Design, synthesis and evaluation of novel, potent DNA alkylating agents and their antibody-drug conjugates (ADCs)

Article abstract of DOI:10.1016/j.bmcl.2019.07.031

Antibody-drug conjugates (ADCs) incorporating potent indolinobenzodiazepine (IGN) DNA alkylators as the cytotoxic payload are currently undergoing clinical evaluation. The optimized design of these payloads consists of an unsymmetrical dimer possessing both an imine and an amine effectively eliminating DNA crosslinking and demonstrating improved tolerability in mice. Here we present an alternate approach to generating DNA alkylating ADCs by linking the IGN monomer with a biaryl system which has a high DNA binding affinity to potentially enhance tolerability. These BIA ADCs were found to be highly cytotoxic in vitro and demonstrated potent antitumor activity in vivo.

Full text of DOI:10.1016/j.bmcl.2019.07.031

Accepted Manuscript
Design, synthesis and evaluation of novel, potent DNA alkylating agents and
their antibody-drug conjugates (ADCs)
Emily E. Reid, Katie E. Archer, Manami Shizuka, Molly A. McShea, Erin K.
Maloney, Olga Ab, Leanne Lanieri, Alan Wilhelm, Jose F. Ponte, Nicholas C.
Yoder, Ravi V.J. Chari, Michael L. Miller
PII:
S0960-894X(19)30487-1
https://doi.org/10.1016/j.bmcl.2019.07.031
BMCL 26572
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Accepted Date:
18 June 2019
19 July 2019
Please cite this article as: Reid, E.E., Archer, K.E., Shizuka, M., McShea, M.A., Maloney, E.K., Ab, O., Lanieri,
L., Wilhelm, A., Ponte, J.F., Yoder, N.C., Chari, R.V.J., Miller, M.L., Design, synthesis and evaluation of novel,
potent DNA alkylating agents and their antibody-drug conjugates (ADCs), Bioorganic & Medicinal Chemistry
Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.07.031
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Design, synthesis and evaluation of novel,
potent DNA alkylating agents and their
antibody-drug conjugates (ADCs)
Leave this area blank for abstract info.
Emily E. Reid, Katie E. Archer, Manami Shizuka, Molly A. McShea, Erin K. Maloney, Olga Ab,
Leanne Lanieri, Alan Wilhelm, Jose F. Ponte, Nicholas C. Yoder, Ravi V.J. Chari, Michael L. Miller*
-
SO3
O
H
N
Lys
N
O
O
S
H
HO3S
S
N
N
O
H
O
S
N
HN
H
N
OMe
31
O

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Design, synthesis and evaluation of novel, potent DNA alkylating agents and
their antibody-drug conjugates (ADCs)
Emily E. Reid, Katie E. Archer, Manami Shizuka, Molly A. McShea, Erin K. Maloney, Olga Ab, Leanne
Lanieri, Alan Wilhelm, Jose F. Ponte, Nicholas C. Yoder, Ravi V.J. Chari, Michael L. Miller*
aImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451, United States
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Antibody-drug conjugates (ADCs) incorporating potent indolinobenzodiazepine (IGN) DNA
alkylators as the cytotoxic payload are currently undergoing clinical evaluation. The optimized
design of these payloads consists of an unsymmetrical dimer possessing both an imine and an
amine effectively eliminating DNA crosslinking and demonstrating improved tolerability in mice.
Here we present an alternate approach to generating DNA alkylating ADCs by linking the IGN
monomer with a biaryl system which has a high DNA binding affinity to potentially enhance
tolerability. These BIA ADCs were found to be highly cytotoxic in vitro and demonstrated potent
antitumor activity in vivo.
Keywords:
Antibody-drug conjugates
DNA alkylation
ADCs
2009 Elsevier Ltd. All rights reserved.
Indolinobenzodiazepines
Antibody-drug conjugates (ADCs) link highly cytotoxic
payloads to a tumor-selective monoclonal antibody in an effort to
increase targeted delivery of the payload and improve its
therapeutic index (TI).1-4 The majority of clinical-stage ADCs
have used tubulin-interacting agents, the maytansinoids and
auristatins,5-6 but more recently payloads with alternative
mechanisms of action have emerged. These include topoisomerase
1 inhibitors, such as the camptothecins, and DNA crosslinkers,
such as the pyrrolobenzodiazepine (PBD) dimers.7-9 The anti-
tumor activity of ADCs of the latter class has been limited, since
clinically achievable doses have been quite low due to cumulative
toxic side effects.10-11
Fig. 1. Structures of DNA Alkylating IGN ADCs and
representative structure of previously reported PBD-biaryl
compound.
To further explore the structure-activity relationship (SAR) of
IGNs and develop new IGN ADCs with a greater therapeutic index
(TI), we considered replacing the IGN monomer subunit bearing
the N-10 amine of our pseudodimer with a DNA binding motif.
-
SO3
H
O
O
S
N
O
N
S
O
N
H
N
N
O
O
10
N
OMe
MeO
O
O
IMGN779 (anti-CD33 DGN462)
We have previously reported on a potent new class of DNA
alkylating indolinobenzodizepine (termed IGN) pseudodimers
purposely designed to eliminate DNA crosslinking.12-13 The
controlled reduction of one of the two imine moieties present in
the dimer effectively changes the mechanism of action from DNA
crosslinking to DNA alkylation. These IGN ADCs show improved
tolerability, with only a slight loss in potency, but were free of the
delayed toxicity in mice observed for their DNA crosslinking
counterparts. As a result of these efforts, multiple IGN ADCs
(IMGN779, IMGN632, and TAK-164) have recently been
advanced into clinical evaluation.
O
O
O
H
N
H
N
HN
N
N
H
S
O
O
O
H
N
N
O
OMe
O
10
N
N
MeO
O
O
IMGN632 (anti-CD123 DGN549)
O
O
H
N
H
N
HN
N
H
O
O
H
N
N
O
O
N
N
OMe
MeO
O
O
TAK-164 (anti-GCC DGN549)
O
N
N
O
N
O
N
H
O
HN
N
N
H
OMe
N
OMe
O
N
O
1
O
H
N
O
O
N
N
N
N
OCH3
O
H
N
N
OMe
2
O
DNA binding motif

Thurston et.al have previously described PBD monomers to which
a DNA binding unit (e.g. either a polyamide (1) or biaryl (2)
system) was attached. The enhanced DNA-binding affinity of
these compounds resulted in PBDs which demonstrated significant
in vitro cytotoxicity improvements in tumor cell lines versus the
PBD monomer itself.14-15 We rationalized that replacing the N-10
amine containing IGN monomer with a DNA binding motif may
lead to increased DNA minor-groove interactions, greater potency,
and potentially improved tolerability as an ADC over that of a
DNA alkylating IGN dimer. Additionally, the removal of the
amine significantly decreases the synthetic complexity associated
with the unsymmetrical IGN pseudodimer. Here, we report on the
design, synthesis, and preclinical evaluation of these IGN
monomers linked with a DNA binding moiety (termed BIAs).
protected THIQ monomer 13. Hydrogenolysis of 13 with Pd/C
followed by coupling with ethyl 4-bromobutanoate and
subsequent hydrolysis of the methyl ester gave the THIQ monomer
14 containing a carboxylic acid.
Using the modified IGN monomer 4 and THIQ monomer 14 we
synthesized a series of BIAs incorporating a variety of aryl
subunits as shown in Figure 2. The modified monomer was
coupled to a biaryl system containing a 5 membered heteroaryl
(i.e., methylpyrrole, methylimidazole, thiazole) ring in place of
unit 1 and a phenyl containing substituent (i.e., benzothiophene,
benzofuran, methyl-3-phenylpyrrole) in place of unit 2. At the
terminal end of the BIA we incorporated a side chain bearing a
variety of functional groups, some of which could be converted to
a moeity used for conjugation to an antibody, such as an N-
hydroxysuccinimide (NHS) ester (A,D) or thiol (B-C). (See
supporting information for synthesis and structures of BIAs 15-28)
The synthesis of a typical BIA (10) suitable for conjugation to
an antibody requires a modified IGN monomer (4) and a DNA
binding group containing a linker handle (9) (Scheme 1).
Carboxylic acid 4 was prepared via coupling IGN monomer 3 with
ethyl 4-bromobutanoate in the presence of potassium carbonate,
followed by hydrolysis of the resulting methyl ester with LiOH.
The synthesis of DNA binding group 9 began with the coupling of
methyl 5-aminobenzo[b]thiophene-2-carboxylate (5) with methyl-
pyrrole carboxylic acid 6 using EDC and DMAP. Hydrolysis of
methyl ester 7 to the corresponding carboxylic acid 8 followed by
condensation with 2-methyl-2-methyldithio-propan-1-amine, and
Boc deprotection with HCl gave the desired DNA binder 9.
Finally, carboxylic acid 4 and amine 9 were coupled in the
presence of EDC and DMAP to generate BIA 10 containing a
methyl disulfide in high yield.
BnO
MeO
NO2
OH
NO2
BnO
MeO
a,b
c,d
N
O
O
11
OH
12
O
N
BnO
MeO
N
O
OH
e,g
N
N
OMe
O
O
13
14
Scheme 2. Synthesis reagents and conditions. a) Oxalyl chloride,
DMF, 100%; (b) (S)-tetrahydroisoquinolin-3-yl)methanol, DCM,
TEA, 98%; (c) Dess-Martin Periodinane, DCM, 91%; (d) Iron
powder, THF, MeOH, water, NH4Cl, 84%; (e) Pd/C, 1,4-
cyclohexadiene, EtOH, 44%; (f) ethyl 4-bromobutanoate , K2CO3,
DMF, 100%; (g) LiOH, MeOH, 53%.
O
N
OH
N
O
OH
a,b
N
OMe
N
OMe
O
O
3
4
O
O
O
O
N
O
unit 1
unit 2
side chain
N
N
O
N
N
d
O
c
OMe
OMe
H
H
S
+
S
BocHN
H2N
BocHN
HN
OH
N
OMe
7
5
6
O
IGN (n = 0)
n
SSMe
O
O
THIQ (n = 1)
H2N
N
N
O
O
SSMe
OH
N
H
e,f
unit 1
unit 2
side chain
S
S
BocHN
H2N
HN
HN
8
9
O
O
O
S
N
N
O
O
HN
SSMe
N
OR
N
N
O
H
O
H
g
benzothiophene
S
N
A
B
H
N
OMe
10
methylpyrrole
O
SR
SR
O
N
N
H
benzofuran
N
O
Scheme 1. Synthesis reagents and conditions. a) ethyl 4-
bromobutanoate, K2CO3, DMF, 100%; (b) LiOH, CH3OH, water,
64%; (c) EDC, DMAP, DMF, 72%; (d) NaOH, CH3OH, 96%; (e)
2-methyl-2-methyldithio-propan-1-amine, EDC, DMAP, DMF,
96%; (f) HCl, dioxane, 100%; (g) 4, EDC, DMAP, DCM, 83%.
methylimidazole
N
N
H
S
C
methyl-3-phenylpyrrole
N
O
thiazole
OR
D
phenyl
Our previous internal studies with the IGN pseudodimer series
Fig. 2. Representative structures of synthesized BIAs.
had shown that replacing the IGN monomer with
a
The in vitro potency for a representative set of BIAs against
three different cell lines (KB, NCI-H2110, and Namalwa) is shown
in Table 1. All BIAs (10, 15-28) were found to be highly potent
towards the cell lines tested. In general, potency of the BIAs was
primarily due to the chosen biaryl systems (unit 1 and 2) and did
not vary greatly based on the side chain that was incorporated. For
example, similar potency was observed for BIA 19, which had no
side chain substituent on the terminal heterocycle, and those with
the same biaryl unit but containing side chains. Incorporation of
the THIQ monomer significantly increased (5-10 fold) in vitro
tetrahydroisoquinoline (THIQ) unit led to improved in vitro
potency. Thus, we wanted to determine if a similar enhanced
potency would be achieved in the BIA series. The synthesis of a
THIQ monomer suitable for attachment of a DNA binding unit is
shown in Scheme 2. Carboxylic acid 11 was converted to its
corresponding acid chloride with oxalyl chloride and then coupled
with (S)-tetrahydroisoquinolin-3-yl)methanol to give the nitro
alcohol 12. Reduction of the nitro group to the amine with iron
powder, followed by oxidation of the primary alcohol with Dess-
Martin periodinane led to spontaneous cyclization to give the

activity (compare 27 and 28) presumably as a result of a stronger
interaction with the DNA.
Table 1. Structure and in vitro potency of BIAs
IC50 pMb
BIA
10
15
16
17
18
19
20
21
22
23
24
25
26
27
28
Monomer
IGN
Unit 1a
Unit 2 a
benzothiophene
benzothiophene
benzothiophene
benzofuran
Side Chain a
B (R = SMe)
D (R = Me)
A (R = Me)
A (R = Me)
B (R = SMe)
-H
KB
80
10
40
200
5
NCI-H2110
300
50
Namalwa
10
methylpyrrole
methylpyrrole
methylpyrrole
methylpyrrole
methylpyrrole
methylimadazole
methylimadazole
methylimadazole
methylimadazole
methylimadazole
methylimadazole
thiazole
IGN
20
IGN
200
500
30
30
IGN
60
THIQ
IGN
benzothiophene
benzothiophene
benzothiophene
benzothiophene
benzofuran
2
40
--
80
20
IGN
A (R = Me)
B (R = SMe)
A (R = Me)
-NH2
--
10
IGN
--
--
30
IGN
80
200
30
100
10
40
6
500
400
400
600
100
60
60
IGN
phenyl
60
IGN
phenyl
C (R = SMe)
C (R = SMe)
B (R = SMe)
B (R = SMe)
B (R = SMe)
30
IGN
phenyl
30
IGN
methylpyrrole
methylimadazole
methylimadazole
Methyl-3-phenylpyrrole
Methyl-3-phenylpyrrole
Methyl-3-phenylpyrrole
30
IGN
10
THIQ
10
1
aSee Figure 2 for definition. bCancer cell lines were incubated with BIAs for 5 days at 37oC. IC50 values were determined using a WST-
based cell viability assay.
-
SO3
O
-
SO3
O
O
H
N
O
PySS
N
N
Lys
O
O
S
HO3S
H
S
N
N
O
O
O
SH
N
HO3S
H
30
O
O
H
N
O
N
S
N
O
H
HN
S
H
N
HN
H
N
OMe
N
31
OMe
Lys
O
29
H2N
HEPES 50mM
O
pH 8.5
a,b
10
Scheme 1
O
O
H
N
HO
O
N
OMe
H
N
N
O
N
O
42
O
NH2
O
h
OH
S
S
BocHN
BocHN
HN
HN
i,j,k
8
41
O
O
c,d
H
N
H
N
O
N
O
N
OR
O
H
S
O
O
N
O
O
HN
H
N
O
N
OMe
N
OMe
43
(R = Me)
H
O
S
H2N
HN
37
l,m
e,f,g
O
O
N
O
O
O
O
H
N
H
N
O
N
O
N
O
O
N
N
O
N
O
O
N
O
H
N
S
O
O
O
N
H
H
HN
O
S
N
O
HN
H
N
OMe
N
44
OMe
38
O
pH 8.5
HEPES 50mM
pH 8.5
HEPES 50mM
Lys
H2N
Lys
H2N
O
O
O
O
N
H
N
N
H
H
N
O
O
N
HO3S
H
O
O
N
Lys
N
HO3S
H
N
N
H
Lys
O
H
H
N
O
S
N
HN
S
N
O
O
HN
H
H
N
OMe
N
OMe
39
45
O
O
Scheme 3. Synthesis reagents and conditions. a) TCEP, 100%; (b) Na2S2O5, isopropanol, water, 13%; (c) -alanine-OMe, EDC, DMAP,
DMF, 73%; (d) HCl, dioxane, 94%; (e) 4, EDC, DMAP, DCM, 65%; (f) Me3SnOH, DCE, 100%; (g) N-hydroxysuccinimide, EDC,
DMAP, DMF, 28%; (h) DPPA, TEA, DMF, 49%; (i) 42, HATU, DIEA, DMF, 59%; (j) HCl, dioxane, 100%; (k) 4, EDC, DMAP, DCM,
24%; (l) LiOH, water, THF, 100%; (m) N-hydroxysuccinimide, EDC, DMAP, DMF, 30%.
We selected DNA binding motif 8 for evaluating BIA ADCs
dipeptide). Methyl disulfide 10, prepared from 8, was treated with
with different linker types (disulfide, non-cleavable amide, and
TCEP to generate the free thiol followed by reversible sulfonation

of the imine (29) using sodium metabisulfite. The introduction of
the sulfonate was found to enhance the aqueous solubility of the
BIA and facilitate conjugation via lysine residues. Conjugation
was performed through in situ treatment of 29 with sulfo-SPDB
(30) and addition to an anti-FR antibody resulting in BIA anti-
FR ADC 31. Disulfide linked anti-FR ADCs 32-36 were
prepared in a similar fashion.
reason to account for this observation. As with the free BIAs
(Table 1) the anti-FR ADCs possessing a THIQ monomer were
found to be more potent (~10-fold) compared with their IGN
monomer containing counterparts (i.e., 31 vs 32). Interestingly, in
the anti-FR ADCs containing a methyl-3-phenylpyrrole group in
unit 2 (34-36) the THIQ monomer was found to have a significant
impact on potency towards the lower antigen expressing T47D
cells line, being up to 32-fold more potent as compared with anti-
FR ADC 31.
A second conjugation approach involved converting the free
acid of intermediate 8 to an NHS ester. This was accomplished by
treating 8 with the methyl ester of -alanine under EDC/DMAP
conditions, followed by Boc deprotection with HCl to give the free
amine 37. Coupling of 37 with the modified IGN monomer 4,
followed by hydrolysis of the terminal methyl ester and treatment
with N-hydroxysuccinimide, in the presence of EDC/DMAP,
generated the desired NHS ester 38. In situ sulfonation of the imine
in 38, using sodium metabisulfite, followed by conjugation with
an anti-FR antibody via lysine residues as described previously
gave the direct amide linked anti-FR ADC 39. Anti-FR ADC
40 was prepared in a similar fashion.
A third method of conjugation was via a cleavable dipeptide
bond generating an amine catabolite with the potential to have
bystander activity. A Curtius rearrangement of intermediate 8 with
diphenylphosphoryl azide (DPPA) gave amino benzothiophene 41
in good yield. Coupling of 41 with Ala-Ala methyladipate (42) in
the presence of HATU, followed by HCl deprotection of the Boc
group and coupling to IGN monomer 4 gave BIA methyl ester 43.
Hydrolysis with LiOH and conversion to the desired NHS ester 44
allowed for conjugation to an anti-FR antibody, after in situ
sulfonation with sodium metabisulfite, to give anti-FR ADC 45.
Anti-FR ADC 46 was prepared in a similar fashion. All BIA
ADCs, regardless of conjugation method, were found to have an
average of 2.3 – 3.1 BIAs per antibody, were >97% monomeric by
SEC and contained <0.5% unconjugated BIA (free drug).
Fig. 3. In vivo antitumor activity of anti-FR ADC 31 and chKTI-
ADC 31 in a cervical KB xenograft in SCID mice.
Encouraged by the potency of a number of FR BIA ADCs, we
selected anti-FR ADC 31 for in vivo evaluation. Treatment of
SCID mice bearing subcutaneous KB cervical tumor xenografts
with a single i.v. dose of 2.5 mg/kg ADC (equivalent to 50 g/kg
linked BIA) resulted in significant tumor growth delay resulting in
6/6 PRs and 4/6 CRs, while a dose of 5 mg/kg ADC produced
complete tumor regressions in all mice lasting more than 60 days
(duration of the experiment). In addition, the non-targeting chKTI
ADC 31 was inactive even at the higher dose of 5 mg/kg indicating
the antitumor activity was antigen specific (Figure 3).
Table 2. In vitro potency of BIA ADCs
IC50 pM
ADC
Anti-FR
31
Linked
BIA
10
KB
T47D
Linker
DAR (3,000k)a (100k)a
B
2.8
2.9
2.5
2.7
2.6
2.3
3.1
2.9
3.1
2.5
40
4
200
30
32
18
B
In conclusion, we have prepared a series of highly potent BIAs
containing either an IGN or THIQ modified monomer, with a
variety of biaryl units that demonstrate a high binding affinity for
DNA. A subset of these BIAs were converted and conjugated, via
antibody lysine residues, to an anti-FR antibody. All of these
anti-FR BIA ADCs were found to be highly potent in vitro on the
high expressing KB cell line whereas a wider range of activity was
observed for the lower expressing T47D cell line. Anti-FR ADC
31 demonstrated antigen-specific highly potent antitumor activity
at the lowest dose tested of 2.5 mg/kg. These data confirm that
potency and antitumor activity can be achieved through the
replacement of the N-10 amine of an IGN dimer with a biaryl DNA
binding moiety. We are continuing to explore these BIAs in an
effort to further expand the TI and utility of this class of molecules.
33
21
B
30
30
100
7
100
2000
1000
60
34
26
B
35
27
B
B
36
28
39
16
A
20
10
5
30
40
20
A
10
45
37
peptide
peptide
400
>3000
46
23
20
aNumber of antibody molecules bound per cell.
The anti-FR ADCs 31-36, 39-40 and 45-46 were evaluated in
vitro against two cell lines which expressed either a high (KB) or
low (T47D) number of folate receptors. As shown in Table 2, all
anti-FR ADCs were found to be highly potent towards the higher
expressing KB cells. There was a much wider range of activity
towards the lower expressing T47D cells, as a few anti-FR ADCs
(34, 35, 46) were considerably less active though there is no clear
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Supplementary Material
Supplementary data (synthetic details, experimental details, and
conjugation methods) associated with this artcle can be found, in
an online version, at .