Design and synthesis of a folate-receptor targeted diazepine-ring-opened pyrrolobenzodiazepine prodrug conjugate

Article abstract of DOI:10.1016/j.bmcl.2020.126987

Pyrrolobenzodiazepines (PBDs) and their dimers (bis-PBDs) have emerged as some of the most potent chemotherapeutic compounds and are currently under development as novel payloads in antibody-drug conjugates (ADCs). However, when used as stand-alone therapeutics or as warheads for small molecule drug conjugates (SMDCs), dose-limiting toxicities are often observed. As an elegant solution to this inherent problem, we designed and synthesized a diazepine-ring-opened bis-PBD prodrug (pro-PBD-PBD) folate conjugate lacking the one of the two imine moieties found in the corresponding free bis-PBD. Upon entering a targeted cell, cleavage of the linker system, including the hydrolysis of an oxazolidine moiety, results in the formation of a reactive intermediate which possesses a newly formed aldehyde as well as an aromatic amine. A fast and spontaneous intramolecular ring-closing reaction subsequently takes place as the aromatic amine adds to the aldehyde with the loss of water to give the imine, and as a result, the diazepine ring, thereby delivering the bis-PBD to the targeted cell. The in vitro and in vivo activity of this conjugate has been evaluated on folate receptor positive KB cells. Sub-nanomolar activity with good specificity and high cure rates with minimal toxicity have been observed.

Full text of DOI:10.1016/j.bmcl.2020.126987

Journal Pre-proofs
Design and synthesis of a folate-receptor targeted diazepine-ring-opened pyr-
rolobenzodiazepine prodrug conjugate
Iontcho R. Vlahov, Longwu Qi, Hari Krishna R. Santhapuram, Albert Felten,
Garth L. Parham, Ning Zou, Kevin Wang, Fei You, Jeremy F. Vaughn, Spencer
J. Hahn, Hanna F. Klein, Paul J. Kleindl, Joe Reddy, Dan Reno, Jeff Nicoson,
Christopher P. Leamon
PII:
S0960-894X(20)30047-0
DOI:
https://doi.org/10.1016/j.bmcl.2020.126987
BMCL 126987
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
18 November 2019
13 January 2020
23 January 2020
Please cite this article as: Vlahov, I.R., Qi, L., Krishna R. Santhapuram, H., Felten, A., Parham, G.L., Zou, N., Wang,
K., You, F., Vaughn, J.F., Hahn, S.J., Klein, H.F., Kleindl, P.J., Reddy, J., Reno, D., Nicoson, J., Leamon, C.P.,
Design and synthesis of a folate-receptor targeted diazepine-ring-opened pyrrolobenzodiazepine prodrug conjugate,
Bioorganic & Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl.2020.126987
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Design and synthesis of a folate-receptor targeted diazepine-ring-opened pyrrolobenzodiazepine prodrug conjugate
Iontcho R. Vlahov*, Longwu Qi, Hari Krishna R. Santhapuram, Albert Felten, Garth L. Parham, Ning Zou, Kevin Wang, Fei
You, Jeremy F. Vaughn, Spencer J. Hahn, Hanna F. Klein, Paul J. Kleindl, Joe Reddy, Dan Reno, Jeff Nicoson, and
Christopher P. Leamon
Endocyte Inc., 3000 Kent Ave., W. Lafayette, IN 47906, USA
Abstract: Pyrrolobenzodiazepines (PBDs) and their dimers (bis-PBDs) have emerged as some of the most potent
chemotherapeutic compounds and are currently under development as novel payloads in antibody-drug conjugates
(ADCs). However, when used as stand-alone therapeutics or as warheads for small molecule drug conjugates (SMDCs),
dose-limiting toxicities are often observed. As an elegant solution to this inherent problem, we designed and synthesized
a diazepine-ring-opened bis-PBD prodrug (pro-PBD-PBD) folate conjugate lacking the one of the two imine moieties found
in the corresponding free bis-PBD. Upon entering a targeted cell, cleavage of the linker system, including the hydrolysis
of an oxazolidine moiety, results in the formation of a reactive intermediate which possesses a newly formed aldehyde as
well as an aromatic amine. A fast and spontaneous intramolecular ring-closing reaction subsequently takes place as the
aromatic amine adds to the aldehyde with the loss of water to give the imine, and as a result, the diazepine ring, thereby
delivering the bis-PBD to the targeted cell. The in vitro and in vivo activity of this conjugate has been evaluated on folate
receptor positive KB cells. Sub-nanomolar activity with good specificity and high cure rates with minimal toxicity have
been observed.
Keywords: pyrrolobenzodiazepine, prodrug, folate-receptor, conjugate, targeted therapy
Pyrrolo[2,1-c][1,4]benzodiazepine (PBD) antibiotics are a class of natural products produced by various actinomycetes
1
bacteria and are known for their antibiotic and antitumor properties. PBDs and their synthetic C-8/C-8’-tethered dimers
(Figure 1) are sequence selective DNA-crosslinking alkylating agents. All PBD-based derivatives bind in a specific manner
to DNA after recognition of a three-base-pair sequence in the minor grove. Insertion of the PBD dimer into the minor
2
groove results in two interstrand cross-linking covalent aminal bonds, formed as the result of the nucleophilic attacks of
both exocyclic N-2/N-2’ amines presented by two guanine (G) bases, integrated in the opposing DNA-strands, on the
electrophilic C-11/C-11’ imines of the PBD dimer (Figure 1B). Two additional hydrogen bonds are formed between the
ring nitrogen N-3 of the 3’-adenines adjacent to the aminal-modified guanine and both N-10, N’-10 protons of the PBD
units. The perfect fit of a PBD-dimer in the DNA minor grove results in negligible distortion of the DNA helix, thus
potentially avoiding DNA-repair mechanisms and the common phenomenon of drug resistance.3
Since the discovery of natural PBDs, various synthetic PBD-monomers, PBD-hybrids, and PBD-dimers have been designed,
4
prepared, and explored, improving our understanding of the mode of action of this important class of compounds. A
4
b
synthetic PBD-dimer, SJG-136 (Figure 1C), has undergone clinical evaluation as a monotherapy. In recent years, synthetic
PBD-dimers have emerged as a new class of warheads in the field of antibody-drug conjugates (ADCs) and small molecule
drug conjugates (SMDCs).1e, 4d, 5
The folate receptor (FR) is a glycosylphosphatidylinositol anchored cell-surface glycoprotein which binds with high affinity
6
to the vitamin folic acid (FA). Upon binding, FA is transported into the cell via FR mediated endocytosis. Several rapidly
growing cancers as well as activated macrophages associated with inflammation have been shown to overexpress the FR.7
Targeting these nefarious cells can be achieved by covalently attaching FA to appropriate biologically active agents.7b, 8
Several years ago, we synthesized a number of SMDCs linking FA through self-immolative linker systems to PBD dimers -
conjugated primarily off one of the C-rings of the dimer (unpublished results). As constructed, these conjugates left PBD
dimers with both of their reactive imines exposed at the end of peptidic chains. Unfortunately, although highly potent,
these conjugates also displayed high (in some cases delayed) toxicities. Modification of the linker or spacer systems in
this series of conjugates did little to improve the very tight therapeutic windows.

A.
1
0
9
1
1
N
8
7
A
11a
1
N C
2
6
O
3
PBD imine form
B.
3
'-Y
C
T
A
H
G
X
Y
HN
H
N
O
N
OMe
O
O
O
MeO
N
N
H
H
NH
5'-X
G
A
T
C
PBD dimer forming non-distorting interstrand
cross-link in the DNA minor groove.
C.
N
O
O
O
O
N
N
N
O
O
SJG-136
tethered PBD dimer)
(
Figure 1. A) General structure of PBD; B) Representation of the insertion of a PBD dimer into the minor groove of DNA; C)
Structure of PBD-dimer SJG-136.
We turned our attention to modifying the PBD dimer within the FA conjugate construct with the hope of reducing the
observed off-target toxicities. Specifically, we believed that by protecting or masking the highly reactive N-10/C-11 imine
of PBD, we could deliver this class of compounds to their targets much more efficaciously. This idea had been explored
earlier. Thurston and Howard synthesized PBD prodrugs (pro-PBDs) and conjugates with cleavable linkers/side-chains at
the N-10 position of PBD.1e, 9 By doing so, they effectively blocked the imine moiety to prevent DNA alkylation or
crosslinking. “Protection” in this manner significantly reduced the toxicities observed in a number of PBD prodrugs. Our
design would find particular inspiration in the work of Jeffrey et al. who have shown that the heterocyclic iminium moiety
in several anthracycline conjugates can be masked by replacing this moiety with its synthetic precursors: namely, an 1,3-
oxazolidine carbamate and an amine.10 The oxazolidine carbamate provided an attachment point for the anthracycline
pro-drug to an antibody through one of several enzymatically labile linkers. A similar strategy could be applied to PDBs
(Scheme 1). Upon entering a targeted cell, endosomal reductive cleavage of a disulfide bond and fragmentation of the
linker system on a pro-PBD folate conjugate triggers the generation of a 1,3-oxazolidine. The latter, being hydrolytically
labile, upon spontaneous reaction with water, forms a reactive aldehyde-amine intermediate. Subsequently, the aromatic
amine in close proximity to the newly generated aldehyde, allows facile formation of the seven-membered carbinolamine
ring. Spontaneous condensation results in the expected imine, thus generating the diazepine ring and providing in situ
the PBD molecule. By applying this strategy, we were able to synthesize folate conjugates of a pro-PDB, a pro-PBD-pro-

PBD (both imine moieties in bis-PBD masked as 1,3-oxazolidine carbamates), a pro-PBD-diazepine-lactam hybrid, and a
pro-PBD/Hoechst dye 33258 hybrid.11
Scheme 1. The concept of pro-PBDs as latent warheads for targeting cancer cells.
Herein, we report the structure, synthesis, and preliminary biological activity of folate-targeted, reductively-labile, pro-
PBD-PBD conjugate 1 (Figure 2). Conjugate 1 delivers a highly active bis-PBD without the added complexity and instability
of our earlier pro-PBD-pro-PBD conjugate which required two linker cleavages and subsequent releases to liberate bis-
PBD.11 The linker/spacer in 1 was optimized. Of note is the addition of a geminal-dimethyl functionality alpha to the
disulfide bond on the linker. This structural feature is designed to stabilize the disulfide bond during circulation by
providing steric hindrance. Once reduced in the endosome, however, the geminal-dimethyl increases the rate of linker
immolation, as described by the Thorpe-Ingold effect.12
HN
NH2
NH
CO2H
CO2H
O
O
O
H
H
H
N
N
N
CO2H
S
O
N
N
N
N
O
H
H
O
H2N
N
O
O
N
N
O
O
H
H
H
O
O
N
O
S
HN
H2N
N
H
N
N
O
N
H
CO2H
CO H
S
O
2
O
N
3
H
N
O
O
O
O
H
O
N
O
O
Folate
Spacer
Linker
pro-PBD-PBD
1
Figure 2. Folate-targeted, reductively-labile pro-PBD-PBD conjugate 1.
Synthesis of 1 began by forming the linker and adjoining PBD C-ring (Scheme 2). 3-thiopropionic acid was treated with
,2’-dithiopyridine to yield the resulting activated disulfanylcarboxylic acid. The acid was then treated with N-
fluorenylmethyloxycarbonylethylenediamine (Fmoc-EDA), benzytriazol-1-oxytripyrrolidinophosphonium
2
hexafluorophosphate (PyBOP), and diisopropylethylamine (DIPEA) to give adduct 3. Treatment of 3 with 2-mercapto-2-
methyl-propanol followed by N,N’-disuccinimidyl carbonate (DSC) and pyridine resulted in activated carbonate 4. (S)-N-
tert-butoxy(Boc)-4-methylene-prolinal (5)11 was treated with ethanolamine in the presence of 3Å molecular sieves
resulting in the formation of the 1,3-oxazolidine which was captured/trapped upon addition of 4 with triethylamine (TEA)
to give 6 as a mixture of enantiomers.10a, 13 The other C-ring in the pro-PBD-PBD was similarly constructed (Scheme 2).
Prolinal 5 was again treated with ethanolamine and magnesium sulfate, resulting in the formation of the 1,3-oxazolidine
which was reacted further with Fmoc-Cl, resulting in adduct 7.10a, 13 Treatment of 7 with trifluoroacetic acid (TFA) followed
1
1
by PyBOP promoted amide bond formation to the tethered bis-A-ring core (9), resulted in the formation of 8. After
removal of the Boc protecting group on 6 with TFA, 8 was coupled with the free amine of 6 to yield adduct 10 as a
diastereomeric mixture (Scheme 3). Treatment of 10 with diethylamine (DEA) resulted in the cleavage of both Fmoc
protecting groups. The loss of the Fmoc moiety on the oxazolidine ring destabilizes the hemiaminal, ultimately resulting
in the formation of the diazapene ring. Further treatment of the remaining free amine with maleimido-Peg -N-
4
hydroxysuccimic acid ester (Mal-Peg -NHS) in the presence of TEA yields the maleimido adduct 11. Michael addition of
4
1
4
folate spacer 12 to compound 11 in a solution of N,N-dimethylformamide (DMF) and ammonium bicarbonate (pH = 7)
buffer (aq.) yields conjugate 1 as a mixture of diastereomers.

H
a, b
HO
SH
N
S
FmocHN
S
N
O
O
2
3
c,d
O
H
N
S
S
O
O
FmocHN
N
O
O
S
O
4
OHC
H
BocN
e,f
NBoc
O
O
H
FmocHN
N
H
S
O
N
O
5
6
Racemate
g,h
H2N
O
O
NH2
O
NFmoc
H
O
i, j
HO2C
OMe
MeO
BocN
Fmoc
H
N
N
O
7
8
Racemate
H2N
HO2C
O
O
NH2
OMe
MeO
CO2H
9
Scheme 2. Synthesis of Intermediate 8. a) 2,2’-dipyridyldithiol (2 eq.), MeOH, 15 min., 68% yield; b) Fmoc-EDA (1 eq.),
PyBOP (1 eq.), DIPEA (3 eq.), DMF, 5 min., 84% yield; c) 2-mercapto-2-methyl-propanol (1.3 eq.), CHCl , MeOH, 4 h., 60 °C,
3
9
0% yield; d) DSC (1.2 eq.), pyridine (1.25 eq.), ACN, 15 h., 95% yield; e) ethanolamine (0.9 eq.), 3Å molecular sieves,
CH Cl , 4 h.; f) 4 (0.75 eq.), TEA (1.1 eq.), CH Cl , 2 h., 70% yield over two steps; g) ethanolamine (1.2 eq.), MgSO (4.6 eq.),
2
2
2
2
4
CH Cl , 1 h.; h) Fmoc-Cl (2 eq.), TEA (3 eq.), CH Cl , 16 h., 70% yield over two steps; i) TFA, CH Cl (1:1), 30 min.; j) 9 (1.8
2
2
2
2
2
2
eq.), PyBOP (1.06 eq.), DIPEA (8 eq.), DMF/CH Cl (1:1), 1 h., 60% yield over two steps.
2
2

H2N
O
O
NH2
O
N
O
O
a,b
OMe
MeO
6
H
Fmoc
H
FmocHN
S
O
N
N
S
O
N
N
O
H
O
10
c, d
H2N
O
O
N
H
N
O
O
O
O
OMe
MeO
N
H
H
O
N
S
O
O
N
N
N
S
O
N
H
4
H
O
O
O
11
HN
NH2
NH
CO2H
CO2H
O
O
e
O
H
H
N
H
N
N
CO2H
SH
N
N
O
N
N
H
H
N
N
O
O
H
O
HN
N
CO2H
2
CO H
H
H2N
12
1
Scheme 3. Synthesis of conjugate 1. a) 30% TFA in CH Cl , 0 °C to rt, 1 h.; b) 8 (1 eq.), PyBOP (2 eq.), DIPEA (3 eq.), DMF,
2
2
3
h., 85% yield over two steps; c) DEA, CH Cl (1 : 2.5), 3h.; d) Mal-Peg -NHS (1.3 eq.), TEA (1.3 eq.), CH Cl , 1 h.; e) 12 (1.3
2
2
4
2
2
eq.), 50 mM NH HCO (pH = 7) buffer (aq.), DMF, 2 h., 24% yield over 3 steps.
4
3
A model chemical release study was performed to verify compound 1 releases PBD dimer under reductive conditions
found in internalized endosomes. 1 was treated with tris(2-carboxyethyl)phosphine (TCEP) in a pH = 6 phosphate buffer
at 37 °C and the reaction was monitored by UPLC-MS (Scheme 4). Within five minutes of treatment with TCEP, 1 was
completely reduced to the thiol terminating folate spacer 13 and pro-PBD-PBD linker remnant 14 as a mixture of
diastereomers/rotamers. Further self-immolative release of the linker/1,3-oxazolidine carbonate from 14 over the course
of 25 minutes resulted in the complete conversion to PBD dimer 15.
Scheme 4. A) Chemical release of PBD dimer 15 from conjugate 1 upon treatment with TCEP; B) Chemical release as
shown by UPLC traces (254 nm) at t = 0, 5, 15, and 30 min. (from bottom to top) post TCEP treatment. At t = 0, 1 is shown
as a mixture of diastereomers. At t = 5 min., TCEP has completely reduced 1. Thiol terminating folate spacer 13 and pro-
PBD-PBD linker remnant 14 as a mixture of diastereomers/rotamers are readily apparent. A small amount of PBD dimer
1
1
5 is also visible at 2.37 min retention time. By t = 30 min., linker immolation is complete. 14 is no longer visible. Spacer
3 and free PBD dimer 15 are the two main remaining components of the reaction mixture.
Conjugate 1 has performed well in pre-clinical biological testing when compared against our previously reported folate-
PBD conjugates (Figure 3).11 For example, 1, 16, 17, and 18 were evaluated in vitro on folate receptor positive KB (FR(+)-
KB) cells with and without added folate competitor (Table 1 and Supplementary Data Figure 4). A sub-100 pM IC was
5
0
obtained for 1 as a stand-alone agent, representing a 12-20 times improvement in activity compared to conjugates 16-18.
In the presence of 100-fold excess folic acid, the IC s for all conjugates increased roughly two orders of magnitude,
5
0
thereby demonstrating the folate receptor targeted activity of these agents. The reduction in the IC observed for 1
5
0
under competitive FA binding conditions was inferior to that observed with compounds 16-18. The activities of 1, 17, and

1
8 against FR(+)-KB xenografts in female nude mice were also evaluated (Figure 4). Curative responses in 100% of the
cohort dosed with 300 nmol/kg of 1 on a single dose a week schedule was observed. Very little weight loss (up to 96 days
post tumor implantation) or major organ tissue degeneration was observed. Conjugates 17 and 18 were dosed at 2
mol/kg on a single dose a week schedule. Even with the increased dose, 17 and 18 only slowed the tumor growth. Some
weight loss was observed with 18.
HN
NH2
NH
CO2H
H
N
O
CO2H
O
O
H
H
H
O
N
N
CO2H
O
O
N
O
N
N
N
N
H N
2
O
O
H
H
H
N
N
O
O
O
S
S
O
N
CO2H
N
N
HN
H2N
N
CO2H
O
H
O
H
O
O
1
6, (pro-PBD-diazepine-lactam hybrid)
CO2H
CO H
H2N
N
O
O
O
O
NH2
2
O
CO2H
O
N
O
O
CO H
O
H
H
N
H
N
2
O
H
N
H
O
N
N
N
H
S
S
O
S
N
H
N
H
O
O
N
N
O
S
H
O
N
N
O
O
O
O
HN
H2N
N
H
N
O
NH
O
O
NH
NH
OH
OH
OH
OH
17, (pro-PBD-pro-PBD)
OH
OH
HO
HO
HO
HO
HO
HO
OH
HO
HO
N
CO2H
CO H
2
N
N
O
CO2H
O
O
O
CO2H
H
H
N
H
N
O
N
N
S
O
N
NH2
O
N
H
O
N
N
H
N
H
S
N
H
H
H
N
N
O
O
O
O
N
H
HN
H2N
N
H
O
O
O
O
N
N
NH
NH
O
NH
OMe
OH
OH
OH
OH
OH
OH
1
8, (pro-PBD/Hoechst dye 33258 hybrid)
HO
HO
HO
HO
HO
HO
OH
HO
HO
Figure 3. Structures of previously reported folate pro-PBD conjugates.
Table 1. Activity of pro-PBD-based folate conjugates on FR(+)-KB cells, 2 h pulse at 37 °C.
Compound IC (nM)
IC with 100 M FA (nM)
50
5
0
1
0.0522
1.12
1.31
3.78
696
184
84.3
1
6
7
8
1
1
0.658
With the synthesis of 1, we have demonstrated another application of the 1,3-oxazolidine carbonate-iminium masking
strategy toward the synthesis of a targeted prodrug form of a bis-PBD. The pre-clinical pharmacological studies which
have been presented here, herald the potential of our approach. Ultimately, the totality of the pre-clinical data led us to
select 1 for clinical development as the first-in-class pro-PBD-based SMDC. Additional information regarding the
optimization of the linker and spacer as well as additional details relating to the activity of 1 in multiple cell lines and on
varying dosing schedules will be reported in due course.

A
1
1
600
200
8
4
00
00
0
6
16 26 36 46 56 66 76 86 96
PTI (Days)
B
3
2
1
5
5
5
5
5
-
-
-
15
25
6
16 26 36 46 56 66 76 86 96
PTI (Days)
Figure 4. Antitumor (A) and weight change (B) effects of 1, 18, and 17 on FR expressing KB tumor nu/nu mice model. KB
6
tumor cells were inoculated subcutaneously into nu/nu mice (1 x 10 cells) and therapy started on randomized animals
3
with tumors in the 100–200 mm (n = 5) range. , untreated controls; , 1, 0.3 mol/kg; , 18, 2 mol/kg; , 17, 2
mol/kg; SIW x 2 weeks. Each curve shows the average volume of 3-5 tumors/animals.
References and Notes.
1
.
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9
4 (2), 433-65; (b) Thurston, D. E. Advances in the study of pyrrolo[2,1-c][1,4]benzodiazepine (PBD) antitumor antibiotics.
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'
Refuin': A non-cytotoxic carcinostatic compound proliferated by a thermophilic actinomycete. Nature 1963, 199, 501; (e)
Mantaj, J.; Jackson, P. J. M.; Rahman, K. M.; Thurston, D. E., From anthramycin to pyrrolobenzodiazepine (PDB) - containing
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2
(
.
Mantaj, J.; Jackson, P. J. M.; Karu, K.; Rahman, K. M.; Thurston, D. E., Covalent bonding of pyrrolobenzodiazepines
PBDs) to terminal guanine residues within duplex and hairpin DNA fragments. PLoS ONE 2016, 11 (4), e0152303/1-
e0152303/26.
(a) Rahman, K. M.; Corcoran, D. B.; Bui, T. T. T.; Jackson, P. J. M.; Thurston, D. E., Pyrrolobenzodiazepines (PBDs)
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3
.

Grzeskowiak, K.; Cascio, D.; Dickerson, R. E., Crystal structure of a covalent DNA-drug adduct: anthramycin bound to C-C-
A-A-C-G-T-T-G-G and a molecular explanation of specificity. Biochemistry 1994, 33 (46), 13593-610.
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.
(a) Hartley, J. A.; Hochhauser, D., Small molecule drugs - optimizing DNA damaging agent-based therapeutics. Curr.
Opin. Pharmacol. 2012, 12 (4), 398-402; (b) Hartley, J. A., The development of pyrrolobenzodiazepines as antitumour
agents. Expert Opin. Invest. Drugs 2011, 20 (6), 733-744; (c) Antonow, D.; Thurston, D. E., Synthesis of DNA-Interactive
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Santhapuram, H.; Felten, A.; Vaughn, J.; Leamon, C. Design, synthesis and early evaluation of hybrids of DNA minor groove
binders and DNA-alkylating agents as warheads for small molecule drug conjugates (SMDCs) for targeted cancer therapies.
2
016, From Abstracts of Papers, 252nd ACS National Meeting & Exposition, Philadelphia, PA, United States, Philadelphia,
PA, United States, August 21-26, MEDI-39.
(a) Saunders, L. R.; Bankovich, A. J.; Anderson, W. C.; Aujay, M. A.; Bheddah, S.; Black, K.; Desai, R.; Escarpe, P. A.;
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Declaration of interests
☒
The authors declare that they have no known competing financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
☐
The authors declare the following financial interests/personal relationships which may be considered as potential
competing interests: