Synthesis of a C2-aryl-pyrrolo[2,1-c][1,4]benzodiazepine monomer enabling the convergent construction of symmetrical and non-symmetrical dimeric analogs

Article abstract of DOI:10.1016/j.tetlet.2015.05.116

Abstract We disclose the synthesis of a versatile C2-aryl-pyrrolo[2,1-c][1,4]benzodiazepine (PBD) monomer that enables the late-stage diversification and synthesis of PBD dimers. The monomer unit was elaborated and dimerized utilizing different tethers to yield symmetric and non-symmetrical C2-aryl-PBD dimers. The dimers were tested for cytotoxic activity on a number of cancer cell lines and showed potent cytotoxicity in the nanomolar to picomolar range. The synthesis of PBD monomers enables the modular construction of PBD free drugs and drug-linkers that may have application for the development of antibody-drug conjugations.

Full text of DOI:10.1016/j.tetlet.2015.05.116

Accepted Manuscript
Synthesis of a C2-Aryl-Pyrrolo[2,1-c][1,4]benzodiazepines Monomer Enabling
the Convergent Construction of Symmetrical and Non-symmetrical Dimeric
Analogs
Robert V. Kolakowski, Thomas D. Young, Philip H. Howard, Scott C. Jeffrey,
Peter D. Senter
PII:
S0040-4039(15)00958-2
DOI:
http://dx.doi.org/10.1016/j.tetlet.2015.05.116
TETL 46388
Reference:
To appear in:
Tetrahedron Letters
Received Date:
Revised Date:
Accepted Date:
1 May 2015
26 May 2015
29 May 2015
Please cite this article as: Kolakowski, R.V., Young, T.D., Howard, P.H., Jeffrey, S.C., Senter, P.D., Synthesis of
a C2-Aryl-Pyrrolo[2,1-c][1,4]benzodiazepines Monomer Enabling the Convergent Construction of Symmetrical
and Non-symmetrical Dimeric Analogs, Tetrahedron Letters (2015), doi: http://dx.doi.org/10.1016/j.tetlet.
2
015.05.116
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Synthesis of a C2-Aryl-Pyrrolo[2,1-c][1,4]benzodiazepines Monomer Enabling the
Convergent Construction of Symmetrical and Non-symmetrical Dimeric Analogs.
1
*
1
2
1
Robert V. Kolakowski , Thomas D. Young , Philip H. Howard , Scott C. Jeffrey , Peter D.
1
Senter .
1
Seattle Genetics, Inc. 21823-30th Drive SE, Bothell, WA 98021.
2
ADC Products Ltd., The QMB Innovation Centre, 42 New Road, London, E1 2AX, United
Kingdom
Keywords. Pyrrolobenzodiazapines, Antibody-Drug Conjugates, Payloads, Drug-Linker, Anti-
cancer
Corresponding author: Robert V. Kolakowski
Postal address; Seattle Genetics, Inc. 21823-30th Drive SE, Bothell, WA 98021.
Phone; 1.425.527.4776
Fax; 1.425.527.4001
E-mail; rkolakowski@seagen.com
Abstract: We disclose the synthesis of a versatile C2-aryl-pyrrolo[2,1-c][1,4]benzodiazepine (PBD)
monomer that enables the late-stage diversification and synthesis of PBD dimers. The monomer unit was
elaborated and dimerized utilizing different tethers to yield symmetric and non-symmetrical C2-aryl-PBD
dimers. The dimers were tested for cytotoxic activity on a number of cancer cell lines and showed potent
cytotoxicity in the nanomolar to picomolar range. The synthesis of PBD monomers enables the modular
construction of PBD free drugs and drug-linkers that may have application for the development of
antibody-drug conjugations.

Introduction: Antibody-drug conjugates, (ADCs) have emerged as important therapeutics for treating
1-4
cancer as evidenced by the FDA approval of Adcetris™, Kadcyla™ and the over 35 ADCs currently in
5-7
clinical trials. A distinctive feature of this approach for cancer therapy is that antibody targeting has the
potential of improving drug activity through antigen mediated delivery, and at the same time, lowering
8
systemic toxicity of the anti-cancer payload. Recent studies have highlighted the impact the payload and
9-10
linker can have on ADC efficacy and tolerability.
The majority of the ADCs currently under evaluation deliver antimitotic agents, either auristatin or
maytansine analogs, as their cytotoxic payloads. While antimitotics hold considerable promise as ADC
payloads, there is a strong rationale for new drug classes with alternative and complementary mechanisms
to further extend ADC technology. Recent work with payloads such as amanitins (RNA-polymerase II
1
1-12
13-14
inhibitors),
topoisomerase II inhibitors)
investigation.
We have recently described a highly potent class of pyrrolobenzodiazepine (PBD) dimers for ADCs.
spliceostatins (spliceosome inhibitors),
and ultrapotent doxorubicin analogs
15-16
(
are a few examples of the many promising chemotypes under active
1
7-18
1
9-22
PBD dimers were designed from monomeric natural products
which include anthramycin 1,
sibiromycin 2 and tomaymycin 3 Figure 1. These natural products are DNA minor groove binders with
GC sequence specificity and potent cytotoxic activity in the nM IC range. They are known to alkylate
5
0
2
3-24
DNA moieties preferentially at guanine residues through their reactive imine functionalities .
Synthetic PBD dimers, such as SJG-136 (4), were shown to be even more potent than the PBD monomers
2
5-27
and to have GATC and the GAAT sequence specificities resulting in DNA cross-linking .
Dimeric
PBDs can have IC values in the pM range. Notably, the C2-aryl derivatives are among the most potent
50
28-32
members in the class.
To better evaluate the properties of this potent class of cytotoxic agents for ADCs, we set out to develop a
modular synthesis of the PBD dimers that would enable us to prepare both symmetric and non-symmetric
analogs. Past syntheses of PBD dimers have introduced a bridging linkage between the monomeric units

3
3-35
early in the synthesis, in order to avoid late-stage protecting group manipulation.
While this method
has utility, it is best suited for symmetrical PBD dimers. Approaches to monomeric PBDs, such as Chen
3
6
et. al., utilize a similar disconnection strategy, but lack the proper protection at the C8 position for late-
37
stage dimerization. Kamal et. al. demonstrated the feasibility of a late-stage dimerization strategy but
their analogs lacked C2 substitution, which was found empirically to be an important element for high
potency. Herein, we demonstrate a synthesis that leverages late-stage monomer diversification via a
Suzuki coupling reaction and subsequent monomer dimerization that can prepare both symmetric and
38-39
non-symmetric substituted analogs.
This synthetic strategy introduces diversity in the final steps of
PBD dimer synthesis, allowing the efficient evaluation of the structure activity relationship (SAR) at the
PBDs point of linker attachment and of the C2 aryl groups.
We began the synthesis of the key intermediate, PBD monomer 18, (Scheme 1) with chlorobenzyl
4
0
protection of methyl vanillate, 5, to give 6 in high yield. The use of claycop, (copper nitrate deposited
on K-10 clay), regioselectively nitrated 6 to provide 7 in a 96 % yield. The methyl ester of 7 was
saponified using an aqueous sodium hydroxide solution in THF to afford the acid 8, in good yield. After
an aqueous wash, 8 was taken on without further purification and treated with oxalyl chloride and
catalytic amount of DMF to convert 8 to the corresponding acid chloride in situ, further treatment of the
acid chloride with TBS protected pyrrolidine diol 9 yielded amide 10. After extensive optimization, then
nitro group of 10 could be reduced and the phenol deprotected under hydrogenating conditions with Pd/C
4
1
and H to provide aniline 11 in a 78 % yield. Intermediate 11 was subjected to a Ley oxidation to give
2
ketone 12 in a 90 % yield which was protected with TBDPSCl in DMF to provide 13. The aniline moiety
could then be capped with TROC-Cl to cleanly yield 14. Acidic removal of the TBS group with PTSA
gave the primary alcohol 15 in an 88% yield. With our primary alcohol 15 in hand, we sought to carry out
an oxidative cyclization of 15 to form the 7-membered diazepine ring system, compound 16. Ley
4
2
43
oxidation with TPAP, Dess-Martin oxidation, and PCC all gave different amounts of over-oxidized
lactam product, instead of the desired lactol, 16. However, the Swern oxidation effectively oxidized the

alcohol to the lactol with one equivalent of oxalyl chloride at – 78 °C. Both acetylation of 16 and enol
triflation of 17 proceeded smoothly to yield our key point of diversity, intermediate 18. The Suzuki
44
coupling of 18 with p-methoxyphenyl boronic acid gave 19 in good yield. Finally, TBAF was employed
to remove the phenolic TBDPS group from 19 to yield the desired monomeric intermediate 20, ready for
dimerization.
To validate this approach, we sought to prepare and evaluate the biological activity of several analogs
4
5
including the well-studied C2-para-methoxy-phenyl dimer SG2202, compound 24 (Scheme 2). To this
end, monomer 20 was alkylated with 0.5 equivalents of 1,3-dibromopropane (21a) using K CO in DMF
2
3
4
6
to yield the 22, in a 86 % yield. Global TROC deprotection with the Pb-Cd couple yielded SG2202 (24)
in a good yield. The synthesis of a symmetric PBD dimer 25, with an aniline group in the para-C2 aryl
position, utilized the para-methoxy monomer 20 which was dimerized with 1,3-bis(bromomethyl)-5-
nitrobenzene (21b) to yield 23. Both the TROC groups and the nitro group could be reduced in a one-pot
procedure utilizing Pb-Cd to yield 25 after silica gel purification.
To demonstrate the feasibility of synthesizing non-symmetrical PBD dimers with this approach, we
converted enol triflate 18 to the PBD monomer 28 (Scheme 3). The para-aniline monomer 26 was
synthesized from the Suzuki coupling of 18 with 4-aminophenylboronic acid pinacol ester. The para-
aniline group of 26 was protected with TROC-Cl and the protected phenol was TBDPS deprotected with
TBAF to yield 28 in a 87 % yield for the two steps. In parallel, the para-methoxy monomer 20 was
alkylated with an excess of 1,3-dibromopropane (21a) to yield the alkyl bromide 27 in a 82 % yield.
Alkylation of 27 with 28 in Cs CO and DMF yielded 80 % of the protected non-symmetric dimer 29.
2
3
Global TROC deprotection with the Pb-Cd couple gave 30 in a 58 % yield.
Compounds, 24, 25, and 30 were tested in an in vitro cellular potency assay (Table 1) on a panel of cancer
4
5
47
cell lines. 24 and 30 gave IC values comparable to literature values, in the nM to pM range. The
50
inclusion of the bridging aniline moiety in the symmetric dimer, 25, did not appear to perturb the

biological potency. Compound 25 was approximately 10-fold more potent than compound 24 and the
increased potency of 25 is consistent with the finding that PBD dimers possessing a 5-carbon connecting
4
8
units are generally more potent than the 3-carbon spaced analogs.
Conclusions: With the synthesis of the versatile monomeric analog 18, we were able to efficiently
synthesize both symmetric and non-symmetrical PBD dimers and the analogs synthesized were highly
potent on cancer cell lines. The method disclosed here enables the evaluation of a variety of PBD dimers
as payloads for ADCs.
ACKNOWLEDGMENTS: We would like to thank Arnaud Tiberghien and Luke Masterson for previous
contributions to the synthesis of PBDs and useful discussions throughout the course of this work.

Abstract Figure
Figure 1. Related pyrrolo[1.4]benzodiazepine natural products and dimers.

Scheme 1:
(
a) p-ClBnBr (1.1 equiv.), K CO , tol., rt, 95 %, (b) Cu(NO ) on K-10 clay 1:1 v/v, DCM, Ac O, 0 °C,
2 3 3 2 2
9
6 %, (c) NaOH, THF:MeOH 1:1, rt, 89%, (d) (COCl) (1.5 equiv.), cat. DMF, DCM, 0 °C, then, rt, 9
2
(
1.0 equiv.) 91 %, (e) 10 % Pd/C, (0.05 equiv.), H , MeOH, DMF, 3 h, rt, 78 %, (f) TPAP (0.05 equiv.),
2
NMO (3.0 equiv.), DCM, rt, 90 %, (g) TBDPSCl (1.2 equiv.), imidazole (equiv 1.2), DMAP (equiv. 0.1),
DMF, rt, 86 %, (h) TROC-Cl (equiv. 1.1), pyr. (5.0 equiv.), DCM, rt, 98 %, (i) PTSA (1.3 equiv), MeOH,
5
0 °C, 88 %, (j) (1.0 equiv.), (COCl) , DCM, -78 °C, then (5 equiv.), Hünig’s Base, -78 °C to rt, 1 h, 72
2
%
, (k) Ac O, pyr. (5.0 equiv.), rt, 84 %, (l) Tf O (3.0 equiv.), 2,6-lutidine (5.0 equiv.), -45 °C, 94 %, (m)
2 2
p-MeO(C H )B(OH) , Pd(P(Ph) ) , (0.01 equiv.), rt, K CO , H O:EtOH:tol (10:1:2), 93 %, (n) TBAF
6
4
2
3 4
2
3
2
(
1.2 equiv.), THF, rt, 91 %.

Scheme 2: (a ) 20 (1.0,equiv.), K CO , DMF, 21 a (0.5 equiv.), rt, 86 % of 22, (a ) 20 (1.0,equiv.),
1
2
3
2
K CO , DMF, 21 b (0.5 equiv.), rt, 74 % of 23, (b ) 22, Pb-Cd 1:10 v/v (5 equiv.), 24, THF:NH OAc (1
2
3
1
4
M, aq.) 1:1, rt, 68 %, (b ) 23, Pb-Cd 1:10 v/v (3 equiv.),THF:NH OAc (1 M, aq.) 1:1, rt, 25, 48 %.
2
4

Scheme 3: (a) NH (C H )B-pinacol ester, Pd(P(Ph) ) , (0.01 equiv.), K CO , H O:EtOH:tol (10:1:2), rt,
2
6
4
3 4
2
3
2
8
4 %, (b) TROC-Cl (equiv. 1.1), DCM, pyr., rt, 97 %, (c) TBAF, THF, 93 %, (d) 27 (1.3 equiv.), Cs CO3,
2
DMF, yields 29 in 80% (e) 29 and Pb-Cd (3 equiv.), THF:NH OAc (1 M, aq.) 1:1, yields 30 in 58 %.
4
compounds
786-0
Caki-1
HEL92.1.7
HL60cy
MCF-7
TF-1a
2
4
0.25
0.11
0.032
0.017
1
1
2
3
5
0
0.030
0.4
0.038
0.2
0.0074
0.024
0.0093
0.013
0.3
1
0.2
1
Table 1: Free drug in vitro activity against cancer cell lines. The cells were treated with the test
articles for 48 h in duplicate. IC values were determined by Cell Titer Glo and are reported in
5
0
nM concentrations.

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