Analogues of DNA minor groove cross-linking agents incorporating aminoCBI, an amino derivative of the duocarmycins: Synthesis, cytotoxicity, and potential as payloads for antibody–drug conjugates

Article abstract of DOI:10.1016/j.bmc.2016.09.068

A Pd-catalysed amination method is used to convert seco-CBI, a synthetic analogue of the alkylating subunit of the duocarmycin natural products, from the phenol to amino form. This allows efficient enantioselective access to the more potent S enantiomer of aminoCBI and its incorporation into analogues of DNA minor groove cross-linking agents. Evaluation in a panel of nine human tumour cell lines shows that the bifunctional agents containing aminoCBI are generally less cytotoxic than their phenolCBI analogues and more susceptible to P-glycoprotein-mediated resistance. However, all bifunctional agents are potent cytotoxins, some in the sub-pM IC₅₀range, with in vitro properties that compare favourably with established microtubule-targeted ADC payloads.

Full text of DOI:10.1016/j.bmc.2016.09.068

Accepted Manuscript
Analogues of DNA minor groove cross-linking agents incorporating aminoCBI,
an amino derivative of the duocarmycins: synthesis, cytotoxicity, and potential
as payloads for antibody-drug conjugates
Anna C. Giddens, Ho H. Lee, Guo-Liang Lu, Christian K. Miller, Jun Guo, Gail
D. Lewis Phillips, Thomas Pillow, Moana Tercel
PII:
S0968-0896(16)30840-9
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.09.068
BMC 13321
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
21 August 2016
26 September 2016
28 September 2016
Please cite this article as: Giddens, A.C., Lee, H.H., Lu, G-L., Miller, C.K., Guo, J., Lewis Phillips, G.D., Pillow,
T., Tercel, M., Analogues of DNA minor groove cross-linking agents incorporating aminoCBI, an amino derivative
of the duocarmycins: synthesis, cytotoxicity, and potential as payloads for antibody-drug conjugates, Bioorganic &
Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.09.068
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Analogues of DNA minor groove cross-linking agents incorporating aminoCBI, an amino
derivative of the duocarmycins: synthesis, cytotoxicity, and potential as payloads for antibody-
drug conjugates
a
a
a
a
b
Anna C. Giddens , Ho H. Lee , Guo-Liang Lu , Christian K. Miller , Jun Guo , Gail D. Lewis
Phillips b, Thomas Pillow b, Moana Tercel a,*
aAuckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, The University
of Auckland, Private Bag 92019, Auckland 1142, New Zealand
bGenentech Inc., 1 DNA Way, South San Francisco, California 94080, USA
*
Corresponding author. Tel: +64 9 373 7599; fax: +64 9 373 7502. E-mail address:
m.tercel@auckland.ac.nz (M. Tercel).
Abbreviations: ADC, antibody-drug conjugate; BINAP, 2,2’-bis(diphenylphosphino)-1,1’-
binaphthalene; CBI, 1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one; EDCI, N-ethyl-N’-(3-
dimethylaminopropyl)carbodiimide hydrochloride; MMAE, monomethyl auristatin E; PBD,
pyrrolobenzodiazepine; Pgp, P-glycoprotein.
Keywords: DNA cross-linking agent; AminoCBI; Duocarmycin; Pyrrolobenzodiazepine; Antibody-
drug conjugate
Abstract
A Pd-catalysed amination method is used to convert seco-CBI, a synthetic analogue of the alkylating
subunit of the duocarmycin natural products, from the phenol to amino form. This allows efficient
enantioselective access to the more potent S enantiomer of aminoCBI and its incorporation into
analogues of DNA minor groove cross-linking agents. Evaluation in a panel of nine human tumour
cell lines shows that the bifunctional agents containing aminoCBI are generally less cytotoxic than
their phenolCBI analogues and more susceptible to P-glycoprotein-mediated resistance. However, all
bifunctional agents are potent cytotoxins, some in the sub-pM IC50 range, with in vitro properties that
compare favourably with established microtubule-targeted ADC payloads.
1. Introduction
Antibody-drug conjugates (ADCs) are an emerging class of antitumour agents in which cytotoxic
small molecules are covalently linked to an antibody that recognises a tumour-associated antigen. 1-3
The recent approvals of AdcetrisÒ (brentuximab vedotin) and KadcylaÒ (ado-trastuzumab
emtansine) have stimulated research in this area, and there are now about 50 ADCs in clinical
development.3 At least 35 of these candidates employ microtubule-targeted compounds as the
cytotoxic species, typically from the auristatin or maytansine classes (see for example monomethyl
auristatin E (MMAE) 1 and the maytansine DM1 2, Figure 1).
More recently there has been interest in exploring payloads with a different mode of action. DNA
minor groove alkylating agents are of particular interest because they tend to be very cytotoxic, active
at all phases of the cell cycle, and insensitive to drug resistance mechanisms. Pyrrolobenzodiazepines
(PBDs, see substructure in Figure 1) are derived from natural products that alkylate in the minor
groove at the C2-NH2 group of guanine. As simple monomeric agents they are not generally
considered cytotoxic enough to be effective ADC payloads, but linking two PBDs together creates
1

O
O
Me
N
SH
O
OH
Me
N
H
H
N
Cl
O
O
N
N
MeO
MeHN
N
Me
O
OMe O
OMe O
O
HO
1
monomethyl auristatin E (MMAE)
N
O
H
2
DM1
OMe
alkylates N3
Cl
Cl
Cl
of adenine
alkylates C2-NH2 of guanine
N
N
N
N
O
H
O
O
O
N
MeO
R
R
OH
CBI
O
5
6
R = OH
R = NH2
3
4
R = OH
R = NH2
PBD
Figure 1.
Structures of some known and proposed ADC payloads. For simplicity CBI is here applied to the
seco
or open-chain form of the alkylating subunit, and the term phenolCBI used to distinguish R = OH from
aminoCBI (R = NH2).
cross-linking agents which can exhibit IC50s in the sub-nM range.4 ADCs with PBD dimer payloads
have shown promising preclinical activity5,6 and six such conjugates recently entered clinical trial.
Duocarmycins are another class of natural products that alkylate DNA in the minor groove, targeting
the N3 position of adenine. Even monoalkylating agents of this type e.g. those employing the
synthetic CBI alkylating subunit7 (substructure in Figure 1) can be highly cytotoxic compounds, and
these have also shown promise as ADC payloads.8,9
As for PBDs, it has been known for some time that linking duocarmycin analogues together to
make dimers can further enhance their already marked cytotoxicity.10 We have investigated bis-CBI
dimers11 and also CBI-PBD heterodimers,12 establishing that cytotoxicity is clearly a function of DNA
cross-linking ability, and that this can be optimised with appropriate linker chain length and
enantiomeric form. Other examples of CBI-PBD heterodimers have also been reported.13,14 Despite
the exceptional potency of particular exemplars (e.g. 311 and 512) there have been few reports on these
agents as ADC payloads.15,16 We have also described aminoCBIs, a structural variant of the CBI
alkylating subunit where the crucial phenol is replaced with an amino substituent. AminoCBIs are the
active form of nitroCBI hypoxia-activated prodrugs which have demonstrated selective activity
against hypoxic cells in human tumour xenografts.17 AminoCBIs and phenolCBIs share the same
mechanism of action and have remarkably similar properties, including equivalent potency as
monoalkylating agents, even though they exhibit quite different stability under physiological
conditions.18 The much higher reactivity of aminoCBIs has potential advantages in an ADC setting in
that rapid hydrolysis might act to limit the toxicity of any systemically released agent, as has been
proposed for a particular phenolCBI payload which undergoes unusually rapid hydrolysis in plasma.8
AminoCBIs also lend themselves to alternative chemical methods for linking to an antibody that may
not be accessible or sufficiently stable with their phenol analogues.
In this study we report the synthesis of two new bifunctional agents 4 and 6 that incorporate
aminoCBI. We compare their cytotoxicity across an extensive cell line panel with that of their
phenolCBI analogues and with microtubule-targeted agents, with a view to the utility of 3-6 as novel
ADC payloads.
2

2. Results and discussion
2.1. Synthesis
For the synthesis of 4 and 6 we needed
access to the S enantiomer of aminoCBI 10
(Scheme 1) which had not been reported
previously. We chose to explore a Pd-catalysed
amination route from the corresponding S-
phenolCBI (-)-S-7 for which an efficient
enantioselective synthesis was available.19 The
method was first tested with racemic 720,21 which
was converted to the triflate 8 and reacted with
benzophenone imine in the presence of catalytic
Pd(OAc)2 and BINAP ligand. A good yield of the
desired 9 was obtained, although using somewhat
higher catalyst loadings and a longer reaction
time than reported.22 Similarly high yields for
both steps were obtained on a multigram scale
starting with the corresponding S-enantiomer (-)-
S-7. Deprotection of the imine via transamination with hydroxylamine22 was slow at room
temperature and messy on heating, although aminoCBI 10 was the major product as determined by
TLC comparison with authentic racemic material.23 Acid-mediated deprotection was explored using
both aqueous HOAc and methanolic HCl, with the latter providing a surprisingly selective reaction in
the presence of the Boc protecting group and providing 10 in almost quantitative yield.
For the preparation of the bifunctional agents 4 and 6 it was found advantageous to leave the
imine in place as a protecting group until the last stage of the synthesis. This required orthogonal
deprotection of the Boc group of (-)-S-9 in the presence of the imine which was achieved under acidic
conditions in non-protic solvents, as shown in Schemes 2 and 3. Firstly however, for the aminoCBI-
phenolCBI dimer 4, a synthesis of acid 12 was required (Scheme 2). This was accomplished by
temporary benzyl protection of phenol (-)-S-7,
removal of the Boc protecting group and
reaction with glutaric anhydride, followed by
hydrogenolysis to cleave the benzyl ether.
Imine (-)-S-9 was treated with HCl in dioxane
and the crude product used directly in an
EDCI
(N-ethyl-N’-(3-
dimethylaminopropyl)carbodiimide
hydrochloride)-mediated coupling with 12 to
give dimer 13 in moderate yield. Final
deprotection of the imine using aqueous
HOAc, followed by HPLC purification, gave
the desired dimer 4.
The preparation of the aminoCBI-PBD
heterodimer 6 used the previously reported
PBD acid 1412 as a starting material (Scheme
3). In this case selective Boc deprotection of (-
3

)-S-9 was performed using TFA rather than
HCl which resulted in a much higher-yielding
and cleaner subsequent amide formation. The
imine and Alloc protecting groups of 15 were
sequentially removed using HCl and a brief
exposure to Pd(PPh3)4 and pyrrolidine
respectively, and the product 6 was isolated
after column chromatography.
The spirocyclic analogue of 5 was also
prepared (18 in Scheme 4). Starting from the
known Alloc-protected phenolCBI-PBD
dimer 1612 spirocyclisation was induced by
exposure to K2CO3 in DMA, followed by
removal of the Alloc protecting group as
described above. The phenolCBI-containing
dimers 3 and 5 were prepared as previously reported.
Some other structural variants of the exceptionally
potent 3 were also considered, namely extending the
polymethylene linker from three to five carbons, or
substituting the central methylene with an NH group.
The synthesis of these dimers in racemic form is
described in the Supporting Information.
2.2. In vitro cytotoxicity
Cytotoxicity was determined by inhibition of
proliferation after 96 h exposure to the compounds in
a panel of nine human tumour cell lines of diverse
origin (Table 1). The established payloads MMAE
(1) and DM1 (examined as the S-methylated
analogue 2-Me) exhibited IC50s in sub-nM range, as
previously reported,24 but for several cell lines an
IC50 could not be established for either compound
because proliferation inhibition reached a plateau at
high compound concentrations. This behaviour,
which could be a consequence of cell cycle arrest
under the assay conditions used, is illustrated for MMAE in Figure 2a. Both microtubule-targeted
payloads were also considerably less active in MES-SA/Dx5, a resistant sub-line of MES-SA that
overexpresses P-glycoprotein (Pgp).25
We previously reported that the phenolCBI dimer 3 exhibits IC50s in the 0.1 to 2 pM range
against a panel of six human tumour cell lines.11 This remarkable cytotoxicity was confirmed in the
present study with 3 showing IC50s generally in the 0.5 to 2 pM range, including in MES-SA/Dx5.
Even in the most ‘resistant’ cell line, HCC1937, the IC50 of 3 was 29 pM (16 pg/mL). The
corresponding compound 4 in which one end of the dimer was changed from phenolCBI to aminoCBI
showed a loss in potency of on average 24-fold (range 2 to >55-fold), and an increased sensitivity to
overexpression of Pgp, but retained low pM-level cytotoxicity in most of the cell lines examined.
We have also reported that the phenolCBI-PBD heterodimer 5 displays IC50s in the pM range
against a selection of human tumour cell lines, particularly after extended (5-day) compound
4

Table 1. In vitro cytotoxicity of known and proposed ADC payloads
Cell line
IC50 (nM)
2-Mea
0.11
>2
0.086
0.023
nd
1
0.67
>30
0.32
0.099
ndb
3
4
5
6
18
MES-SA (uterine, Pgp -ve)
MES-SA/Dx5 (uterine, high Pgp)
BJAB (B-cell)
KPL-4 (breast)
HCC1569 X2 (breast)
T-47D (breast)
0.00054 0.0058 0.033
0.11
0.00063 0.015
0.91 0.017
20.8 0.052
0.0024
0.11
0.011
0.036
0.13
0.016
0.11
1.2
0.0043
0.0015
0.018
0.0021
0.029
0.021
>1
0.081
0.066
0.74 0.016
6.2
2.5
9.5
0.048
0.0077
0.064
nd
nd
nd
nd
HCC1937 (breast)
NCI-H1781 (NSCLC)
SW 900 (NSCLC)
nd
nd
nd
nd
0.0047
0.0088
0.0098 0.0098 0.53 0.0043
0.033 0.051 3.7 0.024
aS-methylated analogue of DM1. bIC50 not determined because of incomplete inhibition of
proliferation.
exposure.12 In the current study 5 was approximately equipotent with 4, and similar levels of
cytotoxicity were observed whether the CBI was in the seco form (5) or spirocyclised prior to cell
treatment (18). This latter observation is consistent with previous reports on CBI monoalkylating
agents and has been attributed to facile spirocyclisation under physiological conditions.7 In all cell
lines, exposure to a sufficiently high concentration of 5 led to complete inhibition of proliferation
(Figure 2b), behaviour that was common to all the bifunctional agents examined and in marked
contrast to 1 and 2-Me. Once again, swapping the phenolCBI end of the dimer to an aminoCBI led to
a reduction in potency, with 6 being on average 85-fold less cytotoxic than 5, and also more sensitive
than 5 to Pgp overexpression (IC50 ratio in MES-SA/Dx5 compared to MES-SA 3.3 for 5 but 23 for
a)
b)
Concentration of MMAE (1) (nM)
Concentration of phenolCBI-PBD (5) (nM)
Figure 2. Representative cell viability curves for a) a microtubule-targeted cytotoxin,
MMAE (1) and b) a DNA minor groove cross-linking agent, phenolCBI-PBD heterodimer
(5). The smaller graphs below reproduce the cell viability curves for the MES-SA and
MES-SA/Dx5 pair of cell lines, illustrating the substantial resistance of the Pgp-
overexpressing subline to 1 (IC50 ratio > 45) but not 5 (IC50 ratio 3.3).
5

6). Overall the cytotoxicity of the bifunctional agents followed the order 3 > 4 ~ 5 > 6 spanning 2-3
orders of magnitude in the pM to nM range.
Given that 3 is clearly the most potent of the cytotoxins examined some other variants of this
structure were also investigated. However it was found that extending the polymethylene linker from
three to five carbons, or substituting the central methylene with an NH group led to a reduction in
potency, as assessed for racemic compounds in the KPL-4 breast cancer cell line (see Supporting
Information). Similar observations have been reported for S,S-enantiomers26 and for an analogue
containing a central N-propargyl unit,27 and these structural modifications were not pursued further.
3. Conclusions
The Pd-catalysed amination method described here gives easy access to the more active S
enantiomer of the aminoCBI alkylating subunit 10. This route is considerably more efficient than
alternative chiral HPLC-based resolutions that have been applied to intermediates in various
aminoCBI and nitroCBI syntheses.28,29 Moreover intermediate (-)-S-9 is synthetically very useful
since highly selective removal of either the imine or the Boc protecting group can be achieved via
appropriate choice of acidic reaction conditions.
Straightforward access to (-)-S-9 has allowed the synthesis of 4 and 6, novel bifunctional agents
that are analogues of the exceptionally cytotoxic CBI dimer 3 and CBI-PBD heterodimer 5.
Surprisingly, given that DNA monoalkylating agents incorporating phenolCBI or aminoCBI are of
equivalent cytotoxicity,18 swapping the phenolCBI of 3 or 5 for the corresponding aminoCBI in 4 and
6 leads to a substantial reduction in potency, ranging from 2- to 190-fold depending on the particular
compound and cell line. The bifunctional agents incorporating aminoCBI are also unexpectedly more
sensitive to overexpression of Pgp than their phenolCBI analogues.
Nevertheless, dimer 4 retains notable low pM level cytotoxicity in most of the cell lines
examined, and, in common with 3, 5, and 6, completely inhibits the proliferation of all cell lines at
sufficiently high concentrations. This stands in contrast to MMAE (1) and S-methylated DM1 (2-Me)
which gave, for example, maximum inhibition of proliferation of only 40-45% in NCI-H1781 and 50-
55% in HCC1569 X2 even at ³10 nM concentration. These observations reinforce the potential of
DNA minor groove alkylating agents, especially ultrapotent bifunctional agents, to overcome some of
the limitations of microtubule-targeted toxins as ADC payloads.
4. Experimental
4.1. Chemistry
Solvents were distilled prior to use by common laboratory methods. Petroleum ether refers to the
fraction with a bp = 40-60 °C. Organic solutions were dried over anhydrous Na2SO4 or MgSO4.
Column chromatography was carried out on silica gel (Merck 230-400 mesh). Melting points were
determined on an Electrothermal 2300 Melting Point Apparatus. NMR spectra were obtained on a
1
Bruker Avance 400 spectrometer at 400 MHz for H and 100 MHz for 13C spectra. HRMS was
performed with a Bruker micrOTOF-QII mass spectrometer. Low resolution mass spectra were
acquired by direct injection of methanolic solutions into a Surveyor MSQ mass spectrometer using
atmospheric pressure chemical ionization (APCI). Combustion analyses were carried out in the
Campbell Microanalytical Laboratory, University of Otago, Dunedin, New Zealand.
4.1.1. tert-Butyl 1-(chloromethyl)-5-(((trifluoromethyl)sulfonyl)oxy)-1H-benzo[e]indole-3(2H)-
carboxylate (8)
6

Trifluoromethanesulfonic anhydride (69 mL, 0.41 mmol) was added dropwise to a solution of
720,21 (125 mg, 0.37 mmol) and Et3N (63 mL, 0.45 mmol) in CH2Cl2 (15 mL) at 0 °C. After 5 min the
mixture was diluted with water and extracted with CH2Cl2 (´2). The extracts were dried and
evaporated and the residue was purified by column chromatography (eluting with EtOAc:petroleum
1
ether 1:9) to give 8 as a very pale yellow oil (160 mg, 92%): H NMR (CDCl3) d 8.31 (v br s, 1H),
8.03 (d, J = 8.5 Hz, 1H), 7.76 (d, J = 8.4 Hz, 1H), 7.60 (dt, J = 7.6, 1.0 Hz, 1H), 7.51 (dt, J = 7.7, 1.0
Hz, 1H), 4.32 (br s, 1H), 4.22-4.14 (m, 1H), 4.09-4.01 (m, 1H), 3.91 (dd, J = 11.2, 2.7 Hz, 1H), 3.51
(dd, J = 11.0, 10.3 Hz, 1H), 1.61 (s, 9H).
4.1.2. (S)-tert-Butyl 1-(chloromethyl)-5-(((trifluoromethyl)sulfonyl)oxy)-1H-benzo[e]indole-
3(2H)-carboxylate (S-8)
The same procedure (20 min reaction time) was used to convert (-)-S-719 (1.70 g) to the
corresponding triflate S-8 (2.20 g, 93%): 1H NMR identical to that for the racemic material.
4.1.3.
carboxylate (9)
A mixture of 8 (170 mg, 0.36 mmol), benzophenone imine (79 mg, 0.44 mmol), Cs2CO3 (0.17 g,
tert-Butyl 1-(chloromethyl)-5-((diphenylmethylene)amino)-1H-benzo[e]indole-3(2H)-
0.52 mmol), Pd(OAc)2 (0.8 mg, 3.6 mmol), and BINAP (2,2’-bis(diphenylphosphino)-1,1’-
binaphthalene) (3.4 mg, 5.5 mmol) in THF (6 mL) was degassed by bubbling with nitrogen and then
stirred at reflux under nitrogen overnight. More portions of Pd(OAc)2 (total 4%) and BINAP (total
6%) were added and the reaction was monitored by TLC until the starting material was consumed.
After 5 days the mixture was diluted with CH2Cl2 and filtered through Celite®. The filtrate was
evaporated and the residue was purified by column chromatography (eluting with EtOAc:petroleum
1
ether 1:9 and then CH2Cl2) to give 9 as a yellow oil (158 mg, 87%): H NMR (CDCl3) d 7.99 (d, J =
8.3 Hz, 1H), 7.87-7.81 (m, 2H), 7.63 (d, J = 8.4 Hz, 1H), 7.54-7.41 (m, 4H), 7.34-7.29 (m, 1H), 7.24-
7.13 (m, 3H), 7.10-7.05 (m, 2H), 4.21-4.13 (m, 1H), 4.04-3.98 (m, 1H), 3.95-3.86 (m, 2H), 3.36 (t, J
13
= 10.7 Hz, 1H), 1.51 (s, 9H), 1H not observed; C NMR d 168.7, 152.3, 149.9, 139.3, 135.9, 130.9,
129.8, 129.5, 128.7, 128.2, 127.8, 127.2, 125.3, 124.2, 123.3, 122.1, 117.7, 104.3, 81.1, 52.6, 46.4,
41.7, 28.4. A sample was recrystallised from MeOH giving a yellow solid: mp 172-174 °C. Anal.
calcd for C31H29ClN2O2: C, 74.91; H, 5.88; N, 5.64. Found: C, 74.79; H, 6.18; N, 5.64.
4.1.4. (-)-(S)-tert-Butyl 1-(chloromethyl)-5-((diphenylmethylene)amino)-1H-benzo[e]indole-
3(2H)-carboxylate ((-)-S-9)
The same procedure (15% Pd(OAc)2 and 15% BINAP, sealed tube, 60-65 °C, 4 days) was used
to convert S-8 (2.15 g) to the corresponding imine (-)-S-9 (2.09 g, 91%): 1H NMR (DMSO-d6) d 7.85
(d, J = 8.4 Hz, 1H), 7.80 (d, J = 8.4 Hz, 1H), 7.75 (d, J = 7.3 Hz, 2H), 7.61-7.57 (m, 1H), 7.54-7.49
(m, 3H), 7.37-7.33 (m, 1H), 7.30-7.23 (m, 3H), 7.06 (d, J = 6.7 Hz, 2H), 4.14-4.02 (m, 2H), 3.99-3.94
(m, 2H), 3.77 (dd, J = 10.8, 7.4 Hz, 1H), 1.46 (s, 9H), 1H not observed. HRMS calcd for
C31H30ClN2O2 (MH+) m/z 497.1990, found 497.1984. [a]D28 = -101.5° (c = 0.995, CH2Cl2).
4.1.5. (S)-tert-Butyl 5-amino-1-(chloromethyl)-1H-benzo[e]indole-3(2H)-carboxylate (10)
(a) Imine deprotection using aqueous HOAc. HOAc (65 mL) was added to a solution of (-)-S-9
(1.30 g, 2.62 mmol) in THF (195 mL) and water (98 mL) and the mixture was stirred at room
temperature for 18 h. The mixture was concentrated under vacuum at <30 °C to remove most of the
THF and then extracted with EtOAc (200 mL). The extract was washed with saturated aqueous
NaHCO3 (´4, until washings were pH 8), dried, and the solvent was removed under vacuum.
Purification of the residue by column chromatography (eluting with petroleum ether and then with
7

EtOAc:petroleum ether 1:9) gave 10 as an unstable solid (503 mg, 58%): 1H NMR (DMSO-d6) d 8.01
(d, J = 8.4 Hz, 1H), 7.64 (d, J = 8.0 Hz, 1H), 7.40 (ddd, J = 8.1, 6.8, 0.9 Hz, 1H), 7.36 (br s, 1H), 7.20
(ddd, J = 8.1, 6.8, 1.1 Hz, 1H), 5.91 (s, 2H), 4.11-3.91 (m, 4H), 3.66 (dd, J = 10.6, 8.2 Hz, 1H), 1.53
(s, 9H); consistent with that previously reported for the racemate.23
(b) Imine deprotection using methanolic HCl. HCl (4M in dioxane, 0.63 mL, 2.5 mmol) was
added to a solution of (-)-S-9 (497 mg, 1.00 mmol) in dry CH2Cl2 (10 mL) and MeOH (10 mL) under
nitrogen and the mixture was stirred at room temperature for 1 h. The solvents were evaporated at 20
°C and the resulting yellow solid was stirred with a mixture of EtOAc-petroleum ether (1:10) (200
mL) for 30 min. The solvents were separated and the process was repeated. The solid was then stirred
at 0 °C with cold aqueous Na2CO3 (2N, 200 mL) and CH2Cl2 (200 mL) for 15 min. The CH2Cl2 layer
was separated, washed with water (100 mL), and then dried and evaporated at 25 °C to give 10 as an
1
unstable solid (322 mg, 97%): H NMR consistent with that previously reported for the racemate.23
HRMS (ESI) calcd for C18H22ClN2O2 (MH+) m/z 333.1364, found: 333.1355; calcd for
C18H21ClN2NaO2 (MNa+) m/z 355.1184, found: 355.1179; calcd for C18H21ClKN2O2 (MK+) m/z
371.0923, found: 371.0920.
4.1.6. (S)-tert-Butyl 5-(benzyloxy)-1-(chloromethyl)-1H-benzo[e]indole-3(2H)-carboxylate (11)
Benzyl bromide (7.13 mL, 60 mmol), KI (50 mg, 0.30 mmol) and K2CO3 (4.14 g, 30 mmol) were
added to a solution of (-)-S-7 (2.00 g, 5.99 mmol) in DMF (5 mL) and the mixture was stirred at room
temperature for 2 h. EtOAc was added, the insoluble material was filtered off, and the filtrate was
partitioned with water. The organic layer was separated and the aqueous phase was extracted with
more EtOAc (´3). The combined organic extracts were dried and evaporated and the residue was
purified by column chromatography (eluting with EtOAc: petroleum ether 1:10) to give 11 as a white
1
solid (1.97 g, 78%): mp 186-188 °C (lit7 mp 184.5-185.5 °C); H NMR (CDCl3) identical to that
reported.7
Further elution (EtOAc: petroleum ether 1:1) gave spirocyclised starting material (8bR,9aS)-tert-
butyl 4-oxo-9,9a-dihydro-1H-benzo[e]cyclopropa[c]indole-2(4H)-carboxylate7 (345 mg, 19%).
4.1.7. (S)-5-(1-(Chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)-5-oxopentanoic acid (12)
HCl in dioxane (4N, 40 mL) was added to a solution of 11 (1.60 g, 3.77 mmol) in CH2Cl2 (15
mL) cooled in an ice bath. The cooling bath was removed and the mixture was stirred for 3 h, then
evaporated to dryness. The residue was stirred with THF (15 mL) in an ice bath and glutaric
anhydride (646 mg, 5.66 mmol), DMAP (46 mg, 0.38 mmol) and pyridine (5 mL) were added. The
cooling bath was removed and the mixture was stirred for 4 h, then evaporated to dryness. The residue
was dissolved in dilute aqueous NaHCO3 and washed 3 times with EtOAc. The aqueous phase was
acidified using 1N HCl to a pH of 2 and extracted with EtOAc (´3). The combined extracts were
washed with brine, dried, and filtered through a silica gel pad washing with a mixture MeOH:EtOAc
(1:10). The solvent was evaporated to give benzyl-protected 12 as an off-white solid (978 mg, 59%).
Aqueous NH4HCO2 (25%, 23 mL) and Pd-C catalyst (10%, 1.5 g) were added to a solution of
benzyl-protected 12 (2.00 g, 4.57 mmol) in THF (60 mL) at -20 °C. The mixture was stirred at -10 to -
15 °C for 22 h, then diluted with MeOH at 0 °C and filtered through Celite®, washing with MeOH.
The solvents were concentrated under vacuum until a solid began to precipitate. Water (150 mL) and
petroleum ether (150 mL) were added and the mixture was stirred at room temperature while being
acidified to pH 1 with conc. HCl. The mixture was stirred for a further 30 min and then the solid was
filtered off, washed with water and petroleum ether, and dried to give 12 as a beige solid (1.43 g,
1
90%): H NMR (DMSO-d6) d 12.07 (br s, 1H), 10.35 (s, 1H), 8.08 (d, J = 8.0 Hz, 1H), 7.98 (s, 1H),
7.77 (d, J = 8.3 Hz, 1H), 7.50-7.46 (m, 1H), 7.33-7.29 (m, 1H), 4.30 (t, J = 10.4 Hz, 1H), 4.14-4.12
8

(m, 2H), 3.98 (dd, J = 10.9, 2.8 Hz, 1H), 3.78 (dd, J = 10.8, 7.8 Hz, 1H), 2.63-2.45 (m, 2H), 2.35 (t, J
= 7.4 Hz, 2H), 1.89-1.78 (m, 2H); LRMS (APCI) calcd for C18H19ClNO4 (MH+) m/z 348.1, found
348.6.
4.1.8. 1-((S)-1-(Chloromethyl)-5-((diphenylmethylene)amino)-1H-benzo[e]indol-3(2H)-yl)-5-((S)-
1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)pentane-1,5-dione (13)
HCl (g) was bubbled through a solution of (-)-S-9 (425 mg, 0.855 mmol) in dry dioxane (12 mL)
at room temperature. A solid precipitated and after 15 min the solvent was removed under vacuum.
To the residue was added 12 (327 mg, 0.941 mmol), EDCI (573 mg, 2.99 mmol), and DMA (10 mL).
The mixture was stirred under nitrogen for 3 days and then the solvent was removed under vacuum.
The residue was purified by column chromatography (first column eluting with CH2Cl2:MeOH 10:0
then 9:1; second column Et2O:CH2Cl2 10:0 to 5:5 to 0:10; third column CH2Cl2:MeOH 99:1 then
98:2) to give 13 (193 mg, 31%): 1H NMR (DMSO-d6) d 10.35 (s, 1H), 8.08 (d, J = 8.1 Hz, 1H), 8.00
(s, 1H), 7.84 (t, J = 9.0 Hz, 2H), 7.79-7.74 (m, 3H), 7.62-7.57 (m, 2H), 7.54-7.46 (m, 4H), 7.40-7.36
(m, 1H), 7.33-7.29 (m, 1H), 7.27-7.22 (m, 3H), 7.09-7.08 (m, 2H), 4.34-4.26 (m, 2H), 4.22-4.12 (m,
4H), 4.01-3.97 (m, 2H), 3.83-3.76 (m, 2H), 2.69-2.50 (m, 4H), 1.93-1.86 (m, 2H). HRMS calcd for
C44H38Cl2N3O3 (MH+) m/z 726.2285; found 726.2264.
4.1.9.
1-((S)-5-Amino-1-(chloromethyl)-1H-benzo[e]indol-3(2H)-yl)-5-((S)-1-(chloromethyl)-5-
hydroxy-1H-benzo[e]indol-3(2H)-yl)pentane-1,5-dione (4)
HOAc (8 mL) was added to a solution of 13 (190 mg, 0.261 mmol) in THF (24 mL) and water
(12 mL) and the mixture was stirred at room temperature. After 19 h the mixture was diluted with
water, causing a solid to precipitate. The THF was removed under vacuum and the aqueous
suspension treated with i-Pr2NEt until neutral. The solid was filtered off, washed with water and
dried. Attempts to purify the crude product by trituration with various solvent mixtures, or by column
chromatography (eluting with CH2Cl2:MeOH 10:0 then 9:1) were only partially successful. The
material was instead purified by preparative HPLC (Synergi-MAX RP 4 μ column, 21.20 ´ 250 mm;
13 mL/min; mobile phase: solvent A: H2O/TFA pH 2.5, solvent B: MeCN/H2O 9:1; solvent A:solvent
1
B 20:80) to give 4 (30 mg, 22%) as a white solid: H NMR (DMSO-d6) d 10.36 (s, 1H), 8.08 (d, J =
8.1 Hz, 1H), 8.04-8.02 (m, 2H), 7.79-7.77 (m, 2H), 7.69 (d, J = 8.3 Hz, 1H), 7.51-7.47 (m, 1H), 7.42
(t, J = 7.5 Hz, 1H), 7.34-7.30 (m, 1H), 7.23 (t, J = 7.6 Hz, 1H), 5.91 (s, 2H), 4.36-4.26 (m, 2H), 4.19-
4.13 (m, 3H), 4.08-4.04 (m, 1H), 4.01-3.93 (m, 2H), 3.79 (dd, J = 10.7, 8.2 Hz, 1H), 3.70 (dd, J =
10.5, 8.9 Hz, 1H), 2.75-2.66 (m, 2H), 2.63-2.54 (m, 2H), 2.00-1.93 (m, 2H). HRMS calcd for
C31H29Cl2N3NaO3 (MNa+) m/z 584.1478, found 584.1456. [a]D28 = -37.6° (c = 0.559, DMSO).
4.1.10.
(11aS)-Allyl
8-((6-((S)-1-(chloromethyl)-5-((diphenylmethylene)amino)-1H-
benzo[e]indol-3(2H)-yl)-6-oxohexyl)oxy)-11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-
1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (15)
Trifluoroacetic acid (2 mL) was added to a solution of (-)-S-9 (166 mg, 0.33 mmol) in dry
CH2Cl2 (2 mL) at 0 °C under nitrogen and the mixture was stirred at this temperature for 2 h 30 min.
The mixture was diluted with cold EtOAc and stirred with excess aqueous Na2CO3 (2N). The EtOAc
layer was separated, washed with water, and then dried and evaporated at 20 °C. To the residue was
added acid 1412 (155 mg, 0.33 mmol), EDCI (128 mg, 0.67 mmol), toluenesulfonic acid (11.5 mg,
0.067 mmol), and dry DMA (1 mL) and the mixture was stirred at room temperature under nitrogen
for 18 h. Water was added and after 15 min the preciptated solid was filtered off, washed with water
and then petroleum ether (3´20 mL) and dried to give 15 as a yellow solid (200 mg, 71%); mp 124-
1
127 °C; H NMR (DMSO-d6) d 7.83 (t, J = 8.0 Hz, 2H), 7.79-7.00 (m, 2H), 7.64-7.47 (m, 5H), 7.37
9

(t, J = 7.6 Hz, 1H), 7.30-7.17 (m, 3H), 7.13-7.09 (m, 3H), 6.77 (s, 1H), 6.49 (poorly resolved d, J =
5.2 Hz, 1H), 5.88-5.72 (m, 1H), 5.45 (dd, J = 8.9 and 6.0 Hz, 1H), 5.15-4.99 (m, 2H), 4.66-4.52 (m,
1H), 4.46-4.33 (m, 1H), 4.32-4.05 (m, 3H), 4.05-3.87 (m, 3H), 3.85-3.71 (m, 4H), 3.53-3.43 (m, 1H),
3.40-3.19 (m, partially obscured by water peak, 2H), 2.61-2.34 (m, partially obscured by DMSO
peak, 2H), 2.10-1.68 (m, 6H), 1.68-1.52 (m, 2H), 1.52-1.35 (m, 2H). HRMS (ESI) calcd for
C49H50ClN4O7 (MH+) m/z 841.3363, found 841.3380; calcd for C49H49ClN4NaO7 (MNa+) m/z
863.3182, found 863.3205. [a]D = +2.1° (c 0.48, CHCl3).
4.1.11. (S)-8-((6-((S)-5-Amino-1-(chloromethyl)-1H-benzo[e]indol-3(2H)-yl)-6-oxohexyl)oxy)-7-
methoxy-2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one (6)
HCl in dioxane (4M, 83 mL, 0.33 mmol) was added to a solution of 15 (56 mg, 0.065 mmol) in
dry CH2Cl2 (1.5 mL) and dry MeOH (1.5 mL) under nitrogen and the mixture was stirred at room
temperature for 1 h 20 min. The mixture was evaporated under reduced pressure at 20 °C to give a
red-brown residue which was triturated with EtOAc:petroleum ether (1:10, 2´25 mL) and then dried.
To this solid were added pyrrolidine (55 mL, 0.65 mmol) and Pd(PPh3)4 (3.8 mg, 9.8% Pd) in dry
CH2Cl2 (5 mL) under nitrogen. After 3 min at room temperature the mixture was transferred directly
to a silica column. Chromatography (eluting with EtOAc:MeOH 50:1), with concentration of product-
containing fractions at 20 °C, gave 6 as a pale yellow solid (27.3 mg, 73%); mp 207-211 °C (dec); 1H
NMR (DMSO-d6) d 7.93 (s, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.70-7.62 (m, 2H), 7.55-7.45 (m, 2H),
7.39-7.31 (m, 1H), 6.81 (s, 1H), 4.48-3.87 (m, 11H, 2H exchange with D2O), 3.87-3.77 (m, 1H), 3.77-
3.68 (m, 1H), 3.64-3.52 (m, 1H), 3.38 (t, J = 10.8 Hz, 1H), 2.67-2.55 (m, 1H), 2.55-2.43 (m, 1H),
2.38-2.25 (m, 2H), 2.14-1.78 (m, 6H), 1.70-1.55 (m, 2H). HRMS (ESI) calc for C32H36ClN4O4 (MH+)
m/z 575.2420, found 575.2409; calcd for C32H35ClN4NaO4 (MNa+) m/z 597.2239, found 597.2254.
[a]D = +516.1° (c 0.062, CHCl3).
4.1.12. (11aS)-Allyl 11-hydroxy-7-methoxy-5-oxo-8-((6-oxo-6-((8bR,9aS)-4-oxo-9,9a-dihydro-1H-
benzo[e]cyclopropa[c]indol-2(4H)-yl)hexyl)oxy)-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-
a][1,4]diazepine-10(5H)-carboxylate (17)
K2CO3 (48 mg, 0.35 mmol) was added to a solution of 1612 (34 mg, 0.05 mmol) in dry DMA (1
mL) and the mixture was stirred at room temperature for 3 h. The mixture was partitioned between
EtOAc (150 mL) and cold aqueous Na2CO3 (1N, 50 mL). The EtOAc layer was separated, washed
with water (2´30 mL), and then dried and evaporated. The glassy solid residue was triturated with
1
EtOAc:petroleum ether (1:1) (2´3 mL) to give 17 as a beige solid (29.2 mg, 91%); H NMR (CDCl3)
d 8.22 (dd, J = 7.8, 1.1 Hz, 1H), 7.52 (td, J = 7.6, 1.4 Hz, 1H), 7.42 (td, J = 8.1, 1.0 Hz, 1H), 7.23 (s,
1H), 6.87 (d, J = 7.5 Hz, 1H), 6.71 (s, 1H), 5.89-5.71 (m, 1H), 5.64 (dd, J = 8.0, 3.4 Hz, 1H), 5.14 (d,
J = 11.6 Hz, 2H), 4.67 (dd, J = 13.9, 5.5 Hz, 1H), 4.52-4.40 (m, 1H), 4.19-3.96 (m, 4H), 3.90 (s, 3H),
3.79 (d, J = 4.5 Hz, 1H), 3.74-3.66 (m, 1H), 3.62-3.45 (m, 2H), 2.83-2.75 (m, 1H), 2.63-2.50 (m, 2H),
2.19-2.10 (m, 2H), 2.10-1.95 (m, 2H), 1.95-1.64 (m, 5H), 1.64-1.50 (m, 2H), 1.44 (t, J = 4.8 Hz, 1H),
1 H not observed. HRMS (ESI) calcd for C36H40N3O8 (MH+) m/z 642.2810, found 642.2823; calcd for
C36H39N3NaO8 (MNa+) m/z 664.2629, found 664.2652. [a]D = +257.1° (c 0.14, CHCl3).
4.1.13.
(8bR,9aS)-2-(6-(((S)-7-Methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-
a][1,4]diazepin-8-yl)oxy)hexanoyl)-9,9a-dihydro-1H-benzo[e]cyclopropa[c]indol-4(2H)-one (18)
Pyrrolidine (35 mL, 0.43 mmol) and Pd(PPh3)4 (2.5 mg, 9.8% Pd) were added to a solution of 17
(27.3 mg, 0.043 mmol) in dry CH2Cl2 (2 mL) under nitrogen and the mixture was stirred at room
temperature. After 3 min the mixture was transferred directly to a silica column. Chromatography
1
(eluting with EtOAc:MeOH 50:1) gave 18 as a glassy solid (20.6 mg, 91%); H NMR (CDCl3) d 8.22
10

(dd, J = 7.9, 1.1 Hz, 1H), 7.66 (d, J = 4.4 Hz, 1H), 7.56-7.46 (m, 2H), 7.40 (td, J = 8.1, 1.1 Hz, 1H),
6.87 (d, J = 7.5 Hz, 1H), 6.80 (s, 1H), 4.18-3.99 (m, 4H), 3.94 (s, 3H), 3.88-3.77 (m, 1H), 3.76-3.69
(m, 1H), 3.63-3.53 (m, 1H), 2.62-2.74 (m, 1H), 2.63-2.49 (m, 2H), 2.39-2.25 (m, 2H), 2.12-1.98 (m,
2H), 1.98-1.72 (m, 4H), 1.67 (dd, J = 7.6, 4.6 Hz, 1H), 1.64-1.50 (m, 2H), 1.43 (t, J = 4.8 Hz, 1H), 1
H not observed. HRMS (ESI) calcd for C32H34N3O5 (MH+) m/z 540.2493, found 540.2504; calcd for
C32H33N3NaO5 (MNa+) m/z 562.2312, found 562.2325. [a]D = +880.8° (c 0.15, CHCl3).
4.2 In vitro cytotoxicity
Cells were seeded in 384-well plates and treated with drug 24 h later. After 4 days of drug
incubation, the cell viability was determined using Promega CellTiter-Glo luminescent reagent, which
measures ATP level (an indirect measure of cell number). The luminescent intensity was measured on
PerkinElmer Envision reader. The relative cell viability was calculated by normalizing to non-drug
treatment control and was graphed using KleidaGraph software package. IC50 value was determined
as the concentration to obtain 50% of the maximum cell killing. Each IC50 was determined by curve-
fitting from 10 data points, and each data point was the average of quadruplicates.
Acknowledgements
We thank Karin Tan and Sisira Kumara for HPLC purification and analyses and Dr Adrian
Blaser for NMR spectrometry. This work was funded by Genentech Inc. and the Auckland Division of
the Cancer Society of New Zealand.
Supplementary Information
Supplementary data associated with this article (describing the synthesis and properties of two
1
racemic phenolCBI dimers and an iminodiacetic acid-linked phenolCBI dimer, and H NMR spectra
of 4, 6, 18, and four compounds of the Supporting Information) can be found, in the online version.
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12

*Graphical Abstract (for review)
alkylates N3
of adenine
Cl
Cl
Cl
alkylates C2-NH2 of guanine
N
N
N
N
O
H
O
O
O
N
MeO
NH2
NH2
OH
O
IC50 (MES-SA) = 5.8 pM
IC50 (MES-SA) = 0.91 nM