Synthesis and evaluation of stable bidentate transition metal complexes of 1-(chloromethyl)-5-hydroxy-3-(5,6,7-trimethoxyindol-2-ylcarbonyl)-2, 3-dihydro-1H-pyrrolo[3,2-f]quinoline (seco-6-azaCBI-TMI) as hypoxia selective cytotoxins

Article abstract of DOI:10.1021/jm9008746

A series of metal complexes were prepared as potential prodrugs of the extremely toxic DNA minor groove alkylator 1-(chloromethyl)-5-hydroxy-3-[(5,6,7- trimethoxyindol-2-yl)carbonyl]-2,3-dihydro-1H-pyrrolo[3,2-f]quinoline (seco-6-azaCBI-TMI) and close analogues. The pyrrolo[3,2-f]quinoline cytotoxins were prepared from 2-methoxy-4-nitroaniline in a nine-step synthesis involving a Skraup construction of a quinoline intermediate, its appropriate functionalization, and a final radical cyclization. The metal complexes were prepared from these and the labile metal complex synthons [Co-(cyclen)(OTf) ₂]⁺, [Cr(acac)₂(H₂O) ₂]⁺, and [Co₂(Me₂dtc) ₅]⁺. The cobalt complexes were considerably more stable than the free effectors and showed significant attenuation of the cytotoxicity of the latter, with IC₅₀ ratios (complex/effector) of 50- to 150-fold, and substantial hypoxic cell selectivity, with IC₅₀ ratios (oxic/hypoxic cells) of 20- to 40-fold. The cobalt complexes were also efficiently activated by ionizing radiation, with G values for loss of the compound close to the theoretical value for one-electron reduction of 0.68 μmol/J. This work extends earlier observations that cobalt cyclen complexes are suitable for both the bioreductive and radiolytic release of potent pyrrolo[3,2-f]quinoline effectors. 2009 American Chemical Society.

Full text of DOI:10.1021/jm9008746

6822 J. Med. Chem. 2009, 52, 6822–6834
DOI: 10.1021/jm9008746
Synthesis and Evaluation of Stable Bidentate Transition Metal Complexes of 1-(Chloromethyl)-
5-hydroxy-3-(5,6,7-trimethoxyindol-2-ylcarbonyl)-2,3-dihydro-1H-pyrrolo[3,2-f]quinoline
(seco-6-azaCBI-TMI) as Hypoxia Selective Cytotoxins
Jared B. J. Milbank,†,§ Ralph J. Stevenson,† David C. Ware,‡ John Y. C. Chang,‡ Moana Tercel,† G-One Ahn,†,
William R. Wilson,† and William A. Denny*,†
†Auckland Cancer Society Research Centre, School of Medical Sciences and ‡Department of Chemistry, The University of Auckland,
Private Bag 92019, Auckland 1142, New Zealand. §Present address: Pfizer Global Research and Development, Ramsgate Road,
Sandwich, Kent CT13 9NJ, U.K. Present address: Department of Radiation Oncology, Stanford University School of Medicine,
269 Campus Drive, CCSR-South, Stanford, CA 94305-5152.
Received June 15, 2009
A series of metal complexes were prepared as potential prodrugs of the extremely toxic DNA minor
groove alkylator 1-(chloromethyl)-5-hydroxy-3-[(5,6,7-trimethoxyindol-2-yl)carbonyl]-2,3-dihydro-
1H-pyrrolo[3,2-f]quinoline (seco-6-azaCBI-TMI) and close analogues. The pyrrolo[3,2-f]quinoline
cytotoxins were prepared from 2-methoxy-4-nitroaniline in a nine-step synthesis involving a Skraup
construction of a quinoline intermediate, its appropriate functionalization, and a final radical cycliza-
tion. The metal complexes were prepared from these and the labile metal complex synthons [Co-
(cyclen)(OTf)2]þ, [Cr(acac)2(H2O)2]þ, and [Co2(Me2dtc)5]þ. The cobalt complexes were considerably
more stable than the free effectors and showed significant attenuation of the cytotoxicity of the latter,
with IC50 ratios (complex/effector) of 50- to 150-fold, and substantial hypoxic cell selectivity, with IC50
ratios (oxic/hypoxic cells) of 20- to 40-fold. The cobalt complexes were also efficiently activated by
ionizing radiation, with G values for loss of the compound close to the theoretical value for one-electron
reduction of 0.68 μmol/J. This work extends earlier observations that cobalt cyclen complexes are
suitable for both the bioreductive and radiolytic release of potent pyrrolo[3,2-f]quinoline effectors.
Introduction
decreased on one-electron reduction of the metal, resulting in
release of the coordinated ligands.13 While these compounds
appear to have insufficient stability to work well in vivo, these
studies demonstrated the potential utility of Co(III) complexes
as bioreductive prodrugs.
Prodrugs capable of selective activation in hypoxic tumor
cells are an attractive concept for tumor-specific chemother-
apy, since hypoxia is known to be more severe and extensive in
human solid tumors than normal tissues.1 Hypoxic cells in
solid tumors are resistant to radiation therapy2 and to some
chemotherapeutic drugs3 and are thus a doubly attractive
target for prodrugs.4
Such prodrugs can potentially be converted to an active form
(effector) in hypoxic tissue by two different methods. In the first
approach, endogenous cellular enzymes such as NADPH-
cytochrome P-450 oxidoreductase are utilized to convert pro-
drugs to a transient a one-electron adduct. While this process
may occur in all tissue, the intermediate can be reoxidized to the
parent prodrug by the molecular oxygen present in normal
tissue, but in the absence of oxygen it can be further converted
or fragmented to toxic species.5,6 A great deal of work has been
reported on bioreductive enzyme prodrugs, with many different
classes of compounds explored.7 Examples include the deacti-
vated nitrogen mustard DNA cross-linking agents PR-104 (1)8
and TH-302 (2),9 and banoxantrone (3)10 (a prodrug of a DNA-
binding topoisomerase inhibitor), all of which have reached
phase II clinical trial. An earlier approach employed transition
metal complexes of well-known DNA alkylating nitrogen
mustards (e.g., 4)11,12 on the premise that the stability of cobalt-
(III) complexes with nitrogen-based ligands is dramatically
In the second approach, the reducing equivalents from
therapeutic ionizing radiation are utilized to activate pro-
drugs (radiation-activated prodrugs). This approach takes
*To whom correspondence should be addressed. Phone: (64 9) 932
6144. Fax: (64 9) 373 7502. E-mail: b.denny@auckland.ac.nz.
r
pubs.acs.org/jmc
Published on Web 10/12/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6823
Scheme 1a
advantage of the ability of modern radiotherapy to deliver
ionizing radiation selectively to the tumor field and has
theoretical advantages over bioreduction.14,15 These include
the additional selectivity provided by radiation targeting, as
well as hypoxia, and the lack of dependence on expression of
reductive enzymes, including an ability to utilize necrotic
regions devoid of enzyme activity.14 This radiation-activated
prodrug approach has been considered for activation of
2-oxopropyl prodrugs of pyrimidines.16 We have also explored
this approach in the context of cobalt(III) and chromium(III)
complexes of 8-hydroxyquinoline (8-HQ) bearing a variety of
different ligands at the auxiliary metal coordination posi-
tions.17 Analogues such as 5, with 1,4,7,10-tetraazacyclodo-
decane (cyclen) as the auxiliary ligand, released 8-HQ very
efficiently on exposure toradiation[G(8-HQ)g 0.46μmol/J in
formate buffer, close to the theoretical value for one-electron
reduction of 0.62 μmol/J in this system]. HPLC studies in
high-density cell cultures showed that 5 was stable under both
aerobic and hypoxic conditions and efficiently masked the
cytotoxicity of the ligand in the complex, being ∼1000-fold
less cytotoxic than 8-HQ itself. These properties suggested the
feasibility of also using such complexes as radiation-activated
prodrugs for the release of more cytotoxic ligands incorporat-
ing a coordinating 8-HQ moiety that could also form stable
metal complexes.
In a search for more potent effectors able to form stable
Co(III) chelates, we considered the CBI-TMI class of com-
pounds, developed following the discovery of the intensely
cytotoxic natural products CC-1065 and the duocarmycins.18
We expected that the unsubstituted aza compound 6 would be
at least as cytotoxic as the “parent” compound 719 (much
more potent than aliphatic mustards) and, as an 8-hydroxy-
quinoline, would be able to form metal complexes. Previous
structure-activity studies on CBI-TMI analogues have
shown that electron-withdrawing groups in the benzoindole
ring provide compounds (e.g., 8) of greater stability and
moderately greater potency than the parent.20 N-Acyl-O-
aminophenol prodrugs of the related duocarmycin, designed
to be released by reducing nucleophiles such as thiols which
are suggested to be at higher levels in hypoxic tumor environ-
ments, have been reported21 but not evaluated for hypoxic cell
selectivity.
The spirocyclic compound (þ)-N-BOC-CPyI (46) has been
shown to have greatly increased reactivity for adenine-N3
alkylation of DNA in the presence of various metal ions, with
therelativeratesofsolvolysis correlating well withthestability
constants of the metal complexes with 8-HQ.22 This was
attributed to metal chelation of the ketoquinoline moiety;
sequential reaction of 46 with Zn(OTf)2 and MeOH gave the
isolated methoxy derivative 48 via the chelate 47 (Scheme 6).
We considered that the aza seco compound 6 and analogues
would form more stable transition metal chelates with appro-
priate polydentate auxiliary ligands, as we have demonstrated
previously for the metal complexes of 8-hydroxyquinoline.17
These complexes of 6 and analogues could thus potentially act
as hypoxia-selective prodrugs, activated by either endogenous
enzymes or ionizing radiation.
We later confirmed this with a study of the cobalt complex
(39) of 1-(chloromethyl)-5-hydroxy-3-[(5,6,7-trimethoxyin-
dol-2-yl)carbonyl]-2,3-dihydro-1H-pyrrolo[3,2-f]quinoline (6).23
Complex 39 was shown to be relatively stable in solution and
demonstrated a G value for loss of the complex in formate buffer
of 0.68 μmol/J and a selectivity of about 20-fold for hypoxic over
aerobic HT29 cells in culture by clonogenic assay.
a Reagents and conditions: (i) glycerol, H2SO4, As2O5; (ii) 48% HBr,
reflux, 65 h; (iii) BnBr, NaI, K2CO3, DMF, 20 °C, 9 h; (iv) Fe, AcOH,
aq EtOH, reflux, 10 min; (v) (BOC)2O, dioxane, reflux, 3 h; (vi) NIS,
MeCN, reflux, 1 h; (vii) BrCH2CHdCH2, NaH, DMF, 20 °C, 3 h; (viii)
Bu3SnH, TEMPO, benzene, under N2, reflux, 3 h.
We report here the syntheses of 39 and related transition
metal complexes of 6 and other potent 8-hydroxyquinoline
derivatives and their evaluation as prodrugs for activation
under hypoxia by cellular (enzymatic) reduction and by
ionizing radiation.
Chemistry
Our initial approach (Scheme 1) to the synthesis of the
(racemic) 6 was based on Boger’s 5-exo-trig radical cyclization
methodology,24 in which cyclization onto an unfunctionalized
alkene and trapping of the primary radical with TEMPO
produce a concise synthesis.25
Conversion of 2-methoxy-4-nitroaniline (9) by the Skraup
reaction gave 8-methoxy-6-nitroquinoline (10); careful appli-
cation of the simplest reported procedure26 allowed the yield
to be improved from the reported 68% to 80%. The methyl
protecting group was then replaced with benzyl to allow for a
more ready removal at the end of the synthesis. Demethyla-
tion of 10didnot requirethe reported27 TFA;whenthesample
was heated with 48% aqueous HBr (5 equiv) for 65 h, the HBr
salt of the quinolinol 11 precipitated from the cooled reaction
mixture (87%yield) and was benzylated directly (BnBr, excess
K2CO3, NaI, DMF, 99%) to give 12. Reduction of 12 with
iron dust in EtOH/AcOH/H2O gave amine 13 quantitatively.
Protection of 13 with di-tert-butyl dicarbonate in refluxing
THF was incomplete, but in refluxing dioxane a 98% yield of
14 was achieved. Iodination of 14 with N-iodosuccinimide
(NIS) in the presence of catalytic acid22 was complicated by
the basic center present in the quinoline, and a preferable
method was treatment with NIS in refluxing acetonitrile.28
This gave iodide 15 regiospecifically in 93% yield. This could
be allylated uneventfully and the allyl derivative 16 cyclized
efficiently in the presence of Bu3SnH and TEMPO to give 17.
Unfortunately, attempts to cleave the N-O bond of 17 with a
variety of reagents (Zn/THF/H2O/AcOH,25 Zn/NaOH/H2O/
EtOH, Na/EtOH, Al/Hg/THF,29 or SmI2/THF30,31) were
unsuccessful.
Of several possible alternative routes, the most attractive
appeared to be cyclization onto a vinyl ether or ester, where
the presence of oxygen on the vinyl group of the starting
material allows a simple reductive cyclization (Scheme 2).
The dimethoxy acetal 18, prepared by alkylation of 15 with

6824 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Milbank et al.
Scheme 2a
Scheme 4a
a Reagents and conditions: (i) Br(CH2)2CH(OMe)2, NaH, 20 °C, 22 h;
(ii) TsOH, Me2CO/water (10:1), reflux, 2.25 h; (iii) Ac2O, Et3N, DMAP,
THF, reflux, 4 h; (iv) Bu3SnH, AIBN, benzene, reflux, 5.5 h; (v) Cs2CO3,
EtOH/water (2:1), reflux, 30 min; (vi) MsCl, Et3N, DCM, 0 °C, 30 min;
(vii) LiCl, DMF, 80 °C, 1 h; (viii) Ph3P, CCl4, DCM, 20 °C, 4 h.
Scheme 3a
a Reagents and conditions: (i) NaH, DMF, then 1,3-dichloropropene,
a Reagents and conditions. (i) HCl(g), dioxane, 20 °C, then RCO2H,
then EDCI, DMA, 20 °C; (ii) NH4HCO2, Pd-C, THF (under N2), 20 °C;
(iii) TFA, 48 h, reflux.
20 °C (under N2), 86 h; (ii) AIBN, Bu3SnH, benzene, reflux (under N2), 3 h.
3-bromo-1,1-dimethoxypropane,32 could be quantitatively
deprotected with TsOH (0.5 equiv) in wet acetone. Since the
substrate is a quinoline, the reagent is effectively an analogue
of PPTS.33 The resulting aldehyde 19 was converted into vinyl
acetate 20 (Ac2O, DMAP, THF, reflux, 81%),34 which under-
went radical cyclization in the presence of Bu3SnH and AIBN
togiveacetate21ingood (77%) yield. Deprotectionof 21with
Cs2CO3 gave alcohol 22, which could be converted either
directly (Ph3P, CCl4, 100%) or via mesylate 23 (MsCl, Et3N,
86%; then LiCl, DMF, 89%) to the desired racemic chlor-
omethylpyrroloquinoline 24. An iodo substituent had initially
been selected because it has been reported24 to give better
yields when TEMPO is used as the radical trap. However, in
simple reductive cyclizations bromine suffices and was used
here. Treatment of 14 with NBS instead of NIS gave bromo-
quinoline 49 in excellent yield, and yields through the remain-
der of the path (49 f 50 f 51 f 52 f 21) were similar or
slightly better (Scheme S1 in Supporting Information).
At this point in the work the synthesis of oxaduocarmycin
involving radical cyclization onto a vinyl chloride was re-
ported.35 Applied to the current work, alkylation of iodide 15
with 1,3-dichloropropene and radical cyclization of the result-
ing vinyl chloride 25 or the bromo equivalent gave 24 in 97%
yield (Scheme 3). A similar process beginning with the corre-
sponding bromide 49 gave 24 in 95% yield (Scheme S2 in
Supporting Information).
made this way for convenience. Resolution of 24 was carried
out by semipreparative HPLC on a ChiralCel OD column,
eluting with hexane/iPrOH (9:1) (R = 1.24). The slower-elu-
ting (-)-enantiomer was assigned the natural S-configuration
on the basis of its conversion, via the (-)-enantiomer of 26, to
the relatively more cytotoxic (-)-enantiomer S-6 (Table 1).
This is consistent with related CBI analogues, where the
slower-eluting enantiomers are also the more potent com-
pounds.36
Preparation of Metal Complexes
Octahedral cobalt(III) complexes are relatively inert as a
result of the d6 low-spin electron configuration at the metal
center and ligand substitution reactions tend to be slow. This
necessitates careful design of the protocol for introducing
ligands into the coordination sphere. All of the pyrrolo[3,2-f]-
quinoline effectors are reactive in solution, undergoing spir-
ocyclization and eventual hydrolysis and decomposition, so
the substitution reaction at cobalt needs to be fast enough that
these reactions cannot compete effectively with coordination
to cobalt. Furthermore, 6 and its analogues are nontrivial to
prepare and so ideally should be introduced into the cobalt
complexes at the last step, with other ligands on cobalt already
in place.
A convenient starting material for the cobalt cyclen series of
complexes is [Co(cyclen)(NO2)2][NO2] (37), which has been
described in the literature.37 The nitro ligands may be repla-
ced by more labile triflato (OTf) ligands by reaction of
[Co(cyclen)(NO2)2]þ with neat triflic acid (HOTf) (Scheme 5).
The nitro groups are protonated and ultimately eliminated as
NOx gases, provided the reaction is carried out under anhy-
drous conditions to prevent coordination of aqua ligands.
Substitution of the weakly basic, labile triflato ligands by 6 is
achieved in acetonitrile or methanol, with iPr2NEt or pyridine
Acid deprotection of 24, followed by EDCI-mediated
coupling with 5,6,7-trimethoxy-, 5-methoxy-, and 5-[2-(di-
methylamino)ethoxy]indole-2-carboxylic acids gave amides
26-28, while coupling with the requisite cinnamic acid gave
29. The 5-benzyloxy intermediates 26-28 were debenzylated
with Pd-C and ammonium formate, and intermediate 29
with TFA, to give the target compounds 6 and 30-32
(Scheme 4). For 6 the alternative route of initial debenzylation
of 24 and coupling the resulting phenol 35 with TMI-2-
carboxylic acid gave a lower yield, but 33 and 34 were also

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6825
Table 1. Growth Inhibitory Properties of Pyrrolo[3,2-f]quinoline Analogues
IC50 (nM)c
IC50 (nM)c,
SKOVb
HCR,d
SKOVb
IC50 (nM), c
A549b
HCR,d
A549b
compd Fm sol.,a mM
R
AA8b
UV4b
EMT6 Vb
(()-6
R-(-)-6
S-(þ)-6
30
A
A
A
A
A
B
B
B
0.03
5,6,7-tri-OMe
5,6,7-tri-OMe
0.14 ( 0.02 0.07 ( 0.02 0.05 ( 0.01 0.26 ( 0.03 0.69 ( 08
22 ( 2 4.6 ( 0.5 7.3 ( 2.8 8.5 ( 0.3
0.07 ( 0.01 0.04 ( 0.01 0.03 ( 0.01 0.11 ( 0.02
0.06 ( 0.01 0.76 ( 0.21
8.8 ( 0.3
0.04 ( 0.01
5,6,7-tri-OMe
5-OMe
0.110
0.032
0.86
0.27 ( 0.10 0.91 ( 0.30 0.08 ( 0.02 1.51 ( 0.06
0.74 ( 0.22 6.9 ( 0.9 0.56 ( 0.10 1.3 ( 0.3 1.11 ( 0.44
31
32
5-O(CH2)2NMe2 4.4 ( 0.8
4-O(CH2)2NMe2 2.6 ( 0.2
4-OMe
1.7 ( 0.2
0.73 ( 0.14 0.58 ( 0.17 1.9 ( 0.3
0.29 ( 0.01
0.65 ( 0.21 0.92 ( 0.14 0.86 ( 0.25
33
34
0.04
0.11
0.09 ( 0.02
0.18 ( 0.03
3-OH, 4-OMe
0.17 ( 0.01
a Solubility in R-MEM þ 5% FCS. b Cell lines: AA8, Chinese hamster fibroblast; UV4, AA8 mutant defective in the incision step of excision repair;
EMT6V, murine breast carcinoma; SKOV3, human ovarian carcinoma; A549, human lung adenocarcinoma. c Concentration for 50% inhibition of cell
proliferation in aerobic log phase cultures following a drug exposure time of 4 h. Results are the mean ( SEM for >2 determinations. d Hypoxic
cytotoxicity ratio=(IC50[aerobic]/IC50[hypoxic]).
Scheme 5a
Scheme 6. Solvolysis of azaCBIs through Transient Two-
Coordinate Metal Complexes (from Reference 22)
added to deprotonate 6. The product [Co(cyclen)(6)]2þ (39)
can be isolated as the perchlorate salt and purified by reverse
phase HPLC. The complexes [Co(cyclen)(31)][OTf]2 (40) and
[Co(cyclen)(32)][OTf]2 (41) were prepared similarly from
[Co(cyclen)(OTf)2]þ, except an added base is not required
presumably because of the presence of the basic side chain on
the ligands.
We have previously developed a route using the binuclear
cobalt(III) precursor [Co2(Me2dtc)5]þ (42)38 for the prepara-
tion of cobalt bis(dithiocarbamate) complexes containing
reactive ligands.39 The binuclear complex efficiently cleaves
to give a neutral Co(Me2dtc)3 complex and a [Co(Me2dtc)2]þ
synthon which can coordinate the target ligand. Reaction of
[Co2(Me2dtc)5][BF4] with 6 proceeded smoothly with added
iPr2NEt as base (Scheme 5), and the product [Co(Me2dtc)2(6)]
(43) was purified by chromatography.
For all of the diamagnetic cobalt(III) effector complexes
coordinationof the effector could be confirmedbytheabsence
of a resonance due to theOHproton in the 1H NMR spectrum
of the complex. For all three effectors this proton appears as a
broad singlet close to 10.0 ppm in the spectrum of the free
ligand. Integration of the peaks arising from the effector
ligand in the complexes compared to those for the auxiliary
ligands confirmed the presence of one effector ligand per
complex.
Chromium(III) complexes also tend to be relatively inert,
and the same design strategy was employed as for cobalt. The
diaqua complex [Cr(acac)2(H2O)2]þ (44) is a suitable precur-
sor, as the aqua ligands are sufficiently labile.40 A mixture of
cis and trans [Cr(acac)2(H2O)2]ClO4 in dry acetonitrile reacts
a Reagents and conditions: (i) excess CF3SO3H, N2(g); (ii) 6 þ 1.5
i
equiv of Pr2NEt, 20 °C; or 6 þ pyridine, 50 °C; or 31, 20 °C; (iii) 32,
20 °C; (iv) 6 þ 2 equiv of iPr2NEt, 20 °C; (v) 6 þ 1.1 equiv of iPr2NEt,
50 °C.

6826 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Milbank et al.
Figure 1. (A, B) Stability of effectors in DMSO at room temperature and in RMEM plus 5% FCS at 37 °C, respectively: 6 (O); 31 (4); 32 (3).
Closed symbols are the corresponding spiro-cyclized products as illustrated with general structure I. (C, D) Stability of the corresponding metal
complexes in formate buffer at room temperature and in RMEM plus 5% FCS at 37 °C, respectively: 39 (O); 40 (4); 41(3). Closed symbols are
the corresponding hydrolysis products as illustrated with general structure II. Each symbol denotes the mean ( range for the duplicate
determinations, by HPLC.
i
with 6 and Pr2NEt to give [Cr(acac)2(6)] (45) which was
purified by flash chromatography on silica gel (50% MeCN/
CHCl3) in 75% yield (Scheme 5).
in turn slowly decomposed in culture medium (Figure 1B).
The complexes 39-41 weremorestable inboth formate buffer
and culture medium, being only slowly converted to the more
polar hydrolysis products (II) shown in Figure 1C,D. The
hydrolysis and spirocyclized products were identified spectro-
scopically and by analogy with previous work.22
Results and Discussion
Biological results for 6 (R, S, and racemate) and a small
series of racemic analogues 30-34 are recorded in Table 1.
While 6 is a very potent cytotoxin with IC50 in the nanomolar
range, it is considerably less cytotoxic than the reported values
for the CBI 719 and the related ester 8,22 at least with the
different drug exposure time and cell lines used (the natural
S-enantiomer of 8 has a reported IC50 of 0.021 nM against
L1210 murine leukemia cells in culture on 72 h of exposure).
The two enantiomers of 6 exhibited striking differences in
cytotoxicity, with the (þ)-enantiomer being on average 330-
fold more toxic than the (-)-enantiomer across the five cell
lines, this differential being larger than that reported for 7 and
its enantiomer (67-fold for the cyclopropyl forms) or for the
enantiomers of 8 (16-fold). The analogues with basic side
chains (31 and 32) were less potent than those with neutral
ones. The two different side chains (indole and cinnamate) did
not result in significant changes in potency (cf. 6 and 31-33).
The ability of 6 and 31-32 to inhibit the growth of hypoxic
cells was confirmed, as demonstrated by HCR values close to
unity (Table 1). The use of basic side chains in effectors 31 and
32 did not give marked increases in aqueous solubility.
The biological properties of the metal complexes are given
in Table 2. The cobalt bis(dithiocarbamate) complex 43
proved too insoluble to work with. The three cobalt/cyclen
complexes 39-41 showed significant attenuation of the cyto-
toxicity of the corresponding free effectors (prodrug/effector
[P/E] ratios of 50- to 150-fold), confirming stable complexes in
each case and demonstrating marked suppression of cytotoxi-
city in the prodrug form. They also showed significant
hypoxic cell selectivity (HCRs in the range of 20- to 40-fold,
although 7.5-fold for 39 in A549 cells). The aerobic toxicities
of these three complexes follow the order of toxicity of the
corresponding pyrrolo[3,2-f]quinoline effectors, suggesting
that they are governed significantly by slow release of the
effector under these conditions, as previously shown23 for 39.
A more extensive study of 39 in a range of cell lines showed
HCRs between 8- and 38-fold (Figure 2). The chromium/acac
complex 45 showed excellentattenuationof the cytotoxicity of
the free effector 6 (P/E ratio of ∼700-fold in both of the cell
lines studied) but showed no differential cytotoxicity between
aerobic and hypoxic cells (HCR 1- to 2-fold). Both of these
properties can likely be attributed to the very low reduction
potential of chromium complexes of this type (E1/2 values of
-211 and -1180 mV, respectively, for the cobalt/cyclen and
chromium/cyclen complexes of 8-HQ).17 This results in a very
The physiochemical properties of these effectors and their
cobalt/cyclen complexes are shown in Figure 1. The effectors
were relatively unstable, converting initially to the corresponding
spirocyclized compounds (I) as shown in Figure 1A,B. These

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6827
Table 2. Biological Properties of Metal Complexes
SKOV3a
A549b
IC50 (nM)d
IC50 (nM)d
G-values
compd
P/Ec
oxic
hypoxic
HCRe
18 ( 8
31 ( 2
26 ( 12
P/Ec
oxic
anoxic
HCRe
G(-P) f
0.68h
G(þE)g
39
40
41
45
152
84
38 ( 14
3.7 ( 1.5
20 ( 2
8.1 ( 5.5
230 ( 30
88
5.0 ( 0.9
130 ( 14
43 ( 3
1.2 ( 0.5
2.9 ( 0.7
2.4 ( 1.5
25 ( 4
7.5 ( 3.2
41 ( 13
35 ( 15
2.0 ( 0.8
0.57h
580 ( 120
100 ( 15
170 ( 30
100
46
54
680
0.61
0.54
1.30 ( 0.13
737
42 ( 14
a SKOV3 human ovarian carcinoma. b A549 human lung adenocarcinoma. c IC50(prodrug)/IC50(effector) under oxic conditions. d For log phase
cultures with drug exposure time of 4 h. e Hypoxic cytotoxicity ratio (oxic IC50/hypoxic IC50); these are intraexperiment ratios and thus may differ from
the ratios of the average oxic/hypoxic values in the table. f G value (μmol/J) for loss of prodrug (complex). g G value (μmol/J) for formation of effector
(both the cyclopropyl and seco forms were detected and were summed to give the total yield of effector). h Data from ref 23.
NADH or glutathione are not high enough to overcome the
low potential.
It was shown previously23 that on exposure to ionizing
radiation in formate buffer the cobalt cyclen complex 39 had a
G value for loss of the compound close to the theoretical value
for one-electron reduction (0.68 μmol/J). This is similar to the
value seen17 for the corresponding cobalt cyclen complex with
8-HQ. In the case of 39, both the cyclopropyl and seco forms
of the free ligand were detected after irradiation, but if these
were summed, the formation of free effector was close to
quantitative. HPLC analysis of irradiated formate solutions
of the cobalt cyclen complex 41 (of the “solubilized” effector
32) showed broadly similar reductive properties (Figure 3),
with a stoichiometry of reduction (G value for prodrug loss of
0.61 μmol/J), and efficient one-electron release on reduction
(G value for effector formation of 0.54 μmol/J, Table 2). This
extends the earlier observation23 of the suitability of the
cobalt/cyclen system for the radiolytic release of CBI-type
effectors.
Figure 2. Cytotoxicity of 39 under oxic and hypoxic conditions
across a human cell line panel, using log phase cultures and a drug
exposure time of 4 h. Results are the mean ( SEM for two to eight
determinations. The numbers above the bars are HCRs as defined in
Table 2. Cell lines are A375 melanoma, A549 NSCLC, C33A
cervical, H460 NSCLC, HCT116 colon, HT29 colon, PC3 prostate,
SiHa cervical, and SKOV3 ovarian.
Conclusions
The 1H-pyrrolo[3,2-f]quinoline analogues 6 and 30-34
prepared here retain the characteristic high and enantiomeri-
cally selective cellular potencies of the broad class of CBI
toxins. They form stable cobalt and chromium complexes
with a variety of ancillary ligands. The corresponding cobalt
cyclencomplexes 39-41weremarkedlylesscytotoxic thanthe
corresponding free effectors and also showed significant
hypoxic cell selective toxicity (7.7- to 40-fold), demonstrating
their utility as hypoxia-activated cytotoxins. Complexes 39
and 41 also showed efficient and close to quantitative release
oftheir effectors on exposuretoionizing radiation, supporting
previous work on the suitability of the cobalt cyclen 1H-
pyrrolo[3,2-f]quinoline complexes for the radiolytic release of
cytotoxins.
Experimental Section
All reagents used were of analytical grade. Analyses were
carried out in the Microchemical Laboratory, University of
Otago, Dunedin, New Zealand. Melting points were determined
on an Electrothermal 2300 melting point apparatus. NMR
spectra were obtained on a Bruker Avance 400 spectrometer at
1
400 MHz for H and 100 MHz for 13C spectra. Spectra were
obtained in (CD3)2SO unless otherwise specified and are refer-
enced to Me4Si. Chemical shifts and coupling constants were
recorded in units of ppm and Hz, respectively. Assignments were
determined using COSY, HSQC, and HMBC two-dimensional
experiments where appropriate. Mass spectra were determined
on a VG-70SE mass spectrometer using an ionizing potential of
70 eV at a nominal resolution of 1000. High-resolution spectra
were obtained at nominal resolutions of 3000, 5000, or 10000 as
appropriate. FABþ spectra used m-nitrobenzyl alcohol as the
Figure 3. Radiolytic reduction of 41 in deoxygenated formate
buffer (pH 7.0), showing the release of effector 32. An equal volume
of isopropanol was added immediately after the radiolysis, and 32
was quantitated as the sum of seco- and cyclopropyl forms.
stablecomplex45, with a very low rateof cellular reduction, as
the redox potentials of possible biological reductants such as

6828 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Milbank et al.
matrix and a xenon atom gun. Accurate mass calculations were
referenced to polyethylene glycol (PEG). Solutions in organic
solvents were dried with anhydrous Na2SO4. Solvents were
evaporated under reduced pressure on a rotary evaporator. Thin-
layer chromatography was carried out on aluminum-backed
silica gel plates (Merck 60 F254), with visualization of components
by UV light (254 nm) or exposure to I2. Column chromatography
was carried out on silica gel (Merck 230-400 mesh). DCM refers
to dichloromethane. DMSO refers to dimethyl sulfoxide. EtOAc
refers to ethyl acetate. MeOH refers to methanol, MeCN refers to
acetonitrile. Petroleum ether refers to petroleum ether, boiling
range 40-60 °C. All solvents were freshly distilled.
129.9, 128.4, 127.7, 127.2, 122.0, 105.8, 103.5, 80.6, 70.6, 28.2.
Anal. (C21H22N2O3) C, H, N.
8-Benzyloxy-6-(tert-butyloxycarbonylamino)-5-iodoquinoline
(15). A mixture of 14 (1.04 g, 3.0 mmol), NIS (0.70 g, 3.1 mmol),
and CH3CN (10 mL) was stirred at reflux for 30 min. Further
NIS (40 mg, 0.18 mmol) was added, and the mixture stirred at
reflux for a further 30 min. The CH3CN was evaporated, and the
residue was taken up in EtOAc (30 mL) and washed with a
solution of Na2S2O5 and Na2CO3 in water (ꢀ3). The aqueous
washes were back-extracted with EtOAc (ꢀ2). The combined
organic extracts were washed with water, dried (brine, MgSO4),
filtered through silica gel, and evaporated to give 15 (1.33 g,
93%), which crystallized from hexane as tan needles: mp
Scheme 1. 8-Hydroxy-6-nitroquinoline Hydrobromide (11).
A solution of 8-methoxy-6-nitroquinoline (10), prepared from
2-methoxy-4-nitroaniline (9) by the reported method.26 (500 g,
0.245 mol), in 48% aqueous HBr (0.205 L, 1.22 mol) was stirred
at reflux for 65 h. The mixture was cooled in ice, and the
precipitate was removed by filtration and dried in a desiccator
to give the hydrobromide salt of 11 (58.0 g, 87%): sublime 140 °C,
mp >230 °C; 1H NMR (CDCl3) δ 10.69 (br s, 2 H), 9.20 (dd, J=
4.9, 1.5 Hz, 1 H), 9.11 (dd, J=8.5, 1.5 Hz, 1 H), 8.64 (d, J=2.4 Hz,
1
118-119 °C; H NMR (CDCl3) δ 8.79 (dd, J = 4.2, 1.4 Hz,
1 H), 8.32 (dd, J=8.6, 1.4 Hz, 1 H), 8.29 (s, 1 H), 7.59 (dd, J=
8.0, 1.7 Hz, 2 H), 7.43 (dd, J=8.6, 4.2 Hz, 1 H), 7.25-7.39 (m,
3 H), 7.24 (br s, 1 H), 5.43 (s, 2 H), 1.57 (s, 9 H); 13C NMR
(CDCl3) δ 155.2, 152.4, 148.1, 139.5, 138.9, 138.3, 136.2, 130.7,
128.5, 128.0, 123.4, 103.9, 81.5, 78.1, 71.0, 28.3. Anal.
(C21H21IN2O3) C, H, N.
8-Benzyloxy-6-[N-(tert-butyloxycarbonyl)-N-(2-propenyl)amino]-
5-iodoquinoline (16). NaH (60% in oil, 0.185 g, 4.62 mmol) was
washed with pentane (2ꢀ2 mL) and then treated with a solution
of 14 (2.00 g, 4.20 mmol) in DMF (20 mL) over 5 min. After
30 min, the effervescence had ceased and the solution had become
deep-yellow. Allyl bromide (0.45 mL, 5.0 mmol) was added, and
the mixture was stirred for 3.25 h. The mixture was poured into
water (100 mL) and extracted with EtOAc (4ꢀ20 mL). The com-
bined extracts were washed with water (ꢀ2), dried (brine,
MgSO4), and evaporated. The residue was triturated with pentane
and the precipitate was collected by filtration and dried to give
1 H), 8.05 (dd, J=8.5, 4.9 Hz, 1 H), 7.90 (d, J=2.4 Hz, 1 H); 13
C
NMR (CDCl3) δ 152.0, 149.4, 146.4, 144.3, 135.4, 128.3, 124.1,
114.5, 106.5. Anal. (C9H6N2O3 HBr) C, H, N.
3
8-Benzyloxy-6-nitroquinoline (12). A mixture of 11 (58.0 g,
0.214 mol), DMF (400 mL), K2CO3 (103.5 g, 0.75 mmol), and
NaI (1.60 g, 10.7 mmol) was stirred at room temperature, while
benzyl bromide (25.4 mL, 0.214 mmol) was added in four
portions at half hourly intervals. A total of 9 h after the first
addition, the mixture was poured onto ice (1.5 kg) and the
precipitate was removed by filtration, washed with water, and
dried. The crude material was dissolved in DCM, and the
solution was filtered through alumina to give 12 (59.55 g,
99%): mp (EtOH) 152-153 °C; 1H NMR (CDCl3) δ 9.13 (dd,
J=4.2, 1.8 Hz, 1 H), 8.35 (d, J=2.3 Hz, 1 H), 8.29 (dd, J=8.4,
1.8 Hz, 1 H), 7.83 (d, J=2.3 Hz, 1 H), 7.59 (dd, J=8.4, 4.2 Hz,
1 H), 7.56 (d, J = 7.6 Hz, 2 H), 7.40 (dd, J = 7.6, 7.2 Hz, 2 H),
7.33 (t, J = 7.2 Hz, 1 H), 5.50 (s, 2 H); 13C NMR (CDCl3) δ
155.4, 152.5, 145.6, 142.6, 137.9, 135.4, 128.8, 128.4, 127.8,
127.5, 123.3, 116.3, 103.1, 71.4. Anal. (C16H12N2O3) C, H, N.
6-Amino-8-benzyloxyquinoline (13). Iron dust (16.0 g, 0.285
mol) was added to a solution of 12 (8.00 g, 28.5 mmol) and
AcOH (16 mL, 0.285 mol) in EtOH-water (5:1, 240 mL) at
reflux. After 10 min, the mixture was carefully poured into
saturated aqueous NaHCO3 (300 mL). The mixture was filtered
through Celite, and the filter cake was washed with water (100 mL),
EtOH (3ꢀ50 mL), and DCM (3ꢀ100 mL). The combined filt-
rates were diluted with water (300 mL), and the aqueous layer
was separated and extracted with DCM (2ꢀ50 mL). The com-
bined extracts were washed with water, dried (Na2SO4), and eva-
porated to give 13 (7.13 g, 100%) as a tan solid: mp 183-185 °C;
1H NMR (CDCl3) δ 8.66 (dd, J=4.2, 1.6 Hz, 1 H), 7.84 (dd, J=
8.3, 1.6 Hz, 1 H), 7.48 (dd, J=8.1, 1.7 Hz, 2 H), 7.23-7.39 (m,
3 H), 7.28 (dd, J=8.3, 4.2 Hz, 1 H), 6.51, 6.48 (2ꢀd, J=2.3 Hz,
2 H), 5.36 (s, 2 H), 3.85 (br s, 2 H); 13C NMR (CDCl3) δ 155.2,
155.7, 144.8, 136.8, 135.9, 133.5, 130.8, 128.6, 127.8, 127.0,
122.0, 102.6, 100.0, 70.6. Anal. (C16H14N2O) C, H, N.
1
16 (2.02 g, 93%) as a cream solid: mp 121-122 °C; H NMR
(CDCl3) major rotamer δ 8.94 (dd, J = 4.1, 1.3 Hz, 1 H), 8.50
(dd, J=8.6, 1.3 Hz, 1 H), 7.52 (dd, J=8.6, 4.1 Hz, 1 H), 7.48 (d,
J = 7.3 Hz, 2 H), 7.35 (dd, J = 7.3, 7.2 Hz, 2 H), 7.30 (t, J =
7.2 Hz, 1 H), 6.83 (br s, 1 H), 5.77 (dddd, J=16.8, 10.4, 7.2, 5.7 Hz,
1 H), 5.49, 5.41 (2ꢀd, J=13.4 Hz, 1 H each), 4.92 (d, J=10.4 Hz,
1 H), 4.87 (d, J=16.8 Hz, 1 H), 4.45 (dd, J=15.0, 5.7 Hz, 1 H),
3.78 (dd, J=15.0, 7.2 Hz, 1 H), 1.26 (s, 9 H); 13C NMR (CDCl3)
major rotamer δ 154.5, 153.5, 150.0, 143.4, 141.1, 140.0, 136.2,
133.0, 131.1, 128.7, 128.0, 126.9, 123.3, 118.3, 112.7, 93.6, 80.6,
70.9, 51.8, 28.2. Anal. (C24H25IN2O3) C, H, N, I.
5-Benzyloxy-3-(tert-butyloxycarbonyl)-1-[(2,2,6,6-tetramethyl-
piperidin-1-yl)oxymethyl]-2,3-dihydro-1H-pyrrolo[3,2-f]quinoline (17).
A solution of 16 (1.03 g, 1.99 mmol) and 2,2,6,6-tetramethylpi-
peridinyloxyl (1.56 g, 10.0 mmol) in benzene (60 mL) under
nitrogen at reflux was treated with Bu3SnH (2.14 mL, 8.0 mmol)
over 1.75 h. The mixture was heated at reflux for a further 1.25 h
before the solvent was evaporated. The residue was taken up in
Et2O and extracted with 0.2 M HCl (ꢀ8). The combined extracts
were washed with Et2O, neutralized with NaHCO3, and ex-
tracted with DCM (ꢀ3). These extracts were dried (brine,
MgSO4), evaporated, and triturated with pentane. The precipi-
tate was collected by filtration, washed with pentane, and dried
to give 17 (0.786 g, 72%) as cream crystals: mp 162-164 °C; 1H
NMR (CDCl3) δ 8.79 (dd, J=4.1, 1.4 Hz, 1 H), 8.10 (dd, J=8.4,
1.4 Hz, 1 H), 8.06 (br s, 1 H), 7.56 (br s, 2 H), 7.33-7.40 (m, 3 H),
7.30 (t, J=7.3 Hz, 1 H), 5.43, 5.38 (2ꢀd, J=12.3 Hz, 1 H each),
4.17 (dd, J=11.2, 2.2 Hz, 1 H), 4.06 (dd, J=11.2, 8.9 Hz, 1 H),
3.97 (dd, J = 8.4, 5.8 Hz, 1 H), 3.84 (dd, J = 8.4, 7.2 Hz, 1 H),
3.76 (dddd, J=8.9, 7.2, 5.8, 2.2 Hz, 1 H), 1.55 (s, 9 H), 1.20-1.50
(m, 6 H), 1.08 (s, 3 H), 1.04 (s, 3 H), 0.99 (s, 6 H); 13C NMR
(CDCl3) δ 154.9, 152.4, 146.9, 141.5 (br), 137.3, 136.6, 131.3,
128.5, 127.9, 127.8, 126.1, 121.7, 115.5 (v br), 100.4 (br), 80.8
(br), 78.5, 70.7, 59.8, 52.7, 39.6, 37.8 (br), 33.0, 28.4, 20.2, 17.0.
Anal. (C33H43N3O4) C, H, N.
8-Benzyloxy-6-(tert-butyloxycarbonylamino)quinoline (14). A
mixture of 13 (7.63 g, 30.5 mmol), BOC2O (8.65 g, 39.6 mmol),
and dioxane (70 mL) was stirred at reflux for 2 h. Further
BOC2O (0.86 g, 4.0 mmol) was added, and the mixture was
heated at reflux for another 1 h. The dioxane was evaporated,
the remaining oil was triturated with pentane, and the resulting
solid was removed by filtration, dissolved in DCM, and filtered
through alumina to give 14 (10.42 g, 98%) as a cream solid: mp
180-181 °C; 1H NMR (CDCl3) δ 8.77 (dd, J=4.2, 1.6 Hz, 1 H),
7.98 (dd, J=8.3, 1.6 Hz, 1 H), 7.55 (d, J=2.1 Hz, 1 H), 7.41 (dd,
J=7.4, 2.2 Hz, 2 H), 7.34 (dd, J=8.3, 4.2 Hz, 1 H), 7.20-7.29
Scheme 2. 8-Benzyloxy-6-[N-(tert-butyloxycarbonyl)-N-(3,3-
dimethoxypropyl)amino]-5-iodoquinoline (18). NaH (60% in oil,
92 mg, 2.3 mmol) under nitrogen was washed with pentane (2ꢀ
2 mL), cooled (ice-water), and treated with a solution of 15
(m, 3 H), 7.02 (d, J=2.1 Hz, 1 H), 5.28 (s, 2 H), 1.49 (s, 9 H); 13
NMR (CDCl3) δ 154.6, 152.7, 147.4, 137.2, 136.8, 136.3, 135.2,
C

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6829
(1.00 g, 2.10 mmol) in DMF (10 mL) over 5 min. The mixture
was allowed to warm to room temperature and stirred for 30 min,
over which time it became bright-yellow and effervescence
ceased. A solution of 3-bromo-1,1-dimethoxypropane (0.69 g,
3.77 mmol) in DMF (0.5 mL) was added, and the mixture was
stirred at room temperature for 22 h. The mixture was poured
into pH 7.4 phosphate buffer (50 mL) and extracted with EtOAc
(3ꢀ20 mL). The combined extracts were washed with water (2ꢀ
50 mL), dried (brine, Na2SO4), evaporated, and purified by dry
flash column chromatography (SiO2, 10-90% EtOAc/hexane)
to give 18 (1.00 g, 83%) as a cream powder: mp 120-121 °C; 1H
NMR (CDCl3) major rotamer δ 8.94 (br d, J=2.9 Hz, 1 H), 8.52
(dd, J = 8.6, 1.5 Hz, 1 H), 7.45-7.58 (m, 3 H), 7.25-7.40 (m,
3 H), 6.96 (br s, 1 H), 5.46 (s, 2 H), 4.40 (t, J=4.7 Hz, 1 H), 3.84
(br ddd, J=14.6, 7.3, 7.3 Hz, 1 H), 3.33 (ddd, J=14.6, 8.2, 5.8
Hz, 1 H), 3.28, 3.25 (2ꢀs, 3 H each), 1.65-1.95 (m, 2 H), 1.23 (br
s, 9 H); 13C NMR (CDCl3) major rotamer δ 154.6, 153.6, 149.9,
143.8, 141.3, 139.8, 136.0, 131.2, 128.7, 128.0, 127.0, 123.4,
112.3, 102.9, 93.3, 80.3, 70.9, 53.1, 52.7, 45.4, 31.2, 28.1. HRMS:
C26H31IN2O5 requires Mþ• 578.1278. Found 578.1257.
collected by filtration to give 21 (0.32 g, 77%), which crystallized
from MeOH as fluorescent pale-yellow rectangular plates: mp
172-173 °C; 1H NMR (CDCl3) δ 8.82 (dd, J=4.1, 1.4 Hz, 1 H),
8.14 (dd, J=8.4, 1.4 Hz, 1 H), 8.07 (br s, 1 H), 7.55 (br s, 2 H),
7.41 (dd, J = 8.4, 4.1 Hz, 1 H), 7.36 (dd, J = 7.3, 7.3 Hz, 2 H),
7.30 (tt, J=7.3, 2.4 Hz, 1 H), 5.44, 5.39 (2ꢀd, J=12.5 Hz, 1 H
each), 4.42-4.52 (m, 1 H), 4.05-4.14 (m, 2 H), 3.82-3.93 (m,
2 H), 2.08 (s, 3 H), 1.57 (s, 9 H); 13C NMR (CDCl3) δ 171.0,
155.2, 152.3, 146.9, 142.0 (br), 137.0, 136.3, 131.1, 128.5, 127.9,
127.7, 126.0, 122.1, 113.3 (v br), 100.4 (br), 81.4 (br), 70.7, 65.8,
52.6, 37.7, 28.4, 20.9. Anal. (C26H28N2O5) C, H, N.
5-Benzyloxy-3-(tert-butyloxycarbonyl)-1-(hydroxymethyl)-
2,3-dihydro-1H-pyrrolo[3,2-f]quinoline (22). A mixture of 21
(0.22 g, 0.50 mmol), Cs2CO3 (0.42 g, 1.29 mmol), and EtOH-
water (2:1, 6 mL) was stirred at reflux for 30 min. The mixture
was diluted with EtOAc (30 mL) and dilute aqueous NaHCO3
(50 mL), and the separated aqueous phase was extracted with
EtOAc (30 mL). The combined extracts were washed with water
(3 ꢀ 50 mL), dried (brine, Na2SO4), and evaporated to give 22
(0.19 g, 95%), which crystallized from MeOH as tiny white
needles: mp 156-157 °C; 1H NMR (CDCl3) δ 8.54 (br s, 1 H),
7.99 (br d, J=8.0 Hz, 1 H), 7.91 (br s, 1 H), 7.55 (d, J=6.6 Hz,
2 H), 7.20-7.40 (m, 4 H), 5.29 (s, 2 H), 4.00-4.22 (m, 2 H),
3.65-3.78 (m, 3 H, H-1), 3.23 (br s, 1 H), 1.56 (s, 9 H); 13C NMR
(CDCl3) δ 154.4, 152.5 (br), 146.2 (br), 142.2 (v br), 136.3, 136.2,
131.3, 128.5, 128.0 (v br), 127.9, 125.9, 121.6, 114.7 (v br), 100.4
(br), 81.0 (br), 70.7, 64.6, 52.3, 40.9 (br), 28.4. Anal.
8-Benzyloxy-6-[N-(tert-butyloxycarbonyl)-N-(3-oxopropyl)-
amino]-5-iodoquinoline (19). A solution of 18 (0.75 g, 1.30 mmol),
TsOH H2O (0.12 g, 0.65 mmol), and water (3.75 mL) in acetone
3
(38 mL) was stirred at reflux for 2.25 h. Most of the acetone was
evaporated, and the residue was diluted with water (50 mL) and
saturated aqueous NaHCO3 (5mL) andextractedwithEtOAc (3ꢀ
20 mL). The combined extracts were washed with water (2 ꢀ
50 mL), dried (Na2SO4), and evaporated to give 19 (0.68g, 99%) as
a pale-yellow foam: 1H NMR (CDCl3) major rotamer δ 9.68 (s,
1 H), 8.97 (dd, J=4.2, 1.5 Hz, 1 H), 8.51 (dd, J=8.6, 1.5 Hz, 1 H),
7.53 (dd, J=8.6, 4.2 Hz, 1 H), 7.47-7.55 (m, 2 H), 7.25-7.40 (m,
3 H), 6.87 (br s, 1 H), 5.49 (s, 2 H), 4.17 (br dt, J= 14.5, 7.1 Hz,
1 H), 3.59 (dt, J=14.5, 6.5 Hz, 1 H), 2.57 (br dd, J=7.1, 6.5 Hz,
2 H), 1.23 (s, 9 H); 13C NMR (CDCl3) major rotamer δ 200.3,
154.8, 153.4, 150.0, 143.0, 141.0, 139.7, 135.9, 131.0, 128.6, 127.9,
127.0, 123.4, 112.1, 93.1, 80.7, 70.7, 42.9, 42.5, 27.9. HRMS:
C24H25IN2O4 requires Mþ• 532.0859. Found 532.0862.
(C24H26N2O4 H2O) C, H, N.
3
5-Benzyloxy-1-(methylsulfonyloxymethyl)-3-(tert-butyloxy-
carbonyl)-2,3-dihydro-1H-pyrrolo[3,2-f]quinoline (23). MsCl (0.06
mL, 0.7 mmol) was added to a cooled (ice-water) solution of 22
(0.17 g, 0.41 mmol) and Et3N (0.2 mL, 1.4 mmol) in DCM (3 mL),
and the mixture was stirred for 30 min. The DCM was evaporated,
and the residue was stirred with water (25 mL) for 10 min. The
mixture was extracted with EtOAc (2 ꢀ 25 mL). The combined
extracts were washed with water (2ꢀ50 mL), dried (Na2SO4), and
evaporated to give 23 (0.17 g, 86%), which crystallized from
MeOH as tiny cream needles: mp 156-157 °C; 1H NMR
(CDCl3) δ 8.80 (dd, J = 4.2, 1.4 Hz, 1 H), 8.02 (dd, J = 8.7, 1.4
Hz, 1 H), 7.97 (br s, 1 H), 7.55 (br d, J=6.9 Hz, 2 H), 7.41 (dd, J=
8.7, 4.2 Hz), 7.25-7.38 (m, 3 H), 5.40 (s, 2 H), 4.46 (dd, J=9.8, 3.7
Hz, 1 H), 3.93-4.24 (m, 4 H), 2.90 (s, 3 H), 1.57 (s, 9 H); 13C NMR
(CDCl3) δ 155.6, 152.1, 147.0, 141.0 (v br), 137.1, 136.1, 130.5,
128.4, 127.9, 127.6 (br), 125.7, 122.3, 112.7 (v br), 100.3, 81.6 (br),
70.7, 69.9, 52.0, 38.2 (br), 37.4, 28.3. Anal. (C25H28N2O6S) C,
H, N, S.
6-[N-(3-Acetoxy-2-propenyl)-N-(tert-butyloxycarbonyl)amino]-
8-benzyloxy-5-iodoquinoline (20). A mixture of 19 (0.62 g, 1.16
mmol), Et3N (0.40 mL, 2.87 mmol), Ac2O (0.25 mL, 2.65 mmol),
DMAP (14 mg, 0.11 mmol), and THF (12 mL) was stirred at
reflux for 2 h. Further Et3N (0.80 mL, 5.74 mmol), Ac2O (0.50 mL,
5.3 mmol), and DMAP (10 mg, 0.08 mmol) were added, and
heating was continued for a further 2 h. The solvent was
evaporated, and the residue was diluted with pH 7.4 phosphate
buffer (50 mL) and extracted with EtOAc (3 ꢀ 20 mL). The
combined extracts were washed with water (50 mL), dilute
aqueous NaHCO3 (50 mL), and water (50 mL) before being
dried (brine, Na2SO4), and evaporated. The residue was purified
by dry flash column chromatography (SiO2, 10-80% EtOAc-
hexane) to give 20 (0.54 g, 81%) as a white foam, which
contained a 1:4 mixture of Z and E isomers: 1H NMR
(CDCl3) major rotamer δ 8.94 (br s, 1 H), 7.45-7.55 (m, 3 H),
7.27-7.40 (m, 3 H), 6.84-7.12 (m, 2 H), 5.36-5.58 (m, 2.8 H),
4.91 (ddd, J=7.6, 6.5, 5.9 Hz, 0.2 H), 4.57 (dd, J=15.0, 5.9 Hz,
0.2 H), 4.39 (dd, J = 14.7, 6.8 Hz, 0.8 H), 4.06 (dd, J = 15.0,
7.6 Hz, 0.2 H), 3.86 (dd, J=14.7, 7.9 Hz, 0.8 H), 2.08 (s, 2.4 H),
1.88 (s, 0.6 H), 1.57 (br s, 1.8 H), 1.26 (br s, 7.2 H); 13C NMR
(CDCl3) major rotamer δ 167.4, 167.0, 154.5, 149.8, 154.3,
149.8, 153.3, 153.1, 142.8, 140.9, 139.7, 138.8, 139.7, 143.1,
135.8, 130.9, 136.0, 127.8, 126.8, 126.7, 128.4, 123.2, 112.1,
112.0, 109.1, 108.2, 93.5, 93.1, 80.9, 80.4, 70.8, 70.7, 46.4, 42.7,
27.9, 28.1, 20.3, 20.1. HRMS: C26H27IN2O5 requires Mþ•
574.0965. Found 574.0962.
5-Benzyloxy-3-(tert-butyloxycarbonyl)-1-(chloromethyl)-2,3-
dihydro-1H-pyrrolo[3,2-f]quinoline (24). Method 1. A mixture
of 23 (50 mg, 0.10 mmol), LiCl (25 mg, 0.59 mmol), and DMF
(0.25 mL) was stirred at 80 °C for 1 h before ice (3 g) was added.
The precipitate was removed by filtration, washed with water,
and taken up in EtOAc (20 mL). This solution was washed
with water (20 mL), dried (Na2SO4), and evaporated to give 24
(39 mg, 89%), which crystallized from MeOH as fluorescent
cream needles: mp 178-179 °C; 1H NMR (CDCl3) δ 8.82 (dd,
J=4.2, 1.5 Hz, 1 H), 8.05 (br s, 1 H), 7.99 (br d, J=8.4 Hz, 1 H),
7.55 (br s, 2 H), 7.41 (dd, J=8.4, 4.2 Hz, 1 H), 7.35 (dd, J=7.3,
7.3 Hz, 2 H), 7.30 (tt, J=7.3, 2.4 Hz, 1 H), 5.42, 5.38 (2ꢀd, J=
12.4 Hz, 1 H each), 4.23 (br d, J=11.7 Hz, 1 H), 4.12 (dd, J =
11.7, 8.9 Hz, 1 H), 3.92 (dddd, J = 10.1, 8.9, 3.2, 2.6 Hz, 1 H),
3.81 (dd, J=11.1, 3.2 Hz, 1 H), 3.45 (dd, J=11.1, 10.1 Hz, 1 H),
1.56 (s, 9 H); 13C NMR (CDCl3) δ 155.5, 152.3, 146.9, 141.9 (br),
137.1, 136.3, 130.3, 128.5, 127.9, 127.7 (br), 125.6, 122.2, 113.4
(v br), 100.4 (br), 81.6 (br), 70.8, 53.0, 46.3, 41.1, 28.4. Anal.
(C24H25ClN2O3) C, H, N, Cl.
1-(Acetoxymethyl)-5-benzyloxy-3-(tert-butyloxycarbonyl)-
2,3-dihydro-1H-pyrrolo[3,2-f]quinoline (21). A solution of 20
(0.54 g, 0.94 mmol), AIBN (15 mg, 0.09 mmol), and Bu3SnH
(0.32 g, 1.13 mmol) in benzene (45 mL) was stirred at reflux
under nitrogen for 5.5 h. The solvent was evaporated, the
residue was triturated with pentane, and the precipitate was
Method 2. CCl4 (0.05 mL, 0.52 mmol) was added to a mixture
of 22 (19 mg, 0.047 mmol), Ph3P (37 mg, 0.14 mmol), and DCM
(0.4 mL), and the mixture was stirred under nitrogen for 4 h. The
mixture was diluted with dilute aqueous NaHCO3 (5 mL) and
extracted with EtOAc (3 ꢀ 5 mL). The combined extracts were

6830 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Milbank et al.
dried (Na2SO4), evaporated, and purified by dry flash column
chromatography (SiO2, 10-90% EtOAc/hexane) to give 24 (20 mg,
100%) identical with the material prepared above.
560.1766. Found (FAB) 558.1770, 560.1786. Anal. (C31H28-
ClN3O5) C, H, N.
THF (10 mL) and then 25% aqueous HCO2NH4 (1.1 mL)
were added to a cooled (ice-water) mixture of 26 (0.25 g, 0.45
mmol) and 10% Pd/C (0.13 g) under nitrogen. The mixture was
stirred at 0 °C for 7.5 h and was then filtered through Celite. The
Celite was washed with a solution of concentrated HCl (2 mL)
and MeOH (40 mL) and then with DCM-MeOH (3:1, 40 mL).
The combined filtrates were diluted with water (40 mL) and
DCM (30 mL) and neutralized with pH 7.4 phosphate buffer.
The lower layer was separated and then diluted with MeOH
(20 mL) and warmed to dissolve the suspended solid. The
aqueous phase was extracted with DCM (2 ꢀ 20 mL). The
extracts were combined, washed with water (100 mL), dried
(Na2SO4), and concentrated to a volume of 20 mL. The con-
centrate was diluted with MeOH (20 mL) and was concentrated
to a volume of 10 mL. The precipitate was removed by filtration
and washed with MeOH to give 6 (0.14 g, 66%) as a pale-yellow
Scheme 3. 8-Benzyloxy-6-[N-(tert-butyloxycarbonyl)-N-(3-
chloro-2-propenyl)amino]-5-iodoquinoline (25). NaH (60% dis-
persion in oil, 0.26 g, 6.5 mmol) under nitrogen was washed with
pentane (3 ꢀ 2 mL), cooled (ice-water), and treated with a
solution of 15 (2.80 g, 5.88 mmol) in DMF (28 mL) over 5 min.
The cooling bath was removed and the mixture was allowed to
stir for 30 min, by which time the solution was deep-yellow
and effervescence had ceased. 1,3-Dichloropropene (0.98 g, 8.82
mmol) was added, and the mixture was stirred for 86 h. The
mixture was diluted with water (150 mL) and extracted with
EtOAc (4 ꢀ 25 mL). The combined extracts were washed with
water (3ꢀ100 mL), dried (brine, Na2SO4), and evaporated. The
residue was triturated with pentane and the precipitate was
collected by filtration to give 25 (3.02 g, 93%) as a tan powder:
mp 115-135 °C consisting of a 1:1 mixture of Z and E isomers;
1H NMR (CDCl3) major rotamer δ 8.95 (br s, 1 H), 8.50 (dd, J=
8.4, 2.5 Hz, 1 H), 7.46-7.55 (m, 3 H), 7.27-7.41 (m, 3 H),
6.79-6.96 (m, 1 H), 5.30-6.03 (m, 4 H), 4.54 (dd, J=15.5, 5.6
Hz, 0.5 H), 4.38 (dd, J=14.8, 6.8 Hz, 0.5 H), 4.18 (dd, J=15.5,
6.9 Hz, 0.5 H), 3.79 (dd, J=14.8, 7.8 Hz, 0.5 H), 1.23-1.82 (m, 9
H); 13C NMR (CDCl3) major rotamer δ 154.7, 155.2, 153.6,
153.3, 150.2, 150.1, 143.2, 142.8, 141.2, 140.2, 136.2, 136.0,
131.13, 131.08, 128.79, 128.73, 128.12, 127.99, 127.2, 126.6,
126.98, 126.90, 123.5, 123.4, 122.0, 121.1, 112.2, 111.9, 93.65,
93.58, 80.90, 80.85, 71.0, 70.9, 48.8, 45.4, 28.4, 28.1. HRMS:
C24H24ClIN2O3 requires Mþ• 550.0520, 552.0491. Found
550.0536, 552.0503. Purification of the mother liquors by dry
flash column chromatography (silica gel, 10-60% EtOAc-
hexane) gave further 25 (0.14 g, 4%).
1
microcrystalline solid: mp >230 °C; H NMR [(CD3)2SO] δ
11.50 (d, J=2.1 Hz, 1 H), 10.03 (br s, 1 H), 8.76 (dd, J=4.1, 1.3
Hz, 1 H), 8.40 (dd, J=8.4, 1.3 Hz, 1 H), 7.97 (s, 1 H), 7.56 (dd,
J=8.4, 4.1 Hz, 1 H), 7.09 (d, J=2.1 Hz, 1 H), 6.97 (s, 1 H), 4.77
(dd, J=11.0, 9.3 Hz, 1 H), 4.48 (dd, J=11.0, 2.0 Hz, 1 H), 4.25
(dddd, J=9.3, 3.9, 3.3, 2.0 Hz, 1 H), 4.03 (dd, J=10.6, 3.3 Hz,
1 H), 3.93, 3.82, 3.80 (3ꢀs, 3 H each), 3.89 (dd, J=10.6, 3.9 Hz,
1 H); 13C NMR ((CD3)2SO) δ 160.3, 153.9, 146.3, 149.1, 142.7,
139.9, 139.0, 136.0, 130.7, 125.4, 124.8, 123.1, 131.6, 122.4,
114.6, 106.2, 102.8, 98.0, 61.0, 60.9, 55.9, 55.0, 47.6, 40.5. Anal.
(C24H22ClN3O5) C, H, N.
1-(Chloromethyl)-3-[(5-methoxy-1H-indol-2-yl)carbonyl]-2,3-
dihydro-1H-pyrrolo[3,2-f]quinolin-5-ol (30). A suspension of 24
(0.10 g, 0.24 mmol) in dioxane (15 mL) was saturated with HCl,
stirred for 5 h, and evaporated. 5-Methoxy-1-H-indole-2-car-
boxylic acid (0.054 g, 0.28 mmol), EDCI (0.23 g, 1.17 mmol),
and DMA (5 mL) were added to the remaining yellow solid, and
the red mixture was stirred for 52 h. The mixture was partitioned
between DCM and cold 5% KHCO3 solution. The aqueous
layer was extracted with DCM (ꢀ3). The organic extracts were
dried (brine, Na2SO4). Flash chromatography (EtOAc/petroleum
ether 7:3) gave 5-(benzyloxy)-1-(chloromethyl)-3-[(5-methoxy-
1H-indol-2-yl)carbonyl]-2,3-dihydro-1H-pyrrolo[3,2-f]quinoline
(27) (0.11 g, 98%) as a yellow solid: mp 186-189 °C; 1H NMR
(CDCl3) δ 9.55 (s, 1 H), 8.88 (dd, J=4.2, 1.7 Hz, 1 H), 8.37 (s,
1 H), 7.99 (dd, J =8.3, 1.6 Hz, 1 H), 7.56 (d, J =7.3 Hz, 2 H),
7.42 (dd, J=8.3, 4.1 Hz, 1 H), 7.33 (m, 4 H), 7.10 (d, J=2.3 Hz,
1 H), 6.99 (m, 2 H), 5.48 (d, J=12.5 hz, 1 H), 5.42 (d, J=12.6
Hz, 1 H), 4.74 (dd, J=10.9, 2.0 Hz, 1 H), 4.61 (dd, J=10.6, 8.7
Hz, 1 H), 4.05 (m, 1 H), 3.85 (s, 3 H), 3.84 (dd, J=11.2, 4.1 Hz,
1 H), 3.45 (dd, J = 11.0, 10.5 Hz, 1 H); 13C NMR (CDCl3) δ
160.7, 155.4, 154.7, 147.9, 142.4, 138.4, 136.4, 131.4, 130.5,
130.2, 128.6, 128.2, 128.0, 127.7, 125.2, 122.4, 117.0, 115.4,
112.7, 106.2, 102.5, 102.4, 70.9, 55.7, 55.2, 45.9, 42.6. Anal.
(C29H24ClN3O3) C, H, N.
THF (6 mL) and then HCO2NH4 (0.14 g, 2.21 mmol) in H2O
(0.7 mL) were added to a cooled (0 °C) mixture of 27 (0.11 g, 0.22
mmol) and 10% Pd/C (0.05 g) under N2. The mixture was stirred
at 0 °C for 5 h and was then filtered through Celite. The Celite
was washed with DCM and water. The aqueous layer was
extracted with DCM (ꢀ3). The organic extracts were dried
(brine, Na2SO4) and evaporated. Precipitation of the residue
from DCM/MeOH gave 30 (0.077 g, 89%) as a gray solid: mp
224-227 °C; 1H NMR [(CD3)2SO] δ 11.66 (s, 1 H), 10.02 (br s, 1
H), 8.77 (dd, J=4.1, 1.3 Hz, 1 H), 8.41 (dd, J=8.4, 1.4 Hz, 1 H),
8.07 (s, 1 H), 7.57 (dd, J=8.4, 4.1 Hz, 1 H), 7.40 (d, J=9.0 Hz, 1
H), 7.16 (d, J=2.4 Hz, 1 H), 7.12 (d, J=1.6 Hz, 1 H), 6.92 (dd,
J=8.9, 2.3 Hz, 1 H), 4.82 (dd, J=10.8, 9.4 Hz, 1 H), 4.57 (dd,
J=11.0, 2.3 Hz, 1 H), 4.30 (m, 1 H), 4.04 (dd, J=11.1, 3.3 Hz,
1 H), 3.91 (dd, J=11.1, 7.2 Hz, 1 H), 3.78 (s, 3 H). HRMS FAB
[MþH] calcd for C22H1935ClN3O3=408.1115. Found, 408.1101.
Calcd for C22H1937ClN3O3 =410.1085. Found, 410.1092.
5-Benzyloxy-3-(tert-butyloxycarbonyl)-1-(chloromethyl)-2,3-
dihydro-1H-pyrrolo[3,2-f]quinoline (24). A solution of 25 (3.00 g,
5.45 mmol), AIBN (89 mg, 0.54 mmol), and Bu3SnH (1.75 g,
6.0 mmol) in benzene (270 mL) was heated at reflux under
nitrogen for 3 h. The benzene was evaporated, the residue was
triturated with pentane, and the precipitate was collected by
filtration to give 24 (2.21 g, 95%), identical with the material
prepared above.
Scheme 4. 1-(Chloromethyl)-5-hydroxy-3-[(5,6,7-trimethox-
yindol-2-yl)carbonyl]-2,3-dihydro-1H-pyrrolo[3,2-f]quinoline (6).
A suspension of 24 (0.65 g, 1.53 mmol) in dioxane (40 mL)
was saturated with HCl, allowed to stand for 1 h, and evapo-
rated. 5,6,7-Trimethoxyindole-2-carboxylic acid (0.38 g, 1.53 mmol),
EDCI (0.88 g, 4.6 mmol), and DMA (25 mL) were added to the
remaining green-yellow solid, and the red mixture was stirred at
room temperature for 39 h. The mixture was poured into a
mixture of ice (60 g) and pH 7.4 phosphate buffer (60 mL). The
precipitate was removed by filtration, washed with water, and
taken up in EtOAc (60 mL). This solution was washed with
water (3ꢀ50 mL), dried (brine, Na2SO4), and evaporated. The
remaining oil was triturated with Et2O. The precipitate was
collected by filtration, purified by flash column chromato-
graphy (silica gel, EtOAc), and triturated with Et2O to give
5-benzyloxy-1-(chloromethyl)-3-[(5,6,7-trimethoxyindol-2-yl)-
carbonyl]-2,3-dihydro-1H-pyrrolo[3,2-f]quinoline (26) (0.38 g,
44%) as a pale-yellow solid: mp 182-184 °C; 1H NMR (CDCl3)
δ 9.59 (s, 1 H), 8.84 (dd, J=4.2, 1.6 Hz, 1 H), 8.37 (s, 1 H), 7.95
(dd, J=8.5, 1.6 Hz, 1 H), 7.58 (br d, J=7.2 Hz, 2 H), 7.38 (dd,
J=8.5, 4.2 Hz, 1 H), 7.36 (dd, J=7.3, 7.2 Hz, 2 H), 7.30 (t, J=
7.3 Hz, 1 H), 6.93 (d, J=2.2 Hz, 1 H), 6.84 (s, 1 H), 5.48, 5.42 (2ꢀ
d, J=12.5 Hz, 1 H each), 4.69 (dd, J=10.8, 1.9 Hz, 1 H), 4.57
(dd, J = 10.8, 8.5 Hz, 1 H), 4.06, 3.93, 3.90 (3 ꢀ s, 3 H each),
4.02 (dddd, J = 10.3, 8.5, 3.2, 1.9 Hz, 1 H), 3.83 (dd, J = 11.4,
3.2 Hz, 1 H), 3.42 (dd, J = 11.4, 10.3 Hz, 1 H); 13C NMR
(CDCl3) δ 160.5, 155.3, 147.8, 150.2, 142.3, 140.6, 138.8,
138.2, 129.5, 125.1, 123.5, 136.4, 130.4, 128.6, 128.0, 127.7,
125.6, 122.3, 115.3, 106.7, 102.3, 97.6, 70.8, 61.4, 61.1, 56.2,
55.1, 45.9, 42.5. C31H28ClN3O5 requires M þ H 558.1796,

Article
1-(Chloromethyl)-3-({5-[2-(dimethylamino)ethoxy]-5-hydroxy-
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6831
and the residue was partitioned between DCM and cold 5%
NaHCO3 solution. The aqueous layer was extracted with DCM
(ꢀ3). The DCM extracts were dried (brine, Na2SO4). Flash
chromatography (DCM/MeOH/NH3, 95:5:trace) gave 32 (0.16 g,
34%): mp 174-180 °C; 1H NMR [(CD3)2SO] δ 9.96 (br s, 1 H),
8.73 (dd, J=4.0, 1.3 Hz, 1 H), 8.36 (dd, J=8.4, 1.3 Hz, 1 H), 8.18
(br s, 1 H), 7.77 (d, J=8.7 Hz, 2 H), 7.66 (d, J=15.2 Hz, 1 H), 7.54
(dd, J=8.5, 4.1 Hz, 1 H), 7.08 (d, J=15.4 Hz, 1 H), 7.02 (d, J=
8.7 Hz, 2 H), 4.54 (dd, J=10.7, 9.5 Hz, 1 H), 4.44 (dd, J=11.0, 2.5
Hz, 1 H), 4.28 (m, 1 H), 4.11 (t, J=5.7 Hz, 2 H), 4.00 (dd, J=11.1,
3.1 Hz, 1 H), 3.88 (dd, J=11.0, 7.4 Hz, 1 H), 2.64 (t, J=5.8 Hz,
indol-2-yl}carbonyl)-2,3-dihydro-1H-pyrrolo[3,2-f]quinoline (31).
A suspension of 24 (0.20 g, 0.47 mmol) in cooled (0 °C) dioxane
(5 mL) was saturated with HCl, allowed to warm to room
temperature over 2 h, and evaporated. 5-[2-(Dimethylamino)-
ethoxy]-1-H-indole-2-carboxylic acid hydrochloride41 (0.13 g,
0.47 mmol), EDCI (0.27 g, 1.42 mmol), and DMA (3 mL) were
added to the remaining yellow solid, and the red mixture was
stirred at room temperature for 20 h. The mixture was parti-
tioned between EtOAc and 5% NaHCO3 solution. The aqueous
layer was extracted with EtOAc (ꢀ3), and the EtOAc extracts
were dried (brine, Na2SO4). Flash chromatography (alumina,
EtOAc/MeOH, 49:1 then 9:1) gave (5-(benzyloxy)-1-(chloro-
methyl)-1H-pyrrolo[3,2-f]quinolin-3(2H)-yl)(5-(2-(dimethylamino)-
ethoxy)-1H-indol-2-yl)methanone (28) (0.22 g, 84%): mp 176-
179 °C; 1H NMR [(CD3)2SO] δ 11.68 (s, 1 H), 8.79 (dd, J=4.1,
1.5 Hz, 1 H), 8.41 (dd, J=8.6, 1.5 Hz, 1 H), 8.29 (s, 1 H), 7.56 (m,
3 H), 7.40 (m, 4 H), 7.17 (d, J=2.3 Hz, 1 H), 7.11 (d, J=1.5 Hz,
1 H), 6.92 (dd, J=9.0, 2.4 Hz, 1 H), 5.32 (s, 2 H), 4.82 (dd, J=
10.7, 9.6 Hz, 1 H), 4.58 (dd, J=10.9, 2.1 Hz, 1 H), 4.32 (m, 1 H),
4.05 (t, J=5.7 Hz, 2 H), 4.04 (m, 1 H), 3.93 (dd, J=11.2, 6.9 Hz,
1 H), 2.65 (t, J = 5.8 Hz, 2 H), 2.23 (s, 6 H); 13C NMR
[(CD3)2SO] δ 160.3, 154.5, 153.0, 147.3, 142.3, 137.4, 136.7,
131.6, 131.3, 130.6, 128.4, 127.9, 127.7, 127.4, 125.1, 122.4,
116.2, 116.0, 113.1, 105.5, 103.1, 102.0, 70.0, 66.9, 66.2, 57.8,
54.9, 47.7, 45.5, 40.7. HRMS FAB [M þ H] calcd for
C32H3735ClN3O3 = 542.2210. Found, 542.2214. Calcd for
C32H3337ClN3O3 =544.2181. Found, 544.2188.
THF (8 mL) and then HCO2NH4 (0.23 g, 3.6 mmol) in H2O
(1 mL) were added to a cooled (0 °C) mixture of 28 (0.20 g, 0.36
mmol) and 10% Pd/C (0.1 g) under N2. The mixture was stirred
at 0 °C for 14 h, and was then filtered through Celite. The Celite
was washed with DCM/H2O. The aqueous layer was extracted
with DCM (ꢀ3). The DCM extracts were dried (brine, Na2SO4)
and passed through a short plug of silica gel to give 31 (0.16 g,
93%): mp 209-215 °C; 1H NMR [(CD3)2SO] δ 11.66 (s, 1 H),
10.02 (br s, 1 H), 8.76 (dd, J=4.1, 1.4 Hz, 1 H), 8.41 (dd, J=8.5,
1.3 Hz, 1 H), 8.07 (s, 1 H), 7.56 (dd, J=8.5, 4.1 Hz, 1 H), 7.40 (d,
J = 8.9 Hz, 1 H), 7.17 (d, J = 2.2 hz, 1 H), 7.11 (d, J =1.2 Hz,
1 H), 6.93 (dd, J=8.9, 2.3 Hz, 1 H), 4.82 (dd, J=10.7, 9.6 Hz,
1 H), 4.57 (dd, J=11.0, 2.1 Hz, 1 H), 4.29 (m, 1 H), 4.06 (t, J=
5.9 Hz, 2 H), 4.04 (m, 1 H), 3.91 (dd, J=11.1, 7.2 Hz, 1 H), 2.64 (t,
J = 5.8 Hz, 2 H), 2.28 (s, 6 H); 13C NMR [(CD3)2SO] δ 160.3,
153.9, 153.0, 146.4, 142.8, 136.1, 131.6, 130.7, 127.4, 124.8, 124.7,
122.5, 116.0, 114.6, 113.1, 105.5, 103.1, 103.0, 66.1, 57.8, 54.9, 47.7,
2 H), 2.22 (s, 6 H). Anal. (C25H26ClN3O3 0.75EtOAc) C, H, N.
3
Alternative Synthesis of 6 from 24. A cooled (ice-water)
mixture of 24 (0.11 g, 0.27 mmol), 10% Pd/C (55 mg), and
THF (5 mL) under nitrogen was treated with 25% aqueous
HCO2NH4 (0.67 mL). The mixture was stirred at 0 °C for 6 h
and was then diluted with EtOAc (20 mL), dried (Na2SO4),
filtered through Celite, evaporated, and purified by dry flash
column chromatography (silica gel, 10-50% EtOAc/hexane)
to give 3-(tert-butyloxycarbonyl)-1-(chloromethyl)-5-hydroxy-
2,3-dihydro-1H-pyrrolo[3,2-f]quinoline (35) (39 mg, 44%) as a
white solid: mp 148-149 °C; 1H NMR (CDCl3) δ 8.61 (dd, J=
4.2, 1.2 Hz, 1 H), 8.01 (dd, J=8.5, 1.2 Hz, 1 H), 7.83 (br s, 1 H),
7.41 (dd, J=8.5, 4.2 Hz, 1 H), 4.26 (dd, J=11.8, 2.2 Hz, 1 H),
4.14 (dd, J=11.8, 8.5 Hz, 1 H), 3.93 (dddd, J=9.8, 8.5, 3.2, 2.2
Hz, 1 H), 3.80 (dd, J=11.1, 3.2 Hz, 1 H), 3.46 (dd, J=11.1, 9.8
Hz, 1 H), 1.61 (s, 9 H); 13C NMR (CDCl3) δ 153.5, 152.3, 145.3,
142.4 (br), 135.0, 130.6, 124.9, 122.6, 112.4 (v br), 100.0, 81.7
(br), 53.0, 46.5, 40.9, 28.4. HRMS: C17H19ClN2O3 requires Mþ•
334.1084, 336.1055. Found 334.1081, 336.1058.
A solution of 35 (0.14 g, 0.43 mmol) in dioxane (9 mL) was
saturated with HCl, allowed to stand for 1 h, and evaporated.
5,6,7-Trimethoxyindole-2-carboxylic acid (0.11 g, 0.43 mmol),
EDCI (0.25 g, 1.28 mmol), and DMA (5 mL) were added to the
remaining yellow solid, and the red mixture was stirred at room
temperature for 22 h. The mixture was poured into a mixture of
ice (20 g) and pH 7.4 phosphate buffer (20 mL). The precipitate
was removed by filtration, washed with water, and taken up in
DCM/MeOH (2:1, 30 mL). Most of the solvent was boiled off,
the remaining mixture was cooled in ice, and the precipitate was
removed by filtration to give 6 (18 mg, 9%) identical to the
material prepared above.
1-(Chloromethyl)-3-[(2E)-3-(4-methoxyphenyl)-2-propenoyl]-
2,3-dihydro-1H-pyrrolo[3,2-f]quinolin-5-ol (33). A suspension of
35 (0.10 g, 0.30 mmol) in dioxane (5 mL) was saturated with
HCl, stirred for 5 h, and evaporated. 4-Methoxycinnamic acid
(0.064 g, 0.36 mmol), EDCI (0.29 g, 1.50 mmol), and DMA
(3 mL) were added to the remaining yellow solid, and the red
mixture was stirred for 3 h. The mixture was partitioned between
DCM and cold 5% KHCO3 solution. The aqueous layer was
extracted with DCM (ꢀ3), and the organic extracts were dried
(brine, Na2SO4) and evaporated. Flash chromatography
(DCM/MeOH, 93:7) gave 33 (0.02 g, 17%) as a yellow solid:
mp (DCM/Et2O) 208-211 °C; 1H NMR [(CD3)2SO] δ 9.96 (br
s, 1 H), 8.73 (d, J=3.3 Hz, 1 H), 8.35 (d, J=7.7 Hz, 1 H), 8.18
(br s, 1 H), 7.78 (d, J=8.7 Hz, 2 H), 7.67 (d, J=15.3, 1 H), 7.54
(dd, J=8.5, 4.1, 1 H), 7.08 (d, J=15.4, 1 H), 7.01 (d, J=8.7, 2
H), 4.54 (dd, J=10.3, 9.5 Hz, 1 H), 4.45 (m, 1 H), 4.27 (m, 1 H),
3.99 (dd, J=11.1, 3.2 Hz, 1 H), 3.88 (dd, J=11.1, 7.3 Hz, 1 H),
3.82 (s, 3 H). C22H20ClN2O3 requires MþH 395.1163, 397.1133.
45.5, 40.7. Anal. (C25H25ClN4O3 0.5DCM) C, H, N.
3
1-(Chloromethyl)-3-((2E)-3-{4-[2-(dimethylamino)ethoxy]phenyl}-
2-propenoyl)-5-hydroxy-2,3-dihydro-1H-pyrrolo[3,2-f]quinoline (32).
Similar reaction of 24 (0.20 g, 0.47 mmol) with HCl/dioxane
followed by treatment with (E)-4-[2-(dimethylamino)ethoxy]-
cinnamic acid hydrochloride42 (0.13 g, 0.47 mmol), EDCI (0.27 g,
1.42 mmol), and DMA (3 mL), and flash chromatography of
the product (alumina, EtOAc/MeOH; 49:1 then 24:1) gave
(E)-1-(5-(benzyloxy)-1-(chloromethyl)-1H-pyrrolo[3,2-f]quino-
lin-3(2H)-yl)-3-(4-(2-(dimethylamino)ethoxy)phenyl)prop-2-en-
1-one (29) (0.18 g, 70%): mp 172-175 °C; 1H NMR [(CD3)2SO]
δ 8.76 (dd, J=4.1, 1.4 hz, 1 H), 8.47 (br s, 1 H), 8.35 (dd, J=8.5,
1.4 Hz, 1 H), 7.76 (d, J=8.7 Hz, 2 H), 7.67 (d, J=15.3 Hz, 1 H),
7.58 (d, J=7.3 Hz, 2 H), 7.54 (dd, J=8.5, 4.1 Hz, 1 H), 7.44 (t,
J=7.2 Hz, 2 H), 7.37 (t, J=7.2 Hz, 1 H), 7.08 (d, J=15.3 Hz,
1 H), 7.02 (d, J=8.7 Hz, 2 H), 5.31 (s, 2 H), 4.55 (dd, J=10.7,
9.5 Hz, 1 H), 4.44 (dd, J=10.9, 2.5 Hz, 1 H), 4.30 (m, 1 H), 4.11
(t, J=5.8 Hz, 2 H), 3.99 (dd, J=11.0, 3.0 Hz, 1 H), 3.91 (dd, J=
11.2, 7.2 Hz, 1 H), 2.64 (t, J = 5.7 Hz, 2 H), 2.23 (s, 6 H); 13C
NMR [(CD3)2SO] δ 164.1, 160.1, 154.6, 147.1, 142.6, 142.2,
137.2, 136.7, 131.1, 130.1, 128.3, 127.83, 127.78, 127.3, 125.1,
122.3, 116.9, 115.7, 114.7, 101.6, 70.0, 65.9, 57.5, 52.9, 47.8, 45.4,
40.1. Anal. (C32H32ClN3O3) C, H, N.
Found (FAB) 395.1161, 397.1169. Anal. (C22H19ClN2O3
0.1DCM) C, H, N.
3
1-(Chloromethyl)-3-[(2E)-3-(3-hydroxy-4-methoxyphenyl)-2-
propenoyl]-2,3-dihydro-1H-pyrrolo[3,2-f]quinolin-5-ol (34). A sus-
pension of 35 (0.10 g, 0.30 mmol) in dioxane (5 mL) was saturated
with HCl, stirred for 5 h, and evaporated. 3-Hydroxy-4-methox-
ycinnamic acid (0.070 g, 0.36 mmol), EDCI (0.29 g, 1.50 mmol),
and DMA (3 mL) were added to the remaining yellow solid, and
the red mixture was stirred for 3 h. The mixture was partitioned
between DCM and cold 5% KHCO3 solution. The aqueous layer
A solution of 29 (0.56 g, 1.03 mmol) was dissolved in CF3-
CO2H (15 mL) and refluxed for 48 h. Solvent was evaporated,

6832 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Milbank et al.
was extracted with DCM (ꢀ3). The organic extracts were dried
(brine, Na2SO4). Flash chromatography (DCM/MeOH, 93:7)
gave 34 (0.01 g, 8%) as a yellow solid: mp (DCM/Et2O) 215-
218 °C; 1H NMR [(CD3)2SO] δ 9.96 (br s, 1 H), 9.13 (s, 1 H), 8.73
(dd, J=4.1, 1.4 Hz, 1 H), 8.36 (dd, J=8.5, 1.4 Hz, 1 H), 8.17 (br s,
1 H), 7.57 (d, J=15.3 Hz, 1 H), 7.54 (dd, J=8.5, 4.1 Hz, 1 H), 7.25
(d, J=2.0 Hz, 1 H), 7.20 (dd, J=8.4, 2.0 Hz, 1 H), 6.99 (d, J=8.1
Hz, 1 H), 6.96 (d, J=15.0 Hz, 1 H), 4.54 (dd, J=10.5, 9.4 Hz, 1 H),
4.44 (dd, J= 11.1, 2.6 Hz, 1 H), 4.00 (dd, J=11.2, 3.3 Hz, 1 H),
3.88 (dd, J=11.1, 7.5 Hz, 1 H), 3.83 (s, 3 H). HRMS FAB [MþH]
calcd for C22H1935ClN2O4 = 411.1112. Found, 411.1127. Anal.
mixture was heated and stirred under nitrogen at 50 °C for
4 days until the mixture was almost free of suspended solid. The
volume of the mixture was reduced to about 1 mL on a rotary
evaporator. Then the suspension was filtered and the filtrate
loaded onto a Sephadex LH-20 gel filtration column with
MeOH as the eluent. The major dark-brown band was collected
and reduced to dryness on a rotary evaporator, followed by
drying on a high vacuum line to give [Co(cyclen)(6)][(OTf)2] (39)
as a dark-brown crystalline solid (34.5 mg, 96%): 1H NMR
[CD3OD] δ 8.85 (br s, 1 H), 8.62 (d, J=4.4 Hz, 1 H), 8.44 (d, J=
8.4 Hz, 1 H), 8.06 (s, 1 H), 7.47 (dd, J =8.4, 5.2 Hz, 1 H), 7.00
(s, 1 H), 6.92 (s, 1 H), 7.05, 6.69, 6.63, 5.50 (br s, 4 H), 4.60 (t, J=
8.4 Hz, 1 H), 4.52 (d, J = 10.8 Hz, 1 H), 4.24 (m, 1 H), 4.04 (s,
3 H), 3.90 (m, 8 H), 3.75 (2 H), 3.64 (2 H), 3.52 (1 H), 3.30 (3 H),
3.12 (2 H), 3.01 (4 H), 2.84 (1 H), 2.66 (1 H), (m, 16 H); 13C NMR
[CD3OD] δ 168.2, 162.4, 151.3, 148.0, 147.0, 146.0, 141.6, 140.1,
136.4, 131.1, 127.2, 125.1, 124.4, 112.5, 109.0, 108.4, 99.3, 62.0,
61.9, 57.9, 57.4, 56.8, 56.3, 51.1, 48.5, 48.4, 42.0. HRMS FABþ
[M - OTf]þ calcd for C37H4735ClCoF3N7O8S = 900.217 94.
Found, 900.215 81. Calcd for C37H4737ClCoF3N7O8S=902.214 99.
Found. 902.214 62.
[[Co(cyclen)(31)][(OTf)2] (40). A solution of 38 (0.101 g, 0.149
mmol) in dry CH3CN (4 mL) was treated with 31 (0.055 g, 0.118
mmol), and the mixture was stirred at room temperature for 8 h
and then cooled overnight at 5 °C. A small amount of unreacted
31 was removed by filtration, and the bright-yellow solid was
washed with cold CH3CN and the washes added to the filtrate.
This dark-brown solution was reduced to ∼2 mL by evaporation
of solvent under reduced pressure at room temperature and then
chromatographed on a short (3.3 mm ꢀ 40 mm) flash silica gel
column (0.32-0.60 μm). Elution was started with MeOH/
CH3NO2 (5%), which was stepwise enriched with MeOH up
to 50%. At this concentration the main band was eluted first
followed closely by a small yellow-brown band. Removal of the
solvent from the main band on a rotary evaporator and then on
a vacuum line gave [Co(cyclen)(31)][(OTf)2] (40) as a brown
glassy residue (0.078 g, 67%): 1H NMR [CD3CN] δ 10.08 (br s,
1 H), 8.71 (d, J=5.2 Hz, 1 H), 8.48 (d, J=8.8 Hz, 1 H), 8.16 (s,
1 H), 7.68 (dd, J =8.4, 5.2 Hz, 1 H), 7.51 (d, J =9.2 Hz, 1 H),
7.25 (s, 1 H), 7.11 (s, 1 H), 7.09 (m, 1 H), 6.42 (br s, 1 H), 5.29 (br
s, 2 H), 5.06 (br s, 1 H), 4.76 (td, J=10.8, 2.0 Hz, 1 H), 4.69 (dd,
J=10.8, 2.4 Hz, 1 H), 4.35 (m, 2 H), 4.26 (m, 1 H), 3.95 (m, 1 H),
3.82 (m, 1 H), 3.56 (m, 6 H), 3.29 (m, 2 H), 3.13 (m, 2H), 2.95 (m,
8 H), 2.89 (m, 4 H), 2.63 (m, 2 H); 13C NMR [CD3CN] δ 168.5,
161.6, 153.4, 147.8, 147.1, 145.9, 136.1, 133.1, 132.2, 129.0,
127.0, 124.3, 117.3, 114.1, 111.7, 107.4, 107.1, 105.0, 62.9,
57.7, 57.4, 56.6, 55.9, 50.6, 48.3, 44.2, 41.9. HRMS FAB [M -
2OTf þe]þ calcd for C33H4435ClCoN8O3 =694.255 69. Found,
694.253 05. Calcd, for C33H4437ClCoN8O3 = 696.252 74. Found,
696.254 01.
(C22H19ClN2O4 0.5DCM) C, H, N.
3
Resolution of Enantiomers of 6. The benzyl intermediate 24
was resolved on a ChiralCel OD semipreparative HPLC column
(2 cm ꢀ 25 cm) in hexane/iPrOH (9:1) (R = 1.24). The slower-
eluting (-)-enantiomer of 24 (mp 159-160 °C; [R]D -7 (c 0.45,
CHCl3)) was assigned the natural S-configuration on the basis
of its conversion via the (-)-enantiomer of 26 (mp 200-201 °C;
[R]D -2 (c 0.40, CHCl3)) to the relatively more cytotoxic (-)-
enantiomer S-6 (mp 224-225 °C; [R]D -16 (c 0.30, CHCl3)).
Similarly, the faster-eluting (þ)-enantiomer of 24 (mp 159-160 °C;
[R]D þ7 (c 0.45, CHCl3)) was assigned the unnatural R-configura-
tion on the basis of its conversion via the (þ)-enantiomer of 26 (mp
200-201 °C; [R]D þ2(c0.40, CHCl3)) to the relatively less cytotoxic
(þ)-enantiomer R-6 (mp 224-225 °C; [R]D þ15 (c 0.30, CHCl3)).
The relative cytotoxicities of the enantiomers are given in Table 1.
Preparation of Metal Complexes (Scheme 5). [Co(cyclen)-
(6)](ClO4)2 (39). Solid [Co(cyclen)(NO2)2][NO2] (37) (1.03 g,
2.79 mmol)37 was cautiously added with stirring to neat triflic
acid (10 mL) cooled in an ice bath. The solution was bubbled
with N2 to remove NOx gas and warmed briefly at 40-50 °C
until reaction was complete. Dry Et2O (250 mL) was added
slowly to the above cold solution (ice bath) with vigorous
stirring, and the resulting precipitate was filtered off, washed
with Et2O (ꢀ4), and dried in a desiccator to give [Co(cyclen)-
(OTf)2][OTf] (38) (1.95 g, 100%). Anal. (C11H24CoF9N4O11S3)
C, H, N. HRMS FAB [M - OTf]þ calcd C10H20CoF6N4O6S2=
529.006 05. Found: 529.004 06.
A solution of 38 (90 mg, 0.132 mmol) in dry CH3CN (3 mL)
was treated with 6 (62 mg, 0.132 mmol), and Pr2NEt (25 mg,
i
1.5 equiv) was then added to the stirred solution. This resulted in
rapid darkening of the solution to a brown color but with
significant amounts of suspended yellow solid (presumed to be
unreacted/undissolved 6) present. The mixture was stirred at
room temperature for 11 days, during which time nearly all the
suspended solid disappeared. The small amount remaining was
removed by filtration through a 0.45 μm membrane filter and
the filtrate made slightly acidic with dilute aqueous HClO4.
Excess aqueous 1 M NaClO4 was added, and the solution was
extracted with CH3NO2 (4ꢀ5 mL). The combined extracts were
evaporated to dryness, the residue was resuspended in dry Et2O
(15 mL), and again evaporated to dryness (first on a rotary
evaporator, finally on a vacuum line) below 20 °C to give crude
product as brown flakes of glassy material (103 mg, 86%).
HRMS FAB [M - ClO4]þ. This material was further purified
by reverse-phase HPLC, (C-18 column, TFA in CH3CN/H2O),
and the pooled pure fractions were concentrated under reduced
pressure, then combined with excess aqueous 1 M NaClO4 and
extracted five times with DCM. The combined organic extracts
were treated as above to give complex [Co(cyclen)(6)][(ClO4)2]
(39) as brownish flakes (70 mg, 59% yield). HRMS FAB [M -
2ClO4 - H]þ calcd for C32H4135ClCoN7O5 =697.218 97. Found,
697.213 27. Calcd for C32H4137ClCoN7O5=699.216 02. Found,
699.216 01.
[[Co(cyclen)(32)][(OTf)2] (41). This was prepared from 38
(0.087 g, 0.128 mmol) and 32 (0.052 g, 0.115 mmol), as for 40
above, to give after flash chromatography on silica gel (20%
MeOH/CH3NO2) [[Co(cyclen)(32)][(OTf)2] (41) as a brown
1
glass (0.089 g, 79%): H NMR [CD3CN] δ 8.70 (d, J = 5.0,
1 H), 8.38 (d, J=8.4 Hz, 1 H), 8.06 (s, 1 H), 7.68 (d, J=8.7 Hz,
2 H), 7.51 (dd, J=8.4, 5.2 Hz, 1 H), 7.38 (d, J=15.2 Hz, 1 H),
7.08 (d, J=8.8 Hz, 2 H), 6.72 (d, J=15.2 Hz, 1 H), 6.53 (br s,
1 H), 5.86 (br s, 1 H), 5.35 (br s, 1 H), 5.15 (br s, 1 H), 4.40-4.36
(m, 4 H), 4.23 (m, 1 H), 3.90 (dd, J=11.2, 3.6 Hz, 1 H), 3.72 (dd,
J=11.2, 7.6 Hz, 1 H), 3.61 (m, 2 H), 3.54 (m, 4 H), 3.35 (m, 2 H),
3.13 (m, 2 H), 2.93 (m, 12 H), 2.72 (dd, J = 13.2, 2.4 Hz, 1 H),
2.62 (d, J = 12.4 Hz, 1 H); 13C NMR [CD3CN] δ 168.4, 165.7,
160.2, 147.5, 146.5, 145.7, 143.6, 135.9, 131.2, 129.3, 126.9,
124.2, 118.1, 116.1, 111.3, 107.4, 62.6, 57.5, 57.4, 56.0, 54.8,
50.7, 48.5, 48.3, 44.3, 41.2. HRMS FABþ [M - 2OTfþe]þ calcd
for C33H4535ClCoN7O3=681.260 44. Found, 681.260 64. Calcd
for C33H4537ClCoN7O3 =683.257 49. Found, 683.260 86.
[Co(Me2dtc)2(6)] (43). Solid [Co2(Me2dtc)5][BF4] (105 mg,
0.1303 mmol) (42)38 was added to a suspension of 6 (46 mg,
Alternative Preparation of [Co(cyclen)(6)](OTf)2] (39). A sus-
pension of 6 (17.2 mg, 0.037 mmol) in MeOH (5 mL) was treated
with a solution of pyridine (9.5 mg, 0.120 mmol) in MeOH
(1 mL). The resulting creamy yellow mixture was purged with
nitrogen and stirred under nitrogen for 5 min. Then a solution of
38 (32.5 mg, 0.048 mmol) in MeOH (5 mL) was added. The

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6833
i
0.0983 mmol) in 5% MeOH/DCM (4 mL). Pr2NEt (25 mg,
0.19 mmol) was added to the stirred suspension in two portions
with the second added 1 day after the first. Stirring was
continued at room temperature for 8 days, by which time very
little suspended/unreacted 6 was evident, and the color of the
solution was the deep-green of the coproduct Co(Me2dtc)3. The
solution was filtered and the filtrate evaporated under reduced
pressure. The residue was taken up in DCM (2 mL) and
chromatographed on a flash silica gel column. Elution began
in DCM, and a large green band of Co(Me2dtc)3 was eluted.
Stepwise enrichment with CH3CN in increments of 10% was
carried out until the product [Co(Me2dtc)2(6)] (43) was eluted
(with ∼50% CH3CN/DCM). The main yellow-green band was
collected, and solvent was removed under reduced pressure to
give the product as a brownish-green amorphous residue (48 mg,
Cytotoxicity in Cell Culture. The cytotoxicity of the effectors
and their metal complexes was determined by assessing inhibi-
tion of cell proliferation following 4 h of exposure under oxic
and hypoxic conditions, using attached cells in 96-well plates, as
previously.2 Compounds were formulated in DMSO and diluted
to <1% DMSO in the cultures.
Irradiation of Metal Complexes. Solutions (30 μM) of com-
plex 41 in 5 mM phosphate buffer (pH 7.0) containing 0.1 M
sodium formate were irradiated using a 60Co source (dose rate
determined by NaCl-modified Fricke dosimetry), as previously
reported22 and analyzed by LC/MS as above.
Acknowledgment. We thank Susan Pullen for undertaking
the IC50 assays and the Auckland Division of the Cancer
Society of New Zealand and the Foundation for Research,
Science and Technology, New Zealand (Grant UOAX0211),
for financial support.
1
63%) free of the cytotoxic ligand 6: H NMR [CDCl3] δ 9.36
(br s, 2 H), 8.76 (d, J=4.9 Hz, 2 H), 8.15 (s, 2 H), 7.95 (dt, J=
8.4 Hz, 2 H), 7.37 (m, 2 H), 6.94 (s, 2 H), 6.84 (s, 2 H), 4.69 (m,
2 H), 4.58 (m, 2 H), 4.06 (s, 6 H), 3.92 (s, 8 H), 3.90 (s, 6 H), 3.80
(dt, J=11.0, 3.3 Hz, 2 H), 3.43 (td, J=11.0, 2.5 Hz, 2 H), 3.36
(s, 3 H), 3.34 (s, 3 H), 3.28 (s, 3 H), 3.27 (s, 3 H), 3.24 (s, 3 H), 3.21
(s, 6 H), 3.19 (s, 3 H); 13C NMR [CDCl3] δ 204.0, 203.9, 171.9,
160.2, 150.0, 147.1, 146.1, 145.8, 140.4, 138.9, 131.1, 130.2,
125.4, 125.3, 123.5, 122.6, 107.2, 107.0, 106.3, 97.6, 61.5, 61.2,
56.2, 55.7, 56.8, 46.2, 42.2, 38.4, 38.2, 38.0, 37.7. HRMS FABþ
[M - e]þ calcd for C30H3335ClCoN5O5S4 =765.038 50. Found,
765.038 56. Calcd. for C30H3335ClCoN5O5S4=767.035 55. Found,
767.037 30.
Supporting Information Available: Synthesis details for
Schemes S1 and S2; combustion analysis results for new com-
pounds. This material is available free of charge via the Internet
at http://pubs.acs.org.
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