Chemistry of pyrrolo[1,2-a]indole- and pyrido[1,2-a]indole-based quinone methides. Mechanistic explanations for differences in cytostatic/cytotoxic properties

Article abstract of DOI:10.1021/jo070866o

(Chemical Equation Presented) In the present study we investigate pyrido[1,2-a]indole- and pyrrolo[1,2-a]indole-based quinones capable of forming quinone methide and vinyl quinone species upon reduction and leaving group elimination. Our goals were to det

Full text of DOI:10.1021/jo070866o

Chemistry of Pyrrolo[1,2-a]indole- and Pyrido[1,2-a]indole-Based
Quinone Methides. Mechanistic Explanations for Differences in
Cytostatic/Cytotoxic Properties
Omar Khdour and Edward B. Skibo*
Department of Chemistry and Biochemistry, Arizona State UniVersity, Tempe, Arizona 85287-1604
eskibo@asu.edu
ReceiVed May 3, 2007
In the present study we investigate pyrido[1,2-a]indole- and pyrrolo[1,2-a]indole-based quinones capable
of forming quinone methide and vinyl quinone species upon reduction and leaving group elimination.
Our goals were to determine the influence of the 6-membered pyrido and the 5-membered pyrrolo fused
rings on quinone methide and vinyl quinone formation and fate as well as on cytostatic and cytotoxic
activity. We used the technique of Spectral Global Fitting to study the fleeting quinone methide intermediate
directly. Conclusions regarding quinone methide reactivity are that carbonyl O-protonation is required
for nucleophile trapping and that the pKa value of this protonated species is near neutrality. The abnormally
high protonated carbonyl pKa values are due to the formation of an aromatic carbocation species upon
protonation. The fused pyrido ring promotes quinone methide and vinyl quinone formation but slows
nucleophile trapping compared to the fused pyrrolo ring. These findings are explained by the presence
of axial hydrogen atoms in the fused pyrido ring resulting in more steric congestion compared to the
relatively flat fused pyrrolo ring. Consequently, pyrrolo[1,2-a]indole-based quinones exhibit more cytostatic
activity than the pyrido[1,2-a]indole analogues due to their greater nucleophile trapping capability.
Introduction
This “bioreductive alkylation” process, first proposed by Moore
for quinone natural products,13,14 has become important in cancer
drug design. The success of bioreductive alkylation is largely
Herein we report on our continuing efforts to design and study
alkylating species activated by cellular quinone reduction.1-12
(8) Skibo, E. B.; Schulz, W. G. J. Med. Chem. 1993, 36, 3050-3055.
(9) Schulz, W. G.; Islam, E.; Skibo, E. B. J. Med. Chem. 1995, 38, 109-
118.
* Address correspondence to this author. Phone: 480-965-3581. Fax: 480-
965-2747.
(1) Skibo, E. B. J. Org. Chem. 1986, 51, 522-527.
(2) Lee, C.-H.; Skibo, E. B. Biochemistry 1987, 26, 7355-7362.
(3) Lemus, R. L.; Skibo, E. B. J. Org. Chem. 1988, 53, 6099-6105.
(4) Lemus, R. L.; Lee, C. H.; Skibo, E. B. J. Org. Chem. 1989, 54, 3611-
3618.
(10) Schulz, W. G.; Nieman, R. A.; Skibo, E. B. Proc. Natl. Acad. Sci.
U.S.A. 1995, 92, 11854-11858.
(11) Skibo, E. S.; Gordon, S.; Bess, L.; Boruah, R.; Heileman, J. J. Med.
Chem. 1997, 40, 1327-1339.
(5) Islam, I.; Skibo, E. B. J. Org. Chem. 1990, 55, 3195-3205.
(6) Islam, I.; Skibo, E. B.; Dorr, R. T.; Alberts, D. S. J. Med. Chem.
1991, 34, 2954-2961.
(12) Khdour, O.; Ouyang, A.; Skibo, E. B. J. Org. Chem. 2006, 71,
5855-5863.
(13) Moore, H. W. Science (Washington, D.C.) 1977, 197, 527-532.
(14) Moore, H. W.; Czerniak, R. Med. Res. ReV. 1981, 1, 249-280.
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10.1021/jo070866o CCC: $37.00 © 2007 American Chemical Society
Published on Web 10/10/2007
8636
J. Org. Chem. 2007, 72, 8636-8647

Pyrrolo- and Pyrido[1,2-a]indole-Based Quinone Methides
CHART 1. Reductive Activation and Double Leaving
Group Elimination
CHART 2. Reductive Activation and Single Leaving Group
Elimination
due to the expression of the 2-electron reducing enzyme DT-
diaphorase in some histological cancer types.15-19 In the present
study we compare the reductive activation of pyrido[1,2-a]indole
(2, n ) 1) and pyrrolo[1,2-a]indole (1, n ) 0) based quinones
(Chart 1). Our goals were to determine the influence of the
6-membered pyrido and the 5-membered pyrrolo fused rings
on quinone methide and vinyl quinone and formation as well
as cytostatic/cytotoxic activity. We developed a new technique
for rapid quinone reduction to the hydroquinone in a Thunberg
(anaerobic) cuvette. As a result, it was possible to carry out
direct kinetic studies of quinone methide formation and fate by
using Spectral Global Fitting.20 The postulate made at the outset
of this study was that reactive species derived from the
6-membered pyrido ring analogues would be less reactive than
those derived from the 5-membered pyrrolo ring analogues. The
presence of axial hydrogens in the fused pyrido ring, in contrast
to the relatively flat fured pyrrolo ring, was the basis for this
postulate. The results of this study would provide an explanation
of the prevalence of the pyrrolo[1,2-a]indole system in naturally
occurring bioreductive alkylating agents, e.g., the mitomycins
and mitosenes.21
been proposed by Moore to be the Michael acceptors formed
upon reductive activation and leaving group elimination from
naturally occurring quinones.13 On the basis of our initial
postulate, we expected the vinyl quinone formation to be favored
in the pyrido-fused system 1, but not in the pyrrolo-fused system
2. Steric congestion in the latter system was expected to favor
the elimination reactions leading to double bond formation.
Chart 2 shows the pyrido[1,2-a]indole- and pyrrolo[1,2-a]-
indole-based latent quinone methide systems 3. Reduction of 3
to the hydroquinone followed by leaving group elimination
would afford the pyrido- and the pyrrolo-fused quinone me-
thides. The plan was to carry out kinetic studies, calculations,
and cytostatic/cytotoxic determinations on 3 to assess the
influence ring size has on chemical and biological properties.
The results of these studies would have a bearing on the
documented lack of antitumor activity exhibited by pyrido[1,2-
a]indole analogues27 as well as the activity of the pyrrolo[1,2-
a]indole-based antitumor agents.
Results and Discussion
Chart 1 shows the pyrido[1,2-a]indole- and pyrrolo[1,2-a]-
indole-based latent vinyl quinone systems 1 and 2, which upon
reduction and elimination of both leaving groups could afford
the vinyl quinone species. The consecutive addition of two
nucleophiles to the vinyl quinone, via a quinone methide
intermediate, will afford a 1,2-bisalkylation adduct. Therefore
the vinyl quinone species could be a 1,2-bisalkylator and as a
result possess antitumor activity. Previously, the reactive vinyl
quinone species has been used as a synthetic intermediate as
well as a monomer.22-26 In addition, vinyl quinone systems have
Synthesis. The synthesis of the pyrido[1,2-a]indole-based
latent vinyl quinone system 1 is outlined in Schemes 1 and 2.
The preparation of the racemic cis-diol key intermediate 8
(Scheme 1) was carried out starting with ketone 4 that was
previously prepared in the laboratory.12 Reduction of the ketone
group of 4 to an alcohol with sodium borohydride was followed
by treatment with methanesulfonyl chloride/DMAP to afford 6
in a one-pot reaction. The racemic cis-diol 7 was conveniently
obtained by catalytic osmium tetroxide oxidation of 6 with
N-methylmorpholine N-oxide to regenerate the oxidizing agent.
Catalytic reduction of the nitro group of 7 to an amine followed
by Fremy oxidation28 of the resulting amine afforded a mixture
of quinone 8 and iminoquinone 9. The iminoquinone 9 is the
actual product of Fremy oxidation that hydrolyses to the quinone
in acidic media. We have previously prepared iminoquinones
by the Fremy oxidation of aromatic amines in neutral buffers.5
Scheme 2 shows the reactions affording the various analogues
of 1 used in mechanistic and cytotoxic studies. Acetylation of
8 with acetic anhydride in the presence of DMAP affords the
(15) Gutierrez, P. L. Free Radical Biol. Med. 2000, 29, 263-275.
(16) Rauth, A. M.; Goldberg, Z.; Misra, V. Oncol. Res. 1997, 9, 339-
349.
(17) Garrett, M. D.; Workman, P. Eur. J. Cancer 1999, 35 (14), 2010-
2030.
(18) Stratford, I. J.; Workman, P. Anti-Cancer Drug. Des. 1998, 13, 519-
528.
(19) Workman, P. Oncol. Res. 1994, 6, 461-475.
(20) Beecham, J. M.; Brand, L. Photochem. Photobiol. 1986, 44, 323-
329.
(21) Tomasz, M.; Palom, Y. Pharmacol. Ther. 1997, 76, 73-87.
(22) Parker, K. A.; Mindt, T. L. Org. Lett. 2001, 3, 3875-3878.
(23) Parker, K. A.; Ruder, S. M. J. Am. Chem. Soc. 1989, 111, 5948-
5949.
(24) Parker, K. A.; Mindt, T. L. Org. Lett. 2002, 4, 4265-4268.
(25) Germeraa, P.; Moore, H. W. J. Org. Chem. 1974, 39, 774-780.
(26) Irngartinger, H.; Lichtenthaler, J.; Herpich, R.; Stadler, B. Mol. Cryst.
Liq. Cryst. Sci. Technol., Section a. A 1996, 276, 349-360.
(27) Orlemans, E. O. M.; Verboom, M. W.; Scheltinga, M. W.;
Reinhoudt, D. N.; Lelieveld, P.; Fiebig, H. H.; Winterhalter, B. R.; Double,
J. A.; Bibby, M. C. J. Med. Chem. 1989, 32, 1612-1620.
(28) Zimmer, H.; Lankin, D. C.; Horgan, S. W. Chem. ReV. 1971, 71,
229-246.
J. Org. Chem, Vol. 72, No. 23, 2007 8637

Khdour and Skibo
SCHEME 1. Preparation of 8 and 9
SCHEME 3. Preparation of Cis/Trans 16
SCHEME 4. Preparation of Analogues of 2
SCHEME 2. Preparation of Analogues of 1
formation. The preparation of 1c involved the treatment of 8
with 2-methoxyethylamine followed by treatment of the product
10 with an excess of acetic anhydride. The cyclic carbonate
leaving group of 1d was prepared by treating 10 with triphos-
gene.
The synthesis of the pyrrolo[1,2-a]indole-based latent vinyl
quinone system 2 is outlined in Schemes 3 and 4. The synthetic
steps leading to series 2 are based on previous reports from
this laboratory29 and elsewhere.30 Annelation of the pyrrolo ring
was carried out by treatment of 1131 with methyl acrylate in
the presence of potassium tert-butoxide. The annelation product
12 was decarboxylated to 13, which was then nitrated to afford
14. Treatment of 14 with cupric bromide/lithium bromide in
DMF afforded a mixture of products including 2-bromo,
desired cis-diacetate 1a. Aziridination of 1a in methanol afforded
1b, which is a potential triple alkylating agent with high
cytotoxic activity. However, the presence of an electron-rich
amine substituent would influence quinone methide fate by
favoring proton trapping over nucleophile trapping. The mecha-
nistic study of 1c would serve to confirm quinone methide
(29) Boruah, R. C.; Skibo, E. B. J. Med. Chem. 1994, 37, 1625-1631.
(30) Allen, G. R., Jr.; Poletto, J. C.; Weiss, M. J. J. Org. Chem. 1965,
30, 2897-2904.
(31) Zhao, S.; Liao, X.; Wang, T.; Flippen-Anderson, J.; Cook, J. M. J.
Org. Chem. 2003, 68, 6279-6295.
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Pyrrolo- and Pyrido[1,2-a]indole-Based Quinone Methides
SCHEME 5. Preparation of Analogues of 3
SCHEME 6. Products Resulting from the Reductive
Activation of 1a
2-hydroxy, and 2-formyloxy derivatives. Aqueous acid workup
afforded the desired 2-hydroxy derivative 15. Borohydride
reduction of 15 afforded the diol 16 as a cis/trans mixture.
Chromatographic separation afforded the cis isomer that was
identified with 2d H NMR. Quinone elaboration from cis-16 to
afford 2a was carried out by nitro group reduction followed by
Fremy oxidation. Other analogues of 2 required for the present
study were prepared as shown in Scheme 5.
The monosubstituted analogue 3a was prepared from 4 as
shown in Scheme 5. Compound 3b was prepared as previously
described.29
Products of Reductive Activation. The product studies
described below provided circumstantial evidence of the forma-
tion of quinone methide species upon the reductive activation
of 1a, 1c, 2b, 3a, and 3b. These quinone methide species can
trap a nucleophile (methanol or water) and an electrophile
(proton). Much of the earlier quinone methide literature has
documented nucleophile and proton trapping reactions.32-35 In
addition, some quinone methides discussed in this report can
form a vinyl quinone by elimination of a second leaving group.
Finally, the vinyl quinone product can undergo prototropic
reactions to afford an aromatized product as well as other
structurally novel products. Scheme 6 shows the structures of
isolated products resulting from the reductive activation of 1a
in anaerobic aqueous buffer (phosphate, pH 7.4) and in
anaerobic methanol. Scheme 7 shows the structures of isolated
products resulting from the reductive activation of the electron-
rich analogue 1c in anaerobic aqueous buffer (phosphate, pH
7.4). The mechanisms associated with the formation of these
isolated products are discussed in conjunction with Schemes
8-13.
SCHEME 7. Products Resulting from the Reductive
Activation of 1c
Reduction of 1a in anaerobic pH 7.4 phosphate buffer affords
17 in high yield along with minor amounts of 18. Inspection of
the anaerobic reaction mixture before aerobic workup revealed
the presence of the red chromophore associated with 17.
Therefore, 17 was formed by the double elimination process
shown in Scheme 8 rather than by oxidation upon aerobic
workup. The vinyl quinone species 17 can undergo aromatiza-
tion to 18H2, which upon air oxadation affords the blue
aromatized quinone 18. Our calculations (Hartree-Fock cal-
culations were carried out with the 3-21G(*) basis set)36 show
that the aromatization of 17 is favorable by 6 kcal/mol. Shown
in Scheme 9 is the mechanism for aromatization via two
prototropic shifts and the calculated relative energies of the
reacting intermediates. Although aromatization is energetically
favored, the presence of a relatively high-energy intermediate
tautomer (labeled 0 kcal/mol) precludes the complete conversion
to the aromatized product.
Reduction of 1a in anaerobic methanol also affords 17 in
high yield upon aerobic workup along with minor amounts of
18. We also isolated and identified trace amounts of 19-21 in
this reaction. The presence of 19 and 21 supports a quinone
methide intermediate that traps methanol and a proton to afford
these respective products, see Scheme 8 for mechanisms. We
(32) Bird, D. M.; Gaudiano, G.; Koch, T. H. J. Am. Chem. Soc. 1991,
113, 308-315.
(33) Taatjes, D. J.; Gaudiano, G.; Resing, K.; Koch, T. H. J. Med. Chem.
1996, 39 (21), 4135-4138.
(34) Pande, P.; Shearer, J.; Yang, J. H.; Greenberg, W. A.; Rokita, S. E.
J Am. Chem. Soc. 1999, 121, 6773-6779.
(35) Rokita, S. E.; Yang, J. H.; Pande, P.; Greenberg, W. A. J. Org.
Chem. 1997, 62, 3010-3012.
(36) Durig, J. R.; Berry, R. J.; Durig, D. T.; Sullivan, J. F.; Little, T. S.
Teubner-Texte Phys. 1988, 20, 54-68.
J. Org. Chem, Vol. 72, No. 23, 2007 8639

Khdour and Skibo
SCHEME 8. Mechanisms of Product Formation upon
Reductive Activation
SCHEME 10. Internal Redox Mechanisms
SCHEME 9. Aromatization Mechanism
SCHEME 11. Reductive Elimination Mechanism
reaction. The excessively electron-rich hydroquinone ring also
favors hydroquinone tautomerization and amine elimination to
afford 23 as illustrated in Scheme 11.
The double elimination reaction did not occur upon reductive
activation of 2b, although an analogue of the pyrroloindole vinyl
quinone has been reported in the literature.37 The major observed
product is the quinone methide water-trapping product, 25
(Scheme 12). The proton-trapping product 26 was observed in
only trace amounts. Apparently the rapid trapping of the quinone
methide intermediate by water prevented the second acetate
elimination.
The apparent stability of 17, judging from its ease of synthesis
and stability in solution, suggests that it would not be an
effective double alkylating agent. The reaction of 17 with
ethylene diamine in methanol merely resulted in displacement
of the methoxy group followed by cyclization to afford 27. The
LUMO of 17 shows high coefficient values in the quinone ring
where nucleophilic attack and displacement actually occur. In
contrast, the alkene center has a negligible wave function
coefficient indicating that nucleophile attack is not likely at this
attribute the formation of 20 to an internal redox reaction
wherein the hydroquinone ring transfers electrons to the double
bond of the fused pyrido ring by means of prototropic shifts
(see the mechanism in Scheme 10). Although 20 was isolated
upon aerobic workup, this compound very likely arises while
the reaction mixture is anaerobic (see the mechanism in Scheme
10).
The internal redox reaction that affords 20 in trace amounts
becomes the predominate process upon reductive activation of
analogue 1c (see Scheme 7 for product structures). The presence
of the amino substituent of 1c results in an excessively electron-
rich hydroquinone ring that favors the observed internal redox
(37) Naruta, Y.; Nagai, N.; Maruyama, K. J. Chem. Soc., Perkin Trans.
1 1988, 1143-8.
8640 J. Org. Chem., Vol. 72, No. 23, 2007

Pyrrolo- and Pyrido[1,2-a]indole-Based Quinone Methides
SCHEME 12. Products of 2b Reductive Activation
SCHEME 14. Products of 3a,b Reductive Activation
SCHEME 13. Nucleophilic Trapping Product of 17 and the
LUMO of 17
The kinetic study of quinone methide formation and fate is
difficult because of the presence of two transient species, the
hydroquinone and quinone methide. Although these species
possess different UV-visible spectra, absorbance vs time
measurements at a single wavelength may not provide rate
constant data for both of these reacting species. To carry out
this kinetic study we used Spectral Global Fitting, a technique
that has been used to visualize and study photochemical
intermediates.20,38 Spectral Global Fitting involves repetitive
UV-visible scanning of reaction mixtures with time resulting
in a surface showing spectral changes associated with the
reaction. Fitting the entire range of wavelengths (Global Fitting)
to a rate law provides accurate rate constants and intermediate
spectra associated with the reaction under study. We recently
reported the use of Spectral Global Fitting to study a transient
intermediate involved in CC-1065 A-ring opening.39
To generate the hydroquinone form of 3a,b in an anaerobic
atmosphere, we utilized a Thunberg cuvette with a quinone
DMSO stock solution in the top port and a buffer solution
containing suspended 5% Pd on Carbon in the bottom port. The
cuvette was purged with hydrogen and sealed. The reduction
reaction was initiated by mixing the ports. Repetitive UV-
visible scanning of the reaction with time afforded an absorbance
vs time vs wavelength surface, an example of which is shown
in Figure 1. The reduction reaction is fast and is complete in a
single scan (∼2 min). The surface shown in Figure 1 was
obtained with 4-fold less catalyst than is used for kinetic studies
so that the initial quinone spectrum can be observed. Global
fitting of the entire surface to a three-exponential rate law (Abs
) Ae-kt + Be-k′t + Ce-k′′t + D) afforded the three rate constants
associated with quinone methide formation from the hydro-
quinone (kobsd), quinone methide disappearance (k′obsd), and the
slow precipitation of the finally divided catalyst (k′′obsd). In this
rate law Abs refers to the total absorbance at a given time and
wavelength and constants A-D refer to the absorbance at this
wavelength for each reacting species. In all reactions, the relative
magnitude of rate constants was quinone methide formation >
quinone methide disappearance . catalyst precipitation. Some
center. Consistent with the presence of an alkylation reaction
involving the quinone ring, 17 possesses modest cytostatic
activity.
Quinone Methide Formation and Fate and Reaction
Kinetics. The goal of these studies was to determine the
reactivity differences between the pyrroloindole and pyridoin-
dole quinone methides by using product studies, kinetic studies,
and calculations.
The results of the isolation studies are shown in Scheme 14.
The dominant quinone methide reaction path for the pyrroloin-
dole quinone methide system at pH 7.4 and 5.0 is the
nucleophilic trapping of water. In contrast, the pyridoindole
quinone methide undergoes significant prototropic reactions
along with the nucleophilic trapping water, particularly at the
pH values of 7.4 and 5. At pH 9.0 the pyridoindole quinone
methide only traps water.
(38) Matheson, I. B. C. Anal. Instrum. 1987, 16, 345-373.
(39) LaBarbera, D. V.; Skibo, E. B. J. Am. Chem. Soc. 2006, 128, 3722-
3727.
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Khdour and Skibo
SCHEME 15. Quinone Methide Formation and Fate
FIGURE 1. Surface produced by reduction of 3a in anaerobic 0.2 M
pH 8.5 boric acid buffer at 30 °C. The reaction mixture was repetitively
scanned 100× from 240 to 600 nm.
reactions exhibited rapid quinone methide formation and we
fit the surface to a two-exponential rate law (Abs ) Ae-k′t
+
Be-k′′t + C). We should point out that fits to three-exponential
rate laws could provide solutions that are not unique. However,
fitting each wavelength from 240 to 600 nm to the same rate
law will provide a unique solution. We also should point out
that casual inspection of the surface shown in Figure 1 does
not show the obvious buildup of a quinone methide species (see
quinone methide arrow in this figure). The Spectral Global
Fitting over the wavelength range studied requires the buildup
of such a transient species early in the reaction.
Equation 1 gives the rate law for quinone methide formation
kobsd ) aHk1 + k0
and eq 2 gives the rate law for quinone methide disappearance.
(aHk2 + Kw[k3 + k4])
k′obsd
)
(aH + Ka)
In Scheme 15, we outline the proposed mechanism for
quinone methide formation and disappearance based on the
studies presented below. Global fitting provided the rates of
quinone methide formation from the hydroquinone forms of 3a,b
that are plotted as log kobsd vs pH in Figure 2. The rate constants
associated with the conversion of reduced 3b to its quinone
methide were fit to the rate law in eq 1. The solid line was
generated with eq 1 where k0 ) 0.09 min-1 and k1 ) 1.5 × 105
M-1 min-1. The mechanism consistent with the pH-rate profile
for reduced 3b is the spontaneous elimination of acetate (k0
process) and the proton assisted elimination of acetate (k1
process) from the electron-rich hydroquinone. The k0 process
is independent of pH and exhibits a zero slope while the k1
process exhibits a -1 slope consistent with acid catalysis. The
rate constants associated with the acid-catalyzed conversion of
reduced 3a to its quinone methide were too large to measure.
We did manage to measure two rate constants at pH 7 and 8,
both with a value of 0.36 min-1. On the basis of the pH-rate
profile obtained for reduced 3b, these rate constants very likely
represent the k0 process of reduced 3a. Even with two data
FIGURE 2. pH-rate profile for quinone methide formation from the
reduced pyridoindole (3aH2) and the pyrroloindole (3bH2).
points, we can safely conclude that acetate is eliminated ∼4-
fold faster from reduced 3b than from reduced 3a.
Equation 2 represents the rate law for quinone methide
disappearance. This equation was derived by using material
balance where reactions occur from an equilibrating mixture
of neutral and protonated quinone metide. Both the protonated
(k2 process) and neutral equivalent (k3 and k4 processes) react
to afford the observed major products shown in Scheme 14.
Alternatively, the quinone methide can be protonated at the
methide carbon center to afford a proton-trapping product (k4
process). The k4 process is expected to parallel the k3 process
on the pH profile because both involve quinone methide
protonation along with the participation of hydroxide. Essentially
the transition states for both k3 and k4 processes consist of neutral
quinone methide and water. In fact, the pyridoindole quinone
methide traps both water and protons by these respective
8642 J. Org. Chem., Vol. 72, No. 23, 2007

Pyrrolo- and Pyrido[1,2-a]indole-Based Quinone Methides
FIGURE 3. pH-rate profile for the decomposition of the pyrroloindole
quinone methide.
FIGURE 5. Potential density maps for the O-protonated pyridoindole
quinone methide and the corresponding neutral species. The charge
density color codes are anionic (red), cationic (blue), and neutral (green).
FIGURE 4. pH-rate profile for the decomposition of the pyridoindole
quinone methide.
FIGURE 6. Comparison of the potential density maps for the
O-protonated pyridoindole and pyrroloindole quinone methides. The
charge density color codes are the same as in Figure 5.
processes only at pH values <pKawhere neutral quinone methide
is the predominant species. The pyrroloindole quinone methide
on the other hand does not exhibit a significant k4 process.
The pH-rate profiles for the disappearance of the pyrroloin-
dole and pyridoindole quinone methides are shown in Figures
3 and 4, respectively. The data in both profiles were fit to eq 2
generating the constants for the pyrroloindole quinone methide
k2 ) 0.032 min-1, Ka ) 2.84 × 10-7 (pKa ) 6.55), k3 ) 8.98
× 104 M-1 min-1 and for the pyridoindole quinone methide k2
) 0.006 min-1, Ka ) 3.5 × 10-8 (pKa ) 7.51), k3 ) 1.9 × 104
M-1 min-1. The small inflection point in Figure 4 is significant
considering the 1.8-fold difference in kobsd for the high and low
pH plateaus and the (10-15% standard deviation for kobsd
values.
The mechanistic interpretation of the pH-rate profiles for
quinone methide disappearance (Figures 3 and 4) relied on
Hartree-Fock calculations with 6-31G basis sets as well as on
product studies. In Figure 5 we show the potential density maps
of the protonated and neutral pyridoindole quinone methide with
negative charge density in red and positive charge density in
blue. Inspection of the methide reacting center of the neutral
species reveals a slight negative charge density (yellow-green
color). We interpret this observation as the combination of
electron release to and electron withdrawal from the methide
reacting center (see resonance structures in Figure 5). This result
suggests that water does not trap the neutral quinone methide
but rather its kinetic equivalent occurs: the hydroxide trapping
of the protonated quinone methide. Thus the rate law in eq 2
contains the autoprotolysis constant for water Kw ) 1.5 × 10-14
at 30 °C. The numerator expression for hydroxide attack on
the protonated quinone methide is aHk3(Kw/aH), which simplifies
to Kwk3 in eq 2, where k3 is the second-order rate constant for
hydroxide attack, aH is the proton activity as measured with a
pH meter, and Kw/aH is the hydroxide concentration at a
particular aH value. Thus the k3 mechanism involves specific
acid catalyzed attack of hydroxide. Although the proton
concentration decreased, the hydroxide concentration has in-
creased. Therefore the second-order rates of decomposition (k3)
are seen to increase with increasing pH. Consistent with this
mechanism, water trapping is the only product observed at pH
values above the quinone methide pKa for both the pyrroloindole
and pyridoindole quinone methide.
Proton transfer to the neutral quinone methide can occur either
to the slightly electron-rich methide center or to the electron-
rich carbonyl oxygen (Figure 5). The former process leads to
the proton-trapping product (k4 in Scheme 15) and the latter
process leads to water trapping (k3 in Scheme 15). Product
studies revealed that the pyridoindole quinone methide traps
both water and protons by these processes at pH values <pKa.
The potential density maps illustrated in Figure 6 show that
the protonated pyrroloindole and the pyridoindole quinone
methides have virtually identical potential density maps. Al-
though the positive potential at the methide centers is nearly
identical for both methides, they exhibit differences in rate
constants k0-k3 and trapping products. The explanation we give
for these differences is the greater steric congestion of the fused
pyrido ring compared to the relatively flat fused pyrrolo ring.
The presence of an axial hydrogen atom in the fused pyrido
ring, illustrated in Figure 6, will promote the elimination of
acetate from reduced 3a due to the relief of steric congestion.
In contrast, the pyrrolo ring of reduced 3b eliminates acetate at
a 4-fold lower rate constant. Conversely, the pyrroloindole
quinone methide traps water and hydroxide with larger rate
J. Org. Chem, Vol. 72, No. 23, 2007 8643

Khdour and Skibo
TABLE 1. In Vitro Assay Results of Pyridoindole and
Pyrroloindole Analogues
the selectivity of antitumor agents, because it provides a measure
of histological specificity.46 The determinations of log values
>-4.00 indicated a lack of activity in the assay results. Thus,
we could not achieve the concentration required for a 50%
cytostatic or cytotoxic effect even when approaching mM
concentration ranges of the compound. Compounds 1b-d, 2c,
and 10 fall into this category and have been left out of Table 1.
In Table 2, we show in vitro assays of 17, 3a, and 3b against
the six human cancer cell lines. These compounds possess only
growth inhibition activity and only the GI50 parameter is shown
in this table.
The data in Table 1 indicate that both 1a and 2b possess low
cytostatic activity and no measurable cytotoxic activity. The
absence of cytotoxic activity exhibited by these compounds is
likely due to their lack of bisalkylation (cross linking) capability.
Cross linking is the cytotoxic event of many alkylating agents
possessing antitumor activity.47 Our product studies suggest that
the reductive activation of 1a and 2b could only result in
monoalkylation. The results of product studies outlined in
Scheme 14 suggest that compound 1a could form 17 upon
cellular reductive activation. The data in Table 2 indicate that
compound 17 possesses low cytostatic activity perhaps due to
its nucleophile-trapping capability illustrated in Scheme 13. The
reductive activation of 2a likewise results in a monoalkylation
event to afford water-trapping product (Scheme 12).
compd
no.
mid-range log
GI50 (range)
mid-range log
TGI nd range
mid-range log
LC50 and range
1a
9
2b
-4.08 (0.06)
-5.69(3.54)
-4.02 (0.06)
-4.01(0.25)
-5.09(3.59)
>-4.00 (0))
>-4.00 (0)
-4.52(3.25)
>-4.00 (0)
constants than those observed for the pyridoindole quinone
methide. Proton trapping of the pyridoindole methide center can
compete with the slowed water trapping affording significant
amounts of 20. Steric congestion can also destabilize biological
adducts of the pyridoindole quinone methide. Previously, we
reported the preparation of an N(7)-pyridoindole adduct of
polydG‚polydC.40 Under mild conditions, the N(7) center was
eliminated from the pyridoindole adduct to afford 17 and native
DNA.
The results enumerated above indicate that pyrroloindole-
based reductive alkylating agents are superior to pyrido-based
analogues, thus the prevalence of the pyrrolo[1,2-a]indole
system in naturally occurring bioreductive alkylating agents such
as the mitomycins and mitosenes.21
Our pH-rate studies and calculated potential density maps
indicate that the quinone methide species must be protonated
to afford a carbocation before it can trap a nucleophile.
Therefore, carbocation pKa values at or near neutrality are
required for nucleophile trapping in a biological system (pH
values ∼7.4). Previously, we reported the pKa for the mitomycin
C carbocation-quinone methide equilibrium to be 7.1,41 a value
similar to those obtained from the pH-rate data shown in
Figures 3 and 4. In contrast, the pKa values for a protonated
carbonyl oxygen of a ketone or ester usually have negative
values. We consider the relatively high pKa values of 6-8 to
be typical for a carbocation-quinone methide equilibrium. The
formation of a resonance-stabilized aromatic carbocation is the
major reason for these high pKa values. Electron-rich carbonyl
compounds likewise possess positive protonated carbonyl pKa
values. Previously we determined that the pKa of an O-
protonated indoloquinone had a value of 1.9 and showed by
means of a Bronsted plot that electron-rich protonated quinones
can have pKa values as high as 5.5.42
Cell Line Assays. Select pyridoindole and pyrroloindole
analogues (1a-d, 2b, 2c, 9, 10, 2b, 17, 3a, and 3b) were
screened for cytostatic and cytotoxic activity in human cancer
cell line panels (Tables 1 and 2). The cytostatic parameters
include GI50 and TGI, which are the concentrations of drug
required for 50% growth inhibition and total growth inhibition,
respectively. The cytotoxic parameter is the LC50, which is the
concentration required for 50% cell kill. These data were
obtained under the NCI’s In Vitro Cell Line Screening
Project.43-45Also provided in Table 1 is the range value, which
represent the log of the maximum concentration difference
between the least sensitive and the most sensitive of the 60 cell
lines. This range parameter has been used to gain insights into
The presence of an amine substituent on the quinone ring of
1c results in mainly proton trapping upon reductive activation
(Scheme 7). Consistent with this result, the amine substituted
analogues 1b, 1c, and 1d lack any detectable cytostatic or
cytotoxic activity.
The iminoquinone derivative 9 is the only analogue that
possesses a high level of cytostatic and cytotoxic activity along
with high cell line specificity (>3 orders of magnitude between
resistant and sensitive cell lines). The histological cancers most
sensitive to 9 include CNS, melanoma, and breast cancers.
COMPARE analysis43,48 was carried out to provide insights into
what might be the molecular target of 9. The COMPARE
analysis correlates mean graph data with known molecular target
levels in cell lines and thereby generates hypotheses concerning
the agent’s mechanism of action. A molecular target is a protein
or enzyme that has been measured in the National Cancer
Institute’s panel of 60 human tumor cell lines. The levels of
over one-thousand biologically relevant molecular targets have
been determined in these tumor cell lines from measurements
of mRNA and enzyme activity levels.49 Compound 9 appears
to target a tyrosine kinase involved in cell division rather than
act as an alkylating agent activated by DT-diaphorase. Signifi-
cantly, the structurally similar quinone analogue 10 lacked
cytostatic and cytotoxic activity. Additional studies will be
carried out to evaluate 9 as an antitumor agent.
Our kinetic and product studies of 3a and 3b correctly
predicted the relative activity of the compounds against the six
human cancer cell lines listed in Table 2. These studies
(46) Xing, C.; Skibo, E. B.; Dorr, R. T. J. Med. Chem. 2001, 44, 3545-
3562.
(40) Ouyang, A.; Skibo, E. B. J. Org. Chem. 1998, 63, 1893-1900.
(41) Boruah, R. C.; Skibo, E. B. J. Org. Chem. 1995, 60, 2232-2243.
(42) Skibo, E. B.; Xing, C. Biochemistry 1998, 37, 15199-15213.
(43) Paull, D. K.; Shoemaker, R. H.; Hodes, L.; Monks, A.; Scudiero,
D. A.; Rubinstein, L.; Plowman, J.; Boyd, M. R. J. Natl. Cancer Inst. 1989,
81, 1088-1092.
(47) Kohn, K. W. Cancer Res. 1996, 56, 5533-5546.
(48) Sausville, E. A.; Zaharevitz, D.; Gussio, R.; Meijer, L.; Louarn-
Leost, M.; Kunick, C.; Schultz, R.; Lahusen, T.; Headlee, D.; Stinson, S.;
Arbuck, S. G.; Senderowicz, A. Pharmacol. Ther. 1999, 82, 285-292.
(49) Ross, D. T.; Scherf, U.; Eisen, M. B.; Perou, C. M.; Rees, C.;
Spellman, P.; Iyer, V.; Jeffrey, S. S.; Van De Rijn, M.; Waltham, M.;
Pergamenschikov, A.; Lee, J. C. E.; Lashkari, D.; Shalon, D.; Myers, T.
G.; Weinstein, J. N.; Botstein, D.; Brown, P. O. Nat. Genet. 2000, 24, 227-
235.
(44) Boyd, M. R. In Principles and Practices of Oncology (PPO updates),
3rd ed.; DeVita, V. T., Hellman, S., Rosenberg, S. A., Eds.; J. B.
Lippincott: Phildelphia, PA, 1989; Vol. 3, pp 1-12.
(45) Boyd, M. R.; Paull, K. D. Drug DeV. Res. 1995, 34, 91-109.
8644 J. Org. Chem., Vol. 72, No. 23, 2007

Pyrrolo- and Pyrido[1,2-a]indole-Based Quinone Methides
TABLE 2. In Vitro Assay Results for 18, 3a, and 3b in Six Human Cancer Cell Lines
BxPC-3
pancreas
MCF-7
breast
SF-268
CNS
H460
NSC lung
KM20L2
colorectal
DU-145
prostrate
parameter
17 log GI50
3a log GI50
3b log GI50
-4.14
>-4
-4.8
-4.75
>-4
-4.95
-4.36
>-4
>-4
-4.81
>-4
-4.95
-4.58
>-4
>-4
-4.36
>-4
-4.96
determined that the pyridoindole quinone methide formed upon
reduction of 3a is a poor nucleophile trap with a tendency to
eliminate the nucleophile. Consistent with these findings, 3a
lacked both cytostatic and cytotoxic activity. In contrast, the
pyrroloindole quinone methide derived from reduction of 3b is
an excellent nucleophile trap and accordingly this compound
exhibits significant cytostatic activity (Table 2).
slows nucleophile trapping compared to a fused pyrrolo ring.
These findings are consistent with the presence of axial hydrogen
atoms in the fused pyrido ring that result in more steric
congestion compared to the relatively flat fused pyrrolo ring.
The elimination of a large biological nucleophile (DNA) from
a pyrroloindole system was demonstrated by a previous study.40
Human cancer cell line cytostatic and cytotoxic studies
validated our conclusion that pyridoindole quinone methides
are unsuitable for alkylating agent design. The pyrroloindole
analogue 3b possessed cytostatic activity but the pyridoindole
analogue 3a was inactive. Likewise, the other pyridoindole
analogues were inactive with some exceptions. Compound 17
possesses modest cytostatic activity perhaps due to alkylation
reactions involving the quinone ring. Compound 9 is the only
analogue that possesses high levels of both cytostatic and
cytotoxic activity. The mechanism of activity likely involves
kinase cellular targets rather than alkylation reactions.
Conclusions
In the present study, we investigate pyrido[1,2-a]indole- and
pyrrolo[1,2-a]indole-based quinones capable of forming quinone
methide and vinyl quinone species upon reduction and leaving
group elimination. Our goals were to determine the influence
of the 6-membered pyrido and the 5-membered pyrrolo fused
rings on the formation and fate of these species as well as on
their cytostatic and cytotoxic activity. Our efforts were aided
by the development of a new technique for rapid quinone
reduction to the hydroquinone in a Thunberg (anaerobic) cuvette.
As result, it was possible to carry out direct kinetic studies of
quinone methide formation and fate by using Spectral Global
Fitting.20 The postulate made at the outset of this study was
that reactive species derived from the 6-membered pyrido ring
analogues would be less reactive than those derived from the
5-membered pyrrolo ring analogues. The presence of axial
hydrogens in the fused pyrido ring, in contrast to the relatively
flat fused pyrrolo ring, was the basis for this postulate.
Experimental Section
Select compounds were screened in the National Cancer Insti-
tute’s 60-cell line screen.43,50 Mean graph results were then analyzed
with COMPARE online (http://itbwork.nci.nih.gov/PublicServer/
jsp/Form_Upload.jsp).43,48 Syntheses of the compounds leading to
series 1, in vitro studies, kinetic studies, and calculations were
carried out as outlined below. The syntheses of series 2 and 3 and
the product isolation studies are described in the Supplemental
Information.
Kinetic studies of quinone methide formation and fate led to
the following conclusions: The pKa of the protonated quinone
methide is near neutrality, only protonated quinone methide traps
nucleophiles, and only neutral quinone methide traps protons.
Typically carbonyl oxygen protonations are associated with pKa
values <0 because the resulting cations are often not stable. In
contrast, O-protonated quinone methides are resonance stabilized
aromatic cations. Potential density maps show that the methide
reacting center has positive charge density only upon protona-
tion. Therefore, nucleophile trapping by a quinone methide
requires a preequilibrium O-protonation. Thus the pKa values
of O-protonated quinone methides near neutrality are largely
responsible for the biological activity of reductive alkylating
agents. Potential density maps also show that the reacting center
of the neutral quinone methide is slightly anionic (yellow color).
As a result, proton transfer to this center competes with proton
transfer to the carbonyl oxygen. Proton trapping only occurs at
pH values at or below the O-protonated quinone methide pKa.
Proton trapping product concentration vs pH data has been used
to estimate O-protonated quinone methide pKa values.41 The
Global Fitting studies described in this report have made it
possible to measure these pKa values directly.
6,7,8,9-Tetrahydro-3,10-dimethyl-2-methoxy-1-nitropyrido-
[1,2-a]indole-9-ol (5). Sodium borohydride (0.5 g, 13.2 mmol) was
added portionwise to a solution of 6,7,8,9-tetrahydro-2-methoxy-
3,10-dimethyl-1-nitro-9-oxo-pyrido[1,2-a]indole (4)12 (1.2 g, 4.16
mmol) in 70 mL of methanol cooled to 0 °C. After addition was
completed, the reaction mixture was stirred at room temperature
for 25 min. The mixture was concentrated in vacuo to dryness and
the residue was suspended in water (100 mL) and extracted with
methylene chloride (3 × 50 mL). The combined organic layers were
dried over Na2SO4, filtered, and evaporated. The remaining solids
were purified by filtration over pad of silica gel with methylene
chloride as the eluent. Compound 5 was crystallized from methylene
chloride/hexane to afford a yellow crystalline solid: 1.1 g (91%)
yield; mp 161-162 °C; IR (KBr pellet) 3408, 3030, 2924, 2866,
1521, 1444, 1371, 1276, 1238 cm-1; 1H NMR (CDCl3) δ 7.17 (1H,
s), 5.15 (1H, t, J ) 3.9 Hz), 4.22 (1H, m), 3.85 (3H, s), 3.77 (1H,
m), 2.43 (3H, s), 2,40 (1H, m), 2.20 (1H, m), 2.17(3H, s), 2.02
(1H, m); HREI MS m/z calcd for C15H18N2O4 290.1267, found
290.1277. Anal. Calcd for C15H18N2O4: C, 62.06; H, 6.25; N, 9.65.
Found: C, 61.90; H, 6.24; N, 9.46.
6,7-Dihydro-2-methoxy-3,10-dimethyl-1-nitropyrido[1,2-a]in-
dole (6). To a stirred solution of 5 (500 mg, 1.8 mmol) in dry
methylene chloride under nitrogen and cooled to 0 °C was added
345 mg (2.8 mmol) of DMAP, followed by 300 µL (3.8 mmol) of
methanesulfonyl chloride. The reaction was stirred for 1 h while
being cooled with an ice bath, and then stirring was continued at
room temperature for 48 h. Solid byproducts were removed by
filtration, the filtrate was evaporated under reduced pressure, and
The comparative kinetic studies of the pyridoindole and the
pyrroloindole quinone methides led to our conclusion that the
former are unsuitable for alkylating agent design. The reason
is that the trapping of a biological nucleophile by a pyridoindole
quinone methide would be reversible due to steric congestion.
Our experimental findings showed that the fused pyrido ring
promotes quinone methide and vinyl quinone formation but
(50) Strumberg, D.; Pommier, Y.; Paull, K.; Jayaraman, M.; Nagafuji,
P.; Cushman, M. J. Med. Chem. 1999, 42, 446-457.
J. Org. Chem, Vol. 72, No. 23, 2007 8645

Khdour and Skibo
then the crude product was purified by flash silica gel chromatog-
raphy with methylene chloride as the eluent. Compound 6 was
crystallized from methylene chloride/hexane to afford the product
as a yellow crystalline solid: 320 mg (68%) yield; mp 126-
127 °C; IR (KBr pellet) 3001, 2920, 1521, 1462, 1325, 1151, 1030,
C15H18N2O4 290.1267, found 290.1277. Anal. Calcd for C15H17-
NO5: C, 62.06; H, 6.25; N, 9.65. Found: C, 61.85; H, 6.35; N,
8.91.
cis-8,9-Diacetoxy-6,7,8,9-tetrahydro-2-methoxy-3,10-dimeth-
ylpyrido[1,2-a]indole-1,4-dione (1a). To a stirred solution of 8
(200 mg, 0.68 mmol) in 15 mL of dry methylene chloride under
nitrogen and cooled to 0 °C was added 50 mg of DMAP, followed
by 0.7 mL of acetic anhydride dropwise. The reaction mixture was
stirred for 10 min at room temperature, and then solvent was
evaporated. Crude product was purified by flash silica gel chro-
matography with methylene chloride as the eluent. Compound 1a
was crystallized from methylene chloride/hexane to afford the
product as a light orange crystalline solid: 215 mg (83%); mp 185-
186 °C; IR (KBr pellet) 3010, 2947, 1737, 1643, 1502, 1369, 1248,
1
754 cm-1; H NMR (CDCl3) δ 7.12 (1H, s), 6.66 (1H, doublet of
triplets, J ) 2.1, 6 Hz), 6.07 (1H, m), 4.04 (2H, t, J ) 6.9 Hz),
3.86 (1H, s), 2.66 (2H, m), 2.43 (3H, s), 2.12 (3H, s); HREI MS
m/z calcd for C15H16N2O3 272.1161, found 272.1153. Anal. Calcd
for C15H16N2O3: C, 66.16; H, 5.92; N, 10.29. Found: C, 66.20;
H, 5.94; N, 10.25.
6,7,8,9-Tetrahydro-2-methoxy-3,10-dimethyl-1-nitropyrido-
[1,2-a]indole-cis-8,9-diol (7). Compound 6 (500 mg, 1.8 mmol)
was added to a mixture consisting of a 0.8 mL solution of osmium
tetroxide (2.5% in 2-methyl-2-propanol), 240 mg of N-methylmor-
pholine N-oxide dihydrate, and 30 mL of t-BuOH/THF/H2O (3:1:
1). The reaction mixture was then stirred at 25 °C for 1 h. Aqueous
saturated NaHSO3 was then added to the reaction mixture which
was stirred for 1 h; this mixture was then extracted with CH2Cl2 (3
× 50 mL). The combined organic layers were dried over Na2SO4,
filtered, and evaporated under reduced pressure. Column chroma-
tography of the crude product was carried out on silica gel with
CH2Cl2/MeOH (99:1) as the eluent. Compound 7 was crystallized
from methylene chloride/hexane to afford the product as a light
yellow crystalline solid: 510 mg (89%) yield; mp g165 °C dec;
IR (KBr pellet) 3296, 3020, 2922, 1743, 1672, 1525, 1361, 1103,
1030 cm-1; 1H NMR (DMSO-d6) δ 7.45 (1H, s), 5.15 (1H, d, J )
5.7 Hz), 4.96 (1H, d, J ) 5.4 Hz), 4.79 (1H, t, J ) 3.9 Hz), 4.23
(1H, m), 3.87 (2H, m), 3.75 (3H, s), 2.38 (3H, s), 2.25 (1H, m),
2.02 (3H, s), 1.91(1H, m); HREI MS m/z calcd for C15H18N2O5
306.1216, found 306.1206. Anal. Calcd for C15H18N2O5: C, 58.82;
H, 5.92; N, 9.15. Found: C, 58.73; H, 5.80; N, 9.07.
6,7,8,9-Tetrahydro-cis-8,9-dihydroxy-2-methoxy-3,10-dimeth-
ylpyrido[1,2-a]indole-1,4-dione (8). 8 was prepared by the fol-
lowing two-step process:
The product obtained above (7, 300 mg, 0.98 mmol) was
catalytically reduced for 45 min in methanol under 50 psi of H2
employing 100 mg of 5% Pd on carbon as catalyst. After complete
reduction, the catalyst was removed by filtration through Celite
and the filtrate concentrated to a residue. No attempt was made to
purify the reactive amino diol product.
1
1024 cm-1; H NMR (CDCl3) δ 6. 37 (1H, t), 5.24 (1H, m), 4.82
(1H, m), 4.11(1H, m), 4.00 (3H, s), 2.46 (1H, m), 2.22 (3H, s),
2.12 (1H, m), 2.09 (3H, s), 2.05 (3H, s), 1.94 (3H, s); HREI MS
m/z calcd for C19H21NO7 375.1318, found 375.1332. Anal. Calcd
for C19H21NO7: C, 60.79; H, 5.64; N, 3.73. Found: C, 60.90; H,
5.59; N, 3.74.
cis-8,9-Diacetoxy-2-aziridinyl-6,7,8,9-tetrahydro-3,10-dimeth-
ylpyrido[1,2-a]indole-1,4-dione (1b). To a stirred solution of 1a
(50 mg, 0.13 mmol) in 10 mL of methanol cooled to 0 °C was
added 300 µL of aziridine dropwise. Reaction was stirred for 1.5
h at room temperature, and then the solvent was evaporated. Crude
product was purified by flash silica gel chromatography (methylene
chloride/ MeOH 98:2) as the eluent. Compound 1b was crystallized
from methylene chloride/hexane to afford the product as a light
yellow crystalline solid: 38 mg (74%); mp 191-193 °C; IR (KBr
pellet) 2947, 1737, 1643, 1369, 1249, 1024 cm-1; 1H NMR (CDCl3)
δ 6.36 (1H, d, J ) 3.3 Hz), 5.25 (1H, m), 4.90 (1H, m), 4.16 (1H,
m), 2.49 (1H, m), 2.28 (4H, s), 2.22 (3H, s), 2.10 (3H, s), 2.09
(1H, m), 2.09 (3H, s), 2.04 (3H, s); MALDI m/z calcd for
C20H23N2O6 (M + 1) 387.1551, found 387.149.
cis-8,9-Dihydroxy-6,7,8,9-tetrahydro-2-(2-methoxyethylamino)-
3,10-dimethylpyrido[1,2-a]indole-1,4-dione (10). To a stirred
solution of 8 (200 mg, 0.68 mmol) in 30 mL of methanol was added
300 µL of 2-methoxyethylamine dropwise. Reaction was stirred
overnight at room temperature, and then product was filtered off.
The crude product was crystallized from methanol to give 10 as a
magenta solid: 185 mg (80%); mp g191 °C dec; IR (KBr pellet)
1
3331, 3254, 2922, 1660, 1599, 1504, 1294, 1011, 952 cm-1; H
This product was dissolved in 2 mL of MeOH to which was
added 30 mL of 0.1 M monobasic potassium phosphate buffer held
at pH 3.20. To this solution was added a solution consisting of 4
g of Fremy salt in 100 mL of the same phosphate buffer. The
reaction mixture was stirred at room temperature for 45 min and
then extracted with 4 × 50 mL of chloroform. The dried extracts
(Na2SO4) were concentrated and the residue purified with flash
column chromatography on silica gel employing CH2Cl2/MeOH
(97:3) as the eluent. Two fractions were collected: The major
compound (8) was crystallized from methylene chloride/hexane to
afford a light orange crystalline solid: 167 mg (58%); mp
g170 °C dec; IR (KBr pellet) 3400, 3296, 3010, 2930, 1601, 1639,
1502, 1446, 1325, 1101, 1001 cm-1; 1H NMR (DMSO-d6) δ 5.16
(1H, d, J ) 5.4 Hz), 4.96 (1H, d, J ) 5.4 Hz), 4.64 (1H, t), 4.43
(1H, m), 4.06 (1H, m), 3.85 (3H, s), 3.81 (1H, m), 2.23 (3H, s),
2.12 (1H, m), 1.82 (3H,s), 1.81 (1H, m); HREI MS m/z calcd for
C15H17NO5 291.1107, found 291.1101. Anal. Calcd for C15H17-
NO5: C, 61.85; H, 5.88; N, 4.81. Found: C, 61.64; H, 5.83; N,
4.77. The minor product 6,7,8,9-tetrahydro-cis-8,9-dihydroxy-2-
methoxy-3,10-dimethylpyrido[1,2-a]indole-1-imino-4-one (9) was
crystallized from methylene chloride/hexane to afford the product
as a light yellow crystalline solid: 57 mg (20%); mp g166 °C
dec; IR (KBr pellet) 3246, 2939, 1751, 1637, 1444, 1296, 1217,-
1095, 1001 cm-1; 1H NMR (DMSO-d6) δ 10.76 (1H, S), 5.10 (1H,
d, J ) 5.4 Hz), 4.93 (1H, d, J ) 5.4 Hz), 4.68 (1H, broad), 4.55
(1H, m), 4.09 (1H, m), 3.83 (2H, m), 3.72 (3H, s), 2.33 (3H, s),
2.16 (2H, m), 1.89 (3H, s), 1.84 (1H, m); HREI MS m/z calcd for
NMR (DMSO-d6) δ 6.11 (1H, NH), 5.04 (1H, d, J ) 5.4 Hz), 4.90
(1H, d, J ) 5.4 Hz), 4.62 (1H, m), 4.45 (1H, m), 3.99 (1H, m),
3.80 (1H, m), 3.63 (2 H, m), 3.48 (2H, m), 3.26 (3H, s), 2.20 (3H,s),
2.07 (1H,m), 1.93 (3 H, s), 1.83(2H, m); HREI MS m/z calcd for
C17H22N2O5 334.1529, found 334.1540. Anal. Calcd for C17H22N2O5
C, 61.07; H, 6.63; N, 8.38. Found: C, 61.08; H, 6.61; N, 8.26.
cis-8,9-Diacetoxy-6,7,8,9-tetrahydro-2-(2-methoxyethylamino)-
3,10-dimethylpyrido[1,2-a]indole-1,4-dione (1c). To a stirred
solution of 10 (100 mg, 0.30 mmol) in 50 mL of dry methylene
chloride cooled to 0 °C was added 35 mg of DMAP, followed by
dropwise addition of 0.5 mL of acetic anhydride. The reaction was
stirred for 45 min at room temperature and then the solvent was
evaporated in vacuo. The residue was suspended in 100 mL of water
and the product was extracted with ethyl acetate (4 × 50 mL). The
combined organic layers were dried over Na2SO4, filtered, and
evaporated. Crude product was purified by flash silica gel chro-
matography with methylene chloride/ MeOH (96:4) as the eluent.
Compound 1c was crystallized from methylene chloride/hexane to
afford a magenta solid: 100 mg (80%) yield; mp 147-148 °C; IR
(KBr pellet) 3323, 3010, 2926, 1737, 1666, 1602, 1508, 1248, 1107
cm-1; 1H NMR (DMSO-d6) δ 6.25 (1H, d, J ) 4.8 Hz), 6.21 (1H,
NH), 5.25 (1H, doublet of triplets, J ) 3.3, 4.8 Hz), 4.75 (1H, m),
4.17 (1H, m), 3.67 (2H, m), 3.49 (2H, m), 3.27 (3H. s), 2.28 (1H,
m), 2.10 (3H, s), 2.08 (1H, m), 2.06 (3H, s), 1.99 (3H, s), 1.95
(3H, s); HREI MS m/z calcd for C21H26N2O7 418.1740, found
418.1736. Anal. Calcd for C21H26N2O7: C, 60.28; H, 6.26; N, 6.69.
Found: C, 60.24; H, 6.25; N, 6.60.
8646 J. Org. Chem., Vol. 72, No. 23, 2007

Pyrrolo- and Pyrido[1,2-a]indole-Based Quinone Methides
cis-8,9-Dihydroxy-6,7,8,9-tetrahydro-2-(2-methoxyethylamino)-
3,10-dimethylpyrido[1,2-a]indole-1,4-dione Carbonic Acid Ester
(1d). Triphosgene (100 mg, 0.33 mmol) in dry methylene chloride
was added dropwise to a stirred solution of 10 (100 mg, 0.3 mmol)
in 0.5 mL of dry pyridine and 10 mL of dry CH2Cl2 cooled to
0 °C. The reaction was stirred at room temperature and the reaction
progress was monitored by thin layer chromatography. After 5 min,
the solution was diluted with water and the two phases were
separated and the aqueous phase was extracted with CH2Cl2 (3 ×
5 mL). The combined organic phases were dried (Na2SO4) and
concentrated to a residue, which was purified by flash chromatog-
raphy on silica gel with CH2Cl2 as the eluent. The isolated carbonate
was recrystallized from CH2Cl2/hexane: 60 mg (56%) yield; mp
g159 °C dec; IR (KBr pellet) 3338, 2926, 1786, 1655, 1599, 1508,
buffer systems were used to hold pH: 1 M acetate buffer (pH 4 to
6.5), 0.2 M phosphate buffer (pH 6.5 to 8.5), and 0.2 M borate
buffer (pH 7.5 to 10). All buffers were maintained at an ionic
strength (µ) of 1.0 with KCl. The temperature of all the kinetic
measurements was maintained at 30.0 ( 0.3 °C with circulating
water from a thermostatic water bath.
The reactions were carried out in Thunberg cuvettes in the
following way: To the bottom port of the Thunberg was added
2.8 mL of buffer and 100 µL of a sonicated suspension consisting
of 50 mg of 5% Pd on carbon in 10 mL of water. The amount of
catalyst added to the bottom port was 0.5 µg. To the top port of
the Thunberg cuvette was added 100 µL of the DMSO stock of 3a
or 3b. The cuvette was sealed with Apiazon L grease and Teflon
tubes from a hydrogen source were threaded into the top and bottom
ports. After the ports were purged with hydrogen gas for 5 min,
the cuvette was sealed and equilibrated at 30 °C in the thermostated
spectrometer. We started the reaction by combining the top and
bottom ports. Reactions were monitored between 240 and 600 nm
with a UV-visible spectrophotometer equipped with Spectral
Global Fitting software. The reaction times varied from 400 to 600
min depending on the pH of the reaction. When the reactions were
complete the pH of the samples was taken and recorded for use on
the pH-rate profile.
Calculations. The absorbance vs time vs wavelength surfaces
obtained as described above was fit with Global fitting software.20,38
These fits provide first-order and two consecutive first-order rate
constants along with intermediate spectra, if any. Log kobsd vs pH
data were fit with Graph Pad Prism 3 software, which was also
used to generate the solid curve shown in pH-rate profiles.
Electrostatic potential maps were obtained with Hartree-Fock
calculations that were carried out with the 3-21G(*) basis set.36
1
1122, 1024 cm-1; H NMR (CDCl3) δ 6.05 (1H, NH), 5.84 (1H,
d, J ) 8.4 Hz), 5.19 (1H, m), 5.13 (1H, t, J ) 4.5 Hz), 4.17 (1H,
m), 3.72 (2H, m), 3.57 (2H, m), 3.38 (3H, s), 2.42 (1H, m), 2.32
(3H, s), 2.07 (1H, m), 2.05 (3H, s); HREI MS m/z calcd for
C18H20N2O6 360.1321, found 360.1338. Anal. Calcd for
C18H20N2O6: C, 59.99; H, 5.59; N, 7.77. Found: C, 58.64; H, 5.47;
N, 7.38.
The NCI in Vitro Screening Procedures. Compounds were
tested against all cell lines included in the 60 cell line panel. All
cell lines are inoculated onto a series of standard 96-well microtiter
plates that consists of cell suspensions that were diluted according
to the particular cell type (expected target cell density 5000-40000
cells per well based on cell growth characteristics). Inoculates were
preincubated for a period of 24 h at 37 °C in the absence of any
test compound. Each test compound was added in 100 µL aliquots
to the microtiter plate wells and evaluated at five 10-fold dilutions
with a high concentration of 10-4 M. Incubation with test
compounds lasted 48 h in an atmosphere of 5% carbon dioxide
with 100% humidity. The cells are assayed by the sulforhodamine
B procedure. Afterward, a plate reader was used to read the optical
densities, and a computer processed the optical densities into the
special concentration parameters GI50, TGI, and LC50.
Acknowledgment. We thank the Arizona State University
College of Liberal Arts and Sciences for their generous support
and Jean-Charles Chapuis for running the assays shown in Table
2.
Kinetic Studies of Quinone Methide Formation and Fate. The
stock solutions of 3a and 3b were prepared by diluting 4 to 5 mg
in a 5 mL volumetric flask with dry dimethyl sulfoxide (DMSO)
to afford solutions with concentrations of 2.5 to 3.3 mM. All kinetic
studies were carried out with buffers prepared from doubly distilled
water. Final concentrations of the 3a and 3b in reaction mixtures
ranged from 83 to 110 µM. DMSO concentrations in reaction
mixtures were kept <2% to minimize solvent effects. The following
Supporting Information Available: Proton NMR spectra of
reported compounds and 13C NMR spectra of select compounds
along with the experimental details for the syntheses of compound
series 2 and 3. This material is available free of charge via the
Internet at http://pubs.acs.org.
JO070866O
J. Org. Chem, Vol. 72, No. 23, 2007 8647