloride was dissolved in THF (30 mL) and
added dropwise to a solution of (S)-methyl 7-hydroxy-1,2,3,4-
tetrahydroisoquinoline-3-carboxylate 8a (1.6 g, 5.21 mmol) and
triethylamine (2.9 mL, 20.8 mmol) in THF (20 mL) at 0 °C. The
reaction was stirred at 0 °C for 2 h before it was quenched with water.
The resulting mixture was extracted with EtOAc (3×). The combined
organic layers were washed with brine, and then dried over sodium
sulfate and concentrated in vacuo. The resulting mixture was then
taken up in MeOH (50 mL). Potassium carbonate (1 g) was added.
The resulting suspension was stirred at room temperature for 1 h
before it was filtered through a pad of Celite and concentrated in
vacuo. The crude product mixture was purified by silica gel
chromatography (220 g column, 0−50% EtOAc/hexane in 15 min)
U
https://dx.doi.org/10.1021/acs.jmedchem.0c01385
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
with water and brine. The organic layer was separated, dried over
sodium sulfate, and concentrated. The resulting mixture was taken up
in MeOH (10 mL), and PPTS (20 mg) was added. The reaction was
stirred at 40 °C for 1 h. The reaction was then concentrated, taken up
in EtOAc, and washed with aqueous sodium bicarbonate and then
brine. The organic layer was separated, dried over sodium sulfate, and
concentrated in vacuo. The crude product was purified by silica gel
chromatography (0−20% EtOAc/hexane, 24 g column) to give 8g as
was taken up in THF/EtOH (1:1, 2 mL) and stirred with aqueous
formic acid (0.05%, 1 mL) at room temperature for 2 h. The mixture
was then neutralized with aqueous NaHCO3 and extracted with
chloroform (3×). The combined organic layers were concentrated
and purified by silica gel chromatography (4 g column, 0−6%
MeOH/DCM) to give the title compound 8 as a white solid (2.1 mg,
2.65 μmol, 38.1% yield). MS (C41H40N5O7) m/z: 714 (M + H)+.
(S)-10-Amino-2-methoxy-3-(3-(((S)-7-methoxy-2-(4-methox-
yphenyl)-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a]-
[1,4]diazepin-8-yl)oxy)propoxy)-7,12-dihydrobenzo[5,6][1,4]-
diazepino[1,2-b]isoquinolin-14(6aH)-one (9). A vial was charged
with 7c (480 mg, 0.907 mmol), 51 (502 mg, 0.907 mmol), and
K2CO3 (125 mg, 0.907 mmol) in DMSO (4 mL) and stirred at room
temperature for 5 h. The reaction was then diluted with EtOAc (30
mL), washed with brine, dried over sodium sulfate, concentrated, and
purified by silica gel chromatography (40 g column, 0−10% MeOH/
DCM) to give 9a as a white solid (830 mg, 0.828 mmol, 91% yield).
MS (C50H68N5O13Si2) m/z: 1002 (M + H)+.
a
white solid (150 mg, 0.163 mmol, 91% yield). MS
(C46H63N4O12Si2) m/z: 919 (M + H)+.
To a solution of 8g (150 mg, 0.163 mmol) in DMSO (2 mL) were
added potassium carbonate (67.7 mg, 0.490 mmol) and iodomethane
(46.3 mg, 0.326 mmol). The mixture was stirred at room temperature
for 16 h. The reaction was then diluted with EtOAc and washed with
water. The organic layer was dried over sodium sulfate and
concentrated in vacuo to give 8h. The crude material was used in
the next step without further purification. MS (C47H65N4O12Si2) m/z:
933 (M + H)+.
To a solution of 8h in DCM (804 μL) and DMSO (804 μL) 0 °C
was added triethylamine (112 μL, 0.804 mmol), followed by sulfur
trioxide pyridine complex (51.2 mg, 0.321 mmol). The reaction was
allowed to warm to room temperature and stirred for 16 h. The
reaction was then diluted with CH2Cl2 (30 mL), washed sequentially
with saturated aqueous NH4Cl (10 mL), H2O (2 × 10 mL), and
saturated aqueous NaHCO3 solution (10 mL). The organic layer was
dried over sodium sulfate, filtered, and concentrated in vacuo. The
crude material was purified by silica gel chromatography (24 g
column; gradient 0−100% EtOAc/CH2Cl2) to provide the title
compound 8i (112 mg, 0.084 mmol, 52.4% yield). MS
(C47H63N4O12Si2) m/z: 931 (M + H)+.
To a solution of oxalyl chloride (0.068 mL, 0.778 mmol) in DCM
(10 mL) at −78 °C was added DMSO (0.110 mL, 1.556 mmol)
dropwise. Stirred for 15 min, then a solution of 9a (0.52 g, 0.519
mmol) in DCM (5 mL) was added dropwise. The solution was stirred
for 10 min before a solution of triethylamine (0.434 mL, 3.11 mmol)
in DCM (3 mL) was added dropwise. The resulting solution was
stirred at −78 °C for 15 min and then slowly warmed to 0 °C. The
reaction was then quenched with ice water and extracted with DCM.
The organic layers were washed with brine, dried, concentrated, and
purified by silica gel chromatography (0−10% MeOH/DCM, 40 g
column) to give 9b as a white solid (0.41 g, 0.410 mmol, 79% yield).
MS (C53H66N5O13Si2) m/z: 1000 (M + H)+.
(S)-3-((5-(((S)-2-(4-Aminophenyl)-7-methoxy-5-oxo-5,11a-
dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)-
pentyl)oxy)-2,10-dimethoxy-7,12-dihydrobenzo[5,6][1,4]-
diazepino[1,2-b]isoquinolin-14(6aH)-one (8). To a solution of 8i
(112 mg, 0.120 mmol) in DCM (2 mL) was added 2,6-lutidine (0.028
mL, 0.241 mmol). The solution was cooled to −78 °C and triflic
anhydride (0.030 mL, 0.180 mmol) was then added dropwise. The
reaction was slowly warmed to 0 °C and stirred for 2 h. The reaction
was then quenched with brine and extracted with DCM. The organic
layer was separated, dried over sodium sulfate, and concentrated in
vacuo. The crude product was purified by silica gel chromatography
(24 g column, 0−10% MeOH/DCM) to give compound 8j (81 mg,
0.076 mmol, 63.3% yield). MS (C48H62F3N4O14SSi2) m/z: 1063 (M +
H)+.
To a solution of 9b (410 mg, 0.410 mmol) in DCM (6 mL) was
added 2,6-lutidine (0.143 mL, 1.230 mmol). The reaction was then
cooled to −78 °C before trifluoromethanesulfonic anhydride (173
mg, 0.615 mmol) was added. The reaction was then slowly warmed to
−10 °C and stirred for 20 min. The reaction was then quenched with
brine, extracted with DCM, dried, concentrated, and purified by silica
gel chromatography (0−10% MeOH/DCM, 40 g column) to give 9c
(330 mg, 0.248 mmol, 60.4% yield). MS (C51H65F3N5O15SSi2) m/z:
1132 (M + H)+.
9c (500 mg, 0.442 mmol), (4-methoxyphenyl)boronic acid (134
mg, 0.883 mmol), and PdCl2(dppf) (32.3 mg, 0.044 mmol) were
combined in a vial. THF (3 mL) was added. The solution was
immediately purged with N2, and potassium phosphate (187 mg,
0.883 mmol) in water (1 mL) was then added. The solution was
purged with N2 again before it was heated to 35 °C for 2 h. The
material was purified by silica gel chromatography (0−5% MeOH/
DCM) to afford 9d as a white solid (412 mg, 0.409 mmol, 93% yield).
MS (C53H68N5O11Si2) m/z: 1006 (M + H)+.
To a solution of 9d (40 mg, 0.040 mmol) in THF (3 mL) at −78
°C was added superhydride (0.238 mL, 0.238 mmol) and allowed to
stir at −78 °C for 1 h. The reaction was then warmed to room
temperature and diluted with dichloromethane (10 mL) and water.
The water layer was washed again with DCM (10 mL), and the
organics were combined and concentrated. The residue was taken up
in DCM/EtOH/water (1:2:1, 4 mL). To this was added silica gel
(300 mg), and the reaction was stirred at room temperature for 3
days. The reaction was filtered to remove the silica gel and then
concentrated to residue. The residue was purified by preparative
HPLC to give the title compound 9 as a white solid (12.2 mg, 39%
yield). MS (C41H40N5O7) m/z: 714 (M + H)+.
(6aS,6a′S)-3,3′-(Propane-1,3-diylbis(oxy))bis(2-methoxy-
6a,7-dihydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinolin-14-
(12H)-one) (10). A suspension of 49 (52 mg, 0.11 mmol) and 1,3-
dibromopropane (12 mg, 0.057 mmol) and potassium carbonate
(47.4 mg, 0.34 mmol) in DMF (1 mL) were stirred at room
temperature for 14 h. The reaction was then diluted with H2O and
extracted with EtOAc (3×). The combined organic layers were
washed with brine, dried over NaSO4, and concentrated in vacuo. The
crude product mixture was purified by silica gel chromatography (12 g
To a vial with a screw-cap top were added 8j (75 mg, 0.071 mmol),
4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (18.54 mg,
0.085 mmol), and [1,1′-bis(diphenylphosphino)ferrocene]-
dichloropalladium(II) (2.58 mg, 3.53 μmol). The vial was degassed
and backfilled with N2, and then THF (1411 μL) and a solution of
aqueous K3PO4 (0.5 M, 705 μL, 0.353 mmol) were added. The
mixture was stirred under N2 at 45 °C for 2 h. The reaction was then
diluted with EtOAc and washed with brine. The organic layer was
separated, dried over sodium sulfate, filtered, and concentrated in
vacuo. The crude material was purified by silica gel chromatography
(12 g column; 0−100% EtOAc/hexane) to provide compound 8k as a
white solid (45 mg, 0.045 mmol, 63.4% yield). MS (C53H68N5O11Si2)
1
m/z: 1006 (M + H)+. H NMR (400 MHz, CDCl3): δ 7.41 (s, 1H),
7.33 (s, 1H), 7.29−7.20 (m, 6H), 6.91−6.80 (m, 2H), 6.68 (d, J = 8.6
Hz, 2H), 5.51 (dd, J = 11.9, 10.0 Hz, 2H), 5.13 (d, J = 15.4 Hz, 1H),
4.75 (dd, J = 16.8, 10.0 Hz, 2H), 4.60 (dd, J = 10.6, 3.4 Hz, 1H), 4.39
(d, J = 15.3 Hz, 1H), 4.33−4.21 (m, 5H), 3.93 (s, 3H), 3.89 (s, 3H),
3.82 (s, 3H), 3.80−3.74 (m, 3H), 3.69 (m, 2H), 3.51 (dd, J = 15.5,
7.5 Hz, 1H), 3.12 (m, 1H), 3.03−2.87 (m, 1H), 2.44 (t, J = 5.9 Hz,
2H), 1.03−0.92 (m, 4H), 0.04 (s, 9H), 0.03 (s, 9H).
To a solution of 8k (7 mg, 6.96 μmol) in THF (1 mL) at −78 °C
was added dropwise a solution of lithium triethylborohydride (0.070
mL, 0.070 mmol, 1 M in THF). The reaction was stirred at −78 °C
for 1 h before it was quenched with brine. The mixture was extracted
with 10% MeOH/chloroform (3×). The combined organic layers
were dried over sodium sulfate and concentrated in vacuo. The residue
V
https://dx.doi.org/10.1021/acs.jmedchem.0c01385
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
column, 0−10% MeOH/DCM) to give compound 10b (23 mg, 21%
yield). MS (C51H65N4O10Si2) m/z: 1049 (M + H)+.
To a solution of 12a (15 mg, 0.015 mmol) in THF (0.7 mL) at
−78 °C was added superhydride (0.153 mL, 0.153 mmol). The
reaction was stirred at −78 °C for 1 h before it was quenched with
water. The mixture was then extracted with EtOAc. The combined
organic layers were dried and concentrated. The crude product was
purified by preparative HPLC to give 12 as a white solid (4.2 mg, 5.50
μmol, 35.9% yield). MS (C39H39N6O6) m/z: 687 (M + H)+.
(6aS,6a′S)-3,3′-(Pentane-1,5-diylbis(oxy))bis(2-methoxy-
7,12-dihydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinolin-14-
(6aH)-one) (13). To a solution of 49 (50 mg, 0.110 mmol), 1,5-
diiodopentane (17.82 mg, 0.055 mmol) in DMF (1 mL) was added
K2CO3 (15.20 mg, 0.110 mmol) and stirred at room temperature for
14 h. The reaction was then diluted with water, extracted with EtOAc,
washed with brine, dried over sodium sulfate, concentrated, and
purified on ISCO (12 g column, 0−10% MeOH/DCM) to give 13b
as a white solid (22 mg, 0.023 mmol, 20.47% yield). MS
(C53H69N4O10Si2) m/z: 977 (M + H)+.
To a solution of 10b (22 mg, 0.023 mmol) in THF/EtOH (1:1, 1
mL) was added a solution of LiBH4 in THF (2 M, 116 μL, 0.232
mmol) at 0 °C. The reaction was slowly warmed to room temperature
and stirred for 15 min before it was quenched with brine. The
resulting mixture was extracted with chloroform. The combined
organic layers were washed with brine, dried over Na2SO4, and
concentrated in vacuo. The crude product was then taken up in
EtOH/chloroform (1:1, 2 mL). Silica gel (700 mg) was added,
followed by water (0.6 mL). The resulting suspension was stirred at
room temperature for 24 h and then filtered, washing with 10%
MeOH/chloroform. The filtrate was concentrated and purified by
preparative HPLC to give 10 as a white solid (8.5 mg, 0.012 mmol,
50.3% yield). MS (C39H37N4O6) m/z: 657 (M + H)+. 1H NMR (400
MHz, CDCl3): δ 7.55 (s, 2H), 7.49 (d, J = 5.3 Hz, 2H), 7.42−7.31
(m, 8H), 6.82 (s, 2H), 5.03 (d, J = 15.6 Hz, 2H), 4.58 (d, J = 15.4 Hz,
2H), 4.26−4.04 (m, 4H), 4.00−3.92 (m, 8H), 3.37−3.25 (m, 2H),
3.21−3.10 (m, 2H), 2.02−1.90 (m, 4H), 1.70 (d, J = 7.3 Hz, 2H).
(S)-10-Amino-2-methoxy-3-(3-(((S)-2-methoxy-14-oxo-
6a,7,12,14-tetrahydrobenzo[5,6][1,4]diazepino[1,2-b]-
isoquinolin-3-yl)oxy)propoxy)-7,12-dihydrobenzo[5,6][1,4]-
diazepino[1,2-b]isoquinolin-14(6aH)-one (11). To a solution of
49 (1 g, 2.200 mmol) in DMSO (5 mL) were added 1,3-
dibromopropane (1.1 g, 5.45 mmol) and K2CO3 (0.608 g, 4.40
mmol). The reaction was stirred for 1 h and then quenched with
water. The mixture was extracted with DCM (3×). The combined
organic layers were washed with brine, dried over sodium sulfate, and
concentrated. The crude product was purified by silica gel
chromatography (0−100% EtOAc/hexane, 40 g column) to give
11a. MS (C27H36BrN2O5Si) m/z: 575 (M + H)+.
To a solution of 51 (77 mg, 0.139 mmol) in DMSO (0.5 mL) were
added 11a (80 mg, 0.139 mmol) and K2CO3 (38.4 mg, 0.278 mmol).
The reaction was stirred at room temperature for 1 h and then
quenched with water. The mixture was extracted with DCM (3×).
The combined organic layers were washed with brine, dried over
sodium sulfate, and concentrated to provide 11b. MS
(C55H70N5O12Si2) m/z: 1048 (M + H)+. This was then taken up in
DCM (10 mL), and morpholine (0.121 mL, 1.391 mmol) was added.
The solution was then purged with N2 and cooled to 0 °C. Pd(Ph3P)4
(4.82 mg, 4.17 μmol) was added. The reaction was then stirred for 2 h
at 0 °C. The crude product was purified by silica gel chromatography
(0−100% EtOAc/hexane, 40 g column) to give 11c as a white solid
(119 mg, 0.123 mmol, 89% yield). MS (C51H66N5O10Si2) m/z: 964
(M + H)+.
To a solution of 13b (22 mg, 0.023 mmol) in THF/EtOH (1:1, 1
mL) was added LiBH4 (225 μL, 0.450 mmol) in THF at 0 °C. The
reaction was slowly warmed to room temperature and stirred for 15
min before it was quenched with brine, extracted with chloroform,
dried, and concentrated. The resulting intermediate was then taken up
in EtOH/chloroform (1:1, 2 mL). Silica gel (700 mg) was added,
followed by water (0.6 mL). That was stirred at room temperature for
24 h and then filtered, washing with 10% MeOH/chloroform. The
filtrate was concentrated and purified by preparative HPLC to give 13
as a white solid (7.5 mg, 9.86 μmol, 43.8% yield). MS (C41H41N4O6)
1
m/z: 685 (M + H)+. H NMR (400 MHz, CDCl3): δ 7.55 (s, 1H),
7.49 (d, J = 5.3 Hz, 1H), 7.42−7.30 (m, 4H), 6.82 (s, 1H), 5.03 (d, J
= 15.6 Hz, 1H), 4.58 (d, J = 15.4 Hz, 1H), 4.11 (d, J = 17.6 Hz, 2H),
4.01−3.91 (m, 4H), 3.36−3.24 (m, 1H), 3.22−3.13 (m, 1H), 2.02−
1.91 (m, 2H), 1.70 (d, J = 7.3 Hz, 1H).
Allyl ((S)-2-Methoxy-3-((5-(((S)-2-methoxy-6,14-dioxo-5-((2-
( t r i m e t h y l s i l y l ) e t h o x y ) m e t h y l ) - 5 , 6 , 6 a , 7 , 1 2 , 1 4 -
hexahydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinolin-3-yl)-
oxy)pentyl)oxy)-6,14-dioxo-5-((2-(trimethylsilyl)ethoxy)-
methyl)-5,6,6a,7,12,14-hexahydrobenzo[5,6][1,4]diazepino-
[1,2-b]isoquinolin-10-yl)carbamate (14a). To a solution of 4b
(100 mg, 0.154 mmol) and 51 (68 mg, 0.123 mmol) in DMSO (3
mL) was added potassium carbonate (50.9 mg, 0.368 mmol). The
resulting suspension was stirred at room temperature for 5 h. The
reaction was then diluted with EtOAc, washed with brine, dried over
sodium sulfate, and concentrated in vacuo. The crude material was
purified by silica gel chromatography (CombiFlash, 12 g column, 0−
10% MeOH/DCM) to give the title compound (124 mg, 0.115
1
mmol, 94% yield). MS (C57H74N5O12Si2) m/z: 1076 (M + H)+. H
To a solution of 11c (10 mg, 10.37 μmol) in THF (0.7 mL) at −78
°C was added superhydride (0.104 mL, 0.104 mmol). The reaction
was stirred at −78 °C for 1 h before it was quenched with water. The
mixture was then extracted with EtOAc. The combined organic layers
were concentrated and treated with silica gel (0.7 g) in CHCl3−
EtOH−water (1:1:1, 3 mL) at room temperature for 24 h and then
filtered, washing with 10% MeOH/chloroform. The filtrate was
concentrated and purified by preparative HPLC to give 11 as a white
solid (3.1 mg, 4.15 μmol, 40% yield). MS (C39H38N5O6) m/z: 672
(M + H)+.
(6aS,6a′S)-3,3′-(Propane-1,3-diylbis(oxy))bis(10-amino-2-
methoxy-7,12-dihydrobenzo[5,6][1,4]diazepino[1,2-b]-
isoquinolin-14(6aH)-one) (12). To a solution of 51 (158 mg, 0.285
mmol) in DMSO (1.5 mL) were added 1,3-dibromopropane (28.8
mg, 0.143 mmol) and K2CO3 (79 mg, 0.571 mmol). The reaction was
stirred for 1 h and then quenched with water. The mixture was
extracted with DCM (3×). The combined organic layers were washed
with brine, dried over sodium sulfate, and concentrated. The crude
mixture was then taken up in DCM (4 mL), and morpholine (0.199
mL, 2.283 mmol) was added. The mixture was purged with N2 and
then cooled to 0 °C. Pd(Ph3P)4 (9.89 mg, 8.56 μmol) was then
added. The reaction was then stirred at 0 °C for 3 h and concentrated.
The crude product was purified by silica gel chromatography (0−
100% EtOAc/hexane, 40 g column) to give 12a (105 mg, 0.107
mmol, 75% yield). MS (C51H67N6O10Si2) m/z: 979 (M + H)+.
NMR (400 MHz, CDCl3): δ 7.30 (d, J = 0.9 Hz, 6H), 7.27−7.18 (m,
5H), 6.89 (s, 1H), 6.01−5.88 (m, 1H), 5.49 (dd, J = 9.9, 4.0 Hz, 2H),
5.34 (dd, J = 17.3, 1.4 Hz, 1H), 5.25 (dd, J = 10.3, 1.1 Hz, 1H), 5.18−
5.04 (m, 2H), 4.71−4.62 (m, 4H), 4.40 (d, J = 15.4 Hz, 2H), 4.32−
4.22 (m, 2H), 4.09−3.97 (m, 4H), 3.90−3.84 (m, 6H), 3.82−3.73
(m, 2H), 3.71−3.44 (m, 5H), 3.08−2.90 (m, 2H), 1.99−1.90 (m,
4H), 1.04−0.91 (m, 4H), 0.02 (s, 9H), 0.02 (s, 9H).
(S)-10-Amino-2-methoxy-3-((5-(((S)-2-methoxy-6,14-dioxo-
5-((2-(trimethylsilyl)ethoxy)methyl)-5,6,6a,7,12,14-
hexahydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinolin-3-yl)-
oxy)pentyl)oxy)-5-((2-(trimethylsilyl)ethoxy)methyl)-7,12-
dihydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinoline-6,14-
(5H,6aH)-dione (14b). To a solution of 14a (124 mg, 0.115 mmol)
in DCM (6 mL) at 0 °C was added morpholine (0.080 mL, 0.922
mmol). After the reaction was purged with N2, Pd(Ph3P)4 (13.31 mg,
0.012 mmol) was added. The reaction was slowly warmed to room
temperature and stirred under N2 for 2 h. The reaction was then
concentrated and purified by silica gel chromatography (CombiFlash,
12 g column, 0−10% MeOH/DCM) to give the title compound 14b
as a white solid (90 mg, 0.091 mmol, 79% yield). MS
(C53H70N5O10Si2) m/z: 992 (M + H)+. 1H NMR (400 MHz,
CDCl3): δ 7.74−7.64 (m, 1H), 7.62−7.44 (m, 1H), 7.37−7.25 (m,
7H), 7.22 (d, J = 2.3 Hz, 2H), 7.09 (d, J = 7.8 Hz, 1H), 6.63 (s, 1H),
5.52 (dd, J = 10.0, 4.1 Hz, 2H), 5.28−5.06 (m, 3H), 4.68 (dd, J =
10.0, 2.2 Hz, 2H), 4.43 (d, J = 15.3 Hz, 1H), 4.34−4.25 (m, 2H), 4.22
W
https://dx.doi.org/10.1021/acs.jmedchem.0c01385
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
(dd, J = 7.7, 6.4 Hz, 1H), 4.16−4.00 (m, 4H), 3.90 (d, J = 0.8 Hz,
6H), 3.81 (m, 2H), 3.76−3.72 (m, 2H), 3.72−3.63 (m, 3H), 3.57
(dd, J = 15.5, 7.5 Hz, 1H), 3.47 (dd, J = 15.4, 7.7 Hz, 1H), 3.07−2.96
(m, 2H), 2.91 (dd, J = 15.4, 6.4 Hz, 1H), 2.63 (s, 1H), 2.47 (d, J = 4.4
Hz, 1H), 1.97 (p, J = 6.7 Hz, 4H), 1.71 (m, 2H), 1.07−0.89 (m, 4H),
0.05 (d, J = 0.8 Hz, 18H).
(0−30% MeOH/DCM) to give dimethylamino compound (77 mg,
85% yield). MS (C59H79N6O12Si2) m/z: 1120 (M + H)+.
To the above material (50 mg, 0.045 mmol) and Pd(PPh3)4 (5.16
mg, 4.47 μmol) in DCM (5 mL) was added pyrrolidine (0.011 mL,
0.134 mmol), and the reaction was stirred at room temperature
overnight. The reaction was taken up in DCM and water. The organic
was collected and purified by silica gel chromatography (0−40%
MeOH/DCM) to give SEM-lactam 15b (40 mg, 86%). MS
(C55H75N6O10Si2) m/z: 1035 (M + H)+. This was taken in THF (3
mL) and cooled to −78 °C in a dry ice/acetone bath. To the reaction
was added superhydride (0.348 mL, 0.348 mmol), and the reaction
was allowed to stir at −78 °C for 2 h. The reaction was quenched with
water and extracted with dichloromethane. The organics were
combined and concentrated to residue. The residue was taken up
in DCM−EtOH−water (1:2:1, 8 mL). To this was added silica gel
(300 mg), and the reaction was stirred at room temperature for 3
days. The reaction was then filtered to remove the silica gel and then
concentrated to residue. The residue was purified by preparative
HPLC to give 15 as a white solid. MS (C43H47N6O6) m/z: 743 (M +
H)+.
(6aS,6a′S)-3,3′-((Oxybis(ethane-2,1-diyl))bis(oxy))bis(2-me-
thoxy-7,12-dihydrobenzo[5,6][1,4]diazepino[1,2-b]-
isoquinolin-14(6aH)-one) (16). To a solution of 49 (50 mg, 0.110
mmol) and 1-iodo-2-(2-iodoethoxy)ethane 16a (17.92 mg, 0.055
mmol) in DMSO (2 mL) was added K2CO3 (22.80 mg, 0.165 mmol)
and allowed to stir at room temperature overnight. The reaction was
then diluted with EtOAc, washed with brine, dried, concentrated, and
purified by silica gel chromatography (0−10% MeOH/DCM, 12 g
column) to give 16b (47 mg, 0.048 mmol, 87% yield). MS
(C52H67N4O11Si2) m/z: 979 (M + H)+.
(S)-10-Amino-2-methoxy-3-((5-(((S)-2-methoxy-14-oxo-
6a,7,12,14-tetrahydrobenzo[5,6][1,4]diazepino[1,2-b]-
isoquinolin-3-yl)oxy)pentyl)oxy)-7,12-dihydrobenzo[5,6][1,4]-
diazepino[1,2-b]isoquinolin-14(6aH)-one (14). To a solution of
14b (89 mg, 0.090 mmol) in THF (3 mL) at −78 °C was added
superhydride (0.448 mL, 0.448 mmol, 1 M in THF). The reaction
was stirred at −78 °C for 1 h and then quenched with water (1 mL)
and extracted with chloroform (3×). The combined organic layers
were dried over sodium sulfate, filtered, and concentrated in vacuo.
The residue was then taken up in chloroform/EtOH (1:1, 2 mL).
Silica gel (0.7 g) was added, followed by water (0.6 mL). The
resulting suspension was stirred at room temperature for 1 day and
then filtered and washed with 10% MeOH/chloroform. The filtrate
was concentrated and purified by preparative HPLC to give the title
compound 14 as a white solid (19 mg, 0.024 mmol, 27.2% yield). MS
(C41H42N5O6) m/z: 700 (M + H)+. 1H NMR (400 MHz, CDCl3): δ
7.54 (d, J = 2.4 Hz, 2H), 7.50 (t, J = 4.7 Hz, 2H), 7.41−7.31 (m, 5H),
7.15 (d, J = 8.6 Hz, 1H), 6.81 (d, J = 1.8 Hz, 2H), 6.73−6.59 (m,
2H), 5.02 (d, J = 15.6 Hz, 1H), 4.92 (d, J = 15.4 Hz, 1H), 4.58 (d, J =
15.6 Hz, 1H), 4.47 (d, J = 15.4 Hz, 1H), 4.22−4.03 (m, 3H), 4.00−
3.70 (m, 9H), 3.34−3.25 (m, 1H), 3.18 (dt, J = 15.4, 4.2 Hz, 2H),
3.09−3.00 (m, 1H), 2.03−1.92 (m, 4H), 1.76−1.63 (m, 2H).
(S)-10-Amino-3-((5-(((S)-10-(dimethylamino)-2-methoxy-14-
oxo-6a,7,12,14-tetrahydrobenzo[5,6][1,4]diazepino[1,2-b]-
isoquinolin-3-yl)oxy)pentyl)oxy)-2-methoxy-7,12-
dihydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinolin-14(6aH)-
one (15). In a flask were added 1,5-diiodopentane (0.276 mL, 1.852
mmol) and 51 (2.154 g, 3.89 mmol) in DMF (10 mL) with potassium
carbonate (0.768 g, 5.56 mmol). The reaction was stirred at room
temperature for 6 h and then quenched with water. This was extracted
with ethyl acetate. The organics were concentrated to residue. The
residue was purified by silica gel chromatography (0−100% ethyl
acetate in hexanes) to give Alloc-dimer (2 g, 92% yield). MS
(C61H79N6O14Si2) m/z: 1175 (M + H)+. This was taken in DCM (3
mL) and treated with pyrrolidine (0.352 mL, 4.25 mmol) and
tetrakis(triphenylphosphine)palladium(0) (0.197 g, 0.170 mmol) at
room temperature for 3 h. The reaction was poured into water and
dichloromethane. The organic was collected and concentrated to
residue. The residue was purified by silica gel chromatography (0−
100% ethyl acetate in hexanes) to give diamine 15a (1.1 g, 64.2%) as
a white solid. MS (C53H71N6O10Si2) m/z: 1007 (M + H)+. 1H NMR
(400 MHz, CDCl3): δ 7.76−7.64 (m, 4H), 7.63−7.53 (m, 2H),
7.53−7.45 (m, 4H), 7.36−7.26 (m, 3H), 7.22 (d, J = 3.5 Hz, 2H),
7.09 (d, J = 7.8 Hz, 2H), 6.63 (d, J = 7.1 Hz, 3H), 5.53 (dd, J = 9.9,
2.8 Hz, 2H), 5.10 (d, J = 15.2 Hz, 2H), 4.72−4.63 (m, 2H), 4.29 (d, J
= 15.2 Hz, 2H), 4.26−4.17 (m, 2H), 4.16−3.99 (m, 4H), 3.95−3.84
(m, 6H), 3.81 (td, J = 9.6, 6.9 Hz, 2H), 3.76−3.59 (m, 4H), 3.47 (dd,
J = 15.4, 7.8 Hz, 2H), 2.95−2.84 (m, 2H), 1.97 (p, J = 6.8 Hz, 4H),
1.79−1.54 (m, 4H), 1.06−0.91 (m, 4H), 0.06 (d, J = 2.6 Hz, 19H).
In a vial was added 15a (330 mg, 0.328 mmol) in THF (10 mL)
with triethylamine (0.183 mL, 1.310 mmol). To this was added allyl
chloroformate (0.035 mL, 0.328 mmol), and the reaction was allowed
to stir at room temperature for 3 h. The reaction was quenched with
water and extracted with dichloromethane. The organic layer was
concentrated and purified by silica gel chromatography (0−10%
MeOH/DCM) to give mono Alloc product (125 mg, 35% yield). MS
(C57H75N6O12Si2) m/z: 1091 (M + H)+. To this in MeOH (1 mL)
and THF (1 mL) were added paraformaldehyde (16.51 mg, 0.550
mmol) and a few drops of acetic acid. The solution was stirred at
room temperature for 10 min before sodium cyanoborohydride (27.6
mg, 0.440 mmol) was added slowly. The reaction was then stirred at
room temperature overnight. The reaction was quenched with water
and extracted with dichloromethane. The organic layer was
concentrated. The residue was purified by silica gel chromatography
To a solution of 16b (27 mg, 0.028 mmol) in THF (2 mL) at −78
°C was added superhydride (138 μL, 0.138 mmol, 1 M THF)
dropwise. The resulting solution was stirred at −78 °C for 1 h. The
reaction was then quenched with brine, warmed to room temperature,
and extracted with chloroform. The organic layers were combined,
dried, and concentrated. The residue was then dissolved in EtOH/
chloroform (1:1, 2 mL), and silica gel (0.6 g) was added, followed by
water (0.5 mL). The resulting slurry was stirred at room temperature
for 36 h and then filtered, washing with 10% MeOH/chloroform. The
filtrate was concentrated and purified by preparative HPLC to give 16
as a white solid (9.5 mg, 0.012 mmol, 45.2% yield). MS
1
(C40H39N4O7) m/z: 687 (M + H)+. H NMR (400 MHz, CDCl3):
δ 7.55 (s, 2H), 7.49 (d, J = 5.3 Hz, 2H), 7.40−7.31 (m, 8H), 6.86 (s,
2H), 5.02 (d, J = 15.4 Hz, 2H), 4.58 (d, J = 15.4 Hz, 2H), 4.36−4.18
(m, 4H), 4.07−4.00 (m, 4H), 3.97−3.92 (m, 8H), 3.35−3.24 (m,
2H), 3.22−3.12 (m, 2H).
(((Allyloxy)carbonyl)azanediyl)bis(ethane-2,1-diyl)bis(4-
methylbenzenesulfonate) (17a). To a suspension of 2,2′-
azanediyldiethanol (5 g, 47.6 mmol, Fluka) and K2CO3 (6.57 g,
47.6 mmol) in acetonitrile (50 mL) at 0 °C was added allyl
chloroformate (5.07 mL, 47.6 mmol) and allowed to stir at room
temperature for 3 h, cooled to 0 °C, and quenched with water (200
mL). The mixture was extracted with EtOAc (3 × 100 mL), and the
combined organic layers were washed with saturated NaHCO3
solution (100 mL) and water (100 mL), followed by brine (100
mL). The organic layer was dried over MgSO4 and concentrated to
afford allyl bis(2-hydroxyethyl)carbamate as a pale yellow oil (2.55 g,
13.48 mmol, 28.3% yield). MS (C8H16NO4) m/z: 190 (M + H)+. 1H
NMR (400 MHz, CDCl3): δ 5.94 (m, 1H), 5.32 (dd, J = 17.6, 1.6 Hz,
1H), 5.24 (dd, J = 10.4, 1.2 Hz, 1H), 4.62 (d, J = 6.8 Hz, 2H), 3.84
(d, J = 12.4 Hz, 2H), 3.52 (s, 2H), 2.86 (s, 2H).
To a solution of allyl bis(2-hydroxyethyl)carbamate (600 mg, 3.17
mmol) and TEA (1.768 mL, 12.68 mmol) in DCM (5 mL) at 0 °C
was added tosyl chloride (1814 mg, 9.51 mmol) in DCM (5 mL), and
the mixture was allowed to stir at room temperature for 1 h. The
reaction mixture was concentrated, and the resulting residue was
purified by silica gel chromatography (CombiFlash, 80 g column, 0−
70% EtOAc/hexane over 25 min) to give the title compound 17a as a
thick oil (854 mg, 54% yield). MS (C22H27NNaO8S2) m/z: 520 (M +
1
Na)+. H NMR (400 MHz, CDCl3): δ 7.78 (m, 4H), 7.38 (m, 4H),
X
https://dx.doi.org/10.1021/acs.jmedchem.0c01385
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
5.82 (m, 1H), 5.22 (m, 2H), 4.48 (m, 2H), 4.13 (m, 4H), 3.52 (m,
4H), 2.47 (s, 6H).
washed sequentially with aq LiCl and brine. The organic layer was
separated, dried over sodium sulfate, and concentrated in vacuo. The
crude material was purified by silica gel chromatography (0−10%
MeOH/DCM, 24 g column) to give 18b. MS (C34H42N3O7Si) m/z:
632 (M + H)+.
A suspension of 18b and 10% Pd/C (25 mg) in MeOH (8 mL)
was stirred under a balloon of H2 at room temperature for 5 h. The
reaction was then purged with N2, filtered through a pad of Celite,
and concentrated to give 18c (150 mg, 0.301 mmol, 86% yield). MS
(C26H36N3O5Si) m/z: 498 (M + H)+. 1H NMR (500 MHz, CDCl3):
δ 7.65 (d, J = 8.2 Hz, 1H), 7.38−7.23 (m, 4H), 7.21 (s, 1H), 7.07 (d,
J = 7.9 Hz, 1H), 6.90−6.64 (m, 3H), 5.41 (d, J = 10.2 Hz, 1H), 5.14
(d, J = 15.3 Hz, 1H), 4.76 (d, J = 10.1 Hz, 1H), 4.44−4.33 (m, 2H),
4.32−4.17 (m, 3H), 3.80 (s, 3H), 3.72−3.50 (m, 4H), 3.39−3.30 (m,
2H), 3.05−2.93 (m, 1H), 0.98−0.87 (m, 2H), 0.00 (s, 9H).
2-(((S)-2-Methoxy-14-oxo-6a,7,12,14-tetrahydrobenzo[5,6]-
[1,4]diazepino[1,2-b]isoquinolin-3-yl)oxy)-N-(2-(((S)-2-me-
thoxy-14-oxo-6a,7,12,14-tetrahydrobenzo[5,6][1,4]diazepino-
[1,2-b]isoquinolin-3-yl)oxy)ethyl)acetamide (18). To a solution
of 18c (65 mg, 0.131 mmol) in DCM (2 mL) at 0 °C were added
triethylamine (0.055 mL, 0.39 mmol) and 2-chloroacetyl chloride
(22.13 mg, 0.196 mmol). The solution was stirred for 1 h and then
diluted with DCM. The organic layer was washed with brine, dried
over sodium sulfate, and concentrated in vacuo to give 18d. MS
(C28H37ClN3O6Si) m/z: 574 (M + H)+. This residue was combined
with 49 (59.4 mg, 0.131 mmol) and treated with potassium carbonate
(4.2 mg, 0.392 mmol) in DMSO (1 mL) at 50 °C for 4 h. After
cooling to room temperature, the reaction was diluted with EtOAc
and washed with brine. The organic layer was separated, dried over
sodium sulfate, and concentrated in vacuo. The crude material was
purified by silica gel chromatography (0−10% Me OH/DCM, 24 g
column) to give the title compound 18e (95 mg, 0.096 mmol, 73.3%
yield). MS (C52H66N5O11Si2) m/z: 992 (M + H)+.
To a solution of 18e (40 mg, 0.040 mmol) in THF (0.6 mL) at
−78 °C was added a solution of lithium triethylborohydride (0.4 mL,
1 M in THF). The reaction was then stirred at −78 °C for 1 h before
it was quenched with water (1 mL). The reaction was then extracted
with chloroform (3×). The combined organic layers were dried over
sodium sulfate, filtered, and concentrated in vacuo. The residue was
then taken up in chloroform/EtOH (1:1, 2 mL). Silica gel (0.8 g) was
added, followed by water (0.6 mL). The resulting suspension was
stirred at room temperature for 1 day and then filtered, washing with
10% MeOH/chloroform. The filtrate was concentrated in vacuo and
purified by preparative HPLC to give the title compound 18 as a
white solid (11 mg, 0.014 mmol, 35.1% yield). MS (C40H38N5O7) m/
z: 700 (M + H)+. 1H NMR (400 MHz, CDCl3): δ 7.57 (d, J = 9.5 Hz,
2H), 7.48 (dd, J = 5.3, 0.9 Hz, 2H), 7.40−7.31 (m, 9H), 6.83 (d, J =
4.0 Hz, 2H), 5.01 (d, J = 15.6 Hz, 2H), 4.64−4.51 (m, 4H), 4.20 (td,
J = 9.4, 5.0 Hz, 2H), 3.96 (s, 3H), 3.95 (s, 3H), 3.91−3.82 (m, 4H),
3.34−3.23 (m, 2H), 3.20−3.11 (m, 2H).
Allyl Bis(2-(((S)-2-methoxy-6,14-dioxo-5-((2-(trimethylsilyl)-
ethoxy)methyl)-5,6,6a,7,12,14-hexahydrobenzo[5,6][1,4]-
diazepino[1,2-b]isoquinolin-3-yl)oxy)ethyl)carbamate (17b).
A solution of 49 (80 mg, 0.176 mmol), bis tosylate 17a (39.8 mg,
0.08 mmol), and K2CO3 (33.2 mg, 0.240 mmol) in DMSO (1.5 mL)
was stirred at 50 °C for 19 h. The reaction mixture was poured into
water (30 mL). The mixture was saturated with brine (10 mL) and
extracted with EtOAc (3 × 15 mL), and the combined organic layers
were concentrated. The resulting residue was purified by silica gel
chromatography (CombiFlash, 24 g column, 0−100% EtOAc/hexane
over 30 min) to give the Alloc dimer 17b as a white solid (54 mg,
0.051 mmol, 63.5% yield). MS (C56H72N5O12Si2) m/z: 1062 (M +
H)+. 1H NMR (400 MHz, CDCl3): δ 7.29−7.17 (m, 12H), 5.88 (m,
1H), 5.44 (d, J = 10.0 Hz, 2H), 5.29−5.11 (m, 4H), 4.70 (dd, J =
18.4, 10.0 Hz, 2H), 4.58 (dd, J = 4.4, 1.2 Hz, 2H), 4.40 (m, 2H), 4.22
(m, 6H), 3.88−3.51 (m, 16H), 2.99 (m, 2H), 0.94 (m, 4H), 0.00 (s,
18H).
(6aS,6a′S)-3,3′-((Azanediylbis(ethane-2,1-diyl))bis(oxy))bis-
(2-methoxy-5-((2-(trimethylsilyl)ethoxy)methyl)-7,12-
dihydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinoline-6,14-
(5H,6aH)-dione) (17c). To a solution of Alloc dimer 17b (54 mg,
0.051 mmol) in THF (4 mL) were added morpholine (0.022 mL,
0.254 mmol) and Pd(Ph3P)4 (2.94 mg, 2.54 μmol). The reaction
mixture was stirred under a nitrogen atmosphere at room temperature
for 2 h and concentrated. The resulting residue was purified by silica
gel chromatography (CombiFlash, 24 g column, 0−8% MeOH/
DCM) to afford compound 17c as a white solid (35 mg, 70% yield).
1
MS (C52H68N5O10Si2) m/z: 978 (M + H)+. H NMR (400 MHz,
CDCl3): δ 7.36−7.17 (m, 12H), 5.46 (d, J = 10.0 Hz, 2H), 5.12 (d, J
= 15.3 Hz, 2H), 4.63 (d, J = 10.0 Hz, 2H), 4.38 (d, J = 15.3 Hz, 2H),
4.25 (dd, J = 7.4, 6.4 Hz, 2H), 4.15 (m, 4H), 3.84 (s, 6H), 3.74 (td, J
= 9.7, 6.9 Hz, 2H), 3.63 (td, J = 9.7, 6.9 Hz, 2H), 3.53 (dd, J = 15.5,
7.4 Hz, 2H), 3.17 (t, J = 5.1 Hz, 4H), 2.97 (dd, J = 15.5, 6.4 Hz, 2H),
1.04−0.87 (m, 4H), 0.00 (s, 18H).
(6aS,6a′S)-3,3′-((Azanediylbis(ethane-2,1-diyl))bis(oxy))bis-
(2-methoxy-7,12-dihydrobenzo[5,6][1,4]diazepino[1,2-b]-
isoquinolin-14(6aH)-one) (17). To a solution of compound 17c
(350 mg, 0.358 mmol) in THF (10 mL) was added superhydride
(1.789 mL, 1.789 mmol) at −76 °C. The reaction mixture was stirred
for 1 h, quenched with cold water (10 mL), and extracted with DCM
(3 × 20 mL). The resulting residue was treated with DCM/EtOH/
water (1:2:1 = 8 mL) and silica gel (2 g) at room temperature for 3
days. This mixture was filtered through a sintered funnel, and the
silica gel was washed with DCM−MeOH (8:2, 100 mL). The filtrate
was concentrated under high vacuum and purified on CombiFlash EZ
Prep 50 g C18 column eluting with 0−50% acetonitrile/water (0.05%
formic acid) over 20 min. Product fractions were filtered through a
basic resin (PL-HCO3 MP-Resin 1.8 mmol/g; Agilent) and washed
with acetonitrile (5 mL). Lyophilization provided the title compound
17 as a white solid (102.5 mg, 0.142 mmol, 39.7% yield). MS
(C40H40N5O6) m/z: 686 (M + H)+. 1H NMR (400 MHz, CDCl3): δ
7.45 (s, 2H), 7.39 (d, J = 5.2 Hz, 2H), 7.23 (m, 8H), 6.75 (s, 2H),
4.92 (d, J = 15.6 Hz, 2H), 4.47 (d, J = 15.6 Hz, 2H), 4.19 (m, 4H),
3.85 (m, 8H), 3.05−3.22 (m, 9H).
(S)-3-(2-Aminoethoxy)-2-methoxy-5-((2-(trimethylsilyl)-
ethoxy)methyl)-7,12-dihydrobenzo[5,6][1,4]diazepino[1,2-b]-
isoquinoline-6,14(5H,6aH)-dione (18c). To a solution of benzyl-
(2-hydroxyethyl)carbamate (Aldrich, 0.781 g, 4 mmol) and triethyl-
amine (1.115 mL, 8.00 mmol) in DCM (20 mL) was added tosyl
chloride (0.915 g, 4.80 mmol). The reaction was stirred at room
temperature for 1 h. The reaction was then concentrated and purified
by silica gel chromatography (0−100% EtOAc/hexane, 40 g column)
to give 2-(((benzyloxy)carbonyl)amino)ethyl 4-methylbenzenesulfo-
nate 18a (0.85 g, 2.43 mmol, 60.8% yield). MS (C17H20NO5S) m/z:
350 (M + H)+.
(S)-10-Hydroxy-2-methoxy-3-((5-(((S)-2-methoxy-6,14-
dioxo-5-((2-(trimethylsilyl)ethoxy) methyl)-5,6,6a,7,12,14-
hexahydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinolin-3-yl)-
oxy)pentyl)oxy)-5-((2-(trimethylsilyl)ethoxy)methyl)-6a,7-
dihydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinoline-6,14-
(5H,12H)-dione (19). To a solution of 4b (135 mg, 0.208 mmol)
and 8f (110 mg, 0.198 mmol) in DMSO (3 mL) was added potassium
carbonate (82 mg, 0.595 mmol). The resulting suspension was stirred
at room temperature for 5 h. The reaction was then diluted with
EtOAc and washed with water. The organic layer was separated, dried
over sodium sulfate, and concentrated in vacuo. The residue was taken
up in MeOH (10 mL), PPTS (15 mg) was added, and the reaction
was stirred at 40 °C for 1 h. The reaction was then concentrated and
purified by silica gel chromatography (0−100% EtOAc/hexane, 24 g
column) to give the title compound 19a (141 mg, 0.142 mmol, 71.6%
1
yield). MS (C53H69N4O11Si2) m/z: 993 (M + H)+. H NMR (400
MHz, CDCl3): δ 7.36−7.29 (m, 4H), 7.27−7.24 (m, 1H), 7.23−7.10
(m, 4H), 6.94 (d, J = 2.2 Hz, 1H), 6.79 (dd, J = 8.1, 2.4 Hz, 1H), 5.51
(t, J = 9.6 Hz, 2H), 5.18 (dd, J = 19.4, 15.2 Hz, 2H), 4.69 (dd, J = 9.9,
7.5 Hz, 2H), 4.42 (d, J = 15.4 Hz, 1H), 4.37−4.26 (m, 2H), 4.22 (dd,
J = 7.7, 6.6 Hz, 1H), 4.09−4.00 (m, 3H), 3.88 (d, J = 2.6 Hz, 6H),
To a solution of 49 (160 mg, 0.352 mmol) and 18a (135 mg, 0.387
mmol) in DMF (3 mL) was added potassium carbonate (97 mg,
0.704 mmol). The resulting suspension was stirred at room
temperature for 2 h. The reaction was then diluted with EtOAc and
Y
https://dx.doi.org/10.1021/acs.jmedchem.0c01385
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
3.80 (dd, J = 7.0, 4.6 Hz, 2H), 3.68 (dd, J = 6.8, 5.1 Hz, 2H), 3.60−
3.43 (m, 2H), 3.10−2.82 (m, 2H), 2.00−1.87 (m, 4H), 1.69 (br s,
2H), 0.98 (td, J = 6.6, 3.3 Hz, 4H), 0.10−0.02 (m, 18H).
EtOAc, washed with water and then brine, dried, and concentrated to
give 22a. MS (C58H79N4O13Si2) m/z: 1095 (M + H)+. This was
dissolved in THF (0.6 mL) and cooled to −78 °C. Superhydride (0.4
mL, 1 M in THF) was then added and stirred at −78 °C for 1 h. The
reaction was quenched with water (1 mL). The reaction was then
extracted with chloroform, dried over sodium sulfate, filtered, and
concentrated. The residue was then taken up in chloroform/EtOH
(1:1, 2 mL). Silica gel (0.8 g) was added, followed by water (0.6 mL),
stirred at room temperature for 1 day, and then filtered, washing with
10% MeOH/chloroform. The filtrate was concentrated and purified
by preparative HPLC to give 22 as a white solid (3.1 mg, 3.47 μmol,
19.18% yield). MS (C46H51N4O9) m/z: 803 (M + H)+.
2-Amino-N-((S)-2-methoxy-3-((5-(((S)-2-methoxy-14-oxo-
6a,7,12,14-tetrahydrobenzo[5,6][1,4]diazepino[1,2-b]-
isoquinolin-3-yl)oxy)pentyl)oxy)-14-oxo-6a,7,12,14-
tetrahydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinolin-10-yl)-
acetamide (23). To a solution of 14 (4.5 mg, 6.43 μmol), Fmoc-
Gly-OH (3.82 mg, 0.013 mmol), and HATU (4.89 mg, 0.013 mmol)
in DMF (0.5 mL) was added Hunig’s base (3.37 μL, 0.019 mmol).
The reaction was stirred at room temperature for 4 h, and then
piperidine (100 μL) was added. The resulting mixture was stirred at
room temperature for 1 h and purified by preparative HPLC to give
23 as a white solid (0.94 mg, 1.180 μmol, 18.35% yield). MS
(C43H45N6O7) m/z: 757 (M + H)+.
(6aS,6a′S)-3,3′-((1,3-Phenylenebis(methylene))bis(oxy))bis-
(2-methoxy-6a,7-dihydrobenzo[5,6][1,4]diazepino[1,2-b]-
isoquinolin-14(12H)-one) (24). To a solution of 50 (68 mg, 0.130
mmol) and [1,3-bis(bromomethyl)benzene (17.10 mg, 0.065 mmol)]
in acetone (0.4 mL) was added Cs2CO3 (42.2 mg, 0.130 mmol) and
stirred at 40 °C for 1 h. That was quenched with 0.1 M citric acid and
extracted with EtOAc. The organic layer was washed with brine, dried
over sodium sulfate, concentrated, and purified by silica gel
chromatography using EtOAc/hexane to give 24b as a white solid
(13.5 mg, 0.012 mmol, 18.09% yield). MS (C64H79N4O12Si2) m/z:
1151 (M + H)+.
To a solution of 19a (18 mg, 0.018 mmol) in THF (0.6 mL) at
−78 °C was added a solution of lithium triethylborohydride (1 M in
THF, 362 μL, 0.362 mmol). The reaction was stirred at −78 °C for 1
h before it was quenched with water (1 mL). The reaction was then
extracted with chloroform (3×). The combined organic layers were
dried over sodium sulfate, filtered, and concentrated in vacuo. The
residue was then taken up in chloroform/EtOH (1:1, 2 mL). Silica gel
(0.8 g) was added, followed by water (0.6 mL). The resulting
suspension was stirred at room temperature for 1 day and then
filtered, washing with 10% MeOH/chloroform. The filtrate was
concentrated in vacuo and purified by preparative HPLC to give the
title compound 19 as a white solid (4.8 mg, 6.16 μmol, 34.0% yield).
MS (C41H41N4O7) m/z: 701 (M + H)+.
(S)-2,10-Dimethoxy-3-((5-(((S)-2-methoxy-14-oxo-
6a,7,12,14-tetrahydrobenzo[5,6][1,4]diazepino[1,2-b]-
isoquinolin-3-yl)oxy)pentyl)oxy)-6a,7-dihydrobenzo[5,6][1,4]-
diazepino[1,2-b]isoquinolin-14(12H)-one (20). To a solution of
19a (18 mg, 0.018 mmol) in DMSO (0.5 mL) were added potassium
carbonate (7.51 mg, 0.054 mmol) and iodomethane (2.53 mg, 0.054
mmol). The reaction was stirred at room temperature for 3 h before it
was diluted with EtOAc and washed with water. The organic layer was
separated, dried over sodium sulfate, and concentrated in vacuo to give
20a. MS (C54H71N4O11Si2) m/z: 1007 (M + H)+. The crude product
was dissolved in THF (0.6 mL) and cooled to −78 °C. A solution of
lithium triethylborohydride (0.4 mL, 1 M in THF) was added
dropwise. The reaction was then stirred at −78 °C for 1 h before it
was quenched with water (1 mL). The reaction was then extracted
with chloroform (3×). The combined organic layers were dried over
sodium sulfate, filtered, and concentrated in vacuo. The residue was
then taken up in chloroform/EtOH (1:1, 2 mL). Silica gel (0.8 g) was
added, followed by water (0.6 mL). The resulting suspension was
stirred at room temperature for 1 day and then filtered, washing with
10% MeOH/chloroform. The filtrate was concentrated in vacuo and
purified by preparative HPLC to give the title compound 20 (4.2 mg,
5.29 μmol, 29.2% yield). MS (C42H43N4O7) m/z: 715 (M + H)+. 1H
NMR (400 MHz, CDCl3): δ 7.55 (d, J = 1.6 Hz, 2H), 7.50 (d, J = 5.3
Hz, 2H), 7.41−7.30 (m, 5H), 6.94−6.86 (m, 2H), 6.81 (m, 1H), 5.01
(t, J = 14.9 Hz, 2H), 4.67−4.47 (m, 2H), 4.24−4.01 (m, 5H), 3.96 (s,
6H), 3.86 (s, 3H), 3.34−3.02 (m, 4H), 2.02−1.88 (m, 4H).
(S)-10-(2-Hydroxyethoxy)-2-methoxy-3-((5-(((S)-2-methoxy-
14-oxo-6a,7,12,14-tetrahydrobenzo[5,6][1,4]diazepino[1,2-b]-
isoquinolin-3-yl)oxy)pentyl)oxy)-6a,7-dihydrobenzo[5,6][1,4]-
diazepino[1,2-b]isoquinolin-14(12H)-one (21). To a solution of
19a (20 mg, 0.020 mmol) in DMF (0.8 mL) was added potassium
carbonate (13.91 mg, 0.101 mmol), followed by 2-bromoethanol
(7.55 mg, 0.060 mmol). The reaction was heated at 80 °C for 4 h, at
which point LCMS showed full conversion to product. The reaction
was then filtered and loaded onto a 24 g silica gel column and eluted
with 0−100% EtOAc/hexane to give 21a. MS (C55H73N4O12Si2) m/z:
1037 (M + H)+. This was dissolved in THF (0.6 mL) and cooled to
−78 °C. Superhydride (0.201 mL, 0.201 mmol) was added and
stirred at −78 °C for 1 h. The reaction was quenched with water (1
mL) and extracted with chloroform, dried over sodium sulfate,
filtered, and concentrated. The residue was taken up in chloroform/
EtOH (1:1, 2 mL). Silica gel (0.8 g) was added, followed by water
(∼0.6 mL) and stirred at room temperature for 1 day and then
filtered, washing with 10% MeOH/chloroform. The filtrate was
concentrated and purified by preparative HPLC to give 21 as a white
solid (3.5 mg, 4.23 μmol, 21% yield over two steps). MS
(C43H45N4O8) m/z: 745 (M + H)+.
To a solution of 24b (13.5 mg, 0.012 mmol) and pyrrolidine in
DCM (0.698 mL, 0.029 mmol, 0.042 M) was added Pd(PPh3)4 (2
mg, 1.731 μmol) and allowed to stir at room temperature for 30 min.
That was diluted with DCM and washed with saturated ammonium
chloride. The aqueous layer was re-extracted with DCM. Combined
organic phases were dried over sodium sulfate, filtered, and
concentrated. The residue was purified by preparative HPLC to
give 24 as a white solid (5.18 mg, 58.4% yield). MS (C44H39N4O6) m/
1
z: 719 (M + H)+. H NMR (500 MHz, CDCl3): δ 7.58 (s, 2H),
7.54−7.31 (m, 12H), 6.85 (s, 2H), 5.25 (d, J = 12.3 Hz, 2H), 5.18 (d,
J = 12.4 Hz, 2H), 5.02 (d, J = 15.5 Hz, 2H), 4.58 (d, J = 15.5 Hz, 2H),
3.99 (s, 6H), 3.94 (m, 2H), 3.28 (dd, J = 15.4, 5.6 Hz, 2H), 3.17 (dd,
J = 15.4, 4.2 Hz, 2H).
(6aS,6a′S)-3,3′-((1,4-Phenylenebis(methylene))bis(oxy))bis-
(2-methoxy-6a,7-dihydrobenzo[5,6][1,4]diazepino[1,2-b]-
isoquinolin-14(12H)-one) (25). To a solution of 50 (119 mg, 0.227
mmol) and 1,4-bis(bromomethyl)benzene 25a (20 mg, 0.076 mmol)
in DMF (2 mL) was added Cs2CO3 (74.1 mg, 0.227 mmol) and
stirred at room temperature for 1 h. The crude material was purified
on silica gel using MeOH/DCM to afford 25b as a white solid (85
mg, 0.069 mmol, 91% yield). MS (C64H79N4O12Si2) m/z: 1151 (M +
1
H)+. H NMR (400 MHz, DMSO-d6): δ 7.45−7.21 (m, 10H), 7.18
(s, 2H), 6.62−6.56 (m, 2H), 5.67 (ddt, J = 16.0, 10.3, 5.0 Hz, 2H),
5.20 (d, J = 9.6 Hz, 3H), 5.09 (dd, J = 26.7, 12.2 Hz, 6H), 4.98 (d, J =
17.0 Hz, 3H), 4.74 (d, J = 15.7 Hz, 3H), 4.49−4.39 (m, 2H), 4.30
(dd, J = 14.2, 6.5 Hz, 4H), 3.84 (s, 6H), 3.52 (s, 1H), 3.09 (dd, J =
15.4, 6.0 Hz, 2H), 0.83 (s, 18H), 0.07 (s, 12H).
The dimer 25b (85 mg, 0.074 mmol) was deprotected using a
procedure similar to compound 24 to afford 25 as a white solid (50
mg, 91% yield). MS (C44H3N4O6) m/z: 719 (M + H)+.
(6aS,6a′S)-3,3′-((1,2-Phenylenebis(methylene))bis(oxy))bis-
(2-methoxy-6a,7-dihydrobenzo[5,6][1,4]diazepino[1,2-b]-
isoquinolin-14(12H)-one) (26). To a solution of O-xylylene
dibromide 26a (10 mg, 0.038 mmol) and 50 (59.6 mg, 0.114
mmol) in DMF (0.1 mL) was added Cs2CO3 (37.0 mg, 0.114 mmol),
and the reaction was stirred at room temperature for 1 h. The crude
(S)-2-Methoxy-3-((5-(((S)-2-methoxy-14-oxo-6a,7,12,14-
tetrahydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinolin-3-yl)-
oxy)pentyl)oxy)-10-(2-(2-methoxyethoxy)ethoxy)-6a,7-
dihydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinolin-14(12H)-
one (22). To a solution of 19a (18 mg, 0.018 mmol) and 1-bromo-2-
(2-methoxyethoxy)ethane (3.32 mg, 0.018 mmol) in DMSO (0.5
mL) was added potassium carbonate (7.51 mg, 0.054 mmol) and
stirred at room temperature for 3 h. The reaction was diluted with
Z
https://dx.doi.org/10.1021/acs.jmedchem.0c01385
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
material was purified on silica gel (30−100% acetone/DCM) to afford
26b as a white solid (50 mg, 0.033 mmol, 88% yield). MS
(C64H79N4O12Si2) m/z: 1151 (M + H)+. 1H NMR (400 MHz,
DMSO-d6): δ 7.61−7.49 (m, 2H), 7.48−7.41 (m, 2H), 7.37 (d, J =
7.0 Hz, 2H), 7.34−7.22 (m, 6H), 7.17 (s, 1H), 6.67 (s, 1H), 5.69 (s,
1H), 5.20 (dd, J = 28.6, 10.5 Hz, 4H), 5.02 (q, J = 16.9, 12.9 Hz, 6H),
4.72 (d, J = 16.2 Hz, 2H), 4.56 (d, J = 15.9 Hz, 1H), 4.40 (m, 6H),
3.81 (s, 6H), 3.54−3.47 (m, 2H), 3.08 (dd, J = 15.2, 5.7 Hz, 2H),
2.93 (s, 1H), 0.70 (d, J = 6.4 Hz, 18H), 0.12−0.03 (m, 12H).
The dimer 26b (16 mg, 0.014 mmol) was deprotected using a
procedure similar to compound 24 to afford 26 as a white solid (4 mg,
5.45 μmol, 39.2% yield). MS (C44H39N4O6) m/z: 719 (M + H)+.
Allyl (3,5-Bis(hydroxymethyl)phenyl)carbamate (27b). A
suspension of 5-nitro-m-xylene-alpha,alpha′-diol 27a (1 g, 5.46
mmol, Aldrich) and 10% palladium on carbon (291 mg, 0.273
mmol) in methanol (50 mL) was stirred under a balloon of H2 at
room temperature for 2 h. The reaction was filtered through Celite
and concentrated in vacuo. This was dissolved in THF (10 mL) and
mixed with potassium carbonate (650 mg, 4.7 mmol). That was
cooled to 0 °C, and allyl chloroformate (0.5 mL, 4.7 mmol) was
added. The reaction was stirred at 0 °C for 30 min before it was
allowed to warm to room temperature and stirred for 3 h. The
reaction was then quenched with water and extracted with EtOAc
(2×). The organic layer was dried and concentrated in vacuo. The
crude product was purified by silica gel chromatography (40 g
column, 0−100% EtOAc/DCM in 15 min) to give 27b (200 mg,
EtOAc (10 mL). The organic layer was washed with saturated
aqueous NaHCO3 and then brine, dried over Na2SO4, filtered, and
concentrated. The crude product was purified by silica gel
chromatography (40 g column, 0−10% methanol/DCM in 15 min)
to give the title compound 27e (160 mg, 87% yield). MS
(C56H68N5O10Si2) m/z: 1026 (M + H)+. 1H NMR (400 MHz,
CDCl3): δ 7.38−7.30 (m, 6H), 7.28 (d, J = 5.1 Hz, 7H), 6.92−6.85
(m, 1H), 6.76 (d, J = 1.1 Hz, 2H), 5.47 (d, J = 10.1 Hz, 2H), 5.18 (d,
J = 15.2 Hz, 2H), 5.08 (d, J = 8.6 Hz, 4H), 4.65 (d, J = 10.1 Hz, 2H),
4.42 (d, J = 15.4 Hz, 2H), 4.30 (d, J = 0.9 Hz, 2H), 3.92 (s, 6H),
3.82−3.50 (m, 8H), 3.09−2.94 (m, 2H), 1.11−0.88 (m, 4H), 0.10−
0.03 (m, 18H).
(6aS,6a′S)-3,3′-(((5-Amino-1,3-phenylene)bis(methylene))-
bis(oxy))bis(2-methoxy-6a,7-dihydrobenzo[5,6][1,4]-
diazepino[1,2-b]isoquinolin-14(12H)-one) (27). To a solution of
27e (22 mg, 0.021 mmol) in THF (233 μL) and EtOH (233 μL) at 0
°C was added a solution of lithium borohydride (214 μL, 0.429
mmol) (2 M in THF). The reaction was slowly warmed to room
temperature and stirred at room temperature for 2 h. The reaction
was quenched with water and extracted with chloroform (2×) and
then chloroform/MeOH (2×). The combined organic extracts were
dried over sodium sulfate and concentrated in vacuo. The residue was
dissolved in chloroform/EtOH/water (1:1:1, 2 mL), silica gel (0.7 g)
was added, and the reaction was stirred at room temperature for 3
days. The reaction mixture was filtered through a Celite plug and
washed with chloroform, and the solution was concentrated. The
residue was purified by preparative HPLC to give the title compound
27 as a pale yellow solid (8.1 mg, 46.3% yield). MS (C44H40N5O6) m/
z: 734 (M + H)+. 1H NMR (400 MHz, CDCl3): δ 7.57 (s, 2H), 7.47
(d, J = 5.3 Hz, 2H), 7.43−7.30 (m, 8H), 6.99−6.79 (m, 2H), 6.73 (s,
2H), 5.40−4.90 (m, 8H), 4.59 (d, J = 15.4 Hz, 2H), 4.17−3.85 (m,
8H), 3.39−3.05 (m, 4H).
1
21.5% yield). MS (C12H15NNaO4) m/z: 260 (M + Na)+. H NMR
(400 MHz, DMSO-d6): δ 7.31 (s, 2H), 6.91 (s, 1H), 5.99 (ddt, J =
17.2, 10.6, 5.4 Hz, 1H), 5.36 (dq, J = 17.2, 1.6 Hz, 1H), 5.24 (dq, J =
10.6, 1.4 Hz, 1H), 5.13 (t, J = 5.7 Hz, 2H), 4.60 (dt, J = 5.4, 1.3 Hz,
2H), 4.44 (d, J = 5.5 Hz, 4H).
(5-(((Allyloxy)carbonyl)amino)-1,3-phenylene)bis-
(methylene) Dimethanesulfonate (27c). A suspension of 27b
(462.5 mg, 1.949 mmol) and triethylamine (0.815 mL, 5.85 mmol) in
DCM (20 mL) was cooled to −10 °C and treated with mesyl chloride
(0.395 mL, 5.07 mmol). The reaction was stirred at −10 °C for 1 h
before it was quenched with ice-cold water and extracted with DCM
(2 × 10 mL). The organic layer was washed with ice-cold water, dried
over sodium sulfate, and concentrated in vacuo to give the title
compound 27c (750 mg, 95% yield). MS (C14H20NO8S2) m/z: 394
(6aS,6a′S)-3,3′-(((5-Azido-1,3-phenylene)bis(methylene))-
bis(oxy))bis(2-methoxy-7,12-dihydrobenzo[5,6][1,4]-
diazepino[1,2-b]isoquinolin-14(6aH)-one) (28). A suspension of
(5-azido-1,3-phenylene)dimethanol31 (0.25 g, 1.40 mmol) and
triethylamine (0.58 mL, 4.19 mmol) in DCM (5 mL) was cooled
on an ice−water bath and treated with mesyl chloride (0.25 mL, 3.2
mmol). The reaction mixture was stirred at the same temperature for
2 h and then quenched by the addition of water. The layers of the
biphasic mixture were separated, and the aqueous layer was extracted
again with a portion of DCM. The combined organics were washed
with cold dilute HCl (0.05 N), followed by brine and then dried over
sodium sulfate. Evaporation of the solvent afforded mesylate 28a as a
white solid, which was used without further purification (0.425 g, 91%
1
(M + H)+. H NMR (400 MHz, CDCl3): δ 7.52 (s, 2H), 7.15 (s,
1H), 7.04 (s, 1H), 6.09−5.87 (m, 1H), 5.39 (dd, J = 17.2, 1.5 Hz,
1H), 5.29 (dd, J = 10.3, 1.3 Hz, 1H), 5.21 (s, 4H), 4.69 (dt, J = 5.7,
1.2 Hz, 2H), 3.02 (s, 6H).
Allyl (3,5-Bis((((S)-2-methoxy-6,14-dioxo-5-((2-
( t r i m e t h y l s i l y l ) e t h o x y ) m e t h y l ) - 5 , 6 , 6 a , 7 , 1 2 , 1 4 -
hexahydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinolin-3-yl)-
oxy)methyl)phenyl)carbamate (27d). A solution of 27c (135 mg,
0.343 mmol) in DMSO (16 mL) was treated with 49 (312 mg, 0.686
mmol) and potassium carbonate (142 mg, 1.029 mmol) at room
temperature for 2 h. The reaction was quenched with water and
extracted with EtOAc (3×). The mixture was then dried and
concentrated in vacuo. The crude product was purified by silica gel
chromatography (40 g column, 0−100% EtOAc/hexane in 15 min) to
give the title compound 27d (250 mg, 66% yield). MS
(C60H72N5O12Si2) m/z: 1110 (M + H)+. 1H NMR (400 MHz,
CDCl3): δ 7.51 (s, 2H), 7.38−7.13 (m, 14H), 6.96−6.75 (m, 1H),
6.12−5.86 (m, 1H), 5.48 (d, J = 10.1 Hz, 2H), 5.43−5.32 (m, 1H),
5.32−5.23 (m, 1H), 5.23−4.99 (m, 6H), 4.74−4.53 (m, 4H), 4.41 (d,
J = 15.2 Hz, 2H), 4.30 (d, J = 0.9 Hz, 2H), 3.96−3.83 (m, 6H), 3.81−
3.46 (m, 6H), 2.98 (s, 2H), 1.14−0.85 (m, 4H), 0.13 to −0.08 (m,
18H).
(6aS,6a′S)-3,3′-(((5-Amino-1,3-phenylene)bis(methylene))-
bis(oxy))bis(2-methoxy-5-((2-(trimethylsilyl)ethoxy)methyl)-
7,12-dihydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinoline-
6,14(5H,6aH)-dione) (27e). To a solution of 27d (200 mg, 0.180
mmol) in THF (5 mL) at 0 °C were added Pd(Ph3P)4 (8.33 mg, 7.20
μmol) and morpholine (0.038 mL, 0.432 mmol). The reaction was
stirred at 0 °C for 2 h. The reaction mixture was quenched with
saturated aqueous ammonium chloride (5 mL) and extracted with
1
yield). H NMR (400 MHz, CDCl3): δ 7.25 (s, 1H), 7.11 (s, 2H),
5.29−5.18 (m, 4H), 3.05 (s, 6H).
A suspension of phenol 50 (94 mg, 0.18 mmol), mesylate 28a (30
mg, 0.089 mmol), and cesium carbonate (73 mg, 0.22 mmol) in
acetone (0.4 mL) was stirred at 40 °C for 1 h. The mixture was
quenched with 0.1 M citric acid and extracted thrice with ethyl
acetate. The combined organics were washed with brine and dried
over sodium sulfate, filtered, and evaporated. The mixture was
purified by silica gel chromatography (30−80% ethyl acetate in
hexanes) to afford dimer 28b (82.6 mg, 77% yield). MS
(C64H78N7O12Si2) m/z: 1192 (M + H)+.
Dimer 28b (41.3 mg, 0.035 mmol) was deprotected using a
procedure similar to compound 24 to afford 28 as a white solid (7.08
mg, 26% yield). HRMS (ESI+): calcd (C44H38N7O6) (M + H)+,
760.2878; found, 760.2872.
Allyl (6aS)-3,6-Dihydroxy-2-methoxy-14-oxo-6,6a,7,12-
tetrahydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinoline-5-
(14H)-carboxylate (29b). To a solution of 66 (600 mg, 1.059
mmol) in DMF−water (50:1, 5 mL) was added LiOAc (69.9 mg,
1.059 mmol) and stirred at room temperature overnight. The mixture
was quenched with 0.1 M citric acid and extracted thrice with ethyl
acetate. The combined organics were washed with brine and dried
over sodium sulfate, filtered, and evaporated to afford 29b as a white
solid (410 mg, 0.999 mmol, 94% yield). MS (C22H23N2O6) m/z: 411
(M + H)+. 1H NMR (400 MHz, CDCl3): δ 7.35−7.23 (m, 13H), 6.77
AA
https://dx.doi.org/10.1021/acs.jmedchem.0c01385
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
(s, 2H), 5.92 (s, 2H), 5.81 (ddt, J = 17.5, 10.8, 5.5 Hz, 2H), 5.37−
5.29 (m, 2H), 5.20−5.12 (m, 4H), 4.84 (d, J = 15.6 Hz, 2H), 4.68−
4.55 (m, 4H), 4.46 (d, J = 13.2 Hz, 2H), 4.15 (q, J = 7.1 Hz, 3H),
3.97 (s, 6H), 3.73 (ddd, J = 9.8, 5.7, 3.8 Hz, 2H), 3.34 (s, 1H), 3.18
(dd, J = 15.4, 3.9 Hz, 2H), 3.10 (dd, J = 15.3, 5.7 Hz, 2H).
8.03−7.95 (m, 1H), 7.51 (dd, J = 5.2, 3.6 Hz, 1H), 7.48−7.39 (m,
2H), 7.36 (dd, J = 10.6, 6.9 Hz, 3H), 7.32−7.17 (m, 6H), 7.12 (d, J =
6.6 Hz, 1H), 6.71 (d, J = 5.3 Hz, 1H), 5.92 (s, 1H), 5.39 (d, J = 9.2
Hz, 1H), 5.32 (d, J = 12.4 Hz, 1H), 5.27−5.19 (m, 1H), 5.18−5.04
(m, 3H), 4.83 (d, J = 15.3 Hz, 1H), 4.64 (d, J = 15.4 Hz, 1H), 4.50
(d, J = 15.3 Hz, 1H), 4.29 (d, J = 9.3 Hz, 1H), 3.92−3.86 (m, 1H),
3.84 (d, J = 4.0 Hz, 2H), 3.72 (d, J = 4.2 Hz, 6H), 3.67−3.61 (m,
1H), 3.57 (dt, J = 9.7, 4.9 Hz, 1H), 3.13−2.95 (m, 5H).
(6aS,6a′S)-3,3′-((Pyridine-2,4-diylbis(methylene))bis(oxy))-
bis(2-methoxy-7,12-dihydrobenzo[5,6][1,4]diazepino[1,2-b]-
isoquinolin-14(6aH)-one) (32). A suspension of phenol 29b (170
mg, 0.414 mmol), 2,4-bis(bromomethyl)pyridine 32a (50 mg, 0.189
mmol), and cesium carbonate (170 mg, 0.522 mmol) in DMF (1.0
mL) was stirred at room temperature for 1 h. The mixture was
quenched with water (20 mL) and filtered. The precipitate was
washed with diethyl ether and air-dried. The material was purified by
silica gel chromatography, eluting with a gradient from 5 to 50%
acetone in dichloromethane to afford 32b (134 mg, 0.138 mmol, 73%
yield). MS (C51H50N5O12) m/z: 924 (M + H)+. 1H NMR (400 MHz,
DMSO-d6): δ 8.59 (d, J = 5.0 Hz, 1H), 7.61 (s, 1H), 7.42 (s, 1H),
7.39−7.23 (m, 8H), 7.16 (d, J = 5.7 Hz, 2H), 6.90 (d, J = 10.3 Hz,
2H), 6.59 (s, 1H), 5.76 (s, 4H), 5.66 (s, 2H), 5.32−5.08 (m, 6H),
5.08−4.84 (m, 4H), 4.69 (d, J = 15.6 Hz, 2H), 4.49−4.26 (m, 6H),
3.85 (s, 3H), 3.84 (s, 3H), 3.48 (s, 2H), 3.04 (m, 4H). Dimer 32b (36
mg, 0.031 mmol) was deprotected using a procedure similar to
compound 24 to afford 32 as a white powder (16.0 mg, 65% yield).
MS (C43H38N5O6) m/z: 720 (M + H)+.
Diallyl 3,3′-(((5-(Bromomethyl)-1,3-phenylene)bis-
(methylene))bis(oxy))(6aS,6a′S)-bis(6-hydroxy-2-methoxy-14-
oxo-6,6a,7,12-tetrahydrobenzo[5,6][1,4]diazepino[1,2-b]-
isoquinoline-5(14H)-carboxylate) (29c). To a mixture of 29b
(120 mg, 0.291 mmol) and cesium carbonate (100 mg, 0.306 mmol)
in DMF (729 μL) was added 1,3,5-tris(bromomethyl)benzene 29a
(52 mg, 0.146 mmol) and stirred at room temperature for 2 h. The
mixture was quenched with 0.1 M citric acid and extracted thrice with
ethyl acetate. The combined organics were washed with brine and
dried over sodium sulfate, filtered, and evaporated. The mixture was
purified by silica gel chromatography, eluting with ethyl acetate/
hexanes to afford dimer 29c (36 mg, 24% yield). MS
1
(C53H51BrN4NaO12) m/z: 1039 (M + Na)+. H NMR (400 MHz,
CDCl3): δ 7.39 (d, J = 15.4 Hz, 3H), 7.30 (d, J = 3.6 Hz, 10H), 6.64
(s, 1H), 5.68 (s, 1H), 5.31 (s, 3H), 5.19−5.02 (m, 8H), 4.82 (dd, J =
15.7, 5.1 Hz, 2H), 4.59 (d, J = 15.4 Hz, 2H), 4.53−4.34 (m, 6H), 3.96
(d, J = 6.1 Hz, 6H), 3.81 (s, 1H), 3.73−3.66 (m, 2H), 3.11 (m, 4H).
(6aS,6a′S)-3,3′-(((5-(Azidomethyl)-1,3-phenylene)bis-
(methylene))bis(oxy))bis(2-methoxy-7,12-dihydrobenzo[5,6]-
[1,4]diazepino[1,2-b]isoquinolin-14(6aH)-one) (29). To a sol-
ution of 29c (30 mg, 0.030 mmol) in DMF (0.1 mL) was added
sodium azide (19.20 mg, 0.295 mmol) and stirred at room
temperature overnight. That was worked-up with EtOAc/water.
The organic layer was dried over sodium sulfate and concentrated to
afford 29d. This was deprotected using a procedure similar to
compound 24 to afford 29 as a white solid (8 mg, 32% yield). MS
(C45H40N7O6) m/z: 774 (M + H)+. 1H NMR (400 MHz, DMSO-d6):
δ 7.68−7.16 (m, 13H), 7.10 (d, J = 3.5 Hz, 1H), 6.95 (d, J = 6.0 Hz,
1H), 6.68 (s, 1H), 5.90 (s, 1H), 5.36 (s, 1H), 5.30−4.97 (m, 4H),
4.82 (d, J = 15.5 Hz, 1H), 4.70−4.41 (m, 4H), 4.27 (s, 1H), 3.83 (s,
3H), 3.71 (s, 3H), 3.59 (d, J = 24.3 Hz, 2H), 3.26 (s, 1H), 3.17−2.87
(m, 3H).
(6aS,6a′S)-3,3′-((Pyridine-2,6-diylbis(methylene))bis(oxy))-
bis(2-methoxy-7,12-dihydrobenzo[5,6][1,4]diazepino[1,2-b]-
isoquinolin-14(6aH)-one) (30). A suspension 50 (60 mg, 0.114
mmol), 2,6-bis(bromomethyl)pyridine 30a, (15 mg, 0.057 mmol) and
cesium carbonate (37 mg, 0.114 mmol) in acetone (0.4 mL) was
stirred at 40 °C for 1 h. The reaction was quenched with 0.1 M citric
acid and extracted thrice with ethyl acetate. The combined organics
were washed with brine and dried over sodium sulfate, filtered, and
evaporated. The mixture was purified by silica gel chromatography,
eluting with a gradient from 30 to 80% ethyl acetate in hexanes to
afford dimer 30b (36.2 mg, 55% yield). MS (C63H78N5O12Si2) m/z:
1153 (M + H)+.
(S)-3-((6-(Bromomethyl)pyridin-2-yl)methoxy)-2-methoxy-
5-((2-(trimethylsilyl)ethoxy)methyl)-7,12-dihydrobenzo[5,6]-
[1,4]diazepino[1,2-b]isoquinoline-6,14(5H,6aH)-dione (33a).
To a solution of 49 (1.830 g, 4.03 mmol) in DMF (20 mL) was
added 2,6-bis(bromomethyl)pyridine 30a (3.2 g, 12.08 mmol),
followed by potassium carbonate (1.669 g, 12.08 mmol). The
reaction was allowed to stir at room temperature overnight and then
poured into water (50 mL). The reaction mixture was extracted with
ethyl acetate and washed with brine. The organic layer was
concentrated and purified by silica gel chromatography (0−100%
ethyl acetate in hexanes) to obtain 33a as a white solid (2.3 g, 89%
1
yield). MS (C31H37BrN3O5Si) m/z: 640 (M + H)+. H NMR (400
MHz, CDCl3): δ 7.77 (t, J = 7.8 Hz, 1H), 7.51 (d, J = 7.8 Hz, 1H),
7.42 (d, J = 7.8 Hz, 1H), 7.36 (s, 1H), 7.34−7.22 (m, 5H), 5.43 (d, J
= 9.9 Hz, 1H), 5.31 (s, 2H), 5.18 (d, J = 15.3 Hz, 1H), 4.66 (d, J = 9.9
Hz, 1H), 4.58 (s, 2H), 4.41 (d, J = 15.3 Hz, 1H), 4.28 (dd, J = 7.6, 6.4
Hz, 1H), 3.95 (s, 3H), 3.75−3.50 (m, 3H), 3.06−2.99 (m, 1H),
1.05−0.88 (m, 2H), 0.05 (s, 9H).
Allyl ((S)-2-Methoxy-3-((6-((((S)-2-methoxy-6,14-dioxo-5-
((2-(trimethylsilyl)ethoxy)methyl)-5,6,6a,7,12,14-
hexahydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinolin-3-yl)-
oxy)methyl)pyridin-2-yl)methoxy)-6,14-dioxo-5-((2-
( t r i m e t h y l s i l y l ) e t h o x y ) m e t h y l ) - 5 , 6 , 6 a , 7 , 1 2 , 1 4 -
hexahydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinolin-10-yl)-
carbamate (33b). To a solution of 33a (2.1 g, 3.29 mmol) and 51
(1.517 g, 2.74 mmol) in DMF (10 mL) was added potassium
carbonate (1.136 g, 8.22 mmol) and stirred at room temperature
overnight. The reaction was poured into dichloromethane and water.
The organic was collected and concentrated. The residue was purified
by silica gel chromatography (0−100% ethyl acetate in hexanes) to
give 33b as a white solid (2.4 g, 79% yield). MS (C59H71N6O12Si2) m/
Dimer 30b (36 mg, 0.031 mmol) was deprotected using a
procedure similar to compound 24 to afford 30 as a white powder
(13.4 mg, 56% yield). HRMS (ESI+): calcd (C43H38N5O6) (M + H)+,
1
720.2817; found, 720.2808. H NMR (500 MHz, CDCl3): δ 7.79−
7.68 (m, 2H), 7.60 (s, 3H), 7.48 (t, J = 6.1 Hz, 5H), 7.41−7.30 (m,
7H), 6.87 (s, 2H), 5.34 (d, J = 1.8 Hz, 4H), 5.02 (d, J = 15.5 Hz, 2H),
4.60 (d, J = 15.5 Hz, 2H), 4.02 (s, 6H), 3.98−3.91 (m, 2H), 3.29 (dd,
J = 15.4, 5.6 Hz, 2H), 3.17 (dd, J = 15.4, 4.3 Hz, 2H).
(6aS,6a′S)-3,3′-((Pyridine-3,5-diylbis(methylene))bis(oxy))-
bis(2-methoxy-7,12-dihydrobenzo[5,6][1,4]diazepino[1,2-b]-
isoquinolin-14(6aH)-one) (31). To a solution of 50 (100 mg, 0.191
mmol), pyridine-3,5-diyldimethanol 31a (26.5 mg, 0.191 mmol), and
polymer-bound triphenylphosphine (200 mg, 0.629 mmol) in THF (2
mL) was added DIAD (0.122 mL, 0.629 mmol). The reaction was
stirred at room temperature for 1.5 h. The solvent was removed, and
the mixture was purified by silica gel chromatography (30−100%
EtOAc/hexane) to afford 31b as a white solid (55 mg, 0.029 mmol,
15.02% yield). MS (C63H77N5NaO12Si2) m/z: 1174 (M + Na)+.
The dimer 31b (15 mg, 0.013 mmol) was deprotected using a
procedure similar to compound 24 to afford 31 (2 mg, 2.64 μmol,
20.28% yield) as a white solid. MS (C43H38N5O6) m/z: 720 (M +
1
z: 1111 (M + H)+. H NMR (400 MHz, CDCl3): δ 7.79 (t, J = 7.8
Hz, 1H), 7.53 (d, J = 7.8 Hz, 2H), 7.39−7.21 (m, 11H), 6.94 (s, 1H),
5.97 (ddt, J = 17.2, 10.4, 5.7 Hz, 1H), 5.48−5.37 (m, 2H), 5.34 (q, J =
1.6 Hz, 1H), 5.28 (d, J = 1.4 Hz, 3H), 5.25 (dd, J = 2.9, 1.6 Hz, 1H),
5.17 (d, J = 15.3 Hz, 1H), 5.11 (d, J = 15.4 Hz, 1H), 4.72−4.62 (m,
3H), 4.40 (d, J = 15.3 Hz, 2H), 4.32−4.21 (m, 2H), 3.93 (s, 6H),
3.76−3.43 (m, 6H), 1.01−0.86 (m, 4H), 0.05 (s, 18H).
(S)-10-Amino-2-methoxy-3-((6-((((S)-2-methoxy-14-oxo-
6a,7,12,14-tetrahydrobenzo[5,6][1,4]diazepino[1,2-b]-
isoquinolin-3-yl)oxy)methyl)pyridin-2-yl)methoxy)-6a,7-
dihydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinolin-14(12H)-
one (33). To a solution of 33b (850 mg, 0.765 mmol) in DCM (10
mL) was added tetrakis(triphenylphosphine)palladium(0) (88 mg,
1
H)+. H NMR (500 MHz, DMSO-d6): δ 8.68 (d, J = 2.2 Hz, 2H),
AB
https://dx.doi.org/10.1021/acs.jmedchem.0c01385
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
0.076 mmol) and pyrrolidine (0.158 mL, 1.912 mmol). The reaction
was stirred at room temperature for 1 h and poured into water and
dichloromethane. The organic was collected and concentrated. The
residue was purified by silica gel chromatography (0−30% MeOH in
DCM) to give 33c as a white solid (500 mg, 63.6% yield).
[1,4]diazepino[1,2-b]isoquinolin-14(12H)-one) (36). To a sol-
ution of dimethyl 4-methoxypyridine-2,6-dicarboxylate33 (0.45 g,
1.998 mmol) in ethanol (20 mL) was added LiBH4 (0.261 g, 16.07
mmol), and the reaction was stirred at room temperature for 5 h. The
reaction was quenched with acetic acid (2 mL, 35 mmol), stirred for
10 min, concentrated, and purified by silica gel chromatography using
MeOH/DCM to give (4-methoxypyridine-2,6-diyl)dimethanol (210
To a solution of 33c (26 mg, 0.025 mmol) in THF (1 mL) at −76
°C was added superhydride (0.127 mL, 0.127 mmol) and stirred for 1
h. The reaction was quenched with cold water (1 mL) and extracted
with DCM (3 × 10 mL). The organic layer was concentrated and
treated with DCM/EtOH/water (1:2:1 = 4 mL) and silica gel (1 g)
for 3 days. This mixture was filtered through a sintered funnel, and the
silica gel was washed with DCM−MeOH (8:2, 100 mL). The filtrate
was concentrated under high vacuum and purified by silica gel
chromatography (0−10% MeOH/DCM) to provide 33 as a white
solid (16 mg, 0.021 mmol, 82% yield). MS (C43H39N6O6) m/z: 735
(M + H)+. 1H NMR (400 MHz, CDCl3): δ 7.64 (t, J = 7.8 Hz, 2H),
7.48 (m, 3H), 7.38 (d, J = 7.7 Hz, 5H), 7.31−7.18 (m, 6H), 7.03 (d, J
= 7.5 Hz, 1H), 6.76 (s, 2H), 6.54 (s, 2H), 5.22 (s, 4H), 4.91 (d, J =
15.5 Hz, 2H), 4.80 (d, J = 15.5 Hz, 2H), 4.48 (d, J = 15.5 Hz, 2H),
4.36 (d, J = 15.4 Hz, 2H), 3.90 (s, 6H), 3.87−3.80 (m, 3H), 3.77 (s,
2H), 3.17 (m, 2H), 3.11−3.01 (m, 4H), 2.91 (m, 3H).
1
mg, 1.241 mmol, 62.1% yield). H NMR (400 MHz, DMSO-d6): δ
6.87 (s, 2H), 5.33 (t, J = 5.5 Hz, 2H), 4.47 (d, J = 4.9 Hz, 4H), 3.84
(s, 3H). This was converted to 36a following 34a procedure (0.155 g,
1
39% yield). H NMR (400 MHz, CDCl3): δ 6.92 (s, 2H), 4.51 (s,
4H), 3.91 (s, 3H).
A mixture of 36a (29.5 mg, 0.3 mmol), 29b (103 mg, 0.25 mmol),
and cesium carbonate (81 mg, 0.46 mmol) in DMF (500 μL) was
added at room temperature for 2.5 h. The mixture was quenched with
0.1 M citric acid and extracted thrice with ethyl acetate. The
combined organics were washed with brine and dried over sodium
sulfate, filtered, and evaporated. The mixture was purified by silica gel
chromatography, eluting with 0−10% MeOH/DCM to afford 36b
(79.4 mg, 0.083 mmol, 83% yield). MS (C52H52N5O13) m/z: 954 (M
+ H)+.
Methyl 2,6-Bis(bromomethyl)isonicotinate (34a). To a
suspension of methyl 2,6-bis(hydroxymethyl)isonicotinate32 (0.5 g,
2.54 mmol) and triethylamine (1.060 mL, 7.61 mmol) in DCM (10
mL) was added mesyl chloride (0.454 mL, 5.83 mmol) at 0 °C. The
reaction was stirred at 0 °C for 2 h. The reaction was then quenched
with water, extracted with DCM, washed with aqueous HCl (0.1 N)
and then brine, dried over sodium sulfate, and concentrated. The
residue was dissolved in DMF (5 mL) and stirred with sodium
bromide (1.3 g, 12.70 mmol) at room temperature for 40 min. That
was diluted with water and extracted with ethyl acetate. The organic
layer was dried over sodium sulfate, filtered, and concentrated to give
34a (730 mg, 2.260 mmol, 89% yield). MS (C9H10Br2NO2) m/z: 322
Dimer 36b (20 mg, 0.021 mmol) was deprotected using a
procedure similar to compound 24 to afford 36 as a white powder
(13.35 mg, 0.017 mmol, 81% yield). MS (C44H40N5O7) m/z: 750 (M
+ H)+.
(6aS,6a′S)-3,3′-(((4-Hydroxypyridine-2,6-diyl)bis-
(methylene))bis(oxy))bis(2-methoxy-6a,7-dihydrobenzo[5,6]-
[1,4]diazepino[1,2-b]isoquinolin-14(12H)-one) (37). To a mix-
ture of 29b (103 mg, 0.25 mmol) and cesium carbonate (81 mg, 0.25
mmol) in DMF (500 μL) was added 4-(allyloxy)-2,6-bis-
(bromomethyl)pyridine 37a (32.1 mg, 0.1 mmol) and stirred at
room temperature for 2.5 h. The mixture was quenched with 0.1 M
citric acid and extracted thrice with ethyl acetate. The combined
organics were washed with brine and dried over sodium sulfate,
filtered, and evaporated. The mixture was purified by silica gel
chromatography, eluting with 0−10% MeOH/DCM to afford 37b as
a white solid (64.4 mg, 0.066 mmol, 65.7% yield). MS (C54H54N5O13)
m/z: 980 (M + H)+.
1
(M + H)+. H NMR (400 MHz, CDCl3): δ 7.95 (s, 2H), 4.61 (s,
4H), 4.06−3.94 (m, 3H).
Methyl 2,6-Bis((((S)-2-methoxy-14-oxo-6a,7,12,14-
tetrahydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinolin-3-yl)-
oxy)methyl)isonicotinate (34). To a mixture of 29b (103 mg, 0.50
mmol) and cesium carbonate (81 mg, 0.25 mmol) in DMF (500 μL)
was added 34a (32.3 mg, 0.1 mmol) and stirred at room temperature
for 1 h. The mixture was quenched with 0.1 M citric acid and
extracted thrice with ethyl acetate. The combined organics were
washed with brine and dried over sodium sulfate, filtered, and
evaporated. The mixture was purified by silica gel chromatography,
eluting with ethyl acetate/hexanes to afford 34b as a white solid (67.2
mg, 0.068 mmol, 68.4% yield). MS (C53H52N5O14) m/z: 982 (M +
H)+. 1H NMR (400 MHz, CDCl3): δ 8.01 (s, 1H), 7.92 (s, 1H), 7.30
(m, 12H), 6.61 (d, J = 27.2 Hz, 1H), 5.66 (m, 2H), 5.30 (m, 6H),
5.16−4.93 (m, 4H), 4.81 (t, J = 17.5 Hz, 3H), 4.60 (t, J = 17.1 Hz,
2H), 4.42 (m, 5H), 4.08−3.89 (m, 9H), 3.74−3.61 (m, 2H), 3.09 (m,
4H). Dimer 34b (22 mg, 0.022 mmol) was deprotected using a
procedure similar to compound 24 to afford 34 as a white solid (9.32
mg, 53.5% yield). MS (C45H40N5O8) m/z: 778 (M + H)+.
Dimer 37b (20 mg, 0.021 mmol) was deprotected using a
procedure similar to compound 24 to afford 37 as a white solid (3.73
mg, 4.82 μmol, 23.60% yield). MS (C43H38N5O7) m/z: 736 (M +
H)+.
(6aS,6a′S)-3,3′-(((4-(Hydroxymethyl)pyridine-2,6-diyl)bis-
(methylene))bis(oxy))bis(2-methoxy-6a,7-dihydrobenzo[5,6]-
[1,4]diazepino[1,2-b]isoquinolin-14(12H)-one) (38). To a sus-
pension of (4-(((tert-butyldimethylsilyl)oxy)methyl)pyridine-2,6-
diyl)dimethanol (120 mg, 0.423 mmol) and TEA (0.177 mL, 1.270
mmol) in DCM (3 mL) was added mesyl chloride (0.082 mL, 1.058
mmol) at 0 °C. The reaction was stirred at 0 °C for 1 h. The reaction
was then quenched with water, extracted with DCM, washed with
cold aqueous HCl (0.05 N) and then brine, dried over sodium sulfate,
and concentrated to give (4-(((tert-butyldimethylsilyl)oxy)methyl)-
pyridine-2,6-diyl)bis(methylene) dimethanesulfonate 38a as an
orange oil (186 mg, 90% yield). MS (C16H30NO7S2Si) m/z: 440
(6aS,6a′S)-3,3′-(((4-Chloropyridine-2,6-diyl)bis(methylene))-
bis(oxy))bis(2-methoxy-7,12-dihydrobenzo[5,6][1,4]-
diazepino[1,2-b]isoquinolin-14(6aH)-one) (35). To a mixture of
29b (123 mg, 0.3 mmol) and cesium carbonate (150 mg, 0.46 mmol)
in DMF (500 μL) was added 2,6-bis(bromomethyl)-4-chloropyridine
35a (90 mg, 0.3 mmol) and stirred at room temperature for 2.5 h.
The mixture was quenched with 0.1 M citric acid and extracted thrice
with ethyl acetate. The combined organics were washed with brine
and dried over sodium sulfate, filtered, and evaporated. The mixture
was purified by silica gel chromatography, eluting with 0−10%
MeOH/DCM to afford 35b (72 mg, 0.075 mmol, 24.99 yield). MS
(C51H49ClN5O12) m/z: 958 (M + H)+.
Dimer 35b (25 mg, 0.026 mmol) was deprotected using a
procedure similar to compound 24 to afford 35 as a white solid (13.7
mg, 66.2% yield). MS (C43H37ClN5O6) m/z: 754 (M + H)+.
(6aS,6a′S)-3,3′-(((4-Methoxypyridine-2,6-diyl)bis-
(methylene))bis(oxy))bis(2-methoxy-6a,7-dihydrobenzo[5,6]-
1
(M + H)+. H NMR (400 MHz, DMSO-d6): δ 7.50 (d, J = 11.6 Hz,
2H), 5.75 (s, 2H), 5.33 (s, 4H), 3.85 (s, 3H), 3.16 (s, 3H), 0.93 (d, J
= 5.7 Hz, 9H), 0.36 (s, 3H), 0.10 (s, 3H).
To a solution of 38a (25 mg, 0.057 mmol) and 50 (90 mg, 0.171
mmol) in DMF (0.3 mL) was added Cs2CO3 (85 mg, 0.261 mmol)
and stirred at room temperature for 1 h. The crude material was
purified by silica gel chromatography (30−100% acetone/DCM) to
afford 38b (70 mg, 0.053 mmol, 93% yield). MS (C70H94N5O13Si3)
m/z: 1296 (M + H)+.
To a solution of 38b (40 mg, 0.031 mmol) in THF (0.3 mL) was
added TBAF (0.037 mL, 0.037 mmol) and stirred at room
temperature for 30 min, quenched with saturated ammonium
chloride, extracted with ethyl acetate (3×), dried over sodium sulfate,
filtered, and concentrated. The residue was purified by silica gel
chromatography (20−50% acetone/DCM) and preparative HPLC to
AC
https://dx.doi.org/10.1021/acs.jmedchem.0c01385
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
afford 38c (14 mg, 0.014 mmol, 46.6% yield). MS (C52H52N5O13) m/
z: 954 (M + H)+.
The dimer 40b (35 mg, 0.030 mmol) was deprotected using a
procedure similar to compound 24 to afford 40 as a white solid (3 mg,
4.08 μmol, 13.44% yield). MS (C42H37N6O6) m/z: 721 (M + H)+.
(6aS,6a′S)-3,3′-((Pyrazine-2,5-diylbis(methylene))bis(oxy))-
bis(2-methoxy-6a,7-dihydrobenzo[5,6][1,4]diazepino[1,2-b]-
isoquinolin-14(12H)-one) (41). A mixture of 2,5-dimethylpyrazine
(200 mg, 1.849 mmol), N-bromosuccinimide (700 mg, 3.93 mmol)
and AIBN (20 mg, 0.122 mmol) in CCl4 (3 mL) was heated in a
microwave vial at 80 °C for 2.5 h. The crude material was purified by
silica gel chromatography (30−100% ethyl acetate/hexane) to afford
2,5-bis(bromomethyl)pyrazine 41a (83 mg, 0.297 mmol, 16.03%
Dimer 38c (14 mg, 0.015 mmol) was deprotected using a
procedure similar to compound 24 to afford 38 as a white solid (5 mg,
1
43% yield). MS (C44H40N5O7) m/z: 750 (M + H)+. H NMR (500
MHz, DMSO-d6): δ 7.55−7.19 (m, 13H), 7.14 (d, J = 10.0 Hz, 1H),
7.00−6.91 (m, 1H), 5.62−5.45 (m, 1H), 5.33−5.18 (m, 3H), 5.18−
5.07 (m, 1H), 4.82 (d, J = 15.2 Hz, 1H), 4.67−4.49 (m, 4H), 3.87 (s,
6H), 3.76 (s, 1H), 3.67 (s, 1H), 3.28 (t, J = 10.0 Hz, 4H), 3.14−2.95
(m, 2H).
(3-Methoxypyridine-2,6-diyl)bis(methylene) Dimethanesul-
fonate (39a). To a suspension of (3-methoxypyridine-2,6-diyl)-
dimethanol (90 mg, 0.532 mmol) and TEA (0.185 mL, 1.330 mmol)
in DCM (3 mL) was added mesyl chloride (0.095 mL, 1.224 mmol)
at 0 °C. The reaction was stirred at 0 °C for 1 h. The reaction was
then quenched with water, extracted with DCM, washed with cold
aqueous HCl (0.05 N) and then brine, dried over sodium sulfate, and
concentrated to give the crude mesylate as an orange oil. The residue
was purified by silica gel chromatography (20−100% ethyl acetate/
hexane) to afford 39a (87 mg, 0.241 mmol, 45.2% yield). MS
1
yield). MS (C6H7Br2N2) m/z: 266 (M + H)+. H NMR (400 MHz,
CDCl3): δ 8.67 (s, 2H), 4.56 (s, 4H).
To a solution of 2,5-bis(bromomethyl)pyrazine 41a (25 mg, 0.094
mmol) and 50 (113 mg, 0.216 mmol) in DMF (3 mL) was added
potassium carbonate (26 mg, 0.188 mmol) and stirred at room
temperature for 16 h. The crude material was dry loaded (Celite) and
purified by silica gel chromatography (2−10% MeOH/DCM) to
afford 41b (115 mg, 0.091 mmol, 97% yield). MS (C62H77N6O12Si2)
m/z: 1154 (M + H)+.
The dimer 41b (60 mg, 0.052 mmol) was deprotected using a
procedure similar to compound 24 to afford 41 as a white solid (10
mg, 0.013 mmol, 25.6% yield). MS (C42H37N6O6) m/z: 721 (M +
H)+.
(6aS,6a′S)-3,3′-((Furan-2,5-diylbis(methylene))bis(oxy))bis-
(2-methoxy-6a,7-dihydrobenzo[5,6][1,4]diazepino[1,2-b]-
isoquinolin-14(12H)-one) (42). To a solution of 50 (100 mg, 0.191
mmol), 2,5-furandimethanol 42a (20 mg, 0.156 mmol), and
triphenylphosphine (100 mg, 0.381 mmol) in THF (2 mL) was
added DIAD (0.074 mL, 0.381 mmol) and stirred at room
temperature for 1.5 h. The solvent was removed, and the mixture
was purified by silica gel chromatography (30−100% ethyl acetate/
hexane) to afford 42b (15 mg, 0.012 mmol, 7.58% yield). MS
(C62H77N4O13Si2) m/z: 1141 (M + H)+.
The dimer 42b (15 mg, 0.013 mmol) was deprotected using a
procedure similar to compound 24 to afford 42 as a white solid (4 mg,
5.08 μmol, 38.7% yield). MS (C42H37N4O7) m/z: 709 (M + H)+.
(6aS,6a′S)-3,3′-((Thiophene-2,5-diylbis(methylene))bis-
(oxy))bis(2-methoxy-6a,7-dihydrobenzo[5,6][1,4]diazepino-
[1,2-b]isoquinolin-14(12H)-one) (43). To a solution of 2,5-
bis(chloromethyl)thiophene 43a (50 mg, 0.276 mmol) and 50 (300
mg, 0.572 mmol) in DMF (0.5 mL) was added K2CO3 (95 mg, 0.690
mmol), and the reaction was stirred at 40 °C for 3 h. The crude
material was dry loaded (Celite) and purified by silica gel
chromatography (25−100% EtOAc/hexane) to afford 43b (135 mg,
0.117 mmol, 42.2% yield). MS (C62H77N4O12SSi2) m/z: 1157 (M +
H)+.
1
(C10H16NO7S2) m/z: 326 (M + H)+. H NMR (400 MHz, DMSO-
d6): δ 7.63 (s, 2H), 5.32−5.28 (m, 2H), 5.28−5.24 (m, 2H), 3.90 (s,
3H), 3.26 (s, 3H), 3.24 (s, 3H).
(6R,6aR)-Allyl 3-((6-((((6S,6aS)-5-((Allyloxy)carbonyl)-6-
((tert-butyldimethylsilyl)oxy)-2-methoxy-14-oxo-
5,6,6a,7,12,14-hexahydrobenzo[5,6][1,4]diazepino[1,2-b]-
isoquinolin-3-yl)oxy)methyl)-3-methoxypyridin-2-yl)-
methoxy)-6-((tert-butyldimethylsilyl)oxy)-2-methoxy-14-oxo-
6a,7,12,14-tetrahydrobenzo[5,6][1,4]diazepino[1,2-b]-
isoquinoline-5(6H)-carboxylate (39b). To a solution of 39a (20
mg, 0.061 mmol) and 50 (97 mg, 0.184 mmol) in DMF (0.3 mL) was
added Cs2CO3 (60.1 mg, 0.184 mmol) and stirred at room
temperature for 3 h. The crude material was purified by silica gel
chromatography (2−10% MeOH−DCM) to afford 39b (67 mg,
0.048 mmol, 77% yield). MS (C64H80N5O13Si2) m/z: 1182 (M + H)+.
1H NMR (400 MHz, DMSO-d6): δ 7.59 (d, J = 8.7 Hz, 1H), 7.55−
7.46 (m, 1H), 7.37 (s, 2H), 7.28 (q, J = 8.9, 7.2 Hz, 6H), 7.16 (d, J =
11.4 Hz, 2H), 6.68 (s, 1H), 6.53 (s, 1H), 5.65 (s, 1H), 5.20 (dd, J =
22.7, 9.3 Hz, 2H), 5.11−4.98 (m, 6H), 4.92 (d, J = 18.3 Hz, 1H), 4.74
(d, J = 16.0 Hz, 2H), 4.51−4.37 (m, 2H), 4.32 (d, J = 17.7 Hz, 4H),
3.87 (s, 3H), 3.81 (d, J = 7.2 Hz, 6H), 3.51 (s, 1H), 3.14−3.05 (m,
2H), 0.90−0.78 (m, 18H), 0.07 (m, 9H).
(6aS,6a′S)-3,3′-(((3-Methoxypyridine-2,6-diyl)bis-
(methylene))bis(oxy))bis(2-methoxy-6a,7-dihydrobenzo[5,6]-
[1,4]diazepino[1,2-b]isoquinolin-14(12H)-one) (39). Dimer 39b
(20 mg, 0.017 mmol) was deprotected using a procedure similar to
compound 24 to afford 39 as a white solid (3 mg, 22% yield). MS
(C44H40N5O7) m/z: 750 (M + H)+. 1H NMR (500 MHz, DMSO-d6):
δ 7.66−7.17 (m, 12H), 7.14−6.92 (m, 2H), 6.74 (d, J = 7.6 Hz, 1H),
6.68 (d, J = 2.0 Hz, 1H), 5.35 (s, 1H), 5.28−4.94 (m, 4H), 4.82 (d, J
= 15.3 Hz, 1H), 4.64 (dd, J = 15.1, 3.9 Hz, 1H), 4.53 (dd, J = 25.5,
15.2 Hz, 2H), 4.33−4.24 (m, 1H), 3.86 (m, 6H), 3.80 (s, 3H), 3.74
(d, J = 2.4 Hz, 2H), 3.67 (d, J = 12.3 Hz, 2H), 3.61−3.49 (m, 2H),
3.12−2.93 (m, 4H).
The dimer 43b (130 mg, 0.112 mmol) was deprotected using a
procedure similar to compound 24 to afford 43 as a white solid (35
mg, 0.046 mmol, 41.3% yield). MS (C42H37N4O6S) m/z: 725 (M +
H)+. 1H NMR (400 MHz, DMSO-d6): δ 7.43−7.13 (m, 16H), 5.37−
5.17 (m, 4H), 4.59−4.47 (m, 4H), 3.97 (d, J = 9.1 Hz, 2H), 3.83 (s,
2H), 3.72 (s, 6H), 3.66−3.55 (m, 2H).
1-(3-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-
N-((S)-1-(((S)-1-(((S)-2-methoxy-3-((5-(((S)-2-methoxy-14-oxo-
6a,7,12,14-tetrahydrobenzo[5,6][1,4]diazepino[1,2-b]-
isoquinolin-3-yl)oxy)pentyl)oxy)-14-oxo-6a,7,12,14-
tetrahydrobenzo[5,6][1,4]diazepino[1,2-b]isoquinolin-10-yl)-
amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobu-
tan-2-yl)-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-amide
(44). To a solution compound 79 (9.6 mg, 0.010 mmol) and 2,6-
lutidine (2.152 mg, 0.020 mmol) in DMSO (0.7 mL) was added
MAL-dPEG8-NHS ester (13.85 mg, 0.020 mmol in 100 μL of
DMSO). The reaction mixture was stirred at room temperature for 1
h and purified by preparative HPLC to give compound 44 as a white
solid (9.4 mg, 5.53 μmol, 55% yield). MS (C78H104N11O21) m/z:
(6aS,6a′S)-3,3′-((Pyrazine-2,6-diylbis(methylene))bis(oxy))-
bis(2-methoxy-6a,7-dihydrobenzo[5,6][1,4]diazepino[1,2-b]-
isoquinolin-14(12H)-one) (40). A mixture of 2,6-dimethylpyrazine
(200 mg, 1.849 mmol), N-bromosuccinimide (700 mg, 3.93 mmol),
and AIBN (20 mg, 0.122 mmol) in CCl4 (3 mL) was heated in a
microwave vial at 80 °C for 6 h. The crude material was purified by
silica gel chromatography (30−100% ethyl acetate/hexane) to afford
2,6-bis(bromomethyl)pyrazine 40a (22 mg, 0.083 mmol, 4.47%
1
yield). H NMR (400 MHz, CDCl3): δ 8.64 (s, 2H), 4.55 (s, 4H).
To a solution of 40a (22 mg, 0.083 mmol) and 50 (100 mg, 0.190
mmol) in DMF (3 mL) was added potassium carbonate (23 mg,
0.165 mmol) and stirred at room temperature for 16 h. The crude
material was dry loaded (Celite) and purified by silica gel
chromatography (2−10% MeOH/DCM) to afford 40b (75 mg,
0.062 mmol, 74.7% yield). MS (C62H77N6O12Si2) m/z: 1154 (M +
H)+.
1
1531 (M + H)+. H NMR (600 MHz, DMSO-d6): δ 9.88 (s, 1H),
8.03 (s, 1H), 7.91 (s, 1H), 7.79 (d, J = 8.8 Hz, 1H), 7.55−7.04 (m,
9H), 6.97 (s, 2H), 6.91 (s, 1H), 6.75 (s, 1H), 6.46 (s, 1H), 5.90 (s,
1H), 5.77 (s, 1H), 5.34 (s, 2H), 5.24 (d, J = 9.7 Hz, 1H), 4.54 (d, J =
15.4 Hz, 1H), 4.43 (q, J = 15.9 Hz, 2H), 4.31 (s, 1H), 4.16 (q, J = 8.8,
AD
https://dx.doi.org/10.1021/acs.jmedchem.0c01385
J. Med. Chem. XXXX, XXX, XXX−XXX