Macrocyclic pyrrolobenzodiazepine dimers as antibody-drug conjugate payloads

Article abstract of DOI:10.1016/j.bmcl.2017.10.028

Macrocyclic pyrrolobenzodiazepine dimers were designed and evaluated for use as antibody-drug conjugate payloads. Initial structure–activity exploration established that macrocyclization could increase the potency of PBD dimers compared with non-macrocyclic analogs. Further optimization overcame activity-limiting solubility issues, leading to compounds with highly potent (picomolar) activity against several cancer cell lines. High levels of in vitro potency and specificity were demonstrated with an anti-mesothelin conjugate.

Full text of DOI:10.1016/j.bmcl.2017.10.028

Accepted Manuscript
Macrocyclic pyrrolobenzodiazepine dimers as antibody-drug conjugate pay-
loads
Andrew F. Donnell, Yong Zhang, Erik M. Stang, Donna D. Wei, Andrew J.
Tebben, Heidi L. Perez, Gretchen M. Schroeder, Chin Pan, Chetana Rao, Robert
M. Borzilleri, Gregory D. Vite, Sanjeev Gangwar
PII:
S0960-894X(17)31013-2
https://doi.org/10.1016/j.bmcl.2017.10.028
BMCL 25357
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
28 August 2017
12 October 2017
14 October 2017
Please cite this article as: Donnell, A.F., Zhang, Y., Stang, E.M., Wei, D.D., Tebben, A.J., Perez, H.L., Schroeder,
G.M., Pan, C., Rao, C., Borzilleri, R.M., Vite, G.D., Gangwar, S., Macrocyclic pyrrolobenzodiazepine dimers as
antibody-drug conjugate payloads, Bioorganic & Medicinal Chemistry Letters (2017), doi: https://doi.org/10.1016/
j.bmcl.2017.10.028
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Macrocyclic pyrrolobenzodiazepine dimers
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Andrew F. Donnell, Yong Zhang, Erik M. Stang, Donna D. Wei, Andrew J. Tebben, Heidi L. Perez, Gretchen
M. Schroeder, Chin Pan, Chetana Rao, Robert M. Borzilleri, Gregory D. Vite, and Sanjeev Gangwar

Bioorganic & Medicinal Chemistry Letters
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Macrocyclic pyrrolobenzodiazepine dimers as antibody-drug conjugate payloads
a, ∗
Andrew F. Donnell , Yong Zhang , Erik M. Stang , Donna D. Wei , Andrew J. Tebben , Heidi L. Perez ,
a
a
a
a
a
a
Gretchen M. Schroeder , Chin Pan , Chetana Rao , Robert M. Borzilleri , Gregory D. Vite , and Sanjeev
b
b
a
a
b
Gangwar
a
Bristol-Myers Squibb Research & Development, Princeton, NJ 08543, USA
b
Bristol-Myers Squibb Research & Development, Redwood City, CA 94063, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Macrocyclic pyrrolobenzodiazepine dimers were designed and evaluated for use as antibody-
drug conjugate payloads. Initial structure–activity exploration established that macrocyclization
could increase the potency of PBD dimers compared with non-macrocyclic analogs. Further
optimization overcame activity-limiting solubility issues, leading to compounds with highly
potent (picomolar) activity against several cancer cell lines. High levels of in vitro potency and
specificity were demonstrated with an anti-mesothelin conjugate.
Revised
Accepted
Available online
Keywords:
2
017 Elsevier Ltd. All rights reserved.
Antibody-drug conjugates
Pyrrolobenzodiazepines
Macrocycles
Keyword_4
Keyword_5
—
∗
——
Corresponding author. Tel.: +1-609-252-5148; e-mail: andrew.donnell@bms.com

Cytotoxic drugs have been widely employed in cancer
chemotherapy, but, in many cases, their therapeutic index is
limited by adverse effects stemming from poor selectivity for
cancer cells. Antibody-drug conjugates (ADCs) have the
potential to mitigate these side-effects by harnessing the high
specificity of antibodies to deliver small-molecule drugs directly
1
,2
to tumor cells. Pyrrolobenzodiazepines (PBDs) are a family of
sequence-selective DNA minor groove binding natural products
whose potent cytotoxic properties make them attractive for use as
3–5
ADC payloads.
PBD dimers such as 1 and 2, formed by
joining two PBD monomers via their C8-phenol groups with
either a three-carbon or five-carbon spacer, inhibit a variety of
cancer cell lines with subnanomolar IC50s. Their high levels of
activity have been attributed to an ability to promote intra- and
interstrand DNA cross-linking by the formation of covalent
aminal linkages between their reactive N10-C11 imine groups
6
groups of guanine bases. Interest in this class of
and the C2-NH
compounds as ADC payloads is exemplified by vadastuximab
talirine, an anti-CD33 conjugate of 1, which has been advanced
2
7
into clinic trials by Spirogen and Seattle Genetics. Recently, an
,8
9
ADC based on 2 has also been described. Both of these
compounds employ
linker
a
lysosomally-cleavable valine-alanine
for attachment of the payload to the antibody. In the
case of 1, the linker is attached directly to the aniline group. For
, the linker is attached to the N10-position of the hemiaminal
form of 2 via a self-immolative p-aminobenzyl carbamate spacer.
10,11
Figure 1. Model of PBD dimers bound to DNA. PBD dimer 3 is
displayed in yellow and a macrocycle formed from 3 using a nine-carbon
linker is displayed in cyan. Modeling was carried out using Maestro
2
(
Schrödinger, LLC, New York, NY) and the image was generated using
PyMOL Molecular Graphics System (Schrödinger, LLC, New York, NY).
1
N
0
1
1
N
H
8
7
O
O
8'
H
1
n
1
1a
N
O
O
N
H
H
N
OMe MeO
N
R1
2
R2
3
O
O
N
N
OMe MeO
O
O
1
: n = 3, R1
=
R2
=
1. macrocyclization linker (R1)
2
2
H N
O
SAR plan:
3
1
2
2. optimal C2-substituents (R , R )
2
3
: n = 5, R = R = Me
: n = 3, R1 = R2 = Me
N
O
O
O
O
N
H
H
N
N
R2
R3
Our interest in these compounds was based on the
examination of a model of PBD dimer 3 bound to DNA (Figure
). The C11a-stereocenter imparts a conformation that is
O
R1
O
Figure 2. Areas for SAR exploration.
1
isohelical with the DNA minor groove, where the compound
binds in a mode that enables the imine moieties to covalently
As an initial test of the tolerability of macrocyclization, we
prepared a series of macrocyclic PBD analogs with alkyl linkers
ranging in length from 7 to 12 carbons, giving 18- to 23-
membered rings. While unsaturation and substitution at the C2-
position of the PBD ring system are known to improve activity,
we focused our early explorations on the more synthetically-
tractable unsubstituted scaffold to establish the feasibility of
macrocyclization as well as gain insight into the optimal ring
size. We began by constructing the known PBD monomer 5 by
adapting a reported five-step sequence starting from methyl 4-
2
bind the NH groups of guanine bases on opposing strands. The
C2-methyl groups project further along the groove, which is
known to tolerate a variety of substituents, the nature of which
1
2
can have a dramatic effect on the activity of the compounds.
This places the C7/C7’-methoxy groups in an exposed
orientation, and we envisioned joining them with a linker to form
macrocycles, potentially exploiting conformational restriction to
improve potency. Modeling of a macrocyclic analog of 3 with a
nine-carbon alkyl chain linking the phenols suggested that linkers
of this length could be tolerated without distorting the helical
binding geometry, although the ideal chain length was not
apparent. Thus, as illustrated in Figure 2, our goals for SAR
exploration were, first, to identify an appropriately-sized linker
for macrocyclization, and, second, to match the macrocyclic PBD
scaffold with suitable C2-substituents.
1
3
(
benzyloxy)-5-methoxy-2-nitrobenzoate (4) (Scheme 1).
Dimerization was then effected by alkylation with 1,3-
dibromopropane, and cleavage of the methyl ether groups
revealed diphenol 6.
We explored several routes for constructing macrocycles from
. Alkylation of the phenol groups with an appropriately-sized ω-
6
bromo olefin set the stage for ring-closing metathesis, which
proceeded in good yield but with by-products arising from olefin
isomerization (this is illustrated in Scheme 2 for the compounds
derived from 5-bromopent-1-ene). That proved useful at this
stage, because we were able to carry the mixtures forward and
isolate both the targeted species and its des-methylene congener

in sufficient quantities for initial biological evaluation.
Compounds 8b–f were made by this approach (see the
Supplementary Data for the full experimental details). However,
in many cases, isomer separation was challenging, and we felt
that a more selective protocol would be needed to efficiently
access larger quantities of material for analog synthesis. Some
improvements in the RCM reaction were realized by carrying out
the reaction in the presence of reported isomerization
1
4
suppressors, such as 2,6-dichlorobenzoquinone, although we
did not fully optimize this approach because alternate methods
for constructing the macrocyclic ring showed promise. For
example, ring-closing alkyne metathesis was effective, but
synthesis of the requisite internal alkyne precursors added to the
overall complexity of the sequence. For many of the analogs, we
relied on alkylation of 6 with a dihalo alkane, such as 1,7-
dibromoheptane. This method was used to prepare compound 8a
and subsequent analogs. To complete the synthesis, the amide
groups were converted to imines by a sequence involving N-
alkylation with SEM-Cl, hydride reduction, and dehydration of
15
the resultant hemiaminals by stirring with silica gel.
To introduce C2-substitution and unsaturation, we used an
alternate synthetic route where we first constructed the
macrocyclic skeleton and then assembled the PBD ring system.
This chemistry is illustrated in Scheme 3. Methyl 4-hydroxy-3-
methoxybenzoate (10) was dimerized using 1,3-dibromopropane,
and then nitration was followed by treatment with refluxing
aqueous sodium hydroxide, which converted the methoxy groups
to phenols with concomitant hydrolysis of the methyl esters. Re-
esterification gave 11, and alkylation with 1,8-diiodooctane
provided the macrocycle 12. Ester hydrolysis followed by
1
6
coupling with the TBS-protected hydroxyproline derivative 13
gave 14. A one-pot nitro reduction/cyclization sequence forged
the central ring of the PBD units, and protection of the resultant
amide groups with SEMCl gave 15. A three-step sequence
converted the TBS ethers to alkenyl triflates 16. From this
intermediate, a variety of C2-substituents were installed using
cross-coupling chemistry, and, finally, the imine moieties were
unmasked to give the targeted compounds 17a–e. Symmetrical
homodimers 17a-b were prepared from bis-triflate 16 by a
Suzuki coupling with excess boronate (2.2 eq.). Heterodimers
1
7c–e were constructed by first reacting 16 with 1 eq. of 4-
aminophenylboronic acid pinacol ester and isolating the mono-
arylated species from the resulting mixture, and then, in the case
2
of 17c and 17d, installing the R aryl group by a subsequent
Suzuki coupling. Installation of a methyl group in 17e was more
effectively carried out by iron-catalyzed coupling with methyl
magnesium bromide.
An analogous macrocycle with an ethylene glycol-based
linker (18) was also prepared from 11 according to the same
sequence, using 1,2-bis(2-iodoethoxy)ethane instead of 1,8-
diiodooctane.

Scheme 1. Reagents and conditions: (a) 2.5 M aq. NaOH, THF, 50 °C, quant.; (b) (COCl)
THF, 0 °C to rt, 72% (2 steps); (d) H (50 psi), Pd(OH) , EtOH, rt; (e) AcOH, MeOH, 80 °C, 71% (2 steps); (f) 1,3-dibromopropane, K
BBr , CH Cl , –78 °C to –5 °C, 33%; (h) 1,7-dibromoheptane, K CO , DMF, 50 °C; (i) for 7b: 5-bromopent-1-ene; for 7c and 7d: 6-bromohex-1-ene; for 7e and
f: 7-bromohept-1-ene, K CO , DMF, rt, 33–77% ; (j) Grubbs-II, DCE, 75 °C; (k) H , 10% Pd/C, MeOH, 64–88% of a mixture of two species (2 steps); (l) NaH,
DMF, 0 °C; SEMCl; (m) LiBH , 1:1 THF–EtOH, 0 °C to rt; silica gel, 1:1 CHCl –EtOH, 4–37% (2 steps).
2
, DMF, THF, rt; (c) L-proline methyl ester hydrochloride, Et
3
N,
2
2
2
CO , DMSO, rt, 78%; (g)
3
3
2
2
2
3
7
2
3
2
4
3
Scheme 2. Ring-closing metathesis proceeded with isomerization by-products. This is illustrated here using the pentene precursor, which gave rise to a
mixture of 7d and 7c. This mixture was carried through the sequence yielding compounds 8c and 8d, which were separable by preparative HPLC. The reagents
and conditions are specified in Scheme 1.
Scheme 3. Reagents and conditions: (a) 1,3-dibromopropane, K
6%; (d) (COCl) , cat. DMF, THF, rt; MeOH, 97%; (e) 1,8-diiodooctane, K
DMF, THF, rt; 13, Et N, THF, 0 °C, 96%; (h) Zn, NH Cl, MeOH, THF, 50 °C, quant.; (i) NaH, DMF, 0 °C; SEMCl, 76%; (j) TBAF, THF, rt, 93%; (k) SO
Et N, DMSO, CH Cl , 0 °C to rt, 81%; (l) Tf O, 2,6-lut., CH
LiBHEt , THF, –78 °C; silica gel, CHCl , EtOH, H O; (o) (4-aminophenyl)boronic acid pinacol ester (1.0 equiv), PdCl
R B(OH) (1.1 equiv), PdCl (dppf), aq. K PO , THF, rt; (q) MeMgBr, Fe(acac) , 20:1 NMP–THF, –30 °C, 51%.
2
CO
3
, DMSO, rt, 52%; (b) SnCl
4
, HNO
3
, CH
2
Cl
2
, –25 °C, 82%; (c) NaOH, H
2
O, 100 °C,
, cat.
•pyr.,
9
2
2
CO , DMF, 100 °C, 47%; (f) NaOH, H
3
2
O, MeOH, 50 °C, 95%; (g) (COCl)
2
3
4
3
3
2
2
2
2
Cl
2
, –78 °C to –20 °C, 65%; (m) RB(OH)
2
(2.2 equiv), PdCl
2
(dppf), aq. K
3
PO
4
, THF, rt; (n)
3
3
2
2
(dppf), aq. K
3
PO
4
, THF, rt, 42%; (p)
2
2
2
3
4
3

The activity of the C2-unsubstituted macrocycles 8a–f is
summarized in Table 1. As a direct comparator, we also tested
macrocycle 17a showed essentially the same level of activity as
its C2-unsubstituted/saturated parent 8b. Further profiling of 17a
showed that it had poor aqueous solubility (<1 µg/mL at pH 5.5),
and we suspected that this was limiting its activity in our assays.
In addition to the three cell lines highlighted earlier, we also
recorded activity against HCT116 colon cancer cells and a related
P-gp-overexpressing HCT116/VM46 cell line. P-gp expression
1
7
the non-macrocyclic analog 9. Inhibitory activity was measured
against three cancer cell lines using a cell proliferation assay.
Consistent with our modeling, macrocyclization was tolerated,
and substantial improvements in potency were noted with several
of the macrocyclic analogs compared with 9. An 8-carbon chain
provided the optimal ring size (compound 8b).
1
8,19
has been linked with reduced activity for some PBD dimers,
although sensitivity to this mechanism depends on the specific
1
structural features. In the case of 17a, potency was attenuated in
0
Table 1. Cell proliferation inhibitory data for C2-
unsubstituted macrocycles 8a–f versus non-macrocyclic
comparator 9
the drug resistant cell line.
We explored several avenues for improving the solubility of
the macrocyclic analogs. For example, the non-macrocyclic
compound 19 has a clogP of 5.1. Addition of the lipophilic
hydrocarbon linker raises the clogP by two orders of magnitude
(
for 17a, the clogP is 7.0). To counter this, we prepared an analog
with an ethylene glycol-derived linker (18). While this change
restored the clogP to the desired level (4.9), solubility remained
poor (<1 µg/mL), and there was no substantial impact on the
biological activity. As an alternate approach to improving the
solubility, we introduced solubilizing groups at the para-position
of the C2-phenyl rings. In an analogous manner to the work of
a
IC50 (nM)
Cmpd
R
12,20,21
H226
70
N87
63
OVCAR3
56
Howard, et al., we introduced 4-methylpiperazine groups
(compound 17b), and we saw a dramatic improvement in both
9
–OMe
solubility and potency: the aqueous solubility of 17b was >698
µg/mL (pH 5.5), and this compound had excellent activity, with
IC50’s below 100 pM in four of the cell lines.
8
8
a
–O(CH
–O(CH
–O(CH
2
2
2
)
)
)
7
8
9
O–
O–
O–
8.1
1.6
8.7
12
4.6
2.7
8.3
22
3.4
b
1.2
8
c
7.3
While the symmetrical homodimer 17b met our potency
criterion, it lacked a suitable handle for linker attachment to
allow us to further evaluate it as an ADC payload. Therefore, we
prepared heterodimer 17c, where we replaced one of the 4-(4-
methylpiperazine)phenyl groups with an aniline. This compound
retained the very high levels of potency seen with 17b. Notably,
this analog also maintained activity against the drug resistant
HCT116/VM46 cells.
8
d
–O(CH
–O(CH
–O(CH
2
)
10O–
11O–
12O–
20
8
e
f
2
2
)
)
69
146
306
78
8
109
136
a
Activity was measured using an MTS cell proliferation assay following 72 h
incubation with the test compound.
3
Varying the R substituent allowed us to tune the potency of
The combination of C2-substitution and unsaturation typically
increases the cytotoxicity of PBDs. For example, the non-
macrocyclic compound 19, which has an endocyclic olefin with
phenyl substitution at C2, displayed a >400-fold improvement in
potency compared with 9 (Table 2). Interestingly, introduction of
these features onto our macrocyclic series did not provide the
expected potency boost: The 4-methoxyphenyl-substituted
the compounds, as shown with analogs 17d and 17e. We felt that
progressing a suite of payloads with a range of potency levels
would allow us to better understand the balance of activity and
tolerability in the context of the full ADCs.

Table 2. Cell proliferation inhibitory data for C2-substituted macrocycles 17a-e and 18 versus non-macrocyclic comparator 19
a
IC50 (nM)
1
2
R
3
R
Compd
R
H226
0.117
N87
OVCAR3
0.126
HCT116
NT
HCT116/VM46
NT
1
9
–OMe
0.152
1
7a
–O(CH
2
)
8
O–
0.993
1.07
0.621
1.54
1.82
1.70
0.167
0.129
1.05
6.44
1
8
–O(CH
2
CH
2
3
O) –
1
7b
–O(CH
2
)
8
8
O–
0.047
0.024
0.034
0.486
0.35
0.006
0.016
0.076
0.015
1
7c
–O(CH
2
)
O–
0.385
1
7d
–O(CH
2
)
8
8
O–
O–
0.355
0.655
0.559
1.36
0.311
1.24
0.063
0.135
0.36
1.22
1
7e
–O(CH
2
)
a
Activity was measured using an MTS cell proliferation assay following 72 h incubation with the test compound.
Scheme 4. Reagents and conditions: (a) Fmoc-Val-Ala-OH, HATU, 2,6-lutidine, DMF, rt; (b) piperidine, THF, rt, 85% (2 steps); (c)
LiBHEt , THF, –78 °C; HCO H, 1:1:1 THF–MeCN–H O, 68%; (d) 22, 2,6-lutidine, DMSO, rt, 37%.
3
2
2
To evaluate these compounds as ADCs, we installed cathepsin
B cleavable valine-alanine linkers and prepared conjugates. This
effort is summarized for macrocyclic analog 17d. Coupling of
aniline 20 with Fmoc-Val-Ala-OH was followed by Fmoc
deprotection, providing 21 (Scheme 4). Subsequent conversion of
the SEM-amides to imines and coupling with the maleimide-
containing activated ester 22 completed the synthesis of payload-
linker compound 23. The maleimide group is the thiol-reactive
site for antibody conjugation, and the short PEG spacer was
included in 22 to balance the lipophilicity of the payload. We
assessed the serum stability of this compound, and no free

payload was observed in mouse, rat, or human serum over an
incubation period of 24 h.
The anti-CD70 ADC control was 90-fold less active (EC50 = 4.4
nM). In 786-0 cells (Figure 3b), which are CD70-positive and
mesothelin-negative, stong activity was seen with the anti-CD70
ADC (EC50 = 0.079 nM), whereas the anti-mesothelin ADC was
We conjugated 23 to an antibody targeting mesothelin, a cell-
2
surface glycoprotein that is highly expressed in many cancers,
2
1
90-fold less active (EC50 = 15 nM). These data suggest that the
and we measured inhibitory activity against the mesothelin-
positive N87 gastric cancer cell line. This cell line does not
express CD70, so anti-CD70 conjugates were prepared as isotype
controls. Conjugation was effected via thiolated lysine residues:
the antibody was treated with 2-iminothiolane to introduce free
thiol groups by modification of the ε-amine of lysine residues,
and subsequent treatment with 23 led to the isolation of anti-
mesothelin-23 in 78% yield with low levels of aggregation and a
drug-antibody ratio (DAR) of 2.5. Similarly, anti-CD70-23 was
obtained in 72% yield with a DAR of 2.2.
observed differential activity derives primarily from antigen-
specific processes.
In summary, the high levels of potency and specificity
observed with conjugates of 23 illustrate the potential of
macrocyclic PBD dimers as novel ADC payloads. While the
increased lipophilicity of the macrocyclic compounds presented
some challenges, combinations with optimal C2-substituents
produced potent analogs that enabled us to demonstrate ADC
activity. Additional studies of the therapeutic possibilities of this
class of highly-potent DNA modifying agents are ongoing.
The in vitro activity of anti-mesothelin-23 and anti-CD70-23
against N87 cells is displayed in Figure 3a. The anti-mesothelin
ADC exhibited potent cell growth inhibition (EC50 = 0.049 nM).
Figure 3. Inhibitory activity of ADCs of PBD macrocycle 23 in (a) N87 gastric cancer cells, and (b) 786-0 cells.
1
2. Antonow, D.; Kaliszczak, M; Kang, G.-D.; Coffils, M.;
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